Metabolic syndrome increases dietary α-tocopherol requirements as assessed using urinary and plasma vitamin E catabolites: a double-blind, crossover clinical trial.

Background: Vitamin E supplementation improves liver histology in patients with nonalcoholic steatohepatitis, which is a manifestation of the metabolic syndrome (MetS). We reported previously that α-tocopherol bioavailability in healthy adults is higher than in those with MetS, thereby suggesting that the latter group has increased requirements.Objective: We hypothesized that α-tocopherol catabolites α-carboxyethyl hydroxychromanol (α-CEHC) and α-carboxymethylbutyl hydroxychromanol (α-CMBHC) are useful biomarkers of α-tocopherol status.Design: Adults (healthy or with MetS; n = 10/group) completed a double-blind, crossover clinical trial with four 72-h interventions during which they co-ingested 15 mg hexadeuterium-labeled RRR-α-tocopherol (d6-α-T) with nonfat, reduced-fat, whole, or soy milk. During each intervention, we measured α-CEHC and α-CMBHC excretions in three 8-h urine collections (0-24 h) and plasma α-tocopherol, α-CEHC, and α-CMBHC concentrations at various times ≤72 h.Results: During the first 24 h, participants with MetS compared with healthy adults excreted 41% less α-CEHC (all values are least-squares means ± SEMs: 0.6 ± 0.1 compared with 1.0 ± 0.1 µmol/g creatinine, respectively; P = 0.002), 63% less hexadeuterium-labeled (d6)-α-CEHC (0.04 ± 0.02 compared with 0.13 ± 0.02 µmol/g creatinine, respectively; P = 0.002), and 58% less d6-α-CMBHC (0.017 ± 0.004 compared with 0.041 ± 0.004 µmol/g creatinine, respectively; P = 0.0009) and had 52% lower plasma d6-α-CEHC areas under the concentration curves [area under the curve from 0 to 24 h (AUC0-24h): 27.7 ± 7.9 compared with 58.4 ± 7.9 nmol/L × h, respectively; P = 0.01]. d6-α-CEHC peaked before d6-α-T in 77 of 80 paired plasma concentration curves. Urinary d6-α-CEHC 24-h concentrations were associated with the plasma AUC0-24 h of d6-α-T (r = 0.53, P = 0.02) and d6-α-CEHC (r = 0.72, P = 0.0003), and with urinary d6-α-CMBHC (r = 0.88, P < 0.0001), and inversely with the plasma inflammation biomarkers C-reactive protein (r = -0.70, P = 0.0006), interleukin-10 (r = -0.59, P = 0.007), and interleukin-6 (r = -0.54, P = 0.01).Conclusion: Urinary α-CEHC and α-CMBHC are useful biomarkers to noninvasively assess α-tocopherol adequacy, especially in populations with MetS-associated hepatic dysfunction that likely impairs α-tocopherol trafficking. This trial was registered at clinicaltrials.gov as NCT01787591.

A rapid HPLC/ESI-MS/MS method for quantitative analysis of isovalerylshikonin in rat plasma.

A new, fast and sensitive high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) method was developed and validated for isovalerylshikonin in rat plasma using emodin as internal standard (IS). The analyte was extracted from rat plasma with ethyl acetate, after 10% HCl treatment and protein precipitated by methanol. The compound was separated on an Ultimate XB-C(18) analytical column using a mobile phase of methanol-10 mM ammonium acetate in water-acetonitrile containing 0.05% formic acid (45 : 10 : 45, v/v/v) with isogradient elution. The analyte was detected in negative ion mode using multiple-reaction monitoring. The method was validated and the specificity, linearity, lower limit of quantitation (LLOQ), precision, accuracy, recoveries and stability were determined. LLOQ was 9 ng/mL for isovalerylshikonin. Correlation coefficient (r) value for the linear range of the analyte was greater than 0.99. The intra-day and inter-day precision and accuracy were better than 8.52%. The relative and absolute recovery was above 86% and no matrix effects were observed for isovalerylshikonin. This validated method provides a modern, rapid and robust procedure for the pharmacokinetic study of the two compounds in rats after intravenous administration to rats (n = 4).

Amino-acid depletion induced by abnormal amino-acid conjugation and protein restriction in isovaleric acidemia.

