Abstract 333: Stable Isotopic Metabolic Labeling with Heavy Water ( ² H ₂ O) to Assess the Kinetics of LDL apoB100 in NASH Patients

Over 90 million Americans suffer from Nonalcoholic Fatty Liver Disease (NAFLD), characterized by excessive liver fat and chronic inflammation. This may progress to nonalcoholic steatosis hepatitis (NASH), liver cirrhosis and hepatocellular carcinoma. Blood lipid metabolic disorders, dyslipidemia, are common in patients with NAFLD. The majority of tracer kinetic studies of apolipoprotein particle metabolism have required in-patient IV tracer administration. Our objective was to develop a simple, safe and clinically useful approach for outpatient assessment of the turnover of apolipoprotein B (ApoB) 100 in NASH, from a single blood draw during 2H2O intake. Very low-density lipoproteins (VLDL) and low-density lipoproteins (LDL) were isolated from plasma for analysis because these are the primary particles that transport triglycerides and cholesterol, respectively, in circulation. The main structural protein, apoB100, was analyzed for label incorporation by mass spectrometry because there is one ApoB100 molecule per particle and its dynamics represent the behavior of the intact particle. We took advantage of an existing clinical study that was designed for assessing de novo lipogenesis in NASH patients. Subjects had been labeled for three days with heavy water prior to a fasting blood draw. Lipoproteins were separated from plasma using ultracentrifugation and native-PAGE. Average plasma triglycerides, apoB, LDL-apoB levels were 208.1 +/- 17.4, 131.8 +/- 8.4, and 78 +/- 6.2 (mg/dl, average +/- SE), respectively. In NASH patients, VLDL-apoB displayed a fractional synthesis value near or at 100%, indicating that this pool was completely replaced after three days. In contrast, LDL-apoB fractional replacement rate (n=50 subjects) was 0.32 +/- 0.03 pools/day and the absolute synthesis rate was 25.1 +/- 2.3 mg/dl/day. In healthy subjects (n=8), the average fractional replacement rate and plasma triglycerides were 0.37 +/- 0.02 pools/day and 93.9 +/- 22.3 mg/dl, respectively. In conclusion, our results show that LDL- apoB kinetics can be measured by use of 2 H 2 O labeling and tandem mass spectrometric analyses in NASH patients. This approach allows exploration of metabolic mechanisms underlying dyslipidemia in this increasingly important condition.