Toxic effects of warfarin in rats fed different diets

TOXICOLOGY AND APPLIED PHARMACOLQGY 28,337-348 (1974)

Toxic Effects of Warfarin in Rats fed Different Diets

H. W. COLVIN, JR. AND W. LEE WANG

Department of Animal Physiology, University of California, Davis 9.5616

Received January 26,1973; accepted January 3,1974

Toxic Effects of Warfarin in Rats Fed Different Diets. COLVIN, H. W., JR. AND WANG W. L. (1974). Toxicol. Appl. Pharmacol. 28, 337-348. Toxic doses of warfarin were administered (0.8 mg/kg, po) daily for 5 days to one group of rats (250-300 g) fed oat groats and to another fed Purina laboratory chow. Five rats from each dietary group were sacrificed at 5,29, 53, 77 and 101 hr after the initial dose. The plasma warfarin concentration increased more rapidly in the oat-fed rats than in those fed chow; however, the liver warfarin : plasma warfarin ratio following the first dose of warfarin indicated that the chow-fed rats were removing the warfarin from the plasma more rapidly. Presumably, the rate of metabolism of warfarin was more rapid in the chow-fed than in the oat-fed rats because the prothrombin times in the former increased at a slower rate. Apparently, less liver damage occurred in the chow-fed rats because they had lower plasma fibrinogen concentrations than those fed oats. There was a 50 ‘A decline in the hematocrit values in both groups. The linear decline in the total plasma protein in each group was a function of linear declines in plasma albumin and a- globulin. In both groups, the b- and y-globulins remained stable until 101 hr, at which time they increased. It was concluded that the response of rats to repeated toxic oral doses of warfarin was influenced by protein in the diet; rats fed higher protein diets were more tolerant to warfarin.

Warfarin, 3-(c+acetonylbenzyl)4-hydroxycoumarin, has been found to have a more potent anticoagulating action in rats than dicumarol, 3,3’-methylenebis-(4-hydroxycoumarin). The time required for warfarin to become effective is only half that of dicumarol and it is approximately 50 times more lethal (Seidman et al., 1950). Clinical comparisons have been made between dicumarol and warfarin (O’Reilly et al., 1964).

Various workers have alluded to the effect of dietary components on the anticoagulant action of warfarin and other coumarin compounds (Weiner, 1962); dietary fat in particular has received attention (Anonymous, 1965; Woods and Penick, 1964). Fyorala (1965) reported that milk-fed rats appeared to be more resistant to the coagulation factor-depressing action of warfarin than those receiving water. Chenkin et al. (1959) found starved guinea pigs to be much more sensitive to the effect of a coumarin derivative anticoagulant than those which were fed. Morrison et al. (1969) and Colvin et al. (1971) have presented evidence which suggests a relationship between dietary protein and the anticoagulant effect of toxic doses of dicumarol force-fed to ground squirrels. In view of these findings, it seemed desirable to examine more closely the relationship between diet and the anticoagulation properties of warfarin, a much more potent anticoagulant than dicumarol. Copyright 0 1974 by Academic Press Inc. 337 All rights of reproduction in any form reserved. Printed in Great Britain

338 COLVIN AND WANG

METHODS

Male Sprague-Dawley rats, weighing 250-300 g, were individually caged in a room maintained at 22°C. One group of 30 rats was fed a diet of Purina laboratory chow,l hereafter called “chow” (proximate analysis: 23.4% protein, 3.78 % fat, 50.58% nitrogen-free extract, 4.86% fiber). Another group of 30 rats received a diet of oat groats, hereafter called “oats”2 (proximate analysis : 12.0 % protein, 6.0 % fat, 65.5 % nitrogen-free extract, 2.0% fiber). A 7-day dietary adjustment period preceded the warfarin administration; during the last 4 days, feed consumption records were begun and continued throughout the experiment. Each rat was weighed at the time of the first dose of warfarin.

After the dietary adjustment period, warfarin3 was administered po every 24 hr for 5 days. The rats were fasted for 5 hr before each dose of warfarin. The warfarin was administered at a dose of 0.8 mg/kg with a rat oral feeding needle as explained previously (Colvin ef al., 1971). The warfarin was dissolved in 0.1 N NaOH at a concentration of 1 .O mg/ml.

