Analysis of biomarkers and metabolic pathways in patients with unstable angina based on ultra­high­performance liquid chromatography­quadrupole time­of­flight mass spectrometry.

Unstable angina (UA) is a coronary disease with a high mortality and morbidity worldwide. The present study aimed to use non­invasive techniques to identify urine biomarkers in patients with UA, so as to provide more information for the early diagnosis and treatment of the disease. Based on metabolomics, urine samples from 28 patients with UA and 28 healthy controls (HCs) were analyzed using ultra­high­performance liquid chromatography­quadrupole time­of­flight mass spectrometry (UPLC­Q­TOF/MS). A total of 16 significant biomarkers that could distinguish between patients with UA and HCs, including D­glucuronic acid, creatinine, succinic acid and N­acetylneuraminic acid, were identified. The major metabolic pathways associated with UA were subsequently analyzed by non­targeted metabolomics. The results demonstrated that amino acid and energy metabolism, fatty acid metabolism, purine metabolism and steroid hormone biosynthetic metabolism may serve important roles in UA. The results of the current study may provide a theoretical basis for the early diagnosis of UA and novel treatment strategies for clinicians. The trial was registered with the Chinese Clinical Trial Registration Center (registration no. ChiCTR­ROC­17013957) at Tianjin University of Traditional Chinese Medicine.

A cross-sectional quantitative analysis of the natural history of free sialic acid storage disease-an ultra-orphan multisystemic lysosomal storage disorder.

PURPOSE: Quantitative definition of the natural history of free sialic acid storage disease (SASD, OMIM 604369), an orphan disorder due to the deficiency of the proton-driven carrier SLC17A5. METHODS: Analysis of published cases with SASD (N = 116) respecting STROBE criteria. MAIN OUTCOME PARAMETERS: survival and diagnostic delay. Phenotype, phenotype-biomarker associations, and geographical patient distribution were explored. RESULTS: Median age at disease onset was 0.17 years. Median age at diagnosis was 3 years with a median diagnostic delay of 2.5 years. Median survival was 11 years. The biochemical phenotype clearly predicted the disease course: patients with a urinary free sialic acid excretion below 6.37-fold or an intracellular free sialic acid storage in fibroblasts below 7.37-fold of the mean of normal survived longer than patients with biochemical values above these thresholds. Cluster analysis of disease features suggested a continuous phenotypic spectrum. Patient distribution was panethnic. CONCLUSION: Combination of neurologic symptoms, visceromegaly, and dysmorphic features and/or nonimmune hydrops fetalis should prompt specific tests for SASD, reducing diagnostic delay. The present quantitative data inform clinical studies and may stimulate and accelerate development of specific therapies. Biomarker-phenotype association is particularly important for both counseling parents and study design.

Evaluation of endogenous urinary biomarkers for indirect detection of urine adulteration attempts by five different chemical adulterants in mass spectrometry methods.

Reliable detection of urine adulteration attempts to circumvent positive drug testing represents a critical step for laboratories in abstinence control settings. An ideal workflow for high-throughput testing would involve simultaneous detection of adulteration attempts in the same run with drug detection. Monitoring of degraded or oxidized endogenous urinary compounds as indirect markers has been previously evaluated for that purpose exemplified for the adulterant potassium nitrite (KNO2 ). Fifteen, previously identified endogenous markers should now be evaluated for their general applicability to detect adulteration attempts for the adulterants hypochlorite-based bleach (NaOCl), peroxidase and peroxide (H2 O2 ), pyridinium chlorochromate (PCC), and iodine (I2 ). Initial experiments revealed similar results for the tested adulterants regarding degradation of indolylacryloylglycine (IAG), uric acid (UA), or UA derivatives. 5-Hydroxyisourate (HIU), the oxidation product of UA, was however only formed by KNO2 , PCC, and H2 O2 . Amino acids showed larger adulterant-dependent differences. All reactions were shown to be influenced by the adulterant concentration and the urinary pH with large variances depending on compound and adulterant. Except for HIU/PCC, all markers were stable within +/- 30% variation for all adulterants at -20°C. Receiver operating characteristics indicated best sensitivity and specificity over all adulterants for IAG (specificity 0.9, sensitivity 1.0) and UA (specificity 1.0, sensitivity 0.9). HIU gave best results for KNO2 , PCC, and H2 O2 and N-acetylneuraminic acid for PCC and H2 O2 , respectively. When integrating a limited number of targets into existing screening methods, monitoring of UA, IAG, N-acetylneuraminic acid, and HIU is recommended.

