2-Aminoadipic acid (2-AAA) as a potential biomarker for insulin resistance in childhood obesity.

Insulin resistance is an important clinical feature of metabolic syndrome, which includes obesity and type 2 diabetes. Increased adipose energy storage in obesity promote insulin resistance and other metabolic adverse effects. To identify a new link between adipocyte and insulin resistance, we performed targeted metabolite profiling of differentiated adipocytes and studied the association between adipogenic metabolites and insulin resistance. We found a correlation between 2-aminoadipic acid (2-AAA) and adipogenic differentiation. Also, circulatory 2-AAA was positively associated with obesity-related factors (fat mass, fat percent, waist circumference, BMI, BMI z-score, triglycerides, insulin, and HOMA-IR) at baseline and after 2 years in the children cohort study. Of these factors, increased BMI z-score and HOMA-IR were the primary independent factors associated with higher 2-AAA levels, and the baseline 2-AAA level was an indicator of the BMI z-score after 2 years. To validate the relationship between 2-AAA and obesity-related factors, we analyzed changes in 2-AAA levels following obesity intervention programs in two independent studies. In both studies, changes in 2-AAA levels during the intervention period were positively correlated with changes in the BMI z-score and HOMA-IR after adjusting for confounders. Moreover, the 2-AAA levels were increased in cell and mouse models of obesity-related insulin resistance. Excess 2-AAA levels led to impaired insulin signaling in insulin-sensitive cells (liver, skeletal muscle and adipose cells) and caused abnormal gluconeogenesis. Our results demonstrate that 2-AAA is associated with adipogenesis and insulin resistance. In this regard, 2-AAA could be a potential biomarker of obesity and obesity-related metabolic disorders.

Lipoxygenase drives lipidomic and metabolic reprogramming in ischemic heart failure.

BACKGROUND: After myocardial infarction (MI), delayed progression or reversal of cardiac remodeling is a prime target to limit advanced chronic heart failure (HF). However, the temporal kinetics of lipidomic and systemic metabolic signaling is unclear in HF. There is no consensus on metabolic and lipidomic signatures that influence structure, function, and survival in HF. Here we use genetic knock out model to delineate lipidomic, and metabolic changes to describe the role of lipoxygenase in advancing ischemic HF driven by leukocyte activation with signs of non-resolving inflammation. Bioactive lipids and metabolites are implicated in acute and chronic HF, and the goal of this study was to define the role of lipoxygenase in temporal kinetics of lipidomic and metabolic reprogramming in HF. MATERIALS AND METHODS: To address this question, we used a permanent coronary ligation mouse model which showed profound metabolic and lipidomic reprogramming in acute HF. Additionally, we defined the lipoxygenase-mediated changes in cardiac pathophysiology in acute and chronic HF. For this, we quantitated systemic metabolic changes and lipidomic profiling in infarcted heart tissue with obvious structural remodeling and cardiac dysfunction progressing from acute to chronic HF in the survival cohort. RESULTS: After MI, lipoxygenase-derived specialized pro-resolving mediators were quantitated and showed lipoxygenase-deficient mice (12/15LOX-/-) biosynthesize epoxyeicosatrienoic acid (EETs; cypoxins) to facilitate cardiac healing. Lipoxygenase-deficient mice reduced diabetes risk biomarker 2-aminoadipic acid with profound alterations of plasma metabolic signaling of hexoses, amino acids, biogenic amines, acylcarnitines, glycerophospholipids, and sphingolipids in acute HF, thereby improved survival. CONCLUSION: Specific lipoxygenase deletion alters lipidomic and metabolic signatures, with modified leukocyte profiling that delayed HF progression and improved survival. Future studies are warranted to define the molecular network of lipidome and metabolome in acute and chronic HF patients.

High sensitivity HPLC method for determination of the allysine concentration in tissue by use of a naphthol derivative.

