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Dihomogammalinolenic acid inhibits platelet aggregation and stimulates platelet prostaglandin E1 production in healthy subjects but not in insulin dependent diabetics
Prog. Lipid Res. Vol. 25, pp. 303-304, 1986 Printed in Great Britain. All rights reserved
, 0!63-7827/86/$0.00 + 0.50 © 1986 Pergamon Journals Ltd
DIHOMOGAMMALINOLENIC ACID INHIBITS PLATELET AGGREGATION AND STIMULATES PLATELET PROSTAGLANDIN E! PRODUCTION IN HEALTHY SUBJECTS BUT NOT IN INSULIN DEPENDENT DIABETICS D. P. MIKHAILIDIS,* S. KIRTLAND, t M. A. BARRADAS*and P. D^NDON^* *Metabolic Unit, Department of ChemicalPathology and Human Metabolism, Royal Free Hospital and School of Medicine, London NW3, U.K. troche Products Limited, WeiwynGarden City, Herts, U.K.
INTRODUCTION The administration of dihomogammalinolenic acid (DHLA), the precursor of prostaglandin E~ (PGE~), has been reported to inhibit platelet aggregation, possibly by increasing the release of PGEt by platelets.4 Since platelet hyperactivity has been reported in diabetes mellitus (DM), 2 we studied the effect of DHLA administration in insulindependent diabetics ODDs). SELECTION OF PATIENTS AND CONTROL SUBJECTS--MATERIALS AND METHODS Patients and Controls
Six healthy volunteers (5 males, 1 female; median age: 36 yr, range: 24-48 yr) and 6 IDD patients of similar age and sex distribution were studied. None of the patients or control subjects had received any drugs (other than insulin for the IDDs) for a period of 2 weeks preceding the study and for the duration of the study. The IDD patients did not have any clinically obvious complications of DM. Design of Study
DHLA (l g, orally, daily) was administered to the control subjects and IDDs. Fasting blood samples were collected on 3 occasions: prior to DHLA; following 21 days of DHLA and 21 days after cessation of DHLA administration. Tests Carried out in Blood Samples
(a) Platelet aggregation was carried out as previously described, the aggregating agent being ADP (1, 2 and 10 ~mol/l). 5 (b) Serum prostanoid measurements were carried out following coagulation, at 37°C, for 1 hr. The level of thromboxane B2 (TxB2, the stable, spontaneous metabolite of TxA 2) was assayed directly from serum using a specific radioimmunoassay; PGEm and PGE2 levels were measured by radioimmunoassay following extraction and isolation by argentation thin layer chromatography. (c) Fatty acid composition of RBC was measured as previously described; ! the procedure involved extraction, derivatization and gas chromatography. RESULTS Following DHLA administration, platelet aggregation in response to low concentrations of ADP was significantly inhibited (p < 0.01 at 2/~mol/l; p < 0.05 at 1/~mol/l) in the control subjects but not in the IDDs. These changes were similar to those previously reported: 303
304
D. P. Mikhailidis et al. TABLE 1. Effect of DHLA on SerumThromboxane, PGEi,PGE2andon the Proportions of DHLA and Araehidonic Acid (AA) in Total Red Cell Lipids Healthy volunteers Before DHLA
Insulin-dependent diabetics
After DHLA
After washout
Before DHLA
After DHLA
After washout
97 (52-238) 241" (156-397) 2.15 (0.7-2.7)
102 (84-290) 30** (25-63) 0.74 (0.3-2.3)
114 (36-252) 19 (ND--60) 1.28 (0.3-1.6)
77 (52-132) 30 (ND-70) 0.79 (0.2-1.0)
68 (24-209) ND 1.40 (0. i-2.9)
2.2** (1.8-2.7) 12.6" (10.0-15.1)
1.6 (0.9--2.1) 14.6" (8.9-19.8)
0.9 (0.7-1.7) 10.5 (4.8-14.8)
2.1" (1.4-2.5) 10.2 (5.1-17.4)
1.2 (0.8-2.9) 12.7 (5.7-16.2)
Serum prostanoids TxB2 (ng/ml) PGE I (pg/ml) PGE: (ng/ml)
76 (44-211) 110 (93-157) 1.10 (0.5-2.0)
Red cell fatty acids DHLA (wt%) AA (wt%)
1.2 (0.9-1.7) 7.9 (5.9-12.0)
*p < 0.004; **p < 0.002; value compared against pre-DHLA value. ND--not detectable.
