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Malondialdehyde concentration in plasma is inversely correlated to the proportion of linoleic acid in serum lipoprotein lipids
ATHEROSCLEROSIS Atherosclerosis 108 (1994) 103-I IO
ELSEVIER
Malondialdehyde concentration in plasma is inversely correlated to the proportion of linoleic acid in serum lipoprotein lipids ohrvall* a, Siv Tengblad”, Bo Ekstrandb, Bengt Vessbya
Margareta
“Department
of Geriatrics,
‘Department
of Clinical
02
Box 2151. S-750 bSwedish Institute
(Received
21 September
,fbr Food Reseurch,
1993; revision
Chemistry,
Uppsalu.
Agneta
Universi/,v
Siegbahn’,
qf Uppsaiu.
Sweden
Gothenhurg,
received 28 February
Siveden
1994; accepted
21 March
1994)
Abstract It has been suggested that the susceptibility of low density lipoprotein (LDL) to oxidative stress depends on the balance between its contents of polyunsaturated fatty acids and antioxidants. In a healthy reference population (n = 103), the plasma concentration of malondialdehyde (MDA) (mean 0.86, range 0.50- 1.27 ~molil) was positively correlated to the serum concentrations of LDL cholesterol (Y= 0.31, P = O.OOl), veiy low density lipoprotein triglycerides (r = 0.25, P = 0.009) and apolipoprotein B (r = 0.23, P = 0.03), and negatively correlated to lipid corrected alpha tocopherol in serum (r = -0.22, P = 0.02) and lipoprotein(a) (Lp(a)) (r = -0.26, P = 0.01). Plasma MDA was negatively correlated to the content of linoleic acid in the serum lipoprotein phospholipids (r = -0.35, P = 0.0008). In a stepwise regression analysis 12% of the variation in plasma MDA was explained by variations in the content of linoleic acid and 27% after addition of Lp(a) and abdominal sagittal diameter. The significant negative relation between plasma MDA and the amount of linoleic acid in the lipoprotein lipids indicates that other factors, e.g. the availability of antioxidants and the lipoprotein metabolism, may be of greater importance for intravascular lipid peroxidation than the proportion of polyunsaturated fatty acids in the lipoprotein lipids. Keywords;
Malondialdehyde;
Fatty acids; Lipoprotein;
Alpha tocopherol
1. Introduction
Lipid oxidation, radicals,
is considered
* Corresponding 17 79 76.
a process mediated by free to be important for the de-
author.
Tel.: +46 I8 17 77 00; Fax: +46 18
velopment of atherosclerosis [I]. After oxidative modification, low density lipoproteins (LDL) are taken up by a scavenger receptor on the macrophages in the vessel wall, forming fatty streaks, the first step in the formation of the atherosclerotic plaque [1,2]. It has been shown that polyunsaturated fatty acids are most susceptible to oxida-
0021-9150/94/$07.00 0 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI
0021-9150(94)05250-M
104
M. dhrvall et al. /Atherosclerosis
tion, and suggested that the susceptibility of LDL to oxidative stress depends on the balance between its contents of polyunsaturated fatty acids and antioxidants, in particular tocopherol (vitamin E) [3]. A way of estimating the free radical activity is to determine the concentration in plasma of malondialdehyde (MDA) - a product of lipid peroxidation. Previously MDA has usually been measured by spectophotometry or fiuorometry, after reaction with thiobarbituric acid (TBA) [4]. A number of biologically significant substances apart from MDA can form complexes with TBA and interfere in the reaction. To minimise this interference an isocratic high performance liquid chromatographit (HPLC) method using fluorimetric detection can be used [4,5]. The aims of this study were to measure the concentration of MDA in the plasma in a healthy reference population and to relate this to the serum concentration of lipids and of antioxidants such as tocopherol, and also to determine whether the plasma concentration of MDA is related to the quality of the dietary fat as reflected by the serum lipid fatty acid composition. 2. Materials All employees of a Swedish telephone company in Uppsala, Sweden, were invited to participate in a health survey regarding risk factors for coronary heart disease. Of the 1006 employees, 906 (90%) participated and from these subjects 108, who were consecutively examined during a number of randomly chosen days, were sampled for detailed analyses of the tocopherol concentration in the serum and for determination of the concentration of MDA in the plasma. Five subjects were excluded from the study because of concurrent diseases, namely insulin-dependent diabetes mellitus, malignant gynaecologic disease, undergone kidney transplantation, cortisone treated bronchiale asthma and untreated hypothyroidism. Of the remaining 103 subjects, one had non-insulindependent diabetes mellitus, two were pregnant, one was receiving postmenopausal hormonal replacement therapy and three were being treated with beta blockers and/or diuretics.
108 (1994)
103-I IO
The mean age of the 103 subjects (75 males and 28 females) was 42.5 years (23-63 years) and their median age was 43 years. Fourteen subjects were supplementing their diet with vitamin preparations containing alpha tocopheryl acetate (4-10 mg daily). The mean body mass index was 24.9 kg/m2 (19.5-35.7 kg!m2) and significantly higher (P = 0.03) in the men than in the women. The men also had greater abdominal adiposity than the women, as indicated by a higher waist to hip ratio and a greater sagittal abdominal diameter (P < 0.0001). The mean serum concentration of cholesterol was 5.55 mmol/l (3.58-9.5 mmol/l) and of triglycerides 1.17 mmol/l (0.21-3.29 mmol/l), and the concentrations were higher in the men than in the women (P = 0.05 and P = 0.02, respectively). HDL cholesterol was lower (P < 0.0001) in the men. The mean concentration of Lp(a) was 333 U/l (17-1964 U/l) and the PAI- activity was 11.2 U/l (O-37 U/l). 3. Methods In the morning of the examinations vigorous physical activity was not allowed and no smoking was permitted in the hour before. Blood samples were drawn after a 12-h overnight fast. Laboratory tests and all measurements, including body weight, height and blood pressure, were made by the same observer. Supine blood pressure was measured after a 10 min rest (mean of two values at 2-min intervals). The waist and hip circumferences were measured with the subject in the supine position. The abdominal sagittal diameter was recorded at the umbilical level as the height of the abdomen measured from the examination couch. A blood sample was taken from the antecubital vein, protected against light and left to coagulate for 2 h at room temperature. Serum was separated by 10 min of low speed centrifugation and then stored frozen at -70°C prior to serum tocopherol determination, which was performed within a few months. Serum alpha, beta and gamma tocopherol concentrations were assayed by HPLC without adding EDTA, using a fluorescence detector as described earlier [6,7]. The serum tocopherol levels are reported both as total serum concentration and as the
