Deuterium-labeled phylloquinone has tissue-specific conversion to menaquinone-4 among Fischer 344 male rats.

Phylloquinone (PK) is converted into menaquinone-4 (MK-4) via side chain removal-addition. Stable isotope use is an effective approach to identify the tissue location of this conversion, which is currently unknown. Following a 14-d PK-deficient diet, male Fischer 344 rats (8 mo; n = 15) were fed 1.6 mg deuterium-labeled PK (L-PK) per kg diet for 0 (control), 1 d (PK-1d), and 7 d (PK-7d). Both L-PK and deuterium-labeled MK-4 (L-MK-4) were detected in tissues in PK-1d and PK-7d, although the results varied. Whereas some tissues had an overall increase in MK-4 in response to L-PK, total brain, testes, and fat MK-4 concentrations did not. In contrast, L-MK-4 concentrations increased in all 3 tissues. The deuterium label was found only on the L-MK-4 naphthoquinone ring, confirming the need for side chain removal for the formation of MK-4. Labeled menadione (MD) was detected in urine and serum in PK-1d and PK-7d, confirming its role as an intermediate. A Caco-2 cell monolayer model was used to study the role of the enterocytes in the conversion process. Neither MK-4 nor MD was detected in Caco-2 cells treated with PK. However, when Caco-2 cells were treated with MD, MK-4 was formed. Similarly, MK-4 was formed in response to MD-treated 293T kidney cells, but not HuH7 liver cells. These data demonstrate that MK-4 is the predominant form of vitamin K in multiple tissues, but there appears to be a tissue-specific regulation for the conversion of PK to MK-4.

Measurement of menadione in urine by HPLC.

Menadione is a metabolite of vitamin K that is excreted in urine. A high performance liquid chromatography (HPLC) method using a C(30) column, post-column zinc reduction and fluorescence detection was developed to measure urinary menadione. The mobile phase was composed of 95% methanol with 0.55% aqueous solution and 5% DI H(2)O. Menaquinone-2 (MK-2) was used as an internal standard. The standard calibration curve was linear with a correlation coefficient (R(2)) of 0.999 for both menadione and MK-2. The lower limit of quantification (LLOQ) was 0.3pmole menadione/mL urine. Sample preparation involved hydrolysis of menadiol conjugates and oxidizing the released menadiol to menadione. Using this method, urinary menadione was shown to increase in response to 3 years of phylloquinone supplementation. This HPLC method is a sensitive and reproducible way to detect menadione in urine.

Vitamin K treatment reduces undercarboxylated osteocalcin but does not alter bone turnover, density, or geometry in healthy postmenopausal North American women.

Low vitamin K status is associated with low BMD and increased fracture risk. Additionally, a specific menaquinone, menatetrenone (MK4), may reduce fracture risk. However, whether vitamin K plays a role in the skeletal health of North American women remains unclear. Moreover, various K vitamers (e.g., phylloquinone and MK4) may have differing skeletal effects. The objective of this study was to evaluate the impact of phylloquinone or MK4 treatment on markers of skeletal turnover and BMD in nonosteoporotic, postmenopausal, North American women. In this double-blind, placebo-controlled study, 381 postmenopausal women received phylloquinone (1 mg daily), MK4 (45 mg daily), or placebo for 12 mo. All participants received daily calcium and vitamin D(3) supplementation. Serum bone-specific alkaline phosphatase (BSALP) and n-telopeptide of type 1 collagen (NTX) were measured at baseline and 1, 3, 6, and 12 mo. Lumbar spine and proximal femur BMD and proximal femur geometry were measured by DXA at baseline and 6 and 12 mo. At baseline, the three treatment groups did not differ in demographics or study endpoints. Compliance with calcium, phylloquinone, and MK4 treatment was 93%, 93%, and 87%, respectively. Phylloquinone and MK4 treatment reduced serum undercarboxylated osteocalcin but did not alter BSALP or NTX. No effect of phylloquinone or MK4 on lumbar spine or proximal femur BMD or proximal femur geometric parameters was observed. This study does not support a role for vitamin K supplementation in osteoporosis prevention among healthy, postmenopausal, North American women receiving calcium and vitamin D supplementation.

Vitamin K deficiency from long-term warfarin anticoagulation does not alter skeletal status in male rhesus monkeys.

