Influence of plasma calcium and vitamin D on bone collagen. Effects on lysine hydroxylation and crosslink formation.

In chick bone collagen the degree of lysine hydroxylation and the magnitude of the crosslink ratio dihydroxylysinonorleucine/hydroxylysinonorleucine were both found to be inversely related to the concentration of plasma calcium. Lysine hydroxylation was also affected by a second factor related to vitamin D status.     

Differences between proline and lysine hydroxylations in their inhibition by zinc or by ascorbate deficiency during collagen synthesis in various cell types

The addition of Zn²⁺ inhibited lysine hydroxylation markedly less effectively than it did proline hydroxylation in chick embryo tendon cells, 3T6 fibroblasts and lysyl hydroxylase-deficient Ehlers-Danlos Syndrome Type VI fibroblasts. With low Zn²⁺ concentrations, a similar difference was also seen in chick embryo cartilage cells, whereas with high concentrations both hydroxylations were affected to the same extent in this cell type. Ascorbate deficiency likewise had a much less effect on lysine than proline hydroxylation when studied with 3T6 fibroblasts. As these two effectors involve quite different mechanisms, it is suggested that relative insensitivity to inhibition may be a property of lysine hydroxylation seen in many cell types with a number of agents.Studies on the mechanism of the difference in the inhibition indicates that the phenomenon is probably not due to differences in the kinetic constants of Zn²⁺ and ascorbate for the two enzymes. Neither is it probably to any major extent due to delayed procollagen triple helix formation nor a difference in the location of the two hydroxylases within the cisternae of the rough endoplasmic reticulum. The difference similarly cannot be explained solely by an excess of lysyl hydroxylase in the cell. It may thus be due either to some other intrracellular property or to the combined effect of several factors.     

Effect of vitamin C deficiency on hydroxylation of trimethylaminobutyrate to carnitine in the guinea pig

The effect of ascorbate deficiency on carnitine biosynthesis was investigated in young male guinea pigs. Liver and skeletal muscle carnitine levels were reduced in scorbutic animals. Heart and kidney concentrations remained unchanged. ¹⁴C-labeled 4-N-trimethylaminobutyrate was administered to control, pair-fed and scorbutic animals and distribution of isotope in compound present in the liver after 30 min was determined. Control and pair-fed animals converted trimethylaminobutyrate to carnitine faster than scorbutic animals. Injection of ascorbate with the [¹⁴C]trimethylaminobutyrate reversed the decline in trimethylaminobutyrate hydroxylase (EC 1.14.11.1) activity in scorbutic animals.     

Effects of phospholipase D during cultured osteoblast mineralization and bone formation

Mammalian phospholipase D (PLD) mostly hydrolyzes phosphatidylcholine producing phosphatidic acid. PLD activity was previously detected in different osteoblastic cell models, and was increased by several growth factors involved in bone homeostasis. To confirm possible actions of PLD isoforms during mineralization process, we analyzed their effects in osteoblastic cell models and during bone formation. PLD1 expression, along with PLD activity, increased during differentiation of primary osteoblasts and Saos-2 cells, and peaked at the onset of mineralization. Subsequently, both PLD1 expression and PLD activity decreased, suggesting that PLD1 function is regulated during osteoblast maturation. In contrast, PLD2 expression was not significantly affected during differentiation of osteoblasts. Overexpression of PLD1 in Saos-2 cells improved their mineralization potential. PLD inhibitor Halopemide or PLD1-selective inhibitor, led to a decrease in mineralization in both cell types. On the contrary, the selective inhibitor of PLD2, did not affect the mineralization process. Moreover, primary osteoblasts isolated from PLD1 knockout (KO) mice were significantly less efficient in mineralization as compared with those isolated from wild type (WT) or PLD2 KO mice. In contrast, bone formation, as monitored by high-resolution microcomputed tomography analysis, was not impaired in PLD1 KO nor in PLD2 KO mice, indicating that the lack of PLD1 or that of PLD2 did not affect the bone structure in adult mice. Taken together, our findings indicate that PLD activity, especially which of PLD1 isoform, may enhance the mineralization process in osteoblastic cells. Nonetheless, the lack of PLD1 or PLD2 do not seem to significantly affect bone formation in adult mice.     

