Quantification of muscle triglyceride synthesis rate requires an adjustment for total triglyceride content.

Intramyocellular triglyceride (imTG) in skeletal muscle plays a significant role in metabolic health, and an infusion of [13C16]palmitate can be used to quantitate the in vivo fractional synthesis rate (FSR) and absolute synthesis rate (ASR) of imTGs. However, the extramyocellular TG (emTG) pool, unless precisely excised, contaminates the imTG pool, diluting the imTG-bound tracer enrichment and leading to underestimation of FSR. Because of the difficulty of excising the emTGs precisely, it would be advantageous to be able to calculate the imTG synthesis rate without dissecting the emTGs from each sample. Here, we tested the hypothesis that the ASR of total TGs (tTGs), a combination of imTGs and emTGs, calculated as "FSR × tTG pool," reasonably represents the imTG synthesis. Muscle lipid parameters were measured in nine healthy women at 90 and 170 min after the start of [13C16]palmitate infusion. While the measurements of tTG content, enrichment, and FSR did not correlate (P > 0.05), those of the tTG ASR were significantly correlated (r = 0.947, P < 0.05). These results demonstrate that when imTGs and emTGs are pooled, the resulting underestimation of imTG FSR is balanced by the overestimation of the imTG content. We conclude that imTG metabolism is reflected by the measurement of the tTG ASR.

Palmitoyl-carnitine production by blood cells associates with the concentration of circulating acyl-carnitines in healthy overweight women.

BACKGROUND: Circulating acyl-carnitines (acyl-CNTs) are associated with insulin resistance (IR) and type 2 diabetes (T2D) in both rodents and humans. However, the mechanisms whereby circulating acyl-CNTs are increased in these conditions and their role in whole-body metabolism remains unknown. The purpose of this study was to determine if, in humans, blood cells contribute in production of circulating acyl-CNTs and associate with whole-body fat metabolism. METHODS AND RESULTS: Eight non-diabetic healthy women (age: 47 ± 19 y; BMI: 26 ± 1 kg·m-2) underwent stable isotope tracer infusion and hyperinsulinemic-euglycemic clamp study to determine in vivo whole-body fatty acid flux and insulin sensitivity. Blood samples collected at baseline (0 min) and after 3 h of clamp were used to determine the synthesis rate of palmitoyl-carnitine (palmitoyl-CNT) in vitro. The fractional synthesis rate of palmitoyl-CNT was significantly higher during hyperinsulinemia (0.788 ± 0.084 vs. 0.318 ± 0.012%·hr-1, p = 0.001); however, the absolute synthesis rate (ASR) did not differ between the periods (p = 0.809) due to ∼30% decrease in blood palmitoyl-CNT concentration (p = 0.189) during hyperinsulinemia. The ASR of palmitoyl-CNT significantly correlated with the concentration of acyl-CNTs in basal (r = 0.992, p < 0.001) and insulin (r = 0.919, p = 0.001) periods; and the basal ASR significantly correlated with plasma palmitate oxidation (r = 0.764, p = 0.027). CONCLUSION: In women, blood cells contribute to plasma acyl-CNT levels and the acyl-CNT production is linked to plasma palmitate oxidation, a marker of whole-body fat metabolism. Future studies are needed to confirm the role of blood cells in acyl-CNT and lipid metabolism under different physiological (i.e., in response to meal) and pathological (i.e., hyperlipidemia, IR and T2D) conditions.

Non-targeted analysis of electronics waste by comprehensive two-dimensional gas chromatography combined with high-resolution mass spectrometry: Using accurate mass information and mass defect analysis to explore the data.