UNLABELLED: Vitamin K (K) inadequacy may cause bone loss. Thus, K deficiency induced by anticoagulants (e.g., warfarin) may be an osteoporosis risk factor. The skeletal impact of long-term warfarin anticoagulation was evaluated in male monkeys. No effect on BMD or bone markers of skeletal turnover was observed. This study suggests that warfarin-induced K deficiency does not have skeletal effects. INTRODUCTION: The skeletal role of vitamin K (K) remains unclear. It is reasonable that a potential role of vitamin K in bone health could be elucidated by study of patients receiving oral anticoagulants that act to produce vitamin K deficiency. However, some, but not all, reports find K deficiency induced by warfarin (W) anticoagulation to be associated with low bone mass. Additionally, epidemiologic studies have found W use to be associated with either increased or no change in fracture risk. Such divergent results may imply that human studies are compromised by the physical illnesses for which W was prescribed. MATERIALS AND METHODS: To remove this potential confounder, we prospectively assessed skeletal status during long-term W anticoagulation of healthy nonhuman primates. Twenty adult (age, 7.4-17.9 yr, mean, 11.7 yr) male rhesus monkeys (Macaca mulatta) were randomized to daily W treatment or control groups. Bone mass of the total body, lumbar spine, and distal and central radius was determined by DXA at baseline and after 3, 6, 9, 12, 18, 24, and 30 mo of W treatment. Serum chemistries, urinary calcium excretion, bone-specific alkaline phosphatase, and total and percent unbound osteocalcin were measured at the same time-points. Prothrombin time and international normalized ratio (INR) were monitored monthly. Serum 25-hydroxyvitamin D was measured at the time of study conclusion. RESULTS: W treatment produced skeletal K deficiency documented by elevation of circulating undercarboxylated osteocalcin (8.3% W versus 0.4% control, p<0.0001) but did not alter serum markers of skeletal turnover, urinary calcium excretion, or BMD. CONCLUSIONS: In male rhesus monkeys, long-term W anticoagulation does not alter serum markers of bone turnover or BMD. Long-term W therapy does not have adverse skeletal consequences in primates with high intakes of calcium and vitamin D.

Vitamin K prophylaxis for premature infants: 1 mg versus 0.5 mg.

We studied babies (22 to 32 weeks gestational age) of mothers wishing to breast-feed. Group 1 received 1 mg of vitamin K and Group 2 received 0.5 mg of vitamin K. The Day 2 plasma levels of vitamin K were 1900 to 2600 times higher on average, and the Day 10 vitamin K levels 550 to 600 times higher on average, relative to normal adult plasma values, whether an initial prophylaxis dose of 0.5 mg or 1 mg was used. We conclude that 0.5 mg as the initial dose of vitamin K intramuscularly or intravenously would likely be more than adequate to prevent hemorrhagic disease of the newborn, and that 0.3 mg/per kg may be used for babies with birth weights below 1000 g. To decrease vitamin K intakes in this population, new preparations of total parenteral nutrition multivitamins are needed.

A high phylloquinone intake is required to achieve maximal osteocalcin gamma-carboxylation.

BACKGROUND: Dietary vitamin K is usually inadequate to maximize serum osteocalcin gamma-carboxylation. Phylloquinone supplementation increases osteocalcin gamma-carboxylation; however, the amount required to maximize carboxylation is not known. OBJECTIVE: This study assessed the ability of various doses of phylloquinone (vitamin K(1)) to facilitate osteocalcin gamma-carboxylation. DESIGN: Healthy adults aged 19-36 y participated in 2 substudies. In an initial dose-finding study (substudy A), 6 women and 4 men received a placebo daily for 1 wk and then phylloquinone daily for 3 wk: 500, 1000, and 2000 micro g during weeks 2, 3, and 4, respectively. Osteocalcin and undercarboxylated osteocalcin were measured at baseline and after each week of supplementation. Subsequently, to further delineate the gamma-carboxylation response of osteocalcin to various doses of vitamin K, 58 women and 42 men were randomly assigned to receive placebo or phylloquinone supplementation (250, 375, 500, and 1000 micro g/d) for 2 wk (substudy B). The percentage of undercarboxylated osteocalcin (%ucOC) was measured at baseline and weeks 1 and 2. RESULTS: In substudy A, %ucOC decreased with phylloquinone supplementation (P < 0.0001); a greater reduction was observed with 1000 and 2000 micro g than with 500 micro g (P < 0.05). In substudy B, %ucOC decreased in all supplemented groups by week 1 (P for the trend < 0.0001), which was sustained through week 2. Phylloquinone supplementation decreased %ucOC dose-dependently; %ucOC was significantly different between the 250- micro g and the placebo groups and between the 1000- and 500- micro g groups but not between the 250-, 375-, and 500- micro g groups. CONCLUSION: A daily phylloquinone intake of approximately 1000 micro g is required to maximally gamma-carboxylate circulating osteocalcin.