354 Anallyst, June, 1968, Vol. 93, +jb. 354-362 An Enzymatic Spectrophotometric Method for the Determination of “Oxypurines” (Hypoxanthine plus Xanthine) in Urine and Blood Plasma BY RONALD A. CHALMERS AND R. W. E. WATTS (The Medical Professorial Unit, St. Bartholomew’s Hospital, West Smithfield, London, E.C. 1) A quantitative method for the determination of “oxypurines” (hypo- xanthine plus xanthine) in the presence of relatively high concentrations of uric acid, as in urine and blood plasma, is described. The oxypurines are separated from the uric acid and, with plasma, from proteins, on a cation-exchange resin, eluted and oxidised enzymatically to uric acid by using xanthine oxidase (xanthine : oxygen oxidoreductase, E.C.1.2.3.2). The uric acid is then determined by the change in the value of E,,,, which occurs when it is oxidised to allantoin by the highly specific enzyme uricase (urate : oxygen oxidoreductase, E.C.1.7.3.3). The accuracy and reproducibility of the method have been evaluated and a comparison with other methods is included in the discussion. THE oxidation of hypoxanthine (6-oxypurine) and xanthine (2,6-dioxypurine) to uric acid (2,6,8-trioxypurine) by xanthine oxidase (xanthine : oxygen oxidoreductase, E.C.1.2.3.2) is the last step in uric acid bi0synthesis.l Blood and urine concentrations of hypoxanthine and xanthine are increased, and the uric acid concentration decreased, when xanthine oxidase is congenitally absent .2 The enzyme can be inhibited in vivo by 4-hydroxypyrazolo(3,4-d)- pyrimidine (allopurinol) and by 4,6-dihydroxypyrazolo(3,4-d)pyrimidine (oxypurinol) , which are structural isomers of hypoxanthine and xanthine, respectively.Allopurinol is used clinically to reduce uric acid production in, for example, g o ~ t , 3 , ~ , ~ and it is sometimes necessary to determine the concentration of xanthine and hypoxanthine in plasma and urine when xanthine oxidase inhibitors are administered, as well as in patients with congenital xanthine oxidase deficiency (xanthinuria) . This paper describes the relatively simple, but precise, methods that we have developed for the determination of the oxypurines in biological fluids; the term “oxypurine concentra- tion” refers, in the present context, to the concentration of hypoxanthine plus xanthine. EXPERIMENTAL Hypoxanthine and xanthine (the “oxypurines”) are separated from almost all of the uric acid that is present in the urine or plasma by a column of cation-exchange resin in the hydrogen form.With plasma, the oxypurines are also separated from the plasma proteins, thus obviating the necessity for de-proteinisation by precipitation, or for dialysis. Uric acid and proteins both pass through the column under the conditions used. The oxypurines are eluted, evaporated to dryness, and the residue dissolved in dilute sodium hydroxide solution. The oxypurines in the solution are determined by initial oxidation to uric acid by xanthine oxidase, and the uric acid so formed is determined by measuring the change in extinction at 292 nm (E,,J that occurs when it is oxidised to allantoin by uricase (urate : oxygen oxidoreductase, E.C.1.7.3.3).Traces of uric acid remaining in the solution are determined separately and subtracted from the value obtained for the oxypurine content. METHOD APPARATUS- Chromatographic colzcmns-All-glass chromatographic columns were fabricated by Howard Rawson Ltd., 22 Middle Street, London, E.C.l, to specifications (i) and (ii) for the analysis of urine and plasma, respectively. (i) 20-mm internal diameter, 50cm long, fitted with a B24 standard ground-glass joint at the upper end. (ii) 11-mm internal diameter, 20 cm long, fused at the upper end to a 30-cm length of 20-mm internal diameter tubing fitted with a B19 standard ground-glass joint. 