Beneficial effect of copper supplementation on deposition of fluoride in bone in fluoride- and molybdenum-fed rabbits.

Fluorosis is a major public health problem in the world, including India. The present study was undertaken to investigate the role of molybdenum (Mo) in the deposition of fluoride (F) in bone and whether copper (Cu) supplementation has any alleviating role when F and Mo are ingested together. For this purpose, four groups of rabbits were used [control (C), fluoride (F), fluoride + molybdenum (F + Mo), and fluoride + molybdenum + copper (F + Mo + Cu)] to find out the effect of these treatments on various bone-related parameters like intact parathyroid hormone (iPTH), alkaline phosphatase and Cu in serum, hydroxyproline and calcium (Ca) in urine, and minerals (F, Cu, manganese, and zinc) in femur bone ash. Bone mineral content (BMC), bone mineral density (BMD) [by dual energy X-ray absorptiometry (DXA)], and strength of femur bones were also assessed. F content in the femur was significantly higher (P < 0.01) in all experimental groups compared to control group. Mo supplementation increased F deposition in femur bone in the F + Mo group, whereas supplementation of Cu reduced F deposition in the F + Mo + Cu group compared to the F + Mo and F groups. Levels of Cu in femurs of the F + Mo and F + Mo + Cu groups were significantly higher (P < 0.05, P < 0.01, respectively) than in the C group, although serum Cu was significantly lower in the F and F + Mo than the C and F + Mo + Cu groups. Magnesium levels in the F + Mo group were significantly higher (P < 0.05) than in the F and F + Mo + Cu groups. Cu supplementation in the F + Mo + Cu group increased deposition of zinc significantly (P < 0.05) compared to the F and F + Mo groups. Serum iPTH, alkaline phosphatase, and urinary hydroxyproline and Ca were significantly higher (P < 0.01) in the F and F + Mo than in the C and F + Mo + Cu groups. However, serum iPTH and urinary hydroxyproline were higher in the F + Mo group than the F group. Alkaline phosphatase was significantly higher in the F + Mo group than the F and F + Mo + Cu groups. Levels of serum Cu in the F and F + Mo groups were lower than in the C group, though serum Cu was significantly higher in the F + Mo + Cu than in all other groups. DXA analysis of femur bone indicated that BMD in the F + Mo group was significantly higher than in the F (P < 0.05), C (P < 0.01), and F + Mo + Cu (P < 0.05) groups. However, there was no significant difference in BMC among the groups. Bone strength was significantly higher (P < 0.05) in the F + Mo group than in the C group. Results of the present study show that ingestion of Mo with F does not create secondary Cu deficiency (due to increased excretion of Cu through urine). However, Cu concentration was decreased in serum in this group (F + Mo) compared to the C and F + Mo + Cu groups. Deposition of F in femur bone was more (22%) when it was given along with Mo compared to F alone, while F deposition in femur bone was less in the F + Mo + Cu group by 80% compared to the F group. Also, deposition of F in the F + Mo + Cu group was 120% less compared to the F + Mo group. The increase in F level due to Mo addition appears to be offset by supplementation with Cu. Supplementation with Cu showed a beneficial effect on bone resorption as well as bone formation.

Additional beneficial effect of tamarind ingestion over defluoridated water supply to adolescent boys in a fluorotic area.

OBJECTIVE: We evaluated the effect of tamarind (Tamarindus indicus) on ingestion and whether it provides additional beneficial effects on mobilization of fluoride from the bone after children are provided defluoridated water. METHODS: A randomized, diet control study was conducted in 30 subjects from a fluoride endemic area after significantly decreasing urinary fluoride excretion by supplying defluoridated water for 2 wk. Subjects were then assigned to one of two groups, with 15 in each group. One group was supplemented with tamarind (experimental group) for 3 wk and the other (control) group was given only defluoridated water for the same period. RESULTS: The mean changes in urinary components after tamarind ingestion (volume, pH, fluoride calcium, copper, and magnesium) in the control and experimental groups were compared. There was a significant increase (P < 0.01) in fluoride excretion and urinary pH and a significant decrease in urinary calcium (P < 0.01) and copper (P < 0.05) excretion in the experimental group as compared with the control group. There was no change in urinary volume between groups. CONCLUSIONS: Tamarind intake appears to have an additional beneficial effect on the mobilization of deposited fluoride from bone, by enhancing urinary excretion of fluoride.

