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Decrease of eicosapentaenoic acid in fatty liver of diabetic subjects
Prostaglandins and Medicine 5: 183-200, 1980
DECREASE OF EICOSAPENTAENOIC ACID IN FATTY LIVER OF DIABETIC SUBJECTS Peter Singer x, Gerhard Honigmann and Volker Schliack. XCentral Institute of Cardiovascular Regulation Research, Academ~ of Sciences of the GDR, DDR-1115 Berlin, and Centre of Diabetes and Metabolic Diseases, 102 Berlin, GDRo (reprint requests to PS)o ABSTRACT Simultaneous biopsies of liver and subcutaneous adipose tissue have been carried out in 228 patients with diabetes mellit-as. In liver trigl~cerides a msmked variability of the fatt~ acid pattern in relation to fatt$ degeneration of liver psmench~ma has been confirmed. In adipose tissue fatt2 acid pattern was relativel2 constant. The most striking finding was a high content of eicosapentaenoic acid in normal liver and its dedrease with the rise of lipid droplet size in the hepatoc~tes. No correlation with the quantit~ of liver fat or inflammator2 liver diseases could be ascertained. W h e n diabetes was associated with b~yperlipoproteinemia (HLP) the percentage of eicosapentaenoic acid was significantl7 less. From the results the suggestion is obvious that a diminution of eicosapentaenoic acid in hepatic trigl2cerides is associated with lipid accumulation in the liver cells. A local mechanism, possibl$ the antilipol~ic potenc7 of prostaglandins, ma~ be responsible for the creation of lipid droplets in liver cells of patients with metabolic disturbances. The alterations of eicosapentaenoic acid should be considered in view of recent data on the antiaggregator2 effect of this fatty acid and its possibl$ preventive role for atherosclerosis. INTRODUCTION Elcosapentaenolc acid (C20:5) has recently been supposed to pla~ an important role in the prevention of cardiovascular diseases (I). It represents the precursor of the antiaggre~ating prostaglandin I~ (PG I3), whereas thromboxane A3 (TXA3) does not induce aggregation (2,3).
183
The low incidence of m2ocardial infarction in Eskimos as well as their prolonged bleeding time are probably due to the high eicosapentaenoic acid and low linoleic and arachidonic acid content of their nutrition and consequently of their tissues (2). Thus, polyunsaturated fatty acids on the whole may be of less relevance to the prevention of Gardiovascular diseases than the individual substance eicosapentaenoic acid (I). Possibl~, the liver is involved in essential fatty acid metabolism (4). Other functions of eicosapentaenoic acid are not ~et clearl2 defined. From human tissues, for example, there is a high percentage of eicosapentaenoic acid in the brain (5) as well as in eruptive xanthomas of patients with h~perlipoproteinemia (6), the latter being no inert deposits but formations of high metabolic activity (6,7). In previous studies distinct alterations in the fatt x acid pattern of liver triglycerides have been described in fatt~ degeneration of the liver (8) which both can be influenced by clofibrate treatment (9). An increase of palmitic and oleic acid as well as a decrease of arachidonic and eicosapentaenoic acid associated with fatty liver have been attributed to the antilipolytic action of PGE 2 and PGE 3 (8,q0). But no distinct concept of the pa~hoph~siological mechanism could be devised at that time. Since the discovery of PG!2 (ql) research work has been focused on the products of the cyolooxygenase cascade. Recently, an antilipolFtic effect of PGI 2 more potent than of PGE 2 has been proved (12). No data, however, dealing with an effect on lipolysis of PGI~ are available. In the present paper the behaviour of eicosapentaenoic acid of liver triglFcerides in relation to lipid droplet size in liver cells and chronic hepatitis will be described.
m m ALs
m HODS In 228 diabetic subjects, 98 of whom had hyperliPoproteinemia (HLP), blood was withdrawn by venous puncture from the antecubital vein after an overnight fast~ Some clinical and biochemical data of the patients are summarized in Table I and 2. Diabetes mellitus was well compensated during the study. ~ P was diagnosed when serum triglycerides were above 250 mg/100 ml and cholesterol was above 300 mg/100 ml. The patients were kept in hospital at least two weeks before the biopsies. 2q % of the diabetics without HLP and 18 % of the d!abet.cs wmth H L P w e r e on i~Tpoglycemlc drugs. All other patients received insulin (Table q).
184
TABLE 1: CLINICAL DATA OF TEE DIABETICS WITHOUT AND WITH HLP J
L
,
without HLP Diabetics (n) Women ~en Axe ' (Mean~SD) Women Men Rel. bod~ weight a Women Men Duration of diabetes ~7poglycemic agents ~ I Insulin therapy
120 72 58 55,2±12,8 59,3~13,6 52,2~14,8 114 ±20 q17 !2fl 111 ±22 6,0± 5,1 21 109
with HI~ 98 73 25 56,O!11,8 6d,2~I0,8 @8,2$16,0 122 ±16 q25 ±18 116 ±20 9,0± 5,8 18 80
a Metropolitan Life Insurance Compan~ Tables - (13). If clinically indicated liver biopsies by Menghini tech nique (17) were performed. One half of the biopsy specimens was fixed in 10 % formalde&Tde for routine histological examination (HE, HS, Sudan III, van Gieson, Turnbull's blue, Fouchet). The other part was carefully cleaned in chilled 0,9 % saline solution and stored at -20°C for gas liquid chromatograph~. About 3 hours later a sample of subcutaneous adipose tissue of the abdominal wall (200-500 mg) was excised after intracutaneous anesthesia (procaine fl %) and a small iofraumbilical incision. Adipose tissue was also stored at -20oc until gas chromatographic analyses. The lipids from serum and tissues were extracted with chloroform-methanol (2:1, v/v) according to Renkonen et al. (18). Total lipids were isolated by thin la~er chromatograph7 on silica gel G (Merck, Darmstadt). The plates were developed in hexane-diethylether-acetic acid (73:25:2, v/v). The fractions were visualized under UV-light after the plates had been sprayed with a solution of 0 , 1 % dichlorfluorescein in ethanol. The triglycerides were scraped into test tubes. The esterification was achieved by 0,5 N sodium methTlate. After evaporation under nitrogen the samples in hexane solution were injected direct&2 into a gas chromatograph (GCHF 18,3, VEB Chromatron, Berlin, flame ionization Aetector). Analyses of fatt2 acid metkTlesters were performed on columns (2 m x 4 mm) of 10 % dieth~leneglycol succinate, stabi-
