Vitamin K-active substances

The classical method for the biological testing of vitamin K-active substances is the measuring of their effect on the blood clotting time of the vitamin K-deficient chick. Dam et al. (1951) have described a modified method, by which a linear relationship between vitamin K dosage and clotting time was obtained. 

Phthiocol was isolated from tubercle bacilli (269a) and since the Johne factor was soluble in fat solvents and water, Woolley and McCarter tested phthiocol for activity. The vitamin K activity of phthiocol suggested a test of other vitamin-K-active substances, and a highly potent concentrate of vitamin K, and also the simple 2-methyl-l, 4-naphthoquinone were thus found to be active for Johne s bacillus. 

Thus vitamin-K-active substances, of which phthiocol is a natural... 

Several substances having vitamin K. activity have been isolated from natural sources. Vitamin Ki from alfalfa oil, is 2-methyl-3-phytyl-1,4-naphthoquinone. 

Vitamin K activity is associated with at least two distinct natural substances, designated as vitamin K, and vitamin Kj. Vitamin K or phylloquinone (phytonadione) is 2-methyl-3-phytyl-l,4-naphthoquinone it is found in plants and is the only natural vitamin K available for therapeutic use. Vitamin K is actually a series of compounds (the mena-quinones) in which the phytyl side chain of phylloquinone has been replaced by a side chain built up of 2 to 13 prenyl units. Considerable synthesis of menaquinones occurs in Gram-positive bacteria indeed, intestinal flora synthesize the large amounts of vitamin K contained in human and animal feces. In animals menaquinone-4 can be synthesized from the vitamin precursor menadione (2-methyl-l,4-naphtho-quinone), or vitamin Kj. Depending on the bioassay system used, menadione is at least as active on a molar basis as phylloquinone. 

Vitamin K activity is associated with at least two distinct natural substances, designated as vitamin A j and vitamin 2- Vitamin or phylloquinone (phytonadione), is 2-methyl-3-phytyl-I,... 

AU naturally occurring compounds that show vitamin K activity (the coagulation vitamin) are derivatives of menadione (2-methyl-1,4-naphthoquinone) with an isoprenoid unsaturated side chain at the C-3 position of the aromatic ring. Today, essentially two types of active substances are recognised. The other compounds with vitamin K activity are synthetic derivatives. 

Vitamin K confers biologic activity upon prothrombin and factors VII, IX, and X by participating in their postribosomal modification. Vitamin K is a fat-soluble substance found primarily in leafy green vegetables. The dietary requirement is low, because the vitamin is additionally synthesized by bacteria that colonize the human intestine. Two natural forms exist vitamins Ki and K2. Vitamin K1 (phytonadione Figure 34-5) is found in food. Vitamin K2 (menaquinone) is found in human tissues and is synthesized by intestinal bacteria. 

Table 6.1 lists the water-soluble vitamins with their structures and coenzyme forms. Certain portions of the coenzymes are especially important in their biological activities, and they are indicated by arrows. For example, in case of coenzyme A, a thiol ester is formed between its -SH residue and the acyl group being transferred. And in the case of pyridoxal phosphate, its carbonyl residue forms a Schiff base with the amino group of the amino acid that is being decarboxylated. Fat-soluble vitamins (Table 6.2) are also transformed into biologically active substances. However, with the possible exception of vitamin K, these do not operate as prosthetic groups or cosubstrates in specific enzyme reactions. 

Active substances that are marketed in the pre-granulated form for direct compression are almost always substances that are difficult to press into tablets and/ or are prone to hydrolysis. Typical examples are the vitamins and acetaminophen [540]. Table 68 contains details on the production of ascorbic acid for direct compression, which is granulated with povidone K 30 as binder in a fluidized bed granulator. 

Unbound factors Ha, IXa, Xa, XIa, and Xlla are inactivated by antithrombin when they migrate to the endothelial cell surface. Heparin and heparin-hke substances present on the surface of endothelial cells enhance the inhibitory capacity of antithrombin. Thrombomodulin binds thrombin and activates protein C. Activated protein C and its cofactor, protein S, are vitamin K-dependent proteins... 

Once the nature of the unsaturation in the C17 chain was established, we considered if this structural feature had an active role in function or could we say nature has just been too lazy to reduce these double bonds to give a saturated polyisoprenoid group. Possibly related in function are the similarly unsaturated, though frequently much longer, isoprenoid chains of ubiquinones, vitamin K s, and related compounds, substances also implicated in electron-transfer and/or oxidative phosphorylation processes. Thus, the C17 group in heme A may be directly related to either electron transfer or coupling of phosphorylation in the oxidase. 

