Oxidation of chlorpromazine by methemoglobin in the presence of hydrogen peroxide. Formation of chlorpromazine radical cation and its covalent binding to methemoglobin

The oxidation of chlorpromazine by methemoglobin plus H2O2 has been studied. The transient formation of the chlorpromazine radical cation in this reaction has been demonstrated by light absorption measurements. Under the experimental conditions complete conversion of chlorpromazine yields approximately 60% chlorpromazine sulfoxide. From studies with 3H-labeled chlorpromazine it appears that the remaining 40% is covalently bound to apohemoglobin. Upon reaction of methemoglobin with H2O2 a stable ferrylhemoglobin is formed. This ferrylhemoglobin is not the reactive species, which accepts the chlorpromazine electron, as its presence is not sufficient to induce chlorpromazine oxidation. For this the presence of H2O2 is a prerequisite. This indicates that a transient species in the formation of the stable ferrylhemoglobin is involved, whether this is a compound I analogue or a ferrylhemoglobin with a free radical on one of the apoprotein residues. Exposition of methemoglobin to H2O2 denatures hemoglobin and induces protein-heme crosslinks, as appears from changes in the visible absorption spectrum and heme retention by the protein after methyl ethyl ketone extraction. Reaction with CPZ partly protects against denaturation and crosslinking.     

Rapid in vitro sulfoxidation of chlorpromazine by human blood: inhibition by an endogenous plasma protein factor.

Sulfoxidation of chlorpromazine is a major metabolic pathway in humans and is clinically significant insofar as chlorpromazine-sulfoxide is thought to be pharmacologically inactive. Sulfoxidizing capacities of hepatic and extrahepatic tissues were examined $\text{in vitro}$. Liver, lung, and gut, but not brain, were found to have differential activities (liver>lung>gut) which were NADPH dependent and had characteristics of enzymatic reactions. NADPH independent sulfoxidation of chlorpromazine is seen in the presence of whole blood. This reaction reaches completion in less than 5 sec. and is considerably more active than that seen with other tissues; the total sulfoxidizing capacity of whole blood can be attributed to hemoglobin.Plasma contains a protein-like factor that actively inhibits blood-catalyzed chlorpromazine sulfoxidation. This factor has a molecular weight of 72,000±6000 daltons. The possible roles of this protein in regulating phenothiazine metabolism are discussed.     

Enzymatic assay of hemoglobin in tissue homogenates with chlorpromazine

An assay of hemoglobin in tissue homogenates is described. Tissues are homogenized with a 40 mM phosphate buffer (pH 8.0) solution without the use of a polytron or sonicator. The assay should be carried out before fractionations such as centrifugation. By measuring the free-radical intermediates of chlorpromazine, which are formed by the addition of hemoglobin, a linear calibration curve can be obtained within the range of hemoglobin contents between 0.8 and 12.0 micrograms. The present method can be applied to the assay of hemoglobin in several kinds of tissues from rats.     

Chlorpromazine induced aggregation of normal human hemoglobin. Electron microscopic evidence.

Normal human hemoglobin exceeding a certain minimum concentration (called critical aggregation concentration) undergoes aggregation in presence of the psychotherapeutic drug chlorpromazine (CPZ). The critical aggregation concentration decreases with the increase of CPZ concentration. Electron micrographs of CPZ-treated hemoglobin clearly indicate that the aggregates of hemoglobin are in filamentous form of average width 75 +/- 8 A. A possible mechanism for such aggregation has been discussed.     

Interaction of chlorpromazine with hemoglobin

Binding of chlorpromazine (CPZ), a widely used antidepressant tranquilizer, with hemoglobin has been studied by equilibrium dialysis method. r/Cf versus r plot was typically concave downwards revealing the positive cooperative nature of binding. Binding parameters, namely the affinity constant (K) and the degree of cooperativity (nH) were determined from the Hill plot. Oxygen was found to be released gradually from hemoglobin with gradual addition of CPZ, the extent of oxygen release depending on the stoichiometric ratio of CPZ: hemoglobin (D/P).     

