ASSUMPTIONS AND DETAILS OF CALCULATIONS FOR FATTY ACID KINETICS

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1 ASSUMPTIONS AND DETAILS OF CALCULATIONS FOR FATTY ACID KINETICS Our hypothesis was that many sources of palmitate (NEFA, lipogenesis, diet) could contribute to newly-synthesized VLDL-TG palmitate in the fed state and thus, our goal was to determine the relative contributions of fatty acids sources to this TG pool. The assumptions for the calculations are as follows. First, serum volume was estimated at.% total body mass (supplemental ref. 1). Second, palmitate (c1:0) serves as a fatty acid marker for the calculations presented below. It is chosen because of its utility in labeling all the pathways and because it is the primary product of de novo lipogenesis. Thus, in addition to administration of the stable isotope of acetate, the free fatty acid ( 1 C palmitate) was also infused IV and the dietary TG was labeled with glyceryl tri-palmitate-d 1. Data of the various sources presented in table reflect percentages and absolute sources of VLDL-TG palmitate in S f 0-00 (as noted in the table) derived from hepatic de novo lipogenesis and reesterification of dietary and adipose fatty acids. The third assumption is that a single fatty acid source can be used to calculate VLDL TGpalmitate turnover if the time frame of its use in the liver is short compared to the total turnover time of the VLDL-TG fraction in the blood (refs. and ; supplemental reference ). In posthoc analysis, eight of the twelve subjects had a sufficient number of fasted and fed-state data points to allow modeling of TG turnover. Because a constant flux of IV 1 C -palmitate to the liver was maintained throughout fasting, the rate of incorporation of this isotope into VLDL-TG palmitate was used to determine VLDL-TG appearance rate from midnight to :00 AM, as has been done previously. Similarly, the isotopic enrichment of the liquid formula was maintained throughout feeding and fed-state VLDL-TG production rate was estimated by analyzing the incorporation of d 1 -palmitate into the VLDL-TG palmitate pool from :0 AM to :00 PM. However, calculation of the fed-state production rate is influenced by at least two factors. First,

2 the presence of chylomicron remnants in the S f 0-00 fraction could cause an overestimation of RaVLDL-TG. Using gradient gel electrophoresis, we found that the average molar ratio of B0 to B in the S f 0-00 fraction was :1 during the fed state. If the d 1 label in this fraction actually came from chylomicrons or chylomicron remnants, rather than from VLDL-TG, this would result in an overestimation of the rate of incorporation of the label into VLDL-TG and a faster calculated turnover rate than was actually occurring. It is notable that in the final analysis, fasted RaVLDL-TG was greater than fed-state RAVLDL-TG, but not significantly so (see table ). Second, we found that in six of the subjects, the chylomicron pool took 1- hours to reach d 1 steady-state enrichments causing a delay in the input of dietary label into the plasma compartment. This resulted in the VLDL-TG d -enrichments occurring within the first hour to be underestimates (e.g., VLDL-TG enrichment would have been higher had the chylomicron pool been fully enriched) and therefore the calculated VLDL-TG turnover would be lower than observed. Whether or not these two factors (chylomicron contamination and a delay in entry of dietary label) canceled each other out is unknown. It should be noted that calculation of the VLDL-TG turnover rate was not among the primary goals of the present study. After the calculation of RaVLDL TG-palmitate, the fatty acid composition was determined in the VLDL- TG fraction by GC and these data were used in the final calculation for the rates of appearance of total VLDL-TG The infusate composition and enrichments were analyzed by GC and GC/MS and the calculations were adjusted for the amount of unlabeled fatty acids present that have been derived from less than 0% purity of the isotope or from fatty acids present on the albumin used in the infusion. The inputs of these fatty acids were subtracted from the final calculated values of

3 1 RaNEFA and dietary spillover. The rate of appearance of fatty acids from adipose-tg lipolysis discussed in this paper has been calculated by taking into account the amount of unlabeled (cold) dietary fatty acid that spilled over into the plasma NEFA pool. Although the term spillover is used to define the appearance of dietary fatty acids in the NEFA pool during the fed state, it is not certain that these dietary fatty acids entered the plasma NEFA pool directly after lipolysis of chylomicron-tg by lipoprotein lipase, or whether these fatty acids entered tissues first and then were re-secreted in a preferential manner compared to preformed tissue-tg fatty acids. This latter process, sometimes referred to as the last in-first out mechanism, could occur if recentlycleared fatty acids are added to the periphery of a cytosolic lipid droplet or reside in an intracellular pool with a very fast turnover. The calculation of the percentage of NEFA from adipose lipolysis and the contribution of adipose FFA to VLDL S f 0-00 are as follows. Equation 1. % NEFA from adipose TG lipolysis: 1 % NEFA from adipose TG lipolysis = % d 1 palmitate in NEFA 1 - x 0 % d 1 palmitate in liquid-formula TG Given equation 1, the percentage of VLDL-TG palmitate derived only from adipose tissue release is calculated by equation. Equation. % VLDL-TG from adipose NEFA: % NEFA from adipose TG lipolysis = % d 1 palmitate in NEFA 1 - x 0 % d 1 palmitate in liquid-formula TG The percentage of VLDL-TG derived from dietary fatty acid spillover takes advantage of the fourth assumption - that all NEFA that clear to the liver will be used in the same manner for

4 VLDL-TG, regardless of whether they were derived from dietary spillover or adipose tissue release. Using the ratio of 1 C to d 1 in the serum NEFA pool, the amount of dietary fatty acids that came through the NEFA pool can be calculated using equation. Equation. % VLDL-TG from diet-derived NEFA: % 1 C palmitate in VLDL-TG % 1 C palmitate in NEFA X % d 1 palmitate in NEFA % d 1 palmitatee in liquid-formula TG Lastly, the fifth assumption used for these calculations is that any excess of dietary label found in VLDL-TG, above that which could have entered through the NEFA pool, was assumed to enter via hepatic uptake of chylomicron-remnants. 1 1 Equation. % VLDL-TG from chylomicron-remnant TG: 1 1 % d 1 palmitate in VLDL-TG % d 1 palmitate in NEFA X % 1 C palmitate in VLDL-TG - % 1 C palmitate in NEFA % d 1 palmitate in liquid-formula TG The presence of chylomicron remnants in the S f 0-00 fraction (discussed above under the third assumption), would result in an overestimation of the percentage of VLDL-TG palmitate that was derived from the diet. The data presented in ancillary figure 1, below, show that the delay time for the initiation of use of both 1 C and the d 1 labels in VLDL-TG was similar at ca. 0 min

5 Supplemental References 1. Dagher FJ, Lyons JH, Finlayson DC, Shamsai J, Moore FD 1 Blood volume measurement: A critical study. Adv Surg 1:-. Packard CJ 1 The role of stable isotopes in the investigation of plasma lipoprotein metabolism. Baillieres Clin Endo Metab :- 1 1 Supplemental Figure 1. Percentages of - 1 C-palmitate (squares) during the fasting state and d 1 -palmitate (diamonds) collected in the fed-state in TG in the S f 0-00 fraction. Each graph represents data from a single subject. Note that the - 1 C palmitate data were collected between midnight and :00 AM (h total) and the zero time point is referenced to midnight. The d 1 - palmitate data were collected between :00 AM an :00 PM (h total), and therefore, the zero time point is referenced to :00 AM.

6 1 C (%) d 1 (%) 1 C (%) d 1 (%) Time (h), relative to start of infusion Time (h), relative to start of infusion