Fluorous derivatization combined with liquid chromatography/tandem mass spectrometry: a method for the selective and sensitive determination of sialic acids in biological samples.

We have developed a novel method for selective and sensitive analysis of sialic acids (N-acetylneuraminic, N-glycolylneuraminic, and 2-keto-3-deoxy-D-glycero-D-galactonononic acid) utilizing liquid chromatography/tandem mass spectrometry (LC/MS/MS) combined with a fluorous derivatization technique. In this method, the carboxylic groups in the sialic acids are derivatized via amidation with heptadecafluoroundecylamine, a commercially available perfluoroalkylamine reagent. This reaction proceeds rapidly and readily at room temperature in the presence of a condensation reagent. Subsequently, the derivatives are retained specifically on an LC column with a perfluoroalkyl stationary phase by means of a fluorophilic or 'fluorous' interaction, and detected by positive electrospray ionization MS/MS. The detection limits of the examined sialic acids are in the range of 60-750 amol on column. We show that the proposed method can be used to analyze trace amounts of sialic acids in biological samples.

Application of a protocol for the detection of disorders of sialic acid metabolism to 124 high-risk Brazilian patients.

Lysosomal storage disorders (LSD) present great clinical variability. Included in this group are sialic acid metabolism disorders (SAMD). In the present study, we describe the application of a 3-step protocol for the diagnosis of SAMD, including (1). oligosaccharide and sialyloligosaccharide chromatography; (2). quantitative determination of sialic acid; and (3). measurement of neuraminidase activity. Application of our protocol to 124 individuals at risk for SAMD led to the diagnosis of five affected patients, two with type I sialidosis, one with type II sialidosis, and two with galactosialidosis. Due to its simplicity and efficiency, we propose the use of this protocol for the diagnostic evaluation of patients with suspected SAMD, which could be specially useful to non-specialized laboratories and to services located in developing countries.

Dominant inheritance of sialuria, an inborn error of feedback inhibition.

"French type" sialuria, a presumably dominant disorder that, until now, had been documented in only five patients, manifests with mildly coarse facies, slight motor delay, and urinary excretion of large quantities (>1 g/d) of free N-acetylneuraminic acid (NeuAc). The basic defect consists of the very rare occurrence of failed feedback inhibition of a rate-limiting enzyme, in this case uridinediphosphate-N-acetylglucosamine (UDP-GlcNAc) 2-epimerase, by a downstream product, in this case cytidine monophosphate (CMP)-NeuAc. We report a new patient with sialuria who has a heterozygous G-->A substitution in nucleotide 848 of the epimerase gene, which results in an R266Q change. The proband's other allele, as expected, had no mutation. However, the heterozygous R266Q mutation was detected in the patient's mother, who has similarly increased urinary levels of free NeuAc, thereby confirming, for the first time, the dominant mode of inheritance of this inborn error. The biochemical diagnosis of the proband was verified by the greatly increased level of free NeuAc in his cultured fibroblasts, the NeuAc distribution, mainly (59%) in the cytoplasm, and by the complete failure of 100 microM CMP-NeuAc to inhibit UDP-GlcNAc 2-epimerase activity in the mutant cells. These findings call for expansion of the phenotype to include adults and for more-extensive assaying of free NeuAc in the urine of children with mild developmental delay. The prevalence of sialuria is probably grossly underestimated.

Occurrence of sialic acids in healthy humans and different disorders.

