1. The synthesis of pent-4-enoyl-l-carnitine, cyclopropanecarbonyl-l-carnitine and cyclobutanecarbonyl-l-carnitine is described. 2. Pent-4-enoate strongly inhibits palmitoyl-l-carnitine oxidation in coupled but not in uncoupled mitochondria. Pent-4-enoyl-l-carnitine strongly inhibits palmitoyl-l-carnitine oxidation in uncoupled mitochondria. Prior intramitochondrial formation of pent-4-enoyl-CoA is therefore necessary for inhibition. 3. There was a small self-limiting pulse of oxidation of pent-4-enoyl-l-carnitine during which the ability to inhibit the oxidation of subsequently added palmitoyl-l-carnitine developed. 4. Pent-4-enoate and pent-4-enoyl-l-carnitine are equally effective inhibitors of the oxidation of all even-chain acylcarnitines of chain length C(4)-C(16). Pent-4-enoyl-l-carnitine also inhibits the oxidation of pyruvate and of 2-oxoglutarate. 5. Pent-4-enoate strongly inhibits the oxidation of palmitate but not that of octanoate. This is presumably due to competition between octanoate and pent-4-enoate for medium-chain acyl-CoA ligase. 6. There was less inhibition of the oxidation of pyruvate by pent-4-enoyl-l-carnitine, and of palmitoyl-l-carnitine by cyclopropanecarbonyl-l-carnitine, after pre-incubation with 10mm-arsenate. This suggests that these inhibitions were caused either by depletion of free CoA or by increase of acyl-CoA concentrations, since arsenate deacylates intramitochondrial acyl-CoA. There was little effect on the inhibition of palmitoyl-l-carnitine oxidation by pent-4-enoyl-l-carnitine. 7. Penta-2,4-dienoate strongly inhibited palmitoyl-l-carnitine oxidation in coupled mitochondria; acrylate only inhibited slightly. 8. Pent-4-enoate (0.1mm) caused a rapid and almost complete decrease in free CoA and a large increase in acid-soluble acyl-CoA when incubated with coupled mitochondria. Cyclopropanecarboxylate caused a similar decrease in CoA, with an equivalent rise in acid-soluble acyl-CoA concentrations. n-Pentanoate caused extensive lowering of CoA and a large increase in acid-soluble acyl-CoA and acetyl-CoA concentrations. Octanoate caused a 50% lowering of CoA and an increase in acid-soluble acyl-CoA and acetyl-CoA concentrations. 9. Cyclopropanecarboxylate and n-pentanoate were less potent inhibitors of palmitate oxidation than was pent-4-enoate. 10. It is concluded that pent-4-enoate causes a specific inhibition of beta-oxidation after the formation intramitochondrially of its metabolites.

Synthesis of [3H]acetylcholine from [3H]acetyl-L-carnitine was demonstrated in vitro by coupling the enzyme systems choline acetyltransferase and carnitine acetyltransferase. Likewise, both [3H] and [14C] labeled acetylcholine were produced when [3H]acetyl-L-carnitine and D-[U-14C] glucose were incubated with synaptosomal membrane preparations from rat brain. Transfer of the acetyl moiety from acetyl-L-carnitine to acetylcholine was dependent on concentration of acetyl-L-carnitine and required the presence of coenzyme A, which is normally produced as an inhibitory product of choline acetyltransferase. These results provide further evidence for a role of mitochondrial carnitine acetyltransferase in facilitating transfer of acetyl groups across mitochondrial membranes, thus regulating the availability in the cytoplasm of acetyl-CoA, a substrate of choline acetyltransferase. They are also consistent with a possible utility of acetyl-L-carnitine in the treatment of age-related cholinergic deficits.