hf. dhrvall et al. /Atherosclerosis 108 (1994) 103-110
concentration corrected for the sum of serum cholesterol and serum triglycerides as suggested by Thurnham et al. [8], who have found the tocopherol:cholesterol + triglyceride ratio to be as useful in identifying vitamin E deficiency as the tocopherol:total lipid ratio. For measurements of MDA an HPLC system was used according to further development of methods described by Young and Trimble [5] and Wong et al. [4]. An HPLC system from Merck Hitachi was used, including a pump and fluorescence detector and a Lichrospher 100 RP-18 (250 x 4 mm) column. Further venous blood samples were collected into dipotassium EDTA and separated within 30 min of collection. A TBA reaction was carried out by mixing 750 ~1 of 0.15 M phosphoric acid, 300 ~1 of water and 250 ~1 of TBA with 200 ~1 plasma. The reaction mixture was incubated in a boiling water bath for 60 min and then cooled in ice. The MDA-TBA complex was mixed with methanol and measured. The fluorescence detector had an excitation wavelength of 532 nm and an emission wavelength of 553 nm and the volume of the injected sample was 20 ~1. The mobile phase contained 200 ml of methanol and 300 ml of 50 mM phosphate buffer. MDA was calibrated with a TEP (1,1,3,3tetraethoxypropan, NR T-9889, Sigma) solution, which had been diluted with ethanol and water to different standard solutions. The coefficient of variation for the method was 10% Very low density lipoprotein (VLDL), LDL and high density lipoprotein (HDL) were isolated by a combination of preparative ultracentrifugation [9] and precipitation with a sodium phosphotungstate and magnesium chloride solution [lo]. Triglyceride and cholesterol concentrations were measured in serum and in the isolated lipoprotein fractions by enzymatic methods, using the IL Test Cholesterol Trinder’s Method 181618-80 and IL Test Enzymatic-calorimetric Method 18 1709-00 for use in a Monarch apparatus (Instrumentation Laboratories, Lexington, MA). The fatty acid composition of the cholesterol esters and of the phospholipids was determined by gas-liquid chromatography as described previously 1111. The concentrations of apolipoprotein B and A-I were determined by turbidimetry in a Multistat III
105
F/LS apparatus, using monospecific polyclonal antibodies against apo B and A-I. The samples were preincubated with triglyceride lipase prior to the assay, as suggested by DaCol and Kostner [ 121. Lipoprotein(a) (Lp(a)) was measured by the Pharmacia Ape(a) RIA method [13]. This is based on the direct sandwich technique in which two monoclonal antibodies are directed against separate antigenie determinants on the apolipoprotein(a) in the sample. The concentration is expressed in units/l (U/l). One U of ape(a) is approximately equal to 0.7 mg Lp(a), according to the manufacturer. Serum insulin was measured by the Phadebas Insulin Test (Pharmacia, Uppsala, Sweden) [ 141. Tissue plasminogen activator inhibitor (PAI-1) activity in plasma was measured with Spectrolyse/pL kits from Biopool AB (Umei, Sweden), using polylysine as a stimulator [ 151. For measurement of tissue plasminogen activator inhibitor (tPA) antigen in plasma, Imulyse 5 t-PA antigen kits (Biopool AB) were used [ 161. 4. Statistics All variables analysed are continuous, on an interval scale and not far from symmetrical distribution. The pair-wise association between variables was examined with the Pearson product-moment correlation. For comparisons of means between factor levels, a one-way analysis of variance was performed (which reduces to Student’s unpaired r-test [17] for factors with two levels). For factors with more than two levels, multiple comparisons were made according to the method of Tukey [18]. For dependent variables with more than one potential independent variable, forward stepwise multiple regression was carried out. The Lp(a) levels had a skewed distribution and were log-transformed before the tests were performed. 5. Results 5. I. Malondialdehyde concentrations The mean concentration of MDA in plasma in all participants was 0.86 pmolil (0.50- 1.27 pmol/l) (Table 1). Men had a significantly higher mean concentration than women (P = 0.0004). Subjects over 40 years of age had a significantly higher mean con-
106
M. iihrvall er al. /Atherosclerosis
Table 1 Concentrations
of lipid-corrected
tocopherol
Alpha tocopherol Beta tocopherol Gamma tocopherol
Malondialdehyde Men (n = 75) Women (n = 28)
(all)
in serumand malondialdehydein
Range (mg/mmol)
S.D.
I.54 0.04 0.20
1.08-2. I2 0.02-0.06 0.07-0.39
0.20 0.01 0.07
(j4mol/l) 0.86 0.89 0.76
(~mol/l) 0.50-1.27 0.52-I .27 0.50-l .06
0.18 0.16 0.16
5.2. Tocopherol concentrations in serum
The concentrations of alpha, beta, and gamma tocopherol are given in Table 1. No significant con-
between
the plasma
concentration
S-chol (mmolil) S-LDL-chol (mmol/l) S-VLDL-TG (mmolil) S-TG (mmol/l) S-Lp(a) (U/l) S-alpha-tocopherol (mg/mmol) Sagittal diameter PAI-I (U/ml) t-PA @g/ml)
(cm)
N.S.. not significant. ‘P < 0.05, (*) P=O.O5;
**p < 0.01; ***p
plasma
Mean (mgimmol)
centration (0.89 pmol/l) than those under 40 years (0.82 pmolll) (P = 0.04 for the difference). There was no difference in the plasma MDA concentration between the 14 participants who were supplementing their diet with vitamin preparations and those who were not. There was no significant difference between cigarette smokers (n = 28) and noncigarette smokers or between tobacco users (including snuff users) (n = 41) and non-tobacco users. Neither was there any difference in the mean plasma concentration of MDA found between subjects with different degree of physical activity.
Table 2 Correlations
I08 (1994) 103-110
of MDA (pmol/l)
centration of delta tocopherol was detected. The concentrations in serum are corrected for the serum lipid concentrations. 5.3. Correlations of the plasma concentration of malondialdehyde to the serum lipoprotein and tocopherol concentrations and to clinical characteristics The concentration of serum cholesterol, LDL cholesterol, serum triglycerides and VLDL triglycerides was positively correlated to the plasma MDA concentration in the whole material (Table 2). Serum apolipoprotein B was positively correlated (r = 0.23, P = 0.03) to plasma MDA. Significant correlations between plasma MDA and serum cholesterol and LDL were found in men but not in women, because of the smaller number. Serum
and clinical characteristics
All n= 103
Men n = 75
Women n = 28
r = +0.32*** r = +0.32** r = +0.26** r = +0.25* r = -0.28** r = -0.22* r = +0.32** r = +0.27** r = +0.33***
r= r= r = r= r = r= r= r = r=
r r r r r r
c 0.001.
+0.26* +0.23(*) +0.17 N.S. +O.l9 N.S. -0.23 N.S. -0.01 N.S. +0.22 N.S. +0.25* +0.21 N.S.
= = = = = =
+0.34 N.S. +0.36 N.S. +0.27 N.S. +0.20 N.S. -0.41; -0.67***
r = +0.23 N.S. r = +0.33 N.S. r = +0.25 N.S.
M. dhrvall et al./Atherosclerosis 108 11994) 103-110
MDA
.
1
MDA
1
1.2
1.2 -
.
