MSACL 2022 Abstract

Teaching an Old Dog New Tricks: Expanding Targeted HDL-proteome Methods to Yield New Insights into Nonalcoholic Fatty Liver Disease Progression Ryan W. Pearce(1), Fernando Bril(2,3), Timothy S. Collier(1), and Michael J McPhaul(4) (1) Quest Cardiometabolic Center of Excellence at Cleveland HeartLab, Cleveland, OH (2) Endocrinology, Diabetes and Metabolism, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL (3) Division of Endocrinology, Diabetes and Metabolism, Birmingham VA Medical Center, Birmingham, AL (4) Quest Diagnostics Nichols Institute, San Juan Capistrano, CA Timothy Collier, PhD (Presenter) Quest Diagnostics

Presenter Bio: Dr. Timothy Collier is Scientific Director of Research & Development for the Quest Cardiometabolic Center of Excellence at Cleveland HeartLab, where his responsibilities include overseeing the identification and development of assays for cardiovascular biomarkers.

INTRODUCTION
A biproduct of the worldwide obesity epidemic is that nonalcoholic fatty liver disease (NAFLD) is now the most common chronic liver condition, ranging from isolated steatosis to more-severe nonalcoholic steatohepatitis (NASH) and NAFLD-related cirrhosis.[1] In addition to liver-related morbidity, NAFLD is associated with other metabolic derangements and increased cardiovascular disease (CVD).[2,3] Because the liver plays a key role in lipoprotein synthesis, the lipoprotein proteome may have utility in assessing the severity of liver disease. We previously developed a method for enrichment of high-density lipoprotein (HDL) particles in human serum that enabled deployment of a multiplexed, quantitative, targeted proteomic method to identify key proteins for estimation of cholesterol efflux capacity and CVD risk.[4,5] In the current study, we applied a similar proteomic approach to assess whether targeted proteins involved in renal and hepatic function could be used to help assess NAFLD severity.

METHODS
Patients (N=185) aged 21 to 70 with no evidence of secondary causes for liver steatosis or use of medications that affect intrahepatic triglyceride content were recruited for this study. 45(24%) did not have NAFLD and were used as controls. The remaining 140 patients underwent percutaneous liver biopsy and were divided based on presence or absence of advanced fibrosis (fibrosis stage 3 or 4).
HDL particles from human serum were enriched by affinity to lipid-free, stable isotope labeled, His-tagged Apolipoprotein A-I as previously described [4]. Enriched fractions were subjected to Lys-C digestion for subsequent LC-MS/MS analysis with a 3-stream Agilent 1260 HPLC coupled to an Agilent 6495 QqQ mass spectrometer using StreamSelect. A multiple-reaction monitoring method was designed to target 28 proteins associated with lipid transport and metabolism in addition to hepatic and renal function.
Comparisons between two groups were performed by t-test or Kruskal-Wallis for continuous measures (depending on distribution) and by chi-squared or Fisher’s exact test for categorical measures. Comparisons among >2 groups were performed by one-way ANOVA. Differences in HDL-bound proteins were adjusted for multiple comparisons, controlling for a false discovery rate of 0.10 by the Benjamini-Hochberg method (p<0.017 was considered statistically significant).

RESULTS
Of 28 proteins quantified, 5 were significantly different between groups with and without advanced fibrosis after adjustment for multiple testing. ApoC-I (p=0.001), ApoC-IV (p=010), ApoM (p=0.015), LCAT (p=0.014), and SAA4 (p=0.015) were significantly reduced in advanced fibrosis patients, even after adjustment for presence of diabetes.
Differences were observed in levels of these 5 proteins with respect to disease progression. While ApoC-I and ApoC-IV demonstrated a stepwise reduction with progression of fibrosis from moderate (stage 2) to advanced (stages 3 and 4), ApoM, LCAT, and SAA4 demonstrated abrupt reduction in advanced fibrosis, but with no apparent reduction in moderate fibrosis.
When comparing patients with and without NAFLD, two proteins (ApoA-I [p=0.001] and PON3 [p<0.001]) were significantly reduced in patients with NAFLD after adjusting for multiple comparisons and for the presence of diabetes. Notably, the same 2 proteins were also independently associated for the presence of diabetes (p<0.001 for both). These results are in-line with previous studies in patients with chronic liver disease resulting from NAFLD and other etiologies, suggesting a mechanistic link between fibrosis and lipoprotein synthesis impairment, and a direct relationship between liver disease and CVD risk.

References
1. Godinez-Leiva, E. et al Curr Hypertens Rev (2021) 17, 94-111.

2. Bril, F. et al J Clin Endocrinol Metab (2016) 101, 644-652.

3. Targher G. et al J Hepatol (2016) 65, 589-600.

4. Collier T. et al J Proteome Res (2018) 17, 1183-93.

5. Jin, Z. et al Clin Chem (2019) 65, 282-290.