RESUMEN
Together with its ß-subunit OSTM1, ClC-7 performs 2Cl-/H+ exchange across lysosomal membranes. Pathogenic variants in either gene cause lysosome-related pathologies, including osteopetrosis and lysosomal storage. CLCN7 variants can cause recessive or dominant disease. Different variants entail different sets of symptoms. Loss of ClC-7 causes osteopetrosis and mostly neuronal lysosomal storage. A recently reported de novo CLCN7 mutation (p.Tyr715Cys) causes widespread severe lysosome pathology (hypopigmentation, organomegaly, and delayed myelination and development, "HOD syndrome"), but no osteopetrosis. We now describe two additional HOD individuals with the previously described p.Tyr715Cys and a novel p.Lys285Thr mutation, respectively. Both mutations decreased ClC-7 inhibition by PI(3,5)P2 and affected residues lining its binding pocket, and shifted voltage-dependent gating to less positive potentials, an effect partially conferred to WT subunits in WT/mutant heteromers. This shift predicts augmented pH gradient-driven Cl- uptake into vesicles. Overexpressing either mutant induced large lysosome-related vacuoles. This effect depended on Cl-/H+-exchange, as shown using mutants carrying uncoupling mutations. Fibroblasts from the p.Y715C patient also displayed giant vacuoles. This was not observed with p.K285T fibroblasts probably due to residual PI(3,5)P2 sensitivity. The gain of function caused by the shifted voltage-dependence of either mutant likely is the main pathogenic factor. Loss of PI(3,5)P2 inhibition will further increase current amplitudes, but may not be a general feature of HOD. Overactivity of ClC-7 induces pathologically enlarged vacuoles in many tissues, which is distinct from lysosomal storage observed with the loss of ClC-7 function. Osteopetrosis results from a loss of ClC-7, but osteoclasts remain resilient to increased ClC-7 activity.
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Canales de Cloruro , Enfermedades por Almacenamiento Lisosomal , Lisosomas , Humanos , Masculino , Canales de Cloruro/genética , Canales de Cloruro/metabolismo , Mutación con Ganancia de Función , Células HEK293 , Enfermedades por Almacenamiento Lisosomal/genética , Enfermedades por Almacenamiento Lisosomal/metabolismo , Enfermedades por Almacenamiento Lisosomal/patología , Lisosomas/metabolismo , Lisosomas/genética , Proteínas de la Membrana , Mutación Missense , Fosfatos de Fosfatidilinositol/metabolismo , Ubiquitina-Proteína Ligasas , Vacuolas/metabolismo , Vacuolas/genética , Vacuolas/patologíaRESUMEN
BACKGROUND: Canavan disease is a devastating neurometabolic disorder caused by accumulation of N acetylaspartate in brain and body fluids due to genetic defects in the aspartoacylase gene (ASPA). New gene therapies are on the horizon but will require early presymptomatic diagnosis to be fully effective. METHODS: We therefore developed a fast and highly sensitive liquid chromatography mass spectrometry (LC-MS/MS)-based method for quantification of N-acetylaspartate in dried blood spots and established reference ranges for neonates and older controls. With this test, we investigated 45 samples of 25 Canavan patients including 8 with a neonatal sample. RESULTS: Measuring N-acetylaspartate concentration in dried blood with this novel test, all Canavan patients (with variable severity) were well separated from the control group (median; range: 5.7; 1.6-13.6 µmol/L [n = 45] vs 0.44; 0.24-0.99 µmol/L [n = 59] (p < 0.05)). There was also no overlap when comparing neonatal samples of Canavan patients (7.3; 5.1-9.9 µmol/L [n = 8]) and neonatal controls (0.93; 0.4-1.8 µmol/L [n = 784]) (p < 0.05). CONCLUSIONS: We have developed a new LC-MS/MS-based screening test for early postnatal diagnosis of Canavan disease that should be further evaluated in a population-based study once a promising treatment becomes available. The method meets the general requirements of newborn screening and should be appropriate for multiplexing with other screening approaches that combine chromatographic and mass spectrometry techniques.
