Studies on the intracellular accumulation process of methotrexate and its correlation with the key protein using an LC-MS/MS method: a novel way to realize prospective individualized medication.

High-dose methotrexate (HDMTX) combined with leucovorin (LV) is the first-line drug therapy for many kinds of malignant tumors. However, the specific treatment plans, such as dosage and duration of administration, are usually formulated according to the clinician's experience and therapeutic drug monitoring (TDM) of methotrexate in patients' plasma, which are responsible for strong individual differences of drug usage. A large number of studies have shown that methotrexate targets the inside of the cell. The key cytotoxic component is the methotrexate polyglutamates (MTXPGs) in the cell. The concentration of methotrexate in plasma does not reflect the efficacy and side effects well. Based on mass spectrometry technology, we developed and validated an accurate, sensitive, and stable method to quantify the intracellular MTX (MTXPG1) and its metabolites MTXPG2-7 simultaneously. The lower limit of quantification was 0.100 ng/ml, and the run time was only 3 min. Moreover, our team has already developed two LC-MS/MS-based methods to respectively quantify methotrexate in plasma samples and two key proteins (γ-glutamyl hydrolase [GGH] and folylpolyglutamate synthetase [FPGS]) in peripheral blood mononuclear cells (PBMC). Through these highly sensitive and accurate approaches, we have gained a deep understanding of the whole pharmacokinetic process of MTX and explored the key factors affecting the accumulation process of intracellular active components (MTXPGs). Based on this research, it is possible to find a more effective way to provide an accurate reference for clinical drug use than traditional therapeutic drug monitoring (TDM).

Mutations in PPCS, Encoding Phosphopantothenoylcysteine Synthetase, Cause Autosomal-Recessive Dilated Cardiomyopathy.

Coenzyme A (CoA) is an essential metabolic cofactor used by around 4% of cellular enzymes. Its role is to carry and transfer acetyl and acyl groups to other molecules. Cells can synthesize CoA de novo from vitamin B5 (pantothenate) through five consecutive enzymatic steps. Phosphopantothenoylcysteine synthetase (PPCS) catalyzes the second step of the pathway during which phosphopantothenate reacts with ATP and cysteine to form phosphopantothenoylcysteine. Inborn errors of CoA biosynthesis have been implicated in neurodegeneration with brain iron accumulation (NBIA), a group of rare neurological disorders characterized by accumulation of iron in the basal ganglia and progressive neurodegeneration. Exome sequencing in five individuals from two unrelated families presenting with dilated cardiomyopathy revealed biallelic mutations in PPCS, linking CoA synthesis with a cardiac phenotype. Studies in yeast and fruit flies confirmed the pathogenicity of identified mutations. Biochemical analysis revealed a decrease in CoA levels in fibroblasts of all affected individuals. CoA biosynthesis can occur with pantethine as a source independent from PPCS, suggesting pantethine as targeted treatment for the affected individuals still alive.

Cytosolic protein ubiquitylation in normal and endotoxin stimulated human peripheral blood mononuclear cells.

The ubiquitin-proteasome pathway is regarded as playing a crucial role in protein breakdown in inflammation and sepsis as well as in the regulation of inflammatory cell responses. In this pathway, ubiquitylation of target proteins is believed to act as a recognition signal for degradation by the 26S proteasome. As yet neither the ubiquitylation rate of cytosolic proteins, as a result of the total ubiquitin-protein ligase (tUbPL) activity, nor the specific ubiquitylation of calmodulin (ubiquitin-calmodulin ligase, uCaM-synthetase) has been determined in human mononuclear cells. Therefore, we studied cytosolic protein ubiquitylation in normal and in endotoxin (LPS)-stimulated human peripheral blood mononuclear cells (PBMNCs).PBMNCs from healthy volunteers were incubated with 0 or 100 ng/ml LPS for 18 h. Cytosolic extracts were obtained by hypotonic lysis and ultracentrifugation. TUbPL was measured as [(125)I]-[CT]-ubiquitin incorporation into the sum of cytosolic proteins. UCaM-synthetase activity was quantified with the fluphenazine (FP)-Sepharose affinity adsorption test. Endotoxin stimulation appears to inhibit tUbPL 3.7 +/- 2.7-fold to 48 +/- 43 fkat/mg (n = 6). UCaM-synthetase in cultures (n = 5) without endotoxin was determined to be 91 +/- 32 fkat/mg +Ca(2+) and 29 +/- 23 fkat/mg -Ca(2+). With endotoxin uCaM-synthetase was 138 +/- 73 fkat/mg +Ca(2+) and 14 +/- 22 fkat/mg -Ca(2+). Ca(2+)-specificity (ratio +/- Ca(2+)) of uCaM-synthetase increases from 3.1 without LPS to 10 after LPS stimulation, which was caused by a 2-fold decrease in minus Ca(2+) activity and a 1.5-fold increase in plus Ca(2+) activity. The data indicate specific regulatory effects of endotoxin on the cytosolic ubiquitylation systems in human PBMNCs.

