The effect of nitrogen containing bisphosphonates, zoledronate and alendronate, on the production of pro-angiogenic factors by osteoblastic cells.

Bisphosphonates (BPs) have been shown to influence angiogenesis. This may contribute to BP-associated side-effects such as osteonecrosis of the jaw (ONJ) or atypical femoral fractures (AFF). The effect of BPs on the production of angiogenic factors by osteoblasts is unclear. The aims were to investigate the effect of (1) alendronate on circulating angiogenic factors; vascular endothelial growth factor (VEGF) and angiopoietin-1 (ANG-1) in vivo and (2) zoledronate and alendronate on the production of VEGF and ANG-1 by osteoblasts in vitro. We studied 18 post-menopausal women with T score⩽-2 randomized to calcium/vitamin D only (control arm, n=8) or calcium/vitamin D and alendronate 70mg weekly (treatment arm, n=10). Circulating concentrations of VEGF and ANG-1 were measured at baseline, 3, 6 and 12months. Two human osteoblastic cell lines (MG-63 and HCC1) and a murine osteocytic cell line (MLO-Y4) were treated with zoledronate or alendronate at concentrations of 10(-12)-10(-6)M. VEGF and ANG-1 were measured in the cell culture supernatant. We observed a trend towards a decline in VEGF and ANG-1 at 6 and 12months following treatment with alendronate (p=0.08). Production of VEGF and ANG-1 by the MG-63 and HCC1 cells decreased significantly by 34-39% (p<0.01) following treatment with zoledronate (10(-9)-10(-6)M). Treatment of the MG-63 cells with alendronate (10(-7) and 10(-6)) led to a smaller decrease (25-28%) in VEGF (p<0.05). Zoledronate (10(-10)-10(-)(6)M) suppressed the production of ANG-1 by MG-63 cells with a decrease of 43-49% (p<0.01). Co-treatment with calcitriol (10(-8)M) partially reversed this zoledronate-induced inhibition. BPs suppress osteoblastic production of angiogenic factors. This may explain, in part, the pathogenesis of the BP-associated side-effects.

Circulating concentrations of vitamin E isomers: Association with bone turnover and arterial stiffness in post-menopausal women.

The effects of vitamin E on cardiovascular and bone health are conflicting with beneficial and detrimental findings reported. To investigate this further, we carried out a cross-sectional study to determine the relationship between circulating concentrations of the 2 vitamin E isomers, α- and γ-tocopherol (TP) with bone turnover and arterial stiffness. Two hundred and seventy eight post-menopausal women with mean age [SD] 60.9 [6.0] years were studied. Fasting serum α-TP and γ-TP, bone turnover markers; procollagen type 1 amino-terminal propeptide (P1NP) and C-terminal telopeptide of type 1 collagen (CTX), parathyroid hormone (PTH), total cholesterol (TC) and triglycerides (TG) were measured. Pulse wave velocity (PWV) and central augmentation index (AI) as markers of arterial stiffness were also determined. A positive correlation was observed between α-TP and γ-TP (r=0.14, p=0.022). A significant negative association between α-TP and P1NP only was seen in multiple linear regression analysis following adjustment for serum TC and TG (p=0.016). In a full multi-linear regression model, following correction for age, years since menopause, smoking habits, alcohol intake, use of calcium supplements, BMI, PTH, serum calcium, and estimated glomerular filtration rate (eGFR), the association between α-TP and P1NP remained significant (p=0.011). We did not observe any significant association between γ-TP or α-TP/γ-TP ratio with P1NP or CTX. P1NP was significantly lower in subjects with α-TP concentrations of >30 µmol/L (α-TP >30 µmol/L; P1NP: 57.5 [20.7], α-TP<30 µmol/L; P1NP: 65.7 [24.9] µg/L, p=0.005). PWV was significantly associated with α-TP/γ-TP ratio (p=0.04) but not with serum α-TP or γ-TP in a full multi-linear regression model adjusting for serum lipids, age, and blood pressure. The data suggest that high serum concentrations of α-TP may have a negative effect on bone formation. The balance of α-TP and γ-TP may be important in maintaining arterial compliance. Longitudinal studies are needed to investigate the impact of the vitamin E isomers on bone and cardiovascular health.

