Natalia Umanskaya

The role of hyperhomocysteinemia as well as folate, vitamin B(6) and B(12) deficiencies in osteoporosis: a systematic review.

Abstract Hyperhomocysteinemia (HHCY) has been suggested as a new risk factor for osteoporosis. Recent epidemiological, clinical and experimental studies provide a growing body of data, which is reviewed in this article. Epidemiological and (randomized) clinical trials suggest that HHCY increases fracture risk, but has minor effects on bone mineral density. Measurement of biochemical bone turnover markers indicates a shift of bone metabolism towards bone resorption. Animal studies confirm these observations showing a reduced bone quality and stimulation of bone resorption in hyperhomocysteinemic animals. Homocysteine (HCY) has been found to accumulate in bone by collagen binding. Cell culture studies demonstrate that high HCY levels stimulate osteoclasts but not osteoblasts, indicating again a shift of bone metabolism towards bone resorption. Regarding B-vitamins, only a few in vivo studies with equivocal results have been published. However, two large cell culture studies confirm the results obtained with exogenous HCY administration. In addition, HHCY seems to have adverse affects on extracellular bone matrix by disturbing collagen crosslinking. In conclusion, existing data suggest that HHCY (and possibly B-vitamin deficiencies) adversely affects bone quality by a stimulation of bone resorption and disturbance of collagen crosslinking. Clin Chem Lab Med 2007;45:1621–32.

Hyperhomocysteinemia induces a tissue specific accumulation of homocysteine in bone by collagen binding and adversely affects bone

BACKGROUND Recently, hyperhomocysteinemia (HHCY) has been suggested to have adverse effects on bone. This study investigated if an experimental HHCY in rats induces an accumulation of homocysteine (HCY) in bone tissue that is accompanied by bone loss and reduced bone strength. MATERIAL AND METHODS HHCY was induced in healthy rats by either a methionine (Meth)- or a homocystine (Homo)-enriched diet and compared with controls. Homocystine is the product of two disulfide linked HCY molecules. Tissue and plasma concentrations of HCY, S-adenosylhomocysteine (SAH) and S-adenosylmethionine (SAM) were measured. Bones were assessed by biomechanical testing, histomorphometry, microCT and the measurement of biochemical bone turnover markers in plasma. RESULTS Meth and Homo animals developed a significant HHCY that was accompanied by a tissue specific accumulation of HCY (1300 to 2000% vs. controls). 65% of HCY in bone was bound to collagen of the extracellular matrix. The SAH / SAM-ratio in bone and plasma of Meth and Homo animals exhibited a tissue specific increase indicating a reduced methylation capacity. Accumulation of HCY in bone was characterized by a distinct reduction of cancellous bone (proximal femur: -25 to -35%; distal femur -56 to -58%, proximal tibia: -28 to -43%). Accordingly, bone strength was significantly reduced (-9 to -12%). CONCLUSION A tissue specific accumulation of HCY in bone may be a promising mechanism explaining adverse effects of HHCY on bone. A reduced methylation capacity of bone cells might be another relevant pathomechanism.

The effect of B-vitamins on biochemical bone turnover markers and bone mineral density in osteoporotic patients: a 1-year double blind placebo controlled trial

Abstract Background: Hyperhomocysteinemia is a new risk factor for osteoporosis. This study analyzed the effect of a homocysteine (HCY)-lowering treatment in osteoporotic individuals. Methods: Osteoporotic subjects (n=47, 55–82 years) were treated with either a combination of 2.5 mg folate, 0.5 mg vitamin B12 and 25 mg vitamin B6 or placebo. Bone mineral density (BMD) at lumbar spine and hip was measured at baseline and after 1 year. Urinary desoxypyridinoline cross-links (DPD) and plasma levels of tartrate resistant acid phosphatase (TRAP), C-terminal cross-links of collagen I (CTx), pro-collagen type I N-terminal peptide (PINP) and osteocalcin (OC) were measured after 0, 4, 8 and 12 months. Results: B-vitamin supplementation significantly reduced HCY (0 vs. 12 months: 13.6±4.8 vs. 8.9±2.4 μmol/L). Placebo treatment had no effect on HCY (0 vs. 12 months: 12.0±3.4 vs. 12.7±3.9μmol/L). BMD, TRAP, CTx, OC and PINP did not change throughout the study in both groups. Vitamin treatment decreased urinary DPD by –13% (p<0.01) after 8 and 12 months. In a sub-group analysis of hyperhomocysteinemic subjects (HCY>15 μmol/L, n=8), B-vitamin treatment tended to increase BMD at the lumbar spine, with a t-score from –2.7 to –1.7, and to decrease OC and PINP by approximately 50%. Conclusions: B-vitamin supplementation had no consistent effects on bone turnover or BMD. However, the situation may be different in patients with hyperhomocysteinemia. Clin Chem Lab Med 2007;45:1785–92.

