Vitamin K-deficient Rats Research Articles

Difference in the metabolism of vitamin K between liver and bone in vitamin K-deficient rats

The difference between vitamin K metabolism in the liver and that in the bone of vitamin K-deficient rats was examined. After 17 d administration of vitamin K-deficient food, vitamin K in the liver was almost depleted, and prothrombin time (PT) was prolonged. Serum total osteocalcin level was slightly decreased by vitamin K deficiency, whereas serum undercarboxylated osteocalcin level did not change. The level of menaquinone (MK)-4 as well as that of phylloquinone was decreased, but approximately 40 % of the initial level still existed in the femur after the 17 d period. A single-dose administration of vitamin K (250 nmol/kg body weight) markedly increased vitamin K level in the liver but not in the femur. These results suggest that the turnover of vitamin K in the bone is slower than that in the liver, and bone metabolism may be little affected by the short period of intake of vitamin K-deficient food. However, intake of a larger amount of vitamin K is required for its accumulation in the bone than in the liver. Furthermore, the counteracting effect of MK-7 on prolonged PT in vitamin K-deficient rats was found to be higher than phylloquinone or MK-4.

Intestinal, hepatic, and circulating vitamin K levels at low and high intakes of vitamin K in rats

The aim of the present study was to assess how high doses of dietary vitamin K influence the intestinal profile of K-vitamins in vitamin K-deficient rats, and whether the induced changes are reflected in the hepatic vitamin K store. Vitamin K-deficient rats were fed for 10 d on diets containing different forms of vitamin K, and it was determined how these diets affected the vitamin K concentration at various sites of the instestine, serum, and the liver. It was found that the absorption of phylloquinone from standard food is not more than 10%, while the absorption of pharmacological doses of oil-solubilized phylloquinone and menaquinone-4 was also far from complete (18 and 55% respectively). High intakes of phylloquinone suppress the colonic production of all higher menaquinones. High menaquinone-4 intake induces very high menaquinone-8 concentrations, both in the colonic contents as well as in the liver. These data suggest that menaquinone-4 may be converted into menaquinone-8 (but not into other menaquinones) via a metabolic pathway which has not been reported previously.

Induction of prothrombin synthesis by K-vitamins compared in vitamin K-deficient and in brodifacoum-treated rats

Vitamin K is a group name for a number of prenylated 2-methyl-1,4-naphtoquinones, which may differ in their ability to function as a cofactor for prothrombin biosynthesis. To quantify the bioactivity of different forms of vitamin K, two experimental animal systems are frequently used: vitamin K-deficient rats and anticoagulated rats. In this paper both models are compared, and it is shown that the results obtained depend on the model used. The main reason for this discrepancy is the difference in recycling of vitamin K-epoxide, which results in a 500 times higher vitamin K requirement in anticoagulated rats. Absorption and hepatic accumulation of long chain menaquinones seem to be restricted to a maximum, whereas also the lipophilic nature of long chain menaquinones may hamper the quinone–quinol reduction in anticoagulated animals. If these data may be extrapolated to patients, food items rich in K 1 and MK-4 would be expected to influence the stability of oral anticoagulation to a much larger extent than food items primarily containing higher menaquinones.

Tissue distribution of K-vitamers under different nutritional regimens in the rat

Two forms of vitamin K [phylloquinone (K 1) and menaquinone-4 (MK-4)] were added to vitamin K-deficient rat food in varying amounts. These diets were given as the sole source of nutrition to rats for one week. The minimal dietary requirements (MDR) to attain maximal prothrombin synthesis were determined to be 0.6 and 6–10 μg/g of food for K 1 and MK-4, respectively. The difference between both vitamers could be explained by the limited hepatic accumulation of MK-4. Next, vitamin K was offered to rats at concentrations ranging between 0.6 and 3000 μg/g of food, and the tissue distribution of vitamin K was investigated after one week of administration. Accumulation of K 1 and MK-4 was found in all tissues investigated, but both the absolute tissue concentration and the ratio between K 1 and MK-4 were tissue-dependent. Highest values were found in liver and in heart, but since the heart contains no γ-glutamylcarboxylase, the function of vitamin K in this tissue remains obscure. High tissue concentrations of MK-4 were also found in pancreas and testis after a diet containing K 1 exclusively. The data indicate that this conversion is tissue-specific, but neither the reason nor its mechanism are known.