Preparation of a molecularly imprinted sensor based on quartz crystal microbalance for specific recognition of sialic acid in human urine.

A novel molecularly imprinted quartz crystal microbalance (QCM) sensor was successfully prepared for selective determination of sialic acid (SA) in human urine samples. To obtain the QCM sensor, we first modified the gold surface of the QCM chip by self-assembling of allylmercaptane to introduce polymerizable double bonds on the chip surface. Then, SA molecularly imprinted polymer (MIP) nanofilm was attached to the modified QCM chip surface. For comparison, we have also characterized the nonmodified and improved surfaces of the QCM sensor by using atomic force microscopy (AFM) and Fourier transform infrared (FTIR) spectroscopy. We then tested the selectivity and detection limit of the imprinted QCM sensor via a series of adsorption experiments. The results show a linear response in the range of 0.025-0.50 µmol L-1 for sialic acid. Moreover, the limit of detection (LOD) of the prepared imprinted QCM sensor was found to be 1.0 nmol L-1 for sialic acid, and high recovery values range from 87.6 to 108.5% with RSD < 8.7 (n = 5) for the spiked urine sample obtained. Overall, this work presents how a novel QCM sensor was developed and used to detect sialic acid in human urine samples. Graphical abstract Specific recognition of sialic acid by the MIP-QCM sensor system.

An impedimetric biosensor for the diagnosis of renal cell carcinoma based on the interaction between 3-aminophenyl boronic acid and sialic acid.

Renal cell carcinoma (RCC) often expresses a high density of sialic acid-rich glycoproteins which helps these late-stage cancer cells to enter the blood stream or urine. Blood diagnosis is a complex and time-consuming process. In this study, we developed a facile and non-invasive electrochemical cytosensor for early detection of RCC in urine samples based on specific recognition by 3-aminophenyl boronic acid (APBA). Polypyrrole (PPy) and bovine serum albumin (BSA)-incorporated Ag submicron particles (Ag@BSA) were co-deposited on a gold electrode (GE) to take advantages of the excellent properties of these biomaterials, including good biocompatibility, low cytotoxicity and excellent electro-conductivity. To further increase the biosensor's sensitivity, APBA molecules were integrated to recognize sialic acid (SA) on the cell surface. Under optimal conditions, the impedimetric cytosensor exhibited a good linear relationship with the logarithm of the cell concentration from 17 to 1.7×106 cellsmL-1, and the low detection limit was 6 cellsmL-1 (S/N=3). Therefore, the electrochemical impedimetric biosensor offers a potential approach to bedside rapid detection of RCC in clinical applications.

Increased Polysialylation of the Neural Cell Adhesion Molecule in a Transgenic Mouse Model of Sialuria.

Sialuria is a rare autosomal dominant disorder of mammalian metabolism, caused by defective feedback inhibition of the UDP-N-acetylglucosamine-2-epimerase N-acetylmannosamine kinase (GNE), the key enzyme of sialic acid biosynthesis. Sialuria is characterized by overproduction of free sialic acid in the cell cytoplasm. Patients exhibit vastly increased urinary excretion of sialic acid and show differently pronounced developmental delays. The physiopathology of sialuria is not well understood. Here we established a transgenic mouse line that expresses GNE containing the sialuria mutation R263L, in order to investigate the influence of an altered sialic acid concentration on the organism. The transgenic mice that expressed the mutated RNA excreted up to 400 times more N-acetylneuraminic acid than wild-type mice. Additionally, we found higher sialic acid concentration in the brain cytoplasm. Analyzing the (poly)sialylation of neural cell adhesion molecule (NCAM) revealed increased polysialylation in brains of transgenic mice compared to wild-type. However, we found only minor changes in membrane-bound sialylation in various organs but, surprisingly, a significant increase in surface sialylation on leukocytes. Our results suggest that the intracellular sialic acid concentration regulates polysialylation on NCAM in vivo; this could play a role in the manifestation of the developmental delays in sialuria patients.

