Jean Engelke

Evaluation of Ergocalciferol or Cholecalciferol Dosing, 1,600 IU Daily or 50,000 IU Monthly in Older Adults

CONTEXT Whether ergocalciferol (D(2)) and cholecalciferol (D(3)) are equally effective to increase and maintain serum 25-hydroxyvitamin D [25(OH)D] concentration is controversial. OBJECTIVE The aim of the study was to evaluate the effect of daily and once monthly dosing of D(2) or D(3) on circulating 25(OH)D and serum and urinary calcium. DESIGN, SETTING AND PARTICIPANTS In a university clinical research setting, 64 community dwelling adults age 65+ were randomly assigned to receive daily (1,600 IU) or once-monthly (50,000 IU) D(2) or D(3) for 1 yr. MAIN OUTCOME MEASURES Serum 25(OH)D, serum calcium, and 24-h urinary calcium were measured at months 0, 1, 2, 3, 6, 9, and 12. Serum PTH, bone-specific alkaline phosphatase, and N-telopeptide were measured at months 0, 3, 6, and 12. RESULTS Serum 25(OH)D was less than 30 ng/ml in 40% of subjects at baseline; after 12 months of vitamin D dosing, levels in 19% of subjects (n = 12, seven receiving daily doses and five monthly doses) remained low, despite compliance of more than 91%. D(2) dosing increased 25(OH)D(2) but produced a decline (P < 0.0001) in 25(OH)D(3). Substantial between-individual variation in 25(OH)D response was observed for both D(2) and D(3). The highest 25(OH)D observed was 72.5 ng/ml. Vitamin D administration did not alter serum calcium, PTH, bone-specific alkaline phosphatase, N-telopeptide, or 24-h urine calcium. CONCLUSIONS 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.

Diet- or warfarin-induced vitamin K insufficiency elevates circulating undercarboxylated osteocalcin without altering skeletal status in growing female rats

To further characterize the skeletal role of vitamin K (K), markers of bone turnover, density, and strength were evaluated in rats with diet‐ or warfarin (W)‐induced K insufficiency. One hundred two, 7‐week‐old, female rats were randomly assigned to low K (phylloquinone [K1], 20 μg/kg diet), control K (K1, 1300 μg/kg diet), low‐dose W (W, 1.5 mg/kg control diet), or high‐dose W plus K (W/K1, 10/100 mg/kg diet). Femur bone mineral content (BMC) and bone mineral density (BMD), plasma prothrombin time (PT) and prothrombin concentration (PC), and serum total alkaline phosphatase (ALP) and skeletal alkaline phosphatase (sALP) were measured at baseline and days 20, 40, 60, and 80. Serum total osteocalcin (OC) and undercarboxylated osteocalcin (ucOC) and femur length (FL) were measured at baseline and day 80. Left femur OC was measured and biomechanical testing of the right femur and third lumbar vertebral body was performed at day 80. Low dietary K elevated circulating ucOC (17% higher than control; p < 0.0001) at day 80. Furthermore, in both W groups, essentially all circulating OC was undercarboxylated and femur OC was lower than control (p < 0.0001). However, there was no change in femur percent ucOC, suggesting deposition of less newly synthesized OC. No between group differences were observed in PT, ALP, sALP, FL, BMC, BMD, or bone strength. In conclusion, skeletal K insufficiency can be induced by W or diet manipulation. This does not hinder peak bone mass attainment in female rats; however, W causes less newly synthesized OC to be deposited in bone. (J Bone Miner Res 2000;15:872–878)

Vitamin K-dependent carboxylase: influence of the "propeptide" region on enzyme activity.

