Epimerase-Deficiency Galactosemia Is Not a Binary Condition
2005; Elsevier BV; Volume: 78; Issue: 1 Linguagem: Inglês
10.1086/498985
ISSN1537-6605
AutoresKimberly K. Openo, Jenny M. Schulz, Claudia A. Vargas, Corey S. Orton, Michael P. Epstein, Rhonda E. Schnur, Fernando Scaglia, Gerard T. Berry, Gary S. Gottesman, Can Fıçıcıoğlu, Alfred E. Slonim, Richard J. Schroer, Chunli Yu, Vanessa E. Rangel, Jennifer C. Keenan, Kerri Lamance, Judith L. Fridovich‐Keil,
Tópico(s)Metabolism, Diabetes, and Cancer
ResumoEpimerase-deficiency galactosemia results from the impairment of UDP-galactose 4′-epimerase (GALE), the third enzyme in the Leloir pathway of galactose metabolism. Originally identified as a clinically benign “peripheral” condition with enzyme impairment restricted to circulating blood cells, GALE deficiency was later demonstrated also to exist in a rare but clinically severe “generalized” form, with enzyme impairment affecting a range of tissues. Isolated cases of clinically and/or biochemically intermediate cases of epimerase deficiency have also been reported. We report here studies of 10 patients who, in the neonatal period, received the diagnosis of hemolysate epimerase deficiency. We have characterized these patients with regard to three parameters: (1) GALE activity in transformed lymphoblasts, representing a “nonperipheral” tissue, (2) metabolic sensitivity of those lymphoblasts to galactose challenge in culture, and (3) evidence of normal versus abnormal galactose metabolism in the patients themselves. Our results demonstrate two important points. First, whereas some of the patients studied exhibited near-normal levels of GALE activity in lymphoblasts, consistent with a diagnosis of peripheral epimerase deficiency, many did not. We detected a spectrum of GALE activity levels ranging from 15%–64% of control levels, demonstrating that epimerase deficiency is not a binary condition; it is a continuum disorder. Second, lymphoblasts demonstrating the most severe reduction in GALE activity also demonstrated abnormal metabolite levels in the presence of external galactose and, in some cases, also in the absence of galactose. These abnormalities included elevated galactose-1P, elevated UDP-galactose, and deficient UDP-glucose. Moreover, some of the patients themselves also demonstrated metabolic abnormalities, both on and off galactose-restricted diet. Long-term follow-up studies of these and other patients will be required to elucidate the clinical significance of these biochemical abnormalities and the potential impact of dietary intervention on outcome. Epimerase-deficiency galactosemia results from the impairment of UDP-galactose 4′-epimerase (GALE), the third enzyme in the Leloir pathway of galactose metabolism. Originally identified as a clinically benign “peripheral” condition with enzyme impairment restricted to circulating blood cells, GALE deficiency was later demonstrated also to exist in a rare but clinically severe “generalized” form, with enzyme impairment affecting a range of tissues. Isolated cases of clinically and/or biochemically intermediate cases of epimerase deficiency have also been reported. We report here studies of 10 patients who, in the neonatal period, received the diagnosis of hemolysate epimerase deficiency. We have characterized these patients with regard to three parameters: (1) GALE activity in transformed lymphoblasts, representing a “nonperipheral” tissue, (2) metabolic sensitivity of those lymphoblasts to galactose challenge in culture, and (3) evidence of normal versus abnormal galactose metabolism in the patients themselves. Our results demonstrate two important points. First, whereas some of the patients studied exhibited near-normal levels of GALE activity in lymphoblasts, consistent with a diagnosis