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Increased arachidonic acid content in platelet phospholipids from diabetic patients
ProstaglandinsLeukotrienesand Medicine 11: 33-41, 1983
INCREASED
ARACHIDONIC
PHOSPHOLIPIDS
ACID CONTENT
FROM DIABETIC
I .Morita,R.Takahashi*,H .Ito*,
IN PLATELET
PATIENTS
H. Orimo*, S .Murota
Department of Pharmacology, Tokyo Metropolitan Institute of Gerontology, * Division of Endocrinology and Metabolism, Tokyo Metropolitan Geriatric Hospital, 35-2 Sakaecho, Itabshi-ku, Tokyo-173, Japan ABSTRACT Platelet abnormalities have been suggested to be linked with vascular Diabetes mellitus has a high incidence of vascular complicadiseases. tions . In the present study gas-liquid chromatography analyses were carried out to investigate the fatty acid composition in platelet phospholipids of Type-2 (non-insulin-dependent) diabetic patients. Fatty acid composition in platelet phospholipids was different between diabetics and Arachidonic acid increased significantly age-matched control subjects. (p
33.
per-ties such as an increased sensitivity to aggregating agents (3). Moreover, the existence of plasma factors that enhance platelet aggregation has also been reported in diabetic subjects (4). Recently eicosapentaenoic acid and eicosatrienoic acid were shown to cause alterations in bleeding time and platelet aggregation (5,6) . With this background, we studied the lipid component of platelets in details and found some significant changes in fatty acid composition of platelet phospholipids in diabetic subjects. In addition, we discovered an interesting relationship in fatty acid composition between plasma total lipids and platelet phospholipids. MATERIALS
AND
METHODS
Subjects Diabetic patients for the study were 6 men and 6 women, hospitalized in the Tokyo Metropolitan Geriatric Hospital, ranging in age between 44 and 88 years (mean : 61 years old). The healthy volunteers, 5 men and 6 women ranging in age between 40 and 81 years (mean : 58 years old), had no history of diabetes mellitus or any other diseases No subject had taken aspirin or that would alter platelet aggregation. any other drugs which might alter prostaglandin metabolism in the previous 2 weeks. No diabetic patient had diabetic nephropathy but 2 patients had background retinopathy . All the subjects took an almost identical diet (amount of dietary lipids, 30-38 g) for i4week prior to the analysis. Chemicals lC-arachidonic acid (58 Ci/mol) was purchased from Radioacid and standard fatty chemical Centre, Amersham, U .K . Arachidonic acid methyl ester for gas chromatography were purchased from Sigma Thin-layer chromatographic plates of silica gel G Chemical Co., U.S.A. 60. 0.25 mm in thickness. were vurchased from Merck, F .R. G. Venous blood was collected from a cubital vein by Isolation of Platelets Final citrate concentration was adiusted to a 1:9 ratio a titrated svrinae. Platelet rich plasma was prepared by between 3.8% ci&ate and blood. The platelet rich plasma was recentrifugation at 1000 rpm for 10 min. The resulcentrifuged at 3000 rpm for 20 sec. to remove erythrocytes. tant platelet rich plasma was gently mixed with 0.1 volumes of 77 mM EDTA solution containing 120 mM NaCl and centrifuged again at 3000 rpm for 15 min. The platelet pellet was rinsed quickly with calcium-free phosphate buffer containing saline and was used for platelet lipid analysis. The supernatant , platelet poor plasma, was used for plasma total lipid analysis. Lipid
Extraction
and Fatty
Acid
Analysis
by Gas-Liquid
Chromatography
The extraction of total lipids from plasma and platelets was conducted by the method of Bligh and Dyer (7). Separation of phospholipids in platelets from other lipids was carried out by thin-layer chromatography in a solvent system of n-hexane/diethyl ether/acetic acid (90:10:1, v/v/v). Analysis of acylated fatty acids in phospholipids and total lipids was done by gas chromatography of their corresponding methyl esters. The samples for the analysis were obtained by transesterifying with 1 ml of 5% methanolic hydrochloric acid solution at 80°C for 2 hrs. Internal standard, heptadecanoic acid (17: 0) , was added before the esterification. The methyl esters were extracted with n-hexane and dried under a N2 gas stream and then dissolved in 20~ 1 of chloroform for gas chromato-
34
graphy analysis. The fatty acid methyl esters were separated on a column of 16%polyethylene glycol succinate coated on Celite 545 at 185OC with a N gas flow rate of 40 ml/min. in a Shimadau Model 7A gas chromatograp%I. The data were computerized by a Shimadzu Chromatopac ElA. RESULTS The levels of plasma lipids, lipoproteins and fasting plasma glucose (FPG) in diabetic and control subjects are shown in Table 1. The levels of FPG, total cholesterol and low density lipoprotein-cholesterol in diabetic subjects were significantly higher than those in control subjects.
