Evaluating new biomarkers for diabetic nephropathy:Role ofα2-macroglobulin,podocalyxin,α-L-fucosidase,retinol-binding protein-4,and cystatin C

BACKGROUND The intricate relationship between type 2 diabetes mellitus(T2DM)and diabetic nephropathy(DN)presents a challenge in understanding the significance of various biomarkers in diagnosis.AIM To elucidate the roles and diagnostic values ofα2-macroglobulin(α2-MG),podocalyxin(PCX),α-L-fucosidase(AFU),retinol-binding protein-4(RBP-4),and cystatin C(CysC)in DN.METHODS From December 2018 to December 2020,203 T2DM patients were enrolled in the study.Of these,115 were diagnosed with DN(115 patients),while the remaining 88 patients were classified as non-DN.The urinary levels ofα2-MG,PCX,and AFU and the serum concentrations RBP-4 and CysC were measured in conjunction with other relevant clinical indicators to evaluate their potential correlations and diagnostic utility.RESULTS After adjustments for age and gender,significant positive correlations were observed between the biomarkers CysC,RBP-4,α2-MG/urinary creatinine(UCr),PCX/UCr,and AFU/UCr,and clinical indicators such as urinary albumin-to-creatinine ratio(UACR),serum creatinine,urea,24-h total urine protein,and neutrophil-to-lymphocyte ratio(NLR).Conversely,these biomarkers exhibited negative correlations with the estimated glomerular filtration rate(P<0.05).Receiver operating characteristic(ROC)curve analysis further demonstrated the diagnostic performance of these biomarkers,with UACR showcasing the highest area under the ROC curve(AUC^(ROC))at 0.97.CONCLUSION This study underscores the diagnostic significance ofα2-MG,PCX,and AFU in the development of DN.The biomarkers RBP-4,CysC,PCX,AFU,andα2-MG provide promising diagnostic insights,while UACR is the most potent diagnostic biomarker in assessing DN.

Purification and characterization ofα-L-fucosidase from human primary hepatocarcinoma tissue

AIM: To purify and characterizeα-L-fucosidase from human liver cancer tissue and to detect the localization ofα-L-fucosidase in tumor tissue. METHODS: Cation exchange chromatography on CM-52 and ultrafiltration were used to separateα-Lfucosidase (AFU) from crude extract of liver cancer tissue. 4-methylumbelliferyl-α-L-fucopyranoside was used as a fluorescent substrate to quantify the purified AFU activity in each step. A polyclonal antibody (pAb) against the purified AFU was obtained by anion exchange chromatography on DEAE-52 after ammonium sulfate fractionation and ultrafiltration. Immuohistochemical staining was used to observe the expression of AFU in malignant and adjacent liver tissues. RESULTS: Humanα-L-fucosidase was purified 74-fold to apparent homogeneity with 15% yield. SDSPAGE indicated the presence of one subunit of molecular weight of 55 Ku. The specific activity of AFU in pooled fraction by chromatography was 10085 IU/mg. Western blot analysis indicated that the pAb could recognize one protein band of molecular weight of 55 Ku. The expression of AFU was observed in cytoplasm membrane of liver cancer tissue but not in that of adjacent tissue. CONCLUSION: The purifiedα-L-fucosidase from primary hepatocarcinoma (PHC) is different in its properties fromα-L-fucosidase in human other organs. The polyclonal antibody prepared in this experiment can be applied to the diagnosis of PHC.

Association between serumα-L-fucosidase andnon-alcoholic fatty liver disease:Cross-sectional study

AIM: To explore the association between serum α-Lfucosidase(Af U) and non-alcoholic fatty liver disease(NAf LD).METHODS: A total of 16473 individuals(9456 men and 7017 women) were included in the current study, who presented for a health examination at the first Affiliated hospital of Zhejiang University School of medicine in 2014. The baseline characteristics of the cohort were compared by NAf LD status. Linear regression analysis and stepwise multiple regression analysis were applied to assess the risk factors for NAf LD. Receiver operating characteristic curve was used to determine the sensitivity and specificity of Af U in the diagnosis of NAf LD.RESULTS: The prevalence rates of NAf LD and metabolic syndrome(met S) were 38.0% and 25.4%, respectively. The NAf LD group had significantly higher Af U levels than the non-NAf LD group(28.7 ± 7.9 U/L vs 26.0 ± 7.3 U/L, P < 0.001) and the prevalence rate of NAf LD increased with progressively higher serum Af U levels. Af U was positively correlated with met S and its five components: central obesity, hypertriglyceridemia, low high-density lipoprotein cholesterol, and elevated blood pressure and fasting glucose. Stepwise multiple logistic regression analysis showed that Af U was associated with an increased risk of NAf LD(OR = 1.009, 95%CI: 1.003-1.014, P < 0.001). The best cut-off value of Af U for the diagnosis of NAf LD was 27.5 U/L. The area under the curve(diagnostic efficacy index) was 0.606. The sensitivity and specificity were 54.6% and 61.8%, respectively. CONCLUSION: Af U level is significantly associated with NAf LD, and elevated Af U level is an independent risk factor for NAf LD.

Evaluation of α-L-Fucosidase in the Diagnosis of Primary Hepatic Carcinoma

α-L-fucosidasc (AFU) activity was determined by referring to Troost’s method in sera, liv-er tissues and lesions from patients with hepatoccllular carcinoma (HCC), other liverspace-occupying lesions (SOL) and diseases. The results showed that the sensitivity and specificityfor the diagnosis of HCC were 81.2% and 83. 3% respectively with the assay of serum AFU activi-ty, and sensitivities in the diagnosis of AFP-negative and early HCC were 76.1% and 70. 8%,respectively. The AFU activity in the HCC tissues and their pericancerous liver tissues was twice asmuch as controls, and consisted with their serum AFU activity (r=0. 742, P【0.01). It is sug-gested that AFU could be a new biologic marker of HCC and have an important value in thediagnosis of HCC.

A NIR ratiometric fluorescent biosensor for sensitive detection and imaging of α-L-fucosidase in living cells and HCC tumor-bearing mice

Detection and imaging of α-L-fucosidase(AFU)is of great value to understand its roles in hepatocellular carcinoma(HCC)and tumor early diagnosis,but ideal assays are still lacking.Herein,a near-infrared(NIR)fluorescent biosensor(α-Fuc-DCM)was elaborately designed and synthesized for rapid and ratiometric detection of AFU activity in cells and HCC tumor mouse models.In the presence of AFU,this biosensor shows an enhancement in NIR emission in a ratiometric manner,which significantly improves the detection accuracy with the limit of detection as low as 4.8 mU/mL.Taking advantage of these merits,the activity of AFU in lysosomes could be visualized using ratiometric and NIR dual modality in living cells.Furthermore,its remarkable application for monitoring of endogenous AFU activity in HCC tumor-bearing mouse model is also demonstrated with bright fluorescence signal,which indicated that the biosensor could clearly monitor the liver tumor in the early stage.Importantly,the α-Fuc-DCM probe can be utilized to detect the AFU in serum from HCC patients.This strategy offers a promising biosensor system for early diagnosis of HCC and studying the roles of AFU in cancers.