a modified copper method for the estimation

7/28/2019 A Modified Copper Method for the Estimation

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A M odified C opper M ethod for the E stim ationof x-A m ino N itrogen in U rine

C . C . Clayton and Betty F. Steele*

The copper m ethod of Pope and Stevens (1 ) for estimation of Z-am ino N has beenmodified by a change in buffer and the use of tetraethylenepentam ine as a colori nt ens if ie r, g iv ing a good c olo rim etric as sa y. a -Amin o N w as m eas ure d s ucc es sfu llyin concentrations of 0.2-2.8 mg./10 m l. In terference due to ammonia has beenm in im ize d, a nd in te rfe re nce d ue to c erta in o th er m ate ria ls h as b ee n d ete rm in ed w iths ug ge stio ns f or its elim ination. The m odified procedure offers the advantages ofs im plic ity an d s pe ed in o bta in in g a n ap pro xim ate d ete rm in atio n o f Z -a min o N in u rin e.

0 NE OF THE SIMPLEST METHODS available for estim ation of -an iino Nis that in which a suspension of copper phosphate is exposed to anam ino acid so lu tion and the copper thus solubilized m easured iod i-metrically (1 , 2) . This method has been modified for colorirnetric meas-urement of the so luble copper-am ino acid complex (3, 4). The colon-metric method presented here is not as sensitive as tile aforementionedcolorim etric procedures, bu t it does have the advantage of greater speedand is quite satisfactory for the nleasurement of am ino acid excretionin urine. Since the solubiiization of copper is not specific for am inoacids, the effect of several other interfering substances which mayoccur in bio logic materials has been studied and the procedure modifiedto m inim ize in terference.

Prom the Department of B iochem istry, M edical College of V irginia, R ichmond, V a. 23219.M any of the data presen ted in th is publication were obtained in the laboratories of D r. C . A .

Baumann, Departm ent of B iochem istry , College of Agricu lture, University of W isconsin .Reference is m ade to the use of the method in the Ph .D . thesis of B . F . Steele entitled Theinfluence of D iet on the Am ino Acid Conten t of B io log ical F luids, University of W isconsin ,1949.

Tetraethylenepentamine (TEP) wa s generously furnished by the Chem ical D ivision of UnionCarbide Corporation, N ew York , N . Y ., fo r the earlier experiments. For later experiments,commercially availab le TEP was obtailled through D istillation Products Industries, D iv.Eastm an Kodak Co., Rochester, N . Y .

Received for publication Ju ly 28, 1966; accepted for publication Sept. 20 , 1966.*presen t address: 208 Henry St., Ann A rbor, M ich .

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50 CLAYTON & STEELE ClinicalChemistryReagents

P ho sp ha te -b or at e b uf fe r 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) in500 m l. of water. This has the same phosphate-borate m olarity as thebuffer of Pope and Stevens (1 ) but results in a pH of approxim ately7 .4 when m ixed w ith tile other reagents.

(J uC l5 s olu tio n 2 7.3 gm . of C uCl 2 . 2H0 made to a liter w ith water.10% (w /v) tetraet1ylenepenIam ine (TEP) 5 gm . TEP m ade to

.10 m l. w ith water.Alanine standard 255 mg. alanine per 100 m l. water (400 1 .g. N

p ei m illiliter)Procedure

An aliquot of 10 m l. or less (2-5 m l. of urine usually adequate) of asolution colltain ing am ino acids is pipetted into a 25-m i. volumetricflask, 2 .5 m l. of CuC i2 solution is added from a buret, followed by 10 m l.of phosphate-borate buffer, and then water to the 25-m i. mark . Them ixture is inverted and shaken 3-4 tim es, allowed to stand 5 mm ., andf il te re dt hr ou gh q ua nt it at iv e f il te rp ap er ( Wh at ma n N o. 4 0 o r 4 2) . F iv emilliliters of the filtrate is p laced in a tube cuvet for use in the Spec-tronic 20 (Bausch & Lomb) colorimeter and 0.1 ml. of 10% TEP isa dd ed . T he i nt en si ty o f t he r es ul ti ng b lu e c ol or i s d et er mi ne d a t 6 63 m j.and the am ount of am ino N in the 25-m i. flask calculated from a stand-ard curve using varying amounts of a standard ized solution of alanineor from a K value derived from the alanine standard. The instrumentwas set at 100% transmittance with 5 ml. of filtratefrom a reagentblank to which the 10% TEP was added. The method could be adaptedr ea di ly f or o th er t yp es o f c ol or im et er s o r s pe ct ro ph ot om et er s.