Bioavailability of phylloquinone and menaquinones after oral and colorectal administration in vitamin K-deficient rats

Rats were made vitamin K-deficient by feeding them a diet devoid of vitamin K and by rigorously preventing coprophagy. After one week, circulating prothrombin concentrations were between 5 and 10% of initial values, and various amounts of phylloquinone. menaquinone-4, and menaquinone-9 were given in a single dose either subcutaneously, orally, or colorectally. The relative ‘vitamin K activities’ of these compounds were assessed by comparing their ability to support prothrombin synthesis after subcutaneous injection. Intestinal and colonic absorption were deduced from the difference between subcutaneous and either oral or colorectal administration of the vitamers. It is concluded that the colonic absorption of all three forms of vitamin K is extremely poor, suggesting that physiological menaquinones in the colon do not contribute substantially to vitamin K status in rats. Furthermore, the stimulation of prothrombin synthesis by menaquinone-9 lasted much longer than that by the two other K-vitamers, resulting in a substantially higher ‘vitamin K activity’ of menaquinone-9.

The relative effects of phylloquinone and menaquinone-4 on the blood coagulation factor synthesis in vitamin K-deficient rats

Rats were made vitamin K-deficient by feeding them a 1:1 (w/w) mixture of a commercial vitamin K-depleted diet and boiled white rice. After one week of treatment the rats had developed severe vitamin K deficiency, resulting in Thrombotest® values of 5–10% of the initial values. In this experimental system the efficacy of phylloquinone (K 1) was compared with that of menaquinone-4 (MK-4) by measuring the extent to which the Thrombotest was normalized after the administration of varying doses of the respective vitamins. Oral administration of the vitamins showed that the efficacy of K 1 was at least two-fold higher than that of MK-4. As comparable results were obtained after subcutaneous administration of the vitamins, we conclude that after oral administration the intestinal absorption had been quick and nearly complete. A less pronounced effect of K 1 and MK-4 was found after colorectal administration. For both forms of vitamin K relatively high amounts (well above the physiological concentration) were required before significant effects on the Thrombotest could be observed. Therefore these data demonstrate the importance of sufficient dietary vitamin K consumption in rats. The efficacy of other menaquinones may be investigated in the same experimental animal model system.

Depression of liver microsomal vitamin K epoxide reductase activity associated with antibiotic-induced coagulopathy

Hypoprothrombinemic changes in blood coagulation parameters, such as prolongation of prothrombin time, increase in the level of plasma protein induced by vitamin K absence, and decrease in plasma prothrombin level, were detected in rats fed a vitamin K-deficient diet. These changes were enhanced by the administration of β-lactam antibiotics containing N-methyltetrazolethiol, thiadiazolethiol or methyl-thiadiazolethiol. Microsomal vitamin K epoxide reductase activity was suppressed with the maximum effect at 1–2 days after the treatment and with recovery, thereafter, gradually to the normal level after 5–7 days. Hypoprothrombinemic alterations in blood coagulation parameters following a single administration of antibiotic to vitamin K-deficient rats were somewhat delayed compared with the change in the epoxide reductase activity, but the effects of the antibiotic on both blood coagulation parameters and the enzyme activity disappeared completely 7 days after the antibiotic treatment. Antibiotic-induced depression of the epoxide reductase activity was observed even in the vitamin K sufficient rats, although the hypoprothrombinemic changes in the blood coagulation parameters did not develop. Vitamin K administration could normalize the blood coagulation parameters in the hypoprothrombinemic rats caused by treatment with the antibiotics but without recovery of the decreased epoxide reductase activity. These results suggest that some antibiotics inhibit liver microsomal vitamin K epoxide reductase, which causes hypoprothrombinemia to develop under vitamin K-deficient conditions.

In vivo effects of β-lactam antibiotics and heterocyclic thiol compounds on vitamin K-dependent carboxylation activity and blood coagulation factors in vitamin K-deficient rats

The vivo effects of heterocyclic thiol compounds, corresponding to the 3′-position substituents of several β-lactam antibiotics, on blood coagulation factors and on liver microsomal γ-glutamylcarboxylation (γ-carboxylation) activity were evaluated in rats maintained on a vitamin K-deficient diet. These rats, when compared to normal control animals, exhibited hypoprothrombinemic changes: prolongation of both prothrombin time and activated partial thromboplastin time, decreases in factor VII and plasma prothrombin, and increases in PIVKA II (descarboxyprothrombin) both in plasma and liver. They also displayed a marked increase in liver microsomal γ-carboxylation activity. These blood coagulation variables could be altered markedly by administering various heterocyclic thiol compounds to the vitamin K-deficient rats, although these compounds did not inhibit γ-carboxylation activity in an assay system using phylloquinone. A similar pattern of alteration was observed when some β-lactam antibiotics were administered. Increased microsomal γ-carboxylation activity in antibiotic-treated vitamin K-deficient rats was normalized by the administration of vitamin K, concomitant with the recovery of blood coagulation variables to the normal range. The results indicate that antibiotic-induced hypoprothrombinemia in vivo is not caused by inhibition of enzymes of the γ-carboxylation system, such as vitamin K reductase and γ-glutamylcarboxylase, but is related to the endogenous vitamin K level.

