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Home , Amphetamine, Rauwolscine, Substituted amphetamine

208 NeuroscienceLetters, 95 (1988) 208-212 Elsevier Scientific Publishers Ireland Ltd.

NSL O5736

Ring-substituted interactions with receptor binding sites in human cortex

Pamela A. Pierce and Stephen J. Peroutka Deparmentsof Neurologyand Pharmacology, Stanford University Medical Center, StanJbrd,CA 94305 (U.S.A.)

(Received 31 May 1988; Revised version received 11 August 1988; Accepted 12 August 1988)

Key words.' Ring-; (_+)-3,4-Methylenedioxyamphetamine; ( +_)-3,4-Methylene- dioxyethamphetamine; (_+)-3,4-Methylenedioxymethamphetamine; Ecstasy; 4-Bromo-2,5- dimethoxyphenylisopropylamine ; Human cortical receptor

The binding affinities of 3 ring-substituted amphetamine compounds were determined at 9 neurotrans- mitter binding sites in human cortex. (_+)-3,4-Methylenedioxyamphetamine (MDAk (_+)-3,4-methylene- dioxyethamphetamine (MDE), and (_+)-3,4-methylenedioxymethamphetamine (MDMA or 'Ecstasy') all display highest affinity (approximately 1 /tM) for the recently identified 'DOB binding site' labeled by ['TBr]R(-)4-bromo-2,5-dimethoxyphenylisopropylamine ([77Br]R(-)DOB). MDA displays moderate affinity (4-5/iM) for the 5-hydroxytryptaminetA (5-HTr^), 5-HT_D,and =,- sites in human cor- tex. MDE and MDMA display lower affinity or are inactive at all other sites tested in the present study. These observations are discussed in relation to the novel psychoactive effects of the ring-substituted .

A series of ring-substituted amphetamine derivatives exist which are structurally related to both amphetamines and . However, such as (+)-3,4- methylenedioxyamphetamine (MDA), (_+)-3,4-methylenedioxyethamphetamine (MDE), and (+)-3,4-methylenedioxymethamphetamine (MDMA or 'Ecstasy') ap- pear to produce unique psychoactive effects which are distinct from the effects of both amphetamines and hallucinogens [13, 15]. These agents have been reported to induce a mild with a sense of enhanced insight in human users. As a result of these properties, ring-substituted amphetamines have been used in both psycho- therapeutic [4] and recreational [8] settings. The human use of these compounds is of concern due to the neurotoxic effects of ring-substituted amphetamines on serotoner- gic nerve terminals in laboratory animals [2, 10, 11, 16].

Corre,_'pomh,nce.'S.J. Peroutka, Department of Neurology, Stanford University Medical Center. Stanford. CA 94305, U.S.A.

0304-3940 885 03 50 _ 1988 Elsevier Scientific Publishers Ireland Ltd. NeuroscienceLetters,95(1988)208-212 209 Elsevier Scientific Publishers Ireland Ltd. The mechanism of action by which ring-substituted amphetamines produce their unique psychoactive effects remains unknown. In the present study, the potencies of MDA, MDE, and MDMA at 8 neurotransmitter binding sites in human cortex were analyzed. In addition, potencies were determined at the recently identified bind- ing site radiolabeled by 4-bromo-2,5-dimethoxyphenylisopropylamine (DOB), a le interactions with relative receptor, Titeler and colleagues first labeled this site in rat brain