Every 24 hr, 5 hr after warfarin administration, 5 rats from each group were anesthe- tized with chloroform. As rapidly as possible, the thorax was opened and blood samples were drawn from the still-beating heart. The first blood sample, 1.5 ml, was heparinized and used for the determination of total plasma protein, hematocrit and plasma protein fractionation. For the second sample, as much blood as possible was drawn and diluted with 0.1 M sodium citrate in 0.9 % NaCl solution (9 parts blood, 1 part citrate). The titrated blood was centrifuged immediately; the plasma was used for the determination of warfarin, prothrombin time and fibrinogen.

Immediately after bleeding, the livers were excised, weighed and frozen. Eventually, the livers were freeze-dried to reduce their water content to less than 5 %.

The method of O’Reilly et al. (1962) was used to determine the warfarin concentration in the titrated plasma. The concentration of warfarin in the liver was estimated by a modification of the procedure reported by O’Reilly et al. (1962) for plasma. Dried liver 1 g, was ground in 10 ml water at high speed for 2 min using a Virtis “23” homogenizer.4 Three ml of liver homogenate were pipetted into a 60-ml glass-stoppered bottle containing 20 ml 1 ,2-dichloroethane5 and shaken in an Eberbach6 laboratory shaker Model 2000 for 20 min at 266 oscillations/min. Following shaking, the contents were transferred to a 40-ml glass centrifuge tube and centrifuged for 10 min at 2000 rpm.’ Fifteen milliliters of the organic phase were transferred to a 60-ml glass-stoppered bottle containing 5 ml 0.5 M phosphate buffer, pH 7.5. This bottle was shaken for 5 min at 176 oscillations/min, the contents were then poured into a 40-ml glass centrifuge tube and centrifuged for 10 min at 2000 rpm. The aqueous phase was aspirated and discarded; 10 ml of the organic phase were transferred to a 60-ml glass-stoppered bottle containing 5 ml 2.5 N NaOH. The bottle was shaken for 5 min at 176 oscillations/min and the

1 Ralston-Purina Co., St. Louis, Missouri. 2 Triangle Milling Co., Portland, Oregon, ’ Sigma Chemical Co., St. Louis, Missouri. 4 Virtis Co., Gardner, New York. ’ MCB No. DX 800, 5636; Matheson Coleman & Bell, Los Angeles, California. 6 Eberbach Corp., Ann Arbor, Michigan. ’ International Centrifuge, Size 1, Model SBR.

TOXIC EFFECTS OF WARFARIN 339

contents transferred to a 40-ml glass centrifuge tube and centrifuged for 10 min at 2000 rpm. Following centrifugation, a sample of the aqueous phase was placed in a quartz cuvette and the absorbance determined at wavelengths of 308 and 360 nm in a spectrophotometer. The net absorbance was determined by subtracting the absorbance observed at 308 nm from the absorbance at 360 nm. The net absorbance was then converted to warfarin concentration by the use of a standard curve.

The warfarin standard curve was prepared by adding known concentrations of warfarin to homogenates (10, 30, 50 pg/ml homogenate) prepared from freeze-dried normal rat livers, 3 ml of which were then analyzed for their warfarin content. The average recovery of the warfarin added to the livers was found to be 91.6 + SE 0.76 7;;.

The Quick one-stage prothrombin time method was used to determine prothrombin complex activity (Quick, 1966). An Emdeco Model 3420 automatic prothrombin timer8 was incorporated into the technique. The thromboplastin reagent was prepared from rat brain acetone powder9 which was kept in a freezer until used (Quick, 1966).

For the determination of plasma fibrinogen, Gram’s method (1921), as modified by Morrison et al. (1969), was used. Hematocrits were determined by using heparinized capillary tubes in an International microcapillary centrifuge.‘O The total plasma proteins were estimated according to the biuret reaction technique as described in the Spectronic 20 Clinical Methods Manual. l1 The plasma protein fractions were determined by electrophoresis.12

All statistical techniques used were those considered in Steel and Torrie (1960).

RESULTS

The lethal dosage of warfarin for rats was reported to be 1 mg/kg (Pyorala, 1965). When this dose was administered po daily to rats for 5 days, it was found to be excessive for the immediate purpose. By further investigation, it was found that most of the rats would survive the experimental period when force-fed 0.8 mg/kg/day.

