Am J Hum Genet 27:748-754, 1975
Galactose Tolerance Studies of Individuals with Reduced Galactose Pathway Activity W. J. MELLMAN,1 B. E. RAWNSLEY,2 C. W. NICHOLS,3 B. NEEDELMAN,1 M. T. MENNUTI,2 J. MALONE,2 AND T. A. TEDESCO4 INTRODUCTION
Galactokinase (GALK)* deficiency is a rare autosomal recessive disorder (GALKG/ GALKG) marked by cataracts in infancy [1]. Monteleone et al. [2] in reporting a case of GALK deficiency noted that apparent heterozygotes in the patient's family had cataracts in the third and fourth decade. Failure to find cataracts among young adults in all families where GALKG is segregating [3] does not necessarily refute this suggestion of Monteleone et al. [2]. The rarity of GALKG genes makes it highly probable that those found in a random population form a heterogeneous group of mutants. In a population sample of 1,700 healthy adult females screened for variations in red cell GALK and galactose-l-phosphate uridylyltransferase (GALT) activity, we identified five women as GALKG heterozygotes [4]. They represent two of 618 white and three of 1,082 black females surveyed. Estimation of the true frequency of the GALKG gene in black populations is complicated by an apparent polymorphic allele in blacks [5] that we have called the Philadelphia variant (GALKP). This report presents studies of galactose metabolism and the occurrence of cataracts among GALK mutant subjects. Evidence will be presented for heterogeneity among GALKG heterozygotes. Crude estimates of galactose tolerance were made by quantitation of urinary galactose excretion and more precise estimates by intravenous galactose tolerance studies. Received January 13, 1975; revised May 16, 1975. This work was supported in part by U.S. Public Health Service grants HD-04861, HD-00588, GM-02138, RR 40, and RR 240. 1Department of Human Genetics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19174. 2 Department of Obstetrics and Gynecology, Hospital of the University of Pennsylvania, Philadelphia, Pennsylvania 19104. 3 Department of Ophthalmology, Hospital of the University of Pennsylvania, Philadelphia, Pennsylvania 19104 4 Department of Pediatrics, University of South Florida, Tampa, Florida 33620. * The symbols used for human alleles in this paper are those recommended by the Human Gene Mapping Nomenclature Committee chaired by E. Giblett. The common galactokinase allele found in European populations is designated GALKA. Q 1975 by the American Society of Human Genetics. All rights reserved.
748
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749
MATERIALS AND METHODS
GALKG heterozygotes were distinguished from individuals with the GALKP variant on the basis of RBC and WBC GALK assays [6]: in the former, GALK activities of both RBC and WBC are reduced, while in the latter, only the RBC activity is low. Intravenous Galactose Tolerance Procedure Galactose was infused intravenously over a 10-min period (0.5 g/kg, maximum 35 g in a 50% wt/vol solution as supplied by Pfanstiehl, Waukegan, Ill.). Heparinized blood samples were obtained before infusion (fasting) and at 10-mmn intervals for 60 min after infusion. For infants a 25% solution of galactose was used by diluting the 50% solution with equal volumes of water. The half-life (to) of infused galactose was determined by a least squares analysis of time after infusion versus the logarithm of the plasma galactose.
Urine and Plasma Galactose Assays Galactose was quantitated in urine (untreated) or plasma (precipitated with equal volumes of 5% perchloric acid) by the following procedure: 100 p.l of sample, 100 I-L of Tris buffer (Trizma, Sigma, St. Louis; 0.5 M, pH 8.6), 50 /.l of NAD solution (Sigma, 4 mM), and 250 i.l of water were placed in 1-ml quartz cuvettes. The reaction was started by adding 5 A.l of galactose dehydrogenase (Boehringer Mannheim, New York) and the 340 nm absorption, as a measure of NADH formation, was followed spectrophotometrically until it reached its maximum. Galactose concentration was directly proportional to 340 nm absorption using standard galactose solutions in the range of 1-50 mg/100 ml. RESULTS
Urine Galactose Excretions Pregnancy. Urine specimens (24 hr) were obtained in the third trimester of gestation from 10 control women (red cell GALK not assayed), from 10 individuals (272 specimens) with low RBC GALK (three with normal, four with low, and three with unknown WBC GALK activity), and from three women (12 specimens) with variant genotypes (two heterozygous and one homozygous for galactosemia, the latter on a milk-free diet [7]). No significant galactose excretions were noted; galactose concentration in the 39 urines averaged 1.0 mg/100 ml, ranging from nondetectable to 9.0 mg/100 ml. Infants. Samples of urine were collected from infants in the nursery reserved for healthy newborn infants with birth weights greater than 2,500 g. The specimens analyzed were untimed collections obtained in plastic bags. An effort was made to remove the bags as soon as voiding had occurred; the samples were promptly transferred to a glass tube and frozen until assay. A total of 10 samples of urine were obtained from the offspring of mothers with low RBC GALK before milk feedings were begun (mean 0.5 mg/100 ml, range nondetectable to 2.9). The collections of urine from control babies were discontinued after three samples were collected (mean 1.2, range nondetectable to 3.7), when only minimal amounts of galactose were found in the study group. Collections were made between 1 and 4 days after beginning formula feeding (containing 7.2 g lactose/100 ml) from 30 control babies (mean 18.9, range 2.0-
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MELLMAN ET AL.