Synthesis of vitamin D3 analogues with A-ring modifications to directly measure vitamin D levels in biological samples

C-3-substituted 25-hydroxyvitamin D₃ analogues were synthesized as tools to directly measure levels of vitamin D in biological samples. The strategy involves vinyloxycarbonylation of the 3β-hydroxy group and formation of a carbamate bond with a hydroxyl or amino group at the end of the alkyl chain. Biotinylated conjugates of synthesized derivatives were generated to be linked with vitamin D binding protein (DBP). The spacer group present in the alkyl chain is important in the binding of antibodies to the analogue–DBP complex. When compared to 25-hydroxyvitamin D₃-DBP, the binding of some antibodies to the analogue–DBP complex of the 25-hydroxyvitamin D₃ derivative 10 that posses an 8-aminoctyl alkyl chain is significantly reduced, but this analogue displaced [26,27-³H]-25-hydroxyvitamin D₃ from DBP. In contrast, the 8-hydroxyoctyl alkyl chain analogue 9 showed less displacement.     

Effects of a calcium/vitamin D/Zinc combination on anti-osteoporosis in ovariectomized rats

BackgroundOsteoporosis is a major health problem in postmenopausal women, and characterized by deteriorated bone mass and micro-architecture. There have been some clinical trials demonstrating the beneficial effects of vitamin-D and some trace elements on calcium absorption and attenuation of osteoporosis development. However, effects of the combination of vitamin-D and zinc on calcium absorption and osteoporosis have not been adequately investigated.MethodsNetwork pharmacology was first performed to explore possible correlations between calcium/vitamin D/zinc and osteoporosis. Forty-nine female Sprague-Dawley rats (6 months old, 250 ± 20 g) were randomized into 7 experimental groups with 7 animals per group for the in vivo study, including one sham surgery control group, one ovariectomizing (OVX) group, and 5 OVX plus treatment groups. At the end of animal experiment, animal tibia and femur leg bones and blood were collected for H&E staining, bone microstructure analysis by a micro-CT, measurement of bone and serum Ca, P and Zn concentrations, and immunohistochemical detection of macrophage-colony stimulating factor receptor (M-CSFR) and receptor activator of nuclear factor-kappa B ligand (RANKL).ResultsThe network pharmacology analysis identified 57 candidate targets that were related to the osteoporosis-Ca/VitD/Zn interconnections. Further pathway analysis suggested that the combined treatment of Ca, VitD and Zn attenuated osteoporosis via modulation of metabolic pathways. We found that a therapy with Ca/VitD-M/Zn-M (73 mg/kg/day Ca, 0.6 g/kg/day VitD3 and 0.6 mg/kg/day zinc citrate) could significantly suppress the progression of osteoporosis in rats. After the Ca/VitD-M/Zn-M treatment, the ratio of bone volume/tissue volume, trabecular number and the trabecular thickness were all significantly elevated while the extent of trabecular separation was significantly reduced. Additionally, both serum calcium and bone calcium levels were significantly upregulated by the Ca/VitD/Zn treatment in a dose-dependent manner. The combination of Ca/VitD-M/Zn-M was superiou to either Ca/VitD-L/Zn-L or Ca/VitD-H/Zn-H treatment for such an effect. Moreover, the osteoporosis-associated M-CSFR and RANKL factors were both significantly downregulated by the Ca/VitD-M/Zn-M treatment in bone tissues of OVX rats.ConclusionsThe combined supplement of VitD and Zn facilitates the Ca(2 +) absorption and attenuates the development of osteoporosis via down-regulation of osteoporosis-associated factors M-CSFR and RANKL, thus potentially constitutes an alternative therapy for the postmenopausal osteoporosis.     

