Specific determination of threonine in biological samples by gas chromatography with electron capture detection

Threonine was oxidized into acetaldehyde at 0 degrees C for 30 min with periodic acid. The acetaldehyde formed was converted to a hydrazone with 2,4-dinitrophenyhydrazine. The hydrazone was extracted with n-heptane and quantified by gas liquid chromatography with electron capture detection. An internal standard, 2-amino-3-hydroxyhexanoic acid, was used. The calibration curve of threonine was linear up to 200 nmol in 200 microl sample solution and the determination limit of threonine was 1 nmol in 200 microl sample solution. The recoveries were 100.0, 94.0 and 100.0% from homogenates of octopus tentacles and blood plasma and rat livers, respectively. This method was applied to the determination of threonine in tissues of rats given threonine and starved octopuses. This threonine determination method has been used for studies on the metabolism of d-lactate.     

Contribution of erythrocytes and plasma in threonine and lysine transfer across the portal drained viscera and the liver in pigs. Effect of threonine and lysine dietary supply

Contributions of erythrocytes and plasma to threonine and lysine transport across the PDV and the liver were determined in growing pigs successively fed a threonine deficient diet and a control well-balanced diet (experiment 1) or a lysine deficient or a well-balanced diet (experiment 2). The animals were surgically prepared for insertion of chronic catheters in the mesenteric vein (MV), the portal vein (PV), a hepatic vein (HV) and the carotid artery (CA). Plasma and whole blood AA concentrations in PV, HV and CA and PV and HV blood flows were determined during 6 hours of para-aminohippuric acid constant infusion. During this period the pigs were continuously fed (1 meal per hour). The contribution of plasma to lysine and threonine transport was higher in pigs fed the well balanced diets. More than 50% of threonine and lysine appearing in the PV and in the HV are transported by the plasma. Our results suggest that erythrocytes are probably little involved in lysine and threonine transfer across the liver and digestive tract of pig continuously fed.     

Influence of high dietary threonine on growth and amino acids in blood and tissues of rats

Summary Diets containing 8 or 15% protein from casein plus limiting amino acids, 25% fat and adequate levels of other nutrients for rat growth were supplemented with 0, 0.5, 1.0, 2.0 or 4% of excess L-threonine. Addition of up to 1% excess threonine had little effect on weight gains or food intakes of weanling rats, but addition of 2 and 4% threonine caused a drastic reduction in weight gains or food intakes (up to 41%); the adverse effect being more severe in rats fed lower protein diets. Addition of graded levels of excess threonine resulted in (5 to 47-fold and 4 to 20-fold) increase in concentration of free threonine in rat plasma and brain, respectively. Addition of excess threonine also caused up to 5-fold increase in plasma level of 3-methylhistidine, suggesting increased muscle protein breakdown.     

L-threonine administration increases glycine concentrations in the rat central nervous system

Intraperitoneal administration of L-threonine increased the glycine and threonine concentrations in rat spinal cord. Glycine contents also increased in synaptosomes prepared from spinal cords from threonine-pretreated animals. These findings suggest that plasma threonine concentrations normally might affect production of glycine by central nervous system neurons, and also that exogenous threonine might be useful in modifying glycinergic transmission.     

Whole blood and plasma amino acid transfers across the portal drained viscera and liver of the pig.

Whole blood (WB) and plasma (P) amino acid transfers across the portal drained viscera and the liver were determined during 6 h of a constant p-aminohippuric acid infusion in three hourly-fed Landrace x Large-White pigs (30.5 kg, mean live weight) surgically prepared with chronically inserted catheters in a mesenteric vein (MV), the portal vein (PV), an hepatic vein (HV) and the carotid artery (CA). Plasma and WB amino acid concentrations were determined in the CA, PV and HV. The plasma/WB ratios showed no significant differences for vessels except for lysine and glutamate for which this ratio is significantly higher in the HV and in the PV for lysine. This suggests that the PV lysine and HV glutamate were preferentially transported in the plasma. In the PV, threonine, valine and alanine are transported by both plasma and red blood cells. These data show that the contribution of plasma and whole blood to amino acid transport can be different between amino acids and between individual tissues.     

