Analytical Investigation of the Profile of Human Chorionic Gonadotropin in Highly Purified Human Menopausal Gonadotrophin Preparations.

Highly purified human menopausal gonadotropin (HP-hMG [Menopur®, Ferring Pharmaceuticals, Saint-Prex, Switzerland]) contains a 1:1 ratio of follicle-stimulating hormone (FSH) and luteinizing hormone (LH). This analysis aimed to assess gonadotropin (FSH, LH and hCG) abundance in HP-hMG and clarify the source of hCG by assessing the presence of sulfated glycans, which are diagnostic for pituitary hCG forms due to their distinct glycosylation patterns. Additionally, the purity of each sample, their specific components, and their oxidation levels were assessed. HP-hMG samples (three of Menopur® and two of Menogon® Ferring Pharmaceuticals, Saint-Prex, Switzerland) were included in the current analyses. Brevactid® (urinary hCG; Ferring Pharmaceuticals, Saint-Prex, Switzerland) and Ovidrel® (recombinant hCG; Merck KGaA, Darmstadt, Germany) were used as control samples. Glycopeptide mapping and analysis of impurities were carried out by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Oxidation was assessed through reducing peptide mapping using LC-MS/MS. The FSH and LH in the HP-hMG samples showed sulfated glycans, while no signals of sulfated glycopeptides were detected on any site of the beta subunit of hCG. HP-hMG test samples presented the same hCG glycan distribution as the control sample (placental hCG, Brevactid®) extracted from the urine of pregnant women, suggesting a non-pituitary source of hCG. Protein impurities were estimated to constitute approximately 20-30% of the entire HP-hMG protein content in the test samples. More than 200 non-gonadotropin proteins were identified in the HP-hMG test samples, of which several were involved in embryonic development or pregnancy. The alpha subunit of the tested samples was strongly oxidized, with a relative abundance of 20% of the total gonadotropin content. Without taking into account all the protein impurities, the beta subunit of LH was detected only in traces (0.9-1.2%) in all tested HP-HMG samples, confirming the data obtained by intact molecule analysis, while high levels of beta hCG (18-47%) were observed. Advanced molecular analysis of HP-hMG indicates a primarily placental origin of hCG, as evidenced by the absence of hCG sulfated glycans and the predominance of placental non-sulfated hCG in LH activity. The analysis revealed 20-30% of protein impurities and a significant presence of oxidized forms in the HP-hMG samples. These findings are critical for understanding the quality, safety, and clinical profile of HP-hMG.

Multiplex Determination of Glycan Profiles on Urinary Prostate-Specific Antigen by Quartz-Crystal Microbalance Combined with Surface-Enhanced Raman Scattering.

Prostate cancer remains a major health concern, with prostate-specific antigen (PSA) being a key biomarker for its detection and monitoring. However, PSA levels often fall into a "gray zone", where PSA levels are not clearly indicative of cancer, thus complicating early diagnosis and treatment decisions. Glycosylation profiles, which often differ between healthy and diseased cells, have emerged as potential biomarkers to enhance the specificity and sensitivity of cancer diagnosis in these ambiguous cases. We propose the integration of two complementary techniques, namely quartz-crystal microbalance with dissipation (QCM-D) and surface-enhanced Raman scattering (SERS) to study PSA glycan profiles. QCM-D offers real-time operation, PSA mass quantification, and label-free detection with high sensitivity, as well as enhanced specificity and reduced cross-reactivity when using nucleic acid aptamers as capture ligands. Complementary SERS sensing enables the determination of the glycosylation pattern on PSA, at low concentrations and without the drawbacks of photobleaching, thereby facilitating multiplexed glycosylation pattern analysis. This integrated setup could retrieve a data set comprising analyte concentrations and associated glycan profiles in relevant biological samples, which may eventually improve early disease detection and monitoring. Prostate-specific antigen (PSA), a glycoprotein secreted by prostate epithelial cells, serves as our proof-of-concept analyte. Our platform allows multiplex targeting of PSA multiplex glycosylation profiles of PSA at "gray zone" concentrations for prostate cancer diagnosis. We additionally show the use of SERS for glycan analysis in PSA secreted from prostate cancer cell lines after androgen-based treatment. Differences in PSA glycan profiles from resistant cell lines after androgen-based treatment may eventually improve cancer treatment.

