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Tandem MS acylcarnitine “profiles” are extremely valuable. Although used appropriately in newborn screening programs to identify patients with possible diseases, their inadequate quantitative accuracy and lack of selectivity is problematic for confirmatory testing. In this report, we show the application of our validated, selective, accurate, precise, and robust UHPLC–MS/MS method for quantitation of acylcarnitines, specifically to C5 acylcarnitines: pivaloyl-, 2-methylbutyryl-, isovaleryl-, and valerylcarnitine. Standardized calibrants were used to generate 13-point, 200-fold concentration range calibration curves. Samples were isolated by solid-phase extraction and derivatized with pentafluorophenacyl trifluoromethanesulfonate. Acylcarnitine pentafluorophenacyl esters were eluted in 14 min chromatograms. Data demonstrating quantitative stability and method robustness over a five year time period are shown and these results validate the method’s accuracy and robustness. Urine from patients with isovaleric acidemia (with the disease marker isovalerylcarnitine) and with pivaloylcarnitine present are shown. These results demonstrate the method’s ability to distinguish true isovaleric acidemia from pivalate derived interference. Our method for acylcarnitine quantitation is shown to be accurate, precise, and robust for selective quantitation of isovalerylcarnitine, and thus is recommended for confirmatory testing of suspected isovaleric acidemia patients. [ABSTRACT FROM AUTHOR]

While selectively quantifying acylcarnitines in thousands of patient samples using UHPLC–MS/MS, we have occasionally observed unidentified branched-chain C8 acylcarnitines. Such observations are not possible using tandem MS methods, which generate pseudo-quantitative acylcarnitine “profiles”. Since these “profiles” select for mass alone, they cannot distinguish authentic signal from isobaric and isomeric interferences. For example, some of the samples containing branched-chain C8 acylcarnitines were, in fact, expanded newborn screening false positive “profiles” for medium-chain acyl-CoA dehydrogenase deficiency (MCADD). Using our fast, highly selective, and quantitatively accurate UHPLC–MS/MS acylcarnitine determination method, we corrected the false positive tandem MS results and reported the sample results as normal for octanoylcarnitine (the marker for MCADD). From instances such as these, we decided to further investigate the presence of branched-chain C8 acylcarnitines in patient samples. To accomplish this, we synthesized and chromatographically characterized several branched-chain C8 acylcarnitines (in addition to valproylcarnitine): 2-methylheptanoylcarnitine, 6-methylheptanoylcarnitine, 2,2-dimethylhexanoylcarnitine, 3,3-dimethylhexanoylcarnitine, 3,5-dimethylhexanoylcarnitine, 2-ethylhexanoylcarnitine, and 2,4,4-trimethylpentanoylcarnitine. We then compared their behavior with branched-chain C8 acylcarnitines observed in patient samples and demonstrated our ability to chromographically resolve, and thus distinguish, octanoylcarnitine from branched-chain C8 acylcarnitines, correcting false positive MCADD results from expanded newborn screening. [ABSTRACT FROM AUTHOR]

Using examples from our analysis of L-carnitine and acyl-L-carnitines, we give specific guidance for the use of mass spectrometry (MS) in quantitative analysis as applied to clinical research and clinical pharmacology. We focus on quantitative accuracy and analytical selectivity as keys to successful implementation of MS methods in clinical applications. [ABSTRACT FROM AUTHOR]

Tandem MS “profiling” of acylcarnitines and amino acids was conceived as a first-tier screening method, and its application to expanded newborn screening has been enormously successful. However, unlike amino acid screening (which uses amino acid analysis as its second-tier validation of screening results), acylcarnitine “profiling” also assumed the role of second-tier validation, due to the lack of a generally accepted second-tier acylcarnitine determination method. In this report, we present results from the application of our validated UHPLC-MS/MS second-tier method for the quantification of total carnitine, free carnitine, butyrobetaine, and acylcarnitines to patient samples with known diagnoses: malonic acidemia, short-chain acyl-CoA dehydrogenase deficiency (SCADD) or isobutyryl-CoA dehydrogenase deficiency (IBD), 3-methyl-crotonyl carboxylase deficiency (3-MCC) or ß-ketothiolase deficiency (BKT), and methylmalonic acidemia (MMA). We demonstrate the assay's ability to separate constitutional isomers and diastereomeric acylcarnitines and generate values with a high level of accuracy and precision. These capabilities are unavailable when using tandem MS “profiles”. We also show examples of research interest, where separation of acylcarnitine species and accurate and precise acylcarnitine quantification is necessary. [ABSTRACT FROM AUTHOR]

