Defective metabolism of long-chain fatty acids and/or their accumulation in nerve may impair nerve function in diabetes by altering plasma or mitochondrial membrane integrity and perturbing intracellular metabolism and energy production. Carnitine and its acetylated derivatives such as acetyl-L-carnitine (ALC) promote fatty acid beta-oxidation in liver and prevent motor nerve conduction velocity (MNCV) slowing in diabetic rats. Neither the presence nor the possible implications of putative ALC deficiency have been definitively established in diabetic nerve. This study explored sciatic nerve ALC levels and the dose-dependent effects of ALC replacement on sciatic nerve metabolites, Na,K-ATPase, and MNCV after 2 and 4 weeks of streptozotocin-induced diabetes (STZ-D) in the rat. ALC treatment that increased nerve ALC levels delayed (to 4 weeks) but did not prevent nerve myo-inositol (MI) depletion, but prevented MNCV slowing and decreased ouabain-sensitive (but not -insensitive) ATPase activity in a dose-dependent fashion. However, ouabain-sensitive ATPase activity was also corrected by subtherapeutic doses of ALC that did not increase nerve ALC or affect MNCV. These data implicate nerve ALC depletion in diabetes as a factor contributing to alterations in nerve intermediary and energy metabolism and impulse conduction in diabetes, but suggest that these alterations may be differentially affected by various degrees of ALC depletion.

Activation of inflammation is an important mechanism in the development of nonalcoholic steatohepatitis (NASH). This study aims to delineate how mitophagy affects NLRP3 inflammasome activation in hepatic lipotoxicity. Mice were fed a high fat/calorie diet (HFCD) for 24 weeks. Primary rat hepatocytes were treated with palmitic acid (PA) for various periods of time. Mitophagy was measured by protein levels of LC3II and P62. NLRP3, caspase-1, interleukin (IL)-18, and IL-1β at mRNA and protein levels were used as indicators of inflammasome activation. Along with steatotic progression in HFCD-fed mice, ratio of LC3II/β-actin was decreased concurrently with increased levels of liver P62, NLRP3, caspase-1, IL-1β, IL-18, and serum IL-1β levels in late-stage NASH. PA treatment resulted in mitochondrial oxidative stress and initiated mitophagy in primary hepatocytes. The addition of cyclosporine A did not change LC3II/Τοmm20 ratios; but P62 levels were increased after an extended duration of PA exposure, indicating a defect in autophagic activity. Along with impaired mitophagy, mRNA and protein levels of NLRP3, caspase-1, IL-18 and IL-1β were upregulated by PA treatment. Pretreatment with MCC950, N-acetyl cysteine or acetyl-L-carnitine reversed inflammasome activation and a pyroptotic cascade. Additionally, mitophagic flux was partially recovered as indicated by increases in LC3II/Tomm20 ratio, parkin, and PINK1 expression, and decreased P62 expression. The findings suggest that impaired mitophagy triggers hepatic NLRP3 inflammasome activation in a murine NASH model and primary hepatocytes. The new insights into inflammasome activation through mitophagy advance our understanding of how fatty acids elicit lipotoxicity through oxidant stress and autophagy in mitochondria.

Oxidative stress is an early and pivotal factor in Alzheimer's disease (AD). The neurotoxic peptide amyloid-beta (Abeta) contributes to oxidative damage in AD by inducing lipid peroxidation, which in turn generates additional downstream cytosolic free radicals and reactive oxygen species (ROS), leading to mitochondrial and cytoskeletal compromise, depletion of ATP, and ultimate apoptosis. Timely application of antioxidants can prevent all downstream consequences of Abeta exposure in culture, but in situ efficacy is limited, due in part to prior damage as well as difficulty in delivery. Herein, we demonstrate that administration of a combination of vitamin E (which prevents de novo membrane oxidative damage), folate (which maintains levels of the endogenous antioxidant glutathione), and acetyl-L-carnitine (which prevents Abeta-induced mitochondrial damage and ATP depletion) provides superior protection to that derived from each agent alone. These findings support a combinatorial approach in Alzheimer's therapy.

OBJECTIVE

L-Carnitine is an endogenous molecule involved in fatty acid metabolism. Secondary carnitine deficiency may develop in patients with end-stage renal disease undergoing long-term hemodialysis because of dialytic loss. In these patients L-carnitine can be administered to restore plasma and tissue levels. The objective of this study was to evaluate the pharmacokinetics of intravenous L-carnitine in patients undergoing long-term hemodialysis.

METHODS

Twelve patients undergoing three dialysis sessions/week received L-carnitine intravenously (20 mg x kg(-1)) at the end of each dialysis session for 9 weeks. Plasma samples were analyzed for L-carnitine, acetyl-L-carnitine, and total carnitine by HPLC.