Identification of ß1-3 galactosylglucose-core free-glycans in human urine.

We previously reported the precise structure of acidic free-glycans in human urine. In the present study, structural analysis of neutral free-glycans in urine was performed in fine detail. Urine samples were collected from 21 healthy volunteers and free-glycans extracted from the creatinine-adjusted urine and then fluorescently labeled with 2-aminopyridine. Neutral glycan profiling was achieved by a combination of high-performance liquid chromatography, mass spectrometry, enzymatic digestion, and periodate cleavage. A total of 79 glycans were identified. Because the ABO-blood group antigen containing urinary neutral glycans are major components, profiling patterns were similar between individuals of the same ABO-group. The neutral glycans were composed of lactose-core (Galß1-4Glc) glycans, type-II N-acetyllactosamine-core (GlcNAcß1-4Glc) glycans, hexose oligomers, N-glycans and to our surprise ß1-3 galactosylglucose-core (Galß1-3Glc) glycans. Although glycans with a ß1-3 galactosylglucose-core were identified as major components in urine, comprising structurally simple isomers of a lactose-core, the core structure has not previously been reported. The major ß1-3 galactosylglucose-core glycans were Fucα1-2Galß1-3(Fucα1-4)Glc, GalNAcα1-3(Fucα1-2)Galß1-3(Fucα1-4)Glc and Galα1-3(Fucα1-2)Galß1-3(Fucα1-4)Glc, corresponding to H-, A-, and B-blood group antigens, respectively. Three lactosamine extended ß1-3 galactosylglucose-core glycans were also detected as minor components. Elucidating the biosynthesis of ß1-3 galactosylglucose will be crucial for understanding the in vivo function of these glycans.

Magnetic porous carbon-dependent platform for the determination of N-glycans from urine exosomes.

A magnetic porous carbon-dependent platform is established to separate and determine N-glycans from urine exosomes of healthy people and patients with gastric cancer. The results of the comparison reveal that 6 N-glycans shared by the two groups are downregulated, most of which present core fucose or bisecting N-acetylglucosamine (GlcNAc) type. In addition, five shared N-glycans including two of sialic acid type are upregulated. These obvious differences indicate the close relationship between glycans and gastric cancer thus permitting early diagnosis. A magnetic porous carbon material (FeMPC) from MIL-101(Fe) was employed to separate and analyze N-glycans from urine exosomes of healthy people and patients with gastric cancer.

Clinically Viable Assay for Monitoring Uromodulin Glycosylation.

Uromodulin, also known as the Tamm-Horsfall protein or THP, is the most abundant protein excreted in human urine. It is associated with the progression of kidney diseases; therefore, changes in the glycosylation profile of this protein could serve as a potential biomarker for kidney health. The typical glycomics analysis approaches used to quantify uromodulin glycosylation involve time-consuming and tedious glycoprotein isolation and labeling steps, which limit their utility in clinical glycomics assays, where sample throughput is important. Herein, we introduce a radically simplified sample preparation workflow, with direct ESI-MS analysis, enabling the quantification of N-linked glycans that originate from uromodulin. The method omits any glycan labeling steps but includes steps to reduce the salt content of the samples, thereby minimizing ion suppression. The method is effective for quantifying subtle glycosylation differences of uromodulin samples derived from different biological states. As a proof of concept, glycosylation from samples that differ by pregnancy status were shown to be differentiable.

An in-depth Comparison of the Pediatric and Adult Urinary N-glycomes.

We performed an in-depth characterization and comparison of the pediatric and adult urinary glycomes using a nanoLC-MS/MS based glycomics method, which included normal healthy pediatric (1-10 years, n = 21) and adult (21-50 years, n = 22) individuals. A total of 116 N-glycan compositions were identified, and 46 of them could be reproducibly quantified. We performed quantitative comparisons of the 46 glycan compositions between different age and sex groups. The results showed significant quantitative changes between the pediatric and adult cohorts. The pediatric urinary N-glycome was found to contain a higher level of high-mannose (HM), asialylated/afucosylated glycans (excluding HM), neutral fucosylated and agalactosylated glycans, and a lower level of trisialylated glycans compared with the adult. We further analyzed gender-associated glycan changes in the pediatric and adult group, respectively. In the pediatric group, there was almost no difference of glycan levels between males and females. In adult, the majority of glycans were more abundant in males than females, except the high-mannose and tetrasialylated glycans. These findings highlight the importance to consider age-matching and adult sex-matching for urinary glycan studies. The identified normal pediatric and adult urinary glycomes can serve as a baseline reference for comparisons to other disease states affected by glycosylation.