.
.
1
.
.. ..
1.1 -
1.1 -
107
+
p=O.O08
.
1.0 0.9 0.8 -
0.8 -
0.7 -
0.7 -
0.6 -
0.6
0.5~ , 16
18
,
,
,
20
22
24
,
,
26
28
.
(
,
30
0.5
f
32
34
’
??
-1 r 17
,.
.
..
=
.
. :
.
,
(
,
18
19
20
.
- .
of MDA
Lp(a) was negatively correlated to plasma MDA when all subjects were analysed together. This negative correlation was stronger in women but was not significant in men. There were no other significant correlations between the plasma concentration of MDA and the serum lipoprotein fractions. Lipid-corrected alpha tocopherol in serum was inversely correlated to the concentration of MDA
Table 3 Correlations between the plasma concentration and cholesterol esters
Phospholipids It?:2 n-6 20:4 n-6 20:5 n-3 Cholesterol l6:l n-7 l8:2 n-6 l8:3 n-6 20:5 n-3
.
. *
,
,
,
,
,
,
,
21
22
23
24
25
26
27
sagittal diameter Fig. 1. Correlations between the plasma concentration (pmolil) and the abdominal sagittal diameter (cm).
. :
18:2 n-6 Fig. 2. Correlations
between the plasma concentration
(pmolll) and the proportion phospholipids (‘XB).
< 0.001
of MDA
of linoleic acid (l8:2 n-6) in the
in plasma in the whole material. This correlation was strong in women but was absent in men. Plasma MDA was positively correlated to weight and all parameters of abdominal adiposity, most strongly to abdominal sagittal diameter (Fig. 1). Age was positively correlated (r = 0.24, P = 0.01) to plasma MDA. The serum activity of PAI- and the serum concentration of tissue plasminogen activa-
of MDA (rmolll) and the relative content (%) of the fatty acids in serum phospholipids
All (n = 103)
Men (n = 75)
Women (n = 28)
r = -0.35*** r = +0.24* r = +0.28**
r = -0.28* r = +0.24 N.S. r = +0.27*
r = -0.41* r = +0.05 r = +0.07
r r r r
r r r r
r r I r
N.S. N.S.
eslers
N.S., not significant. ‘P < 0.05, **p < 0.01. ***p
c 28
= = = =
+0.28** -0.30** +0.29** +0.23*
= = = =
+0.29* -0.27* +0.27* +0.16 N.S.
= = = =
+0.44” -0.40* +0.20 N.S. +O.l3 N.S.
108
hf. ohrvall et al. /Atherosclerosis
tor (t-PA) were positively correlated to plasma MDA. There were no significant correlations between plasma MDA and the serum concentrations of glucose and insulin.
5.4. Correlations between the plasma concentration of malondialdehyde and the fatty acid contents of serum phospholipids and cholesterol esters Serum phospholipids. The expected positive relationship between the concentration of MDA and the proportion of linoleic acid (18:2 n-6) was not found. On the contrary, this fatty acid was inversely correlated to plasma MDA (Fig. 2) and this correlation was also found when men and women were analysed separately (Table 3). Arachidonic acid (20:4 n-6) and eicosapentaenoic acid (20:5 n-3) were positively correlated to MDA in all subjects. Serum cholesterol esters. Also in the cholesterol esters the relative content of linoleic acid was significantly inversely correlated to the plasma concentration of MDA. The contents of palmitoleic acid (16: 1 n-7), gamma-linolenic acid (18:3 n-6) and eicosapentaenoic acid were positively correlated to MDA. There were no significant correlations between the proportion of oleic acid (18: 1) and MDA either in the phospholipids or in the cholesterol esters.
5.5. Regression analysis of the variations in malondialdehyde The relations between the plasma concentration of MDA and the other variables were tested in a multiple regression analysis. The closest correlation was found with the content of linoleic acid in the phospholipids, which explained 12% of the variation in plasma MDA between different subjects. Together, the content of linoleic acid, the serum concentration of Lp(a) and the sagittal diameter explained 27% of the variation in serum MDA. When the women were tested separately in a multiple regression analysis, the concentration of alpha tocopherol explained 42% of the variation in plasma MDA, and together with Lp(a) it explained 52%. In men 10% of the MDA variation was explained by the proportion of palmitoleic acid in the cholesterol esters.
108 (1994) 103-110
6. Discussion
The concentration of MDA in plasma was measured by an isocratic HPLC method using fluorimetric detection, essentially as described previously [4,5]. This method is simple and achieves excellent separation of the MDA-TBA adduct from contaminants. The mean concentration and range of plasma MDA were similar to those reported from other studies. Among 41 healthy persons of the same age, Wong et al. found somewhat lower levels (mean 0.60, range 0.39-0.90 pmol/l) and no significant sexrelated difference [4]. The mean plasma concentration in the study by Young and Trimble among 30 healthy subjects was 0.59 pmol/l[5]. The higher concentrations ‘of MDA in the men of our study can possibly be explained by higher lipid concentrations. High concentrations of lipids, particularly of LDL, may prolong the intravascular circulation and exposure time as a result of down-regulation of the lipoprotein receptors and thus increase the tendency to oxidative modification. Similar effects can be expected in lipoprotein disorders, where an altered composition of the lipoprotein particles may result in a reduced affinity for the B,E receptors, e.g. due to an elevated triglyceride level in patients with hypertriglyceridaemia [ 191.This is compatible with the positive correlations between the plasma concentration of MDA and the serum concentrations of LDL cholesterol and VLDL triglycerides seen in this study. The correlation between plasma MDA and serum Lp(a), which is inverse, does not follow the same pattern as the correlations between MDA and the other lipoproteins. The mechanisms underlying the regulation of the serum level of Lp(a) are poorly understood. Diets rich in monounsaturated fatty acids yield significantly higher concentrations of Lp(a) than those containing more saturated fatty acids [20], which on the other hand tend to increase the LDL cholesterol and apo B concentrations. This may partly explain the inverse correlation, in this study, between MDA and Lp(a) in contrast to the positive correlation between MDA and LDL cholesterol. Lp(a) shows a retarded Cu*+-mediated oxidation with a longer lag phase than LDL in spite of a lower
M. dhrvall et al. /Atherosclerosis