RESULTS

Under baseline conditions, the mean +/- SD predialysis plasma concentration of L-carnitine was 19.5 +/- 5.6 micromol/L, decreasing to 5.6 +/- 1.9 micromol/L at the end of the dialysis session. These concentrations were substantially lower than endogenous levels in healthy human beings. Under baseline conditions the extraction ratios of L-carnitine and acetyl-L-carnitine by the dialyser were 0.74 +/- 0.07 and 0.71 +/- 0.11, respectively. During repeated dosing, there was accumulation of L-carnitine in plasma, and after 9 weeks of dosing, the predialysis and postdialysis plasma levels were 191 +/- 54.1 and 41.8 +/- 13.0 micromol/L, respectively. The predialysis and postdialysis plasma levels of L-carnitine decreased once dosing was ceased but had not returned to pretreatment levels after 6 weeks.

CONCLUSIONS

The study demonstrated that removal of L-carnitine by hemodialysis is extremely efficient and that patients undergoing hemodialysis had plasma concentrations that were substantially lower than normal, particularly during dialysis. During repeated administration of L-carnitine, the predialysis and postdialysis concentrations of the compound increased steadily, reaching an apparent steady state after about 8 weeks. It is proposed that this accumulation arose from the distribution of L-carnitine into a deep tissue pool that includes skeletal muscle.

Degenerative brain disorders (neurodegeneration) can be frustrating for both conventional and alternative practitioners. A more comprehensive, integrative approach is urgently needed. One emerging focus for intervention is brain energetics. Specifically, mitochondrial insufficiency contributes to the etiopathology of many such disorders. Electron leakages inherent to mitochondrial energetics generate reactive oxygen free radical species that may place the ultimate limit on lifespan. Exogenous toxins, such as mercury and other environmental contaminants, exacerbate mitochondrial electron leakage, hastening their demise and that of their host cells. Studies of the brain in Alzheimer's and other dementias, Down syndrome, stroke, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, Huntington's disease, Friedreich's ataxia, aging, and constitutive disorders demonstrate impairments of the mitochondrial citric acid cycle and oxidative phosphorylation (OXPHOS) enzymes. Imaging or metabolic assays frequently reveal energetic insufficiency and depleted energy reserve in brain tissue in situ. Orthomolecular nutrients involved in mitochondrial metabolism provide clinical benefit. Among these are the essential minerals and the B vitamin group; vitamins E and K; and the antioxidant and energetic cofactors alpha-lipoic acid (ALA), ubiquinone (coenzyme Q10; CoQ10), and nicotinamide adenine dinucleotide, reduced (NADH). Recent advances in the area of stem cells and growth factors encourage optimism regarding brain regeneration. The trophic nutrients acetyl L-carnitine (ALCAR), glycerophosphocholine (GPC), and phosphatidylserine (PS) provide mitochondrial support and conserve growth factor receptors; all three improved cognition in double-blind trials. The omega-3 fatty acid docosahexaenoic acid (DHA) is enzymatically combined with GPC and PS to form membrane phospholipids for nerve cell expansion. Practical recommendations are presented for integrating these safe and well-tolerated orthomolecular nutrients into a comprehensive dietary supplementation program for brain vitality and productive lifespan.

Nucleoside analogue reverse transcriptase inhibitors (NRTIs), used as part of highly active antiretroviral therapy for the treatment of HIV and AIDS, disrupt neuronal mitochondrial DNA synthesis, resulting in antiretroviral toxic neuropathy (ATN). Acetyl-L-carnitine (ALC) enhances neurotrophic support of sensory neurons, potentially causing symptom relief and nerve regeneration, and in addition has numerous other effects on metabolic function that might be of benefit in such patients.ALC has been given to HIV patients with symptomatic ATN in a number of clinical studies administered either twice daily intramuscularly or as oral sachets or tablets. It has been shown to significantly reduce a variety of validated pain ratings, and is generally safe and well tolerated. Using a measure of neuronal innervation in standardised skin biopsies of the affected area, cutaneous nerve density has been improved by the administration of ALC in subjects with symptomatic ATN and reduced epidermal and dermal innervation, associated with clinical improvement, which was maintained over a 4-year period. Improvements were seen in both the structure and function of small sensory fibres, which were sustained over time whilst subjects received ALC. Other open-label, non-randomised studies have shown similar benefits in patients with ATN in terms of pain reduction over the short term. Further placebo-controlled studies of both treatment and prophylaxis have been completed and are under analysis to characterise further the usefulness of this pathogenesis-based therapy for ATN.

BACKGROUND

Complementary and Alternative Medicines (CAMs) are frequently given to children and adolescents for reputed benefits in the treatment of hyperkinetic and concentration disorders such as Attention Deficit Hyperactivity Disorder (ADHD). In such vulnerable populations high quality evidence is required to support such claims.

