John T. Matschiner

Isolation and characterization of a new metabolite of phylloquinone in the rat

Abstract The metabolism of radioactive phylloquinone was studied in rats given warfarin. In the presence of warfarin, a new metabolite of vitamin K accumulated in liver. This compound was isolated and characterized as phylloquinone oxide, a substance which has the activity of vitamin K. 6 h after the administration [phytyl-14C]phylloquinone (uniformly 14C-labeled in the phytyl moiety), more than 50% of the radioactivity normally found as phylloquinone, was recovered as the oxide if the rats were given the anticoagulant drug. Evidence was obtained that liver and heart of rats fed radioactive vitamin K without warfarin contained a small percentage of the oxide.

Synthesis and destruction of prothrombin in the rat

Abstract The kinetics of synthesis and destruction of prothrombin in the rat were investigated. In normal rats given cycloheximide or vitamin K antagonists, prothrombin activity declined logarithmically with half-lives ranging from 5.3 to 7.0 hr. The rate of prothrombin inactivation was slightly higher in vitamin K-deficient rats but did not increase with increasing severity of deficiency. The initial response to vitamin K by deficient rats was more rapid than predicted by the theoretical induction curve calculated from the rate of prothrombin destruction. After 1 hr the rate of prothrombin increase was similar to the theoretical rate and required over 15 hr to reach normal prothrombin concentration. In deficient rats given vitamin K in the presence of cycloheximide, a small rise in prothrombin activity was observed whereas in deficient rats given warfarin or the chloro analog of vitamin K response to vitamin K was obliterated. Actinomycin D administered to deficient rats up to 10 hr before vitamin K did not alter response to the vitamin. If the antibiotic was administered to normal rats, the prothrombin level was little changed for 10 hr after which it decreased with a half-life similar to that observed in rats treated with cycloheximide. The effect of actinomycin D on prothrombin concentration in deficient rats was similar to that observed in normal animals. From the data presented in this report we conclude that the inhibition of prothrombin destruction is not the principal mechanism of action of vitamin K. The more rapid than expected appearance of prothrombin in the plasma after the injection of vitamin K into deficient rats suggests that an unsteady state is established at the outset of the action of the vitamin. This could be due to (1) the release of small amounts of stored prothrombin, (2) the completion of a stored prothrombin precursor (on or off ribosomes), (3) the activation of stored or accumulated mRNA for prothrombin biosynthesis. Although de novo protein synthesis appears to be ultimately involved in the maintenance of plasma prothrombin levels under the influence of vitamin K, quantitative studies of the degree of labeling of plasma prothrombin during the initiation of vitamin K action will be required to determine the extent to which vitamin K itself participates in the biosynthesis of prothrombin.

Role of vitamin A in induction of vitamin K deficiency in the rat.

Summary Evidence is presented to indicate that the level of dietary Vit. A is closely related to development of symptoms of Vit. K deficiency in the rat. Under appropriate experimental conditions, 0.5 and 5 i.u. of Vit. A per gram of diet gave reduced prothrombin concentrations which were not observed on corresponding rations deficient in Vit. A. This extends the previously known hemorrhagenic toxicity of Vit. A to physiological levels and emphasizes the need for careful consideration of the level of Vit. A in studies of Vit. K deficiency in the rat. The effect of Vit. A was more severe in male than in female rats. In further studies with male rats, Vit. A acid was markedly more hemorrhagenic than Vit. A acetate.

Effect of sex and sex hormones on plasma prothrombin and vitamin K deficiency.

Summary In rats as in man, pregnancy results in levels of prothrombin higher than observed in normal females. In the rat, prothrombin levels are higher in the female than in the male and the dietary requirement for vitamin K in the female is less. Higher levels of plasma prothrombin in the female are not due to decreased turnover of the protein. Estradiol does not have the activity of vitamin K or otherwise directly stimulate the formation of prothrombin. The effect of estradiol on vitamin K deficiency in male rats occurs slowly over a period of several days. The decreased requirement for vitamin K and the increased circulating levels of prothrombin in female or estrogen-treated rats may be due to separate effects of the hormone. We are indebted to medical students, Ralph Principe and Lynn Hill for their contribution to this report.

