Plasma Long-chain Acylcarnitines Research Articles

Circulatory response to exercise relative to oxygen uptake assessed in the follow-up of patients with fatty acid beta-oxidation disorders.

Patients with fatty acid oxidation disorders (FAODs) experience muscle symptoms due to impaired ATP metabolism and the toxicity of accumulated mitochondrial FAO substrates or intermediates, especially during catabolic states. A major issue is the absence of specific and sensible biomarkers to evaluate metabolic equilibrium. The relationship between cardiac output (Q) and oxygen consumption (VO2) during incremental exercise (dQ/dVO2) provides an indirect surrogate of mitochondrial function. A high dQ/dVO2 slope indicates impaired oxidative phosphorylation in skeletal muscle during exercise. Our study aimed to evaluate dQ/dVO2 as a potential marker of the severity of FAODs. We retrospectively collected clinical, laboratory parameters and treatment data for FAOD patients over 6 years old, including a disease severity score, plasma acylcarnitines and cardiopulmonary exercise tests with Q measurement via thoracic bioelectrical impedance. FAO flux was measured in whole blood and in myoblasts when available. We included 27 FAOD patients followed from 2015 to 2022, with deficiencies in LCHAD (n = 10), CPT2 (n = 6), VLCAD (n = 7), or MADD (n = 4). CPT2 deficient patients with severe scores had the highest C18:1-, C16-, C18-acylcarnitines, and dQ/dVO2. In these patients, dQ/dVO2 was positively correlated with C18:1, C16, and C18 acylcarnitines. In a linear multivariate regression model, dQ/dVO2 was significantly associated with the severity score (B = 0.831, p = 0.008) and triheptanoin treatment (B = -0.547, p = 0.025). dQ/dVO2 and plasma long-chain acylcarnitines might be useful to monitor CPT2D, as these parameters associate with our clinical severity score and could reflect altered mitochondrial functions.

L-Palmitoylcarnitine potentiates plasmin and tPA to inhibit thrombosis

l-Palmitoylcarnitine (L-PC) is an important endogenous fatty acid metabolite. Its classical biological functions are involved in the regulations of membrane molecular dynamics and the β-oxidation of fatty acids. Decreased plasma long-chain acylcarnitines showed the association of venous thrombosis, implying anticoagulant activity of the metabolites and inspiring us to investigate if and how L-PC, a long-chain acylcarnitine, takes part in coagulation. Here we show that L-PC exerted anti-coagulant effects by potentiating the enzymatic activities of plasmin and tissue plasminogen activator (tPA). L-PC directly interacts with plasmin and tPA with an equilibrium dissociation constant (KD) of 6.47 × 10–9 and 4.46 × 10–9 M, respectively, showing high affinities. In mouse model, L-PC administration significantly inhibited FeCl3-induced arterial thrombosis. It also mitigated intracerebral thrombosis and inflammation in a transient middle cerebral artery occlusion (tMCAO) mouse model. L-PC induced little bleeding complications. The results show that L-PC has anti-thrombotic function by potentiating plasmin and tPA.Graphical

Metabolomic exploration of the effects of habituation to livestock trailer and extended transportation in goats.

Goats raised for meat production are often transported long distances. Twelve-month-old male Spanish goats were used to determine the effects of habituation to trailers on plasma metabolomic profiles when transported for extended periods. In a split-plot design, 168 goats were separated into two treatment (TRT; whole plot) groups and maintained on two different paddocks. Concentrate supplement was fed to one group inside two livestock trailers (habituated group, H), while the other group received the same quantity of concentrate, but not inside the trailers (non-habituated, NH). Goats were subjected to a 10-h transportation stress in 4 replicates (n = 21 goats/replicate/TRT) after 4 weeks of habituation period. Blood samples were collected prior to loading, 20min after loading (0h), and at 2, 4, 6, 8, and 10h of transportation (Time; subplot). A targeted quantitative metabolomics approach was employed to analyze the samples. The data were analyzed using R software and MIXED procedures in SAS. Several amino acids (alanine, serine, glycine, histidine, glutamate, trans-hydroxyproline, asparagine, threonine, methylhistidine, ornithine, proline, leucine, tryptophan) were higher (p < 0.05) in the H group compared to the NH group. Six long-chain acylcarnitines were higher (p < 0.05), while free (C0) and short-chain (C3, C5) carnitines were lower (p < 0.05) in the NH goats compared to the H goats. In general, amino acid concentrations decreased and long-chain acylcarnitine (>C10) levels increased with transportation time (p < 0.05). Butyric acid, α-ketoglutaric acid, and α-aminoadipic acid concentrations were lower (p < 0.05) and β-hydroxybutyric acid concentrations were higher in the NH goats compared to the H goats. Plasma glucose, non-esterified fatty acid (NEFA) and urea nitrogen concentrations were significantly influenced by Time (p < 0.01). Plasma NEFA concentrations were significantly lower (p < 0.01) in the H group than the NH group. Habituation to trailers can be beneficial in enhancing stress coping abilities in goats due to higher concentrations of metabolites such as butyrate and certain amino acids that support antioxidant activities and immune function. Plasma long-chain acylcarnitines may be good indicators of stress during long-distance transportation in goats.

