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A simple test for histidinuria and histidinemia
CLINICA CHIMICA ACTA
A SIMPLE
HELEN
TEST
Ii. BERRY
FOR
83
HISTIDINURIA
AND INNA
AND
HISTIDINEMIA
B. PONCET
Children’s Hospital Research Foundation Institute for Developmental Research and the Department of Pediatrics, University ofCincinnati College of Medicine, Cincinnati, Ohio (U.S.A.) (Received
February 9,
1970)
SUMMARY
A screening test for histidinuria and histidinemia is described based on paper chromatographic separation and development with diazotized sulfanilic acid reagent. Mean excretion of histidine by 790 subjects was 210 f 167 pg/ml. Mean histidine concentration in plasma from 127 normal subjects was 0.96 & 0.52 mg/roo ml. Histidine determinations in plasma by paper chromatography and by amino acid analyzer showed a high correlation. Measurement of urinary histidine and detection of histidine metabolites, imidazole lactic acid and imidazole pyruvic acid, permit differentiation of histidinuria associated with histidinemia and histidinuria associated with pathological aminoaciduria.
INTRODUCTION
Histidine is one of the most prominent amino acids found in normal human urine specimens. Elevated excretions of histidine and its metabolites in the urine and elevation of histidine in blood are characteristic features of histidinemia132. The reaction of imidazole pyruvic acid, a metabolite of histidine, with ferric chloride to produce a blue-green color first led to recognition of the inborn error of metabolism. Most of the patients described in the literature were found as a result of screening studies carried out for detection of phenylketonuria. We recently had the opportunity to study a family in which seven members in two generations had histidinuria and histidinemia 3. The reaction with ferric chloride was not positive in all specimens from these individuals and some would have been missed without a separate test for histidine. We have used a simple paper chromatographic screening test for histidinuria and histidinemia based on reaction of imidazole compounds with diazotized sulfanilic acid-potassium carbonate reagent. Data are presented on histidine excretion by 790 subjects including normal individuals and patients with generalized aminoaciduria and on plasma histidine levels in 127 normal subjects. Data obtained from testing patients with histidinemia are shown. Clin. Chim. Acta, 29 (1970) 83-88
84
BERRY,PONCET
EXPERIMENTALPROCEDURE
For most specimens 5 ~1 of urine was used for histidine determination. Urine spots were placed 3 cm apart on Whatman No. I filter paper II inches in height. Histidine standards ranging from 0.1 to 2.5 pg per spot were chromatographed at the same time. The chromatograms were run overnight in a solvent mixture composed of 80 ml butanol, 40 ml 2 N HCl, and 20 ml ethanol. After thorough drying the chromatograms were sprayed with a mixture of equal parts of diazotized sulfanilic acid and 10% potassium carbonate. Histidine appeared as a red spot at RF 0.15, imidazole acetic acid at RF 0.37, and imidazole lactic acid at RF 0.32. Quantitative determinations were made by measuring the density of the spots with a Model 525 photovolt densitometer fitted with a 545-m,u filter and a 4-mm circular aperture. The area of each spot was also measured using a planimeter and the product of area times density for known amounts of histidine was used to prepare a standard curve. Specimens with histidine concentration greater than 500 pg/ml required dilution to bring the concentration into the appropriate range. Plasma or serum was deproteinized with 4 vol. of 95% ethanol and the supernatant solution was used to prepare chromatograms as described for urine. Volumes of the diluted supernatant ranging from 25 to IOO,ul, equivalent to 5 to 20 ,ul of plasma or serum were used for blood histidine determinations. The lower limit for plasma histidine using these volumes was 0.5 mg/roo ml. When lower concentrations of histidine were present as in many normal specimens a miniature ion-exchange column of Dowex 50 was used for deproteinization4. Plasma was passed over the column containing approximately 600 mg of resin previously treated with 2 N HCl. Amino acids were retained on the resin while proteins, electrolytes, sugars and other plasma constituents passed through. The amino acids were eluted in a batch using a 2 N solution of triethylamine in 20% acetone. The eluting solvent was evaporated and the residue taken up in 115 the original volume with distilled water. For chromatography 25 ,ul of the concentrate (corresponding to 0.125 ml plasma) was used as described for urine. During the latter part of the study a Beckman Spinco Amino Acid Analyzer Model 12oC became available. For histidine determination IOO ,ul of the concentrate was aplied to the amino acid analyzer. Urine specimens received for routine amino acid determinations from July, 1967, through June, 1968, were analyzed for histidine concentrations. Subjects included infants and children hospitalized with a variety of illnesses and children seen in outpatient clinics for evaluation of mental retardation or speech retardation. From some subjects blood was obtained at the same time for routine determination of amino acids. Urine and blood specimens obtained from the members of the family with histidinemia were also tested. RESULTS