Is the transformation of fucoidans in human body possible?

Fucoidans are a group of homo-and hetero-polysaccharides, which necessarily contains residues of sulfated α-L-fucose. Fucoidans are found only in brown algae. These polysaccharides exhibit a wide spectrum of biological activity and have a great therapeutic potential. Enzymes capable of catalyzing the degradation of fucoidans are absent in the mammalian enzyme system. The question arises: is the transformation of fucoidan in mammals, particularly in human possible? Studies in vivo (in situ) and in vitro have demonstrated that high molecular weight fucoidans are absorbed across rat intestinal epithelial cells, accumulated by liver macrophages, and characterized by low levels in blood and urine. Using the example of the Okinawa Prefecture (Japan) residents, it was shown that Cladosiphon okamuranus alga is digested and the fucoidan contained in this alga is absorbed in the human body.

Glycans, Glycosite, and Intact Glycopeptide Analysis of N-Linked Glycoproteins Using Liquid Handling Systems.

Aberrant glycosylation has been shown to associate with disease progression, and with glycoproteins representing the major protein component of biological fluids this makes them attractive targets for disease monitoring. Leveraging glycoproteomic analysis via mass spectrometry (MS) could provide the insight into the altered glycosylation patterns that relate to disease progression. However, investigation of large sample cohorts requires rapid, efficient, and highly reproducible sample preparation. To address the limitation, we developed a high-throughput method for characterizing glycans, glycosites, and intact glycopeptides (IGPs) derived from N-linked glycoproteins. We combined disparate peptide enrichment strategies (i.e., hydrophilic and hydrophobic) and a liquid handling platform allowing for a high throughput and rapid enrichment of IGP in a 96-well plate format. The C18/MAX-Tip workflow reduced sample processing time and facilitated the selective enrichment of IGPs from complex samples. Furthermore, our approach enabled the analysis of deglycosylated peptides and glycans from enriched IGPs following PNGase F digest. Following development and optimization of the C18/MAX-Tip methodology using the standard glycoprotein, fetuin, we investigated normal urine samples to obtain N-linked glycoprotein information. Together, our method enables a high-throughput enrichment of glycan, glycosites, and IGPs from biological samples.

N-Linked Glycosylation and Near-Infrared Spectroscopy in the Diagnosis of Prostate Cancer.

BACKGROUND: Performing a prostate biopsy is the most robust and reliable way to diagnose prostate cancer (PCa), and to determine the disease grading. As little to no biochemical markers for prostate tissue exist, we explored the possibilities of tissue N-glycosylation and near-infrared spectroscopy (NIR) in PCa diagnosis. METHODS: Tissue specimens from 100 patients (benign prostate hyperplasia (BPH), n = 50; and PCa, n = 50) were obtained. The fresh-frozen tissue was dispersed and a tissue N-glycosylation profile was determined. Consequently, the formalin-fixed paraffin-embedded slides were analyzed using NIR spectroscopy. A comparison was made between the benign and malignant tissue, and between the various Gleason scores. RESULTS: A difference was observed for the tissue of N-glycosylation between the benign and malignant tissue. These differences were located in the fycosylation ratios and the total amount of bi- and tetra-antennary structures (all p < 0.0001). These differences were also present between various Gleason scores. In addition, the NIR spectra revealed changes between the benign and malignant tissue in several regions. Moreover, spectral ranges of 1055⁻1065 nm and 1450⁻1460 nm were significantly different between the Gleason scores (p = 0.0042 and p = 0.0195). CONCLUSIONS: We have demonstrated biochemical changes in the N-glycan profile of prostate tissue, which allows for the distinction between malignant and benign tissue, as well as between various Gleason scores. These changes can be correlated to the changes observed in the NIR spectra. This could possibly further improve the histological assessment of PCa diagnosis, although further method validation is needed.

Absorption Study of Mozuku Fucoidan in Japanese Volunteers.

