CIinica Chimica Acfq 187 (1990) 221-234
221
Elsevier CCA 04669
Demonstration of a combined deficiency of xanthine oxidase and aldehyde oxidase in xanthinuric patients not forming oxipurinol Sebastian Reiter *, H. Anne Simmonds 3, Nepomuk Zijllner Siegmund L. Braun 2 and Maximilian Knedel 2
I,
I Medizinische Poliklinik and ’ Institut ,t?ir Klinische Chemie, Klinikum Grosshadern der Universitiit Mibchen, (FRG), ’ Purine Laboratory, Guy’s Hospital Medical School, London (UK)
(Received 10 November 1988; revision received 16 August 1988; accepted 27 November 1989) Key words: Xantbine oxidase deficiency; Aldehyde oxidase deficiency; Allopminol metabolism;
Nicotinamide metabolism; Sulfite oxidase
Genetic heterogeneity has been suggested in xanthinuria from the hitherto unexplained ability of some patients with this hereditary disorder to convert allopurinol to its active metabohte oxipurinol - an activity generally attributed to xanthine oxidase. This study provides evidence that the enzyme aldehyde oxidase is also deficient in xanthinuric patients not converting allopu~nol to oxipurinol, whereas a xanthinuric patient with normal formation of oxipurinol had normal aldehyde oxidase activity. It is concluded that the enzyme aldehyde oxidase is the principal enzyme responsible for the formation of oxipurinol in man.
Introduction The uric acid lowering effect of allopurinol in hyperuricemia and gout is due to main metabolite oxipurinol, which inhibits the enzyme xanthine oxidase (EC 1.2.3.2) pseudoirreversibly; despite this inhibition, the metabolic conversion of ~lopu~nol to oxipurinol is still commonly att~buted to xanthine oxidase [1,2]. Aldehyde oxidase (EC 1.2.3.1) a second enzyme capable of converting allopurinol to oxipurinol [3,4] is not inhibited by incubation with allopurinol [5], but nevertheless is usually considered not to have any significant activity against allopurinol in man
its
Correspondence to: Dr. S. Reiter, III. Mediziniscbe Klinik, Klinikum M~~eirn Heidelberg, Wiesbadenerstr. 7-11, 6800 Mannheim 31, FRG. 0009-8981/90/$03.50
0 1990 Elsevier Science Publishers B.V. (Biomedical Division)
der Universitlt
222
[1,3]. This opinion is based on in vitro studies showing a very low activity of aldehyde oxidase in human tissues, which, however, has to be referred to the instability of the enzyme [5,6], the omission of the physiological activator potassium [7] from incubation buffers [5,8,6] and to the use of unphysiological substrates and electron acceptors [9]. The excretion of normal amounts of oxipurinol by several xanthinuric patients given allopurinol was unexpected [lo-131 and aldehyde oxidase was invoked as a possible explanation of this phenomenon [4,1,13-151, alternatively attributed to a mutation of xanthine oxidase altering only its substrate specificity 112,161. The lack of oxipurinol formation in other cases of xanthinuria [17-201 thus could be due either to a double enzyme defect of xanthine oxidase and aldehyde oxidase as suggested by Spector [14] and Krenitsky [15], or to a mutation of xanthine oxidase deleting its activity both with xanthine (and hypoxanthine) and allopurinol. To clarify these hypotheses we have used an indirect method to determine the in vivo activity of aldehyde oxidase in xanthinuric patients by measuring the urinary excretion of its physiological substrate N-methylnicotinamide and its oxidation products IV-methyl-2-pyridone-5-carboxamide(2-pyridone) and N-methyl-4-pyridone-5-carboxamide(4-pyridone) [21-2381. Subjects
Three xanthinuric patients, one of whom was capable of converting allopurinol to oxipurinol were studied. All have been reported in detail previously. Brief clinical data are as follows. Patient L.B. This patient was described by Chalmers et al. in 1969 [24], when 31 yr of age. He was given a daily dose of 3 x 200 mg allopurinol for 3 weeks while on a purine-free diet. The urines analysed in the present report were collected in January and February 1968 and stored at - 20 o C thereafter. Patient B. This patient was 50 yr old when studied by Simmonds et al. in 1974 [19]. He was given a low purine diet and a daily dose of 600 mg allopurinol for 6 days. The urines analysed in the present report were collected in January 1974 and stored at - 20 ’ C. Patient J. 0. This patient was born in 1951. For many years he was aware of his extremely low serum uric acid levels which had been the only pathological finding at several routine check-ups. Although in the best of health he attempted to find an explanation for his constant hypouricemia. The diagnosis of xanthinuria was made by HPLC analysis of several 24-h urines which were collected on an unrestricted diet [20]; demonstration of the enzyme defect in duodenal or liver biopsy was considered to be unnecessary. The patient was given 3 x 200 mg allopurinol/day for 3 days and subsequently nicotinamide 3 x 400 mg/day for 1 day both on unrestricted diet.
223
Twenty four-hour urines were collected without addition of a preservative but kept refrigerated at 4” C over the collection period; aliquots were then stored at - 20 o C until analysed. Controls
Control subjects A and C were healthy male volunteers 38 and 29 yr old; subject D was a 33-yr-old female. All were given 2 X 400 (D) or 3 x 400 (A, C) mg nicotinamide/day for one or several non-consecutive days on an unrestricted diet. Twentyfour-hour urines were collected as described above. Materials and methods Standard substances
Allopurinol and oxipurinol were gifts from the Deutsche Wellcome GmbH, Grossburgwedel, allopurinol-1-riboside from the Dr. G. Henning GmbH, Berlin. Hypoxanthine, xanthine, nicotinamide, nicotinamide-N-oxide and N-methylnicotinamide were obtained from Sigma Chemicals, uric acid from Merck. The 2- and 4-pyridone of N-methylnicotinamide were prepared by enzymatic oxidation [25,7]: 8.8 g liver of a male Sprague-Dawley rat were homogenized in an ice-water cooled Potter-Elvehjem homogenizer with 35 ml of 0.1 mol/l potassium phosphate buffer pH 7.5 (buffer A); the homogenate was centrifuged at 0 o C for 10 min at 37 000 X g. The supematant was filtered through a paper filter and then dialyzed at 0 o C three times for 1 h against 21 of buffer A in a Visking dialysis tubing (Serva, Heidelberg). The complete dialysate (27 ml) was incubated overnight (8.5 h) at 37 o C with 5 ml of a 57.9 mmol/l N-methylnicotinamide solution in buffer A leading to a final concentration of 9 mmol/l. The incubation mixture was then deproteinised by ultrafiltration through Amicon Centriflo membrane cones CF 25 at 2000 rpm in a SorvaIl SS 34 rotor. The 2-pyridone was isolated from the ultrafiltrate by cation exchange chromatography [23]: 16 ml were applied to a column of 10 ml bed volume packed with AG 50 W-X8,200-400 mesh, hydrogen form (Bio-Rad Laboratories) in a 20 ml pipette plugged with glass wool. The column was eluted with water and 10 ml fractions were collected which were analysed by HPLC-system II or IV (see below) and by UV spectrophotometry at neutral and acid pH [26] using a Shimadzu UV-160 spectrophotometer. Pure 2-pyridone was obtained in fractions 4-6. The 4-pyridone being eluted from this column by 1 mol/l HCl simultaneously with unoxidised N-methylnicotinamide was isolated directly from the ultrafiltrate by preparative HPLC using system IV (see below): 50 ~1 were injected repeatedly and the Cpyridone-peak was collected. The collected peak fraction was checked for purity in the same HPLC system and by UV spectrophotometry at neutral and acid PH 1261. The concentration of the pyridone standard solutions was determined spectrophotometrically using the extinction coefficients reported by Bemofsky [26]. The 4-pyridone peak exhibiting considerable tailing concentrations of this metabolite were calculated from the peak height whereas concentrations of the 2-pyridone were calculated from the peak area these latter peaks being often off the page. With this
224
procedure the yield of the enzymatic oxidation of N-methylnicotinamide calculated to be 2.8% of 4-pyridone and 7.5% of 2-pyridone. Preparation
was
of urine samples for HPLC
All purine, allopurinol and nicotinamide metabolites were determined by HPLC after a two step pre-separation of the urine samples by anion exchange chromatography [27]. The effluent plus washings with 2 ml of water for normal urines or 4.5 ml for urines of days with nicotinamide loading contained nicotinamide and its metabolites nicotinamide-N-oxide, N-methylnicotinamide [28] and the 2- and 4pyridone. The oxipurines as well as allopurinol and its metabolites were then eluted with 7.5 ml of 250 mmol/l NaCl in 40 mmol/l HCl. HPLC
analyses
All HPLC analyses were performed with a Hewlett Packard 1084 B Liquid Chromatograph. Elution buffers were prepared using chromatographic grade reagents obtained from Merck or BDH. System Z For the determination of hypoxanthine, xanthine, allopurinol, oxipurinol and allopurinol-1-riboside a Shandon ODS column (250 x 4.6 mm, 5 pm particle size) was eluted isocratically at a flow rate of 1 ml/mm with 7.35 mmol/l KH,PO, adjusted to pH 3; after 14.5 min methanol was introduced by pump B to yield a final concentration of 10% (v/v) in the elution buffer for a further 20 min followed by a 25 min equilibration period without methanol prior to subsequent sample injection [29]. System ZZ For the determination of unmetabolised nicotinamide the Shandon column was eluted isocratically at 1 ml/min with 73.5 mmol/l KH,PO,, pH 6, containing 10% methanol (v/v); for the determination of nicotinamide-N-oxide the content of methanol was reduced to 5% (v/v). System ZZZ N-methylnicotinamide was determined by the method of Carter [28] using a Beckman (Altex) Ultrasphere IP column (250 x 4.6 mm, 5 pm particle size); the elution buffer consisting of 10 mrnol/l KH,PO,, 5 mM 1-octanesulfonate and 10% acetonitrile (v/v) was adjusted to pH 7; the flow rate was 1.5 ml/mm. System IV The 2- and also the 4-pyridone were determined by the method of Carter [28]: the Beckman column was eluted at a reduced flow rate of 1 ml/mm with 10 mmol/l KH,PO,, pH 7, containing 2% acetonitrile (v/v); for the preparative isolation of the 4-pyridone the content of acetonitrile was reduced to 1% (v/v). Peaks were identified by stop flow UV spectra and by co-chromatography with standard substances. Estimation
of sulfite oxidase activity in vivo
The activity of sulfite oxidase (EC 1.8.2.1) was estimated using the Merckoquant 10013 Sulfite and 10019 Sulfate Test strips [30] with freshly voided urine of patient J.O. and proved to be normal.