Stimulation of osteoblast activity by homocysteine.

Homocysteine (HCY) has recently been linked to fragility fractures. Moreover, HCY activates osteoclasts. Little is known about the effect of HCY on activity of human osteoblasts (OBs). We hypothesized that HCY decreases the activity of OBs. Osteoblasts obtained from tra‐becular human bone specimens of eight donors were cultured with conditioned medium. Culture medium was adjusted to 0, 100, 500, 1000 and 2000 μM HCY. After 14 days alkaline phosphatase (AP) activity, pro‐collagen type I N‐terminal peptide (PINP) and osteocalcin (OC) secretion in the supernatant were measured. After 20 days the formation of mineralized matrix was analyzed. HCY‐stimulated AP activity gradually (100 μM HCY: 118%, P= 0.006; 500 μM HCY: 125%, P < 0.001). At 1000 and 2000 μM HCY the increase of AP activity was reversible (1000 μM HCY: 106%, P= 0.317; 2000 μM HCY: 102%, P < 0.737). The PINP secretion was also stimulated by HCY reaching a maximum of 260 ± 154 μg/l at 500 μmol/l versus 205 ± 94 μ,g/l in controls. After 20 days of culture the formation of bone matrix was increased at 100 and 500 μM HCY. OC secretion was not significantly changed. The results of the present study consistently demonstrate a moderate stimulation of primary human OB activity by increasing concentrations of HCY. However, the magnitude of this effect seems to be less pronounced than recent observations on primary human osteoclasts, suggesting a dysbalance between OBs and osteoclasts in favour of osteoclasts

Experimental Folate and Vitamin B12 Deficiency Does Not Alter Bone Quality in Rats

Hyperhomocysteinemia (HHCY) has been linked to fragility fractures and osteoporosis. Folate and vitamin B12 deficiencies are among the main causes of HHCY. However, the impact of these vitamins on bone health has been poorly studied. This study analyzed the effect of folate and vitamin B12 deficiency on bone in rats. We used two groups of rats: a control group (Co, n = 10) and a vitamin‐deficient group (VitDef, n = 10). VitDef animals were fed for 12 wk with a folate‐ and vitamin B12–free diet. Co animals received an equicaloric control diet. Tissue and plasma concentrations of homocysteine (HCY), S‐adenosyl‐homocysteine (SAH), and S‐adenosyl‐methionine (SAM) were measured. Bone quality was assessed by biomechanical testing (maximum force of an axial compression test; Fmax), histomorphometry (bone area/total area; B.Ar./T.Ar.], and the measurement of biochemical bone turnover markers (osteocalcin, collagen I C‐terminal cross‐laps [CTX]). VitDef animals developed significant HHCY (Co versus VitDef: 6.8 ± 2.7 versus 61.1 ± 12.8 μM, p < 0.001) that was accompanied by a high plasma concentration of SAH (Co versus VitDef: 24.1 ± 5.9 versus 86.4 ± 44.3 nM, p < 0.001). However, bone tissue concentrations of HCY, SAH, and SAM were similar in the two groups. Fmax, B.Ar./T.Ar., OC, and CTX did not differ between VitDef and Co animals, indicating that bone quality was not affected. Folate and vitamin B12 deficiency induces distinct HHCY but has no effect on bone health in otherwise healthy adult rats. The unchanged HCY metabolism in bone is the most probable explanation for the missing effect of the vitamin‐free diet on bone.

