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Variations in the fatty acid composition of lipid classes from lipoproteins in elderly women
Atherosclerosis, 98 ( 1993) 24 l-249 0 1993 Elsevier Scientific Publishers Printed and Published in Ireland
ATHERO
241 Ireland,
Ltd. All rights reserved.
0021-9150193/$06.00
04964
Variations
in the fatty acid composition of lipid classes from lipoproteins in elderly women
J. Lecerfa, A. Rossignolb, E. VCricel”, F. Thk”,
M. Farnierb and M. Lagarde”
‘U352
INSERM, Chimie Biologique, INSA, Bar. 406, 20 avenue A. Einstein, 69621 Villeurbanne Cedex. bUA CNRS 33, Faculte des Sciences Gabriel, UniversifP de Dijon, BP 138, 21004 Dijon Cedex and CLaboratoire de Physiologie Animale. FacultP des Sciences Mirande, UniversitP de Dijon, BP 138, 2104 Doon Cedex (France) (Received 23 December, 1991) (Revised, received 29 September, 1992) (Accepted 12 October, 1992)
Summary
Fatty acid composition of lipid classes and NMR spectra of lipoproteins were compared in 6 young (24-35year-old) and 6 elderly (79-90-year-old) women. Cholesteryl ester, triglyceride and protein content of LDL in elderly women were significantly higher (+52-57% and +20% for lipids and proteins, respectively) than those observed in young women, HDL lipid levels were similar in the two groups. The proportion of linoleic acid (mainly in cholesteryl esters and phospholipids) of each lipoprotein species was always lower in octogenarians when compared with young females (lowering of 13-28% and 27-46% for cholesteryl esters and phospholipids, respectively). Conversely, the proportions of mono-unsaturated fatty acids (mainly oleic acid) increased in all lipid classes, although this was only significant in cholesteryl esters from each lipoprotein species. NMR spectra of lipoproteins showed a restricted mobility of acyl chain terminal CHs groups in old women which was significant only in VLDL and HDLs. This suggests that the decrease of linoleic acid could affect the lipid mobility in lipoproteins of elderly women.
Key words: Aging; Lipoproteins;
Fatty acids; Linoleic acid; Cholesterol; NMR
Introduction
Correspondence ro: J. Lecerf, U 352 INSERM, Chimie Biologique, INSA, Bat. 406, 20 avenue A. Einstein, 69621 Villeurbanne Cedex, France.Tel.: 72 43 85 73; Fax: 72 43 85 24. Abbreviarions: CE, cholesteryl esters; FC, free cholesterol; HDL, high-density lipoproteins; LDL, low density lipoproteins; PL, phospholipids; TG, triglycerides; VLDL, very lowdensity lipoproteins. Fatty acids are designated by the number of carbon atoms followed by the number of double bonds.
It is generally assumed that the triglyceride level increases in elderly women [1,2], this increase being enhanced when women are administered sex hormones [3], but age does not affect the cholesterol/triglyceride ratio [4]. The other usual parameters for lipoprotein analysis are similar for those over 70 years compared with young adults [5-71. Even the HDL cholesterol level used as a
242
negative-risk indicator for atherosclerosis, is negatively correlated with peripheral vascular diseases in octo- and nona-genarians [5,6]. In contrast with these similarities, we have previously found [8] that the plasma polyunsaturated fatty acid content (mainly linoleic acid) clearly decreased while those of monounsaturated fatty acids increased in elderly subjects. Since such a change might have physiological consequences, the present study was designed to investigate whether these modifications occur within each class of lipoprotein. In addition, an NMR spectra study of lipoproteins was performed in order to determine whether the fatty acid changes may alter some physical characteristics of lipoproteins.
TABLE 1 DIETARY CHARACTERISTICS OF YOUNG AND OLD WOMEN EXPRESSED IN PERCENTAGE OF TOTAL CALORIC INTAKE Values are the means * S.E. (n = 6).
Proteins Carbohydrates Lipids Fatty acids Saturated Monounsaturated 18:2 18:3
Old women
Young women
17.5 ?? 2 55.8 zt 6 26.5 f 5.3
22.6 * 1.2 42.9 f 3.0 34.3 f 1.9
10.7 ?? 3.8 7.3 f 3.2 2.1 ?? 0.3 0.20 * 0.04
12.0 zt 0.6 13.4 ?? 0.4 3.0 ?? 0.3 0.28 f 0.03
*Signiticant difference at P < 0.05.
Materials and Methods Subjects
The study was performed on blood from 6 young (24-35year-old) and 6 elderly (79-go-yearold) women. All subjects did not take any drug for at least 10 days and fasted overnight before venipuncture. The older women were selected in the Charpennes hospital (Villeurbanne) among patients devoid of evolutive disease or senile dementia. Young subjects were chosen among healthy volunteers, 4 of them taking oral contraceptives. Diet
The dietary habits of each subject were followed for 7 days by the dietitian and results are given in Table 1. Sample preparation
Venous blood from subjects was drawn on CPD anticoagulant mixture (10 vol. collected onto 1.4 vol. CPD: sodium citrate 2.36%, glucose 2.32%, citric acid 0.32% and NaH2P04 0.22% by weight in water and adjusted to pH 5.6). Platelet-rich plasma (PRP) was prepared by centrifuging blood for 10 min at 100 x g. After addition of EDTA (1 mM), PRP was acidified to pH 6.4 by using 0.15 M citric acid and was centrifuged for 15 min at 900 x g in order to pellet platelets and plateletpoor plasma was used for the lipoprotein analysis. Lipoprotein separation
Lipoprotein
classes,
namely
VLDL,
LDL,
HDL2 and HDLs were separated by ultracentrifugation using the NaClKBr density gradient and the procedure described by Chapman et al.
PI. Each lipoprotein class was divided into aliquots for the NMR study, lipid separation and protein and phosphorus determinations. The procedure of Peterson [lo] was used for protein determination. Lipid analysis
Lipoprotein lipids were extracted according to the method of Folch et al. [ 111. Cholesterol esters (CE), triglycerides (TG), and total phospholipids (PL) were separated on columns of silicic acid 100 mesh/super Cell (Mallinkrodt), 2:l w/w, using appropriate volumes of the following mixtures: hexane/CHCls (80:20, v/v), CHCls and CHC13/methanol (l:l, v/v). CE, TG and free cholesterol (FC) were separated by thin layer chromatography using Chromarod S-II quartz rods coated with silica gel and hexane/diethyl ether/formic acid, (54:6:0.08, by vol.) as the solvent system. Quantification of the lipid classes was performed on Iatroscan TH-10 TLC analyser (Iatron Laboratories, Tokyo, Japan) equipped with a flame ionization detector linked to an Enica 2 1 integrator-calculator (Delsi, Paris, France). Cholesteryl acetate was used as an internal standard to determine the response factors [12-141. The phosphorus content of PL was determined by the method of Bartlett [15].
243
'H-NMR
Fatty acids of CE, TG and PL were transmethylated by a mixture of methanoYH2S04 (98:2, v/v) at 100°C for 3 h after addition of heptadecanoic acid as an internal standard. After extraction by hexane, fatty acid methyl esters were analyzed on a Beker-Packard Model 417 gas chromatograph, equipped with a 30 m x 0.4 mm I.D. glass capillary column coated with Carbowax 20 M (Applied Science Labs., State College, PA) at a constant temperature of 195°C and a nitrogen flow-rate of 3 ml/min. Quantitation was performed using a flame ionization detector and an Enica 21 integrator-calculator. Calibration factors were calculated using standard mixtures of fatty acid methyl esters (Nu-Check-Prep, Elysian, MN).
