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BIOCHEMISTRY RESEARCH TRENDS

PALMITIC ACID OCCURRENCE, BIOCHEMISTRY AND HEALTH EFFECTS

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BIOCHEMISTRY RESEARCH TRENDS

PALMITIC ACID OCCURRENCE, BIOCHEMISTRY AND HEALTH EFFECTS

LUCAS F. PORTO EDITOR

New York


Copyright © 2014 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers‘ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works. Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. Additional color graphics may be available in the e-book version of this book.

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Published by Nova Science Publishers, Inc. † New York


CONTENTS Preface

vii

Chapter 1

Fatty Acids in Vascular Health Reggie Hui-Chao Lee, Carl S. Wilkins, Alexandre Couto e Silva, Stephen E. Valido, Celeste Yin-Chieh Wu and Hung Wen Lin

Chapter 2

Occurrence, Biochemical, Antimicrobial and Health Effects of Palmitic Acid Melissa Johnson and Daniel A. Abugri

17

Palmitic Acid: Effect of Diet Supplementation and Occurrence in Animal Origin Food P. G. Peiretti

45

Chapter 3

1

Chapter 4

General Aspects of Palmitic Acid Deusdélia Teixeira de Almeida, Mariana Melo Costa and Sabrina Feitosa

Chapter 5

Palmitic Acid As a Cardiometabolic Risk Factor Danijela Ristić-Medić and Vesna Vučić

105

Chapter 6

Palmitic Acid in Higher Plant Lipids R.A. Sidorov, A.V. Zhukov, V.P. Pchelkin and V.D. Tsydendambaev

125

Chapter 7

Palmitic Acid in Infant Nutrition Fabiana Bar-Yoseph, Yael Lifshitz, Tzafra Cohen and Ita Litmanovitz

145

Chapter 8

Processing of Palmitic Acid and Its Derivatives Using Supercritical Fluids C. E. Schwarz

Chapter 9

Palmitic Acid in Tunisian Olive Oil: Updating and Perspective Ghayth Rigane and Ridha Ben Salem

Index

63

159 211 219



PREFACE This book discusses the occurrence, biochemistry, and health effects of palmitic acid. Chapter 1 - Fatty acids have traditionally been described as artery clogging species that is detrimental to overall health. The most prevalent fatty acid is palmitic acid (PA), a sixteen carbon chain fatty acid that is ubiquitous in biological systems. PA is prevalent in most eukaryotic cell membranes and in the mitochondria derived from the Krebs‘ cycle utilizing acetyl-coenzyme A as its precursor. PA is found in a variety of plants with a high amounts in coconut oil. Many cosmetics, shampoos, and commercialized beauty products contain PA providing structure and substance to the gel or reagent. An emerging field of study is the esterified form of PA or methyl palmitate, as it is involved in biological signaling in the central nervous system. More specifically, methyl palmitate or palmitic acid methyl ester can cause arterial vasodilation and is thought to be involved in neurotransmission, as well as modulate vascular tonicity in cerebral circulation. Methyl palmitate has also been implicated as a neuroprotective agent in both models of focal and global cerebral ischemia; however, the exact mechanism(s) are still unknown. The authors will focus on the known pharmacology, biochemistry, and clinical implications of PA and other related fatty acids (i.e. Non-esterified v. esterified fatty acids) commonly found in daily diets. Additionally, cellular target(s) of PA will be discussed as it relates to improvement of disease states, synthesis, and possible health implications/benefits of methyl palmitate in biological systems. Chapter 2 - Palmitic acid (PA), one of the most abundant saturated fatty acid (SFAs) within plants, humans, animals, microbial (bacteria), fungal, and marine organisms, constitutes ~16 to 45 % of the lipid profile. The impressive abundance of PA throughout nature could be attributed partly to its critical role in membrane lipid structural functionality, formation of subcellular cysteine residue linkages and RNA posttranslational modifications in eukaryotic and prokaryotic cells. PA has been demonstrated to undergo β-oxidation to produce short and medium chain fatty acids to maintain homeostasis in response to endogenous and exogenous cues. Further, non-esterified PA is known to mediate numerous biochemical and antimicrobial pathways towards the betterment of human health. Although the importance of PA in human health and nutrition are established, critics attest that excessive dietary PA may be ―unhealthy‖ and even detrimental. Current microbiological and epidemiological studies suggest that PA produces specific health benefits that are not common to all other SFAs. For example, studies suggest that PA may activate nitric oxide and superoxide, thus functioning as an antimicrobial agent against some strains of bacteria,


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algae, and helminthes and certain foodborne pathogens. This chapter reviews the occurrence, biochemistry, and subsequent health implications of PA. Chapter 3 - In the last few decades, disagreement between opinions and findings concerning the ability of palmitic acid (PA) and other saturated fatty acids (SFAs) to raise cholesterolaemia has led to discussions on whether PA, which has been positively related to high serum cholesterol levels, could increase the risk of cardiovascular diseases. This study aims to review the PA content of meat, dairy products, fish, and other food of animal origin in the human diet and discusses nutritional issues related to the occurrence of this fatty acid (FA) in these foods due to different diet supplementation. Meat and dairy products are considerable dietary sources of SFAs, such as PA. In most industrialized countries, a high meat or dairy intake contributes to a higher than recommended SFA intake. Palmitic and myristic acids are common FAs in meat and dairy products, making up about 30-40% of total FA intake and are the main factors responsible for raising cholesterol levels; indeed, strong evidence indicates that these two SFAs increase serum cholesterol concentrations in humans. Stearic acid is partially converted to oleic acid in vivo and has not been shown to elevate blood cholesterol, while lauric acid is not as potent as PA at raising concentrations of total cholesterol and LDL cholesterol in humans. The occurrence of PA in animal origin food is influenced by both genetic and environmental factors, such as the composition of the animal‘s diet, its digestive system and its biosynthetic processes. The FA profile in food of animal origin mainly reflects dietary lipid sources and has the potential to play a valuable role in human nutrition by manipulating the composition of animal fat through diet. In order to explain the variability in FA composition in food of animal origin, this review examines different nutrition trials that have studied the effects of PA supplementation on the lipid profile of animal origin food. Chapter 4 - Palmitic acid or hexadecanoic acids is the most abundant saturated fatty acid in human nutrition and represents about 17.6g per day in the United Kington diet. It is the first fatty acid produced during the lipogenesis. During this process, glucose is converted to fatty acids, which then react with glycerol to produce triacylglycerols. Palmitic acid mainly occurs as its ester in triglycerides, especially in palm oil (40-44 %) but also in lard (20-30 %), dairy products (25-40 %) and cocoa butter (25-27 %). One of the main applications of palmitic acid in the food industry has been the formulation of interesterified fats, used as a replacement of trans fats. In breast milk, native lard, enzyme-directed and randomly chemically interesterified plant fats, palmitic acid is predominantly esterified to triacylglycerol, center or β-position, in native palm oil and cow´s milk, it is mainly at the external or α-positions. A higher palmitic acid absorption is obtained with formulas rich in palmitic acid esterified in triacylglycerol sn-2 position, than with those containing palmitic acid predominantly esterified in the sn-1,3 positions. These specific fatty acids distributions in triacylglycerol, determine the physical properties of the fat, which affects its absorption, metabolism and distribution into tissues. Many authors claim that a palmitic acid intake may promote increased risk of hypercholesterolemia, liver disease, type 2 diabetes, insulin resistance and toxicity. However, more recent investigations on the topic seem to have reconsidered the negative role of the dietary saturated fatty acids as a risk factor for cardiovascular diseases and show that not only the type of fat, but also that the triglyceride structure plays a role in these diseases. Chapter 5 - Current dietary recommendations are based on a reduced saturated fatty acid (SFA) consumption to prevent cardiovascular disease (CVD). The role of individual SFA in


Preface

ix

metabolic disease is not fully understandable. One type of SFA present in many common foods (dairy, meat, palm and coconut oil) is palmitic acid (16:0). A number of epidemiological studies have shown that the populations who consume large amounts of atherogenic SFA (especially palmitic, myristic, lauric) have elevated levels of LDL and HDLcholesterol. Saturated fatty acid exert their atherogenic and thrombogenic effect through increased production of LDL, very-low-density lipoproteins particles and apolipoproteins A1, with a decrease of LDL- receptors specific activity, and an increase in platelet aggregation. The total cholesterol/ HDL-cholesterol ratio, the best overall indication of potential effects on coronary heart disease (CHD) risk is nonsignificantly affected by consumption of palmitic acid (PA). Compared with lipid effects, the influence of SFA intake on inflammation markers is less well explored. The associations between circulating and tissue PA and dietary intake of PA are diverse and most likely reflecting endogenous metabolism. Status of PA is not in intake–response relationship biomarker, probably partly due to conversion of 16:0 to 16:1 by steaoryl-CoA-desaturase (SCD-1). Increased SFA intake has been associated with increased SCD-1 activity in which may predict mortality. Palmitoylation is the process involved in protein–membrane interactions and signal transduction. Increases in dietary intake of PA decrease fat oxidation and daily energy expenditure with slight increases in adiposity. Evidence for the effects of SFA, particularly PA consumption on insulin resistance, vascular function, type 2 diabetes, and stroke is various. It is considered that circulating PA, as nonesterified fatty acids stimulate insulin resistance by decreasing phosphorylation of the insulin receptor and insulin receptor substrate-1. In muscle cells, PA decrease oxidation of fatty acids and glucose which elevates fatty acid and glucose levels in tissues and blood, and decreases adiponectin production, which may both promote insulin resistance. It was shown that 16:0 and 14:0 stimulate β-cells and endothelial dysfunction. The incidence of type 2 diabetes was associated with total SFA levels of plasma cholesterol esters (also demonstrated for 16:0 levels independently) and phospholipids (also for 16:0 and 18:0). In skeletal muscle phospholipids, PA has been negatively associated with insulin sensitivity and diabetes type 2. Systematic reviews on prospective cohort studies indicated that CHD risk has not been directly associated with SFA intake, although is associated with a dietary habits, high in SFArich foods. Taken together, there is collective convincing evidence for decreased CHD risk when replacing SFA with polyunsaturated fats. Differences in cardiometabolic risk appear greater between food groups and overall dietary patterns rather than between separate SFA. Chapter 6 - Palmitic acid (C16:0) is one of the major fatty acids (FAs) forming virtually all natural lipids. Both in eu-, and prokaryotes, C16:0 forms various lipid classes, which serve either as the lipid background of storage fats and oils, or the hydrophobic matrix of cell membranes, or the components of cuticle waxes and polymers. Non-esterified С16:0 does not occur in living cells, and it is present there only as an acyl residue in various lipid classes, such as mono-, di-, and triacylglycerols, glyco-, phospho-, and sphingolipids, wax and steryl esters etc., where it esterifies the hydroxy groups of glycerol backbone or other alcohols (sphingosine, higher and lower aliphatic alcohols etc.). Palmitic acid is known to be a primary higher FA synthesized in the cell, while nearly all other FAs of natural lipids are the products of its further modification caused by elongation, desaturation, insertion of various functional groups, such as methyl, hydroxy, oxo, epoxy, etc. As a saturated FA, C16:0 is used by the cell for regulating its functional state by shifting the membrane fluidity under adverse environmental conditions and thus providing a necessary molecular species composition of the membrane polar lipids. Among the latter, such classes as phosphatidylinositols,


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Lucas F. Porto

phosphatidylserines, and other highly polar lipids are particularly rich in palmitic acid. In accordance, its content in plant lipids rises as they became less TLC-mobile, more difficultly extractable, or tightly bound. It is evident that further screening of plant lipids as regards this index is of considerable interest. Chapter 7 - Human breast milk provides the optimum nutrition for infants. Designed to provide balanced nutrition, human breast milk naturally meets the needs of growing infants in the first months after birth. In human breast milk, and in most infant formulas, approximately 50% of the energy is supplied to newborns as fat, of which more than 98% is in the form of triglycerides. Triglyceride synthesis occurs in the mammary gland. The fatty acids are specifically positioned to sn1, sn2 or sn3 positions on the glycerol backbone to yield the structure-specific triglycerides that are found in human milk. Palmitic acid (C16:0) is the predominant saturated fatty acid, comprising 17-25% of the fatty acids in mature human milk. Surprisingly, the positioning of palmitic acid is highly conserved across populations, and approximately 70-75% of palmitic fatty acids are esterified to the sn2 position of the triglyceride (sn2 palmitate). Clinical and pre-clinical studies over the last three decades have provided increasing evidence that this specific positioning of palmitic acid on the triglycerides in human milk has a significant holistic effect on optimal infant development and well-being that is related to the increased absorption of both palmitic acid and calcium: softer stools, increased bone strength, increased beneficial gut flora, controlled intestinal health, and reduced infant crying. All of these contribute to the benefits of infant wellbeing. The overall aim of the current review is to expand the understanding of the role of palmitic acid and its specific sn2 position in infant nutrition. Chapter 8 - Palmitic acid, either in its triglyceride form or hydrolysed as a free fatty acid or an ester, needs to be extracted from its source, processed and isolated to obtain useful products. The objective of this work is to consider the use of SCF (supercritical fluid) processing as a method to extract and process palmitic acid and/or its derivatives. A phase behaviour analysis, in supercritical CO2, ethane and propane, at temperatures close to the critical point of the solvent show moderate solubility of palmitic acid and tripalmitin at pressures below 50 MPa and total solubility of methyl and ethyl palmitate at pressures below 25 MPa. Analysis of the phase behaviour considered and studies presented in the literature have shown that SCFs can be widely applied to the processing of palmitic acid containing compounds. In particular SCFs can fractionate a mixture of acids or their derivatives according to the chain length, it can de-acidify an edible oil and it is able to fractionate a mixture containing palmitic acid and other compounds. Additionally, SCFs can also be used to extract palmitic acid containing triglycerides from plant material. SCFs, in particular CO2, are thus excellent alternative solvent to traditional organic solvents and should be considered when processing palmitic acid containing products. Chapter 9 - In this review the major saturated fatty acid, palmitic acid, of Virgin Olive Oil (VOO) was studied. This oil is one of the oldest known vegetable oils and it plays a fundamental role in human nutrition around the Mediterranean basin. This nature juice is the only edible oil of great production obtained by physical methods from the fruit Olea europaea L. Consideration of VOO as a natural functional fat is related to the presence of palmitic acid. Updating of its levels in Virgin olive oils throughout the Tunisian olive oil as well as information on expecting levels in other products from olive tree establish the author‘s view point. Studies on levels palmitic acid upon maturity stage in the oil are also discussed.


Preface

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Major analytical practices are given in brief. Palmitic acid (C16:0) is the principal saturated fatty acid in olive oil, responsible for its figeability at low temperature. Few are the exceptions as palmitic acid content depends heavily on the genetic factor. Palmitic fatty acids, important for the nutritional properties of an olive oil, showed a crucial rule in the characterization of olive oils.



In: Palmitic Acid: Occurrence, Biochemistry and Health Effects ISBN: 978-1-63321-519-1 Editor: Lucas F. Porto © 2014 Nova Science Publishers, Inc.

Chapter 1

FATTY ACIDS IN VASCULAR HEALTH Reggie Hui-Chao Lee1, Carl S. Wilkins2, Alexandre Couto e Silva1, Stephen E. Valido1, Celeste Yin-Chieh Wu1 and Hung Wen Lin1, 1

Cerebral Vascular Disease Laboratories, Department of Neurology, University of Miami Miller School of Medicine, Miami, FL, US 2 Florida International University Herbert Wertheim College of Medicine, Miami, FL, US

ABSTRACT Fatty acids have traditionally been described as artery clogging species that is detrimental to overall health. The most prevalent fatty acid is palmitic acid (PA), a sixteen carbon chain fatty acid that is ubiquitous in biological systems. PA is prevalent in most eukaryotic cell membranes and in the mitochondria derived from the Krebs‘ cycle utilizing acetyl-coenzyme A as its‘ precursor. PA is found in a variety of plants with a high amounts in coconut oil. Many cosmetics, shampoos, and commercialized beauty products contain PA providing structure and substance to the gel or reagent. An emerging field of study is the esterified form of PA or methyl palmitate, as it is involved in biological signaling in the central nervous system. More specifically, methyl palmitate or palmitic acid methyl ester can cause arterial vasodilation and is thought to be involved in neurotransmission, as well as modulate vascular tonicity in cerebral circulation. Methyl palmitate has also been implicated as a neuroprotective agent in both models of focal and global cerebral ischemia; however, the exact mechanism(s) are still unknown. We will focus on the known pharmacology, biochemistry, and clinical implications of PA and other related fatty acids (i.e. Non-esterified v. esterified fatty acids) commonly found in daily diets. Additionally, cellular target(s) of PA will be discussed as it relates to improvement of disease states, synthesis, and possible health implications/benefits of methyl palmitate in biological systems.



Corresponding author: Hung Wen Lin, Ph.D. University of Miami, Miller School of Medicine, Department of Neurology, Cerebral Vascular Disease Research Center, Two Story Laboratory (TSL), Medical Campus, Locator: D4-5, 1420 N.W. 9th Avenue, Miami, FL 33136. E-mail: [email protected].


2

Reggie Hui-Chao Lee, Carl S. Wilkins, Alexandre Couto e Silva et al.

Keywords: Palmitic acid methyl ester, methyl palmitate, stearic acid, stearic acid methyl ester, cerebral ischemia, stroke, vasodilation

INTRODUCTION High fatty acid content has been commonly associated with an increased risk of cardiovascular diseases in humans [1]. The most prevalent of fatty acids are palmitic (16 carbon) and stearic (18 carbon) acids, which are fatty acids ubiquitously present in biological systems abundant in eukaryotic cell membranes. Palmitic acid (PA) is the most common saturated fatty acid found in various organisms. Synthesis of PA is well-known and occurs naturally in mammalian cells via acetyl-coenzyme A (CoA) and malonyl-CoA precursors. Alternatively, the synthesis and function of the esterified form of palmitic and stearic acid, palmitic acid methyl ester (PAME) and stearic acid methyl ester (SAME), has not been welldocumented (Figure 1) [2]. Only recently has PAME and SAME been introduced to the forefront of fatty acid research in biology and disease. Specifically, PAME results in aortic vasodilation and neuroprotection, while SAME causes neuroprotection all in the context of cerebral ischemia. Thus far, PAME and SAME are thought to act as a neurotransmitter/ modulator released from a neuronal source (SCG, superior cervical ganglion SCG) [3].

Figure 1. Structures of palmitic acid, stearic acid, PAME, and SAME.

PALMITIC ACID METHYL ESTER (PAME) PA is the most common saturated fatty acid (16:0) and the main component in many types of cellular membranes [4]. Recent studies suggest that fatty acids may have vasoactive properties involved in circulation. Interestingly, PA alone does not have any vasoactive properties, but methylated PA, also known as methyl palmitate or PAME, has been reported to be a potent vasodilator [5-7]. PAME was first discovered in the superior cervical ganglion (SCG). The SCG is the largest of the three ganglions in the sympathetic chain, and is thought to be the origin of innervations of cerebral blood vessel [3]. Field electrical stimulation of the SCG caused simultaneously release of PAME and SAME [5] further enhanced by the presence of arginine analogs such as L-arginine and nitric oxide synthase (NOS) inhibitor


Fatty Acids in Vascular Health

3

(N-nitro-L-arginine). Subsequent investigations have led to the discovery of PAME in other locale such as perivascular adipose tissue (PVAT), and the retina inducing strong aortic vasodilation [5-7] suggesting that PAME plays a crucial role in regulating systemic circulation and cerebral blood flow (CBF) in the retina and other peripheral circulation in vitro. Since PAME is an abundant component in PVAT and the retina, it is highly likely that PAME is the PVAT-derived relaxing factor as well as the retina-derived relaxing factor, respectively [6, 7]. Furthermore, the EC50 (half maximal effective concentration) for PAMEinduced aortic vasodilation is 1.92 (0.46-7.93) x10-10 M which is much lower than other known nitric oxide (NO, one of the most potent vasodilators) donors such as sodium nitroprusside, 1-[2-(carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate, and N-[4-[1-(3Aminopropyl)-2-hydroxy-2-nitrosohydrazino]butyl]-1,3-propanediamine [5, 8, 9] indicating that PAME is a novel and potent vasodilator. The exact mechanism(s) underlying PAME-induced vasodilation has not been welldefined. However, PAME-induced vasodilation is inhibited by voltage-gated potassium (Kv) channel blockers such as tetraethylammonium and 4-aminopyridine [7] suggesting that PAME may regulate vascular tone via opening of Kv channels hyperpolarizing vascular smooth muscle cells (VSMCs) in blood vessels. The release of PAME from PVAT is significantly reduced in genetically-altered hypertensive animals (spontaneously hypertensive rats, SHR) [7] indicating that PAME may be involved in blood pressure regulation. Focal or global ischemia-induced hypoperfusion that lasts from hours to days is one of the contributors to neuronal cell death and neurocognitive (learning/memory) deficits after ischemia [10, 11].Administration of PAME alleviates focal and global ischemia-induced hypoperfusion (hypoperfusion decreases CBF) to reduce cerebral injury after ischemia due to the fact that PAME causes potent vasodilator [12].

ARACHIDONIC ACID (AA) Besides PAME and SAME, other fatty acids have also been shown to have vasoactive properties. For example, AA is a 20-carbon polyunsaturated -6 fatty acid (20:4 n-6), which is an essential fatty acid found in peanut oil and eggs/meats. AA has been shown to cause endothelium- and K+-independent vasodilation [13] metabolized by cytochrome P450 enzymes in the liver to form epoxyeicosatrienoic acid (EET) (Figure 2). EETs are produced in the vascular endothelium and causes potent vasodilation in renal, mesenteric, coronary, and cerebral arterioles in a variety of mammalian species [14-17]. EETs hyperpolarize VSMCs by activating calcium-activated potassium channels (KCa) channels. Several investigators have proposed that EET may serve as a possible candidate for endothelium-derived hyperpolarizing factor (The exact structure and identity of EDHF is currently unknown) due to the fact that EETs are produced in the endothelium [18]. EETs can lower systemic blood pressure in SHR-hypertensive rats suggesting possible therapeutic validity [19, 20].


4


Figure 2. Structures of arachidonic acid, and epoxyeicosatrienoic acid.

NON-ESTERIFIED FATTY ACIDS (NEFAS) NEFAs are a major component of triglycerides also known as, lauric (C12:0), myristic (C14:0), palmitic (C16:0), stearic (C18:0) or linolenic (C18:3) acid (Figure 3). Some NEFAs have been reported to inhibit endothelium-dependent vasodilation at physiological concentrations [21] indicating that NEFAs may prove crucial in disease processes such as atherosclerosis-mediated endothelium dysfunction [22] and also associated with major risk factors for cardiovascular diseases and metabolic syndrome. Intravenous infusion of NEFAs inhibits vasodilation via attenuation of both endothelium-dependent and independent (bradykinin-mediated) vasodilation, and enhanced vascular α-adrenergic receptor sensitivity [23]. Patients with high plasma concentration of NEFAs have been associated with obesityand type II diabetes (non-insulin-dependent)-mediated hypertension [24].Interestingly, the dihydropyridine family of calcium channel blockers (e.g. nifedipine and amlodipine) and -3 fatty acid supplement (e.g. fish oil) has been reported to either reduce plasma NEFAs concentration or inhibit NEFAs-induced endothelial dysfunction [25, 26] used to treat obesity- and/or type II diabetes-mediated hypertension.

-3 FATTY ACIDS Eicosapentaenoic acid (EPA, 20:5n-3), docosahexaenoic acid (DHA, 22:6n-3) (Figure 4), and α-linolenic acid (ALA, 18:3n-3, rich in plant oils) are three major -3 essential fatty acids involved in cardiovascular function and general circulation. Administration of EPA can 1) induce endothelium-independent aortic and mesenteric vasodilation via activation of K+ATP channels on VSMCs via EPA-derived prostanoids [27], 2) Activation of largeconductance/Ca2+-mediated K+ channels (BK) on VSMCs by EPA metabolite, 17,18-EET [28]. In addition, clinical research studies on the administration of EPA can inhibit the onset and progression of atherosclerosis and decrease the prevalence of myocardial infarction [29].



5

Figure 3. Structures of lauric, myristic, or linolenic acid.

Figure 4. Structures of -3 fatty acids, acidseicosapentaenoic acid and docosahexaenoic acid.

DHA has been shown to induce endothelium-independent vasodilation. The mechanism(s) underlying DHA-induced vasodilation are 1) activation of ATP-sensitive K+ channels in VSMCs by prostanoids (DHA metabolite) [30], 2) inhibition of L-type Ca2+ channel and intracellular calcium release in VSMCs [30]. Ingestion of -3 fatty acid supplements (e.g. fish oil and corn oil) in rats reduced norepinephrine or vasopressin-mediated aortic vasoconstriction and enhanced endothelium-dependent vasodilation via acetylcholine. Furthermore, DHA-mediated vasodilation was prevalent in spontaneous hypertensive rat aorta suggesting that dietary intake of DHA is beneficial to counteract hypertension [30, 31]. Similarly, ALA causes coronary arterial vasodilation via activation of VSMC Na+/K+ATPase-mediated hyperpolarization [32]. Administration of ALA can also increase CBF and vasodilation of rodent basilar artery (via activation of TREK-1 potassium channel) [33] indicating that ALA may also have therapeutic value used to combat stroke/ischemia by increasing cerebral circulation. Dietary intake of -3 fatty acid supplements has been shown to enhance endotheliumdependent brachial arterial vasodilation in hypercholesterolemic patients [34] and reduced forearm vascular resistance to angiotensin II (a potent vasoconstrictor) in normotensive men


6


[35]. Furthermore, dietary intake of fish oil has also shown to enhance the coronary vasomotor activity in patients with coronary artery disease [36] and reduce the number of deaths derived from chronic cardiac failure [29]. Altogether, these data suggest that dietary intake of -3 fatty acid supplements are beneficial to counteract against cardiovascularrelated diseases such as hyperlipidemia, hypertension, atherosclerosis, and myocardial infarction.

LOW-DENSITY LIPOPROTEIN (LDL) V. HIGH-DENSITY LIPOPROTEIN (HDL) LDL is one of the major carriers of cholesterol in circulation. LDL is also known as a “bad” lipoprotein due to the fact that LDL transports cholesterol and other fat molecules to peripheral tissues (i.e. arterial walls) and plays a crucial role in the development of several cardiovascular-related diseases such as atherosclerosis, stroke, and myocardial infarction. Oleic acid and lysophosphatidylcholine (Figure 5) are the major constituents of LDL. Oxidized LDL (ox-LDL) is rapidly engulfed by macrophages to induce foam cell formation in the arterial wall [37]. Therefore, ox-LDL is thought to be one of the major contributors to the development of atherosclerosis. Moreover, ox-LDL can enhance coronary vasospasm (vasoconstriction) via induce endothelium-dependent vasoconstriction consequently preventing vasodilation and increase the activity of protein kinase C isoforms α and ε in VSMCs of porcine coronary arteries [38-40]. HDL is another one of the major lipoproteins in circulation known as the “good” lipoprotein due to the fact that HDL transports cholesterol and fat molecules from peripheral tissues and arterial walls to the liver for excretion. Enhanced plasma HDL-cholesterol concentration lowers the risk of cardiovascular-associated diseases [41] providing healthy endothelial cell function (vasodilation) [42]. HDL enhances the activity of endothelial NOS to induce femoral arterial vasodilation in vitro [43] and enhances myocardial perfusion via NOdependent mechanisms in vivo [44]. Altogether, these data suggest that HDL may reduce the prevalence and may serve as a novel biometric for cardiovascular disease.

FATTY ACID BIOSYNTHESIS Fatty acids are synthesized in the cytosol from acetyl-CoA derived from the Krebs’ cycle. The first step of the Krebs’ cycle in the mitochondria matrix is acetyl-CoA, which condenses with oxaloacetate to form citrate via citrate synthase [45, 46]. However, citrate transferred into the cytosol is converted to acetyl-CoA and oxaloacetate with ATP (adenosine triphosphate) and CoA by ATP-citrate lyase (ATP-CL) [47, 48]. Lipid synthesis begins with the irreversible carboxylation of cytosolic acetyl-CoA to malonyl-CoA. During fatty acid synthesis, (PA or SA), saturated fatty acids are incorporated with a repeated series of reactions. PA is the primary product during fatty acid synthesis and the major precursor to synthesize other fatty acids by an elongation enzyme system. SA is desaturated and elongated via a series of reactions converting to various polyunsaturated fatty acid (Figure 6). The



7

Figure 5. Structures of oleic acid lysophosphatidylcholine.

Modified from Ratledge C. 2004; Catalá A, 2013; Condary R and Yao JK, 2011. Figure 6. Synthesis of unsaturated fatty acids.

elongation reaction occurs in the mitochondria or endoplasmic reticulum [49]. Elongation is achieved via a four-step process. Fatty acyl-CoA adds two-carbon units in each elongation cycle [50-52]. The four sequential enzymatic reactions are 1) fatty acyl-CoA (n) is condensed with malonyl-CoA to generate 3-ketoacyl-CoA by FA elongase (ELOVL1-7, the rate-limiting step) [50, 51, 53], 2) reduction of 3-ketoacyl-CoA to form 3-hydroxyacyl-CoA by 3-ketoacylCoA reductase (KAR) with NADPH as a cofactor [54], 3) 3-hydroxyacyl-CoA is converted to generate trans-2,3-enoyl-CoA using 3-hydroxyacyl-CoA dehydratase (HADC1-4) [55], 4) reduction of trans-2,3-enoyl-CoA to produce elongated fatty acyl-CoA (n+2) by trans-2,3enoyl-CoA reductase (TER) (Figure 7) [54]. PAME and other fatty acid methyl esters (FAMEs) are endogenous compounds [2, 56], formed via fatty acid ethyl ester synthase (FAEES) [2]. The fatty acid esterification catalyzes an oxygen atom from the carboxyl group, then condenses the carboxyl group of an acid and the hydroxyl group of an alcohol. FAME synthesis can also be modulated by exogenous methanol and inhibitors of FAEES [2, 57, 58].


8


Figure 7. Typical fatty acid elongation cycle.

FATTY ACID OXIDATION β-oxidation of fatty acids is a major metabolic process in which fatty acids are degraded in the mitochondria and peroxisome to produce energy [59, 60]. β-oxidation occurs at the βcarbon (C-3) of the fatty acid. However, fatty acids must be activated for degradation before being β-oxidized, because negatively charged fatty acids cannot enter the plasma membrane. Activation of fatty acids are catalyzed by fatty acyl-CoA synthetase (FACS, or called thiokinases) to form fatty acyl-CoA thioester [61]. The net reaction of this activation process is ATP-dependent. Fatty acid + ATP + HS-CoA  Fatty acyl-CoA + AMP + 2 Pi Following the catalytic reaction to form long-chain fatty acyl-CoA, the enzymatic reaction of carnitine palmitoyltranferase I (CPT-1) replaces CoA with carnitine to form fatty acylcarnitine [62]. This conversion allows fatty acids to be transported from the cytoplasm to the inner mitochondrial membrane via carnitine acylcarnitine translocase. Once across the inner mitochondria membrane, fatty acylcarnitine is reversely converted back to long-chain fatty acyl-CoA by carnitine palmitoyltrandferase II (CPT-2) for subsequent β-oxidation [63, 64]. Each β-oxidation cycle removes a two carbon unit and involves four main enzymes: 1) acyl-CoA dehydrogenase, 2) enoyl-CoA hydratase, 3) 3-hydroxyacyl-CoA dehydrogenase, and 4) β-ketothiolase [65]. The net reaction of each β-oxidation pathway is: Fatty acyl-CoA + FAD + NAD+ + CoA + H2O  Fatty acyl-CoA (2C less) + FADH2 + NADH + H+ + acetyl-CoA The net reaction of palmitic acid (C16 fatty acid) requires seven cycles of β-oxidation to convert one molecule of palmitic acid or palmitate into eight molecules of acetyl-CoA.



9

Palmitate + 7FAD + 7NAD+ + 8CoA + 7H2O + ATP  8acetyl-CoA + 7FADH2 + 7NADH + AMP + 2 Pi + 7H+ The large amounts of ATP are generated from FADH2, NADH, and acetyl-CoA by subsequent citric acid cycle (Krebs‘ cycle) and electron transfer chain. It is evident that fatty acid oxidation is a major source of cellular ATP.

THE ROLE OF FATTY ACIDS AND ISCHEMIA/STROKE Ischemic stroke is a condition archetypally resulting from mechanical obstruction of a cerebral artery by an atherosclerotic plaque, or by obstruction from a thrombus causing obstruction. Stroke results in significant morbidity, mortality, and health care costs every year in the United States. According to the American Heart Association, there are 800,000 victims of stroke each year, resulting in about 1 in every 19 deaths [66]. This translates into one stroke victim every 40 seconds and one death attributable to stroke every 4 minutes [66]. Though deaths resulting from stroke fell 35.8% from the year 2000-2010 [66], the prevalence of ischemic stroke is still quite high, so investigations into better therapeutic interventions are important for long-term reduction of stroke and general ischemia. Hypoperfusion after ischemia causes decreased oxygen delivery to neurons resulting in cell death [67]. Pharmacological intervention targeting cerebral vascular regulation after ischemia have gained significant interest as potential therapies, while already existing modalities such as acetylsalicylic acid, heparin, direct thrombin inhibitors (DTI, dabigatran), or inhibitor of factor Xa (rivaroxaban) are not direct targets of cerebral vasculature. However, previous studies have suggested that fatty acids such as PAME promote vasodilation and neuroprotection following global cerebral ischemia as well as in models of ischemic stroke [12]. These fatty acids are endogenously produced hydrocarbon molecules containing a carboxyl functional group, participating in many biological processes involving cellular metabolism, signaling, and structure [68, 69]. Early interests in vasodilation therapy against ischemia involve the use of calcium channel blockers (CCB) to induce vasodilation; however CCBs have little to no efficacy in ischemic stroke treatment [70, 71]. Fatty acids have gained unfair notoriety as harmful agents though they provide many important roles in the body, including emerging data that they could be useful as therapeutic adjuncts for treatment against ischemic stroke. Increased interest in fatty acids is due to previous animal studies suggesting that some fatty acids are endogenously produced and released, causing vasodilation and/or increase in CBF in vivo and in vitro [5, 12]. Though fatty acids do not exhibit any thrombolytic effects, their vasodilation properties offer a novel therapeutic opportunity to restore blood to the ischemic penumbra, bypassing the obstruction resulting in increased chance of survival of hypoxic tissues. Lin et al., 2014 demonstrated that rats pretreated with PAME or SAME can attenuate neuronal cell death in the CA1 region of the rat hippocampus by increasing CBF in order to combat ischemia-induced hypoperfusion after asphyxial cardiac arrest (ACA, global ischemia) and focal ischemia [12]. These results are promising since these FAMEs are endogenously produced suggesting the possibility of low toxicity if pharmacologically administered in vivo.