"'5_:n'_ sites in human thewithnovel[3H]DOBradioligand[7, 17].[7Recently,7Br]R(-)DOBand intothelabelpresentthe 'DOBstudy,bindingour laboratorysite' [19]. Thehas usedpre- sent study represents the first report of ring-substituted amphetamine interactions with neurotransmitter binding sites in human cortex. Radioligand binding studies were performed as described previously [9, 5, 19]. n J. Peroutka Briefly, human cortex samples were thawed in Tris-HCl buffer. Tissues were homoge- nized in 20 vols. Tris-HC1 buffer (pH 7.7 at 25°C) and then centrifuged at 49,000 _ity Medical Center, Stanford, CA 94305 ' for 10 min. The supernatant was discarded and the pellet was resuspended and ust 1988;Accepted 12 August 1988) incubated at 37°C for 10 rain prior to a second centrifugation at 49,000 g for 10 min. The final pellet was resuspended in 80 vols. of Tris-HCl buffer containing 10 #M par- tedioxyamphetamine; (+)-3,4-Methylene- gyline, 4 mM calcium chloride and 0. ! %ascorbic acid. The buffer used for the DOB _ethamphetamine;Ecstasy; 4-Bromo-2,5- site analysis consisted of 50 mM Tris-HC1, 0.5 mM EDTA, 10 mM MgCI:, 0.1% nancorticalreceptor ascorbate, and 10 #M pargyline. Radioligand binding studies consisted of 0.1 mi _pounds were determined at 9 neurotrans- ['_H]radioligand (final concentrations were 0.3 nM [3H]8-OH-DPAT; 1.4 nM [3H]5- ,mphetamine(MDA),(+)-3,4-methylene- HT; 0.4 nM [3H]; 0.5 nM [3H]WB 4101; 1.5 nM [3H]; 0.2 nM thamphetamine (MDMA or 'Ecstasy')all 'q] hydrochloride (DHA); 12-32 pM 77Br-R(-)DOB; 0.08 nM identified 'DOB binding site' labeled by [ H]quinuclidinyl benzilate (QNB); 0.1 nM [3H]flunitrazepam), 0.1 ml buffer or dis- -]R(-)DOB). MDA displays moderate placing drug and 0.8 ml tissue suspension. The presence of 0.1 /_M 8-OH-DPAT Fro,all otherand :_2-adrenergicsites tested insitesthe inpresenthumanstudy.cor- when labeling the 5-HTiD site with [3H]5-HT and the cq-adrenergic site with [3H]WB- ychoactiveeffectsof the ring-substituted 4101 eliminated radioligand binding to 5-HTiA sites. Specific binding was defined as the excess taken over blanks in the presence of 10-5 M 5-HT for 5-HTIA and 5-HT1D sites, 10 _ M for 5-HT2 sites, 10-6 M 4-iodo-2.5-dimethoxyphenylisopro- _ylamine (DOI) for DOB sites, 10-6 M for _t-adrenergic sites, 10 -4 M ttives exist which are structurall? himbine for alpha?adrenergic sites, 10 -6 M for/_-adrenergic sites, j 0 M for muscarinic cholinergic sites, and 10 6 M diazepam for ben- However, drugs such as (2)-3,4- ·-methylenedioxyethamphetamine zodiazepine sites. The KD values for the various radioligands in general have not been determined tmine (MDMA or 'Ecstasy') ap- h are distinct from the effects of in human cortex so that calculating K i values is not possible at this time. Final radio- tese agents have been reported to concentrations used in the assays were less than one-half the KD values _sight in human users. As a result reported for the radioligands in rat brain. have been used in both psycho- Radiolabeled drugs were obtained from Dupont-New England Nuclear except man use of these compounds is of H]8-OH-DPAT from Research Products International Corp. and [77Br]R(-)DOB '_75 Ci mmol), a generous gift from Dr. Chester A. Mathis. Drugs were obtained uted amphetamines on serotoner- 16]. 1¥omcommercial sources except MDMA, MDE, MDA, and DOI which were obtained from the National Institute on Drug Abuse, Bethesda, MD. Drug interactions with 5-hydroxytryptamine binding site subtypes. Drug competi- FordUniversity MedicalCenter,Stanford. lion studies were performed to determine the IC50 values of 3 ring-substituted amphe- tamine compounds at 5-HT binding sites in human cortex (Table I). All 3 of the psy-

nd Ltd. 210

TABLEI 'I RING-SUBSTITUTED AMPHETAMINE INTERACTIONS WITH HUMAN 5-HT BINDING SITE p SUBTYPES 1

Radioligandbindingassayswereperformedusing human cortex membranesas describedinthe text. ICs0 I values weredetermined using log probit analysis. Values shown are means + S.E.M. of 3-5 experiments. eachperformedintriplicate, p,

