Highly sensitive and accurate measurement of underivatized phosphoenolpyruvate in plasma and serum via EDTA-facilitated hydrophilic interaction liquid chromatography-tandem mass spectrometry.

Phosphoenolpyruvate (PEP) is an essential intermediate metabolite that is involved in various vital biochemical reactions. However, achieving the direct and accurate quantification of PEP in plasma or serum poses a significant challenge owing to its strong polarity and metal affinity. In this study, a sensitive method for the direct determination of PEP in plasma and serum based on ethylenediaminetetraacetic acid (EDTA)-facilitated hydrophilic interaction liquid chromatography-tandem mass spectrometry was developed. Superior chromatographic retention and peak shapes were achieved using a zwitterionic stationary-phase HILIC column with a metal-inert inner surface. Efficient dechelation of PEP-metal complexes in serum/plasma samples was achieved through the introduction of EDTA, resulting in a significant enhancement of the PEP signal. A PEP isotopically labelled standard was employed as a surrogate analyte for the determination of endogenous PEP, and validation assessments proved the sensitivity, selectivity, and reproducibility of this method. The method was applied to the comparative quantification of PEP in plasma and serum samples from mice and rats, as well as in HepG2 cells, HEK293T cells, and erythrocytes; the results confirmed its applicability in PEP-related biomedical research. The developed method can quantify PEP in diverse biological matrices, providing a feasible opportunity to investigate the role of PEP in relevant biomedical research.

Plasma metabolomics reveals a diagnostic metabolic fingerprint for mitochondrial aconitase (ACO2) deficiency.

Mitochondrial respiratory chain dysfunction has been identified in a number of neurodegenerative disorders. Infantile cerebellar-retinal degeneration associated with mutations in the mitochondrial aconitase 2 gene (ACO2) has been recently described as a neurodegenerative disease of autosomal recessive inheritance. To date there is no biomarker for ACO2 deficiency and diagnosis relies on genetic analysis. Here we report global metabolic profiling in eight patients with ACO2 deficiency. Using an LC-MS-based metabolomics platform we have identified several metabolites with affected plasma concentrations including the tricarboxylic acid cycle metabolites cis-aconitate, isocitrate and alpha-ketoglutarate, as well as phosphoenolpyruvate and hydroxybutyrate. Taken together we report a diagnostic metabolic fingerprint for mitochondrial aconitase 2 deficiency.

31Phosphorus magnetic resonance spectroscopy of the liver for evaluating inflammation and fibrosis in autoimmune hepatitis.

BACKGROUND: Liver biopsy is the gold standard in evaluating inflammation and fibrosis in autoimmune hepatitis. AIMS: In search of non-invasive follow-up tools in autoimmune hepatitis, we evaluated 31phosphorus magnetic resonance spectroscopy (31P MRS). METHODS: Twelve consecutive AIH patients (mean age 42.8 years, 10 women) underwent liver biopsy, routine laboratory liver function tests, which were compared to findings in 31P MRS and transient elastography (TE). RESULTS: Phosphoenolpuryvate (PEP) correlated with the grade of inflammation (r = 0.746, p = .005) and thromboplastin time (r = 0.592, p = .043). It also differentiated patients with active inflammation from patients without (t = 3.781, p = .009). There was no correlation between PEP and aminotransferase or immunoglobulin G levels. The phosphoethanolamine (PE)/phosphocholine (PC) ratio, PE/glyserophosphoethanolamine (GPE) ratio and PC/[total phosphomonoester (PME) + phosphodiester (PDE)] ratios correlated with immunoglobulin G (r = 0.764, p = .006; r = 0.618, p = .043; and r= -0.636, p = .035, respectively). PME/PDE and PE/GPE correlated with fibrosis (r = 0.668, p = .018 and r = 0.604, p = .037). PE/GPE also differentiated F3 from F0-2 patients (t = 3.810, p = .003). Phosphorus metabolites did not correlate with TE results and TE did not correlate with liver histology or laboratory parameters. CONCLUSIONS: 31P MRS seems to detect active inflammation and advanced fibrosis in AIH patients. TE was ineffective in fibrosis quantification.

Adrenergic regulation of Rana balcanica erythrocyte pyruvate kinase.