0 SAC and the authors.CHALMERS AND WATTS 355 Both types of column had sintered-glass discs (porosity 1) and removable spring-loaded Micro $i$ettes-Constriction pipettes, to deliver 0.05 and 0.1 ml, with a quoted accuracy Rotary Jilm evaporator-A suitable rotary film evaporator, EU050 (Wright Scientific Ltd.) ; Spectrophotometer-A Unicam SP500 spectrophotometer with matched silica cells of 1-cm stopcocks at their lower ends.of k0-001 ml, were used; obtainable from H. J. Elliot Ltd., E-Mi1 Works, Glamorgan. obtainable through Gallenkamp Ltd. light path and 4-ml nominal capacity was used. REAGENTS- Analytical-grade reagents were used whenever possible. Xanthine oxidase-A suspension of milk xanthine oxidase in ammonium sulphate solution was used; obtainable from Worthington Biochemical Co., Freehold, New Jersey, U.S.A., through Cambrian Chemicals Ltd., 73, Cherry Orchard Road, Croydon.The stated activity of this material was 10 units per ml, one unit being that amount which forms 1 pmole of uric acid per minute at 25" C from 20 pg of hypoxanthine per ml; 0-05 M phosphate buffer at pH 7.5.6 Portions of the xanthine oxidase were diluted to 1 in 40 with "Tris" [2-amino-2- (hydroxymethyl) -1,3-propanediol or tris (hydroxymethyl) amino- methane] buffer, pH 8-2, before use, both the dilution and suspension being stored at -20" C when not in use. Uricase-This was supplied by Leo Laboratories Ltd., Hayes Gate House, Uxbridge Road, Hayes, Middlesex. The contents of four ampoules were dissolved in 2 ml of Tris buffer (pH 8.2) to give a solution containing 150 units per ml, which was stored at -20" C when not in use.One unit of uricase is the amount that oxidises 1 pmole of uric acid per minute to allantoin at 25" C in 0.1 M borate buffer (pH 9.4) containing 20 pg of uric acid per m1.6 Xanthine-Obtained from British Drug Houses Ltd. Hypoxmthine-Obtainable from Sigma London Chemical Co. Ltd., 12, Lettice Street, London, S.W.6. Uric acid-This was further purified by recrystallisation, as described by Liddle, Seeg- miller and Laster.' Ion-exchange resin-Dowex AG 50W x 12, analytical-grade cation-exchange resin (200 to 400 mesh, H+ form) ; obtainable from Biorad Laboratories, Richmond, California, U.S.A., through V. A. Howe & Co. Ltd., 46 Pembridge Road, London, W.11. The resin was washed with several portions of 6 N hydrochloric acid, transferred into the chromatographic column, and washed with de-ionised water until the eluate was neutral, before use.The used resin was regenerated in the same way. "T~is" bu@r working solution (PH &2), 0.05 M-A 0.2 M stock solution of Tris buffer was first prepared by dissolving 2.423 g of tris(hydroxymethy1)aminomethane in 100 ml of de-ionised water. The working solution was prepared by adding 22.5 ml of 0.1 N hydrochloric acid to 25.0 ml of 0.2 M Tris buffer stock solution and diluting to 100.0 ml with water. The solutions were stored at 4" C. Hydrochloric acid, 6 and 0.1 N. Ammonia solution, 5 and 1 N. Sodium hydroxide solution, 0-01 N. De-ionised water was used throughout the determinations and was prepared by passing distilled water through an Elgastat Deioniser (Elga Products Ltd., Buckinghamshire).COLLECTION AND PRESERVATION OF SAMPLES- Urine-Collect the urine in a polythene bottle containing 10 ml of toluene as preservative. Store 24-hour collections of urine at room temperature between voidings to avoid loss of uric acid by precipitation. If the analysis is not performed immediately, add saturated lithium carbonate (0.5 ml per 50 ml of urine) and store at -10" C; thaw the sample in a warm water bath and then cool to room temperature before analysis. Samples stored in this way showed no loss of uric acid or oxypurines over a period of 6 months. Blood-Collect 20 ml of venous blood into a heparinised syringe through a needle filled with heparin solution (5000 units per ml). Transfer the blood into a dry centrifuge