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Life Soiencsa Vol . 16, pp" 1577-153a Printed in the O .S .A .

TI~ I~FECT OF PROB13rLCID OA CATECHOLAMIAE MITABOLITES ITI HUbIAF1 CEREIIROSPINAL FLUID AAALYZI~ BY MASS FRAG2dENTOGRAPHY l:dna K, Gordon, Jerry Oliver and Irwin J . Ropin Laboratory of Clinical Science Tlational Institute of Diental Health Bethasch, P4aryland 20014 (Received in final form April 28, 1975) SUA44ARY A gas chromatography-mass fragme~ography (GC-MS) method was used to measure homovanillic acid (IIVA), vanillylmandelic acid (VMA) and 3-metho~çv-4-hydro~çrphenet}~vlene glycol (MHI~) in lumbar cerebrospinal Fluid (CSF) of 31 patients before and after treatment with probenecid . INA values increased from 24 .6 + 2 .6 5 .1: .M, to 210 _+ 17 ng/ml . The increase in VMA was from 1 .06 +0,23~to 2 .22 + 0 .17 ng/ml and that of MHPG Was from 12,2 + 1 .08 to 15.6 + 1 .27 ng/ml. All increases were significant (p = < ,O1) . The results for MHPG and HVA are consistent with results of earlier studies using different methods . VMA concentrations increased significantly but at a rate much lower than those of INA : Ilamovanillic acid (HVA), the major metabolite of dopamine found in lumbar cerebrospinal fluid (CSF) is derived mainly Prop brain rather than spinal cord (1,2,3) . Since transport of organic acids out of CSF is blocked by probenecid (4,5,6) the accumulation of HVA in human lumbar CSF after treatment with this drug has been studied extensively es as index of brain dopamine metabolism (7,8,°) . The major metabolite of norepinephrine in brain is 3-methoxy-4hydrox;rphenvl-glycol (MILPG), which in same species is largely conjugated to form the sulfate (TS}iP( : " S0~) (10,11,12,13) . As a result of probenecid treat ment levels of this caml?ound and its conjugate increase only slightly in human CSF (14) so that norepinephrine turnover c8nnot be studied by the probenecid technique which has proved so useful for the study of dopamine metabolism . Chase et al ., (15) have reported that more [ 1 ~C] VMA than [ 1 ~C] IgiPG was excreted inthe urine of dogs after the animals had been in,~ected intraof [ H] norepinephrine ventriculr~rly with [ 1 ~C] dopamine . Similar injections resulted in the excretion of more [~H] MHPG than [ s H] VMA . This suggests that endogenous norepinephrine formed Pram dopamine may be metabolized to VMA to a greater extent than is exogenous norepinephrine . Compared to MHPG, small quantities of VAfA are found in lumbar CSF (16,17,18) . -Removal of VMA Pram CSF b3" the same active transport mechanism that removes 5 HIAA and HVA from CSF could also result in low VTtA levels, even if more VMA than MFD'( : was being formed Pram norepinerhrine . The development of a sensitive gas chromatographymass fragmentography method (17) for the simultaneous analysis of RVA, VMA and DIIiP(: in the same CSF sample made it possible to measure and compare the accumulation in CSF after probenecid treatment and thereby determine the importance of VblA as a metabolite in human brain . In sa®e patients levels of INA and VP1A in blood were also examined . 1527

1528

$ffect of Probenecid on V1~1 in CS!

