Ultrasound-Induced Formation of S-Nitrosoglutathione and S-Nitrosocysteine in Aerobic Aqueous Solutions of Glutathione and Cysteine

S-Nitrosocompounds are formed when aqueous solutions of cysteine or glutathione are exposed to ultrasound (880 kHz) in air. The yield of the S-nitrosocompounds was as high as 10% for glutathione and 4% for cysteine of the initial thiol concentrations (from 0.1 to 10 mM) in the aqueous solutions. In addition to the formation of S-nitrosocompounds, thiol oxidation to disulfide forms was observed. After the oxidation of over 70% of the sulfhydryl groups, formation of peroxide compounds as well as cysteic acid derivatives was recorded. The formation of the peroxide compounds and peroxide radicals in the ultrasound field reduced the yield of S-nitrosocompounds. S-Nitrosocompounds were not formed when exposing low-molecular-weight thiols to ultrasound in atmospheres of N₂ or CO. In neutral solutions, ultrasound-exposed cysteine or glutathione released NO due to spontaneous degradation of the S-nitrosocompounds. N₂O₃, produced due to the spontaneous degradation of the S-nitrosocompounds in air, nitrosylated sulfhydryl groups of glutathione manifested in the appearance of new absorption bands at 330 and 540 nm. The nitrogen compounds formed in an ultrasound field modified the sulfhydryl groups of apohemoglobin and serum albumin. The main target for ultrasound-generated oxygen free radicals were cystine residues oxidized to cysteic acid residues.     

Secretion in yeast of mutant human lysozymes with and without glutathione bound to cysteine 95

A mutant human lysozyme C77A, in which Cys-77 is replaced with Ala, was secreted by Saccharomyces cerevisiae as two proteins (C77A-a and C77A-b) with different specific activities. A peptide fragment from Val93 to Ala108 was obtained from C77A-a by pepsin digestion, and examined by fast atom bombardment mass spectrometry and amino acid analysis. The results showed that glutathione was attached to the thiol group of Cys95 of the fragment through a disulfide linkage. This observation was confirmed by quantitative formation of free glutathionesulfonic acid from C77A-a by performic acid treatment. In contrast, there was no modification in the case of C77A-b. These results indicate that C77A-a contained a mixed disulfide with glutathione attached to cysteine residue 95. In C77A-b, there appears to be a free thiol of Cys95 surrounded by many side chains, which was not modified by iodoacetic acid under native conditions, suggesting that the attachment of glutathione occurs during folding. These findings further suggest that in the oxidation step of disulfide bond formation in human lysozyme secreted by yeast, mixed disulfides are formed with glutathione and that posttranslational modification with glutathione can occur even in a protein secreted by yeast.     

Screening of Thiol Compounds: Depolarization-Induced Release of Glutathione and Cysteine from Rat Brain Slices

Superfusates from rat brain slices were screened for thiol compounds after derivatization with monobromobimane by reversed-phase HPLC. Only glutathione and cysteine were detected. The Ca(2+)-dependent release of these compounds from slices of different regions of rat brain was investigated, applying a highly sensitive and reproducible quantification method, based on reduction of superfusates with dithiothreitol, reaction of thiols with iodoacetic acid, precolumn derivatization with o-phthalaldehyde reagent solution, and analysis with reversed-phase HPLC. This methodology allowed determination of reduced and total thiols in aliquots of the same superfusates. Mostly reduced glutathione and cysteine were released upon K+ depolarization and the Ca2+ dependency suggests that they originate from a neuronal compartment. The GSH release was most prominent in the mesodiencephalon, cortex, hippocampus, and striatum and lowest in the pons-medulla and cerebellum. This underscores a physiologically significant role for glutathione in CNS neurotransmission.     