tube,356 CHALMERS AND WATTS : AN ENZYMATIC SPECTROPHOTOMETRIC [Analyst, VOl.93 chill on ice immediately, and separate the plasma by centrifugation for 10 minutes at 760 g (Gave measured at the centre of the centrifuge tube) and 5" C. Plasma may be stored at -10" C and any film formed on thawing separated by centri- fugation. In most of the present work the determinations were made after an 8-hour fast by the patient. Immediate cooling of the blood and prompt separation of the cells and plasma are essential to minimise the effect of the liberation of hypoxanthine from the cells, which begins immediately blood is drawn. PROCEDURE AND CALCULATION URINE- Adjust the pH of 25.0 ml of urine to between 1.5 and 2 by dropwise addition of 6 N hydrochloric acid, with narrow-range pH papers.Pass it through a column (19 x 130 mm) of cation-exchange resin (Dowex AG 50W x 12, 200 to 400 mesh) in the hydrogen cycle, and wash the beaker and column with four 25-ml portions of water, discarding the effluent and washings. Elute the oxypurines from the column with 400ml of 1 N ammonia solution (this step can conveniently be carried out overnight by using a 500-ml separating funnel as a reservoir). Evaporate 100 to 150-ml portions of the eluate to dryness in a long-necked, 250-ml round- bottomed flask (Quickfit and Quartz, FR250/3U) at a temperature of about 35" C by using a rotary film evaporator. Dissolve the residue in 5 ml of 0.01 N sodium hydroxide, warming slightly if necessary, and transfer the solution into a 25-ml calibrated flask with the aid of further portions of 0.01 N sodium hydroxide, finally diluting to 25.0 ml with water.Determine the oxypurine content of this solution by measuring the change in the extinction (E) at 292 nm resulting from the enzymatic reactions described below. Cuvettes additional to the assay cuvette are required to correct for changes in optical density that result from factors other than the enzymatic reactions. (a) Assay cuvette-To 24ml of Tris buffer (0.05 M, pH 8.2) in a silica cuvette, add 0.1 ml of the test solution and 0-1 ml of dilute xanthine oxidase. Mix by covering the cuvette with Parafilm (Gallenkamp Ltd.) and inverting several times, and read the value of E292 at 1 to 2-minute intervals until the reaction is complete (5 to 10 minutes). Record the final value of E292 [reading (i)].Add 0.05 ml of dilute uricase, mix and read the value of E29, after 5 minutes, 30 minutes, and every 10 minutes thereafter until the reaction is complete (usually 40 to 50 minutes). Record the final value of E292 [reading (ii)]. (b) Enzyme bZank czwette-Mix 2.9 ml of Tris buffer and 0.1 ml of dilute xanthine oxidase and read the value of E292 at the same time intervals as the assay cuvette during the xanthine oxidase reaction, recording the final figure [reading (iii)]. Add 0.05ml of dilute uricase, mix, and read at the same time intervals as the assay cuvette during the uricase reaction, recording the final value of E292 [reading (iv)]. (c) Reference cuvette-Use Tris buffer to set the spectrophotometer to zero. Concurrently determine the residual uric acid content (if any) of the test solution by the following modification of the method of Liddle, Seegmiller and Laster.' (a) Assay czcvette-Mix 2.9 ml of Tris buffer and 0.1 ml of the test solution, and record the value of E292 [reading (v)].Add 0.05 ml of dilute uricase and read the value of E292 every 10 minutes until the reaction (if any) is complete, recording the final value of E292 [reading (vi)]. (b) Enzyme blank cuvette-Mix 3.0 ml of Tris buffer and 0.05 ml of dilute uricase and read the value of E292 at the same time intervals as the assay cuvette, recording the final value of E292 [reading (vii)]. (c) Reference cuvette-This is the same as for the oxypurine determination. CALCULATION- (i) Residual uric acid in cuvette. AE292 = reading (v) - [reading (vi) - reading (vii)] = A .June, 19681 METHOD FOR THE DETERMINATION OF “OXYPURINES” 357 (ii) Oxypurines + residual uric acid.The true initial reading for uricase reaction = reading (i) - reading (iii) = (a). The true final reading for uricase reaction = reading (ii) - reading (iv) = (b). :. AE292 (oxypurines + residual uric acid) = (a - b) = B. (iii) Oxypurines in cuvette. AE292 (oxypurines) = B - A . Therefore, oxypurines in cuvette ( ~ 0 . 