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BACKGROUND: There is near-global consensus that all newborns be given parenteral vitamin K1 (VK1 ) at birth as prophylaxis against VK deficiency bleeding (VKDB). Breastmilk has a low VK content and cases of late VKDB are reported in exclusively breastmilk-fed preterm infants despite VK prophylaxis at birth.OBJECTIVES: To assess the prevalence of functional VK insufficiency in preterm infants based on elevated under-γ-carboxylated (Glu) species of Gla-proteins, factor II (PIVKA-II) and osteocalcin (GluOC), synthesized by liver and bone respectively.PATIENTS/METHODS: Prospective, multi-center, observational study in preterm infants born RESULTS: Post discharge, breastmilk-fed babies had significantly lower serum VK1 (0.15 vs. 1.81 μg/L), higher PIVKA-II (0.10 vs. 0.02 AU/mL) and higher %GluOC (63.6% vs. 8.1%) than those receiving a formula/mixed-feed diet. Pre-discharge (based on elevated PIVKA-II), only 1 (2%) of 45 breastmilk-fed infants was VK insufficient. Post-discharge, 8 (67%) of 12 exclusively breastmilk-fed babies were VK insufficient versus only 1 (4%) of 25 formula/mixed-fed babies.CONCLUSIONS: Preterm infants who remain exclusively or predominantly human breastmilk-fed post neonatal unit discharge are at high risk of developing subclinical VK deficiency in early infancy. Routine post-discharge VK1 supplementation of breastfed infants to provide intakes comparable to those from formula milks should prevent this deficiency.

Vitamin K deficiency bleeding (VKDB) is a rare haemostatic disorder that occurs in the first 6 months of life and is subclassified, according to age of presentation, as either early, classical or late VKDB. Late VKDB (day 8 onwards) is the most insidious form, typically presenting between 3 weeks and 8 weeks in exclusively breastfed infants as an intracranial haemorrhage (ICH). Estimations of the prevalence of ICH as the first presenting symptom of late VKDB range from 40% to 80% in different populations1 and so preventing it is a major objective of global healthcare. Vitamin K prophylaxis was first introduced in the 1940s for the treatment and prevention of bleeding in the first week of life (classical VKDB). Because the early use of menadione (vitamin K3) often provoked neonatal haemolytic anaemia and kernicterus, it was replaced in the late 1950s by synthetic phylloquinone (vitamin K1), the major dietary form of vitamin K. By 1961, the American Academy of Pediatrics had concluded that a single intramuscular dose of 0.5–1.0 mg (or oral dose of 1.0–2.0 mg) vitamin K1 was ‘probably adequate for prophylaxis’. In Europe, the 1970s saw a decline in universal vitamin K prophylaxis in favour of selective prophylaxis for infants considered at risk (eg, preterms), a trend no doubt influenced by a supportive 1978 Editorial in the Lancet . This all changed with a spate of case reports from across the world (particularly in the Far East) of what came to be defined as late VKDB …

Warfarin, acenocoumarol, phenprocoumon, and fluindione are commonly prescribed oral anticoagulants for the prevention and treatment of thromboembolic disorders. These anticoagulants function by impairing the biosynthesis of active vitamin K–dependent coagulation factors through the inhibition of vitamin K epoxide reductase (VKOR). Genetic variations in VKOR have been closely associated with the resistant phenotype of oral anticoagulation therapy. However, the relative efficacy of these anticoagulants, their mechanisms of action, and their resistance variations among naturally occurring VKOR mutations remain elusive. Here, we explored these questions using our recently established cell-based VKOR activity assay with the endogenous VKOR function ablated. Our results show that the efficacy of these anticoagulants on VKOR inactivation, from most to least, is: acenocoumarol > phenprocoumon > warfarin > fluindione. This is consistent with their effective clinical dosages for stable anticoagulation control. Cell-based functional studies of how each of the 27 naturally occurring VKOR mutations responds to these 4 oral anticoagulants indicate that phenprocoumon has the largest resistance variation (up to 199-fold), whereas the resistance of acenocoumarol varies the least (

BACKGROUND: Phylloquinone is the primary form of vitamin K in the diet and circulation. Large intra- and interindividual variances in circulating phylloquinone have been partially attributed to age. However, little is known about the nondietary factors that influence phylloquinone absorption and metabolism. Similarly, it is not known if phylloquinone absorption is altered by the individual's existing vitamin K status. OBJECTIVE: The purpose of this secondary substudy was to compare plasma response with deuterium-labeled phylloquinone intake in older and younger adults after dietary phylloquinone depletion and repletion. METHODS: Forty-two older [mean ± SD age: 67.2 ± 8.0 y; body mass index (BMI; in kg/m(2)): 25.4 ± 4.6; n = 12 men, 9 women] and younger (mean ± SEM age: 31.8 ± 6.6 y; BMI: 25.5 ± 3.3; n = 9 men, 12 women) adults were maintained on sequential 28-d phylloquinone depletion (∼10 µg phylloquinone/d) and 28-d phylloquinone repletion (∼500 µg phylloquinone/d) diets. On the 23rd d of each diet phase, participants consumed deuterated phylloquinone-rich collard greens ((2)H-phylloquinone). Plasma and urinary outcome measures over 72 h were compared by age group, sex, and dietary phase via 2-factor repeated-measures ANOVA. RESULTS: The plasma (2)H-phylloquinone area under the curve (AUC) did not differ in response to phylloquinone depletion or repletion, but was 34% higher in older than in younger adults (P = 0.02). However, plasma (2)H-phylloquinone AUC was highly correlated with the serum triglyceride (TG) AUC (r(2)( )= 0.45). After adjustment for serum TG response, the age effect on the plasma (2)H-phylloquinone AUC was no longer significant. CONCLUSIONS: Plasma (2)H-phylloquinone response did not differ between phylloquinone depletion and repletion in older and younger adults. The age effect observed was explained by the serum TG response and was completely attenuated after adjustment. Plasma response to phylloquinone intake, therefore, seems to be a predominantly lipid-driven effect and not dependent on existing vitamin K status. More research is required to differentiate the effect of endogenous compared with exogenous lipids on phylloquinone absorption. This trial was registered at clinicaltrials.gov as NCT00336232.