New observation of sialuria prompts detection of liver tumor in previously reported patient.

UNLABELLED: Sialuria, a rare inborn error of metabolism, was diagnosed in a healthy 12-year-old boy through whole exome sequencing. The patient had experienced mild delays of speech and motor development, as well as persistent hepatomegaly. Identification of the 8th individual with this disorder, prompted follow-up of the mother-son pair of patients diagnosed over 15years ago. Hepatomegaly was confirmed in the now 19-year-old son, but in the 46-year-old mother a clinically silent liver tumor was detected by ultrasound and MRI. The tumor was characterized as an intrahepatic cholangiocarcinoma (IHCC) and DNA analysis of both tumor and normal liver tissue confirmed the original GNE mutation. As the maternal grandmother in the latter family died at age 49years of a liver tumor, a retrospective study of the remaining pathology slides was conducted and confirmed it to have been an IHCC as well. The overall observation generated the hypothesis that sialuria may predispose to development of this form of liver cancer. As proof of sialuria in the grandmother could not be obtained, an alternate cause of IHCC cannot be ruled out. In a series of 102 patients with IHCC, not a single instance was found with the allosteric site mutation in the GNE gene. This confirms that sialuria is rare even in a selected group of patients, but does not invalidate the concern that sialuria may be a risk factor for IHCC. SYNOPSIS: Sialuria is a rare inborn error of metabolism characterized by excessive synthesis and urinary excretion of free sialic acid with only minimal clinical morbidity in early childhood, but may be a risk factor for intrahepatic cholangiocarcinoma in adulthood.

Assay of urinary protein-bound sialic acid can differentiate steroidsensitive nephrotic syndrome from steroid-resistant cases.

The protein selectivity index as measured from the ratio of urinary immunoglobulin to albumin failed to differentiate between steroid-sensitive (SS) and steroid-resistant (SR) cases of nephrotic syndrome (NS). Sialic acid contributes negative charges to many plasma proteins. The negative charge is a determinant of protein excretion rate. The prognostic significance of assay of urinary excretion of protein-bound sialic acid in NS has not been evaluated. Hence, the present study was designed to evaluate whether measurement of urinary protein bound sialic acid (UPBSA) can be used as a marker to differentiate SS from SR cases of NS. The urine samples of 70 (47 SS and 23 SR) pediatric NS children were assayed for UPBSA by Aminoff's method. The levels were compared and the receiver-operator curve was drawn to determine the optimum cutoff point to differentiate among the groups before starting the therapy. The excretion of UPBSA in SR cases of NS was significantly higher than that of SS cases (P<0.05). The optimum cutoff limit for UPBSA was 2.71 µg/mg of proteins with 75% sensitivity and 75.5% specificity for differentiating SS cases from SR cases (area under the plasma- concentration time curve=0.814, P=0.009). We conclude that UPBSA can differentiate SR cases from SS cases of NS in pediatric patients and may help in predicting the response to steroid therapy.

An evaluation of Tamm-Horsfall protein glycans in kidney stone formers using novel techniques.

Tamm-Horsfall protein (THP) is theorized to play a critical role in preventing kidney stone formation. There is conflicting literature on THP analysis in kidney stone patients; therefore, this study was conducted using sensitive and specific bio-analytical techniques to better understand differences in THP, which play a potential role in nephrolithiasis pathogenesis. THP was isolated from urine samples of 34 male and 19 female kidney stone patients and 30 male and 24 female control subjects using diatomaceous earth. Protein was quantified by Superdex-200 size-exclusion chromatography. Sialic acid was determined by 1,2-diamino-4,5-methylenedioxybenzene high-performance liquid chromatography. Neutral and amino sugars were determined by high pH anion-exchange chromatography (HPAEC) with pulsed amperometric detection. THP N-glycans were derivatized with 2-aminobenzamide (2-AB) and profiled by HPAEC with fluorescence detection. N-glycan structures were confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Results indicate that kidney stone patients had 32% lower protein content compared to controls, while sialic acid content was lower by 29 and 24% in male and female kidney stone patients, respectively, compared to controls. The neutral and amino sugars were also lower by 18 and 20% for male and female kidney stone patients, respectively, compared to controls. All results were statistically significant (p<0.001). These results are supported by 2-AB profiling of THP N-glycans and by MALDI-TOF MS of highly sialylated N-glycans in the range of m/z 3000-6000. This study demonstrates quantitative and qualitative differences in THP, which can be crucial contributing factors for nephrolithiasis.