OBJECTIVE

The aim of the paper is to assess the current evidence of herbal and nutritional interventions for ADHD using a systematic search of clinical trials meeting an acceptable standard of evidence.

METHODS

PubMed, PsycINFO, Cochrane Library and CINAHL were searched up to May 26th, 2011 for randomised, controlled clinical trials using CAM products as interventions to treat ADHD. A quality analysis using a purpose-designed scale, and an estimation of effect sizes (Cohen's d) where data were available, were also calculated.

RESULTS

The review revealed that 16 studies met inclusion criteria, with predominant evidentiary support found for zinc, iron, Pinus marinus (French maritime pine bark), and a Chinese herbal formula (Ningdong); and mixed (mainly inconclusive) evidence for omega-3, and l-acetyl carnitine. Current data suggest that Ginkgo biloba (ginkgo), and Hypercium perforatum (St. John's wort) are ineffective in treating ADHD.

CONCLUSIONS

The research suggests only some CAMs may be beneficial in ADHD, thus clinicians need to be aware of the current evidence. Promising candidates for future research include Bacopa monniera (brahmi) and Piper methysticum (kava), providing potential efficacy in improving attentional and hyperkinetic disorders via a combination of cognitive enhancing and sedative effects.

Rat hepatic mitochondrial function, including oxidative phosphorylation, fatty acid oxidative capacity, kinetic parameters of carnitine palmitoyltransferase I (CPT I), and sensitivity of CPT I to malonyl-CoA inhibition were studied in vitro in isolated mitochondria following Escherichia coli lipopolysaccharide (LPS). The hepatic mitochondrial CPT I in LPS-treated rats showed a lower apparent maximum velocity (Vmax) for palmitoyl-CoA and Ki for malonyl-CoA without changes in apparent Km for palmitoyl-CoA. The rate of oxygen consumption or end-product formation of palmitoyl-L-carnitine and octanoate was not altered, but the rate of CPT I-dependent palmitoyl-CoA (plus L-carnitine) oxidation was reduced by LPS, when acetyl-CoA produced via beta-oxidation was directed toward citrate. When acetyl-CoA was directed to acetoacetate, the oxygen consumption rates of palmitoyl-L-carnitine and palmitoyl-CoA (plus L-carnitine) were decreased by LPS, although mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase activity was not altered. These results indicate that hepatic mitochondria isolated from LPS-treated rats show lower ketogenic and long-chain acyl-CoA oxidative capacity than those of fasted controls, and inhibition of ketogenesis is elicited at a site distal to CPT I in addition to reduction in CPT I activity.

Oxygen free radicals and other reactive oxygen species (ROS) are common products of normal aerobic cellular metabolism, but high levels of ROS lead to oxidative stress and cellular damage. Increased production of ROS favors vascular dysfunction, inducing altered vascular permeability and inflammation, accompanied by the loss of vascular modulatory function, the imbalance between vasorelaxation and vasoconstriction, and the aberrant expression of inflammatory adhesion molecules. Inflammatory stimuli promote oxidative stress generated from the increased activity of mitochondrial nicotinamide adenine dinucleotide phosphate oxidase, particularly of the Nox4 isoform, with the consequent impairment of mitochondrial β-oxidation. Vascular dysfunction due to the increase in Nox4 activity and ROS overproduction leads to the progression of cardiovascular diseases, diabetes, inflammatory bowel disease, and neurological disorders. Considerable research into the development of effective antioxidant therapies using natural derivatives or new synthetic molecules has been conducted. Antioxidants may prevent cellular damage by reducing ROS overproduction or interfering in reactions that involve ROS. Vitamin E and ascorbic acid are well known as natural antioxidants that counteract lipid peroxidative damage by scavenging oxygen-derived free radicals, thus restoring vascular function. Recently, preliminary studies on natural antioxidants such as goji berries, thymus, rosemary, green tea ginseng, and garlic have been conducted for their efficacy in preventing vascular damage. N-acetyl-cysteine and propionyl-L-carnitine are synthetic compounds that regulate ROS production by replacing endogenous antioxidants in both endothelial and smooth muscle cells. In this review, we consider the molecular mechanisms underlying the generation of oxidative stress-induced vascular dysfunction as well as the beneficial effects of antioxidant therapies.

OBJECTIVE

Loss of skeletal muscle mass, also known as muscle wasting or muscle atrophy, is a common symptom of several chronic diseases, such as cancer and infectious diseases. Due to the strong negative impact of muscle loss on patient's prognosis and quality of life, the development of efficacious treatment approaches to combat muscle wasting are of great importance. In order to evaluate the suitability of L-carnitine (LC) as an anti-wasting agent for clinical purposes the present review comprehensively summarizes the results from animal and clinical studies showing the effects of supplementation with LC or LC derivatives (acetyl-LC, propionyl-LC) on critical mechanisms involved in skeletal muscle loss under pathologic conditions, such as increased proteolysis, impaired protein synthesis, myonuclear apoptosis, inflammation, oxidative stress, and mitochondrial dysfunction.