The regulation of pyruvate kinase activity by noradrenaline was investigated in Rana balcanica red cells. Thirty minutes of noradrenaline incubation induced a significant increase in the Vo/Vmax ratio of pyruvate kinase. The S0.5 for phosphoenolpyruvate of the enzyme significantly increased in the presence of noradrenaline while the Km for ADP decreased. In response to hormonal stimulation the Na +/H+ exchange was activated as was shown by the increase in Na+ and cyclic adenosine monophosphate from the 3rd min of incubation. All these effects were specific to alpha1 and beta antagonists. High concentrations of fructose diphosphate significantly activated the enzyme in the presence of noradrenaline but not in its absence. Furthermore, the presence of noradrenaline partially released the inhibition of the enzyme by adenosine triphosphate, inorganic phosphate and 2,3-diphosphoglycerate. The results suggest that noradrenaline stimulates glycolysis through pyruvate kinase activation. The mechanism of stimulation may is through Na+/H+ exchange activation, cyclic adenosine monophosphate concentration and Na(+)-K(+)-ATPase activation.

Erythrocyte pyruvate kinase deficiency. The influence of physiologically important metabolites on the function of normal and defective enzymes.

The dependence of the erythrocyte pyruvate kinase (PK)-catalyzed reaction on the glycolytic intermediates glucose-6-phosphate (Gluc-6-P), 2,3-diphosphoglycerate (2,3-DPG) and the nucleotides ADP and ATP was studied in normal individuals and 14 patients with PK deficiency. The Gluc-6-P concentrations in the erythrocytes are markedly elevated (4- to 6-fold) in 9 patients with severe hemolytic anemia compared to those 5 exhibiting a mild clinical course (up to 2-fold increased). 2,3-DPG is elevated up to 2 times compared to the controls whereas the measured ADP and ATP only slightly deviate from the normal range. Control experiments showed that these elevations of Gluc-6-P and 2,3-DPG do not depend on the number of reticulocytes. In enzyme kinetic terms, Gluc-6-P shifts the Hill coefficient to smaller values, i.e. suppresses the positive cooperativity (sigmoidal reaction kinetics), found in normal and some of the mutant enzymes and shift the noncooperative enzymes of some patients to an enzyme exhibiting negative cooperativity. The negative cooperativity already present in the enzymes of some of the patients suffering from severe hemolytic anemia becomes more pronounced upon addition of Gluc-6-P. Apparently 2,3-DPG acts as an antagonist to Gluc-6-P in increasing the Hill coefficient, i.e. enhancing the positive cooperativity of the normal enzyme. It shifts the hyperbolic patients' enzymes to a sigmoidal reaction type and the enzymes of those patients with negative cooperativity to a hyperbolic type. ADP and ATP show a similar behavior as 2,3-DPG, but additionally inhibit the enzyme at higher concentrations. The influence of all four phosphates on the Michaelis constant varies depending on the type of cooperativity, in some cases increasing and in some cases decreasing K0.5 PEP. With 7 of the patients, all of them with severe clinical course, a genetic analysis of their R-type PK gene was performed and genetic defects have been identified in the coding sequence. The found changes in the amino acid sequence and their corresponding location in the tertiary structure of the PK subunit can satisfactorily explain the alterations of the regulatory properties of the mutant enzymes thus allowing to establish a good correlation between altered structural and functional properties of the deficient enzyme and the severeness of the course of the disease.

Band 3-Memphis is associated with a lower transport rate of phosphoenolpyruvate.

Band 3-Memphis is the most common variant of erythrocyte band 3 protein, in which a single amino acid substitution (Lys56-->Glu) in a cytoplasmic domain of band 3 has been found. We showed here that the prevalence of the variant was particularly higher in the Japanese population than that in other populations. The calculated gene frequency of the variant in Japanese was 0.156, which was about 4 times higher than that in Caucasian. Although it has been generally accepted that the cytoplasmic domain of band 3 bears no relation to anion transport activity, we found that the transport rate of phosphoenolpyruvate in erythrocytes of homozygotes was decreased to about 80% of that in control cells, and that of heterozygotes was at an intermediate level. The evidence indicates that some structural changes in the cytoplasmic domain of band 3 may have influence on the conformation of anion transport system.

Pyruvate kinase Greensboro. A four-generation study of a high K0.5s (phosphoenolpyruvate) variant.

