Pediat. Res. 3: 279-286 (1969) Erythrocytes galactose- 1-phosphate galactose uridine diphosphate galactose galactosemia
Formation of Galactose-1-Phosphate from Uridine Diphosphate Galactose in Erythrocytes from Patients with Galactosemia'"'
RICHARDGITZELMANN[~~'
Laboratory for Metabolic Research, University Pediatric Department, Kinderspital Zurich, Switzerland
Extract
Erythrocyte lysates of galactosemic patients were found to form galactose-1-phosphate from uridine diphosphate galactose. The enzymatic reaction most likely responsible for this conversion is uridine diphosphate galactose pyrophosphorylase, an enzyme believed to be absent from human erythrocytes, but other reactions might be involved.
Speculation
Galactose-1-phosphate concentration in red cells of galactosemic infants may prove useless as an indicator of galactose uptake from foodstuffs.
Introduction red cells were incubated for two days in a glucose- containing galactose-free medium, and disappear- In 1966, a galactosemic baby (R.W.) was admitted to ance of galactose-1-P was followed. For comparison, this hospital on the third day of life. A galactose- erythrocytes of the patient's 20-month-old galacto- restricted diet had been prescribed for the mother dur- semic sister (B.W.) were exposed in uitro to galactose ing the last six months of pregnancy. In the baby's cord and, after galactose-1-P accumulation, were incu- blood, no galactose-1-phosphate uridyltransferase was bated in the same manner. The red cells of both detectable; the amount of galactose-1-P was 8 mg/100 children behaved in a strikingly similar way (fig. 2). ml erythrocytes. The baby was given a soybean for- When the medium was renewed at 2 to 3-hour inter- mula thought to be free of galactose. Unexpectedly, vals, galactose-1-P content dropped rapidly; however, at age 10 days, red cell galactose-1-P had risen to 22 when the medium was not changed during 14-hour mg/100 ml and continued to oscillate between 9 and intervals and the glucose content was depleted, the 21 mg/100 ml during the next 8 weeks. HARRISand galactose-1-P content remained constant in the first ISBELL[ll] have reported a similar case. Galactose glucose 'starvation' period. It rose slightly in the second contamination of the soybean formula was never ruled such period, although the medium contained no galac- out, and the presence of galactose-1-P could have tose. stemmed from galactose via the galactokinase reaction The possible formation of galactosc-1-P from uridine (fig. 1). Formation of red cell galactose-1-P from pre- diphosphate galactose in galactosemic red cells was cursors other than galactose was also considered. also investigated. Synthesis of the phosphate ester The ability of the red cells of the baby to dispose of would necessarily involve a reaction or reactions other accumulated galactose-I-P was investigated. Washed than that catalyzed by galactose-1-phosphate uridyl- 280 GITZELMANNFormation
transferase, since this enzyme was absent in the pa- Galactose tients. Experimental conditions were especially design- ed to test the occurrence of the UDP-galactose pyro- 1p2; phosphorylase reaction [l, 121. The results of these investigations are presented in this study. Galactose-1 -P
Materials and Methods
Fifteen hemolysates were obtained from five typical galactosemic patients older than 9 months of age. Re- peated assays [2, 51 of these patients revealed no de- tectable erythrocyte galactose-1-phosphate uridyl- transferase activity, and approximately 50 % activity UTP was found in their parents. Erythrocytes were washed three times with cold 0.154 M NaCl, packed, lysed by freezing, and used immediately or within 5 weeks Fig.1. Some reactions of intermediary galactose me- of storage at -20'. Sources of the chemicals and tabolism, catalized by: 1. galactokinase, 2. galactose- enzymes used are listed under 'References and Notes' 1-phosphate uridyltransferase (this reaction is blocked [27]. Descending paper chromatography of hexoses in galactosemia), 3. UDP-glucose 4-epimerase, 4. and hexose phosphates was performed with the follow- UDP-glucose pyrophosphorylase, 5. UDP-galactose ing solvents: n-propanollconcentrated ammonia/ pyrophosphorylase. water, 60/30/10 by volume [lo], solvent A; tert- butanol/O.l N hydrochloric acid, 80120 v/v [24], Abbreviations : solvent B; and ethyl acetate/pyridine/water, 10/4/3 by Galactose- 1-P a-D-galactose-1 -phosphate volume, solvent C. Sugars were visualized with aniline Glucose-1 -P a-D-glucose-1 -phosphate phthalate [19], with ammonium molybdate reagent UDP-galactose uridine diphosphate galactose [lo], or by autoradiography. Gas liquid chromato- UDP-glucose uridine diphosphate glucose graphy of permethyl-silylated sugars was performed as ATP adenosine triphosphate described previously [8]. ADP adenosine diphosphate Erythrocyte galactose-1-P was estimated enzymati- UTP uridine triphosphate cally [14]. Galactose was measured according to the UDP uridine diphosphate method of ROTHet al. [21], using 0.04 M tris buffer at PPi inorganic pyrophosphate pH 8.1. Inorganic and total phosphates were deter- mined by using the method of BAGINSKIet al. [3]. De- ionization was carried out by passing probes through glass columns filled with alternating layers of Amber- lite resin IR-45-AG (-OH)- and IR-120-AG (-H) +
