The ability of an LC-MS/MS-based erythrocyte GALT enzyme assay to predict the phenotype in subjects with GALT deficiency.

BACKGROUND: GALT deficiency is a rare genetic disorder of carbohydrate metabolism. Due to the decreased activity or absence of the enzyme galactose-1-phosphate uridylyltransferase (GALT), cells from affected individuals are unable to metabolize galactose normally. Lactose consumption in the newborn period could potentially lead to a lethal disease process with multi-organ involvement. In contrast to the newborn-stage disease, however, a galactose-restricted diet does not prevent long-term complications such as central nervous system (CNS) dysfunction with speech defects, learning disability and neurological disease in addition to hypergonadotropic hypogonadism or primary ovarian insufficiency (POI) in females. As the literature suggests an association between GALT enzyme activity and the long-term complications, it is of importance to have a highly sensitive assay to quantify the GALT enzyme activity. To that end, we had developed a sensitive and accurate LC-MS/MS method to measure GALT enzyme activity. Its ability to predict outcome is the subject of this report. MATERIALS AND METHODS: The GALT enzyme activity in erythrocytes from 160 individuals, in which 135 with classic, clinical variant or biochemical variant galactosemia, was quantified by LC-MS/MS. Individuals with GALT deficiency were evaluated for the long-term complications of speech defects, dysarthria, ataxia, dystonia, tremor, POI, as well as intellectual functioning (full scale IQ). The LC-MS/MS results were compared to a variety of assays: radioactive, [14C]-galactose-1-phosphate, paper chromatography with scintillation counting, enzyme-coupled assays with spectrophotometric or fluorometric readout or high-pressure liquid chromatography with UV detection of UDP-galactose. RESULTS: The LC-MS/MS method measured GALT activity as low as 0.2%, whereas other methods showed no detectable activity. Largely due to GALT activities that were over 1%, the LC-MS/MS measurements were not significantly different than values obtained in other laboratories using other methodologies. Severe long-term complications were less frequently noted in subjects with >1% activity. Patients with a p.Q188R/p.Q188R genotype have no residual enzyme activity in erythrocytes. CONCLUSION: Our LC-MS/MS assay may be necessary to accurately quantify residual GALT activities below 5%. The data suggest that patients with >1% residual activity are less likely to develop diet-independent long-term complications. However, much larger sample sizes are needed to properly assess the clinical phenotype in patients with residual enzyme activities between 0.1 and 5%.

Multiplex tandem mass spectrometry assay for newborn screening of X-linked adrenoleukodystrophy, biotinidase deficiency, and galactosemia with flexibility to assay other enzyme assays and biomarkers.

All States screen for biotinidase deficiency and galactosemia, and X-linked adrenoleukodystrophy (X-ALD) has recently been added to the Recommended Uniform Screening Panel (RUSP).We sought to consolidate these tests by combining them into a single multiplex tandem mass spectrometry assay as well as to improve the current protocol for newborn screening of galactosemia.A 3 mm punch of a dried blood spot (DBS) was extracted with organic solvent for analysis of the C26:0-lysophosphatidylcholine biomarker for X-ALD.An additional punch was used to assay galactose-1-phosphate uridyltransferase (GALT) and biotinidase.All assays were combined for a single injection for analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) (2.3 min per sample).The GALT LC-MS/MS assay does not give a false positive for galactosemia if glucose-6-phosphate dehydrogenase is deficient.The multiplex assay shows acceptable reproducibility and provides for rapid analysis of X-ALD, biotinidase deficiency, and galactosemia.The throughput and ease of sample preparation are acceptable for newborn screening laboratories.We also show that the LC-MS/MS assay is expandable to include several other diseases including Pompe and Hurler diseases (enzymatic activities and biomarkers).Because of consolidation of assays, less manpower is needed compared to running individual assays on separate platforms.The flexibility of the LC-MS/MS platform allows each newborn screening laboratory to analyze the set of diseases offered in their panel.

