MEASUREMENTOF PROSTAGLANDINFzcrLEVELS IN HUMAN CEREDROSPINALFLUID IN NORMAL AND PATHOLOGICALCONDITIONS
L. S. Wolfe and 0. A. Memer Donner Laboratoryof ExperimentalNeurochemistry,Montreal NeurologicalInstitute,McGill Universityand the.Mass SpectrometryLaboratory,Royal VictoriaHospital,Montreal, Canada H3A 2B4.
ABSTRACT
ProstaglandinFee concentrationswere measured in human cerebrospinal fluid by the gas-chromatography-mass spectrometrictechnique *II-PGF as internal standard and carrier. Normal levels of $%34.4 p&Z were found. Considerableincreasesin PGFsc concentrationswere found in patients with epilepsy,meningitis or followingcerebrovascularaccidentsor neurosurgicalremovals of brain tissue. The results agree in general with recent measurementsusing radioimmunoassay.
INTRODCCTION The activity of prostsglandincatabolizingenzymes in brain tissue appears to be very low (1,2). The data availableat present indicate that prostaglandinssynthesizedin brain -in vivo appear in the cerebrospinalfluid(c.s.f.) and are actively taken up by the choroid plexus and transportedinto the venous circulationand degraced by extraneuraltissues (3,4). Thus c.s.f. in contrast to plasma (5) mi&t be expected to contain measurable levels of prostaglandins. Prostaglandin-likeactivity has been measured by bioassay methods in c.s.f. from experimentalanimals (6,7) and shown to be inoreased in febrile animals (8). It is only very recently that prostsglandinFsc (PGFzc) conoentrationshave been measured in human c.s.f. by radioimmunoassay(9). This paper reports PGFsc contents for human c.s.f. obtained freshly during diagnosticpneumoencephalography or lumbar puncture and during neurosurgery. The analyses were done after purificationby gas-chromatography-mass fragmentography utilizing *HJ-PGFecas internal standardend carrier (10,ll). Accepted Deceuber 2
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PROSTAGLANDINS
MATERIALSAlVDMETHODS Humen c.s.f. samples obtained from patientsundergoing diagnosticpneumoencephalography or lumbar puncture were immediately centrifu@;ed at 1000 rpm in a clinical centrifugeand the supernataut immediatelyfrozen and stored at -2OOC in sealed containersuntil extractionand purification. The samples were numbered and the diagnosiswas not known until the clinical charts were examined after the analyses. The initial few drops of c.s.f. were always discarded. Samples from traumaticlumbar punctureswere not included. 3,3,4, 4-2H4-pGFzcI ad XFz Q standardswere kindly provided by Dr. John E. Pike, The Upjohn Company, Kalamazoo,Michigan. 3H8-PGF2. was obtained from NEN Canada. Purificationand Derivatisation The c.s.f. samples were thawed and 1 ale; of the 2H4-PGFzacarrier and internal standardadded togetherwith approximately15,000 cpm of the high specific activity 3H8-PGFzU (less than 0.03 ng) to monitor the elution of the prostaglandinsand determinerecoveries. The samples were acidified to pH 3.5 and then passed through freshly preparedAmberlite X0-2 columns (1.7x10 cm). The columns were eluted first with 100 ml of distilledwater and then 100 ml of absolute ethanol. All the radioactivitywas found in the ethanol fractions. The ethanol fractionswere evaporatedto dryness end the residue dissolved in a small volume of chlorofoxm:methanol (2:l by vol.) end applied as a band to 0.5 mm silica Gel G thin layer plates developedwith chloroform: methanol:glacialacetic:water(90:9:1:0.65 by vol.). A standard of PGFia was run on either side of the sample band which was covered with a glass plate anI the standardsdetectedby a lO$ phosphomolybdicacid in ethanol spray heated locally by an air gun. The narrow zone of silica gel correspondingto the standardswas scraped off into small glass columns fitted with sinteredglass discs and eluted with 90% ethanol. The eluates were evaporatedto dryness, 2 ml of methanol added, and then 30 ml of water, 80 ml of redistilled diethyl ether and acidified with 0.1 ml of lN HCl. The upper phase w&s separatedend the lower phase extractedagain with 80 ml of diethyl ether. The etherphase was washed to neutralitywith distilledwater. The combined ether extracts were evaporatedto dryness, redissolvedin a small volume of 6% methanol in chloroformand introducedinto small Pasteur pipette columns of BioSil HA (Calbiochem.). The silicic acid columns were eluted first with 15 ml of 6% methanol in chloroformand then with 15 ml of 2% methanol in chloroform. All the radioactivityand PGFz. appeared in the latter fraction. This fractionwas evaporatedto dryness and methylatedwith 0.5 ml of freshly distilledethereal diazomethane:methsnol (9:l by vol.) for 60 min at room temperature. The solventswere evaporatedby a stream of nitrogen. The me lated prostsglandinswere transferredto small glass tubes (0.2x2.5cm th? fitted with a small selNm septa. Immediatelybefore introductioninto the gas chromatographof th.eLKB9000 mass spectrometer,the samples were silylatedwith 10 ~1 of Trisil Z (PierceChemical Co.) at 600 for 5 min. The recoveriesof PGFza based on counts of the added 3H8-PGFz.were between 70 and 8%.