The proband with lifelong hemolytic anemia has a high K0.5s phosphoenolypyruvate (PEP) erythrocyte pyruvate kinase (PK) variant substantially but incompletely normalized by the allosteric modifier fructose-1,6-diphosphate (F-1,6-P2) with conversion of sigmoidal to hyperbolic kinetics. Heterozygotes in four generations express qualitatively identical but less severely abnormal kinetics and lack overt hemolysis. Kinetic abnormalities are closely mimicked by sulfhydryl modification of normal PK. Three distinct clinical and metabolic phenotypes characterize the proband and two sisters: variant PK and hemolytic anemia, variant PK without clinical manifestations or hemolysis, and complete normality. Their mother, whose red cell PK is entirely normal except for a questionably slightly low Vmax, is postulated to express the gene products of nonidentical alleles, one encoding a product with mildly less favorable catalytic characteristics. At low PEP concentrations, the proband and heterozygotes for the PK mutant express only a very small fraction of normal PK activity despite apparent inheritance of one normal allele in the latter. Evidence suggests that disproportionately lowered PK activity may be a property of a heterotetrameric PK. Illusory abnormalities in nucleotide specificity are artifacts of diminished substrate affinity characterizing the mutant PK.

Metabolism of 3-phosphoglyceroyl phosphate in phosphoenolpyruvate-enriched human erythrocytes.

The concentration of 3-phosphoglyceroyl phosphate in erythrocytes was increased by more than 100-fold when red cells were incubated with extracellular phosphoenolpyruvate at 37 degrees C. Since these elevated levels were maintained for 60 min, the metabolism of 3-phosphoglyceroyl phosphate and related compounds could be investigated in phosphoenolpyruvate-treated erythrocytes. 2,3-Bisphosphoglycerate synthesis was not affected by intracellular pH when the 3-phosphoglyceroyl phosphate level was constant but did vary with 3-phosphoglyceroyl phosphate concentration. On the other hand, the relationship between the rate of 2,3-bisphosphoglycerate synthesis and 3-phosphoglyceroyl phosphate concentration was not straightforward. At relatively low concentrations of 3-phosphoglyceroyl phosphate, the observed rate of 2,3-bisphosphoglycerate synthesis agreed with a rate calculated from a formula incorporating kinetic parameters of purified 2,3-bisphosphoglycerate synthase (Rose, Z.B. (1973) Arch. Biochem. Biophys. 158, 903-910). However, at high concentrations of 3-phosphoglyceroyl phosphate, the observed rate of 2,3-bisphosphoglycerate synthesis was lower than the calculated value. The concentration of glucose 1,6-bisphosphate did not increase even when 3-phosphoglyceroyl phosphate was elevated to 200 microM. Elevated levels of intracellular 2,3-bisphosphoglycerate did not inhibit glycolytic activity in these erythrocytes. These results suggest that incubation of erythrocytes with phosphoenolpyruvate is a useful technique to investigate the effect of metabolic perturbations at the intermediate stages of glycolysis.

The active center of transport for phosphoenolpyruvate and inorganic phosphate in the human erythrocyte membrane.

Phosphoenolpyruvate is transported into erythrocytes by a carrier-mediated transport system. The transport is competitively inhibited by inorganic phosphate and pyridoxal phosphate, which are transported across the cell membrane by Band 3. The pH profiles for the transport of inorganic phosphate and phosphoenolpyruvate suggest that a histidine residue exposed at the inner surface of the membrane participates in both of the transport. A hydrophobic peptide which may be a part of the active center for the transport is purified from the CNBr fragments of Band 3.

Red-cell metabolism in patients with iron deficiency.

18 components of metabolism were determined in the red cells of iron-deficient patients and data were expressed per 10(12) red cells to avoid the complicating effects of hypochromia and microcytosis. Glucose consumption, AMP and ATP, glycolytic intermediates except 2,3-bisphosphoglycerate (2,3-DPG) and phosphoenolpyruvate (PEP), red-cell Na+ and the net passive leakage of Na+ and K+ at 4 degrees C were all normal. Creatine, 6-phospho-D-gluconate: NADP oxidoreductase (6PGD) activity and fresh red-cell K+ were raised, suggestive of a young cell population. However, ATP: D-fructose-6-phosphate 1-phosphotransferase (PFK) activity and ADP were low. An elevated 2,3-DPG level was attributable to the anaemia present but the somewhat raised PEP level is unexplained. It is concluded that red cells in iron deficiency show some characteristics of a young cell population; in other respects they appear normal, but in containing a low PFK activity they are abnormal.

