Lipid Metabolism

Metabolic Solutions offers project design assistance and a mass spectrometry service to help researchers study lipid metabolism using stable isotope methods. Stored triglycerides in the body can be mobilized from fat cells. 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 (palmitate, glycerol) can be used to quantify the rate of appearance of fatty acids and glycerol into the blood stream.

New Research Methods in Lipid Metabolism

The obesity epidemic has focused much research attention to fat metabolism studies. Fat metabolism can be traced with isotope-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. Palmitate oxidation is often measured with ¹³C- or ¹⁴C-palmitate to labeled CO₂ but the calculations of oxidation require an acetate recovery factor.

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

List of Lipid Metabolism Services

• Fatty acid oxidation
• Glycerol kinetics to detect fatty acid triglyceride cycling
• Rates of lipogenesis
• Fatty acid turnover

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. Fatty acid flux can underestimate the rate of lipolysis except under fasting conditions because of reesterification. Fatty acids can become reesterified within adipocytes which prevents 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. The stable isotope approach is advantageous compared to radioactive tracer methods because gas chromatography-mass spectrometry (GC/MS) methods measure isotopic glycerol directly. Specific activity of glycerol is difficult to measure because glycerol must be isolated from glucose before counting the radioactivity. GC/MS methods can also be used to accurately measure blood concentrations of glycerol in the same analysis.

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 and glycerol tracers will provide an index of the relative rate of fatty acid reesterification. Three fatty acids are released per glycerol molecule released. If triglycerides are hydrolyzed within adipocytes and subsequently fatty acids are reesterified and do not enter the blood stream, then this results in intracellular recycling. The intracellular recycling will be equal to 3 times the flux of glycerol minus the free fatty acid flux. Recycling can also occur when free fatty acids are released into the bloodstream and eventually reesterified. This would be classified as extracellular recycling. Extracellular recycling is calculated as the free fatty acid flux minus the total fat oxidation. Therefore, total recycling equals 3 times the glycerol flux minus total fat oxidation.

Fatty Acid Oxidation

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 the obtainment of a steady-state level of ¹³C-fatty acid in the bloodstream and in expired ¹³C-labeled carbon dioxide. Using priming doses ¹³C-sodium bicarbonate before the continuous infusion of tracers will allow isotopic equilibrium by 60 minutes.

If you need protocol information on how to conduct fatty acid isotope tracer studies, the following technical paper is available: Fatty Acid Metabolism measured with Stable Isotope Tracers

Amino Acid Metabolism and Protein Turnover

Metabolic Solutions offers project design assistance and a mass spectrometry service to help researchers to study amino acid metabolism and protein turnover using stable isotope methods.

Amino Acid Metabolism

The quantification of various aspects of specific amino acid metabolism in human subjects is achieved with stable isotope tracers. Initially, protein and amino acid nutrition was based on the nitrogen (N) balance technique. However, it is widely accepted that a major portion of body protein is continuously degraded and resynthesized, but remains in N balance. Stable isotope techniques can dissect the relative contributions of protein synthesis and degradation in relation to body N balance. Furthermore, stable labeled amino acid tracers can be utilized to determine how amino acid metabolism is affected by various nutritional and non-nutritional factors.

Applications

List of Amino Acid Metabolism Services

• Metabolic fate of dietary amino acids
• Amino acid kinetics for requirement studies
• Oxidation
• Transamination
• Extracellular intermediates

Download our technical paper on conducting Amino Acid Isotope Tracer Studies.

Protein Turnover

Tracer methods using isotopically-labeled compounds are ideal to explore dynamic aspects of protein metabolism in the whole organism. The infusion of a tracer is coupled with the measurement of end products of nitrogen metabolism (Picou and Taylor-Roberts Model) or with the determination of the enrichment of isotope (¹⁵N or ¹³C) in plasma following administration of a labeled amino acid (Waterlow Model). When this latter approach is combined with measurements of ¹³C in expired carbon dioxide or ¹⁵N in urinary urea or ammonia, the components of whole body amino acid flux and whole body protein synthesis and catabolism can be determined.

New Techniques of Protein Turnover

Metabolic Solutions can determine protein turnover using a deuterium oxide labeling method. Oral doses of deuterium oxide are administered to subjects. The alpha-hydrogen of alanine of protein is labeled during metabolism with alanine transaminase. Previs et al. (Am J Physiol 286:E665-E672, 2004) have demonstrated that deuterium oxide administration can be used to quantitate protein synthetic rates in humans.

List of Protein Turnover Services

• Whole body protein synthesis/breakdown assessment
• Flooding dose turnover techniques
• Lipoprotein synthesis and kinetics
• Muscle protein synthesis

Download our technical paper on conducting Protein Turnover Isotope Tracer Studies.