Serum levels of TxB2 and PGE: (Table 1) were unchanged following DHLA, in both controls and IDDs. In contrast, serum levels of PGEI increased significantly in the controls but not in the IDDs, following DHLA administration. PGEI levels were consistently higher in the controls, whatever the time of sampling (see Table 1). DHLA content of RBC increased significantly in both controls and IDDs following supplementation; arachidonic acid (AA) content, however, only increased significantly in the controls (see Table 1). DISCUSSION
The present findings confirm that DHLA administration to healthy subjects is associated with inhibition of platelet aggregation and increased PGE~ production. 4 However, the present study indicates that these changes may not occur in IDDs. This lack of inhibition of platelet aggregation may relate to the low concentrations of PGE~ (an inhibitor of platelet aggregation 4) observed in the serum samples of IDDs. The low concentrations of PGEI detected do not necessarily preclude a biologically relevant role for this PG, since intracellular and local "microenvironment" concentrations may be more relevant than the amounts added in vitro to produce significant inhibition of aggregation. The mechanism responsible for the decreased PGEI production by the IDDs does not appear to involve a deficiency of substrate, since DHLA levels in the RBC were very similar to the control values; a defect involving the steps between DHLA in the cell membrane phosphotipids and PGE~ release from platelets (and possibly other cells) therefore seems to be responsible. Future studies clearly need to establish whether the composition of RBC fatty acids reflects the fatty acid composition of other cells. The increase in DHLA and AA content of RBC from controls, but of DHLA only in the RBC from IDDs, is in agreement with the presence of a A5 enzyme defect which has been demonstrated in experimental DM in animals? The use of DHLA in DM does not appear to result in any obvious beneficial effects, but further studies involving higher doses (perhaps administered for more prolonged periods) may overcome the "defects" which prevented DHLA from mediating its anti-aggregatory effects. REFERENCES 1. BLIGH, E. G. and DWYER, W . J. Can. ,I. Bioehem. Pharmacol. 37, 911-917 (1959). 2. GENSlNI, G. F., ABBA1"E,R., FAVlLLA, S. and N ~ SF.a~I, G. G. Thromb. ltaem. 42, 983-993 (1979). 3. HOLMAN,R. T., JOHNSON,S. B., G ~ t ~ g D , J. M., MASS,r, S. M., KtYPOaO-SANaEgG, S. and BROWN, D. M. Proc. Natl. Acad. Sei. USA go, 2375--2379 0983). 4. KERNOFF,e. B. A., WILLIS, A. L., STONE,K. J., DAVI~, J. A. and MACNICOL,G. Br. Med. J. 2, 1441-1444 (1979). 5. M1KH~LIDIS, D. P., J ~ , J. Y., B ~ A S , M. A., Ggr~N, N. and DANOONA, P. Br. Med. J. 287, 1495-1498 (1983).
, 0!63-7827/86/$0.00 + 0.50 © 1986 Pergamon Journals Ltd
DIHOMOGAMMALINOLENIC ACID INHIBITS PLATELET AGGREGATION AND STIMULATES PLATELET PROSTAGLANDIN E! PRODUCTION IN HEALTHY SUBJECTS BUT NOT IN INSULIN DEPENDENT DIABETICS D. P. MIKHAILIDIS,* S. KIRTLAND, t M. A. BARRADAS*and P. D^NDON^* *Metabolic Unit, Department of ChemicalPathology and Human Metabolism, Royal Free Hospital and School of Medicine, London NW3, U.K. troche Products Limited, WeiwynGarden City, Herts, U.K.
INTRODUCTION The administration of dihomogammalinolenic acid (DHLA), the precursor of prostaglandin E~ (PGE~), has been reported to inhibit platelet aggregation, possibly by increasing the release of PGEt by platelets.4 Since platelet hyperactivity has been reported in diabetes mellitus (DM), 2 we studied the effect of DHLA administration in insulindependent diabetics ODDs). SELECTION OF PATIENTS AND CONTROL SUBJECTS--MATERIALS AND METHODS Patients and Controls
Six healthy volunteers (5 males, 1 female; median age: 36 yr, range: 24-48 yr) and 6 IDD patients of similar age and sex distribution were studied. None of the patients or control subjects had received any drugs (other than insulin for the IDDs) for a period of 2 weeks preceding the study and for the duration of the study. The IDD patients did not have any clinically obvious complications of DM. Design of Study
DHLA (l g, orally, daily) was administered to the control subjects and IDDs. Fasting blood samples were collected on 3 occasions: prior to DHLA; following 21 days of DHLA and 21 days after cessation of DHLA administration. Tests Carried out in Blood Samples
