De Novo Lipogenesis

Metabolic Solutions helps researchers study de novo lipogenesis and lipid kinetics using carbon-13 and deuterium stable isotope methods. Dysregulation of de novo lipogenesis and lipid pathways is associated with obesity, diabetes, and non-alcoholic liver and cardiovascular diseases. De novo lipogenesis is a complex regulated process in which circulating carbohydrates are converted to fatty acids. Lipid synthesis from these biochemical processes is thought to contribute to the pathogenesis of non-alcoholic fatty liver disease, metabolic syndrome with consequent insulin residence.

We offer de novo lipogenesis analytical services to measure incorporation of 13C-acetate (Mass Isotopomer Distrubution Analysis (MIDA)) or deuterium labeling into plasma or hepatic triglycerides.

We also offer lipid kinetic services to determine fatty acid turnover rates. The process of triglyceride breakdown or lipolysis results in the release of fatty acids and glycerol. Fatty acids can serve as energy substrates while glycerol can act as a gluconeogenic precursor. Isotopic tracers (1-13C-palmitate and D5-glycerol) can be used to quantify the rate of appearance of fatty acids and glycerol into the blood stream.

List of Lipid Analytical Services

  • Rates of De Novo Lipogenesis
  • Rates of lipolysis
  • Fatty acid turnover
  • Fatty acid oxidation
  • Glycerol kinetics

De Novo Lipogenesis (13C-acetate Tracer)

Sodium [1-13C]acetate (10g/day) is continuously infused after an overnight fast for 10 hours to establish hepatic stores of 13C-acetyl CoA for monitoring fractional de novo lipogenesis. To increase de novo lipogenesis, subjects are given a high fructose liquid diet for the 10 hours. De novo lipogenesis is calculated with a mathematical technique called mass isotopomers distribution analysis (MIDA) based on the mathematics of combinatorial probabilities.

De Novo Lipogenesis (Deuterium oxide Tracer)

Labeling with deuterated water is 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.

Deuterium labeling studies are easy to conduct. Only a single oral dose of deuterium oxide (1 gram/kg BW) is required. Blood samples are collected for lipid enrichment prior to dosing and after 4-24 hours. The precursor enrichment of total body water is easily measured with plasma, saliva or urine.

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 separates 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.

Fatty Acid Turnover

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 isotope. 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.

Glycerol Turnover

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 into triglycerides because glycerol kinase is absent within adipocytes.

A stable isotope tracer of glycerol (typically, D5-glycerol) is continuously infused. A priming dose of tracer is used to achieve steady-state levels quickly.

Rates of Fatty Acid Futile Cycle

Lypolysis and subsequent reesterification of released free fatty acid represent a futile cycle. This futile cycle allows the adipocyte to rapidly adjust free fatty acid levels in meeting energy demands. Simultaneous isotopic infusions of labeled fatty acid, example 1-13C-palmitate and D5-glycerol, provides an index of the relative rate of fatty acid reesterification.

Fatty Acid Oxidation

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.

Applications

The following examples offer protocols useful for studying various aspects of lipid kinetics.

ApplicationReference
Fatty Acid OxidationImpaired fatty acid oxidation in type 2 diabetics
Fatty Acid OxidationIncrease in fat oxidation on high fat diet
Fatty Acid TurnoverAcute IL-6 treatment increases fatty acid turnover
Fatty Acid TurnoverEndurance training increases fatty acid turnover
Glycerol TurnoverGlycerol turnover with growth hormone receptor deficiency

If you need protocol information on how to conduct lipid kinetic tracer studies, the following technical paper is available: Fatty acid metabolism measured with stable isotope tracers.

Published Lipid Kinetic Studies Analyzed By Metabolic Solutions

1. Verbruggen SC, Coss-BU J, Wu M, Schierbeek H, Joosten KF, Dhar A, van Goudoever JB, and Castillo L. Current recommended parenteral protein intakes do not support protein synthesis in critically ill septic, insulin-resistant adolescents with tight glucose control. Crit Care Med. 39(11):2518-25, 2011.

“Conducted a primed, constant, 7-hr stable isotope tracer infusion with [1,1,2,3,3-2H5]glycerol in combination with a hyperinsulinemic euglycemic clamp.”

2. Van Pelt RE, Gozansky WS, Hickner RC, Schwartz RS, and Kohrt WM. Acute modulation of adipose tissue lipolysis by intravenous estrogens. Obesity 14(12):2163-72, 2006.

“Assessed whole-body lipolysis by 2H5-glycerol rate of appearance and abdominal and femoral lipolysis by subcutaneous microdialysis.”