Not only substances of the tocopherol series, but also dimethyl- and trimethylbenzoquinones with isoprenoid side chains, ubiquinones, and members of the vitamin K family were effective in preventing respiratory decline. The activity of these substances, all of them supplemented in the... 

The fact that synthetic, substituted benzoquinones, and also substances of the vitamin K series and ubiquinones are similar in activity to vitamin E or its oxidation products constitutes another indication against the antioxidant hypothesis of vitamin E action. It would hardly be feasible to class all the active compounds as antioxidants. 

Vitamin E is in the NADH cytochrome c reductase system. When the system was extracted with isoectane, NADEl oxidation was blocked normal oxidation rates can be restored by adding a-tocopherol, but many other lipid substances are also active in that respect e.g., vitamin K, ubiquinone, phytol. Of course, such observations do not exclude the possibility that in vivo vitamin E is in fact the only substance capable of maintaining the integrity of the NADH cytochrome c reductase system. 

There is little doubt that the sjanptoms produced by vitamin K deficiency and by poisoning with Dicumarol and related substances are very similar. In both cases, administered vitamin Ki acts by normalizing the prolonged blood clotting time. Menadiones, however, are only as active as vitamin Ki in nutritional vitamin Ki deficiency and are almost without effect as antidotes to Dicumarol. 

Assuming therefore that only vitamin K substances with a typical side chain, as in vitamin Ki and the forms of vitamin Kj, are active in metabolism, the above-mentioned differences in the biological activity of vitamin Ki and of menadione can be explained. Menadiones, necessary only in very small doses to cure nutritional vitamin K deficiency, may be quantitatively transformed into the active vitamin Ka form. This bio thesis, being limited, however, as described in Section VI, is insufficient to provide the high amount of metabolically active vitamin Kj needed to counteract Dicumarol poisoning. 

A criterion for a substance to be a member of the respiratory chain is its ability to reactivate an enzymatic system from which this component has been removed. Such a system has been described by Nason and Lehman (1956), who used a rat skeletal muscle cytochrome c reductase inactivated by extraction with isooctane. In such a test vitamin E (Nason and Lehman, 1956) and vitamin K (Deul et ah, 1958) were found to be active. In an extensive study Weber et ah (1958a, b) showed, however, that this reactivation is not related to the redox system but to the isoprene-like side chain. Esterified tocopherol and the diacetates of the dihydro forms of vitamin Ki, Kj, and of ubiquinone(50) are as active as the free forms. Moreover, isolated side chains, such as phytol and squalene, showed exactly the same activity as the corresponding menadione derivatives, compared on a molar basis. The authors speculate that in this experiment a special function of the isoprene-like side chain, namely the binding of this vitamin to the fat material of the mitochondria, is demonstrated. 

Vitamin K3 (menadione, MK-0,2-methyl-1,4-naphthoquinone) is a synthetic substance. The product of menadione reduction, known as menadiol (2-methylnaphthalene-l,4-diol), and derived compounds, such as the fat-soluble menadiol diacetate or menadiol dibutyrate and the water-soluble sodium salt of menadiol diphosphate, are referred to as vitamin K4. Activity of vitamin K was also detected in the synthetic monoamino analogues and diamino analogues of menadiol, for example, in l-amino-4-hydroxy-3-methylnaphthalene (vitamin K5), l,4-diamino-2-methylnaphthalene (vitamin Kg) and l-amino-4-hydroxy-2-methylnaphthalene (vitamin K ). 

Meat and meat products have a moderately high vitamin K content. Liver is high in vitamin K (Table 5.7). In pigs liver, for instance, more than ten active substances have been identified, of which vitamin Kj o) (0.012 mg/kg), MK-4 (0.011 mg/kg), MK-7 (0.016 mg/kg), MK-8 (0.025 mg/kg), MK-9 (0.006 mg/kg) and MK-10 (0.008 mg/kg) occur in significant quantities. Menaquinones MK-10 to MK-13, originally synthesised by bacteria in the rumen and subsequently absorbed, have been found in beef liver. 

It is essential for a successful assay that the vitamins be quantitatively extracted from the food matrix in a form that can be accurately measured by the particular HPLC technique to be used. An effective extraction procedure serves to homogenize and concentrate the sample, isolate the vitamin analyte from its association with protein, eliminate as far as possible known interfering substances, and destroy any indigenous enzyme activity. The vitamin-rich fraction thus obtained may require some form of cleanup before the vitamins can be measured, particularly when measuring the trace amounts of naturally occurring vitamins D and K. 

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