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.

Stereoselective analysis of 2-hydroxysebacic acid in urine of patients with Zellweger syndrome and of premature infants fed with medium-chain triglycerides.

The chiral metabolite 2-hydroxysebacic acid (2-HS) is considered to be an important diagnostic marker for peroxisomal disorders. The pathway of formation of 2-HS, excreted in increased amounts in patients with peroxisomal diseases, is not absolutely clear. Moreover, there is no information about the enantiomeric distribution of 2-HS in human urine. Here, we describe the stereodifferentiation of 2-HS in urine samples of nine patients with Zellweger syndrome (ZS), and for the first time in urine samples of premature infants fed a medium-chain triglyceride (MCT)-containing diet. Using enantioselective multidimensional gas chromatography-mass spectrometry, an increased excretion of 2R-HS was observed in all investigated ZS patients. 2-HS was also present in urine samples of premature infants fed MCT. Analogously to the ZS patients, a dominant 2R-HS excretion in the urine samples of the premature infants was identified. The formation of 2-HS is expected to result from the same or similar pathways as described for ZS patients. Additionally, we determined the absolute configuration of urinary 3-hydroxysebacic acid (3-HS) in the cases investigated. The enantioselective analysis provides further information for the diagnosis and treatment of patients with impaired peroxisomal fatty acid oxidation. Further insight into the metabolic origin and the biochemical pathway leading to these urinary metabolites is provided.

Elevated urinary excretion of nitric oxide metabolites in young infants with Zellweger syndrome.

BACKGROUND: Children with Zellweger syndrome (ZS), a rare peroxisome deficiency disorder, excrete into the urine highly elevated amounts of urinary metabolites of the arachidonic acid cascade. This pathway may interact in vivo with the L-arginine/nitric oxide (NO) pathway. The aim of this study was to investigate NO production in ZS. METHODS: We studied 11 infants aged 2-12 months with ZS and 30 healthy controls (HC) aged 1-12 months. Urinary excretion of nitrite plus nitrate (U(NO(x))), which is a reliable measure of whole body NO formation, was determined by gas chromatography-mass spectrometry (GC-MS) and corrected for creatinine excretion. RESULTS: In the subjects aged 1-6 months, U(NO(x)) was more than twofolds higher in ZS (median, 666 micromol/mmol creatinine) as compared to HC (median, 257 micromol/mmol creatinine) (P=0.014 by Mann-Whitney U-test). In children aged 7-12 months, U(NO(x)) was similar for ZS subjects and HC (P=0.96). U(NO(x)) correlated negatively with age in ZS (Kendall's rank correlation coefficient, tau=-0.75, P=0.001). By contrast, no such correlation was found in HC (tau=0.06, P=0.6). CONCLUSIONS: NO production is highly elevated during the first 6 months of life in infants with ZS and falls to normal levels within the following 6 months, suggesting a dramatic decrease in NO synthesis in ZS.

Simultaneous enantioselective analysis of chiral urinary metabolites in patients with Zellweger syndrome.

Enantio-MDGC-MS analysis with heptakis-(2,3-di-O-methyl-6-O-tert.-butyl-dimethylsilyl)-beta-cyclodextrin as the chiral main column is a powerful tool for the separation of chiral compounds. This paper reports on the simultaneous stereodifferentiation of 2-hydroxyisocaproic acid (HICA), 3-methyladipic acid (3-MA), 2-hydroxyglutaric acid (2-HG), 3-(4-hydroxyphenyl)-lactic acid (HPLA), 2-hydroxysebacic acid (2-HS) and 3-hydroxysebacic acid (3-HS) in a single chromatographic run. These chiral urinary metabolites are useful in the diagnosis of peroxisomal diseases such as Zellweger syndrome (ZS). In this investigation, urine samples from nine patients with ZS were analysed in order to reveal the enantiomeric ratio of these chiral metabolites. The stereodifferentiation of the analysed chiral compounds may provide important information on their biochemical origin.

Increased urinary excretion of LTB4 and omega-carboxy-LTB4 in patients with Zellweger syndrome.