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Mass spectrometrioanalysis Ions of m/e 423 and 427 (M+-(TMSiOH+C$Ill)) are prominent in the mass spectrum of the trimethylsilylether'derivativesof the methyl esters of PGFec and 2H@GF,c respectively(constituting3 percent of the total ionization)and were the ones chosen for monitoringby the multiple ion detectorunit (AVA) of the LICB-9000 gas chromatographmass spectrometer. Operatingconditionsfor the gas chromatography were l$ SE-30 on Gas Chrom Q, 6 ft glass column at 220° and for the mass spectrometer,electron energy 20 eV, source temperature290°, trap current 120 @, gain setting 10 or 11. The followingprocedure was used for focusing. A partial spectrom of perfluorokerosene was obtained and a good fragment ion just above m/e 423 chosen and focused carefully. The reading on the mass marker was noted. Watching the most sensitivegalvsnometeron the osoillographrecorder, ions were counted down to m/e 423 and focused again carefully. !Chereading on the mass marker was again noted and maintainedunchanged as the acceleratingvoltage altered with the fine adjustmentto focus exactly on m/e 427. Then 200 ng of a standardmixture of lH/2H PGFec (2$ of the protium form) was injected into the gas chromatographand after the solvent peak had passed, the inlet valve was opened and the acceleratingvoltage alternatorstarted. As the prostaglaudinpeek of the standardmixture appeared on the recorder (retentiontime about 6 min) the m/e 423 and 427 were again preciselyre-focusedand the reading on the mass marker again noted. We found very little drift over a 60 minute period and minor re-focusingadjustmentswere made after every 2-3 samples. Once the focusingprocedurehad been oompleted then the order of sample injectionswas 2H4-carrierPGFec followedby standardmixtures containing0.1, 0.5, 1.0 and 2.0 percent PGFzclin the deuteratedcarrier and then the unknown samples interspersed with standardsevery 2-3 samples. In our experience,no more than 6 unknownsamples should be run consecutivelybefore a waiting period to prevent build-up of contaminatingpeaks and an unacceptable rise in background. For the very low levels of the protium foim, the amounts of deuteratedcarrier injected should be small (100 ng) to prevent increasesin the m/e 423 due to the skirt of the nearby carrier frsgment ion at m/e 427. With injectionsof 100-200 ng of carrier alone the m/e 423/427 was 4-6 parts per 1000. Following the runs of standardsand samples the frsgmentogramrecords were exsmined end the peak heights of the protium snd deuteriumions determinedand a standard line plotted of the ratio of the 1H/2H4 x 1000 against the ng of PGF,&g of 2H -PGF,, carrier after subtractionof the small proportionof m/e 429 in the deuteriumcarrier alone. The slope obtained was always very close to the theoreticalof 45” indicatingno loss of deuterium during fragmentation(11). In our experience,since little deviationfrom the theoreticalline was found, all calculations of unknowns reported here were based on the theoreticalline.