RESULTS

Evidence from both animal and clinical studies exists that LC supplementation causes an improved nitrogen balance either due to increased protein synthesis or reduced protein degradation, an inhibition of apoptosis and an abrogation of inflammatory processes under pathologic conditions. Furthermore, strong evidence has been provided, at least from animal studies, that LC supplementation prevents oxidative stress and ameliorates mitochondrial function, whereas results from a very low number of available clinical studies in this regard are inconclusive.

CONCLUSIONS

In conclusion, LC supplementation beneficially influences several critical mechanisms involved in pathologic skeletal muscle loss that may at least partially explain the anti-catabolic effects and the improvement of fatigue-related parameters following LC supplementation in patients with chronic diseases. However, more suitable clinical trials (double-blinded, randomized, placebo-controlled, large-scale) are necessary in order to establish LC supplementation as strategy for anti-wasting therapy.

OBJECTIVE

What is the effect of a combination of three antioxidants (Acetyl-L-Carnitine, N-Acetyl-L-Cysteine and α-Lipoic Acid), present in IVF medium during mouse oocyte and sperm collection, on fertilization and subsequent IVF embryo development?

UNASSIGNED

A combination of antioxidants resulted in faster developmental times from the 2-cell stage through to expanded blastocyst stage, accompanied by a significant increase in blastocyst cell number and a reduction of intracellular hydrogen peroxide (H2O2) levels.

UNASSIGNED

The antioxidant combination Acetyl-L-Carnitine, N-Acetyl-L-Cysteine and α-Lipoic Acid, when present in embryo culture media, has a significant beneficial effect on in vitro fertilized mouse pronucleate oocyte development, especially under oxidative stress.

UNASSIGNED

IVF was conducted with combined antioxidants supplemented in IVF medium that was used for mouse oocyte collection and fertilization (oocyte IVF medium, 4 h exposure) and sperm collection and preparation (sperm IVF medium, 1 h exposure).

UNASSIGNED

IVF was conducted under 20% oxygen, in the presence or absence of a combination of antioxidants (10 μM Acetyl-L-Carnitine, 10 μM N-Acetyl-L-Cysteine, 5 μM α-Lipoic Acid) and resultant embryos cultured with and without antioxidants under 20% oxygen. Subsequently, the effects of antioxidants on either oocytes or sperm was evaluated. Embryo development was analysed through time-lapse microscopy followed by differential nuclear staining to determine cell allocation in the blastocyst. Intracellular levels of H2O2 were assessed using an aryl boronate probe after 4 h of incubation with antioxidants. Controls were gametes and embryos that had no antioxidants in the medium. In a separate series of experiments, pronucleate oocytes were collected in handling medium with and without antioxidants for 20 min and subsequent cell numbers analysed.

UNASSIGNED

Antioxidant treatment during both IVF and culture resulted in significantly faster development times to two cell cleavage (P < 0.01), which continued through to the expanded blastocyst stage (P < 0.05). Resultant blastocysts had a significant increase in both trophectoderm (TE) cell numbers, inner cell mass (ICM) and total cell numbers (P < 0.001). The addition of antioxidants to IVF medium or embryo culture media exclusively also resulted in a significant increase in both blastocyst TE and ICM numbers leading to an increase in total cell numbers (P < 0.001). Antioxidant supplementation of either oocyte IVF medium alone, or in both oocyte and sperm IVF medium, lead to significantly faster times to two cell cleavage, which continued through to the expanded blastocyst stage. Blastocyst cell number in both these groups had significantly higher TE cell numbers resulting in an increase in total cell numbers. In contrast, there were no differences in embryo developmental rates and blastocyst cell number when antioxidants were present only in the sperm IVF medium. Levels of H2O2 were significantly reduced in pronucleate oocytes that were cultured in the presence of antioxidants (P < 0.001) compared to control, untreated embryos. Similarly, pronucleate oocytes treated with the combined antioxidants during pronucleate oocyte collection resulted in significantly increased blastocyst ICM numbers compared with controls (P < 0.05).

UNASSIGNED

Embryo development was only examined in the mouse.

UNASSIGNED

These findings suggest that supplementation of antioxidants to the IVF medium, as well as to embryo culture media, may further assist in maintaining the viability of human embryos in ART, conceivably through the reduction of oxidative stress.

UNASSIGNED

This work was funded by a research grant from Vitrolife AB (Sweden). The authors have no conflict of interest to declare.

