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DATE DUE DATE DUE DATE DUE _-::J—- fiF—Tm chnNfimwvoActimEmd Opponunlty ltylmtltuion flHiE EFFECT OF PENICILLIN AND DICOUMAROL ON BLOOD COAGULATION AND DPN CYTOCHROME-c REDUCTASE ACTIVITY IN THE RAT by Sister Mary Romana McDermott, S.N.J.M. A THESIS fiubmitted to the School of Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Foods and Nutrition College of Home Economics 1959 tha 811i Ca] ACKNOWLEDGEMENT This worker wishes to eXpress her sincere thanks to Dr. Dorothy Arata for her encouragement, guidance and constructive criticism; and to Catherine Carroll for her assistance in checking data. TABLE OF CONTENTS INTRODUCTION........................................... 1 REVIEW OF LITERATURE................................... 3 The Effect of Vitamin K on Blood Coagulation........ 3 Site of Action of Vitamin K......................... 5 Effect of Penicillin on Blood Coagulation........... 6 Role of Vitamin K in Electron Transport.............lO EXPERIMENTAL PROCEDURE.................................l4 RESULTS................................................l9 DISCUSSION.............................................22 SUMMARY................................................27 LITERATURE CITED.......................................31 APPENDIXOOO.........OOOOOOO00............OOOOOOOOOOOOOO i Number I II III IV VII VIII TABLES Title Page Supplements to Basal Ration.............. 14 Schedule for Sacrificing Animals......... 15 Reaction Mixture for DEN Cytochrome—o Re— ductaseOOOOOOO0.000000000000000000000000 17 Food Intake, Weight Gain and Liver Weight of Experimental Groups.................. 29 Coagulation Time, Liver Nitrogen and DPN Cytochrome—c Reductase Activity of Ex- perimental Groups....................... 50 Appendix Data for Individual Rats in Group 1...... 1 Data for Individual Rats in Group II..... iii Data for Individual Rats in Group III.... v Data for Individual Rats in Group IV..... vii Data for Individual Rats in Group V...... ix Data for Individual Rats in Group VI..... xi Data for Individual Rats in Group VII....xiii Data for Individual Rats in Group VIII... xv INTRODUCTION us ce. th in: the and blo on 1 this betw We. 88 a] a PM INTRODUCTION Antibiotics have come to play an important role in the lives of the present generation. The medical pro- fession recognized the usefulness of antibiotics in the treatment of disease. The nutritionist discovered their ability to improve the growth and quality of livestock. The antibiotics were widely used before reports began to appear which indicated possible unfavorable reactions to their wanton use. In the case of penicillin, one of the most widely used of the antibiotics, conflicting reports appeared con- cerning the effect of this compound on the coagulation time of the blood. Some laboratories reported an observed increase in coagulation time in patients under penicillin therapy, while other workers reported the opposite effect, and still others could measure no effect of penicillin on blood clotting time. Obviously, any effect of penicillin on blood coagulation would seriously restrict the use of this antibiotic in certain clinical conditions. In contemplating the possible interrelationship between penicillin and blood coagulation, vitamin K is immediately implicated. Vitamin K has long been recognized as an essential factor in the synthesis of prothrombin, a protein necessary for the normal coagulation of blood. latj res; In ( redt act: Sume for anin beg stud Prac The fact that the sulfa drugs exert many of their proper- ties by virtue of their action on microflora and thus in- directly on the synthesis of several vitamins, lends sup- port to the association of penicillin with vitamin K. In recent years several workers have been con- cerned with functions of vitamin K other than prothrombin formation. It has been suggested that the vitamin may play a role in cell metabolism, and that this role may be equally as important as that of catalyzing the synthesis of prothrombin. Recent findings indicate that vitamin K3 may function as a hydrogen carrier in the respiratory chain. This experiment was undertaken to study the re- lationship, if any, between penicillin and vitamin K with respect to both blood coagulation and cellular metabolism. In order to measure the latter, the enzyme DPN cytochrome-c reductase was chosen for two reasons: (1) it is an enzyme active in electron transport where vitamin K is also pre- sumed to function; and (2) a relatively simple assay method for this system is known. The albino rat was chosen as the experimental animal for obvious reasons. It was hOped that data would be collected which would provide suggestions for further study in human subjects with ultimate implications for practical clinical application. REVIEW OF LITERATURE REVIEW OF LITERATURE Effect of Vitamin K on Blood Coagulation Prothrombin is a protein which functions as a precursor for thrombin -- a substance necessary for the formation of a clot. The liver is intimately concerned with the production of prothrombin. Two main lines of experimentation have been the basis of this evidence: (1) the effect of liver poisons and liver injuries on the prothrombin level in the plasma; and (2) the influence exerted by vitamin K on the formation of prothrombin (Char- gaff 1945). It is probable that prothrombin is present in most of the body fluids, but satisfactory evidence as regards its distribution in tissues is lacking since the material is easily contaminated with blood. From calcu- lations based on the volume of blood, it was shown that vitamin K was not part of the prothrombin molecule but was concerned with the mechanism that produced prothrom- bin. It was suggested (Kemmerer 1952) that vitamin