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1975 VOL. 9 NO. 2
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PROSTAGLANDINS
RESULTS AND DISCUSSION Table I shows the precisionend accuracy of the deuteriumisotope dilutionmethod for gas-liquidchromatography-mass spectrometrio measurementof PGFZU. Repetitiveinjectionsof 50-100 ng 2H4-PGFsa containingPGF,. in amounts of l-100 ng/pg of deuteratedPGFZcr indicated that 100 pg could be detectedwith an accuracy of about 2% but at higher amounts the accuracy was considerablybetter (3-6%). Since the recovery during extractionand purificationis 70-80 percent end there is some loss on the SE-30 column, a 1 ml sample of o.s.f. would have to contain more than 100 p&l to be detectable. Fortunately,during pneumoencephalography usually more than 10 ml of c.s.f. is withdrawn,thus enablingmeasurementswith reasonable precisionbelow 100 pg/ml. The samples of c.s.f. were classifiedinto 5 groups. Group 1, the so-called 'normal'group, included 16 patients who had normal pneumoenoephalograms and no evidence of central nervoufjsystem disease. Patients in this group had non-specificdiagnosesof depression, anxiety neurosis, tension headache,benign hypertension,neck pain, facial pain or spasmodictortioollis. Group 2 included seven patients with temporal lobe epilepsy, two with generalizedepilepsy,one with a diffuse electroencephalographic disturbanceand one with focal seizures due to cerebral infarction. None of these patients had had previousneurosurgery. Group 3 contains a small group of patients with meningitisor meningoenoephalitis of viral origin. Group 4 representsfive patients in which several samples of c.s.f. were obtained post-operatively. In all these patients,removal of brain tissue (temporallobectomy,multiple excisions,etc.) was carried out for the treatmentof epilepsy. Group 5 includespatients with vascular lesions and subaraohnoidhemorrhsge. The PGFZ. concentration in the c.s.f. of these five patient groups is shown in Tables II-IV. The mean 'normal'PGFZa concentrationin c.s.f. was 71.6 pg/ml 2 34.7 (Table II). The high standard deviationmay be due in part to the considerablevariabilityof the method at the lowest rsnge of sensitivity(see Table I) since total pGFz. in all the 'nonnall samples snalyzed was between 1 and 5 ng. La Torre and coworkers (9) reported mean normal values of 37.7 pg/ml by radioimmunoassaybut found at these levels a &30 pg/ml variation on repeated analyses. Cerebrospinalfluid thus contains considerablyhigher concentrations of PGFza than blood plasma (6). PGFZcrcannot be detected in 10 ml of human plasma by the describedGC-MS method. Significantincreasesin c.s.f. FGFZ. concentrationswere found in patients with epilepsy and in meningoencephalitis(Table II). But it should be noted that the standard deviationis very high. This is in accord with the known increasesin biosynthesisof prostsglandinsfollowingcortical stimulation and in inflammation(12,13). Consistentlyhigher levels of PGFs. were found in o.s.f. samples t&en serially from patients followingneurosurgicalremovals of brain tissue (Table III). In
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three patients values in the ng/ml.range were found. These high levels most likely are due to the increasedbiosynthesisand release of FGFZ. due to the surgical trauma. The vascular lesion group (Group 5, Table IV) is as yet too small to make any firm conolusions. Only one patient showed olear radiologioalevidence of vasospasmand in this case the PGF2. was 720 ng/ml. In two patients with rupture of cerebral aneurysms,PGFza was undetectable,but these patients were comatose, subsequentlydied, and c.s.f. samples were obtainedmany days after the subarachnoidhemorrhage. La Torre and coworkers (9) could not find any correlationbetween c.s.f. PGFz. and the appearance of cerebralvasospasm following subarachnoidhemorrhage. Their values ranged from less than 50 pg/ml to over 1.4 ng/ml. It is difficultto obtain clearly matched patients in this group end many more analyses will be requiredbefore any connectionbetween PGFZa concentrations in c.s.f. and cerebralvasospasm ten be made. Furthermore,FGF2. is just as likely to derive from damaged brain tissue adjacent to the hemorrhageas from blood platelets. In this connection,two patients with stroke showed lumbar c.s.f. PGFza concentrationsgreater than lng/ml. In conclusion,human c.s.f. from normal subjects contains concentrations of FGFz. less than 100 p&l. Considerableincreaseswere found in several pathologicalsituations. However, the range from one patient to another and even in day-to-dayserial measurementsin one patient is large. The results obtainedby La Torre and coworkers (9) by radioimunm oassay are in general agreementwith the gas chromatographic-massspectrometricmethod. The latter procedure,however, is expensiveand time-consumingand since the c.s.f. of many more patients must be analyzed,particularlyin the subarachnoidhemorrhageand stroke group, it seems that it would be more prudent to use the radiodoassay method for FGF2. measurementsin studies of this type in the future.
This research was supportedby grants W-1345 and MA-3719from the Medical Research Council of Canada. We wish to thank Dr. Romeo Ethier and Dr. Terence Myles for their help at the clinical level in obtaining the cerebrospinalfluid samples.
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Table I. Accuracy of the GLC-MS method for PGFZo,determinedby repetitive injectionsof standardmixtures of FGFZ. and 3,3,4,4-2H4
PGFZ. as the
trimethylsilylether derivativesof the methyl ester. Ions monitored were m/e 423 and 427. ng Deuterated carrier injected
Parts per 1000 ng H-form of H-form added measured to carrier Mean + S.D.$
100
1.0
ng H-form No. of injected injections
1.09 + 22.9
0.1
8
100
5.0
5.12 f: 6.5
0.5
8
100
10.0
11.07 2 3.0
1.0
8
100
20.0
20.51 -I5.1
2.0
8
100 50
50.0 100.0
51.50 ? 2.9 104.69 + 5.1
2.5 5.0
5 6
Table II. PGFZc,concentrationsin cerebrospinalfluid.in patients classifiedin Groups l-3. The analyses were performed on c.s.f. obtained from patients undergoingdiagnosticpneumoencephalography or lumbar puncture. Disgnosis
PGF2
No.
a
(Pdml) mean + S.D. -
c.s.f. volume analyzed (ml)
Group 1
'Normals',no evidence of CNS pathology
71.6 + 34.7 (mwe: 30-139)
1.6 15-80
Group 2
Epilepsy,before operation
559 t 411 (range: 124-1230)
11
12-54
Group 3
Meningoencephalitis
954 + 783 (range: 252-2196)
5
S-34
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270
44-Q
Right
Right temporal lobectomy
Focal seizures - multiple removals
3.
