Evidence for function of UDP galactose pyrophosphorylase in mice with absent galactose-1-phosphate uridyltransferase.

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.

Metabolic fate of administered [13C]galactose in tissues of galactose-1-phosphate uridyl transferase deficient mice determined by nuclear magnetic resonance.

The pattern of distribution of galactose and its metabolites was determined in tissues of mice deficient in galactose-1-phosphate uridyl transferase (G/G) 4 h after the administration of 1mg/g of [13C]galactose. Labeled galactose was found in all the tissues examined, the highest amounts in liver and kidney. Each of the tissues had its own pattern of labeling of galactose-1-phosphate (gal-1-P), galactitol and galactonate. [13C]gal-1-P and galactonate concentration was highest in liver while [13C]galactitol was higher in kidney and heart than in other tissues. Muscle had the lowest amounts of these compounds. In contrast, no galactose was found in tissues of normal mice (N/N) except for a minute amount in muscle. No [13C]gal-1-P was found in liver, kidney or brain and only minute amounts in heart and muscle of N/N animals. Barely detectible, labeled galactitol was observed in these tissues except liver, where none was found. [13C]Galactonate was formed in liver comparable to G/G mice. Almost all of the accumulating 13C isotope was found in liver and kidney glucose and lactate in the normal animals. [13C]Glucose and lactate was also found in liver of the G/G animals, but to a lesser extent than in normals, indicating the presence of a pathway in G/G animals for circumventing the block at GALT for the normal conversion of galactose to glucose.

Urinary galactitol and galactonate quantified by isotope-dilution gas chromatography-mass spectrometry.

BACKGROUND: Measurements of urine galactitol have been used to monitor the adequacy of diet therapy in the treatment of galactosemia. We have devised a gas chromatographic mass spectrometry (GC/MS) isotope-dilution method for the simultaneous quantification of urine galactitol and another alternate pathway product, galactonate. METHODS: We prepared trimethylsilyl (TMS) derivatives and used D-[UL-13C]galactitol and D-[UL-13C]galactonate as the internal standard for GC/MS. Results obtained with this method were compared with those determined by the established GC method for galactitol and the NMR method for galactonate. Thirty-three normal urine specimens were analyzed by the isotope dilution technique for galactitol and galactonate. Results of galactitol in 6 of these urine specimens along with 18 from classic galactosemics and 19 variant galactosemics were compared with the established GC method. Results for galactonate in 15 urine specimens from galactosemics were compared to the established NMR technique. RESULTS: The method was linear up to 200 nmol with lower limits of detection of 1.1 nmol (1.75 mmol/mol creatinine) (Cr) and 0.8 nmol (1.28 mmol/mol Cr) for galactitol and galactonate, respectively. Intra- and Interassay imprecision ranged from 2.1-6.7% for galactitol and 3.5-8.0% for galactonate. The excretion of both metabolites was age dependent in both normal and galactosemics. In 12 normal urines from subjects under 1 year, values for galactitol ranged from 8-107 mmol/mol Cr, and in 7 over age 6, ranged from 2-5 mmol/mol Cr. Under 1 year, the range for galactonate was non-detectable to 231 and in the over 6 years group non-detectable to 25 mmol/mol Cr. In galactosemics under 1 year, the value for galactitol ranged from 397-743 and for galactonate 92-132 mmol/mol Cr while in nine patients over age 6 the range was 125-274 mmol/mol Cr for galactitol and 17-46 mmol/mol Cr for galactonate. CONCLUSIONS: The GC/MS method enables the simultaneous determination of urine galactitol and galactonate and is precise and useful over the wide range of concentrations needed to assess the galactose burden in patients with galactosemia.

Pathways of galactose metabolism by galactosemics: evidence for galactose conversion to hepatic UDPglucose.