Corneal endothelial status in the subtypes of primary angle closure glaucoma.

PURPOSE: To study the corneal endothelium and pachymetry in eyes with different subtypes of primary angle closure glaucoma (PACG), as compared to controls. METHODS: A cross-sectional study was conducted on 30 consecutive patients in each subtype of PACG, subacute, acute and chronic, and 30 age and refraction matched controls. The parameters recorded included gonioscopy, optic disc evaluation, applanation tonometry, specular microscopy and central ultrasonic pachymetry. RESULTS: The mean endothelial cell counts in the four groups were as follows: subacute PACG 2396 +/- 271 cells/mm2, acute PACG 1597 +/- 653 cells/mm2, chronic PACG 2229 +/- 655 cells/mm2 and controls 2461 +/- 321 cells/mm2. The mean endothelial cell count in the fellow eyes of subacute PACG, acute PACG and chronic PACG patients was 2294 +/- 305 cells/mm2, 2388 +/- 226 cells/mm2 and 2108 +/- 203 cells/mm2, respectively (NS). The acute PACG patients had significantly lower endothelial cell counts (P < 0.001) as compared to the other three groups. Eyes in which the acute attack of angle closure persisted for less than 72 h had a mean endothelial cell count of 2016 +/- 306 cells/mm2, as compared to 759 +/- 94.4 cells/mm2 in eyes with an attack lasting for 72 h or more (P < 0.001). The endothelial count was also significantly lower in eyes with chronic PACG as compared to control eyes (P < 0.001). There was increased pleomorphism and polymegathism of the corneal endothelial cells seen in eyes with resolved acute and chronic PACG. The mean central corneal thickness was 531.4 +/- 25.3 microm in eyes with subacute PACG, 567.9 +/- 37.3 microm in eyes with acute PACG, 526.4 +/- 31.9 microm in eyes with chronic PACG and 525 +/- 12.6 microm in control eyes. The acute PACG eyes had a significantly higher corneal thickness (P < 0.001) when compared to all the other groups. CONCLUSION: There is a significant decrease in the corneal endothelial cell density in eyes that have had an acute attack of angle closure glaucoma and in eyes with chronic PACG. The endothelial cell population in eyes with sub-acute PACG and in the fellow eyes of all subtypes of PACG is not significantly different from the normal population.

Insect wing conjunctival granuloma.

PURPOSE: To report a case of conjunctival granuloma formation in response to lodgment of an insect wing. METHOD: Case report and review of the literature. RESULTS Granuloma formation in the conjunctiva can occur following lodgment of an insect wing. This can cause the patient chronic redness, discharge, and discomfort. CONCLUSIONS: Small foreign bodies with relatively little mass and large surface area can evade the normal protective mechanisms for removal of a foreign body and give rise to a chronic inflammatory response.

Effect of tamarind ingestion on fluoride excretion in humans.

OBJECTIVE: To evaluate the effect of tamarind (Tamarindus indicus) ingestion on excretion of fluoride in school children. DESIGN: Randomized, diet-control study. SUBJECT: Twenty healthy boys were included and 18 of them completed the study. INTERVENTIONS: Each subject consumed 10 g tamarind daily with lunch for 18 days at the social welfare boys' hostel. The nutrient composition of the daily diet was constant throughout the experimental period. RESULTS: Tamarind intake led to significant increase (P<0.001) in the excretion of fluoride in 24 h urine (4.8+/-0.22 mg/day) as compared to excretion on control diet (3.5+/-0.22 mg/day). However, excretion of magnesium and zinc decreased significantly (7.11+/-1.48 mg of Mg and 252.88+/-12.84 microg of Zn per day on tamarind diet as compared to 23.39+/-3.68 mg of Mg and 331.78+/-35.31 microg Zn per day on control diet). Excretion of calcium and phosphorous were not significantly different while creatinine excretion decreased with tamarind intake (225.66+/-81 mg creatinine/day with tamarind and 294.5+/-78.76 mg creatinine/day without tamarind). CONCLUSION: Tamarind intake is likely to help in delaying progression of fluorosis by enhancing urinary excretion of fluoride.

