Urinary excretion of purines, pyrimidines and pyrazolopyrimidines in patients treated with allopurinol or oxipurinol

CLINICA

353

CHIMICA ACTA

URINARY

EXCRETION

PYRAZOLOPYRIMIDINES

OF PURINES,

PYRIMIDINES

IN PATIENTS TREATED

AND

WITH ALLOPURINOL

OR OXIPURINOL

H. ANNE

SIMMONDS

Wellcome Research Laboratories (Received

September

(Biological

Division)

Beckenham,

Kent (U.K.)

27. 1968)

SUMMARY

When allopurinol was administered to three patients with good renal function, two suffering from gout and one a xanthinuric, the metabolites recovered in the urine (76% of the dose) consisted of oxipurinol (73.6%), allopurinol (10.4~/~), allopurinol riboside (12.5%) and oxipurinol riboside (3.5%). Only 36 ‘A of an equivalent dose of oxipurinol was recovered daily from the urine of the same patients, chiefly in the form of unchanged oxipurinol (94.6%), th e remainder being oxipurinol riboside. In the two gouty patients the urinary excretion of xanthine and hypoxanthine was increased during drug treatment and the ratio of xanthine to hypoxanthine excreted was markedly altered. Prior to treatment these two patients had a xanthine/ hypoxanthine ratio of 0.68 which increased to 4.15 on allopurinol and I.75 on oxipurinol. The xanthine/hypoxanthine ratio in the patient with xanthinuria was approximately 5 and not markedly altered throughout the study. In all three patients the pattern of excretion of other urinary purines was not altered by treatment with allopurinol or oxipurinol. A dietary origin is suggested for and 5-acetylamino-6-amino-3-methyluracil r-methylxanthine, 7-methylxanthine since these compounds were not excreted when purine intake was restricted. The excretion of pseudouridine was excessive in two patients, irrespective of diet. A remarkable finding in these two patients was the replacement of urinary pseudouridine by an equivalent amount of uracil during separate periods of several consecutive days.

Until recently, few studies had been carried out on the excretion of purines other than uric acid in man, largely owing to the difficulty of isolation and identification of these compounds. Since the development of more sensitive techniques, investigations of both purine and pyrimidine excretion have been reported in health and in a variety of diseases by a number of workers l-12. These techniques involve either a number of steps or the use of isotopes and are not convenient for the investigation of the daily excretion of these compounds. Long term studies with allopurinol, 4-hydroxypyrazolo-(3,4_d)pyrimidine, which acts on the last enzyme of purine catabolism, xanthine oxidase, have resulted Clin. Chim. Acta, 23 (1969) 353-364

354

SI~lMONDS

in the widespread investigation of the effect of this drug on the excretion of the oxypurines, xanthine, hypoxanthine and uric acid 13--20.Allopurinol is rapidly oxidised in vivo to oxipurinol, 4,6-dihydroxypyrazolo-(3,4-d)pyrimidine, and is excreted in man as this metabolite, together with small amounts of the ribosides of allopurinol and oxipurinol 21--23 . However, no long term investigation of the 24-h urinary excretion of these metabolites has so far been reported, possibly again due to the tedious nature of the reported isolation procedures. The development of a simpler method”* has made possible investigation of the variation in 24-h urinary excretion not only of purinesz6 but also of pyrimidines and of pyrazolopyrimidines in patients treated with xanthine oxidase inhibitors. The present report is concerned with the application of this method to the investigation of the urinary excretion of these compounds in patients being treated with allopurinol and oxipurinol, during periods of restricted and unrestricted purine intake. CLINICAL AND BIOCHEMICAL

