BIOCHEMICAL
MEDICINE
AND
METABOLIC
BIOLOGY
47,
199-204 (1992)
Effect of Diazepam on the Fatty Acid Composition of Plasma and Liver Phospholipids in Rat VANJA RISTI~ AND SNEZANA
R. VRBA~KI
Institute for Medical Research, P.O. Box 721, 11001 Beograd, Yugoslavia Received December 27, 1991 Male Wistar rats (2 months old) were maintained on a nutritionally adequate diet, and diazepam was administered at a dose of 10 mg/kg/day. After 24 weeks the effects on the fatty acid composition of plasma and liver phospholipids were studied. Increased levels of palmitic (16:0), palmitoleic (16: ln-7) stearic (18:0), and oleic (18: ln-9) acids were found in plasma phospholipids. In contrast, the levels of docosapentanoic (22:5n-3) and docosahexanoic (22:6n-3; DHA) acids were drastically decreased by diazepam. A significant decrease produced by diazepam was also found in levels of DHA in liver phosphohpids. o 199~ Academic
Press. Inc.
Diazepam (DZP, 7-chloro-l,3-dihydro-l-methyi-5-phenyl-2H-l,4-benzodiazepin-2(W)-one; 1 ,Cbenzodiazepin) is widely used therapeutically as an anxiolytic, anticonvulsant, muscle relaxant, and psychosedative (1). The presence of highaffinity binding sites for this drug has been demonstrated in the CNS (2), and these receptors have been shown to be functionally coupled to both the y-aminobutyric acid receptor and the chloride ionophore (3). In addition, low-affinity binding sites for benzodiazepins appear to be present in some peripheral tissues, such as the kidney, lung, liver (4), heart, skeletal muscle, smooth muscle, mast cells (5), and even in the brain (6). It has been demonstrated that the so-called “benzodiazepin peripheral recognition sites” are pharmacologically and biochemically distinct from the centraltype receptors (7,8). Anholt (9) showed that these peripheral benzodiazepin binding sites are associated with the mitochondrial outer membrane, and suggested that these receptors may represent an important control site for modulation of intermediary (cellular) metabolism. However, the precise identity and physiological role of these peripheral-type receptors are still unclear. Some observations suggested that diazepam influences lipid and carbohydrate metabolism (10-12). Our previous study demonstrated that chronic diazepam treatment provokes changes in triacylglycerol and phospholipid levels in the liver of rats (13,14). The aim of the present investigation was to determine possible biochemical 199 0885-4505192 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
200
RISTIC AND VRBASKI
alterations in the fatty acid composition after chronic diazepam treatment.
of liver and plasma phospholipids
in rats
METHODS Animals and Treatment
Male Wistar rats (2 months old, approx. 210 g) were housed individually in two groups of eight rats each. Over a 24-week period, both groups were fed a pelleted cereal-based diet (21% protein, 60% carbohydrate, 7% fat, 0.25% vitamin premix, 2.25% mineral mixture; Veterinarski Zavod, Zemun). The fatty acid composition of the diet was 0.4% 14:0, 8.7% 16:0, 0.7% 16:ln-7, 4.9% 18:0, 26.3% 18:ln-9, 54.3% 18:2n-6, 2.8% 18:3n-3; and 1.9% 20:4n-6. All animals were matched for initial body weight and were weighed at weekly intervals thereafter. One experimental group (diazepam) was offered a diazepam (Krka, Novo Mesto) solution instead of drinking water at a constant dose of 10 mg/kg/day (15). The control group had free access to tap water. All rats of both groups were offered 16 g of diet. After 24 weeks of treatment, the animals were fasted for 24 h, and approx. 54 h after the last dose of diazepam they were sacrificed under nembutal narcosis. Biochemical Analysis
