Measurement of precursor enrichment for calculating intramuscular triglyceride fractional synthetic rate.

Our goal was to assess the validity of the enrichments of plasma free palmitate and intramuscular (IM) fatty acid metabolites as precursors for calculating the IM triglyceride fractional synthetic rate. We infused U-¹³C₁₆-palmitate in anesthetized rabbits for 3 h and sampled adductor muscle of legs using both freeze-cut and cut-freeze approaches. We found that IM free palmitate enrichment (0.70 ± 0.07%) was lower (P < 0.0001) than IM palmitoyl-CoA enrichment (2.13 ± 0.17%) in samples taken by the freeze-cut approach. The latter was close (P = 0.33) to IM palmitoyl-carnitine enrichment (2.42 ± 0.16%). The same results were obtained from the muscle samples taken by the cut-freeze approach, except the enrichment of palmitoyl-CoA (2.21 ± 0.08%) was lower (P = 0.02) than that of palmitoyl-carnitine (2.77 ± 0.17%). Plasma free palmitate enrichment was ∼2-fold that of IM palmitoyl-CoA enrichment and palmitoyl-carnitine enrichment (P < 0.001). These findings indicate that plasma free palmitate overestimated IM precursor enrichment owing to in vivo IM lipid breakdown, whereas IM free palmitate enrichment underestimated the precursor enrichment because of lipid breakdown during muscle sampling and processing. IM palmitoyl-carnitine enrichment was an acceptable surrogate of the precursor enrichment because it was less affected by in vitro lipid breakdown after sampling.

liquid nitrogen using a metal net to dunk the samples. All frozen muscle samples were transferred into cryogenic tubes for storage in a Ϫ 80°C freezer. The freeze-cut and cut-freeze muscle sampling was performed twice in each leg in the sequence from the distal to the proximal site of the adductor muscle.
Mean arterial blood pressure, heart rate, and rectal temperature were maintained stable by adjusting the infusion rates of anesthetics and physiological saline, and by using a heating blanket. These vital signs were recorded every 30 min during the 3 h tracer infusion. At the end of the experiment, the rabbits were euthanized by intravenous injection of 5 ml saturated KCl under general anesthesia.

Sample analysis
Plasma FFA and TG were processed for palmitate enrichment using GC-MS (MSD, Agilent Technologies, Palo Alto, CA) ( 14,17,18 ). Ions were selectively monitored at m/z 270, 285, and 286 for palmitate. Heptadecanoic acid and triheptadecanoin were added to the plasma samples as internal standards for calculating palmitate concentrations in plasma FFA and TG. Seven fatty acids in plasma FFA and TG were measured using a GC system with fl ame ionization (FI) detection (model 6890, Agilent Technologies). The percentage of palmitate in the seven fatty acids was used to convert palmitate concentration to total FFA and TG concentrations in plasma. The seven fatty acids were myristate (14: Five representative frozen muscle samples from concomitant other experiments were thawed and observed under a stereo microscope (10-40×, Konus microscopes). No overt adipose tissue was found under the microscope, so the muscle samples taken by the same procedure were considered free of fat contamination. Frozen muscle samples were pulverized with a mortar and pestle prechilled in liquid nitrogen. Heptadecanoyl-CoA (Sigma Chemical, St. Louis, MO) or d 3 -PalCn (CIL, Andover, MA) were added as internal standards for calculating the concentration of PalCoA or PalCn, respectively. Fatty acyl-CoA was extracted with KH 2 PO 4 and 2-propranol, and fatty acyl-carnitine was extracted with KH 2 PO 4 and acetronitrile/methanol from 40-50 mg tissue ( 15,16 ). The enrichments of PalCoA and PalCn along with percentage composition of seven fatty acyl-CoAs and seven fatty acyl-carnitines were measured on an Agilent 1100 series liquid chromatograph-1956B SL single quadrupole mass spectrometer (Agilent Technologies) as previously described ( 15,16 ). The seven fatty acids were the same as measured in plasma FFA and TG.