We performed an oral administration study of fucoidan in 396 Japanese volunteers and investigated significant factors concerning the absorption of fucoidan. Urine samples were collected at 0, 3, 6, and 9 h after ingestion of 3 g of fucoidan. Fucoidan was detected in urine after ingestion in 385 out of 396 subjects. The maximum value (mean ± standard deviation (SD)) of urinary fucoidan was 332.3 ± 357.6 µg/gCr in subjects living in Okinawa prefecture, compared with 240.1 ± 302.4 µg/gCr in subjects living outside Okinawa. Compared with the estimated urinary excretion of fucoidan by place of residence, those of subjects living in Okinawa prefecture were significantly higher than those living outside Okinawa prefecture (p < 0.01). In addition, subjects living in Okinawa prefecture consumed significantly greater amounts of mozuku compared with those living outside Okinawa prefecture (p < 0.01). Multiple regression analysis showed that having Okinawa prefecture as a place of residence was a significant factor (p < 0.01) contributing to the estimated urinary excretion of fucoidan. Because the habit of eating mozuku was significantly higher (p < 0.01) in subjects living in Okinawa prefecture than in those living outside Okinawa prefecture, the habit of eating mozuku was speculated to be a factor in the absorption of fucoidan.

Identification of Novel Urinary Biomarkers for Predicting Renal Prognosis in Patients With Type 2 Diabetes by Glycan Profiling in a Multicenter Prospective Cohort Study: U-CARE Study 1.

OBJECTIVE: Because quantifying glycans with complex structures is technically challenging, little is known about the association of glycosylation profiles with the renal prognosis in diabetic kidney disease (DKD). RESEARCH DESIGN AND METHODS: In 675 patients with type 2 diabetes, we assessed the baseline urinary glycan signals binding to 45 lectins with different specificities. The end point was a decrease of estimated glomerular filtration rate (eGFR) by ≥30% from baseline or dialysis for end-stage renal disease. RESULTS: During a median follow-up of 4.0 years, 63 patients reached the end point. Cox proportional hazards analysis revealed that urinary levels of glycans binding to six lectins were significantly associated with the outcome after adjustment for known indicators of DKD, although these urinary glycans, except that for DBA, were highly correlated with baseline albuminuria and eGFR. Hazard ratios for these lectins were (+1 SD for the glycan index) as follows: SNA (recognizing glycan Siaα2-6Gal/GalNAc), 1.42 (95% CI 1.14-1.76); RCA120 (Galß4GlcNAc), 1.28 (1.01-1.64); DBA (GalNAcα3GalNAc), 0.80 (0.64-0.997); ABA (Galß3GalNAc), 1.29 (1.02-1.64); Jacalin (Galß3GalNAc), 1.30 (1.02-1.67); and ACA (Galß3GalNAc), 1.32 (1.04-1.67). Adding these glycan indexes to a model containing known indicators of progression improved prediction of the outcome (net reclassification improvement increased by 0.51 [0.22-0.80], relative integrated discrimination improvement increased by 0.18 [0.01-0.35], and the Akaike information criterion decreased from 296 to 287). CONCLUSIONS: The urinary glycan profile identified in this study may be useful for predicting renal prognosis in patients with type 2 diabetes. Additional investigation of glycosylation changes and urinary glycan excretion in DKD is needed.

New insights into early and late onset subgroups of preeclampsia from longitudinal versus cross-sectional analysis of urinary inositol-phosphoglycan P-Type.

Most pre-eclampsia (PE) studies have used cross-sectional data to derive conclusions regarding the pathophysiology of the condition. This has led to the concept that there exists early (<34 weeks) and late-onset (>34 weeks) disease according to gestational age at diagnosis. Survival time models have predicted that if the pregnancy was to continue indefinitely, all women would develop PE. In this study we have performed a longitudinal analysis of the urinary biomarker, inositol phosphoglycan (IPG), in a cohort of women giving birth in Mauritius (n-920). We have analysed the PE data in the traditional cross-sectional manner for n = 77 women who developed PE and also then looked at the longitudinal data for 71/77 of the same women. The data allows us to use longitudinal values to calculate a date of onset (first presence of biomarker in urine) and compare that to date of clinical diagnosis (cross sectional). We find two populations for both analysis consistent with an early and late stage subgroup. The calculated date of onset had subgroups (early and late) at 28.4 ±â€¯0.41 weeks and 35.37 ±â€¯0.26 weeks and for clinical date of diagnosis, 32.3 ±â€¯0.59 weeks and 37.04 ±â€¯0.62 weeks, respectively. The presence of the same biomarker in both subgroups and its ability to predict clinical onset 2-4 weeks prior to clinical diagnosis suggest that both groups may have similar aetiology.