K serves as the prosthetic group which unites with the apoenzyme to form the active synthesizing enzyme. Bernheim and Bern- heim (1940) have shown that naphthoquinone derivatives are able to catalyze the formation of the -S—S- groups in prothrombin from -SH groups of cysteine in vitro. Hypoprothrombinemia is produced by a number of compounds which act as metabolic antagonists to vitamin K; the best known of these is dicoumarol. The administra- tion of vitamin K counteracts the action of dicoumarol. Hypoprothrombinemia has been observed clinically, particularly in cases of jaundice and sprue where there is interference with the absorption of fat, and thus of the fat soluble vitamin K. According to Reich gt a; (1947) the newborn are particularly susceptible to vitamin K de- ficiency, and hence to hypoprothrombinemia, due to a re- latively inactive microflora. In an effort to isolate the specific role played by vitamin K in the synthesis of prothrombin Dam (1942) observed that when vitamin K is given intravenously it is possible to study the effect at different time inter- vals from the moment of its introduction into the blood stream. Using this procedure, it was shown that the ac- tion of the vitamin was not immediate. Approximately 5 hours were required to increase the prothrombin content of the blood of a K-avitaminous chick following injection of vitamin K1.1 The prothrombin level reached a normal value in most cases 24 hours after the injection. Thereafter IT__S§V€EEITf6fims of vitamin K are known. In this paper reference will be made to 3 forms of the vitamin: (l) vitamin K which is found in green plants; (2) vitamin K which is s nthesized by bacteria; and (3) vitamin K (menadione which is a synthetic form of the vitamIn. it decreased unless large quantities of vitamin K1 were given, in which case the prothrombin level remained normal for a few days before a marked decrease was observed. If smaller quantities were injected than were necessary to bring the prothrombin up to the normal value, then the decrease occurred more rapidly. Similar results have been obtained in experiments with oral administration of the vitamin. Dam (1942) noted further that the prothrombin content of the blood from a K-avitaminous animal does not increase when vitamin K is added in zitgg, even if the vitamin remains in contact with the blood for 5 or 6 hours at body temperature. This observation suggests that the action of the vitamin takes place in tissues other than the blood. SITE OF ACTION OF VITAMIN K Several workers have studied the various organs in an attempt to define the site of action of vitamin K in the synthesis of prothrombin. Dam (1942) proposed, as a method of examining the importance of a given organ to the action of vitamin K1, the removal of the organ from a K—avitaminous animal, introduce vitamin K intravenously, and follow the rate at which prothrombin is synthesized. With this technique it has been shown that the spleen is not essential to the action of vitamin K. The work of Andrus gt a; (1941) showed that the removal of a portion of the liver of normal dogs resulted in a decrease in the level of prothrombin in the blood. This observation cor— roborated that made by Warner 23 al (1938) who reported a decrease in prothrombin after removal of two-thirds of the liver in rats. More recent studies with labeled vitamin K have supported these observations. Taylor 23 a; (1956) admin- istered vitamin Kl-Cl4 by various routes to rats. The liver showed the greatest amount of radioactivity. In 14 wa further work by Taylor 23 a; (1957) vitamin Kl-C s found concentrated in the liver of the rat. EFFECT OF PENICILLIN ON BLOOD COAGULATION Reports in the literature of the effect of peni- cillin on blood coagulation have been contradictory. Mol- davsky, Hesselbrobk, and Cateno (1945) found that the co- agulation time of the blood was decreased following the administration of penicillin. Further work from this same laboratory (Moldavsky gt a; 1953) showed that the coagu- lation time in human subjects of apparent good health was consistently decreased after administration of penicillin. There was a direct relationship between the clotting time and the concentration of penicillin in the blood. Analy- sis of blood samples obtained at fifteen minute intervals after intramuscular injection of penicillin revealed no change in the hemoglobin, red blood cell count, the sedi- mentation rate and hematocrit. Those elements associated with coagulation, i.e., plasma fibrinogen and serum cal— cium and prothrombin also remained unaffected by the pre- sence of penicillin in the blood. However, the number and size of platelets in the circulating blood during this period increased. There was greater platelet fragility, and a more pronounced tendency to aggregate and agglutinate. Since it is known that the spleen is associated with the maintenance of normal platelets (Olef 1936, 1937) it was concluded that this organ was implicated in the effect of penicillin on blood coagulation. Moldavsky 2£.