[Biological assay for galactose-1 phosphate measurement application in subjects with galactosemia]. / Dosage du galactose-1 phosphate et application chez des sujets galactosémiques.

Congenital galactosemia is a hereditary, autosomal recessive and metabolic disease. It is linked to an enzyme deficiency, more commonly known by the deficiency of galactose-1- phosphate uridyltransferase (GALT), which is responsible for an accumulation of galactose-1- phosphate in the blood. Clinical symptoms appear early in infancy from the second week of life. They generally manifested by some disorders within liver, kidney, eye, gastrointestinal, neurological and also with cataracts. Currently, the clinical diagnosis remains difficult hence the importance of further investigations based on effective biological assessments to highlight the disease. The diagnosis of galactosemia is made by the laboratory test. The latter includes the determination of Gal-1-P which is done by a fluorometric method spot test. This study was conducted in order to assess the repeatability, reproducibility, accuracy, and effectiveness of the techniques used. We have found the CV for a repeatability (CV = 5 %), reproducibility (CV = 4 %) which confirms the accuracy of the method proceeded in this study. This method allows us to have a degree of inaccuracy less than 1%. According to the study of the effectiveness of "spot test", we found that our technique is specific (Sp = 93 %) and sensitive (Se = 83 %).

Monitoring of biochemical status in children with Duarte galactosemia: utility of galactose, galactitol, galactonate, and galactose 1-phosphate.

BACKGROUND: Duarte galactosemia (DG) is frequently detected in newborn-screening programs. DG patients do not manifest the symptoms of classic galactosemia, but whether they require dietary galactose restriction is controversial. We sought to assess the relationships of selected galactose metabolites (plasma galactose, plasma galactitol, erythrocyte (RBC) galactitol, RBC galactonate, and urine galactitol and galactonate) to RBC galactose 1-phosphate (Gal-1-P), dietary galactose intake, and neurodevelopmental/clinical outcomes in DG children. METHODS: We studied 30 children 1-6 years of age who had DG galactosemia and were on a regular diet. All participants underwent a physical and ophthalmologic examination and a neurodevelopmental assessment. RBC galactitol, RBC galactonate, RBC Gal-1-P, plasma galactose, plasma galactonate, and urine galactitol and galactonate concentrations were measured. RESULTS: RBC galactitol and galactonate concentrations were about 2 and 6 times higher, respectively, than control values. Plasma galactose and galactitol concentrations were also about twice the control values. The mean values for RBC Gal-1-P and urine galactitol were within the reference interval. We found a relationship between plasma and urine galactitol concentrations but no relationship between RBC galactose metabolites and urine galactitol. There was a significant relationship between galactose intake and RBC galactose metabolites, especially RBC galactitol (P < 0.0005) and RBC galactonate (P < 0.0005). Galactose intake was not related to the urine galactitol, plasma galactose, or plasma galactitol concentration. RBC galactitol, RBC galactonate, plasma galactose, plasma galactitol, and urine galactonate concentrations showed no relationship with clinical or developmental outcomes. CONCLUSIONS: DG children on a regular diet have RBC Gal-1-P concentrations within the reference interval but increased concentrations of other galactose metabolites, including RBC galactitol and RBC galactonate. These increased concentrations correlate with galactose intake and neither cause any developmental or clinical pathology during early childhood nor oblige a lactose-restricted diet.

Lithium-mediated suppression of morphogenesis and growth in Candida albicans.

Hyphal development in Candida albicans contributes to virulence, and inhibition of filamentation is a target for the development of antifungal agents. Lithium is known to impair Saccharomyces cerevisiae growth in galactose-containing media by inhibition of phosphoglucomutase, which is essential for galactose metabolism. Lithium-mediated phosphoglucomutase inhibition is reverted by Mg(2+). In this study we have assessed the effect of lithium upon C. albicans and found that growth is inhibited preferentially in galactose-containing media. No accumulation of glucose-1-phosphate or galactose-1-phosphate was detected when yeasts were grown in the presence of galactose and 15 mM LiCl, though we observed that in vitro lithium-mediated phosphoglucomutase inhibition takes place with an IC(50) of 2 mM. Furthermore, growth inhibition by lithium was not reverted by Mg(2+). These results show that lithium-mediated inhibition of growth in a galactose-containing medium is not due to inhibition of galactose conversion to glucose-6-phosphate but is probably due to inhibition of a signaling pathway. Deletion of the Ser-Thr protein phosphatase SIT4 and treatment with rapamycin have been shown to inhibit filamentous differentiation. We observed that C. albicans filamentation was inhibited by lithium in solid medium containing either galactose as the sole carbon source or 10% fetal bovine serum. These results suggest that suppression of hyphal outgrowth by lithium could be related to inhibition of the target of rapamycin (TOR) pathway.

