High-performance liquid chromatographic determination of the imino acids (opines) meso-alanopine and d-strombine in muscle extract of invertebrates

A sensitive HPLC method is presented for the determination of the imino acids alanopine and strombine, anaerobic metabolites that are formed in muscle tissue of several species of invertebrates. The separation of alanopine and strombine was achieved using the Alltech OA 2000 cation-exchange column. The analysis of the two opines does not require any complicated derivatization and can be performed in a pH neutralized sulphuric acid solution. The sensitivity of this method is in the range of 100 pmol at least 10 nmol for both investigated opines. For the first time opines were demonstrated in the bivalves Macoma balthica and Cerastoderma edule.     

Gas-liquid chromatography and enzymatic determination of alanopine and strombine in tissues of marine invertebrates

Gas-liquid chromatography (GLC) and enzymatic assays were developed for quantitating the imino acids, alanopine and strombine, alternate products of anaerobic glycolysis (replacing lactate) in the tissues of many marine invertebrates. For GLC analysis, d-strombine (2-methyliminodiacetic acid) and meso-alanopine (2,2′-iminodipropionic acid) were chromatographeo as N-trifluoroacetyl isobutyl esters. Modifications of techniques used for GLC analysis of amino acids were required to overcome steric hindrance in the acylation reaction caused by the presence of imino, rather than amino, groups. Both imino acids were separated from each other and from all amino acids by GLC. Detection limit of the technique was 0.05 μg imino acid. Enzymatic determination of imino acids made use of the alanopine-specific alanopine dehydrogenase (ADH) purified from the periwinkle, Littorina littorea, and the strombine/alanopine utilizing strombine dehydrogenase (SDH) from the clam, Mercenaria mercenaria, with assay conditions: 300 mm hydrazine buffer, pH 9.0, 5 mm NAD, and 0.3 unit ADH or 1.0 unit SDH. Enzymatic determinations of mixtures of alanopine and strombine in tissue samples required a dual analysis using both enzymes. Production of alanopine and strombine during anoxic stress in two species of marine molluscs was quantitated.     

Quantitative HPLC analysis of acidic opines by phenylthiocarbamyl derivatization

A new method for the quantitative analysis of acidic opines--alanopine, strombine, tauropine, and beta-alanopine--is presented. The method is based on formation of phenylthiocarbamyl (PTC) derivatives of the acidic opines after partial purification by ion-exchange chromatography. The PTC acidic opines are separated by reverse-phase high-performance liquid chromatography (HPLC) and detected within 20 min by ultraviolet absorbance. This HPLC method gives higher sensitivity, 10-30 pmol minimum detection, and better reproducibility than the high-voltage paper electrophoresis method. There is also good agreement for the three acidic opines (alanopine, strombine, and tauropine) when compared by HPLC and electrophoresis methods. Accumulation of beta-alanopine was observed for the first time in the adductor muscle of blood shell, Scapharca broughtonii, during aerial exposure by application of the HPLC detection method.     

Pyruvate reductases catalyze the formation of lactate and opines in anaerobic invertebrates

The recent discovery of several enzymes, other than lactate dehydrogenase, with pyruvate reductase activity together with studies on the formation of end products of glycolysis during environmental and functional anaerobiosis have made it clear that anaerobic glycolysis in invertebrates is more important than previously thought. The presence of pyruvate reductase activity guarantees the continuous flux of glycolysis and, consequently, a constant supply of ATP by maintaining a low NADH/NAD⁺ ratio during exercise and hypoxia as well as in the subsequent recovery period. This review summarizes distribution, physicochemical, catalytic and regulative parameters of lactate-, octopine-, strombine- and alanopine dehydrogenase. In the second part, details are given on the formation of the end products lactate, octopine, strombine and alanopine as well as an evaluation of the biological role of the pyruvate reductases.     

