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.

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.

A GC/MS/MS screening method for multiple organic acidemias from urine specimens.

A gas chromatography tandem mass spectrometry method using an ion trap GC/MS system was developed to quickly screen urine samples for 14 organic acids associated with multiple organic acidemias. The following organic acids are used as diagnostic markers: methylmalonic acid, glutaric acid, 2-ketoisocaproic acid, succinylacetone, 3-methylcrotonylglycine, tiglylglycine, isovalerylglycine, fumaric acid, butyrylglycine, propionylglycine, hexanoylglycine, adipic acid, suberic acid, and sebacic acid. 2-ketocaproic acid is used as an internal standard. The samples are prepared using a solid-phase extraction and converted to trimethylsilyl derivatives. The extraction efficiency for the 14 compounds is between 57 and 106%. A derivatized standard mixture of the 14 markers is run prior to the patient samples to determine the accurate absolute and relative retention times. The samples are then injected and the product ion spectra monitored. For data analysis, one characteristic product ion plot is extracted for each of the 14 marker compounds, and the presence of a peak with the expected retention time is determined. The areas of the product ion peaks are compared with the reference range determined from 30 normal controls. Ten samples of patients with known organic acidemias were measured. For all patients, diagnostic peaks at the expected retention times of at least five times the upper limit of the reference range were detected. The method, with its relatively fast sample preparation, short 10.0 min run time and simple data analysis, is suitable for use as a quick metabolic screen of very sick patients in whom there is concern regarding the possibility of a treatable inborn error.

Influence of carbon chain length on the hepatic effects of perfluorinated fatty acids. A 19F- and 31P-NMR investigation.

Using nuclear magnetic resonance (NMR) spectroscopy, we investigated the importance of carbon chain length with regard to the hepatic effects associated with perfluoro-n-carboxylic acids. Male F-344 rats were administered a single intraperitoneal dose of either perfluoro-n-heptanoic acid (C7-PFA), perfluoro-n-nonanoic acid (C9-PFA), or perfluoro-n-undecanoic acid (C11-PFA). Data from previous studies involving perfluoro-n-octanoic acid (C8-PFA) and perfluoro-n-decanoic acid (C10-PFA) are included for comparison. Food consumption/body weight was monitored daily for all groups. C9- and C11-PFA treatment yields a prolonged hypophagic response while C7-PFA shows a more acute response. Fluorine-19 NMR spectra of urine and bile samples show no evidence of fluorometabolites and suggest that the distribution of perfluorocarbons into urine or bile is dependent upon carbon chain length. The aqueous solubility of C7-PFA appears to facilitate rapid urinary excretion, similar to that observed for C8-PFA. The relative hydrophobicity of C9- and C11-PFA appears to favor biliary enterohepatic recirculation, yielding a more protracted toxicity, similar to C10-PFA. Phosphorus-31 NMR studies of liver in vivo and liver extracts show that perfluorocarbons of > or = C9 carbons produce a significant increase in liver phosphocholine concentration. These data are discussed with regard to the impact of these chemicals on hepatic phospholipid metabolism. Hepatic peroxisomal fatty acyl CoA-oxidase activity (FAO) was measured to determine if C7-, C9-, and C11-PFA are peroxisome proliferators. Data indicate that the induction of peroxisomal enzyme activity by perfluorocarbons requires a chain length greater than seven carbons. In general, these results demonstrate the significance of carbon chain length in the hepatotoxic response and provide clues toward understanding the processes involved in the biological activities associated with exposure to these compounds.

Excretion of a radiolabelled anticancer biodegradable polymeric implant from the rabbit brain.