Involvement of a histidine residue in inorganic phosphate and phosphoenolpyruvate transport across the human erythrocyte membrane.

The transport of phosphoenolpyruvate across the erythrocyte membrane was compared with the transport of inorganic phosphate in resealed ghosts of human erythrocytes. Two conditions were employed: in one, the external pH (pHe) was varied from 6.0 to 7.5 while internal pH (pHi) was maintained at 7.2 or 6.2, whereas in the other, the internal and external pH were adjusted to have the same value over a pH range of 6.0 to 7.5. At a constant pHi of 7.2, the pH profile for the transport of phosphoenolpyruvate was different from that of inorganic phosphate; both pH profiles were similar to those previously observed in intact erythrocytes (Deuticke, B. (1970) Naturwissenschaften 57, 172-179; Hamasaki, N., et al. (1978) Biochem. J. 170, 39-46). In the absence of a transmembrane pH gradient, the pH profile for phosphoenolpyruvate transport was bell-shaped with the maximum at pH 6.8, and essentially the same pattern was seen with inorganic phosphate transport. The different pH profiles obtained under the two conditions suggests that one or more residues exposed at the inner surface of the membrane participate in the transport of inorganic phosphate and phosphoenolpyruvate. One of the most likely candidates is a histidine residue of the transport protein.

Red cell glycolytic intermediates and adenosine triphosphate in preterm infants on the first day of life.

Red cell glycolytic intermediates and ATP were evaluated in 47 appropriate for gestational age preterm infants on the 1st day of life who were divided into three groups on the basis of gestational age: 28-30, 31-33, and 34-36 wk. The results were compared to those previously obtained in term infants. The concentrations of glucose-6-phosphate, total triose phosphates, and ATP were significantly higher than in term infants but appeared to be appropriately elevated for the young mean age of the red cell population. The concentration of red cell 2,3-diphosphoglycerate (2,3-DPG) was significantly decreased when compared to term infants and was lowest at 28-30 wk gestation. The content of red cell 3-phosphoglycerate was increased in term infants and was inappropriately elevated for the age of the red cell population at 28-30 wk gestation. This pattern of glycolytic intermediates was suggestive of a young red cell population metabolizing at an increased glycolytic rate with increased flow through the phosphoglycerate kinase step rather than the 2,3-DPG bypass in "normal" preterm infants. Two preterm infants of 28-30 wk gestation with low red cell intracellular pH were also evaluated and had markedly decreased concentrations of red cell 2,3-DPG and ATP and all phosphorylated intermediates distal to the phosphofructokinase reaction, indicative of a cross-over at the phosphofructokinase step secondary to acidosis.(ABSTRACT TRUNCATED AT 250 WORDS)

2,3-Bisphosphoglycerate inhibits the transport of phosphoenolpyruvate across the erythrocyte membrane.

Mono- and bisphosphoglycerate competitively inhibited the transport of phosphoenolpyruvate across the erythrocyte membrane, although phosphoglycerates were impermeable to the cell membrane. The Ki values of 2-phosphoglycerate, 3-phosphoglycerate and 2,3-bisphosphoglycerate were 8.8 mM, 10.4 mM and 4.3 mM, respectively, whereas glucose 6-phosphate had almost no effect on the transport of phosphoenolpyruvate.

[Status of several types of tissue metabolism at rest and during functional tests in patients in the initial stage of heart failure]. / Sostoianie nekotorykh vidov tkanevogo obmena v pokoe i v usloviiakh funktsional'nykh prob u bol'nykh s nachal'noi stadiei serdechnoi nedostatochnosti.

A series of 217 patients with chronic coronary heart disease at an early stage of heart failure were examined. A comprehensive examination of the cardiovascular system and tissue metabolism makes it possible to diagnose cardiac failure at an early stage. Additional diagnostic signs of an early stage of heart failure were shown to include arteriolar hypoxemia, venous hyperoxia, a reduction in the arteriovenous difference with relation to oxygen and in tissue oxygen utilization which increased following a hydrostatic test, as well as an excessive accumulation of blood lactate and manifestations of disordered acid-base balance.

Human red cell glycolysis in high altitude chronic hypoxia.