Vitamin K may affect bone mineral density and fracture incidence. Since publication of a previous systematic review the integrity of some of the previous evidence has been questioned and further trials have been published. Therefore an update to the systematic review was required.This systematic review was designed to assess the effectiveness of oral vitamin K supplementation for increasing bone mineral density and reducing fractures in adults.MEDLINE, EMBASE, CENTRAL, CINAHL, clinicaltrials.gov, and WHO-ICTRP were searched for eligible trials. Randomised controlled trials assessing oral vitamin K supplementation that assessed bone mineral density or fractures in adult populations were included. A total of 36 studies were identified. Two independent reviewers extracted data using a piloted extraction form.For post-menopausal or osteoporotic patients, meta-analysis showed that the odds of any clinical fracture were lower for vitamin K compared to controls (OR, 0.72, 95%CI 0.55 to 0.95). Restricting the analysis to low risk of bias trials reduced the OR to 0.76 (95%CI, 0.58 to 1.01). There was no difference in vertebral fractures between the groups (OR 0.96, 95%CI 0.83 to 1.11). In the bone mineral density meta-analysis, percentage change from baseline at the lumbar spine was higher at 1 year (MD 0.93, 95%, CI - 0.02 to 1.89) and 2 years (MD 1.63%, 95%CI 0.10 to 3.16) for vitamin K compared to controls; however, removing trials at high risk of bias tended to result in smaller differences that were not statistically significant. At 6 months, it was higher in the hip (MD 0.42%, 95%CI 0.01 to 0.83) and femur (MD 0.29%, 95%CI 0.17 to 0.42). There was no significant difference at other anatomical sites.For post-menopausal or osteoporotic patients, there is no evidence that vitamin K affects bone mineral density or vertebral fractures; it may reduce clinical fractures; however, the evidence is insufficient to confirm this. There are too few trials to draw conclusions for other patient groups.

Vitamin K (VK) is an essential cofactor for the post-translational conversion of peptide-bound glutamate to γ-carboxyglutamate. The resultant vitamin K–dependent proteins are known or postulated to possess a variety of biological functions, chiefly in the maintenance of hemostasis. The vitamin K cycle is a cellular pathway that drives γ-carboxylation and recycling of VK via γ-carboxyglutamyl carboxylase (GGCX) and vitamin K epoxide reductase (VKOR), respectively. In this review, we show how novel molecular biological approaches are providing new insights into the pathophysiological mechanisms caused by rare mutations of both GGCX and VKOR. We also discuss how other protein regulators influence the intermediary metabolism of VK, first through intestinal absorption and second through a pathway that converts some dietary phylloquinone to menadione, which is prenylated to menaquinone-4 (MK-4) in target tissues by UBIAD1. The contribution of MK-4 synthesis to VK functions is yet to be revealed.

Vitamin K deficiency bleeding (VKDB) in infancy is a serious but preventable cause of mortality or permanent disability. Lack of epidemiologic data for VKDB in sub-Saharan Africa hinders development and implementation of effective prevention strategies. We used convenience sampling to consecutively enroll mothers delivering in a southwestern Uganda Hospital. We collected socio-demographic and dietary information, and paired samples of maternal venous and neonatal cord blood for the immunoassay of undercarboxylated prothrombin (PIVKA-II), a sensitive marker of functional vitamin K (VK) insufficiency. We used univariable and multivariable logistic regression models to identify predictors of VK insufficiency. We detected PIVKA-II of ≥0.2 AU (Arbitrary Units per mL)/mL (indicative of VK insufficiency) in 33.3% (47/141) of mothers and 66% (93/141) of newborns. Importantly, 22% of babies had PIVKA-II concentrations ≥5.0 AU/mL, likely to be associated with abnormal coagulation indices. We found no significant predictors of newborn VK insufficiency, including infant weight (AOR (adjusted odds ratio) 1.85, 95% CI (confidence interval) 0.15-22.49), gender (AOR 0.54, 95% CI 0.26-1.11), term birth (AOR 0.72, 95% CI 0.20-2.62), maternal VK-rich diet (AOR 1.13, 95% CI 0.55-2.35) or maternal VK insufficiency (AOR 0.99, 95% CI 0.47-2.10). VK insufficiency is common among mothers and newborn babies in southwestern Uganda, which in one fifth of babies nears overt deficiency. Lack of identifiable predictors of newborn VK insufficiency support strategies for universal VK prophylaxis to newborns to prevent VKDB.

In contrast to other fat-soluble vitamins, dietary vitamin K is rapidly lost to the body resulting in comparatively low tissue stores. Deficiency is kept at bay by the ubiquity of vitamin K in the diet, synthesis by gut microflora in some species, and relatively low vitamin K cofactor requirements for γ-glutamyl carboxylation. However, as shown by fatal neonatal bleeding in mice that lack vitamin K epoxide reductase (VKOR), the low requirements are dependent on the ability of animals to regenerate vitamin K from its epoxide metabolite via the vitamin K cycle. The identification of the genes encoding VKOR and its paralog VKOR-like 1 (VKORL1) has accelerated understanding of the enzymology of this salvage pathway. In parallel, a novel human enzyme that participates in the cellular conversion of phylloquinone to menaquinone (MK)-4 was identified as UbiA prenyltransferase-containing domain 1 (UBIAD1). Recent studies suggest that side-chain cleavage of oral phylloquinone occurs in the intestine, and that menadione is a circulating precursor of tissue MK-4. The mechanisms and functions of vitamin K recycling and MK-4 synthesis have dominated advances made in vitamin K biochemistry over the last five years and, after a brief overview of general metabolism, are the main focuses of this review.

