Chain Acylcarnitine Levels Research Articles

Short and medium chain acylcarnitines as markers of outcome in diabetic and non-diabetic subjects with acute coronary syndromes

Abstract Background Dietary carnitine that is not absorbed can serve as a precursor for gut microbiota-dependent generation of trimethylamine N-oxide (TMAO), a pro-atherogenic and pro-thrombosis promoting metabolite. Gut microbiome-derived metabolites of dietary carnitine, including TMAO and g-butyrobetaine, may accelerate atherosclerosis, increase platelet reactivity and in vivo thrombosis. Carnitine metabolism also produces numerous molecular species of short, medium and long chain acylcarnitines, which play important roles in energy metabolism and intracellular fatty acid transport. Purpose We sought to evaluate the differences between diabetics and non-diabetics presenting with ACS with respect to acylcarnitines, and to explore their relationship with incident cardiovascular outcomes. Methods Using a large, prospectively recruited cohort of patients presenting to the cardiac cath lab with suspected acute coronary syndromes, we measured levels of plasma acylcarnitines, carnitine and its gut microbial-derived metabolites to assess their relationship with independently adjudicated major adverse cardiac events (MACE = myocardial infarction, stroke or TIA, need for revascularization or all-cause mortality) amongst diabetics and non-diabetics. Results We analysed 1683 patients who presented with ACS, were treated according to current guidelines and had undergone acylcarnitine analysis. There were 294 diabetics and 1389 non-diabetics. Diabetics had significantly higher plasma levels of all acyl carnitine metabolites than non-diabetics (P<0.001), but not of carnitine itself. Baseline plasma levels of all gut microbiome derived carnitine metabolites (TMAO, g-butyrobetaine and crotonobetaine) were also significantly higher in those who subsequently experienced a MACE. All carnitine metabolites, apart from octenoylcarnitine, were significantly associated with MACE on univariate analysis, while acetylcarnitine and crotonobetaine were independently associated with MACE after multivariate adjustment. Conclusion Serum short- and medium- chain acylcarnitine levels are significantly higher in diabetic patients presenting with ACS and predict MACE. After multivariate adjustment, acetylcarnitine and crotonobetaine remained an independent predictor of MACE. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): Zurich Heart House - Foundation for Cardiovascular Research

Impaired acylcarnitine profile in transfusion-dependent beta-thalassemia major patients in Bangladesh

Patients with beta-thalassemia major (BTM) suffer from fatigue, poor physical fitness, muscle weakness, lethargy, and cardiac complications which are related to an energy crisis. Carnitine and acylcarnitine derivatives play important roles in fatty acid oxidation, and deregulation of carnitine and acylcarnitine metabolism may lead to an energy crisis. The present study aimed to investigate carnitine and acylcarnitine metabolites to gain an insight into the pathophysiology of BTM. Dried blood spots of 45 patients with BTM and 96 age-matched healthy controls were analyzed for free carnitine and 24 acylcarnitines by using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Although medium chain acylcarnitine levels were similar in the patients with BTM and healthy controls, free carnitine, short chain acylcarnitines, long chain acylcarnitines, and total acylcarnitine levels were significantly lower in patients with BTM than in the healthy controls (P < 0.05). Moreover, an impaired fatty acid oxidation rate was observed in the patients with BTM, as manifested by decreased fatty acid oxidation indicator ratios, namely C2/C0 and (C2 + C3)/C0. Furthermore, an increase in the C0/(C16 + C18) ratio indicated reduced carnitine palmitoyltransferase-1 (CPT-1) activity in the patients with BTM compared with that in the healthy controls. Thus, a low level of free carnitine and acylcarnitines together with impaired CPT-1 activity contribute to energy crisis-related complications in the patients with BTM.

