Energy expenditure, insulin, and VLDL-triglyceride production in humans

Hypertriglyceridemia is considered a cardiovascular risk factor in diabetic and nondiabetic subjects. In this study, we aimed to determine potential regulators of very low density lipoprotein-triglyceride (TG) production. VLDL-TG kinetics were measured in 13 men and 12 women [body mass index [mean (range)]: 24.8 (20.2-35.6) kg/m(2)]. VLDL-TG production was assessed from the plasma decay of a bolus injection of ex vivo labeled VLDL particles ([1-(14)C]triolein-VLDL-TG). Similar VLDL-TG production (micromol/min) was found in men and women. VLDL-TG production was not significantly correlated with palmitate flux ([9,10-(3)H]palmitate) (r = 0.09, P = 0.67) or palmitate concentration (r = -0.29, P = 0.2) but was correlated significantly with fasting insulin concentration (r = 0.46, P < 0.05) and resting energy expenditure (REE) (r = 0.45, P < 0.05). The latter correlation improved when adjusted for sex. The best multivariate model with VLDL-TG production as the dependent variable and REE, body composition, hormones, and substrate levels as independent variables included fasting insulin (P = 0.02) and REE (P = 0.02) (r(2) = 0.32, P < 0.001). We conclude that VLDL kinetics are similar in men and women and that REE and plasma insulin are significant independent predictors of VLDL-TG production. FFA availability and body fat distribution are unrelated to VLDL production. We suggest that REE plays a greater role in VLDL-TG production than previously anticipated. REE and insulin should be taken into account when VLDL-TG production comparisons between groups are made.     

Use of stable isotopically labeled tracers to measure very low density lipoprotein-triglyceride turnover

Tracer methods for VLDL-TG kinetics vary in their ability to account for the effect of tracer recycling, which can influence the calculation of VLDL-TG fractional catabolic rates (FCRs). We evaluated a novel approach, involving stable isotopically labeled glycerol or palmitate tracers in conjunction with compartmental modeling, for measuring VLDL-TG kinetics in normolipidemic human subjects. When administered as a bolus simultaneously, both tracers provided identical VLDL-TG FCRs when the data were analyzed by a compartmental model that accounted for hepatic lipid tracer recycling, but not by non-compartmental analysis. The model-derived FCR was greater than that determined using a non-compartmental approach, and was 2- to 3-fold higher than that usually reported by using a bolus of radioactive [3H]glycerol. When palmitate tracer was given as a constant infusion, VLDL-TG turnover appeared 5-fold slower, because tracer recycling through hepatic lipid pools could not be resolved with the infusion protocol. We conclude that accounting for tracer recycling, particularly the contribution of hepatic glycerolipid pools, is essential to accurately measure VLDL-TG kinetics, and that bolus injection of stable isotopically labeled glycerol or palmitate tracers in conjunction with compartmental modeling analysis offers a reliable approach for measuring VLDL-TG kinetics.     

Effects of exercise on VLDL-triglyceride oxidation and turnover

Lipids are important substrates for oxidation at rest and during exercise. Aerobic exercise mediates a delayed onset decrease in total and VLDL-triglyceride (TG) plasma concentration. However, the acute effects of exercise on VLDL-TG oxidation and turnover remain unclear. Here, we studied the acute effects of 90 min of moderate-intensity exercise in healthy women and men. VLDL-TG kinetics were assessed using a primed constant infusion of ex vivo labeled [1-(14)C]triolein VLDL-TG. Fractional VLDL-TG-derived fatty acid oxidation was measured from (14)CO(2) specific activity in expired air. VLDL-TG concentration was unaltered during exercise and early recovery, whereas non-VLDL-TG concentration decreased significantly.VLDL-TG secretion rate decreased significantly during exercise and remained suppressed during recovery. Total VLDL-TG oxidation rate was unaffected by exercise. However, the contribution of VLDL-TG oxidation to total energy expenditure fell from 14 ± 9% at rest to 3 ± 4% during exercise. We conclude that VLDL-TG fatty acids are quantitatively important oxidative substrates under basal postabsorptive conditions but remain unaffected during 90-min moderate-intensity exercise and, thus, become relatively less important during exercise. Lower VLDL secretion rate during exercise may contribute to the decrease in TG concentrations during and after exercise.     