40.1) and from 12 babies born to mothers with low RBC GALK (mean 18.8, range nondetectable to 71.3). There were 35 samples in the latter group. In this group five mothers had normal, three had low, and four had unknown levels of WBC GALK. Six specimens were also obtained from three babies born to mothers who were transferase mutants (mean 18.9, range 2.0-26.5). The galactose content was determined in random samples of urine from babies who were followed in a well baby clinic over the first 5 months of life; nine samples were obtained from six babies of mothers with low RBC galactokinase (one with normal, four with low, and one with unknown levels of WBC GALK in the mother; mean 5.7, range undetectable to 9.2), and single specimens from 48 control babies (mean 10.8, range 1.3-40.7). Three of the four infants born to mothers with low WBC GALK were determined subsequently to have reduced WBC GALK also. No differences in urinary galactose concentration were found between the control group and the variant groups. Intravenous Galactose Tolerance The half-life (t%) of infused galactose (0.5 g/kg) was determined for four healthy adults with normal levels of RBC GALK and GALT, six adults with transferase (GALT) variant genotypes, and 14 adults with low RBC GALK (table 1). Individuals with GALT variant genotypes included two heterozygous for the Duarte variant (GALTD/GALTA), two heterozygous for the galactosemic gene (GALTG/GALTA), one homozygous for the Duarte gene (GALTD/GALTD), and one person heterozygous for both variant alleles (GALTD/GALTG). Of the 14 with reduced RBC GALK activity, two were obligate heterozygotes for the deficiency gene (GALKG/GALKA) [8], and the rest were identified in a population survey [4]. Of these, 10 subjects are black: four with normal, four with low, and two with unknown WBC GALK activity. The other two are a mother and daughter who are white females with low RBC and WBC GALK levels. The mean (± SD) galactose half-lives of the group of 24 subjects who were studied was 16.3 + 5.25 min. The half-lives galactose in a white mother and her daughter (natives of Cuba) were significantly prolonged, 32.2 and 31.7 min. Their tolerance tests were performed on different days and were assayed separately. When these two are removed from the group of 24, the statistics for the remaining group of 22 are 14.8 ± 2.16 min. Four newborn infants of mothers with low RBC GALK were found to have galactose tolerances similar to adults (mean t%, 13.5; range 11.0-16.2 min). These values are comparable to those of Hjelm and Sjolin [9] for both normal adults and newborn infants more than 50 hr old. Haworth and Ford [10] suggested that newborns have a greater ability to metabolize galactose than older infants and children. Their contention was later supported by Ng et al. [11] who found that red blood cells of newborns have three times as much GALK activity as those of adults. We have confirmed the finding of increased RBC GALK activity in newborns but have noted from an analysis of RBC and WBC GALK activity in the same sample of cord blood that the age-related difference found by Ng et al.
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751
TABLE 1 INTRAVENOUS GALACTOSE TOLERANCE TESTS
Controls ................................
Transferase variants: GALTD/GALTA .....
.........
Sex
Race
t112(min)
M M M F
W W W W
12.8
F M
W W W B W W
13.7 13.9 13.3 13.1 18.9 17.4
W W W W B B B B B B B B B B
16.7* 14.4* 32.2 31.7 16.8 19.8 14.4 15.5
M F GALTD/GALTD .......................... M
GALTG/GALTA ...
........
GALTD/GALTG .......................... M Galactokinase variants: GALKG/GALKA (low WBC GALK) ....... M F F F F M F F GALKP variants (normal WBC GALK) .... F F F F Mixed mutants (unknown WBC GALK ..... F F
NOTE.-Mean t/2 + SD: (N = 24) 16.3 + 5.25 min; (N GALKG/GALKA samples with prolonged half-lives). * Obligate heterozygotes (GALKG/GALKA).
