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A mass fragmentographic method for the quantitative evaluation of brain prostaglandin biosynthesis
A MASS FRAGMENTOGRAPHIC METHOD FOR THE QUANTITATIVE EVALUATION OF BRAIN PROSTAGLANDIN BIOSYNTHESIS
S. Nicosia and G. Galli I n s t i t u t e of Pharmacology and Pharmacognosy, University of Milan, 20129 Milan, I t a l y .
ABSTRACT A method for the evaluation of PGF, and PGE. biosynthesis in rat cerebral cortex is described. TiSSue slice~ were incubated without any added precursor for d i f f e r e n t lengths of time. The analytical procedure involves prostaglandin extraction, p u r i f i c a t i o n and q u a n t i t a t i v e determination by mass fragmentography. S i g n i f i c a n t amounts of both prostaglandins were synthesized. The biosynthesis reached a plateau a f t e r 30 minutes and the r a t i o of PGF2~ to PGE2 was approximately 3.
ACKNOWLEDGMENTS We are grateful to Dr. J. Pike for generous supplies of protonated and deuterated prostaglandins and to Ono Pharmaceutical Co. for the g i f t of t r i t i a t e d PGE2. S. Nicosia is supported by a research fellowship from the University of Milan. The authors are greatly indebted to Professor R. Paoletti for helpful discussion and for his i n t e r e s t in this work. The s k i l f u l technical assistance of Mr. A. Toia is gratef u l l y acknowledged. Accepted February 5
PROSTAGLANDINS MARCH 1975
VOL. 9 NO. 3
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PROSTAGLANDINS
INTRODUCTION Prostaglandins are physiological components of the mammalian brain tissue, where they may play a role in the modulation of the neuronal response. However, few data are available on prostaglandin biosynthesis in brain (1,2,3), and the quantitative analysis of these compounds has been performed with bloassay, a very sensitive but not s u f f i c i e n t l y specific and reproducible procedure. A very sensitive and specific method for prostagIandin determination at picomole l e v e l , based on mass fragmentography, has been recently developed by us (4). This method involves the reaction of prostaglandin-MEs with N-trimethylsilylimidazole (TSIM) in the presence of piperidine, which allows the quantitative conversion of PGE-MEs into the corresponding mono-TMS derivatives of PGB-MEs and of PGF-MEs into the corresponding tris-TMS derivatives. The reaction is instantaneous and, in a l l cases, a single derivative with excellent gas chromatographic properties is formed; the presence of very prominent peaks in the mass spectra of the derivatives makes possible an optimal u t i l i z a t i o n of mass fragmentography for t h e i r determination at picomole levels. We have u t i l i z e d this method to study the formation of PGE. and PGF~ from endogenous precursors in slices of rat cerebral co~ex. MATERIALS AND METHODS Materials. PGE~, PGF. , ~,3,4,4-D,]-PGE~, ~ , 3 , 4 , 4 - D , ] PGF2~ were generously supplied by UpjohB Company, KalamazOo, U.S.A. T r i t i a t e d PGE2 w@§ a g i f t of Ono Pharmaceutical Co., Ltd., Osaka, Japan, l-~C-Arachidonic acid was obtained from Radiochemical Centre, Amersham, England. N - t r i m e t h y l sily|imidazole (TSIM) was purchased from Pierce Chemical Co., Rockford, U.S.A. Piperidine and the other solvents were purchased from Carlo Erba, Milan, I t a l y and were freshly d i s t i ] l e d before use. S i l i c i c acid was obtained from Bio-Rad Laboratories, Richmond, U.S.A. Collection of brains and incubation. Male rats, approximately 200 g in weight, were k i l l e d by decapitation, the brain was immediately removed and the hemispheres were separated. Cerebral cortex slices were rapidly obtained with
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PROSTAGI.,ANDINS
a manual microtome (2-3 slices per hemisphere) and placed in Kr~bs-Ringer bicarbonate at pH 7.4 (8-9 slices = 0.2-0.3 g in lO ml). The incubation was performed in a metabolic shaker at 37°C in the a i r and was ended by a c i d i f i c a t i o n at pH 3.5 with hydrochloric acid. Basal levels (time 0') were obtained placing the s l i c e s in the medium at pH 3.5 immediately a f t e r eparation_=. After the~ncubation~ a known amount (1,5 pg) of ,3,4,4-DA~-PGE- and L3,3,4,4-D. I-PGF^ was added to dach sample which wasZthen homogenize~withL~ Polytron homogenizer at maximum speed for I minute.