The excretion of histidine by 790 subjects is shown in Table I. Median excretion was 174 pg/ml. 90% of the subjects excreted less than 500 ,ug/ml. To avoid weighting the mean with the specimens from histidinemia patients, the upper 2% of values were excluded from the calculation. Mean excretion was 210 + 167 ,ug/ml. The results of plasma histidine determinations carried out on 127 specimens received for routine Clin. Chim. Acta, zg (1970) 83-88
SIMPLE TEST FOR HISTIDINURIA TABLE
AND HISTIDINEMIA
I
DISTRIBUTION
OF URINARY
Concn. (pguglml) O-25 26-50
HISTIDINE
301-32.5 326-350
24 21
Total number Mean Standard deviation
790 2Iopg/ml
Percentiles
Concn. (pg/ml)
126-150 151-17.5 176-200 201-225 226-250
36 82
50 75 90 95
I74 309 492 607
TABLE
17 =4 7 3 4 16
701-750 751-800 #So1
167
25
IO
I9 19 ‘4 13 I4 6 16
351-375 376-400 401-425 426-450 451-475 476-500 501-550 55 I-600 601-650 651-700
251-275 276-300
51-75 76-100
Number
Number 49 70 64 52 57 59 4.5 40 50 34 33 28
101-125
EXCRETION
II
DISTRIBUTION
OF PLASMA
HISTIDINE
CONCENTRATIONS
IN NORMAL
1NDIvIDUAI.S
Number
Concn.
0.01-0.215 0.26-0.50
I 18
1.26-1.50 1.51-1.75
17 6
0.51-0.75 0.76-1.00
24 27
1.76-2.00 2.01-2.25
3 I
1.01-1.25
28
2.26-2.50
2
(mgl~oo ml)
Concn.
Total
number
Mean Standard
deviation
Percentiles
127 0.96
ml)
Number
ml
0.52
Golacn. (rngl~oo ml)
25
0.40 0.63
50 (median) 75
0.93 I.22
90 95
I.35 I.71
IO
mg/Ioo
(mElI
amino acid analysis are shown in Table II. Mean concentration was 0.96 mg/roo ml. A much narrower range for histidine was observed in plasma than in urine. Only 6 specimens (5%) showed histidine concentrations greater than 1.73 mg/roo ml. Results of histidine measured using paper chromatography and the Amino Acid Analyzer in 15 normal and 4 histidinemic subjects are compared in Table III. Plasma
G&k.CMm.
A&,
29 (1970) 83-88
BERRY, PONCET
56
was deproteinized using the ion-exchange column and concentrated as described for these determinations. Pearson product moment correlation using only values in the normal range was 0.92. If values for histidinemia patients are included, the correlation is 0.99. Histidine concentration in plasma and urine of selected patients with histidine excretion in excess of 500 ,ug/ml are shown in Fig. I together with data from members of the family with histidinemia. It was not possible to obtain blood from all patients with elevated histidine excretion. Elevated plasma hi&dine levels, ranging from 2.0 to 19.5 mg/xoo ml were found only in individuals from the family with histidinemia. One member of the histidillemic family had elevated urinary histidine in the presence of normal plasma levels. Two members of the family in whom urinary histidine excretions were below 500,ug/ml had plasma histidine levels of z mg/roo ml, approximately twice the normal mean. These three individuals and an additional family member whose plasma histidine was 4 mg/roo ml excreted imidazoIe lactic acid, but their urine specimens were negative for imidazole pyruvic acid using Phenistix or ferric chloride. Members of the histidinemic family who had plasma histidine concentrations over 6 mg/Ioo ml excreted imidazole lactic acid and in addition showed positive tests for imidazole pyruvic acid. Imidazole lactic acid was not detected in specimens from patients with normal plasma histidine concentration in spite of the marked elevation of urinary histidine in some instances. Among the patients with urinary histidine excretions over 5oo pg/ml and normal plasma histidine concentrations were three with Lowe’s disease, four with Hartnup disease, three with renal tubular disorders resembling Fanconi syndrome and two with untreated galactosemia. It was not surprising to find elevated histidine excretions in patients with gross generalized aminoacidurias such as these. Among the high histidine excretors were also a number of patients with TABLE
III
COMPARISOII‘OF HISTIDINE AXD
BECKMAN
SPINCO
DETERMINATIONS
AMINO
Number
ACID
Concn. (mglIo0 Paper
Normal
individltals 0.68
0.49 0.65 0.31 1.12 ‘-74 0.56 0.56 1.24 0.93 I.02 0.65 I.55 1.27 1.05 8.20 7.82
5.65 6.08 C&n. Chim.
