Characterization of F8 defects in haemophilia A in Pakistan: investigation of correlation between mutation type and the in vitro thrombin generation assay.

Hereditary haemophilia A is an X-linked bleeding disorder caused by mutations in the coagulation factor VIII gene (FVIII abbreviates protein, gene symbol F8). The mutation spectrum has been reported in various populations but not in Pakistan. The aims of this study were to (i) characterize F8 mutations in a large haemophilia A cohort from Pakistan and to (ii) investigate whether in vitro thrombin generation (TG) differs according to mutation type (null compared with missense) in severe haemophilia A. One hundred individuals diagnosed with haemophilia A and 100 healthy controls were recruited in Pakistan. Phenotypic measurements were re-evaluated in Cardiff; the essential regions of F8 were screened for the causative defect. A diagnosis of haemophilia A was confirmed for 92 individuals, 7 were found to have haemophilia B and 1 did not have haemophilia. The F8 defects were characterized for 80 of the 92 haemophilia A individuals and comprised point mutations, inversions (intron 22 and intron 1) and frameshifts. Point mutations (41%) were the most frequent, followed by the intron 22 inversion (20%). Thirty novel variants were identified. Comparison of in vitro TG parameters [velocity index (VI) and peak] was made between severe individuals who had a null mutation (no FVIII) and those with a missense change (dysfunctional FVIII), no significant difference was observed. The spectrum of F8 defects in Pakistan is heterogenous; VI and peak in severe haemophilia A are not influenced by whether the underlying mutation gives rise to dysfunctional FVIII or no coagulation factor at all.

Joint health scores in a haemophilia A cohort from Pakistan with minimal or no access to factor VIII concentrate: correlation with thrombin generation and underlying mutation.

Haemophilia A is associated with recurrent joint bleeding which leads to synovitis and debilitating arthropathy. Coagulation factor VIII level is an important determinant of bleed number and development of arthropathy . The aim of this study was to compare the haemophilia joint health score (HJHS) and Gilbert score with severity, age, thrombin generation (TG) and underlying mutation in a haemophilia A cohort which had minimal access to haemostatic replacement therapy. Ninety-two haemophilia A individuals were recruited from Pakistan. Age, age at first bleed, target joints, haemophilic arthropathy joints, HJHS and Gilbert score were recorded. A strong correlation was found between HJHS and Gilbert score (r = 0.98), both were significantly higher in severe (n = 59) compared with non-severe (n = 29) individuals before the age of 12 years (P ≤ 0.01) but not thereafter. When individuals were divided according to developmental age (<12 years, 12-16 years and >16 years), both HJHS and Gilbert score were significantly lower in the youngest group (P ≤ 0.001), there was no difference between 12-16 years and >16 years. In severe individuals there was no correlation between in vitro TG and joint score, whereas in non-severe individuals there was a weak negative correlation. In the severe group, no significant difference was observed for either joint score according to the underlying mutation type (inversion, missense, nonsense, frameshift). In this cohort of haemophilia A individuals with minimal access to haemostatic treatment, haemophilic arthropathy correlated with severity and age; among severe individuals, joint health scores did not relate to either the underlying mutation or in vitro TG.

Bees wax polyphenols as suppressor of CC1--induced oxidative stress in rats.

Bee's wax produced by honeybees is rich in polyphenols. As the polyphenols are thought to protect cell constituents against oxidative damage through scavenging of free radicals, the present work was undertaken to evaluate the effects of polyphenols extracted from bees wax on the oxidative stress induced by carbon tetrachloride (CCl4) in rats. The polyphenols extracted by 80% methanol from bee wax (PBW) were fed to Wistar rats at 100 mg/kg body weight and 200 mg/kg body weight for 14 days in order to study its antioxidative and antihepatotoxic effects against CCl4 (1.5 ml/kg body weight)-induced stress. On 15th day all the rats were sacrificed, blood was collected for serum and organs/tissues were excised for biochemical analysis. The results showed a significant decrease in hepatic antioxidant enzyme activities viz. catalase, glucose-6-phosphate dehydrogenase (G-6-PDH), glutathione peroxidase (GSH-Px), glutathione reductase, superoxide dismutase (SOD) and a significant increase in glutathione S-transferase (GST) and gamma-glutamyl transpeptidase (GGT) by CCl4, probably due to the peroxidative effects. The prophylactic use of PBW at 200 mg/kg level resulted in a significant increase in CCl4-induced reduction in catalase, G-6-PDH, GSSGR and SOD. The hepatic levels of lipid peroxides viz. malondialdehyde, conjugated dienes and lipid hydroperoxides, enhanced by the administration of CCl4 were brought down by the ingestion of PBW at a level of 200 mg/kg. The hepatotoxicity caused by the administration of CCl4 was reduced significantly. Hence, it is concluded that the polyphenols from bees wax exhibit hepatoprotective and antioxidative properties in

Effects of photodynamic treatment on biological antioxidant systems in rats.