~
Extraction and p u r i f i c a t i o n . An aliquot of the homogenate was used for protein content determination according to Lowry (5), while the remainder was extracted with ethyl acetate (8 ml for three times). The combined extracts were evaporated to dryness under nitrogen flow and treated with a f r e s h l y prepared ethereal solution of diazomethane. After conversion to dryness, the methylated dry residue was applied on a s i l i c i c acid column (l g), from which neutral l i p i d s were eluted with ethyl ether (5 ml), and prostaglandins with ethyl acetate (5 ml). The ethyl acetate fraction was then concentrated and applied on a S i l i c a Gel HF plate with authentic PGEI and PGFI. methyl esters as reference standards, and the plate developed with CHCI3:CH~OH (85:15 v : v ) . The PGE and PGF zone was i d e n t i f i e d under UV Tight, scraped and eluted with ethyl acetate. Preparation of prostaglandin derivatives (4). The residue remaining a f t e r evaporation of the eluate is dissolved in benzene ( l S ) u l ) and then treated with a mixture of t r i m e t h y l s i l y l i m i d a z d l e and piperidine, l : l v:v ( 3 5 p I ) . An aliquot of the solution can be d i r e c t l y injected i dto the gas chromatograph-mass spectrometer.
Gas chromatography-mass spectrometry. An LKB gas chromatograph-mass spectrometer, Model 9000, was used. Analysis were carried out on a 2 m/3mm glass silanized column, packed with I% SE 30 on Gas Chrom P (I00-200 mesh). The working conditions were as follows : oven 220°C; i n j e c t o r 270°C; separator 280°C; ion source 290°C; electron energy 70 eV; trap current 60pA; electron m u l t i p l i e r 3.7 kV; f i l t e r 20 cps; helium flow r a t # 4 0 ml/min. S l i t width was adjusted to achieve a resolution power of 400 with I0% v a l l e y . Focusing was checked time by time.
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PROSTAGLANDINS
Ions monitored for mass fragmentography were : m/e 307 and 333 for PGF~ , m/e 311 for tetradeuterated PGF~ , m/e 321 and 349 forL~&~, m/e 325 for tetradeuterated ~E~ (4). Values were Corrected by subtraction of the "blank" created by the deuterated prostaglandins. Radio~as-chromatography. A radiogas-chromatograph Carlo Erba-Nuclear Chicago, Model 4998 was used. Gas chromatographic conditions were as described above. RESULTS AND DISCUSSION Prostaglandins are beleived to play an important role in the modulation of neuronal transmission and may well be involved in pathological processes. I t is therefore very interesting to evaluate the basal levels of prostaglandins i n brain and the biosynthetic a c t i v i t y of this tissue. I t has been demonstrated that, when exogenous arachidonic acid is incubated with ox and rabbit brain homogenates, i t s conversion into prostaglandins is exceedingly small (2,3), whereas prostaglandin biosynthesis from endogenous precursors in ox cerebral cortex homogenates is much higher (3). However, in a l l the studies with endogenous precursors, the methodology used for the characterization and quantitative determination of prostaglandins was based only on t h e i r behaviour in d i f f e rent chromatographic systems and on t h e i r c o n t r a c t i l e a c t i v i t y on smooth muscle preparations. These methods have a high degree of s e n s i t i v i t y but lack of s p e c i f i c i t y , and this may account for the great v a r i a b i l i t y of results obtained in d i f ferent laboratories. The method for q u a n t i t a t i v e prostaglandin analysis developed by us involves the extraction from tissue homogenates, p u r i f i c a t i o n of the extract with column and t h i n - l a y e r chromatography, formation of ME-TMS ethers and mass fragmentographic determination (4), as described in the Methods. This analytical procedure, based on mass fragmentography, shows not only a high s e n s i t i v i t y , but also a great s p e c i f i c i t y , which allow the evaluation of any single prostaglandin of the E and F series at levels of 0.2 picomoles. The reaction necessary for gas phase analysis offers d e f i n i t e advantages with respect to the other reported micromethods (6-9): i t is instantaneous and gives rise to a single derivative for each prostaglandin, which is stable in the reaction mixture and under gas chromatographic conditions.