TABLE
spectra
Lipoprotein fractions were dialysed against PBS buffer (pH 7.4) for 48 h at 4°C and concentrated by ultrafiltration through Centricon 30 microconcentrators (Amicon Corp., Danvers, MA) at 1500 x g for 30 min to obtain final volumes of 0.4 ml. Samples were prepared in j-mm NMR tubes by mixing 0.4 ml of lipoproteins with 0.1 ml of D20. Peaks were referenced to sodium 3-trimethylsilyl-[D4]propionate (TMSP) as an internal standard. Fourier transform ‘H spectra were obtained at 20°C on a Bruker WM 400 spectrometer system operating at a proton frequency of 400.13 MHz and equipped with an Aspect 2000 computer. The
2
LIPOPROTEIN
COMPOSITION
IN YOUNG
AND OLD WOMEN
Values are means f SE (n = 6). Young
women
mg/lOO ml
Old women weight %
mg/lOO ml
weight %
VLDL CE TG FC PL
9.1 39.8 3.5 13.6 10.2
f 0.8 f 9.9 ?? 0.3 f 0.9 f 0.9
11.5 ?? 1.2 52.1 f 5.2 4.4 ?? 0.2 18.8 f 3.2 13.3 * 1.2
11.9 41.8 5.0 13.0 10.6
f 1.5 ZIZ7.5 * 0.9 f 0.7 zt 2.8
14.7 50.1 6.0 16.7 2.5
f f + f f
0.8 2.9 0.5 1.7 1.9
zk 14.0* f 5.0: f 5.3 zt 8.1 f 7.4*
43.7 8.2 8.2 17.0 22.9
f & it f f
1.2 0.5 0.8 1.7 0.4
zt f f + zt
3.5 0.7 0.3 2.9 3.2
25.0 5.4 4.5 32.2 32.9
+ 3.1 ?? 1.1 + 0.6 f 3.4 zt 1.6
zt 3.7 f 1.0 z+ 0.5 zt 3.2 f 7.0
14.1 3.4 2.4 24.3 55.8
f f zt f zt
LDL CE TG FC PL
123.3 23.5 24.7 67.4 17.5
f f f f f
16.6 2.5 3.0 5.2 4.6
38.3 7.6 7.7 21.7 24.7
zt 2.9 zt 0.9 ?? 0.5 f 2.0 + 1.0
187.6 37.1 36.0 72.2 93.4
f f f f f
3.1 0.6 0.7 2.6 2.4
19.2 4.0 3.3 24.1 25.1
f f f zt f
1.3 0.3 0.1 2.3 2.8
27.4 6.7 4.6 45.8 105.9
HDL, CE TG FC PL Proteins
f 2.7
18.1 3.5 4.5 25.0 26.0
f 0.7 f 3.2 zt 4.5
22.6 4.6 5.5 31.8 35.6
36.3 5.0 4.4 51.8 116.9
zt 7.5 f 0.7 ?? 0.7 zt 6.6 + 6.5
17.4 2.4 2.0 26.6 54.6
?? 0.4
HDL, CE TG FC PL Proteins ‘Significant
difference
between
young
and old women
at P < 0.05.
1.3 0.4 0.3 1.8 1.7
244
water resonance was suppressed by a presaturation pulse at the frequency of the water resonance at 400.13 MHz. Sixteen free-induction decays were signal-averaged and Fourier transform was performed. Tr values were measured according to Vold et al. [ 161 by using the (a, 7, 7r/2) inversion recovery sequence. T, values are accurate within 2% which is in the range reported previously [17,18]. Statistical analysis
Young women were compared with old women by using Student’s I-test [19] or the t’-test [20] when sample variances were not equal. Results
There was no significant difference between the dietary characteristics of young and old women (Table 1). However, it seems that the consumption
TABLE
3
FATTY ACID COMPOSITION YOUNG AND OLD WOMEN
OF CHOLESTERYL
Values are mol % of total fatty acids, means Fatty
acid
Cholesteryl Young women
14:o 16:O 16:l 18:O 18:l 18:2 18:3 2o:o 20:3 20:4 20:5 22:o 22:1 22:4 22:5 22:5 22:6 24:0 24:1
of lipids and particularly that of monounsaturated fatty acids of young women tended to be higher than that of the old women. Table 2 shows that the proportions (expressed as weight %) of the different lipid classes and proteins were not significantly different between the two groups. However, the absolute level (expressed as mg/lOO ml) of LDL was higher in the elderly compared with young subjects. This difference is mainly due to higher levels of CE, TG and proteins in the LDL from old women. The total lipid/ protein ratio in LDL from aged women (3.37 f 0.09) was not significantly higher than that from young women (3.16 A 0.20), suggesting that the number of LDL particles was increased in the aged group. The fatty acid composition of CE, TG and PL of VLDL, LDL, HDLz and HDL3 are reported in Tables 3, 4, 5 and 6, respectively. The proportion of linoleic acid (18:2) in CE and PL from all the
(n-6)
(n-6) (n-6) (n-3) (n-9) (n-6) (n-6) (n-3) (n-3)
0.3 16.5 6.4 3.8 25.0 39.1 1.6 1.0 0.4 3.4 1.5 -
f zt + f f f f f * zt zt
0.1 2.5 1.0 0.5 1.4 2.1 0.4 0.5 0.1 0.6 0.6
TRIGLYCERIDES
* S.E. for 6 determinations
(SE.
Young women
Old women
2.3 15.8 1.9 3.0 26.5 34.0 1.4 1.0 0.3 4.6 1.9 -
0.4 34.0 5.0 7.0 30.6 16.2 1.9 0.6 0.6 1.5 0.7
0.4 31.4 7.4 5.3 38.9 10.9 1.4 0.5 0.2 1.7 0.6
f 0.0 zt 6.5 f 0.6 ?? 1.3 f 4.7 f 5.1 zt 0.6 + 0.1 zt 0.4 f 0.4 f 0.4
f f f f f f f f f f f
0.1 2.4 1.1 0.9 1.1 1.1* 0.1 0.0 0.0 0.3 0.2
0.6 zt 0.3 0.6 f 0.1 -
0.8 f 0.4 0.8 * 0.2
OF VLDL FROM
Phospholipids
Old women f 1.8 f 2.1 ?? 1.5 f 0.4 zt 2.1 * 2.1* f 0.2 f 0.1 f 0.1 f 1.6 zt 0.6
AND PHOSPHOLIPIDS
< 0.1 are noted as 0.0).
Triglycerides
esters
0.8 f 0.6 0.2 f 0.2
ESTERS,
0.4 f 0.1 1.0 f 0.1
(n-9)
*Significant difference between young and old women at P < 0.05. When the position represents the sum of all the isomers.
Young women
Old women
1.1 28.9 6.0 11.0 22.1 16.8 1.1 0.9 1.2 4.2 0.8 0.9 0.2 0.3 0.5 0.6 2.1 0.7 0.4
0.8 32.3 7.1 11.4 22.9 11.3 0.8 0.7 1.5 4.0 1.1 0.8 0.2 0.4 0.1 0.8 2.4 0.6 0.5
f zt f f f f f f f zt zt f * f f f + f
0.3 1.0 1.2 0.9 2.7 1.3 0.3 0.1 0.3 0.9 0.1 0.1 0.0 0.1 0.4 0.1 0.6 0.1 ?? 0.1
f zt f f zt f zt f f zt f f * f f f zt f *
0.2 1.4 1.8 1.1 2.8 1.3’ 0.1 0.1 0.4 0.6 0.2 0.1 0.1 0.1 0.1 0.2 0.4 0.4 0.3
of the last double bond is omitted,
the value
245 TABLE 4 FATTY ACID COMPOSITION OF CHOLESTERYL ESTERS, TRIGLYCERIDES YOUNG AND OLD WOMEN Values are mol % of total fatty acids, means Fatty acid
14:o 16:0 16:l 18:O 18:l 18:2 (n-6) 18:3 2o:o 20:3 (n-6) 20:4 (n-6) 20:5 (n-3) 22:o 22:l (n-9) 22:4 (n-6) 22:5 (n-6) 22:5 (n-3) 22:6 (n-3) 24:0 24:l (n-9)
?? S.E.
for 6 determinations (S.E. < 0.1 are noted as 0.0).