A validated quantitative method for the determination of free and total carnitine, butyrobetaine, and acylcarnitines is presented. The versatile method has four components: (1) isolation using strong cation-exchange solid-phase extraction, (2) derivatization with pentafluorophenacyl trifluoromethanesulfonate, (3) sequential ion-exchange/reversed-phase (ultra) high-performance liquid chromatography [(U)HPLC] using a strong cation-exchange trap in series with a fused-core HPLC column, and (4) detection with electrospray ionization multiple reaction monitoring (MRM) mass spectrometry (MS). Standardized carnitine along with 65 synthesized, standardized acylcarnitines (including short-chain, mediumchain, long-chain, dicarboxylic, hydroxylated, and unsaturated acyl moieties) were used to construct multiple-point calibration curves, resulting in accurate and precise quantification. Separation of the 65 acylcarnitines was accomplished in a single chromatogram in as little as 14 min. Validation studies were performed showing a high level of accuracy, precision, and reproducibility. The method provides capabilities unavailable by tandem MS procedures, making it an ideal approach for confirmation of newborn screening results and for clinical and basic research projects, including treatment protocol studies, acylcarnitine biomarker studies, and metabolite studies using plasma, urine, tissue, or other sample matrixes. [ABSTRACT FROM AUTHOR]

Background: In [16,16,16-d3]palmitic acid (M+3) perfused working hearts, M+3 stearoylcarnitine is formed. Results: Rat hearts chain-elongate palmitate to stearate and arachidate. Furthermore, rat heart mitochondria catalyze the malonyl/acetyl-CoA-dependent chain elongation of palmitoyl-CoA. Conclusion: The two-carbon units for fatty acid chain elongation are derived from mitochondrial fatty acid β-oxidation. Significance: This pathway may represent the molecular basis for heart-specific fatty acid remodeling. Rat hearts were perfused with [1,2,3,4-13C4]palmitic acid (M+4), and the isotopic patterns of myocardial acylcarnitines and acyl-CoAs were analyzed using ultra-HPLC-MS/MS. The 91.2% 13C enrichment in palmitoylcarnitine shows that little endogenous (M+0) palmitate contributed to its formation. The presence of M+2 myristoylcarnitine (95.7%) and M+2 acetylcarnitine (19.4%) is evidence for β-oxidation of perfused M+4 palmitic acid. Identical enrichment data were obtained in the respective acyl-CoAs. The relative 13C enrichment in M+4 (84.7%, 69.9%) and M+6 (16.2%, 17.8%) stearoyl- and arachidylcarnitine, respectively, clearly shows that the perfused palmitate is chain-elongated. The observed enrichment of 13C in acetylcarnitine (19%), M+6 stearoylcarnitine (16.2%), and M+6 arachidylcarnitine (17.8%) suggests that the majority of twocarbon units for chain elongation are derived from β-oxidation of [1,2,3,4-13C4]palmitic acid. These data are explained by conversion of the M+2 acetyl-CoA to M+2 malonyl-CoA, which serves as the acceptor for M+4 palmitoyl-CoA in chain elongation. Indeed, the 13Cenrichment in mitochondrial acetyl-CoA (18.9%)andmalonyl- CoA (19.9%) are identical. No 13C enrichment was found in acylcarnitine species with carbon chain lengths between 4 and 12, arguing against the simple reversal of fatty acid β-oxidation. Furthermore, isolated, intact rat heart mitochondria 1) synthesize malonyl-CoA with simultaneous inhibition of carnitine palmitoyltransferase1band2) catalyze the palmitoyl-CoA-dependent incorporation of 14C from [2-14C]malonyl-CoA into lipid-soluble products. In conclusion, rat heart has the capability to chain-elongate fatty acids using mitochondria-derived two-carbon chain extenders. The data suggest that the chain elongation process is localized on the outer surface of the mitochondrial outer membrane. [ABSTRACT FROM AUTHOR]

Abstract: The measurement of acyl-CoA dehydrogenase activities is an essential part of the investigation of patients with suspected defects in fatty acid oxidation. Multiple methods are available for the synthesis of the substrates used for measuring acyl-CoA dehydrogenase activities; however, the yields are low and the products are used without purification. In addition, the reported characterization of acyl-CoAs focuses on the CoA moiety, not on the acyl group. Here we describe the synthesis of three medium-chain acyl-CoAs from mixed anhydrides of the fatty acids using an aqueous–organic solvent mixture optimized to obtain the highest yield. First, cis-4-decenoic acid and 2,6-dimethylheptanoic acid were prepared (3-phenylpropionic acid is commercially available). These were characterized by gas chromatography/mass spectrometry (GC/MS), 1H nuclear magnetic resonance (NMR), and 13C NMR. Then cis-4-decenoyl-CoA, 3-phenylpropionyl-CoA, and 2,6-dimethylheptanoyl-CoA were synthesized. These were then purified by ion exchange solid-phase extraction using 2-(2-pyridyl)ethyl-functionalized silica gel, followed by reversed-phase semipreparative high-performance liquid chromatography with ultraviolet detection (HPLC–UV). The purified acyl-CoAs were characterized by analytical HPLC–UV followed by data-dependent tandem mass spectrometry (MS/MS) analysis on the largest responding MS mass (HPLC–UV–MS–MS/MS) and 13C NMR. The yields of the purified acyl-CoAs were between 75% and 78% based on coenzyme A trilithium salt (CoASH). Acyl-CoA dehydrogenase activities were measured in rat skeletal muscle mitochondria using, as substrates, the synthesized cis-4-decenoyl-CoA, 3-phenylpropionyl-CoA, and 2,6-dimethylheptanoyl-CoA. These results were compared with the results using our standard substrates butyryl-CoA, octanoyl-CoA, and palmitoyl-CoA. [Copyright &y& Elsevier]