Osteocalcin (OC) is a vitamin K-dependent protein found in bone and in circulation. High serum γ-carboxylated OC reflects a high, and high uncarboxylated OC (ucOC) reflects a low vitamin K status. A revolutionary hypothesis is that ucOC acts as a hormone improving glucose handling and reducing fat mass. The objective was to test the logical extrapolation of the ucOC hormone hypothesis to humans that elevated ucOC is associated with higher body weight, BMI and fat mass. In a cross-sectional analysis, the associations of vitamin K status with circulating adiponectin and body composition were investigated in 244 postmenopausal women (study I). The effects of vitamin K treatment on adiponectin, body weight and BMI were investigated in archived samples from forty-two young men and women who received varying doses of menaquinone-7 during 12 weeks (study II) and from a cohort of 164 postmenopausal women who participated in a 3-year placebo-controlled trial on 45 mg menaquinone-4 (MK-4) (study III). No association was found between vitamin K status and circulating adiponectin before or after vitamin K supplementation. A higher carboxylation of OC was significantly correlated with lower body weight, BMI and fat mass of the trunk. Women taking MK-4 maintained their baseline body weight and BMI, whereas women taking placebo showed significant increases in both indices. These findings demonstrate that a high vitamin K status of bone has no effect on circulating adiponectin in healthy people and long-term vitamin K supplementation does not increase weight in healthy postmenopausal women.

In 2001, the US Food and Nutrition Board concluded that there were insufficient data with which to establish a RDA for vitamin K, in large part because of a lack of robust endpoints that reflected adequacy of intake. Knowledge of the relative bioavailability of multiple vitamin K forms was also poor. Since then, stable isotope methodologies have been applied to the assessment of the bioavailability of the major dietary form of vitamin K in its free state and when incorporated into a plant matrix. There is a need for stable isotope studies with enhanced sensitivity to expand knowledge of the bioavailability, absorption, disposition, and metabolism of different molecular forms of vitamin K. Another area for future research stems from evidence that common polymorphisms or haplotypes in certain key genes implicated in vitamin K metabolism might affect nutritional requirements. Thus far, much of this evidence is indirect via effects on warfarin dose requirements. In terms of clinical endpoints, vitamin K deficiency in early infancy continues to be a leading cause of intracranial bleeding even in developed countries and the reasons for its higher prevalence in certain Asian countries has not been solved. There is universal consensus for the need for vitamin K prophylaxis in newborns, but the effectiveness of any vitamin K prophylactic regimen needs to be based on sound nutritional principles. In contrast, there is still a lack of suitable biomarkers or clinical endpoints that can be used to determine vitamin K requirements among adults.

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.

The vitamin K external quality assurance scheme (KEQAS) aims to assist in the harmonization of phylloquinone (vitamin K1) 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. Copyright © 2009 John Wiley & Sons, Ltd.

Vitamin K (as phylloquinone and menaquinones) is an essential cofactor for the conversion of peptide-bound glutamate to gamma-carboxy glutamic acid (Gla) residues in a number of specialized Gla-containing proteins. The only unequivocal deficiency outcome is a bleeding syndrome caused by an inability to synthesize active coagulation factors II, VII, IX, and X, although there is growing evidence for roles for vitamin K in bone and vascular health. An adult daily intake of about 100 microg of phylloquinone is recommended for the maintenance of hemostasis. Traditional coagulation tests for assessing vitamin K status are nonspecific and insensitive. Better tests include measurements of circulating vitamin K and inactive proteins such as undercarboxylated forms of factor II and osteocalcin to assess tissue and functional status, respectively. Common risk factors for vitamin K deficiency in the hospitalized patient include inadequate dietary intakes, malabsorption syndromes (especially owing to cholestatic liver disease), antibiotic therapy, and renal insufficiency. Pregnant women and their newborns present a special risk category because of poor placental transport and low concentrations of vitamin K in breast milk. Since 2000, the Food and Drug Administration has mandated that adult parenteral preparations should provide a supplemental amount of 150 microg phylloquinone per day in addition to that present naturally, in variable amounts, in the lipid emulsion. Although this supplemental daily amount is probably beneficial in preventing vitamin K deficiency, it may be excessive for patients taking vitamin K antagonists, such as warfarin, and jeopardize their anticoagulant control. Natural forms of vitamin K have no proven toxicity.

Vitamin K deficiency bleeding (VKDB) in infants is a rare but serious worldwide problem, particularly in Southeast Asia. Apart from exclusive breast feeding, little is known of the maternofetal risk factors that predispose infants to VKDB.To assess (a) the relationships between functional vitamin K insufficiency in a large cohort of Thai mothers to that of their newborn infants and (b) the importance of delivery risk factors and maternal intakes of vitamin K as determinants of neonatal vitamin K status.Vitamin K status was assessed by measuring undercarboxylated prothrombin (protein induced by vitamin K absence/antagonist-II (PIVKA-II)) in 683 mothers and in the cord blood of their babies by sensitive immunoassay. Dietary phylloquinone (vitamin K(1); K(1)) intakes were assessed in 106 of these mothers by food frequency questionnaire.Babies were categorised as 'normal' (n=590) or 'high risk' (n=93) according to birth weight and delivery type. PIVKA-II was detectable (0.15 arbitrary units (AU)/ml) in 85 mothers (12.4%) and 109 babies (16.0%) with median levels of 0.78 and 1.04 AU/ml in mothers and babies, respectively. 'High-risk' babies had a higher median detectable PIVKA-II concentration than 'normal-risk' babies (3.1 vs 1.0 AU/ml, p=0.02) and a higher prevalence of clinically relevant (5.0 AU/ml) concentrations (p=0.006). Mothers with K(1) intakes below the US recommended 'adequate intake' for pregnancy (90 microg/day) had a higher prevalence of detectable PIVKA-II (18.8%) than those with adequate intakes (3.3%) (p=0.01).Functional, clinically relevant, vitamin K insufficiency was more common in 'high-risk' than 'normal-risk' newborns. Vitamin K insufficiency in mothers was linked to lower dietary K(1) intakes during pregnancy.