Peroxisomal disorders: clinical and biochemical studies in 15 children and prenatal diagnosis in 7 families.

We describe the main clinical and biochemical findings in 15 patients with peroxisomal disorders, together with the results of 11 prenatal investigations for Zellweger syndrome. The initial laboratory diagnosis depended in most cases on demonstration of elevated very long chain fatty acids in plasma, but follow-up studies using cultured fibroblasts were essential for complete classification. The patient group comprises nine cases of Zellweger syndrome, one of neonatal adrenoleucodystrophy, two of infantile Refsum disease, one of bifunctional protein deficiency, and two of rhizomelic chondrodysplasia punctata. The study illustrates the clinical and biochemical variability of this group of patients and the detailed studies that are required for classification.

Raised concentration of plasma creatine kinase BB isoenzyme in myelodysplasia.

A 72 year old woman presented with a suspected myocardial infarction. An echocardiograph showed no acute changes but her plasma creatine kinase (CK) activity was increased at 343 U/l (< 175 normal range). The apparent creatine kinase-MB activity by a CK-M subunit immunoinhibition assay was 350 U/l. In view of the discrepancy between the total creatine kinase and CK-MB activity plasma creatine kinase electrophoresis studies were performed which showed not only a band of creatine kinase-MM but also a band of creatine kinase-BB, 53% of the total creatine kinase activity. No band of CK-MB was seen. It later transpired that the woman had myelodysplasia. It is suggested that premalignant and malignant haematological conditions should be considered in patients with an unexplained increase in plasma CK-BB.

Larvicidal properties of water bug Diplonychus indicus Venkatesan & Rao and its use in mosquito control.

Males, females and egg carrying males of water bug D. indicus were collected at different sites from Chetpet pond, Madras, India. The bugs showed differences in their predatory efficiencies in relation to density, size and type of prey. The bug showed a typical predatory response in relation to hunger level.

Interrelationships of Rotylenchulus reniformis with Rhizoctonia solani on Cotton.

The interrelationships between reniform nematode (Rotylenchulus reniformis) and the cotton (Gossypium hirsutum) seedling blight fungus (Rhizoctonia solani) were studied using three isolates of R. solani, two populations of R. reniformis at multiple inoculum levels, and the cotton cultivars Dehapine 90 (DP 90) and Dehapine 41 (DP 41). Colonization of cotton hypocotyl tissue by R. solani resulted in increases (P

Influence of Rhizoctonia solani on Egg Hatching and Infectivity of Rotylenchulus reniformis.

The effects of culture filtrates of Rhizoctonia solani and root exudates of R. solani-infected cotton (Gossypium hirsutum) seedlings on hatching of eggs and infectivity of females of Rotylenchulus reniformis were evaluated in an attempt to account for the enhanced nematode reproduction observed in the presence of this fungus. Crude filtrates of R. solani cultures growing over sterile, deionized distilled water did not affect egg hatching. Exudates from roots of cotton seedlings increased hatching of R. reniformis eggs over that observed in water controls. Exudates from cotton seedling roots not infected or infected with R. solani did not differ in their effect on egg hatching. However, infection of cotton seedlings by reniform females was increased in the presence of R. solani, resulting in the augmented egg production and juvenile population densities in soil observed in greenhouse studies.

Changes in Dickkopf-1 (DKK1) and Sclerostin following a Loading Dose of Vitamin D 2 (300,000 IU).

Background. Vitamin D is important for bone health, although high loading doses have been associated with an increase in fracture risk. The mechanisms remain uncertain. Aim. We hypothesize that supraphysiological concentrations of 1,25 (OH)2 vitamin D may inhibit formation by increasing the production of Wnt inhibitors: sclerostin and DKK1. Subjects and Methods. We measured serum sclerostin and DKK1 in 34 patients (21 F, 13 M) aged mean (SD) 61.3 (15.6) years with vitamin D deficiency/insufficiency treated with a loading dose of vitamin D2 (300,000 IU) intramuscularly. Blood samples were taken at baseline and serially up to 3 months. Results. Serum 1,25 (OH)2 vitamin D increased markedly at 3 months (mean (SD) baseline 116 (63), 3 months : 229 (142) pmol/L, P < 0.001). There was a significant correlation between sclerostin and DKK1 at baseline (r = 0.504, P = 0.002) and at 3 months (r = 0.42, P = 0.013). A significant inverse correlation was observed between sclerostin and eGFR at 3 months (r = -0.494, P = 0.007). Sclerostin increased significantly at 3 months (P = 0.033). In a multilinear regression analysis with % change in sclerostin and DKK1 as dependent variable, a positive significant association was observed with % change in 1,25 (OH)2 vitamin D (P = 0.038), independent of changes in PTH and following correction for confounders such as age, gender, BMI, BMD and eGFR. Conclusions. Supraphysiological concentration in 1,25 (OH)2 vitamin D achieved following a loading dose of vitamin D increases sclerostin and may inhibit Wnt signalling. This may have detrimental effects on bone.

