8009281
GERKEN, DIANE KAY FUHRHOP
AN IN VITRO AND IN VIVO STUDY OF THE EFFECT OF ANTICONVULSANTS ON THE VITAMIN K DEPENDENT CLOTTING FACTOR, PROTHROMBIN, IN RATS AND CATS
The Ohio State University PH.D. 1979
University Microfilms International300 N. Zeeb Road, Ann Arbor, MI 48106 18 Bedford Row, London WC1R 4EJ, England AN IN VITRO AND IN VIVO STUDY OP THE EFFECT OF
ANTICONVULSANTS ON THE VITAMIN K DEPENDENT
CLOTTING FACTOR, PROTHROMBIN, IN RATS AND CATS
DISSERTATION
Presented in Partial Fulfillment of the Requirements for
the Degree Doctor of Philosophy in the Graduate
School of The Ohio State University
By
Diane K. Gerken, B.S., D.V.M. *****
The Ohio State University
1979
Reading Committee: Approved by
Dr. Roger A. Yeary
Dr. Gary Kociba
Dr. Syed Saiduddin .SO] Depafrttfent tof Veterinary Physiology and Pharmacology To Mark ACKNOWLEDGMENTS
Words cannot express the feelings of love and appre ciation I have for my husband, Jon. His patience, his philosophy of life and his love were supportive and essen tial during the long nights of studying, weekends of research, and the joys and disappointments in these five years.
To my advisor, Dr. Roger Yeary, I express my gratitude and my admiration. Without his guidance, advice, and end less patience, this degree could not have been completed.
Most of all, I appreciate his friendship all these years and sincerely hope that in the years to come, this friend ship will remain as it is today.
My coworkers in the toxicology laboratory have been very special to me. Recognition and personal thanks are expressed to Dr. David Davis, who, as a personal friend, helped me with the research and helped me to keep the proper perspective;
Mrs. Kay Lee, whose technical help was appreciated; and
Miss Karen Yeary whose animal restraint expertise was essen tial to this project.
Many thanks are expressed to other members of my family and members of the Department of Veterinary Physiology and
Pharmacology for their support.
iii VITA
October 26, 19^7 ...... Born - Napoleon, Ohio
1969 ...... Bachelor of Science, Capital University, Columbus, Ohio
197^ ...... Doctor of Veterinary Medicine, The Ohio State University, Columbus, Ohio
197^-1977 ...... Graduate Teaching Associate, Department of Veterinary Physiology and Pharmacology, The Ohio State University, Columbus, Ohio
1977-1979 ...... Instructor, Department of Veter inary Physiology and Pharmacology, The Ohio State University, Columbus, Ohio
PUBLICATIONS
Yeary, R.A. and Gerken, D.P.: "Hepatic Drug Metabolism In Vitro in the Horse", Biochemical Pharmacology, Vol. 20, 3219-3221, 1971.
Yeary, R.A., Gerken, D.F. and Davis, D.R.: "Postnatal Development of Hepatic Drug Metabolizing Enzymes in the Gunn Rat", Biology of the Neonate, Vol. 23, 371-380, 1973.
Kociba, G.J., Mansmann, R.A. and Gerken, D.F.: "Acquired Hemostatic Defects in Horses", First International Symposium on Equine Hematology, 55^-559, May 1975.
FIELDS OF STUDY
Major Field: Pharmacology and Toxicology TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS...... iii
VITA ...... iv
LIST OF T A B L E S ...... vi
LIST OF FIGURES...... vii
INTRODUCTION...... 1
Chapter
I. LITERATURE REVIEW ...... k
II. MATERIALS AND METH O D S ...... 26
III. RESULTS...... kZ
IV. DISCUSSION ...... 69
V. SUMMARY AND CONCLUSIONS...... 75
APPENDIX ...... 78
BIBLIOGRAPHY ...... 83
v LIST OF TABLES
Page
1. Effect of phenobarbital, phenytoin, primidone on in vitro prothrombin production in vitamin K deficient rat liver homogenate ...... 44
2. In vitro prothrombin production in rat liver slices with phenobarbital or phenytoin added to the incubation ...... 48
3. Prothrombin synthesis in vitro in rats pre treated for two days before sacrifice with phenobarbital, primidone or phenytoin ...... 50
4. Percent prothrombin produced jLn vitro by fetal rat livers from dams treated with phenobarbital (80 mg/kg body weight) or phenytoin (100 mg/kg body weight) during days 8-20 of g e s t a t i o n ...... 52
5 . Fetal rat liver prothrombin production (percentage) in vitro from dams treated with 100 mg phenytoin/kg body weight from day 8-20 of gestation...... 54
6. Weekly prothrombin values in normal adult cats over a seven week period ...... 55
7 . Plasma prothrombin values in adult cats dosed orally with 10 mg phenytoin/kg body weight or 10 mg phenobarbital/kg bodywe i g h t ...... 57
8. Weekly plasma concentrations over a three week phenytoin treatment and a four week phenobarbi tal treatment in cats dosed at 10 mg phenytoin or phenobarbital/kg body weight ...... 59
9- Plasma prothrombin in control cats and cats treated for three days with 20 mg phenytoin/ kg body weight and four days with 10 mg phenytoin/kg body weight ...... 64
10. Plasma prothrombin values in cats given pheno barbital (10 mg/kg body weight) for one week followed by phenytoin (20 mg/kg body weight) for one w e e k ...... 66 vi LIST OF FIGURES
Page
1. Modified clotting scheme ...... 6
2. Prothrombin synthesis and degradation ...... 18
3. Thrombin standard c u r v e ...... *4-3
Prothrombin production in vitro in rat liver homogenate at varying vitamin K concentrations . . A-6
5* Body weights of cats given oral phenytoin for three weeks or phenobarbital for four weeks . . . 58
6. Mean body weights and standard error of the mean for five cats given oral phenobarbital for four weeks and phenytoin...... 63
7« Mean weights and standard error of weights for five cats given phenobarbital and phenytoin . 67 INTRODUCTION
Hemorrhage during the neonatal period is often a life threatening event. Chadd"*" stated that about 40-50$ of human perinatal mortalities were caused by hemorrhage and thrombosis. This high mortality rate may be the result of inappropriate therapy due to poor understanding of hemosta sis in the newborn infant.
One of the most documented causes of newborn bleeding is vitamin K deficiency. Generally, hemorrhage due to this deficiency occurs during the second or third day after birth and occurs mainly in the gastrointestinal tract. Low plasma concentrations of vitamin K-dependent clotting factors
(Factors II, VII, IX, and X) are characteristic and usually return to normal after treating the neonate with vitamin K.
This syndrome is commonly referred to as classical hemorr hagic disease of the newborn.
A similar vitamin K deficient-like state has been des cribed in infants whose mothers were treated with certain anti convulsant drugs during pregnancy. The newborn whose hemorrhagic condition was anticonvulsant-induced has charac teristic low plasma concentrations of Factors II, VII, IX, and X, and usually responds to vitamin K treatment. Time
1 2
of onset of hemorrhage and site of hemorrhage with the anti
epileptic-induced syndrome differs from the classical
hemorrhagic disease of the newborn. Usually, hemorrhage is
noticed within the first 24 hours after birth and is seen
in the organs of the abdomen, thorax, and head. Therefore, 2 Bleyer and Skinner recommended special precautions and
additional treatments with vitamin K before and after
delivery.
Very little is known about the anticonvulsant-induced
hemorrhage in the newborn other than it can be treated
successfully with vitamin K. All of the most common anti
convulsants (phenytoin, primidone, or any barbiturate,
singularly or in combination) have been reported to cause
neonatal hemorrhage. This syndrome has been reported by 4 5 Solomon et al. ’ to have been produced in cats using
either phenytoin, phenobarbital, or primidone. Solomon and workers also demonstrated using an in vitro rat liver slice
system that liver production of Factor VII was inhibited by
phenobarbital and phenytoin. From these reports, they con
cluded that an inverse dose-response relationship exists between anticonvulsant serum concentrations and the vitamin
K-dependent clotting factor depression in the experimental
animals.
The objective of this research was to investigate in
further detail the site and mechanism of the drug induced 3 coagulopathy. The three anticonvulsants chosen as possible vitamin K antagonists were phenobarbital, phenytoin, and primidone. One of the aims of the research was to develop an in vitro test to aid in the detection of other structurally similar vitamin K antagonistic drugs. CHAPTER I
LITERATURE REVIEW
The literature review will include clinical, biochemical, and pharmacological aspects of clotting to provide the back ground to understand the rationale for research, on the problem of anticonvulsant-induced neonatal hemorrhage. A summary of recent vitamin K research will be presented as it pertains to this research, but no attempt will be made to historically review the literature concerning vitamin K.
References to pharmacological antagonists such as the coumarin derivatives and vitamin K analogs will be made to aid in the understanding of the action of vitamin K and the possible mechanisms of antagonism of vitamin K by anticonvulsants.
Physiology of Clotting with Emphasis on the Vitamin K-Dependent Factors
The knowledge of the blood clotting systems in the mam malian species has grown and changed considerably in the last
20 years. Of course, hemostasis is not just limited to the clotting system, but also requires responses from blood ves sels and platelets.
A universally acceptable scheme for the exact interaction of the blood coagulation factors has not been established.
k 5
There are many theories, and the one presented here is an outline of a "cascade" hypothesis.
Traditionally, blood clotting has been divided into an intrinsic and extrinsic system. Activators of these two systems can be physiological (intrinsic) or pathological
(extrinsic). Once the clotting process is activated, a series of enzymatic reactions occur that result in a stable fibrin clot. Figure 1 represents an abbreviated version of the clotting cascade. Terms used in Figure 1 are those recom mended by the International Committee on Thrombosis and
Hemostasis in 1 9 7 7 For a glossary of definitions and synonyms, the reader is referred to page 2*K
Those factors requiring vitamin K for synthesis by the liver are prothrombin, Factor VII, Factor IX, and Factor X.
Vitamin K deficiency is rarely seen in the adult as the result of decreased vitamin availability. Not only is the vitamin found in many foods such as green leafy plants, but vitamin K is synthesized in large quantities by intestinal micro-organisms. Since vitamin K is a fat soluble vitamin, bile salts are required for its absorption. Therefore, protracted antibiotic therapy and numerous malabsorptive disorders could result in absorption of insufficient quan tities of vitamin K. Either hepatocellular disease or pharma cological antagonism such as seen with coumarin or indanedione derivatives may result in decreased utilization of vitamin K.® Intrinsic Pathway Extrinsic Pathway
activating enzyme Factor XII------> Xlla Tissue Factor
Factor XI- ■♦XI a
Factor IX' ■*IXa Factor VIII Ca+2 Phospholipids
Factor X Xa
Factor V Phospholipids + 2 Ca
Prothromhin -> Thrombin
+2 Ca
Fibrinogen ♦Fibrin
Factor XIII
Insoluble Fibrin
Figure 1. Modified clotting scheme from Esnouf (1977).^ In utero vitamin K is supplied to the fetus through the
maternal circulation. Since human fetal blood has the o ability to clot by 10-12 weeks of gestation, the liver must,
by this time, be capable of synthesizing clotting factors
including the vitamin K-dependent factors. Holmberg et al.10
reported that in human fetuses 12-24 weeks gestational age,
the vitamin K-dependent clotting factor concentrations were
between 12$ and 35f° of adult values. It was thought that
this difference was due to liver immaturity. Infants that
are premature reportedly have vitamin K-dependent clotting
factor concentrations between 28$ and 30$ of adult concentra
tions, and term infants have 30$ to 60$ of adult values.^
Generally, neonatal vitamin K deficiency appears to be one
of the main causes of the low vitamin K-dependent clotting
factors seen at birth. Administration of vitamin K to the
newborn or to the mother during labor usually results in an
increase in concentration of low clotting factors. The pre mature infant may not respond as dramatically as the term
infant to the supplemental vitamin K because of hepatic 11 immaturity. Vitamin K therapy is recommended routinely for
this physiological vitamin K deficiency at birth, but it is
also recommended for the prevention of the normal decline
in vitamin K-dependent clotting factors seen two to four days after birth. Prothrombin may decrease to as low as 5%
of normal adult values two to four days after birth. Since 8
clinical evidence of hemorrhage is seen when clotting factor
concentrations decrease to 10fo of normal adult concentrations,
it is not surprising that these newborns have a great risk of
coagulopathy.