Acta.
29
(1970)
83-88
IN
BLOOD
BY
PAPER
CHROMATOGRAPHY
ANALYZER
ml)
chromatograph>
A milao acid analyses 0.78 0.47 0.71
0.40 1.24 I.61
0.53 0.65 1.02
1.15 0.81 0.56 1.15
1.30 I.05
8.22
7.32 5.13 6.27
SIMPLETEST FOR HISTIDINURIAAND HISTIDINEMIA
1600 t
A
600 A 400 I
I
A
A 2
4
6 SERUM
8
IO
HISTIOINE
12
14
16
18
,
20
- mg/lOOml
Fig. I. Relation of urinary histidinc to serum histidine concentrations in normal individuals and members of a family with histidincmia.
anemia, with severe seizures, and with degenerative diseases of the central nervous system. The latter is suggestive of the imidazoleaminoaciduria described in cerebromacular degeneration by Bessman and Raldwin 6. There was no apparent relation of histidine excretion to age in the group studied, and particularly no evidence of histjdinuria in infants as a reflection of renal immaturity. DISCUSSION
Most of the chemical tests described for histidine determination are non-specific. The cuprizone method used by Gerber and Gerber& in a screening test for histidinu~a reacts both with the methyl histidines and histidine, resulting in many false positive tests. The procedure described by Baldridge and Greenberg’ based on absorption of the enol-borate complex produced when histidine reacts with L-amino acid oxidase has the required specificity, but is expensive and time consuming for use in a screening method. By combining the reaction of imidazole compounds with diazotized sulfanilic acid (Pauly reagent) and paper chromatographic separation, a simple screening procedure for histidinuria was obtained. The methylated histidines, often present in large amounts in urine following ingestion of meat, do not interfere. The simultaneous
detection of imidazole lactic acid in specimens with elevated histidine may indicate that blood histidine levels have risen as a result of a block in metabolism of histidine.
Clin.
Chim.
Acta,
29 (1970)
83-88
88
BERRY,
PONCET
ACKNOWLEDGMENT
This work was supported in part by Grant HDoo324 from the National Institute of Child Health and Human Development, U.S. Public Health Service. REFERENCES
I H. GHADIMI,M.W.PARTINGTON AND A.HuNTER,N~~E~~I. J. Med.,265(1961)221. 2 V.H.AUERBACH,A.M.DIGEORGE,R.C.BALDRIDGE,C.D.TOURTELI.OTTEANDM.P.BRIGHAM, J. Pediat., 60 (1962) 487. 3 C. BRUCKMAN,H. K.BERRY
AND R.J.DASENBROCK, Am.J.DiseasesChiZdren, 4 C. K. HARRIS,E. TIGANE AND C. S. HANES,&Z~. J. Physiol., 3g (1961) 439. 5 S. P. BESSMAN AND R. BALDWIN, Science, 135 (1962)789. 6 M. G. GERBER AND D. A. GERBER, Pediatrics, 43 (1969) 40. 7 R. C. BALDRIDGE AND N. GREENBERG, J. Lab.Clin. Med., 61 (1963) 700. Clin.Chim.