GALT Deficiency Galactosemia.

Galactosemia is an inborn error of galactose metabolism that results from a deficiency in one of three enzymes, uridine diphosphate galactose 4'epimerase, galactokinase, or galactose-1-phosphate uridyltransferase (GALT). This article focuses on classical, clinical variant, and biochemical variant (Duarte) galactosemias caused by GALT enzyme deficiency. A brief overview of galactosemia and newborn screening is presented, followed by detailed information about each of the conditions. Confirmatory testing, acute and long-term management, and outcome for these galactosemia types are discussed as well as the importance of genetic counseling and testing for the infant and family to refine reproductive risk.

Accurate prediction of gestational age using newborn screening analyte data.

BACKGROUND: Identification of preterm births and accurate estimates of gestational age for newborn infants is vital to guide care. Unfortunately, in developing countries, it can be challenging to obtain estimates of gestational age. Routinely collected newborn infant screening metabolic analytes vary by gestational age and may be useful to estimate gestational age. OBJECTIVE: We sought to develop an algorithm that could estimate gestational age at birth that is based on the analytes that are obtained from newborn infant screening. STUDY DESIGN: We conducted a population-based cross-sectional study of all live births in the province of Ontario that included 249,700 infants who were born between April 2007 and March 2009 and who underwent newborn infant screening. We used multivariable linear and logistic regression analyses to build a model to predict gestational age using newborn infant screening metabolite measurements and readily available physical characteristics data (birthweight and sex). RESULTS: The final model of our metabolic gestational dating algorithm had an average deviation between observed and expected gestational age of approximately 1 week, which suggests excellent predictive ability (adjusted R-square of 0.65; root mean square error, 1.06 weeks). Two-thirds of the gestational ages that were predicted by our model were accurate within ±1 week of the actual gestational age. Our logistic regression model was able to discriminate extremely well between term and increasingly premature categories of infants (c-statistic, >0.99). CONCLUSION: Metabolic gestational dating is accurate for the prediction of gestational age and could have value in low resource settings.

Predicting gestational age using neonatal metabolic markers.

BACKGROUND: Accurate gestational age estimation is extremely important for clinical care decisions of the newborn as well as for perinatal health research. Although prenatal ultrasound dating is one of the most accurate methods for estimating gestational age, it is not feasible in all settings. Identifying novel and accurate methods for gestational age estimation at birth is important, particularly for surveillance of preterm birth rates in areas without routine ultrasound dating. OBJECTIVE: We hypothesized that metabolic and endocrine markers captured by routine newborn screening could improve gestational age estimation in the absence of prenatal ultrasound technology. STUDY DESIGN: This is a retrospective analysis of 230,013 newborn metabolic screening records collected by the Iowa Newborn Screening Program between 2004 and 2009. The data were randomly split into a model-building dataset (n = 153,342) and a model-testing dataset (n = 76,671). We performed multiple linear regression modeling with gestational age, in weeks, as the outcome measure. We examined 44 metabolites, including biomarkers of amino acid and fatty acid metabolism, thyroid-stimulating hormone, and 17-hydroxyprogesterone. The coefficient of determination (R(2)) and the root-mean-square error were used to evaluate models in the model-building dataset that were then tested in the model-testing dataset. RESULTS: The newborn metabolic regression model consisted of 88 parameters, including the intercept, 37 metabolite measures, 29 squared metabolite measures, and 21 cubed metabolite measures. This model explained 52.8% of the variation in gestational age in the model-testing dataset. Gestational age was predicted within 1 week for 78% of the individuals and within 2 weeks of gestation for 95% of the individuals. This model yielded an area under the curve of 0.899 (95% confidence interval 0.895-0.903) in differentiating those born preterm (<37 weeks) from those born term (≥37 weeks). In the subset of infants born small-for-gestational age, the average difference between gestational ages predicted by the newborn metabolic model and the recorded gestational age was 1.5 weeks. In contrast, the average difference between gestational ages predicted by the model including only newborn weight and the recorded gestational age was 1.9 weeks. The estimated prevalence of preterm birth <37 weeks' gestation in the subset of infants that were small for gestational age was 18.79% when the model including only newborn weight was used, over twice that of the actual prevalence of 9.20%. The newborn metabolic model underestimated the preterm birth prevalence at 6.94% but was closer to the prevalence based on the recorded gestational age than the model including only newborn weight. CONCLUSIONS: The newborn metabolic profile, as derived from routine newborn screening markers, is an accurate method for estimating gestational age. In small-for-gestational age neonates, the newborn metabolic model predicts gestational age to a better degree than newborn weight alone. Newborn metabolic screening is a potentially effective method for population surveillance of preterm birth in the absence of prenatal ultrasound measurements or newborn weight.

