Ion-pairing high-performance liquid chromatographic method for the detection of N-acetylaspartate and N-acetylglutamate in cerebral tissue extracts

An ion-pairing high-performance liquid chromatographic method for the determination of N-acetylaspartate and N-acetylglutamate using a C-18 column and a UV detection at 210 nm wavelength, by means of a diode array detector, is presented. A buffer containing 2.8 mM tetrabutylammonium hydroxide, 25 mM KH(2)PO(4), 1.25% methanol, pH 7. 00, is utilized for the isocratic separation of these N-acetylated amino acids, at a flow rate of 1 ml/min and a column temperature of 23 degrees C. The suitability of this chromatographic separation (without additional chromatographic steps prior to HPLC assay) to monitor variations both of N-acetylaspartate and of N-acetylglutamate in perchloric acid brain extracts from rats subjected to the impact acceleration model of diffuse brain injury is also reported. According to the data presented, this HPLC method allows the separation of the two N-acetylated amino acids considered from the many possible interfering compounds, commonly present in extracts of cerebral tissue, which have high extinction coefficients at 210 nm wavelength. Values of N-acetylaspartate and N-acetylglutamate determined by this method showed that cerebral trauma negatively affects both compounds, according to the severity of trauma itself.     

Analysis of intracellular nucleoside triphosphate levels in normal and tumor cell lines by high-performance liquid chromatography

A reversed-phase ion-pair high-performance liquid chromatographic method for the direct and simultaneous determination of ribonucleoside triphosphates and their corresponding deoxyribonucleoside triphosphates in trichloroacetic acid cell extracts is presented. Using this system, high resolution of nine acid-soluble compounds, including ADP, CTP, dCTP, GTP, UTP, dGTP, dTTP, ATP and dATP in 16 normal or tumor cell lines, is obtained. The method is based on an extraction of nucleotides from cells with a solution of 6% trichloroacetic acid followed by neutralization with the addition of 5 M K(2)CO(3) just prior to HPLC analysis. Chromatographic separations were performed using a Symmetry C(18) 3.5 micrometer (150x4.6 mm) column (Waters) equipped with a NovaPak C(18) Sentry guard column with UV detection at 254 nm. The HPLC columns were kept at 27 degrees C. The mobile phase was delivered at a flow-rate of 1.0 ml/min, with the following stepwise gradient elution program: A-B (60:40) at 0 min-->(40:60) at 30 min-->(40:60) at 60 min. Solvent A contained 10 mM tetrabutylammonium hydroxide, 10 mM KH(2)PO(4) and 0.25% MeOH, and was adjusted to pH 6.9 with 1 M HCl. Solvent B consisted of 5.6 mM tetrabutylammonium hydroxide, 50 mM KH(2)PO(4) and 30% MeOH, and was neutralized to pH 7.0 with 1 M NaOH. The calibration curves (r>0.99) of the components in cell extracts were established with their aqueous standards. The average within-day precision for the nine compounds was 0.9%, and the average day-to-day precision was 5.0%. The detection limits (pmol) of the nine reagents were 1.39 (ADP), 4.32 (CTP), 15.5 (dCTP), 2.38 (GTP), 4.42 (UTP), 9.45 (dGTP), 14.6 (dTTP), 2.44 (ATP) and 11.8 (dATP). The recovery of this method for the standards ranged from 82.4 to 120.5%. The results for the detection of nucleotide pools in 16 normal and tumor cell lines were presented. In conclusion, this simplified analytical method enables the simultaneous quantitation of NTP and dNTP in cell or tissue extracts and may represent a valuable tool for the detection of minute alterations of intracellular NTP/dNTP pools induced by anticancer/antiviral drugs and diseases.     

Extraction and measurement of myocardial nucleotides, nucleosides, and purine bases by high-performance liquid chromatography

Nucleotides, nucleosides, and purine bases were extracted from human endomyocardial biopsies, freeze-clamped rat hearts, and porcine coronary sinus plasma. Perchloric acid extracts were neutralized with Freon-trioctylamine and analyzed at 250 nm by reverse-phase ion-pairing high-performance liquid chromatography. To achieve the sensitivity necessary for analyzing small (1-3 mg wet wt) tissue samples, a small-bore, 2.1-mm-internal-diameter, C18, 5-micron reverse-phase column and a flow rate of 0.2 ml/min were used. All of the myocardial nucleotides and AMP degradation products were resolved in a total separation time of 27 min with 30 mM KH2PO4, 7.5 mM tetrabutylammonium phosphate buffers, and binary pH and acetonitrile gradients.     