[Testing the activity of enzymes responsible for biosynthesis of purine nucleotides AIR-carboxylase and SAICAR-synthetase in human cell extracts]. / Testirovanie aktivnosti fermentov biosinteza purinovykh nukleotidov AIR-karboksilazy i SAIKAR-sintetazy v ékstraktakh kletok cheloveka.

Activities of AIR-carboxylase (ES 4.1.1.21) and SAICAR-synthetase (EC 6.3.2.6) were found in lysates of human erythrocytes, thrombocytes and leukocytes and in homogenate of the stomach biopsy sample. However, these activities were absent in blood plasma and bile. The human erythrocyte enzyme preparation, which had both activities, was isolated and purified about 200 times. The copurification of both activities and properties of the enzyme preparation suggest that two consecutive reactions of purine biosynthesis de novo (from AIR to SAICAR) in human cells are catalyzed by one bifunctional enzyme which is probably encoded by one gene.

Improved assay of the enzymes of glutathione synthesis: gamma-glutamylcysteine synthetase and glutathione synthetase.

New methods for the estimation of red cell gamma-glutamylcysteine synthetase and glutathione synthetase have been developed. gamma-32P ATP is allowed to equilibrate until the gamma and beta phosphate groups are equally labelled. The amount of 32Pi released in the presence of glutamic acid and cysteine, the substrates for GC-S or in the presence of gamma-glutamylcysteine and glycine, the substrates of GSH-S, is measured. This is accomplished by extraction of the phosphomolybdate complex into isobutanol-benzene. The methods are linear with time and hemolysate concentration. Normal values are presented.

Significance of glutathione S-conjugate for glutathione metabolism in human erythrocytes.

The significance of glutathione S-conjugate in the regulation of glutathione synthesis was studied using human erythrocyte gamma-glutamylcysteine synthetase. Feedback inhibition of the enzyme by reduced glutathione was released by the addition of the glutathione S-conjugate (S-2,4-dinitrophenyl glutathione). A half-maximal effect of glutathione S-conjugate on gamma-glutamylcysteine synthetase activity was obtained at approximately 1 microM; 50 microM glutathione S-conjugate in the presence of 10 mM glutathione actually increased the enzyme activity twofold above uninhibited levels. Glutathione S-conjugate had no effect on the enzyme activity in the absence of glutathione. When erythrocytes were exposed to the electrophile 1-chloro-2,4-dinitrobenzene, which forms a glutathione S-conjugate by the catalytic reaction of glutathione S-transferase, the level of glutathione synthesis increased. These data suggest that glutathione S-conjugate plays a role in stimulating the synthesis of glutathione.

Glutathione, glutathione reductase and glutathione S-transferase activities in erythrocytes and lymphocytes in chronic renal disease.

Erythrocyte and lymphocyte reduced glutathione (GSH) levels, and glutathione reductase (GR) and glutathione S-transferase (GST) activities have been investigated in uremic patients pre- and post-dialysis and normal subjects of the same age span. GSH levels and GST activities in erythrocytes and lymphocytes and GR activity in lymphocytes from uremic patients were higher as compared to the corresponding controls. Dialysis resulted in a 17% decrease in GSH levels in erythrocytes. Hemodialysis did not significantly alter erythrocyte GST and GR activities. Hemodialysis produced a 50% decrease in lymphocyte GST activity and a 32% decrease in lymphocyte GR activity. Elevated levels of GSH and increased activities of GST and GR in blood cells of uremic patients may be associated with a compensatory protective mechanism against accumulating toxic wastes in uremic plasma.

A reappraisal of the gamma-glutamylcysteine synthetase activity in haemolysates from normal erythrocytes by two different methods.

gamma-Glutamylcysteine synthetase catalyses the combination of L-glutamate and L-cysteine to form gamma-glutamylcysteine with a stoichiometric conversion of ATP to ADP and inorganic phosphate (Pi). During the estimation of this enzyme in haemolysates from normal erythrocytes it was found that the Pi released was more than the amount of gamma-glutamylcysteine synthesised. Furthermore, the activity estimated by analysing either product was higher than the corresponding values reported in the literature. An investigation into these discrepancies resulted in improvements of the assay methods which produced two substantially different normal ranges for the gamma-glutamylcysteine synthetase activity in haemolysates: one derived from the Pi released and the other from the gamma-glutamylcysteine synthesised during the enzymatic reaction.