Table 1.
Plasma levels of fasting glucose (FPG) and lipids
control (n=Il)
age
58k
5
FPG
88k
3*
total cholesterol
192 +
6
triglycerides
121 f 16
HDL cholesterol
46+
4
LDL cholesterol
143 *
7
diabetes (n=12) 61 +
significance
5
n.s.
135 ?
6*
p < 0.001
220 f
5
p < 0.01
141 + 19 41 +
2
173 + 6
* values represent the mean + S. E. (mg8).
n.s. n.s. p < 0.01
n. s. ; not significant.
The fatty acids were identified by gas-liquid chromatography in plasma total lipids and platelet phospholipids. In human platelet phosp holipids, the predominant fatty acids were palmitic, stearic, oleic, linoleic, a-linolenic and eicosapolyunsaturated fatty acids, As shown in Table 2, a tendency to low level of eicosatrienoic acid and eicosapentaenoic acid was found in diabetic platelet phospholipids. On the contrary, the level of arachidonic acid in platelet phospholipids was increased significantly (p
35
Table 2
Fatty acid compositionof plateletphospholipids
control (n=ll)
diabetics(n=l2) %
% 16:0 16:l 18:0 18:l 18:2 18:3 20:o 2O:l 20:3 20:4 20:5 24:0 unknown
PIS ratio
22.75 1.15 16.81 16.90 6.18 1.24 1.13 0.16 2.64 19.74 2.20 3.76 5.35
21.54 kO.15 +0.57 kO.71 +0.38 iO.18 + 0.07 +0,05 +0.99 + 0.76" kO.32 ~0.58 21.07
23.12 50.52 0.16 20.05 17.02 kO.55 16.92 ? 0.42 6.71 kO.21 1.44 20.24 0.98 ? 0.22 0.09 kO.06 1.59 20.27 23.57 f 0.41* 1.53 20.21 3.00 to.38 3.88 ~0.31
0.72 to.08
0.79 +0.07
* p< 0.001
20:3 m
20:5 m
a2
03 *P*OAIOl
*
PcOf301
Fig, 1 Ratio of eicosatrienoic acid or eicosapentaenoicacid to arachidonic acid in plateletphospholipids.
36
Next, we tried to estimate the correlation between the levels of fatty A sigacids in platelet phospholipids and those in plasma total lipids. nificant correlation between fatty acid composition of platelet phospholipids and of plasma total lipids was found for palmitic acid (p
37
Table
3
Fatty
acid
composition
of
control 16:0 16:l 18:0 18:l 18:2 18:3 20:3 20:4 20:5 22:4 22:6 24:0
23.79 2.76 9.02 17.42 24.37 1.12 1.14 6.96 3.32 1.22 6.25 1.93
plasma (n=ll
+ 0.60 ?r 0.38 + 0.39 + 0.60 A 1.04 _+ 0.09 + 0.12 k 0.41 * 0.44 + 0.05 f 0.34 + 0.15
20~4
Fig.
2
Correlation and plasma
total
diabetes
)
%
lipids
25.78 1.82 8.78 17.22 25.14 1.15 1.44 7.13 2.57 1.14 5.93 2.03
r f ? + ik * + ?r + ?r f
(n=12) %
0.94 0.39 0.23 0.61 1.44 0.13 0.20 0.38 0.42 0.10 0.43 0.25
20~5
between fatty total lipids.
acid
38
content
-.-
x Diabetic
-----
??