The relationship between intact PTH and biointact PTH (1-84) with bone and mineral metabolism in pre-dialysis chronic kidney disease (CKD).

OBJECTIVES: Abnormalities in PTH are implicated in the pathogenesis of bone abnormalities in chronic kidney disease (CKD)-mineral bone disorder (CKD-MBD). PTH concentrations are important in clinical decision and management. This emphasises the importance of providing an assay which measures biologically active PTH. We compared concentrations of intact PTH with biointact PTH (1-84) in CKD and end stage renal disease (ESRD) and investigated the relationship between the 2 PTH assays with bone and mineral laboratory parameters and bone mineral density (BMD) in CKD. DESIGN AND METHODS: We assessed 140 patients (61 in ESRD and 79 with CKD stages 1-4) in this cross-sectional study. We measured biointact PTH (1-84) as well as routine biochemical parameters on all subjects. In the CKD cohort, bone turnover markers; bone alkaline phosphatase (BAP) and tartrate resistant acid phosphatase (TRACP)-5b and bone mineral density (BMD) were also determined. RESULTS: In ESRD, intact PTH concentration was significantly higher compared to biointact PTH (1-84) (422 [443] v/s 266 [251] pg/mL, (p<0.001) with an average bias of 60%. In CKD, intact PTH concentration was also higher compared to biointact PTH (1-84) (79[55] v/s 68[49] pg/mL p<0.001) with an average bias of 18%. Only the biointact PTH (1-84) assay showed any significant correlation with serum calcium concentrations (r=-0.26, p<0.05) and phosphate (r=0.25, p<0.05) in CKD. Following multilinear regression analysis and adjustment for all significant co-variables, only eGFR, BAP and 25 (OH)vitamin remained significantly associated with intact PTH and biointact PTH (1-84). The strength of association was stronger between BAP and biointact PTH (1-84) (biointact PTH (1-84): p=0.007, intact PTH: p=0.01). In adjusted analyses, only biointact PTH (1-84) was significantly associated with BMD at the fore-arm (FARM) (p=0.049). CONCLUSIONS: The study confirms the differences between intact PTH and biointact PTH (1-84) in ESRD. Whilst there may be similarities in the diagnostic ability of both intact and biointact PTH (1-84), our data suggest that biointact PTH (1-84) assay may better reflect bone metabolism and BMD in CKD. Further longitudinal studies are needed.

First case report of testosterone assay-interference in a female taking maca (Lepidium meyenii).

A young female with prolonged intermenstrual bleeding was found to have raised total plasma testosterone of 25.8 nmol/l (NR<2.9 nmol/l) using the Roche Elecsys Testosterone I immunoassay without clinical features of virulisation. Few months ago investigations for lethargy and low libido had shown normal total testosterone of 0.8 nmol/l. Further history revealed that she was using maca extract to improve her lethargy and low libido. Maca is traditionally used for its aphrodisiac and fertility-enhancing properties. Maca use has not been shown to affect serum testosterone in mice and human studies. Immunoassay interference with maca was suspected. Testosterone immunoassays use monoclonal antibodies specifically directed against testosterone. They are prone to interference from androgenic compounds. Reanalysis of the original serum sample using Elecsys Testosterone II assay, a higher affinity assay, revealed a total testosterone level of 2.9 nmol/l. It is important to exclude assay interference when testosterone level is greater than 5 nmol/l without supportive clinical signs.

A common polymorphism in the methylenetetrahydrofolate reductase gene, homocysteine, and ischemic cerebrovascular disease.