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Ácido Aspártico , Enfermedad de Canavan , Pruebas con Sangre Seca , Tamizaje Neonatal , Espectrometría de Masas en Tándem , Humanos , Enfermedad de Canavan/diagnóstico , Enfermedad de Canavan/sangre , Enfermedad de Canavan/genética , Recién Nacido , Tamizaje Neonatal/métodos , Pruebas con Sangre Seca/métodos , Espectrometría de Masas en Tándem/métodos , Ácido Aspártico/análogos & derivados , Ácido Aspártico/sangre , Cromatografía Liquida , Femenino , Masculino , Lactante , Preescolar , Cromatografía Líquida con Espectrometría de Masas , AmidohidrolasasRESUMEN
Newborn screening (NBS) for metachromatic leukodystrophy (MLD) is based on first-tier measurement of sulfatides in dried blood spots (DBS) followed by second-tier measurement of arylsulfatase A in the same DBS. This approach is very precise with 0-1 false positives per â¼30,000 newborns tested. Recent data reported here shows that the sulfatide molecular species with an α-hydroxyl, 16carbon, mono-unsaturated fatty acyl group (16:1-OH-sulfatide) is superior to the original biomarker 16:0-sulfatide in reducing the number of first-tier false positives. This result is consistent across 4 MLD NBS centers. By measuring 16:1-OH-sulfatide alone or together with 16:0-sulfatide, the estimated false positive rate is 0.048% and is reduced essentially to zero with second-tier arylsulfatase A activity assay. The false negative rate is predicted to be extremely low based on the demonstration that 40 out of 40 newborn DBS from clinically-confirmed MLD patients are detected with these methods. The work shows that NBS for MLD is extremely precise and ready for deployment. Furthermore, it can be multiplexed with several other inborn errors of metabolism already tested in NBS centers worldwide.
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Cerebrósido Sulfatasa , Pruebas con Sangre Seca , Leucodistrofia Metacromática , Tamizaje Neonatal , Sulfoglicoesfingolípidos , Humanos , Leucodistrofia Metacromática/diagnóstico , Leucodistrofia Metacromática/sangre , Recién Nacido , Sulfoglicoesfingolípidos/sangre , Tamizaje Neonatal/métodos , Cerebrósido Sulfatasa/sangre , Cerebrósido Sulfatasa/genética , Pruebas con Sangre Seca/métodos , Reacciones Falso Positivas , Biomarcadores/sangreRESUMEN
Renal proximal tubulopathy in Fanconi-Bickel syndrome is caused by impaired basolateral glucose transport via GLUT2 and consequently, intracellular accumulation of glucose and glycogen. SGLT2 inhibitors act on apical glucose reabsorption of renal proximal tubular cells. The purpose of this study was to retrospectively describe the first experiences with repurposing the SGLT2 inhibitor empagliflozin to treat the generalized tubulopathy in Fanconi-Bickel syndrome. A case series was conducted of seven persons from five families (five males, two females; three children, who were 14y5m, 2y9m, and 1y6m old) with genetically confirmed Fanconi-Bickel syndrome, off-label treated with empagliflozin. Median (range) age at start of empagliflozin was 27 years (1y6m - 61y) and duration of follow-up under empagliflozin treatment was 169 days (57-344). Under empagliflozin (up to 25 mg/d), biochemical parameters of tubular cell integrity (urinary N-acetyl-glucosaminidase) and/or tubular functions (including urinary α1-microglobulin) improved in all persons with Fanconi-Bickel syndrome, albeit to varying degrees. Clinically, supplementations (i.e., phosphate, alkali, carnitine, and alfacalcidol) could be completely discontinued in the three children, whereas results in the four adult patients were more variable and not as significant. Empagliflozin was well-tolerated and no symptomatic hypoglycemia was observed. In conclusion, SGLT2 inhibitors such as empagliflozin shift the metabolic block in Fanconi-Bickel syndrome, that is, they intervene specifically in the underlying pathophysiology and can thus attenuate renal proximal tubulopathy, especially when started in early childhood.
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The aim of our study was to assess the current state of newborn screening (NBS) in the region of southeastern Europe, as an example of a developing region, focusing also on future plans. Responses were obtained from 11 countries. Phenylketonuria screening was not introduced in four of 11 countries, while congenital hypothyroidism screening was not introduced in three of them; extended NBS programs were non-existent. The primary challenges were identified. Implementation of NBS to developing countries worldwide should be considered as a priority.