I-
cctntrol
of platelet
phospholipids
different dietary intake of fatty acids. In our present study, however, both groups took the same dietary lipids for 1 week prior to blood sampling . Under these conditions, we clearly showed a difference in arochidonic acid composition between platelet phospholipids and plasma total lipids. Norddy et al. ( 21) found significant positive correlations in free fatty acid levels between plasma and platelets. Fatty acid analysis in their report, however, did not include arachidonic acid and eicosapentaenoic acid. In the present study, the contents of predominant fatty acids in platelet phospholipids show a significant correlation with those in plasma total lipids, with the exception of arachidonic acid. The high levels of arachidonic acid content in platelet phospholipids in diabetes mellitus are independent of the arachidonic acid content in the plasma total lipids. Possible mechanisms of the high levels of arachidonic acid in platelet phospholipids of diabetic subjects may be as follows ; 1) increased uptake of arachidonic acid into platelets, 2) decreased A 6-desaturase activity in platelets, 3) activation of an arachidonic acid specific reacylation system, discovered by Wilson et al. (22) in human platelets. Regarding A 6-desaturase, Poisson et al. ( 23) reported that alloxan-induced diabetic rats showed depressed microsomal desaturation of linoleic acid to y-linoFurthermore, Horrobin ( 24) reviewed the lenic acid in liver tissue. evidence that diabetes, alcohol, radiation and aging inhibit A 6-desaturase activity. If A 6-desaturase activity in platelets from diabetes mellitus was depressed as in liver, or if platelets had less A 6-desaturase metabolic products available to them, the presence of fatty acids such as 20: 3 (I\6 and 20: 5 w 3 would be reduced, possibly leading to replacement by dietary arachidonic acid. The data in this paper could then be explained. Further investigations along these lines are underway in our laboratory, ACKNOWLEDGEMENTS The authors wish to thank Mrs. T. Kawata, A.Matsuda for their technical assistances.
and Mrs.Y. Saito
REFERENCES 1.
Ross R, Glomset JA. The pathogenesis Engl.J.Med. 295:369, 1976
2.
Sinzinger H, Schernthaner G, Kaliman J. Sensitivity of platelets to prostaglandins in coronary heart disease and angina pectoris. Prostaglandins . 22 : 773, 1981
3.
Colwell JA, Halushka PV, Sarji K, Sage1 J, Nair RMG. Altered platelet function in diabetes mellitus. Diabetes 25:826, 1976
4.
Colwell JA, Zile JV, Kilpatrick JM, Nair RMG, Virella factors and platelet aggregation in diabetes mellitus. Metab. Res. 11:l. 1981
39
of atherosclerosis.
N,
G. Plasma Horm.
5.
Sanders TAB, Naismith DJ, Haines AP, Vickens M. Cod-liver oil, platelet fatty acids, and bleeding time. Lancet i: 1189, 1980
6.
Willis AL, Comai K, Kuhn DC, Paulsrud J. Dihomo-y-linolenate supresses platelet aggregation when administrated in vitro or in vivo. Prostaglandins 25: 509, 1974
7.
Bligh EG, Dyer and purification.
8.
Colwell JA, Halushka PV. Platelet Br.J.Haematol. 44:521, 1978
9.
WJ. A rapid method of total lipids extraction Can.J.Biochem.Physiol. 37:911, 1959 function
in diabetes
Preston FE, Ward JD, Marcola BH, Porter NR, O’Malley BC . Elevated B-thrombogloblin levels platelet aggregated in diabetic microangiopathy. 1978
mellitus.
Timperley WR, and circulating Lancet i: 238,
10.
Bensoussan D, Levy-Toledano S, gation and increased plasma level diabetic retinopathy . Diabetologia
11.
Dembinska-Kiec A, Gryglewska T, Zmuda A, Gryglewski RJ. The generation of prostacyclin by arteries and by the coronary vascular bed is reduced in experimental atherosclerosis in rabbits. Prostaglandins 14: 1025, 1977
12.
Jugdutt BI. Prostaglandins in myocardial infarction emphasis on myocardial presevation. Prostaglandins icine 7:109, 1981
13.