225
Results Metabolism of allopurinol in xanthinuric patients and in normal controls
The metabolism of allopurinol in our xanthinuric patients and in normal controls has been reported in previous publications [10,19,20]. The results are compiled in Table I and clearly demonstrate the fundamental difference between the two types of xanthinuric patients: patient L.B. represents the group with a completely normal pattern of allopurinol metabolites; these patients excrete up to about 10% of the daily allopurind dose as allopurinol and allopurinol-1-riboside, and after an equiIibration period of 3-5 days up to 60% as o~pu~nol. Patients B. and J.O. on the other hand produced no oxipurinol, leading to a greatly increased excretion of unmetabolised allopurinol and allopurinol-1-riboside; allopurinol and allopurinol1-riboside being much more rapidly excreted than oxipurinol [18] the recovery amounted to about 70% of the dose already on the first day. An observation time of a few days is thus sufficient to confirm the lack of oxipurinol formation. The absence of uric acid and oxipurinol in the urine of patient J.O. is demonstrate in Fig. 1 which compares the urinary excretion of purines and allopurinol metabolites with control subject A. Activity of aldehyde oxidase in xanthinuric patients and in normal controls
The activity of aldehyde oxidase in vivo was determined by measuring the ratio of its physiolo~c~ substrate ~-methylni~otina~de to its oxidation products, the 2and cl-pyridone, in 24-h urines. Fig 2 shows the ratio of these metabolites using HPLC system II in control subject A and patient J-0. with and without a nicotinamide load of 3 X 400 mg/day. In control A a small amount of the 2-pyridone (retention time (RT) 10.20) and a trace of the 4-pyridone (RT 9.74) were found under normal conditions (Fig. 2a); following the ingestion of 3 X 400 mg nicotinamide a large increase in both pyridone peaks was observed {Fig. 2b).
TABLE I Mean 24-h urinary excretion of allopurinol, oxipurinol and allopurinol-1-riboside (mmol/day and % of dose) in our xanthinnric patients and control subjects receiving 3 x 200 mg (3 X 1.47 mmol) ailop~oI/ day. Source
Days of treatment
Oxipurinol
Allopurinol
AllopurinolI -riboside
Total
ll- 17 206-211 4,8
2.6(59%) 2.59(58.7%) 2.21(50.1%)
0.3(6.8%) 0.37(8.4%) 0.35(7.9X)
0.33(7.5%} 0.42(9.5%) 0.51(11.6X)
3.23(73.2%) 3.38(76.6%) 3.07(69.6%)
WI patient control control t191 patient
LB. B. G. B.
0-
6
n.d.
1.15(26.1%)
2.13(48.3%)
3.28(74.4%)
o7-
3 9
n.d. 2.91(65.9%)
1.7qlO.38) 0.4 (9%)
1.36(30.8%) 0.33(7.5%)
3.14(71.1%) 3.63(82.4%)
VOI patient J.O. control. A. n.d. = not detected.
226
t
t
0;
i
:, 0:
t f-4 N
Y
parlent
3.0.
Fig. 1. Determination of urinary excretion of purine and allopurinol metabolites. Chromatograms of 24-h urine samples of control subject A (day 8, 2030 ml) and patient J.O. (day 2, 2450 ml) receiving 600 mg allopurinol per day. Sample dilution 1: 15 with NaCl/HCl solution by pre-separation. HPLC system I. 254 nm, attenuation 2 t 5, injection volume 50 ~1. Peak identification: uric acid 6.60/6.62; hypoxanthine 7.85/7.84; xanthine 9.56/9.54; oxipurinol 12.29; allopurinol 14.49/14.44; allopurinol-l-riboside 24.13/23.98.
N-methylnicotinamide was not detected under normal conditions (Fig. 2a); after nicotinamide administration it appeared in a flat and broad peak covered with small shoulder peaks (Fig. 2b, RT 6.0, 7.51, 8.09). In patient J.O. no pyridones were detectable, either under normal conditions (Fig. 2c) or following the administration of nicotinamide (Fig. 2d); in the latter case a large peak of N-methylnicotinamide (RT 5.34) substituted for the missing pyridones. The ratio of pyridones to N-methylnicotinamide corresponding to the activity of aldehyde oxidase in vivo could thus be estimated to be high in the normal control and extremely low in patient J.O. For an exact determination of this ratio an HPLC system yielding quantifiable peaks of N-methylnicotinamide and a higher sensitivity especially for urines collected under normal conditions were required. Excretion of the 2- and 4-pyridone of N-methylnicotinamide A more sensitive pyridone determination was achieved by using the method of Carter [28] with a reduced flow rate (HPLC system IV) allowing for the simultaneous determination of the 2- and the 4-pyridone, the latter not being found by Carter. The results are
221
b
C
d
Fig. 2. Estimation of aldehyde oxidase activity in vivo. Chromatograms of 24-h urine samples of: control subject A before nicotinamide administration (965 ml) (a); control subject A after 3 X 400 mg nicotinamide (1670 ml) (b); patient J.O. before nicotinamide administration (1240 ml) (c); patient J.O. after 3 x400 mg nicotinamide (2030 ml) (d). Sample dilution 1: 5.3 with water by pre-separation. HPLC system II with 5% methanol. 254 nm, attenuation 2f6, injection volume 20 ~1. Peak identification: creatinine 3.79/3.80; nicotinamide-N-oxide 4.39; N-methylnicotinamide 5.34; 4-pyridone 9.14/9.75; 2-pyridone 10.20/10.19.
shown in Table II and Fig. 3. Using this more sensitive method, an increased injection volume (50 ~1) and a decreased attenuation (2 t 4) still no 4-pyridone but a small peak of 2-pyridone could be found in the urines of patient J.O.: the urine before nicotinamide administration (Fig. 3c) contained up to 14.5 pmol 2pyridone/day, the maximum excretion of this patient, surpassing the excretion on the day with nicotinamide administration (12.6 pmol/day; Fig. 3d). Patient B. not forming oxipurinol either excreted no 4-pyridone but a very little 2-pyridone (average 1.9 pmol/day; Table II). By contrast patient L.B. with normal formation of oxipurinol excreted an average of 95.7 pmol 2-pyridone/day together with 12.3 pmol 4-pyridone/day, values within the ranges of the control subjects which averaged 142.4 pmol/day for the 2-pyridone and 19.3 pmol/day for the 4-pyridone (Table II). The chromatogram of control subject A corresponds to a basal excretion of 139 pmol 2-pyridone and 18.9 pmol 4-pyridone per day (Fig. 3a). On the day of nicotinamide administration the sum of pyridones excreted in control subject A
II
4
x0-, oxip’ L.B.
n-d. = not detected.
Controls A A A A A C C D D
4 6 1
x0-, oxipB. J.O. J.O.
