Concentrations of plasma triglycerides, cholesterol. total lipids and lipid per- oxides in patients with atherosclerotic lesions of peripheral arteries, who were.
215
Chnrca Chrmrca Acra. 155 (1986) 275-284 Elsevier
CCA
03427
The relationship between plasma triglycerides, cholesterol, total lipids and lipid peroxidation products during human atherosclerosis Andrzej
Ledwoiyw
‘. Jerzy Michalak Adam Kgdzidka
b, Adam a.*
Stepieh
b and
’ Department of Parhophysiologr’, Institute a/ Phvsrologrcal Sciences. Agrrcultural Academ!, Akademrcka 12. N-033 Luhhn und h Clinic of Vascular Surgey. (Received
February
Kq,‘ ward.x A rherosclerosis:
Medical Academ,*. Sluszrca 16. 20.OHI Luhlm (Poland)
27th. 1985; revision December
28th. 1985)
Lipid peroxrdes: Trcg(vcerrdes; Cholesterol: Total l1prd.s
Summary
Concentrations of plasma triglycerides, cholesterol. total lipids and lipid peroxides in patients with atherosclerotic lesions of peripheral arteries, who were divided into groups according to the extent and intensity of the lesions. were estimated, as well as lipid peroxide levels in the arterial wall. Statistically highly significant increases of the estimated compounds were found in all groups in comparison with the controls. The existence of a positive correlation between the lipid peroxide concentration and other investigated components in plasma and between the lipid peroxide level in plasma and in the arterial wall was found. Possible mechanisms for lipid peroxide involvement in the process of originating atherosclerotic lesions are discussed.
Introduction
The etiology of atherosclerotic lesions in arteries is complex, and has not been fully explained. Lately. more and more attention has been focused on lipid peroxides as one of the factors impairing the function of producing arterial endothelial cells and primary lesions in the vascular wall [l-3]. Many authors have found increased levels of lipid peroxides during the atherosclerotic process [4-61. These compounds peroxidise polyunsaturated fatty acids of cell membranes under the influence of free radical O;- and free hydroxyl
l
To whom correspondence
0009-8981/86/$03.50
should be addressed
8 1986 El sevier Science Publishers B.V. (Biomedical
Division)
276
OH -, the intermediate products in the process of reduction of molecular oxygen to H,O [7-91. These radicals are cytotoxic and genotoxic [lO,ll]; they disturb correct protein and phospholipid structure of cell membranes causing changes in their permeability. The mechanism of their activity in the atherosclerotic process was clarified by the discovery of the role of balance between the level of prostacyclin (PGI,) in the vascular wall [12] and the level of thromboxane (TXA,) in blood platelets [13]. Both PGI Z and TXA z rise from cyclic endoperoxide, prostaglandin Hz (PGH Z) as a result of adequate synthetases action. It was demonstrated that high lipid peroxide concentrations (above 1 x 10m6 mol/l) inhibit synthetases of both PGI, [12,14,15] and PGH, [16]. Because prostaglandin G, (PGG,), a substrate of these enzymes, is also a lipid peroxide, PGI,- and TXA,-synthetases may have self-regulatory and self-deactivatory properties [7-9,161. Hence, the balance between lipid peroxide production and catabolism maintains an adequate prostacyclin level [14,17], but increased levels of lipid peroxides may cause reorientation of arachidonic acid cascade towards intensified thromboxane synthesis [3]. Because hypercholesterolemia and hypertriglyceridemia are regarded as risk factors in atherosclerosis, it was decided to investigate the relationship between levels of triglycerides, cholesterol, total lipids and malondialdehyde (MDA), which is a product of reduction in the lipid peroxidation processes, in the course of peripheral atherosclerosis in man, and the degree of lesion advancement. Materials and methods The following samples were examined: platelet-rich plasma (PRP) from 26 patients with intermittent claudication, who had been treated pharmacologically (group I), PRP from 31 patients on whom reconstruction of iliac or femoral arteries had been performed (group II) and PRP from 15 patients who had their limb amputated at thigh height because of the extent of lesions. The patients were classified into a particular group on the basis of clinical examination and arteriography by the Seldinger technique [29]. The patients chosen for the experiment were free from thyroid dysfunction, diabetes, liver and kidney diseases; for 2 wk before the experiment they had not been treated with beta-blockers nor with hypolipidemizing agents. The patients were men 35-65 yr old, and the average age was 52 yr. None was obese. Blood was obtained from the cubital vein after overnight fasting and 0.13 mol/l trisodium citrate solution in the ratio 1 vol of citrate + 9 vol of blood was used as anticoagulant. The blood was centrifuged for 15 min at 200 x g and PRP was obtained. PRP of 15 clinically healthy men of similar age, obtained in the same way was used as the control. From patients of groups II and III segments of arteries with atherosclerotic lesions were obtained. A 500-mg sample of arterial tissue after careful removal of an adventitia and washing in the cold (277 K) 0.154 mol/l sodium chloride solution was homogenized in 5 vol of 0.2 mol/l phosphate buffer, pH 7.4, with a Waring Blendor homogenizer. The homogenate was transferred quantitatively to an all-glass Potter homogenizer with motor-driven pestle (clearance 0.15 mm), homogenized