Effects of Acute Aerobic Exercise on Acylcarnitine Metabolomics in Skeletal Muscle of Lean vs Overweight/Obese Men

Aerobic exercise improves skeletal muscle mitochondrial function and completeness of fatty acid beta oxidation, which contribute to reductions in lipid accumulation and improved insulin sensitivity. The aim of this study was to determine the acute effects of exercise on the completeness of beta oxidation in lean vs. overweight and obese (Ov/Ob) skeletal muscle. 30 men, aged 19–30 years, were recruited and divided into two groups: lean (BMI&lt;25, 18.5–24.1 kg/m2, n=15) and Ov/Ob (BMI≥25, 25.5–36.9 kg/m2, n=15). Four hours after eating a standardized high‐carbohydrate breakfast (7 kcal/kg; 60% carbohydrate, 25% fat, 15% protein), participants performed an acute bout of cycling exercise (50% VO2max, spending ~650 kcal). Vastus lateralis muscle biopsy samples were collected prior to (Pre) and immediately following (Post) the acute exercise bout. In skeletal muscle, metabolomics of acylcarnitines was determined by tandem MS and data were analyzed using JMP. Pairwise correlation maps were generated for all skeletal muscle acylcarnitines, Pre and Post, in lean and Ov/Ob. Strong (correlation coefficient greater than 0.6) positive correlations were observed between long and medium chain acylcarnitine species in lean Pre and between long and short chain species in lean Post. In Ov/Ob, strong positive correlations were observed among long chain species Pre and between long, medium and short chain species Post. Ov/Ob Pre exhibited a smaller number of positive correlations between long and medium/short chain acylcarnitines compared to lean Pre, indicating more incomplete beta oxidation in Ov/Ob Pre compared to lean Pre. Ov/Ob Post exhibited a higher number of positive correlations between long and medium/short chain fatty acids compared to lean Post, indicating a shift Post exercise to more complete beta oxidation in Ov/Ob Post than in lean Post. Prior to exercise, C2, C10‐OH, C12‐OH, C18:1, C18:2‐OH, C22:4, C22:3 were increased in Ov/Ob compared to lean. Acute exercise resulted in significantly increased levels of short chain acylcarnitnes C2, C3, C4, C5:1, C5, C4‐OH and free carnitine in lean and C2 and C4‐OH in Ov/Ob, with other short chain acylcarnitines tending to increase in Ov/Ob. Medium chain acylcarnitines, C6, C8:1, C7‐DC, C10 and C8‐DC increased in lean Post compared to lean Pre; whereas, C8:1, C7‐DC, C10 and C12 increased in Ob/Ov Post compared to lean Post. There no changes in long chain acylcarnitine levels in lean Post vs. lean Pre; however, C14, C18:1, C20:2, C20:1, C20, C22:3, C22:2 and C22:1 increased in Ov/Ob Post compared to Ov/Ob Pre. Thus, exercise may increase skeletal muscle mitochondrial beta oxidation of fatty acids in both lean and Ov/Ob and may be more effective in increasing completeness of beta oxidation in Ov/Ob.Support or Funding InformationThis research was funded by a Ralph W. and Grace M. Showalter Research Trust grant and Indiana CTSI funds to TMH.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract LB-200: Integrating dynamic 18F-FDG PET-CT, tumor metabolomics and functional genomics to understand metformin's pharmacodynamic effects in breast cancer: results of a phase 0 clinical trial

