Rapid and sensitive HILIC-MS/MS analysis of carnitine and acetylcarnitine in biological fluids.

Monitoring carnitine and acetylcarnitine levels in biological fluids is a powerful tool for diagnostic studies. Research has recently shown that the analysis of carnitine and related compounds in clinical samples can be accomplished by different analytical approaches. Because of the polar and ionic nature of the analytes and matrix complexity, accurate quantitation is a highly challenging task. Thus, sample processing factors, preparation/cleanup procedures, and chromatographic/ionization/detection parameters were evaluated. On the basis of the results obtained, a rapid, selective, sensitive method based on hydrophilic interaction liquid chromatography-tandem mass spectrometry for the analysis of carnitine and acetylcarnitine in serum and urine samples is proposed. The matrix effect was assessed. The proposed approach was validated, the limits of detection were in the nanomolar range, and carnitine and acetylcarnitine levels were found within the micromolar range in both types of sample.

Oxidative stress parameters in urine from patients with disorders of propionate metabolism: a beneficial effect of L:-carnitine supplementation.

Propionic (PA) and methylmalonic (MMA) acidurias are inherited disorders caused by deficiency of propionyl-CoA carboxylase and methylmalonyl-CoA mutase, respectively. Affected patients present acute metabolic crises in the neonatal period and long-term neurological deficits. Treatments of these diseases include a protein restricted diet and L: -carnitine supplementation. L: -Carnitine is widely used in the therapy of these diseases to prevent secondary L: -carnitine deficiency and promote detoxification, and several recent in vitro and in vivo studies have reported antioxidant and antiperoxidative effects of this compound. In this study, we evaluated the oxidative stress parameters, isoprostane and di-tyrosine levels, and the antioxidant capacity, in urine from patients with PA and MMA at the diagnosis, and during treatment with L: -carnitine and protein-restricted diet. We verified a significant increase of isoprostanes and di-tyrosine, as well as a significant reduction of the antioxidant capacity in urine from these patients at diagnosis, as compared to controls. Furthermore, treated patients presented a marked reduction of isoprostanes and di-tyrosine levels in relation to untreated patients. In addition, patients with higher levels of protein and lipid oxidative damage, determined by di-tyrosine and isoprostanes levels, also presented lower urinary concentrations of total and free L: -carnitine. In conclusion, the present results indicate that treatment with low protein diet and L: -carnitine significantly reduces urinary biomarkers of protein and lipid oxidative damage in patients with disorders of propionate metabolism and that L: -carnitine supplementation may be specially involved in this protection.

Diagnosis of isovaleric acidaemia by tandem mass spectrometry: false positive result due to pivaloylcarnitine in a newborn screening programme.

Tandem mass spectrometric analysis of acylcarnitines and amino acids has been applied in newborn screening programmes for the detection of several inborn errors of metabolism. We report a false positive result for isovaleric acidaemia in a newborn screening programme using this method. The newborn screening sample showed a very prominent signal corresponding to the mass of isovalerylcarnitine. Repeat samples (age 6 days) of blood and urine showed similar results. However, urine organic acids were normal. Acylcarnitine analysis in blood, breast milk and urine of the mother also showed a prominent signal of the same mass. Gas chromatography-mass spectrometry of the methyl esters demonstrated that the signal in the patient's urine was due to the presence of pivaloylcarnitine, which is isomeric with isovalerylcarnitine. The patient's mother was receiving an antibiotic containing a derivative of pivalic acid to treat a urinary tract infection. Follow-up samples in the patient and the mother confirmed a decrease in the levels of pivaloylcarnitine, concomitant with the discontinuation of the treatment. We conclude that pivaloylcarnitine can give a false positive result for isovaleric acidaemia in newborns whose mothers are on treatment with pivoxilsulbactam-containing antibiotics.

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.

Renal handling of carnitine in ill preterm and term neonates.