Trials in free-living populations involving increased consumption of fruit and vegetables are difficult to monitor. We evaluated biomarkers for assessing fruit and vegetable intake and compliance in a 2-year trial. Postmenopausal women were randomised to 300 g additional fruit and vegetables per d (n 66), placebo (n 70) or potassium citrate (n 140). They completed dietary checklists (3-monthly) and food diaries or FFQ (yearly). We measured whole-blood folate, plasma vitamin C and homocysteine (yearly), serum vitamin E and carotenoids (at 12 months) and urinary vitamin K metabolites (yearly). Plasma vitamin C was associated with fruit and vegetable intake at baseline (r +0·31; P r +0·43; P P = 0·07) but was not significantly associated with fruit and vegetable intake; vitamin E, β-carotene and β-cryptoxanthin were higher compared with the non-fruit and vegetable group (P 500 g/d, whereas whole-blood folate, β-carotene and β-cryptoxanthin continued to increase. Concentrations of vitamin C, folate and β-cryptoxanthin were lower and the 7C-aglycone metabolite of vitamin K higher, in smokers compared with non-smokers. Suitable markers for monitoring fruit and vegetable compliance include β-carotene and β-cryptoxanthin. Plasma vitamin C and whole-blood folate may be suitable for monitoring intakes in populations but for monitoring compliance the former may be restricted to low intakes of fruit and vegetables and the latter to vegetable intake.

Nutrition is important to bone health, and a number of minerals and vitamins have been identified as playing a potential role in the prevention of bone diseases, particularly osteoporosis. Despite this, there is currently no consensus on maximum levels to allow in food or as dietary supplements. The benefits of supplementation of populations at risk of osteoporosis with Ca and vitamin D are well established. Prolonged supplementation of Ca and vitamin D in elderly has been shown to prevent bone loss, and in some intervention studies to prevent fragility fractures. Although P is essential to bone health, the average intake is considered to be more than sufficient and supplementation could raise intake to adverse levels. The role of vitamin K in bone health is less well defined, though it may enhance the actions of Ca and vitamin D. Sr administered in pharmacological doses as the ranelate salt was shown to prevent fragility fractures in postmenopausal osteoporosis. However, there is no hard evidence that supplementation with Sr salts would be beneficial in the general population. Mg is a nutrient implicated in bone quality, but the benefit of supplementation via foodstuffs remains to be established. A consensus on dietary supplementation for bone health should balance the risks, for example, exposure of vulnerable populations to values close to maximal tolerated doses, against evidence for benefits from randomised clinical trials, such as those for Ca and vitamin D. Feedback from community studies should direct further investigations and help formulate a consensus on dietary supplementation for bone health.

The more recent introduction of techniques to assess vitamin K status is also leading to a deeper understanding of the nutritional and clinical role of vitamin K. Progress has been particularly rapid in the last decade and is the purpose of this annotation to review the advances in different areas

SummaryNaturally occurring vitamin K compounds comprise a plant form, phylloquinone (vitamin K1) and a series of bacterial menaquinones (MKs) (vitamin K2). Structural differences in the isoprenoid side chain govern many facets of metabolism of K vitamins including the way they are transported, taken up by target tissues, and subsequently excreted. In the post-prandial state, phylloquinone is transported mainly by triglyceride-rich lipoproteins (TRL) and long-chain MKs mainly by low-density lipoproteins (LDL). TRL-borne phylloquinone uptake by osteoblasts is an apoE-mediated process with the LRP1 receptor playing a predominant role. One K2 form, MK-4, has a highly specific tissue distribution suggestive of local synthesis from phylloquinone in which menadione is an intermediate. Both phylloquinone and MKs activate the steroid and xenobiotic receptor (SXR) that initiates their catabolism, but MK-4 specifically upregulates two genes suggesting a novel MK-4 signalling pathway. Many studies have shown specific clinical benefits of MK-4 at pharmacological doses for osteoporosis and cancer although the mechanism(s) are poorly understood. Other putative non-cofactor functions of vitamin K include the suppression of inflammation, prevention of brain oxidative damage and a role in sphingolipid synthesis. Anticoagulant drugs block vitamin K recycling and thereby the availability of reduced vitamin K. Under extreme blockade, vitamin K can bypass the inhibition of Gla synthesis in the liver but not in the bone and the vessel wall. In humans, MK-7 has a greater efficacy than phylloquinone in carboxylating both liver and bone Gla proteins. A daily supplement of phylloquinone has shown potential for improving anticoagulation control.

