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.

Molecular Profiling Reveals a Common Metabolic Signature of Tissue Fibrosis

“…We previously reported a method to measure newly synthesized collagen in mouse liver by using stable isotope labeling and targeted peptide quantitation by liquid chromatography-mass spectrometry (LC-MS). 31 While comparable stable isotope labeling was achieved in Sham and UUO mice, there was a dramatic increase in newly synthesized Collagen 1A1 fragment (GAAGPpGATGFpGAAGR) in UUO kidneys at day 7 following the surgery ( Figure 2 B, left panel), as well as its relative abundance ( Figure 2 B, right panel). The synthesis of collagen remained unchanged in sham-operated kidneys but was accelerated in UUO kidneys, consistent with histological observations of collagen accumulation.…”

“…To maintain a stable enrichment of body water for the 14-day duration of UUO time course, mice were given a bolus of 2 H-labeled water (Thermo Fisher Scientific, #AC166300010) through intraperitoneal injection (10 mL/kg), and then allowed free access to food and 10% 2 H-labeled drinking water. The collagen extract was prepared as described previously 31 . Kidney and/or liver tissue samples were homogenized in ∼20X volume of 0.1 M NaOH (in ddH2O).…”

Stable isotope-based flux studies in nonalcoholic fatty liver disease

“…In addition, because of the slow turnover of collagen (02–0.6%/day) (Decaris, et al, 2015), it is critical to consider an appropriate labeling protocol to achieve “sufficient” product (collagen) labeling for accurate modeling of the data. For instance, it was recently shown that the heterogeneity in hepatic collagen pool may result in erroneous interpretations of turnover results if one does not take into account appropriate sampling time and a curve fitting model (linear vs. exponential) (Zhou, et al, 2015). Although it is always desirable to perform the turnover studies in the shortest possible time, it appears that the heterogeneity of collagen pools may require extra considerations when designing labeling protocols for accurate quantification of collagen turnover.…”

Enhancing Studies of Pharmacodynamic Mechanisms via Measurements of Metabolic Flux: Fundamental Concepts and Guiding Principles for Using Stable Isotope Tracers

“…They rely instead on a surrogate measure of intracellular amino acid labeling as a proxy for the precursor labeling. A main takehome message is that one can obtain different estimates of FSR depending on what analyte is used to represent the precursor labeling; some experimentation may be required to build confidence in one's choice (Zhou et al, 2015).…”