Gestational dating by metabolic profile at birth: a California cohort study.

BACKGROUND: Accurate gestational dating is a critical component of obstetric and newborn care. In the absence of early ultrasound, many clinicians rely on less accurate measures, such as last menstrual period or symphysis-fundal height during pregnancy, or Dubowitz scoring or the Ballard (or New Ballard) method at birth. These measures often underestimate or overestimate gestational age and can lead to misclassification of babies as born preterm, which has both short- and long-term clinical care and public health implications. OBJECTIVE: We sought to evaluate whether metabolic markers in newborns measured as part of routine screening for treatable inborn errors of metabolism can be used to develop a population-level metabolic gestational dating algorithm that is robust despite intrauterine growth restriction and can be used when fetal ultrasound dating is not available. We focused specifically on the ability of these markers to differentiate preterm births (PTBs) (<37 weeks) from term births and to assign a specific gestational age in the PTB group. STUDY DESIGN: We evaluated a cohort of 729,503 singleton newborns with a California birth in 2005 through 2011 who had routine newborn metabolic screening and fetal ultrasound dating at 11-20 weeks' gestation. Using training and testing subsets (divided in a ratio of 3:1) we evaluated the association among PTB, target newborn characteristics, acylcarnitines, amino acids, thyroid-stimulating hormone, 17-hydroxyprogesterone, and galactose-1-phosphate-uridyl-transferase. We used multivariate backward stepwise regression to test for associations and linear discriminate analyses to create a linear function for PTB and to assign a specific week of gestation. We used sensitivity, specificity, and positive predictive value to evaluate the performance of linear functions. RESULTS: Along with birthweight and infant age at test, we included 35 of the 51 metabolic markers measured in the final multivariate model comparing PTBs and term births. Using a linear discriminate analyses-derived linear function, we were able to sort PTBs and term births accurately with sensitivities and specificities of ≥95% in both the training and testing subsets. Assignment of a specific week of gestation in those identified as PTBs resulted in the correct assignment of week ±2 weeks in 89.8% of all newborns in the training and 91.7% of those in the testing subset. When PTB rates were modeled using the metabolic dating algorithm compared to fetal ultrasound, PTB rates were 7.15% vs 6.11% in the training subset and 7.31% vs 6.25% in the testing subset. CONCLUSION: When considered in combination with birthweight and hours of age at test, metabolic profile evaluated within 8 days of birth appears to be a useful measure of PTB and, among those born preterm, of specific week of gestation ±2 weeks. Dating by metabolic profile may be useful in instances where there is no fetal ultrasound due to lack of availability or late entry into care.

Galactose-1-phosphate uridyltransferase dried blood spot quality control materials for newborn screening tests.