No effect of menstrual cycle phase on basal very-low-density lipoprotein triglyceride and apolipoprotein B-100 kinetics

Dyslipidemia, manifested by increased plasma triglyceride (TG), increased total and LDL-cholesterol concentrations and decreased HDL-cholesterol concentration, is an important risk factor for cardiovascular disease. Premenopausal women have a less atherogenic plasma lipid profile and a lower risk of cardiovascular disease than men, but this female advantage disappears after menopause. This suggests that female sex steroids affect lipoprotein metabolism. The impact of variations in the availability of ovarian hormones during the menstrual cycle on lipoprotein metabolism is not known. We therefore investigated whether very-low-density lipoprotein (VLDL)-TG and VLDL-apolipoprotein B-100 (apoB-100) kinetics are different during the follicular (FP) and luteal phases (LP) of the menstrual cycle. We studied seven healthy, premenopausal women (age 27 +/- 2 yr, BMI 25 +/- 2 kg/m(2)) once during FP and once during LP. We measured VLDL-TG, VLDL-apoB-100, and plasma free fatty acid (FFA) kinetics by using stable isotope-labeled tracers, VLDL subclass profile by nuclear magnetic resonance spectroscopy, whole body fat oxidation by indirect calorimetry, and the plasma concentrations of lipoprotein lipase (LPL) and hepatic lipase (HL) by ELISA. VLDL-TG and VLDL-apoB-100 concentrations in plasma, VLDL-TG and VLDL-apoB-100 secretion rates and mean residence times, VLDL subclass distribution, FFA concentration and rate of appearance in plasma, whole body substrate oxidation, and LPL and HL concentrations in plasma were not different during the FP and the LP. We conclude that VLDL-TG and VLDL-apoB-100 metabolism is not affected by menstrual cycle phase.     

Mechanism of avian estrogen-induced hypertriglyceridemia: evidence for overproduction of triglyceride.

Relying on methods other than the determination of turnover rate of triglyceride from the curve of plasma triglyceride radioactivity after administration of labeled precursor, we have confirmed that the endogenous hypertriglyceridemia induced by estrogenization of the chick is accompanied by increased production of triglyceride. Chicks estrogenized with diethylstilbestrol became grossly hypertriglyceridemic and had elevated levels of plasma free fatty acid. Within 5 min of administration of labeled palmitate, estrogenized hypertriglyceridemic birds converted approximately 10 times more plasma free fatty acid to hepatic triglyceride than did controls. In addition, 2 hr after intraperitoneal injection of [14-C]acetate or [U-14-C]glucose, the specific activity of very low density lipoprotein triglyceride (VLDL-TG) of estrogenized birds reached or exceeded that of the untreated controls, and the rapid enrichment of the vastly expanded plasma VLDL-TG pool with labeled triglyceride further indicated that increased production of triglyceride occurs with estrogenization. Furthermore, [14-C]acetate incorporation into VLDL-TG was calculated to be 1.6 and 6.6% of the injected dose in estrogenized birds compared with 0.1 and 0.2% in untreated birds. Increased production of plasma VLDL-TG was confirmed by a kinetic study of VLDL-TG metabolism, employing reinjected, endogenously prepared [14-C]triglyceride-labeled VLDL. The fractional turnover rate of VLDL-TG in estrogenized hypertriglyceridemic birds was substantially less than that in untreated controls (0.32 plus or minus 0.03 vs 0.71 plus or minus 0.03/hr), but the total turnover rate was nearly 50 times greater (244 plus or minus 52 vs. 5 plus or minus 1 mg/hr).     