Abstract: Rat hearts perfused for up to 60min in the working mode with palmitate, but not with glucose, resulted in substantial formation of palmitoylcarnitine and stearoylcarnitine. To test whether lipolysis of endogenous lipids was responsible for the increased stearoylcarnitine content or whether some of the perfused palmitate underwent chain elongation, hearts were perfused with hexadecanoic-16,16,16-d 3 acid (M+3). The pentafluorophenacyl ester of deuterium labeled stearoylcarnitine had an M+3 (639.4 m/z) compared to the unlabeled M+0 (636.3 m/z) consistent with a direct chain elongation of the perfused palmitate. Furthermore, the near equal isotope enrichment of palmitoyl- (90.2±5.8%) and stearoylcarnitine (78.0±7.1%) suggest that both palmitoyl- and stearoyl-CoA have ready access to mitochondrial carnitine palmitoyltransferase and that most of the stearoylcarnitine is derived from the perfused palmitate. [Copyright &y& Elsevier]

Abstract: We present a validated high-performance liquid chromatography/mass spectrometry (HPLC/MS) method for the quantification of malonyl-coenzyme A (CoA) in tissues. The assay consists of extraction of malonyl-CoA from tissue using 10% trichloroacetic acid, isolation using a reversed-phase solid-phase extraction column, HPLC separation, and detection using electrospray MS. Quantification was performed using an internal standard ([13C3]malonyl-CoA) and multiple-point standard curves from 50 to 1000pmol. The procedure was validated by performing recovery, accuracy, and precision studies. Recoveries of malonyl-CoA were determined to be 28.8±0.9, 48.5±1.8, and 44.7±4.4% (averages±SD, n =5) for liver, heart, and skeletal muscle, respectively. Accuracy was demonstrated by the addition of known amounts of malonyl-CoA to tissue samples. The malonyl-CoA detected was compared with the malonyl-CoA added, and the resulting relationships were linear with slopes and regression coefficients equal to 1. Precision was demonstrated by repetitive analysis of identical samples. These showed a within-run variation between 5 and 11%, and the interbatch repeatability was essentially the same. This procedure was then applied to rat liver, heart, and skeletal muscle, where the malonyl-CoA contents were found to be 1.9±0.6, 1.3±0.4, and 0.7±0.2nmol/g wet weight, respectively, for these tissues. This analytical approach can be extended to the quantification of other acyl-CoA species with no significant modification. [Copyright &y& Elsevier]

Background: Carnitine palmitoyltransferase-II deficiency (CPT-II deficiency) is a rare disorder of lipid metabolism, in which the accumulation of long-chain acylcarnitines is a diagnostic marker. HPLC with fluorescence detection is an attractive analysis method due to its favorable combination of sensitivity, specificity, ease of analysis and minimal capital equipment costs. Methods: Long-chain acylcarnitines were isolated from tissue homogenates (0.5–2 mg wet weight) or plasma (50 μl) using silica gel columns and derivatized with 2-(2,3-naphthalimino)ethyl trifluoromethanesulfonate. Quantitation was by HPLC and fluorescence detection with standard curves (0.0–5.0 nmol/ml) for myristoyl-, palmitoleoyl-, palmitoyl-, oleoyl- and stearoylcarnitine using heptadecanoylcarnitine as the internal standard. Results: Significantly greater amounts of long-chain acylcarnitines were quantified in patients with CPT-II deficiency when compared to controls; e.g. (nmol/ml in patient plasma, controls mean±standard deviation): myristoylcarnitine (0.3, not detectable), palmitoleoylcarnitine (0.5, 0.1±0.1), palmitoylcarnitine (0.9, 0.1±0.0), oleoylcarnitine (3.0, 0.2±0.1), stearoylcarnitine (0.4, not detectable). Conclusions: This method can be used to quantitate long-chain acylcarnitines, illustrating their accumulation in CPT-II deficiency. The analysis was accomplished using inexpensive and widely available instrumentation and is appropriate for research investigators who require precise quantitation of long-chain acylcarnitines in complex biological samples. [Copyright &y& Elsevier]

Two isomers of malonyl-coenzyme A (malonyl-CoA) were detected in a commercial preparation of malonyl-CoA. These compounds were separated by preparative high-performance liquid chromatography (HPLC) and characterized by HPLC/ultraviolet (UV)/mass spectrometry. Both compounds had a UV absorbance maximum at 259–260 nm. Both compounds underwent negative electrospray ionization to produce a [M-H]−quasi-molecular ion at m/z 852 and both compounds underwent collision-induced dissociation to produce a characteristic fragment at m/z 808, all consistent with the structure of malonyl-CoA. Nuclear magnetic resonance spectrometry showed that the two chromatographically distinguishable malonyl-CoAs are structural isomers: the major component is the naturally occurring malonyl-CoA and the contaminant is 3′-dephospho-2′-phospho-coenzyme A. [Copyright &y& Elsevier]

