Two dimensional thin-layer high-voltage electrophoresis and chromatography for the separation of urinary purines, pyrimidines and pyrazolopyrimidines

CLINICA

CHIMICA ACTA

TWO DIMENSIONAL

319

THIN-LAYER

AND CHROMATOGRAPHY PURINES.

H. ANNE

Wellcome (Received

PYRIMIDINES

HIGH-VOLTAGE

FOR THE SEPARATION

ELECTROPHORESIS OF URINARY

AND PYRAZOLOPYRIMIDINES

SIMMONDS

Research

Laboratories,

September

Biological

Division,

Bechenham,

Kent

(Great Britain)

27, 1968)

SUMMARY

A method, which is both rapid and quantitative, is described for the separation of purines, pyrimidines, and pyrazolopyrimidines by two dimensional thin-layer high-voltage electrophoresis and chromatography, following anion exchange. The observed electrophoretic and chromatographic mobilities, as well as the spectral data calculated for these bases from the behaviour of authentic compounds in the three systems, have been tabulated. The experimental conditions necessary to ensure the reproducibility of the method are defined.

INTRODUCTION

In recent years many drugs have been developed whose action is to inhibit one or more of the enzymes involved in the metabolism of purines and pyrimidines. The xanthine oxidase inhibitor, allopurinol (4-hydroxypyrazolo(3,4-d)pyrimidine) used in hyperuricaemia and gout, exemplifies drugs of this type. The excretion of purines and pyrimidines, in patients treated with such drugs, is of great interest and it is important therefore to develop a method by which these compounds may be separated simply and efficiently. A method which would enable the simultaneous study of the metabolic fate of the drug itself would be of even greater value. In the course of attempting to fractionate a mixture of purines and other U.V.absorbing compounds obtained by anion exchange’, the possibility of achieving effective separation of these compounds by high-voltage electrophoresis on thin layers, rather than filter paper, was investigated. This technique has been applied successfully to the separation of amino acidsa, and appears to have distinct advantages over the methods described for their separation on filter paper. This paper describes a method for the separation, identification and quantitation of purines, pyrimidines and pyrazolopyrimidines in three simple steps. The separation can be completed within 24 hours and the equipment used is available, or may be constructed, in most laboratories. The first step consists of the isolation C&z. Chim. Acta,

23 (1969) 3Ig-33o

SIMMONDS

320

and concentration of these bases into three separate fractions by anion exchange. Efficient separation of the majority of purines, pyrimidines and pyrazolopyrimidines is achieved in the next step by thin-layer high voltage electrophoresis of the three fractions at a pH of 8.65. Thin-layer chromatography following electrophoresis of individual fractions is used in the final step for the separation and concentration of bases which are not separated in the electrophoretic system. MATERIALANDMETHODS Step

Separation by mien exchange This procedure has been reported in detail in a previous paper’. A suitable aliquot of urine up to 25 ml (e.g. 5 ml in patients on allopurinol, 25 ml in untreated patients) was applied to the column at pH 10.0. After washing with IO ml of 0.1 N NH,OH and IO ml distilled water, the column was eluted with 0.01 N HCI and the U.V. absorption of the effluent monitored at 254 m,u using an LKB Uvicord I. The fractions were collected at a flow rate of 0.6-1.0 ml/min, using a Watson-Barlow flow inducer attached to a 15 mm x 45 cm QVF column containing 15 cm of Dowex I x 8 (IOO-zoo mesh) resin in the acetate form. The first U.V. absorbing peak in Fig. I consisted mainly of creatinine and was discarded. All the fractions from each of the next three peaks were combined in separate pools, A, B, and C, then concentrated under reduced pressure at 30-4o”, and the dry residue in each case dissolved in 0.5-2.0 ml of 0.1 N NH&OH with warming to 40’ approximately. I:

Step 2: Separation

by high-voltage

electrophoresis

Thin-layer plates were prepared, as described by Troughton et aL2 at a thickness of 250 ,L!.,and stored in a desiccator for at least one week before use. A perspex tank of the type described by Bieleski% was constructed to take either 20 x 20 cm or 20 x 30 cm plates. The tank was cooled by Esso “White Spirit Ioo”. The buffer was 0.04 M sodium borate-boric acid of pH 8.65. zo-120,~~l of each of the concentrates from Step I was applied with a Drummond microcap pipette (IO ~1) in three narrow bands corresponding to A, B and C, at the centre of the thin-layer plate. A strip of Whatman No. 3 filter paper, I cm wide, was placed over these bands to protect them while the borate buffer was sprayed evenly onto the thin layer up to each side of the strip. Joining of the two buffer fronts then served to condense the applied concentrates into very narrow bands. The wicks used were of 3 mm Whatman chromatography paper (20 x 6 cm). One edge of each wick was covered with cellophane tubing to prevent the onrush of buffer (dialysis tubing cut down the centre was used) and the wicks applied by this edge to opposite ends of the thin layer. A thin glass rod was used to hold each wick firmly in place. The temperature of the White Spirit was adjusted to 20~ and highvoltage electrophoresis carried out for 30 min at 75 V/cm. After drying in a stream of warm air the thin layers were examined with a U.V. light at 254 m/c, and the U.V.-absorbing or fluorescing areas outlined with a soft pencil. These areas and adjacent blank areas were scraped off into conical Pyrex centrifuge tubes, and eluted in 3 ml of 0.01 N HCI for 30 min with constant shaking. The tubes were then centrifuged at 3000 rev./min for IO min. Cl&Z.Chim. Acta, 23

(1969)

31’+330

SEPARATION

OF URINARY

PURINES

AND

321

PYRIMIDINES

The U.V. spectra of the supematants were recorded automatically using a Unicam SP 800 Spectrophotometer and silica cuvettes with a r-cm light path. The pH was altered to 12.by the addition of sodium hydroxide and the spectra recorded without removing the sample from the instrument. Concentrations of the purine and pyrimidine compounds eluted from the thin layer after ele~trophoresis were calculated from the extinction coefficients (E,, I mg/ IOO ml) at pH 2.0. The wave length calibration of the.Unicam SP 800 Spectrophotometer was verified with a Holmium filter. In patients where xanthine and hypoxanthine excretion was likely to be increased, such as those on treatment with allopurinol, only a few ml of urine was required. In these patients the excretion of xanthine and hypoxanthine as well as that of allopurinol, oxipurinol and its metabolites could be calculated directly from the absorbance of the eluates from the thin layer, since most of the other urinary purines excreted at levels of only a few mg/24 h would not be detected at this concentration. These minor urinary purines and pyrimidines and any other components not separated in the above steps were isolated and identified as follows. Step 3: Chromatography fo~~o~~~gHugh-voltageelectyophores~s In this case 30-60 ,ul of only one concentrate, i.e. A or B or C from Step I, was applied as a single narrow band approximately 3 cm in length at the centre of the thin-layer plate and 3 cm from the left hand side. After electrophoresis at 75 V/ cm as described, the plate was dried for 30 min in a stream of warm air and the purines located by examination under U.V. light. ~hromato~aphy was then carried out at right angles to the direction of electrophoresis using a solvent system consisting of: n-propanol-methylethylketone-water-ammonia (40: 30 : 20 :IO, v/v). When the solvent had reached the top edge (2-3 h), the plate was removed and dried in a stream of air. The U.V.-absorbing or fluorescing areas outlined under U.V. light were scraped off into conical centrifuge tubes and eluted in I ml of 0.01 N HCl with frequent shaking. After 30 min the tubes were centrifuged for IO min at 3000 rev./ min and the spectra of the eluted purines determined in the supematant at pH 2 and 12 using semimicro silica cuvettes with a r-cm light path, in an SF’ 800 spectrophotometer. Radioactive allopurinol and oxi~zcrinol determination by isotope dilution These experiments were carried out as described by Elion et al.&. Measured quantities of [6-1*C]allopurinol and [6-%]oxipurinol were added to aliquots of urine and determination of the oxypurines and pyrazolopyrimidines carried out as before. The radioactivity of the eluate from each band separated by high-voltage electrophoresis was measured by liquid scintillation counting in a Packard Tricarb counter. Radioactivity was found only in the two bands corresponding to allopurinol and oxipurinol. The amount of a~opu~nol or oxipurinol present in the sample was calculated from the dilution of the specific activity of the [14C]allopurinol and [“*C]oxipurinol and compared with the amount of these compounds found in an identical aliquot of urine by the chemical method.