The trabecular meshwork in acute and chronic angle closure glaucoma.

PURPOSE: To determine the effect of acute and chronic primary angle closure glaucoma (PACG) on the trabecular meshwork. METHODS: Trabecular specimens of 16 consecutive patients with primary angle closure glaucoma (PACG)--6 acute PACG eyes, and 10 chronic PACG eyes without an acute attack--were studied by light and electron microscopy. RESULTS: Acute PACG: The trabecular meshwork revealed a generalised oedema and an accumulation of pigment in the widened trabecular spaces and Schlemm's canal. Attenuated trabecular endothelial cells appeared to be devoid of subcellular components. Chronic PACG: In chronic PACG eyes the trabecular architecture had lost its regular arrangement, with fewer and narrower trabecular spaces and fusion of the trabecular beams in areas. There were numerous electron-dense bodies in the trabecular tissues, both within the trabecular beams and in the extracellular spaces, which had a banded fibrillar structure. An overall loss of endothelial cells was noted; the remaining cells were crowded together and were polymorphic. Melanin pigment was present both within the stroma and in the endothelial cells. CONCLUSIONS: Pigment accumulation in the trabecular spaces and within the cells and a noninflammatory degeneration appeared to be the primary changes in the trabecular meshwork after acute angle closure glaucoma. In chronic PACG eyes, there was evidence of loss of endothelial cells and reactive repair processes. These changes were present in areas away from visible peripheral anterior synechiae. A gonioscopic evaluation of the extent of peripheral anterior synechiae alone may not reflect the extent of trabecular meshwork damage in acute and chronic PACG. Patients experiencing an acute attack of PACG require a long-term follow up, because the intraocular pressure (IOP) may rise later, due to ongoing changes compromising the outflow facility, or due to the effects of aging in the trabecular meshwork.

Osseous choristoma of the choroid.

Osseous choristoma of the choroid, also called choroidal osteoma, is a very rare and unusual form of intraocular ossification. We report a case of choroidal osteoma in a young healthy male that to the best of our knowledge is the first report from India.

Ocular parameters in the subgroups of angle closure glaucoma.

PURPOSE: This study was conducted to compare anatomical parameters, thought to be responsible for causing angle closure glaucoma (ACG), among eyes having acute, subacute or chronic ACG. METHODS: Ninety consecutive patients diagnosed with a subgroup of ACG, and 30 age, sex and refraction matched controls, provided a total of 240 eyes for a prospective study. The refractive error, corneal diameter, keratometry, pachymetry, lens thickness and axial length were measured and the relative lens position was calculated. The data were analysed by paired t-test, ANOVA, signed rank test and multivariate analysis. RESULTS: Acute ACG eyes were mildly hyperopic. All the ACG subgroups had similarly short eyeballs and a steeper corneal curvature compared to control eyes. Acute ACG lenses were thicker than all the other groups (P < 0.001), but all ACG eyes had thicker lenses than the controls. Corneal diameters and anterior chamber depths were decreased in acute and chronic ACG eyes compared with subacute ACG and controls (P < 0.001). The uninvolved fellow eyes in each subgroup differed from affected eyes only in having more posteriorly positioned lenses. CONCLUSIONS: There was a spectrum of anatomical variations seen in the subgroups of ACG. Acute ACG eyes expressed an extreme shift of anatomical features away from normal, especially, smaller corneal diameters, leading to a large mobile lens in an already crowded anterior segment. This predisposed them to a severe relative pupillary block, and to a form of ciliary block glaucoma. Chronic ACG eyes were less divergent from normal and therefore could have suffered a milder form of the same kind of angle closure, but over a more prolonged period. Subacute ACG eyes deviated least from controls, and therefore exhibited mild signs and spontaneous resolution. Further work is required to elucidate completely the pathophysiology that leads to ACG.

