Prostaglandins in cerebrospinal fluid of healthy human volunteers, abstinent alcoholics and rhesus monkeys

PROSTAGLANDINS

PROSTAGLANDINS IN CEREBROSPINAL FLUID OF HEALTHY HUMAN VOLUNTEERS, A~STINENT ALCOHOLICS AND R H ~ U S MONKEYS J.A.

Y e r g e y ~, ~ . W . Karania4n~ , N. S a l e m ~, M.P. H e y e s ~, B. R a v i t z ~, and M. L i n n o i l a ~

1 Laboratory of Clinical Studies, DICBR, NIAAA, Bethesda, MD 20892 2 Laboratory of Neurophysiology, NIMH, Bethesda MD ~0892

ABSTRACT A sensitive and selective assay for measuring prostaglandins in cerebrospinal fluid has been developed, based on the selected-ion-monitoring, electron-capture negative ionization GC/MS detection for the MO-PFB-TMS derivatives of prostaglandins E2, El, F2a, Fla, and 6-keto-Flct. Improvements over previuously published assay procedures have been made, and the new assay has been applied to measurement of prostaglandin concentrations in lumbar CSF of healthy human volunteers, abstinent alcoholic patients, in cistemal CSF of Rhesus monkeys, and continuously sampled lumbar CSF of awake Rhesus monkeys. Resuhs indicated that the concentrations of PGE2, PGE1, PGFla, and 6-keto-PGF1ct were below 15 pg/mL CSF in lumbar CSF of healthy humans and abstinent alcoholics, and in cisternal CSF of Rhesus monkeys. In contrast, continuously sampled lumbar CSF of awake Rhesus monkeys contained more than 200 pg/mL of PGE2, PGF2ct, and 6-keto-PGFla, probably present as a result of local production. INTRODUCTION The ability of central nervous system tissue to produce prostaglandins in vitro has been known for quite some time, and their association with a wide variety of brain functions has been previously reviewed (1-3). In recent years, a relationship between the central nervous system effects of ethanol and central production of prostaglandins has been suggested, based primarily upon the finding that behavioral effects of ethanol can be modulated by pre-administration of prostaglandin synthetase inhibitors (4-8). It has also been hypothesized that the etiology of alcoholism may be linked to aberrant fatty acid and prostaglandin metabolism (9). In order to obtain evidence for possible ethanolinduced changes in production of prostaglandins in the brain, we have chosen to measure the prostaglandins in cerebrospinal fluid (CSF). Although literature values have been quite variable, measurement of prostaglandins in CSF has often been regarded as indicative of production of these substances in the brain (10-15). We began by measuring prostaglandins in the lumbar CSF of healthy humans and abstinent alcoholics in order to establish a baseline for planned measurements in drinking and acutely withdrawing alcoholics. Rhesus monkey CSF samples were also obtained in order to investigate a non-human primate source of CSF, which offered the availability of larger sample sizes and continuous sampling. We have developed a sensitive and specific assay based on selected-ion-monitoring, electron-capture negative ionization GC/MS detection of the N-methyl methoxime, pentafluorobenzyl ester, tris-trimethylsilyl ether derivatives (MO-PFB-TMS) of prostaglandins E2, El, F2a, Flct, and 6-keto-Flc~. We report here several