§l (1945) proposed that the penicillin must mediate its effect by stimulation of the spleen since this antibiotic had no effect on blood coagulation when the spleen was removed from human subjects. A similarity was noted between the effects of penicillin on the blood platelets as described by Moldavsky gt a; (1945, 1953) and the effects of adrena- line as described by Olef (1935, I936, I937). Adrenaline empties the blood reservoirs in the spleen and produces an increase in the number of platelets. The fact that penicillin increased blood coagulation was also noted by Frada (1946). He observed an increased incidence of em- bolism of the large blood vessels occurring in the course of penicillin therapy in acute and subacute endocarditis. The opposite effect of penicillin on blood co- agulation in the human was observed by Lewitus (1948). He observed a prothrombinopenic effect of penicillin. Lewitus has suggested that in the combined dicoumarol-peni- cillin therapy for infective thrombosis, less dicoumarol would be needed than would be necessary in the absence of penicillin. In contrast to these reports, Lewis (1946) was unable to demonstrate any effect of penicillin on blood coagulation in normal or hemophiliac subjects. Likewise, Weiner 23 g; (1948) found that when therapeutic quantities of penicillin were administered to humans the coagulability of the blood did not change to a significant degree. Tri— antaphyIIOpoulos and Waisbren (1952) carried out experi- ments lg 1133 and in‘zitgg on the effect of varying con- centrations of crystalline penicillin on the prothrombin time, coagulation time, and clot retraction of normal hu- man blood. Penicillin had no effect on these processes. These data were in confirmation of previous studies which had failed to show an effect of penicillin on these fac- tors, and in conflict with those which indicated a rela- tionship between penicillin and blood coagulation. may be observ meat 1 fectiw Gilli] F was warke fluen panic line 0f b] the a ing] A possible explanation of these conflicting data may be found in the work of Macht and Ostro (1947) who observed that the type of penicillin used in the experi- ment was of marked importance. Penicillin X was most ef— fective for increasing the coagulability of blood. Peni- cillins K and G ranked second and third, while penicillin F was least effective in increasing coagulability. These workers also found that the purity of the penicillin in- fluenced the results. They observed that the amorphous penicillin increased coagulability, but that the crystal- line penicillin caused no increase in the coagulation time of blood. They suggested that the impurities present in the amorphous product were responsible for the conflict- ing reports in the literature. Another suggestion has been advanced to explain the contradictory results on coagulation time obtained when penicillin is administered. Lewis (1946) stated that an increase in prothrombin time occurred in the first few hours following the administration of penicillin and there- after decreased below normal. According to this thesis, if an insufficient period of time is allowed following -penicillin treatment, erroneous data regarding blood co- agulation time may be obtained. 10 ROLE OF VITAMIN K IN ELECTRON TRANSPORT Several workers have noted that vitamin K1 and vitamin K3 apparently do not follow the same metabolic pathway. Vitamin K1 has been found to be more effective than vitamin K3 in the prevention or treatment of the ef- fects of anticoagulants in humans, rats, and chicks (Boyer 1955). Weber 2£.§l (1958) found that Mycobaoterium phlei cells exhibited specific dependence on vitamin K1 for coup- led oxidative phosphorylation. This is in agreement with the work of Martius and Nitz-Litzow (1955) who found that vitamin K1 derivatives, but not menadione, increased the oxidative phosphorylation of mitochondrial preparations for vitamin K deficient chicks. Thus vitamin K1 appears to be intimately involved with oxidative phosphorylation. Vitamin K3 (menadione) has also been shown to be involved in electron transport, though in a different capacity than is vitamin K1. Schulz and Goss (1956) found that menadione inhibited phosphorylation by promoting elec- tron transport via a pathway which by-passed the phospho- rylative mechanism. Martins and Nitz-Litzow (I954) carried on exten- sive investigation of the uncoupling effect of oxidative phosphorylation by dicoumarol. According to these work- ers, this uncoupling action is directly related to its ll antagonism to vitamin K. If vitamin K3 (menadione) is Inot involved in oxidative phosphorylation, presumably the uncoupling action of dicoumarol must be a function of its antagonism to vitamin K1. In this study we have been concerned primarily with the role of vitamin K3 (menadione). The specific role of this form of vitamin K in the respiratory chain has not been fully elucidated. Two hypotheses have been presented. Uehara £3 El (1956) showed that the enzymatic oxidation of ethyl alcohol as well as lactate, requiring DPN as cofactor, was promoted by vitamin K3. According to this theory, vitamin K3 functions as a hydrogen acceptor from diphosphOpyridine nucleotide or triphosphopyridine nucleotide. It was suggested that a yellow enzyme requir— ing FMN as a coenzyme might participate in the reduction of vitamin K3 by DPN. Substrate DPN (or TPN) Methylnaphtho- hydroquinone XX: . PNH (or TPNH) Oxidized Substrate Dehydrogenase K3 reductase 12 However, further purification of the yellow enzyme is ne— cessary in order to determine whether K3 reductase is iden— tical with the flarburg yellow enzyme. Colpa—Boonstra and Slater (1958) have suggested a scheme which fits the data in this report better than that proposed by Uehara 33 a1. According to their scheme, vitamin K is an alternate pathway for the transport of 3 hydrogen in the respiratory chain. KBHZ J) (l) (2) (3) (4) (5) DPNH-——a pr ———9faotor-——a>cyt.cl————>cyt.c-———9cyt.a is) (11) (lo) (7) O2 +—————cyt.a3 succinate;::: prI;::::cyt.b (pr and prI represent the flavoproteins, diaphorase and succinate dehydrogenase, respectively) The oxidase systems are comprised of the following reac- tions: (A) DPNd oxidase ... (l) to (7); (B) succinate oxidase ... (8), (9), (10) followed by (3) to (7); and (c) KBH oxidase ... (12) followed by (2) to (7). 