The role of coenzyme A in the biotransformation of 2-arylpropionic acids

A number of 2-arylpropionic acid non-steroidal anti-inflammatory drugs (‘profens’) undergo highly enantioselective inversion from the (R)- to (S)-enantiomer. The mechanism of this inversion reaction involves the initial enantioselective formation of a coenzyme A thioester followed by epimerization and finally hydrolysis to regenerate free acids. Long-chain fatty acyl-CoA synthetase appears to mediate the initial thioester formation and an epimerase of an unknown physiologic function effects the second step. The hydrolases mediating the final step are poorly defined. Available evidence suggests that the liver is quantitatively the most important tissue site of inversion but local tissue inversion may influence the pharmacological and toxicological response of a given organ. Data from isolated rat hepatocytes indicate that other xenobiotics can modulate the formation and hydrolysis of ibuprofenyl-CoA by influencing inversion pathways, non-inversion pathways or both. Interactions between xenobiotics may therefore accentuate inter-individual variability in response to 2-arylprpionic acids. The formation of 2-arylpropionyl-CoA thioesters in vivo has the potential to disrupt numerous biochemical pathways in addition to enhancing individual exposure to the potent anti-inflammatory (S)-enantiomers.     

Determination of the epimeric composition of ibuprofenyl-CoA

Ibuprofen [racemic2-(4-isobutylphenyl)propionic acid] is a 2-arylpropionic acid nonsteroidal anti-inflammatory drug which undergoes unidirectional, R to S chiral inversion in vivo. It has been proposed that this chiral inversion phenomenon occurs via a coenzyme A (CoA) thioester intermediate. To characterize the formation and metabolism of this metabolic intermediate, ibuprofenyl-CoA, reference standards were needed and thus the CoA derivatives of (R)-, (S)-, and racemic ibuprofen were chemically synthesized. An HPLC assay employing a C18 reverse-phase column was developed to quantitate "total" ibuprofenyl CoA. Samples collected from this assay were then analyzed for ibuprofenyl-CoA epimeric composition by chiral chromatography employing a Chiral-AGP alpha 1-acid glycoprotein column. The applicability of these methods was demonstrated by assessing (R)- and (S)-ibuprofenyl-CoA hydrolysis and epimerization following incubation with rat liver homogenates. Rat liver homogenate catalyzed the complete and rapid epimerization of ibuprofenyl-CoA and the rate constants for (R)- and (S)-ibuprofenyl-CoA hydrolysis were equal. ATP and CoA were found to inhibit rat liver-catalyzed ibuprofenyl-CoA hydrolysis by 70-80% with no effect on epimerization. Additionally, it was demonstrated that traditional indirect ibuprofenyl-CoA assays which employ basic hydrolysis result in erroneous epimeric ratio determinations due to chemical epimerization.     

Stereoselective arylpropionyl-CoA thioester formation in vitro

The inversion from R- to S-enantiomer that occurs for some arylpropionic acids may have both toxicological and therapeutic implications. To characterize some properties of this inversion, arylpropionyl-CoA thioester formation was studied in rat tissue homogenates and subcellular fractions for the enantiomers of fenoprofen, ibuprofen, and flurbiprofen. Thioesters were formed from (R)-fenoprofen (64%) and (R)-ibuprofen (33%) but not from the corresponding S-enantiomers or the enantiomers of flurbiprofen. This correlates with the extensive inversion of fenoprofen and ibuprofen and lack of inversion of flurbiprofen in vivo. Subcellular fractions from rat liver showed thioester formation to occur in mitochondria and microsomes but not cytosol. Once formed, the thioesters were readily racemized by whole rat liver homogenate, mitochondria, and cytosol, but only partially inverted (S:R = 0.3) in microsomes. Thioester formation from fenoprofen and ibuprofen was studied in tissue homogenate obtained from liver, diaphragm, kidney, lung, skeletal muscle, smooth muscle, fat, caecum, and intestines. The liver was at least 50-fold more efficient than the other tissues studied and would be expected to be a major organ of enantiomeric inversion. Our data support the hypothesis that R- to S-enantiomeric inversion of arylpropionic acids proceeds via the stereoselective formation of CoA thioesters followed by enzymatic racemization and hydrolysis of the thioesters to regenerate free acid.     

Peroxisomal oxidases and catalase in liver and kidney homogenates of normal and di(ethylhexyl)phthalate-fed rats.