Triglycerides
Cholesteryl esters
AND PHOSPHOLIPIDS OF LDL FROM
Phospholipids
Young women
Old women
Young women
Old women
Young women
Old women
0.1 f 0.0 14.1 f 1.8 3.5 f 0.4 1.5 f 0.1 21.3 + 1.1 52.5 zt 2.2 0.9 f 0.1 0.2 f 0.0 0.5 f 0.1 4.6 zk 0.6 0.4 f 0.1
0.5 ?? 0.4 15.8 + 0.9 6.2 f 0.7’ 1.1 f 0.1 25.7 zt 1.0’ 41.8 f 1.4* 1.4 ?? 0.2 0.1 f 0.0 0.6 f 0.1 5.2 f 0.7 1.2 ?? 0.5
0.3 ?? 0.1 27.6 + 1.7 6.3 f 0.5 6.0 zt 0.5 32.1 zt 2.0 19.1 ?? 1.9 1.5 ?? 0.2 0.7 f 0.1 0.3 ?? 0.0 3.5 f 0.4 0.6 zt 0.3
0.3 ?? 0.1 28.6 f 1.6 6.7 zt 0.5 4.9 f 0.7 37.3 * I.78 12.2 f 1.1* 1.4 f 0.1 0.4 f 0.0 0.3 f 0.1 3.8 f 0.9 1.8 f 0.6
0.4 ?? 0.1 29.9 * 0.6 3.7 f 1.1 11.9 f 0.5 15.1 f 1.7 18.8 f 1.6 0.6 zt 0.1 0.9 f 0.1 1.8 f 0.3 6.1 f 0.9 0.7 f 0.1 2.0 f 0.3 0.1 f 0.0 1.1 ?? 0.2 0.2 zk 0.1 0.5 f 0.1 2.5 f 0.2 1.5 f 0.2 2.2 f 0.4
0.4 f 0.1 32.4 f 0.8 3.0 f 0.4 11.5 f 0.6 15.4 ?? 1.1 13.7 f 1.5* 0.5 f 0.1 0.9 f 0.1 1.9 f 0.3 6.4 f 0.2 1.2 f 0.3 1.6 f 0.1 0.1 f 0.0 1.1 f 0.1 0.2 ?? 0.1 1.0 f 0.2* 3.9 * 0.5: 1.3 f 0.1 3.2 zt 0.8
-
0.1 f 0.0 0.3 * 0.1
-
-
0.9 f 0.4 1.0 ?? 0.3 -
0.8 f 0.4 1.5 ?? 0.3 -
*Significant difference between young and old women at P < 0.05. When the position of the last double bond is omitted, the value represents the sum of all the isomers.
lipoproteins studied was significantly lower in the elderly than in young women. This was also found in TG but only from VLDL and LDL. Conversely, the proportion of oleic acid (18:l) in old women was higher in CE from LDL, HDL2 and HDLs as well as in LDL-TG and HDLs-PL. The higher proportion of 18:l tended to be associated with a higher proportion of palmitoleic acid (16: l), and reached significance in LDL-CE. The percentage of palmitic acid (16:0) also tended to rise in some lipid classes of lipoproteins from aged women. In PL from VLDL and HDL2 the lowered proportion of 18:2 was not significantly balanced by any particular fatty acid. The proportions of polyunsaturated fatty acids other than 18:2 were not affected by aging, except 22:5(n-3) and 22:6(n-3) which were higher in PL of LDL and HDLs from aged women. Such an increase was also found for 22:5(n-3) in HDL,-PL.
T, values obtained for resonances of the different chemical groups were not significantly different with the exception of those of the methyl groups which were significantly decreased in VLDL and HDLs from elderly women (672.8 zt 29.3 versus 580.2 f 10:0 and 357.8 f 4.3 vs. 326.8 f 3.2 ms for VLDL and HDLs of young and old women respectively). Discussion The lipoprotein composition of the two groups of subjects agrees with the values obtained by the method of separation used elsewhere [9]. The lack of differences between the HDLs cholesterol content of the two groups agrees with data reported for octo- and nonagenarians [5-71 but not with the decreased lipid/protein ratio reported in HDL2 of old subjects [6].
246
TABLE
5
FATTY ACID COMPOSITION YOUNG AND OLD WOMEN
OF CHOLESTERYL
Values are mol % of total fatty acids, means Fatty
acid
140 160 161 18:O 18:l 18:2 (n-6) 18:3 20:o 20:3 (n-6) 20:4 (n-6) 20~5 (n-3) 22:o 22:1 (n-9) 22:4 (n-6) 22:5 (n-6) 22:5 (n-3) 22:6 (n-3) 240 241 (n-9)
Cholesteryl
esters Old women
0.4 15.1 5.3 3.3 19.5 46.1 1.9 1.2 0.4 5.2 0.4 -
0.5 20.5 1.2 5.0 24.1 33.3 0.9 0.7 0.5 5.2 1.2
0.2 ?? 0.0 0.6 zt 0.1 -
TRIGLYCERIDES
f S.E. for 6 determinations
?? 0.2 ?? 3.1
* 1.2 ?? 2.1
f zt * f f zt f
2.0* 5.2* 0.2 0.3 0.1 1.7 0.5
0.4 0.4 -
?? 0.2 ?? 0.3
Old women
0.6 29.5 1.4 12.2 29.3 12.0 1.5 1.4 0.3 3.0 0.6
0.5 26.9 8.3 1.2 31.4 12.9 1.6 0.9 0.3 6.3 1.8
-
-
1.7 ?? 0.2 0.3 f 0.0 -
0.7 1.0 -
f 0.2 zt 2.9 + 0.9 ?? 0.5 f 2.5 zt 1.6 zt 0.1 ?? 0.2 f 0.1 zt 0.7* f 0.6
?? 0.4 ?? 0.1*
‘Significant difference between young and old women at P < 0.05. When the position represents the sum of all the isomers.
Although it is known that contraceptive hormones can modify the proportions of plasma triglycerides [3,4] no difference was observed between plasma profiles of young females taking or not taking hormones. However, as the levels of TG, CE and apolipoproteins vary throughout the menstrual cycle [21], this was probably due to the small number of each group of young women. On the other hand, the proportions of 18:2 were similar in these groups. This is not surprising since our previous report [8] showed that age-induced lowering of plasma 18:2 affected both genders and therefore this phenomenom did not seem directly related to sex hormones. Consequently, data from both groups of young females were pooled. The low proportion of 18:2 in the aged women agrees with our previous results obtained in whole plasma [8], although no reduction of arachidonic
OF HDL,
FROM
Phospholipids
Young women zt 0.2 f 1.7 f 1.3 ?? 3.5 f 1.5 f 1.4 f 0.1 ?? 0.1 f 0.1 f 0.8 zt 0.3
AND PHOSPHOLIPIDS
(S.E. < 0.1 are noted as 0.0).
Triglycerides
Young women ?? 0.1 zt 1.6 ?? 0.3 f 1.3 ?? 1.1 * 4.6 ?? 1.0 ?? 0.7 f 0.1 f 0.6 ?? 0.1
ESTERS,
Young women
Old women
0.9 31.6 4.9 13.3 17.0 16.2 0.6 0.8 1.3 5.4 0.8 1.4 0.2 0.6 0.2 0.5 2.2 1.1 0.8
1.4 26.1 4.6 16.3 19.6 11.3 1.0 1.4 1.1 6.0 1.4 1.6 0.4 0.6 0.4 1.7 2.3 1.2 1.4
f 0.2 zt 1.2 f 0.9 ?? 1.0 f 1.6 zt 1.3 f 0.1 zt 0.1 f 0.1 ?? 0.4 f 0.1 f 0.1 f 0.1 zt 0.1 f 0.1 f 0.1 * 0.5 ?? 0.1 f 0.2
f zt zt f f f f f f f
0.8 5.9 1.1 3.3 0.9 0.7* 0.2 0.5 0.4 0.6 ?? 0.2 ziz0.4 f 0.2 f 0.0 f 0.2 zk 0.8* f 0.4 * 0.5 f 0.9
of the last double bond is omitted,
the value
acid (20:4) was found in the present work, but rather an increase in TG of HDL2 and HDL3. Although the 18:2 intake by elderly women was lower than that of the young women, it was nevertheless not statistically different and in the range of values commonly accepted as adequate to prevent essential fatty acid deficiency [22]. Moreover, the low proportion of 18:2 in plasma of old women was not associated with any appearance of 20:3(n9), the biological marker of essential fatty acid deficiency, which was also hardly detectable in the control group. The lowering of plasma 18:2 does not seem directly related with the 18:2 intake since the consumption of 18:2 by old people investigated in our previous work [8] was twofold higher than in the present study though the plasma proportion of 18:2 from elderly people was similar in the two studies. On the other hand, we found an increased
24-l
TABLE
6
FATTY ACID COMPOSITION YOUNG AND OLD WOMEN
OF CHOLESTERYL
Values are mol % of total fatty acids, means Fatty
140 16:0 16:l 18:0 18:l 18:2 18:3 20:o 20:3 20:4 20:5 22:o 22:l 22:4 22:5 22:5 22:6 24:0 24:l
acid
(n-6)
(n-6) (n-6) (n-3)
Cholesteryl
esters
ESTERS,
f S.E. for 6 determinations
(SE.
