Hexosediphosphatase from spinach chloroplasts: Purification,crystallization and some properties

Hexosediphosphatase (d-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) has been isolated, purified, and crystallized, from previously isolated spinach chloroplasts. The effects of various anions, cations, and sulfhydryl compounds were tested, and activation by Mg²⁺, glycine, HCO₃^?, and sulfhydryl compounds is described. The purified enzyme is very specific for fructose 1,6-diphosphate and does not attack sedoheptulose-1,7-bisphosphate. The s₂₀ value of the enzyme was 7.7, from which the molecular weight of the enzyme was estimated as 140 000.     

A modulating role of mercurials-sensitive sulfhydryl groups in synaptic GABA receptor binding

The specific binding of GABA (γ-aminobutyric acid) agonist ³H-muscimol, to synaptic membranes from the rat brain showed a significant increase, when the membranous preparations were treated with a low concentration (10^?4–10^?5M) of mercurial sulfhydryl reagents such as p-chloromercuribenzoate and mercuric chloride. This activation in GABA receptor binding was bicuculline-sensitive, and was partially restored by subsequent treatments with 10 mM cysteine, penicillamine, or mercaptoethanol. Scatchard analysis of the binding revealed that this activation was due to the increase in the affinity of both high and low affinity bindings sites but not in the B_max values. On the other hand, the treatment of synaptic membranes with hydrophilic sulfhydryl reagents such as N-ethylmaleimide and iodoacetate had no effect on the binding. These hydrophilic sulfhydryl reagents, however, induced an increase of the binding following the pretreatment of synaptic membranes with 0.01% Triton X-100 or 0.5 U/mg prot. of phospholipase A₂ (EC 3.1.1.4.). These results suggest that mercurials-sensitive sulfhydryl groups, which are normally masked by membrane lipids, may play a modulating role in GABA receptor binding at central synapses.     

Controlled Administration of Penicillamine Reduces Radiation Exposure in Critical Organs during 64Cu-ATSM Internal Radiotherapy: A Novel Strategy for Liver Protection

Purpose

⁶⁴Cu-diacetyl-bis (N ⁴-methylthiosemicarbazone) (⁶⁴Cu-ATSM) is a promising theranostic agent that targets hypoxic regions in tumors related to malignant characteristics. Its diagnostic usefulness has been recognized in clinical studies. Internal radiotherapy (IRT) with ⁶⁴Cu-ATSM is reportedly effective in preclinical studies; however, for clinical applications, improvements to reduce radiation exposure in non-target organs, particularly the liver, are required. We developed a strategy to reduce radiation doses to critical organs while preserving tumor radiation doses by controlled administration of copper chelator penicillamine during ⁶⁴Cu-ATSM IRT.

Methods

Biodistribution was evaluated in HT-29 tumor-bearing mice injected with ⁶⁴Cu-ATSM (185 kBq) with or without oral penicillamine administration. The appropriate injection interval between ⁶⁴Cu-ATSM and penicillamine was determined. Then, the optimal penicillamine administration schedule was selected from single (100, 300, and 500 mg/kg) and fractionated doses (100 mg/kg×3 at 1- or 2-h intervals from 1 h after ⁶⁴Cu-ATSM injection). PET imaging was performed to confirm the effect of penicillamine with a therapeutic ⁶⁴Cu-ATSM dose (37 MBq). Dosimetry analysis was performed to estimate human absorbed doses.

Results

Penicillamine reduced ⁶⁴Cu accumulation in the liver and small intestine. Tumor uptake was not affected by penicillamine administration at 1 h after ⁶⁴Cu-ATSM injection, when radioactivity was almost cleared from the blood and tumor uptake had plateaued. Of the single doses, 300 mg/kg was most effective. Fractionated administration at 2-h intervals further decreased liver accumulation at later time points. PET indicated that penicillamine acts similarly with the therapeutic ⁶⁴Cu-ATSM dose. Dosimetry demonstrated that appropriately scheduled penicillamine administration reduced radiation doses to critical organs (liver, ovaries, and red marrow) below tolerance levels. Laxatives reduced radiation doses to the large intestine.

Conclusions

We developed a novel strategy to reduce radiation exposure in critical organs during ⁶⁴Cu-ATSM IRT, thus promoting its clinical applications. This method could be beneficial for other ⁶⁴Cu-labeled compounds.     