Thermo‐sensitive mitochondrial trifunctional protein deficiency presenting with episodic myopathy

Mitochondrial trifunctional protein (MTP) is involved in long‐chain fatty acid β‐oxidation (lcFAO). Deficiency of one or more of the enzyme activities as catalyzed by MTP causes generalized MTP deficiency (MTPD), long‐chain hydroxyacyl‐CoA dehydrogenase deficiency (LCHADD), or long‐chain ketoacyl‐CoA thiolase deficiency (LCKATD). When genetic variants result in thermo‐sensitive enzymes, increased body temperature (e.g. fever) can reduce enzyme activity and be a risk factor for clinical decompensation. This is the first description of five patients with a thermo‐sensitive MTP deficiency. Clinical and genetic information was obtained from clinical files. Measurement of LCHAD and LCKAT activities, lcFAO‐flux studies and palmitate loading tests were performed in skin fibroblasts cultured at 37°C and 40°C. In all patients (four MTPD, one LCKATD), disease manifested during childhood (manifestation age: 2–10 years) with myopathic symptoms triggered by fever or exercise. In four patients, signs of retinopathy or neuropathy were present. Plasma long‐chain acylcarnitines were normal or slightly increased. HADHB variants were identified (at age: 6–18 years) by whole exome sequencing or gene panel analyses. At 37°C, LCHAD and LCKAT activities were mildly impaired and lcFAO‐fluxes were normal. Remarkably, enzyme activities and lcFAO‐fluxes were markedly diminished at 40°C. Preventive (dietary) measures improved symptoms for most. In conclusion, all patients with thermo‐sensitive MTP deficiency had a long diagnostic trajectory and both genetic and enzymatic testing were required for diagnosis. The frequent absence of characteristic acylcarnitine abnormalities poses a risk for a diagnostic delay. Given the positive treatment effects, upfront genetic screening may be beneficial to enhance early recognition.

Acylcarnitines Are Novel Anticoagulant Lipids That Target Factor Xa and That Are Reduced in Plasma of Venous Thrombosis Patients Based on Untargeted and Targeted Metabolomics

