PROSTAGLANDINS
DETERMINATION
OF PROSTAGLANDIN
F2e
,
IN HUMAN C E R E B R O S P I N A L M.S. Abdel-Halim,
J. Ekstedt
E2, D 2 and 6-keto-Fle FLUID
and E. ~ngg~rd
Department of Alcohol and Drug A d d i c t i o n Research, Department of Pharmacology, K a r o l i n s k a institutet, 104 01 Stockholm and the Department of Neurology, Ume~ University, 104 85 Ume&, Sweden. ABSTRACT P r o s t a g l a n d i n (PG) F2~ , E~, D~ and 6-keto-F. were deterZ Z . l~ mined in human cerebrosplnal fluid by a mass spectrometric technique. The samples were obtained from 12 patients with suspected intracranial disease. A 64 fold variation in PG levels was observed. The major PG was 6 - k e t o ~ (0.12-15 ng/ml). PGF^ and PGE_ were present in lower concentrations PGD 2 ~ s b e l o w ~he level of detection (0.05 ng/ml) except in one patient w i t h extremely high total levels of PGs. INTRODUCTION Using mass spectrometric techniques P G F ~ has been detected in human cerebrospinal fluid (CSF) ~ , 2 ) . Other methods have indicated the presence of PGE 2 (3). We have recently shown PGD 2 to be the major PG in homogenates of r a t b r a i n (4). C o n s i d e r a b l e species differences were found to exist with respect to the prostanoids formed from endogenous p r e c u r s o r (5). In addition p r o s t a c y c l i n (PGI 9) has been discovered in recent years. PGI^ is metabolically labile and is usually analysed as i~s metabolite 6-keto-PGFle (6). It therefore seemed desirable to reexamine human CSF for presence of PGs, p a r t i c u l a r l y with respect to PGD 2 and 6-keto-PGFl . MATERIALS CSF samples were obtained from patients admitted to Ume~ U n i v e r s i t y hospital for suspected intracranial disease (Table i). All patients were subjected to CSF hydrodynamic investigation (7). Lumbar puncture was made in the sitting position and the recording of the CSF resting pressure was made in the supine p o s i t i o n during one hour. CSF was then c o n t i n u o u s l y w i t h d r a w n at a pressure of 0.0 KP~. CSF samples (20-70 ml) were collected and stored at -20vC until analysed.
MARCH
1 9 7 9 V O L . 17 N O . 3
405
PROSTAGLANDINS
D e u t e r i u m (D) labelled and u n l a b e l l e d PGs were obtained from the Upjohn Company, Kalamazoo, through the courtesy of Dr U. Ax~n. Thin layer plates coated with silica gel 60 were obtained from Merck, Darmstadt, West Germany. S o d i u m b o r o h y d r i d e (NaBH 4) was obtained from Alfa Inorganics, Beverly, Massachussetts. M e t h o x y a m i n e hydrochloride from Applied Science Laboratories, State College, P e n n s y l v a n i a and N,O-bis (trimethylsilyl) trifluoroacetamide (BSTFA) from Pierce Chemicals Co., Rockford, Illinois. All solvents were of analytical grade. METHODS P u r i f i c a t i o n and derivatization: Samples were thawed and 250 ng of the following d e u t e r i u m (D) labelled PGs were added to each sample as internal standards and carriers: PGF. -D , PGE_-D., 6-keto-PGF. -D.. Samples were freezez 4 z drie~, redissolved in 5 ml o f ± ~ d i ~ t i l l e d water, centrifuged at 5000 rpm for 5 min. The supernatant was acidified to pH3, using 2 M formic acid and PGs were extracted in ethyl acetate. The s a m p l e ~ a s concentrated and subjected to preparative thin layer chromatography. Authentic PGF_ , PGE^, PGD_ and 6-keto-PGF. standards were applied ~ a n a rzr o w band z . Is of the plate. on both sldes The plate was developed in the organic phase of the solvent system (Ethylacetate:acetic acid:trimethylpentane: wate r 110:20:30:100). The standard PGs were v i s u a l i z e d by local spraying with 10% p h o s p h o m o l y b d i c acid in ethanol followed by local heating. Three areas were then localized and scraped off. A lower one contained 6 - k e t o - P G F ] {Rf=0.23), a middle one contained PGF~ (Rf=0.33) and a~ ~ UDDer wider area contained PGE 2 + P G D ~ a t o g e t h e r (Rf PGEg=0.50, Rf PGD2=0.67). Each was then processed and analysed by GC-MS separately as follows. 6 - k e t o - P G F l fraction was eluted from the silica gel by ethyl acetate. After evaporation samples were r e d i s s o l v e d into 0.i ml pyridine; containing 2 mg m e t h o x y a m i n e h y d r o c h l o r i d e and kept in room temperature overnight. On the next day samples were dried under a stream of nitrogen, m e t h y l a t e d with diazomethane, evaporated, then silylated with 50 ~I BSTFA for 30 min at room temperature, dried and redissolved in 25 ul ethyl acetate. The PGF. was eluted from t h e silica gel by methanol, concentrate~ under a stream of nitrogen, then treated with d i a z o m e t h a n e and BSTFA as above. PGE 9 + PGD 2 fraction was extracted in methanol, reduced into PGF 2 and P G F ~ using NaBH 4 (4), then methylated and sily~ated as ~ s c r i b e d above. GC-MS anal[sis: meter was used.