The syndrome commonly referred to as "hemorrhagic disease of the newborn" has been reported to occur in less 12 than 1 7 of all newborn infants. The usual sites of hemorrhage are the skin and intestinal tract. The causes of hemorrhagic disease of the newborn include: (l) low fetal
stores of vitamin K; (2) sterile intestine at birth; and
(3) breast feeding (human milk is very low in vitamin K O content). Not all workers agree that a lack of intestinal
organisms is a cause for vitamin K deficiency at birth.
Since most of the vitamin K producing organisms are located in the large intestine and not higher in the gastrointestinal tract, some researchers question the ability of the human 13 large intestine to absorb vitamin K. J
It has been reported that healthy term babies may not have lower than normal adult quantities of vitamin K-dependent Ik it u clotting factors. ’ Gobel J reported that clotting factor content in babies fed within 2k hours' of birth did not differ from those babies given vitamin K prior to birth. Most authors agree that the post parturient deficiency of vitamin
K-dependent clotting factors decreases further two to four 9 days after birth and then increases slowly until adult con- 9 l6 centrations are reached at one year of age. The temporal pattern of vitamin K-dependent coagulation factors in the human newborn differs from that of newborn laboratory animals. Vitamin K-dependent clotting factors were low at birth in laboratory animals, but progressively increased after birth until adult quantities were reached. In experi- 17 ments on newborn animals, Hathaway et al. included the following species: cow, cat, dog, guinea pig, rabbit, and
Pig- Pharmacological Antagonists of Vitamin K and Anticonvulsants
In addition to the "physiologically" low levels of vita min K-dependent clotting factors at birth and two to four days after birth, pharmacologic agents may affect the utili zation of vitamin K in the newborn infant and further depress synthesis of clotting factors. Exposure to pharmacologic agents may occur in utero as the result of therapy for maternal disorders.
Thromboembolic disease is a very common and serious com plication of pregnancy. Treatment in nonobstetric patients usually involves the use of the coumarin or indanedione anti coagulants. Unfortunately, these anticoagulants cross the placenta and are a serious threat to the fetus. It has been estimated that there is about 15% fetal mortality in pregnant o women taking the coumarin-like anticoagulants. 10
Fetal vitamin K-dependent clotting factors appear also
to be affected by anticonvulsants used to treat various
seizure disorders. As the most widely used anticonvulsants,
the barbiturates and phenytoin have been incriminated as
depressants of the fetal vitamin K-dependent clotting factors. 1 fi In 1970, Mountain et al. published a prospective study on
neonates from normal women and neonates from epileptic women
treated with various anticonvulsant combinations. Coagulation
tests showed that half of the babies from treated epileptic
mothers had abnormally low quantities of Factor II, VII, IX,
and X but only two of these babies showed clinical hemorrhage.
None of the babies from unmedicated mothers had a similar
clotting defect. All epileptic mothers used in this study
had been treated with phenytoin and some were treated with
phenytoin plus a barbiturate or primidone. Bleyer and
Skinner in 1976, published a summary of reported cases of
neonatal hemorrhage related to exposure to anticonvulsants
in utero. There were eight fatalities in the 22 cases
reported. Eleven of the 22 babies began hemorrhaging within
2k hours after birth. The most common sites of hemorrhage were skin, intracranial, intrathoracic, and intra-abdominal.
Sites of hemorrhage and onset of bleeding differentiated
this syndrome from classical hemorrhagic disease of the
newborn. No anticonvulsant or combination of anticonvulsants 11 could be singled out as being the cause since hydantoins, barbiturates, and primidone were used individually and in combinations. Bleyer and Skinner recommended prophylactic vitamin K therapy to the epileptic mothers prior to parturi tion and to the infants immediately after birth. Clotting
factor concentrations increased in most infants treated by this therapeutic regime and clinical hemorrhage was avoided. O I O 1 Q Barbiturates have been reported-^ ’ ' y to prolong pro thrombin times in the neonate when used exclusively during labor and delivery. The hydantoins and the barbiturates are known to cross the placenta and high concentrations of these drugs have been found in the fetal liver. Excretion of these drugs occurs very slowly in the newborn. The mechanism of the antagonistic action of these drugs on the formation of vitamin K-dependent clotting factors in the fetal liver is Ll unknown. Solomon et al. were able to decrease vitamin K- dependent clotting factors in adult cats by giving phenytoin intraperitoneally daily for three to four weeks. The degree of depression of clotting factors appeared to be dose-related.
Adult cats, receiving 5 nig phenytoin/kg body weight, showed an approximate 50fo decrease of these clotting factors after nine days. Clinical signs such as bleeding with upper respir atory infections and gastroenteritis were not seen in adult cats until the highest dosage, 10 mg phenytoin/kg body weight, 12 was given. The liver function tests on these cats were nor mal. When the adult cats were given vitamin K and phenytoin
daily, the depressed vitamin K-dependent clotting factors
returned to normal. Pregnant queens were given 5 mg pheny
toin/kg body weight daily for the last two weeks of gestation.
Newborn kittens from the treated queens showed depression of the vitamin K-dependent clotting factors. Solomon and Zj, workers reported an _in vitro study using rat liver slices that showed that phenytoin, like warfarin, inhibited Factor
VII production. In a subsequent paper by Solomon et al.-^, phenobarbital was used instead of phenytoin in in vitro experiments. The results showed that phenobarbital also depressed vitamin K-dependent clotting factor production in cats _in vivo and in rat liver in vitro. Depression of clot- 20 ting factors appeared to be dose related. Fahim et al. reported that progeny of female rats given daily doses of phenobarbital exhibited subcutaneous hemorrhages in which the incidence appeared to be dose related. k Solomon et al. '^ theorized that the anticonvulsants, phenobarbital and phenytoin, directly interferred with the synthesis of the vitamin K-dependent clotting factors similar to the coumarin anticoagulants. Their work suffered from absence of statistical analysis and has not been confirmed. 2i Contradictory results were obtained by Sadowski et al. who found an increase in prothrombin synthesis in vitamin K- deficient rat livers when the rats were treated with 1 mg 13 phenobarbital/ml of drinking water for 10 days. Phenobarbi
tal pretreatment increased not only liver microsomal protein and cytochrome P ^5° content, but also vitamin K epoxidase ik activity and ( C) protein carboxylation. Induction of microsomal enzymes by phenobarbital has been recognized for many years so the increase in cytochrome P ^50 content -was not surprising. The increase in the carboxylation and epoxidation activities was significant in that the epoxida- tion of vitamin K is not mediated through cytochrome P .
When cytochrome P *4-5° inhibitors were added to the in vitro system, no effect was seen on vitamin K epoxidation or pro thrombin synthesis. The enzymes responsible for epoxidation and carboxylation are probably among those microsomal enzymes that are increased due to proliferation of the endoplasmic reticulum.
In addition to a possible effect on vitamin K, phenytoin, phenobarbital, and primidone have been reported to have effects on other vitamins such as folic acid and vitamin D.
Even in adult humans, those anticonvulsants may have caused 22 a folic acid deficiency which led to megaloblastic anemia.
In the pregnant epileptic, phenytoin therapy has lowered folic acid concentrations to the point of overt megaloblastic anemia. There are two possible hypotheses for this effects phenytoin may (l) block intestinal absorption, and (2 ) increase folic acid metabolism. Paradoxically, folic acid supplemen tation, which is often prescribed during pregnancy, lowers serum phenytoin concentrations. Possible breakthrough seizures may become a hazard in treating the pregnant epi leptic. The exact mechanism of how folic acid affects phenytoin serum concentrations is unknown. Several recent 23 2k 25 papers by Blake et al. and Collins et al. J have found that although liver weight increased, the ability to metabo lize phenytoin was reduced by 50% in the term pregnant rat.
They concluded that folic acid inhibited the normal decrease
(turnover) of phenytoin hydroxylase. This hepatic enzyme metabolizes phenytoin to 5-(p-hydroxyphenyl)-5-phenyl hydan- 2k toin. According to this hypothesis, folic acid supplemen tation would result in lower serum phenytoin levels during pregnancy due to increased enzymatic activity of the stabilized enzyme system. This effect of folic acid on serum phenytoin 26 concentrations appeared to be dose related.
Biochemistry of Vitamin K
Since vitamin K was discovered by Henrik Dam in 1929, workers have tried to solve the mystery of exactly how vitamin K participates in clotting factor synthesis. To this day, the precise mechanism of action is unknown. However, during the last 50 years, an abundant literature about vitamin K has contributed greatly to the knowledge of clot ting factor synthesis. 15
The existence of the vitamin was discovered by accident when chicks were fed a fat-free diet and subcutaneous hemorr- 27 hages developed. For many years, vitamin K was thought to be required only for prothrombin synthesis. As more infor mation became available about physiological processes of
clotting, it become apparent that this vitamin was required for the production of Factors VII, IX, and X.
A number of other vitamin K-dependent proteins have been discovered in the last 10 years. These other proteins
P fi have been found in bone, kidney, and plasma. The exact purpose of the proteins is unknown, but it is thought that 29 they may be involved in calcium metabolism. Osteocalcin, the vitamin K-dependent bone protein,- has been found in the fetus. The amount of osteocalcin present appears to corre late with the degree of mineralization found in the bone.
Fetuses exposed by maternal ingestion of warfarin have been reported to have skeletal malformations. The present theory is that extrahepatic vitamin K-dependent proteins may be the regulators of calcium ion concentration in bone, kidney, and plasma. There may be many other tissues that are affected by vitamin K since metabolism of vitamin K has also been SO found in the spleen, lung, and placenta.
Most of the in-depth research on the function of vitamin
K has been concerned with vitamin K-dependent clotting factor 16
production in the liver. One of the first theories was that
since vitamin K was a naphthoquinone, it might be involved
in electron transport in mitochondrial respiration. No link
to oxidative phosphorylation was found. Then in the 1950's
and 1960's, vitamin K was hypothesized as a regulator of
transcription due to gene repression. After much work to prove his gene repressor theory, Olson withdrew his original
hypothesis and concluded from his experiments that vitamin
K probably functioned as a regulatory protein at the ribo-
somal level.