Malondialdehyde production and ascorbate decrease are associated to the reperfusion of the isolated postischemic rat heart.

Isolated Langendorff-perfused rat hearts after 20 min of normoxic perfusion in the presence of 2.5 mM Ca++ and 11 mM glucose were subjected to 30 min of global normothermic ischemia followed by 30 min of normoxic reperfusion with the starting buffer. At the end of each perfusion condition, hearts were freeze-clamped and deproteinized by 0.6 M HClO4. Two-hundred microL of the neutralized tissue extracts were analyzed by a recently developed high-performance liquid chromatography (HPLC) method for the simultaneous determination of malondialdehyde (MDA), ascorbic acid, and adenine nucleotides. By means of this analytical technique, it was possible to demonstrate that MDA is undetectable in control hearts. In contrast, 30 min of ischemia induced a modest production of MDA (0.012 mumol/g dw), while a large amount of MDA (0.103 mumol/g dw) was observed in reperfused hearts. Values referring to ascorbic acid showed that the concentration of this antioxidant progressively decreased from 1.190 (control hearts) to 0.837 (ischemic hearts) and to 0.595 mumol/g dw (reperfused hearts). The overall conclusions of this study are that reperfusion induces an oxidative stress to the isolated myocardium, a decrease of ascorbate, and an increase of lipid peroxidation. Therefore, by means of a proper analytical method, MDA may represent a valid biochemical parameter to demonstrate the relationship between myocardial reperfusion and a detectable tissue damage.     

Thin-layer chromatographic separation of intermediates of the pyridine nucleotide cycle

A rapid thin-layer chromatographic procedure for separation of the compounds comprising the intermediates in the salvage pathway known as the pyridine nucleotide cycle plus quinolinic acid and the reduced forms of nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate is described. The method utilizes silica gel high-performance thin-layer plates and a mobile phase of methanol, tetrabutylammonium hydroxide, and acetonitrile. The time required for analysis is greatly reduced and results in greater than 96% purity of each migrating compound.     

Flocculation and adsorption of enzymes during growth of a moderate halophile, Micrococcus varians var. Halophilus.

Flocculation of a moderate halophile, Micrococcus varians ATCC 2197, occurred during growth in complex medium containing 3 M NaCl and a concentration of MgSO4 and KH2PO4 greater than 40 and 14 mM, respectively. Extracellular nuclease activity was absent in the flocculated cultures. Repeated washing of flocs by Mg2+-free Tris buffer containing 3 M NaCl, lowering of pH value of floc suspension below 6.3, or addition of ethylenediaminetetraacetic acid resulted in complete dissociation of the flocs and release of Mg2+ ions as well as nuclease and amylase. Inhibition of extracellular enzyme production accompanied by flocculation appeared to be the result of adsorption of enzyme proteins to surfaces of the flocs, but not of inhibition of biosynthesis. Floc formation could also occur in media containing 18 mM CaCl2 and 3.0 mM KH2PO4, but the Ca flocs were not deflocculated by washing with Ca2+-free buffer, suggesting that the affinity of Ca2+ for cell envelopes was stronger than that of Mg2+. It was also observed that most halophilic Planococcus and Micrococcus flocculated in the presence of MgSO4 and phosphate but halophilic Pseudomonas, Acinetobacter, and Bacillus did not.     

High-performance liquid chromatographic determination of 6-aminopenicillanic acid by postcolumn alkaline degradation

A high-performance liquid chromatographic method has been developed for the determination of 6-aminopenicillanic acid in amino acid mixtures and human serum. The separation of 6-aminopenicillanic acid was carried out on a C18 column using sodium heptylsulfonate or tetrabutylammonium bromide as an ion-pairing agent and methanol as an organic mobile phase modifier. Detection was based on a postcolumn reaction with sodium hydroxide, mercury(II) chloride, and ethylenediaminetetraacetic acid disodium salt followed by measurement of ultraviolet absorbance (at 300 nm) of the reaction product(s). The method is quantitative for 6-aminopenicillanic acid concentrations down to 0.1 microgram/ml in human serum samples with a 20-microliter injection. At a concentration of 2 micrograms/ml, the within- and between-run precisions (relative standard deviation) were 1.29-3.91% and 2.30%, respectively.     