1. Activities of peroxisomal oxidases and catalase were assayed at neutral and alkaline pH in liver and kidney homogenates from male rats fed a diet with or without 2% di(2-ethylhexyl)phthalate (DEHP) for 12 days. 2. All enzyme activities were higher at alkaline than at neutral pH in both groups. 3. The effect of the DEHP-diet on the peroxisomal enzymes was different in kidney and liver. Acyl-CoA oxidase activity was raised three- and sixfold in kidney and liver homogenates, respectively. The activity of D-amino acid oxidase decrease in liver, but increased in kidney homogenates. In liver homogenates, urate oxidase activity was not affected by the DEHP diet. The catalase activity was twofold induced in liver, but not in kidney. 4. The differences suggest that the changes of peroxisomal enzyme activities by DEHP treatment are not directly related to peroxisome proliferation. 5. DEHP treatment caused a marked increase of total and peroxisomal fatty acid oxidation in rat liver homogenates. 6. In the control group the rate of peroxisomal fatty acid oxidation was higher at alkaline pH than at neutral pH. 7. This rate was equal at both pH values in the DEHP-fed group, in contrast to the acyl-CoA oxidase activity. These results indicate that after DEHP treatment other parameters than acyl-CoA oxidase activity become limiting for peroxisomal beta-oxidation.     

Immunocytochemical localization of urate oxidase, fatty acyl-CoA oxidase, and catalase in bovine kidney peroxisomes

We investigated the localization of urate oxidase, peroxisomal fatty acyl-CoA oxidase, and catalase in bovine kidney by immunoblot analysis and protein A-gold immunocytochemistry, using the respective polyclonal monospecific antibodies raised against the enzymes purified from rat liver. By immunoblot analysis, these three proteins were detected in bovine kidney and bovine liver homogenates. Subcellular localization of these three enzymes in kidney was ascertained by protein A-gold immunocytochemical staining of Lowicryl K4M-embedded tissue. Peroxisomes in bovine kidney cortical epithelium possessed crystalloid cores or nucleoids, which were found to be the exclusive sites of urate oxidase localization. The limiting membrane, the marginal plate, and the matrix of renal peroxisomes were negative for urate oxidase staining. In contrast, catalase and fatty acyl-CoA oxidase were found in the peroxisome matrix. These results demonstrate that, unlike rat kidney peroxisomes which lack urate oxidase, peroxisomes of bovine kidney contain this enzyme as well as peroxisomal fatty acyl-CoA oxidase.     

Topical administration of racemic ibuprofen

In vitro experiments to investigate possible stereoselective aspects of the topical administration of ibuprofen have been conducted. Incubation of ibuprofen with rat skin homogenates in the presence of coenzyme A, ATP, and magnesium provided no evidence for the formation of ibuprofenyl coenzyme A (the initial intermediate in the metabolic inversion of [R]- to [S]-ibuprofen). Similar incubation studies gave no indication of a change in the enantiomeric ratios of ibuprofen over the time course of the experiments. Percutaneous penetration studies of ibuprofen gel through porcine skin indicated that the ibuprofen enantiomer levels in the reservoir solutions were consistent with racemic ibuprofen having traversed the skin with no metabolic inversion. These results suggest that, in the models studied, skin metabolism does not result in the chiral inversion of (R)- to (S)-ibuprofen and that the topical administration of ibuprofen will result in the delivery of 50% “isomeric ballast.” Chirality 9:313–316, 1997. © 1997 Wiley-Liss, Inc.     

Sex- and species-related differences in the biotransformation of isosorbide dinitrate by various tissues of the rabbit and rat

The biotransformation of isosorbide dinitrate (ISDN) by various tissues of the rabbit and rat was examined. Incubation of 2 X 10(-7) M ISDN at 37 degrees C with tissue homogenates of liver, lung, kidney, intestine, skeletal muscle, aorta, and erythrocytes from the rabbit and rat resulted in a significant disappearance of ISDN after a 30-min incubation (also, 5-min incubation for liver). The disappearance of ISDN in each tissue homogenate was accompanied by an equimolar production of the mononitrate metabolites, isosorbide-2-mononitrate (2-ISMN) and isosorbide-5-mononitrate (5-ISMN), with the exception of liver homogenates where the loss of ISDN could not be accounted for by mononitrate formation. The relative rate of ISDN disappearance in various tissue homogenates was for the male rabbit, liver greater than lung approximately intestine greater than kidney greater than erythrocytes approximately skeletal muscle approximately aorta; for the female rabbit, liver greater than kidney approximately lung approximately intestine greater than erythrocytes approximately skeletal muscle approximately aorta; and for the male rat, liver greater than intestine greater than erythrocytes greater than skeletal muscle greater than lung approximately kidney. A sex difference in the percent disappearance of ISDN was observed in homogenates of lung and intestine from male and female rabbits. In addition, a sex difference in the ratio of metabolite (2-ISMN/5-ISMN) formed by denitration of ISDN was seen in homogenates of lung, skeletal muscle, and erythrocyte lysate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolic chiral inversion of ibuprofen in isolated rat hepatocytes