BACKGROUND: Previously, we detected 19 'new' amino-acid conjugates in the urine of patients with isovaleric acidemia. There is currently a poor understanding of the relationship between the clinical symptoms and the excreted metabolites occurring in these patients, owing to insufficient metabolite characterization and quantification. Consequently, controversial treatment protocols exist, particularly pertaining to dietary protein restriction. OBJECTIVE: To determine the effect of the previously identified amino-acid conjugates and conventional dietary protein restriction therapy, on the free amino-acid concentrations in isovaleric acidemia patients, to better explain the clinical symptoms and develop more effective therapy. DESIGN: Free amino-acid quantification via liquid chromatography mass spectrometry (LC-MS-MS) was performed on pre- and post-treatment urine or serum samples collected from six isovaleric acidemia patients, previously investigated for the presence of new induced N-isovaleryl and N-acetyl-amino-acid conjugates. RESULTS: Depleted amino-acid concentrations were detected in varying degrees in all six patients and did not recover after conventional treatment. CONCLUSIONS: The 19 potentially toxic metabolites previously identified and the consequent amino-acid depletions detected in this study, may explain many of the clinical symptoms associated with isovaleric acidemia. Furthermore, the occurrence of amino-acid depletions in these patients, steers away from the controversial dietary protein restriction treatment protocols, and towards dietary leucine restriction alone with essential amino-acid supplementation, in combination with glycine and L-carnitine supplementation.

Improvement in exercise tolerance in isovaleric acidaemia with L-carnitine therapy.

The effect of 4 weeks' treatment with oral-L-carnitine (100 mg/kg per day) on carnitine status and metabolic parameters during an incremental ramp exercise test in a 12-year-old girl with isovaleric acidaemia was examined to determine its possible therapeutic role. The maximum work rate achieved increased from 110 to 120 watts; oxygen consumption at anaerobic threshold from 600 to 800 L/min; peak oxygen consumption from 1270 to 1450 L/min; and oxygen pulse, a measure of cardiac output, from 7.0 to 8.1 L/beat. These changes were associated with increases in plasma and urinary free and acyl carnitine concentrations but no change in physical activity. This observed effect of L-carnitine on exercise performance may be on cardiac or skeletal muscle function or both. We conclude that, in this single patient with isovaleric acid-aemia, L-carnitine supplementation had objective benefits and further studies on more patients are warranted.

Isovaleric acidemia: response to a leucine load after three weeks of supplementation with glycine, L-carnitine, and combined glycine-carnitine therapy.

OBJECTIVE: To assess the effectiveness of glycine and carnitine therapy on isovaleryl conjugate excretion in isovaleric acidemia (IVA). STUDY DESIGN: Urinary isovalerylglycine (IVG) and isovalerylcarnitine (IVC) were measured from 12-hour urine specimens collected overnight from an 8-year-old patient with IVA (who had no residual activity of isovaleryl-CoA dehydrogenase in fibroblasts) before and during 3-week courses of supplementation with glycine alone (250 mg/kg per day), L-carnitine alone (100 mg/kg per day) therapy, and both of these agents combined, with a 2 gm leucine challenge performed at the end of each treatment period. RESULTS: Isovalerylglycine was the predominant metabolite excreted throughout the study, and its mean value doubled with glycine treatment. Isovalerylcarnitine excretion was minimal without carnitine supplementation. L-Carnitine therapy was associated with a 50% decline in excretion of IVG without a fully compensatory increase in IVC. The readdition of glycine to the carnitine regimen resulted in an increase in IVG excretion. Leucine challenge resulted in a 2.7- and 2.4-fold increase of IVG and IVC excretion, respectively, during L-carnitine therapy but not during glycine supplementation, and a 3.5- and 4-fold increase in excretion of both metabolites during glycine plus L-carnitine therapy. Total conjugate excretion was highest after a leucine load during combined glycine and L-carnitine therapy. CONCLUSIONS: Combined glycine and L-carnitine therapy maximally increases isovaleryl conjugate excretion during metabolic stress but not under stable conditions.

Effect of carnitine administration on glycine metabolism in patients with isovaleric acidemia: significance of acetylcarnitine determination to estimate the proper carnitine dose.

In isovaleric acidemia (IVA), accumulated isovaleryl-CoA in the mitochondrion induces variable metabolic disturbances. To remove intramitochondrial isovaleryl groups, glycine therapy has been advocated primarily. On the other hand, secondary carnitine deficiency has been documented in this disorder and carnitine supplementation alone has been reported to be effective. In the present study, we administered carnitine and glycine to patients with IVA, and investigated serum carnitine and urinary excretion of total and free carnitine, acylcarnitine profile (i.e., isovalerylcarnitine and acetylcarnitine), and isovalerylglycine. By adding carnitine to glycine supplementation, more isovalerylglycine, not only isovalerylcarnitine, was excreted in the urine. Acetylcarnitine was detected in the urine only when sufficient carnitine was supplemented. We concluded that combined therapy of glycine and carnitine is more effective and safer to eliminate isovaleryl-CoA in IVA than conventional therapy using either glycine or carnitine. Urinary acetylcarnitine concentration might be a good marker indicating the optimal dose of L-carnitine supplementation.