Intracellular distribution of vitamin K in the rat

The intracellular distribution of vitamin K in rat liver was studied with [6,7-3H2]-phylloquinone of high specific activity. After the injection of 3 μg of the labelled vitamin to normal or deficient rats, radioactivity was found principally in the microsomal fraction of liver while mitochondria, ribosomes and purified nuclei contained only small amounts of 3H. A similar distribution was observed in rats receiving 0.02 μg or fed a maintenance level of the radioactive vitamin. The administration of warfarin or 2-chloro-3-phytyl-I,4-naphthoquinone did not greatly affect the intracellular distribution observed after the injection of radioactive vitamin K. Chromatographic analysis of extracts of subcellular fractions revealed that 49% or more of the 3H in each fraction was present in metabolites more polar than phylloquinone. These findings do not substantiate proposals that vitamin K acts at the nuclear, mitochondrial or ribosomal level; however, radioactivity originally bound to ribosomes may be have been released by deoxycholate. The present data indicate that vitamin K and its principal metabolites in liver cells are associated with the endoplasmic reticulum.

Separation of vitamin K and associated lipids by reversed-phase partition column chromatography.

Abstract A reversed-phase partition chromatographic procedure is described that effectively separates isoprenologs of vitamin K and ubiquinone, other fat-soluble vitamins, and associated lipids. The method is adaptable to a wide range of fat-soluble substances and conveniently used for preparative purification.

Effect of Indigestible Oils on Vitamin K Deficiency in the Rat

Studies of indigestible oils such as mineral oil and squalene were undertaken to determine their effect on vitamin K deficiency in the rat. Severe hypo- prothrombinemia occurred in male rats fed a vitamin K-deficient diet containing as little as 0.5% of the oil and the effect was reversed by feeding vitamin K. Female rats were partially resistant to the treatment. Dietary squalene also interrupted the assimilation of vitamin K in chicks where 0.5% of dietary oil inhibited the utiliza tion of 0.1 fig of phylloquinone/g of diet. Similar results were obtained in chicks fed retinyl acetate and retinole acid.

Naturally Occurring Bile Acids and Alcohols and Their Origins

The first scientific phase of bile acid investigation began in the middle of the nineteenth century when these compounds were first obtained in crystalline form. Earlier observations of bile and its properties are often cited but the efforts of the early writers, Hippocrates, Galen, Paracelsus, and others, were confined to the subjective and speculative considerations of their age. The first scientific phase, characterized by uncertainty and confusion concerning the constituents of bile, terminated around the end of the nineteenth century and gave rise to a productive period during which much information was obtained. The beginning and improvements in methods of isolation and characterization of bile acids during this period provided a strong background of structural knowledge concerning these compounds. Studies were stimulated by the recognized relationship between the bile acids and cholesterol, and, later, the steroid hormones. The contributions of Wieland and his collaborators to the structure of the bile acids began in 1912 and are reviewed in Fieser’s monograph on the steroids (1).

Identification of phylloquinone in horse liver

Abstract Extraction of horse liver with acetone yielded lipid containing 3.3 μg/g of vitamin K as determined by chick bioassay. The fat-soluble vitamin was purified by silicic acid and partition column chromatography, and finally by thin-layer chromatography. The final product was identified as phylloquinone by ultraviolet spectroscopy and mass spectrometry. An isolation procedure using Claisens alkali confirmed the presence of phylloquinone in horse liver. Evidence for the presence of a more polar form of vitamin K was also obtained.

Characterization of vitamin K from pig liver and dog liver

Abstract Extraction of pig liver and dog liver with acetone yielded lipid containing the equivalent of 10 and 16.6 μg of phylloquinone/g, respectively, as determined by bioassay. The vitamins were purified by Chromatographic techniques and by extraction with Claisens alkali. Mass spectrometry of purified samples from pig liver gave molecular ions for MK-7, MK-7 (H 2 ), MK-8, MK-8 (H 2 ), MK-8 (H 4 ), MK-8 (H 6 ), MK-9, MK-9 (H 2 ), MK-9 (H 4 ), MK-9 (H 6 ), MK-10, and MK-10 (H 2 ). After chromatographic purification alone or after preparation of the vitamin with Claisens alkali, phylloquinone and 2-methyl-3-Δ6-dehydrophytyl-1,4-naphthoquinone were not detected. The maximum amount of these compounds which could have been present in pig liver but remain undetected was estimated to be 10–11% of the biological activity present in the extracted lipid. Mass spectrometry of the purified samples from dog liver gave molecular ions for MK-6, MK-6 (H 2 ), MK-7, MK-7 (H 2 ), MK-7 (H 4 ), MK-7 (H 6 ),MK-8,MK-8 (H 2 ),MK-8 (H 4 ),MK-8 (H 6 ),MK-9,MK-9 (H 2 ), MK-9 (H 4 ), MK-9 (H 6 ), MK-10, MK-10 (H 2 ), MK-11, MK-12, and MK-13. The principal forms present were MK-9, MK-10, and multiply saturated forms of MK-8 and MK-9.