Days
-
3x400
Dose of nicotinamide (mg)
139 301.9 4845.5 4952.6 4653.4 151.5 4141.2 77.1 3816.1
96 (61
-162)
2 (1.3- 2.4) 6 (3.5-14.5) 12.6
2-Pyridone
Urinary excretion (pmol/day) of 2-pyridone, 4-pyridone, patients (X0-) without (oxip-) and with normal oxipurinol latter with the range for the number of days indicated
TABLE
18.9 26.4 458.5 491.6 467.8 21.6 408.7 10.2 372.3
12.3 (9-18)
n.d. n.d. n.d.
CPyridone
20.4 53.5 2024.5 2127.3 2322.5 37 2557.1 19.3 1898.2
17 (lo-
33)
178 (M-235) 205 (49-348) 7762.2
N-methylnicotinamide
N-methylnicotinamide, nicotinamide-N-oxide formation (oxip+ ) and in control subjects.
145.8 216.1 n.d. 281.8 n.d. 87.8
2.6 2.2 4.7 1.8 4.5 2.2
-7.6)
n.d. n.d. 158.2
(5.4
n.d. 177.4
Nicotinamide-Noxide
7.7 4.3 2.6
6.8
0.013 (0.006-0.024) 0.038 (0.015-0.072) 0.0016
ratio pyridones: N-methylnicotinamide
88.5
n.d.
n.d. 383.6
Nicotinamide
and unmetabolised nicotinamide in xanthinuric Results given are either single or mean values, the
229
a
b
Fig. 3. Determination of 2- and Cpyridone excretion. Chromatograms of 24-h urine samples of: control subject A before nicotinamide administration (965 ml) (a); control subject A after 3 x 400 mg nicotinamide (1670 ml) (b); patient J.O. before nicotinamide administration (1240 ml) (c); patient J.O. after 3 x 400 mg nicotinamide (2030 ml) (d). Sample dilution 1: 10.7 with water by pre-separation. HPLC system IV with 2% acetonitrile. 258 nm, attenuation 2f4, injection volume 50 pl. Peak identification: Cpyridone 10.69/10.67; 2-pyridone 11.07-11.24.
amounted to 5304 pmol/day (Fig. 3b) corresponding to an excretion 420-fold that of patient J.O. Similar amounts were found in controls C and D (Table II). The average ratio of 2-pyridone to Cpyridone was 7.5 in patient L.B. and the controls on days without and 10.2 in the controls on days with nicotinamide administration (Table II).
Excretion of N-methylnicotinamide Using the ion-pairing HPLC method of Carter [28] the basal urinary excretion of N-methylnicotinamide was easily quantifiable in all urine samples: it amounted to 20.4 pmol/day in control subject A (Fig. 4a) corresponding to one tenth of the excretion of patient J.O. (Fig. 4c; Table II). A high average basal excretion of 178 pmol N-methylmcotinamide per day was found also in patient B., the second xanthinuric patient not forming oxipurinol, whereas xanthinuric patient L.B. with normal formation of oxipurinol showed a very low average value of 17 pmol/day within the range of the healthy controls (Table II). On the day of nicotinamide administration, although a strong increase in N-methylnicotinamide excretion was observed in control subject A (2024.5 pmol/ day; Fig. 4b), the increment was much higher in patient J.O. (7762.2 pmol/day; Fig. 4d; Table II) thus compensating for the lack of pyridone formation.
230
a
b
C
Fig. 4. Determination of N-methylnicotinamide excretion. Chromatograms of 24-h urine samples of: control subject A before nicotinamide administration (965 ml) (a); control subject A after 3 x 400 mg nicotinamide (1670 ml) (b); patient J.O. before nicotinamide administration (1240 ml) (c); patient J.O. after 3 x 400 mg nicotinamide (2030 ml) (d). Sample dilution 1: 10.7 with water by pre-separation. HPLC system III. 264 nm, attenuation 2 t 4, injection volume 20 pl. Peak identification: N-methylmcotinamide 7.21-7.36.
Ratio of pyridone to N-methylnicotinamide excretion The absolute values of pyridone and N-methyhkotinamide excretion obviously strongly depend on the dietary supply of nicotinamide. However, the ratio of pyridone to N-methylnicotinamide excretion corresponding to the activity of aldehyde oxidase was rather constant under normal conditions ranging from 4.3 to 7.7 in the control subjects (Table II); in xanthimuic patient L.B. with normal formation of oxipurinol the same ratio was found ranging from 5.4 to 7.6 thus indicating a normal activity of aldehyde oxidase. On the other hand in the xanthinuric patients B. and J.O. the ratio of pyridone to N-methylnicotinamide excretion was extremely low ranging from 0.006 to 0.072 with an average of 0.013 in patient B. and 0.038 in patient J.O. corresponding to less than 1% of the normal value. This difference became even more pronounced following
231
the administration of 3 X 400 mg nicotinamide: in the control subjects the ratio of pyridone to N-methylnicotinamide excretion decreased a little to an average value of 2.3, whereas it was much further reduced in patient J.O. to 0.0016 corresponding to 0.07% of the normal value (Table II). Excretion of nicotinamide-N-oxide and unmetabolised nicotinamide Nicotinamide-N-oxide originates from direct oxidation of nicotinamide catalysed by a microsomal mixed function oxidase [31]. The urinary excretion of nicotinamide-Noxide was measured using HPLC system II with 5% methanol (v/v; Fig. 2): nicotinamide-N-oxide was undetectable under normal conditions in control subject A (Fig. 2a) as well as in patient J.O. (Fig. 2~); on the day of nicotinamide administration a small peak of nicotinamide-N-oxide (RT 4.39) was observed both in control subject A (Fig. 2b) and patient J.O. (Fig. 2d) corresponding to 1.5-2.2 and 1.8X, respectively, of the dose (Table II). Unmetabolised nicotinamide, determined using HPLC system II with 10% methanol (v/v), was not detectable in urines of control subject A or patient J.O. while on normal diet. On the day of nicotinamide ingestion 0.9% of the dose were recovered unmetabolised in control subject A and 3.9% in patient J.O. (Table II). These results confirm our above observation that the main pathway of nicotinamide metabolism, the conversion to N-methylmcotinamide, is not influenced by aldehyde oxidase deficiency. Discussion
In order to evaluate the role of aldehyde oxidase in the metabolism of allopurinol we have used an indirect method to estimate the activity of this enzyme in normal and xanthinuric individuals in vivo thus avoiding the difficulties encountered using tissue biopsies: following the suggestion of Stanulovic and Chaykin [8,25] and of Krenitsky et al. [9] we have determined the conversion of N-methylmcotinamide, a physiological substrate of aldehyde oxidase, into the 2- and 4-pyridone. These metabolites of nicotinic acid and nicotinamide are excreted in appreciable amounts in human urine on a normal diet [32,28,33]. Terry and Simon [33] gave normal range estimates of 6-51.3 mg (39.5-337.5 pmol) 2-pyridone/24 h, 1.6-14.8 mg (9.3-85.7 pmol) N-methylnicotinamide/24 h and of a 2-pyridone: N-methyhricotinamide weight ratio of 1.76-5.9 corresponding to a molar ratio of 2-6.7. Considering this ratio as a measure of aldehyde oxidase activity in vivo we found that xanthinuric patient L.B. who formed oxipurinol had a normal activity of aldehyde oxidase: the average ratio being 6.8 : 1 as compared to 5.3 : 1 in the control subjects on a normal diet. The two other xanthinuric patients B. and J.O. not forming oxipurinol exhibited average ratios of 0.013 : 1 and 0.038 : 1 corresponding to 0.25% and 0.72% of the control value and thus indicating an almost complete deficiency of aldehyde oxidase. Lower than normal values for the 2-pyridone : N-methylmcotinamide ratio have been observed in nicotinic acid and tryptophan deficiency [34], the lowest molar ratio reported being 0.13 in a child suffering from pellagra; following nicotinamide
232