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Enfermedades Genéticas Congénitas/diagnóstico , Tamizaje Neonatal , Hipotiroidismo Congénito/diagnóstico , Hipotiroidismo Congénito/epidemiología , Europa (Continente) , Enfermedades Genéticas Congénitas/epidemiología , Humanos , Recién Nacido , Tamizaje Masivo/economía , Tamizaje Masivo/métodos , Tamizaje Neonatal/economía , Tamizaje Neonatal/métodos , Fenilcetonurias/diagnóstico , Fenilcetonurias/epidemiologíaRESUMEN
OBJECTIVE: Maple syrup urine disease (MSUD), a life-threatening metabolic disorder, is included in newborn screening (NBS) programs worldwide. The study aims to evaluate the impact of NBS on the long-term outcome of MSUD patients. METHODS: We performed a prospective, national, multicenter, observational study. RESULTS: In the studied NBS cohort (N = 33; 22 classic MSUD [cMSUD], 11 variant MSUD [vMSUD]; median age at last visit 10.4 years), 32 (97%) patients survived, 58% of them had normal cognitive functions (median IQ 87). Initial peak leucine increased linearly with age in cMSUD (median: 1712 µmol/L), but not in vMSUD. Global IQ correlated inversely with the initial peak leucine concentration (P = .04; ß = -0.0081) and the frequency of decompensations (P = .02; ß = -9.133). A cluster analysis identified 2 subgroups differing in their long-term metabolic control (median leucine concentration: 162 vs 278 µmol/L; P < .001). In cMSUD, lower leucine concentrations were associated with a higher IQ (95.5 vs 80; P = .008). Liver transplantation (median age 5.8 years) was not associated with better cognitive outcome. NBS is highly sensitive for cMSUD, but vMSUD might be missed (N = 2 missed by NBS). CONCLUSIONS: NBS and the early start of treatment improve survival and long-term outcome in individuals with cMSUD. Disease severity is an important modifier of outcome; however, the time to NBS report and the quality of long-term metabolic control had an independent impact on cognitive outcome, highlighting the importance of an early diagnosis and the quality of treatment.
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Enfermedad de la Orina de Jarabe de Arce , Tamizaje Neonatal , Humanos , Enfermedad de la Orina de Jarabe de Arce/diagnóstico , Enfermedad de la Orina de Jarabe de Arce/terapia , Tamizaje Neonatal/métodos , Recién Nacido , Masculino , Femenino , Estudios Prospectivos , Niño , Resultado del Tratamiento , Preescolar , Leucina/sangre , Adolescente , LactanteRESUMEN
We report on liver transplantation in two patients with GSD Ib on treatment with empagliflozin. The use of this SGLT2 inhibitor resulted in a marked decrease of 1,5-anhydroglucitol which has an important role in the development of neutropenia in this condition. As intended, this caused a significant rise of neutrophil numbers. Liver transplantation alone did not produce the desired effect and our observation argues for continuing SGLT2 inhibitor treatment after transplantation.
RESUMEN
Newborn screening (NBS) for isovaleric acidemia (IVA) is performed by flow injection tandem mass spectrometry quantifying C5 carnitines (C5). Isovalerylcarnitine, however, is isomeric with pivaloylcarnitine which can be present in blood due to maternal use of pivaloylester-containing antibiotics, available in Germany since late 2016. During a 36-month period (January 19-December 21), all newborns screened in Hamburg with a C5 above cutoff (NeoGram®: 0.50 µmol/L or Neobase®2: 0.45 µmol/L) were included in the study. As a second-tier test, a simple ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed to differentiate the C5 isomers pivaloyl-, 2-methylbutyryl-, isovaleryl-, and valerylcarnitine. Out of 156 772 newborns tested, one turned out to have genetically proven IVA while 99 were false positive (C5: 0.5-8.2 µmol/L) due to the presence of pivaloylcarnitine. These cases have increased year by year and show local clusters. Retrospective analysis of another 39 cases from 287 206 neonates tested at the NBS center in Heidelberg with C5 elevation (0.9-10.6 µmol/L) but clinical and biochemical exclusion of IVA yielded evidence of pivaloylcarnitine in all cases. Inclusion of a second-tier test into NBS significantly reduces the high and increasing false-positive rate of IVA screening. This avoids further diagnostic steps, prevents unnecessary stress and anxiety of parents in a remarkably high number of cases. If Hamburg data of 2021 are extrapolated to all of Germany, one can assume around 800 (1) false-positive cases in comparison to an average of two classic IVA cases per year. Unless licensing of pivaloylester-containing drugs for use during pregnancy is reconsidered, a second-tier test for C5 determination is indispensable.
