Development and Evaluation of Novel ELISA for Determination of Urinary Pentosidine

Abstract

Pentosidine is the most well-characterized advanced glycation end product (AGE). It has been measured by HPLC, although this approach cannot be adapted to analyze many clinical samples and is also time-consuming. Furthermore, the detection of pentosidine using a reported ELISA kit and HPLC system requires pretreatment by heating, which generates artificial pentosidine leading to overestimation. We developed a novel pentosidine ELISA system that don't require sample pretreatment for analyzing urine samples. We then analyzed the accuracy, precision, and reliability of this system. Urinary samples for analysis were obtained from healthy volunteers and stored urinary samples from the participants of the Nagano cohort study were also used. The LoB and LoD were 4.25 and 6.24 pmol/mL, respectively. Intra-and inter-assay coefficients of variation were less than 5%. The spiking and dilution recoveries were 101.4% and 100.5%, respectively. Analysis of the cross-reactivities against seven compounds representative of AGEs and structurally similar to pentosidine showed no significant cross-reactivity. The correlation coefficient between the concentrations of pentosidine obtained from HPLC and ELISA for the same urine samples was r50.815. The urinary excretion of pentosidine upon overnight fasting was lower than that after a meal, suggesting the presence of diurnal variation in urinary pentosidine. In contrast, day-today variation was not observed. These results indicate that the ELISA system has sufficient reliability, accuracy, and precision for measuring urinary pentosidine. Sampling of fasting urine is suitable for minimizing variation. In conclusion, this ELISA system is promising to evaluate the effect of AGE on lifestyle-related diseases.

Pentosidine and carboxymethyl-lysine associate differently with prevalent osteoporotic vertebral fracture and various bone markers

“…Urinary PEN level was determined by an ELISA method 6,18 without the need for sample pre-treatment with heat hydrolysis; this method was preferred over PEN measurement in serum samples, which would have required heat hydrolysis and possible inclusion of artificial error. The assay performance of this ELISA system for urinary PEN was reported previously 18 . The sensitivity of the assay system was ≥ 6.24 pmol/mL, and the intra-and inter-assay variations in human urine samples were less than 5% and 4%, respectively.…”

Urinary pentosidine level is associated with grip strength and gait speed in community-dwelling adults: a cross-sectional study

“…Furthermore, the detection of pentosidine using a reported ELISA kit and an HPLC system requires heat pretreatment, which generates artificial pentosidine, leading to overestimation. A novel pentosidine ELISA system that does not require sample pretreatment for analyzing urine samples has been developed [19]. In this study, the urinary pentosidine level was measured using this new ELISA system.…”

“…An analysis of crossreactivity against seven compounds representative of AGEs and with structures close to pentosidine revealed no significant cross-reactivity. The comparability between the values obtained from high-performance liquid chromatography (HPLC) and ELISA (in the same urine samples) was r = 0.815 [19]. Serum creatinine, HbA1c, eGFR, and IGF-1 levels were measured in August 2016 [13]; serum PTH and 25(OH)D levels were measured in June 2018; urinary pentosidine levels were measured in January 2019.…”

Is the Novel Slot Blot a Useful Method for Quantification of Intracellular Advanced Glycation End-Products?

“…Furthermore, many commercial ELISA kits can be used to analyze major proteins. ELISA can be performed on various samples such as serum [5,[61][62][63][64][65][66][67], plasma [68], urine [69], saliva [5], supernatants [9,62], cell lysates [8,70], and tissue lysates [8]. The target in ELISA is generally one protein, such as interleukin (IL)-1β, IL-6, IL-17, IL-18, or tumor necrosis factor (TNF)-α [61,62,70].…”

“…The type of ELISA measurement of AGEs can be divided into (i) one free type of AGEs (e.g., pentosidine) or one type of AGE-modified protein (e.g., CML-modified human serum albumin (HSA)), and (ii) crude AGE-modified proteins. Interestingly, pentosidine, a free AGE, was measured using a novel competitive ELISA by Kashiwabara et al [69]. They used biotin-conju- Further, LeWinter et al investigated the location of CML in the cardiac tissue of patients with coronary bypass grafting; moreover, they quantified the CML count per µm 2 in the cytoplasm and mitochondria in the cardiac tissue of brain-dead patients [7].…”