Dominic J. Harrington

Compilation of a provisional UK database for the phylloquinone (vitamin K1) content of foods.

This paper reports the compilation of a food composition database for phylloquinone (vitamin K1) derived from the direct analysis of foods, recipe calculation and the assignment of values based on food similarities. All the basic and other food items used in these calculations had been analysed by HPLC and about 170 of the items had been obtained and assayed in the UK. Recipe calculations took account of the cooking method and changes in water and fat content. Currently, approximately 1501 food items with Royal Society of Chemistry/Ministry of Agriculture, Fisheries and Food food codes have been allocated a vitamin K1 value, and a further 282 new recipe codes are included in the database. Representative values from each food group are reported together with an indication of the potential variation. Detailed examples of some recipe calculations are included, and also the impact of changing the type of fat in recipes. Vitamin K1 is associated with, and most abundant in, photosynthetic tissues of plants. Accordingly, the highest concentrations (3000-6000 micrograms/kg) are found in dark-green leafy vegetables and herbs, such as kale, parsley, spinach and green cabbage. Intermediate concentrations (1000-2000 micrograms/kg) are found in plants with paler leaves such as white cabbage and lettuce or in green, non-leafy vegetables such as broccoli and brussel sprouts. Fats and oils contain variable amounts of vitamin K1 with the highest concentrations (300-1300 micrograms/kg) in soyabean, rapeseed and olive oils and the margarines based on them. Other foods such as dairy products, meat dishes and cereal-based foods (bread, biscuits, cakes, desserts etc.), although not in themselves particularly rich in vitamin K1 (< 200 micrograms/kg), may contribute significantly to intakes when consumption of green vegetables is poor. Within the scope of this present study, it has not been possible to address issues such as inter-sample variability, losses during storage or the bioavailability from different foods and further work on these aspects is needed.

Pharmacodynamic resistance to warfarin associated with a Val66Met substitution in vitamin K epoxide reductase complex subunit 1

The gene encoding vitamin K epoxide reductase complex subunit 1 (VKORC1), a component of the enzyme that is the therapeutic target site for warfarin, has recently been identified. In order to investigate the relationship betweenVKORC1 and warfarin dose response, we studied the VKORC1 gene (VKORC1) in patients with warfarin resistance. From a study group of 820 patients, we identified 4 individuals who required more than 25 mg of warfarin daily for therapeutic anticoagulation. Three of these had serum warfarin concentrations within the therapeutic range of 0.7-2.3 mg/l and showed wild-type VKORC1 sequence. The fourth warfarin resistant individual had consistently high (> or =5.7 mg/l) serum warfarin concentrations, yet had no clinically discernible cause for warfarin resistance. VKORC1 showed a heterozygous 196G-->A transition that predicted aVal66Met substitution in the VKORC1 polypeptide. This transition was also identified in 2 asymptomatic family members who had never received warfarin. These individuals had normal vitamin-K dependent coagulation factor activities and undetectable serum PIVKAII and vitamin K1 2,3 epoxide suggesting that their basal vitamin K epoxide reductase activity was not adversely affected by the VKORC1 Val66Met substitution. The association between a nucleotide transition in VKORC1 and pharmacodynamic warfarin resistance supports the hypothesis that VKORC1 is the site of action of warfarin and indicates thatVKORC1 sequence is an important determinant of the warfarin dose response.

Pharmacodynamic resistance to warfarin is associated with nucleotide substitutions in VKORC1

Summary.  Background: Vitamin K epoxide reductase subunit 1 (VKORC1) is the molecular target of coumarin anticoagulants and mutations in VKORC1 have been identified previously in individuals who required high warfarin doses. Objective: Detailed characterization of the relationship between variation in VKORC1 and the warfarin resistance phenotype. Patients and methods: Serum warfarin concentration and coagulation parameters were determined in 289 subjects who required warfarin doses >20 mg day−1. The VKORC1 sequence was studied in selected study subjects. Results: Twenty‐eight out of 289 (10%) subjects had serum warfarin >2.3 mg L−1 during stable therapeutic anticoagulation indicating pharmacodynamic warfarin resistance. Detailed analysis of 15 subjects from this group showed that eight out of 15 (53%) had nucleotide substitutions in VKORC1 predictive of p.V66M, p.L128R, p.V54L or p.D36Y. VKORC1 was normal in the remaining seven out of 15 (47%) subjects and in nine out of nine (100%) subjects with high warfarin dose requirement not caused by pharmacodynamic resistance. At referral, subjects with VKORC1 mutations received a median warfarin dose of 32 mg day−1 (range 22–55) and had a median serum warfarin concentration of 4.6 mg L−1 (range 2.6–9.0). VKORC1 substitutions were associated with a requirement for high warfarin doses but not with adverse clinical events. Family members with VKORC1 nucleotide substitutions and not receiving warfarin had undetectable PIVKA‐II and K1 epoxide (K1O). Conclusions: Nucleotide variations in VKORC1 are a common cause of pharmacodynamic warfarin resistance but are not associated with adverse outcome during anticoagulation. Mutations associated with warfarin resistance do not cause a discernible defect in VKORC1 reductase function.