OBJECTIVES: We aimed to prepare dried-blood-spot (DBS) quality control (QC) materials for galactose-1-phosphate uridyltransferase (GALT), to evaluate their stability during storage and use, and to evaluate their performance in five DBS GALT test methods. DESIGN AND METHODS: We prepared and characterized GALT-normal and GALT-deficient DBS materials and compared GALT activities in DBSs after predetermined storage intervals at controlled temperatures and humidities. External evaluators documented the suitability of the DBS QC materials for use in five GALT test methods. RESULTS: GALT activity losses from DBSs stored in low (<30%) humidity for 14 days at 45°C, 35 days at 37°C, 91 days at room temperature, 182 days at 4°C, and 367 days at -20°C were 54%, 53%, 52% 23%, and 7% respectively. In paired DBSs stored in high humidity (>50%) for identical intervals, losses were: 45°C-68%; 37°C-79%; room temperature-72%, and 4°C-63%. GALT activities in DBSs stored at 4°C were stable throughout 19 excursions to room temperature. Twenty-five of 26 external evaluators, using five different GALT test methods, classified the GALT-deficient DBSs as "outside normal limits". All evaluators classified the GALT-normal DBSs as "within normal limits". CONCLUSIONS: Most of the GALT activity loss from DBSs stored at elevated or room temperature was attributable to the effects of storage temperature. Most of the loss from DBSs stored at 4°C was attributable to the effects of elevated humidity. Loss from DBSs stored at -20°C was insignificant. The DBS materials were suitable for monitoring performance of all five GALT test methods.

Galactosemia: when is it a newborn screening emergency?

Classic galactosemia is an autosomal recessive disorder of carbohydrate metabolism, due to a severe deficiency of the enzyme, galactose-1-phosphate uridyltransferase (GALT), that catalyzes the conversion of galactose-1-phosphate and uridine diphosphate glucose (UDPglucose) to uridine diphosphate galactose (UDPgalactose) and glucose-1-phosphate. Upon consumption of lactose in the neonatal period, the affected infants develop a potentially lethal disease process with multiorgan involvement. Since the advent of newborn screening (NBS) for galactosemia, we rarely encounter such overwhelmingly ill newborns. After ascertainment that the positive NBS indicates the possibility of galactosemia due to GALT deficiency, the critical question for the physician is whether the infant has the classic or a variant form of GALT deficiency, as classic galactosemia is a medical emergency. However, there are over 230 GALT gene mutations that have been detected around the world. Yet, most positive NBS tests are due to the Duarte biochemical variant condition or a simple false positive. In order to make the correct decision as well as provide informative counseling to parents of infants with a positive NBS, I utilize a relatively simple classification scheme for GALT deficiency. There are three basic forms of GALT deficiency: 1) classic galactosemia; 2) clinical variant galactosemia; and 3) biochemical variant galactosemia. The classic genotype is typified by Q188R/Q188R, the clinical variant by S135L/S135L and the biochemical variant by N314D/Q188R. In classic galactosemia, the erythrocyte GALT enzyme activity is absent or markedly reduced, the blood galactose and erythrocyte galactose-1-phosphate levels are markedly elevated, and the patient is at risk to develop potentially lethal E. coli sepsis, as well as the long-term diet-independent complications of galactosemia. Patients with the clinical variant form require treatment but do not die from E. coli sepsis in the neonatal period. If the clinician suspects galactosemia, even if based on clinical findings alone, then the infant should be immediately placed on a lactose-restricted diet. The purpose of this review is to help the clinician make the correct therapeutic decision after an NBS test has returned positive for galactosemia.

The stability of markers in dried-blood spots for recommended newborn screening disorders in the United States.

OBJECTIVE: We aimed to measure separately the contributions of heat and humidity to changes in levels of 34 markers of inborn disorders in dried-blood-spot (DBS) samples. DESIGN AND METHODS: We stored paired sets of DBSs at 37°C for predetermined intervals in low-humidity and high-humidity environments. Marker levels of all samples in each complete sample set were measured in a single analytic run. RESULTS: During the 30 ± 5 day studies, galactose-1-phosphate uridyltransferase and biotinidase lost almost 65% of initial activities in low-humidity storage; most of the degradation in 27 other markers was attributable to adverse effects of high-humidity storage; seven markers in DBSs stored at high humidity lost more than 90% of initial levels by the end of the study and 4 of the 7 lost more than 50% of initial levels within the first week of storage. CONCLUSIONS: Minimizing both humidity and temperature in DBS transportation and storage environments is essential to maintaining sample integrity.