Characterization of a new plasma membrane-associated ecto-5'-phosphodiesterase/nucleotide-pyrophosphatase from rat hepatocarcinoma AS-30D cells

We have identified in plasma membrane fractions isolated from rat hepatocarcinoma AS-30D ascites cells three glycoproteins of 125 kDa, 115 kDa and 105 kDa (gp125, gp115 and gp105) which become adenylylated using ATP as substrate, most readily in the presence of EDTA. The gp115 becomes also phosphorylated. The adenylylation of these tumor glycoproteins was much lower than that of a group of analogous adenylylatable glycoproteins (gp130, gp120-gp110 dimer and gp100) present in normal rat liver plasma membrane. The tumor glycoproteins were reversibly O-adenylylated at threonine residues, as was the case for their normal rat liver counterparts. The tumor gp115, and the gp120-gp110 dimer from normal rat liver were both isolated using either ATP-affinity chromatography and/or AMP-affinity chromatography. The gp120-gp110 dimer from normal rat liver was identified as the plasma cell differentiation antigen-1 (PC-1 protein), an ecto-5' phosphodiesterase/ nucleotide-pyrophosphatase (5'-PDE/NPPase). The gp115 from tumor cells also exhibited Zn2+-stimulated 5'-PDE and NPPase activities in alkaline conditions, although it appears to be distinct from the PC-1 protein. We have determined that the gp115 is an ecto-enzyme that catalyzes the hydrolysis of extracellular ATP, since its adenylylation and phosphorylation were detected in intact cells using extracellularly added [alpha-32P]ATP or [gamma-32P]ATP, respectively, in the absence of any permeabilizing agent.     

Characterization of a new plasma membrane-associated ecto-5'-phosphodiesterase/nucleotide-pyrophosphatase from rat hepatocarcinoma AS-30D cells

We have identified in plasma membrane fractions isolated from rat hepatocarcinoma AS-30D ascites cells three glycoproteins of 125 kDa, 115 kDa and 105 kDa (gp125, gp115 and gp105) which become adenylylated using ATP as substrate, most readily in the presence of EDTA. The gp115 becomes also phosphorylated. The adenylylation of these tumor glycoproteins was much lower than that of a group of analogous adenylylatable glycoproteins (gp130, gp120-gp110 dimer and gp100) present in normal rat liver plasma membrane. The tumor glycoproteins were reversibly O-adenylylated at threonine residues, as was the case for their normal rat liver counterparts. The tumor gp115, and the gp120-gp110 dimer from normal rat liver were both isolated using either ATP-affinity chromatography and/or AMP-affinity chromatography. The gp120-gp110 dimer from normal rat liver was identified as the plasma cell differentiation antigen-1 (PC-1 protein), an ecto-5′ phosphodiesterase/nucleotide-pyrophosphatase (5′-PDE/NPPase). The gp115 from tumor cells also exhibited Zn²⁺-stimulated 5′-PDE and NPPase activities in alkaline conditions, although it appears to be distinct from the PC-1 protein. We have determined that the gp115 is an ecto-enzyme that catalyzes the hydrolysis of extracellular ATP, since its adenylylation and phosphorylation were detected in intact cells using extracellularly added [α-³²P]ATP or [γ-³²P]ATP, respectively, in the absence of any permeabilizing agent.     