185
lized (Analabs, North Haven, USA) on Gas Chrom P (q00-120 mesh), isothermal at 200~C° The components were identified bF their retention time relative to authentic standards (Applied Science Laboratories, State College, USA). The quantit~ of each fatt~ acid was estimated from the product of peak height and retention time. The method has been published previousl2 in detail (19). Histological findings were classified into 4 categories according to thelipid droplet size independent of the amount of fat: no visible fat, prevailing small 'lipid droplets (mean±SD: 1,74-0,26/um, range O-2,5/um), medium-sized droplets (9,65±6,60/um, range 2,5-14,5/um) and big lipid droplets (19,06±8,1~/um, over 14,dpam)° This classification has been proved b~ mbrphometric sthdies(8). In t~pe IIa HLP no or small lipid droplets were found but in t y p e I V big droplets were predominant in the liver (Table 3)° Mixed forms were subdivided into small to medium-sized, medium-sized to big and small tp big lipid droplets in hepatocFtes (Table 2). The significance of the results was assessed bF Student's t-test. TABLE 2: BIOCHEMICAL DATA OF DIABETICS WITHOUTAND WITH HLP IN RELATION TO LIPID DROPLET SIZE IN LIVER C E L ~ a) without HLP Size of lipid droplet s not visible small medium-sized big small to medium-sized medium-sized to big small to big
Fasting a blood sugar (mg/100 ml)
n
TriglT- b cerides (mg/lO0 ml)
Bromsulfalein retention (%)
20 17 11 29
138329 154~43 166m40 q60±38
1133 12q z 138~ q#5 z
30 27 25 31
4 ,0~3,4 + #,7~2,5 3,7~2,4 6,4~3,3
9
144±39
114± 29
#,8±3,3
16 28
fl53~36 q55m38
133~ 35 q#3 ~ 39
5,4~2,8 4,4~3,q
153
132
130
±38
"I"
34
I4 , 9 ± 3 ,
3
b) with HLP -,
i
not
i
ii
i
visible
9
'135~51'
- 677~625 '+ ..........
small medium-sized big •small to medium-sized medium-sized
13 10 25
132437 158z49 160±37
1103~951 9885680 88flz547
3,6~2,3 4,2Zl,6 4,5~2,1 5,7~2,8
4
flfl4!38
695±576
4,2±0,8
to
2#
q#6~29 q62z23
806±4#2 979±774
6,q~3,8 5,8~3,0
1#8I±I~6 '
890±614
5,2{2,9 ....
big
small to big .
.
.
.
q3 9 8
....
a Lange et al. (14). b Stolz et al. (15). 186
TABLE~: TYPES OF HIP IN RELATION TO THE SIZE OF HEPATIC LIPID DROPLETS; COLUMNS REFER TO THE FREDRICESON CLASSIFICATION OF HYPERLIPOPROTEINEMIAS (16). • Size of without lipid droplets H]~P lla lib III IV V not visible small medium-sized big small to medium-sized medium-sized to big small to big ,
i
L
20 17
1 0 1
0 0 0 0
4 7 9 19
2 3
29
2 2 0 0
9
1
1
0
1
1
16 28
0 0
2 4
0 I
18 6
4 2
1_30
5
10
1
6L~
18
11
1
1
5
i
RESULTS The percentage of eicosapentaenoic acid in diabetics with noymal livers is high (about 30 per cent). In adipooe tissue it is low (under I per cent) whereas in serum trigl~cerides it lies between liver trigl~cerides and depot fat (about 3 per cent). The high percentage of eicosapentaenoic acid in triglycerides of normal liver decreases with rise of fat droplet size in hepatocytes (Fig. I). In diabetics without HLP this decrease is more pronounced than in diabetics with HLP (Fig. 2 and 3). On the other hand, palmitic and oleic acid increased with the size of hepatic lipid droplets. In spite of some differences between diabetics without and with I~LP the tendencies of the major fatty acids were the same (Fig. 2 and 3). In diabetics with kT~P significantly higher percentages of palmitic and oleic acids as well as lower percentages of arachidonic and eicosapentaenoic acids in liver trigl~cerides could be observed when compared with diabetics without HLP. This might be due to thepre v a l e n c e of fatty degeneration with big lipid droplets in diabetics with HLP (Table 3). Standard deviations and significance levels have been omitted in Fig. 2 and 3 because onl~ eicosapentaenoic acid is the topic of this presentation. In livers containing big lipid droplets the fatty acid pattern of triglycerides became similar to the depot fat, i.e. adipocytes. There was no evidence of other factors being involved in this correlation. Since in free fatty acids (FFA) and in serum trigly~erides of normal subjects eicosapentaenoic acid is low (Table 4) this fatty acid must derive from local synthesis or its deterioration in the liver.
187
percent I o) Liver
40
z~ =p.~o.ol o = P':O.05
30 2O
10 ....
L-.- 0 ~
L . . - g~---J k-.. ~ . , . . ~ L . . - A - . - l k - - ~ - - - J
L - -
L~
. J
per cent
lO:
b) Serum
L
~
0
[
n
20
'
17
normo/ Smo//
.
j ~_..- ~, . ~ J
0
J
II
29
medium,
sized
big
9
16
smoJ/to medium-
28 srno//
medium- sized tob/9 sized tobig
Figure I. Eicosapentaenoic acid of triglFcerides in liver, serum and adipose tissue in relation to lipid droplet size in hepatoc~tes (abscissa)
188
Liver
Adipose tissue
C
&ze of
hp/ddroplets ¢12
CI~: 1
C76 - = 7 6 . 0 ~
22..a ~,0
Ceo.$~ 6 . o = n
Figure 2.
20
17
o.6 1I
29
130
Fatt~ acid pattern of trigl~cerides in hepatoc2tes in relation to lipid droplet size as well as in adipose tissue of diabetics without HIP.
189
Ad,~osetissue
Liver ci ,..1
-=20.$~ C,,:, . ~ 3 7 . 2 ~ I
~6
C16
~7
~6
c/8..3~ / . 3 = c20.3~ 0 . 4 ~ .
43
C2o:,~ o . 8 = .