Oxidative folding of reduced and denatured huwentoxin-I

Huwentoxin-I, a neurotoxic peptide with 33 ammo acid residues and three disulfide bonds, was used to investigate the pathway of reduction/denaturation and of oxidative folding in small proteins with multiple disulfide bonds. Titration of thiol groups, reversed-phase HPLC, 1D NMR spectroscopy, and biological activity assays were used to monitor the extent of reduction/denaturation and renaturation of the toxin. The reduction and denaturation of huwentoxin-I resulted in a 100% loss of bioactivity as measured in a mouse phrenic nerve-diaphragm preparation. About 90% of full biological activity could be restored under optimized conditions of oxidative refolding of the reduced peptide. Several reaction conditions employing air oxidation, oxidized and reduced glutathione (GSSG and GSH), and cystine/cysteine were investigated in order to find optimal conditions for renaturation of huwentoxin-I. The best renaturation yield was achieved in 0.1 mM GSSG and 1 mM GSH at pH 8.5 and 4°C over 24 hr. High concentrations of glutathione and high temperatures reduced renaturation yields. Oxidative refolding of huwentoxin-I in air requires about 6 days for maximal yields and is inhibited by EDTA.     

Effect of oxidizing agents and sulfhydryl group reagents on beta toxin from Clostridium perfringens type C

Purified beta toxin from Clostridium perfringens type C was inactivated by the oxidizing agents o-iodosobenzoate (OIBA), oxidized glutathione, and ferricyanide, and by the sulfhydryl group regents 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimide, iodoacetamide, and iodoacetic acid, causing loss of activity in various degrees depending on the concentration used. The activity of the toxin was not influenced by exposure to 1.0 mM of p-chloromercuribenzoate. The toxin treated by OIBA or DTNB was reactivated by incubation with 2-mercaptoethanol and dithiothreitol. The data suggest that beta toxin contains thiol groups which are essential for the activity.     

High Performance Liquid Chromatography Analysis of Reduced and Oxidized Glutathione in Woody Plant Tissues

A rapid, and sensitive high performance liquid chromatographymethod for quantifying both the reduced and oxidized forms ofglutathione simultaneously in woody plant tissues is reported.Samples were extracted in 10% perchloric acid with 1 mM bathophenanthrolinedisulfonic acid, homogenized, and centrifuged. The supernatantwas carboxy-methylated with iodoacetic acid, and derivatizedwith 2,4-dinitro-l-fluorobenzene. A 3-aminopropyl-Spherisorbcolumn was used, for separations. Dinitrophenol derivativeswere detected at 365 nm. The retention times were 13.9 min and15.1 min and the lowest limits of detection were 10 pmol and5 pmol for reduced and oxidized glutathione, respectively. This HPLC method was applicable to a variety of woody plantspecies and different tissues. Total glutathione content inthe bark tissue of seven of the woody plant species tested werelower during the period of active plant growth, and higher duringthe dormant stage. Such change was not observed in grape. Reducedglutathione always represented a high percentage of the totalglutathione. All tested tissues of peach plants contained glutathione.The highest and lowest levels were found in the leaves and roots,respectively. Both cherry and sugar pine seeds contained highlevels of reduced glutathione. ¹Oregon Agricultural Experiment Station Technical paper No.9560. (Received April 3, 1991; Accepted August 17, 1991)     

Clinical assay of four thiol amino acid redox couples by LC–MS/MS: Utility in thalassemia

The total concentrations of four sulfur amino acid (SAA) metabolite redox couples (reduced and oxidized forms of homocysteine, cysteine, glutathione, and cysteinylglycine) in human blood are assayed with a simple and sensitive method by liquid chromatography–electrospray positive ionization–tandem mass spectrometry. To prevent ex vivo thiol oxidation, iodoacetamide (IAM) is used immediately following the blood draw. To selectively enrich for S-carboxyamidomethylated SAA, and other cationic amino acids metabolites, proprietary strong cation-exchange solid phase extraction tips are used. Analytes are further derivatized with isopropylchloroformate (IPCF) to esterify the amino and the carboxylic groups. Double derivatization with IAM and IPCF improves the reverse phase liquid chromatography separation of SAA metabolites. The use of detection mode of multiple-reaction monitoring (MRM) allows sensitive and specific simultaneous detection of SAA. The internal standards used to account for the matrix effects of human plasma and erythrocytes were plant glutathione analogue, homoglutathione, and stable isotopes of cystine and homocystine. The method was validated for its linearity, accuracy, and precision. Excellent linearity of detection (r² > 0.98) was observed over relevant ranges for plasma and erythrocyte samples, and the limits of detection were established to be between 5 and 20 nM. Relative standard deviations were <9% for within-day variations and <15% for between-day variations. The method was used to assess thiol redox states in plasma and erythrocytes isolated from healthy subjects and thalassemia patients.     