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A BH(4) loading test was performed in 36 patients from 34 unrelated families. The patients had 29 different genotypes, and previous data on only eight of them were found in the BIOPKU database. Thirteen patients were classified as classic PKU (35.1%), 14 as mild PKU (37.8%) and 9 as MHP (27.0%). Blood Phe levels were shown to reach a plateau after three full days of increased natural protein ingestion. Measuring the 24-hour blood Phe levels (T(-24), T(-16), T(0)) on the fourth day of increased protein ingestion before BH(4) administration showed that within 24h Phe on average increased by 2.4% in MHP patients, decreased by 2.7% in mild PKU patients and increased by 9.7% in classic PKU patients (NS for all comparisons); Phe only slightly decreased in responders by 0.2% but increased in non-responders by 7.8% (P>0.05). Altogether, 16 of 36 (44.4%) patients represented by 12 of 29 (41.4%) different genotypes were proven to be BH(4) responders, and four (10.8%) were slow-responders. Responders were 6/9 (66.7%) MHP patients, 10/14 (71.4%) mild PKU patients and 0/13 classic PKU patients. Twenty of the 29 (68.9%) genotypes harbored at least one mutation with a known PRA of 10% or more but only 11 (55%) of them were BH(4)-responsive. Spontaneous reduction of blood Phe levels within 24h on the fourth day of natural protein loading was observed only in mild PKU patients and was shown not to be an important part of the BH(4)-response. 73.3% of genotypes containing at least one allele with a PRA of at least 30% were found to be BH(4) responsive; a PRA of at least 15.5% was needed for the responder genotype in our population.
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Biopterinas/análogos & derivados , Fenilalanina Hidroxilasa/deficiencia , Fenilalanina/sangre , Fenilcetonurias/sangre , Fenilcetonurias/tratamiento farmacológico , Adolescente , Biopterinas/uso terapéutico , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Mutación , Fenilalanina Hidroxilasa/genética , Fenilalanina Hidroxilasa/metabolismo , Resultado del Tratamiento , Adulto JovenRESUMEN
Fanconi-Bickel syndrome (FBS) is a very rare but distinct clinical entity with the combined features of hepatic glycogen storage disease, generalized proximal renal tubular dysfunction with disproportionately severe glucosuria, and impaired galactose tolerance. Here, we report five cases (out of 93 diagnosed in our lab) with pathogenic variants on both GLUT2 (SLC2A2) alleles. They come from 3 families and presented with an exceptionally mild clinical course. This course was correlated to data from old and most recent expression and transport studies in Xenopus oocytes. GLUT2 genotype in patients 1 and 2 was p.[153_4delLI];[P417R] with the first variant exhibiting normal membrane expression and partially retained transport activity (5.8%) for 2-deoxyglucose. In patient 3, the very first GLUT2 variant ever detected (p.V197I) was found, but for the first time it was present in a patient in the homozygous state. This variant had also shown unaffected membrane expression and remarkable residual activity (8%). The genotype in patient 4, p.[153_4delLI];[(E440A)], again included the 2-amino-acid deletion with residual transporter function, and patient 5 is the first found to be homozygous for this variant. Our results provide further evidence for a genotype-phenotype correlation in patients with GLUT2 variants; non-functional variants result in the full picture of FBS while dysfunctional variants may result in milder presentations, even glucosuria only, without other typical signs of FBS.