Metal binding drugs induce synthesis of four proteins in normal cells

The synthesis of four proteins in chick embryo cells in culture is induced by exposure to several metal binding drugs and several cations. We reported previously that two chelating agents, kethoxal bis(thiosemicarbazone) and disulfiram induce these proteins. We now extend these findings to include 8-hydroxyquinoline, ortho-phenanthroline, and thiosemicarbazide. The non-chelating analogues of these latter three compounds; 4-hydroxyquinoline, metaphenanthroline, and semicarbazide, do not induce. Other chelating agents, such as thenoyl trifluoroacetone, mercaptopyridine N-oxide, mercaptobenzothiazole, and methimazole induce. However, not all chelators induce since EDTA, penicillamine, α,α-dipyridyl, and several others are ineffective. Several cations, such as copper, zinc, cadmium, and mercuric ions induce, but cobalt, nickel, manganese, iron, lead, and platinum do not. The anions arsenite and arsenate induce, but bromide, dichromate, fluoride, metabisulfite, molybdate, nitrite, permanganate, phosphate, sulfite, and sulfate ions do not. The chelating agents that induce the proteins are ionophores for⁶⁴Cu. The chelating agents that do not induce, such as EDTA, penicillamine, and the nonchelating analogs of the inducers, are not ionophores for copper. Since arsenite induces but is not a copper ionophore, we hypothesize that the common mechanism for both these compounds and the inducing cations is an interaction with a sulfhydryl group site.     

Purification and Some Properties of Extracellular Protease of Euglena gracilis Z

The extracellular protease of Euglena gracilis z was purified to a single protein. It was an endopeptidase as found by the Nunokawa’s method, and showed optimum pH for the proteinase, esterase and amidase activities at 7.3, 7.0 and 6.3, respectively. It had a molecular weight of 41,000 and isoelectric point of 8.3. The bleached mutant of E. gracilis produced higher activity of extracellular protease than the wild strain, and supplementation of peptone to the growth medium augmented the enzyme production in both green and bleached cells. The Euglena extracellular protease was markedly inhibited by diisopropylfluorophosphate and Streptomyces subtilicin inhibitor, and to lesser extents by EDTA and p-chloromercuribenzoate. The enzyme was potentiated by some sulfhydryl compounds, activated greatly by Fe²⁺ and stabilized by Ca²⁺ and K⁺.     

Effect of sulfhydryl compounds on ATP-stimulated H+ transport and Cl− uptake in rabbit renal cortical endosomes

Summary The vacuolar H⁺ ATPase is inhibited by N-ethylmaleimide (NEM), a sulfhydryl compound, suggesting the involvement of a sulfhydryl group in this transport process. We have examined the effects of several sulfhydryl-containing compounds on the vacuolar H⁺ ATPase of rabbit renal cortical endosomes. A number of such compounds were effective inhibitors of endosomal H⁺ transport at 10^–5–10^–6 m, including NEM, mersalyl, aldrithiol, 5,5 dithiobis (2-nitrobenzoic acid),p-chloromercuribenzoic acid (PCMB) andp-chloromercuriphenyl sulfonic acid (PCMBS). NEM, mersalyl, aldrithiol and PCMBS had no effect on pH-gradient dissipation, whereas PCMB decreased the pH gradient faster than control. In the absence of ATP, PCMB (10^–4 m) stimulated endosomal³⁶Cl^– uptake, particularly in the presence of an inside-alkaline pH gradient (pH_in=7.6/pH_out=5.5.). This result was not an effect of PCMB on the Cl^–-conductive pathway. The less permeable PCMBS did not stimulate³⁶Cl^– uptake. The effects of PCMB were concentration dependent and were prevented by dithioerithritol,. ATP-dependent³⁶Cl^– uptake was decreased by addition of PCMB. Finally, PCMB had no effect on⁴⁵Ca²⁺ uptake. These results support the presence of two functionally important sulfhydryl groups in this endosomal preparation. One such group is involved with ATP-driven H⁺ transport and must be located on the cytoplasmic surface of the endosomal membrane. The second sulfhydryl group must reside on the internal surface of the endosomal membrane and relates to a PCMB-activated Cl^–/OH^– exchanger that is functional both in the presence and absence of ATP. This endosomal transporter is similar to the PCMB-activated Cl^–/OH^– exchanger recently described in rabbit renal brush-border membranes.     

