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Vitamin D and rheumatoid arthritis
Abstracts
Prevalence of asymptomatic vertebral fracture and deficit of vitamin D in osteoporotic women who are overweight or obese M.S. Larroudé, M.S. Moggia, G. Lichtcajger, Z. Man Centro TIEMPO, Buenos Aires Argentina Objective: To evaluate the prevalence of asymptomatic vertebral fractures (VF) and the lack of vitamin D (VD) in patients with osteoporosis (OP) who are overweight (OW) or obese (O). Material and methods: 66 female patients, average age 67.6 years with OP diagnosed by DXA on lumbar spine (LS). They were questioned about history of VF. Exclusion criteria: liver or renal disease or treated with corticosteroids, anticonvulsants and anticoagulants. We performed lateral thoracic and LS X-ray. VF was considered with a 20% decreased. Body mass index (BMI) of OW was 25–30 and for O, BMI was over 30. Vitamin 25(OH) D was measured by RIA, DiaSorin kit, divided into <20 ng/ml, between 20–30 ng/ml and 30–40 ng/ml. Results: 19.7% (13/66) patients presented O, a mean age 67.61 years of which 15.4% (2/13) had VF. 61.5% (8/13) had VD value <20 ng/ml, 23% (3/13) between 20 and 30 ng/ml and 15.5% (2/13) had levels >30 ng/ml. VF were also found in patients who had low VD. 80.3% (53/66) were OW, average age 67.6 years of which 15.09% (8/53) had VF. 43.4% (23/53) had values <20 ng/ml for VD, 41.5% (22/53) between 20 and 30 ng/ml and 15.1% (8/53) higher values to 30 ng/ml. 7 patients with VF had vitamin D insufficiency. 42.2% (49/116) had a normal BMI of which 12.24% (6/49) had VF. 32.7% (16/49) had VD value <20 ng/ml, 42.8% (21/49) between 20 and 30 ng/ml, 24.5% (12/49) > 30 ng/ml. 5 of the patients with VF had values <30 ng/ml. 15% had VF and 90% (9/10) had levels lower values 30 ng/ml for VD. Conclusion: Patients who were O or OW may have OP and VF, have VD deficient values. It is important to assess VD deficiency, which may contribute to decreased bone mass and VF. This article is part of a Special Issue entitled AAOMM 2011 Abstracts. doi:10.1016/j.bone.2011.09.011
Vitamin D and rheumatoid arthritis M.S. Larroudé, A.M. Capuccio, L. Naftal, Z. Man, D. Scublinsky Centro TIEMPO, Hospital Dr. Cesar Milstein, Ciudad de Buenos Aires, Argentina Objective: To study the prevalence of VD sufficiency in patients with rheumatoid arthritis (RA) and its relation to disease activity and functional capacity and bone mineral density (BMD), and immunosuppressants. Material and methods: patients with RA, ACR87 criteria. The activity as measured by DAS28, functional capacity by HAQ. VD was measured with a DiaSorin kit (March and June). VD deficiency <10 ng/ml, insufficiency 10–20 ng/ml, hypovitaminosis 20–30 ng/ml and sufficiency value > 30 ng/ml. Measured BMD with DXA. Statistics: Student's t tests, ANOVA, Pearson, Spearman, Kruskal Wallis and chi square and logistic regression. Significance was set at p < 0.05. Results: 73 patients (70 female, 3 male) mean age 59 years (49–75), with a duration of 12 years (6–19). VD deficiency: 4.5%, insufficiency 33.33%, hypovitaminosis 36.40% and sufficiency 25.80%. Significant difference was detected in the disease activity between patients with VD deficiency and those with values greater than 20 ng/ml. (Student's t: p = 0.03) and VD levels of 21 to 30 ng/ml versus greater than 30 ng/ml. (Student's t: p = 0.05). Patients with a longer history had higher VD deficiency. (KW, p = 0.04). Patients with VD levels below 30 ng/ml have a significant regression with the HAQ moderate/severe (p = 0.02), while in patients with VD levels greater than 30 was observed significant regression when they received anti-TNF agents versus MTX alone (p = 0.01). No significant differences in VD in patients with and without steroids (logistic regression p = 0.57) nor in BMD at lumbar spine and femoral neck in patients with values > or <30 ng/ml. (p = 0.78, p = 0.63, p = 0.53 and p = 0.16 respectively). Conclusion: 74% of our RA patients had inadequate vitamin D values that are associated with longer disease progression and deterioration of the same. This article is part of a Special Issue entitled AAOMM 2011 Abstracts. doi:10.1016/j.bone.2011.09.012