4.
5.
370
Focal seizures - multiple removals
2.
temporal lobectomy
Sub-total hemispherectomy for uncontrolled epilepsy
3
1.
Case
118
5
340
6
-
1300
7
post-operation
lumbar c.s.f. in volumes from lo-18 ml.
1950
336
-
1800
1560
8
treatment of epilepsy). The enalyses were performed on
classified as Group 4 (post-operative for the surgical
PGF,a concentrations in oerebrospinal fluid from patients
Table III.
2180
9
1147
1089
Rupture aneurysm - anterior commun. artery - comatose
Cerebrovasculaxaccident, right side
Cerebrovascularaccident, left side
5.
6.
7.
* Volume of c.8.f. used for analyses 9-15 ml.
less than 50
Rupture aneurysm of anterior cerebralartery - comatose
4.
430
less than 50
Rupture aneurysm - posterior comm. artery - comatose
720
740
208
406
284
796
pg/ml
PGFza
3.
2 8 9
Intracerebellar hemorrhage Arterio-venousmalformation DsYl
Diagnosis *
(vascularlesions and subarachnoidhemorrhage)
Lumbar fluid
Lumbar fluid
L.P. drain in O.R. on day of SBB
1215 rbcls/mm3
Ventricular drain 14 days after SAR
Ventricular fluid day 5 post-op.
43,100 rbc's/mm3
Ventricular drain
comments
concentrationsin cerebrospinalfluid from patients classified in Group 5
Internalcarotid aneurysm severe vasospasm
a
2.
Case
=32
Table IV.
PROSTAGLANDINS
HEFEBENCES 1.
Nakano, J., A. V. Pracan and S. E. Moore. Metabolism of prosta-
glandin q in cerebral cortex and cerebellumof the dog 1972. and rat. Brain Hes. 39:545-54.8, 2. Pappius, H. M., K. Hostworowskiand L. S. Wolfe. Biosynthesisof prostaglsndlnsFE, and E, by brain tissue -in vitro. Trans. Amer. Sot. Neurochem. 5:119, 1974 and unpublishedresults. 3.
Bito, L. Z. Accumulationand apparent active transportof prostaglandins by some rabbit tissues-in vitro. J. Physiol. (London)221:371-387,1972.
4.
Bito, L. Z. Comparativestudy of concentrativeprostaglandin accumulationby various tissues of mammals snd marine vertebratesand invertebrates. Comp. Biochem. Physiol. 43A: 65-82, 1972.
5.
Samuelsson,B. Quantitativeaspects of prosts&ndin man. Adv. Biosciences9:7-14, 1973.
6.
Ramwell, P. W. The action of oerebrospinalfluid on the frog rectus abdominismuscle and other isolated tissue preparatkons. J. Physiol. (London)170:21-38,1964.
7.
Holmes, S. W. The spontaneousrelease of prostaglandinsinto the cerebral ventriclesof the dog and the effect of external factors on this release. Brit. J. Phazmacol. 37:653-658,197(
0.
Feldberg,W. and K. P. Gupta. Pyrogen fever and prostaglandin-like activity in cerebrospinalfluid. J. Physiol. (London)228: 41-53, 1973.
9.
La Terre, E., C. Patrono,A. Fortuna and D. Grossi-Belloni. Hole of prostsglandinF, in human cerebralvasospasm. J. Neurosurg. 41:293-299,1974.
synthesisin
10. Axen, U., K. Gden, D. %rlin and B. Samuelsson. Mass spectrometric determinationof picomole amounts of prostaglandi.ns E, end Fzcrusing syntheticdeuteriumlabelled carriers. Biochem. Biophys. Hes. Comm. 45:419-525,1971. 11. G&en, K., E. GranstG&m,B. Samuelssonand U. Axen. Methods for QusntitativeAnalysis of PGFza, PGs, 9a,lla-Dihydroq-15Keto-Prost-5-Enoic Acid and 9a,lla,15-Trihydroxy-Prod-5Enoic Acid from Body Fluids Using DeuteratedCarriers and Gas Chromatography-Mass Spectrometry. Anal. Biochem. 54: 434-453, 1973.
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1975
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PROSTAGLANDINS
12. Bradley, P. B., G. M. R. Samuels and J. E. Shaw. Correlation of prostaglandinrelease from the cerebral cortex of cats with electro-corticogram followingstimulationof the reticularformation. Brit. J. Phazmacol.37:151-157, 1969. 13.