Fig. 2. Galactose-I-P levels in erythrocytes of galactosemic siblings R. W. (m) and B. W. (e) during in uitro incubation in the presence of glucose as the sole carbohydrate. Erythrocytes were washed 3 times with cold saline, suspended in 3 volumes of Krebs-Ringer bicarbonate buffer, pH 7.4, containing 100 mg glucose/100 ml. The suspensions were transfered into Erlenmeyer flasks which were continually gassedwith humidified CO,/O, (95 %/5 %) and gently moved in a metabolic shaker at 37 "C. At indicated intervals (A), the buffer was renew- ed. Erythrocyte aliquots were withdrawn for enzy- matic determination of galactose-1-P, and incubation continued with the remaining red cells in renewed medium. Glucose in the supernatant medium was within a 60-100 mg1100 ml range after the shorter Time Ih) intervals and 0-15 mg/100 ml after the longer intervals. from uridine diphosphate galactose in erythrocytes from patients with galactosemia 281
[28] or with Retardion 1lA8, 50-100 mesh [29], as standard conditions, a newly formed radioactive inter- suggested by FENTON[6]. mediate thought to be galactose-1-P was observed and characterized as follows. In Vitro Incubation of Hemolysates with UDP-Galactose Deproteinized reaction mixtures were subjected to In order to deplete NAD and thus prevent epimerase ascending paper chromatography (solvent D), and reaction [14], hemolysates were preincubated at 37' sections containing the presumed galactose-1-P were for 10 minutes. Fourteen different incubations were eluted withH,O in the cold. Eluates were concentrated carried out using UDP-~-galactose-~~Cand PP; as in vacuo and served as the substrate in the modified substrates. When the reverse reaction was tested, either Warburg procedure [26], using as the enzyme source UTP (one experiment) or galactose-1-P (three experi- whole blood lysates of the following: healthy controls, ments) was 14C-labeled. Typical reaction mixtures a galactokinase-deficient man [7], and several galacto- contained UDP-galactose-14C, 10 pc/0.55 pmoles; PP;, semic patients. Production of 14C0,was significant in 20 pmoles; MgCl,, 10 pmoles; tris buffer pH 7.5, 60 the vials containing blood of healthy controls and of the pmoles; and 0.2 ml of hemolysate containing a small galactokinase-deficient man, but was approximately amount (0.03-0.05 pmoles) of galactose-1-P, if any, 10 times smaller in vials containing blood of galactose- in one ml. The reaction was started by addition of the mic patients, that is, of individuals with expected in- hemolysate. To terminate the reaction, tubes were ability to metabolize galactose-1-P. placed in a boiling water bath for 90 seconds and then Similarly prepared eluates containing negligible kept in ice. Supernatants were obtained by centrifu- amounts of UV-absorbing material (at 260 nm) were gation, and the reaction products were separated by subjected to paper chromatography analysis, using chromatography on either paper or ion exchange co- solvents A or B. Radioactivity co-chromatographed lumns or both. with added labeled galactose-1-P, but not with galac- Ascending chromatography was performed on tose-6-P or 1,6-diphosphate. Whatman No.4 paper strips, prewashed with the sol- When initial reaction products were separated by vent, and developed with 7.5 volumes of 96 % ethanol column chromatography on Dowex (fig.3), fractions plus 3 volumes of 1 M ammonium acetate, pH 3.8, containing the presumed galactose-1-P were pooled, solvent D [18] for a distance of 22 cm from the starting concentrated to a few ml in a rotary evaporator at 38O, point. The strips were air dried and scanned for radio- diluted and reconcentrated several times. They were activity. Sections containing the labeled products were then subjected to a high vacuum overnight, and finally cut out and placed into vials for liquid scintillation made up to a volume of 0.5 or 1.0 ml with water. These counting [22]. Ion exchange chromatography [4] was preparations are termed concentrates. Two-dimen- performed on 1 x 17 cm columns containing Dowex 1 sional chromatograms of concentrates were developed (formate form, prepared from Dowex 1-X8 chloride, using solvents A and B. Radioactivity was limited in a 200-400 mesh) with a nonlinear gradient of zero to single spot that co-chromatographed with galactose- 0.55 N ammonium formate pH 