VLDL-triglyceride kinetics during hyperglycemia-hyperinsulinemia: effects of sex and obesity

We have previously shown that sex and obesity independently affect basal very low density lipoprotein (VLDL)-triglyceride (TG) kinetics. In the present study, we investigated the effect of hyperglycemia-hyperinsulinemia on VLDL-TG kinetics in lean and obese men and women (n = 6 in each group). VLDL-TG kinetics were measured during basal, postabsorptive conditions and during glucose infusion (5.5 mg x kg FFM(-1) x min(-1)) by using [(2)H(5)]glycerol bolus injection in conjunction with compartmental modeling analysis. Basal VLDL-TG secretion in plasma was greater in obese than in lean men (7.8 +/- 0.6 and 2.9 +/- 0.4 micromol x l plasma(-1) x min(-1); P < 0.001) but was not different in lean and obese women (5.0 +/- 1.1 and 5.9 +/- 1.1 micromol x l plasma(-1) x min(-1)). Glucose infusion decreased the VLDL-TG secretion rate by approximately 50% in lean and obese men and in lean women (to 1.5 +/- 0.4, 4.0 +/- 0.6, and 2.2 +/- 0.4 micromol x l plasma(-1) x min(-1), respectively; all P < 0.05) but had no effect on the VLDL-TG secretion rate in obese women (4.9 +/- 1.0 micromol x l plasma(-1) x min(-1)). These results demonstrate that both sex and adiposity affect the regulation of VLDL-TG metabolism. Glucose and insulin decrease VLDL-TG production in both lean men and lean women; obesity is associated with resistance to the glucose- and insulin-mediated suppression of VLDL-TG secretion in women, but not in men.     

Reproducibility of stable isotope-labeled tracer measures of VLDL-triglyceride and VLDL-apolipoprotein B-100 kinetics

To gain insight into the mechanisms regulating plasma lipid homeostasis, FFA, VLDL-triglyceride (TG), and VLDL-apolipoprotein B-100 (apoB-100) kinetics are commonly assessed using stable isotope-labeled tracer methods. The reproducibility of these measurements, which is critical for the experimental design, is unknown. Therefore, we investigated the repeatability of plasma FFA, VLDL-TG, and VLDL-apoB-100 kinetics in eight healthy men using stable isotope-labeled tracer techniques. There were no systematic differences in plasma FFA, VLDL-TG, and VLDL-apoB-100 concentrations and kinetics between the two studies. Intraindividual day-to-day variability for various outcome variables ranged from 15% to 25%, and almost all of this was of biological origin. The most robust outcome variables were FFA rate of appearance and hepatic VLDL-TG and VLDL-apoB-100 secretion rates; the least robust were VLDL-TG and VLDL-apoB-100 plasma clearance rates and mean residence times. Overall, physiologically meaningful differences in mean values (i.e., 25-30% in magnitude) can be obtained with a sample size of 6-10 subjects for paired studies and 12-20 subjects per group for cross-sectional studies, assuming a type I error rate of 0.05 and a type II error rate of 0.20 (i.e., 80% power). These findings will be useful for future studies investigating FFA, VLDL-TG, and VLDL-apoB-100 kinetics with the methods described.     

Thematic review series: patient-oriented research. Recent advances in liver triacylglycerol and fatty acid metabolism using stable isotope labeling techniques

Isotopic measurement of biosynthetic rates of lipids in VLDL particles has long posed difficult technical problems. In this review, key methodologic issues and recent technical advances are discussed. A common problem for all biosynthetic measurements is the requirement to measure isotopic labeling of the true intracellular biosynthetic precursor pool. Two techniques that address this problem for lipid biosynthesis, and that are applicable to humans, have been developed-the combinatorial probability method (or mass isotopomer distribution analysis) and (2)H(2)O incorporation. The theoretical basis and practical application of these methods, both of which involve mass spectrometry, are described. Issues relevant to specific lipid components of VLDL, such as differences in the labeling of the various particle lipids (phospholipid, cholesterol, etc.), and the contribution of an intrahepatic cytosolic triacylglycerol (TG) storage pool to VLDL-TG are discussed. In summary, advances in stable isotope-mass spectrometric techniques now permit accurate measurement of liver-TG synthesis and flux. In vivo regulation of the synthesis, assembly, and secretion of VLDL-TG in humans is thereby accessible to direct investigation. Patient-oriented research in conditions such as dyslipidemia and hepatic steatosis is made feasible by these scientific advances.     