Exploratory studies of some drug charge transfer interactions

Conductivity and capacitance titrations yield minima for the chlorpromazine hydrochloride-heparin interaction, confirming clinical suspicions of its occurrence. The effective dosage of heparin thus is reduced if administered in conjunction with chlorpromazine. The interaction is interpreted as charge transfer complex formation, occurring as an (electrode) surface reaction. It is suggested that the charge transfer complexing capability of heparin preparations, as evidenced by conductance and/or capacitance changes, evaluated against a well defined donor such as chlorpromazine hydrochloride, may be adapted as a more precise method of measuring heparin activity than coagulation time determinations. Phenytoin and chlorpromazine likewise yield conductance and capacitance minima; voltammetry indicates new peaks at +250mV and −300mV vers.SCE supporting the suggestions that an uncharged 1∶1 complex is being formed, again in a type of surface reaction. Phenytoin and lignocain form a precipitate at 0.002 equimolar; in conductance and capacitance titrations phenytoin behaves as a weak electron donor against iodine though as a weak acceptor against lignocain. Lignocain and chlorpromazine conductance and capacitance titrations using gold electrodes fail to show any evidence for their previously reported interaction on Pt/Pt electrodes. Voltammetry on Pt/Pt electrodes indicates 2 new peaks at zero and at −750mV vers.SCE. It is thought that these two compounds interact only on catalytically highly active surfaces, where they form a weak surface charge transfer complex. Adrenalin, in conductance and capacitance titrations, behaves amphoteric, i.e. as an electron acceptor against the strong donor chlorpromazine and as a donor against the strong acceptor tetramethyl-p-phenylenediamine. Voltammograms of the above listed interactions are interpreted as of the ECE type exhibiting mainly irreversible behaviour.     

Application of differential flow microcalorimetry for study of drug interactions in the blood system

A compact differential flow microcalorimeter has been developed to investigate biomolecular reactions, especially drug interactions in the blood system. The calorimeter is an adiabatic type and consists of a twin-cell structure, each mixing part having a volume of 60 microliters. Both the precision and accuracy of the instrument have been evaluated by dilution of sucrose solutions to be 0.1-0.5% at a heat effect of 100-10 microW. The resolution is approximately 0.5 microW (less than 10(-3) Torr). The heat produced in erythrocyte hemolysis induced by chlorpromazine hydrochloride (CPZ) and the binding heat of CPZ to human blood components viz., intact erythrocytes, erythrocyte membranes, serum albumin and plasma were measured. The heat effect of hemolysis was endothermic and related to the quantity of free hemoglobin released from erythrocytes. The overall binding of CPZ to blood components was, however, an exothermic process. The thermodynamic and binding parameters were computed directly from the calorimetric data by use of a nonlinear least squares regression method, assuming a one-class binding model, and the stoichiometry of the binding reaction was determined.     

The effects of amphiphilic cationic drugs and inorganic cations on the activity of phosphatidate phosphohydrolase.

1. Phosphatidate phosphohydrolase from the particle-free supernatant of rat liver was assayed by using emulsions of phosphatidate as substrate. 2. The inhibition of the phosphohydrolase by chlorpromazine was of a competitive type with respect to phosphatidate. The potency of various amphiphilic cationic drugs as inhibitors of this reaction was related to their partition coefficients into a phosphatidate emulsion. 3. The effect of chlorpromazine on the phosphohydrolase activity was complementary rather than antagonistic towards Mg2+. Chlorpromazine stimulated the phosphohydrolase activity in the absence of added Mg2+ and was able to replace the requirement for Mg2+. However, at optimum concentrations of Mg2+, chlorpromazine inhibited the reaction, as did Ca2+. The phosphohydrolase activity was also stimulated by Co2+ and to a lesser extent by Mn2+, Fe2+, Fe3+, Ca2+, spermine and spermidine when Mg2+ was not added to the assays. 4. It is concluded that the inhibition of phosphatidate phosphohydrolase by amphiphilic cations can largely be explained by the interaction of these compounds with phosphatidate, which changes the physical properties of the lipid, making it less available for conversion into diacylglycerol. 5. The implications of these results to the effects of amphiphilic cations in redirecting glycerolipid synthesis at the level of phosphatidate are discussed.     

On the mechanism of inhibition of the bovine heart F1-ATPase by local anesthetics

The rate of inactivation of the mitochondrial F₁-ATPase by dicyclohexylcarbodiimide is slowed by concentrations of chlorpromazine, dibucaine, or tetracaine which have been shown by others (B. Chazotte, G. Vanderkooi, and D. Chignell (1982)$\text{Biochim. Biophys. Acta}\text{680}$, 310–316) to inhibit the hydrolytic reaction catalyzed by the enzyme. The order of effectiveness of the drugs as protectors of the enzyme against inactivation by dicyclohexylcarbodiimide is: chlorpromazine > dibucaine > tetracaine. Examination of the steady state kinetics showed the chlorpromazine inhibits the ATPase competitively at concentrations up to 18.5 μM while complex kinetic behavior is exhibited at chlorpromazine concentrations from 25–50 μM. These results suggest that the drugs inhibit the F₁-ATPase by interacting with the catalytic site of the enzyme and not by promoting its dissociation.     