Many investigators in the mid-1960's reported evidence that vitamin K may be involved in conversion of a protein precursor. Suttie,-^2 in 1970, published that prothrombin appeared too quickly in blood (within 60 minutes) after vitamin K administration to vitamin K deficient rats to attribute regulation of protein synthesis as the function of vitamin K. Shah and Suttie-^ subsequently reported that when vitamin K deficient rats were given cycloheximide, vitamin K, and radiolabelled amino acids, no radioactivity was found in the resulting prothrombin produced in the liver.
From that report and others, the concept that vitamin K was instrumental in post-translational conversion of prothrombin precursor to prothrombin, gained acceptance.
The precursor theory was strengthened with the discovery of a plasma protein similar to prothrombin in the blood of 17 of coumarin anticoagulated patients. The protein was anti- genetically similar to prothrombin but displayed no biologi cal activity. The abnormal prothrombin or prothrombin precursor was also found to exist in large quantities in the plasma of the coumarin-treated bovine, but not in the coumarin-treated rat. Several laboratories extensively studied the characteristics of the abnormal prothrombin.
Suttie3^ reviewed the properties of abnormal bovine prothrom bin as compared to normal bovine prothrombin. The two pro teins had almost identical properties except for biological activity, ability to bind calcium, and absorption to barium salts. The abnormal prothrombin was found to have very low or no biological activity, very low calcium binding activity, and very low ability to absorb to barium salts.
The relationship between these three properties in the abnormal prothrombin molecule became apparent when Stenflo et al.35 discovered that the actual chemical difference between abnormal prothrombin and prothrombin was a newly discovered amino acid, tf-carboxyglutamic acid (GLA). It is hypothesized that the abnormal prothrombin molecule or pre cursor molecule contains glutamic acid (Glu) residues that are carboxylated in the presence of vitamin K to make the biologically active prothrombin molecule as illustrated in
Figure 2. The carboxylation of glutamic acid (Glu) residues occurs on the amino terminal end of the prothrombin molecule?^ AMINO ACIDS 18
protein synthesis in ribosomes
33jU Glu Glu Glu Glu Prothrombin JL JL precursor nh2 C COOH Glu Gl*u Giu Giu Glu Jp
carboxylation requires active form of vitamin K (occurs in rough microsomal fraction)
GLA GLA GLA GLA GLA
Prothrombin COOH GLA G U GLA GLA GLA
Hepatocyte
Blood t Activation of Clotting System
GLA GLA GLA GLA G U — I I I 1-- 1_ n Phospholipid + Ca++ + Factor X + NH [ COOH G U G U G U G U G U
I enzymatic attack of Xfi G U G U G U G U G U __l___ I____ » l I ‘1_____ nh2 C COOH —I--- 1---- 1-- 1--- r - G U G U G U G U G U I I I I I I I I I I Ca++Ca++Ca'v+Ca++Ca++
Factor V to accelerate reaction GU GU GU GU GU — 1--- »___ I___ I___ L_ NH, ~ i — i— i— i— r d T COOK G U G U G U G U G U t T Fragment I Fragment II Two-chained thrombin
Figure 2. Prothrombin synthesis and degradation. 19
This is supported by the fact that the amino terminal end
will bind calcium in the biologically active prothrombin,
whereas the abnormal prothrombin will not bind calcium.37
Calcium binding is required for phospholipid-prothrombin
interaction. This interaction permits the correct spatial
configuration of prothrombin for cleavage to thrombin by
activated Factor X .
Shah and Suttie^® in 197^. developed an in vitro
method using rat liver in which the response to added vita
min K was the production of prothrombin from its precursor
protein. This helped to further research on the biochemical
function of the vitamin. Rats are the best species to inves
tigate the carboxylation reaction in vitro. Rats and per
haps the rabbit are the only two species that significantly
store the prothrombin precursor in the liver when made 19 vitamin K deficient or treated with dicumarol anticoagulants.
The mouse, guinea pig, hamster, and dog store some prothrombin precursor, but significant quantities of the abnormal pro thrombin are released into the plasma. In the cow and human, almost all of the abnormal prothrombin is found circulating in plasma. 39
In Shah and Suttie's system, 38 a postmitochondrial
supernatant, an ATP generating system, and vitamin K were 20
incubated in an atmosphere of 95% and 5$ COg. They were
able to show inhibition with the vitamin K analog, Chloro K
(2-chloro-3-phytyl-l, 4-naphthoquinone), but not with war
farin. Modifications of the method by Houser et al.^0
showed that warfarin was an inhibitor when much lower con
centrations of vitamin K were added to the system. In
Houser's experiments, concentrations of vitamin K over the
range of 2 ng/ml to 2 pg/ml without warfarin added displayed
a semilogarithmic dose-response.
Subsequently, many in vitro systems have been developed
in an attempt to understand the mechanism of vitamin K. The
actual peptide sequence required by the vitamin K-dependent
carboxylase was the short peptide sequences adjacent to the
glutamic acid residues.^ This was discovered by Suttie
et al.^1 using a Triton X-100 solubilizedsystem that required vitamin K + NADH or vitamin KHg. Dithiothreitol used as a
reducing agent for vitamin K was more effective in stimulat ing carboxylation when NADH and vitamin K were used than when vitamin KHg was used. Sodium bicarbonate was required by the system, but not oxygen.
Humans treated with the coumarin drugs have large con- 42 centrations of vitamin K-2, 3-epoxide in their plasma.
This was found to affect the urinary metabolites of vitamin
K found in man and is probably due to a decrease in vitamin
K glucuronides and an increase in vitamin K-2, 3-epoxide 21
glucuronides.^ Matschiner et al.^ found that vitamin K accumulated in rat liver as a new metabolite, phylloquinone-
2,3-epoxide in the presence of warfarin. During normal physiological processes, vitamin K is oxidized to the epoxide, and subsequently reduced in the liver. It is excreted as a glucuronide conjugate (Figure 3)* prothrombin precursor----- cark°xylation » prothrombin
|epoxidase J vitamin K ^ vitamin K epoxide reductase i t 1 , excretion vitamin K-2,3- epoxide glucuronide vitamin K glucuronide Figure 3* Fate of vitamin K in the liver as modified from Willingham and Matschiner. ->
Since warfarin causes prothrombin precursor and vitamin
K epoxide accumulation in rat liver, vitamin K epoxide was thought to be a competitive inhibitor of vitamin K . ^ in contrast to this, Bell et al.^? published that warfarin inhibition caused a change in the vitamin K-2,3-epoxide to vitamin K ratio in the liver. When the ratio of oxidesK became large enough, inhibition of prothrombin synthesis occurred. This was refuted by Goodman et al.^® who found no change in prothrombin synthesis in normal rats at very high vitamin K oxide:vitamin K ratios. 22
Direct inhibition of vitamin K epoxidase and inhibition of prothrombin synthesis by tetrachloro-4-pyridinol (^--TCP) and 2-chloro-3-phytyl-l,4-naphthoquinone (Chloro-K), two noncoumarin anticoagulants, was reversed by large concentra tions of vitamin K . ^ From this, there appeared to be an association between epoxidation of vitamin K and carboxyla- 21 tion of prothrombin precursor. Sadowski et al. hypothe sized that there may be an obligatory coupling of the epoxi dation process and the carboxylation process since one appeared simultaneously with the other, and both processes depended on the same cellular requirements.
The separation of the two processes was postulated by
Willingham and Matschiner when they were able to carboxy- late prothrombin precursor without the simultaneous epoxida tion of vitamin K. The uncoupling of the two processes led to the proposal that possibly carboxylation required an intermediate form of vitamin K and that the vitamin K-2,
3-epoxide resulted from that intermediate form. ^ Esnouf et al. suggested that perhaps the superoxide form of vitamin K was the active form since the enzyme, superoxide dismutase, inhibited both the carboxylation and the epoxide formation in their in vitro liver system. Their work is in agreement with Willingham and Matschiner since vitamin K-2,
3-epoxide formation was inhibited when catalase was added in vitro. 23
It was not until W a l l e n 5 2 separated the carboxylation enzyme system and the epoxidation enzyme system by affinity chromatography that two separate enzymatic systems were des cribed. Wallen isolated and purified NAD(P)H dehydrogenase
(DT-diaphorase). With this enzyme, carboxylation in the presence of vitamin K + NADH occurred without vitamin K epoxidation. Wallen's theory is that in the presence of
NAD(P)H dehydrogenase, vitamin K;j_ is converted to vitamin semiquinone. The semiquinone is then the active form of the vitamin and promotes carboxylation of prothrombin precursor.
The semiquinone is then acted on by vitamin K epoxidase resulting in vitamin K epoxide. There appears to be some association between carboxylation and epoxidation since the presence of the epoxidase increases the incorporation of 1 h H CO^ in prothrombin. More research is needed to identify the exact part the active form of vitamin K plays in the carboxylation process. GLOSSARY
The following nomenclature has been recommended by the
International Committee on Thrombosis and Hemostasis in
1977*^ The recommended name appears first followed by
either a definition or previously used synonym. The
recommended nomenclature will be used throughout the remain
der of the text.
Prothrombin Factor II
Factor III Tissue factor
Ca++ Factor IV
Factor V Proaccelerin
Factor VII Proconvertin
Factor VIII Antihemophilic factor (AHF); antihemo philic globulin (AHG)
Factor IX Plasma thromboplastin component (PTC); Christmas factor
Factor X Stuart-Prower factor
Factor XI Plasma thromboplastin antecedent (PTA)
Factor XII Hageman factor
Factor XIII Fibrin-stabilizing factor Fletcher factor (prekallikrein)
2k 25
Acarboxyprothrombin "dicoumarol induced PIVKA (Protein Induced by Vitamin K Absence), abnormal prothrombin (in some instances, prothrombin precursor is synonymous)
Factor (*)a Activation of blood clotting factor so that it then becomes an enzyme Example: Factor Xa
* Any Roman numeral denoting a blood clotting zymogen
Thrombin Factor IIa CHAPTER II
MATERIALS AND METHODS
To determine the effect of the anticonvulsants on vita min K-dependent clotting factors, the initial approach was to investigate the production of prothrombin in an in vitro system.
I. The Effect of Phenobarbital, Phenytoin, and Primidone on In Vitro Production of Prothrombin in Liver Homogenate
Animals. Male Sprague Dawley rats (125-150 gm) were housed in suspended wire cages to reduce coprophagy. Rats were fed a vitamin K-deficient diet,a and were given 0.5$ •j_ neomycin sulfate in their drinking water for 9-18 days to produce vitamin K deficiency.
Drugs. Phenobarbital, phenytoin, and primidone were added to the reaction mixture to achieve a concentration of 10 M. Stock solutions were made daily and kept at o c 37 C until used. The stock solution of phenobarbital was
Vitamin K deficient diet (Grimenger et al., J. Nutr. 70» 361, i960) ICN Pharmaceuticals, Inc. Life Sciences Group, Cleveland OH. b Neomycin sulfate, USP (6^9 mg neomycin/gm neomycin sulfate) Upjohn Co., Kalamazoo MI.
