- Email: [email protected]
A simple rapid method for the determination of α amino acids in urine
CLINICACHIMICAACTA
A SIMPLE RAPID
27
METHOD FOR THE DETERMINATION
OF a AMINO
ACIDS IN URINE M. G. WELLS Biochemistry
Laboratory, Edgeware Geneva1 Hospital, Edgeware, Middx. (U.K.)
(Received December20, 1968)
SUMMARY A simple rapid method is described for the determination of total a amino acids in urine without the use of ion exchange resins, based on the chelation of cc amino acids by cupric ionslps and subsequent estimation by production of the highly specific and stable yellow complex with 2,9-dimethyl-r,ro-phenanthroline (“Neocuproine”). “Normal” ranges are provided from a study of 125 subjects. The method has been found of particular value for screening urines prior to chromatographic and other techniques.
REAGENTS chloride 0.03 M (2) Disodiz~m hydrogen orthophosphate bufler 0.1 M. Add 0.5 ml of chloroform per litre and shake well, and store at 4’. (3) Reducing solutiofi. 34 g of “A.R.” hydroxyammonium chloride and 50 g of “A.R.” sodium citrate dihydrate per 500 ml in distilled water. If high “blank” values are observed due to the presence of cuprous impurities in this reagent then these may be removed by the addition of 2 vol. of neocuproine per IOO vol. of reagent and extracting the complex formed into chloroform in a separating funnel. This step is usually unnecessary if high quality reagents are used. (4) z,g-Dimethyl-r,ro-phenantlzroline (“Neocuproine”). 0.1 g of a,g-dimethylr,ro-phenanthroline per IOO ml in ethanol. It should be noted that industrial spirit containing pyridine causes a low degree of colour development and should be avoided. (5) Stock glycilze standard 2 N. 150.14 g of glycine and 0.2 g of phenyl mercuric acetate per litre in distilled water. (6) Working glycine stalzdard. I ml of stock standard per roe ml distilled water. This solution contains 20 mequiv. glycine per litre (280 mg CCamino acid N perlitre). (I) Cupric
METHOD To 0.5 ml of urine add 0.5 ml of cupric chloride, agitate the tubes then add 9 ml Clin. Chim. Acta, 25 (1969)
27-29
WELLS
28
of buffer, mix by inversion and allow the tubes to stand for 15 min at room temperature, then centrifuge hard. To 1.0 ml of supernatant add 7 ml of reducing solution, z ml of neocuproine solution, mix well and stand for at least 3 min to allow the bubbles to clear and the colour to stabilize; then read the absorbance of the solution at 457 rnp or suitable violet filter, e.g. Ilford 601. The colour now has good stability for many hours. 0.5 ml of working standard or water may be treated in the samemanner as urine in order to produce the standard and blank respectively. Tests of above 50 mequiv. a amino acid per litre should be repeated on diluted urine unless differential studies are to be made. Normal
range The “normal ranges” suggested for use with this method 12.5-38 mequiv. amino acids per 24 h. Adults (175-530 mg a amino acid nitrogen) 2-36.5 mequiv. amino acids per litre. Children (28-510
Yre@aration
qf
standard
are:
mg a amino acid nitrogen) came
A standard curve may be plotted over the range of o-50 mequiv.11 by making appropriate dilutions of stock glycine and treating in the same manner as urine. This should obey the Beer-Lambert relationship. Collection and storage 0-f specinaens Twenty-four-hour urines may be collected in bottles containing by volume hydrochloric acid. Owing to the difficulties in the collection
5 ml of 50~~ of 24-h urines
from children, normal ranges have been produced based on single early morning urine specimens. These urines should be adjusted to the region of pH 6 if necessary by dropwise addition of concentrated hydrochloric acid before starting the test. Specimens may be preserved by refrigeration: storage a.t 4’ is adequate over short periods time but they may be stored in a frozen state for many weeks.