Erythrocyte gamma-glutamylcysteine synthetase from normal and low-glutathione sheep.

gamma-Glutamylcysteine synthetase (L-glutamate:L-cysteine gamma-ligase (ADP-forming), EC 6.3.2.2) was purified from the erythrocytes of normal and low-glutathione sheep. The molecular weight (78 000), pH optimum (pH 7), substrate specificity, inhibition constant for glutathione (0.44-0.50 mM), electrophoretic mobility, and heat stability were similar for the purified enzyme from both sources. Using immunological techniques, the specific activity of gamma-glutamylcysteine synthetase from low-glutathione sheep was lower than that from normal sheep.

Cystine metabolism in human fibroblasts. Comparison of normal, cystinotic, and gamma-glutamylcysteine synethetase-deficient cells.

Incubation of normal human skin fibroblasts or fibroblasts derived from patients with erythrocyte deficiency of gamma-glutamylcysteine synthetase (gamma-glutamylcysteine synthetase-deficient) in culture medium containing L-[35S]cystine resulted in incorporation of radioactivity into protein, cysteine, and glutathione, gamma-Glutamylcysteine synthetase-deficient fibroblasts synthesized glutathione from [35S]cystine at 30% the rate of normal cells and contained 30% the normal amount of glutathione. Cystinotic fibroblasts incorporated [35S]cystine into the large intracellular cystine pool not found in normal or gamma-glutamylcysteine synthetase-deficient cells and also appeared to synthesize glutathione more slowly than normal cells. However, the radioactivity recovered as cystine was reduced greatly and the rate of [35S]cystine incorporation into glutathione increased if cystinotic cells were first depleted of their intracellular cystine pool before incubation in [35S]cystine. This suggests that the apparent reduced rate of glutathione synthesis observed in untreated cystinotic cells was a secondary effect caused by dilution of the [35S]cystine by the large pool of nonradioactive cystine. Cystinotic cells depleted of cystine by treatment with mercaptoethylamine reaccumulate 30 to 50% of their initial cystine in 24 hours in the absence of extracellular cystine. Both normal and cystinotic cells lose more than 90% of their intracellular glutathione in 24 hours in cystine-free medium. Both cell types can reutilize cysteine from glutathione for protein synthesis.

On the mechanism of 5-oxoproline overproduction in 5-oxoprolinuria.

The primary metabolic defect in 5-oxoprolinuria (pyroglutamic aciduria) is the lack of glutathione synthetase. The mechanism of the concomitant overproduction of 5-oxoproline was studied using cell-free extracts of erythrocytes from control individuals and from patients with 5-oxoprolinuria. Such extracts catalyzed the synthesis of 5-oxoproline from L-glutamate. Addition of ATP, Mg ions and alpha-aminobutyrate was needed for optimal activity. The conversion of glutamate to 5-oxoproline occurred in two steps, catalyzed by gamma-glutamyl-cysteine synthetase and gamma-glutamyl cyclotransferase, respectively. Extracts of erythrocytes from control subjects and patients with 5-oxoprolinuria had identical capacity to synthesize 5-oxoproline. The conversion of glutamate to 5-oxoproline was markedly inhibited by reduced glutathione, which exerted its effect on the gamma-glutamyl-cysteine synthetase step. The following mechanism is postulated for the overproduction of 5-oxoproline in 5-oxoprolinuria: the deficiency of glutathione synthetase causes a lack of glutathione which is an essential feed-back inhibitor in the initial step of its biosynthesis. Therefore gamma-glutamyl-cysteine is produced in excessive amounts and it is subsequently converted to 5-oxoproline (and cysteine) by gamma-glutamyl cyclotransferase. This overproduction of 5-oxoproline exceeds the capacity of the 5-oxoprolinase and 5-oxoproline accumulates in body fluids.

[Glutathionestatus of Plasmodium vinckei parasitized erythrocytes in correlation to the intraerythrocytic development of the parasite (author's transl)]. / Glutathionstatus in Plasmodium vinckei parasitierten Erythrozyten in Abhängigkeit vom intraerythrozytären Entwicklungsstadium des Parasiten

The glutathione status of Plasmodium vinckei parasitized erythrocytes of mice was determined in correlation to the intraerythrocytic stage of maturation of the parasite. The different stages of blood schizogony were separated by discontinuous Dextran-density-centrifugation. The changes of protein content, glutathione concentration (reduced/oxidized and bound/free glutathione) and in the specific activities of the following enzymes: gamma-glutamyl-cysteine-synthetase (GC-synthetase), glutathione-reductase (GR), glucose-6-phosphate dehydrogenase (Gl-DH), glutathione-peroxydase (G-POD) and catalase were investigated in dependence of the intraerythrocytic stage of development. The following changes of the investigated metabolic parameters were observed during the schizogony: - the protein content decreased to about one half, - the glutathione concentration increased about 10-fold, while the relations reduced/oxidized and free/bound glutathione remained constant, - Gl-DH activity appeared and increased steeply, - the specific activities of GC-synthetase and of GR increased more than 2-fold, while G-POD remained almost constant, - and the activities of G-6-PDH and catalase showed a significant, strong decrease to about 25% of the original values. It is tried to relate the observed changes to the growing parasite or to the host cell. The significance of the results for the metabolism of malaria parasites and for a possible adaptation to the mosquito by a GSH mediated protection of the malaria parasite against an enzymatic defence-reaction of the mosquito, is discussed.