192
Vane, J. FL Inhibitionof prostaglandinbiosynthesisas the mechanism of action of aspirin-likedrugs. Adv. Biosciences 9:395-411, 1973.
FEBRUARY
1975
VOL. 9 NO. 2
L. S. Wolfe and 0. A. Memer Donner Laboratoryof ExperimentalNeurochemistry,Montreal NeurologicalInstitute,McGill Universityand the.Mass SpectrometryLaboratory,Royal VictoriaHospital,Montreal, Canada H3A 2B4.
ABSTRACT
ProstaglandinFee concentrationswere measured in human cerebrospinal fluid by the gas-chromatography-mass spectrometrictechnique *II-PGF as internal standard and carrier. Normal levels of $%34.4 p&Z were found. Considerableincreasesin PGFsc concentrationswere found in patients with epilepsy,meningitis or followingcerebrovascularaccidentsor neurosurgicalremovals of brain tissue. The results agree in general with recent measurementsusing radioimmunoassay.
INTRODCCTION The activity of prostsglandincatabolizingenzymes in brain tissue appears to be very low (1,2). The data availableat present indicate that prostaglandinssynthesizedin brain -in vivo appear in the cerebrospinalfluid(c.s.f.) and are actively taken up by the choroid plexus and transportedinto the venous circulationand degraced by extraneuraltissues (3,4). Thus c.s.f. in contrast to plasma (5) mi&t be expected to contain measurable levels of prostaglandins. Prostaglandin-likeactivity has been measured by bioassay methods in c.s.f. from experimentalanimals (6,7) and shown to be inoreased in febrile animals (8). It is only very recently that prostsglandinFsc (PGFzc) conoentrationshave been measured in human c.s.f. by radioimmunoassay(9). This paper reports PGFsc contents for human c.s.f. obtained freshly during diagnosticpneumoencephalography or lumbar puncture and during neurosurgery. The analyses were done after purificationby gas-chromatography-mass fragmentography utilizing *HJ-PGFecas internal standardend carrier (10,ll). Accepted Deceuber 2
PROSTAGLANDINS FEBRUARY
1975
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PROSTAGLANDINS
MATERIALSAlVDMETHODS Humen c.s.f. samples obtained from patientsundergoing diagnosticpneumoencephalography or lumbar puncture were immediately centrifu@;ed at 1000 rpm in a clinical centrifugeand the supernataut immediatelyfrozen and stored at -2OOC in sealed containersuntil extractionand purification. The samples were numbered and the diagnosiswas not known until the clinical charts were examined after the analyses. The initial few drops of c.s.f. were always discarded. Samples from traumaticlumbar punctureswere not included. 3,3,4, 4-2H4-pGFzcI ad XFz Q standardswere kindly provided by Dr. John E. Pike, The Upjohn Company, Kalamazoo,Michigan. 3H8-PGF2. was obtained from NEN Canada. Purificationand Derivatisation The c.s.f. samples were thawed and 1 ale; of the 2H4-PGFzacarrier and internal standardadded togetherwith approximately15,000 cpm of the high specific activity 3H8-PGFzU (less than 0.03 ng) to monitor the elution of the prostaglandinsand determinerecoveries. The samples were acidified to pH 3.5 and then passed through freshly preparedAmberlite X0-2 columns (1.7x10 cm). The columns were eluted first with 100 ml of distilledwater and then 100 ml of absolute ethanol. All the radioactivitywas found in the ethanol fractions. The ethanol fractionswere evaporatedto dryness end the residue dissolved in a small volume of chlorofoxm:methanol (2:l by vol.) end applied as a band to 0.5 mm silica Gel G thin layer plates developedwith chloroform: methanol:glacialacetic:water(90:9:1:0.65 by vol.). A standard of PGFia was run on either side of the sample band which was covered with a glass plate anI the standardsdetectedby a lO$ phosphomolybdicacid in ethanol spray heated locally by an air gun. The narrow zone of silica gel correspondingto the standardswas scraped off into small glass columns fitted with sinteredglass discs and eluted with 90% ethanol. The eluates were evaporatedto dryness, 2 ml of methanol added, and then 30 ml of water, 80 ml of redistilled diethyl ether and acidified with 0.1 ml of lN HCl. The upper phase w&s separatedend the lower phase extractedagain with 80 ml of diethyl ether. The etherphase was washed to neutralitywith distilledwater. The combined ether extracts were evaporatedto dryness, redissolvedin a small volume of 6% methanol in chloroformand introducedinto small Pasteur pipette columns of BioSil HA (Calbiochem.). The silicic acid columns were eluted first with 15 ml of 6% methanol in chloroformand then with 15 ml of 2% methanol in chloroform. All the radioactivityand PGFz. appeared in the latter fraction. This fractionwas evaporatedto dryness and methylatedwith 0.5 ml of freshly distilledethereal diazomethane:methsnol (9:l by vol.) for 60 min at room temperature. The solventswere evaporatedby a stream of nitrogen. The me lated prostsglandinswere transferredto small glass tubes (0.2x2.5cm th? fitted with a small selNm septa. Immediatelybefore introductioninto the gas chromatographof th.eLKB9000 mass spectrometer,the samples were silylatedwith 10 ~1 of Trisil Z (PierceChemical Co.) at 600 for 5 min. The recoveriesof PGFza based on counts of the added 3H8-PGFz.were between 70 and 8%.