Surgical removal of a free floating cyst of the iris pigment epithelium causing disturbing visual symptoms.

Pigmented cysts in the anterior chamber, fixed or free floating, are considered to be unusual but not very infrequent. However, most of these cases usually do not need any treatment other than a periodic observation. We report the surgical removal of an iris pigment epithelial cyst floating freely in the anterior chamber. The reason for surgical removal was, disturbance in near vision being caused by movement of the cyst across the visual axis. This specific symptom of disturbed near vision, to the best of our knowledge, is a rare indication for surgery that has not been pointed out earlier. Histopathological confirmation of the clinical diagnosis was also obtained.

Inhibition of xanthine oxidase-xanthine-iron mediated lipid peroxidation by eugenol in liposomes.

The effect of eugenol on xanthine oxidase (XO) xanthine(X)-Fe+3-ADP mediated lipid peroxidation was studied in liver microsomal lipid liposomes. Eugenol inhibited the lipid peroxidation in a dose dependent manner as assessed by formation of thiobarbituric acid reactive substances. When tested for its effect on XO activity per se, (by measuring uric acid formation) eugenol inhibited the enzyme to an extent of 85% at 10 microm concentration and hence formation of O2.- also. However, the concentration of eugenol required for XO inhibition was more in presence of metal chelators such as EDTA, EGTA and DETAPAC, but not in presence of deferoxamine, ADP and citrate. The antiperoxidative effect of eugenol was about 35 times more and inhibition of XO was about 5 times higher as compared to the effect of allopurinol. Eugenol did not scavenge O2.- generated by phenazine methosulfate and NAD but inhibited propagation of peroxidation catalyzed by Fe2+ EDTA and lipid hydroperoxide containing liposomes. Eugenol inhibits XO-X-Fe+3 ADP mediated peroxidation by inhibiting the XO activity per se in addition to quenching various radical species.

The protective effects of eugenol on carbon tetrachloride induced hepatotoxicity in rats.

Our earlier studies in vitro have shown that eugenol inhibits liver microsomal monooxygenase activities and carbon tetrachloride (CCl4)-induced lipid peroxidation (Free Rad. Res. 20, 253-266, 1994). The objective of the present investigation was to study the in vivo protective effect of eugenol against CCl4 toxicity. Eugenol (5 or 25 mg/kg body wt) given orally for 3 consecutive days did not alter the levels of serum glutamic oxalacetic transaminase (SGOT), microsomal enzymes such as cytochrome P450 reductase, glucose-6-phosphatase (G-6-Pase) xenobiotic-metabolizing enzymes (aminopyrine-N-demethylase, N-nitrosodimethylamine-demethylase and ethoxyresorufin-O-deethylase) and liver histology. Doses of eugenol (5 or 25 mg/kg) administered intragastrically to each rat on three consecutive days i.e. 48 hr, 24 hr and 30 min before a single oral dose of CCl4 (2.5 ml/kg body wt) prevented the rise in SGOT level without appreciable improvement in morphological changes in liver. Eugenol pretreatment also did not influence the decrease in microsomal cytochrome P450 content, G-6-Pase and xenobiotic-metabolizing enzymes brought about by CCl4. Since eugenol is metabolized and cleared rapidly from the body, the dose schedule was modified in another experiment. Eugenol (0.2, 1.0, 5.0 or 25 mg/kg) when given thrice orally i.e. prior to (-1 hr) along with (0 hr) and after (+3 hr) the i.p. administration of CCl4 (0.4 ml/kg) prevented significantly the rise in SGOT activity as well as liver necrosis. The protective effect was more evident at 1 mg and 5 mg eugenol doses. However, the decrease in microsomal G-6-Pase activity by CCl4 treatment was not prevented by eugenol suggesting that the damage to endoplasmic reticulum is not protected. The protective effect of eugenol against CCl4 induced hepatotoxicity is more evident when it is given concurrently or soon after rather than much before CCl4 treatment.