Ck.

c!+n.

Acta, 23 (I&Q) 319-330

322

SIMMONDS

Chromatographic

behaviour and source of authentic compounds

The method was standardised with a known mixture of purines, pyrimiclines and pyrazolopyrimidines. Three different synthetic mixtures were prepared for this purpose from xanthine, hypoxanthine, allopurinol, oxipurinol, adenine, guanine, 7methylxanthine, pseudouricline, uracil, 5-acetylamino-6-amino-3-methyluracil and the compounds listed below: I-methylxanthine, guanine, 7-methylguanine, N2-methylguanine,

1/7dmethylxanthine,

I-methyl-

I-methylhypoxanthine and allopurinol I-ribosicle. These latter compounds plus the [Xlallopurinol and [14C]oxipurinol were the generous gift of Dr. G. B. Elion and Dr. G. H. Hitchings. I am also

indebted to Dr. K. Fink for the gift of the 5-acetylamino-6-amino-3-methyluracil used in this study. Other purines and pyrimidines were obtained commercially. RESULTS

Step

I:

E&ion

from

anion exchange resin

Fig. I shows the typical elution sequence, recorded at 254 mp, following the application of an aliquot of urine from a patient on treatment with allopurinol, to

90 100 g

I 5 z cj 6

EFFLUENT

VOLUME

(ml

I

Fig. I. Chromatographic separation of purines, pyrimidines and pyrazolopyrimidines on a Dowex-1 acetate column. Elution diagram at 254 m,u obtained from the urine of a patient treated with allopurinol.

the anion exchange resin. Hypoxanthine has been shown, by comparison with authentic compounds, to be elutecl in peak A; xanthine and allopurinol formed the major constituents of peak B, and oxipurinol was elutecl in peak C. Elution volumes in a given specimen were entirely reproducible in duplicate runs. A discussion of this step has been given in a previous publication’. The exact purine, pyrimidine and pyrazolopyrimidine content of peaks A, B and C, was determined by highvoltage electrophoresis in the next step. High voltage electrophoresis on thin layers A. Determination of experimental conditions. A series of experiments was carried out to determine the ideal conditions for the separation, by high-voltage electroStep

2:

SEPARATION

OF URINARY

PURINES AND PYRIMIDINES

323

phoresis on thin layers, of the urinary bases isolated in Step I. 0.04 M sodium borate/ boric acid buffer at pH 8.65 gave the best results

and the minimum

time required

for the separation of the bases at 75 V/cm was 30 min. The ideal temperature rise was found to be between 20-28’, with an associated increase in current from 20-38 mA. A much greater rise in temperature and current occurred, with cracking and lifting of the layer, when the thickness of the layer exceeded 250 ,LL The amount of concentrate applied was also a critical factor, and either irregular spreading of the bands or little movement from the origin resulted when the concentration of the individual bases applied to the thin layer was in excess of IO //g/cm. Providing all the conditions mentioned above were adhered to, excellent separation without spreading was obtained, not only for most of the purine bases, but for the pyrimidines and pyrazolopyrimidines

as well, as demonstrated

in Fig. 2.

Fig. 2. Photograph in U.V. light following thin-layer high-voltage electrophoresis of three fractions A, B, and C, which were obtained by anion exchange of the urine from a patient treated with allopurinol. The electrophoretic pattern was obtained in 0.04 M sodium borate/boric acid buffer pH 8.65 after electrophoresis at 75 V/cm for 30 min.