C Mallinkrodt Chemical Works, St. Louis MO.
26 27
made by dissolving phenobarbital in 250 mM sucrose-25 mM
imidazole buffer (pH-7.2). A small quantity of sodium j hydroxide was used to dissolve the phenytoin and 250 mM
sucrose-25 mM imidazole buffer (pH-7-2) made up the remain
ing volume. Primidonee was dissolved in hot absolute
ethanol.
Prothrombin Synthesis. Rats were killed by decapitation
and a postmitochondrial supernatant made according to the
method of Shah and Suttie.^® The following was added to
10 ml Erlenmeyer flasks: 2.0 ml postmitochondrial super
natant, 1 mM ATP, 10 mM phosphocreatine, 25 pg creatine
phosphokinase in 0.1% albumin, 50 mM KC1, and 2.5 mM Mg
(Acjg. The remainder of the 2.5 ml volume was made up by
the 250 mM sucrose-25 mM imidazole buffer (pH=7.2). Post mitochondrial supernatant from one rat was added to five f flasks (one without vitamin K added; one with vitamin K ,
50 ug in ethanol, added) and three with vitamin K plus one
of each of the anticonvulsants (10-^ M) added. The flasks were placed under an atmosphere of 95$ ^2^ ^ ° ^ 2 ^ ^ ^ e r / min and incubated for 10 minutes at 37°C in a Dubnoff meta bolic shaking incubator. The incubated mixtures were chilled
Aldrich Chemical Co., Milwaukee W I .
9 Gift from Ayerst Laboratories Inc., New York NY.
^ Aqua MEPHYT0N, Merck, Sharp and Dohme, West Point PA. 28 on ice to terminate the reaction and then centrifuged at
96,592 x g for 60 minutes. The surface of the microsomal pellet was washed with 1 ml of 25 mM imidazole buffer, pH=7-2. The microsomes were resuspended in 1.0 ml of imidazole buffer by homogenization at slow speed in a
Potter-Elvehjem tissue homogenizer with a motor driven teflon pestle. To the microsomal suspension, 0.35 nil of
1$ Triton X-100 in 25 mM imidazole buffer (pH-7.2) was added, and the suspension was solubilized after being shaken for 10 minutes at ^°C with a wrist-action shaker.
The resulting suspension was frozen and capped in poly ethylene tubes. These microsomal suspensions were thawed at a later date, and two-stage prothrombin assays were performed.
Prothrombin Assay. The modification by Shapiro and
W a u g h ^ 3 of the two-method of Ware and Seegers^ was the basic method used to analyze prothrombin.
Reagents:
a. Acacia solution: 15$ in saline
b. Imidazole buffer: 1.72 gm imidazole dissolved in 90 ml of 0.1N HGl and made to final volume of 100 ml with water (pH=7.2-7.^).
c. Thromboplastin reagent: Dade Activated Throm boplastin of Rabbit Brain Origin®
® Dade Diagnostics, Inc., Miami FL. 29
■y. d. Fibrinogen: 1$ solution of bovine fibrinogen in saline was made daily. The solution was hydrated for two hours at room temperature and the nondissolved material was removed before use.
e. Ac-globulin: bovine blood was collected and allowed to clot at room temperature. If complete clotting did not occur within three hours, blood was warmed at 35°C in a water bath for two hours and then refrigerated. Serum was decanted off and centrifuged. Approximately 200 mg of barium carbonate was mixed with 5 ml of serum to make a paste, then 35 ml of serum was added to the paste and agitated for 10 minutes at room temperature. After centrifugation, the clear straw-colored serum was divided into aliquots and frozen in glass vials. Ac-globulin was kept frozen until the day of usage, then thawed and kept cold during use.
Procedure. The following were mixed in a glass tube:
1.1*4- ml of saline, 0.68 ml of 15$ acacia, and 0.18 ml of
imidazole buffer, and then kept in a 30°C water bath until
used. In a separate tube, 0.3 ml of thawed, cold micro
somal preparation was placed in the 30°C water bath for
one minute. Due to the instability of the Ac-globulin after
dilution, 0.1 ml of cold bovine serum was diluted with 7.5 ml of saline just prior to use; 0.7 ml of this dilution was
added to the warmed microsomal preparation. One ml of throm boplastin was added to the saline, acacia, and imidazole buffer solution and mixed. This was the reaction mixture.
The 1 ml of diluted bovine serum-microsomal preparation was
Bovine Fibrinogen, 95$ clottable, Miles Research Products, Elkhart IN. 30 then added to the reaction mixture. After mixing, the solu tion was allowed to incubate for five minutes at 30°C . At the end of five minutes, a 0.24 ml aliquot was taken and placed in a coagulation cup.1 The timer of the fibrometer^ was started after 0.06 ml of a 1% fibrinogen solution was added to the coagulation cup. The fibrometer automatically timed the end point or fibrin strand formation. Duplicates were determined on each sample and the times for fibrin formation were averaged.
It A known concentration of human thrombin was obtained from the Bureau of Biologies, Food and Drug Administration,
Bethesda MD, and was used to develop a standard curve.
Since it is generally accepted that one prothrombin unit is equal to one thrombin unit, the prothrombin activity was calculated from the formula obtained from a least squares regression analysis of the curve.
To evaluate the experimental data, a one-way analysis of variance for repeated measures was done. The Newman-
Kuels test was used to evaluate the pattern of differences.
II. XU Vitro Prothrombin Response to Warfarin and Phenytoin at Various Concentrations of Vitamin K
Animals. A vitamin K deficiency was produced in male
Sprague Dawley rats (125-150 gm) by feeding a vitamin K
1 FibroTube Disposable Coagulation Cups, BBL, Div. Becton, Dickinson and Co., Cockeysville MD
J Fibrometer Precision Coagulation Timer, BBL, Cockeysville MD k Sigma Chemical Co., St. Louis MO. 31
deficient diet and adding 0 .5$ neomycin sulfate to their
drinking water for 13 days. These rats were housed in sus
pended wire mesh cages to reduce coprophagy.
Drugs. 3-(p^-acetonylbenzyD-^-hydroxycoumarin1 was
dissolved in ethanol such that the addition of 0.05 ml to
the _in vitro reaction resulted in a final concentration
of 10"-5 M. _ ri Phenytoin solution (for a final 10 J M concentration)
and three concentrations of vitamin K (Aqua MEPHYTON) were
also prepared in ethanol. The vitamin K solutions were made up so that the addition of 0.05 ml would result in concentrations of ^.44 x 10-^ M, 4.4^ x 10-^ M, and 2.22 x
10“^ M in the reaction mixture.
NADH was dissolved in 250 mM sucrose-25 mM imidazole buffer (pH-7 .2 ) and added to the reaction mixture to achieve a concentration of 1 mg NADH/ml.
Procedure. Rats were decapitated and their livers were chilled in 250 mM sucrose. A 33$ (w/v) homogenate was made as described in experiment I . The homogenate was centri fuged at 27,000 x g for 20 minutes. Two ml of this post mitochondrial supernatant was added to 10 ml Erlenmeyer flasks containing 1 mM ATP, 10 mM phosphocreatine, 25 jag creatine phosphokinase in 0.1$ albumin, 50 mM KCl, 2.5 mM
1 Courtesy of Dr. David Aronson, Bureau of Biologies, FDA, Bethesda MD. 32
Mg (Ac)^, and 2.5 mg NADH.The homogenate from one rat liver was distributed to each of the following 10 reaction flasks: a blank flask (without vitamin K ) ; three flasks containing one of each concentration of vitamin K (4.44 x
10"9 M, 4.44 x 10-7 and 2.22 x 10“^ M) ; three flasks containing 10 ^ M phenytoin plus one of each concentration of vitamin K; and three flasks containing 10~^ M warfarin plus one of each concentration of vitamin K. The rest of the procedure for synthesis and assay of prothrombin was the same as in experiment I . The data were analyzed by using a two-way analysis of variance for repeated measures for both factors followed by the Newman-Kuels test to deter mine the differences.
Ill. Effect of Phenytoin or Sodium Phenobarbital on Production of Prothrombin Using Rat Liver Slices
Animals and Drugs. Same as described in experiment I.
Procedure. Vitamin K deficient rats were killed by decapitation and the liver was exposed. A small cut was made in the portal vein about 5 mm from the liver. A 16 gauge Luer sub adapter™ on a 10 ml syringe was used to catheterize the portal vein and perfuse the liver with 10 ml of cold 250 mM sucrose. The posterior vena cava was com pletely severed, and 20 ml of cold 250 mM sucrose was m Intramedic Luer Stub Adapter, Clay-Adams, Inc., NY. 33 perfused through the liver via the catheterized portal vein.
The liver was removed and chilled in 250 mM sucrose. Liver slices (0.5 mm thick) were made with a Stadie-Riggs micro tome.11 Three to four liver slices were incubated in 50 ml beakers with the same incubation mixture used in experiment
I except primidone was not used. The beakers were incubated for 30 minutes in a Dubnoff metabolic shaking incubator under an atmosphere of 95% 0^/5% C02 (l liter/min). The reactions were terminated by placing the beakers on ice.
The incubation mixture and liver slices were then homogenized.
Microsomes were prepared and assayed for prothrombin as described previously. A portion of the microsomal prepara tion was used to determine protein content by the method of
Lowry et al.^5 a one-way analysis of variance for repeated measures was used to analyze the data and the differences were determined by using the Newman-Kuels test.
IV. The Effect of Phenobarbital, Phenytoin, and Primidone Pretreatment on Prothrombin Production in vitro in Vitamin K Deficient Rats
Animals. Male Sprague Dawley rats (125-15° gm) were fed a vitamin K deficient diet and given 0.5% neomycin sul fate drinking water for 7-15 days.
Procedure. Individual rats were treated daily for two days with either 200 mg primidone/kg body weight, 200 mg n Arthur H. Thomas Co., Philadelphia PA. sodium phenytoin°/kg body weight, 80 mg phenobarbital/kg
body weight, or 10$ acacia (which served as a control).
Primidone and phenytoin were suspended in 10$ acacia.
Individual primidone and sodium phenytoin suspensions were
made up for each rat so that a consistent 2 ml volume was
given to deliver the required dose. Primidone, phenytoin,
and acacia suspensions were given by stomach tube.^ Pheno
barbital was administered intraperitoneally in a volume of
1 ml. The rats were fasted 12 hours prior to treatment and
four hours after treatment. Twenty-four hours after the
final treatment, the rats were decapitated. The livers
were analyzed for prothrombin as described previously
(experiment I). Because the drugs were administered in vivo,
the postmitochondrial supernatant was incubated in two
flasks, one without vitamin K added and one with vitamin K
added. The Dunnett's t test was used following the one-way
analysis of variance for repeated measures in the analysis of
these data.
V. The Effect of Phenobarbital or Phenytoin in Fetuses from Pregnant Rats Treated from Day 8 Through Day 20 of Gestation
Animals. Female time-mated Sprague Dawley rats were
obtained at the fifth to seventh day of gestation. They
0 Sigma Chemical Co., St. Louis MO.
^ K31 Feeding Tube, Size 8 French, Pharmaseal, Inc., Toa Alta, Puerto Rico. 35
were housed in suspended wire cages and given 0 .5$ neomycin
sulfate drinking water. Until the fifteenth day of gesta
tion, the rats were fed Purina Rat Chow, and then, from
day 15 to day 21 of gestation, they were fed a vitamin K
deficient diet.