Vitamin B12 deficiency

#### Summary points Vitamin B12 is an essential cofactor that is integral to methylation processes important in reactions related to DNA and cell metabolism, thus a deficiency may lead to disruption of DNA and cell metabolism and thus have serious clinical consequences.1 Intracellular conversion of vitamin B12 to two active coenzymes, adenosylcobalamin in mitochondria and methylcobalamin in the cytoplasm, is necessary for the homeostasis of methylmalonic acid and homocysteine, respectively.2 3 Methylmalonic acid is converted into succinyl-CoA, of which vitamin B12 is a cofactor for the reaction. Homocysteine is biosynthesised from methionine then resynthesised into methionine or converted into amino acid cysteine. Vitamin B12 (also referred to as cobalamin) deficiency is relatively common, with important and variable clinical consequences. This review presents a concise summary of the most up to date evidence on how to diagnose and manage vitamin B12 deficiency. #### Sources and selection criteria We searched PubMed and Google Scholar using the terms “vitamin B12 deficiency” and “cobalamin deficiency”, and hand selected the most relevant and appropriate articles. We also used evidence based guidelines from the British Committee for Standards in Haematology; however, evidence, especially in the form of randomised controlled trials, is lacking.4 Foods containing vitamin B12 are …

Plasma phylloquinone (vitamin K 1 ) concentration and its relationship to intake in a national sample of British elderly people

Plasma phylloquinone (vitamin K1) concentration was examined according to season, socio-demographic and lifestyle factors and phylloquinone intake in a nationally representative sample of British people aged 65 years and over from the 1994-5 National Diet and Nutrition Survey. Values for both plasma phylloquinone concentration and phylloquinone intake were available from 1076 participants (561 men, 515 women). Eight hundred and thirty-four were living in private households, 242 in residential or nursing homes. Weighted geometric mean plasma phylloquinone concentrations were 0.36 (inner 95% range [corrected] 0.06, 2.01) and 0.24 (inner 94% range [corrected] 0.06, 0.96) nmol/l in free-living and institution samples respectively. Plasma phylloquinone concentrations did not generally differ between men and women, although values in free-living people were significantly lower during autumn and winter (October to March). Plasma phylloquinone concentration was not significantly associated with age. Plasma phylloquinone concentrations were positively correlated with phylloquinone intake in free-living men and women (r 0.18 and 0.30 respectively, both P<0.001). Stepwise multiple regression analysis found that 11 % of the variation in plasma phylloquinone concentration was explained by phylloquinone intake, season and plasma triacylglycerol concentration. After adjustment for age and corresponding nutrient intakes, plasma phylloquinone concentration was significantly associated (each P<0.01) with plasma concentrations of triacylglycerol, cholesterol, retinol and 25-hydroxyvitamin D in free-living women but not men, and with plasma concentrations of carotenes, alpha- and gamma-tocopherols and lutein in free-living men and women. The possibility of concurrent low fat-soluble vitamin status in elderly populations may be a cause for concern.

An audit of holotranscobalamin (“Active” B12) and methylmalonic acid assays for the assessment of vitamin B12 status: Application in a mixed patient population

BACKGROUND Vitamin B12 insufficiency/deficiency is common in mixed patient populations. However there is no single marker which can reliably diagnose B12 insufficiency/deficiency. Elevated concentrations of methylmalonic acid (MMA) are considered the most representative marker of metabolic vitamin B12 insufficiency, but poor assay availability limits clinical utility. Low concentrations of serum vitamin B12 are often used to assess vitamin B12 status but this approach generates a high rate of false negative results. Emerging evidence indicates that holotranscobalamin (holoTC) may be a more reliable indicator of vitamin B12 status. AIMS AND METHODS We substituted serum vitamin B12 measurement with holoTC, supported by MMA in patients referred for assessment of vitamin B12 status. A service evaluation was undertaken of the pattern of MMA values obtained for patients with holoTC 25-50 pmol/L (an indeterminate result). MMA cut-offs of 280 and 360 nmol/L were applied for patients ≤ 65 or >65 years respectively. RESULTS A total of 4,175 consecutive patients were investigated and MMA was analysed for 19% of patients. The incidence of elevated MMA was 41% (holoTC, 25-29 pmol/L), 32% (30-34 pmol/L), 33% (35-39 pmol/L), 30% (40-44 pmol/L), and 26% (45-50 pmol/L). CONCLUSIONS Our results indicate that in the clinical setting a holoTC between 25 and 50 pmol/L is a poor predictor for the concentration of MMA provided the goal is to identify patients with MMA values above the limits used in the present study. Further studies are needed to evaluate to what extent holoTC <25 and >50 pmol/L reflect circulatory MMA concentrations.

