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1.
Anal Biochem ; 641: 114427, 2022 03 15.
Artículo en Inglés | MEDLINE | ID: mdl-34688604

RESUMEN

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.


Asunto(s)
Polisacáridos/orina , Femenino , Glicósido Hidrolasas/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Polisacáridos/metabolismo
2.
Mikrochim Acta ; 188(3): 66, 2021 02 04.
Artículo en Inglés | MEDLINE | ID: mdl-33543311

RESUMEN

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.


Asunto(s)
Carbono/química , Exosomas/química , Polisacáridos/orina , Neoplasias Gástricas/orina , Orina/citología , Adsorción , Humanos , Hierro/química , Fenómenos Magnéticos , Estructuras Metalorgánicas/síntesis química , Estructuras Metalorgánicas/química , Polisacáridos/química , Porosidad , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Neoplasias Gástricas/diagnóstico
3.
J Am Soc Mass Spectrom ; 32(2): 436-443, 2021 Feb 03.
Artículo en Inglés | MEDLINE | ID: mdl-33301684

RESUMEN

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.


Asunto(s)
Polisacáridos/análisis , Espectrometría de Masa por Ionización de Electrospray/métodos , Uromodulina/metabolismo , Femenino , Fetuínas/metabolismo , Glicosilación , Humanos , Polisacáridos/metabolismo , Polisacáridos/orina , Embarazo , Reproducibilidad de los Resultados , Uromodulina/análisis , Uromodulina/orina
4.
Mol Cell Proteomics ; 19(11): 1767-1776, 2020 11.
Artículo en Inglés | MEDLINE | ID: mdl-32737218

RESUMEN

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.


Asunto(s)
Glicómica/métodos , Polisacáridos/análisis , Polisacáridos/orina , Espectrometría de Masas en Tándem/métodos , Adulto , Niño , Preescolar , Cromatografía Liquida , Estudios de Cohortes , Femenino , Fucosa/orina , Glicosilación , Humanos , Lactante , Masculino , Manosa/metabolismo , Persona de Mediana Edad
5.
Int J Biol Macromol ; 142: 778-781, 2020 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-31622701

RESUMEN

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.


Asunto(s)
Phaeophyceae/química , Extractos Vegetales/química , Extractos Vegetales/metabolismo , Polisacáridos/química , Polisacáridos/metabolismo , Animales , Células Epiteliales/efectos de los fármacos , Microbioma Gastrointestinal/genética , Humanos , Absorción Intestinal , Hígado , Macrófagos , Metaboloma , Peso Molecular , Extractos Vegetales/sangre , Extractos Vegetales/orina , Polisacáridos/sangre , Polisacáridos/orina
6.
Anal Chem ; 92(2): 1680-1686, 2020 01 21.
Artículo en Inglés | MEDLINE | ID: mdl-31859482

RESUMEN

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.


Asunto(s)
Glicopéptidos/orina , Glicoproteínas/química , Polisacáridos/orina , Automatización , Glicosilación , Humanos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectrometría de Masas en Tándem
7.
Int J Mol Sci ; 20(7)2019 Mar 29.
Artículo en Inglés | MEDLINE | ID: mdl-30934974

RESUMEN

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.


Asunto(s)
Neoplasias de la Próstata/diagnóstico por imagen , Neoplasias de la Próstata/diagnóstico , Espectroscopía Infrarroja Corta , Glicosilación , Humanos , Masculino , Ácido N-Acetilneuramínico/metabolismo , Clasificación del Tumor , Polisacáridos/orina
8.
Mar Drugs ; 16(8)2018 Jul 30.
Artículo en Inglés | MEDLINE | ID: mdl-30061499

RESUMEN

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.


Asunto(s)
Conducta Alimentaria/fisiología , Absorción Gastrointestinal/fisiología , Polisacáridos/farmacocinética , Algas Marinas/química , Administración Oral , Adulto , Anciano , Voluntarios Sanos , Humanos , Japón , Masculino , Persona de Mediana Edad , Polisacáridos/administración & dosificación , Polisacáridos/sangre , Polisacáridos/orina , Eliminación Renal , Adulto Joven
9.
Diabetes Care ; 41(8): 1765-1775, 2018 08.
Artículo en Inglés | MEDLINE | ID: mdl-29930140

RESUMEN

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.