13.7 12.1 12.4
17.2 14.8 12.1 13.9 16.0 13.5
22) 14.8 + 2.16 min (after elimination of two
[11] is restricted to the red cells (table 2). Our findings that newborn WBC GALK activity and intravenous galactose tolerance are similar to those of adults suggest that total body galactose metabolism in newborns is not different from adults. Eye Findings in Galactokinase-Variant Individuals Slit-lamp and ophthalmoscopic examinations were performed by a single ophthalmologist (C. W. Nichols) on a group of GALK variant individuals including all those identified with low WBC GALK who were detected in the screening of 1,700 pregnant women. This group of 20 adults includes 18 black individuals with low RBC GALK (seven with normal WBC GALK, five with low WBC GALK, six with unknown WBC GALK) and two white females, the mother and daughter described above with low WBC GALK and galactose intolerance (table 1). The obligate heterozygotes (GALKG/GALKA) reported in table 1 were examined by slit lamp previously and have normal lenses (R. R. Howell, personal communication). No functionally significant cataracts were found in the group examined in detail. The older of the two women in this study with prolonged intravenous galactose
752
MELLMAN ET AL. TABLE 2
RBC AND WBC GALACTOKINASE (GALK) OF MATERNAL AND CORD BLOOD WBC
GALK Activity (nmol/hr/mg protein)
Maternal (N = 9) Cord
(N = 10)
RBC GALK/GALT
GALK Activity (,mol/hr/ml RBC)
GALK/GALT
Mean
SD
Mean
SD
Mean
SD
Mean
SD
....
58.1
20.1
0.321
0.029
0.224
0.076
0.033
0.008
...
51.9
15.0
0.322
0.050
1.037
0.350
0.150
0.041
NOTE.-Samples from eight black and one white mothers and their cord bloods (one black mother had twin pregnancy). GALT-galactose-l-phosphate uridylyltransferase.
tolerance times was noted to have scattered punctate nuclear opacities probably congenital in origin. In addition there were five individuals who had a few scattered punctate cortical opacities and one with several unilateral zonular opacities. We interpret these findings to be similar to what might be observed in detailed examinations of a random group of healthy young adults. DISCUSSION
This study has looked for disturbances of galactose metabolism and cataract formation among individuals detected in a population survey with variations in the activity levels of RBC GALK and GALT. Galactose tolerance was assessed crudely by measuring urinary galactose excretion during periods when galactose metabolism was potentially under stress-during late pregnancy in adult females and in the newborn and early infancy period. No unusual excretion patterns were observed. The minimal galactose excretions observed in pregnancy contrast with the significant elevations of plasma and urine lactose found after the eighteenth week of pregnancy [12]. Studies have shown that circulating lactose is not converted to glucose and galactose in either man or experimental animals [13]. Galactose dehydrogenase used to assay galactose in our studies, unlike galactose oxidase, does not react with lactose [14]. The galactose concentrations measured in the urines of newborns were similar to those reported by Dahlqvist and Svenningsen [14] who used essentially the same procedure for collection and analysis. It is apparent that healthy infants on lactose-containing feedings excrete easily measured amounts of galactose. Age and diet-matched control data, therefore, are necessary to decide when galactosuria is significant. A more critical test of metabolic competence, the intravenous galactose tolerance test, was made on 24 adults. Only two of these 24 individuals, a mother and daughter ages 53 and 22, respectively, were found to have significantly prolonged galactose disappearance times. Both women have RBC and WBC GALK activities in the half-normal range and are presumed to be galactokinase-deficient heterozygotes (GALKG/GALKA). Their RBC and WBC GALK activities are com-
GALACTOSE TOLERANCE STUDIES
753
parable to those of the heterozygotes in a family where there was a homozygous GALK deficient girl with undetectable RBC and WBC GALK activities. In this latter family the obligate heterozygotes (parents of the homozygote) have normal intravenous galactose tolerances. This is also the case with three black women from two different families detected in our population survey who have reduced RBC and WBC GALK levels and are presumptive GALKG heterozygotes [4, 6]. This interfamilial difference in the galactose tolerance of GALKG heterozygotes can be explained by heterogeneity in the GALKG mutations that occur in the general population. Support for this thesis comes from the intrafamilial consistencies we have observed (i.e., both GALKG/GALKA adults in one family have abnormal galactose tolerances and in other families all have normal tolerances). Because cataracts are held to be the major consequence of GALK deficiency in the homozygote, we have looked carefully for cataracts in adults heterozygous for GALK deficiency (GALKG/GALKA). If such individuals have some compromise of galactose utilization, cataracts might be a consequence, although possibly only in adult life. This has been suggested by Monteleone et al. [2]. In their report the form of cataracts they observed in GALKG heterozygotes was not noted; in their homozygous patient (GALKG/GALKG) the cataracts were of the embryonal nucleus. The