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Stanton Segal - One of the best experts on this subject based on the ideXlab platform.
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The rate of de novo Galactose synthesis in patients with Galactose-1-phosphate uridyltransferase deficiency
Molecular Genetics and Metabolism, 2020Co-Authors: Gerard T. Berry, Cong Ning, Robert Reynolds, Claire Yager, Peter J. Moate, Raymond C. Boston, Stanton SegalAbstract:Abstract Using both a continuous infusion of isotopically labeled [1-13C]Galactose with a steady-state analysis and a single injection kinetic approach, we have calculated the apparent Galactose appearance rate (GAR) in patients with Galactose-1-phosphate uridyltransferase deficiency and control subjects. With the steady-state protocol, the GAR in 18 patients less than 18 years of age was 1.34 ± 0.53 mg/kg/h (mean ± SD) and was significantly greater than the mean of 0.56 ± 0.01 mg/kg/h (p=0.004) in five patients above 18 years of age. Patients who were given a priming dose of [1-13C]Galactose had a reduced GAR compared to those without a priming dose, 0.73 ± 0.05 (n=9) vs 1.46 ± 0.62 (n=14) mg/kg/h (p=0.005). The GAR in controls was lower than in patients ranging from 0.58 to 0.68 mg/kg/h in children and 0.07–0.09 mg/kg/h in adults. In the single bolus studies the plasma [13C]Galactose enrichment decreased in a biexponential pattern suggesting at least a two-compartment system. The calculated GAR in three adult patients was similar to that found in them by the continuous infusion technique. The GAR in patients suggests the source of Galactose for the continued elevation of Galactose metabolites as well as the basis for the long-term complications in Galactosemia despite restricted dietary Galactose intake.
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Evidence for function of UDP Galactose pyrophosphorylase in mice with absent Galactose-1-phosphate uridyltransferase
Molecular Genetics and Metabolism, 2007Co-Authors: Suzanne L. Wehrli, Robert Reynolds, Stanton SegalAbstract:Abstract Mice with deletion of the Galactose-1-phosphate uridyltransferase (GALT) gene were examined for their ability to form 13 C labeled hepatic UDP glucose from administered 1- 13 C Galactose. NMR analysis of urinary acetaminophen glucuronide, which is derived from hepatic UDP glucose showed 13 C enrichment after concomitant administration of 13 C Galactose and acetaminophen. The finding is consistent with the function of UDP Galactose pyrophosphorylase as an alternate pathway of Galactose metabolism.
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UDP-Galactose pyrophosphorylase in mice with Galactose-1-phosphate uridyltransferase deficiency.
Molecular Genetics and Metabolism, 2005Co-Authors: Nancy D. Leslie, Claire Yager, Robert Reynolds, Stanton SegalAbstract:Abstract UDP-glucose pyrophosphorylase (E.C. 2.7.7.9), encoded by ugp , provides UDP-glucose which is critical to the synthesis of glycogen, and also catalyzes the reaction between UTP and Galactose-1-phosphate, yielding UDP-Galactose. This activity of UDP-gal pyrophosphorylase (UDP-galPP) suggests a role in an alternate pathway for Galactose metabolism in patients with deficiency of Galactose-1-phosphate uridyltransferase (GALT). We examined the effects of GALT deficiency and dietary Galactose on UDP-glucose pyrophosphorylase (UDP-gluPP) and UDP-Galactose pyrophosphorylase activity and ugp expression in liver of mice with homozygous deletion of the critical regions of galt. Activity with glucose-1-phosphate as substrate was significantly higher than that with Galactose-1-phosphate. In liver from mice with GALT deficiency (G/G), UDP-galPP activity appeared to be lower than that measured in liver from control (N/N) animals. This difference disappeared when the N/N tissue homogenate was dialyzed to remove residual UDP-glucose, confirming that careful elimination of residual GALT activity is necessary, since GALT has 1000-fold greater activity toward Galactose-1-phosphate than that of UDP-galPP in liver homogenates. Prior exposure to conventional mouse chow, high Galactose chow, and high glucose chow did not alter UDP-glu PP or UDP-galPP activity. Steady state UGP mRNA levels were determined in tissues from normal and G/G animals. UGP expression was highest in liver, and did not differ by genotype or exposure to high Galactose chow. UDP-galPP activity may account for unexplained ability to oxidize Galactose in animals with no GALT activity, but is insufficient to alter accumulation of Galactose metabolites.