Drugs. Sodium phenytoin was suspended in 10$ acacia
and phenobarbital was dissolved in ethanol and 0.05 M KPO^
buffer (pH=7-^)- Each drug was weighed according to the
rat's weight so that each rat would either receive 2 ml of
sodium phenytoin (100 mg sodium phenytoin/kg body weight) via stomach tube or 1 ml of phenobarbital (80 mg phenobar- bital/kg body weight) subcutaneously. A group of control
rats for each treatment group were given 2 ml of 10$ acacia by stomach tube or 1 ml of 0 .0$ K KPO^ buffer subcutaneously.
The rats were weighed four times during the treatment period and the concentration adjusted to keep the volume constant.
Procedure. After being treated with either phenobarbi tal, phenytoin, acacia or KPO^ buffer from the eighth day through the twentieth day of gestation, the rats were anes thetized with ether on the twenty-first day of gestation.
Maternal blood samples were obtained by cardiac puncture and mixed with 3 -2$ sodium citrate solution in the ratio of one part anticoagulant to nine parts blood. The blood was centrifuged for 20 minutes, and the plasma was decanted off. 36
The plasma was frozen for determination of phenobarbital or
phenytoin content. Rat pups were delivered by Cesarean
section. Blood samples were obtained from the rats after
decapitation. The serum samples from each litter were pooled after centrifugation and frozen for drug analysis.
Liver specimens from each litter were pooled and treated as
one sample. Two prothrombin determinations were made on each liver sample, one without vitamin K added and one with vitamin K added to the incubation mixture. These data were analyzed using the student's t test.
Phenytoin and Phenobarbital Analyses. Concentrations of the anticonvulsants in maternal plasma samples or fetal serum samples were determined by gas chromatography using a Varian Model 2100 Gas Chromatograph.^- A modified procedure of the method published by Supelco in Bulletin 768^ Was used. After the plasma or serum sample was acidified to a pH of 1-2 with HC1, the anticonvulsants were extracted into redistilled spectrograde chloroform. The samples were then evaporated to dryness and reconstituted to 100 pi of chloro form for a 2.-3 pi injection. Standards for the calibration curve for phenobarbital or phenytoin were from EMIT AEDr or
Anticonvulsant Calibration Standards.3
^ Varian Instruments, Sunnyvale CA. r EMIT AED, Syva Corp., Palo Alto C A . c* Supelco, Inc., Beliefonte PA. 37
Separation of the anticonvulsants and the internal
standard, 5-p-(methylpheny])-5-phenylhydantoin, was accom
plished using GP 2% SP-2510 D.A. on 100/120 Supelcoport •jj column packing in a 6ft x 2 mm I.D. glass column. The
carrier gas was nitrogen at the rate of 30-^0 ml/min flow.
Column temperature was programmed to run from 200°C-260°C with 15°C/min increases. The flame ionization detector temperature was held at 280°C and the temperature at the injection port was 3°°°C.
VI. Hepatic Prothrombin Production in Fetuses from Pregnant Rats Fed a Vitamin K-Folic Acid Deficient Diet and Treated with Phenytoin
The identical procedure described in experiment V was followed except the pregnant rats were fed a vitamin K deficient diet which was specially prepared to also be folic acid deficient. The student's t test was used for the data analysis.
VII. Determination of Weekly Variation of Prothrombin Concentrations in Cats Using the Two-Stage Prothrombin Assay
Animals. Four adult cats (three males and one female) were housed in individual cages and given Purina Cat Chow and water free choice. The cats were vaccinated for feline panleukopenia and then allowed to stabilize for four weeks before blood samples were drawn.
^ Supelco, Inc., Bellefonte PA. 38
Procedure. Blood samples were drawn at weekly inter vals for five weeks. A 1 ml volume of placebo (lk%
ethanol) was given to each cat daily for the first two weeks of the experiment. Blood samples were drawn from the jugular vein and mixed with 3.2% sodium citrate solu tion in the ratio of one part anticoagulant to nine parts blood. Care was taken to perform venipunctures without trauma to avoid contamination of the blood sample with tissue thromboplastin. Blood was centrifuged for 20 minutes and the plasma was frozen in capped polyethylene tubes for the two-stage prothrombin determinations at a later time.
Prothrombin concentrations were determined as previously described (experiment I) except for sample dilutions and concentration of bovine Ac-globulin used. One-tenth ml of plasma was added to 0.1 ml of saline and warmed for one minute in a 30°C water bath. Then, 0.1 ml of the warmed solution was diluted with 0.1 ml of saline. From the twice diluted plasma, 0.1 ml aliquot was added to 0.9 ml of diluted Ac-globulin (0.1 ml Ac-globulin previously diluted in 7*5 ml of 0.9$ NaCl). The entire 1.0 ml of the diluted plasma-diluted Ac-globulin mixture was then mixed with the thromboplastin-saline-acacia and imidazole buffer solution.
Evaluation of the data was accomplished using the one-way analysis of variance for repeated measures. 39
VIII. Plasma Prothrombin Concentration in Cats Given Daily Oral Treatments With Phenobarbital and Phenytoin
Animals. Ten adult female cats were housed in separate
cages and were given Purina Cat Chow and water ad libitum.
The cats were stabilized for a minimum of four weeks prior
to the experiment and vaccinated for feline rhinotracheitis •
and feline calicivirus at the beginning of the experiment.
Drugs. Sodium phenobarbital was prepared in lk% ethanol
to make a concentration of 25 mg phenobarbital/ml. Sodium
phenytoin was suspended in ikfo ethanol in 5% acacia at a
concentration of 5° rag phenytoin/ml.
Procedure. Five cats were allocated to each of the two
treatment groups. They received either 10 mg phenobarbital/
kg body weight or 10 mg phenytoin/kg body weight. The cats
were weighed and treated with phenobarbital daily for four
weeks and treated with phenytoin daily for three weeks.
Adjustments in dose were made weekly. Blood samples were
drawn at weekly intervals and were obtained 2k hours after
the preceeding dose. The blood samples were processed and
analyzed for prothrombin as described in experiment VII.
An aliquot of the plasma sample was used for analytical
determination of the anticonvulsant. Both prothrombin data
and weight data were analyzed using the two-way analysis of
variance for repeated measures for two factors. The Newman-
Kuels test was used to evaluate the differences. *J-0
IX. Plasma Prothrombin Activity in Cats Given an Initial Phenytoin Dosage of 20 mg Phenytoin /kg Body Weight for Three Days Followed by 10 mg Phenytoin/kg Body Weight
The five cats that received phenobarbital were crossed
over to phenytoin one week after the last phenobarbital
treatment. Blood was drawn for prothrombin analysis as a pretreatment control. The cats were treated with 20 mg phenytoin/kg body weight daily for three days and then with
10 mg phenytoin/kg body weight daily for the following four days. Blood samples were drawn after one week of treatment and weekly for the subsequent two weeks and analyzed for prothrombin. Plasma phenytoin concentration was determined at the end of the phenytoin treatment period. The two-way analysis of variance for repeated measures for one factor was used to analyze the prothrombin data, and the two-way analysis of variance for repeated measures for two factors was used to analyze the weight data. The Newman-Kuels test was used to evaluate the differences.
X . Effect of Phenobarbital Pretreatment on Plasma Prothrombin in Cats Given Phenytoin
Four of the five cats used in experiment IX were used after a one month rest period. A fifth cat (male), pre viously used as a control, was added to this treatment group.
After a pretreatment blood sample was taken, these cats were treated orally with 10 mg phenobarbital/kg body weight daily 41 for one week. The blood samples drawn after the phenobar bital pretreatment were analyzed for prothrombin and pheno barbital concentrations. Sodium phenytoin was then given daily for one week at the dosage of 20 mg phenytoin/kg body weight. The cats were weighed and blood was drawn at three day intervals during the second treatment week. Plasma pro thrombin and anticonvulsant concentrations were determined.
The prothrombin data was analyzed using two-way analysis for repeated measures for one factor followed by the Newman-Kuels test. The weight data was analyzed by the one-way analysis of variance for repeated measures followed by the Dunnett’s t test.
XI. Plasma Prothrombin Activity of Cats Given Warfarin and Phenytoin Simultaneously
Animals. Four of the adult cats used in experiment X
(three females and one male) were used after a 23-day recovery period.
Drugs. 3-(t*-acetonylbenzyl)-4-hydroxycoumarin (war farin) was suspended in 14$ ethanol in % acacia with a final concentration of 3 mg warfarin/ml.
Procedure. A pretreatment blood sample was drawn for prothrombin determination. The four cats were dosed with
0.5 mg warfarin and 20 mg phenytoin/kg body weight. Blood samples were taken every three days and analyzed for pro thrombin content until prothrombin activity decreased to below 12 units/ml plasma. CHAPTER III
RESULTS
Prothrombin Standard Curve
Human thrombin standard was used to develop a standard curve using 1% fibrinogen. The results are shown in Figure
3. All subsequent prothrombin data were obtained from the regression line formula: ln(y)= -1.5644 ln(x)+2.65188 which described the best fit for this curve.
I. The Effect of Phenobarbital, Phenytoin, and Primidone on In Vitro Production of Prothrombin in Liver Homogenate
Phenobarbital, phenytoin, or primidone at the concen- tration of 10 M did not significantly affect prothrombin production when added in vitro to rat liver homogenate
(Table 1). To ensure vitamin K deficiency, only those samples were included in the data where the activity in the control incubation was less than 1.97 units of prothrombin/ gm of liver. The low concentration of prothrombin in the incubation where vitamin K was not added reflected depletion of vitamin K when rats were fed the vitamin K deficient diet and received 0 .5$ neomycin sulfate in their drinking water.
Feeding rats a vitamin K deficient diet alone did hot produce
42 Figure 3. Thrombin standard curve - dilutions of human of dilutions - curve standard Thrombin 3.Figure units of thrombin 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.10 thrombin standard and standard thrombin \ i o cj n o in c\j o in c\j o o ltigtm (sec) time clotting o l H rl 1% irngn ee used. were fibrinogen o o o TABLE 1
Effects of Phenobarbital, Phenytoin, Primidone on In Vitro Prothrombin Production in Vitamin K Deficient Rat Liver Homogenate (Experiment l)
Incubation conditions Units of prothrombin/ gm of liver______without vitamin K 1.97+ 0.00 with vitamin K 12.^7 + 1.59 vitamin K and 10 J M phenobarbital 12.^6 + rH i—1 vitamin K arrilO-^ M phenytoin 13-22 + 1.16 vitamin K and 10“^ M primidone 11.89+ 1.16
* Statistically different at P<0.05*
Each value represents the mean and standard error of the mean of seven animals. 45 the low prothrombin concentration in the liver reported here.