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improvements in published assay procedures (16-22) as well as prostaglandin concentrations in lumbar CSF of healthy human volunteers and abstinent alcoholic patients, cisternal CSF of Rhesus monkeys, and in continuously sampled lumbar CSF of awake Rhesus monkeys. MATERIALS AND METHODS Stock Solutions and Standards: Stock solutions of unlabeled prostaglandins were made by dissolving 100-250 I~g each of solid PGE2, PGE1, PGF2a, PGFI~, and 6-ketoPGF]a (Sigma Chemical, St. Louis, MO) in ethanol to make 1.0 ~tg/mL solutions, which were stored at -70°C. The stock solution of mixed tetradeuterated internal standards was made by dilution of purchased ethanol solutions of 3,3,4,4-2H4-PGE2,, 3,3,4,4-2H4-PGF2cx,, and 3,3,4,4-2H4-6-keto-PGF]ct (MSD Isotopes, St.Louis, MO) to approximately 250 pg each in 100 mL of ethanol. Standard curves were constructed using solutions containing 0, 10, 30, 100, 300 and 1000 pg of each unlabeled prostaglandin in 2 mL of phosphate buffered saline (PBS) plus 100 lxL of tetradeuterated internal standard solution. Human Samples: Human lumbar CSF samples were drawn on ice and the 22-23 mL aliquot of a 30 mL total volume was used for this study. Initially, sampling tubes contained indomethacin at a final concentration of 0.1 mM in order to inhibit prostaglandin synthesis. This was later found unnecessary, as described below, and was, therefore, discontinued. Exactly 2.0 mL of CSF was transferred to a tube containing 100 p.L of tetradeuterated internal standard solution and the samples were then frozen and stored at -70°C. Rhesus Monkev Samples: An adult male Rhesus monkey was familiarized over a seven week period to voluntarily getting into and sitting in a plexiglass restraining chair. A polyethylene catheter (PE20) was inserted 6 cm into the lumbar space of the ketamine anesthetized monkey for the continuous collection of CSF. The monkey was placed in the restraining chair and fed monkey chow, fruit and vegetables, and water a d libitum on the usual feeding schedule and was carefully monitored. Samples of CSF (0.6 mL/h) were collected over a three day period. Cisternal CSF was collected from a separate group of 6 Rhesus monkeys while anesthetized with ketamine. Samples were frozen at -70°C and deuterated internal standards were added just prior to extraction. Derivatization and Extraction: Thawed samples were oximated in a modification of the procedure of Kelly, et. al. (17) by treating at room temperature overnight with twice the sample volume of 0.2% methoxyamine-hydrochloride (MOX-HC1, Sigma) dissolved in 0.1M potassium mono-phosphate buffer (pH 5-7). Solid phase extraction, using 1 mL Bond-Elut Ct8 cartridges (Analytichem, Harbor City, CA), was accomplished with a modification of the method described by Powell, e t al. (18). Cartridges were conditioned with 2 mL of methanol followed by 2 mL of distilled water, and the aqueous samples were applied and rinsed with 2 mL of 80:20 water:methanol. The cartridges were then dried completely with vacuum (5-10 min) and rinsed with 2 mL of dichloromethane. The prostaglandins were eluted with 1 mL of methanol into 1.5 mL silanized glass vials, and dried with a stream of nitrogen. Cartridge flow rates were individually controlled, and maintained in the range of 1-3 mL/min throughout the extraction procedure. Samples were then converted to the pentafluorobenzyl esters (PFBB) in a modification of the procedure first noted by Wickramasinghe, et al. (19),