Deep vein thrombosis and pulmonary embolism (VTE) are common causes of morbidity and mortality. No genetic or acquired biomarkers or risk factors can be identified in many VTE patients. Thus, there is a major unmet need to identify new biomarkers and new causal risk factors in VTE patients. Metabolomics is an unexplored frontier for VTE research. Thus, to discover novel plasma metabolite biomarkers for VTE risk, we performed liquid chromatography-mass spectrometry (LC-MS)-based untargeted metabolomics in a pilot study to analyze plasma metabolites without any specific targeting of known metabolites. Untargeted metabolomics data for 40 male idiopathic adult VTE cases and 40 age-matched male controls recorded 9,400 metabolic features for each plasma sample. Data analysis revealed two plasma long-chain acylcarnitines (ACs) (10:1 and 16:1) to be significantly decreased in VTE patients compared to controls, suggesting the association of plasma long-chain ACs levels with the risk of VTE. To validate the association of reduced plasma levels of long-chain ACs with the VTE risk, LC-MS-based targeted metabolomics was used to determine long-chain ACs levels. Data from targeted metabolomics additionally showed that several long-chain ACs (10:1, 12:0, 12:2 and 18:2) were lower in VTE patient plasmas than in control plasmas for subjects in our pilot VTE case-control study.ACs, which heretofore have no known function in coagulation reactions, consist of a hydrophobic side chain of an acyl moiety which is linked to the carnitine moiety. ACs circulate in plasma and also play key roles in mitochondrial energy metabolism. Studies were initiated to define any procoagulant or anticoagulant properties of ACs. Remarkably, ACs inhibited factor Xa-initiated clotting assays which were triggered by adding either purified factor Xa or RVV-X to plasma. The anticoagulant activities of various ACs were both dose-dependent and acyl chain length-dependent, where ACs with longer acyl chains were more potent procoagulants than ACs with shorter acyl chains (18, 16 > 14, 10 > 6 acyl chain carbons). However, thrombin-induced clotting was not inhibited by ACs, suggesting that ACs were acting on the prothrombinase complex.For purified prothrombinase reactant systems containing prothrombin, factor Xa, factor Va, and phospholipid vesicles [phosphatidylcholine:phosphatidylserine (PC/PS) 90:10 w/w], thrombin generation was dose-dependently inhibited by 16:0-AC (IC50 = 13 µM). For reactant mixtures lacking either phospholipids or factor Va, prothrombin activation by factor Xa was still dose-dependently inhibited by 16:0-AC (IC50 = 5.6 µM and 5.8 µM, respectively). Prothrombin activation by factor Xa in the absence of both factor Va and phospholipids was still inhibited by 16:0-AC (IC50 = 11 µM). In controls, 16:0-AC did not inhibit the amidolytic activity of either factor Xa or thrombin. These findings indicate that neither factor Va nor phospholipid was required for the anticoagulant property of 16:0-AC and that this lipid did not inhibit the enzyme active sites, suggesting that this lipid disrupts interactions between factor Xa and prothrombin. Although canonical coagulation paradigms emphasize key roles for lipid binding sites that are localized in the amino terminal Gla domain of vitamin K dependent clotting factors, Gla-domainless (DG)-prothrombin activation by factor Xa and prothrombin activation by DG-factor Xa were each similarly inhibited by 16:0-AC with IC50values of 11 and 7.0 μM, respectively. For surface plasmon resonance (SPR) binding studies, biotinylated-Glutamyl-Glycyl-Arginyl-chloromethyl ketone (BEGR) was used to label the active site of factors Xa, IXa, and VIIa. BEGR-factor Xa and BEGR-DG-factor Xa bound with similar affinities to 16:0-AC (10 µM and 23 µM, respectively) whereas no binding of 16:0-AC (60 μM) to BEGR-factor VIIa or BEGR-factor IXa was observed. Thus, these data suggest that a 16:0-AC binding site on factor Xa that is located outside the Gla domain mediates this lipid’s anticoagulant activity.In summary, first, untargeted and targeted metabolomics data for a pilot VTE case-control study identified ACs as potential biomarkers for VTE, and, second, detailed mechanistic studies show that the AC, 16:0 acyl-carnitine, has anticoagulant activity in the absence of factor Va or phospholipids that is related to its ability to bind factor Xa outside the Gla domain. DisclosuresNo relevant conflicts of interest to declare.

Altered body composition and energy expenditure but normal glucose tolerance among humans with a long-chain fatty acid oxidation disorder

The development of insulin resistance has been associated with impaired mitochondrial fatty acid oxidation (FAO), but the exact relationship between FAO capacity and glucose metabolism continues to be debated. To address this controversy, patients with long-chain 3-hydroxy acyl-CoA dehydrogenase (LCHAD) deficiency underwent an oral glucose tolerance test (OGTT) and measurement of energy expenditure, body composition, and plasma metabolites. Compared with controls, patients with LCHAD deficiency had a trend toward higher total body fat and extramyocellular lipid deposition but similar levels of intramyocelluar and intrahepatic lipids. Resting energy expenditure was similar between the groups, but respiratory quotient was higher and total energy expenditure was lower in LCHAD-deficient patients compared with controls. High-molecular-weight (HMW) adiponectin levels were lower and plasma long-chain acylcarnitines were higher among LCHAD-deficient patients. Fasting and post-OGTT levels of glucose, insulin, and ghrelin, along with estimates of insulin sensitivity, were the same between the groups. Despite decreased capacity for FAO, lower total energy expenditure and plasma HMW adiponectin, and increased plasma acylcarnitines, LCHAD-deficient patients exhibited normal glucose tolerance. These data suggest that inhibition of the FAO pathway in humans is not sufficient to induce insulin resistance.