METHODS

The two patients, B. and G., included in this report are patients with gout in whom clinical studies of the effect of allopurinol and oxipurinol on total oxypurine excretion have already been published. (Gout patients I and 2 respectively, Chalmers et ~l.~~.) Both patients were given a low purine diet throughout the study which included four consecutive periods. Patient B. who had been on allopurinol for 207 days when the low purine diet was introduced continued on allopurinol for a further 6 days. He then had IZ days without xanthine oxidase inhibitors, 12 days of oxipurinol therapy and 14 days without xanthine oxidase inhibitors. Patient G. had a 5-day control period, 7 days of oxipurinol treatment, II days without treatment and finally 8 days on allopurinol. Their inulin clearances were 89 ml/min/r.73 ma in the case of B., and 66 mljmin/r.73 m2 in the case of G.28. The third patient, L.B., a xanthinuric, was given ailopnrinol for 3 weeks whiie on a pm-me-free diet, followed by a z-week control period on an unrestricted diet, after which oxipurinol was given for a further two weeks. The inulin clearance, as determined by Chalmers et al., was III ml/min/r.73 m2. All three patients who received allopurinol and oxipurinol in doses of 600 mg per day (4.51 and 3.94 mmoles respectively) during each period of study, were patients of Dr. R. W. E. Watts, The Medical Professorial Unit, St. Bartholomew’s Hospital, London. A detailed investigation was carried out in the case of patient B. Specimens were analysed daily during the two periods of drug therapy and at 3-4-day intervals throughout the control period. In the case of G. and L.B., specimens were investigated towards the middle and at the end of each period of therapy. Aliquots of 24-h urine specimens, collected as describedaa, were stored at --IO’ until required for use. The compounds investigated have been found to be stable under these conditions for periods of at least 12 months2*. The purines, pyrimidines and pyrazolopyrimidines in these specimens were separated by specific adsorption of from 5-25 ml of urine onto an anion exchange resin and were quantitatively eluted in three separate U.V.-absorbing peaks, A, B, and Clin.

Chim.

Acta, 23 (1969) 353-364

URINARY

PURINES

IN PURINOL-TREATED

PATIENTS

355

CSQtas.Pooled fractions of these peaks were concentrated and separated into the constituent purine, pyrimidine and pyrazolopyrimidine components by thin-layer highvoltage electrophoresis in sodium borate/boric acid buffer at pH 8.65 for 30 min at 75 V/cm. Chromatography in the second dimension was then used to separate any compounds not already separated by the previous techniques. RESULTS

The 24-h urinary excretion of pyrazolopyrimidines in the two patients with gout and one with xanthinuria has been studied during periods of therapy with either allopurinol or oxipurinol and is reported in Tables I and II. il. The excretiolz of metabolites of allopurinol. The mean 24-h urinary excretion of allopurinol and its metabolites in the three patients was as follows: allopurinol 0.34 mmole, allopurin~l riboside 0.42 mmole, oxipurinol 2.46 mmoles, oxipurinol riboside 0.12 mmole. This represents a mean total excretion of 3.34 mmoles, the equivalent of approximately 767; of the dose of allopurinol administered. The principal excretion product was oxipurinol (mean 56% of the dose), the excretion of which increased for the first four days and then remained at about 400 mg/24 h (2.60 mmoles). Allopurinol and its riboside were excreted in approximately equimolar amounts; together they amounted to 18% of the allopurinol dose administered. When TABLE

I

24-h EXCRETION

OF ALLOPURINOL,

OXIPURINOL,

AND

THEIR

RIBOSIDES

IN

THREE

PATIENTS

TREATED

WITH

ALLO-

PURINOL

Patient

Treatment Duration,

B.’

--

Al~o~uy~no~ Dosage

mg mmole

days

mgl24

206

600

60

4.41 mmoles

56 53 42

207 208 209 7.10

46 46

Mean Nil

Nil Nil Nil

600 4.41 mmoles

39 55

Mean L.B.**

II

600

14

4.41

16

Mean Mean overall

* Low-puke ** Puke-free

33 mi-llOk?S

26

‘5 57

I7

Oxipzlrinol

mmole mg

mg

---

Ox~purimol

Total

riboside mmole

mg

mmole

mmoles

I.93 2.77 2.92

39 2;

3.01 3.80

2.86 2.58 2.45 2.59

67 46 4’

0.14 0.15 0.24 0.24 0.16 0.14

0.70

27 %

0.10 0.08

r8.2 25.5

0.06 0.09

h

211

c.*

AE~o@~~~oE riboside

0.44 0.41

133 126

0.50 0.47

0.39 0.31

114

o-43 0.42

0.34

87 82

0.34 0.37

112

0.32 0.31 0.42

Nil Nil Nil

0.29 0.40 0.35

106 I67

0.24 0.19 0.11

IO1 50 66

0.38 0.18 0.25

0.42 0.30

132

0.49 0.33

0.34

0.40 0.62 0.51

0.42

294 422 444 435 392 372 107 38 ‘3

0.25 0.09

306 365

2.01 2.40

0.18

0.08

2.21

420

2.76

45

0.16

320 325 515

2.11

32

O.II

2.14 3.39

gc~t deZZed

3.98 3.82 3.40 3.24 3.54

2.76 3.51 3.14

2.60

0.11

3.54 2.59 2.57 4.30 3.25

2.46

0.12

3.34

diet. diet. C&z. Chim. Acta, 23 (1969) 353-364

356

SIMMONDS

TABLE 24-h

II

EXCRETION

OF OXIPURINOL,,

AND ITS RIBOSIDE

IN THREE

PATIENTS

TREATED

Patient -.-.B.*

days 2

600

128 218

9

3.94 mmoles

232

II

222

I2

221

Mean hTil

I

2.

