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Enzymatic synthesis of anandamide through N-acylphosphatidylethanolamine pathway in rat brain and testis
264 EFA & Eicosanoids 1997 - Edinburgh
Poster Presentations Thursday 24 July
P205
P206
The Effect of Administration of Thyme and Lovage Volatile Oils upon the Percentage Composition of Fatty Acids in a rat Model HJ.D. Dorman & S.G. Deans. Scottish Agricultural college, Biochemical Sciences Department, Ayr KA6 5HW, Scotland
A Preliminary Study into the Effects of the Oral Administration of Oregano Volatile Oil upon the Percentage Composition of Fatty Acids in a Rat Model HJD Dorman & SG Deans. Scottish Agricultural College, Biochemical Sciences Department, Ayr KA6 5HW, Scotland.
Volatile oils extracted from aromatic and medicinal plants have been used since antiquity by man for their biological activities. The bioactivities of these extracts include antibacterial, antifungal and ant±oxidant properties. Volatile oils extracted by hydrodistillation from Thyme (Thymus vutgarus) and Lovage (Levisticum officinalis) were administered to pregnant rats in two feeding trials to investigate their capacities to alter the fatty acid composition in cholesteryl ester, triglyceride, free fatty acid and phospholipid fractions. These were extracted from a number of organs, including muscle tissue. In the muscle, thyme and lovage volatile oils changed the percent composition of C17:0 from 3.32+-0.18 n=8 to 4.59+-0.31"* n=4 and CI2:0 from 3.32-+0.18 n=4 to 1.89-+0.23~* n=4 respectively in the cholesteryl ester fraction. In the triacylglyceride fraction, the effects of both oils were more dramatic, significant changes being found in saturated and unsaturated fatty acids, see table below. ~he Effect of the AAmini~tration of Thyme and Lovage Volatile Oils on the% ._Compositionof Fatty Acids in the Triacvl$1ycer!..deFraction of Maternal MuscleTiss~ FFA CONTROL THYME LOVAGE
Oregano volatile oil (Origanurn vulgate ssp. birtum) was administered at three concentration levels:165(a), 334(b) and 835mg\kg(c), to pregnant rats to investigate its capacity to change the fatty acid (FA) % composition in the cholesteryl ester (CE), triacylglyceride (TG), free fatty acid (FFA) and phospholipid (PL) fractions in a number of organs. In the kidney, C20:4(n-6) changed in the CE fraction at the highest treatment level, 33.60+10.80 n=3 to 10.98+-1.21 n=3. C18:1(n-7) changed from 1.41+_0.76 n=4 to 0.09-+0.01*** n=3 in the TG fraction. C14:0 changed at concentrations Co) and (c), from 0.79-+0.04 n=2 to 1.19+-0.09~ n=2 and 1.30+-0.06"* n=2 respective!y. In the PL fraction, C20:5(n-3) changed from 0.72+-0.01n=2 to 0.90_+0.01 n=2 at (a). In liver CE and TG fractions, C18:2(n-6) showed changes at (a), 20.93--1.35 n=3 to 14.45-+2.00" n=2 and from 32.01-+1.15 n~3 to 25.17+_L55" n=3 respectively. In the TG fraction, C20:1(n-9) changed from 0.17+-0.03 n=3 to 0.39+-0.07" n=2 at the (b) level. Modifications were limited in the FFA fraction to C22:4(n-6) at concentration (a), from 0.65+_0.05 n=2 to 0.43_+0.06 n=2. Both C17:0 and C22:4(n-6) changed significantly in the PL fraction at the (c) treatment level from 0 36-+0.02 n=4 to 0 52-+0 02"~ n=2 and from 0.50-+0.02n=4 to 0.97-+0.02""n=2. In the heart, all the lipid fractions showed significant changes. In the CE fraction, C18:2(n-6) changed from 6.12-+0.81 n=2 to 13.10+~.63"" n=4 at (b). C18:0, C20:4(n-6) and C22:6(n-3) all changed at the two lowest concentration levels. C18:2(n-6), C18:3(n-3), C20:3(n-6), C20:4(n-6) and C22:5(n-3) all showed changes at (b) in the TG fraction. In the FFA fraction, C20:0, C22:5(n-3), C22:6(n-3) all changed at (a) level. C22:6(n-3) was altered at the highest concentration, as did C18:2(n-6). C17:1(n-7), D.24+0.04n=3 to 0.60-+0.08" n=5 at (b); C18:2(n-6), 13.61-+0.63 n=3 to 16.14±0.04" n=2 at (c). These results suggest that oregano volatile oil may cause significant changes in the FA % composition profile of essential organs in different lipid fractions at the concentration levels tested in this experiment.
