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Objective: Polycystic ovary syndrome (PCOS) appears to be a common endocrine disorder of women in reproductive age. Adipose tissue (AT) is known as an active tissue in the metabolism of branched-chain amino acids (BCAA; Valine, Leucine, and Isoleucine) that they have associated with blood BCAA levels is a prognostic factor for insulin-resistant. Although the crucial roles of AT in women suffering from PCOS was reported, little information exists on the BCAA metabolism in AT of PCOS women. The aim was to assess and compare the expression of BCAAs metabolism pathway genes in abdominal subcutaneous AT of pregnant women with PCOS and non-PCOS pregnant women. Materials and methods: AT samples from 13 PCOS were compared with samples collected from 6 non-PCOS women, all of whom underwent caesarean. Quantitative real-time PCR technique was used for gene expression of branched chain aminotransferase 2 mitochondrial (BCAT2), branched chain ketoacid dehydrogenase E1-alpha (BCKDHA), branched chain ketoacid dehydrogenase E1-Beta (BCKDHB), dihydrolipoamide branched chain transacylase E2 (DBT), dihydrolipoamide dehydrogenase E3 (DLD), branched chain ketoacid dehydrogenase kinase (BCKDK), Data were analyzed using t-test or U-test. Results: No significant differences were found in age and body mass index (BMI) between non-PCOS and PCOS women. The mRNA level of BCAT2 and DLD in PCOS group was not significantly different from non-PCOS group whereas mRNA level of BCKDHB and DBT was significantly increased in PCOS group (P

Background Dihydrolipoamide dehydrogenase deficiency (DLDD) is a rare metabolic disorder inherited in an autosomal recessive manner. This heterogeneous disease has a variable clinical presentation, onset, and biochemical markers. Materials and methods We retrospectively reviewed the clinical and molecular diagnosis of eight cases with DLDD from four referral centers in Saudi Arabia. Results Remarkably, we found hepatic involvement ranging from acute hepatic failure to chronic hepatitis in five patients. In addition, neurological disorders in the form of seizures, developmental delay, ataxia, hypotonia and psychomotor symptoms were found in five patients, two of them with a combination of hepatic and neurological symptoms. In addition, only one patient had recurrent episodes of hypoglycemia. While most patients had the hepatic form of homozygous variant c.685G > T in the DLD gene, one patient was found to have a novel variant c.623C > T that had neurological and hepatic symptoms. Conclusions We describe the largest reported DLDD cohort in the Saudi population. Clinical, biochemical, radiological, and molecular characterization was reviewed and no clear genotype-phenotype correlation was found in this cohort.

A significant problem in the oxidative breakdown of lignin is the tendency of phenolic radical fragments to re-polymerise to form higher molecular weight species. In this paper we identify an extracellular flavin-dependent dehydrolipoamide dehydrogenase from Thermobifida fusca that prevents oxidative dimerization of a dimeric lignin model compound, which could be used as an accessory enzyme for lignin depolymerisation.

Dihydrolipoamide dehydrogenase deficiency, also known as maple syrup urine disease (MSUD) type III, is caused by the deficiency of the E3 subunit of branched chain alpha-ketoacid dehydrogenase (BCKDH), α-ketoglutarate dehydrogenase (αKGDH), and pyruvate dehydrogenase (PDH). DLD deficiency variably presents with either a severe neonatal encephalopathic phenotype or a primarily hepatic phenotype. As a variant form of MSUD, it is considered a core condition recommended for newborn screening. The detection of variant MSUD forms has proven difficult in the past with no asymptomatic DLD deficiency patients identified by current newborn screening strategies. Citrulline has recently been identified as an elevated dried blood spot (DBS) metabolite in symptomatic patients affected with DLD deficiency. Here we report the retrospective DBS analysis and second-tier allo-isoleucine testing of 2 DLD deficiency patients. We show that an elevated citrulline and an elevated allo-isoleucine on second-tier testing can be used to successfully detect DLD deficiency. We additionally recommend that DLD deficiency be included in the "citrullinemia/elevated citrulline" ACMG Act Sheet and Algorithm.