of peripheral epimerase deficiency, many did not. We detected a spectrum of GALE activity levels ranging from 15%–64% of control levels, demonstrating that epimerase deficiency is not a binary condition; it is a continuum disorder. Second, lymphoblasts demonstrating the most severe reduction in GALE activity also demonstrated abnormal metabolite levels in the presence of external galactose and, in some cases, also in the absence of galactose. These abnormalities included elevated galactose-1P, elevated UDP-galactose, and deficient UDP-glucose. Moreover, some of the patients themselves also demonstrated metabolic abnormalities, both on and off galactose-restricted diet. Long-term follow-up studies of these and other patients will be required to elucidate the clinical significance of these biochemical abnormalities and the potential impact of dietary intervention on outcome. In both prokaryotes and eukaryotes, galactose is metabolized predominantly via the Leloir pathway, a series of reactions catalyzed by the enzymes galactokinase (GALK [EC 2.7.1.6]), galactose-1-phosphate uridylyltransferase (GALT [EC 2.7.7.12]), and UDP-galactose 4′-epimerase (GALE [EC 5.1.3.2]) (fig. 1) (Holton et al. Holton et al., 2000Holton JB Walter JH Tyfield LA Galactosaemia.in: Scriver CR Beaudet AL Sly SW Valle D Childs B Kinzler KW Vogelstein B Metabolic and molecular bases of inherited disease. McGraw-Hill, New York2000: 1553-1587Google Scholar). Impairment of any of these three enzymes in humans results in galactosemia, although the symptoms and severity depend on the identity of the enzyme impaired and the extent of the deficiency, among other factors. The first clinically recognized form of galactosemia was profound transferase deficiency (MIM 230400), reported by Goppert (Goppert, 1917Goppert Galaktosurie nach Milchzuckergabe bei angeborenem, familiaerem chronischem Leberleiden.Klin Wschr. 1917; 54: 473-477Google Scholar). This so-called classic galactosemia occurs with a frequency of 1/30,000 to 1/60,000 live births in the United States and in other multiethnic populations, and it is panethnic. If untreated, classic galactosemia can lead to rapid neonatal demise. Symptoms progress from vomiting and diarrhea to cataracts and failure to thrive to hepatomegaly, liver dysfunction with bleeding diathesis, and Escherichia coli sepsis, culminating in neonatal death (Holton et al. Holton et al., 2000Holton JB Walter JH Tyfield LA Galactosaemia.in: Scriver CR Beaudet AL Sly SW Valle D Childs B Kinzler KW Vogelstein B Metabolic and molecular bases of inherited disease. McGraw-Hill, New York2000: 1553-1587Google Scholar). Classic galactosemia is often detected presymptomatically in most industrialized nations by newborn screening and is treated by lifelong dietary restriction of galactose. Although this intervention resolves the acute and potentially lethal symptoms of classic galactosemia, unfortunately, a spectrum of complications persist, including ataxia, learning disabilities, and verbal dyspraxia in >30% of patients and primary ovarian failure in >80% of females (Waggoner et al. Waggoner et al., 1990Waggoner DD Buist NRM Donnell GN Long-term prognosis in galactosemia: results of a survey of 350 cases.J Inher Metab Dis. 1990; 13: 802-818Crossref PubMed Scopus (429) Google Scholar). The pathophysiology of classic galactosemia remains unclear, hindering the development of more-effective therapies. The second form of galactosemia to be recognized clinically was GALK deficiency (MIM 230200), first reported by Gitzelmann (Gitzelmann, 1967Gitzelmann R Hereditary galactokinase deficiency, a newly recognized cause of juvenile cataracts.Pediatr Res. 1967; 1: 14-23Crossref Scopus (102) Google Scholar). Patients with GALK deficiency demonstrate none of the potentially lethal developmental, cognitive, or ovarian complications seen in classic galactosemia but do present with neonatal cataracts, which generally self-resolve upon dietary restriction of galactose. On the basis of biochemical ascertainment of carriers in a select white population, GALK deficiency was estimated to affect 1/40,000 to 1/50,000 newborn infants (Mayes and Guthrie Mayes and Guthrie, 1968Mayes J Guthrie R Detection of heterozygotes for galactokinase deficiency in a human population.Biochem Genet. 