Plasma and liver lipids were extracted with a chloroform-methanol mixture (16,17) and were separated by thin-layer chromatography using hexane-diethyl ether-acetic acid (87: 12: 1, v/v/v). The phospholipid fraction which remained at the origin was removed from the plate by scraping and eluted with hexane. Methyl esters of phospholipid fatty acids were prepared by transesterification with 2 M NaOH-methanol (heating for 1 h) and 1 M sulfuric acid-methanol (heating for 2 h). A Varian GC (Model 3400, Varian Associates) equipped with flame ionization detector and 30 x 0.53 mm SP-2380 fused silica capillary column (Supelco, Bellefonte, PA) was used to analyze the fatty acid methyl esters. The oven temperature was held at 130°C for 3 min, programmed to 190°C at 3”C/min, and then held at 190°C for 15 min. The chromatograph was operated with a column flow rate of 2.5 ml nitrogen/min, and injection temperature 220°C. The detector was maintained at 250°C. Individual fatty acid methyl esters in the sample were identified from the retention times of authentic standards (Sigma Chemical Co.) and/or the PUFA-2 standard mixture (Supelco). Peak areas were determined with a Varian 4290 integrator, and the areas were reported as weight percentages. Statistical Analysis
All results were expressed as means + SD. The significance of differences in liver and plasma lipids after diazepam treatment was analyzed by Student t test. RESULTS
During the study the average food intake per rat was 16 g or 270 kJ per day in both the control and diazepam groups. The initial and final body weights and
DIAZEPAM
Mean (*SD)
AND FATTY
201
ACIDS
TABLE 1 BodyWeight and Daily Body Weight Gain in Rats of the Control and Diazepam Group
Initial body weight (g) Final body weight (g) Daily body weight (g)
Control
Diazepam
209 -+ 23.8 339 + 20.4 0.77 k 0.1
226 + 21.0 333 f 12.3 0.63 f 0.1
daily body weight gain for both groups are summarized in Table 1. Body weight gain did not differ significantly between the groups. Significant differences were observed in plasma phospholipid fatty acids after diazepam treatment (Table 2). The percentage of 16:0, 16: ln-7, 18:0, and 18: ln9 acids was higher in the diazepam group, while the percentage of 22:5n-3 and 22:6n-3 was lower. Total saturated and monounsaturated fatty acids in plasma phospholipids were similarly affected by diazepam and were increased by about 10%. The polyunsaturated n-3 fatty acids were reduced by about 34%. After diazepam treatment the fatty acid composition of liver phospholipids showed a significant decrease of 22 : 6n-3, i.e., PUFA n-3 (Table 3).
TABLE 2 Influence of Diazepam on the Fatty Acid Composition of Plasma Phospholipids” Fatty acids
Control
16:O 16: ln-7 18:O 18: ln-9 18 : ln-7 18:2n-6 18:3n-3 20 : 3n-9 20: 3n-6 20:4n-6 20:5n-3 22:4n-6 22 :5n-3 22:6n-3
21.52 0.19 30.37 5.05 1.29 15.90 0.30 0.17 0.47 20.15 0.20 0.54 0.33 3.50
SFAb MUFA PUFA n-6 n-3
f 1.11 + 0.07 k 0.96 +- 0.25 f 0.08 k 1.20 f 0.05 * 0.05 f 0.06 f 2.82 k 0.03 k 0.06 2 0.05 5 0.40
Diazepam 23.70 0.37 33.40 5.68 1.23 14.57 0.26 0.15 0.29 17.17 0.27 0.59 0.11 2.21
Significance
f 1.12 2 0.10 k 1.98 f 0.24 f 0.09 2 2.19 f 0.12 * 0.07 + 0.04 + 3.76 + 0.12 f 0.09 k 0.05 -r- 0.30
0.01 0.01 0.01 0.001 ns ns ns ns ns ns ns
51.90 ‘- 2.10 6.53 t 0.30
57.10 k 3.00 7.28 2 0.33
0.01 0.001
37.06 4.33 ft 4.14 0.50
32.55 2.85 k” 6.08 0.50
O.&
0.k 0.001
Note. Means k SD for eight rats. a Values are the weight percentages of total identified fatty acids. b SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated acids.