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Other fatty acids such as alpha-linolenic acid (ALA), have been shown to increase CBF and vasodilation in vivo and in vitro [33]. ALA, PA, or saline was administered onto rat carotid or basilar arteries resulting in ALA-induced vasodilation in basilar arteries. This was also confirmed by laser-Doppler flowmetry suggesting an increase in CBF further supporting ALA as a vasodilator [33] thought to be activated by the TREK-1 K+ channel. Additionally, ALA has been shown to provide some degree of neuroprotection in animal models following transient global ischemia [72]. Interestingly, Wang et al., 2006 and 2007 suggested that treatment with SA alone has been shown to mitigate neuronal cell death due to ischemia-induced oxidative stress [73, 74]. More research with SAME, the esterified form of SA, is necessary to prove therapeutic efficacy under ischemic conditions. Moreover, since PAME and SAME were co-released from a neuronal source, it would be interesting to co-administer PAME/SAME and observe the possible effects of CBF and neuroprotection [5]. Taken together, it is important to note that the esterified from of PA produces consistent vasodilation while, PA alone does not possess any vasodilatory properties.These results illustrate the need for further investigation into PAME regarding its potential receptor/binding site, synthesis, and localization of stored PAME. Assuming that further studies of PAME, SAME, and other fatty acid signaling molecules reinforce data already available, this class of endogenous molecules should be highly considered for interventional therapy against stroke and general ischemia.

ACKNOWLEDGMENTS This work was supported by National Institutes of Health grant NS073779-03, American Heart Association-Philips grant AHA-13SDG13950014 and ASA-14BFSC17690007, and Miami Evelyn F. McKnight Brain Institute. Disclosure/Conflict of Interest: The authors have no conflict of interest in this manuscript.

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[69] Vrablik, T. L., Watts, J. L. (2012) Emerging roles for specific fatty acids in developmental processes. Genes Dev. 26: 631-637. [70] Horn, J., Limburg, M. (2001) Calcium antagonists for ischemic stroke: a systematic review. Stroke 32: 570-576. [71] Zhang, J., Yang, J., Zhang, C., Jiang, X., Zhou, H., et al. (2012) Calcium antagonists for acute ischemic stroke. Cochrane Database Syst. Rev. 5: CD001928. [72] Lauritzen, I., Blondeau, N., Heurteaux, C., Widmann, C., Romey, G., et al. (2000) Polyunsaturated fatty acids are potent neuroprotectors. EMBO J. 19: 1784-1793. [73] Wang, Z. J., Li, G. M., Nie, B. M., Lu, Y., Yin, M. (2006) Neuroprotective effect of the stearic acid against oxidative stress via phosphatidylinositol 3-kinase pathway. Chem. Biol. Interact. 160: 80-87. [74] Wang, Z. J., Liang, C. L., Li, G. M., Yu, C. Y., Yin, M. (2007) Stearic acid protects primary cultured cortical neurons against oxidative stress. Acta Pharmacol. Sin. 28: 315-326.




Chapter 2

OCCURRENCE, BIOCHEMICAL, ANTIMICROBIAL AND HEALTH EFFECTS OF PALMITIC ACID Melissa Johnson*1 and Daniel A. Abugri2† 1

Department of Agricultural and Environmental Sciences, College of Agriculture, Environment and Nutrition Sciences, Tuskegee University, Tuskegee AL, US 2 Department of Chemistry, College of Arts and Sciences, Tuskegee University, Tuskegee, AL, US

ABSTRACT Palmitic acid (PA), one of the most abundant saturated fatty acid (SFAs) within plants, humans, animals, microbial (bacteria), fungal, and marine organisms, constitutes ~16 to 45 % of the lipid profile. The impressive abundance of PA throughout nature could be attributed partly to its critical role in membrane lipid structural functionality, formation of subcellular cysteine residue linkages and RNA posttranslational modifications in eukaryotic and prokaryotic cells. PA has been demonstrated to undergo β-oxidation to produce short and medium chain fatty acids to maintain homeostasis in response to endogenous and exogenous cues. Further, non-esterified PA is known to mediate numerous biochemical and antimicrobial pathways towards the betterment of human health. Although the importance of PA in human health and nutrition are established, critics attest that excessive dietary PA may be ―unhealthy‖ and even detrimental. Current microbiological and epidemiological studies suggest that PA produces specific health benefits that are not common to all other SFAs. For example, studies suggest that PA may activate nitric oxide and superoxide, thus functioning as an antimicrobial agent against some strains of bacteria, algae, and helminthes and certain foodborne pathogens. This chapter reviews the occurrence, biochemistry, and subsequent health implications of PA.

Keywords: Palmitic acid, saturated fatty acid, biochemistry, health implications

* †

[email protected]; [email protected]; [email protected].


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Melissa Johnson and Daniel A. Abugri

1.1. INTRODUCTION The most common and abundant saturated fatty acid (SFA) in nature, as well as within plants, animals and humans, palmitic acid (PA) has many critical functions (Figure 1). Serving as the primary storage form of excess dietary carbohydrates and the precursor for longer chain fatty acids, palmitic acid occupies a critical role in long-term energy storage, the synthesis of other biomolecules (e.g. glycolipids, phospholipids, vitamins, prostaglandins, prostacyclins, thromboxanes, etc.) and cellular membrane structural constituents. Palmitic acid has also been evaluated based on its antimicrobial, antifungal, antibacterial contributions, when bound to specific proteins. Applications within the health and beauty (e.g. cosmetics, soap production), food (e.g. additive, texturing agent), pharmaceutical (e.g. palmate ester as a carrier medium and releasing agent) and biotechnology industries further add credence to the virtual diversity of this fatty acid to function as a ―multipurpose‖ entity and challenges one to consider this fatty acid beyond the conventional understanding as a fatty acid whose primarily purpose for existence is to serve as a fuel source. Palmitic acid and its bioactive metabolites and successors exhibit unique absorption, transport, metabolic, site-specific distribution(s), mechanisms of action and implications (Figure 1). These characteristics contribute to localized and generalized observed and subsequent potential repercussions- both acute and chronic. The ability of palmitic acid to influence protein-protein interactions, hydrophobicity, membrane association, and subcellular trafficking within and between membrane constituents, exemplifies the capability of palmitic acid to purposefully engage in covalent binding to specific protein residues. The occurrence, biochemistry and health effects of palmitic acid within the micro and macro bio-arenas of plants, animals, humans and other microorganisms accentuate the transformative nature of this fatty acid and its influence on the lipidome, metabolome, proteome, transcriptome and genome. This chapter seeks to integratively explore the multi-faceted nature of palmitic acid and provide the reader with an interactive learning experience, with appropriate visual aids to abridge concepts and aid in comprehension, which allows for the enhanced appreciation of one of the most abundant fatty acids in nature.

1.2. DEFINITION, IMPORTANCE AND POTENTIAL OF PALMITIC ACID 1.2.1. Standard Definition of Palmitic Acid Palmitic acid (PA) or hexadecanoic acid is one of the most plentiful fatty acids in nature and tissues, is characterized by a 16 Carbon atom chain with the absence of a C-C double bond (C16:0). It encompasses both hydrophobic and hydrophilic regions, containing carboxyl acid (-COOH) and a methyl group end (-CH3) functional groups respectively. This unique feature allows palmitic acid to exist both as a free fatty acid and in complex with other lipids (e.g. diglyceride, triglycerides), carbohydrate (e.g. glycolipid), and protein (e.g. lipoprotein) entities.


*Excess carbohydrates (CHOs) are converted to palmitic acid; negative feedback (inhibition) by acetyl-CoA carboxylase (Benoit et al. 2009) the enzyme catalyzing the conversion of acetyl-CoA to malonyl-CoA; positive feedback (synthesis) catalyzing the synthesis of palmitic acid; CVD- cardiovascular disease. Figure 1. Summary of the occurrence, biochemical courses of action and implications of palmitic acid.


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1.2.2. Importance of Palmitic Acid Palmitic acid is used as the precursor of long chain fatty acids lipogenesis and can also undergo beta oxidation to produce short and medium chain fatty acids. For instance, palmitic acid is first used to synthesized palmitoleic acid under cellular condition using the de novo pathways which is further use to make C18:0 (stearic acid). From the C18:0 fatty acid, a C18:1n9 and C18:2n6 and C18:3n3 can also be synthesized from the 18 carbon saturated fatty acid (C18:0). Palmitic acid serves as a precursor for longer chain fatty acid synthesis e.g. Triacylglycerol (Figure 5). From these C18 carbons the omega 6 and omega 3 from C18 and high chain fatty acids can then be made for cellular utilization. The C16:0 fatty acid presences in the cell play a major role in posttranslational modification of mRNA (Cooper, 2000). Here, the acid is known to bind with cysteine residues which render the cysteine residue favorable for any modification of the protein to be expressed for cellular use (Corvi et al., 2000). Basically, palmitic acid under cellular condition binds to sulfur atoms from the side chains of the internal amino acid called cysteine residues during posttranslational modification. In addition, palmitoylation, palmitic acid has the ability to contribute greatly to lipid rafts associated with cytosolic and plasmic cell signaling mechanisms (Dykstra et al., 2003). This is very important in understanding the pathogenesis of diseases and their causative agents (Corvi et al., 2012). This type of phenomena is unique to palmitic acid due to thioester bond formation between the cysteine amino acid. This in turn, permits soluble proteins to interact with cellular membranes, mediate and initiate localization at the surface of mammalian cell membranes (Resh, 2006). It has been proposed that the ability of palmitic acid to dictates protein function is due to its reversibility property on like other lipid modification that cannot be reversible (Blanc, Blaskovic, & Goot, 2013). It is important to point out that most protein hydrophobicity among most fatty acids modification (e.g. myristic) is not as efficient as the one formed by palmitic acid. When palmitic acid is involved, it increases membrane proteins interaction in the cell. Also aside the, palmitoylation, they are good sources for generation of ATP during exogenous and endogenous activities in the cell. Palmitic acid ability to under modification creates several opportunities for soluble and integral membrane proteins such as signaling proteins, enzymes, scaffolding proteins, ion channels, cell adhesion molecules and neurotransmitter receptors (Blanc, Blaskovic and Goot, 2013).

1.2.3. Potentials of Palmitic Acid In addition to exhibiting vast importance to biological systems, palmitic acid, palmitic acid is of importance to the food, cosmetics, pharmaceutical, biotechnology and biodiesel industries (Carmo Jr et al., 2009; Houde, Kademi, & Leblanc, 2004; Jaeger & Reetz, 1998; Keng et al., 2009; Metzger & Bornscheuer, 2006; Pitto et al., 2002; Quintavalla & Vicini, 2002; Schmelzle et al., 2003). As outlined, in the figure 2 below palmitic acid have many functions, extending beyond the obvious cellular functions of serving as a precursor for longer chain fatty acid synthesis and as an energy source during β-oxidation.


Occurrence, Biochemical, Antimicrobial and Health Effects of Palmitic Acid

21

Figure 2. Functions and contributions of palmitic acid. (Image Sources: en.wikipedia.org; Microsoft office clipart).

1.3. OCCURRENCE OF PALMITIC ACID 1.3.1. Distribution in Nature (Plant Sources, Algae, Fungus, Etc.) In plants, algae, fungus, yeast and bacteria have also shown similar predominant saturated fatty acid to be palmitic. Palmitic acid distribution varies both within species and among species (Table 1). In recent reports PA content can be influence by the environment in which such plant, fungus, bacteria and yeast are found (Griffiths et al. 2003). Most of the factors contributing to this variation are soil pH, nutrient-ion interaction, age, water and host interaction. For instance variation has been observed in mushrooms, yeast, algae, viruses and other plants PA content (Table 1). It is important that in considering chemotaxonomy these above factors need to be critical considered to avoid biasness in data.

1.3.2. Distribution in Human Tissue Palmitic acid was found to be the second most abundant fatty acid in abdominal adipose tissue following oleic acid (Garaulet et al., 2001). With increasing concentrations of palmitic acid, increases in storage within visceral adipocytes have been observed; palmitic acid was able to significantly increase linoleic acid accumulation in visceral fat (adipocytes) and to a lesser degree in subcutaneous adipocytes (Sabin et al., 2007). Further, in free fatty acid form,


22


lipid levels increased in both visceral and subcutaneous adipocytes, in comparison to primary accumulation in visceral adipocytes when in triglyceride form.

1.3.3. Distribution in Animal Tissue PA is the most abundant saturated fatty acid found in most animal products, for example meat. Palmitic acid has been reported to account for approximately 27% of the saturated fatty acid content of beef (Whetsell et al., 2003). PA occurrences in animal‘s tissues are likely to be influence by environmental and nutritional factors. This unique feature makes PA an essential chemotaxonomic tool for identification of species of animals and poultry and even plant related species. Table 1. Approximate palmitic acid distribution in selected human, animal, plant, fungal, viral and bacterial sources Source Human Milk Sebum (from back) Hair Animal Adipose tissue Plasma (mice) Brain (mice) Brain (rat)

Liver (SHR) Liver (mice) Fungal Agaricus bisporus Cortinarius glaucopus Hygrophoropsis aurantiaca Hypholoma capnoides Laccaria laccata Lactarius salmonicolor Lespista inversa Turkey tail

% PA

References

20-25% 17.6-30.1% 36.0%

(Innis, Dyer, & Nelson, 1994) (Boughton & Wheatley, 1959) (Weitkamp, Smiljanic, & Rothman, 1947)

23.4-23.8% 19.5-19.6% 19.8-21.5% 69% (Phosphoglycerides) 3% (Monoacylglycerides) 8% (Cholesterol) 4% (Free fatty acids) 6% (1,2-Diacylglyceride) 3% (1,3-Diacylglyceride) 2%(Cholesterol ester) 6% (Triglyceride) 16-21% 21.0-21.5%

(Rule, 1997) (Shirai, Suzuki, & Wada, 2005) (Shirai, Suzuki, & Wada, 2005)

13-14%

(Abugri, McElhenney, & Willian, 2012) (Heleno et al., 2009) (Heleno et al., 2009)

12% 10% 16% 12% 7.4% 16.4% 23%

(Johnson et al., 2013) (Shirai, Suzuki, & Wada, 2005)

(Heleno et al., 2009) (Heleno et al., 2009) (Heleno et al., 2009) (Heleno et al., 2009) (Abugri, McElhenney, & Willian, 2012)


Occurrence, Biochemical, Antimicrobial and Health Effects of Palmitic Acid Source Tinder polypore

% PA 14%-15.1%

Artis Conk

17.3% -17.4%

Algae Phaeodactylum tricornutum Thalassiosira weissflogii

14.7-26.8% 28.8-36.6%

Dunaliella primolecta

21.8-26.0%

Nannochloris sp.

15.1-17.8%

Parietochloris incisa

10.0-19.8%

Nostoc commune

25.3-43.5%

Synechocystis sp.

18.8-26.5%

Pavlova lutheri Emiliana huxleyi

11.1-23.6% 10.3-17.7%

Heterosigma akashiwo

40.0-46.3%

Yeast Nadsonia fulvescens and N. commutata R. bisporidii and R. dibovatum, R.Toruloides Bacteria Lactobacillus arabinosus

References (Abugri, McElhenney, & Willian, 2012) (Abugri, McElhenney, & Willian, 2012) (Lang et al., 2011; Tonon et al., 2002; Viso & Marty, 1993) (Lang et al., 2011; Viso & Marty, 1993) (Lang et al., 2011; Viso & Marty, 1993) (Lang et al., 2011; Viso & Marty, 1993) (Bigogno et al., 2002; Lang et al., 2011) (Lang et al., 2011; Temina et al., 2007) (Lang et al., 2011; Viso & Marty, 1993) (Lang et al., 2011; Tonon et al., 2002) (Lang et al., 2011; Viso & Marty, 1993) (Lang et al., 2011; Viso & Marty, 1993)

14-15%

Botha and Kock,1993

14-19.0%

Westhuizen et al., 1987

18.7%

(Hofmann et al., 1955; Hofmann, Lucas, & Sax, 1952) (Hofmann et al., 1955; Hofmann & Sax, 1953) (Hofmann, Henis, & Panos, 1957)

Lactobacillus casei

24.3%

Lactobacillus delbrueckii Streptococcus sp

27.3%

Clostridium butyricum Escherichia coli Argrobacterium tumefaciens Plant Malva sylvestris (leaves,flowers,immature fruits, leafy flowered stems) Sweet potatoes (Ipomoea batatasi) leaves Capsicum chinnese

49.0% 25-38.6% 8.2%

26.6%

(Hofmann et al., 1955; Hofmann & Tausig, 1955a; Hofmann & Tausig, 1955b) (Goldfine & Bloch, 1961) (Shaw & Ingraham, 1965) O‘ Leary, 1959

10% (leaves) 17.2% (flowers) 20%(immature fruits) 13% (leafy flowers stems) 10-21%% 16-21%

(Barros, Carvalho, & Ferreira, 2010)

(Almazan & Adeyeye, 1998; Karmakar, Muslim, & Rahman) Abugri et al., 2014 unpublished data


23

24

Melissa Johnson and Daniel A. Abugri Table 1. (Continued) Source Sorghum bicolor red leaves Rice seeds (Hassawi and CV. Hassa No.2)

% PA 16.2%

References (Abugri et al., 2013)

16 %-29.20%

(Abdulaziz & Ai-Bahrany, 2002)

1.4. PHYSICAL, CHEMICAL, STUCTURAL AND FUNCTIONAL PROPERTIES OF PALMITIC ACID PA, written as, CH3CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2COOH, is solid at room temperature and has a melting point of approximately 63 oC, boiling point of about 351 oC. The fatty acid has a density of about 853 kg/m3. Palmitic acid, the most common fatty acid within plants, humans, animals and microorganisms, is a 16 carbon unsaturated fatty acid (Figures 3a, 3b) that appears as white crystalline scales or colorless needles. Palmitic acid has a molecular mass and melting point of 256.42 g mol-1 (O'Neil, 2013). Furthermore, palmitic acid has a low fluidity as compared to unsaturated fatty acid and other long chain saturated fatty acids, but has high fluidity than other saturated fatty acid ranged from C3.0 to C15:0. The bonds are mostly carbon-carbon and carbon-hydrogen single bonds. There is also carbon-oxygen double bond. It is important to point out that PA has negative feed backs inhibition on acetyl-CoA carboxylase (Benoit et al. 2009) enzyme which is responsible for converting acetyl-ACP to malonyl-ACP on the growing acyl chain, thus resulting in the further generation of palmitate. The physical and chemical properties of palmitic acid facilitate the biochemical actions of palmitic acid (Figure 4)

Source: http://en.wikipedia.org/wiki/File:Palmitic-acid-3D-balls.png. Figure 3a. Ball-and-stick 3D structure of palmitic acid molecule.

Source: http://en.wikipedia.org/wiki/File:Palmitic_acid.svg. Figure 3b. Linear structure of palmitic acid molecule.



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Source: http://ercfre86.wordpress.com/2012/03/19/applications-of-palmitic-acid/ Figure 3c. Physical appearance of palmitic acid.

Figure 4. Summary of biochemical actions of palmitic acid.

1.4.3. Antimicrobial, Antibacterial and Antifungal Properties and Mechanisms Action of Palmitic acid Antimicrobial Agents Palmitic acid inhibits and kills bacteria, virus and other pathogens (i.e. fungus) directly or via indirect mechanism (Yff et al., 2002; McGraw et al. 2002; Seidal and Taylor, 2004; Fluhr et al., 2005; Takigawa et al., 2005; Orhan et al. 2011). If PA is in its free form it makes condition not favorable for growth of some type of fungus and bacteria on the surface of the skin because of its acidity property. This conjecture is supported by (Fluhr et al., 2001; Takigawa et al., 2005). Another route use by fatty acids to inhibit pathogens is their ability for interaction with cell membrane. For instance, in fungus, the cell membrane is expected to maintain the cell organelles in order as well as the cell integrity (Pohl, Kock, & Thibane, 2011). Furthermore, fatty acids can either interacts with the membrane directly resulting in the insertion into the lipid bio-layers of the fungal and any pathogens membranes which cause a physical disturbance of the membrane integrity (Pohl et al. 2011). This consequently, creates high fluidity of the membrane. The higher the fluidity within the cell, the greater the


26


cell will experience disruption in the cell membranes which could lead to greater conformational changes within membrane proteins and their functionality (Liu et al. 2008; Avis & Belanger, 2001; Altieri et al., 2007; Pohl et al. 2011). Other effects meditated by PA at high fluidity could be the release of intracellular components, cytoplasmic disorder and eventually cell disintegration and apoptosis (Liu et al. 2008; Avis & Belanger, 2001; Altieri et al., 2007; Pohl et al. 2011). Fatty acids are known in nature to function as anionic surface agents and these roles make them nonfunctional at certain physiological pH conditions (Armstrong, 1957; Kabara et al., 1972; Scharff & Beck, 1959). Palmitic acid acts as antimicrobial agents against bacteria (Kabara et al., 1972), fungal and other pathogens. For instance, palmitic acid has been demonstrated to greatly inhibit Alernaria solani (Liu et al., 2008), Aspergillus niger (Altieri et al., 2007), Aspergillus terreus (Altieri et al., 2007), Cucumerinum lagenarium (Liu et al., 2008), Emericella nidulans (Altieri et al., 2007) and Fusarium oxysporum (Liu et al., 2008). With this knowledge about palmitic acid, its biochemical pathways of inhibition of the enzymatic secretory pathways used by such microorganisms are speculated to be either disruption of mitochondrial machinery resulting in much electron transport influx or disruption of the cell membrane integrity (Kabara et al., 1972; Desbois & Smith, 2010; Pohl et al., 2011).

Selected Possible Mechanisms of Action of Palmitic Acid Generally, fatty acid either in a free form or in an esterified form has antimicrobial properties. These properties are depended on several factors such as the temperature, ions of fatty acids, pH, functional groups (COOCH3, COOH, and NCOCH2), carbon chain length and the degree of unsaturation (Kabara et al., 1972; Sikkema, De Bont, & Poolman, 1995). Furthermore, lipid classes (phospholipids and sphingolipids) also have influence on the degree of inhibitory, biostatic, killing, DNA and protein synthesis disruption, cell membrane permeability, influx of ion concentration, electron transfer and mitochondria energy expenditure production ( Desbois and Smith, 2010; Liu et al. 2008; Avis & Belanger, 2001; Altieri et al., 2007; Pohl et al., 2011 ). Willett and Morse (Willett & Morse, 1966) have demonstrated the unique potency of PA against certain group of bacteria. The target organelle of palmitic acid is possibly the cell membrane of the bacteria, fungi, virus or any parasite (Pohl et al., 2011; Liu et al. 2008; Avis & Belanger, 2001; Altieri et al., 2007). When the cell membrane is disrupted as a result of the detergent permeability properties of palmitic acid, it might result in disruption of the intracellular organelles functionality. The acid might be able to perform this role due to its amphipathic nature. The good thing is that the amphipathic property of palmitic acid will permit it to interact with the cell wall and cell membranes resulting in the creation of small pores throughout the cell membranes. This consequently affects the cell delimiting ability to prevent the introduction of foreign materials into the inner contents or organelles of the microorganism. Hence, no regularly movements of substances into and out of the cell will exist, resulting in osmotic shock in which the parasite is kill internally. The different processes that palmitic acid might be using to achieve this antimicrobial activity include cell lysis, inhibition of enzymes activity, impartment of nutrient uptake and release, release of toxic peroxidation and autoxidation products (Desbois & Smith, 2010; Pohl et al.2011).



27

A. Cell Lysis Fluidity of fatty acid depends on the chain length and the degree of unsaturation, therefore because of these unique property, fatty acids have the ability to insert themselves into the inner membranes of pathogens resulting in more fluidity and permeability (Chamberlain et al., 1991; Greenway & Dyke, 1979). PA mechanism of cell lysising may be due to high permeability of membrane due to the insertion of PA, and has inner consequence such as allowing of internal contents to leak from cells which can result in growth inhibition and death (Galbraith & Miller, 1973; Shin et al., 2007; Wang & Johnson, 1992; Boyaval et al. 1995; Liu et al. 2008; Avis & Belanger, 2001; Altieri et al., 2007). The higher the fluidity in the cell the greater the cell tendency to lysising or bursting because of the issue of unbalance membrane fluidity of the cell (Desbois & Smith, 2010). B. Inhibition of Enzymatic Activity and Disruption of Electron Transport Chains Minor and major biochemical processes in the body depend upon enzymes for catalysis to bring about homeostasis. However, these enzymes can be hampered by the physiological conditions. Palmitic acid just like any other fatty acid might have the capability to penetrate through the cell wall, which could cause irreversible deformation of the cell membranes (Pohl et al., 2011; Liu et al. 2008; Avis & Belanger, 2001; Altieri et al., 2007). Since these are protected by membrane proteins, the conformational forms of these proteins could be disrupted depending on the levels require in bring such effect within the cell. When the enzymes are inhibited it results in the disruption of the ATP synthesis due to decoupling effect on the energy chain which affects the ATP synthase responsible for production and regulation of ATPs in the cell (Desbois & Smith, 2010; Harold, 1972). Palmitic acid might cause a direct binding to the electron carriers, insertion between carriers preventing interaction, complete displacement of carriers from membranes and prevention of carriers‘ interaction by reducing fluidity of the membranes (Pohl et al., 2011; Liu et al. 2008; Avis & Belanger, 2001; Altieri et al., 2007). However, further studies will be needed to actually conclude its mechanism of action. C. Impartment of Nutrient Uptake and Release Every microorganism has well defined coordination systems that allow nutrient uptake into the cellular level for the cell growth and bioenergetics purposes. However, if the cell membranes and its associated proteins that is performing this role is disrupted then the coordination becomes poor for the cell to do its selectivity in terms of nutrient uptake. It has been proposed by (Desbois & Smith, 2010) that the presence of holes created by free fatty acids cause‘s leakages which cause impairment of active nutrient uptake by either indirectly or direct on transport proteins. In most nutrient uptake the effect of this fatty acid is directly influence by the inability for the transport protein to carry their function properly due to conformational changes during cell membrane and its associated machinery disruption. On the part of the indirect route used by free fatty acids to destabilized proper nutrient uptake into vital parts of the cell could be attributed to the not favorable condition received by the cell due to leaking, unregulated exchange of palmitic acid between inner and out membranes machinery which results in the cells inability to produce ATP. The interesting biochemistry of these effects is that in larger animals the cell organization is totaling different and that helps prevent leakages in humans and associated mammalian species.


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D. Release of Toxic Peroxidation and Autoxidations Products During dietary intake of PA and metabolic activities within the cell certain toxic peroxidation and autoxidations metabolites can result. These products when not cleared in a fast manner could result in deleterious effect on bacteria and other related pathogens. E. Palmitoylation and Cell Signaling Fatty acid generally have the ability to acylate with protein, during posttranslational modification makes them unique for up-regulating and down-regulating of certain cell signaling in the cell (Resh, 2004; Smotrys and Linder, 2004). Fatty acids are usually covalent bonded to proteins during posttranslational modification (Drisdel et al. 2006). This is common in eukaryotic cell than in prokaryotic cell (Drisdel et al. 2006). The mechanisms employed by fatty acids to link up with proteins in the cellular levels are diverse in nature and not only that, they involve several different processing events depending on the fatty acid of interest. Palmitolylation, mysristolylation and prenylation are the most common once observed in posttranslational modification (Resh, 2004; Smotrys & Linder, 2004). The ability for palmitic acid to undergo posttranslational modification in a form of palmitoylation is very important for cell signaling and binding to membrane proteins (Ross, 1995). For instance, palmitolylation aid in proper binding to particular proteins and enhancement of membrane attachment (Ross, 1995). This allows for specificity in location of G-proteins because of changes in receptors proteins in the cells (Ross, 1995). This may help the body of the host to trafficked these proteins that might have been invaded by pathogens for survival and replication (Ross, 1995). F. Phospholipids Synthesis Palmitic acids play an important role in the synthesis of phospholipids which are the key class of lipids for cell membrane, brain and nerves coordination and integrity (Greseth & Traktman, 2014). In some prokaryotes, PA is required for the phospholipids to be synthesis which helps them to survive; for example, viral phospholipids biosynthesis for survival (Greseth & Traktman, 2014). G. PA Induced Proton Permeability of the Inner Mitochondrial Membrane In 1950s, studies have testified the distribution of mitochondria energy coupling potential by lipophilic extracts obtained from microsomes and other organic derived materials (Wojtczak & Wieckowski, 1999). According to these authors, the uncoupling effects and phosphorylation yield within the mitochondrial were observed to be controlled when an isolated mitochondria using serum albumin with high binding ability for fatty acids was tested (e.g. Palmitic acid) (Wojtczak, 1976; Wojtczak & Schönfeld, 1993). Naturally, uncoupling compounds have been identified to include nonesterified fatty acids forms in which palmitic acid could be one of them (Borst et al., 1962; Pressman & Lardy, 1956; Wojtczak & Lehninger, 1961). Further evidence, on fatty acid been accumulated in larger amounts inside or trapped by any isolated mitochondria has been reported to be responsible for poor P/O ratios in aged rat liver mitochondria (Chefurka & Dumas, 1966). These fatty acids turn to create swelling of the organelle (Mitochondria) resulting in



29

improperly disruption of cell energy dispensation (Lehninger & Remmert, 1959; Wojtczak & Lehninger, 1961; Lehninger, 1962). The possible causes for these swelling in the mitochondrial depended on the chain length and degree of unsaturation. This potential is similar to the features used to disrupt the energy coupling processes in cellular (Pressman & Lardy, 1956).

Adapted from (Fatima, 2012 #103). Figure 5. Fatty acid and triacylglycerol biosynthetic pathways.


30


There have been strong correlations between fatty acids and stimulation of mitochondrial respiration and their potentials as protonophoric properties (Cunarro & Weiner, 1975). Furthermore, the longer the chain of the fatty acid, the more the proton conductance will occur in the phospholipid bilayer membranes (Gutknecht, 1988). This implies that multiple studies are in agreement with the observation (Gutknecht, 1988). Palmitic acid may be using a similar mechanism as any other fatty acids which are able to facilitate transmembrane flux of some other cations, namely monovalent alkali metal cations ( Liu et al. 2008; Avis & Belanger, 2001; Altieri et al., 2007; Zeng, Han, & Gross, 1998). However, research is needed to draw a conclusive mechanism of PA under the membrane flux potential. Most of the monovalent elements that palmitic can help in exchange between membranes are potassium, sodium and lithium (Zborowski & Wojtczak, 1963). The mechanism is made possible by (Pressman & Lardy, 1956; Zborowski & Wojtczak, 1963; Schonfeld, Schild & Kunz, 1989). The capacity of palmitic acid in its undissociated form to perform flip-flop in the inner mitochondrial membrane (Zborows; ki & Wojtczak, 1963) makes it potent for interfering with the pathogens monovalent machinarys. The second possible way palmitic acid is utilizing in inhibiting or killing pathogens could be due to its ability to exist in the anionic form. This form is transferred by the adenine nucleotide translocator as well as other proteins. It is important to note that the process mentioned above is possible based on size and the structural nature of the fatty acid molecule in question. Many authors have found out that fatty acid has highest potency to uncouple oxidative phosphorylation (Pressman & Lardy, 1956; Schönfeld, Schild, & Kunz, 1989), to induce mitochondrial swelling (Zborowski & Wojtczak, 1963), and to promote energy-dependent accumulation of monovalent cations in mitochondria (Wojtczak, 1974). This could possibly be the same as what has been observed in palmitic acid in fungus inhibition and killing (Wojtczak, 1974; Choi et al. 2010).

1.5. BIOCHEMISTRY OF PALMITIC ACID Metabolism of Palmitic Acid The metabolism of palmitic acid may be broadly characterized as anabolic (i.e. synthesis) or catabolic (i.e. degradation) (Figure 6). The primary route of palmitic acid metabolism is via oxidative degradation or β-oxidation within the mitochondria. During this process palmitic acid is converted to acetyl-coA, which then enters the Citric Acid Cycle (or Kreb‘s Cycle), during which energy is generated (Figure 7). Depending upon the homeostatic needs, palmitic acid may also be converted to other fatty acids such as stearic, oleic, palmitoleic and myristic acids within the liver and intestinal mucosa. Further, palmitic acid may undergo omegaoxidation, a metabolic pathway often employed during episodes of increased hepatic fatty acid influx and as a compensatory mechanism in the presence of a fatty acid oxidation disorder (Hoek-van den Hil et al., 2013; Wanders, Komen, & Kemp, 2011). Palmitic acid has been demonstrated to be metabolized utilizing different pathways than other fatty acids. For example, PA was found to be metabolized in human skeletal muscle cells in a mechanism quite different from that of oleic acid (Bakke et al., 2012). In addition, cellular uptake, the incorporation into cellular phospholipids, desaturation and oxidation of palmitic acid was found to differ from that of oleic, as well as stearic acids in hamster



31

hepatocytes (Bruce & Salter, 1996). Others found palmitic acid to be metabolized at a much lower rate than myristic acid in rat hepatocytes; myristic acid exhibited a significantly greater rate of β-oxidation and elongation (Rioux, Lemarchal, & Legrand, 2000). In the presence of other fatty acids, the rates of gluconeogenesis, palmitic acid metabolism and the metabolism of long-chain fatty acids is adapted accordingly in both humans and animals (Emken, 1994; Mashek, Bertics, & Grummer, 2002). Palmitic acid also influences the metabolism of lipids, lipoproteins, total and HDL cholesterol (Mensink et al., 2003; Ramamoorthy, Gupta, & Khosla, 2000; Sanders et al., 2011; Snook et al., 1999). However, others found diets high in palmitic acid to alter neither fasting nor postprandial levels of homocysteine or other inflammatory biomarkers (e.g. TNF-α, IL-1β, IL-6 and IL-8, high-sensitivity C-reactive protein and interferon-γ) (Voon et al., 2011). Although researchers found palmitic acid supplementation to facilitate an increase in plasma cholesterol concentrations in a metabolic-diet study in comparison to lauric acid (Denke & Grundy, 1992), conflicting findings were presented elsewhere, with dietary palmitic acid resulting in lower serum cholesterol concentrations than a lauric-myristic acid combination (Sundram, Hayes, & Siru, 1994). In a study examining the effect of palmitic acid intake (high vs low) on the endogenous synthesis of cholesterol and plasma lipoprotein cholesterol levels, palmitic acid was unable to illicit a significant effect on lipoprotein profiles, when recommended intakes of dietary linoleic acid (C18:2n6) were achieved (Clandinin et al., 2000). Researchers have found increasing dietary palmitic acid to decrease fatty acid oxidation and daily energy expenditure (Kien, Bunn, & Ugrasbul, 2005).