Only when 0.5$ neomycin sulfate was added to the drinking water in addition to the vitamin K deficient diet was the deficiency detected. From Table 1, it can be seen that when vitamin K was added to the incubation, a significant quantity of prothrombin was produced when compared to the incubation where no vitamin K was added. Experiments where NADH and dithiothreitol were added to the incubation did not result in any increase in prothrombin production. Greater concen trations of phenytoin, phenobarbital, or primidone were not added to the incubation because the water solubility of the anticonvulsants at pH =7.2 was the limiting factor.
II. XU Vitro Prothrombin Response to Warfarin and Phenytoin at Various Concentrations of Vitamin K
In this experiment, varying concentrations of vitamin K were added to the in vitro incubation of vitamin K deficient rat liver homogenate. Prothrombin production was directly related to the concentration of vitamin K added to the system
(Fig. 4). None of the prothrombin values for the incubation containing phenytoin and vitamin K differed from the vitamin
K controls (Appendix, Table 11). The prothrombin response was significantly different (P^O.05) from the controls and phenytoin plus vitamin K at the 200 ng/ml and 1000 ng/ml concentrations of vitamin K when warfarin was added to the incubation. Warfarin at the concentration of lO-^ m appeared to be an inhibitor at 200 ng/ml and 1000 ng/ml of vitamin
K. units of prothrombin (xl0-2 )/gm of liver 100C- 1100 Figure 4. Prothrombin production iri production Prothrombin liver ratin vitro 4.Figure 20C 30C- 70c- 90C- IOC 60C 80C- - 100
Each point represents the mean of fiveratsof the mean represents point Each homogenate at varying vitamin K concentrations K at vitamin varying homogenate and in the presence of phenytoin or warfarin. of phenytoin presencethein and n h etclbr ersn h standardthe represent barsthe vertical and error of the mean (ExperimentII). oftheerrormean 200
300 400 ng of ng K/mlvitamin 500
600
700
800
900
1000
46
III. Effect of Phenytoin or Sodium Phenobarbital on Production of Prothrombin Using Rat Liver Slices
There was a significant effect (P>0.05) of vitamin K
on prothrombin synthesis in liver slices (Table 2). When phenytoin plus vitamin K or phenobarbital plus vitamin K were added to the system, there was no difference (PC0.05). in the effect of vitamin K on prothrombin synthesis.
Because there was no observable effect on prothrombin pro duction when neither of the two anticonvulsants were added to the vitamin K deficient rat liver in vitro, the anticon vulsants were given in vivo in the remainder of the experi ments. IV. The Effect of Phenobarbital, Phenytoin, and Primidone Pretreatment on Prothrombin Production In Vitro in Vitamin K Deficient Rats
Rats that were vitamin K deficient did not tolerate
200 mg primidone or 200 mg phenytoin/kg body weight. Many rats either were not sufficiently vitamin K deficient (as determined by the prothrombin concentration in the in vitro system without vitamin K added) or the rats died within the two-day treatment period with nonspecific necropsy findings.
The dosage and method of administration of phenobarbital was changed because of the rat mortality rate. Originally, the rats were dosed at 200 mg phenobarbital/kg body weight orally as a suspension. Because of the mortality with this regime and the known enzyme inducibility at a lower dosage, TABLE 2
In Vitro Prothrombin Production in Rat Liver Slices with Phenobarbital or Phenytoin Added to the Incubation (Experiment III)
Incubation conditions Units of prothrombin (xl0-3)/mg protein without vitamin K added 13.07 + 1.58* with vitamin K added 23.81 + 3-21 vitamin K + 10-^ M phenytoin added 22.67 + 3.96 vitamin K + 10”^ M phenobarbital added 23.36 + 4.19
* Statistically different at P<0.05.
Each value is the mean and standard error of the mean of six animals. the dosage was lowered to 80 mg phenobarbital/kg body weight.
Phenobarbital was sufficiently soluble at this decreased dosage to administer intraperitoneally. The amount of prothrombin present without added vitamin K (in vivo pro thrombin) was subtracted from the total quantity of pro thrombin present in the vitamin K fortified sample to give the amount of prothrombin actually produced in vitro. The percentage of prothrombin produced was calculated as the prothrombin produced in vitro divided by the total pro thrombin present after the incubation (Table 3). There was no difference (P^O.05) in prothrombin production between the treated and the control rats.
The original coagulopathy was described as an effect on the neonates of anticonvulsant-treated mothers. Since vitamin K antagonism had not been produced in male rats either in vitro or in vivo, anticonvulsant treatment during pregnancy in rats was investigated.
V. The Effect of Treatment of Pregnant Rats with Phenobarbital or Phenytoin from Day 8 through Day 20 of Gestation on Fetal Prothrombin Production
When the rats were dosed at 200 mg phenytoin/kg body weight, many rats died. The rats appeared to die as a result of central nervous system depression, and the necropsy obser vations were nondescript. The dosage was then reduced to 50
TABLE 3
Prothrombin Synthesis In Vitro in Rats Pretreated for Two Days Before Sacrifice with Phenobarbital, Primidone or Phenytoin (Experiment IV) In Vitro Prothrombin Synthesis “T$ of Total Prothrombin)a
Acacia Primidone Phenytoin Phenobarbital
1^.6$ 68.0$ 76.4$ 62. 6$
69.1% 84-. 2$ 5^-7% 7^.9$*
77 .0$ 70.7$ 72.3$ 71.2$
73.6% 66. 6$ 70.6$ 72.9$
68.0$ 71.7$
6?. 1$ x=6l .57$ x=72.i|$ x= 68.5$ x=70.66$
Each value represents the results from one rat. a $ prothrombin produced in vitro =
Incubation with _ Incubation without vitamin K added “ vitamin K added Incubation with vitamin K added 51
100 mg phenytoin/kg body weight. There were no deaths in
the rats dosed at 100 mg phenytoin/kg body weight.
The means and standard error of the means for the blood phenytoin concentrations in the dams and the fetuses were
5.01 jug/ml plasma (+ 1.97) and 95.^2 pg/ml serum (± 37.^8), respectively. These values represent the blood concentra tions of phenytoin 2k hours after the last dose was given to the dam. Phenytoin appeared to accumulate in the rat fetus as indicated by the high fetal blood concentration as compared to the maternal blood concentration after two weeks of daily treatment. Even though the fetal blood concentra tion of phenytoin was high, there was no effect on percentage prothrombin production in the livers of fetuses from pheny- toin-treated dams (Table k).
In vitro prothrombin synthesis of fetuses from pheno- barbital-treated dams was not significantly different (P<0.05) from its control group (Table *0. The mean maternal pheno barbital blood concentrations 2k hours after the last dose was 22.85 pg/ml plasma (± 5.22 as the standard error of the mean). The mean fetal phenobarbital blood concentration 2k hours after the last dose was 32.25 jug/ml serum (+ M.51 as the standard error of the mean). 52
TABLE 4
Percent Prothrombin Produced In Vitro by Fetal Rat Livers from Dams Treated with Phenobarbital (80 mg/kg body weight) or Phenytoin (100 mg/kg body weight) During Days 8-20 of Gestation (Experiment V)
In Vitro Prothrombin Synthesis ~T# of Total Prothrombin)a
Control Phenytoin Control Phenobarbital
82 .4# 66. 3# 61.1# 58.1#
71.1# 65.9# 53-3# 63. 6fo
46.0# 68.9# 48.6# 59.5#
45. 8# 52.3# 60.8# 58.3#
60.4-# 64. 1# 45.1# 57.9#
70.0# 55.3# 44.0# 69.2#
65.0$ 82 .0# 54.9# 54.3#
67. 2# 43. 0# 62. 8# 59.9# x =63•5# X=62 .2fa x=53.8# x= 60.1%
Each value is from pooled fetal livers from one dam. a # prothrombin produced in vitro =
Incubation with Incubation without vitamin K added vitamin K added Incubation with vitamin K added V I . Hepatic Prothrombin Production in Fetuses from Pregnant Rats Fed a Vitamin K-Folic Acid Deficient Diet and Treated with Phenytoin
There was no difference in prothrombin synthesis between
the phenytoin-treated and acacia-treated pooled fetal livers
(Table 5)* Twenty-four hours after the last dose, the mean
maternal phenytoin blood concentration was 7.?6 pg/ml plasma
(+ *J-. 93, standard error of the mean). Again, phenytoin
appeared to accumulate in the fetus as the mean serum con
centration 2k hours after the last dose was 289.73 Mg/ml
serum (± 109.95, standard error of the mean).
VII. Determination of Weekly Variation of Prothrombin Concentrations in Cats Using the Two-Stage Prothrombin Assay
This experiment was designed to determine the variation
of normal plasma prothrombin concentration between adult
cats and within each cat over a seven week period. Using the two-stage prothrombin method, the grand mean of the four control cats over the seven weeks was 57*75 units of pro thrombin/ml plasma (Table 6). The prothrombin concentration varied very little from week to week in the same cat. There was a greater variation between individual cats in their prothrombin concentration.
VIII. Plasma Prothrombin Concentration in Cats Given Daily Oral Treatments with Phenobarbital and Phenytoin
For this experiment, there were two controls. A pre treatment prothrombin determination was made on each of the 54
TABLE 5
Fetal Rat Liver Prothrombin Production (Percentage) In Vitro from Dams Treated with 100 mg Phenytoin/ kg Body Weight from Day 8-20 of Gestation (Experiment VI)
In Vitro Prothrombin Synthesis ~T% of Total Prothrombin)a
Control Phenytoin
71.6% 69.3$
42.2% 34.6%
58.7% 68.7%
x= 57.57$ x= 57•5%
Each value obtained from pooled fetal livers from one dam. a % prothrombin produced in vitro =
Incubation with Incubation without vitamin K added ~ vitamin K added Incubation with vitamin K added
56 55
TABLE 6
Weekly Prothrombin Values in Normal Adult Cats Over a Seven Week Period (Experiment VII)
Week Number Units of Prothrombin/ml of Plasma
.1 58.49 ± 6.06
2 58.42 ± 4.43
3 57.05 + 4.53
4 54.68 ± 3.33
5 59.27 1 I .70
6 60.51 + 3.03
7 55.82 + 2.11
x=57•75 + 8.59
Each value represents the mean and standard error of the mean of four cats. Grand mean and standard error of the grand mean are at the bottom of the column.
57 phenytoin-treated or phenobarbital-treated cats which served
as one control. Prothrombin concentrations from the drug-
treated cats during treatment were also compared to the
prothrombin concentrations from a separate group of control
cats. The only difference observed was an increase in pro
thrombin in the phenytoin-treated cats at the end of the
fourth week (Table ?)• Therefore, evidence of vitamin K
antagonism was not observed.
The two toxic effects observed with phenytoin were
anorexia and ataxia. A graph of the mean weekly body weights
of the two treatment groups is shown in Figure 5* The
actual mean values and standard error of the mean which are
plotted in Figure 5 are found in Table 12 in the Appendix.
From this, it can be seen that the mean weights of the two
treatment groups did not differ significantly (P>0.05) at
the beginning of the experiment, but did differ significantly
(P<0.05) each of the following four weeks.
There was a small fluctuation in weight in the pheno barbital-treated group. The cats accepted oral treatment of phenobarbital without much objection and did not appear to
show any signs of toxicity during the treatment period.
Weekly blood concentrations in the cats are given in Table 8.