fatty
202
RISTIC AND VRBASKI TABLE 3 Influence of Diazepam on the Fatty Acid Composition of Liver Phospholipids”
Fatty acids
Control
Diazepam
16:O 16: ln-7 18:O 18: ln-9 18: ln-7 18 :2n-6 20 : 3n-6 20:4n-6 22 : 4n-6 22 : 5n-3 22 : 6n-3
14.60 0.33 28.25 3.34 1.34 15.50 0.27 29.55 0.31 0.35 6.16
f k 2 IL 2 2 k f f f 2
0.90 0.05 1.22 0.46 0.14 1.53 0.03 1.79 0.06 0.06 1.00
15.62 0.28 27.24 3.17 1.33 15.39 0.26 31.59 0.28 0.37 4.47
f k f 2 2 2 + f f k k
1.00 0.03 1.37 0.39 0.13 0.85 0.05 2.35 0.03 0.06 0.71
SFAb MUFA PUFA n-6 PUFA n-3
42.85 5.01 45.63 6.51
k k f f
2.15 0.66 3.11 1.06
42.86 4.78 47.52 4.84
k -c -c +
1.90 0.55 3.00 0.90
Significance ns ns ns ns ns ns ns ns ns Ofb”l IIS
ns OnoSl
Note. Means f SD for eight rats. * Values are the weight percentages of total identified fatty acids. b SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids.
DISCUSSION The main interest in our investigation was to determine possible alterations in phospholipid fatty acid composition in rat plasma and liver homogenate after 24 weeks of diazepam treatment. It would appear that this is the first report of a phenomenon of this kind concerning the effects of diazepam. The duration of chronic treatment (6 months) was longer than is generally used in such studies and is probably more relevant to the clinical situation (18). The contents of saturated and monounsaturated fatty acids in plasma phospholipids were elevated by diazepam. On the other hand, diazepam produced a drastic decrease of PUFA n-3, 22: 5n-3, and 22: 6n-3. These significant changes in plasma phospholipid fatty acid composition indicate a disturbance in fatty acid metabolism. Diazepam also caused a significant decrease of 22: 6n-3 in liver phospholipids. It is evident that diazepam markedly affected the metabolism of docosahexanoic acid (DHA). In the liver, intermediary metabolism leads to secretion of DHA in the form of lipoprotein phospholipids, which are then a major source of the DHA that is required by the brain. DHA is the most prominent fatty acid in the aminophospholipids, phosphatidylethanolamine and phosphatidylserine, of brain membranes and has a more specific biochemical function in neuronal transmission and the visual process (19,20). It may be supposed that the DHA deficiency in liver and plasma phospholipids causes changes in brain phospholipid composition and is associated with the decrease of phosphatidylethanolamine and phosphatidylserine in brain induced by diazepam (21).
DIAZEPAM
AND FATTY
ACIDS
203
Thus, the change in brain phospholipid composition produced by diazepam might alter the transmission of information through the brain’s visual pathways as well as affect the photoreceptive process in the retina (22). The exact mechanism by which diazepam affects fatty acid metabolism is not clear and will require further research. ACKNOWLEDGMENT This study was supported by the Medical Research Fund of Serbia.
REFERENCES 1. Skolnick P, Paul SM. The mechanism(s) of action of the benzoidiazepines. Med Res Rev 1:3-22, 1981. 2. Miihler H, Okada TC. Benzodiazepine receptor: Demonstration in the central nervous system. Science
198~849-851,
1977.
3. Olsen RW. Drug interaction at the GABA receptor-ionophore complex. Annu Rev Pharmacol Toxic01 22~245-277, 1982. 4. Braestrup C, Squires RE. Specific benzodiazepine receptors in rat brain characterized by high affinity [Hldiazepam binding. Proc Nat1 Acad Sci USA 74~3805-3809, 1977. 5. Taniguchi T, Wang JK, Spector S. [H]Diazepam binding sites on rat heart and kidney. Biochem Pharmacol31:589-600,
1982.