Abstract: A novel procedure for the quantitative isolation and purification of acyl-coenzyme A esters is presented. The procedure involves two steps: (1) tissue extraction using acetonitrile/2-propanol (3+1, v+v) followed by 0.1M potassium phosphate, pH 6.7, and (2) purification using 2-(2-pyridyl)ethyl-functionalized silica gel. Recoveries determined by adding radiolabeled acetyl-, malonyl-, octanoyl-, oleoyl-, palmitoyl-, or arachidonyl-coenzyme A to powdered rat liver varied 93–104% for tissue extraction and 83–90% for solid-phase extraction. The procedure described allows for isolation and purification, with high recoveries, of acyl-coenzyme A esters differing widely in chain length and saturation. [Copyright &y& Elsevier]

Heinen et al reported the use of [U-13C6]gluconolactone to trace the PPP in microorganisms (Appl. Environ. Microbiology 07/2006, p 4743). We adapted this technique for use in perfused hearts and livers from rats. The tissue was extracted in acetonitrile:water (50:50) and the supernatant applied to an ion exchange solid phase extraction cartridge. The sugar phosphates were eluted with 50 mM ammonium formate in 50% methanol-water (recovery was 90%). We used a hybrid triple quadrupole/linear ion trap mass spectrometer (Applied Biosystems) coupled with HPLC to monitor the sugar phosphates using the enhanced resolution mode. We used hydrophilic interaction liquid chromatography to avoid ion pairing reagents and to increase the detection sensitivity. The flux through the PPP is calculated as (uptake of [U-13C6]gluconolactone)/(M6 enrichment of 6-P-gluconate). The latter can be used in tracer amounts because of low background of analytes at M6 and M5. The production of NADPH in the PPP is equal to twice its flux. The contribution of the oxidative branch of the PPP is the enrichment ratio (M5 ribose-5-P)/(M6 6-P-gluconate). [ABSTRACT FROM AUTHOR]

Hepatic mitochondrial fatty acid oxidation and ketogenesis increase during starvation. Carnitine palmitoyltransferase I (CPT-I) catalyses the rate-controlling step in the overall pathway and retains its control over β-oxidation under fed, starved and diabetic conditions. To determine the factors contributing to the reported several-fold increase in fatty acid oxidation in perfused livers, we measured the Vmax and Km values for palmitoyl-CoA and carnitine, the Ki (and IC50) values for malonyl-CoA in isolated liver mitochondria as well as the hepatic malonyl-CoA and carnitine contents in control and 48 h starved rats. Since CPT-I is localized in the mitochondrial outer membrane and in contact sites, the kinetic properties of CPT-I also was determined in these submitochondrial structures. After 48 h starvation, there is: (a) a significant increase in Ki and decrease in hepatic malonyl-CoA content; (b) a decreased Km for palmitoyl-CoA; and (c) increased catalytic activity (Vmax) and CPT-I protein abundance that is significantly greater in contact sites compared with outer membranes. Based on these changes the estimated increase in mitochondrial fatty acid oxidation is significantly less than that observed in perfused liver. This suggests that CPT-I is regulated in vivo by additional mechanism(s) lost during mitochondrial isolation or/and that mitochondrial oxidation of peroxisomal β-oxidation products contribute to the increased ketogenesis by bypassing CPT-I. Furthermore, the greater increase in CPT-I protein in contact sites as compared to outer membranes emphasizes the significance of contact sites in hepatic fatty acid oxidation. [ABSTRACT FROM AUTHOR]

Aging selectively decreases the rate of oxidative phosphorylation in the interfibrillar population of cardiac mitochondria (IFM) located between the myofibers. In contrast, subsarcolemmal mitochondria (SSM), located below the plasma membrane, remain unaffected. IFM from elderly (24-month-old) Fischer 344 rats have a decreased specific activity of complexes III and IV. Complexes III and IV require an inner mitochondrial membrane lipid environment enriched in the oxidatively sensitive phospholipid cardiolipin for maximal activity. We asked if aging decreases the content or alters the composition of cardiolipin as a potential mechanism of the aging defect in IFM. The content and composition of mitochondrial phospholipids were measured in SSM and IFM from adult and aging rat hearts. Aging did not alter the content of mitochondrial phospholipids, including cardiolipin, in either population of mitochondria. The composition of cardiolipin based on characterization of both acyl group and the individual molecular species of cardiolipin was also unaltered by aging. Lipid-mediated oxidative modification of complex III subunits was not detected, making cardiolipin-derived oxidative damage to complex III unlikely. Thus, alterations in cardiolipin are not the mechanism for the aging defect in IFM in Fischer 344 rats. [ABSTRACT FROM AUTHOR]

Presents a study which examined skeletal muscle mitochondrial oxidative metabolism in the aged rat. Materials and methods used; Mitochondrial content and yield; Respiratory properties of skeletal muscle mitochondria isolated from adult and elderly Fischer 344 rats.