Karpen CW, Pritchard KA, Merola JAJ, Panganamala RV. Alterations of the prostacyclin-thromboxane ratio in streptozotocin induced diabetic rats. Prostaglandins and Medicine 8 : 93, 1982
14.
Seyberth HW, Oelz 0, Kennedy T. Increased arachidonate on prostaglandin lipids after adminstration to man : Effects Clin.Pharmacol.Ther. 18: 521, 1975 synthesis.
15.
Spector AA, Hoak JC, Fry CL, acid modification of prostacyclin J.Clin.Invest. endothelial cells.
16.
Takahashi R, Morita unpablished data
17.
Dyerberg J, Bang HO. unsaturated fatty acids
18.
in prostaglandin formation. Role of phospholipase A Vogt w. 5r-d ed (Galli et al.eds) ~89 in Adv.PG and TX research. Raven Press, New York, 1978
19.
Gulp BR, synthesis.
I,
Murota
Passa P, Platelet of von Willebrand 11: 307, 1975
hyperaggrefactor in
: with and Med-
Denning GM. Effects of fatty production by cultured human 65: 1003, 1980 S,
Orimo
H,
Ito
H.
Haemostatic function and platelet in Eskimos. Lancet ii: 433,1979
Titus BJ, Land Prostaglandins
WEM. Inhibition of prostaglandin and Medicine 3: 269, 1979
40
in bio-
poly-
bio-
20.
Kalofoutis A, Lekakis J. Changes of platelet phospholipids 21: 540, 1981 diabetes mellitus . Diabetologia
21.
NordQy A, in patients 133, 1970
22.
Wilson DB, Prescott SM. Majerus PW. Discover of an arachidonyl coenzyme A synthetase in human platelets. J.Biol.Chem. 257: 3510, 1982 Poisson JP, Lemarchal P, Blond JP. Influence of alloxan diabetes on the conversion of linoleic and gamma-linolenic acids to arachidonic acid in the rat in vivo. Diabetes Metab. 4:39, 1978
23.
24.
Horrobin in aging.
Rddset JM. Platelet with ishemia heart
phospholipids disease. Acta
and their function med. stand. 188:
DF. Loss of delta-6-desaturase activity Medical Hypotheses 7: 1121, 1981
41
in
as a key
factor
INCREASED
ARACHIDONIC
PHOSPHOLIPIDS
ACID CONTENT
FROM DIABETIC
I .Morita,R.Takahashi*,H .Ito*,
IN PLATELET
PATIENTS
H. Orimo*, S .Murota
Department of Pharmacology, Tokyo Metropolitan Institute of Gerontology, * Division of Endocrinology and Metabolism, Tokyo Metropolitan Geriatric Hospital, 35-2 Sakaecho, Itabshi-ku, Tokyo-173, Japan ABSTRACT Platelet abnormalities have been suggested to be linked with vascular Diabetes mellitus has a high incidence of vascular complicadiseases. tions . In the present study gas-liquid chromatography analyses were carried out to investigate the fatty acid composition in platelet phospholipids of Type-2 (non-insulin-dependent) diabetic patients. Fatty acid composition in platelet phospholipids was different between diabetics and Arachidonic acid increased significantly age-matched control subjects. (p
33.