The physiological function and putative health roles of vitamin K-dependent proteins now extend beyond their classical role in hemostasis and include bone mineralization, arterial calcification, apoptosis, phagocytosis, growth control, chemotaxis, and signal transduction. Current assessments of vitamin K status do not reflect the variety of molecular forms of vitamin K. We assessed whether urinary excretion of 2-methyl-3-(5'-carboxy-3'-methyl-2'-pentenyl)-1,4-naphthoquinone (7C-aglycone) and 2-methyl-3-(3'-3'-carboxymethylpropyl)-1,4-naphthoquinone (5C-aglycone), vitamin K metabolites common to both phylloquinone and the menaquinone series, reflect dietary vitamin K intake. In a randomized crossover study, 9 adults resided in a metabolic unit for two 30-d periods separated by a free-living period of > or = 4 wk. During each residency, subjects consumed 3 sequential diets: a control diet (93 microg phylloquinone/d) for 5 d, a phylloquinone-restricted diet (11 microg/d) for 15 d, followed by a randomly assigned repletion diet for 10 d with either phylloquinone (206 microg/d) or dihydrophylloquinone (240 microg/d). During the second residency, the alternative repletion diet was assigned. Urinary excretion of the 5C- and 7C-aglycones was measured in sequential 24-h collections. The 5C-aglycone accounted for approximately 75% of total excretion and declined in response to phylloquinone restriction (P = 0.001) to approximately 30% of that during the control diet period. Repletion with phylloquinone and dihydrophylloquinone doubled the excretion rate of the major 5C-aglycone by 24 h (P < 0.001), and tripled excretion by 4 d. There was a linear relationship between the logarithm of total urinary excretion and dietary vitamin K intake (r = 0.699, P < 0.001). We conclude that the urinary excretion of vitamin K metabolites reflects dietary phylloquinone intake and offers the first candidate marker of global vitamin K status.

Phylloquinone is converted into menaquinone-4 and accumulates in extrahepatic tissues. Neither the route nor the function of the conversion is known. One possible metabolic route might be the release of menadione from phylloquinone by catabolic activity. In the present study we explored the presence of menadione in urine and the effect of vitamin K intake on its excretion. Menadione in urine was analysed by HPLC assay with fluorescence detection. Urine from healthy male volunteers was collected before and after administration of a single dose of K vitamins. Basal menadione excretion in non-supplemented subjects (n 6) was 5·4 (sd 3·2) μg/d. Urinary menadione excretion increased greatly after oral intake of the K vitamins, phylloquinone and menaquinone-4 and -7. This effect was apparent within 1–2h and peaked at about 3h after intake. Amounts of menadione excreted in 24h after vitamin K intake ranged, on a molar basis, from 1 to 5% of the administered dose, indicating that about 5–25% of the ingested K vitamins had been catabolized to menadione. Menadione excretion was not enhanced by phylloquinone administered subcutaneously or by 2′,3′-dihydrophylloquinone administered orally. In archived samples from a depletion/repletion study (Booth et al. (2001)Am J Clin Nutr74, 783–790), urinary menadione excretion mirrored dietary phylloquinone intake. The present study shows that menadione is a catabolic product of K vitamins formed after oral intake. The rapid appearance in urine after oral but not subcutaneous administration suggests that catabolism occurs during intestinal absorption. The observations make it likely that part of the menaquinone-4 in tissues results from uptake and prenylation of circulating menadione

We describe a method for the determination of the two major urinary metabolites of vitamin K as the methyl esters of their agyclone structures, 2-methyl-3-(3-3-carboxymethylpropyl)-1,4-naphthoquinone (5C-side-chain metabolite) and 2-methyl-3-(5-carboxy-3-methyl-2-pentenyl)-1,4-naphthoquinone (7C-side-chain metabolite), by HPLC with electrochemical detection (ECD) in the redox mode. Urinary salts were removed by reversed-phase (C18) solid phase extraction (SPE) and the predominately conjugated vitamin K metabolites hydrolysed with methanolic HCl. The resultant carboxylic acid aglycones were quantitatively methylated with diazomethane and fractionated by normal-phase (silica) SPE. Final analysis was by reversed-phase (C18) HPLC with a methanol-aqueous mobile phase. Metabolites were detected by amperometric, oxidative ECD of their quinol forms, which were generated by post-column coulometric reduction at an upstream electrode. The assay gave excellent linearity (r2 typically = 0.999) and high sensitivity with an on-column detection limit of

Background/Aims: In patients with severe acute liver dysfunction, i.v. phylloquinone (vitamin K-1) may be given to exclude vitamin K deficiency, rather than impaired hepatic synthesis of coagulation factors alone, as the cause of the coagulopathy. However, there have been no studies of the pharmacokinetics or efficacy of i.v. or oral K-1 in such patients.Methods: 49 adults with severe acute liver disease were randomised double-blind to a single 10 mg dose of i.v. or oral mixed-micellar K-1, or placebo. Serum levels of phylloquinone and undercarboxylated prothrombin (PIVKA-II) were assessed before and after treatment.Results: At admission, 13 patients (27 %) had either low serum K-1 levels or elevated PIVKA-II concentrations, indicative of subclinical vitamin K deficiency. In the 16 patients who received i.v. K-1, there was one (6 %) treatment failure (K-1 rise < 10 ng/ml above baseline), compared with 12 of the 15 (80 %) who received oral K, (P < 0.0001). One patient in the placebo group developed overt vitamin K deficiency.Conclusions: A minority of patients with severe acute liver dysfunction have subclinical vitamin K deficiency at the time of presentation, which is corrected by a single dose of i.v. K-1. The intestinal absorption of mixed-micellar K, is unreliable in adults with severe acute liver dysfunction. (c) 2004 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

SummaryThe 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 theVKORC1 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 ( ≥ 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 K 1 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 that VKORC1 sequence is an important determinant of the warfarin dose response.