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Síndrome de Fanconi/genética , Transportador de Glucosa de Tipo 2/genética , Mutación , Fenotipo , Adolescente , Adulto , Animales , Síndrome de Fanconi/patología , Femenino , Genotipo , Glucosa/metabolismo , Transportador de Glucosa de Tipo 2/metabolismo , Homocigoto , Humanos , Lactante , Masculino , Linaje , XenopusRESUMEN
The Sava River is the biggest tributary to the Danube River. As a part of the 6th FW EU project, Sava River Basin: Sustainable Use, Management and Protection of Resources (SARIB), ecological status of sediments was investigated. In order to assess the geographical distribution in sediment contamination of the Sava River, inorganic and persistent organic pollutants were analyzed in sediments at 20 selected sampling sites along the Sava River from its spring to its outfall into the Danube River. For comparability of data to other river basins the sediment fraction below 63 microm was studied. Due to complexity of the work performed, the results are published separately ("Part I: Selected elements" and "Part II: Persistent organic pollutants"). In the present study, the extent of pollution was estimated by determination of the total element concentrations and by the identification of the most hazardous highly mobile element fractions and anthropogenic inputs of elements to sediments. To assess the mobile metal fraction extraction in 0.11 mol L(-1), acetic acid was performed (first step of the Community Bureau of Reference extraction procedure), while anthropogenic inputs of elements were estimated on the basis of normalization to aluminum (Al) concentration. According to the Water Framework Directive, the following elements were investigated in sediments: cadmium (Cd), lead (Pb), nickel (Ni), and mercury (Hg). Furthermore, copper (Cu), zinc (Zn), chromium (Cr), arsenic (As), and phosphorous (P) were determined. The analyses of sediments demonstrated slightly elevated values for Hg, Cr, and Ni in industrially exposed sites (concentrations up to 0.6, 380, and 210 mg kg(-1), respectively). However, the latter two elements exist in sparingly soluble forms and therefore do not represent an environmental burden. P concentrations were found in elevated concentrations at agricultural areas and big cities (up to 1,000 mg kg(-1)).
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Sedimentos Geológicos/química , Contaminantes del Agua/análisis , Espectrometría de Masas/métodos , EsloveniaRESUMEN
A new analytical procedure using separation support based on Convective Interaction Media (CIM) was developed for speciation of Al in human serum at normal concentration levels. The separation of proteins was performed on a weak anion exchange CIM diethylamine monolithic column. Isocratic elution with buffer A (0.05 M tris(hydroxymethyl)aminomethane-hydrochloric acid + 0.03 M sodium hydrogen carbonate) was applied for 5 min, followed by linear gradient elution from 100% buffer A to 100% buffer B (buffer A + 1 M ammonium chloride) for the next 40 min. Separation of proteins was followed by UV detection at 278 nm. Separated Al species were detected online by inductively coupled plasma mass spectrometry. It was experimentally proven that 91 +/- 7% of Al in human serum was eluted under the transferrin peak. Transferrin was identified on the basis of the retention volume and by ACQUITY ultraperformance liquid chromatography-electrospray ionization mass spectrometry. The problem of extraneous contamination with Al was successfully overcome by using efficient cleaning procedures of eluents and chromatographic supports. The efficient cleaning was of paramount importance to perform Al speciation at extremely low concentration levels. The repeatability of measurement tested for six consecutive separations of unspiked serum was +/-8.6%. The limits of detection and quantification (based on 3 and 10 s of the blank) were 0.15 and 0.49 ng mL(-1) Al bound to transferrin, respectively. This is the first report on quantitative and reliable speciation of Al in human serum at normal concentration levels.