Muscle TG was extracted from 30-50 mg of muscle powder overnight at 4°C in 1:2 (v/v) methanol:chloroform solution containing 0.05 mg/ml butylated hydroxytoluene. Heptadecanoic acid was added to the samples as an internal standard for calculating IM FFA concentration. After extraction, the samples were centrifuged, and the supernatant was dried under nitrogen gas. The samples were reconstituted with 50 µl chloroform for TLC (Partisil LK5D, Silica Gel 150 Å, Schleicher and Schuell, Maidstone, United Kingdom) isolation of TG and FFA. The isolation was processed in a tank with a mixture of hexane:ethyl ether:acetic acid (70:30:1 in ml). The isolated TG was hydrolyzed in HNO 3 to get fatty acids and glycerol. Fatty acids from the IM FFA fraction were derivatized to their methyl esters for measurement of palmitate enrichment by GC-MS; fatty acid concentrations were measured by GC-FI. Fatty acids from IM TG were derivatized to their methyl esters; palmitate enrichment was measured on a GC-combustion-isotopic ratio MS (Finnigan, MAT, Bremen, Germany). The measured 13 C enrichment was multiplied by 17/16 to convert to the enrichment of the U-13 C 16 -palmitate as 16 of 17 total carbons in the palmitate methyl ester molecule have a chance to be labeled. vitro lipid breakdown during muscle biopsy and processing. We have established the stable isotope and mass spectrometry methods of measuring both PalCoA and PalCn, as well as more conventionally used surrogates, as precursors for the measurement of IM TG synthesis ( 15,16 ). The current study was performed to assess the relationship of the potential surrogates for precursor enrichment with the measured enrichment of PalCoA, which we considered the true value.

Animal preparation
Adult male New Zealand white rabbits (Myrtle's Rabbitry, Thompson Station, TN), weighing ‫ف‬ 4.5 kg, were used for this study. The rabbits were housed in individual cages and were given 150 g/day of unpurifi ed diet (Lab Rabbit Chow 5326, Purina Mills, St. Louis, MO) for weight maintenance. This protocol complied with the Public Health Service Policy on Humane Care and Use of Laboratory Animals, incorporated in the Institute for Laboratory Animal Research Guide for Care and Use of Laboratory Animals, and it was approved by the Animal Care and Use Committee of the University of Texas Medical Branch at Galveston.
The animals were studied after overnight food deprivation with free access to water. Surgery was performed to insert catheters into the carotid artery and jugular vein under general anesthesia ( 14,17 ). The arterial line was used for drawing blood and monitoring arterial blood pressure and heart rate; the venous line was used for infusion of anesthetics and saline. An additional venous line was installed in a marginal ear vein by means of a Tefl on-top needle (24 G 3/4 Introcan Safety, B. Braum Medical, Inc., Bethlehem, PA), which was used exclusively for tracer infusion. Tracheotomy was performed for placement of a tracheal tube, which was connected to a hood fi lled with oxygen-enriched room air.

Stable isotope tracer infusion
After surgery, we observed blood pressure, heart rate, and rectal temperature for 20-30 min to ensure stable physiological conditions before the start of tracer infusion. U-13 C 16 -palmitate (99% enriched; Cambridge Isotope Laboratories), bound to 5% albumin, was infused continuously for 3 h at the dose of ‫ف‬ 0.1 µmol/ kg/min after a priming dose of 1.0 µmol/kg. The tracer was infused into the marginal ear vein using a Harvard Syringe pump (Harvard Apparatus, Boston, MA) set at 15 ml/h. Blood samples were taken from the carotid artery catheter at 0, 5, 30, 60, 90, 120, 150, and 180 min. After centrifugation, plasma was separated and stored at Ϫ 20°C for later analysis. The adductor muscle of both legs was sampled at 0, 5, 60, 120, and 180 min. At each sampling time except the zero time (background enrichment), a pair of muscle samples were taken: the fi rst muscle sample was taken using the freeze-cut approach and the second one using the cutfreeze approach. The freeze-cut approach involved the in situ clamping of a piece of isolated adductor muscle with a metal clamp prechilled in liquid nitrogen. The tissue was held with forceps while the frozen muscle was excised. The samples were immediately put in liquid nitrogen. After thorough freezing in liquid nitrogen for >1 min, extra tissue not included in the part of the sample that was freeze clamped was trimmed. Immediately following the freeze-cut, a second piece of muscle was taken using scissors (the cut-freeze approach) from the muscle adjacent to the freeze-cut site. The samples were briefl y washed with icecold saline if blood was visible. The samples were then frozen in reached a plateau after 1 h of tracer infusion ( Fig. 1 ). Plasma enrichment of TG-bound palmitate increased gradually during tracer infusion ( Fig. 1 ). The mean plasma free palmitate enrichment was 4.56 ± 0.95 mol% excess (MPE), which was calculated from the 5, 30, 60, 90, 120, 150, and 180 min enrichments.
