Five years of screening for galactosaemia in South Africa: Pitfalls of using Benedict's test and thin layer chromatography to screen for galactosaemia in a developing country.

BACKGROUND: The objective of the study was to investigate the effectiveness of screening for hereditary galactosaemia with Benedict's test and thin layer chromatography (TLC) in a tertiary laboratory from a developing country. METHODS: We retrospectively analysed the results of tests done in suspected galactosaemia patients including Benedict's test, thin layer chromatography, GALT activity and DNA analysis. RESULTS: 878 paediatric patients were screened with Benedict's test; the age range was 5 days to 19 years. 48% tested positive/trace on the Benedict's test of which 52% of these had galactosuria evident on TLC. 22% of this sample had pathologically low GALT results on follow-up. 8 patients from the screened population were confirmed to have galactosaemia, in addition to 6 more patients diagnosed with galactosaemia without screening tests performed. Median ages at which the diagnoses were made in the screened and non-screened samples were 2 months and 6 months respectively. Confirmatory DNA testing was performed in 2 patients, whom were found to be heterozygous for S135L mutation. CONCLUSION: Inadequate performance of Benedict's test and TLC was demonstrated by false positive and false negative results leading us to conclude that screening test results require interpretation with caution.

Novel Mutation in GALT Gene in Galactosemia Patient with Group B Streptococcus Meningitis and Acute Liver Failure.

Classic galactosemia is an autosomal recessive disorder caused by the deficiency of the enzyme galactose-1-phosphate uridyltransferase (GALT) involved in galactose metabolism. Bacterial infections are a known cause of early morbidity and mortality in children with classic galactosemia. The most common agent is Escherichia coli, but in rare situations, other bacteria are incriminated. We report a case of a three-week-old female patient with galactosemia, who presented with Group B Streptococcus (GBS) meningitis/sepsis. She received treatment with antibiotics, supportive therapy, and erythrocyte transfusion, but after a short period of improvement, she presented acute liver failure with suspicion of an inborn error of metabolism. Rapid nuclear magnetic resonance (NMR) spectroscopy from urine showed highly elevated values of galactose and galactitol. Under intensive treatment for acute liver failure and with a lactose-free diet, her clinical features and laboratory parameters improved considerably. Genetic testing confirmed compound heterozygous status for GALT mutations: c.563 A>G [p.Q188R] and c. 910 C>T, the last mutation being a novel mutation in GALT gene. In countries without an extensive newborn screening program, a high index of suspicion is necessary for early diagnosis and treatment of galactosemia.

Simultaneous analysis of carbohydrates, polyols and amines in urine samples using chemical ionization gas chromatography with tandem mass spectrometry.

A simple method for the simultaneous derivatization of carbohydrates, polyols, amines and amino acids using hexamethyldisilazane and N,O-bis(trimethylsilyl)trifluoroacetamide was developed. This method allows the direct derivatization of urine samples without sample pretreatment before derivatization. The method was successfully used for analysis of the selected metabolites in urine samples of healthy individuals and neonates suffering from galactosemia. The limits of detection by positive chemical ionization gas chromatography with tandem mass spectrometry analysis were in the range of 1.0 mgL-1 for mannitol to 4.7 mg/L for glucose.

Vertically-Oriented and Shape-Tailored Electrocatalytic Metal Nanowire Arrays for Enzyme-Free Galactosemia Rapid Diagnosis.

Metallic catalytic nanowires such as nickel and copper nanowires (NWs) for electrochemical detection of carbohydrates involved in metabolic rare diseases are proposed. NWs were electrodeposited using a polycarbonate membrane template, which was cut with the desired shape, stuck in double-sided adhesive tape, pasted into a non-conductive substrate and in situ removed. This simple and versatile approach allowed to obtain NWs vertically oriented (v-NWs), which are contained in the double-sided adhesive tape, becoming highly versatile. The high specific surface of working electrode in which the transduction is supported exclusively by the nanomaterial yielded a high analytical performance [extremely low fouling for galactose (RSD<2 %; n=25)]. Likewise, v-NWs exhibited a superior analytical performance with respect to commercial sputtered thick-film electrodes showing also a clear advantage related with the price, as well as with non-need clean room facilities. The analytical potency of the new approach was clearly demonstrated towards the fast and reliable diagnosis of galactosemia using precious newborn urine samples compared to standard clinical diagnosis. These results revealed new opportunities for future enzyme-free diagnosis and development of future point-of-care applications.