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Aluminio/sangre , Cromatografía Líquida de Alta Presión/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , Humanos , Unión Proteica , Espectrometría de Masa por Ionización de Electrospray/instrumentación , Espectrofotometría Ultravioleta , Transferrina/análisisRESUMEN
PURPOSE: CLN2 disease is a genetic disorder caused by dysfunction of the lysosomal enzyme tripeptidyl peptidase 1 (TPP1) that belongs to the neuronal ceroid lipofuscinoses (NCL) and leads to epilepsy, dementia, and death in young persons. CLN2 disease has recently become treatable by enzyme replacement, which can only be effective when the disease is diagnosed early. We have investigated the reliability of a test for TPP1 deficiency in dried blood specimens (DBS) to detect CLN2 disease. RESULTS: During a 12-year period we have received 3882 samples for testing TPP1. Quality of samples was checked by measuring two additional lysosomal enzyme activities. For 50 samples with subnormal TPP1 activity and good sample quality, we obtained adequate clinical and molecular genetic data. All 50 patients had doubtless evidence of CLN2 disease (including seven atypical patients) as shown by clinical findings and the presence of known pathogenic CLN2 variants. Our institution is a major reference center for NCL, and we have never received information that a patient with a normal DBS test was later diagnosed with CLN2 disease. CONCLUSIONS: We consider our TPP1 test on DBS to be a reliable, convenient and inexpensive tool for a first diagnostic step in suspected CLN2 disease.
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Aminopeptidasas/sangre , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/sangre , Pruebas con Sangre Seca/métodos , Fluorometría/métodos , Lipofuscinosis Ceroideas Neuronales/sangre , Lipofuscinosis Ceroideas Neuronales/enzimología , Serina Proteasas/sangre , Femenino , Humanos , Masculino , Lipofuscinosis Ceroideas Neuronales/genética , Fenotipo , Factores de Tiempo , Tripeptidil Peptidasa 1RESUMEN
OBJECTIVES: Precise quantification of amino acids (AAs) is mandatory for successful diagnosis and monitoring of patients with metabolic diseases. We compared ion-exchange chromatography (IEC) and liquid chromatography with tandem mass spectrometry (LC-MS/MS), the two methods most commonly used in clinical laboratories for the quantification of AAs in physiological samples. DESIGN & METHODS: 123 apparently healthy children were selected for the study. The plasma samples for LC-MS/MS were prepared accordingly to the aTRAQ Kit for Physiological Fluids on Sciex 3200 Qtrap, for IEC according to the protocol from Pickering laboratories on the AA analyzer Pinnacle PCX. Results were interpreted using the Pearson correlation coefficient and the percent difference Bland-Altman test. RESULTS: The Spearman correlation coefficients of the 14 AAs that we evaluated varied from 0.67 in Tau to 0.89 in Leu and Thr. The mean differences in measurements (IEC compared to LC-MS/MS) of 11 AAs complied with our acceptance criterion of <15%, the differences of Ser and Tyr were higher (19.5% and -19.0%, respectively), and the measured concentrations of Cit were much lower in LC-MS/MS than IEC (31% difference). CONCLUSION: The two methods are sufficiently comparable for most AAs and the reference values for individual AAs did not have to be refined, with the exception of citrulline. For the monitoring of patients on therapy (e.g. patients with phenylketonuria), it is still advisable to always use the same analytical method for the quantification of AAs.
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Aminoácidos/sangre , Aminoácidos/química , Análisis Químico de la Sangre/métodos , Cromatografía por Intercambio Iónico/métodos , Cromatografía Liquida/métodos , Ninhidrina/química , Espectrometría de Masas en Tándem/métodos , Adolescente , Niño , Preescolar , Femenino , Humanos , MasculinoRESUMEN
The potential use of filter dust in asphalt composites for road construction was investigated. Filter dust contains high concentrations of metals, of which Cr(VI) and Pb are leached with water. Compact and ground asphalt composites with addition of 2% of filter dust by mass were studied. In order to evaluate their environmental impact, leachability tests were performed using water and salt water as leaching agents. The concentrations of Cr(VI) and Pb were determined in leachates over a time period of 182 days. The results indicated that Pb was not leached with leaching agents from asphalt composites. Cr(VI) was also not leached with leaching agents from compact asphalt composites. However, in ground asphalt composites, Cr(VI) was leached with water in concentrations up to 220 microg L(-1) and in salt water up to 150 microg L(-1). From the physico-mechanical and environmental aspects, filter dust can be used as a component in asphalt mixtures.