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by reaction with 100 I~L of 0.1% pentafluorobenzylbromide (Pierce Chemical, Rockford, IL) and 10% diisopropylethylamine (DIEA, Pierce) in acetonitrile at 60°C for 15 min. Samples were dried with nitrogen and treated with 50 ~tL each of acetonitrile and bis(trimethylsilyl)-trifluoroaceteamide (BSTFA, Pierce) at 60°C for 15 min to produce the trimethylsilyl ether (TMS). Dried samples were dissolved in 10 ktL of heptane. All solvents were of HPLC or pesticide grade, and were used without further purification. GC/MS Determination: Gas chromatographic separations were performed on a 30 m, 0.25 mm i.d., 0.25 ktm film thickness SPB-5 column (Supelco, Bellefonte, PA) using helium carrier gas (99.999%, Matheson Gas, Dorsey, MD) at a linear velocity of 50 cm/sec. Samples (1-3 ~tL aliquots) were injected into the Carlo-Erba 4160 gas chromatogi~aph using the on-column injector, with the column oven at 110°C. The column was immediately heated to 315°C at 50°C/min, and the prostaglandins eluted between 11 and 14 min. The column was fed through a transfer line maintained at 225°C directly into the ion source of a Kratos MS-80 mass spectrometer which was kept at 250°C. The mass spectrometer was operated in selected-ion-monitoring, electroncapture negative ionization mode using methane reagent gas (99.99%, Matheson) at a source pressure of approximately 0.3 torr. The [M-PFB]- ions of the prostaglandins and internal standards were monitored. Quantification was based on peak area ratios with 2H4-PGE2 as internal standard for PGE2 and PGE1, with 2I-I4-PGF2ct as standard for PGF2c~ and PGFlc~, and with 2H4-6-keto-PGFlc~ as standard for 6-keto-PGFlc~. Stability and Assay Recovery: The stability of the prostaglandins in cerebrospinal fluid and their recovery through the extraction and derivatization process was established using 3H-PGE2, 3H-PGF2ct and 3H-6-keto-PGFlc~ (100-200 Ci/mmol, New England Nuclear). About lxl05 dpm of 3H-labelled standard was used for these experiments, corresponding to approximately 100 pg of prostaglandin; this was within the range of expected experimental values. Solid-phase extraction recoveries were assessed by spiking pooled human lumbar CSF with 3H-labelled standards and counting each extraction fraction, adjusted to uniform solvent composition. Prostaglandin stabilities and derivatization reaction recoveries were monitored using HPLC separation and radiometfic detection. Separations were performed on a Beckman Model 347 HPLC system (Beckman Instruments, Berkeley, CA), equipped with a 10 cm Whatman Partisil-5 ODS-3 column (Whatman Chemical Separations Inc., Clinton, NJ) and Radiomatic Flo-One detection system (Radiomatic Instrument & Chemical Co., Tampa, FL). Net assay recoveries were assessed by spiking CSF with 3H-labelled standard, proceeding through the extraction and derivatization procedure, and extracting the final product from BSTFA/acetonitrile with 2 x 1 mL aliquots of heptane. GC/MS - RIA Comparison: Concentrations of prostaglandins in human lumbar CSF were also measured with radioimmunoassay (RIA), using commercially available 1251 RIA kits (New England Nuclear, Boston, MA). A set of duplicate CSF samples was obtained which had been stored without prior addition of tetradeuterated internal standards. The second set of samples was spiked with 13 pg/mL of PGE 2 and 6-ketoPGFlw Both treated and untreated samples were then extracted and split in half for assay by GC/MS and RIA for PGE2 and 6-keto-PGFla. Tetradeuterated internal standards for the GC/MS assay were added following extraction and sample splitting, and methoximation reactions were performed in pyridine (0.1% MOX-HC1).

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PROSTAGLANDINS RESULTS Stability of Prostaglandins in CSF: Prostaglandins were stable in cerebrospinal fluid, both at room temperature for short periods and following extended storage at -70°C. The stability at room temperature (25-30°C) was evidenced by the lack of change in the HPLC chromatographic peak size and retention for 3H-PGE2 added to CSF and allowed to stand for 96 hr. Stability upon extended storage at -70°C was measured indirectly, by comparison of the absolute GC/MS peak areas for deuterated standards that were stored in CSF versus those stored in ethanol and used to generate standard curves. No measurable difference between the absolute peak areas for deuterated internal standards was apparent, even upon storage at -70°C for more than 18 months. Derivatization and Extraction: Aqueous oximation proceeded quantitatively (>95%) for reaction times of 12 hr. An example of the HPLC separation of the starting material and reaction product for 3H-PGE2 is shown in Figure 1. By-products of the reaction were efficiently removed in the subsequent solid-phase extraction step.