The external quality assurance of phylloquinone (vitamin K1) analysis in human serum

The vitamin K external quality assurance scheme (KEQAS) aims to assist in the harmonization of phylloquinone (vitamin K(1)) analysis in order to improve the comparability of clinical and nutritional studies. Serum samples were despatched to 17 groups from eight countries during 2000-2006. Using pilot data (1996-1999), an analytical performance target of 20% absolute difference from the all-laboratory trimmed mean (ALTM) was assigned and formed the basis for interlaboratory comparison. Assay specificity, analytical bias and assay performance were evaluated. From 21 batches of samples distributed, 414 results were reported of which 2.7% were outliers. The mean interlaboratory absolute difference from the ALTM was 21.7% with 47% of groups consistently meeting the performance target. The mean interlaboratory coefficient of variation was 29.6%. The false positive rate for phylloquinone depleted samples was high at 35%. Bias was found to be independent of HPLC-detector type (fluorescence vs electrochemical). Assay characteristics for the measurement of phylloquinone in human serum compare favourably with methods for analytes at equivalent concentrations. The high proportion of false positive results suggest that poor assay specificity at low phylloquinone concentrations is a common problem, which in the clinical setting could lead to underreporting of vitamin K deficiency.

Vitamin K metabolism: Current knowledge and future research

Vitamin K is an essential fat-soluble micronutrient that is required for the post-translational γ-carboxylation of specific glutamic acid residues in hepatic and extra-hepatic proteins involved in blood coagulation and preventing cartilage and vasculature calcification. In humans, sources of vitamin K are derived from plants as phylloquinone and bacteria as the menaquinones. Menadione is a synthetic product used as a pharmaceutical but also represents an intermediate in the tissue-specific conversion of vitamin K to menaquinone-4, which preferentially resides in tissues such as brain. Research into vitamin K metabolism is essential for the understanding of vitamin K biology in health and disease. Progress in this area, driven by knowledge of vitamin K and the availability of markers of vitamin K status, has already proved beneficial in many areas of medicine and further opportunities present themselves. Areas of interest discussed in this review include prophylactic administration of vitamin K1 in term and preterm neonates, interactions between vitamins K and E, the industrial conversion of vitamin K to dihydro-vitamin K in foods, tissue-specific conversion of vitamin K to menaquinone-4, the biological activity of the five and seven carbon metabolites of vitamin K and circadian variations.

Vitamin E decreases extra-hepatic menaquinone-4 concentrations in rats fed menadione or phylloquinone.

SCOPE The mechanism for increased bleeding and decreased vitamin K status accompanying vitamin E supplementation is unknown. We hypothesized that elevated hepatic α-tocopherol (α-T) concentrations may stimulate vitamin K metabolism and excretion. Furthermore, α-T may interfere with the side chain removal of phylloquinone (PK) to form menadione (MN) as an intermediate for synthesis of tissue-specific menaquinone-4 (MK-4). METHODS AND RESULTS In order to investigate these hypotheses, rats were fed phylloquinone (PK) or menadione (MN) containing diets (2 μmol/kg) for 2.5 weeks. From day 10, rats were given daily subcutaneous injections of either α-T (100 mg/kg) or vehicle and were sacrificed 24 h after the seventh injection. Irrespective of diet, α-T injections decreased MK-4 concentrations in brain, lung, kidney, and heart; and PK in lung. These decreases were not accompanied by increased excretion of urinary 5C- or 7C-aglycone vitamin K metabolites, however, the urinary α-T metabolite (α-CEHC) increased ≥ 100-fold. Moreover, α-T increases were accompanied by downregulation of hepatic cytochrome P450 expression and modified expression of tissue ATP-binding cassette transporters. CONCLUSION Thus, in rats, high tissue α-T depleted tissue MK-4 without significantly increasing urinary vitamin K metabolite excretion. Changes in tissue MK-4 and PK levels may be a result of altered regulation of transporters.

Urinary excretion of vitamin K metabolites in term and preterm infants: relationship to vitamin K status and prophylaxis.

Little is known about the metabolic turnover and excretion of vitamin K in healthy newborn infants and the metabolic consequences of prophylactic regimens designed to protect against vitamin K deficiency bleeding (VKDB). We measured the excretion of two urinary metabolites (≤24 h) of vitamin K (5C- and 7C-aglycones) in term infants before (n = 11) and after (n = 5) a 1000 μg i.m. dose of vitamin K1 (K1) and in preterm infants after 200 μg i.m. (n = 4), 500 μg i.m. (n = 4), or 200 μg i.v. (n = 5). In preterm infants, we also measured serum K1, vitamin K1 2,3-epoxide, and PIVKA-II at 5 d postpartum. Before prophylaxis, the rate of 5C- and 7C-aglycone excretion was 25 times lower than adults, reflecting low vitamin K stores at birth. After prophylaxis, the excretion rate correlated to K1 dose (r = 0.6) but was two orders of magnitude lower than that in adults, probably reflecting the immaturity of neonatal catabolism. All term and 10 of 13 preterm infants mainly excreted 5C-aglycone. We present evidence that increased excretion of the 7C-aglycone was associated with metabolic overload because of the exposure to high-tissue K1 concentrations. Measurement of the 5C- and 7C-aglycones may facilitate longitudinal studies of vitamin K status in neonates and aid the development of improved prophylactic regimens.