Alzheimer disease (AD) is an age-related neurodegenerative disease characterized by the presence of three pathological hallmarks: synapse loss, extracellular senile plaques (SP) and intracellular neurofibrillary tangles (NFTs). The major component of SP is amyloid β-peptide (Aβ), which has been shown to induce oxidative stress. The AD brain shows increased levels of lipid peroxidation products, including 4-hydroxy-2-nonenal (HNE). HNE can react covalently with Cys, His, or Lys residues on proteins, altering structure and function of the latter. In the present study we measured the levels of the HNE-modified lipoic acid in brain of subjects with AD and age-matched controls. Lipoic acid is a key co-factor for a number of proteins including pyruvate dehydrogenase and α-ketoglutarate dehydrogenase, key complexes for cellular energetics. We observed a significant decrease in the levels of HNE-lipoic acid in the AD brain compared to that of age-matched controls. To investigate this phenomenon further, the levels and activity of lipoamide dehydrogenase (LADH) were measured in AD and control brains. Additionally, LADH activities were measured after in-vitro HNE-treatment to mice brains. Both LADH levels and activities were found to be significantly reduced in AD brain compared to age-matched control. HNE-treatment also reduced the LADH activity in mice brain. These data are consistent with a two-hit hypothesis of AD: oxidative stress leads to lipid peroxidation that, in turn, causes oxidative dysfunction of key energy-related complexes in mitochondria, triggering neurodegeneration. This study is consonant with the notion that lipoic acid supplementation could be a potential treatment for the observed loss of cellular energetics in AD and potentiate the antioxidant defense system to prevent or delay the oxidative stress in and progression of this devastating dementing disorder., Graphical abstract The NADH-dependent oxido-reductase enzyme lipoamide dehydrogenase (LADH) is an important member of the mitochondrial energy generation complex. Alteration of the structure and activity of LADH by elevated reactive oxygen species (ROS) may hamper energy metabolism and ATP production. Lipoic acid (LA) must be in the reduced form as part of its co-factor function for mitochondrial TCA complexes such as α-ketoglutarate dehydrogenase. However, oxidized LADH is unable to reduce LA to DHLA, and therefore HNE is unable to bind to DHLA efficiently. Consequently, in this study, decreased LA-HNE binding was observed in Alzheimer disease brain. Severe effects on learning, memory, and higher executive functioning, all significantly lost in AD patients, would be expected. Supplementation of LA conceivably may protect LADH from ROS or end products of ROS (e.g., HNE) by self-sacrifice mechanism, potentially providing protection against dementia or slowing the rate of progression of AD. Highlights ► HNE-bound lipoic acid (HNE-LA) levels were decreased in the IPL region of AD brain. ► The TCA enzyme LADH converts inactive lipoic acid to its active-antioxidant form. ► LADH levels and activity were deceased in AD in IPL brain region. ► In-vitro HNE-treatment to mouse brain reduced LADH activity. ► The results fit the two-hit hypothesis of AD.