older of the two GALKG/GALKA women in our study with abnormal galactose tolerance interestingly enough has minor changes in her lens nuclei. This may be merely a coincidental finding, since we believe such changes are not unusual in the general population. In this particular family the GALKG/ GALKA genotype cannot alone be responsible for this congenital lens anomaly, since the daughter and her 2-year-old grandson have similar WBC GALK activity with normal lenses. Therefore, in order to ascribe any phenotypic significance to the GALKG/GALKA genotype in terms of lens abnormalities in this family, additional factors, such as the role of nutrition in pregnancy and other environmental variables, must be invoked. Although this study is mainly a negative one, we would suggest that genetic heterogeneity may explain the observed interfamilial differences in the metabolic consequences of the heterozygous state for GALK deficiency. The intravenous galactose tolerance test may be a useful test of this genetic heterogeneity. The galactose half-life appears to be a highly reproducible test with small variance, and, in the absence of serious liver disease, should detect families with normal and abnormal galactose tolerance. If certain GALKG/GALKA individuals are predisposed to cataracts, as has been suggested, we would predict that those families with abnormal tolerances would be those which are cataract prone. SUMMARY
The galactose tolerance of individuals with mutant genotypes affecting the activities of galactokinase (GALK) and galactose-1-phosphate uridylyltransferase (GALT) was examined. Genotypes studied were heterozygotes for the GALK and GALT forms of galactosemia, the Duarte-variant GALT, and Philadelphia-variant GALK alleles. The measurements used were urinary concentration of galactose during pregnancy in adults and in infants from the newborn period through the
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first 5 months of life; the rate of elimination of an intravenous infusion of galactose; and slit-lamp examination of the lens for evidence of cataracts. No unusual urinary excretions of galactose were noted in any of the age groups studied. Intravenous galactose tolerance tests were normal in all but two women, a mother and daughter heterozygous for the GALK-deficient form of galactosemia (GALKG/GALKA). Six other GALKG/GALKA subjects had normal tolerance studies. The intrafamilial consistency and interfamilial differences in the galactose tolerance of GALKG/GALKA individuals suggest heterogeneity of the genes responsible for the GALK-deficient form of galactosemia. Although subclinical cataracts were observed in several individuals, their significance relative to the mutant genotype cannot be resolved with the available data. ACKNOWLEDGMENTS The diligence of the nursing staffs of the obstetric and pediatric departments of the Hospital of the University of Pennsylvania in obtaining specimens at critical times was essential to this study. REFERENCES 1. KALCKAR HM, KINOSHITA JH, DONNELL GN: Galactosemia: biochemistry, genetics, pathophysiology and developmental aspects, in Biology of Brain Dysfunction, edited by GAULL GE, New York, Plenum, 1973, pp 31-38 2. MONTELEONE JA, BEUTLER E, MONTELEONE PL, UTZ CL, CASEY EC: Cataracts, galactosuria and hypergalactosemia due to galactokinase deficiency in a child: studies of a kindred. Am J Med 50:403-407, 1971 3. OLAMBIWONNU NO, MCVIE R, FRASIER SD, DONNELL GN: Galactokinase deficiency in twins: clinical and biochemical studies. Pediatrics 53:314-318, 1974 4. TEDESCO TA, MILLER KL, RAWNSLEY BE, MENNUTI MT, MELLMAN WJ: Human erythrocyte galactokinase and galactose-1-phosphate uridylyltransferase: a population survey. Am J Hum Genet 27:737-747, 1975 5. TEDESCO TA, BONow R, MILLER K, MELLMAN WJ: Galactokinase: evidence for a new racial polymorphism. Science 178:176-178, 1972 6. TEDESCO TA, MILLER KL, RAWNSLEY BE, ADAMS MC, BOEDECKER HJ, ORKWISZEWSKI KG, MELLMAN WJ: Characterization of the Philadelphia variant of galactokinase. Submitted for publication 7. TEDEScO TA, MORROW G, MELLMAN WJ: Normal pregnancy and childbirth in a galactosemic woman. J Pediatr 81:1159-1161, 1972 8. PICKERING WR, HOWELL RR: Galactokinase deficiency: clinical and biochemical findings in a new kindred. J Pediatr 81:50-55, 1972 9. HJELM M, SJOLIN S: Changes in the elimination rate from blood or intravenously injected galactose during the neonatal period. Scand J Clin Lab Invest 18, suppl. 92:126, 1966 10. HAWORTH JC, FORD JD: Variation of the oral galactose tolerance test with age. J Pediatr 63:276-282, 1963 11. NG WG, DONNELL GN, BERGREN WR: Galactokinase activity in human erythrocytes of individuals at different ages. J Lab Clin Med 66:115-121, 1965 12. DATE JW: The excretion of lactose and some monosaccharides during pregnancy and lactation. Scand J Clin Lab Invest 16:589-596, 1964 13. WESER E, SLEISENGER MH: Metabolism of circulating disaccharides in man and the rat. J Clin Invest 46:499-507, 1967 14. DAHLQVIST A, SVENNINGSEN NW: Galactose in the urine of newborn infants. J Pediatr 75:454-462, 1969
Galactose Tolerance Studies of Individuals with Reduced Galactose Pathway Activity W. J. MELLMAN,1 B. E. RAWNSLEY,2 C. W. NICHOLS,3 B. NEEDELMAN,1 M. T. MENNUTI,2 J. MALONE,2 AND T. A. TEDESCO4 INTRODUCTION