The mean enrichments of IM free palmitate, PalCoA, and PalCn were calculated from the 5, 60, 120, and 180 min muscle samples. Because the enrichment of plasma free palmitate was much greater ( P < 0.0001) than of the enrichment of IM free palmitate, IM PalCoA, or IM PalCn ( Table 1 ), the plasma free palmitate was not an acceptable surrogate of IM precursor for lipid synthesis. The following comparisons were made among the three IM metabolites using ANOVA. In the muscle samples taken by the freeze-cut approach, IM PalCoA enrichment was not signifi cantly ( P = 0.33) lower than PalCn enrichment ( Fig. 2A and Table 1 ). In the muscle samples taken using the cutfreeze approach, the enrichment of PalCoA was lower ( P = 0.02) than that of PalCn ( Fig. 2B ). In both cases, IM free palmitate enrichment was much lower ( P < 0.0001) than

Calculations
The concentrations of palmitate in plasma and muscle lipids were calculated by the internal standard method. Palmitate (or its metabolites) concentrations were divided by percentage of palmitate in the seven fatty acids to obtain total fatty acid concentrations.
FSR of IM TG was calculated by the tracer incorporation method, which is based on the precursor-product principle ( 18 ). The equation is : is the enrichment increment of TG-bound palmitate from t 1 to t 2 , and E P (t 2 Ϫ t 1 ) is the average precursor enrichment from t 1 to t 2 .
The percentage contributions from plasma FFA, plasma TG, and IM lipids to IM precursor were calculated using the following equations ( 14 ): where ECoA 0 and EPal 0 are enrichments of IM PalCoA (represented by PalCn) and plasma free palmitate, respectively, at the beginning of tracer infusion when plasma TG-bound palmitate enrichment is zero.
where ECoA p and EPal p are enrichments of IM PalCoA (represented by PalCn) and plasma free palmitate at the plateaus, and (ECoA p / EPal p ) represents the percentage contribution from plasma FFA and TG.

Statistics
Values are expressed as means ± SEM. Differences comparing two parameters from same muscle samples were tested by the paired t -test. Differences among three parameters were compared with ANOVA. The Tukey adjustment was used to adjust for multiple comparisons. We used SAS ® 9.2 software (SAS Institute, Cary, NC) for the analysis. A P value less than 0.05 was considered statistically signifi cant.

RESULTS
The body weight of rabbits was 4.5 ± 0.1 kg. During the 3 h tracer infusion, the rabbits were maintained under stable physiological conditions and suffi cient depth of anesthesia; mean rectal temperature was 37.7 ± 0.4°C; heart rate was 153 ± 2 beats/min; and mean arterial blood pressure was 63 ± 2 mmHg. Plasma concentrations of FFA and TG were 335 ± 71 and 191 ± 31 nmol/ml, respectively; palmitate accounted for 42 ± 7% and 47 ± 6% of the seven fatty acids measured, respectively.
The enrichment of plasma free palmitate increased rapidly after the start of U-13 C 16 -palmitate infusion, and it  IM free palmitate enrichment as the precursor enrichment were greater ( P < 0.0001) than the corresponding values measured using PalCoA enrichment or PalCn enrichment as the precursor enrichment in muscle samples taken by both the freeze-cut and cut-freeze approaches ( Table 1 ). However, the FSR values calculated using IM PalCn enrichment as the precursor enrichment were not signifi cantly ( P = 0.64 for freeze-cut and P = 0.94 for cut-freeze) different from the corresponding FSR values calculated using IM Pal-CoA as the precursor enrichment.
In the muscle samples taken by the cut-freeze approach, IM FFA concentration was greater than that in muscle samples taken by the freeze-cut approach ( Table 2 ). The concentrations of IM fatty acyl-CoA and fatty acyl-carnitine were comparable between the muscle samples taken by the freeze-cut and cut-freeze approaches ( Table 2 ).