Acta,
zg (1970)
83-88
119(1970)221.
A SIMPLE
HELEN
TEST
Ii. BERRY
FOR
83
HISTIDINURIA
AND INNA
AND
HISTIDINEMIA
B. PONCET
Children’s Hospital Research Foundation Institute for Developmental Research and the Department of Pediatrics, University ofCincinnati College of Medicine, Cincinnati, Ohio (U.S.A.) (Received
February 9,
1970)
SUMMARY
A screening test for histidinuria and histidinemia is described based on paper chromatographic separation and development with diazotized sulfanilic acid reagent. Mean excretion of histidine by 790 subjects was 210 f 167 pg/ml. Mean histidine concentration in plasma from 127 normal subjects was 0.96 & 0.52 mg/roo ml. Histidine determinations in plasma by paper chromatography and by amino acid analyzer showed a high correlation. Measurement of urinary histidine and detection of histidine metabolites, imidazole lactic acid and imidazole pyruvic acid, permit differentiation of histidinuria associated with histidinemia and histidinuria associated with pathological aminoaciduria.
INTRODUCTION
Histidine is one of the most prominent amino acids found in normal human urine specimens. Elevated excretions of histidine and its metabolites in the urine and elevation of histidine in blood are characteristic features of histidinemia132. The reaction of imidazole pyruvic acid, a metabolite of histidine, with ferric chloride to produce a blue-green color first led to recognition of the inborn error of metabolism. Most of the patients described in the literature were found as a result of screening studies carried out for detection of phenylketonuria. We recently had the opportunity to study a family in which seven members in two generations had histidinuria and histidinemia 3. The reaction with ferric chloride was not positive in all specimens from these individuals and some would have been missed without a separate test for histidine. We have used a simple paper chromatographic screening test for histidinuria and histidinemia based on reaction of imidazole compounds with diazotized sulfanilic acid-potassium carbonate reagent. Data are presented on histidine excretion by 790 subjects including normal individuals and patients with generalized aminoaciduria and on plasma histidine levels in 127 normal subjects. Data obtained from testing patients with histidinemia are shown. Clin. Chim. Acta, 29 (1970) 83-88
84
BERRY,PONCET
EXPERIMENTALPROCEDURE
For most specimens 5 ~1 of urine was used for histidine determination. Urine spots were placed 3 cm apart on Whatman No. I filter paper II inches in height. Histidine standards ranging from 0.1 to 2.5 pg per spot were chromatographed at the same time. The chromatograms were run overnight in a solvent mixture composed of 80 ml butanol, 40 ml 2 N HCl, and 20 ml ethanol. After thorough drying the chromatograms were sprayed with a mixture of equal parts of diazotized sulfanilic acid and 10% potassium carbonate. Histidine appeared as a red spot at RF 0.15, imidazole acetic acid at RF 0.37, and imidazole lactic acid at RF 0.32. Quantitative determinations were made by measuring the density of the spots with a Model 525 photovolt densitometer fitted with a 545-m,u filter and a 4-mm circular aperture. The area of each spot was also measured using a planimeter and the product of area times density for known amounts of histidine was used to prepare a standard curve. Specimens with histidine concentration greater than 500 pg/ml required dilution to bring the concentration into the appropriate range. Plasma or serum was deproteinized with 4 vol. of 95% ethanol and the supernatant solution was used to prepare chromatograms as described for urine. Volumes of the diluted supernatant ranging from 25 to IOO,ul, equivalent to 5 to 20 ,ul of plasma or serum were used for blood histidine determinations. The lower limit for plasma histidine using these volumes was 0.5 mg/roo ml. When lower concentrations of histidine were present as in many normal specimens a miniature ion-exchange column of Dowex 50 was used for deproteinization4. Plasma was passed over the column containing approximately 600 mg of resin previously treated with 2 N HCl. Amino acids were retained on the resin while proteins, electrolytes, sugars and other plasma constituents passed through. The amino acids were eluted in a batch using a 2 N solution of triethylamine in 20% acetone. The eluting solvent was evaporated and the residue taken up in 115 the original volume with distilled water. For chromatography 25 ,ul of the concentrate (corresponding to 0.125 ml plasma) was used as described for urine. During the latter part of the study a Beckman Spinco Amino Acid Analyzer Model 12oC became available. For histidine determination IOO ,ul of the concentrate was aplied to the amino acid analyzer. Urine specimens received for routine amino acid determinations from July, 1967, through June, 1968, were analyzed for histidine concentrations. Subjects included infants and children hospitalized with a variety of illnesses and children seen in outpatient clinics for evaluation of mental retardation or speech retardation. From some subjects blood was obtained at the same time for routine determination of amino acids. Urine and blood specimens obtained from the members of the family with histidinemia were also tested. RESULTS