Inhibition of microsomal lipid peroxidation and monooxygenase activities by eugenol.

Previously we reported that eugenol (4-allyl-2-methoxyphenol) inhibits non-enzymatic peroxidation in liver mitochondria (E. Nagababu and N. Lakshmaiah, 1992, Biochemical Pharmacology. 43, 2393-2400). In the present study, we examined the effect of eugenol on microsomal mixed function oxidase mediated peroxidation using Fe+3-ADP-NADPH, carbon tetrachloride (CCL4)-NADPH and cumene hydroperoxide (CumOOH) systems. In the presence of eugenol the formation of thiobarbituric acid reactive substances (TBARS) was decreased in all the systems (IC50 values: 14 microM for Fe+3-ADP-NADPH, 4.0 microM for CCl4-NADPH and 15 microM for CumOOH). Oxygen uptake was also inhibited to a similar extent with Fe+3-ADP-NADPH and CumOOH systems. A comparative evaluation with other antioxidants showed that in Fe+3-ADP-NADPH and CumOOH systems, the antioxidant efficacy was in the order: butylated hydroxytoluene (BHT) > eugenol > alpha-tocopherol, while in CCl4-NADPH system the order was alpha-tocopherol > BHT > eugenol. Time course of inhibition by eugenol indicated interference in initiation as well as propagation of peroxidation. Eugenol did not inhibit cytochrome P-450 reductase activity but it inhibited P-450 - linked monooxygenase activities such as aminopyrine-N-demethylase, N-nitrosodimethylamine demethylase, benzo(a)pyrene hydroxylase and ethoxyresorufin-O-deethylase to different extents. However, CumOOH supported monooxygenases (aminopyrine-N-demethylase and benzo(a)pyrene hydroxylase) required much higher concentrations of eugenol for inhibition. The concentration of eugenol required to inhibit monooxygenase activities was more than that required to inhibit peroxidation in all the systems. Eugenol elicited type 1 changes in the spectrum of microsomal cytochrome P-450. These results suggest that the inhibitory effect of eugenol on lipid peroxidation is predominantly due to its free radical quenching ability. Eugenol significantly protected against the degradation of cytochrome P-450 during lipid peroxidation with all the systems tested. These findings suggest that eugenol has the potential to be used as a therapeutic antioxidant. Further evaluation may throw more light on this aspect.

Inhibitory effect of eugenol on non-enzymatic lipid peroxidation in rat liver mitochondria.

The anti-peroxidative activity of eugenol on Fe(2+)-ascorbate- and Fe(2+)-H2O2-induced lipid peroxidation was studied using rat liver mitochondria. Eugenol inhibited thiobarbituric acid reactive substance (TBARS) formation induced by both the systems in addition to oxygen uptake and mitochondrial swelling induced by Fe(2+)-ascorbate. Time course studies on TBARS formation indicated the ability of eugenol to inhibit initiation and propagation reactions. There was no measurable chemical modification of eugenol during the course of mitochondrial peroxidation by both the systems. Mitochondrial peroxidation by Fe(2+)-H2O2 was inhibited by hydroxyl radical (OH) scavengers like mannitol, benzoate, formate and dimethyl sulfoxide apart from eugenol. The OH scavenging ability of eugenol was evident from its inhibitory effect on OH-mediated deoxyribose degradation. The second-order rate constant for the reaction of OH with eugenol was about 4.8 x 10(10) M-1 sec-1. Eugenol reduced Fe3+ ions and Fe3+ chelated to citrate or ADP but it did not exhibit pro-oxidant activity in OH-mediated deoxyribose degradation. Incubation of mitochondria with eugenol resulted in the uptake of small but significant quantities of eugenol which inhibited subsequent lipid peroxidation by acting as a chain breaking antioxidant.

