Calbindin-D9K immunolocalization and vitamin D-dependence in the bone of growing and adult rats

This report presents evidence for the presence of the vitamin D-dependent calcium-binding protein, calbindin-D9K, in bone cells and matrix. In undecalcified frozen sections of growing and adult rat bone, calbindin-D9K was immunohistochemically localized in trabecular bone of the epiphysis and metaphysis and in cortical bone of the diaphysis. It was found within the cytoplasm of osteocytes, of osteoblasts lining the osteoid, and osteoblasts inside the osteoid seams. It was also found in the osteoblast processes and the anastomosed reticulum of the processes connecting the osteocytes with each other. Extracellularly, calbindin-D9K immunoreactivity was present in compact cortical bone in the areas of the mineralized matrix surrounding the osteocyte lacunae, and in the pericanalicular walls containing the cell processes. Calbindin-D9K immunoreactivity was low or absent from the cytoplasm of osteocytes in trabecular bone from severely vitamin D-deficient rats and restored in vitamin D-deficient rats given a single dose of 1,25(OH)2-VitD3. Thus, the synthesis of immunoreactive calbindin-D9K by osteoblasts and osteocytes in trabecular bone is vitamin D-dependent. The presence of immunoreactive calbindin-D9K in the osteocytes and their cell processes suggests that this calcium-binding protein is involved in the calcium fluxes regulating bone calcium homeostasis. Its localization in osteoblasts involved in bone formation and in their cell processes suggests that it has a role in the calcium transport from these cells towards the sites of active bone mineralization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vitamin K deficiency inhibits mineralization and enhances deformity in vertebrae of haddock (Melanogrammus aeglefinus L.)

Vitamin K has been known to regulate bone formation through osteocalcin synthesis by osteoblasts, which is important for mineralization and bone structure. The mechanism underlying the relationship of vitamin K with the changes of microanatomy is not fully understood, and our goal is to test whether bone deformities develop in association with vitamin K deficiency. Fish were fed a semi-purified diet containing either devoid (0.00 mg/kg diet) or adequate (40.0 mg/kg diet supplemented but 20.8 mg/kg analyzed) levels of vitamin K (menadione sodium bisulphite) for 20 weeks. At the end of 8 and 20 weeks, fish were subjected to gross examination and X-ray, and mineral content of the vertebrae was measured. The vertebrae were also subjected to histological, histomorphometric and enzyme histochemical examinations to determine the bone formation and resorption. Vitamin K deficiency primarily decreased bone mineralization and subsequently a decrease in bone mass thus resulted in an increased susceptibility to bone deformity. The occurrence of bone deformities coincided with an increased amount of osteoid tissue and decreased bone mineral content. Number of osteoblasts and osteoclasts were not affected by dietary vitamin K. In conclusion, vitamin K deficiency can impair bone mineralization and enhances bone deformities.     

Vitamin K therapy for cortical bone fragility caused by reduced mechanical loading in a child with hemiplegia

Fractures frequently occur at cortical bone sites in children with cerebral palsy, but there is no established therapy. We previously found that treatment with vitamins D and K increased cortical bone mass in children with severe physical disability, and have hypothesized that vitamin K could play a significant role in pediatric cortical bones under conditions with reduced mechanical loading. In the present case report, we treated a right hemiplegic ambulant eight-year-old boy with oral vitamin K (15 mg per day) for eight months. Cortical bone geometries at mid-diaphyseal sites in bilateral tibiae were evaluated before and after the treatment. The cross-sectional total, bone and marrow areas of non-hemiplegic tibia increased by 8.8%, 7.4% and 12.0%, respectively, while those of hemiplegic tibia changed by 9.0%, 14.9% and -3.4%, respectively. As a result, the polar moment of inertia, an indicator of the resistance to torsion forces, increased by 13.0% in the non-hemiplegic tibia and by 63.7% in the hemiplegic tibia. Vitamin K may restrict cortical bone fragility, caused by reduced mechanical loading, through its actions at the endosteal bone marrow interface. Further studies are needed to confirm these findings and to clarify the mechanisms involved.     