The elimination of a clinically used anticancer biodegradable polymer implant (Gliadel) in the rabbit brain was studied. The implant is composed of N,N-bis(2-chloroethyl)-N-nitrosourea (BCNU) (1.6 wt%) dispersed in a copolyanhydride matrix of 1,3-bis(p-carboxyphenoxypropane) (CPP) and sebacic acid (SA) in a 20:80 molar ratio. Four groups of rabbits were implanted with wafers loaded with BCNU, one in a 14C-SA-labelled polymer, another in a 14C-CPP-labelled polymer and two groups with 14C-BCNU in a non-labelled polymer, one for BCNU disposition study and one for residual drug study. In the rabbits implanted with the 14C-SA-labelled polymer, approximately 10% of the radioactivity was found in the urine and 2% in the faeces, and about 10% remained in the device 7 d after implantation. In contrast, only 4% of the radioactivity of the 14C-CPP-labelled polymer was found in urine and faeces during this period. However, a drastic increase in the CPP excretion was found after 9 d, and at 21 d, 64% of the implanted 14C-CPP was found in the urine and faeces, and 29% was still in the recovered wafers. Approximately 50% of the BCNU in the wafers was released in 3 d, and over 95% was released after 6 d in the rabbit brain. This study demonstrates that BCNU-loaded polyanhydride is biodegradable and is excreted from the body primarily through the renal system. The water-soluble components SA and BCNU were rapidly excreted, while the insoluble CPP was gradually eliminated after a lag time of 9 d.

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.

Azelaic and pimelic acids: metabolic intermediates or artefacts?

Azelaic and pimelic acids are excreted in elevated amounts in urine in disorders of mitochondrial beta-oxidation and disorders of peroxisomal beta-oxidation, for which they are of significant diagnostic value. We have detected the presence of azelaic, pimelic and even-chain-length dicarboxylic acids (adipic, suberic and sebacic acids) arising artefactually as a result of storage of small sample volumes in plastic containers. Storage of samples for organic acid analysis in glass containers is recommended.

Tracer study of metabolism and tissue distribution of sebacic acid in rats.

The present study investigates the metabolic disposition of sebacic acid in rats. Three groups of experimental animals received different doses of disodium sebacate with 25 microCi of 14C-labeled molecule by intravenous injection. In the first group radioactivity plasma elimination curves were examined for two administered doses (80 and 160 mg). In the second group, expired 14CO2, urine tracer and feces tracer were counted after intravenous administration of 160 mg of sebacate. The animals of the third group were sacrificed at different times after intravenous administration of 160 mg of sebacate, and tracer elimination curves were obtained for several organs. The plasma half-life of sebacate is 38.71 min; about 35% of the administered tracer was excreted in the urine as unchanged sebacate; about 25% was eliminated as 14CO2 in expired air. Disposition of sebacate was complete within 4 h of administration. The sebacate half-life is longest in adipose tissue (135 min) and in liver (74 min), sites of likely transformation. In all other organs examined, the sebacate half-life is similar to that in plasma.

Tissue uptake and oxidation of disodium sebacate in man.

In order to better ascertain its possible use as an alternative fuel substrate in total parenteral nutrition, sebacate (Sb) metabolism was studied in seven overnight-fasting healthy male volunteers, who received a constant iv infusion (99 mmoles over 8 hours) of disodium sebacate. Sb oxidation rate was determined using an isotopic sebacate (disodic salt of (1-10)14C-sebacic acid) infusion (100 mu Ci from the fourth to the eighth hour of the cold sebacate infusion). Blood samples were collected during and after sebacate infusion at intervals of 30 minutes and Sb serum concentrations were determined by high performance liquid chromatography. Excreted radioactivity (mu Ci/min) was measured by bubbling the expired air into an apparatus containing 3 mEq hyamine to trap CO2 from a 20-L Douglas-bag. CO2 production and O2 consumption were measured before and at 4 and 8 hours after starting the infusion. Twenty-four hour nitrogen excretion with urine was obtained. The RQ and the percent of calories derived from lipid oxidation were calculated by indirect calorimetry. The Sb serum level at the plateau phase was (mean +/- SD) 4.54 +/- 0.71 mumole/mL, the overall rate of tissue uptake was 180.89 +/- 4.50 mumole/min, and the percent oxidation was 6.14 +/- 0.44%. At the end of Sb infusion the RQ dropped to 0.839 +/- 0.043, the percent of calories due to sebacate oxidation was 1.59 +/- 0.52%, and the calories derived from lipids increased to 37.77 +/- 12.90%. These data show that a definite amount of the sebacate infused is oxidized in human tissues.