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TABLE l PGE.2 RECOVERY AFTER EXTRACTION AND PURIFICATION PROCEDURE Incubation Extraction Column Cethyl acetate] Thin layer c h r o m a t o graphy
DPM 623.600 604.800 540.400 517.600
% lO0 96.9 86.6 83.0
The recovery achieved with the p u r i f i c a t i o n method has been checked by adding a known amount of t r i t i a t e d PGEp to the mixture before incubation. Table l shows the percentag~ of r a d i o a c t i v i t y recovered a f t e r each p u r i f i c a t i o n step : 83% of the i n i t i a l r a d i o a c t i v i t y is recovered at the end of the procedure and i t is associated with PGE2, since the radiochemical p u r i t y of the prostaglandin has been controlled by radiogas-chromatography. In any case, the deuterated prostaglandins acting as i n t e r n a l standard are added to the mixture as early as possible, immediately a f t e r the end of the incubation, so that no correction for the losses during the p u r i f i c a t i o n is needed. We have improved the s p e c i f i c i t y inherent to the mass fragmentographic method by monitoring two ions for each prostaglandin contemporarily, so that each compound was i d e n t i f i e d by four parameters: the retention time, the presence of the f i r s t ion, the presence of the second ion, the r a t i o between these two ion i n t e n s i t i e s . The studies on prostaglandin biosynthesis have been performed incubating slices of rat cerebral cortex without any added precursor. Prostaglandin basal levels have been evaluated by homogenizing the cortex slices immediately a f t e r t h e i r preparation in a medium at pH 3.5, to avoid subsequent biosynthesis; some synthesis can indeed take place during the homogenization of a number of tissues ( l O , l l ) . In one experiment, the deuterated prostaglandins were added before the incubation to v e r i f y i f any degradation occurred during shaking in a i r ; the amount of deuterated
MARCH 1975
VOL. 9 NO. 3
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PROSTAGLANDINS
prostaglandin recovered a f t e r extraction and p u r i f i c a t i o n has been evalueted by mass fragmentography, and i t is almost identical in samples incubated for d i f f e r e n t lenghts of time and in non-incubated samples. These results indicate that non-enzymatic degradation does not o c ~ r in the conditions we have used. In some experiments, l C-arachidonic acid ( l ) J g ; s p e c i f i c a c t i v i t y 58 mC/mmole) was incubated with the boiled enzyme in the described conditions. No radioactive prostaglandin, as s p e c i f i c a l l y determined by radiogas-chromatography, was whatsoever detected. Table 2 shows PGE9 and PGF~_ levels before and a f t e r incubation of slices o~ rat cer~i}ral cortex, without any addition of precursors. The basal levels of PGE2 are extremely low, (<10 rig/g), since not detectable with t h i s method; the brain cortex s l i c e s produce both PGF~ and PGEp during the f i r s t 30 minutes, when the biosynthesis reache~ a plateau and the r a t i o of PGF2a to PGE2 is approximately 3. TABLE 2 PROSTAGLANDIN BIOSYNTHESIS IN SLICES OF RAT CEREBRAL CORTEX
Incubation Time O' 15' 30' 60'
PGF2a ng/g tissue I15 643 814 790
+ ¥ ¥ ¥
12 19 71 62
PGE2 ng/g tissue
PGF2 /PGE2
not detectable 45 + 5 294 ¥ 44 266 ¥ 39
.... 14.3 2.8 2.9
Each figure represents the mean of 5 experiments + S.E. Incubation in Krebs Ringer pH = 7.4, 37°C, shaking in air. These data r e p r e s e n t the q u a n t i t a t i v e d e t e r m i n a t i o n of the rate of formation of PGE~ and PGFga prostaglandins in mammalian brain. I t is of i n t e r e s t to'note that mainly PGF~ is accumulated. Other brain regions are now being investigated and the effects of transmitter substances on the t o t a l synthesis and r a t i o between the two prostaglandins w i l l be evaluated.