Molecular design of boronic acid-functionalized squarylium cyanine dyes for multiple discriminant analysis of sialic acid in biological samples: selectivity toward monosaccharides controlled by different alkyl side chain lengths.

We designed a new series of boronic acid-functionalized squarylium cyanine dyes (SQ-BA) with different lengths of alkyl chain residues, suitable for multiple discriminant analysis (MDA) of sialic acid (Neu5Ac) in biological samples. The SQ-BA dyes form aggregates based on hydrophobic interactions, which result in quenched fluorescence in aqueous solutions. When the boronic acid binds with saccharides, the fluorescence intensity increases as a result of dissociation to the emissive monomeric complex. We inferred that different dye aggregate structures (H-aggregates and J-aggregates) were induced depending on the alkyl chain length, so that monosaccharides would be recognized in different ways (especially, multipoint interaction with J-aggregates). A distinctive emission enhancement of SQ-BA dyes with shorter-alkyl-chains in the presence of Neu5Ac was observed (2.4-fold fluorescence enhancement; with formation constant 10(1.7) M(-1)), with no such enhancement for SQ-BA dyes with longer-alkyl-chain. In addition, various enhancement factors for other monosaccharides were observed depending on the alkyl chain length. Detailed thermodynamic and NMR studies of the SQ-BA complexes revealed the unique recognition mechanism: the dye aggregate with a shorter-alkyl-chain causes the slipped parallel structure and forms a stable 2:1 complex with Neu5Ac, as distinct from longer-alkyl-chain dyes, which form a 1:1 monomeric complex. MDA using the four SQ-BA dyes was performed for human urine samples, resulting in the successful discrimination between normal and abnormal Neu5Ac levels characteristic of disease. Thus, we successfully controlled various responses to similar monosaccharides with a novel approach that chemically modified not the boronic acid moiety itself but the length of the alkyl chain residue attached to the dye in order to generate specificity.

The early detection of Salla disease through second-tier tests in newborn screening: how to face incidental findings.

We describe here a 34 months child, practically asymptomatic which presented with high levels of free sialic acid in urine by biochemical detection in second-tier tests newborn screening and with two disease causing mutations in SLC17A5 gene. SLC17A5 mutation analysis showed p.Tyr306* previously described and the novel mutation p.Leu167Pro. This early onset diagnosis allowed us to perform a fast and accurate genetic counseling to the family, helped to better understanding the natural history of this rare disease and probably it could promote cost reduction in future diagnostic tests in the hypothetic case of starting symptoms without diagnosis established. Moreover, an early diagnosis could save family from a long period of time until achieving a definitive diagnostic and to develop an early symptomatic and supportive management of patient to attenuate, as much as possible, disease complications. But, above all, this case illustrates the huge ethical dilemma which arises from any secondary finding (second tier) in newborn screening.

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.

Noninvasive urinary metabolomic profiling identifies diagnostic and prognostic markers in lung cancer.