Vitamin K Reversible Hypoprothrombinemia in Rats II. Efficacy of Vitamin K on Latamoxef-Induced Coagulopathy in Rats

Feeding of a vitamin K-deficient diet caused the development of hypo-prothrombinemic changes in rats such as prolongation of prothrombin time (PT) and activated partial thromboplastin time (APTT), decreases in plasma prothrombin and clotting factor VII levels, and an increase in the descarboxyprothrombin (PIVKA) level in both plasma and liver. Successive administrations of latamoxef (LMOX) to the vitamin K-deficient rats resulted in the further enhancement of these changes. After the development of hypoprothrombinemia with LMOX, a single subcutaneous injection of vitamin K1 normalized most of these abnormalities in blood coagulation parameters within 6 hr. When vitamin Κ was given at 200 μg/kg, PT, APTT and the plasma PIVKA level showed normal values for at least 8 days even when the animals were fed a vitamin K-deficient diet and treated with LMOX during the recovery period. The amount of vitamin Κ required to maintain most of the blood coagulation parameters in the normal range was about 3 μg/kg/day. The plasma level of vitamin Κ was higher than 0.3-0.5 ng/ml when the blood coagulation parameters were maintained in the normal range.

Vitamin K reversible hypoprothrombinemia in rats. II. Efficacy of vitamin K on latamoxef-induced coagulopathy in rats.

Feeding of a vitamin K-deficient diet caused the development of hypoprothrombinemic changes in rats such as prolongation of prothrombin time (PT) and activated partial thromboplastin time (APTT), decreases in plasma prothrombin and clotting factor VII levels, and an increase in the descarboxyprothrombin (PIVKA) level in both plasma and liver. Successive administrations of latamoxef (LMOX) to the vitamin K-deficient rats resulted in the further enhancement of these changes. After the development of hypoprothrombinemia with LMOX, a single subcutaneous injection of vitamin K1 normalized most of these abnormalities in blood coagulation parameters within 6 hr. When vitamin K was given at 200 micrograms/kg, PT, APTT and the plasma PIVKA level showed normal values for at least 8 days even when the animals were fed a vitamin K-deficient diet and treated with LMOX during the recovery period. The amount of vitamin K required to maintain most of the blood coagulation parameters in the normal range was about 3 micrograms/kg/day. The plasma level of vitamin K was higher than 0.3-0.5 ng/ml when the blood coagulation parameters were maintained in the normal range.

Production of hypoprothrombinaemia by cefazolin and 2-methyl-1,3,4-thiadiazole-5-thiol in the rat.

Recent findings have suggested that the 1-methyltetrazole-5-thiol (MTT) group contained in several beta-lactam antibiotics may be responsible for the hypoprothrombinaemia associated with these drugs. In order to determine if the hypoprothrombinaemia associated with cefazolin is due to the presence of the structurally related 2-methyl-1,3,4,-thiadiazole-5-thiol (MTD) group which it possesses, the ability of MTD and cefazolin to produce hypoprothrombinaemia in rats was examined. Female rats maintained on a vitamin K-deficient diet for ten days developed hypoprothrombinaemia after the intravenous administration of cefazolin for two subsequent days. Hypoprothrombinaemia was also produced by the oral administration to vitamin K-deficient rats of either cefazolin or MTD, while the oral administration of cefotaxime, which does not contain a thiol group, had no effect. In a rat liver microsomal system, MTD was found to be a much more potent inhibitor than cefazolin of the vitamin K-dependent step in clotting factor synthesis, the gamma-carboxylation of glutamic acid. These results suggest that the hypoprothrombinaemia associated with cefazolin may be due to the MTD group.