Ibuprofen was used to demonstrate that isolated rat hepatocytes offer a suitable in vitro model to investigate the metabolic chiral inversion of anti-inflammatory 2-arylpropionic acids (profens). The inversion of the pharmacologically inactive (-)-(R)-ibuprofen to the active (+)-(S)-ibuprofen was shown to obey apparent first-order kinetics during 5 h and to increase linearly with increasing hepatocyte concentration up to 4 x 10(5) cells/ml. No elimination of (R)-ibuprofen by routes other than inversion was seen, whereas the elimination of (S)-ibuprofen appeared to be saturable.     

In Vitro Metabolic Stability of Exendin-4: Pharmacokinetics and Identification of Cleavage Products

The aim of this study was to investigate the metabolic stability and cleavage sites of exendin-4 in rat tissue homogenates, as well as to identify the types of proteases involved in exendin-4 degradation. The stability of exendin-4 in kidney and liver homogenates from rats was evaluated using liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) with gradient elution. Furthermore, we used a combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and LC-ESI-MS/MS to identify the structures of the major degradation products of exendin-4, and peptidase inhibitors were used to characterize exendin-4 degradation in rat liver and kidney homogenates and to identify the proteases involved in exendin-4 metabolism. Exendin-4 had a half-life of 7.8 and 100.9 min in the kidney and liver homogenate, respectively. The enzymes most likely to be involved in the degradation of exendin-4 were aminopeptidases, serineproteases, and metalloproteases. Exendin-4(15-39) and exendin-4(16-39) were the predominant direct exendin-4 metabolites in the kidney, and the main product of exendin-4 metabolism in the liver was exendin-4(12-39). Our results indicated that the metabolism of exendin-4 involved an initial endoproteolytic cleavage and subsequent exoproteolytic digestion. The degradation of exendin-4 in the kidney and liver homogenates followed distinct patterns, and the primary cleavage sites of exendin-4 degradation in rat kidney homogenates were located after AA-14, and -15, whereas those in rat liver homogenates were located after AA-11.     

Acyl-CoA oxidase activity and peroxisomal fatty acid oxidation in rat tissues

Acyl-CoA oxidase, the first enzyme of the peroxisomal β-oxidation, was proved to be rate-limiting for this process in homogenates of rat liver, kidney, adrenal gland, heart and skeletal muscle. Acyl-CoA oxidase activity, based on H₂O₂-dependent leuko-dichlorofluorescein oxidation in tissue extract, was compared with radiochemically assayed peroxisomal β-oxidation rates. Dichlorofluorescein production was a valid measure of peroxisomal fatty acid oxidation only in liver and kidney, but not in adrenal gland, heart or skeletal muscle. Production of ¹⁴C-labeled acid-soluble products from 1-¹⁴C-labeled fatty acids in the presence of antimycin-rotenone appears to be a more accurate and sensitive estimate of peroxisomal β-oxidation than the acyl-CoA oxidase activity on base of H₂O₂ production. Chain-length specificity of acyl-CoA oxidase changed with the acyl-CoA concentrations used. Below 80 μM, palmitoyl-CoA showed the highest activity of the measured substrates in rat liver extract. No indications were obtained for the presence in rat liver of more forms of acyl-CoA oxidase with different chain-length specificity.     

Comparison of the activities of some peroxisomal and extraperoxisomal lipid-metabolizing enzymes in liver and extrahepatic tissues of the rat.