Old women
Young women
0.2 13.9 4.3 1.8 19.0 51.8 1.0 0.4 0.6 5.6 0.8
0.9 17.3 7.0 1.7 25.2 38.8 1.0 0.2 0.5 5.1 1.5
0.4 31.0 7.9 7.5 32.8 15.0 1.6 0.9 0.1 1.6 0.2
f f f f zt f f
0.7 1.1 1.1 0.4 1.6; 3.2* 0.2 ?? 0.1 * 0.1 ?? 1.5 f 0.7
(n-9) (n-6) (n-6) (n-3) (n-3)
0.2 f 0.1 0.5 f 0.1
0.2 * 0.1 0.5 f 0.3
(n-9)
_
-
f zt f f * zt zt f f f f
0.2 2.7 0.6 0.5 1.9 3.2 0.3 0.0 0.1 0.2 0.1
0.5 f 0.2 0.4 f 0.1
OF HDL,
FROM
Phosphohpids Old women
Young women
Old women
0.5 30.0 8.1 6.6 31.9 12.6 1.6 0.9 0.4 4.3 1.2
0.6 30.5 2.7 12.2 13.6 21.0 0.5 0.6 1.8 6.5 2.0 1.4 0.3 0.6 0.3 0.6 2.6 1.2 1.1
0.6 31.6 4.0 11.9 16.6 13.9 0.5 0.7 2.0 7.7 1.3 0.9 0.1 0.6 0.2 1.2 4.2 0.6 I.1
f f zt f zt zt zt
0.1 4.3 0.8 1.3 2.9 3.4 0.3 ?? 0.2 f 0.1 + 0.8* f 0.4
0.7 ?? 0.4 0.9 f 0.3
‘Significant difference between young and old women at P < 0.05. When the position represents the sum of all the isomers.
proportion of n-3 long chain fatty acids such as 20:5 and 22:5 as observed previously in total plasma [S], in addition to an enhanced 22:6 in the present work. These different changes of fatty acid composition are analogous to the age-induced changes in fatty acid composition of several rat tissues [23]. In aorta, liver and adipose tissue, the 18:2 proportion was lowered, that of 20:4 unchanged and those of n-3 fatty acids enhanced. Moreover, in adipose tissue the proportions of n-9 fatty acids were significantly enhanced. All these data suggest that the effects of age on the plasma fatty acid composition affect similarly humans and other mammals. On the other hand, a parallel could be established between the plasma 18:2 decrease associated with the slight increase of LDL cholesterol and the recent observation showing that plasma 18:2 is negatively correlated to LDL cholesterol in European men [24].
AND PHOSPHOLIPIDS
< 0.1 are noted as 0.0).
Triglycerides
Young women f 0.1 f 2.2 f 0.6 f 0.6 f 1.7 f 4.5 f 0.1 f 0.2 zk 0.0 zt 0.5 ZIZ0.1
TRIGLYCERIDES
zt f f f f + f f zt zt f f f f f zt zt f
0.2 1.4 0.4 0.6 1.2 0.8 0.1 0.0 0.2 1.2 1.2 0.2 0.2 0.1 0.1 0.1 0.6 0.4 ?? 0.2
+ zt zt f f + f f f f f f f zt f f
0.1 0.9 0.6 0.5 0.6’ 0.6; 0.1 0.1 0.3 0.4 0.2 0.1’ 0.0 0.0 0.1 0.1* ?? 0.3* f 0.1 f 0.3
of the last double bond is omitted,
the value
The lowered proportion of 18:2 in elderly women was most often reciprocally accompanied by higher proportions of 18:l and 16:1, although not significantly with exception of cholesteryl esters. It is unlikely that such an increase would be related to the diet since the monounsaturated fatty acid intake of old women tended to be lower than that of young women. Since the proportion of 18:1 was enhanced in cholesteryl esters, it is expected to observe the same increase of 18:l from HDLJ phospholipids, part of this lipid class acting as a substrate for 1ecithin:cholesterol acyl transferase [25]. A replacement of 18:2 by monounsaturated fatty acids in cholesteryl esters might suggest a reduction of lipoprotein fluidity, particularly in LDL which carry the main part of this lipid class. Moreover, it has been reported that dietary 18:2 specifically influences LDL fluidity [26]. Relaxation times (rt) of chemical lipid groups revealed
248
that only the motion of CHs groups tended to be restricted in all the lipoproteins from elderly people, and this reached significance in VLDL and HDLs. NMR spectra were performed at 20°C below the midpoint transition temperature of cholesteryl ester mixtures [27] and below the temperatures of the two main phase transitions of core lipids [28]. Under these conditions the methyl groups, associated with the cholesterol ring structure, do not show measurable signals [28,29], the T, changes reflecting essentially those of the terminal methyl groups of acyl chains. This result suggests that the physical behaviour of lipid within VLDL and HDLs could be affected by the changes of fatty acid composition in the old women. However, further detailed NMR and other physical studies are necessary to confirm this.
8
9
10
11
12
13
Acknowledgments 14
This research was supported by the Ministere de la Recherche et de 1’Enseignement Supkrieur Grant MRES 87 G 0556.
15
References
16
Hoover, J.J., Walden, C.E., Bergelin, R.O., Wahl, P-W., Albers, J.J., Hazzard, W.R. and Knopp, R.H., Cholesterol and triglyceride distribution in an adult employee population: the Pacific Nothwest Bell Telephone Company Health Survey, Lipids, 15 (1980) 895. Manila, K.S., Mamiemi, J., Miiki, J. and Juva, K., Lipids, lipoproteins and apolipoproteins in the elderly, Stand. J. Clin. Lab. Invest., 46 (1986) 136. Wahl, P., Wamick, G.R., AIbers, J.J., Hoover, J.J., Walden, C.E., Bergelin, R.O., Ogilvie, J.T., Hazzard, W.R. and Knopp, R.H., Distribution of lipoprotein triglyceride and lipoprotein cholesterol in an adult population by age, sex and hormone use, Atherosclerosis, 39 (1981) 111. Knopp, R.H., Wamick, G.R., Walden, C.E., Wahl, P:W., Hoover, J.J., Bergelin, R.O., Ogilvie, J.T., Albers, J.J. and Hazzard, W.R., Relationship of gender, sex hormone use and age with lipoprotein cholesterol, Atherosclerosis, 39 (1981) 133. Schneider, J., High-density lipoproteins and peripheral vascular disease in octo- and nonagenarians, J. Am. Geriatr. Sot., 28 (1980) 215. Ron, D., Aviram, M. and Brooks, J.G., High-density lipoprotein in octogenarians, Biochem. Med., 30 (1983) 253. Schneider, J., Boldt, J., Leyhe, A. and Kaffarnick, H.,
17 18
19
20
21
22
23
HDL cholesterol, peripheral and coronary vascular disease in high age groups, Atherosclerosis, 35 (1980) 487. Vericel, E., Croset, M., Perrot, L., Renaud, S. and Lagarde, M., Platelets and aging II -Plasma lipoproteins and fatty acid profiles, Thromb. Res., 49 (1988) 451. Chapman, M.J., Goldstein, S., Lagrange, D. and Laplaud P.M., A density gradient ultracentrifugal procedure for the isolation of the major lipoprotein classes from human serum, J. Lipid Res., 22 (1981) 339. Peterson, G.L., A simplification of protein assay of Lowry et al. which is more generally applicable, Anal. B&hem., 83 (1977) 346. Folch, J., Less, M. and Sloane-Stanley, G.H., A simple method for the isolation and purification of total lipids from animal tissues, J. Biol. Chem., 226 (1957) 497. Kaitaranta, J.K. and Nicholaides, N., Response and linearity of different lipid compounds when analyzed by thin-layer chromatography with flame ionization detection, J. Chromatogr., 205 (1981) 347. Van Tornout, P., Vercaemst, R., Caster, H., Lievens, M.J., De Keersgiester, W., Soetewey, F. and Rosseneu, M., Use of I-octadecanol as an internal standard for plasma lipid quantitation on chromarods, J. Chromatogr., 164 (1979) 222. Vandamme, R., Blaton, V. and Peeters, H., Screening of plasma lipids by thin-layer chromatography with flame ionization detection on chromarods, J. Chromatogr., 145 (1978) 151. Bartlett, G.R., Phosphorus assay in column chromatography, J. Biol. Chem., 234 (1959) 466. Vold, R.L., Waugh, J.S., Klein, M.P. and Phelps, D.E., Measurement of spin relaxation in complex systems, J. Chem. Phys., 48 (1968) 3831. Damadian, R., Tumor detection by nuclear magnetic resonance, Science, 171 (1971) 1151. Kroon, P.A., Kainosho, M. and Chan, S.I., Proton magnetic resonance studies of bilayer membranes. Experimental determination of inter- and intramolecular nuclear relaxation rates in sonicated phosphatidylcholine bilayer vesicles, Biochim Biophys. Acta, 443 (1976) 282. Snedecor, G.W. and Cochran, G., Statistical Methods, 7th Edn., The Iowa State University Press, Ames, IA, 1980, p. 54. Snedecor, G.W. and Cochran, G., Statistical Methods, 7th Edn., The Iowa State University Press, Ames, IA, 1980 p. 96. Lussier-Cacan, S., Xhignesse, M., Desmarais, J.L., Davignon, J., Kafrissen, M.E. and Chapdelaine A., Cyclic fluctuations in human serum lipid and apolipoprotein levels during the normal menstrual cycle: comparison with changes occurring during oral contraceptive therapy, Metabolism, 40 (1991) 849. Kinsella, J.E., Linolenic acid: functions and effects on linoleic acid metabolism and eicosanoid-mediated reactions. II Essential fatty acids, Adv. Food Nutr. Res., 35 (1991) 7. Takahashi, R. and Horrobin, D.F., Linoleic acid-induced fatty acid changes in platelet and aorta of the rat: effect of age and cholesterol, Lipids, 23 (1988) 885.