The blood was drawn 2k hours following the last dose. 57
TABLE 7
Plasma Prothrombin Values in Adult Cats Dosed Orally with 10 mg Phenytoin/kg Body Weight or 10 mg Phenobarbital/kg Body Weight (Experiment VIII)
Prothrombin Units/ml Plasma
Week Control Phenytoin Phenobarbital
Pretreatment 58.49 + 6.06 50.16 + 4.43 57.40 + 5.45
1 58.42 + 4.43 51.92 ± 4.66 50.49 + 5.45
2 57-05 + 4.53 55-63 ± 4.71 50.26 + 3 •66
3 54.68 ± 3.33 55-41 + 7.29 54.81 + 1.79
4 59.2? + 1.70 70.99 ± 7•61* 53.36 + 4.28
Each value in the treatment groups represents the mean with the standard error of the mean for five animals.
* Significantly different (P<0.05) from the control and phenobarbital-treated rats. Each control value represents the mean with the standard error of the mean for four animals. iue 5 Figure weight in kg 2.0 2.2 2A 2 2.8 3.0 3.2 . 6
______ramn ...... treatment weeks. Each point represents the mean weights themean represents point Each weeks. phenobarbital (10 mg/kg body weight) for four for (10 weight)body mg/kg phenobarbital Body weights of cats that were given oralpheny given that ofcatswere Body weights of five cats. The vertical bars represent the represent bars vertical The cats.fiveof or three for weeks (10 body weight) mg/kg toin tnad ro ftema. (ExperimentVIII) ofthemean. error standard r- 4 3 2 1 pre- , hnbria ramn ^ treatment phenobarbital 4, peyontetet J, treatment phenytoin | i ______1 ______phenobarbital treatment phenobarbital iemweeks m time l ______phenytoin treatment phenytoin 1 ______1 58
59
TABLE 8
Weekly Plasma Concentrations Over a Three Week Phenytoin Treatment and a Four Week Phenobarbital Treatment in Cats Dosed at 10 mg Phenytoin or Phenobarbital/kg Body Weight (Experiment VIII)
Drug Concentration (pg/ml plasma)
Week Phenytoin Phenobarbital
1 16.25 + 2.88 18.14- ± 2.4-7
2 13.23 t 5-91 12.16 + 1.84-
3 13.10 ± 5-85 18.4-3 + 4-.4-6
4- 2.70 + 1.21 18.75 ± 3*20
Each value represents the mean with the standard error of the mean for five animals.
Blood samples were taken 24- hours following the last dose. Cats responded to the oral phenytoin treatment with immediate salivation. The nature of the salivation was similar to the salivation seen after sympathetic stimula tion in cats. After one week of phenytoin treatment, the cats began refusing food. They drank water, but progres sively lost weight. The degree of weight loss can be seen in Figure 5- There was an acute loss in weight until the phenytoin treatment was stopped at the third week. After the phenytoin treatment was stopped, the appetites of all the cats returned in about one to two days, except one which had to be fed via stomachtube. Since the appetite of this cat did not return, the cat was euthanatized.
During the third week of phenytoin treatment, two of the five cats showed signs of ataxia. The anorexia and ataxia were probably due to phenytoin toxicity. The plasma phenytoin concentrations were presented in Table 8 and were taken 2k hours following the last dose.
IX. Plasma Prothrombin Activity in Cats Given an Initial Phenytoin Dosage of 20 mg Phenytoin/kg Body Weight for Three Days Followed by 10 mg Phenytoin/kg Body Weight
After one week of no treatment, the group of cats pre viously treated with phenobarbital were initially given 20 mg phenytoin/kg body weight which was two times higher than the phenytoin dosage in experiment VIII. The anorexia produced in these cats was so severe that the dosage was lowered to 6 1
10 mg phenytoin/kg body weight after three days of treat ment at the initial dosage. After four days of the 10 mg phenytoin/kg body weight treatment, the treatment was stopped and the cats were monitored for the following two weeks. The impressive weight loss is graphed in Figure 6.
Means and standard error of the mean are recorded in Table
13 in the Appendix. There was a significant difference
(P<0.05) between the grand mean weight during the pheno barbital treatment (3*00 kg) and the grand mean weight during the one week phenytoin treatment and two weeks post treatment (2.46 kg). Because of small sample size, the weekly differences between the two treatment groups in weight were not significant, but were just at the edge of being significant. There were no other toxicity signs noted. The mean and standard error of the mean of the plasma phenytoin concentration for these cats after one week of phenytoin treatment was 22.5 J^g/ml plasma (± 4.33)-
This plasma concentration is that level of phenytoin present
24 hours after dosing.
There was no significant difference in plasma prothrom bin concentration between the pretreatment period and after one week of phenytoin (Table 9). Nor was there any differ ence between the plasma prothrombin concentrations after one week of phenytoin treatment and the subsequent two weeks post treatment. When all those values were compared with a similar Figure 6. Mean body weights and standard error of the mean for five cats given oral phenobarbital (10 mg/kg body weight) for four weeks followed by one week of drug withdrawal before a cross-over to oral phenytoin (20 mg/kg body weight) and 10 mg/kg body weight during one week Experiment IX).
62 ON 8 7 L ______5 treatment phenytoin I i -J- -- time weeks time in 3 3 T •phenobarbital treatment •phenobarbital 2.2 2.0 2.6 2.k 2.8 3.0 3.2
weight in kg 64
TABLE 9
Plasma Prothrombin in Control Cats and Cats Treated for Three Days with 20 mg Phenytoin/kg Body Weight and Then Four Days with 10 mg Phenytoin/kg Body Weight. These cats were Previously Treated with Phenobarbital (Experiment IX)
Units of Prothrombin/ml Plasma
Phenytoin Controls
Pretreatment 50.54 ± 7.09a 1st week 58.49 ± 6 .06b
1st week 40.75 ± 6.08 2nd week 58.42 ± 4.43 (phenytoin treatment)
2nd week 47.65 + 6.52 3rd week 57.05 1 4.53 (one week post-treatment)
3rd week 63.63 +10.69 4th week 54.68 ± 3.33 (second week post-treatment) g Values represent the mean and standard error of five cats. b Values represent the mean and standard error of the mean of four cats. 65
four week period in the control cats, there was also no
difference between the prothrombin concentration in con
trols and treated cats.
X. Effect of Phenobarbital Pretreatment on Plasma Prothrombin in Cats Given Phenytoin
Plasma prothrombin concentrations did not differ sig
nificantly (P>0.05) during the phenobarbital treatment or
the phenytoin treatment (Table 10), nor were they different
from a control group of cats. The means and standard error
of the means for the 24- hour plasma drug concentrations
were 15-80 jug phenobarbital/ml plasma (± 1.4-4-) on the
seventh day, 11.4-5 Pg phenytoin/ml plasma (+ O.65), and
6-3 pg phenobarbital/ml plasma (± 1.4-8) on the tenth day,
and 36.99 pg phenytoin/ml plasma (± 4-.23) on the thirteenth
day.
The anticonvulsants did have an effect on the weight of
the cats. A graph of their weights is presented in Figure 7
and the data plotted can be found in Table 14- of the Appen
dix. Each mean weight is significantly different (P<0.05)
from the pretreatment weight. After the 10 mg phenobarbital/
kg body weight treatment, the cats' weights increased, but
then decreased sharply when 20 mg phenytoin/kg body weight was given for seven days. Again, no other toxicity signs were seen except the prominent anorexia. 66
TABLE 10
Plasma Prothrombin Values in Cats Given Phenobarbital (10 mg/kg Body Weight) for One Week Followed by Phenytoin (20 mg/kg Body Weight) for One Week (Experiment X)
Time Units of Prothrombin/ml Plasma
Pretreatment 50.5^ ± 7.95 7th day (phenobarbital treatment) *4-0.75 ± 6.81
10th day (phenytoin and phenobarbital treatment) *17.65 + 7.31
13th day (phenytoin treatment) 63.63 ±11.98
Each value represents the mean and standard error of the mean for five animals. weight in kg Figure 7- Each point represents the mean weight with its with theweight mean represents point Each 7-Figure 3-5 .0 phenobarbital treatment-^— phenytoin treatment--^ phenytoin treatment-^— phenobarbital ment. Each cat was given 10 phenobarbital/ given mg catwas Each ment. gbd egt rlyfrsvndy n then andseven daysfor orally weight body mg treated with orally for sixfordays.orally five cats at daysatfivecats of ofthe weights ofthe error mean standard 20 ie in TimeDays mg phenytoin/kg body body weight phenytoin/kg mg 0 , 7 , 10 and 13 fte experi ofthe
6
?
68
XI. Plasma Prothrombin Activity of Cats Given Warfarin and Phenytoin Simultaneously
Since administering the anticonvulsants to cats had no detectable effect on the plasma prothrombin concentration, warfarin was given as a positive control to verify that the cats were indeed sensitive to a hypoprothrombinemic drug.
The cats were given 0.5 mg warfarin/kg body weight and 20 mg phenytoin/kg body weight. Prothrombin concentrations were measured every three days. Treatment was stopped when the plasma prothrombin concentration dropped below 12 pro thrombin units/ml plasma. Three of the four cats had less than 12 prothrombin units/ml plasma after six days of treat ment, and one cat had below 12 prothrombin units/ml after three days of treatment. The pre-experiment prothrombin concentrations ranged from 5^ units to 82 units/ml plasma. CHAPTER IV
DISCUSSION
This report does not support the results of Solomon I f. c et al. ’-'in any way except for the loss of appetite and ataxia seen with the phenytoin treatment in cats. From the results presented in this paper, there was no evidence of anticonvulsant antagonism of vitamin K in prothrombin syn thesis. Neither use of phenobarbital, phenytoin, or primidone had any effect on _in vitro prothrombin production when fetal or adult rat livers were utilized. Prothrombin concentrations in adult cats were similarly unaffected by treatment with either phenobarbital or phenytoin. A decrease in prothrombin quantities was observed with war farin in both species.
In attempts to resolve the contradictory results, it is observed that in the reports of Solomon et al., the numbers of animals used in the experiments were not indicated, and they did not present a statistical evaluation of their data.
Thus, the validity of their conclusions can be questioned.
To date, the only published experimental evidence for the
69 anticonvulsant-induced depression of the vitamin K depen
dent clotting factors are the original two reports of
Solomon et al., in 1972 and 197^- It is of interest that
they have not reported on further investigations of this
syndrome. In their rat experiments, Solomon et al. analyzed
the effects of the anticonvulsants on Factor VII synthesis
using the in vitro liver slice method of Pool and Robinson.57
The rats they used were not vitamin K deficient, so it is difficult to predict if there was accumulation of Factor
VII precursor in the liver before the incubation. Since this method requires a four or five hour incubation, the anticonvulsants could have affected both Factor VII protein synthesis or degradation and not just the vitamin K depen dent step. In contrast, the in vitro results presented in this paper had measured the vitamin K dependent conversion of prothrombin precursor to prothrombin. Evidence of pro thrombin precursor accumulation in the liver of vitamin K deficient rats is well documented.2?
There was no evidence of any effect on prothrombin con centrations in cats treated with phenobarbital or phenytoin.