Anaerobiosis and acid-base status in marine invertebrates: a theoretical analysis of proton generation by anaerobic metabolism

Summary In animals, various organic acids are accumulated during hypoxia or anoxia as products of anaerobic energy metabolism. The diversity of such acids is largest in marine invertebrates where succinate, propionate, acetate, lactate, alanine, octopine, strombine, and alanopine, are produced mainly from glycogen and aspartate. The effect of these substances on the acid-base status was assessed by a theoretical analysis of the respective metabolic pathways. This resulted in a general rule which was applied to evaluate the proton balance of the reactions in energy metabolism: net changes in the number of carboxyl groups or changes in the degree of dissociation of other groups (e.g. phosphate or ammonia) determine the net amount of H⁺ ions released or bound by the substrates and the metabolic end products.For marine invertebrates the results of the analysis can be summarized as follows: In glycogenolysis one mol of protons per mol of end products is released during cytosolic glycolysis, independent of the type of metabolic end product (lactate, octopine, alanopine, strombine, or alanine). The same applies for mitochondrial production of propionate and acetate, whereas formation of succinate results in dissociation of two mol H⁺ per mol. Fermentation of aspartate, however, diminishes the amount of protons which is produced in the succinate-propionate pathway. Net metabolisation of Mg ATP^2– yields extra protons, whereas the cleavage of phosphagens (e.g. creatine phosphate, arginine phosphate) consumes protons.Additionally the break-down of energy-rich phosphates to inorganic phosphate has to be taken into account because of the shift of the intracellular buffer curve caused by changes of the respective effective pK values.     

The anaerobic molluscan heart: adaptation to environmental anoxia. Comparison with energy metabolism in vertebrate hearts

The hearts of many bivalve and gastropod molluscs are resistant to exposure to hypoxic and anoxic conditions. Glycogen and aspartate are simultaneously fermented leading to the accumulation of alanine, succinate and alanopine/strombine. Lactate is not a major end product of anaerobic metabolism in molluscan hearts. In contrast, vertebrate hearts respond to hypoxia by the fermentation of glycogen leading to lactate formation. There is some evidence for aspartate and glutamate breakdown in vertebrate hearts during anoxia. However, the quantitative contribution of this process to energy production is small. The differences in modes of energy production in molluscan and vertebrate hearts may reflect adaptations to long-term as opposed to short-term anoxia.     

Multiple forms of octopine dehydrogenase in Strombus luhuanus (Mollusca,Gastropoda, Strombidae): Genetic basis of polymorphism,properties of the enzymes,and relationship between the octopine dehydrogenase phenotype and the accumulation of anaerobic end products during exercise

Octopine dehydrogenase (ODH) is electrophoretically polymorphic in the gastropod mollusk Strombus luhuanus. The frequencies of the six electrophoretic phenotypes in the Heron Island population, together with the molecular weight values of 38,000 obtained for each of the three forms of the enzyme, demonstrate that the monomeric enzyme is encoded by three codominant alleles at a single locus. The purified allozymes are indistinguishable in terms of K _m values for substrates, product inhibition by octopine and NAD, pH optima, and substrate inhibition by pyruvate. No statistically significant correlations were found between the ODH phenotype and the maximum activities of ODH or alanopine dehydrogenase, the capacity for anaerobic muscle work, or the accumulation of octopine or strombine/alanopine during exercise. It would appear that the ODH allozymes may be functionally equivalent both in vitro and in vivo.     

Ammonia assimilating enzymes from cyanobacteria: in situ and in vitro assay using high-performance liquid chromatography

Assay systems for ammonia assimilating enzymes in cyanobacteria are reported. Glutamine synthetase, glutamate synthase, and glutamate dehydrogenase can be easily assayed in situ, after the cells are made permeable to the reagents, or in vitro. The method is based upon the quantitation of glutamine or glutamate after the separation, when needed, of their o-phthaldialdehyde derivatives by reverse-phase high-performance liquid chromatography on a C18 column. The isocratic elution and the fluorometric detection of the amino acid derivatives make the method fast, simple, sensitive, and free of the assay artifacts which can be produced in coupled assays or when spectrophotometric measurements are carried out in the turbid samples employed for in situ assays.     

