Accepted Manuscript Moderate doses of commercial preparations of Ginkgo biloba do not alter markers of liver function but moderate alcohol intake does: A new approach to identify and quantify biomarkers of ‘adverse effects’ of dietary supplements Harris R. Lieberman, Mark D. Kellogg, Victor L. Fulgoni, III, Sanjiv Agarwal PII:
S0273-2300(16)30392-0
DOI:
10.1016/j.yrtph.2016.12.010
Reference:
YRTPH 3745
To appear in:
Regulatory Toxicology and Pharmacology
Received Date: 28 September 2016 Revised Date:
21 December 2016
Accepted Date: 22 December 2016
Please cite this article as: Lieberman, H.R., Kellogg, M.D., Fulgoni III., , V.L., Agarwal, S., Moderate doses of commercial preparations of Ginkgo biloba do not alter markers of liver function but moderate alcohol intake does: A new approach to identify and quantify biomarkers of ‘adverse effects’ of dietary supplements, Regulatory Toxicology and Pharmacology (2017), doi: 10.1016/j.yrtph.2016.12.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Moderate doses of commercial preparations of Ginkgo biloba do not alter markers of liver
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function but moderate alcohol intake does: A new approach to identify and quantify
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biomarkers of ‘adverse effects’ of dietary supplements
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Harris R. Lieberman; Mark D. Kellogg; Victor L. Fulgoni, III; Sanjiv Agarwal
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HRL - Military Nutrition Division, U.S. Army Research Institute of Environmental Medicine,
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Natick, MA 01760, USA, email
[email protected]
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MDK - Department of Laboratory Medicine, Children’s Hospital Boston, Harvard Medical
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School, Boston, MA 02115, USA,
[email protected]
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VLF - Oak Ridge Institute for Science and Education, Belcamp, MD 21017, USA; Henry M.
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Jackson Foundation, Bethesda, MD 20817, USA,
[email protected]
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SA - Oak Ridge Institute for Science and Education, Belcamp, MD 21017, USA,
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[email protected]
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Corresponding author: Harris R. Lieberman, PhD, U.S. Army Research Institute of
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Environmental Medicine, 10 General Greene Ave., Building 42, Natick, MA 01760-5007, Tel:
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508-233-4856, Fax: 508-233-5854, Email:
[email protected]
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Abstract It is difficult to determine if certain dietary supplements are safe for human consumption. Extracts of leaves of Ginkgo biloba trees are dietary supplements used for various purported
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therapeutic benefits. However, recent studies reported they increased risk of liver cancer in
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rodents. Therefore, this study assessed the association between ginkgo consumption and liver
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function using NHANES 2001-2012 data (N=29,684). Since alcohol is known to adversely affect
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liver function, association of its consumption with liver function was also assessed. Alcohol and
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ginkgo extract intake of adult consumers and clinical markers of liver function (alkaline
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phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma glutamyl transferase,
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lactate dehydrogenase, bilirubin) were examined. Moderate consumers of alcohol (0.80±0.02
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drinks/day) had higher levels of aspartate aminotransferase and gamma glutamyl transferase than
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non-consumers (P0.01) in levels of markers of liver
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function in 616 ginkgo consumers (65.1±4.4 mg/day intake) compared to non-consumers. While
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moderate alcohol consumption was associated with changes in markers of liver function, ginkgo
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intake as typically consumed by U.S. adults was not associated with these markers. Biomarkers
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measured by NHANES may be useful to examine potential adverse effects of dietary
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supplements for which insufficient human adverse event and toxicity data are available.
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Trial registration number: Not applicable, as this is secondary analysis of publicly released
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observational data (NHANES 2001-2012).
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Keywords: NHANES, alanine aminotransferase, aspartate aminotransferase, gamma glutamyl
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transferase, lactate dehydrogenase, bilirubin
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1. Introduction Dietary supplements (DS) are widely used as alternatives to traditional medical therapies.