B. Detection and identification. Thin-layer high-voltage electrophoresis of the three urinary concentrates from Step I, under these conditions, resulted in the selective migration of the constituent bases in each concentrate toward either the anode or the cathode. Examination of the thin layer under U.V. light showed a series of narrow fluorescing or U.V.-absorbing bands at different distances from the origin in the case of concentrates A and B, while C usually showed a single fluorescing band. Fig. 2, a photograph taken in U.V. light, demonstrates the appearance of the thin layer following high-voltage electrophoresis of aliquots of the three concentrates Clin.

Chim.

Acta,

23 (1969) 319-330

SIMMONDS

324

Fig. 3. Electrophoretic mobility of authentic standards traced in U.V. light after thin-layer highvoltage electrophoresis at 75. V/cm for 30 min. Three mixtures were applied at the centre of a 20 cm x zo cm mate. Kev: r-Methylhypoxanthine rMeHx $acetylamino-6-amino-3-methyluracil AMeU Pseudouridine PSU Ad Adenine Oxipurinol OX Al Allopurinol Oxipurinol riboside OxR AlR Allopurinol riboside Xanthine X Guanine G I-Methylxanthine rMeX r-Methylguanine rMeG 7-Methylxanthine 7MeX 7-Methylguanine 7MeG I /7_Dimethylxanthine I /7MeX iV*-Methylguanine N*MeG Uracil U 8 .OH .7MeG S-Hydroxyq-methylguanine Hypoxanthine Hx

(A, B, and C) obtained pyrimidines separated

from the anion exchange procedure (Step I). The purines and in each concentrate were identified by comparison of their

behaviour in the electrophoretic system with that obtained for standard substances. Fig. 3 is a tracing of a thin-layer plate in U.V. light following the high-voltage electrophoresis of three different mixtures of authentic compounds. The mobility of each base was determined in separate experiments. The mixtures illustrated in this figure were chosen to demonstrate as effectively as possible the mobility of the individual bases and are not related to A, B and C in Fig. 2. The electrophoretic mobilities calculated for these compounds are given in Table I. From a comparison of the mobility of the authentic compounds with the mobility obtained, in a series of experiments, for the urinary bases contained in concentrates A, B and C it was found that the chief constituents of A were always hypoxanthine and pseudouridine, which were readily separated since the former migrated toward the cathode, the latter toward the anode. Adenine, guanine, the methylated guanines and xanthines, uracil and 5-acetylamino-6-amino-3-methylClin.

Chim.

Acta,

23 (1969) 319-330

32.5

SEPARATION OF URINARY PURINES AND PYRIMIDINES TABLE

I

SPECTROPHOTOMETRIC, TION

OF URINARY

ELECTROPHORETIC

PURINES,

Compound

PYRIMIDINES

Absorption

_

m,u pH N-Methyl-a-pyridone-5-carboxamide Adenine 5-Acetylamino-6amino-3-methyl uracil Guanine I-Methylguanine 7-Methylguanine Na-Methylguanine 7-Methylxanthine Hypoxanthine 1-Methylhypoxanthine Uracil Pseudouridine 6-Succinoaminopurine z-Dimethylamino-6hydroxy purine 7-Ribosyloxipurinol 1-Ribosylallopurinol I /7_Dimethylxanthine Allopurinol Xanthine I-~~ethylxanthine Oxipurinol 8-Hydroxy-7-methylguanine

2.0

AND

CHROMATOGRAPHIC

AND

PYRAZOLOPYRIMIDINES

rna~~rna

_~ 1% E max mp pH 1cm

12.0

258/&o-300

DATA

FOR THE

IDENTIFICATION

Peak of elution E~ect~opho~ei~c from Dowex r rn~b~~~~y in RF’“* borate buffer acetate column at pH 8.65

AND

ESTIMA-

V&des

f,K

262.j

0.95

269

Eluted with urea creatinine prior to peak A A +4.7 - 65 -

263.5 248.512755 251/274 249.51273 2511278 268 248.5

0.68 o.6g5 0.62~ 0.62~ o-748 0.61 0.80

265.5 2461273.5 2561276.5 244 1280 2451276 238/28g 259.5

A A A A A A A

-to.5 $4.6 +5.9 f5.3 +5-3 Nil +4.o

54 45 56 50 54 57 56

8.0s 9.213 10.4’ 9.4’

