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At least 1,315 records · Page 73Linked to original sources
PubMed1975

Assay of galactose-1-phosphate uridyl transferase in cultured amniotic cells for prenatal diagnosis of galactosaemia.

We report studies designed to establish optimal conditions for the assay of amniotic cell galactose 1-phosphate uridyl transferase (Gal-PUT) for early prenatal diagnosis of galactosaemia. Methods based on linkage of the reaction to cause of non-specific reactions occurring even in the absence of Gal-1-P. In the final method, sonicates of confluent cultures are incubated with (14-C) Gal-1-P is degraded by treatment with alkaline phosphatase. Gal-PUT specific activities of both control and galactosaemic amniotic cells are higher in non-confluent that confluent cultures.

Amniocentesis↗
PubMed1976

Galactose-1-phosphate uridyltransferase in cultured cells.

A method to measure the enzyme galactose-1-phosphate uridyltransferase in cultured cells is described. The optimun pH was 8.7 and no enzyme activity was found without preincubation with dithiothreitol. The KM values for Gal-1-P and UDPG for the wild type enzyme were found to be 0.2 mM and 0.08 mM, respectively. Values for the Duarte variant were found to be identical. No significant change in enzyme activity with time after subculture was found in either cultured skin fibroblasts or cultured amniotic fluid cells. Different transferase genotypes were clearly distinguished and reference range established. Transferase levels found in normal cultured amniotic fluid cells were the same as in normal cultured skin fibroblasts. The results of a prenatal diagnosis was obtained within 3 weeks of amniocentesis.

Amniocentesis↗
PubMed1977

Evaluation of metabolic pathway activity in cultured skin fibroblasts and blood leukocytes.

Screening for a variety of blocked catabolic mutants can be achieved in cultured skin fibroblasts and in peripheral blood leukocytes with a simple radioisotope method that is reliable and convenient. The method measures the incorporation into trichloroacetic acid (TCA) insoluble macromolecules of a 14C-labelled intermediate in the pathway under question; incorporation of a 3H-labelled metabolite, not in the same pathway, is used as an internal control of metabolism in the cell population. The 14C/3H incorporation ratio is decreased in blocked pathways; use of the ratio method eliminates the delay required by radioautography (used in earlier adaptations of this method), the problems involved in CO2 collection, and the need to standardize cultures for cell number. This method has been used to identify cells with biochemical lesions in the oxidation of propionate, galactose, hypoxanthine and pyruvate; it has allowed us to identify a new variant of methylmalonicaciduria; we believe it can be extended to include other metabolites and pathways.

Acidosis↗
PubMed1978

An improved quantitative assay of galactose-1-phosphate uridyltransferase activity in erythrocytes based on the determination of glucose 1-phosphate generation.

An improved quantitative method for measuring galactose-1-phosphate uridyltransferase (EC 2.7.7.12, Gal-PUT) activity in erythrocytes was developed based on the detection of glucose 1-phosphate generated under the catalytic influence of the enzyme. This is achieved by incubating the enzyme with galactose 1-phosphate and uridyldiphosphoglucose during 15 min, followed by deproteinisation. The glucose 1-phosphate generated is quantitated subsequently by measuring NADPH formation from added NADP+ in a second incubation step with added phosphoglucomutase (EC 2.7.5.1) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49). Gal-PUT activity is calculated from the increment in absorption at 340 nm. Because it is technically a simple assay that is sensitive, specific and not affected by UDPgalactose-4-epimerase (EC 5.1.3.2) activity in the erythrocytal lysates it is suggested to be the method of choice for measuring Gal-PUT activity. Activities in erythrocytes of controls varied from 264 to 556 U/kg hemoglobin; in obligate heterozygotes from 53 to 190 U/kg Hb and in homozygous deficient patients less than 5 U/kg Hb was measured at 37 degrees C.

Erythrocytes↗
PubMed1982

A new microfluorometric method for the measurement of galactose-1-phosphate in erythrocytes.