per-ties such as an increased sensitivity to aggregating agents (3). Moreover, the existence of plasma factors that enhance platelet aggregation has also been reported in diabetic subjects (4). Recently eicosapentaenoic acid and eicosatrienoic acid were shown to cause alterations in bleeding time and platelet aggregation (5,6) . With this background, we studied the lipid component of platelets in details and found some significant changes in fatty acid composition of platelet phospholipids in diabetic subjects. In addition, we discovered an interesting relationship in fatty acid composition between plasma total lipids and platelet phospholipids. MATERIALS
AND
METHODS
Subjects Diabetic patients for the study were 6 men and 6 women, hospitalized in the Tokyo Metropolitan Geriatric Hospital, ranging in age between 44 and 88 years (mean : 61 years old). The healthy volunteers, 5 men and 6 women ranging in age between 40 and 81 years (mean : 58 years old), had no history of diabetes mellitus or any other diseases No subject had taken aspirin or that would alter platelet aggregation. any other drugs which might alter prostaglandin metabolism in the previous 2 weeks. No diabetic patient had diabetic nephropathy but 2 patients had background retinopathy . All the subjects took an almost identical diet (amount of dietary lipids, 30-38 g) for i4week prior to the analysis. Chemicals lC-arachidonic acid (58 Ci/mol) was purchased from Radioacid and standard fatty chemical Centre, Amersham, U .K . Arachidonic acid methyl ester for gas chromatography were purchased from Sigma Thin-layer chromatographic plates of silica gel G Chemical Co., U.S.A. 60. 0.25 mm in thickness. were vurchased from Merck, F .R. G. Venous blood was collected from a cubital vein by Isolation of Platelets Final citrate concentration was adiusted to a 1:9 ratio a titrated svrinae. Platelet rich plasma was prepared by between 3.8% ci&ate and blood. The platelet rich plasma was recentrifugation at 1000 rpm for 10 min. The resulcentrifuged at 3000 rpm for 20 sec. to remove erythrocytes. tant platelet rich plasma was gently mixed with 0.1 volumes of 77 mM EDTA solution containing 120 mM NaCl and centrifuged again at 3000 rpm for 15 min. The platelet pellet was rinsed quickly with calcium-free phosphate buffer containing saline and was used for platelet lipid analysis. The supernatant , platelet poor plasma, was used for plasma total lipid analysis. Lipid
Extraction
and Fatty
Acid
Analysis
by Gas-Liquid
Chromatography
The extraction of total lipids from plasma and platelets was conducted by the method of Bligh and Dyer (7). Separation of phospholipids in platelets from other lipids was carried out by thin-layer chromatography in a solvent system of n-hexane/diethyl ether/acetic acid (90:10:1, v/v/v). Analysis of acylated fatty acids in phospholipids and total lipids was done by gas chromatography of their corresponding methyl esters. The samples for the analysis were obtained by transesterifying with 1 ml of 5% methanolic hydrochloric acid solution at 80°C for 2 hrs. Internal standard, heptadecanoic acid (17: 0) , was added before the esterification. The methyl esters were extracted with n-hexane and dried under a N2 gas stream and then dissolved in 20~ 1 of chloroform for gas chromato-
34
graphy analysis. The fatty acid methyl esters were separated on a column of 16%polyethylene glycol succinate coated on Celite 545 at 185OC with a N gas flow rate of 40 ml/min. in a Shimadau Model 7A gas chromatograp%I. The data were computerized by a Shimadzu Chromatopac ElA. RESULTS The levels of plasma lipids, lipoproteins and fasting plasma glucose (FPG) in diabetic and control subjects are shown in Table 1. The levels of FPG, total cholesterol and low density lipoprotein-cholesterol in diabetic subjects were significantly higher than those in control subjects.
Table 1.
Plasma levels of fasting glucose (FPG) and lipids
control (n=Il)
age
58k
5
FPG
88k
3*
total cholesterol
192 +
6
triglycerides
121 f 16
HDL cholesterol
46+
4
LDL cholesterol
143 *
7
diabetes (n=12) 61 +
significance
5
n.s.
135 ?
6*
p < 0.001
220 f
5
p < 0.01
141 + 19 41 +
2
173 + 6
* values represent the mean + S. E. (mg8).
n.s. n.s. p < 0.01
n. s. ; not significant.
The fatty acids were identified by gas-liquid chromatography in plasma total lipids and platelet phospholipids. In human platelet phosp holipids, the predominant fatty acids were palmitic, stearic, oleic, linoleic, a-linolenic and eicosapolyunsaturated fatty acids, As shown in Table 2, a tendency to low level of eicosatrienoic acid and eicosapentaenoic acid was found in diabetic platelet phospholipids. On the contrary, the level of arachidonic acid in platelet phospholipids was increased significantly (p
35
Table 2
Fatty acid compositionof plateletphospholipids
control (n=ll)
diabetics(n=l2) %
% 16:0 16:l 18:0 18:l 18:2 18:3 20:o 2O:l 20:3 20:4 20:5 24:0 unknown
PIS ratio
22.75 1.15 16.81 16.90 6.18 1.24 1.13 0.16 2.64 19.74 2.20 3.76 5.35
21.54 kO.15 +0.57 kO.71 +0.38 iO.18 + 0.07 +0,05 +0.99 + 0.76" kO.32 ~0.58 21.07
23.12 50.52 0.16 20.05 17.02 kO.55 16.92 ? 0.42 6.71 kO.21 1.44 20.24 0.98 ? 0.22 0.09 kO.06 1.59 20.27 23.57 f 0.41* 1.53 20.21 3.00 to.38 3.88 ~0.31
0.72 to.08
0.79 +0.07
* p< 0.001
20:3 m
20:5 m
a2
03 *P*OAIOl
*
PcOf301
Fig, 1 Ratio of eicosatrienoic acid or eicosapentaenoicacid to arachidonic acid in plateletphospholipids.