402
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PROSTAGLANDINS
REFERENCES l) Kataoka, K., P.W. Ramwell and S. Jessup, Prostaglandins: Localization in Subcellular Particles of Rat Cerebral Cortex, Science 157:1187, 1967. 2) Wolfe, L.S., F. Coceani and C. Pace-Asciak, in Prostaglandins, Nobel Symposium 2 (BergstrSm S. and B. Samuelsson, Editors), Almqvist and Wiksell, Stockholm, 1967, p. 265. 3) Flower, R.J. and J.R. Vane, I n h i b i t i o n of Prostaglandin Synthetase in Brain explains the A n t i - p y r e t i c A c t i v i t y of Paracetamol (4-Acetamidophenol), Nature 240:410, 1972 4) Nicosia Zaraga, S. and G. G a l l i , A Rapid Gas Chromatographic-Mass Spectrometric Method for Prostaglandin Analysis at Picomole Levels, Anal. Biochem. 61:192, 1974. 5) Lowry, H., N.J. Rosenbrough, A.L. Farr and R.J. Randall, Protein Measurement with the Folin Phenol Reagent, J. Biol. Chem. 193:265, 1951 6) Green, K., E. Granstr~m and B. Samuelsson, Method for Quantitative Analysis of PGF~ , PGE., 9~, lla-Dihidroxy-15Keto-Prost-5-Enoic Acid and §~, lla~ 15-Trihydroxy-Prost5-Enoic Acid from Body Fluids Using Deuterated Carriers and Gas Chromatography-Mass Spectrometry, Anal. Biochem., 54:434, 1973 7) Axen, U., L. Baczynskyj, D.J. Duchamp, K.T. Kirton and J.F. Z i e s e r l , Jr. D i f f e r e n t i a t i o n between Endogenous and Exogenous (Administered) Prostaglandins in Biological Fluids, Advances in Biosciences 9:109, 1973 8) Sweetman, B.J., J.C. Fr~lich and J.T. Watson, Quantitative Determination of Prostaglandins A, B and E in the Sub-nanogram Range, Prostaglandins 3:75, 1973 9) Kelly, R.W., Methods for the Measurement of Prostaglandin Fo in Biological Fluids by Gas Chromatography-Mass Spectrometr~ Anal. Chem. 45:2079, 1973 I0) Jouvenaz, G.H., D.H. Nugteren, R.K. Beerthuis and D.A. van Dorp, A sensitive Method for the Determination of Prostaglandins by Gas Chromatography with Electron-Capture Detection, Biochim. Biophys. Acta 202:231, 1970 l l ) Angg~rd, E., S.O. Bohman, J.E. G r i f f i n I l l , C. Larsson and A.B. Maunsbach, Subcellular Localization of the Prostaglandin System in the Rabbit Renal Papilla, Acta Physiol. Scand. 84:231, 1972
M A R C H 1975
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S. Nicosia and G. Galli I n s t i t u t e of Pharmacology and Pharmacognosy, University of Milan, 20129 Milan, I t a l y .
ABSTRACT A method for the evaluation of PGF, and PGE. biosynthesis in rat cerebral cortex is described. TiSSue slice~ were incubated without any added precursor for d i f f e r e n t lengths of time. The analytical procedure involves prostaglandin extraction, p u r i f i c a t i o n and q u a n t i t a t i v e determination by mass fragmentography. S i g n i f i c a n t amounts of both prostaglandins were synthesized. The biosynthesis reached a plateau a f t e r 30 minutes and the r a t i o of PGF2~ to PGE2 was approximately 3.
ACKNOWLEDGMENTS We are grateful to Dr. J. Pike for generous supplies of protonated and deuterated prostaglandins and to Ono Pharmaceutical Co. for the g i f t of t r i t i a t e d PGE2. S. Nicosia is supported by a research fellowship from the University of Milan. The authors are greatly indebted to Professor R. Paoletti for helpful discussion and for his i n t e r e s t in this work. The s k i l f u l technical assistance of Mr. A. Toia is gratef u l l y acknowledged. Accepted February 5
PROSTAGLANDINS MARCH 1975
VOL. 9 NO. 3
397
PROSTAGLANDINS