High-throughput analysis of N-glycans using AutoTip via glycoprotein immobilization.

Analysis of a large number of samples requires an efficient, rapid and reproducible method. Automation is an ideal approach for high-throughput sample preparation. Multi-plexing sample preparation via a 96-well plate format becomes popular in recent years; however, those methods lack specificity and require several cleanup steps via chromatography purification. To overcome these drawbacks, a chemoenzymatic method has been developed utilizing protein conjugation on solid-phase. Previously, sample preparation was successfully performed in a snap-cap spin-column (SCSC) format. However, sample preparation using SCSC is time-consuming and lacks reproducibility. In this work, we integrated the chemoenzymatic technique in a pipette tip (AutoTip) that was operated by an automated liquid handler. We established a multi-step protocol involving protein immobilization, sialic acid modification, and N-glycan release. We first optimized our automated protocol using bovine fetuin as a standard glycoprotein, and then assessed the reproducibility of the AutoTip using isobaric tags for relative N-linked glycan quantification. We then applied this methodology to profile N-glycans from 58 prostate cancer patient urine samples, revealing increased sialyation on urinary N-glycans derived from prostate cancer patients. Our results indicated AutoTip has applications for high-throughput sample preparation for studying the N-linked glycans.

Detection of Fucoidan in Urine after Oral Intake of Traditional Japanese Seaweed, Okinawa mozuku (Cladosiphon okamuranus Tokida).

Seaweed has been considered an indigestible food. Fucoidan, which is found abundantly in seaweed, especially in Cladosiphon okamuranus (Okinawa mozuku), has a high molecular weight and has been long believed to be hardly absorbed in the human digestive system due to a lack of certain digestive enzymes. We previously reported that fucoidan can be detected in serum and urine after oral intake of purified fucoidan in humans and rats. However, it is unclear whether the fucoidan in mozuku can be absorbed after digestion of mozuku. Therefore, we attempted to detect fucoidan in urine before and after mozuku intake. We determined the fucoidan concentration in urine after oral intake of Okinawa mozuku and urinary fucoidan was detected in several volunteers. In conclusion, these results suggest that fucoidan in mozuku can be absorbed after ingestion of mozuku.

Antithrombotic activity of oral administered low molecular weight fucoidan from Laminaria Japonica.

INTRODUCTION: Fucoidans extracted from brown algae have been documented to have excellent antithrombotic activity when administered by either intravenous or subcutaneous route in animal models. However, it is unknown if the fucoidans also have antithrombotic activity when administered orally, a highly desirable feature of oral antithrombotic agents. In the present study, we compared the oral absorption, bioavailability and antithrombotic activity of two fucoidan fractions from Laminaria japonica with different molecular weight by oral administration in an electricity induced arterial thrombosis model and the underlying molecular mechanisms. RESULTS AND CONCLUSIONS: After a single dose of oral administration, the fucoidan content in plasma and urine in rats was assessed using the reverse-phased HPLC analysis of 1-phenyl-3-methyl-5-pyrazolone (PMP)-labeled fucose. The fucose content in the low molecular weight (LMW) fucoidan-treated rats increased up to 2-fold and peaked at 15h, indicating that the LMW fucoidan had much better absorption and bioavailability than the MMW fucoidan in vivo. Oral administration of the LMW fucoidan at 400 and 800mg/kg for 30days inhibited the arterial thrombosis formation effectively induced by electrical shock in rats, accompanied by moderate anticoagulation activity, regulation on TXB2 and 6-keto-PGF1α, significant antiplatelet activity and effective fibrinolysis. The LMW fucoidan showed better oral absorption and antithrombotic activity in addition to different antithrombotic mechanisms compared to those of the medium molecular weight (MMW) fucoidan. Thus, the LMW fucoidan has a potential to become an oral antithrombotic agent.

Comparative Assessment of Glycosylation of a Recombinant Human FSH and a Highly Purified FSH Extracted from Human Urine.