406
LKB-2091 gas c h r o m a t o g r a p h - m a s s spectroThe column was 5 feet of 1% SE-30 and ope-
MARCH 1979 VOL. 17 NO. 3
PROSTAGLANDINS
rated at 235°C. The helium flow rate was 20-25 ml/min, the electron energy was 22 5 eV and the ion source temperature was 280 C. For determlnatlon of PGF~ the instrument was focused at the mass fragments 494Z~nd 498 for the unlabelled and d e u t e r i u m labelled PGF^ derivatives respectlvely. These fragments correspond ~o the m~lecular ion minus one trimethyl silyl ether group (M--90). The same fragments were also used for determination of PGE 2 and PGDg, after reduction into PGFg~ and PGF?~(4,8). For 6-keto-PGF]e, 2 fragments were monitored simuI£aneously for eac~ derivative. Fragments 598 and 508 were selected for the unlabelled, and 602 and 512 for the deute~ated derivatives. These fragments correspond to the (M--31) and (M - ( 3 1 + 9 0 ) r e s p e c t i v e l y (9). O
"
.
•
The intensities of the peaks were entered into a standard curve constructed by mixing 1 ~g of the d e u t e r i u m labelled PG with varying amounts of non labelled PG ranging from 0-1000 ng. The standard samples were processed and analysed in parallel with the CSF samples. The d e u t e r i u m labelled standards added to the samples were found to contain non labelled background which ranged between 0.5-1%. We assigned that ratio to be the limit of detection. In all determinations reported, PGF~ , 6-keto-PGF. and PGE 2 levels were higher than 2% of the z~ added standards. RESULTS AND DISCUSSION The levels of PGF. , PGE^, PGD and 6-keto-PGF~ in CSF obtai n e d from 12 patients are s~own in Table i. The variation in "total PG" between these patients were considerable with range of 0.296 to 19.0 ng/ml. One patient, suffering from intracranial h y p e r t e n s i o n had p a r t i c u l a r l y high levels, 16 times higher than the mean of the rest. In all patients 6-keto-PGF 1 was found to occur in higher concentrations than any of ~he other PGs. The difference was sometimes considerable, with 6-keto-PGF_ levels tenfold higher than those of PGE and P G F . . ~ D . was below the level of detection (0~05 n g / m l ~ i n al~ patients except one. It is thus evident that in this group of patients 6-keto-PGFle is dominant PG in CSF. Previous levels of PGF_ in human CSF determined by GCMS have been reported 6~ Wolfe and Mamer (i) and C o r e y e t a l. (2).The values reported were 71.6 ± 34.7 pg/ml for normal and much higher levels for patients w i t h epilepsy or meningoencephalitis. Our values on PGF_ in CSF from patients with definite CNS abnormalities ~ e in general agreement with these workers and in addition give data on
MARCH 1979 VOL. 17 NO. 3
407
Z O
< o r
=
>
Tra%~atic brain injury (before operation)
Tral~naticbrain injury (after operation)
Papilloede~a, sarcoidosis
Acquired hydrocephalus
Cerebral contusion
M/58
M/58
M/65
M/63
M/58
7
8
9
Post traumatic cranial defect
Intracranial hypertension
M/36
~V25
ii
12
Acquired hydrocephalus
Nephropathy, polyneuritis and papilloedema
M/60
F/I
Intracranial hvDertension
i0
Presenile dementia
F/62
logy 1.8
0.i
0.i
0.i
Paracetamol,indfm~thacin, aspirin, brcrnhexin
Chloramphenicol, am picillin
Paracetamol, pentazocin
N.T.
0.i
0.i
0.2
0.i
0.4
0.4
0.i
salbutamol 0.i
Thiazide, aspirin
Digoxin,
Nitrazepam
N.T.
