A Modified Copper Method for the Estimation of x-Amino Nitrogen in Urine

C. C. Clayton and Betty F. Steele*

The copper method of Pope and Stevens (1) for estimation of Z-amino N has been modified by a change in buffer and the use of tetraethylenepentamine as a color intensifier, giving a good colorimetric assay. a-Amino N was measured successfully in concentrations of 0.2-2.8 mg./10 ml. Interference due to ammonia has been minimized, and interference due to certain other materials has been determined with suggestions for its elimination. The modified procedure offers the advantages of simplicity and speed in obtaining an approximate determination of Z-amino N in urine.

OF THE SIMPLEST METHODS available for estimation of -aniino N is that in which a suspension of copper phosphate is exposed to an amino acid solution and the copper thus solubilized measured iodimetrically (1, 2). This method has been modified for colorirnetric measurement of the soluble copper-amino acid complex (3, 4). The colonmetric method presented here is not as sensitive as tile aforementioned colorimetric procedures, but it does have the advantage of greater speed and is quite satisfactory for the nleasurement of amino acid excretion in urine. Since the solubiiization of copper is not specific for amino acids, the effect of several other interfering substances which may occur in biologic materials has been studied and the procedure modified to minimize interference.
NE
Prom the Department of Biochemistry, Medical College of Virginia, Richmond, Va. 23219. Many of the data presented in this publication were obtained in the laboratories of Dr. C. A. Baumann, Department of Biochemistry, College of Agriculture, University of Wisconsin. Reference is made to the use of the method in the Ph.D. thesis of B. F. Steele entitled The influence of Diet on the Amino Acid Content of Biological Fluids, University of Wisconsin,

1949.
Tetraethylenepentamine

(TEP)

was

generously

furnished

by

the

Chemical

Division

of

Union

Carbide Corporation, New York, N. Y., for the earlier experiments. For later experiments, commercially available TEP was obtailled through Distillation Products Industries, Div. Eastman Kodak Co., Rochester, N. Y. Received for publication July 28, 1966; accepted for publication Sept. 20, 1966. *present address: 208 Henry St., Ann Arbor, Mich.
49

50

CLAYTON &

STEELE

ClinicalChemistry

Reagents
Phosphate-borate buffer 3.0 gm. boric acid (0.1 M), 16.4 gm. Na2HPO4 7H0 (0.13 M), and 3.8 gm. and Na3PO4. 121120 (0.02 M) in 500 ml. of water. This has the same phosphate-borate molarity as the buffer of Pope and Stevens (1) but results in a pH of approximately 7.4 when mixed with tile other reagents. (JuCl5 solution 27.3 gm. of CuCl2 2H0 made to a liter with water. 10% (w/v) tetraet1ylenepenIamine (TEP) 5 gm. TEP made to .10 ml. with water. Alanine standard 255 mg. alanine per 100 ml. water (400 1.g. N
. .

pei

milliliter)

Procedure
An aliquot of 10 ml. or less (2-5 ml. of urine usually adequate) of a solution colltaining amino acids is pipetted into a 25-mi. volumetric flask, 2.5 ml. of CuCi2 solution is added from a buret, followed by 10 ml. of phosphate-borate buffer, and then water to the 25-mi. mark. The mixture is inverted and shaken 3-4 times, allowed to stand 5 mm., and filtered through quantitative filter paper (Whatman No. 40 or 42). Five milliliters of the filtrate is placed in a tube cuvet for use in the Spectronic 20 (Bausch & Lomb) colorimeter and 0.1 ml. of 10% TEP is added. The intensityof the resultingblue color is determined at 663 mj. and the amount of amino N in the 25-mi. flask calculated from a standard curve using varying amounts of a standardized solution of alanine or from a K value derived from the alanine standard. The instrument was set at 100% transmittance with 5 ml. of filtrate from a reagent blank to which the 10% TEP was added. The method could be adapted readily for other types of colorimeters or spectrophotometers.

Results
A linear relationship of absorbance and c(-amino N was obtained with the alanine standard from 0.2 to 2.8 mg. of N (0.5-7.0ml. standard). When differentconcentrations of an amino acid mixture (based on the amino acid composition of casein (5)) were analyzed according to this procedure, the recoveries were satisfactory (92-108%) for the concentrations studied (0.3-2.0 mg. of tx-amino N per sample). When individual amino acids were analyzed by this method, the recovery varied from 26% with cysteine to 138% with threonine. The recoveries of most of the amino acids were in the range of 70-115%. Alanine, glycine, and a mixture of amino acids (as found in casein) gave 100% recovery. Similar results were found for the individual amino acids when they