Microchip in situ electrosynthesis of silver metallic oxide clusters for ultra-FAST detection of galactose in galactosemic newborns' urine samples.

This work describes for the first time the coupling of microfluidic chips (MC) to electrosynthetized silver metallic oxide clusters (AgMOCs). As an early demonstration of this novel approach, the ultrafast detection of galactose in galactosemic newborns' urine samples is proposed. AgMOCs were in situ electrosynthetized on integrated microchip platinum electrodes using a double pulse technique and characterized in full using scanning electronic microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), X-ray photoelectron spectroscopy (XPS) and electrochemical techniques revealing the presence of silver oxides and electrocatalysis towards galactose as a galactosemia biomarker. Galactose detection in galactosemic newborns' urine samples proceeded in less than 30 s, differentiating between ill and healthy urine samples and requiring negligible urine sample consumption. The significance of the newborns' urine samples confirmed the analytical potency of the MC-AgMOCs approach for future implementation of screening for rare disease diagnosis such as galactosemia.

Copper nanowires immobilized on the boards of microfluidic chips for the rapid and simultaneous diagnosis of galactosemia diseases in newborn urine samples.

Galactosemia is a rare disease that is diagnosed through the identification of different metabolite profiles. Therefore, the specific detection of galactose 1-phosphate (Gal 1-P), galactose (Gal), and uridyl diphosphate galactose (UDP-Gal) confirms type I, II, and III galactosemia diseases. Because of the low prevalence of galactosemia, sample availability is very scarce and screening methods to diagnose the illness are not commonly employed around the world. This work describes the coupling of microfluidic chips (MCs) to copper nanowires (CuNWs) as electrochemical detectors for the fast diagnosis of galactosemia in precious newborn urine samples. Conceptually speaking, we hypothesize that the inherent selectivity and sensitivity of CuNWs, toward galactosemia metabolites detection in connection with MC selectivity could allow the fast and simultaneous detection of the three galactosemia biomarkers, which implies the fast diagnosis of any galactosemia type in just one single analysis. Electrosynthesized CuNWs show a well-defined shape, with an average length of 6 µm and a width of 300 nm. The modified electrodes exhibited an enhanced electroactive surface area twice as high as the nonmodified ones. Very good intraelectrode repeatability with relative standard deviations (RSDs) of <8% (n = 10) and interelectrode reproducibility with RSDs of <12% (n = 5) were obtained, indicating an excellent stability of the nanoscaled electrochemical detector. Under optimum chemical (3 mM NaOH, pH 11.5), electrokinetic (separation voltage +750 V, injection +1500 V for 5 s) and electrochemical (E = +0.70 V in 3 mM NaOH, pH 11.5) conditions, galactosemia diseases were unequivocally identified, differentiating between type I, II, and III, using selected precious ill diagnosed newborn urine samples. Detection proceeded within less than 350 s, required negligible urine sample consumption, and displayed impressive signal-to-noise characteristics (ranging from 14 to 80) and micromolar limits of detection (LODs) much lower than the cutoff levels (Gal 1-P > 0.4 mM and Gal > 1.4 mM). Excellent reproducible recoveries (93%-107%, RSDs <6%) were also achieved, revealing the reliability of the approach. The significance of the newborn urine samples studied confirms the analytical potency of MC-CuNWs approach, enhancing the maturity of the microchip technology and opening new avenues for future implementation of screening applications in the field.

Differential glycomics of epithelial membrane glycoproteins from urinary exovesicles reveals shifts toward complex-type N-glycosylation in classical galactosemia.