Comprehensive two-dimensional gas chromatography (GC×GC) and high-resolution mass spectrometry (HRMS) offer the best possible separation of their respective techniques. Recent commercialization of combined GC×GC-HRMS systems offers new possibilities for the analysis of complex mixtures. However, such experiments yield enormous data sets that require new informatics tools to facilitate the interpretation of the rich information content. This study reports on the analysis of dust obtained from an electronics recycling facility by using GC×GC in combination with a new high-resolution time-of-flight (TOF) mass spectrometer. New software tools for (non-traditional) Kendrick mass defect analysis were developed in this research and greatly aided in the identification of compounds containing chlorine and bromine, elements that feature in most persistent organic pollutants (POPs). In essence, the mass defect plot serves as a visual aid from which halogenated compounds are recognizable on the basis of their mass defect and isotope patterns. Mass chromatograms were generated based on specific ions identified in the plots as well as region of the plot predominantly occupied by halogenated contaminants. Tentative identification was aided by database searches, complementary electron-capture negative ionization experiments and elemental composition determinations from the exact mass data. These included known and emerging flame retardants, such as polybrominated diphenyl ethers (PBDEs), hexabromobenzene, tetrabromo bisphenol A and tris (1-chloro-2-propyl) phosphate (TCPP), as well as other legacy contaminants such as polychlorinated biphenyls (PCBs) and polychlorinated terphenyls (PCTs).

Triglycerides produced in the livers of fasting rabbits are predominantly stored as opposed to secreted into the plasma.

OBJECTIVE: The liver plays a central role in regulating fat metabolism; however, it is not clear how the liver distributes the synthesized triglycerides (TGs) to storage and to the plasma. MATERIALS AND METHODS: We have measured the relative distribution of TGs produced in the liver to storage and the plasma by means of U-(13)C(16)-palmitate infusion in anesthetized rabbits after an overnight fast. RESULTS: The fractional synthesis rates of TGs stored in the liver and secreted into the plasma were not significantly different (stored vs. secreted: 31.9 ± 0.8 vs. 27.7 ± 2.6%∙h(-1), p > 0.05). However, the absolute synthesis rates of hepatic stored and secreted TGs were 543 ± 158 and 27 ± 7 nmol∙kg(-1)∙min(-1) respectively, indicating that in fasting rabbits the TGs produced in the liver were predominately stored (92 ± 3%) rather than secreted (8 ± 3%) into the plasma. This large difference was mainly due to the larger pool size of the hepatic TGs which was 21 ± 9-fold that of plasma TGs. Plasma free fatty acids (FFAs) contributed 47 ± 1% of the FA precursor for hepatic TG synthesis, and the remaining 53 ± 1% was derived from hepatic lipid breakdown and possibly plasma TGs depending on the activity of hepatic lipase. Plasma palmitate concentration significantly correlated with hepatic palmitoyl-CoA and TG synthesis. CONCLUSION: In rabbits, after an overnight fast, the absolute synthesis rate of hepatic stored TGs was significantly higher than that of secreted due to the larger pool size of hepatic TGs. The net synthesis rate of TG was approximately half the absolute rate. Plasma FFA is a major determinant of hepatic TG synthesis, and therefore hepatic TG storage.

Acute hyperinsulinemia and reduced plasma free fatty acid levels decrease intramuscular triglyceride synthesis.