Galactokinase (GALK)* deficiency is a rare autosomal recessive disorder (GALKG/ GALKG) marked by cataracts in infancy [1]. Monteleone et al. [2] in reporting a case of GALK deficiency noted that apparent heterozygotes in the patient's family had cataracts in the third and fourth decade. Failure to find cataracts among young adults in all families where GALKG is segregating [3] does not necessarily refute this suggestion of Monteleone et al. [2]. The rarity of GALKG genes makes it highly probable that those found in a random population form a heterogeneous group of mutants. In a population sample of 1,700 healthy adult females screened for variations in red cell GALK and galactose-l-phosphate uridylyltransferase (GALT) activity, we identified five women as GALKG heterozygotes [4]. They represent two of 618 white and three of 1,082 black females surveyed. Estimation of the true frequency of the GALKG gene in black populations is complicated by an apparent polymorphic allele in blacks [5] that we have called the Philadelphia variant (GALKP). This report presents studies of galactose metabolism and the occurrence of cataracts among GALK mutant subjects. Evidence will be presented for heterogeneity among GALKG heterozygotes. Crude estimates of galactose tolerance were made by quantitation of urinary galactose excretion and more precise estimates by intravenous galactose tolerance studies. Received January 13, 1975; revised May 16, 1975. This work was supported in part by U.S. Public Health Service grants HD-04861, HD-00588, GM-02138, RR 40, and RR 240. 1Department of Human Genetics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19174. 2 Department of Obstetrics and Gynecology, Hospital of the University of Pennsylvania, Philadelphia, Pennsylvania 19104. 3 Department of Ophthalmology, Hospital of the University of Pennsylvania, Philadelphia, Pennsylvania 19104 4 Department of Pediatrics, University of South Florida, Tampa, Florida 33620. * The symbols used for human alleles in this paper are those recommended by the Human Gene Mapping Nomenclature Committee chaired by E. Giblett. The common galactokinase allele found in European populations is designated GALKA. Q 1975 by the American Society of Human Genetics. All rights reserved.
748
GALACTOSE TOLERANCE STUDIES
749
MATERIALS AND METHODS
GALKG heterozygotes were distinguished from individuals with the GALKP variant on the basis of RBC and WBC GALK assays [6]: in the former, GALK activities of both RBC and WBC are reduced, while in the latter, only the RBC activity is low. Intravenous Galactose Tolerance Procedure Galactose was infused intravenously over a 10-min period (0.5 g/kg, maximum 35 g in a 50% wt/vol solution as supplied by Pfanstiehl, Waukegan, Ill.). Heparinized blood samples were obtained before infusion (fasting) and at 10-mmn intervals for 60 min after infusion. For infants a 25% solution of galactose was used by diluting the 50% solution with equal volumes of water. The half-life (to) of infused galactose was determined by a least squares analysis of time after infusion versus the logarithm of the plasma galactose.
Urine and Plasma Galactose Assays Galactose was quantitated in urine (untreated) or plasma (precipitated with equal volumes of 5% perchloric acid) by the following procedure: 100 p.l of sample, 100 I-L of Tris buffer (Trizma, Sigma, St. Louis; 0.5 M, pH 8.6), 50 /.l of NAD solution (Sigma, 4 mM), and 250 i.l of water were placed in 1-ml quartz cuvettes. The reaction was started by adding 5 A.l of galactose dehydrogenase (Boehringer Mannheim, New York) and the 340 nm absorption, as a measure of NADH formation, was followed spectrophotometrically until it reached its maximum. Galactose concentration was directly proportional to 340 nm absorption using standard galactose solutions in the range of 1-50 mg/100 ml. RESULTS
Urine Galactose Excretions Pregnancy. Urine specimens (24 hr) were obtained in the third trimester of gestation from 10 control women (red cell GALK not assayed), from 10 individuals (272 specimens) with low RBC GALK (three with normal, four with low, and three with unknown WBC GALK activity), and from three women (12 specimens) with variant genotypes (two heterozygous and one homozygous for galactosemia, the latter on a milk-free diet [7]). No significant galactose excretions were noted; galactose concentration in the 39 urines averaged 1.0 mg/100 ml, ranging from nondetectable to 9.0 mg/100 ml. Infants. Samples of urine were collected from infants in the nursery reserved for healthy newborn infants with birth weights greater than 2,500 g. The specimens analyzed were untimed collections obtained in plastic bags. An effort was made to remove the bags as soon as voiding had occurred; the samples were promptly transferred to a glass tube and frozen until assay. A total of 10 samples of urine were obtained from the offspring of mothers with low RBC GALK before milk feedings were begun (mean 0.5 mg/100 ml, range nondetectable to 2.9). The collections of urine from control babies were discontinued after three samples were collected (mean 1.2, range nondetectable to 3.7), when only minimal amounts of galactose were found in the study group. Collections were made between 1 and 4 days after beginning formula feeding (containing 7.2 g lactose/100 ml) from 30 control babies (mean 18.9, range 2.0-
750
MELLMAN ET AL.