In preliminary experiments, it was found that 3 hr after oral dosing, the plasma warfarin concentration attained its maximum and remained at this level for about 6 hr at which time it began to decline. As the result of these findings, 5 hr after each po administration of warfarin, specific rats were sacrificed for analysis. A 5-hr fast preceded the force-feeding of warfarin in order to minimize the variability caused by the nutritional state of the rats.

The differences in feed consumption (Fig. 1) between the 2 dietary groups before and after warfarin administration were statistically significant (p < 0.01). Feed consumption declined in both groups of rats after warfarin administration began; the difference between the rates of decline was not statistically significant. There is some indication that feed consumption was maintained nearer normal levels for a longer period of time in the chow-fed rats when compared with those fed oats after warfarin administration was begun, i.e., 2 days for the chow-fed versus 1 day for the oat-fed.

8 EMDECO, Subsidiary of Coleman Instruments, Inc., Maywood, Illinois. 9 Type 2, fine ground, sodium citrate, desiccator dried; Pel-Freez Biologicals, Inc., Rogers, Arkansas. lo International Equipment Co., Needham Heights, Massachusetts. I1 Bausch & Lomb, Rochester, New York. I2 Model R-101 Microzone Electrophoresis Cell, Beckman Instruments, Inc., Fullerton, California.

340 COLVIN AND WANG

FIG. 1. Effect of diet and repeated toxic oral doses of warfarin (0.8 mg/kg/day) on the feed consumption of rats. Warfarin administration began on the fourth day. The dashed lines refer to the regression equations.

In the oat-fed rats there was a sharp increase in the plasma warfarin concentration 5 hr following the initial dose of warfarin (Fig. 2A). This plasma concentration of warfarin was sustained by doses of warfarin which were administered every 24 hr. There appeared to be a maximum at 77 hr (6.29 + SE 1.15 pg/ml titrated plasma); however, significant differences between the various means could not be shown.

For the chow-fed rats (Fig. 2B), the difference between the means for the 5-hr (2.71 + SE 0.22 pg/ml titrated plasma) and 29-hr (5.61 f SE 0.60) plasma warfarin concentrations was highly significant (p < 0.01). The differences between the means calculated for the other time periods were not statistically significant.

A comparison of Figs. 2A and 2B indicates that the nature of the diet had an influence on the rate at which plasma warfarin reached its maximum concentration. The difference between the 5-hr means for the rats on the 2 diets was statistically significant (p < 0.05). The titrated plasma warfarin values for each diet were pooled and a mean for each diet calculated; the difference between the means was not statistically significant.

FIG. 2. Effect of diet and repeated toxic oral doses of warfarin on the warfarin concentration of titrated rat plasma. Warfarin (0.8 mg/kg) was administered at 0,24,28,72 and 96 hr.

TOXIC EFFECT.9 OF WARFARIN 341

The mean liver warfarin concentration for the oat-fed rats (Fig. 3A) reached the maximum at 53 hr (41.7 + SE 3.05 pg/g dried liver). The differences between the 53-hr mean and the means calculated for the 5-hr (31.9 f SE 2.7) and 29-hr (26.8 + SE 1.52) periods were statistically significant at the 5 % and 1% levels, respectively.

The maximum liver warfarin concentration for the chow-fed rats (45.1 -t SE 2.54 pg/g dried liver) was reached at 77 hr. The difference between the means calculated for the 53-hr (31.9 + SE 2.35 pg/g dried liver) and 77-hr time intervals was statistically significant (p < 0.05).

The liver warfarin values from each diet were pooled and a mean calculated for each diet; the difference between these means was not statistically significant. However, the difference between the 53-hr means for the rats on the 2 diets was statistically significant (p < 0.05); therefore, these data suggest that the livers of the oat-fed rats reached the maximum warfarin concentration 24 hr faster than the chow-fed rats.

FIG. 3. The effect of diet and repeated toxic oral doses of warfarin on the warfarin concentration of dried rat liver. Warfarin (0.8 mg/kg) was administered at 0, 24, 48, 72 and 96 hr.

The control rat prothrombin time was found to be 14.4 + SE 0.36 sec. For the purpose of this experiment, if the plasma had not clotted by 240 set, it was assigned this value as the prothrombin time irrespective of how long the prothrombin time might have been. The rats on the oats diet had a more rapid increase in prothrombin time than the chow-fed rats (Fig. 4). The difference between the means at 53 hr (oat-fed 151 f SE 34.0 set, chow-fed 57.1 ) 5.69 set) was statistically significant (p < 0.01).