The case for improving vitamin D status

Evidence from both physiological experiments and randomized trials demonstrates that elevating vitamin D status above levels prevailing in the North American and European adult populations improves calcium absorption and reduces fall risk and osteoporotic fractures. Additionally observational data suggest that raising vitamin D status protects against various cancers and autoimmune disorders as well. Hence a strong case can be made for immediate improvement in vitamin D status of the general population.     

Stimulation of spd-glucose transport A novel effect of vitamin D on intestinal membrane transport

Vitamin D stimulates absorption of spd-glucose in chick jejunum and ileum by a specific action on the maximal velocity of Na⁺-gradient driven spd-glucose transport across the brush-border membrane of intestinal cells. Induction of spd-glucose transport by either vitamin D-3 or 1,25-dihydroxyvitamin D-3 in embryonic intestine can be blocked by inhibitors of RNA and protein synthesis.     

The effects of vitamin D deficiency on proteoglycan and hyaluronate constituents of chick bone

The effect of vitamin D deficiency on proteoglycan and hyaluronate constituents of cortical diaphyseal chick bone was studied. Proteoglycans in rachitic bone showed no significant change with respect to their size, composition, or amount relative to other extractable macromolecular components. In contrast, bone hyaluronate levels were raised in chicks fed on diets that were either vitamin D-deficient or depleted in calcium or phosphate, a 7-fold increase being seen in hypocalcaemic vitamin D-deficient chicks. This increase in hyaluronate was not directly related either to the absence of vitamin D or to abnormal levels of blood calcium or phosphate per se; hyaluronate levels are probably regulated by another factor, not yet identified, that is responsive to changes in vitamin D and mineral metabolism.     

Active vitamin D possesses beneficial effects on the interaction between muscle and bone

Vitamin D deficiency and advanced glycation end products (AGEs) are suggested to be involved in the pathogenesis of osteoporosis and sarcopenia. However, the effects of vitamin D and AGEs on myogenesis and the interaction between muscle and bone remains still unclear. We previously showed that osteoglycin (OGN) is secreted from myoblasts and stimulates osteoblastic differentiation, suggesting that it plays important roles in the interaction between muscle and bone. The aim of this study is thus to examine the effects of vitamin D and AGEs on myoblastic differentiation of C2C12 cells and osteoblastic differentiation of osteoblastic MC3T3-E1 cells through OGN expression. 1α,25-dihydroxyvitamin D₃ (1,25D) and eldecalcitol, an active vitamin D analog, induced the expression of MyoD, myogenin and OGN, and these effects were abolished by vitamin D receptor (VDR) suppression by siRNA in C2C12 cells. Moreover, conditioned medium from 1,25D-pretreated C2C12 cells stimulated the expression of type 1 collagen and alkaline phosphatase in MC3T3-E1 cells, compared to control medium from 1,25D-untreated C2C12 cells. In contrast, conditioned medium from VDR-suppressed and 1,25D-pretreated C2C12 cells showed no effects. AGE2 and AGE3 suppressed the expression of MyoD, myogenin and OGN in C2C12 cells. Moreover, 1,25D blunted the AGEs’ effects. In conclusion, these findings showed for the first time that active vitamin D plays important roles in myogenesis and muscle-induced osteoblastogenesis through OGN expression. Active vitamin D treatment may rescue the AGEs-induced sarcopenia as well as – suppressed osteoblastic differentiation via OGN expression in myoblasts.     

