The Impact of Vitamin D, Calcium, Protein Supplementation, and Physical Exercise on Bone Metabolism After Bariatric Surgery: The BABS Study.

Laparoscopic Roux-en-Y gastric bypass (RYGB) and sleeve gastrectomy (SG) are common and effective methods to treat severe obesity, but these procedures can adversely influence bone metabolism and areal bone mineral density (aBMD). This was a prospective 24-month single-center interventional two-arm study in 220 women and similarly aged men (median age 40.7 years) with a body mass index (BMI) >38 kg/m(2) after RYGB and SG procedures. Patients were randomized into: 1) an intervention group receiving: 28,000 IU cholecalciferol/wk for 8 weeks before bariatric surgery, 16,000 IU/wk and 1000 mg calciummonocitrate/d after surgery, daily BMI-adjusted protein supplementation and physical exercise (Nordic walking, strength perseverance, and equipment training); 2) a non-intervention group: no preoperative loading, nutritional supplementation, or obligatory physical exercise. At study endpoint, when comparing the intervention group to the non-intervention group, the relative percentage changes of serum levels of sclerostin (12.1% versus 63.8%), cross-linked C-telopeptide (CTX, 82.6% versus 158.3%), 25-OH vitamin D (13.4% versus 18.2%), phosphate (23.7% versus 32%, p < 0.001 for all), procollagen type 1 amino-terminal propeptide (P1NP, 12% versus 41.2%), intact parathyroid hormone (iPTH, -17.3% versus -7.6%), and Dickkopf-1 (-3.9% versus -8.9%, p < 0.05 for all) differed. The decline in lumbar spine, total hip and total body aBMD, changes in BMI, lean body mass (LBM), as well as changes in trabecular bone score (TBS) values (p < 0.005 for all) were less, but significantly, pronounced in the intervention group. We conclude that vitamin D loading and ongoing vitamin D, calcium, and BMI-adjusted protein supplementation in combination with physical exercise decelerates the loss of aBMD and LBM after bariatric surgery. Moreover, the well-known increases of bone turnover markers are less pronounced.

Atypical Femoral Fractures-Ongoing and History of Bone-Specific Therapy, Concomitant Diseases, Medications, and Survival.

Although atypical femoral fractures (AFFs) are generally rare events; several studies have indicated a potential link between AFF and long-term bone-specific therapies (BSTs). The aim of this study was to analyze the frequency of AFF and potential associations with prior or ongoing BST. A total of 8851 Caucasian female and male patients with de novo hip fractures treated in the largest Austrian level 1 trauma center from 2000 to 2013 were selected. Of the total, 194 patients with a de novo low-traumatic subtrochanteric or shaft fractures were identified: 35 atypical and 159 typical fractures. Of these patients, concomitant diseases, medication, previous fractures, and survival data were retrieved and analyzed. Female patients in both groups were significantly older. The median survival was significantly shorter in patients with AFF (9 vs 18 months; p < 0.0001). Cardiovascular disease, sarcopenia, chronic kidney disease, type 2 diabetes, smoking (past or current history), and prevalent fragility fractures were more frequent in AFF patients, as well as the concomitant use of phenprocoumon, furosemide, and sulfonylurea. Although the number of patients with current BST was less in (14.5%) both groups, more patients in the AFF group were previously treated with BST (71% vs 49%; p = 0.016), and they received these therapies for a longer time period. A combination of severe comorbidities, long-term pharmaceutical therapies, and a history of previous or ongoing BST was associated with an increased individual risk for AFF.

Sensitization of erythrocytes to suicidal erythrocyte death following water deprivation.