Common to all fibrotic and metastatic diseases is the uncontrollable remodeling of tissue that leads to the accumulation of fibrous connective tissue components such as collagen and elastin. Build-up of fibrous tissue occurs through the cross-linking of collagen or elastin monomers, which is initiated through the oxidation of lysine residues to form α-aminoadipic-δ-semialdehyde (allysine). To provide a measure of the extent of collagen oxidation in disease models of fibrosis or metastasis, a rapid, sensitive HPLC method was developed to quantify the amount of allysine present in tissue. Allysine was reacted with sodium 2-naphthol-7-sulfonate under conditions typically applied for acid hydrolysis of tissues (6M HCl, 110°C, 24h) to prepare AL-NP, a fluorescent bis-naphthol derivative of allysine. High performance liquid chromatography was applied for analysis of allysine content. Under optimal reaction and detection conditions, successful separation of AL-NP was achieved with excellent analytical performance attained. Good linear relationship (R2=0.994) between peak area and concentration for AL-NP was attained for 0.35-175pmol of analyte. A detection limit of 0.02pmol in the standard sample with a 20µL injection was achieved for AL-NP, with satisfactory recovery from 88 to 100% determined. The method was applied in the quantification of allysine in healthy and fibrotic mouse lung tissue, with the fibrotic tissue showing a 2.5 fold increase in the content of allysine.

Protein oxidation during frozen storage and subsequent processing of different beef muscles.

This study examined the relationship between protein and lipid oxidation and the impairment of the water holding capacity (WHC), redness and instrumental hardness occurring during 20 weeks of frozen storage (-18 °C) and subsequent processing (cooking, chilled storage) of psoas major, quadriceps femoris and longissimus dorsi beef patties. Patties were analyzed at sampling times upon thawing (weeks 0, 4, 8, 12 and 20) for lipid (TBARS, hexanal) and protein oxidation products (α-aminoadipic and γ-glutamic semialdehydes, α-aminoadipic acid, Schiff bases). A significant impact of frozen storage on protein oxidation was found, which occurred concomitantly with a loss of WHC, redness and significant changes in the hardness of cooked patties. Heme-iron, endogenous antioxidant enzymes and to a lower extent, fatty acid composition, played a role in the oxidative stability of patties. Plausible mechanisms by which particular protein oxidation changes may lead to loss of WHC and impaired quality traits were discussed.

Oncosecretomics coupled to bioenergetics identifies α-amino adipic acid, isoleucine and GABA as potential biomarkers of cancer: Differential expression of c-Myc, Oct1 and KLF4 coordinates metabolic changes.

Bioenergetic profiling of tumors is a new challenge of cancer research and medicine as therapies are currently being developed. Meanwhile, methodological means must be proposed to gather information on tumor metabolism in order to adapt these potential therapies to the bioenergetic specificities of tumors. Studies performed on tumors and cancer cell lines have shown that cancer cells bioenergetics is highly variable. This profile changes with microenvironmental conditions (eg. substrate availability), the oncogenes activated (and the tumor suppressors inactivated) and the interaction with the stroma (i.e. reverse Warburg effect). Here, we assessed the power of metabolic footprinting (MFP) to unravel the bioenergetics and associated anabolic changes induced by three oncogenes, c-Myc, KLF4 and Oct1. The MFP approach provides a quantitative analysis of the metabolites secreted and consumed by cancer cells. We used ultra performance liquid chromatography for quantifying the amino acid uptake and secretion. To investigate the potential oncogene-mediated alterations in mitochondrial metabolism, we measured oxygen consumption rate and ATP production as well as the glucose uptake and lactate release. Our findings show that c-Myc deficiency initiates the Warburg effect along with a reduction of mitochondrial respiration. KLF4 deficiency also stimulated glycolysis, albeit without cellular respiration impairment. In contrast, Oct1 deficiency reduced glycolysis and enhanced oxidative phosphorylation efficiency. MFP revealed that c-Myc, KLF4 and Oct1 altered amino acid metabolism with specific patterns. We identified isoleucine, α-aminoadipic acid and GABA (γ-aminoisobutyric acid) as biomarkers related. Our findings establish the impact of Oct1, KLF4 and c-Myc on cancer bioenergetics and evidence a link between oncosecretomics and cellular bioenergetics profile.

