Urine acylcarnitine analysis by ESI-MS/MS: a new tool for the diagnosis of peroxisomal biogenesis disorders.

BACKGROUND: Patients with peroxisomal biogenesis disorders (PBDs) have an abnormal profile of circulating acylcarnitines (i.e. elevated C16:0-DC-, C18:0-DC-, C24:0-, C26:0-carnitine). We developed an ESI-MS/MS method for quantification of urine acylcarnitines and tested its reliability for the diagnosis of PBDs. METHODS: Urine from 7 patients with PBDs (5 Zellweger syndrome, 2 infantile Refsum disease), from 2 patients with D-bifunctional protein (D-BP) deficiency, and from 130 healthy controls were analysed by ESI-MS/MS, using a multiple reactions monitoring (MRM) method, and quantified with labelled internal standards. Acylcarnitine levels between groups were analyzed by the STATA statistics data analysis and compared by the non parametric Mann-Whitney test. RESULTS: In PBDs, the urinary excretion of long-chain dicarboxylylcarnitines (C14:0-DC-, C16:0-DC-, and C18:0-DC-carnitine), and of very long-chain monocarboxylylcarnitines (C22:0-, C24:0-, C26:0-carnitine) were significantly elevated compared to controls (p<0.0001). Interestingly, among PBDs the most severe abnormalities of acylcarnitine profile were observed in patients with Zellweger syndrome. One patient with D-BP showed similar abnormalities to PBDs, while in the other only C16:0-DC-carnitine was markedly elevated. CONCLUSIONS: This study shows that MRM ESI-MS/MS acylcarnitine analysis unequivocally discriminates patients with PBDs and D-BP deficiency from controls, representing a reliable and sensitive method for the diagnosis that requires a short-time analysis with high sample through-put.

Identification of the peroxisomal beta-oxidation enzymes involved in the degradation of leukotrienes.

Leukotrienes (LTs) are metabolically inactivated via omega-oxidation and subsequent beta-oxidation from the omega-end. This beta-oxidation process takes place in peroxisomes. In this study we investigated the role of different enzymes involved in peroxisomal beta-oxidation in the degradation of LTs. We analyzed LTB(4), LTE(4), and their oxidation products in urine of patients with Infantile Refsum's disease (IRD), d-bifunctional protein (DBP) deficiency, Rhizomelic Chondrodysplasia Punctata (RCDP) type 1, and X-linked adrenoleukodystrophy (XALD). We found that patients with IRD and DBP deficiencies excrete increased amounts of LTB(4), LTE(4), omega-carboxy-LTB(4), and omega-carboxy-LTE(4) in their urine, whereas the beta-oxidation products were not detectable. These results show that DBP plays an essential role in the degradation of LTs. In urine of patients with XALD and RCDP type 1 we found normal levels of LTB(4), LTE(4), and their oxidation products, indicating that the adrenoleukodystrophy protein and peroxisomal 3-ketoacyl-CoA thiolase are not involved in the metabolic inactivation of LTs.

Excretion of 3,6-epoxydicarboxylic acids in peroxisomal disorders.

The urinary excretions of several organic acids were quantitatively studied by gas chromatography/mass spectrometry in subjects with disorders of peroxisome biogenesis (n = 8) and controls (n = 26). The excretion of 3,6-epoxtetradecanedioic acid was significantly elevated in all subjects with disorders of peroxisome biogenesis (1.8-20.8; controls, not detected-0.5, mumol/mmol of creatinine). 3,6-Epoxydodecanedioic acid excretion was usually elevated (1.4-19.8; controls, not detected-4.2) and 3,6-epoxyoctanedioic acid excretion was not elevated not detected-8.8; controls, 0.6-9.5 mumol/mmol of creatinine). It is suggested that measurement of 3,6-epoxydicarboxylic acids may be useful for the diagnosis of these disorders.

Stable isotope dilution analysis of very long chain fatty acids in plasma, urine and amniotic fluid by electron capture negative ion mass fragmentography.