GSH biosynthesis in glutathione deficient erythrocytes from Finnish landrace and Tasmanian merino sheep.

1. The maximum activities of the enzymes for the biosynthesis of GSH (gamma-glutamyl-cysteine synthetase and GSH synthetase) have been assayed in high GSH and low GSH erythrocytes from Tasmanian Merino and Finnish Landrace sheep. 2. For the Merinos, the activities (mumol product/g haemoglobin per min +/- S.E.M. (n)) in the high and low GSH erythrocytes respectively were: gamma-glutamyl-cysteine synthetase: 0.776 +/- 0.065 (11) and 0.375 +/- 0.063 (13); and GSH synthetase: 0.069 +/- 0.003 (11) and 0.066 +/- 0.002 (13). 3. For the Finnish Landrace sheep the activities in the high and low GSH erythrocytes respectively were: gamma-glutamyl-cysteine synthetase: 0.595 +/- 0.063 (12) and 0.555 +/- 0.033 (10) and gamma-glutamyl-cysteine synthetase: 0.073 +/- 0.002 (12) and 0.070 +/- 0.002 (10). 4. gamma-Glutamyl-cysteine synthetase was markedly inhibited by physiological GSH concentrations. No evidence was found for the presence of an inhibitor of GSH biosynthesis (other than GSH) in low GSH erythrocytes from Finnish Landrace sheep. 5. Although for the Merinos the low GSH trait can be explained in terms of a diminished activity of gamma-glutamyl-cysteine synthetase, no such explanation is tenable for the Finnish Landrace sheep.

Coenzyme A requirement of malaria parasites: enzymes of coenzyme A biosynthesis in normal duck erythrocytes and erythrocytes infected with Plasmodium lophurae.

Normal duck erythrocytes and erythrocytes infected with Plasmodium lopharae have all of the enzymes for coenzyme A biosynthesis, whereas parasites freed from their host cells have non. Since erythrocytefree cultivation of P. lophurae requires an exogenous source of coenzyme A, this parasite must obtain its coenzyme A entirely from the host cell during infection.

[Biosynthesis of glutathione. VI. Ocurrence and level of gamma-glutamylcysteine in human red cells (author's transl)]. / Glutathionbiosynthese, VI. Der gamma-Glutamylcystein-Spiegel in menschlichen Erythrozyten

The concentration of the glutathione precursor gamma-glutamylcysteine in hemolysates of human red cells was determined using three different methods. a) The [14C]N-ethylmaleimide derivatives of the intracellular sulfhydryl compounds were separated on a cation exchange column. The amount of [14C]N-ethylmaleimide glutamycysteine was radiochemically determined. b) With purified glutathione synthetase, an enzymatic endpoint metabolite determination was performed with [14C]glycine as one substrate. The amount of labelled glutathione formed from the gamma-glutamylcysteine present was measured radiochemically.

[Assay and normal levels of L-glutamate-L-cysteine-gamma-ligase (E.C. 6.3.2.2) in human erythrocytes; biosynthesis of glutathione V]. / Aktivitätsbestimmung und Normalwerte von L-Glutamat-L-Cystein-gamma-Ligase (EC 6.3.2.2) in menschlichen Erythrocyten; Glutathionbiosynthese V)

For the determination of the activity of glutamate: cysteine-gamma-ligase one method was optimized and a new micromethod was developed: a) Utilizing [14C] glutamate and cysteine as substrates, the product [14C] gamma-glutamylcysteine was isolated as its cadmium mercaptide and determined by liquid scintillation counting gamma-glutamyl-[14C] aminobutyrate, which was synthesized from glutamate and [14C] alpha-aminobutyrate was separated from remaining radioactive substrate by paper electrophoresis and counted. Both methods were used to determine the activity of the enzyme in human red blood cells. For 20 parallel determinations a standard deviation of +/- 9% and +/- 5% was obtained for method a and b, respectively. The specific activity of the enzyme in erythrocytes of adults was found to be 0.40 +/- 0.05 U/g hemoglobin with method a, and 0.50 +/- 0.05 U/g hemoglobin with method b.