Hormonal regulation of mitochondrial function. Description of a system capable of mimicking several effects of glucagon

Isolated rat liver mitochondria were incubated at 0 degrees C in a medium consisting of 225 mM sucrose, 10 mM KCl, 1 mM EDTA, 10 mM KH2PO4, 5 mM MgCl2 and 10 mM Tris-HCl, pH 7.4 (buffer 1) for 10 min, centrifuged and resuspended in 0.3 M sucrose. This treatment resulted in a stimulation of mitochondrial functions, mimicking several of the effects that follow glucagon treatment of the intact rat or isolated hepatocytes. Both phosphate and potassium are required for this effect; the addition of magnesium serves to enhance it. Mitochondrial respiration is essential for the development of the activated state as the stimulation is blocked by increasing concentrations of rotenone in the incubation. The intramitochondrial ATP/ADP ratio is increased, but when this increase was prevented by including low levels of rotenone or oligomycin in buffer 1, the stimulation of mitochondrial function was not diminished, thus demonstrating that an increased ATP/ADP ratio is not essential for activation. The rate of citrulline formation was unaffected by buffer 1 treatment unless glutamate was also included in the medium, indicating that control of this mitochondrial function differs from other functions studied.     

Fluorometric determination of 2-oxoadipic acid, a common metabolite of tryptophan and lysine, by high-performance liquid chromatography with pre-chemical derivatization

2-Oxoadipic acid, a key metabolite of tryptophan and lysine, reacted with 1,2-diamino-4,5-methylenebenzene in an acidic solution to produce a fluorescent derivative. The reaction product was separated using a Tosoh ODS-80Ts column with 20 mmol/L of KH?PO?-K?HPO? buffer (pH 7.0) containing 26% methanol at a flow rate 0.8 mL/min. The excitation wavelength of detection was 367 nm, and the emission wavelength was 446 nm. The limit of quantification was 1 pmol per injection, sufficiently sensitive for the determination of 2-oxoadipic acid in human and experimental animal urine.     

An improved method for tissue long-chain acyl-CoA extraction and analysis

We report an extensively modified method for the extraction, solid-phase purification, and HPLC analysis of long-chain acyl-CoAs from tissues. Tissue samples were homogenized in a glass homogenizer in KH2PO4 buffer (100 mM, pH 4.9) and again after the addition of 2-propanol. Acyl-CoAs were then extracted from the homogenate with acetonitrile (ACN). The acyl-CoAs in the extract were bound to an oligonucleotide purification column and eluted using 2-propanol. This eluent was concentrated and then loaded onto a C-18 column and eluted using a binary gradient system in which solvent A was KH2PO4 (75 mM, pH 4.9) and solvent B was ACN containing 600 mM glacial acetic acid. Initial flow rate was 0.5 or 0.25 ml/min depending upon the tissue used. The HPLC eluent was monitoring at 260 nm. Our modifications increased the recovery of the extraction procedure to 70-80%, depending upon tissue, with high reproducibility and significantly improved separation of the most common unsaturated and saturated acyl-CoAs. We also report, for the first time, the mass (nanomoles per gram wet weight) of the most common polyunsaturated acyl-CoAs in rat heart, kidney, and muscle tissues. The modifications and high recovery permit the use of tissue samples of less than 100 mg, making this method useful for the analysis of small tissue amounts associated with mice.     