To determine if classic galactosemics have residual galactose-1-phosphate uridyltransferase (GALT) activity to explain their considerable ability to oxidize galactose over 24 h, we devised a method for assessing their ability to form hepatic UDPglucose (UDPglu), an intermediate in the normal Leloir pathway of galactose metabolism. The protocol involved the single oral administration of 7 mg/kg [2-13C]galactose concomitant with multiple small doses of acetaminophen with measurement of the extent of labeling of urinary acetaminophen glucuronide, the glucuronide moiety being formed from hepatic UDPglu. We performed the study lasting 24 h in two normal subjects and three classic galactosemics, two homozygous for the Q188R mutation and one compound for the Q188R/K258N mutation. The labeling and total excretion of acetaminophen glucuronide was measured in urine by nuclear magnetic resonance techniques. Concomitant with determination of label in the glucuronide measurement was made of galactose oxidation to 13CO2 and the 13C enrichment of plasma glucose. All of the galactosemic patients formed 13C enriched acetaminophen glucuronide indicating that they had converted the labeled galactose to [13C]UDPglu and that residual GALT or another pathway that forms UDPglu is present in hepatic tissue. Compared to the normal whose glucuronide labeling was rapid and short-lived that of the galactosemics was delayed and extended for a long period over 10 h. The extent of isotopic enrichment of glucuronide by galactosemics was comparable to the normals, resulting in a much greater conversion of galactose to UDPglu by the galactosemics. The labeling of the UDPglu pool was reflected by the rate of 13CO2 formation being rapid in the normal with peak labeling at 2-3 h with total oxidation of over 70% in 24 h. The oxidation of the galactosemics was slow with a broad peak of 13CO2 at 10 h and a total excretion of 25-39% of the [13C]galactose administered. The normal subjects formed highly enriched plasma glucose within 30 min while no enrichment of plasma glucose was detected until after 300 min in galactosemics. The exact pathway(s) of galactose metabolism by galactosemics to UDPglu remain to be determined. Their delineation may contribute to new approaches to therapeutic strategies for this enigmatic disorder.

UDP-galactose pyrophosphorylase in mice with galactose-1-phosphate uridyltransferase deficiency.

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.

Galactitol and galactonate in red blood cells of children with the Duarte/galactosemia genotype.

We measured galactitol, galactonate, and galactose-1-phosphate in the red blood cell (RBC) to elucidate the biochemical phenotype of infants with a Duarte/galactosemia (D/G) genotype by isotope dilution GC/MS. The RBC galactonate, galactitol and Gal-1-P were quantified in 14 D/G newborns on a lactose containing formula or breast milk, eight D/G newborns on a galactose-free formula, and 18 D/G children between 1 and 2 years of age that were on a regular diet. The results were compared with those of non-galactosemic subjects of comparable age. In the D/G newborns on regular formula/breast milk, the levels of RBC galactitol, galactonate, and Gal-1-P were significantly higher than those of D/G newborns on diet treatment and non-galactosemic newborns. There was no difference in the levels of RBC galactitol, galactonate, and Gal-1-P between D/G newborns on a lactose-restricted diet and the control group. There appears to be two different responses to dietary galactose intake in D/G children. The first group of D/G children placed on a regular diet after a year of lactose restriction had higher RBC galactitol, galactonate levels than those of non-galactosemic children. The mean level of RBC galactonate was higher and the mean value of RBC galactitol was as high as that of galactosemic (G/G) patients on diet treatment. The second group of D/G children on a regular diet had normal levels of RBC galactitol and galactonate. The levels of RBC Gal-1-P were normal in both groups of D/G patients. The alternative pathway products may reflect galactose intake better than RBC Gal-1-P in D/G children.

Extended [13C]galactose oxidation studies in patients with galactosemia.