Drug-protein interactions: binding of chlorpromazine to calmodulin, calmodulin fragments, and related calcium binding proteins

The quantitative binding of a phenothiazine drug to calmodulin, calmodulin fragments, and structurally related calcium binding proteins was measured under conditions of thermodynamic equilibrium by using a gel filtration method. Plant and animal calmodulins, troponin C, S100 alpha, and S100 beta bind chlorpromazine in a calcium-dependent manner with different stoichiometries and affinities for the drug. The interaction between calmodulin and chlorpromazine appears to be a complex, calcium-dependent phenomenon. Bovine brain calmodulin bound approximately 5 mol of drug per mol of protein with apparent half-maximal binding at 17 microM drug. Large fragments of calmodulin had limited ability to bind chlorpromazine. The largest fragment, containing residues 1-90, retained only 5% of the drug binding activity of the intact protein. A reinvestigation of the chlorpromazine inhibition of calmodulin stimulation of cyclic nucleotide phosphodiesterase further indicated a complex, multiple equilibrium among the reaction components and demonstrated that the order of addition of components to the reaction altered the drug concentration required for half-maximal inhibition of the activity over a 10-fold range. These results confirm previous observations using immobilized phenothiazines [Marshak, D.R., Watterson, D.M., & Van Eldik, L.J. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 6793-6797] that indicated a subclass of calcium-modulated proteins bound phenothiazines in a calcium-dependent manner, demonstrate that the interaction between phenothiazines and calmodulin is more complex than previously assumed, and suggest that extended regions of the calmodulin molecule capable of forming the appropriate conformation are required for specific, high-affinity, calcium-dependent drug binding activity.     

Nitric oxide formation from the reaction of nitrite with carp and rabbit hemoglobin at intermediate oxygen saturations

The nitrite reductase activity of deoxyhemoglobin has received much recent interest because the nitric oxide produced in this reaction may participate in blood flow regulation during hypoxia. The present study used spectral deconvolution to characterize the reaction of nitrite with carp and rabbit hemoglobin at different constant oxygen tensions that generate the full range of physiological relevant oxygen saturations. Carp is a hypoxia-tolerant species with very high hemoglobin oxygen affinity, and the high R-state character and low redox potential of the hemoglobin is hypothesized to promote NO generation from nitrite. The reaction of nitrite with deoxyhemoglobin leads to a 1 : 1 formation of nitrosylhemoglobin and methemoglobin in both species. At intermediate oxygen saturations, the reaction with deoxyhemoglobin is clearly favored over that with oxyhemoglobin, and the oxyhemoglobin reaction and its autocatalysis are inhibited by nitrosylhemoglobin from the deoxyhemoglobin reaction. The production of NO and nitrosylhemoglobin is faster and higher in carp hemoglobin with high O(2) affinity than in rabbit hemoglobin with lower O(2) affinity, and it correlates inversely with oxygen saturation. In carp, NO formation remains substantial even at high oxygen saturations. When oxygen affinity is decreased by T-state stabilization of carp hemoglobin with ATP, the reaction rates decrease and NO production is lowered, but the deoxyhemoglobin reaction continues to dominate. The data show that the reaction of nitrite with hemoglobin is dynamically influenced by oxygen affinity and the allosteric equilibrium between the T and R states, and that a high O(2) affinity increases the nitrite reductase capability of hemoglobin.     

Microwave effect upon chlorpromazine-inhibited kidney ATPase

Presence of chlorpromazine, a non-active-site inhibitor of Na(+)-K(+)-ATPase catalytic activity, in a reaction system exposed to 9.14 GHz CW radiation, resulted in approximately 23% inhibition. This effect was temperature-independent within the normal range for this protein. A low-level microwave field also inhibited the enzyme catalytic rate. Loci of chlorpromazine inhibition and of low-level microwave inhibition appear to be distinct and non-interactive under the conditions of this study. Use of enzyme reaction systems as models for microwave causation of leukemia and the possible involvement of pharmacological agents, such as ouabain and chlorpromazine, in this process has been considered.     