Metabolism of very low density lipoproteins in genetically lean or fat lines of chicken

Metabolism of very low density lipoproteins (VLDL) has been compared in fat (FL) and lean (LL) lines of chicken. When refed after fasting, plasma triglyceride concentration reached a significantly higher plateau in FL, although their feed consumption was lower than in LL. Newly synthesized VLDL were studied using anti-lipoprotein lipase antibodies. VLDL triglyceride (TG) concentrations were increased by antibody injection and reached a higher concentration in FL plasma than in LL. Newly synthesized VLDL exhibited a similar lipid composition. Fatty acid profiles were also similar when birds ingested a very low fat diet. Comparison of in vitro affinity of lipoprotein lipase and VLDL from both genotypes did not reveal any difference in Km and Vmax. [14C]labelled VLDL from fat or lean donors were prepared and were injected into chickens from both genotypes. Fractional rate constants did not differ between lines. However, as plasma VLDL-TG pools were very different, plasma turnover was higher in FL than in LL. About 3-fold more VLDL-TG were incorporated in abdominal fat of FL than in LL. Difference in fattening between both genotypes seem to be due to both increased VLDL secretion and VLDL removal from plasma without difference in VLDL characteristics.     

Effects of CETP inhibition with anacetrapib on metabolism of VLDL-TG and plasma apolipoproteins C-II,C-III,and E

Cholesteryl ester transfer protein (CETP) mediates the transfer of HDL cholesteryl esters for triglyceride (TG) in VLDL/LDL. CETP inhibition, with anacetrapib, increases HDL-cholesterol, reduces LDL-cholesterol, and lowers TG levels. This study describes the mechanisms responsible for TG lowering by examining the kinetics of VLDL-TG, apoC-II, apoC-III, and apoE. Mildly hypercholesterolemic subjects were randomized to either placebo (N = 10) or atorvastatin 20 mg/qd (N = 29) for 4 weeks (period 1) followed by 8 weeks of anacetrapib, 100 mg/qd (period 2). Following each period, subjects underwent stable isotope metabolic studies to determine the fractional catabolic rates (FCRs) and production rates (PRs) of VLDL-TG and plasma apoC-II, apoC-III, and apoE. Anacetrapib reduced the VLDL-TG pool on a statin background due to an increased VLDL-TG FCR (29%; P = 0.002). Despite an increased VLDL-TG FCR following anacetrapib monotherapy (41%; P = 0.11), the VLDL-TG pool was unchanged due to an increase in the VLDL-TG PR (39%; P = 0.014). apoC-II, apoC-III, and apoE pool sizes increased following anacetrapib; however, the mechanisms responsible for these changes differed by treatment group. Anacetrapib increased the VLDL-TG FCR by enhancing the lipolytic potential of VLDL, which lowered the VLDL-TG pool on atorvastatin background. There was no change in the VLDL-TG pool in subjects treated with anacetrapib monotherapy due to an accompanying increase in the VLDL-TG PR.     