6. Marangos PJ, Pate1 J, Boulenger JP, Clark-Rosenberg R. Characterization of peripheral-type benzo-diazepine binding sites in brain using [H]Ro 5-4864. Mol PharmacoZ22:26-32, 1982. 7. Skerritt H, Chow SC, Johnston GAR, Davies LP. Purines interact with central but not peripheral benzodiazepine binding sites. Neurosci Lett 34:63-68, 1982. 8. Trifilotti RR, Verma A, Snyder SH. Molecular identification of periferal type benzodiazepine receptors. Sot Neurosci Abstr ti666, 1986. 9. Anholt RR. Mitochondrial benzodiazepine receptors as potential modulators of intermediary metabolism. Trends Pharmacol Sci 7:507-512, 1986. 10. Cuparencu B, Horak J, Cucuianu M, Hancu N, Seusan E, Vincze J. Effects of some benzodiazepine derivatives on librinolysis and serum lipids in normolipimic rats and in humans. Atherosclerosis 31:435-441, 1978. 11. Cuparencu B, Madar J, Horak J. Effects of the intraperitoneal administration of diazepam on the streptozotocin-induced diabetes in rats. Curr Ther Res 38~30-39, 1985. 12. Madar J, Cuparencu D, Horak J, Oildan N, Mohammad HE, Marmo C. Improvement of acute intravenous glucose tolerance in streptozotocin-induced diabetes in rats with intraperitoneal administration of diazepam. Curr Ther Res 41: 961-966, 1987. 13. RistiC V, VrbaSki S, Petrovic G, RistiC M. The effect of long-term diazepam treatment on liver and plasma phospholipids in rats. Actu Pharm Jugoslavica 38:151-155, 1988. 14. RistiC V, VrbaSki S, Petrovic G, RistiC M. Liver and plasma lipids after long-term diazepam treatment. Arch Biol Nauka 41:19-24, 1989. 15. Fuch V, Burbes E, Coper H. The influence of haloperidol and aminooxyacetic acid on etonitazene, alcohol, diazepam and barbital composition. Drug Alcohol Depend l&179-186, 1984. 16. Folch J, Lees M, Stanley-Sloane GH. A simple method for isolation and purification of total lipids from animal tissue. J Biol Chem 226:497-503, 1957. 17. Alling C, Liljequist S, Engel J. The effect of chronic ethanol administration on lipids and fatty acids in subcellular fractions of rat brain. Med Biol 60:149-154, 1982. 18. Swift CG, Stevenson IH. Benzodiazepines in the elderly. In The Benzodiazepines (Costa E, Ed.). New York: Raven Press, 1983, pp 225-236. 19. Carlson SE, Salem N. Essentiality of n-3 fatty acids in growth and development of infants. In Health Effects of n-3 Polyunsaturated Fatty Acids in Seafoods (Simopoulos AP, Kifer RR, Martin RE, Barlow SM, Eds.), Vol. 66. Basel: Karger 1991, pp 74-86. 20. Bourre J-M, Dumont 0, Piciotti M, Clement M et al. Essentialiaty of n-3 fatty acids for brain
204
RJSTIC AND VRBASKI
structure and function. In Health Effects of n-3 Polyunsaturated Fatty Acids in Seafoods. (Simopoulos AP, Kifre RR, Martin RE, Barlow SM. Eds.), Vol. 66. Base]: Karger, 1991, pp 103-117. 21. VrbaF;ki S, RistiC VI, PetroviC CT, RistiC MS. Brain lipids in rat after chronic diazepam treatment. J Biochem 105:705-707, 1989. 22. Connor WE, Neuringer M, Reisbick S. Essentiality of n-3 fatty acids: Evidence from the primate model and implications for human nutrition. In Health Effects n-3 Polyunsaturated Fatty Acids in Seafoods. (Simopoulos AP, Kifer RR, Martin RE, Barlow SM. Eds.), Vol. 66. Basel: Karger, 1991, pp 118-132.