0.i
0.2
2.8
0.4
0.2
0.8
0.8
0.i
0.1
15.3
3.6
0.i
1.5
0.8
0.i
0.05
0.i
DETERMINATION
OF PROSTAGLANDIN
F2e
,
IN HUMAN C E R E B R O S P I N A L M.S. Abdel-Halim,
J. Ekstedt
E2, D 2 and 6-keto-Fle FLUID
and E. ~ngg~rd
Department of Alcohol and Drug A d d i c t i o n Research, Department of Pharmacology, K a r o l i n s k a institutet, 104 01 Stockholm and the Department of Neurology, Ume~ University, 104 85 Ume&, Sweden. ABSTRACT P r o s t a g l a n d i n (PG) F2~ , E~, D~ and 6-keto-F. were deterZ Z . l~ mined in human cerebrosplnal fluid by a mass spectrometric technique. The samples were obtained from 12 patients with suspected intracranial disease. A 64 fold variation in PG levels was observed. The major PG was 6 - k e t o ~ (0.12-15 ng/ml). PGF^ and PGE_ were present in lower concentrations PGD 2 ~ s b e l o w ~he level of detection (0.05 ng/ml) except in one patient w i t h extremely high total levels of PGs. INTRODUCTION Using mass spectrometric techniques P G F ~ has been detected in human cerebrospinal fluid (CSF) ~ , 2 ) . Other methods have indicated the presence of PGE 2 (3). We have recently shown PGD 2 to be the major PG in homogenates of r a t b r a i n (4). C o n s i d e r a b l e species differences were found to exist with respect to the prostanoids formed from endogenous p r e c u r s o r (5). In addition p r o s t a c y c l i n (PGI 9) has been discovered in recent years. PGI^ is metabolically labile and is usually analysed as i~s metabolite 6-keto-PGFle (6). It therefore seemed desirable to reexamine human CSF for presence of PGs, p a r t i c u l a r l y with respect to PGD 2 and 6-keto-PGFl . MATERIALS CSF samples were obtained from patients admitted to Ume~ U n i v e r s i t y hospital for suspected intracranial disease (Table i). All patients were subjected to CSF hydrodynamic investigation (7). Lumbar puncture was made in the sitting position and the recording of the CSF resting pressure was made in the supine p o s i t i o n during one hour. CSF was then c o n t i n u o u s l y w i t h d r a w n at a pressure of 0.0 KP~. CSF samples (20-70 ml) were collected and stored at -20vC until analysed.
MARCH
1 9 7 9 V O L . 17 N O . 3
405
PROSTAGLANDINS
D e u t e r i u m (D) labelled and u n l a b e l l e d PGs were obtained from the Upjohn Company, Kalamazoo, through the courtesy of Dr U. Ax~n. Thin layer plates coated with silica gel 60 were obtained from Merck, Darmstadt, West Germany. S o d i u m b o r o h y d r i d e (NaBH 4) was obtained from Alfa Inorganics, Beverly, Massachussetts. M e t h o x y a m i n e hydrochloride from Applied Science Laboratories, State College, P e n n s y l v a n i a and N,O-bis (trimethylsilyl) trifluoroacetamide (BSTFA) from Pierce Chemicals Co., Rockford, Illinois. All solvents were of analytical grade. METHODS P u r i f i c a t i o n and derivatization: Samples were thawed and 250 ng of the following d e u t e r i u m (D) labelled PGs were added to each sample as internal standards and carriers: PGF. -D , PGE_-D., 6-keto-PGF. -D.. Samples were freezez 4 z drie~, redissolved in 5 ml o f ± ~ d i ~ t i l l e d water, centrifuged at 5000 rpm for 5 min. The supernatant was acidified to pH3, using 2 M formic acid and PGs were extracted in ethyl acetate. The s a m p l e ~ a s concentrated and subjected to preparative thin layer chromatography. Authentic PGF_ , PGE^, PGD_ and 6-keto-PGF. standards were applied ~ a n a rzr o w band z . Is of the plate. on both sldes The plate was developed in the organic phase of the solvent system (Ethylacetate:acetic acid:trimethylpentane: wate r 110:20:30:100). The standard PGs were v i s u a l i z e d by local spraying with 10% p h o s p h o m o l y b d i c acid in ethanol followed by local heating. Three areas were then localized and scraped off. A lower one contained 6 - k e t o - P G F ] {Rf=0.23), a middle one contained PGF~ (Rf=0.33) and a~ ~ UDDer wider area contained PGE 2 + P G D ~ a t o g e t h e r (Rf PGEg=0.50, Rf PGD2=0.67). Each was then processed and analysed by GC-MS separately as follows. 6 - k e t o - P G F l fraction was eluted from the silica gel by ethyl acetate. After evaporation samples were r e d i s s o l v e d into 0.i ml pyridine; containing 2 mg m e t h o x y a m i n e h y d r o c h l o r i d e and kept in room temperature overnight. On the next day samples were dried under a stream of nitrogen, m e t h y l a t e d with diazomethane, evaporated, then silylated with 50 ~I BSTFA for 30 min at room temperature, dried and redissolved in 25 ul ethyl acetate. The PGF. was eluted from t h e silica gel by methanol, concentrate~ under a stream of nitrogen, then treated with d i a z o m e t h a n e and BSTFA as above. PGE 9 + PGD 2 fraction was extracted in methanol, reduced into PGF 2 and P G F ~ using NaBH 4 (4), then methylated and sily~ated as ~ s c r i b e d above. GC-MS anal[sis: meter was used.