Behavior of the enzymes of the salvage pathway of purine bases in leukemia lymphocytes

The Authors present a procedure for the determination of adenine phosphoribosyltransferase (APRT) and hypoxanthine phosphoribosyltransferase (HPRT) in lymphocytes which exhibits high sensitivity and requires low quantities of lymphocytes. 5 normal subjects and 4 patients affected by chronic lymphocytic leukemia (CLL) were considered. Human lymphocytes were prepared and treated as previously reported. To the incubation mixtures buffered with 50 mM TRIS-HCl pH 7.4 either 14C-adenine or 14C-hypoxanthine was added: after deproteinization and neutralization we followed the formation of either 14C-adenylic acid (AMP) or 14C-inosinic acid (IMP) by HPLC. A Supelcosil C18 5 microns (250 X 4.5 mm) column was used: IMP was eluted with 20 mM KH2PO4 pH 5.5 while AMP with a linear gradient to 40% B in 20 min., where A was 20 mM KH2PO4 pH 5.5 and B methanol/water 60:40. Evaluation of AMP and IMP formed was carried out by determination of the radioactivity of the collected peaks. The values of APRT in leukemic patients were enhanced when referred to the proteins and those of HGPRT decreased: the Authors propose to complete the study evaluating the intracellular content of adenine and hypoxanthine.     

Myocardial triglyceride turnover during reperfusion of isolated rat hearts subjected to a transient period of global ischemia.

Triglyceride turnover in reperfused/ischemic rat hearts was investigated. Hearts were initially perfused under aerobic conditions for a 1-h "pulse" perfusion with 1.2 mM [1-14C]palmitate to label the endogenous lipid pools, followed by a 30-min period of no-flow ischemia or a 10-min period of retrograde perfusion (control). Hearts were then reperfused under aerobic conditions with buffer containing 1.2 mM [9,10-3H]palmitate. All buffers contained 11 mM glucose and 500 microunits/ml insulin. Rates of endogenous triglyceride lipolysis and synthesis were measured during reperfusion, whereas rates of exogenous palmitate oxidation were measured both prior to ischemia and during reperfusion following ischemia. During reperfusion of ischemic hearts, a 20% increase in exogenous fatty acid oxidation rates was seen compared with pre-ischemic rates. Despite an initial burst of endogenous fatty acid oxidation, no acceleration of steady state endogenous triglyceride lipolysis was seen compared with their nonischemic hearts. In contrast, a significant increase in triglyceride synthesis was observed. Triglyceride turnover was also measured in a series of hearts reperfused following ischemia in the absence of exogenous fatty acids. A significant enhancement of functional recovery was seen compared with hearts reperfused with 1.2 mM palmitate. In addition, a significant increase in fatty acid oxidation from endogenous triglyceride lipolysis was observed. We conclude that the heart quickly recovers its ability to oxidize exogenous fatty acids during reperfusion and that although triglyceride lipolysis is not accelerated during reperfusion of ischemic hearts in the presence of 1.2 mM palmitate, a significant increase in triglyceride synthesis does occur.     

Simultaneous determination of two antioxidants, uric and ascorbic acid, in animal tissue by high-performance liquid chromatography.

A rapid and simple method for the simultaneous analysis of uric and ascorbic acid in extracts of animal tissue is described. The method uses reversed-phase ion-pair chromatography with ultraviolet detection. The technique allows efficient separation of both acids while showing high selectivity, recovery, reproducibility, and sample stability. Calculated levels of both substances in mouse liver tissue were 1.00 +/- 0.05 mumol ascorbic acid/g and 130 +/- 5 nmol uric acid/g.     

Simultaneous analysis of dextromethorphan and its three metabolites in human plasma using an improved HPLC method with fluorometric detection

A simple and improved HPLC method with fluorometric detection for simultaneous determination of dextromethorphan (DM) and its three metabolites (dextrorphan (DX), 3-methoxymorphinan (MM), 3-hydroxymorphinan (HM)) in human plasma was developed and validated. The method involved a simple and efficient extraction protocol using an n-heptane/ethyl acetate (1:1) solvent mixture that achieved recoveries of 70-90% with an insignificant interference from the plasma matrix. The analysis was performed on a phenyl column with isocratic elution, a mobile phase composed of 20% methanol, 30% acetonitrile, and 50% KH2PO4 buffer (10mM, with adding 0.02% of TEA; adjusted with phosphoric acid to pH 3.5), and a run time of only 15 min. Linear calibration curves were constructed in the concentration range of 1-200 nM for DM and its three metabolites. The lower limit of quantitation (LLOQ) in human plasma was 1 nM for each compound. The coefficient of variation and RSE% of the intraday and interday analyses for DM and its three metabolites all complied with USFDA requirements. This analytical method was preliminarily applied to determine the polymorphic functions of CYP2D6 and CYP3A4 in the metabolic pathway of DM to DX and then to HM.     