1968; 2: 219-230Crossref PubMed Scopus (39) Google Scholar). One large population study, however, indicated that the frequency of detectable GALK deficiency at birth is significantly <1/100,000 (Levy Levy, 1980Levy H Screening for galactosemia.in: Burman D Holton JB Pennock CA Inherited disorders of carbohydrate metabolism. MTP Press, Lancaster1980: 133-139Crossref Google Scholar). The third and most poorly understood form of galactosemia is epimerase deficiency (MIM 230350). First hypothesized by Kalckar (Kalckar, 1965Kalckar HM Galactose metabolism and cell “sociology”.Science. 1965; 150: 305-313Crossref PubMed Scopus (67) Google Scholar) and first reported by Gitzelmann (Gitzelmann, 1972Gitzelmann R Deficiency of uridine diphosphate galactose 4-epimerase in blood cells of an apparently healthy infant.Helv Paediat Acta. 1972; 27: 125-130PubMed Google Scholar), epimerase-deficiency galactosemia was originally described as a benign condition in which human GALE (hGALE) impairment was restricted to the circulating red blood cells (RBC) and white blood cells (Gitzelmann Gitzelmann, 1972Gitzelmann R Deficiency of uridine diphosphate galactose 4-epimerase in blood cells of an apparently healthy infant.Helv Paediat Acta. 1972; 27: 125-130PubMed Google Scholar; Gitzelmann and Steimann Gitzelmann and Steimann, 1973Gitzelmann R Steimann B Uridine diphosphate galactose 4-epimerase deficiency.Helv Paediat Acta. 1973; 28: 497-510PubMed Google Scholar). Fibroblasts, liver, phytohemagglutinin (PHA)-stimulated leukocytes, and Epstein Barr virus (EBV)–transformed lymphoblasts from these patients all demonstrated normal or near-normal levels of hGALE activity (Gitzelmann Gitzelmann, 1972Gitzelmann R Deficiency of uridine diphosphate galactose 4-epimerase in blood cells of an apparently healthy infant.Helv Paediat Acta. 1972; 27: 125-130PubMed Google Scholar; Gitzelmann and Steimann Gitzelmann and Steimann, 1973Gitzelmann R Steimann B Uridine diphosphate galactose 4-epimerase deficiency.Helv Paediat Acta. 1973; 28: 497-510PubMed Google Scholar; Mitchell et al. Mitchell et al., 1975Mitchell B Haigis E Steinmann B Gitzelmann R Reversal of UDP-galactose 4-epimerase deficiency of human leukocytes in culture.Proc Nat Acad Sci. 1975; 72: 5026-5030Crossref PubMed Scopus (20) Google Scholar; Gitzelmann et al. Gitzelmann et al., 1977Gitzelmann R Steinmann B Mitchell B Haigis E Uridine diphosphate galactose 4′-epimerase deficiency. IV. Report of eight cases in three families.Helv Paediat Acta. 1977; 31: 441-452PubMed Google Scholar), which lead to the designation of this condition as “peripheral” epimerase deficiency (Holton et al. Holton et al., 2000Holton JB Walter JH Tyfield LA Galactosaemia.in: Scriver CR Beaudet AL Sly SW Valle D Childs B Kinzler KW Vogelstein B Metabolic and molecular bases of inherited disease. McGraw-Hill, New York2000: 1553-1587Google Scholar). Soon thereafter, however, a second form of epimerase-deficiency galactosemia became apparent when Holton and colleagues (Holton et al., 1981Holton JB Gillett MG MacFaul R Young R Galactosemia: a new severe variant due to uridine diphosphate galactose-4-epimerase deficiency.Arch Dis Child. 