Male circumcision has been shown to reduce the risk of heterosexually-acquired human immunodeficiency virus (HIV) infection in men, and male circumcision in early infancy (defined as 1–60 days of life by the World Health Organization [WHO]) is recommended by the WHO as part of HIV prevention strategies because of its relative ease and safety (http://libdoc.who.int/publications/2007/9789241595988_eng.pdf) compared with the procedure in older ages. Given the potential scale-up of early infant male circumcision in resource-limited settings, more attention must be paid to the prevention and treatment of potential postcircumcision bleeding associated with vitamin K deficiency. Those who perform circumcision for religious or traditional reasons should also be aware of this potential cause of postcircumcision bleeding. Vitamin K deficiency bleeding can occur in infants up to 6 months of age, although it most commonly occurs either in the first week of life (classical) or at 3–8 weeks (late). Infants are at risk of vitamin K deficiency because they are born with low liver stores of vitamin K and subsequently may not be able to obtain sufficient amounts from breast milk. Before neonatal vitamin K prophylaxis became the standard of care in the United States, vitamin K deficiency bleeding had been reported after circumcisions done within the first week of life at Cincinnati General Hospital, and bleeding in this population was most common among the breastfed neonates.1 Postcircumcision bleeding is of particular concern in the developing world where the majority of infants are breastfed and thus at increased risk of vitamin K deficiency bleeding. In a recent report from Kenya, one of the reasons given by parents for not wanting to circumcise an infant was fear of bleeding2 and in a recent trial of circumcision in Zambia, most of the adverse events were excessive bleeding with one case requiring a transfusion.3 Therefore, the use of vitamin K to prevent or treat postcircumcision bleeding deserves urgent attention as countries in sub-Saharan Africa are poised to scale-up early infant male circumcision as part of HIV-prevention interventions.

A modified high-performance liquid chromatography (HPLC) method, based on Davidson and Sadowski [Meth. Enzymol. 282 (1997) 408], with fluorescence detection after zinc postcolumn reduction was developed and validated for the analysis of phylloquinone (vitamin K1) in plasma or serum samples. Compensation for procedural losses of vitamin K1 was made by the method of internal standardization using a proprietary vitamin K derivative. Increased sensitivity of detection by the use of a high-sensitivity Waters 440 fluorescence detector and optimized chromatography conditions increased the sensitivity to 4 fmol vitamin K1. The response was linear and free from interfering peaks and from baseline drift. It is therefore adequately sensitive for 0.25 ml or less of plasma sample. Long-term reproducibility of quality assurance (QA) samples was verified over a period of 4 months. The intraassay precision estimates of the QA samples within-run with mean vitamin K1 concentrations of 0.4, 1.4 and 3.4 nmol/l were 5.2% (n=6), 8.2% (n=6) and 3.0% (n=12), respectively, while interassay precision estimates between runs were 16% (n=22), 12% (n=21) and 8.1% (n=15), respectively. The assay accuracy was validated by comparing the results we obtained for 14 samples from the Vitamin K External Quality Assessment Scheme (KEQAS) with the consensus of the results from the other participating laboratories. Good agreement was obtained, with y=1.06x-0.09, R2=0.99. Validation also included linearity of response, absence of interference and confirmation of vitamin K1 peak purity.

Objective: To compare the pharmacokinetics and efficacy of oral versus intravenous mixed micellar vitamin K prophylaxis in infants with cholestatic liver disease, a known risk factor for vitamin K deficiency bleeding. Design: Prospective randomised controlled study. Setting: Paediatric Liver Unit. Patients: Forty four infants less than 6 months of age with conjugated hyperbilirubinaemia. Main outcome measures: Serum concentrations of vitamin K 1 and undercarboxylated prothrombin (PIVKA-II; a sensitive functional indicator of vitamin K status) before and for up to four days after a single dose of mixed micellar K 1 1 mg intravenously or 2 mg orally. Comparison of K 1 levels 24 hours after oral K 1 with those from 14 healthy newborns given the same dose. Results: At admission, 18 infants (41%) had elevated levels of serum PIVKA-II and eight (18%) had low K 1 concentrations, indicative of subclinical vitamin K deficiency. Median serum K 1 concentrations were similar in the oral and intravenous groups at baseline (0.92 v 1.15 ng/ml), rising to 139 ng/ml six hours after intravenous K 1 but to only 1.4 ng/ml after oral administration. In the latter group, the low median value (0.95 ng/ml) and wide range ( 1 compared unfavourably with the much higher levels (median 77, range 11–263 ng/ml) observed in healthy infants given the same oral dose, and suggested impaired and erratic intestinal absorption in cholestatic infants. The severity of malabsorption was such that only 4/24 (17%) achieved an incremental rise in serum K 1 > 10 ng/ml. Conclusions: The intestinal absorption of mixed micellar K 1 is unreliable in infants with conjugated hyperbilirubinaemia. Given the strong association between cholestasis and late vitamin K deficiency bleeding, these data provide an explanation for the failure of some oral vitamin K 1 prophylaxis regimens in infants with latent cholestasis.

Intake and sources of phylloquinone (vitamin K1) were examined according to socio-demographic and lifestyle factors in free-living British people aged 65 years and over, from the 1994-5 National Diet and Nutrition Survey. Complete 4-d weighed dietary records were obtained from 1152 participants living in private households. Using newly-available, mainly UK-specific food content data, the weighted geometric mean intake of phylloquinone was estimated at 65 (95 % CI 62, 67) microg/d for all participants, with higher intakes in men than in women (70 v. 61 microg/d respectively, P

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 (95 % CI 0·06, 2·01) and 0·24 (95 % CI 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 (r0·18 and 0·30 respectively, bothPP

Little is known of how the fat components of diets influence the absorption and metabolism of vitamin K and the possible consequences to the synthesis of vitamin K-dependent (VKD) proteins in different target organs. We have evaluated the effects of two diets on circulating phylloquinone (K1) and triacylglycerols (TAG). One diet was enriched with corn oil (CO) (also rich in gamma-tocopherol) and the other with an olive/sunflower (O/SO) mixture (rich in alpha-tocopherol). Effects on gamma-carboxylation were assessed from coagulation assays and sensitive assays for undercarboxylated prothrombin (ucFII) and osteocalcin (ucOC). Total plasma matrix Gla-protein (MGP) was also measured. After an initial adjustment diet, 26 healthy young men were fed, in a crossover design, the O/SO or CO diet for 2 weeks. Mean intakes of K1 during consumption of adjustment, O/SO, and CO diets were 225 microg/day, 291 microg/day, and 291 microg/day, respectively. Mean fasting levels of TAG and K1 were both significantly reduced by the CO diet, but not by the O/SO diet. Neither diet reduced FII activity but ucFII became detectable in nine subjects, eight of whom showed this abnormality with both diets. The CO diet induced a rise in ucOC (P < 0.05), which was negatively correlated to ucFII (r = -0.71, P < 0.03). The CO but not O/SO diet induced a decrease of total circulating MGP. We conclude that both oils, notably CO, affected vitamin K absorption and/or metabolism which may increase the requirements for gamma-carboxylation. The mechanism is unclear but may result from interactions of vitamin K with PUFA and/or other lipid components such as vitamin E.