Pyruvate decarboxylase-negative (Pdc(-)) mutants of Saccharomyces cerevisiae require small amounts of ethanol or acetate to sustain aerobic, glucose-limited growth. This nutritional requirement has been proposed to originate from (i) a need for cytosolic acetyl coenzyme A (acetyl-CoA) for lipid and lysine biosynthesis and (ii) an inability to export mitochondrial acetyl-CoA to the cytosol. To test this hypothesis and to eliminate the C(2) requirement of Pdc(-) S. cerevisiae, we attempted to introduce an alternative pathway for the synthesis of cytosolic acetyl-CoA. The addition of L-carnitine to growth media did not restore growth of a Pdc(-) strain on glucose, indicating that the C(2) requirement was not solely due to the inability of S. cerevisiae to synthesize this compound. The S. cerevisiae GLY1 gene encodes threonine aldolase (EC 4.1.2.5), which catalyzes the cleavage of threonine to glycine and acetaldehyde. Overexpression of GLY1 enabled a Pdc(-) strain to grow under conditions of carbon limitation in chemostat cultures on glucose as the sole carbon source, indicating that acetaldehyde formed by threonine aldolase served as a precursor for the synthesis of cytosolic acetyl-CoA. Fractionation studies revealed a cytosolic localization of threonine aldolase. The absence of glycine in these cultures indicates that all glycine produced by threonine aldolase was either dissimilated or assimilated. These results confirm the involvement of pyruvate decarboxylase in cytosolic acetyl-CoA synthesis. The Pdc(-) GLY1 overexpressing strain was still glucose sensitive with respect to growth in batch cultivations. Like any other Pdc(-) strain, it failed to grow on excess glucose in batch cultures and excreted pyruvate when transferred from glucose limitation to glucose excess.

OBJECTIVE

Fibromyalgia (FMS) is a chronic syndrome characterized by widespread pain, troubled sleep, disturbed mood, and fatigue. Several analgesic strategies have been evaluated but the results are moderate and inconsistent. Antidepressant agents are now considered the treatment of choice in most patients. It has been recently suggested that FMS may be associated with metabolic alterations including a deficit of carnitine. In this multicenter randomized clinical trial we evaluated the efficacy of acetyl L-carnitine (LAC) in patients with overt FMS.

METHODS

One hundred and two patients meeting the American College of Rheumatology criteria for FMS were randomized into the study. The treatment consisted of 2 capsules/day of 500 mg LAC or placebo plus one intramuscular (i.m.) injection of either 500 mg LAC or placebo for 2 weeks. During the following 8 weeks the patients took 3 capsules daily containing either 500 mg LAC or placebo. The patients were seen during treatment after 2 (visit 3), 6 (visit 4) and 10 weeks (visit 5). The patients were also visited 4 weeks after treatment discontinuation (follow-up visit). Outcome measures included the number of positive tender points, the sum of pain threshold (kg/cm2 or "total myalgic score"), the Short Form 36 (SF36), a 100 mm visual analog scale (VAS) for self-perceived stiffness, fatigue, tiredness on awakening, sleep, work status, depression, and muscular-skeletal pain, and the Hamilton depression scale.

RESULTS

The "total myalgic score" and the number of positive tender points declined significantly and equally in both groups until the 6th week of treatment. At the 10th week both parameters remained unchanged in the placebo group but they continued to improve in the LAC group with a statistically significant between-group difference. Most VAS scores significantly improved in both groups. A statistically significant between-group difference was observed for depression and musculo-skeletal pain. Significantly larger improvements in SF36 questionnaire were observed in LAC than in placebo group for most parameters. Treatment was well-tolerated.

CONCLUSIONS

Although this experience deserves further studies, these results indicate that LAC may be of benefit in patients with FMS, providing improvement in pain as well as the general and mental health of these patients.

Successful embryo and fetal development is dependent on the quality of the oocyte from which it was derived. Several studies to date have demonstrated the link between appropriate metabolism and sufficient ATP production with oocyte quality and preimplantation embryo development. Metabolism of fatty acids for the purpose of synthesizing ATP occurs within mitochondria via β-oxidation and entry of fatty acids into this organelle is the rate-limiting step in this process. Transport of activated fatty acids into mitochondria is catalyzed by carnitine palmitoyl transferase-I (CPTI) which also requires the metabolite carnitine. Once inside the mitochondrial matrix, fatty acids are broken down into acetyl CoA molecules which are further metabolized via the TCA cycle and electron transport chain to produce ATP. The potential to improve oocyte quality by modulating fatty acid metabolism and β-oxidation with carnitine in culture media formulations or via dietary supplementation has received little attention. This review summarizes studies to date investigating the developmental importance of β-oxidation through the use of metabolic inhibitors and whether regulation by carnitine, in vitro or in vivo, has beneficial effects on oocyte and embryo development. Overall, there is little evidence to date that dietary carnitine can improve oocyte quality or female fertility; however inclusion of l-carnitine to in vitro oocyte maturation and embryo growth media improves embryo outcomes, most likely by supplying the oocyte and embryo with an essential co-factor required to utilize fatty acids.