2 This investigation showed that K3H2 does not 13 act between DPNH and cytochrome b in the electron chain as Uehara §j_gl (1956) had proposed. However, Colpa-Boon- stra and Slater do suggest that K3H2 enters the respiratory chain in the vicinity of the flavoproteins or cytochrome b. Ernster gt_gl (1955) suggest that K332 enters the res- piratory chain by reaction with succinate dehydrogenase rather than with diaphorase. Thus it is seen that the flavoproteins can be reduced by a) DPNH, b) succinate, or c) K3H2. Vitamin K then, can act as an electron shunt around DPNH. 5! EXPERIMENTAL PROCEDURE EXPERIMENTAL PROCEDURE Forty weanling, male, albino rats of the Sprague- Dawley strain were divided into eight groups of five ani- mals each. The average weight per group was 52 grams. The animals were housed in wire mesh, raised bottom cages and allowed food and water fig libitum for a two week ex- perimental period. Food intake and weight records were kept. The basal diet consisted of sucrose 725 gm., vita- min free casein 180 gm., salts W 40 gm., vitamin mix2 2.5 gm., choline 1.5 gm., and corn oil3 50 gm. per kilogram of diet. The basal diet was supplemented with penicillin G, dicoumarol, and vitamin K3 singly or in combination (Table I). All supplements were added at the expense of sucrose. TABLE I SUPPLEMENTS TO BASAL RATION GROUP I II III IV V VI VII VIII LPENICILLIN G gm/kg 0.5 0.5 - - 0.5 0.5 - - LDICOUMAROL mg/kg 3.0 - 3.0 - 3.0 - 3.0 - ‘VITAMIN K3 mg/kg 3.8 3.8 5.8 3.8 - — - — 2. Vitamin.mix provided thiamine 5 mg, riboflavin 5 mg, niacin 10 mg, pyridoxine HCl 2.5 mg, Ca pantothenate 20 mg, inositol 100 mg, folic acid 0.2 mg, B1 0.02 mg, biotin 0.1 mg, vitamin A 100 mg, vitamin B 1.8 mg, and sucrose 2.25 gm per 2.5 gm of mix. j}. Containing 75 mg.(—tocopherol acetate. 15 At the end of the two week experimental period the rats were killed according to the schedule in Table II. The animals were sacrificed by administering a sharp blow on the head followed by decapitation. The blood sam- ples were collected in screw cap bottles which had been oxalated with 0.1 ml of 0.1 M neutral sodium oxalate. The bottles were swirled to mix the blood with the oxalate. They were capped and stored in the refrigerator until the coagulation time of the blood could be determined. TABLE II SCHEDULE FOR SACRIFICING ANIMALS GROUP I II III IV V VI VII VIII On the 11th day - - 2 -. - _ - _ On the 14th day l 1 — 1 On the 15th day5 - - - - On the 16th day 2 2 - 2 On the 17th day l l l l +4 +4 l4 +1 +4 +4 +4 +4 +4 +4 +4 +4 l4 +I +4 +4 r4 +» +4 I4 On the 18th day l l l l 4. Two rats in Group III appeared moribund on the 11th day of the experiment. They were killed before the 14th day and enzyme activity was determined. 5. One rat in Group III was found dead. Autopsy revealed an enlarged cecum filled with gas, and multiple intes- tinal hemorrhages. 16 The liver was removed from the carcass as quick- ly as possible, rinsed with distilled water, chilled for a few seconds in chipped ice, blotted dry with filter pa- per and weighed. Total liver weight was recorded. One part of liver was homogenized in 19 parts of chilled so- dium—potassium phosphate buffer (0.093 M) in a Potter-E1- vehjem homogenizer which had been chilled in chipped ice. A measured volume of the chilled homogenate was added to the main compartment of chilled Warburg flasks which had been prepared previously by pipetting the required amounts of each solution into the flasks (Table III). DPN Cytochrome-c reductase was determined in duplicate for each rat. The flasks were seated on the manometers and immersed in a water bath maintained at 37° C. A ten minute period was allowed for temperature equilibra- tion. The manometers were removed from the water bath, the substrate (0.2 ml of 0.5% DPN) tipped into the main compartment from the side arm, swirled gently and returned to the water bath. The manometers were set at 15 and the stop cocks closed. Readings were taken every 5 minutes for one-half hour. Calculations were based on 20 minute readings; the activity of the enzyme decreased after this interval of time. The remaining portion of liver was weighed, fro— zen and stored. When all the enzyme determinations were 17 completed the frozen liver samples were thawed and homo- genized. An aliquot containing 1 gm of liver (wet weight) was taken for nitrogen analysis by the Macro-Kjeldhal method. TABLE III REACTION MIXTURE FOR DPN CYTOCHROME-c REDUCTASE Flasks 1 2 3 ml ml ml KOH 10% 0.w.6 0.2 0.2 0.2 H20 M.C.7 0.2 - .- Sodium—potassium M.C. 0.8 0.8 0.8 phosphate buffer 0.1 M pH 7.4 Nicotinamide 0.1 p M.C. 0.3 0.3_ 0.3 Na Glutamate 0.5 M M.C. 0.3 0.3 0.3 Cytochrome-c 4xlO-4 p M.C. 0.3 0.3 0.3 Crude Malic Dehydrogenase M.C. 0.6 0.6 0.6 Na Malate 0.5 g S.A.8 0.3 0.3 0.3 DPN 005% Ser - 002 0.2 Liver Homogenate 5% in sodium-potassium phosphate buffer M.C. 0.2 . 0.2 0.2 Crude malic dehydrogenase was isolated from sheep liver according to the method of Potter (1946). 6: C.W.= Center well of Warburg flask. 7. M.C.: Main compartment of Warburg flask. 8. S.A.= Side arm of Warburg flask. 18 The determination of coagulation time on the oxalated blood samples was accomplished by adding back an excess of Ca 012. The addition of 0.1 ml of 0.1 Q Ca Cl2 to 0.1 m1 of oxalated blood on a glass slide proved to be satisfactory. The slide containing the recalcified blood was kept in a moist atmosphere over a water bath in an effort to reduce the error caused by evaporation of moisture from the sample (Todd and Sanford 1943). The coagulation time was taken as the time, in seconds, required for the first appearance of fibrin threads. RESULTS RESULTS Results from this study are summarized in Tables IV and V; data from individual animals are compiled in the appendix. FOOD CONSUMPTION AND GROWTH RATE (TABLE IV) The animals receiving menadione + penicillin (Group II) consumed less food than those animals receiving penicillin (Group VI) and penicillin + dicoumarol (Group V). The difference was significant in both instances. Because of the non-specific infection in Group III which necessitated early sacrifice of two rats in this group and the death of a third, growth and food consumption are not considered reliable for this group and will be omitted. No significant difference in growth rates between groups was observed. This was not unexpected in view of the short feeding trial used in this experiment.) (Wost- man, Knight, and Reynier 1958). Liver size was not altered by the composition of the diet; no significant difference was noted among the ‘various groups. 