Growth hormone-induced insulin resistance is associated with increased intramyocellular triglyceride content but unaltered VLDL-triglyceride kinetics

The ability of growth hormone (GH) to stimulate lipolysis and cause insulin resistance in skeletal muscle may be causally linked, but the mechanisms remain obscure. We investigated the impact of GH on the turnover of FFA and VLDL-TG, intramuscular triglyceride content (IMTG), and insulin sensitivity (euglycemic clamp) in nine healthy men in a randomized double-blind placebo-controlled crossover study after 8 days treatment with (A) Placebo+Placebo, (B) GH (2 mg daily)+Placebo, and (C) GH (2 mg daily)+Acipimox (250 mgx3 daily). In the basal state, GH (B) increased FFA levels (P<0.05), palmitate turnover (P<0.05), and lipid oxidation (P=0.05), but VLDL-TG kinetics were unaffected. Administration of acipimox (C) suppressed basal lipolysis but did not influence VLDL-TG kinetics. In the basal state, IMTG content increased after GH (B; P=0.03). Insulin resistance was induced by GH irrespective of concomitant acipimox (P<0.001). The turnover of FFA and VLDL-TG was suppressed by hyperinsulinemia during placebo and GH, whereas coadministration of acipimox induced a rebound increase FFA turnover and VLDL-TG clearance. We conclude that these results show that GH-induced insulin resistance is associated with increased IMTG and unaltered VLDL-TG kinetics; we hypothesize that fat oxidation in muscle tissue is an important primary effect of GH and that circulating FFA rather than VLDL-TG constitute the major source for this process; and the role of IMTG in the development of GH-induced insulin resistance merits future research.     

A single 1-h bout of evening exercise increases basal FFA flux without affecting VLDL-triglyceride and VLDL-apolipoprotein B-100 kinetics in untrained lean men

Our group (Magkos F, Wright DC, Patterson BW, Mohammed BS, Mittendorfer B, Am J Physiol Endocrinol Metab 290: E355-E362, 2006) has recently demonstrated that a single, prolonged bout of moderate-intensity cycling (2 h at 60% of peak oxygen consumption) in the evening increases basal whole-body free fatty acid (FFA) flux and fat oxidation, decreases hepatic VLDL-apolipoprotein B-100 (apoB-100) secretion, and enhances removal efficiency of VLDL-triglyceride (TG) from the circulation the following day in untrained, healthy, lean men. In the present study, we investigated the effect of a single, shorter-duration bout of the same exercise (1 h cycling at 60% of peak oxygen consumption) on basal FFA, VLDL-TG, and VLDL-apoB-100 kinetics in seven untrained, healthy, lean men by using stable isotope-labeled tracer techniques. Basal FFA rate of appearance in plasma and plasma FFA concentration were approximately 55% greater (P < 0.05) the morning after exercise than rest, whereas resting metabolic rate and whole-body substrate oxidation rates were not different after rest and exercise. Exercise had no effect on plasma VLDL-TG and VLDL-apoB-100 concentrations, hepatic VLDL-TG and VLDL-apoB-100 secretion rates, and VLDL-TG and VLDL-apoB-100 plasma clearance rates (all P > 0.05). We conclude that in untrained, healthy, lean men 1) the exercise-induced changes in basal whole-body fat oxidation, VLDL-TG, and VLDL-apoB-100 metabolism during the late phase of recovery from exercise are related to the duration of the exercise bout; 2) single sessions of typical recreational activities appear to have little effect on basal, fasting plasma TG homeostasis; and 3) there is a dissociation between systemic FFA availability and VLDL-TG and VLDL-apoB-100 secretion by the liver.     

Dissociation between rate of hepatic lipoprotein secretion and hepatocyte microtubule content