The metabolic inactivation of leukotrienes proceeds by beta-oxidation from the omega-end. We investigated the importance of peroxisomes and mitochondria in LTB4 oxidation in vivo. LTB4 and its oxidation products were analysed after high-performance liquid chromatography separation by immunoassays and gas chromatography-mass spectrometry in the urine of patients with Zellweger syndrome, patients with long-chain acyl CoA dehydrogenase deficiency, and healthy controls. LTB4 (median 97; range 35-238 nmol/mol creatinine) and its omega-oxidation product omega-carboxy-LTB4 (median 898; range 267-4583 nmol/mol creatinine) were present and significantly increased in the urine of all patients with Zellweger syndrome compared to the controls (P <0.01). In contrast, LTB4 and omega-carboxy-LTB4 were below the detection limit (< 5 nmol/ mol creatinine) in patients with long-chain acyl CoA dehydrogenase deficiency and healthy controls. The beta-oxidation product omega-carboxy-tetranor-LTB3 was neither detectable in the urine of patients with Zellweger syndrome, patients with long-chain acyl CoA dehydrogenase deficiency nor in the controls (< 5 nmol/mol creatinine). Analysis of urinary leukotrienes represents an additional diagnostic tool in peroxisome deficiency disorders. Furthermore, these results clearly underline the essential role of peroxisomes in the oxidation of LTB4 in humans.

Specific and rapid quantification of 8-iso-prostaglandin F2alpha in urine of healthy humans and patients with Zellweger syndrome by gas chromatography-tandem mass spectrometry.

8-iso-Prostaglandin F2alpha (8-iso-PGF2alpha) is currently discussed as a potential index parameter of oxidative stress in vivo. We describe in this article a fully validated gas chromatographic-tandem mass spectrometric method for the quantitative determination of 8-iso-PGF2alpha in human urine. The method is highly specific and requires a single thin-layer chromatographic step for sample purification. Inter- and intraday imprecision were below 8%. Mean inaccuracy was 5.3% for added levels of 8-iso-PGF2alpha up to 2000 pg/ml of urine. We measured highly elevated excretion of 8-iso-PGF2alpha in the urine of children with peroxisomal beta-oxidation deficiency, i.e. Zellweger syndrome, (63.3+/-16.6 ng/mg creatinine) compared to that of healthy children (0.51+/-0.16 ng/mg creatinine) (mean+/-S.D., both n=5). The method could be useful for diagnosing Zellweger syndrome and for investigating the utility of 8-iso-PGF2alpha as a novel marker for oxidative stress in vivo in man.

Biochemical features of a patient with Zellweger-like syndrome with normal PTS-1 and PTS-2 peroxisomal protein import systems: a new peroxisomal disease.

The peroxisomal disorders represent a group of inherited metabolic disorders that derive from defects of peroxisomal biogenesis and/or from dysfunction of single or multiple peroxisomal enzymes. We described earlier an 8 1/2 year-old with a history of progressive developmental delay, micronodular cirrhosis, and elevated very long chain fatty acids in plasma and skin fibroblasts. These findings were felt to be compatible with both neonatal adrenoleukodystrophy (nALD) and Zellweger syndrome (ZS). This patient is now 21 years old and his clinical course, inconsistent with either nALD or ZS, led us to examine his peroxisomal status in light of a possible new peroxisomal disease. The normal levels of bile acid precursors found in this patient suggest that peroxisomal beta-oxidation is functional. The activities of dihydroxyacetone phosphate acyltransferase and oxidation of lignoceric acid and phytanic acid were 14, 17, and 15% of the control, respectively. This partial activity for oxidation and the normal levels of bile acid precursors suggests that this patient has peroxisomes containing beta-oxidation enzymes. Western blot analysis of subcellular organelles showed that beta-oxidation enzyme proteins are present at normal levels in catalase-negative peroxisomes of density equivalent to normal peroxisomes. The presence of acyl-CoA oxidase and 3-ketoacyl-CoA thiolase in catalase-negative peroxisomes suggests that both peroxisomal targeting signal-1 (PTS-1), and peroxisomal targeting signal-2 (PTS-2)-mediated protein transport processes into peroxisomes are normal in this patient. These findings of catalase-negative peroxisomes of normal density and normal PTS-1 and PTS-2 import machinery with partial peroxisomal functions clearly demonstrate that this patient differs from those with known disorders of peroxisomal biogenesis.

Studies on the urinary excretion of thromboxane B2 in Zellweger patients and control subjects: evidence for a major role for peroxisomes in the beta-oxidative chain-shortening of thromboxane B2.