Explores the mitochondrial activity of aging skeletal muscle. Decrease in the aerobic capacity of the muscle; Reduction in uncoupling protein-3 abundance; Display of respiratory control ratios.

The period around bariatric surgery offers a unique opportunity to characterize metabolism responses to dynamic shifts in energy, gut function, and anesthesia. We analyzed plasma acylcarnitines in obese women (n = 17) sampled in the overnight fasted/postabsorptive state approximately 1-2 wk before surgery (condition A), the morning of surgery (prior restriction to a 48-h clear liquid diet coupled in some cases a standard polyethylene glycol gut evacuation: condition B), and following induction of anesthesia (condition C). Comparisons tested if 1) plasma acylcarnitine derivatives reflective of fatty acid oxidation (FAO) and xenometabolism would be significantly increased and decreased, respectively, by preoperative gut preparation/negative energy balance (condition A vs. B), and 2) anesthesia would acutely depress markers of FAO. Acylcarnitines associated with fat mobilization and FAO were significantly increased in condition B: long-chain acylcarnitines (i.e., C18:1, ~70%), metabolites from active but incomplete FAO [i.e., C14:1 (161%) and C14:2 (102%)] and medium- to short-chain acylcarnitines [i.e., C2 (91%), R-3-hydroxybutyryl-(245%), C6 (45%), and cis-3,4-methylene-heptanoyl-(17%), etc.]. Branched-chain amino acid markers displayed disparate patterns [i.e., isobutyryl-(40% decreased) vs. isovaleryl carnitine (51% increased)]. Anesthesia reduced virtually every acylcarnitine. These results are consistent with a fasting-type metabolic phenotype coincident with the presurgical "gut preparation" phase of bariatric surgery, and a major and rapid alteration of both fat and amino acid metabolism with onset of anesthesia. Whether presurgical or anesthesia-associated metabolic shifts in carnitine and fuel metabolism impact patient outcomes or surgical risks remains to be evaluated experimentally. [ABSTRACT FROM AUTHOR]

Mitochondrial enoyl-CoA isomerase (ECU) is an auxiliary enzyme involved in unsaturated fatty acid oxidation. In contrast to most of the other enzymes involved in fatty acid oxidation, a deficiency of ECI1 has yet to be identified in humans. We used wild-type (WT) and Ecil-deficient knockout (KO) mice to explore a potential presentation of human ECU deficiency. Upon food withdrawal, Ecil-deficient mice displayed normal blood β-hydroxybutyrate levels (WT 1.09 mM vs. KO 1.10 mM), a trend to lower blood glucose levels (WT 4.58 mM vs. KO 3.87 mM, P=0.09) and elevated blood levels of unsaturated acylcarnitines, in particular C12:l acylcarnitine (WT 0.03 µM vs. KO 0.09 µM, P<0.01). Feeding an olive oil-rich diet induced an even greater increase in C12:l acylcarnitine levels (WT 0.01 oM vs. KO 0.04 µM, P<0.01). Overall, the phenotypic presentation of Ecil-deficient mice is mild, possibly caused by the presence of a second enoyl-CoA isomerase (Eci2) in mitochondria. Knockdown of Eci2 in Ecil-deficient fibroblasts caused a more pronounced accumulation of C12:l acylcarnitine on incubation with unsaturated fatty acids (12-fold, P<0.05). We conclude that Eci2 compensates for Eci1 deficiency explaining the mild phenotype of Eci1-deficient mice. Hypoglycemia and accumulation of C12:1 acylcarnitine might be diagnostic markers to identify ECU deficiency in humans. [ABSTRACT FROM AUTHOR]

Abstract: Several mouse models for mitochondrial fatty acid β-oxidation (FAO) defects have been developed. So far, these models have contributed little to our current understanding of the pathophysiology. The objective of this study was to explore differences between murine and human FAO. Using a combination of analytical, biochemical and molecular methods, we compared fibroblasts of long chain acyl-CoA dehydrogenase knockout (LCAD−/−), very long chain acyl-CoA dehydrogenase knockout (VLCAD−/−) and wild type mice with fibroblasts of VLCAD-deficient patients and human controls. We show that in mice, LCAD and VLCAD have overlapping and distinct roles in FAO. The absence of VLCAD is apparently fully compensated, whereas LCAD deficiency is not. LCAD plays an essential role in the oxidation of unsaturated fatty acids such as oleic acid, but seems redundant in the oxidation of saturated fatty acids. In strong contrast, LCAD is neither detectable at the mRNA level nor at the protein level in men, making VLCAD indispensable in FAO. Our findings open new avenues to employ the existing mouse models to study the pathophysiology of human FAO defects. [Copyright &y& Elsevier]

