Simple and sensitive galactose monitoring based on capillary SERS sensor.

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 (H2O2) generated during galactose oxidation by galactose oxidase (GOx). A new SERS signal was observed, which was generated by H2O2 produced when galactose and GOx reacted. Our strategy yielded a quantitative change in the SERS signal, specifically in the band intensity ratio of 998 to 1076 cm-1 (I998/I1076) as the galactose concentration increased. Our capillary-based SERS biosensor provides a promising platform for early galactosemia diagnosis.

A founder noncoding GALT variant interfering with splicing causes galactosemia.

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.

Receptor-mediated attenuation of insulin-like growth factor-1 activity by galactose-1-phosphate in neonate skin fibroblast cultures: Galactosemia pathogenesis.

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.

Developmental Outcomes in Duarte Galactosemia.

: media-1vid110.1542/5849572227001PEDS-VA_2018-2516Video 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.

Fertility in classical galactosaemia, a study of N-glycan, hormonal and inflammatory gene interactions.

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 (rs = - 0.68) and (rs = - 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.

Effect of genotype on galactose-1-phosphate in classic galactosemia patients.

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.

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.

Biochemical changes and clinical outcomes in 34 patients with classic galactosemia.

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.

Downregulation of Insulin-Like Growth Factor-1 via Nitric Oxide Production in a Hypergalactosemic Model of Neonate Skin Fibroblast Cultures.

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.

The Rapid and Sensitive Quantitative Determination of Galactose by Combined Enzymatic and Colorimetric Method: Application in Neonatal Screening.

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.

Classical galactosaemia: novel insights in IgG N-glycosylation and N-glycan biosynthesis.

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.

Galactose metabolism and health.

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.

Genetic and functional studies reveal a novel noncoding variant in GALT associated with a false positive newborn screening result for galactosemia.

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.

Capillary bedside blood glucose measurement in neonates: missing a diagnosis of galactosemia.

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.

Rapid screening of classic galactosemia patients: a proof-of-concept study using high-throughput FTIR analysis of plasma.

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.

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.

Endogenous galactose formation in galactose-1-phosphate uridyltransferase deficiency.

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.

[What disorders suspect following an increase of phenylalanine on newborn screening?]. / Quelles pathologies suspecter suite à une augmentation de la phénylalanine lors du dépistage néonatal ?

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.

An interference-free two-step enzyme assay with UPLC-tandem mass spectrometric product measurement for the clinical diagnosis of uridine diphosphate galactose-4-epimerase deficiency.

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.

Liquid chromatography-tandem mass spectrometry enzyme assay for UDP-galactose 4'-epimerase: use of fragment intensity ratio in differentiation of structural isomers.

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.