Perioperative Management of a Child With Glucose Transporter Type 1 Deficiency Syndrome: A Case Report.

Glucose transporter type 1 deficiency syndrome (GLUT1DS) causes central nervous system dysfunction including intractable epilepsy caused by impaired glucose transport to the brain. To prevent convulsions and maintain an energy source for the brain in patients with GLUT1DS, the maintenance of adequate ketone body concentrations, compensation of metabolic acidosis, and reduction of surgical stress are essential. We here report the perioperative management of a child with GLUT1DS.

MALDI-QTOFMS/MS identification of glycoforms from the urine of a CDG patient.

Identification of single glycoconjugate components in a complex mixture from the urine of a patient suffering from a congenital disorder of glycosylation was probed by MALDIMS analysis on a hybrid quadrupole time-of-flight instrument. In negative ion mode, complex maps containing more than 50 ionic species were obtained and a number of molecular ions directly as-signed using a previously developed computer-assisted algorithm. To confirm the data and determine the carbohydrate sequence, single molecular ions were selected and submitted to fragmentation experiments. Interpretation of fragmentation spectra was also assisted by the soft-ware using alignment with spectra generated in silico. According to fragmentation data, the majority of glycoconjugate ionic species could be assigned to free oligosaccharides along with ten species tentatively assigned to glycopeptides. Following this approach for glycan identification by a combination of MALDI-QTOFMS and MS/MS experiments, computer-assisted assignment and fragment analysis, data for a potential glycan data base are produced. Of high benefit for this approach are two main factors: low sample consumption due to the high sensitivity of ion formation, and generation of only singly charged species in MALDIMS allowing interpretation with-out any deconvolution. In this experimental set-up, sequencing of single components from the MALDI maps by low energy CID followed by computer-assisted assignment and data base search is proposed as a most efficient strategy for the rapid identification of complex carbohydrate structures in clinical glycomics.

Detection of transaldolase deficiency by quantification of novel seven-carbon chain carbohydrate biomarkers in urine.

Transaldolase deficiency, a recently discovered disorder of carbohydrate metabolism with multisystem involvement, has been diagnosed in 6 patients. Affected patients have abnormal concentrations of polyols in body fluids and in all patients we have previously found increased amounts of a seven-carbon chain carbohydrate which we suspected of being sedoheptulose. We report development of a liquid chromatography-tandem mass spectrometry method for quantitation of the seven-carbon carbohydrates sedoheptulose and mannoheptulose in urine. Additionally, other seven-carbon chain carbohydrates were characterized in urine, including sedoheptitol, perseitol and sedoheptulose 7-phosphate. Transaldolase-deficient patients had significantly increased urinary sedoheptulose and sedoheptulose 7-phosphate, associated with subtle elevations of mannoheptulose, sedoheptitol and perseitol. Our findings reveal novel urinary biomarkers for identification of transaldolase deficiency.

Micellar electrokinetic chromatographic and capillary zone electrophoretic methods for screening urinary biomarkers of human disorders: a critical review of the state-of-the-art.

Human urine plays a central role in clinical diagnostic being one of the most-frequently used body fluid for detection of biological markers. Samples from patients with different diseases display patterns of biomarkers that differ significantly from those obtained from healthy subjects. The availability of fast, reproducible, and easy-to-apply analytical techniques that would allow identification of a large number of these analytes is thus highly desiderable since they may provide detailed information about the progression of a pathological process. From among the variety of methods so far applied for the determination of urinary metabolites, capillary electrophoresis, both in the capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) modes, represents a robust and reliable analytical tool widely used in this area. The aim of the present article is to focus the interest of the reader on recent applications of MEKC and CZE in the field of urinary biomarkers and to discuss advantages and/or limitations of each mode.

L-Arabinosuria: a new defect in human pentose metabolism.

A female patient, the first child of healthy non-consanguineous parents, presented at the age of 16 months with delayed motor development and facial dysmorphism. In addition she displayed a palatoschizis and multiple skeletal abnormalities as hypoplastic scapulae, hypoplastic os ilea, and an extreme cervical kyphosis. Biochemical investigation of urine revealed no abnormalities except for the presence of large amounts of reducing sugars. The sugar was identified as L-arabinose, which mainly originated from fruit formula in her diet. In addition highly elevated levels of L-arabitol were found in urine, plasma, and cerebrospinal fluid. Although little is known about human arabinose metabolism, we presume that L-arabitol dehydrogenase is deficient in our patient. As polyols are potentially toxic to the central nervous system there could be deleterious long-term effects of this disorder. Withdrawal of dietary fruit led to normalization of polyol levels. The above-mentioned clinical abnormalities are probably not related to this new inborn error of metabolism and should be considered as a separate entity.