Pre-freezing raw hams affects quality traits in cooked hams: potential influence of protein oxidation.

The influence of protein carbonylation and lipid oxidation on colour and texture changes in cooked hams from fresh and pre-frozen (frozen/thawed) raw material was studied. Samples from three muscles, biceps femoris (BF) quadriceps femoris (QF) and semimembranosus (SM) were analysed for the gain of specific protein carbonyls, α-aminoadipic and γ-glutamic semialdehydes, the gain of TBA-RS and their colour and texture properties by instrumental and sensory techniques. The formation of protein carbonyls occurred concomitantly with an intense loss of redness and increase of hardness. Both phenomena were found to be more intense in QF and SM muscles in cooked hams elaborated from frozen material. Lipid oxidation played a negligible role on the impaired quality traits observed in cooked hams as a result of pre-freezing. Plausible mechanisms by which protein carbonylation may be implicated in the loss of quality in cooked hams produced from pre-frozen material are discussed.

Dog rose (Rosa canina L.) as a functional ingredient in porcine frankfurters without added sodium ascorbate and sodium nitrite.

The effect of dog rose (Rosa canina L.; RC), rich in polyphenols and ascorbic acid, on lipid and protein oxidation, colour stability and texture of frankfurters was investigated. Four treatments were prepared: with 5 or 30 g/kg RC extract and without sodium ascorbate and sodium nitrite (5RC and 30RC, respectively), a positive control (with sodium ascorbate and sodium nitrite; PC) and a negative control (without sodium ascorbate, sodium nitrite or RC extract; NC). Hexanal values were much higher throughout storage in NC compared to RC and PC frankfurters (P<0.001). The RC extracts protected against protein oxidation, but not as efficiently as PC (P<0.05). In the RC treated frankfurters, lower a* values were measured compared to PC due to the lack of sodium nitrite. In conclusion, dog rose can act as a natural antioxidant in frankfurters, but not as full replacer for sodium nitrite.

Analysis of protein oxidation markers alpha-aminoadipic and gamma-glutamic semialdehydes in food proteins using liquid chromatography (LC)-electrospray ionization (ESI)-multistage tandem mass spectrometry (MS).

To elucidate the formation of protein oxidation biomarkers alpha-aminoadipic semialdehyde (AAS) and gamma-glutamic semialdehyde (GGS) in food proteins was the main purpose of the present study. Food proteins, namely, myofibrillar proteins, alpha-lactalbumin, and soy proteins, as well as bovine serum albumin (BSA), were suspended in a piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES) buffer and oxidized by Fe(3+) and H(2)O(2) while kept in an oven for 14 days at 37 degrees C. For the analysis of semialdehydes, a derivatization procedure with p-aminobenzoic acid (ABA) and NaCNBH(3) followed by liquid chromatography (LC)-electrospray ionization (ESI)-multistage tandem mass spectrometry (MS) was performed. For comparative purposes, the dinitrophenylhydrazine (DNPH) method was also employed as a routine method to assess carbonyl gain. Both semialdehydes were specifically and accurately detected by LC-MS in all oxidized proteins proving that GGS and AAS are formed as a consequence of the oxidation of lysine, proline, and arginine amino acid residues from BSA and other food proteins. Proteins from an animal source and, particularly, BSA were more susceptible to undergo oxidative reactions than soy proteins. The results from the present paper highlight the significance of using both semialdehydes as protein oxidation indicators in meat and dairy products. The analysis of GGS and AAS in real food systems would contribute to the understanding of the precise mechanisms involved in food protein oxidation and shed light on the fate of oxidizing amino acids during food processing and storage.

A novel type of lysine oxidase: L-lysine-epsilon-oxidase.