/,2
C;o..s~ 3 . n
9
13
/=. I0
O.7
25
Figure 3. Fatt2 acid pattern of trigl2cerides in hepatoc2tes in relation to lipid droplet size as well as in adipose tissue of diabetics with HIE>o
190
TABLE
4 :
FATTY ACID PATTERN OF F F A A N D SERUM TRIGLYCERIDES IN NORMAL SUBJECTS (YET UNPUBLISHED DATA). FFA n = 11
Tri~l~cerides n = 11
C12
-
0,2+0,4
C14
0,6+0,6
1,7+1,0
C16 016:1 C18 018:1 C18:2 C18:3
24,3+2,2 5,9+1,5 12,3+2,1 38,1~,6 8,7+1,6 fl,2+0,5
21,6+1,5 4,7+1,3 3,9+0,4 ~,2+1,8 12,7+2,0 1,2+0,7
C20:4
2,6.T-O,9 1,7+1,2 1,1+1,8
2,6.@4D,5 1,6+0,6
020:5 C22 TABLE ~:
0,3+0,1
SELECTED FATTY ACIDS OF LIVER TRIGLYCERIDES AT A LOW (a) AND HIGH (b) DEGRk~ OF STEATOSIS IN DIABETICS WITHOUT ANDWITHHI~P.
Without HLP n
a) 1~ b) 12
C16
27,2±#,3 27,7±~,#
Cfl8:1
41,3+3,3 42,3+3,2
C18:2
C20:#
4,9+2,1 5,1+2,1
0,8+0,3 O, 7.T-O,5
C18:2
C20:4
C20:5
5,7+2,2 6,1+2,9
With KLP n a)
11
b) 12
C16
27,7±2,6 28,3~1,9
C18:1
#7,8!1,8 48,2±2,2
6,5±1,7 6,0!1,4
C20:5
0,8.~D,3 3,3±2,1 0,7±0,# 2,851,3
a) = big lipid droplets in less than 30 per cent of liver cells b) = big lipid droplets in more than 50 per cent of liver cells.
191
TABLE 6: SELECTED FATTY ACIDS OF LIVER TRIGLYCERIDES
IN CHRONIC HEPATITIS Without HLP n C16 No hepatitis 54 Chronic persist. hepatitis 45 Chr on. -aggr e s s. hepatitis 8 Fibrosis 23
C18:1
C20:5
22,8!5,5
35,6±7,2
15.7±10,0
23,1±5,2
37,4±8,0
13,3+12,8
22,4+6,3 24,1±6,0
35,4±9,5 39,5±7,5
11,7 + 8,0 12,1 + 9,7
With HLP n No hepatitis 42 Chronic persist. hepatitis 42 Chronic-aggress. hepatitis 0 Fibrosis 14 ,
C16
C18:1
C20:5
25,2±5,6
42,5±6,9
I0,0! 9,4
25,8±4,7
44,6~,4
7,1± 5,0
27,0+3,2
45,4+3,1
6,0! 3,2
,
,
By comparison of a low degree ~big lipid droplets in less than 30 per cent of liver cells) and a high degree of steatosis (more than 50 per cent big droplets in hepatocytes) no differences in the fatty acid pattern of triglycerides could be confirmed (Table 5). Thus, not the absolute amount of liver fat but the lipid droplet size seems to determine the fatty acid pattern of hepatic triglycerides. Other liver diseases, like chronic hepatitis and fibrosis, did not exercise an influence on the fatty acid pattern (Table 6). The standard deviations werewide, possibly because of the irregular distribution of the different lipid droplet populations over the groups with inflammatorF liver diseases. Again, in diabetics with HLP a significant higher percentage of palm±tic and oleic acid as well as a lower content of eicosapentaenoic acid than in diabetics without HLP can be demonstrated (Table 5 and 6).
192
DISCUSSION
In general, it is important to stress the differences between the fatt2 acid pattern of triglFcerides in serum and the tissues studied. Thus, no conclusions from the fatty acid composition of serum lipids can be drawn which are necessaril2 relevant to the tissues (20,2q). Contrar~ to its levels in serum and depot fat, the high percentage of eicosapentaenoic acid in liver indicates an extraordinarF role in this organ, the relevance of which is not ~et clearl2 understood, Since the C20:#/C20:5-ratio, for example, increases with lipid~droplet size from 0,08z0,06 in small lipid droplets to 0,34-D,2# in big droplets the decrease of eicosapentaenoic acid is more pronounced than that of arachidonic acid (Table 7). TABLE 7:C20:~/C20:5 -RATIO IN LIVER, SERUN AND ADIPOSE TISSUE OF DIABETICS IN RELATION TO THE SIZE OF HEPATIC LIPID DROPLETS Size of lipid droplets not visible small medium-sized big small to medium-sized medium-sized to big small to big
n
Adipose tissue
0,13~0,09 0,77~0,50 0,08±0,067 ~ 0,72±0,70 0,15!0,1~a~ 1,62±q,09
q3
o,13±o,o9~
0,41±0,18
~,63~4,06
~0 41
o,3q±o,27] o,27~o,2o
1,06~0,87 0,7#~0,67
3,56~3,04 3,90!3,44
0,23~0,21
0,95~0,82
#,30!3,85
i
a= P~O,05,
Serum
29 30 21 5~
228 i
Liver
o,34±o,243jI,42±o,97
5,1~!5,09 5,33-+4,92 6,o4!5,91 3,39!2,78
, i
b= PLO,Oq
Thus, a relative lower proportion of all polFunsaturated fatty acids due to an increase in palmitic and oleic acid can be excluded. On the. other hand,, since the C20:4./C20..5-ratio was • ° about 1:5 in llver, 1.• I in serum and nearly #.1 in depot fat the question arises of whether the relation of these two PG precursors might be more relevant for triglyceride accumulation than the individual fatty acids alone, comparable
193
to the balance of PG precursors involved in aggregation of platelets (2,3). The PG content of human liver in relation to fatty degeneration has not been studied till now. Therefore, no comment on the s~nthesis of PG from eicosapentaenoic acid in liver parenchxma is possible. Chronic administration of PGE resulted in fat infiltration in liver of animals (22,23), although lipolysis in adipose tissue was inhibited (24,25). Besides, in the rat pancreas PGE 2 increases insulin secretion (26) which favours hepatic lipogenesis. In the triglyceride-rich droplets of rat hepatocytes palmitic and oleic acid were augmented (27) in agreement with our findings in diabetic patients. In experimental fatty liver of rats (28,29) the percentages of eicosapentaenoic and docosahexaenoic acids, which both are representatives of the linolenic acid family (30), were also decreased whereas palmitic and oleic acid were increased. In vitro studies (31) of isolated rat liver mitochondria revealed a continuous time-related release of FFA after anoxia which was reversible b2 oxygen exposure. The rise of arachidonic, eicosapentaenoic and docosapentaenoic acids was higher in comparison to other fatty acids. This release of FFA may be due to an increased activity of lipases and to an inhibition of B-oxydation in anoxic cells (32-3A). Since liver has an enzymatic complement for PG s~nthesis, the accumulation of PG precursors might be a prerequisite step for substrate suppLT. This could be Consistent with our data