3 4 5 9 13

0.84 1.43 1.53 I .46 I.45 1.47

izl

0.05 -

5 51 40

0.02

= 45

I20 102

0.79 0.67

39 19 5

::

0.48 0.41 0.13 0.06

41 Nil Nil

20

g 600

128

0.84

3.94 mmoles

153

I.01

IO.4

187

1.23

12.1

IO

Nil

Nil

I2

600 3.94 mmoles

215

4-3

1.12

--

0.14 0.07

0.89 1.43 1.55 1.64 1.59 1.55

0.02 0.02

3 5 8

Xean overall

0.18 0.14 0.11

220

Mean L.B.**

OXIPURINOL

Total mmoles

5

G.*

WITH

-

0.02

0.86

0.04 0.04 0.04

1.05 1.27

0.08

I.50

0.08

1.42

1.16

Nil 1.42

1.34

22

* Low-purine diet. ** Purine-free diet.

the drug was withdrawn, the excretion of allopurinol and its riboside ceased immediately while the output of oxipurinol fell gradually to zero in x0--14 days. B. Tke excretiolz of ~etab~~~tes of ~x~~~r~no~. In the period when oxipurinol therapy was given, an average 1.34 mmoles of the drug was recovered unchanged per 24 h. The mean excretion of oxipurinol plus its riboside, the only metabolite, represented 36% of the oxipurinol dose administered daily. As with allopurinol, the excretion of oxipurinol increased during 4-5 days to reach approximately 200 mg/z4 h (1.3 mmoles), or about 33% of the dose; the excretion then decreased slowly after the drug was withdrawn. In patient B, there was still some excretion of oxipurinol even after IO days (see Fig. I). C. The identification. of oxi@.~i~zod in the urine of a patient with xanthinuria. During preliminary anion exchange fractionation of urine excreted by the xanthinuric patient L.B. while on allopurinol, the W.V. elution trace at 254 m/c consistently recorded the three peaks A, B and C. Peak C, which is predominantly oxipurinolz~ and consequently found only in the anion exchange eluate of urine from patients treated with either allopurinol or oxipurinoi, would not be expected in the urine of a patient with xanthinuria. Thin-layer high-voltage electrophoresis of the concentrate obtained from the pooled fractions of this peak produced a single compound which migrated 5.1 cm toward the anode and fluoresced in U.V. light. (Allopurinol by contrast is eluted in peak B, is U.V.-absorbing and migrates towards the cathodea4). The identity of this metabolite as oxipurinol was established unequivocally by comparison of its infra-red spectrum with that of authentic oxipurinol, as demonstrated in Fig. 2. %?a. Chim. Acta, 23 (1969) 353-364

URINARY tLow

PURINES IN PURINOL-TREATED

PATIENTS

357

putinediet

ALLOPURINOL

RISOSIDE

OXlPURlNOL

ALLOPURINOL

-i

OXIPURINOL

PSEUDO URIDINE

Fig. I. The q-h urinary excretion of purines, pyrimidines and pyrazolopyrimidines in the gouty patient B. treated with allopurinol or oxipurinol, 600 mg/day, for the periods indicated. The patient was on a low-purine diet throughout. Vertical base lines represent two-day intervals. The units employed for total oxypurine excretion are not to scale. These values are computed from the xanthine/hypoxanthine values recorded in this study and the uric acid values of Chalmers et al.rs, and are included purely for comparison. 2. Purine

(a)

excretion The excretion of xalzthine and hypoxanthitie in $atients treated with allo$winol

and oxipu~inol. Mean xanthine and hypoxanthine excretion levels have been calculated for the two gouty patients from a series of estimations of which only a few representative values are recorded in Table III. The ratio of the mean excretion of xanthine to hypoxanthine was 0.68 before treatment. The output of both xanthine and hypoxanthine was increased during allopurinol treatment, but the increment of xanthine was about six times that of hypoxanthine (xanthine/hypoxanthine ratio 4.15). During oxipurinol treatment, on the other hand, the increase in xanthine excretion was only 2.5 times the increase in hypoxanthine excretion (xanthine/hypoxanthine ratio 1.75). In patient 3. the excretion of xanthine deciined rapidly in the first 3 days after allopu~nol was stopped, while the excretion of hypoxarlthine continued relative-