An evaluation of Figs. 1 and 4 indicates an inverse relationship between feed consumption after the beginning of warfarin administration (Fig. 1) and prothrombin time (Fig. 4). Correlation coefficients were calculated using the average values for feed consumption shown in Fig. 1 and the average values for prothrombin time shown in Fig. 4. Between these 2 parameters, the correlation coefficients were found to be -0.943 and -0.997 for the rats on the oats and chow diets, respectively; both of these coefficients were statistically significant (p < 0.01).

For the fibrinogen values in the rats on the oats diet, there were no statistical differences between the means (Fig. 5). The mean fibrinogen concentration calculated for the lOl-hr time period for the rats fed chow was significantly different (p < 0.01) from the mean fibrinogen concentration of other chow-fed rats. The difference between the mean 0-hr (control) fibrinogen concentration for the oat-fed rats (232 k SE 14.4 mg/lOO ml

342 COLVIN AND WANG

titrated plasma) and the mean of the chow-fed rats at 0-hr (202 f SE 6.80) approached significance (0.05 < p < 0.10). The difference between the mean fibrinogen values at the 5-hr time period (oat-fed, 222 f SE 7.00 mg/lOO ml titrated plasma; chow-fed

225 -

200 -

175 - 150 -

125 -

OAT GROATS OIET

$ 175-

150 -

125 -

100 -

75 -

I 1 L 5 29 53 17 101

TINE AFTER INITIAL WARFARIN ADMINISTRATION blRS1

FIG. 4. Effect of diet and repeated toxic oral doses of warfarin on the prothrombin time of titrated rat plasma. Warfarin (0.8 mg/kg) was administered at 0,24,48,72 and 96 hr.

320 - ,

300 - (A) _

280 -

“, IGO-

OAT GROATS DIET " l4D-

F I I I

c IGO-

160 -

1 53 77 ID1

TIME AGFTER INI::~ WARFARIN ADYINISTRATION (HRS)

FIG. 5. Effect of diet and repeated toxic oral doses of warfarin on the fibrinogen concentration of titrated rat plasma. Warfarin (0.8 mg/kg) was administered at 0,24,48,72 and 96 hr.

201 f SE 4.00) was statistically significant (p c 0.05). Mean fibrinogen values were calculated from the pooled data for the rats on each diet (oat-fed, 230 a SE 11.1 mg fibrinogen/100 ml titrated plasma; chow-fed, 203 + SE 8.2); the difference between the means was statistically significant (p < 0.05).


The repeated oral administration of warfarin to the rats on both diets caused a marked decline in hematocrit (Fig. 6). Significant regressions (p < 0.01) were found for both sets of data; the difference in the slopes was not statistically significant.

FIG. 6. Effect of diet and repeated toxic oral doses of warfarin on the hematocrit of heparinized rat blood. Warfarin (0.8 mg/kg) was administered at 0,24,48,72 and 96 hr. The dashed lines refer to the regression equations.

In both groups of rats there was a decline in the plasma albumin fractions (Tables 1 and 2). The following regressions were found to be statistically significant (p < 0.05) but the difference between the slopes lacked statistical significance: oat-fed rats, Y = 3.41 - 0.0086 X, + SE 0.31; chow-fed rats, Y = 3.46 - 0.0049 1, + SE 0.35; where Y refers to the g albumin/100 ml plasma, and Xto time in hr. The difference between the means calculated from the pooled albumin observations from the rats on each diet approached significance (0.05 < p < 0.10).

The a-globulin fraction declined with time following warfarin administration in both groups of rats (Tables 1 and 2). The following regressions were found to be statistically significant (p < 0.01) but the difference between the slopes was found not be be statistically significant: oat-fed rats, Y = 1.26 - 0.0075 X, + SE 0.22; chow-fed rats, Y = 1.20 - 0.0077 X, f SE 0.15; where Y refers to the g a-globulin/100 ml plasma, and X to time in hr. The difference between the means calculated from the pooled a-globulin observations from the rats on each diet was not statistically significant.