The enrichments of PalCn were 53.3 ± 2.3%, 58.9 ± 2.9%, and 60.8 ± 3.8% of the corresponding plasma free palmitate enrichment at 1, 2, and 3 h, respectively. The increasing percentages of IM PalCn enrichment to plasma free palmitate enrichment over time were explained by the gradual increase of plasma TG-bound palmitate enrichment ( Fig. 1 ), which contributed labeled palmitate to the IM precursor via the action of lipoprotein lipase. If we extrapolate to 0 h using linear curve fi tting, the percentage contribution of IM PalCn enrichment to plasma free palmitate enrichment was 50.2%, which represented the contribution of plasma FFA to IM precursor (see Eq. 2). At 3 h, the percentage of IM PalCn enrichment to plasma free palmitate enrichment was 60.8%, so plasma TG contribution was 10.6% (60.8% Ϫ 50.2%) at 3 h. However, plasma TG-bound palmitate enrichment did not reach a plateau at 3 h. Based on exponential curve fi tting, the plateau enrichment should be 3.88%, which was 69% greater than the enrichment at 3 h (2.29%). Thus, the maximal contribution of plasma TG to IM precursor was estimated to be 17.9%, which was the sum of 10.6% at 3 h plus a further 69% increase at the plateau of plasma TG-bound palmitate enrichment (see Eq. 3). The contribution of IM lipids to precursor was therefore 31.9% (see Eq. 4).
The enrichment of IM TG-bound palmitate, which was measured from the muscle samples taken by the freeze-cut approach, increased over time ( Fig. 3 ). The enrichment increase appeared to be faster later than earlier during tracer infusion. FSRs calculated using the enrichments of IM free palmitate, PalCoA, and PalCn as the precursor enrichment were compared using ANOVA. The FSRs calculated using Fig. 2. IM free palmitate, PalCoA, and PalCn enrichment. A: In muscle samples taken by the freeze-cut approach, IM free palmitate enrichment was lower ( P < 0.0001) than were PalCoA and PalCn enrichments. IM PalCoA enrichment was not signifi cantly ( P = 0.33) lower than PalCn enrichment. B: In muscle samples taken by the cut-freeze approach, the same results were obtained, except the enrichment difference between PalCoA and PalCn was signifi cant ( P = 0.02).  Values are means ± SEM. Unit is nmol/g for concentrations. Palmitate in the seven fatty acids measured is expressed as percentage. a P < 0.05 versus freeze-cut by paired t -test.
Tracer dilution could occur at several steps of muscle sampling and processing. When we excised a muscle sample, several seconds were required to submerge it in liquid nitrogen, and freezing is not instantaneous in liquid nitrogen. We used a metal net to freeze the muscle samples when using the cut-freeze approach. This procedure eliminated the time span needed to transfer the sample to a cryogenic tube, and it shortened the time span of being frozen. However, there was still an unavoidable time delay from muscle excision to freezing. Further, when we weighed the muscle powder on a digital scale and added internal standards to the sample vial, the muscle samples might no longer have been in a completely frozen state. Although we made every possible effort to minimize the time span in which the samples were not frozen (such as using dry ice to keep samples cold during the procedure), it was not possible to completely avoid exposing the samples to a temperature change during processing.
The magnitude of dilution of the IM free palmitate can be explained by the large concentration difference between IM lipid-bound palmitate and IM free palmitate. In our previous experiment, IM TG was measured to be 11.6 ± 2.4 mol/g in muscle from lean rabbits ( 14 ). In another experiment, we found that palmitate accounted for ‫ف‬ 30% of total fatty acids in muscle TG (unpublished data). Thus, the concentration of IM TG-bound palmitate would be 10.44 mol/g. If we regard the PalCn enrichment represented in vivo precursor enrichment, the IM free palmitate concentration in Table 2 overestimated the in vivo concentration by the amount of unlabeled palmitate released from in vitro lipolysis. We estimated that the true in vivo IM free palmitate to be ‫ف‬ 10 nmol/g [measured free palmitate concentration × (measured free palmitate enrichment / PalCn enrichment)]. This means that the pool size (or concentration) of IM TGbound palmitate was approximately 1000-fold that of IM free palmitate. If we included the possible in vitro breakdown of muscle phospholipids and diacylglycerol, the difference between total IM lipid-bound palmitate and free palmitate would be even greater. Thus, it is not surprising that in vitro lipid breakdown markedly diluted IM free palmitate enrichment if the lipolytic process was not instantly quenched during muscle biopsy. On the other hand, the concentration difference between IM free palmitate and PalCoA and between PalCoA and PalCn were much smaller (3-to 7-fold, Table 2 ). Therefore, the dilution effect of free palmitate on PalCoA, and of Pal-CoA on PalCn decreased in sequence. In the muscle samples taken by the freeze-cut approach, the enrichments of PalCoA and PalCn were not signifi cantly different ( Table  1 ), indicating minimal effect of in vitro lipid breakdown. In the muscle samples taken by the cut-freeze approach, IM PalCoA enrichment was lower than PalCn enrichment ( P < 0.05; see Table 1 and Fig. 2 ). Thus, IM PalCn is an acceptable surrogate for precursor enrichment. Because of severe trauma caused by the freeze-cut approach, the acceptability of PalCn enrichment as the precursor enrichment is meaningful when the freeze-cut technique is not applicable.