250 258 263

0.715 0.76 0.31

260 283 286

A A A

+6-4 +7.1 -2.9

64 61 31

9.1’ 9.513 9.69

27611

0.50~1 275rr

256/285r6 252** 25 I

0.24* 0.31

-3.6** -4.4

36** 41

8.6ra 8.83ia

269 250 267 267 252

+3.o +6.2

8.5’0 9.343’ 7-44’” 7.7l” 7.74=

248/2g4**

8.6’

258/280-300”

+ Migration towards cathode. - Migration towards anode.

9.813

Not available

245/27Q” 268** 271/286

Not available A & B** A&B

o.535 0.56 0.66 0.705 0.40

238/28Q 2521261

A&B B _

240.5[277.5

6

-6.0

2421277 242.5/267.5

B C

-4.2 -5.1

66 66 50 59 57

0.85~

24s/zs7**

c**

+3_6**

49**

* Arbitrary value, see text. * ** Solvent system : n-propanol-methyl** No authentic compound availethylketone-water-ammonia able for comparison. 40:30:20:10.

uracil, occurred also in fraction A, but in the experiment illustrated in Fig. 2, they were not present in sufficient quantity to be detected. Fraction B generally contained mostly xanthine, and in patients treated with allopurinol, allopurinol plus its ribosides. I- and x,+methylxanthine also occurred in this fraction when present in the urine. C usually contained oxipurinol only. Oxipurind riboside is recorded as present in A and B from spectral data alone, but no authentic compound was available for direct comparison. Likewise, what was assumed to be B-hydroxy-7-methylguanine was found in C in concentrated specimens, but no authentic compound was available for comparison, and both these compounds are indicated by dotted lines in Fig. 3 and 4. The purine and pyrimidine bases separated above appeared as dark U.V. absorbing bands on the thin-layer plate. Oxipurinol and its riboside, on the other hand, both fluoresced at 254 rnp. All other fluorescing compounds isolated on the thin-layer plate did not absorb U.V. light and have been disregarded. Clin. Chim. Acta, 23 (1969) 319-330

SIMMONDS

326

CATHODE

Fig. 4. Mobility of authentic compounds traced in U.V. light after chromatography for z’/* h in n-propanol-methylethylketone-ammonia-water following thin-layer high-voltage electrophoresis. The mixture was applied at the left hand centre of a 20 cm x 20 cm plate. Key: as for Fig. 3.

C. Spectrophotometric measurements and calculations. Experiments to determine the efficiency of elution from the thin layer in 0.01 N HCl, using authentic standards in the electrophoretic system alone, gave virtually 100% recoveries. Absorption maxima and extinction coefficients used for calculation were also determined when possible by the use of authentic compounds in the system, and the results recorded in Table I at pH 2 were measured in the 0.01 N HCl in which the bases were eluted from the thin layer. Where this was not done the data employed were derived from N o pure synthetic sample of oxipurinol other sources as indicated in Table 16~8~11*16~17. riboside was available and an arbitrary value was chosen for the extinction coefficient, by comparison with the values obtained from allopurinol and its riboside, to permit expression

of the results in mg.

Step 3: Chromatography

following

high-voltage

electrophoresis

Fig. 4, a tracing taken from a thin-layer plate examined under U.V. light, shows the position of authentic compounds before and after chromatography performed following thin-layer high-voltage electrophoresis. The time taken was 2-3 h. RF values obtained for these bases in the chromatographic system are listed in Table I. Investigation of the urines from patients not on treatment with any drug usually resulted in the appearance of many more bands in fraction A; this necessitated chromatography following high-voltage electrophoresis of this fraction alone. The urinary compounds were identified by comparison with the authentic compounds in Fig. 4. Table I gives a complete summary of the chromatographic and electrophoretic mobilities under the experimental conditions described, of most of the purine and Clin.