Peroxisomal (acyl-CoA oxidase and peroxisomal dihydroxyacetone-phosphate acyltransferase) and extraperoxisomal (mitochondrial fatty acid oxidation, extraperoxisomal dihydroxyacetone-phosphate acyltransferase, mitochondrial and microsomal glycerophosphate acyltransferases) lipid-metabolizing enzymes were measured in homogenates from rat liver and from seven extrahepatic tissues. Except for jejunal mucosa and kidney, extrahepatic tissues contained very little acyl-CoA oxidase activity. Peroxisomal dihydroxyacetone-phosphate acyltransferase, taken as the activity that was not inhibited by 5 mM-glycerol 3-phosphate, was present in all tissues examined, and its specific activity in liver and extrahepatic tissues was roughly of the same order of magnitude. Clofibrate treatment increased the activity of acyl-CoA oxidase in liver, and to a smaller extent also in kidney, but did not influence the activity of peroxisomal dihydroxyacetone-phosphate acyltransferase. Comparison of the activities of peroxisomal and extraperoxisomal lipid-metabolizing enzymes in extrahepatic tissues and in liver, an organ in which the contribution of peroxisomes to fatty acid oxidation and to glycerolipid synthesis has been estimated previously, suggests that, as in liver, peroxisomal long-chain fatty acid oxidation is of minor quantitative importance in extrahepatic tissues, but that in these tissues (micro)-peroxisomes are responsible for most of the dihydroxyacetone phosphate acylation and, consequently, for initiating ether glycerolipid synthesis.     

Metabolic stability of long-acting luteinizing hormone-releasing hormone antagonists

Long-acting luteinizing hormone-releasing hormone (LHRH) antagonists designed to be protease resistant consisted of a series of novel decapeptides structurally similar to LHRH. The aim of this study was to evaluate the in vitro metabolic stability of the LHRH decapeptides using pancreatin and homogenates models and identify the metabolites in rat liver homogenate for the purpose of illustrating the metabolic features of the decapeptides. The major metabolites in rat liver homogenate were identified by LC-ESI-MS(n). The half-lives of the 11 LHRH decapeptides were from 44 to 330?min in the pancreatin model. The half-lives of the five decapeptides in rat liver, kidney and lung homogenates were between 8 and 462?min. The most stable decapeptides were the LY616 and LY608 peptides with half-lives of 36?min in liver homogenate. Two major cleavage sites were found by analysing the metabolites of the LY618 peptide in rat liver homogenate, between the Pal(3)-Ser(4) and the Leu(7)-Ilys(8) peptide bonds. The major metabolites were produced via cleavages of peptide bonds at these sites, and further metabolic reactions such as hydroxylation, oxidative dechlorination, alcohol dehydration and isopropyl dealkylation were also observed.     

Long-chain acyl-CoA dehydrogenase is a key enzyme in the mitochondrial beta-oxidation of unsaturated fatty acids

The first reaction of mitochondrial beta-oxidation, which is catalyzed by acyl-CoA dehydrogenases, was studied with unsaturated fatty acids that have a double bond either at the 4,5 or 5,6 position. The CoA thioesters of docosahexaenoic acid, arachidonic acid, 4,7,10-cis-hexadecatrienoic acid, 5-cis-tetradecenoic acid, and 4-cis-decenoic acid were effectively dehydrogenated by both rat and human long-chain acyl-CoA dehydrogenases (LCAD), whereas they were poor substrates of very long-chain acyl-CoA dehydrogenases (VLCAD). VLCAD, however, was active with CoA derivatives of long-chain saturated fatty acids or unsaturated fatty acids that have double bonds further removed from the thioester function. Although bovine LCAD effectively dehydrogenated 5-cis-tetradecenoyl-CoA (14:1) and 4,7,10-cis-hexadecatrienoyl-CoA, it was nearly inactive toward the other unsaturated substrates. The catalytic efficiency of rat VLCAD with 14:1 as substrate was only 4% of the efficiency determined with tetradecanoyl-CoA, whereas LCAD acted equally well on both substrates. The conclusion of this study is that LCAD serves an important, if not essential function in the beta-oxidation of unsaturated fatty acids.     

Metabolic stability of human parathyroid hormone peptide hPTH (1–34) in rat tissue homogenates: kinetics and products of proteolytic degradation

The present study aim to investigate the metabolic stability and degradation of cleavage sites of human parathyroid hormone peptide, hPTH (1–34), in rat tissue homogenate, and to identify the types of proteases involved in hPTH (1–34) processing degradation. The stability of hPTH (1–34) in rat kidney, lung and liver homogenates was evaluated by LC–ESI–MS, and the structures of the major degradation products were identified by MALDI–TOF MS and LC–ESI–MS/MS. The ability of protease inhibitors to inhibit hPTH (1–34) degradation was used to identify the class of proteases involved in the metabolism of hPTH (1–34). hPTH (1–34) peptide was readily degraded in rat kidney, liver, and lung homogenates, with half-lives of 5.7, 32.2, and 18.9 min, respectively. The degradation of hPTH (1–34) in each tissue can be inhibited by inhibitors of serine and metalloproteases. The major degradation products of hPTH (1–34) are similar in each tissue and suggest that hPTH (1–15) and hPTH (16–34) appear as the major degradation products. The degradation patterns of hPTH (1–34) incubated in rat kidney, liver and lung homogenates are largely overlapping, and a majority of the fragments are generated via cleavages at sites of Leu15–Asn16 peptide bond.     