treatment the ratio increased to 1.5 [34]. Thus in cases with decreased excretion of both pyridones and N-methyhricotinamide, resulting in a ratio of less than 1.0, nicotinamide should be administered to provide sufficient substrate for aldehyde oxidase. If high doses of nicotinamide are given it has to be taken into account that N-methylnicotinamide shows a considerably higher renal clearance than the pyridones [32]. Therefore the ratio of pyridones : N-methylnicotinamide will be lower in the first 24-h urine collected after a single administration of nicotinamide than in a 48- or 72-h urine collection [32] or after long-term daily ingestion [35]. Consequently a decrease of the ratio by about 50% was observed in the 24-h urines of our control subjects collected during nicotinamide administration. Nevertheless this nicotinamide load greatly exacerbated the difference between the control subjects and patient J.O.: the latter excreting 76.9% of the nicotinamide dose as N-methylnicotinamide, with still only a trace of 2-pyridone, and a decrease in the pyridone: Nmethylnicotinamide ratio to 0.0016 corresponding to 0.07% of the control value. This result confirms the almost complete lack of aldehyde oxidase in patient J.O. Our patients B. and J.O. represent the first cases of a proven combined deficiency of xanthine and aldehyde oxidase. This double enzyme defect in our patients seems not to have had any serious consequences, thus confirming the view that the symptoms of molybdenum cofactor deficiency are entirely attributable to the lack of the third molybdoenzyme sulfite oxidase [36] which was shown to have normal activity in our patient J.O. However, problems might arise following the administration of drugs which depend on aldehyde oxidase for detoxification. Considering the drugs known to be metabolised by aldehyde oxidase [37,38] no danger seems to exist at present. Other xanthinuric patients have also been reported who were unable to form oxipurinol from allopurinol which suggests that a similar combined deficiency of xanthine and aldehyde oxidase exists in these patients [17,18,39]. The patient reported by Higashino et al. [39] was also unable to oxidise pyrazinamide to 5-hydroxypyrazinamide in contrast to 2 other xanthinuric patients forming both oxipurinol and 5-hydroxypyrazinamide and thus corresponding to the patient of Auscher et al. [16,11]. This difference was attributed either to different mutations of xanthine oxidase altering its substrate specificity or to an alternative oxidase [16,38], presumably aldehyde oxidase [40]. From our results we suggest that the xanthimnic patients with normal oxipurinol formation possess a normal activity of aldehyde oxidase which obviously is able to convert pyrazinamide to 5-hydroxypyrazinamide at a normal rate [16]. The normal oxipurinol formation brought about by a normal activity of aldehyde oxidase in patient L.B. indicates that aldehyde oxidase can fully substitute for xanthine oxidase in the conversion of allopurinol to oxipurinol. Further support for this conclusion is the linear increase in the formation of oxipurinol with increasing allopurinol dosage in the presence of a decreasing formation of uric acid, indicating progressive inhibition of xanthine oxidase; this phenomenon has been observed by Sweetman [41] in children given increasing allopurinol doses up to 100 mg/kg per day. Saturation of oxipurinol formation was reached at doses of about 80 mg/kg per day. The maximum metabolic conversion of allopurinol to oxipurinol thus
233
coincided with an almost complete inhibition of xanthine oxidase and therefore has to be attributed to aldehyde oxidase which is not inhibited by allopurinol or oxipurinol. We conclude from these observations that aldehyde oxidase as in rodents [7] is the main oxipurinol forming enzyme in man. Acknowledgements
We thank Mrs. H. Deharde for her technical assistance. The HPLC apparatus was given by the Deutsche Forschungsgemeinschaft. These studies were supported by the Arthritis and Rheumatism Council, UK. The kind and helpful cooperation of patient J.O. is gratefully acknowledged. After this work had been finished we were given a sample of 2-pyridone synthesised chemically by Dr. J. de Vries, Abteilung Klinische Pharmakologie, Universitat Heidelberg; by HPLC analysis and UV spectrophotometry this compound was identical with our 2-pyridone standard obtained by enzymatic oxidation of N-methylnicotinamide. References 1 Elion GB. Allopurinol and other inhibitors of urate synthesis. In: Kelley WN, Weiner IM, eds. Uric acid. Berlin: Springer Verlag, 1978;485-513. 2 Murrell GAC, Rapeport WG. Clinical pharmacokinetics of allopurinol. Clin Pharmacokinet 1986;11:343-353. 3 Johns DG, Spector T, Robins RK. Studies on the mode of oxidation of pyrazolo(3,4-d)pyrimidine by aldehyde oxidase and xanthine oxidase. Biochem Pharmacol 1969;18:2371-2383. 4 Krenitsky TA, Neil SM, Elion GB, Hitchings GH. A comparison of the specificities of xanthine oxidase and aldehyde oxidase. Arch Biochem Biophys 1972;150:585-599. 5 Johns DG. Human liver aldehyde oxidase: differential inhibition of oxidation of charged and uncharged substrates. J Clin Invest 1967;46:1492-1505. 6 Duley JA, Harris 0, Holmes RS. Analysis of human alcohol- and aldehyde-metabolizing isozymes by electrophoresis and isoelectric focusing. Alcoholism: Clin Exp Res 1985;9:263-271. 7 Huh K, Yamamoto I, Gohda E, Iwata H. Tissue distribution and characteristics of xanthine oxidase and allopurinol oxidizing enzyme. Jpn J Pharmacol 1976;26:719-724. 8 Stanulovic M, Chaykin S. Metabolic origins of the pyridones of N’-methylmcotinamide in man and rat. Arch Biochem Biophys 1971;145:35-42. 9 Krenitsky TA, Tuttle JV, Cattau EL Jr, Wang P. A comparison of the distribution and electron acceptor specificities of xanthine oxidase and aldehyde oxidase. Comp Biochem Physiol 1974;498:687-703. 10 Simmonds HA. Urinary excretion of purines, pyrimidines and pyrazolopyrimidines in patients treated with allopurinol or oxipurinol. Clin Chim Acta 1969;23:353-364. 11 Auscher C, Pasquier C, Mercier N, Delbarre F. Urinary excretion of 6 hydroxylated metabolite and oxypurines in a xanthinuric man given allopurinol or thiopurinol. Adv Exp Med Biol 1974;4IB:663-667. 12 Kojima T, Nishina T, Kitamura M, Hosoya T, Nishioka K. Biochemical studies on the purine metabolism of four cases with hereditary xanthinuria. Clin Chim Acta 1984;137:189-198. 13 Carpenter TO, Lebowitz RL, Nelson D, Bauer S. Hereditary xanthinuria presenting in infancy with nephrolithiasis. J Pediatr 1986;109:307-309. 14 Spector T. Inhibition of urate production by allopurinol. Biochem Pharmacol 1977;26:355-358. 15 Krenitsky TA. Aldehyde oxidase and xanthine oxidase - functional and evolutionary relationships. Biochem Pharmacol 1978;27:2763-2764. 16 Auscher C, Pasquier C, Pehuet P, Delbarre F. Study of urinary pyrazinamide metabolites and their
234
17
18 19 20 21 22 23 24
25 26 21
28 29 30 31 32 33
34
35 36
31 38 39 40 41
action on the renal excretion of xamhine and hypoxanthine in a xanthinuric patient. Biomedicine 1978;28:129-133. Engelman K, Watts RWE, Khnenberg JR, Sjoerdsma A, Seegmiller JE. Clinical, physiological and biochemical studies of a patient with xanthinuria and pheochromocytoma. Am J Med 1964;37:839-861. Elion GB, Kovensky A, Hitchings GH, Metz E, Rundles RW. Metabolic studies of allopurinol, an inhibitor of xanthine oxidase. Biochem Pharmacol 1966;15:863-880. Simmonds HA, Levin B, Cameron JS. Variations in allopurinol metabolism by xanthinuric subjects. Clin Sci Molec Med 1974;47:173-178. Reiter S, Zollner N, Braun S, Knedel M. Allopurinol induced oroticaciduria in a xanthinuric patient not forming oxipurinol. KJm Wochenschr 1987;65(suppl X):13-14. Knox WE. The quinine-oxidizing enzyme and liver aldehyde oxidase. J Biol Chem 1946;163:699-711. Knox WE, Grossman WI. A new metabolite of nicotinamide. J Biol Chem 1946;166:391-392. Chang MLW, Johnson BC. N-methyl-4-pyridone-5-carboxamide as a metabolite of nicotinic acid in man and monkey. J Biol Chem 1961;236:2096-2098. Chalmers RA, Parker R, Simmonds HA, Snedden W, Watts RWE. The conversion of Chydroxypyrazolo(3,4-d)pyrimidine(allopurinol) into 