LC-MS/MS quantification of N-acetylneuraminic acid, N-glycolylneuraminic acid and ketodeoxynonulosonic acid levels in the urine and potential relationship with dietary sialic acid intake and disease in 3- to 5-year-old children.

Red meat and dairy products contain high sialic acid (Sia) levels, but the metabolic fate and health impact in children remain unknown. The aims of the present study were to quantify the levels of urinary Sia N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc) and ketodeoxynonulosonic acid (KDN) and to determine their relationship with dietary Sia intake. Spot urine samples were collected from 386 healthy children aged 3 (n 108), 4 (n 144) and 5 (n 134) years at 06.30-07.00, 11.30-12.00 and 16.30-17.00 hours. Food intake levels were recorded on the day of urine sample collection. Sia levels were quantified using LC-MS/MS with [13C3]Sia as an internal standard. We found that (1) total urinary Sia levels in healthy pre-school children ranged from 40 to 79 mmol Sia/mol creatinine; (2) urinary Sia levels were independent of age and consisted of conjugated Neu5Ac (approximately 70·8 %), free Neu5Ac (approximately 21·3 %), conjugated KDN (approximately 4·2 %) and free KDN (approximately 3·7 %); Neu5Gc was detected in the urine of only one 4-year-old girl; (3) total urinary Sia levels were highest in the morning and declined over time in 4- and 5-year-old children (P< 0·05), but not in 3-year-old children; (4) Sia intake levels at breakfast and lunch were approximately 2·5 and 0·16 mg Sia/kg body weight; and (5) there was no significant correlation between dietary Sia intake levels and urinary Sia levels. Urinary Sia levels varied with age and time of day, but did not correlate with Sia intake in 3- to 5-year-old children. The difference in urinary Sia levels in children of different age groups suggests that the metabolism and utilisation rates of dietary Sia are age dependent.

Can glycans unveil the origin of glycoprotein hormones? - human chorionic gonadotrophin as an example -.

Doping with (glyco)protein hormones represent an extremely challenging, analytical problem as nearly all are constitutively present at low concentrations that fluctuate according to circadian or alternative periodical, or external stimuli. Thus the mere concentration in a biological sample is only resolutive when this surpasses extreme values. As the vast majority of these molecules are produced by recombinant DNA technology it is believed that the exogenous molecules could bear the signature of the host cell. In particular, these could comprise structural differences originated from co or post-translational differences. In this study we have employed both proteomics and glycomics strategies to compare recombinant and urinary human chorionic gonadotrophin in order to evaluate this hypothesis. As anticipated the recombinant hormone could be shown to contain N-glycolyl neuraminic acid, a sialic acid that cannot be produced by humans. Furthermore, differences were observed in the overall glycosylation, in particular the presence of abundant hybrid-type glycans that were much less pronounced in the recombinant species. These differences were determined to occur predominantly in the alpha-subunit for which antidoping strategies focussed on these elements could be used for both chorionic gonadotrophin and lutrophin as they share the same alpha-subunit.

Yunis-Varon syndrome: evidence for a lysosomal storage disease.

We present clinical and neuropathological findings in a female infant with Yunis-Varon syndrome (YVS) comprising absence of thumbs and halluces, aphalangia of fingers and toes, hypoplasia of clavicles, severely undermineralized skeleton (especially skull), microcephaly, and multiple nonskeletal anomalies. The patient also had a Dandy-Walker malformation, hydrocephalus, and hypertension, which were not reported previously in YVS. The infant excreted an abnormal unidentified oligosaccharide. The patient died at day 108 with severe neurological impairment. Autopsy showed prominent intraneuronal inclusions with vacuolar degeneration, mainly in the thalamic, dentate nuclei, cerebellar cortex, and inferior olivary nuclei. No storage phenomena were observed in other tissues. These findings strongly suggest that a lysosomal storage disorder is involved in the pathogenesis of Yunis-Varon syndrome.

Excretion of neuraminic acid-containing trisaccharides in the urine during pregnancy.

The excretion of neuraminic acid-containing trisaccharides in the urine during the course of normal pregnancy was studied. Neuraminyl-lactose and neuraminyl-galactosyl-(l led to 4)-N-acetylglucosamine were identified, and their excretion patterns were analyzed by thin-layer and gas chromatography. A progressive increase in the outputs of these acidic oligosaccharides was observed during the course of pregnancy in all cases studied. Neuraminyl-lactose excretion increased threefold, from 13.7 +/- 1.75 to 37.1 +/- 2.56 mg/24 h, and neuraminyl-galactosyl-N-acetylglucosamine twofold, from 8.4 +/- 1.27 to 15.3 +/- 2.21 mg/24 h (Mean +/- S.E.).

Quantitative determination and confirmatory analysis of N-acetylneuraminic and N-glycolylneuraminic acids in serum and urine by solid-phase extraction on-line coupled to liquid chromatography-tandem mass spectrometry.

N-acetylneuraminic acid (Neu5Ac) and N-acetylglycolylneuraminic acid (Neu5Gc), two acylated derivatives of 9-C carboxylated monosaccharides, are involved in a number of biological processes as modulators of glycoconjugates. A partially automated method is here presented for determination of these sialic acids in the two most important biofluids for clinical analysis: serum and urine. For this purpose, a solid-phase extraction (SPE) workstation was on-line connected to an LC-MS/MS triple quadrupole mass detector. Hydrolysis to release sialic acids bound to glycoconjugates and derivatization were the two steps implemented as sample preparation prior to SPE-LC-MS/MS analysis. Following thorough optimization of the SPE and LC-MS/MS conditions, the analytical method was validated using the standard addition approach to assess the presence of matrix effects. The proposed method affords detection limits of 0.03ng/mL and 0.04ng/mL for Neu5Ac and Neu5Gc, respectively. The precision (expressed as relative standard deviation) was 1.7 and 4.6% for within-day variability, and 4.8 and 7.2% for between-days variability. Accuracy, estimated using spiked (between 1 and 50ng/mL) and non-spiked samples of both biofluids, ranged from 95.2 to 99.6%. The method was applied to human serum and urine of healthy volunteers, thus showing its suitability for application in both clinical and research laboratories.