Comparative study on the thermostability of collagen I of skin and bone: Influence of posttranslational hydroxylation of prolyl and lysyl residues

Pepsin-solubilized collagen I from skin and bone was analyzed with regard to its thermal stability as a triple helical molecule in solution and afterin vitro fibril formation. Collagen I from human control bone was compared with samples showing deficiencies or surplus in the degree of hydroxylation of lysine. The helix to coil transitions were studied by circulardichroism measurements and limited trypsin digestion. Melting of fibrils from standardizedin vitro self-assembly was investigated turbidimetrically. Human control bone collagen I has a maximum transition rate (T _m ) at 43.3°C in 0.05% acetic acid. This is 1.9°C above control skin (T _m =41.4°C), most likely, due to a higher degree of prolyl hydroxylation—0.48 in bone vs. 0.41 in skin collagen I. Lysyl overhydroxylation of human and mouse bone collagen I appears to reduce theT _m slightly (1°C). Underhydroxylated bone collagen has aT _m which is 2°C below control. Melting temperatures ofin vitro formed fibrils are an indication for higher thermostability in parallel with an increase of lysyl hydroxylation. Accordingly, the melting temperature of such fibrils from human control skin, 49.3°C, exceeds control bone by 1.4°C. The degree of lysyl hydroxylation in these samples is 0.14 and 0.10, respectively. Further underhydroxylation (0.06) reduced it down to 45.4°C, while extensive overhydroxylation did not continue to increase the thermal stability of fibrils.     

Circannual rhythm of plasmatic vitamin D levels and the association with markers of psychophysical stress in a cohort of Italian professional soccer players

Adequate plasmatic Vitamin D levels are crucial to maintain calcium homeostasis and bone metabolism both in the general population and in athletes. Correct dietary supply and a regular sun exposure are fundamental for allowing the desired and effective fitness level. Past studies highlighted a scenario of Vitamin D insufficiency among professional soccer players in several countries, especially in North Europe, whilst a real deficiency in athletes is rare. The typical seasonal fluctuations of Vitamin D are wrongly described transversally in athletes belonging to teams that play at different latitudes and a chronobiologic approach studying the Vitamin D circannual rhythm in soccer players has not been described yet. Therefore, we studied plasma vitamin D, cortisol, testosterone, and creatin kinase (CK) concentrations in three different Italian professional teams training at the same latitude during a period of two consecutive competitive seasons (2013 and 2014). In this retrospective observational study, 167 professional soccer players were recruited (mean age at sampling 25.1 ± 4.7 years) and a total of 667 blood drawings were carried out to determine plasma 25(OH)D, serum cortisol, serum testosterone and CK levels. Testosterone to cortisol ratio (TC) was calculated based as a surrogate marker of overtraining and psychophysical stress and each athlete was drawn until a maximum of 5 times per season. Data extracted by a subgroup of players that underwent at least 4 sample drawings along a year (N = 45) were processed with the single and population mean cosinor tests to evaluate the presence of circannual rhythms: the amplitude (A), acrophase (Φ) and the MESOR (M) are described. In total, 55 players (32.9%) had an insufficient level of 25(OH)D during the seasons and other 15 athletes (9.0%) showed, at least once, a deficiency status of Vitamin D. The rhythmometric analyses applied to the data of Vitamin D revealed the presence of a significant circannual rhythm (p < 0.001) with the acrophase that occurred in August; the rhythms of Vitamin D levels were not different neither among the three soccer teams nor between competitive seasons. Cortisol, testosterone and TC showed significant circannual rhythms (p < 0.001): cortisol registered an acrophase during winter (February) while testosterone and TC registered their peaks in the summer months (July). On the contrary, CK did not display any seasonal fluctuations. In addition, we observed weak but significant correlations between 25(OH)D versus testosterone (r = 0.29 and p < 0.001), cortisol (r = ?0.27 and p < 0.001) and TC (r = 0.37 and p < 0.001). No correlation was detected between Vitamin D and CK. In conclusion, the correct chronobiologic approach in the study of annual variations of Vitamin D, cortisol and testosterone could be decisive in the development of more specific supplementation and injury prevention strategies by athletic trainers and physicians.     

Effect of vitamin D on the content of the stable crosslink, pyridinoline, in chick bone collagen.