The excretion of histidine by 790 subjects is shown in Table I. Median excretion was 174 pg/ml. 90% of the subjects excreted less than 500 ,ug/ml. To avoid weighting the mean with the specimens from histidinemia patients, the upper 2% of values were excluded from the calculation. Mean excretion was 210 + 167 ,ug/ml. The results of plasma histidine determinations carried out on 127 specimens received for routine Clin. Chim. Acta, zg (1970) 83-88
SIMPLE TEST FOR HISTIDINURIA TABLE
AND HISTIDINEMIA
I
DISTRIBUTION
OF URINARY
Concn. (pguglml) O-25 26-50
HISTIDINE
301-32.5 326-350
24 21
Total number Mean Standard deviation
790 2Iopg/ml
Percentiles
Concn. (pg/ml)
126-150 151-17.5 176-200 201-225 226-250
36 82
50 75 90 95
I74 309 492 607
TABLE
17 =4 7 3 4 16
701-750 751-800 #So1
167
25
IO
I9 19 ‘4 13 I4 6 16
351-375 376-400 401-425 426-450 451-475 476-500 501-550 55 I-600 601-650 651-700
251-275 276-300
51-75 76-100
Number
Number 49 70 64 52 57 59 4.5 40 50 34 33 28
101-125
EXCRETION
II
DISTRIBUTION
OF PLASMA
HISTIDINE
CONCENTRATIONS
IN NORMAL
1NDIvIDUAI.S
Number
Concn.
0.01-0.215 0.26-0.50
I 18
1.26-1.50 1.51-1.75
17 6
0.51-0.75 0.76-1.00
24 27
1.76-2.00 2.01-2.25
3 I
1.01-1.25
28
2.26-2.50
2
(mgl~oo ml)
Concn.
Total
number
Mean Standard
deviation
Percentiles
127 0.96
ml)
Number
ml
0.52
Golacn. (rngl~oo ml)
25
0.40 0.63
50 (median) 75
0.93 I.22
90 95
I.35 I.71
IO
mg/Ioo
(mElI
amino acid analysis are shown in Table II. Mean concentration was 0.96 mg/roo ml. A much narrower range for histidine was observed in plasma than in urine. Only 6 specimens (5%) showed histidine concentrations greater than 1.73 mg/roo ml. Results of histidine measured using paper chromatography and the Amino Acid Analyzer in 15 normal and 4 histidinemic subjects are compared in Table III. Plasma
G&k.CMm.
A&,
29 (1970) 83-88
BERRY, PONCET
56
was deproteinized using the ion-exchange column and concentrated as described for these determinations. Pearson product moment correlation using only values in the normal range was 0.92. If values for histidinemia patients are included, the correlation is 0.99. Histidine concentration in plasma and urine of selected patients with histidine excretion in excess of 500 ,ug/ml are shown in Fig. I together with data from members of the family with histidinemia. It was not possible to obtain blood from all patients with elevated histidine excretion. Elevated plasma hi&dine levels, ranging from 2.0 to 19.5 mg/xoo ml were found only in individuals from the family with histidinemia. One member of the histidillemic family had elevated urinary histidine in the presence of normal plasma levels. Two members of the family in whom urinary histidine excretions were below 500,ug/ml had plasma histidine levels of z mg/roo ml, approximately twice the normal mean. These three individuals and an additional family member whose plasma histidine was 4 mg/roo ml excreted imidazoIe lactic acid, but their urine specimens were negative for imidazole pyruvic acid using Phenistix or ferric chloride. Members of the histidinemic family who had plasma histidine concentrations over 6 mg/Ioo ml excreted imidazole lactic acid and in addition showed positive tests for imidazole pyruvic acid. Imidazole lactic acid was not detected in specimens from patients with normal plasma histidine concentration in spite of the marked elevation of urinary histidine in some instances. Among the patients with urinary histidine excretions over 5oo pg/ml and normal plasma histidine concentrations were three with Lowe’s disease, four with Hartnup disease, three with renal tubular disorders resembling Fanconi syndrome and two with untreated galactosemia. It was not surprising to find elevated histidine excretions in patients with gross generalized aminoacidurias such as these. Among the high histidine excretors were also a number of patients with TABLE
III
COMPARISOII‘OF HISTIDINE AXD
BECKMAN
SPINCO
DETERMINATIONS
AMINO
Number
ACID
Concn. (mglIo0 Paper
Normal
individltals 0.68
0.49 0.65 0.31 1.12 ‘-74 0.56 0.56 1.24 0.93 I.02 0.65 I.55 1.27 1.05 8.20 7.82
5.65 6.08 C&n. Chim.