Hydroperoxide-dependent folic acid degradation by cytochrome c.

Folic acid is degraded by cytochrome c in the presence of hydrogen peroxide/tert-butyl hydroperoxide at the C9-N10 bond. The degradation is increased with increasing temperature. When guanidine HCl or benzoate are included in the reaction medium, the amount of folic acid degradation is enhanced. Catalase, formate, and thiourea inhibited hydrogen peroxide-dependent folic acid degradation only, and not tert-butyl hydroperoxide dependent degradation. Cyanide and azide markedly inhibited both the hydroperoxide-dependent degradations. Superoxide dismutase, EDTA, ethanol, mannitol, and dimethyl sulfoxide did not inhibit the degradation. The mechanism of cytochrome c-catalyzed folic acid degradation is discussed.

Studies on hydroperoxide-dependent folic acid degradation by hemin.

Hemin (ferric protoporphyrin IX chloride) in the presence of hydrogen peroxide or tert-butyl hydroperoxide was found to cleave folic acid at the C9-N10 bond. The ferrous form of hemin was not involved in hydroperoxide-dependent folic acid degradation, as indicated by the lack of inhibition by carbon monoxide. Molecular oxygen was not required for the degradation. GSH-Mn(II) or NAD(P)H in the presence of molecular oxygen did not support hemin-mediated folic acid degradation. The degradation increased as the temperature was elevated from 10 to 70 degrees C. Ascorbic acid and azide were potent inhibitors. Superoxide dismutase and hydroxyl radical quenchers, such as ethanol, mannitol, benzoate, and dimethyl sulfoxide did not inhibit the reaction. Catalase inhibited hydrogen peroxide-supported degradation but not the tert-butyl hydroperoxide-dependent one. Thiol compounds, such as thioglycolic acid, thiourea, glutathione, cysteine, and 2-mercaptoethanol, inhibited the hydrogen peroxide-dependent degradation but supported the tert-butyl hydroperoxide-mediated one. N5-formyl tetrahydrofolic acid, but not N10-formyl folic acid, was degraded by hemin in the presence of H2O2 or TBHP. The data obtained are suggestive of a mechanism similar to N-demethylation reactions catalyzed by cytochrome P-450 and some peroxidases.

Half-life and metabolism of 3H-folic acid in oral contraceptive treated rats.

Half-life and tissue distribution of injected tritium labelled folate was examined in oral contraceptive (OCA) treated and control rats to elucidate the mechanism by which OCA treated rats show increased urinary excretion as well as tissue levels of folate. Urinary excretion of folate within the first 12 hours of injection was higher but subsequent excretion lower in OCA treated rats. Faecal excretion of folate was also lower in treated rats. Thus, the half-life of the rapid turnover labile pool of folate appears to be reduced, whereas that of the slow turnover stable pool is raised by OCA treatment. Concentration of labelled folate was higher in the liver and kidneys of treated rats but it was not affected in tissues such as intestine, bone and brain. The concentration of soluble folate binders in the kidney was raised by OCA treatment and the activity of dihydrofolate reductase in the liver (a folate binder) also tended to be higher though the difference was not significant. The observation suggest that OCA may alter folate turnover by changing the concentration of folate binders in the tissues.

Effect of oral contraceptives on folate economy--a study in female rats.

Daily administration of one tenth of a tablet of Ovulen-50 (ethinodiol diacetate, 1 mg, ethinylestradiol, 50 microgram) to adult female rats resulted in an increase in the liver and kidney folate levels, increased urinary excretion of folate and a fall in serum folate while red blood cell folate levels remained unaffected. The tissue folate levels did not indicate adverse effect of OCA on folate economy of the body.