Determination of Creatine and Creatinine in Urine LELAND C. CLARK, JR., AND HASKELL L. THOMPSOS Fels Research I n s t i t u t e f o r t h e S t u d y of H u m a n Development, Antioch College, Yellow Springs, Ohio An improved method for the determination of creatine and creatinine in urine using the Jaffe reaction is described. Accurate results for the determination of creatinine depend upon carefully controlling the hydrogen ion concentration of the reaction mixture, the temperature, and the heating time. The authors conclude that many of the disputes regarding the optimum conditions for the determination of creatine in urine may be resolved in the light of the very marked effect of hydrogen ion concentration.
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H E literature contains many conflicting reports regarding the optimum conditions necessary for the determination of creatine and creatinine. The present study discusses the various factors that must be controlled in order to obtain accurate, reproducible determinations of creatine and creatinine in urine. The methods described have given very satisfactory results over a period of 2 years in the analysis of over 600 urine specimens. EXPERIMENTAL
Instruments. The instruments used were the Beckman spectrophotometer, Model DU, Beckman pH meter, and the Evelyn colorimeter (Rubicon Company). REAGENTS
Picric Acid Solution. Picric acid ( c . P . ' ~ is rrcrystallized twice from glacial acetic acid and dried at room temperature, following 4 1,17y0 solution is used, folthe suggestion of Benedict ( 2 ) . ' lowing the recommendation of Peters (11). Sodium Picrate Butler. One liter of the 1.17% picric acid solution is adjusted to pH 2.0 * 0.05 by the addition of approximately 20 ml. of 2 N sodium hydroxide. I t is advisable to check the pH of the solution after several hours, because the freshly prepared solution tends to drift in pH. Sodium Hydroxide Solution. One hundred grams of C.P. sodium hydroxide are dissolved in 1 liter of water and stored in a Pyrex bottle.
diluted with 15 ml. of water, and mixed thoroughly, and the optical density is measured a t a suitable wave length. The color is stable for several hours. 'CTrine samples are usually diluted 1 to 10 before pipetting. A reagent blank, substitub ing water for the creatinine standard, is carried through each batch of determinations. It is desirable to run duplicate determinations on all samples, and triplicate determinations on the standard. For the routine procedure, optical measurements are made in 1-cm. Corex cells with the Beckman spectrophotometer a t 510 mp wave length, using a slit width of 0.05 mm. After a preliminary reading of the reagent blank the instrument is set, to 100% transmittance against the ],lank. Measurements in the Evelyn colorimeter are made with a No. 520 filter. Comparison of Results Obtained with Evelyn Colorimeter and Beckman Spectrophotometer. Simplification of both calculation and laboratory manipulation is possible if conditions can be controlled so that a colorimetric procedure follows the BeerLambert law. Figure 1 illustrates the dependency of the creatinine measurement upon the optical conditions of measurement. The wide band of the filter-type colorimeter apparently causes wide deviations from linearity between creatinine concentration and optical density, particularly with higher creatinine values. Greater sensitivity is obtainrd a t 510 mp than at 520 mp T
STANDARD
Creatinine Standard. Creatinine (Pfanstiehl) is recrystallized from water a?d dried over phosphorus pentoxide a t 80 C. in vacuo. ANALYSIS O F CREATININE (CaH7N,O) USED. Calculated, C 42.47, H 6.24, K 37.15T0. Found, C 42.16, H 6.28, N 37.14%. A stock solution containing 1 gram per liter of 0.1 S hydrochloric acid is diluted 1 to 10 with water to give a standard of 0.1 mg. per ml. Creatine Standard. Creatine (Pfanstiehl) is recrystallized from water and dried over Dhosuhorus Dentoxide a t 80" C. in vacuo. ANALYSIS 'OF CREATINE ( C ~ H A - ~ OUSED. ~) Calculated, C 36.63, H 6.92, N 32.05%. Found, C 36.60. H 7.05. N 31.82%. A stock solution containing -1 gram per liter of water is diluted 1 to 10 with water to give a working standard of 0.1 mg. per ml. All standards are maintained a t 4" C. using an overlayer of toluene as a preservative.