if the decrease of eicosapentaenoic acid in the triglyceride fraction is associated with an increase of free eicosapentaenoic acid, the direct precursor of PGI3, via hxdrolysis. In the present study the individual FFA could not be estimated because of the small material obtained from one half ' of the liver biopsy specimens. In addition, although the patients were informed as to the kind and purpose of the examination the expectation and performance of biopsies ma~ lead to stress, which not only provokes an elevation of FFA but also a change in their fatty acid pattern (35,36), thus confusing the interpretation because of the short half life time of the FFA. The different mitochondrial resistance to anoxia and the cellular autonom x of PG might result in i r r e ~ I s m fat infiltration of hepatocytes (37). This m8.7 be the reason wh7 in some liver cells no or small fat droplets and in others medium-sized or big droplets can be observed. The synthesis and release of hepatic PG m&y be regarded as a defensive mechanism of ischemic cells (32), which also has been assumed in other tissues (38-#0). This oxygen dependent
194
regulation might not onl2 protect ischemic cells but ma~ also signal the local environment and t h e s F s t e m i c circulation of the ischemic state of the cells. This is conceivable because of PG receptors being present in the plasma membrane of different cells (4q). In this respect, the local s~nthesis of trigLycerides maF be integrated in the regulation of PG formation which alread2 has been assumed for lipid droplets of renal papillae (42). It must be conceded, however, that our results were obtained in patients with overt diabetes mellitus, although there is n o e v i d e n c e of differences in the appearance of fat infiltration in the liver of diabetic and non-diabetic subjects. Thus, the problem remains to be pursued whether the histological-biochemical correlation described is a common pathoph~siological phenomenon or is restricted to diabetics. It might be relevant that alterations of the fatt2 acid pattern in fattF liver could be restored bF clofibrate treatment in relation to a diminution of the lipid droplet size in liver parench~ma. AccordinglF, linoleic, arachidonic acid and predominantl~ eicosapentaenoicacid were increased in control biops~ specimens after one ~ear of clofibrate therapF (9). The results suggest that clofibrate lowers serum trigl~ceride levels bF increasing the rate of fatt~ acid oxidation in the liver (43). Further studies are recommended to clarif2 the pathogenetic mechanism of these clinical observations~ Although the true incidence of fattF liver cannot be ascertained b2 means of biopsies or autopsies, respectively, its frequenc2 seems to be of interest in populations in which the dietar2 content of eicosapentaenoic acid is high (44). From the data available (2,45,46) it is tempting to speculate that enrichment of tissue lipids with eicosapentaenoic acid maF reduce not onl~ the development of thrombosis and atherosclerosis (47,48) but also of fattF liver. The correlation of hepatic steatosis with cardiovascular diseases and their metabolic risk factors is alread~ well ~..
"
°
.
°
•
•
°
diseases has been reviewed (5q,52). Finall~, a reduced percentage of eicosapentaenoic acid can not onl2 be observed in liver, but also in serum (53) of diabetics with HLP when compared to diabetics without HLP. That ma~ explain the increased platelet aggregation and adhesiveness in HLP and alimentar2 h~perlipaemia (54;55).
vet
195
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37.
38.
Levine RA. p. 75 in Prostaglandins and CFclic AMP, Biological Actions and Clinical Applications. (RH Kahn, W E M Lands eds.) Academic Press Inc., New York, 1973. Pek S, Tai TY, Elster A, Fajans SS° Stimulation b2 prostaglandin E2 of glucagon and insulin release from isolated rat pancreas. Prostaglandins 10: 493, 1975. Diaugustine RP, Schaefer JM, Fours JR. Hepatic lipid droplets. Biochem. J. I~2: 323, 1973. Viviani R, Sechi AM, Lenaz G. Fatt2 acid composition of portal fattF liver in 12sine- and theronine - deficient rats. J. Lipid Res. 5: 52, 1964. Viviani R, Sechi AM, Lenaz G. Lipid metabolism in fattF liver of 1Fsine- and threonine - deficient rats. J. Lipid Res. 7: 473, 1966. Brenner RR. The oxidative desaturation of unsaturated fatt2 acids in animals. Molec & Cell. Biochem. ~: 41, 1974. Markelonis G°, Garbus J. Elaboration of medium chain free fattF acids and long chain fatt2 acid prostaglandin precursors b2 isolated anoxic rat liver mitochendria. FEBS Lett. 51: 7, 1975. Chefurka W, Dumas T. Oxidative phosphorFlation in vitro aged mitochondria. II. Dinitrophenol-stimulated adenosine triphosphate activitF and fattF acid content of mouse liver mitochondria. Biochemistr2 5: 3904, 1966. Boime J, Smith E~:, Hunter FE. The role of fatt~ acids in mitochondrial changes during liver ischemia. Arch. Biochem. BiophFSo 1 ~ : 425, 1970. Lehninger AL. W a t e r uptake and extrusion b2 mitochondria in relation to oxidative phosphor~lation° PhTsiol. Rev. 42: 467, 1962o Taggart P, Carruthers M. Endogenous h~perlipidaemia induced b2 emotional stress of racing driving° Lancet I: 363, 1971. Hurter R, Swale J, Penman MA, Barnett CWHo Some immediate a n d long-term effects of exercise on the plasma lipids. Lancet II: 671, 1972. Beskid M, Mojdecki T, Marciniak M° The effect of ethanol on the activit2 of some oxidative enzymes and on the ultrastructure of liver cells in the dog. Folia histochem, c~tochem. 10: 155, 1972. Jaffe BM, Parker CN, Marshall GR, Needleman P. Renal concentrations of prostaglandin E in acute and chronic renal ischemia. Bi0chemo Bioph~s. Res. Commun. 49: 799, 1972.
198
39.
40.
41.
42°
43.
#4.
45.
46. 47. 48. 49. 50. 51.
52. 53.
