uricosuric effect of dicoumarol

Acta Medica Scandinavica. Vol. 175, fasc. 4,1964

From the First University Clinic of Internal Medicine (Head: C. Holten, M. D.), Aarhus Kommunehospital, and the Institute of Hygiene (Head: B. Lambert, M. D.),

University of Aarhus, Aarhus, Denmark

Uricosuric Effect of Dicoumarol

BY

FLEMMING CHRISTENSEN

It has been known for some time that certain drugs, which are chemically cou- marins and indandiones, and which are used clinically as anticoagulants, produce a uricosuric effect in man. I t has thus been demonstrated that several of these anticoagulants on administration to human beings give rise to an increase in the urinary excretion of uric acid and a decrease in the plasma concentration of uric acid.

Such a uricosuric effect of a coumarin preparation was first described by Sougin- Mibashan and Horwitz (11) after administration of ethyl biscoumacetate (3,3’- carboxyethylmethylene -bis- (4- hydroxycoumarin) Tomexan@) in human beings, and this effect has since been confirmed by Ogryzlo and Harrison (8) and Thomp- son et al. (12). A similar uricosuric effect of dicoumarol (3,3’-methylene-bis-(4- hydroxycoumarin)) was reported by Hansen and Holten (6) and Dreyfuss and Czaczkes (4). Thompson et al. performed a comparative study of the uricosuric effect of four different coumarin and indandione drugs. They administered the Submitted for publication September 11, 1963.

drugs in a dosage such that the effects as assessed on the basis of blood-coagulation studies were nearly identical. Adminis- tered in this way, ethyl biscoumacetate and, to a lesser degree, phenylindandione (2-phenyl-indandione- 1,3) had a pronounced uricosuric effect. O n the other hand, the two other drugs studied - acenocoumarin (3-( a-p-nitrophenyl-B- acetyl-ethyl)-4-hydroxycoumarin, Sin- tram@) and anisindione (2-(p-methoxy- pheny1)-indandione-l,3, MiradonB) - were without demonstrable uricosuric effect. Thus, no evidence was disclosed in favour of the assumption that the uricosuric action varies in parallel with the anticoagulant action. Pasero (9) also reported that no correlation seems to exist between the uricosuric and anticoagulant actions of coumarin drugs.

The mechanism of the uricosuric action of anticoagulants is largely unknown. In the light of the results of the aforementioned studies by Sougin-Mi- bashan and Horwitz and Thompson et al., it is, however, reasonable to assume that the effect is due to a decreased re-

46 1

462 FLEMMING CHRISTENSEN

absorption or an increased secretion of uric acid by the renal tubules and not to an enhanced filtration rate i n the glomeruli. It is uncertain if the effect on the kidneys is referable to a n anti-vitamin K effect; Pasero mentioned a number of factors which weigh against this explana- tion.

Apart from the inhibitory action on blood coagulation, anticoagulant drugs of the coumarin or indandione group exert few pharmacodynamic effects on the organism. T h e uricosuric action must be included among these effects. At the present time, it appears doubtful if this effect of anticoagulants is of any significance in connexion with their therapeutic use. Both Sougin-Mibashan & Horwitz and Hansen & Holten have, however, suggested such a possibility.

Thus, the uricosuric effect of anticoagulants seems, for several reasons, to be of interest, and further studies of this effect may contribute to our under- standing of the mode of action of these drugs in the organism.

T h e present paper is a report of the results of an investigation on the uricosuric action of dicoumarol as determined by clearance studies in four subjects. I n addition, attempts were made to throw light on the relationship of this action to the plasma concentration and urinary excretion of dicoumarol.

Material and methods The investigation reported was performed

on four men admitted to the First University Clinic of Internal Medicine, Aarhus Kom- munehospital. The four subjects, who are hereafter referred to as A, B, C and D, presented no clinical signs of hepatic, renal or cardiac disease. Furthermore, none of the subjects studied had any known predis- positions to disorders of uric acid metabolism.