Assay for aliphatic amino acid decarboxylases by high-performance liquid chromatography

A sensitive and rapid assay for aliphatic amino acid decarboxylases based on separation of the product from the substrate by ion-pairing reversed-phase high-performance liquid chromatography and subsequent fluorometric detection has been developed. The resolution of substrates and products of seven amino acid decarboxylases, namely, arginine, aspartate, 2,6-diaminopimelate, histidine, glutamate, lysine, and ornithine decarboxylase, is complete within 15 to 35 min of isocratic elution. The limit of detection for the product is 40 pmol. The applicability of the procedure was assessed with glutamate decarboxylase. The formation of the product 4-aminobutyrate proved to be linear with time and protein concentration. The method allows the time course of the reaction to be followed in a single assay and works well with crude extracts of bacteria or tissues.     

Quantitative liquid chromatographic determination of sanguinarine in cell culture medium and in rat urine and plasma

Sanguinarine is a quaternary benzo[c]phenanthridine alkaloid, extracted from the argemone oil, which produced severe human intoxications. To investigate the sanguinarine biotransformation, we develop a simple extraction process and a high performance liquid chromatographic separation coupled to a sensitive fluorometric detection of sanguinarine in cell culture medium, as well as in rat urine and plasma. After extraction with an acidified organic solvent, sanguinarine elution is performed within 15 min on a Nucleosil C18 column with a gradient using 0.2% formic acid/water/acetonitrile as mobile phase. Extracted and standard sanguinarine are characterized by mass spectrometry. The extraction recovery of sanguinarine is about 80% in cell culture medium and in rat urine, but lower in plasma. This convenient high performance liquid chromatography (HPLC) method allows to quantify sanguinarine over concentrations ranged 10-2000 ng ml(-1). The limit of fluorometric detection is 0.5 ng. Under these conditions, the lower limit of quantification of sanguinarine is 50 ng ml(-1) in cell culture medium and in rat urine and 100 ng ml(-1) in rat plasma. This analytical HPLC method is specific, linear and reproducible in all media and is suitable for quantitative determination of sanguinarine in biological fluids.     

A fluorometric assay for peptidyl alpha-amidation activity using high-performance liquid chromatography

A rapid and sensitive method for the determination of peptidyl alpha-amidation activity has been developed and is based on reverse-phase high-performance liquid chromatographic separation and fluorometric detection. A dansylated tripeptide, N-dansyl-Tyr-Val-Gly-OH, is used as the substrate in the assay and the amount of alpha-amidation activity is determined by quantitating the extent of its conversion to product, N-dansyl-Tyr-Val-NH2. Both product and substrate can be detected in a single assay in quantities as low as 5 fmol by isocratic elution using C-18 reverse-phase columns. The method yields highly reproducible results and requires less than 3 min per sample for separation and quantitation. The assay procedure is applicable to the screening of a large number of samples under different pH conditions and is readily adaptable for use in a variety of studies. For example, the procedure is ideal for detecting alpha-amidation activity in various tissues, monitoring activity at the different stages during purification of a particular alpha-amidation enzyme, determining kinetic parameters of the purified enzyme, and identifying both competitive and noncompetitive inhibitors.     

Measurement of DNA single-strand breaks by alkaline elution and fluorometric DNA quantification

The method presented is based on the alkaline elution procedure for the determination of DNA single-stand (ss) breaks developed by Kohn and on the principles of DNA quantification after binding with the dye Hoechst 33258. In the present study, modification of the alkaline elution procedure with regard to the elution solution volume was performed. The influences of the DNA strandedness, the ethylenediaminetetraacetate/tetraethylammonium hydroxide denaturation and elution solution presence, the DNA solution pH, the dye amount, and the incubation time for the formation of the dye-ssDNA complex on the DNA fluorometric quantification were also studied. The modified DNA alkaline elution procedure followed by the optimized fluorometric determination of the ssDNA was applied on liver tissue from both untreated and treated (N-nitroso-N-methylurea- administered) Wistar rats. The criteria for the selection of the appropriate estimator and statistical analysis of the obtained results are also presented. The method of the DNA alkaline elution followed by fluorometric determination of ssDNA as modified and evaluated is an accurate and reliable approach for the determination of in vivo induced ssDNA strand breaks.     

