Prenatal diagnosis of infantile sialic acid storage disease in a twin pregnancy

Summary A diagnosis of infantile sialic acid storage disease was made in an infant who died aged 17 months. In the mother's next pregnancy no morphological or biochemical abnormality was found in chorionic villi, amniotic fluid, cultured amniotic fluid cells or fetal blood and a normal boy was born. In the subsequent pregnancy an ultrasound scan revealed a twin pregnancy. Chorionic villus samples were obtained from both twins and microscopic and biochemical analysis indicated one twin to be affected with sialic acid storage disease. Selective fetocide was performed. The unaffected twin proceeded to term.     

The effect ofd-(+)-glucosamine on levels of freeN-acetylneuraminic acid and UDP-N-acetylhexosamines in infantile sialic acid storage disease (ISSD) fibroblasts

An impairment in the regulation ofN-acetylneuraminic acid (NANA) biosynthesis might potentially contribute to accumulation of free NANA in fibroblasts of patients with sialic acid storage disease (ISSD). By the use of a glucosamine loading test an increase in uridine-diphosphate-N-acetyl-hexosamines (UDP-HexNAc) but not in free NANA was found. NANA biosynthesis therefore appears to be under normal regulatory control in ISSD.     

Biochemical characterization of patients and prenatal diagnosis of sialic acid storage disease for three families

Summary Modifications of the assay method of Aminoff (1961) for the determination of sialic acid levels in urine, amniotic fluid, cultured cell homogenates and tissue homogenates, which reduce the interference from proteins by precipitation and from interfering chromogens by second derivative spectroscopy are described.Biochemical profiles of patients from three families with different clinical forms of sialic acid storage disease were made using data obtained with the new method. A family with two patients with the clinically severe, early-onset form is described, in which a 9-fold elevation of sialic acid can be detected in the skin fibroblasts and a 12-fold elevation in the urine. A patient from the second family is described with very severe clinical features and with 160-fold and 16-fold elevations of sialic acid in the urine and skin fibroblasts respectively. A patient from a third family is described with mild clinical features but with a 160-fold and 6-fold elevation of sialic acid in urine and skin fibroblasts respectively. The data obtained in this study are compared with data obtained in other laboratories on other patients.The method was used to assess the levels of sialic acid present in amniotic cells and chorionic villus cells obtained prenatally from pregnancies in each of the three families. In one case, in which amniotic cells were elevated in sialic acid and were vacuolated, the pregnancy was terminated. Follow-up studies confirmed the diagnosis. Pregnancies from the other two families were assessed to be not affected.     

Storage material from urine and tissues in the nephropathic phenotype of infantile sialic acid storage disease

Summary We analysed urine and tissue specimens from two nephrotic infantile sialic acid storage disease patients (nISSD) for free and bound sialic acids in comparison to non-nephrotic ISSD patients (ISSD), patients with minimal change nephrosis (nControl) and normal controls (Control). No differences in the excretion of urinary free sialic acid could be detected between ISSD and nISSD urines. Sialyloligosaccharide fractions were only slightly elevated and of apparently normal composition. Owing to glomerular dysfunction, measurable quantities of protein-bound sialic acids were present in nISSD and nControl.In nISSD tissues, free sialic acid was elevated 18–100-fold above control and 3–12-fold above Niemann-Pick A (NPA) samples. The storage of membrane-bound sialic acid was slightly increased compared to control tissues, but equal to those from NPA, thus reflecting an unspecific increase of membranes due to lysosomal storage.According to these results no major biochemical differences were detectable between ISSD and nISSD. The nephrotic syndrome in nISSD could not be related to a general deficit in the sialylation of glycoproteins. Nevertheless, a cell membrane-specific alteration in sialoglycoproteins of glomerular cells might still be possible.     

Peroxisomes in infantile phytanic acid storage disease: a cytochemical study of skin fibroblasts

Cultured skin fibroblasts from six patients demonstrating clinical signs and biochemical characteristics of infantile phytanic acid storage disease (IPSD) were examined by electron microscopy, using cytochemical procedures for the demonstration of peroxisomal catalase activity. In four of the six fibroblast cell lines peroxisomes strongly reactive for catalase were present in numbers similar to those found in normal fibroblasts. Liver biopsy tissue from one of these patients showed no typical hepatic peroxisomes, but did show small, marginally reactive bodies. In two other IPSD fibroblast cell lines peroxisomes with appreciable cytochemical reactivity were rare or absent. It seems, therefore, that infantile phytanic acid storage disease is heterogeneous with respect to the presence of cytochemically recognizable peroxisomes, at least in the cases studied here. Furthermore, peroxisomes may be markedly affected in one cell type — liver — and yet not affected in another — skin fibroblasts — within a single individual.     