Before and during the experiments, the subjects were given an ordinary hospital diet, and during the experimental periods no changes occurred in their daily activities, except those necessitated by the experiments. The clearance studies were performed by collection of 2-hour urinary samples, and blood samples were withdrawn exactly in the middle of each 2-hour period. In all the experiments, spontaneously voided urine was used, and the subjects had been told beforehand that it was important that the evacuation of the bladder should be as complete as possible. In order to ensure a suitable urinary output during the individual clearance periods, the subjects ingested a t least 0.5 1 of water during the first half hour of each period. After measurement of the volume, the total urinary specimen was stored at - 20" C until the analysis was performed. The heparinised blood samples were immediately centrifuged, and the plasma was pipetted off. The urine and plasma were analysed for their content of uric acid, creatinine and dicoumarol. However, dicoumarol determinations were performed only on the urine from B and C. The clearance values for uric acid and creatinine were calculated in the usual way, and the values obtained were corrected to a body surface of 1.73 sq.m. The body surfaces of the subjects were determined by the use of a nomogram based on Du Bois' height-weight formula (2).

The first clearance period - early in the morning of the first day - was used as a control period. Immediately after the end of the control period, each subject was given a single oral dose of dicoumarol. The doses were 400 mg in A and B and 500 mg in C and D. Dicoumarol was invariably given in the form of 50 mg tablets.

The clearance studies were performed at suitable intervals during the period immediately following the administration of dicoumarol. These studies were continued until the plasma concentration of dicoumarol had reached a suitably low level, usually for 120 hours, However, in subject A, the experiment was interrupted after the lapse of 72 hours.

The plasma concentration of dicoumarol was assessed by means of ultraviolet spectro- photometry by the method of Axelrod et al. (1). This method is based upon extraction of

URICOSURIC EFFECT OF DICOUMAROL 463

dicoumarol from acidified plasma or urine by means of n-heptane. The substance is re- extracted from this by 2.5 N NaOH. The content of dicoumarol, which has a distinct absorption maximum at a wavelength of 315 mp, is then determined spectrophoto- metrically. I t is reported that the presence of dicoumarol metabolites in the plasma does not interfere with the determination. In the present study, this method was used with the slight modification that the acidification of the plasma was done with 1 N HC1 instead of with 3 N HCl as originally stated. With this slightly modified technique, a number of experiments were performed in which the re- recovery of dicoumarol added to human plasma in amounts ranging from 10 to 40 pg/ml was studied. The mean recovery rate obtained was 97.9 % (n = 8), and the standard error of this mean was calculated as & 1 yo. The results of the plasma-dicoumarol determinations given are not corrected for this incomplete recovery. All dicoumarol determinations were performed in duplicate, and the mean value of the two determinations was taken as the result. The individual determinations rarely differed more than 4 yo from the mean value.

The dicoumarol determinations on urine were performed according to the same method as was used in the plasma-dicoumarol determinations. The specificity of dicoumarol determinations in urine seems to be lower than in plasma, since urine samples from un- treated persons revealed appreciable amounts of apparent dicoumarol. This fact coupled with the low concentration of dicoumarol in the urine of dicoumarol-treated persons rendered the determinations on urine fairly uncertain.

Uric acid in the plasma and urine was estimated by the uricase method described by Prztorius and Poulsen (10). The results are given as the mean value of duplicate determinations. The individual determinations rarely differed more than 2 yo from the mean value.

Creatinine in the plasma and urine was determined by the method of Owen et al. (7). Experiments with recovery of creatinine added to human plasma in amounts from 5 to 15 pg/ml showed a mean recovery rate of 98.0 yo (n = 6). The standard error of this mean was found to be + 0.9 %.