As major components of neuronal membranes, omega-3 polyunsaturated acids (n-3 PUFA) exhibit a wide range of regulatory functions, modulating from synaptic plasticity to neuroinflammation, from oxidative stress to neuroprotection. Recent human and animal studies indicated the n-3 PUFA neuroprotective properties in aging, with a clear negative correlation between n-3 PUFA levels and hippocampal deficits. The present multidimensional study was aimed at associating cognition, hippocampal neurogenesis, volume, neurodegeneration and metabolic correlates to verify n-3 PUFA neuroprotective effects in aging. To this aim 19 month-old mice were given n-3 PUFA mixture, or olive oil or no dietary supplement for 8 weeks during which hippocampal-dependent mnesic functions were tested. At the end of behavioral testing morphological and metabolic correlates were analyzed. n-3 PUFA supplemented aged mice exhibited better object recognition memory, spatial and localizatory memory, and aversive response retention, without modifications in anxiety levels in comparison to controls. These improved hippocampal cognitive functions occurred in the context of an enhanced cellular plasticity and a reduced neurodegeneration. In fact, n-3 PUFA supplementation increased hippocampal neurogenesis and dendritic arborization of newborn neurons, volume, neuronal density and microglial cell number, while it decreased apoptosis, astrocytosis and lipofuscin accumulation in the hippocampus. The increased levels of some metabolic correlates (blood Acetyl-L-Carnitine and brain n-3 PUFA concentrations) found in n-3 PUFA supplemented mice also pointed toward an effective neuroprotection. On the basis of the present results n-3 PUFA supplementation appears to be a useful tool in health promotion and cognitive decline prevention during aging.

Acetyl-L-carnitine (ALC) has gained clinical interest for its analgesic effect in different forms of neuropathies associated with chronic pain, such as diabetic and HIV-related peripheral neuropathies. The antinociceptive effect of ALC has been confirmed in several experimental models of neuropathic pain, including streptozotocin- and chemotherapy-induced neuropathy, and the sciatic nerve chronic constriction injury model. In these models, prophylactic administration of ALC has proven to be effective in preventing the development of neuropathic pain. In addition, ALC is known to produce a strong antinociceptive effect when given after neuropathic pain has been established. ALC can also improve the function of peripheral nerves by increasing nerve conduction velocity, reducing sensory neuronal loss, and promoting nerve regeneration. Analgesia requires repeated administrations of ALC, suggesting that the drug regulates neuroplasticity across the pain neuraxis. Recent evidence indicates that ALC regulates processes that go beyond its classical role in energy metabolism. These processes involve the activation of muscarinic cholinergic receptors in the forebrain, and an increased expression of type-2 metabotropic glutamate (mGlu2) receptors in dorsal root ganglia neurons. Induction of mGlu2 receptors is mediated by acetylation mechanisms that involve transcription factors of the nuclear factor (NF)-kappaB family.

BACKGROUND

Recent evidence indicates that insulin resistance in skeletal muscle may be related to reduce mitochondrial number and oxidation capacity. However, it is not known whether increasing mitochondrial number and function improves insulin resistance. In the present study, we investigated the effects of a combination of nutrients on insulin resistance and mitochondrial biogenesis/function in skeletal muscle of type 2 diabetic Goto-Kakizaki rats.

RESULTS

We demonstrated that defect of glucose and lipid metabolism is associated with low mitochondrial content and reduced mitochondrial enzyme activity in skeletal muscle of the diabetic Goto-Kakizaki rats. The treatment of combination of R-alpha-lipoic acid, acetyl-L-carnitine, nicotinamide, and biotin effectively improved glucose tolerance, decreased the basal insulin secretion and the level of circulating free fatty acid (FFA), and prevented the reduction of mitochondrial biogenesis in skeletal muscle. The nutrients treatment also significantly increased mRNA levels of genes involved in lipid metabolism, including peroxisome proliferator-activated receptor-alpha (Ppar alpha), peroxisome proliferator-activated receptor-delta (Ppar delta), and carnitine palmitoyl transferase-1 (Mcpt-1) and activity of mitochondrial complex I and II in skeletal muscle. All of these effects of mitochondrial nutrients are comparable to that of the antidiabetic drug, pioglitazone. In addition, the treatment with nutrients, unlike pioglitazone, did not cause body weight gain.

CONCLUSIONS

These data suggest that a combination of mitochondrial targeting nutrients may improve skeletal mitochondrial dysfunction and exert hypoglycemic effects, without causing weight gain.