Bone markers during a 6-month space flight: effects of vitamin K supplementation

Rapid bone loss is a serious health problem for astronauts during long lasting missions in space. We have recorded the changes of biochemical markers for bone metabolism in one of the astronauts during the 6-month space flight of the EUROMIR-95 mission. Immediately after launch both bone resorption markers and urinary calcium excretion increased about two fold, whereas bone formation markers remained unchanged. After 12 1/2 weeks the astronaut received vitamin K1 (10 mg/day for 6 weeks). Vitamin K is known to be involved in the formation of gamma-carboxyglutamate (Gla) in proteins, such as the calcium-binding bone Gla-proteins osteocalcin and matrix Gla-protein. Concomitant with the start of vitamin K treatment, the calcium-binding capacity of osteocalcin increased, and so did the urinary excretion of free Gla. This is suggestive for a subclinical vitamin K-deficiency in the astronaut before vitamin K-supplementation. During periods of high vitamin K status markers for bone formation (osteocalcin and bone alkaline phosphatase) had increased as compared to the first part of the flight. The mean increases were 14 and 23%, respectively. Our data suggest that increased intake of vitamin K may contribute to counteracting microgravity-induced loss of bone mass during long lasting space missions, but need confirmation in more astronauts.     

Vitamin K in combination with other biochemical markers to diagnose osteoporosis.

The significance of a multiparametric classification approach of vitamin K is analysed to differentiate premenopausal (CTRL), postmenopausal non-osteoporotic (nOSP) and osteoporotic (OSP) women. Data records of women between 28 and 74 years of age were used for evaluation. Bone mineral density was determined by quantitative computed tomography of the lumbar spine using the T-score to diagnose osteoporosis. Vitamin K and biochemical markers of bone formation and resorption--alkaline phosphatase (AP), bone alkaline phosphatase (bAP), osteocalcin (OC), undercarboxylated osteocalcin (ucOC), procollagen type I carboxyterminal propeptide (PICP), pyridinoline (PYD), deoxypyridinoline (DPD), N-terminal cross-linked telopeptide of type I collagen (NTx) and bone sialo protein (BSP)--were analysed in all women on days 1 and 42. Vitamin K was significantly lower in the OSP group versus nOSP and CTRL. The odds ratio results revealed the following: vitamin K, 16.7; PYD, 7.5; NTx, 6.0; DPD, 2.7; and ucOC, 2.7. Vitamin K represented a sensitivity rate of 64% and a specificity rate of 82%. In the receiver operating curve analysis, vitamin K reached the highest area under curve (AUC) score. The combination of vitamin K and AP, bAP and PYD resulted in increased AUC scores (>0.9). The parameter combination of vitamin K/PYD and vitamin K/bAP demonstrated a sensitivity rate of 75-88%, with a specificity rate of more than 82%. The data suggests that a combination of vitamin K with other biochemical bone indices might be a useful tool for assessing bone metabolism, especially in metabolic bone diseases such as osteoporosis.     

A potential biotechnological process for the sustainable production of vitamin K1

The primary objective of this review is to propose an approach for the biosynthesis of phylloquinone (vitamin K₁) based upon its known sources, its role in photosynthesis and its biosynthetic pathway. The chemistry, health benefits, market, and industrial production of vitamin K are also summarized. Vitamin K compounds (K vitamers) are required for the normal function of at least 15 proteins involved in diverse physiological processes such as coagulation, tissue mineralization, inflammation, and neuroprotection. Vitamin K is essential for the prevention of Vitamin K Deficiency Bleeding (VKDB), especially in neonates. Increased vitamin K intake may also reduce the severity and/or risk of bone fracture, arterial calcification, inflammatory diseases, and cognitive decline. Consumers are increasingly favoring natural food and therapeutic products. However, the bulk of vitamin K products employed for both human and animal use are chemically synthesized. Biosynthesis of the menaquinones (vitamin K₂) has been extensively researched. However, published research on the biotechnological production of phylloquinone is restricted to a handful of available articles and patents. We have found that microalgae are more suitable than plant cell cultures for the biosynthesis of phylloquinone. Many algae are richer in vitamin K₁ than terrestrial plants, and algal cells are easier to manipulate. Vitamin K₁ can be efficiently recovered from the biomass using supercritical carbon dioxide extraction.     