Publisher Summary This chapter discusses the metabolic role and antioxidant properties of α-lipoate and its reduced form, dihydrolipoate and the effects of this antioxidant in modulating the redox-sensitive transcription factor nuclear factor KB (NF- κ B). Because NF- κ B is involved in a wide variety of acute inflammatory responses, and other aspects of rapid responses in cells, this system has been chosen to explore the action of α-lipoate and dihydrolipoate on transcription factors. α-Lipoic acid is found as lipoamide in five proteins in eukaryotes, where it is covalently attached to a lysyl residue. Four of these proteins are found in the three α-keto acid dehydrogenase complexes, the pyruvate dehydrogenase complex (PDC), the branched chain keto acid dehydrogenase complex, and the α-ketoglutarate dehydrogenase complex. Three are in the E2 enzyme, dihydrolipoyl acyltransferase, which is different in each of the complexes and specific for the substrate of the complex. These three enzymes catalyze five reactions that oxidatively decarboxylate their substrates. The chapter discusses protein X, glycine cleavage system; there is argument on dihydrolipoamide dehydrogenase serving a purpose outside of multienzyme complexes, exogenous lipoate reduction, and the metabolic effects of exogenous α-lipoate, the antioxidant properties of lipoate, dihydrolipoate, and lipoic acid as a metabolic antioxidant. The chapter highlights NF- κ B role in normal and abnormal cell function. In responses to stresses such as injury and infection, various genes must be activated, and a central, coordinating factor in this activation of rapid response gene is NF- κ B. NF- κ B also helps modulate gene expression during growth and development. NF- κ B has far-reaching significance for a variety of pathological conditions in which inflammation, growth, or viral activation occurs. The chapter details the role of redox balance in modulating NF- κ B activation, effects of lipoate and dihydrolipoate on NF- κ B, possible therapeutic roles for lipoate involving modification of NF- κ B activity such as in AIDs, atherosclerosis, and diabetes.

Publisher Summary This chapter discusses the structure and posttranslational modification of lipoyl domain of 2-oxo-acid dehydrogenase multienzyme complexes. 2-oxo-acid dehydrogenase complexes catalyze the oxidative decarboxylation of a specific 2-oxo-acid and the subsequent acylation of coenzyme A, with the concomitant formation of NADH. A typical pyruvate dehydrogenase complex consists of multiple copies of three enzymes that act sequentially: pyruvate decarboxylase, dihydrolipoyl acetyltransferase, and dihydrolipoyl dehydrogenase. The E2 component is a multidomain polypeptide chain that aggregates with octahedral (24-mer) or icosahedral (60-mer) symmetry, depending on the source. The symmetry is dictated by the large C-terminal domain that also houses the acyltransferase active site. The N-terminal half of the polypeptide chain comprises one or more lipoyl domains and a peripheral subunit (E1/E3)-binding domain joined together by long and highly flexible linker regions of polypeptide chain. 2-oxo-acid dehydrogenase complexes specific for the oxidative decarboxylation of other 2-oxo acids—such as 2-oxoglutarate and branched-chain 2-oxo acids derived from the transamination of valine, leucine, and isoleucine—follow similar structural patterns. This chapter describes the purification of the lipoyl domain of the B. stearothermophilus pyruvate dehydrogenase complex and summarizes its solution structure. The chapter also describes the mechanism of posttranslational modification of the lipoyl domain, in particular, of the selection of the target lysine residue for lipoylation.

Publisher Summary This chapter discusses ATP synthesis under the influence of R - and S -enantiomers of α lipoic acid. The S -enantiomer is not a naturally occurring compound. The reduced form of the racemate and the R - and S -forms (oxidized) also differ in their properties in a wild strain of E. coli compared to the dihydrolipoyl dehydrogenase mutant strain JRG 1174. The main metabolic difference in JRG 1174 is an increase in ATP synthesis when the substrate is succinate. In the wild strain, glutamate–malate–pyruvate (GMP) yields higher rates of ATP synthesis. Dihydrolipoic acid is the only substance that increases ATP synthesis in JRG 1174 in combination with GMP substrate. ATP synthesis with succinate as substrate increases by dihydrolipoic acid in the wild strain. With the S -form of lipoic acid, the wild strain of E. coli exhibits a steep decrease in ATP synthesis, by about 2 nmol/mg protein, whereas in the presence of the R -form, the decrease is much less rapid (substrate GMP). JRG 1174 reveals a transient small increase of up to 5 nmol/mg protein with the S -form, and, thereafter, a concentration-dependent decrease with both R- and S-α- lipoic acid (substrate succinate). Mitoplasts reveal decisive differences in ATP synthesis as well as in ATPase activities in the presence of R - vs S -enantiomers.