The melanogenic marine bacterium M. mediterranea synthesizes marinocine, a protein with antibacterial activity. We cloned the gene coding for this protein and named it lodA [P. Lucas-Elío, P. Hernández, A. Sanchez-Amat, F. Solano, Purification and partial characterization of marinocine, a new broad-spectrum antibacterial protein produced by Marinomonas mediterranea. Biochim. Biophys. Acta 1721 (2005) 193-203; P. Lucas-Elío, D. Gómez, F. Solano, A. Sanchez-Amat, The antimicrobial activity of marinocine, synthesized by M. mediterranea, is due to the hydrogen peroxide generated by its lysine oxidase activity. J. Bacteriol. 188 (2006) 2493-2501]. Now, we show that this protein is a new type of lysine oxidase which catalyzes the oxidative deamination of free L-lysine into 6-semialdehyde 2-aminoadipic acid, ammonia and hydrogen peroxide. This new enzyme is compared to other enzymes related to lysine transformation. Two different groups have been used for comparison. Enzymes in the first group lead to 2-aminoadipic acid as a final product. The second one would be enzymes catalyzing the oxidative deamination of lysine releasing H2O2, namely lysine-alpha-oxidase (LalphaO) and lysyl oxidase (Lox). Kinetic properties, substrate specificity and inhibition pattern show clear differences with all above mentioned lysine-related enzymes. Thus, we propose to rename this enzyme lysine-epsilon-oxidase (lod for the gene) instead of marinocine. Lod shows high stereospecificity for free L-lysine, it is inhibited by substrate analogues, such as cadaverine and 6-aminocaproic acid, and also by beta-aminopropionitrile, suggesting the existence of a tyrosine-derived quinone cofactor at its active site.

New method for the quantitative determination of major protein carbonyls, alpha-aminoadipic and gamma-glutamic semialdehydes: investigation of the formation mechanism and chemical nature in vitro and in vivo.

Alpha-aminoadipic semialdehyde (AAS) and gamma-glutamic semialdehyde (GGS) are identified as the major carbonyl products in oxidized proteins. To elucidate the formation pathway of AAS and GGS in vivo, we developed and validated a new quantification method. AAS and GGS in proteins were derivatized by reductive amination with NaCNBH(3) and p-aminobenzoic acid, a fluorescent reagent, followed by acid hydrolysis. It is noteworthy that the fluorescent derivatives were completely stable during acid hydrolysis. The present method permitted the specific, accurate, and sensitive quantification of both semialdehydes by fluorometric high-performance liquid chromatography. Analysis of proteins oxidized by various oxidation systems revealed that AAS and GGS are notably generated by the reaction of proteins with (*)OH, which is produced by metal-catalyzed oxidation (MCO). Furthermore, exposure of transferrin and human plasma to ascorbic acid and H(2)O(2) significantly promoted the formation of AAS and GGS in vitro, suggesting that both semialdehydes can be generated by MCO in vivo. We also demonstrated their generation through oxidative stress induced by acute iron overload in vivo. In this paper, we describe this analytical technique for simple and precise measurement of AAS and GGS and discuss their formation mechanism in vivo.

Formation of alpha-aminoadipic and gamma-glutamic semialdehydes in proteins by the maillard reaction.

Recent research has demonstrated that nonenzymatic glycation (the Maillard reaction) lead to the formation of carbonyl groups and advanced glycation end products (AGEs) in proteins. Such oxidative modifications are a major contributing factor to diabetic complications and aging. alpha-Aminoadipic semialdehyde (AAS) and gamma-glutamic semialdehyde (GGS) have been identified as the major carbonyl products in oxidized proteins both in vitro and in vivo. AAS is an oxidative deamination product of lysine residue, while GGS originates from arginine and proline residues. To evaluate oxidative damage to proteins by the Maillard reaction, we developed a method of detecting AAS and GGS by high-performance liquid chromatography (HPLC). The aldehydic residues in proteins were derivatized by reductive amination with NaCNBH3 and p-aminobenzoic acid (ABA), a fluorescence regent. After acid hydrolysis of the ABA-derivatized protein, ABA-AAS and ABA-GGS were measured by fluorometric HPLC. Thus, AAS and GGS could be detected in various proteins such as human plasma protein using the present method. Accumulation of both aldehydic residues was observed in oxidized proteins by reactive oxygen species. Furthermore, AAS and GGS were markedly formed in the incubation of BSA with ascorbic acid. The formation of both aldehydic residues was also observed in the incubation of BSA with 100 mM glucose or 1.0 mM methylglyoxal in the absence and presence of 100 microM Fe3+ for 2 weeks. These results suggest that the Maillard reaction can contribute to the formation of AAS and GGS in vivo.