cPMI |

0

d

|

15 Time (min)

11

30

30

Time (min) FIGURE 1: Radio-chromatographic monitoring of methoximation; before reaction (top) and after 12 hour reaction at room temperature (bottom). HPLC mobile phase: 50:50 methanol:water, 1 ml/min. Extraction recoveries were similar for 3H-PGE2, 3H-PGF2a and 3H-6-keto-PGFlc~ and their methoximated products, and an example for a triplicate analysis of 3H-PGE2 at various pH values is presented in Table 1. Losses upon application of sample (5% ethanol) were insignificant at moderate flow rates (1-3 mL/min) and independent of pH in the range of pH 3-7. Significant losses of sample in the dichloromethane fraction were observed when columns were not dried following the 80:20 water:methanol rinse. Note that while greater than 95% of the material eluting from the cartridges was found in the methanol fraction, approximately 15% of the total radioactivity was not recovered. Attempts to use larger elution volumes (up to 20 mL), other elution solvents (methyl formate, 50:50 water:acetonitrile, ethyl acetate), or other manufacturers' C18 cartridges (J.T. Baker, Supelco) in order to improve recovery were unsuccessful.

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pit 3.0 5.0 7.0

TABLE 1: Solid-Phase Extraction Recovery for PGE2 CPM 1 5% Ethanol 80:20 Dichloromethane Methanol 285 + 98 329 + 8 335 + 26

556 + 11 668 + 50 703 + 34

1Mean + SD, n=3

394 + 23 331 + 25 286 + 11

50249 + 2099 50717 + 2807 49507 + 2201

% Recovery in Methanol 2 85.2 + 3.6% 86.0 + 4.8% 83.9 + 3.7%

2Based upon total applied = 59000 cpm

Pentafluorobenzyl esterification proceeded with 95% recovery in a 15 min reaction at 60°C. An example of the HPLC separation of the starting material and reaction product for 3H-PGF2c~is shown in Figure 2. Similar recoveries were obtained by reaction with a 10% PFBB and 10% DIEA in acetonitrile solution for 5 min at room temperature, as has been noted by others (20). We observed,however, that reducing the concentration of PFBB from 10% to 0.1%, while maintaining the elevated reaction temperature, allowed a much greater reduction in the total amount of interfering by-products, as measured in both full scan and selected-ion GC/MS determinations.

A CPM

Time(min)

L

t~

Time (min) FIGURE 2: Radio-chromatographic monitoring of pentafluorobenzyl esterification; before reaction (top), and after 15 minute reaction at 60°C (bottom). HPLC mobile phase: 60:40 acetonitrile:water, 1 mL/min. It was also observed, using HPLC monitoring of reaction products, that the correct reaction order for methoximation and esterification was critical to achieving maximum recovery. When prostaglandins were esterified first, the reaction proceeded quantitatively but the ester product was lost upon subsequent methoximation; the net yield for the PFB-MO product was less than 50%. The need for reversing the reaction order was first noted by Claeys and coworkers (21), but has not been universally documented or adopted. Overall assay recoveries for duplicate determinations of 105

