Rat liver gamma-butyrobetaine hydroxylase catalyzed reaction: influence of potassium, substrates, and substrate analogues on hydroxylation and decarboxylation

Interaction of rat liver gamma-butyrobetaine hydroxylase (EC 1.14.11.1) with various ligands was studied by following the decarboxylation of alpha-ketoglutarate, formation of L-carnitine, or both. Potassium ion stimulates rat liver gamma-butyrobetaine hydroxylase catalyzed L-carnitine synthesis and alpha-ketoglutarate decarboxylation by 630% and 240%, respectively, and optimizes the coupling efficiency of these two activities. Affinities for alpha-ketoglutarate and gamma-butyrobetaine are increased in the presence of potassium. gamma-Butyrobetaine hydroxylase catalyzed decarboxylation of alpha-ketoglutarate was dependent on the presence of gamma-butyrobetaine, L-carnitine, or D-carnitine in the reaction and exhibited Km(app) values of 29, 52, and 470 microM, respectively. gamma-Butyrobetaine saturation of the enzyme indicated a substrate inhibition pattern in both the assays. Omission of potassium decreased the apparent maximum velocity of decarboxylation supported by all three compounds by a similar percent. beta-Bromo-alpha-ketoglutarate supported gamma-butyrobetaine hydroxylation, although less effectively than alpha-ketoglutarate. The rat liver enzyme was rapidly inactivated by 1 mM beta-bromo-alpha-ketoglutarate at pH 7.0. This inactivation reaction did not show a rate saturation with increasing concentrations of beta-bromo-alpha-ketoglutarate. None of the substrates or cofactors, including alpha-ketoglutarate, protected the enzyme against this inactivation. Unlike beta-bromo-alpha-ketoglutarate, beta-mercapto-alpha-ketoglutarate did not replace alpha-ketoglutarate as a cosubstrate. Both beta-mercapto-alpha-ketoglutarate and beta-glutathione-alpha-ketoglutarate were noncompetitive inhibitors with respect to alpha-ketoglutarate.     

gamma-butyrobetaine in tissues and serum of fed and starved rats determined by an enzymic radioisotopic procedure.

A method for the determination of picomole quantities of gamma-butyrobetaine and its application for the determination of gamma-butyrobetaine distribution in tissues are described. The method is based on the quantitative conversion of gamma-butyrobetaine into carnitine by using a 50-60%-satd.-(NH4)2SO4 fraction of rat liver supernatant as the source of gamma-butyrobetaine hydroxylase [4-trimethylaminobutyrate,2-oxoglutarate:oxygen oxidoreductase (3-hydroxylating), EC 1.14.11.1]; the carnitine formed is then measured enzymically. The mean gamma-butyrobetaine content, as nmol/g wet wt. of tissue, ranged from a low of 4.6 in livers to a high of 12.3 in hearts of normal fed male adult rats. Starvation for 48 h did not affect the gamma-butyrobetaine concentration in serum, liver and brain, but that in skeletal muscles, kidney and heart was increased. These data are in line with the present views that most tissues are able to produce gamma-butyrobetaine, and show that starvation enhances the synthesis and/or the retention of this compound in many tissues. The observed high affinity of gamma-butyrobetaine hydroxylase for gamma-butyrobetaine (Km 7 microM), the high activity of this enzyme and the low concentration of gamma-butyrobetaine in liver indicate that gamma-butyrobetaine availability is one of the factors that normally limit carnitine synthesis.     

Gamma-butyrobetaine hydroxylase: stereochemical course of the hydroxylation reaction