Regulatory sulfhydryl groups, conformational change, and allosteric inhibition in rabbit muscle fructose diphosphatase

The 16 sulfhydryl groups of native, homogeneous rabbit muscle fructose diphosphatase can all react with 5,5′-dithiobis-(2-nitrobenzoic acid). High concentrations of substrate (1–2 mm) decrease the reaction rate of the sulfhydryl groups, while concentrations up to 70 μm have no effect. After titration of the four most rapidly reacting sulfhydryl groups there is a marked desensitization toward the allosteric inhibitor AMP. In the presence of 30 μm AMP only 4–5 sulfhydryl groups/tetramer react with 5,5′-dithiobis-(2-nitrobenzoic acid), and the enzyme again becomes desensitized toward AMP inhibition. Together with a 3.5-fold increase in the I₅₀ for AMP inhibition, the K_m for Mg²⁺ or Mn²⁺ ions is also increased. In the presence of 7 mm MgCl₂ or 0.28 mm MnCl₂ only 6–8 sulfhydryl groups are modified. The rapid reaction of 4 sulfhydryl groups again results in desensitization. There is neither a protection by the substrate against inactivation, nor a protection by the allosteric inhibitor against desensitization. It is concluded that AMP and the divalent cations induce conformational changes in the protein molecule making 11–12 or 8–10 sulfhydryl groups inert for 5,5′-dithiobis-(2-nitrobenzoic acid), respectively. The K_m for fructose-1,6-diphosphate is not changed after the modification of 4–5 sulfhydryl groups.     

Phenylethynyl-Butyltellurium Inhibits the Sulfhydryl Enzyme Na+, K+-ATPase: An Effect Dependent on the Tellurium Atom

Organotellurium compounds are known for their toxicological effects. These effects may be associated with the chemical structure of these compounds and the oxidation state of the tellurium atom. In this context, 2-phenylethynyl-butyltellurium (PEBT) inhibits the activity of the sulfhydryl enzyme, δ-aminolevulinate dehydratase. The present study investigated on the importance of the tellurium atom in the PEBT ability to oxidize mono- and dithiols of low molecular weight and sulfhydryl enzymes in vitro. PEBT, at high micromolar concentrations, oxidized dithiothreitol (DTT) and inhibited cerebral Na⁺, K⁺-ATPase activity, but did not alter the lactate dehydrogenase activity. The inhibition of cerebral Na⁺, K⁺-ATPase activity was completely restored by DTT. By contrast, 2-phenylethynyl-butyl, a molecule without the tellurium atom, neither oxidized DTT nor altered the Na⁺, K⁺-ATPase activity. In conclusion, the tellurium atom of PEBT is crucial for the catalytic oxidation of sulfhydryl groups from thiols of low molecular weight and from Na⁺, K⁺-ATPase.     

Effects of various compounds on growth of yeast cells of Histoplasma capsulatum

Summary A number of compounds were screened for their effects on growth of the yeast cells ofHistoplasma capsulatum. Included were penicillin and related compounds, sulfhydryl inhibitors, various organic sulfur compounds recently synthesized for the first time, and compounds structurally related to the required metabolites, thiamine and cystine or cysteine. Cephalothin was the only one of the penicillin related compounds which inhibited growth. This occurred only when a high concentration (8.3 × 10^–4 M) was used. Of the analogues of cystine tested, allylglycine had the greatest inhibitory effect on growth of the yeast cells in the synthetic medium, but it failed to inhibit growth in a complex medium containing peptones and plasma. Among the sulfhydryl inhibitors, the maleimides were the most effective, producing complete inhibition of growth in the peptone medium at 10µg/ml or less. At subinhibitory concentrations the cultures tended to become mycelial. The action of the maleimides was reversed by cystine over a range of concentrations. At low concentrations, some of the disulfide derivatives of thiamine stimulated growth equally as well as thiamine, but at concentrations of 100 to 150µg/ml, they completely inhibited growth. On the basis of results obtained to date, three classes of the new organic sulfur compounds being tested offer promise as sources of potentially useful chemotherapeutic agents. These classes, which differ widely in structure, are as follows: the benzyl decylaminoethyl disulfides, the acyl disulfides, and the trithiopercarbamates.This investigation was supported by Public Health Service Research Grant AI-03524 from the National Institute of Allergy and Infectious Diseases.     