Diet in human health is no longer simple nutrition, but in light of recent research, especially nutrigenomics, it is linked via evolution and genetics to cell health status capable of modulating apoptosis, detoxification, and appropriate gene response. Nutritional deficiency and disease especially lack of vitamins and minerals is well known, but more recently, epidemiological studies suggest a role of fruits and vegetables, as well as essential fatty acids and even red wine (French paradox), in protection against disease. In the early 1990s, various research groups started considering the use of antioxidants (e.g., melatonin, resveratrol, green tea, lipoic acid) and metabolic compounds (e.g., nicotinamide, acetyl-L-carnitine, creatine, coenzyme Q10) as possible candidates in neuroprotection. They were of course considered on par with snake oil salesman (women) at the time. The positive actions of nutritional supplements, minerals, and plant extracts in disease prevention are now mainstream and commercial health claims being made are subject to regulation in most countries. Apart from efficacy and finding, the right dosages, the safety, and especially the level of purification and lack of contamination are all issues that are important as their use becomes widespread. From the mechanistic point of view, most of the time these substances replenish the body's deficiency and restore normal function. However, they also exert actions that are not sensu stricto nutritive and could be considered pharmacological especially that, at times, higher intake than recommended (RDA) is needed to see these effects. Free radicals and neuroinflammation processes underlie many neurodegenerative conditions, even Parkinson's disease and Alzheimer's disease. Curcumin, carotenoids, acetyl-L-carnitine, coenzyme Q10, vitamin D, and polyphenols and other nutraceuticals have the potential to target multiple pathways in these conditions. In summary, augmenting neuroprotective pathways using diet and finding new natural substances that can be more efficacious, i.e., induction of health-promoting genes and reduction of the expression of disease-promoting genes, could be incorporated into neuroprotective strategies of the future.

Modulation of endogenous cellular defense mechanisms via the stress response signaling represents an innovative approach to therapeutic intervention in diseases causing chronic damage, such as neurodegeneration and cancer. Protein thiols play a key role in redox sensing, and regulation of cellular redox state is crucial mediator of multiple metabolic, signaling, and transcriptional processes. Maintenance of optimal long-term health conditions is accomplished by a complex network of longevity assurance processes that are controlled by vitagenes, a group of genes involved in preserving cellular homeostasis during stressful conditions. Vitagenes encode for heat shock proteins (Hsp) Hsp32, Hsp70, the thioredoxin, and the sirtuin protein systems. Dietary antioxidants, such as polyphenols and L-carnitine/acetyl-L-carnitine, have recently been demonstrated to be neuroprotective through the activation of hormetic pathways, including vitagenes. The hormetic dose-response, challenges long-standing beliefs about the nature of the dose-response in a low dose zone, having the potential to affect significantly the design of pre-clinical studies and clinical trials as well as strategies for optimal patient dosing in the treatment of numerous diseases. Given the broad cytoprotective properties of the heat shock response, there is now strong interest in discovering and developing pharmacological agents capable of inducing these responses. In this review we discuss the most current and up-to-date understanding of the possible signaling mechanisms by which acetylcarnitine by activating vitagenes can differentially modulate signal transduction cascades inducing apoptosis/cell death in abnormal cancer cells but at the same time enhancing defensive enzymes to protect against carcinogenesis and neurodegeneration in normal cells. (c) 2009 International Union of Biochemistry and Molecular Biology, Inc.

OBJECTIVE

Despite considerable microsurgical innovation in peripheral nerve repair, the outcome has improved little since the 1940s, reflecting surgical inability to adequately address the complex neurobiology of nerve injury and regeneration. Axotomy-induced neuronal death is potentially the most fundamental problem, and given recently published data, a review is timely.

METHODS

Initial review of relevant doctoral theses from the University of Umeå, and Blond-McIndoe Research Laboratories, the University of Manchester, plus initial PubMed search including terms 'neuron death' and 'neuroprotection', subsequently expanded to relevant quoted articles.

RESULTS

Various factors related to patient (principally age) and injury (Sunderland grade, proximity to cell body and mechanism) determine the extent of neuronal death, the mechanism of which is reviewed. A considerable proportion of sensory neurons (particularly small cutaneous afferents) die after distal injury and death is more widespread after proximal injury. Motor neurons are susceptible to post-ganglionic plexus and spinal root level injury. Root avulsion causes the greatest cell death. The time course of neuronal death is fortuitously slow and mainly occurs by a process akin to apoptosis. A therapeutic window therefore exists, as do potential neuroprotective targets. Nerve repair is partly neuroprotective, but must be performed early. Exogenous neurotrophic factor administration (e.g. in tissue engineered conduits) is beneficial, but not practical for various reasons. In contrast, adjuvant neuroprotective pharmacotherapy is practical, and two clinically safe agents are reviewed. Acetyl-L-carnitine arrests sensory neuronal death and speeds up regeneration. N-acetyl-cysteine provides comparable sensory neuronal protection via mitochondrial preservation and protects motor neurons. Both agents are well characterized experimentally and highly effective even after clinically relevant delays between injury and treatment. Barriers to translational research are being addressed.