Human ICsovalues(uM) binding site Radioligand MDA MDE MDMA

DOB [7?Br]R(-)DOB 1.2 _+0.3 0.88 _+0.2 1.4 _+0.1 5-HTi^ [3H]8-OH-DPAT 4.7+ 0.3 11+ 2 Il + 1 5-HTm [3H]5-HT 4.1_+0.6 70+ 10 11+ 1 5-HT2 [3H]Spiperone 33+ 3 36+ 7 18+ 6

choactive agents display the highest affinity for the 'DOB binding site' labeled by [TVBr]R(-)DOB. MDA is at least 3-fold more potent at the DOB site (1.2_+0.3/tM) than at any of the other sites tested. MDE (0.88+_0.2/iM) and MDMA (1.4_+0.1 /tM) are essentially equipotent with MDA at the 'DOB binding site' and are at least 10-fold more potent at this site than at the other sites tested. MDA displays an affinity below 5/aM for both the 5-HTtA site labeled by [3H]S- OH-DPAT and the 5-HTiD site labeled by [3H]5-HT in the presence of 0.1/aM 8-OH- DPAT. Both MDE and MDMA display affinities greater than 10/aM for the 5-HTiA and 5-HTiD sites. MDE is notably weak at the 5-HTm site (70/aM). All 3 agents are relatively weak at the 5-HT2 site labeled by [3H]spiperone, displaying affinities of > 18/aM. Drug interactions with other neurotransmitter binding sites. The affinities of MDA. MDE, and MDMA for various other neurotransmitter binding sites in human cortex are shown in Table II. MDA displays moderate affinity (5.2_+0.1 uM) for the =,- adrenergic site labeled by [3H]rauwolscine. MDA is weaker at the :q-adrenergic site (38_+9/aM) labeled by [3H]WB-4101 in the presence of 0.1 /tM 8-OH-DPAT and essentially inactive at the fi-adrenergic site labeled by [3H]DHA, the muscarinic choli- nergic site labeled by [3H]QNB, and the site labeled by [3H]flunitraze- pam. MDE and MDMA are relatively weak (> 10/aM) at all of the above sites and are inactive at the benzodiazepine site. The major finding of the present study is that the ring-substituted amphetamine compounds, MDA, MDE, and MDMA all display relatively high affinity (approxi- mately I /tM) for the recently identified 'DOB binding site' in human cortex. MDA is at least 3-fold, and MDE and MDMA at least 10-fold, more potent at the 'DOB binding site' than at any of the other 8 sites analyzed in this study. Although previous studies have reported MDA and MDMA interactions with brain 5-HT receptors in the rat [1. 6], the present study is the first report of ring-substituted amphetamine interactions with neurotransmitter binding sites in human cortex. These results are 211

TABLE II 'ITH HUMAN 5-HT BINDING SITE RING-SUBSTITUTEDAMPHETAMINE INTERACTIONS WITH HUMANNEUROTRANSMIT- TER BINDING SITES nembranes as described in the text. IC50 Radioligand binding assays were performed using human cortex membranes as described in the text. IC50 'e means + S.E.M. of 3-5 experiments, alues were determined using log probit analysis. Values shown are the means + S.E.M. of 3-5 experi- ments, each performed in triplicate.

IC50values_M) Human ICs0values(uM) binding MDE MDMA site Radioligand MDA MDE MDMA

