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PLASMA CREATININE by MARY RALSTON from the Department of Chemical Pathology, Postgraduate Medical School, London

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PLASMA CREATININE by MARY RALSTON from the Department of Chemical Pathology, Postgraduate Medical School, London J Clin Pathol: first published as 10.1136/jcp.8.2.160 on 1 May 1955. Downloaded from J. clin. Path. (1955), 8, 160. PLASMA CREATININE BY MARY RALSTON From the Department of Chemical Pathology, Postgraduate Medical School, London (RECEIVED FOR PUBLICATION JANUARY 25, 1955) The non-specificity of reactions for creatinine is agreement between the Lloyd's adsorption method a well-recognized drawback in most methods for and the specific enzymatic method. It was con- its determination. The Jaffe colour reaction which sidered that another approach to checking its forms the basis of most procedures is given by specificity would be to investigate the Lloyd's numerous substances other than creatinine, over adsorbed material chromatographically. This 30 being listed by Hunter (1928). They include, method has given results which are lower than those apart from substances related to creatinine, acetone, obtained with the ceric sulphate method; and aceto-acetic acid, and pyruvic acid. chromatograms of the eluates from the Lloyd's Various methods of estimating true creatinine as adsorption have shown only one Jaff-positive spot, opposed to the total Jaffe-positive chromagen have identical with that for creatinine. We conclude been proposed. Miller and Dubos (1937), using that the Lloyd's procedure is, in fact, specific for suspensions of a creatininase-producing bacterium, creatinine in plasma. carried out the Jaffe reaction on plasma filtrates before and after incubation with a suspension of Method the organism, and thus calculated the true creatinine the Reagents.-The following are required: by difference. According to this technique, Sodium Tungstate.-10%. true plasma creatinine constituted 80-100% of the Sulphuric Acid.-j N. total chromagen. Methods employing adsorption Lloyd's Reagent.-Fuller's earth B.D.H.: Haugen and on Lloyd's reagent have also been claimed to give Blegen (1953) point out that not all samples of Fuller's http://jcp.bmj.com/ true creatinine values. This substance is a hydrated earth are suitable. Each batch should be tested for aluminium silicate. In acid solution it adsorbs satisfactory adsorption. creatinine which can subsequently be eluted by Oxalic Acid.-Saturated solution. alkali. Sodium Hydroxide.-2.5 N-NaOH. Recently and 8onka (1952) published a Picric Acid.-Saturated solution. Kogtir Alkaline Picric Solution.-Picric acid solution (27.5 ml.) method for creatinine determination based on the made to 100 ml. with observation that pyruvic acid is a Jaff&-positive plus 2.5 N-NaOH (5.5 ml.) up water; prepared freshly for use. on October 1, 2021 by guest. Protected copyright. substance which can be oxidized by ceric sulphate Standard Creatinine Solution.-1 mg./100 ml. in water. without destroying creatinine. Normal values from 0.3 to 0.6 mg./ Procedure.-To 3 ml. of plasma is added 4.5 ml. of found by this method were water, 1.5 ml. of sodium tungstate, and 3 ml. of i N sul- 100 ml. with a mean of 0.48 mg./100 ml. A series phuric acid. After thorough mixing and standing for of determinations with ceric sulphate showed a 30 min., the mixture is filtered through a No. 1 Whatman reduction of 60% on a parallel series without ceric filter paper. sulphate treatment. In our hands normal plasmas To 100 mg. (±2 mg.) Lloyd's reagent in a centrifuge (11) treated with ceric sulphate by this method gave tube is added 5 ml. of filtrate (equivalent to 1.25 ml. a range of 0.77 to 1.21 mg./100 ml., with a mean of plasma) and 0.5 ml. of saturated oxalic acid. The tube 0.97 mg./100 ml. This was a reduction of 16% on is stoppered and shaken intermittently for 10 min. (As the values obtained on the same plasmas without an alternative to weighing individual lots of the Lloyd's was reagent, a suspension of 10 g. in 100 ml. of water may ceric sulphate treatment. This reduction signi- be made and 1 ml. of this solution taken after thorough ficant but was not of the magnitude found by shaking.) The tube is then centrifuged at 3,000 r.p.m. Kostiir and gonka. for 20 min. The supernatant is decanted and the tube We have also investigated the adsorption proce- allowed to drain. dure with Lloyd's reagent (Fuller's earth) of Haugen To the packed deposit is added 7.5 ml. of freshly and Blegen (1953) as modified by Owen, Iggo, prepared alkaline picrate solution. The deposit is Scandrett, and Stewart (1954), who found good stirred, the tube stoppered and shaken intermittently for J Clin Pathol: first published as 10.1136/jcp.8.2.160 on 1 May 1955. Downloaded from PLASMA CREATININE 161 Solvent _ ferred as completely as possible front to chromatogram paper (No. 1 Whatman). With each sample of plasma a second sample of the same plasma, with added creatinine, was run. The creatinine (10 pg. in 1 ml. of water) was added to the 5 ml. of filtrate, and adsorbed on Lloyd's reagent. The chromatograms were run 16 Indicator hours in butanol-acetic acid-water spots .4 bi solvent (in the proportions 4: 1: 5) and then sprayed with a freshly mixed Jaffe + ve .1. alkaline picrate solution (equal parts spots of a 3 % NaOH solution and a satur- ated solution of purified picric acid dilution in the proportion of 7.5 to 2.5 ml. of water). Starting I I I I line .,L -.2 . Results Pl. Pi-+ Pt. PL.