CONCLUSIONS

The future of peripheral nerve repair lies in modulating neurobiology at the time of injury, repair and during regeneration. Neuroprotection may be an essential component of that therapeutic package.

Although the mechanisms of neurodegeneration in Parkinson's disease are not fully understood, mitochondrial dysfunction, oxidative stress and environmental toxins may be involved. The current research was directed to investigate the protective role of two bioenergetic antioxidants, acetyl-L-carnitine and α-lipoic acid, in rotenone-parkinsonian rats. Ninety six male rats were divided into five groups. Group I is the vehicle-injected group, group II is the disease control group and was injected with six doses of rotenone (1.5 mg/kg/48 h, s.c.). Groups III, IV and V received rotenone in addition to acetyl-L-carnitine (100 mg/kg/day, p.o.), α-lipoic acid (50 mg/kg/day, p.o.) or their combination, respectively. Results showed that rotenone-treated rats exhibited bradykinesia and motor impairment in the open-field and square bridge tests. In addition, ATP level was decreased whereas lipid peroxides and protein carbonyls increased in the striata of rotenone-treated rats as compared to vehicle-treated rats. Treatment with acetyl-L-carnitine or α-lipoic acid improved the motor performance and reduced the level of lipid peroxides in rat brains as compared to rotenone group. Further, ATP production was enhanced along with acetyl-L-carnitine treatments (p≤0.05). Taken together, our study reinforces the view that acetyl-L-carnitine and α-lipoic acid are promising candidates for neuroprotection in Parkinson's disease.

Acute acoustic trauma (AAT) results in oxidative stress to the cochlea through overproduction of cellular reactive oxygen, nitrogen, and other free radical species appearing from 1 h to 10 days after noise exposure. It has been shown that N-acetyl-L-cysteine (NAC), a glutathione prodrug, and acetyl-L-carnitine (ALCAR), a mitochondrial biogenesis agent, are effective in reducing noise-induced hearing loss. Phenyl N-tert-butylnitrone (PBN), a nitrone-based free radical trap, appears to suppress oxidative stress in a variety of disorders and several biological models. In this study, we tested whether 4-hydroxy PBN (4-OHPBN), a major metabolite of PBN, administered 4 h after noise exposure is effective in treating noise-induced hearing loss and whether a combination of antioxidant drugs (4-OHPBN plus NAC and 4-OHPBN plus NAC plus ALCAR) provides greater efficacy in attenuating AAT since each agent addresses different injury mechanisms. Chinchilla were exposed to a 105 dB octave-band noise centered at 4 kHz for 6 h. 4-OHPBN and combinations of antioxidant drugs were intraperitoneally administered beginning 4 h after noise exposure. Hearing threshold shifts in auditory brainstem responses and missing outer hair cell counts were obtained. 4-OHPBN reduced threshold shifts in a dose-dependent manner while both drug combinations showed greater effects. These results demonstrate that 4-OHPBN and combinations of antioxidants can effectively treat acute acoustic trauma and drug combinations may increase the effectiveness of treatment and decrease the required individual medication dose.

OBJECTIVE

We investigated potential analgesics for chemotherapy-evoked neuropathic pain using rats treated with paclitaxel.

METHODS

Drugs were tested in a repeated dosing paradigm (four daily injections). Topiramate was tested with a long-term treatment paradigm (12 days). A literature search was performed to summarize prior data.

METHODS

Mechanical stimulation of the hind paw was used to assay antiallodynic and antihyperalgesic effects acutely and 24 hours after injection.

RESULTS

Amitriptyline produced significant analgesia, but this was not apparent until after the second injection. Baclofen produced significant effects, but the response varied erratically. Mexiletine and NMED-126 (a mixed N- and T-type calcium channel blocker) produced consistent, significant analgesia when tested acutely, but the pain relief did not persist at 24 hours postinjection. Oxcarbazepine had no effect at any time. Tramadol produced consistent, near-complete analgesia when tested acutely, but the analgesia did not persist to 24 hours postinjection. Topiramate produced significant effects that were first evident after 6-8 days of dosing.