Effect of sex hormones on hypoprothrombinemia induced by N-Methyltetrazolethiol in rats

N-Methyltetrazolethiol (NMTT) increased prothrombin time (PT) and decreased plasma factor VII and prothrombin levels only in vitamin K-deficient male rats. In female rats identical treatment with NMTT did not produce hypoprothrombinemia. Conventional and germ-free rats displayed no significant difference in the manifestation of hypoprothrombinemia, but the increase of PT in NMTT-treated vitamin K-deficient rats was greater in the germ-free males. Estradiol administration or castration of male rats retarded manifestation of vitamin K deficient syndromes such as increases of PT and activated partial thromboplastin time (APTT), decreases of plasma factor VII and prothrombin levels, and increases of plasma and liver descarboxyprothrombin (PIVKA) levels, and testosterone injection to the castrated rats restored these changes. In female rats testosterone treatment or castration enhanced the manifestation of hypoprothrombinemia and estradiol treatment to the castrated females retarded it. Gamma-glutamyl-carboxylase activity was increased by vitamin K-deficiency but not inhibited by testosterone or NMTT. These data suggest that estrogen protects the rat against manifestation of hypoprothrom-binemia even with NMTT treatment, while androgen enhances vitamin K deficiency, and supplementation of vitamin K prevents its deficiency in NMTT-treated rats.

Vitamin K-dependent carboxylase.

Vitamin K-dependent carboxylase.

Behavioral perturbations in the vitamin K-deficient rat

Anecdotal observations of the behavior of rats with a vitamin K-deficiency suggested that this deficiency was associated with hypoactivity, general malaise, and a lack of exploratory behavior. These observations were pursued by assessing locomotor activity in a circular photocell-monitored track, open-field activity, and radial-arm maze performance in rats rendered vitamin K-deficient by dietary depletion or by warfarin treatment. There was a significant reduction (approximately 25% at the median) in the locomotor activity of dietary vitamin K-deficient rats compared with rats fed a control diet. In the open-field, warfarin administration was associated with a significant shift from more exploratory behaviors to less exploratory behaviors. Consistent with these findings, radial-arm maze assessment showed a comparative reduction in locomotor activity in the dietary vitamin K-deficient rats with no alteration in performance, i.e., short-term memory. These animal behavioral studies suggest that sub-clinical and clinical vitamin K-deficiency may contribute to physical and psychiatric symptomatology.

Hypercalciuria during experimental vitamin K deficiency in the rat.

Vitamin K promotes the formation of gamma-carboxylated glutamate (GLA) in several protein species. GLA residues have a high affinity for the Ca ion. In the present study, we tested the hypothesis that experimental vitamin K deficiency in rats could induce changes in Ca metabolism. Vitamin K depletion, which was associated with a reduction in urinary GLA excretion, induced within 7 days a significant increase in cumulative urinary Ca excretion that persisted throughout the 21 days of study. The hypercalciuria of vitamin K-deficient rats was corrected on vitamin K supplementation. No concomitant changes were observed in intestinal Ca absorption determined by a balance technic or of skeletal resorption and apposition rates determined by bone histomorphometry. Plasma Ca, but not total protein concentration, of vitamin k-depleted rats showed a transient decrease at day 15 that disappeared at day 21. plasma sodium, phosphate and 1,25(OH)2 vitamin D concentration, and urinary phosphate, sodium, and creatinine excretion remained unchanged. In conclusion, vitamin k deficiency in the rat induced hypercalciuria that could be of renal origin. Its possible relationship to vitamin K-dependent renal GLA protein remains to be clarified.

The interaction of salicylate and vitamin K in synthesis of factor II (prothrombin)

The effects of sodium salicylate on synthesis of a vitamin K-dependent plasma coagulation protein, Factor II (prothrombin) was studied using the isolated rat liver perfused for 10 hours in vitro . Cumulative synthesis of Factor II was measured by a standard coagulation assay, by activation with E. carinatus venom, and by rocket immunoelectrophoresis. When sodium salicylate 5 mg or 25 mg was added to the liver perfusate (volume 100 ml) at the outset of the perfusion, cumulative synthesis of both coagulation activity and immunoreactive protein was significantly less than that seen in control perfusions containing no salicylate. The inhibitory effect of salicylate was prevented by pretreatment of rat liver donors with supplemental vitamin K injected 24 hours before sacrifice. Although some interaction between salicylate and vitamin K was apparent from these experiments, the results from vitamin K-deficient rat liver donors were quite different from those containing salicylate. There was no assayable Factor II coagulation activity produced in 10 hours of perfusion of vitamin K-deficient rat livers, but cumulative synthesis of immunoreactive Factor II was quite comparable to that seen in control perfusions.