3.67 buffer at 1 or 2 ml/ 1-P and was clearly separated from galactose-6-P and min. Fractions of 15 ml were collected. For identifica- 1,6-diphosphate; glucose-1-P, glucose-6-P, glucose- tion of radioactive products, 3 to 5 mg of UDP-galac- 1,6-diphosphate; and fructose-1-P. tose and galactose-1-P were added to the filtrates prior Concentrates were hydrolyzed for 10 minutes in to chromatography and were demonstrated in the 0.05 N HCl at 100"; aliquots were withdrawn, chilled, effluent with the anthrone reaction [15] and by UV- neutralized, and deionized by passage through Retar- absorbance. Measurements of radioactivity in the dion columns. Radioactivity was measured in the effluents were made by liquid scintillation spectro- effluents. Apparent hydrolysis of the probe proceeded metry [20], using toluene-14C internal standards for at the same rate as that of the separately treated authen- determining counting efficiencies. Reaction velocity tic galactose-1-P. At this step, deionization was omit- was calculated from the proportions of radioactivity ted, and the effect of hydrolysis was examined by deter- in the reaction products. mining inorganic phosphate. Galactose-6-P was not hydrolyzed appreciably under the same conditions. Acid hydrolysis in 0.05 N HC1 at 100" was also per- Results formed after addition of unlabeled galactose-1-P to the concentrates. Figure 4 shows a typical result. The par- Formation of Galactose-I-Pfrom UDP-Galactose by allel appearance of galactose and radioactivity indi- Galactosemic Hemolysates; Characteri
Formation of Galactose-I-P from UDP-Galactose by Galactosemic Hemolysate; Reaction Conditions Formation of galactose-1-P proceeded in the manner of an enzymatic reaction. Omission of the hemolysate prevented the reaction, but addition of hemolysate started the reaction immediately. The time course of Fraction no. such an experiment is shown in figure 5. In most experi- ments, the apparent reaction equilibrium was reached Fig. 3. Dowex 1 (formate) elution pattern of reaction within 60 to 100 minutes; at this point, addition of mixtures deproteinized after incubation of a patient's more pyrophosphate or uridine diphosphate galactose erythrocyte lysate with UDP-galactose and PPi as did not carry the reaction any farther, possibly because substrates, for 20 sec (a) and 20 min (b). 0.03 pmoles of enzyme inactivation. Apparent initial reaction rate of galactose-1-P from 0.2 ml of hemolysate were present was proportional to the amount of hemolysate present initially in one ml of standard reaction mixture. Prior in the reaction mixture (fig. 6). Pyrophosphate con- to chromatography, 5 mg of non-labeled UDP-galac- centration was critical. When PPi was excluded from tose and of galactose-1-P were added to each filtrate. the reaction mixtures, no galactose-I-P was formed, Fraction volume was 15 ml. Radioactivity was followed but an unidentified product containing 14C-labeled throughout, anthrone reaction (E,,,) and UV-absorp- galactose (5-15 % of the radioactivity present initially) tion through the radioactive regions. appeared within the first 20 seconds of incubation; this component was eluted from Dowex 1 (formate) columns immediately after galactose-1-P and had slightly higher mobility than galactose-1-P on paper Fig. 5. Formation of galactose-1-P from UDP-galactose chromatograms (solvent D). Doubling the standard by erythrocyte lysate of a galactosemic patient during PPi concentration of 2 x lo-, M reduced the activity a 4 hours incubation. One ml of reaction mixture con- to approximately 20 %, and at 0.2 x M, galactose- tained UDP-galactose-14C4 pcI0.55 /imoles, PPi 20 1-P formation was reduced to 1.5 %. When Mg+ + was pmoles, MgCI, 10 ,umoles; Tris-HC1 (pH 7.5) 60 omitted from the reaction mixture, galactose-1-P for- pmoles; and 0.2 ml lysate containing no galactose-1-P. mation was barely detectable. At pH 5.1 (0.06 M so- Reaction products were separated by paper chromato- dium acetate buffer), the reaction proceeded very graphy. from uridine diphosphate galactose in erythrocytes from patients with galactosemia 283
0 0.2 0.4 0.6 0.8 1.0 Hemolysafe concentration
Fig. 6. Formation of galactose-1-P from UDP-galactose by erythrocyte lysate of a galactosemic patient. 'En- 10 2 0 Time (min) zyme' velocity curve. Various amounts of hemolysate free of galactose-1-P in standard reaction mixture were Fig. 4. Acid hydrolysis (0.05 N HCl, 100") of reaction used. Incubations were carried out over 5 % min; product prepared by ion exchange chromatography on 4 measurements were taken at 90 sec intervals. Reac- Dowex 1 (formate) followed by 2-dimensional paper tion products were separated by ion exchange chro- chromatography. Prior to hydrolysis, non-labeled matography. galactose-1-P was added. Galactose (A) and radioac- tivity (a) were measured.