0.88+ 0.2 1.4+ 0.1 :q-Adrenergic [3H]WB-4101 38__+9 36+ 6 18+ 5 I14-2 Il 4-l x,-Adrenergic [3Hlrauwolscine 5.2± 0.1 22+_2 134-2 704- 10 11 + I 'J-Adrenergic [3H]DHA > 100 33 + 2 37 + 3 364-7 18+ 6 iusca_rinic cholinergic [3H]QNB 92 4- 4 65 + 10 24 + 2 Benzodiazepine [3H]flunitrazepam > 100 > 100 > 100 'DOB binding site' labeled by t at the DOB site (1.24-0.3 p,M) ).2/aM) and MDMA (1.4 + 0.1 of potential interest since the mechanism of action of ring-substituted amphetamines )B binding site' and are at least remains unknown. tested. The results of the present study suggest the 'DOB binding site' might mediate some te 5-HTt^ site labeled by [3H]8- the psychoactive effects of the ring-substituted amphetamines. The 3 compounds in the presence of 0.1/tM 8-OH- are essentially equipotent at the 'DOB binding site' in human cortex. Similarly, the ater than 10 #M for the 5-HTjA usual human doses of these 3 drugs are also equivalent: i.e. 100-150 mg [12, 14]. Mi- lTm site (70 #M). All 3 agents cromolar blood levels of these agents would be expected from such doses since a single !]spiperone, displaying affinities oral dose of 50 mg MDMA has been reported to result in a plasma MDMA level of 0.45 ,aM (106/ag/l) [18]. A plasma level of 30/aM (7000/ag/l) MDMA has also ng sites. The affinities of MDA, been documented in a recreational MDMA user although the exact amount of er binding sites in human cortex MDMA ingested by the individual was not known [31.These data suggest that recrea- 5nity (5.2+0.1 uM) for the :_,_- nal human doses o.f the ring-substituted amphetamines interact with the 'DOB weaker at the =l-adrenergic site _mding site' in whereas, at higher doses, the drugs are likely to interact :e of 0.1 /aM 8-OH-DPAT and ,. with multiple other neurotransmitter receptors. Clearly, further studies are needed [3H]DHA, the muscarinic choli- to determine the functional significance of the 'DOB binding site' in the human cen- ne site labeled by [3H]flunitraze- tral nervous system and its possible relationship to the psychoactive effects of MDA, M) at all of the above sites and MDE and MDMA. The mechanism of action by which ring-substituted amphetamines produce their ring-substituted amphetamine psychoactive effects is of importance due to their neurotoxic effects in laboratory ani- relatively high affinity (approxi- ils and their recent recreational popularity [8]. Conceivably, their unique psychoac- ng site' in human cortex. MDA ,,. e effects may be independent of their toxic effects on serotonergic nerve terminals. -fold, more potent at the 'DOB If so, then the possibility exists for the development of non-toxic analogues that may in this study. Although previous have useful psychotherapeutic applications. On the other hand, the neurotoxic effects ns with brain 5-HT receptors in of the currently available agents suggests that human use of these compounds should f ring-substituted amphetamine be avoided. roman cortex. These results are 212

This work was supported by the Pharmaceutical Manufacturers Association (PAP), John A. and George L. Hartford Foundation, the McKnight Foundation, the Alfred P. Sloan Foundation and NIH Grants NS 12151-13 and NS 23560-02.