Glutathione is a target in tellurite toxicity and is protected by tellurite resistance determinants in Escherichia coli

Tellurite (TeO3(2-)) is highly toxic to most microorganisms. The mechanisms of toxicity or resistance are poorly understood. It has been shown that tellurite rapidly depletes the reduced thiol content within wild-type Escherichia coli. We have shown that the presence of plasmid-borne tellurite-resistance determinants protects against general thiol oxidation by tellurite. In the present study we observe that the tellurite-dependent depletion of cellular thiols in mutants of the glutathione and thioredoxin thiol:redox system was less than in wild-type cells. To identify the type of low-molecular-weight thiol compounds affected by tellurite exposure, the thiol-containing molecules were analyzed by reverse phase HPLC as their monobromobimane derivatives. Results indicated that reduced glutathione is a major initial target of tellurite reactivity within the cell. Other thiol species are also targeted by tellurite, including reduced coenzyme A. The presence of the tellurite resistance determinants kilA and ter protect against the loss of reduced glutathione by as much as 60% over a 2 h exposure. This protection of glutathione oxidation is likely key to the resistance mechanism of these determinants. Additionally, the thiol oxidation response curves were compared between selenite and tellurite. The loss of thiol compounds within the cell recovered from selenite but not to tellurite.     

Determination of total aminothiols and neuroactive amino acids in plasma by high performance liquid chromatography with fluorescence detection

This paper describes a simple and sensitive reversed-phase HPLC method for the determination of total homocysteine, total cysteine, total glutathione (GSH + GSSG), and neuroactive amino acids (Asp, Glu, Tau, GABA) using precolumn derivatization with ortho-phtaldialdehyde and fluorimetric detection at 360 and 470 nm for emission and excitation, respectively. Derivatization was performed with ortho-phthaldialdehyde in the presence of 2-mercaptoethanol after alkylation of free sulfhydryl groups with iodoacetic acid. For determination of total aminothiols, the disulfide bonds were reduced and protein-bound thiols were released by addition of dithiothreitol to the plasma sample. The advantage of this method is the simultaneous determination of both homocysteine/cysteine/glutathione and neuroactive amino acids in the sample. The plasma levels of studied compounds were determined in 14 healthy volunteers (20–45 years old) and 55 patients with chronic hepatitis C (20–49 years old) and the resulting values were in a good agreement with results published earlier. The calibration curves were linear over a concentration range of 5–100 μM in plasma (r ² = 0.985−0.996). The intraday and interday coefficients of variation were 3–6% and 4–7%, respectively. The recovery of the standards added to the plasma samples ranged from 94 to 102%. The limits of detection (LOD) were 0.2–0.5 ng per 10 μl of the injection volume (signal-to-noise ratio of 3).     

Incorporation of ascorbic acid and α-tocopherol to the extender media to enhance antioxidant system of cryopreserved sea bass sperm

Despite the overwhelming application of sperm cryopreservation in aquaculture and broodstock management, its detrimental effects on sperm quality must be taken into account. Imbalance of reactive oxygen species is considered one of the main triggers of cell damage after cryopreservation, because the spermatozoa antioxidant system is decimated during this process, mainly because the natural antioxidants present in seminal plasma diminish when sperm is diluted in extenders. It has been demonstrated that the addition of antioxidants to the extender improves the quality of thawed sperm. Thus, the aim of the present work was to evaluate the status of the antioxidant system in cryopreserved sea bass sperm, and the possibility of enhancing this system to reduce oxidation of the membrane compounds by extender supplementation with vitamins. To do this, sperm from European sea bass (Dicentrarchus labrax) was cryopreserved using an extender control (NAM), supplemented with 0.1 mm α-tocopherol or 0.1 mm ascorbic acid. Sperm motility (computer assisted sperm analysis (CASA) parameters), viability (SYBR Green/propidium iodide (PI)), lipid peroxidation (malondialdehyde (MDA) levels) and protein oxidation (DNPH levels) were analyzed, as well as the status of the sperm antioxidant system by determining glutathione peroxidase, glutathione reductase and superoxide dismutase (GPX, GSR and SOD) activity. The results demonstrated that extenders containing vitamins significantly increased sperm motility. Total motility, velocity and linearity increased from 31.2 ± 3.0 μm/sec, 18.3 ± 1.7 μm/sec and 46.9 ± 2.0% in extender containing 0.1 mm α-tocopherol or 30.6 ± 3.9 μm/sec, 19.5 ± 1.6 μm/sec and 47.9 ± 2.2% in extender containing 1 mm ascorbic acid respect to the extender control (20.7 ± 3.3 μm/sec, 13.8 ± 1.7 μm/sec and 37.3 ± 4.1%). However, viability and levels of lipid peroxidation and protein oxidation were not affected by the presence of these antioxidants, suggesting that membrane impairment could be more associated to osmotic shock or membrane destabilization than oxidative damage. The increased activity of both GPX and GSR after cryopreservation showed that the antioxidant system of sea bass sperm must play an important role in preventing oxidation of the membrane compounds. In conclusion, the addition of α-tocopherol and ascorbic acid to the extender media, together with the antioxidant system of the spermatozoa improved sea bass sperm motility, which is one of the impairment parameters most affected by cryopreservation.     

