Abstract 538: Metabolism of Multiple Apolipoproteins Across HDL Size in Humans

Introduction: Studying the kinetics of several HDL apolipoproteins together may help elucidate their function in HDL metabolism. We present multicompartmental analysis describing the metabolism of several apolipoproteins across 5 HDL size fractions. Methods and Results: Three participants were infused with a bolus of D3-Leu tracer, and blood was collected for 70 hrs. ApoA-I-HDL was prepared by immunoaffinity purification, separated into 5 size fractions, preβ, α3, α2, α1, and α0, by ND-PAGE, and in-gel trypsinized for mass spectrometry. We monitored 7 proteins that likely affect HDL metabolism - apoA-I, apoA-II, apoE, apoM, apoC-III, apoA-IV, and apoD. We used stable isotope labeled peptide standards to quantify pool size and high resolution parallel reaction monitoring to measure tracer enrichment in these 7 proteins in the 5 HDL size fractions. These data were then used for multicompartmental analysis to describe the kinetic behaviors across HDL size for each apolipoprotein, with the exception of apoD. All size fractions with discernible enrichment curves for at least 2 participants were included in the final model for each apolipoprotein (Fig.1). The majority of apoA-I α HDL originated form the source compartment, presumably the liver and/or small intestine, while apoA-I preβ HDL came primarily from α3. Size expansion pathways (preβ to α1/2, and α3 to α2) contributed only slightly to apoA-I metabolism. Similarly to apoA-I on the α sizes, the majority of the other apolipoproteins appear on each HDL size fraction directly from the source compartment. Only minor flux pathways from smaller to larger HDL sizes were identified: apoA-II, α3 to α2; apoE, α3 to α2, and α3 to α1. Conclusions: This study supports the new model of HDL metabolism in which HDL and its attached apolipoproteins are metabolized mainly within each HDL size fraction. Pathways between HDL sizes only provide a small contribution to the metabolism of each apolipoprotein.

Thyroidal and peripheral production of 3,5,3'-triiodothyronine in humans by multicompartmental analysis

Multicompartmental analysis of thyroxine (T4) and 3,5,3'-triiodothyronine (T3) kinetics based on the plasma disappearance curves of the two tracer hormones (J. J. DiStefano III, M. Jang, T. K. Malone, and M. Broutman. Endocrinology 110: 198-213, 1982 and J. J. DiStefano III, T. K. Malone, and M. Jang. Endocrinology 111: 108-117, 1982) was extended to include additional experimental data, namely, the appearance curve in plasma of labeled T3 generated in vivo from precursor T4. Kinetic analysis of data obtained in 14 studies carried out in normal subjects by using a composite six-pool model made it possible to quantify the contributions of the thyroid (3.3 micrograms.day-1.m-2) and the periphery (12.7 micrograms.day-1.m-2) to T3 production. T4 monodeiodination occurred mainly in peripheral tissues rapidly exchanging with plasma (10.7 micrograms T3.day-1.m-2), whereas only 2.0 micrograms T3.day-1.m-2 arose in slowly exchanging tissues. In contrast, if plasma disappearance curves only were analyzed, a value of 10.9 micrograms T3.day-1.m-2 was calculated for peripheral conversion in slowly exchanging tissues; this underscores the need for additional data, such as the [125I]T3 plasma appearance curve for the partition of central and peripheral production of T3.

Liver and adipose tissue contributions to newly formed fatty acids in an ascites tumor

We determined the contribution from host hepatic and extrahepatic tissues to newly synthesized fatty acids (FA) in the Ehrlich ascites tumor (EAT). We administered 3H2O (subcutaneously) and [14C]glucose (in a test meal) and measured the appearance of radioactivity in plasma triglyceride fatty acids (TGFA) and free fatty acids (FFA) and in tumor total lipid fatty acids (TLFA). Using [14 C]FFA, we selectively labeled epididymal fat TGFA to estimate the FA transport rate from intraperitoneal adipose tissue directly to the tumor. Contributions of four major pathways to newly synthesized FA in EAT were determined by multicompartmental analysis. De novo FA synthesis by EAT accounted for more than 93% of the TLFA radioactivity found in the tumor. Contributions from liver TGFA via plasma TGFA (less than 0.5%), adipose tissue TGFA via plasma FFA (less than 6%), and adipose tissue TGFA via direct intraperitoneal transport of FFA (less than 1%) accounted for less than 7% of all TLFA radioactivity measured in the EAT. Thus the present study establishes that practically all labeled esterified FA in the EAT is derived from de novo synthesis by tumor cells.

[13C]bicarbonate kinetics in humans: intra- vs. interindividual variations

A comprehensive multicompartmental analysis of HCO-3 kinetics was carried out on five normal, resting, fasted adults on three separate occasions at 1-wk to 1-mo intervals to obtain a set of bicarbonate kinetic parameters and estimates of their inter- and intraindividual variations for use in the design and analysis of future nutrient oxidation studies. Following a rapid bolus of NaH13CO3 (10 mumol . kg-1 iv), the decay of 13C enrichment of breath CO2 could be described by a three-exponential decay process and a linearly time-dependent term that accounted for changes in the 13C enrichment of metabolic fuels. The data were fitted subsequently to a mammillary multicompartmental model that consisted of a central pool and two peripheral pools of 3,310, 3,490, and 8,070 mumol . kg-1 HCO-3-CO2. Labeled CO2 was eliminated from the central pool by respiratory and nonrespiratory routes at rates of 101 and 97 mumol . kg-1 . min-1, respectively. The within-subject and among-subject variances were similar for the amount of freely exchangeable bicarbonate (14,870 mumol . kg-1), CO2 output (101 mumol . kg-1 . min-1), bicarbonate flux (198 mumol . kg-1 . min-1), and the fraction of administered bicarbonate recovered in breath (0.51). Comparison of variances associated with the assignment of a population mean value to a new subject and the variance associated with the use of a value obtained in the same individual on another day indicated that there was no advantage to making a determination on an alternate day over using a population value. Efforts should be made to compile population values for bicarbonate kinetics in different age groups and metabolic, nutritional, and pathological states for use in interpretation of nutrient oxidation data.