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PROSTAGLANDINS cpm 3H-PGE2, 3H-PGF2a and 3H-6-keto-PGFlct, added to 2 mL of pooled human CSF and processed using these improved methods, were 67.0+_5.2%, 75.9-&-0.4%, and 68.6+9.0% (Mean~_+SD),respectively. GC/MS - RIA Comoarison: Similar results were obtained by both methods for the assay of PGE/ and-6-keto-PGFla in human lumbar CSF subjected to solid-phase extraction. In both cases there were no prostaglandins observed in the untreated sample and the values obtained for spiked samples were in good agreement with the amount added. The results for PGE2 are presented in Table 3 and an example of the results for the spiked human CSF in this comparative study are shown in Figure 3. Quantification of PGE2 is based on the peak area for the major isomer of the two formed during methoximation, as noted on the GC/MS trace. TABLE 3: Comparison of PGE2 Levels (pg]mL) in Human Lumbar CSF as Measured b~¢RIA and GC/MS Untreated Treated (13 pg/mL) Sample RIA GC/MS RIA GC/MS 1 nd nd 12 12 2 nd nd 13 16 3 nd nd 17 8 4 nd nd 14 11 5 nd nd 11 12 6 nd nd 14 9 7 nd nd 14 15 8 nd nd 14 12 9 nd nd 14 10 Mean+ SD 14+2 12+3 Human and Rhesus Monkey CSF: Prostaglandins E2, E], Flct, and 6-keto-Fla are not present at a concentration of 15 pg/mL or greater in lumbar CSF of healthy human volunteers, abstinent alcoholics or cisternal CSF of Rhesus monkeys. Easily quantifiable concentrations of the 2-series prostaglandins were found in continuously sampled lumbar CSF of Rhesus monkeys. Data are summarized for the single animal which we were able to study over several days. We observed similiarly high values for single samples taken in the first hours following recovery from anesthisia for animals used subsequently in other protocols (n=4, data not shown). An example of the GC/MS chromatographic results for human CSF are presented in Figure 4, and results for all groups are summarized in Table 4. The limit of detection for pure standards was below 300 fg (1 fmol injected on-column), while sample recovery and chemical background restrict the limit of quantification for the assay to 6 pg injected (15 pg in a 2 mL CSF sample). Chemical interferences were present in all CSF samples on the rn]z 569 trace which are sufficiently close in retention time that reliable quantification of P G F 2 a was not possible. These peaks were not present in blanks or standards. Evidence for the lack of formation of prostaglandins in CSF following collection was obtained by analyzing 4 lumbar monkey CSF samples, obtained at the start of the continuous collection protocol, only two of which were treated with 0.1M indomethacin; no variation was observed in the prostaglandin concentrations (data not

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PROSTAGLANDINS

rn/z 618.40 2H4-6-keto-PGFla 1 2 5 ~

m/z 614.37

09

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© Z

350 10:47

400 11:19

450 11:52

500 12:25

550 12:57

600 13:30

RETENTIONTIME (rain)

Figure 3: GC/MSselected-ion-chromatogramfor spikedhumanCSF (13 pg/mLeach of PGE and 6-keto-PGF). Eachm/z u'aceis individuallynormalized.

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PROSTAGLANDINS

t

m/z 618.40 2H4-6-keto-PGFla 125 pg/mL

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J

PGF2c~ ? (see text)

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Figure 4: GC/MS selected-ion-chromatogram for human CSF sample. Each rn/z trace is individually normalizexl.

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PROSTAGLANDINS

shown). Further evidence was obtained by demonstrating that prostaglandins remained undetectable in human CSF collected into tubes not containing indomethacin (n=7). TABLE 4: Prostaglandin Concentrations in Cerebrospinal Fluid of Humans and Rhesus Monke~,s (pg,/mL) Sample (n) PGE2 PGE1 PGF2c~ PGFlct 6-keto-PGFlc~ Healthy volunteers I (16) nd Abstinent alcoholics 1 (16) nd Monkey 2 (9) 400 + 109 Monkey 3 (6) nd

nd nd nd nd

? ? >200 ?

