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Competing Interests: Declaration of Interests T.B. has nothing to disclose. R. Kastury has nothing to disclose. R. Kavarthapu has nothing to disclose. R.B. has nothing to disclose. M.D.L.L.S. has nothing to disclose. H.L. has nothing to disclose. C.A. has nothing to disclose. J.C.Y. has nothing to disclose. V.G.-L. has nothing to disclose.

Galactosemia, a severe genetic metabolic disorder, results from the absence of galactose-degrading enzymes, leading to harmful galactose accumulation. In this study, we introduce a novel capillary-based surface-enhanced Raman spectroscopy (SERS) sensor for convenient and sensitive galactose detection. The developed sensor enhances SERS signals by introducing gold nanoparticles (Au NPs) onto the surface of silver nanoshells (Ag NSs) within a capillary, creating Ag NSs with Au NPs as satellites. Utilizing 4-mercaptophenylboronic acid (4-MPBA) as a Raman reporter molecule, the detection method relies on the conversion of 4-MPBA to 4-mercaptophenol (4-MPhOH) driven by hydrogen peroxide (H 2 O 2 ) generated during galactose oxidation by galactose oxidase (GO x ). A new SERS signal was observed, which was generated by H 2 O 2 produced when galactose and GO x reacted. Our strategy yielded a quantitative change in the SERS signal, specifically in the band intensity ratio of 998 to 1076 cm -1 (I 998 /I 1076 ) as the galactose concentration increased. Our capillary-based SERS biosensor provides a promising platform for early galactosemia diagnosis., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

Galactosemia is a rare, treatable hereditary disorder of carbohydrate metabolism. We investigated the etiology of decreased GALT enzyme activity in a cohort of newborns referred by the Florida Newborn Screening Program with no detectable GALT variants in diagnostic molecular tests. Six affected individuals from four families with Guatemalan heritage were included. GALT enzyme activity ranged from 20% to 34% of normal. Clinical findings were unremarkable except for speech delay in two children. Via genome sequencing followed by Sanger confirmation we showed that all affected individuals were homozygous for a deep intronic GALT variant, c.1059+390A>G, which segregated as an autosomal recessive trait in all families. The intronic variant disrupts splicing and leads to a premature termination and is associated with a single haplotype flanking GALT, suggesting a founder effect. In conclusion, we present a deep intronic GALT variant leading to a biochemical variant form of galactosemia. This variant remains undiagnosed until it is specifically targeted in molecular testing., (© 2020 SSIEM.)

Background: The pathogenesis of classical galactosemia, a rare metabolic disorder associated with developmental complications in neonates and children due to inherited deficiency of galactose-1-phosphate (Gal-1-P) uridylyltransferase (GALT), is known to be mediated by elevated Gal-1-P levels and involves a cascade of cytokines, reactive oxygen species (ROS) and growth factors., Objectives: To examine ex vivo the effect of Gal-1-P on the mitogenic activity of different growth factors, particularly insulin-like growth factor-1 (IGF-1), known to regulate growth and development from the fetal stage to adulthood., Material and Methods: Fibroblasts derived from the foreskin of 3-8-day-old healthy neonates were cultured for 1-14 days with 0-20 mM galactose or 0-10 mM Gal-1-P and then stimulated with 5% fetal bovine serum (FBS) or 50 ng/mL of platelet-derived growth factor (PDGF) or fibroblast growth factor (FGF) or IGF-1 for 24 h. DNA synthesis was measured and protein expression of PDGFR, FGFR and IGF-1R was assessed with western blotting., Results: Supra-physiological concentrations of galactose significantly decreased FBSand IGF-1-induced BrdU incorporation. The presence of Gal-1-P (5-10 mM) in culture medium for 7-14 days significantly (p < 0.01) decreased IGF-1-, PDGFand FBS-stimulated DNA synthesis. While treatment with Gal-1-P selectively and significantly (p < 0.01) reduced the protein expression of IGF-1 receptor, galactose treatment did not have any marked effect on examined growth factor receptors., Conclusions: This study demonstrates that Gal-1-P impairs IGF-1 activity through IGF-1-receptor impairment, thereby providing a new insight into the molecular mechanisms of galactosemia pathogenesis.

: media-1vid110.1542/5849572227001PEDS-VA&#95;2018-2516 Video Abstract OBJECTIVES: For decades, infants with Duarte galactosemia (DG) have been identified by newborn screening (NBS), but whether they should be treated with dietary restrictions of galactose has remained unknown. To clarify, we conducted a study of dietary and developmental outcomes in 206 children with DG (case patients) and 144 controls, all of whom were 6 to 12 years old., Methods: We recruited case patients from states where they were identified by NBS; unaffected siblings served as controls. Diet in infancy was ascertained by retrospective parent surveys; developmental outcomes were assessed in 5 domains, yielding 73 outcome measures for each child. We divided subjects randomly into independent discovery ( n = 87) and validation ( n = 263) sets. We tested the discovery set to order the 73 outcome measures by ascending P values and tested the 10 outcomes with the lowest P values for possible association with DG in the validation set. We also tested these same 10 outcomes for possible association with milk exposure in infancy among case patients in the validation set., Results: None of the 73 outcomes tested in the discovery set revealed significant association with DG, and none of the 10 outcomes tested in the validation set revealed either significant association with DG or significant association with milk exposure among children with DG., Conclusions: Through our results, we demonstrated that there were no significant differences in outcomes tested between case patients and controls or among case patients as a function of milk exposure in infancy. In this study, we provide a long-needed foundation of knowledge for health care providers, families, and NBS professionals seeking to make evidence-based decisions about DG., Competing Interests: POTENTIAL CONFLICT OF INTEREST: The authors have indicated they have no potential conflicts of interest to disclose., (Copyright © 2019 by the American Academy of Pediatrics.)

