Ultra fast and sensitive liquid chromatography tandem mass spectrometry based assay for galactose-1-phosphate uridylyltransferase and galactokinase deficiencies.

The diagnosis of transferase and galactokinase deficiency galactosemia usually involves the measurement of erythrocyte galactose-1-phosphate uridylyltransferase (GALT) and galactokinase (GALK) enzyme activity, respectively. The current gold standard assays for these enzymes are radioactive assays, which are laborious and/or incapable of measuring low enzyme activities. To further our knowledge of genotype-phenotype relationships, we had developed an assay for GALT activity alone using LC-MS/MS. In this study we generated a robust and sensitive LC-MS/MS based GALT and GALK assay using a novel normal phase chromatographic condition. We improved upon our earlier assay by drastically reducing the instrument run time and eliminating the use of an ion pairing reagent. Stable isotope labeled substrates were utilized in the GALT and GALK assays. The enzymatic products ([(13)C(6)]-uridine diphosphate galactose in GALT assay and [(13)C(6)]-galactose-1-phosphate in GALK assay) were quantified in a 3 min LC-MS/MS run. The assays were sensitive enough to allow for the quantification of enzyme activities as low as 0.2% and 0.3% of normal control values in the GALT and GALK assays, respectively. Thirty-three samples from non-galactosemic patients were assayed to have erythrocyte GALT activity of 23.4±4.2 and GALK activity of 1.8±0.47 (mean±SD) µmol⋅(g Hgb)(-1) h(-1). Erythrocyte GALT activities in a cohort of 16 patients with classic or severe galactosemia were measured: 4 patients had GALT activity less than 1% of normal control values and the remaining 12 had no detectable GALT activity. No GALK activity was detected in a GALK deficient sample we analyzed. Lastly, we tested the feasibility of adapting this LC-MS/MS based GALT/GALK assay as a newborn screening (NBS) test.

The clinical and molecular spectrum of galactosemia in patients from the Cape Town region of South Africa.

BACKGROUND: The objective of this study was to document the clinical, laboratory and genetic features of galactosemia in patients from the Cape Town metropolitan region. METHODS: Diagnoses were based on thin layer chromatography for galactosuria/galactosemia and assays of erythrocyte galactose-1-phosphate uridyltransferase (GALT) and galactokinase activities. Patients were screened for the common S135L and Q188R transferase gene mutations, using PCR-based assays. Screening for the S135L mutation in black newborns was used to estimate the carrier rate for galactosemia in black South Africans. RESULTS: A positive diagnosis of galactosemia was made in 17 patients between the years 1980 to 2001. All had very low or absent galactose-1-phosphate uridyltransferase (GALT) activity, and normal galactokinase levels. The mean age at diagnosis was 5.1 months (range 4 days to 6.5 months). A review of 9 patients showed that hepatomegaly (9/9), and splenomegaly, failure to thrive, developmental delay, bilateral cataracts (6/9) were the most frequent features at diagnosis. Six had conjugated hyperbilirubinemia. Four experienced invasive E. coli infection before diagnosis. Ten patients were submitted to DNA analysis. All 4 black patients and 2 of mixed extraction were homozygous for the S135L allele, while all 3 white patients were homozygous for the Q188R allele. The remaining patient of mixed extraction was heterozygous for the Q188R allele. The estimated carrier frequency of the S135L mutation in 725 healthy black newborns was 1/60. CONCLUSIONS: In the absence of newborn screening the delay in diagnosis is most often unacceptably long. Also, carrier frequency data predict a galactosemia incidence of approximately 1/14 400 for black newborns in the Cape Metropole, which is much higher than the current detection rate. It is thus likely that many patients go undetected.

Disturbed galactose metabolism in elderly and diabetic humans is associated with cataract formation.

Lactose consumption has been associated with a high incidence of cataract in northern Indian and southern Italian populations. Galactose absorbed after hydrolysis of lactose from milk in individuals with normal lactase activity is considered responsible. However, lactase-deficient subjects who often avoid drinking milk are able to digest lactose and absorb free galactose in fermented milk and yogurt. This study was conducted to evaluate the relationships between milk and yogurt consumption, galactose metabolism and cataract risk. Milk ingestion was dose-related with cataract risk in lactose digesters (particularly in diabetics) but not in lactose maldigesters. Conversely, yogurt intake had a protective dose-effect on cataract formation for the whole population. Maximal galactose concentrations after an oral galactose test increased exponentially with age. Red blood cell galactokinase activity was significantly lower in elderly subjects (> 60 y) than in young individuals (P < 0.05), and galactose-1-phosphate uridyl-transferase activity was significantly lower in institutionalized subjects and in home-living elderly with cataract than in healthy elderly subjects (P < 0.05). We conclude that the cataractogenic action of milk lactose is dependent on the disturbance of galactose metabolism in elderly subjects and that yogurt is not cataractogenic, although the mechanism of the protective effect of yogurt remains unknown.