MEDICINE
AND
METABOLIC
BIOLOGY
47,
199-204 (1992)
Effect of Diazepam on the Fatty Acid Composition of Plasma and Liver Phospholipids in Rat VANJA RISTI~ AND SNEZANA
R. VRBA~KI
Institute for Medical Research, P.O. Box 721, 11001 Beograd, Yugoslavia Received December 27, 1991 Male Wistar rats (2 months old) were maintained on a nutritionally adequate diet, and diazepam was administered at a dose of 10 mg/kg/day. After 24 weeks the effects on the fatty acid composition of plasma and liver phospholipids were studied. Increased levels of palmitic (16:0), palmitoleic (16: ln-7) stearic (18:0), and oleic (18: ln-9) acids were found in plasma phospholipids. In contrast, the levels of docosapentanoic (22:5n-3) and docosahexanoic (22:6n-3; DHA) acids were drastically decreased by diazepam. A significant decrease produced by diazepam was also found in levels of DHA in liver phosphohpids. o 199~ Academic
Press. Inc.
Diazepam (DZP, 7-chloro-l,3-dihydro-l-methyi-5-phenyl-2H-l,4-benzodiazepin-2(W)-one; 1 ,Cbenzodiazepin) is widely used therapeutically as an anxiolytic, anticonvulsant, muscle relaxant, and psychosedative (1). The presence of highaffinity binding sites for this drug has been demonstrated in the CNS (2), and these receptors have been shown to be functionally coupled to both the y-aminobutyric acid receptor and the chloride ionophore (3). In addition, low-affinity binding sites for benzodiazepins appear to be present in some peripheral tissues, such as the kidney, lung, liver (4), heart, skeletal muscle, smooth muscle, mast cells (5), and even in the brain (6). It has been demonstrated that the so-called “benzodiazepin peripheral recognition sites” are pharmacologically and biochemically distinct from the centraltype receptors (7,8). Anholt (9) showed that these peripheral benzodiazepin binding sites are associated with the mitochondrial outer membrane, and suggested that these receptors may represent an important control site for modulation of intermediary (cellular) metabolism. However, the precise identity and physiological role of these peripheral-type receptors are still unclear. Some observations suggested that diazepam influences lipid and carbohydrate metabolism (10-12). Our previous study demonstrated that chronic diazepam treatment provokes changes in triacylglycerol and phospholipid levels in the liver of rats (13,14). The aim of the present investigation was to determine possible biochemical 199 0885-4505192 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
200
RISTIC AND VRBASKI
alterations in the fatty acid composition after chronic diazepam treatment.
of liver and plasma phospholipids
in rats
METHODS Animals and Treatment
Male Wistar rats (2 months old, approx. 210 g) were housed individually in two groups of eight rats each. Over a 24-week period, both groups were fed a pelleted cereal-based diet (21% protein, 60% carbohydrate, 7% fat, 0.25% vitamin premix, 2.25% mineral mixture; Veterinarski Zavod, Zemun). The fatty acid composition of the diet was 0.4% 14:0, 8.7% 16:0, 0.7% 16:ln-7, 4.9% 18:0, 26.3% 18:ln-9, 54.3% 18:2n-6, 2.8% 18:3n-3; and 1.9% 20:4n-6. All animals were matched for initial body weight and were weighed at weekly intervals thereafter. One experimental group (diazepam) was offered a diazepam (Krka, Novo Mesto) solution instead of drinking water at a constant dose of 10 mg/kg/day (15). The control group had free access to tap water. All rats of both groups were offered 16 g of diet. After 24 weeks of treatment, the animals were fasted for 24 h, and approx. 54 h after the last dose of diazepam they were sacrificed under nembutal narcosis. Biochemical Analysis