Impaired activity of galactose-1-phosphate uridyltransferase (GALT) causes classic galactosemia (OMIM 230400), characterized by the accumulation of galactose-1-phosphate (GAL1P) in patients' red blood cells (RBCs). Our recent study demonstrated a correlation between RBC GAL1P and long-term outcomes in galactosemia patients. Here, we analyze biochemical and molecular results in 77 classic galactosemia patients to evaluate the association between GALT genotypes and GAL1P concentration in RBCs. Experimental data from model organisms were also included to assess the correlation between GAL1P and predicted residual activity of each genotype. Although all individuals in this study showed markedly reduced RBC GALT activity, we observed significant differences in RBC GAL1P concentrations among galactosemia genotypes. While levels of GAL1P on treatment did not correlate with RBC GALT activities (p = 0.166), there was a negative nonlinear correlation between mean GAL1P concentrations and predicted residual enzyme activity of genotype (p = 0.004). These studies suggest that GAL1P levels in RBCs on treatment likely reflect the overall functional impairment of GALT in patients with galactosemia., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Classical Galactosaemia (CG) (OMIM #230400) is a rare inborn error of galactose metabolism caused by deficiency of the enzyme galactose-1-phosphate uridylyltransferase (GALT). Long-term complications persist in treated patients despite dietary galactose restriction with significant variations in outcomes suggesting epigenetic glycosylation influences. Primary Ovarian Insufficiency (POI) is a very significant complication affecting females with follicular depletion noted in early life. We studied specific glycan synthesis, leptin system and inflammatory gene expression in white blood cells as potential biomarkers of infertility in 54 adults with CG adults (27 females and 27 males) (age range 17-51 yr) on a galactose-restricted diet in a multi-site Irish and Dutch study. Gene expression profiles were tested for correlation with a serum Ultra-high Performance Liquid Chromatography (UPLC)-Immunoglobulin (IgG)-N-glycan galactose incorporation assay and endocrine measurements., Results: Twenty five CG females (93%) had clinical and biochemical evidence of POI. As expected, the CG female patients, influenced by hormone replacement therapy, and the healthy controls of both genders showed a positive correlation between log leptin and BMI but this correlation was not apparent in CG males. The strongest correlations between serum leptin levels, hormones, G-ratio (galactose incorporation assay) and gene expression data were observed between leptin, its gene and G-Ratios data (r s  = - 0.68) and (r s  = - 0.94) respectively with lower circulating leptin in CG patients with reduced IgG galactosylation. In CG patients (males and females analysed as one group), the key glycan synthesis modifier genes MGAT3 and FUT8, which influence glycan chain bisecting and fucosylation and subsequent cell signalling and adhesion, were found to be significantly upregulated (p < 0.01 and p < 0.05) and also the glycan synthesis gene ALG9 (p < 0.01). Both leptin signalling genes LEP and LEPR were found to be upregulated (p < 0.01) as was the inflammatory genes ANXA1 and ICAM1 and the apoptosis gene SEPT4 (p < 0.01)., Conclusions: These results validate our previous findings and provide novel experimental evidence for dysregulation of genes LEP, LEPR, ANXA1, ICAM1 and SEPT4 for CG patients and combined with our findings of abnormalities of IgG glycosylation, hormonal and leptin analyses elaborate on the systemic glycosylation and cell signalling abnormalities evident in CG which likely influence the pathophysiology of POI.

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Impaired activity of galactose-1-phosphate uridyltransferase (GALT) causes galactosemia, an autosomal recessive disorder of galactose metabolism. Early initiation of a galactose-restricted diet can prevent or resolve neonatal complications. Despite therapy, patients often experience long-term complications including speech impairment, learning disabilities, and premature ovarian insufficiency in females. This study evaluates clinical outcomes in 34 galactosemia patients with markedly reduced GALT activity and compares outcomes between patients with different levels of mean galactose-1-phosphate in red blood cells (GAL1P) using logistic regression: group 1 (n = 13) GAL1P ≤1.7 mg/dL vs. group 2 (n = 21) GAL1P ≥ 2 mg/dL. Acute symptoms at birth were comparable between groups (p = 0.30) with approximately 50% of patients presenting with jaundice, liver failure, and failure-to-thrive. However, group 2 patients had significantly higher prevalence of negative long-term outcomes compared to group 1 patients (p = 0.01). Only one of 11 patients >3 yo in group 1 developed neurological and severe behavioral problems of unclear etiology. In contrast, 17 of 20 patients >3 yo in group 2 presented with one or more long-term complications associated with galactosemia. The majority of females ≥15 yo in this group also had impaired ovarian function with markedly reduced levels of anti-Müllerian hormone. These findings suggest that galactosemia patients with higher GAL1P levels are more likely to have negative long-term outcome. Therefore, evaluation of GAL1P levels on a galactose-restricted diet might be helpful in providing a prognosis for galactosemia patients with rare or novel genotypes whose clinical presentations are not well known.

Classical galactosaemia (OMIM #230400), a rare disorder of carbohydrate metabolism, is caused by a deficient activity of galactose-1-phosphate uridyltransferase (EC 2.7.7.12). The pathophysiology of the long-term complications, mainly cognitive, neurological and female fertility problems remains poorly understood. The lack of validated biomarkers to determine prognosis, monitor disease progression and responses to new therapies, pose a huge challenge. We report the detailed analysis of an automated robotic hydrophilic interaction ultra-performance liquid chromatography N-glycan analytical method of high glycan peak resolution applied to serum IgG. This has revealed specific N-glycan processing defects observed in 40 adult galactosaemia patients (adults and adolescents), in comparison with 81 matched healthy controls. We have identified a significant increase in core fucosylated neutral glycans (P<0.0001) and a significant decrease in core fucosylated (P<0.001), non-fucosylated (P<0.0001) bisected glycans and, of specific note, decreased N-linked mannose-5 glycans (P<0.0001), in galactosaemia patients. We also report the abnormal expression of a number of related relevant N-glycan biosynthesis genes in peripheral blood mononuclear cells from 32 adult galactosaemia patients. We have noted significant dysregulation of two key N-glycan biosynthesis genes: ALG9 upregulated (P<0.001) and MGAT1 downregulated (P<0.01) in galactosaemia patients, which may contribute to its ongoing pathophysiology. Our data suggest that the use of IgG N-glycosylation analysis with matched N-glycan biosynthesis gene profiles may provide useful biomarkers for monitoring response to therapy and interventions. They also indicate potential gene modifying steps in this N-glycan biosynthesis pathway, of relevance to galactosaemia and related N-glycan biosynthesis disorders.

The quantitative measurement of galactose in blood is essential for the early diagnosis, treatment, and dietary monitoring of galactosemia patients. In this communication, we aimed to develop a rapid, sensitive, and cost-effective combined method for galactose determination in dry blood spots. This procedure was based on the combination of enzymatic reactions of galactose dehydrogenase (GalDH), dihydrolipoyl dehydrogenase (DLD), and alkaline phosphates with a colorimetric system. The incubation time and the concentration of enzymes used in new method were also optimized. The analytical performance was studied by the precision, recovery, linearity, and sensitivity parameters. Statistical analysis was applied to method comparison experiment. The regression equation and correlation coefficient (R (2)) were Y = 0.0085x + 0.032 and R (2) = 0.998, respectively. This assay exhibited a recovery in the range of 91.7-114.3 % and had the limit detection of 0.5 mg/dl for galactose. The between-run coefficient of variation (CV) was between 2.6 and 11.1 %. The within-run CV was between 4.9 and 9.2 %. Our results indicated that the new and reference methods were in agreement because no significant biases exist between them. Briefly, a quick and reliable combined enzymatic and colorimetric assay was presented for application in newborn mass screening and monitoring of galactosemia patients.