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.

Direct non-radioactive assay of galactose-1-phosphate:uridyltransferase activity using high performance liquid chromatography.

BACKGROUND: Galactose-1-phosphate:uridyltransferase (GALT) catalyses the conversion of galactose-1-phosphate (Gal-1-P) and UDP-glucose (UDP-Glc) into glucose-6-phosphate and UDP-galactose (UDP-Gal). Complete, or near complete, deficiency of GALT causes classic galactosaemia. The diagnosis is confirmed by measuring GALT activity in erythrocytes. The most commonly used assays require radio labelled substrates or indirect coupled assays. METHODS: GALT activity was measured in erythrocyte lysates using optimal concentrations of the substrates galactose-1-phosphate and UDP-Glc. UDP-Gal and UDP-Glc were separated using reversed-phase high performance liquid chromatography with UV detection. Clinical validity was assessed using blood samples from galactosaemic patients. RESULTS: UDP-Gal and UDP-Glc were separated with HPLC. The assay was linear with incubation times up 80 min and between 0 and 42.5 nmol haemoglobin. Within-day and between-day imprecision at 50, 75 and 100% enzyme activity was <1.4% and <2.4%, respectively. Mean GALT activity in 33 individuals was 601+/-79 nmol UDP-Gal/(micromol Hb.h) (range 492-697). Patients with classical galactosaemia were easily detected by their extremely low activity. CONCLUSIONS: We have developed a reliable and convenient direct method to measure GALT activity in human erythrocytes using HPLC with UV detection.

Newborn screening for galactosemia: a review of 5 years of data and audit of a revised reporting approach.

BACKGROUND: Availability of the galactose-1-phosphate uridyltransferase (GALT) assay for newborn (NB) screening has improved identification of classic galactosemia. Previously defined critical cutoffs for total galactose (Gal), typically 1.110 mmol/L (20 mg/dL), are still in use in laboratories measuring total Gal for the diagnosis of nonclassic galactosemias. Urgent notification/referral to a treatment center follows, although few of the NBs will need treatment. METHODS: We reviewed all NB galactosemia-screening results and their corresponding clinical outcomes over a 5-year period (first phase, 1.32 x 10(6) NBs) and then over a 2-year period (second phase, 274 960 NBs). Each NB was screened for Gal and GALT. When Gal was increased and/or GALT was deficient, testing for percentage galactose-1-phosphate and/or DNA testing for common GALT mutations were performed. RESULTS: Of 209 reported positive results, 89% did not indicate GALT deficiency. These non-GALT-deficient results represented mostly clinically benign cases with a Gal threshold of > or = 1.110 mmol/L (> or = 20 mg/dL). The positive predictive value of a GALT cutoff of < or = 40 micromol/L was 83%. After a protocol change that redefined a critical result as a GALT value < or = 40 micromol/L and/or a Gal value > or = 1.665 mmol/L (> or = 30 mg/dL), results were monitored for an additional 2 years. The new protocol dramatically reduced the number of urgent calls/referrals and reduced the total number of referrals by nearly half. CONCLUSIONS: Use of a GALT cutoff of < or = 40 micromol/L/L and a Gal cutoff of > or = 1.665 mmol/L (> or = 30 mg/dL) for urgent notification/referral dramatically reduces false positives and unnecessary follow-up, thereby reducing the stress on healthcare resources.

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.

Functional analysis of the human galactose-1-phosphate uridyltransferase promoter in Duarte and LA variant galactosemia.