(a) Platelet aggregation was carried out as previously described, the aggregating agent being ADP (1, 2 and 10 ~mol/l). 5 (b) Serum prostanoid measurements were carried out following coagulation, at 37°C, for 1 hr. The level of thromboxane B2 (TxB2, the stable, spontaneous metabolite of TxA 2) was assayed directly from serum using a specific radioimmunoassay; PGEm and PGE2 levels were measured by radioimmunoassay following extraction and isolation by argentation thin layer chromatography. (c) Fatty acid composition of RBC was measured as previously described; ! the procedure involved extraction, derivatization and gas chromatography. RESULTS Following DHLA administration, platelet aggregation in response to low concentrations of ADP was significantly inhibited (p < 0.01 at 2/~mol/l; p < 0.05 at 1/~mol/l) in the control subjects but not in the IDDs. These changes were similar to those previously reported: 303
304
D. P. Mikhailidis et al. TABLE 1. Effect of DHLA on SerumThromboxane, PGEi,PGE2andon the Proportions of DHLA and Araehidonic Acid (AA) in Total Red Cell Lipids Healthy volunteers Before DHLA
Insulin-dependent diabetics
After DHLA
After washout
Before DHLA
After DHLA
After washout
97 (52-238) 241" (156-397) 2.15 (0.7-2.7)
102 (84-290) 30** (25-63) 0.74 (0.3-2.3)
114 (36-252) 19 (ND--60) 1.28 (0.3-1.6)
77 (52-132) 30 (ND-70) 0.79 (0.2-1.0)
68 (24-209) ND 1.40 (0. i-2.9)
2.2** (1.8-2.7) 12.6" (10.0-15.1)
1.6 (0.9--2.1) 14.6" (8.9-19.8)
0.9 (0.7-1.7) 10.5 (4.8-14.8)
2.1" (1.4-2.5) 10.2 (5.1-17.4)
1.2 (0.8-2.9) 12.7 (5.7-16.2)
Serum prostanoids TxB2 (ng/ml) PGE I (pg/ml) PGE: (ng/ml)
76 (44-211) 110 (93-157) 1.10 (0.5-2.0)
Red cell fatty acids DHLA (wt%) AA (wt%)
1.2 (0.9-1.7) 7.9 (5.9-12.0)
*p < 0.004; **p < 0.002; value compared against pre-DHLA value. ND--not detectable.
Serum levels of TxB2 and PGE: (Table 1) were unchanged following DHLA, in both controls and IDDs. In contrast, serum levels of PGEI increased significantly in the controls but not in the IDDs, following DHLA administration. PGEI levels were consistently higher in the controls, whatever the time of sampling (see Table 1). DHLA content of RBC increased significantly in both controls and IDDs following supplementation; arachidonic acid (AA) content, however, only increased significantly in the controls (see Table 1). DISCUSSION
The present findings confirm that DHLA administration to healthy subjects is associated with inhibition of platelet aggregation and increased PGE~ production. 4 However, the present study indicates that these changes may not occur in IDDs. This lack of inhibition of platelet aggregation may relate to the low concentrations of PGE~ (an inhibitor of platelet aggregation 4) observed in the serum samples of IDDs. The low concentrations of PGEI detected do not necessarily preclude a biologically relevant role for this PG, since intracellular and local "microenvironment" concentrations may be more relevant than the amounts added in vitro to produce significant inhibition of aggregation. The mechanism responsible for the decreased PGEI production by the IDDs does not appear to involve a deficiency of substrate, since DHLA levels in the RBC were very similar to the control values; a defect involving the steps between DHLA in the cell membrane phosphotipids and PGE~ release from platelets (and possibly other cells) therefore seems to be responsible. Future studies clearly need to establish whether the composition of RBC fatty acids reflects the fatty acid composition of other cells. The increase in DHLA and AA content of RBC from controls, but of DHLA only in the RBC from IDDs, is in agreement with the presence of a A5 enzyme defect which has been demonstrated in experimental DM in animals? The use of DHLA in DM does not appear to result in any obvious beneficial effects, but further studies involving higher doses (perhaps administered for more prolonged periods) may overcome the "defects" which prevented DHLA from mediating its anti-aggregatory effects. REFERENCES 1. BLIGH, E. G. and DWYER, W . J. Can. ,I. Bioehem. Pharmacol. 37, 911-917 (1959). 2. GENSlNI, G. F., ABBA1"E,R., FAVlLLA, S. and N ~ SF.a~I, G. G. Thromb. ltaem. 42, 983-993 (1979). 3. HOLMAN,R. T., JOHNSON,S. B., G ~ t ~ g D , J. M., MASS,r, S. M., KtYPOaO-SANaEgG, S. and BROWN, D. M. Proc. Natl. Acad. Sei. USA go, 2375--2379 0983). 4. KERNOFF,e. B. A., WILLIS, A. L., STONE,K. J., DAVI~, J. A. and MACNICOL,G. Br. Med. J. 2, 1441-1444 (1979). 5. M1KH~LIDIS, D. P., J ~ , J. Y., B ~ A S , M. A., Ggr~N, N. and DANOONA, P. Br. Med. J. 287, 1495-1498 (1983).