Anaerobiosis and metabolic plasticity of Pinna nobilis: Biochemical and ecological features

Changes in the energetic metabolism were studied in the fan mussel Pinna nobilis L. exposed to environmental and anthropic stress. The high polymorphism of enzymes suggests an adaptation of the fan mussel to environmental variability peculiar of transitional waters with respect to the same species living in exposed coastal sea. The electrophoretic patterns showed a predominance of LDH-A4 and the presence of both mitochondrial and cytosolic MDH isozymes. Moreover, in all the analyzed tissues and organs, MDH activity was greater than the LDH one. Metabolic plasticity of the fan mussel is further highlighted by octopine dehydrogenase and superoxide dismutase electrophoretic patterns, showing the presence of many isoforms. These evidences are also confirmed by spectroscopic determinations of alanopine, tauropine, strombine and octopine dehydrogenase activity characterized by a specific trend due to environmental variability. Specific variations in anaerobic capacity of P. nobilis L. are discussed in relation to their distribution according to the marine-brackish gradient.     

Determination of meso-alanopine and D-strombine by high pressure liquid chromatography in extracts from marine invertebrates

meso-Alanopine and D-strombine are separated by high pressure liquid chromatography using a cation exchange resin and 2.5 X 10(-5) M sulfuric acid as eluant, at a flow rate of 1.0 ml/min, 20 degrees C column temperature and a pressure of 4 500 kPa. Both opines were detected by conductivity. Separation and quantitation was possible in the range of 0.05 to 25 nmol of meso-alanopine and D-strombine. Chemically or enzymatically synthesized opines were quantitated using alanopine/strombine dehydrogenase from Crassostrea angulata. The enzyme was purified by ammonium sulfate precipitation, Sephadex G-100 filtration and fast-protein-liquid chromatography. Specific activity of the final preparation was 500 U/mg protein with glycine as substrate. The formation of meso-alanopine and D-strombine was demonstrated in neutralized perchloric acid extracts from muscle tissue of Arenicola marina L. following enhanced muscular activity and in Mytilus edulis L., Nucula nitida and Crassostrea angulata after 24 h of anoxia.     

Enzymatic fluorometric assay for myo-inositol trisphosphate

The determination of myo-inositol trisphosphate by an enzymatic fluorometric assay is presented. The method involves the acid extraction of water-soluble inositol polyphosphates followed by separation by anion-exchange chromatography. Samples are subsequently neutralized by passage over a Dowex Cl- resin and elution with lithium chloride. Samples are then desalted with ethanol. Following dephosphorylation with alkaline phosphatase, free myo-inositol is measured enzymatically via the NAD-dependent oxidation to scyllo-inosose with myo-inositol dehydrogenase. The efficiency of recovery, assay specificity, and an application to the measurement of inositol polyphosphates in hormone-stimulated tissue are discussed.     

Identification of octopine dehydrogenase from Mytilus galloprovincialis

A cDNA encoding the putative octopine dehydrogenase (OcDH) from the mussel Mytilus galloprovincialis was cloned and sequenced. The complete coding region was expressed in the bacteria Escherichia coli and the recombinant protein was purified. The M. galloprovincialis OcDH appears to have the highest affinity for the amino acid substrate L-arginine (88.22%), compared to L-alanine (9.04%) and glycine (2.74%). This enzyme showed no activity when taurine or β-alanine was used as substrate. These data strongly support that this recombinant enzyme is octopine dehydrogenase and not another opine dehydrogenase such as alanopine or strombine dehydrogenases. The superimposition of the theoretical three-dimensional model of the M. galloprovincialis OcDH and the crystal structure of its homologous counterpart from the great scallop Pecten maximus showed interesting changes in the amino acid binding site which could explain the differences found in the substrate affinity between the two molluscs. A phylogenetic analysis was performed comparing M. galloprovincialis OcDH and annotated sequences representing the five opine dehydrogenase (OpDH) protein family members. The phylogenetic tree which was obtained clustered the OpDH enzymes according to the evolutionary relationships of the species and not to the biochemical reaction catalysed. Octopine dehydrogenase has been identified in the Mytilidae family for the first time, having previously only been established in one other marine invertebrate (P. maximus).     