Effects of dietary vitamin k levels on bone quality in broilers

This study was conducted to evaluate the effects of dietary vitamin K (menadione) on bone quality in cage-raised broilers. Three hundred and sixty male broilers were randomly allotted to one of six treatments, with six replicate pens per treatment and 10 chicks per pen. Broilers were fed one of six diets including a control diet or the control diet plus graded levels of vitamin K (0.5 mg/kg, 2 mg/kg, 8 mg/kg, 32 mg/kg and 128 mg/kg). Water and feed were provided ad libitum during the 7-week experimental period. Results indicated that vitamin K supplementation of broilers diets significantly effected bone quality and feed efficiency. The treatment containing vitamin K at 8 mg/kg improved growth performance (during weeks 6 - 7) and bone quality (during weeks 0 - 3). In our study, hydroxyapatite binding capacity of serum osteocalcin (during weeks 0 - 3), bone breaking strength, bone flexibility, bone ash weight increased linearly (P < 0.05) and bone mineral density, bone mineral content increased quadratically (P < 0.05) with increasing supplementation of vitamin K. In conclusion, to gain optimum bone quality and broiler performance, our studies suggest that the concentration of vitamin K in broilers diets should be 8 mg/kg, 2 mg/kg, and 2 mg/kg, for the starter, grower and finisher phases, respectively. Furthermore, it was shown that the starter period is an important phase for improving bone quality. In addition, this study validated the mechanism of vitamin K effects on bone quality. Vitamin K boosts the carboxylation of osteocalcin and decreases the concentration of serum undercarboxylated osteocalcin enhancing hydroxyapatite binding capacity of serum osteocalcin and improving bone quality.     

Glutamyl substrate-induced exposure of a free cysteine residue in the vitamin K-dependent gamma-glutamyl carboxylase is critical for vitamin K epoxidation.

The vitamin K-dependent carboxylase catalyzes the posttranslational modification of glutamic acid to gamma-carboxyglutamic acid in the vitamin K-dependent proteins of blood and bone. The vitamin K-dependent carboxylase also catalyzes the epoxidation of vitamin K hydroquinone, an obligatory step in gamma-carboxylation. Using recombinant vitamin K-dependent carboxylase, purified in the absence of propeptide and glutamic acid-containing substrate using a FLAG epitope tag, the role of free cysteine residues in these reactions was examined. Incubation of the vitamin K-dependent carboxylase with the sulfhydryl-reactive reagent N-ethylmaleimide inhibited both the carboxylase and epoxidase activities of the enzyme. This inhibition was proportional to the incorporation of radiolabeled N-ethylmaleimide. Stoichiometric analyses using [(3)H]-N-ethylmaleimide indicated that the vitamin K-dependent carboxylase contains two or three free cysteine residues. Incubation with propeptide, glutamic acid-containing substrate, and vitamin K hydroquinone, alone or in combination, indicated that the binding of a glutamic acid-containing substrate to the carboxylase makes accessible a free cysteine residue that is important for interaction with vitamin K hydroquinone. This is consistent with our previous observation that binding of a glutamic acid-containing substrate activates vitamin K epoxidation and supports the hypothesis that binding of the carboxylatable substrate to the enzyme results in a conformational change which renders the enzyme catalytically competent.     

Synthesis of novel vitamin K derivatives with alkylated phenyl groups introduced at the ω-terminal side chain and evaluation of their neural differentiation activities

Vitamin K is an essential cofactor of γ-glutamylcarboxylase as related to blood coagulation and bone formation. Menaquinone-4, one of the vitamin K homologues, is biosynthesized in the body and has various biological activities such as being a ligand for steroid and xenobiotic receptors, protection of neuronal cells from oxidative stress, and so on. From this background, we focused on the role of menaquinone in the differentiation activity of progenitor cells into neuronal cells and we synthesized novel vitamin K derivatives with modification of the ω-terminal side chain. We report here new vitamin K analogues, which introduced an alkylated phenyl group at the ω-terminal side chain. These compounds exhibited potent differentiation activity as compared to control.     