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CREATININE DETERMINATION
Procedure. test tube are standard, 4 and 0.3 ml. The solution
Into a 125 X 25 mm. Pyres pipetted 1 ml. of the creatinine ml. of sodium picrate buffcr, of sodium hydroxide solution. is allowed to stand 20 minutes,
MICROGRAMS OF CREATININE
Figure 1. Comparison of Evelyn Colorimeter and Beckman Spectrophotometer in Determination of Creatinine
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two different concentrations of creatinine. The curves illustrate that it is necessary to iiieasure the colored reaction product using a very narrow nave band because of the stiong absorption of the reagent itself a t only a slightly shorter wave length. Interfering Substances. Various drugs, glucose, acetone, acetoacetic acid, etc., may affect the reaction. All urines analyzed in this laboratory by this method are tested qualitatively for acetone and glucose. If either of these abnormal substances is presc a n t it should be eliminated. CREATINE DETERMINATIOY
Procedure. The procedure for creatine measurement is identical to that used for creatinine except that the samples are heated, under controlled conditions, between the additions of sodium picrate buffer and sodium hydroxide. Triplicate creatine standards are analyzed with each batch of samples, For routine procedure, the samples are heated for 30 minutes a t 120 ' C. in an autoclave. Because evaporation is negligible under the conditions used here, it is not necessary to stopper the tubes, although t h y
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0-KCI- HCI Buffer (IOOy Creatme) e - N a Citrate Buffer (100~Creatine) &-picrate Buffer ( 5 0 7 Creatine) A-picrate Buffer (150 Creatine)
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ANALYTICAL CHEMISTRY
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TITRATION OF PICRIC ACID
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cwrtstion of crt~atinc~ which is usually, though not always, niarkcdly reduced. Thc hcalthy adult males tested excreted betwetw 0 and 0.070 gram of creatine per day. A dvtailed analysis of these data is in progress and will be r r p o r t d olsrwhrre.
niscussIoN
The factors rcgulating the conversioii ~ ) t ' creatinrx to creatinint, and those affecting t Jaffe color reaction (9) have been thc subjcsct of numerous studies during the past 40 yc~ti~s. The resrarch has been directed toward : i i i understanding of the reaction kinetics, :i tit,scriptioil of the precise nature of the colot~ reaction, and tht: dcwlopment of optini:tl analytical conditions. Space does not pc,riiiit a conipl(1te reviv\r. and detailing of t 1iv ( ~ 1 1 0 1 ~ mous literaturc, which has been far too oftc'ii overlooked by iuvestigat,ors attempting t n devise niethods for the analysis of crratinv t t i i r l I N NaOH creatinine in hiological fluids, Thi, c w l i c ~ r rnl literature has bwii reviewed by Hunt(br (8). 0 " ~ 1 ' 1 " ~ ' ' I ~ I ' l ' I ' I ' I ' S II ' l ' 0.2 0.4 0.6 0.0 1.0 12 14 16 I8 20 22 2.4 2 6 2.8 The reports of Edgar and eo-workers (4, 5 ) established the fundamental roltb of hydroFigure 1.. Titration of 50 MI. of l . l i 5 qo Picric i c i d with 1~1'Sorliirni grn ion concentration in establishing t ht> final Hydroxide c,quilihrium bctwetxn creatine and crtvitininc' in aqutaous solution and indicate, as wcsll, tli:tt t h(h hydrogen ion conwntration controls the rate of conlcrsioir. minutes at 120" hside froni the, practical advantages of the shortened time, it would seem generally drsirable to conduct a reaction under its optimuni conditions. Yield of Creatine