Manfred Kiese

Formation of ferrihaemoglobin with aminophenols in the human for the treatment of cyanide poisoning

Abstract Experiments with several aminophenols showed that 4-dimethylaminophenol and 4-methylaminophenol most rapidly produced controlled amounts of ferrihaemoglobin in the blood of various species in vitro and in vivo. The rapid reaction of 4-dimethylaminophenol and 4-methylaminophenol was also observed after intravenous injection in humans. Doses which oxidize 30 to 40% of the haemoglobin produce the half-maximal ferrihaemoglobin concentration in 1 and 2 min respectively without any immediate effect on the cardiovascular system. Sodium nitrite in doses of 4 mg/kg recommended for the treatment of cyanide poisoning was found to oxidize slowly about 7% of the haemoglobin, 10 min being taken for producing half the maximal amount of ferrihaemoglobin, i.e. 3.5% of the total haemoglobin. Nitrite caused a decrease in arterial blood pressure in all experiments and orthostatic collapse of several test persons. The rate of ferrihaemoglobin reduction in humans was determined. An amount of ferrihaemoglobin equal to about 8% of the total haemoglobin is reduced each hour, when the ferrihaemoglobin concentration amounts to more than 10% of the total haemoglobin.

The fate of phenylhydroxylamine in human red cells.

SummaryPhenylhydroxylamine added to human red cells under aerobic conditions and in the presence of glucose was partly reduced to aniline. About half the hydroxylamine was recovered as amine after a 2-hr incubation. The aniline, after acetylation, was identified as acetanilide by melting point, Rf-value in TLC as well as UV, IR, and NMR spectroscopy.The fate of the remaining phenylhydroxylamine was followed by use of 14C-labeled phenylhydroxylamine. About 30% of the total radioactivity was bound to hemoglobin or other proteins and about 20% was found in highly polar low-molecular substances which were insoluble in organic solvents.The elucidation of the sites at which phenylhydroxylamine was bound to hemoglobin was complicated by the lability of the bonds. When purified human hemoglobin had reacted with radioactive phenylhydroxylamine, large proportions of the radioactivity bound to hemoglobin were removed by treatment with acid or with PMB for separation of α- and β-chains. The radioactive compound liberated from hemoglobin by acid was found to be aniline.After reaction with phenylhydroxylamine the number of SH groups titrable with PMB was found to be diminished. Pretreatment of hemoglobin with N-ethylmaleimide or PMB decreased the amount of phenylhydroxylamine bound to hemoglobin but did not fully prevent the reaction.Tryptic digestion of hemoglobin after reaction with radioactive phenylhydroxylamine yielded tryptic peptides with lower specific activity than that of hemoglobin. Chymotryptic digestion of the tryptic core yielded a core with specific activity much higher than that of hemoglobin. Fingerprinting of the tryptic or chymotryptic hydrolyzates showed the presence of peptides with high and other ones with low or no radioactivity and of radioactive compounds which did not react with ninhydrin.In the covalent binding of phenylhydroxylamine to globin the SH group β93 plays an important role, but other yet unknown sites are also reactive.

Pharmacokinetics of cyanide in poisoning of dogs, and the effect of 4-dimethylaminophenol or thiosulfate.

Cyanide in blood, plasma, and urine of dogs after administration of K14CN was determined with the isotope dilution technique. The addition of large amounts of inactive KCN as soon as possible to a sample to be analyzed inhibited the decrease of the original cyanide concentration.After administration of several lethal doses of cyanide into the stomach or by slow intravenous infusion a concentration of about 40 μM cyanide in plasma was found at the moment of respiratory arrest. Since 60% of the cyanide in plasma was bound to proteins the concentration of free cyanide which stopped respiration was about 16 μM.Quick formation of ferrihemoglobin by i.v. injection of 4-dimethylaminophenol after plasma cyanide had risen to or above 40 μM decreased the cyanide concentration in plasma and restored respiration, while cyanide was accumulated in red cells by formation of ferrihemoglobin cyanide.Equilibrium constants calculated for the reaction between ferrihemoglobin and cyanide in vivo indicated that the reaction approached equilibrium in a few minutes.Up to 60% of the radioactive cyanide absorbed was found as non-cyanide radioactivity in the urine.

