Development of a Sandwich Chemiluminescence Immunoassay for the Detection of Intact Procollagen Type I N Propeptide with Magnetic Nanosphere Carrier Technology

The metabolic product of type I collagen synthesis, intact procollagen type I N propeptide (intact PINP), is a potential marker of bone formation and osteoporosis, which is not affected by kidney function. We sought to establish a chemiluminescent immunoassay method for the detection of serum intact PINP with previously prepared paired monoclonal antibodies and to evaluate the diagnostic value of the assay in osteoporosis. Using the capture molecule and monoclonal antibody as detection molecule, a diagnostic reagent was developed to detect intact PINP in serum with magnetic nanosphere carriers by the chemiluminescence method, and its analytical performance in the laboratory was evaluated. Serum intact PINP was measured in 142 healthy people and 115 osteoporosis patients. Results were matched with results of a similar test kit, Roche total PINP Elecsys Chemiluminescent Immunoassay Assay. Compared with the performance of the Roche PINP assay product, our method had higher sensitivity (0.02 ng/mL), wider linear range (0.02-1500 ng/mL), and anti-interference. Serum intact PINP values in osteoporosis patients were significantly higher than in healthy subjects (p < 0.001). Our method had good consistency compared with the Roche PINP assay (r = 0.9794). This chemiluminescence method for detecting serum intact PINP (CLIA-intact PINP) with magnetic nanosphere carrier technology meets the requirements of a clinical testing reagent and is expected to have clinical application after further evaluation and can compete with expensive imported kits on the market.

Serum markers of fibrosis, cardiovascular and all-cause mortality in hemodialysis patients: the AURORA trial

Background Biomarkers of fibrosis are associated with outcome in several cardiovascular diseases. However, their relevance to chronic kidney disease and dialysis is uncertain, as it remains unclear how the kidneys and the dialysis procedure itself affect their elimination and degradation. We aimed to investigate the relationship of the blood levels of two markers associated with fibrosis: procollagen type I C-terminal pro-peptide (PICP) and galectin-3 (Gal-3) with mortality in dialysis patients. Methods Procollagen type I C-terminal pro-peptide and galectin-3 were measured at baseline in 2773 patients enrolled in the AURORA trial, investigating the effect of rosuvastatin on cardiovascular outcomes, in patients on hemodialysis, and their interaction with CV death or all-cause mortality using survival models. The added prognostic value of these biomarkers was assessed by the net reclassification improvement (NRI). Results The median follow-up period was 3.8 years. Blood concentrations of PICP and Gal-3 were significantly associated with CV death [adjusted HR per 1 SD = 1.11 (1.02–1.20) and SD = 1.20 (1.10–1.31), respectively] and all-cause mortality (all adjusted p < 0.001). PICP and Gal-3 had a synergistic effect with regard to CV death and all-cause mortality (interaction p = 0.04 and 0.01, respectively). Adding PICP, Gal-3 and their interaction on top of clinical and biological covariates, resulted in significantly improved prognostic accuracy NRI = 0.080 (0.019–0.143) for CV death. Conclusion In dialysis patients, concomitant increase in PICP and Gal-3 concentrations are associated with higher rates of CV death. These results suggest that concomitantly raised PICP and Gal-3 may reflect an activated fibrogenesis relevant to risk stratification in dialysis, raising the hypothesis that anti-fibrotic therapy may be beneficial for cardiovascular protection in such patients. Graphic abstract

EXPRESS: Assessing bone formation in patients with chronic kidney disease using procollagen type I N-terminal propeptide (PINP): the choice of assay makes a difference