Sialic acid (SA), N-acetylated derivatives of neuraminic acid, play a central role in the biomedical functioning of humans. The normal range of total sialic acid (TSA) level in serum/plasma is 1.58-2.22 mmol L-1, the free form of SA only constituting 0.5-3 mumol L-1 and the lipid-associated (LSA) forms 10-50 mumol L-1. Notably, considerably higher amounts of free SA are found in urine than in serum/plasma (approximately 50% of the total SA). In inherited SA storage diseases such as Salla's disease, SA levels are elevated many times over, and their determination during clinical investigation is well established. Furthermore, a number of reports describe elevated SA levels in various other diseases, tentatively suggesting broader clinical utility for SA markers. Increased SA concentrations have been reported during inflammatory processes, probably resulting from increased levels of richly sialylated acute-phase glycoproteins. A connection between increased SA levels and elevated stroke and cardiovascular mortality risk has also been reported. In addition, SA levels are slightly increased in cancer, positively correlating with the degree of metastasis, as well as in alcohol abuse, diabetes, chronic renal failure and chronic glomerulonephritis. Several different mechanisms are assumed to underlie the elevated SA concentrations in these disorders. The apparent non-specificity of SA to a given disease limits the potential clinical usefulness of SA determination. In addition, some non-pathological factors, such as aging, pregnancy and smoking, may cause changes in SA concentrations. The absolute increases in SA levels are also rather small (save those in inherited SA storage disorders); this further limits the clinical potential of SA as a marker. Tentatively, SA markers might serve as adjuncts, when combined with other markers, in disease screening, disease progression follow-up, and in the monitoring of treatment response. To become clinically useful, however, the existing SA determination assays need to be considerably refined to reduce interferences, to be specific for certain SA forms, and to be more easy to use.

Sialuria in a Portuguese girl: clinical, biochemical, and molecular characteristics.

Sialuria, a disorder of sialic acid (NeuAc) metabolism characterized by increased free NeuAc in the cytoplasm of cells, is due to failure of CMP-Neu5Ac to feedback inhibit UDP-N-acetylglucosamine (UDP-GlcNAc) 2-epimerase. We now describe the fifth patient in the world with sialuria, a 7-year-old Portuguese girl with developmental delay, hepatomegaly, coarse facies, and urinary excretion of 19 micromol of free NeuAc/mg creatinine. The patient's fibroblasts stored excess free NeuAc in the cytosolic fraction, and fibroblast UDP-GlcNAc 2-epimerase activity was only 26% inhibited by 100 microM CMP-Neu5Ac (normal, 79%). The patient's UDP-GlcNAc 2-epimerase gene displayed an R266Q mutation in only one allele, consistent with known sialuria mutations and with the proposed dominant nature of this disorder. Extensive description of sialuria patients will help to define the clinical and biochemical spectrum of this disease.

Determination of sialic acids in biological fluids using reversed-phase ion-pair high-performance liquid chromatography.

A simple, rapid and sensitive reversed-phase ion-pair high-performance liquid chromatographic method for the determination of N-acetylneuraminic acid and 2-deoxy-2,3-dehydro-N-acetylneuraminic acid in biological fluids is described. Determination of N-acetylneuraminic acid released by acidic hydrolysis, in serum, urine and saliva, and 2-deoxy-2,3-dehydro-N-acetylneuraminic acid in urine, without hydrolysis, was accomplished by injecting the sample without derivatization, into the chromatograph. Measurements were carried out isocratically within 6 min using a C18 column and a mobile phase of aqueous solution of triisopropanolamine, as ion-pair reagent, 60 mM, pH 3.5 at room temperature with UV absorbance detection. The present method is reported for the first time for the determination of sialic acids in biological fluids. Recoveries in serum, urine and saliva ranged from 90 to 102% and the limits of detection were 60 nM and 20 nM for the two sialic acids, respectively. The method has been applied to normal and pathological sera from patients with breast, stomach, colon, ovarian and cervix cancers, to normal urine and urine from patient with sialuria and to normal saliva.

Analysis of sialyllactoses in blood and urine by high-performance liquid chromatography.

A sensitive and highly selective high-performance liquid chromatography (HPLC)-based method has been developed for the analysis of oligosaccharides in biological fluids. In this method, a sample of biological fluid, such as blood serum or urine, is filtered through a 10,000 molecular weight cutoff filter cartridge to remove large molecules such as proteins and lipids. The carbohydrates in the filtrate are then derivatized with 1-phenyl-3-methyl-5-pyrazolone (PMP) as described previously [Anal. Biochem. 180, 351-357, (1989)]. The derivatized carbohydrates are separated by reverse-phase HPLC and monitored by UV absorbance at 245 nm. Quantitative analysis of the carbohydrates can be achieved based on their integration values relative to a standard calibration curve. Since neutral and acidic carbohydrates can be separated by using Dowex 1-X8 anion exchange resin, this method can be used specifically to analyze neutral, acidic, and total carbohydrates in the biological fluids. Because PMP specifically reacts with reducing aldoses, interference from noncarbohydrate components present in the biological fluids is essentially eliminated. This method has proven to be highly sensitive, requiring as little as 5 pmol of analyte for reliable analysis. It has also been used successfully for pharmacokinetic analysis of carbohydrate drugs in human blood and urine samples.