Table I . Urinary excretion of uric acid in pguglminl 1.73 sq.m body surjace in the four subjects A , B, C and D before and after oral administration of dicoumarol

Periods (hours after ingestion)

Control 2-4 6-8

11-13 23-25 31-33 47-49 55-57 71-73

119-121 167-169

- A

200 340 515 48 1 883 567 394 45 1 294 - -

- B

252 250 427 624 559 345 329 306 310 244 -

- C

320

504 389 549 635 3 72

502 429 333

-

-

- D

371

5 76 393 574 405 384

344 376

-

-

-

Mean values

286

506 472 641 488 370

363

-

-

(350) -

Results The results of the experiments appear

from fig. 1 and from table I. I t should be noted that the results from the control periods are extrapolated to zero time, and that the values for the uric acid clearances and urinary excretion of uric acid are indicated by dots corresponding to the middle of the individual periods.

In the periods following adminstration of dicoumarol, the urinary excretion of uric acid showed increasing values (table I). The maximum values for the urinary excretion of uric acid were found in the four experiments from about 12 to 36 hours after the administration, and were from slightly less than two to about four times as high as the values recorded for the corresponding control periods. Thus, while the average excretion of uric acid in the control periods was found to be 286 pglmin., the four subjects showed an average maximum excretion of 641 pgl min. 24 hours after the administration of

FLEMMINO CHRISTENSEN 464

20- cluric acid lClcreatinine

Ol

4 24 48 72 96 hours I I 1

Plasma uric acid

4 o l

r Plasma dicoumarol

c'uric acid

L r O L

Plasma uric acid

z 40

Plasma dicoumarol

c h i c acid /Clcrca t ininc

OL 6 or Plasma uric acid

4oL Plasma dicoumarol

1 I I b 1

24 48 72 96 hoi

:: 10

CIuric acid

OL Plasma uric acid

40L Plasma dicoumarol

20

B

D

Fig. 1. The concentrations of dicoumarol and uric acid in the plasma and the clearance of uric acid after a single oral dose of dicoumarol (400-500 mg) given to each of the subjects A, B, C and D. The ratios of uric acid clearance to creatinine clearance are also given. The values for uric acid clearance are calculated for a surface area of 1.73 sq.m.


dicoumarol. At 72 hours the average excretion of uric acid had fallen to 363 pug/ min.

The plasma concentrations of uric acid in the four subjects tended to fall after administration of dicoumarol. In three of them, the fall was most pronounced at about 32 hours after the administration, while the minimum occurred as early as at 12 hours in the fourth (D) . The mean value of the plasma concentration of uric acid in the control periods was found to be 56.0 ,ug/ml; at 32 hours after the administration of dicoumarol it was 45.0 ,ug/ml, and at the end of the experiments it had increased

The values of the uric acid clearances increased during the first 12 to 36 hours after administration of dicoumarol, after which time decreasing values were obtained. During the control periods before the administration of dicoumarol, the mean value of the uric acid clearances was 5.1 ml/min., while the average of the maximum values recorded was 14.1 ml/min. At 24 hours the mean value of the uric acid clearances reached a maximum of 11.6 ml/min., and at the end of the experiments the mean value recorded was 5.8 ml/min.

The uric acid/creatinine clearance ratio also showed an increase after dicoumarol administration. Peak values were recorded during the period from 12 to 36 hours after administration. For the control periods this ratio expressed as a percentage averaged 6.2, while the average of the maximum values recorded was 13.8. At 24 hours the mean value was 10.2, a t 32 hours 10.8 and at the end of the experiments 6.8.

In the four subjects, the plasma concentration of dicoumarol was found to reach a maximum within the first 12

to 53.8 pg/ml.

hours after the administration of dicoumarol. The peak concentrations of dicoumarol observed in A, B and C ranged from about 26 to about 21 pg/ml, while D, who also revealed the least uricosuric effect after dicoumarol, showed a peak concentration of only 16.9 pg/ml. When the maximum plasma concentration of dicoumarol had been recorded, the concentrations decreased with time as in a first-order reaction. After the lapse of 120 hours, the plasma concentration of dicoumarol had fallen to between 1 and 2 pg/ml in the subjects B, C and D. As already stated, the experiment was interrupted in A at 72 hours after administration; at this time the plasma concentration of dicoumarol was still about

The urinary excretion of dicoumarol, which was studied in all periods in the subjects B and C, was found to be very low. The maximum excretion demonstrated amounted to approx. 1 puglmin., and the total excretion was calculated as less than 1 % of the dose administered.

