Development of a Highly Sensitive, High-Throughput, Mass Spectrometry-Based Assay for Rat Procollagen Type-I N-Terminal Propeptide (PINP) To Measure Bone Formation Activity

2007 ◽  
Vol 6 (11) ◽  
pp. 4218-4229 ◽  
Author(s):  
Bomie Han ◽  
Marci Copeland ◽  
Andrew G. Geiser ◽  
Laura V. Hale ◽  
Anita Harvey ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Bone Formation ◽  
High Throughput ◽  
Type I ◽  
Procollagen Type ◽  
Highly Sensitive ◽  
Procollagen Type I

scholarly journals Comparison of an Enzyme Immunoassay versus Mass Spectrometry-based Assay for the Quantitative Determination of the Procollagen Type I N-terminal Propeptide in Rat Serum

2009 ◽  
Vol 2 ◽  
pp. PRI.S3454 ◽  
Author(s):  
Monika Dzieciatkowska ◽  
Marci Copeland ◽  
Jinsam You ◽  
Jean-Pierre Wery ◽  
Mu Wang
Keyword(s):  
Mass Spectrometry ◽  
Enzyme Immunoassay ◽  
Dynamic Range ◽  
High Specificity ◽  
Specific Protein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Type I ◽  
Rat Serum ◽  
Procollagen Type ◽  
Procollagen Type I

Traditionally, antibody-based assays, such as enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA), are the primary tool for the targeted quantification of a specific protein. An antibody-based assay can be run at high-throughput and has extraordinary sensitivity and specificity. In the cases where antibody-based assays exist, the process of validating biomarker candidates can be relatively straightforward. However, the antibody-based approach is limited by the lack of availability of antibodies with high specificity. The development of a high quality antibody-based assays can be costly, time-consuming and a resource-intensive effort. Another disadvantage of antibody-based assays is that they often do not discriminate closely related isoforms. While the antibody development is central to the success of antibody-based platform, mass spectrometry (MS) provides alternative and complementary approach to existing antibody-based assays. The MS-based assays are becoming very popular for quantitative candidates proteins detection in a complex biological mixture. In the present paper, an in-house developed mass spectrometry (MS)-based assay was compared to a commercially available EIA in reproducibility, measurement accuracy, and dynamic range using rat procollagen type-I N-terminal propeptide (P1NP) as a model.

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

2021 ◽  
pp. 000456322110255
Author(s):  
Andreas Tridimas ◽  
Anna Milan ◽  
Eileen Marks
Keyword(s):  
Chronic Kidney Disease ◽  
Kidney Disease ◽  
Bone Formation ◽  
Type I Collagen ◽  
Method Comparison ◽  
Type I ◽  
Positive Bias ◽  
Procollagen Type ◽  
Metabolic Bone ◽  
Procollagen Type I

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/1.73m<sup>2</sup>) and instead an intact assay used as the total assay overestimates PINP levels due to monomeric PINP accumulation.

Abstract 74: The Inflammatory Phenotype Of Mesenchymal Fibroblasts And Its Role In Aging Dependent Cardiac Fibrosis- A Target For Statins?

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Katarzyna A Cieslik ◽  
JoAnn Trial ◽  
Mark L Entman
Keyword(s):  
Endothelial Cells ◽  
Myeloid Cell ◽  
Transendothelial Migration ◽  
Mouse Heart ◽  
Type I ◽  
Aged Mouse ◽  
Vitro Assay ◽  
Procollagen Type ◽  
Procollagen Type I ◽  
Inflammatory Phenotype