Publisher Summary This chapter discusses the structure–antioxidant activity relationships of dihydrolipoic acid (DHLA). α-Lipoic acid/DHLA is effective as a therapeutic agent. In many cases, the effects of α-lipoic acid appear to be linked to its ability to influence oxidative stress-mediated processes. For example, α-lipoic acid dietary supplementation successfully prevents myocardial damage induced by ischemia-reperfusion; α-lipoic acid prevents macromolecular alterations induced by high concentrations of glucose; preincubation of cultured human T cells with α-lipoic acid inhibits activation of NF-κB, a transcription factor responsible for the activation of human immunodeficiency virus (HIV). Such findings on the effects of α-lipoic acid may relate to the antioxidant properties of DHLA, and thus, the development of DHLA-based antioxidants may have significant clinical ramifications. Success in such a strategy may require a more comprehensive approach based on the structure–function relationships of a compound, involving (1) determination of structure–activity relationships, (2) application of concepts derived from structural studies in cellular systems to elucidate the mechanism of action, and (3) verification of information in intact animals.

Publisher Summary This chapter discusses the pathways of central metabolism in the archaebacteria. Halophilic archaebacteria are chemo-organotrophs and many use amino acids and proteins as sole sources of carbon. However, a number of carbohydrate-utilizers have been isolated, and from a detailed study of one such halophile, Halobacteriurn saccharovorurn, glucose catabolism has shown to proceed via a modified Entner-Doudoroff pathway. Whether heterotrophic or autotrophic, all archaebacteria possesses the capacity to carry out gluconeogenesis from C3 or C4 compounds. In both halophiles and methanogens, it appears that gluconeogenesis proceeds via a reversal of the Embden-Meyerhof glycolytic pathway. The enzyme appears not to be a part of any multi-enzyme system in the halophiles, but purification and subsequent characterization showed it to be very similar to its eubacterial and eukaryotic counterpart in both its dimeric nature and its catalytic mechanism. The presence of the enzyme and co-factor are co-incident, indicating that lipoic acid may indeed be the true substrate of the archaebacterial dihydrolipoamide dehydrogenase. Interestingly, their presence can be correlated with the organisms' phylogenetic positions within the archaebacteria.

Publisher Summary The pyruvate dehydrogenase complex is composed of three enzymes in a multienzyme complex similar to the mammalian and Escherichia coli enzyme complex. The three enzymes are pyruvate dehydrogenase, lipoate transacetylase, and dihydrolipoyl dehydrogenase. This chapter describes the assay method, purification procedure, and properties of pyruvate dehydrogenase complex (PDC) isolated from broccoli and cauliflower. The spectrophotometric assay is based on the reduction of nicotinamide adenine dinucleotide kinase (NAD + ) and the resultant increase of absorbance at 340 nm. The isolation method of cauliflower and broccoli mitochondria is also described in the chapter. The steps involved in the purification procedure are (1) extraction, (2) protamine sulfate treatment, (3) ultracentrifugation, (4) polyethylene glycol fractionation, and (5) glycerol gradient. All steps are performed at 0–4°, unless otherwise noted. The enzyme is kept as concentrated as possible owing to inactivation at dilute protein concentrations. The cauliflower complex is sensitive to dilution and every effort must be made to maximize protein concentration during purification and storage.