Disposition of perfluorodecanoic acid in male and female rats.

The elimination, tissue distribution, and metabolism of [1-14C]PFDA were examined in male and female rats for 28 days after a single ip dose (9.4 mumol/kg, 5 mg/kg). A sex difference in the fecal elimination of perfluorodecanoic acid (PFDA) was observed with 51 and 24% of the administered 14C being recovered in the feces of male and female rats, respectively, by 28 days post-treatment. The cumulative excretion of PFDA-derived 14C in the urine in 28 days was less than 5% of the administered dose in both sexes. The sex-related difference in the rate of fecal elimination resulted in the observed difference in whole body elimination t1/2 of PFDA in males (t1/2 = 23 days) and females (t1/2 = 45 days). The liver contained the highest concentration of PFDA-derived 14C in both males and females, followed by the plasma and kidneys. The heart, fat pads, testes, and gastrocnemius muscle of males, and the ovaries of females contained much lower concentrations of PFDA. The reason for the high percentage of the ip dose of [1-14C]PFDA in the liver (53% males and 41% females, 2 hr post-treatment) was further examined using an in situ nonrecirculating liver perfusion technique. It was shown that approximately 25% of the [14C]PFDA in the perfusate was extracted by the liver in a single pass. The basis for the sex difference in fecal elimination of PFDA does not appear to be due to a sex difference in biliary excretion. In a 6-hr period, male and female rats with kidneys ligated eliminated essentially the same percentage dose of [14C]PFDA into bile. We had hypothesized that the persistence of PFDA in rats was due to formation of a PFDA-containing lipid (i.e., a [14C]PFDA-containing mono-, di-, or triacylglycerol, cholesteryl ester, methyl ester, or phospholipid). However, no evidence that PFDA is conjugated to form persistent hybrid lipids was obtained, nor were polar metabolites of PFDA detected in urine or bile. In addition, daily urinary excretion of fluoride in male and female rats before and after PFDA treatment was similar, suggesting that the parent compound is not defluorinated. Thus, the disposition of PFDA in male and female rats is characterized by an apparent lack of biotransformation.

[Clinical and biochemical studies in a case of acute encephalopathy associated with calcium hopanthenate administration].

A case with acute disturbance of consciousness associated with calcium hopanthenate (HOPA) administration was reported. He was a 3-year-old boy with autistic developmental delay, had orally taken 1.5 g of HOPA daily for 3 months. Clinical manifestations consisted of fever, vomiting and coma. Laboratory examination revealed severe hypoglycemia and metabolic acidosis, but there were no hepatic enzyme abnormalities. Analysis of urinary organic acid profile showed that very large amounts of medium and long chain dicarboxylic acids and omega-1 hydroxy-fatty acids were excreted. In particular, 2-hydroxysebacic acid, the accumulation of which has only been reported in the urine of patients with Zellweger syndrome and neonatal adrenoleukodystrophy (NALD), was observed. Analysis of urinary acylcarnitines showed that acetylcarnitine was predominant and C6-C10 dicarboxylic acylcarnitines were also excreted. He was treated with and rapidly responded to intravenous glucose and bicarbonate. After withdrawal of the drug he has had no problems and dicarboxylic aciduria disappeared. A CT scan showed symmetric, low density areas in periventricular white matter, especially around the posterior horns of the lateral ventricles. A T2-weighted MRI scan revealed high-intensity signal in the white matter corresponding to areas of low density on CT scan. We conclude that that a large amount of HOPA administration may cause encephalopathy by the inhibition of both mitochondrial and peroxisomal beta-oxidation.

Mass spectrometric identification of 2-hydroxy-sebacic acid in the urines of patients with neonatal adrenoleukodystrophy and Zellweger syndrome.