Determination of alpha-aminoadipic acid in brain, peripheral tissues, and body fluids using GC/MS with negative chemical ionization.

alpha-Aminoadipic acid (alphaAA) is a structural homolog of the excitatory amino acid glutamate and a natural product of lysine metabolism in mammalian cells. Under experimental conditions, alphaAA can influence various elements of glutamatergic neurotransmission. Moreover, as a selective inhibitor of kynurenine aminotransferase II, alphaAA is capable of decreasing the levels of the neuroinhibitory metabolite kynurenic acid in the brain. We now describe the identification of this potential endogenous neuromodulator in tissues and body fluids by gas chromatography/mass spectrometry (GC/MS) analysis of its pentafluorobenzyl (PFB) derivative. alphaAA was recovered from the GC column with a retention time of approximately 7 min. Subsequent MS analysis using electron capture with negative ionization revealed two separate ions for alphaAA (m/z 520, approximately 45% and m/z 322, approximately 55%). Both of these ions were positively identified with two different GC methodologies. In the rat, alphaAA levels ranged from 5 to 30 microM in various brain areas and from 8 to 40 microM in peripheral organs, whereas serum and urine contained only 1-2 microM alphaAA. Levels in the human brain were 18.7+/-2.4 microM (cortex) and 18.0+/-1.7 microM (striatum) alphaAA (n=9 each), and the mouse forebrain contained 8.3+/-1.9 microM alphaAA (n=6). Neuronal depletion, caused in rats by an intrastriatal injection of NMDA (300 nmol/2.5 microl), did not alter the striatal content of alphaAA, indicating that brain alphaAA resides at least in part in glial cells. alphaAA may therefore function as a glia-derived modulator of excitatory neurotransmission.

Nonprotein amino acids in edible lentil and garden pea seedlings.

Commercial edible seedlings of garden pea (Pisum sativum L.) and lentil (Lens culinaris L.) contain high concentration of nonprotein amino acids and trigonelline. Both seedlings grown in the laboratory or purchased in a supermarket were studied by HPLC. Samples from both origins contained trigonelline, alpha-aminoadipic acid, homoserine, beta-(isoxazolin-5-on-2-yl)-alanine (BIA), and gamma-glutamyl-BIA. Garden pea seedlings also contained a uracil-alanine derivative (isowillardiine) in substantial amount. Some of these compounds such as BIA and alpha-aminoadipic acid have neurotoxic activity.

High-performance liquid chromatographic quantification of allysine as bis-p-cresol derivative in elastin.

The first step in normal cross-linking in elastin is the formation of alpha-aminoadipic-delta-semialdehyde, allysine, through oxidative deamination of specific peptidyl lysine by the enzyme lysyl oxidase (EC 1.4.3.13). For the analysis of allysine, allysine was derivatized with p-cresol. The derivatization was carried out by acid hydrolysis (6N HCl containing 5% (w/v) p-cresol at 110 degrees C for 48 h) accompanied with the hydrolysis of elastin. A bis-p-cresol derivative of allysine was isolated from bovine ligamentum nuchae elastin hydrolysates, and was characterized by UV, FAB-MS and NMR. This derivative was identified as 2-amino-6,6-bis(2-hydroxy-5-methylphenyl)hexanoic acid. A rapid, sensitive reverse-phase high-performance liquid chromatographic method with UV detection was developed for the quantitative determination of allysine as its bis-p-cresol derivative. The lower limit of detection of the bis-p-cresol derivative was 58 pmol in the standard sample with a 20-microl injection at a signal-to-noise ratio of 3. This method was applied to the determination of allysine in bovine ligamentum nuchae, aorta, lung, and rat aorta elastin. The allysine content in rat aorta elastin dramatically increased from 1 week to 2 weeks of age.

Two new elastin cross-links having pyridine skeleton. Implication of ammonia in elastin cross-linking in vivo.