Background: Galactosemia is a severe metabolic disorder known to cause hepatosplenomegaly, jaundice and cataracts in neonates, and many patients develop later complications such as mental retardation, disorders of motor function or speech, and hypergonadotrophic hypogonadism. The pathogenetic mechanisms of classical galactosemia are unclear; however, nitric oxide (NO) has been suggested to play a role., Objectives: Insulin-like growth factor-1 (IGF-1) is important for the growth and development of children, and the aim of this study was to examine the association of NO production with IGF-1 gene expression under galactosemic conditions., Methods: Serum levels of IGF-1 and nitrite were measured in 15 galactosemia patients and 15 age- and gender-matched healthy controls. Fibroblast cultures established from postcircumcision foreskin of 3- to 8-day-old healthy neonates were treated for 72 h with 0-10 mM of galactose or 0-5 mM of galactose-1-phosphate (Gal-1-P) in the presence or absence of NO synthase inhibitor (L-NAME), and inducible NO synthase (iNOS) protein was measured using Western blot analysis. RT-PCR was performed to assess the IGF-1 gene expression., Results: Galactosemia patients were observed to have significantly (p < 0.01) elevated serum nitrites and markedly decreased levels (p < 0.01) of serum IGF-1 as compared to healthy controls. The cotreatment of neonate skin fibroblast cultures with galactose and Gal-1-P significantly (p < 0.01) increased cellular levels of NO and iNOS protein expression, and decreased (p < 0.01) IGF-1 mRNA levels. Treatment with L-NAME, a NOS inhibitor, significantly (p < 0.05) alleviated a galactose/Gal-1-P-induced decrease in IGF-1 mRNA levels., Conclusion: These results suggest that NO mediates the downregulation of IGF-1 by Gal-1-P/galactose, thereby providing a new molecular mechanism and possible therapeutic insight for galactosemia-related complications., (© 2016 S. Karger AG, Basel.)

Purpose of Review: Galactose - a key source of energy and a crucial structural element in complex molecules - is particularly important for early human development. However, galactose metabolism might be important not only for fetal and neonatal development but also for adulthood, as evidenced by the inherited disorders of galactose metabolism. The purpose of this review is to summarize the current evidence of galactose metabolism in health and disease., Recent Findings: The biological importance of galactose goes beyond its importance as a nutrient and a metabolite. Galactose has been selected by evolutionary pressure to exert also a crucial structural role in macromolecules. Additionally, galactose has recently been reported as beneficial in a number of diseases, particularly in those affecting the brain., Summary: Galactose is crucial for human metabolism, with an established role in energy delivery and galactosylation of complex molecules, and evidence for other roles is emerging.

Background: Classic galactosemia (CG) is a potentially lethal genetic disorder that results from profound loss of galactose-1-phosphate uridylyltransferase (GALT). CG is detected by newborn screening (NBS) in many countries; however, conclusive diagnosis can be complex due to broad and overlapping ranges of GALT activity. Molecular studies can also be complex due to allelic heterogeneity at the GALT locus., Methods: We conducted both biochemical and molecular follow-up studies for an infant flagged by NBS for possible galactosemia. To clarify the diagnosis we also conducted biochemical and RNA studies of lymphoblasts prepared from the child and one parent., Results: We identified a novel noncoding GALT variant, c.377+17C>T, that was homozygous in the child and heterozygous in both parents. The child and both parents also showed diminished GALT activity in red blood cells, and transformed lymphoblasts from the child and one parent further showed diminished GALT activity. However, qRT-PCR studies demonstrated apparently normal GALT mRNA levels in lymphoblasts, and Gal-1P values measured in the child following galactose exposure in infancy and at 1 year were normal., Conclusions: These results highlight the existence of rare but apparently benign variants in GALT and underscore the need for functional studies to distinguish pathogenic from benign variants., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Classic galactosemia is an autosomal recessive metabolic disease involving the galactose pathway, caused by the deficiency of galactose-1-phosphate uridyltransferase. Galactose accumulation induces in newborns many symptoms, such as liver disease, cataracts, and sepsis leading to death if untreated. Neonatal screening is developed and applied in many countries using several methods to detect galactose or its derived product accumulation in blood or urine. High-throughput FTIR spectroscopy was investigated as a potential tool in the current screening methods. IR spectra were obtained from blood plasma of healthy, diabetic, and galactosemic patients. The major spectral differences were in the carbohydrate region, which was first analysed in an exploratory manner using principal component analysis (PCA). PCA score plots showed a clear discrimination between diabetic and galactosemic patients and this was more marked as a function of the glucose and galactose increased concentration in these patients' plasma respectively. Then, a support vector machine leave-one-out cross-validation (SVM-LOOCV) classifier was built with the PCA scores as the input and the model was tested on median, mean and all spectra from the three population groups. This classifier was able to discriminate healthy/diabetic, healthy/galactosemic, and diabetic/galactosemic patients with sensitivity and specificity rates ranging from 80% to 94%. The total accuracy rate ranged from 87% to 96%. High-throughput FTIR spectroscopy combined with the SVM-LOOCV classification procedure appears to be a promising tool in the screening of galactosemia patients, with good sensitivity and specificity. Furthermore, this approach presents the advantages of being cost-effective, fast, and straightforward in the screening of galactosemic patients.

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., (Copyright © 2014 The Canadian Society of Clinical Chemists. All rights reserved.)

A number of factors may lead to inaccuracy in measurement of capillary blood glucose with a glucometer. Measurement of other carbohydrate molecules such as galactose and fructose along with glucose can potentially be a cause of error. We report a newborn patient who was referred to our hospital with conjugated bilirubinemia, hepatomegaly and high capillary blood glucose levels measured with a glucometer. Simultaneous biochemical measurements revealed normal blood glucose levels. Further investigation led to a diagnosis of classical galactosemia. Capillary blood glucose level measured with glucometer also dropped to normal values following cessation of breastfeeding and initiation of feeding with a lactose-free formula.

Patients with classical galactosaemia (galactose-1-phosphate uridyltransferase (GALT) deficiency) manifest clinical complications despite strict dietary galactose restriction. Therefore the significance of endogenous galactose production has been assessed. Previous in vivo studies primarily focused on patients homozygous for the most common genetic variant Q188R but little is known about other genetic variants. In the present study the endogenous galactose release in a group of non-Q188R homozygous galactosaemic patients (n = 17; 4-34 years) exhibiting comparably low residual GALT activity in red blood cells was investigated. Primed continuous infusion studies with D-[1-(13)C]galactose as substrate were conducted under post-absorptive conditions and in good metabolic control. The results demonstrate that all patients exhibiting residual GALT activity of <1.5% of control showed a comparable pathological pattern of increased endogenous galactose release irrespective of the underlying genetic variations. Possible implications of the findings towards a more differentiated dietary regimen in galactosaemia are discussed.