High-performance liquid chromatographic separation of hyaluronan and four proteoglycans produced by human bone cell cultures

Four proteoglycans and hyaluronan synthesized by cultured human bone cells were isolated using a two-step high-performance liquid chromatography system involving desalting and buffer exchange with a TSK-GEL HW 40(S) column followed by ion-exchange separation on a Nucleogen 4000-10 DEAE column. The desalting of 4 M guanidinium HCl extracts by a TSK-GEL HW 40(S) column equilibrated in a formamide:KH2PO4 buffer produces greater than 95% recoveries, enables quantitation of label incorporation and requires only 40 min to complete. The Nucleogen 4000-10 DEAE column utilizes the same buffer system and requires only 100 min for the resolution of four distinct types of proteoglycans. The formamide:KH2PO4 buffer system is compatible with a previously developed polyacrylamide gel system for the electrophoretic profiling of proteoglycans. After separation by charge density, proteoglycans were further resolved by size distribution using a calibrated TSK-GEL HW 75(F) column which also enabled the estimation of the apparent Mr of hyaluronan produced by the bone cells. The same TSK-GEL HW 40(S) resin is used to exchange pooled proteoglycans into buffers for analyzing enzyme digests of glycosaminoglycan chains and core proteins. The technique has been applied to the analysis of biosynthetically labeled proteoglycans produced in culture by fetal and adult human bone cells. A distinct pattern of proteoglycan size and secretion for both cell types could be shown using this method. The method of analysis is useful for high yield and rapid screening of various cell types for both biosynthetic rate studies and analysis of patterns of proteoglycan synthesis.     

Analysis of free amino acids in mammalian brain extracts

An optimized method for analysis of free amino acids using a modified lithium-citrate buffer system with a Hitachi L-8800 amino acid analyzer is described. It demonstrates clear advantages over the sodium-citrate buffer system commonly used for the analysis of protein hydrolysates. A sample pretreatment technique for amino acid analysis of brain extracts is also discussed. The focus has been placed on the possibility of quantitative determination of the reduced form of glutathione (GSH) with simultaneous analysis of all other amino acids in brain extracts. The method was validated and calibration coefficient (K _GSH) was determined. Examples of chromatographic separation of free amino acids in extracts derived from different parts of the brain are presented.     

Inhibition of ascorbate autoxidation by a dialyzed,heat-denatured extract of plant tissues

Preparations obtained from various plant sources were analyzed for their effect on the autoxidation of ascorbic acid and norepinephrine. The former reaction was followed by spectro-photometric detection of ascorbic acid at 265 nm, the latter one by measuring the formation of noradrenochrome at 480 nm. Extracts were prepared from $\text{Philodendron}$ leaves and the edible portion and seeds of green peppers ($\text{Capsicum Annuum}$). The tissues were minced, homogenized in 10 volumes of 16 mM Na-phosphate buffer pH 7.4 and centrifuged at 35,000g for 30 min. The supernatant was dialyzed in 12,000 m.w. cut-off tubing, denatured in boiling water and centrifuged at 10,000g for 10min. Aliquots (5–50 ul) of the supernatant were assayed in 5 ml 16 mM Na-phosphate buffer pH 7.4 containing 100 uM ascorbate or norepinephrine. The denatured extracts had marked dose-dependent inhibitory effect on the autoxidation of ascorbic acid, with negligible influence on the formation of noradrenochrome. EDTA inhibited both reactions. The selectiveness of the extract toward the autoxidation of ascorbic acid makes it unlikely that the inhibitory effect is based on sequestering metal-ions.     

