Nutritional bone disease in Indian population.

Syndromes of bone disease and deformities consequent to disorders of nutrition, bone and mineral metabolism constitute a serious national health problem. The studies on this subject are scanty. Data on nutritional bone disease are described and discussed. We had surveyed 337.68 million population residing in 0.39 million villages in 22 States of India during the period 1963 to 2005. Of the 4,11,744 patients identified with the disorders of bone and mineral metabolism, 2,13,760 (52%) had nutritional bone disease, 1,77,200 (43%) had endemic skeletal fluorosis and 20,784 (5%) had metabolic bone disease and in 41 patients (0.19%) the bone disease was rare, mixed or unidentified. Vitamin D deficiency osteomalacia and rickets caused by inadequate exposure to sunlight (290-315 nm), dietary calcium deficiency (<300 mg/day) and fluoride interaction syndromes, calcium deficiency induced osteoporosis and calcium and vitamin D deficiency induced osteoporosis in the elderly, were the commonest disorders responsible for bone disease and deformities, besides caused by endemic skeletal fluorosis as a single entity in endemic fluorosis villages. Calcium deficiency per se dose not cause rickets, as revealed in our long-term follow up study on 47,500 calcium deficient children. Only mothers with severely depleted bone mineral and vitamin D stores gave birth to their babies with congenital rickets. Vitamin D deficiency rickets in children and osteomalacia in the mothers are the commonest disorders prevalent in the rural population of India. These disorders and the syndromes of calcium deficiency and fluoride interactions are largely responsible for the morbidity and mortality in the young and promising individuals, with economic consequences.

Chitosan as a macroaffinity ligand purification of chitinases by affinity precipitation and aqueous two-phase extractions.

(1) Chitosan was found to be a suitable macroaffinity ligand for affinity precipitation of chitinase from Neurospora crassa, cabbage and puffballs. (2) The activity recoveries of 85, 82 and 90% with concomitant fold purifications in terms of specific activities were 27, 15 and 30 with N. crassa, cabbage and puffballs and were obtained with affinity precipitation. These results were obtained with clarified extracts/homogenates as the starting materials. (3) The incorporation of chitosan in poly(ethylene glycol) (PEG)-salt aqueous two-phase system allowed purification of chitinases from these sources directly from unclarified extracts/homogenates. (4) The 96% (w/v) chitosan (of initially introduced into the aqueous two-phase system) partitioned into PEG-phase and this enhanced the partitioning of chitinases into PEG-phase. The chitosan, free as well as bound to chitinases, could be separated from PEG-phase by increasing the pH to 7. (5) By the process of desorption with 2.0 M MgCl2, 86, 80 and 88% activity recoveries (% expressed in terms of total units of enzyme activities in the crude extract) were obtained in the case of N. crassa, cabbage and puffballs, respectively. The corresponding fold purifications in terms of specific activities were 34, 20 and 38. (6) The purified preparations gave single bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the estimated molecular masses agreed with the reported values in the literature.

Purification of phospholipase D by two-phase affinity extraction.

An aqueous two-phase system of polyethylene glycol (PEG)-salt was used for purification of phospholipase D (PLD) from peanuts and carrots. Alginate, a known macroaffinity ligand for PLD, was incorporated in the PEG phase and resulted in 91 and 93% of the enzyme activity (from peanuts and carrots, respectively) getting partitioned in the PEG phase. The elution of the enzyme from alginate was facilitated by exploiting the fact that the latter can be reversibly precipitated in the presence of Ca2+. The enzyme was eluted from the polymer by using 0.5 M NaCl. Peanuts and carrots PLD could be purified 78- and 17-fold with 82 and 85% activity recovery, respectively. The purified enzyme from both sources gave a single band on sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis.

One-step purification of glucoamylase by affinity precipitation with alginate.

It was found that alginate binds to glucoamylase, presumably through the recognition of starch binding domain of the latter. The present work exploits this for purification of glucoamylases from commercial preparation of Aspergillus niger and crude culture filtrate of Bacillus amyloliquefaciens by affinity precipitation technique in a single-step protocol. Glucoamylase is selectively precipitated using alginate as macroaffinity ligand and later eluted with 1.0 M maltose. In the case of A. niger, 81% activity is recovered with 28-fold purification. The purified glucoamylase gave a single band on SDS-PAGE corresponding to 78 kDa molecular weight. The developed affinity precipitation process also works efficiently for purification of Bacillus amyloliquefaciens glucoamylase from its crude culture filtrate, giving 78% recovery with 38-fold purification. The purified preparation showed a major band corresponding to 62 kDa and a faint band about 50 kDa on SDS-PAGE. The latter corresponds to the molecular weight for alpha-amylase of Bacillus amyloliquefaciens.

Reversibly soluble macroaffinity ligand in aqueous two-phase separation of enzymes.