Abstract There is growing interest in the anticancer effect of the diabetes drug, metformin, and over 100 clinical trials are underway worldwide. However it is still not determined as to whether metformin has significant direct effects on cancer cells or solely indirect effects via modulation of host metabolism. We recruited 41 non-diabetic patients with primary breast cancer to a neoadjuvant window trial. Patients received an escalating dose of metformin (500mg up to 1500mg) for 2 weeks with pre- and post-metformin pharmacodynamic assessments including, dynamic 18F-FDG PET-CT scans, serum metabolic markers, and tumour biopsies for whole transcriptome RNASeq, tumour metabolomics and immunohistochemistry. Assessment of tumour FDG kinetics using a classic 2-tissue compartment model with three rate constants showed a 1.3 fold change (FC) post-metformin in the composite 18F-FDG flux constant, Kflux (p = 0.041, on paired t-test). There was a non-statistically significant 1.1 fold increase in the variable, metabolic rate of glucose utilization (MRglu) (p = 0.141). Mass spectrometry analysis revealed a decrease in intra-tumoral levels of the short-chain acyl-carnitines, propionylcarnitine (FC -0.50, p = 0.039) and acetylycarnitine (FC -0.40, p = 0.046) consistent with inhibition of fatty acid oxidation (wilcoxon rank test). This effect on fatty acid oxidation was validated in pre-clinical in vitro and in vivo breast cancer cell line models. There was a statistically significant positive correlation between the actual difference in Kflux and intratumoral levels of acetylcarnitine (p = 0.012, Spearman's rank test). Small but consistent falls in the levels of serum glucose (p = 0.032), c-peptide (p = 0.001), insulin (p = 0.005), IGF1 (p = 0.048) and IGF2 (p = 0.040) were observed following metformin treatment (paired t-test) but there was no correlation with change in Kflux or short chain acyl-carnitine levels. Pathway annotation analysis using GeneCodis revealed several pathways associated with mitochondrial and fatty acid metabolism that were upregulated. Immunohistochemical intratumoral nuclear expression of pAMPK increased 1.5 fold (p = 0.037, paired t-test) but this did not correlate with change in Kflux or levels of short-chain acyl-carnitines. Peak serum metformin levels correlated with intratumoral metformin levels (p = 0.012, Spearman's rank test) but did not correlate with change in Kflux or short-chain acyl-carnitine levels. Our data shows that metformin treatment alters FDG kinetics, inhibits fatty acid oxidation and leads to altered gene expression in the mitochondrial (nuclear encoded) transcriptome but that these effects do not correlate with changes in host metabolism. This data provides strong evidence that metformin has a direct effect on breast cancer metabolism at clinical doses. Citation Format: Simon Lord, Daniel Liu, Syed Haider, Edoardo Gaude, Eugene Teoh, Patel Neel, Qifeng Zhang, Fergus Gleeson, Michael Wakelam, Christian Frezza, Francesca Buffa, Fenwick John, Adrian Harris. Integrating dynamic 18F-FDG PET-CT, tumor metabolomics and functional genomics to understand metformin's pharmacodynamic effects in breast cancer: results of a phase 0 clinical trial. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-200.

Abstract 69: Characterization of the Role of Glycerol-3-phosphate Acyltransferase-3 in Intestinal Lipid Absorption

Glycerol-3-phosphate acyltransferases (GPAT) catalyze the first step in the synthesis of glycerolipids and glycerophospholipids. Microsomal GPAT, the major GPAT activity, is encoded by at least two closely related genes, GPAT3 and GPAT4 (also known as AGPAT9 and 8, respectively). The physiological role of GPAT3 is poorly understood. GPAT3 mRNA was found to be abundantly expressed in small intestine which was confirmed by immunohistochemistry. The GPAT3 protein was highly enriched in enterocytes suggesting a potential role in intestinal lipid absorption. To further investigate this possibility, we generated Gpat3-deficient mice (Gpat3-/-). The Gpat3-/- mice were viable and fertile, and exhibited no obvious metabolic abnormalities on standard laboratory chow. As expected, Gpat3-/- mice had reduced intestinal GPAT activity compared to wild-type animals and when challenged with an oral lipid bolus, Gpat3-/- mice displayed attenuated plasma triglyceride excursion. Histological and biochemical analysis of the small intestine revealed dramatically increased lipid deposition in the intestinal mucosa of Gpat3-/- mice. Long chain acylcarnitine levels were elevated in the jejunum of Gpat3-/- suggesting a compensatory increase in mitochondrial fatty acid oxidation. Lipid flux studies performed in primary cultures of enterocytes derived from wild-type and Gpat3-/- mice reveal a defect in triacylglycerol trafficking suggesting a role for GPAT3 in the post-prandial secretion of lipid in the intestine. Finally, gene expression analysis revealed dysregulation of multiple genes involved in lipid absorption, chylomicron assembly, and secretion. Taken together, our results demonstrate a hitherto unappreciated role for GPAT3 in lipid absorption.

Clinical and environmental influences on metabolic biomarkers collected for newborn screening

ObjectivesIdentifying common clinical and environmental factors that influence newborn metabolic biomarkers will improve the utilization of metabolite panels for clinical diagnostic medicine. Design and methodsEnvironmental effects including gender, season of birth, gestational age, birth weight, feeding method and age at time of collection were evaluated for over 50 metabolites collected by the Iowa Neonatal Metabolic Screening Program on 221,788 newborns over a six year period. ResultsWe replicated well known observations that low birth weight and preterm infants have higher essential amino acids and lower medium and long chain acylcarnitine levels than their term counterparts. Smaller, but still significant, differences were observed for gender and timing of sample collection, specifically the season in which the infant was born. Most intriguing were our findings of higher thyroid stimulating hormone in the winter months (P<1×10−40) which correlated with an increased false positive rate of congenital hypothyroidism in the winter (0.9%) compared to summer (0.6%). Previous studies, conducted globally, have identified an increased prevalence of suspected and confirmed cases of congenital hypothyroidism in the winter months. We found that the percentage of unresolved suspected cases were slightly higher in the winter (0.3% vs. 0.2%). ConclusionsWe identified differences in metabolites by gestational age, birth weight, gender and season. Some are widely reported such as gestational age and birth weight, while others such as the effect of seasonality are not as well studied.