Threonine Entry into Rat Brain After Diet-Induced Changes in Plasma Amino Acids

Abstract: Passage of amino acids across the blood-brain barrier is modified by the amino acid composition of the blood. Because blood amino acid concentrations respond to changes in protein intake, we have examined associations among diet, plasma amino acid patterns, and the rate of entry of threonine into the brain. Rats were adapted for 8 h/ day for 7–10 days to diets containing 6, 18 , or 50% casein before receiving a single, independently varied, final meal of a diet containing 0, 6, 18 , or 50% casein. After 4–7 h, they were anesthetized and infused intravenously with [¹⁴C]threonine for 5 min before plasma and brain samples were taken for determination of radioactivity and amino acid content. Plasma and brain threonine concentrations decreased as protein content increased in the diets to which the rats had been adapted. Plasma threonine concentrations increased twofold, from 1.6 to 3.0 m M , when rats adapted to 6% casein meals received a single 50% casein meal rather than a nonprotein meal; a fivefold increase, from 0.13 to 0.69 m M , occurred when rats had been previously adapted to 50% casein meals. Increasing the protein content of the final meal did not increase brain threonine concentrations. Highest and lowest rates of threonine entry into the brain occurred, respectively, in rats adapted to 6 and 50% casein meals. Changes in plasma threonine concentrations and threonine flux into brain reflected protein content of both pretreatment and final meals.     

Chemoattraction of infective larvae of Ancylostoma braziliense to rodent plasmas and to salts

Infective larvae of Anyclostoma braziliense were tested for orientational response to rat plasma, to mouse plasma, to rat plasma fractions, and to salts. A high percentage of larvae accumulated at sources of rat plasma, mouse plasma, rat plasma diffusate, concentrated rat plasma dialysate, and some salts, notably sodium chloride. Because sodium chloride is present at an effective concentration in mammalian plasma, and because this salt may form a gradient between the blood and the skin surface, sodium chloride from the blood may direct the penetrating larvae through host's skin. Preliminary tracking of the larvae in gradients of rat plasma and of sodium chloride suggests that orientation to sources of these attractants was via a taxis, possibly a klinotaxis, whereas accumulation at these sources was via a klinokinesis.     

The occurence of cystinylglycine in blood plasma

Cystinylglycine has been identified as a minor ninhydrin-positive component of deproteinized blood plasma from human, bovine, rat and rabbit blood. The amount present in human blood plasma is approximately one-fifth of that of cystine and is not significantly correlated with age or with the cystine level.     

The occurrence of cystinylglycine in blood plasma.

Cystinylglycine has been identified as a minor ninhydrin-positive component of deproteinized blood plasma from human, bovine, rat and rabbit blood. The amount present in human blood plasma is approximately one-fifth of that of cystine and is not significantly correlated with age or with the cystine level.     

Metabonomic phenotype and identification of “heart blood stasis obstruction pattern” and “qi and yin deficiency pattern” of myocardial ischemia rat models

The traditional Chinese medicine concepts of “Xinxueyuzuzheng (heart blood stasis obstruction pattern)” and “Qiyinliangxuzheng (qi and yin deficiency pattern)” for myocardial ischemia rat models were constructed in the present study. Endogenous metabolites in rat plasma were analyzed using the GC/TOF-MS-based metabonomic method. Significant metabolic differences were observed between the control and two model groups, and the three groups were distinguished clearly by pattern recognition. Compared with those of the control, the levels of hydroxyproline, threonic acid, glutamine and citric acid were strikingly up- or down-regulated in model rats. The metabolites contributing most to the classification between the two “pattern” rats were identified, such as valine, serine, threonine, ornithine, hydroxyproline, lysine, 2-hydroxybutanoic acid, 3-hydroxybutanoic acid, galactofuranose and inositol. These compounds were indicated as the potential biomarkers. The results suggested that the two “patterns” are involved in dysfunction in oxidative stress, energy metabolism and amino acid metabolism. These findings also provided the substantial foundation for exploring the scientific connotation of these two “Zhengxing (pattern types)” of myocardial ischemia, and “Bianzheng (pattern identification)”.     