Figure 6. Summary of palmitic acid metabolism.


32


Figure 8. Simplified illustration outlining the catabolism of palmitic acid as indicated by: 1) β-oxidation of palmitic acid into acetyl CoA; 2) acetyl CoA entry into the Citric Acid Cycle; and 3) electron transfer via the Electron Transport Chain.

Palmitic acid serves as the major metabolic fuel, particularly in the brain, which utilizes fatty acids as a major fuel source during fatty acid oxidation and active transport into cerebral microvessels (Williams et al., 1997). Up on transport across the palmitic is incorporated into brain phospholipids, contributing to ~29% of phospholipids (μmol/g) in rat brain (Rapoport, 2001). The uptake of palmitic acid is enhanced with the expression of the fatty acid transport protein, which facilitates the transcellular transversion of fatty acids across the blood-brain



33

barrier (Mitchell & Hatch, 2011). During metabolic conditions of starvation and diet-induced obesity, palmitic acid in triglyceride form but not free fatty acid form, when administered intravenously was able to inhibit the transport of leptin across the blood-brain barrier (Banks et al., 2004). This suggests the role of increased triglyceride levels and subsequent hypertriglyceridemia during starvation and conditions such as diabetes in mediating the homeostasis of hormones such as leptin, which regulates fat storage and energy expenditure and in certain cases inducing the resistance of certain hormones to enter the brain.

1.6. HEALTH IMPLICATIONS OF PALMITIC ACID As with any dietary constituent, the evidence on the influence of palmitic acid on health is variable. Rather than identifying the influence of palmitic acid on health as ―effects‖, it may be more appropriate to associate the mechanisms, actions, and interactions of palmitic acid as ―potential implications‖. As palmitic acid is one of the most abundant fatty acids, functioning as potent uncoupling agents of cell communication it is not surprising that it occupies a substantial role in metabolism, health and disease (Kremmyda et al., 2011; Tvrzicka et al., 2011). Palmitic acid has been identified as a potential dietary agent for increased cardiovascular disease (CVD) risk (Siri-Tarino et al., 2010). Palmitic acid has been implicated as an important fatty acid in growth and development. For example, the exposure of mice blastocyte cells to increasing concentrations of palmitic acid resulted in increased apoptosis, decreased proliferation, suggesting the long-term effects of palmitic acid exposure on embryonic metabolism and growth (Jungheim et al., 2011). In addition, mice blastocyte exposure to palmitic acid resulted in fetal growth restriction, which offspring were later able to overcome and reach weights that caught up with or surpassed control mice. The ability of palmitic acid to induce endoplasmic reticulum stress and cellular apoptosis has also been observed in hepatoma cells (Zhang et al., 2012), human granulosa cells (Mu et al., 2001), rat cardiomyocytes (Dyntar et al., 2001) and rat testicular Leydig cells (Lu et al., 2003). Palmitic acid is one of the major agents of free fatty acid-induced apoptosis (i.e. lipoapoptosis) and toll-like receptor activation implicated in innate immune response (Malhi & Gores, 2008). Increases in dietary palmitic acid have been associated with decreases in fat oxidation and daily energy expenditure in adults, which may increase the risk for obesity and insulin resistance (Kien, Bunn, & Ugrasbul, 2005). Palmitic acid was positively associated with risk of heart failure (Matsumoto et al., 2013). As a major participant in the de novo lipogenesis pathway, palmitic acid and its successor palmitoleic acid have the potential to offer a significant contribution to the risk of cardiovascular disease – as influenced by the endogenous fatty acid pool and lipid profile (Wu et al., 2011). In addition to modulating tissue lipid profile, PA influences glucose metabolism via its actions on the hypothalamus (Benoit et al., 2009), thus influencing insulin and leptin secretion, both key hormones in appetite signaling, glucose metabolism, weight regulation, which may have implications on risk for obesity, insulin resistance and diabetes mellitus. Palmitic acid, via the interruption of β-cell function, turnover and subsequent apoptosis, promotes the lipotoxicity and glucotoxicity contributing to diabetes, the effects of which were ameliorated with palmitoleic acid (Maedler et al., 2003; Maedler et al., 2001). A fatty acid profile characteristic of elevated


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palmitoleic acid content have been observed in metabolic disorders such as anorexia nervosa, obesity, metabolic syndrome (Kremmyda et al., 2011). Recently it has been suggested that not all saturated fats increase the risk for cardiovascular and other diseases, but that the triglyceride structure of the fatty acid is of more importance when assessing disease risk (Fattore & Fanelli, 2013). Studies investigating the influence of palm oil, which is comprised of ~50% palmitic acid, 40% oleic acid and 10% linoleic acid, have yielded conflicting evidence in the relationship between palm oil and cardiovascular disease risk. However, the replacement of trans fatty acids with palm oil more favorably influenced biomarkers for disease (Fattore et al., 2014). The inclusion of palm oil into the diets of moderately hyperlipidemic individuals adversely altered plasma lipid profiles, more noticeable increases LDL-cholesterol concentrations (Vega-López et al., 2006). Insulin sensitivity was reduced, HDL- and total cholesterol significantly increased and fatty acid oxidation was moderately increased in individuals consuming diets enriched in saturated fatty acids (9% palmitic acid) compared to those consuming diets enriched in monounsaturated fatty acids (9% oleic acid) (Lovejoy et al., 2002). Dietary monounsaturated fatty acids have been proposed to provide protection from the risks associated with metabolic syndrome and cardiovascular diseases (Gillingham, Harris-Janz, & Jones, 2011). Palmitoleic acid (C16:1n7), the monounsaturated complement of palmitic acid (C16:0), is believed to provide such protection.

Role of Palmitic acid in Inflammatory Pathways The consumption of excessive energy intake prompts the conversion of excess carbohydrates into fatty acids for storage and an enhanced adipocyte inflammatory response, observed during the initiation and progression of obesity, insulin resistance and metabolic syndrome (Kennedy et al., 2009). Palmitic acid, as a modulator of immune responses, functions as an anti-inflammatory agent capable of binding to the active site of phospholipase A2 (PLA2) and thus interfere with its enzyme kinetics and catalytic function inhibiting the synthesis of the inflammatory eicosanoids resulting from the catalytic hydrolysis of arachidonic acid (Aparna et al., 2012). However others found palmitic acid (when administered to elicit a substantial hyperproliferation response) to induce the production of proinflammatory cytokines in keratinocytes by inducing an upregulation of IL-6, TNF-α, IL1β secretion and NF-κβactivation and translocation (Zhou et al., 2013). In addition, palmitic acid, isolated from Sargassum fusiforme, when bound to a novel pocket on the CD4+ cell receptor was found to obstruct the entry of HIV into the cell and subsequent infection (Lee et al., 2009; Lin et al., 2011; Paskaleva et al., 2014; Paskaleva et al., 2010).

CONCLUSION This chapter emphasized palmitic acid, its physical, chemical and structural features as pertaining to its functional characteristics, occurrence and percent distribution in plants, algae, fungus, human and animal tissues. The antimicrobial mechanisms of action, metabolism, general biochemistry and health implications of palmitic acid provide insight into the versatile



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nature of this fatty acid. Palmitic acid, one of the most abundant saturated fatty acids in nature and within living systems, offers more than meets the eye. Palmitic acid is a master and universal saturated fatty acid that has several benefits and effects when in high concentration. Both de novo and salvage pathways ultilze palmitic acid for the biochemical synthesis of short, medium, and long(er) chain fatty acids as well as other phospholipids, which are required for cell mebrane structure and function, brain functionality and neuronal stability. Palmitic acid is the only fatty acid posttranslation modification is reversible and because of this unique feature cell signalling and trafficking is effect in the cell. Although, palmitic acid has unique benefits, its biochemistry is still not explored into in both prokaryotes and eukaryotes. Also because of the mechanisms exerted by palmitic acid for instance cell signalling and trafficking, there is a possibility of it been used in theraupeutics and as nutraceuticals for human microbial infection prevention. Although the evidence is conflicting regarding the role of palmitic acid in health and its implications for health promotion and disease prevention, there exists the potential for palmitic acid to serve as a functional and bioactive endogenous and exogenous constituent of homeostasis.

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Sanders, T. A. B., Filippou, A., Berry, S. E., Baumgartner, S., & Mensink, R. P. (2011). Palmitic acid in the sn-2 position of triacylglycerols acutely influences postprandial lipid metabolism. Am J Clin Nutr, 94(6), 1433-1441. Scharff, T. G., & Beck, J. L. (1959). Effects of surface-active agents on carbohydrate metabolism in yeast. Exp Biol Med, 100(2), 307-311. Schmelzle, H., Wirth, S., Skopnik, H., Radke, M., Knol, J., Böckler, H. M., & Fusch, C. (2003). Randomized Double-Blind Study of the Nutritional Efficacy and Bifidogenicity of a New Infant Formula Containing Partially Hydrolyzed Protein, a High β-Palmitic Acid Level, and Nondigestible Oligosaccharides. J Pediatr Gastroenterol Nutr, 36(3), 343-351. Schönfeld, P., Schild, L., & Kunz, W. (1989). Long-chain fatty acids act as protonophoric uncouplers of oxidative phosphorylation in rat liver mitochondria. Biochimica et Biophysica Acta (BBA)-Bioenergetics, 977(3), 266-272. Seidel, V. & Taylor P.W. (2004). In vitro activity of extracts and constituents of Pelagonium against rapily growing mycobacteria. International Journal of Antimicrobial Agents, 23, 613-619. Shaw, M. K., & Ingraham, J. L. (1965). Fatty acid composition of Escherichia coli as a possible controlling factor of the minimal growth temperature. J Bacteriol, 90(1), 141146. Shin, S. Y., Bajpai, V. K., Kim, H. R., & Kang, S. C. (2007). Antibacterial activity of eicosapentaenoic acid (EPA) against foodborne and food spoilage microorganisms. LWTJ Food Sci Technol, 40(9), 1515-1519. Shirai, N., Suzuki, H., & Wada, S. (2005). Direct methylation from mouse plasma and from liver and brain homogenates. Anal Biochem, 343(1), 48-53. Sikkema, J., De Bont, J. A., & Poolman, B. (1995). Mechanisms of membrane toxicity of hydrocarbons. Microbiol Rev, 59(2), 201-222. Siri-Tarino, P. W., Sun, Q., Hu, F. B., & Krauss, R. M. (2010). Saturated fat, carbohydrate, and cardiovascular disease. Am J Clin Nutr, 91(3), 502-509. Smotrys, J. E., & Linder, M. E. (2004). Palmitoylation of intracellular signaling proteins: regulation and function. Annu Rev Biochem, 73(1), 559-587. Snook, J., Park, S., Williams, G., Tsai, Y., & Lee, N. (1999). Effect of synthetic triglycerides of myristic, palmitic, and stearic acid on serum lipoprotein metabolism. Eur J Clin Nutr, 53(8), 597-605. Sundram, K., Hayes, K. C., & Siru, O. H. (1994). Dietary palmitic acid results in lower serum cholesterol than does a lauric-myristic acid combination in normolipemic humans. Am J Clin Nutr, 59(4), 841-846. Takigawa, H., Nakagawa, H., Kuzukawa, M., Mori, H., & Imokawa, G. (2005). Deficient production of hexadecenoic acid in the skin is associated in part with the vulnerability of atopic dermatitis patients to colonization by Staphylococcus aureus. Dermatology, 211(3), 240-248. Temina, M., Rezankova, H., Rezanka, T., & Dembitsky, V. M. (2007). Diversity of the fatty acids of the Nostoc species and their statistical analysis. Microbiol Res, 162(4), 308-321. Tonon, T., Harvey, D., Larson, T. R., & Graham, I. A. (2002). Long chain polyunsaturated fatty acid production and partitioning to triacylglycerols in four microalgae. Phytochemistry, 61(1), 15-24.


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Tvrzicka, E., Kremmyda, L.-S., Stankova, B., & Zak, A. (2011). Fatty acids as biocompounds: their role in human metabolism, health and disease-a review. Part 1: classification, dietary sources and biological functions. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub, 155(2). Vega-López, S., Ausman, L. M., Jalbert, S. M., Erkkilä, A. T., & Lichtenstein, A. H. (2006). Palm and partially hydrogenated soybean oils adversely alter lipoprotein profiles compared with soybean and canola oils in moderately hyperlipidemic subjects. Am J Clin Nutr, 84(1), 54-62. Viso, A.-C., & Marty, J.-C. (1993). Fatty acids from 28 marine microalgae. Phytochemistry, 34(6), 1521-1533. Voon, P. T., Ng, T. K. W., Lee, V. K. M., & Nesaretnam, K. (2011). Diets high in palmitic acid (16: 0), lauric and myristic acids (12: 0+ 14: 0), or oleic acid (18: 1) do not alter postprandial or fasting plasma homocysteine and inflammatory markers in healthy Malaysian adults. Am J Clin Nutr, 94(6), 1451-1457. Wanders, R. J. A., Komen, J., & Kemp, S. (2011). Fatty acid omega-oxidation as a rescue pathway for fatty acid oxidation disorders in humans. FEBS Journal, 278(2), 182-194. doi: 10.1111/j.1742-4658.2010.07947.x Wang, L.-L., & Johnson, E. A. (1992). Inhibition of Listeria monocytogenes by fatty acids and monoglycerides. Appl Environ Microbiol, 58(2), 624-629. Weitkamp, A. W., Smiljanic, A. M., & Rothman, S. (1947). The free fatty acids of human hair fat. J Am Chem Soc, 69(8), 1936-1939. Whetsell, M. S., B, R. E., & D, L. J. (2003). Human health effects of fatty acids in beef. Fact sheet. In W. V. S. U. U. A. R. Service (Ed.), Extension Service West Virginia University Willett, N. P., & Morse, G. E. (1966). Long-chain fatty acid inhibition of growth of Streptococcus agalactiae in a chemically defined medium. J Bacteriol, 91(6), 2245-2250. Williams, W. M., Chang, M. C. J., Hayakawa, T., Grange, E., & Rapoport, S. I. (1997). In Vivo oncorporation from plasma of radiolabeled palmitate and arachidonate into rat brain microvessels. Microvasc Res, 53(2), 163-166. Wojtczak, L. (1974). Effect of fatty acids and acyl-CoA on the permeability of mitochondrial membranes to monovalent cations. FEBS letters, 44(1), 25-30. Wojtczak, L. (1976). Effect of long-chain fatty acids and acyl-CoA on mitochondrial permeability, transport, and energy-coupling processes. J Bioenerg Biomembr, 8(6), 293311. Wojtczak, L., & Lehninger, A. L. (1961). Formation and disappearance of an endogenous uncoupling factor during swelling and contraction of mitochondria. Biochimica et biophysica acta, 51(3), 442-456. Wojtczak, L., & Schönfeld, P. (1993). Effect of fatty acids on energy coupling processes in mitochondria. Biochimica et Biophysica Acta (BBA)-Bioenergetics, 1183(1), 41-57. Wojtczak, L., & Wie¸ckowski, M. R. (1999). The Mechanisms of Fatty Acid-Induced Proton Permeability of the Inner Mitochondrial Membrane. Journal of Bioenergetics and Biomembranes, 31(5), 447-455. doi: 10.1023/A:1005444322823 Wu, J. H. Y., Lemaitre, R. N., Imamura, F., King, I. B., Song, X., Spiegelman, D., & Mozaffarian, D. (2011). Fatty acids in the de novo lipogenesis pathway and risk of coronary heart disease: the Cardiovascular Health Study. Am J Clin Nutr, 94(2), 431-438.



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Yff, B.T.S., Lindsey, K.L., Taylor, M.B., Erasmus, D.G., & Jager, A.K.(2002). The pharmacological screening of Pentanisia prunelloides and the isolation of the antibacterial compound palmitic acid. Journal of Ethnopharmacology, 79, 101-107. Zborowski, J., & Wojtczak, L. (1963). Induction of swelling of liver mitochondria by fatty acids of various chain length. Biochimica et biophysica acta, 70, 596-598. Zeng, Y., Han, X., & Gross, R. W. (1998). Phospholipid subclass specific alterations in the passive ion permeability of membrane bilayers: separation of enthalpic and entropic contributions to transbilayer ion flux. Biochem, 37(8), 2346-2355. Zhang, Y., Xue, R., Zhang, Z., Yang, X., & Shi, H. (2012). Palmitic and linoleic acids induce ER stress and apoptosis in hepatoma cells. Lipids Health Dis, 11(1), 1. Zhou, B.-R., Zhang, J.-A., Zhang, Q., Permatasari, F., Xu, Y., Wu, D., & Luo, D. (2013). Palmitic Acid Induces Production of Proinflammatory Cytokines Interleukin-6, Interleukin-1, and Tumor Necrosis Factor- via a NF-B-Dependent Mechanism in HaCaT Keratinocytes. Mediators Inflamm, 2013, 11. doi: 10.1155/2013/530429.




Chapter 3

PALMITIC ACID: EFFECT OF DIET SUPPLEMENTATION AND OCCURRENCE IN ANIMAL ORIGIN FOOD P. G. Peiretti* Institute of Sciences of Food Production, Italian National Research Council, Grugliasco, Italy

ABSTRACT In the last few decades, disagreement between opinions and findings concerning the ability of palmitic acid (PA) and other saturated fatty acids (SFAs) to raise cholesterolaemia has led to discussions on whether PA, which has been positively related to high serum cholesterol levels, could increase the risk of cardiovascular diseases. This study aims to review the PA content of meat, dairy products, fish, and other food of animal origin in the human diet and discusses nutritional issues related to the occurrence of this fatty acid (FA) in these foods due to different diet supplementation. Meat and dairy products are considerable dietary sources of SFAs, such as PA. In most industrialized countries, a high meat or dairy intake contributes to a higher than recommended SFA intake. Palmitic and myristic acids are common FAs in meat and dairy products, making up about 30-40% of total FA intake and are the main factors responsible for raising cholesterol levels; indeed, strong evidence indicates that these two SFAs increase serum cholesterol concentrations in humans. Stearic acid is partially converted to oleic acid in vivo and has not been shown to elevate blood cholesterol, while lauric acid is not as potent as PA at raising concentrations of total cholesterol and LDL cholesterol in humans. The occurrence of PA in animal origin food is influenced by both genetic and environmental factors, such as the composition of the animal‘s diet, its digestive system and its biosynthetic processes. The FA profile in food of animal origin mainly reflects dietary lipid sources and has the potential to play a valuable role in human nutrition by manipulating the composition of animal fat through diet. In order to explain the variability in FA composition in food of animal origin, this review examines different

*

Corresponding author: P. G. Peiretti. Tel.: +39 11 6709230; fax: +39 11 6709297. [email protected].


E-mail address:

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P. G. Peiretti nutrition trials that have studied the effects of PA supplementation on the lipid profile of animal origin food.

Keywords: Fatty acid, oilseed, meat, milk, cheese, fish, egg

INTRODUCTION Palmitic acid (PA) or n-hexadecanoic acid is the most common saturated fatty acid (SFA) found in plants, animals, and microorganisms (Gunstone et al., 2012). As the name suggests, PA is characteristic of palm oil and its saponified form was discovered and isolated for the first time by Frémy (1840). The major difference between palm oil and other vegetable oils is the higher proportion of PA (Table 1). The impact of excess SFA in the diet on cardiovascular diseases has been studied and discussed both in animal and human studies (Crawford, 1968; Keys, 1970; Temme et al., 1996). In the past, because of its high PA content, palm oil has been attacked as ―highly saturated oil‖ and accused of raising blood cholesterol and increasing the risk of cardiovascular disease (Mukherjee and Mitra, 2009). Several studies attack SFAs with regard to their hypercholesterolaemic and atherogenic effects, which adversely affect cardiovascular risk (Kromhout et al., 1989; Menotti et al., 1989; Verschuren et al., 1995). Lauric and myristic acid are the main cholesterol-raising SFAs, whereas PA and stearic acid have much weaker cholesterol-raising potential (Sundram, 1994). Table 1. Palmitic acid content of oils and fats of vegetable sources (expressed as percentage mass-fraction of total fatty acids) Palm oil Cottonseed oil Cocoa butter Illipe fat Olive oil Oat bran oil Avocado oil Rice brain oil Wheat germ oil Corn oil Tomato seed oil Peanut oil Soya bean oil Pistachio nut oil Grapeseed oil Babassu fat Poppyseed oil Sesame oil

40.1-47.5 21.4-26.4 25.4 18.0-22.0 7.5-20.0 17.4 17.2 16.9 16.6 8.6-16.5 12.0-15.5 8.3-14.0 8.0-13.3 11.6 5.5-11.0 5.2-11.0 10.6 7.9-10.2


Palmitic Acid: Effect of Diet Supplementation and Occurrence … Palm kernel fat Macadamia nut oil Coconut oil Walnut oil Linseed oil Almond oil Pecan nut oil Safflower oil Linola oil Cashew nut oil Rapeseed oil Sunflower oil Hazelnut oil Canola oil

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6.5-10.0 8.9 8.2 3.9-7.2 4.0-7.0 6.5 6.4 4.8-6.2 6.0 4.0-6.0 1.5-6.0 5.4-5.9 5.2 4.0

Adapted from Beare-Rogers et al., 2001.

The negative effect attributed to lauric and myristic acid explains why foods rich in SFA should be consumed in moderation and there is ―convincing evidence‖ that PA increases the risk of cardiovascular disease (World Health Organization, 2003). Palm oil raises plasma cholesterol only when the diet contains excess dietary cholesterol, in which case the risk of coronary heart disease may rise (Jones, 1989). Temme et al. (1996) reported that both PA and lauric acid are hypercholesterolaemic compared with oleic acid. Lauric acid raises total cholesterol concentrations more than PA, which is partly due to a greater rise in HDL cholesterol. Enig (1993) reported that PA increased the level of blood cholesterol more than other SFAs, including lauric acid and myristic acid, which are more abundant in palm kernel oil and coconut oil than in palm oil. Clarke et al. (1997) concluded that, compared to carbohydrates, PA raises blood cholesterol levels. However, some reviews do not seem to support these conclusions (Edem, 2002; Ong and Goh, 2002; Sundram et al., 2003; Oguntibeju et al., 2009; McNamara, 2010) and indicate that the effect of PA (found mainly in palm oil) on blood cholesterol is relatively neutral when compared to other fats and oils. We must therefore realize, although it may seem simplistic, that what matters is the dietary context, rather than the individual nutrient. In a balanced diet, in fact, PA is usually harmless (it is even synthesized by the body), but can become dangerous when consumed as part of frequent caloric excesses or is consumed in particularly large quantities. Hayes and Khosla (1992) suggested that PA may be neutral in normocholesterolaemic subjects if the diet contains little cholesterol and linoleic acid intake is adequate. Fattore and Fanelli (2013) reviewed the scientific literature on the evidence of the relationship between palm oil and adverse effects on human health and concluded that there is no clear evidence of a negative role of PA on health and much less of native palm oil, which is a complex alimentary matrix, in which PA is only one of its components. However, more recent lipid research on the topic seems to have reconsidered the negative role of dietary SFAs as a risk factor for cardiovascular diseases. For instance, lauric acid and myristic acid have a greater total cholesterol-raising effect than PA, whereas stearic acid has a neutral effect on the concentration of total serum cholesterol, including no apparent impact on either LDL or HDL (Daley et al., 2010).


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P. G. Peiretti

Fattore and Fanelli (2013) showed that not only the type of fat, but also its triglyceride structure, play a role in cholesterolaemia. PA is located at the Sn-1 position of the three principal triglyceride species in palm oil (Small, 1991) and a high fraction of PA in palm oil is bound at the Sn-1 or Sn-3 position of the glycerol molecule (Mu and Høy, 2004). This location confers the non-hypercholesterolaemic property to the oil (Ng, 1997). This is in contrast with the major triglyceride species in animal fats such as butter, which contains PA in the Sn-2 position with resultant hypercholesterolaemic and atherogenic effects (Ng, 1997). Evidence is now growing that the molecular structures of dietary triacylglycerols play an important role in the development of atherosclerosis (Patsch, 1994), because triacylglycerols, enriched with SFA at the Sn-2 position, exhibit different metabolic behaviour than triacylglycerols with SFA at the Sn-1/3 position (Redgrave et al., 1988; Tuten et al., 1993; Carnielli et al., 1995). The enzymatic hydrolysis of dietary triglycerides by pancreatic and lipoprotein lipases preferentially targets fatty acids (FAs) in the Sn-1/3 position rather than those esterified to the Sn-2 position, which are substantially preserved in chylomicrons (Karupaiah and Sundram, 2007). These authors showed that the positioning of unsaturated versus SFAs in the Sn-2 position may explain the modulatory effects on atherogenicity and thrombogenicity. Kritchevsky (2000) reported a higher degree of absorption of PA at the Sn-2 position in rabbit models and this could be related to the increased atherogenicity of interesterified palm oil, in comparison with the native one. The predominant SFAs that occur naturally in animal fats and the main products of cytosolic FA synthetase multienzyme complex in lipogenesis are stearic acid and PA, which can be biosynthesized de novo by all known organisms, including fish (Sargent et al., 1989). Some authors have determined enzyme activities in subcutaneous adipose tissue. StearoylCoA or Δ9 desaturase is the terminal step in the desaturation and conversion of PA, myristic and stearic acid into the Δ9 monounsaturated FAs palmitoleic, myristoleic, and oleic acid, respectively (De Smet et al., 2004). Kazala et al. (1999) suggested that FA elongation was unable to keep pace with the de novo production of PA in animals that deposited greater amounts of intramuscular fat. However, based on tissue incubations, it has been suggested that Δ9 desaturation and not elongation is the rate-limiting step for the conversion of PA to oleic acid in bovine subcutaneous adipose tissue (St. John et al., 1991). This review examines the occurrence and variation of PA content in food of animal origin in relation to different diets and different dietary regimens.

OCCURRENCE IN ANIMAL ORIGIN FOOD PA and myristic acid are common FAs in dairy products and meat, contributing in total about 30–40% of FAs (Valsta et al., 2005). Approximately 60% of the SFAs in the US diet are obtained from meat, poultry, fish and dairy products (Dupont et al., 1991). In a more detailed analysis from the late 1980s of the FA consumption pattern of Americans, it was shown that PA was the predominant SFA in the US diet at the time, contributing 52–57% of SFA intake (Jonnalagadda et al., 1995). Of the SFAs, short chain FAs and lauric and myristic acid are obtained from dairy products, while the predominant sources of PA and stearic acid are meat, poultry, fish and blended foods (Valsta et al., 2005).


Palmitic Acid: Effect of Diet Supplementation and Occurrence …

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FAT, MEAT AND MEAT PRODUCTS In most industrialized countries, fat is an unpopular constituent of meat for consumers, being considered unhealthy. This is because meat is seen to be a major source of fat in the diet and especially of SFAs, which have been implicated in diseases associated with modern life, especially in developed countries, such as various cancers and especially coronary heart disease (Wood et al., 2003). High meat intake contributes to a higher than recommended total and saturated fat and cholesterol intake and may replace sources of other important nutrients in the diet (Valsta et al., 2005). Yet fat and FAs, whether in adipose tissue or muscle, contribute significantly to various aspects of meat quality and are central to the nutritional value of meat (Wood et al., 2008). One important source of PA is meat, including poultry, beef and game meats (Table 2). The amount of PA found in meat depends on its source, as well as on the method used to prepare the food. Fatty cuts of red meat, as well as skin-on poultry, typically contain relatively high levels of SFA and contain large amounts of PA. Pork containing an increased proportion of unsaturated FAs and less PA would be better for consumer health, provided that there is no concomitant increase in fat and cholesterol content (Kouba and Mourot, 1999). These authors examined the influence of a diet high in unsaturated fat on lipogenic enzyme activities, lipid content and FA composition of muscles and adipose tissue in pigs. They concluded that diets with a high content of linoleic acid lead to the production of pork enriched in this FA without any modification in intramuscular fat content and PA percentage, thus conserving a totally acceptable level of cholesterol from a human nutritional point of view. Table 2. Palmitic acid content of meat and meat products (expressed as percentage mass-fraction of total fatty acids) Suet Lard Salami Goose Pancetta Mortadella Ham Wurstel Speck Veal fillet (cooked) Whole chicken with skin (raw) Pork loin Lamb Rabbit thigh Pork steak Pork shoulder Beef belly Horse meat

24.02 21.07 5.73-7.55 7.41 5.67-5.99 5.70 3.93-4.93 4.03 3.71 2.89 2.19 2.06 0.58-1.99 1.95 1.90 1.63-1.77 1.66 1.65


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P. G. Peiretti Whole turkey with skin (raw) Whole rabbit Beef meat Sheep meat Goat meat Ostrich meat Deer meat

1.54 1.22 0.31-1.14 0.58 0.40 0.16 0.12

Adapted from Italian food composition tables - National Research Institute for Food and Nutrition (INRAN).

A survey conducted by Enser et al. (1996) illustrated the differences in fat content and FA profile of muscle between lamb, pork and beef; in particular, PA content, expressed as mg per 100 g muscle, decreased in the order lamb>beef>pork, while PA, expressed as percentage of total FAs, decreased in the order beef>pork>lamb. According to Banskalieva et al. (2000), FA composition of fat depots in goats appears to be in the range typical for ruminants, with average percentages of PA in goat muscles being similar to those for other ruminant species. The PA concentration in kidney fat is similar in goats and lambs, but lower for goat meat than in beef. Hilditch and Williams (1964) noted that land animals tend to have a relatively constant amount of PA in fat depots.

MILK AND DAIRY PRODUCTS Another dietary source of PA is dairy. Cow's milk naturally contains SFAs, so both the milk itself and foods made from the milk typically contain PA. Foods such as ice cream or butter can prove especially rich sources of FAs, as these foods contain high levels of dairy fat (Table 3). Table 3. Palmitic acid content of milk and dairy product (expressed as percentage mass-fraction of total fatty acids) Butter Parmesan cheese Fontina cheese Cream Gelato Ricotta cheese (cow) Ricotta cheese (sheep) Cow's milk (condensed and sweetened) Sheep‘s whole milk Goat‘s whole milk Cow's whole milk Yogurt Semi-skimmed milk Skimmed milk

20.86 8.04 7.31 5.72 4.10 3.49 2.85 1.97 1.58 1.34 0.92 0.92 0.45 0.05




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The FA composition of milk varies in relation to the influence of several factors related to the animal and its environment (Palmquist et al., 1993; Perdrix et al., 1996). These include numerous highly significant factors such as the nature of the food (Grummer, 1991; Coulon et al., 1994), the type of ration, mode of administration, energy concentration and energy level of the ration, the physical state of the food and the entire ration, quantity, quality and length of the fiber, the type, shape and physical treatment of cereals, etc.

FISH AND OTHER FOOD OF ANIMAL ORIGIN Gruger et al. (1965) studied the FA composition of oils from 21 species of marine fish, freshwater fish and shellfish, and found that PA content varies widely among species from 9.5 to 33.4% of total FA. Steffen (1997) reported the FA composition of several freshwater fish lipids and PA content ranged from 10.8 and 24.0% of total FA. Özogul and Özogul (2007) found that PA ranged from 15.5 to 20.5% of total FAs and it was the primary SFA, contributing 53-65% of the total SFA content of lipids in the flesh of eight commercially important fish species from the Mediterranean, Aegean and Black Seas. Milinsk et al. (2003) found a percentage of PA that ranged from 21.7 to 22.8% of total FA in yolk lipids from eggs of White Lohman hens after 16 weeks of feeding with diets enriched with different oils (canola, flaxseed, soybean or sunflower). Organic eggs are reported to have higher (Hidalgo et al., 2008) or similar (Cherian et al., 2002) levels of SFAs as compared with eggs from other production systems. Samman et al. (2009) reported that n-3 polyunsaturated fatty acid (PUFA) egg yolk contained lower percentages of PA compared with conventional and organic eggs (22.7 vs 25.1 and 25.5% of total FA, respectively). Table 4 reports the PA content of the most common eggs.

