Arch Dis Child: first published as 10.1136/adc.49.7.545 on 1 July 1974. Downloaded from Archives of Disease in Childhood, 1974, 49, 545. Histidinaemia in mouse and man GRAHAME BULFIELD and HENRIK KACSER From the Department of Genetics, University of Edinburgh Bulfield, G., and Kacser, H. (1974). Archives of Disease in Childhood, 49, 545. Histidinaemia in mouse and man. A recently discovered mutant in the mouse was found to have very low levels of histidase. It is an autosomal recessive. In its enzymic and metabolic properties it appears to be a homologue ofhuman histidinaemia. While the homozygous mouse mutants show no overt abnormalities, offspring of histidinaemic mothers display a balance defect resulting in circling behaviour. This is associated with vestibular damage during in utero development. Mental retardation caused by human maternal phenylketonuria may have a similar aetiology. There are well recognized advantages in studying histidine and its derivatives. The enzymic and animal models in the investigation of human metabolic changes in both mouse and human diseases. The increasing number of disorders in mutants appear to be the same. In addition, man which are reported to have a genetic basis maternal histidinaemia in the mouse has a terato- (McKusick, 1971; Raine, 1972) makes it desirable to genic effect on the developing inner ear which results find and investigate animal homologues with the in abnormal behaviour of the affected offspring. same genic lesions. The genetically most in- These findings may be relevant to the aetiology of vestigated laboratory mammal is the mouse, with human histidinaemia. over 500 known mutants (Green, 1966; Searle, 1973). Some of these mouse mutants show disorders similar to human syndromes such as Genetic and metabolic characterization Histidinaemic mice have high levels ofhistidine in maturity-onset diabetes (Mayer, 1960), the pituitary http://adc.bmj.com/ dwarf (Elftman and Wegelius, 1959), and Chediak- urine, liver, plasma, brain, and skin, and high Higashi syndrome (Padgett et al., 1967). Only a concentrations of imidazole derivatives in urine and few, however, have been shown to be enzymically more moderate ones in liver. Table I summarizes identical in the two species. These are albinism the quantitative observations. (tyrosinase: Coleman, 1962), acatalasaemia (catalase: Any of the metabolite pools in all the tissues Feinstein, 1970), and glycogen storage disease type studied gave a consistent nonoverlapping difference VIa (phosphorylase kinase: Huijing, 1970). between 'normal' and 'high' values. Classification Most of the classical inborn errors of metabolism of histidinaemic individuals is therefore possible on on September 30, 2021 by guest. Protected copyright. have therefore yet to be shown in a mouse mutant. any one of the tissue determinations. A further One of the reasons for this may be the lack of a consequence of low histidase activity is a reduced concerted effort to screen the relevant biochemical concentration of the product of the reaction, i.e. parameters. In an attempt to produce such urocanic acid. This is evident in the skin, as seen in evidence, a project was established based on Table II, which shows the comparison of the two automated column chromatography and automated classes in the skin of 7-day-old mice. enzyme assays. The first major abnormality Considerable variation was found in the absolute detected by this programme-histidinaemia-is histidine levels within the high (histidinaemic) discussed here. group. This variation, however, was in general In brief, the histidinaemia displayed in the mouse reflected in all the tissues of a single animal. Fig. is caused by an autosomal recessive gene (designated 1-3 show these relations. It should be pointed out his) resulting in low histidase activity with less than that the plasma, liver, and brain values represent the 5% of normal (Kacser, Bulfield, and Wallace, 1973). metabolites at the same time point, while urines As a consequence, there are large accumulations of were collected days (and sometimes weeks) before sacrifice of the animal. This may account for the Received 15 November 1973. poorer correlation in Fig. 3. 545 Arch Dis Child: first published as 10.1136/adc.49.7.545 on 1 July 1974. Downloaded from 546 Bulfield and Kacser TABLE I Biochemical characterization. Mean values at 7 weeks Tissue Histidinaemic Normal R = H/N Liver Histidase* 0*013 0 *280 0*046 Histidine 20 *9 0 *880 23 *7 Ac histidine + Im pyruvatet 0*111 0 *0082 13 *5 Im lactate 0*102 0*0069 14-8 Im acetate 0*017 <0*0044 > 3*7 Urine Histidine 0*86 0*04 21*5 Ac histidine + Im pyruvatet 3*19 0*11 29 *0 Im lactate 1*21 0 *07 17*3 Im acetate 1*29 0*05 25 *8 Plasma Histidine 3*30 0*115 28 *7 Ac histidine > 0 I + Im pyruvatet 0-016 <0-0008 20 0 Plasma (,umol /ml) Im lactate 0*012 <0*001 > 12*0 Im acetate 0*008 <0*001 >8*0 FIG. 1.