The liver microsomal vitamin K-dependent carboxylase catalyzes the post-translational conversion of specific glutamyl to gamma-carboxyglutamyl (Gla) residues in precursor forms of a limited number of proteins. These proteins contain an amino-terminal extension (propeptide) that is presumed to serve as an enzyme recognition site to assure their normal processing. The free, noncovalently bound propeptide has also been shown to stimulate the in vitro activity of this enzyme. This peptide has now been shown to lower the app Km of a low-molecular-weight Glu site substrate while having no influence on the app Km of the other substrates, vitamin KH2, O2, and CO2/HCO3-. Propeptide addition was shown to have no influence on the ratio of the two products of the enzyme, Gla and vitamin K-2,3-epoxide. Stimulation of carboxylase activity by the propeptide from human factor X was observed in a number of rat tissues and in the liver of a number of different species. Stability of the enzyme in crude microsomal preparations was greatly enhanced by the presence of propeptide. These observations are consistent with the hypothesis that this region of the protein substrates for the carboxylase not only serves an enzyme recognition or docking function but also modulates the activity of the enzyme by altering the affinity for one of its substrates.

Bone loss detection in rats using a mouse densitometer.

Estrogen‐depletion bone‐loss studies often use ovariectomized (ovx) rats and measure bone mineral density in vivo or ex vivo using DXA. Recently, a portable densitometer (PIXImus) was developed for mouse research; however, its use in rats is unclear. This study compared the ability of PIXImus and a standard densitometer (DPXL) to detect ovx‐induced bone loss in rats both in vivo and ex vivo. Additionally, instrument accuracy was assessed by comparing measured bone mass with ash weight. Finally, the use of two distal femur regions of interest (ROI) to detect ovx‐induced bone loss was evaluated. Twenty‐three 6‐month‐old nulliparous female Sprague‐Dawley rats were randomly assigned to sham or ovx groups. Distal femur bone mineral density was assessed at baseline and at 1 and 2 months postoperatively, using a PIXImus and DPXL densitometer. At 3 months postoperatively, all animals were killed, and ex vivo femur scans obtained. Distal femur bone loss was demonstrable by 1 month post‐ovx using either densitometer. With the PIXImus, a 4‐mm ROI demonstrated greater bone loss (p < 0.05) than an 8‐mm ROI. Using the 4‐mm ROI, similar amounts of bone loss were detected by the PIXImus and DPXL: 22.2% and 22.4%, respectively, at 2 months post‐ovx. Total femur bone mineral content was overestimated by the PIXImus but highly correlated with the DPXL measurement (r = 0.988) and ash weight (r = 0.998). Given its comparability to standard DXA plus its rapid scan speed and portability, the PIXImus is useful in evaluating ovx‐induced osteopenia in rats.

Vitamin K Supplementation Does Not Affect Ovariectomy-induced Bone Loss in Rats

Vitamin K may be important in bone metabolism. Notably, high-dose menaquinone-4 (menatetrenone, MK4) has been reported to reduce ovariectomy (ovx)-induced bone loss in rats and to decrease osteoporotic fracture in postmenopausal women. However, it is unclear whether these beneficial effects reflect a physiologic effect of vitamin K, or indicate direct pharmacologic activity of MK4. To further evaluate this, 60 6-month-old nulliparous Sprague-Dawley rats were randomized by distal femur bone mineral density (BMD) in a 3:1 ratio to ovx or sham groups. The sham and one ovx groups diet contained 1% calcium and 1300 microg/kg of vitamin K1, phylloquinone. Diets of the other two ovx groups were supplemented with 882 mg phylloquinone or MK4 per kilogram chow. Distal femur bone mineral density (DFBMD) in an 8 mm region of interest was measured at baseline, 1 and 3 months postoperatively, utilizing dual-energy X-ray absorptiometry (DXA). All animals were killed at 3 months, their right femurs excised, ex vivo BMD measured by DXA, and biomechanical testing performed. No effect of phylloquinone or MK4 supplementation on ovx-induced bone loss was observed. Specifically, DFBMD declined 10.5%, 9.2%, and 11.2% at 1 month and 14.4%, 10.6%, and 13.9% at 3 months in the ovx control, high phylloquinone, and high MK4 groups, respectively. In addition, serum osteocalcin was elevated by ovx; this was not altered by phylloquinone or MK4. Finally, femoral biomechanical properties were not affected by phylloquinone or MK4. To conclude, in this study, neither high-dose phylloquinone nor MK4 reduced the ovx-associated increase in bone turnover or decline in DFBMD.