Acid:Coenzyme A Ligase in Brain: Fatty Acid Specificity in Cellular and Subcellular Fractions

Abstract: We measured long-chain fatty acid:coenzyme A (CoA) ligase (EC 6.2.1.3) activity with four fatty acids in brain homogenates, and cellular and subcellular fractions to determine whether there are differences in activity that could be correlated with differences in fatty acid composition and metabolism. In rat brain homogenates, ligase activity varied appreciably with the four acids, with 18:2 > 18:1 > 16:0 > 22:1 (nmol acyl-CoA formed/min/mg protein; 1.46, 1.20, 0.96, and 0.57, respectively). This order was similar under all incubation conditions tested, including variable pH and fatty acid concentrations. The relative specific activities (RSA, 16:0 = 1.0) with the four substrates were similar in rat brain homogenate, mitochondria, and microsomes, with the highest specific activities in the latter fraction. The RSA were also similar in ox brain homogenates, in rabbit brain microsomes prepared from gray and white matter, in neurons isolated from rat brain, and in cultured neuroblastoma cells. Rat liver homogenates had a significantly different pattern of RSA. These results indicate that the ligase(s) has a preference for certain fatty acids, but suggest that the major control of fatty acid composition and metabolism is a function of subsequent metabolic steps.     

Stereoselective disposition of tiaprofenic acid enantiomers in rats

The pharmacokinetics and metabolic chiral inversion of the S(+)‐ and R(−)‐enantiomers of tiaprofenic acid (S‐TIA, R‐TIA) were assessed in vivo in rats, and in addition the biochemistry of inversion was investigated in vitro in rat liver homogenates. Drug enantiomer concentrations in plasma were investigated following administration of S‐TIA and R‐TIA (i.p. 3 and 9 mg/kg) over 24 hr. Plasma concentrations of TIA enantiomers were determined by stereospecific HPLC analysis. After administration of R‐TIA it was found that 1) there was a time delay of peak S‐TIA plasma concentrations, 2) S‐TIA concentrations exceeded R‐TIA concentrations from ∼2 hr after dosing, 3) C_max and AUC_(0‐∞) for S‐TIA were greater than for R‐TIA following administration of S‐TIA, and 4) inversion was bidirectional but favored inversion of R‐TIA to S‐TIA. Bidirectional inversion was also observed when TIA enantiomers were incubated with liver homogenates up to 24 hr. However, the rate of inversion favored transformation of the R‐enantiomer to the S‐enantiomer. In conclusion, stereoselective pharmacokinetics of R‐ and S‐TIA were observed in rats and bidirectional inversion in rat liver homogenates has been demonstrated for the first time. Chiral inversion of TIA may involve metabolic routes different from those associated with inversion of other 2‐arylpropionic acids such as ibuprofen. Chirality 11:103–108, 1999. © 1999 Wiley‐Liss, Inc.     

Interactions between the omega- and beta-oxidations of fatty acids

Long-chain monocarboxylic, omega-hydroxymonocarboxylic and dicarboxylic acids were activated approximately at the same rate by rat liver homogenates into their CoA esters (2-3 U/g liver). These acyl-CoA were substrates for rat liver peroxisomal beta-oxidation. The distribution of the peroxisomal oxidation of these substrates was also studied in various tissues. Rat liver mitochondria were capable of oxidizing long-chain monocarboxyl- and omega-hydroxymonocarboxylyl-CoAs but not dicarboxylyl-CoAs. When the mitochondrial preparations were incubated in coupling conditions, the addition of either free decanoic acid or free 10-hydroxydecanoic acid resulted in an increase of the oxygen uptake conversely to the addition of decanedioic acid. The comparative study of the chain-length substrate specificity of peroxisomal fatty acyl-CoA oxidase and mitochondrial fatty acyl-CoA dehydrogenase activities revealed that, actually, both types of organelles, peroxisomes and mitochondria, contain "oxido-reductases" active on long-chain monocarboxylyl-CoAs, omega-hydroxymonocarboxylyl-CoAs and dicarboxylyl-CoAs.     