Efficient synthesis and biological evaluation of omega-oxygenated analogues of vitamin K2: study of modification and structure-activity relationship of vitamin K2 metabolites

Novel omega-oxygenated vitamin K2 analogues, which are candidates for metabolites of vitamin K2 homologues, were efficiently synthesized and their apoptosis-inducing activity was evaluated. We revealed that some of those analogues were biologically active and the side-chain part played an important role in apoptosis-inducing activity. Our results can provide useful information to develop the structure-activity relationship of vitamin K2 analogues for new drugs based on vitamin K.     

Effects of vitamin K2 administration on calcium balance and bone mass in young rats fed normal or low calcium diet

OBJECTIVE: The purpose of this study was to examine the effects of vitamin K2 administration on calcium balance and bone mass in young rats fed a normal or low calcium diet. METHODS: Forty female Sprague-Dawley rats, 6 weeks of age, were randomized by stratified weight method into four groups with 10 rats in each group: 0.5% (normal) calcium diet, 0.1% (low) calcium diet, 0.5% calcium diet + vitamin K2 (menatetrenone, 30 mg/100 g chow diet), and 0.1% calcium diet + vitamin K2. After 10 weeks of feeding, serum calcium and calciotropic hormone levels were measured, and intestinal calcium absorption and renal calcium reabsorption were evaluated. Bone histomorphometric analyses were performed on cortical bone of the tibial shaft and cancellous bone of the proximal tibia. RESULTS: Feeding a low calcium diet induced hypocalcemia, increased serum parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D [1,25(OH)2D] levels with decreased serum 25-hydrovyvitamin D [25(OH)D] level, stimulated intestinal calcium absorption and renal calcium reabsorption, and reduced cortical bone mass as a result of decreased periosteal bone gain and enlarged marrow cavity, but did not significantly influence cancellous bone mass. Vitamin K2 administration in rats fed a low calcium diet stimulated renal calcium reabsorption, retarded the abnormal elevation of serum PTH level, increased cancellous bone mass, and retarded cortical bone loss, while vitamin K2 administration in rats fed a normal calcium diet stimulated intestinal calcium absorption by increasing serum 1,25(OH)2D level, and increased cortical bone mass. CONCLUSION: This study clearly shows the differential response of calcium balance and bone mass to vitamin K2 administration in rats fed a normal or low calcium diet.     

Vitamin D in the aging musculoskeletal system: an authentic strength preserving hormone

Until recently, vitamin D was only considered as one of the calciotrophic hormones without major significance in other metabolic processes in the body. Several recent findings have demonstrated that vitamin D plays also a role as a factor for cell differentiation, function and survival. Two organs, muscle and bone, are significantly affected by the presence, or absence, of vitamin D. In bone, vitamin D stimulates bone turnover while protecting osteoblasts of dying by apoptosis whereas in muscle vitamin D maintains the function of type II fibers preserving muscle strength and preventing falls. Furthermore, two major changes associated to aging: osteoporosis and sarcopenia, have been also linked to the development of frailty in elderly patients. In both cases vitamin D plays an important role since the low levels of this vitamin seen in senior people may be associated to a deficit in bone formation and muscle function. In this review, the interaction between vitamin D and the musculoskeletal components of frailty are considered from the basic mechanisms to the potential therapeutic approach. We expect that these new considerations about the importance of vitamin D in the elderly will stimulate an innovative approach to the problem of falls and fractures which constitutes a significant burden to public health budgets worldwide.     

Bone mineral density in patients with apolipoprotein E type 2/2 and 4/4 genotype

The peak bone mass and the rate of bone loss are in part genetically determined. It has been suggested that bone mineral density (BMD) may be related to allelic variation in the apolipoprotein E (ApoE) gene locus. ApoE is important in the receptor-mediated clearance of chylomicron particles from the plasma, Apo E4 having the highest and Apo E2 the lowest receptor affinity. Chylomicrons are the main carrier of vitamin K in the plasma; vitamin K plays an important role in the carboxylation of osteocalcin. We have tested the hypothesis that persons with E4 variant would have lower BMD and increased bone turnover than those with E2 variant. A total of 18 ApoE 2/2 and ApoE 4/4 homozygotes were selected from 873 patients who were examined for the ApoE genotype. BMD in lumbar vertebral, femoral neck and distal forearm was measured and plasma concentrations of osteocalcin and C-terminal fragments of collagen (CTx) were determined. BMD values (expressed as T-score) at the three specified sites were -0.12+/-1.72, -0.52+/-1.32 and -0.52+/-0.81 in ApoE 2/2 group and -0.24+/-1.22, 0.00+/-0.84 and -0.17+/-1.07 in the ApoE 4/4 group. Plasma osteocalcin and CTx were within normal limits in both groups. In conclusion, we did not observe any association of ApoE genotype with BMD and biochemical markers of bone metabolism in ApoE 2/2 and ApoE 4/4 homozygotes.     