20 COAGULATION TIME (TABLE V) Addition of penicillin to the basal ration (Group VI) did not alter coagulation time as compared with the control group (VIII). No significant difference in coagu- lation time was observed except when dicoumarol was added to the basal ration (Group VII). The addition of 0.3 mg % dicoumarol increased clotting time approximately 45% over the controls. The addition of penicillin (Group V) and/or menadione (Group III) to the ration containing dicoumarol reversed this effect and returned coagulation time to a normal value. LIVER NITROGEN (TABLE V) When the experimental groups are compared with respect to the presence or absence of menadione (Group I vs V; II vs VI; III vs VII; and IV vs VIII) it should be noted that in every-case the addition of menadione to the ration significantly lowered liver nitrogen. These results cannot be explained on the basis of available data. The addition of dicoumarol (Group VII) to the basal diet also reduced the nitrogen of the liver below that of the control. This decrease was less marked than that observed with menadione, but the difference was sig- nificant. 21 - ENZYME DATA (TABLE V) Enzyme data are reported as ul Oz/hr/IOO mg fresh weight liver tissue. Enzyme activity expressed as ul 02/hr/IO mg nitrogen may be found in the appendix. The former unit of measure is used in this report because the fluctuations in DPN cytochrome-c reductase activity are independent of the fluctuations in total liver nitrogen. Group V (penicillin + dicoumarol) and Group VI (penicillin) showed a significantly higher DPN cytochrome-c reductase activity than did any other group; no signifi- cant difference was observed between these two groups. The addition of menadione to the ration containing peni- cillin or penicillin + dicoumarol (Group V vs I, and Group VI vs II) significantly decreased the activity of the en- zyme. The addition of dicoumarol to any ration had no significant effect on the activity of DPN cytochrome-c reductase. DISCUSSION DISCUSSION It was noted that the food consumption among the groups varied only slightly. Animals receiving mena- dione + penicillin (Group II) consumed a significantly lower quantity of food than those animals in any other group. This difference in food intake was not reflected in the growth rate. A greater difference in growth might have been noted had the feeding trial been of longer dura- tion. The inclusion of penicillin.pgg‘§g in the ration had no measurable effect on food intake or growth. Sev- eral workers (Phillips and Constant 1954; and Guerrant and Steel 1958) have found that penicillin supplementation increased food intake and growth rate only when animals were fed diets sub-optimum in certain nutrients. In view of the fact that the animals in this experiment were re- ceiving an 18% casein diet in which only vitamin K was deficient, it was not expected that a difference would be observed in food intake or growth rate. The deficiency of vitamin K in the diet for such a short period of time did not impose a marked stress on the animal since the rat is able to synthesize this vitamin. The effect of penicillin on coagulation time 23 observed in this study is somewhat puzzling. The fact that penicillin alone was ineffective in altering blood coagulation time from the control group is in agreement with severalcxher workers (Lewis 1946; Weiner 33 a; 1948; Triantaphyllopoulos and Waisbren 1952). The penicillin used in this study was crystalline, thus lending support to the theory of Macht and Ostro (1947) that the impuri- ties present in amorphous preparations were responsible for the recorded effects on coagulation time. However, although penicillin alone did not af- fect coagulation time, the antibiotic reversed the anti- coagulant action of dicoumarol. In fact, the action of penicillin was identical with that of menadione with re- spect to counteracting the effect of dicoumarol. The mech- anism of this action is obscure. A possible explanation. is that penicillin increases the synthesis of vitamin K2, probably in the intestinal tract. These data suggest that the clinical use of crys— talline penicillin would not present a problem with respect to the coagulation of blood. On the contrary, if penicil- lin functions at all in the blood coagulation scheme, it functions to maintain a normal coagulation time, even in the presence of the anticoagulant dicoumarol. The use of penicillin may be contra-indicated in cases of embolism if this effect is observed in human subjects. 