ResultsA linear relationsh ip of absorbance and c(-am ino N was obtained w ith

the alanine standard from 0.2 to 2.8 mg. of N (0.5-7.0ml. standard).When different concentrations of an amino acid mixture (based on theam ino acid composition of casein (5)) were analyzed according to thisprocedure, the recoveries were satisfactory (92-108% ) for the con-centrations stud ied (0.3-2.0 mg. of tx-am ino N per sam ple). W hen in-d iv idual am ino acids were analyzed by th is m ethod, the recovery variedfrom 26% with cysteine to 138% w ith threonine. The recoveries of mostof the am ino acids were in the range of 70-115% . A lanine, g lycine, anda m ixture of am ino acids (as found in casein) gave 100% recovery.S im ilar results were found for the indiv idual am ino acids when they


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vol. 13, No. I. 1967 a-AM INO NITROGEN IN URINE 51

were determ ined iodimetrically by the method of A lbanese and Irby(2 ) and have been reported previously (3, 4).W hen samples of urine were hydrolyzed with acid and the hydroly-

sates analyzed m icrobio logically for the individual am ino acids, chem i-cally for the tx-am ino N by the titrim etric ninhydrin m ethod (6), an dby the copper method as applied by A lbanese and Irby to urine (2), itw as found that there was an increased amount of apparen t tx -am ino Ncontent by the copper method. Good agreement between the 3 methodswas obtained when the am ino N of protein h drol sates w as deter-m ined . S ince the most abundant nitrogenous substance in hydro lyzedurine is ammonia, its effect on the copper method was stud ied further.It w as found that pH had a decided effect upon the so lubilizing ofcopper by ammonium ions. A s the pH of the solution, as filtered , w asincreased from 7 to 9 .5, the apparen t tx-am ino N content of the urineincreased. Further, when 100 mg. N114C1 was added to 10 m l. of urine,the apparent tx-am ino N increased w ith the h igher p11; at pH 9.5 th isammonium salt concentration increased the apparent tx-am ino N by 4.00mg. while at pH 7 it increased it on ly by 0 .20 mg.

Since recoveries of the am ino acids them selves cease to be satis-factory at a pH lower than 7, the buffer m ixture was made so that alld et erm ina tio ns i n th e m od ifi ed p ro ce dur e w er e do ne at ap pro xim at el ypH 7.4 , the pH of the copper phosphate suspension resulting from theaddition of 10 m l. of the phosphate-borate buffer to 2.5 m l. of the CuC l2solution . Between the pH range of 6.1-10 .4, 1 m l. of 0.1 N acid or basechanged the pH of 12.5 m l. of tile suspension by 0 .15 pH units, and thusthe pH of most samples, unless markedly acidic or basic, would notinflu en ce th e determ ination.

V /hen the CuCL solution was added to the am ino acid m ixture beforethe addition of buffer, the filtrates w ere invariably clear, but when thebuffer was added first, turbid filtrates sometimes resulted , althoughthis turbidity cleared upon the addition of the TEP. The interval oftim e betw een the addition of the copper to the am ino acids and theaddition of the buffer had no effect on the final results; nor was thereany difference whether the sample was filtered 0.5 or 10 mm . after theaddition of the reactants. However, recoveries were low when thesamples were filtered immediately. Therefore, in routine experim entsat least 5 m iii. elapsed before filtration . A t tim es, a turb id solutionresulted w ith a precipitation of copper on pro longed standing afterfiltration unless the TEP was added. It is advisab le, therefore, to addthe TEP within an hour, and preferably less, after the tim e of filtration.

