Biochemical investigation of histidinemia in schizophrenic patients

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Biochemical Investigation of Histidinemia in Schizophrenic Patients A. Lucca, M. Catalano, R. Valsasina, C. Fara, and E. Smeraldi

Our results suggest that the association between the clinical diagnosis of schizophremc c:isorder and heterozygosis for histidinemia is not a chance one. The real meaning of this ,elation~hip has to be further investigated.

Introduction We studied plasma and urinary amino acid patterns of 21 sctuzophremc outpatiems and their parents (Catalano et al. 1988) to look for heterozygosifies in an attempt to test the hypothesis that the genetic susceptibility for sc~zophrenic disorder might coinc'~de with heterozygosity for more than one of the loci for aminoacidopathies. Our finding of a greater frequency of hetero- or polybeterozygotes in schizophrenic patients than in the general population seems to support the possibility t~at this kind of association is not a chance one. However, a simple analysis of basal amino acid patterns, even when the parents of each patient are included, is not sufficiently pzecise or conclusive. A normal concentratio~i of a single amino acid car,not exclude per s e a heterozygous condition, as m~ay heterozygotes may have normal values under basal conditions, and display abnorElallti~ o~]y under stress or in disease. Consequently there is a real risk of too many false negatives This is probably because enzymatic activities in heterozygotes for autosomal recessive diseases are reduced to only about 50% of nozmal, and this activity may be sutficient to maintain adequate function under basal conditions (Motulski 1982; Vogel 1982; Vogel ~ ~tppin~: 1982). For ~ese reasons~ we ha:'e given oral ioading tests wi*.h ?urifi¢.d amin9 acids to schizophrenic patients and h,~althy controls. E-w sever~ reasons, the first metal.lie disorder we investigated in this way wzs histidine~. First, in fi~is disease the metJlbolic defect in heterozygotes leads, almost invariably, ~o ~tered plasma concentrations and urinary excretio~t of histid~ne after a single oral load, p,.~viding easy ~ d reliable detection of heterozygotes. Second, in the previous stuay, 3/2i (~.3%) ~f the schizophrenic subj0~cts and their parents had elevated basal plasma levels of histidine. Third, both homczygous and heterozygous subjects for hi~tidinemia mey have mental, cognitive, and speech imp,&nzents (Lott et al. 1970; Neville et al. 1972; Wadman et al. 1967; Wit.~op ~:,d Hynr~, 1963).

From the Chau" m Clinical Psychiatry Ill, Unive~i~'- ~,f Milan, Isfituto S•iendfico Hospital Sa.~ P.=~fa:~e ~A.[., M.C ~E.S.); and the Chair of Clinical Pediatry V, University of Milan Biemedic~ $::~e,lces Institute, Ht,ow~,d Salt PaGlo. '3,.V.~, Milan, I',aly Address repmat requests to Prof. Enrico Smeraldi, H San Raffaele, Dipartimento ,~i S:ienze e Tecn,:,lo~e Biomediche, Clinica Poichiatrica, Via Luigi Prinem 29, 20127 Milano, Italy. Received July 7, 198.7 revised March 6, 1989. f) 1990 Society of Biological Psychiatry

0006-3223/90/$03.56

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Material a n d M e t h o d s

Sample Twenty-four psychiatric patients (19 outpatients and 5 inpatients, 15 men and 9 women), aged 19-43 years (mean 30.0 +- 6.8 so), fulfilling the DSM III-R (APA, 1987) criteria for schizophrenia, were included in this study. Seven of them had already been investigated in the previous study and 1 was a probable beterozygote for histidinenua, l'he others were newly admitted patients. All were medicated with neuroleptic drugs because these do not seem to have significant effects on amino acid pa~erns, even after an exogenous load, as we have repeatedly observed. We also considered some clinical variables such as subtype (according to DSM m-R criteria), age of onset (defined as the patient's age when he or she first fulfilled the DSM III-R criteria for schizophrenia), and positive or negative family history for schizophrenia. A limited number of patients also had a WAIS IQ test (for details see Klonoff et al. 1970), and a smooth pursuit eyes movement (SPEM) evaluation (for details see Scarone et al. 1987). We used 14 fellows of e,ar clinic (8 men and 6 women), aged 24-41 years (mean 28.6 ± 4.5 SD), ~1! without any psychiatric disorder, as the healthy control group. All subjects gave imbrmed consent. Routine analysis excluded any renal or liver diseases.