5-Methyltetrahydrofolate (5-MTHF) is the predominant form of folate and a strong determinant of homocysteine concentrations. There is evidence that suboptimal 5-MTHF availability is a risk factor for cardiovascular disease independent of homocysteine. The analysis of folates remains challenging and is almost exclusively limited to the reporting of “total” folate rather than individual molecular forms. The purpose of this study was to establish the reference intervals of 5-MTHF in plasma and red cells of healthy adults who had been prescreened to exclude biochemical evidence of functional deficiency of folate and/or vitamin B12. Functional folate and vitamin B12status was assessed by respective plasma measurements of homocysteine and methylmalonic acid in 144 healthy volunteers, aged 19–64 years. After the exclusion of 10 individuals, values for 134 subjects were used to establish the upper reference limits for homocysteine (13 μmol/L females and 15 μmol/L males) and methylmalonic acid (430 nmol/L). Subjects with values below these cutoffs were designated as folate and vitamin B12replete and their plasma and red cell 5-MTHF reference intervals determined,N=126: 6.6–39.9 nmol/L and 223–1041 nmol/L, respectively. The application of these intervals will assist in the evaluation of folate status and facilitate studies to evaluate the relationship of 5-MTHF to disease.

Aim: To investigate the vitamin K status of preterm infants who have a prolonged prothrombin time (PT) in the first month of life. Methods: Measures of vitamin K status were assessed in 21 preterm infants who were found to have an abnormal PT, despite 0.2-0.5 mg vitamin K 1 prophylaxis at birth. Results: All infants had normal or supraphysiological vitamin K 1 concentrations and undetectable or, in one infant, insignificant PIVKA-II, indicating adequate vitamin K status. Conclusion: In preterm infants born at < 32 wk gestation who received ≥ 0.2 mg vitamin K 1 after delivery, a prolonged PT in the first month of life is unlikely to be due to vitamin K deficiency.

Notes that vitamin K has long been known to be required for blood clotting, and more recent evidence suggests a role in bone mineralization, atherosclerosis, and apoptosis. Guidelines for vitamin K intake were set in 1991; however, the food compositional data and intake levels in the UK were not known and, as such, no dietary reference values were set. Since then, work in the field has progressed enormously, with a provisional food composition database being developed, both in the UK and USA, and a healthy population reference plasma range for K1 has been established in the UK in parallel with detailed estimates of K1 intake. Plasma K1 and the proportion of undercarboxylated prothrombin and osteocalcin have been identified as sensitive functional markers of vitamin K status. Current work on aspects of vitamin K and bone health in the elderly will further assist with the setting of dietary reference values for the future.

Molecules with vitamin K activity are important for optimal bone health. The major compound of this group in bone is vitamin K1 (phylloquinone), which is derived exclusively from plant foods in the diet. Vitamin K1 is absorbed along with dietary fat from the small intestine and transported by chylomicrons in blood. In serum obtained after an overnight fast from healthy men more than half of the vitamin K1 was recovered from the density fraction that contains chylomicrons and chylomicron remnants (CR), and only a quarter was associated with the major lipoprotein in serum, low density lipoprotein. The concentration of vitamin K1 in serum is closely related to the triglyceride concentration. Another determinant of vitamin K1 concentration in serum is the presence of specific variants of apolipoprotein E (apoE). ApoE is a small protein through which the vitamin K-rich CR bind to lipoprotein receptors. The three most common variants of apoE promote CR clearance from circulation with very different efficiency, in the order E2>E3>E4. The variant that promotes CR clearance best is associated with low vitamin K1 concentration in serum and increased response to vitamin K antagonists. Vitamin K1 concentration in serum is linked to vitamin K status of bone. The bone protein osteocalcin tends to be less completely carboxylated in people with low vitamin K concentrations in serum. Many hemodialysis patients with a history of bone fractures have indications of poor vitamin K status. The same patients also appear to have a greatly increased prospective bone fracture risk.

Vitamin K antagonists (VKA) have been the mainstay of oral anticoagulant therapy for over 60years. In this review we critically assess the evidence for the importance of vitamin K nutrition during VKA therapy; the methodologies for measuring dietary intakes; the vitamin K intake data in patients on VKA and healthy people; and the experimental evidence for the influence of vitamin K intakes and biochemical measures of vitamin K status on VKA response. Several studies show that dietary intakes of phylloquinone (vitamin K1) are associated to the sensitivity and stability of anticoagulation during initiation and maintenance dosing with low habitual intakes associated with greater instability of the INR and risk of sub-therapeutic anticoagulation. Preliminary evidence suggests that the stability of anticoagulation therapy may be improved by daily vitamin K supplementation, but further studies are needed to find out whether this, or other dietary interventions, can improve anticoagulant control in routine clinical practice.

See also Watzka M, Geisen C, Bevans CG, Sittinger K, Spohn G, Rost S, Seifried E, Mnller CR, Oldenburg J. Thirteen novel VKORC1 mutations associated with oral anticoagulant resistance: insights into improved patient diagnosis and treatment. J Thromb Haemost 2011; 9: 109–18.