Oral administration of glutamine prevents oxidative stress triggered by menadione in duodenal enterocytes G.E. Díaz de Barboza, M.M. Benedetto, N.G. Tolosa de Talamoni Lab. de Metabolismo Fosfocálcico y Vitamina D “Dr Cañas”, Cátedra de Bioquímica y Biología Molecular, Fac de Cs Médicas, Universidad Nacional de Córdoba, Argentina We have previously demonstrated that menadione (MEN) decreases intestinal calcium absorption triggering oxidative stress and apoptotic death of enterocytes. The aminoacid glutamine (GLN) has restorative and protective properties of the redox state in tissues exposed to oxidant drugs. In this work we studied the possible role of GLN on the oxidative stress induced by MEN in enterocytes. Chickens (4 weeks old Cobb-Harding) were fed a commercial diet. Controls were treated with vehicle and the treated group with 1 g GLN/kg bw per os and with MEN 12.5 μmol/kg bw intraperitoneally (60 or 90 min later). Both drugs acted together for 30 min. Glutathione (GSH) content was quantified by enzymatic method. The activity of catalase (CAT) and superoxide dismutase (SOD) was determined in duodenal
1377
mucosa homogenates by spectrophotometry. GLN administration 60 or 90 min before MEN injection increased 30–35% GSH levels, restoring the control values. GLN prevented the induction of CAT activity by MEN (Control 14.06 ± 0.69; MEN 23.51 ± 1.94*; GLN60 min + MEN 17.15 ± 0.96 and GLN90 min + MEN 17.89 ± 1.04 U/mg of protein; *p < 0.001 vs Control). Similarly, GLN avoided the increase in SOD activity produced by MEN (Control 10.95 ± 1.11, MEN 20.34 ± 1.87*, GLN60 min + MEN 12.66 ± 1.08 and GLN90 min + MEN 11.06 ± 2.15 U/mg protein, *p < 0.05 vs Control). The data suggest that GLN is capable of preventing the oxidative effect induced by MEN administration in duodenal enterocytes, as shown by the restoration of control values in intracellular GSH content and in the activity of CAT and SOD. Future studies should evaluate if cellular redox state restoration may prevent the loss of intestinal Ca absorption caused by MEN. This article is part of a Special Issue entitled AAOMM 2011 Abstracts. doi:10.1016/j.bone.2011.09.013
Alterations in the viability, cell cycle and TRPV6 gene expression induced by calcitriol and BSO in breast cancer cells L. Bohl, A. Liaudat, G. Picotto, A. Marchionatti, V. Rodríguez, C. Narvaez, J. Welsh, N. Tolosa de Talamoni Lab Cañas, Cát de Bioq y Biol Mol, FCM, UNC, Córdoba Argentina, University at Albany, USA Calcitriol has been evaluated as anticancer agent in many cancer cells. This effect may be increased with glutathione-depleting drugs such as L-Buthionine-S,R-Sulfoximine (BSO), which may be applied as an alternative to avoid bone complications associated with breast cancer metastasis. Our aim was to investigate calcitriol and BSO actions, alone or in combination, on breast cancer cells. MCF-7 cells were treated with 100 nM calcitriol, 20 μM BSO or both at different times. Cell growth was evaluated by crystal violet staining. GSH content was measured by spectrophotometry, while reactive oxygen species (ROS) and cell cycle by flow citometry. Apoptosis was analyzed by phase contrast microscopy and immunocytochemistry and TRPV6 gene expression by qPCR. Results were analyzed using ANOVA and Bonferroni. BSO, calcitriol and the combination reduced MCF-7 proliferation at 96 h. GSH levels were also reduced by BSO, calcitriol or the combination in a time-dependent way. ROS levels were increased in cells treated with calcitriol or calcitriol + BSO (96 h). Calcitriol and the combination augmented the proportion of cells in G1 phase (72 h) and induced DNA fragmentation and changes in Cit c localization (96 h). TRPV6 gene expression resulted 6-fold higher in cells treated with calcitriol and with the combination (96 h). Altogether, these results may indicate that calcitriol and BSO inhibited MCF-7 cell growth altering cell redox state due to GSH reduction and ROS production. Cell arrest in G1 phase may contribute to the reduced cellular growth. DNA fragmentation and Cit c release from the mitochondria suggest apoptotic effects of calcitriol and calcitriol + BSO. Finally, calcitriol and its combination with BSO induced TRPV6 gene expression, which implies a possible role of calcium underlying these effects. This article is part of a Special Issue entitled AAOMM 2011 Abstracts. doi:10.1016/j.bone.2011.09.014