content of alpha tocopherol in Lp(a) [21]. Despite the atherogenic properties of Lp(a), Rath and Pauling have suggested that ape(a) is effective as an antioxidant when it acts as a surrogate for ascorbate and may prevent peroxidation of lipids in Lp(a) [22]. They also suggest that ascorbate deficiency increases plasma Lp(a). The inverse relationship between Lp(a) and MDA in this study could possibly be related to the suggested antioxidative capacity of ape(a). One of the most powerful antioxidants in the serum is alpha tocopherol. In this study we found an inverse correlation between plasma MDA and serum alpha tocopherol in women. We have previously observed an inverse correlation between the serum concentration of lipid-corrected alpha tocopherol and the serum concentrations of cholesterol and triglycerides [6]. The consumption of tocopherol may increase with increasing serum lipid levels, leading to higher plasma MDA. Another possible explanation is that subjects with hyperlipoproteinaemia may have a lower intake of tocopherol in their diet and hence poorer antioxidative protection. The composition of fatty acids in the serum mirrors the content of fatty acids in the diet. There were significant inverse correlations between the concentration of MDA in the plasma and the relative content of linoleic acid, the most important polyunsaturated fatty acid in the diet, in both the serum cholesterol esters and the phospholipids. This indicates that subjects on a diet rich in linoleic acid, which is also rich in antioxidants such as alpha tocopherol [23], have a capacity for protection of these very susceptible fatty acids from oxidation. Studies in vitro have shown a lower degree of oxidation of monounsaturated than of polyunsaturated fatty acids [3]. Although polyunsaturated fatty acids have an increased susceptibility to non-enzymatic oxidation in vitro, it seems that the amount of antioxidants in vivo is great enough to protect these fatty acids from oxidation. The data from the present study do not seem to contradict established dietary advice concerning the advantages of a moderately increased intake of polyunsaturated fatty acids (up to 10 energy %) to improve the lipid concentrations in the serum. We have previously found an inverse correlation
108 (1994)
103-110
109
between parameters for abdominal adiposity and the serum concentration of alpha tocopherol [6]. In the present study we observed a positive correlation between abdominal adiposity and the plasma concentration of MDA. In the multiple regression analysis the sagittal abdominal diameter was the most explanatory clinical parameter, suggesting that abdominal adiposity is associated with an increased tendency to oxidation of lipoproteins and an increased risk of development of atherosclerosis and coronary heart disease. In conclusion, we found that the amount of products of oxidative damage, measured as MDA, increases with increasing blood lipids and decreasing concentrations of antioxidants such as alpha tocopherol. That patients with hyperlipoproteinaemia, with an increased risk for coronary heart disease, have elevated levels of MDA in the plasma is compatible with the suggestion that oxidated lipoproteins are of importance for the occurrence of atherosclerosis [ 11. The significant negative relationship between the plasma MDA concentration and the amount of linoleic acid in the lipoprotein lipids indicates that other factors, e.g. the availability of antioxidants and lipoprotein metabolism, may be of greater importance for the risk of intravascular lipid peroxidation than the lipoprotein content of polyunsaturated fatty acids. References Steinberg, D. and Witztum, J., Lipoproteins and atherogenesis, J. Am. Med. Assoc., 264 (1990) 3047. Aviram. M.. Modified forms of low density lipoprotein and atherosclerosis, Atherosclerosis, 98 (1993) I. Esterbauer, H.. Puhl, H., Dieber-Rotheneder, M., Waeg, G. and Rabl, H., Effect of antioxidants on oxidative modification of LDL, Ann. Med., 23 (1991) 573. Wong, S., Knight, J.. Hopfer, S.. Zaharia. O., Leach. C.. and Sunderman, W., Lipid peroxides in plasma as measured by liquid-chromatographic separation of malondialdehyde-thiobarbituric acid adduct, Clin. Chem., 33 (2) (1987) 214. Young, I.S. and Trimble, E.R., Measurement of malondialdehyde in plasma by high performance liquid chromatography with fluorimetric detection, Ann. Clin. Biochem., 28 (1991) 504. ijhrvall. M., Tengblad, S. and Vessby, B., Lower tocopherol serum levels in subjects with abdominal adiposity, J. Int. Med.. 234 (1993) 53. Driskell, W.J.. Measurement of vitamin A and vitamin E
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M. dhrvall et al. /Atherosclerosis
in human serum by high-performance liquid chromatography, J. Chromatogr., 231 (1982) 439. 8 Thurnham. D.I., Davies, J.A., Crump, B.J., Situnayake, R.D. and Davis, M., The use of different lipids to express serum tocopheroklipid ratios for the measurements of vitamin E status, Ann. Clin. Biochem., 23 (1986) 514. 9 Havel, R.J., Eder, H.A. and Bragdon. J.H., The determination and chemical composition of ultracentrifugally separated lipoproteins in human serum, J. Clin. Invest., 34 (1955) 1345. IO Seigler, L. and Wu, W.T., Separation of serum highdensity lipoprotein for cholesterol determination: ultracentrifugation vs. precipitation with sodium phosphotungstate and magnesium chloride, Clin. Chem.. 27 (198 I) 838. II Boberg, M., Croon. L.-B., Gustafsson, L-B. and Vessby, B., Platelet fatty acid composition in relation to fatty acid composition in plasma and to serum lipoprotein lipids in healthy subjects with special reference to the linoleic acid pathway, Clin. Sci., 68 (1985) 581. 12 DaCol, P. and Kostner, GM.. Immunoquantitication of total apolipoprotein B in serum by nephelometry: influence of lipase treatment and detergents, Clin. Chem., 29 (1983) 1045. 13 Pharmacia Diagnostics AB. Uppsala, Sweden, 1985, Pharmacia (a) RIA. 14 Wide, L., Axen, R. and Porath, J., Radioimmunosorbent assay of proteins, chemical couplings of antibodies to insoluble dextran, Immunochemistry, 4 (1967) 38 1.
I5
108 (1994)
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Eriksson, E., Rinby, M., Gyzander. E. and Risberg. B., Determination of plasminogen activator inhibitor in plasma using t-PA and a chromogenic single point poly-Dlysine stimulated assay, Thromb. Res.. 50 (1988) 90. 16 R&rby, M., Bergsdorf, N.. Nilsson, T.. Mellbring, G., Winblad, B. and Bucht G., Age dependence of tissue plasminogen activator concentrations in plasma, as studied by an improved enzyme-linked immunosorbent assay, Clin. Chem.. 32 (12) (1986) 2160. 17 Brownlee, K.A., Statistical Theory and Methodology in Science and Engineering, Wilney Publications, USA, 1965, pp. 297-305. I8 Tukey, J.W.. Exploratory Data Analysis. AddisonWesley. Reading, MA, 1977. I9 Hiramatsu, K., Bierman, E.L. and Chait, A., Metabolism of low density lipoprotein from patients with diabetic hypertriglyceridemia by cultured human skin fibroblasts, Diabetes, 34 (1985) 8. 20 Mensink, R.. Zock, P., Katan, M. and Hornstra. G.. Effect of dietary cis and trans fatty acids on serum lipoprotein(a) levels in humans, J. Lipid Res., 33 (1992) 1493. 21 Sattler, W., Kostner, G.M., Waeg, G. and Esterbauer, H., Oxidation of lipoprotein Lp(a). A comparison with lowdensity lipoproteins, Biochim. Biophys. Acta, 1081 (I) (1991) 65. 22 Rath, M. and Pauling, L., Hypothesis: lipoprotein (a) is a surrogate for ascorbate, Proc. Natl. Acad. Sci. USA, 87 ( 1990) 6204. 23 Horwitt, M.K.. Status of human requirements for vitamin E, Am. J. Clin. Nutr., 27 (1974) 1182.