249 24
Seidell, J.C., Cigolini, M., Deslypere, J.P., Charzewska, J. and Ellsinger, B.M., Polyunsaturated fatty acids in adipose tissue in european men aged 38 years in relation to serum lipids, smoking habits and fat distribution, Am. J. Epidemiol., 134 (1991) 583. 25 Jonas, A., Lecithin cholesterol acyltransferase. In: Gotto, A.M., Jr. (Ed.), Plasma Lipoproteins, Elsevier, Amsterdam, 1987, p. 229. 26 Berlin, E., Judd, J.T., Marshall, M.W. and Kliman, P.G., Dietary linoleate increase fluidity and influence chemical composition of plasma low density lipoprotein in adult man, Atherosclerosis, 66 (1987) 215.
27
Kroon, P.A. and Krieger, M., The mobility of cholesteryl esters in native and reconstituted low density lipoprotein as monitored by nuclear magnetic resonance spectrometry, J. Biol. Chem., 256 (1981) 5340. 28 Kroon, P.A., The order-disorder transition of the core cholesteryl esters of human plasma low-density lipoprotein, J. Biol. Chem., 256 (1981) 5332. 29 Chapman, D., Leslie, R.B., Hirz, R. and Scanu, A.M., High resolution NMR spectra of high-density serum lipoproteins, Biochim. Biophys. Acta, 176 (1969) 524.
ATHERO
241 Ireland,
Ltd. All rights reserved.
0021-9150193/$06.00
04964
Variations
in the fatty acid composition of lipid classes from lipoproteins in elderly women
J. Lecerfa, A. Rossignolb, E. VCricel”, F. Thk”,
M. Farnierb and M. Lagarde”
‘U352
INSERM, Chimie Biologique, INSA, Bar. 406, 20 avenue A. Einstein, 69621 Villeurbanne Cedex. bUA CNRS 33, Faculte des Sciences Gabriel, UniversifP de Dijon, BP 138, 21004 Dijon Cedex and CLaboratoire de Physiologie Animale. FacultP des Sciences Mirande, UniversitP de Dijon, BP 138, 2104 Doon Cedex (France) (Received 23 December, 1991) (Revised, received 29 September, 1992) (Accepted 12 October, 1992)
Summary
Fatty acid composition of lipid classes and NMR spectra of lipoproteins were compared in 6 young (24-35year-old) and 6 elderly (79-90-year-old) women. Cholesteryl ester, triglyceride and protein content of LDL in elderly women were significantly higher (+52-57% and +20% for lipids and proteins, respectively) than those observed in young women, HDL lipid levels were similar in the two groups. The proportion of linoleic acid (mainly in cholesteryl esters and phospholipids) of each lipoprotein species was always lower in octogenarians when compared with young females (lowering of 13-28% and 27-46% for cholesteryl esters and phospholipids, respectively). Conversely, the proportions of mono-unsaturated fatty acids (mainly oleic acid) increased in all lipid classes, although this was only significant in cholesteryl esters from each lipoprotein species. NMR spectra of lipoproteins showed a restricted mobility of acyl chain terminal CHs groups in old women which was significant only in VLDL and HDLs. This suggests that the decrease of linoleic acid could affect the lipid mobility in lipoproteins of elderly women.
Key words: Aging; Lipoproteins;
Fatty acids; Linoleic acid; Cholesterol; NMR
Introduction
Correspondence ro: J. Lecerf, U 352 INSERM, Chimie Biologique, INSA, Bat. 406, 20 avenue A. Einstein, 69621 Villeurbanne Cedex, France.Tel.: 72 43 85 73; Fax: 72 43 85 24. Abbreviarions: CE, cholesteryl esters; FC, free cholesterol; HDL, high-density lipoproteins; LDL, low density lipoproteins; PL, phospholipids; TG, triglycerides; VLDL, very lowdensity lipoproteins. Fatty acids are designated by the number of carbon atoms followed by the number of double bonds.
It is generally assumed that the triglyceride level increases in elderly women [1,2], this increase being enhanced when women are administered sex hormones [3], but age does not affect the cholesterol/triglyceride ratio [4]. The other usual parameters for lipoprotein analysis are similar for those over 70 years compared with young adults [5-71. Even the HDL cholesterol level used as a
242
negative-risk indicator for atherosclerosis, is negatively correlated with peripheral vascular diseases in octo- and nona-genarians [5,6]. In contrast with these similarities, we have previously found [8] that the plasma polyunsaturated fatty acid content (mainly linoleic acid) clearly decreased while those of monounsaturated fatty acids increased in elderly subjects. Since such a change might have physiological consequences, the present study was designed to investigate whether these modifications occur within each class of lipoprotein. In addition, an NMR spectra study of lipoproteins was performed in order to determine whether the fatty acid changes may alter some physical characteristics of lipoproteins.
TABLE 1 DIETARY CHARACTERISTICS OF YOUNG AND OLD WOMEN EXPRESSED IN PERCENTAGE OF TOTAL CALORIC INTAKE Values are the means * S.E. (n = 6).
Proteins Carbohydrates Lipids Fatty acids Saturated Monounsaturated 18:2 18:3
Old women
Young women
17.5 ?? 2 55.8 zt 6 26.5 f 5.3
22.6 * 1.2 42.9 f 3.0 34.3 f 1.9
10.7 ?? 3.8 7.3 f 3.2 2.1 ?? 0.3 0.20 * 0.04
12.0 zt 0.6 13.4 ?? 0.4 3.0 ?? 0.3 0.28 f 0.03
*Signiticant difference at P < 0.05.
Materials and Methods Subjects
The study was performed on blood from 6 young (24-35year-old) and 6 elderly (79-go-yearold) women. All subjects did not take any drug for at least 10 days and fasted overnight before venipuncture. The older women were selected in the Charpennes hospital (Villeurbanne) among patients devoid of evolutive disease or senile dementia. Young subjects were chosen among healthy volunteers, 4 of them taking oral contraceptives. Diet
The dietary habits of each subject were followed for 7 days by the dietitian and results are given in Table 1. Sample preparation
Venous blood from subjects was drawn on CPD anticoagulant mixture (10 vol. collected onto 1.4 vol. CPD: sodium citrate 2.36%, glucose 2.32%, citric acid 0.32% and NaH2P04 0.22% by weight in water and adjusted to pH 5.6). Platelet-rich plasma (PRP) was prepared by centrifuging blood for 10 min at 100 x g. After addition of EDTA (1 mM), PRP was acidified to pH 6.4 by using 0.15 M citric acid and was centrifuged for 15 min at 900 x g in order to pellet platelets and plateletpoor plasma was used for the lipoprotein analysis. Lipoprotein separation
Lipoprotein
classes,
namely
VLDL,
LDL,
HDL2 and HDLs were separated by ultracentrifugation using the NaClKBr density gradient and the procedure described by Chapman et al.