Use of alginate as a free bioligand incorporated in an aqueous two-phase system of polyethylene glycol 6000-salt resulted in considerable purification of wheat germ alpha-amylase and sweet potato beta-amylase from their crude extracts. The elution of the enzyme from the free bioligand was facilitated by exploiting the fact that alginate can be reversibly precipitated in the presence of Ca2+. alpha-Amylase could be purified 42-fold with 92% activity recovery. beta-Amylase on the other hand could be purified 43-fold with 90% recovery. Both purified enzymes showed a single band on sodium dodecylsulfate-polyacrylamide gel electrophoresis.

Free polymeric bioligands in aqueous two-phase affinity extractions of microbial xylanases and pullulanase.

Two reversibly soluble-insoluble polymers (viz. Eudragit S-100 and alginate) were used as free macroaffinity bioligands in polyethylene glycol (PEG)/salt two-phase systems for separation of enzymes. Incorporation of Eudragit S-100 and alginate in the PEG phase led to considerable selectivity in separation of microbial xylanases and pullulanase, respectively. Xylanase from Aspergillus niger was recovered 93% with 56-fold purification, whereas the enzyme from Trichoderma reesei and Bacillus amyloliquefaciens was obtained with 93% activity recovery (31-fold purification) and 90% activity recovery (32-fold purification), respectively. From Bacillus acidopullulyticus pullulanase, 85% enzyme activity recovery with 44-fold purification was obtained. The approach described here shows the potential of developing into a general approach for use of reversibly soluble-insoluble macroaffinity ligand in two-phase affinity extraction.

An efficient purification process for sweet potato beta-amylase by affinity precipitation with alginate.

beta-amylases are used in production of maltose syrup. It is shown that sweet potato beta-amylase can be purified by affinity precipitation with alginate with 80% activity yield and 44 fold purification. SDS-PAGE of the purified protein showed a single band and a subunit weight of 50 kDa. Preliminary data with soybean and barley enzymes indicate that this may be a general method for purification of beta-amylases.

Purification of alpha-amylases using magnetic alginate beads.

Magnetic alginate beads were used to purify alpha-amylases from porcine pancreas, starchzyme, BAN 240L (a commercial purification from Bacillus subtilis), and wheat germ. The beads bound a significant level of alpha-amylase activity from porcine pancreas, BAN 240L, and wheat germ. In each case, the enzyme activity could be eluted by using 1.0 M maltose, a known competitive inhibitor of alpha-amylase. In the case of BAN 240L, 3.6-fold purification with 72% recovery of activity was observed. In the case of wheat germ enzyme, starting from the crude extract, 48-fold purification with 70% activity recovery was observed. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis also indicated considerable purification in the latter case.

A laboratory assessment of enamel hypoplasia of teeth with varying severities of dental fluorosis.

The effect of lifelong exposure to drinking-water containing fluoride on tooth enamel microhardness was investigated. Dental fluorosis of teeth from adult subjects, who lived continuously since birth in areas characterized by the hot climate of India, supplied by drinking-water containing between 0.5 and 8.7 parts/10 F, was estimated by the Dean score. Tooth enamel sections were examined from the enamel surface towards the dentino-enamel junction (DEJ) for microhardness. Separated enamel crowns were pulverized and analysed for fluoride. Regarding the distribution of the fluoride concentrations in the drinking-water, we selected a partition below and above 0.8 parts/10 F as a criterion for comparison between the results. Tooth enamel of humans from Israel living in a drinking-water area containing about 0.5 parts/10 F was also examined. Positive associations were evident between fluoride in drinking-water, fluoride concentrations in the bulk enamel and fluorosis selection. A significantly high inverse relationship was evident between the fluoride concentration of the drinking-water and the subsurface enamel microhardness. In similar fluoride-concentration drinking-water areas of India and Israel the microhardness of the subsurface enamel was less for the Indian teeth. The microhardness of the enamel near the DEJ for the three different water fluoride-concentration areas was not significantly different.

Endemic chronic fluoride toxicity and dietary calcium deficiency interaction syndromes of metabolic bone disease and deformities in India: year 2000.

Epidemiological studies during 1963-1997 were conducted in 45,725 children exposed to high intake of endemic fluoride in the drinking water since their birth. Children with adequate (dietary calcium > 800 mg/d) and inadequate (dietary calcium < 300 mg/d) calcium nutrition and with comparable intakes of fluoride (mean 9.5 +/- 1.9 mg/d) were compared. The toxic-effects of fluoride were severe and more complex and the incidence of metabolic bone disease (rickets, osteoporosis. PTH bone disease) and bony leg deformities (genu valgum, genu varum, bowing, rotational and wind-swept) was greater (> 90%) in children with calcium deficiency as compared to < 25% in children with adequate calcium who largely had osteosclerotic form of skeletal fluorosis with minimal secondary hyperparathyroidism. The syndrome of skeletal fluorosis and associated metabolic bone disease and deformity is a unique clinical entity classified as a variant of osteosclerotic form of skeletal fluorosis. This syndrome chiefly results from the biological impact of excess fluoride, low calcium, high PTH and 1,25 (OH)2D3 separately and through their interactions on bone structure and metabolism as studied by radiology, bone scanning, bone histomorphometry and relevant metabolic and endocrine laboratory investigations. Metabolically active and vascular bones of children accumulate fluoride at faster and greater rate than adults (at the sites of active growth). In calcium deficient children the toxic effects of fluoride manifest even at marginally high (> 2.5 mg/d) exposures to fluoride. Fluoride toxicity also exaggerates the metabolic effects of calcium deficiency on bone. The findings strongly suggest that children with calcium deficiency rickets reported in the literature should be re-investigated for possible fluoride interactions. Deep bore drinking water supply with fluoride < 0.5 ppm and improvement of calcium nutrition provide 100% protection against the toxic effects of fluoride and are recommended as the cost effective and practical public health measures for the prevention and control of endemic fluorosis.