Since patients with galactose-1-phosphate uridyltransferase (GALT) deficiency have considerable endogenous galactose formation and only limited urinary excretion of galactose metabolites, there must be mechanisms for disposal of the sugar. Otherwise, a steady-state could not be maintained and there would be continuous body accumulation of galactose and alternate pathway products. Previous studies quantitating the amount of galactose handled by oxidation to CO2 focused on short collection periods of expired air after administering isotopically labeled galactose mainly designed for discerning differences in the capacity to oxidize the sugar in relation to genotype. Assuming that there may be more extensive oxidation than that observed in short-term studies in order to dispose the daily galactose burden, we have examined the amount of [1-13C]galactose oxidized to 13CO2 over a 24-h period after either a single bolus or continuous IV administration by 11 patients with classic galactosemia including patients homozygous for the Q188R gene mutation. As much as 58% of the administered galactose was oxidized to 13CO2 in 24 h. The pathways involved remain to be determined but a significant amount may be metabolized by non-GALT pathways since a patient homozygous for gene deletion had an oxidative capability. We conclude that classic patients have the ability to slowly oxidize galactose to CO2 in 24 h in amounts comparable to that which a normal handles in approximately one-fifth the time. This capacity enables the galactosemic to maintain a balance of galactose disposal with the galactose burden imposed by endogenous formation and dietary intake.

The rate of de novo galactose synthesis in patients with galactose-1-phosphate uridyltransferase deficiency.

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.

Galactitol and galactonate accumulation in heart and skeletal muscle of mice with deficiency of galactose-1-phosphate uridyltransferase.

Under conditions of dietary galactose loading, mice deficient in galactose-1-phosphate uridyltransferase (GALT) accumulate large amounts of galactitol and galactonate in heart and skeletal muscle. In contrast to liver, brain, and kidney, which form little galactitol when GALT-deficient animals (G/G) ingest a 40% galactose diet, heart and skeletal muscle galactitol reaches 22.90+/-1.62 (M+/-SE) and 38.88+/-2.62 micromol/g tissue, respectively, levels 40-100 times that of galactose-1-phosphate (Gal-1-P). Sixteen-day-old suckling G/G mice accumulate galactitol in heart and to a lesser extent, in skeletal muscle. Heart and skeletal muscle of G/G mice also form galactonate, with levels comparable to that of liver, which was presumed previously to be the only tissue capable of converting galactose to galactonate under conditions of loading. The data suggest that heart and skeletal muscle play a role in disposition of galactose when GALT activity is impaired, contributing a large share to urinary galactitol and galactonate excretion. The ability of heart and muscle to form galactonate may also contribute to the G/G mouse's ability to slowly oxidize galactose to CO2, since the compound is an intermediate in an alternate route for galactose disposition.

Galactitol and galactonate in red blood cells of galactosemic patients.

The red blood cell (RBC) concentration of galactitol and galactonate was measured in 27 patients with galactose-1-phosphate uridyltransferase (GALT) deficiency galactosemia and 19 non-galactosemic subjects by a newly devised isotope dilution gas chromatography/mass spectrometry (GC/MS) method. The method utilizing UL[13C]galactitol and UL[13C]galactonate was reproducible with excellent precision and recovery of 99%. The RBC galactitol in galactosemic patients on galactose-restricted diets averaged 5.98+/-1.2 microM (M+/-SD) with a range of 3.54-8.81 microM. The mean in non-galactosemic patients was 0.73+/-0.31 microM with a range of 0.29-1.29 microM. The mean of RBC galactonate in the same galactosemic patients was 4.16+/-1.32 microM (M+/-SD) with a range of 0.68-6.47, while the mean in non-galactosemic subjects was 1.94+/-0.96 (M+/-SD) with a range of 0.69-3.84. In galactosemic RBC the galactitol was higher than galactonate while this was reversed in non-galactosemic cells. RBC galactose-1-phosphate (Gal-1-P) measured at the same time as galactitol and galactonate was 30 times the level of the other two metabolites. There was no relationship between RBC Gal-1-P and galactitol or galactonate. The ability to measure all three galactose metabolites in the same procedure offers the possibility of augmented monitoring of the galactose metabolic status of patients. The measurement of RBC galactitol and galactonate presents a new means of characterizing galactosemic patients and their levels monitored over time may provide new insight in the development of long-term complications observed in afflicted patients.