ELSEVIER
Malondialdehyde concentration in plasma is inversely correlated to the proportion of linoleic acid in serum lipoprotein lipids ohrvall* a, Siv Tengblad”, Bo Ekstrandb, Bengt Vessbya
Margareta
“Department
of Geriatrics,
‘Department
of Clinical
02
Box 2151. S-750 bSwedish Institute
(Received
21 September
,fbr Food Reseurch,
1993; revision
Chemistry,
Uppsalu.
Agneta
Universi/,v
Siegbahn’,
qf Uppsaiu.
Sweden
Gothenhurg,
received 28 February
Siveden
1994; accepted
21 March
1994)
Abstract It has been suggested that the susceptibility of low density lipoprotein (LDL) to oxidative stress depends on the balance between its contents of polyunsaturated fatty acids and antioxidants. In a healthy reference population (n = 103), the plasma concentration of malondialdehyde (MDA) (mean 0.86, range 0.50- 1.27 ~molil) was positively correlated to the serum concentrations of LDL cholesterol (Y= 0.31, P = O.OOl), veiy low density lipoprotein triglycerides (r = 0.25, P = 0.009) and apolipoprotein B (r = 0.23, P = 0.03), and negatively correlated to lipid corrected alpha tocopherol in serum (r = -0.22, P = 0.02) and lipoprotein(a) (Lp(a)) (r = -0.26, P = 0.01). Plasma MDA was negatively correlated to the content of linoleic acid in the serum lipoprotein phospholipids (r = -0.35, P = 0.0008). In a stepwise regression analysis 12% of the variation in plasma MDA was explained by variations in the content of linoleic acid and 27% after addition of Lp(a) and abdominal sagittal diameter. The significant negative relation between plasma MDA and the amount of linoleic acid in the lipoprotein lipids indicates that other factors, e.g. the availability of antioxidants and the lipoprotein metabolism, may be of greater importance for intravascular lipid peroxidation than the proportion of polyunsaturated fatty acids in the lipoprotein lipids. Keywords;
Malondialdehyde;
Fatty acids; Lipoprotein;
Alpha tocopherol
1. Introduction
Lipid oxidation, radicals,
is considered
* Corresponding 17 79 76.
a process mediated by free to be important for the de-
author.
Tel.: +46 I8 17 77 00; Fax: +46 18
velopment of atherosclerosis [I]. After oxidative modification, low density lipoproteins (LDL) are taken up by a scavenger receptor on the macrophages in the vessel wall, forming fatty streaks, the first step in the formation of the atherosclerotic plaque [1,2]. It has been shown that polyunsaturated fatty acids are most susceptible to oxida-
0021-9150/94/$07.00 0 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI
0021-9150(94)05250-M
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M. dhrvall et al. /Atherosclerosis
tion, and suggested that the susceptibility of LDL to oxidative stress depends on the balance between its contents of polyunsaturated fatty acids and antioxidants, in particular tocopherol (vitamin E) [3]. A way of estimating the free radical activity is to determine the concentration in plasma of malondialdehyde (MDA) - a product of lipid peroxidation. Previously MDA has usually been measured by spectophotometry or fiuorometry, after reaction with thiobarbituric acid (TBA) [4]. A number of biologically significant substances apart from MDA can form complexes with TBA and interfere in the reaction. To minimise this interference an isocratic high performance liquid chromatographit (HPLC) method using fluorimetric detection can be used [4,5]. The aims of this study were to measure the concentration of MDA in the plasma in a healthy reference population and to relate this to the serum concentration of lipids and of antioxidants such as tocopherol, and also to determine whether the plasma concentration of MDA is related to the quality of the dietary fat as reflected by the serum lipid fatty acid composition. 2. Materials All employees of a Swedish telephone company in Uppsala, Sweden, were invited to participate in a health survey regarding risk factors for coronary heart disease. Of the 1006 employees, 906 (90%) participated and from these subjects 108, who were consecutively examined during a number of randomly chosen days, were sampled for detailed analyses of the tocopherol concentration in the serum and for determination of the concentration of MDA in the plasma. Five subjects were excluded from the study because of concurrent diseases, namely insulin-dependent diabetes mellitus, malignant gynaecologic disease, undergone kidney transplantation, cortisone treated bronchiale asthma and untreated hypothyroidism. Of the remaining 103 subjects, one had non-insulindependent diabetes mellitus, two were pregnant, one was receiving postmenopausal hormonal replacement therapy and three were being treated with beta blockers and/or diuretics.
108 (1994)
103-I IO
The mean age of the 103 subjects (75 males and 28 females) was 42.5 years (23-63 years) and their median age was 43 years. Fourteen subjects were supplementing their diet with vitamin preparations containing alpha tocopheryl acetate (4-10 mg daily). The mean body mass index was 24.9 kg/m2 (19.5-35.7 kg!m2) and significantly higher (P = 0.03) in the men than in the women. The men also had greater abdominal adiposity than the women, as indicated by a higher waist to hip ratio and a greater sagittal abdominal diameter (P < 0.0001). The mean serum concentration of cholesterol was 5.55 mmol/l (3.58-9.5 mmol/l) and of triglycerides 1.17 mmol/l (0.21-3.29 mmol/l), and the concentrations were higher in the men than in the women (P = 0.05 and P = 0.02, respectively). HDL cholesterol was lower (P < 0.0001) in the men. The mean concentration of Lp(a) was 333 U/l (17-1964 U/l) and the PAI- activity was 11.2 U/l (O-37 U/l). 3. Methods In the morning of the examinations vigorous physical activity was not allowed and no smoking was permitted in the hour before. Blood samples were drawn after a 12-h overnight fast. Laboratory tests and all measurements, including body weight, height and blood pressure, were made by the same observer. Supine blood pressure was measured after a 10 min rest (mean of two values at 2-min intervals). The waist and hip circumferences were measured with the subject in the supine position. The abdominal sagittal diameter was recorded at the umbilical level as the height of the abdomen measured from the examination couch. A blood sample was taken from the antecubital vein, protected against light and left to coagulate for 2 h at room temperature. Serum was separated by 10 min of low speed centrifugation and then stored frozen at -70°C prior to serum tocopherol determination, which was performed within a few months. Serum alpha, beta and gamma tocopherol concentrations were assayed by HPLC without adding EDTA, using a fluorescence detector as described earlier [6,7]. The serum tocopherol levels are reported both as total serum concentration and as the
hf. dhrvall et al. /Atherosclerosis 108 (1994) 103-110