OBJECTIVE: To investigate the effect of acute hyperinsulinemia and the resulting decrease in plasma free fatty acid (FFA) concentrations on intramuscular TG synthesis. MATERIALS/METHODS: U-(13)C(16)-palmitate was infused for 3 h in anesthetized rabbits after overnight food deprivation. Arterial blood and leg muscle were sampled during the tracer infusion. Plasma samples were analyzed for free and TG-bound palmitate enrichments and concentrations. The enrichments and concentrations of palmitoyl-CoA and palmitoyl-carnitine as well as the enrichment of palmitate bound to TG were measured in muscle samples. Fractional synthetic rate (FSR) of intramuscular TG was calculated using the tracer incorporation method. The rabbits were divided into a control group and a hyperinsulinemic euglycemic clamp group. Insulin infusion decreased the rate of appearance of plasma free palmitate (2.00±0.15 vs 0.68±0.20 µmol⋅kg(-1)⋅min(-1); P<.001), decreased plasma FFA concentration (327±61 vs 72±25 nmol/mL; P<.01), decreased the total concentration of intramuscular fatty acyl-CoA plus fatty acyl-carnitine (12.1±1.6 vs 7.0±0.7 nmol/g; P<.05), and decreased intramuscular TG FSR (0.48±0.05 vs 0.21±0.06%/h; P<.01) in comparison with the control group. Intramuscular TG FSR was correlated (P<.01) with both plasma FFA concentrations and intramuscular fatty acyl-CoA concentrations. CONCLUSIONS: Fatty acid availability is a determinant of intramuscular TG synthesis. Insulin infusion decreases plasma and intramuscular fatty acid availability and thereby decreases TG synthesis.

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.

Enternal [corrected] arginine supplementation stimulates DNA synthesis in skin donor wound.

BACKGROUND & AIMS: Arginine infusion has been demonstrated to increase wound protein deposition; however, the effects of its enteral supplementation on wound cell proliferation have not been studied. METHODS: Skin donor wound was created on the back of rabbits. The rabbits were randomly assigned to receive a control enteral diet, or the control enteral diet with supplemental arginine. On day 5 L-[ring-(13)C(6)]phenylalanine and D-[U-(13)C(6)]glucose were infused to measure the fractional synthetic rates of DNA (reflecting cell proliferation) and protein in the wound. RESULTS: In the arginine group (n = 6) plasma arginine concentration was increased to 2.8 fold that in the control group (n = 8), which was a less increase than that of 6.4 fold for ornithine. Wound DNA fractional synthetic rate was 5.37 ± 0.21%/day in the arginine group, greater (p < 0.05) than that of 4.27 ± 0.35%/day in the control group. Protein fractional synthetic rates in the wound were comparable between the two groups. CONCLUSIONS: Enternal arginine supplementation increased wound DNA synthesis, which is anticipated to promote cell proliferation for wound healing. The failure of enteral arginine to stimulate protein synthesis is explained by limited increase in plasma arginine. Decreased availability of essential amino acids, especially branched chain amino acids, may also contribute to the failure to stimulate protein synthesis.

Informatics for cross-sample analysis with comprehensive two-dimensional gas chromatography and high-resolution mass spectrometry (GCxGC-HRMS).

This paper describes informatics for cross-sample analysis with comprehensive two-dimensional gas chromatography (GCxGC) and high-resolution mass spectrometry (HRMS). GCxGC-HRMS analysis produces large data sets that are rich with information, but highly complex. The size of the data and volume of information requires automated processing for comprehensive cross-sample analysis, but the complexity poses a challenge for developing robust methods. The approach developed here analyzes GCxGC-HRMS data from multiple samples to extract a feature template that comprehensively captures the pattern of peaks detected in the retention-times plane. Then, for each sample chromatogram, the template is geometrically transformed to align with the detected peak pattern and generate a set of feature measurements for cross-sample analyses such as sample classification and biomarker discovery. The approach avoids the intractable problem of comprehensive peak matching by using a few reliable peaks for alignment and peak-based retention-plane windows to define comprehensive features that can be reliably matched for cross-sample analysis. The informatics are demonstrated with a set of 18 samples from breast-cancer tumors, each from different individuals, six each for Grades 1-3. The features allow classification that matches grading by a cancer pathologist with 78% success in leave-one-out cross-validation experiments. The HRMS signatures of the features of interest can be examined for determining elemental compositions and identifying compounds.

The synthetic rate of muscle triglyceride but not phospholipid is increased in obese rabbits.