Purification, characterization, and cDNA cloning of opine dehydrogenases from the polychaete rockworm Marphysa sanguinea

Alanopine dehydrogenase (AlDH) and three isoforms of strombine/alanopine dehydrogenase (St/AlDH) were purified from muscle tissue of the polychaete rockworm Marphysa sanguinea. The four enzymes, which can be distinguished by the isoelectric point, are monomeric 42 kDa proteins, possess similar pH-activity profiles, and display specificity for pyruvate and NAD(H). The three isoforms of St/AlDH show equivalent Km and Vmax for glycine and L-alanine and for D-strombine and meso-alanopine. Free amino acid levels in the muscle and D-strombine accumulation in vivo during muscle activity suggest that St/AlDHs function physiologically as StDH. AlDH shows specificity for L-alanine and meso-alanopine, but not for glycine or D-strombine. The amino acid sequences of AlDH and one of the St/AlDH isoforms were determined by a combination of amino acid sequence analysis and cDNA cloning. St/AlDH cDNA consisted of 1586 bp nucleotides that encode a 399-residue protein (43,346.70 Da), and AlDH cDNA consisted of 1587 bp nucleotides that encode a 399-residue protein (43,886.68 Da). The two amino acid sequences deduced from the cDNA displayed 67% amino acid identity, with greatest similarity to that of tauropine dehydrogenase from the polychaete Arabella iricolor.     

High-performance liquid chromatographic determination of 1,2,3,4-tetrahydroisoquinoline in rat brain with fluorescence detection

A high-performance liquid chromatographic method for the fluorometric determination of 1,2,3,4-tetrahydroisoquinoline in rat brain is described. 1,2,3,4-Tetrahydroisoquinoline and 4-phenylpiperidine (internal standard) are isolated by liquid-liquid extraction, and then converted into the corresponding fluorescent derivatives with 3,4-dihydro-6,7-dimethoxy-4-methyl-3-oxoquinoxaline-2-carbonyl chloride, a fluorescence derivatization reagent for amines. The derivatives are separated within 60 min on a reversed-phase column, TSK gel ODS-120T, with isocratic elution, and detected fluorometrically. The detection limit of 1,2,3,4-tetrahydroisoquinoline is 1.0 pmol/g in rat brain (S/N = 3).     

Simultaneous analyses of phenethylamine, phenylethanolamine, tyramine and octopamine in rat brain using fluorescamine

A fluorometric method for the simultaneous analyses of phenethylamine, phenylethanolamine, tyramine and octopamine has been developed. The method involves ion-exchange chromatography, derivatization with fluorescamine, solvent extraction and then separation by thin-layer chromatography. The fluorescent spots are then quantitated by scanning. The detection limits of this method are about 10 pmoles for phenethylamine, phenylethanolamine and tyramine, and 20 pmoles for octopamine. The method was used for simultaneous analyses of putative neurotransmitter amines in whole rat brain.     

An enzymatic fluorometric assay for pyridoxal with high specificity and sensitivity

An enzymatic fluorometric assay for pyridoxal with pyridoxal dehydrogenase was developed. The detection limit was about 10 pmol: the calibration curve of pyridoxal showed high linearity (r=0.993). The values obtained by this method correlated well with those by the HPLC method. The enzyme had a high specificity for pyridoxal, and thus animal samples could be directly analyzed without separation of pyridoxal 5'-phosphate by column chromatography.