Human galactose-1-phosphate uridyltransferase (hGALT) is an evolutionarily conserved enzyme central to D-galactose metabolism. The impairment of hGALT causes galactosemia. One missense mutation, an aspartate to asparagine substitution at amino acid 314 (N314D), impairs 50% activity in the homozygous state in some patients but gives near normal activity in others. The former condition is called Duarte (D) and the latter, Los Angeles (LA). The D allele is linked to hGALT polymorphisms including a deletion 5'to the translation start site (-119 to -116delGTCA), g1391G --> A and g1105G --> C. The LA allele is linked to a g1721C --> T transition. To investigate possible mechanisms for differences in hGALT activity between the D and LA alleles, we sequenced 3951 nucleotides of genomic DNA 5' to the hGALT translation start site. Using a dual-luciferase reporter system to express deletion constructs of the hGALT promoter, we noted both positive and negative regulatory regions. Two putative positive regulatory domains overlap with the naturally occurring -119 to -116delGTCA linked to Duarte. One is an E-box motif (CACGTG) at -117 to -112 bp. The second is an AP-1 motif (TCAGTCAG) at -124 to -119 bp. The delGTCA mutation confers reduced luciferase activity to transfected cell lines derived from human ovarian and liver neoplasms. Additionally, human lymphoblasts derived from patients with the Duarte allele have reduced GALT mRNA. We conclude that the human GALT gene is regulated in the first -165 bp of its promoter region by positive regulators of GALT gene expression. The -119 to -116delGTCA reduces hGALT transcription resulting in reduced GALT activity in the Duarte allele.

Risk factors for premature ovarian failure in females with galactosemia.

UNLABELLED: The risk for premature ovarian failure (POF) in females with galactosemia can be predicted by analyzing 3 areas of risk pathology: the patient's molecular genotype for galactose-1-phosphate uridyltransferase (GALT), alternate pathways for galactose metabolism, and the patient's environment at diagnosis and during treatment. STUDY DESIGN: Retrospective cross-sectional information was collected on 53 females with classic galactosemia, and their ovarian function was analyzed by determination of serum follicle-stimulating hormone and luteinizing hormone levels and by clinical observation. The associations were analyzed between POF and the mutations in GALT, the highest erythrocyte galactose-1-phosphate (Gal-1-P) level at diagnosis, the age at which dietary treatment was initiated, mean erythrocyte Gal-1-P level during treatment, and whole-body carbon 13-labeled galactose oxidation to (13)CO(2). RESULTS: The most prevalent mutation, Q188R, had a significant effect of genotype category (Q188R/Q188R, Q188R/Other, Other/Other) on POF (P =.04, Fisher exact test and an odds ratio of 8.3). Mean erythrocyte Gal-1-P level during treatment was a significant risk factor for POF (P =.04). Also, all patients studied with less than 5% total body oxidation of galactose to (13)CO(2) had POF, whereas those with more than 5% did not have POF (P =.008, Fisher exact test). CONCLUSION: The development of POF in females with galactosemia is more likely if the patient's genotype is Q188R/Q188R, if the mean erythrocyte Gal-1-P is >3.5 mg/dL during therapy, and if the recovery of (13)CO(2) from whole-body (13)C-galactose oxidation is reduced below 5% of administered (13)C-galactose.

Plasma galactose and galactitol concentration in patients with galactose-1-phosphate uridyltransferase deficiency galactosemia: determination by gas chromatography/mass spectrometry.