36
Next, we tried to estimate the correlation between the levels of fatty A sigacids in platelet phospholipids and those in plasma total lipids. nificant correlation between fatty acid composition of platelet phospholipids and of plasma total lipids was found for palmitic acid (p
37
Table
3
Fatty
acid
composition
of
control 16:0 16:l 18:0 18:l 18:2 18:3 20:3 20:4 20:5 22:4 22:6 24:0
23.79 2.76 9.02 17.42 24.37 1.12 1.14 6.96 3.32 1.22 6.25 1.93
plasma (n=ll
+ 0.60 ?r 0.38 + 0.39 + 0.60 A 1.04 _+ 0.09 + 0.12 k 0.41 * 0.44 + 0.05 f 0.34 + 0.15
20~4
Fig.
2
Correlation and plasma
total
diabetes
)
%
lipids
25.78 1.82 8.78 17.22 25.14 1.15 1.44 7.13 2.57 1.14 5.93 2.03
r f ? + ik * + ?r + ?r f
(n=12) %
0.94 0.39 0.23 0.61 1.44 0.13 0.20 0.38 0.42 0.10 0.43 0.25
20~5
between fatty total lipids.
acid
38
content
-.-
x Diabetic
-----
??
I-
cctntrol
of platelet
phospholipids
different dietary intake of fatty acids. In our present study, however, both groups took the same dietary lipids for 1 week prior to blood sampling . Under these conditions, we clearly showed a difference in arochidonic acid composition between platelet phospholipids and plasma total lipids. Norddy et al. ( 21) found significant positive correlations in free fatty acid levels between plasma and platelets. Fatty acid analysis in their report, however, did not include arachidonic acid and eicosapentaenoic acid. In the present study, the contents of predominant fatty acids in platelet phospholipids show a significant correlation with those in plasma total lipids, with the exception of arachidonic acid. The high levels of arachidonic acid content in platelet phospholipids in diabetes mellitus are independent of the arachidonic acid content in the plasma total lipids. Possible mechanisms of the high levels of arachidonic acid in platelet phospholipids of diabetic subjects may be as follows ; 1) increased uptake of arachidonic acid into platelets, 2) decreased A 6-desaturase activity in platelets, 3) activation of an arachidonic acid specific reacylation system, discovered by Wilson et al. (22) in human platelets. Regarding A 6-desaturase, Poisson et al. ( 23) reported that alloxan-induced diabetic rats showed depressed microsomal desaturation of linoleic acid to y-linoFurthermore, Horrobin ( 24) reviewed the lenic acid in liver tissue. evidence that diabetes, alcohol, radiation and aging inhibit A 6-desaturase activity. If A 6-desaturase activity in platelets from diabetes mellitus was depressed as in liver, or if platelets had less A 6-desaturase metabolic products available to them, the presence of fatty acids such as 20: 3 (I\6 and 20: 5 w 3 would be reduced, possibly leading to replacement by dietary arachidonic acid. The data in this paper could then be explained. Further investigations along these lines are underway in our laboratory, ACKNOWLEDGEMENTS The authors wish to thank Mrs. T. Kawata, A.Matsuda for their technical assistances.
and Mrs.Y. Saito
REFERENCES 1.
Ross R, Glomset JA. The pathogenesis Engl.J.Med. 295:369, 1976
2.
Sinzinger H, Schernthaner G, Kaliman J. Sensitivity of platelets to prostaglandins in coronary heart disease and angina pectoris. Prostaglandins . 22 : 773, 1981
3.