We present a robust clinical assay for the measurement of red blood cell uridine diphosphate galactose-4-epimerase enzyme activity for the diagnostic confirmation of patients positive for a newborn screen for inherited galactosemia in whom galactose-1-phosphate uridyltransferase activity is normal. Previous assays required the use of ion-pairing reagents and frequent need for system maintenance that was not appropriate for heavy clinical use where patient results should be quickly available. We have designed a two-step enzyme assay which converts stable-isotope-labeled UDP-galactose to isotope-labeled-UDP-glucose which is converted in the second reaction to the final product of [(13)C6]-UDP-glucuronic acid. Measurement conditions t remove potential interference from endogenous UDP-glucose and UDP-galactose. We also report a significant ion suppression effect of the red cell preparation for which we have optimized assay sample volume to minimize this effect., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Distinction between asymptomatic and potentially clinically significant forms of galactosemia due to UDP-galactose 4'-epimerase (GALE) deficiency requires enzyme measurement in erythrocytes and other cells. We sought to develop a GALE assay using a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method., Methods: The reversible GALE assay was conducted with UDPGal as a substrate. The coeluting reaction product, uridine diphosphate glucose (UDPGlc), and its isomeric substrate, uridine diphosphate galactose (UDPGal), were detected by MS/MS at mass transitions 565 > 280, 565 > 241 and 565 > 403. The UDPGal was enriched in mass transition 565 > 403 compared with UDPGlc, whereas the UDPGlc was enriched in the mass transition 565 > 241 compared with UDPGal. The percentage of UDPGal in the reaction mixture was calculated by use of the ratio of ion intensities of the 2 daughter ions and a fourth-order polynomial calibrator curve., Results: The method yielded a mean (SD) GALE activity of 9.8 (2.2) μmol · g(-1) hemoglobin · h(-1) in erythrocyte extracts from 27 controls. The apparent Km of the substrate, UDPGal, was 0.05 mmol/L. The GALE activity ranged from 433 to 993 μmol · g(-1) protein · h(-1) in control lymphoblast extracts. In a blinded test of 22 subjects suspected of GALE deficiency, we identified 6 individuals whose residual activities were below the range of controls, compatible with intermediate GALE deficiency., Conclusions: This assay can be used to distinguish the different forms of GALE deficiency. From an analytical standpoint, differentiating isomers on the basis of fragment intensity ratios should also prove useful for analogous enzymatic studies involving substrates and products that are structural isomers.

Screening for PKU, in France, is made on the 3rd day of life by measuring the concentration of phenylalanine in dried blood spot samples. In this study, the goal was to examine the final diagnosis of patients who showed a hyperphenylalaninemia during newborn screening laboratory. Over a period of 11 years from 1 February 2002 to 31 January 2013, all newborns with a phenylalanine concentration increase (>180 μmol/L) have been identified and the cause of this increase was noted. Of the 165,113 newborns screened, hyperphenylalaninemia was identified in 90 patients during the newborn screening laboratory. During this period 35% of cases were due to classical phenylketonuria or hyperphenylalaninemia. In 4.4% of cases, increase concentrations were due to other diseases (biopterine deficiency, galactosemia, MSUD). However, 48.9% of high concentrations have not been confirmed by a second sample and 11% were children who died rapidely during their first days of life. The positive predictive value (PPV) of the test with a threshold of positivity >180 μmol/L was 40%. Our study showed that the positivity threshold of 180 μmol/L proposed by the Association française pour le dépistage et la prévention des handicaps de l'enfant (AFDPHE) provides a comprehensive detection of all phenylketonuria cases as well as mild hyperphenylalaninemia permanent and transient cases. Eventhough the use of a higher threshold would have the advantage of increasing the PPV of the test, none the less we would have missed out on some cases to follow.

A new cell-based galactose assay system, which is comprised of two bioluminescent Escherichia coli strains immobilized within an agarose gel arrayed on a well plate, has been developed. For this purpose, a galT knockout strain [galT(-) cell] of E. coli was genetically constructed so that cell growth is not promoted by galactose but rather by glucose present in a sample. Another E. coli W strain (normal cell), which grows normally in the presence of either glucose or galactose, was employed. A luminescent reporter gene, which produces luminescence as cells grow, was inserted into both of the E. coli strains, so that cell growth could be monitored in a facile manner. The two strains were separately grown for 4 h on gel arrays to which test samples were individually supplied. The relative luminescence unit (RLU) values caused by cell growth were determined for each array, one of which is resulted by glucose only and the other of which is resulted by both glucose and galactose present in the sample. By employing this protocol, galactose concentrations present in the test sample are reflected in the differences between the RLU values for each array. The practical utility of the new assay system was demonstrated by its use in determining galactose levels in clinical blood spot specimens coming from newborn babies. Because it can be employed to diagnosis of galactosemia in newborn babies in a more rapid, convenient, and cost-effective manner, this cell-based solid-phase galactose assay system should become a powerful alternative to conventional methods, which require labor-intensive and time-consuming procedures and/or complicated and expensive equipment.

Previous studies examining reproductive parameters in men with galactosemia have inconsistently demonstrated abnormalities. We hypothesized that men with galactosemia may demonstrate evidence of reproductive dysfunction. Pubertal history, physical examination, hormone levels and semen analyses were examined in 26 males with galactosemia and compared to those in 46 controls. The prevalence of cryptorchidism was higher in men with galactosemia than in the general population [11.6% vs. 1.0% (95%CI: 0.75-1.26; p <0.001)]. Testosterone (461±125 vs. 532± 33 ng%; p=0.04), inhibin B (144±66 vs. 183±52 pg/mL; p=0.002) and sperm concentration (46±36 vs. 112±75×10(6) spermatozoa/mL; p=0.01) were lower and SHBG was higher (40.7±21.5 vs 26.7±14.6; p=0.002) in men with galactosemia compared to controls. Semen volume was below normal in seven out of 12 men with galactosemia. Men with galactosemia have a higher than expected prevalence of cryptorchidism and low semen volumes. The subtle decrease in testosterone and inhibin B levels and sperm count may indicate mild defects in Sertoli and Leydig cell function, but does not point towards severe infertility causing reproductive impairment. Follow-up studies are needed to further determine the clinical consequences of these abnormalities.