24 As suggested earlier, the enzyme data collected in this experiment demand the use of the "shunt hypothe- sis" for vitamin K3 (Colpa-Boonstra and Slater 1958). According to this theory, the stream of electrons may be transported gig DPN or TPN down the electron chain, or as an alternative to this pathway, electrons may be accepted by vitamin K3 and be introduced into the chain at a point below the pyridine nucleotides. One may assume that the enzyme DPN cytochrome-c reductase would be operative only for those electrons transported‘zig DPN, since DPN must serve as substrate for this enzyme. Under normal conditions, a balance of electrons streaming into the electron chain from these various routes must be attained. The control group (VIII) establishes the measure of this normal balance. As factors operate to shift this balance, such a shift must be reflected in the activity of DPN cytochrome-c reductase. If the supply of electrons normally coming into the chain gig vitamin K3 is markedly reduced, and if the total number of elec- trons streaming through the chain is maintained, then a greater number of electrons must pass through the pyridine nucleotides, with a concomitant increase in the activity of DPN cytochrome-c reductase. Conversely, if factors operate to diminish the number of electrons normally pass- ing through the pyridine nucleotide system, the activity 25 of DPN cytochrome-c reductase will be reduced. And, still a third possibility, if this electron balance is undisturbed by dietary factors, the DPN cytochrome-c reductase acti- vity will be unchanged as compared with the control. Since the addition of dicoumarol to any of the diets had no effect on the activity of DPN cytochrome-c reductase the action of this metabolite cannot be centered on the function of vitamin K in the electron transport 5 chain. These data are in agreement with those of Martius and Nitz—Litzow (1954) who postulated the action of dicou- marol to be more specifically mediated through vitamin K1 rather than vitamin K3. The blood coagulation data pre- sented in this paper also support this thesis. The addition of penicillin to the diet markedly increased the activity of DPN cytochrome-c reductase. Therefore, this antibiotic must exert a profound influence on the balance of electron transport along the electron chain. The net result of penicillin administration is an increased flow of electrons through DPN. Based on the assumptions made earlier, penicillin must function either as an inhibitor of the vitamin K3 shunt into the electron chain, or as a stimulator of the pathway gig DPN. Since the addition of menadione to this penicil- lin-containing ration reduced the activity of DPN cytochrome—c reductase to that of the control group, the former action 26 of penicillin is implicated. These data suggest that peni- cillin exerts a metabolic block in the oxidation-reduction of vitamin K3 and by including vitamin K in the diet, 5 this inhibition is overcome. Additional work is necessary to support this theory. The presence of dicoumarol in either of these rations did not alter the pattern. Dicoumarol, as already noted, is inactive in this system. SUMMARY SUMMARY Forty male, weanling, albino rats were fed an 18% casein diet containing penicillin, dicoumarol and mena- dione either singly or in combination. The control animals were fed the basal diet (18% casein) unsupplemented. Food and water were allowed ad libitum for a period of 2 weeks. Records of food intake and weight gain were kept. At the end of the experimental feeding period the rats were killed, and the livers were analyzed for nitrogen and DPN cytochrome-c reductase activity. Coagu- lation time was determined on blood samples taken immedi- ately after the animals were sacrificed. No significant difference in growth was observed. It was felt that this was due, in part, to the short feed- ing trial used in this experiment, and in part to the high nutritive quality of the ration. Dicoumarol was found to have significantly in- creased the coagulation time of blood. This effect was reversed by menadione. These data are in agreement with many published reports. Penicillin, when fed alone, had no effect on blood coagulation time. However, when penicillin was fed with dicoumarol, the antibiotic behaved in a manner simi- lar to menadione. Penicillin reversed the anti-coagulant 28 effect of dicoumarol. Possible clinical implications in penicillin therapy are discussed. Dicoumarol was found to be ineffective in alter- ing the DPN cytochrome-c reductase activity. Penicillin increased the activity of DPN cytochrome—c reductase 45% over that of the control. Menadione was able to counter- act this effect of penicillin. When menadione was added to a diet containing penicillin, the activity of DPN cy— tochrome-c reductase was identical with the control. These relationships are discussed. The sugges- tion was made that penicillin exerts a metabolic block in the oxidation-reduction of vitamin K3. Under the conditions of this experiment, dicou- marol appeared to be a more specific antimetabolite for 'vitamin K1 than for vitamin K3. This theory was supported by blood coagulation data and by enzyme data. 29 TABLE IV GROUP SUPPLEMENT FOOD WEIGHT LIVER INTAKE GAIN WEIGHT (em/week) (em/week) (em) I menadione 67:39 33129 5.919.?9 penicillin dicoumarol II menadione 56:4 30:2 5.5:O.l penicillin III menadione 36_+_l8lO 20:810 4. 710.8 dicoumarol IV menadione 67:2 32:6 5.9:O.4 V penicillin 7414 50:9 5.0iO.5 dicoumarol VI penicillin 74:4 29:2 5.5:O.5 VII dicoumarol 68:3 26:4 5.6:0.02 VIII - 68:3 28:1 5.6:0.0l 9. Standard error of mean. 10. Due to infection and death in this group, these data are not considered reliable. 30 TABLE V GROUP SUPPLEMENT COAGULATION N/lOO mg DPN Cyto- TIME LIVER chrome-c (sec) (mg) Reductase (ul 0 /hr/ 100 m LIVER) I menadione 28:31]” 2 . 84iO . 