TABLE 1 EFFECT OF REPEATED TOXIC ORAL DOSES OF WARFARIN (0.8 mg/kg) ON THE PLASMA PROTEIN

FRACTIONS OF RATS FED A DIET OF OAT GROATS

Plasma protein fractions (g/100 ml + SE)

Time” Albumin

0 3.44 _+ 0.10 5 3.30 + 0.10

29 3.24 + 0.08 53 2.90 f 0.17 77 2.80 + 0.29

101 2.53 f 0.13

a-Globulin

1.26 + 0.10 1.10 + 0.06 1.17 & 0.10 0.97 f 0.12 0.53 + 0.14 0.48 + 0.07

/?-Globulin

0.70 * 0.03 0.69 + 0.03 0.65 + 0.02 0.68 t 0.05 0.70 + 0.15 0.93 + 0.06

y-Globulin

2.05 k 0.19 1.92 & 0.10 1.98 * 0.17 1.96 + 0.16 2.04 + 0.21 2.45 5 0.28

’ Refers to hours after the initial warfarin administration.

344 COLVIN AND WANG

The plama /?-globulin fraction during warfarin administration did not change until the IOl-hr time period (Tables 1 and 2). The difference between the 0-hr and lOl-hr means for each experimental group was statistically significant (p < 0.01). The difference between the lOl-hr means for the 2 groups of rats was not statistically significant. The difference between the means calculated from the pooled /?-globulin observations from the rats on each diet was not statistically significant.

TABLE 2

EFFECT OF REPEATED TOXIC ORAL D~SESOF WARFARIN(O.S mg/kg) ON THEPLASMA PROTEIN FRACTIONS OF RATS FED A Dm OF PURINA LAB CHOW

Plasma protein fractions (g/100 ml f SE)

Time” Albumin a-Globulin B-Globulin y-Globulin

0 3.63 f 0.11 1.14 L- 0.07 0.66 f 0.03 1.89 + 0.13 5 3.32 + 0.11 1.23 & 0.03 0.64 * 0.05 2.08 f 0.25

29 3.26 + 0.02 1.01 + 0.06 0.70 f 0.03 2.00 + 0.10 53 3.15 f 0.22 0.80 + 0.10 0.69 + 0.08 2.16 f 0.27 77 3.04 f 0.29 0.47 + 0.04 0.55 * 0.07 1.94 + 0.26

101 3.07 + 0.12 0.54 f 0.04 1 .oo + 0.01 2.67 f 0.15

’ Refers to hours after the initial warfarin administration.

The plasma y-globulin fraction did not appear to bc influenced by the nature of the diet or the warfarin administration until the lOl-hr time period, at which time this fraction increased in both groups of rats (Tables 1 and 2). The difference between the zero-hour and lOl-hr means for the chow-fed rats was statistically significant (p < 0.01) ; however, this was not the case for the oat-fed rats. All the plasma y-globulin observations were pooled for each dietary group and means calculated; the difference between these means was not statistically significant.

DISCUSSION

The reason why the rats on the chow diet should consume more feed than those fed oats is unclear (Fig. 1); however, the same difference has been observed in all of our experiments where these two diets have been used. The lab chow may have been more palatable, or, since their original diet was pelleted, this form was more acceptable. Other explanations might include the essential amino acid deficiencies (Warner, 1962), and amino acid imbalance (Harper et al., 1964) of the oat groats and the glucostat theory of satiety.

It is important to recognize that food intake in the chow-fed rats was maintained only slightly below normal levels for 3 of the 5 days of warfarin administration, whereas food consumption declined within 2 days in the oat-fed rats. The significance of food intake on the stress imposed by the conditions of these experiments is indicated by the high correlation coefficients found between feed consumption and prothrombin time. Presumably, the rats on the chow ration were in a better dietary position to maintain an amino acid pool for plasma protein synthesis.


In a previous study, Morrison et al. (1969) found the plasmadicumarol concentration to increase more rapidly in chow-fed than in oat-fed squirrels. The diet-warfarin response found in the work reported here with rats was just the opposite (Fig. 2). The difference might be explained by species and/or compound differences. Species differences in the metabolism of coumarin compounds have been previously reported (Pyorala, 1965). Differences in gastrointestinal pH induced by diet or differences in the volume and chemical composition of the ingesta which might influence warfarin ab- sorption could be responsible for the initial difference in plasma warfarin concentrations between the two groups of rats; more work is needed in this area.