Galactosemia is an autosomal recessive disorder with a wide range of clinical abnormalities. Cellular oxidative stress is considered as one of the pathogenic mechanisms of galactosemia. In this study, we examined the activity of NADPH oxidase (NOX), a major superoxide-generating enzyme system, in peripheral blood lymphocytes (PBL) from galactosemia patients. PBL were isolated from galactosemia patients and healthy control subjects and used for cell culture studies and biochemical assays. PBL were cultured in the presence or absence of galactose or galactose-1-phosphate (Gal-1-P), and enzyme activities and/or gene expression of NOX, catalase, superoxide dismutase (SOD) and glutathione peroxidase (GPx) were measured in the cell homogenates. PBL isolated from galactosemia patients showed significantly reduced (P < 0.01) activities of catalase and GPx; however SOD activity remained unaltered. Galactosemia patients were found to have significantly (P < 0.01) increased levels of malondialdehyde (MDA) in blood lymphocytes. Enzymatic activity of NOX was significantly (P < 0.001) reduced in galactosemia patients; however, Western blotting revealed that NOX-1 protein was not significantly altered. Interestingly, levels of NOX activity in lymphocytes isolated from galactosemia patients significantly increased but remained subnormal when cultured in galactose-deficient medium for two weeks, indicating a galactose-mediated inhibition of NOX. Lymphocytes isolated from control subjects were found to have significantly (P < 0.01) reduced NOX activity when cultured in the presence of galactose or Gal-1-P for two weeks. These results show that galactose-induced cellular oxidative stress is not NOX mediated. However, impairment of the NOX system might be responsible for some of the clinical complications in galactosemia patients.

Summary: This study evaluated bone health in adults with galactosemia. Associations between bone mineral density (BMD) and nutritional and biochemical variables were explored. Calcium level predicted hip and spine BMD, and gonadotropin levels were inversely associated with spinal BMD in women. These results afford insights into management strategies for these patients., Introduction: Bone loss is a complication of galactosemia. Dietary restriction, primary ovarian insufficiency in women, and disease-related alterations of bone metabolism may contribute. This study examined relationships between clinical factors and BMD in patients with galactosemia., Methods: This cross-sectional sample included 33 adults (16 women) with classic galactosemia, mean age 32.0 ± 11.8 years. BMD was measured by dual-energy X-ray absorptiometry, and was correlated with age, height, weight, fractures, nutritional factors, hormonal status, and bone biomarkers., Results: There was a significant difference in hip BMD between women and men (0.799 vs. 0.896 g/cm(2), p = 0.014). The percentage of subjects with BMD-Z <-2.0 was also greater for women than men [33 vs. 18 % (spine), 27 vs. 6 % (hip)], and more women reported sustaining fractures. Bivariate analyses yielded correlations between BMI and BMD-Z [at the hip in women (r = 0.58, p < 0.05) and spine in men (r = 0.53, p < 0.05)]. In women, weight was also correlated with BMD-Z (r = 0.57, p < 0.05 at hip), and C-telopeptides (r = -0.59 at spine and -0.63 hip, p < 0.05) and osteocalcin (r = -0.71 at spine and -0.72 hip, p < 0.05) were inversely correlated with BMD-Z. In final regression models, higher gonadotropin levels were associated with lower spinal BMD in women (p = 0.017); serum calcium was a significant predictor of hip (p = 0.014) and spine (p = 0.013) BMD in both sexes., Conclusions: Bone density in adults with galactosemia is low, indicating the potential for increased fracture risk, the etiology of which appears to be multifactorial.

Classic galactosemia is a potentially lethal metabolic disorder that results from profound impairment of the enzyme galactose-1-phosphate uridylyltransferase (GALT); despite decades of research, the underlying mechanism of pathophysiology remains unclear. Previous studies of plasma and tissue samples from patients with classic galactosemia have revealed defects of protein and lipid glycosylation, however, the underlying bases for these defects and their clinical significance, if any, has remained unclear. As a step toward addressing these questions we characterized both the N- and O-linked glycomes of plasma proteins from neonates, infants, children, and adults with galactosemia using mass spectrometry and asked (1) whether similar or disparate defects exist for N-linked and O-linked modifications, (2) what factors correlate with the severity of these defects in different patients, and perhaps most important, (3) whether there is any apparent relationship between chronic glycosylation defects and long-term outcome in patients. We found that some but not all of the galactosemic neonates tested exhibited abnormal N- and O-linked glycosylation of plasma proteins. The types of abnormalities seen were similar between N- and O-linked moieties, but the extent of the defects varied between patients. Age, gender, GALT genotype, and predicted residual GALT activity all failed to explain the extent of the glycosylation defect in the samples studied. Dietary galactose restriction markedly normalized both the N- and O-linked glycosylation patterns for all infants tested; however, any remaining glycosylation defects evident in the plasma of older children or adults on galactose-restricted diets showed no correlation with clinical outcome. These data cannot rule out the possibility that subtle or localized glycosylation defects, not detectable by our methods or not reflected in plasma, may contribute to acute or long-term outcome severity., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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., (Copyright © 2012. Published by Elsevier Inc.)

A colorimetric method for quantification of galactose, which utilizes a nanostructured multi-catalyst system consisting of Fe(3)O(4) magnetic nanoparticles (MNPs) and galactose oxidase (Gal Ox) simultaneously entrapped in large pore sized mesocellular silica, is described. Gal Ox, immobilized in a silica matrix, promotes reaction of galactose to generate H(2)O(2) that subsequently activates MNPs in silica mesopores to convert a colorimetric substrate into a colored product. By using this colorimetric method, galactose can be specifically detected. Along with excellent reusability via application of simple magnetic capturing, enhanced operational stability was achieved by employing a cross-linked enzyme aggregate (CLEA) method for Gal Ox immobilization. This protocol leads to effective prevention of enzyme leaching from the pores of mesocellular silica. The analytical utility of the new colorimetric biosensor was demonstrated by its use in diagnosing galactosemia, a genetic metabolic disorder characterized by the inability to utilize galactose, through analysis of clinical dried blood spot specimens. A microscale well-plate format was employed that possesses a multiplexing capability. The multi-catalyst system entrapping Gal Ox and MNPs represents a new approach for rapid, convenient, and cost-effective quantification of galactose in human blood and it holds promise as an alternative method for galactosemia diagnosis, replacing the laborious procedures that are currently in use.