Background: Measurement of procollagen type I N-terminal propeptide (PINP) concentration in serum reflects the rate of type I collagen synthesis and can therefore be used as a bone formation marker. There are two methods of PINP quantification; the first measures the trimeric propeptide (intact PINP) and the second measures both the trimeric and monomeric propeptides (total PINP). Trimeric PINP is excreted via hepatic endothelial cells whereas monomeric PINP is cleared renally. Therefore in renal failure the total assay has a positive bias with respect to the intact assay, due to monomeric PINP accumulation. The aim of this study was to compare the performance of both assays across all stages of chronic kidney disease (CKD). Methods: Serum was taken from male (n=111) and female (n=105) patients attending a metabolic bone clinic and these were partitioned into stages of CKD 1-5. Each serum sample was analysed using the Roche electrochemiluminescence immunoassay for total PINP and the Immunodiagnostic Systems chemiluminescence immunoassay for intact PINP. Results: Passing-Bablok regression analysis comparing both methods showed that with advancing CKD there was a proportional positive bias affecting the total assay when compared to the intact assay. This proportional positive bias was statistically significant for CKD stages 3b, 4 and 5. Conclusions: Based on this method comparison study, usage of the total PINP assay should be avoided in CKD stages 3b, 4 & 5 (eGFR <u><</u>44 ml/min/1.73m²) and instead an intact assay used as the total assay overestimates PINP levels due to monomeric PINP accumulation.

Molecular and Histological Evidence Detailing Clinically Observed Skin Improvement Following Cryolipolysis

Background In addition to body contouring, there is anecdotal and supportive clinical evidence of reduced laxity and skin tightening after cryolipolysis. 10,11 Objectives The nature by which cryolipolysis triggers dermal changes has not been established. This study investigated fundamental mechanisms behind clinically observed dermal changes using molecular and immunohistochemistry methods. Methods This feasibility study involved n=7 subjects that received cryolipolysis treatment. Tissue samples were harvested from 3 days to 5 weeks after treatment. RNA-Sequencing examined differential gene expression of major collagens. RNA In Situ Hybridization (RNA-ISH) investigated the distribution of one of the gene markers for collagen Type I (COL1A1). Immunohistochemistry for Procollagen Type I, heat shock protein 47 (HSP47), transforming growth factor beta (TGF-β and Tropoelastin was performed and quantified. Results Gene expression analysis highlighted a gradual upregulation of collagen mRNA genes. RNA-ISH confirmed upregulation of COL1A1 mRNA and showed a homogenous distribution through the dermis. Immunohistochemistry showed increases in protein expression. Quantification revealed 3.62-fold increase of Procollagen Type I (p&lt;0.0071) and 2.91-fold increase of TGF-β (p&lt;0.041); 1.54-fold increase of HSP47 (p&lt;0.007); and 1.57-fold increase of Tropoelastin (p&lt;0.39) compared to untreated areas. Conclusions This study revealed significant induction of molecular and protein markers of Type I collagen, which supports neocollagenesis and may play an essential role in clinically relevant skin improvement. A dermal remodeling process driven by increased TGF-β and higher expression of HSP47 was observed. Overall, these data provide the first evidence of dermal remodeling and clarify the mechanism by which cryolipolysis may induce skin improvement.