Alkylthiolation. Evidence for involvement in cell division

Several malignant hematopoietic cells from mice, previously shown to require sulfhydryl compounds and serum for proliferation $\text{in vitro}$, are shown to grow well in medium lacking both of these components if methylthio compounds are provided at appropriate concentrations. The methylthio groups can be provided as the mixed disulfides of methyl mercaptan and various thiols. The optimal concentration of the various disulfides is between 1×10^?5 and 1×10^?4 M. The cells can be maintained indefinitely in serum-free medium containing cysteine-methyl disulfide and one of several purified proteins. The evidence is consistent with the theory that these cells require an exogenous source of alkylthio groups for division, that serum is a source of these groups, and that sulfhydryl compounds act as carriers to transport the groups into the cells. A new diffusion analysis is described for quantitative measurement of disulfides of volatile mercaptans.     

Effects of cations,sugars, detergents,sulfhydryl compounds,and cryoprotective agents on the colorimetric determination of N-acetylglucosamine by the method of reissig

The monovalent cations, K⁺ and Na⁺, and the detergents, Triton X-100, Hyamine 2389, and sodium dodecyl sulfate, as well as the sugars, sucrose and α-methylglucoside, do not interfere with the colorimetric determination of N-acetylglucosamine. On the other hand, divalent cations, cryoprotective agents, and sulfhydryl compounds do influence the test of Reissig. By order of increasing effectiveness in reducing color production we find, among the cations, Mg²⁺ < Ca²⁺ < Mn²⁺ ≤ Zn²⁺; among the cryoprotective agents, dimethylsulfoxide < polyethylene glycol < glycerol; and among the sulfhydryl compounds, mercaptoethanol < dithiothreitol < cysteine. Not every interfering agent exerts its effect through the same mechanism. Only the first step of the test of Reissig is impaired by the divalent ions and dimethylsulfoxide while both steps are affected by the other agents. By using the appropriate calibration curve it is possible to determine the real effect of any interfering agent on an enzyme whose activity is monitored by the test of Reissig. By doing so, we have been able to show that Ca²⁺ does have an effect on bull acrosomal hyaluronidase; it activates the enzyme.     

The in vitro loss of penicillamine in plasma,albumin solutions,and whole blood: Implications for pharmacokinetic studies of penicillamine

The recent development of a high performance liquid chromatography assay method for the analysis of penicillamine in biological samples such as plasma, whole blood, and urine has provided a specific and sensitive assay method to aid in the study of penicillamine pharmacokinetics. Several investigators have reported measuring the plasma concentration of penicillamine. Some of these investigators have indicated that the plasma must be assayed immediately. However, such restrictions can limit the feasibility of a pharmacokinetic study. The results of this paper demonstrate the instability of penicillamine in plasma, albumin solutions, and whole blood. The rate of loss of penicillamine was shown to be influenced by the concentration of albumin. As a result of the significant loss of penicillamine over a short period of time, plasma or whole blood samples must be deproteinated immediately upon collection to avoid the loss of reduced penicillamine. Methods are presented for the preparation of biological samples so that the oxidation of penicillamine is prevented and the samples can be held for several days prior to analysis.     

DNA biosynthesis in rat liver mitochondria: Inhibition by sulfhydryl compounds and stimulation by cytoplasmic proteins

The sulfhydryl compounds, 2-mercaptoethanol, dithiothreitol, cysteine. and glutathione inhibit the incorporation of [³H]dTTP or [³H]dATP into mitochondrial DNA by rat liver mitochondria in vitro. The lack of inhibition by non-SH-containing analogs indicates that the SH group is responsible for the inhibition.The inhibition does not result from an effect of the sulfhydryl compounds on precursor permeability, ATP formation, or respiration, or the action of the thiol on the outer mitochondrial membrane. An intact inner membrane is not required for the action of the inhibitor. Furthermore, SH compounds do not appear to exert their effect by activation of a mitochondrial nuclease, chemical breakdown of high molecular-weight mitochondrial DNA or dissociation of membrane-bound DNA from the inner mitochondrial membrane. Incorporation of labeled precursor into DNA by mitochondrial DNA polymerase, when removed from the inner mitochondrial membrane, is not inhibited by SH compounds.Cytoplasmic extracts prepared from rat and mouse tumors and 22-h regenerating rat liver contain a protein(s) not detectable in normal rat liver which can reverse the inhibition by SH compounds of the synthesis of mitochondrial DNA in rat liver mitochondria in vitro.More importantly, when the stimulatory protein(s) is partially purified by affinity chromatography on DNA-cellulose, it is possible to demonstrate that this protein(s) also stimulates the synthesis of mitochondrial DNA by normal rat liver mitochondria in vitro in the absence of the sulfhydryl inhibitor.     

The Source for the Difference Between Sulfhydryl and Hydroxyl Anions in Their Nucleophilic Addition Reaction to a Carbonyl Group: A DFT Approach.