C&z. Chim. A&,

23 (1969) 353-364

SIMMONDS

358

Fig. 2. Infra-red spectra, in two concentrations in KC1 discs, of the metabolite recovered from the urine of a patient with xanthinuria during therapy with allopurinol. The spectra were measured in a Unicam SP IOO infra-red spectrometer from 650-3670 cm-i (change of scale at aooo cm-i). The metabolite was isolated and identified by the method described in the text and was recrystallised twice in 0.01 N HCl. The first spectra are reference spectra of authentic oxipurinol at comparable concentrations.

ly unchanged. The 24-h excretion levels of both xanthine and hypoxanthine fell slowly thereafter, to reach pretreatment figures in the next seven days. For the first 4 days after oxipurinol was stopped, on the other hand, both xanthine and hypoxanthine continued to be excreted in the urine at the levels attained during oxipurinol therapy. The daily excretion of xanthine and hypoxanthine subsequently fell to pretreatment levels again over a period of seven days. Neither allopurinol nor oxipurinol caused any marked change in the ratio of xanthine to hypoxanthine excretion in the xanthinuric patient L.B., after 2 to 3 weeks on either drug. The daily excretion of “oxypurines” (xanthine plus hypoxanthine) as determined by thin-layer high-voltage electrophoresis in this study, has been recalculated in terms of uric acid and compared with results obtained on the same specimens by an enzymatic method (Chalmers et ~1.~~). Good agreement was obtained, as shown in Table III. (b) The excretiolz of @urines other than xanthine and hypoxantlaine. The urinary purines other than xanthine, hypoxanthine and uric acid were also separated by the technique of two-dimensional thin-layer high-voltage electrophoresis used in this study, and their excretion was investigated simultaneously. A detailed investigation of the daily excretion of these purines was carried out in the case of patient B. The methylated guanines (go% of which was 7-methylguanine) made up approximately 800/, of the daily excretion of these “non-uric acid” purines. The remaining 20% Clin.

Chim.

Acta,

23 (1969) 353-364

2 w

;;

Ox

Nil Al Al Al

Nil r Ox OX 1 Al I Al

I

Run in on diet Purinefree diet ( Nonrestricted

Lowpurine diet

Al Al Al Nil OX OX Nil Nil

Al

I2

4 II I4 17

5 3 5 4 8

1340

965 1320

1490 1360

1530

3070 2260 1580 2180

3140 3020 3280

3580 2gEo

1960

3480 2900

206

1870

207 209 3 9 II 4 9

II0

EXCRETION

Volume

276

264 253 193 322 92

47.5 45.2 19.3 58

23 29.8

'I3 128.8

7.4

6.5 7.4 18 20.1

17.5 14.5 19.6

22 34 21.3

3.7 35 41.5

46 28.6

180 25.5

THREE

X+Hx**

418

351 336 239 427

71 153 179

13

61

226 248 256 64 46 57 48 I7

283

h) IN

26 45

53

Hz*

(mg/2q.

‘75 I74

196

X

* X resp. Hx = Xanthine resp. hypoxanthine. ** Calculated as uric acid. *** 5-acetylamino-6.amino-3-methyluracil.

G.

I

Nonrestricted Run in on diet Lowpurine diet I

Day’

PYRIMIDINE

Drug

AND

B.

PURINE

Diet

URINARY

Patient

III

TABLE ON

61

409

325 324 214 399

I69 183

‘4 48

236 66 74 94 42 ‘7

322

X+-H%**=

PATIENTS

37.2

54.5 Nil Nil Nil

16.6 21.5 35.0 27.0 36.6

19.6 17.0 14.0 IO.1 7.5 9.8 7.3 9.8

9.0

15.2 11.5 IO.3 9.5 14.6 10.6

21.0

14.2

12.6

Methylated guanines

ALLOPURINOL

7-Methyl xanthine

WITH

AJD

OXIPURINOL

99

82 99 78 Nil

32 49.5 44.5 52.0 62.0

Nil Nil I34 128 Nil 48.8 I34

129

126

Pseudouridine

9.7

13.7 10.7 II.5 51.7

3.7 I3 7.3 13.5 10.9

12.3 75.0 65.0 11.8 10.5 63 44.5 5.6

18.7

Uracil

Nil

Nil Nil Nil Nil

Nil Nil Nil Nil Nil

56 Nil Nil Nil Nil Nil Nil Nil

134

A MeU***

1 Refers to length of time on particular form of treatment. 2 Determinations carried out by Chalmers et al. using an enzymatic method.