I Battaglia, G., Kuhar, M. and De Souza, E., MDA and MDMA (Ecstasy) interactions with brain sero- tonin receptors and uptake sites: studies, Soc. Neurosci. Abstr., 12 (1986) 1234. 2 Battaglia, G., Yeh, S., O'Hearn. E., Molliver, M., Kuhar, M. and De Souza, E., 3,4-Methylenedioxy- , and 3,4-methylenedioxyamphetamine destroy terminals in rat brain: quantification of by measurement of pH]-labeled serotonin uptake sites, J, Pharmacol. Exp. Ther., 242 (1987) 911-916. 3 Brown, C. and Osterloh J., Multiple severe complications from recreational ingestion of MDMA ('Ec- stasy'), J. Am. Med. Assoc., 258 (1987) 780-781. 4 Greer, G. and Tolbert, R., Subjective reports of the effects of MDMA in a clinical setting, J. Psychoac- tive Drugs, 18 (1986) 31%327. 5 Heuring, R. and Peroutka, S., Characterization of a novel 3H-5-hydroxytryptamine binding site sub- type in bovine brain membranes, J. Neurosci., 7 (1987) 894-903. 6 Lyon, R., Glennon, R. and Titeler, M., 3,4-Methylenedioxymethamphetamine (MDMA): stereoselec- rive interactions at brain 5-HTj and 5-HT2 receptors, , 88 (1986) 525 526. 7 Lyon, R., Davis, K. and Titeler, M,, 3H-DOB (4-bromo-2,5-dimethoxyphenylisopropylamine) labels a guanyl nucleotide-sensitive state of cortical 5-HT2 receptors, Mol. Pharmacol, 31 (1987) 194-199. 8 Peroutka, S.. Incidence of the recreational use of 3,4-methylenedimethoxymethamphetamine IMDMA; Ecstasy) on an undergraduate campus, New Engl. J. Med., 317 (1987) 1543-1544. 9 Peroutka. S. and Snyder, S., Multiple serotonin receptors: differential binding of _H-5-hydroxytrypta- mine, 3H-lysergic acid diethylamide and 3H-spiroperidol, Mol. Pharmacol., 16 (1979) 687-699. 10 Ricaurte, G., Bryan, G., Strauss, L., Seiden, L. and Schuster. C., Hallucinogenic amphetamine selecti rely destroys brain serotonin nerve terminals, Science, 229 (1985) 986-988. 11 Schmidt, C., Wu, L. and Lovenberg, W.. Methylenedioxymethamphetamine: a potentially neurotoxic amphetamine analogue, Eur. J. Pharmacol., 124 (1986) 175-178. 12 Shulgin, A., drugs: structure activity relationships. In L. lverson. S. lverson and S. Snyder (Eds.), Handbook of Psychopharmacology, Vol. 11, Plenum, New York, 1978, pp. 243-333. 13 Shulgin, A., The background and chemistry of MDMA, J. Psychoactive Drugs, 18(1986) 291 304. 14 Shulgin, A. and Nichols, D., Characterization of three new psychotomimetics. In R. Stillman and R. Willene (Eds.), The PharmacoLogy of Hallucinogens, Pergamon, New York. 1978. pp. 74- 83. 15 Snyder, S., Drugs and the Brain, Freeman, New York, 1986, pp. 179 205. 16 Stone, D., Stahl, D.. Hanson, G. and Gibb. J., The effects of 3,4-methylenediox_methamphetamme IMDMA) and 3,4-methylenedioxyamphetamine (MDA) on systems in the rat brain, Eur. J. Pharmacol., 128 (1986)41-48. 17 Titeler, M., Herrick, K., Lyon, R., McKenney, J. and Glennon, R., ['H]DOB: a specific radioli- gand for 5-HT2 serotonin receptors, Eur. J. Pharmacol., 117 (1985) 145-146. 18 Verebey. K.. Alrazi, J. and Jaffe, J., The complications of 'Ecstasy' (MDMA). J. Am. Med. Assoc., 259 t 1988) 164%1650. 19 Wang, S.. Mathis, C. and Peroutka, S., R(-)-2,5-Dimethoxy-4-7*bromoamphetamine [77Br-Rt - )DOB}: a novel radioligand which labels a 5-HT binding site subtype, Psychopharmacology, 94 (1988) 431 432. MDMAand MDA Cross Reactivity Observed with sensitive for this compound. In fact, the sensitivity of the I Abbott TDx Amphetamine/Methamphetamine Reagents, Abbott antibody with MDA is approximately equal to its i'i. james M. Ramos, Jr. (Dept. of Pathol., Med. College of reactivity with equivalent concentrations of the d- and l- Virginia; current address: 4th Medical Group/SGHL, of amphetamine and methamphetamine. In one Seymour Johnson AFB, NC 27531), Robert L. Fitzgerald reported case of nonfatal overdose with MDMA, concentra- (Dept. of Pharmacol. and Toxicol., Med. College of tions of the drug in urine were 410 p.g/mL i h after Virginia), and Alphonse Poklis (Dept. of Pathol., Med. admission to the hospital and 816 /_g/mL the day after admission (4). In MDA-related deaths, urine concentrations College of Virginia, Virginia Commonwealth University, have ranged from 2 to 175 /zg/mL (5). Clearly, these Box 597 MCV Station, Richmond, VA 23298) concentrations would have yielded positive results with the Abbott Laboratories has recently introduced a fluorescent Abbott reagents, given the data in Table 1. polarization kit for detection of amphetamine Although MDMA was classified as a schedule I controlled and methamphetamine in urine for use on the Abbott TDx. substance in 1985 and MDA was so classified years earlier, i With these reagents, Abbott has successfully eliminated they continue to be widely abused (6) and have