Hypoglycemic effect of Prunus amygdalus seeds in albino rabbits.

On administration of 2.5 g almond seed and its proportionate fractions, viz. 1.22 g defatted seed and 1.28 g oil to three groups of albino rabbits, they showed a definite hypoglycemic action during a two months study. The active factor seems to be a non oil fraction which is only partly soluble in ethyl ether.

Nutritional and metabolic rickets.

Nutritional rickets is caused by vitamin D deficiency due to lack of exposure to sunlight. Neonatal rickets occurs only in infants born to mothers with very severe osteomalacia. Calcium deficiency alone does not cause mineralisation defects. It only causes osteoporosis and secondary hyperparathyroidism with raised plasma, 1,25 (OH)2D and osteocalcin. Low 25-OHD, increased IPTH, increased alkaline phosphatase in plasma and decreased calcium and increased hydroxyproline in urine are diagnostic of rickets. Low or undetectable plasma levels of 25-OHD, in presence of high plasma 1,25(OH)2D and IPTH are often observed during treatment with vitamin D. Even the marginal intakes of fluoride (> 2.5 mg/day) cause rickets in calcium deficient children. Indian children often need high dose of vitamin D due to severely depleted D stores, high IPTH and severe bone disease (radiologic and histomorphometric) for treatment.

Effect of Prunus amygdalus seeds on lipid profile.

The effect of Prunus amygdalus, which is rich in unsaturated fat, fibre diet and low cholesterol was studied in 36 normolipidemic male albino rabbits for 60 days. Three different doses i.e. whole seeds defatted and oil showed a decrease in serum total cholesterol, triglyceride, LDL cholesterol, VLDL cholesterol and an increase in phospholipid, faecal sterol and HDL cholesterol. The maximum effects where shown by whole seeds followed by oil and defatted.

Reduction of femoral arterial bleeding post catheterization using percutaneous application of fibrin sealant.

The number of cardiac catheterizations performed yearly is growing with correspondingly increasing amounts of morbidity, complications, and hospital costs. This study suggests that fibrin sealant instillation via an arterial sheath at the completion of femoral catheterization may improve hemostasis. Results using fibrin sealant in 12 unheparinized dogs documented significant reductions (McNemar's exact test) versus control for groin ecchymoses (1 versus 8, P = .008) and radiolabeled hematoma formation (0 versus 7, P = .016). Also swelling was less in the fibrin sealant treated groins when compared to control groins (1 versus 6, P = .125), but failed to reach statistical significance. Results in eight heparinized dogs (activated clotting time 374 +/- 22, mean +/- SEM) revealed a statistically significant reduction in signs of gross bleeding in the fibrin sealant-treated groins (1 versus 8, P = .016). This method may contribute to reduced morbidity, complications, and length of hospitalization. It may also allow for earlier patient mobilization after cardiac catheterization.

Metabolic studies in congenital vitamin D deficiency rickets.

Congenital rickets in 3 newborns of mothers with advanced nutritional osteomalacia, healed with maternal breast milk feeding when mothers alone were given calcium supplements and 7.5 mg of intravenous D2 and the mother baby pair protected from sunlight. Maternal plasma biochemistry indicated more severe vitamin D deficiency compared to their newborns (intrauterine foetal priority). The first dose of 7.5 mg of vitamin D3 and calcium supplements to mother healed osteomalacia but did not appear to heal the rickets of their breast fed infants (extrauterine maternal priority for vitamin D). A second dose given at 3 months interval healed the rickets in their infants and the biochemistry of the mother and baby returned towards normal. Congenital rickets developed when maternal bone mineral and vitamin D stores had been completely exhausted. Raised IPTH levels in the newborn suggested that foetal parathyroids were responsive to hypocalcaemic stimulus.

Handigodu disease: a radiological study. A new variety of spondyloepi(meta)physeal dysplasia of the autosomal dominant type.

Handigodu disease is a new syndrome of familial spondyloepi(meta)physeal dysplasia. It is inherited as an autosomal dominant trait. The disease is prevalent in a localised area of South India. On the basis of detailed clinical, anthropometric and radiological investigations of 234 affected individuals, it has been observed that different clinical presentations reflect variation in the severity of the disease. All of them could be explained as being caused by defective development of bones as a result of monogenic disorder.