Plasma and liver lipids were extracted with a chloroform-methanol mixture (16,17) and were separated by thin-layer chromatography using hexane-diethyl ether-acetic acid (87: 12: 1, v/v/v). The phospholipid fraction which remained at the origin was removed from the plate by scraping and eluted with hexane. Methyl esters of phospholipid fatty acids were prepared by transesterification with 2 M NaOH-methanol (heating for 1 h) and 1 M sulfuric acid-methanol (heating for 2 h). A Varian GC (Model 3400, Varian Associates) equipped with flame ionization detector and 30 x 0.53 mm SP-2380 fused silica capillary column (Supelco, Bellefonte, PA) was used to analyze the fatty acid methyl esters. The oven temperature was held at 130°C for 3 min, programmed to 190°C at 3”C/min, and then held at 190°C for 15 min. The chromatograph was operated with a column flow rate of 2.5 ml nitrogen/min, and injection temperature 220°C. The detector was maintained at 250°C. Individual fatty acid methyl esters in the sample were identified from the retention times of authentic standards (Sigma Chemical Co.) and/or the PUFA-2 standard mixture (Supelco). Peak areas were determined with a Varian 4290 integrator, and the areas were reported as weight percentages. Statistical Analysis
All results were expressed as means + SD. The significance of differences in liver and plasma lipids after diazepam treatment was analyzed by Student t test. RESULTS
During the study the average food intake per rat was 16 g or 270 kJ per day in both the control and diazepam groups. The initial and final body weights and
DIAZEPAM
Mean (*SD)
AND FATTY
201
ACIDS
TABLE 1 BodyWeight and Daily Body Weight Gain in Rats of the Control and Diazepam Group
Initial body weight (g) Final body weight (g) Daily body weight (g)
Control
Diazepam
209 -+ 23.8 339 + 20.4 0.77 k 0.1
226 + 21.0 333 f 12.3 0.63 f 0.1
daily body weight gain for both groups are summarized in Table 1. Body weight gain did not differ significantly between the groups. Significant differences were observed in plasma phospholipid fatty acids after diazepam treatment (Table 2). The percentage of 16:0, 16: ln-7, 18:0, and 18: ln9 acids was higher in the diazepam group, while the percentage of 22:5n-3 and 22:6n-3 was lower. Total saturated and monounsaturated fatty acids in plasma phospholipids were similarly affected by diazepam and were increased by about 10%. The polyunsaturated n-3 fatty acids were reduced by about 34%. After diazepam treatment the fatty acid composition of liver phospholipids showed a significant decrease of 22 : 6n-3, i.e., PUFA n-3 (Table 3).
TABLE 2 Influence of Diazepam on the Fatty Acid Composition of Plasma Phospholipids” Fatty acids
Control
16:O 16: ln-7 18:O 18: ln-9 18 : ln-7 18:2n-6 18:3n-3 20 : 3n-9 20: 3n-6 20:4n-6 20:5n-3 22:4n-6 22 :5n-3 22:6n-3
21.52 0.19 30.37 5.05 1.29 15.90 0.30 0.17 0.47 20.15 0.20 0.54 0.33 3.50
SFAb MUFA PUFA n-6 n-3
f 1.11 + 0.07 k 0.96 +- 0.25 f 0.08 k 1.20 f 0.05 * 0.05 f 0.06 f 2.82 k 0.03 k 0.06 2 0.05 5 0.40
Diazepam 23.70 0.37 33.40 5.68 1.23 14.57 0.26 0.15 0.29 17.17 0.27 0.59 0.11 2.21
Significance
f 1.12 2 0.10 k 1.98 f 0.24 f 0.09 2 2.19 f 0.12 * 0.07 + 0.04 + 3.76 + 0.12 f 0.09 k 0.05 -r- 0.30
0.01 0.01 0.01 0.001 ns ns ns ns ns ns ns
51.90 ‘- 2.10 6.53 t 0.30
57.10 k 3.00 7.28 2 0.33
0.01 0.001
37.06 4.33 ft 4.14 0.50
32.55 2.85 k” 6.08 0.50
O.&
0.k 0.001
Note. Means k SD for eight rats. a Values are the weight percentages of total identified fatty acids. b SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated acids.