Lung cancer remains the most common cause of cancer deaths worldwide, yet there is currently a lack of diagnostic noninvasive biomarkers that could guide treatment decisions. Small molecules (<1,500 Da) were measured in urine collected from 469 patients with lung cancer and 536 population controls using unbiased liquid chromatography/mass spectrometry. Clinical putative diagnostic and prognostic biomarkers were validated by quantitation and normalized to creatinine levels at two different time points and further confirmed in an independent sample set, which comprises 80 cases and 78 population controls, with similar demographic and clinical characteristics when compared with the training set. Creatine riboside (IUPAC name: 2-{2-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)-oxolan-2-yl]-1-methylcarbamimidamido}acetic acid), a novel molecule identified in this study, and N-acetylneuraminic acid (NANA) were each significantly (P < 0.00001) elevated in non-small cell lung cancer and associated with worse prognosis [HR = 1.81 (P = 0.0002), and 1.54 (P = 0.025), respectively]. Creatine riboside was the strongest classifier of lung cancer status in all and stage I-II cases, important for early detection, and also associated with worse prognosis in stage I-II lung cancer (HR = 1.71, P = 0.048). All measurements were highly reproducible with intraclass correlation coefficients ranging from 0.82 to 0.99. Both metabolites were significantly (P < 0.03) enriched in tumor tissue compared with adjacent nontumor tissue (N = 48), thus revealing their direct association with tumor metabolism. Creatine riboside and NANA may be robust urinary clinical metabolomic markers that are elevated in tumor tissue and associated with early lung cancer diagnosis and worse prognosis.

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.

Increased inflammatory response and imbalance in blood and urinary oxidant-antioxidant status in South Indian women with gestational hypertension and preeclampsia.

OBJECTIVE: The objective of this study is to assess the association of blood and urinary oxidative stress parameters and inflammatory markers in women with gestational hypertension and preeclampsia. DESIGN AND METHODS: Malondialdehyde, protein bound sialic acid and C-reactive protein were estimated in serum and urine of pregnant women diagnosed with preeclampsia (n=30) and gestational hypertension (n=30) and the results were compared with 30 normal pregnant women. RESULTS: Whole blood glutathione level was reduced, and malondialdehyde and C-reactive protein levels were significantly higher and correlated with each other in preeclampsia (p<0.05). Urinary malondialdehyde significantly correlated with urinary protein bound sialic acid in preeclampsia (r=0.412; p=0.02). Receiver operating curve analysis of serum protein bound sialic acid and serum malondialdehyde showed reasonable cutoff values for the differential diagnosis of preeclampsia. CONCLUSIONS: Oxidative stress and inflammatory response are greater in women with preeclampsia in comparison to pregnant women with gestational hypertension and there is an association between oxidative stress and inflammatory response.

Measurement of free and total sialic acid by isotopic dilution liquid chromatography tandem mass spectrometry method.

The measurement of urine sialic acid (N Acetylneuraminic Acid: Neu5Ac) is useful for screening sialic acid storage disorders. We developed a new LC MS/MS method for the determination of a sialic acid. Urine samples were analyzed, after an HCl n-Butanol derivatization step, by a reverse phase based high-performance liquid chromatography method using 1,2,3-(13)C(3) N-Acetyl-D-neuraminic Acid ((13)C-Neu5Ac) as an internal standard. Selective detection was performed by tandem mass spectrometry using an electrospray source operating in positive ionization mode employing multiple reactions monitoring to monitor N-Acetylneuraminic Acid and the internal standard. The transitions m/z 366→330 and 369→333 for Neu5Ac and (13)C-Neu5Ac were respectively monitored. The limit of the method quantification was 1.40 µM of N-Acetylneuraminic Acid and the calibration curve showed a good linearity up to 1000 µM. The within assay precision and accuracy of the method ranged from 3.22 to 5.95% and 98.69 to 109.18%, respectively and the between assay precision and accuracy ranged, respectively, from 5.15 to 7.65% and 96.14 to 102.30%. The method can be applied for the determination of N-Acetylneuraminic Acid concentrations in urine and other biological fluids (e.g., amniotic and peritoneal fluids).

Significance of serum and urine neuraminidase activity and serum and urine level of sialic acid in diabetic nephropathy.