Procedure We followed the method proposed by Holton et al. (1964), with some modification. All the subjects 0 e . , schizophrenic probands, one of them probably heterozygous for histidinemia, and healthy controls), were fasted overnight and drank 100 ml of water every half hr starting 1 hr before the histidine load (15 g of L-histidine monohydrochloride monohydrate) to 8 hr after. Blood samples were drawn before histidine ingestion (0 time) and at 1-, 2-. 3-, and 4-hr intervals thereafter. Urines were collected at 2-hr hitervals up to 8 hr z#,er He oral load. AE heparinized blood samples were deproteinized by 1:~ 5 (v/v) dilution with a 5% solution of sulfosalicyllc acid containing norleucine and propionic ~acidin known amounts as internal standards. They were then frozen and stored at - 20°(2 until assayed. Immediately before assay, all the after-load samples were diluted 1:10 within lithium citrate buffer (Li + 0.2 N, pH 2.2) to bring the concentrations within the .range of the assay method. Aliquots of the 2-hr urines were processed in the same way af:er measurement of the total volumes. Amino acid analyses were performed by column ion-exchange chromatography, with postcolumn ninhydfir~ derivatizatien, using the KonIron Liquimat III Aminoacid Analyzer. All amino acid concentrations were expressed as mg/100 ml for plasma and as mg/2 hours for urine~ and areas under the peaks were calculated by comparison with a known standard (Pierce Calibration Mixture. 25 nmol/100 VLI) Results for patients and controls were analyzed statistically by SPSS/PC + ~nalys!~ of variance (ANOVA), the Mann-Whitney nonparamelric test, and the i-lierarchic,~l Cluster Analysis subprograms (1986).

Results Plasma Values Table 1 lists the results of ANOVA with the peak time as the dependent variable (as heterozygotes are expect6d to show a higher ~ld more prolonged elevation of plasma histidine levels), with diagnosis (i.e., controls versus schizophrenics) and gender as main effects.

Histidinemia and Schizophrenia

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Table 1. Analysis of Variance in Conuols and Schizophrenics Source of Variation

Sum of Squares

Degrees of Freedom

Mean Square

Covariates 0 time Main effects Diagnosis Gender 2-Way interactions Diagnosis Gender Explained Residual Total

7.350 7.350 8275.972 8275.601 69.318 926.070 926.070 9209.393 46116.923 55326.316

1 1 2 1 ! 1 1 4 33 37

7.350 7.350 4137.986 8275.601 69.318 926.070 926.070 2302.348 1397.483 1495.306

F

Significance of F

0.005 0.005 2.961 5.922 0.050 0.663 0.663 ! .647

0.943 0.943 0.066 0.021 0.825 0.421 0.421 0.186

Peak time is the decadent variable.

Diagnosis had a significant effect (p = 0.021). Moreover, hierarchical cluster analysis of these data without the variable of diagnosis showed the great .importance of peak time as a clustering factor for 4 subjects; a Mann-Whitney test of the same data showed the greatest significant differences in histidine levels be.tween schizophrenics and controls at 180 and 240 rain (p = 0.029 and p = 0.047, respectively). Consequently, we compared all the subjects' individual curves graphically. In this way, we detected two dift%rent clusters: one including healthy controls and 20 schizophrenic subjects, and one including 4 schizophrenic subjects (1 of whom had been previously diagnosed as a probable heterozygote) whose di_agrams were clearly different from those of the other group (Figure 1).

Urine Values We thought it better to analyze the urinary excretion of histidine as absolute amounts o f amino acid per time interval, rather than as mg/lO0 ml of ~,'ine, in order to avoid problems connected with possible interindividual differences in diuresis per hr. In this case, too, the 4 schizophrenic subjects with abnormal plasma values were dearly different from the other subjects, with higher and more prolonged histidine excretion and a maximal difference of 4 hr after the oral load (Figure 2).