Colwell JA, Halushka PV, Sarji K, Sage1 J, Nair RMG. Altered platelet function in diabetes mellitus. Diabetes 25:826, 1976
4.
Colwell JA, Zile JV, Kilpatrick JM, Nair RMG, Virella factors and platelet aggregation in diabetes mellitus. Metab. Res. 11:l. 1981
39
of atherosclerosis.
N,
G. Plasma Horm.
5.
Sanders TAB, Naismith DJ, Haines AP, Vickens M. Cod-liver oil, platelet fatty acids, and bleeding time. Lancet i: 1189, 1980
6.
Willis AL, Comai K, Kuhn DC, Paulsrud J. Dihomo-y-linolenate supresses platelet aggregation when administrated in vitro or in vivo. Prostaglandins 25: 509, 1974
7.
Bligh EG, Dyer and purification.
8.
Colwell JA, Halushka PV. Platelet Br.J.Haematol. 44:521, 1978
9.
WJ. A rapid method of total lipids extraction Can.J.Biochem.Physiol. 37:911, 1959 function
in diabetes
Preston FE, Ward JD, Marcola BH, Porter NR, O’Malley BC . Elevated B-thrombogloblin levels platelet aggregated in diabetic microangiopathy. 1978
mellitus.
Timperley WR, and circulating Lancet i: 238,
10.
Bensoussan D, Levy-Toledano S, gation and increased plasma level diabetic retinopathy . Diabetologia
11.
Dembinska-Kiec A, Gryglewska T, Zmuda A, Gryglewski RJ. The generation of prostacyclin by arteries and by the coronary vascular bed is reduced in experimental atherosclerosis in rabbits. Prostaglandins 14: 1025, 1977
12.
Jugdutt BI. Prostaglandins in myocardial infarction emphasis on myocardial presevation. Prostaglandins icine 7:109, 1981
13.
Karpen CW, Pritchard KA, Merola JAJ, Panganamala RV. Alterations of the prostacyclin-thromboxane ratio in streptozotocin induced diabetic rats. Prostaglandins and Medicine 8 : 93, 1982
14.
Seyberth HW, Oelz 0, Kennedy T. Increased arachidonate on prostaglandin lipids after adminstration to man : Effects Clin.Pharmacol.Ther. 18: 521, 1975 synthesis.
15.
Spector AA, Hoak JC, Fry CL, acid modification of prostacyclin J.Clin.Invest. endothelial cells.
16.
Takahashi R, Morita unpablished data
17.
Dyerberg J, Bang HO. unsaturated fatty acids
18.
in prostaglandin formation. Role of phospholipase A Vogt w. 5r-d ed (Galli et al.eds) ~89 in Adv.PG and TX research. Raven Press, New York, 1978
19.
Gulp BR, synthesis.
I,
Murota
Passa P, Platelet of von Willebrand 11: 307, 1975
hyperaggrefactor in
: with and Med-
Denning GM. Effects of fatty production by cultured human 65: 1003, 1980 S,
Orimo
H,
Ito
H.
Haemostatic function and platelet in Eskimos. Lancet ii: 433,1979
Titus BJ, Land Prostaglandins
WEM. Inhibition of prostaglandin and Medicine 3: 269, 1979
40
in bio-
poly-
bio-
20.
Kalofoutis A, Lekakis J. Changes of platelet phospholipids 21: 540, 1981 diabetes mellitus . Diabetologia
21.
NordQy A, in patients 133, 1970
22.
Wilson DB, Prescott SM. Majerus PW. Discover of an arachidonyl coenzyme A synthetase in human platelets. J.Biol.Chem. 257: 3510, 1982 Poisson JP, Lemarchal P, Blond JP. Influence of alloxan diabetes on the conversion of linoleic and gamma-linolenic acids to arachidonic acid in the rat in vivo. Diabetes Metab. 4:39, 1978
23.
24.
Horrobin in aging.
Rddset JM. Platelet with ishemia heart
phospholipids disease. Acta
and their function med. stand. 188:
DF. Loss of delta-6-desaturase activity Medical Hypotheses 7: 1121, 1981
41
in
as a key
factor