INTRODUCTION Prostaglandins are physiological components of the mammalian brain tissue, where they may play a role in the modulation of the neuronal response. However, few data are available on prostaglandin biosynthesis in brain (1,2,3), and the quantitative analysis of these compounds has been performed with bloassay, a very sensitive but not s u f f i c i e n t l y specific and reproducible procedure. A very sensitive and specific method for prostagIandin determination at picomole l e v e l , based on mass fragmentography, has been recently developed by us (4). This method involves the reaction of prostaglandin-MEs with N-trimethylsilylimidazole (TSIM) in the presence of piperidine, which allows the quantitative conversion of PGE-MEs into the corresponding mono-TMS derivatives of PGB-MEs and of PGF-MEs into the corresponding tris-TMS derivatives. The reaction is instantaneous and, in a l l cases, a single derivative with excellent gas chromatographic properties is formed; the presence of very prominent peaks in the mass spectra of the derivatives makes possible an optimal u t i l i z a t i o n of mass fragmentography for t h e i r determination at picomole levels. We have u t i l i z e d this method to study the formation of PGE. and PGF~ from endogenous precursors in slices of rat cerebral co~ex. MATERIALS AND METHODS Materials. PGE~, PGF. , ~,3,4,4-D,]-PGE~, ~ , 3 , 4 , 4 - D , ] PGF2~ were generously supplied by UpjohB Company, KalamazOo, U.S.A. T r i t i a t e d PGE2 w@§ a g i f t of Ono Pharmaceutical Co., Ltd., Osaka, Japan, l-~C-Arachidonic acid was obtained from Radiochemical Centre, Amersham, England. N - t r i m e t h y l sily|imidazole (TSIM) was purchased from Pierce Chemical Co., Rockford, U.S.A. Piperidine and the other solvents were purchased from Carlo Erba, Milan, I t a l y and were freshly d i s t i ] l e d before use. S i l i c i c acid was obtained from Bio-Rad Laboratories, Richmond, U.S.A. Collection of brains and incubation. Male rats, approximately 200 g in weight, were k i l l e d by decapitation, the brain was immediately removed and the hemispheres were separated. Cerebral cortex slices were rapidly obtained with
398
MARCH 1975
VOL. 9 NO. 3
PROSTAGI.,ANDINS
a manual microtome (2-3 slices per hemisphere) and placed in Kr~bs-Ringer bicarbonate at pH 7.4 (8-9 slices = 0.2-0.3 g in lO ml). The incubation was performed in a metabolic shaker at 37°C in the a i r and was ended by a c i d i f i c a t i o n at pH 3.5 with hydrochloric acid. Basal levels (time 0') were obtained placing the s l i c e s in the medium at pH 3.5 immediately a f t e r eparation_=. After the~ncubation~ a known amount (1,5 pg) of ,3,4,4-DA~-PGE- and L3,3,4,4-D. I-PGF^ was added to dach sample which wasZthen homogenize~withL~ Polytron homogenizer at maximum speed for I minute.
~
Extraction and p u r i f i c a t i o n . An aliquot of the homogenate was used for protein content determination according to Lowry (5), while the remainder was extracted with ethyl acetate (8 ml for three times). The combined extracts were evaporated to dryness under nitrogen flow and treated with a f r e s h l y prepared ethereal solution of diazomethane. After conversion to dryness, the methylated dry residue was applied on a s i l i c i c acid column (l g), from which neutral l i p i d s were eluted with ethyl ether (5 ml), and prostaglandins with ethyl acetate (5 ml). The ethyl acetate fraction was then concentrated and applied on a S i l i c a Gel HF plate with authentic PGEI and PGFI. methyl esters as reference standards, and the plate developed with CHCI3:CH~OH (85:15 v : v ) . The PGE and PGF zone was i d e n t i f i e d under UV Tight, scraped and eluted with ethyl acetate. Preparation of prostaglandin derivatives (4). The residue remaining a f t e r evaporation of the eluate is dissolved in benzene ( l S ) u l ) and then treated with a mixture of t r i m e t h y l s i l y l i m i d a z d l e and piperidine, l : l v:v ( 3 5 p I ) . An aliquot of the solution can be d i r e c t l y injected i dto the gas chromatograph-mass spectrometer.
Gas chromatography-mass spectrometry. An LKB gas chromatograph-mass spectrometer, Model 9000, was used. Analysis were carried out on a 2 m/3mm glass silanized column, packed with I% SE 30 on Gas Chrom P (I00-200 mesh). The working conditions were as follows : oven 220°C; i n j e c t o r 270°C; separator 280°C; ion source 290°C; electron energy 70 eV; trap current 60pA; electron m u l t i p l i e r 3.7 kV; f i l t e r 20 cps; helium flow r a t # 4 0 ml/min. S l i t width was adjusted to achieve a resolution power of 400 with I0% v a l l e y . Focusing was checked time by time.