The urines of children with neonatal adrenoleukodystrophy and Zellweger syndrome contained an excess of unusual even- and odd-numbered dicarboxylic acids with a chain length of from 5 to 15 carbon atoms, as well as 2-hydroxy-compounds, including 2-hydroxy-isocaproate, 2-hydroxy-glutarate and 2-hydroxy-sebacate. The latter product, not previously found in metabolic diseases, appears as an additional useful marker of these peroxisomal disorders.

Medium- and long-chain dicarboxylic aciduria in patients with Zellweger syndrome and neonatal adrenoleukodystrophy.

This study reports that patients with neonatal adrenoleukodystrophy and Zellweger syndrome excrete a very peculiar pattern of organic acids. Dicarboxylic acids with an even number of carbon atoms (adipic, suberic, sebacic, 2- and 3-hydroxy-sebacic, hexadecanedioic), as well as with an odd number of carbon atoms (pimelic, azelaic, un-, tri-, and pentadecanedioic) were found in excess in the urines of six patients with neonatal adrenoleukodystrophy and one with Zellweger syndrome. The accumulation of dicarboxylic acids, reflecting an impairment of their beta-oxidation in mitochondria and/or peroxisomes, thus appears as an additional useful marker of these peroxisomal diseases.

General (medium-chain) acyl-CoA dehydrogenase deficiency (non-ketotic dicarboxylic aciduria): quantitative urinary excretion pattern of 23 biologically significant organic acids in three cases.

Urinary analysis of the pattern of 23 organic acid metabolites derived from fatty acids in three patients with general (medium-chain) acyl-CoA dehydrogenase deficiency was performed. Although there exist quantitative differences in the excreted amounts of the different metabolites in the three patients the qualitative picture was the same. The excretion of adipic, suberic and sebacic acids was substantial, whereas that of dodecanedioic acid was within or just above control limit. The monounsaturated C6-C10-dicarboxylic acid excretion was only marginally or not increased. 5-OH-hexanoic acid and hexanoylglycine were excreted in excessive amounts, whereas 7-OH-octanoic acid, 9-OH-decanoic acid, octanoylglycine and decanoylglycine were excreted in limited amounts. The excreted amounts of 6-OH-hexanoic, 8-OH-octanoic and 10-OH-decanoic acids were not or only marginally elevated compared to controls. In one of the patients the excretion of ethylmalonic and methylsuccinic acids was enhanced, whereas the excretion of these two acids in the two other patients was comparable to that in controls. The urinary excretion of hexanoic, octanoic, decanoic and dodecanoic acids was just a little above the control limit, whereas the esterified hexanoic and octanoic acids were excreted in appreciable amounts. It is argued that the microsomal omega- and omega-1-oxidation systems are involved in the dicarboxylic and omega-1-OH-monocarboxylic acids formation at C10 and C12 level and that the C8-C6-dicarboxylic and omega-1-OH-monocarboxylic acids are formed from higher chained acids by beta-oxidation in both mitochondria and peroxisomes.

C6-C10-dicarboxylic aciduria: biochemical considerations in relation to diagnosis of beta-oxidation defects.