In the aging mouse (C57BL/6) myocardium fibrosis steadily increases after 14 months of age and is accompanied by elevated numbers of myeloid derived fibroblasts. Recently, we proposed a mechanism by which inflammatory mesenchymal fibroblasts (IMF) derived from mesenchymal stem cells secrete monocyte chemoattractant protein-1 (MCP-1) necessary for myeloid fibroblast induction in the aging heart. The current study extends the characterization of this inflammatory phenotype by describing elevated interleukin-6 (IL-6) secretion and increased expression of IL-6 receptor (IL-6R) in IMF. Since IL-6R lacks an intracellular domain it requires a co-receptor gp130 (generally expressed) to induce an intracellular signal. Thus, generation of an IL-6R soluble receptor allows IL-6 signaling on cells that do not express IL-6R (or expression is low), such as endothelial cells. We investigate the function of IL-6 and IL-6R in the promotion of transendothelial migration of monocytes through cardiac endothelium and their maturation into myeloid fibroblasts in in vitro assay. Treatments with IL-6 and more extensively IL-6+IL-6R resulted in a 3-5 fold increase (above the control level) in myeloid cell migration and maturation into myeloid fibroblasts. Thus IMF can contribute both IL-6 and IL-6R to endothelial cells and facilitate myeloid cell transendothelial migration. In agreement with these data, analysis of the aged mouse heart revealed the presence of fibroblasts expressing IL-6 (procollagen type I + IL-6 + cells), M1 macrophages (CD86 + cells) and M2 macrophages (CD301 + procollagen type I + cells) that were absent in hearts from young mice. The mechanisms by which expression of these factors is upregulated in IMF are being investigated; our data suggest that MCP-1 and IL-6 expression are controlled by the farnesyltransferase (FTase)-Ras-Erk1/2 pathway. Interestingly, since atorvastatin interferes with farnesyl synthesis it also reduced MCP-1 and IL-6 expression in IMF. These data may introduce a new use of this class of drugs in the prevention of the age-related fibrosis.

Effects of fluoride on human bone cells in vitro: differences in responsiveness between stromal osteoblast precursors and mature osteoblasts

1994 ◽  
Vol 130 (4) ◽  
pp. 381-386 ◽  
Author(s):  
Moustapha Kassem ◽  
Leif Mosekilde ◽  
Erik F Eriksen
Keyword(s):  
Bone Marrow ◽  
Alkaline Phosphatase ◽  
Sodium Fluoride ◽  
Bone Cells ◽  
Human Bone ◽  
Type I ◽  
Procollagen Type ◽  
Procollagen Type I ◽  
Osteoblast Precursors

Kassem M, Mosekilde L, Eriksen EF. Effects of fluoride on human bone cells in vitro: differences in responsiveness between stromal osteoblast precursors and mature osteoblasts. Eur J Endocrinol 1994;130:381–6. ISSN 0804–4643 The cellular effects of sodium fluoride (NaF) on human bone cells in vitro have been variable and dependent on the culture system used. Variability could be attributed to differences in responsiveness to NaF among different populations of cells at various stages of differentiation in the osteoblastic lineage. In this study we compared the effects of NaF in serum-free medium on cultures of more differentiated human osteoblast-like (hOB) cells derived from trabecular bone explants and on osteoblast committed precursors derived from human bone marrow, i.e. human marrow stromal osteoblast-like (hMS(OB)) cells. Sodium fluoride (10−5 mol/l) increased proliferation of hMS(OB) cells (p<0.05, N = 10) but was not mitogenic to hOB cells (p>0.05, N= 10). Alkaline phosphatase (AP) production increased in both hMS(OB) (p<0.05, N=9) and hOB cells (p<0.05, N=9). No significant effects on procollagen type I propeptide production were obtained in either culture. In the presence of 1,25-dihydroxycholecalciferol (10−9 mol/l), NaF enhanced alkaline phosphatase (p<0.05, N=8), procollagen type I propeptide (p<0.05, N=7) and osteocalcin (p<0.05, N=7) production by hMS(OB) cells but not by hOB cells. Our results suggest that osteoblast precursors are more sensitive to NaF action than mature osteoblasts and that the in vivo effects of NaF on bone formation may be mediated by stimulating proliferation and differentiation of committed osteoblast precursors in bone marrow. M Kassem, Mayo Clinic, Endocrine Research Unit, W-Joseph 5-164, Rochester, MN 55904, USA

Elevated Procollagen Type I Carboxyterminal Propeptide Production in Cultured Scleroderma Fibroblasts

Dermatology ◽  
1995 ◽  
Vol 190 (2) ◽  
pp. 104-108 ◽  
Author(s):  
K. Kikuchi ◽  
T. Kadono ◽  
M. Fujimoto ◽  
H. Ihn ◽  
S. Sato ◽  
...  
Keyword(s):  
Type I ◽  
Procollagen Type ◽  
Procollagen Type I

Abstract 1957: The Carboxyterminal Telopeptide of Procollagen type I and Procollagen type III Aminoterminal Peptide (PIIINP) are Strong Predictors of Cardiovascular Morbidity and Mortality in the Elderly; The Cardiovascular Health Study

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Eddy Barasch ◽  
John S Gottdiener
Keyword(s):  
Prognostic Value ◽  
Cardiovascular Health ◽  
Health Study ◽  
Cardiovascular Health Study ◽  
Type I ◽  
Elderly Individuals ◽  
Procollagen Type ◽  
Procollagen Type I ◽  
Cv Disease ◽  
Carboxyterminal Telopeptide