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Galactose Metabolism in Mice with Galactose-1-Phosphate Uridyltransferase Deficiency: Sucklings and 7-Week-Old Animals Fed a High-Galactose Diet
Molecular Genetics and Metabolism, 2001Co-Authors: Cong Ning, Robert Reynolds, Jie Chen, Claire Yager, Gerard T. Berry, Nancy D. Leslie, Stanton SegalAbstract:Abstract Mice deficient in Galactose-1-phosphate uridyltransferase (GALT) demonstrate abnormal Galactose metabolism but no obvious clinical phenotype. To further dissect the pathways of Galactose metabolism in these animals, Galactose oxidation and metabolite levels were studied in 16-day-old sucklings and the effect of a 4 week prior exposure to a 40% glucose or 40% Galactose diet was determined in 7-week-old mice. Suckling GALT-deficient (G/G) mice slowly oxidized [1- 14 C]Galactose to 14 CO 2 , 4.0% of the dose when fed and 7.9% when fasted compared to normal animals 38.3 and 36.4% in 4 h, respectively. Plasma of G/G sucklings contained 11.1 mM Galactose and erythrocyte Galactose 1-phosphate levels were 28.2 and 31.9 mg/dl packed cells. Galactose, galactitol, galactonate, and Galactose 1-phosphate were found in G/G suckling mouse tissues. The tissue Galactose concentrations were 10% or less of that in plasma, suggesting that there was limited cellular entry of Galactose. In 7-week-old fasted mice with 4 weeks prior exposure to glucose or Galactose-containing diet, 4-h oxidation was 12.9 and 15.0% of the administered radiolabeled Galactose, respectively. Normal animals oxidized 33.9 and 37.9% of the dose when fed the same diets, respectively. The ability of G/G mice to oxidize Galactose in the absence of GALT activity suggests the presence of alternate metabolic pathways for Galactose disposition. G/G mice fed the Galactose-free 40% glucose diet had erythrocyte Galactose 1-phosphate levels ranging from 6.4 to 17.7 mg/dl packed cells and detectable Galactose and Galactose metabolites in tissues, suggesting that these animals endogenously produced Galactose. The plasma of 40% Galactose-fed G/G mice contained 9.1 mM Galactose with red blood cell Galactose 1-phosphate averaging 43.6 mg/dl. Tissues of these animals also contained high levels of Galactose and Galactose 1-phosphate. Liver contained over 4 μmol/g galactonate but little galactitol. Despite the elevated Galactose and Galactose 1-phosphate, the animals tolerated the high-Galactose diet and were indistinguishable from normal animals, exhibiting no manifestations of Galactose toxicity seen in human GALT-deficient Galactosemia. The data suggest that high Galactose 1-phosphate levels do not cause Galactose toxicity and that high galactitol in combination with Galactose 1-phosphate may be a prerequisite. Absence of GALT appears necessary but insufficient to produce human Galactosemic phenotype.
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Evidence for Alternate Galactose Oxidation in a Patient with Deletion of the Galactose-1-Phosphate Uridyltransferase Gene
Molecular Genetics and Metabolism, 2001Co-Authors: Gerard T. Berry, Claire Yager, Robert Reynolds, Nancy D. Leslie, Stanton SegalAbstract:Abstract The persistent, dietary-independent elevation of Galactose metabolites in patients with Galactose-1-phosphate uridyltransferase (GALT) deficiency is probably secondary to de novo synthesis of Galactose. Relatively constant steady-state levels of Galactose metabolites in patients also suggest that non-GALT metabolic pathways must function to dispose of the Galactose synthesized each day. The discovery of a patient with a rare deletion of the GALT gene provided a unique opportunity to examine the availability of any alternate Galactose oxidative capacity both in vivo and in vitro. Utilizing genomic DNA from the patient, Southern blot data demonstrated that 10 of the 11 GALT exons were homozygously deleted. By measurement of 13CO2 in expired air for up to 24 h after an oral bolus of [1-13C]Galactose, it was demonstrated that 17% of the Galactose was metabolized, a value comparable to the 3-h elimination rate in a control subject. Furthermore, lymphoblasts prepared from the patient could also convert [1-14C]Galactose to 14CO2. This unique study provides the first unambiguous evidence that another pathway exists in man that can be responsible for Galactose disposal. Further knowledge of this alternate Galactose oxidative route and its regulation may aid in formulating new strategies for the treatment of Galactosemia.
Perry A Frey - One of the best experts on this subject based on the ideXlab platform.