Publisher Summary This chapter discusses the regulation of pyruvate dehydrogenase (PDH) by phosphorylation/dephosphorylation. The PDH complex of animal tissues contain three component enzymes—pyruvate decarboxylase (PDC), dihydrolipoyl acetyltransferase (LAT) and dihydrolipoyl dehydrogenase (DLD). They catalyze the elementary reactions as shown in the chapter. SDS polyacrylamide disc gel electrophoresis of bovine kidney or heart, or pig heart PDH complexes shows four subunits. These are α and β subunits of pyruvate decarboxylase (molecular weights 40,600 and 35,100); subunit of dihydrolipoyl acetyltransferase (molecular weight 76,100); and subunit of dihydrolipoyl dehydrogenase (molecular weight 58,200). The molecular weights are for the pig heart complex. The molecular weights of the component pig heart enzymes indicate that the decarboxylase is a tetramer (α2β2); that dihydrolipoyl acetyltransferase contains 24 subunits; and that dihydrolipoyl dehydrogenase is a dimer. In this chapter, the mechanisms that regulate the PDH complex are examined in detail. Stoichiometry of phosphorylations has been discussed. Amino acid sequence around phosphorylation sites has also been described.

Multiple forms of lipoamide dehydrogenase (NAD + -NADH: lipoamide oxidoreductase, EC 1.6.4.3) have been isolated from a variety of mammalian tissues. Sixmain enzymically active anodal species can be separated of which three are associated with the pyruvate dehydrogenase complex and three with the α-ketoglutarate dehydrogenase complex. Whether or not these isozymes exist in vivo or arise as a consequence of the experimental conditions employed in the isolation of lipoamide dehydrogenase has beenthe subject of several previous investigations. Further experimental efforts, includingthe use of the proteolytic enzyme inhibitor, pepstatin, during enzyme purification and isolation of the isozymes from sonicated fresh pig heart mitochondria, provide evidenceagainst a solely artifactual origin.

Publisher Summary This chapter describes the assay method and properties of pyruvate dehydrogenase complex isolated from Hansenula . The multienzyme complexes, collectively called “pyruvate dehydrogenase complexes (PDC),” are resolved into three component enzymes—namely,pyruvate dehydrogenase, dihydrolipoyl transacetylase, and dihydrolipoyl dehydrogenase. These component enzymes catalyze a series of five reactions that constitute the overall reaction. The amount of nicotinamide adenine dinucleotide dehydrogenase (NADH) formed is proportional to the concentration of the multienzyme complex. The steps involved in the purification procedure of the enzyme are (1) the preparation of cell-free extract, (2) protamine sulfate fractionation, (3) first and second ultracentrifugation, and (4) ammonium sulfate fractionation. All operations are carried out at 0–5°C and solutions are prepared with deionized water. The amount of wet cells used for each purification experiment is 500 g; this is obtained from about 200 liters of culture broth. The samples of purified PDC have an ability to form α-acetolactate and acetoin from pyruvate and acetaldehyde.

Publisher Summary Asparagusate dehydrogenase and lipoyl dehydrogenase are essential components of asparagus pyruvate dehydrogenase complexes, which catalyze a CoA- and NAD-linked oxidative decarboxylation of pyruvate. These two enzymes are flavoproteins and catalyze the oxidoreduction of asparagusate and lipoate. Asparagusate dehydrogenase and lipoyl dehydrogenase activities are measured spectrophotometrically by the decrease in absorption at 340 nm due to NADH oxidation in the presence of asparagusate or lipoate. Assays are carried out in cuvettes of 10-mm light path maintained at 25° and containing 0.05 ml each of sodium phosphate buffer and EDTA and 0.02 ml each of NADH, NAD, and asparagusic acid or DL-lipoic acid. The reaction is started by adding enzyme. A unit of enzyme activity is defined as that amount of enzyme which oxidizes 1 nmol of NADH per minute under the conditions of assay specified above. Specific activity is defined as units per milligram of protein; protein is determined by the method of Lowry et al ., using crystalline bovine serum albumin as a standard.