Measurement of bone resorption (BR) and formation (BF) in rats using the urinary pharmacokinetic of fluoride (F) M. Lupo, B. Garcia, H. Moreno, A. Rigalli Bone Biology Laboratory, Rosario, Argentina A methodology to measure bone remodeling through the pharmacokinetic parameters of F has been developed. This method has been validated in different biological models but there were some incongruent results. The aim of this job was to develop and validate a more precise methodology from fluoruria samples. The pharmacokinetic model has three parameters: Ke: rate constant of plasma F clearance, Ku: rate constant of urinary F excretion, Ko: rate constant of bone F uptake. This method requested obtaining urine samples for 1 hour. After this, an intravenous F dose is injected (1 μmol NaF/100 g bw) and urine sampling continued through intra urethral catheterism for 4 h. The experiment was carried out under ketamine anaesthesia and rectal hydration. Fluoruria was determined and a mathematical model was developed to calculate Ke, Ko and Ku. BF and BR are obtained with these values and basal plasma F level. The model has been validated in Sprague Dawley rats with different bone remodeling (n = 5 per group). BR is shown as mean±SEM and differences were considered significant when p < 0.05 ANOVA and Newman Keuls test. Normal rats: 0.014 ± 0.013 μmol/l·min, ovariectomized (ovx) rats: 0.046 ± 0.040, ovx with hypercalcic diet: 0.018 ±0.010, ovx with hipocalcic diet: 0.153 ±0.030 and ovx with zoledronate treatment: 0.010 ±0.059. Similar results were obtained with BF (data not shown). Conclusions: 1) In 100% of the cases the pharmacokinetic parameters obtained were compatible with the described model. 2) BF and BR values were coincident with the biological models. 3) The methodology is more precise than the previous one that depends on plasma F levels. Current experiments are being carried out to determine the sensitivity and specificity of this new method. This article is part of a Special Issue entitled AAOMM 2011 Abstracts. doi:10.1016/j.bone.2011.09.015
Prevalence of asymptomatic vertebral fracture and deficit of vitamin D in osteoporotic women who are overweight or obese M.S. Larroudé, M.S. Moggia, G. Lichtcajger, Z. Man Centro TIEMPO, Buenos Aires Argentina Objective: To evaluate the prevalence of asymptomatic vertebral fractures (VF) and the lack of vitamin D (VD) in patients with osteoporosis (OP) who are overweight (OW) or obese (O). Material and methods: 66 female patients, average age 67.6 years with OP diagnosed by DXA on lumbar spine (LS). They were questioned about history of VF. Exclusion criteria: liver or renal disease or treated with corticosteroids, anticonvulsants and anticoagulants. We performed lateral thoracic and LS X-ray. VF was considered with a 20% decreased. Body mass index (BMI) of OW was 25–30 and for O, BMI was over 30. Vitamin 25(OH) D was measured by RIA, DiaSorin kit, divided into <20 ng/ml, between 20–30 ng/ml and 30–40 ng/ml. Results: 19.7% (13/66) patients presented O, a mean age 67.61 years of which 15.4% (2/13) had VF. 61.5% (8/13) had VD value <20 ng/ml, 23% (3/13) between 20 and 30 ng/ml and 15.5% (2/13) had levels >30 ng/ml. VF were also found in patients who had low VD. 80.3% (53/66) were OW, average age 67.6 years of which 15.09% (8/53) had VF. 43.4% (23/53) had values <20 ng/ml for VD, 41.5% (22/53) between 20 and 30 ng/ml and 15.1% (8/53) higher values to 30 ng/ml. 7 patients with VF had vitamin D insufficiency. 42.2% (49/116) had a normal BMI of which 12.24% (6/49) had VF. 32.7% (16/49) had VD value <20 ng/ml, 42.8% (21/49) between 20 and 30 ng/ml, 24.5% (12/49) > 30 ng/ml. 5 of the patients with VF had values <30 ng/ml. 15% had VF and 90% (9/10) had levels lower values 30 ng/ml for VD. Conclusion: Patients who were O or OW may have OP and VF, have VD deficient values. It is important to assess VD deficiency, which may contribute to decreased bone mass and VF. This article is part of a Special Issue entitled AAOMM 2011 Abstracts. doi:10.1016/j.bone.2011.09.011