By comparing the plasma dicoumarol concentrations in the individual cases (A, B, C and D) with the corresponding values of uric acid clearances, the ratios of uric acid clearance to creatinine clearance and with the plasma uric acid, it is evident that no simple relationship (i. e. proportionality) exists between the plasma dicoumarol concentration and the uricosuric action measured by these quantities. I t should be noted that the maximum concentration of dicoumarol in cases A and C was observed at least 12 hours before the maximum uricosuric effect was obtained. I t should also be noted that there did not seem to be any definite correlation between the urinary excretion of dicoumarol and the observed uricosuric effect.

5 pg/ml.

30--1343003. A d a M e d . Scand. Vol. 175

466 FLEMMINO CHRISTENSEN

Discussion

From the results reported above it appears that the administration of dicoumarol in man affects the urinary excretion of uric acid. After oral administration of the drug in doses ranging from 400 to 500 mg, the urinary excretion rose to a mean maximum value of about twice the value recorded in the control period. The uric acid clearance also showed an increase which was almost of the same order of magnitude. In comparison, it may be stated that Hansen and Holten ( 6 ) determined the urinary excretion of uric acid during 24-hour periods after oral administration of 500 mg dicoumarol in two persons. In these experiments, they observed an increase in the clearance of uric acid of 45 and 31 yo as compared with the preceding control periods. However, these figures cannot be directly compared with the results reported in this paper, because of the difference in the lengths of the clearance periods used in the two studies. If this difference is taken into account, the results obtained do not seem to be di- vergent.

In order to place dicoumarol among other uricosuric anticoagulants the present results may be compared with those obtained by others in studies on the uricosuric effect of ethyl biscoumacetate and phenylindandione. Sougin-Mibashan and Horwitz (11) found in two persons, after oral administration of 1.8 and 1.5 g ethyl biscoumacetate, an increase in the uric acid clearance to 6 and 8 times the values recorded during the preceding control periods. In these experiments, short clearance periods of about 30 minutes were used. Thompson et al. (12) found in four subjects, who had each ingested 2,000 mg ethyl biscoumacetate,

an increase in the uric acid clearance. The mean value of the recorded maximum uric acid clearance here was 4 times greater than the mean value during the control periods. After oral administration of 350 mg phenylindandione to each subject, the mean value of the uric acid clearance increased to 1.5 times the value which had been recorded in the preceding control period. In both experiments, the clearance periods extended over 3 hours. The doses of ethyl biscoumacetate and phenylindandione just mentioned and those of dicoumarol used in the present study are frequently used as therapeutic initial doses of these anticoagulants. As a rule, these doses will exert roughly the same effect on blood coagulation. When the uricosuric action of these three anticoagulants is compared on the basis of these doses, it is seen that the effect of ethyl biscoumacetate is considerably greater than those of phenylindandione and dicoumarol, while the latter two seem to have approximately the same effect. If the size of the doses administered is taken into account, the difference in the uricosuric action of the three anticoagulants is less pronounced.

As stated in the section on the results, a fall in the plasma level of uric acid was observed after administration of dicoumarol. On an average, this fall amounted to 11.0 ,ug/ml after the lapse of 32 hours as compared with the mean value of the plasma uric acid inthe control periods. In comparison, it may be stated that Hansen and Holten found an average fall in the serum level of uric acid of 1.8 mg/100 ml after oral adminstration of dicoumarol in doses ranging from 500 to 700 mg. After oral administration of 2,000 mg ethyl biscoumacetate to four persons, Thompson et al. observed an average decrease in the serum level of


uric acid of 2.0 mg/100 ml, and after oral administration of 350 mg phenylindandione a similar average fall of 1.5 mg/100 ml. Judging from these figures, therapeutic doses of dicoumarol seem to have a somewhat lower uricosuric effect than corresponding doses of ethyl biscoumacetate and an effect of almost the same magnitude as therapeutic doses of phenylindandione.