C17:0 0.35+0.02 4.59_+0.31 3.64_+0.41 C18:0 4.25_+0.15 8.64-+0.83 "'~ 12.565:1.81.** C18:1(N-9) 26,04±0.47 10,50-+1.19 *'~ 10,55±1.09"~ C18:26N-6) 26.13-+0.83 21.09_+4,51 12.01_+0.72"*" C18:3[N-3) 1.61_+0.09 1.97±0.46 2.54-+0.66*~ C20:1(N-9) 0.40-+0.02 2.05-+0.67 ~* rod. C22:6(N-3) 1.00+_0.08 n.d. 4.74_+2.02" n=10 for control values, n -> 4 for treatments except C18:30W-3) and C20:1(N-9) where n = 2. n.d. = no values were detected in the sample. Significance was determined using a Student t-test (* P <0,0S, *'~P< 0.01, '~'~*P< 0,001).
These results demonstrate that the administration of these volatile oils are capable of changing the percent composition of specific fatty acids in the cholesteryl ester and triacylglyceride fractions in muscle tissue of pregnant rats. These changes are consistent with other organs in both mother and pup tissues.
P207
P208
Cell Type Distribution And Subcellular Location Of Enzymes Involved In The Metabolism Of Acyl Ethanaloamines. A.A.Makda, M.A.Elmore, M.E.HilI, A.Stamps, S.Tejura And M.J.Finnen. Yamanouchi Research institute, Oxford, Littlemore Park Ox4 4SX.
ENZY~IATIC SYNTHESIS OF ANANDA/~IDETI~OUGH NACYLPHOSPHATIDYLETHANOLAY~NEPATHWAY IN RAT BRAIN AND TESTIS T. S u g i u r a , S. Kondo, S. Nakane, h. ¥ a m a s h i t a and K. Waku. F a c u l t y of P h a r m a c e u t i c a l S c i e n c e s , T e i k y o U n i v e r s i t y , Sagamiko, Kanagawa 199-01, J a p a n
Two N-acyl ethanlolamines, arachidonyl ethanolamine (AEA or anandamide) and palmitoyt ethanolamine (PEA) have been shown to be the endogenous ligands for the cannabinoid receptors. The CB1 receptor is found primarily in the CNS but also in the periphery. Unlike CB1, CB2 receptors are found only in the peripheral system with a relatively high distribution on B lymphocytes and macmphages. This study was undertaken to determine which cell types are capable of metabolising these N-acyl ethanolamines via the condensation and hydrolysis pathways. Synthesis of PEA was found mainly in monocyte/macmphage cell types (PBMC monocytes 33; U937 53.8; Monomac 56.9 pmol/mg protein/rain) with far less PEA synthesis activity detectable in T cells (PBMC lymphocytes 4.2; Jurkat 0.5 pmol/mg protein/min). In contrast AEA was synthesised to comparable extents by both monocytes and T cells (PBMC monocytes 6.3; PBMC ~ymphocytes 1.0; U937 0.68; Monomac 1.0 pmol/mg/min). On subcellular fractionation of U937 cells the highest specific activity for PEA and AEA synthesis was in the P3 microsomal fraction. Parallel studies on the degradation of PEA and AEA showed similar cell type and sub cellular distribution to synthesis. Degradation rates 'for PEA and AEA were at levels commensurate with the rates of synthesis.
