DNA linkage based diagnosis of Wilson disease in asymptomatic siblings.

BACKGROUND & OBJECTIVES: Wilson disease (WD) is an autosomal recessive disorder caused by defects in ATP7B gene located in chromosome 13q14, and manifested as hepatolenticular degeneration as a result of accumulation of copper. No information on the mutation in the ATP7B gene and haplotypes using linked markers is available for WD patients in India. Hence, the present study was undertaken to identify, by a PCR-based molecular diagnostic test, presymptomatic siblings of WD affected individuals in families with multiple offspring. METHODS: Genomic DNA was prepared from the peripheral blood of the patients, siblings and his/her first degree relatives. The repeat-markers flanking WD locus were amplified by PCR using fluorescent labeled primers. Amplified DNA fragments were analyzed by polyacrylamide gel electrophoresis in ABI 377 DNA sequencing system. Genotypes of the samples were determined using Genescan software. Haplotypes were determined based on segregation of the alleles in the families under study. RESULTS: Among 15 WD affected families with multiple children, 4 cases were identified where younger siblings shared same genotype as the patient at all three markers analyzed. Further, eight different haplotypes were detected in the four patients. INTERPRETATION & CONCLUSION: The siblings of the WD patients carrying the same genotype at the markers linked to WD locus were presymptomatically diagnosed individuals. Presence of eight different haplotypes in the four patients suggested mutational heterogeneity at the WD locus. The test helps clinicians for therapeutic intervention in suspect WD cases by copper chelating agents prior to manifestation of overt clinical symptoms.

Ontogenic profile of some antioxidants and lipid peroxidation in human placental and fetal tissues.

Reactive oxygen species (ROS) pose a serious threat to maternal and fetal health during pregnancy. However, there is little information on the oxidative damage caused by ROS and its protection during prenatal life. The present study highlights the status of various antioxidants in human placental and fetal tissues at different phases of gestation. The activity profile of scavenging enzymes, superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase as well as the concentrations of non-enzymatic antioxidants, ascorbic acid, alpha-tocopherol, bilirubin and glutathione have been determined in human placental whole homogenate, placental brush border membrane and fetal liver over gestational periods ranging from 6 weeks of pregnancy till birth. The ontogenic profile of lipid peroxidation, a marker of oxidative damage has also been investigated in the feto-placental system. Catalase, superoxide dismutase and glutathione reductase activities increased significantly, but glutathione peroxidase activity remained almost the same throughout development. Except alpha-tocopherol and bilirubin, the concentrations of other non-enzymic scavengers followed a significant increasing trend with advancement of pregnancy. Results indicate that there is gradual suppression of lipoperoxide formation with the progress of gestation to protect the fetus against oxygen toxicity.

Antioxidant status and lipid peroxidation in human feto-placental unit.

The human feto-placental unit being in the environment of the materno-fetal circulation is very much susceptible to the danger of reactive oxygen species-induced oxidative damage. The present study reports the status of various antioxidants in the feto-placental system at birth. The activities of superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase as well as concentrations of non-enzymic antioxidants viz, ascorbic acid, alpha-tocopherol, bilirubin and glutathione have been determined in term placental brush border membrane (BBM) and umbilical cord (UC) blood. The extent of lipid peroxidation in developing and term BBM and UC blood has been studied by estimation of malondialdehyde and conjugated dienes, both being the end products of lipid peroxidation. The results indicate the presence of a protective mechanism against oxygen toxicity in the feto-placental system at the time of parturition.

Modulation of bilayer fluidity by lipid peroxidation of human placental syncytiotrophoblast membranes during embryogenesis.

Placental syncytiotrophoblast while regulating the passage of nutrients from maternal blood to the fetal circulation exposes itself to the risk of oxidative attack by the oxygen free radicals. Extent of lipid peroxidation (LPO) investigated in placental brush border membrane (BBM) and basal membrane (BM) revealed a decreasing trend with gestational progress. Steady-state fluorescence anisotropy measurement of 1,6-diphenyl-1,3,5-hexatriene (DPH)-labeled malondialdehyde treated placental membrane vesicles suggested modulation of BBM and BM fluidity by lipid peroxidation in all gestational ages. alpha-tocopherol content in both the placental membranes which increased as gestation progressed has been proposed to play a significant role in decreasing LPO of placental membranes during intrauterine development.