from Creatinine. \$-hen pure standards are used, the converPion of creatine and cwatininr has a mean value Table I. Recover) of Creatine Added to Lrine of 98.5% a.ith a standard deviation of 1.09. Thew values were Total Creatine Creatine Creatine obtained from 42 routine checks distributed over a period of 2 Added Found" Recovered Recovery vears. / Y f 9 Recovery of Creatine Added to Urine. Table I lists the recovery of creatine added to urine, using the present nlethod of . These wsults illustrate that ercLatine can be measured quantitativcly i n the presencr of urinv hy thc procedure developed. Mean ... ... 90,o Identity of Reaction Product. 111 Figurcx 6 are shown the * 'Phis urine baiiiple conrainrcl 0.792 nip. o l creatinine prr n i l . I r \vas absorption spectra of the colored rt:action products obtained from diliited 1 to 20 before nsc. creat,inine, creatints converted to creatininv, and a urincs sample treat,ed for the determination of creatinv. The instrument was set to 1 0 0 ~ otransmittance using the reagent blank. The similarity of these curves is evidence that the final product measured in all cases is creatinine. Routine spectrophotomet,ric measurr0 400 nients arc made at 510 mp rather than a t the masimum (478 mp) because the strong absorption of picric acid itself at thr, maximum would necessitate subtrac0.300 tion of an unnecessarily high reagent blank. Creatinine and Creatine Content of Urine from Healthy Individuals. An inspection of the data obtained during the past year indicates a \vide range of excretion of creatine and creatinine. For esample, 0 200 the creatinine excretion of 212 children between the ages of 6 and 18 years ranged from 0.281 to 2.05 grams in 24 hours. The creatinine excretion is probably 0 IO0 best related to the muscle mass of the healthy child ( 1 2 ) . These same children excreted between 0 and 0.428 gram of creatine per day. I n the last 132 prenatal samples analyzed creatinine excretion in 24 0 000 hours ranged between 1.09 and 1.61 grams, while 5 io 15 20 25 3C 35 '0 45 50 55 60 TIME IN MINUTES creatine excretion varied between 0.005 and 1.07 grams. Urine samples obtained within 3 months Figure 5. Effect of pH on Conversion of Creatine to Creatinine postpartum show a similar range in creatinints. but an a t Various Temperatures 1 x 9
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part of thv Confusion iritimtiuC~dinto the literature in rcwnt y~'ai.s
tc~mincdfrom the use of filter-type colorimrtrrs for this restud!-. Rtbgarding the detr~rminationof creatinine using picric acid, it has been repeatedly found (10, 11) that sw('rt' dc%viations f r o m t h r Brer-1,anibert (quation occur and, furthermore', that t w o supposedly standardiztd instruments of the sanit. manufacturc~ may differ considcrably fi.om one another (11). Thus I,ainh(~i,t found that, in ordvr to stay within the relatively short rangtl of linearity using tho Ilvvlyn instrunient, it was necessary t o dilutv urint samples, first t o a constant specific gravity, and thr:n again (about 1 to 20). B y using the, more precise Beckman instrunwnt the range of linearity is so extended that a single dilution sufiws for over 90% of all urines tested. Furthwmorr, it has Iwc.n possible to study the convcrsion of creatine to creatinine, as wrll as the details of thta color reaction itsplf, with much grcatcsr prt.ckiori. This study, as rcported above, has confirnied thc original statc~mcntsof Folin ( 6 , 7 ) regarding the usefulness of thv .Jaff(. reaction and has dt>fined the analytical conditions in tht. mor(' I)rc&cb tclrnis possibk with modern instruments.