Dual s y n t h e t i c p a t h w a y s have b e e n p r o p o s e d f o r t h e b i o s y n t h e s i s o f a n a n d a m i d e . One i s t h e e n z y m a t i c c o n d e n s a t i o n o f f r e e a r a c h i d o n i c a c i d and e t h a n o l a m i n e , and t h e o t h e r i s t h e e n z y m a t i c hydrolysis of N-arachidonoylphosphatidylethanolamine ( N - a r a c h i d o n o y l P E ) . I n t h i s s t u d y , we i n v e s t i g a t e d enzyme a c t i v i t i e s i n v o l v e d i n t h e s e p a t h w a y s and t h e t i s s u e l e v e l s o f p r e c u r s o r s . We f o u n d t h a t r a t b r a i n and t e s t i s c o n t a i n s m a l l a m o u n t s of anandamide ( 4 . 3 p m o l / g t i s s u e and 6 . 0 p m o l / g t i s s u e ) b e s i d e s relatively large amounts of N-palmitoylethanolamine and N - s t e a r o y l e t h a n o l a m l n e ( 1 , 2 ) . We a l s o f o u n d t h a t r a t b r a i n and t e s t i s c o n t a i n s i g n i f i c a n t a m o u n t s of Na r a c h i d o n o y l P E ( 5 0 . 2 p m o l / g t i s s u e and 274.3 p m o l / g t i s s u e ) b e s i d e s N - p a l m i t o y l P E and N - s t e a r o y l P E . The fatty acyl moiety of N-acylethanolamine resemble that o f N - a c y l m o i e t y o f N-acylPE r a t h e r t h a n f r e e f a t t y a c i d s . We c o n f i r m e d t h a t b r a i n or t e s t i s m i c r o s o m e s c o n t a i n a p h o s p h o d i e s t e r a s e a c t i v i t y which c a t a l y z e s the hydrolysis of N-arachidonoylPE to release a n a n d a m i d e . We a l s o c o n f i r m e d t h a t b r a i n or t e s t i s m i c r o s o m e s c o n t a i n a t r a n s a c y l a s e a c t i v i t y which c a t a l y z e s t h e t r a n s f e r o f a r a c h i d o n i c a c i d f r o m t h e 1p o s i t i o n o f PC t o t h e amino g r o u p of PE t o y i e l d Na r a c h i d o n o y l P E . T h e s e r e s u l t s s u g g e s t t h a t N-acylPE p a t h w a y i s c r u c i a l l y i m p o r t a n t i n p r o d u c i n g anandamide in t h e s e t i s s u e s ( 1 , 2 } . 1. T. S u g i u r a e t a l . (1996) Biochem. B i o p h y s . Res. C o ~ u n . 218, 1 1 3 - 1 1 7 . 2. T. S u g i u r a e t a l . (1996} E u r . J . Biochem. 240, 5 3 - 6 2 .
Poster Presentations Thursday 24 July
P205
P206
The Effect of Administration of Thyme and Lovage Volatile Oils upon the Percentage Composition of Fatty Acids in a rat Model HJ.D. Dorman & S.G. Deans. Scottish Agricultural college, Biochemical Sciences Department, Ayr KA6 5HW, Scotland
A Preliminary Study into the Effects of the Oral Administration of Oregano Volatile Oil upon the Percentage Composition of Fatty Acids in a Rat Model HJD Dorman & SG Deans. Scottish Agricultural College, Biochemical Sciences Department, Ayr KA6 5HW, Scotland.
Volatile oils extracted from aromatic and medicinal plants have been used since antiquity by man for their biological activities. The bioactivities of these extracts include antibacterial, antifungal and ant±oxidant properties. Volatile oils extracted by hydrodistillation from Thyme (Thymus vutgarus) and Lovage (Levisticum officinalis) were administered to pregnant rats in two feeding trials to investigate their capacities to alter the fatty acid composition in cholesteryl ester, triglyceride, free fatty acid and phospholipid fractions. These were extracted from a number of organs, including muscle tissue. In the muscle, thyme and lovage volatile oils changed the percent composition of C17:0 from 3.32+-0.18 n=8 to 4.59+-0.31"* n=4 and CI2:0 from 3.32-+0.18 n=4 to 1.89-+0.23~* n=4 respectively in the cholesteryl ester fraction. In the triacylglyceride fraction, the effects of both oils were more dramatic, significant changes being found in saturated and unsaturated fatty acids, see table below. ~he Effect of the AAmini~tration of Thyme and Lovage Volatile Oils on the% ._Compositionof Fatty Acids in the Triacvl$1ycer!..deFraction of Maternal MuscleTiss~ FFA CONTROL THYME LOVAGE