Progress in expanded newborn screening for metabolic conditions by LC-MS/MS in Tuscany: update on methods to reduce false tests

We report on our 6-year experience of expanded newborn screening by tandem mass spectrometry in Tuscany (Italy), the first Italian Region to screen all newborns for more than 40 inborn errors of metabolism: organization, diseases observed and updates on methods to reduce false-positive and false-negative tests are described. Blood collection is recommended between 48 and 72 h of life. Blood spots are sent daily by courier to laboratory. When a positive result occurs, two subsequent procedures are followed: for disorders with possible acute metabolic decompensation, the baby is immediately recalled and clinical examinations and confirmatory tests are performed; for the other disorders, the nursery provides for a second blood spot. If the test is positive, clinical examinations and confirmatory tests are performed. In both cases, if confirmatory tests are positive, a treatment and a follow-up programme are started. Up to now, spots from 160 000 infants have been analysed and 80 affected patients have been identified (disorders of amino acids, organic acids and fatty acids metabolism). We describe adjustments to cut-off values, the introduction of a second-tier test for propionic acidaemia and for methylmalonic aciduria, the inclusion of succinylacetone in the panel of metabolites, and protocols for premature infants and for newborns on parenteral nutrition or transfused. These changes resulted in a reduction in recalls from 1.37% to 0.32% and consequently of working time and parental stress. Avoiding false-negatives by using more specific markers and minimizing the false-positive rate with second-tier testing is important for a successful newborn screening programme.     

Diagnosis of Gaucher's disease in cultured skin fibroblasts and leucocytes

Washing skin fibroblasts or leucocytes in 0.25 mol/l sucrose increases the activity of-glucosidase at acid pH. This effect is primarily due to removal of low levels of sodium chloride, which inhibit acid-glucosidase. A secondary factor for skin fibroblasts is the removal of residual phosphate buffer pH 7.3 used to wash the cells following trypsinization. As the-glucosidase activity of water-lysed leucocytes is higher at acid pH than that of a saline suspension of leucocytes, the former are better for the diagnosis of Gaucher's disease. However, more reliable results still may be obtained by assay of this enzyme in cultured skin fibroblasts.     

Synthesis of underpolymerized hyaluronic acid by fibroblasts cultured from rheumatoid and non-rheumatoid synovitis

Summary Fibroblast cultures were started from synovial tissue of normal controls and patients with seronegative and seropositive rheumatoid arthritis, juvenile rheumatoid arthritis and chronic non-rheumatoid synovitis. Glycosaminoglycan synthesis of the cultures was studied by metabolic labeling with ³H-glucosamine. Molecular weight of hyaluronic acid produced and secreted into culture medium by these fibroblast strains was studied by gel filtration in a Sepharose 2 B column. All fibroblast strains from joints with active inflammation produced hyaluronate with decreased molecular weight. The synthesis of underpolymerized hyaluronate thus appears to be a property of activated synovial fibroblasts common to rheumatoid and non-rheumatoid inflammation.     

Quantification of <Emphasis Type="Italic">N</Emphasis>-acetylaspartic acid in urine by LC-MS/MS for the diagnosis of Canavan disease

Summary Canavan disease is an autosomal recessive leukodystrophy characterized by excessive excretion of N-acetylaspartic acid (NAA) in urine. The disease is caused by deficiency of aspartoacylase, the enzyme responsible for the hydrolysis of NAA into acetate and l-aspartate. Patients, who are often asymptomatic in their early months, show a wide spectrum of clinical presentation thereafter that includes macrocephaly, poor head control, seizures, abnormal muscle tone, optic atrophy, significant developmental delay and death. In this work, we describe a simple liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for the determination of NAA in urine. The internal standard d₃-NAA was added to untreated urine and the mixture was injected into the LC-MS/MS system operated in the negative ion mode. Detection was achieved in multiple reaction monitoring (MRM) mode by monitoring m/z 174 → 88, 174 → 130 and 174 → 58 for NAA and 177 → 89 for the internal standard. Separation was carried out on a C₈ column (2.1 × 150 mm) using a mixture of acetonitrile and water (1:1 v/v) containing 0.05% formic acid at a flow rate of 0.25 ml/min. NAA was eluted at 1.6 min and the run time was approximately 2 min. Using spiked urine, the assay was linear up to 2 mmol/L with limit of quantification at 1 μmol/L (S/N = 12). NAA in patients’ urine (n = 17) ranged between 366 and 21235 mmol/mol creatinine compared to controls of <39 mmol/mol creatinine (n = 159). This LC-MS/MS method for NAA as described involved no extraction and no derivatization, showed no interference, and gave excellent recovery with low variability and short analytical time. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Online citation: JIMD Short Report #057 (2007) Online     

The role of sialic acid in the dysfibrinogenemia associated with liver disease: distribution of sialic acid on the constituent chains