L-Carnitine treatment reduces severity of physical and mental fatigue and increases cognitive functions in centenarians: a randomized and controlled clinical trial

Background: Centenarians are characterized by weakness, decreasing mental health, impaired mobility, and poor endurance. l-Carnitine is an important contributor to cellular energy metabolism. Objective: This study evaluated the efficacy of l-carnitine on physical and mental fatigue and on cognitive functions of centenarians. Design: This was a placebo-controlled, randomized, double-blind, 2-phase study. Sixty-six centenarians with onset of fatigue after even slight physical activity were recruited to the study. The 2 groups received either 2 g levocarnitine once daily (n = 32) or placebo (n = 34). Efficacy measures included changes in total fat mass, total muscle mass, serum triacylglycerol, total cholesterol, HDL cholesterol, LDL cholesterol, Mini-Mental State Examination (MMSE), Activities of Daily Living, and a 6-min walking corridor test. Results: At the end of the study period, the levocarnitine-treated centenarians, compared with the placebo group, showed significant improvements in the following markers: total fat mass (−1.80 compared with 0.6 kg; P < 0.01), total muscle mass (3.80 compared with 0.8 kg; P < 0.01), plasma concentrations of total carnitine (12.60 compared with −1.70 μmol; P < 0.05), plasma long-chain acylcarnitine (1.50 compared with −0.1 μmol; P < 0.001), and plasma short-chain acylcarnitine (6.0 compared with −1.50 μmol; P < 0.001). Significant differences were also found in physical fatigue (−4.10 compared with −1.10; P < 0.01), mental fatigue (−2.70 compared with 0.30; P < 0.001), fatigue severity (−23.60 compared with 1.90; P < 0.001), and MMSE (4.1 compared with 0.6; P < 0.001). Conclusions: Our study indicates that oral administration of levocarnitine produces a reduction of total fat mass, increases total muscular mass, and facilitates an increased capacity for physical and cognitive activity by reducing fatigue and improving cognitive functions.

L-Carnitine treatment reduces severity of physical and mental fatigue and increases cognitive functions in centenarians: a randomized and controlled clinical trial

Centenarians are characterized by weakness, decreasing mental health, impaired mobility, and poor endurance. L-Carnitine is an important contributor to cellular energy metabolism. This study evaluated the efficacy of L-carnitine on physical and mental fatigue and on cognitive functions of centenarians. This was a placebo-controlled, randomized, double-blind, 2-phase study. Sixty-six centenarians with onset of fatigue after even slight physical activity were recruited to the study. The 2 groups received either 2 g levocarnitine once daily (n = 32) or placebo (n = 34). Efficacy measures included changes in total fat mass, total muscle mass, serum triacylglycerol, total cholesterol, HDL cholesterol, LDL cholesterol, Mini-Mental State Examination (MMSE), Activities of Daily Living, and a 6-min walking corridor test. At the end of the study period, the levocarnitine-treated centenarians, compared with the placebo group, showed significant improvements in the following markers: total fat mass (-1.80 compared with 0.6 kg; P < 0.01), total muscle mass (3.80 compared with 0.8 kg; P < 0.01), plasma concentrations of total carnitine (12.60 compared with -1.70 mumol; P < 0.05), plasma long-chain acylcarnitine (1.50 compared with -0.1 micromol; P < 0.001), and plasma short-chain acylcarnitine (6.0 compared with -1.50 micromol; P < 0.001). Significant differences were also found in physical fatigue (-4.10 compared with -1.10; P < 0.01), mental fatigue (-2.70 compared with 0.30; P < 0.001), fatigue severity (-23.60 compared with 1.90; P < 0.001), and MMSE (4.1 compared with 0.6; P < 0.001). Our study indicates that oral administration of levocarnitine produces a reduction of total fat mass, increases total muscular mass, and facilitates an increased capacity for physical and cognitive activity by reducing fatigue and improving cognitive functions.

Inhibitory effect of 3-hydroxyacyl-CoAs and other long-chain fatty acid beta-oxidation intermediates on mitochondrial oxidative phosphorylation.