1981; 56: 885-887Crossref PubMed Scopus (78) Google Scholar) reported a patient who, despite normal GALT activity, presented with symptoms reminiscent of classic galactosemia and demonstrated severely impaired GALE activity in both RBCs and fibroblasts. This patient, whose acute clinical symptoms responded to dietary restriction of galactose, was said to have “generalized” epimerase deficiency. Subsequent studies revealed four additional patients with generalized epimerase-deficiency galactosemia, ostensibly derived from two different families (Sardharwalla et al. Sardharwalla et al., 1988Sardharwalla IB Wraith JE Bridge C Fowler B Roberts SA A patient with severe type of epimerase deficiency galactosemia.J Inher Metab Dis. 1988; 11: 249-251Crossref PubMed Scopus (37) Google Scholar; Walter et al. Walter et al., 1999Walter JH Roberts REP Besley GTN Wraith JE Cleary MA Holton JB MacFaul R Generalised uridine diphosphate galactose-4-epimerase deficiency.Arch Dis Child. 1999; 80: 374-376Crossref PubMed Scopus (62) Google Scholar), although all five patients later proved to be homozygotes for the same mutation, V94M (Walter et al. Walter et al., 1999Walter JH Roberts REP Besley GTN Wraith JE Cleary MA Holton JB MacFaul R Generalised uridine diphosphate galactose-4-epimerase deficiency.Arch Dis Child. 1999; 80: 374-376Crossref PubMed Scopus (62) Google Scholar; Wohlers et al. Wohlers et al., 1999Wohlers TM Christacos NC Harreman MT Fridovich-Keil JL Identification and characterization of a mutation, in the human UDP galactose-4-epimerase gene, associated with generalized epimerase-deficiency galactosemia.Am J Hum Genet. 1999; 64: 462-470Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), raising the suspicion that they might share common ancestry. Together, these reports supported the conclusion that epimerase-deficiency galactosemia was a binary condition, with a benign peripheral form occurring at a population frequency of 1/6,700 to <1/60,000, depending on the racial group (Alano et al. Alano et al., 1998Alano A Almashanu S Chinsky JM Costeas P Blitzer MG Wulfsberg EA Cowan TM Molecular characterization of a unique patient with epimerase-deficiency galactosaemia.J Inher Metab Dis. 1998; 21: 341-350Crossref PubMed Scopus (44) Google Scholar), and a severe generalized form occurring at an extremely low frequency (Holton et al. Holton et al., 2000Holton JB Walter JH Tyfield LA Galactosaemia.in: Scriver CR Beaudet AL Sly SW Valle D Childs B Kinzler KW Vogelstein B Metabolic and molecular bases of inherited disease. McGraw-Hill, New York2000: 1553-1587Google Scholar). The conclusion that hGALE deficiency is a binary condition held until the 1990s, when groups in both Europe and the United States reported individual patients with partial or tissue-specific GALE impairment and who exhibited isolated clinical abnormalities. For example, Shin and colleagues (Endres and Shin Endres and Shin, 1990Endres W Shin YS Cataract and metabolic disease.J Inher Metab Dis. 1990; 13: 509-516Crossref PubMed Scopus (19) Google Scholar; Schulpis et al. Schulpis et al., 1993Schulpis KH Papakonstantinou ED Koidou A Michelakakis H Tzamouranis J Patsouras A Shin Y UDP galactose-4-epimerase deficiency in a 5.5-year-old girl with a unilateral cataract.J Inher Metab Dis. 1993; 16: 903-904Crossref PubMed Scopus (6) Google Scholar; Shin et al. Shin et al., 2000Shin YS Korenke GC Huddke P Knerr I Podskarbi T UDPgalactose epimerase in lens and fibroblasts: activity expression in patients with cataracts and mental retardation.J Inherit Metab Dis. 2000; 23: 383-386Crossref PubMed Scopus (11) Google Scholar) described patients with GALE impairment in the lens who presented with cataracts, and both Quimby et al. [Quimby et al., 1997Quimby BB Alano A Almashanu S DeSandro AM Cowan TM Fridovich-Keil JL Characterization of two mutations associated with epimerase-deficiency galactosemia using a yeast expression system for human UDP-galactose-4-epimerase.Am J Hum Genet. 