Cl~dcal Variables We searched unsuccessfully for a correlation between the clinical variables and the condition of heterozygosity or nonheterozygosity. We found no significant differences in such clinical variables as subtvve, age of onset, family histou,, or M-W test results, between the 6 heterozygous subjc~.'.: (2 from the previous study and 4 from the present one) and the ofiiei schizopmeiiics, although the former group shewed a trend toward earlier age of onset (Table 2). With other relevant variables such as I.Q. ~'ad SPEM, we flso failed to find any items of significance.

Discussion ~'~t~ loading test confir_~,led the suspecte~ he!erozygosi~ for | case. and detected 3 heterozygous subjects who had normal basal plasma values of histidine. We also wish to

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1990;27:69-75

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Figure 1 Plasma levels of histidine after a 15 g load Dven orally (mean ___SEM).

emphasize '.he importance of peak time per se as a clustering factor for the 2 groups ol patients (i.e., heterozygous and nonheterozygous). The finding by both methods of 6 subjects heterozygotic for histidinemia i~. a group of 38 patieuts (!5.8%) is of great importance. As frequencies of homozygotes for histidinemia in genc~i populations ;ange between 1:20000 and !:7400 (?din et al. 198|; I.e,,~j et al. 1974), the e ~ c t e d heterozygote frequencies should range, according to the Hardy-Weinberg equilibrium, t~:,:,een 1.4% and 2.3%, definitely lower than the frequency for our group. This higher frequency suggests that me association between clinical diagnosis and the h~,terozygous condition is not a chance finding. At this point it is hard to say how this all may relate to the pathogenesis of schizophrenic disorders. As we know of no neuropsychological correlates or any other linked marker of this heterozygous condition, we can only theorize about any possible relationship to etiopathogenesis. In spite of that, we think that this type of study may prove to be useful for more accurate diagnoses of conditions that present only phenomenologica! features such as homoge~;eous and reproducible elements, and thus remair~ completely equivocal from a medical-biological point of view. It is very probable that, up to now, we have focused our interest on a heterogeneous group of diseases that present us with a common symptomatology, which makes it difficult to draw any definite etiopathogenetic conclusions. As a consequence, the first step after these preliminary studies should be an accurate investigation of methodological problem~ to promote reliable and simple methods for ,nass screening of large numbers of subjects (e.g., enzyme assays). This might make it possible for us to assess more than one amino acid pattern at the same time, not only • under basal conditions, and to obtain more precise evaluation of possible polyheterozygosities.

Histidinemia and Schizophrenia

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1990,27:69-75

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Figure 2. Urinary excretion of histidir,e after a 15 g ~oad given orally (mean ± SEM).

In fact, the 4 subjects w i ~ abnormal responses in the histidine load~g test are not heterozygous for other aminoacidopathies, whereas in the pre,,inus study, one of the two heterozygotes for histidinen'da was also heterozygeus for hyperorniminemia. Therefore, positivity for heterozygosity to histidinemia does not necessarily imply negative heterozygosity for another amino acid: to avoid false-negatives, loading tests with several amino acids should be done. As it is feasible to perform tests with several amino acids in the same subject, we may be able to clarify this problem, but for the present we think this low frequency of polyheterozygosity has little influence on the initial hypothesis. At the presen t state of our knowledge, we should not rule out the possibility that "d~e condition of a single heterozygote for some aminoacidopathies may constitute either per se or by interaction with other defects with different ways of t;ansmission and phenotypical

Table 2. Clinical Variables in Schizophrenic Patients: R~sult from the Mann-Whitney Test Mean Ranks Variable

Schizo NH (20)

S~ii:,zo SH (6)

p

Significance

Family history Subtype diagnosis Age of onset

12.77 12.23 13.75

11.13 13.88 6.25

0.682 0.682 0.056

n.s. n.s. n.s.