Ab initio calculations show that sulfhydryl anion has a significantly lower potential than the hydroxide anion for stabilizing the products of its attack on carbonyl moieties - the tetrahedral complexes (TC). In this paper we analyze the factors that contribute to this phenomenon. Quantum mechanical MO ab initio calculations were used for studies of two reaction series, one for the attack of hydroxyl and one for the attack of sulfhydryl anion on different carbonyl compounds and their analogs. All of the anionic TCs formed by HS^- are characterized by higher charge transfer, but are significantly less stable than the relevant TC of HO^-. To explain the phenomenon we used a simple qualitative model based on Density Functional Theory (DFT). The crucial role of the occupied valence MOs is demonstrated in the process of electronegativity equalization between the donor and acceptor fragments in the final TC product. The sulfhydryl anion has significantly lower potential to stabilize TC products in comparison with the hydroxide anion because of the larger extent of electron back-donation from the electrophiles HOMO_A to the nucleophiles LUMO_D. This electron back-donation thus reduces the stability of the anionic TC in the case of HS^- and may account for the calculational results. Applications of this work to enzyme reactions help in understanding the differences in mechanisms of serine and cysteine proteases and may be used to guide the design of inhibitors for these enzymes. In perspective, the back-donation phenomenon discussed here may be applied to the study of electron transfer processes involving oxidation-reduction enzymes.     

Evaluation of Factors Affecting the Survival of Escherichia coli in Sea Water: VI. Cysteine

The relationship between death of cells of Escherichia coli in artificial sea water and time was established as linear, and statistical tests demonstrated that the most suitable measure of survival was log per cent after 24 hr. Survival of E. coli in water supplemented with cysteine at levels of 0.284 × 10^-6 to 284 × 10^-6m was increased greatly over that in untreated water. To provide an insight into the mode of action of cysteine, the effect of concentration of various sulfhydryl and disulfide compounds was measured, and the influence of several compounds that lack a functional sulfur group but which are capable of affecting oxidation-reduction potential was determined. Moreover, a number of substances related structurally to cysteine were tested to ascertain their influence on the survival of cells of E. coli in artificial sea water. It appeared that the beneficial effect of cysteine was not due to the sulfhydryl group of the amino acid or to the ability of the compound to influence oxidation-reduction potential. Some sulfhydryl compounds had no favorable effect and, in general, disulfides were more active than the corresponding sulfhydryl compounds. Substances that lack a functional sulfur group but influence oxidation-reduction potential had no significant activity. The beneficial effect of a number of compounds related structurally to cysteine indicates that both an amino and carboxyl group are required for activity. It is suggested that cysteine and other amino acids act to increase survival of cells of E. coli in sea water by a chelation mechanism.     

Studies on the Induced Synthesis of Maleate cis-trans Isomerase by Malonate

Maleate cis-trans isomerase in Alcaligenes faecalis IB–14 was induced by malonate and purified about 100-fold over the crude cell-free extract by treatments of ammonium sulfate fractionation, Sephadex G–100 gel filtration, DEAE-cellulose and DEAE-Sephadex A–50 column chromatography. The preparation was shown to be monodisperse on ultracentrifugal analysis and Svedberg value was found to be 3.84 S.

The enzyme was most active at pH value around 8.3 and was stable over the range of pH 5.0 to 7.0 in the presence of dithiothreitol (DTT) for a few weeks, but in the absence of it, the enzyme activity was markedly decreased, especially in the alkaline region. The enzyme activity was inhibited by various sulfhydryl reagents and oxidizing agents, whereas it was not affected by metal chelating agents. The inhibition by Hg²⁺ and PCMB was overcome by the addition of sulfhydryl compounds such as DTT, 2-mercaptoethanol, l-cysteine and glutathione. It was observed that the enzyme did not require co-factor for its function.

Kinetic studies showed that Michaelis constant for maleate was 2.8×10^?3 m and the enzyme did not catalyze the reverse reaction.     