Vitamin D and rheumatoid arthritis M.S. Larroudé, A.M. Capuccio, L. Naftal, Z. Man, D. Scublinsky Centro TIEMPO, Hospital Dr. Cesar Milstein, Ciudad de Buenos Aires, Argentina Objective: To study the prevalence of VD sufficiency in patients with rheumatoid arthritis (RA) and its relation to disease activity and functional capacity and bone mineral density (BMD), and immunosuppressants. Material and methods: patients with RA, ACR87 criteria. The activity as measured by DAS28, functional capacity by HAQ. VD was measured with a DiaSorin kit (March and June). VD deficiency <10 ng/ml, insufficiency 10–20 ng/ml, hypovitaminosis 20–30 ng/ml and sufficiency value > 30 ng/ml. Measured BMD with DXA. Statistics: Student's t tests, ANOVA, Pearson, Spearman, Kruskal Wallis and chi square and logistic regression. Significance was set at p < 0.05. Results: 73 patients (70 female, 3 male) mean age 59 years (49–75), with a duration of 12 years (6–19). VD deficiency: 4.5%, insufficiency 33.33%, hypovitaminosis 36.40% and sufficiency 25.80%. Significant difference was detected in the disease activity between patients with VD deficiency and those with values greater than 20 ng/ml. (Student's t: p = 0.03) and VD levels of 21 to 30 ng/ml versus greater than 30 ng/ml. (Student's t: p = 0.05). Patients with a longer history had higher VD deficiency. (KW, p = 0.04). Patients with VD levels below 30 ng/ml have a significant regression with the HAQ moderate/severe (p = 0.02), while in patients with VD levels greater than 30 was observed significant regression when they received anti-TNF agents versus MTX alone (p = 0.01). No significant differences in VD in patients with and without steroids (logistic regression p = 0.57) nor in BMD at lumbar spine and femoral neck in patients with values > or <30 ng/ml. (p = 0.78, p = 0.63, p = 0.53 and p = 0.16 respectively). Conclusion: 74% of our RA patients had inadequate vitamin D values that are associated with longer disease progression and deterioration of the same. This article is part of a Special Issue entitled AAOMM 2011 Abstracts. doi:10.1016/j.bone.2011.09.012
Oral administration of glutamine prevents oxidative stress triggered by menadione in duodenal enterocytes G.E. Díaz de Barboza, M.M. Benedetto, N.G. Tolosa de Talamoni Lab. de Metabolismo Fosfocálcico y Vitamina D “Dr Cañas”, Cátedra de Bioquímica y Biología Molecular, Fac de Cs Médicas, Universidad Nacional de Córdoba, Argentina We have previously demonstrated that menadione (MEN) decreases intestinal calcium absorption triggering oxidative stress and apoptotic death of enterocytes. The aminoacid glutamine (GLN) has restorative and protective properties of the redox state in tissues exposed to oxidant drugs. In this work we studied the possible role of GLN on the oxidative stress induced by MEN in enterocytes. Chickens (4 weeks old Cobb-Harding) were fed a commercial diet. Controls were treated with vehicle and the treated group with 1 g GLN/kg bw per os and with MEN 12.5 μmol/kg bw intraperitoneally (60 or 90 min later). Both drugs acted together for 30 min. Glutathione (GSH) content was quantified by enzymatic method. The activity of catalase (CAT) and superoxide dismutase (SOD) was determined in duodenal
1377
mucosa homogenates by spectrophotometry. GLN administration 60 or 90 min before MEN injection increased 30–35% GSH levels, restoring the control values. GLN prevented the induction of CAT activity by MEN (Control 14.06 ± 0.69; MEN 23.51 ± 1.94*; GLN60 min + MEN 17.15 ± 0.96 and GLN90 min + MEN 17.89 ± 1.04 U/mg of protein; *p < 0.001 vs Control). Similarly, GLN avoided the increase in SOD activity produced by MEN (Control 10.95 ± 1.11, MEN 20.34 ± 1.87*, GLN60 min + MEN 12.66 ± 1.08 and GLN90 min + MEN 11.06 ± 2.15 U/mg protein, *p < 0.05 vs Control). The data suggest that GLN is capable of preventing the oxidative effect induced by MEN administration in duodenal enterocytes, as shown by the restoration of control values in intracellular GSH content and in the activity of CAT and SOD. Future studies should evaluate if cellular redox state restoration may prevent the loss of intestinal Ca absorption caused by MEN. This article is part of a Special Issue entitled AAOMM 2011 Abstracts. doi:10.1016/j.bone.2011.09.013