Tegumental Ca-stimulated adenosine triphosphatase activity in adult Schistosoma mansoni worms

A Ca-stimulated ATPase activity (pH 9.5) associated with the tegumental membrane enriched (TME) fraction of Schistosoma mansoni adults was partially inhibited by NAP-taurine or by increasing concentrations of chlorpromazine; endogenous calmodulin was found associated with the TME fraction. A similar activity (pH 8.6) was histochemically visualized within the tegument of fixed worms on the cytoplasmic leaflet of both the double surface membrane and the basement membrane; this reaction was inhibited by 1 microM chlorpromazine and it was also observed on the inner side of double membrane vesicles present in the TME fraction. No ATPase activity could be seen at alkaline pH with added Mg or Na/K ions. Without ATP, the addition of external Ca to the fixed worms induced the appearance of lead precipitates on the tegumental discoid bodies; this reaction was inhibited by molybdate and not by chlorpromazine. The intrategumentary regulation of calcium by the systems described and the possible use of phenothiazines against schistosomes are discussed.     

CHLORPROMAZINE ALONE AND WITH RESERPINE—Use in the Treatment of Mental Diseases

One hundred psychiatric patients were treated with chlorpromazine, alone or combined with reserpine. Fifty-six per cent of patients with chronic schizophrenic reactions showed moderate or pronounced improvement when treated with chlorpromazine alone. The results of treatment with the combined drugs were not so good as that. Indications are that treatment of patients with chronic schizophrenic reactions is more efficacious with these drugs than with other forms of somatic therapy.Complications of treatment were far greater with combined use of chlorpromazine and reserpine. For this reason, the combination appears to have limited usefulness. The Parkinson syndrome was the most frequent complication of large doses of these drugs. It appears to be a toxic reaction, requiring reduction in dosage. Jaundice appears to be neither a frequent nor a serious complication of treatment.     

Chlorpromazine alone and with reserpine; use in the treatment of mental diseases

One hundred psychiatric patients were treated with chlorpromazine, alone or combined with reserpine. Fifty-six per cent of patients with chronic schizophrenic reactions showed moderate or pronounced improvement when treated with chlorpromazine alone. The results of treatment with the combined drugs were not so good as that. Indications are that treatment of patients with chronic schizophrenic reactions is more efficacious with these drugs than with other forms of somatic therapy. Complications of treatment were far greater with combined use of chlorpromazine and reserpine. For this reason, the combination appears to have limited usefulness. The Parkinson syndrome was the most frequent complication of large doses of these drugs. It appears to be a toxic reaction, requiring reduction in dosage. Jaundice appears to be neither a frequent nor a serious complication of treatment.     

The reaction of deoxy-sickle cell hemoglobin with hydroxyurea.

In addition to its capacity to increase fetal hemoglobin levels, other mechanisms are implicated in hydroxyurea's ability to provide beneficial effects to patients with sickle cell disease. We hypothesize that the reaction of hemoglobin with hydroxyurea may play a role. It is shown that hydroxyurea reacts with deoxy-sickle cell hemoglobin (Hb) to form methemoglobin (metHb) and nitrosyl hemoglobin (HbNO). The products of the reaction as well as the kinetics are followed by absorption spectroscopy and electron paramagnetic resonance (EPR) spectroscopy. Analysis of the kinetics shows that the reaction can be approximated by a pseudo-first order rate constant of 3.7x10(-4) (1/(s.M)) for the disappearance of deoxy-sickle cell hemoglobin. Further analysis shows that HbNO is formed at an observed average rate of 5.25x10(-5) (1/s), three to four times slower than the rate of formation of metHb. EPR spectroscopy is used to show that the formation of HbNO involves the specific transfer of NO from the NHOH group of hydroxyurea. The potential importance of this reaction is discussed in the context of metHb and HbNO being able to increase the delay time for sickle cell hemoglobin polymerization and HbNO's vasodilating capabilities through conversion to S-nitrosohemoglobin.     

A microcytofluorometric method for quantitative and qualitative evaluation of CFU-E and BFU-E colonies

