The main pathway of galactose metabolism in humans is the conversion of galactose to glucose, without disruption of the carbon skeleton. The name 'galactosemia' has been associated with a syndrome of toxicity associated with the administration of galactose to patients with an inherited disorder of galactose utilization, leading to multiple clinical manifestations, including malnutrition, mental retardation, liver disease, and cataracts. The clinical manifestations are linked to specific enzymatic defects. Thus the term 'galactosemia' should be qualified by the specific defect. Three enzymatic steps are required to metabolize galactose to UDP-glucose. Two alternate pathways, oxidation and reduction, are used in the absence of enzymes of the main route.
Step 1: Galactokinase
Galactose is phosphorylated by galactokinase with ATP to form galactose 1-phosphate. The equilibrium is far in the direction of sugar phosphoryla-tion, but the reaction is reversible. Galactokinase has been studied in detail in human red cells, leucocytes, fibroblasts, placenta, liver, and various human fetal tissues. It is detectable in fetal liver from 10 weeks of gestation onwards and the activity of the enzyme in liver and red cells is higher in the second and third trimester. Its activity is higher in red blood cells from human infants than in cells from adults, and in reticulocytes than with aged red cells. Cultured human fibroblasts show enhanced galactokinase activity when grown in the presence of galactose, whereas in the liver the activity does not appear to be regulated by dietary galactose. The red cell enzyme, like that of the liver, undergoes substrate and product inhibition.
The assignment of the gene for galactokinase has been made to human chromosome 17, and its regional localization of the chromosome has been assigned to band q21-22.
Step 2: Transferase
Galactose 1-phosphate reacts with UDP-glucose to produce UDP-galactose and glucose 1-phosphate. This step is catalyzed by galactose-1-phosphate uridyltransferase, an enzyme present in bacteria and most mammalian tissues. Like galactokinase, galac-tose-1-phosphate uridyltransferase is detectable in fetal liver from 10 weeks of gestation, with the liver enzyme-specific activity being highest at 28 weeks of gestation. The rate of reaction may be regulated by substrate concentration and limited by UDP-glucose substrate inhibition of transferase. Glucose 1-phosphate is a potent inhibitor of the enzyme. Uridine nucleotides such as uridine di- and triphosphate are powerful competitive inhibitors of substrate UDP-glucose.
Galactose-1-phosphate uridyltransferase deficiency is the most commonly reported defect in galactosemic patients. In the young infant galactose is a major energy source and its metabolism to glucose 1-phosphate is essential, but this is not the case in the fetus in whom glucose is the main energy source. However, the metabolism of galactose in the fetus is important to prevent accumulation of toxic galactose metabolites. Thus in galactose-1-phosphate uridyl-transferase deficiency the fetus could be at a disadvantage as early as the 10th week of gestation. Dietary and hormonal influences on the liver enzyme have not been reported. In the rat a galactose-rich diet increases transferase activity.
Galactose-1-phosphate uridyltransferase is localized on chromosome 9p13. At least 32 variants in the nucleotide sequence of the galactose-1-phosphate uridyltransferase gene have been identified, with the most frequent being change in amino acid codon position 188 in which an arginine is substituted for a glutamine, the Q188R mutation. This Q188R mutation is associated with 'classical' galactosemia with virtually no galactose-1-phosphate uridyl-transferase activity detectable. However, there are other variant forms of the enzyme which have diminished but detectable activity, known as Duarte, Indiana, Rennes, Los
Angeles, Miinster, and Chicago. Heterozygotes for normal and Duarte alleles are presumed to have 75% of normal galactose-1-phosphate uridyltrans-ferase activity. Homozygotes for the Duarte allele could have 50% activity, and compound heterozygotes for the Duarte allele and the classical galac-tosemia allele have 25% activity in peripheral erythrocytes.
Step 3: Epimerase
The UDP-galactose is converted to UDP-glucose by UDP-galactose 4'-epimerase. The UDP-glucose thus formed can then enter the reaction again in a cyclical fashion until all the free galactose coming into the pathway is converted to glucose 1-phosphate. This enzyme is responsible for the inversion of the hydroxyl group at the C-4 carbon of the hexose chain to form glucose from galactose; it is also important for the conversion of UDP-glucose to UDP-galactose when only glucose is available and galactose is required as a constituent of complex polysaccharides. The epimerase maintains a cellular equilibrium of UDP-glucose to UDP-galactose in a ratio of about 3:1.