Processing of N-linked carbohydrate chains in a patient with glucosidase I deficiency (CDG type IIb).

Recently, we reported a novel congenital disorder of glycosylation (CDG-IIb) caused by severe deficiency of the glucosidase I. The enzyme cleaves the alpha1,2-glucose residue from the asparagine-linked Glc(3)-Man(9)-GlcNAc(2) precursor, which is crucial for oligosaccharide maturation. The patient suffering from this disease was compound-heterozygous for two mutations in the glucosidase I gene, a T-->C transition in the paternal allele and a G-->C transition in the maternal allele. This gives rise in the glucosidase I polypeptide to the substitution of Arg486 by Thr and Phe652 by Leu, respectively. Kinetic studies using detergent extracts from cultured fibroblasts showed that the glucosidase I activity in the patient's cells was < 1% of the control level, with intermediate values in the parental cells. No significant differences in the activities of other processing enzymes, including oligosaccharyltransferase, glucosidase II, and Man(9)-mannosidase, were observed. By contrast, the patient's fibroblasts displayed a two- to threefold higher endo-alpha1,2-mannosidase activity, associated with an increased level of enzyme-specific mRNA-transcripts. This points to the lack of glucosidase I activity being compensated for, to some extent, by increase in the activity of the pathway involving endo-alpha1,2-mannosidase; this would also explain the marked urinary excretion of Glc(3)-Man. Comparative analysis of [(3)H]mannose-labeled N-glycoproteins showed that, despite the dramatically reduced glucosidase I activity, the bulk of the N-linked carbohydrate chains (>80%) in the patient's fibroblasts appeared to have been processed correctly, with only approximately 16% of the N-glycans being arrested at the Glc(3)-Man(9-7)-GlcNAc(2) stage. These structural and enzymatic data provide a reasonable basis for the observation that the sialotransferrin pattern, which frequently depends on the type of glycosylation disorder, appears to be normal in the patient. The human glucosidase I gene contains four exons separated by three introns with exon-4 encoding for the large 64-kDa catalytic domain of the enzyme. The two base mutations giving rise to substitution of Arg486 by Thr and Phe652 by Leu both reside in exon-4, consistent with their deleterious effect on enzyme activity. Incorporation of either mutation into wild-type glucosidase I resulted in the overexpression of enzyme mutants in COS 1 cells displaying no measurable catalytic activity. The Phe652Leu but not the Arg486Thr protein mutant showed a weak binding to a glucosidase I-specific affinity resin, indicating that the two amino acids affect polypeptide folding and active site formation differently.

Glycoscreening by on-line sheathless capillary electrophoresis/electrospray ionization-quadrupole time of flight-tandem mass spectrometry.

An analytical approach based on sheathless on-line coupling of capillary electrophoresis (CE) and electrospray ionization (ESI) quadrupole time-of-flight (QTOF) mass spectrometry (MS) has been developed for providing new insight into the characterization of carbohydrate mixtures. The home-built sheathless CE/

In vivo and in vitro NMR spectroscopy reveal a putative novel inborn error involving polyol metabolism.

In vivo NMR spectroscopy was performed on the brain of a patient with a leukoencephalopathy, revealing unknown resonances between 3.5 and 4.0 ppm. In addition, urine and CSF of the patient were measured using high-resolution NMR spectroscopy. Also in these in vitro spectra, unknown resonances were observed in the 3.5-4.0 ppm region. Homonuclear (1)H two-dimensional J-resolved spectroscopy (JRES) and (1)H-(1)H correlation spectroscopy (COSY) were performed on the patient's urine for more accurate assignment of resonances. The NMR spectroscopic studies showed that the unknown resonances could be assigned to arabinitol and ribitol. This was confirmed using gas chromatography. The arabinitol was identified as D-arabinitol. The patient is likely to suffer from an as yet unknown inborn error of metabolism affecting D-arabinitol and ribitol metabolism. The primary molecular defect has not been found yet. Urine spectra of patients suffering from diabetes mellitus or galactosemia were recorded for comparison. Resonances outside the 3.2-4.0 ppm region, which are the most easy to recognize in body fluid spectra, allow easy recognition of various sugars and polyols. The paper shows that NMR spectroscopy in body fluids may help identifying unknown resonances observed in in vivo NMR spectra.

A novel disorder caused by defective biosynthesis of N-linked oligosaccharides due to glucosidase I deficiency.