C rumpler (7) has reported that 10 m l. of a 2% solution of TEP in100 m l. of a copper solu tion furnished an adequate excess of the reagent


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52 C LA YTON & STEELE C lin ic al C he mis tryand that the reagent itself did not interfere w ith the colorim etric de-term ination at this concentration. In the presen t study, 0 .1 m l. of 10%TEP per 5 m l. of copper solu tion was found to furnish at least a 100%excess of the co lor intensifier for the highest concentration of am inonitrogen that could be determ ined accurately i)y th is m ethod. A s m uchas 1.0 nIl, of the TEP solution per 5 m l. did not affect tile in tensity ofcolor or the accuracy of the reading . The color w as found to be stablefor at least 48 hr. It w as found unnecessary to distill the TEP unless itw as so dark as to result in the 10% aqueous solu tions being highlycolored.

Certain d i- and tri-pep tides were tested to determ ine their in fluenceon the so lubilization of the copper. A t pH 9.5 (as in the method ofA lbanese and Irby) recoveries were about 200% when it w as assumedthat on ly 1 N atom per peptide was complexing w ith copper. By themodified procedure at pH 7.4, recoveries for dipeptides were still about200% , but the recoveries for tripeptides were somewhat less. Theselim ited tests would ind icate that peptides do illterfere w ith the determ i-nation, bu t this interference would be less w ith the m odified procedure.

No in terference was observed w ith glucose or urea at concentrationsof 250 mg. in 23 m l. of the suspension solution or w ith 25 mg. ofg lycerol, sodium acetate, sodium succinate, or hippuric acid in the25-m i. solution . C reatin ine, lactate, and glycocholate d id not interferew ith the test in concentrations higher than those usually encounteredin urine samples (Table 1). Some organic acids, however, did interfereto varying degrees. TT ric acid , ascorbic acid, m alic acid , and salts ofcitric and tartrate did form copper complexes which were measurableand caused appreciable interference (Table 1). This interference wasreduced somewhat by the fo llow ing procedure: The sample was pipettedinto a 25-m i. volumetric flask, and 0 .5 m l. of 0.2% KMn0 was added ,followed immediately by 0.2 m l. of glacial acetic acid . The m ixturewas allowed to stand for 1 mm., 0.2 m l. of 3% 11902 was added todecolorize the KM nO4, and 1 m l. of 3.5 N NaOH was added to bring thepH to near neutrality. For most samples this resulted in a dark pre-cipitate of Mn0. The CuC10 and buffer w ere then added and the -am ino N determ ined as before. The oxidation did not interfere w iththe recovery of am ino acids in the recovery m ixture; nor d id theresu lting m anganese components in terfere w ith the determ ination. Theinterference due to ascorbic acid or tartrate was decreased greatly bythis procedure; tile in terference due to citric acid was decreased onlym oderately. The treatment had little effect on uric acid, while inter-ference due to the malic acid was increased slightly.

Kober and Siguira (8 ) used a solution of MS0 to precipitate uricacid urior to the determ ination of tx-am ino N by the copper method .V alues for the tx-am ino N content of urine were essentially the sam e,


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Vol. 13 , No . I, 1967 a-AM INO NITROGEN IN UR INE

regardless of whether 0 .1 m l. of saturated MgSO4 was added per 25 m l.of reaction m ixture, probably because the uric acid content of the urinewas low . However, 5 m g. of uric acid as potassium urate added to therecovery m ixture resulted in high recoveries which were reduced to

Table 1 . ENFECT OF VARIOUS COM POUNDS UPON THE COLORIM ETRIC DETERM INATION OF a-AM INO N

Cone ,A pp are nt c on c. ofa-amino N Recovery

Substance tested (mg./25 ml.) (pg.) (% ) Interference- 780 95 -

Uric Acid 830 104 -2 885 110 +5 1065 133 +

Creatin ine 4 813 1028 805 10 1

20 853 106NH ,cI 6.3 (2)* 805 101 -

12.6 (4) 825 103 -31.5 (10) 866 108 +

(NH4),So4 7 .8 (2) 820 102 -15 .6 (4) 843 105 -39.0 (10) 877 109 +66. (21) 916 114 +132. (42) 1035 129 +330 . (105) 1271 159 +