CONCLUSIONS

The present data and data from the literature review suggest that there are several potential treatments for chemotherapy-evoked neuropathic pain. Nonsteroidal anti-inflammatory drugs have little or no efficacy. Opioids have an effect, but probably only with high doses. At least some antidepressants are analgesic in these conditions. Some, but clearly not all, anticonvulsants and sodium channel blockers have efficacy. Tramadol is a particularly promising candidate. Topiramate, acetyl-L-carnitine, carbamazepine, and venlafaxine may have protective or restorative effects. Clinical trials of these candidates are needed to advance the treatment of chemotherapy-evoked pain.

Although exogenously administered acetyl-l-carnitine (ALCAR, (2-acetoxy-3-carboxypropyl)-trimethylammonium) and l-carnitine (LC, (3-carboxy-2-hydroxypropyl)-trimethylammonium) prevent brain damage in several ischemic models, the protective mechanism of these compounds remains unclear. Here, we evaluated the effect of ALCAR and LC in primary cultured neurons from the cerebral cortex, striatum and thalamus of 18-day-old rat embryos. Deprivation of the serum from cultured medium for 3 days reduced the number of viable cells and mitochondrial activity and induced cell death with characteristics of apoptosis such as DNA fragmentation, nuclear condensation and histone-DNA release into the cytoplasm. ALCAR (1 - 100 microM) and LC (1 - 100 microM) promoted neuronal survival and mitochondrial activity in a concentration-dependent manner. Moreover, these compounds attenuated DNA fragmentation and nuclear condensation in cultured neurons and significantly decreased histone-DNA release into the cytoplasm. These results indicate that anti-apoptotic actions of ALCAR and LC contribute to their neuroprotective effect.

Acylcarnitines, a biologically important group of metabolites which have thus far eluded characterization by mass spectrometry, exhibit very intense fast atom bombardment mass spectra dominated by parent cations. The combination of high resolution selected ion detection and daughter ion analysis using the linked scan at constant B/E ratio has enabled the unequivocal identification of the acylcarnitines in the urine of children with propionic acidemia, methylmalonic aciduria and Reye's Syndrome. Quantitative analysis of acetylcarnitine and propionylcarnitine in selected samples was accomplished by isotope dilution, utilizing (2H3)acetyl- and (2H5)propionylcarnitines as internal standards. These techniques will allow assessment of the therapeutic use of L-carnitine in these disorders.

Matrix-assisted laser desorption/ionization (MALDI) based mass spectrometric imaging (MSI) is increasingly being used as an analytical tool to evaluate the molecular makeup of tissue samples. From the direct analysis of a tissue section, the physical integrity of sample is preserved; thus, spatial information of a compound's distribution may be determined. One limitation of the technique, however, has been the inability to determine the absolute concentration from a tissue sample. Here we report the development of a quantitative MSI technique in which the distribution of acetyl-L-carnitine (AC) in a piglet brain sample is quantified with MALDI MSI. An isotopically labeled internal standard was applied uniformly beneath the tissue section, and wide-isolation tandem mass spectrometry was performed. Normalizing the analyte ion signal by the internal standard ion signal resulted in significant improvements in MS images, signal reproducibility, and calibration curve linearity. From the improved MS images, the concentration of AC was determined and plotted producing a concentration-scaled image of the distribution of AC in the piglet brain section.

The attention deficit/hyperactivity disorder (ADHD) can affect human infants and adolescents. One important feature of this disorder is behavioural impulsivity. This study assessed the ability of chronic acetyl-L-carnitine (ALC, saline or 100 mg/kg SC, plus 50 mg/kg orally) to reduce impulsivity in a validated animal model for ADHD. Food-restricted rats were tested during adolescence (postnatal days, pnd, 30-45) in operant chambers with two nose-poking holes, one delivering one food pellet immediately, and the other five pellets after a delay. Delay length was increased over days (from 0 to 80 s). Individual differences in the preference-delay curve emerged, with the identification of two distinct subpopulations, i.e. one with a nearly horizontal curve and another with a very steep ("impulsive") slope. The impulsivity profile was slightly but consistently reduced by chronic ALC administration. Consistent results were also obtained with methylphenidate (MPH, saline or 3 mg/kg IP twice daily). Impulsive rats exhibited a lower metabolite/serotonin (5HIAA/5HT) ratio in the medial frontal cortex (MFC) and lower noradrenaline (NA) levels in the MFC and cingulate cortex (CC) when compared with the other subgroup. The ALC treatment increased NA levels in the CC and the 5HIAA/5HT ratio in both CC and MFC. Present data suggest that ALC, a drug devoid of psychostimulant properties, may have some beneficial effects in the treatment of ADHD children.