40.1) and from 12 babies born to mothers with low RBC GALK (mean 18.8, range nondetectable to 71.3). There were 35 samples in the latter group. In this group five mothers had normal, three had low, and four had unknown levels of WBC GALK. Six specimens were also obtained from three babies born to mothers who were transferase mutants (mean 18.9, range 2.0-26.5). The galactose content was determined in random samples of urine from babies who were followed in a well baby clinic over the first 5 months of life; nine samples were obtained from six babies of mothers with low RBC galactokinase (one with normal, four with low, and one with unknown levels of WBC GALK in the mother; mean 5.7, range undetectable to 9.2), and single specimens from 48 control babies (mean 10.8, range 1.3-40.7). Three of the four infants born to mothers with low WBC GALK were determined subsequently to have reduced WBC GALK also. No differences in urinary galactose concentration were found between the control group and the variant groups. Intravenous Galactose Tolerance The half-life (t%) of infused galactose (0.5 g/kg) was determined for four healthy adults with normal levels of RBC GALK and GALT, six adults with transferase (GALT) variant genotypes, and 14 adults with low RBC GALK (table 1). Individuals with GALT variant genotypes included two heterozygous for the Duarte variant (GALTD/GALTA), two heterozygous for the galactosemic gene (GALTG/GALTA), one homozygous for the Duarte gene (GALTD/GALTD), and one person heterozygous for both variant alleles (GALTD/GALTG). Of the 14 with reduced RBC GALK activity, two were obligate heterozygotes for the deficiency gene (GALKG/GALKA) [8], and the rest were identified in a population survey [4]. Of these, 10 subjects are black: four with normal, four with low, and two with unknown WBC GALK activity. The other two are a mother and daughter who are white females with low RBC and WBC GALK levels. The mean (± SD) galactose half-lives of the group of 24 subjects who were studied was 16.3 + 5.25 min. The half-lives galactose in a white mother and her daughter (natives of Cuba) were significantly prolonged, 32.2 and 31.7 min. Their tolerance tests were performed on different days and were assayed separately. When these two are removed from the group of 24, the statistics for the remaining group of 22 are 14.8 ± 2.16 min. Four newborn infants of mothers with low RBC GALK were found to have galactose tolerances similar to adults (mean t%, 13.5; range 11.0-16.2 min). These values are comparable to those of Hjelm and Sjolin [9] for both normal adults and newborn infants more than 50 hr old. Haworth and Ford [10] suggested that newborns have a greater ability to metabolize galactose than older infants and children. Their contention was later supported by Ng et al. [11] who found that red blood cells of newborns have three times as much GALK activity as those of adults. We have confirmed the finding of increased RBC GALK activity in newborns but have noted from an analysis of RBC and WBC GALK activity in the same sample of cord blood that the age-related difference found by Ng et al.
GALACTOSE TOLERANCE STUDIES
751
TABLE 1 INTRAVENOUS GALACTOSE TOLERANCE TESTS
Controls ................................
Transferase variants: GALTD/GALTA .....
.........
Sex
Race
t112(min)
M M M F
W W W W
12.8
F M
W W W B W W
13.7 13.9 13.3 13.1 18.9 17.4
W W W W B B B B B B B B B B
16.7* 14.4* 32.2 31.7 16.8 19.8 14.4 15.5
M F GALTD/GALTD .......................... M
GALTG/GALTA ...
........
GALTD/GALTG .......................... M Galactokinase variants: GALKG/GALKA (low WBC GALK) ....... M F F F F M F F GALKP variants (normal WBC GALK) .... F F F F Mixed mutants (unknown WBC GALK ..... F F
NOTE.-Mean t/2 + SD: (N = 24) 16.3 + 5.25 min; (N GALKG/GALKA samples with prolonged half-lives). * Obligate heterozygotes (GALKG/GALKA).