FEBRUARY
1975
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PROSTAGLANDINS
Mass spectrometrioanalysis Ions of m/e 423 and 427 (M+-(TMSiOH+C$Ill)) are prominent in the mass spectrum of the trimethylsilylether'derivativesof the methyl esters of PGFec and 2H@GF,c respectively(constituting3 percent of the total ionization)and were the ones chosen for monitoringby the multiple ion detectorunit (AVA) of the LICB-9000 gas chromatographmass spectrometer. Operatingconditionsfor the gas chromatography were l$ SE-30 on Gas Chrom Q, 6 ft glass column at 220° and for the mass spectrometer,electron energy 20 eV, source temperature290°, trap current 120 @, gain setting 10 or 11. The followingprocedure was used for focusing. A partial spectrom of perfluorokerosene was obtained and a good fragment ion just above m/e 423 chosen and focused carefully. The reading on the mass marker was noted. Watching the most sensitivegalvsnometeron the osoillographrecorder, ions were counted down to m/e 423 and focused again carefully. !Chereading on the mass marker was again noted and maintainedunchanged as the acceleratingvoltage altered with the fine adjustmentto focus exactly on m/e 427. Then 200 ng of a standardmixture of lH/2H PGFec (2$ of the protium form) was injected into the gas chromatographand after the solvent peak had passed, the inlet valve was opened and the acceleratingvoltage alternatorstarted. As the prostaglaudinpeek of the standardmixture appeared on the recorder (retentiontime about 6 min) the m/e 423 and 427 were again preciselyre-focusedand the reading on the mass marker again noted. We found very little drift over a 60 minute period and minor re-focusingadjustmentswere made after every 2-3 samples. Once the focusingprocedurehad been oompleted then the order of sample injectionswas 2H4-carrierPGFec followedby standardmixtures containing0.1, 0.5, 1.0 and 2.0 percent PGFzclin the deuteratedcarrier and then the unknown samples interspersed with standardsevery 2-3 samples. In our experience,no more than 6 unknownsamples should be run consecutivelybefore a waiting period to prevent build-up of contaminatingpeaks and an unacceptable rise in background. For the very low levels of the protium foim, the amounts of deuteratedcarrier injected should be small (100 ng) to prevent increasesin the m/e 423 due to the skirt of the nearby carrier frsgment ion at m/e 427. With injectionsof 100-200 ng of carrier alone the m/e 423/427 was 4-6 parts per 1000. Following the runs of standardsand samples the frsgmentogramrecords were exsmined end the peak heights of the protium snd deuteriumions determinedand a standard line plotted of the ratio of the 1H/2H4 x 1000 against the ng of PGF,&g of 2H -PGF,, carrier after subtractionof the small proportionof m/e 429 in the deuteriumcarrier alone. The slope obtained was always very close to the theoreticalof 45” indicatingno loss of deuterium during fragmentation(11). In our experience,since little deviationfrom the theoreticalline was found, all calculations of unknowns reported here were based on the theoreticalline.