We show that mitochondrial function in the majority of hepatocytes isolated from old rats (24 mo) is significantly impaired. Mitochondrial membrane potential, cardiolipin levels, respiratory control ratio, and overall cellular O2 consumption decline, and the level of oxidants increases. To examine whether dietary supplementation of micronutrients that may have become essential with age could reverse the decline in mitochondrial function, we supplemented the diet of old rats with 1% (w/v) acetyl-L-carnitine (ALCAR) in drinking water. ALCAR supplementation (1 month) resulted in significant increases in cellular respiration, mitochondrial membrane potential, and cardiolipin values. However, supplementation also increased the rate of oxidant production, indicating that the efficiency of mitochondrial electron transport had not improved. To counteract the potential increase in oxidative stress, animals were administered N-tert-butyl-alpha-phenyl-nitrone (30 mg/kg) (PBN) with or without ALCAR. Results showed that PBN significantly lowered oxidant production as measured by 2,7'-dichlorofluorescin diacetate (DCFH), even when ALCAR was coadministered to the animals. Thus, dietary supplementation with ALCAR, particularly in combination with PBN, improves mitochondrial function without a significant increase in oxidative stress.

Phenyllactate dehydratase from Clostridium sporogenes grown anaerobically on L-phenylalanine catalyses the reversible syn-dehydration of (R)-phenyllactate to (E)-cinnamate. Purification yielded a heterotrimeric enzyme complex (130 +/- 15 kDa) composed of FldA (46 kDa), FldB (43 kDa) and FldC (40 kDa). By re-chromatography on Q-Sepharose, the major part of FldA could be separated and identified as oxygen insensitive cinnamoyl-CoA:phenyllactate CoA-transferase, whereas the transferase depleted trimeric complex retained oxygen sensitive phenyllactate dehydratase activity and contained about one [4Fe-4S] cluster. The dehydratase activity required 10 microM FAD, 0.4 mM ATP, 2.5 mM MgCl2, 0.1 mM NADH, 5 microM cinnamoyl-CoA and small amounts of cell-free extract (10 microg protein per mL) similar to that known for 2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans. The N-terminus of the homogenous FldA (39 amino acids) is homologous to that of CaiB (39% sequence identity) involved in carnitine metabolism in Escherichia coli. Both enzymes are members of an emerging group of CoA-transferases which exhibit high substrate specificity but apparently do not form enzyme CoA-ester intermediates. It is concluded that dehydration of (R)-phenyllactate to (E)-cinnamate proceeds in two steps, a CoA-transfer from cinnamoyl-CoA to phenyllactate, catalysed by FldA, followed by the dehydration of phenyllactyl-CoA, catalysed by FldB and FldC, whereby the noncovalently bound prosthetic group cinnamoyl-CoA is regenerated. This demonstrates the necessity of a 2-hydroxyacyl-CoA intermediate in the dehydration of 2-hydroxyacids. The transient CoA-ester formation during the dehydration of phenyllactate resembles that during citrate cleavage catalysed by bacterial citrate lyase, which contain a derivative of acetyl-CoA covalently bound to an acyl-carrier-protein (ACP).

BACKGROUND

Analysis of carnitine and acylcarnitines by tandem mass spectrometry (MS/MS) has limitations. First, preparation of butyl esters partially hydrolyzes acylcarnitines. Second, isobaric nonacylcarnitine compounds yield false-positive results in acylcarnitine tests. Third, acylcarnitine constitutional isomers cannot be distinguished.

METHODS

Carnitine and acylcarnitines were isolated by ion-exchange solid-phase extraction, derivatized with pentafluorophenacyl trifluoromethanesulfonate, separated by HPLC, and detected with an ion trap mass spectrometer. Carnitine was quantified with d(3)-carnitine as the internal standard. Acylcarnitines were quantified with 42 synthesized calibrators. The internal standards used were d(6)-acetyl-, d(3)-propionyl-, undecanoyl-, undecanedioyl-, and heptadecanoylcarnitine.