The stereochemical course of the aliphatic hydroxylation of gamma-butyrobetaine by calf liver and by Pseudomonas sp AK1 gamma-butyrobetaine hydroxylases has been determined. With [3(RS)-3-3H]-gamma-butyrobetaine or [3(R)-3-3H]-gamma-butyrobetaine as substrate, a rapid and significant loss of tritium to the medium occurred. On the other hand, with [3(S)-3-3H]-gamma-butyrobetaine, only a negligible release of tritium to the aqueous medium was observed. Indeed, on hydroxylation of [3(S)-3-2H]-gamma-butyrobetaine by either the calf liver or bacterial hydroxylase, the isolated product L-carnitine was found to have retained all of the deuterium initially present in the 3(S) position. Since the absolute configuration of the product L-carnitine has been determined to be R, such results are only compatible with a hydroxylation reaction that proceeded with retention of configuration. With [methyl-14C,3(R)-3-3H]-gamma-butyrobetaine as substrate for the calf liver hydroxylase, the percentage of tritium retained in the [methyl-14C]-L-carnitine product was determined as a function of percent reaction. The results of these studies indicated that pro-R hydrogen atom abstraction exceeded 99.9%. Experiments using racemic [methyl-14C,3(RS)-3-3H]-gamma-butyrobetaine as substrate yielded similar results and additionally allowed us to estimate alpha-secondary tritium kinetic isotope effects of 1.10 and 1.31 for the bacterial and calf liver enzymes, respectively. These results are discussed within the context of the radical mechanism for gamma-butyrobetaine hydroxylase previously proposed [Blanchard, J. S., & Englard, S. (1983) Biochemistry 22, 5922], and the required topographical arrangement of enzymic oxidant and substrate is illustrated.     

OCTN3: A Na+-independent L-carnitine transporter in enterocytes basolateral membrane

L-carnitine transport has been measured in enterocytes and basolateral membrane vesicles (BLMV) isolated from chicken intestinal epithelia. In the nominally Na+-free conditions chicken enterocytes take up L-carnitine until the cell to medium L-carnitine ratio is 1. This uptake was inhibited by L-carnitine, D-carnitine, gamma-butyrobetaine, acetylcarnitine, tetraethylammonium (TEA), and betaine. L-3H-carnitine uptake into BLMV showed no overshoot, and it was (i) Na+-independent, (ii) trans-stimulated by intravesicular L-carnitine, and (iii) cis-inhibited by TEA and cold L-carnitine. L-3H-carnitine efflux from L-3H-carnitine preloaded enterocytes was also Na+-independent, and trans-stimulated by L-carnitine, D-carnitine, gamma-butyrobetaine, acetylcarnitine, TEA, and betaine. Both, uptake and efflux of L-carnitine were inhibited by verapamil and unaffected by either extracellular pH or palmitoyl-L-carnitine. RT-PCR with specific primers for the mouse OCTN3 transporter revealed the existence of OCTN3 mRNA in mouse intestine, which was confirmed by in situ hybridization studies. Immunohystochemical analysis showed that OCTN3 protein was mainly associated with the basolateral membrane of rat and chicken enterocytes, whereas OCTN2 was detected at the apical membrane. In conclusion, the results demonstrate for the first time that (i) mammalian small intestine expresses OCTN3 mRNA along the villus and (ii) that OCTN3 protein is located in the basolateral membrane. They also suggest that OCTN3 could mediate the passive, Na+ and pH-independent L-carnitine transport activity measured in the three experimental conditions.     

gamma-Butyrobetaine hydroxylase and the protective role of glutathione peroxidase

The selenoenzyme glutathione peroxidase in the presence of GSH effectively replaced catalase in the in vitro assay for gamma-butyrobetaine hydroxylase. Quantitatively, glutathione peroxidase was an order of magnitude more efficient than catalase, with maximal activity at less than 0.1 microM glutathione peroxidase in a standard reaction. Glutathione peroxidase prevented the loss of gamma-butyrobetaine hydroxylase during preliminary incubation with ferrous ions but without other substrates as well as in the course of the reaction. Regardless of whether glutathione peroxidase or catalase was present in the assay, the ascorbate concentrations needed to achieve half-maximal rates were similar (about 1 mM). Phosphate stimulated the rate of L-carnitine synthesis. Ferrous ion saturation indicated a pronounced effect of phosphate on the maximal velocity of the enzyme-catalyzed reaction, but its mechanism of action remains to be elucidated. Based on the subcellular distribution of gamma-butyrobetaine hydroxylase, catalase, and glutathione peroxidase, the role of glutathione peroxidase assumes importance. However, initial studies indicated that the assayable activity of liver gamma-butyrobetaine hydroxylase and L-carnitine concentrations in liver, blood plasma, and muscle were not significantly altered in selenium-deficient rats.     