Among the abnormal biochemical findings, a mild to severe lactic acidaemia (Treem et al 1994) is frequently described in patients with a long-chain fatty acid β-oxidation disorder, together with a markedly increased plasma long-chain acylcarnitine fraction, abnormal excretion of C 6 -C 10 dicarboxylic acids and an accumulation of medium- and long-chain 3-hydroxydicarboxylic acids (Duran et al 1991). Our aim was to explain the lactic acidosis found in these patients and to associate this with the accumulation of long-chain fatty acid derivatives. Previously (Ventura et al 1995) we have reported that long-chain (C 14 -C 20 ) acyl-CoA esters, both saturated and unsaturated, inhibit the synthesis of ATP in digitonin-permeabilized normal human fibroblasts (Wanders et al 1993). Using the same system we investigated in this work the effects of palmitoyl-CoA, α,β-unsaturated palmitoyl-CoA, 3-hydroxy- and 3-ketopalmitoyl-CoA on the oxidative phosphorylation system.

3-Hydroxydicarboxylic aciduria due to long-chain 3-hydroxyacyl-coenzyme A dehydrogenase deficiency associated with sudden neonatal death: protective effect of medium-chain triglyceride treatment

Two siblings were found to be affected by long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency, one of which died suddenly and unexpectedly on the 3rd day of life suffering from extreme hypoketotic hypoglycaemia. The younger sibling started to have feeding problems, lowered consciousness, and liver dysfunction at the age of 5 months. Her urine contained large amounts of C6-C14 3-hydroxydicarboxylic acids and conjugated 3-hydroxyoctanoic acid, as verified by gas chromatography/mass spectrometry. Plasma long-chain acylcarnitine was increased. A clue to the diagnosis was given by the results of a phenylpropionic acid loading test. This revealed small, but significant amounts of conjugated 3-hydroxyphenylpropionic acid (phenylhydracrylic acid) in the patient's urine. Subsequently, the activity of long-chain 3-hydroxyacyl-CoA dehydrogenase was found to be deficient in cultured skin fibroblasts. Based on the findings obtained by a medium-chain triglyceride load, a diet enriched in this type of fat was prescribed. On this regimen the patient started to thrive, signs of cardiomyopathy disappeared, and her liver function normalized.

Plasma carnitine in alcoholism.

Carnitine is essential for the beta-oxidation of long-chain fatty acids in the mitochondria and probably has a major role in modulating the acyl-coenzyme A/reduced coenzyme A ratio in the matrix of mitochondria. Plasma carnitine concentrations are elevated in several conditions and reflect changes at the cellular level. We previously had reported elevated plasma carnitine in patients with alcoholic liver disease compared to healthy control subjects. In this study we measured plasma carnitine in a third group, alcoholic patients without overt liver disease. The alcoholic patients (n = 20) had significantly elevated plasma long-chain acylcarnitine (P less than 0.01) compared to 32 healthy men of identical age and significantly lower short-chain (P less than 0.01) and long-chain acylcarnitine (P less than 0.01) than 28 men with alcoholic liver disease. We conclude that alcoholism is another condition in which carnitine homeostasis is altered.

Abnormal plasma carnitine derivatives reflecting an altered metabolic state in anorexic women at rest and following maximal effort treadmill exercise

Five women diagnosed as having classical anorexia nervosa were studied at rest and following a maximal effort treadmill test prior to (AN-1) and after hospital treatment (AN-II). A group of 14 lean women (matched for age, percent body fat, height, weight, and fitness level as closely as possible) were studied as lean controls (LC). Before treatment, AN-I had significantly less plasma long-chain acyl carnitines than LC (4.59 versus 6.16 μM), whereas short-chain acyl carnitines were nearly twice as high (9. 01 versus 4. 91 μM, AN-1 versus LC) at rest. Following maximal exercise LC women demonstrated a sharp increase in short-chain acyl carnitines (from 4. 19 to 8. 83 μM), whereas the AN-1 group showed no change in these derivaties relative to their resting level (from 9.01 to 10. 49 μM). Long-chain acyl carnitines, however, were reduced in AN-1 compared with LC after exercise (2.37 versus 6.55 μM, respectively). Following hospital treatment short-chain acyl carnitines at rest were reduced toward normal (6.68 μM) in the AN-II, although the long-chain acyl forms still remained lower (3.2 1 μM) than in the LC (6.16 μM). However, even aiter hospital treatment the AN-II group did not show the typical increase in short-chain acyl carnitines with an acute bout oi exercise.