Diagnosis of inherited disorders of galactose metabolism.

Galactose metabolism occurs through an evolutionarily conserved pathway in which galactose and uridine diphosphoglucose are converted to glucose-1-phosphate and uridine diphosphogalactose through the action of three sequential enzymes: galactokinase (GALK, EC 2.7.1.6), galactose-1-phosphate uridyltransferase (GALT, EC 2.7.7.12), and uridine phosphogalactose 4'-epimerase (GALE, EC 5.1.3.2). Inborn errors of galactose metabolism occur with impaired activity for each of the enzymes. Classical galactosemia is the most common and the most severe of these diseases and is caused by deficiency of the GALT enzyme, affecting from approximately 1 in 10,000 to 1 in 30,000 live births. Deficiency of GALE is the rarest of the three diseases. Assays for galactitol and galactose-1-phosphate and methods for assaying enzyme activities of GALT, GALK, and GALE are provided here. Interpretation of diagnostic results for screen-positive newborns or symptomatic patients, as well as therapeutic interventions based on biochemical phenotype and molecular genotype, are also included as decision trees.

Galactose metabolism in normal human lymphoblasts studied by (1)H, (13)C and (31)P NMR spectroscopy of extracts.

The development of tools to follow and quantitate the fate of galactose in mammalian cells is crucial to the study and understanding of the inherited disorders of galactose metabolism. In this study we incubated normal human lymphoblasts with 1- or 2-(13)C galactose for 2.5 or 5 h and prepared TCA extracts of the cells. The various galactose metabolites were identified and quantified using a combination of proton, carbon and phosphorus NMR spectra. Galactose-1-phosphate (gal-1P), uridine diphosphogalactose, uridine diphosphoglucose and galactitol were present in the extracts. Average levels for gal-1P were around 10 nmol/mg protein and for uridine diphosphoglucose, uridine diphosphogalactose and galactitol in the range of 0.5-2 nmol/mg protein. Galactonate was never found in any conditions. Percentage labeling could be estimated for gal-1P and for the ribose carbons of AMP. The labeling agrees with a conversion of galactose to glucose through the Leloir pathway.

Transient galactosemia detected by neonatal mass screening.

BACKGROUND: The Paigen method has detected not only persistently galactosemic patients, but also many children with transient galactosemia during the neonatal period. The diagnosis and clinical course of 389 patients with transient galactosemia detected by neonatal mass-screening from 1986 to 1996 in the Hiroshima prefecture were evaluated. METHODS: Enzyme assays for galactose metabolism, measurement of blood galactose levels, erythrocyte galactose-1-phosphate levels, serum total bile acid (TBA) levels and liver function tests were performed at the first visit by patients to our hospital. Liver function and the mental and physical development of patients were evaluated during the follow-up period (approximately 1 year). RESULTS: The diagnoses were classified as follows: 253 patients with unknown cause, 128 heterozygotes and two homozygotes for galactose enzyme deficiency (galactose-1-phosphate uridyltransferase, galactokinase, UDP-galactose 4-epimerase) and six heterozygotes for Duarte variant. Twelve patients showed high serum levels of TBA (> 80 mumol/L), which suggests the presence of portal-systemic shunts during the neonatal period causing galactosemia. Most patients showed normal mental and physical development during infancy. However, of 25 patients with mild to moderate abnormal liver function tests of unknown etiology after the neonatal period, five showed poor weight gain coincident with liver dysfunction. In almost all patients, levels of transferase decreased to the normal range by 1 year of age. CONCLUSION: We found that the prognosis of transient galactosemia was almost always favorable. However, patients should be followed for at least 1 year, because late liver dysfunction, which might cause poor weight gain, occurred in 6% of our patients.

Clinical and biochemical evidence of skeletal muscle involvement in galactose-1-phosphate uridyl transferase deficiency.

