Labeled breath tests in patients with NASH: Octanoate oxidation relates best to measures of glucose metabolism

**Background:** Non-alcoholic fatty liver disease (NAFLD) and its advanced form non-alcoholic steatohepatitis (NASH) are highly prevalent liver conditions that currently require invasive liver biopsy for diagnosis and monitoring. Mitochondrial dysfunction is believed to play a key role in NASH progression, but validated non-invasive methods to assess hepatic mitochondrial function in vivo are lacking. Carbon-labeled breath tests using substrates like ¹³C-octanoate, a medium-chain fatty acid that bypasses the carnitine transport system and is oxidized directly in liver mitochondria, have shown conflicting results in prior NAFLD studies. No previous investigations had compared octanoate oxidation (OctOx) with measures of liver glucose metabolism in biopsy-proven NASH, nor evaluated how OctOx changes with lifestyle intervention.

**Methods:** Twenty-five subjects (9 men, 16 women; mean age 47±10 years; mean weight 113±23 kg) with biopsy-confirmed NASH (NAFLD activity score ≥4/8) were recruited. All subjects were sedentary (<60 min/week structured physical activity), aged 22–65 years, consumed <20 g alcohol/day, and had metabolic syndrome characteristics. Within 3.0±1.8 months of diagnostic liver biopsy, subjects underwent a controlled 3-day pre-study diet followed by a 12-hour fast and an oral ¹³C₄-octanoate breath test (23.4 mg in orange juice). Breath samples were collected over 135 minutes and analyzed by isotope ratio mass spectrometry. Fasting endogenous glucose production (EGP) was measured within 2 weeks using a primed-continuous IV infusion of [U-¹³C₆]-glucose. Twenty-two subjects returned after 10.2±1.0 months for repeat testing (15 received supervised lifestyle intervention with diet and exercise counseling; 7 received standard care with one dietitian visit). Liver histology was scored by a single blinded pathologist using the NAFLD activity score (0–8).

**Key Results:** At baseline, subjects oxidized 23.4±3.9% of the octanoate dose (range: 14.9%–31.5%) over 135 minutes, with peak oxidation occurring at 53±20 minutes. OctOx (% dose) was significantly negatively correlated with fasting plasma glucose (r=−0.474, p=0.017), HbA1c (r=−0.441, p=0.028), and fasting EGP relative to fat-free mass (r=−0.441, p=0.028). No association was found between OctOx and NAFLD activity score, fibrosis, or liver enzymes at baseline. At follow-up, subjects in the lifestyle arm showed significant reductions in NAFLD activity score (p=0.002), inflammation (p=0.003), ballooning (p<0.001), and fibrosis score (p=0.045). Across all subjects, steatosis was significantly reduced (p=0.007) and OctOx (% dose/kg) was significantly increased (p=0.044). The increase in OctOx tended to correlate with reductions in steatosis (r=−0.411, p=0.058) and with reductions in fasting EGP (r=−0.401, p=0.064). Changes in OctOx also tended to correlate with reductions in fasting glucose (r=−0.371, p=0.090) and ALT (r=−0.405, p=0.068).

**Clinical Implications:** This study provides evidence that the ¹³C-octanoate breath test may serve as a non-invasive indicator of hepatic steatosis and glucose metabolism in patients with NASH. The finding that lower OctOx was associated with worse glycemic control (higher fasting glucose, HbA1c, and EGP) suggests that mitochondrial fat oxidation capacity is linked to hepatic insulin resistance. The longitudinal data showing that increased OctOx tended to accompany improvements in steatosis and EGP raises the possibility that this breath test could be used to monitor metabolic improvements following lifestyle interventions without requiring repeated liver biopsies. However, the authors appropriately caution that these relationships require verification in larger studies across a broader range of NAFLD severity. Limitations include the modest sample size, the absence of healthy controls, the use of a single bicarbonate correction factor rather than individual measurements, and the potential confounding effects of portal blood flow and gastric emptying. The non-invasive nature of the test represents a key strength, as current methods for assessing mitochondrial function require invasive liver biopsies.