Modification of the erythrocyte membrane by sulfhydryl group reagents

Summary In this study, the consequences of modification of human erythrocyte membrane sulfhydryl groups by N-ethyl maleimide (NEM), 5,5dithiobis-(2-nitrobenzoic acid) (DTNB) andp-hydroxymercuriphenyl sulfonate (PHMPS) were investigated. These reagents differ in chemical reactivity, membrane penetrability and charge characteristics.Results of sulfhydryl modification were analyzed in terms of inhibitory effects on activities of five membrane enzymes; Mg⁺⁺- and Na⁺, K⁺-ATPase, K⁺-dependent and independentp-nitrophenyl phosphatase (NPPase) and DPNase. Structural considerations involved in the sulfhydryl-mediated inhibition were evaluated by studying the changes in susceptibility to sulfhydryl alteration produced by shearing membranes into microvesicles and by the addition of the membrane modifiers, Mg⁺⁺ and ATP.Conclusions from the data suggest that the effects of NEM appeared to result from modification of a single class of sulfhydryls; DTNB interacted with two different sulfhydryl classes. Increasing concentrations of PHMPS resulted in the sequential modification of many types of sulfhydryls, presumably as a result of increasing membrane structural disruption. DTNB and PHMPS caused solubilization of about 15% of membrane protein at concentrations giving maximal enzyme inhibition.In contrast to the usually observed parallels between Na⁺, K⁺-ATPase and K⁺-dependent NPPase, activities of Mg⁺⁺-ATPase, Na⁺, K⁺-ATPase and K⁺-dependent NPPase varied independently as a result of sulfhydryl modification. We suggest complex structural and functional relationships exist among these components of the membrane ATP-hydrolyzing system.Our studies indicate that the effects of sulfhydryl group reagents on these membrane systems should not be ascribed to sulfhydryl modificationper se, but rather to the resulting structural perturbations. These effects depend upon the structural characteristics of the particular membrane preparation studied and on the chemical characteristics of the sulfhydryl group reagent used.     

Use of N-ethylmaleimide to prevent interference by sulfhydryl reagents with the glucose oxidase assay for glucose

Interference with the glucose oxidase-peroxidase method of glucose determination by the sulfhydryl agents cysteine and reduced glutathione can be overcome simply by adding N-ethylmaleimide to the assay system. A 30-fold molar excess of N-ethylmaleimide over the amount of glucose present produced no interference of its own and completely prevented the effects of cysteine and glutathione. It is suggested that this agent be added to the reaction mixture whenever it is suspected that low molecular weight sulfhydryl compounds may be present in samples to be analyzed for glucose.     

Determination of total aminothiols and neuroactive amino acids in plasma by high performance liquid chromatography with fluorescence detection

This paper describes a simple and sensitive reversed-phase HPLC method for the determination of total homocysteine, total cysteine, total glutathione (GSH + GSSG), and neuroactive amino acids (Asp, Glu, Tau, GABA) using precolumn derivatization with ortho-phtaldialdehyde and fluorimetric detection at 360 and 470 nm for emission and excitation, respectively. Derivatization was performed with ortho-phthaldialdehyde in the presence of 2-mercaptoethanol after alkylation of free sulfhydryl groups with iodoacetic acid. For determination of total aminothiols, the disulfide bonds were reduced and protein-bound thiols were released by addition of dithiothreitol to the plasma sample. The advantage of this method is the simultaneous determination of both homocysteine/cysteine/glutathione and neuroactive amino acids in the sample. The plasma levels of studied compounds were determined in 14 healthy volunteers (20–45 years old) and 55 patients with chronic hepatitis C (20–49 years old) and the resulting values were in a good agreement with results published earlier. The calibration curves were linear over a concentration range of 5–100 μM in plasma (r ² = 0.985−0.996). The intraday and interday coefficients of variation were 3–6% and 4–7%, respectively. The recovery of the standards added to the plasma samples ranged from 94 to 102%. The limits of detection (LOD) were 0.2–0.5 ng per 10 μl of the injection volume (signal-to-noise ratio of 3).     

Metabolism of caffeine by mouse liver microsomes: GSH or cytosol causes a shift in products from 1, 3, 7-trimethylurate to a substituted diaminouracil

Incubation of [¹⁴C] caffeine with hepatic microsomes from male AKR/J mice resulted in the formation of several metabolites including 1, 3, 7-trimethylurate and 6-amino-5-(N-formylmethyl-amino)-1, 3-dimethyluracil. These two compounds comprised about 60% of products and are major urinary metabolites in several animals. When cytosol was included during incubation, there was a 14-fold increase in yield of the uracil at the expense of the urate; the combination of the two metabolites remained about 60% of total products. Cytosol alone was catalytically inert. Glutathione and other sulfhydryl compounds reproduced the effect of cytosol, and the action of cytosol was accounted for quantitatively by its sulfhydryl content. We propose that an oxidized intermediate of caffeine en route to trimethylurate is reduced by glutathione to the ring-opened uracil derivative.