The fact that colchicines inhibits hepatic secretion of very low density lipoprotein (VLDL) particles has been interpreted to mean that microtubules are involved in hepatic VLDL secretion. To further define this relationship, we have attempted to see if changes in hepatic VLDL secretion are associated with changes in hepatocyte microtubule or tubulin content. Accordingly, hepatic secretion of VLDL was increased in rats, and the hepatocyte content of both microtubules (using quantitative morphometric methods) and tubulin (using a time-decay colchicine binding assay) was determined. In acute experiments, VLDL secretion was increased by perfusion of isolated rat livers for 2 h with varying concentrations of free fatty acids (FFA). Results indicate that hepatic VLDL triglyceride (TG) secretion at perfusate FFA levels of 0.7 μEq/ml is threefold greater (P < 0.01) than when livers are perfused without added FFA. However, no differences are observed in the content of microtubules in these livers: specifically, microtubules occupy 0.029 percent of hepatocyte cytoplasm in livers perfused without FFA and 0.030 percent of cytoplasm in livers perfused with FFA. In chronic experiments, rats were fed for 1 wk with either standard rat chow or a hyperlipidemic (sucrose/lard) diet. With the experimental diet, plasma triglyceride levels increase threefold over controls, and liver VLDL-TG production, as determined by [(3)H]glycerol turnover studies, is 55 percent greater (P < 0.01) than controls. However, microtubules occupy 0.027 percent of the cytoplasm of hepatocyte cytoplasm whether rats are on standard or hyperlipidemic diets. Furthermore, the tubulin content of isolated hepatocytes does change, and represents 1 percent of hepatocyte soluble protein, irrespective of diet. These results suggest that increases in hepatic VLDL secretion can occur without any demonstrable change in hepatocyte assembled microtubule or tubulin content, and raise questions as to the role played by microtubules in hepatic VLDL secretion.     

Nonuniform radiolabeling of VLDL apolipoprotein B: implications for the analysis of studies of the kinetics of the metabolism of lipoproteins containing apolipoprotein B

Radiolabeling of whole lipoproteins or individual apolipoproteins has been an essential tool for the determination of the kinetics of apolipoprotein metabolism in vivo. Mathematical analysis of specific radioactivity (SA) or total radioactivity data has demonstrated the existence of significant complexity in the plasma decay curves of several apolipoproteins. Results obtained during development of methods to study the metabolism of apolipoprotein B (apoB) in very low density lipoprotein (VLDL) subclasses isolated according to flotation (Sf) rates from whole radiolabeled (d less than 1.006 g/ml) VLDL suggested nonuniform radiolabeling of apoB in the three Sf subclasses being studied. We therefore determined apoB SA in VLDL Sf subclasses in ten hypertriglyceridemic and five normal subjects. After radioiodination of apoB in whole VLDL, different apoB SA were found in Sf 400-100, Sf 100-60, and Sf 60-20. The pattern of labeling was quite variable among subjects. On average, apoB SA in the VLDL tracer was greatest in Sf 400-100, and least in Sf 60-20. Nonuniform labeling could also be demonstrated in five studies in which samples were obtained 3 min after intravenous injection of the tracer into subjects with a wide range of plasma triglycerides. Nonuniform labeling of apoB in whole VLDL was also demonstrated in two of the subjects by isolating subclasses of their VLDL that did not bind to an anti-apolipoprotein E immunoaffinity column. These results indicate that the usual assumption of homogeneous labeling of apoB may be erroneous. We have derived a simple mathematical formula to study the consequences of this assumption in estimating kinetic parameters. It is shown that an erroneous assumption of homogeneous tracer labeling may significantly underestimate or overestimate the true production rate, even in a simple two-pool model. Identification of labeling characteristics and incorporation of this information into the mathematical analysis of the plasma radioactivity data can improve the accuracy of the analysis as well as the sensitivity of compartmental models generated by such data.     