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Cromo/química , Materiales de Construcción/análisis , Polvo/análisis , Hidrocarburos/química , Filtración , Solubilidad , Factores de Tiempo , Contaminantes Químicos del AguaRESUMEN
A new analytical procedure using anion-exchange separation support based on convective-interaction media (CIM) was developed for the speciation of Al in human serum. The separation of proteins was performed on a weak anion-exchange CIM diethylamine (DEAE) fast-monolithic disk. To prevent co-elution of low molecular mass (LMM) Al species with high molecular mass (HMM) Al compounds on CIM disk serum proteins were first separated from LMM-Al species by the use of size exclusion chromatography (SEC). For this purpose 1 mL of serum was injected onto SEC (Superdex 75 HR 10/30) column. Isocratic elution using 0.05 M TRIS-HCl+0.03 M NaHCO(3) was applied and separation of proteins was followed by UV detection at 278 nm. It was experimentally proven that proteins were eluted in 5.5 mL peak that was collected into a polyethylene cup. A 0.1 mL of the sample aliquot was then injected onto the CIM DEAE disk. The separation of serum proteins was obtained in 10 min by applying linear gradient elution from 100% buffer A (0.05 M TRIS-HCl+0.03 M NaHCO(3)) to 100% buffer B (A+1M NH(4)Cl) and followed by UV detection at 278 nm. Separated Al species were detected on-line by inductively coupled plasma mass spectrometry (ICP-MS). Well-resolved protein peaks were obtained. It was experimentally proven that 90+/-3% of Al in spiked serum of renal patient was eluted under the transferrin peak. The proposed speciation procedure removes LMM-Al species and enables reliable determination of the concentration and composition of Al bound to proteins by CIM DEAE-ICP-MS when the concentration of Al in serum is higher than 5 ng mL(-1). In comparison to chromatographic columns CIM disks enable faster separation and simpler manipulation during cleaning procedure and coupling to ICP-MS.
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Aluminio/sangre , Cromatografía Líquida de Alta Presión/métodos , Espectrometría de Masas/métodos , Cromatografía por Intercambio Iónico , Humanos , Estándares de Referencia , Espectrofotometría UltravioletaRESUMEN
BACKGROUND: Short-chain acyl-CoA dehydrogenase deficiency (SCADD) is a rare inherited mitochondrial fatty acid oxidation disorder associated with variations in the ACADS (Acyl-CoA dehydrogenase, C-2 to C-3 short chain) gene. SCADD has highly variable biochemical, genetic and clinical characteristics. Phenotypes vary from fatal metabolic decompensation to asymptomatic individuals. SUBJECT AND METHODS: A Romani boy presented at 3 days after birth with hypoglycaemia, hypotonia and respiratory pauses with brief generalized seizures. Afterwards the failure to thrive and developmental delay were present. Organic acids analysis with gas chromatography-mass spectrometry (GS/MS) in urine and acylcarnitines analysis with liquid chromatography-tandem mass spectrometry (LC-MS/MS) in dried blood spot were measured. Deoxyribonucleic acid (DNA) was isolated from blood and polymerase chain reactions (PCRs) were performed for all exons. Sequence analysis of all exons and flanking intron sequences of ACADS gene was performed. RESULTS: Organic acids analysis revealed increased concentration of ethylmalonic acid. Acylcarnitines analysis showed increase of butyrylcarnitine, C4-carnitine. C4-carnitine was 3.5 times above the reference range (<0.68 µmol/L). Confirmation analysis for organic acids and acylcarnitine profile was performed on the second independent sample and showed the same pattern of increased metabolites. Sequence analysis revealed 3-bp deletion at position 310-312 in homozygous state (c.310_312delGAG). Mutation was previously described as pathogenic in heterozygous state, while it is in homozygous state in our patient. CONCLUSIONS: In our case clinical features of a patient, biochemical parameters and genetic data were consistent and showed definitely SCAD deficiency.