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the leloir pathway a mechanistic imperative for three enzymes to change the stereochemical configuration of a single carbon in Galactose
The FASEB Journal, 1996Co-Authors: Perry A FreyAbstract:The biological interconversion of Galactose and glucose takes place only by way of the Leloir pathway and requires the three enzymes galactokinase, Galactose-1-P uridylyltransferase, and UDP-Galactose 4-epimerase. The only biological importance of these enzymes appears to be to provide for the interconversion of galactosyl and glucosyl groups. Galactose mutarotase also participates by producing the galactokinase substrate alpha-D-Galactose from its beta-anomer. The galacto/gluco configurational change takes place at the level of the nucleotide sugar by an oxidation/reduction mechanism in the active site of the epimerase NAD+ complex. The nucleotide portion of UDP-Galactose and UDP-glucose participates in the epimerization process in two ways: 1) by serving as a binding anchor that allows epimerization to take place at glycosyl-C-4 through weak binding of the sugar, and 2) by inducing a conformational change in the epimerase that destabilizes NAD+ and increases its reactivity toward substrates. Reversible ...
Robert Reynolds - One of the best experts on this subject based on the ideXlab platform.
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The rate of de novo Galactose synthesis in patients with Galactose-1-phosphate uridyltransferase deficiency
Molecular Genetics and Metabolism, 2020Co-Authors: Gerard T. Berry, Cong Ning, Robert Reynolds, Claire Yager, Peter J. Moate, Raymond C. Boston, Stanton SegalAbstract:Abstract Using both a continuous infusion of isotopically labeled [1-13C]Galactose with a steady-state analysis and a single injection kinetic approach, we have calculated the apparent Galactose appearance rate (GAR) in patients with Galactose-1-phosphate uridyltransferase deficiency and control subjects. With the steady-state protocol, the GAR in 18 patients less than 18 years of age was 1.34 ± 0.53 mg/kg/h (mean ± SD) and was significantly greater than the mean of 0.56 ± 0.01 mg/kg/h (p=0.004) in five patients above 18 years of age. Patients who were given a priming dose of [1-13C]Galactose had a reduced GAR compared to those without a priming dose, 0.73 ± 0.05 (n=9) vs 1.46 ± 0.62 (n=14) mg/kg/h (p=0.005). The GAR in controls was lower than in patients ranging from 0.58 to 0.68 mg/kg/h in children and 0.07–0.09 mg/kg/h in adults. In the single bolus studies the plasma [13C]Galactose enrichment decreased in a biexponential pattern suggesting at least a two-compartment system. The calculated GAR in three adult patients was similar to that found in them by the continuous infusion technique. The GAR in patients suggests the source of Galactose for the continued elevation of Galactose metabolites as well as the basis for the long-term complications in Galactosemia despite restricted dietary Galactose intake.
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Evidence for function of UDP Galactose pyrophosphorylase in mice with absent Galactose-1-phosphate uridyltransferase
Molecular Genetics and Metabolism, 2007Co-Authors: Suzanne L. Wehrli, Robert Reynolds, Stanton SegalAbstract:Abstract Mice with deletion of the Galactose-1-phosphate uridyltransferase (GALT) gene were examined for their ability to form 13 C labeled hepatic UDP glucose from administered 1- 13 C Galactose. NMR analysis of urinary acetaminophen glucuronide, which is derived from hepatic UDP glucose showed 13 C enrichment after concomitant administration of 13 C Galactose and acetaminophen. The finding is consistent with the function of UDP Galactose pyrophosphorylase as an alternate pathway of Galactose metabolism.
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UDP-Galactose pyrophosphorylase in mice with Galactose-1-phosphate uridyltransferase deficiency.