BACKGROUND: Prospective studies have reported associations among various markers of inflammation and the incidence of diabetes, and it has been proposed that inflammation has a causal role in the development of diabetes. The objective of this study was to investigate the significance of serum and urine neuraminidase activity (NA) and serum and urine sialic acid (SA) level in patients with Diabetic nephropathy. METHODS: In a prospective study, 190 diabetic patients with established diabetic nephropathy, 30 type 2 diabetes patients without any diabetic related nephropathy, and 36 non-diabetic patients with diagnosed nephropathy were enrolled. Two hundred and forty healthy individuals without diabetes or kidney disease were also enrolled as control group. Fasting venous blood samples and urine samples were collected and checked for serum and urine NA and SA level. RESULTS: In the diabetic nephropathy group, the mean value of serum and urine NA was 64.6 ± 2.6 and 11.7 ± 1.2 mU/ml, respectively, and mean values of serum and urine SA were 93.2 ± 3.6 and 17.7 ± 1.4 mg/dl, respectively. Serum and urine NA and SA levels were significantly higher in patient with diabetic nephropathy when compared to the other groups (P < 0.001). CONCLUSIONS: Our study suggests that there is a strong association between elevated serum and urine NA and serum and urine SA levels with the presence of diabetic nephropathy in type 2 diabetic patients. Further investigations are needed on the diagnostic and prognostic significance of these two inflammatory markers.

Metabolome-wide association study identifies multiple biomarkers that discriminate north and south Chinese populations at differing risks of cardiovascular disease: INTERMAP study.

Rates of heart disease and stroke vary markedly between north and south China. A (1)H NMR spectroscopy-based metabolome-wide association approach was used to identify urinary metabolites that discriminate between southern and northern Chinese population samples, to investigate population biomarkers that might relate to the difference in cardiovascular disease risk. NMR spectra were acquired from two 24-h urine specimens per person for 523 northern and 244 southern Chinese participants in the INTERMAP Study of macro/micronutrients and blood pressure. Discriminating metabolites were identified using orthogonal partial least squares discriminant analysis and assessed for statistical significance with conservative family wise error rate < 0.01 to minimize false positive findings. Urinary metabolites significantly (P < 1.2 × 10(-16) to 2.9 × 10(-69)) higher in northern than southern Chinese populations included dimethylglycine, alanine, lactate, branched-chain amino acids (isoleucine, leucine, valine), N-acetyls of glycoprotein fragments (including uromodulin), N-acetyl neuraminic acid, pentanoic/heptanoic acid, and methylguanidine; metabolites significantly (P < 1.1 × 10(-12) to 2 × 10(-127)) higher in the south were gut microbial cometabolites (hippurate, 4-cresyl sulfate, phenylacetylglutamine, 2-hydroxyisobutyrate), succinate, creatine, scyllo-inositol, prolinebetaine, and trans-aconitate. These findings indicate the importance of environmental influences (e.g., diet), endogenous metabolism, and mammalian-gut microbial cometabolism, which may help explain north-south China differences in cardiovascular disease risk.

Elevated CSF N-acetylaspartylglutamate in patients with free sialic acid storage diseases.

OBJECTIVE: To investigate body fluids of patients with undiagnosed leukodystrophies using in vitro (1)H-NMR spectroscopy (H-NMRS). METHODS: We conducted a cross-sectional study using high-resolution in vitro H-NMRS on CSF and urine samples. RESULTS: We found a significant increase of free sialic acid in CSF or urine in 6 of 41 patients presenting with hypomyelination of unknown etiology. Molecular genetic testing revealed pathogenic mutations in the SLC17A5 gene in all 6 patients. H-NMRS revealed an increase of N-acetylaspartylglutamate in the CSF of all patients with SLC17A5 mutation (range 13-114 micromol/L, reference <12 micromol/L). CONCLUSION: In patients with undiagnosed leukodystrophies, increased free sialic acid in CSF or urine is a marker for free sialic acid storage disorder and facilitates the identification of the underlying genetic defect. Because increase of N-acetylaspartylglutamate in CSF has been observed in other hypomyelinating disorders, it can be viewed as a marker of a subgroup of hypomyelinating disorders.

Free sialic acid storage disease without sialuria.

We performed high-resolution in vitro proton nuclear magnetic resonance spectroscopy on cerebrospinal fluid and urine samples of 44 patients with leukodystrophies of unknown cause. Free sialic acid concentration was increased in cerebrospinal fluid of two siblings with mental retardation and mild hypomyelination. By contrast, urinary excretion of free sialic acid in urine was normal on repeated testing by two independent methods. Both patients were homozygous for the K136E mutation in SLC17A5, the gene responsible for the free sialic acid storage diseases. Our findings demonstrate that mutations in the SLC17A5 gene have to be considered in patients with hypomyelination, even in the absence of sialuria.