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A. Lucca et al.

expression, a contributing facto., to genetic susceptibility to schizophrenic disorders. The h~-~othesis of polyheterozygosity for aminoacidopathy is also strengthened by the assu~nption that the susceptibility to schizophrenic disorders must be a sum of inborn error~ each with small effects. However, it would certainly be premature to exclude a priori the possibility that increased vulnerability of the CNS could also result from a single e."ror. An inborn error involving metabolic steps crucial to the CNS or to its development might by itself entail consequences that create an increased susceptibility. The WernickeKors',tkoff syndrome is a good example. In this case, a single enzymatic impairment (a genetically determined defect of transketolase) interacts with an environmental factor (thiamine deficiency) to create the clinical disease (Blass 1982).

References Aim J, Holmgren G, Larsson A, Schimpfessel L (1981): Histidinaemia in Sweden: Rep,,~: on a neonatal screening programme. C~in Genet 20:229--233. American Psychiatric Association (1987): Diagnostic and S~atistical Manual of Mental Disorders (ed 3), revised. Washington, DC: APA. Blass JP (1982): Inborn errors, inborn predispositions, and normal variation: Some implications for psychiatric research. In Usdin E, Hanin I (eds), Biological i~iarkers in Psychiatry and Neurology. Oxford, New York, Toronto, Sydney, Paris, Frankfuv" Pergamon Press, pp 473482. Catalano M, Lucca A, Della Maggiore P, Valsasiaa R, Ghezzi R, Smeraldi E (1988): Ti,.e possible relationship between heterozygosity for ammoacidopathies in genetic susceptibility for sehizophrelSr dlsorder~ In Smeraldi E, Bellodi L (eds), A Genetic Perspective for Schizophre~:ic and Related Disorders. Milano: Edi Ermes, pp 67-78. Holton .IB, Lewis FJW, Moore GR (1964): Biochemical investigation of histi "d:_naemJa. J Cl!n Patha', 17:671--675. Klonoff H, Fibiger CH, HuRon GH (1970): Neurops~claoiogical patterns in chrome schizophrenia. J Nerv Ment Dis 150:291-300. Levy HL, Shin VE, Madigan PM (1974): Roatine ne,vborn screening for histidinemia: Clinical ~,~.ndbiochemical results. N Engl J Medl 291:1214-1219. Lott IT, Wheelden JA, l.,c'O/HL (1970): Speech and histidinemia: Methodology and evaluation e.f four cases. Dev Med Child Neurol 12:596-603. Motulski AG (1982): Genetic approaches to common diseases. In Bonn¢-Tamir B, Cohen T, Goodman RM (eds), Human Genetics, Part 2: Medical Aspects. New Yor}:: .Man R. Liss, Inc, pp 89-95. Neville BGR, Bentov ~, ~,, Clayton BE, Sheperd J (1972): Histidinaemia: Study of relation between clinical and biological findings in 7 subjects Arch Dis Child 47:190-200. Scarone S, Garnbini O, HMele E, Bellodi L, Smerald~ E (1987): Neurofunctionai assessment of schizophrenia: n, preliminary investigation of the presence of eye-tracking (SPEMs) and quality estinction test (QET) abnormalities in a sample of schizophrenic patients. Biol Psychology 24:253-259. SPSS/PC+ (1986): SPSSfor the IBM PCIXT/AT. Chicago: SPSS Inc. Vogel F (1982): Biological basis of behavior: Chairman's introductien. In rlonne~-Tamir B, Cchen T, Goodman RM (eds), Human Genetics, Part A: The Unfolding Genorne. New Yo~: Alan R. L ' ~ Inc pp 409--416. Vogel F, Propping i ~ (1982): Genetic variability and its infl,~ence on the risk to sehizophr~ni~,. In

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Theoretical Problems of Modern Psychiatry. International Symposium, Moscow, May I 1-12, 1982. Sandoz, pp 59-74. Wadman SK, Van Sprang FJ, Van Stekelenburg GK, DeBree PK (1967): ~x'hree new cases of histidiaemia. Clinical and biochemical data. Acta Paediatr Scand 56:485-492. Witkop CJ, Henry FV (1963): SjOrgen-Larssonsyndrome and histidinemia: Hereditary biochemical diseases with defects of speech and oral functions. J Speech Hear Disord 28:109-123.