DISCUSSION
We found that IM PalCoA and PalCn enrichments were approximately 50% of plasma free palmitate enrichment. This refl ects that there must have been active intracellular lipid breakdown whereby released unlabeled palmitate diluted the precursor enrichment. IM lipid breakdown was calculated to account for 31.9% of IM precursor, and plasma FFA and TG accounted for 50.2% and 17.9% of precursor, respectively. Because the palmitate enrichment in IM TG could be regarded as fundamentally unlabeled in relation to the precursor enrichment ( Table 1 ), and palmitate enrichment in plasma TG was lower than that of plasma free palmitate ( Fig. 1 ), the lower enrichment of the IM precursor can only be attributed to intracellular lipid breakdown. Thus, the use of intracellular free palmitate enrichment as the precursor enrichment would have overestimated TG FSR by a minimum of approximately 300%, and perhaps even more, because of the difference in the free IM palmitate enrichment as compared with the PalCn enrichment.
The lower enrichment of IM free palmitate as compared with those of PalCoA and PalCn indicates that there was continuous lipid breakdown during muscle sampling and processing. Muscle sampling instantly stopped the blood supply (and delivery of tracer) to the tissue. Whereas entry of labeled palmitate into the tissue was stopped with excision of the tissue, the process of lipid breakdown and release of unlabeled palmitate into the IM compartment was not necessarily stopped. Continued lipolysis could explain a greater reduction in free palmitate enrichment than either PalCoA or PalCn due to the more rapid turnover time of the free IM palmitate pool. It is also possible that if PalCoA and PalCn continued to be formed after sampling, then the enrichment of those compounds would also have been diluted.
Theoretically, the true precursor for IM TG synthesis is fatty acyl-CoA ( 18 ). When fatty acids enter the intracellular pool, the fi rst step is to be converted to metabolically active fatty acyl-CoA, catalyzed by fatty acyl-CoA synthase and synthetase ( 19,20 ). Our recent results from a complementary experiment demonstrated that the transport of fatty acids into the IM compartment and their subsequent conversion to fatty acyl-CoA and fatty acyl-carnitine occurred very fast, with the peak enrichment within 2 min after a bolus palmitate tracer injection (unpublished data). Thus, the diluted enrichment of IM free palmitate would readily dilute PalCoA enrichment. Because PalCn is a downstream metabolite of PalCoA ( 21 ), PalCn is less affected by in vitro lipid breakdown than IM free palmitate. The same explanation applies to the observation that Pal-CoA enrichment was lower than PalCn enrichment from the muscle samples taken by the cut-freeze approach. The freeze-cut technique decreased the enrichment difference to an insignifi cant level ( Fig. 2 and Table 1 ), indicating that the in vitro lipid breakdown was responsible for the difference in enrichment in the cut-freeze samples. This fi nding is consistent with our previous report in which the freeze-cut technique eliminated enrichment difference between PalCoA and PalCn ( 14 ). and cut-freeze approaches ( Table 1 ). However, FSRs calculated using PalCoA enrichment were not significantly different ( P = 0.94 for freeze-cut and P = 0.64 for cut-freeze) than the corresponding values from PalCn enrichment. The variable enrichments in the IM TGbound palmitate (i.e., product enrichment) eliminated the signifi cant difference between IM PalCoA and PalCn enrichments in the muscle taken by the cut-freeze approach, so the calculated FSRs were not signifi cantly different ( P = 0.64).
Note that the enrichments of IM PalCoA and PalCn increased over time during a constant tracer infusion ( Fig.  2A, B ). Such a precursor enrichment pattern was consistent with that of product enrichment, which increased faster later than earlier during tracer infusion ( Fig. 3 ). This can be explained by the increase in plasma TG-bound palmitate enrichment over time ( Fig. 1 ). At the beginning of tracer infusion, plasma free palmitate was the only source of tracer entering the IM precursor pool. Thereafter, plasma TG-bound palmitate enrichment increased gradually, which also contributed labeled palmitate to the IM precursor pool through the action of lipoprotein lipase ( 22,23 ). Thus, it was proper to calculate the mean precursor enrichment over the 3 h infusion period. In contrast, if only one muscle sample is taken at the end of tracer infusion as some authors reported ( 10,24 ), the measured precursor enrichment would overestimate the precursor enrichment to an unknown extent, which depends on the activity of lipoprotein lipase. This potential error was overlooked in previous publications in which the one biopsy approach was used ( 10,24 ).