In this paper we studied the urinary excretion of thromboxane B2 and its beta-oxidation product 2,3-dinor-thromboxane B2 in urines from control subjects and four Zellweger patients, which lack morphologically distinguishable peroxisomes. In the urine of three classical Zellweger patients we found a ratio of 2,3-dinor-thromboxane B2/thromboxane B2 of 0.35, 0.48 and 0.62 respectively, whereas in healthy children and adults values were found of 3.1-10 and 5.5-40 respectively. These data strongly suggest that peroxisomes are a major site for beta-oxidation of thromboxane B2.

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.

Stereochemistry of pipecolic acid found in the urine and plasma of subjects with peroxisomal deficiencies.

Recently it was found that normal adults excrete pipecolic acid primarily as the D-enantiomer even though it is present in the blood stream mainly as the L-enantiomer (i.e. > 98% L). This study of pipecolic acid stereochemistry was extended to subjects with peroxisomal deficiencies since they are known to have high levels of pipecolic acid in their physiological fluids. Also, pipecolic acid stereochemistry was examined in young normal subjects since this group was not considered previously. It was found that the stereochemical composition of pipecolic acid in plasma was very similar for all subjects tested (i.e. > 98% of the L-enantiomer). However, the stereochemical composition of excreted pipecolic varied considerably. Urine samples from subjects with the most severe peroxisomal deficiency, i.e. cerebralhepatorenyl (Zellweger) syndrome (CHRS) contained little D-pipecolic acid. In fact the enantiomeric ratios for pipecolic acid in the urine and plasma of these subjects were very similar. This was not the case for normal subjects. Levels of D-pipecolic acid in the urine of subjects with 'less severe' peroxisomal deficiencies tended to be somewhat higher but they did not approach the levels found in normal adults. Several possible reasons for these results are discussed.

Peroxisomal chain-shortening of thromboxane B2: evidence for impaired degradation of thromboxane B2 in Zellweger syndrome.

We have shown that rat liver peroxisomes can chain-shorten prostaglandins to dinor- and tetranor-metabolites. In a recent in vivo study we could demonstrate that peroxisomes are of major importance for chain-shortening of prostaglandin F2 alpha in humans (1991, Diczfalusy et al. J. Clin. Invest. 88:978-984). This was shown by identifying the major urinary metabolites of radiolabeled prostaglandin F2 alpha given intravenously to a patient lacking functional peroxisomes (Zellweger syndrome). In the present investigation we have studied the peroxisomal chain-shortening of thromboxane B2, a compound structurally related to prostaglandins. Isolated rat liver peroxisomes oxidized thromboxane B2 to a chain-shortened metabolite in an NAD(+)-dependent reaction. The metabolite was identified as 9,11,15-trihydroxy-2,3,4,5-tetranor-thromb-13-enoic acid (tetranor-thromboxane B1). The urinary excretion of the major beta-oxidized metabolites of thromboxane B2 and prostacyclin was determined in three Zellweger patients and six age-matched controls. The controls excreted on an average 1.7 and 1.1 ng/mg creatinine of 2,3-dinorthromboxane B2 and 2,3-dinor-6-keto-prostaglandin F1 alpha, respectively. In none of the three Zellweger patients could these dinor-metabolites be detected, i.e., the urinary excretion was less than 0.2 ng/mg creatinine. This shows that peroxisomes play an important role in the degradation of the carboxyl side chain of thromboxane B2 in vivo.

Impaired degradation of leukotrienes in patients with peroxisome deficiency disorders.