Effects of photodynamic treatments on inherent antioxidant metabolites and cellular defence enzymes have been investigated in rats. Wistar rats were grouped into untreated controls, light controls, hematoporphyrin derivative (Hpd) (treated with 5 and 10 mg Hpd/kg body weight and kept in dark) and sets treated with both Hpd and red light (dose 172 and 344 j/m2 ). After 2, 24, 48 and 72 hr of Hpd injection the rats sacrificed, livers quickly excised to analyze Hpd uptake, activities of enzymes like catalase, GSH-Px and antioxidants like GSH, vitamin A, vitamin E and vitamin C. The results showed that the loss of Hpd from liver as a function of post- injection time was non- linear. An increased generation of lipid radicals was observed in the groups treated with 5 mg Hpd and higher dose of light and in groups treated with 10 mg Hpd at both the doses of light. Combination of light and Hpd reduced hepatic GSH content with a concomitant reduction in GSH-Px. At higher doses of Hpd and light, there was a significant reduction in hepatic vitamin A levels. Combination of Hpd and light in all doses reduced vitamin E content in liver. The decreased biological antioxidant contents and GSH-Px may be attributed to their utilization for the scavenging of free radicals generated by Hpd and light in tissues. However, no change in catalase activity and vitamin C content in liver was noted in experimental rats. The results suggest that exposure to higher doses of Hpd with light alters oxidant stress system and TBARS content in rat.

Anticarcinogenic effects of curry leaves in dimethylhydrazine-treated rats.

Curry leaves are one of the spices used in Indian dishes for aroma and preservation. There are no reports on the antioxidant properties of curry leaves. In this study, the antioxidant potential of curry leaves in rats treated with a known chemical carcinogen, dimethylhydrazine hydrochloride (DMH) was investigated. Food intake was reduced in the rats fed curry leaf-supplemented diet but the body and the organ weights were not affected. Vitamin A content in the liver was significantly increased whereas glutathione (GSH) content was not altered. A 50% reduction was seen in the micronuclei induced by DMH and a 30% reduction in the activity of gamma-glutamyl transpeptidase when the rats were fed a curry leaf-supplemented diet. These results indicate that curry leaves have high potential as reducer of the toxicity of DMH.

Dietary fiber content of commonly fresh and cooked vegetables consumed in India.

Legumes, leafy vegetables, roots and tubers, gourds and other vegetables were analyzed for total (TDF), soluble (SDF) and insoluble (IDF) dietary fiber contents, both before and after cooking either by a conventional open-pan method or by pressure cooker. Data revealed a significant increase in SDF fraction with a concomitant decrease in the IDF fraction upon cooking by both the methods employed. Although the decrease in IDF matched the increase in SDF values in some cases, it was found to be more in vegetables categorized as 'other'. The dietary fiber values have also been reported on a fresh weight basis which may serve as a guideline for calculating dietary intake of each component by the consumer.

Effects of feeding fresh garlic and garlic oil on detoxifying enzymes and micronuclei formation in rats treated with azoxymethane.

The effect of feeding a fresh garlic or garlic oil-supplemented diet was studied in rats for a period of 23 weeks with or without the treatment of a carcinogen azoxymethane (AOM), on the modulation of detoxification enzymes and micronuclei formation. The results showed that feeding fresh garlic or garlic oil-supplemented diets tended to reduce hepatic lipid peroxidation, though not to significant levels. Glutathione content was also not altered. The catalase activity in liver of rats fed a fresh garlic-supplemented diet was reduced compared to that of the control diet; however, the activity was not affected by AOM treatment. Ingestion of garlic caused a 40 percent increase in the hepatic glutathione peroxidase activity, whereas carcinogen treatment reduced it. The activity of hepatic glutathione-S-transferase was unaffected by the feeding regimen, while it was lowered in the garlic oil diet group treated with AOM. The gamma glutamyl transpeptidase activity was elevated more than sevenfold, in the kidney of rats treated with AOM, while it was reduced almost to half when the AOM-treated rats were fed fresh garlic or garlic oil. Micronuclei formation was increased fourfold, in rats exposed to AOM whereas the increase was reduced to half when AOM-injected groups had either fresh garlic or garlic oil in their diet. From these studies, it is concluded that long-term feeding of garlic, fresh or oil, reduced the toxic effect of AOM in rats.

Cellular accumulation and biological activity of hematoporphyrin derivative(L) in comparison with photofrin II.