Herbacz~nska - Cedro K, Vane JR. Prostaglandins as mediators of reactive hTperemia in kidne2. Nature 24~: #92, 1974. Mc Giff JC, Crowshaw K, Terragno NA, Lonigro AJ, Strand JC, WilliamsonMA, Lee JB, N g K K F 4 Prostaglandin - like substances appearing in canine renal venous blood during renal ischemia° Circular. Res~ 27: 765, 1970. Smigel M, Fleischer S~ Characterization and localization of prostaglandin E 1 receptors in rat liver plasma membranes. Biochim. BiophTs. Acta 332: 358, 1974. Bojesen J. Quantitative and qualitative analyses of isolated lipid droplets from interstitial cells in renal papillae from various species. Lipids ~: 835, 1974. Lazarow PB. Rat liver peroxisomal s2stem of fatt~ acid B-oxidation: elevation b~ clofibrate and other hTpolipidemic drugs, p 15 in International Conference on Atherosclerosis (LA Carlson ed) Raven Press, New York, fl978. Dzerberg J, Bang HO, Hj~rne N. Fatt2 acid composition of the plasma lipids in Greenland Eskimos. Am. J. clin. Nutr. 2_~8: 958, 1975. Bang HO, Dzerberg J, Hj~rne N. The composition of food consumed b~ Greenland Eskimos. Acta med. scand. 200: 69, q976. Peifer JJ. p 322 in Fish Oils (M E Stansb~ ed). The Avi Publishing Compan2, Westport, Connecticut, 1967. Vane J. Fats and atheroma: an inquest. Lancet ~:484, 1979. Dzerberg J, Bang HO. Dietar2 fat and thrombosis. Lancet I: 152, 1978. Gromotka Ro Uber Leberver&uderungen bei allgemeiner Arteriosklerose. Acta hepatosplenol. ~: 409, 1962. Barlow KA. HTperlipidemia in primar~ gout. Metabolism q7: 289, 1968. Davies JA, McNicol GP. Blood coagulation in pathological thrombus formation and the detection in blood of a thrombotic tendenc2. Brit. Med. Bull. 34: 113, 1978. Bloom AL~ Intravascular coagulation and the liver. Brit. J. Haematol. 30: 1, 1975. Gnauck G, Singer P, Thoelke H, Honigmann G, 8chliack V. Das Fetts~uremuster der Trigl~ceride im Serum von Patienten mit HTperlipoprotein~mie sowie Diabetes mellitus im Vergleich zu Stoffwechselgesunden. Dr. Gesundh.Wesen 30: 1510, 1975.
199
54. Nord~x A, R~dseth JM. The influence of dietar~ fats on platelets in man. Acta reed. scand. 189: 385, 1971. 55. N o r d ~ A, StrMm E, Gjesdal K. The effect of alimentar~ hyperlipaemia and primar~ h~pertrigLTceridemia on plate lets in man. Scand. J. Haemat. 12: 329, 1974.
200
DECREASE OF EICOSAPENTAENOIC ACID IN FATTY LIVER OF DIABETIC SUBJECTS Peter Singer x, Gerhard Honigmann and Volker Schliack. XCentral Institute of Cardiovascular Regulation Research, Academ~ of Sciences of the GDR, DDR-1115 Berlin, and Centre of Diabetes and Metabolic Diseases, 102 Berlin, GDRo (reprint requests to PS)o ABSTRACT Simultaneous biopsies of liver and subcutaneous adipose tissue have been carried out in 228 patients with diabetes mellit-as. In liver trigl~cerides a msmked variability of the fatt~ acid pattern in relation to fatt$ degeneration of liver psmench~ma has been confirmed. In adipose tissue fatt2 acid pattern was relativel2 constant. The most striking finding was a high content of eicosapentaenoic acid in normal liver and its dedrease with the rise of lipid droplet size in the hepatoc~tes. No correlation with the quantit~ of liver fat or inflammator2 liver diseases could be ascertained. W h e n diabetes was associated with b~yperlipoproteinemia (HLP) the percentage of eicosapentaenoic acid was significantl7 less. From the results the suggestion is obvious that a diminution of eicosapentaenoic acid in hepatic trigl2cerides is associated with lipid accumulation in the liver cells. A local mechanism, possibl$ the antilipol~ic potenc7 of prostaglandins, ma~ be responsible for the creation of lipid droplets in liver cells of patients with metabolic disturbances. The alterations of eicosapentaenoic acid should be considered in view of recent data on the antiaggregator2 effect of this fatty acid and its possibl$ preventive role for atherosclerosis. INTRODUCTION Elcosapentaenolc acid (C20:5) has recently been supposed to pla~ an important role in the prevention of cardiovascular diseases (I). It represents the precursor of the antiaggre~ating prostaglandin I~ (PG I3), whereas thromboxane A3 (TXA3) does not induce aggregation (2,3).
183
The low incidence of m2ocardial infarction in Eskimos as well as their prolonged bleeding time are probably due to the high eicosapentaenoic acid and low linoleic and arachidonic acid content of their nutrition and consequently of their tissues (2). Thus, polyunsaturated fatty acids on the whole may be of less relevance to the prevention of Gardiovascular diseases than the individual substance eicosapentaenoic acid (I). Possibl~, the liver is involved in essential fatty acid metabolism (4). Other functions of eicosapentaenoic acid are not ~et clearl2 defined. From human tissues, for example, there is a high percentage of eicosapentaenoic acid in the brain (5) as well as in eruptive xanthomas of patients with h~perlipoproteinemia (6), the latter being no inert deposits but formations of high metabolic activity (6,7). In previous studies distinct alterations in the fatt x acid pattern of liver triglycerides have been described in fatt~ degeneration of the liver (8) which both can be influenced by clofibrate treatment (9). An increase of palmitic and oleic acid as well as a decrease of arachidonic and eicosapentaenoic acid associated with fatty liver have been attributed to the antilipolytic action of PGE 2 and PGE 3 (8,q0). But no distinct concept of the pa~hoph~siological mechanism could be devised at that time. Since the discovery of PG!2 (ql) research work has been focused on the products of the cyolooxygenase cascade. Recently, an antilipolFtic effect of PGI 2 more potent than of PGE 2 has been proved (12). No data, however, dealing with an effect on lipolysis of PGI~ are available. In the present paper the behaviour of eicosapentaenoic acid of liver triglFcerides in relation to lipid droplet size in liver cells and chronic hepatitis will be described.
m m ALs
m HODS In 228 diabetic subjects, 98 of whom had hyperliPoproteinemia (HLP), blood was withdrawn by venous puncture from the antecubital vein after an overnight fast~ Some clinical and biochemical data of the patients are summarized in Table I and 2. Diabetes mellitus was well compensated during the study. ~ P was diagnosed when serum triglycerides were above 250 mg/100 ml and cholesterol was above 300 mg/100 ml. The patients were kept in hospital at least two weeks before the biopsies. 2q % of the diabetics without HLP and 18 % of the d!abet.cs wmth H L P w e r e on i~Tpoglycemlc drugs. All other patients received insulin (Table q).