The effect of host blood in the in vitrotransformation of bloodstream trypanosomes by tsetse midgut homogenates

Abstract. Midgut homogenates prepared from Glossina morsitans morsitans , that had previously been fed on different host blood samples, were tested for their abilities to transform bloodstream Trypanosoma brucei into procyclic (midgut) forms in vitro. Compared to rat and goat blood samples, eland blood had the least capacity to support trypanosome transformation, whereas buffalo blood showed intermediate capacity. Fractionation of rat blood showed the importance of the cellular portion since both rat and eland red blood cells (RBCs) supported the process. Virtually no transformation was observed in rat and eland plasma or serum fractions. Suspending rat blood cells in eland plasma led to a reduction in parasite transformation rates. Further experiments showed that the RBC membranes were also capable of supporting the process. These results clearly show the important role played by blood, especially the red blood cells, in the transformation of bloodstream trypanosomes. In addition, the low transformation rates observed in eland blood is due to an inhibitory factor(s) present in the plasma fraction.     

Influence of glucogenic amino acids on the hepatic metabolism of threonine.

The supplementation of a low-protein diet with L-threonine leads to a marked accumulation of threonine in plasma and liver, whereas increasing dietary protein generally leads to an induction of threonine dehydratase in the liver, hence depressed availability for extrasplanchnic tissues. The aim of the present study was, thus, to further investigate the factors which control the utilization of threonine by the liver. Increasing the dietary supply of threonine led to parallel increases in the afferent and hepatic concentrations and in the rate of utilization by the liver; however, the fractional extraction tended to decrease. It appears that the addition of a mixture of glucogenic amino acids to the diet prevented the accumulation of threonine in plasma induced by exogenous threonine. The glucogenic amino acids increased the fractional hepatic uptake of threonine, and counteracted its accumulation in the liver. These effects reflect the fact that the glucogenic amino acids elicited a potent induction of the threonine dehydratase, whereas threonine alone was uneffective. Our results suggest that, besides the well-established effect of glucogenic conditions, the availability of some glucogenic amino acids is an important factor in the control of threonine catabolism.     

Blood amino acid composition of poikilothermic vertebrate under prolonged starvation

Free amino acids in the blood plasma of lamprey (Lampetra fluviatilis) and frog (Rana temporaria) have been determined quantitatively for the periods of deprivation of the exogenous feed. The lamprey's total amino acid pool increased by 74% from November to April and reached the lower limit known for the mammals. The amount of free amino acids in frogs decreased by 40% in the spring as compared with the autumn values. The difference is accounted for by certain features of the living cycles of these animals. A more energetic proteolysis in the lamprey tissues as compared with that in the frog tissues has been confirmed by quantitative determining of leucine, isoleucine and valine in the blood of these animals. Apart from the above, alanine, glycine, lysine, threonine and, in certain periods, tyrosine have been found to be quantitatively significant in the plasma of both animal species. The composition and proportion of the amino acids in blood plasma of these animals are due to specific features of their metabolism and connected with the energy state of the liver cells under starvation.     

Effect of starvation and sampling time on plasma alkaline phosphatase activity and calcium homeostasis in the rat

The effect of starvation and sampling time on plasma alkaline phosphatase activity, total plasma calcium concentration and whole blood ionized calcium concentration was determined in the rat. Starvation caused a significant fall in total and ionized calcium concentrations as well as in alkaline phosphatase activity. These changes were accompanied by a fall in whole blood pH and an increase in the anion gap and a decrease in urinary excretion of calcium. These indices were restored to normal following refeeding. There was no change in serum 25-OH vitamin D concentrations following starvation for 3 days. Alkaline phosphatase activity showed a pattern compatible with the presence of a circadian rhythm when sampling took place between 0800 and 1800 h. Total and ionized calcium concentrations did not show such a rhythm when animals were fed the present diet.     