Hematoporphyrin derivative, a drug used in the photodynamic therapy of solid tumours was synthesized in the laboratory and was called Hpd(L). Physico-chemical and biological properties of this drug have been compared with Photofrin II, the commercially available drug. Both Hpd(L) and Photofrin II possess similar properties qualitatively. Quantitatively, Hpd(L) was half as active as Photofrin II in its efficacy in causing photodynamic cytotoxicity or in the optical densities at the absorption peaks. These differences could be due to the differences in the compositions. Hpd(L) is a non-purified complex mixture of a number of porphyrin derivatives whereas Photofrin II is a relatively purer compound consisting of di- and tri-hematoporphyrins linked through ether or ester bonds. In vitro cellular uptake and retention of these drugs has been found to be a passive process not involving energy expenditure. pH and temperature of the incubation media have been found to profoundly influence these processes, while a complex relation seems to exist between physiological state of a cell and accumulation of these photosensitizers.

Effects of photofrin II and light on cellular adenine nucleotides and their modulation.

Effects of photofrin II (PII) and light on the intra cellular nucleotide levels have been investigated using BHK-21 cell line. Results indicate that lower concentrations of photofrin II in dark increases ATP levels in a non linear manner, however, there has been no change in energy charge and levels of other nucleotides. Photoirradiation of PII-treated cells leads to a significant reduction in ATP levels and energy charge along with an increase in ATP breakdown products like ADP and AMP. The phosphorylation potential [ATP]/[ADP][Pi] also reduces upon photoirradiation of PII treated cells. Incubation conditions like pH of the medium and temperature modulate the cellular responses to a great extent.

Effect of hematoporphyrin derivative on hematological parameters in rats.

Wistar rats were injected with hematoporphyrin derivative (Hpd) intraperitoneally and kept in the dark. Rats were sacrificed 2,24,48 and 72 h after injection. It was observed that Hpd in the dark did not affect the hemoglobin content and number of erythrocytes, while the leukocyte count was increased and blood pH decreased. Blood levels of glucose and lactate were increased significantly. Because the food intake was similar in all the groups, glycogenolysis was suspected to be the source of increased glucose levels in blood. However, a significant increase in the glycogen content of the livers of Hpd-treated rats was observed, which rules out glycogenolysis. Hyperglycemia may result due to a number of reasons such as stimulation of the central nervous pathways innervating the liver and adrenal medulla, excessive glucogenesis in liver from glycogen and noncarbohydrate sources, emotional stress, anesthesia and hormonal effects. The present study rules out hyperglycemia due to anesthesia and glucogenesis in the liver. Maintenance of blood glucose levels is a highly complex mechanism. Further investigations to understand these mechanisms are in progress.

Effects of hematoporphyrin derivative on the cellular energy metabolism in the absence and presence of light.

Effects of Photofrin II on energy metabolism and metabolic viability were studied in a mammalian transformed cell line (BHK-21) in dark and after photo-irradiation with visible light. Cells were allowed to accumulate Photofrin by incubating for 4 h in buffer containing Photofrin (5-60 micrograms/ml). The results show that Photofrin significantly affects the cellular energy metabolism even in the absence of light; activity of cytochrome c oxidase is decreased and glucose utilization and lactate production (glycolysis) are increased. Irradiation with light resulted in a significant decrease in the activity of cytochrome c oxidase, glycolysis, ATP content, energy charge, ratios of adenine nucleotides like ATP/ADP, ATP/AMP and cell viability (dye exclusion test). Presence of inhibitors of energy metabolism, potassium cyanide (respiration) and 2-deoxyglucose (glycolysis), further enhanced the cytotoxic effects induced by hematoporphyrin derivative and light.

Use-effectiveness of standard-dose and low-dose oral contraceptives in Dacca.

Five hundred women accepting oral contraceptives at an urban clinic were alternately prescribed standard-dose (50 microgram of estrogen) or low-dose (30 microgram of estrogen) combined pills. Life-table continuation rates at 12 months were approximately 40% for both groups, with no significant differences between or within the two groups after controlling for sociodemographic variables and nutritional status. Approximately three fifths of those who discontinued using both formulations cited medical reasons, especially dizziness. The low-dose oral contraceptive appeared to be as acceptable to these urban Bangladeshi women as the higher-dose pill.

PIP: Results of an urban comparative study of continuation rates and reasons for discontinuation of standard-dose and low-dose oral contraceptives are presented. 500 females were alternately given standard-dose (0.5 mg of DL-norgestrel with 50 ug of ethinyl estradiol) or low dose (0.15 mg of levonorgestrel with 30 ug of ethinyl estradiol) pills. Nutritional status was measured according to Huffman's method. 110 women were excluded from the analysis for various reasons, leaving 192 standard-dose and 198 low-dose acceptors. Cumulative life-table rates show a slightly higher, but statistically insignificant continuation rate for low-dose acceptors at 3, 6, 9 and 12 months (40% for both groups). When age, parity and nutritional status were controlled, no significant differences in continuation rates were observed either between or within the 2 groups. Medical reasons, especially dizziness, accounted for nearly 1/2 of standard-dose discontinuation, and for about 1/3 of low-dose discontinuation. Low-dose pills were just as acceptable as the standard dose in this urban setting.