Alterations in the viability, cell cycle and TRPV6 gene expression induced by calcitriol and BSO in breast cancer cells L. Bohl, A. Liaudat, G. Picotto, A. Marchionatti, V. Rodríguez, C. Narvaez, J. Welsh, N. Tolosa de Talamoni Lab Cañas, Cát de Bioq y Biol Mol, FCM, UNC, Córdoba Argentina, University at Albany, USA Calcitriol has been evaluated as anticancer agent in many cancer cells. This effect may be increased with glutathione-depleting drugs such as L-Buthionine-S,R-Sulfoximine (BSO), which may be applied as an alternative to avoid bone complications associated with breast cancer metastasis. Our aim was to investigate calcitriol and BSO actions, alone or in combination, on breast cancer cells. MCF-7 cells were treated with 100 nM calcitriol, 20 μM BSO or both at different times. Cell growth was evaluated by crystal violet staining. GSH content was measured by spectrophotometry, while reactive oxygen species (ROS) and cell cycle by flow citometry. Apoptosis was analyzed by phase contrast microscopy and immunocytochemistry and TRPV6 gene expression by qPCR. Results were analyzed using ANOVA and Bonferroni. BSO, calcitriol and the combination reduced MCF-7 proliferation at 96 h. GSH levels were also reduced by BSO, calcitriol or the combination in a time-dependent way. ROS levels were increased in cells treated with calcitriol or calcitriol + BSO (96 h). Calcitriol and the combination augmented the proportion of cells in G1 phase (72 h) and induced DNA fragmentation and changes in Cit c localization (96 h). TRPV6 gene expression resulted 6-fold higher in cells treated with calcitriol and with the combination (96 h). Altogether, these results may indicate that calcitriol and BSO inhibited MCF-7 cell growth altering cell redox state due to GSH reduction and ROS production. Cell arrest in G1 phase may contribute to the reduced cellular growth. DNA fragmentation and Cit c release from the mitochondria suggest apoptotic effects of calcitriol and calcitriol + BSO. Finally, calcitriol and its combination with BSO induced TRPV6 gene expression, which implies a possible role of calcium underlying these effects. This article is part of a Special Issue entitled AAOMM 2011 Abstracts. doi:10.1016/j.bone.2011.09.014
Measurement of bone resorption (BR) and formation (BF) in rats using the urinary pharmacokinetic of fluoride (F) M. Lupo, B. Garcia, H. Moreno, A. Rigalli Bone Biology Laboratory, Rosario, Argentina A methodology to measure bone remodeling through the pharmacokinetic parameters of F has been developed. This method has been validated in different biological models but there were some incongruent results. The aim of this job was to develop and validate a more precise methodology from fluoruria samples. The pharmacokinetic model has three parameters: Ke: rate constant of plasma F clearance, Ku: rate constant of urinary F excretion, Ko: rate constant of bone F uptake. This method requested obtaining urine samples for 1 hour. After this, an intravenous F dose is injected (1 μmol NaF/100 g bw) and urine sampling continued through intra urethral catheterism for 4 h. The experiment was carried out under ketamine anaesthesia and rectal hydration. Fluoruria was determined and a mathematical model was developed to calculate Ke, Ko and Ku. BF and BR are obtained with these values and basal plasma F level. The model has been validated in Sprague Dawley rats with different bone remodeling (n = 5 per group). BR is shown as mean±SEM and differences were considered significant when p < 0.05 ANOVA and Newman Keuls test. Normal rats: 0.014 ± 0.013 μmol/l·min, ovariectomized (ovx) rats: 0.046 ± 0.040, ovx with hypercalcic diet: 0.018 ±0.010, ovx with hipocalcic diet: 0.153 ±0.030 and ovx with zoledronate treatment: 0.010 ±0.059. Similar results were obtained with BF (data not shown). Conclusions: 1) In 100% of the cases the pharmacokinetic parameters obtained were compatible with the described model. 2) BF and BR values were coincident with the biological models. 3) The methodology is more precise than the previous one that depends on plasma F levels. Current experiments are being carried out to determine the sensitivity and specificity of this new method. This article is part of a Special Issue entitled AAOMM 2011 Abstracts. doi:10.1016/j.bone.2011.09.015