PI. Each lipoprotein class was divided into aliquots for the NMR study, lipid separation and protein and phosphorus determinations. The procedure of Peterson [lo] was used for protein determination. Lipid analysis
Lipoprotein lipids were extracted according to the method of Folch et al. [ 111. Cholesterol esters (CE), triglycerides (TG), and total phospholipids (PL) were separated on columns of silicic acid 100 mesh/super Cell (Mallinkrodt), 2:l w/w, using appropriate volumes of the following mixtures: hexane/CHCls (80:20, v/v), CHCls and CHC13/methanol (l:l, v/v). CE, TG and free cholesterol (FC) were separated by thin layer chromatography using Chromarod S-II quartz rods coated with silica gel and hexane/diethyl ether/formic acid, (54:6:0.08, by vol.) as the solvent system. Quantification of the lipid classes was performed on Iatroscan TH-10 TLC analyser (Iatron Laboratories, Tokyo, Japan) equipped with a flame ionization detector linked to an Enica 2 1 integrator-calculator (Delsi, Paris, France). Cholesteryl acetate was used as an internal standard to determine the response factors [12-141. The phosphorus content of PL was determined by the method of Bartlett [15].
243
'H-NMR
Fatty acids of CE, TG and PL were transmethylated by a mixture of methanoYH2S04 (98:2, v/v) at 100°C for 3 h after addition of heptadecanoic acid as an internal standard. After extraction by hexane, fatty acid methyl esters were analyzed on a Beker-Packard Model 417 gas chromatograph, equipped with a 30 m x 0.4 mm I.D. glass capillary column coated with Carbowax 20 M (Applied Science Labs., State College, PA) at a constant temperature of 195°C and a nitrogen flow-rate of 3 ml/min. Quantitation was performed using a flame ionization detector and an Enica 21 integrator-calculator. Calibration factors were calculated using standard mixtures of fatty acid methyl esters (Nu-Check-Prep, Elysian, MN).
TABLE
spectra
Lipoprotein fractions were dialysed against PBS buffer (pH 7.4) for 48 h at 4°C and concentrated by ultrafiltration through Centricon 30 microconcentrators (Amicon Corp., Danvers, MA) at 1500 x g for 30 min to obtain final volumes of 0.4 ml. Samples were prepared in j-mm NMR tubes by mixing 0.4 ml of lipoproteins with 0.1 ml of D20. Peaks were referenced to sodium 3-trimethylsilyl-[D4]propionate (TMSP) as an internal standard. Fourier transform ‘H spectra were obtained at 20°C on a Bruker WM 400 spectrometer system operating at a proton frequency of 400.13 MHz and equipped with an Aspect 2000 computer. The
2
LIPOPROTEIN
COMPOSITION
IN YOUNG
AND OLD WOMEN
Values are means f SE (n = 6). Young
women
mg/lOO ml
Old women weight %
mg/lOO ml
weight %
VLDL CE TG FC PL
9.1 39.8 3.5 13.6 10.2
f 0.8 f 9.9 ?? 0.3 f 0.9 f 0.9
11.5 ?? 1.2 52.1 f 5.2 4.4 ?? 0.2 18.8 f 3.2 13.3 * 1.2
11.9 41.8 5.0 13.0 10.6
f 1.5 ZIZ7.5 * 0.9 f 0.7 zt 2.8
14.7 50.1 6.0 16.7 2.5
f f + f f
0.8 2.9 0.5 1.7 1.9
zk 14.0* f 5.0: f 5.3 zt 8.1 f 7.4*
43.7 8.2 8.2 17.0 22.9
f & it f f
1.2 0.5 0.8 1.7 0.4
zt f f + zt
3.5 0.7 0.3 2.9 3.2
25.0 5.4 4.5 32.2 32.9
+ 3.1 ?? 1.1 + 0.6 f 3.4 zt 1.6
zt 3.7 f 1.0 z+ 0.5 zt 3.2 f 7.0
14.1 3.4 2.4 24.3 55.8
f f zt f zt
LDL CE TG FC PL
123.3 23.5 24.7 67.4 17.5
f f f f f
16.6 2.5 3.0 5.2 4.6
38.3 7.6 7.7 21.7 24.7
zt 2.9 zt 0.9 ?? 0.5 f 2.0 + 1.0
187.6 37.1 36.0 72.2 93.4
f f f f f
3.1 0.6 0.7 2.6 2.4
19.2 4.0 3.3 24.1 25.1
f f f zt f
1.3 0.3 0.1 2.3 2.8
27.4 6.7 4.6 45.8 105.9
HDL, CE TG FC PL Proteins
f 2.7
18.1 3.5 4.5 25.0 26.0
f 0.7 f 3.2 zt 4.5
22.6 4.6 5.5 31.8 35.6
36.3 5.0 4.4 51.8 116.9
zt 7.5 f 0.7 ?? 0.7 zt 6.6 + 6.5
17.4 2.4 2.0 26.6 54.6
?? 0.4
HDL, CE TG FC PL Proteins ‘Significant
difference
between
young
and old women
at P < 0.05.
1.3 0.4 0.3 1.8 1.7
244
water resonance was suppressed by a presaturation pulse at the frequency of the water resonance at 400.13 MHz. Sixteen free-induction decays were signal-averaged and Fourier transform was performed. Tr values were measured according to Vold et al. [ 161 by using the (a, 7, 7r/2) inversion recovery sequence. T, values are accurate within 2% which is in the range reported previously [17,18]. Statistical analysis
Young women were compared with old women by using Student’s I-test [19] or the t’-test [20] when sample variances were not equal. Results
There was no significant difference between the dietary characteristics of young and old women (Table 1). However, it seems that the consumption
TABLE
3
FATTY ACID COMPOSITION YOUNG AND OLD WOMEN
OF CHOLESTERYL
Values are mol % of total fatty acids, means Fatty
acid
Cholesteryl Young women
14:o 16:O 16:l 18:O 18:l 18:2 18:3 2o:o 20:3 20:4 20:5 22:o 22:1 22:4 22:5 22:5 22:6 24:0 24:1
of lipids and particularly that of monounsaturated fatty acids of young women tended to be higher than that of the old women. Table 2 shows that the proportions (expressed as weight %) of the different lipid classes and proteins were not significantly different between the two groups. However, the absolute level (expressed as mg/lOO ml) of LDL was higher in the elderly compared with young subjects. This difference is mainly due to higher levels of CE, TG and proteins in the LDL from old women. The total lipid/ protein ratio in LDL from aged women (3.37 f 0.09) was not significantly higher than that from young women (3.16 A 0.20), suggesting that the number of LDL particles was increased in the aged group. The fatty acid composition of CE, TG and PL of VLDL, LDL, HDLz and HDL3 are reported in Tables 3, 4, 5 and 6, respectively. The proportion of linoleic acid (18:2) in CE and PL from all the
(n-6)
(n-6) (n-6) (n-3) (n-9) (n-6) (n-6) (n-3) (n-3)
0.3 16.5 6.4 3.8 25.0 39.1 1.6 1.0 0.4 3.4 1.5 -
f zt + f f f f f * zt zt
0.1 2.5 1.0 0.5 1.4 2.1 0.4 0.5 0.1 0.6 0.6
TRIGLYCERIDES
* S.E. for 6 determinations
(SE.
Young women
Old women
2.3 15.8 1.9 3.0 26.5 34.0 1.4 1.0 0.3 4.6 1.9 -
0.4 34.0 5.0 7.0 30.6 16.2 1.9 0.6 0.6 1.5 0.7
0.4 31.4 7.4 5.3 38.9 10.9 1.4 0.5 0.2 1.7 0.6
f 0.0 zt 6.5 f 0.6 ?? 1.3 f 4.7 f 5.1 zt 0.6 + 0.1 zt 0.4 f 0.4 f 0.4
f f f f f f f f f f f
0.1 2.4 1.1 0.9 1.1 1.1* 0.1 0.0 0.0 0.3 0.2
0.6 zt 0.3 0.6 f 0.1 -
0.8 f 0.4 0.8 * 0.2
OF VLDL FROM
Phospholipids
Old women f 1.8 f 2.1 ?? 1.5 f 0.4 zt 2.1 * 2.1* f 0.2 f 0.1 f 0.1 f 1.6 zt 0.6
AND PHOSPHOLIPIDS
< 0.1 are noted as 0.0).