A new and sensitive assay for measuring galactose-1-phosphate in erythrocytes is described. Galactose-1-phosphate is determined by mixing an aliquot of deproteinized hemolysate with a reagent containing uridine diphosphoglucose, NADP+, hexose-1-phosphate uridylyltransferase, phosphoglucomutase, glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase and measuring the NADPH formed fluorometrically. Under the conditions of this assay 2 mol of NADPH are formed per mol of galactose-1-phosphate. The assay is linear from 0 to 1160 micrograms of galactose-1-phosphate per gram of hemoglobin. Recovery of galactose-1-phosphate added to four hemolysates averaged 99%. Galactose-1-phosphate concentrations were measured in erythrocytes from five heterozygous subjects not under dietary control and seven transferase-deficient galactosemic individuals who were receiving galactose restricted diets. In all samples from the heterozygous individuals, the galactose-1-phosphate concentrations were normal. Of the samples from galactosemic subjects, two showed extreme elevations of galactose-1-phosphate, four showed moderate elevations, and one was normal. Galactose-1-phosphate levels are used to monitor the degree of dietary control in the transferase-deficient galactosemic individual.

Adolescent↗
PubMed1993

The investigation of UDPGlucose and UDPGalactose concentration in red blood cells of patients with classical galactosaemia.

UDPGlucose (UDPGlc) and UDPGalactose (UDPGal) are nucleotide sugars formed via the galactose metabolic pathway and are essential cofactors for the incorporation of galactose and glucose into complex glycoproteins and glycolipids. It has been proposed that in classical galactosaemia, where the enzyme galactose-1-phosphate uridyl transferase is deficient, the reaction product UDPGal is reduced leading to the long-term complications associated with the disease. We have measured the concentration of UDPGal and UDPGlc in red blood cells by high performance liquid chromatography (HPLC) in 16 children and 15 adult galactosaemics and compared the results with 30 and 27 control children and adults, respectively. The results indicate that UDPGal levels were found to be significantly reduced in galactosaemic patients and UDPGlc/UDPGal ratios significantly increased.

Adolescent↗
PubMed1993

Contraction and relaxation of aortas from galactosaemic rats and the effects of aldose reductase inhibition.

Rats were fed for 10 days with a 40% galactose diet, in order to chronically stimulate the polyol pathway. Thoracic aorta contraction and relaxation were studied. Compared to controls, galactosaemia did not influence contractions to phenylephrine or serotonin. Acetylcholine produced concentration-dependent relaxation of aortic rectangles precontracted with phenylephrine; galactosaemia caused a 25% deficit in maximum relaxation to acetylcholine (P < 0.01) and a 168% increase in EC50. There was a similar 25% reduction in relaxation to 3 microM calcium ionophore A23187 (P < 0.05). By contrast, there were no significant differences in endothelium-independent relaxation to nitroglycerine or cromakalim. The abnormalities in endothelium-dependent relaxation were completely prevented by treating galactosaemic rats with the aldose reductase inhibitor, ponalrestat. Thus, the data demonstrate that elevated polyol pathway activity contributes to reduced endothelium production, release or the action of nitric oxide in experimental galactosaemia, and suggest that this mechanism could also contribute to the vascular defects seen in diabetes mellitus.

Aldehyde Reductase↗
PubMed1989

Prevention or moderation of some ultrastructural changes in the RPE and retina of galactosemic rats by aldose reductase inhibition.

