Overall, D(3) is slightly, but significantly, more effective than D(2) to increase serum 25(OH)D. One year of D(2) or D(3) dosing (1,600 IU daily or 50,000 IU monthly) does not produce toxicity, and 25(OH)D levels of less than 30 ng/ml persist in approximately 20% of individuals. Substantial between-individual response to administered vitamin D(2) or D(3) is observed.

Four weeks of oral vitamin D(2) supplementation effectively and safely normalizes serum 25(OH)D in nursing home residents.

A daily phylloquinone intake of approximately 1000 micro g is required to maximally gamma-carboxylate circulating osteocalcin.

Background: Subclinical vitamin K insufficiency, manifested by under-␥-carboxylation of the bone matrix protein osteocalcin, may be common. Objective: Our objective was to delineate the prevalence of submaximal ␥-carboxylation as assessed by response to phylloquinone supplementation and to evaluate the effect of this intervention on skeletal turnover in healthy North American adults. Design: Healthy subjects (n = 219), approximately equally distributed by sex and age (18-30 y and ≥ 65 y), received daily phylloquinone (1000 g) or placebo for 2 wk. Serum undercarboxylated osteocalcin (ucOC) and total osteocalcin, N-telopeptides of type I collagen (NTx), bone-specific alkaline phosphatase (BSAP), and phylloquinone concentrations were measured at baseline and after weeks 1 and 2. Results: At baseline, the mean serum phylloquinone concentration was lower in the young than in the old group; there was no effect of sex. Concomitantly, baseline %ucOC was highest in the young and lowest in the old men (P < 0.0001) but did not differ significantly by age in women. After supplementation, serum phylloquinone concentration increased Ϸ10-fold (P < 0.0001) at week 1 (from 0.93 ± 0.08 to 8.86 ± 0.70 nmol/L, x -± SEM); this was sustained through week 2. Among all supplemented groups, mean %ucOC decreased from 7.6% to 3.4% without significant differences by age or sex; 102 of 112 subjects had a > 1% decrease.

Vitamin D insufficiency (VDI) is widely reported. In patients with normal PTH, the diagnosis rests on increases in fractional calcium absorption (FCA) when 25(OH)D increases above 30 ng/ml. However, estimates of increased FCA after correction of VDI vary dramatically, depending on study methods. We used a dual stable calcium isotope to clarify the impact of vitamin D repletion on FCA in postmenopausal women with VDI. We hypothesized that FCA would increase with vitamin D repletion. We studied postmenopausal women with VDI [25(OH)D ‫ס‬ 16-24 ng/ml] and an estimated calcium intake Յ1100 mg daily. Exclusion criteria included hypercalcemia, hypercalciuria, renal insufficiency, nephrolithiasis, gastrointestinal disorders, osteomalacia, prior adult fragility fracture, baseline T-score < −3.0, and use of medications known to interfere with vitamin D or calcium metabolism. Each woman underwent inpatient FCA studies before and after correction of VDI. We used ergocalciferol 50,000 IU/d for 15 days to achieve vitamin D repletion. During each study, women consumed their typical diet. They ingested 44 Ca orally with breakfast and received 42Ca intravenously. We collected urine for 24 h and measured its calcium isotope content by mass spectrometry. Eighteen women completed the study; all but two had normal PTH. During the first and second FCA studies, their mean 25(OH)D level was 22 ± 4 and 64 ± 21 ng/ml, respectively (p < 0.001). Subjects' average FCA was 24 ± 7% initially and 27 ± 6% after vitamin D repletion (p ‫ס‬ 0.04). Thus, FCA increased by 3 ± 1% with correction of VDI. Postmenopausal women with VDI experience small FCA increments with vitamin D therapy. In existing literature, this small change in FCA does not associate with lower fracture rates or consistently higher bone mass. Future studies should ascertain whether small FCA increments favorably affect the skeleton.

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.