Comparison of sialic acids excretion in spot urines and 24-hour-urines of children and adults.

Sialic acids comprise all N- and O-acyl derivatives of neuraminic acid and are components of glycoproteins and glycolipids. Their concentrations vary physiologically with age but also in diseases such as inflammation, neoplastic tumours or in inborn genetic disorders causing abnormal sialic acid metabolism. Determination of free and bound sialic acids in urine using the thiobarbituric acid method according to Warren (J Biol Chem 1959; 234:1971-5) was shown to be useful for the diagnosis of diseases that involve sialic acid metabolic disorders. This test-also used for the diagnosis of inborn errors of metabolic diseases, such as sialidosis, infantile sialic acid storage disease, Salla's disease, neuraminidase deficiency and others-should be included in the selective screening for storage diseases. With the reported number of mild, juvenile and adult forms of genetic disorders increasing, this diagnosis will also be useful for teenagers and adults. We therefore considered it important not to confine our investigation to children and compared the diagnostic value of 24-hour and spot urines. As shown in 24-hour urines (n = 242, 128 males, 114 females) the average excretion of sialic acids increases constantly during life, from 67.6 mumol to 444.0 mumol per day, as does the free (27.5 mumol to 217.1 mumol) and bound fraction (40.1 mumol to 226.9 mumol). The relative proportion of free and bound sialic acid shows only slight lifetime variations, the free fraction increases from about 40 percent the first few years to about 53 percent of total in the fifth decade. In the spot urines, the mean ratio of total free sialic acids and urinary creatinine (mmol/mol) decreases constantly during the first few decades, with a sharp drop during the first years of life (from 3 months-2 years: from 203.9 to 94.2 and 82.1 to 42.3 respectively; with 10 years: 52.3 and 22.4 respectively; in the sixth decade: 44.8 and 21.9). Similar findings could also be observed in the investigated 24-hour urines (correlation coefficient of ratios, R = +0.981). The comparison of 24-hour urines and spot urines confirms the reliability of results for spot urines, however, the urine collection over an extended period yields additional information.

The spectrum of free neuraminic acid storage disease in childhood: clinical, morphological and biochemical observations in three non-Finnish patients.

N-acetylneuraminic acid (sialic acid) storage disease is a rare autosomal recessive lysosomal disorder. Clinically two major forms exist, an infantile type with severe progression leading to early death, and a milder form (Salla disease) with a protracted course. Intermediate forms may also exist. Diagnosis rests on the determination of an excessive excretion of sialic acid in urine and concomitant storage in fibroblasts, the severe forms exhibiting the highest excretion and storage. We present clinical, morphological, and biochemical data on three non-Finnish patients with sialic acid storage disease. Patient 1 was a preterm infant with neonatal ascites, coarse face, hepatosplenomegaly, pale skin, and wispy hair. Vacuolated lymphocytes were abundant in a peripheral blood smear and he excreted large amounts of free sialic acid. High levels of free sialic acid were also found in cultured skin fibroblasts. He died at age 6 months from progressive respiratory insufficiency. Patient 2 was an 11-month-old Egyptian girl with coarse face, frequent upper respiratory tract infections, hepatosplenomegaly, and severe psycho-motor retardation. Sialic acid excretion was elevated, likewise the storage in fibroblasts. Histological investigations documented vacuolar storage in a skin biopsy and in iliac crest tissue. Patient 3 was a 16-year-old girl with slightly coarse face, severe generalized muscular hypotonia, ataxia, and kyphoscoliosis originally diagnosed as having post-partum asphyxia. She suffered progressive motor function loss and had dysarthria. Urinary sialic acid was elevated and a skin biopsy demonstrated vacuolization. The clinical variability of sialic acid storage disease is exemplified by these three cases. Simple urinary screening for free sialic acid facilitates the diagnosis. The degree of urinary excretion may indeed correlate with clinical presentation and progression.

A Japanese case of infantile sialic acid storage disease.