Changes in lipid composition and fluidity of human placental basal membrane and modulation of bilayer protein functions with progress of gestation.

Human placental syncytiotrophoblast basal membrane plays an important role in transfer of nutrients from the mother to the growing fetus all throughout gestation. The membrane lipid composition together with the bilayer fluidity is found to be the major index in modulation of these transport processes. In the present study, the effects of changing lipid composition on the placental basal membrane fluidity and the modulating influence of the latter on membrane enzyme and transport functions with progress of gestation,were investigated. Steady-state fluorescence analysis using 1,6-diphenyl- 1,3,5 hexatriene as the probe, indicated a decrease in fluorescence anisotropy of both labeled native membrane vesicles and liposomes prepared from lipids extracted from the basal membrane vesicles, signifying increased bilayer fluidity with progress of gestation. This in turn, was successfully correlated to the lowering of cholesterol content and enhanced phospholipid concentration with a steady decrease in cholesterol/phospholipid ratio during placental development. Enhanced Na+-K+-ATPase activity and steady-state glucose uptake across basal membrane with gestational progress suggested modulation of membrane protein functions by the fluidity, which was further corroborated by the increased bilayer fluidity and enzyme activity in benzyl alcohol treated basal membrane in each gestational age group.

Role of fatty acid binding protein in the modulation of inhibitory effect of fatty acids on fatty acid synthase and ATP-citrate lyase in developing human brain.

Inhibition of the activities of fatty acid synthase and ATP-citrate lyase (ATP-CL) by fatty acids and their CoA esters has been studied. Purified fatty acid binding protein from human fetal brain reverses this inhibition. This protein also activates the enzyme when added alone. ATP-citrate lyase and fatty acid synthase activity gradually increased with the advancement of gestation showing a relationship between high demand of fatty acid synthesis in developing brain and supply of its precursors.

Increase in fluidity of human placental syncytiotrophoblastic brush-border membrane with advancement of gestational age: a fluorescence polarization study.

The syncytiotrophoblastic brush-border membrane (BBM) of human placenta plays a vital role in the exchange of metabolites between mother and developing fetus. The fluidity of this membrane renders its selective permeable character. To meet the changing needs of the growing fetus with the progress of gestational age changes in the composition and properties of BBM occur. In the present study, decrease in cholesterol/phospholipid ratio of BBM indicated an increase in fluidity with advancement of gestational age. Measurement of the steady state fluorescence anisotropy of labeled liposomes prepared from lipid extract of BBM as well as that of labeled BBM vesicles prepared from native BBM shows decrease in restriction of the bound dye molecule with increased gestational age. Decrease in transition temperature of BBM and enhanced glucose transport through it have been observed with advancement of pregnancy.

Inhibition of two HMP shunt pathway enzymes by fatty acids and their CoA esters in developing human brain: role of fatty acid binding protein.

Inhibitory effects of fatty acids and their CoA esters on glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities of human fetal brain cytosol have been studied. Purified human fetal brain fatty acid binding protein reverses the inhibitory effects of palmitoyl-CoA and oleic acid on glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities in human fetal brain cytosol. This protein, when added alone, activates the enzymes. Levels of fatty acid binding proteins as well as the activities of these two HMP shunt pathway enzymes, which provide cofactors like NADPH for reductive biosynthesis, increase with gestation. These results indicate that a relationship exists between the high demand for fatty acids and synthesis of cofactors for lipid biosynthesis in developing brain.

Effect of fatty acid binding proteins on developing human placental malate dehydrogenase activity.

Role of fatty acid binding proteins (FABPs) in modulating inhibition of human placental malate dehydrogenase by palmitoyl-CoA and oleate has been studied. Activity of human placental cytosolic malate dehydrogenase is detected throughout the gestation, showing a peak at midgestation (20-25 weeks). Inhibition (50%) of the enzyme activity is obtained by 20 microM palmitoyl-CoA or 35 microM oleate. FABPs enhance the activity of malate dehydrogenase in absence of palmitoyl-CoA or oleate and also protect against palmitoyl-CoA or oleate inhibition. Such a modulatory effect of FABP may be due to the binding of long chain fatty acyl-CoA or fatty acid rather than a direct effect of FABPs on the enzyme.