1997; 61: 590-598Abstract Full Text PDF PubMed Scopus (51) Google Scholar] and Alano et al. [Alano et al., 1998Alano A Almashanu S Chinsky JM Costeas P Blitzer MG Wulfsberg EA Cowan TM Molecular characterization of a unique patient with epimerase-deficiency galactosaemia.J Inher Metab Dis. 1998; 21: 341-350Crossref PubMed Scopus (44) Google Scholar] described a patient with partial GALE deficiency who was clinically well as an infant but who gradually manifested notable developmental/cognitive delay, starting after his first year of life. This patient exhibited only ∼15% of normal levels of GALE activity in his transformed lymphoblasts (Quimby et al. Quimby et al., 1997Quimby BB Alano A Almashanu S DeSandro AM Cowan TM Fridovich-Keil JL Characterization of two mutations associated with epimerase-deficiency galactosemia using a yeast expression system for human UDP-galactose-4-epimerase.Am J Hum Genet. 1997; 61: 590-598Abstract Full Text PDF PubMed Scopus (51) Google Scholar). It is important to point out that, although lymphoblasts are clearly derived from peripheral cells, with regard to GALE activity they more closely resemble nonperipheral cells (Mitchell et al. Mitchell et al., 1975Mitchell B Haigis E Steinmann B Gitzelmann R Reversal of UDP-galactose 4-epimerase deficiency of human leukocytes in culture.Proc Nat Acad Sci. 1975; 72: 5026-5030Crossref PubMed Scopus (20) Google Scholar) and, as such, offer a convenient tool for distinguishing peripheral from nonperipheral forms of epimerase-deficiency galactosemia. While these cases clearly raised concern that epimerase deficiency might not be a binary condition, the broader clinical significance of these isolated findings remained unclear. We report here the biochemical and genetic studies of a cohort of 10 additional and unrelated epimerase-deficient patients accrued over a 4-year period and characterized, relative to controls, with regard to epimerase activity in transformed lymphoblasts as well as metabolic sensitivity of those lymphoblasts to galactose exposure in culture. Using these samples, we asked two fundamental questions: (1) Do all infants/children who receive the diagnosis of hemolysate epimerase deficiency exhibit normal GALE activity in their nonperipheral tissues, as represented by transformed lymphoblasts, and (2) is there any abnormal metabolic consequence to partial impairment of hGALE activity in patients or patient lymphoblasts exposed to environmental galactose? The answer to the first question was “no.” Our data clearly demonstrated that, at least in biochemical terms, epimerase deficiency is not binary; it is a continuum disorder. We observed GALE activity levels in lymphoblasts from patients that ranged from a low of ∼15% of control levels to a high of 64% of control levels. The answer to the second question was “yes.” Our results demonstrated that lymphoblasts with as much as 35% residual GALE activity nonetheless exhibited metabolic abnormalities upon exposure to environmental galactose. Furthermore, some of the patients from whom those lymphoblasts were derived also demonstrated abnormal galactose metabolites. The relationship between these metabolic abnormalities and the possibility of clinical abnormalities later in life remains unclear. Long-term clinical follow-up studies of significantly larger numbers of patients with partial GALE deficiency will be required to address this question. All patients were ascertained by referral, and we obtained their consent in accordance with Emory University Institutional Review Board Protocol 618–99 (primary investigator: Fridovich-Keil). Controls were ascertained as anonymous blood samples from nongalactosemic individuals. All hemolysate biochemical data were generated in clinical labs, as noted, and all clinical follow-up data were reported by referring health care professionals. EBV-transformed lymphoblasts were prepared and cultured from patient or control blood samples as described elsewhere (Neitzel Neitzel, 1986Neitzel H A routine method for the establishment of permanent growing lymphoblastoid cell lines.Hum Genet. 