Four hours after the administration of a single iv dose of warfarin (approximately 4.4 mg/kg) to rats, Lin (1955) found the concentration of warfarin in the plasma (per ml) to be about twice as great as the warfarin concentration in the liver (per g). Five hours after the first oral dose of warfarin we determined the mean liver :plasma ratio to be g.6 + SE 0.7 and 13.4 +_ SE 1.5 for the rats on the oats and chow diets, respectively. The difference between these means is statistically significant (p < 0.05). Using rat livers perfused with dicumarol, Levy and Nagashima (1969) found liver concentrations of dicumarol up to 3 times higher than in perfusion fluids. They commented that this difference might be due to the interaction of dicumarol with proteins and/or lipids in the liver, but that it could also reflect the existence of an active transport process. Pre- sumably, the rate of warfarin metabolism was more rapid in the chow-fed rats than in those fed oats because their prothrombin times did not increase as rapidly (Fig. 4).

As with squirrels (Morrison et al., 1969), the rats being fed the chow resisted the influence of the anticoagulant when compared with those fed oat groats (Fig. 4). Morrison et al. (1969) suggested that the level of protein in the diet may have been an important factor. Oat groats offer quantitatively an amount of protein which exceeds the maintenance requirements for rats but qualitatively they are deficient in lysine and methionine and borderline for tryptophan (Warner, 1962). Whether these amino acids can be limiting in the synthesis of the coagulation factors of the prothrombin complex under the condition of warfarin stress is not yet known.

The factors of the prothrombin complex begin to be synthesized when the plasma warfarin concentration in rats decreases to about 2 pg/ml (Pyorala, 1965). Conversely, when the plasma concentration of warfarin in humans reached 9 pg/ml the synthesis rate of the prothrombin complex factors was reduced to zero (O’Reilly ef al., 1970). In rats, the plasma warfarin concentration necessary to inhibit the synthesis of the prothrombin complex factors is about 4-5 pg/ml (Figs. 2 and 4).

In terms of liver concentrations of warfarin in the rat, it would appear that when the concentration is approximately 35 pg/g dried liver, there is a complete suppression of the synthesis of the factors of the prothrombin complex (Figs. 3 and 4).

There is the possibility that the level of vitamin K available to the two groups of rats might have been different and explain the differences in the prothrombin times. How- ever, it has not been possible to make rats deficient in vitamin K without preventing coprophagy (Mameesh and Johnson, 1959; Warner, 1962); we made no attempt to do this in our experiments.

There remains the possibility that hemodilution could have been involved in the observed difference in prothrombin times between the rats on the two diets (Fig. 4). Working with the concentration of prothrombin in human plasma, Quick ( 1966) has

346 COLVIN AND WANG

shown that when the prothrombin concentration was reduced to 30 ‘A the clotting time increased from 12 to 19 set, approximately. The hematocrit values in our rats declined by 50 %, which indicates a hemodilution that cannot explain the dramatic increase in the prothrombin times observed, if the events following the dilution of human plasma can be extrapolated to rats.

According to Field and Dam (1946), growing chickens fed on a high-protein diet had less plasma fibrinogen than their controls. Similar results have also been reported in dogs by Zeldis et al. (1945), who proposed that an elevated plasma fibrinogen reflects a metabolic disturbance in the liver when the animals have inadequate dietary protein, qualitatively or quantitatively. Our data appear to be in agreement with these findings (Fig. 5).

Nakamura et al. (1964) reported that long-term nontoxic dosages of warfarin in humans did not influence plasma fibrinogen. Recently, Forbes et al. (1973) found no significant differences in plasma fibrinogen in warfarin poisoning studies with dogs. Also working with dogs, Irish and Jaques (1945) reported that large doses of dicumarol tended to lower the plasma fibrinogen concentration and smaller doses raise it. In ground squirrels force-fed toxic dosages of dicumarol, Morrison et al. (1969) observed a slight increase in the plasma fibrinogen concentration. It has been suggested that slight hepatic dysfunction stimulates fibrinogen synthesis whereas severe damage retards it (Foster and Whipple, 1922; Irish and Jaques, 1945); our results are in agreement with these suggestions (Fig. 5).

There seems little doubt that the decline in hematocrit values (Fig. 6) was a function of the warfarin administration and ensuing hemorrhage. Similar results have been reported by others (Armour and Barnett, 1950; Morrison et al., 1969).

