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. 1989 Dec 15;264(3):829–835. doi: 10.1042/bj2640829

Non-invasive detection of protein metabolism in vivo by n.m.r. spectroscopy. Application of a novel 19F-containing residualizing label.

A Daugherty 1, N N Becker 1, L A Scherrer 1, B E Sobel 1, J J Ackerman 1, J W Baynes 1, S R Thorpe 1
PMCID: PMC1133660  PMID: 2482736

Abstract

Protein residualizing labels facilitate localization of tissue sites of protein catabolism and the quantification of protein accumulation because of their prolonged intracellular retention of protein accumulation because of their prolonged intracellular retention times. Radioiodinated residualizing labels have been used to define the metabolism of a wide variety of proteins, but this has necessitated destructive analysis. Here we describe the implementation and validation of a novel 19F-containing residualizing label for protein, NN-dilactitol-3,5-bis(trifluoromethyl)benzylamine (DLBA), that permits the non-invasive assessment of protein accumulation and catabolism by n.m.r. spectroscopy in vivo. DLBA comprises a reporter molecule containing six equivalent 19F atoms. 19F is strongly n.m.r.-active, has 100% natural abundance, and is present in minimal background concentrations in soft tissues. We validated the use of DLBA as a protein-labelling compound by coupling to asialofetuin (ASF), a protein that is recognized exclusively by hepatic tissue via a saturable receptor-mediated process. Coupling of DLBA to ASF by reductive amination had no effect on the physiological receptor-mediated uptake of the protein in rat liver in vivo. The 19F-n.m.r. spectrum of DLBA exhibited a single peak that was subject to a small chemical-shift change and broadening after coupling to ASF. Pronase digestion of DLBA-ASF was performed to simulate intracellular degradation products, and resulted in a narrower set of resonances, with chemical shifts intermediate between those of uncoupled DLBA and DLBA-ASF. Intravenous administration of DLBA-ASF to rats followed by quantification of 19F in homogenates of liver tissue indicated that the half-life of residence time of degradation products from DLBA-ASF in liver was approx. 2 days. This intracellular half-life was comparable with that described for similar residualizing labels that contain radioiodide as a reporter. Similar results for the half-life of retention were obtained non-destructively and non-invasively in situ with the use of a whole-body radio-frequency antenna to acquire sequential spectra over 80 h after intravenous administration of DLBA-ASF. Quantification of these spectra demonstrated an initial accumulation of DLBA-ASF in liver followed by an expected gradual loss of 19F-labelled degradation products. The approach developed offers promise for the sequential and longitudinal characterization of metabolism of specific proteins in individual experimental animals and ultimately in human subjects.

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Selected References

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  1. Ackerman J. J., Ewy C. S., Becker N. N., Shalwitz R. A. Deuterium nuclear magnetic resonance measurements of blood flow and tissue perfusion employing 2H2O as a freely diffusible tracer. Proc Natl Acad Sci U S A. 1987 Jun;84(12):4099–4102. doi: 10.1073/pnas.84.12.4099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Arnold W., Kalden J. R., von Mayersback H. Influence of different histologic preparation methods on preservation of tissue antigens in the immunofluorescent antibody technique. Ann N Y Acad Sci. 1975 Jun 30;254:27–34. doi: 10.1111/j.1749-6632.1975.tb29153.x. [DOI] [PubMed] [Google Scholar]
  3. Ashwell G., Harford J. Carbohydrate-specific receptors of the liver. Annu Rev Biochem. 1982;51:531–554. doi: 10.1146/annurev.bi.51.070182.002531. [DOI] [PubMed] [Google Scholar]
  4. Baynes J. W., Maxwell J. L., Rahman K. M., Thorpe S. R. Purification of residualizing glycoconjugate labels for protein by reversed-phase high-pressure liquid chromatography. Anal Biochem. 1988 May 1;170(2):382–386. doi: 10.1016/0003-2697(88)90647-1. [DOI] [PubMed] [Google Scholar]
  5. Bretthorst G. L., Kotyk J. J., Ackerman J. J. 31P NMR Bayesian spectral analysis of rat brain in vivo. Magn Reson Med. 1989 Feb;9(2):282–287. doi: 10.1002/mrm.1910090214. [DOI] [PubMed] [Google Scholar]
  6. DULBECCO R., VOGT M. Plaque formation and isolation of pure lines with poliomyelitis viruses. J Exp Med. 1954 Feb;99(2):167–182. doi: 10.1084/jem.99.2.167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Daugherty A., Thorpe S. R., Lange L. G., Sobel B. E., Schonfeld G. Loci of catabolism of beta-very low density lipoprotein in vivo delineated with a residualizing label, 125I-dilactitol tyramine. J Biol Chem. 1985 Nov 25;260(27):14564–14570. [PubMed] [Google Scholar]
  8. Maxwell J. L., Baynes J. W., Thorpe S. R. Inulin-125I-tyramine, an improved residualizing label for studies on sites of catabolism of circulating proteins. J Biol Chem. 1988 Oct 5;263(28):14122–14127. [PubMed] [Google Scholar]
  9. McFeeters R. F. A manual method for reducing sugar determinations with 2,2'-bicinchoninate reagent. Anal Biochem. 1980 Apr;103(2):302–306. doi: 10.1016/0003-2697(80)90614-4. [DOI] [PubMed] [Google Scholar]
  10. Moldoveanu Z., Epps J. M., Thorpe S. R., Mestecky J. The sites of catabolism of murine monomeric IgA. J Immunol. 1988 Jul 1;141(1):208–213. [PubMed] [Google Scholar]
  11. Pittman R. C., Carew T. E., Glass C. K., Green S. R., Taylor C. A., Jr, Attie A. D. A radioiodinated, intracellularly trapped ligand for determining the sites of plasma protein degradation in vivo. Biochem J. 1983 Jun 15;212(3):791–800. doi: 10.1042/bj2120791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Pittman R. C., Taylor C. A., Jr Methods for assessment of tissue sites of lipoprotein degradation. Methods Enzymol. 1986;129:612–628. doi: 10.1016/0076-6879(86)29094-1. [DOI] [PubMed] [Google Scholar]
  13. Strobel J. L., Baynes J. W., Thorpe S. R. 125I-glycoconjugate labels for identifying sites of protein catabolism in vivo: effect of structure and chemistry of coupling to protein on label entrapment in cells after protein degradation. Arch Biochem Biophys. 1985 Aug 1;240(2):635–645. doi: 10.1016/0003-9861(85)90071-2. [DOI] [PubMed] [Google Scholar]
  14. Strobel J. L., Cady S. G., Borg T. K., Terracio L., Baynes J. W., Thorpe S. R. Identification of fibroblasts as a major site of albumin catabolism in peripheral tissues. J Biol Chem. 1986 Jun 15;261(17):7989–7994. [PubMed] [Google Scholar]
  15. Yedgar S., Carew T. E., Pittman R. C., Beltz W. F., Steinberg D. Tissue sites of catabolism of albumin in rabbits. Am J Physiol. 1983 Jan;244(1):E101–E107. doi: 10.1152/ajpendo.1983.244.1.E101. [DOI] [PubMed] [Google Scholar]

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