L-carnitine supplementation in breeding pigeons: impact on zootechnical performance and carnitine metabolism

In the first experiment (Exp1), three consecutive breeding rounds were performed by two groups of six pigeon couples in order to study the impact of L-carnitine supplementation (80 mg x d(-1)) of parent pigeons on zootechnical performance. Both in the second and third experiments (Exp2, Exp3), one breeding round was performed by two groups of six pigeon couples to reveal the biochemical background of the increase in squab growth, the limitation of body weight decrease in male parent birds and the tendency for an improved cumulative feed efficiency due to L-carnitine supplementation in Exp1. Growth improvement of the squabs with L-carnitine was only seen when the parent pigeons were supplemented, together with a marked rise in the body weight of the parent birds around hatching. Based on the results of the crop milk analysis, growth improvement was probably due to a quantitative impact on crop milk production. The crop milk from the supplemented groups in both Exp2 and Exp3 had increased levels of carnitine. Carnitine, gamma-butyrobetaine and acetylcarnitine were increased in plasma samples of the supplemented parent pigeons. No differences were present in the squabs' plasma for these parameters. In the squabs of Exp3, no changes were seen in the proportional growth or the protein content of the heart, breast muscle and liver, but the breast muscle of the squabs from the supplemented group in Exp3 showed a considerable rise in carnitine and a marked decrease in gamma-butyrobetaine.     

一株假单胞菌(Pseudomonas sp.)L-1菌株γ-丁基甜菜碱羟化酶基因bbh 的克隆、表达及酶学性质

【目的】γ-丁基甜菜碱羟化酶是生物体内合成L-肉碱的关键酶。从假单胞菌(Pseudomonas sp.)L-1中克隆γ-丁基甜菜碱羟化酶基因,实现其在大肠杆菌(Escherichia coli)中的高效表达,并对表达产物进行酶学性质分析,为生物转化生产L-肉碱奠定基础。【方法】通过PCR克隆γ-丁基甜菜碱羟化酶基因,并将其开放阅读框(ORF)克隆至融合表达载体pET-15b;表达产物经His.Bind Resin纯化后对BBH进行酶学性质及三维空间结构分析;并以静止细胞进行L-肉碱的转化。【结果】成功地克隆了一个γ-丁基甜菜碱羟化酶基因bbh(GenBank:JQ250036),并实现了其在E.coli中的高效表达。融合蛋白以同源二聚体的形式存在,单个亚基的分子量约46.5 kDa,最适反应温度为30℃,最适反应pH为7.5。该酶在45℃以下稳定。在pH6.0时该酶有最高的pH稳定性。以表达bbh基因的重组大肠杆菌静止细胞转化L-肉碱,L-肉碱产量可达12.7mmol/L。【结论】Pseudomonas sp.L-1γ-丁基甜菜碱羟化酶与现有报道的bbh基因有较大的差异。由该基因表达的γ-丁基甜菜碱羟化酶能有效地转化γ-丁基甜菜碱生成L-肉碱。本研究不仅丰富了γ-丁基甜菜碱羟化酶基因资源,而且为L-肉碱的生物转化提供了一种新的转化方案。     

Catabolic pathways for high-dosed L(-)- or D(+)-carnitine in germ-free rats?

Gnotobiotic rats received up to 3 mmol L-carnitine/day with the drinking water during 9 days. They excreted about a quarter of the administered dose with the urine, partially in form of acetyl-L-carnitine, but trimethylamine, trimethylamine N-oxide or gamma-butyrobetaine were not detectable in urine or faeces in contrast to conventional animals. After oral loading with D-carnitine the unphysiological isomer was absorbed and either excreted unchanged in urine or metabolized to acetonyltrimethylammonium. With regard to the development of carnitine deficiency syndromes and the degradation of nutritional carnitine the conclusion has to be drawn, that the bacteria of the gastro-intestinal tract, but not the tissues of the mammals, are responsible for the metabolization of L-carnitine to gamma-butyrobetaine or trimethylamine.     