fatty
202
RISTIC AND VRBASKI TABLE 3 Influence of Diazepam on the Fatty Acid Composition of Liver Phospholipids”
Fatty acids
Control
Diazepam
16:O 16: ln-7 18:O 18: ln-9 18: ln-7 18 :2n-6 20 : 3n-6 20:4n-6 22 : 4n-6 22 : 5n-3 22 : 6n-3
14.60 0.33 28.25 3.34 1.34 15.50 0.27 29.55 0.31 0.35 6.16
f k 2 IL 2 2 k f f f 2
0.90 0.05 1.22 0.46 0.14 1.53 0.03 1.79 0.06 0.06 1.00
15.62 0.28 27.24 3.17 1.33 15.39 0.26 31.59 0.28 0.37 4.47
f k f 2 2 2 + f f k k
1.00 0.03 1.37 0.39 0.13 0.85 0.05 2.35 0.03 0.06 0.71
SFAb MUFA PUFA n-6 PUFA n-3
42.85 5.01 45.63 6.51
k k f f
2.15 0.66 3.11 1.06
42.86 4.78 47.52 4.84
k -c -c +
1.90 0.55 3.00 0.90
Significance ns ns ns ns ns ns ns ns ns Ofb”l IIS
ns OnoSl
Note. Means f SD for eight rats. * Values are the weight percentages of total identified fatty acids. b SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids.
DISCUSSION The main interest in our investigation was to determine possible alterations in phospholipid fatty acid composition in rat plasma and liver homogenate after 24 weeks of diazepam treatment. It would appear that this is the first report of a phenomenon of this kind concerning the effects of diazepam. The duration of chronic treatment (6 months) was longer than is generally used in such studies and is probably more relevant to the clinical situation (18). The contents of saturated and monounsaturated fatty acids in plasma phospholipids were elevated by diazepam. On the other hand, diazepam produced a drastic decrease of PUFA n-3, 22: 5n-3, and 22: 6n-3. These significant changes in plasma phospholipid fatty acid composition indicate a disturbance in fatty acid metabolism. Diazepam also caused a significant decrease of 22: 6n-3 in liver phospholipids. It is evident that diazepam markedly affected the metabolism of docosahexanoic acid (DHA). In the liver, intermediary metabolism leads to secretion of DHA in the form of lipoprotein phospholipids, which are then a major source of the DHA that is required by the brain. DHA is the most prominent fatty acid in the aminophospholipids, phosphatidylethanolamine and phosphatidylserine, of brain membranes and has a more specific biochemical function in neuronal transmission and the visual process (19,20). It may be supposed that the DHA deficiency in liver and plasma phospholipids causes changes in brain phospholipid composition and is associated with the decrease of phosphatidylethanolamine and phosphatidylserine in brain induced by diazepam (21).
DIAZEPAM
AND FATTY
ACIDS
203
Thus, the change in brain phospholipid composition produced by diazepam might alter the transmission of information through the brain’s visual pathways as well as affect the photoreceptive process in the retina (22). The exact mechanism by which diazepam affects fatty acid metabolism is not clear and will require further research. ACKNOWLEDGMENT This study was supported by the Medical Research Fund of Serbia.
REFERENCES 1. Skolnick P, Paul SM. The mechanism(s) of action of the benzoidiazepines. Med Res Rev 1:3-22, 1981. 2. Miihler H, Okada TC. Benzodiazepine receptor: Demonstration in the central nervous system. Science
198~849-851,
1977.
3. Olsen RW. Drug interaction at the GABA receptor-ionophore complex. Annu Rev Pharmacol Toxic01 22~245-277, 1982. 4. Braestrup C, Squires RE. Specific benzodiazepine receptors in rat brain characterized by high affinity [Hldiazepam binding. Proc Nat1 Acad Sci USA 74~3805-3809, 1977. 5. Taniguchi T, Wang JK, Spector S. [H]Diazepam binding sites on rat heart and kidney. Biochem Pharmacol31:589-600,
1982.
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