been implicated in several deaths (5-7). In addition, MDMA and i_ m0StamiandthenesOcrf OSSsimilarreactivitYcomftepon°uthernodbssWithervedSympathother°mimetiCiMnDA abuse no longer seems to be confined to only certain [_ immunoassays (1). However, we have noted that the TDx geographical areas. Thus we believe that the cross reactivity of the Abbott reagents With these compounds is actually a amphetamine/methamphetamine kit does cross react signif- desirable feature; certainly, the presence of MDMA or MDA icantly with 3,4-methylenedioxymethamphetamine in a sample may be a significant finding. It is important, (MI)MA), also known as "Ecstasy," and 3,4-methylenedi- however, that analysts be aware that a positive result With oxyamphetamine (MDA). A 25-year-old woman was brought to the emergency room Abbott TDx amphetamine/methamphetamine reagents may by her husband, who admitted that his wife had been using actually be due to the presence of MDMA or MDA. ii Ecstasy. During the drug screen for this patient, results of References i an amphetamine/methamphetamine fluorescent polariza- 1. Abbott Laboratories. TDx system assays: amphetamine/meth- tion were positive. GC/MS analysis of an alkaline extract of amphetamine. Abbott Laboratories Diagnostics Division, 03/10/87. t the urine sample (2) confirmed the presence of MDMA and, 2. AggarwalV, Santinga P. GC for drugs of abuse. In: Sunshine I, in addition, ruled out the presence of amphetamine or ed. Methodologyfor analytical toxicology.Cleveland: CRC.Press, i methamphetamine. 1975:448-58. To confirm this suspected cross reactivity of TDx reagents 3. Fitzgerald RL, Blanke RV, Narasimhachari N, Glennon J, with MDMA, we tested, With the Abbott kit, urine from a RosecransJ. Identification of3,4-methylenedioxyamphetamine as a majormetabolite of3,4-methylenedioxyamphetamine in rat plasma rat given 40 mg of MDMA per kilogram, subcutaneously, and urine. NIDA Res Monogr, in press. MDA is reportedly a major metabolite of MDMA in rats and 4. BrownC, Osterloh J. Multiple severecomplications from recre- is suspected to be so in humans as well (3). This rat urine ational ingestion of MDMA ("Ecstasy"). J Am Med Assoc contained both the parent MDMA and its metabolite MDA, 1987;258:780-1. and yielded a "HI" positive With the kit. 5. Baselt RC. Dispositionof toxic drugs and chemicalsin man. 2nd To determine the percent cross reactivity of both MDMA ed. Davis, CA:Biomedical Publications, 1982:521-2. and MDA with the Abbott reagents, we supplemented blank 6. Climko R, Roehrich H, Sweeney D, Al-RaziJ. Ecstasy: a review human urine With MDMA or MDA in the concentrations of MDMAand MDA.Int J Psychiatry Med 1986--87;16:359-67. listed in Table 1, calibrated the instrument, and assayed the 7. DowlingG, McDonoughE, Bost R."Eve" and "Ecstasy": a report samples. Percent cross reactivity was calculated as follows: of fivedeaths associated with the use of MDEAand MDMA.J Am Med Assoc 1987;257:1615-7. (concn. detected by TDx/actual concn of drug) x 100 = percent cross reactivity As Table 1 shows, the percent cross reactivity for MDMA tends to be greater for lower concentrations--the cross- reactivity pattern seen in all TDx drug-of-abuse assays (1). Limitations of a Sensitive Assay for Thyrotropin in ForMDA, however, the cross reactivity is apparently very Managing Patients with , J. A. high at all concentrations, indicating that the assay is very Franklyn, E. G. Black, E. M. Wilson, J. R. E. Davis, and _, M.C. Sheppard (Dept. of Med., Queen Elizabeth .-7'-- Hospital, Edgbaston, Birmingham, B15 2TH, U.K.) · Table 1. Cross Reactivity of MDMA and MDA with Abbott TDx Amphetamine/Methamphetamine Kit a Here we extend our preliminary evaluation of a sensitive _.____ Concn,/*g/mL thyrotropin (TSH) assay (1) in a further study of its value in ': * : Detected by % cross first-line assessment of thyroid function. Added TOx reactivity First, we examined 97 consecutive thyroid-clinic patients Mi?MA suspected of having thyroid disease. We used an immuno- i_:_:_1.36 0.91 66.9 metric method for serum TSH based on enhanced chemilu- '_4.! 10 .. 1.53 13.9 minescence (Amerlite, Amersham International plc, Amer- ,_DA,,'_101.0 2.15 2.1 sham, Bucks, U.K.) (1) and measured free thyroxin (FT4) and free triiodothyronine (FT3) concentrations with Amer- . :'0 54 0.35 64.8 lex-M kits (Amersham). Results of these tests were not _..[__.i:'141'1507 >3.000.81 >66.69.27 known to the managing physician. A diagnosis ofhyperthy- " roidism, hypothyroidism, or euthyroidism was made in each _'_ta:l_leaofh'n = 3 determinations. case on the basis of clinical features and from the results of

, CLINICALCHEMISTRY,Vol.34,:.No._.. ;,1988991