A sensitive and selective stable isotope dilution electron capture negative ion chemical ionization mass fragmentography method applying pentafluorobenzyl derivatives was developed for the accurate quantitation of very long chain fatty acids. This technique allowed detection of 1-5 pg of each compound and was applied to plasma (100 microliters), amniotic fluid (1 ml) and urine (1 ml). Normal concentrations were established and the concentrations in samples of selected patients with classified peroxisomal disorders were determined. In plasma samples of all patients the C26:0/C22:0 ratios were elevated (range 0.03-0.43), compared to the control ratios (range 0.003-0.021). The ratio C26:0/C22:0 was elevated in four of five amniotic fluid samples from fetuses with peroxisomal disorders (range 0.18-0.54) when compared with controls (range 0.05-0.25). An elevation of the ratio C26:1/C22:0 was observed in all five amniotic fluid samples (range 0.22-0.60 vs. 0-0.08 in controls). Urinary C26:0 concentrations were lower than in plasma and amniotic fluid and diagnostic ratios were not elevated in patients with peroxisomal disorders.

Rapid diagnosis of Zellweger syndrome and infantile Refsum's disease by fast atom bombardment--mass spectrometry of urine bile salts.

A method is described for the rapid determination of urinary bile salt profiles by fast atom bombardment--mass spectrometry (FAB-MS). Urine was passed through a reverse-phase octadecylsilane bonded silica cartridge and the bile salts eluted with methanol. Negative ion FAB spectra could be obtained from the equivalent of 10 microliter of urine loaded onto the target probe with glycerol as matrix. In samples from normal infants and children bile salt peaks were rarely detectable above the background whereas peaks produced by steroid sulphates and glucuronides and bile alcohol glucuronides could usually be identified. In samples from infants and children with cholestasis the major peaks were produced by the taurine and glycine conjugates of di-, tri- and tetrahydroxycholanoic acids (and their monosulphates). In samples from patients with Zellweger syndrome and infantile Refsum's disease, a unique ion at m/z 572 indicated the presence of taurine-conjugated tetrahydroxy-cholestanoic acid(s). The amide linkage to taurine was cleaved by alkaline hydrolysis but not by cholylglycine hydrolase. Capillary gas chromatography--mass spectrometry (GC-MS) of the bile acids liberated by alkaline hydrolysis indicated the presence of at least two nuclear-tetrahydroxylated cholestanoic acids, probably the 6 alpha- and 1 beta-hydroxylated derivatives of 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestan-26-oic acid.

2,6-Dimethyloctanedioic acid--a metabolite of phytanic acid in Refsum's disease.

The urine of two patients with Refsum's disease consistently contained 2,6-dimethyloctanedioic acid, a compound not normally found in human urine. In addition, their urines contained increased concentrations of 3-methylhexanedioic acid. These two compounds may be formed from phytanic acid by an initial omega-oxidation and subsequent beta-oxidations. It was calculated that this oxidation pathway may metabolize at least 30 mg of phytanic acid per day.

Occurrence of novel branched-chain fatty acids in Refsum's disease.

Two novel branched-chain fatty acids, which appear to be unsaturated analogs of phytanic acid, have been observed in sera and urine of patients with Refsum's disease. They occur in both phospholipids and neutral lipids, and have been isolated and characterized.

Localization of the oxidative defect in phytanic acid degradation in patients with Refsum's disease.

The rate of oxidation of phytanic acid-U-(14)C to (14)CO(2) in three patients with Refsum's disease was less than 5% of that found in normal volunteers. In contrast, the rate of oxidation of alpha-hydroxyphytanic acid-U-(14)C and of pristanic acid-U-(14)C to (14)CO(2), studied in two patients, while somewhat less than that in normal controls, was not grossly impaired. These studies support the conclusion that the defect in phytanic acid oxidation in Refsum's disease is located in the first step of phytanic acid degradation, that is, in the alpha oxidation step leading to formation of alpha-hydroxyphytanic acid. The initial rate of disappearance of plasma free fatty acid radioactivity after intravenous injection of phytanic acid-U-(14)C (t(1/2) = 5.9 min) was slower than that seen with pristanic acid-U-(14)C (t(1/2) = 2.7 min) or palmitic acid-1-(14)C (t(1/2) = 2.5 min). There were no differences between patients and normal controls in these initial rates of free fatty acid disappearance for any of the three substrates tested. There was no detectable lipid radioactivity found in the plasma 7 days after the injection of palmitic acid-1-(14)C or pristanic acid-U-(14)C in either patients or controls. After injection of phytanic acid-U-(14)C, however, the two patients showed only a very slow decline in plasma lipid radioactivity (estimated t(1/2) = 35 days), in contrast to the normals who had no detectable radioactivity after 2 days. Incorporation of radioactivity from phytanic acid-U-(14)C into the major lipid ester classes of plasma was studied in one of the patients; triglycerides accounted for by far the largest fraction of the total present between 1 and 4 hr.