The plasma concentration of galactose and galactitol was measured in 27 patients with galactose-1-phosphate uridyltransferase (GALT) deficiency galactosemia on a lactose-restricted diet, 17 infants on lactose-free formula, and 21 infants and children on a normal diet, by a newly devised isotope dilution gas chromatograph/mass spectrometry (GC/MS) method. The method was linear in the range of 0.1 to 10 micromol/L for galactose and 1 to 20 micromol/L for galactitol with good reproducibility and a coefficient of variation less than 3%. The mean plasma galactose in 15 patients who were homozygous for the most common Q188R mutation of the GALT gene was 2.72 +/- 0.70 micromol/L (mean +/- SE) with a range of 0.58 to 3.98 in specimens obtained at regular clinic visits. In 12 patients with other GALT mutations, it was 2.45 +/- 0.75 micromol/L. The mean value in nongalactosemic subjects on lactose-free formula was 0.52 +/- 0.08 micromol/L, with a range of 0.12 to 1.25. The range in 21 normal subjects without diet restriction was 0.11 to 6.33 micromol/L, with a mean of 1.48 +/- 0.32. The plasma galactitol level was 11.63 +/- 0.46 and 10.85 +/- 1.38 micromol/L in the 2 galactosemic groups. There was no relationship between plasma galactose and galactitol levels, with variable ratios of the two substances in the galactosemic patients. Galactitol was not detectable in the plasma of normal subjects. The red blood cell galactose-1-phosphate level was also measured in the galactosemic patients, and no relationship between plasma galactose and red blood cell galactose-1-phosphate was found. The galactose-1-phosphate concentration was 28 to 54 times higher than the ambient galactose. The low galactose concentration in the plasma of galactosemics on galactose-restricted diets in relation to the higher plasma galactitol and red blood cell galactose-1-phosphate is a metabolic enigma. The ability to measure plasma galactose accurately presents a new way of characterizing the galactosemic patient and the levels monitored over time may provide insight into the development of long-term complications associated with the disorder.

Quantitative Beutler test for newborn mass screening of galactosemia using a fluorometric microplate reader.

BACKGROUND: The Beutler enzyme spot test is an effective assay for newborn mass screening of galactosemia, but it is qualitative and relies on visual interpretation. We describe a quantitative, instrumental modification of the assay. METHODS: We modified the macroscopic visual Beutler enzyme spot test by adding extraction of blood components from filter paper, deproteinization with acetone-methanol, and quantification and recording by a fluorescent microplate reader and personal computer. All handling was performed in microplates. The measurement time was 90 min. RESULTS: Fluorescence intensity (FI) of healthy controls correlated with hematocrit and galactose-1-phosphate uridyltransferase (GALT) activity. Patients with GALT deficiency were distinguished clearly from healthy subjects and heterozygous carriers by FI. FI decreased to 75% of the initial activity after storage at 25 degrees C for 3 days and to 40% after storage at 37 degrees C for 7 days. Screening of 46 742 newborns yielded 1 false-positive result (in a heterozygous carrier), 1 patient with glucose-6-phosphate dehydrogenase deficiency, and no apparent false negatives as judged by concurrent measurements of galactose and galactose-1-phosphate. CONCLUSIONS: The quantitative Beutler test can provide precise GALT activity in newborn mass screening, and can take into consideration the influence of high temperature and humidity, duration between sampling and testing, and anemia. This method is clinically useful, simple, automated, and highly reliable for newborn mass screening of galactosemia.

Newborn screening for galactosemia: ultramicro assay for galactose-1-phosphate-uridyltransferase activity.

An enzymatically optimized, miniaturized (20 microl) fluorimetric assay of galactose-1-phosphate-uridyltransferase using dried blood spots for newborn screening is presented. The Beutler reaction principle has been adapted to the microtiter plate technology and acetone/methanol was used for complete deproteinization. A special ultramicro multiwell screening plate resistant to organic solvents has been developed and employed. The assay is simple, sensitive and inexpensive, due to small reagent volumes and the low prices of ultramicro screening plates. The reaction is linear with galactose-1-phosphate-uridyltransferase activity up to 120 min of incubation time. It shows low imprecision and good correlation to a quantitative validation test. For standardization the use of plate means or medians of activity or fluorescence values is proposed. Individual blank measurement prevents false negative assessments.