There is clear evidence that inadequate dietary protein can rapidly reduce the concentration of plasma albumin (Fleck et al., 1971; Kelman et al., 1972) and the half-life of the liver ribosomal RNA (Nordgren and Stenram, 1972). Our results indicate that the oat-fed rats had a lower plasma albumin concentration than those fed chow (Tables 1 and 2). The possible interaction of dietary protein, plasma albumin concentration, warfarin binding and liver enzymes concerned with warfarin metabolism has not been investigated.

The y-globulin fraction is not of hepatic origin and is not directly affected by liver insult of the type inflicted by our experiment. Others (Hoffenberg et al., 1964) have found the plasma content of y-globulin to be little influenced by non-protein diets or dietary protein deprivation. At the time of the last blood sample in our experiment, there was an actual increase in the y-globulin fraction in both groups of rats which may have reflected the general debilitated state of the animals.

In summary, we are of the opinion that dietary protein has an influence on the response of animals to which toxic levels of warfarin have been administered. At present we see no need to implicate vitamin K in the explanation of our results because of the difficulty in producing vitamin K deficiency in rats. A possible interpretation of our data implicates the influence of dietary protein on the plasma albumin concentrations. Diets quantitatively and/or qualitatively deficient in protein have been shown to de- press the total albumin mass. It is known that warfarin is tightly bound to plasma albumin (O’Reilly et al., 1963), and if this plasma protein is depressed, the possibility of a higher concentration of free warfarin will exist in the plasma. In turn, this free warfarin


can move more readily into the liver. If at the same time there is a depression of those enzymes involved with warfarin metabolism caused by dietary protein deficiency, the anticoagulant will be more effective in reducing the synthesis of the proteins of the prothrombin complex. Thus, dietary protein deficiency may augment the depression of the prothrombin complex factors induced by warfarin by depriving the synthetic machinery of essential amino acids.

REFERENCES

ANONYMOUS. (1965). Warfarin and a thrombogenic diet in rats. Nutr. Rev. 23,112-l 15. ARMOUR, C. J. AND BARNETT, S. A. (1950). The action of dicumarol on laboratory and wild

rats, and its effect on feeding behaviour. J. Hyg. 48,158-172. CHENKIN, T., DAYTON, P. G., WEISBERG, L. G. AND WEINER, M. (1959). Effect of starvation,

acenocoumarin and vitamin K on the coagulation pattern of the guinea pig. Exp. Med. Surg. 17,219-226.

COLVIN, H. W., JR., SKAIFE, D. D. AND MORRISON, J. T. (1971). Liver and fecal bishydroxycoumarin following repeated toxic oral bishydroxycoumarin administration in ground squirrels. Toxicol. Appl. Pharmacol. 19, 155-163.

FIELD, J. B. AND DAM, H. (1946). Influence of diet on plasma fibrinogen in the chick. J. Nutr. 31,509-523.

FLECK, A., WUNNER, W. H., HENDERSON, A. R., BALLANTYNE, F. C. AND TILSTONE, W. J. (1971). Response of the liver to protein feeding. Proc. Nutr. Sot. 39,4246.

FORBES, C. D., THOMSON, C., PRENTICE, C. R. M., MCNICOL, G. P., AND MCEWAN, A. D. (1973). Experimental warfarin poisoning in the dog. Platelet function, coagulation and fibrinolysis. J. Comp. Pathol. 83, 173-180.

FOSTER, D. P., AND WHIPPLE, G. H. (1922). Blood fibrin studies. II. Normal fibrin values and the influence of diet. Amer. J. Physiol. 58, 379-392.

GRAM, H. C. (,1921). A new method for the determination of the fibrin percentage in blood and plasma. J. Biol. Chem. 49, 279-295.

HARPER, A. E., LEUNG, P., YOSHIDA, A. AND ROGERS, Q. R. (1964). Some new thoughts on amino acid imbalance. Fed. Proc., Fed. Amer. Sot. Exp. Biol. 23, 1087-1092.

HOFFENBERG, R., BLACK, E., AND BROCK, J. F. (1966). Albumin and gamma globulin tracer studies in protein depletion states. J. Clin. Znvest. 45, 143-152.