Fat is a major energy source for skeletal muscle, and disruption of normal trafficking of fatty acids in muscle is linked to insulin resistance. We quantified muscle triglyceride (TG) and phospholipid (PL) synthesis in lean and obese rabbits by means of l-[U-(13)C(16)]palmitate infusion. Intramyocellular palmitoyl-coenzyme A was used as the precursor for rates of TG and PL synthesis, which were compared with the rates calculated using plasma nonesterified palmitate as the precursor. The muscle of obese rabbits had a greater (P < .05) combined pool of fatty acyl-coenzyme A plus fatty acyl-carnitine than lean rabbits (40.9 +/- 3.7 vs 28.6 +/- 5.3 nmol/g). Although the fractional synthetic rates of muscle TG were almost identical (0.095%/h +/- 0.016%/h vs 0.092%/h +/- 0.019%/h), the absolute synthetic rates were greater (P < .01) in the obese than in lean rabbits (39.7 +/- 9.5 vs 10.1 +/- 2.5 nmol g(-1) h(-1)) because of greater TG content in the muscle of obese rabbits. Plasma nonesterified fatty acids and TG accounted for 51% to 55% of the true precursor pool for muscle lipid synthesis in both groups, and the rest was derived from fatty acids recycled within the muscle. In contrast, the fractional and absolute synthetic rates of muscle PL as well as PL contents were comparable in the 2 groups. In conclusion, the content and synthetic rate of muscle TG rather than PL were increased in obese rabbits, which might be linked to insulin resistance. Plasma lipids and muscle lipolysis were the 2 predominate contributors to the intramyocellular fatty acyl-coenzyme A pool for lipid synthesis.

Determination of phenylalanine isotope ratio enrichment by liquid chromatography/time- of-flight mass spectrometry.

The application of time-of-flight mass spectrometry to isotope ratio measurements has been limited by the relatively low dynamic range of the time-to-digital converter detectors available on commercial LC/ToF-MS systems. Here we report the measurement of phenylalanine isotope ratio enrichment by using a new LC/ToF-MS system with wide dynamic range. Underivatized phenylalanine was injected onto a C18 column directly with 0.1% formic acid/acetonitrile as the mobile phase. The optimal instrument parameters for the time-of-flight mass spectrometer were determined by tuning the instrument with a phenylalanine standard. The accuracy of the isotope enrichment measurement was determined by the injection of standard solutions with known isotope ratios ranging from 0.02% to 9.2%. A plot of the results against the theoretical values gave a linear curve with R2 of 0.9999. The coefficient of variation for the isotope ratio measurement was below 2%. The method is simple, rapid, and accurate and presents an attractive alternative to traditional GC/MS applications.

Fractional synthesis rates of DNA and protein in rabbit skin are not correlated.

We developed a method for measurement of skin DNA synthesis, reflecting cell division, in conscious rabbits by infusing D-[U-(13)C(6)]glucose and L-[(15)N]glycine. Cutaneous protein synthesis was simultaneously measured by infusion of L-[ring-(2)H(5)]phenylalanine. Rabbits were fitted with jugular venous and carotid arterial catheters, and were studied during the infusion of an amino acid solution (10% Travasol). The fractional synthetic rate (FSR) of DNA from the de novo nucleotide synthesis pathway, a reflection of total cell division, was 3.26 +/- 0.59%/d in whole skin and 3.08 +/- 1.86%/d in dermis (P = 0.38). The de novo base synthesis pathway accounted for 76 and 60% of the total DNA FSR in whole skin and dermis, respectively; the contribution from the base salvage pathway was 24% in whole skin and 40% in dermis. The FSR of protein in whole skin was 5.35 +/- 4.42%/d, which was greater (P < 0.05) than that in dermis (2.91 +/- 2.52%/d). The FSRs of DNA and protein were not correlated (P = 0.33), indicating that cell division and protein synthesis are likely regulated by different mechanisms. This new approach enables investigations of metabolic disorders of skin diseases and regulation of skin wound healing by distinguishing the 2 principal components of skin metabolism, which are cell division and protein synthesis.