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In most countries DS are not regulated as extensively as drugs and definitive evidence regarding
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their efficacy and safety, including results of pre-clinical animal safety testing and adverse event
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(AE) reports, is not always available (Bagchi, 2014; Devala et al., 2011; IOM, 2005; Marcus and
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Grollman, 2012). Serious adverse health events resulting from DS use must be reported by DS
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manufacturers to the U.S. Food and Drug Administration (FDA) but for non-serious AE,
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manufacturers are only required to keep records for six months and there is no formal
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requirement to report minor adverse health events (Clauson et al., 2008; deBoer and Sherker,
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2016; FDA, 2013; Pawar and Grundel, 2016). The FDA recently announced it will begin posting
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AE report data associated with foods, DS and cosmetics on the internet (DHHS, 2016). It is
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estimated that less than 1 in 100 AE that occur are reported to regulatory authorities, and reports
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that are filed are difficult to aggregate and evaluate (Walji et al., 2009; Woo, 2007). Scientists
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and regulatory officials from a number of countries, including the U.S. National Academy of
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Sciences, have called for improvements in the methods used for ensuring the safety of DS and
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detection of AE from consumption of DS (Borg et al., 2011; Gardiner et al., 2008; IOM, 2005;
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USGAO, 2009; Walji et al., 2009).
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The tree Ginkgo biloba L. has been cultivated in China for several thousand years and
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extracts of the leaves are used as dietary supplements (Stromgaard et al., 2010). The most
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common therapeutic effects attributed to ginkgo extract are improved blood circulation and brain
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function, resulting in various cognitive benefits including memory enhancement in healthy
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individuals and those suffering from various illnesses including Alzheimer’s disease (DeKosky
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et al., 2008; Ihl, 2013; Mohanta et al., 2014; NCCIH, 2016; Solomon et al., 2002; Stromgaard et
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al., 2010), claims that are not supported by the literature (Popa, 2002). In Germany and China,
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countries where herbal DS are particularly popular, ginkgo extract is the most commonly
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prescribed herbal supplement (Popa, 2002). Gingko extract is a popular herbal supplements and
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it is estimated that in 2007, 11.3% of Americans using non-vitamin, non-mineral supplements
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were using it (Barnes et al., 2008; Diamond and Bailey, 2013; Kleijnen and Knipschild, 1992).
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Herbal supplements are particularly popular in the U.S. among certain groups of individuals such
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as those with various illnesses and college students (Farina et al., 2014; Lieberman et al., 2015).
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Supplements containing ginkgo extracts are widely believed to be safe and to only cause
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minor side effects such as stomach upset, headache, dizziness, constipation, and allergic skin
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reactions (NCCIH, 2016; Stromgaard et al., 2010). However, several animal studies have
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reported toxic effects (Koch et al., 2013; Rider et al., 2014) including a large animal study using
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standardized toxicology testing methods that reported high doses of an extract of ginkgo biloba
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leaves increased risk of liver and thyroid cancer in mice and rats (NTP, 2013). Few human
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studies have examined the adverse effects of commercially-available DS containing Ginkgo
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biloba, and the sample size of these trials in aggregate is modest compared to the sample size of
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clinical trials typically required for approval of prescription drugs (Carlson et al., 2007; Dodge et
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al., 2008; Gavrilova et al., 2014; Ihl et al., 2011; Kellermann and Kloft, 2011; Koehler et al.,
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2004; Le Bars and Kastelan, 2000; Le Bars et al., 1997; Pittler and Ernst, 2004).
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The National Health and Nutrition Examination Survey (NHANES) is designed to
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monitor the health and nutritional status of the U.S. population. The survey examines a
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nationally representative sample of approximately 5,000 adults every year and includes an in-
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home survey and a mobile laboratory physical examination. Using standardized, state-of-the-art
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techniques, NHANES assesses food consumption and dietary supplement use, multiple measures
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of blood chemistry, body composition, cardiovascular fitness and numerous other health-related
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parameters of volunteers. Data from these surveys are used to determine prevalence of diseases
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and their risk factors, assess nutritional status and its association with health promotion and
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disease prevention, and assist with formulation of national standards and public health policy
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(CDC, 2016). Because NHANES surveys are conducted using the same standardized procedures,
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very large data sets can be obtained by combining multiple years of data. By combining multiple
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years of NHANES a large number of reported DS consumers can be studied to determine if there
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is evidence of specific AE associated with use of a particular DS.
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The primary objective of this study was, using NHANES 2001-2012 data, to determine if
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there is an association of consumption of commercial preparations containing extracts of Ginkgo
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biloba with alterations in clinical markers of liver function in U.S. adults. In addition, as a
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positive control it was determined if the method used could detect changes in markers of liver
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function associated with alcohol intake, which is known to have adverse effects on the liver in
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high doses. It was hypothesized consumption of ginkgo preparations typical of those consumed
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by the U.S. population could alter these markers of liver function since recent animal
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toxicological studies have reported gingko extract intake has toxic effects on the liver (NTP,
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2013; Rider et al., 2014).