184
TABLE 1: CLINICAL DATA OF TEE DIABETICS WITHOUT AND WITH HLP J
L
,
without HLP Diabetics (n) Women ~en Axe ' (Mean~SD) Women Men Rel. bod~ weight a Women Men Duration of diabetes ~7poglycemic agents ~ I Insulin therapy
120 72 58 55,2±12,8 59,3~13,6 52,2~14,8 114 ±20 q17 !2fl 111 ±22 6,0± 5,1 21 109
with HI~ 98 73 25 56,O!11,8 6d,2~I0,8 @8,2$16,0 122 ±16 q25 ±18 116 ±20 9,0± 5,8 18 80
a Metropolitan Life Insurance Compan~ Tables - (13). If clinically indicated liver biopsies by Menghini tech nique (17) were performed. One half of the biopsy specimens was fixed in 10 % formalde&Tde for routine histological examination (HE, HS, Sudan III, van Gieson, Turnbull's blue, Fouchet). The other part was carefully cleaned in chilled 0,9 % saline solution and stored at -20°C for gas liquid chromatograph~. About 3 hours later a sample of subcutaneous adipose tissue of the abdominal wall (200-500 mg) was excised after intracutaneous anesthesia (procaine fl %) and a small iofraumbilical incision. Adipose tissue was also stored at -20oc until gas chromatographic analyses. The lipids from serum and tissues were extracted with chloroform-methanol (2:1, v/v) according to Renkonen et al. (18). Total lipids were isolated by thin la~er chromatograph7 on silica gel G (Merck, Darmstadt). The plates were developed in hexane-diethylether-acetic acid (73:25:2, v/v). The fractions were visualized under UV-light after the plates had been sprayed with a solution of 0 , 1 % dichlorfluorescein in ethanol. The triglycerides were scraped into test tubes. The esterification was achieved by 0,5 N sodium methTlate. After evaporation under nitrogen the samples in hexane solution were injected direct&2 into a gas chromatograph (GCHF 18,3, VEB Chromatron, Berlin, flame ionization Aetector). Analyses of fatt2 acid metkTlesters were performed on columns (2 m x 4 mm) of 10 % dieth~leneglycol succinate, stabi-
185
lized (Analabs, North Haven, USA) on Gas Chrom P (q00-120 mesh), isothermal at 200~C° The components were identified bF their retention time relative to authentic standards (Applied Science Laboratories, State College, USA). The quantit~ of each fatt~ acid was estimated from the product of peak height and retention time. The method has been published previousl2 in detail (19). Histological findings were classified into 4 categories according to thelipid droplet size independent of the amount of fat: no visible fat, prevailing small 'lipid droplets (mean±SD: 1,74-0,26/um, range O-2,5/um), medium-sized droplets (9,65±6,60/um, range 2,5-14,5/um) and big lipid droplets (19,06±8,1~/um, over 14,dpam)° This classification has been proved b~ mbrphometric sthdies(8). In t~pe IIa HLP no or small lipid droplets were found but in t y p e I V big droplets were predominant in the liver (Table 3)° Mixed forms were subdivided into small to medium-sized, medium-sized to big and small tp big lipid droplets in hepatocFtes (Table 2). The significance of the results was assessed bF Student's t-test. TABLE 2: BIOCHEMICAL DATA OF DIABETICS WITHOUTAND WITH HLP IN RELATION TO LIPID DROPLET SIZE IN LIVER C E L ~ a) without HLP Size of lipid droplet s not visible small medium-sized big small to medium-sized medium-sized to big small to big
Fasting a blood sugar (mg/100 ml)
n
TriglT- b cerides (mg/lO0 ml)
Bromsulfalein retention (%)
20 17 11 29
138329 154~43 166m40 q60±38
1133 12q z 138~ q#5 z
30 27 25 31
4 ,0~3,4 + #,7~2,5 3,7~2,4 6,4~3,3
9
144±39
114± 29
#,8±3,3
16 28
fl53~36 q55m38
133~ 35 q#3 ~ 39
5,4~2,8 4,4~3,q
153
132
130
±38
"I"
34
I4 , 9 ± 3 ,
3
b) with HLP -,
i
not
i
ii
i
visible
9
'135~51'
- 677~625 '+ ..........
small medium-sized big •small to medium-sized medium-sized
13 10 25
132437 158z49 160±37
1103~951 9885680 88flz547
3,6~2,3 4,2Zl,6 4,5~2,1 5,7~2,8
4
flfl4!38
695±576
4,2±0,8
to
2#
q#6~29 q62z23
806±4#2 979±774
6,q~3,8 5,8~3,0
1#8I±I~6 '
890±614
5,2{2,9 ....
big
small to big .
.
.
.
q3 9 8
....