Use of a questionnaire to assess vitamin D status in young adults

OBJECTIVE We hypothesized that young adults would commonly have vitamin D deficiency and that a questionnaire could help identify subjects with the condition. DESIGN Between January and May 2004, we administered a questionnaire to a convenience sample of young adults. We measured each participants serum level of 25-hydroxyvitamin D (25(OH)D) using a chemiluminescent assay and defined deficiency as serum 25(OH)D < 16 ng/ml. SETTING AND SUBJECTS We recruited young adults living in Madison, Wisconsin without pre-existing conditions affecting vitamin D and/or Ca metabolism. RESULTS One hundred and eighty-four adults (mean age 24 years, 53 % women, 90 % Caucasian) participated in the study. Nearly three in four adults (71 %) had 25(OH)D level <30 ng/ml and 26 % were vitamin D-deficient. In multivariate analysis, persons reporting a suntan (OR = 0.24, 95 % CI 0.09, 0.63, P = 0.004), tanning booth use (OR = 0.09, 95 % CI 0.02, 0.43, P = 0.002) and daily ingestion of two or more servings of milk (OR = 0.21, 95 % CI 0.09, 0.48, P < 0.001) were less likely to be deficient. These three questions provided a sensitivity and specificity of 79 % and 78 %, respectively, for the presence of deficiency. CONCLUSIONS The questionnaire is moderately useful to identify young adults likely to be vitamin D-deficient. Additional revisions of the questionnaire may improve its ability to predict vitamin D deficiency.

Between‐Meal Risedronate Does Not Alter Bone Turnover in Nursing Home Residents

OBJECTIVES: To assess the effect of between‐meal weekly risedronate and daily calcium 630 mg and vitamin D 400 IU on bone turnover markers.

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

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.

Effect of N-methyl-thiotetrazole on rat liver microsomal vitamin K-dependent carboxylation.

The use of a number of antibiotics which contain an N-methyl-thiotetrazole (NMTT) side chain has been reported to be associated with an increased incidence of hypoprothrombinemia. The suggested role of NMTT as an inhibitor of the liver microsomal vitamin K-dependent carboxylase has been investigated. In standard incubations, NMTT had no effect on carboxylation when vitamin KH2 was a substrate but was a weak inhibitor when [vitamin K + NADH] was a substrate. Microsomal vitamin K reductases, however, were not inhibited by NMTT. Preincubation of the incubation mixture with NADH and NMTT resulted in inhibition of carboxylase activity when either vitamin KH2 or [vitamin K + NADH] was the substrate. A fraction of the microsomal membrane which was not readily solubilized by dilute detergent protected the enzyme from this inhibition. The data suggest that NMTT is metabolized to an active inhibitor or is able to covalently inactivate the enzyme in the presence of NMTT. The vitamin K responsiveness of the clinically observed hypoprothrombinemia suggests that it is not related to this in vitro inhibition of the vitamin K-dependent carboxylase.

Vitamin K-dependent carboxylase: Synthesis of an inhibitor of the glutamyl binding site

Liver microsomes contain a vitamin K and O2‐dependent carboxylase that converts peptide‐bound glutamyl residues to γ‐carboxyglutamyl residues. The peptide Boc‐O‐phospho—Ser‐O‐phospho—Ser—Leu‐OMe has now been synthesized. This peptide inhibits the carboxylation of endogenous protein precursors by a detergent‐solubilized preparation of the carboxylase and is an apparent competitive inhibitor of the carboxylation of Phe—Leu—Glu—Glu—Leu.