OBJECTIVE: To investigate the renal handling of carnitine in preterm and term ill neonates. METHODS: We studied the fractional tubular reabsorption of carnitine and the proximal renal tubular function of infants in the first week of life who were receiving very little or no carnitine in their diets. RESULTS: Mean plasma levels were low: total carnitine was 16.4 +/- 7.0 mumol/L, free carnitine was 9.2 +/- 5.0 mumol/L, and acylcarnitine was 7.2 +/- 4.1 mumol/L. The most premature group of neonates (gestation age, 26 to 31 weeks) had a fractional tubular reabsorption rate of free carnitine of 94.3% +/- 3.3%, which was lower than in the other two groups (98.1% +/- 2.4% for gestational age 32 to 36 weeks, p = 0.001; and 99.2% +/- 0.6% for gestational age 37 to 42 weeks, p = 0.002). In all patients the fractional tubular reabsorption of acylcarnitine was lower than that of free carnitine, indicating possible tubular secretion of acylcarnitine. It correlated with the total plasma carnitine levels (r = 0.53; p = 0.002). The fractional tubular reabsorption of free carnitine also correlated with gestational age (r = 0.60; p < 0.001). CONCLUSIONS: Ill neonates have a fractional tubular reabsorption rate of free carnitine within the normal range. It increases with gestational age, and has the same maturation rate as the other known indexes of proximal tubular function.

Glutaric aciduria type I: unusual biochemical presentation.

We describe a patient with glutaryl-coenzyme A dehydrogenase deficiency, demonstrated by a residual enzyme activity of only 1% in cultured fibroblasts. Although the clinical presentation was typical of glutaric aciduria type I, the urine concentrations of glutaric, glutaconic, and 3-hydroxyglutaric acids remained normal, even during episodes of clinical decompensation. An increased free glutarate level was demonstrated only in cerebrospinal fluid.

Quantitation of urinary carnitine esters in a patient with medium-chain acyl-coenzyme A dehydrogenase deficiency: effect of metabolic state and L-carnitine therapy.

Urinary carnitine esters were quantitated in an infant with medium-chain acylcoenzyme A dehydrogenase deficiency by means of a highly sensitive and specific radioisotopic exchange high-pressure liquid chromatography method. During fasting, the excretion of free carnitine and of acetylcarnitine, octanoylcarnitine, and hexanoylcarnitine was increased. The fractional tubular reabsorption of free carnitine was decreased, suggesting a renal leak of free carnitine. In the symptom-free, fed state, only minor amounts of free carnitine and of short-chain acylcarnitine, octanoylcarnitine, and hexanoylcarnitine were present in urine, and carnitine loss occurred in the form of "other" carnitine esters not exceeding that of control subjects. During L-carnitine therapy, the excretion of free carnitine, short-chain acylcarnitine, octanoylcarnitine, and hexanoylcarnitine, and particularly of "other" carnitine esters, was increased, suggesting a possible detoxifying effect of administered carnitine that is not confined to the elimination of octanoic and hexanoic acids. The employed method detects very low urinary concentrations of octanoylcarnitine and hexanoylcarnitine (less than 1 mumol/L) characteristic of medium-chain acyl-coenzyme A dehydrogenase deficiency and may be useful in screening for this disease, which has been associated with sudden infant death.

Enhanced lipid utilization in infants receiving oral L-carnitine during long-term parenteral nutrition.