1111 144351611 penicillin dicoumarol II menadione 27:2 2.71:0.05 136:8 penicillin III menadione 2613 2.90:0.08 152:8 dicoumarol IV menadione 23:6 2.98:9.06 133:14 V penicillin 2213 3.34i0.0l 190:20 dicoumarol VI penicillin 23:2 5.55:0.03 165:15 VII dicoumarol 42:9 3.16:0.05 134113 VIII - 29:4 5.55:0.16 144314 11. Standard error of the mean. LITERATURE CITED LITERATURE CITED Andrus, W. D. 1941 The newer knowledge of vitamin K. Bulletin N. Y. Academy of Medicine 31: 116-134 Bernheim, F. and Bernheim, M. 1940 Action of 4-amino- 2-methy1naphthol on the oxidation of certain sulfhydryl groups. Journal of Biological Chem- istry 334: 457-458 Boyer, P. D. 1955 The Fat Soluble Vitamins. Annual Re- view of Biochemistry g1: 447—449 Chargaff, Erwin 1946 The coagulation of the blood. Ad- vances in Enzymologyl3: 31-65 Colpa-Boonstra, J. P. and Slater, E. C. 1958 The role of vitamin K in the respiratory chain. Biochi- mica et Biophysica Acta 21; 122-133 Dam, H. 1942 Vitamin K, its chemistry and physiology. Advances in Enzymology g: 285-324 Ernster, L., Jalling, 0., Low, H. and Lindberg, O. 1955 Alternate pathways of mitochondrial reduced di- phosphopyridine nucleotide (DPNH) on oxidation studied with amytal. Experimental Cell Research, Supplement 3: 124-135 Frada, G. 1946 Large embolism in bacterial endocarditis in course of penicillin therapy. Giornale di Medicina 3: 95-138 cited from Journal of Ameri- can Medical Association 333; 354 Guerrant, N. B. and Steel, J. M. 1958 Some effects of aureomycin and penicillin on thiamine and ribo- flavin metabolism in growing rats. Proceedings Society for EXperimental Biology and Medicine 93; 542-545 Kemmerer, A. R. 1952 Fat-soluble vitamins. Annual Re- view of Biochemistry g3: 333-354 Lewis, J. H. 1946 Effect of penicillin on blood coagu- lation. Proceedings Society for Experimental Biology and Medicine 33: 538-540 52 Lewitus, Z. A. 1948 Prothrombinopenic effect of penicil— lin. Archives of Internal Medicine 82: 625 Macht, D. I. and Ostro, M. 1947 Thromboplastic proper- ties of penicillin and streptomycin. Science 105: 313-314 Martius, C. and Nitz-Litzow, D. 1954 Oxydative phosphory- lierung und vitamin K mangel. Biochimica et Biophysica Acta 13: 152-153 Chemical Abstracts 18: 5315i Martius, C. and Nitz—Litzow, D. 1955 Mechanism of action of vitamin K. Biochemische Zeitschrift 327: 1—5 Chemical Abstracts 39; 51100 Moldavsky, L. F., Crowley, J. H. and Hesselbrock, Wm. B. 1953 Penicillin effects on blood coagulation. Ohio State Medical Journal 49; 111-112 Moldavsky, L. F., Hesselbrock, Wm. B. and Cateno, C. 1945 Penicillin effects on blood coagulation. Sci- ence 102: 38-40 Olef, L. 1955 The enumeration of blood platelets. Jour- nal of Laboratory and Clinical Medicine 29: 416 1936 The rate of disintegration of platelets. ibid 21: 128 1937 The determination of platelet volume. ibid g3: 166 Phillips, P. H. and Constant, M. A. 1954 Antibiotics in Nutrition. Annual Review of Biochemistry g3: 333—334 Potter, V. R. 1946 The assay of animal tissues for res— piratory enzymes. The Malic dehydrogenase sys- tem. Journal of Biological Chemistry 165: 311- 324 Reich, 0., McCready, R. L., Chaplin, H. and Lipkin, R. 1947 Parenteral vitamin K therapy during ante— partum period and its effects on the infants' prothrombin levels. American Journal of Obstet— rics and Gynecology 33: 300-302 33 Schulz, A. R. and Goss, H. 1956 Menadione as a link in non-phosphorylative respiration in mitochondria. Biochimica et Bi0physica Acta 21: 578-579 Taylor, J. D., Miller, G. J., Jaques, L. B., Spinks, J. W. T. 19564 The distribution of administered vitamin K C in rats. Canadian Journal of Biochemistry and Physiology‘ji: 1143-1152 Taylor, J. D., Miller, G. J. and Wood, R. J14 1957 A com- parison of the concentration of C in the tis— sues of pregnant and non-pregnant female rats following the intravenous administration of vi- tamin K -C and vitamin K —C . Canadian Jour- nal of Biochemistry and Physiology 22: 691—697 Todd, J. C. and Sanford, A. H. 1943 Clinical diagnosis by laboratory methods. 10th edition W. B. Saun- ders Co. Philadelphia. p. 198 Triantaphyllopoulos, D. C. and Waisbren, B. A. 1952 Lack of influence of penicillin on blood coagulation. Archives of Internal Medicine 99: 653-659 Uehara, K, Muramatsu, I. and Makita, M. 1956 Role of vitamin K in biological oxidations. Journal of Vitamigology g: 44-50 Warner, E. D., Brinkhous, K. M. and Smith, H. P. 1938 Bleeding tendency of obstructive jaundice: pro- thrombin deficiency and dietary factors. Pro- ceedings Society for Experimental Biology and Medicine 21: 628-630 Weber, M. M., Brodie, A. F., and Merselis, J. E. 1958 Possible role for vitamin K in electron trans- port. Science 128: 896-897 Weiner, M., Zeltmacher, K. and Shapiro, S. 1948 The ef- fect of penicillin upon the coagulation of the blood. Experimental Medicine and Surgery 6: 181-188 Wostman, B. S., Knight, L. and Reynier, J. A. 1958 The influence of orally administered penicillin upon the growth and liver thiamine of growing germ- free and normal stock rats fed a thiamine defi- cient diet. Journal of Nutrition 66: 577-586 APPEND IX TABLE I Data for Individual Rats in Group I Fed the Basal Diet (18% Casein) Supplemented with Menadione, Penicillin, and Dicoumarol. RAT # INITIAL FINAL WEIGHT FOOD LIVER WEIGHT WEIGHT GAIN INTAKE WEIGHT (gm) (gm) (gm/wk) (gm/wk) (gm) I1 52 136 33 61 7.5 12 49 106 24 67 4.9 I3 55 159 57 75 7.6 14 56 154 54 75 5.6 15 48 120 36 58 4.0 AVERAGE 52 127 3312 6713 5.910.? ii TABLE I (Continued) RAT # N/lOO mg COAGULATION DPN CYTOCHROME-c REDUCTASE LIVER TIME ul 02/hr/1OO mg ul 0 (m8) liver hr/la mg NITROGEN II 2.56 25 83 324 12 2.82 26 173 613 I3 2.71 29 152 561 I4 2.90 28 147 507 I5 3.22 33 164 509 AVERAGE 2.84:0.11 2813 144:16 503148 iii TABLE II Data for Individual Rats in Group II Fed the Basal Diet (18% Casein) Supplemented with Menadione and Penicillin. RAT # INITIAL FINAL WEIGHT FOOD LIVER WEIGHT WEIGHT GAIN INTAKE WEIGHT (8m) (gm) (gm/wk) (gm/wk) (gm) IIl 51 155 52 69 5.9 112 51 107 23 48 4.8 113 57 141 37 63 6.4 II4 50 112 27 49 5.4 II5 50 114 32 52 4.8 AVERAGE 52 121 30:2 56:4 5.5:0.1 RAT # II II II II II iv TABLE II (Continued) N/lOO mg COAGULATION DPN CYTOCHROME-c LIVER TIME ul 02/hr/100 mg (mg) (sec) LIVER 2.67 24 141 2.71 26 114 2.82 30 122 2.53 25 156 2.81 28 146 AVERAGE 2.71:0.05 2712 