-Relation of plasma histidine to liver histidine. Brain The values ranging from 2 to 5 pmol/ml plasma represent Histidine 2*16 0*169 12 *8 individual histidinaemic animals. The cluster of low values Ac histidine is representative of the range for normal animals. + Im pyruvatet 0*0044 <0*0006 >7*3 Im lactate 0*0204 < 0 * 0007 >29*1 Im acetate <0 004 < 0 0006 - 0S Note: All values are the means of between 5-10 animals. Liver and brain metabolites: imol/g wet tissue. Plasma and urine 25 0 metabolites: ,tmol/ml. All determinations by column chromato- graphy and ninhydrin or Pauly reaction, respectively. was determined the 0 *Histidase (E.C. 4.3.1.3) activity by I so appearance of urocanic acid at 277 nm. The assay system was 65 L 0~~~~ http://adc.bmj.com/ mmol/l. phosphate buffer pH 9 4, 5 mmol/l. reduced glutathione, 133 @0 mmol/l. L-histidine HC1, 005 ml enzyme, final volumn 1 ml. The 20 liver was extracted in a Tri-R homogenizer 3:1 v/w with a solution of i- 1- s 0 25 mol/l. sucrose, 1 mmol/l. EDTA sodium salt, 1 mmol/l. E * 0 dithiothreitol, 50 mmol/l. tris-HCl (pH 7 4). The homogenate was -. centrifuged at 60,000gav and the supernatant used to assay histidase 10 as g±mol urocanate min7l g-1 at 30 °C. tIn our buffer system these two substances are not completely I*II/ resolved. The values reported are the sum of both. Im, imidazole. 05 I Matings between histidinaemic animals produce S on September 30, 2021 by guest. Protected copyright. exclusively histidinaemic offspring. Matings 0 2 3 s between known heterozygotes give an unambiguous Plasma (,,mol/ml) 3:1 ratio of normals to histidinaemics (158 51; FIG. 2.-Relation of plasma histidine to brain histidine. X2 = 0 0399). From this it is evident that the his Animals as for Fig. 1. TABLE II Histidine/urocanate relations in skin of littermates at 7 days of age Note: Histidine and urocanate were determined by column chromatography and Pauly reaction. They are expressed as ,mol/g wet tissue. Histidase activity in ,imol urocanate/min per g at 30 'C. The activities of the normals at 7 days are about 1/5 of the adult (7-week) values. Arch Dis Child: first published as 10.1136/adc.49.7.545 on 1 July 1974. Downloaded from Histidinaemia in mouse and man 547 values are overlapping, there is a small but . significant difference in mean values between homozygous 1- 2 ] normals and heterozygotes. Loading tests can distinguish the two genotypes. Addition of 6 0o histidine to the normal mouse diet (Dixon's -E FFG/LAC) when given to heterozygotes for 4 days --0- E raises the urine histidine and imidazoles to levels equal to, or above, those ofhistidinaemics. Normal oeX 0 * homozygotes show little or no rise. 044- In many human recessive metabolic disorders, a 0 normal substrate concentration in heterozygotes is . 4 associated with approximately half the enzyme 0C activity compared with the normal homozygotes (Harris, 1970). This is also the case with histidase O 21 3 4 Plosma ,Usmol/ml activity in mouse liver. As expected, there are three classes of activity which segregate 1 :2:1 from FIG. 3.-Relation of plasma histidine to urine histidine. crosses, Animals as for Fig. 1. heterozygous with the intermediate class having values about half-way between the other two. All known heterozygotes had enzyme activities in allele is recessive with complete penetrance and that this intermediate range, though the male the histidine values of fall heterozygous carriers distributions showed some overlap. Table III within the normal range. This range is from 0 5 to * shows the enzyme activities of a number of males 1 - I.umol/g liver and contains animals of the two and females matched for age, with their associated genotypes, homozygous normals and heterozygotes. histidine levels. This shows that almost complete These two genotypes were produced separately by dominance at the histidine level is accompanied by and their histidine values were appropriate matings intermediacy at the enzyme level. The small but determined (Fig. 4). This shows that, though the significantly higher heterozygote levels show that there is no 'threshold' at one-half the enzyme concentration. This observation is in agreement Homozygous Heterozygous with general considerations of the nature of multi- enzyme systems (Kacser and Burns, 1973). http://adc.bmj.com/ 0 Comparison with human data E The plasma concentrations of 51 and 1 *77 mg/100 ml for the two mouse phenotypes, respectively, can be directly compared to the ranges reported for 05Ii Ob 0.7 08 0*9 10 human subjects of 5 to 25 and 0 4-3 0 mg/100 ml.