We report a 4-year-old Japanese girl with infantile sialic acid storage disease. She presented with failure to thrive, coarse facial features, hepatosplenomegaly, severe mental retardation and spastic quadriplegia. Electron microscopic examination of cultured skin fibroblasts revealed multiple vacuoles and inclusion material representing distended lysosomes, thus suggesting a lysosomal storage disorder. A high concentration of free sialic acid was present in the urine and cultured fibroblasts, but bound sialic acid was not increased. The activity of a variety of lysosomal enzymes was not diminished. The MRI findings included brain atrophy and a diffuse high signal in the cerebral white matter and low signal in the basal ganglia on T2-weighted images. To our knowledge, this is the first case of infantile sialic acid storage disease described in a non-Caucasian family.

Determination of the novel sialic acid analog GG167 (GR121167X) in human urine by liquid chromatography: direct injection with column switching.

GG167 is a novel compound which selectively inhibits viral neuraminidase and has demonstrated activity against influenza A and B. A liquid chromatography (LC) method for the determination of GG167 in human urine has been developed and validated. The method allows direct injection of urine (7 microliters) using LC column switching followed by UV detection. Initial chromatography is performed using a Nucleosil-Diol column (7 microns, 250 mm x 4.6 mm), eluted with 20 mM phosphate buffer (pH 2.5):acetonitrile (18:82, v/v) at 2.0 ml min-1. GG167 is "heart-cut" to a Spherisorb-SCX column (5 microns, 100 mm x 4.6 mm) and eluted with 35 mM phosphate buffer (pH 2.5):acetonitrile (50:50, v/v) at 1.5 ml min-1 for final separation. GG167 is detected by UV absorbance at lambda = 238 nm. UV detection and peak shape are enhanced at pH < 2.5. The quantitation range of the assay is 0.3-100 micrograms ml-1. The method has demonstrated sufficient ruggedness to be used in support of GG167 clinical trials.

The urinary concentration of sialic acid is increased in non-insulin-dependent diabetic patients with microangiopathy: a possible useful marker for diabetic microangiopathy.

In order to investigate the relationship between urinary excretion of sialic acid and the severity of diabetic microangiopathy, urinary levels of sialic acid were determined in patients with non-insulin-dependent diabetes mellitus. The urinary molar ratio of sialic acid to creatinine in the diabetic patients was significantly higher than in the healthy controls (p < 0.01). Moreover, the urinary ratio was found to be gradually increased with the degree of diabetic microangiopathy. Urine molar ratio of sialic acid to creatinine in patients with proliferative diabetic retinopathy was significantly higher than in patients without retinopathy (p < 0.01). Urinary excretion in patients with macroproteinuria was also significantly higher than in patients without nephropathy (p < 0.01). Since urinary levels of sialic acid are proportionally increased with the severity of diabetic microangiopathy, the measurement of urinary sialic acid could become a useful biochemical means to monitor the degree of diabetic microangiopathy.

Urinary excretion of sialic acid in patients with bladder tumors.

The pre- and post-treatment urinary total sialic acid/creatinine (TSA/Cr) ratios of patients with bladder tumor (n = 60) were determined. We found a significant increase in the mean urinary TSA/Cr ratio in patients with bladder tumors than in healthy people (99.80 +/- 15.60 micrograms/g Cr, 52.57 +/- 15.60 micrograms/g Cr, P < 0.001). We determined that the mean post-treatment TSA/Cr ratio of 44 patients was significantly lower than their pretreatment ratio and this value also decreased to the level in healthy people. (TSA/Cr; healthy people, 52.57 +/- 15.60 micrograms/g Cr, P < 0.001). The patients with decreased TSA/Cr ratio in the post-treatment period showed complete or partial regression of their disease. In 8 patients, urinary TSA/Cr ratio in the post-treatment period increased to 105 +/- 14.5 micrograms/g Cr value. In clinical and pathologic evaluation, it was shown that disease progressed in all of these 8 patients. The mean post-treatment TAS/Cr ratio of 8 patients did not differ from the pretreatment ratio (87.44 +/- 20.20 micrograms/Cr) and it was shown that their clinical status did not change. These findings show that urinary excretion of TSA correlates with the clinical status of bladder tumor and it could be used to follow the course of the disease, and follow-up treatment.

Plasma and urine sialic acid in non-insulin dependent diabetes mellitus.