Background: Classical galactosemia is a rare but very severe disease characterized by a deficiency of the galactose-1-phosphate uridyltransferase enzyme. The confirmed galactosemic patients are treated with a galactose-restricted diet. Nevertheless, metabolites such as galactose-1-phosphate can accumulate in red blood cells of treated patients and its measurement is a standard practice for their monitoring. At present, no commercial methods for measuring galactose-1-phosphate in erythrocytes are available., Methods: In this study, we will describe the optimization and laboratory validation of a previously published quantitative gas chromatographic-mass spectrometric method and its clinical validation on normal donors and galactosemic patients both at the diagnosis and during the follow-up., Results: The method was technically optimized and validated for its clinical use on normal donors and galactosemic newborns, children and adults. The method was suitable for the monitoring of dietary compliance. Galactose-1-phosphate levels were found to be well correlated with the clinical signs in the galactosemic patients at the follow-up., Conclusions: This paper provides information on the measurement of Galactose-1-phosphate levels that can be very useful for the management of classical galactosemia.

Background: Methionine (Met) in blood and urine is a useful diagnostic marker for homocystinuria (HCU). However, galactosemia could be misdiagnosed as HCU when Met is used as the sole marker, since elevated excretion of Met presents in both galactosemia and HCU. Use of a more specific diagnostic marker in addition to Met is therefore necessary for reduction of false positive results for HCU as well as confirmative diagnosis of HCU., Methods: Chromatographic separation was performed using an anion-exchange column. The levels of Met and galactose (Gal) on blood were measured and Met/Gal ratios were calculated from blood spot samples from 300 normal volunteers, eight galactosemia patients, and three HCU patients., Results: The Met/Gal ratio ranged 0-4.95 for normal blood spots (n=300), 0-0.22 for galactosemia samples (n=8), and >1250 for HCU patient samples., Conclusions: Separation, extraction, and deproteinization procedures were established for Met and Gal in blood spots. And Met/Gal ratio allowed HCU to clearly distinguish from galactosemia. As a way of second tier confirmative analysis, the ratio is the best way to reduce false positives. The assay is most appropriate to reduce false positives in labs that do not screen for galactosemia., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Congenital galactosemia is a hereditary, autosomal recessive and metabolic disease. It is linked to an enzyme deficiency, more commonly known by the deficiency of galactose-1- phosphate uridyltransferase (GALT), which is responsible for an accumulation of galactose-1- phosphate in the blood. Clinical symptoms appear early in infancy from the second week of life. They generally manifested by some disorders within liver, kidney, eye, gastrointestinal, neurological and also with cataracts. Currently, the clinical diagnosis remains difficult hence the importance of further investigations based on effective biological assessments to highlight the disease. The diagnosis of galactosemia is made by the laboratory test. The latter includes the determination of Gal-1-P which is done by a fluorometric method spot test. This study was conducted in order to assess the repeatability, reproducibility, accuracy, and effectiveness of the techniques used. We have found the CV for a repeatability (CV = 5 %), reproducibility (CV = 4 %) which confirms the accuracy of the method proceeded in this study. This method allows us to have a degree of inaccuracy less than 1%. According to the study of the effectiveness of "spot test", we found that our technique is specific (Sp = 93 %) and sensitive (Se = 83 %).

Renal Fanconi syndromes are both clinically challenging and physiologically fascinating. The diagnosis requires a certain index of suspicion to correctly identify the clinical symptomatology and pursue the appropriate laboratory evaluations. The renal Fanconi syndrome (FS) is a defect of proximal tubular function attributable to different rare inherited diseases or acquired disorders caused by a multitude of exogenous agents. It can manifest as complete or incomplete FS, characterized by low molecular weight proteinuria, glucosuria, aminoaciduria, and loss of electrolytes, bicarbonate and lactate. Implementation of new methods and recent findings from urinary proteome pattern in patients with renal FS has led to the identification of new markers for proximal tubular dysfunction. Future combined proteomic and metabonomic studies will provide additional potential biomarkers and may help to gain novel insights in the diagnosis and differentiation of the various forms of FS. Moreover, the observation of poor renal uptake of 99 mTc-DMSA in patients with tubular proteinuria, which is not fully understood yet, may also help to elucidate the individual basis of FS in early stages. This review focuses on the new advances in the evaluation of proximal tubular dysfunction in various forms of Fanconi syndrome.

The paper describes a case of hereditary thesaurismosis - galactosemia in neonate infant with mutation of GALT-gene (Q188R/N) and vascular malformation of the internal organs.

Objective: To determine if girls with Duarte variant galactosemia (DG) have an increased risk of developing premature ovarian insufficiency based on prepubertal anti-Müllerian hormone (AMH) levels., Design: Cross-sectional study., Setting: University research laboratory., Patient(s): Study volunteers included 57 girls with DG, 89 girls with classic galactosemia (GG), and 64 control girls between the ages of <1 month and 10.5 years., Intervention(s): Blood sampling., Main Outcome Measure(s): We determined AMH and FSH levels in study volunteers with and without Duarte variant or GG., Result(s): FSH levels were significantly higher and AMH levels significantly lower in girls with GG than in age-stratified control girls, but there was no significant difference between FSH and AMH levels in girls with DG and control girls., Conclusion(s): Although >80% of girls with GG in this study demonstrated low to undetectable AMH levels consistent with diminished ovarian reserve, 100% of girls with DG in our study demonstrated no apparent decrease in AMH levels or increase in FSH levels, suggesting that these girls are not at increased risk for premature ovarian insufficiency., (Copyright © 2011. Published by Elsevier Inc.)

Female classic galactosemia patients suffer from primary ovarian insufficiency (POI). The cause for this long-term complication is not fully understood. One of the proposed mechanisms is that hypoglycosylation of complex molecules, a known secondary phenomenon of galactosemia, leads to FSH dysfunction. An earlier study showed less acidic isoforms of FSH in serum samples of two classic galactosemia patients compared to controls, indicating hypoglycosylation. In this study, FSH isoform patterns of five classic galactosemia patients with POI were compared to the pattern obtained in two patients with a primary glycosylation disorder (phosphomannomutase-2-deficient congenital disorders of glycosylation, PMM2-CDG) and POI, and in five postmenopausal women as controls. We used FPLC chromatofocussing with measurement of FSH concentration per fraction, and discovered that there were no significant differences between galactosemia patients, PMM2-CDG patients and postmenopausal controls. Our results do not support that FSH dysfunction due to a less acidic isoform pattern because of hypoglycosylation is a key mechanism of POI in this disease.