EFFECT OF DIET SUPPLEMENTATION Manipulation of the FA composition of animal tissues has been of great interest in recent years in order to produce meat with desirable technological and nutritional qualities (Wood et al., 2003). Table 4. Palmitic acid content of eggs (expressed as percentage mass-fraction of total fatty acids) Hen egg (whole powdered) Hen egg (yolk) Duck egg Goose egg Turkey egg Hen egg (whole)

9.96 5.98 3.00 2.98 2.72 1.90



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Scollan et al. (2001) examined the effects of different sources of dietary n-3 PUFA on the FA composition of muscle and adipose tissue in beef cattle. These authors found that the proportion of PA was decreased by the linseed diet in both neutral lipids and phospholipids of muscle. However, fish oil significantly increased the proportion of PA in neutral lipids, while Mandell et al. (1997) also found that fish meal feeding increased PA content, contrary to the lack of effect observed by Mills et al. (1992) on feeding fish meal and Ashes et al. (1992) who fed ruminally-protected fish oil. Furthermore, there was a lower proportion of PA in phospholipids as a result of feeding the linseed diet compared with the control diet. However, compared to the control diet, fish oil did not alter the proportion of PA, but produced lower proportions of stearic and oleic acid (Scollan et al., 2001). Nuernberg et al. (2005) examined the effects of feeding system and breed on the content of the beneficial n-3 PUFA and conjugated linoleic acids in beef muscle, finding that the percentage of PA decreased significantly with the grass-based system, while there was no influence of breed on the percentages of PA. Daley et al. (2010) report that grass-finished cattle are typically lower in total fat as compared to grain-fed contemporaries. Interestingly, there is no consistent difference in total SFA content between these two feeding regimens. Those SFAs considered to be more detrimental to serum cholesterol levels, i.e., PA and myristic acid, were higher in grain-fed beef as compared to grass-fed contemporaries in 60% of the studies reviewed. Grass-finished meat contains elevated concentrations of stearic acid, the only SFA with a net neutral impact on serum cholesterol. Thus, grass-finished beef tends to produce a more favorable SFA composition, although little is known of how grass-finished beef would ultimately impact serum cholesterol levels in hyper-cholesterolaemic patients as compared to grain-fed beef (Daley et al., 2010). Diet is known to affect the FA composition of pig adipose tissue, particularly perirenal and back fat (Fontanillas et al., 1997; Larick et al., 1992) and to improve the FA profile of carcass fat in pigs (Morgan et al., 1992; Van Oeckel and Boucqué, 1992). In particular, dietary oils affected total FA content in pig longissimus muscle (Corino et al., 2002; Kouba and Mourot, 1999; Van Oeckel et al., 1996). Teye et al. (2006) evaluated the effects of palm oil, palm kernel oil and soyabean oil, in combination with high and low protein levels, on the FA composition of the longissimus dorsi muscle in pigs; they found that palm kernel oil increased the concentrations of PA and other SFAs, while it decreased linoleic acid levels (Figure 1). Wood et al. (2004) examined the effects of diet, breed and muscle on FA composition in pigs and concluded that PA and myristic acid in neutral lipids were higher in Berkshire and Tamworth than in Duroc and Large White, while the dietary effects on these FAs were small. Dietary FA modification is considered a viable method of adding value to poultry products for the health-conscious consumer (Hargis and Vanelswyk, 1993). The profile of dietary FAs is of importance because it influences the quality of the fat deposited on the broiler carcass (Figure 2). PA was the predominant SFA in the adipose tissue of birds (Kang et al., 2001). These authors found that dietary supplementation of palm oil resulted in a significant increase in PA in the liver and adipose tissue of broilers. According to Rodriguez et al. (2002), palm oil or mixtures of palm oil, and FAs distilled from palm and calcic soap are sources of vegetal oils with an FA profile that might replace animal fats without any kind of negative impact on carcass quality. Consistent parallels between dietary fat and abdominal fat in broilers have been described by Scaife et al. (1994). The FA pattern of abdominal fat



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represents the FA pattern in the dietary fat sources very well and the key FAs which occur at a somewhat constant rate in broiler adipose tissue are PA, stearic acid, and to some extent, oleic acid (Zollitsch et al., 1997). Smink et al. (2008) found that the feeding of randomized instead of native palm oil significantly raised the PA content of breast meat and abdominal fat and lowered the ratio of unsaturated fatty acids to SFA.

Figure 1. Relationship between dietary palmitic acid percentage and muscle palmitic acid percentage in pig nutritional trials: Peiretti et al. (2013; ■), Teye et al. (2006; ▲), and Corino et al. (2002; ♦).

Figure 2. Relationship between dietary palmitic acid percentage and muscle palmitic acid percentage in poultry nutritional trials: Zollitsch et al. (1997; ■), Kang et al. (2001; ▲), and Smink et al. (2010; ♦).

Rabbits, like other monogastric animals, are able to directly incorporate dietary FAs into adipose and intramuscular tissue lipids, thus making it possible to modify the FA profile of


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rabbits through the strategic use of unsaturated dietary fat sources (Dalle Zotte, 2002). FA levels vary a great deal on the basis of the nature of the rabbit diets. The influence of the FA profile in the diet seems to be more pronounced on the FA composition of adipose tissue than intramuscular fat (Xiccato, 1999). Peiretti (2012) showed that FA profile was clearly influenced by diet composition and it was possible to linearly characterize the incorporation of certain FAs, while PA failed to show a good correlation, both in muscle fat and in perirenal fat, with its percentage in feed (Figures 3 and 4).

Figure 3. Regressions of palmitic acid percentage in rabbit muscle fat according to its contents in feed.

Lactation responses to dietary fat supplementation have been variable and have been dependent on fat source, stage of lactation, and dry matter intake (Coppock and Wilks, 1991). Several studies have examined the effects of PA supplementation on milk FA profile. Grummer (1991) demonstrated that de novo FA synthesis decreased linearly as supplementation of dietary fat increased, and that the changes in stearic acid and PA were dependent on the ratio in the added fat. Steele and Moore (1968) reported reductions in yield and concentration of short and medium-chain FAs (from butyric to myristic acids) and dramatic increases in PA with increased dietary intake of PA; the concentration of PA in milk increased from 38.7% of total FA in controls to 60.7% of total FA in cows supplemented with PA. Noble et al. (1969) reported similar changes in milk FAs when diet was supplemented with PA at 10%, they found that short- and medium-chain FAs decreased when compared with a no-fat control, while milk PA increased from 36.4% of total FA in controls to 49.8% of total FA in PA-treated cows. Banks et al. (1976) also observed decreases in short- and medium-chain FAs in milk, with increases observed in concentrations of PA, palmitoleic, and oleic acids. Using duodenal infusions of 500 g of PA, Enjalbert et al. (2000) reported that concentrations of PA in milk increased 30% compared with controls. Mosley et al. (2007) determined the optimum feeding level of a by-product rich in PA (86.6%) on dry matter intake, milk yield, milk components, and milk FA profile in dairy cattle. They found that milk FA concentrations were affected by the addition of this by-product. As the intake of PA increased with the supplemented diets, milk PA concentrations increased. When 1.5 kg/d of this by-product was consumed, milk PA concentration increased by 50% compared with the



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control. This increase was countered by a general decrease in the weight percentage of many other FAs, with the major difference being an increase in SFAs driven by the linear increase in PA.

Figure 4. Regressions of palmitic acid percentage in rabbit perirenal fat according to its contents in feed.

Chouinard et al. (1998) found that dietary Ca salts had no effect on the proportion of PA in milk fat, but that dietary Ca salts of FA from palm oil typically increased PA (Atwal et al., 1990; Klusmeyer and Clark, 1991; Elmeddah et al., 1994). Chouinard et al. (1998) reported two possible explanations for the higher concentration of PA observed for dietary Ca salts of FA from palm oil. First, palm oil contains a high proportion of PA (>40%) that may be directly incorporated in milk fat. Second, exogenous PA stimulated synthesis and incorporation of PA into triacylglycerols by dispersed mammary gland epithelial cells in vitro, as found by Hansen and Knudsen (1987). Chouinard et al. (1998) reported that PA of dietary origin might not have been sufficient to compensate for the depression in the de novo synthesis that occurs, as observed by Grummer (1991), when a fat supplement containing long-chain FA is fed. Several researchers (Yu et al., 1977; Mugrditchian et al., 1981; Greene and Selivonchick, 1990; Ng et al., 2000) have reported that fish maintained a constant level of total SFA regardless of the amount in their diet. Ng et al. (2001; 2004) have shown that palm oil has some advantages compared to other vegetable oils when used in feeds for warm-water fish species such as tilapia and catfish. Ng et al. (2007) found that FA composition of Atlantic salmon fillet total lipid showed close correlations with dietary palm oil inclusion, such that the concentrations of PA, oleic acid, linoleic acid, SFAs and monounsaturated FAs increased linearly with increasing dietary oil supplementation. Bell et al. (2002), studying the FA composition of muscle total lipid in Atlantic salmon post-smolts fed diets containing increasing levels of crude palm oil, found that the concentration of PA and other FAs increased linearly with increasing dietary palm oil (Figure 5).


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Figure 5. Relationship between dietary palmitic acid percentage and muscle palmitic acid percentage in Atlantic salmon (Bell et al., 2002; ■) and in African catfish (Ng et al., 2003;▲).

Similarly, Ng et al. (2003) found that FA composition in the muscle lipids of African catfish were strongly influenced by dietary treatments. In general, the FAs found in high concentrations in the diet were also the most abundant in muscle and the converse was true of the least abundant FAs. In particular, the concentration of PA was generally high in muscle lipid irrespective of diet. PA was present in low concentrations in the sunflower oil and crude palm kernel oil diets (7.2% and 7.7% of total FA, respectively) and at high concentrations in the refined, bleached, deodorized palm olein and crude palm oil diets (44.3% and 49.5% of total FA, respectively), but its concentration in muscle remained somewhat constant at a mean of about 26%. Nevertheless, relatively higher concentrations of PA were still found in the muscle of fish fed with diets having higher PA levels. PA was found in muscle lipids at a relatively uniform concentration of 19.4–30.2% of total FA despite being fed with diets containing varied levels of PA at concentrations of 7.2–49.5% of total FA. The conservation of PA levels might be because this FA is the major FA in phosphatidylcholine, found in the Sn-1 position. The amounts of SFA and unsaturated FAs in egg yolk could be altered by dietary manipulation (Milinsk et al., 2003). These authors found that there is a decrease in the percentage of PA and stearic acid in egg yolk produced by hens fed diets enriched with canola, flaxseed, soybean or sunflower oils in comparison with those of hens fed a control diet. Kang et al. (2001) found that PA composition of egg yolk was not influenced by hen diets containing different levels of palm oils and their PA percentage ranged from 21.8 to 24.0 % of total FA.

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Kazala, E.C., Lozeman, F.J., Mir, P.S., Laroche, A., Bailey, D.R.C. and Weselake, R.J. (1999). Relationship of fatty acid composition to intramuscular fat content in beef from crossbred Wagyu cattle. Journal of Animal Science, 77, 1717-1725. Keys, A. (1970). Coronary heart disease in seven countries. Circulation 41, 186-195. Klusmeyer, T. H., Lynch, G.L., Clark, J.H. and Nelson, D.R.. (1991). Effects of calcium salts of fatty acids and protein source on ruminal fermentation and nutrient flow to duodenum of cows. Journal of Dairy Science, 74, 2206-2219. Kouba, M. and Mourot, J. (1999). Effect of feeding linoleum acid diet on lipogenic enzyme activities and on the composition of the lipid fraction of the fat and lean tissues in the pig. Meat Science, 52, 39-45. Kritchevsky, D. (2000). Overview: dietary fat and atherosclerosis. Asia Pacific Journal of Clinical Nutrition, 9, 141-145. Kromhout, D., Keys, A., Aravanis, C., Buzina, R., Fidanza, F., Giampaoli, S., Jansen, A., Menotti, A., Nedeljkovic, S. and Pekkarinen M. (1989). Food consumption patterns in the 1960s in seven countries. American Society for Clinical Nutrition, 49, 889-894. Larick, D. K., Turner, B.E., Shoenherr, W.D., Coffey, M.T. and Pilkington, D.H. (1992). Volatile compound content and fatty acid composition of pork as influenced by linoleic acid content of diet. Journal of Animal Science, 70, 1397-1403. Lindsey, S., Benattar, J., Pronczuk, A. and Hayes, K.C. (1990). Dietary palmitic acid enhances HDL-cholesterol and LDL receptor mRNA abundance in hamsters. Experimental Biology and Medicine, 195, 261-269. Mandell, I.B., Buchanan-Smith, J.G., Holub, B.J. and Campbell, C.P. (1997). Effects of fish meal in beef cattle diets on growth performance, carcass characteristics and fatty acid composition of longissimus muscle. Journal of Animal Science, 75, 910-919. Milinsk, M.C., Murakami, A.E., Gomes, S.T.M., Matsushita, M. and de Souza, N.E. (2003). Fatty acid profile of egg yolk lipids from hens fed diets rich in n-3 fatty acids. Food Chemistry, 83, 287-292. Mills, E.W., Comerford, J.W., Hollender, C., Harpster, H.W., House, B. and Henning, W.R. (1992). Meat composition and palatability of Holstein and beef steers as influenced by forage type and protein source. Journal of Animal Science, 70, 2446-2451. McNamara, D.J. (2010). Palm oil and health: a case of manipulated perception and misuse of science. Journal of the American College of Nutrition, 29, 240S-244S. Menotti, A., Keys, A., Aravanis, C., Blackburn, H., Dontas, A., Fidanza, F., Karvonen, M.J., Kromhout, D., Nedeljkovic, D., Nissinen, A., Pekkanen, J., Punsar, S., Seccareccia, F. and Toshima, H. (1989). Seven countries study. First 20-year mortality data in 12 cohorts of six countries. Annals of Medicine, 21, 175-179. Morgan, C.A., Noble, R.C., Cocchi, M. and McCartney, R. (1992). Manipulation of the fattyacid composition of pig meat lipids by dietary means. Journal of the Science of Food and Agriculture, 58, 357-368. Mosley, S.A., Mosley, E.E., Hatch, B., Szasz, J.I., Corato, A., Zacharias, N., Howes, D. and McGuire, M.A. (2007). Effect of varying levels of fatty acids from palm oil on feed intake and milk production in Holstein cows. Journal of Dairy Science, 90, 987-993. Mu, H. and Høy, C.E. (2004). The digestion of dietary triacylglycerols. Progress in Lipid Research, 43, 105-133.


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Perdrix, M.F., Sutter, F. and Wenk, C. (1996). Facteurs de variation de la composition en acides gras de la matière grasse du lait de vache. Revue Suisse d’Agriculture, 28, 71-76. Redgrave, T.G., Kodali, D.R. and Small, D.M. (1988). The effect of triacyl-sn-glycerol structure on the metabolism of chylomicrons and triacylglycerol-rich emulsions in the rat. Journal of Biological Chemistry, 263, 5118-5123. Rodríguez, M.A., Crespo, N.P., Cortés, M., Creus, E. and Medel, P. (2002). Efecto del tipo de grasa de la dieta en la alimentación del broiler, con enfasis en los productos derivados del aceite de palma. Selecciones Avícolas, 44, 693-702. Samman, S., Kung, F.P., Carter, L.M., Foster, M.J., Ahmad, Z.I., Phuyal, J.L. and Petocz, P. (2009). Fatty acid composition of certified organic, conventional and omega-3 eggs. Food Chemistry, 116, 911-914. Sargent, J.R., Henderson, R.J. and Tocher, D.R. (1989). The lipids. In: Halver, J.E. (Ed.), Fish Nutrition, (pp.153-218). New York: Academic Press. Scaife, J.R.. Moyo, J., Galbraith, M., Michie, M. and Campbell, V. (1994). Effect of different dietary supplemental fats and oils on the tissue fatty acid composition and growth of female broilers. British Poultry Science, 35, 107-118. Scollan, N.D., Choi, N.-J., Kurt, E., Fisher, A.V., Mike Enser, M. and Wood J.D. (2001). Manipulating the fatty acid composition of muscle and adipose tissue in beef cattle. British Journal of Nutrition, 85, 115-124. Small, D.M. (1991). The effect of glyceride structure on absorption and metabolism. Annual Review of Nutrition, 11, 413-434. Smink, W., Gerrits, W.J.J., Hovenier, R., Geelen, M.J.H., Lobee, H.W.J., Verstegen, M.W.A. and Beynen, A.C. (2008). Fatty acid digestion and deposition in broiler chickens fed diets containing either native or randomized palm oil. Poultry Science, 87, 506-513. Smink, W., Gerrits, W.J.J., Hovenier, R., Geelen, M.J.H., Verstegen, M.W.A. and Beynen, A.C. (2010). Effect of dietary fat sources on fatty acid deposition and lipid metabolism in broiler chickens. Poultry Science, 89, 2432-2440. Steele, W. and Moore, J.H. (1968). The effects of a series of saturated fatty acids in the diet on milk-fat secretion in the cow. Journal of Dairy Research, 35, 361-369. Steffens, W. (1997). Effects of variation feeds on nutritive in essential fatty acids in fish value of freshwater fish for humans. Aquaculture, 151, 97-119. St. John, L.C., Lunt, D.K. and Smith, S.B., (1991). Fatty acid elongation and desaturation enzyme activities of bovine liver and subcutaneous adipose tissue microsomes. Journal of Animal Science, 69 1064-1073. Storry, J.E., Hall, A.J. and Johnson, V.W. (1968). The effect of increasing amounts of dietary red palm oil on milk fat secretion in the cow. British Journal of Nutrition, 22, 609-614. Sundram, K. (1994). Dietary palmitic acid results in lower serum cholesterol than does a lauric-myristic acid combination in normolipidemic humans. American Journal of Clinical Nutrition, 54, 841-846. Sundram, K., Sambanthamurthi, R. and Tan, Y.A. (2003). Palm fruit chemistry and nutrition. Asia Pacific Journal of Clinical Nutrition, 12, 355-362. Temme, E., Mensink, R.P. and Hornstra, G. (1996). Comparison of the effects of diets enriched in lauric, palmitic, or oleic acids on serum lipids and lipoproteins in healthy women and men. American Journal of Clinical Nutrition, 63, 897-903.


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Teye, G.A., Sheard, P.R., Whittington, F.M., Nute, G.R., Stewart, A. and Wood, J.D. (2006) Influence of dietary oils and protein level on pork quality. 1. Effects on muscle fatty acid composition, carcass, meat and eating quality. Meat Science, 73, 157-165. Tholstrup, T., Marckmann, P., Jespersen, J., Vessby, B., Jart, A. and Sandstrom, B. (1994). Effect on blood lipids, coagulation, and fibrinolysis of a fat high in myristic acid and a fat high in palmitic acid. American Journal of Clinical Nutrition, 60, 919-925. Tuten, T., Robinson, K.A. and Sgoutas, D.S. (1993). Discordant results for determinations of triglycerides in pig sera. Clinical Chemistry, 39, 125-128. Valsta, L.M., Tapanainen, H. and Männisto, S. (2005). Meat fats in nutrition. Meat Science, 70, 525-530. Van Oeckel, M.J. and Boucque, C.V. (1992). Omega-3-fatty-acids in pig nutrition – A review. Landbouwtijdschrift – Revue de l’Agriculture, 45, 1177-1192. Van Oeckel, M.J., Casteels, M., Warnants, N., Van Damme, L. and Boucqué, C.V. (1996). Omega 3 fatty acid in pigs nutrition: implication for the intrinsic and sensory and quality of meat. Meat Science, 44, 55-63. Verschuren, W.M., Jacobs, D.R., Bloemberg, B.P., Kromhout, D., Menotti, A., Aravanis, C., Blackburn, H., Buzina, R., Dontas, A.S., Fidanza, F., Karvonen, M.J., Nedelijković, S., Nissinen, A. and Toshima, H. (1995). Serum total cholesterol and long-term coronary heart disease mortality in different cultures. Twenty-five-year follow-up of the seven countries study. Journal of American Medical Association, 274, 131-136. Wood, J.D., Richardson, R.I., Nute, G.R., Fisher, A.V., Campo, M.M., Kasapidou, E., Sheard, P.R. and Enser, M. (2003). Effects of fatty acids on meat quality: a review. Meat Science, 66, 21-32. Wood, J. D., Nute, G. R., Richardson, R. I., Whittington, F. M., Southwood, O., Plastow, G., Mansbridge, R., da Costa, N. and Chang, K.C. (2004). Effects of breed, diet and muscle on fat deposition and eating quality in pigs. Meat Science, 67, 651-667. Wood, J.D., Enser, M., Fisher, A.V., Nute, G.R., Sheard, P.R., Richardson, R.I., Hughes, S.I. and Whittington, F.M. (2008). Fat deposition, fatty acid composition and meat quality: A review. Meat Science, 78, 343-358. World Health Organization (2003). Diet, nutrition and the prevention of chronic diseases. In: Technical Report Series 916 (pp. 1-104). Geneva, Switzerland: WHO. Yu, T.C., Sinnhuber, R.O. and Putnam, G.B. (1977). Effect of dietary lipids on fatty acid composition of body lipid in rainbow trout (Salmo gairdneri). Lipids, 12, 495-499. Zollitsch, W., Knaus, W., Aichinger, F. and Lettner, F. (1997). Effects of different dietary fat sources on performance and carcass characteristics of broilers. Animal Feed Science and Technology, 66, 63-73.



Chapter 4

GENERAL ASPECTS OF PALMITIC ACID Deusdélia Teixeira de Almeida, Mariana Melo Costa and Sabrina Feitosa Federal University of Bahia (UFBA), Brazil

ABSTRACT Palmitic acid or hexadecanoic acids is the most abundant saturated fatty acid in human nutrition and represents about 17.6g per day in the United Kington diet. It is the first fatty acid produced during the lipogenesis. During this process, glucose is converted to fatty acids, which then react with glycerol to produce triacylglycerols. Palmitic acid mainly occurs as its ester in triglycerides, especially in palm oil (40-44 %) but also in lard (20-30 %), dairy products (25-40 %) and cocoa butter (25-27 %). One of the main applications of palmitic acid in the food industry has been the formulation of interesterified fats, used as a replacement of trans fats. In breast milk, native lard, enzyme-directed and randomly chemically interesterified plant fats, palmitic acid is predominantly esterified to triacylglycerol, center or β-position, in native palm oil and cow´s milk, it is mainly at the external or α-positions. A higher palmitic acid absorption is obtained with formulas rich in palmitic acid esterified in triacylglycerol sn-2 position, than with those containing palmitic acid predominantly esterified in the sn-1,3 positions. These specific fatty acids distributions in triacylglycerol, determine the physical properties of the fat, which affects its absorption, metabolism and distribution into tissues. Many authors claim that a palmitic acid intake may promote increased risk of hypercholesterolemia, liver disease, type 2 diabetes, insulin resistance and toxicity. However, more recent investigations on the topic seem to have reconsidered the negative role of the dietary saturated fatty acids as a risk factor for cardiovascular diseases and show that not only the type of fat, but also that the triglyceride structure plays a role in these diseases.

Keywords: Palmitic acid, palm oil, triacylglycerol, saturated fatty acid, dietetic fatty acid


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D. Teixeira de Almeida, M. Melo Costa and S. Feitosa

LIST OF ABBREVIATIONS 3HB – 3-Hydroxybutyrate; AcAc – Cetoacetate; ACC – Acetyl-Coenzyme-A Carboxylase; ACP – Acyl Carrier Protein; ACSL – Long-Chain Acyl-CoA Synthetase; AMP – Adenosine Monophosphate; apo – Apolipoproteins; ARA – Arachadonic Acid; ATP – Adenosine Triphosphate; BC – Biotin Carboxylase; CACT – Carnitine/Acylcarnitine Translocase; CB – Cocoa Butter; CBE – Cocoa Butter Equivalents; CBR – Cocoa Butter Replacers; CBS – Cocoa Butter Substitutes; CoA – Coenzyme-A; CPT – Carnitine Palmitoyltransferase; CT – Carboxyltransferase; DHA – Docosahexaenoic Acid; DPA – Docosapentaenoic Acid; ELOVL – Elongation of Very Long-Chain Fatty Acids; EPA – Eicosapentaenoic Acid; ETC – Electron Transport Chain; FA – Fatty Acids; FAD – Flavin Adenine Dinucleotide; FADH – Reduced Flavin Adenine Dinucleotide; FAS – Fatty Acid Synthase; FABP – Fatty Acid-Binding Protein; FABPpm – Plasma Membrane Fatty Acid-Binding Protein; FAT/CD36 – Fatty Acid Translocase/Cluster of Differentiation; FAO – Fatty Acid Oxidation; FATP – Fatty Acid Transport Protein; GPx – Glutathione Peroxidase; GTP – Guanosine-5'-Triphosphate; HADC – 3-Hydroxyacyl-CoA Dehydratase HDL – High Density Lipoprotein; HMF – Human Milk Fat; HMG-CoA – 3-Hydroxy-3-Methyl-Glutaryl Coenzyme A; IL – Interleukins; JNK – Jun Amino Terminal Kinase; KAR – 3-Ketoacyl-CoA Reductase; LCAD – Long-Chain Acyl Coenzyme A Dehydrogenase; LCFA – Long-Chain Fatty Acids;


General Aspects of Palmitic Acid LDL – Low Density Lipoprotein; LRAT – Lecithin Retinol Acyltransferase; MAPK – Mitogen-Activated Protein Kinase; MCP – Monocyte Chemoattractant Protein; MG- Monoacylglycerols; mRNA – Messenger Ribonucleic Acid; MUFA – Monounsaturated Fatty Acid; NAD – Nicotinamide Adenine Dinucleotide; NADP – Nicotinamide Adenine Dinucleotide Phosphate; NAFLD – Nonalcoholic Fatty Liver Disease; NASH – Nonalcoholic Steatohepatitis; NFkB – Nuclear Factor Kappa B; OAA – Oxaloacetate; OOL- Dioleoyl- Linoleoyl-Glycerol; OOO – Trioleoyl Glycerol; OOS – Dioleoyl Stearoyl Glycerol; OPL – 1-Oleoyl-2-Palmitoyl-3-Linoleoyl-sn-Glycerol; OPO – 1,3-Dioleoyl-2-Palmitoylglycerol; PA – Palmitic Acid; Pi – Inorganic Phosphate; PKA – Protein Kinase; PKB – Protein Kinase B; PKC – Protein Kinase C; PLO – Palmitoyl-Linoleoyl-Oleoyl-Glycerol; PLP – Dipalmitoyl Linoleoyl Glycerol; POMF – Palm Oil Mid-Fraction; POO – Palmitoyl Dioleoyl Glycerol; POP – Dipalmitoyl Oleoyl Glycerol; POS – Glycerol-1-Palmitate-2-Oleate-3-Stearate; PPP – Tripalmitoil Glycerol; PUFA – Polyunsaturated Fatty Acid; RBD – Bleached and Deodorized; RDI – Recommended Daily Intake; RNA – Ribonucleic Acid; RNS – Reactive Nitrogen Species; ROS – Reactive Oxygen Species; RP – Retinyl Palmitate; RPB – Retinol-Binding Protein; SCD – Stearoyl-CoA Desaturase; SER – Smooth Endoplasmic Reticulum; SFA – Saturated Fatty Acid; SN – Stereospecific Numbering; SOD – Superoxide Dismutase; SOS – Glycerol-1,3-Distearate-2-Oleate; SPO – 1-Stearoyl-2-Palmitoyl-3-Oleoyl Glycerol; SREBP1c – Sterol Regulatory Element-Binding Protein;


65

66

D. Teixeira de Almeida, M. Melo Costa and S. Feitosa SSS – Tri-Saturated Fatty Acids; SSU – Di-Saturated Fatty Acids; SUS – Di-Saturated Fatty Acids; SUU – Diunsaturated; TAG – Triglyceride or Triacylglycerol; TC – Total Cholesterol; TCA – Tricarboxylic Acid Cycle; TER – trans-2,3-Enoyl-CoA Reductase; TFA – trans-Fatty Acid; TLR – Toll-Like Receptors; TNF-α – Alpha Tumoral Necrosis Factor; USU – Diunsaturated; UUU – Triunsaturated Fatty Acids; VLDL – Very-Low-Density Lipoprotein.

INTRODUCTION Fatty acids (FAs) have attracted the attention of the scientific community owing to their striking fundamental properties which are interesting for science and technology. Fatty acids are required not only for membrane synthesis, modifications of proteins and carbohydrates, construction of various structural elements in cells and tissues, production of signaling compounds, and energy storage, but also for solubilizing a variety of nonpolar and poorly soluble cellular and extracellular constituents [1, 2]. All fats and oils are esters of glycerol and fatty acids. It is commonly referred to as triglyceride or triacylglycerol (TAG) because of the glycerol molecule has three hydroxyl groups where a fatty acid can be attached. All TAGs have the same glycerol units, and then fatty acids contribute to the different properties. Fatty acids are often categorized into short chain (up to 6 carbons), medium chain (8 to 12 carbons), or long chain (>12 carbons). Although hydrocarbon chain length is an important determinant of function, fatty acids are often classified based on whether or not the fatty acid carbon chain contains no double bonds (saturated fatty acid – SFA), one double bond (monounsaturated fatty acid – MUFA), or more than one double bond (polyunsaturated fatty acid – PUFA), as well as the configuration of the double bonds (cis or trans). In addition, PUFAs are often further classified based on the position of the first double bond from the fatty acid methyl terminus, creating n-3 and n-6 fatty acids [3, 4]. It is the differences in chain length and saturation status that dictate their performance in food and cooking, as well as their role in the body and impact on human health and disease risk [5]. The major types of FAs in the circulation and in the tissues of mammals are the longchain and very-long-chain FAs with many degrees of saturation. These include palmitic acid (C16:0), palmitoleic acid (C16:1), stearic acid (C18:0), oleic acid (C18:1n-9), linoleic acid (C18:2n-6) and, particularly in smaller mammals, arachidonic acid (20:4n-6) and docosahexaenoic acid (22:6n-3) [6]. The variety of fatty acid in common fats and oils is provided in Table 1.


Table 1. Fatty acid profiles (%)a of select animal and vegetable fats and oils

a

b

c

d

LIPID

SFA

8:0

10:0

12:0

14:0

16:0

18:0

MUFA

18:1

PUFA

18:2

18:3

Avocado oil Beef tallow Butterj Canola oil Coconut oil Corn oil Flaxseed oil Grapeseed oil Lard Olive oil Palm oil Palm kernel oil Rice bran Salmon oil Soybean oil

11.9 46.8 53.6 7.6 11.8 12.9 9.0 9.6 36.9 13.7 49.3 81.5 19.7 19.9 15.7

0.0 0.0 5.1 0.0 1.0 0.0 0.0 0.0 0.0 0.0 0.0 3.3 0.0 — 0.0

0.0 0.0 2.6 0.0 0.8 0.0 0.0 0.0 0.1 0.0 0.0 3.7 0.0 — 0.0

0.0 0.9 2.7 0.0 6.1 0.0 0.0 0.0 0.2 0.0 0.1 47.1 0.0 — 0.0

0.0 3.5 7.8 0.0 2.3 0.0 0.1 0.1 1.3 0.0 1.0 16.4 0.7 3.3 0.0

11.3 23.5 22.6 4.4 1.1 10.6 5.1 6.7 22.4 11.2 43.5 8.1 16.9 9.9 10.4

0.7 17.8 10.4 6.8 0.4 1.8 3.4 2.7 12.7 1.9 4.3 2.8 1.6 4.3 4.4

72.6 39.3 22.0 65.1 0.8 27.6 18.5 16.1 42.5 72.4 37.0 11.4 39.3 29.0 22.8

69.9 33.9 20.8 63.5 0.8 27.4 18.3 15.8 38.8 70.7 36.6 11.4 39.1 17.0 22.6

13.9 3.8 3.2 29.0 0.2 54.7 67.9 69.9 10.5 10.4 9.3 1.6 35.0 40.3 57.7

12.9 2.9 2.9 19.6 0.2 53.5 14.3 69.6 9.6 9.7 9.1 1.6 33.4 1.5 51.0

1.0 0.6 0.4 9.4 0.0 1.2 53.4 0.1 1.0 0.7 0.2 0.0 1.6 1.0 7.1

EPAe DPAf DHAg 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 31.3 0.0

ARAh

TFAi

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.7 0.0

0.0 0.0 0.0 0.4 0.0 0.3 0.1 — 0.0 — — — — — 0.5

Listed as percent of total fatty acid content, based on 13.6 g fatty acids/tablespoon. Cells without numbers did not have data in United States Department of Agriculture Nutrient Database; bSFA ═ saturated fatty acid; cMUFA ═ monounsaturated fatty acid; dPUFA ═ polyunsaturated fatty acid; eEPA ═ eicosapentaenoic acid; fDPA ═ docosapentaenoic acid; gDHA ═ docosahexaenoic acid; hARA═ arachadonic acid; iTFA ═ trans-fatty acid; jButter contains 16 % water and therefore the percentages are unable to be directly compared with percentages of the other fats/oils [5].


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PALMITIC ACID The word 'palmitic' is French in origin, derived from the word palmitique which refers to the pith of the palm tree. The palmitic acid (PA) (C16:0, hexadecanoic acid) is one of the most common saturated fatty acids found in animals and plants with chemical formula CH3─(CH2)14─COOH. It is a white solid that melts at 63-64 °C, have a boiling point of 351352 ºC, a mass of 256.42 g·mol and a density of 0.853 g·cm3 at 62 °C. PA is water-insoluble (7.2 x 10-4 g/100 g) at 20 ºC and slightly soluble in ethanol and iced acetone, but it is highly soluble in alcohol, heated acetone and chloroform with 4.76 ± 0.02 pKa value [7]. It is constituted of carbon atoms linked to each other through single bonds as shown in Figure 1. The molecules are arranged as dimers through O─H═O hydrogen bonds. These dimers are packed in bilayers with terminal methyl groups at both external faces and these layers are parallel to the crystallographic (100) plane [8]. PA mainly occurs as its ester in TAG, especially in palm oil but also in lard, dairy products, avocado oil, butter and beef tallow (Table 1). Palmitate is a term for the salts and esters of palmitic acid. The palmitate anion is the observed form of palmitic acid at basic pH. Aluminum salts of palmitic acid and naphthenic acid were combined during World War II to produce napalm. The word "napalm" is derived from the words naphthenic acid and palmitic acid [9].

Figure 1. Unit cell and molecular structure of palmitic acid [8].

Beyond PA presence in feed, it has utilities ranging from inks antioxidants application, waterproofing in textile industry, candle manufacture together with paraffin and liquid crystal, widely used in electronic industry [7]. Palmitic acid can participates in several chemical reactions as other acids of this same class, which include: - Neutralization reactions: salts of palmitic acid are formed through this reaction. The palmitic acid reacts with a hard base, forming a salt of palmitic acid and water. Using


69

General Aspects of Palmitic Acid

sodium hydroxide as a base, for example, sodium palmitate is obtained as shown in Equation 1 (Figure 2) [7].

Figure 2. Equation 1. Neutralization reaction.

Those salts, especially lithium, sodium and potassium, have vast application as ink constituent, lubricants and insulations that protect against water corrosive action [7]. - Esterification reaction: directly reaction of palmitic acid and a monohydric alcohol obtaining an ester (Figure 3. Equation 2), or by a reaction between a salt of palmitic acid and a haloalkane (Figure 3. Equation 3) [7].