Glycosylation is an important PTM and is critical for the manufacture and efficacy of therapeutic glycoproteins. Glycan significantly influences the biological properties of human follicle-stimulating hormone (hFSH). Using a glycoproteomic strategy, this study compared the glycosylation of a putative highly purified FSH (uhFSH) obtained from human urine with that of a recombinant human FSH (rhFSH) obtained from Chinese hamster ovary (CHO) cells. Intact and subunit masses, N-glycans, N-glycosylation sites, and intact N- and O-glycopeptides were analyzed and compared by mass spectrometry. Classic and complementary analytical methods, including SDS-PAGE, isoelectric focusing, and the Steelman-Pohley bioassay were also employed to compare their intact molecular weights, charge variants, and specific activities. Results showed that highly sialylated, branched, and macro-heterogeneity glycans are predominant in the uhFSH compared with those in rhFSH. The O-glycopeptides of both hFSHs, which have not been described previously, were characterized herein. A high degree of heterogeneity was observed in the N-glycopeptides of both hFSHs. The differences in glycosylation provide useful information in elucidating and in further investigation the critical glycan structures of hFSH.

Urinary Excretion of Myo-Inositol and D-Chiro-Inositol in Early Pregnancy Is Enhanced in Gravidas With Gestational Diabetes Mellitus.

The effects of gestational diabetes mellitus (GDM) were determined on urinary excretion of putative components of insulin signaling. Random urine samples were collected from 375 gravidas at 6 to 14 weeks' gestation, 22 to 32 weeks' gestation, and ∼6 weeks' postpartum. Gestational diabetes mellitus developed in 35 women who were matched with 59 normal gravidas. Urinary concentrations of myo-inositol (MI) and D-chiro-inositol (DCI) were measured by gas chromatography/mass spectrometry and normalized to creatinine levels. Compared to postpartum values, urinary excretion of MI and DCI was increased 2.9-fold and 2-fold, respectively, in early pregnancy, and 5.5-fold and 4.5-fold, respectively, in later gestation. Gravidas with GDM had significantly greater MI and DCI excretion than controls in the first trimester but not subsequently. The results suggest that gravidas destined to develop GDM have altered synthesis, metabolism, and/or renal excretion of MI and DCI in early pregnancy.

Determination of the active transport of fucoidan derived from Okinawa Mozuku across the human intestinal Caco-2 cells as assessed by size-exclusion chromatography.

INTRODUCTION: In order to clarify the mechanism of fucoidan transport, we developed the chromatographic determination method. METHOD: A size-exclusion chromatography (SEC) method for the determination of Okinawa-fucoidan using Develosil 300 Diol-5 (60×8.0mm I.D., 30nm pore-diameter) with the eluent containing 1% non-ionic detergent is developed. Determination range (UV at 210nm) is from 0 to 100ng of fucoidan with the linear calibration line inserting to zero. RESULTS: A transport activity of fucoidan is demonstrated by using Caco-2 cells (model of gut transport system); i.e., the initial transport velocity 12nmol/h/mg of protein (25-fold slower rate as compared to a bacterial l-alanine active-transport activity 300nmol/h/mg of protein) is found to occur. Since this fucoidan transport is inhibited by 10mM sodium azide (respiration inhibitor) and 0.05mM FCCP (uncoupler), this transport by Caco-2 cells is found to be an active one requiring energy-source. On the other hand, colchicine (inhibitor of phagocytosis/pinocytosis) and mannitol (putative competitive-inhibitor of tight-junction transport) cannot inhibit the fucoidan transport at all. CONCLUSION: We firstly report that the active transport occurs for such a high molecular-weight sulphated-polyfucose of fucoidan in vitro using Caco-2 cells.

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.

[Research progresses of pharmacokinetics of polysaccharides].

Pharmacokinetic analysis has attracted more and more attentions in the research field of bioactive natural product. However, there is limited study on the pharmacokinetics of polysaccharides. This paper focused on the research progresses of pharmacokinetics of polysaccharide, summarized the applications of chromatography, isotope labeling method, spectrophotometry, fluorospectrophotometry and biological assay in the analysis of polysaccharide pharmacokinetics, elucidated the behaviors of absorption, distribution, degradation and excretion of polysaccharide in experimental animals, and revealed the effects of physicochemical characteristic, administration dose and route on the pharmacokinetic properties of polysaccharide, which could be served as a reference for the related works.