Rats were maintained on a 50% galactose diet with and without the aldose reductase inhibitor, Sorbinil, or on a normal diet of rat chow, and the retinas and retinal pigment epithelium were examined ultrastructurally at time points ranging from 4 weeks to 20 months. Several ultrastructural changes were observed in the retinal pigment epithelium and retinal capillaries of galactosemic rats that were not seen in rats on a normal diet. Only some of these changes are similar to those that have been previously noted in diabetic (glucosemic) rats. As in diabetics, galactosemic rats demonstrated thickening of the basal laminae of the retinal pigment epithelium and retinal capillaries. They also manifested vacuolization and degenerative foci in the retinal pigment epithelium that were similar, but not identical, to changes seen in diabetic rats. Each of these changes was significantly inhibited by Sorbinil. Unlike diabetics, galactosemic rats did not have dilated basal infoldings, but did have outer retinal folds and a significant increase in large-lipofuscin-like aggregates in the retinal pigment epithelium, both of which were partly prevented by Sorbinil. These data suggest that multiple mechanisms may be involved in retinal and retinal pigment epithelium changes in diabetic and galactosemic rats, and that enhanced polyol metabolism is likely to be involved in some of the changes in both.

Aldehyde Reductase↗
PubMed1990

Metabolic studies of galactosemic cataract.

Experimental diabetic and galactosemic animal models are widely used to study diabetes-induced complications. Galactose feeding can rapidly produce cataract, retinopathy and nephropathy; it is therefore favored over the diabetic model. Although the common feature for both models is the activation of aldose reductase, there are substantial differences between the two--not only does the rate of cataract progression differ but the metabolic patterns are far more complex than for polyol production alone. We here present the result of a comparison between diabetic and galactosemic lenses and show the differences in phosphorus and aldose metabolism, cell integrity and osmotic environment.

Animals↗
PubMed1990

Effects of long-term diabetes and galactosaemia upon lens and retinal mRNA levels in the rat.

The levels of mRNAs encoding the alpha 1 chain of collagen IV and the B1 chain of laminin were assayed in the lenses and retinas of long-term (28-week) diabetic and galactosaemic rats in order to gain some insight into the effects on basement membrane (BM) synthesis in these tissues. mRNAs coding for beta-actin, glucose transporter protein and the alpha 2 catalytic subunit of Na+,K(+)-ATPase were also assayed to determine whether any effects on BM-coding mRNA levels were specific. Long-term diabetes had no significant effect on the levels of alpha 1 (IV) collagen mRNA but caused a significant reduction in the laminin B1 message in the lens. In the same samples, the level of the glucose transporter protein mRNA was found to be elevated significantly in the diabetic tissue, whereas the mRNAsen coding beta-actin and alpha 2 Na+,K(+)-ATPase were unaffected in comparison with age-matched controls. Long-term galactosaemia resulted in significant increases in the levels of all mRNAs assayed when expressed per micrograms total RNA used for each analysis. However, this effect appeared to be due to a specific loss of ribosomal RNA from these severely cataractous lenses. When related to the beta-actin mRNA internal control, the levels of mRNA in the galactosaemic lenses were very similar to that found in the diabetics. Laminin B1 mRNA levels were decreased significantly.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1990

Contradictory effects of uncomplicated versus complicated abdominal surgery on the hepatic capacity for urea synthesis in rats.

Female Wistar rats weighing 217 g were subjected to two types of surgical stress: uncomplicated (hysterectomy) and complicated (spleen and uterus ligated, crushed, and left in situ). Liver function as assessed by amino-N conversion was measured as the capacity for urea-N synthesis preoperatively (control animals) and on Days 1, 3, and 6 postoperatively. Uncomplicated surgery transiently increased the capacity for urea-N synthesis by 30% the first postoperative day (P less than 0.001). Complicated surgery decreased the capacity for urea-N synthesis to 55% throughout the investigation period (P less than 0.001). This was not due to a general change in liver mass since galactose elimination capacity remained constant. The increase in the capacity for urea-N synthesis after uncomplicated surgery is probably due to glucagon since plasma glucagon increased whereas plasma insulin and blood glucose remained unchanged after amino acid loading. The persistent decrease in the capacity for urea-N synthesis in complicated surgery is not due to changes in these regulators: glucagon increased, insulin decreased, and the rats were hypoglycemic. All changes are expected to increase the capacity for urea-N synthesis. The mechanism for the emergence of these two distinct metabolic patterns is not known. The phenomenon is probably important for interpretation of metabolic data on clinical stress.

Amino Acids↗