Ion-pair reverse-phase high-performance liquid chromatography. Application to the study of chicken liver NAD+ kinase

An ion-pair, reverse-phase, high-performance liquid chromatography method of assay was developed and used in a series of rate studies carried out with the enzyme chicken liver NAD+ kinase (ATP:NAD+ 2'-phosphotransferase, EC 2.7.1.23). Complete separation of all products and reactants was achieved within 15 min. ATP, NAD+, ADP, and NADP+ were monitored at 260 nm as they eluted from a Zorbax (Dupont) ODS (4.6 X 250-mm) column using an acetonitrile and 0.01 mM NH4(H2PO4)/0.005 M tetrabutylammonium phosphate (pH 7.0) gradient. The enzyme shows a marked preference for ATP (and dATP) and Mg2+ (or Mn2+) relative to other trinucleotides and divalent metal ions. It exhibits residual adenylate kinase and ATPase activity, but no NADH kinase activity. When polyphosphate replaced ATP, NADP+ production dropped to 2.5%. The addition of Ca2+ and/or bovine brain calmodulin did not significantly enhance the rate of NADP+ production.     

Single-sample preparation for simultaneous cellular redox and energy state determination

A simple and reliable method for the preparation of biological samples for the evaluation of biochemical parameters representative of the redox and energy states, such as glutathione (GSH), oxidized glutathione (GSSG), oxidized nicotinamide adenine dinucleotide (NAD+), reduced nicotinamide adenine dinucleotide (NADH), oxidized nicotinamide adenine dinucleotide phosphate (NADP+), reduced nicotinamide adenine dinucleotide phosphate (NADPH), coenzyme A (CoASH), oxidized CoASH, ascorbate, malondialdehyde, oxypurines, nucleosides, and energy metabolites, is presented. Fast deproteinization under nonoxidizing conditions is obtained by tissue homogenization in ice-cold, nitrogen-saturated CH3CN + 10 mM KH2PO4 (3:1; v:v), pH 7.40. After sample centrifugation to pellet precipitated proteins, organic solvent removal is performed on clear supernatants by three washings with large volumes of high-performance liquid chromatography (HPLC)-grade chloroform. The remaining aqueous phase, free of solvent and any lipid-soluble substances that may interfere with the further metabolite analysis, is used for the simultaneous ion-pairing HPLC determination of 39 compounds by means of a Kromasil C-18, 250 x 4.6-mm, 5-microm-particle-size column with tetrabutylammonium hydroxide as the pairing reagent. Results obtained by using the present method to prepare different rat tissue extracts demonstrate that it is possible to perform a single tissue preparation only for monitoring, in the same sample, compounds representative of the redox state (through the direct determination of GSH, GSSG, NAD+, NADH, NADP+, NADPH, CoASH, and oxidized CoASH) and of the cell energy state (by the analysis of oxypurines, nucleosides, and energy metabolites). Applicability of this sample processing procedure to quantify variations of the aforementioned compounds under pathological conditions was effected in rats subjected to moderate closed-head trauma.     

The relative contribution of glucose and fatty acids to ATP production in hearts reperfused following ischemia

High levels of fatty acids decrease the extent of mechanical recovery of hearts reperfused following a transient period of severe ischemia. Glucose oxidation rates during reperfusion are low under these conditions, which can result in a decreased recovery of mechanical function. Stimulation of glucose oxidation with the carnitine palmitoyl transferase I inhibitor, Etomoxir, or by directly stimulating pyruvate dehydrogenase activity with dichloroacetate (DCA) results in an improvement in mechanical function during reperfusion of previously ischemic hearts. Addition of DCA (1 mM) to hearts perfused with 11 mM glucose and 1.2 mM palmitate results in an increase in contribution of glucose oxidation to overall ATP production from 6 to 23%, with a parallel decrease in that of fatty acid oxidation from 90 to 69%. In aerobic hearts, endogenous myocardial triglycerides are an important source of fatty acids for -oxidation. Using hearts in which the myocardial triglycerides were pre-labeled, the contribution of both endogenous and exogenous fatty acid oxidation to myocardial ATP production was determined in hearts perfused with 11 mM glucose, 1.2 mM palmitate and 500 µU/ml insulin. In hearts reperfused following a 30 min period of global no flow ischemia, 91.9% of ATP production was derived from endogenous and exogenous fatty acid oxidation, compared to 87.7% in aerobic hearts. This demonstrates that fatty acid oxidation quickly recovers following a transient period of severe ischemia. Furthermore, therapy aimed at overcoming fatty acid inhibition of glucose oxidation during reperfusion of ischemic hearts appears to be beneficial to recovery of mechanical function.