Body composition determines direct FFA storage pattern in overweight women

Direct FFA storage in adipose tissue is a recently appreciated pathway for postabsorptive lipid storage. We evaluated the effect of body fat distribution on direct FFA storage in women with different obesity phenotypes. Twenty-eight women [10 upper body overweight/obese (UBO; WHR >0.85, BMI >28 kg/m(2)), 11 lower body overweight/obese (LBO; WHR <0.80, BMI >28 kg/m(2)), and 7 lean (BMI <25 kg/m(2))] received an intravenous bolus dose of [9,10-(3)H]palmitate- and [1-(14)C]triolein-labeled VLDL tracer followed by upper body subcutaneous (UBSQ) and lower body subcutaneous (LBSQ) fat biopsies. Regional fat mass was assessed by combining DEXA and CT scanning. We report greater fractional storage of FFA in UBSQ fat in UBO women compared with lean women (P < 0.01). The LBO women had greater storage per 10(6) fat cells in LBSQ adipocytes compared with UBSQ adipocytes (P = 0.04), whereas the other groups had comparable storage in UBSQ and LBSQ adipocytes. Fractional FFA storage was significantly associated with fractional VLDL-TG storage in both UBSQ (P < 0.01) and LBSQ (P = 0.03) adipose tissue. In conclusion, UBO women store a greater proportion of FFA in the UBSQ depot compared with lean women. In addition, LBO women store FFA more efficiently in LBSQ fat cells compared with UBSQ fat cells, which may play a role in development of their LBO phenotype. Finally, direct FFA storage and VLDL-TG fatty acid storage are correlated, indicating they may share a common rate-limiting pathway for fatty acid storage in adipose tissue.     

Turnover of free fatty acids during recovery from exercise.

The turnover of plasma free fatty acid (FFA) was studied during the recovery from exercise with the aid of a continuous infusion of 14C-labeled oleic acid. Arterial FFA reached a maximum of twice the exercise value after 6 min of recovery and was still 75% above the basal level after 20 min. Within 2 min after exercise, plasma radioactivity had increased and the specific activity of plasma oleic acid had fallen. The rate of uptake of FFA from the plasma pool rsoe by 40% during the first minutes after exercise. The rate of release of FFA to the plasma pool showed a peak 2 min after exercise and was thereafter about 40 mumol/min lower than the rate of uptake. The fractional turnover of FFA decreased to resting levels within 5-10 min after exercise. It is concluded that the postexercise peak in arterial FFA is a consequence of augmented release of FFA into the plasma pool above the level during exercise, possibly related to the release of sympathetic vasoconstrictor tone. As a consequence, the rate of removal of FFA rises at the end of exercise and remains augmented above the basal level for as long as the arterial concentration is increased.     

Taurine kinetics assessed using [1,2-13C2]taurine in healthy adult humans

To assess the dynamics of taurine metabolism in vivo, two sets of studies were carried out in healthy volunteers. First, pilot studies were carried in a single human subject to determine the time course of plasma and whole blood isotope enrichment over the course of an 8-h, unprimed continuous infusion of [1,2-(13)C(2)]taurine. Second, five healthy adult males received two tracer infusions on separate days and in randomized order: 1) a 6-h continuous infusion of [1,2-(13)C(2)]taurine (3.1 +/- 0.2 micromol x kg(-1) x h(-1)) and 2) a bolus injection of [(13)C(2)]taurine (3.0 +/- 0.1 micromol/kg). Isotope enrichments in plasma and whole blood taurine were determined by gas chromatography-mass spectrometry. The pilot experiments allowed us to establish that steady-state isotope enrichment was reached in plasma and whole blood by the 5th h of tracer infusion. The plateau enrichment reached in whole blood was lower than that obtained in plasma taurine (P < 0.02). In the second set of studies, the appearance rate (R(a)) of plasma taurine, determined from continuous infusion studies was 31.8 +/- 3.1 micromol x kg(-1) x h(-1). After a bolus injection of tracer, the enrichment decay over the subsequent 2 h was best fitted by a two-exponential curve. Taurine R(a) was approximately 85% higher when determined using the bolus injection technique compared with continuous infusion of tracer. We conclude that 1) taurine R(a) into plasma is very low in healthy postabsorptive humans, and, due to taurine compartmentation between the extra- and intracellular milieus, may represent only interorgan taurine transfer and merely a small fraction of whole body taurine turnover; and 2) the bolus injection technique may overestimate taurine appearance into plasma. Further studies are warranted to determine whether alterations in bile taurine dynamics affect taurine R(a).     