Isolation and structure analysis of two amino acids from bovine ligamentum nuchae elastin hydrolysates revealed the presence of pyridine cross-links in elastin. The structures of these amino acids were determined to have 3,4,5- and 2,3,5-trisubstituted pyridine skeletons both with three carboxylic acids and a mass of 396 (C(18)H(28)N(4)0(6)) identified as 4-(4-amino-4-carboxybutyl)-3,5-di-(3-amino-3-carboxypropyl)-pyridine and 2-(4-amino-4-carboxybutyl)-3,5-di-(3-amino-3-carboxypropyl)-pyridine. We have named these pyridine cross-links desmopyridine (DESP) and isodesmopyridine (IDP), respectively. Structure analysis of these pyridine cross-links implied that the formation of these cross-links involved the condensation reaction between ammonia and allysine. The elastin incubated with ammonium chloride showed that DESP and IDP levels increased as the allysine content decreased. DESP and IDP were measured by high pressure liquid chromatography (HPLC) with UV detection and were found in a variety of bovine tissues. The DESP/desmosine (DES) and IDP/isodesmosine (IDE) ratios in aorta elastin were higher than in other tissues. DESP and IDP contents in human aorta elastin were found to be gradually increased with age. The concentration of IDP was significantly elevated in aorta elastin of rat with chronic liver cirrhosis induced by carbon tetrachloride (mean +/- S.D.; 11.1 +/- 0.9 nmol/mg elastin) when compared with normal rats (5.9 +/- 1.5 nmol/mg elastin). Although DESP and IDP are present at only trace concentrations in the tissue elastin, these pyridine cross-links may be useful biomarkers for the aortic elastin damaged by ammonia.

Bisphenol derivative of allysine for high-performance liquid chromatographic analysis of allysine residue of proteins.

Allysine is the most important precursor of physiologically essential cross-links formation in collagen and elastin and is formed by enzymatic oxidative deamination of lysine residues. Because it is a highly reactive aldehyde, many cross-linking amino acid residues may arise from its reaction with other allysine residues or lysine or even histidine residues. We purified and isolated an allysine bisphenol derivative, 1-amino-1-carboxy-5,5-bis-p-hydroxyphenylpentane (ACPP), from the reaction products of phenol and allysine residue of bovine ligamentum nuchae by acid hydrolysis in 6 M HCl. The structure of ACPP was verified by UV, fast atom bombardment-MS, 1H- and 13C-nuclear magnetic resonance spectroscopies. The optimal reaction condition for ACPP synthesis accompanied by hydrolysis of such proteins was investigated and an ion-paired high-performance liquid chromatographic method for determination of allysine as ACPP was also developed.

Accumulation and metabolism of pipecolic acid in the developing brain of the mouse.

In newborn mice, following i.p. injections of D,L-[3H]PA (pipecolic acid, 28 micrograms/kg), accumulation of radioactivity continues to increase up to 24 h. In adults, radioactivity peaks at 5 min and remains approximately constant up to 5 h, and then declines slowly to 24 h. Fifteen-day-old mice follow the newborn pattern, while 30-day-old mice show the same trend as the adult. Radioactivity in plasma shows essentially the same pattern of accumulation in adult and newborn animals with some quantitative differences. Secretion of radioactivity in the urine is significantly higher in the adult than in the newborn during the interval between 10 min and 5 h. Accumulation of radioactivity at 24 h in the newborn brain shows a preferential localization to the olfactory bulb, the anterior telencephalon and the diencephalon. Two hours after the i.p. injection, approximately 70% of the radioactivity recovered in brain is due to PA. This percentage increases to 75% and 87% at 5 and 24 h respectively. Alpha-aminoadipic acid (alpha-Aaa) a major metabolite of PA was identified in brain extracts at 5 h. The maximal formation of alpha-Aaa in relation to PA occurs approximately at 5 h. No other brain metabolites of PA could be identified with this chromatographic system. The present results show that access of PA to the brain is easier in the newborn mouse than in the adult. In addition, our results demonstrate, for the first time, the presence of PA metabolism in the newborn mouse.