BACKGROUND AND PURPOSE: A common polymorphism (T/t) in the gene encoding the methylenetetrahydrofolate reductase (MTHFR) enzyme has been associated with elevated serum homocysteine, itself a risk factor for stroke. Some studies have reported an association with ischemic heart disease, but no published studies have examined its relationship with stroke. METHODS: We determined the TT genotype frequency and T allele frequency in 345 patients with ischemic cerebrovascular disease (CVD) and 161 control subjects. In a subgroup we also determined serum homocysteine and folate concentrations. RESULTS: In the patient group there was a significant relationship between TT genotype and homocysteine concentration after we controlled for other risk factors. Controlling for serum folate weakened this relationship, and folate itself was independently related to serum homocysteine. There was no difference between patients and control subjects in either TT genotype frequency (10.7% versus 13.7%; P = .34) or T allele frequency (0.68 versus 0.67; P = .67). There was no association when analysis was limited to individuals deficient in folate (serum folate < 25th centile) or to younger individuals (< 65 years). There was no association between TT genotype and any stroke subtype or with degree of carotid stenosis. CONCLUSIONS: In patients with CVD we confirmed a relationship between the MTHFR genotype and serum homocysteine concentration and an interaction with serum folate concentration. We found no association between CVD and genotype. However, the interaction with serum folate suggests that the genotype could still be a risk factor in populations with a low folic acid intake.

Transport of phytanic acid on lipoproteins in Refsum disease.

Patients with Refsum disease accumulate significant quantities of phytanic acid in adipose and neural tissue. The accumulation can be reversed by following a diet low in phytanic acid, yet the mechanism of transport of this fatty acid is obscure. We investigated the distribution of phytanic acid in different lipoprotein subfractions in 11 patients with Refsum disease and 9 unaffected siblings. Plasma phytanic acid was distributed on VLDL (16.2% +/- 12.2%), IDL (1.77% +/- 1.64%), LDL (34.8% +/- 12.6%) and HDL (14.3% +/- 7.87%). No correlations with any parameter were seen with total phytanic acid content. Weak nonsignificant correlations were found with the fractional distribution of phytanic acid and VLDL triglyceride (r = 0.35; p = 0.12) and plasma HDL-cholesterol (r = 0.32; p = 0.16) and with LDL:HDL cholesterol ratio (r = 0.33; p = 0.14). Significant correlation of the fractional distribution of phytanic acid on lipoprotein particles was noted with the ratio of apolipoprotein B: apolipoprotein A1-containing particles (r = 0.46; p = 0.03) and apolipoprotein B: apolipoprotein A1 in HDL2 (r = 0.53; p = 0.01). This suggests that the import-export balance for phytanic acid in plasma is related to forward and reverse cholesterol transport on lipoprotein particles, and only weakly to plasma cholesterol and triglycerides. These ratios of apolipoprotein particles may play a significant role in determining the rate of phytanic acid elimination in patients with Refsum disease.

Influence of plasma phytanic acid levels in Refsum's disease on the behaviour of the erythrocyte membrane sodium-lithium countertransporter.

BACKGROUND: Abnormal behaviour of the erythrocyte membrane sodium-lithium countertransporter (SLC) is associated with plasma triglyceride concentrations. Refsum's disease is characterized by progressive neurological dysfunction and accumulation of phytanic acid, an isoprenoid fatty acid, in fat-containing tissues. METHODS: This study explored the effects of plasma phytanic acid on SLC kinetics in nine Caucasian patients with Refsum's disease and in age- and sex-matched Caucasian control subjects. RESULTS: A dose-dependent association was seen between countertransporter maximal velocity and phytanic acid content of low-density lipoprotein (LDL)-cholesterol (r = -0.61, r = -0.65 respectively; P = 0.05, P = 0.04) and high-density lipoprotein (HDL)-cholesterol (r = -0.81, -0.82 respectively; P = 0.005, P = 0.003). No significant association was seen with the sodium affinity of the transporter (r = -0.44, P = 0.20, for LDL; and -0.43, P = 0.21, for high-density lipoprotein). CONCLUSION: These findings suggest that phytanic acid may alter the behaviour of the sodium-lithium countertransporter.