OBJECTIVE

Patients with diabetic neuropathy (DN) demonstrate variable degrees of nerve regeneration and degeneration. Our aim was to identify risk factors associated with sural nerve degeneration in patients with DN.

METHODS

Demographic, anthropometric, biochemical, and anatomical data of subjects with DN from a 52-week trial of acetyl-L-carnitine were retrospectively examined. Based on the change in sural nerve myelinated fiber density (ΔMFD%), subjects were divided into three groups: regenerator (top 16 percentiles, n = 67), degenerator (bottom 16 percentiles, n = 67), and intermediate (n = 290), with dramatically increased, decreased, and steady ΔMFD%, respectively. ANOVA, Fisher exact test, and multifactorial logistic regression were used to evaluate statistical significance.

RESULTS

ΔMFD%s were 35.6 ± 17.4 (regenerator), -4.8 ± 12.1 (intermediate), and -39.8 ± 11.0 (degenerator). HbA1c at baseline was the only factor significantly different across the three groups (P = 0.01). In multifactorial logistic regression, HbA1c at baseline was also the only risk factor significantly different between regenerator (8.3 ± 1.6%) and degenerator (9.2 ± 1.8%) (odds ratio 0.68 [95% CI 0.54-0.85]; P < 0.01). Support Vector Machine classifier using HbA1c demonstrated 62.4% accuracy of classifying subjects into regenerator or degenerator. A preliminary microarray experiment revealed that upregulated genes in the regenerator group are enriched with cell cycle and myelin sheath functions, while downregulated genes are enriched in immune/inflammatory responses.

CONCLUSIONS

These data, based on the largest cohort with ΔMFD% information, suggest that HbA1c levels predict myelinated nerve fiber regeneration and degeneration in patients with DN. Therefore, maintaining optimal blood glucose control is likely essential in patients with DN to prevent continued nerve injury.

Little information is available in the literature on the effect of L-carnitine to improve glucose disposal in healthy control subjects and type 2 diabetic patients. No data are reported on the pharmacological properties of acetyl-L-carnitine (ALC) in type 2 diabetes mellitus. The present study evaluates glucose uptake and oxidation rates with either ALC or placebo administration in 18 type 2 diabetic patients. On different days, each patient received both a primed-constant infusion of ALC (5 mg/kg body weight [BW] priming bolus and either 0.025, 0.1, or 1.0 mg/kg BW/min constant infusion) and a comparable placebo formulation. During the infusion period, continuous indirect calorimetric monitoring and a euglycemic-hyperinsulinemic clamp (EHC) study were performed. The total end-clamp glucose tissue uptake (M value) was significantly increased by the administration of ALC (from 3.8 to 5.2 mg/kg/min, P = .006), and the dose dependence of this effect reached borderline statistical significance (P = .037). The increase in the M/I ratio was also highly significant after ALC administration (from 3.9 to 5.8 x 10(-2) mg/kg/min/(microUI/mL, P < .001), while no statistically significant effect was attributable to the different dosages. The increase in the M value was related to increased glucose storage (highly significant effect of ALC) rather than increased glucose oxidation (no statistical significance). In conclusion, the effect of ALC on glucose disposal has no relationship to the amount administered. This could be due to an effect of ALC on the enzymes involved in both the glycolytic and gluconeogenetic pathways, and a possible reversibility of glycogen synthase inhibition in diabetic subjects.

The present study was designed to characterize cardiac autonomic neuropathy in streptozotocin-induced (45 mg/kg i.v.) diabetic rat by analysis of heart rate variability (HRV), and to assess, in this model, the effects of treatment with acetyl-L-carnitine (ALC). Heart rate was reduced in diabetic rats (332+/-22 vs. 411+/-35 beat per min; P<0.0001). This bradycardia was partly reversed with ALC (369+/-52 beat per min; P<0.05 vs. untreated). Both time- and frequency-domain parameters of HRV were significantly reduced in diabetic rats. The reduction of spectral power was around 50% at high frequencies and about 70% at low frequencies, suggesting a decrease of parasympathetic activity. Low/high frequency ratio was significantly decreased in diabetic rats suggesting decreased sympathetic tone, while nonlinear analysis indicated a reduction of the chaotic complexity of heart rate dynamics in diabetic rats. Standard deviation of heart rate in ALC-treated rats was significantly higher than in untreated diabetic rats (P<0.0001). ALC counteracts the reduction of the power spectrum observed in diabetic animals (P<0.0005) normalizing the spectra profile. ALC restored chaotic complexity of heart rate dynamics. These results on the whole indicate that both sympathetic and parasympathetic cardiac tone were reduced significantly in diabetic rats and that ALC treatment prevents the development of autonomic neuropathy in streptozotocin-induced diabetes in rats.