Asunto(s)
Biomarcadores/orina , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/diagnóstico , Nefropatías Diabéticas/diagnóstico , Polisacáridos/orina , Urinálisis/métodos , Anciano , Albuminuria/complicaciones , Albuminuria/diagnóstico , Albuminuria/orina , Biomarcadores/análisis , Estudios de Cohortes , Diabetes Mellitus Tipo 2/orina , Nefropatías Diabéticas/orina , Progresión de la Enfermedad , Femenino , Tasa de Filtración Glomerular , Glicosilación , Humanos , Riñón/fisiopatología , Fallo Renal Crónico/diagnóstico , Fallo Renal Crónico/etiología , Fallo Renal Crónico/orina , Masculino , Persona de Mediana Edad , Polisacáridos/análisis , Pronóstico , Diálisis Renal/efectos adversos
10.
J Reprod Immunol ; 125: 64-71, 2018 02.
Artículo en Inglés | MEDLINE | ID: mdl-29253795

RESUMEN

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.


Asunto(s)
Fosfatos de Inositol/orina , Polisacáridos/orina , Preeclampsia/diagnóstico , Segundo Trimestre del Embarazo/inmunología , Tercer Trimestre del Embarazo/inmunología , Adulto , Biomarcadores/orina , Estudios de Casos y Controles , Estudios Transversales , Femenino , Humanos , Fosfatos de Inositol/inmunología , Estudios Longitudinales , Mauricio/epidemiología , Polisacáridos/inmunología , Preeclampsia/epidemiología , Preeclampsia/inmunología , Preeclampsia/orina , Valor Predictivo de las Pruebas , Embarazo , Segundo Trimestre del Embarazo/orina , Tercer Trimestre del Embarazo/orina , Pronóstico , Estudios Prospectivos , Factores de Tiempo , Adulto Joven
11.
Sci Rep ; 7(1): 10216, 2017 08 31.
Artículo en Inglés | MEDLINE | ID: mdl-28860471

RESUMEN

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.


Asunto(s)
Glicoproteínas/metabolismo , Polisacáridos/análisis , Neoplasias de la Próstata/metabolismo , Animales , Bovinos , Ensayos Analíticos de Alto Rendimiento/instrumentación , Humanos , Proteínas Inmovilizadas/metabolismo , Masculino , Ácido N-Acetilneuramínico/química , Polisacáridos/orina , Neoplasias de la Próstata/orina , Reproducibilidad de los Resultados
12.
J Nutr Sci Vitaminol (Tokyo) ; 63(6): 419-421, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-29332904

RESUMEN

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.


Asunto(s)
Dieta , Phaeophyceae , Polisacáridos/orina , Algas Marinas , Adulto , Anciano , Anciano de 80 o más Años , Disponibilidad Biológica , Digestión , Femenino , Humanos , Absorción Intestinal , Masculino , Persona de Mediana Edad , Phaeophyceae/química , Polisacáridos/farmacocinética , Algas Marinas/química
13.
Thromb Res ; 144: 46-52, 2016 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-27289072

RESUMEN

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.


Asunto(s)
Anticoagulantes/uso terapéutico , Coagulación Sanguínea/efectos de los fármacos , Polisacáridos/uso terapéutico , Trombosis/tratamiento farmacológico , Administración Oral , Animales , Anticoagulantes/administración & dosificación , Anticoagulantes/sangre , Anticoagulantes/orina , Disponibilidad Biológica , Cromatografía de Fase Inversa , Laminaria/química , Masculino , Peso Molecular , Extractos Vegetales/administración & dosificación , Extractos Vegetales/sangre , Extractos Vegetales/uso terapéutico , Extractos Vegetales/orina , Polisacáridos/administración & dosificación , Polisacáridos/sangre , Polisacáridos/orina , Ratas Wistar , Trombosis/sangre
14.
J Proteome Res ; 15(3): 923-32, 2016 Mar 04.
Artículo en Inglés | MEDLINE | ID: mdl-26812091

RESUMEN

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.


Asunto(s)
Hormona Folículo Estimulante Humana/orina , Polisacáridos/orina , Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Animales , Células CHO , Conformación de Carbohidratos , Secuencia de Carbohidratos , Cricetinae , Cricetulus , Hormona Folículo Estimulante Humana/química , Hormona Folículo Estimulante Humana/aislamiento & purificación , Glicosilación , Humanos , Peso Molecular , Polisacáridos/química , Polisacáridos/aislamiento & purificación , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo
15.
Reprod Sci ; 23(3): 365-71, 2016 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-26355118

RESUMEN

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.


Asunto(s)
Diabetes Gestacional/diagnóstico , Diabetes Gestacional/orina , Número de Embarazos/fisiología , Fosfatos de Inositol/orina , Inositol/orina , Polisacáridos/orina , Primer Trimestre del Embarazo/orina , Adulto , Biomarcadores/orina , Estudios de Casos y Controles , Femenino , Cromatografía de Gases y Espectrometría de Masas/métodos , Humanos , Periodo Posparto/orina , Embarazo , Distribución Aleatoria
16.
Artículo en Inglés | MEDLINE | ID: mdl-26118620

RESUMEN

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.