The ability of male rats to accumulate menaquinone-4 (MK-4) in tissues when fed a vitamin K-deficient diet supplemented with intraperitoneal phylloquinone (K) as the sole source of vitamin K for 14 d was assessed. In both conventionally housed controls and gnotobiotic rats, supplementation with the equivalent of 1500 microg vitamin K/kg diet increased (P < 0.001) tissue MK-4 concentrations above those of controls fed a vitamin K-deficient diet. MK-4 concentrations were approximately 5 ng/g (11 pmol/g) in liver, 14 ng/g in heart, 17 ng/g in kidney, 50 ng/g in brain and 250 ng/g in mandibular salivary glands of gnotobiotic rats. MK-4 concentrations in conventionally housed rats were higher than in gnotobiotic rats in heart (P < 0.01), brain (P < 0.01) and kidney (P < 0.05) but lower in salivary gland (P < 0.05). Cultures of a kidney-derived cell line (293) converted K to the expoxide of MK-4 in a manner that was dependent on both time of incubation and concentration of vitamin K in the media. A liver-derived cell line (H-35) was less active in carrying out this conversion. These data offer conclusive proof that the tissue-specific formation of MK-4 from K is a metabolic transformation that does not require bacterial transformation to menadione as an intermediate in the process.

The activity and extent of adenylylation of glutamine synthetase was examined in both free-living and bacteroid forms of Rhizobium japonicum in the presence of excess ammonia. Ammonia caused an apparent repression of glutamine synthetase in free-living R. japonicum and adenylylation of the enzyme was also increased. In contrast, neither the activity nor the extent of adenylylation of the bacteroid enzyme was consistently affected by ammonium treatment of bacteroid suspensions. Similar results were obtained after ammonium treatment of soybean plants even though nitrogenase activity was reduced markedly. We have been unable to demonstrate ammonium repression of nitrogenase activity in R. japonicum-Glycine max symbiotic association that is mediated through bacteroid glutamine synthetase. This result is in contrast to the situation in nitrogen-fixing strains of Klebsiella where a role of glutamine synthetase in the regulation of nitrogenase has been reported.The inhibitory effects of fixed nitrogen on nodulation of and N2 fixation by legumes have been well documented (1, 12, 16) but details of how nitrogenase synthesis is regulated in legumeRhizobium associations are unknown. The mechanism of regulation of nitrogenase synthesis in free-living bacteria is beginning to be understood. Evidence by Tubb (22) and by Streicher et al. (20) indicates that catalytically active glutamine synthetase is necessary for nitrogenase synthesis by Klebsiella pneumoniae. They have also demonstrated that strains of K. aerogenes, and K. pneumoniae that exhibit the Gln C-mutant phenotype (constitutive synthesis of glutamine synthetase) and carry the nif operon(s) were partially derepressed for nitrogenase synthesis when cultured in the presence of NH4+. These results suggest that glutamine synthetase acts as a positive control element for nitrogenase synthesis in a fashion similar to that proposed for histidase synthesis in K. aerogenes (17). The 3 To whom all correspondence should be addressed.speculate that the regulatory system for nitrogen fixation by the Rhizobium-legume system may be similar to that in Klebsiella.We have investigated the effects of NH4+ on glutamine synthetase in both free-living R. japonicum and the bacteroid forms of the microorganisms in root nodules. Our experiments have led to the unexpected conclusion that neither activity nor the extent of adenylylation of bacteroid glutamine synthetase is consistently influenced by NH4+ even under conditions where nitrogenase activity was inhibited markedly. Glutamine synthetase in freeliving R. japonicum, on the other hand, behaves like the enzyme in Escherichia coli (13,24) showing repression and adenylylation when excessive NH4+ is supplied. MATERIALS AND METHODSCulture of Free-living Bacteria. Rhizobium japonicum strain OSR-2 was cultured in 1-liter flasks containing 500 ml of medium containing the following components dissolved in 1 liter of water: K2HPO4, 0.23 g; MgSO4-7H2O, 0.10 g; sodium glutamate, 1.10 g; glycerol, 4 g; CaC12, 5 mg; H3B03, 145 ,ug; FeSO4...