MO734CONTRIBUTION OF SOLUBLE ST2 TO THE EFFECT OF RIGHT VENTRICULAR DYSFUNCTION ON MORTALITY IN HEMODIALYSIS PATIENTS

Background and Aims Right ventricular dysfunction (RVD) has been shown to predict mortality in patients with kidney failure. It has been proposed that RVD in these patients is mediated by inflammatory and fibrotic mechanisms, which may be enhanced by hemodialysis (HD). The present study aimed to investigate the potential associations between RVD and circulating biomarkers of myocardial inflammation and fibrosis with all-cause mortality in HD patients. Method We performed a retrospective single-centre cohort study of prevalent patients admitted in a chronic HD program for more than 3 months. Clinical characteristics and echocardiographic parameters were assessed in all patients. Pre-dialysis blood samples for measurement of inflammatory (e.g., C reactive protein, interleukin-1, interleukin-18) and fibrotic (e.g., soluble suppression of tumorigenesis-2 [sST2], galectin-3, C-terminal pro-peptide of procollagen type I and N-terminal pro-peptide of procollagen type III) biomarkers were collected. Right ventricular dysfunction (RVD) was defined using tricuspid annular plane systolic excursion (TAPSE) &lt;1.7 cm or pulsed Doppler peak annular velocity (S´) &lt;9.5 cm/s. The ability of sST2 to discriminate between mortality was assessed using AuROC curve. Results We enrolled 48 patients (mean age 74 [64–79] years, 62.5% males) followed over a period of 1.4 years. Mortality was higher 45.5% (log-rank, p=0.003) in patients with RVD as diagnosed by S' than in patients without RVD. No difference in mortality was observed for RVD defined by TAPSE. There were no differences in the morphology and function parameters of the left ventricle between patients with and without RVD. From all biomarkers measured only sST2 was associated with RVD. Indeed, an age- and sex-adjusted analyses showed that doubling of sST2 was inversely associated with a decreased in S´ (estimate=-2.03, 95% CI [-3.04 to -1.00] cm/s; P=0.002). Mortality was increased in patients with sST2 ≥40.45 ng/mL compared to patients with sST2 &lt;40.45 ng/mL (66.7% vs. 18.9%, log-rank; p=0.004). Crude analysis showed that patients presenting with S’ &lt;9.5 cm/s and sST2 ≥40.45 ng/mL exhibited higher mortality (log-rank; p=0.001) than patients with S’ &gt;9.5 cm/s and sST2 &lt;40.45pg/mL. Conclusion Albeit preliminary these findings suggest that an excess of sST2 may be involved in RVD and on its effect on mortality in HD patients. The myocardial pro-remodeling effect of sST2 among HD patients with RVD warrants further investigation.

Could procollagen type I N-terminal propeptide (PINP) and bone alkaline phosphatase (B-ALP) be valid alternative diagnostic markers to dual X-ray absorptiometry (DEXA) in elderly females with osteoporosis? An Egyptian radiological and laboratory monocentric study

Background Osteoporosis is a major health problem of elders. Dual X-ray absorptiometry (DEXA) is the commonly used modality for diagnosis osteoporosis; serum markers have been suggested for predicting osteoporosis and discriminate osteoporotic from healthy subjects. We aimed to analyze the status of some bone turnover biochemical markers namely PINP, B-ALP, estrogen, and progesterone in the elderly osteoporotic and osteopenic women as probable markers for the discrimination between patients and healthy individual in diagnosing osteoporosis, and also, to detect the impact of osteoporosis on quality of life of patients using assessment of the quality of life for osteoporosis (ECOS-16). Post-menopausal 108 females were involved in the current study, divided into two groups (osteoporotic group (60 with BMD˂-2.5), osteopenic group (48 with BMD between − 1 and − 2.5)), and 60 healthy elderly females as control group were involved in the study. Serum levels of procollagen type I N-terminal propeptide (PINP), bone alkaline phosphatase (B-ALP), estrogen, and progesterone were measured by ELISA technique. Results PINP and B-ALP significantly differ between studied groups. Also, PINP and B-ALP levels had high sensitivity and specificity to discriminate osteoporotic patients from healthy individuals. PINP and B-ALP significantly correlated with bone mineral density (BMD) and with ECOS-16. Estrogen differs significantly between osteoporotic and osteopenic groups and significantly correlated with bone mineral density of femur (BMD-F) and bone mineral density of spine (BMD-S) in the osteopenic group. Progesterone differed significantly between patients and controls and significantly correlated with BMD-F in the osteoporotic group. Conclusion We can consider PINP and B-ALP as biomarkers for early detection then monitoring of osteoporosis. Measuring these serum markers can replace the assessment of BMD if not available. Also, it could replace the gap between BMD subsequently spaced assessment or could be of value in cases with severe spondylosis, DISH syndrome, old spondylarthritis, and/or previous spinal surgery. Sex hormones could not differentiate the normal from the osteoporotic/osteopenic patients, so they cannot be used as diagnostic or prognostic markers. Validation of this assumption needs large and longitudinal studies.