4,6-dihydroxypyrazolo(3,4-d)pyrimidine(oxipurinol) in vivo in the absence of xanthine-oxygen oxidoreductase. Biochem J 1969;112:527-532. Stanulovic M, Chaykin S. Aldehyde oxidase: catalysis of the oxidation of N’-methylnicotinamide and pyridoxal. Arch B&hem Biophys 1971;145:27-34. Bemofsky C. New synthesis of the 4- and 6-pyridones of 1-methylnicotinamide and 1-methylnicotinic acid (trigonelline). Anal Biochem 1979;96:189-200. Reiter S, Simmonds HA, Webster DR, Watson AR. On the metabolism of allopurinol. Formation of allopurinol-1-riboside in purine nucleoside phosphorylase deficiency. Biochem Pharmacol 1983;32:2167-2174. Carter EGA. Quantitation of urinary niacin metabolites by reversed-phase liquid chromatography. Am J Clin Nutr 1982;36:926-930. Reiter S, Liiffler W, Grijbner W, Zijllner N. Urinary oxipurinol-1-riboside excretion and allopurinolinduced oroticaciduria. Adv Exp Med Biol 1986;195 A:453-460. Duran M, Beemer FA, v.d. Heiden C, et al. Combined deficiency of xanthine oxidase and sulphite oxidase: a defect of molybdenum metabolism or transport? J Inher Metab Dis 1978;1:175-178. Kirchner J. Metabolism of nicotinamide. PhD thesis. University of California, Davis, 1965. Holman WIM, De Lange DJ. Methods for the determination of N-methyl-2-pyridone-5-carboxylamide and of N-methyl-2-pyridone-3-carboxylamide in human urine. Biochem J 1949;45:559-563. Terry RC, Simon M. Determination of niacin metabolites 1-methyl-5-carboxylamide-2-pyridone and N-1-methylnicotinamide in urine by high-performance liquid chromatography. J Chromatogr 1982;232:261-274. Prinsloo JG, Du Plessis JP, Kruger H, De Lange DJ, De Villiers LS. Protein nutrition status in childhood pellagra. Evaluation of nicotinic acid status and creatinine excretion. Am J Clin Nutr 1968;21:98-106. Mrochek JE, Jolley RL, Young DS, Turner WJ. Metabolic response of humans to ingestion of nicotinic acid and nicotinamide. Clin Chem 1976;22:1821-1827. Johnson JL, Waud WR, Rajagopalan KV, Duran M, Beemer FA, Wadman SK. Inborn errors of molybdenum metabolism: combined deficiencies of sulfite oxidase and xanthine dehydrogenase in a patient lacking the molybdenum cofactor. Proc Nat1 Acad Sci USA 1980;77:3715-3719. Beedham C. Molybdenum hydroxylases as drug-metabolizing enzymes. Drug Metab Rev 1985;16:119-156. Bee&am C, Bruce SE, Critchley DJ, Al-Tayib Y, Rance DJ. Species variation in hepatic aldehyde oxidase activity. Eur J Drug Metab Pharmacokinet 1987;12:307-310. Higashino K, Yamamoto T, Hada T, et al. Studies on the metabolism of pyrazinamide and allopurinol in patients with hereditary xanthinuria. Pediatr Res 1988;24:120. Yamamoto T, Moriwaki Y, Takahashi S, Hada T, Higashino K. S-Hydroxypyrazinamide, a human metabolite of pyrazinamide. Biochem Pharmacol 1987;36:2415-2416. Sweetman L. Urinary and cerebrospinal fluid oxypurine levels and allopurinol metabolism in the Lesch-Nyhan syndrome. Fed Proc 1968;27:1055-1058.
221
Elsevier CCA 04669
Demonstration of a combined deficiency of xanthine oxidase and aldehyde oxidase in xanthinuric patients not forming oxipurinol Sebastian Reiter *, H. Anne Simmonds 3, Nepomuk Zijllner Siegmund L. Braun 2 and Maximilian Knedel 2
I,
I Medizinische Poliklinik and ’ Institut ,t?ir Klinische Chemie, Klinikum Grosshadern der Universitiit Mibchen, (FRG), ’ Purine Laboratory, Guy’s Hospital Medical School, London (UK)
(Received 10 November 1988; revision received 16 August 1988; accepted 27 November 1989) Key words: Xantbine oxidase deficiency; Aldehyde oxidase deficiency; Allopminol metabolism;
Nicotinamide metabolism; Sulfite oxidase
Genetic heterogeneity has been suggested in xanthinuria from the hitherto unexplained ability of some patients with this hereditary disorder to convert allopurinol to its active metabohte oxipurinol - an activity generally attributed to xanthine oxidase. This study provides evidence that the enzyme aldehyde oxidase is also deficient in xanthinuric patients not converting allopu~nol to oxipurinol, whereas a xanthinuric patient with normal formation of oxipurinol had normal aldehyde oxidase activity. It is concluded that the enzyme aldehyde oxidase is the principal enzyme responsible for the formation of oxipurinol in man.
Introduction The uric acid lowering effect of allopurinol in hyperuricemia and gout is due to main metabolite oxipurinol, which inhibits the enzyme xanthine oxidase (EC 1.2.3.2) pseudoirreversibly; despite this inhibition, the metabolic conversion of ~lopu~nol to oxipurinol is still commonly att~buted to xanthine oxidase [1,2]. Aldehyde oxidase (EC 1.2.3.1) a second enzyme capable of converting allopurinol to oxipurinol [3,4] is not inhibited by incubation with allopurinol [5], but nevertheless is usually considered not to have any significant activity against allopurinol in man
its
Correspondence to: Dr. S. Reiter, III. Mediziniscbe Klinik, Klinikum M~~eirn Heidelberg, Wiesbadenerstr. 7-11, 6800 Mannheim 31, FRG. 0009-8981/90/$03.50
0 1990 Elsevier Science Publishers B.V. (Biomedical Division)
der Universitlt
222
[1,3]. This opinion is based on in vitro studies showing a very low activity of aldehyde oxidase in human tissues, which, however, has to be referred to the instability of the enzyme [5,6], the omission of the physiological activator potassium [7] from incubation buffers [5,8,6] and to the use of unphysiological substrates and electron acceptors [9]. The excretion of normal amounts of oxipurinol by several xanthinuric patients given allopurinol was unexpected [lo-131 and aldehyde oxidase was invoked as a possible explanation of this phenomenon [4,1,13-151, alternatively attributed to a mutation of xanthine oxidase altering only its substrate specificity 112,161. The lack of oxipurinol formation in other cases of xanthinuria [17-201 thus could be due either to a double enzyme defect of xanthine oxidase and aldehyde oxidase as suggested by Spector [14] and Krenitsky [15], or to a mutation of xanthine oxidase deleting its activity both with xanthine (and hypoxanthine) and allopurinol. To clarify these hypotheses we have used an indirect method to determine the in vivo activity of aldehyde oxidase in xanthinuric patients by measuring the urinary excretion of its physiological substrate N-methylnicotinamide and its oxidation products IV-methyl-2-pyridone-5-carboxamide(2-pyridone) and N-methyl-4-pyridone-5-carboxamide(4-pyridone) [21-2381. Subjects
Three xanthinuric patients, one of whom was capable of converting allopurinol to oxipurinol were studied. All have been reported in detail previously. Brief clinical data are as follows. Patient L.B. This patient was described by Chalmers et al. in 1969 [24], when 31 yr of age. He was given a daily dose of 3 x 200 mg allopurinol for 3 weeks while on a purine-free diet. The urines analysed in the present report were collected in January and February 1968 and stored at - 20 o C thereafter. Patient B. This patient was 50 yr old when studied by Simmonds et al. in 1974 [19]. He was given a low purine diet and a daily dose of 600 mg allopurinol for 6 days. The urines analysed in the present report were collected in January 1974 and stored at - 20 ’ C. Patient J. 0. This patient was born in 1951. For many years he was aware of his extremely low serum uric acid levels which had been the only pathological finding at several routine check-ups. Although in the best of health he attempted to find an explanation for his constant hypouricemia. The diagnosis of xanthinuria was made by HPLC analysis of several 24-h urines which were collected on an unrestricted diet [20]; demonstration of the enzyme defect in duodenal or liver biopsy was considered to be unnecessary. The patient was given 3 x 200 mg allopurinol/day for 3 days and subsequently nicotinamide 3 x 400 mg/day for 1 day both on unrestricted diet.