Acta.
29
(1970)
83-88
IN
BLOOD
BY
PAPER
CHROMATOGRAPHY
ANALYZER
ml)
chromatograph>
A milao acid analyses 0.78 0.47 0.71
0.40 1.24 I.61
0.53 0.65 1.02
1.15 0.81 0.56 1.15
1.30 I.05
8.22
7.32 5.13 6.27
SIMPLETEST FOR HISTIDINURIAAND HISTIDINEMIA
1600 t
A
600 A 400 I
I
A
A 2
4
6 SERUM
8
IO
HISTIOINE
12
14
16
18
,
20
- mg/lOOml
Fig. I. Relation of urinary histidinc to serum histidine concentrations in normal individuals and members of a family with histidincmia.
anemia, with severe seizures, and with degenerative diseases of the central nervous system. The latter is suggestive of the imidazoleaminoaciduria described in cerebromacular degeneration by Bessman and Raldwin 6. There was no apparent relation of histidine excretion to age in the group studied, and particularly no evidence of histjdinuria in infants as a reflection of renal immaturity. DISCUSSION
Most of the chemical tests described for histidine determination are non-specific. The cuprizone method used by Gerber and Gerber& in a screening test for histidinu~a reacts both with the methyl histidines and histidine, resulting in many false positive tests. The procedure described by Baldridge and Greenberg’ based on absorption of the enol-borate complex produced when histidine reacts with L-amino acid oxidase has the required specificity, but is expensive and time consuming for use in a screening method. By combining the reaction of imidazole compounds with diazotized sulfanilic acid (Pauly reagent) and paper chromatographic separation, a simple screening procedure for histidinuria was obtained. The methylated histidines, often present in large amounts in urine following ingestion of meat, do not interfere. The simultaneous
detection of imidazole lactic acid in specimens with elevated histidine may indicate that blood histidine levels have risen as a result of a block in metabolism of histidine.
Clin.
Chim.
Acta,
29 (1970)
83-88
88
BERRY,
PONCET
ACKNOWLEDGMENT
This work was supported in part by Grant HDoo324 from the National Institute of Child Health and Human Development, U.S. Public Health Service. REFERENCES
I H. GHADIMI,M.W.PARTINGTON AND A.HuNTER,N~~E~~I. J. Med.,265(1961)221. 2 V.H.AUERBACH,A.M.DIGEORGE,R.C.BALDRIDGE,C.D.TOURTELI.OTTEANDM.P.BRIGHAM, J. Pediat., 60 (1962) 487. 3 C. BRUCKMAN,H. K.BERRY
AND R.J.DASENBROCK, Am.J.DiseasesChiZdren, 4 C. K. HARRIS,E. TIGANE AND C. S. HANES,&Z~. J. Physiol., 3g (1961) 439. 5 S. P. BESSMAN AND R. BALDWIN, Science, 135 (1962)789. 6 M. G. GERBER AND D. A. GERBER, Pediatrics, 43 (1969) 40. 7 R. C. BALDRIDGE AND N. GREENBERG, J. Lab.Clin. Med., 61 (1963) 700. Clin.Chim.
Acta,
zg (1970)
83-88
119(1970)221.