concentration corrected for the sum of serum cholesterol and serum triglycerides as suggested by Thurnham et al. [8], who have found the tocopherol:cholesterol + triglyceride ratio to be as useful in identifying vitamin E deficiency as the tocopherol:total lipid ratio. For measurements of MDA an HPLC system was used according to further development of methods described by Young and Trimble [5] and Wong et al. [4]. An HPLC system from Merck Hitachi was used, including a pump and fluorescence detector and a Lichrospher 100 RP-18 (250 x 4 mm) column. Further venous blood samples were collected into dipotassium EDTA and separated within 30 min of collection. A TBA reaction was carried out by mixing 750 ~1 of 0.15 M phosphoric acid, 300 ~1 of water and 250 ~1 of TBA with 200 ~1 plasma. The reaction mixture was incubated in a boiling water bath for 60 min and then cooled in ice. The MDA-TBA complex was mixed with methanol and measured. The fluorescence detector had an excitation wavelength of 532 nm and an emission wavelength of 553 nm and the volume of the injected sample was 20 ~1. The mobile phase contained 200 ml of methanol and 300 ml of 50 mM phosphate buffer. MDA was calibrated with a TEP (1,1,3,3tetraethoxypropan, NR T-9889, Sigma) solution, which had been diluted with ethanol and water to different standard solutions. The coefficient of variation for the method was 10% Very low density lipoprotein (VLDL), LDL and high density lipoprotein (HDL) were isolated by a combination of preparative ultracentrifugation [9] and precipitation with a sodium phosphotungstate and magnesium chloride solution [lo]. Triglyceride and cholesterol concentrations were measured in serum and in the isolated lipoprotein fractions by enzymatic methods, using the IL Test Cholesterol Trinder’s Method 181618-80 and IL Test Enzymatic-calorimetric Method 18 1709-00 for use in a Monarch apparatus (Instrumentation Laboratories, Lexington, MA). The fatty acid composition of the cholesterol esters and of the phospholipids was determined by gas-liquid chromatography as described previously 1111. The concentrations of apolipoprotein B and A-I were determined by turbidimetry in a Multistat III
105
F/LS apparatus, using monospecific polyclonal antibodies against apo B and A-I. The samples were preincubated with triglyceride lipase prior to the assay, as suggested by DaCol and Kostner [ 121. Lipoprotein(a) (Lp(a)) was measured by the Pharmacia Ape(a) RIA method [13]. This is based on the direct sandwich technique in which two monoclonal antibodies are directed against separate antigenie determinants on the apolipoprotein(a) in the sample. The concentration is expressed in units/l (U/l). One U of ape(a) is approximately equal to 0.7 mg Lp(a), according to the manufacturer. Serum insulin was measured by the Phadebas Insulin Test (Pharmacia, Uppsala, Sweden) [ 141. Tissue plasminogen activator inhibitor (PAI-1) activity in plasma was measured with Spectrolyse/pL kits from Biopool AB (Umei, Sweden), using polylysine as a stimulator [ 151. For measurement of tissue plasminogen activator inhibitor (tPA) antigen in plasma, Imulyse 5 t-PA antigen kits (Biopool AB) were used [ 161. 4. Statistics All variables analysed are continuous, on an interval scale and not far from symmetrical distribution. The pair-wise association between variables was examined with the Pearson product-moment correlation. For comparisons of means between factor levels, a one-way analysis of variance was performed (which reduces to Student’s unpaired r-test [17] for factors with two levels). For factors with more than two levels, multiple comparisons were made according to the method of Tukey [18]. For dependent variables with more than one potential independent variable, forward stepwise multiple regression was carried out. The Lp(a) levels had a skewed distribution and were log-transformed before the tests were performed. 5. Results 5. I. Malondialdehyde concentrations The mean concentration of MDA in plasma in all participants was 0.86 pmolil (0.50- 1.27 pmol/l) (Table 1). Men had a significantly higher mean concentration than women (P = 0.0004). Subjects over 40 years of age had a significantly higher mean con-
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M. iihrvall er al. /Atherosclerosis
Table 1 Concentrations
of lipid-corrected
tocopherol
Alpha tocopherol Beta tocopherol Gamma tocopherol
Malondialdehyde Men (n = 75) Women (n = 28)
(all)
in serumand malondialdehydein
Range (mg/mmol)
S.D.
I.54 0.04 0.20
1.08-2. I2 0.02-0.06 0.07-0.39
0.20 0.01 0.07
(j4mol/l) 0.86 0.89 0.76
(~mol/l) 0.50-1.27 0.52-I .27 0.50-l .06
0.18 0.16 0.16
5.2. Tocopherol concentrations in serum
The concentrations of alpha, beta, and gamma tocopherol are given in Table 1. No significant con-
between
the plasma
concentration
S-chol (mmolil) S-LDL-chol (mmol/l) S-VLDL-TG (mmolil) S-TG (mmol/l) S-Lp(a) (U/l) S-alpha-tocopherol (mg/mmol) Sagittal diameter PAI-I (U/ml) t-PA @g/ml)
(cm)
N.S.. not significant. ‘P < 0.05, (*) P=O.O5;
**p < 0.01; ***p
plasma
Mean (mgimmol)
centration (0.89 pmol/l) than those under 40 years (0.82 pmolll) (P = 0.04 for the difference). There was no difference in the plasma MDA concentration between the 14 participants who were supplementing their diet with vitamin preparations and those who were not. There was no significant difference between cigarette smokers (n = 28) and noncigarette smokers or between tobacco users (including snuff users) (n = 41) and non-tobacco users. Neither was there any difference in the mean plasma concentration of MDA found between subjects with different degree of physical activity.
Table 2 Correlations
I08 (1994) 103-110
of MDA (pmol/l)
centration of delta tocopherol was detected. The concentrations in serum are corrected for the serum lipid concentrations. 5.3. Correlations of the plasma concentration of malondialdehyde to the serum lipoprotein and tocopherol concentrations and to clinical characteristics The concentration of serum cholesterol, LDL cholesterol, serum triglycerides and VLDL triglycerides was positively correlated to the plasma MDA concentration in the whole material (Table 2). Serum apolipoprotein B was positively correlated (r = 0.23, P = 0.03) to plasma MDA. Significant correlations between plasma MDA and serum cholesterol and LDL were found in men but not in women, because of the smaller number. Serum
and clinical characteristics
All n= 103
Men n = 75
Women n = 28
r = +0.32*** r = +0.32** r = +0.26** r = +0.25* r = -0.28** r = -0.22* r = +0.32** r = +0.27** r = +0.33***
r= r= r = r= r = r= r= r = r=
r r r r r r
c 0.001.
+0.26* +0.23(*) +0.17 N.S. +O.l9 N.S. -0.23 N.S. -0.01 N.S. +0.22 N.S. +0.25* +0.21 N.S.
= = = = = =
+0.34 N.S. +0.36 N.S. +0.27 N.S. +0.20 N.S. -0.41; -0.67***
r = +0.23 N.S. r = +0.33 N.S. r = +0.25 N.S.
M. dhrvall et al./Atherosclerosis 108 11994) 103-110
MDA
.
1
MDA
1
1.2
1.2 -
.
.
.
1
.
.. ..
1.1 -
1.1 -
107
+
p=O.O08
.