1 ml of urine) = as uric acid. (iv) Thus, oxypurine content of urine x - X- mg per ml, as uric 1000 0.1 ‘ I (B - A ) x 3.05 1 = [- 0.0745 acid, mg per ml, expressed as hypo- 1000 xm 168 (B - A ) x 3-05 1 X - 0.0745 xant hine. The results are normally expressed as milligrams per 24 hours. PLASMA- Dilute 4.0ml of plasma, contained in a 50-ml beaker, to about 20ml with water and adjust the pH to between 1.5 and 2 by the dropwise addition of 6 N hydrochloric acid, with narrow-range pH papers. Pass the diluted, acidified plasma through a column (11 x 100 mm) of cation-exchange resin (Dowex AG 50W x 12, 200 to 400 mesh) in the hydrogen form, wash the beaker and column with four 25-ml portions of water, and discard the effluent and washings.Elute the oxypurines with 200 ml of N ammonia solution and evaporate the eluate as described for urine. Dissolve the dry residue in 4.0 ml of 0.01 N sodium hydroxide, warming slightly if necessary. Determine the oxypurine content of the solution by a similar procedure to that described for urine, with the cuvettes below. (a) Assay cuvette-To 1.9 ml of Tris buffer in a silica cuvette, add 1-0 ml of the test solution and 0.1 ml of dilute xanthine oxidase, and mix.Read the value of E292 at 1 to 2-minute intervals until the reaction is complete, recording the final value of E292 [reading (i)]. Add 0.05 ml of dilute uricase, mix, and read the value of E292 after 5 minutes, 20 minutes, and every 10 minutes thereafter until the reaction is complete, recording the final value of E292 [reading (ii)]. The procedures for (b), enzyme blank and (c), reference cuvette are the same as those described for urine. CALCULATION- 1.0 ml of plasma), is calculated as described in paragraphs (i) to (iii) above for urine. The oxypurine content of the cuvette, from 1.0ml of the test solution (equivalent to Thus, the oxypurine content of the plasma x 100 mg per 100 ml, as uric acid, 1 = [ 0-0745 1000 (B - A ) x 3.05 X - mg per 100 ml, as hypoxanthine.168 (23 - A ) x 3.05 X- 0.0745 1000 RESULTS Preliminary experiments showed that the oxypurine-containing fraction from blood plasma was protein free, the plasma proteins being washed completely through the resin, together with almost all (more than 99 per cent.) of the uric acid, with three 25-ml portions of water. The recoveries of xanthine and hypoxanthine from aqueous solutions, urine and plasma were determined and found to be satisfactory (Tables I and 11).358 CHALMERS AND WATTS : AN ENZYMATIC SPECTROPHOTOMETRIC [AutdySt, VOl. 93 In Table I, results axe corrected, where necessary, for the original urine oxypurine content, determined by the same method.Urine analyses were carried out with a 24-hour collection of urine from a normal subject. Uric acid and oxypurine content (expressed as uric acid) of the urine was 608 and 13 mg per 24 hours, respectively. In Table 11, results are corrected for the original plasma oxypurine content determined by the same method. These analyses were carried out with plasma from a normal subject, with uric acid content of 6.6 mg per 100 ml and oxypurine content of 0.04 mg per 100 ml, both expressed as uric acid. The plasma contained 65.5mg per ml of protein (determined by Warburg and Christian’s method) .8 The reading method was calibrated with standard oxypurine solutions and found to give rwults ranging from 98-5 to 101.8 per cent. w/w (mean 9943 per cent.)