1986; 73: 320-326Crossref PubMed Scopus (532) Google Scholar), with a substitution of interleukin-2 in place of cyclosporine. Transformed lymphoblasts were maintained in RPMI 1640 medium containing 11.1 mM glucose (2 g/liter) and 0.3 g/liter l-glutamine and were supplemented with penicillin (100 U/ml), streptomycin (100 μg/ml), 25 mM HEPES (Cellgro), and 10% (v/v) fetal bovine serum (Gibco). All cells were maintained at 37°C in a humidified 5% CO2 incubator (NuAire). Soluble protein lysates were prepared from 0.15 g transformed lymphoblast cell pellets as follows. Cells were harvested by centrifugation, were washed with 1× PBS to remove residual medium, and were resuspended in 300 μl of 100-mM glycine buffer (pH 8.7) containing protease inhibitor cocktail (Roche). Cells were disrupted on ice by three 15-s exposures to a Misonix Sonicator 3000 with microtip at output level 1.0. Insoluble debris was removed from each sample by centrifugation in an Eppendorf 5415D at 13.2 rpm at 4°C, and the supernatant was passed over a P-30 Bio-Spin Column (Bio-Rad) to remove small molecules before further analysis. Protein concentrations were determined using the Bio-Rad protein assay reagent with BSA as the standard, as recommended by the manufacturer. Both GALT and GALE activities were measured in samples of each cell lysate by use of enzymatic assay procedures, essentially as described elsewhere (Ross et al. Ross et al., 2004Ross KL Davis CN Fridovich-Keil JL Differential roles of the Leloir pathway enzymes and metabolites in defining galactose sensitivity in yeast.Mol Genet Metab. 2004; 83: 103-116Crossref PubMed Scopus (61) Google Scholar; Schulz et al. Schulz et al., 2005Schulz J Ross K Malmstrom K Krieger M Fridovich-Keil J Mediators of galactose sensitivity in UDP-galactose 4′-epimerase-impaired mammalian cells.J Biol Chem. 2005; 280: 13493-13502Crossref PubMed Scopus (23) Google Scholar), with separation and quantification of reactants and products achieved by high-performance liquid chromatography (HPLC). GALE assays were performed at 37°C with the use of both UDP-galactose (UDP-Gal) and UDP-N-acetylgalactosamine (UDP-GalNAc) substrates, with a 30-min incubation time and 7.5–30 μg total protein in a final volume of 12.5 μl. UDP-Gal GALE activity assays contained 40 mM glycine, 0.4 mM UDP-Gal, and 8 mM NAD+. UDP-GalNAc GALE activity assays contained 40 mM glycine, 0.4 mM UDP-GalNAc, and 8 mM NAD+. GALT assays were performed at 37°C for 60 min with the use of 50–100 μg total protein in 125 mM glycylglycine (pH 7.5) and 4.1 mM UDP-glucose (UDP-Glc) in a final volume of 50 μl. GALT activity assays for each sample were performed in both the presence and absence of 1 mM galactose 1-phosphate (Gal-1P) to reveal background production of UDP-Gal (from UDP-Glc) by epimerase. To determine GALT activity, formation of UDP-Gal in the absence of Gal-1P was subtracted from the amount of UDP-Gal produced in the presence of Gal-1P. All reactions were initiated by the addition of protein and were monitored to ensure linearity. GALE and GALT reactions were stopped by the addition of 237.5 μl or 450 μl, respectively, of chilled sterile water and were then filtered (0.2−μm nylon filters [Alltech]) and subjected to HPLC separation and quantification of substrates and products. Enzyme activity was defined by picomole of product formed per microgram of protein per minute, under the conditions described. HPLC separation and quantification of enzymatic substrates and products was achieved as described elsewhere (Ross et al. Ross et al., 2004Ross KL Davis CN Fridovich-Keil JL Differential roles of the Leloir pathway enzymes and metabolites in defining galactose sensitivity in yeast.Mol Genet Metab. 