BACKGROUND/AIMS: Klotho deficiency results in excessive formation of 1,25(OH)2D3, accelerated ageing and early death. Moreover, klotho deficiency enhances eryptosis, the suicidal erythrocyte death characterized by phosphatidylserine exposure at the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca(2+)-activity ([Ca(2+)]i), glucose depletion, hyperosmotic shock and oxidative stress. Klotho expression is decreased and 1,25(OH)2D3-formation enhanced by dehydration. The present study thus explored whether dehydration influences eryptosis. METHODS: Blood was drawn from hydrated or 36h dehydrated mice. Plasma osmolarity was determined by vapour pressure method, plasma 1,25(OH)2D3 and aldosterone concentrations using ELISA, and plasma Ca(2+)-concentration utilizing photometry. Erythrocytes were exposed to Ca(2+)-ionophore ionomycin (1 µM, 30 min), energy depletion (12 h glucose removal), hyperosmotic shock (500 mM sucrose added, 2 h) and oxidative stress (100 µM tert-butyl-hydroperoxide, 30 min) and phosphatidylserine exposure at the erythrocyte surface estimated from annexin V binding. RESULTS: Dehydration increased plasma osmolarity and plasma 1,25(OH)2D3 and aldosterone concentrations. Dehydration did not significantly modify phosphatidylserine-exposure of freshly drawn erythrocytes but significantly enhanced the increase of phosphatidylserine-exposure under control conditions and following treatment with ionomycin, glucose-deprivation, hyperosmolarity or tert-butyl-hydroperoxide. CONCLUSIONS: Dehydration sensitizes the erythrocytes to spontaneous eryptosis and to the triggering of eryptosis by excessive Ca(2+)-entry, energy depletion, hyperosmotic shock and oxidative stress.

Azathioprine favourably influences the course of malaria.

BACKGROUND: Azathioprine triggers suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and exposure of phosphatidylserine at the erythrocyte surface. Eryptosis may accelerate the clearance of Plasmodium-infected erythrocytes. The present study thus explored whether azathioprine influences eryptosis of Plasmodium-infected erythrocytes, development of parasitaemia and thus the course of malaria. METHODS: Human erythrocytes were infected in vitro with Plasmodium falciparum (P. falciparum) (strain BinH) in the absence and presence of azathioprine (0.001 - 10 microM), parasitaemia determined utilizing Syto16, phosphatidylserine exposure estimated from annexin V-binding and cell volume from forward scatter in FACS analysis. Mice were infected with Plasmodium berghei (P. berghei) ANKA by injecting parasitized murine erythrocytes (1 x 106) intraperitoneally. Where indicated azathioprine (5 mg/kg b.w.) was administered subcutaneously from the eighth day of infection. RESULTS: In vitro infection of human erythrocytes with P. falciparum increased annexin V-binding and initially decreased forward scatter, effects significantly augmented by azathioprine. At higher concentrations azathioprine significantly decreased intraerythrocytic DNA/RNA content (>or= 1 microM) and in vitro parasitaemia (>or= 1 microM). Administration of azathioprine significantly decreased the parasitaemia of circulating erythrocytes and increased the survival of P. berghei-infected mice (from 0% to 77% 22 days after infection). CONCLUSION: Azathioprine inhibits intraerythrocytic growth of P. falciparum, enhances suicidal death of infected erythrocytes, decreases parasitaemia and fosters host survival during malaria.

Inhibition of Na+/H+ exchanger activity by parvovirus B19 protein NS1.

Infection with parvovirus B19 (B19) may induce apoptosis resulting in anemia, acute fulminant liver failure, placental insufficiency and myocarditis. Apoptosis has been attributed to proapoptotic activity of the non-structural viral protein NS1, which is known to trigger a signaling cascade eventually leading to activation of caspases. In several cell types apoptosis was found to be paralleled by profound cytosolic acidification, which may be secondary to inhibition of the Na+/H+ exchanger. The acidification has been considered to support the activation of pH sensitive caspases and endonucleases. However, nothing is known about the effect of NS1 on Na+/H+ exchanger activity and cytosolic pH. The present study thus explored whether NS1 expression affects cytosolic pH (pHi) and Na+-dependent realkalinization (DeltapHi) following acidification by an ammonium pulse. According to FACS analysis, overexpression of NS1 in RXR-10SW cells led within 72 hours to activation of caspase 3 and DNA fragmentation. NS1 overexpression resulted within 24 hours in a significant decline of pHi from 6.93 +/- 0.03 (n = 6) to 6.78 +/- 0.04 (n = 7), and to a significant decrease of DeltapHi from 0.159 +/- 0.017 (n = 6) to 0.039 +/- 0.004, (n = 7). The decrease of pHi and of DeltapHi following NS1 expression could be significantly blunted by inhibition of caspase 3 with zVAD. Western blot analysis revealed degradation of NHE1 following NS1 expression. In vitro, caspase 3, but not caspase 6, caspase 7 and caspase 8 degraded NHE1 protein of cell lysates. In conclusion, overexpression of NS1 triggers a signaling cascade eventually leading to activation of caspase 3 and subsequent degradation of NHE1. The effect contributes to cytosolic acidification which may in turn favor activation of caspases and endonucleases and thus participate in the pathophysiology of B19-infection.