Summary A new method has been developed for the precise identification of human bone marrow colony forming unit erythroid (CFU-E) and burst forming unit erythroid (BFU-E) colonies, and for determination of the hemoglobin contents using microcytofluorometry. The method relies on a photochemical reaction in which intracellular hemoglobin is converted into fluorescent porphyrin under violet light (=405 nm) in the presence of an SH-donor (mercaptoethylamine hydrochloride). The CFU-E and BFU-E colonies showed red fluorescence with two spectrum peaks at 600 and 650 nm when illuminated by violet light. These two peaks are consistent with those of porphyrin fluorescence. The porphyrin fluorescence was not inducible in colony forming unit granulocyte-macrophage (CFU-GM) colonies, while 20% of the CFU-GM colonies were false positive with respect to the conventional benzidine reaction. The photochemically inducible fluorescence began to appear in BFU-E colonies on the 4th day of culture, while the same colonies started to be positive for the benzidine reaction on the 9th day. Therefore, the photochemical reaction was more specific and sensitive than the benzidine reaction for the identification of CFU-E and BFU-E colonies. In addition, this method enabled us to measure the hemoglobin level in the cells forming the colonies because the intensity of the fluorescence was proportional to the amount of hemoglobin when the photochemical reaction was carried out for 50 min. As a result of qualitative and quantitative analysis of CFU-E colonies by this method, it was possible to detect the hemoglobin levels in the colonies from 1 of 4 cases of untreated acute nonlymphocytic leukemia and from 2 of 4 cases of myelodysplastic syndrome in which the hemoglobin levels were too low to be detected by the benzidine reaction. These cases, where the CFU-E colonies showed very low levels of hemoglobin, were associated with poor prognosis. Thus, our method is useful for identifying CFU-E colonies, determining their hemoglobin synthesis, and as a cue to predict the clinical course of the patients.     

The Nernst equation applied to oxidation-reduction reactions in myoglobin and hemoglobin. Evaluation of the parameters

Analyses of the binding of oxygen to monomers such as myoglobin employ the Mass Action equation. The Mass Action equation, as such, is not directly applicable for the analysis of the binding of oxygen to oligomers such as hemoglobin. When the binding of oxygen to hemoglobin is analyzed, models incorporating extensions of mass action are employed. Oxidation-reduction reactions of the heme group in myoglobin and hemoglobin involve the binding and dissociation of electrons. This reaction is described with the Nernst equation. The Nernst equation is applicable only to a monomeric species even if the number of electrons involved is greater than unity. To analyze the oxidation-reduction reaction in a molecule such as hemoglobin a model is required which incorporates extensions of the Nernst equation. This communication develops models employing the Nernst equation for oxidation-reduction reactions analogous to those employed for hemoglobin in the analysis of the oxygenation (binding of oxygen) reaction.     

Calmodulin inhibitors protect against cadmium-induced testicular damage in mice

Recent reports showing that cadmium can interact with calmodulin and activate calmodulin-sensitive enzymes have lead us to examine the effects of calmodulin inhibitors on cadmium-induced testicular toxicity in mice. Male CF-1 mice were pretreated with the various calmodulin inhibitors or inactive analogs and then, one hour later, given CdCl2 X 2 1/2 H2O (32 mumoles/kg). After 24 hours, the mice were killed and the testes were removed and weighed. The extent of hemorrhaging was quantified by determining the absorbance of hemoglobin at 414 nm in the soluble fraction of testicular homogenates. Exposure to Cd2+ increased the mean testicular weight from 118 +/- 5 mg to 146 +/- 4 mg and the hemoglobin absorbance from 0.096 +/- 0.006 to 0.767 +/- 0.138. Pretreatment with the calmodulin inhibitors, trifluoperazine (40 mumoles/kg), chlorpromazine (40 mumoles/kg) or W-7 (140 mumoles/kg) greatly attenuated the CD2+-induced increase in both parameters, whereas pretreatment with chlorpromazine sulfoxide (140 mumoles/kg), pentobarbital (140 mumoles/kg), verapamil (80 mumoles/kg) or ethylenediaminetetraacetate (140 mumoles/kg) did not. These results indicate that calmodulin inhibitors can protect against certain toxic effects of cadmium and are consistent with the hypothesis that some of the effects of cadmium may result from the improper activation of calmodulin-dependent enzymes.     

Kinetic study of the transient phase of a second-order chemical reaction coupled to an enzymic step: application to the oxidation of chlorpromazine by peroxidase-hydrogen peroxide

Peroxidase-hydrogen peroxide assay with chlorpromazine as substrate was kinetically analysed as a system of a chemical reaction of second-order coupled to an enzymic step. This system follows a mechanism defined as enzymic-chemical second-order with substrate regeneration (EzC2-S.R.). The rate constant of the chemical step and the enzymic reaction rate have been evaluated from the progress curves of the accumulation of the cation radical intermediate (CPZ+.) and the non-linear regression analysis of these curves. The presteady-state rate of the accumulation of the cation radical intermediate (CPZ+.) and the level of the steady-state plateau were dependent on the rate constant of the chemical step and the enzyme concentration. The rate constant of the chemical reaction was dependent on the proton, buffer and chlorpromazine concentrations.