Erythrocyte galactokinase assay with high performance liquid chromatography.

An assay for erythtocyte galactokinase based on high performance liquid chromatographic determination of galactose 1-phosphate (Gal-1-P) is described. The determination of Gal-1-P was applied to a post-column fluorometric detection of reducing sugars using arginine. This method is as sensitive and accurate as conventional radioisotopic methods, but needs no radioisotopic facilities. It requires only a small blood sample and is suitable as a follow-up test in neonatal screening.

[Measurement of the erythrocyte galactokinase activity in patients with presenile and senile idiopathic cataract. Preliminary study]. / Valutazione dell'attività galattochinasica eritrocitaria in pazienti con cataratta idiopatica presenile e senile. Studio preliminare.

The activity of galactokinase in red blood cells, has been assayed in 17 patients with idiopathic senile and presenile cataract and in 12 age-matched subjects with perfectly transparent lenses. 3 of the idiopathic cataract patients (17.6%) showed low erythrocytes GK activity, while nobody in the control group showed reduced GK activity. Although preliminary, our results seem to support the possibility that a chronic disorder of galactose metabolism may be involved in the pathogenesis of the idiopathic senile and presenile cataract.

[Studies on blood galactokinase of patients with presenile and senile cataracts].

Whole blood galactokinase (GK) activities were determined in 278 individuals including 77 patients with senile cataracts, 60 with presenile cataracts and 141 normal controls. No significant difference was found among the GK levels of the three groups. The frequency of GK heterozygocity in the presenile cataract group, however, was significantly higher than that in the normal control group (P less than 0.01), suggesting that even a partial decrease in GK activity might facilitate cataract formation in relatively early stages of life. The intake of milk and its products should be limited, both in GK homo- and heterozygotes.

[Identification of inborn errors of galactose metabolism in patients with cataracts]. / Identificación de errores innatos del metabolismo de la galactosa en pacientes con cataratas.

133 patients with congenital or idiopathic cataracts were studied (94 patients had ages between 1 month and 14 years; 10 patients had ages between 16 and 50 years and 29 patients did not have an age registry) along with 18 patients with a clinical diagnosis of classic galactosemia. The activity of galactokinase (GALAK) and that of erythrocyte galactose-1-phosphate uridyl transferase (GALT) was measured. There were no individuals with a total deficiency of GALK or GALT. The cataract patients of ages between 1 monthly and 14 years, 3 (3.19%) and 4 (4.25%) showed GALK and GALT levels in the range corresponding to the respective heterozygotes. As compared with the expected incidence of heterozygotes in the general population (0.2% for GALK and 0.8% for GALT) we found a significant rise of individuals with low levels of enzymes for the metabolism of galactose. The possibility that heterozygote galactosemic states contribute a risk factor in the development of cataracts and its therapeutic implications are discussed.

Idiopathic presenile cataract formation and galactosaemia.

Five hundred patients undergoing cataract surgery were prospectively examined, and 46 Caucasian patients were found to have strictly idiopathic cataracts severe enough to warrant surgery on or before age 55. In a masked fashion we determined the activity of galactokinase (GK) and galactose-1-phosphate uridyl transferase (GPUT) in these patients as well as on 53 age matched controls. With respect to GK no cataract patient had an enzyme level of less than 2 standard deviations below the control mean. However, 3 of 45 (6.7%) patients in the cataract group had a GPUT level less than 2 standard deviations below the mean for controls, and were presumably heterozygotes for this enzyme. In comparison with the expected population rate of 0.8% this is highly significant (p = 0.006). Abnormalities in galactose pathway enzymes may therefore predispose to development of presenile cataracts. In affected people there is a possibility of treating these patients clinically by dietary restriction of dairy products or by using aldose reductase inhibitors to prevent or reverse cataract formation.

Strain difference in galactokinase level and susceptibility to the teratogenic effect of dietary galactose in mice: I. Teratogenic and embryopathic effect.