(2)

(3) Figure 3. Equation 2 and 3. Esterification reactions.

RETYNIL PALMITATE The two most abundant retinoid forms that are present in the diet are retinol and retinyl esters - a fatty acyl group is esterified to the hydroxyl terminus of retinol [10]. Dietary retinol is taken up directly by mucosal cells. Nevertheless, dietary retinyl esters are cleaved in the intestine by the pancreatic triglyceride lipase and intestinal brush border enzyme, phospholipase B [11]. The free retinol taken up by the enterocyte is complexed with cellular retinol-binding protein type 2 and the complex serves as a substrate for reesterification of the retinol by the enzyme lecithin retinol acyltransferase (LRAT). The resulted retynyl ester are incorporated with other neutral lipid esters (i.e., triacylglycerols and cholesteryl esters) into chylomicrons and absorbed via the lymphatics [11, 12]. In the vascular compartment, much of the chylomicron triacylglycerol is hydrolyzed by lipoprotein lipase in extrahepatic tissues, resulting in the production of a ―chylomicron remnant‖ that contains most of the newly absorbed retinyl esters [13]. Under conditions of adequate vitamin A nutrition, the liver is the main site of vitamin A storage, with more than 95 % of the total neutral retinoid being present


70


as retinyl esters, predominately retinyl palmitate and stearate [12, 14, 15]. According to Ihara et al. [12] the explanation for the use of those fatty acids would be that the retinyl esters together cholesterol present in its particle would be release into the liver cells via LDL receptor wherein saturated fatty acid regulate it pathway largely mRNA level. Once the cells has met it requirements for retinol, saturated fatty acids inhibit receptor activity, so that the receptor is no longer able to internalize retynil esters. In the response to cellular requirements the liver release retinol in forms of a retinol-binding protein (RPB4), target cells, a cell surface receptor for retinol RBP4 remove retinol from RPB4. Esterification techniques using palmitic acid have yielded more stable esters in the form of retinyl palmitate which has been used successfully as a supplement as well as a way to fortify numerous foods, including vegetable oil, rice, monosodium glutamate, cereal flours and sugar [review 16, 17]. This application is due to its high stability in relation to vitamin A and its low cost [16]. The oil matrix protects against the oxidation of vitamin A during storage, improves stability of the retinol and facilitates the vitamin‘s absorption by the body [18]. The advantages of oil fortified with retinyl palmitate have historically been utilized by food aid programs, where a daily intake of 16 g of oil provided approximately 50 % of the recommended daily intake (RDI) of an adult male [19]. Surman et al. [20] reports that retinyl palmitate it also can be associated with toxicities at high doses. The precise human dose required to ensure efficacy without toxicity remains a point of controversy. Also, retinyl palmitate (RP) is widely used in pharmaceutical and cosmetics products to improve the skin elasticity [21, 22].

OCCURRENCE OF PALMITIC ACID Palm Oil Elaeis guineensis palm tree fruits are yellow or orange ovoid little coconuts, with variable-size and seeds that produce two types of oil: the palm oil from fruit mesocarp and palm kernel oil from seed [23, 24]. The palm oil has become the major edible oil in world, markets accounting for 57 % worldwide vegetable oils exports and approximately 62 million tons of palm oil is projected to 2015 against 45.5 million tons in 2010 [25]. The major palm oil producers are Malaysia, Indonesia and Nigeria [26, 27]. Palm oil and palm kernel oil differentiate to physico-chemical properties as their intended applications [28]. Palm kernel oil is minority and produces 50 % of oil with dark coloration and high contents of lauric and myristic FAs. It is used in manufacturing of confectionery products, ice cream, soaps, detergents, among others [23; 29; 30]. Palm oil contains 6-10 % of tri-saturated fatty acids (SSS), especially tripalmitoil glycerol (PPP). Saturated fraction corresponds to 1-5 % of lauric and myristic acid and 17-23 % to palmitic acid (C16:0) in sn-2 position, while the myristic (C14:0) and the stearic (C18:0) in 1 and 3 position [23]. The unsaturated fraction represents 44-50 % of di-unsaturated fatty acids (SSU or SUS): dipalmitoyl oleoyl glycerol (POP) and dipalmitoyl linoleoyl glycerol (PLP); 38-42 % diunsaturated (SUU or USU): POO and PLO, palmitoyl dioleoyl glycerol and



71

palmitoyl-linoleoyl-oleoyl-glycerol, respectively; and 5-8 % of triunsaturated fatty acids (UUU), especially trioleoyl glycerol (OOO) and dioleoyl- linoleoyl-glycerol (OOL) [23, 30]. Palm oil physico-chemical properties allow it to be the most widely fractioned oil (Table 2). Fractioning involves physical or chemical refine applying high temperatures, desodorisation and deacidification of oil under vacuum, in both cases. Physical deacidification accurs at 250-270 ºC under vacuum up to 3-5 Torr, whereas chemical uses 220-240 ºC. The high temperatures and vacuum are necessary to remove undesirable compounds as traces of metals, free fatty acids, oxidation and decomposition products. Nevertheless, those procedures also remove some tocopherols and tocotrienols, and all carotenoids presented in the oil [30, 31, 32, 33, 34]. Two types of oil with different physical and chemical properties are obtained from the fractioning process: the olein (65-70 %), liquid phase, melting point at 18-20 ºC; and stearin (30-35 %), solid, melting point at 48-50 ºC. Refining industry obtain the others fractions from those two, applying in the manufacture of several products [23; 34; 35]. Malaysia Palm Oil Research Institute patented the physical refine method that involves degumming and bleaching, followed by clarification and desodorisation by molecular distillation using lower temperatures (< 170 ºC) and pressure (100 mTorr), obtaining an oil comparable to the RBD (bleached and deodorized) with less than 0.1 % of acidity and impurities, but with vitamin E retention and 80 % of carotenoids, called carotino [28, 29, 33, 34]. It is also observed the use of crude palm oil in Bahia (Brazil) and Africa, being part of many culinary dishes [36]. The crude palm oil is reddish because it contains a high amount of β-caroten (500-1000 mg·kg–1) [37]. Palm oil liquid phase, olein (65-70 %), is liquid at room temperature and can present TGAs precipitation by higher melting point in case of cold storage. It is extensively used as cooking oil and blended with other oils. The stearin is a co-product of olein that is used in vegetable fats manufacturing as margarines, pastas and bakery products [31]. Palm olein and super olein have higher contents of linoleic and oleic FAs and lower content of palmitic acid than the stearin fraction [23]. Whereas the middle fraction present properties between olein and stearin, with 60 % of palmitic acid and 40 % of oleic acid, being used as cocoa butter substitute [23; 35]. Palm oil composition shows that most triglycerides are esterified in sn-2 position with unsaturated fatty acids (> 58.25 % of oleic acid and > 18.41 % of linoleic acid) and high proportion of palmitic acid [38]. Edem [23] points out some characteristics that made palm oil an important food industry ingredient. These characteristic include: (a) a high solid glyceride content giving required consistency without hydrogenation, (b) resistance to oxidation and therefore, long shelf life (c) high melting point triglycerides together with relatively low solids content at 10 ºC, which is helpful in formulation of products with a wide plastic range, that are suitable for hot climates and some industrial applications, (d) a competitive price, (e) use in limited quantities in margarine specifying a high polyunsaturated fatty acid (PUFA) level because of its linoleic acid content (10–11 %), (f) relatively slow melting properties because of the wide plastic range and (g) slow crystallization properties capable of leading to structural hardness and a tendency for recrystallization.


72

D. Teixeira de Almeida, M. Melo Costa and S. Feitosa Table 2. Fatty acid composition of palm oil and its fractions

Fatty acid 12:0 14:0 16:0 16:1 18:0 18:1 18:2 18:3 20:0 Saturates Monounsaturates Polyunsaturates [23].

Red palm oil 0–0.2 0.8–1.3 43.1–46.3 Trace–0.3 4.0–5.5 36.7–40.8 9.4–11.9 0.1–0.4 0.1–0.4 50.2 39.2 10.5

Palm olein 0.1–0.2 0.9–1.0 39.5–40.8 Trace–0.2 3.9–4.4 42.7–43.9 10.6–11.4 0–0.4 0.1–0.3 45.8 42.5 11.6

Super olein 0.4 1.4 31.5 – 3.2 49.2 13.7 0.3 0.4 36.6 49.2 14.0

Super stearin 0.1–0.2 1.0–1.3 46.5–68.9 Trace–0.2 4.4–5.5 19.9–38.4 4.1–9.3 0.1–0.2 0.1–0.3 52.1–76.2 19.9–38.6 4.2–9.5

Butter Butter or buttermilk is an important edible fat in northern Europe, North America and Brazil, being about the third product of world‘s milk production. It is a yellow-to-white solid and an emulsion of fat globules, water and inorganic salts produced by churning the cream from cows‘ milk. Butter has high energy (~715 Kcal/100 g), cholesterol (~215 mg/100 g) and a major content of FAs, respectively: palmitic (25-32 %, C16:0), oleic (22-29 %, C18:1), myristic (C14:0) and stearic (C18:0) about same proportions (8-13 %), linoleic (C18:2) and lauric (C12:0) with less than 4.5 %. In despite of high SFAs contents, MUFAs are higher than PUFAs in buttermilks [39, 40]. Verardo et al. [40] determined the fatty acid composition of different samples of butter and the samples manufactured by a traditional method showed higher levels of MUFAs and PUFAs compared with industrial samples. Palmitic acid presented the higher fatty acids contents, 29.8 to 31.1 % and 30.3 to 33.6 % from traditional and industrial process, respectively.

Cocoa Butter The generic name of cocoa is Theobroma belonging to the family of Sterculiaceae, also called ‗‗Food of God‘‘. It contains about 30–50 beans, covered with pulp. About 500 years ago, cocoa beans were originated from Latin America and within a few years it was spread to Europe [41, 42]. Cocoa butter (CB) is a highly valued ingredient primarily used in the confectionery industry due to its specific physical and chemical properties. CB is solid at room temperature (below 25 °C), and liquid at body temperature (~37 °C) [43]. Furthermore, the predominant presence of symmetrical TAG, about 90 % of the TAG species in CB, is mainly responsible for the functionality of this fat [44]. The major FAs of cocoa butter are palmitic acid (C16) 25–33.7 %, stearic acid (C18:0) 33.7–40.2 %, oleic acid (C18:1) 26.3–35 % and linoleic acid



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(C18:2) 1.7–3 % which contribute about 98 % of the total fatty acid [45]. Regarding the palmitic acid composition of natural cocoa butter produced from various countries ranged from 24.1 to 27.9 % [43]. CB fat contains significantly higher amount of saturated acid leadings to triglycerides of glycerol-1,3-dipalmitate-2-oleate (POP), glycerol-1-palmitate-2oleate-3-stearate (POS) and glycerol-1,3-distearate-2-oleate (SOS). Among these three TAGs, POS is the major leading triacylglycerol component present in cocoa butter with range 42.5– 46.4 % yield followed by SOS (27.8–33.0 %) and POP (18.9–22.6 %). Therefore, palmitic acid occupies mostly the sn-1 position in cocoa butter [45].

Avocado Oil Avocado (Persea americana Mill) is an important tropical fruit and a good source of lipophilic phytochemicals such as monounsaturated fatty acids, carotenoids, vitamin E and sterols [46]. It has several cultivars that present great variation on time of fruit production and oil content in the pulp. Studies have indicated that the avocado oil is similar to olive oil and can be used in cosmetics and also for human consumption [47, 48]. New Zealand, Mexico, Chile United States and South America are among the main avocado oil producers. Avocado oil has the advantage that can be obtained from the fruit by means of a cold extraction methods, which is an easy and low technology that allow maintain in the oil significant amounts of the bioactive phytochemicals present in the fruit [47]. In comparison to other vegetable sources, avocado oil is characterized by its contents of palmitic (C16:0), linoleic (C18:2), palmitoleic (16:1) and alpha-linolenic (18:3) FAs that are 13.5, 12.6, 3.26 and 1.0 % of total fatty acids, respectively. Stearic (18:0), tridecanoic (13:0), tetradecanoic (14:0), cis-10-heptadecenoic (17:1) and cis-13-16-eicosenoic (20:2) FAs are present in trace amounts [49]. Ozdemir and Topuz [50] showed in Fuerte and Hass avocado varieties present a reduction of palmitc acid according to fruit ripening, with variations of 22.4-12 % to Fuerte variety and 23.3-16.8 % to Hass variety. Yanty et al. [51] studying three Malaysia avocados varieties, found oleic acid as the major fatty acid (43.65–51.22 %) followed by palmitic (26.41–30.37 %) and linoleic (12.75–17.45 %) FAs. Oils of avocado fruits are generally found to have extremely low amounts of stearic acid (0.27–1.56 %).

Beef Tallow Beef tallow is one residual material from slaughterhouses which main destination is the soap industry, however because of its high melting point (45 ºC) and low level of polyunsaturated fatty acids (< 3 %) [52, 53] beef tallow is considered as a less valuable fat not suitable for direct human consumption [54]. From the nutritional point of view, vegetable oils are preferred over animal fats, because contain a high proportion of saturated fatty acids and low proportion of polyunsaturated fatty acids. Regarding fatty acids composition, tallow has about 29 % palmitic acid, 25-37 % stearic acid and 23-31 % oleic acid. Thus, saturated fatty acid content is responsible for over 50 % of total fatty acids in the beef tallow [52, 53]. The higher stearic and palmitic acid content of beef tallow are accounted for the unique properties of high melting point and high viscosity [55]. In relation to TAG, beef tallow and other bovine adipose tissues have nearly 50% of the fatty acids in the sn-2 position, which are


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oleic acid (unsaturated fatty acid), palmitic and stearic acids. Nevertheless, oleic acids are the most fatty acids in the sn-1,3 positions [52, 56].

Lard Animal fats such as lard and tallow have long been recognized as raw material for food and industrial applications. Lard has exceptional properties compared to other vegetable oils such as firmness and special flavor values. Nevertheless, lard has lost its significance to numerous substitutes such as hydrogenated cottonseed and soybean oil due to its negative nutritional values such as low digestibility, high calories and high content of saturated fatty acids. Nonetheless, lard remains a major ally in the meat product industry due to its positive contributions in flavor and texture [57, 58]. Lard contains about 28.4 % palmitic acid (C16:0), 21 6 % stearic acid (C18:0), 31.4 % oleic acid (C18: 1) 11.1 % linoleic acid (C18: 2). It is unique among animal depot fats, because it has a strong predominance of saturated fatty acids in the sn-2 position [52]. In lard, C16:0 is located exclusively at the sn-2 position, with an unsaturated fatty acid at sn-3 but the fatty acid occupying the sn-1 position is highly variable, as in SPO, OPL and OPO TAGs (species dominants in lard) [56].

Milk Bovine milk and dairy products have long traditions in human nutrition. Its fat fractions are widely used in a variety of food products such as liquid milk, cream, butter, cheese and ice cream due to many favorable physical, chemical and nutritional properties of milk fat [59]. Milk contains, in average, about 33 g total lipid/L, being 95-98 % of triacylglycerols composed of fatty acids of different length and saturation, less than 0.5 % of cholesterol, about 1 % of phospholipids and less than 0.5 % of free fatty acids. SFA represent more than half of total milk fat, about 19 g/L especially lauric (C12:0), myristic (C14:0) and palmitic (C16:0). Furthermore it is rich in oleic acid (about 25 %), however, a relatively poor source of polyunsaturated fatty acids as linoleic (C18:2) and alpha linolenic (C18:3), with contents in the order of 3 % and 2 %, respectively [59; 60; 61]. In relation to TAG molecules, the most probable sn-position of the main fatty acids in milk fat are: sn-position 1 C16:0, C18:0 and C18:1 with 44.1 %, 54.0 % and 37.3% respectively; sn-position 2 C12:0 (62.9 %), C14:0 (65.6 %) and C16:0 (45.4 %); and snposition 3 98.1 % of C4:0, 93.0 % of C6:0, 34.5 % of C8:0 and 41.5 % of C18:1. Therefore, the most probable sn-position of palmitic acid in TAG molecules of milk are in sn-1 and sn-2 [61].

Human Milk The human mammary gland has evolved with unusual pathways for acylation of fatty acids into triglycerides for secretion in milk, and major portion of milk fat is comprised by



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these molecules that represent 98 % of total fat [61, 62]. The average fat content of human milk is about 3.8–3.9 g/100 ml, but it varies widely [61]. The predominant fraction are saturated fatty acids, followed by a relatively high proportion of monounsaturated fatty acids such as oleic acid (18:1n 9) [59]. Palmitic acid is the qualitatively and quantitatively major SFA in human milk and constitutes approximately one fourth of this with a concentration highly conserved, regardless of ethnic origin or the nutritional status of the woman [62]. PA comprises 17 % to 25 % of the total FAs [63] and it is an important source of energy thereby contributing 10–12 % of breast-fed infants' dietary energy intake [64]. The stereospecific numbering (sn) designates the location of fatty acids within the triglyceride molecule. If the glycerol is drawn with the first and third hydroxyl groups to the right and the second to the left, the first carbon is termed sn-1; the second, sn-2, and the third sn-3 [61]. Most of the 16:0 in human milk is located in the sn-2 position of the triacylglycerol molecules (70 % to 75 %) [63], in contrast to cow‘s milk and vegetable oils which have 40 % and 5 % to 20 %, respectively, of the 16:0 in the sn-2 position [62]. The major unsaturated fatty acid in human milk is oleic acid (18:1n-9) and this is mostly esterified at the triglyceride sn-1,3 (outer) positions, with the result that triglycerides with the structure 18:1n-9—16:0— 18:1n-9 (1,3-dioleoyl-2-palmitoylglycerol, OPO) are a major triglyceride species in human milk and represent an estimated 11.8 % of the total triglyceride species [65]. Since lipolytic enzymes will cleave the FA in sn-1 and sn-3-positions, human milk palmitic acid will appear primarily in the remaining monoglyceride which has a higher polar than free palmitic acid [61]. Hence, most of the 16:0 is absorbed as the sn-2 and this structure is preserved through and beyond the intestinal wall.

SPECIAL OILS Pequi Tree Pequi tree (Caryocar brasiliensis Camb.) is a member of the Central and South American family Caryocaraceae [66, 67]. It stands out by high occurrence in Brazilian Cerrado and extensive period of fruit production, which can be collected from September to February in Cerrado of Goias (Brazil) [68]. It is a Brazilian oleaginous fruit, rich in A, E, C and B2 vitamins in both edible parts: pulp and kernel [69, 70]. The oil is extracted by rendering the mesocarp and kernel of pequi fruit and the oil is generally used to cook rice with the objective of adding specific flavors and a light-yellow color to the final product [71]. The fruit is rich in β-carotene and selenium. It is even used to produce fermented liquor. Pequi has a considerable economic importance in some parts of Brazil and has a substantial ecological impact on the country. In relation to fatty acids composition, kernel and pulp present high content of palmitic acid (35.17 % and 43.76 %) and oleic (43.59 % and 55.87 %), respectively. The TAG composition of pequi oil is also relatively simple with trioleoyl glycerol (OOO, 56 g·kg−1), palmitoyl dioleoyl glycerol (POO, 466 g·kg−1) and dipalmitoyl oleoyl glycerol (POP, 452 g·kg−1) comprising 974 g·kg−1 of the total. Dioleoyl stearoyl glycerol (OOS) was found in small amounts (5.2 g·kg−1). The relatively simple composition of pequi oil may be of interest for selected applications.


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It appears that is has some promise as a less expensive chocolate substitute upon fractionation [23, 38].

Pili Nut Oil The pili (Canarium ovatum) is a tropical tree, native to the Philippines, with about 600 species in the Burseraceae family. The pili fruit is a drupe with 4 to 7 cm long and 2.3 to 3.8 cm in diameter and weighs 15.7 to 45.7 g which consists of a pulp (68 % by weight), a shell (25 % by weight) and a seed (7 % by weight) [72]. The fibrous pulp is edible and is usually consumed as an appetizer or dessert. Already the pili pulp oil is comparable to coconut oil and can be used for cooking. It can also be used as fuel for lighting, and for the manufacture of soaps, perfumes and other cosmetic products. Nevertheless, the kernel or known as pili nut is the most important part of the pili fruit, with a taste comparable to that of walnut and almond, has been used in chocolates, baked goods and ice cream. It contains about 70–75 % oil which can be used for domestic and industrial purposes [73]. In the pili nut oil the fatty acids saturates (palmitic and stearic FAs) account for 33.3 and 10.9 %, respectively and the oleic acid represents 44.7 % of pili nut oil. Furthermore, this oil is very low in polyunsaturated fatty acids (18:2 and 18:3), with the combination of linoleic and linolenic less than 11 % [72]. With regard to triacylglycerol (TAG) composition in the roasted pili nut oil, 54.3 % is of 1-palmitoyl-2-oleyl-3-oleyl-sn-glycerol (POO), 13 % of 1,3-dipalmitoyl-2-oleoyl-glycerol (POP), 8.2 % of triolein (OOO) and 6.1 % of 1-palmitoyl-2-oleyl-3-stearoyl glycerol (POS), whereas unrosted pili nut oil has about 61.1 % of 1-palmitoyl-2-oleyl-3-oleyl-sn-glycerol (POO), 12.5 % of 1,3-dipalmitoyl-2-oleoyl-glycerol (POP), 7.7 % of Triolein (OOO) and 6.2 % of 1-palmitoyl-2-oleyl-3-stearoyl glycerol (POS) respectively. Thus, the pili nut oil not contain the trisaturated TAGs [74].

Occurrence of Palmitic Acid Interesterified Foods Oils and fats industry has been looking for alternatives to replace the called hydrogenated fats which are rich in trans fatty acids, because of the human health implications [75, 76, 77]. One of the methods that have been applied in this way is the interesterification of fats. In this reaction, the fatty acids remain unchanged occurring a redistribution of them in triglyceride molecule, resulting in a modification of triglyceride composition. The final characteristic is completely determined by initial fatty acids composition of raw material. Intesterification can be carried out chemically or enzimatically. A chemical, such as sodium methoxide, is used as a catalyst in chemical interesterification which produces complete positional randomization of the acyl group in the TAG. On the other hand, enzymatic interesterification uses microbiological lipases as catalyst [78; 79]. Commonly used interesterified fats, which provide suitable functionality for the food industry, include fats that are rich in the long-chain SFA, palmitic acid (16:0) and stearic acid (18:0) [77]. Chemical and enzymatic interesterification has been specially used in the formulation of margarines and shortenings to provide products with no TFA but that still maintain physical properties, taste, and stability [80].



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Shortenings and Margarines For preparation of various food products industries take into account the polymorphism of fats, defined as the ability of a chemical compound to form different crystalline or liquid crystalline structures and three forms of α, β' and β are typical polymorphic forms of fat [81]. Polymorph β', the meta-stable form, is used in margarine and shortening because of its optimal crystal morphology and fat crystal networks which give rise to optimal rheological and texture properties [82]. Because of the high content of palmitic acid, palm oil is β' tending, and hence, palm oil plays the role of a β' in margarines and shortenings. It was related that margarines at 15 ºC with palmitic acid below 11 % are in β form, while those with 17 % and above in β' crystal form [80]. This phenomenon leads to the production of smooth, continuous and homogenous products.

Cocoa Butter Alternatives Food industries are looking for alternative fats to cocoa butter (CB) from natural matrices that are denoted as cocoa butter replacers (CBRs), cocoa butter equivalents (CBEs) and cocoa butter substitutes (CBSs) fat [41; 83]. CBRs are defined as non-lauric fats that could replace cocoa butter either partially or completely in the chocolate or other food products. On the other hand, a cocoa butter equivalent (CBE) is a type of fat that has a very similar chemical composition, but its triglycerides derive from other source than cocoa beans, such as palm kernel oil, palm oil, mango seed fat, kokum butter, sal fat, shea butter, illipé butter, soya oil, rape seed oil, cotton oil, ground nut oil and coconut oil [43]. Replacing the cocoa butter either partially or wholly with other natural fats has been investigated due to the technological and economic advantages. CBE contains approximately 40 % 1-palmito, 2-olein, 3-sterin glycerol (POS), 27 % of 1,3distearin-monooleate glycerol (SOS) and 21 % of 1,3 dipalmitin-2-monooleato glycerol (POP) and minor amounts of other triglycerides. A suitable raw material for the production of CBE in terms of cost, availability and composition is palm oil mid-fraction (POMF), which is obtained by double fractionation of palm oil. POMF contains approximately 73 % POP, 13 % POS, 2 % SOS and 12 % of other triglycerides [84].

Palmitic Acid in Infant Formulas Developing infant milk fat similar to human milk fat (HMF) is of great interest and a challenge to food processors. Infant formulas have been designed to provide infants with the required for optimal growth and development [62; 85; 86]. The quantity of fat in its formulas provides an infant with 40–50 % of its total daily energy intake [87]. Complex mixtures of oils that contain modest levels of long-chain saturated fatty acid can be used in developing formulas with fatty acid profiles closer to that of human milk [86; 87] but not the triacylglycerol structure [62; 88; 89]. Simple mixtures of unsaturated vegetable oils (e.g., corn or soy oil) and fats that contain a predominance of lauric acid or shorter saturated fatty acids (e.g., coconut oil and palm kernel) are common fat blends in some formulas [90]. Several types of enzymatic reactions appear in the literature to synthesize structured TAGs rich in PA


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at position 2 and in other fatty acids at positions 1 and 3 (oleic acid, caprylic acid, etc.). These fat compositions can be obtained by subjecting fatty mixtures comprising glycerides consisting substantially of more saturated 2-palmitoyl glycerides to a rearrangement catalyst, such as a lipase, which is regiospecific in activity in the 1- and 3-positions of the glycerides. Under the influence of the catalyst, unsaturated fatty acid residues may be introduced into the 1- and 3-positions of the 2-palmitoyl glycerides by exchange with unsaturated free fatty acids or their alkyl esters. Unfortunately, such formulas have resulted in poor absorption of fats and minerals, particularly when studied in infants during the first few weeks of life [62, 91, 92]. This is because PA is present in the sn-2 position (human breast milk, lard native, enzyme-directed and randomly chemically interesterified fats plant) compared with the sn-1 and sn-3 positions (bovine milk, randomly chemically interesterified lard or crude palm oil). It was found the relative absorption of palmitic acid and full fat was linearly related to the proportion of palmitic acid in the sn-2 position of the TAG in human infants. Pancreatic lipase selectively hydrolyses the fatty acids at the sn-1 and sn-3 positions, yielding free fatty acids and monoacylglycerols (MGs). The TAG sn-2 position is absorbed more efficiently than free palmitic acid and it is conserved through the digestion, absorption and chylomicron TAG synthesis [63, 91]. Nonetheless, after digestion, the free PA solidify in the intestine because of their high melting temperature, creating insoluble and indigestible complexes with dietary minerals (eg, calcium), and causing hard stools [87]. Quinlan et al. [93] were able to relate stool hardness to stool composition. They concluded that differences in the triacylglycerol palmitate content of formula and breast milk resulted in more calcium soap formation in formula- fed infants and thus in harder stools. HMF containing palmitic acid at the sn-2 position yields 2-MG during digestion which does not lead to the formation of calcium soaps. Consequently, both calcium and 2-MG become bioavailable for the infant [94]. Yaron et al. [63] studying two infant formulas demonstrates that β-palmitate may affect the intestinal microbiota composition during the first weeks of life by increasing Lactobacillus and Bifidobacteria abundance in the stool, and thus may provide beneficial effects for the health and well-being of formula-fed infants. They concluded that the effects of the infant formulas of the gut microflora are due to lipid estructure. It is therefore important to synthesize TAGs with a composition and distribution of fatty acids similar to those of human milk. In particular there is considerable interest in the synthesis of 1,3-diolein-2-palmitin (OPO), which is the most abundant TAG in human milk.

BIOCHEMISTRY OF PALMITIC ACID Lipogenesis Fats used by or stored in animal tissues come from two sources: enzymatic synthesis and diet. When a cell or organism has more than enough metabolic fuel available to meet its energetic needs, the excess is generally converted to FAs and stored as lipids such as TAGs [95]. Insulin stimulates fatty acid synthesis in adipose tissue, liver and lactating mammary glands along with formation and storage of TAG in adipose tissue and liver [96, 97].



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In glycolysis, glucose (C6H12O6) is converted to two molecules of pyruvate [97]. The pyruvate dehydrogenase complex contributes to transforming pyruvate into acetyl-CoA by a process called pyruvate decarboxylation. When energy needs in a cell are not high, citrate, the condensation product of oxaloacetate and acetyl-CoA in the glicolysis, builds up in the mitochondrial matrix. Nonetheless, mitochondria do not contain an acetyl CoA transporter, therefore a shuttle system, called the citrate shuttle, is required to move the C2 units across the membrane citrate transport out of the mitochondria provides a mechanism to stimulate fatty acid synthesis in the cytosol where acetil Co-A is cleaved back to oxaloacetate and acetyl-CoA by the ATPcitrate lyase (Figure 4) [98, 99]. The oxaloacetate (OAA) is converted to malate by cytosolic malate dehydrogenase The malate is then either transported to the mitochondrial matrix where it is reduced by mitochondrial malate dehydrogenase or oxidized and decarboxylated by malic enzyme in the cytosol to form pyruvate, NADPH and CO2 [100]. The production of cytosolic NADPH by malic enzyme provides additional reducing equivalents for fatty acid synthesis and supplements the NADPH generated by the pentose phosphate pathway. The pyruvate formed by malic enzyme is transported back into the mitochondrial matrix where it is carboxylated by pyruvate carboxylase to form oxaloacetate. Note that while malic enzyme generates NADPH for fatty acid synthesis, the pyruvate carboxylase reaction consumes an ATP in the matrix to generate OAA. The liver cells can still run the glycolytic pathway as the NADH/NAD+ ratio is low in the cytoplasm while NADPH/NADP+ ratio is high [98]. The acetyl-CoA can be converted to malonyl-CoA via the action of acetyl-CoA carboxylase (ACC; biotin-dependent enzyme) [101]. Two isoforms have been identified, ACC1 and ACC2, with ACC1 being principally expressed in lipogenic tissues such as adipose tissue and liver, while ACC2 is predominantly expressed in oxidative tissues like heart and skeletal muscle [102]. This reaction, which proceeds in two half-reactions, a biotin carboxylase (BC) reaction and a carboxyltransferase (CT) reaction is the first committed step in fatty acid biosynthesis and is the rate limiting reaction for the pathway [103]. The ACC is controlled by three global signals – glucagon, epinephrine and insulin – that correspond to the overall energy status of the organism. Insulin stimulates FA synthesis by activating the ACC, whereas glucagon and epinephrine have the reverse effect. The levels of citrate, palmitoyl CoA and adenosine monophosphate (AMP) within a cell also exert control. Citrate, a signal that building blocks and energy are abundant, activates the carboxylase. Palmitoyl CoA and AMP, in contrast, lead to the inhibition of the carboxylase [102, 103, 104]. The remaining series of reactions of fatty acid synthesis in eukaryotes is catalyzed by fatty acid synthase (FAS) is a homodimeric and multifunctional complex of 250 kDa and contains seven different enzymatic activities plus a domain that covalently binds a molecule of 4′-phosphopantetheine, of the acyl carrier protein (ACP) [105, 106]. PA, the most abundant acid, is synthesized de novo from acetyl-CoA as a primer, malonyl-CoA as a carbon donor, and NADPH as a reducing equivalent according to the following reaction [104, 105, 107].


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Figure 4. Origin of cytoplasmic acetyl-CoA: carnitine shuttle.

Figure 5. Acetyl-coenzyme-A carboxylase (ACC) has critical roles in fatty acid metabolism. The ACCcatalyzed biotin carboxylase (BC) and carboxyltransferase (CT) reactions [103].

Basically, this reaction consists of elongating the acetyl group by C2 units derived from malonyl-CoA, so that each step takes place by condensation, reduction, dehydration and further reduction [99] (Figura 6): 1. Transfer of the acetyl group of acetyl-CoA to ACP-catalyzed by acetyl-CoA-ACP transacylase;



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2. Next, this two-carbon fragment is transferred to a temporary holding site, the thiol group of a cysteine residue on the enzyme; 3. The now-vacant ACP accepts a three-carbon malonate unit from malonyl CoA catalyzed by malonyl CoA-ACP transacylase; 4. The acetyl group on the cysteine residue condenses with the malonyl group on ACP as the CO2 originally added by acetyl CoA carboxylase is released. The result is a four-carbon unit attached to the ACP domain. The loss of free energy from the decarboxylation drives the reaction catalyzed by 3-Ketoacyl-ACP synthase; 5. Reduction of the Beta-keto group to a Beta-hydroxyl group with NADPH catalyzed by Beta-keto-ACP reductase; 6. Dehydration between the alpha and Beta by Beta-hydroxyacyl-ACP dehydrase. A molecule of water is removed to introduce a double bond between carbons 2 and 3 (the α- and β-carbons); 7. Reduction of the trans double bond by NADPH catalyzed by enoyl-ACP reductase).

Figure 6. Reaction sequence for biosynthesis of fatty acids de novo by the animal FAS.

The result of these seven steps is production of a four-carbon compound (butyryl) whose three terminal carbons are fully saturated, and which remains attached to the ACP. These seven steps are repeated, beginning with the transfer of the butyryl chain from the ACP to the Cys residue, the attachment of a molecule of malonate to the ACP (3), and the condensation of the two molecules liberating CO2 (4). The carbonyl group at the β-carbon is then reduced (5), dehydrated (6), and reduced (7), generating hexanoyl-ACP. This cycle of reactions is repeated five more times, each time incorporating a two-carbon unit (derived from malonyl CoA) into the growing fatty acid chain at the carboxyl end. When the fatty acid reaches a length of 16 carbons, the synthetic process is terminated with palmitoyl-S-ACP. All the carbons in PA have passed through malonyl CoA except the two donated by the original acetyl CoA, which are found at the methyl-group (ω) end of the fatty acid [98, 99, 105, 106].