M A R C H 1975
VOL. 9 NO. 3
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PROSTAGLANDINS
Ions monitored for mass fragmentography were : m/e 307 and 333 for PGF~ , m/e 311 for tetradeuterated PGF~ , m/e 321 and 349 forL~&~, m/e 325 for tetradeuterated ~E~ (4). Values were Corrected by subtraction of the "blank" created by the deuterated prostaglandins. Radio~as-chromatography. A radiogas-chromatograph Carlo Erba-Nuclear Chicago, Model 4998 was used. Gas chromatographic conditions were as described above. RESULTS AND DISCUSSION Prostaglandins are beleived to play an important role in the modulation of neuronal transmission and may well be involved in pathological processes. I t is therefore very interesting to evaluate the basal levels of prostaglandins i n brain and the biosynthetic a c t i v i t y of this tissue. I t has been demonstrated that, when exogenous arachidonic acid is incubated with ox and rabbit brain homogenates, i t s conversion into prostaglandins is exceedingly small (2,3), whereas prostaglandin biosynthesis from endogenous precursors in ox cerebral cortex homogenates is much higher (3). However, in a l l the studies with endogenous precursors, the methodology used for the characterization and quantitative determination of prostaglandins was based only on t h e i r behaviour in d i f f e rent chromatographic systems and on t h e i r c o n t r a c t i l e a c t i v i t y on smooth muscle preparations. These methods have a high degree of s e n s i t i v i t y but lack of s p e c i f i c i t y , and this may account for the great v a r i a b i l i t y of results obtained in d i f ferent laboratories. The method for q u a n t i t a t i v e prostaglandin analysis developed by us involves the extraction from tissue homogenates, p u r i f i c a t i o n of the extract with column and t h i n - l a y e r chromatography, formation of ME-TMS ethers and mass fragmentographic determination (4), as described in the Methods. This analytical procedure, based on mass fragmentography, shows not only a high s e n s i t i v i t y , but also a great s p e c i f i c i t y , which allow the evaluation of any single prostaglandin of the E and F series at levels of 0.2 picomoles. The reaction necessary for gas phase analysis offers d e f i n i t e advantages with respect to the other reported micromethods (6-9): i t is instantaneous and gives rise to a single derivative for each prostaglandin, which is stable in the reaction mixture and under gas chromatographic conditions.
400
MARCH 1975
VOL. 9 NO. 3
PROSTAGLANDINS
TABLE l PGE.2 RECOVERY AFTER EXTRACTION AND PURIFICATION PROCEDURE Incubation Extraction Column Cethyl acetate] Thin layer c h r o m a t o graphy
DPM 623.600 604.800 540.400 517.600
% lO0 96.9 86.6 83.0
The recovery achieved with the p u r i f i c a t i o n method has been checked by adding a known amount of t r i t i a t e d PGEp to the mixture before incubation. Table l shows the percentag~ of r a d i o a c t i v i t y recovered a f t e r each p u r i f i c a t i o n step : 83% of the i n i t i a l r a d i o a c t i v i t y is recovered at the end of the procedure and i t is associated with PGE2, since the radiochemical p u r i t y of the prostaglandin has been controlled by radiogas-chromatography. In any case, the deuterated prostaglandins acting as i n t e r n a l standard are added to the mixture as early as possible, immediately a f t e r the end of the incubation, so that no correction for the losses during the p u r i f i c a t i o n is needed. We have improved the s p e c i f i c i t y inherent to the mass fragmentographic method by monitoring two ions for each prostaglandin contemporarily, so that each compound was i d e n t i f i e d by four parameters: the retention time, the presence of the f i r s t ion, the presence of the second ion, the r a t i o between these two ion i n t e n s i t i e s . The studies on prostaglandin biosynthesis have been performed incubating slices of rat cerebral cortex without any added precursor. Prostaglandin basal levels have been evaluated by homogenizing the cortex slices immediately a f t e r t h e i r preparation in a medium at pH 3.5, to avoid subsequent biosynthesis; some synthesis can indeed take place during the homogenization of a number of tissues ( l O , l l ) . In one experiment, the deuterated prostaglandins were added before the incubation to v e r i f y i f any degradation occurred during shaking in a i r ; the amount of deuterated