Congenital cataracts have been noted to occur in infants when either the mother or the mother and infant have reduced activity of the enzymes galactokinase (GK) or galactose-1-phosphate uridyl transferase (GALT). Studies were undertaken to elucidate the possible genetic and dietary fetomaternal interactions leading to the presumptive galactose teratogenesis noted in two inbred strains of mice differing in GK activity. Pregnant A/J (high erythrocyte GK activity 134.18 +/- 17.89 mU/gm Hb) and C57BL/6J (low erythrocyte GK activity 55.05 +/- 11.39 mU/gm Hb) mice were treated with a diet containing either 25% or 50% galactose throughout gestation. A significantly higher percentage of C57BL offspring (92.3%) were observed to have lens opacities when their mothers were fed a high-galactose diet, whereas no increase in lens pathology was observed in the offspring of similarly treated A/J mothers. Additionally, reciprocal matings were carried out so that all offspring were genetically equivalent in terms of GK activity. However, when the mother had low GK activity, a significant incidence of lens opacities was present in their offspring; this was not found when the mother had high GK activity. After weaning, no difference in the incidence of lens opacities was observed when the 25% galactose diet was introduced to the offspring of these reciprocal crosses, providing additional support for a maternal dietary influence on the development of lens opacities.

The Philadelphia variant of galactokinase in human erythrocytes: physicochemical and catalytic properties.

The Philadelphia variant of galactokinase (GALKP) is responsible for an asymptomatic disorder of galactose metabolism. Individuals with GALKP phenotype are common among black people. They exhibit reduced galactokinase (GALK) activity in their red blood cells but normal activity in their white blood cells. We explored the biochemical characteristics of hemolysates from individuals with the GALKP phenotype and from controls. In mixed hemolysates from a control and a proband, the GALK activity measured did not suggest the presence of an inhibitor. We observed that the catalytic properties, pI and thermolability in hemolysates from controls and GALKP individuals were identical. Thus, the Philadelphia variant of galactokinase seems not to alter biochemical properties of the red blood cell enzyme. A silent amino acid substitution, or the dysfunction of a regulatory gene might be likely suggested to explain the reduced enzyme activity.

The Philadelphia variant of galactokinase: impaired [1-14C]galactose oxidation by intact erythrocytes.

Probands with the Philadelphia variant of galactokinase (GALKP) are black people who exhibit reduced galactokinase (GALK) activity in their red blood cells (RBC), but normal activity in their white blood cells (WBC). This reduced RBC GALK was demonstrated in disrupted erythrocytes. To investigate the possibility of a missing cofactor in hemolysates from individuals with GALKP phenotype, we compared [1-14C]galactose oxidation by intact erythrocytes, with the direct GALK assay in disrupted erythrocytes. The rate of [1-14C]glucose oxidation was also measured in order to differentiate an impaired galactose metabolism from a defect further along the pentose phosphate pathway. A good correlation (p less than 0.001) was found between the direct GALK assay and [1-14C]galactose oxidation in control subjects, which indicates that this method can be used effectively for the detection of GALK defects. This was further supported by studies on samples from heterozygotes and homozygotes for the GALKG deficient gene. For all the probands with a GALKP phenotype, diminished CO2 production from galactose was observed in the absence of impaired glucose metabolism. This allowed us to confirm the existence of a GALK deficiency in intact erythrocytes due to the GALKP variant. Further studies of RBC GALK catalytic properties are needed to investigate the molecular basis of this GALK deficiency.

Possible role of galactose-1-P-uridyl transferase activity deficiency in red blood cells in the development of the presenile and senile cataract.

The activity of red blood cells galactose-1-P-uridyl transferase in 64 patients with presenile and senile cataracts (nondiabetics) and in 41 age-matched controls was investigated. All control subjects examined have shown normal enzymatic levels, while 21.9% of patients with presenile cataracts and 21.7% of patients with senile cataracts presented a statistically significant reduced enzymatic activity (mean +/- 2 SD in controls).

Purification of human galactokinase and evidence for its existence as a monomer form.

A procedure for preparing a highly purified galactokinase (ATP:D-galactose 1-phosphotransferase, EC 2.7.1.6) from human erythrocytes and placenta is described, involving DEAE-Sephacel, ammonium sulfate fractionation, gel-filtration and a subsequent chromatography step on Blue Sepharose CL-6B. The final chromatography step yields a homogeneous preparation of high specific activity. The subunit molecular weight was determined to be 38 000 for both placental and erythrocyte galactokinases. Both active preparations of the native enzyme eluted from a gel filtration column gave a molecular weight of 37 000-38 000, thus suggesting the enzyme to be present in monomeric form. The isoelectric points for both crude and their respective purified enzymes was determined to be at pH 5.7. This method for purifying human galactokinase from placenta and erythrocytes represents a significant improvement over that previously reported and contradicts past evidence for the enzyme existing as a dimer.