Background: The fibrillar myocardial extracellular matrix is mainly composed of type I and III fibrillar collagen and their turnover are reflected in the serum level of carboxyl-terminal propeptide type I (PIP) and procollagen type III aminoterminal peptide (PIIINP). The prognostic value of these biomarkers in elderly individuals with heart failure (HF) or other cardiovascular disease (CVD) and in healthy subjects is largely unknown. Aim: To determine the predictive value of PIP, its degradation metabolite, carboxyterminal telopeptide of procollagen type I (CTIP), and PIIINP serum level for the incident CV morbidity and mortality in a nested case control study of community-dwelling elderly individuals enrolled in the Cardiovascular Health Study (CHS). Methods: In 880 participants (ppts) enrolled in the CHS (mean age 77 ± 6 yrs, 52 % males, 79 % white), 310 with HF, 287 controls (no HF but other CVD) and 283 healthy ppts, serum levels of PIIINP, PIP and CTIP were measured by radioimmunoassay. The number of incident CV disease and death were recorded. Wilcoxon rank sum test, Kruskal-Wallis test, and Cox proportional hazards regression were used as appropriate. Results: Age, gender and race and fully adjusted analyses are presented in the table : Conclusions: In this large elderly cohort, there is a strong association between CTIP, PIIINP and incident CVD and death. Elevated CTIP level increases the risk of death more than 50% and of symptomatic PVD by almost two-fold. Whereas PIIINP has a lower predictive power than CTIP, PIP was not associated with incident CVD or death. Serum CTIP and PIIINP have a good prognostic value for both incident CVD and death in elderly individuals with or without known CV disease.

Procollagen type I gene expression and cell proliferation are increased in lipodermatosclerosis

2005 ◽  
Vol 152 (2) ◽  
pp. 242-249 ◽  
Author(s):  
A.M. deGiorgio-Miller ◽  
L.J. Treharne ◽  
R.J. McAnulty ◽  
P.D. Coleridge Smith ◽  
G.J. Laurent ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Type I ◽  
Procollagen Type ◽  
Procollagen Type I ◽  
I Gene

R432 – Gene Expression of the Remodeling Rat Vocal Fold Wound

2008 ◽  
Vol 139 (2_suppl) ◽  
pp. P189-P189
Author(s):  
Tsunehisa Ohno ◽  
Lesley C. French ◽  
Bernard Rousseau
Keyword(s):  
Gene Expression ◽  
Extracellular Matrix ◽  
Messenger Rna ◽  
Vocal Fold ◽  
Molecular Mechanisms ◽  
Type I ◽  
Post Injury ◽  
Time Points ◽  
Procollagen Type ◽  
Procollagen Type I

Problem The authors investigated the expression of key extracellular matrix genes after vocal fold wounding in a rat model to better understand the reparative mechanisms of tissue repair during the remodeling phase of vocal fold injury. Methods Bilateral vocal fold wounds were created in 30 rats. Injured vocal fold specimens were harvested 1, 3, 7, 14, 28, and 56 days after wounding. 5 unwounded rats were used to establish baseline for polymerase chain reaction (PCR). The authors used real-time PCR to quantify messenger RNA expression of procollagen type I, III, interleukin-1 beta (IL-1 beta), decorin, and hyaluronan synthase (HAS) −1, −2, and −3. Analysis of variance was used to detect main effects for gene expression. Post-hoc tests were used to make comparisons between time points. Results Procollagen type I expression was decreased from baseline on post-injury day 1, 28, and 56. Procollagen type III was decreased on post-injury day 1 and 56, and increased from baseline on post-injury day 14. IL-1 beta expression was increased from baseline on post-injury day 1, 3, and 7. Decorin expression was decreased from baseline on post-injury day 1, 3, 7, and 56. HAS-1 expression was decreased from baseline at all post-injury time points. HAS-2 expression was increased from baseline on post-injury day 3, and decreased from baseline on post-injury day 14, 28, and 56. HAS-3 expression was decreased from baseline on post-injury day 1, 28, and 56. Conclusion Findings provide temporal changes in the expression of key extracellular matrix genes during a remodeling phase of vocal fold injury in a rat wound model. Significance Vocal fold wound models provide a means for investigating tissue reparative processes and molecular mechanisms controlling synthesis and degradation of the vocal fold extracellular matrix. Support Vanderbilt University Medical Center.

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