of
ExPERIMENTAL AND DISCUSSION The specific diseases of amino acid metabolism have been widely discussed%%5. Many workers have elaborated complex or time-consuming techniques for the determination of total a amino acids in an attempt to increase specificity, although for clinical purposes these results are of limited value unless differential studies are carried out. “Normal” ranges from 50 adults between 15 and 65 years showed a range of 12.5-38 mequiv. per 24 hours with a mean (2) of 23.8 (2 for females was found to be 0.6 mequiv. higher than for males). In 50 children from 0-15 years, the range was 2-36.5 mequiv/l, X = 20.4. From all these subjects, normal z-way chromatograms were obtained, using 50 ,~il of urine. An analysis of results from 25 patients with possible abnormalities in arnino acid excretion indicated by clinical findings, showed II patients within the normal ranges and 2 subjects within 15% of the upper limits. Urines from all these patients gave normal paper chromatograms. Paper chromatography of the remaining 12 subjects showed 8 patients to have a generalized increase, Clin.
Chim.
Acta,
25 (1969)
27-29
O! AMINO
ACIDS
IN URINE
29
2 pregnant patients and I known phenylketonuric gave typical patterns for these conditions and I known homocystinuric showed no abnormality without prior oxidation of the urine. All these subjects had urinary amino acid concentrations of more than 18.594 above the “normal range”. The cc amino acid concentrations were then determined on all of the 125 urines after preliminary isolation of the urine on Amberlite IRC 50 ion exchange resin. Subjects who were previously within the normal ranges remained “normal” and vice versa, without a single exception, suggesting that for practical purposes the ion exchange step was an unnecessary complication. REFERENCES I A. A. ALBANESE AND V. IRBY, J. Biol. Chem., 153 (1944) 583. 2 M. L. EFRON, New Engl. J. Med., 272 (1965) 1107. 3 C. G. POPE AND M. F. STEVENS, Biochem. J.. 33 (1939) 1070. 4 S. E. SNYDERMAN AND L. E. HOLT, Jr., Advan. Pediat., II (1960) 209. 5 C. E. DENT, Lectures on the Scienci~c Basis of Medicine, Vol. 2, Athlone I,. 213-234.
Press, London,
1954,
Clin. Chim. Acta, 25 (1969) 27-29
A SIMPLE RAPID
27
METHOD FOR THE DETERMINATION
OF a AMINO
ACIDS IN URINE M. G. WELLS Biochemistry
Laboratory, Edgeware Geneva1 Hospital, Edgeware, Middx. (U.K.)
(Received December20, 1968)
SUMMARY A simple rapid method is described for the determination of total a amino acids in urine without the use of ion exchange resins, based on the chelation of cc amino acids by cupric ionslps and subsequent estimation by production of the highly specific and stable yellow complex with 2,9-dimethyl-r,ro-phenanthroline (“Neocuproine”). “Normal” ranges are provided from a study of 125 subjects. The method has been found of particular value for screening urines prior to chromatographic and other techniques.
REAGENTS chloride 0.03 M (2) Disodiz~m hydrogen orthophosphate bufler 0.1 M. Add 0.5 ml of chloroform per litre and shake well, and store at 4’. (3) Reducing solutiofi. 34 g of “A.R.” hydroxyammonium chloride and 50 g of “A.R.” sodium citrate dihydrate per 500 ml in distilled water. If high “blank” values are observed due to the presence of cuprous impurities in this reagent then these may be removed by the addition of 2 vol. of neocuproine per IOO vol. of reagent and extracting the complex formed into chloroform in a separating funnel. This step is usually unnecessary if high quality reagents are used. (4) z,g-Dimethyl-r,ro-phenantlzroline (“Neocuproine”). 0.1 g of a,g-dimethylr,ro-phenanthroline per IOO ml in ethanol. It should be noted that industrial spirit containing pyridine causes a low degree of colour development and should be avoided. (5) Stock glycilze standard 2 N. 150.14 g of glycine and 0.2 g of phenyl mercuric acetate per litre in distilled water. (6) Working glycine stalzdard. I ml of stock standard per roe ml distilled water. This solution contains 20 mequiv. glycine per litre (280 mg CCamino acid N perlitre). (I) Cupric