We have found that glycolysis in human red blood cells under the hypoxic conditions found at high altitudes is connected with changes in enzyme activities and levels of various metabolic intermediates. The sensitivity of the four kinases to hypoxia results in 1) glycolytic hyperactivity leading to a higher intracellular energy state, and 2) accumulation of 2-3 DPG, whose role in the adaptation of red blood cell respiration to high altitude has been shown by previous research. PEP, 3PG , and G6P appear to be the main regulating intermediates in glycolysis in this system. The reason for the very large increase in G1- 6DP is still not clear.

Erythrocyte pyruvate kinase deficiency: characterization of a new variant (PK "Aarau").

A new PK variant with moderate hemolytic anemia is described. The enzymes of the nonanemic parents show sigmoidal reaction kinetics, with normal kinetic parameters, but differ with respect to nucleotide specificity, thermostability, and the concentrations of the glycolytic intermediates in the erythrocytes. The most characteristic features of the patient's (daughter) enzyme are a 30% activity, hyperbolic reaction kinetics and only two bands in the SDS-gel electrophoresis instead of three bands observed with the parental enzymes. Moreover, the pH-optimum is shifted to the acidic range, the affinity for PEP and ADP is decreased, ATP inhibition is negligible and FDP-activation is roughly ten times smaller than with controls. The concentrations of 2,3-DPG, 2-PG and PEP in the erythrocyte are increased, but ATP decreased. As there is no consanguinity in the parents and their enzymes are different this PK mutant can be considered to be compound-heterozygous for two different mutant PK alleles.

Characterization of phosphoenolpyruvate transport across the erythrocyte membrane. Evidence for involvement of band 3 in the transport system.

Phosphoenolpyruvate was found to be transported across the erythrocyte membrane by a carrier-mediated transport system. The transport of phosphoenolpyruvate was competitively inhibited by inorganic phosphate (Ki = 24 mM) and pyridoxal 5-phosphate (Ki = 0.2 mM), whereas the transport was noncompetitively inhibited by L(+)-lactate (Ki = 37 mM). Specific inhibitors for the inorganic anion transport system such as 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and 4,4'-dinitrostilbene-2,2'-disulfonic acid strongly inhibited the phosphoenolpyruvate transport. The transport was irreversibly inhibited by treating erythrocytes with pyridoxal 5-phosphate and NaBH4. Transport activities of phosphoenolpyruvate and inorganic phosphate in the treated cells were similarly inhibited by pyridoxal 5-phosphate depending on its concentrations. 4,4'-Dinitrostilbene-2,2'-disulfonic acid protected both transport activities against the pyridoxal 5-phosphate/NaBH4 treatment. The major integral membrane protein, band 3, was preferentially labelled by treating erythrocytes with pyridoxal 5-phosphate and NaB[3H]H4. The radioactive incorporation into band 3 was confirmed by two-dimensional gel electrophoresis combining isoelectric focusing in the first dimension and sodium dodecyl sulfate/polyacrylamide gel electrophoresis in the second dimension. Taken together, these results suggest that band 3 mediates the transport of phosphoenolpyruvate as well as inorganic phosphate.

Studies with human erythrocyte pyruvate kinase (PK): effects of modification of sulfhydryl groups.

Cysteinyl residues of red cell pyruvate kinase (PK; ATP: pyruvate phosphotransferase, EC 2.7.1.40) were modified with methylmethanethiosulfonate (MMTS), p-nitrophenoxycarbonyl methyl disulfide (NPCMD), and sodium tetrathionate (NaTT). At pH greater than 7 . 0, K0.5 s phosphoenol-pyruvate (PEP) was markedly increased. Fructose-1,6-diphosphate (FDP) increased affinity for PEP, but K0.5 s (PEP) remained elevated and hyperbolic kinetics were not achieved. Inhibition by negative effectors ATP and alanine was not reversed by PEP and FDP concentrations far greater than those abolishing inhibition of unmodified enzyme. At pH less than 7 . 0, PEP affinity was reduced, and FDP markedly increased Vmax and diminished K0.5 s (PEP). MMTS greatly impaired the thermostability of PK. Acid pH alone and the simultaneous presence of Mg++, K+ and PEP prior to MMTS treatment protected against the effects on PEP kinetics, but did not alter the induction of thermolability. No MMTS effect on the FDP binding site, on ADP kinetics or on the relative effectiveness of GDP, UDP or CDP cofactors was demonstrated. The MMTS-induced alterations closely resembled those observed with certain PK mutants associated with haemolytic anaemia.