223
Twenty four-hour urines were collected without addition of a preservative but kept refrigerated at 4” C over the collection period; aliquots were then stored at - 20 o C until analysed. Controls
Control subjects A and C were healthy male volunteers 38 and 29 yr old; subject D was a 33-yr-old female. All were given 2 X 400 (D) or 3 x 400 (A, C) mg nicotinamide/day for one or several non-consecutive days on an unrestricted diet. Twentyfour-hour urines were collected as described above. Materials and methods Standard substances
Allopurinol and oxipurinol were gifts from the Deutsche Wellcome GmbH, Grossburgwedel, allopurinol-1-riboside from the Dr. G. Henning GmbH, Berlin. Hypoxanthine, xanthine, nicotinamide, nicotinamide-N-oxide and N-methylnicotinamide were obtained from Sigma Chemicals, uric acid from Merck. The 2- and 4-pyridone of N-methylnicotinamide were prepared by enzymatic oxidation [25,7]: 8.8 g liver of a male Sprague-Dawley rat were homogenized in an ice-water cooled Potter-Elvehjem homogenizer with 35 ml of 0.1 mol/l potassium phosphate buffer pH 7.5 (buffer A); the homogenate was centrifuged at 0 o C for 10 min at 37 000 X g. The supematant was filtered through a paper filter and then dialyzed at 0 o C three times for 1 h against 21 of buffer A in a Visking dialysis tubing (Serva, Heidelberg). The complete dialysate (27 ml) was incubated overnight (8.5 h) at 37 o C with 5 ml of a 57.9 mmol/l N-methylnicotinamide solution in buffer A leading to a final concentration of 9 mmol/l. The incubation mixture was then deproteinised by ultrafiltration through Amicon Centriflo membrane cones CF 25 at 2000 rpm in a SorvaIl SS 34 rotor. The 2-pyridone was isolated from the ultrafiltrate by cation exchange chromatography [23]: 16 ml were applied to a column of 10 ml bed volume packed with AG 50 W-X8,200-400 mesh, hydrogen form (Bio-Rad Laboratories) in a 20 ml pipette plugged with glass wool. The column was eluted with water and 10 ml fractions were collected which were analysed by HPLC-system II or IV (see below) and by UV spectrophotometry at neutral and acid pH [26] using a Shimadzu UV-160 spectrophotometer. Pure 2-pyridone was obtained in fractions 4-6. The 4-pyridone being eluted from this column by 1 mol/l HCl simultaneously with unoxidised N-methylnicotinamide was isolated directly from the ultrafiltrate by preparative HPLC using system IV (see below): 50 ~1 were injected repeatedly and the Cpyridone-peak was collected. The collected peak fraction was checked for purity in the same HPLC system and by UV spectrophotometry at neutral and acid PH 1261. The concentration of the pyridone standard solutions was determined spectrophotometrically using the extinction coefficients reported by Bemofsky [26]. The 4-pyridone peak exhibiting considerable tailing concentrations of this metabolite were calculated from the peak height whereas concentrations of the 2-pyridone were calculated from the peak area these latter peaks being often off the page. With this
224
procedure the yield of the enzymatic oxidation of N-methylnicotinamide calculated to be 2.8% of 4-pyridone and 7.5% of 2-pyridone. Preparation
was
of urine samples for HPLC
All purine, allopurinol and nicotinamide metabolites were determined by HPLC after a two step pre-separation of the urine samples by anion exchange chromatography [27]. The effluent plus washings with 2 ml of water for normal urines or 4.5 ml for urines of days with nicotinamide loading contained nicotinamide and its metabolites nicotinamide-N-oxide, N-methylnicotinamide [28] and the 2- and 4pyridone. The oxipurines as well as allopurinol and its metabolites were then eluted with 7.5 ml of 250 mmol/l NaCl in 40 mmol/l HCl. HPLC
analyses
All HPLC analyses were performed with a Hewlett Packard 1084 B Liquid Chromatograph. Elution buffers were prepared using chromatographic grade reagents obtained from Merck or BDH. System Z For the determination of hypoxanthine, xanthine, allopurinol, oxipurinol and allopurinol-1-riboside a Shandon ODS column (250 x 4.6 mm, 5 pm particle size) was eluted isocratically at a flow rate of 1 ml/mm with 7.35 mmol/l KH,PO, adjusted to pH 3; after 14.5 min methanol was introduced by pump B to yield a final concentration of 10% (v/v) in the elution buffer for a further 20 min followed by a 25 min equilibration period without methanol prior to subsequent sample injection [29]. System ZZ For the determination of unmetabolised nicotinamide the Shandon column was eluted isocratically at 1 ml/min with 73.5 mmol/l KH,PO,, pH 6, containing 10% methanol (v/v); for the determination of nicotinamide-N-oxide the content of methanol was reduced to 5% (v/v). System ZZZ N-methylnicotinamide was determined by the method of Carter [28] using a Beckman (Altex) Ultrasphere IP column (250 x 4.6 mm, 5 pm particle size); the elution buffer consisting of 10 mrnol/l KH,PO,, 5 mM 1-octanesulfonate and 10% acetonitrile (v/v) was adjusted to pH 7; the flow rate was 1.5 ml/mm. System IV The 2- and also the 4-pyridone were determined by the method of Carter [28]: the Beckman column was eluted at a reduced flow rate of 1 ml/mm with 10 mmol/l KH,PO,, pH 7, containing 2% acetonitrile (v/v); for the preparative isolation of the 4-pyridone the content of acetonitrile was reduced to 1% (v/v). Peaks were identified by stop flow UV spectra and by co-chromatography with standard substances. Estimation
of sulfite oxidase activity in vivo
The activity of sulfite oxidase (EC 1.8.2.1) was estimated using the Merckoquant 10013 Sulfite and 10019 Sulfate Test strips [30] with freshly voided urine of patient J.O. and proved to be normal.
225
Results Metabolism of allopurinol in xanthinuric patients and in normal controls
The metabolism of allopurinol in our xanthinuric patients and in normal controls has been reported in previous publications [10,19,20]. The results are compiled in Table I and clearly demonstrate the fundamental difference between the two types of xanthinuric patients: patient L.B. represents the group with a completely normal pattern of allopurinol metabolites; these patients excrete up to about 10% of the daily allopurind dose as allopurinol and allopurinol-1-riboside, and after an equiIibration period of 3-5 days up to 60% as o~pu~nol. Patients B. and J.O. on the other hand produced no oxipurinol, leading to a greatly increased excretion of unmetabolised allopurinol and allopurinol-1-riboside; allopurinol and allopurinol1-riboside being much more rapidly excreted than oxipurinol [18] the recovery amounted to about 70% of the dose already on the first day. An observation time of a few days is thus sufficient to confirm the lack of oxipurinol formation. The absence of uric acid and oxipurinol in the urine of patient J.O. is demonstrate in Fig. 1 which compares the urinary excretion of purines and allopurinol metabolites with control subject A. Activity of aldehyde oxidase in xanthinuric patients and in normal controls
The activity of aldehyde oxidase in vivo was determined by measuring the ratio of its physiolo~c~ substrate ~-methylni~otina~de to its oxidation products, the 2and cl-pyridone, in 24-h urines. Fig 2 shows the ratio of these metabolites using HPLC system II in control subject A and patient J-0. with and without a nicotinamide load of 3 X 400 mg/day. In control A a small amount of the 2-pyridone (retention time (RT) 10.20) and a trace of the 4-pyridone (RT 9.74) were found under normal conditions (Fig. 2a); following the ingestion of 3 X 400 mg nicotinamide a large increase in both pyridone peaks was observed {Fig. 2b).
TABLE I Mean 24-h urinary excretion of allopurinol, oxipurinol and allopurinol-1-riboside (mmol/day and % of dose) in our xanthinnric patients and control subjects receiving 3 x 200 mg (3 X 1.47 mmol) ailop~oI/ day. Source
Days of treatment
Oxipurinol
Allopurinol
AllopurinolI -riboside
Total
ll- 17 206-211 4,8
2.6(59%) 2.59(58.7%) 2.21(50.1%)
0.3(6.8%) 0.37(8.4%) 0.35(7.9X)
0.33(7.5%} 0.42(9.5%) 0.51(11.6X)
3.23(73.2%) 3.38(76.6%) 3.07(69.6%)
WI patient control control t191 patient
LB. B. G. B.
0-
6
n.d.
1.15(26.1%)
2.13(48.3%)
3.28(74.4%)
o7-
3 9
n.d. 2.91(65.9%)
1.7qlO.38) 0.4 (9%)
1.36(30.8%) 0.33(7.5%)
3.14(71.1%) 3.63(82.4%)
VOI patient J.O. control. A. n.d. = not detected.
226
t
t
0;
i
:, 0:
t f-4 N
Y
parlent
3.0.
Fig. 1. Determination of urinary excretion of purine and allopurinol metabolites. Chromatograms of 24-h urine samples of control subject A (day 8, 2030 ml) and patient J.O. (day 2, 2450 ml) receiving 600 mg allopurinol per day. Sample dilution 1: 15 with NaCl/HCl solution by pre-separation. HPLC system I. 254 nm, attenuation 2 t 5, injection volume 50 ~1. Peak identification: uric acid 6.60/6.62; hypoxanthine 7.85/7.84; xanthine 9.56/9.54; oxipurinol 12.29; allopurinol 14.49/14.44; allopurinol-l-riboside 24.13/23.98.