The cats used in the experiments described in this paper had blood phenytoin concentrations between 13*1 pg/ml and 36.9
Hg/ml. The blood phenytoin concentrations reported by 71
Solomon et al. ranged approximately from 5 /ug/ml to 30 jug/ml.
Although the blood concentrations of phenytoin were com parable, they reported a greater than 3°% decrease in pro thrombin concentration after one week of phenytoin treat ment .
In this report, phenobarbital blood concentrations in cats ranged from 12.2 pg/ml to 18.8 jag/ml while, in the work of Solomon et al., blood phenobarbital concentrations in cats treated with the same dosage (10 mg phenobarbital/ kg body weight) were approximately 25 pg/ml. They also found no effect on prothrombin concentrations at the 10 mg phenobarbital/kg body weight dosage. However, in cats given
40 mg phenobarbital/kg body weight, they observed a 50% decrease in prothrombin content after three weeks and signs of neurologic toxicity such as ataxia, loss of righting reflexes, appetite loss, and marked lethargy. The presence of neurologic toxicity is understandable since blood pheno barbital concentrations ranged from approximately 60 jug/ml to 70 pg/ml. Due to the toxicity signs in cats treated with 40 mg phenobarbital/kg body weight, this dosage was not used in the experiments described for this dissertation.
The neurotoxic signs in the phenytoin-treated cats observed in this research and in the report of Solomon et al. 72
were ataxia and loss of appetite. Loss of appetite was very
pronounced and was reflected in the weight loss data. In
this research,the appetite loss was observed without other
neurotoxic signs and is believed to be caused by phenytoin
depression of the central nervous system. 21 Sadowski et al.. did not observe an anti-vitamin K
effect with phenobarbital in rats; in fact, an increase in
prothrombin content was observed. The increase in prothrom
bin production is understandable as phenobarbital is a known
microsomal enzyme inducer. Although there was no observed
increase in prothrombin in this research, the phenobarbital
treatment period in male rats was only two days which may
not have been sufficient time to observe the inductive
effect.
The results of hepatic in vitro prothrombin synthesis were similar to those found by Shah and Suttie ^8 ancj Houser
et al. ^0 confirming the reproducibility of the method.
Because anticonvulsant antagonism of vitamin K was not demonstrated in vitro or in vivo using male rats, pregnant female rats were given a vitamin K deficient diet along with a daily phenytoin or phenobarbital treatment. In as much as no effect was seen in prothrombin production in the in vitro fetal liver incubations when compared with their controls, pregnant female rats were then given a vitamin K- folic acid deficient diet. Folic acid supplements have 2 6 been shown to lower serum phenytoin levels. Folic acid supplement in the vitamin K deficient diet (80 pg/day) exceeded the daily folic acid treatment of approximately
25 jug/day, shown to have stabilized hepatic phenytoin 2b 2b hydroxylase activity. Blake et al. demonstrated that with folic acid supplementation in rats, the normal decrease in phenytoin hydroxylase activity during pregnancy was not observed. Consequently, phenytoin metabolism was not decreased and the possible fetal exposure to higher concen trations of phenytoin was avoided. By giving the folic acid deficient diet to the dams, the rat fetuses were ex posed to higher concentrations of unmetabolized phenytoin.
This was confirmed in this paper when the fetal phenytoin blood concentrations from the dams fed the vitamin K deficient diet (95*^ ± 37*5 pg/ml) are compared with those from dams fed the vitamin K-folic acid deficient diet
(289.7 ± 37*5 pg/ml). Although folic acid measurement was not made, the differences in fetal phenytoin suggest that folic acid deficiency did result in dams fed the folic acid deficient diet. There was no effect on fetal prothrom bin production regardless of the diet used. 7^
Perhaps the anticonvulsants precipitate an earlier and
greater postnatal decrease of the vitamin K dependent fac
tors in human newborns. If the preceeding statement were
feasible, then this problem might be investigated using a
species such as nonhuman primates, which also may have a normal decrease in vitamin K dependent factors after birth.
Most of the literature reporting the effects of the
anticonvulsants on the vitamin K dependent clotting factors
in newborn humans has been published outside the United
States. Very few cases have been documented in this country although thousands of women taking anticonvulsants have given birth. This may be because it has not been recognized before, or it may be that prenatal practices in other countries may affect this syndrome. Furthermore, most of the reports are of a clinical nature and the therapeutic use of vitamin K does not always correct this hemorrhagic problem. If this were true, correlation observed by chance may have supported an etiology for a syndrome that may not exist. CHAPTER V
SUMMARY AND CONCLUSIONS
The investigation of the anticonvulsant-induced depres
sion of the vitamin K dependent clotting factors resulted
in the following observations:
1. Prothrombin production in vitamin K deficient rat
liver in vitro was not affected by the presence of 10"^ M _3 _3 phenytoin, 10 M phenobarbital, or 10 ^ M primidone in the
incubation. There was no difference in the results whether
liver homogenate or liver slices were used.
2. Prothrombin production varied directly with increas
ing concentrations of vitamin K added ini vitro to vitamin K
deficient rat liver homogenate. No difference in the
response was observed when 10 ^ M phenytoin was added.
Prothrombin synthesis was significantly decreased when 10“^
M warfarin was added.
3. In vivo treatments with 80 mg phenobarbital, 200 mg primidone, or 200 mg phenytoin/kg body weight for two days in vitamin K deficient male rats had no effect on prothrom bin synthesis in vitro. Likewise, there was no effect
75 76
observed on in vitro prothrombin synthesis in fetal liver when 80 mg phenobarbital subcutaneously or 100 mg phenytoin
orally/kg body weight was given to their dams during the
last two weeks of gestation.
Phenytoin blood concentrations were observed to be about 20 times higher in vitamin K deficient fetal rats than in their vitamin K deficient dams on the 21st day of gestation after a two-week oral treatment with 100 mg phenytoin/kg body weight. Fetal phenytoin blood concentra tions were ^40 times higher than maternal blood phenytoin concentrations on the 21st day of gestation when dams were given a vitamin K-folic acid deficient diet and 100 mg phenytoin/kg body weight orally.
5. Plasma prothrombin concentrations in cats were un affected by oral treatment with phenytoin (10 or 20 mg/kg body weight) or phenobarbital (10 mg/kg body weight). Pre treatment with 10 mg phenobarbital/kg body weight and treatment with 20 mg phenytoin/kg body weight in cats resulted in no change in prothrombin concentration when compared to controls.
6 . Oral warfarin at 0.5 mg/kg body weight and oral phenytoin at 20 mg/kg body weight given separately each day to cats did significantly depress the plasma prothrombin concentration in three to six days. 11
1. Severe anorexia and weight loss was observed in the
cats in each experiment when phenytoin (10 mg or 20 mg
phenytoin/kg body weight) was administered. Mean plasma
phenytoin concentrations ranged from 11.5 Mg/ml to 36.9
pg/ml in those experiments.
The coumarin-like effect of the anticonvulsants on the
vitamin K dependent clotting factors reported in the litera
ture was not confirmed by the results presented in this
paper. Phenobarbital, primidone, or phenytoin had no
effect on prothrombin production in either fetal or adult
rat liver. No coagulopathy in adult cats was observed with
phenytoin or phenobarbital treatment. Inability to repro
duce the anticonvulsant depression of prothrombin creates
questions about the validity of the unconfirmed work of
Solomon et al. A more controlled clinical study would
be helpful to clearly establish if there is a relationship
between the maternal anticonvulsant therapy and "hemorr
hagic disease of the newborn".
Phenobarbital (10 mg/kg body weight) treatment appeared
to have no neurological contraindications to its use in the
feline species. Due to the severe anorexia and weight loss,
the use of phenytoin therapy at daily dosages of 10 mg phenytoin/kg body weight in cats is not recommended. APPENDIX 79
TABLE 11
Prothrombin Activity in Rat Liver Homogenate at Varying Vitamin K Concentrations with Phenytoin or Warfarin Added to the Incubation (Experiment II)
Prothrombin (units x 10-2/gm liver)
Incubation 2 ng Vitamin 200 ng Vitamin 1000 ng Vitamin Conditions K added K added K added
Control 57-1 + 28.0 459-9 ± 33.7 763.8 + 68.3
Phenytoin added (10~3m ) 75*9 ± 21.8 575.6 ±132.23 877.7 ±172.9
Warfarin added (10~3m ) 63.8 ± 24.1 97.2 ± 15.9* 369.O ± 40.2*
Values are significantly different (P<0.05) from other values in that column. Each value represents the mean and the standard error of the mean of five rats. 80
TABLE 12
Body Weights of Cats Treated with 10 mg Phenytoin/kg Body Weight for Three Weeks or 10 mg Phenobarbital /kg Body Weight for Four Weeks (Experiment VIII)
Body Weight (kg)
Time Phenytoin Phenobarbi tal
Pretreatment 3.09 + 0.21 2.99 + 0.19
1st week 2.73 + 0 .16* 3.02 + 0 .16*
2nd week 2.55 + 0 .11* 3.02 + 0.15*
3rd week 2.37 + 0.13* 2.98 + 0.17*
4th week 2.48 + 0.15* 2.98 + 0.18*
* Significantly different (P<0.05) from the other values recorded in that week.
Each value represents the mean and the standard error of the mean for four cats. 81
TABLE 13
Body Weights of Cats Given Phenytoin (20 mg/kg Body Weight for Three Days and 10 mg/kg Body Weight for Four Days) After Previously Being Treated with Phenobarbital for Four Weeks (Experiment IX)
Time Body Weight in kg
Pretreatment 2.69 + 0.18
1st week (phenytoin treatment) 2.37 ± 0.16
2nd week (one week post-treatment) 2.32 + 0.23
3rd week (second week post-treatment) 2.^7 + 0.25
Pretreatment 2.99 ± 0.19
1st week (previous phenobarbital treatment) 3*02 ± 0.16
2nd week (previous phenobarbital treatment) 3*02 + 0.15
3rd week (previous phenobarbital treatment) 2.98 + 0.17
Each value represents the mean and the standard error of the mean for four cats. 82
TABLE 14
Body Weights of Cats After Seven Days of 10 mg Phenobarbital/kg Body Weight Treatment Followed by a Six Day Treatment With 20 mg Phenytoin/kg Body Weight (Experiment X)
Time Body Weight in kg
Pretreatment 3.35 + 0.39
7th day (phenobarbital treatment) 3.48 ± o.4l*
10th day (phenytoin and phenobarbital treatment) 3.25 ± 0 .38*
13th day (phenytoin treatment) 2.98 + O.36*
* Significantly different (P<0.05) from pretreatment value.
Each value represents the mean and the standard error of the mean of five animals. BIBLIOGRAPHY
1. Chadd, M.A. (1973) Coagulation status of the neonate. Bib. Anat., 1 9 7 3 '. #12, 77-82.
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3. Zellweger, H. (1974) Anticonvulsants during pregnancy: Danger to developing fetus? Clin. Ped., 12: #4, 338-346.
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5* (1974) Phenobarbital-induced coagulation defects in cats. Neurology, 24: #10, 920-924.
6. Jackson, C.M. (1977) Recommended nomenclature for blood clotting zymogens and zymogen activation products of the International Committee on Thrombosis and Hemostasis. Thrombo. Haemostas., ^8: 567-577*
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