Time (min) Fig.7. Inhibition of galactose-1-P formation from UDP- galactose in galactosemic hemolysates. Standard reac- tion mixtures were employed. Initial concentrations 1 2 4 were 0.5 x 10-3 M UDP-galactose and 2.0 x M Time (h) PP;. Inhibitors were added after 5 min to a final con- Fig. 5. (Legend see page 282.) centration of 5.0 x M UDP and 5.8 x M Pi. 284 GITZELMANNFormation of galactose- 1-phosphate slowly in the presence of Mg+ +, but did not take place free medium (fig.2), it can be assumed that erythro- at all when Mg+ + was replaced by Co+ +. cytes of galactosemic patients will also, under suitable Since, at this point, it seemed likely that the in vitro conditions, form galactose-1-P from UDP-galactose production of galactose-1-P was due to pyrophosphoro- in vivo. Since UDP-glucose 4-epimerase is active from lysis of UDP-galactose, possible inhibitors [l, 251 of the early embryonic stages in man [30], UDP-galactose is UDP-galactose pyrophosphorylase reaction were test- also supplied as the precursor of such galactose-1-P. ed. Inorganic phosphate and UDP were added to In the present study, the reaction of UDP-galactose standard mixtures after the reaction had proceeded for to galactose-1-P proceeded at an initial rate of up to 5 minutes and were found to reduce markedly the rate 4.8 pmoles/h/ml erythrocytes, calculated from the ex- of galactose-1-P formation (fig.7). MgUDP and periment shown in figure 5. Several possible enzymatic MgADP were also inhibitory. When equimolar con- reactions by which galactose-1-P could arise from centrations of UDP-glucose were present at the start UDP-galactose must be considered. First, galactose-l- of the incubation, appearance of galactose-1-P from phosphate uridyltransferase action can be ruled out. UDP-galactose was reduced by 55 percent. The presence of residual transferase activity in galac- In control experiments, erythrocyte lysates of normal tosemic hemolysates had been reported by NGet al. [16] subjects incubated under identical conditionsproduced to explain formation of small amounts of UDP-hexose 14C-galactose-1-P from UDP-galactose-14C at about from galactose-1-P. If, in the experiments performed the same rate as did the hemolysates of galactosemic in the present study, reversal of the galactose-l-phos- patients. The effects of omission of PP; or Mg++ were phate uridyltransferase reaction had produced the not tested. galactose-1-phosphate, substantial transferase activity should have been found in the hemolysates. Reversal of the Reaction Direct sugar release from guanosine nucleotide has In only a few experiments, erythrocyte lysates of been described by SONNINOet al. [23] ; this novel hydro- galactosemic patients and of normal subjects were lytic reaction in yeast proceeded according to the fol- reacted with 14C-galactose-1-Pduring a one-hour peri- lowing formula : od. One ml of reaction mixture contained galactose- (1) GDP-glucose +H,O + GDP +glucose 1-P, 5 pmoles; UTP, 5 pmoles; MgCI,, 15 pmoles; tris buffer pH 7.5, 30 pmoles; and 0.25 ml of hemolysate. In the galactosemic hemolysates, UDP-galactose Reaction products were again separated by ion ex- could not have been hydrolyzed similarly, since phos- change column chromatography. Galactosemic ly- phorylation of liberated galactose would not have ex- sates produced a number of labeled intermediates, one ceeded a rate of 0.5 pmoles/h/ml red blood cells, the of which was eluted from the column in the UDP- normal rate of galactokinase reaction in red cells [7, hexose region but remained on the column somewhat 171. Furthermore, free I4C-labeled galactose would longer than did authentic UDP-galactose and over- have been detected. lapped with it. This fraction contained only a small Another type of nucleoside diphosphate sugar hydro- percent of the total radioactivity recovered. Normal lase