N-methylnicotinamide was not detected under normal conditions (Fig. 2a); after nicotinamide administration it appeared in a flat and broad peak covered with small shoulder peaks (Fig. 2b, RT 6.0, 7.51, 8.09). In patient J.O. no pyridones were detectable, either under normal conditions (Fig. 2c) or following the administration of nicotinamide (Fig. 2d); in the latter case a large peak of N-methylnicotinamide (RT 5.34) substituted for the missing pyridones. The ratio of pyridones to N-methylnicotinamide corresponding to the activity of aldehyde oxidase in vivo could thus be estimated to be high in the normal control and extremely low in patient J.O. For an exact determination of this ratio an HPLC system yielding quantifiable peaks of N-methylnicotinamide and a higher sensitivity especially for urines collected under normal conditions were required. Excretion of the 2- and 4-pyridone of N-methylnicotinamide A more sensitive pyridone determination was achieved by using the method of Carter [28] with a reduced flow rate (HPLC system IV) allowing for the simultaneous determination of the 2- and the 4-pyridone, the latter not being found by Carter. The results are
221
b
C
d
Fig. 2. Estimation of aldehyde oxidase activity in vivo. Chromatograms of 24-h urine samples of: control subject A before nicotinamide administration (965 ml) (a); control subject A after 3 X 400 mg nicotinamide (1670 ml) (b); patient J.O. before nicotinamide administration (1240 ml) (c); patient J.O. after 3 x400 mg nicotinamide (2030 ml) (d). Sample dilution 1: 5.3 with water by pre-separation. HPLC system II with 5% methanol. 254 nm, attenuation 2f6, injection volume 20 ~1. Peak identification: creatinine 3.79/3.80; nicotinamide-N-oxide 4.39; N-methylnicotinamide 5.34; 4-pyridone 9.14/9.75; 2-pyridone 10.20/10.19.
shown in Table II and Fig. 3. Using this more sensitive method, an increased injection volume (50 ~1) and a decreased attenuation (2 t 4) still no 4-pyridone but a small peak of 2-pyridone could be found in the urines of patient J.O.: the urine before nicotinamide administration (Fig. 3c) contained up to 14.5 pmol 2pyridone/day, the maximum excretion of this patient, surpassing the excretion on the day with nicotinamide administration (12.6 pmol/day; Fig. 3d). Patient B. not forming oxipurinol either excreted no 4-pyridone but a very little 2-pyridone (average 1.9 pmol/day; Table II). By contrast patient L.B. with normal formation of oxipurinol excreted an average of 95.7 pmol 2-pyridone/day together with 12.3 pmol 4-pyridone/day, values within the ranges of the control subjects which averaged 142.4 pmol/day for the 2-pyridone and 19.3 pmol/day for the 4-pyridone (Table II). The chromatogram of control subject A corresponds to a basal excretion of 139 pmol 2-pyridone and 18.9 pmol 4-pyridone per day (Fig. 3a). On the day of nicotinamide administration the sum of pyridones excreted in control subject A
II
4
x0-, oxip’ L.B.
n-d. = not detected.
Controls A A A A A C C D D
4 6 1
x0-, oxipB. J.O. J.O.
Days
-
3x400
Dose of nicotinamide (mg)
139 301.9 4845.5 4952.6 4653.4 151.5 4141.2 77.1 3816.1
96 (61
-162)
2 (1.3- 2.4) 6 (3.5-14.5) 12.6
2-Pyridone
Urinary excretion (pmol/day) of 2-pyridone, 4-pyridone, patients (X0-) without (oxip-) and with normal oxipurinol latter with the range for the number of days indicated
TABLE
18.9 26.4 458.5 491.6 467.8 21.6 408.7 10.2 372.3
12.3 (9-18)
n.d. n.d. n.d.
CPyridone
20.4 53.5 2024.5 2127.3 2322.5 37 2557.1 19.3 1898.2
17 (lo-
33)
178 (M-235) 205 (49-348) 7762.2
N-methylnicotinamide
N-methylnicotinamide, nicotinamide-N-oxide formation (oxip+ ) and in control subjects.
145.8 216.1 n.d. 281.8 n.d. 87.8
2.6 2.2 4.7 1.8 4.5 2.2
-7.6)
n.d. n.d. 158.2
(5.4
n.d. 177.4
Nicotinamide-Noxide
7.7 4.3 2.6
6.8
0.013 (0.006-0.024) 0.038 (0.015-0.072) 0.0016
ratio pyridones: N-methylnicotinamide
88.5
n.d.
n.d. 383.6
Nicotinamide
and unmetabolised nicotinamide in xanthinuric Results given are either single or mean values, the
229
a
b
Fig. 3. Determination of 2- and Cpyridone excretion. Chromatograms of 24-h urine samples of: control subject A before nicotinamide administration (965 ml) (a); control subject A after 3 x 400 mg nicotinamide (1670 ml) (b); patient J.O. before nicotinamide administration (1240 ml) (c); patient J.O. after 3 x 400 mg nicotinamide (2030 ml) (d). Sample dilution 1: 10.7 with water by pre-separation. HPLC system IV with 2% acetonitrile. 258 nm, attenuation 2f4, injection volume 50 pl. Peak identification: Cpyridone 10.69/10.67; 2-pyridone 11.07-11.24.
amounted to 5304 pmol/day (Fig. 3b) corresponding to an excretion 420-fold that of patient J.O. Similar amounts were found in controls C and D (Table II). The average ratio of 2-pyridone to Cpyridone was 7.5 in patient L.B. and the controls on days without and 10.2 in the controls on days with nicotinamide administration (Table II).
Excretion of N-methylnicotinamide Using the ion-pairing HPLC method of Carter [28] the basal urinary excretion of N-methylnicotinamide was easily quantifiable in all urine samples: it amounted to 20.4 pmol/day in control subject A (Fig. 4a) corresponding to one tenth of the excretion of patient J.O. (Fig. 4c; Table II). A high average basal excretion of 178 pmol N-methylmcotinamide per day was found also in patient B., the second xanthinuric patient not forming oxipurinol, whereas xanthinuric patient L.B. with normal formation of oxipurinol showed a very low average value of 17 pmol/day within the range of the healthy controls (Table II). On the day of nicotinamide administration, although a strong increase in N-methylnicotinamide excretion was observed in control subject A (2024.5 pmol/ day; Fig. 4b), the increment was much higher in patient J.O. (7762.2 pmol/day; Fig. 4d; Table II) thus compensating for the lack of pyridone formation.
230
a
b
C
Fig. 4. Determination of N-methylnicotinamide excretion. Chromatograms of 24-h urine samples of: control subject A before nicotinamide administration (965 ml) (a); control subject A after 3 x 400 mg nicotinamide (1670 ml) (b); patient J.O. before nicotinamide administration (1240 ml) (c); patient J.O. after 3 x 400 mg nicotinamide (2030 ml) (d). Sample dilution 1: 10.7 with water by pre-separation. HPLC system III. 264 nm, attenuation 2 t 4, injection volume 20 pl. Peak identification: N-methylmcotinamide 7.21-7.36.