Inefficient muscle long-chain fatty acid (LCFA) combustion is associated with insulin resistance, but molecular links between mitochondrial fat catabolism and insulin action remain controversial. We hypothesized that plasma acylcarnitine profiling would identify distinct metabolite patterns reflective of muscle fat catabolism when comparing individuals bearing a missense G304A uncoupling protein 3 (UCP3 g/a) polymorphism to controls, because UCP3 is predominantly expressed in skeletal muscle and g/a individuals have reduced whole-body fat oxidation. MS analyses of 42 carnitine moieties in plasma samples from fasting type 2 diabetics (n = 44) and nondiabetics (n = 12) with or without the UCP3 g/a polymorphism (n = 28/genotype: 22 diabetic, 6 nondiabetic/genotype) were conducted. Contrary to our hypothesis, genotype had a negligible impact on plasma metabolite patterns. However, a comparison of nondiabetics vs. type 2 diabetics revealed a striking increase in the concentrations of fatty acylcarnitines reflective of incomplete LCFA beta-oxidation in the latter (i.e. summed C10- to C14-carnitine concentrations were approximately 300% of controls; P = 0.004). Across all volunteers (n = 56), acetylcarnitine rose and propionylcarnitine decreased with increasing hemoglobin A1c (r = 0.544, P < 0.0001; and r = -0.308, P < 0.05, respectively) and with increasing total plasma acylcarnitine concentration. In proof-of-concept studies, we made the novel observation that C12-C14 acylcarnitines significantly stimulated nuclear factor kappa-B activity (up to 200% of controls) in RAW264.7 cells. These results are consistent with the working hypothesis that inefficient tissue LCFA beta-oxidation, due in part to a relatively low tricarboxylic acid cycle capacity, increases tissue accumulation of acetyl-CoA and generates chain-shortened acylcarnitine molecules that activate proinflammatory pathways implicated in insulin resistance. [ABSTRACT FROM AUTHOR]

Inefficient muscle long-chain fatty acid (LCFA) combustion is associated with insulin resistance, but molecular links between mitochondrial fat catabolism and insulin action remain controversial. We hypothesized that plasma acylcarnitine profiling would identify distinct metabolite patterns reflective of muscle fat catabolism when comparing individuals bearing a missense G304A uncoupling protein 3 (UCP3 g/a) polymorphism to controls, because UCP3 is predominantly expressed in skeletal muscle and g/a individuals have reduced whole-body fat oxidation. MS analyses of 42 carnitine moieties in plasma samples from fasting type 2 diabetics (n = 44) and nondiabetics (n = 12) with or without the UCP3 g/a polymorphism (n = 28/genotype: 22 diabetic, 6 nondiabetic/genotype) were conducted. Contrary to our hypothesis, genotype had a negligible impact on plasma metabolite patterns. However, a comparison of nondiabetics vs. type 2 diabetics revealed a striking increase in the concentrations of fatty acylcarnitines reflective of incomplete LCFA β-oxidation in the latter lie, summed C10-to C14-carnitine concentrations were ~300% of controls; P = 0.004). Across all volunteers (n = 56), acetylcarnitine rose and propionylcarnitine decreased with increasing hemoglobin A1c(r= 0.544, P< 0.0001; and r= -0.308, P < 0.05, respectively) and with increasing total plasma acylcarnitine concentration. In proof-of-concept studies, we made the novel observation that C12-C14 acylcarnitines significantly stimulated nuclear factor κ-B activity (up to 200% of controls) in RAW264.7 cells. These results are consistent with the working hypothesis that inefficient tissue LCFA β-oxidation, due in part to a relatively low tricarboxylic acid cycle capacity, increases tissue accumulation of acetyl-C0A and generates chain-shortened acylcarnitine molecules that activate proinflammatory pathways implicated in insulin resistance. [ABSTRACT FROM AUTHOR]

Aims: Heart failure is associated with decreased myocardial fatty acid oxidation capacity and has been likened to energy starvation. Increased fatty acid availability results in an induction of genes promoting fatty acid oxidation. The aim of the present study was to investigate possible mechanisms by which high fat feeding improved mitochondrial and contractile function in heart failure. [ABSTRACT FROM PUBLISHER]

Abstract: We previously showed that, in the perfused rat heart, the capacity of n-fatty acids to generate mitochondrial acetyl-CoA decreases as their chain length increases. In the present study, we investigated whether the oxidation of a long-chain fatty acid, oleate, is inhibited by short-chain fatty acids, acetate or propionate (which do and do not generate mitochondrial acetyl-CoA, respectively). We perfused rat hearts with buffer containing 4 mM glucose, 0.2 mM pyruvate, 1 mM lactate, and various concentrations of either (i) [U-13C]acetate, (ii) [U-13C]acetate plus [1-13C]oleate, or (iii) unlabeled propionate plus [1-13C]oleate. Using mass isotopomer analysis, we determined the contributions of the labeled substrates to the acetyl moiety of citrate (a probe of mitochondrial acetyl-CoA) and to malonyl-CoA. We found that acetate, even at low concentration, markedly inhibits the oxidation of [1-13C]oleate in the heart, without change in malonyl-CoA concentration. We also found that propionate, at a concentration higher than 1 mM, decreases (i) the contribution of [1-13C]oleate to mitochondrial acetyl-CoA and (ii) malonyl-CoA concentration. The inhibition by acetate or propionate of acetyl-CoA production from oleate probably results from a competition for mitochondrial CoA between the CoA-utilizing enzymes. [Copyright &y& Elsevier]