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Palmitic acid can be further elongated by the addition of two-carbon units in the smooth endoplasmic reticulum (SER). Elongation involves the addition of two-carbon units to a fatty acyl-CoA, employing malonyl-CoA as the donor and NADPH as the reducing agent. In mammals, the initial and rate-controlling condensation reaction is catalysed by the elongase enzymes referred to as elongation of very long-chain fatty acids (ELOVLs) [108]. To date, seven ELOVL proteins (ELOVL1-7) have been identified, with ELOVL1, ELOVL3, ELOVL6 and ELOVL7 preferring saturated and monounsaturated fatty acids as substrate; and ELOVL2, ELOVL4 and ELOVL5 being selective for PUFAs [109, 110]. The process of elongation requires four separate enzymatic reactions: condensation between the fatty acylCoA and malonyl-CoA to yield 3-ketoacyl-CoA; reduction of 3-ketoacyl-CoA to generate 3hydroxyacyl-CoA; dehydration of 3-hydroxyacyl-CoA to produce trans-2-enoyl-CoA, and; reduction of trans-2-enoyl-CoA to form the two-carbon elongated acyl-CoA (Figure 7). [108, 109, 111].

Figure 7. Enzymatic steps in long-chain fatty acid elongation. Enzymatic steps of microsomal fatty acyl chain elongation. ELOVL, elongation of very-long-chain fatty acids; KAR, 3-ketoacyl-CoA reductase; HADC, 3-hydroxyacyl-CoA dehydratase; TER, trans-2,3-enoyl-CoA reductase [108].



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Fatty acids are also formed in endoplasmic reticulum and the reaction is catalyzed by enzymatic systems, generically designated as Acyl-CoA desaturases [112]. Acyl-coenzymeA (CoA) desaturases introduce a double bond at a specific position on the acyl chain of longchain fatty acids, thereby influencing several of the key biological properties of the fatty acid itself and of more complex lipids containing this acyl chain. Mammalian cells express Δ9, Δ6 and Δ5-desaturase activities [108]. The desaturation process involves an oxidoreductase chain (including cytochrome b5) that O2 works as last oxidant of Acyl-CoA and NADPH (or NADH). The PA product of FAS and its metabolite produced by Stearoyl-Coa Desaturase (SCD-1), C16:1n-7, can both be further elongated by ELOVL6 to yield stearic acid (C18:0) and vaccenic acid (C18:1, n−7), respectively [108; 109].

Lipolisis In the fasted state, most tissues, except the brain and red blood cells, rely heavily on the direct utilization of FA to generate energy. The prime pathway for the degradation of fatty acids is mitochondrial fatty acid β-oxidation (FAO), a key metabolic pathway for energy homoeostasis in organs [113, 114]. Long-chain fatty acids (LCFA) (C16-18) in tissues exist as components of TAG or phospholipids. Adipose tissue TAG storages are the primary source of fatty acids used for FAO during fasting conditions [115]. TAGs are first hydrolyzed by the action of endothelium-bound lipoprotein lipase release free FAs, which are transported to tissues via the bloodstream. The uptake of FAs seems to be largely mediated by membrane proteins, although passive uptake probably also occurs. This implies that several transport steps are necessary before fatty acids are oxidized [115, 116, 117]. The solubility of LCFA in aqueous solutions is extremely low, so the fatty acids must cross the cell membrane via a protein-mediated mechanism. Membrane-associated fatty acidbinding proteins (‗fatty acid transporters‘) are small (15kDa) cytosolic proteins that enhance the uptake of long chain and very long chain fatty acids into cells. Through their control of fatty acid transport, metabolism and storage, FABPs are proposed to be central regulators of lipid metabolism, inflammation and energy homeostasis. In humans, FATPs comprise a family of six highly homologous proteins, FATP1–FATP 6, which are found in all FAs utilizing tissues of the body [118, 119]. Besides FATPs, FAT/CD36 (fatty acid translocase /Cluster of Differentiation 36) has been shown to function as a plasma membrane LCFA transporter in various tissues, including skeletal muscle, heart, liver, adipose tissue and the small intestine [120, 121]. Other mechanism is fatty acid uptake plasma membrane fatty acidbinding protein (FABPpm) which is associated with the plasma membrane in many tissues including liver, adipose tissue, cardiac muscle and vascular [122, 123, 124, 125]. After transport across the plasma membrane, FAs must be esterified to coenzyme A, on the outer mitochodrial membrane by long chain acyl-CoA synthetase activity (ACSL; C12 to C20) before they can undergo oxidative degradation. This reaction is coupled with two ATP hydrolysis to AMP and 2Pi. The mitochondrial membrane is not permeable to long chain acyl-CoA (i.e., C16-C18), therefore requires the initial conversion of acyl-CoA to an ester acylcarnitine, followed by transport of the acylcarnitine across the inner mitochondrial membrane into the mitochondrial matrix and subsequent delivery of acyl-CoA [126]. This process is referred to as ―carnitine shuttle‖ and requires the concerted action of 3 proteins 6:


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carnitine palmitoyltransferase I (CPT I), carnitine/acylcarnitine translocase (CACT), and carnitine palmitoyltransferase II (CPT II). [116, 127, 128, 129]. The FAO pathway as described above is displayed in Figure 8: 1. CPT I converts acyl-CoA compounds to their acylcarnitine metabolites at the outer mitochondrial membrane and further transported across the inner mitochondrial membrane by CACT. 2. The enzyme CPT2 is responsible for the conversion of the acylcarnitines back to the corresponding acyl-CoAs, the true substrates of the FAO pathway.

Figure 8. The mitochondrial carnitine shuttle. Abbreviations: CPT1, carnitine-palmitoyl transferase 1; CPT2, carnitine-palmitoyl-transferase 2; carnitine/acylcarnitine translocase (CAC or CACT) [127].

The complete oxidation PA is achieved in the following three steps (Figure 9): betaoxidation of fatty acid chain yielding acyl-CoA; the oxidation of acetyl CoA to CO2 and production FADH2 and NADH2 in citric acid cycle; the transfer of electron from reduced electrons carries FADH2 and NADH2 to mitochondrial respiratory chain resulting into ATPs [130]. This process involves a variety of enzymes: long-chain acyl coenzyme dehydrogenase (LCAD), enoyl-CoA hydratase, hydroxyacyl-CoA dehydrogenase and ketoacyl-CoA thiolase [117, 131]; 1. In the first step, LCAD, catalyzes oxidation of the fatty acid moiety of acyl-CoA to produce a double bond is introduced into a carboxylic acid between the α and β carbons, FAD is the electron acceptor, and electrons from the reaction ultimately enter the respiratory chain and are carried to O2 with the concomitant synthesis of two ATP molecules per electron pair; 2. In the second step of the fatty acid oxidation cycle, water is added to the double bond of the trans-Δ2-enoyl-CoA to form the L stereoisomer of β-hydroxyacyl-CoA. This reaction, catalyzed by enoyl-CoA hydratase; 3. In the third step, the L-β-hydroxyacyl-CoA is dehydrogenated to form β-ketoacylCoA by the action of β-hydroxyacyl-CoA dehydrogenase; NAD+ is the electron acceptor. The NADH formed in this reaction donates its electrons to NADH



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dehydrogenase an electron carrier of the respiratory chain. Three ATP molecules are generated from ADP per pair of electrons passing from NADH to O2 via the respiratory chain; 4. Finally hydroxy-acyl-CoA is dehydrogenated to 3-keto-acyl-CoA. Then, thiolytic cleavage of the 3-keto-acyl-CoA produces a two-carbon chain-shortened acyl-CoA plus acetyl-CoA. Each cycle yields an acyl-CoA shortened by two carbon atoms, an acetyl-CoA, and one nicotinamide adenine dinucleotide (NADH) and one flavin adenine dinucleotide (FADH2) as electron carriers (or reducing equivalents). The PA undergoes seven passes through this oxidative sequence, in each pass losing two carbons as acetyl-CoA. At the end of seven cycles the last two carbons of palmitate (originally C-15 and C-16) are left as acetyl-CoA. Generally, the total ATP yield due to the complete oxidation of palmitic acid in the following equation [99, 130]: Palmitic acid + 8 Coenzyme A + 7 FAD+ + 7NAD+ + ATP → 8 CH3CO-SCoA (AcetylCoA) + 7FADH2 + 7 (NADH + H+) + AMP + PPi. If one acetyl –CoA involved in TCA cycle gives = 10 ATPs; ATPs due 8 acetyl-CoA = 8 x 10 = 80; ATPs due to 7 FADH2 = 1.5 x 7= 10.5; ATPS due 7 (NADH + H+) = 2.5 x 7.5 = 17.5. The total of number ATPs produced 108. During the initiation of the β- oxidation pathway a 2 ATPs converts into a 2 AMP and 2 Pi for the activation of fatty acid. So, net ATPs produced by palmitic acid are 106. These calculations assume that mitochondrial oxidative phosphorlation produces 1.5 ATPs/FADH2 oxidized and 2.5 ATP/NADH2 oxidized. The Guanosine-5'-triphosphate (GTP) produced directly in the acid citric cycle yields ATP in the reaction catalyzed by nucleoside diphosphate kinase [99, 130].

Figure 9. The β-oxidation of saturated fatty acids involves a cycle of four enzyme-catalyzed reactions. Each cycle produces single molecules of FADH2, NADH, and acetyl-CoA and yields a fatty acid shortened by two carbons.


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Furthermore, the produced acetyl-CoA during PA β-oxidation goes to TCA or to ―ketone bodies‖ production. The oxaloacetate availability to make acetyl-CoA get in the citric acid will determinate which metabolic pathway acetyl-CoA will follow [132]. In some situations, as fasting or catecholaminergic stress, oxaloacetate molecules will be intended to glucose synthesis by gluconeogenesis. With this, oxaloacetate availability content will be decreased, and consequently, less acetyl-CoA will get into TCA, thereby having Ketogenesis [133]. In this process, ―ketone bodies‖ are formed. These are small water-soluble molecules, easily transportable form of acetyl units that can be oxidized to carbon dioxide and water to yield energy. ―Ketone bodies‖ include acetoacetate (AcAc), 3-hydroxybutyrate (3HB) and acetone, which are an important source of energy for peripheral tissues, supply up to 50% of basal energy requirements these tissues and up to 70% for the brain which cannot derive energy from other sources when blood glucose levels are low [134, 135].

HEALTH EFFECTS OF PALMITIC ACID Effects of Palmitic Acid in Serum Lipids and Lipoproteins Dietary and endogenous fats are carried to the target organs by different lipoproteins, i.e. chylomicrons, LDL and HDL. These particles contain a core of TAG liposoluble vitamins and cholesteryl esters, surrounded by a phospholipid and free cholesterol layer. These also contain, by the way, specific proteins, called apolipoproteins (apo) which act as enzyme cofactors or receptor ligands. Exogenous fat is transported in chylomicrons from the intestinal epithelium to the peripheral cells, reaching the bloodstream via the lymphatic system [11]. After entry in the blood stream the chylomicrons are hydrolyzed by the endothelial-bound lipoprotein lipase with apo C-I as a co-factor, allowing the delivery of free FAs to muscle and adipose tissue. The chylomicron remnants are rapidly taken up into the liver via especial receptor. ApoE is the moiety required for rapid hepatic removal. Its activity is inhibited by C apolipoproteins, especially apoC-I. The liver utilizes the exogenous fat and can release surplus lipids via VLDL into the blood. The VLDL is another substrate for lipoprotein lipase. The remaining VLDL remnants can either be taken up into the liver or are hydrolyzed to LDL. These last delivers cholesterol to all body cells via its receptor [136]. Moreover other type of lipoprotein denominated as high–density protein (HDL) is an important scavenger of surplus cholesterol transporting it from cell membranes to the liver, where it is degraded or converted into biliary salts, an then eliminated by the entero-hepatic cycle [137]. Keys et al. [138] and Hegsted et al. [139] showed from mathematical equations that serum cholesterol concentration of a person would be predicted by the diet fat consumption. Furthermore, SFAs were two times more effective in raising cholesterol, and PUFA reducing them. In this context, SFA can not correspond with more than 1/3 of fat diet intake. Nevertheless, there are great reservations about those equations, due to they were made with middle-aged men who consumed high fats contents and, therefore, they‘re not necessarily valid to other populations in different diet conditions. In addition, in 1960, when the biochemical and dietary analyses were done, trans and n-3 PUFA could not be identified and hence only information on saturated monounsaturated and polyunsaturated fats were report.



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Keys et al. [138]: TC = 2.76 SFA – 1.356 PUFA 1 1.56D C1/2 Where TC is the total cholesterol, SFA is the proportion of SFA (% of the total energy amount), UFA is the proportion of unsaturated FA (% of the total energy amount) and C is the dietary cholesterol (in mg per 1000 kcal/day). Hegsted et al. [139]: TC = 2.166 SFA – 1.356 PUFA 1 1.56 C‘ Where C‘ is the dietary cholesterol (in mg/day). Much effort has gone into proving that SFA is the major dietary factor for hypercholesterolemia, although the mechanism by which this would occur still remains unclear. Some hypotheses have been cited to explain its effect. On the one hand, they might be responsible for a decrease in the LDL receptor expression on the hepatocyte surface, and on the other hand, their incorporation into the cell membrane phospholipids might impair its fluidity, thus disturbing the receptor action. A diet rich in SFAs may inhibit, to some extent, the hepatic esterification of cholesterol, thus raising the concentration of unesterified cholesterol. In response to raised unesterified cholesterol, the hepatic concentration of LDL receptor mRNA is decreased, thus reducing LDL receptor activity, resulting in raised plasma LDL cholesterol levels [140]. Nonetheless, the debate over what constitutes the "ideal" fat is controversial, since it uses the term saturated fat without distinguishing individual SFA. In this sense, even a cursory analysis of some of the so-called saturated fats (e.g., palm oil, lard, tallow, butter, coconut oil) reveals that they have distinct profiles and empirically exert different metabolic effects. Accordingly, research in recent years has shifted toward elucidating the effects of specific dietary fatty acids in TAG, as opposed to specific classes of fats, on plasma lipids and lipoprotein metabolism [141]. Scientific studies point out an association between saturated fat and blood lipids increase due to the presence of three principal FAs: myristic, lauric and palmitic. Since PA is the principal saturated acid in the diet, it is the chief saturated component used to develop the diet equations [138; 139], has meant that the cholesterol-raising property of SFA has generally been attributed to their palmitic acid content. Researches that assess your action in many body functions had been based on human and animals studies, especially, with palm oil and its fractions as principal source of PA [review 142]. Whereas most studies have shown a cholesterol-raising effect of this FA [140, 143, 144, 145], some others have demonstrated relative neutrality [141]. According to Grundy and Denke [140], three nutritional factors would be responsible for raising serum LDL levels; these are SFA, cholesterol itself and excess caloric intake. These authors point out the major cholesterol-raising SFA in the diet would be the PA. In the same way, Sun et al. [146] reviewed 25 trials of at least 2 weeks that compared the effects of palm oil consumption with natural highly unsaturated and partially hydrogenated vegetable oils and animal fat. These authors concluded that palm oil consumption results in higher LDL cholesterol levels than other natural unsaturated vegetable oils and it may be preferable to trans-fat rich oils based on its effect on HDL cholesterol and suggested that more studies are needed to evaluate the effects of palm oil consumption on incidence of coronary heart diseases. Contrary to the predictions of the Keys et al. [138] and Hegsted et al. [139] equations, the review about the cholesterolaemic effects of palm oil by Wai [review 142], indicates that the


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substitution of palm oil or its liquid fractions (palm olein, super olein) for habitual fats in the diet does not result in an elevation of total serum cholesterol. In another review, Edem [23] concluded that, in animal experiment and human studies, palm oil administration with approximately 50 % of saturated fatty acid, does not behave as saturated oil, reducing the blood levels of total cholesterol, LDL cholesterol. A review by Sundram [29] also concluded that high levels of palmitic acid in the diet do not significantly affect serum total and LDL cholesterol levels. Khosla and Hayes‘ [141] conclusions about cholesterolaemic effects of the saturated fatty acid of palm oil suggest that not all SFAs are cholesterol-raising. According to authors, when fatty acids contents are similar, the palmitic acid appears to have no impact on the plasma cholesterol in normocholesterolaemic subjects. Above 400 mg of dietary cholesterol intakeed per day, PA might be cholesterol increasing, even more than myristic acid and quite neutral underneath this value. Nevertheless, if cholesterol consumption exceeds the critical value or when hypercholesterolaemic subjects are studied, the PA appears to increase the plasma cholesterol. Furthermore, authors linked the different PA actions to the differences in LDLreceptor status. It seems that more studies are needed to explain these inconclusive results. Free FAs absorption rate depends on the type of fatty acid and intestine emulsifier environment. An important explanation of why palm oil does not "follow‖ the Keys [138] and Hegsted [139] model is due to unsaturated fatty acids are in sn-2 position (> 58.25 % of oleic acid and > 18.41 % of linoleic acid) and a high proportion of PA is in sn-1 and 3 positions (17-23 %) [23]. Therefore, as already mentioned in this text, fatty acids in sn-2 position are preferentially absorbed at bowel wall and, thereby, more bioavailable than the fatty acids in sn-1 and 3 positions [87]. On the assumption that all SFA localized at sn-1 and 3 positions in palm oil are preferentially absorbed, whilst saturated are faecal excreted as salts, therefore only 8 % of SFA localized at sn-2 position would be absorbed as consequence there is a less caloric intake and a lower serum TGA content. Another explanation to palm oil hypocholesterolemic action would be the presence of tocotrienols. They are admittedly considered hypocholesterolemic once they regulate cholesterol synthesis through 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inactivation – enzyme that primarily synthesizes the cholesterol [147]. In a review of Hayes and Khosla [148], the authors concluded that cholesterolemic effects of PA are large extent determined by the concomitant level of linoleic FAs. It is why C18:2 regulate numerous lipogenic genes involved in fatty acid synthesis. PUFA maximally inhibit hepatic gene transcription when they provide 20 % of the dietary calories. Nonetheless, as little as 5% of calories as PUFA will inhibit gene expression 50 %. In this way, once 18:2 intake is above ―threshold‖ (5–7 %) the detrimental effects of the SFA on LDL are no longer observed in part because LDL receptors are maximally up-regulated and sterol regulatory element-binding protein (SREBP1c; that group of proteins uptake cholesterol and FAs biosynthesis) is inhibited, resulting in decreased FAs synthesis and decreased VLDL secretion. As a consequence, PA ―appears‖ neutral above the threshold requirement for linoleic FAs (Figure 10). Simply stated it implies that a certain level of 18:2 is required by an individual to prevent certain SFA-rich fats from raising the serum LDL level. If you drop below your 18:2 threshold, LDL will increase, with the increase being most severe during consumption of 12:0 1 14:0-rich fats. Also note that if 18:2 intake is high enough (6–10 % en depending on the LP ―setpoint‖), SFA no longer have a significant cholesterol-raising effect. Simply stated it implies that a certain level of 18:2 is required by an



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individual to prevent certain SFA-rich fats from raising the serum LDL level. If you drop below your 18:2 threshold, LDL will increase, with the increase being most severe during consumption of 12:0 1 14:0-rich fats. Also note that if 18:2 intake is high enough (6–10 % depending on the LP ―setpoint‖), SFA no longer have a significant cholesterol-raising effect [148].

Figure 10. The scheme depicts the 18:2/SFA ratio hypothesis, which can be applied to data in Simply stated it implies that a certain level of 18:2 is required by an individual to prevent certain SFA-rich fats from raising the serum LDL level. If you drop below your 18:2 threshold, LDL will increase, with the increase being most severe during consumption of 12:0 1 14:0-rich fats. Also note that if 18:2 intake is high enough (6–10 % depending on the lipids ―setpoint‖), SFA no longer have a significant cholesterolraising effect [148].

Palmitic Acid and Fatty Liver Disease The liver is a central organ in metabolism that serves multiple functions such as protein synthesis, glycogen storage, hormone production, and detoxification [149]. Fatty liver is the earliest and the most common form of both nonalcoholic steatohepatitis (NASH) and nonalcoholic fatty liver disease (NAFLD) [150, 151]. Hepatocellular injury, inflammation and fibrosis are hallmarks of NASH, which are observed in only a fraction of subjects with NAFLD, although the exact mechanisms leading from NAFLD to NASH are still largely unknown [152]. Several lines of evidence indicate the importance of both quantitative and qualitative (e.g. saturated vs unsaturated) changes in dietary FAs as relevant mechanisms for the development of NAFLD both in rodent models and in humans [150, 153, 154, 155]. NAFLD develops when consumption of energy exceeds the combustion of calories and the unburnt energy is conserved in the form of TAG in liver [150]. Donnelly‘s et al. [156] research with obese patients showed that TAG accounted for in liver, 59.0 % of TAG arose from nonesterified fatty acid; 26.1 % from de novo lipogenesis and 14.9 % from the diet. Patients under those conditions develop with a high production of reactive oxygen/nitrogen species (ROS/RNS) that promote hyper-stimulation of Ito cells, increasing collagen production in hepatic parenchyma with a consequent fibrosis and cirrhosis that can lead to hepatocellular carcinoma [157, 158]. Oxidative stress refers to an imbalanced cellular state in which the production of ROS/RNS are increased to an extent that overrides the normal operating free radical clearing mechanisms; such as glutathione peroxidase (GPx), superoxide dismutase (SOD) and catalase [159]. This process plays a significant role in the


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development of hepatic steatosis to NASH. The increase in FA oxidation in the steatotic state could potentially induce an increased electron flux through the electron transport chain (ETC), which may lead a major production of reactive oxygen/nitrogen specie (ROS/RNS). These reactive lipid derivatives have the potential to amplify intracellular damage by mediating the diffusion of ROS/RNS into the extracellular space thus causing tissue damage [160]. Enzymatic and no enzymatic systems are also capable to avoid hepatic damage, inducing an inflammatory process initiation. Damage and lipid peroxidation products induce an inflammatory response with up-regulation of pro-inflammatory cytokines including alpha tumoral necrosis factor (TNF-α), interleukins 6 and 1 (IL-6 and IL-1). These cytokines are of major importance for directing polymorphonuclear and mononuclear leukocytes into inflamed tissues [158]. The greater cytokines pro-inflammatory production, especially TNF-α and IL-6 and IL-1, can contribute to peripheral and hepatic of insulin, which induce to an infiltration in the hepatic parenchyma, in a vicious cycle that promotes more tissue injury. This mechanism is described as the ‗two hit‘ model with the ‗first hit‘ being steatosis and insulin resistance, and the ‗second hit‘ needed to initiate NASH requiring ‗other‘ factor(s) that promote lipid peroxidation, inflammatory cascade, oxidative stress, tissue injury and inflammatory process [161]. Moreover a ‗multiple parallel hits‘ model has been suggested to promote progression of steatosis to NASH because of failure of the antilipotoxic protection systems of the liver and multiple hits from the gut and/or adipose tissue [162]. Palmitic acid roles in NAFLD installation and development have been discussed. PA overloading is known to induce apoptotic cell death and a large number of molecular mechanisms have been implicated in this action: nitric oxide (NO) synthesis, suppression of antiapoptotic factors such as Bcl-2 [163, 164] reactive oxygen species generation, endoplasmic reticulum stress [165], nuclear factor-kB activation [166]. Apoptosis can be triggered by mitochondrial damage, which is followed by the release of cytochrome c and the caspase cascade [167]. The BAX protein activates mitochondrial release of cytochrome c [168], while Bcl-2 is a mitochondrial protein inhibits the apoptotic process and promotes cell survival [169]. Ji et al. [163] research showed that occur a decrease in mitochondrial Bcl-2 and a markedly increase in mitochondrial level of Bax in the HepG2 cells treated with PA from 200 to 400 mM concentrations. The authors suggest that this mechanism can contribute to NASH and NAFLD installation, and especially may play an important role in the transition from steatosis to steatohepatitis in human. Other studies [170] on the effect of FAs-induced steatosis on cellular apoptosis have demonstrated that palmitic and oleic FA mixtures-induced steatosis is associated with apoptosis in hepatocyte cell cultures. Joshi-Barve‘s et al. [171] studies also showed that exposure to excess palmitic acid induces apoptosis and IL-8 production in hepatocytes in a relation of dose-dependent and time dependent manner, via activation of c-Jun amino terminal kinase (JNK/AP-1), and nuclear factor kappa B (NF-B) transcription factors for IL-8 expression.

Palmitic Acid and Diabetes Diabetes is a group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both [172, 173]. The vast majority of cases of



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diabetes fall into two broad etiopathogenetic categories. In one category, type 1 diabetes, the cause is an absolute deficiency of insulin secretion. Individuals at increased risk of developing this type of diabetes can often be identified by serological evidence of an autoimmune pathologic process occurring in the pancreatic islets and by genetic markers. In the other, much more prevalent category, type 2 diabetes, is associated with a combination of pancreatic β-cell dysfunction and insulin resistance [174]. The chronic hyperglycemia produces ―glucotoxicity‖ characterized by β-cell function gradual deterioration and insulin resistance aggravation. It is similar to the paradoxically deleterious effects of chronic hyperglycemia, if ―lipotoxicity‖ is produced, once the free FAs which are essential fuels in the normal state, become toxic when they are chronically present in excessive levels [164, 175]. Under diabetic conditions, oxidative stress and endoplasmic reticulum stress are induced in various tissues [173, 176, 177, 178, 179]. Moreover, the β-cells have very low levels of antioxidative enzymes, becoming them more susceptible to the stress [172]. ROS can function as signaling molecules to activate a number of cellular stress-sensitive pathways that cause cellular damage, and are ultimately responsible for the late complications of diabetes. Evidence suggests that common stress-activated signaling pathways such as nuclear factor nuclear factor-κB (NF-κB) [180, 181, 182], p38 mitogen-activated protein kinase (MAPK) [183], protein kinase C (PKC) [184], toll-like receptors (TLRs) [185, 186], and c-Jun Nterminal kinase (JNK) [187; 188] underlie the development of these diabetic complications. Nuclear factor kappa-B (NFkB), a redox-sensitive transcription factor regulating a battery of inflammatory genes, has been indicated to play a role in the development of numerous pathological states [189]. Activation of NFkB induces gene programs leading to transcription of factors that promote inflammation, such as leukocyte adhesion molecules, cytokines and chemokines [181]. Mitogen-activated protein kinases (MAPKs) are a family of serine/threonine kinases and consist, among others, Jun N-terminal protein kinase (JNK), p38s MAP kinase, cyclic AMP dependent protein kinase (PKA), protein kinase B (PKB) and protein kinase C (PKC) [190]. It is involved in the regulation of a wide range of cellular responses, including cell proliferation, differentiation, and survival [191]. It‘s also well established that p38 and JNK play important roles in mediating apoptosis caused by various stimuli [190]. There are three isozymes of JNK: JNK1, JNK2 and JNK3, and that only JNK1 has been shown to be implicated in type 2 diabetes [192] probably to reduce insulin gene expression [187]. The TLR family is known to consist of 10 members (TLR1-TLR10) that are pattern recognition receptors which initiate innate immune responses upon recognition of a wide range of pathogen-associated molecular patterns [185, 193]. In vitro studies have shown that β-cells are vulnerable to palmitate, in the presence of high glucose concentration [194, 195]. Many authors report that the palmitate induces β-cell dysfunction in vivo by activating inflammatory processes within islets, for example: activation of nuclear factor-kB, resulted in increased expression of several proinflammatory cytokines (TNF-α, IL-1β, IL-6, MCP1) in rat liver as well as an increase in circulating MCP1 levels. The rise in plasma MCP1 is particularly interesting because MCP1 is well established to regulate macrophage recruitment to sites of inflammation [196]. Additionally, palmitate increased the expression and secretion of inflammatory cytokines (e.g. IL-6 and TNF-α) and impaired insulin sensitivity via an NFkB/PKC pathway in muscle cells [197, 198]. Too increased intracellular concentration of PA producing diacylglycerol


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have been shown to activate and cause cellular redistribution of protein kinase C isoforms [199], which result in the induction of inflammatory pathways via NF-κB activation [200]. Jiang [201] studies with PA showed to induce endothelial progenitor cells apoptosis via p38 and JNK mitogen activated protein kinases MAPK pathways in a time-and-dose-dependent manner. Eguchi et al. [186], through a combination of in vivo and in vitro studies, reported that SFA palmitate induces β-cell dysfunction. According to authors this cell responds to palmitate via the TLR4/MyD88 pathway and produce chemokines that recruit M1-type proinflammatory monocytes/macrophages to the islets. Depletion of M1-type cells protected mice from palmitate-induced β-cell dysfunction. After insulin binds to insulin receptor on cell surface, insulin receptor and its substrates are phosphorylated, which leads to activation of various insulin signaling pathways [202]. Reynoso et al. [203] evaluated several aspects of the insulin resistance induced by palmitic acid in rats and found that after treatment with 0.09 g/kg of palmitic acid there is a delay in the curve of tolerance to glucose. The authors concluded that occur an increase in the phosphorylations in serine of the insulin receptor after the treatment with palmitate, suggesting that PKC has a role as negative regulator of the insulin receptors activation in the insulin resistance induced by palmitic acid.

CONCLUSION Palmitic acid is the most abundant saturated fatty acid in human nutrition. It is a major component of palm oil, but can also be found in beef tallow, lard, cocoa butter, human, cow‘s milk and interesterification food. PA is the first fatty acid produced during fatty acid synthesis and the precursor to longer fatty acid. Through this bioprocesses, glucose is converted to fatty acids, which then react with glycerol to produce triacylglycerols. Furthermore, palmitic acid can participates in several chemical reactions as other acids of this same class, being attached to the alcohol form of vitamin A which has been used successfully as a supplement due to its high stability in relation to vitamin A and low cost. Several studies have documented that palmitic acid position in TGC molecule has a great importance in fatty acids action in several human metabolic bioprocesses. In addition, a higher palmitic acid absorption is obtained with formulas rich in palmitic acid esterified in triacylglycerols sn-2 position, than those predominantly esterified in sn-1,3 positions. Some authors suggest that palm oil does not behave as saturated oil, reducing the blood levels of total cholesterol, LDL cholesterol due to TGC sn position. In the other hand, if cholesterol consumption exceeds the critical value or when hypercholesterolaemic subjects are studied, the palmitic acid appears to increase those cholesterol levels. There are still other researches that observed adverse healthy effects by the use of palmitic acid. Moreover, also several studies have documented roles in NAFLD installation and insulin resistance higher levels development in diets rich in palmitic acid. Nonetheless, those studies have still much divergent results, being necessary more researches to clarify the real participation of PA in these processes.



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[198] Jové, M; Planavila, A; Sánchez, RM; Merlos, M; Laguna, JC; Vázquez-Carrera, M. Palmitate Induces Tumor Necrosis Factor-α Expression in C2C12 Skeletal Muscle Cells by a Mechanism Involving Protein Kinase C and Nuclear Factor-κB Activation. Endocrinol, 2006; 147(1): 552–61. [199] Itani, SI; Ruderman, NB; Schmieder, F; Boden, G. Lipid-induced insulin resistance in human muscle is associated with changes in diacylglycerol, protein kinase C, and IkappaB-alpha, Diabetes, 2002; 51: 2005–11. [200] Weigert, C.; Brodbeck, K; Staiger, H; Kausch, C; Machicao, F; Haring, HU; et al. Palmitate, but not unsaturated fatty acids, induces the expression of interleukin-6 in human myotubes through proteasome dependent activation of nuclear factor-kappaB. J Biol Chem, 2004; 279: 23942–52. [201] Jiang, H; Liang, C; Liu, X; Jiang, Q; He, Z; Wu, J; et al. Palmitic acid promotes endothelial progenitor cells apoptosis via p38 and JNK mitogen-activated protein kinase pathways Atherosclerosis, 2010; 210: 71–7. [202] Pitocco, D; Zaccardi, F; Di Stasio, E; Romitelli, F; Santini, SA; Zuppi, C; et al. Oxidative Stress, Nitric Oxide, and Diabetes. Rev Diabet Stud, 2010; 7(1): 15–25. [203] Reynoso, R; Salgado, LM; Calderón, V. High levels of palmitic acid lead to insulin resistance due to changes in the level of phosphorylation of the insulin receptor and insulin receptor substrate-1. Vascular Biochem Mol Cell Biochem: Int J Chem Biol Health Disease, 2003; 41: 155–62.



Chapter 5

PALMITIC ACID AS A CARDIOMETABOLIC RISK FACTOR Danijela Ristić-Medić* and Vesna Vučić Centre of Research Excellence in Nutrition and Metabolism, Institute for Medical Research, University of Belgrade, Belgrade, Serbia

ABSTRACT Current dietary recommendations are based on a reduced saturated fatty acid (SFA) consumption to prevent cardiovascular disease (CVD). The role of individual SFA in metabolic disease is not fully understandable. One type of SFA present in many common foods (dairy, meat, palm and coconut oil) is palmitic acid (16:0). A number of epidemiological studies have shown that the populations who consume large amounts of atherogenic SFA (especially palmitic, myristic, lauric) have elevated levels of LDL and HDL-cholesterol. Saturated fatty acid exert their atherogenic and thrombogenic effect through increased production of LDL, very-low-density lipoproteins particles and apolipoproteins A1, with a decrease of LDL- receptors specific activity, and an increase in platelet aggregation. The total cholesterol/ HDL-cholesterol ratio, the best overall indication of potential effects on coronary heart disease (CHD) risk is nonsignificantly affected by consumption of palmitic acid (PA). Compared with lipid effects, the influence of SFA intake on inflammation markers is less well explored. The associations between circulating and tissue PA and dietary intake of PA are diverse and most likely reflecting endogenous metabolism. Status of PA is not in intake–response relationship biomarker, probably partly due to conversion of 16:0 to 16:1 by steaoryl-CoA-desaturase (SCD-1). Increased SFA intake has been associated with increased SCD-1 activity in which may predict mortality. Palmitoylation is the process involved in protein–membrane interactions and signal transduction. Increases in dietary intake of PA decrease fat oxidation and daily energy expenditure with slight increases in adiposity. Evidence for the effects of SFA, particularly PA consumption on insulin resistance, vascular function, type 2 diabetes, and stroke is various. It is considered that circulating PA, as nonesterified *

Corresponding author: Danijela Ristić-Medić, MD. PhD. Institute for Medical Research, Centre of Research Excellence in Nutrition and Metabolism, University of Belgrade, Tadeusa Koscuska 1, 11129 Belgrade, Serbia. Tel:+38111303-1997; Fax: +381 11 2030-169; e-mail: [email protected].