FEBRUARY
1975 VOL. 9 NO. 2
185
PROSTAGLANDINS
RESULTS AND DISCUSSION Table I shows the precisionend accuracy of the deuteriumisotope dilutionmethod for gas-liquidchromatography-mass spectrometrio measurementof PGFZU. Repetitiveinjectionsof 50-100 ng 2H4-PGFsa containingPGF,. in amounts of l-100 ng/pg of deuteratedPGFZcr indicated that 100 pg could be detectedwith an accuracy of about 2% but at higher amounts the accuracy was considerablybetter (3-6%). Since the recovery during extractionand purificationis 70-80 percent end there is some loss on the SE-30 column, a 1 ml sample of o.s.f. would have to contain more than 100 p&l to be detectable. Fortunately,during pneumoencephalography usually more than 10 ml of c.s.f. is withdrawn,thus enablingmeasurementswith reasonable precisionbelow 100 pg/ml. The samples of c.s.f. were classifiedinto 5 groups. Group 1, the so-called 'normal'group, included 16 patients who had normal pneumoenoephalograms and no evidence of central nervoufjsystem disease. Patients in this group had non-specificdiagnosesof depression, anxiety neurosis, tension headache,benign hypertension,neck pain, facial pain or spasmodictortioollis. Group 2 included seven patients with temporal lobe epilepsy, two with generalizedepilepsy,one with a diffuse electroencephalographic disturbanceand one with focal seizures due to cerebral infarction. None of these patients had had previousneurosurgery. Group 3 contains a small group of patients with meningitisor meningoenoephalitis of viral origin. Group 4 representsfive patients in which several samples of c.s.f. were obtained post-operatively. In all these patients,removal of brain tissue (temporallobectomy,multiple excisions,etc.) was carried out for the treatmentof epilepsy. Group 5 includespatients with vascular lesions and subaraohnoidhemorrhsge. The PGFZ. concentration in the c.s.f. of these five patient groups is shown in Tables II-IV. The mean 'normal'PGFZa concentrationin c.s.f. was 71.6 pg/ml 2 34.7 (Table II). The high standard deviationmay be due in part to the considerablevariabilityof the method at the lowest rsnge of sensitivity(see Table I) since total pGFz. in all the 'nonnall samples snalyzed was between 1 and 5 ng. La Torre and coworkers (9) reported mean normal values of 37.7 pg/ml by radioimmunoassaybut found at these levels a &30 pg/ml variation on repeated analyses. Cerebrospinalfluid thus contains considerablyhigher concentrations of PGFza than blood plasma (6). PGFZcrcannot be detected in 10 ml of human plasma by the describedGC-MS method. Significantincreasesin c.s.f. FGFZ. concentrationswere found in patients with epilepsy and in meningoencephalitis(Table II). But it should be noted that the standard deviationis very high. This is in accord with the known increasesin biosynthesisof prostsglandinsfollowingcortical stimulation and in inflammation(12,13). Consistentlyhigher levels of PGFs. were found in o.s.f. samples t&en serially from patients followingneurosurgicalremovals of brain tissue (Table III). In
186
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three patients values in the ng/ml.range were found. These high levels most likely are due to the increasedbiosynthesisand release of FGFZ. due to the surgical trauma. The vascular lesion group (Group 5, Table IV) is as yet too small to make any firm conolusions. Only one patient showed olear radiologioalevidence of vasospasmand in this case the PGF2. was 720 ng/ml. In two patients with rupture of cerebral aneurysms,PGFza was undetectable,but these patients were comatose, subsequentlydied, and c.s.f. samples were obtainedmany days after the subarachnoidhemorrhage. La Torre and coworkers (9) could not find any correlationbetween c.s.f. PGFz. and the appearance of cerebralvasospasm following subarachnoidhemorrhage. Their values ranged from less than 50 pg/ml to over 1.4 ng/ml. It is difficultto obtain clearly matched patients in this group end many more analyses will be requiredbefore any connectionbetween PGFZa concentrations in c.s.f. and cerebralvasospasm ten be made. Furthermore,FGF2. is just as likely to derive from damaged brain tissue adjacent to the hemorrhageas from blood platelets. In this connection,two patients with stroke showed lumbar c.s.f. PGFza concentrationsgreater than lng/ml. In conclusion,human c.s.f. from normal subjects contains concentrations of FGFz. less than 100 p&l. Considerableincreaseswere found in several pathologicalsituations. However, the range from one patient to another and even in day-to-dayserial measurementsin one patient is large. The results obtainedby La Torre and coworkers (9) by radioimunm oassay are in general agreementwith the gas chromatographic-massspectrometricmethod. The latter procedure,however, is expensiveand time-consumingand since the c.s.f. of many more patients must be analyzed,particularlyin the subarachnoidhemorrhageand stroke group, it seems that it would be more prudent to use the radiodoassay method for FGF2. measurementsin studies of this type in the future.
This research was supportedby grants W-1345 and MA-3719from the Medical Research Council of Canada. We wish to thank Dr. Romeo Ethier and Dr. Terence Myles for their help at the clinical level in obtaining the cerebrospinalfluid samples.
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PROSTAGLANDINS
Table I. Accuracy of the GLC-MS method for PGFZo,determinedby repetitive injectionsof standardmixtures of FGFZ. and 3,3,4,4-2H4
PGFZ. as the
trimethylsilylether derivativesof the methyl ester. Ions monitored were m/e 423 and 427. ng Deuterated carrier injected
Parts per 1000 ng H-form of H-form added measured to carrier Mean + S.D.$
100
1.0
ng H-form No. of injected injections
1.09 + 22.9
0.1
8
100
5.0
5.12 f: 6.5
0.5
8
100
10.0
11.07 2 3.0
1.0
8
100
20.0
20.51 -I5.1
2.0
8
100 50
50.0 100.0
51.50 ? 2.9 104.69 + 5.1
2.5 5.0
5 6
Table II. PGFZc,concentrationsin cerebrospinalfluid.in patients classifiedin Groups l-3. The analyses were performed on c.s.f. obtained from patients undergoingdiagnosticpneumoencephalography or lumbar puncture. Disgnosis
PGF2
No.
