title: Tracer-based estimates of protein flux in cases of incomplete product renewal: evidence and implications of heterogeneity in collagen turnover.
abstract: The synthesis of various molecules can be estimated by measuring the incorporation of a labeled precursor into a product of interest. Unfortunately, a central problem in many studies has been an inability to estimate the intracellular dilution of the precursor and therein correctly calculate the synthesis of the product; it is generally assumed that measuring the true product labeling is straightforward. We initiated a study to examine liver collagen synthesis and identified an apparent problem with assumptions regarding measurements of the product labeling. Since it is well known that collagen production is relatively slow, we relied on the use of [(2)H]H2O labeling (analogous to a primed infusion) and sampled animals over the course of 16 days. Although the water labeling (the precursor) remained stable and we observed the incorporation of labeled amino acids into collagen, the asymptotic protein labeling was considerably lower than what would be expected based on the precursor labeling. Although this observation is not necessarily surprising (i.e., one might expect that a substantial fraction of the collagen pool would appear "inert" or turn over at a very slow rate), its implications are of interest in certain areas. Herein, we discuss a novel situation in which tracers are used to quantify rates of flux under conditions where a product may not undergo complete replacement. We demonstrate how heterogeneity in the product pool can lead one to the wrong conclusions regarding estimates of flux, and we outline an approach that may help to minimize errors surrounding data interpretation.
authors.affiliation: Cardiometabolic Disease, Merck Research Laboratories, Kenilworth, New Jersey;
authors.forename: Haihong
authors.initials: H
authors.lastname: Zhou
authors.affiliation: Cardiometabolic Disease, Merck Research Laboratories, Kenilworth, New Jersey;
authors.forename: Sheng-Ping
authors.initials: SP
authors.lastname: Wang
authors.affiliation: Cardiometabolic Disease, Merck Research Laboratories, Kenilworth, New Jersey;
authors.forename: Kithsiri
authors.initials: K
authors.lastname: Herath
authors.affiliation: Department of Gastroenterology and Hepatology, Cleveland Clinic, Cleveland, Ohio; and.
authors.forename: Takhar
authors.initials: T
authors.lastname: Kasumov
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas.
authors.forename: Rovshan G
authors.initials: RG
authors.lastname: Sadygov
authors.affiliation: Cardiometabolic Disease, Merck Research Laboratories, Kenilworth, New Jersey; stephen_previs@merck.com.
authors.forename: Stephen F
authors.initials: SF
authors.lastname: Previs
authors.affiliation: Cardiometabolic Disease, Merck Research Laboratories, Kenilworth, New Jersey;
authors.forename: David E
authors.initials: DE
authors.lastname: Kelley
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D003094:Collagen / Q000378:metabolism*; D003903:Deuterium / Q000493:pharmacokinetics*; D006801:Humans; D007202:Indicators and Reagents / Q000493:pharmacokinetics; D007553:Isotope Labeling / Q000379:methods*; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism; D021381:Protein Transport; D014867:Water / Q000378:metabolism
chemical_list: D000596:Amino Acids; D007202:Indicators and Reagents; D011498:Protein Precursors; D014867:Water; D003094:Collagen; D003903:Deuterium
keywords: collagen; kinetics; mass spectrometry; protein turnover; stable isotopes