A variety of genetic variations in the galactose-1-phosphate uridyltransferase (GALT) gene cause profound activity loss of the enzyme and acute toxic effects mediated by accumulating metabolic intermediates of galactose in newborns induced by dietary galactose. However, even on a severely galactose-restricted diet, patients develop serious long-term complications of the CNS and ovaries, which may result from damaging perturbations in cell biology caused by endogenously synthezised galactose. Under galactose stress, the cosubstrate of GALT, galactose-1-phosphate, accumulates and disturbs catabolic and anabolic pathways of the carbohydrate metabolism with potential effects on protein glycosylation and membrane localization of glycoprotein receptors, like the epidermal growth factor receptor. To address this issue in view of a cellular pathomechanism, we performed a differential semiquantitative N-glycomics study of membrane proteins. A suitable noninvasive cellular material derived from epithelial plasma membranes was found in urinary exovesicles and in the shed Tamm-Horsfall protein. By applying matrix-assisted laser ionization mass spectrometry on permethylated, PNGaseF released N-glycans, we demonstrate that GALT deficiency is associated with dramatic shifts from prevalent high-mannose-type glycans found in healthy subjects toward complex-type N-linked glycosylation in patients. These N-glycosylation shifts were observed on exosomal N-glycoproteins but not on the Tamm-Horsfall glycoprotein, which showed predominant high-mannose-type glycosylation with M6.

Long-term complications in Estonian galactosemia patients with a less strict lactose-free diet and metabolic control.

The main aim of our study was to retrospectively evaluate long-term complications and measure urinary galactose and galactitol excretion in classical galactosemia patients in Estonia who have been treated with a less restricted lactose-free diet and metabolic control. Our study group consisted of five classical galactosemia patients aged 7-14 years and diagnosed since 1996 in Estonia. Their diet eliminates lactose present in dairy foods, but we did not restrict the consumption of mature cheeses, fruits and vegetables. All patients had normal growth, except for one patient who was overweight at the last evaluation. In three patients mental and speech development was normal. One patient, number 1, who was diagnosed latest (at 6 weeks of age), had moderate mental retardation, verbal dyspraxia, extrapyramidal signs and bilateral cataracts. In both patients with developmental problems, a brain MRI showed bilateral subcortical changes in the cerebral white matter. Of four females, only patient 4 (p.Q188R homozygote) has premature ovarian insufficiency. Urinary galactose and galactitol content were retrospectively measured using high-performance liquid chromatography and refractive-index detection from urinary samples that were preserved during the years 1996-2009. Galactose ranged from 60 to 600 mmol/mol creatinine (normal=4-6), and galactitol ranged from 70 to 1200 mmol/mol creatinine (normal=2-4), which was 10-100 and 17-300 times higher than the respective reference ranges for galactose and galactitol. We conclude that a less strict lactose-free diet and metabolic control performed in Estonian classical galactosemia patients does not change long-term outcome compared to previously published studies.

Novel techniques and newer markers for the evaluation of "proximal tubular dysfunction".

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.

Monitoring of biochemical status in children with Duarte galactosemia: utility of galactose, galactitol, galactonate, and galactose 1-phosphate.

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.

Vertical sandwich-type continuous/evaporative TLC with fixed mobile phase volume for separating sugars of clinical relevance in paper-borne urine and blood samples in newborn screening.

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.

Electrokinetic probes for single-step screening of polyol stereoisomers: the virtues of ternary boronate ester complex formation.

Electrokinetic probes based on the differential migration of ternary boronate ester complexes permit the selective analysis of micromolar levels of UV-transparent polyol stereoisomers in urine samples via dynamic complexation-capillary electrophoresis that is applicable to single-step screening of in-born errors of sugar metabolism, such as galactosemia.

Urinary galactitol and galactonate quantified by isotope-dilution gas chromatography-mass spectrometry.