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Danijela Ristić-Medić and Vesna Vučić fatty acids stimulate insulin resistance by decreasing phosphorylation of the insulin receptor and insulin receptor substrate-1. In muscle cells, PA decrease oxidation of fatty acids and glucose which elevates fatty acid and glucose levels in tissues and blood, and decreases adiponectin production, which may both promote insulin resistance. It was shown that 16:0 and 14:0 stimulate β-cells and endothelial dysfunction. The incidence of type 2 diabetes was associated with total SFA levels of plasma cholesterol esters (also demonstrated for 16:0 levels independently) and phospholipids (also for 16:0 and 18:0). In skeletal muscle phospholipids, PA has been negatively associated with insulin sensitivity and diabetes type 2. Systematic reviews on prospective cohort studies indicated that CHD risk has not been directly associated with SFA intake, although is associated with a dietary habits, high in SFA-rich foods. Taken together, there is collective convincing evidence for decreased CHD risk when replacing SFA with polyunsaturated fats. Differences in cardiometabolic risk appear greater between food groups and overall dietary patterns rather than between separate SFA.

INTRODUCTION Previously, low fat intakes were traditionally recommended in the prevention of cardiovascular disease (CVD) as a component of a health promoting diet, without much attention to the quality of fat. However, current dietary guidelines generally put more emphasis on the quality of fat [1-4]. Imbalances in the amounts of individual fatty acids in the diet may have an impact on the occurrence of dyslipidemia, atherosclerosis, thrombosis, hypertension and obesity. Saturated fatty acids (SFA) have shown to be particularly important for development of the above mentioned diseases. However, in spite of an increasing body of new data, the role of individual dietary SFA in metabolic diseases is not fully clarified (Micha 2010). The reachest dietary sources of SFA include fast foods, processed foods, high-fat dairy products, red meats, and pork [1,5]. One of the most abundant SFA in many common foods (dairy, meat, palm and coconut oil) is palmitic acid (PA, 16:0). The amount of PA is the highest in palm oil (around 50%), but significant amounts of PA (25-26%) can also be found in butter, chicken fat, lard, beef and lamb fat, as well as in cocoa butter. Even olive oil, which is one of basic components of the healthy Mediterranean diet contains around 16% of PA [6]. Furthermore, PA is present in human milk with 20-25% of total fats. Overall, PA and stearic acid (18:0) are the most common dietary SFA and therefore they are also the major SFA in human plasma and tissues. Their concentration in serum/plasma phospholipids and cholesterol esters reflect dietary high fat intake. Dietary saturated fats are of particular scientific interest because of their association with CVD. In some countries, e.g. in Finland, there has been a decline in coronary heart disease (CHD) mortality along with the decreased intake of saturated and total fats [7]. Some epidemiological studies showed that total dietary fats intake is positively associated with metabolic syndrome [8-11]. De Oliveira et al [11] have recently reported that saturated fat intake greater than 10% of total caloric value represented a double risk for metabolic syndrome diagnosis, with odds ratio (OR) 2.0 (1.04-3.84). This association is mostly attributed to palmitic acid, due to the fact that excessive intake of PA increases the visceral adipose tissue in greater proportion than other fat types [12]. Metabolic syndrome or cardiometabolic risk refers to a cluster of metabolic abnormalities including disturbances in


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glucose and insulin metabolism, central obesity, dyslipidemia (high triglyceride levels, low HDL-cholesterol and high levels of small dense LDL-particles) and hypertension [13,14]. Central to the etiology of metabolic syndrome is an interrelated triad comprising inflammation, obesity (particularly abdominal), and aberrations in fatty acid metabolism [15,16].

PALMITIC ACID INTAKE, ADIPOSITY AND INFLAMMATION Low-grade systemic inflammation is an important component of most chronic noncommunicable diseases, including metabolic syndrome, CVD, diabetes type 2 and cancer [17]. Although polyunsaturated fatty acids n-3 and n-6 series play dominant role in inflammation, as shown by several papers (review by Ristic-Medic 18), increased serum SFA has also been closely connected to low grade tissue inflammation. The primary cell-intrinsic dysfunctions include lipid dysregulation (e.g. accumulation of intracellular diacylglycerols, SFA, and ceramides) [19]. Studies in vitro indicated direct proinflammatory and pro-oxidant effects of fatty acids [20,21]. Importantly, these effects were mostly attributed to saturated fats, with palmitate commonly employed [21]. Mechanisms proposed by Kennedy et al [12] related to SFA effects which contributed to the cardiometabolic risk include: 1) accumulation of diacylglycerol (DAG) and ceramide; 2) activation of nuclear factor-kB (NFkB), protein kinase C (PKC), and mitogen-activated protein kinases (MAPK), and subsequent induction of inflammatory genes in white adipose tissue (WAT), immune cells, and myotubes; 3) decreased activation of peroxisome proliferator-activated receptor-γ (PPARγ) coactivator-1 α/ß and adiponectin production, which decreases the oxidation of glucose and fatty acids; and 4) recruitment of immune cells such as macrophages, neutrophils, and bone marrow-derived dendritic cells (BMDC) to WAT and muscles. Overconsumption of SFA (especially PA) enhances WAT expansion and adipocyte hypertrophy and subsequent death [12]. These processes led to increased inflammatory signaling and recruitment and activation of macrophages, neutrophils, and bone marrowderived dendritic cell. As a consequence, inflammation, impaired insulin signaling, and insulin resistance in multiple tissues, particularly in WAT and muscles are occurred [22,23,24].

Effect of Palmitic Acid on White Adiposity Tissue Function Therefore, excessive palmitate intake expands WAT, but it also increases inflammation and apoptosis through oxidative or endoplasmic reticulum stress, generation of ceramide and reactive oxygen species (ROS), and PKC signaling. Palmitic acid induces cytokine production in adipocytes, via activation of PKC, NFkB, and MAPK signaling with induced interleukin-6 (IL-6) and tumor necrosis factor (TNF)-α expression [12,25]. It is well known that SFA activated toll-like receptor (TLR) signaling in murine adipocytes [26,27] and macrophages [28,29], led to NFkB and c-jun-NH2-terminal kinase (JNK) activation and cytokine production. Consistent with these data, TLR-4 deficiency selectively protects against obesity induced by diets rich in palmitic acid [30]. In the conditions where palmitic acid is a


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major component in the diet, palmitate strongly increased the expression and secretion of TNFα and IL-10 in murine 3T3–L1 adipocytes, compared with monounsaturated oleic acid and polyunsaturated docosahexanoic acid (DHA) [31]. It has been considered that in adipocytes, palmitic acid modulated intracellular signaling and induced endoplasmic reticulum stress by increasing C/EBP homologous protein and glucose regulatory protein 78. Furthermore, palmitic acid alters phosphorylation of eIF2α and increases phosphorylation of JNK and extracellular receptor kinase [32]. Experimental studies [33,34] have shown that high level of palmitic acid in the diet impaired insulin sensitivity by reducing adiponectin secretion and impairing insulin signaling pathways required for glucose uptake. Adiponectin is an insulin-sensitizing protein produced by adipocytes. Reducing the levels of adiponectin appears to be the mechanism by which palmitate caused insulin resistance in isolated rat adipocytes [35]. In accordance, experiments in mice fed a high-fat diet showed that overexpression of adiponectin decreased insulin resistance [36]. Furthermore, PA leads to insulin resistance due to changes of phosphorylation level of the insulin receptor and insulin receptor substrate. Saturated fatty acid induced inflammatory response in the interaction between adipocytes and macrophages by the TLR4/NFkB signaling patway [26]. Supplementation with palmitic acid induced IP-10 inflammatory gene expression in human macrophages (U937) by an NFkB-dependent mechanism [37]. It was shown that adipocytes containing SFA have the capability to activate macrophages to a greater extent than smaller adipocytes. It is even more pronounced when compared adipocytes containing SFA with adipocytes enriched in unsaturated fatty acid. For instance, SFA increased TNFα mRNA levels in cultures of adipocytes and murine macrophages, whereas unsaturated fatty acid had no effect. Factors secreted from macrophages increase adipocyte inflammation and insulin resistance [38,39] and high-fat feeding in mice increased TLR4 signaling in macrophages and adipocytes and impaired insulin signaling effects [29].

Effect of Palmitic Acid in Muscle Cells In muscle cells (for example C2C12) [40,41], palmitic acid also induce inflammation by increasing the expression and secretion of IL-6 and TNFα. Furthermore, PA leads to glucose transporter 4 down-regulation and impaired insulin sensitivity via an NFkB/PKCθ pathway. Palmitate-mediated down regulation of PPARγ coactivator-1 (PGC-1α) in skeletal muscle cells involves MEK1/2 and NFkB activation [42]. It is well established that PGC-1α promotes oxidative phosphorylation, mitochondrial gene expression, and insulin stimulated glucose uptake [43]. However, PGC-1α expression is reduced in obesity [44]. In addition, higher palmitic acid intake in C2C12 myotubes, increased p38 MAPK signaling, reducing PGC-1 α /1ß expression and activity, that would lead to decreased oxidation of fatty acid and glucose, with increasing their accumulation in tissues and blood. Palmitate also caused the accumulation of DAG and ceramide and reduced insulin stimulated glucose uptake in murine L6 myotubes [45]. Taken together, these studies showed that palmitic acid are particularly potent in recruiting and activating immune cells in WAT, increasing inflammation and demonstrate the adverse effects of SFA on glucose uptake and utilization in muscle.



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DIETARY PA -EFFECTS ON CARDIOVASCULAR RISK FACTORS Several mechanisms mediate between dietary fatty acid intake and cardiovascular disease risk. The main mediating mechanism is probably concentration of blood cholesterol [6, 46,47]; whereas other mechanisms are related to insulin resistance, inflammation, and endothelial function [48,49].

Lipids and Lipoproteins The quality of dietary fats has been shown to have a significant effect on serum lipid profile. According to several publications, substituting SFA with unsaturated fats convincingly decreased concentrations of serum/plasma total and LDL-cholesterol in randomized control studies (RCTs) [49-57].However, in seven of these studies concentration of serum/plasma triglycerides did not change. Other serum or plasma lipoproteins were also affected. Thus consumption of polyunsaturated (PUFA) or monounsaturated fatty acids (MUFA) in place of SFA leads to lowering of serum/plasma total cholesterol, LDLcholesterol, and ApoB; slight lowering (for PUFA) of HDL-cholesterol and ApoA1; little effect on triglycerides; and lowering of the total cholesterol/ HDL-cholesterol. Effects of SFA consumption on serum lipids and lipoproteins further vary according to which specific SFA is consumed [6,58,59]. Palmitic acid intake raised serum/plasma total cholesterol and LDL-cholesterol. However, all SFA increased HDL-cholesterol as well, but HDL-raising effects are greater as SFA chain-length decreases. This has been shown by metaanalysis of RCT studies, when different chain-length SFA were used as an isocaloric replacement for carbohydrates [60]. The total cholesterol/HDL-cholesterol ratio, the best overall indication of potential effects on coronary heart disease (CHD) risk is nonsignificantly affected by consumption of palmitic acid. Epidemiological studies have shown that the population who consume large amounts of SFA (especially those of 12-16 carbon: lauric, myristic, and palmitic) have elevated levels of LDL-cholesterol [61,62]. Saturated FA exert their atherogenic and thrombogenic effect through increased production of very low-density lipoproteins (VLDL)-particles and Apo A1, with a decrease of LDL-receptors specific activity, and an increase in platelet aggregation [63]. Collective evidence suggests that SFA of 12-16 carbon, rather than stearic acid (18:0), are the major activators of clotting factor VII [64]. Stearic acid has also been considered highly atherogenic for years, but recent studies testify in favor of antiaterogenic and even anti- carcinogenic effect of stearic acid [59,65,66]. For these reasons, recommendation by the WHO / FAO experts for SFA (butter, cream, full-fat dairy products) intake is limited to less than 10% of daily energy needs [1,4]. The most abundant SFA in milk fat is palmitic acid, which make up about 36% of total fats and 44-51% of the total SFA in milk fat [67,68]. For this reason, milk and dairy products are usually considered unhealthy, especially for people with dyslipidemia. In spite of this fact, it has been well established that milk fat raises serum HDL-C, helping to maintain a good HDL-cholesterol/total cholesterol ratio that is inversely related to CVD [60]. Furthermore, palmitic and stearic acids in milk fat occupy the sn-2 position of triglycerides, which is typically the position of unsaturated fatty acids in plant oils [69,70]. The selectivity of


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pancreatic lipase to hydrolyze triglycerides at the sn-1 and sn-3positions leads to the production of free fatty acids and 2-monoglyceride [69]. In this way, the unique position of SFA in milk fat may affect postprandial metabolism, leading to prevention of hypercholesterolemia and hypertriglyceridemia that would otherwise be associated with consumption of saturated fat [69,68,70]. The beneficial effect of milk fat on serum lipids may partially explain why milk fat, despite its contribution of SFA to the diet, has not been consistently associated with higher incidence of CVD [69,72] or risk factors for cardiometabolic syndrome [72,73]. In a recent cohort study, butter and dairy intake did not predict all-cause and ischemic heart disease mortality in men, and slightly increased risk in women, whereas fermented full-fat milk was inversely associated with mortality in both men and women [74] Moreover, similar findings are reported for palm oil, which also does not raise blood cholesterol as expected based on the content of PA. It may be explained by the position of palmitic acid in palm oil triacilglycol (10% of total PA is in the middle position). Because triacilglycols with SFA in the sn-2 position may be absorbed more efficiently and cleared from circulation more slowly than triacilglycols with SFA in the sn-1 and sn-3 positions, intake of these dietary triacilglycols often leads to a more pronounced postprandial lipemia, which is an independent risk factor for CHD [75]. Therefore, it can be assumed that PA esterified to the sn-2 position is more atherogenic than when esterified to the sn-1 and sn-3 positions. Studies in animals and in human infants have supported this assumption since they reported higher plasma triglycerides levels after diets with PA in the sn-2 position than after diets containing PA in the sn-1/3 positions [76]. However, no significant differences were found in one adult trial [77], whereas another study [78] reported larger LDL-cholesterol concentrations caused by diets including palmitic acid in the sn-2 position in men but not in women. Thus further research is needed to elucidate these relationships.

Insulin Resistance and Diabetes Quality and amount of dietary fats significantly affects insulin resistance (IR), which is a key player in development of metabolic syndrome and diabetes [79]. According to the Vessby et al [15,49], amount can be considered more important, since excessive intake of total fat (> 37% of daily energy intake) independently on the FA composition, may worse IR. Furthermore, they reported that an exchange of dietary saturated for monounsaturated fats improved insulin sensitivity. The underlying mechanism is not clarified yet, but it likely includes interference with binding of insulin to its receptors and accumulation of triglycerides in skeletal muscle [80]. On the contrary, total fat intake up to 30% of daily energy intake differently influences IR, in term of the type of fatty acid consumed [15,49]. Observational studies assessing fatty acid composition in serum or tissues (which correlates with dietary intake) suggest that IR is associated with relatively high intakes of saturated fat (e.g. palmitic acid) and low intakes of polyunsaturated fat (e.g. linoleic acid), findings that are supported by recent clinical data [81,82] The effects of SFA and MUFA on IR are different. Saturated fats increase IR when compared to monounsaturated fats [83]. Hotamisligil et al [84] reported that reason for this increase is that SFA activates serine kinases, thus inhibiting the insulin phosphorylation cascade, decreasing glucose uptake and increasing glycemia. Gaster et al [85] have recently found that PA and monounsaturated oleic acid are identically utilized in diabetic and control



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myotubes, although oxidation of PA is reduced in diabetic myotubes. Furthermore, there is a difference in handling of PA and oleic acid in myotubes: PA accumulates in form of di- and triacylglycerols, whereas oleic acid accumulates as intracellular free FA. Diacylglycerols from palmitic acid activate protein kinase C, which has been shown to increase IR and thus decrease glucose uptake in human skeletal muscle cells [86]. There is also a link between PA and myristic acid (14:0) and insulin synthesis. These SFA (in particular PA) have been demonstrated to stimulate β-cells in pancreas. When SFA content in pancreatic islets increases, nitric oxide and ceramide are synthesized. A link between serum ceramides, IR and inflammation is related to the inflammatory marker IL-6 [87]. As a consequence increased oxidative stress, inflammation and endoplasmic reticulum stress are occurred, that lead to β-cell apoptosis [88]. At the same time, insulin gene expression is inhibited [89] Taking into account the positive association between PA intake and IR, effect of PA on diabetes is expected. In a recent report by FAO, SFA was considered to have a possible positive relationship with increased risk of diabetes mellitus type 2 [1,61]. Nevertheless, in a systematic review by van Dam et al [90] no clear associations were found. This systematic review has shown that SFA intake was associated with a higher risk of diabetes mellitus type 2, but this association was not independent of body mass index. Few research studies have explored the relationship between the amount of SFA in the diet and glycemic control and CVD risk in people with diabetes. A systematic review by Wheeler et al [91] found just one small 3-week study that compared a low-SFA diet (8% of total kcal) versus a high-SFA diet (17% of total kcal) and found no significant difference in glycemic control and most CVD risk measures [59,91].

DIETARY PA IN RELATION WITH CARDIOVASCULAR DISEASE RISK Expert panel reached the conclusion that the evidence from epidemiologic, clinic and mechanistic studies is consistent in the finding that CVD is reduced when SFAs are replaced with PUFA in diet [92]. It is considered that replacement of only 1% of energy from SFA with PUFA lowers LDL- cholesterol and is likely to produce a reduction in CHD incidence of 2–3% [93,94]. Individual SFA may have different cardiovascular effects and the effect of particular foods on CHD cannot be predicted solely by their content of total SFA because major SFA food sources contain other constituents that could influence CHD risk [92]. As already mentioned, SFA with different chain length have different atherosclerotic potential. Meta-analyses of cohort studies with self-reported SFA intakes are not associated with CHD, stroke, or CVD [95, 96]. Two systematic reviews of prospective cohort studies and meta-analyses, the first including 9 cohorts [97] and the second including 16 cohorts [98], with self-reported SFA intakes found no significant association between SFA intake and CHD risk. However, multivariable adjusted pooled analysis of individual-level data from 11 prospective cohorts over 4–10 years of follow-up [97] indicated that SFA consumption was associated with higher CHD risk only in comparison to PUFA intake. In this direction, metaanalysis of eight RCTs, including participants with CHD events, found that CHD risk was lowered by 10% when 5% energy intake of SFA substituted with PUFA [93]. That, SFA consumption was associated with trend to lowered risk of stroke compared the highest to the



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lowest category of SFA intake has been shown in meta-analyses of eight prospective cohort studies [98]. Results from Women Health initiative trial [99], indicated that reduction in SFA consumption does not appear to increase risk of stroke over 8 years. Some links between dietary PA and cardiometabolic risk factors are presented in Figure 1.

Palmitic acid Dietary sources: Palm oil, dairy, meat

Elevated LDL Elevated HDLcholesterol

Tissue inflammation Weight gain

Increased risk for diabetes

Increased risk for CHD Endotel dysfunction

Beta cell disfunction

Figure 1. Relationship between dietary intake of palmitic acid and cardiometabolic risk.

PALMITIC ACID PROPORTION IN SERUM OR TISSUES IN RELATION TO CARDIOMETABOLIC RISK Fatty acid composition in serum lipids reflects dietary intake and metabolic processes. High proportions of palmitic, palmitoleic (16:1), and dihomo-γ-linoleic (DHLA, 20:3n−6) acids and a low proportion of linoleic acid (LA, 18:2n−6) in serum/plasma lipids predicts type 2 diabetes [100,101,102], myocardial infarction [103,104], stroke [105], left ventricular hypertrophy [106], and the metabolic syndrome [107,108.109]. In a Swedish cohort, 14:0 and 16:0 in serum cholesterol esters independently predicted cardiovascular and all-cause mortality over 33 years [110]. Patients with incident hypertensives had higher levels of palmitic acid compared to normotensives person [111]. In overweight adolescents plasma fatty acid composition is associated with the metabolic syndrome and low-grade inflammation [112]. PA status in blood of patients with cardiometabolic risk from available literature data are shown in Table 1 [15, 111-121]. As comparison, Table 2 [117,122,123] presents PA status in plasma and erythrocytes phospholipids in healthy subjects depending on the age and gender. For total SFA, the associations between tissue or circulating fatty acids and dietary intake are diverse and often weak [124,125] possibly reflecting endogenous metabolism. Plasma 16:0 levels increase with dietary intake of SFA, although not as much as expected and not in a dose–response manner, probably partly due to conversion of 16:0 to 16:1 by steaoryl-CoAdesaturase (SCD) [126]. Thus, the ratio of 16:1 to 16:0, which represents the estimated SCD activity, may also be used as a marker of dietary 16:0 intake [83]. Additionally, circulating and tissue levels of 14:0, 16:0 and 18:0 can be affected by high intakes of carbohydrate or alcohol [124]. Warensjö et al [110] showed that PA, but not stearic acid, was significantly associated with increased mortality, especially cardiovascular disease mortality. This is in line with



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experimental data suggesting that palmitic acid has unique effects on several cellular functions, such as apoptosis [127], endoplasmic reticulum stress [128], and up-regulation of SCD-1 [129]. The lipogenic enzyme SCD catalyzes the synthesis of MUFAs, eg, oleic and palmitoleic acids. Estimated SCD activity (16:1/16:0 ratio), together with palmitoleic acid, has been considered as a strong predictor of mortality [126]. It may be associated with increased lipogenesis [129], ectopic fat deposition and thereby insulin resistance [130,131,132]. Accordingly, the estimated SCD ratio was established as an independent predictor of directly measured insulin sensitivity over 20 years [81]. In contrast, our data on patients with non-Hodgkin lymphoma showed very low proportion of 16:1 and activity of SCD, especially in patients with progression of disease, but the role of SDC in cancer should be further investigated [133]. Table 1. Palmitic acid status in patients with cardiometabolic risk Cardiometabolic risk patients 1.Hyperlipidemic patients (n=29) [113] 2. Hyperlipidemic patients (n=39) [114] Obesity women (n=30) [15] I-NGT group (n=12) II-IR group (n=18) DM type 2 with hyperlipidemia (n=28) [115] I-IHTG (n=14) II-CHL (n=14) 1. Hemodialysis patients (n=35) [116] 2. Hemodialysis patients (n=37) [117]

Alcoholic cirrhosis (n=20) [118] I. Patient with incident hypertension (n=413) [119] II. Patient with no incident hypertension (n=1965) III. Patient with prevalent hypertension (n=698) IV. Patient with no prevalent hypertension (n=2383)

PA status (source of FA, mol%) 1.serum PL 30.35 ± 5.94 Er 25.58 ± 4.15 2.serum PL 30.30 ± 1.39 Er 23.64 ± 0.90 Er 22.63±1.39 I-Er 22.49±1.67 II-Er 22.73±1.21 serum PL 30.01 ± 2.70 I-serum PL 29.05 ±1.43 II-serum PL 30.71 ± 3.67 1.serum PL 29.93 ± 3.52 2. serum PL 28.09 ± 3.34 controls 26.46 ± 2.44 Er 21.63 ± 1.85 control 22.42 ±2.59 serum PL 30.76 ±4.75 control 26.53 ± 2.44 I. serum CE 10.07± 0.77 II. serum CE 9.91± 0.77 p< 0.001 III. serum CE 10.21± 0.83 IV. serum CE 9.94± 0.77 p< 0.001

Stroke (n=20) [120]

serum PL 29.2 ± 2.57 control 37.2 ± 2.13 p< 0.001



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Table 1. (Continued) Cardiometabolic risk patients

PA status (source of FA, mol%) I. Patients with incident CHD (n=282) [101] I- serum PL 25.5 ± 1.5 II-Patients with no incident CHD (n=3309) II-serumCE 10.02 ± 0.8 I- serum PL 25.4 ± 1.7 II-Serum CE 10.0 ± 0.8 p < 0.01 Patients with no MS (n=640) [121] Er 22.7 ± 1.2 Patients with MS (n=396) Er 23.1 ± 1.22 p 0.997 in all cases), therefore illustrating the linear relationship between temperature and the phase transition pressure. Coorens et al. [31] also published vapour-liquid-liquid equilibrium data showing that the system has a three phase region between 349 and 370 K. However, compositions were not included.


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Comparing Figure 19 and Figure 20, it can be seen that the phase behaviour for the VSE and VLE is significantly different. As for the CO2/palmitic acid and CO2/tripalmitin systems, the VSE data shows an increase in solubility with temperature while the converse is true for the VLE data. Additionally, the solubility of the solid in propane is again much lower than that of the liquid in propane. Thus, while the absolute values of the pressure differ (a detailed analysis is presented below) the same trends as for the CO2/tripalmitin system are present.

Analysis of Phase Equilibrium Data Many of the comments made for the phase behaviour of palmitic acid and its derivatives in CO2 and in ethane are also valid for propane as SC solvent. Figure 21 shows a comparison of the propane/tripalmitin, propane/palmitic acid, propane/methyl palmitate and propane/ethyl palmitate systems. 10

Pressure (MPa)

8

Tripalmitin Palmitic acid

6

Methyl palmitate

4 2 0 0.0

0.2

0.4

w2

0.6

0.8

1.0

Figure 21. Comparison of the pressure – composition (w2) for the systems propane (1)/palmitic acid (2) [28], propane (1)/methyl palmitate (2) [59], propane (1)/ethyl palmitate (2) [30] and propane (1)/tripalmitin (2) [31] systems at 393 K.

As for CO2 and ethane as SC solvents, the same trends are observed for propane as SC solvent. Methyl palmitate and ethyl palmitate behave very similarly and are the most soluble, followed by palmitic acid. Tripalmitin is the least soluble in propane, yet even for tripalmitin only moderate pressures are required for total solubility. Despite the high temperatures, relatively low pressures are required for total solubility, therefore decreasing the capital cost investment. Propane should thus be regarded as a good substitute or co-solvent to CO2 to reduce the processing pressures.


Processing of Palmitic Acid …

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Comparison of Supercritical Solvents A comparison of the phase behaviour of palmitic acid, methyl palmitate, ethyl palmitate and tripalmitin in SC CO2, ethane and propane is shown in Figure 22, Figure 23, Figure 24 and Figure 25, respectively. With exception of the tripalmitin data, the ethane and CO2 data are at the same temperature with the propane at a similar reduced solvent temperature. 25

Pressure (MPa)

20 Propane

15

Ethane

10

Carbon dioxide

5 0 0.0

0.2

0.4

w2

0.6

0.8

1.0

Figure 22. Comparison, at similar reduced temperatures (Tr ~ 1.11), of the pressure – composition (w2) phase behaviour of the CO2 (1)/palmitic acid (2) at 338 K [24], ethane (1)/palmitic acid (2) at 338 K [27] and propane (1)/palmitic acid (2) at 410 K [28] systems.

15

Pressure (MPa)

12 9

Propane Ethane

6

Carbon dioxide

3 0 0.0

0.2

0.4

w2

0.6

0.8

1.0

Figure 23. Comparison, at similar reduced temperatures (Tr ~ 1.11), of the pressure – composition (w2) phase behaviour of the CO2 (1)/methyl palmitate (2) at 323 K [48,49], ethane (1)/methyl palmitate (2) at 323 K [29] and propane (1)/methyl palmitate (2) at 393 K [59] systems.


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C. E. Schwarz 15

Pressure (MPa)

12 9

Propane Ethane

6

Carbon dioxide

3 0 0.0

0.2

0.4

w2

0.6

0.8

1.0

Figure 24. Comparison, at similar reduced temperatures (Tr ~ 1.06), of the pressure – composition (w2) phase behaviour of the CO2 (1)/ethyl palmitate (2) at 323 K [26,51], ethane (1)/ethyl palmitate (2) at 323 K [30] and propane (1)/ethyl palmitate (2) at 393 K [30] systems.

35

Pressure (MPa)

30 25 Ethane

20

Carbon dioxide

15

Propane 436 K

10

Propane 429 K

5 0 0.0

0.2

0.4

w2

0.6

0.8

1.0

Figure 25. Comparison, at similar reduced temperatures of the pressure – composition (w2) phase behaviour of the CO2 (1)/tripalmitin (2) at 353 K (Tr = 1.16) [25,33,52,53], ethane (1)/tripalmitin (2) at 360 K (Tr = 1.18) (data generated using the ethane/n-alkane correlations for the system ethane/ntetrapentacontane, as proposed by Schwarz et al. [57]) and propane (1)/tripalmitin (2) at 429 (Tr = 1.16) and 436 K (Tr = 1.18) [31] systems.

The results show that, as expected from the analysis above, the phase transition pressure of propane is the lowest and as such its solubility the highest. Additionally, for palmitic acid, methyl palmitate and ethyl palmitate the phase transition pressures are lower for propane than for ethane, and CO2 has the highest phase transition pressure and thus the lowest solubility.



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For tripalmitin, it should be noted that the CO2 and ethane data are not at the same temperature not at the same reduced temperature. The CO2 and propane at 429 K data are at similar reduced solvent temperatures and the ethane and propane at 436 K data are at similar reduced solvent temperatures. Tripalmitin is thus considerably more soluble in propane than in ethane or CO2. It should be recalled (See Figure 8) that significant scatter exists for the CO2/tripalmitin data. Before an outcome regarding a comparison of the solubility of tripalmitin in CO2 and ethane can be given, issues relating to this scatter need to be resolved. The accuracy of the ethane/tripalmitin approximation also needs to be verified. In all likelihood, additional CO2/tripalmitin as well as ethane/tripalmitin data need to be measured. The low molecular mass alkanes are thus able to dissolve palmitic acid and its derivatives at lower pressures than CO2. This is in agreement with Münüklü et al. [53] who compared the solubility of CO2 and in propane in a hardened rape seed oil and found that propane is able to dissolve in the oil a lot better than the CO2. While ethane and in propane are not toxic, they are highly flammable and as such care is required if they are used as alternative solvents or even in high concentrations as co-solvents. However, despite the flammability issues associated with ethane and propane, these two solvents are non-toxic and excellent alternatives to CO2. As they are both also SC solvents, the solvent residue would be similar than when using CO2 as solvent and the solvent recycle systems are of similar complexity to using CO2.

The Influence of a Co-Solvent on the Phase Behaviour in Supercritical Solvents As seen above, at certain operating conditions high pressures (> 25 MPa) are required for significant solubility of tripalmitin and palmitic acid in CO2. These high pressures are as a result of CO2 not being a very good solvent for these systems. In order to circumvent the low solubility of these components in SC CO2, either an alternative solvent to or a co-solvent with CO2 can be used. Ethane and propane were considered in sections 3.2 and 3.3 above as alternative solvents or co-solvents. However, in the literature other volatile organic compounds have also been studied. Table 14 presents a summary of the main studies involving the phase behaviour of palimitic acid and its derivatives in a SC CO2 in the presence of a co-solvent. The majority of the studies involve ethanol as a co-solvent. This may be due to the fact that it is generally a good co-solvent compared to the other components considered [36,60]. Additionally, as it is often difficult to remove residual co-solvent from the high molecular mass product, ethanol is more suitable than most other organic solvents as small amounts of ethanol are not harmful to humans. The effect of the co-solvent will now be investigated by firstly considering the effect that the quantity of solvent has on the phase behaviour and secondly comparing some co-solvents, as shown in Figure 26 to Figure 29. Figure 26 shows that the inclusion of even 20 % (on a molar basis) of ethanol to the solvent significantly increases the solubility of palmitic acid in the CO2. Figure 27 shows a reduction in pressure due to the co-solvent for ethyl palmitate as solute and further indicates than in increase in the molar ratio of ethanol to CO2 leads to an increase in solubility. However, Gaschi et al. [51] found that while the addition of ethanol as an entrainer reduced


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the phase transition pressure in the region of the critical point (as shown in Figure 27), at low CO2 compositions this observation is reversed. Similar phase behaviour was observed for the CO2/biodiesel/ethanol system [62]. Table 14. Literature data phase equilibria of palmitic acid or its derivatives (2) in CO2 (1) in the presence of a co-solvent (3)

a

Reference

System

Purity

Temperature range

Pressure range

Composition range (w2, w3) a

Brandt et al. [34]

(2) palmitic acid (3) ethanol

(1) 99.995 % (2) 99 % (3) 99.8 %

313 K

8.21 to 24.61 MPa

w2 = 0.010 to 0.1271; w3 = 0.00525 to 0.0948 (11)

Brandt et al. [34]

(2) palmitic acid (3) 2propanol

(1) 99.995 % (2) 99 % (3) 99.8 %

313 K

10.90 to 20.69 MPa

w2 = 0.0326 to 0.1272 w3 = 0.0110 to 0.118 (9)

Garlapati and Madras [36]


(2) 98 % (3) 99.9 %

308 an 318 K

12.8 to 22.6 MPa

w2 = 3.07E-3 to 0.0349; w3 = 7.57E-3 to 0.0429 (20)

Garlapati and Madras [36]

(2) palmitic acid (3) 3methyl-1butanol

(2) 98 % (3) 98 %

308 an 318 K

12.8 to 22.6 MPa

w2 = 2.39E-3 to 0.0145; w3 = 0.0144 to 0.0387 (15)

Gaschi et al. [51]

(2) ethyl palmitate (3) ethanol

(1) 99.9 % (2) 95 % (3) 99.5 %

303.15 to 353.15 K

3.52 to 19.05 MPa

w2 = 0.0530 to 0.700; w3 = 0.0140 to 0.343 (108)

Iwai et al. [40]

(2) palmitic acid (3) water

(1) 99.9 % (2) 99 % (3) Ultrapure

313.2 K

15.0 MPa

w2 = 0.00659 and 0.00568; w3 = 0.00352 and 0.00391 (2)

Koga et al. [60]


(1) 99.9 % (2) 99 % (3) 99.5 %

308.2 K

9.9 and 19.7 MPa

w2 = 8.91E-3 to 0.0622; w3 = 0 to 0.0879 (22)

Koga et al. [60]

(2) palmitic acid (3) octane

(1) 99.9 % (2) 99 % (3) 98 %

308.2 K

9.9 and 19.7 MPa

w2 = 8.91E-4 to 0.0331; w3 = 0 to 0.0184 (22)

Rosso Comin et al. [61]


(1) 99.9 % (2) 99 % (3) 99.9 %

313 to 343 K

8.34 to 19.94 MPa

w2 = 0.105 to 0.705; w3 = 0.0611 to 0.186 (34)

Value in brackets indicates the number of data points published



189

25

Pressure (bar)

20 With Entrainer 333 K Without Entrainer 333K With Entrainer 343 K Without Entrainer 343 K

15 10 5 0 0.0

0.2

0.4

w2

0.6

0.8

1.0

Figure 26. Pressure – composition (w2) plot for the system CO2 (1)/palmitic acid (2)/ethanol (3) at 323 and 343 K without ethanol [24] and for an ethanol to CO 2 molar ratios of 0.25 [61].