*CREATININE 100rnicmgrams
\CKYOWLEDGIIEYT
Tht, authote ~ 1 4 t1o acknoii lcdyr the technical assistancta of Jacobstm and ICleanor c'lai k during the early stages of tht. work. Dt.tt,rminations ot carbon, hldiogen, and nitrogen \\('I ( * donr. IIV thti Clat h llicroanalytical I.ahoratorv, Urhana, Ill. \Vtmcii
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3pectral thsorption Curves LITERATURE CITED
Thrl aut how' i'sptt i i i i h , i i t s N Y I ' ( ~ dt'siyii(d priniarily to discovcxrth(8 optinium p H at whichquantitative conversion of creatine to crcxatininv occurred. Independrnt of the type of buffer, this optimum point was found to lie nt'ar pH 2, a finding which could not h a w 1 ) c ~ i predicted i froni tht! work of Icdgar. I n thv light of this infor( I ) proccdurcl has dccrcmcd, ratht>r than ty of t h c b original Folin method, as has b t ~ > n pointed out by Lambert ( I O ) . However, contrary to thc statc~tiient of 1,ambcrt. the effrrt is due, riot t o the presencca of picric acid p c ~ but to the hydrogen ion concentration of picric acid solutions. \Vith tht. advent 01' nioclcrn photoelwtric colori~netersand Fpc'ctrophotoineterb, it is natural and appropriate> that the J a f f ~ i ' c ~ t cion t roctudird i n mot'(' prc.rist1 terms. Curiousl>- c~liouyh. ~
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( I 1 Aibanese, -1..I., and Wangeriii, D. SI.,Science, 100, 68 (1944). (2) Benedict, S. 11.. J . B i d . Chem., 82, 1 (1929). ( 3 ) Clark, \T. &I., "Determination of Hydrogen Ions." Baltimore, Williams and n'ilkins Co.. 1922. (1) Edgar. G., arid Shivel,, H. E.. J . A m . C h r m . Soc.. 47, 1 1 i O (1926). (5) Edgar. G.. and Wakefield, I t . A , , Ibid., 45, 2242 (1923). (6) Foliu. 0..J . Bioi. C'hem., 17, 469 (1914). ( i ) Folin, 0.. arid Doiry. 12. A , , I b i d . , 28, 349 ( 1 9 1 i ) . ( 8 ) Hunter, .I.,"Ci.eatine and Creatinine," New York. I2oiigiiritir~, Green and C o . , 1928. (9) Jaffe. &I.. 2. ~ h u s i o l C'hrm.. . 10. 391 (1886). (10i Lambert: G . ' F , J . Riol. Chem., 161, 678 (1945). (11) Peters, J. H., Itlid., 146, 179 (1942). (12) Iteynolds. E. L.. n t ~ dC'Iai,k. I>.C . , Chi'idDrJwiop.. 18, I55 i i ) I i r R E C E I V E lDl a r c h 9,1940
Separation of Calcium from Magnesium by Oxalate Method A Critical Study 'roKE
t LOL T H 1
Cfreniicul Znstitrcte, Oslo Zniuersitj., Oslo.
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1115 osalate mrrhod for deterniin:ition of Calcium an(\ its sopnr:~tionfrom niagnesium dates from the earliest times of (?I. Fresenius (4)in 1868 st:ited that thtl prwipitato of enlciuni oxalate is always contaminated 1))- magnfxsium oxalate. It is tlic'refore neeessay?. t o dissolve the precipitittr in hydrochloric arid, and reprecipit:ite calcium oxalate with :iinmoni:i. Richard? a n d co1l:il)or:itol~s ( 1 6 ) in 1901 a h o ~ v c dthat iiiidei, certain conditioiis :L single precipitation oi' c:ilc4uni i, 1 I'w,iant s d d r e r b , 5-orweginn Defense Kcv?ari,li E ~ t a h l i s h m e n t ,Cli \ i - i o n Kieller pr. 1.ilIe.troiu S o r w a y .
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sufhcieiit. In thr, niethotl tlesCritwl, :I large :mount oi :minioniurn chloi,ide i h :idtl(d. Cnlcium oxalate is precipitated 11y drop\\-ise addition ot' :immonia to a calcium chloride solution cont:tining oxalic. avid find hydrochloric acid. After t h r precipitation an us(*essof :ammonium oxalate is added. Fischer ( 3 ) in 1926 pul)lished a paper, disparaging the work of 1iich:ircts. He shon.ec1 th:it magnesium oxalate is not precipitated f r o m supers:ttui,:itecl solutiolis when special precautions are taken. I n the determinatioii of c.nlciuni, Fischcr usod but ii slight o \ c r s s of ammonium os:il:it