MARCH 1975
VOL. 9 NO. 3
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PROSTAGLANDINS
prostaglandin recovered a f t e r extraction and p u r i f i c a t i o n has been evalueted by mass fragmentography, and i t is almost identical in samples incubated for d i f f e r e n t lenghts of time and in non-incubated samples. These results indicate that non-enzymatic degradation does not o c ~ r in the conditions we have used. In some experiments, l C-arachidonic acid ( l ) J g ; s p e c i f i c a c t i v i t y 58 mC/mmole) was incubated with the boiled enzyme in the described conditions. No radioactive prostaglandin, as s p e c i f i c a l l y determined by radiogas-chromatography, was whatsoever detected. Table 2 shows PGE9 and PGF~_ levels before and a f t e r incubation of slices o~ rat cer~i}ral cortex, without any addition of precursors. The basal levels of PGE2 are extremely low, (<10 rig/g), since not detectable with t h i s method; the brain cortex s l i c e s produce both PGF~ and PGEp during the f i r s t 30 minutes, when the biosynthesis reache~ a plateau and the r a t i o of PGF2a to PGE2 is approximately 3. TABLE 2 PROSTAGLANDIN BIOSYNTHESIS IN SLICES OF RAT CEREBRAL CORTEX
Incubation Time O' 15' 30' 60'
PGF2a ng/g tissue I15 643 814 790
+ ¥ ¥ ¥
12 19 71 62
PGE2 ng/g tissue
PGF2 /PGE2
not detectable 45 + 5 294 ¥ 44 266 ¥ 39
.... 14.3 2.8 2.9
Each figure represents the mean of 5 experiments + S.E. Incubation in Krebs Ringer pH = 7.4, 37°C, shaking in air. These data r e p r e s e n t the q u a n t i t a t i v e d e t e r m i n a t i o n of the rate of formation of PGE~ and PGFga prostaglandins in mammalian brain. I t is of i n t e r e s t to'note that mainly PGF~ is accumulated. Other brain regions are now being investigated and the effects of transmitter substances on the t o t a l synthesis and r a t i o between the two prostaglandins w i l l be evaluated.
402
MARCH 1975
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PROSTAGLANDINS
REFERENCES l) Kataoka, K., P.W. Ramwell and S. Jessup, Prostaglandins: Localization in Subcellular Particles of Rat Cerebral Cortex, Science 157:1187, 1967. 2) Wolfe, L.S., F. Coceani and C. Pace-Asciak, in Prostaglandins, Nobel Symposium 2 (BergstrSm S. and B. Samuelsson, Editors), Almqvist and Wiksell, Stockholm, 1967, p. 265. 3) Flower, R.J. and J.R. Vane, I n h i b i t i o n of Prostaglandin Synthetase in Brain explains the A n t i - p y r e t i c A c t i v i t y of Paracetamol (4-Acetamidophenol), Nature 240:410, 1972 4) Nicosia Zaraga, S. and G. G a l l i , A Rapid Gas Chromatographic-Mass Spectrometric Method for Prostaglandin Analysis at Picomole Levels, Anal. Biochem. 61:192, 1974. 5) Lowry, H., N.J. Rosenbrough, A.L. Farr and R.J. Randall, Protein Measurement with the Folin Phenol Reagent, J. Biol. Chem. 193:265, 1951 6) Green, K., E. Granstr~m and B. Samuelsson, Method for Quantitative Analysis of PGF~ , PGE., 9~, lla-Dihidroxy-15Keto-Prost-5-Enoic Acid and §~, lla~ 15-Trihydroxy-Prost5-Enoic Acid from Body Fluids Using Deuterated Carriers and Gas Chromatography-Mass Spectrometry, Anal. Biochem., 54:434, 1973 7) Axen, U., L. Baczynskyj, D.J. Duchamp, K.T. Kirton and J.F. Z i e s e r l , Jr. D i f f e r e n t i a t i o n between Endogenous and Exogenous (Administered) Prostaglandins in Biological Fluids, Advances in Biosciences 9:109, 1973 8) Sweetman, B.J., J.C. Fr~lich and J.T. Watson, Quantitative Determination of Prostaglandins A, B and E in the Sub-nanogram Range, Prostaglandins 3:75, 1973 9) Kelly, R.W., Methods for the Measurement of Prostaglandin Fo in Biological Fluids by Gas Chromatography-Mass Spectrometr~ Anal. Chem. 45:2079, 1973 I0) Jouvenaz, G.H., D.H. Nugteren, R.K. Beerthuis and D.A. van Dorp, A sensitive Method for the Determination of Prostaglandins by Gas Chromatography with Electron-Capture Detection, Biochim. Biophys. Acta 202:231, 1970 l l ) Angg~rd, E., S.O. Bohman, J.E. G r i f f i n I l l , C. Larsson and A.B. Maunsbach, Subcellular Localization of the Prostaglandin System in the Rabbit Renal Papilla, Acta Physiol. Scand. 84:231, 1972
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