The decrease in the plasma level of uric acid is presumably due to the increased urinary- excretion. O n the basis of the present investigation it cannot be decided if other factors also contribute to the induction of this effect.

The mechanism of the uricosuric action of dicoumarol and other anticoagulants is at present unknown. The increase in the ratio of uric acid clearance to creatinine clearance observed in this study after ingestion of dicoumarol and also described after ingestion of other anticoagulants (1 1, 12) indicates a tubular mechanism as a cause of the uricosuric action. An increase in the amount of uric acid filtered by the renal glomeruli might contribute to this effect. As the amount of uric acid filtered in the glomeruli is equal to the product of the concentration of filterable uric acid in the plasma and the rate of glomerular filtration, it is evident that this amount would increase if one of the aforementioned factors increased. I t is a widely accepted view that all the uric acid in the plasma is freely filterable in the glomeruli (5, 15). An increase in the filtered amount of uric acid should then be possible only if the rate of glomerular filtration increased. On the assumption that the size of creatinine clearance reflects this filtration rate, it is possible from the present study to judge the significance of this factor. After ingestion of dicoumarol the mean values

for creatinine clearance in the various periods showed a certain but low increase (5-15 %) in comparison with the value for the control periods. In one period a somewhat higher value (about 30 yo) was observed. From this it may be concluded that the uricosuric action of dicoumarol is mainly or purely of tubular origin, and that increased filtration by the glomeruli is of only minor importance, if of any. Onthe basis of the studies of Gutman et al. (5) and others it may be said that it is highly probable that uric acid is both reabsorbed and secreted by the tubular cells. Dicoumarol must exert its uricosuric action through one of these factors, but a t present it is not clear which of these is of importance. Among the various possibilities of the mechanism of action of dicoumarol on the renal tubules two will be mentioned. First, the possibility that dicoumarol and uric acid should compete for a common system of transport in the tubules seems unlikely as it is known that dicoumarol is not filtered by the glomeruli because of extensive binding to the plasma proteins, especially the albumin fraction ( 13), and as stated, no excretion of unchanged dicoumarol occurs in the urine. Secondly, the possibility that dicoumarol in the organism might be metabolised to a uricosurically active substance should be mentioned. Such a metabolic process would be assumed to be time-consuming and a certain time lag between maximal dicoumarol concentration in the plasma and maximal uricosuric action observed should be expected. Such an observation was actually made in experiments A and C. However, as the metabolic trans- formation of dicoumarol in man is unknown (14), it is not possible to test this hypothesis further a t present. The observation made by Burns et al. (3) that a

468 FLEWMING CHRISTENSEN

metabolite of a phenylbutazon analogue (G-26671) showed a much more pronounced uricosuric effect than the parent drug indicates that such a mechanism would not be unreasonable.

Summary

The uricosuric effect of a single oral dose of dicoumarol (400-500 mg) was studied in four subjects. Urinary excretion of uric acid, uric acid clearance and the ratio of uric acid clearance to creatinine clearance all showed increasing values after ingestion of dicoumarol. Maximum mean values were obtained after the lapse of about 24 hours and were about twice the mean values obtained in the control periods. The plasma uric acid concentration decreased to a minimum at about 32 hours after the administration of dicoumarol. The average decrease was 11 pglml. No simple relationship was found between the dicoumarol concentration in the plasma, which showed a maximum value at 8- 12 hours after dicoumarol ingestion, and the uricosuric effect, but nevertheless a certain relationship between the maximum concentration of dicoumarol in the plasma and the maximum uricosuric effect may exist. The urinary excretion of dicoumarol was studied in two persons and was found to be very low. No relationship between urinary dicoumarol excretion and uricosuric action was established.

Some problems concerning the mechanism of the uricosuric action are dis- cussed. The possibility that the uricosuric action of dicoumarol might be ascribed to a metabolite of this drug is mentioned.

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uricosuric effect of dicoumarol

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