Oregano volatile oil (Origanurn vulgate ssp. birtum) was administered at three concentration levels:165(a), 334(b) and 835mg\kg(c), to pregnant rats to investigate its capacity to change the fatty acid (FA) % composition in the cholesteryl ester (CE), triacylglyceride (TG), free fatty acid (FFA) and phospholipid (PL) fractions in a number of organs. In the kidney, C20:4(n-6) changed in the CE fraction at the highest treatment level, 33.60+10.80 n=3 to 10.98+-1.21 n=3. C18:1(n-7) changed from 1.41+_0.76 n=4 to 0.09-+0.01*** n=3 in the TG fraction. C14:0 changed at concentrations Co) and (c), from 0.79-+0.04 n=2 to 1.19+-0.09~ n=2 and 1.30+-0.06"* n=2 respective!y. In the PL fraction, C20:5(n-3) changed from 0.72+-0.01n=2 to 0.90_+0.01 n=2 at (a). In liver CE and TG fractions, C18:2(n-6) showed changes at (a), 20.93--1.35 n=3 to 14.45-+2.00" n=2 and from 32.01-+1.15 n~3 to 25.17+_L55" n=3 respectively. In the TG fraction, C20:1(n-9) changed from 0.17+-0.03 n=3 to 0.39+-0.07" n=2 at the (b) level. Modifications were limited in the FFA fraction to C22:4(n-6) at concentration (a), from 0.65+_0.05 n=2 to 0.43_+0.06 n=2. Both C17:0 and C22:4(n-6) changed significantly in the PL fraction at the (c) treatment level from 0 36-+0.02 n=4 to 0 52-+0 02"~ n=2 and from 0.50-+0.02n=4 to 0.97-+0.02""n=2. In the heart, all the lipid fractions showed significant changes. In the CE fraction, C18:2(n-6) changed from 6.12-+0.81 n=2 to 13.10+~.63"" n=4 at (b). C18:0, C20:4(n-6) and C22:6(n-3) all changed at the two lowest concentration levels. C18:2(n-6), C18:3(n-3), C20:3(n-6), C20:4(n-6) and C22:5(n-3) all showed changes at (b) in the TG fraction. In the FFA fraction, C20:0, C22:5(n-3), C22:6(n-3) all changed at (a) level. C22:6(n-3) was altered at the highest concentration, as did C18:2(n-6). C17:1(n-7), D.24+0.04n=3 to 0.60-+0.08" n=5 at (b); C18:2(n-6), 13.61-+0.63 n=3 to 16.14±0.04" n=2 at (c). These results suggest that oregano volatile oil may cause significant changes in the FA % composition profile of essential organs in different lipid fractions at the concentration levels tested in this experiment.
C17:0 0.35+0.02 4.59_+0.31 3.64_+0.41 C18:0 4.25_+0.15 8.64-+0.83 "'~ 12.565:1.81.** C18:1(N-9) 26,04±0.47 10,50-+1.19 *'~ 10,55±1.09"~ C18:26N-6) 26.13-+0.83 21.09_+4,51 12.01_+0.72"*" C18:3[N-3) 1.61_+0.09 1.97±0.46 2.54-+0.66*~ C20:1(N-9) 0.40-+0.02 2.05-+0.67 ~* rod. C22:6(N-3) 1.00+_0.08 n.d. 4.74_+2.02" n=10 for control values, n -> 4 for treatments except C18:30W-3) and C20:1(N-9) where n = 2. n.d. = no values were detected in the sample. Significance was determined using a Student t-test (* P <0,0S, *'~P< 0.01, '~'~*P< 0,001).
These results demonstrate that the administration of these volatile oils are capable of changing the percent composition of specific fatty acids in the cholesteryl ester and triacylglyceride fractions in muscle tissue of pregnant rats. These changes are consistent with other organs in both mother and pup tissues.
P207
P208
Cell Type Distribution And Subcellular Location Of Enzymes Involved In The Metabolism Of Acyl Ethanaloamines. A.A.Makda, M.A.Elmore, M.E.HilI, A.Stamps, S.Tejura And M.J.Finnen. Yamanouchi Research institute, Oxford, Littlemore Park Ox4 4SX.