Protein adhesion on dental surfaces—a combined surface analytical approach

Protein adsorption is a field of huge interest in a number of application fields. Information on protein adhesion is accessible by a variety of methods. However, the results obtained are significantly influenced by the applied technique. The objective of this work was to understand the role of adhesion forces (obtained by scanning force spectroscopy, SFS) in the process of protein adsorption and desorption. In SFS, the protein is forced to and retracted from the surface, even under unfavorable conditions, in contrast to the natural situation. Furthermore, adhesion forces are correlated with adhesion energies, neglecting the entropic part in the Gibbs enthalpy. In this context, dynamic contact angle (DCA) measurements were performed to identify the potential of this method to complement SFS data. In DCA measurements, the protein diffuses voluntarily to the surface and information on surface coverage and reversibility of adsorption is obtained, including entropic effects (conformational changes and hydrophobic effect). It could be shown that the surface coverage (by DCA) of bovine serum albumin on dental materials correlates well with the adhesion forces (by SFS) if no hydrophobic surface is involved. On those, the entropic hydrophobic effect plays a major role. As a second task, the reversibility of the protein adsorption, i.e., the voluntary desorption as studied by DCA, was compared to the adhesion forces. Here, a correlation between low adhesion forces and good reversibility could be found as long as no covalent bonds were involved. The comparative study of DCA and SFS, thus, leads to a more detailed picture of the complete adsorption/desorption cycle.

Folate is related to phosphorylated neurofilament‐H and P‐tau (Ser396) in rat brain

J. Neurochem. (2011) 117, 1047–1054.

Cleaning of biomaterial surfaces: Protein removal by different solvents

The removal of biofilms or protein films from biomaterials is still a challenging task. In particular, for research investigations on real (applied) surfaces the reuse of samples is of high importance, because reuse allows the comparison of the same sample in different experiments. The aim of the present study was to evaluate the cleaning efficiency of different solvents (SDS, water, acetone, isopropanol, RIPA-buffer and Tween-20) on five different biomaterials (titanium, gold, PMMA (no acetone used), ceramic, and PTFE) with different wettability which were covered by layers of two different adsorbed proteins (BSA and lysozyme). The presence of a protein film after adsorption was confirmed by transmission electron microscopy (TEM). After treatment of the surfaces with the different solvents, the residual proteins on the surface were determined by BCA-assay (bicinchoninic acid assay). Data of the present study indicate that SDS is an effective solvent, but for several protein-substrate combinations it does not show the cleaning efficiency often mentioned in literature. RIPA-buffer and Tween-20 were more effective. They showed very low residual protein amounts after cleaning on all examined material surfaces and for both proteins, however, with small differences for the respective substrate-protein combinations. RIPA-buffer in combination with ultrasonication completely removed the protein layer as confirmed by TEM.

Albumin-lysozyme interactions: Cooperative adsorption on titanium and enzymatic activity

The interplay of albumin (BSA) and lysozyme (LYZ) adsorbed simultaneously on titanium was analyzed by gel electrophoresis and BCA assay. It was found that BSA and lysozyme adsorb cooperatively. Additionally, the isoelectric point of the respective protein influences the adsorption. Also, the enzymatic activity of lysozyme and amylase (AMY) in mixtures with BSA was considered with respect to a possible influence of protein-protein interaction on enzyme activity. Indeed, an increase of lysozyme activity in the presence of BSA could be observed. In contrast, BSA does not influence the activity of amylase.

Enzymology and Ultrastructure of the in situ Pellicle in Caries-Active and Caries-Inactive Patients

Aim: The present study aimed to evaluate the impact of caries activity on the key enzymes and the ultrastructure of the in situ pellicle. Methods: Pellicle formation was performed on bovine enamel slabs. Intraoral exposure (3, 30, and 120 min) was accomplished by 14 caries-active (DMFS: 22.7 ± 12.1) and 13 caries-inactive (DMFS: 1.5 ± 1.8) individuals. The enzyme activities (lysozyme, peroxidase, α-amylase, glycosyltransferase [GTF]) in the in situ pellicle and resting saliva of all participants were analyzed directly after oral exposure. In addition, a simultaneous visualization of these enzymes, extracellular glucans, and adherent bacteria was carried out. Fluorescent patterns were analyzed with fluorescence labeling and 4′,6-diamidino-2-phenylindole/concanavalin A staining. In addition, the distribution of GTF B, C, and D and the ultrastructure of the pellicle were examined by gold immunolabeling and transmission electron microscopy with selected samples. Results: Enzyme activities of amylase, peroxidase, lysozyme, and GTF were detected on all enamel slabs in an active conformation. Neither exposure time nor caries activity had an impact on the enzyme activities. Gold immunolabeling indicated that the pellicle of caries-active subjects tends to more GTF D molecules. The pellicles of caries-inactive and -active individuals revealed a similar ultrastructural pattern. Conclusion: The enzyme activities as well as the pellicles ultrastructure are of high similarity in caries-active and -inactive subjects. Thereby, oral exposure time has no significant influence. This reflects a high uniformity during the initial phase of bioadhesion (3-120 min) concerning enzymatic functions. However, there is a tendency towards more GTF D in caries-active individuals.