Viruses occurring in white clover (Trifolium repens L.) from permanent pastures in Britain

The uptake of captan from aqueous solution by conidia of Neurospora crassa can be markedly reduced by pretreatment of the cells with thiol reagents such as iodoacetic acid (IOA). The nature of this reaction has been investigated and it is shown that IOA largely reacts with the soluble thiol pool of the spores. Captan, however, reacts with both soluble and insoluble thiols and it is suggested that whilst the former is a detoxication process the latter may provide the key to its toxic action. Using glutathione as a model soluble thiol the molar ratio and pH dependence of the captan detoxication process has been determined and compared with the cellular reactions. Assay of ³⁵S-labelled captan has shown that captan is almost completely decomposed by the spores and that one-third of the accumulated captan is converted to sulphur compounds fixed to insoluble cell entities.     

Ribonuclease inhibitor from human placenta. Purification and properties

A soluble ribonuclease inhibitor from the human placenta has been purified 4000-fold by a combination of ion exchange and affinity chromatography. The inhibitor has been isolated in 45% yield (about 2 mg/placenta) as a protein that is homogeneous by sodium dodecyl sulfate-gel electrophoresis. In common with the inhibitors of pancreatic ribonuclease from other tissues that have been studied earlier, the placental inhibitor is an acidic protein of molecular weight near 50,000; it forms a 1:1 complex with bovine pancreatic RNase A and is a noncompetitive inhibitor of the pancreatic enzyme, with a Ki of 3 X 10(-10) M. The amino acid composition of the protein has been determined. The protein contains 30 half-cystine plus cysteine residues determined as cysteic acid after performic acid oxidation. At pH 8.6 the nondenatured protein alkylated with iodoacetic acid in the presence of free thiol has 8 free sulfhydryl groups. The inhibitor is irreversibly inactivated by sulfhydryl reagents and also by removal of free thiol from solutions of the protein. Inactivation by sulfhydryl reagents causes the dissociation of the RNase - inhibitor complex into active RNase and inactive inhibitor.     

Assay of thiols and disulfides based on the reversibility of N-ethylmaleimide alkylation of thiols combined with electrolysis

A simple and specific method for analyzing thiols and disulfides on the basis of the reversibility of N-ethylmaleimide (NEM) alkylation of thiols is described. When the adduct of NEM and glutathione (GSH) was electrolyzed at neutral pH, all of the GSH was recovered. When the adduct was exposed to pH 11.0 for 15 min at 30°C before electrolysis, GSH was not detected. The same behavior was observed after protein thiols reacted with NEM. This pH-dependent production of thiol from the adduct was used to assay GSH and oxidized glutathione in yeast cells, to assay sulfhydryl groups and disulfide bonds in authentic proteins, and to protect thiols from oxidation during enzymatic digestion of protein. This method is useful for assay of thiols and disulfides of both small and large molecules and can be used to identify labile thiols in biological samples that are oxidized during extraction procedures.     