Nil

Nil Nil Nil Nil

Nil Nil Nil Nil Nil

Nil Nil Nil Nil Nil Nil Nil Nil

40

r-Methyl xanthine

TREATMENT

360

SIMMONDS

consisted cheifly of guanine, adenine, 8hydroxy-7-methylguanine and 6-succinoaminopurine, at excretion levels of less than 2 mg/24 h. Identification of the last two compounds has been based on their U.V. spectra alone as no authentic compound was available for comparison. Calculation of the daily excretion of these compounds was confined to the urine specimens investigated in the earlier part of the study. As no gross abnormality was noted, only the absorption spectra and extinction maxima have been recorded for these purines in the latter part of the study, in order to watch for any possible

alteration

in their 24-h excretion.

3. Urinary compounds of dietary origin Purines of dietary origin were also readily separated and identified by the method used. The daily excretion of these compounds is shown in Table III. 7-Methylxanthine was excreted throughout the study by patients B. and G., although both were on low purine diets, and was also excreted by patient L.B. during the period of stabilisation on a purine-free diet. 7-Methylxanthine was not excreted by patient L.B. during the period of maintenance on a purine-free diet, but reappeared in the urine when he later reverted to an unrestricted diet. I-Methylxanthine was excreted in large amounts by patient B. on an unrestricted diet, but was not found in the urine of B., G. or L.B. when their purine intake was restricted. In patient B., 5acetylamino-6-amino-3methyluracil was excreted in large amounts when the purine intake was not restricted. Its excretion fell during the period of stabilisation on a low-purine diet, and it was not excreted at all during dietary restriction. This compound was not present in the urine of patient B. ,who had been stabilised on a lowpurine diet before

the investigation,

even on an unrestricted

or in the urine of the xanthinuric

patient L.B.

diet.

4. Pyrimidine excretion Urinary pyrimidine excretion by the 3 patients is shown in the last three columns of Table III. In patient G., the excretion of pseudouridine 46 mg/24 h, was within the normal range of 24-75 mg/24 h a7, but in the two patients B. and L.B. the 24-h urinary excretion was raised to 134 and 90 mg respectively. The level of 24-h urinary pseudouridine excretion was relatively constant in each patient, independent of diet. An unusual finding in patients B, and L.B. was the replacement of pseudouridine by uracil, as shown in Table III, and by the more detailed study of patient B. shown in Fig. I. In this patient the substitution of pseudouridine excretion (0.55 mmole) by uracil (0.54 mmole) occurred on two occasions while on a low purine diet. This occurred on the first occasion during allopurinol therapy and continued for several days. Pseudouridine excretion began to revert to normal before the last day of therapy, and the urine on this occasion contained equal amounts of pseudouridine and uracil. Pseudouridine excretion reverted completely to normal within the next 24 h. The same pattern occurred in this patient during oxipurinol therapy, but the substitution of uracil in this instance continued for several days after oxipurinol therapy had ceased and, as on the previous occasion, pseudouridine excretion reverted to normal in 48 h. In patient L.B., on a purine-free diet, uracil (0.42 mmole) again replaced pseudouridine (0.37 mmole) during a period of allopurinol therapy.

Clin. Chim. Acta.

23 (19691 353-364

URINARY

PURINES

IN PURINOL-TREATED

PATIENTS

361

DISCUSSION

The excretion of allopurinol and its metabolites in the urine of 3 patients with good renal function, at a remarkably constant daily level approximating 76% of the dose, is indicative of the relatively efficient absorption of this compound from the gastrointestinal tract. The recovery of only 36 “/ of an equal oral dose of oxipurinol as urinary metabolites, suggests that oxipurinol was not as efficiently absorbed from the gastrointestinal tract as was allopurinol. Chalmers et dZ6 investigated the effectiveness of both drugs in reducing the uric acid excretion of the same two gouty patients included in this study and found that oxipurinol was two thirds as effective as allopurinol in equal oral dosage. However, the above results for urinary pyrazolopyrimidine excretion suggest that only half the amount of oxipurinol was actually available for xanthine oxidase inhibition during oxipurinol, as compared with allopurinol, therapy. Allopurinol was readily oxidised and excreted chiefly as oxipurinol in these patients. The small quantity of allopurinol riboside excreted daily approximated to the amount of unchanged allopurinol and these two compounds together constituted 23% of the urinary pyrazolopyrimidine excretion. Despite widely differing periods of allopurinol therapy the relative proportion in which the drug and its metabolites were excreted in the urine was comparable in all three patients so that no decrease in the rate of conversion of allopurinol to oxipurinol was observed with time as has been suggested in other report+Tz3. A solubility in urine of 20 mg/roo ml has been reported for oxipurinoll@, so that patients constantly excreting a fairly acid urine, and a mean 56% of the dose of allopurinol as oxipurinol, could exceed this limit, unless drug dosage and fluid intake are controlled. The absence of allopurinol and its riboside from the urine 3 days after allopurino1 therapy was stopped, indicates that both these compounds were cleared more rapidly by the kidney than was oxipurinol, which could be detected in the urine up to