The purified enzyme is a dimer of identical subunits that consists of a mixture of catalytically active subunits (epimerase-NAD+) and inactive subunits (epimerase-NADH-uridine nucleotide). The NAD binds to the enzyme and induces a conformational change resulting in enzymatic activity. For liver enzyme activity, exogenous NAD is required and NADH is a potent inhibitor of the enzyme. Any process disturbing the NAD/ NADH ratio, such as ethanol metabolism which generates NADH, will impair galactose utilization. Cellular levels of UDP-glucose and other uridine nucleotides may also exert rate-regulating effects. Cells not exposed to free galactose form the sugar from glucose in adequate amounts to satisfy normal growth and development. Epimerase activity of the intestinal mucosa increases with age, whereas human red cells have a higher activity in newborns than adults. The intestinal enzyme activity can be enhanced by feeding diets high in glucose or galactose content. Less information is available on fetal levels of UDP-galactose 4'-epimerase, but one fetus of 16 weeks' gestation had liver enzyme activity comparable with that of children and adults. In epimerase deficiency, when the amount of entering galactose is low, an elevated level of galactose 1-phosphate in red blood cells may be reduced to normal but the UDP-galactose level stays elevated. The gene for epimerase has been assigned to human chromosome 1.
Alternative Pathway: Reduction
The polyol pathway was first identified in placenta and seminal vesicles and is responsible for the fructose content of seminal fluid. Two enzymatic reactions involving aldose reductase and sorbitol dehydrogenase catalyze the conversion of glucose to fructose with sorbitol as the intermediate. In certain cells, such as renal collecting duct cells, retinal pigment epithelial cells, and renal glomerular endothelium, and under certain conditions, aldose reductase functions to produce sorbitol which acts as an intracellular osmolyte. The acyclic polyols such as sorbitol, galactitol, and mannitol are the end product of metabolism and have osmotic properties. The presence of galactitol in the urine and plasma of patients with transferase-, galactokinase-, and epimerase-deficiency galactosemia is suggestive of the importance of the reduction of galactose as an alternative pathway. However, the high Km of this enzyme indicates that reduction will occur only when galactose levels in tissues are very high.
Patients with classical galactosemia have markedly elevated levels of galactitol in plasma and urine, which remain above age-matched control levels after treatment with galactose-free diet, whereas high urinary galactose levels return to normal in all patients. Aldose reductase has been localized to the Schwann cells of peripheral nerves and to renal paptillae cells. Kinetic studies suggest that neither glucose nor galactose are preferred substrates. Only when tissue levels of galactose are much elevated would reduction be important. Aldose reductase activity of lens and other tissue is stimulated by sulfate ions and ATP and is inhibited by various keto acids, fatty acids, and ADP. Increased production of galactitol is felt to play an important role in the pathogenesis of cataracts in the infant with galactose-1-phosphate uridyltransfer-ase, galactokinase, and UDP-galactose epimerase deficiency. The toxicity of polyols in the ocular lens is probably related to their ability to act as osmotically active particles within the lens cells, which leads to accumulation of water and eventually cell dysfunction.
Cataracts are the primary manifestation of disease in untreated patients with galactokinase deficiency, who manifest accumulation of galactitol but not galactose 1-phosphate in tissues. Thus the galactose 1-phosphate and not galactitol toxicity is probably a necessary mediator in both transferase and epimer-ase deficiencies for expression of hepatic disease, renal tubular dysfunction, and increased red blood cell turnover.
Alternative Pathway: Oxidation
In the absence of galactose-1-phosphate uridyltrans-ferase activity, galactose 1-phosphate and galactose accumulate behind the block. The second alternate pathway, besides reduction of galactose to sugar alcohol, galactitol, is the oxidation of galactose to sugar acid, galactonate. Galactonate, for example, appears in the urine of transferase-deficient individuals. Galactonate can be further metabolized to xylulose, a sugar capable of further metabolism. This pathway accounts for about 50% of oxidation of galactose by galactosaemic patients. Patients with transferase-deficient galactosemia excrete galactonate in urine after galactose is administered, and galactonate has been found in the liver of a transferase-deficient subject.
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