nd nd nd nd

nd nd 200 + 87 nd

1Lumbar CSF 2Continuous (4 day) lumbar CSF samples from a single animal; Mean + SD 3Acute cisternal CSF samples ? - Reliable quantification not possible (see text). DISCUSSION Assay Development: Because cerebrospinal fluid is a relatively clean biological matrix, it has been possible to extract the prostaglandins efficiently from CSF with a single-step solid-phase extraction process, eliminating more tedious HPLC or TLC clean-up steps. We found that both underivatized and methoximated prostaglandins were extracted efficiently over a wide pH range, and with relatively low solvent volumes in comparison to the commonly used solid-phase extraction method (18). It should be noted, however, that about 15% of the added quantity of prostaglandins was not recovered from the extraction cartridge. Use of HPLC separations, coupled with radiometric detection of labelled prostaglandins, was extremely useful in addressing several questions regarding assay development. This technique allowed us to observe that methoximation must precede esterification in order to avoid losing the esterified product. Having reversed the order of these reactions, aqueous oximation was attempted both to avoid using pyridine, a solvent difficult to maintain free of contamination, and to allow methoximation byproducts to be separated from prostaglandins during solid-phase extraction. Reaction conditions were readily determined, and it was found unnecessary to use a pyridinium salt to catalyze the reaction as previously reported (17). By-products of the esterification reaction, which are themselves efficient electron-capturing agents in the GC/MS ion source, have usually necessitated an additional extraction after the esterification reaction (16,20,22). Monitoring the reaction using HPLC allowed us to determine that elevating the reaction temperature and using 100-fold less pentafluorobenzyl bromide produced the maximum yield of the esterified product while minimizing the interference in the GC/MS determination. Prostaglandins Concentrations in Human and Monkey CSF: Our results clearly indicate that cerebrospinal fluid from healthy human volunteers contains very low concentrations of prostaglandins, that is, less than 15 pg/mL of PGE2, PGE1, PGF]ct, and 6-keto-

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PROSTAGLANDINS

PGFI~. These data have been confirmed in our laboratory by comparisons of human lumbar CSF assayed by both GC/MS and RIA. The possibility that low levels in the lumbar CSF are the result of a concentration gradient along the spinal column is not likely since similarly low levels were observed in monkey cisterna magna samples. Moreover, there is clear evidence in the literature that prostaglandins are not catabolized in the brain (23-26), suggesting that they must first be removed from the brain. Our data suggest that there are not significant levels of the 15-keto or 13,14-dihydro metabolites in lumbar CSF. This was evidenced by the lack of any peaks at the corresponding gas chromatographic relative retention times for these metabolites, which have been characterized by others (22). The absence of measurable prostaglandins is conceivably the result of low brain synthesis rates or the result of transport phenomena which limit their bulk CSF concentration. The latter possibility is supported by the observations of Bito and coworkers who demonstrated that exogenously administered prostaglandins are rapidly removed from the CSF by an organic acid transport system (27,28). One explanation for the low bulk CSF concentration of prostaglandins is, therefore, that the organic transport system facilitates removal of the prostaglandins directly from the extracellular space of the brain into the microvasculature. Literature reports for prostaglandin concentrations in CSF of normal humans have ranged from undetectable to as high as 1 ng/mL (10-15). Problems with earlier methodologies are one explanation for the contrast with our observations. Only recently have iodinated RIA methodologies been employed for measuring prostaglandins, and earlier tritium labelled assays had much poorer sensitivity. Non-specific cross reactivities, especially for unextracted CSF, might also explain some of the higher values previously obtained by RIA. Another explanation for the higher levels reported in the literature might relate to the difficulty in obtaining CSF samples from healthy human volunteers to serve as controls in many studies. These results indicate that future studies of prostaglandins in human CSF should include samples from healthy volunteers serving as controls, in which appropriately low levels are observed. Cisternal CSF from Rhesus monkeys is similar to human lumbar CSF in having less than 15 pg/mL of these primary prostaglandins. However, continuously sampled Rhesus monkey lumbar CSF contains measurable concentrations of PGE2, PGF2ct, and 6-keto-PGFla (> 200 pg/mL). While it is not possible to be certain of the origin of these prostaglandins, it is feasible that local irritation from the indwelling catheter stimulates their production. Hsu and coworkers demonstrated that local injury to the spinal column of the cat could produce significant increases in prostaglandin production (29). It appears, therefore, that continuously sampling the lumbar space of the Rhesus monkey may not be useful as an index of brain prostaglandin production. We observed that, similar to healthy controls, alcoholics sampled following a three week period of abstinence had lumbar CSF prostaglandin levels below 15 pg/mL. If arachidonic acid metabolism is disturbed by ethanol exposure, our data suggest that the effect is reversible after three weeks of abstinence, or that any change occurs at levels which remain immeasurable. Studies are in progress to measure prostaglandin concentrations in CSF of drinking alcoholics and alcoholics during acute withdrawal. Earlier studies suggest that any changes in prostaglandin concentrations should be most apparent in these groups (5-9). Indeed, elevated prostaglandin levels were reported for unipolar depressive patients using a GC/MS assay; the only literature report using this technique which examined patients that were not suspected to have structural damage to