has been characterized by GLASERet al. [9]. In hemolysates produced only traces of the same inter- some bacteria, this UDP-sugar hydrolase catalyzes the mediate. When 14C-UTPwas used as the substrate, a breakdown of UDP-glucose : similar intermediate was eluted from the column. Be- ~glucose-I-P (2) UDP-glucose-tI3 + UDP-glucose-E cause of the small amount of this labeled intermediate, \ E-UMP no attempts at purification and analysis were made. Thus, reversal of the reaction was not demonstrated Such a reaction has not been described in mammals, convincingly. but it could have occurred in the experiments described above. The fact that at pH 5.1, formation of galactose- 1-P was minimal and that at this pH, Co++ could not Discussion replace Mg+ + as it does in the bacterial enzyme, does not preclude this possibility since a mammalian en- As had been suspected, hemolysates of galactosemic zyme could have different properties. patients, that is, individuals lacking appreciable ga- Still, theUDP-galactosepyrophosphorylasereaction: lactose-l-phosphate uridyltransferase activity, proved capable of forming galactose-1-P from UDP-galactose in vitro. The identity of the labeled reaction product would provide the most likely explanation for the for- with galactose-1-P was established. In view of the mation of galactose-1-P from UDP-galactose in galac- ready formation of this phosphate ester by hemolysates tosemic hemolysates as reported here. There was pyro- and of the observed rise of galactose-1-P in galactose- phosphate dependence as well as a Mg++ require- from uridine diphosphate galactose in erythrocytes from patients with galactosemia 285 ment; UDP, Pi and UDP-glucose were inhibitory; 9. GLASER,L.; MELO,A. and PAUL,R. : Uridine di- at pH 5.1, the reaction proceeded very slowly. phosphate sugar hydrolase. Purification of enzyme In human red cells, UDP-glucose pyrophosphory- and protein inhibitor. J. biol. Chem. 242: 1944 lase is active [2], but UDP-galactose pyrophosphory- (1967). lase has been believed to be absent [13]. Recently, 10. HANES,C. S. and ISHERWOOD,F. A. : Separation of however, evidence for the common identity of these the phosphoric esters on the filter paper chromato- enzymes, at least in calf liver, has been presented [25]. gram. Nature, Lond. 164: 1107 (1949). In view of this new evidence and in order to adequately 11. HARRIS,R. C. and ISBELL,S.V. : Galactose-l- explain the results of the present study, reexamination phosphate accumulation in red cells of a neonate. of human red cells for the presence of UDP-galactose Abstr. Soc. Pediat. Res.; Pediat. Res. 1: 200 (1967). pyrophosphorylase is necessary. 12. ISSELBACHER,K. J. : A mammalian uridinediphos- Elevated levels of erythrocyte galactose-1-P in treat- phate galactose pyrophosphorylase. J. biol. Chem. ed galactosemic patients have been believed to indicate 232: 429 (1958). that galactose has been taken up from foodstuffs. It 13. ISSELBACHER,K.J. : Galactosemia; in : The meta- now appears that galactose-1-P in erythrocytes may bolic basis of inherited disease (ed. STANBURY,J. B. ; have an endogenous origin. The possibility cannot be WYNGAARDEN,J. B. and FREDRICKSON,D. S.), p. 178 excluded that galactose deprivation in the mother (McGraw-Hill, New York 1966). during pregnancy may create in the mother and the 14. KIRKMAN,H. N. and MAXWELL,E. S. : Enzymatic susceptible infant a peculiar metabolic situation mani- estimation of erythrocytic galactose-1-phosphate. fest after birth as a high rate of UDP-hexose to galac- J.Lab. clin. Med. 56: 161 (1960). tose-1-P conversion in the red cells of the newborn. 