2004; 83: 103-116Crossref PubMed Scopus (61) Google Scholar; Schulz et al. Schulz et al., 2005Schulz J Ross K Malmstrom K Krieger M Fridovich-Keil J Mediators of galactose sensitivity in UDP-galactose 4′-epimerase-impaired mammalian cells.J Biol Chem. 2005; 280: 13493-13502Crossref PubMed Scopus (23) Google Scholar). Cells were cultured in the absence of galactose, as described above, until time zero, at which point (t=0) the samples were harvested for both external and internal metabolites, and galactose was added to the remainder of the culture for a final concentration of 0.5 mM. Cultures were harvested 24 h after galactose addition. At each time point, 50 μl of cell suspension was removed for protein determination by use of the BioRad DC system, as recommended by the manufacturer. To monitor external galactose, 250 μl of culture medium was added directly into 500 μl 60% MeOH at −20°C, was mixed, and was centrifuged briefly at 13,200 rpm at 4°C in an Eppendorf 5415C microcentrifuge to remove cellular debris. Then, 375 μl of supernatant was dried under vacuum without heat, was rehydrated in 390 μl sterile Milli-Q water, and was filtered before HPLC fractionation. Internal metabolites were extracted from 10-ml samples of cell suspension harvested by centrifugation at 3,500 rpm in Sorvall RT6000B at room temperature and were washed once with PBS before further manipulation. Samples were prepared using a modified form of the procedure described elsewhere by our laboratory (Ross et al. Ross et al., 2004Ross KL Davis CN Fridovich-Keil JL Differential roles of the Leloir pathway enzymes and metabolites in defining galactose sensitivity in yeast.Mol Genet Metab. 2004; 83: 103-116Crossref PubMed Scopus (61) Google Scholar; Schulz et al. Schulz et al., 2005Schulz J Ross K Malmstrom K Krieger M Fridovich-Keil J Mediators of galactose sensitivity in UDP-galactose 4′-epimerase-impaired mammalian cells.J Biol Chem. 2005; 280: 13493-13502Crossref PubMed Scopus (23) Google Scholar) and originally described by Smits et. al (Smits et al., 1998Smits HP Cohen A Buttler T Nielsen J Olsson L Cleanup and analysis of sugar phosphates in biological extracts by using solid-phase extraction and anion-exchange chromatography with pulsed amperometric detection.Anal Biochem. 1998; 261: 36-42Crossref PubMed Scopus (70) Google Scholar). In brief, each washed cell pellet was resuspended in 1.1 ml cold PBS, of which 100 μl were used for protein determination via the Bio-Rad DC protein assay, as recommended by the manufacturer. Cells from the remaining 1 ml of suspension were collected by high-speed centrifugation at 4°C in an Eppendorf 5415C microcentrifuge. Intracellular metabolites were extracted by vigorous agitation of the cells for 45 min at 4°C in a final volume of an 875−μl 4:2:1 mixture of CHCl3/MeOH/water. The aqueous layer was collected after repeated high-speed centrifugation for 10 min at 4°C. The remaining organic phase was back-extracted a second time with 125 μl MeOH and 125 μl water. Aqueous layers were combined and dried under vacuum without heat (for ∼6 h). Each dried metabolite pellet was rehydrated with a volume of sterile Milli-Q water calculated from the protein level measured for that cell suspension, with the use of 1 μl of water for every 5 μg of protein detected. Rehydrated samples were filtered through 0.2−μm nylon filters (Alltech) before being loaded into the HPLC autosampler. HPLC analysis was performed using a DX600 or ISC2500 HPLC system (Dionex), each consisting