..v/0 50 100 150 200 250 300 350 400 ’ 0 mg per 24 hours (expressed as uric acid) The reproducibility of the method for the determination of the Fig. 1. oxypurines (hypoxanthine plus xanthine) content of urine The reproducibility of the methods for urine and plasma are shown graphically in Figs. 1 and 2, respectively. The standard deviations (Note 1) for the duplicate determinations on urine and plasma were 1-92 mg per 24 hours, with a standard error of the mean of k0-16 (70 paired observations in the range 6-3 to 420.5 mg per 24 hours) and 0-0092 mg per 100 ml, with a standard error of the mean of + l o 0 (40 paired observations in the range 0.016 to 1.157 mg per 100 ml), respectively. NOTE 1- Standard dmdation = 0 = dz where d is the difference between duplicate determinations, and Standard error of the mean =(I where u is the standard deviation.n is the number of pairs of determinations.* 4%TABLE I RECOVERIES FROM AQUEOUS SOLUTIONS AND FROM URINE The urine contained 608 mg of uric acid and 13 mg of oxypurine (expressed as uric acid) per 24 hours Oxypurine Oxypurine added per 25 ml of water or urine, mg found per 25 ml Number of of final solution, duplicate Total, expressed expressed as Experiment determinations Hypoxanthine Xanthine as uric acid uric acid, mg A A queous solutions- 1 to 6 6 2.506 0 3.097 3.093 7 to 12 6 0 2.379 2.628 2.650 Uvine- 1 to 4 4 2.615 0 3.1 1 3.097 5 to 8 4 0 2.532 2.80 2.795 9 1 0.252 0.253 0.59 0.61 10 1 0.765 0.760 1.77 1-82 11 1 1.258 1.266 2-96 2.94 12 1 2.62 2.63 5-91 5.87 TABLE I1 Recovery, Mean recovery per cent.w/w and range - 99.9 - 100.9 (98.2 to 101.1) (99.3 to 102.6) - 99-6 - 99.8 (98.4 to 102.9) (96.8 to 103.9) 103.3 - 102.8 99.3 101.2 99.3 (99.3 to 103.3) Over-all mean recovery and range 3 8 - E 100.2 z ! U 100.4 r cl tJ (98.2 to 102.6) (96.8 to 103.9) Z 5 ij 1: 0 r 0- RECOVERIES OF OXYPURINES ADDED TO PLASMA The plasma contained 6.6 mg of uric acid and 0-04 mg of oxypurine (expressed as uric acid) per 100 ml, and 66.5 mg of protein per ml x Ox ypurine Oxypurines added per 4.0 ml of plasma, pg foundper4*0-d Number of A of final solution, r duplicate Total, expressed expressed as Recovery, Experiment determinations Hypoxanthine Xanthine as uric acid uric acid, pg per cent. w/w 1 to 4 4 20-0 0 24.7 25-18 - 5 to 8 4 0 20.0 22- 1 2146 - 9 to 10 2 10.0 10.0 234 23.5 100.6 11 to 12 2 20.0 20.0 46.8 46.0 98.4 Over-all mean Mean recovery recovery and and range range 101.9 (101.2 to 102.9) 98.9 100.1 (97.3 to 101.8) 994 (97.7 to 101.7) (97-3 to 102.9) W860 CHALMERS AND WATTS : AN ENZYMATIC SPECTROPHOTOMETRIC 0.9 0.8 - n s 0-7 - .- L 3 0.6- W E I k 0.5 - 9) v - E 0 04- E! L 0.3 - 2 0.2 - 04 - [Analyst, VOl.93 0 0.1 0.2 0.3 0.4 0.5 0.6 0-7 0.8 0.9 mg per 100 ml (expressed as uric acid) Fig. 2. The reproducibility of the method for the determination of the oxypurines (hypoxanthine plus xanthine) content of plasma DISCUSSION The determination of “oxypurines” (hypoxanthine plus xanthine) in urine or blood plasma usually has to be undertaken in the presence of relatively high concentrations of uric acid.This could theoretically be overcome by determining the uric acid content before, and after, oxidation of the oxypurines with xanthine oxidase, the oxypurine content being represented by the difference between these two values. Such a difference method has been used for urine,1°~11~12 but, in our experience, this is only reliable if the oxypurine excretion is considerably increased. It is more satisfactory to remove the uric acid, and this is most con- veniently done chromatographically, rather than by preliminary incubation with uricase.l3J* In the latter procedure, the uricase must be completely destroyed by the alkalinisation of the reaction mixture before the oxypurines can be oxidised with xanthine oxidase, and the uric acid