Intravenous L-carnitine and acetyl-L-carnitine in medium-chain acyl-coenzyme A dehydrogenase deficiency and isovaleric acidemia.

The purpose of this study was to determine whether treatment with L-carnitine or acetyl-L-carnitine enhances the turnover of lipid or branched-chain amino acid oxidation in patients with inborn errors of metabolism. Increasing i.v. doses of L-carnitine and acetyl-L-carnitine were given to one patient with medium-chain acyl-CoA dehydrogenase deficiency and to another with isovaleric acidemia. Both patients were in stable condition and receiving oral L-carnitine supplements. The excretion of carnitine and disease-specific metabolites was measured. The incorporation of L-carnitine in the intracellular pool was demonstrated using stable isotopes and mass spectrometry. Increasing doses of either i.v. L-carnitine or acetyl-L-carnitine did not stimulate the excretion of octanoylcarnitine in the patient with medium-chain acyl-CoA dehydrogenase deficiency, nor did it raise the plasma levels of either cis-4-decenoate or octanoylcarnitine. Similarly, increasing doses of either i.v. L-carnitine or acetyl-L-carnitine did not enhance the excretion of isovalerylcarnitine in a patient with isovaleric acidemia. The excretion of isovalerylglycine actually decreased. We conclude that there was no evidence of enhanced fatty acid beta-oxidation or enhanced branched-chain amino acid oxidation in vivo by the administration of high doses of L-carnitine or acetyl-L-carnitine in these two patients. Because only one individual with each disorder was studied, the data are only indicative and may not necessarily be representative of all individuals with these disorders. Definite settlement of this issue will require further studies in additional subjects.

Effect of increasing ruminal butyrate on milk yield and blood constituents in dairy cows fed a grass silage-based diet.

The effects of increased ruminal supply of butyrate on milk yield, milk composition, and blood metabolites were studied in four lactating cows in a 4 x 4 Latin square design. The basal diet comprised grass silage, hay, and concentrate (34:22:42, DM basis) and was supplemented with isoenergetic VFA infusions (3.58 Mcal/d). A 3:1 molar mixture of acetate and propionate was replaced gradually with butyrate at the rates of 0, 200, 400, or 600 g/d. When the amount of infused butyrate increased, isobutyrate, butyrate, and isovalerate in plasma and acetoacetate and beta-hydroxybutyrate in whole blood increased linearly, but plasma glucose concentration decreased. The latter was associated with a trend toward higher plasma urea concentration, suggesting that more AA were used for gluconeogenesis as the supply of propionate decreased and that of butyrate increased. Milk yield was not changed. The concentrations of milk fat and protein increased, and that of lactose decreased linearly, with the rate of butyrate infusion. Milk fat yield increased, and lactose yield tended to decrease, with increased butyrate infusion. These results indicate that changes in the supply of butyrate do not affect markedly milk yield in cows yielding less than 20 kg/d but cause marked changes in milk composition. The increase in ruminal butyrate supply increased ketogenesis and decreased gluconeogenesis in the liver of lactating dairy cows.

Long-term L-carnitine treatment in isovaleric acidemia.

A 5-year-old girl with isovaleric acidemia was treated with long-term L-carnitine and no supplemental glycine. Clinical and laboratory data are presented. Following diagnosis and treatment at age 2 years, the frequency of acute exacerbations of metabolic acidosis was reduced and she resumed normal growth and development. L-carnitine supplementation and protein restriction may be sufficient for effective therapy of isovaleric acidemia.

The treatment of isovaleric acidemia with glycine supplement.

Although dietary leucine restriction and supplemental glycine are used to treat patients with isovaleric acidemia [deficient isovaleryl-CoA-dehydrogenase (E.C.1.3.99.10)], little quantitative information is available regarding their optimum relationship. Herein we compare different glycine supplements and quantitate isovalerylglycine produced in two patients with clinically different forms of isovaleric acidemia during restricted leucine intake and during oral leucine loading. We found that under stable conditions of leucine restriction, 150 mg glycine/kg/day is an optimum glycine supplement and that glycine supplements of more than 250 mg/kg/day may result in reduced isovalerylglycine production; that when isovaleric acid accumulation is increased, glycine supplements to 600 mg/kg/day will increase isovalerylglycine production; and that the phenotype of isovaleric acidemia is related not only to the extent of impaired isovaleryl-CoA dehydrogenase, but also the ability to detoxify accumulated isovaleryl CoA to isovalerylglycine.