277
again (1000 t-pm, 10 strokes) and then centrifuged for 15 min at 2000 X g. The sediment was discarded and the supernatant was used for MDA estimation. Arterial segments from 11 patients with no atherosclerotic lesions obtained during artery-vein anastomosis surgery were used as controls. Triglycerides in plasma were estimated by the method of Carlson [18], cholesterol that of Abel1 et al [19], total lipids by that of Friedman [20] and MDA by the thiobarbituric acid reaction as follows: 0.5 ml of PRP or tissue homogenate supernatant was mixed with 2.5 ml of 1.22 mol/l tri-chloroacetic acid in 0.6 mol/l HCl and allowed to stand for 15 min. To this mixture 1.5 ml of thiobarbituric acid solution was added (obtained by dissolving 500 mg of thiobarbituric acid in 6 ml 1 mol/l NaOH and then adding 69 ml H,O) and thereafter heating for 30 min in a boiling water bath. After cooling to room temperature 4 ml of n-butanol was added and the mixture was shaken vigorously for 3 min and centrifuged 10 min at 1500 X g. The organic layer was removed and its absorbance was measured at 532 nm, and also at 515 and 555 nm, so that an Allen correction could be made for non-specific absorption by other tissue components. Precision of the duplicate samples was good. Variation ranged from l-6% for 10 tissue or PRP samples. Recovery of MDA added either to PRP or tissue homogenate supernatant ranged from 85594% by this method. Ten replicate analyses from the same piece of artery had a mean & SD of 7.18 & 0.40 nmol/g. The coefficient of variation (CV) was 5.2%. The thiobarbituric acid complex extracted with butanol and standard thiobarbituric acid derivatives were chromatographed on Silica Gel G thin-layer plates (E. Merck, Darmstadt, FRG) in butanol-acetic acid-water 4: 1 : 5 v/v/v. Only one spot with an B, value of 0.80 was observed for both the extracts and standards after 4 h. The number of platelets in blood was determined using a Coulter counter S-Plus VI (Coulter Electronics Ltd., Northwell Dr, Luton, Beds, UK). The platelet aggregate ratio was estimated as described by Wu and Hoak [21]. The results were analysed statistically [22]. The MDA standard curve was obtained by using malondialdehyde bis-dimethylacetal (Dr. Theodor Schuchardt, Hohenbrunn, FRG); 2-thiobarbituric acid was purchased from E. Merck, all other reagents, analytical grade, were obtained from PoCh, Gliwice, Poland. Results MDA, cholesterol, triglycerides and total lipid concentrations in plasma and MDA concentrations in arterial tissue are presented in Table I. It is necessary to note a statistically highly significant increase of MDA concentrations in all tested groups in relation to that control group, and also between particular groups ( p < 0.001). Cholesterol behaves similarly except for the plasma levels in the control group and group I. A significant increase in triglyceride concentration was observed in the test groups compared with control and in group Ill compared with group I ( p < 0.001). Differences between groups II and I and groups Ill and II were also significant ( p < 0.05). For total lipids note the significant increase both in tested groups compared with the control and between groups ( p < O.OOl), except for group II
278 TABLE
I
Concentrations (mean_IsD) of MDA, cholesterol, total lipids and triglycerides in plasma and MDA in the arterial wall in the control group and in patients with different stages of atherosclerosis (in parentheses, the number of cases examined) Total lipids plasma
( kt moI/I)
Cholesterol plasma (mmol/l)
(g/I)
Triglycerides plasma (mmol/l)
Control
0.94 f 0.09
5.19+0.28
8.20 f 0.20
0.82 f 0.07
3.42kO.15
I
(15) 2.14kO.24
(15) 5.30*0.46
(15) 9.76 f 0.84
(15) 1.51 kO.35
(11) _
II
(26) 3.22kO.18
(26) 5.91 f 0.51
(26) 10.29 f 0.73
(26) 1.71*0.36
7.11+0.59
III
(31) 4.20 k 0.16
(31) 6.88 + 0.86
(31) 12.45 f 0.72
(31) 1.97 k 0.28
(31) 7.38 + 0.40
(15)
(15)
(15)
(15)
(15)
MDA plasma
Group
TABLE
II
Significances of differences in plasma MDA, cholesterol triglycerides of MDA concentrations in the arterial wall in all cases investigated
Plasma
MDA
Plasma cholesterol
Plasma
Plasma
Arterial
triglycerides
total lipids
wall MDA
ns, difference
TABLE
MDA artery (nmoI/g)
statistically
I II III I II III I II III I II 111 1 II III
and total lipid concentrations
Control
I
11
III
(P)
(P)
(P)
(P)
< 0.001
< 0.001
x
< 0.001
< < ns i < i < < < <