14 days after the cessation of either allopurinol or oxipurinol therapy. The slow rate of elimination of oxipurinol from the body is compatible with the finding reported by others that oxipurinol is handled by the human kidney in a manner similar to uric acid21,“3. From a study of the excretion of IO different urinary purines in the 2 gouty patients reported in this paper it appears that therapy with allopurinol or oxipurinol affected only the excretion of the 3 urinary purines, xanthine, hypoxanthine and uric acid, and this to differing degrees with either drug. Several interesting points emerge from a comparison of the present results with those obtained in the same patients by Chalmers et aLz6. They found that the increase in xanthine plus hypoxanthine excretion produced by allopurinol was approximately 56% of the decrease in uric acid excretion. The increase produced by oxipurinol, on the other hand, was only 29% of the uric acid decrease so that a net deficit in total oxypurine excretion of equal magnitude actually resulted with either drug. Other workers have also found a net deficit in total oxypurine excretion during allopurinol therapy13~14~17~33.Reutilisation of xanthine and hypoxanthine by the body salvage mechanismsas+, or inhibition of de novo purine synthesis by accumulating nucleotides s1 has been postulated to account for this deficit. These pathways are shown schematically in Fig. 3. The existence of a more efficient salvage mechanism for hypoxanthine32, associated with stimulation of Clin. Chim.

Acta,

23 (1969)

353-364

362

SIMMONDS

the irreversible pathway for xanthine formation via guanine (Fig. 3) has been postulated to account for the five-fold excess of urinary xanthine which has been reported in patients on allopurinol and also in xantllinuriaS2p33. It is well established by these and other experiments that allopurinol is rapidly converted to oxipurino121~23 so that qualitative differences in the effects of the two drugs would not be expected. The finding of a qualitative difference in the ratio of xanthine/hypoxanthine increments caused by the two drugs is, therefore, of interest.

au?

.

T

DNA RNA

‘-.

-.I---

-

1

I

I ’

\ ]

‘-

”

GMP

G”*nir

)

I

HYFOXANTHINE‘XANTHINE,-1

G"AN,NE

1

Fig. 3. Schematic representation of the pathways for purine metabolism in man. Dark arrows represent major pathways, dotted arrows indicate the site of action of nucleotide feedback control mechanisms. t,, t, and t, are the enzymes responsible for purine salvage. = Glutamine phosphoribosyl amidotransferase ; ASMP = Adenylosuccinic acid; t, = Adenine phosphoribosyltransferase ; IMP = Inosinic acid; t2 = Hypoxanthine-guanine phosphoribosyltransferase; XMP = Xanthylic acid; t, = Xanthine phosphoribosyltransferase ; GMP = Guanylic acid. t4 AMP = Adenylic acid;

The logical explanation for this difference is that the greater degree of enzyme inhibition produced by allopurinol would make more xanthine and hypoxanthine available for reutilisation by the above salvage mechanisms. Certainly the net increment in xanthine/hypoxanthine production during oxipurinol therapy was only two thirds that produced by allopurinol. However, the results of Chalmers et al., show that 71% and44% of this xanthine/hypoxanthine increment must have been reutilised while on oxipurinol and allopurinol therapy respectively, so that an equal amount of xanthine plus hypoxanthine was actually reutilised with both drugs. No qualitative difference in the effects of the two drugs on xanthine/hypoxanthine ratios would thus be expected if the salvage pathways alone were considered to account for the equal deficits of total oxypurine excretion. One possible explanation is that oxipurinol, either absorbed as such, or derived from allopurinol, could activate the salvage pathways to produce the appropriate purine nucleotides in the ratio critical for feedback control of de novo Clin.