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the brain (30). It is important to consider, however, that rapid transport of prostaglandins from the extracellular space of brain tissue probably still reduces bulk CSF prostaglandin concentrations in these patients, even if much larger changes occur at the cellular level. REFERNECES 1.

Gross, H.A., D.L. Dunner, D. Lafleur, H.L. Meltzer, H.L. Muhlbauer, R.R. Fieve. Prostaglandins: A review of neurophysiology and psychiatric implications. Arch. Gen. Psychiatry 34:1189-1196. 1977.

2.

Wolfe, L.S., F. Coceani. The role of prostaglandins in the central nervous system. Ann. Rev. Physiol. 4._!1:669-84. 1979.

3.

Chiu, E.K.Y., J.S. Richardson. Behavioral and neurochemical aspects of prostaglandins in brain function. Gen. Pharmac. 16:163-175. 1985.

4.

Grupp, L.A., J. Elias, E. Perlanski, R.B. Stewart. Modification of ethanolinduced motor impairment by diet, diuretic, mineralocorticoid, or prostaglandin synthetase inhibitor. Psychopharm. 87:20-24. 1985.

5.

Wescott, J.Y. and A.C. Collins. Brain arachidonic acid metabolites, function and interactions with ethanol. In: Recent Developments in Alcoholism (M. Galanter, ed.), Plenum Press, New York, 1985, pp. 143-152.

6.

Minocha, A., J.T. Barth, D.A. Herold, D.A. Gideon, D.A. Spyker. Modulation of ethanol-induced central nervous system depression by ibuprofen. Clin. Pharm. Ther. 39:123-127. 1986.

7.

Anton, R. and C.L. Randall. Central nervous system prostaglandins and ethanol. Alcoholism 11:10-18. 1987.

.

9.

Segarnick, D. and J. Rotrosen. Essential fatty acids, prostaglandins, and nonsteroidal antiinflammatory agents: physiological and behavioral interactions. Alcoholism 11:19-24. 1987. Horrobin, D.F. Essential fatty acids, prostaglandins, and alcoholism: overview. Alcoholism 11:2-9. 1987.

an

10. Wolfe, L.S., O.A. Mamer. Measurement of prostaglandin F2et levels in human cerebrospinal fluid in normal and pathological conditions. Prostaglandins 9:183192. 1975. 11. Egg, D., M. Herold, E. Rumpl, R. Gunther. Prostaglandin F2et levels in human cerebrospinal fluid in normal and pathological conditions. J. Neurol. 222:239-248. 1980.