Enzymatic tumour markers in ovarian cancer: a multiparametric study.

The serum concentration of some enzymes, namely placental alkaline phosphatase, lactate dehydrogenase, 5' nucleotidase and amylase, was determined by commercially available kit in 50 ovarian cancer patients and 31 patients with benign gynaecological disease before initiation of any treatment. The values were compared with those of 30 healthy women. Multivariate analysis showed a statistically significant difference between healthy women and ovarian cancer patients. These results indicate that by using discriminating function of the above four enzyme variables it is possible to screen ovarian cancer in outpatient obstetric and gynaecological clinics (sensitivity 96%, specificity 83.3%, relative risk 11.7). Hence this system can serve as a suitable marker for ovarian cancer.

Characterization and binding properties of human fetal lung fatty acid-binding proteins.

When delipidated Mr > 10,000 cut-off human fetal lung cytosol was separated on gel filtration and ion-exchange chromatography on Auto-FPLC system, two fatty acid-binding proteins (FABPs) of pI 6.9 and pI 5.4 were purified to homogeneity. On Western blotting analysis with the anti-human fetal lung pI 6.9 FABP, these two proteins showed immunochemical cross reactivity with each other and with purified hepatic FABPs but not with cardiac or gut FABP. These two FABPs have identical molecular mass of 15.2 kDa, which is slightly higher than that of the hepatic proteins (14.2 kDa). Carbohydrate covalently linked to FABPs, that may substantially add to the molecular mass, was not detected in the purified protein preparations. Amino acid analysis revealed that both the proteins have same amino acid composition each containing one Trp residue that is lacking in hepatic FABP. Different isoforms of lung FABP exhibited different binding ability for their natural ligands. These proteins bind palmitoyl CoA with higher affinity than oleic acid. pI 6.9 FABP can more rapidly and efficiently transfer fatty acid than can pI 5.4 FABP from unilammelar liposomes. Thus these FABPs may play a critical role in fatty acid transport during human fetal lung development.

Characterization of cardiac fatty-acid-binding protein from human placenta. Comparison with placenta hepatic types.

When a 105,000 x g supernatant of human placenta was incubated with [1-14C]oleate and subjected to Sephadex G-75 gel filtration and HPLC, two fatty-acid-binding protein (FABP) peaks were obtained. One of these, when further purified by carboxymethyl-cellulose, gave one 15.3-kDa FABP with pI5.3. The other, when chromatographed on DEAE cellulose, separated into two 14.2-kDa FABP with pI6.9 and 5.4. Purity of the proteins was checked by SDS/PAGE. Molecular mass, pI, immunochemical properties and amino acid compositions all indicated that 15.3-kDa FABP was of the cardiac type, whereas both 14.2-kDa FABP were of the hepatic type. Cardiac FABP did not cross-react with hepatic proteins. When tested for the acceptor/donor properties of these FABP, hepatic types were found to be better candidates than cardiac in uptaking fatty acids from liposomes. Cardiac FABP, on the other hand, acted in a more efficient way as a donor, indicating a distinct role of these proteins in human placenta, which furnishes a multiorgan system for the developing fetus.

Role of rusticyanin in the electron transport process in Thiobacillus ferrooxidans.

Effect of diethyl dithiocarbamate (DEDC), an antimicrobial agent, on growth of Thiobacillus ferrooxidans, possibly by inhibiting rusticyanin present in the periplasmic space of the microorganism, has been studied to gain more insight into the electron transport chain in the bioleaching process. DEDC is found to form a stable complex with rusticyanin in solution and also in polyacrylamide gel. The spectrum of the complex is identical to that of Cu-DEDC complex, suggesting binding of DEDC with copper moiety of rusticyanin and resulting in inhibition of growth. In vitro reduction of purified rusticyanin by Fe(II) in absence of acid-stable cytochrome c is very slow, indicating the importance of cytochrome c in electron transport. Thus, in the iron oxidation process, acid-stable cytochrome c is the primary acceptor of electron, transferring the electron to rusticyanin at pH 2.0, which, in turn, affects electron transfer to iron-cytochrome c reductase around pH 5.5.