RESULTS

Example recoveries [mean (SD)] were 69.4% (3.9%) for total carnitine, 83.1% (5.9%) for free carnitine, 102.2% (9.8%) for acetylcarnitine, and 107.2% (8.9%) for palmitoylcarnitine. Example imprecision results [mean (SD)] within runs (n = 6) and between runs (n = 18) were, respectively: total carnitine, 58.0 (0.9) and 57.4 (1.7) micromol/L; free carnitine, 44.6 (1.5) and 44.3 (1.2) micromol/L; acetylcarnitine, 7.74 (0.51) and 7.85 (0.69) micromol/L; and palmitoylcarnitine, 0.12 (0.01) and 0.11 (0.02) micromol/L. Standard-addition slopes and linear regression coefficients were 1.00 and 0.9998, respectively, for total carnitine added to plasma, 0.99 and 0.9997 for free carnitine added to plasma, 1.04 and 0.9972 for octanoylcarnitine added to skeletal muscle, and 1.05 and 0.9913 for palmitoylcarnitine added to skeletal muscle. Reference intervals for plasma, urine, and skeletal muscle are provided.

CONCLUSIONS

This method for analysis of carnitine and acylcarnitines overcomes the observed limitations of MS/MS methods.

A study of the appearance of liver apoptosis after ochratoxin A (OTA) administration was performed in male mice. Administration of OTA twice a week for one or two weeks period results in the occurrence of apoptosis in mice"s liver. The presence of intracellular apoptosis bodies was detected at two weeks after toxin treatment. Light microscopic examination demonstrated the presence of eosinophilic globules, often containing apoptotic bodies. They were found within the cytoplasm of intact hepatic cells. The number of apoptotic bodies was further enhanced at two weeks, resulting in 8 fold increases in liver over the control values. No evidence of cell necrosis could be observed by histological and biochemical analysis at one week. However, centrilobular necrosis was evident at two weeks. The ability of the combined antioxidants: Coenzyme Q 10 (CoQ 10), L-carnitine, Zn, Mg, N-acetyl cysteine, vitamin C, vitamin E and selenium or tamoxifen to intervene in apoptosis induced in livers of mice by OTA was also investigated. The inhibition by these scavengers was more clear in mice treated with OTA for one week than those mice treated for two weeks. Treatment with tamoxifen, known in restoration of tumor suppressor function and on induction of programmed cell death (apoptosis), after OTA administration, had no significant inhibition effect on the incidence of apoptotic bodies in liver. Because hepatic glutathione represents the major defence against toxic liver injury, we studied the activity of tissue reduced glutathione (GSH), known to inhibit apoptosis. Our finding showed that two weeks after treatment, OTA caused a decrease of the GSH activity. However, treatment of mice with the combined antioxidants could enhance hepatic antioxidant/detoxification system, as indicated by increase in hepatic reduced glutathione level. In the light of these results, apoptosis was observed after two weeks of OTA administration. We also suggest that use of the combined antioxidants may be of interest in conditions were certain toxin-mediated forms of cell death and/or apoptosis contribute significantly to toxicity.

BACKGROUND

Increasing evidence points toward the efficacy of nutritional modifications in delaying cognitive decline and mood/behavioral difficulties in Alzheimer's disease (AD). Nutritional supplementation with individual agents has shown varied results suggesting the need for combinatorial intervention.

OBJECTIVE

We set out to determine whether nutritional intervention could positively impact cognitive performance and behavioral difficulties for individuals diagnosed with AD.

METHODS

A double-blind, multi-site, phase II study (ClinicalTrials.gov NCT01320527; Alzheimer's Association Trialmatch) was conducted in which 106 individuals with AD were randomized to a nutraceutical formulation (NF; folate, alpha-tocopherol, B12, S-adenosyl methioinine, N-acetyl cysteine, acetyl-L-carnitine) or placebo for 3 or 6 months, followed by an open-label extension where participants received NF for 6 additional months.

RESULTS

The NF cohort improved versus the placebo cohort within 3 months (Clox-1 p = 0.0083, 95%CI [0.4481, 2.9343]; Dementia Rating Scale p = 0.0266, 95%CI [0.1722, 2.7171]). Caregivers reported non-significant improvements in Neuropsychiatric Inventory. Both cohorts improved or maintained baseline performance during open-label extensions. Activities of Daily Living did not change for either cohort.