Fourteen infants requiring long-term total parenteral nutrition but able to tolerate small quantities of enteral feedings were randomized into carnitine treatment and placebo control groups. All infants had received nutritional support devoid of carnitine. Plasma carnitine levels and observed plasma lipid indices were not different before supplementation. Under standardized, steady-state conditions, 0.5 g/kg fat emulsion (intralipid) was administered intravenously over 2 hours both before and after infants received 7 days of continuous nasogastric or gastric tube L-carnitine (50 mumol/kg/day) or placebo. Plasma triglyceride, free fatty acid, acetoacetate, beta-hydroxybutyrate, and carnitine concentrations were observed at 0 (start of lipid infusion), 2, and 4 hours for pre- and post-treatment periods, and in addition at 6 and 8 hours after carnitine supplementation. Infants receiving carnitine had significantly greater beta-hydroxybutyrate plasma concentrations (P less than 0.05) and carnitine (P less than 0.001) at 0, 2, 4, 6, and 8 hours, and greater plasma acetoacetate concentrations (P less than 0.05) at 2, 4, 6, and 8 hours, compared with controls. Twenty-four-hour urinary carnitine excretion was very low for both groups before supplementation; after supplementation, excretion was higher (P less than 0.05) in the carnitine group. No significant differences were found between groups for plasma triglyceride or free fatty acid concentrations at any observation period. This study demonstrated enhanced fatty acid oxidation, as evidenced by increased ketogenesis, with L-carnitine supplementation in infants receiving long-term total parenteral nutrition.

Renal handling of carnitine in children with carnitine deficiency and hyperammonemia associated with valproate therapy.

Free and acylcarnitine in serum and urine samples were measured in five patients with hyperammonemia associated with anticonvulsant therapy including sodium valproate, of whom three had a Reye-like syndrome. All had considerable reduction in serum free carnitine and slight increase of acylcarnitine concentrations, suggesting increased conversion of free to acylcarnitine by valproate administration. Urinary excretion of both free and acylcarnitine was increased, accompanied by depressed reabsorption of free carnitine and decreased acylcarnitine/free carnitine clearance ratio. These results indicate a decreased threshold for free carnitine. The combination of these several factors may be responsible for carnitine deficiency in patients with hyperammonemia taking valproate.

Recognition of medium-chain acyl-CoA dehydrogenase deficiency in asymptomatic siblings of children dying of sudden infant death or Reye-like syndromes.

The medium-chain acyl-CoA dehydrogenase (MCAD) deficiency of mitochondrial beta oxidation has been identified in two asymptomatic siblings in a family in which two previous deaths had been recorded, one attributed to sudden infant death syndrome and the other to Reye syndrome. Recognition of this disorder in one of the deceased and in the surviving siblings was accomplished by detection of a diagnostic metabolite, octanoylcarnitine, using a new mass spectrometric technique. This resulted in early treatment with L-carnitine supplement in the survivors, which should prevent metabolic deterioration. Further studies suggest that breast-feeding may be protective for infants with MCAD deficiency. Families with children who have had Reye syndrome or in which sudden infant death has occurred are at risk for MCAD deficiency. We suggest that survivors and asymptomatic siblings should be tested for this treatable disorder.

Octanoic acidemia and octanoylcarnitine excretion with dicarboxylic aciduria due to defective oxidation of medium-chain fatty acids.

Five patients aged 7 to 21 months are described who developed attacks of coma after a short prodromal illness with diarrhea or vomiting or both. Four had concomitant hypoglycemia, and all had hypoketonemia, with excessive urinary excretion of medium-chain dicarboxylic acids, medium-chain (omega-1)-hydroxyacids, suberylglycine, hexanoylglycine, and octanoylcarnitine. All patients accumulated octanoic acid, decanoic acid, and cis-4-decenoic acid in plasma. Fibroblasts from three patients showed a decreased rate of octanoate oxidation (10%, 12%, and 29% of control values, respectively). These findings suggest a deficiency of medium-chain acyl-CoA dehydrogenase, most probably an autosomal recessive inherited metabolic disorder. Two of the patients died during an acute attack, and a third had severe neurologic sequelae; the two remaining patients recovered. Plasma free carnitine levels were low, but total carnitine was normal. The three surviving patients underwent a fasting test, which did not lead to hypoglycemia, although hypoketonemia, dicarboxylic aciduria, and excessive mobilization of fatty acids did occur. The surviving patients were maintained on frequent carbohydrate-enriched meals.