a Lange et al. (14). b Stolz et al. (15). 186
TABLE~: TYPES OF HIP IN RELATION TO THE SIZE OF HEPATIC LIPID DROPLETS; COLUMNS REFER TO THE FREDRICESON CLASSIFICATION OF HYPERLIPOPROTEINEMIAS (16). • Size of without lipid droplets H]~P lla lib III IV V not visible small medium-sized big small to medium-sized medium-sized to big small to big ,
i
L
20 17
1 0 1
0 0 0 0
4 7 9 19
2 3
29
2 2 0 0
9
1
1
0
1
1
16 28
0 0
2 4
0 I
18 6
4 2
1_30
5
10
1
6L~
18
11
1
1
5
i
RESULTS The percentage of eicosapentaenoic acid in diabetics with noymal livers is high (about 30 per cent). In adipooe tissue it is low (under I per cent) whereas in serum trigl~cerides it lies between liver trigl~cerides and depot fat (about 3 per cent). The high percentage of eicosapentaenoic acid in triglycerides of normal liver decreases with rise of fat droplet size in hepatocytes (Fig. I). In diabetics without HLP this decrease is more pronounced than in diabetics with HLP (Fig. 2 and 3). On the other hand, palmitic and oleic acid increased with the size of hepatic lipid droplets. In spite of some differences between diabetics without and with I~LP the tendencies of the major fatty acids were the same (Fig. 2 and 3). In diabetics with kT~P significantly higher percentages of palmitic and oleic acids as well as lower percentages of arachidonic and eicosapentaenoic acids in liver trigl~cerides could be observed when compared with diabetics without HLP. This might be due to thepre v a l e n c e of fatty degeneration with big lipid droplets in diabetics with HLP (Table 3). Standard deviations and significance levels have been omitted in Fig. 2 and 3 because onl~ eicosapentaenoic acid is the topic of this presentation. In livers containing big lipid droplets the fatty acid pattern of triglycerides became similar to the depot fat, i.e. adipocytes. There was no evidence of other factors being involved in this correlation. Since in free fatty acids (FFA) and in serum trigly~erides of normal subjects eicosapentaenoic acid is low (Table 4) this fatty acid must derive from local synthesis or its deterioration in the liver.
187
percent I o) Liver
40
z~ =p.~o.ol o = P':O.05
30 2O
10 ....
L-.- 0 ~
L . . - g~---J k-.. ~ . , . . ~ L . . - A - . - l k - - ~ - - - J
L - -
L~
. J
per cent
lO:
b) Serum
L
~
0
[
n
20
'
17
normo/ Smo//
.
j ~_..- ~, . ~ J
0
J
II
29
medium,
sized
big
9
16
smoJ/to medium-
28 srno//
medium- sized tob/9 sized tobig
Figure I. Eicosapentaenoic acid of triglFcerides in liver, serum and adipose tissue in relation to lipid droplet size in hepatoc~tes (abscissa)
188
Liver
Adipose tissue
C
&ze of
hp/ddroplets ¢12
CI~: 1
C76 - = 7 6 . 0 ~
22..a ~,0
Ceo.$~ 6 . o = n
Figure 2.
20
17
o.6 1I
29
130
Fatt~ acid pattern of trigl~cerides in hepatoc2tes in relation to lipid droplet size as well as in adipose tissue of diabetics without HIP.
189
Ad,~osetissue
Liver ci ,..1
-=20.$~ C,,:, . ~ 3 7 . 2 ~ I
~6
C16
~7
~6
c/8..3~ / . 3 = c20.3~ 0 . 4 ~ .
43
C2o:,~ o . 8 = .
/,2
C;o..s~ 3 . n
9
13
/=. I0
O.7
25
Figure 3. Fatt2 acid pattern of trigl2cerides in hepatoc2tes in relation to lipid droplet size as well as in adipose tissue of diabetics with HIE>o
190
TABLE
4 :
FATTY ACID PATTERN OF F F A A N D SERUM TRIGLYCERIDES IN NORMAL SUBJECTS (YET UNPUBLISHED DATA). FFA n = 11
Tri~l~cerides n = 11
C12
-
0,2+0,4
C14
0,6+0,6
1,7+1,0
C16 016:1 C18 018:1 C18:2 C18:3
24,3+2,2 5,9+1,5 12,3+2,1 38,1~,6 8,7+1,6 fl,2+0,5
21,6+1,5 4,7+1,3 3,9+0,4 ~,2+1,8 12,7+2,0 1,2+0,7
C20:4
2,6.T-O,9 1,7+1,2 1,1+1,8
2,6.@4D,5 1,6+0,6
020:5 C22 TABLE ~:
0,3+0,1
SELECTED FATTY ACIDS OF LIVER TRIGLYCERIDES AT A LOW (a) AND HIGH (b) DEGRk~ OF STEATOSIS IN DIABETICS WITHOUT ANDWITHHI~P.
Without HLP n
a) 1~ b) 12
C16
27,2±#,3 27,7±~,#
Cfl8:1
41,3+3,3 42,3+3,2
C18:2
C20:#
4,9+2,1 5,1+2,1
0,8+0,3 O, 7.T-O,5
C18:2
C20:4
C20:5
5,7+2,2 6,1+2,9
With KLP n a)
11
b) 12
C16
27,7±2,6 28,3~1,9
C18:1
#7,8!1,8 48,2±2,2
6,5±1,7 6,0!1,4
C20:5
0,8.~D,3 3,3±2,1 0,7±0,# 2,851,3
a) = big lipid droplets in less than 30 per cent of liver cells b) = big lipid droplets in more than 50 per cent of liver cells.
191
TABLE 6: SELECTED FATTY ACIDS OF LIVER TRIGLYCERIDES
IN CHRONIC HEPATITIS Without HLP n C16 No hepatitis 54 Chronic persist. hepatitis 45 Chr on. -aggr e s s. hepatitis 8 Fibrosis 23
C18:1
C20:5
22,8!5,5
35,6±7,2
15.7±10,0
23,1±5,2
37,4±8,0
13,3+12,8
22,4+6,3 24,1±6,0
35,4±9,5 39,5±7,5
11,7 + 8,0 12,1 + 9,7
With HLP n No hepatitis 42 Chronic persist. hepatitis 42 Chronic-aggress. hepatitis 0 Fibrosis 14 ,
C16
C18:1
C20:5
25,2±5,6
42,5±6,9
I0,0! 9,4
25,8±4,7
44,6~,4
7,1± 5,0
27,0+3,2
45,4+3,1
6,0! 3,2
,
,
By comparison of a low degree ~big lipid droplets in less than 30 per cent of liver cells) and a high degree of steatosis (more than 50 per cent big droplets in hepatocytes) no differences in the fatty acid pattern of triglycerides could be confirmed (Table 5). Thus, not the absolute amount of liver fat but the lipid droplet size seems to determine the fatty acid pattern of hepatic triglycerides. Other liver diseases, like chronic hepatitis and fibrosis, did not exercise an influence on the fatty acid pattern (Table 6). The standard deviations werewide, possibly because of the irregular distribution of the different lipid droplet populations over the groups with inflammatorF liver diseases. Again, in diabetics with HLP a significant higher percentage of palm±tic and oleic acid as well as a lower content of eicosapentaenoic acid than in diabetics without HLP can be demonstrated (Table 5 and 6).