Reactions of 4-dimethylaminophenol with hemoglobin, and autoxidation of 4-dimethylaminophenol

Abstract The reactions between 4-dimethylaminophenol and hemoglobin were studied with 4-dimethylaminophenol 14C-labelled either in the methyl groups or in C1 of the ring. In the absence of oxygen 4-dimethylaminophenol was stable in red cell suspensions or hemoglobin solutions. In the presence of oxygen oxyhemoglobin rapidly oxidized 4-dimethylaminophenol. The following reaction products were found in incubates of 4-dimethylaminophenol with red cells or hemoglobin: ferrihemoglobin, formaldehyde, dimethylamine, and hemoglobin with derivatives of 4-dimethylaminophenol covalently bound to its protein moiety. 4-Dimethylaminophenol catalytically transferred electrons from ferrohemoglobin to oxygen. It was oxidized by oxyhemoglobin, and oxidized 4-dimethylaminophenol was reduced to 4-dimethylaminophenol by ferrohemoglobin with formation of ferrihemoglobin. Hydrolysis of oxidized 4-dimethylaminophenol, N,N-dimethylquinonimine, and its covalent binding to globin limited the catalytic ferrihemoglobin formation by 4-dimethylaminophenol to an average between 50 and 100 electron transfers per molecule of 4-dimethylaminophenol, when 4-dimethylaminophenol concentration was low and hemoglobin concentration was high. Since 4-dimethylaminophenol reduced ferrihemoglobin to ferrohemoglobin, though more slowly than the catalytic cycle produced it, the increase in ferrihemoglobin content does not indicate the amount of ferrihemoglobin produced. In red cell suspensions at 37° 4-dimethylaminophenol, 0.58 mM, disappeared in 10 min, but dimethylamine continued to be formed, obviously from protein-bound derivative(s) of 4-dimethylaminophenol. The rate of autoxidation of 4-dimethylaminophenol was found to be much lower that the rate of oxidation of 4-dimethylaminophenol by oxyhemoglobin. After autoxidation of 4-dimethylaminophenol several products were isolated and identified which were not detected in incubates of 4-dimethylaminophenol with oxyhemoglobin, namely hydroquinone, 4-methylaminophenol, 4-aminophenol, 2-dimethylamino-1, 4-benzoquinone, a purple and a yellow dye. Nuclear magnetic resonance (NMR), mass spectroscopy, and synthesis from 1,4-benzoquinone and 4-methylaminophenol proved the purple dye to be 2-(N- methyl-N-(p-hydroxyphenyl)-amino-1,4-benzoquinone. The structure of the yellow dye, which is produced also by oxidation of the purple dye with hydrogen peroxide, was not proved unequivocally. IR, NMR spectra and the product of hydrogenation with Pd-charcoal and acetylation showed the compound to be an epoxide of 2-(N-methyl-N-(p-hydroxyphenyl)-amino)-benzoquinone.

Biotransformation of 4-dimethylaminophenol: reaction with glutathione, and some properties of the reaction products.

4-Dimethylaminophenol (DMAP) forms ferrihemoglobin by catalytic transfer of electrons from ferrohemoglobin to oxygen. In solutions of purified human hemoglobin, quick binding of oxidized DMAP to the globin moiety of hemoglobin terminates this reaction. Reduced glutathione in high concentrations, as in the red cell, substantially diminished binding of oxidized DMAP to hemoglobin by formation of S,S,S-(2-dimethylamino-5-hydroxy-1,3,4-phenylene)-tris-glutathione (tris-(GS)-DMAP), which does not form ferrihemoglobin. In the presence of reduced glutathione, DMAP disappeared more rapidly from hemoglobin solutions than in its absence. The formation of tris(GS)-DMAP in red cells was found to be of importance for the termination of catalytic ferrihemoglobin formation by DMAP in vivo. With low concentrations of GSH, DMAP in hemoglobin solutions formed another conjugate, (GS)-DMAP, S,S(2-dimethylamino-5-hydroxy-1,3-phenylene)-bis-glutathione. Similar to DMAP, bis(GS)-DMAP catalyzed the formation of ferrihemoglobin. As the oxidized bis(GS)-DMAP was bound to hemoglobin more slowly and to a lesser extent, it produced more ferrihemoglobin than DMAP. In contrast to the reactions of DMAP with hemoglobin, hydrogen peroxide and superoxide radicals are involved in the ferrihemoglobin formation by bis(GS)-DMAP. The radicals accelerate the oxidation of bis(GS)-DMAP and thereby the ferrihemoglobin formation.