Publisher Summary The chapter describes assay method, purification procedure, and properties of the α- ketoglutarate dehydrogenase enzyme. This enzyme has been isolated from pig heart muscle, Escherichia coli, and beef kidney mitochondria as multienzyme complexes with molecular weights of several million. The E. coli α-ketoglutarate dehydrogenase complex is separated in three enzymes, α-ketoglutarate dehydrogenase (E1), dihydrolipoyl transsuccinylase (E2), and dihydrolipoyl dehydrogenase (E3), and the complex is reconstituted from the isolated enzymes. There is as no sensitive and convenient assay for the a-ketoglutarate dehydrogenase complex that may be used at all levels of purity of the enzyme complex. The DPN reduction assay described is the only reliable assay for the intact complex however it is not suitable for crude preparations that contain DPNH oxidase. Assays for the three component enzymes of the a-ketoglutarate dehydrogenase complex are described, but these assays do not distinguish between the free and combined enzymes. Thus, the ferricyanide reduction assay described provides an estimate of the activity of the α-ketoglutarate dehydrogenase component (E1) of the complex.

Publisher Summary This chapter discusses the assay method, purification procedure, and properties of lipoamide dehydrogenases. Lipoamide dehydrogenase is an essential component of the pig heart α -keto acid dehydrogenase complexes, which catalyze a coenzyme A (CoA)- and nicotinamide adenine dinucleotide (NAD)-linked oxidative decarboxylation of pyruvate and 2-oxoglutarate. This enzyme is a flavoprotein and catalyzes the oxidation of protein-bound dihydrolipoic acid. Nicotinamide adenine dinucleotide hydrogen (NADH)-lipoamide oxidoreductase assay is based on the spectrophotometric determination of the rate of NADH oxidation in the presence of lipoamide. The catalytic activities of the enzymes are summarized in the chapter in tabulated form. The enzymes differ only with respect to their diaphorase activity. The natural substrate of the two mammalian lipoamide dehydrogenases is the dihydrolipoyl moiety, which is covalently bound to lipoate acyltransferase, presumably to the є-amino group of a lysine residue. However, the enzyme also catalyzes reaction with lipoic acid and lipoic acid derivatives.

Friedreich ataxia (FA) is a cardioneurodegenerative disease caused by deficient frataxin expression. This mitochondrial protein has been related to iron homeostasis, energy metabolism, and oxidative stress. Previously, we set up a cardiac cellular model of FA based on neonatal rat cardiac myocytes (NRVM) and lentivirus-mediated frataxin RNA interference. These frataxin-deficient NRVMs presented lipid droplet accumulation, mitochondrial swelling and signs of oxidative stress. Therefore, we decided to explore the presence of protein thiol modifications in this model. With this purpose, reduced glutathione (GSH) levels were measured and the presence of glutathionylated proteins was analyzed. We observed decreased GSH content and increased presence of glutahionylated actin in frataxin-deficient NRVMs. Moreover, the presence of oxidized cysteine residues was investigated using the thiol-reactive fluorescent probe iodoacetamide-Bodipy and 2D-gel electrophoresis. With this approach, we identified two proteins with altered redox status in frataxin-deficient NRVMs: electron transfer flavoprotein-ubiquinone oxidoreductase and dihydrolipoyl dehydrogenase (DLDH). As DLDH is involved in protein-bound lipoic acid redox cycling, we analyzed the redox state of this cofactor and we observed that lipoic acid from pyruvate dehydrogenase was more oxidized in frataxin-deficient cells. Also, by targeted proteomics, we observed a decreased content on the PDH A1 subunit from pyruvate dehydrogenase. Finally, we analyzed the consequences of supplementing frataxin-deficient NRVMs with the PDH cofactors thiamine and lipoic acid, the PDH activator dichloroacetate and the antioxidants N-acetyl cysteine and Tiron. Both dichloroacetate and Tiron were able to partially prevent lipid droplet accumulation in these cells. Overall, these results indicate that frataxin-deficient NRVMs present an altered thiol-redox state which could contribute to the cardiac pathology. This work has been funded by project SAF2017-83883-R from Ministerio de Economía y Empresa (MINECO, Spain). We thank Isabel Sánchez (Proteomic services, UdL) and Roser Pané for technical assistance