ENZY~IATIC SYNTHESIS OF ANANDA/~IDETI~OUGH NACYLPHOSPHATIDYLETHANOLAY~NEPATHWAY IN RAT BRAIN AND TESTIS T. S u g i u r a , S. Kondo, S. Nakane, h. ¥ a m a s h i t a and K. Waku. F a c u l t y of P h a r m a c e u t i c a l S c i e n c e s , T e i k y o U n i v e r s i t y , Sagamiko, Kanagawa 199-01, J a p a n
Two N-acyl ethanlolamines, arachidonyl ethanolamine (AEA or anandamide) and palmitoyt ethanolamine (PEA) have been shown to be the endogenous ligands for the cannabinoid receptors. The CB1 receptor is found primarily in the CNS but also in the periphery. Unlike CB1, CB2 receptors are found only in the peripheral system with a relatively high distribution on B lymphocytes and macmphages. This study was undertaken to determine which cell types are capable of metabolising these N-acyl ethanolamines via the condensation and hydrolysis pathways. Synthesis of PEA was found mainly in monocyte/macmphage cell types (PBMC monocytes 33; U937 53.8; Monomac 56.9 pmol/mg protein/rain) with far less PEA synthesis activity detectable in T cells (PBMC lymphocytes 4.2; Jurkat 0.5 pmol/mg protein/min). In contrast AEA was synthesised to comparable extents by both monocytes and T cells (PBMC monocytes 6.3; PBMC ~ymphocytes 1.0; U937 0.68; Monomac 1.0 pmol/mg/min). On subcellular fractionation of U937 cells the highest specific activity for PEA and AEA synthesis was in the P3 microsomal fraction. Parallel studies on the degradation of PEA and AEA showed similar cell type and sub cellular distribution to synthesis. Degradation rates 'for PEA and AEA were at levels commensurate with the rates of synthesis.
Dual s y n t h e t i c p a t h w a y s have b e e n p r o p o s e d f o r t h e b i o s y n t h e s i s o f a n a n d a m i d e . One i s t h e e n z y m a t i c c o n d e n s a t i o n o f f r e e a r a c h i d o n i c a c i d and e t h a n o l a m i n e , and t h e o t h e r i s t h e e n z y m a t i c hydrolysis of N-arachidonoylphosphatidylethanolamine ( N - a r a c h i d o n o y l P E ) . I n t h i s s t u d y , we i n v e s t i g a t e d enzyme a c t i v i t i e s i n v o l v e d i n t h e s e p a t h w a y s and t h e t i s s u e l e v e l s o f p r e c u r s o r s . We f o u n d t h a t r a t b r a i n and t e s t i s c o n t a i n s m a l l a m o u n t s of anandamide ( 4 . 3 p m o l / g t i s s u e and 6 . 0 p m o l / g t i s s u e ) b e s i d e s relatively large amounts of N-palmitoylethanolamine and N - s t e a r o y l e t h a n o l a m l n e ( 1 , 2 ) . We a l s o f o u n d t h a t r a t b r a i n and t e s t i s c o n t a i n s i g n i f i c a n t a m o u n t s of Na r a c h i d o n o y l P E ( 5 0 . 2 p m o l / g t i s s u e and 274.3 p m o l / g t i s s u e ) b e s i d e s N - p a l m i t o y l P E and N - s t e a r o y l P E . The fatty acyl moiety of N-acylethanolamine resemble that o f N - a c y l m o i e t y o f N-acylPE r a t h e r t h a n f r e e f a t t y a c i d s . We c o n f i r m e d t h a t b r a i n or t e s t i s m i c r o s o m e s c o n t a i n a p h o s p h o d i e s t e r a s e a c t i v i t y which c a t a l y z e s the hydrolysis of N-arachidonoylPE to release a n a n d a m i d e . We a l s o c o n f i r m e d t h a t b r a i n or t e s t i s m i c r o s o m e s c o n t a i n a t r a n s a c y l a s e a c t i v i t y which c a t a l y z e s t h e t r a n s f e r o f a r a c h i d o n i c a c i d f r o m t h e 1p o s i t i o n o f PC t o t h e amino g r o u p of PE t o y i e l d Na r a c h i d o n o y l P E . T h e s e r e s u l t s s u g g e s t t h a t N-acylPE p a t h w a y i s c r u c i a l l y i m p o r t a n t i n p r o d u c i n g anandamide in t h e s e t i s s u e s ( 1 , 2 } . 1. T. S u g i u r a e t a l . (1996) Biochem. B i o p h y s . Res. C o ~ u n . 218, 1 1 3 - 1 1 7 . 2. T. S u g i u r a e t a l . (1996} E u r . J . Biochem. 240, 5 3 - 6 2 .