Influence of age on Cu/Zn-superoxide dismutase and indole 2,3-dioxygenase activities in rat tissues

The aim of this study was to investigate in vitro the variations with age of the activities of the two antioxidant enzymes Cu/Zn-superoxide dismutase (SOD) and indole 2,3-dioxygenase (IDO) in metabolically active tissues of rats of various ages. In rats aged one week and 2-3 months the highest Cu/Zn-SOD activity was found in the liver and the lowest in the small intestine. At 12 and 18 months of age, the activity was higher in the brain and kidneys, when compared to the small intestine, lungs and liver. Cu/Zn-SOD activity decreased significantly after 2-3 months of age with advancing age in all tissues examined. In newborn rats IDO activity was present only in the small intestine. In the group of rats aged 2-3 months, the highest specific activity was observed in the small intestine and the lowest in the lungs and kidneys, whereas at 12 months of age, the highest IDO activity was found in the brain, with kidneys presenting the lowest activity. At 18 months, IDO returned to be more elevated in the small intestine. At 12 months of age the values of IDO in the tissues varied slightly, while at 18 months similar activities were found between the lungs and brain and between the small intestine and kidneys. In relation to age, IDO specific activity declined in the small intestine, after 2-3 months of age. In the lungs, the activity remained unchanged; in the brain and in the kidneys activity decreased significantly from 2-3 to 18 months of age. In conclusion, this study demonstrates an age-related decline in Cu/Zn-SOD and IDO activities, the two enzymes responsible for scavenging O2*-.     

Regulation of NAD- and NADP-linked isocitrate dehydrogenase by hydrocortisone in the brain and liver of male rats of various ages

The activity and hormonal regulation of NAD- and NADP-linked isocitrate dehydrogenase (EC 1.1.1.41 and 1.1.1.42, respectively) in the brain and liver of rats of various ages were investigated. The activity of NAD-linked isocitrate dehydrogenase of the brain was greater than cytoplasmic or mitochondrial NADP-linked isocitrate dehydrogenase. In contrast, the cytoplasmic NADP-isocitrate dehydrogenase of the liver predominates over both NAD- and mitochondrial NADP-isocitrate dehydrogenases at the three ages studied. The activity of NAD-isocitrate dehydrogenase increased in the brain (139%) and liver (17%) of rats upt o 33 weeks of age and decreased (57 and 39%, respectively) in old rats (85-week-old). The activity of cytoplasmic NADP-isocitrate dehydrogenase was maximum in immature (6-week-old) rat brain and decreased as the age of the rats increased; whereas, in liver, the activity of this enzyme was found to be maximum in adult rats (33-week-old). Brain mitochondrial NADP-isocitrate dehydrogenase activity increased (64%) in adult rats, but in liver it decreased (45 and 33% in 33- and 85-week-old rats, respectively). In both tissues, adrenalectomy and hydrocortisone treatment showed differential age-dependent response. Hydrocortisone-mediated induction of the level of enzymes was inhibited by actinomycin D.     

Isocitrate dehydrogenase activity and its regulation by estradiol in tissues of rats of various ages

The activity and hormonal regulation of NAD- and NADP-linked isocitrate dehydrogenase (EC.1.1.1.41 and EC.1.1.1.42, respectively) in the brain, liver and kidney cortex of female rats of various ages was investigated. The activity of NAD-ICDH of brain was greater than extramitochondrial (-c) or intramitochondrial (-m) NADP-ICDH. In contrast, liver c-NADP-ICDH was much higher than NAD- or m-NADP-ICDH, whereas in kidney cortex the activity of m-NADP-ICDH is dominant over both NAD- and c-NADP-ICDH in all the age group of rats studied. The activity of the NAD-ICDH of brain and all the enzymes of liver and kidney cortex increases until adulthood (33-weeks) and decreases thereafter in old rats (85-weeks). In brain c-NADP-ICDH was much higher in immature (6-weeks) rats and decreases with increasing age of the animal, whereas m-NADP-ICDH showed no significant change with the age of the rats. Bilateral ovariectomy decreases the level of all the three forms of enzyme in all the tissues of 6-, 13- and 33-week rats but failed to show any significant effect in 85-week old rats. Exogenous administration of estradiol induces all the three forms of enzyme in all the tissues of ovariectomized rats. The degree of response is tissue- and age-specific.     