Chim.

Acta, 23 (1969) 319-330

SEPARATION

OF URINARY

PURINES AND PYRIMIDINES

327

pyrimidine bases which are known to occur in human urine or to be excreted as a result of treatment with allopurinol. The pK values listed in the last column of the table have been extracted from the literature as indicateds*7+13. RECOVERY

EXPERIMENTS

Non-radioactive

recovery eqberiments

Table II shows the results when a synthetic mixture consisting of allopurinol, oxipurinol, xanthine and hypoxanthine was subjected to anion exchange followed by high-voltage electrophoresis. In addition, recoveries obtained when these purines were added to an aliquot of urine from a gouty patient before and during treatment with allopurinol, are presented. Recoveries for xanthine, hypoxanthine, allopurinol and oxipurinol ranged from 85%-98%. Radioactive recovery experiments

Results obtained are shown also in Table II and are in agreement with the results obtained by the chemical recovery experiments. Mean recoveries for xanthine, hypoxanthine, allopurinol and oxipurinol from the combined experiments were 92.0%, 94.4%, 92.6% and g3.3o/o respectively. Autoradiography of the thin-layer plates showed that allopurinol was eluted exclusively in peak B and migrated towards the cathode while oxipurinol was eluted exclusively in peak C and migrated towards the anode. These two sets of experiments demonstrate that efficient separation and recovery of xanthine, hypoxanthine and the pyrazolopyrimidine bases allopurinol and oxipurinol may be obtained by the method described. REPRODUCIRILITY

OF RESULTS

Good agreement between individual results was obtained when specimens were analysed in triplicate or quadruplicate as indicated in Table III. No change in the level of oxypurines or pyrazolopyrimidines was found when these specimens were examined after 12 months storage at -IO’. Other purines and pyrimidines, TABLE

III

REPRODUCIBILITY

OF METHOD

An example of replicate determination of purine excretion in mg/z4 h Treatment

Allopurinol

Oxipurinol Nil

Allopurinol

* Determinations

54.8 55.5 55 54.5*

Oxipurinol

Xanthine

Hy@wanthine

156 150

44 39

20.2

lg.8

157*

43.5*

22.3*

370 361 363

4.9 4.9 5.1 127 127.5 132

5.5 5.8 5.1 29.3 30.1 30.0

367*

139’

32.4*

carried out after the specimens had been stored

12

months at -IO’.

Clin. Chim.

Acta,

23 (1969)

319-330

Clin. Chim.

Ada,

23 (xglig)

319-330

SEPARATION

OF URINARY

PURINES

AND PYRIMIDINES

329

and the ribosides of allopu~nol and oxipurinol were also determined and showed no change in the measured level after 12 months storage. DISCUSSION

Methods employed in the study of purine excretion generally use cation exchange techniques5r7ps and are either time consuming or involve the use of isotopes, while methods for the estimation of pyrimidine excretion usually employ anion exchange technique9. The methods described are generally unsuitable for the routine investigation of any variation in the daily excretion of these bases over an extended period. The present work is an extension of a method developed to study the effect of the hypoxanthine analogue, allopurinol, (4 hydrox~yrazolo(3,4-~)pyrimidine) on the excretion of xanthine and hypoxanthine I. The method described here, which enables the simultaneous study of the effect of this drug on the excretion of purines and pyrimidines, as well as investigation of the metabolic fate of the drug itself, utilises properties common to all these compounds for their isolation and identification. Purines, pyrimidines and pyrazolop~rrimidi~les are all heterocyclic bases which contain both keto and amino groups capable of ionisation. For the initial separation of these bases from other urinary constituents by anion exchange, use is made of their ability to form anions in strongly alkaline solutions. In the electrophoretic system, the direction and distance of migration of a particular base depends on the degree of dissociation of ionisable groups at any fixed pH. Ribosides on the other hand, form complexes with borate and migrate towards the anode. Buffers of different composition, varying in pH from 1.8-9.6, were originally tried for the high-voltage electrophoretic separation of a selection of the urinary compounds it was desired to isolate. Borate buffer 0.03 M, pH 9.2, gave the best results in initial experiments designed to measure the ratio of xanthine to hypoxanthine excretion in gouty uraemic patients .r9. In this system, however, it was found that hypoxanthine migrated first towards the cathode and then commenced to move slightly toward the anode resulting in spreading of this band. Marked deterioration, resulting in cracking and lifting of the layer, also occurred owing to the high voltage and the considerable rise in temperature which sometimes accompanied it, in the 45 min required for separation. The present buffer system which was designed to overcome all these difficulties has produced a much more efficient separation of the urinary bases with a considerable reduction in the time required to do so. More than 30 different solvent systems described by different workers were investigated for the separation of the bases by thin-layer chromatography following high voltage electrophoresis 4sr2J6. Spreading, or poor separation, resulted with the majority of solvents, probably due to the presence of borate in the thin-layer. Good separationwasobtainedwithtert. butanoI-methylkethylketone-water-ammonials, but the time taken was 4-5 h. The present system was developed and found to give comparable separation in 2-3 11.