Urinary excretion of N-hydroxy derivatives of some aromatic amines by rabbits, guinea pigs, and dogs☆

Abstract The N-hydroxy derivatives of p-ethylaniline, p-chloroaniline, p- and maminopropiophenone, 4-aminobiphenyl, and 2-aminofluorene were found in the urine after injection. Rabbits excrete 30 per cent of the p-aminopropiophenone and 20 per cent of the 4-aminobiphenyl as N-hydroxy derivative. The N-hydroxy derivatives of the other amines appear in the urine to a much smaller extent. Guinea pigs excrete a smaller proportion of the amines as N-hydroxy derivative than rabbits; 15 per cent of p-amino-propiophenone was found in the urine as N-hydroxy derivative. Dogs excrete only 1 per cent or less of the amines tested as N-hydroxy derivative. N-Hydroxy-p-amino-propiophenone is excreted to a large extent as a conjugate which is split in acid solution. The fraction of p-aminopropiophenone excreted as N-hydroxy derivative is the same over a wide range of doses. The relationship between the concentration of N-hydroxy derivative, and nitroso analogue, in the blood and urine of rabbits is quite different from that observed in dogs.

The absorption of p-toluenediamine through human skin in hair dyeing

Abstract After the hair of 5 persons had been dyed with a simple dye, prepared by oxidizing a mixture of 2.5 g of p -toluenediamine sulfate and 2.5 g resorcinol with hydrogen peroxide, an average of 3.7 mg N,N′ -diacetyl- p -toluenediamine appeared in the urine. A similar amount of N,N′ -diacetyl- p -toluenediamine, namely 4.5 mg, was found in the urine when 5.5 mg of p -toluenediamine was subcutaneously injected. From these data it is calculated that about 4.6 mg of p -toluenediamine was either absorbed during the hair dyeing or produced in vivo from a compound formed during the preparation of the hair dye and absorbed through the skin while the dye was applied to the hair and scalp.

N-oxygenation of N-alkyl- and N,N-dialkylanilines by rabbit liver m1crosomes

Abstract The N -oxygenation of N -ethylaniline to the N -hydroxy derivative and of N , N -dimethylaniline to the N -oxide in rabbit liver microsomes were studied. Both oxygenating systems had a low affinity for oxygen and were not inhibited by carbon monoxide. They were not stimulated by the treatment of rabbits with phenobarbital. p -Chloromercuribenzoate, N -ethylmaleimide, and 8-hydroxyquinoline were found to affect the N -oxygenation of N -ethylaniline and of N , N -dimethylaniline differently.

FURTHER FACTORS AFFECTING THE HYDROXYLATION OF ANILINE AND SOME OF ITS DERIVATIVES BY LIVER MICROSOMES.

I n an earlier s tudy of the hydroxyla t ion of anil ine and N-ethylanil ine by •ADPH-dependen t enzymes in rabb i t liver microsomes we found tha t some substances which are known to react with SILgroups of enzymes affect the Nand C-hydroxyla t ion of anil ine differently (KAMPFFMEYER and KIES~). These and other observat ions pointed to the existence of different enzymes involved in the Nand C-hydroxylat ion of aniline. The following invest igat ions were unde r t aken to elucidate fur ther the na tu re of the reactions differently affecting various enzymic hydroxyla t ions and to get fur ther in format ion on the hydroxyla t ion reactions and their catalysts.

The formation in vivo of p-hydroxylaminopropiophenone from p-aminopropiophenone and its action in vivo and in vitro

SummaryThe N-hydroxylation of p-aminopropiophenone in dogs was demonstrated by the spectrum of p-nitrosopropiophenone in carbon tetrachloride extracts taken from the blood of dogs after injecting p-aminopropiophenone.The N-hydroxylation of p-aminopropiophenone proceeded rather rapidly. The p-hydroxylaminopropiophenone proved to be more active than phenylhydroxylamine in the enzymic cycle of hemiglobin formation in the red cell.By comparing the velocity of hemiglobin formation and the concentration of p-hydroxylamino- and p-nitrosopropiophenone in the blood of dogs following the injection of p-aminopropiophenone or the slow infusion of p-hydroxylaminopropiophenone it was ascertained that the hemiglobin formation following the injection of p-aminopropiophenone was mainly due to p-hydroxylaminopropiophenone.