a
(Pdml) mean + S.D. -
c.s.f. volume analyzed (ml)
Group 1
'Normals',no evidence of CNS pathology
71.6 + 34.7 (mwe: 30-139)
1.6 15-80
Group 2
Epilepsy,before operation
559 t 411 (range: 124-1230)
11
12-54
Group 3
Meningoencephalitis
954 + 783 (range: 252-2196)
5
S-34
188
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270
44-Q
Right
Right temporal lobectomy
Focal seizures - multiple removals
3.
4.
5.
370
Focal seizures - multiple removals
2.
temporal lobectomy
Sub-total hemispherectomy for uncontrolled epilepsy
3
1.
Case
118
5
340
6
-
1300
7
post-operation
lumbar c.s.f. in volumes from lo-18 ml.
1950
336
-
1800
1560
8
treatment of epilepsy). The enalyses were performed on
classified as Group 4 (post-operative for the surgical
PGF,a concentrations in oerebrospinal fluid from patients
Table III.
2180
9
1147
1089
Rupture aneurysm - anterior commun. artery - comatose
Cerebrovasculaxaccident, right side
Cerebrovascularaccident, left side
5.
6.
7.
* Volume of c.8.f. used for analyses 9-15 ml.
less than 50
Rupture aneurysm of anterior cerebralartery - comatose
4.
430
less than 50
Rupture aneurysm - posterior comm. artery - comatose
720
740
208
406
284
796
pg/ml
PGFza
3.
2 8 9
Intracerebellar hemorrhage Arterio-venousmalformation DsYl
Diagnosis *
(vascularlesions and subarachnoidhemorrhage)
Lumbar fluid
Lumbar fluid
L.P. drain in O.R. on day of SBB
1215 rbcls/mm3
Ventricular drain 14 days after SAR
Ventricular fluid day 5 post-op.
43,100 rbc's/mm3
Ventricular drain
comments
concentrationsin cerebrospinalfluid from patients classified in Group 5
Internalcarotid aneurysm severe vasospasm
a
2.
Case
=32
Table IV.
PROSTAGLANDINS
HEFEBENCES 1.
Nakano, J., A. V. Pracan and S. E. Moore. Metabolism of prosta-
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4.
Bito, L. Z. Comparativestudy of concentrativeprostaglandin accumulationby various tissues of mammals snd marine vertebratesand invertebrates. Comp. Biochem. Physiol. 43A: 65-82, 1972.
5.
Samuelsson,B. Quantitativeaspects of prosts&ndin man. Adv. Biosciences9:7-14, 1973.
6.
Ramwell, P. W. The action of oerebrospinalfluid on the frog rectus abdominismuscle and other isolated tissue preparatkons. J. Physiol. (London)170:21-38,1964.
7.
Holmes, S. W. The spontaneousrelease of prostaglandinsinto the cerebral ventriclesof the dog and the effect of external factors on this release. Brit. J. Phazmacol. 37:653-658,197(
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Feldberg,W. and K. P. Gupta. Pyrogen fever and prostaglandin-like activity in cerebrospinalfluid. J. Physiol. (London)228: 41-53, 1973.
9.
La Terre, E., C. Patrono,A. Fortuna and D. Grossi-Belloni. Hole of prostsglandinF, in human cerebralvasospasm. J. Neurosurg. 41:293-299,1974.
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10. Axen, U., K. Gden, D. %rlin and B. Samuelsson. Mass spectrometric determinationof picomole amounts of prostaglandi.ns E, end Fzcrusing syntheticdeuteriumlabelled carriers. Biochem. Biophys. Hes. Comm. 45:419-525,1971. 11. G&en, K., E. GranstG&m,B. Samuelssonand U. Axen. Methods for QusntitativeAnalysis of PGFza, PGs, 9a,lla-Dihydroq-15Keto-Prost-5-Enoic Acid and 9a,lla,15-Trihydroxy-Prod-5Enoic Acid from Body Fluids Using DeuteratedCarriers and Gas Chromatography-Mass Spectrometry. Anal. Biochem. 54: 434-453, 1973.
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1975
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PROSTAGLANDINS
12. Bradley, P. B., G. M. R. Samuels and J. E. Shaw. Correlation of prostaglandinrelease from the cerebral cortex of cats with electro-corticogram followingstimulationof the reticularformation. Brit. J. Phazmacol.37:151-157, 1969. 13.
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Vane, J. FL Inhibitionof prostaglandinbiosynthesisas the mechanism of action of aspirin-likedrugs. Adv. Biosciences 9:395-411, 1973.
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1975
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