Urinary excretions of albumin, glycosaminoglycans (GAGS), total sialic acid (TSA), and lipid associated sialic acid (LASA) were measured in 78 non-insulin dependent diabetic patients (NIDDM) and 28 healthy subjects. TSA excretion was significantly higher in normoalbuminuric and microalbuminuric diabetic subjects than the control subjects and TSA excretion was correlated with urinary albumin excretion rate (AER). In normoalbuminuric diabetics, the duration of diabetes correlated significantly with both sialicaciduria and albuminuria. Although serum TSA levels were significantly higher in both diabetic groups than the control subjects, there was no correlation between serum and urinary TSA levels.

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.

Elevation of ratio of urinary N-acetylneuraminlactose to free sialic acid in some advanced cancer patients.

We estimated the levels of free sialic acid and sialylated oligosaccharides excreted in the urine of normal donors (n = 10) and patients with gastric cancer (n = 6) and colorectal cancer (n = 4). The total sialic acid level in cancer patients was similar to that in normal donors. However, the ratios of glycosidically bound sialic acids to free sialic acid were higher in some advanced cancer patients than in the normal donors. A major component of sialylated oligosaccharides was N-acetylneuraminyl alpha (2-->3) lactose. The elevation of the urinary ratio of this sialylated oligosaccharide to free sialic acid observed in some advanced cancer patients in this study may reflect the elevation of sialyltransferase activity in tumor tissues.

Diagnostic and prognostic role of plasma and urinary sialic acid in human carcinoma of uterine cervix.

In different stages of carcinoma of uterine cervix, the plasma total sialic acid (TSA), lipid bound sialic acid (LSA) and urinary sialic acid levels were studied before and after radiation treatment. Glycocomponents of glycoproteins were found to be increased progressively as the disease advances. The increased levels of glycocomponents were found to be decreased to near normal levels after radiotherapy. This may be a better indicator of the disease in conjunction with the studies of cervical smear rather than the latter alone.

Urinary excretion of sialic acid in patients with superficial bladder tumors.

Urinary total sialic acid/creatinine (TSA/Cr) ratio was determined in 73 patients with superficial bladder tumors and 34 healthy volunteers. The mean TSA/Cr value in the tumor group was higher than the control group (P < 0.001) and this difference was significant. Comparing the urinary TSA/Cr ratio of Tl, Ta, grade I-II, grade III, single, multiple, primary and recurrent tumors, with the control group revealed significant results (P < 0.001). Therefore the urinary TSA/Cr ratio determination in the follow-up of these patients may be used as a non-invasive procedure.

Report on two patients with Costello syndrome and sialuria.

We report on 2 unrelated patients with Costello syndrome. The first is a 5-year-old girl with "coarse" face, nasal papillomata, redundant skin of feet and hands, hyperextensible hand and finger joints, curly hair, feeding problems due to oral motor apraxia, growth and psychomotor retardation. The second is a 3-year-old boy with "coarse" face, loose skin on hands and feet, curly hair, oral motor apraxia, severe growth and psychomotor retardation. In both patients urine sialic acid levels were found to be repeatedly high. The meaning of this biochemical abnormality is discussed.

[Relation between dampness-heat syndrome of glomerulonephritis and sialic acid and N-acetyl-beta-D-glucosaminidase (NAG)].

Plasmic and urinary sialic acid and urinary N-acetyl-beta-D-glucosaminidase (NAG) of 87 glomerulonephritic patients with and without Dampness-Heat Syndrome were measured, and the influence of clearing up Dampness-Heat therapy on above-mentioned parameters was investigated. The results showed that Psa, Usa and UNAG of Dampness-Heat Syndrome were significantly higher than those of non-Dampness-Heat Syndrome (P < 0.05-0.01). The further analysis indicated that the patients with acute onset of chronic nephritis manifested as Dampness-Heat, showed marked positive correlation between Usa and UNAG as well as between UNAG and proteinuria respectively (r = 0.75 and 0.722, P < 0.001). With the treatment of Abelmoschus manihot which could remove the Dampness-Heat, the amount of proteinuria, Usa and UNAG were all significantly decreased (P < 0.05-0.001). It suggested that Usa and UNAG might be as diagnostic and curative parameters of Dampness-Heat of glomerulonephritis.