The response to L-carnitine and glycine therapy in isovaleric acidaemia.

The profound metabolic disturbances which occur in isovaleric acidaemia are due to the intramitochondrial accumulation of isovaleryl coenzyme A (CoA) with a consequent reduction in the availability of free CoA. Secondary carnitine insufficiency is also a feature of this and other disorders of organic acid metabolism. A patient who presented at 2.5 years of age was diagnosed using capillary GC-MS as having isovaleric acidaemia. She showed the full spectrum of abnormal organic acids previously associated with the 'neonatal' form of the disease despite her late presentation, indicating that it is inappropriate to refer to acute early and late onset forms of isovaleric acidaemia. Instead, a spectrum of disease exists, determined by environmental factors, residual enzyme activities and modifying effects of different phenotypes in different individuals. She also showed evidence of carnitine insufficiency. An oral challenge with L-carnitine resulted in the excretion of large amounts of urinary acylcarnitines which were shown by use of fast atom bombardment mass spectrometry to be primarily isovalerylcarnitine. Regular glycine supplementation caused no significant increase in urinary isovalerylglycine and had to be stopped because of side-effects after 5 days. An oral L-carnitine challenge during glycine supplementation resulted in a marked increase in isovalerylglycine excretion, again associated with the excretion of large amounts of isovalerylcarnitine. Carnitine acts by removing (detoxifying) intramitochondrial isovaleryl groups and, in the presence of glycine, it promotes the formation of isovalerylglycine. We believe L-carnitine supplementation is of value in the treatment of isovaleric acidaemia and that, in the present case, L-carnitine together with a moderate dietary restriction has proved to be the optimum form of therapy.

Alanine decreases the protein requirements of infants with inborn errors of amino acid metabolism.

Supplementation with alanine was found to increase growth in weight and nitrogen balance in 5 infants with a variety of inborn errors of amino acid metabolism receiving diets restricted in protein. The addition of alanine to the regimen led to a mean increase in weight of 15 g/day. This and the increased nitrogen balance of 15 mg/kg/day were highly significant statistically. In addition a dose-response effect of alanine was observed. The effect of alanine was compared with that of a supplemental mixture of essential and non-essential amino acids, lacking only those considered to be toxic in these patients. Alanine at 0.05 g/kg was as effective in promoting growth in weight as 1.05 g/kg of the amino acid mixture, while 0.25 g/kg of alanine was more effective than 0.70 g/kg of the amino acid mixture. The protein sparing anabolic effect of alanine is thought to be a reflection of the alanine glucose cycle.

L-carnitine therapy in isovaleric acidemia.

Isovaleric acidemia, resulting from isovaleryl-coenzyme A dehydrogenase deficiency, is associated with marked reduction of free carnitine in both plasma and urine. Fast atom bombardment-mass spectrometry, hydrolysis, and gas chromatography/mass spectrometry have unequivocally identified the existence of isovalerylcarnitine, a new metabolite specific for this disorder. Administration of equimolar amounts of glycine or L-carnitine separately with leucine demonstrated that isovaleryl-coenzyme A is removed by supplemental L-carnitine in the form of isovalerylcarnitine as effectively as it is by glycine, in the form of isovalerylglycine. When L-carnitine is given alone, excretion of isovalerylglycine decreases in preference to enhanced excretion of isovalerylcarnitine and hippurate. Treatment with L-carnitine alone has proven effective in preventing further hospitalizations in a patient with this genetic disorder.

Hudson memorial lecture. Neonatal management of organic acidurias. Clinical update.

Therapeutic guidelines have been obtained from a retrospective review of 41 patients affected with organic acidaemias, 16 patients with neonatal maple syrup urine disease (MSUD), 11 methylmalonic acidaemia, (MMA) seven propionic acidaemias (PA) and seven isovaleric acidaemias (IVA), and by comparing this personal series with similar reported cases. The emergency treatment of these organic acidurias in the neonate has to main goals: toxin removal and anabolism. Anabolism is always promoted by early diet therapy. The best method of toxin removal depends on the nature of the defect; peritoneal dialysis with exchange transfusions or multiple or prolonged exchange transfusions in MSUD and in PA, diuresis and exchange transfusions in MMA and glycine supplementation in IVA. Vitamin supplementation (thiamine 20 mg, biotin 10 mg, B12 2 mg and riboflavin 100 mg) should be tried in all cases although the neonatal forms of these defects are very rarely vitamin responsive. Additional treatments such as carnitine or insulin may prove to be useful.