50 Hz magnetic fields activate mussel immunocyte p38 MAP kinase and induce HSP70 and 90

We studied the effects of a sublethal concentration of pyrethroid insecticide fenvalerate on metabolic enzymes, RNA and protein of brain, liver and skeletal muscle of the freshwater catfish, Clarias batrachus. Exposure to fenvalerate gradually decreased the activity of citrate synthase (CS), glucose 6-phosphate dehydrogenase (G6-PDH) and lactate dehydrogenase (LDH) in brain, liver and skeletal muscle up to 21 days. The maximum decrease in enzyme activity was 23-47%. Withdrawal of fenvalerate from the medium for 21 days restored enzyme activity to their control level in all three tissues. RNA and protein content in brain, liver and skeletal muscle decreased significantly with exposure of fenvalerate up to 21 days. The maximum decrease in RNA and protein was 22-32%. Withdrawal of fenvalerate from the medium for 21 days restored the RNA and protein contents to control levels. The present study suggests that fenvalerate impairs cellular metabolism and its biochemical effects are reversible after withdrawal of fenvalerate.     

Comparison of melatonin, vitamin E and L-carnitine in the treatment of neuro- and hepatotoxicity induced by thioacetamide

This study was designed to evaluate and compare the effect of melatonin, vitamin E and L-carnitine on brain and liver oxidative stress and liver damage. Oxidative stress and hepatic failure were produced by a single dose of thioacetamide (TAA) (150 mg kg(-1)) in Wistar rats. A dose of either melatonin (3 mg kg(-1)) vitamin E (20 mg kg(-1) ) or L-carnitine (100 mg kg(-1)) was used. Blood samples were taken from the neck vasculature in order to determine ammonium, blood urea nitrogen (BUN) and liver enzymes. Lipid peroxidation products, glutathione (GSH) content and antioxidative enzymes were determined in cerebral and hepatic homogenates. The results showed a decrease in BUN and in the antioxidant enzymes activities and GSH in the brain and liver. Likewise, TAA induced significant enhancement of lipid peroxidation products levels in both liver and brain, as well as in ammonia values. Melatonin, vitamin E and L-carnitine, although melatonin more significantly, decreased the intensity of the changes produced by the administration of TAA alone. Furthermore melatonin combined with TAA, decreased the ammonia levels and increased the BUN values compared with TAA animals. Also it was more effective than vitamin E or L-carnitine in these actions. These data show the protective effect of these agents, especially melatonin, against oxidative stress and hepatic damage present in fulminant hepatic failure.     

Stability of the anti-oxidative enzymes in aqueous and detergent solution

Activities of the anti-oxidative enzymes, superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase were studied in rat tissues to determine the ability of detergents both to solubilize the enzymes and also to stabilize enzyme activity. Rat brain, heart and liver were homogenized in 0.1M KCl, 0.1% sodium dodecyl sulfate, 0.1% lubrol, or 0.1% cetyl-trimethylammonium bromide. In general lubrol was more effective than the other solutions in solubilizing GPx and catalase. Lubrol and 0.1M KCl were equally effective in solubilizing SOD. The highest enzyme activities were (1) SOD: 2484 ng/mg (brain), 2501 ng/mg (heart), and 5586 ng/mg (liver); (2) GPx: 224 mU/mg (brain), 1870 mU/mg (heart), and 7332 mU/mg (liver); (3) catalase: 2.8 mU/mg (brain), 10.6 mU/mg (heart), and 309 mU/mg (liver). While cetyl trimethylammonium bromide is marginally better than sodium dodecyl sulfate in solubilizing active enzyme, neither ionic detergent has any advantage over lubrol or 0.1M KCl. For catalase and GPx, enzyme activity loss with time is biphasic. After initial, rapid activity loss (1–5 days for GPx and 7–10 days for catalase) the differences noted among the homogenizing solutions disappear and very little if any activity loss is noted over the next 2–3 weeks. For catalase and GPx, only baseline enzyme activity from t = 0 – 3 weeks is found in the most chaotropic solution, 0.1% sodium dodecyl sulfate while biphasic activity loss is most pronounced in 0.1% lubrol. These results may indicate active GPx and catalase species stabilized by a lipid-like environment. Correlatingin vitro catalase or GPx measurements within vivo anti-oxidative protection may underestimate tissue defences.     