BACKGROUND: Measurements of urine galactitol have been used to monitor the adequacy of diet therapy in the treatment of galactosemia. We have devised a gas chromatographic mass spectrometry (GC/MS) isotope-dilution method for the simultaneous quantification of urine galactitol and another alternate pathway product, galactonate. METHODS: We prepared trimethylsilyl (TMS) derivatives and used D-[UL-13C]galactitol and D-[UL-13C]galactonate as the internal standard for GC/MS. Results obtained with this method were compared with those determined by the established GC method for galactitol and the NMR method for galactonate. Thirty-three normal urine specimens were analyzed by the isotope dilution technique for galactitol and galactonate. Results of galactitol in 6 of these urine specimens along with 18 from classic galactosemics and 19 variant galactosemics were compared with the established GC method. Results for galactonate in 15 urine specimens from galactosemics were compared to the established NMR technique. RESULTS: The method was linear up to 200 nmol with lower limits of detection of 1.1 nmol (1.75 mmol/mol creatinine) (Cr) and 0.8 nmol (1.28 mmol/mol Cr) for galactitol and galactonate, respectively. Intra- and Interassay imprecision ranged from 2.1-6.7% for galactitol and 3.5-8.0% for galactonate. The excretion of both metabolites was age dependent in both normal and galactosemics. In 12 normal urines from subjects under 1 year, values for galactitol ranged from 8-107 mmol/mol Cr, and in 7 over age 6, ranged from 2-5 mmol/mol Cr. Under 1 year, the range for galactonate was non-detectable to 231 and in the over 6 years group non-detectable to 25 mmol/mol Cr. In galactosemics under 1 year, the value for galactitol ranged from 397-743 and for galactonate 92-132 mmol/mol Cr while in nine patients over age 6 the range was 125-274 mmol/mol Cr for galactitol and 17-46 mmol/mol Cr for galactonate. CONCLUSIONS: The GC/MS method enables the simultaneous determination of urine galactitol and galactonate and is precise and useful over the wide range of concentrations needed to assess the galactose burden in patients with galactosemia.

Pathways of galactose metabolism by galactosemics: evidence for galactose conversion to hepatic UDPglucose.

To determine if classic galactosemics have residual galactose-1-phosphate uridyltransferase (GALT) activity to explain their considerable ability to oxidize galactose over 24 h, we devised a method for assessing their ability to form hepatic UDPglucose (UDPglu), an intermediate in the normal Leloir pathway of galactose metabolism. The protocol involved the single oral administration of 7 mg/kg [2-13C]galactose concomitant with multiple small doses of acetaminophen with measurement of the extent of labeling of urinary acetaminophen glucuronide, the glucuronide moiety being formed from hepatic UDPglu. We performed the study lasting 24 h in two normal subjects and three classic galactosemics, two homozygous for the Q188R mutation and one compound for the Q188R/K258N mutation. The labeling and total excretion of acetaminophen glucuronide was measured in urine by nuclear magnetic resonance techniques. Concomitant with determination of label in the glucuronide measurement was made of galactose oxidation to 13CO2 and the 13C enrichment of plasma glucose. All of the galactosemic patients formed 13C enriched acetaminophen glucuronide indicating that they had converted the labeled galactose to [13C]UDPglu and that residual GALT or another pathway that forms UDPglu is present in hepatic tissue. Compared to the normal whose glucuronide labeling was rapid and short-lived that of the galactosemics was delayed and extended for a long period over 10 h. The extent of isotopic enrichment of glucuronide by galactosemics was comparable to the normals, resulting in a much greater conversion of galactose to UDPglu by the galactosemics. The labeling of the UDPglu pool was reflected by the rate of 13CO2 formation being rapid in the normal with peak labeling at 2-3 h with total oxidation of over 70% in 24 h. The oxidation of the galactosemics was slow with a broad peak of 13CO2 at 10 h and a total excretion of 25-39% of the [13C]galactose administered. The normal subjects formed highly enriched plasma glucose within 30 min while no enrichment of plasma glucose was detected until after 300 min in galactosemics. The exact pathway(s) of galactose metabolism by galactosemics to UDPglu remain to be determined. Their delineation may contribute to new approaches to therapeutic strategies for this enigmatic disorder.

High tolerance for oral galactose in classical galactosaemia: dietary implications.

AIM: To study the relevance of restricting the exogenous intake of small amounts of galactose, such as from fruit and vegetables, in patients with classical galactosaemia. METHODS: For a period of six weeks, increasing doses of oral galactose to a maximum of 600 mg per day, were added to a very strict galactose restricted diet in three adolescent patients homozygous for the Q188R mutation. During the study, physical examination, including an extended ophthalmic examination, and laboratory studies were performed on a weekly basis. RESULTS: No significant change in any of the studied clinical or biochemical parameters was observed. CONCLUSIONS: These findings provide further evidence that attempts to exclude trace amounts of galactose from the diet are not justified. Once the diet is made more liberal, a long term follow up study will be necessary.