Potentially conflicting metabolic demands of diving and exercise in seals

Metabolic replacement rates (Ra) for glucose and free fatty acids (FFA) were determined during rest, exercise, and diving conditions in the gray seal using bolus injections of radiotracers. In the exercise experiments the seal swam at a metabolic rate elevated twofold over resting Ra for glucose and FFA while resting were similar to values found in terrestrial mammals and other marine mammal species. During exercise periods glucose turnover increased slightly while FFA turnover changes were variable. However, the energetic demands of exercise could not be met by the increase in the replacement rates of glucose or FFA even if both were completely oxidized. Under diving conditions the tracer pool displayed radically different specific activity curves indicative of the changes in perfusion and metabolic rate associated with a strong dive response. Since the radiotracer curves during exercise and diving differed qualitatively and quantitatively, it is possible that similar studies on freely diving animals can be used to assess the role of the diving response during underwater swimming in nature.     

Use of Intralipid for kinetic analysis of HDL apoC-III: evidence for a homogeneous kinetic pool of apoC-III in plasma

Apolipoprotein C-III (apoC-III) is an important regulator of lipoprotein metabolism. Radioisotope and stable isotope kinetic studies show differing results in relation to the kinetics of apoC-III in HDL. Kinetic analysis of HDL apoC-III may be difficult because of its low concentration, as well as the presence of other apoproteins at higher concentration, in the HDL fraction. We used Intralipid(R) (IL), known to preferentially extract apoC proteins from plasma, as a means of extracting apoC-III from HDL before apoprotein separation by isoelectric focusing gel electrophoresis for the measurement of tracer enrichment. Protein purity was assessed by an isoleucine-to-leucine (Ile/Leu) ratio, as apoC-III contains no isoleucine. We compared apoC-III kinetics in 14 men using a bolus infusion of deuterated leucine. The Ile/Leu ratio for IL-extracted HDL (IL-HDL) apoC-III (3.0 +/- 0.7%) was not different from that of VLDL apoC-III (2.6 +/- 0.6%) but was significantly lower than that of untreated HDL apoC-III (9.0 +/- 2.9%) (P < 0.001). The isotopic enrichment curves and fractional catabolic rates (FCRs) for IL-HDL apoC-III were not different from those of VLDL apoC-III. In contrast, HDL apoC-III had significantly lower isotopic enrichments and FCRs than IL-HDL apoC-III (P < 0.001). In conclusion, this simple IL method can be used to isolate apoC-III from HDL with minimal interference from other HDL apoproteins, and it demonstrates that the kinetics of apoC-III in VLDL and HDL are similar, supporting the concept of a single kinetically homogeneous pool of apoC-III in plasma.     

Quantification of triglyceride transport in blood plasma: a critical analysis.

Reliable and precise quantification of endogenous triglyceride transport in man has not been possible with simple means to date. Direct measurement of net splanchnic secretion of triglyceride fatty acids (TGFA) in very low density lipoproteins (VLDL) provides the must unambiguous information, but precision is low. Coupling infusion of labeled fatty acid with sampling of arterial and hepatic venous blood increases precision; however, the contribution of precursors other than plasma free fatty acids (FFA) must be assessed. Measurement of the rate of hydrolysis of plasma triglycerides after displacing lipases into the blood with heparin holds promise as a simple, nonisotopic method, but it has not been carefully validated and heparin itself alters FFA and triglyceride transport. Multicompartmental analysis following pulse injection of labeled fatty acid offers a practical approach, but uncertainties about the number and location of interacting compartments have made it impossible to determine an absolute value for transport. Reinjection of biologically labeled plasma VLDL is impractical for large scale use, and validity of this approach remains uncertain because of heterogeneity of VLDL-triglycerides and their complex metabolic behavior. Methods to label VLDL-triglycerides in vitro deserve more study as does labeling of other components, such as the B-apoprotein. Such approaches will require rigorous comparison with biologically labeled material as well as careful assessment of alterations in kinetic behavior that may occur when VLDL are separated from blood plasma.