Glucosidase I is an important enzyme in N-linked glycoprotein processing, removing specifically distal alpha-1,2-linked glucose from the Glc3Man9GlcNAc2 precursor after its en bloc transfer from dolichyl diphosphate to a nascent polypeptide chain in the endoplasmic reticulum. We have identified a glucosidase I defect in a neonate with severe generalized hypotonia and dysmorphic features. The clinical course was progressive and was characterized by the occurrence of hepatomegaly, hypoventilation, feeding problems, seizures, and fatal outcome at age 74 d. The accumulation of the tetrasaccharide Glc(alpha1-2)Glc(alpha1-3)Glc(alpha1-3)Man in the patient's urine indicated a glycosylation disorder. Enzymological studies on liver tissue and cultured skin fibroblasts revealed a severe glucosidase I deficiency. The residual activity was <3% of that of controls. Glucosidase I activities in cultured skin fibroblasts from both parents were found to be 50% of those of controls. Tissues from the patient subjected to SDS-PAGE followed by immunoblotting revealed strongly decreased amounts of glucosidase I protein in the homogenate of the liver, and a less-severe decrease in cultured skin fibroblasts. Molecular studies showed that the patient was a compound heterozygote for two missense mutations in the glucosidase I gene: (1) one allele harbored a G-->C transition at nucleotide (nt) 1587, resulting in the substitution of Arg at position 486 by Thr (R486T), and (2) on the other allele a T-->C transition at nt 2085 resulted in the substitution of Phe at position 652 by Leu (F652L). The mother was heterozygous for the G-->C transition, whereas the father was heterozygous for the T-->C transition. These base changes were not seen in 100 control DNA samples. A causal relationship between the alpha-glucosidase I deficiency and the disease is postulated.

Age-related reference values for urinary excretion of sialic acid and deoxysialic acid: application to diagnosis of storage disorders of free sialic acid.

We have established by HPLC age-related reference intervals for sialic acid urinary excretion in 364 control individuals to assist in evaluating the clinical significance of the free sialic acid concentration in urine. In addition, an HPLC method for quantitative analysis of free deoxysialic acid was developed, and age-related reference intervals for excretion of this compound in urine were established. In patients with storage disorders of free sialic acid (n = 11) the sialic acid excretion was increased 2- to 35-fold, compared with the mean value of the control subjects in the corresponding age group, and exceeded the interval in each case. The excretion of deoxysialic acid was within the reference interval in all of the patients, indicating that its metabolism was not affected in the disorders. The age-related reference values assist in evaluating the excretion of free sialic acid in the diagnosis of storage disorders of free sialic acid, especially in young children.

CT and MR of the brain in the diagnosis of organic acidemias. Experiences from 107 patients.

The results of CT and/or MRI of the brain in 107 patients with different types of organic acidemia are presented. The CSF spaces were wide in more than two-thirds of the patients, in 46 slightly-to-moderately and in 26 markedly-to-severely dilated. Marked widening of the operculae was found in all 5 patients with glutaric acidemia type 1, but open opercula was also found in other organic acidemias. White matter changes were found in about half the patients, in 28 mildly-to-moderately pronounced, in another 28 marked or severe. Basal ganglia or central pathway pathology was seen in a total of 34 patients, i.e. 32%. These changes in 25 patients involved the caudate and/or lentiform nuclei: in 14 cases the T2 signal was increased and volume loss was present, in 9 cases increased T2 signal with preserved volume was found (in one of these the changes were transient). In 2 patients, both with ethylmalonic aciduria (cause unknown), only small high T2 spots were seen in the caudate heads and the putamina. In 4 patients, all suffering from methylmalonic acidemia, only the globus pallidus was affected. In 3 patients, all with beta-ketothiolase deficiency, high T2 intensity changes were seen only in the postero-lateral putamina. The remaining 8 patients represent a variety of different locations of lesions. The CT or MRI findings in many patients with organic acidemias should alert the radiologist that a neurometabolic disorder may be present; in some cases the location and appearance of the lesions may even suggest the correct diagnosis.

Molecular basis of essential fructosuria: molecular cloning and mutational analysis of human ketohexokinase (fructokinase).

Essential fructosuria is one of the oldest known inborn errors of metabolism. It is a benign condition which is believed to result from deficiency of hepatic fructokinase (ketohexokinase, KHK, E.C.2.7.1.3). This enzyme catalyses the first step of metabolism of dietary fructose, conversion of fructose to fructose-1-phosphate. Despite the early recognition of this disorder, the primary structure of human KHK and the molecular basis of essential fructosuria have not been previously defined. In this report, the isolation and sequencing of full-length cDNA clones encoding human ketohexokinase are described. Alternative mRNA species and alternative KHK isozymes are produced by alternative polyadenylation and splicing of the KHK gene. The KHK proteins show a high level of sequence conservation relative to rat KHK. Direct evidence that mutation of the KHK structural gene is the cause of essential fructosuria was also obtained. In a well-characterized family, in which three of eight siblings have fructosuria, all affected individuals are compound heterozygotes for two mutations Gly40Arg and Ala43Thr. Both mutations result from G-->A transitions, and each alters the same conserved region of the KHK protein. Neither mutation was seen in a sample of 52 unrelated control individuals. An additional conservative amino acid change (Val49IIe) was present on the KHK allele bearing Ala43Thr.