Galactonate determination in urine by stable isotope dilution gas chromatography-mass spectrometry.

A stable isotope dilution assay was developed for the sensitive determination of D-galactonic acid. D-[U-13C(6)]galactono-1,4-lactone was prepared as internal standard. Unlabelled and U-13C-labelled D-galactonic acid species were converted to the N-(1-butyl)galactonamide pentaacetate derivatives and assessed by gas chromatography-mass spectrometry (GC-MS). Positive chemical ionisation and monitoring of the [MH-60](+)-ions in the galactonate chromatographic peak at m/z 402 and m/z 408 were used for quantification. The procedure was applied to study the variability of D-galactonate excretion in healthy subjects and galactosemic patients and to monitor the D-galactonate-D-galactitol ratio in human urine.

Renal excretion of galactose and galactitol in patients with classical galactosaemia, obligate heterozygous parents and healthy subjects.

The age dependence of galactose and galactitol excretion was assessed in overnight-fasted galactose-1-phosphate uridyltransferase-deficient patients under dietary treatment (ages 4-34 years; n = 51), obligate heterozygous parents (ages 25-71 years; n = 49) and healthy subjects (ages 3-58 years; n = 215). Urine concentrations were analysed by stable-isotope dilution gas chromatography mass spectrometry. There was considerable interindividual variability. The intraindividual variation, however, was not age-dependent and was rather low. Excretion estimates were calculated from the creatinine-related concentrations using weight-, age- and sex-related creatinine excretion rates. Experimental evidence is presented underscoring the problems inherent in random sampling and substantiating the primary endogenous origin of galactose and galactitol in postabsorptive urine samples. Age-dependent excretion estimates were best fitted to a simple growth-related model assuming an exponential decrease with age until adulthood. According to the model, mean postabsorptive galactose and galactitol excretion in healthy subjects was similar and decreased exponentially from about 1.2 micromol/kg body weight per day in infants to about 0.2 micromol/kg body weight per day in adults. Excretion in heterozygotes was normal. In galactosaemic patients, galactose excretion was in the normal range. Galactitol excretion, however, was enhanced over 50-fold and decreased from a mean estimate of about 64 micromol/kg body weight per day in infants to about 23 micromol/kg body weight per day in adults. The results are discussed with respect to the significance of galactose and galactitol excretion for whole-body galactose removal and with respect to the applicability of urinary galactitol analysis for metabolic monitoring in galactosaemia.

Galactosemia: presentación de un caso clínico, revisión y actualización / Galactosemia: report of a clinical case, review and updating

Presentamos un caso de galactosemia en un recién nacido de pretérmino, treinta y seis semanas de edad gestacional por examen físico, que a partir del tercer día de vida comienza con sintomatología (Ictericia), recibiendo alimentación desde su ingreso con fórmula de inicio y posteriormente al pecho; hasta el 6§ día de vida que debe ser ingresado en UTI en delicado estado clínico. Realizado el diagnóstico tras completar los estudios, con indicación de fórmula libre de lactosa egresa del hospital a los 33 días de vida, continuando su seguimiento por consultorio externo de clínica pediátrica y seguimiento de especialistas en metabolopatías.

Galactosemia: presentación de un caso clínico, revisión y actualización / Galactosemia: report of a clinical case, review and updating

Presentamos un caso de galactosemia en un recién nacido de pretérmino, treinta y seis semanas de edad gestacional por examen físico, que a partir del tercer día de vida comienza con sintomatología (Ictericia), recibiendo alimentación desde su ingreso con fórmula de inicio y posteriormente al pecho; hasta el 6º día de vida que debe ser ingresado en UTI en delicado estado clínico. Realizado el diagnóstico tras completar los estudios, con indicación de fórmula libre de lactosa egresa del hospital a los 33 días de vida, continuando su seguimiento por consultorio e