1.0 0.9 0.8 -
0.8 -
0.7 -
0.7 -
0.6 -
0.6
0.5~ , 16
18
,
,
,
20
22
24
,
,
26
28
.
(
,
30
0.5
f
32
34
’
??
-1 r 17
,.
.
..
=
.
. :
.
,
(
,
18
19
20
.
- .
of MDA
Lp(a) was negatively correlated to plasma MDA when all subjects were analysed together. This negative correlation was stronger in women but was not significant in men. There were no other significant correlations between the plasma concentration of MDA and the serum lipoprotein fractions. Lipid-corrected alpha tocopherol in serum was inversely correlated to the concentration of MDA
Table 3 Correlations between the plasma concentration and cholesterol esters
Phospholipids It?:2 n-6 20:4 n-6 20:5 n-3 Cholesterol l6:l n-7 l8:2 n-6 l8:3 n-6 20:5 n-3
.
. *
,
,
,
,
,
,
,
21
22
23
24
25
26
27
sagittal diameter Fig. 1. Correlations between the plasma concentration (pmolil) and the abdominal sagittal diameter (cm).
. :
18:2 n-6 Fig. 2. Correlations
between the plasma concentration
(pmolll) and the proportion phospholipids (‘XB).
< 0.001
of MDA
of linoleic acid (l8:2 n-6) in the
in plasma in the whole material. This correlation was strong in women but was absent in men. Plasma MDA was positively correlated to weight and all parameters of abdominal adiposity, most strongly to abdominal sagittal diameter (Fig. 1). Age was positively correlated (r = 0.24, P = 0.01) to plasma MDA. The serum activity of PAI- and the serum concentration of tissue plasminogen activa-
of MDA (rmolll) and the relative content (%) of the fatty acids in serum phospholipids
All (n = 103)
Men (n = 75)
Women (n = 28)
r = -0.35*** r = +0.24* r = +0.28**
r = -0.28* r = +0.24 N.S. r = +0.27*
r = -0.41* r = +0.05 r = +0.07
r r r r
r r r r
r r I r
N.S. N.S.
eslers
N.S., not significant. ‘P < 0.05, **p < 0.01. ***p
c 28
= = = =
+0.28** -0.30** +0.29** +0.23*
= = = =
+0.29* -0.27* +0.27* +0.16 N.S.
= = = =
+0.44” -0.40* +0.20 N.S. +O.l3 N.S.
108
hf. ohrvall et al. /Atherosclerosis
tor (t-PA) were positively correlated to plasma MDA. There were no significant correlations between plasma MDA and the serum concentrations of glucose and insulin.
5.4. Correlations between the plasma concentration of malondialdehyde and the fatty acid contents of serum phospholipids and cholesterol esters Serum phospholipids. The expected positive relationship between the concentration of MDA and the proportion of linoleic acid (18:2 n-6) was not found. On the contrary, this fatty acid was inversely correlated to plasma MDA (Fig. 2) and this correlation was also found when men and women were analysed separately (Table 3). Arachidonic acid (20:4 n-6) and eicosapentaenoic acid (20:5 n-3) were positively correlated to MDA in all subjects. Serum cholesterol esters. Also in the cholesterol esters the relative content of linoleic acid was significantly inversely correlated to the plasma concentration of MDA. The contents of palmitoleic acid (16: 1 n-7), gamma-linolenic acid (18:3 n-6) and eicosapentaenoic acid were positively correlated to MDA. There were no significant correlations between the proportion of oleic acid (18: 1) and MDA either in the phospholipids or in the cholesterol esters.
5.5. Regression analysis of the variations in malondialdehyde The relations between the plasma concentration of MDA and the other variables were tested in a multiple regression analysis. The closest correlation was found with the content of linoleic acid in the phospholipids, which explained 12% of the variation in plasma MDA between different subjects. Together, the content of linoleic acid, the serum concentration of Lp(a) and the sagittal diameter explained 27% of the variation in serum MDA. When the women were tested separately in a multiple regression analysis, the concentration of alpha tocopherol explained 42% of the variation in plasma MDA, and together with Lp(a) it explained 52%. In men 10% of the MDA variation was explained by the proportion of palmitoleic acid in the cholesterol esters.
108 (1994) 103-110
6. Discussion
The concentration of MDA in plasma was measured by an isocratic HPLC method using fluorimetric detection, essentially as described previously [4,5]. This method is simple and achieves excellent separation of the MDA-TBA adduct from contaminants. The mean concentration and range of plasma MDA were similar to those reported from other studies. Among 41 healthy persons of the same age, Wong et al. found somewhat lower levels (mean 0.60, range 0.39-0.90 pmol/l) and no significant sexrelated difference [4]. The mean plasma concentration in the study by Young and Trimble among 30 healthy subjects was 0.59 pmol/l[5]. The higher concentrations ‘of MDA in the men of our study can possibly be explained by higher lipid concentrations. High concentrations of lipids, particularly of LDL, may prolong the intravascular circulation and exposure time as a result of down-regulation of the lipoprotein receptors and thus increase the tendency to oxidative modification. Similar effects can be expected in lipoprotein disorders, where an altered composition of the lipoprotein particles may result in a reduced affinity for the B,E receptors, e.g. due to an elevated triglyceride level in patients with hypertriglyceridaemia [ 191.This is compatible with the positive correlations between the plasma concentration of MDA and the serum concentrations of LDL cholesterol and VLDL triglycerides seen in this study. The correlation between plasma MDA and serum Lp(a), which is inverse, does not follow the same pattern as the correlations between MDA and the other lipoproteins. The mechanisms underlying the regulation of the serum level of Lp(a) are poorly understood. Diets rich in monounsaturated fatty acids yield significantly higher concentrations of Lp(a) than those containing more saturated fatty acids [20], which on the other hand tend to increase the LDL cholesterol and apo B concentrations. This may partly explain the inverse correlation, in this study, between MDA and Lp(a) in contrast to the positive correlation between MDA and LDL cholesterol. Lp(a) shows a retarded Cu*+-mediated oxidation with a longer lag phase than LDL in spite of a lower
M. dhrvall et al. /Atherosclerosis