Vitamin K-dependent carboxylase: Effect of detergent concentrations, vitamin K status, and added protein precursors on activity

Activity of the rat liver microsomal vitamin K-dependent carboxylase has been studied at various concentrations of detergent. The activity which could be solubilized by 0.25% Triton X-100 was low but could be greatly increased if vitamin K-deficient rats were given vitamin K a few minutes before they were killed. At higher concentrations of Triton, more activity was solubilized and this effect was not seen. In vitro carboxylation of endogenous microsomal proteins was decreased by 80–90% if vitamin K was administered 1 min before rats were killed, but the amount of assayable prothrombin precursor was decreased by only 20%. Decarboxylated vitamin K-dependent rat plasma proteins were not substrates for the carboxylase and did not influence peptide carboxylase activity significantly. Purified microsomal prothrombin precursors did, however, stimulate carboxylation of peptide substrate and were used as a substrate for the carboxylase in a preparation from precursor depleted vitamin K-deficient rats.

A Plasma Factor Enhances Activity of Vitamin K-Dependent Coagulation Proteins

A plasma factor, "coagulopoietin", present in animals with depleted vitamin K-dependent coagulation factors, appears to enhance activity of these factors in normal animals. We have investigated the effects of "coagulopoietin" on synthesis of certain coagulation proteins by the isolated rat liver perfused for eight hours. Liver donor rats received plasma injections from vitamin K-deficient rats or from normal rats 24 hr before sacrifice. Coagulation activity of Factor VII and Factor II in liver perfusate samples was measured with a coagulation assay; Factor II synthesis was also measured by rocket immunoelectrophoresis and by activation with E. carinatus venom. Cumulative hepatic synthesis of Factor VII coagulation activity was increased by 43% when rat liver donors received vitamin K-deficient rat plasma compared to normal rat plasma. Cumulative synthesis of Factor II coagulation activity was increased by 51%, but synthesis of the protein measured immunologically or by activation with venom was not affected. The "coagulopoietin" factor in these studies appears to increase measurable coagulation factor activity without increasing total protein synthesis.

A new detergent for the solubilization of the vitamin K-dependent carboxylation system from liver microsomes: Comparison with triton X-100

A new nondenaturing zwitterion detergent has been used to solubilize the vitamin K-dependent carboxylating system from rat liver microsomes. The optimal conditions for solubilization were 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) and 1 m NaCl while maximum carboxylating activity was achieved with 0.5% CHAPS, 0.75 m NaCl for a microsomal protein concentration varying from 7.5 to 10 mg/ml. Using CHAPS, high levels of carboxylase activity can be found not only in liver microsomes from vitamin K-deficient rats, but also in microsomes prepared from the livers of normal animals, normal animals supplemented with vitamin K 1, or vitamin K-deficient animals supplemented with vitamin K 1, while little if any activity is detected after Triton X-100 solubilization of the microsomes of such livers. Thus, the new zwitterion detergent proves to be more effective and less denaturing in solubilizing the vitamin K-dependent carboxylase from rat liver microsomes than other detergents used in this system.

Characterization of vitamin K-dependent carboxylase from the livers from the adult ox and dicoumarol-treated calf.

The properties of the microsomal vitamin K-dependent carboxylase from the livers of the adult ox and dicoumarol-treated calf were investigated. The enzymes from both sources utilized glutamic residues of synthetic peptides as substrates and could be solubilized with Triton X-100 similarly to the enzyme from vitamin K-deficient rat liver. Under the optimal assay conditions, the microsomes from calf liver had peptide carboxylase activity comparable with that of the rat liver microsomes and 6.5-fold that of adult ox liver microsomes. The apparent Km for reduced vitamin K and the ionic strength optima of the calf and adult ox enzyme clearly differ from those of the rat enzyme. Pyridoxal phosphate activated the adult ox carboxylase only slightly, whereas the calf enzyme was activated by pyridoxal phosphate as effectively as was the enzyme from the vitamin K-deficient rat. Mn2+ activated the adult ox enzyme 9-fold and calf enzyme 22-fold under optimal conditions (no KCl). Three other divalent metal cations (Ca2+, Ba2+, and Mg2+) activated the adult ox and calf enzymes to about half the extent caused by Mn2+, KCl inhibited this activation. The vitamin K-dependent carboxylase from the dicoumarol-treated calf is apparently more tightly bound to the microsomal membrane than is the adult ox enzyme. In many other respects (pH optimum), temperature optimum, Km values for peptide substrate, substrate specificity, inhibitor effects), the properties of the adult ox and calf enzymes resemble closely those of the rat enzyme.