The metabolic effects of two different lipid emulsions used in parenterally fed premature infants — A randomized comparative study

ObjectiveTo compare the effects of two different lipid emulsions, based on soybean oil and olive oil respectively on plasma lipid concentrations and acylcarnitine profile of very low birth weight infants. DesignRandomized comparative study. Patients and methodsForty very low birth weight infants, ≤32weeks of gestational age and receiving at least 40% of the calorie taken by parenteral nutrition from lipid solution at 14th day of life were evaluated. Group I (n=20) received soybean oil based lipid emulsion (Intralipid®) and Group II (n=20) received olive oil based lipid emulsion (Clinoleic®). Main outcome measuresPlasma lipid concentrations and acylcarnitine profile were assessed. ResultsTriglyceride, cholesterol, high and low density lipoprotein levels, liver function tests were similar between two groups whereas very low density lipoprotein level was statistically lower in Group I (p<0.05). Free carnitine levels were 15.73±10.67 in Group I and 34.25±22.18μM in Group II (p=0.012) and hexanoyl carnitine levels 2.18±2.10 in Group I and 0.38±0.12μM in Group II, respectively (p=0.005). Plasma medium chain acylcarnitine levels were significantly higher in Group I. ConclusionsLow levels of very low density lipoprotein in Group I may be a way of hemostasis to keep the serum triglyceride within normal levels. Lower free carnitine levels in soybean oil-based group is the result of carnitine need during the mitochondrial transport of long chain fatty acids. In Group I, due to the inefficient transport of medium chain fatty acids into the mitochondria, medium chain acylcarnitines accumulate in plasma. This may be the reason of lower carnitine levels in Group I. We suggest that higher levels of hexanoyl carnitine, reflecting defective mitochondrial transport of hexanoyl which leads immunsupression, may be the cause of higher sepsis risk in Group I.

Reduced carnitine palmitoyl transferase activity and altered acyl-trafficking in red blood cells from hemodialysis patients

We measured carnitine palmitoyl transferase activity, free carnitine, and long chain acyl carnitine levels in erythrocytes from 15 uremic patients and 25 controls. Carnitine palmitoyl transferase levels in patients were significantly lower than in controls. The levels of free carnitine and long chain acyl carnitines as well as the long chain acyl carnitine/free carnitine ratio were significantly higher in patients than in controls. Our results suggest that hemodialysis causes alteration in the acyl-trafficking in red blood cells membrane.

Response of isolated working hearts to fatty acids and carnitine palmitoyltransferase I inhibition during reduction of coronary flow in acutely and chronically diabetic rats.

The effects of palmitate on mechanical failure of ischemic hearts were studied in acutely (48-hour) and chronically (6-week) streptozotocin diabetic rats. Coronary flow was reduced by 50% in isolated working hearts perfused at a 15 cm H2O preload and 100 mm Hg afterload by the one-way ball valve model of ischemia. Peak systolic pressure (PSP) and cardiac output (CO) decreased 40% by 4 minutes in control hearts perfused with 11 mM glucose and paced at 280 beats/min, compared with 50% in hearts from acutely diabetic rats. Addition of 1.2 mM palmitate to the perfusate accelerated failure rates, with PSP and CO decreasing 65% and 80% by 4 minutes in control and acutely diabetic rat hearts, respectively. In chronically diabetic rats, mechanical function could not be maintained in palmitate-perfused hearts paced at 280 beats/min, even in the absence of ischemia. If these hearts were paced at 250 beats/min and subjected to ischemia, PSP and CO decreased 90% by 4 minutes, regardless of whether palmitate was added to the perfusate. Under these conditions, PSP decreased less than 10% by 4 minutes in both palmitate- or glucose-perfused control hearts. Etomoxir (10(-9) M), a carnitine palmitoyltransferase I inhibitor, markedly decreased the rate of mechanical failure in both acutely and chronically diabetic rat hearts, in the presence and absence of palmitate. The beneficial effect of Etomoxir on mechanical function did not occur as a result of a decrease in either myocardial long chain acyl-coenzyme A or long chain acylcarnitine levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucose oxidation is stimulated in reperfused ischemic hearts with the carnitine palmitoyltransferase 1 inhibitor, Etomoxir.