13.7 12.1 12.4
17.2 14.8 12.1 13.9 16.0 13.5
22) 14.8 + 2.16 min (after elimination of two
[11] is restricted to the red cells (table 2). Our findings that newborn WBC GALK activity and intravenous galactose tolerance are similar to those of adults suggest that total body galactose metabolism in newborns is not different from adults. Eye Findings in Galactokinase-Variant Individuals Slit-lamp and ophthalmoscopic examinations were performed by a single ophthalmologist (C. W. Nichols) on a group of GALK variant individuals including all those identified with low WBC GALK who were detected in the screening of 1,700 pregnant women. This group of 20 adults includes 18 black individuals with low RBC GALK (seven with normal WBC GALK, five with low WBC GALK, six with unknown WBC GALK) and two white females, the mother and daughter described above with low WBC GALK and galactose intolerance (table 1). The obligate heterozygotes (GALKG/GALKA) reported in table 1 were examined by slit lamp previously and have normal lenses (R. R. Howell, personal communication). No functionally significant cataracts were found in the group examined in detail. The older of the two women in this study with prolonged intravenous galactose
752
MELLMAN ET AL. TABLE 2
RBC AND WBC GALACTOKINASE (GALK) OF MATERNAL AND CORD BLOOD WBC
GALK Activity (nmol/hr/mg protein)
Maternal (N = 9) Cord
(N = 10)
RBC GALK/GALT
GALK Activity (,mol/hr/ml RBC)
GALK/GALT
Mean
SD
Mean
SD
Mean
SD
Mean
SD
....
58.1
20.1
0.321
0.029
0.224
0.076
0.033
0.008
...
51.9
15.0
0.322
0.050
1.037
0.350
0.150
0.041
NOTE.-Samples from eight black and one white mothers and their cord bloods (one black mother had twin pregnancy). GALT-galactose-l-phosphate uridylyltransferase.
tolerance times was noted to have scattered punctate nuclear opacities probably congenital in origin. In addition there were five individuals who had a few scattered punctate cortical opacities and one with several unilateral zonular opacities. We interpret these findings to be similar to what might be observed in detailed examinations of a random group of healthy young adults. DISCUSSION
This study has looked for disturbances of galactose metabolism and cataract formation among individuals detected in a population survey with variations in the activity levels of RBC GALK and GALT. Galactose tolerance was assessed crudely by measuring urinary galactose excretion during periods when galactose metabolism was potentially under stress-during late pregnancy in adult females and in the newborn and early infancy period. No unusual excretion patterns were observed. The minimal galactose excretions observed in pregnancy contrast with the significant elevations of plasma and urine lactose found after the eighteenth week of pregnancy [12]. Studies have shown that circulating lactose is not converted to glucose and galactose in either man or experimental animals [13]. Galactose dehydrogenase used to assay galactose in our studies, unlike galactose oxidase, does not react with lactose [14]. The galactose concentrations measured in the urines of newborns were similar to those reported by Dahlqvist and Svenningsen [14] who used essentially the same procedure for collection and analysis. It is apparent that healthy infants on lactose-containing feedings excrete easily measured amounts of galactose. Age and diet-matched control data, therefore, are necessary to decide when galactosuria is significant. A more critical test of metabolic competence, the intravenous galactose tolerance test, was made on 24 adults. Only two of these 24 individuals, a mother and daughter ages 53 and 22, respectively, were found to have significantly prolonged galactose disappearance times. Both women have RBC and WBC GALK activities in the half-normal range and are presumed to be galactokinase-deficient heterozygotes (GALKG/GALKA). Their RBC and WBC GALK activities are com-
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parable to those of the heterozygotes in a family where there was a homozygous GALK deficient girl with undetectable RBC and WBC GALK activities. In this latter family the obligate heterozygotes (parents of the homozygote) have normal intravenous galactose tolerances. This is also the case with three black women from two different families detected in our population survey who have reduced RBC and WBC GALK levels and are presumptive GALKG heterozygotes [4, 6]. This interfamilial difference in the galactose tolerance of GALKG heterozygotes can be explained by heterogeneity in the GALKG mutations that occur in the general population. Support for this thesis comes from the intrafamilial consistencies we have observed (i.e., both GALKG/GALKA adults in one family have abnormal galactose tolerances and in other families all have normal tolerances). Because cataracts are held to be the major consequence of GALK deficiency in the homozygote, we have looked carefully for cataracts in adults heterozygous for GALK deficiency (GALKG/GALKA). If such individuals have some compromise of galactose utilization, cataracts might be a consequence, although possibly only in adult life. This has been suggested by Monteleone et al. [2]. In their report the form of cataracts they observed