Stable Isotope Assays for
De Novo Lipogenesis and Lipid Kinetics

Sodium [1-¹³C]acetate (10 g/day) is continuously infused after an overnight fast for 10 hours to establish hepatic ¹³C-acetyl CoA pools. To stimulate lipogenesis, subjects receive a high-fructose liquid diet during the infusion. Fractional DNL is calculated using Mass Isotopomer Distribution Analysis (MIDA), which applies combinatorial probability mathematics to tracer incorporation patterns.

Labeling with deuterated water is a more convenient and less costly alternative for measuring de novo lipogenesis. The basis of the technique is that deuterium oxide rapidly equilibrates with total body water. The deuterium atom labels acetyl-CoA through various enzymatic reactions in the Krebs cycle.

Gas chromatography pyrolysis isotope ratio mass spectrometry (GCP-IRMS) is used to measure very low levels of deuterium in plasma or tissue lipids. The GCP-IRMS analysis involves the separation of derivatized fatty acids and cholesterol by gas chromatography. The lipid peaks are then combusted to hydrogen gas at 1450°C, which is analyzed by isotope ratio mass spectrometry.

We can detect enrichment levels of 0.001% of deuterium with accuracy and precision. The fractional de novo lipogenesis rate is calculated from the average enrichment of deuterium in fatty acids or cholesterol.

A stable isotope labeled fatty acid, typically ¹³C-palmitate, is continuously infused intravenously in tracer amounts. The rate of appearance of endogenous unlabeled fatty acids into the bloodstream can be determined by calculating the dilution of infused isotopes.

Upon reaching steady-state, the rate of appearance equals the rate of disappearance or uptake. Therefore, the rate of appearance is equal to the flux or turnover rate of the substrate.

The rate of appearance of glycerol is a direct index of lipolysis.
Measurement of glycerol appearance is useful since fatty acid flux underestimates the rate of lipolysis because of reesterification. Fatty acids can become reesterified within adipocytes, which prevent release of fatty acids into the bloodstream despite active lipolysis.

However, Glycerol cannot be reincorporated because adipocytes lack glycerol kinase, making it a reliable marker of lipolysis. Continuous infusion with a primed dose of D₅-glycerol rapidly achieves steady state, allowing calculation of glycerol appearance as a direct index of lipolysis.

Fatty acid oxidation can be measured two ways.
The rate of fatty acid oxidation can be estimated by infusing a ¹³C-fatty acid and measuring the rate of excretion of expired ¹³CO₂ in the breath. The procedure requires a steady-state level of ¹³C-fatty acid in the bloodstream and in expired ¹³C-labeled carbon dioxide. Using priming doses of ¹³C-sodium bicarbonate before the continuous infusion of tracers will allow isotopic equilibrium by 60 minutes.

Fat metabolism can also be traced with deuterium-labeled fatty acids. Votruba et al. have validated a method using deuterated palmitate to measure dietary fat oxidation. The method involves administration of 20 mg/kg D₃₁-palmitate in a meal. As palmitate is oxidized, each deuterium atom is lost to water. Urine or plasma can be sampled to measure the labeling in total body water.
The advantage of the deuterium label method is that no recovery factor is needed. Westerterp et al. has verified the results of Votruba and found a mean dietary fat oxidation of 16 ± 6%. This compares similarly to other published studies.
Westerterp found that dietary fat oxidation was negatively correlated with body mass index. The obese subjects had lower fat oxidation while the lean subjects had higher fat oxidation. It was hypothesized that dietary fat oxidation may play a role in human obesity.

The futile cycle occurs when fatty acids released via lipolysis are re-esterified within adipocytes. This cycle allows rapid adjustment of fatty acid availability to meet energy demands.
Simultaneous isotopic infusions of ¹³C-palmitate and D₅-glycerol quantify both lipolysis and re-esterification, providing an index of futile cycling.