Relationship between fatty acid synthesis, transport and total lipid content during human fetal lung development.

Levels of fatty acid binding proteins (FABPs), lipids as well as activities of fatty acid synthesizing enzymes such as fatty acid synthase and ATP-citrate lyase increase with gestation showing maximum at term in human fetal lung. However, the activity of ATP-citrate lyase showed the same trend up to 30 weeks of gestation before declining slightly at term. These results indicate the importance of supply and/or synthesis of fatty acids when lung surfactant synthesis begins; thereby showing a correlation between the FABPs, lipid pattern and the activities of fatty acid synthesizing enzymes during prenatal lung development.

Biochemical studies following ovulation induction with clomiphene citrate and human menopausal gonadotropin in infertile Indian women.

Biochemical studies were done in 65 anovulatory women before and following induction of ovulation with ethinyl estradiol/clomiphene citrate/human chorionic gonadotropin and also clomiphene citrate/human menopausal gonadotropin/human chorionic gonadotropin to determine the metabolic adverse effects, if any, of such therapy. Significant increases occurred in lipids and lipoprotein levels, while minor changes were seen in liver function enzymes and fasting blood sugar. Sialic acid decreased significantly after therapy in both the regimens, and both induction therapies seem to be free of adverse metabolic effects of hormones. On comparison with normal ovulatory women, this study suggests that some of the biochemical parameters can serve as reliable indicators of ensuing ovulation.

Purification and characterization of fatty acid-binding proteins from human fetal lung.

Fatty acid-binding protein (FABP) was isolated, purified, and characterized from developing human fetal lung cytosol by gel filtration and ion-exchange chromatography. FABP exists in three immunochemically identical forms, DE-I, DE-II, and DE-III, having Mr 15,200 +/- 200 each and isoelectric pH 7.8, 6.9, and 5.4, respectively. DE-I is almost lipid-free, DE-II binds mainly long-chain unsaturated fatty acids, and DE-III is an arachidonic acid carrier. One mole of DE-II and DE-III each binds 1 mol of fatty acids noncovalently. Concentrations of all these FABPs increase gradually from early gestation to term. Defatted lung FABP reverses the inhibitory effect of palmitoyl coenzyme A (CoA) (PAL-CoA) on lung glucose-6-phosphate dehydrogenase (G6PD), a key enzyme of the hexose monophosphate (HMP) shunt pathway. This protein when added alone activates the enzyme, suggesting that the original submaximal activity is probably due to the presence of endogenous long-chain fatty acyl CoA esters in the cytosols. As FABP is present in relatively high concentration in most mammalian cells, the potent inhibitory effects of long-chain acyl CoA esters on the HMP shunt pathway in vitro are not seen in intact cells.

Human fetal liver fatty acid binding proteins. Role on glucose-6-phosphate dehydrogenase activity.

Fatty acid binding proteins (FABPs) may play an important role in the transport and metabolism of fatty acids during human embryogenesis. Three fractions of FABP, namely, DE-I, DE-II and DE-III, having Mr 14,200 Da each and pI values 7.8, 6.9 and 5.4, respectively, have been detected in human fetal liver. These proteins were purified by heat and butanol precipitation of fetal liver supernatant as well as by gel filtration and ion-exchange chromatography. Fetal liver FABPs are immunochemically identical to each other. Concentrations of DE-I, DE-II and DE-III increase gradually from early gestation to term. DE-I is almost lipid-free, DE-II binds long-chain fatty acids nonspecifically and DE-III transports mainly arachidonic acid. DE-II and DE-III protect glucose-6-phosphate dehydrogenase, which furnishes NADPH for fatty acid synthesis, from the feed-back inhibition exerted by added palmitoyl-CoA and oleate. In the absence of exogenous inhibitors, this enzyme is stimulated by FABPs. DE-I has no effect on such inhibition. Thus, FABPs play a regulatory role in critical aspects of cellular physiology during human embryogenesis.