in GALKG heterozygotes was not noted; in their homozygous patient (GALKG/GALKG) the cataracts were of the embryonal nucleus. The older of the two GALKG/GALKA women in our study with abnormal galactose tolerance interestingly enough has minor changes in her lens nuclei. This may be merely a coincidental finding, since we believe such changes are not unusual in the general population. In this particular family the GALKG/ GALKA genotype cannot alone be responsible for this congenital lens anomaly, since the daughter and her 2-year-old grandson have similar WBC GALK activity with normal lenses. Therefore, in order to ascribe any phenotypic significance to the GALKG/GALKA genotype in terms of lens abnormalities in this family, additional factors, such as the role of nutrition in pregnancy and other environmental variables, must be invoked. Although this study is mainly a negative one, we would suggest that genetic heterogeneity may explain the observed interfamilial differences in the metabolic consequences of the heterozygous state for GALK deficiency. The intravenous galactose tolerance test may be a useful test of this genetic heterogeneity. The galactose half-life appears to be a highly reproducible test with small variance, and, in the absence of serious liver disease, should detect families with normal and abnormal galactose tolerance. If certain GALKG/GALKA individuals are predisposed to cataracts, as has been suggested, we would predict that those families with abnormal tolerances would be those which are cataract prone. SUMMARY
The galactose tolerance of individuals with mutant genotypes affecting the activities of galactokinase (GALK) and galactose-1-phosphate uridylyltransferase (GALT) was examined. Genotypes studied were heterozygotes for the GALK and GALT forms of galactosemia, the Duarte-variant GALT, and Philadelphia-variant GALK alleles. The measurements used were urinary concentration of galactose during pregnancy in adults and in infants from the newborn period through the
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first 5 months of life; the rate of elimination of an intravenous infusion of galactose; and slit-lamp examination of the lens for evidence of cataracts. No unusual urinary excretions of galactose were noted in any of the age groups studied. Intravenous galactose tolerance tests were normal in all but two women, a mother and daughter heterozygous for the GALK-deficient form of galactosemia (GALKG/GALKA). Six other GALKG/GALKA subjects had normal tolerance studies. The intrafamilial consistency and interfamilial differences in the galactose tolerance of GALKG/GALKA individuals suggest heterogeneity of the genes responsible for the GALK-deficient form of galactosemia. Although subclinical cataracts were observed in several individuals, their significance relative to the mutant genotype cannot be resolved with the available data. ACKNOWLEDGMENTS The diligence of the nursing staffs of the obstetric and pediatric departments of the Hospital of the University of Pennsylvania in obtaining specimens at critical times was essential to this study. REFERENCES 1. KALCKAR HM, KINOSHITA JH, DONNELL GN: Galactosemia: biochemistry, genetics, pathophysiology and developmental aspects, in Biology of Brain Dysfunction, edited by GAULL GE, New York, Plenum, 1973, pp 31-38 2. MONTELEONE JA, BEUTLER E, MONTELEONE PL, UTZ CL, CASEY EC: Cataracts, galactosuria and hypergalactosemia due to galactokinase deficiency in a child: studies of a kindred. Am J Med 50:403-407, 1971 3. OLAMBIWONNU NO, MCVIE R, FRASIER SD, DONNELL GN: Galactokinase deficiency in twins: clinical and biochemical studies. Pediatrics 53:314-318, 1974 4. TEDESCO TA, MILLER KL, RAWNSLEY BE, MENNUTI MT, MELLMAN WJ: Human erythrocyte galactokinase and galactose-1-phosphate uridylyltransferase: a population survey. Am J Hum Genet 27:737-747, 1975 5. TEDESCO TA, BONow R, MILLER K, MELLMAN WJ: Galactokinase: evidence for a new racial polymorphism. Science 178:176-178, 1972 6. TEDESCO TA, MILLER KL, RAWNSLEY BE, ADAMS MC, BOEDECKER HJ, ORKWISZEWSKI KG, MELLMAN WJ: Characterization of the Philadelphia variant of galactokinase. Submitted for publication 7. TEDEScO TA, MORROW G, MELLMAN WJ: Normal pregnancy and childbirth in a galactosemic woman. J Pediatr 81:1159-1161, 1972 8. PICKERING WR, HOWELL RR: Galactokinase deficiency: clinical and biochemical findings in a new kindred. J Pediatr 81:50-55, 1972 9. HJELM M, SJOLIN S: Changes in the elimination rate from blood or intravenously injected galactose during the neonatal period. Scand J Clin Lab Invest 18, suppl. 92:126, 1966 10. HAWORTH JC, FORD JD: Variation of the oral galactose tolerance test with age. J Pediatr 63:276-282, 1963 11. NG WG, DONNELL GN, BERGREN WR: Galactokinase activity in human erythrocytes of individuals at different ages. J Lab Clin Med 66:115-121, 1965 12. DATE JW: The excretion of lactose and some monosaccharides during pregnancy and lactation. Scand J Clin Lab Invest 16:589-596, 1964 13. WESER E, SLEISENGER MH: Metabolism of circulating disaccharides in man and the rat. J Clin Invest 46:499-507, 1967 14. DAHLQVIST A, SVENNINGSEN NW: Galactose in the urine of newborn infants. J Pediatr 75:454-462, 1969