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12. Math6, A.A., F.A. Wiesel, G. Sedvall, H. Nybiick. Increased content of immunoreactive prostaglandin E in cerebrospinal fluid of patients with schizophrenia. Lancet Jan 5, 1980. 13. Gerner, R.H., J.E. Merill. Cerebrospinal fluid prostaglandin E in depression, mania, and schizophrenia compared to normals. Biol. Psych. 18:565-569. 1983. 14. Kosfi6, V.S., B.M. Djuri~i6, B.B. Mr~ulja. Cerebrospinal fluid prostaglandin F in stroke patients: No relationship to degree of neurological deficit. Eur. Neurol 23:291-295. 1984. 15. Romero, S.D., D. Chyatte, D.E. Byer, J. C. Romero, T.L. Yaksh. Measurement of prostaglandins in cerebrospinal fluid in cat, dog, and man. J. Neurochem. 4_33:1642-1649. 1984. 16. Waddell, K.A., I.A. Blair and J. Welby. Combined capillary column gas chromatography negative ion chemical ionization mass spectrometry of prostanoids. Biomed. Mass Spectrom. 10:83-88. 1983. 17. Kelly, R.W., M.H. Abel. The measurement of 13,14-Dihydro-15-keto prostaglandin E2 by combined gas chromatography mass spectrometry. Biomed. Mass Spectrom. 10:276-279. 1983. 18. Powell, W.S. Rapid extraction of oxygenated metabolites of arachidonic acid from biological samples using octadecylsilyl silica. Prostaglandins 20:947-957. 1980. 19. Wickramasinghe, J.A.F., W. Morozowich, W.E. Hamlin and S.R. Shaw. Detection of prostaglandin F2ct as pentafluorobenzyl ester by electron-capture GLC. J. Pharm. Sci. 6_22:1428-1431. 1973. 20. Strife, R.J. and R.C. Murphy. Preparation of pentafluorobenzyl esters of arachidonic acid lipoxygenase metabolites: Analysis by gas chromatography and negative-ion chemical ionization mass spectrometry. J. Chrom. 305:3-12. 1984. 21. Claeys, M., C. Van Hove, A. Duchateau, A.G. Herman. Quantitative determination of 6-oxo-PGFla in biological fluids by gas chromatography mass spectrometry. Biomed. Mass Spectrom. 7:544-548. 1980. 22. Pace-Asciak, C.R. and S. Micallef. Gas chromatographic-mass spectrometric profiling with negative-ion chemical ionization detection of prostaglandins and their 15-keto and 15-keto-13,14-dihydro catabolites in rat blood. J. Chrom. 310:233242. 1984. 23. Nakano, J., A.V. Prancan, S.E. Moore. Metabolism of prostaglandin E1 in the cerebral cortex and cerebellum of the dog and rat. Brain Res. 3_.99:545-548. 1972. 24. Wolfe, L.S., K. Rostworowski, H.M. Pappius. The endogenous biosynthesis of prostaglandins by brain tissue in vitro. Can. J. Biochem. 5._44:629-640. 1976.

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25. Pace-Asciak, C.R., G. Rangaraj. Prostaglandin biosynthesis and catabolism in the developing sheep brain. J. Biol. Chem. 251:3381-3385. 1976. 26. Abdel-Halim, M.S., E. Angg~d. Regional and species differences in endogenous prostaglandin biosythesis by brain homogenates. Prostaglandins 17:411-418. 1979. 27. Bito, L. Absorptive transport of prostaglandins and other eicosanoids across the blood-brain barrier system and its physiological significance. In: The Blood-brain Barrier in Health and Disease (A.J. Suckling, M.G. Rumsby, M.W.B. Bradbury, eds.) VCH Publishers, Deerfietd Beach, FL, 1986, pp. 109-121. 28. Bito, L.Z., H. Davson, J.R. Hollingsworth. Facilitated transport of prostaglandins across the blood-brain barriers. J. Physiol. 2~6:273-285. 1976. 29. Hsu, C.Y., P.V. Halushkam, E.L. Hogan, N.L. Banik, W.A. Lee, P.L. Perot, Jr. Alteration of thromboxane and prostacyclin levels in experimental spinal cord injury. Neurol. 35:1003-1009. 1985. 30. Linnoila, M., R. Whorton, D.R. Rubinow, R.W. Cowdry, P.T. Ninan, R.N. Waters. CSF prostaglandin levels in depressed and schizophrenic patients. Arch. Gen. Psych. 40:405-406. 1983. Editor: F. C o c e a n i

APRIL 1989 VOL. 37 NO. 4

Received: 11-21-88

Accepted:2-28-89

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