vol. 13, No. I. 1967

a-AMINO

NITROGEN

IN

URINE

51

were determined iodimetrically by the method of Albanese and Irby (2) and have been reported previously (3, 4). When samples of urine were hydrolyzed with acid and the hydrolysates analyzed microbiologically for the individual amino acids, chemically for the tx-amino N by the titrimetric ninhydrin method (6), and by the copper method as applied by Albanese and Irby to urine (2), it was found that there was an increased amount of apparent tx-amino N content by the copper method. Good agreement between the 3 methods was obtained when the amino N of protein h drol sates was determined. Since the most abundant nitrogenous substance in hydrolyzed urine is ammonia, its effect on the copper method was studied further. It was found that pH had a decided effect upon the solubilizing of copper by ammonium ions. As the pH of the solution, as filtered, was increased from 7 to 9.5, the apparent tx-amino N content of the urine increased. Further, when 100 mg. N114C1 was added to 10 ml. of urine, the apparent tx-amino N increased with the higher p11; at pH 9.5 this ammonium salt concentration increased the apparent tx-amino N by 4.00 mg. while at pH 7 it increased it only by 0.20 mg. Since recoveries of the amino acids themselves cease to be satisfactory at a pH lower than 7, the buffer mixture was made so that all determinations in the modified procedure were done at approximately pH 7.4, the pH of the copper phosphate suspension resulting from the addition of 10 ml. of the phosphate-borate buffer to 2.5 ml. of the CuCl2 solution. Between the pH range of 6.1-10.4, 1 ml. of 0.1 N acid or base changed the pH of 12.5 ml. of tile suspension by 0.15 pH units, and thus the pH of most samples, unless markedly acidic or basic, would not influence the determination. V/hen the CuCL solution was added to the amino acid mixture before the addition of buffer, the filtrates were invariably clear, but when the buffer was added first, turbid filtrates sometimes resulted, although this turbidity cleared upon the addition of the TEP. The interval of time between the addition of the copper to the amino acids and the addition of the buffer had no effect on the final results; nor was there any difference whether the sample was filtered 0.5 or 10 mm. after the addition of the reactants. However, recoveries were low when the samples were filtered immediately. Therefore, in routine experiments at least 5 miii. elapsed before filtration. At times, a turbid solution resulted with a precipitation of copper on prolonged standing after filtration unless the TEP was added. It is advisable, therefore, to add the TEP within an hour, and preferably less, after the time of filtration. Crumpler (7) has reported that 10 ml. of a 2% solution of TEP in 100 ml. of a copper solution furnished an adequate excess of the reagent

52

CLAYTON & STEELE

Clinical

Chemistry

and that the reagent itself did not interfere with the colorimetric determination at this concentration. In the present study, 0.1 ml. of 10% TEP per 5 ml. of copper solution was found to furnish at least a 100% excess of the color intensifier for the highest concentration of amino nitrogen that could be determined accurately i)y this method. As much as 1.0 nIl, of the TEP solution per 5 ml. did not affect tile intensity of color or the accuracy of the reading. The color was found to be stable for at least 48 hr. It was found unnecessary to distill the TEP unless it was so dark as to result in the 10% aqueous solutions being highly colored. Certain di- and tri-peptides were tested to determine their influence on the solubilization of the copper. At pH 9.5 (as in the method of Albanese and Irby) recoveries were about 200% when it was assumed that only 1 N atom per peptide was complexing with copper. By the modified procedure at pH 7.4, recoveries for dipeptides were still about 200%, but the recoveries for tripeptides were somewhat less. These limited tests would indicate that peptides do illterfere with the determination, but this interference would be less with the modified procedure. No interference was observed with glucose or urea at concentrations of 250 mg. in 23 ml. of the suspension solution or with 25 mg. of glycerol, sodium acetate, sodium succinate, or hippuric acid in the 25-mi. solution. Creatinine, lactate, and glycocholate did not interfere with the test in concentrations higher than those usually encountered in urine samples (Table 1). Some organic acids, however, did interfere to varying degrees. TTric acid, ascorbic acid, malic acid, and salts of citric and tartrate did form copper complexes which were measurable and caused appreciable interference (Table 1). This interference was reduced somewhat by the following procedure: The sample was pipetted into a 25-mi. volumetric flask, and 0.5 ml. of 0.2% KMn0 was added, followed immediately by 0.2 ml. of glacial acetic acid. The mixture was allowed to stand for 1 mm., 0.2 ml. of 3% 11902 was added to decolorize the KMnO4, and 1 ml. of 3.5 N NaOH was added to bring the pH to near neutrality. For most samples this resulted in a dark precipitate of Mn0. The CuC10 and buffer were then added and the amino N determined as before. The oxidation did not interfere with the recovery of amino acids in the recovery mixture; nor did the resulting manganese components interfere with the determination. The interference due to ascorbic acid or tartrate was decreased greatly by this procedure; tile interference due to citric acid was decreased only moderately. The treatment had little effect on uric acid, while interference due to the malic acid was increased slightly. Kober and Siguira (8) used a solution of MS0 to precipitate uric acid urior to the determination of tx-amino N by the copper method. Values for the tx-amino N content of urine were essentially the same,

Vol. 13, No. I, 1967

a-AMINO

NITROGEN IN URINE

regardless of whether 0.1 ml. of saturated MgSO4 was added per 25 ml. of reaction mixture, probably because the uric acid content of the urine was low. However, 5 mg. of uric acid as potassium urate added to the recovery mixture resulted in high recoveries which were reduced to
Table 1. ENFECT OF VARIOUS COMPOUNDS

UPON

THE COLORIMETRIC conc. of

DETERMINATION

OF a-AMINO

Apparent
Cone, Substance tested

a-amino N
ml.) (pg.)