C&-z. China. Acta, 23 (1969) 31g-330

SIBIMONIX

330 ACKNOWLEDGEMENTS

I am indebted to Dr. T. Hanley for much valuable discussion, to Dr. J. D. Wilson and to Mrs. R. St. C. Lindop for helpful advice and to Dr. W. I;. Duncombe who kindly performed the radioisotope experiments. The urine specimens of patients receiving allopurinol or oxipurinol were made available through the co-operation of Dr. R. W. E. Watts. Miss J. Ansell and Miss M. Streeton typed the manuscript. REFERENCES I 2 3 4 5 6 7 8 9 IO II 12

H. A. SIMMONDS AND J, D. WILSON, Clin. Chim. Acta, 16 (1967) 155. W. D. TROUGHTON, R. ST. C. BROWN AND N. A. TURNER, Am. J. Cl&. Pathol., 46 (1966) 139. R. L. BIELESKI, Anal. Biochem., 12 (1965) 230. G. B. ELION, A. KOVENSKY, G. H. HITCHINGS, E. METZ AND R. W. RLJNDLES, Biochem. Phawnacol., 15 (1966) 863. B. M. BOLLARD, R. H. CULPAX, N. MARKS, H. MCILWAIN AND M. SHEPHERD, J. Mental Sci., 106 (1960) 1250. K. FINK, W. S. ADAMS AR‘D W. A. PFLEIDERER, J. Biol. Chem., 239 (1964) 4250. B. WEISSMAN, P. A. BROMBERG AND A. B. GUTMAN, J. Biol. Chem., 224 (1957) 407. B. WEISSMAN, P. A. BROMBERG AND A. B. GUTMAPS,J. Biol. Chem., 224 (1957) 423. IV. E. COHN, J. Biol. Chew, 235 (1960) 1488. A. G. OGSTON, J. Chem. Sot., (1935) 1376. C. E. CARTER, J. Biol. Chew, 223 (1956) 139. T. A. KRENITSKY, G. B. ELION, R. A. STRELITZ AXY~)G. H. HITCHINGS, J. Biol. Chem., 242

(1967) 2675. 13 K. BURTON, in R. M. C. DAU’SON, D. C. ELLIOTT, W. H. ELLIOTT AND K. M. JONES (Eds.), Data for Biochemical Research, Oxford lrniversity Press, London, 1959, p. 74. 14 J. D. WILSON, H. A. SIMMONDS AND J. D. K. NORTH, Ann. Rheumatic Diseases, 26 (1967) 136. 15 K. FINK AND W. S. ADAMS J. Chromatog., 22 (1966) 118. 16 K. FINK, W. S. ADAMS, F. W. DAVIS AND M. NAKATANI, Cancer Res., 23 (1963) 1824. 17 W. F. ADAMS, S. DAVIS AND M. NAKATASI, Am. J. Med., 28 (1960) 726. Clin. Chim.

Acta,

23 (1969)

319-330