Traditionally, cardiovascular disease (CVD) risk factors have been determined and measured in the fasting state. However in recent years there has been an increasing awareness of the importance of elevated postprandial lipemia as an independent risk for CVD.1 Indeed, the efficiency with which the body manages incoming dietary macronutrients such as lipid can modulate disease risk in chronic conditions such as obesity2, type 2 diabetes,3,4 and non-alcoholic fatty liver disease.5 Interest in the postprandial state has led to the development of products and technologies designed to quantitate the kinetics of dietary fat (triacylglycerols, TG) absorption, assimilation, and disposal. Development of isotope methodology has expanded the investigation of human lipid metabolism by allowing for a multitude of applications for measurement of in vivo lipid kinetics. Although radioactive isotopes have been used to elucidate seminal findings in the area of fat metabolism,6 the present review will focus on the use of stablely-labeled compounds in human dietary research.
The most obvious use of dietary lipid tracers is for investigation of lipid absorption after a test meal. The labeled fat can be observed as lipids, fatty acids (FA) and TG, in plasma, chylomicrons (CM), and very low-density lipoprotein (VLDL) particles. Dietary TG absorption reaches its maximum between 3-4 hours after the meal (Figure 1). In studies of normal physiology, isotopically-labeled dietary FA can be used to determine if absorption depends on fat quality, such as structure of the FA7,8 or TG,9 and how absorbed FA are partitioned into lipid fractions other than TG.10,11 Alternatively, tracers can be used to determine if absorption is affected by diseases or stage of life. Such applications have been used in studies of obesity,12,13 diabetes,4 malnourishment,14 and aging.15 In addition, because stable isotopes (and stable isotopically-labeled compounds) are naturally-occurring, these compounds are safe for use in many populations, including infants,16 children,14 pregnant or lactating women,7 and the elderly.15
The use of dietary tracers lends itself to a wide variety of analytical applications beyond the measurement of absorption. Labeled precursors, such as the essential FA linoleic and linolenic acid, have allowed the determination of FA elongation and desaturation into longer-chain products.17,18 Combining tracer technologies with other compartment analyses can further expand the analytical possibilities of dietary tracers. For example, tissue biopsies are used to assess tissue uptake of dietary fat into muscle, adipose, or liver.2,19,20 Lipid oxidation can be determined by measuring 13CO2 in breath or D2O in urine.8,21-23 Therefore, with the many applications possible, investigators can create and define new avenues of exploration in postprandial lipid metabolism.24
Figure 1. Healthy males (n=6, mean ± SEM) consumed two meals (0700 and 1200) of identical composition (32% fat, 54% carbohydrate, 14% protein), with each meal representing 1/3 of daily energy needs. D31-tripalmitin (1.6g) was mixed with the liquid formula containing 40 g of dietary fat/meal as described previously.26, 30 Lipoprotein TG-palmitate was analyzed by GC/MS. The d31-palmitate percentages (tracer/tracee) in the two lipoprotein fractions were compared to the TG enrichment in the liquid meal to calculate the percentage of lipoprotein-TG derived from the meal (Parks & Timlin unpublished data).
Common measurement techniques for analysis of metabolized labeled compounds are gas chromatography coupled with either mass spectrometry (MS) or isotope ratio mass spectrometry (IRMS). Choice of analytical technique will depend on availability, cost, and sensitivity required for the experiment, and will influence the dose of tracer to be used. For example, the higher sensitivity of IRMS allows for detection at lower levels of tracer enrichment; therefore smaller doses of tracer can be administered.
As shown in Table 1, the choices of dietary tracers can include FA fed in the free form (e.g. Na+-palmitate) or as a TG (glyceryl trihexadecanoate-D31, or tripalmitin-D31). The degree of isotope labeling will depend on the desired application, and cost, as some compounds are time-consuming to synthesize. Tracers can be combined using different delivery methods in order to investigate multiple pathways of lipid metabolism. Using the multiple stable isotope approach, Donnelly et al. utilized IV infusion of K+-13C4-palmitate on albumin to measure non-esterified FA metabolism, and a meal containing D31-tripalmitin to concurrently measure dietary FA metabolism to determine the lipid contributors to VLDL-TG.5 If multiple tracers are to be used, either different isotopic atoms should be chosen for each compound (e.g. Donnelly (2005) used a carbon-labeled FA and a deuterium-labeled TG) or it must be ensured that each tracer differs by enough mass to enable accurate and separate measurement of the products of interest. For example, in order to investigate effects of sham feeding and multiple meals, Chavez-Jaurequi et al. incorporated 13C2-triolein into the evening meal and 13C7-triolein into the breakfast meal.13 Alternatively, different FA containing the same isotopic label (e.g. 13C-oleate and 13C-palmitate) can be used;25 however there is some debate as to whether this approach is appropriate, because differences in absorption8,11 or oxidation8,23 between different FA could influence results and interpretation.