Molecular Genetics and Metabolism, 2005Co-Authors: Nancy D. Leslie, Claire Yager, Robert Reynolds, Stanton SegalAbstract:Abstract UDP-glucose pyrophosphorylase (E.C. 2.7.7.9), encoded by ugp , provides UDP-glucose which is critical to the synthesis of glycogen, and also catalyzes the reaction between UTP and Galactose-1-phosphate, yielding UDP-Galactose. This activity of UDP-gal pyrophosphorylase (UDP-galPP) suggests a role in an alternate pathway for Galactose metabolism in patients with deficiency of Galactose-1-phosphate uridyltransferase (GALT). We examined the effects of GALT deficiency and dietary Galactose on UDP-glucose pyrophosphorylase (UDP-gluPP) and UDP-Galactose pyrophosphorylase activity and ugp expression in liver of mice with homozygous deletion of the critical regions of galt. Activity with glucose-1-phosphate as substrate was significantly higher than that with Galactose-1-phosphate. In liver from mice with GALT deficiency (G/G), UDP-galPP activity appeared to be lower than that measured in liver from control (N/N) animals. This difference disappeared when the N/N tissue homogenate was dialyzed to remove residual UDP-glucose, confirming that careful elimination of residual GALT activity is necessary, since GALT has 1000-fold greater activity toward Galactose-1-phosphate than that of UDP-galPP in liver homogenates. Prior exposure to conventional mouse chow, high Galactose chow, and high glucose chow did not alter UDP-glu PP or UDP-galPP activity. Steady state UGP mRNA levels were determined in tissues from normal and G/G animals. UGP expression was highest in liver, and did not differ by genotype or exposure to high Galactose chow. UDP-galPP activity may account for unexplained ability to oxidize Galactose in animals with no GALT activity, but is insufficient to alter accumulation of Galactose metabolites.
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Galactose Metabolism in Mice with Galactose-1-Phosphate Uridyltransferase Deficiency: Sucklings and 7-Week-Old Animals Fed a High-Galactose Diet
Molecular Genetics and Metabolism, 2001Co-Authors: Cong Ning, Robert Reynolds, Jie Chen, Claire Yager, Gerard T. Berry, Nancy D. Leslie, Stanton SegalAbstract:Abstract Mice deficient in Galactose-1-phosphate uridyltransferase (GALT) demonstrate abnormal Galactose metabolism but no obvious clinical phenotype. To further dissect the pathways of Galactose metabolism in these animals, Galactose oxidation and metabolite levels were studied in 16-day-old sucklings and the effect of a 4 week prior exposure to a 40% glucose or 40% Galactose diet was determined in 7-week-old mice. Suckling GALT-deficient (G/G) mice slowly oxidized [1- 14 C]Galactose to 14 CO 2 , 4.0% of the dose when fed and 7.9% when fasted compared to normal animals 38.3 and 36.4% in 4 h, respectively. Plasma of G/G sucklings contained 11.1 mM Galactose and erythrocyte Galactose 1-phosphate levels were 28.2 and 31.9 mg/dl packed cells. Galactose, galactitol, galactonate, and Galactose 1-phosphate were found in G/G suckling mouse tissues. The tissue Galactose concentrations were 10% or less of that in plasma, suggesting that there was limited cellular entry of Galactose. In 7-week-old fasted mice with 4 weeks prior exposure to glucose or Galactose-containing diet, 4-h oxidation was 12.9 and 15.0% of the administered radiolabeled Galactose, respectively. Normal animals oxidized 33.9 and 37.9% of the dose when fed the same diets, respectively. The ability of G/G mice to oxidize Galactose in the absence of GALT activity suggests the presence of alternate metabolic pathways for Galactose disposition. G/G mice fed the Galactose-free 40% glucose diet had erythrocyte Galactose 1-phosphate levels ranging from 6.4 to 17.7 mg/dl packed cells and detectable Galactose and Galactose metabolites in tissues, suggesting that these animals endogenously produced Galactose. The plasma of 40% Galactose-fed G/G mice contained 9.1 mM Galactose with red blood cell Galactose 1-phosphate averaging 43.6 mg/dl. Tissues of these animals also contained high levels of Galactose and Galactose 1-phosphate. Liver contained over 4 μmol/g galactonate but little galactitol. Despite the elevated Galactose and Galactose 1-phosphate, the animals tolerated the high-Galactose diet and were indistinguishable from normal animals, exhibiting no manifestations of Galactose toxicity seen in human GALT-deficient Galactosemia. The data suggest that high Galactose 1-phosphate levels do not cause Galactose toxicity and that high galactitol in combination with Galactose 1-phosphate may be a prerequisite. Absence of GALT appears necessary but insufficient to produce human Galactosemic phenotype.