Galactosemia is one of the most important inherited metabolic disorders detected by newborn screening tests. Abnormal results during screening should be confirmed by enzyme activity assays. Recently, we developed a multiplex enzyme assay for galactosemia in erythrocytes using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). In this study, we proposed a second-tier multiplex enzyme assay for galactosemia that can be directly applied to dried blood spots (DBSs). Supernatants from two rehydrated-punched 3.2-mm DBSs were incubated with a reaction mixture containing [¹³C6]galactose, [¹³C2]galactose-1-phosphate, and UDP-glucose as substrates for three galactose-metabolizing enzymes. After a 4-hour incubation, the end products from the combined reaction mixture, [¹³C6]galactose-1-phosphate, UDP-[¹³C2]galactose, and UDP-galactose, were simultaneously measured using UPLC-MS/MS. Substrates, products, and internal standards from the mixture of the three enzyme reactions were clearly separated in the UPLC-MS/MS system, with an injection cycle time of 10 min. Intra- and inter-assay imprecisions of the UPLC-MS/MS were 8.4-14.8% and 13.2-15.7% CV, respectively. Enzyme activities in DBSs from 37 normal individuals and 10 patients with enzyme deficiencies were analyzed. DBSs from galactosemia patients showed consistently lower enzyme activities as compared to those of normal individuals. In conclusion, multiplex enzyme assays using UPLC-MS/MS can be successfully applied to DBS analysis. This method allows a fast and effective second-tier test for newborns showing abnormal screening results.

Background: To describe a simple, rapid, quantitative ultramicrotest (UMTEST) based on the fluorometric method introduced by Fujimura et al. adapted to an Ultra Micro Analytic System (SUMA) for the detection of total galactose (GAL) in dried blood specimens., Methods: The assay uses 3 mm discs of dried blood on Whatman 903 filter paper and small volumes of each reagent. A methanol/acetone/water solution is used for deproteination, and a specially designed 96-well polystyrene opaque ultramicroplates, with a maximum capacity of 30 μL per well, are used for the reading., Results: The UMTEST GAL is completed in 2 h, with measuring range of 0.28-3.92 mmol/L. The intra- and inter-assay coefficients of variation were 2.3%-8.9% and 6.8%-11.1%, respectively, depending on the total GAL concentrations. Percentage recovery ranged from 97.7% to 103%. Limit of detection and limit of quantitation were 0.06 and 0.16 mmol/L, respectively. The mean GAL concentration, in 2510 dried blood samples from the National Neonatal Screening Program was 0.23 mmol/L. Our assay showed high concordance correlations with the commercially available ICN Immuno-Chem™ GAL-MW EA kit., Conclusions: The analytical performance characteristics of this assay is suitable for mass newborn screening of galactosemia in Cuba.

Unlabelled: Introduction Newborn screening for phenylketonuria (PKU) can reveal other conditions which lead to an increased blood spot phenylalanine (Phe) concentration. We have investigated the proportion of blood spot samples that gave a positive screen due to clinically significant conditions other than PKU, compared the positive predictive value (PPV) of our referral Phe cut-off with that recommended by the UK Newborn Screening Programme Centre (UKNSPC) (>210 and >240 μmol/L, respectively) and evaluated the effectiveness of reflex testing for galactosaemia using a lower blood spot Phe cut-off concentration of 130 μmol/L., Methods: All blood spot samples that screened positive, for an increased Phe concentration, between April 2001 and March 2008, were identified from the records of the Sheffield Newborn Screening Laboratory and the diagnoses noted. In addition, all cases of galactosaemia detected in or notified to our screening laboratory within this time were also examined and the screened Phe concentrations compared., Results: Out of 438,674 babies who were screened, 67 had Phe concentration >210 μmol/L (15 per 100,000). Of these, 40 had PKU or persistent hyperphenylalaninaemia with a Phe concentration identified by screening between 270 and 2350 μmol/L. A further 11 were diagnosed with another clinically significant disorder: galactosaemia (n = 8), biopterin defects (n = 2), tyrosinaemia Type 1 (n = 1). In addition, 16 had transient elevations in Phe. In total, nine cases of galactosaemia were identified, of whom, three had Phe concentrations <240 μmol/L with one asymptomatic individual having a concentration <210 μmol/L., Conclusions: Adoption of the UKNSPC recommended cut-off (>240 μmol/L) will not affect the detection rate of classical PKU, but will improve the PPV from 76% to 80%. The use of a lower cut-off (130 μmol/L) for reflex galactosaemia testing enables the timely identification of asymptomatic cases that benefit particularly from early treatment, without prompting any unnecessary clinical referrals or delaying any referrals. This intervention may reduce mortality in this vulnerable group.

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., (Copyright 2010 Elsevier B.V. All rights reserved.)

Background: Duarte galactosemia (DG) is frequently detected in newborn-screening programs. DG patients do not manifest the symptoms of classic galactosemia, but whether they require dietary galactose restriction is controversial. We sought to assess the relationships of selected galactose metabolites (plasma galactose, plasma galactitol, erythrocyte (RBC) galactitol, RBC galactonate, and urine galactitol and galactonate) to RBC galactose 1-phosphate (Gal-1-P), dietary galactose intake, and neurodevelopmental/clinical outcomes in DG children., Methods: We studied 30 children 1-6 years of age who had DG galactosemia and were on a regular diet. All participants underwent a physical and ophthalmologic examination and a neurodevelopmental assessment. RBC galactitol, RBC galactonate, RBC Gal-1-P, plasma galactose, plasma galactonate, and urine galactitol and galactonate concentrations were measured., Results: RBC galactitol and galactonate concentrations were about 2 and 6 times higher, respectively, than control values. Plasma galactose and galactitol concentrations were also about twice the control values. The mean values for RBC Gal-1-P and urine galactitol were within the reference interval. We found a relationship between plasma and urine galactitol concentrations but no relationship between RBC galactose metabolites and urine galactitol. There was a significant relationship between galactose intake and RBC galactose metabolites, especially RBC galactitol (P < 0.0005) and RBC galactonate (P < 0.0005). Galactose intake was not related to the urine galactitol, plasma galactose, or plasma galactitol concentration. RBC galactitol, RBC galactonate, plasma galactose, plasma galactitol, and urine galactonate concentrations showed no relationship with clinical or developmental outcomes., Conclusions: DG children on a regular diet have RBC Gal-1-P concentrations within the reference interval but increased concentrations of other galactose metabolites, including RBC galactitol and RBC galactonate. These increased concentrations correlate with galactose intake and neither cause any developmental or clinical pathology during early childhood nor oblige a lactose-restricted diet.

We developed a simultaneous diagnostic method for phenylketonuria (PKU) and galactosemia through simultaneous determination of phenylalanine (Phe) and galactose (Gal) by high-performance liquid chromatography (HPLC) with pulsed amperometric detection (PAD). The intra- and inter-day precisions were <5.8%, with satisfactory mean recoveries (98.2-105%). For all PKU-positive samples, Phe levels were above the cut-off value (>30.0 mg/L), but Gal levels were nearly zero. For 77% of galactosemia-positive samples, Phe levels were above the cut-off value, but Gal levels were above the cut-off value (>80.0 mg/L) for all samples. Our HPLC-PAD method can reduce the false-positive rate of misdiagnosis for PKU and galactosemia., (Copyright 2010 Elsevier B.V. All rights reserved.)