18 15 Pressure (bar)

12

No entrainer 1:1

9

1:3

6 3 0 0.0

0.2

0.4

w2

0.6

0.8

1.0

Figure 27. Pressure – composition (w2) plot for the system CO2 (1)/ethyl palmitate (2)/ethanol (3) at 333.15 K for various ethanol to ethyl palmitate molar ratios [51].


190

C. E. Schwarz 24 0.73 mol % 2Me3BuOH 1.98 mol % 2Me3BuOH

Pressure (bar)

18

0.73 mol % EtOH

12

1.98 mol % EtOH 4.16 mo l% EtOH

6

No entrainer 0 0.000

0.010

0.020

0.030

w2

0.040

Figure 28. Pressure – composition (w2) plot for the system CO2 (1)/palmitic acid (2)/co-solvent (3) at 318 K for 3-methyl-1-butanol and ethanol as co-solvents at a range of co-solvent concentrations [36].

0.07 0.06 0.05 Ethanol, 9.9 MPa

w2

0.04

Ethanol, 19.7 MPa Octane, 9.9 MPa

0.03 0.02 0.01 0.00 0.00

0.05

0.10

w3

0.15

0.20

Figure 29. Palmitic acid (w2) – co-solvent (w3) concentration plot at 308 K and constant pressure for the system CO2 (1)/palmitic acid (2)/co-solvent(3) for ethanol and octane as co-solvent [60].

Garlapati and Madras [36] and Koga et al. [60] compared various co-solvents and found the increase in solubility to be more significant using ethanol compared to 3-methyl-1-butanol and octane, respectively. Iwai et al. [40] considered the effect of water and found that the solubility of palmitic acid increased with increasing molarity of water, especially near the saturation point of water. They also found that the solubility of palmitic acid in CO2 saturated with water was 16 times higher than that of pure CO2. (It should be noted that only 2 data points are tabulated; the remaining data is all presented in figures.)



191

Güçlü-Üstündağ and Temelli [63] reviewed the effect of a co-solvent on the phase behaviour of lipids in SC CO2. They found that physical interactions between the solutes and co-solvent, such as dipole – dipole, dipole – induced dipole or induced dipole – induced dipole (dispersion) interactions and specific interactions such as H-bonding and charge transfer complexes, are important contributors to the co-solvent effect. The use of a cosolvent may also lead to a change in selectivity. The magnitude of the effect of the co-solvent is thus a combination of the solvent, the co-solvent, the solute and the operating conditions. The use of a co-solvent can therefore significantly reduce the phase transition pressure and thus increase the solubility of palmitic acid and its derivatives in a SC solvent. However, while this reduction leads to a decrease in operating pressure, it comes at the cost of a more complicated solvent recycling system, a more complicated control philosophy and increased solvent residue in the products. The choice of the use of a co-solvent thus needs to take these aspects, as well as the nature of the co-solvent itself, into account when the use of a cosolvent is evaluated.

SCFF OF MIXED FATTY ACIDS AND THEIR DERIVATIVES Palmitic acid and its derivatives have, depending on the temperature and pressure, reasonable solubility in SCFs. In particular, liquid phase components have an acceptable solubility. In nature, palmitic acid does not often occur as a pure component. In fact, it is usually present as a triglyceride but even tripalmitin is seldom encountered. Usually palmitic acid is present in combination with similar high molecular mass acids in the form of triglycerides. Hydrolysis of these triglycerides results in a mixture of fatty acids or their esters, which in turn need to be fractionated to obtain palmitic acid or its ester, usually ethyl palmitate or methyl palmitate. This section of the chapter focusses the use of SCFs as a mass transfer agent to fractionate fatty acids and their esters. The SCF thus behaves in a similar manner to a liquid organic solvent where it preferentially dissolves one component above another. This section will start by considering the phase behaviour of various fatty acids in SCFs, followed by considering typical set-ups and concludes with a summary of the fractionation of plant oils and fish oils to obtain products of palmitic acid and its derivatives.

Phase Behaviour Analysis As mentioned above, in order for separation to occur, a difference in phase behaviour is required. Figure 30 and Figure 31 below consider the phase behaviour of a range of saturated fatty acids in SC CO2 and ethane, respectively. Propane shows a similar trend to that of ethane, albeit at lower pressures [28]. Figure 32 considers the phase behaviour of various tryglycerides in SC CO2. Although no such comparison is possible for ethane or propane due to a lack of data, similar trends are expected. Figure 33 shows that phase behaviour of a range of saturated fatty acid ethyl esters in SC CO2. Schwarz and co-workers [29,30] studied the


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C. E. Schwarz

phase behaviour of methyl and ethyl esters in SC ethane and propane and found similar results. 30

Pressure (MPa)

25 20

Stearic acid Palmitic acid

15

Myristic acid Lauric acid

10

Decanoic acid 5 0 0.0

0.2

0.4

w2

0.6

0.8

1.0

Figure 30. Pressure – Composition (w2) of various CO2 (1)/linear saturated acid (2) systems at 353 K [24].

30

Pressure (MPa)

25 Behenic acid

20

Stearic acid 15

Palmitic acid Myristic acid

10

Lauric acid Capric acid

5 0 0.0

0.2

0.4

w2

0.6

0.8

1.0

Figure 31. Pressure – Composition (w2) of various ethane (1)/linear saturated acid (2) systems at 353 K [27].



193

30

Pressure (MPa)

25 20 Tristearin Tripalmitin

15

Trilaurin 10

Tricaprylin

5 0 0.0

0.2

0.4

w2

0.6

0.8

1.0

Figure 32. Pressure – Composition (w2) of various CO2 (1)/triglyceride (2) systems at 353 K [25,33].

20

Pressure (MPa)

16 Ethyl stearate

12 8

Ethyl palmitate

4

Ethyl myristate

0 0.0

0.2

0.4

w2

0.6

0.8

1.0

Figure 33. Pressure – Composition (w2) of various CO2 (1)/ethyl ester (2) systems at 333 K [26].

The phase behaviour clearly shows that SCFs are able to distinguish between molecules based on the number of carbon atoms present. Bharath et al. [33] found, from their phase equilibria study, that that fatty acid and triglycerides can be fractionated based on their carbon number using SC CO2. This is in agreement with their previous study [64] that considered the phase behaviour of palm kernel oil and sesame oil in SC CO2, where they found that SC CO2 is able to selectively dissolve triglycerides based on their carbon number. Numerous other studies came to similar conclusions [48,65–67]. Additionally, Soares et al. [68] compared the solubility of various fats and oils in SC CO2 and they found, in agreement with the trends noted above, that oils with more lower molecular mass acids have a higher solubility.


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As temperature and pressure affect the solubility, these effects can also be varied to optimise the difference in solubility. Liang and Yeh [50] studied the separation of ethyl palmitate, ethyl oleate, eicosapentaenoic acid (EPA) ethyl ester and docosahexaenoic acid (DHA) ethyl ester. They considered the phase behaviour and used the determined coefficients for the Chrastil equation to estimate the separation efficiency that can be attained. In general better separation efficiencies were achieved at higher temperatures and lower pressures. They verified their qualitative prediction through extraction of esterified fish oil and found the results correlated well. While it is believed that solute-solute interactions are not as large as solute-solvent interactions, there is clear evidence that some type of solute-solute interaction is present in SCF/high molecular mass systems. Lockemann [49] studied the phase behaviour of the ternary system CO2/methyl myristate/methyl palmitate and found that these two components can be separated using SC CO2. However, the separation factor, which dictates the degree or difficulty of separation, is dependent on the composition of the feed to be separated and the operating pressure and temperature. They found that while the composition does not significantly affect the separation factor, better separation can be achieved at lower composition of the component to be extracted. In addition to saturated fatty acids with varying chain length, palmitic acid and its derivatives usually occur in the presence of unsaturated fatty acids and their derivatives, in particular those with 18 carbon atoms (oleic acid, linoleic acid and linolenic acid). Figure 34 compares the phase behaviour of various methyl esters of C18 acids in SC CO2. Phase behaviour shown in Figure 34 suggests that it is also possible to achieve separation according to the degree of saturation, but comparing Figure 33 and Figure 34, it is noted that the difference in phase behaviour due to unsaturation is less than due to variations in the chain length. Separation according to unsaturation, while possible, will be more difficult than according to the chain length. The higher the degree of saturation the lower the phase transition pressure and thus the higher the solubility. Liong et al. [65] and Nilsson et al. [67] came to similar conclusions. Normal counter-current fractionation may not be sufficient to separate compounds only differing in hydrocarbon backbone length and an additional stationary phase (such as the column in a SCF chromatograph) may be required to achieve such separations. Güçlü-Üstündağ and Temelli [66] as well as Zou et al. [70] studied the ternary system CO2/oleic acid/linoleic acid. Güçlü-Üstündağ and Temelli found that depending on the initial composition, SC CO2 can also distinguish between the two acids. The interactions are, however, complex and there are indications that solute-solute interactions are present. These interactions can both enhance and decrease the partition coefficients determined based on the binary data, depending on the temperature, pressure and acid ratios. Interestingly Zou et al. found that while the ratio of the components in the liquid phase remains essentially the same due to the fact that the majority of the components are in the liquid phase, the ratio is the vapour phase is significantly different, but the difference depends on the molar ratio of the components.



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15

Pressure (MPa)

12 9

Methyl linoleate

6

Methyl oleate

3

Methyl stearate

0 0.0

0.2

0.4

w2

0.6

0.8

1.0

Figure 34. Pressure – Composition (w2) of various CO2 (1)/C18 ethyl ester (2) systems at 313 K [48,69].

Therefore, provided the correct operating conditions are applied, SC CO2 and other solvents are able to distinguish between acids of the same chain length but different degrees of saturation. The analysis presented above has focused primarity on CO2 as SC solvent, mainly due to an abundance of information on this solvent and a lack of information on other SC solvents. However, qualitatively similar trends are expected in other SC solvents and as such it is expected that these solvents could also achieve the desired separation.

Separation Set-Up The phase behaviour analysis presented above shows that SC CO2 can be used to fractionate fatty acids and their derivatives primarily according to their chain length, but also according to their degree of saturation. Most sources of palmitic acid contain other acids of both longer and shorter chain length and as such a two-step separation process is required where, in one step, components that are more soluble (generally those with less than 16 carbon atoms) are removed while, in the other step, components with that are less soluble (generally those with more than 16 carbon atoms) are removed. Two process options, shown in Figure 35 and Figure 36 are therefore possible. A decision as to the better process option would depend on the feed stock composition and the associated technical and economic analysis.


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Figure 35. Possible set-up for fractionation of acids or esters according to their hydrocarbon backbone where the light fraction is removed in the first column and heavy fraction removed in the second column.

Figure 36. Possible set-up for fractionation of acids or esters according to their hydrocarbon backbone where the heavy fraction is removed in the first column and the light fraction removed in the second column.



197

In the setup shown in Figure 35 the light fraction (i.e. components with less than 16 carbon atoms) is first removed and the raffinate from the first column is fed to the second column where the C16 component is extracted and the raffinate ideally contains components with 18 and more carbon atoms. By and large this option would be preferred as the solvent usage would be less. However, there may be occasions where it is more viable to first remove the heavy components (C18 and greater) after which the extract is fractionated into the light components (C14 and less) and the resultant product (C16). Such a typical process is shown in Figure 36. It should be noted that these setups were compiled where only palmitic acid or its derivative is the only desired product. However, in many cases palmitic acid occurs in combination with other valuable fatty acids (e.g. oleic acid, linoleic acid, stearic acid and even DHA and EPA) and as such more complicated separation sequences are likely.

SCFF of Palmitic Acid and Its Derivatives from Plant Extracts A large number of plants extracts, especially from the seeds, contain palmitic acid and/or its derivatives. These sources may include soybean oil [1], cocoa butter [2], palm kernel oil [3], wheat bran oil [4], pumpkin seed oil [5], to name but a few. By and large the palmitic acid occurs as triglycerides, in most cases in mixed triglycerides where the other acids present range from C12 to generally C18 but these may be as high as C22 or even C24. In order to obtain the palmitic acid, the triglycerides are hydrolysed to either FFA or their methyl or ethyl esters. Therefore, in order to obtain palmitic acid or its derivatives, the mixtures need to be fractionated, usually according the chain length. In addition to hydrolysed triglycerides, palm fatty acid distillates also contains a significant amount of palmitic acid. Palm fatty acid distillates is a byproduct of the palm oil refining process and contains predominantly FFA (palmitic, oleic and linoleic, and possibly some stearic acid)[71]. This byproduct, as well as similar byproducts from other crude oil refining processes, are thus also potential sources of palmitic acid. The analysis presented above shows that SCFs is able to achieve this separation. Fractionation set-ups similar to that presented above can therefore be used to fractionate the feed material resulting in a product rich in palmitic acid. An excellent example of the implementation of SCFF to obtain palmitic acid from a plant source is the work of Brunner and Machado [7,72]. They conducted a detailed analysis on the fractionation of fatty acids from palm fatty acid distillates (99 % FFA (mainly palmitic, oleic and linoleic acid) , 0.9 % squalene and 0.1 tocopherol) starting with a phase equilibrium analysis through to pilot plant studies and experimental verification of the separation. They postulated, from the phase equilibrium studies, that squalene and palmitic acid would be preferentially extracted and verified their postulation experimentally. They also considered a pseudo-binary mixture separation where palmitic acid is to be separated from oleic and linoleic acid and showed, using separation factors that this is possible. On pilot plant scale they showed that such a separation is feasible and balanced yield and extract quality. At their optimum conditions (373 K, 29 MPa, extract to raffinate ratio of 1.2) they obtained an extract where the palmitic acid content was enriched from 52.5 % in the feed to 74.4 % in the extract and the oleic and linoleic acid content enriched from 46.3 % in the feed to 59.0 % in the raffinate. Squalene was also enriched in the extract from 0.6 % in the feed to 1.2 % in the


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extract. They thus showed it is possible to separate high molecular mass acids based on the number of carbon atoms on the hydrocarbon backbone. Using the same concept, only applying it to triglycerides Asep et al. [2] fractionated cocoa butter according to the molecular mass of the triglyceride. Their triglycerides contained mainly palmitic, stearic and oleic acid and triglycerides containing more palmitic acid were preferentially extracted. Acetone, ethanol and isopropanol were used as co-solvents to increase the extraction yield. Interestingly, an increase in co-solvent concentration also showed an increase in palmitic acid selectivity while it did not influence the selectivity of the other acids significantly. Ethanol was found to be the best polar co-solvent.

Palmitic Acid As a By-Product from Fish Oil Fractionation Fish oils are also rich in fatty acids. Gruger et al. [8] studied the composition of various fish oils in terms of their fatty acid content. In general, fish oils contain slightly longer chain fatty acids than those of plant material origin. The study if Gruger et al. showed that the oils have between 9.5 and 33.4 % of palmitic acid with palmitic acid being the lowest major constituent in fish oils. The majority of literature information on the fractionation of fatty acids from fish oils is centred on obtaining the polyunsaturated fatty acids (or their esters). However, while the main focus of the fractionation of fish oils is to obtain unsaturated fatty acids, in particular EPA and DHA [73], fractionation of fish oils can produce palmitic acid as a by-product. Staby et al. [74] considered the phase behaviour of fish oil in SC CO2 and determined Kvalues for the various constituents. They found that the K-values depend primarily on the operating temperature and pressure as well as the hydrocarbon backbone chain length but not so specifically on the number of double bonds present. This is in agreement with phase equilibria observations where there is a much larger difference in the phase behaviour between the acids of differing chain length than acids of the same chain length and differing degrees of saturation. Staby and Mollerup [75] conducted a review on the fractionation of fish oils according to their molecular mass using SC CO2. They proposed a process whereby the fish oil can be fractionated into 4 fractions. Three extraction columns are used in series and each column should contain a stripping and an enrichment section. The lightest fraction obtained in their process contains C10 to C16 acids. One could modify this set-up slightly through the addition of another column and in such also obtain a C16 fraction, as suggested in Figure 37. The fish oil could be fractionated into five fractions, of which a low chain length, a stream rich in palmitic acid, a stream rich in stearic, oleic and linoleic acid (C18 acids) and a stream rich in EPA (C20 acid) and DHA (C22 and C24 acids) are produced. It should, however, be noted that although the set-up proposed in Figure 37 is probably the most suitable in general, there may be cases where the column sequencing would be different, such as the difference between the two sequences shown in Figure 35 and Figure 36. The optimum column sequencing would be feed stream and separation sequence specific and as such would not be discussed further here. Riha and Brunner [76,77] considered the separation of a pseudo 5 component system of fatty acid ethyl esters of fish oil origin. The focus of the fractionation process was to separate



199

low molecular mass esters (C14 to C18) from high molecular mass esters (C18 to C20). The high molecular mass ethyl ester products were achieved at 95 % recovery and 95 % purity. They thus showed that practically fatty acid ethyl esters can also be separated according to their molecular mass using SC fluids.

Figure 37. Possible set-up for obtaining palmitic acid or the ester thereof from fish oil using SCFF (Modification of process proposed by Staby and Mollerup [75]).

SCFF TO SEPARATE PALMITIC AND OTHER FATTY ACIDS AND THEIR DERIVATIVES FROM OTHER COMPONENTS SCFF of oils containing palmitic acid and/or its derivatives is not limited only to studies involving the fractionation of these oils according to their fatty acid content. In fact, as far back as 1949 Passino [78] published one of the early works on the processing of oils using high pressure/liquefied gases. In particular he used high pressure propane to process Mahendra oil, Sardine oil, Soybean oil, Linseed oil, Cod-liver oil and Tall oil. The oils were improved by removing the colour molecules and ash, and, where applicable, the oil was then fractionated. In 1978, Peter and Brunner [18] showed experimentally that SC propane, in the


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presence of acetone as an entrainer, is able to separate mono-, di- and triglycerides. Later Peter and Ender [79] showed that monoglycerides can be separated from a mixture of glycerides using CO2 with propane as an co-solvent. A product containing 99.5 % monoglycerides was obtained from a glyceride mixture containing 55 % monoglycerides. Outlined below is a brief discussion on how SCFs can be used to achieve fractionations involving palmitic acid where the required separation is not (only) between fatty acids of differing chain length, but rather obtaining palmitic acid from a mixture of a wide range of compounds. In particular, this part of the chapter will focus on the reduction of FFA content of oils and the separation of palmitic acid from tocopherols, sterols and other components.

Reduction in Free Fatty Acid Content of Oils In general, edible oils are preferred to have a low FFA content. In fact, for olive oils the International Olive Council has included the maximum FFA content in extra virgin (< 0.8 mass %), virgin (< 2.2 mass %) and ordinary olive oil (< 3.3 mass %). SCFF is an excellent method to reduce the FFA content of oil and as such to improve the quality of the oil increasing its retail value. SCFF is highly suitable due to the fact that the SC solvent is able to preferentially dissolve the FFA above the triglycerides present. Such a process would operate at low temperatures thus limiting thermal degradation and using SC CO2 would omit the use of organic solvents and unacceptable solvent residues.

Phase Behaviour Analysis The FFAs (amongst others, palmitic acid) are to be separated from the oil, which contains predominantly triglycerides. Figure 10, for CO2 as solvent, and Figure 21, for propane as solvent, show that palmitic acid and tripalmitin have a significantly different solubility in the SCFs and that the acid preferentially dissolves compared to the triglyceride. While the plots in Figure 10 and Figure 21 illustrate the difference for palmitic acid and tripalmitin, similar difference are noted for other fatty acids and their corresponding triglycerides. Published Studies A number of studies have, on pilot plant scale, shown that this separation is indeed possible. Bondoli et al. [80] considered the upgrading of olive oil where the primary aim was to remove the FFA from the oil. While they did not consider the individual components they did show that a product with a significant reduction in FFA can be attained. In the extract high enrichment ratios for squalene, FFA and monoglycerides were obtained. Similarly List et al. [81] showed experimentally that the FFA content of soybean oil can be reduced using SC CO2. Ziegler and Liaw [82] used near and SC CO2 to remove FFA from edible oil. A synthetic mixture containing refined soybean oil with added oleic, linoleic and palmitic acid as well as pyrazine and its derivatives was used. The acids were concentrated in the extract with the highest degree of concentration being that of palmitic acid. Ooi et al. [83] considered the fractionation of palm oil using SC CO2 to produce an improved product. Their results show that a raffinate with a reduced FFA, monoglyceride and diglyceride content is achieved and slight fractionation of the triglycerides are obtained. Similar results were obtained with the use of ethanol as a co-solvent with the exception that



201

the process operated at a lower pressure. They did not consider a detailed acid content of the triglycerides, but did show fractionation is possible by separating them into fractions with 48, 50, 52 and 54 carbon atoms. While literature studies on the removal of FFAs have focussed almost exclusively on CO2 as SC solvent, the work by Peter and co-workers [18,79] on the fractionation of mono-, diand triglycerides using propane or CO2 with propane as co-solvent, shows that it would also be possible to remove FFAs from oils using other SC solvents.

Separation of Fatty Acids from Tocopherol, Sterols and Other Components Palmitic acid and other fatty acids very seldom occur in a mixture that does not contain other chemical compounds. In fact, components such as squalene, tocopherols and sterols are often present in combination with the fatty acid. The question now arises as to the presence of these compounds and their distribution between the extract and raffinate phases.

Phase Behaviour Analysis Figure 38 shows the phase behaviour of palmitic acid in combination with other compounds often found together with palmitic acid. Importantly, Figure 38 shows that there exists a significant difference in the phase behaviour between the various compounds (or the groups of molecules that they represent). Separation would this be possible using a two-step process where the components more soluble than palmitic acid are removed in the one column and palmitic acid removed from the components that are less soluble in a second column. Similar set-ups such as those shown in Figure 35 and Figure 36 may be used to achieve these separations. 30

Pressure (MPa)

25 20

Cholesterol

15

alpha-tocopherol Palmitic acid

10

Squalene 5 0 0.00

0.01

w2

0.02

0.03

Figure 38. Comparison of the pressure – composition (w2) phase behaviour of the CO2 (1)/squalene (2) [84], CO2 (1)/palmitic acid (2) [24], CO2 (1)/alpha-tocopherol (2) [85] and CO2 (1)/cholesterol (2) [86] systems at 333 K.


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Published Studies Araujo et al. [6] modelled the SCFF with SC CO2 of Soybean Oil distillates, containing FFAs, squalene, tocopherols, sterols and triglycerides, using the Peng Robinson equation of state. Their results show that squalene and the FFAs present themselves in the extract, the remainder of the product in the raffinate. However, the squalene has a much higher partition coefficient and therefore separation from the FFAs is possible in a second column. In their study the FFAs were lumped together and they did not consider palmitic acid alone. However, the concept should be applicable to palmitic acid, as suggested by Figure 38. Stoldt and Brunner [87] considered the phase behaviour of various deacidified palm oils and soybean oil deodoriser distillates. They focused their study in the compounds other than the acids/triglycerides present and showed that the other components can all be concentrated either in the extract or the raffinate of a SCF process. However, improved purity palmitic acid fractions are obtained as by-products in their process, showing that palmitic acid can also be recovered from these sources.

SCFE OF PALMITIC ACID CONTAINING OILS FROM SOLID MATRICES In the above sections, the focus of the applications of SCF processing was to fractionate a liquid-like stream using SCFs as extraction medium, similar to liquid-liquid extraction. However, SCFs has another, often larger, application, namely the extraction of oils and other compounds from a solid matrix. In fact, one of the earliest commercial applications of SCF processing is the SCFE of caffeine from coffee to produce decaffeinated coffee [88]. Traditionally components are extracted from solid matrices using liquid organic solvents such as hexane and methylene chloride. However, these organic solvents result in an unacceptable solvent residue and thermal degradation occurs during solvent removal. SCF extraction, especially using SC CO2, is an attractive alternative method.

SCFE as a Possible Extraction Medium Both solid palmitic acid and solid tripalmitin have acceptable, although not high, solubilities in SC CO2 and therefore it is possible to obtain extracts of these compounds using SC CO2. A semi-batch setup, as proposed in Figure 2, can thus be used to obtain oil containing fatty acids, amongst others palmitic acid, from solid matrices. Salgin and Korkmaz [5] considered the SCFE of pumpkinseed oil. They found that the oil they obtained did not differ significantly from that obtained using hexane extraction in terms of fatty acid content. They came to the conclusion that SCFE is a green process and can be used to recover healthy oil from pumpkin seeds. Jung et al. [4] conducted extraction with near and SC CO2 of wheat bran and found that the major fatty acids present in the extracted oil were palmitic, oleic, linoleic and -linolenic acids. They only considered the yield change with time, not the compositional change. However, as seen in 6.2 below, partial fractionation of the oil may be possible.



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Özkal and co-workers [89,90] studied the extraction of palmitic acid containing oil from apricot kernels. They studied the effect of, amongst others, temperature, pressure and CO2 flow rate on the extraction yield and built a mass transfer model to describe the extraction. Their results show that irrespective of temperature, pressure or solvent flow rate, at very long extraction times the same yield is obtained. However, particle size is important as higher yields at long extraction times were obtained for smaller particles. Özkal and co-workers concentrated their efforts on the extraction yields and, save the optimum point, did not consider the composition of their extracts. The operating parameters may influence the sequence in which the components are extracted and would thus warrant a separate study.

Possibility of Partial Fractionation Although fatty acids occur in nature predominantly in the form of mixed triglycerides, some degree of fractionation of the triglycerides based on their total mass may be achieved using SCFE if the extraction conditions are correctly chosen and varied. Additionally, a prerequisite for fractionation during extraction would also be that the triglycerides present vary in molecular mass. Fractionation would thus achieved by the fact that the lower molecular mass triglycerides are more soluble in SC CO2 and are thus preferentially extracted. Salgin and Korkmaz [5] found in their study in the SCFE of pumpkinseed oil that fatty acid profile of the oil obtained did not differ significantly as the process conditions changed. This may be due to how the triglycerides are combined or could even be attributed to the experimental conditions. Partial fractionation is thus not always possible. There are, however, a number of studies that have shown that partial fractionation is indeed possible. In most of these studies the temperature and pressure have been kept constant. Hassan et al. [3] used SC CO2 extraction to obtain fractions of oils, containing lauric acid, myristic acid, palmitic acid and oleic acid amongst others, from Palm kernels. Their results show that at the beginning of the extraction lower acids are removed and later higher acids are removed. Jokić et al. [1] extracted soybean oil from soybeans and analysed the oil for the acid content. Statistically significant changes in the acid content were observed as the experiment continued. The palmitic acid content of the earlier fractions were higher than that of the latter fractions while the reverse was true for the higher acids. Nik Norulaini et al. [91] considered the extraction of palm kernels with SC CO2 and analysed the fractions for their acid content. They found that SCFE can be applied in such a way that the earlier fractions are richer in lower molecular mass acids and the later fractions are richer in higher molecular mass acids.

Use of Entrainers As for SCFF entrainers can be used in SCFE to increase the solubility and/or decrease the operating pressure. SCFE is by and large more concerned with extraction rather than


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fractionation and thus the loss of selectivity sometimes encountered with the use of entrainers is far out-weighed by the increase in solubility and/or decrease in operating pressure. A large number of studies have been published on the SCFE using a co-solvent with a selection of these focussing on products that contain palmitic or similar acids (or rather their triglycerides). Bimakr et al. [92] considered the extraction of seed oil from winter melon using SC CO2 and ethanol as a co-solvent with a 10:1 CO2 to ethanol mass ratio. While they did not investigate the effect of the co-solvent per se, they did conclude that their optimised SCFE process yields similar results to that of traditional solvent extraction using ethanol. Their optimised the SCFE process did yield a slightly lower saturated fatty acid contents, with palmitic acid being approximately 40 % less. However, only the yield was optimised and the only sample for which analysis was presented was that of the optimised condition. The process was not optimised for a specific component and it may thus be that higher palmitic acid content, possibly even higher than that of the traditional ethanol extraction, may be obtained. Mendes et al. [93] used ethanol as co-solvent (10 mol %) to increase the lipid yield during the SC extraction of Spirulina. The CO2 modified with ethanol gave lipid yields comparable with those of traditional organic solvents. Mendes et al. focussed their research on the extraction of -linoleinic acid but did also analyse the palmitic acid content of their samples. However, no comments regarding palmitic acid were given and too little information is presented to provide definitive conclusions. Özkal et al. [89] investigated the effect of parameters on the SCFE of apricot kernels. Amongst others, they considered the effect of the use of and the amount of ethanol as cosolvent. They found that an increase in the amount of co-solvent lead to an increase in the fatty acid yield. The fatty acid contents was not presented for all samples, only the maximum yield sample and a composition similar to that using traditional organic solvent extraction was obtained. In another study on the SC CO2 of extraction of apricot kernels Özkal et al. [90] developed a mass transfer model. They used the model to predict, amongst others, the effect of the co-solvent and found that while the co-solvent initially increases the extraction yield at a very long times the extraction yield with or without the co-solvent was the same. The effect of the co-solvent therefore appears to be to reduce the amount of solvent required and thus also the extraction time rather than the absolute maximum extraction that can be attained.

CONCLUSION This chapter has shown that SCF processing is a viable method to process products containing palmitic acids and its derivatives (tripalmitin, ethyl palmitate and methyl palmitate). This study was conducted by investigating the solubility of these compounds in SCFs, in particular CO2, ethane and propane, followed by an investigation as to how SCFF and SCFE can be applied based on the phase behaviour and verified using published studies. This study concludes that SCF processing is able to (i) extract palmitic acid and/or its derivatives from a solid matrix, (ii) fractionate a fatty acid mixture or a mixture of its derivatives mainly according to the chain length and (iii) provided sufficient difference in the



205

phase behaviour is present, is able to separate palmitic acid from other components. The fundamental basis for the possibility of the extraction and separation is that palmitic acid and its derivatives have a reasonable solubility in SC solvent. While the solubility in CO2 may be low at times, the solubility can be improved through the use of co-solvents, or ethane or propane may be used as alternative solvents. This study has shown that on technical level SCF processing of palmitic acid containing products is a viable method. In particular, a more environmentally friendly and generally regarded as safe solvent is used resulting in a product with minimal, if any, solvent residue. This study was conducted by conceptually considering the separation of palmitic acid or its derivatives. While examples were mentioned, a more global approach was taken to prove the technical viability. In order to implement SCF processing technology, the separation required needs to be investigated based on the raw materials. In addition, an economic and energy analysis would be required to provide a final outcome as to the viability of the process as a whole.

NOMENCLATURE Details pertaining to the acids (and their derivatives) studied in this works are presented in Table 15. Table 15. Nomenclature and structure of acids mentioned in this work Common name

IUPAC ID

Number of carbon atoms

Degree of unsaturation

Capric acid

Decanoic acid

10

None

Lauric acid

Dodecanoic acid

12

None

Myristic acid

Tetradecanoic acid

14

None

Palmitic acid

Hexadecanoic acid

16

None

Stearic acid

Octadecanoic acid

18

None

Oleic acid

Octadecenoic acid

18

Single double bond in varying positions

Linoleic acid

Octadecadienoic acid

18

Two double bonds, usually in position 9 and 12

Linolenic acid

Octadecatrienoic acid

18

Three double bonds

EPA

Eicosapentaenoic_a cid

20

Five double bonds, usually from position 3 onwards

Behenic acid

Docosanoic acid

22

None

DHA

Docosahexaenoic acid

22

Six double bonds, usually from position 3 onwards


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Chapter 9

PALMITIC ACID IN TUNISIAN OLIVE OIL: UPDATING AND PERSPECTIVE Ghayth Rigane1,2, and Ridha Ben Salem1,† 1

Laboratoire de Chimie Organique-Physique UR11ES74, Faculté des Sciences de Sfax, Département de Chimie, Sfax, Université de Sfax, Tunisie 2 Département de Physique-Chimie, Faculté Des Sciences et Techniques de Sidi Bouzid, Sidi Bouzid, Université de Kairouan, Tunisie

ABSTRACT In this review the major saturated fatty acid, palmitic acid, of Virgin Olive Oil (VOO) was studied. This oil is one of the oldest known vegetable oils and it plays a fundamental role in human nutrition around the Mediterranean basin. This nature juice is the only edible oil of great production obtained by physical methods from the fruit Olea europaea L. Consideration of VOO as a natural functional fat is related to the presence of palmitic acid. Updating of its levels in Virgin olive oils throughout the Tunisian olive oil as well as information on expecting levels in other products from olive tree establish our view point. Studies on levels palmitic acid upon maturity stage in the oil are also discussed. Major analytical practices are given in brief. Palmitic acid (C16:0) is the principal saturated fatty acid in olive oil, responsible for its figeability at low temperature. Few are the exceptions as palmitic acid content depends heavily on the genetic factor. Palmitic fatty acids, important for the nutritional properties of an olive oil, showed a crucial rule in the characterization of olive oils.

Keywords: Palmitic acid, olive oil, saturated fatty acid composition, storage olive oil  Ghayth Rigane: Laboratoire de Chimie Organique-Physique UR11ES74, Faculté des Sciences de Sfax, Département de Chimie, B.P « 1171 » 3038, Sfax, Université de Sfax, Tunisie. † Corresponding author: Ridha Ben Salem. Laboratoire de Chimie Organique-Physique UR11ES74, Faculté des Sciences de Sfax, B.P « 1171 » 3038 Sfax, Université de Sfax, Tunisie. E-mail : [email protected]


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1. INTRODUCTION Olives (Olea europea L.) are the most widespread and valuable plant in Mediterranean countries. Chehab et al. (2013) mentioned that world-wide production of olive oil during the last 20 years increased by almost 70% (from 1.7 to 2.8 million tons). Olive oils makes up a small proportion (