Chim.

Acta,

23 (1969) 353-364

URINARY

PURINES

IN PURINOL-TREATED

PATIENTS

363

purine synthesi9 without markedly altering xanthine/hypoxanthine excretion ratios. It is also conceivable that unchanged allopurinol and/or its riboside could produce a secondary effect on the kidney, perhaps by stimulation of kidney guanase which together with the greater degree of xanthine oxidase inhibition could result in the excretion of xanthine in marked excess. Allopurinol has been given previously to one of the four well documented cases of xanthinuriazl. This patient had a demonstrated absence of the enzyme xanthine oxidase34, yet readily converted administered hypoxanthine to xanthine, a reaction which must have occurred via the alternative nucleotide pathway. The fact that allopurinol was excreted substantially unchanged by this patient, suggested that xanthine oxidase was obligatory for the oxidation of allopurinol to oxipurinolzr. Possible interpretations of the unexpected finding of oxipurinol as a urinarymetabolite in this study of a xanthinuric patient treated with allopurinol will be discussed in a more detailed clinical and biochemical account of this patient now in preparation by Chalmers et al. Pseudouridiue (5-ribosyluracil) is a recently identified urinary pyrimidine which probalby occurs only in transfer RNA ll. Since it is extremely stable, being neither reutilised nor catabolised by the body35, its excretion has been thought to represent the breakdown of body transfer RNA I1. The excretion of pseudouridine is increased in diseases involving the excessive turnover of nucleic acids and in some cases of goutI’>%‘. No studies of its excretion in xanthinuria have been reported previously. Pseudouridine excretion was increased in the xanthinuric patient and in one of the two patients with gout studied here. An interesting finding in these two patients was the replacement, on an equimolar basis, of pseudouridine by uracil, for a period of several days on separate occasions. The reason for this change is not clear at present, and is being investigated. Certainly it occurred when the body was dependent mainly on endogenous sources for its supply of purines and pyrimidines and it began during therapy with either allopurinol or oxipurinol. The reasons why it had already started to revert to normal before ~lopurinol therapy was stopped, or why it should have continued several days after oxipurinol therapy ceased are obscure, but the relatively constant level of excretion of either pseudouridine or uracil is certainly compatible with their being of endogenous origin. The pyrimidine, 5-acetylamino-G-amino-3-methyluracil, has been isolated recently from human urine and a partly endogenous origin has also been suggested for this compoundra. Its excretion, together with the methylated xanthines, on an unrestricted diet and the total absence of these compounds from the urine during restricted purine intake, suggests the dietary origin of all these bases. I-Methylxanthine has been demonstrated in the urine of a xanthinuric patient on a methyl purinefree diet3”, and it was suggested that the excretion of this compound might be related to the enzyme defect, since other workers had previously demonstrated a dietary origin for this con~pound2~3.I-Methylxanthine was not detected in the urine of the xanthinuric patient in this study. The absence of 5-acetylamino-6-amino-3-methyluracil from the urine of the xanthinuric patient, irrespective of diet, may be related either to the fact that this patient’s normal diet did not include the possible precursors of this compound or that the enzyme defect was again involved. Clin. Ckim. Acta, 23 (1969) 353-364