Ratio of pyridone to N-methylnicotinamide excretion The absolute values of pyridone and N-methyhkotinamide excretion obviously strongly depend on the dietary supply of nicotinamide. However, the ratio of pyridone to N-methylnicotinamide excretion corresponding to the activity of aldehyde oxidase was rather constant under normal conditions ranging from 4.3 to 7.7 in the control subjects (Table II); in xanthimuic patient L.B. with normal formation of oxipurinol the same ratio was found ranging from 5.4 to 7.6 thus indicating a normal activity of aldehyde oxidase. On the other hand in the xanthinuric patients B. and J.O. the ratio of pyridone to N-methylnicotinamide excretion was extremely low ranging from 0.006 to 0.072 with an average of 0.013 in patient B. and 0.038 in patient J.O. corresponding to less than 1% of the normal value. This difference became even more pronounced following
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the administration of 3 X 400 mg nicotinamide: in the control subjects the ratio of pyridone to N-methylnicotinamide excretion decreased a little to an average value of 2.3, whereas it was much further reduced in patient J.O. to 0.0016 corresponding to 0.07% of the normal value (Table II). Excretion of nicotinamide-N-oxide and unmetabolised nicotinamide Nicotinamide-N-oxide originates from direct oxidation of nicotinamide catalysed by a microsomal mixed function oxidase [31]. The urinary excretion of nicotinamide-Noxide was measured using HPLC system II with 5% methanol (v/v; Fig. 2): nicotinamide-N-oxide was undetectable under normal conditions in control subject A (Fig. 2a) as well as in patient J.O. (Fig. 2~); on the day of nicotinamide administration a small peak of nicotinamide-N-oxide (RT 4.39) was observed both in control subject A (Fig. 2b) and patient J.O. (Fig. 2d) corresponding to 1.5-2.2 and 1.8X, respectively, of the dose (Table II). Unmetabolised nicotinamide, determined using HPLC system II with 10% methanol (v/v), was not detectable in urines of control subject A or patient J.O. while on normal diet. On the day of nicotinamide ingestion 0.9% of the dose were recovered unmetabolised in control subject A and 3.9% in patient J.O. (Table II). These results confirm our above observation that the main pathway of nicotinamide metabolism, the conversion to N-methylmcotinamide, is not influenced by aldehyde oxidase deficiency. Discussion
In order to evaluate the role of aldehyde oxidase in the metabolism of allopurinol we have used an indirect method to estimate the activity of this enzyme in normal and xanthinuric individuals in vivo thus avoiding the difficulties encountered using tissue biopsies: following the suggestion of Stanulovic and Chaykin [8,25] and of Krenitsky et al. [9] we have determined the conversion of N-methylmcotinamide, a physiological substrate of aldehyde oxidase, into the 2- and 4-pyridone. These metabolites of nicotinic acid and nicotinamide are excreted in appreciable amounts in human urine on a normal diet [32,28,33]. Terry and Simon [33] gave normal range estimates of 6-51.3 mg (39.5-337.5 pmol) 2-pyridone/24 h, 1.6-14.8 mg (9.3-85.7 pmol) N-methylnicotinamide/24 h and of a 2-pyridone: N-methyhricotinamide weight ratio of 1.76-5.9 corresponding to a molar ratio of 2-6.7. Considering this ratio as a measure of aldehyde oxidase activity in vivo we found that xanthinuric patient L.B. who formed oxipurinol had a normal activity of aldehyde oxidase: the average ratio being 6.8 : 1 as compared to 5.3 : 1 in the control subjects on a normal diet. The two other xanthinuric patients B. and J.O. not forming oxipurinol exhibited average ratios of 0.013 : 1 and 0.038 : 1 corresponding to 0.25% and 0.72% of the control value and thus indicating an almost complete deficiency of aldehyde oxidase. Lower than normal values for the 2-pyridone : N-methylmcotinamide ratio have been observed in nicotinic acid and tryptophan deficiency [34], the lowest molar ratio reported being 0.13 in a child suffering from pellagra; following nicotinamide
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treatment the ratio increased to 1.5 [34]. Thus in cases with decreased excretion of both pyridones and N-methyhricotinamide, resulting in a ratio of less than 1.0, nicotinamide should be administered to provide sufficient substrate for aldehyde oxidase. If high doses of nicotinamide are given it has to be taken into account that N-methylnicotinamide shows a considerably higher renal clearance than the pyridones [32]. Therefore the ratio of pyridones : N-methylnicotinamide will be lower in the first 24-h urine collected after a single administration of nicotinamide than in a 48- or 72-h urine collection [32] or after long-term daily ingestion [35]. Consequently a decrease of the ratio by about 50% was observed in the 24-h urines of our control subjects collected during nicotinamide administration. Nevertheless this nicotinamide load greatly exacerbated the difference between the control subjects and patient J.O.: the latter excreting 76.9% of the nicotinamide dose as N-methylnicotinamide, with still only a trace of 2-pyridone, and a decrease in the pyridone: Nmethylnicotinamide ratio to 0.0016 corresponding to 0.07% of the control value. This result confirms the almost complete lack of aldehyde oxidase in patient J.O. Our patients B. and J.O. represent the first cases of a proven combined deficiency of xanthine and aldehyde oxidase. This double enzyme defect in our patients seems not to have had any serious consequences, thus confirming the view that the symptoms of molybdenum cofactor deficiency are entirely attributable to the lack of the third molybdoenzyme sulfite oxidase [36] which was shown to have normal activity in our patient J.O. However, problems might arise following the administration of drugs which depend on aldehyde oxidase for detoxification. Considering the drugs known to be metabolised by aldehyde oxidase [37,38] no danger seems to exist at present. Other xanthinuric patients have also been reported who were unable to form oxipurinol from allopurinol which suggests that a similar combined deficiency of xanthine and aldehyde oxidase exists in these patients [17,18,39]. The patient reported by Higashino et al. [39] was also unable to oxidise pyrazinamide to 5-hydroxypyrazinamide in contrast to 2 other xanthinuric patients forming both oxipurinol and 5-hydroxypyrazinamide and thus corresponding to the patient of Auscher et al. [16,11]. This difference was attributed either to different mutations of xanthine oxidase altering its substrate specificity or to an alternative oxidase [16,38], presumably aldehyde oxidase [40]. From our results we suggest that the xanthimnic patients with normal oxipurinol formation possess a normal activity of aldehyde oxidase which obviously is able to convert pyrazinamide to 5-hydroxypyrazinamide at a normal rate [16]. The normal oxipurinol formation brought about by a normal activity of aldehyde oxidase in patient L.B. indicates that aldehyde oxidase can fully substitute for xanthine oxidase in the conversion of allopurinol to oxipurinol. Further support for this conclusion is the linear increase in the formation of oxipurinol with increasing allopurinol dosage in the presence of a decreasing formation of uric acid, indicating progressive inhibition of xanthine oxidase; this phenomenon has been observed by Sweetman [41] in children given increasing allopurinol doses up to 100 mg/kg per day. Saturation of oxipurinol formation was reached at doses of about 80 mg/kg per day. The maximum metabolic conversion of allopurinol to oxipurinol thus
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coincided with an almost complete inhibition of xanthine oxidase and therefore has to be attributed to aldehyde oxidase which is not inhibited by allopurinol or oxipurinol. We conclude from these observations that aldehyde oxidase as in rodents [7] is the main oxipurinol forming enzyme in man. Acknowledgements
We thank Mrs. H. Deharde for her technical assistance. The HPLC apparatus was given by the Deutsche Forschungsgemeinschaft. These studies were supported by the Arthritis and Rheumatism Council, UK. The kind and helpful cooperation of patient J.O. is gratefully acknowledged. After this work had been finished we were given a sample of 2-pyridone synthesised chemically by Dr. J. de Vries, Abteilung Klinische Pharmakologie, Universitat Heidelberg; by HPLC analysis and UV spectrophotometry this compound was identical with our 2-pyridone standard obtained by enzymatic oxidation of N-methylnicotinamide. References 1 Elion GB. Allopurinol and other inhibitors of urate synthesis. In: Kelley WN, Weiner IM, eds. Uric acid. Berlin: Springer Verlag, 1978;485-513. 2 Murrell GAC, Rapeport WG. Clinical pharmacokinetics of allopurinol. Clin Pharmacokinet 1986;11:343-353. 3 Johns DG, Spector T, Robins RK. Studies on the mode of oxidation of pyrazolo(3,4-d)pyrimidine by aldehyde oxidase and xanthine oxidase. Biochem Pharmacol 1969;18:2371-2383. 4 Krenitsky TA, Neil SM, Elion GB, Hitchings GH. A comparison of the specificities of xanthine oxidase and aldehyde oxidase. Arch Biochem Biophys 1972;150:585-599. 5 Johns DG. Human liver aldehyde oxidase: differential inhibition of oxidation of charged and uncharged substrates. J Clin Invest 1967;46:1492-1505. 6 Duley JA, Harris 0, Holmes RS. Analysis of human alcohol- and aldehyde-metabolizing isozymes by electrophoresis and isoelectric focusing. Alcoholism: Clin Exp Res 1985;9:263-271. 7 Huh K, Yamamoto I, Gohda E, Iwata H. Tissue distribution and characteristics of xanthine oxidase and allopurinol oxidizing enzyme. Jpn J Pharmacol 1976;26:719-724. 8 Stanulovic M, Chaykin S. Metabolic origins of the pyridones of N’-methylmcotinamide in man and rat. Arch Biochem Biophys 1971;145:35-42. 9 Krenitsky TA, Tuttle JV, Cattau EL Jr, Wang P. A comparison of the distribution and electron acceptor specificities of xanthine oxidase and aldehyde oxidase. Comp Biochem Physiol 1974;498:687-703. 10 Simmonds HA. Urinary excretion of purines, pyrimidines and pyrazolopyrimidines in patients treated with allopurinol or oxipurinol. Clin Chim Acta 1969;23:353-364. 11 Auscher C, Pasquier C, Mercier N, Delbarre F. Urinary excretion of 6 hydroxylated metabolite and oxypurines in a xanthinuric man given allopurinol or thiopurinol. Adv Exp Med Biol 1974;4IB:663-667. 12 Kojima T, Nishina T, Kitamura M, Hosoya T, Nishioka K. Biochemical studies on the purine metabolism of four cases with hereditary xanthinuria. Clin Chim Acta 1984;137:189-198. 13 Carpenter TO, Lebowitz RL, Nelson D, Bauer S. Hereditary xanthinuria presenting in infancy with nephrolithiasis. J Pediatr 1986;109:307-309. 14 Spector T. Inhibition of urate production by allopurinol. Biochem Pharmacol 1977;26:355-358. 15 Krenitsky TA. Aldehyde oxidase and xanthine oxidase - functional and evolutionary relationships. Biochem Pharmacol 1978;27:2763-2764. 16 Auscher C, Pasquier C, Pehuet P, Delbarre F. Study of urinary pyrazinamide metabolites and their
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