The effect of the carnitine palmitoyltransferase 1 (CPT 1) inhibitor, Etomoxir, on glucose oxidation rates was determined in ischemic hearts reperfused in the presence of fatty acids. Isolated working rat hearts were perfused with 11 mM (14C)-glucose and 1.2 mM palmitate at a 15 cm H2O preload, 80 mm Hg afterload. Hearts were subjected to either 60 min aerobic perfusion, or 15 min work followed by 25 min global ischemia then 60 min of aerobic reperfusion. Steady state glucose oxidation rates in reperfused ischemic hearts were not significantly different from non-ischemic hearts. If 10(-9) M Etomoxir was added immediately prior to reperfusion no significant change in glucose oxidation occurred. Addition of 10(-8) M and 10(-6) M Etomoxir, however, significantly increased glucose oxidation. Etomoxir also significantly improved recovery of mechanical function at a concentration of 10(-8) M or greater. As we previously reported, no significant improvement of function was seen when 10(-9) M Etomoxir was added to the perfusate (Lopaschuk GD et al., Circ Res 63: 1036-1043, 1988). Long chain acylcarnitine levels were significantly reduced in the presence of both 10(-9) M and 10(-8) M Etomoxir. These data demonstrate that the beneficial effect of Etomoxir on reperfusion recovery of ischemic hearts is not due to a lowering of long chain acylcarnitine levels. Etomoxir may improve recovery of function by overcoming fatty acid inhibition of glucose oxidation.

Antiarrhythmic and myocardial metabolic effects of verapamil during coronary artery reperfusion.

The accumulation of metabolic intermediates subsequent to impaired beta-oxidation of free fatty acids has been suggested to be a cause of cellular damage and ventricular arrhythmias in the ischemic heart. The effects of verapamil on ventricular arrhythmias and free fatty acids metabolism during coronary artery reperfusion in experimental dogs were evaluated over a period of 40 minutes and followed by reperfusion for 15 minutes. One tenth mg/kg/min of verapamil was administered for 5 minutes before occlusion and followed by an infusion of 0.01 mg/kg/min to the end of the experiment. Myocardial samples were obtained from both the non-ischemic and ischemic areas after coronary artery reperfusion and then ATP, free carnitine, long chain acyl carnitine and long chain acyl CoA were measured. In the control group, 3 dogs (27%) had ventricular fibrillation and 2 dogs (18%) had ventricular tachycardia during coronary occlusion. In addition, 2 dogs (25%) developed ventricular fibrillation after reperfusion. On the other hand, all 6 dogs treated with verapamil had neither ventricular fibrillation nor tachycardia during both coronary artery occlusion and reperfusion. ATP and free carnitine levels in the ischemic area were significantly higher in the verapamil group than in the control group (ATP: p less than 0.01, free carnitine: p less than 0.05), while long chain acyl carnitine levels in the ischemic area were significantly lower in the verapamil group than in the control group (p less than 0.01). However, there was no significant change in long chain acyl CoA levels between the control and verapamil groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Lack of effect of thyroid hormone on diabetic rat heart function and biochemistry.

Cardiac functional abnormalities are frequently seen in diabetics and diabetes is also known to produce a state of mild hypothyroidism. To study the degree of involvement of diabetes-induced hypothyroidism on altered myocardial function, thyroid replacement therapy was carried out in streptozotocin-diabetic rats. Triiodothyronine (T3) treatment was initiated 3 days after the rats were made diabetic and was carried out for 6 weeks thereafter. Isolated perfused hearts from diabetic rats exhibited a depression in left ventricular developed pressure and positive and negative dP/dt at higher filling pressures as compared with controls. The depression could not be prevented by thyroid treatment. Calcium uptake activity in the cardiac sarcoplasmic reticulum (SR) was also depressed as a result of diabetes and this depression also was not prevented by thyroid treatment. Long chain acyl carnitine levels were found to be elevated in diabetic cardiac SR and could not be lowered by T3 treatment. The results indicate that the myocardial dysfunction observed in diabetic rats is due to factors other than the induced hypothyroidism.