Direct determination of selenium in rat blood plasma by Zeeman atomic absorption spectrometry

The method was developed to be applied for direct determination of selenium in rat plasma by graphite-furnace atomic absorption spectrometry with Zeeman background correction. Blood was obtained from CD rats of both sexes 2h after dosing in weeks 7 and 13 in order to acquire data on the levels of selenium in these animals during 13-week gavage administration of l-seleno-methylselenocysteine (SeMC), a new candidate chemopreventive agent under development. Application of the commonly used method of standard addition was found to be unsuitable to calculate the selenium content in rat plasma (within-run and between-run accuracy and precision parameters were less than 85%). Therefore, a new analytical method was developed. In this method, samples of rat plasma (50 microL) were diluted 10-fold with a reducing agent containing l-ascorbic acid, a modifier solution containing palladium chloride and Triton X-100. Samples were atomized in pyrolytically coated graphite tubes and peak height signals were measured. Selenium concentrations were determined by linear least squares regression analysis based on the standard curve generated in pooled rat blank plasma. Since selenium is normally present in plasma, a three-step approach was used to calculate selenium plasma levels. Initially selenium levels were determined based on the standard curve with selenium-spiked pool plasma. In the second step, background selenium levels in the pooled plasma were determined based on the same standard curve. In the third step, background level was added to the previously derived number. The relative errors were in the range from -4.6 to 11.4% (intra-day assay) and from -0.4 to 8.8% (inter-day assay) which proved good accuracy. The relative standard deviations were in the range from 1.88 to 4.70% (intra-day precision) and from 3.28 to 5.38% (inter-day precision). In rat plasma, the following dose-dependent selenium levels (mean+/-S.D.) in males and females, respectively, were observed at 13 weeks: 655.5+/-48.8 and 595.8+/-43.9 ng/mL (control group), 927.9+/-85.3 and 859.3+/-164.3 ng/mL (0.4 mg/kg per day dose group), 1238.9+/-182.4 and 1169.9+/-112.6 ng/mL (0.8 mg/kg per day dose group), and 1476.5+/-138.1 and 1320.1+/-228.6 ng/mL (2.0mg/kg per day dose group). No significant sex differences in selenium plasma levels were seen in the SeMC-treated groups. No significant differences in selenium plasma levels were seen between mean plasma levels at 7 and 13 weeks. The described method is simple, rapid, accurate, precise and can be easily applied in other laboratories for a large number of samples.     

Effect of the composition of branched chain amino acids, taurine, and tryptophan on the amino acid metabolism in experimental models of alcoholism

Ethanol withdrawal after forced alcoholization of rats according to Majchrowicz led to the development of amino acid imbalance in the pool of free amino acids in the liver (increasing levels of alanine, aspartate, glutamate, glutamine and histidine, decreasing levels of glycine, lysine, threonine and taurine) and blood plasma (increasing levels of tyrosine and alanine, decreasing levels of most glycogen aminoacids, branched-chain aminoacids and Lys). Less profound changes were observed after prolonged alcohol intoxication (decreasing levels of alanine, ornitine, citrulline and increasing level of Glu in liver, increasing levels of sulfur-containing compounds, Asp and Lys in blood plasma). Amino acid mixture which contained branched-chain amino acids, taurine and tryptophan administered intragastrically was found to correct levels of sulfur-containing amino acids, threonine, lysine and isoleucine after ethanol withdrawal and to eliminate disorders in urea cycle, exchange of threonine, glycine and phenylalanine after prolonged alcohol intoxication.     

Isocratic high-performance liquid chromatographic method for studying the metabolism of blood plasma pyrimidine nucleosides and bases: concentration and radioactivity measurements

A rapid and efficient isocratic high-performance liquid chromatographic method for studying the metabolism of blood plasma cytosine, uracil, thymine, cytidine, deoxycytidine and uridine has been elaborated. For each compound this method can measure concentrations in the range 0.5–200 μM and determine radioactivity. All the pyrimidine compounds can be eluted in less than 18 min, and the total time elapsed between collection of the blood and completion of the analysis need to exceed 3 h. All measurements can be performed on 0.25-ml blood samples. Blood plasma pyrimidine concentrations were determined for the rat, the rabbit, the guinea pig, the dog and the healthy human. This method could be well applied to experimentation on small animals using radiolabelled pyrimidine derivatives, in order to study the metabolic pathways of nucleotides and nucleic acids. It could also be used to characterize certain illnesses or cases of toxicity created by a chemotherapy affecting the plasma level of pyrimidine bases on nucleosides.