136:8 REDUCTASE ul 0 hr /10 fig NITROGEN 528 421 455 617 520 504136 TABLE III Data for Individual Rats in Group III Fed the Basal Diet (18% Casein) Supplemented with Menadione and Dicoumarol. RAT # INITIAL FINAL WEIGHT FOOD LIVER WEIGHT WEIGHT GAIN INTAKE WEIGHT (gm) (8m) (gm/wk) (gm/wk) (gm) IIIl 51 147 57 60 6.5 III2 51 122 29 75 5.5 III 5412 - - - - 3 1114 5413 64 6 5 3.5 III5 5113 63 8 4 3.3 AVERAGE 52 99 20:8 36:18 4.7:0.8 12. No further data could be collected because of the 13. death of this animal. Animals 4 and 5 of Group III were sacrificed on the 11th day. RAT # III III III III III AVERAGE 2.90:0.08 N/lOO mg LIVER (mg) 2.91 5.04 2.96 2.68 vi TABLE III (Continued) COAGULATION DPN CYTOCHROME-c TIME ul 02/hr/100 mg (sec) LIVER 25 141 25 152 28 141 29 174 26:3 152:8 REDUCTASE ul 0 hr /10 6g NITROGEN 485 500 476 649 528:41 vii TABLE IV Data for Individual Rats in Group IV Fed the Basal Diet (18% Casein) Supplemented with Menadione. RAT # INITIAL FINAL WEIGHT FOOD LIVER WEIGHT WEIGHT GAIN INTAKE WEIGHT (gm) (gm) (gm/wk) (gm/wk) (gm) IV1 51 144 56 76 6.8 IV2 50 130 55 67 6.9 IV3 54 128 32 63 5.9 IV4 54 112 25 64 5.5 IV5 55 117 32 66 4.5 AVERAGE 52 126 32:6 67:2 5.9:O.4 viii TABLE IV (Continued) RAT # N/1OO mg COAGULATION DPN CYTOCHROME—c REDUCTASE LIVER TIME ul 02/hr/100 mg ul 0 /hr/ (mg) (sec) LIVER 10 NITROGEN IVl 2.78 24 148 532 IV2 3.10 25 180 581 IV3 3.07 20 125 407 IV4 2.92 21 102 349 IV5 3.04 25 110 362 AVERAGE 2.98:9.06 23:6 133:14 446:13 ix TABLE V Data for Individual Rats in Group V Fed the Basal Diet (18% Casein) Supplemented with Penicillin and Dicoumarol. RAT # INITIAL FINAL WEIGHT FOOD LIVER WEIGHT WEIGHT GAIN INTAKE WEIGHT (am) (am) (gm/wk) (gm/wk) (gm) V1 50 107 22 60 4.2 V2 53 121 28 71 5.9 V3 55 158 57 80 4.8 V4 55 ' 150 56 85 6.2 V5 50 106 28 78 3.7 AVERAGE 52 120 30:9 74:4 5.0:0.5 TABLE V (Continued) RAT # N/lOO mg COAGULATION DPN CYTOCHROI‘IiE-c REDUCTASE LIVER TIME ul 02/hr/100 mg ul 0 /hr/10 mg (mg) (sec) LIVER NITROGEN Vl 3.60 20 189 525 V2 2.84 18 143 504 V3 3.31 25 189 571 V4 5.41 25 165 484 V5 3.56 24 264 714 AVERAGE 3.34:9.01 22:3 190:20 560:41 xi TABLE VI Data for Individual Rats in Group VI Fed the Basal Diet (18% Casein) Supplemented with Penicillin. RAT # INITIAL FINAL WEIGHT FOOD LIVER WEIGHT WEIGHT GAIN INTAKE WEIGHT (am) (am) (gm/wk) (em/wk) (gm) VIl 53 126 28 71 5.4 VI2 54 115 25 63 4.8 VI3 55 115 27 70 4.0 V14 51 120 32 82 6.9 V15 49 115 55 85 5.5 AVERAGE 52 118 29:2 74:4 5.3:O.5 xii TABLE VI (Continued) RAT # N/lOO mg COAGULATION DPN CYTOCHROME-c REDUCTASE LIVER TIME ul O2/hr/1OO mg ul 0 /hr/1O mg (mg) (sec) LIVER NIT OGEN VI1 3.47 22 191 346 VI2 5.56 21 180 556 VI3 5.57 25 150 417 VI4 3.10 20 115 565 VI5 3.23 25 189 585 AVERAGE 3.35:9.03 23:2 165:15 488:4? xiii TABLE VII Data for Individual Rats in Group VII Fed the Basal Diet (18% Casein) Supplemented with Dicoumarol. RAT # INITIAL FINAL WEIGHT FOOD LIVER WEIGHT WEIGHT GAIN INTAKE WEIGHT (am) (am) (gm/wk) (gm/wk) (gm) VIIl 52 121 27 74 5.2 VII2 54 95 16 64 4.6 VII3 50 140 40 76 6.6 VII4 54 94 19 60 6.0 VII5 50 111 31 66 5.5 AVERAGE 52 112 26:4 68:3 5.6:0.01 xiv TABLE VII(Continued) RAT # N/lOO mg COAGULATION DPN CYTOCHROHE—c REDUCTASE LIVER TIME ul 0 /hr/100 mg ul 0 /hr/10 mg (mg) (sec) 2LIVER NITROGEN Vii 5.15 55 125 590 VII2 3.13 34 165 527 VII3 3.12 57 131 420 VII4 3.07 45 92 300 V115 5.55 40 158 472 AVERAGE 3.16:9.05 42:9 134:13 422:38 XV TABLE VIII Data for Individual Rats in Group VIII Fed the Unsupple- mented Basal Diet (18% Casein). RAT # INITIAL FINAL WEIGHT FOOD LIVER WEIGHT WEIGHT GAIN INTAKE WEIGHT (am) (am) (gm/wk) (gm/wk) (gm) VIIIl 52 124 28 67 5.0 VIII2 53 117 26 57 5.7 VIII3 53 120 29 76 5.4 VIII4 55 113 27 68 6.0 VIII5 49 106 29 73 5.3 AVERAGE 52 116 28:1 68:3 5.5:0.01 RAT # N/lOO mg COAGULATION DPN CYTOCHROME—c /hr/100 mg ul 0 VIII VIII VIII VIII VIII AVERAGE 3.35:9.16 LIVER (mg) 5.81 5.06 5.11 5.67 3.12 xvi TABLE VIII (Continued) TIME (sec) 25 50 52 55 25 29:4 ul 0 2 LIVER 192 155 147 111 119 144:14 REDUCTASE NIT /hr/10 mg ROGEN 504 500 475 502 581 4323540 THE EFFECT OF PENICILLIN AND DICOUMAROL ON BLOOD COAGULATION AND DPN CYTOCHROME-c REDUCTASE ACTIVITY IN THE RAT by Sister Mary Romana McDermott, S.N.J.M. AN ABSTRACT Submitted to the School of Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Foods and Nutrition College of Home Economics 1959 Approved _Jfigzr «p, r? 7. ABSTRACT Forty weanling, male, albino rats were divided into eight groups and fed an 18% casein diet containing penicillin, dicoumarol and menadione either singly or in combination. The control group received no supplement. Food and water were allowed gd libitum throughout the 2 week experimental period. The animals were sacrificed by decapitation. Livers were analyzed for nitrogen and DPN cytochrome-c reductase activity. Blood samples were collected when the animals were killed and coagulation time was deter- mined. Dicoumarol increased to a significant degree the coagulation time of the blood. Menadione was found to counteract this effect of dicoumarol. Penicillin alone had no effect on blood coagulation; however, when penicil- lin was added to rations supplemented with dicoumarol, an effect similar to that of menadione was Observed. It was suggested that if penicillin does function in the blood coagulation scheme it Operates to maintain a normal coagu- lation time. The activity of DPN cytochrome-c reductase was unaffected by dicoumarol. Penicillin was found to increase significantly the activity of this enzyme. The addition of menadione to the rations containing penicillin returned DPN cytochrome-c reductase activity to normal. From these data it was suggested that penicillin may function as an inhibitor of the vitamin K3 shunt around DPN in electron transport. cs": ewr r243“, 14" v- -. "1111411MAN