We previously showed that a fraction of the acetyls used to synthesize malonyl-CoA in rat heart derives from partial peroxisomal oxidation of very long and long-chain fatty acids. The 13C labeling ratio (malonyl-CoA)/(acetyl moiety of citrate) was >1.0 with 13C-fatty acids, which yields [13C]acetyl-CoA in both mitochondria and peroxisomes and <1.0 with substrates, which yields [13C]acetyl-CoA only in mitochondria. In this study, we tested the influence of 13C-fatty acid concentration and chain length on the labeling of acetyl-CoA formed in mitochondria and/or peroxisomes. Hearts were perfused with increasing concentrations of labeled docosanoate, oleate, octanoate, hexanoate, butyrate, acetate, or dodecanedioate. In contrast to the liver, peroxisomal oxidation of 1-13C-fatty acids in heart does not form [1-13C]acetate. With [1-13C]docosanoate and [1,1213C2]dodecanedioate, malonyl-CoA enrichment plateaued at 11 and 9%, respectively, with no detectable labeling of the acetyl moiety of citrate. Thus, in the intact rat heart, docosanoate and dodecanedioate appear to be oxidized only in peroxisomes. With [1-13C]oleate or [1-13C]octanoate, the labeling ratio >1 indicates the partial peroxisomal oxidation of oleate and octanoate. In contrast, with [3-13C]octanoate, [1-13C]hexanoate, [1-13C]butyrate, or [1,2-13C2]acetate, the labeling ratio was <0.7 at all concentrations. Therefore, in rat heart, (i) n-fatty acids shorter than 8 carbons do not undergo peroxisomal oxidation, (ii) octanoate undergoes only one cycle of peroxisomal β-oxidation, (iii) there is no detectable transfer to the mitochondria of acetyl-CoA from the cytosol or the peroxisomes, and (iv) the capacity of C2-C18 fatty acids to generate mitochondrial acetyl-CoA decreases with chain length. [ABSTRACT FROM AUTHOR]

Ischemia and reperfusion result in mitochondrial dysfunction, with decreases in oxidative capacity, loss of cytochrome c, and generation of reactive oxygen species. During ischemia of the isolated perfused rabbit heart, subsarcolemmal mitochondria, located beneath the plasma membrane, sustain a loss of the phospholipid cardiolipin, with decreases in oxidative metabolism through cytochrome oxidase and the loss of cytochrome c. We asked whether additional injury to the distal electron chain involving cardiolipin with loss of cytochrome c and cytochrome oxidase occurs during reperfusion. Reperfusion did not lead to additional damage in the distal electron transport chain. Oxidation through cytochrome oxidase and the content of cytochrome c did not further decrease during reperfusion. Thus injury to cardiolipin, cytochrome c, and cytochrome oxidase occurs during ischemia rather than during reperfusion. The ischemic injury leads to persistent defects in oxidative function during the early reperfusion period. The decrease in cardiolipin content accompanied by persistent decrements in the content of cytochrome c and oxidation through cytochrome oxidase is a potential mechanism of additional myocyte injury during reperfusion. [ABSTRACT FROM AUTHOR]

Features a study which showed that ischemia decreased the content of cardiolipin in rabbit heart subsarcolemmal mitochondria with a selectivity corresponding to the decrease in oxidative phosphorylation through cytochrome oxidase. Materials and methods; Rabbit heart model of ischemia; Discussion.

According to current concepts, GNG from lactate involves the transfer of mitochondrial oxaloacetate to the cytosol via the glutamate-aspartate shuttle. This concept was established by showing that AOA, an inhibitor of aminotransferases, markedly decreases GNG from lactate but not from pyruvate (in GNG from pyruvate, oxaloacetate leaves mitochondria as malate). In rat livers perfused with non-recirculating buffer containing 5 mM lactate ± 0.5 mM AOA, we found that AOA decreased the tissue pyruvate concentration 20-fold. We suspected that AOA (carboxymethoxylamine) forms a carboxymethoxamate with pyruvate. We synthesized this methoxamate and set up its assay by LC-MS-MS. In the effluent of livers perfused with 5 mM lactate ± 0.3 mM AOA, we found pyruvate carboxymethoxamate in the presence of AOA (18 ± 3 µM), but not in its absence. When unlabeled lactate was replaced by [13C3]lactate, M3 [13C3]pyruvate carboxymethoxamate (21 µM) was formed in the presence of AOA. In perfusions with 2 mM pyruvate, GNG was not inhibited by 0.5 mM AOA, but we found pyruvate carboxymethoxamate in the effluent perfusate (216 ± 29 µM). The excess pyruvate prevented the inhibition of GNG by AOA. In conclusion, the inhibition of GNG from lactate by AOA involves the trapping of pyruvate as carboxymethoxamate. This trapping occurs upstream of the inhibition of the glutamate-aspartate shuttle by AOA. [ABSTRACT FROM AUTHOR]