Cloning and characterization of an intracellular isoamylase gene from Pectobacterium chrysanthemi PY35

The melibiose transport carrier of Escherichia coli (coded by melB gene) is a cotransport system which couples the transport of a-galactosides to protons, sodium, or lithium ions. The charged amino acid residues in membrane-spanning helices are of considerable interest because many of them have important function in substrate recognition. In most cases changing these charged residue to an uncharged residue (cysteine) results in total loss of activity. In this communication we describe experiments in which the cysteine substitution for a charged residue was chemically changed by sulfhydryl reagents (MTSEA and MTSET to restore a positive charge and MTSES a negative charge) or by iodoacetic acid or through oxidation by hydrogen peroxide so as to regain the original negative charge. In two cases (D55C and D124C) the reconstructed negative charges via the oxidation of the thiol to the sulfinic and/or sulfonic acid resulted in partial recovery of transport: D55C up to 27% of the normal and D124C up to 4% of the normal in melibiose accumulation; D55C up to 36% of the normal and D124 up to 4.5% of the normal in downhill transport. Sulfhydryl reagents and iodoacetic acid failed to recover transport in all cases. We infer that the configurations of the charges as well as the structure of the side chains that carry them are critical in the maintenance of the transport.     

Detection of reversible protein thiol modifications in tissues

Oxidation/reduction reactions of protein thiol groups (PSH) have been implicated in many physiological and pathological processes. Although many new techniques for separation and identification of modified cysteinyl residues in proteins have been developed, critical assessment of reagents and sample processing often are overlooked. We carefully compared the effectiveness of N-ethylmaleimide (NEM), iodoacetamide (IAM), and iodoacetic acid (IAA) in alkylating protein thiols and found that NEM required less reagent (125 vs. 1000 mol:mol excess), required less time (4 min vs. 4h), and was more effective at lower pHs (4.3 vs. 8.0) in comparison with IAM and IAA. The relative efficacy of dithiothreitol (DTT) and tris(2-carboxyethyl)phosphine (TCEP) for reducing protein disulfides suspended in NaPO(4) buffer or MeOH was assessed, and no differences in total normalized fluorescence were detected at the concentrations tested (10-100mM); however, individual band resolution appeared better in samples reduced with DTT in MeOH. In addition, we found that oxidation ex vivo was minimized in tissue samples that were homogenized in aqueous buffers containing excess molar quantities of NEM compared with samples homogenized in MeOH containing NEM. Using NEM for thiol alkylation, DTT for disulfide reduction, and mBBr for labeling the reduced disulfide and fluorimetric detection, we were able to generate an in-gel standard curve and quantitate total disulfide contents within biological samples as well as to identify changes in specific protein bands by scanning densitometry. We demonstrated that reagents and techniques we have identified for disulfide detection in complex samples are also applicable to two-dimensional electrophoresis separations.     

Liquid chromatography-tandem mass spectrometry assay of reduced and oxidized glutathione and main precursors in mice liver

A liquid chromatography/tandem mass spectrometry assay of glutathione (GSH), glutathione disulfide (GSSG) and of precursors (gamma-glutamyl-cysteine, cysteinyl-glycine, cysteine, cystine, homocysteine and homocystine) was developed to study glutathione synthesis in mice liver. After iodoacetic acid derivatization, the analytes were analyzed using reversed-phase gradient HPLC and detected using multiple reaction monitoring. Linear calibrations were performed over the concentrations range of 100-10,000 ng/mL for the thiol-containing precursors and extended up to 100,000 ng/mL for GSH and GSSG. The method was validated for each compound with inter-day accuracy below 11.9% and with precision below 15%. The method showed low limits of quantitation of 100 ng/mL for each thiol-containing compound and GSSG and of 200 ng/mL for other disulfides.     

Identification and quantitation of unique fatty acid oxidation products in human atherosclerotic plaque using high-performance liquid chromatography