Propofol is a widely used general anesthetic. A growing body of data suggests that perinatal exposure to general anesthetics can result in long-term deleterious effects on brain function. In the developing brain there is evidence that general anesthetics can cause cell death, synaptic remodeling, and altered brain cell morphology. Acetyl-L-carnitine (L-Ca), an anti-oxidant dietary supplement, has been reported to prevent neuronal damage from a variety of causes. To evaluate the ability of L-Ca to protect against propofol-induced neuronal toxicity, neural stem cells were isolated from gestational day 14 rat fetuses and on the eighth day in culture were exposed for 24h to propofol at 10, 50, 100, 300 and 600 μM, with or without L-Ca (10 μM). Markers of cellular proliferation, mitochondrial health, cell death/damage and oxidative damage were monitored to determine: (1) the effects of propofol on neural stem cell proliferation; (2) the nature of propofol-induced neurotoxicity; (3) the degree of protection afforded by L-Ca; and (4) to provide information regarding possible mechanisms underlying protection. After propofol exposure at a clinically relevant concentration (50 μM), the number of dividing cells was significantly decreased, oxidative DNA damage was increased and a significant dose-dependent reduction in mitochondrial function/health was observed. No significant effect on lactase dehydrogenase (LDH) release was observed at propofol concentrations up to 100 μM. The oxidative damage at 50 μM propofol was blocked by L-Ca. Thus, clinically relevant concentrations of propofol induce dose-dependent adverse effects on rat embryonic neural stem cells by slowing or stopping cell division/proliferation and causing cellular damage. Elevated levels of 8-oxoguanine suggest enhanced oxidative damage [reactive oxygen species (ROS) generation] and L-Ca effectively blocks at least some of the toxicity of propofol, presumably by scavenging oxidative species and/or reducing their production.

In vitro neuronal preparations are used to study the action mechanism of substances which are active in normal and pathological brain aging. One major concern with in vitro assays is that the use of embryonic or adult neurons may hamper an appreciation of the relevance of these substances on aged nervous tissue. In the present study for the first time cultures of aged dorsal root ganglia from 24-months-old rats were maintained in vitro up to 2 weeks. This model was used to investigate the neurotrophic/neuroprotective action of nerve growth factor and acetyl-L-carnitine. A large population of aged dorsal root ganglia neurons was responsive to nerve growth factor (100 ng/ml). Nerve growth factor induced an increase of initial rate of axonal regeneration and influenced the survival time of these neurons. Acetyl-L-carnitine (250 microM) did not affect the axonal regeneration but substantially attenuated the rate of neuronal mortality. A significant difference was evident between the acetyl-L-carnitine-treated and the untreated neurons from the first cell counting (day 3 in culture). After 2 weeks the number of aged neurons treated with acetyl-L-carnitine was almost double that of the controls. The effects of acetyl-L-carnitine on aged DRG neurons potentially explain the positive effects in clinical and in vivo experimental studies.

Acetyl-L-carnitine (ALC), a physiological component of the L-carnitine family, has been proposed for treating Alzheimer's disease in pharmacological doses. As this condition requires prolonged therapy, its kinetics has been examined after a multiple dose regimen, involving different routes of administration, in 11 patients suffering from Senile Dementia of Alzheimer Type. The study design comprised a 3-day basal observation period, sham treatment with repeated blood sampling; treatment with 30 mg.kg-1 i.v. [corrected] for 10 days (plasma kinetics was studied on the 7th day), and 50 days of 1.5 g/day [corrected] p.o. given in three daily doses. Total acid soluble L-carnitine, L-carnitine and acetyl-L-carnitine in plasma and CSF were evaluated using an enantioselective radioenzyme assay. Short chain L-carnitine esters were calculated as the difference between total and free-L-carnitine. The plasma concentrations of individual components of the L-carnitine family did not change during the three days of the basal period, nor were they affected during the sham therapy period. Following the i.v. bolus injections, the plasma concentrations showed a biphasic curve, with average t1/2 of 0.073 h and 1.73 h, respectively. At the end of oral treatment, plasma acetyl-L-carnitine and L-carnitine short chain esters were significantly higher than during the run-in phase. The CSF concentrations paralleled those in plasma, suggesting that ALC easily crosses the blood-brain barrier. It is concluded that i.v. and oral administration of multiple doses of ALC can increase its plasma and CSF concentration in patients suffering from Alzheimer's disease.