so formed finally determined by the further addition of uricase; this involves several adjustments of the pH of the reaction mixture, which introduces further potential sources of error.Chromatographic separation of oxypurines and uric acid can be carried out by anion or cation-exchange methods. The use of an anion-exchange resin for this purpose16 is more complicated, and the resin cannot be regenerated. In acidic solution, the oxypurines act as cations because of their ability to take up protons on to the C=N-C groups to form the cationic C=N-C group. This is inhibited where the C-N=C group occurs because of I H tautomerism between this configuration and C-N-C. One (hypoxanthine) and two (xanthine) nitrogen-containing groups are inhibited in this manner in the oxypurines, and nearly complete inhibition occurs with uric acid, which has three tautomeric groups.Hypo- xanthine and xanthine are thus retained on the cation-exchange resin, while the extremely weakly cationic uric acid is washed through by water (H,O+). A cation-exchange resin was chosen for the present method, as it is not only suitable for the determination of oxypurines in urine but also for their measurement in blood plasma. These compounds are removed + I OH I ll H OJune, 19681 METHOD FOR THE DETERMINATION OF “OXYPURINES” 361 directly from the plasma by the resin, and the plasma proteins washed through the column with the uric acid. Preliminary experiments showed that this method of de-proteinisation provides a high degree of accuracy compared with methods that involve the use of protein precipitants.Trichloracetic and perchloric acids are subject to co-precipitation errors, and have to be neutralised or removed before chromatography or enzymatic analysis. The method is also considerably simpler than methods that involve dialysis,12 which take longer and may require larger samples. The absorption spectra of xanthine, hypoxanthine and uric acid overlap, as shown in Fig. 3; thus xanthine, or a mixture of the two oxypurines, cannot be determined by direct oxidation to uric acid and measurement of the increase in E292, because of the contribution of xanthine to the initial value of E292. Oxidation of xanthine to uric acid is accompanied by two changes in E292, a rise caused by the formation of uric acid and a fall by the removal of xanthine; hence, the over-all rise in E292, which occurs during the reaction, is lowered because of the opposing changes taking place.This overlap could explain the low recoveries of added xanthine that some previous workers have reported.3 Xanthine oxidase also oxidises a wide range of purines, pteridines and aldehydes, and the oxidation of some of these in urine and plasma could be accompanied by changes in E292. 0.300 0.250 - c / - , I \ 0 Wavelength, rnp Fig. 3. Absorption spectra of A, hypoxanthine; B, xanthine; and C , uric acid (concentration is 20 pmolar) in Tris buffer (0.50 M, pH 8.2) These difficulties can be overcome by determining the uric acid formed from xanthine and hypoxanthine by xanthine oxidase. This is effected by measuring the decrease in E,, that occurs when uric acid is oxidised to allantoin by uricase, this enzyme being highly specific with respect to uric acid.16 Tris buffer (pH 8.2) was chosen for use as the reaction medium because, at this pH, xanthine oxidase is reacting at its optimum pH, and uricase is sufficiently active to allow the reaction to go to completion in a reasonable length of time.In addition, the extinction coefficient of uric acid at 292 nm is the same at pH 8.2 as at pH 9.4, normally used for the assay of uric acid,’ and no variation in this factor for the calculations is required. No difference was observed between recoveries of uric acid carried out by using Tris buffer (pH 8.2) and glycine buffer (pH 9.4). The use of a single buffer and pH