Functional Characterization of Intestinal L-Carnitine Transport

The carnitine transporter OCTN2 is responsible for the renal reabsorption of filtered L-carnitine. However, there is controversy regarding the intestinal L-carnitine transport mechanism(s). In this study, the characteristics of L-carnitine transport in both, isolated chicken enterocytes and brush-border membrane vesicles (BBMV) were studied. In situ hybridization was also performed in chicken small intestine. Chicken enterocytes maintain a steady-state L-carnitine gradient of 5 to 1 and 90% of the transported L-carnitine remains in a readily diffusive form. After 5 min, L-Carnitine uptake into BBMV overshot the equilibrium value by a factor of 2.5. Concentrative L-carnitine transport is Na+-, membrane voltage-and pH-dependent, has a high affinity for L-carnitine (Km 26 - 31 microM ) and a 1:1 Na+: L-carnitine stoichiometry. L-Carnitine uptake into either enterocytes or BBMV was inhibited by excess amount of cold L-carnitine > D-carnitine = acetyl-L-carnitine = gamma-butyrobetaine > palmitoyl-L-carnitine > betaine > TEA, whereas alanine, histidine, GABA or choline were without significant effect. In situ hybridization studies revealed that only the cells lining the intestinal villus expressed OCTN2 mRNA. This is the first demonstration of the operation of a Na+/L-carnitine cotransport system in the apical membrane of enterocytes. This transporter has properties similar to those of OCTN2.     

Significance of renal gamma-butyrobetaine hydroxylase for carnitine biosynthesis in man

Carnitine biosynthesis was studied in man and rat. Three healthy adult men were given intravenous injections of 1 mCi of [methyl-3H]epsilon-N-trimethyl-L-lysine, a precursor of carnitine. Labeled metabolites of this compound were monitored in serum and urine at 2, 6, 12, 24, and 48 h. At least nine radioactive metabolites were detected. For each collecton period, the specific activity of urinary carnitine exceeded the average serum specific activity. In man, the amount of labeled carnitine in urine was 2 to 8 times greater than labeled gamma-butyrobetaine (the immediate precursor of carnitine). In similar experiments in rats (intravenous injection of 0.1 mCi of [methyl-3H]epsilon-N-trimethyl-L-lysine), the specific activity of carnitine in urine was always lower than the corresponding average specific activity in serum. Between 0 and 2 h after administration of labeled precursor, the animals excreted large amounts of labeled gamma-butyrobetaine but little labeled carnitine. Significant gamma-butyrobetaine, 2-oxoglutarate dioxygenase (EC 1.14.11.1) activity was found in human kidney but this activity was absent in rat kidney. The results indicate that in man and rat the kidney accumulates intravenously administered [methyl-3H]epsilon-N-trimethyl-L-lysine. This compound is metabolized predominantly to gamma-butyrobetaine in rat kidney and to carnitine in human kidney. In both species, the synthesized products are at least partially leaked (either by secretion or by passive diffusion down a concentration gradient) into the renal tubular lumen from which they are either reabsorbed into the circulation for distribution to other tissues or excreted.     

Differential Changes in Superoxide Dismutase Activity in Brain and Liver of Old Rats and Mice

Superoxide dismutase (SOD) activity was measured in the brain and liver of 24–26- and 3-month-old rats. No significant age-related differences in Cu/Zn-SOD activity were found in any of the tissues studied. A small but significant increase in total SOD activity was observed in the whole brain (10-20%), cerebral cortex (11%), and hypothalamus (18%) of old rats, whereas a much more important increase in Mn-SOD activity was found in the whole brain (48%), cerebral cortex (70%), striatum (60%), and hypothalamus (30%). The increase of Mn-SOD activity in the brain of old rats suggests the enzyme may play an important role in the process of aging. Mn-SOD is found only in the mitochondrion, which could be an important site of oxygen free radical production, and a significant increase in the enzyme activity was also found in the lung of hypoxic rats. A significant decrease in total SOD and Mn-SOD activity was observed in the liver of old rats. Preliminary experiments in 23–24-month-old mice similarly showed an increase and a decrease in total SOD and Mn-SOD activity, respectively, in the whole brain and liver. These results suggest that the regulatory mechanisms of Mn-SOD in the brain and liver vary differentially with age.     