Incubation of carnitine acetyltransferase with low concentrations of bromoacetyl-l-carnitine causes a rapid and irreversible loss of enzyme activity; one mol of inhibitor can inactivate one mol of enzyme. Bromoacetyl-d-carnitine, iodoacetate or iodoacetamide are ineffective. l-Carnitine protects the transferase from bromoacetyl-l-carnitine. Investigation shows that the enzyme first reversibly binds bromoacetyl-l-carnitine with an affinity similar to that shown for the normal substrate acetyl-l-carnitine; this binding is followed by an alkylation reaction, forming the carnitine ester of a monocarboxymethyl-protein, which is catalytically inactive. The carnitine is released at an appreciable rate by spontaneous hydrolysis, and the resulting carboxymethyl-enzyme is also inactive. Total acid hydrolysis of enzyme after treatment with 2-[(14)C]bromoacetyl-l-carnitine yields N-3-carboxy[(14)C]methylhistidine as the only labelled amino acid. These findings, taken in conjunction with previous work, suggest that the single active centre of carnitine acetyltransferase contains a histidine residue.

Carnitine acetyltransferase was purified from the supernatant obtained after centrifugation of human liver homogenate to a final specific activity of 78.75 unit.mg-1 with acetyl-CoA as a substrate. Human carnitine acetyltransferase is a monomer of 60.5 kDa with maximum activity in the presence of propionyl-CoA and a pH optimum of 8.7. Apparent Km values for acetyl-CoA are three times lower than for decanoyl-CoA. Km values for L-carnitine in the presence of acetyl-CoA are six times lower than in the presence of decanoyl-CoA. Km values for acetylcarnitine are three times lower than for octanoylcarnitine. The polyclonal antibodies against human carnitine acetyltransferase recognize a 60.5-kDa peptide in the purified preparation of human liver and brain homogenates and in immunoblots of mitochondrial and peroxisomal fractions from human liver. Immunoprecipitation and SDS/PAGE analysis of 35S-labelled proteins produced by human fibroblasts indicate that mitochondrial carnitine acetyltransferase is synthesized as a precursor of 65 kDa. We also purified carnitine acetyltransferase from the pellet obtained after centrifugation of liver homogenate. The pellet was extracted by sonication in the presence of 0.5% Tween 20. The chromatographic procedures for the purification and the kinetic, physical and immunological properties of pellet-extracted carnitine acetyltransferase are similar to those of carnitine acetyltransferase purified from the supernatant of human liver homogenate.

BACKGROUND

l-carnitine is an endogenous substance, vital in the transport of fatty acids across the inner mitochondrial membrane for oxidation. Disturbances in carnitine homeostasis can have a significant impact on human health; therefore, it is critical to define normal endogenous concentrations for l-carnitine and its esters to facilitate the diagnosis of carnitine deficiency disorders. This study was conducted to determine the normal concentrations of a number of carnitines in healthy adults using three analytical methods. The impact of age and gender on carnitine concentrations was also examined.

METHODS

Blood samples were collected from 60 healthy subjects of both genders and various ages. Plasma samples were analysed for endogenous carnitine concentrations by radioenzymatic assay, high-performance liquid chromatography and electrospray tandem mass spectrometry.

RESULTS

Precision and accuracy of results obtained for each assay were within acceptable limits. Average endogenous concentrations obtained from the three analytical methods in this study were in the range of 38-44, 6-7 and 49-50 mumol/L for l-carnitine, acetyl-l-carnitine and total carnitine, respectively. Comparison of results between the genders indicated that males had significantly higher endogenous plasma l-carnitine and total carnitine concentrations than females. Age was found to have no impact on plasma carnitine concentrations.

CONCLUSIONS

These results are useful in the evaluation of biochemical or metabolic disturbances and in the diagnosis and treatment of patients with carnitine deficiency.

Peripheral nerve trauma remains a major cause of morbidity, largely due to the death of approximately 40% of innervating sensory neurons, and to slow regeneration after repair. Acetyl-L-carnitine (ALCAR) is a physiological peptide that virtually eliminates sensory neuronal death, and may improve regeneration after primary nerve repair. This study determines the effect of ALCAR upon regeneration after secondary nerve repair, thereby isolating its effect upon neuronal regenerative capacity. Two months after unilateral sciatic nerve division 1 cm nerve graft repairs were performed (n=5), and treatment with 50 mg/kg/day ALCAR was commenced for 6 weeks until harvest. Regeneration area and distance were determined by quantitative immunohistochemistry. ALCAR treatment significant increased immunostaining for both nerve fibres (total area 264% increase, P<0.001; percentage area 229% increase, P<0.001), and Schwann cells (total area 111% increase, P<0.05; percentage area 86% increase, P<0.05), when compared to no treatment. Regeneration into the distal stump was greatly enhanced (total area 2,242% increase, P=0.008; percentage area 3,034% increase, P=0.008). ALCAR significantly enhances the regenerative capacity of neurons that survive peripheral nerve trauma, in addition to its known neuroprotective effects.