Ascorb ic acid 1 .5 916 114 +3.0 1260 157 +7.5 2700 338 +

Lactate 2 .0 795 994.0 801 100

10.0 838 105Citrate 3 .0 1204 150 +

6.0 1638 203 +15.0 3118 389 +

Tartrate 25.0 3440 430 +Na glyeocholate 2 .0 820 102

4 .0 820 10210.0 830 104Mal ie acid 6 .2 1110 139 +

12 .5 1343 168 +Concentration of a-am ino N in each test is 800 pg . supplied by a m ix ture of am ino acids

based on casein.*Figures in parentheses show m illigrams of NH4 per 25 m l.


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CLAYTON & STEELE C linical Chem is try

100% when 0.1 ml. of the MgSO4 solution w as added to the mixture.Concentrations of MgSO4 (saturated solution) above 0.1 ml./25 ml.reaction m ixture resu lted in turbidity when TEP was added . Inter-ference due to p igments in urine was usually not appreciable at thedilutions used in determ ining tx-am ino N ; h ighly p igmented urine gaveonly a few m icrogram s of apparen t am ino nitrogen.

DiscussionThe method as modified has the advantages of ease and speed. It is

possible to estim ate the tx-am ino N in 20 sam ples in 2 hr. w ithout d if-ficulty, and checks betw een duplicate sam ples and recoveries are good .The accuracy is adequate w ith the range of 0.2-2.8 m g. per sam ple. Thevalues obtained on hydrolyzed protein agree well w ith the total -am ino N calculated from the results of m icrobio logic assays for thei nd iv id ua l a mi no a ci ds .

In crude m aterials, substances frequently m ay be presen t that in-validate the method. Ammonia could easily interfere in the determ ina-tion of tx-am ino N in a sample of acid-hydrolyzed urine, but the extentof in terference would be m inim ized by the modified method. U ric acid,ascorbic acid, and citric acid usually occur in normal urine at levelslower than those which cause appreciable interference. However, forspecial samples MgSO4 can be used to elim inate uric acid, and KMnO4would remove ascorbic acid , although citric acid m ight still be a sourceof error.

The method should not he used w ithout due regard for its lim itations.However , it can be used for samples in which the presence of inter-fering com pounds is unlikely or m inim al, and it m ight also be valuablein a prelim inary estimation of concentration prior to th e use of th emore precise but more tim e-consum ing methods.

References1. Pope, C . G ., and S tevens, M . F., The determ ination of am ino-n itrogen using a coppermethod. Biochem . J. 33 , 1070 (1939).2. A lbanese, A . A ., and Irby , V ., D eterm ination of urinary am ino-n itrogen by the copper

method . J. Biol. Chem . 153, 583 (1944).3. Sobel, C. , Henry, H. J ., C hiam or i, N ., and Seg alove, M., De termin ation of alp ha-ami no

acid nitrogen in urine. Proc. Soc. Exp. B io. Med. 95 , 808 (1957).4. Kekki, M ., Microdeterm ination of am ino nitrogen as copper complexes. A modification forplasma and urine. Scand. J. Chin . Lab. Inve.st. 11, 311 (1959).5 . Steele, B . F ., Sauberlich, H . E ., Reynolds, M . S., and Baumann, C . A ., M edia for leuconostocmesenteroidcs P-60 and leucomostoc citrovorum 8081 . J. B ioh. Chern. 177, 533 (1949).

6. Van Slyke, D. D ., MacFadyen, D . A., and Hamilton, P. B. , Gasometric determination ofcarboxyl group in free amino acids. J. B iol. Che,n . 141, 671 (1941).

7 . C ru mp le r, T . B ., Tetraethy lenepen tam ine as a co lorimetric reagen t for copper. Anal. C hein.19, 325 (1947).

8. Kober, P . A ., and S iguira , K ., A m icro-chem ical m ethod for the determ ination of a- an da-am ino acids and certain derivatives in proteolysis, blood and urin. J. Am . Clsein.. Soc.35 , 1 546 (1 91 3).

a modified copper method for the estimation

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