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Evidence for Alternate Galactose Oxidation in a Patient with Deletion of the Galactose-1-Phosphate Uridyltransferase Gene
Molecular Genetics and Metabolism, 2001Co-Authors: Gerard T. Berry, Claire Yager, Robert Reynolds, Nancy D. Leslie, Stanton SegalAbstract:Abstract The persistent, dietary-independent elevation of Galactose metabolites in patients with Galactose-1-phosphate uridyltransferase (GALT) deficiency is probably secondary to de novo synthesis of Galactose. Relatively constant steady-state levels of Galactose metabolites in patients also suggest that non-GALT metabolic pathways must function to dispose of the Galactose synthesized each day. The discovery of a patient with a rare deletion of the GALT gene provided a unique opportunity to examine the availability of any alternate Galactose oxidative capacity both in vivo and in vitro. Utilizing genomic DNA from the patient, Southern blot data demonstrated that 10 of the 11 GALT exons were homozygously deleted. By measurement of 13CO2 in expired air for up to 24 h after an oral bolus of [1-13C]Galactose, it was demonstrated that 17% of the Galactose was metabolized, a value comparable to the 3-h elimination rate in a control subject. Furthermore, lymphoblasts prepared from the patient could also convert [1-14C]Galactose to 14CO2. This unique study provides the first unambiguous evidence that another pathway exists in man that can be responsible for Galactose disposal. Further knowledge of this alternate Galactose oxidative route and its regulation may aid in formulating new strategies for the treatment of Galactosemia.
J P Latge - One of the best experts on this subject based on the ideXlab platform.
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overlapping and distinct roles of aspergillus fumigatus udp glucose 4 epimerases in Galactose metabolism and the synthesis of Galactose containing cell wall polysaccharides
Journal of Biological Chemistry, 2014Co-Authors: Fabrice N Gravelat, Robert P Cerone, Stefanie D Baptista, Paolo Campoli, Sein Choe, Ilia Kravtsov, Evgeny Vinogradov, Carole Creuzenet, Albert M Berghuis, J P LatgeAbstract:The cell wall of Aspergillus fumigatus contains two Galactose-containing polysaccharides, galactomannan and galactosaminogalactan, whose biosynthetic pathways are not well understood. The A. fumigatus genome contains three genes encoding putative UDP-glucose 4-epimerases, uge3, uge4, and uge5. We undertook this study to elucidate the function of these epimerases. We found that uge4 is minimally expressed and is not required for the synthesis of Galactose-containing exopolysaccharides or Galactose metabolism. Uge5 is the dominant UDP-glucose 4-epimerase in A. fumigatus and is essential for normal growth in Galactose-based medium. Uge5 is required for synthesis of the galactofuranose (Galf) component of galactomannan and contributes Galactose to the synthesis of galactosaminogalactan. Uge3 can mediate production of both UDP-Galactose and UDP-N-acetylgalactosamine (GalNAc) and is required for the production of galactosaminogalactan but not galactomannan. In the absence of Uge5, Uge3 activity is sufficient for growth on Galactose and the synthesis of galactosaminogalactan containing lower levels of Galactose but not the synthesis of Galf. A double deletion of uge5 and uge3 blocked growth on Galactose and synthesis of both Galf and galactosaminogalactan. This study is the first survey of glucose epimerases in A. fumigatus and contributes to our understanding of the role of these enzymes in metabolism and cell wall synthesis.
Jean Marc Nicaud - One of the best experts on this subject based on the ideXlab platform.
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Awakening the endogenous Leloir pathway for efficient Galactose utilization by Yarrowia lipolytica
Biotechnology for Biofuels, 2015Co-Authors: Zbigniew Lazar, Anne-Marie Crutz- Coq, Heber Gamboa-Meléndez, Cécile Neuvéglise, Jean Marc NicaudAbstract:BackgroundProduction of valuable metabolites by Yarrowia lipolytica using renewable raw materials is of major interest for sustainable food and energy. Galactose is a monosaccharide found in galactomannans, hemicelluloses, gums, and pectins.ResultsYarrowia lipolytica was found to express all the Leloir pathway genes for Galactose utilization, which encode fully functional proteins. Gene organization and regulation in Y. lipolytica resembles filamentous fungi rather than Saccharomyces cerevisiae. After Y. lipolytica was grown on mixture of glucose and Galactose, it was then able to metabolize Galactose, including when glucose concentrations were higher than 4 g/L. However, glucose was still the preferred carbon source. Nonetheless, a strain overexpressing the four ylGAL genes of the Leloir pathway was able to efficiently use Galactose as its sole carbon source. This mutant was used to produce citric acid and lipids from Galactose; the yields were comparable to or greater than that obtained for the parental strain (W29) on glucose.ConclusionsThe construction of a Y. lipolytica strain able to produce citric acid and lipids from Galactose is a very important step in bypassing issues related to the use of food-based substrates in industrial applications.