Elucidation of the mechanism producing menaquinone-4 in osteoblastic cells

Vitamin K is an essential nutrient and a cofactor for the carboxylation of specific glutamyl residues of proteins to γ-glutamyl residues, which activates osteocalcin related to bone formation. Among vitamin K homologues, menaquinone-4 (MK-4) is the most active biologically, up-regulating the gene expression of bone markers, and thus has been clinically used in the treatment of osteoporosis in Japan. Recently, we confirmed that MK-4 was converted from dietary phylloquinone (PK), and then accumulated in various tissues at high concentrations. This system should play an important role in biological functions including bone formation, however, the pathway by which MK-4 is converted remains unclear. In this study, we studied the mechanism of MK-4’s conversion with chemical techniques using deuterated analogues.     

Warfarin administration reduces synthesis of sulfatides and other sphingolipids in mouse brain

The modulation of phosphosphingolipid synthesis by vitamin K depletion has been observed in the vitamin K-dependent microorganism, Bacteriodes levii. When cultured briefly without the vitamin, a reduction occurred in the activity of the first enzyme of the sphingolipid pathway, 3-ketodihydrosphingosine synthase. In this report, 16-day-old mice were treated with the vitamin K antagonist, warfarin. Brain microsomes from these animals showed a 19% reduction in synthase activity. Mice treated with warfarin for 2 weeks showed a major reduction in sulfatide level (42%), with a lesser degree or no reduction in levels of gangliosides and cerebrosides. In further experiments, mice were treated with warfarin for 2 weeks and a group was then injected with vitamin K1 (aquamephyton) for 3 days. Enzyme activity returned to a normal level within 2-3 days. Sulfatide levels had increased 33% in the vitamin K-injected group and ganglioside levels also increased, where levels of cerebrosides and sphingomyelin declined. Sulfatide synthesis determined by [35S] sulfate incorporation, showed a 52% increase in incorporation following administration of vitamin K for 3 days. These results suggest a role for vitamin K in the biosynthesis of sulfatides and other sphingolipids in brain. This putative role could be by post-translational protein modification analogous to the role of vitamin K in other systems.     

Exposure of the pregnant rat to warfarin and vitamin K1: an animal model of intraventricular hemorrhage in the fetus

Pregnant Sprague-Dawley rats were given daily oral doses of sodium warfarin (100 mg/kg) and concurrent intramuscular injections of vitamin K1 (10 mg/kg). This dosing regimen did not have any apparent deleterious effect on the dams and did not affect the fetuses when administered from day 1 to day 12 of pregnancy. However, similar treatment from day 9 to 20 caused hemorrhage in the fetuses examined on day 21 of gestation. There were no hemorrhages in the control fetuses from dams receiving vitamin K1 only. The lowest effective dose of warfarin, in conjunction with daily doses of vitamin K1, was 3 mg/kg. This dose caused hemorrhage in 28% of fetuses; the incidence of affected fetuses was not further increased by doses of warfarin up to 100 mg/kg. Hemorrhages affected the fetal brain, face, eyes, and ear and occasionally the limbs. Brain hemorrhages were frequently intraventricular and caused various degrees of hydrocephaly. Bony defects were not a feature of prenatal exposure to warfarin. These results show that prenatal exposure of the rat to warfarin and vitamin K duplicates the hemorrhagic abnormalities and pathology associated with prenatal exposure to warfarin in the human. It did not induce bony or facial defects probably because the vitamin K-dependent components of bone development occur postnatally in the rat. This model should allow detailed determination of the role of vitamin K-dependent proteins in development.