CONCLUSIONS

These findings extend phase I studies where NF maintained or improved cognitive performance and mood/behavior.

OCTN2 (SLC22A5), an organic cation/carnitine transporter, is widely distributed throughout the body, including the brain. In the present study, the involvement of OCTN2 in acetyl-L-carnitine (ALCAR) permeation across the blood-brain barrier (BBB) was examined using a microdialysis method in mouse. OCTN2 function was examined by comparison of wild-type mice with jvs mice, which express defective OCTN2 and are considered a model for primary systemic carnitine deficiency. Zero-net-flux method analysis indicated higher in vivo recovery of ALCAR and lower physiological ALCAR concentration in thalamus extracellular fluid (ECF) in jvs mice compared with wild-type mice. Externally added ALCAR showed significantly slower initial uptake across the BBB in jvs mouse. These results indicated that OCTN2 is functionally involved in ALCAR transfer across the BBB. Total radioactivity in ECF after i.v. administration of radiolabelled ALCAR remained constant for the rest of the experimental period. Accordingly, our results indicate that ALCAR is transported from blood to brain ECF by OCTN2 at least in part, and its concentration in brain ECF is regulated by other events such as protein binding and anabolic reactions in the brain, as well as by transport across the BBB.

A randomized, double-blind, placebo-controlled, parallel-group clinical trial was carried out to compare 24-week periods of treatment with 1 g acetyl-l-carnitine twice daily and placebo in the treatment of patients with dementia of the Alzheimer type. A total of 36 patients entered the trial, of whom 20 patients (7 active, 13 placebo) completed the full 24 weeks. Whilst several of the efficacy indices showed little change in either group during the trial, there was an apparent trend for more improvement in the acetyl-l-carnitine group in relation to the Names Learning Test and a computerized Digit Recall Test, both related to aspects of short-term memory. Similarly, there was a trend for reaction time in the computerized classification test to show less deterioration in the active treatment group. Changes within groups, and changes between groups, failed to reach statistical significance, at least partially because of the small number of patients available for analysis. Two indices of overall therapeutic benefit showed a trend for less deterioration in the active-treatment group than in the placebo group. Nausea and/or vomiting occurred in 5 patients in the acetyl-l-carnitine group. Laboratory tests revealed no signs of drug toxicity. The results suggest that acetyl-l-carnitine may have a beneficial effect on some clinical features of Alzheimer-type dementia, particularly those related to short-term memory.

Recent advances in the development and administration of chemotherapy for malignant diseases have been rewarded with prolonged survival rates. The cost of progress has come at a price and the nervous system is frequently the target of chemotherapy-induced neurotoxicity. Unlike more immediate toxicities that effect the gastrointestinal tract and bone marrow, chemotherapy-induced neurotoxicity is frequently delayed in onset and may progress over time. In the peripheral nervous system, the major brunt of the toxicity is directed against the peripheral nerve, resulting in chemotherapy-induced peripheral neuropathy (CIPN). Chemotherapeutic agents used to treat hematologic and solid tumors target a variety of structures and functions in the peripheral nervous system, including the neuronal cell body, the axonal transport system, the myelin sheath, and glial support structures. Each agent exhibits a spectrum of toxic effects unique to its mechanism of toxic injury, and recent study in this field has yielded clearer ideas on how to mitigate injury. Combined with the call for a greater recognition of the potentially devastating ramifications of CIPN on quality of life, basic and clinical researchers have begun to investigate therapy to prevent neurotoxic injury. Preliminary studies have shown promise for some agents including glutamine, glutathione, vitamin E, acetyl-L-carnitine, calcium, and magnesium infusions, but final recommendations await prospective confirmatory studies.

Beagle dogs between 7.6 and 8.8 years of age administered a twice daily supplement of alpha-lipoic acid (LA) and acetyl-L-carnitine (ALC) over approximately 2 months made significantly fewer errors in reaching the learning criterion on two landmark discrimination tasks compared to controls administered a methylcellulose placebo. Testing started after a 5 day wash-in. The dogs were also tested on a variable delay version of a previously acquired spatial memory task; results were not significant. The improved performance on the landmark task of dogs supplemented with LA + ALC provides evidence of the effectiveness of this supplement in improving discrimination and allocentric spatial learning. We suggest that long-term maintenance on LA and ALC may be effective in attenuating age-associated cognitive decline by slowing the rate of mitochondrial decay and cellular aging.