Inhibition of cation channels and suicidal death of human erythrocytes by zidovudine.

Zidovudine, a drug widely used in the treatment of AIDS, has been shown to influence cytosolic calcium activity in HIV-infected lymphocytes. Thus, zidovudine may modify the activity of Ca(2+)-permeable ion channels. In erythrocytes, activation of Ca(2+)-permeable cation channels stimulates eryptosis, the suicidal erythrocyte death. Eryptosis is characterized by cell shrinkage (apparent from a decrease of forward scatter) and phosphatidylserine (PS) exposure (apparent from annexin V-binding) at the erythrocyte surface. Triggers of eryptosis include isotonic cell shrinkage (Cl(-) replacement by gluconate), energy depletion (removal of glucose) or exposure to a variety of drugs including azathioprine. The present study explored, whether zidovudine influences the activity of erythrocytic Ca(2+)-permeable cation channels and eryptosis. Whole-cell patch-clamp recordings indeed revealed that zidovudine blocked the Ca(2+)-permeable cation channels activated by Cl(-) removal. In the presence of Cl(-) and glucose, the percentage of annexin V-binding cells was low and not significantly modified by the presence of zidovudine. Both, Cl(-) removal and glucose depletion increased annexin V-binding and decreased forward scatter, effects significantly blunted by zidovudine (2 microg/ml). According to Fluo3 fluorescence, zidovudine (2 microg/ml) did not significantly modify cytosolic Ca(2+) concentration under control conditions, but significantly blunted the increase in cytosolic Ca(2+) activity following glucose depletion. Furthermore, zidovudine significantly inhibited azathioprine-induced eryptosis. The present observations disclose a completely novel effect of zidovudine, i.e. its inhibitory influence on Ca(2+) entry and subsequent suicidal erythrocyte death during isotonic cell shrinkage or energy depletion.

Azathioprine-induced suicidal erythrocyte death.

BACKGROUND: Azathioprine is widely used as an immunosuppressive drug. The side effects of azathioprine include anemia, which has been attributed to bone marrow suppression. Alternatively, anemia could result from accelerated suicidal erythrocyte death or eryptosis, which is characterized by exposure of phosphatidylserine (PS) at the erythrocyte surface and by cell shrinkage. METHODS: The present experiments explored whether azathioprine influences eryptosis. According to annexin V binding, erythrocytes from patients indeed showed a significant increase of PS exposure within 1 week of treatment with azathioprine. In a second series, cytosolic Ca2+ activity (Fluo3 fluorescence), cell volume (forward scatter), and PS-exposure (annexin V binding) were determined by FACS analysis in erythrocytes from healthy volunteers. RESULTS: Exposure to azathioprine (> or =2 microg/mL) for 48 hours increased cytosolic Ca2+ activity and annexin V binding and decreased forward scatter. The effect of azathioprine on both annexin V binding and forward scatter was significantly blunted in the nominal absence of extracellular Ca2+. CONCLUSIONS: Azathioprine triggers suicidal erythrocyte death, an effect presumably contributing to azathioprine-induced anemia.

Stimulation of suicidal erythrocyte death by amantadine.

Amantadine is an effective drug for treatment of both, Parkinson's disease and viral infections. Side effects of amantadine include anemia, which may limit its therapeutic use. The cause of amantatine induced anemia is ill defined. At least in theory, the anemia could partially result from suicidal erythrocyte death or eryptosis, which accelerates the clearance of circulating erythrocytes. Eryptosis is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine exposure at the cell surface. Triggers of erythrocyte membrane scrambling include an increase of cytosolic Ca2+ concentration ([Ca2+]i) resulting from activation of Ca2+-permeable cation channels. The present study has been performed to test for an effect of amantadine on eryptosis. Erythrocytes from healthy volunteers were exposed to amantadine and annexin V binding (disclosing phosphatidylserine exposure), forward scatter (reflecting cell volume), and Fluo3-dependent fluorescence (reflecting [Ca2+]i) were determined by flow cytometry. Exposure of erythrocytes to amantadine (> or =0.2 microg/ml) increased [Ca2+]i and triggered annexin V binding, and increased forward scatter. The effect on annexin V binding was virtually abolished in the absence of extracellular Ca2+. The present observations disclose mechanisms presumably contributing to amantadine induced anemia.