Oxidation of lipoproteins, particularly low-density lipoprotein, is thought to play a major role in the development of atherosclerosis. We set out to identify and quantitate the major fatty acid oxidation products in human atherosclerotic plaque obtained from individuals undergoing carotid endarterectomy. Oxidized lipids were extracted from plaque homogenate under conditions to prevent artifactual oxidation. Identification and quantitation was performed using HPLC and GC-MS. High levels of hydroxyoctadecanoic acids (0.51 +/- 0.17 ng/microg of linoleic acid), 15-hydroxyeicosatetranoic acid (HETE) (0.66 +/- 0.24 ng/microg of arachidonic acid), and 11-HETE (0.84 +/- 0.24 ng/microg of arachidonic acid) were detected in all atherosclerotic plaques (n = 10). Low levels of 9-oxo-octadecanoic acid (oxoODE) (0.04 +/- 0.01 ng/microg of linoleic acid), were present in all samples, while 13-oxoODE (0.01 +/- 0.008 ng/microg of linoleic acid) was present in only 4 of the 10 plaque samples. Of interest was the identification of two previously unidentified compounds in atherosclerotic plaque, 11-oxo-eicosatetranoic acid in 9 of the 10 samples and 5,6-dihydroxyeicosatetranoic acid in 3 samples. Chiral analysis revealed that all the major compounds identified in this study are of a nonenzymatic origin. This study is the first to provide a convenient HPLC method to quantify all the products of both linoleic acid and arachidonic acid oxidation in human atherosclerotic plaque. The quantitation of lipid peroxidation products in plaque may be important given the potential biological activity of these compounds and their possible relationship to plaque pathogenesis and instability.     

Assay of Malondialdehyde in Biological Fluids by Gas Chromatography-Mass Spectrometry

Malondialdehyde (MDA) is assayed in femtomole quantities in biological samples by gas chromatography-mass spectrometry (GC-MS). The MDA trapped in protein as a Schiff base is released by H₂SO₄, the protein precipitated using Na₂WO₄, and the MDA derivatized with pentafluorophenylhydrazine to form the stable adduct, N-pentafluorophenylpyrazole. Negative chemical ionization (NCI) capability allows the sensitive detection of this MDA adduct in biological samples at a level of 5 nM on-column. A stable-isotope-labeled MDA, [²H₂]MDA, was used as an internal standard for quantitation. MDA recovery from plasma was 76%. This assay provides two forms of confirmation of the analyte, retention time and mass ion, thus minimizing error due to interfering compounds. The commonly used thiobarbituric acid assay for MDA overestimates the MDA levels by over 10-fold, possibly resulting from cross-reactivity with other aldehydes and artifactual oxidation due to 100°C temperature conditions. In our assay, all steps were performed at room temperature thereby suppressing artifactual oxidation of the sample. We have successfully applied this assay to biological samples including plasma, tissue homogenates, and sperm.     

Determination of the in vivo redox status of cysteine, cysteinylglycine, homocysteine, and glutathione in human plasma.

An assay that measures the reduced, oxidized, and protein-bound forms of cysteine, cysteinylglycine, homocysteine, and glutathione in human plasma is described. Oxidized and protein-bound thiols are converted to their reduced counterparts by the use of NaBH4, and, following derivatization with monobromobimane (mBrB), the thiol-bimane adducts are quantified by reversed-phase ion-pair liquid chromatography and fluorescence detection. The presence of 50 microM dithioerythritol provides linearity of the standard curves at very low thiol concentrations. Selective determination of the oxidized forms was accomplished by blocking free sulfhydryl groups with N-ethylmaleimide (NEM) and excess NEM is inactivated by the subsequent addition of NaBH4. The reduced forms of the thiols in plasma were trapped with minimal oxidation by derivatizing blood samples at the time of collection. This was attained by drawing blood directly into tubes containing isotonic solutions of mBrB or NEM. The assay is sufficiently sensitive (less than 2 pmol) to detect the various forms of the four thiol compounds in human plasma. The analytical recovery of cysteine, cysteinylglycine, homocysteine, and glutathione was close to 100%, and the within-day precision corresponded to a coefficient of variation of 7, 8, 6, and 7%, respectively. The assay has been used to determine the various forms of the four thiol compounds in human plasma.     

Detection of Cystathionine Ketimine and Lanthionine Ketimine in Human Brain

The sulfur containing imino acids cystathionine ketimine (CK) and lanthionine ketimine (LK) have been detected in the human brain by an HPLC procedure. The HPLC procedure takes advantage of the selective absorbance at 380 nm of the phenylisothiocyanate-ketimine adduct. Quantitation of cystathionine ketimine and lanthionine ketimine indicates a mean concentration (mean ± SD, n = 4) of 2.3 ± 0.8 nmol/g for CK and of 1.1 ± 0.3 nmol/g for LK in four human cerebral cortex samples of neurosurgical source. The identification of these cyclic ketimine derivatives of L-cystathionine and L-lanthionine as normal human metabolites in human nervous tissue may have interesting metabolic and physiological implications.