406
LKB-2091 gas c h r o m a t o g r a p h - m a s s spectroThe column was 5 feet of 1% SE-30 and ope-
MARCH 1979 VOL. 17 NO. 3
PROSTAGLANDINS
rated at 235°C. The helium flow rate was 20-25 ml/min, the electron energy was 22 5 eV and the ion source temperature was 280 C. For determlnatlon of PGF~ the instrument was focused at the mass fragments 494Z~nd 498 for the unlabelled and d e u t e r i u m labelled PGF^ derivatives respectlvely. These fragments correspond ~o the m~lecular ion minus one trimethyl silyl ether group (M--90). The same fragments were also used for determination of PGE 2 and PGDg, after reduction into PGFg~ and PGF?~(4,8). For 6-keto-PGF]e, 2 fragments were monitored simuI£aneously for eac~ derivative. Fragments 598 and 508 were selected for the unlabelled, and 602 and 512 for the deute~ated derivatives. These fragments correspond to the (M--31) and (M - ( 3 1 + 9 0 ) r e s p e c t i v e l y (9). O
"
.
•
The intensities of the peaks were entered into a standard curve constructed by mixing 1 ~g of the d e u t e r i u m labelled PG with varying amounts of non labelled PG ranging from 0-1000 ng. The standard samples were processed and analysed in parallel with the CSF samples. The d e u t e r i u m labelled standards added to the samples were found to contain non labelled background which ranged between 0.5-1%. We assigned that ratio to be the limit of detection. In all determinations reported, PGF~ , 6-keto-PGF. and PGE 2 levels were higher than 2% of the z~ added standards. RESULTS AND DISCUSSION The levels of PGF. , PGE^, PGD and 6-keto-PGF~ in CSF obtai n e d from 12 patients are s~own in Table i. The variation in "total PG" between these patients were considerable with range of 0.296 to 19.0 ng/ml. One patient, suffering from intracranial h y p e r t e n s i o n had p a r t i c u l a r l y high levels, 16 times higher than the mean of the rest. In all patients 6-keto-PGF 1 was found to occur in higher concentrations than any of ~he other PGs. The difference was sometimes considerable, with 6-keto-PGF_ levels tenfold higher than those of PGE and P G F . . ~ D . was below the level of detection (0~05 n g / m l ~ i n al~ patients except one. It is thus evident that in this group of patients 6-keto-PGFle is dominant PG in CSF. Previous levels of PGF_ in human CSF determined by GCMS have been reported 6~ Wolfe and Mamer (i) and C o r e y e t a l. (2).The values reported were 71.6 ± 34.7 pg/ml for normal and much higher levels for patients w i t h epilepsy or meningoencephalitis. Our values on PGF_ in CSF from patients with definite CNS abnormalities ~ e in general agreement with these workers and in addition give data on
MARCH 1979 VOL. 17 NO. 3
407
Z O
< o r
=
>
Tra%~atic brain injury (before operation)
Tral~naticbrain injury (after operation)
Papilloede~a, sarcoidosis
Acquired hydrocephalus
Cerebral contusion
M/58
M/58
M/65
M/63
M/58
7
8
9
Post traumatic cranial defect
Intracranial hypertension
M/36
~V25
ii
12
Acquired hydrocephalus
Nephropathy, polyneuritis and papilloedema
M/60
F/I
Intracranial hvDertension
i0
Presenile dementia
F/62
logy 1.8
0.i
0.i
0.i
Paracetamol,indfm~thacin, aspirin, brcrnhexin
Chloramphenicol, am picillin
Paracetamol, pentazocin
N.T.
0.i
0.i
0.2
0.i
0.4
0.4
0.i
salbutamol 0.i
Thiazide, aspirin
Digoxin,
Nitrazepam
N.T.
0.i
0.2
2.8
0.4
0.2
0.8
0.8
0.i
0.1
15.3
3.6
0.i
1.5
0.8
0.i
0.05
0.i