content of alpha tocopherol in Lp(a) [21]. Despite the atherogenic properties of Lp(a), Rath and Pauling have suggested that ape(a) is effective as an antioxidant when it acts as a surrogate for ascorbate and may prevent peroxidation of lipids in Lp(a) [22]. They also suggest that ascorbate deficiency increases plasma Lp(a). The inverse relationship between Lp(a) and MDA in this study could possibly be related to the suggested antioxidative capacity of ape(a). One of the most powerful antioxidants in the serum is alpha tocopherol. In this study we found an inverse correlation between plasma MDA and serum alpha tocopherol in women. We have previously observed an inverse correlation between the serum concentration of lipid-corrected alpha tocopherol and the serum concentrations of cholesterol and triglycerides [6]. The consumption of tocopherol may increase with increasing serum lipid levels, leading to higher plasma MDA. Another possible explanation is that subjects with hyperlipoproteinaemia may have a lower intake of tocopherol in their diet and hence poorer antioxidative protection. The composition of fatty acids in the serum mirrors the content of fatty acids in the diet. There were significant inverse correlations between the concentration of MDA in the plasma and the relative content of linoleic acid, the most important polyunsaturated fatty acid in the diet, in both the serum cholesterol esters and the phospholipids. This indicates that subjects on a diet rich in linoleic acid, which is also rich in antioxidants such as alpha tocopherol [23], have a capacity for protection of these very susceptible fatty acids from oxidation. Studies in vitro have shown a lower degree of oxidation of monounsaturated than of polyunsaturated fatty acids [3]. Although polyunsaturated fatty acids have an increased susceptibility to non-enzymatic oxidation in vitro, it seems that the amount of antioxidants in vivo is great enough to protect these fatty acids from oxidation. The data from the present study do not seem to contradict established dietary advice concerning the advantages of a moderately increased intake of polyunsaturated fatty acids (up to 10 energy %) to improve the lipid concentrations in the serum. We have previously found an inverse correlation
108 (1994)
103-110
109
between parameters for abdominal adiposity and the serum concentration of alpha tocopherol [6]. In the present study we observed a positive correlation between abdominal adiposity and the plasma concentration of MDA. In the multiple regression analysis the sagittal abdominal diameter was the most explanatory clinical parameter, suggesting that abdominal adiposity is associated with an increased tendency to oxidation of lipoproteins and an increased risk of development of atherosclerosis and coronary heart disease. In conclusion, we found that the amount of products of oxidative damage, measured as MDA, increases with increasing blood lipids and decreasing concentrations of antioxidants such as alpha tocopherol. That patients with hyperlipoproteinaemia, with an increased risk for coronary heart disease, have elevated levels of MDA in the plasma is compatible with the suggestion that oxidated lipoproteins are of importance for the occurrence of atherosclerosis [ 11. The significant negative relationship between the plasma MDA concentration and the amount of linoleic acid in the lipoprotein lipids indicates that other factors, e.g. the availability of antioxidants and lipoprotein metabolism, may be of greater importance for the risk of intravascular lipid peroxidation than the lipoprotein content of polyunsaturated fatty acids. References Steinberg, D. and Witztum, J., Lipoproteins and atherogenesis, J. Am. Med. Assoc., 264 (1990) 3047. Aviram. M.. Modified forms of low density lipoprotein and atherosclerosis, Atherosclerosis, 98 (1993) I. Esterbauer, H.. Puhl, H., Dieber-Rotheneder, M., Waeg, G. and Rabl, H., Effect of antioxidants on oxidative modification of LDL, Ann. Med., 23 (1991) 573. Wong, S., Knight, J.. Hopfer, S.. Zaharia. O., Leach. C.. and Sunderman, W., Lipid peroxides in plasma as measured by liquid-chromatographic separation of malondialdehyde-thiobarbituric acid adduct, Clin. Chem., 33 (2) (1987) 214. Young, I.S. and Trimble, E.R., Measurement of malondialdehyde in plasma by high performance liquid chromatography with fluorimetric detection, Ann. Clin. Biochem., 28 (1991) 504. ijhrvall. M., Tengblad, S. and Vessby, B., Lower tocopherol serum levels in subjects with abdominal adiposity, J. Int. Med.. 234 (1993) 53. Driskell, W.J.. Measurement of vitamin A and vitamin E
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M. dhrvall et al. /Atherosclerosis
in human serum by high-performance liquid chromatography, J. Chromatogr., 231 (1982) 439. 8 Thurnham. D.I., Davies, J.A., Crump, B.J., Situnayake, R.D. and Davis, M., The use of different lipids to express serum tocopheroklipid ratios for the measurements of vitamin E status, Ann. Clin. Biochem., 23 (1986) 514. 9 Havel, R.J., Eder, H.A. and Bragdon. J.H., The determination and chemical composition of ultracentrifugally separated lipoproteins in human serum, J. Clin. Invest., 34 (1955) 1345. IO Seigler, L. and Wu, W.T., Separation of serum highdensity lipoprotein for cholesterol determination: ultracentrifugation vs. precipitation with sodium phosphotungstate and magnesium chloride, Clin. Chem.. 27 (198 I) 838. II Boberg, M., Croon. L.-B., Gustafsson, L-B. and Vessby, B., Platelet fatty acid composition in relation to fatty acid composition in plasma and to serum lipoprotein lipids in healthy subjects with special reference to the linoleic acid pathway, Clin. Sci., 68 (1985) 581. 12 DaCol, P. and Kostner, GM.. Immunoquantitication of total apolipoprotein B in serum by nephelometry: influence of lipase treatment and detergents, Clin. Chem., 29 (1983) 1045. 13 Pharmacia Diagnostics AB. Uppsala, Sweden, 1985, Pharmacia (a) RIA. 14 Wide, L., Axen, R. and Porath, J., Radioimmunosorbent assay of proteins, chemical couplings of antibodies to insoluble dextran, Immunochemistry, 4 (1967) 38 1.
I5
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103-110
Eriksson, E., Rinby, M., Gyzander. E. and Risberg. B., Determination of plasminogen activator inhibitor in plasma using t-PA and a chromogenic single point poly-Dlysine stimulated assay, Thromb. Res.. 50 (1988) 90. 16 R&rby, M., Bergsdorf, N.. Nilsson, T.. Mellbring, G., Winblad, B. and Bucht G., Age dependence of tissue plasminogen activator concentrations in plasma, as studied by an improved enzyme-linked immunosorbent assay, Clin. Chem.. 32 (12) (1986) 2160. 17 Brownlee, K.A., Statistical Theory and Methodology in Science and Engineering, Wilney Publications, USA, 1965, pp. 297-305. I8 Tukey, J.W.. Exploratory Data Analysis. AddisonWesley. Reading, MA, 1977. I9 Hiramatsu, K., Bierman, E.L. and Chait, A., Metabolism of low density lipoprotein from patients with diabetic hypertriglyceridemia by cultured human skin fibroblasts, Diabetes, 34 (1985) 8. 20 Mensink, R.. Zock, P., Katan, M. and Hornstra. G.. Effect of dietary cis and trans fatty acids on serum lipoprotein(a) levels in humans, J. Lipid Res., 33 (1992) 1493. 21 Sattler, W., Kostner, G.M., Waeg, G. and Esterbauer, H., Oxidation of lipoprotein Lp(a). A comparison with lowdensity lipoproteins, Biochim. Biophys. Acta, 1081 (I) (1991) 65. 22 Rath, M. and Pauling, L., Hypothesis: lipoprotein (a) is a surrogate for ascorbate, Proc. Natl. Acad. Sci. USA, 87 ( 1990) 6204. 23 Horwitt, M.K.. Status of human requirements for vitamin E, Am. J. Clin. Nutr., 27 (1974) 1182.