of a Dionex AS50 autosampler, a Dionex GP50 gradient pump, and a Dionex ED50 electrochemical detector. As described elsewhere, carbohydrates were separated on a CarboPac PA10 column (250 × 4 mm) with an amino-trap (50 × 14 mm) placed before the analysis column and a borate-trap (50 × 4 mm) placed before the injector port to remove trace amounts of borate from the mobile phase buffers (Ross et al. Ross et al., 2004Ross KL Davis CN Fridovich-Keil JL Differential roles of the Leloir pathway enzymes and metabolites in defining galactose sensitivity in yeast.Mol Genet Metab. 2004; 83: 103-116Crossref PubMed Scopus (61) Google Scholar; Schulz et al. Schulz et al., 2005Schulz J Ross K Malmstrom K Krieger M Fridovich-Keil J Mediators of galactose sensitivity in UDP-galactose 4′-epimerase-impaired mammalian cells.J Biol Chem. 2005; 280: 13493-13502Crossref PubMed Scopus (23) Google Scholar). For all samples, 20 μl were injected into a 25−μl injection loop. Samples were maintained at 4°C in the autosampler tray, and the chromatography was performed at room temperature. Mobile phase buffers for the separation of carbohydrates were used as described above. Flow rate was maintained at 0.8–1.0 ml/min. To prevent carbonate contamination of the analysis column, a 50% sodium hydroxide solution containing <0.04% sodium carbonate was used to prepare mobile phase buffers (Fisher). Buffers were degassed and maintained under a helium atmosphere. All carbohydrate metabolites were separated as described elsewhere (Ross et al. Ross et al., 2004Ross KL Davis CN Fridovich-Keil JL Differential roles of the Leloir pathway enzymes and metabolites in defining galactose sensitivity in yeast.Mol Genet Metab. 2004; 83: 103-116Crossref PubMed Scopus (61) Google Scholar; Schulz et al. Schulz et al., 2005Schulz J Ross K Malmstrom K Krieger M Fridovich-Keil J Mediators of galactose sensitivity in UDP-galactose 4′-epimerase-impaired mammalian cells.J Biol Chem. 2005; 280: 13493-13502Crossref PubMed Scopus (23) Google Scholar). In brief, hexoses and hexose-phosphates were separated using buffers A (15 mM NaOH) and B (50 mM NaOH/1 M NaAC) mixed using a low-salt gradient procedure with flow rate of 1 ml/min. The sample injection occurred at t=0. Gradient 1: 98% A and 2% B (−10 to 8 min), a linear increase of B to 30% (8–15 min), a linear increase of B to 50% (15–25 min), hold 50% A and 50% B (25–30 min), and a linear decrease of B to 2% (30–35 min). External galactose was determined using a low-salt gradient procedure with a flow rate of 1 ml/min. Gradient 2: 98% B and 2% B (−5 to 5 min), a linear increase of B to 50% (5–10 min), hold 50% A and 50% B (10–15 min), and a linear decrease of B to 2% (15–17 min). UDP-hexoses were separated using a high-salt gradient procedure with a flow rate of 0.8 ml/min. Gradient 3: 50% A and 50% B for (−5 to 1 min), a linear increase of B to 70% (1–22 min), hold 30% A and 70% B (22–27 min), and a linear decrease of B to 50%. Carbohydrates were detected and were quantified as described elsewhere (Ross et al. Ross et al., 2004Ross KL Davis CN Fridovich-Keil JL Differential roles of the Leloir pathway enzymes and metabolites in defining galactose sensitivity in yeast.Mol Genet Metab. 2004; 83: 103-116Crossref PubMed Scopus (61) Google Scholar; Schulz et al. Schulz et al., 2005Schulz J Ross K Malmstrom K Krieger M Fridovich-Keil J Mediators of galactose sensitivity in UDP-galactose 4′-epimerase-impaired mammalian cells.J Biol Chem. 2005; 280: 13493-13502Crossref PubMed Scopus (23) Google Scholar). Genomic DNA was isolated from lymphoblast cells, as described elsewhere, by use of phenol/chloroform extraction (Davis et al. Davis et al., 1994Davis L Kuehl M Battey J Basic methods in molecular biology. 2nd
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