The aim of the study was to develop a method for fast and reliable diagnosis of peroxisomal diseases and to facilitate differential diagnosis of cholestatic hepatopathy. For the quantification of bile acids and their conjugates as well as C(27) precursors di- and trihydroxycholestanoic acid (DHCA, THCA), in small pediatric blood samples we combined HPLC separation on a reverse-phase C18 column with ESI-MS/MS analysis in the negative ion mode. Analysis was done with good precision (CV 3,7%-11.1%) and sufficient sensitivity (LOQ: 11-91 nmol/L) without derivatization. Complete analysis of 17 free and conjugated bile acids from dried blood spots and 10 microL serum samples, respectively, was performed within 12 min. Measurement of conjugated primary bile acids plus DHCA and THCA as well as ursodeoxycholic acid was done in 4.5 min. In blood spots of healthy newborns, conjugated primary bile acids were found in the range of 0.01 to 2.01 micromol/L. Concentrations of C(27) precursors were below the detection limit in normal controls. DHCA and THCA were specifically elevated in cases of peroxysomal defects and one Zellweger patient.

Long-term outcomes of classic galactosemia (GAL) remain disappointing. It is unclear if the complications result mainly from prenatal-neonatal toxicity or persistent glycoprotein and glycolipid synthesis abnormalities. We performed gene expression profiling (T transcriptome) to characterize key-altered genes and gene clusters of four patients with GAL with variable outcomes maintained on a galactose-restricted diet, compared with controls. Significant perturbations of multiple cell signaling pathways were observed including mitogen-activated protein kinase (MAPK) signaling, regulation of the actin cytoskeleton, focal adhesion, and ubiquitin mediated proteolysis. A number of genes significantly altered were further investigated in the GAL cohort including SPARC (osteonectin) and S100A8 (S100 calcium-binding protein). The whole serum N-glycan profile and IgG glycosylation status of 10 treated patients with GAL were compared with healthy control serum and IgG using a quantitative high-throughput analytical HPLC platform. Increased levels of agalactosylated and monogalactosylated structures and decreases in certain digalactosylated structures were identified in the patients. The persistent abnormal glycosylation of serum glycoproteins seen with the microarray data indicates persisting metabolic dyshomeostasis and gene dysregulation in "treated" GAL. Strict restriction of dietary galactose is clearly life saving in the neonatal period; long-term severe galactose restriction may contribute to ongoing systemic abnormalities.

We describe the history and current implementation of an inexpensive thin layer chromatography (TLC) method, vertical sandwich-type continuous/evaporative TLC with fixed mobile phase volume, that is convenient for detecting and identifying reducing sugars of clinical relevance in the paper-borne blood and urine samples collected in neonatal screening programmes. This method facilitates screening by providing a considerable degree of standardization of chromatographic results. Among some 555,000 newborns to which it has been applied, it has detected 10 cases of classical galactosaemia, 7 cases of galactokinase deficiency, 2 cases of glucosuria, and 3 cases of transitory neonatal diabetes mellitus; the only false negatives we are aware of were two cases of galacto-4-epimerase deficiency detected by tandem mass spectrometry. Screening for sugars in urine has allowed the detection of galactosaemia when the accompanying blood sample was invalid because of transfusion or parenteral feeding. The conclusion is that this inexpensive procedure is very useful for the detection of relevant metabolopathies in circumstances where others fail., ((c) 2010 Wiley-Liss, Inc.)

Objective: To determine whether premature ovarian insufficiency (POI) associated with classic galactosemia results from a true impairment of ovarian function or from aberrant FSH., Design: Cross-sectional study., Setting: University research laboratory., Patient(s): Study subjects included 35 girls and women with galactosemia and 43 control girls and women between the ages of <1 and 51 years., Intervention(s): Blood sampling and medical and reproductive histories were obtained., Main Outcome Measurement(s): We determined FSH and anti-Müllerian hormone (AMH) levels in subjects with and without classic galactosemia. FSH bioactivity was measured in a subset of girls and women with and without galactosemia who were not on hormone therapy., Result(s): FSH levels were significantly higher and AMH levels were significantly lower in our galactosemic cases relative to controls. FSH bioactivity did not significantly differ between cases and controls., Conclusion(s): Close to 90% of girls and women with classic galactosemia have a profound absence of ovarian function, a deficit that is evident shortly after birth, if not before. These patients have no evidence of abnormally functioning FSH. AMH levels can be assessed before menarche or after initiation of hormone therapy and may supplement FSH as a useful blood biomarker of ovarian function for patients with classic galactosemia.

Objective: To report a pregnancy in a patient with classic galactosemia despite signs of no ovarian reserve to draw attention to the limited predictive value of ovarian reserve tests in these patients., Design: Case report., Setting: Secondary and tertiary care center., Patient(s): A patient with classic galactosemia with premature ovarian failure and two previous pregnancies., Intervention(s): Exogenous FSH ovarian reserve test and anti-Müllerian hormone (AMH) measurement., Main Outcome Measure(s): 17beta-Estradiol response, AMH level., Result(s): Pregnancy despite undetectable AMH (<0.1 microg/L) and no E(2) response (exogenous FSH ovarian reserve test)., Conclusion(s): Fluctuating premature ovarian failure makes fertility counseling of patients with classic galactosemia difficult. Commonly used ovarian function and reserve tests seem to have no significance.

Background: Variable increases in the plasma activity of different lysosomal enzymes have been reported in patients with congenital disorders of glycosylation (CDG). In particular, elevated plasma aspartylglucosaminidase activity (AGA) has been found in the majority of CDG type I patients. We report on the plasma activity of AGA and other lysosomal enzymes in patients with different types of primary and secondary CDG defects., Methods: AGA, alpha-mannosidase, beta-mannosidase and beta-hexosaminidase activities were assayed in the plasma of patients with CDGI (4CDGIa, 4CDGIx) and CDGIIx (5, all with a combined N- and O-glycosylation defect), classical galactosemia (GALT) (n=3) and hereditary fructose intolerance (HFI) (n=2)., Results: Increased AGA and beta-hexosaminidase activities were found in all and 7/8 of the GDGI patients respectively. All enzymic activities were normal in the CDGIIx patients. Elevated AGA and beta-hexosaminidase activity was also seen in GALT and HFI patients before treatment, when transferrin isoelectric focusing (TfIEF) patterns were also abnormal., Conclusions: Increased AGA plasma activity, although a consistent finding in CDGI patients, is not specific to this group of disorders since it is also observed in untreated cases of GALT and HFI. Furthermore, plasma AGA activity cannot serve as a marker for CDGII disorders. In conjunction with TfIEF it could be used in the follow up of GALT and HFI patients.