METHOD To 0.5 ml of urine add 0.5 ml of cupric chloride, agitate the tubes then add 9 ml Clin. Chim. Acta, 25 (1969)
27-29
WELLS
28
of buffer, mix by inversion and allow the tubes to stand for 15 min at room temperature, then centrifuge hard. To 1.0 ml of supernatant add 7 ml of reducing solution, z ml of neocuproine solution, mix well and stand for at least 3 min to allow the bubbles to clear and the colour to stabilize; then read the absorbance of the solution at 457 rnp or suitable violet filter, e.g. Ilford 601. The colour now has good stability for many hours. 0.5 ml of working standard or water may be treated in the samemanner as urine in order to produce the standard and blank respectively. Tests of above 50 mequiv. a amino acid per litre should be repeated on diluted urine unless differential studies are to be made. Normal
range The “normal ranges” suggested for use with this method 12.5-38 mequiv. amino acids per 24 h. Adults (175-530 mg a amino acid nitrogen) 2-36.5 mequiv. amino acids per litre. Children (28-510
Yre@aration
qf
standard
are:
mg a amino acid nitrogen) came
A standard curve may be plotted over the range of o-50 mequiv.11 by making appropriate dilutions of stock glycine and treating in the same manner as urine. This should obey the Beer-Lambert relationship. Collection and storage 0-f specinaens Twenty-four-hour urines may be collected in bottles containing by volume hydrochloric acid. Owing to the difficulties in the collection
5 ml of 50~~ of 24-h urines
from children, normal ranges have been produced based on single early morning urine specimens. These urines should be adjusted to the region of pH 6 if necessary by dropwise addition of concentrated hydrochloric acid before starting the test. Specimens may be preserved by refrigeration: storage a.t 4’ is adequate over short periods time but they may be stored in a frozen state for many weeks.
of
ExPERIMENTAL AND DISCUSSION The specific diseases of amino acid metabolism have been widely discussed%%5. Many workers have elaborated complex or time-consuming techniques for the determination of total a amino acids in an attempt to increase specificity, although for clinical purposes these results are of limited value unless differential studies are carried out. “Normal” ranges from 50 adults between 15 and 65 years showed a range of 12.5-38 mequiv. per 24 hours with a mean (2) of 23.8 (2 for females was found to be 0.6 mequiv. higher than for males). In 50 children from 0-15 years, the range was 2-36.5 mequiv/l, X = 20.4. From all these subjects, normal z-way chromatograms were obtained, using 50 ,~il of urine. An analysis of results from 25 patients with possible abnormalities in arnino acid excretion indicated by clinical findings, showed II patients within the normal ranges and 2 subjects within 15% of the upper limits. Urines from all these patients gave normal paper chromatograms. Paper chromatography of the remaining 12 subjects showed 8 patients to have a generalized increase, Clin.
Chim.
Acta,
25 (1969)
27-29
O! AMINO
ACIDS
IN URINE
29
2 pregnant patients and I known phenylketonuric gave typical patterns for these conditions and I known homocystinuric showed no abnormality without prior oxidation of the urine. All these subjects had urinary amino acid concentrations of more than 18.594 above the “normal range”. The cc amino acid concentrations were then determined on all of the 125 urines after preliminary isolation of the urine on Amberlite IRC 50 ion exchange resin. Subjects who were previously within the normal ranges remained “normal” and vice versa, without a single exception, suggesting that for practical purposes the ion exchange step was an unnecessary complication. REFERENCES I A. A. ALBANESE AND V. IRBY, J. Biol. Chem., 153 (1944) 583. 2 M. L. EFRON, New Engl. J. Med., 272 (1965) 1107. 3 C. G. POPE AND M. F. STEVENS, Biochem. J.. 33 (1939) 1070. 4 S. E. SNYDERMAN AND L. E. HOLT, Jr., Advan. Pediat., II (1960) 209. 5 C. E. DENT, Lectures on the Scienci~c Basis of Medicine, Vol. 2, Athlone I,. 213-234.
Press, London,
1954,
Clin. Chim. Acta, 25 (1969) 27-29