BRIEF REPORT Clinical Infectious Diseases 542 • CID 2019:69 (1 August) • BRIEF REPORT Received 6 September 2018; editorial decision 28 November 2018; accepted 22 December 2018; published online December 24, 2018. Correspondence: R. Loomba, NAFLD Research Center; Division of Gastroenterology and Epidemiology, University of California, San Diego, 9500 Gilman Drive, MC 0887, La Jolla, CA 92093 (roloomba@ucsd.edu). Clinical Infectious Diseases ® 2019;69(3):542–5 © The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com. DOI: 10.1093/cid/ciy1100 Discovery of Half-life of Circulating Hepatitis B Surface Antigen in Patients With Chronic Hepatitis B Infection Using Heavy Water Labeling Rohit Loomba, 1 Martin Decaris, 2 Kelvin W. Li, 2,3 Mahalakshmi Shankaran, 3 Hussein Mohammed, 2,3 Marcy Matthews, 2,3 Lisa M. Richards, 1 Phirum Nguyen, 1 Emily Rizo, 1 Barbara Andrews, 1 Robin Soto, 1 Thomas Angel, 3 Vithika Suri, 4 Kathryn M. Kitrinos, 5 Dwight Barnes, 6 Gregg Czerwieniec, 6 Kathy Brendza, 6 G. Mani Subramanian, 4 Anuj Gaggar, 4 and Marc K. Hellerstein 2,3 1 NAFLD Research Center, University of California, San Diego, La Jolla, 2 KineMed, Inc., Emeryville, 3 Department of Nutritional Sciences and Toxicology, University of California Berkeley, 4 Clinical Research, 5 Biomarkers, and 6 Biology, Gilead Sciences, Inc., Foster City, California. In a pilot study, heavy water labeling was used to determine hepatitis B surface antigen (HBsAg) turnover rates in chronic hepatitis B (CHB) patients. Te mean (standard deviation) half-life of HBsAg in blood was 6.7 (5.5) days, which refects recent production in the liver and supports strategies aimed at reducing HBsAg production in CHB patients. Keywords. HBV; deuterated water; hepatitis B surface an- tigen; half-life. Te hepatitis B virus (HBV) releases a large quantity of viral proteins into the infected patient’s circulation. Te most clin- ically important of these subviral particles is the hepatitis B surface antigen (HBsAg), as its elimination from circulation is regarded as functional cure of chronic HBV [1]. HBsAg is composed of 3 glycosylated proteins: small, middle, and large proteins. Circulating levels of HBsAg can be used to monitor the natural history of the disease and have recently been shown to be predictive of response to treatment [2]. Currently approved an- tiviral agents are highly efective in suppressing viral replication and reducing liver fbrosis but have minimal efect in reducing the levels of HBsAg and other plasma proteins [3]. In recent years, researchers have increasingly targeted elimination of HBsAg as an endpoint in clinical trials. However, the production rate and the elimination rate of HBsAg have not been defnitively quantifed. Importantly, the question of whether the persistence of blood HBsAg refects ongoing production by the liver or very slow clearance of the protein or a subpopulation of protein molecules from blood remains unknown and has implications for therapeutic attempts to eliminate HBsAg. We conducted a small pilot study to determine the half-life of HBsAg, with the aim of increasing our understanding of disease biology and aiding the development of future therapeutic strategies. Heavy water ( 2 H 2 O) labeling is based on the incorporation of tracer levels of deuterium ( 2 H) into covalent C-H bonds of amino acids, which occurs only in newly synthesized proteins, allowing for sensitive measurements of in vivo protein synthesis [4–6]. Te distribution of 2 H label in body water is rapid and to all tissues following oral ingestion of 2 H 2 O and persists in the body with a half-life of 7–10 days [6]. Elevated 2 H levels in body water label the intracellular pool of amino acids in a predictable manner [5, 6], thereby labeling newly synthesized proteins. Using tandem mass spectrometric analysis of peptides from proteins afer trypsin hydrolysis, the fraction of newly synthesized proteins in the pool can be quantifed and protein synthesis rates can be calculated by kinetic modeling [6]. Tis method has several important advantages over traditional la- beling methods, including high sensitivity of tandem mass spectrometry for proteins, the defnitive identifcation and pu- rity of peptides assigned to parent proteins, and the fact that heavy water can be administered safely at low doses over days, weeks, or months, an appropriate timeframe for the measure- ment of proteins spanning a wide range of turnover rates. Te half-lives (t 1/2 ) of large numbers of human serum proteins as well as individual proteins in blood have been determined using this methodology [7–12]. Here, we describe the utilization of heavy water labeling with tandem mass spectrometric anal- ysis to measure the turnover of circulating HBsAg in patients infected with HBV. METHODS Deuterated water (70%) was administered to all patients orally over 21 days according to the following regimen: 50 mL 3 times a day for 4 days (doses taken 3 or more hours apart) followed by 50 mL twice a day for the remaining 17 days, in order to maintain enrichment of 1.0%–2.0% 2 H in body water. Plasma and saliva samples were drawn at 6 timepoints: baseline and days 3, 7, 10, 14, and 21 for determination of body water en- richment (from saliva samples) with immunoprecipitation of HBsAg from plasma for mass spectrometric analysis of isotopic labeling changes over time. Briefy, HBsAg was immunoprecipitated using the HBsAg antibody (GS-645730) with 30 μg/mg of epoxy-coated mag- netic beads and then separated by molecular weight via sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Gel bands containing the 3 HBsAg subtypes (small [20–25 kD], medium Downloaded from https://academic.oup.com/cid/article/69/3/542/5259172 by guest on 11 June 2022