Urinary oligosaccharide excretion in nephrotic syndrome.

Urinary oligosaccharides from nephrotic syndrome patients were investigated by Bio-Gel P-4 column chromatography and methylation analysis to clarify the electrostatic defects and size barrier defects in the glomerular basement membrane of nephrotic syndrome patients. The levels of urinary oligosaccharides (nmol/mg creatinine) in nephrotic syndrome patients were about 25 times the levels in controls. The ratio of neutral oligosaccharides to sialyl oligosaccharides was about 1:1 in the urine of nephrotic syndrome patients. The levels of neutral oligosaccharides in the urine of nephrotic syndrome patients were about 13 times that of controls. The proportion of trisialylated oligosaccharides in the urine of nephrotic syndrome patients was higher than that in galactosialidosis patients. These findings suggest that electrostatic defects and size barrier defects in the glomerular basement membrane might contribute to the characteristic oligosacchariduria of nephrotic syndrome patients. In addition, the structures of 11 different kinds of oligosaccharides that have not been previously reported were identified in the urine of nephrotic syndrome patients.

Diagnosis and therapy of organic acidurias.

Organic acidurias are a group of numerous inherited metabolic disorders with an involvement of different pathways of the amino acid, carbohydrate and lipid metabolism. Many diseases have their onset with an acute metabolic crisis in the newborn period. The most important diagnostic investigation is organic acid analysis of urine by gas chromatography-mass spectrometry. Because specific clinical signs are lacking, a selective screening procedure is recommended. Diagnosis and therapy should be aggressive, since prognosis of many disorders is related to early diagnosis and specific therapeutic measures. These include long term management with protein restriction, supplementation with special amino acid mixtures and carnitine. In our experience continuous arteriovenous hemofiltration has turned out to be a very effective detoxification method during acute metabolic crisis of the newborn. Long term follow up and therapy control is very important for the outcome of the patients. Measurement of odd long chain fatty acids (OLCFA) in erythrocyte membranes has proved to be a good control parameter of the metabolic condition of children with disorders of the branched-chain amino acid metabolism.

Essential fructosuria: increased levels of fructose 3-phosphate in erythrocytes.

Erythrocytes of 3 adult siblings with essential fructosuria contained 45-200 mumol/l fructose 3-phosphate (Fru-3-P), i.e. 3-15 times the concentration in normal controls. Sorbitol 3-phosphate was also increased, but to a lesser degree. An oral load with 50 g of fructose produced an additional 40 mumol/l increase of erythrocyte Fru-3-P after 5 h. The rate of Fru-3-P formation by red cells in vitro was normal. HbA1 and HbA1c were normal. The suspected pathogenetic role of Fru-3-P in diabetic complications is questioned.

High-performance liquid chromatography of urinary oligosaccharides in the diagnosis of glycoprotein degradation disorders.

Urinary oligosaccharides can be separated by high-performance anion-exchange chromatography using a Dionex CarboPac PA1 column, elution with aqueous sodium hydroxide and sodium acetate solutions and detection by pulsed amperometry. Each of the urines of patients with glycoprotein degradation disorders yielded a pattern of oligosaccharide excretion unique for that disorder, facilitating an unambiguous diagnosis. The method is sensitive (10 microliters of urine required) and fast (40 min).

D(+)-glyceric aciduria: etiology and clinical consequences.

A family comprising mother, father, and five children is described. Four of the children were found to excrete massive amounts of D(+)-glyceric acid in their urine. This was verified by gas chromatography-mass spectrometry and the configuration determined by capillary gas chromatography of O-acetylated menthyl esters. The excretion ranged from 10.8 to 19.9 mmol/24 h. The remaining child and the parents showed no evidence of this unusual metabolite. The virtual absence of clinical manifestations in this family was particularly interesting. Only two of the children showed any clinical abnormality and this was limited to mild microcephaly and speech delay; the other two children found to excrete large amounts of D(+)-glycerate were healthy and developmentally normal at 7 y and 9 y of age. There was a marked increase in the excretion rate of D(+)-glycerate in response to both oral fructose and serine loading. These results are consistent with a deficiency of D(+)-glycerate kinase and indicate the potentially benign nature of this disorder.