Dose of the tracer will depend on level of enrichment desired, metabolite of interest, and dietary fat content (Table 1A). The tracer-to-tracee enrichment, or rather the comparison of amount of tracer FA fed to the amount of unlabeled dietary FA fed, can be as high as 10-20%.5,26 We have chosen a high level of enrichment in meals to be able to assess the early absorption of the label. The ratio of the tracer (e.g. 100-800 mg) to the amount of total dietary fat (35-40 g) may be much lower (<3%) depending how much dietary fat is fed.25,27 The greater the amount fed in the meal, the more accurate assessment at earlier postprandial timepoints.
Capsules have been used previously to deliver tracer lipids,8,13 but it has been reported that the recovery of 13C in breath is 10 times higher when the tracer is fed in the meal compared to capsule form.23 This presents a problem for incorporation into a food if the tracer is a FA bound to a salt such as sodium or potassium; this is because the FA salt has physical properties as a soap. Therefore, if the FA salt is solubilized in water, it must be handled gently or it will create foam and result in a food product more difficult to ingest as well as absorb.28 The incorporation of labeled FA into food (Table 1B) for administration has typically been achieved by emulsifying the FA into a sugar-protein complex, vegetable oil, or butter, and placed into food or a “milkshake” type beverage for consumption.10,11,29 The vehicle for the tracer often must be heated or sonicated in order to efficiently incorporate the tracer. Barrows et al. used a homogenizer readily available in food processing labs (Model 110Y microfluidizer, Microfluidics, Newton, MA).30 Triglyceride tracers are more easily incorporated into test vehicles if the tracer is an oil (such as triolein), whereas tripalmitin is a crystal which must be heated for complete solubilization.
Rate of Delivery and Meal Consumption
Early fat tolerance tests performed without isotopes were designed to provide a dose that would challenge the body’s lipid handling systems.31 Later, postprandial studies were designed to better mimic normal patterns of eating by including carbohydrate and protein with the fat,32,33 and feeding solid foods with the addition of isotopes to meals. Investigators have used both liquid26 and solid5,22 meals (Table 1B). Liquid meals have been provided as either continuous feedings via periodically sipping a beverage34 or by duodenal administration30 (Table 1B). However, feeding the meal as a typical bolus is preferred because it produces greater label incorporation and greater fat oxidation8 compared to continuous feeding.30 The exception is studies in hospitalized infants, where set meal times are not practical.35 The choice of the amount of dietary fat fed in the test meal will depend on the increase in postprandial plasma lipids desired. Increasing the amount of dietary fat leads to higher postprandial plasma TG concentrations,36 which dilutes the appearance of the label but can also amplify plasma lipid concentrations to a level sufficient for effective analysis.
Many investigators have used only one meal containing the tracer to measure postprandial metabolism. However, a delay in absorption has been demonstrated in the appearance of dietary tracer in the CM and VLDL TG pools13,25,26,37 (Figure 1). The delay can occur from 4h to 13h after the first labeled meal. This so-called “Second Meal Effect” is required to see maximum appearance of the dietary tracer in plasma lipid compartments,13,25 such that the consumption of a subsequent labeled meal is associated with more label appearance in blood (Figure 1). After two meals, 100% of the CM-TG pool contains the same enrichment as the meal, leading to all of the TG in CM particles being derived from the tracer-containing meal. This second meal does not need to contain fat, as a simple glucose drink has been shown to induce a CM lipid response.38 The Second Meal Effect also indicates that the food consumed the evening before the test influences postprandial metabolism.39 The pre-test meal composition should therefore be controlled by providing standardized meals at least the evening before, if not for 1-3 days leading up to the testing day.30 If using a 13C-labeled tracer, some investigators also instruct subjects to avoid foods naturally rich in 13C, such as corn products and cane sugar.4 Other lifestyle aspects to take into consideration include asking participants to avoid strenuous exercise and alcohol 2-3 days before the testing period.4,30
With the growing interest in postprandial metabolism and wide variety of applications, stable isotopes are an invaluable tool for investigating human metabolism. A large body of literature utilizing isotopes to measure the absorption and disposal of dietary fat exists. Standardized protocols have been published to aid researchers interested in studying the impact of dietary fat using stable isotopes. Given the role of fat intake on the development of obesity and its related disorders, stable isotope and mass spectrometry methods can provide powerful tools for further characterization of normal and dysregulated physiological processes. Such investigations will help to unravel how postprandial metabolism contributes to disease pathology and create novel clinical tools for screening and treatment. Examination of postprandial lipid metabolism in obesity may pave the way for development of pharmaceutical or nutritional interventions for beneficial modification of postprandial lipemia to reduce disease risk.