192
DISCUSSION
In general, it is important to stress the differences between the fatt2 acid pattern of triglFcerides in serum and the tissues studied. Thus, no conclusions from the fatty acid composition of serum lipids can be drawn which are necessaril2 relevant to the tissues (20,2q). Contrar~ to its levels in serum and depot fat, the high percentage of eicosapentaenoic acid in liver indicates an extraordinarF role in this organ, the relevance of which is not ~et clearl2 understood, Since the C20:#/C20:5-ratio, for example, increases with lipid~droplet size from 0,08z0,06 in small lipid droplets to 0,34-D,2# in big droplets the decrease of eicosapentaenoic acid is more pronounced than that of arachidonic acid (Table 7). TABLE 7:C20:~/C20:5 -RATIO IN LIVER, SERUN AND ADIPOSE TISSUE OF DIABETICS IN RELATION TO THE SIZE OF HEPATIC LIPID DROPLETS Size of lipid droplets not visible small medium-sized big small to medium-sized medium-sized to big small to big
n
Adipose tissue
0,13~0,09 0,77~0,50 0,08±0,067 ~ 0,72±0,70 0,15!0,1~a~ 1,62±q,09
q3
o,13±o,o9~
0,41±0,18
~,63~4,06
~0 41
o,3q±o,27] o,27~o,2o
1,06~0,87 0,7#~0,67
3,56~3,04 3,90!3,44
0,23~0,21
0,95~0,82
#,30!3,85
i
a= P~O,05,
Serum
29 30 21 5~
228 i
Liver
o,34±o,243jI,42±o,97
5,1~!5,09 5,33-+4,92 6,o4!5,91 3,39!2,78
, i
b= PLO,Oq
Thus, a relative lower proportion of all polFunsaturated fatty acids due to an increase in palmitic and oleic acid can be excluded. On the. other hand,, since the C20:4./C20..5-ratio was • ° about 1:5 in llver, 1.• I in serum and nearly #.1 in depot fat the question arises of whether the relation of these two PG precursors might be more relevant for triglyceride accumulation than the individual fatty acids alone, comparable
193
to the balance of PG precursors involved in aggregation of platelets (2,3). The PG content of human liver in relation to fatty degeneration has not been studied till now. Therefore, no comment on the s~nthesis of PG from eicosapentaenoic acid in liver parenchxma is possible. Chronic administration of PGE resulted in fat infiltration in liver of animals (22,23), although lipolysis in adipose tissue was inhibited (24,25). Besides, in the rat pancreas PGE 2 increases insulin secretion (26) which favours hepatic lipogenesis. In the triglyceride-rich droplets of rat hepatocytes palmitic and oleic acid were augmented (27) in agreement with our findings in diabetic patients. In experimental fatty liver of rats (28,29) the percentages of eicosapentaenoic and docosahexaenoic acids, which both are representatives of the linolenic acid family (30), were also decreased whereas palmitic and oleic acid were increased. In vitro studies (31) of isolated rat liver mitochondria revealed a continuous time-related release of FFA after anoxia which was reversible b2 oxygen exposure. The rise of arachidonic, eicosapentaenoic and docosapentaenoic acids was higher in comparison to other fatty acids. This release of FFA may be due to an increased activity of lipases and to an inhibition of B-oxydation in anoxic cells (32-3A). Since liver has an enzymatic complement for PG s~nthesis, the accumulation of PG precursors might be a prerequisite step for substrate suppLT. This could be Consistent with our data if the decrease of eicosapentaenoic acid in the triglyceride fraction is associated with an increase of free eicosapentaenoic acid, the direct precursor of PGI3, via hxdrolysis. In the present study the individual FFA could not be estimated because of the small material obtained from one half ' of the liver biopsy specimens. In addition, although the patients were informed as to the kind and purpose of the examination the expectation and performance of biopsies ma~ lead to stress, which not only provokes an elevation of FFA but also a change in their fatty acid pattern (35,36), thus confusing the interpretation because of the short half life time of the FFA. The different mitochondrial resistance to anoxia and the cellular autonom x of PG might result in i r r e ~ I s m fat infiltration of hepatocytes (37). This m8.7 be the reason wh7 in some liver cells no or small fat droplets and in others medium-sized or big droplets can be observed. The synthesis and release of hepatic PG m&y be regarded as a defensive mechanism of ischemic cells (32), which also has been assumed in other tissues (38-#0). This oxygen dependent
194
regulation might not onl2 protect ischemic cells but ma~ also signal the local environment and t h e s F s t e m i c circulation of the ischemic state of the cells. This is conceivable because of PG receptors being present in the plasma membrane of different cells (4q). In this respect, the local s~nthesis of trigLycerides maF be integrated in the regulation of PG formation which alread2 has been assumed for lipid droplets of renal papillae (42). It must be conceded, however, that our results were obtained in patients with overt diabetes mellitus, although there is n o e v i d e n c e of differences in the appearance of fat infiltration in the liver of diabetic and non-diabetic subjects. Thus, the problem remains to be pursued whether the histological-biochemical correlation described is a common pathoph~siological phenomenon or is restricted to diabetics. It might be relevant that alterations of the fatt2 acid pattern in fattF liver could be restored bF clofibrate treatment in relation to a diminution of the lipid droplet size in liver parench~ma. AccordinglF, linoleic, arachidonic acid and predominantl~ eicosapentaenoicacid were increased in control biops~ specimens after one ~ear of clofibrate therapF (9). The results suggest that clofibrate lowers serum trigl~ceride levels bF increasing the rate of fatt~ acid oxidation in the liver (43). Further studies are recommended to clarif2 the pathogenetic mechanism of these clinical observations~ Although the true incidence of fattF liver cannot be ascertained b2 means of biopsies or autopsies, respectively, its frequenc2 seems to be of interest in populations in which the dietar2 content of eicosapentaenoic acid is high (44). From the data available (2,45,46) it is tempting to speculate that enrichment of tissue lipids with eicosapentaenoic acid maF reduce not onl~ the development of thrombosis and atherosclerosis (47,48) but also of fattF liver. The correlation of hepatic steatosis with cardiovascular diseases and their metabolic risk factors is alread~ well ~..
"
°
.
°
•
•
°
diseases has been reviewed (5q,52). Finall~, a reduced percentage of eicosapentaenoic acid can not onl2 be observed in liver, but also in serum (53) of diabetics with HLP when compared to diabetics without HLP. That ma~ explain the increased platelet aggregation and adhesiveness in HLP and alimentar2 h~perlipaemia (54;55).
vet
195
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