The effect of vitamin D on the content of a crosslink, pyridinoline, in chick bone collagen was studied. One-day-old chicks were fed a synthetic diet with or without vitamin D for 6 weeks. No significant difference was observed between vitamin D-deficient and -supplemented chicks till 4 weeks, but at 5 and 6 weeks, the pyridinoline content in vitamin D-deficient chicks markedly increased as compared with that in vitamin D-supplememted chicks. Concomitantly, the collagen fiber of vitamin D-deficient chicks became less susceptible to proteolytic enzymes such as pepsin and papain. A possible relationship of these observations to the disturbance of bone remodeling in vitamin D deficiency was discussed.     

Effects of vitamin D supplementation and circuit training on indices of obesity and insulin resistance in T2D and vitamin D deficient elderly women

[Purpose]

The purpose of this study was to investigate the effects of vitamin D supplementation and circuit training on body composition, abdominal fat, blood lipids, and insulin resistance in T2D and vitamin D deficient elderly women.

[Methods]

Fifty-two elderly women were randomly assigned to either the vitamin D supplementation with circuit training group (D+T: n = 15), the circuit training group (T: n = 13), the vitamin D supplementation group (D: n = 11), or the control group (CON: n = 13). The subjects in D took vitamin D supplements at 1,200 IU per day for 12 weeks; the subjects in T exercised 3 to 4 times per week, 25 to 40 minutes per session for 12 weeks; and the subjects in D+T participated in both treatments. Subjects in CON were asked to maintain normal daily life pattern for the duration of the study. Body composition, abdominal fat, blood lipids, and surrogate indices for insulin resistance were measured at pre- and post-test and the data were compared among the four groups and between two tests by utilizing two-way ANOVA with repeated measures. The main results of the present study were as follows:

[Results]

1) Body weight, fat mass, percent body fat, and BMI decreased significantly in T, whereas there were no significant changes in the variables in D and CON. Lean body mass showed no significant changes in all groups. 2) TFA and SFA decreased significantly in T, whereas there were no significant changes in the variables in D and CON. The other abdominal fat related variables showed no significant changes in all groups. 3) TC, TG, HDL-C, and LDL-C showed improvements in T, whereas there were no significant changes in the variables in D and CON. 4) Fasting glucose, fasting insulin, and HOMA-IR tended to be lower in D+T.

[Conclusion]

It was concluded that the 12 weeks of vitamin D supplementation and circuit training would have positive effects on abdominal fat and blood lipid profiles in T2D and vitamin D deficient elderly women. Vitamin D supplementation was especially effective when it was complemented with exercise training.     

维生素D及其受体在帕金森病中的作用

帕金森病(Parkinson's disease,PD)是一种常见的中枢神经系统退行性疾病,引起帕金森病的发病机制至今尚未明确。帕金森病患者及老年人普遍存在维生素D缺乏,这可能是帕金森病的重要发病机制之一。由于维生素D具有免疫调节,抗氧化,调节神经营养因子,降低神经毒性的功能,能同时针对几种导致神经退行性病变因素发挥作用,特别是老年人纠正维生素D缺乏可能会阻止神经元的损失和PD相关的认知功能下降。因此补充维生素D可能成为治疗PD的方法。近年来研究发现,维生素D受体基因多态性与帕金森病的发病有相关性。该文就维生素D及其受体在帕金森病中可能发生的保护作用及其机制作一综述。     

Rapid intracellular degradation of newly synthesized collagen by bone cells Effect of dichloromethylenebisphosphonate

Experiments were carried out to determine whether bone cells isolated from rat calvaria degrade newly synthesized collagen intracellularly prior to secretion and to assess the effect of dichloromethylenebisphosphonate, a compound shown to stimulate collagen synthesis during this event. The findings indicate that isolated bone cells grown in culture degraded a proportion (average 16%) of newly synthesizes collagen prior to secretion. This process was markedly reduced by exposure to dichloromethylenebisphosphonate in a dose-related manner. Concomitantly with the observed decrease of degradation, an increase of collagen synthesis was detected as determined by the incorporation of [³H]proline into collagenase-digestible proteins or by the conversion of [³H]proline into [³H]hydroxyproline. No similar enhancement on total non-collagenous protein synthesis was evident. Dichloromethylenebisphosphonate did not influence the extracellular degradation of collagen. Although the reduction in intracellular degradation accounted only for part of the bisphosphonate mediated increase in net collagen synthesis, it is conceivable that the rate of collagen synthesis is regulated, at least in part, by mechanisms that modulate the level of intracellular degradation.     