Asunto(s)
Cromatografía en Gel/métodos , Polisacáridos/análisis , Polisacáridos/farmacocinética , Adolescente , Anciano , Transporte Biológico Activo , Células CACO-2 , Humanos , Masculino , Persona de Mediana Edad , Neoplasias/metabolismo , Polisacáridos/química , Polisacáridos/orina
17.
Urolithiasis ; 43(4): 303-12, 2015 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-25935139

RESUMEN

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.


Asunto(s)
Nefrolitiasis/orina , Uromodulina/orina , Adulto , Anciano , Estudios de Casos y Controles , Femenino , Humanos , Masculino , Persona de Mediana Edad , Monosacáridos/orina , Ácido N-Acetilneuramínico/orina , Polisacáridos/orina , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Adulto Joven
18.
Yao Xue Xue Bao ; 49(4): 443-9, 2014 Apr.
Artículo en Chino | MEDLINE | ID: mdl-24974459

RESUMEN

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.


Asunto(s)
Absorción Intestinal , Polisacáridos/farmacocinética , Administración Oral , Animales , Inyecciones , Tasa de Depuración Metabólica , Peso Molecular , Polisacáridos/administración & dosificación , Polisacáridos/análisis , Polisacáridos/orina , Distribución Tisular
19.
Anal Chem ; 86(13): 6277-84, 2014 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-24766348

RESUMEN

Differences in ionization efficiency among neutral and sialylated glycans prevent direct quantitative comparison by their respective mass spectrometric signals. To overcome this challenge, we developed an integrated chemical strategy, Dual Reactions for Analytical Glycomics (DRAG), to quantitatively compare neutral and sialylated glycans simultaneously by MALDI-MS. Initially, two glycan samples to be compared undergo reductive amination with 2-aminobenzoic acid and 2-(13)[C6]-aminobenzoic acid, respectively. The different isotope-incorporated glycans are then combined and subjected to the methylamidation of the sialic acid residues in one mixture, homogenizing the ionization responses for all neutral and sialylated glycans. By this approach, the expression change of relevant glycans between two samples is proportional to the ratios of doublet signals with a static 6 Da mass difference in MALDI-MS and the change in relative abundance of any glycan within samples can also be determined. The strategy was chemically validated using well-characterized N-glycans from bovine fetuin and IgG from human serum. By comparing the N-glycomes from a first morning (AM) versus an afternoon (PM) urine sample obtained from a single donor, we further demonstrated the ability of DRAG strategy to measure subtle quantitative differences in numerous urinary N-glycans.


Asunto(s)
Fetuínas/química , Inmunoglobulina G/química , Ácido N-Acetilneuramínico/análisis , Polisacáridos/análisis , Polisacáridos/orina , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Animales , Secuencia de Carbohidratos , Bovinos , Humanos , Datos de Secuencia Molecular , Polisacáridos/química
20.
J Reprod Immunol ; 101-102: 148-152, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-23962712

RESUMEN

Preeclampsia and eclampsia account for major pregnancy complications in Mauritius, an emerging country (maternal mortality rate of 60 per 100,000 deliveries). This prospective longitudinal study was carried out in the main regional hospital in the north of the island, to measure inositol phosphoglycan-P type (IPG-P) in the urine of pregnant women (using an ELISA-based assay). Women had approximately 10 prenatal visits per pregnancy and a complete follow-up in this same referral centre after the first trimester of pregnancy. Urine samples were collected every 1-4 weeks in all women. In a cohort of 416 patients, preeclampsia (PE) was diagnosed in 34 women. In established PE (hypertension and proteinuria), the assay as a diagnostic test showed a positive likelihood ratio of 18.73, a negligible negative likelihood ratio with area under the curve (AUC) of 0.99, sensitivity of 96.7%, specificity of 94.8% and remained negative in control women (n=312), women with gestational hypertension (without proteinuria (n=56), and gestational diabetic mothers (n=14). Moreover, as a predictive screening test two weeks before the diagnosis of PE, the assay showed sensitivity of 84.2% and specificity of 83.6%. Detection of urinary inositol phosphoglycan-P type in pregnant women can be a useful confirmatory marker of PE, as well as a predictive marker, two weeks before the onset of the disease.


Asunto(s)
Biomarcadores/orina , Fosfatos de Inositol/orina , Polisacáridos/orina , Preeclampsia/diagnóstico , Adulto , Diagnóstico Precoz , Femenino , Estudios de Seguimiento , Humanos , Estudios Longitudinales , Tamizaje Masivo , Mauritania , Sistemas de Atención de Punto , Valor Predictivo de las Pruebas , Embarazo , Pronóstico , Estudios Prospectivos , Sensibilidad y Especificidad , Adulto Joven
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