Sites and regulation of carnitine biosynthesis in mammals

Although the pathway of carnitine biosynthesis in mammals is known, the location of active synthesis of carnitine and regulation of the pathway have not been clearly defined. Studies in several laboratories have shown that the enzymes that collectively convert epsilon-N-trimethyllysine (epsilon-N-TML) to gamma-butyrobetaine are found in all tissues studied in rats and humans, but distribution of the final enzyme of the pathway, gamma-butyrobetaine, 2-oxoglutarate dioxygenase (gamma-butyrobetaine hydroxylase) is variable from one species to another. Evidence from studies in rats and humans indicates that uptake and metabolism of epsilon-N-TML by the kidney is necessary for carnitine biosynthesis from circulating epsilon-N-TML. Limited data now available suggest that some of the intracellularly derived epsilon-N-TML is metabolized to gamma-butyrobetaine and carnitine in the tissue of origin, and some is released into the circulation. epsilon-N-TML in mammals is apparently derived from lysine residues in proteins, which are methylated and later released by protein hydrolysis. This source probably provides sufficient substrate for carnitine biosynthesis. Carnitine biosynthesis from epsilon-N-TML is not regulated by end-product feedback mechanisms. Hepatic gamma-butyrobetaine hydroxylase activity in rats and humans is developmentally regulated, and is increased by dietary L-thyroxine in adult rats. No other mechanisms for regulation of carnitine biosynthesis have been identified.     

Characterization of the antioxidant status of the heterozygous manganese superoxide dismutase knockout mouse

The antioxidant status of several tissues (liver, kidney, lung, brain, heart, muscle, stomach, and spleen) from heterozygous manganese superoxide dismutase (MnSOD) mutant mice (Sod2-/+) was characterized. The activity of MnSOD was decreased (30 to 80%) in all tissues examined. The levels of mRNA coding for the major antioxidant enzymes (CuZnSOD, catalase, and glutathione peroxidase) were not significantly altered in liver, kidney, heart, lung, or brain in the Sod2-/+ mice. The activities of the enzymes were not altered in any of these tissues, with the exception of a decrease in glutathione peroxidase activity in muscle in the Sod2-/+ mice compared to the Sod2+/+ mice. Thus, there was no up-regulation of the activities of the major antioxidant enzymes to compensate for the decrease in MnSOD activity. Reduced glutathione levels were 30 to 50% lower in the lung, brain, and muscle of the Sod2-/+ mice compared to the wild-type Sod2+/+ mice. In addition, the ratio of GSH/GSSG was decreased approximately 50% in Sod2-/+ muscle, indicating that the decrease in MnSOD activity in the Sod2-/+ mice results in some degree of oxidative stress in this tissue.     

L-carnitine increases liver alpha-tocopherol and lowers liver and plasma triglycerides in aging ovariectomized rats

The objective of this study was to determine whether dietary L-carnitine can influence the status of alpha-tocopherol, retinol and selected lipid parameters in aging ovariectomized rats, an animal model for the menopausal state. Fourteen Fisher-344 female rats 18 months old were acclimated for 4 weeks and ovarectomized. Seven rats per treatment were assigned to either a control group fed ad libitum AIN-93M diet or a carnitine group fed the same diet supplemented with L-carnitine. After an 8-week feeding period, blood and selected tissues were taken for analyses. No differences were noted in food intake, body weight, or organ weights due to L-carnitine. Dietary carnitine significantly increased liver alpha-tocopherol and tended to increase plasma alpha-tocopherol (P<.09). No changes in alpha-tocopherol were observed in other tissues including the brain, lungs and retroperitoneal fat. Retinol levels in plasma and tissues were not affected by supplemental L-carnitine. Significant decreases in liver and plasma triglyceride (TG) levels were noted, suggesting increased utilization of fatty acids. No differences were observed in the fatty acid profile of tissues. The results provide evidence that dietary supplementation of L-carnitine enhances the alpha-tocopherol status and improves the utilization of fat leading to lowering of the liver and plasma levels of TG in aging ovariectomized rats. Whether supplemental L-carnitine may be of benefit to postmenopausal women in lowering plasma TG and improving the antioxidant status remains to be studied.