Triglycerides
esters
0.8 f 0.6 0.2 f 0.2
ESTERS,
0.4 f 0.1 1.0 f 0.1
(n-9)
*Significant difference between young and old women at P < 0.05. When the position represents the sum of all the isomers.
Young women
Old women
1.1 28.9 6.0 11.0 22.1 16.8 1.1 0.9 1.2 4.2 0.8 0.9 0.2 0.3 0.5 0.6 2.1 0.7 0.4
0.8 32.3 7.1 11.4 22.9 11.3 0.8 0.7 1.5 4.0 1.1 0.8 0.2 0.4 0.1 0.8 2.4 0.6 0.5
f zt f f f f f f f zt zt f * f f f + f
0.3 1.0 1.2 0.9 2.7 1.3 0.3 0.1 0.3 0.9 0.1 0.1 0.0 0.1 0.4 0.1 0.6 0.1 ?? 0.1
f zt f f zt f zt f f zt f f * f f f zt f *
0.2 1.4 1.8 1.1 2.8 1.3’ 0.1 0.1 0.4 0.6 0.2 0.1 0.1 0.1 0.1 0.2 0.4 0.4 0.3
of the last double bond is omitted,
the value
245 TABLE 4 FATTY ACID COMPOSITION OF CHOLESTERYL ESTERS, TRIGLYCERIDES YOUNG AND OLD WOMEN Values are mol % of total fatty acids, means Fatty acid
14:o 16:0 16:l 18:O 18:l 18:2 (n-6) 18:3 2o:o 20:3 (n-6) 20:4 (n-6) 20:5 (n-3) 22:o 22:l (n-9) 22:4 (n-6) 22:5 (n-6) 22:5 (n-3) 22:6 (n-3) 24:0 24:l (n-9)
?? S.E.
for 6 determinations (S.E. < 0.1 are noted as 0.0).
Triglycerides
Cholesteryl esters
AND PHOSPHOLIPIDS OF LDL FROM
Phospholipids
Young women
Old women
Young women
Old women
Young women
Old women
0.1 f 0.0 14.1 f 1.8 3.5 f 0.4 1.5 f 0.1 21.3 + 1.1 52.5 zt 2.2 0.9 f 0.1 0.2 f 0.0 0.5 f 0.1 4.6 zk 0.6 0.4 f 0.1
0.5 ?? 0.4 15.8 + 0.9 6.2 f 0.7’ 1.1 f 0.1 25.7 zt 1.0’ 41.8 f 1.4* 1.4 ?? 0.2 0.1 f 0.0 0.6 f 0.1 5.2 f 0.7 1.2 ?? 0.5
0.3 ?? 0.1 27.6 + 1.7 6.3 f 0.5 6.0 zt 0.5 32.1 zt 2.0 19.1 ?? 1.9 1.5 ?? 0.2 0.7 f 0.1 0.3 ?? 0.0 3.5 f 0.4 0.6 zt 0.3
0.3 ?? 0.1 28.6 f 1.6 6.7 zt 0.5 4.9 f 0.7 37.3 * I.78 12.2 f 1.1* 1.4 f 0.1 0.4 f 0.0 0.3 f 0.1 3.8 f 0.9 1.8 f 0.6
0.4 ?? 0.1 29.9 * 0.6 3.7 f 1.1 11.9 f 0.5 15.1 f 1.7 18.8 f 1.6 0.6 zt 0.1 0.9 f 0.1 1.8 f 0.3 6.1 f 0.9 0.7 f 0.1 2.0 f 0.3 0.1 f 0.0 1.1 ?? 0.2 0.2 zk 0.1 0.5 f 0.1 2.5 f 0.2 1.5 f 0.2 2.2 f 0.4
0.4 f 0.1 32.4 f 0.8 3.0 f 0.4 11.5 f 0.6 15.4 ?? 1.1 13.7 f 1.5* 0.5 f 0.1 0.9 f 0.1 1.9 f 0.3 6.4 f 0.2 1.2 f 0.3 1.6 f 0.1 0.1 f 0.0 1.1 f 0.1 0.2 ?? 0.1 1.0 f 0.2* 3.9 * 0.5: 1.3 f 0.1 3.2 zt 0.8
-
0.1 f 0.0 0.3 * 0.1
-
-
0.9 f 0.4 1.0 ?? 0.3 -
0.8 f 0.4 1.5 ?? 0.3 -
*Significant difference between young and old women at P < 0.05. When the position of the last double bond is omitted, the value represents the sum of all the isomers.
lipoproteins studied was significantly lower in the elderly than in young women. This was also found in TG but only from VLDL and LDL. Conversely, the proportion of oleic acid (18:l) in old women was higher in CE from LDL, HDL2 and HDLs as well as in LDL-TG and HDLs-PL. The higher proportion of 18:l tended to be associated with a higher proportion of palmitoleic acid (16: l), and reached significance in LDL-CE. The percentage of palmitic acid (16:0) also tended to rise in some lipid classes of lipoproteins from aged women. In PL from VLDL and HDL2 the lowered proportion of 18:2 was not significantly balanced by any particular fatty acid. The proportions of polyunsaturated fatty acids other than 18:2 were not affected by aging, except 22:5(n-3) and 22:6(n-3) which were higher in PL of LDL and HDLs from aged women. Such an increase was also found for 22:5(n-3) in HDL,-PL.
T, values obtained for resonances of the different chemical groups were not significantly different with the exception of those of the methyl groups which were significantly decreased in VLDL and HDLs from elderly women (672.8 zt 29.3 versus 580.2 f 10:0 and 357.8 f 4.3 vs. 326.8 f 3.2 ms for VLDL and HDLs of young and old women respectively). Discussion The lipoprotein composition of the two groups of subjects agrees with the values obtained by the method of separation used elsewhere [9]. The lack of differences between the HDLs cholesterol content of the two groups agrees with data reported for octo- and nonagenarians [5-71 but not with the decreased lipid/protein ratio reported in HDL2 of old subjects [6].
246
TABLE
5
FATTY ACID COMPOSITION YOUNG AND OLD WOMEN
OF CHOLESTERYL
Values are mol % of total fatty acids, means Fatty
acid
140 160 161 18:O 18:l 18:2 (n-6) 18:3 20:o 20:3 (n-6) 20:4 (n-6) 20~5 (n-3) 22:o 22:1 (n-9) 22:4 (n-6) 22:5 (n-6) 22:5 (n-3) 22:6 (n-3) 240 241 (n-9)
Cholesteryl
esters Old women
0.4 15.1 5.3 3.3 19.5 46.1 1.9 1.2 0.4 5.2 0.4 -
0.5 20.5 1.2 5.0 24.1 33.3 0.9 0.7 0.5 5.2 1.2
0.2 ?? 0.0 0.6 zt 0.1 -
TRIGLYCERIDES
f S.E. for 6 determinations
?? 0.2 ?? 3.1
* 1.2 ?? 2.1
f zt * f f zt f
2.0* 5.2* 0.2 0.3 0.1 1.7 0.5
0.4 0.4 -
?? 0.2 ?? 0.3
Old women
0.6 29.5 1.4 12.2 29.3 12.0 1.5 1.4 0.3 3.0 0.6
0.5 26.9 8.3 1.2 31.4 12.9 1.6 0.9 0.3 6.3 1.8
-
-
1.7 ?? 0.2 0.3 f 0.0 -
0.7 1.0 -
f 0.2 zt 2.9 + 0.9 ?? 0.5 f 2.5 zt 1.6 zt 0.1 ?? 0.2 f 0.1 zt 0.7* f 0.6
?? 0.4 ?? 0.1*
‘Significant difference between young and old women at P < 0.05. When the position represents the sum of all the isomers.