364

SUMMONDS

ACKNOWLEDGEMENTS

I am deeply indebted to Dr. T. Hanley who contributed in so many ways to this research, to Drs. A. J. Woiwod and D. C. Edwards who assisted in the criticism of this work and also to Dr. P. Torkington who kindly measured the infra-red spectra. I wish to thank Dr. R. W. E. Watts and Mr. R. A. Chalmers for their co-operation in supplying specimens from their patients on treatment with allopurinol and oxipurinol and also for making their results in these patients available prior to publication. I am grateful to Miss M. Streeton and Miss C. M. Jones for secretarial assistance. REFERENCES B. WEISSMANN, P. A. BROMBERG AND A. B. GUTMAN, Proc. Sot. Exptl.Biol. Med., 87 (1954) 257. B, WEISSMANN, P. A. BROMBERGAND A. B. GUTMAN, J.Biol.Chem., 224 (1957)407. B. WEISSMANN, P. A. BROMBERGAND A. B. GUTMAN, J.Biol.Chem., 224(1957) 423. B. WEISSMANN AND A. B. GUTMAN, J.Biol.Chem., 229 (1957) 239. D. L. HORRIGAN, J. Clin. Invest., 33 (1954) gor. W.S. ADAMS, F. DAVIS AND M.NAKATANI, Am.J.Med., 28(196o) 726. R. W. PARK, J. F. HOLLAND AND A. JENKINS,&~C~V Res., 22(1962)469. B.M. BOLLARD, R. H.CULPAN, N.MARKS, H. MCILWAIN AND M. SHEPHERD, J. Mental Sci.. 106(196o) 1250. 9 K. FINK, W. S. ADAMS, F.W. DAVIS AND M. NAKATANI,~~~~~~ Res., 23(1963) 1824. ICI W. E. COHN, J. Biol. Chem., 235 (1960) 1488. II S. M. WEISSMANN, J. Am. Med. Assoc., 195 (1966) 117. 239(1964) 4250. I* K. FINK, W. S. ADAMS AND W. A. PFLEIDERER, J.Biol.Chem., H.HITCHINGS, G. B. ELION AND H. R. SILBERMAN, ‘3 R. W. RUNDLES, J. B. WYNGAARDEN,G. Trans. Assoc. Am. Physicians, 76 (1963) 126. I4 R. W. RUNDLES, Ann. Rheumatic Diseases, 25 (1966) 615. 62 (1965) I5 J. R. KLINENBERG, S.E. GOLDFINGER AND J. E. SEEGMILLER, Ann. IntevnalMed.. 639. 16 N. W. LEVIN AND 0. L. ABRAHAMS, Ann. Rheumatic Diseases, 25 (1966) 681. AND J. DANCIS, Science,156(1967) 1122. I7 M.E. BALIS, I.H. KRAKOFF, P.H.BERMAN 136. 18 J. D. WILSON, H. A. SIMMONDS AND J. D. K. NORTH, Ann. Rheumatic Diseases, 26(1967) AND W.L.NYHAN, Nature, 215 (1967) 859. I9 L. SWEETMANN M. ROSENBLOOM, J.MILLER AND J. E. SEEGMILLER, N.Engl. J. Med., 278 20 W.N. KELLEY,F. (1968) 287. G. H. HITCHINGS, E. METZ AND R. W. R~~~~~s,Biochem.Phar21 G. B. ELION, A. KOVENSKY, macol., 15 (1966) 863. J.Biol. Chem., 242 22 T. A. KRENITSKY, G. B. ELION, R. A. STRELITZ AND G. H. HITCHING% (1967) 2675. AND G. H. HITCHING% Am. J. Med., 45 (1968) 69. 23 G. B. ELION, T. F. Yii, A. B. GUTMAN 24 H. A. SIMMONDS, Clin. Chim. Acta, 23 (1969) 319. AND J. D. WILSON, Clin. Chim. Acta, 16 (1967) 155. 25 H. A. SIMMONDS ANDR. W. E. WATTS,CZ~~. Sci.,35(1968) 353. 26 R. A. CHALMERS, J. KRBNER, J.T. SCOTT A. Z. EISEN AND M. KARON, J. Lab.Clin. Med., 59 (1962) 852. 27 S. M. WEISSMANN, 28 G. B. ELION, Ann. Rheumatic Diseases 25 (1966) 605. Biochim.Biophys.Acta, 72(1963) 29 R. POMALES,S.BIEBER,R.FRIEDMANANDG.H.HITCHINGS, 119. 30 R. POMALES, G. B. ELION AND G. H. HITCHING% Biochim.Biophys.Acta,95(1965) 505. 31 J. B.~YNGAARDEN AND D. M. ASHTON, J.Biol. Chem., 234(1959) 1492. 32 M.J.BRADFORD,I.H.KRAKOFF,R.LEEPERANDM.E.BALIS, J.Clin.Znvest.. 47(1968) 1325. 33 J.B. WYNGAARDEN,~~ J.B.STANBURY,J.B.WYNGAARDEN AND D.S.FREDERlcKsox (Eds.). The MetabolicBasis ofInherited Disease, McGraw-Hill, New York, 1966. pp. 687. 711 and 733. 34 K. ENGELMAN, R. W. E. WATTS,J. R. KLINENBERG, A. SJOERDSMA AND J. E. SEEGMILLER. Am. J. Med., 37 (1964) 839. 35 S. M. WEISSMANN, A. Z. EISEN,M.LEWIS AND M. KARON, J.Lab.Clin. Med., 60 (1962) 40. 36 J. H. AYVAZIAN AND S. SKUPP, J. CZin. Invest., 44 (1965) 1248. I

Clin. Chim. Acta, 23 (1969) 353-364