The human vitamin K requirement is not known precisely, but the minimal requirement is often assumed to be between 0.5 and 1 x g/kg body weight. In the present study we addressed the question to what extent circulating vitamin K concentrations are influenced by the form in which the vitamer is consumed.The experimental group consisted of five healthy volunteers who received phylloquinone after an overnight fast. On the first day of three successive weeks the participants consumed 1 mg (22 pmol) phylloquinone, either in the form of a pharmaceutical preparation (Konakion"), or in the form of spinach + butter, or as spinach without added fat. Circulating phylloquinone levels after spinach with and without butter were substantially lower (75-and 24.3-fold respectively) than those after taking the pharmaceutical concentrate. Moreover, the absorption of phylloquinone from the vegetables was 1.5 times slower than from Konakion. In a second experiment in the same five volunteers it was shown that relatively high amounts of menaquinone-4 enter the circulation after the consumption of butter enriched with this vitamer. It is concluded that the bioavailability of membrane-bound phylloquinone is extremely poor and may depend on other food components, notably fat. The bioavailability of dietary vitamin K (phylloquinone + menaquinones) is lower than generally assumed, and depends on the form in which the vitamin is ingested. These new insights may lead to a revision of the recommended daily intake for vitamin K.

Vitamin K is involved in blood coagulation and in bone metabolism via the carboxylation of glutamate residues in (hepatic) blood coagulation factors and (osteoblastic) bone proteins. The bioavailability of nutritional vitamin K depends on the type of food, the dietary fat content, the length of the aliphatic side chain in the K-vitamer and probably also the genetically determined polymorphism of apolipoprotein E. Although undercarboxylation of blood coagulation factors is very rare, undercarboxylated osteocalcin (bone Gla-protein) is frequently found in postmenopausal women. Supplementation of these women with extra vitamin K causes the markers for bone formation to increase. In parallel, a decrease of the markers for bone resorption is frequently seen. Insufficient data are available to conclude that the regular administration of vitamin K concentrates will reduce the loss of bone mass in white women at risk for developing postmenopausal osteoporosis.

SummaryOsteocalcin (bone Gla-protein) is a vitamin K-dependent protein synthesized by osteoblasts. Its hydroxylapatite binding capacity (HBC) is generally used to estimate the Gla-content of circulating osteocalcin. Here we have used the HBC of serum osteocalcin as a marker for the vitamin K-status in pregnant women and their offspring. For all cases investigated the HBC values in the cord samples were substantially lower than in the corresponding maternal ones. Babies from mothers who had been treated with vitamin K during the last 6 weeks prior to delivery, had significantly higher HBC values than those from a placebo group. The results presented in this paper are indicative for a generally occurring vitamin K deficiency in newborns. At delivery the HBC in untreated women was low as well. In both the placebo- and the vitamin K-group a good correlation was found between the HBC values in paired samples from mother and child. Whether the maternal HBC value may be used as a prenatal marker for estimating the fetal vitamin K-status remains to be seen.

Abstract-Matrix Gla protein (MGP) is synthesized in a vitamin K-dependent way in smooth muscle cells of the healthy vessel wall, and its mRNA transcription is substantially upregulated in atherosclerotic lesions. Here we report the preparation of a monoclonal antibody against human MGP and its use in an enzyme-linked immunosorbent assay. The intra-assay and interassay coefficients of variation in serum samples were 5.4% and 12.6%, respectively, and the lower detection limit was 8.5% of the normal serum value. Individual within-day variations were Ͻ11% and did not show a distinct circadian pattern. Day-to-day variations in fasting morning samples were Ͻ8%. In a first explorative survey, serum MGP concentrations were found to be significantly increased in patients with severe atherosclerosis, whereas these values were normal in those with low bone mass and osteoporosis. This finding is consistent with the high MGP mRNA expression observed in atherosclerotic vessels and plaques. More elaborate studies are required to assess the potential clinical utility of this newly developed assay.

In this article we describe the isolation of a 4-carboxyglutamic acid (Gla)-containing protein from calcified human atherosclerotic plaques. The protein was extracted from pulverized calcified plaques by demineralization with ethylenediaminetetraacetate and was subsequently purified by anion-exchange fast protein and highperformance liquid chromatography by using ion-exchange and gel-filtration columns. The protein was designated as plaque Gla protein (PGP) and has an apparent mass of 23 kD as estimated from sodium dodecyl sulfate-polyacrylamide gel analysis. By determining the sequence of its first six amino acid residues, the protein was unequivocally demonstrated to be not related to any other known protein.Moreover, no immunological relationship (as tested by Western blot analysis) was found between PGP and other known Gla-containing proteins. (Arteriosclerosis 10:991-995, November/December 1990)

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In a subgroup of postmenopausal women vitamin K induced a decrease of the urinary calcium loss. This effect was significant (P < 0.0001) in the so-called fast losers of calcium (calcium/creatinine ratio > 0.5). To find out whether vitamin K antagonists would have an opposite effect, a study was started among 141 persons on long-term oral anticoagulant therapy. In this population the number of fast losers was recorded, and compared to that in a group of age- and sex-matched non-treated controls. Notably in young men the fraction of fast losers was significantly higher in the anticoagulant-treated group than in the control group (25 v 0%, P < 0.02). Differences between treated and nontreated groups may also be found in other markers for calcium and bone metabolism, notably in serum osteocalcin concentration and in urinary hydroxyproline excretion. The conclusion of our study is that oral anticoagulant treatment must be regarded as a potential risk factor for a high loss of urinary calcium.

Given the growing interest in ascorbic acid (AA), there is a need for a reliable and reproducible method to measure AA status in the human body. Serum AA concentrations do not correlate well with tissue levels, but AA levels in leukocytes do. However, a standard method for clinical application is lacking. This present study describes a method to measure AA in the peripheral blood mononuclear cells (PBMCs) with hydrophilic interaction liquid chromatography (HILIC). The method can also be used in plasma and other leukocyte subsets. The measurements of AA in PBMCs and plasma were performed with HPLC with HILIC separation and UV detection. The sample preparation involved the isolation of PBMCs and lysis and precipitation with acetonitrile. European Medicine Agency guidelines for bioanalytic method validation were followed for the evaluation. A highly precise execution of the method was found with intra- and inter-assay variations at a maximum of 7.8%. In 40 healthy donors, a mean intracellular AA concentration of 7.9 microgram/108 cells was found in PBMCs. A correlation between plasma and PBMC AA concentration was not present (r = 0.22). In conclusion, we developed a convenient, reliable, and reproducible method for the quantitative determination of AA within PBMCs and plasma from human blood.