+ PL. PLl.+ Std. Cre. Cre. re. Cre. Ten normal plasmas, each as plasma FIG. I.-Chromatogram of eluates of Lloyd's adsorbed plasma (P1.), plasma plus alone and as plasma plus creatinine, 'creatinine (P1. + Cre.), and of creatinine standard solution (Std. Cre.). Run showed a single Jaffe-positive spot. for 16 hours in butanol-acetic acid-water solvent, then sprayed with alkaline was picrate solution. That for plasma plus creatinine considerably darker and of the same 10 min. It is centrifuged for 10 min., and then placed Rf value a'Ls the Jaffe-positive spot for plasma alone. in a water bath (20°-24° C.) and allowed to reach the The meth5 yl orange indicator (contained in the test bath temperature. .solutions) ran in advance of the Jaffe-positive spot. The solution is then read in a spectrophotometer at a . s) tsidered that the results of these chromato- wavelength of 520 n,p. Satisfactory results have also It con Is licate that Lloyd's reagent will adsorb been obtained with the Ilford bright spectrum green grams md http://jcp.bmj.com/ light filter No. 624 in a photo-electric colorimeter. Solvent A blank consisting of 5 ml. of water, and two 5 ml. front standards containing 0.01 mg. and 0.025 mg. (equivalent to 0.8 mg. (0.01 x 100/1.25) and 2 mg. creatinine/100 ml. plasma), are prepared in a similar manner, i.e., treated with Lloyd's reagent and alkaline picrate like the filtrate. Experiments Elution of Creatin'ine and Chromatography.- on October 1, 2021 by guest. Protected copyright. Plasma was deproteinized, the filtrate allowed to stand in contact with Lloyd's reagent, the latter then centrifuged down and the tube drained. Sodium hydroxide, however, could not be used for elution as this interferes with obtaining satisfactory chromatograms. Hence other alkalis which could be removed before running the chromatogram were investigated. Ammonium hydroxide and potassium hydroxide proved unsuitable. A satur- ated solution of barium hydroxide, i.e., approxi- mately N/3, was satisfactory. This was subsequently Starting exactly neutralized by sulphuric acid using methyl line orange as indicator. The precipitate of barium ultra Acetone Creatirvme in filtrate filItrate water sulphate was spun down, the supernatant decanted FiG;. 2.-Chromatogram of plasma ultrafiltrate and of an acetone and evaporated to dryness at reduced pressure at extract of plasma. Run for 16 hours mn butanol-acetic acid- a temperature not exceeding 370 C. It was then water solvent, then sprayed with alkaline picrate solution. Two Jaff-positive spots were discerned in plasma together with taken up in 0.1 ml. of distilled water and trans- streaking- salt effect J Clin Pathol: first published as 10.1136/jcp.8.2.160 on 1 May 1955. Downloaded from 162 MARY RALSTON creatinine alone of the Jaffe-positive substances with either ceric sulphate or Lloyd's reagent, i.e. present in plasma (Fig. 1). total plasma creatinine; (B) treated with ceric Chromatograms were also run on ultrafiltrates sulphate; (C) treated with Lloyd's reagent; of plasma and on plasma deproteinized with (D) treated with Lloyd's reagent followed by ceric acetone. In these, salts are of course present and sulphate. these interfere with the obtaining of clear-cut No further reduction in the plasma creatinine chromatograms. However, at least two spots were value was obtained (Table I). detectable (Fig. 2). Attempts at de-salting without destruction of the creatinine were unsuccessful. Summary and Conclusions TABLE I An investigation of certain methods of plasma PLASMA CREATININE VALUES BY DIFFERENT creatinine determination is described. Treatment PROCEDURES (MG./100 ML.) with ceric sulphate was found to give a slight D reduction only of the " total" creatinine. The A B Reduc- C Reduc- Treated Reduc- Lloyd's reagent adsorption procedure was investi- "Total" After tion Treated tion with tion (Untreated Ceric (% B with (% C Lloyd's (% D gated chromatographically and only one Jaffe- Plasma) Sulphate on A) Lloyd's on A) and on A) Reagent Ceric positive substance, identical with that of creatinine Sulphate similarly treated, was obtained. 1 29 1-13 12 0 86 33 0 82 36 It is concluded that the Jaffe-positive material 1-27 1-00 21 0-77 39 0-78 39 method is 1 34 1-00 25 0.91 32 0-92 31 measured by the Lloyd's adsorption 1.11 0 92 17 0 80 28 0 84 24 creatinine and only creatinine.
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