for both enzymatic reactions avoids the necessity for adjusting the pH of the solution between successive stages of the reaction, which has made some previous methods cumbersome in operation.12~13s14s16 The presence of uric acid in the final oxypurine-containing solution is checked as a matter of routine to confirm the efficiency of the chromatographic separation.Although at very low concentration, when present, its determination is of particular importance when low concentrations of oxypurines are being determined, the amounts in these instances often being about the same. It has been shown1’ that allopurinol and its metabolite, oxypurinol, occur in the urine, and presumably the plasma, of patients undergoing treatment with allopurinol. Both of362 CHAL.MERS AND WATTS these compounds are xanthine oxidase inhibitors and might be expected to interfere with the enzymatic reaction in the assay procedure. Preliminary experiments showed that there is complete recovery of hypoxanthine added to the urine and plasma of a patient being treated with allopurinol and that suflticient xanthine oxidase is present to overcome any possible inhibitory effects of the drugs (Note 2).It has also been ~ h 0 ~ n l 7 that allopurinol is retained on Dowex 50W x 12 and eluted with the oxypurines, and that oxypurinol passes through the column with the uric acid. More than 90 per cent. of the allopurinol is excreted as its metabolite1’ and is, therefore, removed during the chromatographic separation pro- cedure; this reduces the possibility of enzyme inhibition during the assay.The addition of xanthine oxidase and uricase to solutions containing only allopurinoll0 and oxypurinol, in the present investigation, did not alter Emz; this excludes the possibility of error caused by reaction between the drugs and enzymes. NOTE 2- The recoveries of 2 and 4-pg amounts of hypoxanthine in the presence of about 5 pg of oxypurines were determined on the contents of the assay cuvettes during the measurement of the oxypurine content of urine from a patient who was receiving 600 mg of allopurinol per 24 hours. The recoveries obtained were 100 and 104 per cent., respectively, indicating that little or no inhibition of the enzyme had occurred. The present work has been concerned with the determination of hypoxanthine and xanthine together, but in certain studies it may be necessary to determine these purines separately.It would be expected that, with interference caused by uric acid removed, the oxypurine-containing solutions from the present chromatographic separation would be suitable for the separate determinations of hypoxanthine and xanthine by differential spectr~photometry,~~ and this is being investigated. CONCLUSION his int collect grants 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 16. 16. 17. The enzymatic spectrophotometric determination of the oxypurines (hypoxanthine $Zus xanthine) described above for the analysis of urine and blood plasma is accurate, reproducible and highly specific. The method has given better recoveries than those previously reported for other methods, is reasonably economic and requires a minimum of working time, and is suitable for routine determinations.The method may be extended by further study to enable the separate determinations of xanthine and hypoxanthine to be carried out. We are pleased to acknowledge our gratitude to Dr. Gertrude B. Elion, who kindly made information available to us in advance of publication, to Professor E. F. Scowen for :rest, and to the Governors of St. Bartholomew’s Hospital for their generous research Our thanks are also given to the staff of our metabolic ward for their help in the on of the numerous samples for this investigation. REFERENCES Wyngaarden, J. B., in Stanbury, J. B., Wyngaarden, J. 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