A limitation in this experiment was that the IM TG FSR value might refl ect mixed TG in both intracellular and interstitial pools. We dissected the adductor muscle before taking biopsies and checked fi ve representative muscle specimens under the stereomicroscope to ensure no adipose tissue contamination occurred during our biopsy procedure. However, we did not dissect the muscle specimens to remove the interstitial tissue when measuring IM TG-bound palmitate enrichment. We did not perform dissection because it would have required a longer period during which the sample was not frozen, thereby inducing rapid changes in intracellular enrichment.
In summary, active breakdown in the muscle complicates the measurement of intracellular concentration and enrichment of lipids and lipid products. Lipid breakdown releases unlabeled palmitate, which results in an enrichment gradient from plasma to the IM compartment. Consequently, plasma free palmitate overestimates the IM precursor enrichment. During muscle sampling and processing, the IM lipid breakdown may continue to release unlabeled palmitate to dilute the precursor enrichment in the sequence of IM free palmitate, PalCoA, and PalCn. The optimal approach to minimizing the in vitro lipid breakdown is to use the freeze-cut technique for muscle sampling and to avoid sample thawing during processing. If the direct measurement of IM PalCoA enrichment is problematic, IM PalCn enrichment is an acceptable surrogate for the precursor enrichment because PalCn is less affected by Our data do not allow us to exclude the possibility of IM fatty acid compartmentation. If plasma-derived palmitate favors PalCoA formation and if IM lipid-derived palmitate favors IM free palmitate, the IM free palmitate enrichment could be substantially lower than PalCoA and PalCn enrichments. However, compartmentation could not explain the enrichment difference between PalCoA and PalCn when the cut-freeze approach was used ( Fig. 2B ), nor could it explain the elimination of the enrichment difference by the freeze-cut approach ( Fig. 2A ). Thus, the tracee dilution from in vitro lipid breakdown during sampling and processing is a reasonable explanation. Fatty acid compartmentation is also possible. Neither of these explanations supports the use of IM free palmitate enrichment as a surrogate of precursor enrichment. The fatty acid metabolic pathways and dilution are depicted in Fig. 4 .
The acceptability of PalCn as a surrogate precursor was confi rmed by the FSR values calculated using different IM enrichments as the precursor enrichment ( Table 1 ). IM TG FSRs calculated using IM free palmitate enrichment as the precursor enrichment were greater ( P < 0.0001) than the corresponding value calculated using PalCoA enrichment or PalCn enrichment as the precursor enrichment in muscle samples from both freeze-cut Fig. 4. Fatty acid metabolic pathways and dilution. When the bloodstream passes through the muscle bed, plasma FFA enters the IM compartment directly, whereas plasma TG requires lipoprotein lipase to hydrolyze into FFA before entering the IM compartment. The IM FFA pool receives fatty acids not only from plasma but also from IM lipid breakdown. During palmitate tracer infusion, plasma free palmitate is highly labeled, plasma TG-bound palmitate is labeled over time, and palmitate released from IM lipid breakdown is not labeled. Therefore, IM precursor enrichment is lower than plasma free palmitate enrichment. During muscle sampling and processing, continued IM lipid breakdown may dilute the enrichment of IM free palmitate, PalCoA, and PalCn in sequence. IM fatty acid compartmentation may also explain the lower enrichment of IM free palmitate than that of PalCoA and PalCn if the IM lipid breakdown favors the IM FFA pool. Fatty acyl-CoA either can be used as a precursor for lipid synthesis or can be converted to fatty acyl-carnitine for mitochondrial oxidation. If not oxidized in time, fatty acyl-carnitine is converted back to fatty acyl-CoA. Because of rapid equilibrium between fatty acyl-CoA and fatty acylcarnitine, PalCn enrichment can be used to represent PalCoA enrichment. FA-Cn, fatty acyl-carnitine; FA-CoA, fatty acyl-CoA; PL, phospholipids.
in vitro lipid breakdown. When plasma free palmitate enrichment is used as the precursor enrichment, the resultant FSR represents the use of plasma FFA for synthesizing IM TG, which does not include the portion of TG synthesis that uses fatty acids derived from IM lipid breakdown and from plasma TG breakdown through the action of lipoprotein lipase.