The effect of calcium and vitamin D supplementation on osteoporotic rabbit bones studied by vibrational spectroscopy

Fourier transform infrared spectroscopy is utilized to examine the effects of increased calcium, vitamin D, and combined calcium-vitamin D supplementation on osteoporotic rabbit bones with induced inflammation. The study includes different bone sites (femur, tibia, humerus, vertebral rib) in an effort to explore possible differences among the sites. We evaluate the following parameters: mineral-to-matrix ratio, carbonate content, and non-apatitic species (labile acid phosphate and labile carbonate) contribution to bone mineral. Results show that a relatively high dose of calcium or calcium with vitamin D supplementation increases the bone mineralization index significantly. On the other hand, vitamin D alone is not as effective in promoting mineralization even with high intake. Mature B-type apatite was detected for the group with calcium supplementation similar to that of aged bone. High vitamin D intake led to increased labile species concentration revealing bone formation. This is directly associated with the suppression of pro-inflammatory cytokines linked to induced inflammation. The latter is known to adversely alter bone metabolism, contributing to the aetiopathogenesis of osteoporosis. Thus, a high intake of vitamin D under inflammation-induced osteoporosis does not promote mineralization but suppresses bone resorption and restores metabolic balance.     

Evaluation of serum parameters relevant to vitamin D status in tamarins

Serum levels of 25(OH)D, alkaline phosphatase (AP), and parathormone (PTH) were evaluated to investigate the vitamin D requirement of Saguinus fuscicollis. Diets with various vitamin D content were fed 4 weeks and longer. The values of 25(OH)D (30-300 nmol/l), AP (less than 300 U/l), and PTH (less than or equal to 1,000 equl/l) considered as normal were obtained with 2,000 IU D3/kg diet, or 33 IU/animal/day, which we regard as the level required. Animals depleted of vitamin D for 215 days developed a secondary hyperparathyroidism.     

Effects of vitamin D2-fortified shiitake mushroom on bioavailability and bone structure

Bioavailability and bone loss inhibitory effects of vitamin D₂ derived from UV-irradiated shiitake mushroom were determined in vivo. The effect of the absence of ovaries on the bioavailability of vitamin D₂ and bone structure was also investigated. Sham operated (sham) and ovariectomized (OVX) rats were divided in 3 groups according to their diets, i.e. control: only vitamin D-deficient diets; UV(X): vitamin D-deficient diets with non-irradiated mushroom powder; UV(O): vitamin D-deficient diets with irradiated mushroom powder. The obtained results showed that vitamin D₂ from shiitake mushroom was able to increase bone mineral density and trabecular bone structure of femur bone as well as its bioavailability. The absence of estrogen induced adverse effects not only on bioavailability of vitamin D₂ but also on trabecular bone. In conclusion, vitamin D₂-fortified shiitake mushroom might help postmenopausal women increase vitamin D₂ bioavailability and retard trabecular bone loss.

Abbreviations: OVX: ovariectomized; 25(OH)D: 25-hydroxyvitamin D; 1,25(OH)2D: 1,25-dihydroxyvitamin D; BMD: bone mineral density; micro-CT: micro computed tomography; RSM: response surface methodology; RP-HPLC: Reverse phase-high performance liquid chromatography; MS/MS: tandem mass spectrometry; E2: estradiol; NTx: N-terminal telopeptide of type I collagen; BV/TV: bone volume/total volume; BS/BV: bone surface/bone volume; Tb.Th: trabecular thickness; Tb.Sp: trabecular separation.