15. MORRIS,D. L. : Quantitative determination of carbohydrates with Dreywood's anthrone reagent. Science 107: 254 (1948). References and Notes 16. NG, W. G.; BERGREN,W. R. and DONNELL,G. N. : Galactose-1-phosphate uridyl transferase activity 1. ALBRECHT,G. J. ; BASS,S.T. ; SEIFERT,L. L. and in galactosemia. Nature, Lond. 203: 845 (1964). HANSEN,R. G. : Crystallization and properties of 17. NG, W. G.; DONNELL,G. N. and BERGREN,W. R. : uridine diphosphate glucose pyrophosphorylase Galactokinase activity in human erythrocytes of from liver. J. biol. Chem. 241: 2968 (1966). individuals at different ages. J. Lab. clin. Med. 66: 2. ANDERSON,E. P. ; KALCKAR,H.M. ; KURAHASHI,K. 115 (1965). and ISSELBACHER,K. J. : A specific enzymatic assay 18. PALADINI,A. C. and LELOIR,L. F. : Studies on uri- for the diagnosis of congenital galactosemia. J.Lab. dine-diphosphate-glucose. Biochem. J. 51: 426 clin. Med. 50: 469 (1957). (1952). 3. BAGINSKI,E. S.; FoA, P. P. and ZAK,B. : Micro- 19. PARTRIDGE,S. M. : Aniline hydrogen phthalate as determination of inorganic phosphate, phospho- a spraying reagent for chromatography of sugars. lipids, and total phosphate in biologic materials. Nature, Lond. 164: 443 (1949). Clin. Chem. 13: 326 (1967). 20. PATTERSON,M. S. and GREENE,R. C. : Measure- 4. BARTLETT,G. R. : Methods for the isolation of gly- ment of low energy beta-emitters in aqueous solu- colytic intermediates by column chromatography tion by liquid scintillation counting of emulsions. with ion exchange resins. J. biol.Chem. 234: 459 Anal. Chem. 37: 854 (1965). (1959). 2 1. ROTH,H.; SEGAL,S. and BERTOLI,D. : The quan- 5. BEUTLER,E. and BALUDA,M. C. : Improved meth- titative determination of galactose. An enzymic od for measuring galactose-1-phosphate uridyl method using galactose oxidase, with applications transferase activity of erythrocytes. Clin. chim. to blood and other biological fluids. Anal. Biochem. Acta 13: 369 (1966). 10: 32 (1965). 6. FENTON,J. C. B. : The estimation of galactose-l- 22. SHERMAN,J.R.: Rapid enzyme assay technique phosphate in red cells. Clin. chim. Acta 11: 205 utilizing radioactive substrate, ion-exchange paper, (1965). and liquid scintillation counting. Anal.Biochem. 7. GITZELMANN,R. : Hereditary galactokinase defi- 5: 548 (1963). ciency, a newly recognized cause of juvenile cata- 23. SONNINO,S.; CARMINATTI,H. and CABIB,E. : Direct racts. Pediat.Res. 1: 14 (1967). release of sugar from a sugar nucleotide. A novel 8. GITZELMANN,R. ; CURTIUS,H. C. and MULLER,M. : enzymatic reaction. J. biol. Chem. 241: 1009 (1966). Galactitol in the urine of a galactokinase deficient 24. STEINITZ,K. : Detection and identification or fruc- man. Biochem. biophys. Res. Commun. 22: 437 tose 1-phosphate by paper chromatography. Anal. (1966). Biochem. 2: 497 (1961). 25. TING,W. K. and HANSEN,R. G. : Uridine diphos- calf intestinal alkaline phosphatase, Boehringer, phate galactose pyrophosphorylase from calf ljver. Mannheim, Germany. Proc. Soc. exp. Biol. N.Y. 127: 960 (1968). 28. British Drug Houses, Poole, England. 26. WEINBERG,A. N. : Detection of congenital galac- 29. Biorad, Los Angeles, Cal. tosemia and the carrier state using galactose C14 30. Unpublished observation. and blood cells. Metabolism 10: 728 (1961). 3 1. Supported in part by Julius Klaus-Stiftung, Zurich, 27. UDP-~-galactose-~~C(D-galactose-14C, u.1.) and and by Hartmann Muller Stiftungfur medizinische Uridine-14C (u.1.)- 5'triphosphate, New England Forschung, Zurich. Nuclear Corp., Boston, Mass. ; D-galactose-1- 32. I wish to thank Drs. S. SEGALand R. G.HANSEN phosphate-14C (u.l.), International Chemical and for valuable advice and comments. Nuclear Corp., City of Industry, California; a-D- 33. Requests for reprints should be addressed to: RI- galactose-1-P, UDP-galactose, UTP and tetra- CHARD GITZELMANN,M.D., Laboratory for Meta- sodium pyrophosphate, Sigma Chemical Co., St. bolic Research, University Pediatric Department, Louis, Mo. ; mold galactose oxidase, Miles Labora- Steinwiesstrasse 75, 8032 Zurich (Switzerland). tories, Elkha.rt, Ind. ; horse radish peroxidasc and