We present a new LC-MS-MS technique for measuring the concentration and stable isotope enrichment of reduced and oxidized glutathione (GSH and GSSG) in biological fluids. Because of differences in signal sensitivity and stability of GSH and GSSG, we assay both compounds as similar GSH thioethers. The assay involves (i) spiking the sample with homoglutathione internal standard (glutamate-cysteine-alanine), (ii) reaction with iodoacetate to form a stable thioether with extant GSH, (iii) reaction with mercaptoethanol to reduce GSSG to GSH, and to trap excess iodoacetate (iv) reaction with excess iodoacetonitrile to form a stable thioether nitrile with GSH derived from GSSG reduction, and (v) electrospray-ionization mass spectrometry of thioethers on a 4000 QTrap (Applied Biosystems) coupled to Agilent HPLC. In addition to enhanced mass spectra, ions monitored are 366.1/237.2 for GSH, 347.2/272.1 for GSH derived from GSSG reduction, and 380.1/251.2 for homoglutathione internal standard (carboxymethyl derivative). The method was applied to the measuring of the turnover of plasma GSH/GSSG in rats whose body fluids were 2% 2H-enriched for 2.5 days. GSH and GSSG showed similar patterns of enrichment with half lives of 21 and 23 hr, respectively. [ABSTRACT FROM AUTHOR]

GHB is both a brain metabolite and a drug of abuse (date rape drug). We conducted a metabolomic study of GHB metabolism liver, using GCMS, GC-combustion-IRMS, and LC-MS-MS. Rat livers were perfused with recirculating albuminated buffer containing 4 mM glucose and 2 mM of either unlabeled GHB, [U-13C4]GHB, or [²H6]GHB. In the perfusate, we identified by GC-MS 2,4-dihydroxybutyrate, 3,4-dihydroxybutyrate, and 3-hydroxypropionate. The mass isotopomer distribution of these compounds in livers perfused with GHB, [U-13C4]GHB, or [²H6]GHB confirms that they are derived from the partial β-oxidation of GHB. 3-Hydroxypropionate may derive from the omega-oxidation of 3,4-dihydroxybutyrate (or its CoA ester). GC-combustion-IRMS detected 5 unknown metabolites of [U-13C4]GHB (to be identified). In livers perfused with [U13C4]GHB, acetyl-CoA was unlabeled (even in the absence of glucose), confirming the incomplete β-oxidation of GHB. Citric acid cycle intermediates were labeled from [U-13C4]GHB consistent with entry into the cycle via succinate. LC-MS-MS of liver acyl-CoAs detected an unknown compound (m/z 934 with CoA signature daughter ion at m/z 428). This compound, which becomes M4 with [U-13C4]GHB, and M6 with [²H6]GHB, contains the 4 C and the 6 H of GHB. In livers perfused with unlabeled GHB and either [U-13C6]glucose or [1,2-13C3]acetate, the CoA ester was not labeled. Based on the above and on its rapid migration on the reverse phase LC column, the unknown CoA ester was tentatively identified as 4-phospho-GHB-CoA. [ABSTRACT FROM AUTHOR]

Dietary therapy with triheptanoin, a precursor of anaplerotic propionyl-CoA, is used for the chronic treatment of long-chain fatty acid oxidation disorders (Roe, JCI 110: 252, 2002) and pyruvate carboxylase deficiency (Mochel, Mol Gen Metab 84: 305, 2005). Heptanoate is partially converted in liver to C5-ketone bodies (β-ketopentanoate and β-hydroxypentanoate. These are precursors of propionyl-CoA in peripheral tissues. We tested whether heptanoate can be taken up as such by the brain. Anesthetized mice were infused with [5,6,7-13C3]heptanoate (20 to 100 nmol/g x min) for 30 min before freezing the brains. We assayed the concentration and mass isotopomer distribution of brain acyl-CoAs by LC-MS-MS. We found that the brain concentrations of [5,6,7-13C3]heptanoyl-CoA, [3,4,5-13C3]pentanoyl-CoA, and [1,2,3-13C3]propionyl-CoA increased with the rate of infusion of [5,6,7-13C3]heptanoate. The spectrum of triply-labeled acyl-CoAs demonstrates that heptanoate is taken up as such by the brain, and that it is anaplerotic in this organ. Our data support the hypothesis, presented in (Mol Gen Metab 84: 305, 2005) that the neurological status of a patient with pyruvate carboxylase deficiency was improved by anaplerosis from heptanoate in the brain. These investigations are extended to mice deficient in very long-chain acyl-CoA dehydrogenase deficiency. [ABSTRACT FROM AUTHOR]