The degradation of leukotrienes by beta-oxidation from the omega-end proceeds in peroxisomes (Jedlitschky et al. J. Biol. Chem. 1991. 266:24763-24772). Peroxisomal degradation of leukotrienes was studied in humans by analyses of endogenous leukotrienes in urines from eight patients with biochemically established peroxisome deficiency disorder and eight age- and sex-matched healthy infant controls. Leukotriene metabolites were separated by high-performance liquid chromatography, quantified by radioimmunoassays, and identified as well as quantified by gas chromatography-mass spectrometry. Urinary leukotriene E4 (LTE4) and N-acetyl-LTE4 excretions, relative to creatinine, were increased > 10-fold in the patients in comparison to healthy infants. The beta-oxidation product omega-carboxy-tetranor-LTE3 averaged 0.05 mumol/mol creatinine in the controls but was not detectable in the patients. However, omega-carboxy-LTE4 (median 13.6 mumol/mol creatinine) was significantly increased in the patients' urine, whereas LTB4 (median 0.07 mumol/mol creatinine) and omega-carboxy-LTB4 were detected exclusively in the urines of the patients. These data indicate an impairment of the inactivation and degradation of both LTE4 and LTB4 in patients with peroxisomal deficiency. The increased levels of the biologically active, proinflammatory mediators LTE4 and LTB4 might be of pathophysiological significance in peroxisome deficiency disorders. This is the first and so far only condition with a pronounced urinary excretion of omega-carboxy-LTE4, omega-carboxy-LTB4, and LTB4. This impaired catabolism of leukotrienes and the altered pattern of metabolites may be of diagnostic value. These findings underline the essential role of peroxisomes in the catabolism of leukotrienes in humans.

Urinary polyamine and metabolite excretion by children with Zellweger's syndrome.

In order to investigate whether, due to a lack of peroxisomes, polyamine degradation is altered in patients with the cerebro-hepato-renal syndrome of Zellweger, we determined total, free and acetylated polyamines and some of their catabolites in urines of six patients and age-matched healthy children. The normal polyamine excretion patterns of the patients, compared to the control group, suggest that either the intracellular localisation of the polyamine degrading enzyme, polyamine oxidase, is not exclusively limited to peroxisomes or that the enzyme is located in the peroxisomal matrix.

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.

Identification of 3 alpha, 6 alpha, 7 alpha, 12 alpha-tetrahydroxy-5 beta-cholestanoic acid in Zellweger's syndrome.

In order to confirm the occurrence of 3 alpha, 6 alpha, 7 alpha, 12 alpha-tetrahydroxy-5 beta-cholestanoic acid in Zellweger's syndrome, the nature of tetrahydroxycholestanoic acids present in a patient with this disease was studied. Urinary bile acids were extracted with a Sep-pak C18 cartridge and methylated after alkaline hydrolysis. The methyl esters were purified by silica gel column chromatography, and the methyl tetrahydroxycholestanoate fraction was analyzed by gas liquid chromatography-mass spectrometry. Along with already known side chain hydroxylated derivatives of 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid, 3 alpha, 7 alpha, 12 alpha, 24- and 3 alpha, 7 alpha, 12 alpha, 26-tetrahydroxy-5 beta-cholestanoic acids, three nuclear hydroxylated derivatives of 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid were found. One of them was identified as 3 alpha, 6 alpha, 7 alpha, 12 alpha-tetrahydroxy-5 beta-cholestanoic acid by direct comparison with the authentic standard which was chemically synthesized from 3 alpha, 6 alpha, 7 alpha, 12 alpha-tetrahydroxy-5 beta-cholanoic acid by side chain elongation.

Identification of (22R)-3 alpha,7 alpha,12 alpha,22- and (23R)-3 alpha,7 alpha,12 alpha,23-tetrahydroxy-5 beta-cholestanoic acids in urine from a patient with Zellweger's syndrome.

The nature of two novel C27 bile acids present as the taurine conjugates in urine from a patient with Zellweger's syndrome was studied. Bile acids conjugated with taurine were isolated from unconjugated and glycine-conjugated bile acids by means of ion-exchange chromatography. After alkaline hydrolysis of the taurine conjugates, the hydrolysate was acidified and extracted with ether; the extract was again subjected to ion-exchange chromatography to separate neutral from acidic compounds. The neutral fraction, which consisted mainly of two steroidal lactones, was treated with lithium aluminum hydride, and the reduction products were identified as (22R)-5 beta-cholestane-3 alpha,7 alpha,12 alpha,22,26-pentol and (23R)-5 beta-cholestane-3 alpha,7 alpha,12 alpha,23,26-pentol by direct comparison of their gas-liquid chromatographic behaviors and mass spectral data with those of chemically synthesized authentic samples. Thus, the chemical structure of two native bile acids present in urine from a patient with Zellweger's syndrome should be formulated as (22R)-3 alpha,7 alpha,12 alpha,22-tetrahydroxy-5 beta-cholestanoic acid and (23R)-3 alpha,7 alpha,12 alpha,12 alpha,23-tetrahydroxy-5 beta-cholestanoic acid, respectively.