An 8-year-old boy with galactose-1-phosphate uridyl transferase (GALT) deficiency presented with hypotonia, muscle hypotrophy, hepatomegaly, bilateral cataract and mild mental retardation. Two brothers showed a GALT activity consistent with a homozygotic condition and both parents were found to be heterozygotes for this defect. Histological and ultrastructural examination of muscle biopsy specimens showed several necrotic fibres. GALT activity was undetectable in skeletal muscle and muscle tissue cultures; myotubes converted galactose to CO2 at a lower rate than controls. Galactose-1-phosphate was increased in the patient's red cells and muscle tissue. GALT deficiency, not previously described in muscle, may be of pathogenic relevance in determining the myopathic features present in GALT deficiency syndrome.

Determination by heteronuclear NMR spectroscopy of the complete structure of the cell wall polysaccharide of Streptococcus sanguis strain K103.

Although complete structures of complex polysaccharides have traditionally been determined by chemical degradative methods, a number of recent developments in instrumentation have greatly facilitated this task. We illustrate the application of several of these methods in a determination of the complete covalent structure of the polysaccharide from Streptococcus sanguis K103, which is composed of an octasaccharide repeating subunit linked by phosphodiester bonds. Carbohydrate analysis by HPAE-PAD and by reverse-phase chromatography of benzoylated derivatives of the hydrolysis products of the polysaccharide gave glucose (3 mol), galactose (1 mol), rhamnose (2 mol), N-acetylglucosamine (1 mol), and galactose 6-phosphate (1 mol). Circular dichroism of the O-benzoylated monosaccharides showed the absolute configurations to be D for all residues except for rhamnose, which is L. The 1H NMR spectrum was completely assigned by two-dimensional homonuclear methods (DQF-COSY, NOESY, HOHAHA). The stereochemistry of pyranosides was assigned from 3JHH coupling constant values determined from these experiments. The 13C spectrum was assigned by 1H-detected heteronuclear multiple-quantum correlation (1H[13C] HMQC) and by the hybrid method of HMQC-COSY. The glycosidic linkage positions of the polymer were determined by 1H-detected multiple-bond correlation (1H[13C] HMBC) and by 2D-NOESY spectra. The position of the phosphodiester linkage was determined by splitting observed in the 13C resonances due to 31P couplings leading to the overall structure given in Chart I.

HPLC analysis of hexosamine phosphates in biological samples.

Galactosamine is quickly metabolized to galactosamine 1-phosphate in rats treated with this compound. An HPLC method to quantify hexosamine phosphates in biological samples is described, modified from the o-phthaldialdehyde amino acid analysis procedure. o-Phthaldialdehyde derivatives of hexosamines and hexosamine-phosphates can be eluted from a reverse-phase column at different retention times, with a total analysis time of 30 min and without overlapping with free amino acids at physiological concentrations. The standard curves are linear between 1 and 40 nmol. This simple method is more selective and sensitive than previous enzymatic analyses of hexosamine phosphorylation.

Newborn screening for galactosemia: a new method used in Manitoba.

In July 1983, the Manitoba Perinatal Screening Programme modified its existing procedure for neonatal screening for galactosemia by introducing quantitation of total galactose plus galactose-1-phosphate from dried blood spots using the Multistat centrifugal analyzer. The first 4 years of experience with this method in combination with the Beutler spot test for galactose-1-phosphate uridyl transferase activity is the subject of this report. Of 70,336 newborns screened, 142 (0.20%) met the criteria for clinical follow up. Of these, one child was confirmed to have classical galactosemia and nine children were found to be Duarte/galactosemia genetic compounds. This method of galactosemia screening has proven to be rapid, sensitive, efficient, and the method of choice for mass screening of disorders of galactose metabolism.

Further observations in a case of uridine diphosphate galactose-4-epimerase deficiency with a severe clinical presentation.

The red-cell concentrations of galactose-1-phosphate and uridine diphosphate galactose have been studied in relation to dietary galactose in a case of uridine diphosphate galactose-4-epimerase deficiency (McKusick 23035). Uridine diphosphate galactose accumulates rapidly in response to very small amounts of galactose but the concentration of galactose-1-phosphate increases proportionately to galactose intake. The significance of the observation is discussed with respect to the pathogenesis and treatment of the disease.