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3-Hidroxiacil-CoA Deshidrogenasas/genética , Acil-CoA Deshidrogenasa/deficiencia , Secuencia de Bases , Errores Innatos del Metabolismo Lipídico/sangre , Errores Innatos del Metabolismo Lipídico/genética , Eliminación de Secuencia , Acil-CoA Deshidrogenasa/sangre , Acil-CoA Deshidrogenasa/genética , Análisis Mutacional de ADN , Humanos , Lactante , Errores Innatos del Metabolismo Lipídico/patología , Masculino , Reacción en Cadena de la PolimerasaRESUMEN
INTRODUCTION: Newborn screening in whole Slovenia started in 1979 with screening for phenylketonuria (PKU). Congenital hypothyroidism (CH) was added into the programme in 1981. The aim of this study was to analyse the data of neonatal screening in Slovenia from 1993 to 2012 for PKU, and from 1991 to 2012 for CH. METHODS: Blood samples were collected from the heels of newborns between the third and the fifth day after birth. Fluorometric method was used for screening for PKU, CH screening was done by dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA). RESULTS: From 1993 to 2012, from 385,831 newborns 57 were identified with PKU. 184 newborns out of 427,396 screened from 1991 to 2012, were confirmed for CH. Incidences of PKU and CH in the periods stated are 1:6769 and 1:2323, respectively. CONCLUSIONS: Successful implementation of newborn screening for PKU and CH has helped in preventing serious disabilities of the affected children. Adding screening for new metabolic diseases in the future would be beneficial.
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BACKGROUND: Inborn errors of metabolism (IEM) are disorders with a block in the metabolic pathway caused by a genetic defect of a specific enzyme. Although each of these diseases is quite rare, as a group they account for a significant proportion of newborn and childhood morbidity and mortality. Early diagnosis is important to prevent complications or even death of the child. Selective screening is an important diagnostic tool for the diagnosis of IEM. METHODS: In Slovenia, symptomatic patients with suspected IEM are referred to the University Children's Hospital Ljubljana. Techniques used for selective screening are gas chromatography-mass spectrometry, ion exchange chromatography-post-column derivatization, liquid chromatography-tandem mass spectrometry and isoelectric focusing. Fluorimetric method is used for enzyme activity measurement. RESULTS: There are 168 patients with amino and organic acidemias, 5 patients with disorders in fatty acids metabolism, 1 patient with a congenital disorder of glycosylation, 42 patients with Fabry disease (of which 37 are adult) and 20 patients with Gaucher disease (of which 18 are adult) in the Slovenian Register for Rare Diseases. CONCLUSIONS: In Slovenia, management of patients with IEM is centralized at the University Children's Hospital, with the exception of adult patients with Fabry and Gaucher disease. The team work is well organized with close cooperation between the laboratory and pediatricians specialized in metabolic disorders. According to the known frequencies of IEM from the literature, we would expect more positive results than obtained. To evaluate these results, we are planning to perform a pilot study on expanded newborn screening.
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OBJECTIVES: Regular and accurate monitoring of blood phenylalanine (Phe) and tyrosine (Tyr) levels is prerequisite for a successful management of patients with hyperphenylalaninemia (HPA). We aimed to compare the tandem mass spectrometry (MS/MS) and the amino acid analyzer (AAA) as methods to measure blood Phe and Tyr levels and Phe/Tyr ratio. METHODS: Venous blood samples were collected for the AAA analysis, using Pinnacle PCX (Pickering Laboratories), with HPLC Series 1200 (Agilent). Capillary blood was spotted directly on filter paper (Whatman 903) for the MS/MS analysis, using 3200 QTrap AB SCIEX and Perkin Elmer Series 200 HPLC system. The Bland-Altman test was used to compare agreement between the methods and Pearson correlation coefficient to assess the association between the methods. RESULTS: 207 pairs of measurements were performed. The Phe levels (range 0-2500µM) obtained by the MS/MS were on average 26.1% (SD 13.9%) lower compared to those obtained by the AAA. The Tyr levels by the MS/MS were on average 15.5% (SD 20.6%) lower. The Phe/Tyr ratio by the MS/MS was on average 10.6% (SD 15.9%) lower. The Pearson correlation coefficients for Phe (range 0-2500µM), Tyr and the Phe/Tyr ratio were 0.984 (p<0.001), 0.841 (p<0.001) and 0.987 (p<0.001) respectively. CONCLUSIONS: When monitoring blood Phe and Tyr levels in patients with HPA, clinicians need to be informed about the method used. Due to the considerable inter-assay variability, a single method is preferable for long-term follow-up of patients. When using MS/MS, on average 26% lower blood Phe levels were obtained as compared to the AAA. The guidelines and recommendations on HPA management should take into consideration the differences in laboratory methods.