By means of gas chromatographic methods substantial amounts of the C6-C10-dicarboxylic acids, i.e. adipic, suberic and sebacic acids, have been found in the urine from children with unexplained attacks of lethargy and hypotonia, presumably related to episodes of fever and/or insufficient food intake. The course have once been fatal and is often characterized by severe hypoglycemia without ketonuria. Systematic gas chromatographic/mass spectrometric determinations of selected organic acid metabolites in the urine, together with enzymatic measurements in fibroblasts and clinical data from 4 patients of this category, have shown that the biochemical basis of this syndrome can be inborn errors of the beta-oxidation of fatty acids, localized to the medium-chain acyl-CoA dehydrogenation system. The biosynthesis of adipic, suberic and sebacic acids was studied using ketotic rats as the model, since ketosis in rats and humans is accompanied by excessive urinary excretion of adipic and suberic acids. A probable pathway for the production of the three dicarboxylic acids was found to be an initial omega-oxidation of the medium-chain C10-C14-monocarboxylic acids followed by beta-oxidation of the resulting medium-chain dicarboxylic acids. It is argued that the source of the omega-oxidizable monocarboxylic acids in ketosis most probably is the fat deposites, and it is speculated that the patients with beta-oxidation defects supplement this source with beta-oxidation intermediate medium-chain monocarboxylic acids, accumulated as a result of the defect. The ratio between the excreted amounts of adipic acid and sebacic acid in the urine from the patients with beta-oxidation defects is less than 50. This is in contrast to the ratio in urine from ketotic patients, where it is greater than 100. Adipic acid/sebacic acid ratio-measured by means of a gas chromatographic analysis-is therefore suggested as a tool in the diagnosis of dicarboxylic acidurias. Based on the clinical picture and the pattern of a series of organic acids in the urinary metabolic profile our four patients can be divided in two types of dicarboxylic aciduria. The two types have different therapeutic implications.

Urinary excretion of C4--C10-dicarboxylic acids and antiketogenic properties of adipic acid in ketogenic-stimulated rats due to diabetes, long-chain and short-chain monocarboxylic acids.

The urinary excretion of C4--C10-dicarboxylic acids (succinic, adipic, suberic and sebacic acids) and the antiketogenicity of adipic acid have been studied in ketogenic-stimulated rats in three biochemically different states: diabetes, fat-feeding (long-chain monocarboxylic acids) and feeding of hexanoic acid (short-chain monocarboxylic acid). In diabetic rats urinary excretions of adipic and suberic acids were elevated before the rise in urinary excretions of 3-hydroxybutyric acid, i.e. before ketosis appeared. In severe diabetic ketosis sebacic acid was below normal values, whereas the excretion of succinic acid was unaltered. Rats, in which ketosis was provoked by hexanoic acid, had preketotic high urinary excretions of adipic and succinic acids. After ketosis the excretions of succinic acid declined again whereas the excretion of adipic acid rose further, together with that of suberic acid. Moreover, when rats which were ketotic due to treatment with long-chain triacylglycerol or hexanoic acid received 500 mg of adipic acid the urinary excretion of succinic acid rose significantly. However, no changes in succinic acid excretion were seen in diabetic ketotic rats treated with the same amount of adipic acid. Exogenously administered adipic acid was strongly antiketogenic towards ketosis caused by long-chain or short-chain monocarboxylic acids, but had no effect on diabetic ketosis.

C6--C10-dicarboxylic aciduria in starved, fat-fed and diabetic rats receiving decanoic acid or medium-chain triacylglycerol. An in vivo measure of the rate of beta-oxidation of fatty acids.

Administration of decanoic acid to rats resulted not only in elevated urinary excretions of the C10-dicarboxylic acid (sebacic acid), but also in highly elevated excretions of the beta-oxidation products C8- and C6-dicarboxylic acids (suberic and adipic acids). Activation of the lipid metabolism by starvation, fat-feeding and experimental diabetes increased the excretions of adipic acid and decreased the excretions of sebacic acid, i.e. the rate of oxidation of fatty acids was correlated to the adipic : sebacic acid ratio in urine. Compared with nondiabetic unstarved rats the adipic : sebacic acid ratio was elevated 2--3-, 8--16-, 5--19-, and 22--88-times in rats which were, respectively, starved for 2 days, 4 days, on a fat-diet for 4 days, and ketotic due to streptozotocin-induced diabetes. All rats with ratios above 10 were ketotic (urinary excretions of 3-hydroxybutyric acid over 500 microgram/mg creatinine) and all rats with ratios below 4 were nonketotic, while ketosis was a variable finding in rats with intermediary ratios. Similar changes in the ratio of excreted dicarboxylic acids were found when medium-chain triacylglycerols were fed instead of decanoic acid.