IRISH, U. D. AND JAQUES, L. B. (1945). The effect of dicumarol upon plasma fibrinogen. Amer. J. Physiol. 143, 101-104.

KELMAN, L., SAUNDERS, S. J., FRITH, L., WICHT, S. AND CORRIGAL, A. (1972). Effects of dietary protein restriction on albumin synthesis, albumin catabolism, and the plasma aminogram. Amer. J. Clin. Nutr. 25, 1174-1178.

LEVY, G. AND NAGASHIMA, R. (1969). Comparative pharmacokinetics of coumarin anti- coagulants. VI. Effect of plasma protein binding on the distribution and elimination of bishydroxycoumarin by rats. J. Pharm. Sci. 58, 100-1004.

LIN, T. H. (1955). Studies on Warfarin. Ph.D. Thesis, University of Wisconsin, Madison, Wisconsin.

MAMEESH, M. S. AND JOHNSON, B. C. (1959). Production of dietary vit. K deficiency in the rat. Proc. Sot. Exp. Biol. Med. 101,467468.

MORRISON, J. T., COLVIN, H. W., JR., SKAIFE, D. D. AND MARSH, R. E. (1969). Effect of bishydroxycoumarin and diet on blood coagulation in ground squirrels. Amer. J. Physiol. 216,429-435.

NAKAMURA, R. M., KUNITAKE, G. M., FUJITA, K. AND REILLY, E. B. (1964). Plasma-fibrinogen levels after long-term warfarin therapy. Lancet 2, (7363), 821-822.

NORDGREN, H. AND STENRAM, U. (1972). Decreased half-life of the RNA of free and mem- brane-bound ribosomes in the liver of protein-deprived rats. Hoppe-Seyler’s Z. Physiol. Chem. 353, 1832-1836.

348 COLVIN AND WANG

O’REILLY, R. A., AGGELER, P. M., HOAG, M. S. AND LEONG, L. (1962). Studies on the coumarin anticoagulant drugs: The assay of warfarin and its biologic application.. Thromb. Diath. Haemorrh. 8, 82-95.

O’REILLY, R. A., AGGELER, P. M. AND LEONG, L. S. (1963). Studies on the coumarin anticoagulant drugs. The pharmacodynamics of warfarin in man. J. Clin. Invest. 42,1542-1551.

O’REILLY, R. A., AGGELER, P. M. AND LEONG, L. S. (1964). Studies on the coumarin anticoagulant drugs. A comparison of the pharmacodynamics of dicumarol and warfarin in man. Throm. Diath. Haemorrh. 11, l-22.

O’REILLY, R. A., LEVY, G. AND KEECH, G. M. (1970). Kinetics of the anticoagulant effect of bishydroxycoumarin in man. Clin. Pharmacol. Ther. 11, 378-384.

PYORALA, K. (1965). Determinants of the clotting factor response to warfarin in the rat. Ann. Med. Exp. Biol. Fenn. 43, Suppl. 3, l-99.

QUICK, A. J. (1966). Hemorrhagic Disease and Thrombosis. Lea & Febiger, Philadelphia, Pennsylvania.

SEIDMAN, M., ROBERTSON, D. N. AND LINK, K. P. (1950). Studies on 4-hydroxy-coumarins. X. Acylation of 3-a-phenyl-j?-acetylethyl-4-hydroxycoumarin. J. Amer. Chem. Sot. 72, 5193-5194.

STEEL, R. G. D. AND TORRIE, J. H. (1960). Principles andprocedures ofstatistics. McGraw-Hill, New York.

WARNER, R. G. (1962). Nutrient requirements of the laboratory rat. Nutrient Requirements of Laboratory Animals. NAS-NRC Publ. 990, pp. 51-95.

WEINER, M. (1962). Pharmacological considerations of antithrombotic therapy. Advan. Pharmacol. 1,277-307.

WOODS, J. W. AND PENICK, G. D. (1964). Warfarin and diet induced lipidosis in rats. Arch. Pathol. 78,234-244.

ZELDIS, L. J., ALLING, E. L., MCCOORD, A. B. AND KULKA, J. P. (1945). Plasma protein metabolism-electrophoretic studies. Chronic depletion of circulating proteins during low protein feeding. J. Exp. Med. 82, 157-179.

Toxic effects of warfarin in rats fed different diets

Documents

Transcript of Toxic effects of warfarin in rats fed different diets