Recovery

(mg./25
-

(%)
95 104 110 133

Interference

780 830 885 1065

Uric

Acid 2 5

Creatinine

4
8 20

813
805 853 805 825 866 820 843 877 916 1035 1271 916 1260 2700 795 801 838 1204 1638 3118 3440 820 820 830 1110 1343

102 101
106 101 103 108 102 105 109 114 129 159 114 157 338 99 100 105 150 203 389 430 102 102 104 139 168 by a mixture +
-

NH,cI

6.3 (2)* 12.6 (4) 31.5 (10) 7.8 (2) 15.6 (4) 39.0 (10) 66. (21) 132. 330. (42) (105)

+
-

(NH4),So4

+ + + + + + +

Ascorbic

acid

1.5 3.0 7.5

Lactate

2.0 4.0 10.0

Citrate

3.0 6.0 15.0 25.0 2.0 4.0 10.0 6.2 12.5

+ + + +

Tartrate Na glyeocholate

Malie

acid

+
of amino acids

Concentration of a-amino N in each test is 800 pg. supplied based on casein. *Figures in parentheses show milligrams of NH4 per 25 ml.

CLAYTON

& STEELE

Clinical

Chemistry

100% when 0.1 ml. of the MgSO4 solution was added to the mixture. Concentrations of MgSO4 (saturated solution) above 0.1 ml./25 ml. reaction mixture resulted in turbidity when TEP was added. Interference due to pigments in urine was usually not appreciable at the dilutions used in determining tx-amino N; highly pigmented urine gave only a few micrograms of apparent amino nitrogen.

Discussion
The method as modified has the advantages of ease and speed. It is possible to estimate the tx-amino N in 20 samples in 2 hr. without difficulty, and checks between duplicate samples and recoveries are good. The accuracy is adequate with the range of 0.2-2.8 mg. per sample. The values obtained on hydrolyzed protein agree well with the total amino N calculated from the results of microbiologic assays for the individual amino acids. In crude materials, substances frequently may be present that invalidate the method. Ammonia could easily interfere in the determination of tx-amino N in a sample of acid-hydrolyzed urine, but the extent of interference would be minimized by the modified method. Uric acid, ascorbic acid, and citric acid usually occur in normal urine at levels lower than those which cause appreciable interference. However, for special samples MgSO4 can be used to eliminate uric acid, and KMnO4 would remove ascorbic acid, although citric acid might still be a source of error. The method should not he used without due regard for its limitations. However, it can be used for samples in which the presence of interfering compounds is unlikely or minimal, and it might also be valuable in a preliminary estimation of concentration prior to the use of the more precise but more time-consuming methods.

References
1. 2. 3. Pope, C. G., and Biochem. Albanese, A. A., method. J. Biol. Sobel, C., Henry, acid nitrogen in

method.

Stevens, M. F., The determination of amino-nitrogen J. 33, 1070 (1939). and Irby, V., Determination of urinary amino-nitrogen Chem. 153, 583 (1944). H. J., Chiamori, N., and Segalove, M., Determination urine. Proc. Soc. Exp. Bio. Med. 95, 808 (1957).
of amino nitrogen as copper complexes.

using by

a the

copper copper

of alpha-amino
modification for

4. Kekki,
5.

M.,

Microdetermination

plasma and urine. Scand. J. Chin. Lab. Inve.st. 11, 311 (1959). Steele, B. F., Sauberlich, H. E., Reynolds, M. S., and Baumann, C. A., Media for leuconostoc mesenteroidcs P-60 and leucomostoc citrovorum 8081. J. Bioh. Chern. 177, 533 (1949).

6. Van Slyke, D. D., MacFadyen, D. A., and Hamilton, P. carboxyl group in free amino acids. J. Biol. Che,n. 141, 7. Crumpler, T. B., Tetraethylenepentamine as a colorimetric

B.,

Gasometric

determination

of

671 (1941). reagent for

copper.

Anal.

Chein.

19,

325

(1947).

8. Kober,
a-amino

P. A., and Siguira,

acids and certain

K., A micro-chemical
derivatives

in proteolysis,

method for the determination blood and urin. J. Am.

of a- and Clsein.. Soc.

35, 1546

(1913).