To further evaluate the role of sialic acid in the dysfibrinogenemia associated with liver disease, we studied the effect of removal of excess sialic acid residues from the fibrinogen of five patients with liver disease on the thrombin time and fibrin monomer aggregation. Patient fibrinogens containing 1.4-3.4 residues of sialic acid per molecule in excess of normal controls, with thrombin times 12-22 sec longer than normal and with abnormal fibrin monomer aggregation, were stripped of their excess sialic acid by incubation with Vibrio cholerae neuraminidase, followed by rapid removal of the enzyme by antineuraminidase antibody affinity chromatography. These partially desialylated patient fibrinogens, with a normal number of sialic acid residues remaining, exhibited normal thrombin times and normal fibrin monomer aggregation. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of reduced normal, patient, and partially desialylated patient (sialyl-3H)-fibrinogen exhibited 60% of the radioactivity in the B beta chain and 40% in the gamma chain. There was no radioactivity detectable in the A alpha chain. These studies provide additional evidence that the increased sialic acid content of the acquired dysfibrinogenemia of liver disease is responsible for its functional defect and that the excess sialic acid is distributed on the B beta chain and gamma chains of the fibrinogen.     

Retinoic acid inhibition of hyaluronate synthesis in cultured human skin fibroblasts

The effects of all-trans retinoic acid on glycosaminoglycan (GAG) accumulation were determined in cultured primary human skin fibroblasts. Confluent cultures treated with retinoic acid accumulated less [3H]GAG than those without the compound, an effect with an apparent threshold of 10 nM which was dose dependent in the concentration range tested (0-10 microM). At 10 microM, the inhibition was 54%. Greater than 80% of the labeled macromolecular material was streptomyces hyaluronidase digestible in cultures labeled with [3H]acetate. The incorporation of H2[35S]O4 into chondroitin sulfate and dermatan sulfate was unaffected, as was total protein synthesis. Retinol also inhibited accumulation of [3H]GAG, but was far less potent. T3 and dexamethasone can inhibit [3H]hyaluronate synthesis. When retinoic acid was added to cultures treated with either of these hormones at concentrations that maximally inhibit [3H] GAG accumulation, there was a further decrease in the rate of macromolecular accumulation. The retinoic acid effect evolved over 24-48 h after addition to the culture medium. A pulse-chase study failed to demonstrate any effect on [3H]GAG degradation.     

Significance of serum sialic acid in patients with Crohn's disease.

Serum sialic acid was measured to evaluate the activity of Crohn's disease. The sialic acid levels of patients with Crohn's disease in remission (CRP 0.0 mg/dl) were significantly higher than those of healthy subjects and postoperative patients with Crohn's disease. In patients in remission, serum sialic acid was significantly correlated with hemoglobin, hematocrit, platelet, and rapid turnover protein. Correlations with platelet, retinol-binding protein, and prealbumin were especially strong. From these findings, it was concluded that serum sialic acid level provides a useful index of the activity of Crohn's disease.     

LC-MS/MS测定小鼠肝脏中10种胆汁酸浓度的方法与应用

目的建立LC-MS/MS法同时测定小鼠肝脏中10种游离型与结合型胆汁酸的浓度。方法肝组织样品匀浆经乙腈沉淀蛋白后进行LC-MS/MS分析,空白基质经活性炭(100 mg/m L)吸附获得,色谱柱为Zorbax Eclipse XDB-C18柱(5μm,150 mm×4.60 mm),流动相为乙腈-含0.05%甲酸和4 mmol/L乙酸铵溶液,梯度洗脱,流速1 m L/min,进样10μL,采用电喷雾离子源(ESI)、负离子的多级反应模式(MRM)监测。结果在定量范围内,各胆汁酸的线性关系良好,检测时间较短(14 min),精密度、准确度、提取回收率等均符合要求。结论建立的方法灵敏度高、专属性好,可用于肝组织样品中胆汁酸浓度的测定。     

Glucose-free medium exacerbates microvesicular steatosis in cultured skin fibroblasts of genetic defects of fatty acid oxidation. A novel screening test

Skin fibroblasts from patients with various fatty acid oxidation defects (FAOD) and four normal controls were subcultured in standard glucose-containing medium or in glucose-free medium simulating fasting. The FAOD fibroblasts developed microvesicular steatosis, which was greatly exacerbated in glucose-free medium. Rescue treatment with glucose-containing medium was performed in the short-chain L-3-hydroxyacyl-CoA dehydrogenase-deficient (SCHADD) fibroblasts and resulted in a partial resolution of the steatosis and improved cellular viability. Transmission electron microscopy of autopsy specimens from the SCHADD patient demonstrated that most renal interstitial fibroblasts and 50% of fibroblasts in the heart had microvesicular steatosis. The demonstration of microvesicular steatosis in parenchymal and/or cultured skin fibroblasts may provide important and cost-effective screening tools for the detection of genetic defects of fatty acid oxidation.