Macular deposits in galactokinase deficiency.

Deficiency of the enzyme, galactokinase, is a recognized cause of "juvenile" lens opacities; these opacities are felt to be its only clinical expression. The deficiency itself is inherited as an autosomal recessive and as such is expected to be clinically manifest in the homozygote. We have recently demonstrated cataracts and associated bilateral macular deposits in a white male who is heterozygous for the deficiency of the enzyme and whose dietary intake of milk and its products is extremely high. To our knowledge, intraretinal deposits have not previously been described in patients with galactokinase deficiency, and their clinical significance and biochemical makeup can only be speculative. Dietary restriction of galactose is recommended for all individuals proven to be deficient in this enzyme, whether homozygous or heterozygous.

Red blood cell galactokinase activity and presenile cataracts.

Red blood cell galactokinase activity was measured in 70 patients with cataracts to assess a possible correlation between galactokinase activity levels and risk of cataract development. Among all, 15 patients developed cataracts during the first year of life, 25 patients under the age of 50 and 30 later in life. No cases of total or partial galactokinase deficiency were found. These results, taken together with the absence of cataracts in 9 patients with partial galactokinase deficiency render less certain the cause and effect relationship between partial galactokinase deficiency and the appearance of cataracts.

Adult and fetal galactokinases in human red blood cells.

This paper reports the biochemical properties of galactokinase from fetal and adult human red blood cells. The specific activity of galactokinase is three times higher in the fetal red cells than in adult cells, shows a significant difference in the Michaelis constant toward galactose, and is more thermostable. On the other hand, no differences were found in molecular weight, electric charge, temperature and pH dependence between the two enzymes partly purified from fetal and adult erythrocytes. The possibility that these differences could be due to the shorter lifespan of the fetal erythrocytes (which could result in a higher proportion of young cells in the blood samples utilized) was investigated. Fetal and adult red blood cells were separated into fractions of different mean age by ultracentrifugation through density gradients. The kinetic properties and thermostability of galactokinase from fetal erythrocytes do not show any similarity with the same properties of the enzyme from young red blood cells. These results indicate that galactokinase from fetal erythrocytes show some biochemical properties that are typical signs distinguishing a fetal enzyme.

Galactosemic enzyme levels in presenile cataracts.

Galactose-1-phosphate uridyl transferase and galactokinase activities have been measured in the red blood cells of a group of patients with "idiopathic" presenile cataract and of a group of nondiabetic patients with senile cataract. The activity of both galactosemic enzymes was found to be within the normal range in all the patients with presenile cataract. In the group of patients with senile cataract, galactokinase activity was normal in all 24 subjects examined, and galactose-1-phosphate uridyl transferase activity was moderately reduced in 3 of 14.

[Determination of the galactokinase activity of erythrocytes of swine of various ages]. / Bestimmung der Galaktokinase-Aktivität in den Erythrozyten von Schweinen unterschiedlichen Alters.

In view of the fact that the ability of pig erythrocytes to use galactose will decrease with growing age, the galactokinase activity in pig erythrocytes of different age was determined by means of a spectrophotometrical method. A total of 5 pig fetuses (112th day of gestation) and 45 piglets at the age of 8 hours to 35 days belonging to different broods as well as 10 pigs for slaughter whose blood was collected after slaughtering out were included in the examination. In 5 piglets of one brood a progress examination was made during the suckling period. In the erythrocytes of all examined animals of different age groups the activity of the enzyme could be identified. There are great differences in the galactokinase activity of haemolysates of pig erythrocytes in different broods irrespective of their age. Therefore, an enzyme age dependence can only be recognized as a tendency from the comparison of the enzyme activities in erythrocytes of animals taken from different broods. In the erythrocyte haemolysate of 5 piglets of one brood the age dependence on galactokinase activity during the suckling period was determined in such a way that newborn piglets show a 30% higher enzyme activity than piglets at an age of 35 days. The enzyme activity in them, however, is still 1.6 times higher than in pigs for slaughtering. Thus, it may be concluded tht a postnatal decrease of galactokinase activity will occur in pig erythrocytes similarly to human erythrocytes. However, it is not responsible for a decrease of galactose utilization because no direct relations exist between both procedures. Parallel to glucose the decrease of galactose membrane permeability is assumed to play the decisive role here.