Vascular calcification (VC) is highly prevalent in CKD and leads to increased vascular stiffness and cardiovascular disease (CVD). Non-traditional cardiovascular risk factors include abnormal bone turnover and/or dysregulation of the calcification inhibitors, although their relative contribution remains unclear. We investigated the association between bone turnover, the calcification inhibitors (matrix gla protein; MGP and Fetuin-A), and the phosphate regulating hormone; fibroblast growth factor-23 (FGF-23) and arterial stiffness in pre-dialysis CKD patients. One hundred and forty-five patients with CKD stages 1-4 (74 M, 71 F) aged (mean [SD]) 53 [14] years were studied. Bone turnover markers (bone-specific alkaline phosphatase (BALP) and tartrate-resistant acid phosphatase (TRACP)) and MGP, Fetuin-A and FGF-23 were determined. BMD was measured at the lumbar spine (LS), femoral neck (FN), forearm (FARM) and total hip (TH). Arterial stiffness was assessed by contour analysis of digital volume pulse (SI(DVP)). There was a significant positive correlation between TRACP:BALP ratio and SI(DVP) ( r=0.19, p=0.023). Following multi-linear regression analysis, significant associations were seen between serum BALP (p=0.037), TRACP (p=0.009) and TRACP:BALP ratio (p=0.001) and SI(DVP) independently of traditional CVD risk factors. No significant relationship between SI(DVP) and MGP, Fetuin-A and FGF-23 was observed. A significant negative correlation was seen between BMD at the FARM and SI(DVP) in CKD stage 4 (r=-0.35, p=0.024). The association remained significant following correction for age, gender and cardiovascular risk factors (p=0.029). Our data suggest a link between imbalances in bone turnover and arterial stiffness in pre-dialysis CKD. Longitudinal studies are needed to evaluate the clinical usefulness of these bone turnover markers as predictors of CVD in CKD.

SummaryAssessments of the vitamin K status in newborns and their mothers by means of des-γ-carboxy-prothrombin (PIVKA II) measurement have given equivocal results. Part of the variability could be attributed to differences in sensitivity (i.e. the ability to detect small concentrations) and validity (i.e. ability to detect vitamin K deficiency) of the methods applied. None of these methods have yet been validated with respect to plasma vitamin K1. In 22 healthy mother/infant pairs PIVKA II was determined using three different assays including ratio Xa/ecarin (Xa/ec), crossed immunoelectrophoresis (CIE), and an ELISA with a monoclonal antibody (MAB). The results were compared with conventional clotting tests and plasma vitamin K1. The following results were obtained:Cord blood: Clotting tests within age-related normal ranges; PIVKA II detection rates: 0/22 (Xa/ec), 1/22 (CIE), 4/22 (MAB); plasma vitamin K1: undetectable in 20/22.Mothers: Clotting tests all within normal range; PIVKA II detection rates: 1/22 (Xa/ec), 0/22 (CIE), 5/22 (MAB); plasma vitamin K1 (pg/ml) for all mothers (median; range): 186; 55–833; for PIVKA II positive mothers: 213; 59–699.PIVKA II detectability in newborns and mothers was not correlated. The results show an increase in sensitivity for PIVKA II detection in the order of MAB ≫CIE >Xa/ec. Due to the very low plasma vitamin K1 at birth, no correlation was possible between cord PIVKA II detectability and plasma vitamin K1. However, in mothers at term PIVKA II MAB appears to be unrelated to the vitamin K status.

Dietary fluctuations of vitamin K are detrimental to oral anticoagulant control. Attempts to improve control through the avoidance of vitamin K-rich foods (mainly green vegetables) may inadvertently compromise folate status, itself a risk factor for thromboembolism. We evaluated the effect of a 6-month period of warfarin therapy on folate status in 114 patients using measurements of red-cell folate and 5-methyltetrahydrofolate and plasma folate and total homocysteine. Circulatory levels of phylloquinone, vitamin B12 and methylmalonic acid were also determined. A subset of 45 patients completed 7-day food diaries at the beginning and end of their treatment. There was a significant decrease in total erythrocyte folate (P = 0.005) and 5-methyltetrahydrofolate (P = 0.002) during the study. A concurrent increase in plasma phylloquinone (P = 0.003) was attributed to warfarin-induced perturbation of vitamin K metabolism. No other longitudinal changes were observed. Folate and phylloquinone intakes correlated with each other at baseline (P = 0.024) and after treatment (P = 0.011). Based on robust measurements of erythrocyte folates, patients showed a significant impairment in folate status after 6-month therapy with warfarin. The majority of patients had intakes of folate and phylloquinone below the national average or UK guidelines. The study highlights the need for improved dietary management of patients taking oral anticoagulants.

Vitamin K deficiency bleeding (VKDB) is a rare and potentially life-threatening bleeding disorder of early infancy. Vitamin K stores are low at birth; thereafter breast-fed infants are at risk because of low concentrations in human milk. Classical VKDB occurs in the first week of life, is related to delayed or inadequate feeding and is readily prevented by small doses of vitamin K at birth. Late VKDB peaks at 3-8 weeks, typically presents with intracranial haemorrhage often due to undiagnosed cholestasis with resultant malabsorption of vitamin K. Diagnosis can be difficult but PIVKA-II measurements can provide confirmation even several days post-treatment. Without vitamin K prophylaxis, the incidence of late VKDB in Europe is 4-7 cases per 10(5) births; it is higher in SE Asia where in rural, low-income areas some 0.1% of affected infants may suffer intracranial bleeding. Late VKDB is largely preventable with parenteral vitamin K providing the best protection. The efficacy of oral prophylaxis is related to the dose and frequency of administration. Most multi-dose oral regimens provide protection for all except a small reservoir of infants with undetected hepatobiliary disease. Targeted surveillance of high-risk groups (e.g. biliary atresia) offers a novel approach to assess efficacy of prophylaxis.