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METHODS Lumbar CSF was obtained from 31 psychiatric and 13 neurologic patients hospitalized on research units at the Rational Institute of Mental Health . The psychiatric subjects had been off all medications for at least 2 weeks before the initial CSF was drawn but were in continuous individual and group psychotherapy throughout hospitalization . Patients had been told of the ongoing lumbar puncture studies before voluntary admission to the hospital and informed consent was obtained . Lumbar punctures were done at 9:00 r. .M . after nice hours of bed rest, and at 3 :00 P .M, the following day, after 18 hours of repeated probenecid administration (100 mg/kg), as described in greater detail elsewhere (19) . CSF was mixed with ascorbic acid (2 mg/ml) and stored at -40°C until essayed. Lumbar CSF from neurologic patients was drawn nt 9 :00 A.M . and stored in the same manner . Blood samples were drawn before and 6 hours after probenecid treatment, centrifuged immediately, the plasma removed and stored at -~~0 °C until seas"" . In human CSF, INA and VttA have always been Pound in the unconjugated form but t~~C. occurs both as the free coapound and as the sulfate conjugate (20,21,22) . All meanurementa were made on 2 ml portions of lumbar CSF after hydrolysis with "glusulase" (Fndo Laboratories, Garden City, ti .Y .) by the method of Cordon et _al., (17) . This procedure is quantified by adding deuterium labelledinternal standards to the samples and measuring, the relative amounts of the endogenous hydroPen and the added deuterated form of a major fra~ent of the trifluoroacetyl derivative of each compound . Since the amounts of added deuterated metabolites are known and react is the same manner as the endogenous (I~vdrogen) forma of the corresponding metabolites, the amount of the endogenous cosipound can be calculated from the relative amounts of deute!rated and naturally occuring forma . The retention times on the gas chromatographic column assure specificity of assay. Probenecid also resets with diazomethane and trifluoroacetic anY~dride in this procedure but retention time and mesa spectra of the product are unlike those of the oampounda being measured and it does not interfere with the assay . Since donemine has been reported in "glusulase" (23) and both dopamine and INA are found in enzymes prepared from Helix pomatia (24) "glusulase" was assayed by mesa fragmentography for the presence of RVA, MHPG and VMA . All three metabolites occur in small amounts which are roughly proportional to the sulfatase content of the Preparation. The amount of "glusulase" used (250 units of aulfatase) did not contain sufficient quantities of the metabolites to interfere with the procedure. RYSULTS Baseline levels of the three metabolites in the lumbar CSF of psychiatric patients gets similar to those of the neurologic patiente receiving a variety of drugs but no probenecid . All three metabolites show significant increases (n a < .O1) after 18 hours of probenecid treatment (Table 1) . HVA increased more than 7 fold, VAtA levels doubled, but AiFiP(: increased only 28~. All of the increases are statistically significant but the increase in VItA (109,0 is clearly much lower than that of INA (75kw) . The levels of INA and ttFlf(* found before and after probenecid in the current study are similar to those retorted in the earlier study (14) . In that studv INA was measured b~" the fluorimetric procedure of Gerbode and 3owers (25) and ttITG b"," the Fas chromatographic method of Cordon and Oliver (2G) . Patients in that study were treated with probenecid and lumbar CSF obtained in the same manner but soar o° the patients had also received a variety of drops . These values are shrn m in Table 1 .for comparison . ~t is possible that the higher levels of acid metabolites in blood (increased as a result of renal blockade by nroheneci3) ms"," affect C^f levels of these acids. Therefore, levels of VT-V', sad I:VA were measured in the plasma of three Patients before and E hours

Vol . 16, No. 10

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1529

Bffect of Probenacid on ~ in C3F

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1530

Effect of Probenecid on VMA in CSF

Vol . 16, No . 10

after administration of rrobenecid (Table 2) . TABLR 2 Effect of FYobenecid on Plasma Levels of Acidic Catecholamine l'+tetabolites a

Patient #

Aefore

t{VA

After

Before

VMA

After

1

18

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13

31

2

8

11

11

14

3

4

9

7

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81laluea are expressed as ng/ml and r."ere obtained from Flasma of blood drawn before sad 6 hours after administration of probenecid . After probenecid administration plasma levels of HVA and VMA increase, presumably as a result of diminished urinary excretion of these acidic metabolites . The levels of üVA in plasma do not rise to the point where it ie possible that the elevation of BVA in CSF can be a result of entry of HVA from nlasma, The levels of Vt4A, however, are higher in plasma than in CSF and the possibilty cannot be excluded that same VMH in CSF is derived from plasma . llISCU3SI0N Watson and Wilk (16) using a gas chromatographic method, report an average level of 0 .5 ng VAtA/ml in human CSF . Sjôquist (18) using mass fragmentography found 1 .9 ng/ml . These results are consistent with levels found in our patients . The levels of iIVA and MHPG in Mbar CSF are consistent with previous reports (14,16,17,18), The accumulation, in CSF, of acidic metabor liter of bioeenic amines following blockade of their active transport by administration of probenecid has been used as an index of amine utilization in brain . The differences in the rates of acc~ulation may be due to real differences in their rates of formation or may be a consequence of differences in the efficacy of blockade, by probenecid, of transport of their metabolites . The rates of accumulation in CSF of AVA and MHPG (or ite sulfate) after probenecid are in accord with the results of earlier studies . The relatively low levels and the lower (1gOK) increase in VMA than HVA (754) after probenecid treatment suggest that VMA is not a major metabolite of norepinephrine metabolism in the brain . Furthermore, it is possible that same of this metabolite in CSF ie derived iY~am plasma . If this is the case, even less VMA may be formed in brain than would be concluded on the basis of nngl;"eis of lumbar CRF, ACKNOWLEDGMEA7.S We wish to thank Drs . F,K, Goodwin, R,M . Post and T,N, Chaee for the cerebrospinal fluid semples, Robert Sherman provided excellent technical assiateace,

Vol . 16, No . 10

Effect of Probenecid on V1~41 in CSF

]531

REFEREACES 1. 2. 3. 4, 5. 6, 7. 8. 9. 10 . 11 . 12 . 13 . llr . 15 . 16 . 17 . 18 . 19 . 20 . 21 . 22 . 23 . 24 . 25 . 26 .

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