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2. Methods
2.1. Study population: NHANES data are collected on a nationally representative
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sample of non-institutionalized Americans by the National Center for Health Statistics (NCHS)
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of the Center for Disease Control and Prevention (CDC), and released every two years (CDC,
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2016). All participants or proxies provided written informed consent and the Research Ethics
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Review Board at the NCHS approved the survey protocol. Data from NHANES 2001-2002,
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2003-2004, 2005-2006, 2007-2008, 2009-2010 and 2011-2012 were combined for these
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analyses. The combined sample included 29,684 adults (age 19 years and older) excluding
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pregnant or lactating females. Participants completed an in-person health examination in a
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Mobile Examination Center that included a 24-h dietary recall and provided blood and urine
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specimen for laboratory analyses. Detailed description of the survey design and the data
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collection procedures are available elsewhere (CDC, 2016).
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2.2. Estimates of gingko and alcohol intake: In conducting the NHANES 2001–2012, a dietary supplement questionnaire was administered in person during the household interview to
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assess use of vitamins, minerals, botanicals, and other dietary supplements over the past 30 days
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(CDC, 2016). The interviewer also examined each dietary supplement container and recorded
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complete product information so that every reported dietary supplement could be matched or
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entered into a dietary supplements database. The NCHS maintains an extensive dietary
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supplement database that contains product label information including the labeled dosage or
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serving size, ingredients, and the amounts of ingredients per serving (CDC, 2016). Ginkgo intake
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in milligrams from each ginkgo-containing commercial preparation used was calculated from the
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NHANES dietary supplement intake files that contained 42 different dietary supplement codes
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(different manufacturer forms and concentration levels) and ginkgo consumers were defined as
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those consuming any amount of a ginkgo preparation over the past 30 days. A complete list and a
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detailed description of these preparations can be derived using the public NHANES data files
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associated with each 2-year cycle examined that are available on the CDC website (CDC, 2016).
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Alcohol intake was calculated as average number of drinks (12 oz of beer, 5 oz of wine and/or
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1.5 oz of liquor) per day over the last year using the NHANES 2001-2012 alcohol questionnaire
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and alcohol consumers were defined as those consuming any amount of alcohol (CDC, 2016).
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2.3. Outcome variables for markers of liver function: Laboratory test values for the following liver enzymes in blood samples from NHANES 2001-2012 were compared across
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consumers and non-consumers of ginkgo or alcohol: alkaline phosphatase (ALP), alanine
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aminotransferase (ALT), aspartate aminotransferase (AST), gamma glutamyl transferase (GGT),
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lactate dehydrogenase (LDH) and bilirubin levels. The activities of ALP, ALT, AST, GGT and
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LDH were measured spectrophotometerically using their respective kinetic enzymatic methods
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and bilirubin levels were measured spectrophotometerically using a timed-endpoint diazo
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method (CDC, 2016). ALT and AST are aminotransferases that, when elevated, are indicative of
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reduced hepatocyte integrity. GGT is a marker of cholestasis and LDH is a marker of ischemic
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liver damage (CDC, 2016; Limdi and Hyde, 2003; Woreta and Alqahtani, 2014). LDH is also a
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marker of heart disease and lung or kidney tumors (CDC, 2016). Bilirubin is used as a marker of
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liver function as hepatobiliary diseases will affect its uptake, storage and excretion (CDC, 2016;
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Limdi and Hyde, 2003; Woreta and Alqahtani, 2014).
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2.4. Statistical analysis: Analyses were conducted using SAS 9.2 (SAS Institute, Inc.;
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Cary, NC) and SUDAAN release 11.0 (Research Triangle Institute; Research Triangle Park,
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NC). Appropriate weighting factors were used to adjust for oversampling of selected groups,
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survey non-response of some individuals, and day of the week when the interview was
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conducted. Least square means and standard errors (SE) of laboratory parameters were
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determined using PROC REGRESS of SUDAAN after adjustment for various covariates.
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Outcome variables were adjusted for gender, race-ethnicity, age, physical activity (categorized as
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sedentary, moderate, or vigorous based on responses to questions on activity), poverty income
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ratio, smoking habits (yes/no), energy intake and BMI. In a separate set of regression analyses,
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alcohol intake was added as an additional covariate to those described above and we assessed
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whether there was an interaction of ginkgo consumption and alcohol intake. Differences in least
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square means of consumers and non-consumers were determined. Significance was set at
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P