Although it is known that contraceptive hormones can modify the proportions of plasma triglycerides [3,4] no difference was observed between plasma profiles of young females taking or not taking hormones. However, as the levels of TG, CE and apolipoproteins vary throughout the menstrual cycle [21], this was probably due to the small number of each group of young women. On the other hand, the proportions of 18:2 were similar in these groups. This is not surprising since our previous report [8] showed that age-induced lowering of plasma 18:2 affected both genders and therefore this phenomenom did not seem directly related to sex hormones. Consequently, data from both groups of young females were pooled. The low proportion of 18:2 in the aged women agrees with our previous results obtained in whole plasma [8], although no reduction of arachidonic
OF HDL,
FROM
Phospholipids
Young women zt 0.2 f 1.7 f 1.3 ?? 3.5 f 1.5 f 1.4 f 0.1 ?? 0.1 f 0.1 f 0.8 zt 0.3
AND PHOSPHOLIPIDS
(S.E. < 0.1 are noted as 0.0).
Triglycerides
Young women ?? 0.1 zt 1.6 ?? 0.3 f 1.3 ?? 1.1 * 4.6 ?? 1.0 ?? 0.7 f 0.1 f 0.6 ?? 0.1
ESTERS,
Young women
Old women
0.9 31.6 4.9 13.3 17.0 16.2 0.6 0.8 1.3 5.4 0.8 1.4 0.2 0.6 0.2 0.5 2.2 1.1 0.8
1.4 26.1 4.6 16.3 19.6 11.3 1.0 1.4 1.1 6.0 1.4 1.6 0.4 0.6 0.4 1.7 2.3 1.2 1.4
f 0.2 zt 1.2 f 0.9 ?? 1.0 f 1.6 zt 1.3 f 0.1 zt 0.1 f 0.1 ?? 0.4 f 0.1 f 0.1 f 0.1 zt 0.1 f 0.1 f 0.1 * 0.5 ?? 0.1 f 0.2
f zt zt f f f f f f f
0.8 5.9 1.1 3.3 0.9 0.7* 0.2 0.5 0.4 0.6 ?? 0.2 ziz0.4 f 0.2 f 0.0 f 0.2 zk 0.8* f 0.4 * 0.5 f 0.9
of the last double bond is omitted,
the value
acid (20:4) was found in the present work, but rather an increase in TG of HDL2 and HDL3. Although the 18:2 intake by elderly women was lower than that of the young women, it was nevertheless not statistically different and in the range of values commonly accepted as adequate to prevent essential fatty acid deficiency [22]. Moreover, the low proportion of 18:2 in plasma of old women was not associated with any appearance of 20:3(n9), the biological marker of essential fatty acid deficiency, which was also hardly detectable in the control group. The lowering of plasma 18:2 does not seem directly related with the 18:2 intake since the consumption of 18:2 by old people investigated in our previous work [8] was twofold higher than in the present study though the plasma proportion of 18:2 from elderly people was similar in the two studies. On the other hand, we found an increased
24-l
TABLE
6
FATTY ACID COMPOSITION YOUNG AND OLD WOMEN
OF CHOLESTERYL
Values are mol % of total fatty acids, means Fatty
140 16:0 16:l 18:0 18:l 18:2 18:3 20:o 20:3 20:4 20:5 22:o 22:l 22:4 22:5 22:5 22:6 24:0 24:l
acid
(n-6)
(n-6) (n-6) (n-3)
Cholesteryl
esters
ESTERS,
f S.E. for 6 determinations
(SE.
Old women
Young women
0.2 13.9 4.3 1.8 19.0 51.8 1.0 0.4 0.6 5.6 0.8
0.9 17.3 7.0 1.7 25.2 38.8 1.0 0.2 0.5 5.1 1.5
0.4 31.0 7.9 7.5 32.8 15.0 1.6 0.9 0.1 1.6 0.2
f f f f zt f f
0.7 1.1 1.1 0.4 1.6; 3.2* 0.2 ?? 0.1 * 0.1 ?? 1.5 f 0.7
(n-9) (n-6) (n-6) (n-3) (n-3)
0.2 f 0.1 0.5 f 0.1
0.2 * 0.1 0.5 f 0.3
(n-9)
_
-
f zt f f * zt zt f f f f
0.2 2.7 0.6 0.5 1.9 3.2 0.3 0.0 0.1 0.2 0.1
0.5 f 0.2 0.4 f 0.1
OF HDL,
FROM
Phosphohpids Old women
Young women
Old women
0.5 30.0 8.1 6.6 31.9 12.6 1.6 0.9 0.4 4.3 1.2
0.6 30.5 2.7 12.2 13.6 21.0 0.5 0.6 1.8 6.5 2.0 1.4 0.3 0.6 0.3 0.6 2.6 1.2 1.1
0.6 31.6 4.0 11.9 16.6 13.9 0.5 0.7 2.0 7.7 1.3 0.9 0.1 0.6 0.2 1.2 4.2 0.6 I.1
f f zt f zt zt zt
0.1 4.3 0.8 1.3 2.9 3.4 0.3 ?? 0.2 f 0.1 + 0.8* f 0.4
0.7 ?? 0.4 0.9 f 0.3
‘Significant difference between young and old women at P < 0.05. When the position represents the sum of all the isomers.
proportion of n-3 long chain fatty acids such as 20:5 and 22:5 as observed previously in total plasma [S], in addition to an enhanced 22:6 in the present work. These different changes of fatty acid composition are analogous to the age-induced changes in fatty acid composition of several rat tissues [23]. In aorta, liver and adipose tissue, the 18:2 proportion was lowered, that of 20:4 unchanged and those of n-3 fatty acids enhanced. Moreover, in adipose tissue the proportions of n-9 fatty acids were significantly enhanced. All these data suggest that the effects of age on the plasma fatty acid composition affect similarly humans and other mammals. On the other hand, a parallel could be established between the plasma 18:2 decrease associated with the slight increase of LDL cholesterol and the recent observation showing that plasma 18:2 is negatively correlated to LDL cholesterol in European men [24].
AND PHOSPHOLIPIDS
< 0.1 are noted as 0.0).
Triglycerides
Young women f 0.1 f 2.2 f 0.6 f 0.6 f 1.7 f 4.5 f 0.1 f 0.2 zk 0.0 zt 0.5 ZIZ0.1
TRIGLYCERIDES
zt f f f f + f f zt zt f f f f f zt zt f
0.2 1.4 0.4 0.6 1.2 0.8 0.1 0.0 0.2 1.2 1.2 0.2 0.2 0.1 0.1 0.1 0.6 0.4 ?? 0.2
+ zt zt f f + f f f f f f f zt f f
0.1 0.9 0.6 0.5 0.6’ 0.6; 0.1 0.1 0.3 0.4 0.2 0.1’ 0.0 0.0 0.1 0.1* ?? 0.3* f 0.1 f 0.3
of the last double bond is omitted,
the value
The lowered proportion of 18:2 in elderly women was most often reciprocally accompanied by higher proportions of 18:l and 16:1, although not significantly with exception of cholesteryl esters. It is unlikely that such an increase would be related to the diet since the monounsaturated fatty acid intake of old women tended to be lower than that of young women. Since the proportion of 18:1 was enhanced in cholesteryl esters, it is expected to observe the same increase of 18:l from HDLJ phospholipids, part of this lipid class acting as a substrate for 1ecithin:cholesterol acyl transferase [25]. A replacement of 18:2 by monounsaturated fatty acids in cholesteryl esters might suggest a reduction of lipoprotein fluidity, particularly in LDL which carry the main part of this lipid class. Moreover, it has been reported that dietary 18:2 specifically influences LDL fluidity [26]. Relaxation times (rt) of chemical lipid groups revealed
248
that only the motion of CHs groups tended to be restricted in all the lipoproteins from elderly people, and this reached significance in VLDL and HDLs. NMR spectra were performed at 20°C below the midpoint transition temperature of cholesteryl ester mixtures [27] and below the temperatures of the two main phase transitions of core lipids [28]. Under these conditions the methyl groups, associated with the cholesterol ring structure, do not show measurable signals [28,29], the T, changes reflecting essentially those of the terminal methyl groups of acyl chains. This result suggests that the physical behaviour of lipid within VLDL and HDLs could be affected by the changes of fatty acid composition in the old women. However, further detailed NMR and other physical studies are necessary to confirm this.
8
9
10
11
12
13
Acknowledgments 14
This research was supported by the Ministere de la Recherche et de 1’Enseignement Supkrieur Grant MRES 87 G 0556.
15
References
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