Metabolism of 13C galactose by lymphoblasts from patients with galactosemia determined by NMR spectroscopy.

In order to assess the pathways by which galactose is metabolized by galactose-1-phosphate uridyltransferase (GALT) deficient cells, lymphoblasts from 10 galactosemic patients with defined genotypes (six Q188R homozygotes, two S153L homozygotes, and two with homozygous deletions) were incubated with 1mM 1- or 2-13C galactose for 2.5 and 5 h. The 13C-labeled metabolites were identified and quantified using nuclear magnetic resonance and the results were compared to that obtained with cells from eight normal individuals. Cells from galactosemic patients formed two to three times the galactose-1-phosphate (Gal-1P) in normal cells, no difference being observed between the various genotypes. Galactitol formation was not significantly different from normal cells. No labeled galactonate was detected. Cells with the Q188R and S135L mutations formed both labeled uridine diphosphogalactose (UDPgal) and uridine diphosphoglucose (UDPglu), but to a lesser extent than normals, whereas cells with the GALT deletion did not. The pattern of 13C enrichment of the ribose carbons of adenosine monophosphate upon incubation of the normal cells with 1-13C galactose paralleled that found for incubations with 1-13C glucose, which is consistent with galactose disposition through the Leloir pathway to glucose and its subsequent metabolism to ribose. Cells with the GALT deletion formed no detectable labeled ribose, whereas cells from a patient homozygous for Q188R mutation formed labeled ribose in a pattern similar to normal albeit with lower enrichment. The results suggest that there is residual GALT activity and function of the Leloir pathway in the presence of the Q188R as well as S135L mutation.

Identification of galactitol and galactonate in red blood cells by gas chromatography/mass spectrometry.

BACKGROUND: Because the products of alternate pathways of galactose metabolism, galactitol and galactonate are important in galactosemia, we sought to identify these compounds in red blood cells (RBC). METHODS: RBC extracts were trimethylsilylated (TMS) and analyzed by gas chromatography/mass spectrometry (GC/MS). RESULTS: The presence of both galactitol and galactonate was identified in RBC of 15 galactosemic and 13 normal subjects by their mass spectra and chromatographic comparisons with both unlabeled and 13C labeled standards. The levels in RBC of galactosemics appear to be much higher than those of normal subjects. CONCLUSION: The determination of these compounds in RBC along with galactose-1-phosphate (gal-1-P) in the same procedure provides the potential for their use in better monitoring of diet therapy in galactosemic patients.

Erythrocyte galactose 1-phosphate quantified by isotope-dilution gas chromatography-mass spectrometry.

BACKGROUND: Measurements of alpha-D-galactose 1-phosphate (Gal-1-P) in erythrocytes are used to monitor the adequacy of dietary therapy in the treatment of galactosemia. We have devised a gas chromatography-mass spectrometry (GC/MS) isotope-dilution method for quantification of Gal-1-P. METHODS: We prepared trimethylsilyl (TMS) derivatives and used alpha-D-[2-(13)C]Gal-1-P as the internal standard for GC/MS. Results obtained with this method were compared with those determined by the established enzymatic method for samples from 23 healthy individuals (11 children and 12 adults), 9 suspected patients with galactosemia, 12 galactosemic patients on diet therapy, and 2 newly diagnosed toxic neonates. RESULTS: The method was linear up to 2.5 mmol/L with a lower limit of detection of 2.1 nmol (0.55 mg/L). Intra- and interassay imprecision (CVs) was 2.2-8.8%. In the 23 healthy individuals, values ranged from nondetectable to 9.2 micromol/L (2.4 mg/L of packed erythrocytes). Galactosemic patients on diet therapy had values of 10.9-45 mg/L of packed erythrocytes, whereas the newly identified patients had values of 166 and 373 mg/L. CONCLUSIONS: The GC/MS method is precise and useful over the wide range of concentrations needed to assess the galactose burden in patients with galactosemia.