Xenobiotic acyl-CoA formation: evidence of kinetically distinct hepatic microsomal long-chain fatty acid and nafenopin-CoA ligases

Multiplicity of hepatic microsomal coenzyme A ligases catalyzing acyl-CoA thioester formation is an important factor for consideration in relation to the metabolism of xenobiotic carboxylic acids. In this study the kinetic characteristics of rat hepatic microsomal nafenopin-CoA ligase were studied and compared with those of long-chain fatty acid (palmitoyl) CoA ligase. The high affinity component of palmitoyl-CoA formation was inhibited by nafenopin (K_i 53 μM) and ciprofibrate (K_i 1000 μM). Analagous to palmitoyl-CoA, nafenopin-CoA formation was catalyzed by an apparent high affinity low capacity isoform (K_m 6 ± 2.5 μM, (V_max 0.33 ± 0.12 nmol/mg per min) which was inhibited competitively by palmitic acid (mean K_i 1.7 μM, n = 5) and R-ibuprofen (mean K_i 10.8 μM, n = 5) whilst ciprofibrate and clofibric acid were ineffective as inhibitors. The intrinsic metabolic clearance of nafenopin to nafenopin-CoA (V_max/K_m 0.057 ± 0.011 nmol/mg/min ± M) was similar to that reported recently for the formation of ibuprofenyl-CoA by rat liver microsomes. Evidence of both a substantial difference between the K_m and K_i for nafenopin and lack of commonality with regard to xenobiotic inhibitors suggests that the high affinity microsomal nafenopin-CoA and long-chain fatty acid-CoA ligases are kinetically distinct. Thus until the current ‘long-chain like’ xenobiotic-CoA ligases are fully characterised in terms of substrate specificity, inhibitor profile, etc, it will be impossible to rationalize (and possibly predict) the metabolism and hence toxicity of xenobiotic carboxylic acids forming acyl-CoA thioester intermediates.     

Epimerization in peptide thioester condensation

Peptide segment couplings are now widely utilized in protein chemical synthesis. One of the key structures for the strategy is the peptide thioester. Peptide thioester condensation, in which a C‐terminal peptide thioester is selectively activated by silver ions then condensed with an amino component, is a powerful tool. But the amino acid adjacent to the thioester is at risk of epimerization. During the preparation of peptide thioesters by the Boc solid‐phase method, no substantial epimerization of the C‐terminal amino acid was detected. Epimerization was, however, observed during a thioester–thiol exchange reaction and segment condensation in DMSO in the presence of a base. In contrast, thioester–thiol exchange reactions in aqueous solutions gave no epimerization. The epimerization during segment condensation was significantly suppressed with a less polar solvent that is applicable to segments in thioester peptide condensation. These results were applied to a longer peptide thioester condensation. The epimer content of the coupling product of 89 residues was reduced from 27% to 6% in a condensation between segments of 45 and 44 residues for the thioester and the amino component, respectively. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Focal neurometabolic alterations in mice deficient for succinate semialdehyde dehydrogenase

Metabolite profiling in succinate semialdehyde dehydrogenase (SSADH; Aldh5a1-/-) deficient mice previously revealed elevated gamma-hydroxybutyrate (GHB) and total GABA in urine and total brain and liver extracts. In this study, we extend our metabolic characterization of these mutant mice by documenting elevated GHB and total GABA in homogenates of mutant kidney, pancreas and heart. We quantified beta-alanine (a GABA homolog and putative neurotransmitter) to address its potential role in pathophysiology. We found normal levels of beta-alanine in urine and total homogenates of mutant brain, heart and pancreas, but elevated concentrations in mutant kidney and liver extracts. Amino acid analysis in mutant total brain homogenates revealed no abnormalities except for significantly decreased glutamine, which was normal in mutant liver and kidney extracts. Regional amino acid analysis (frontal cortex, parietal cortex, hippocampus and cerebellum) in mutant mice confirmed glutamine results. Glutamine synthetase protein and mRNA levels in homogenates of mutant mouse brain were normal. We profiled organic acid patterns in mutant brain homogenates to assess brain oxidative metabolism and found normal concentrations of Kreb's cycle intermediates but increased 4,5-dihydroxyhexanoic acid (a postulated derivative of succinic semialdehyde) levels. We conclude that SSADH-deficient mice represent a valid metabolic model of human SSADH deficiency, manifesting focal neurometabolic abnormalities which could provide key insights into pathophysiologic mechanisms.