Cola-colored dialysate as a gastrointestinal sequelae of cardiac surgery in a patient who underwent peritoneal dialysis.

The discoloration of effluent peritoneal dialysate, which is transparent in origin, is seen in some particular conditions including chyloperitoneum, calcium channel blocker usage, hemoperitoneum, perforated cholecystitis, iron administration, and hemorrhagic pancreatitis. We report a case of a 60-year-old woman who underwent peritoneal dialysis for 3 years and presented with conspicuous cola-colored (brownish-black) dialysate after a cardiac surgery. The findings of the dialysate analysis and the abdominal computed tomography showed that this discoloration could be due to the presence of methemalbumin caused by pancreatitis (not hemorrhagic) combined with intra-abdominal bleeding-both of which are rare gastrointestinal complications of cardiac surgery. She eventually died of pulseless electrical activity due to severe sepsis with profound shock. Therefore, the rare event of cola-colored peritoneal dialysate could present as severe gastrointestinal sequelae of cardiac surgery and may indicate a poor prognosis.

Methaemalbumin formation in sickle cell disease: effect on oxidative protein modification and HO-1 induction.

Normally, cell free haemoglobin is bound by haptoglobin and efficiently cleared. However, the chronic haemolysis in sickle cell disease (SCD) overwhelms haptoglobin binding capacity and protein turnover, resulting in elevated cell free haemoglobin. Cell free haemoglobin acts as both a scavenger of vasoactive nitric oxide and a pro-oxidant. In addition, methaemoglobin (metHb) releases the haem moiety, which can bind to albumin to form methaemalbumin (metHSA). This study used electron paramagnetic resonance to detect metHSA in SCD plasma and demonstrated that haptoglobin prevents haem transfer from metHb to HSA. MetHSA may either provide a second line of defence against haemoglobin/haem-mediated oxidation or contribute to the pro-oxidant environment of SCD plasma. We demonstrated that HSA inhibited oxidative protein modification induced by metHb. Additionally, we showed that while metHb induced haem oxygenase 1 (HO-1), an indicator of oxidative stress, HSA attenuated metHb induction of this enzyme, thereby limiting the potential benefits of HO-1. Furthermore, HO-1 induction by metHSA was less than HO-1 induction by equimolar metHb not bound to albumin. Our findings confirm the presence of metHSA in SCD and suggest that haem transfer from metHb to HSA reduces the oxidative effects of free haemoglobin/haem on endothelium with both beneficial (reduced protein oxidation) and potentially harmful (reduced HO-1 induction) outcomes.

[Antioxidant activity of oxygen-containing aromatic compounds].

Inhibition efficiency (antioxidant activity) of 26 oxygen-containing aromatic compounds was studied in methemalbumin-H202-o-phenylenediamine (PDA) or tetramethylbenzidine (TMB) pseudoperoxidase system at 20 degrees C in buffered physiological solution (pH 7.4) containing 6% DM F and 0.25% DMSO. The inhibitor's efficiency was quantitatively characterized by the inhibition constants (Ki, microM) or the inhibition degree (%). Ki values varied in the range of4 to 500 microM and were influenced by a substrate, the structure of an inhibitor, hydroxyl groups, electron-donating substituents in aromatic ring, and steric hindrances. The type of inhibition at cooxidation of eight pairs was noncompetitive, and that of five pairs was mixed and determined by the substrate nature and the inhibitor structure. Lignin phenolic compounds ofguaiacyl and syringal series exhibited high antioxidant activity (Ki in the range of 10-300 microM), and their efficiency decreased in the following order: caffeic acid > synapaldehyde > syringic acid > coniferyl aldehyde > para-hydroxycou maric acid.

Purification and viral inactivation of hemoglobin from human placenta blood.

A new procedure was developed to obtain high-quality polymerized human hemoglobin by modifying purified hemoglobin with PLP and polymerized with GDA. Comparing polymerized hemoglobin products obtained from different methods, the product from the new procedure has similar physical, chemical, and biological properties in the molecular distribution, methemoglobin concentration, oxygen carrier capacity, P(50) and spectral analysis. Furthermore, the new procedure of modification after polymerization can save PLP greatly, and significantly reduce the cost. So the procedure of modification after polymerization is a better way in research and production.

Binding of heme to human serum albumin: steady-state fluorescence, circular dichroism and optical difference spectroscopic studies.

The binding of monomeric heme to human serum albumin (HSA) was investigated using steady-state fluorescence, circular dichroism (CD) and optical difference spectroscopic (ODS) techniques. The existence of one strong binding site for heme on HSA was confirmed by titrating heme with HSA and following the quenching of tryptophan (Trp214) fluorescence emission intensity that occurred due to energy transfer. Up to around 1:1 stoichiometric ratio of HSA/heme, the quenching was observed to be very strong, however at higher ratios the quenching progressed very weakly. Similarly, the negative CD band centered at -397 nm, which appeared on adding heme to HSA, increased in intensity on sequential addition of heme up to [heme]/[HSA] = 1. Titration of HSA with heme was followed by ODS and the dissociation constant K(D) = (4.0 +/- 1.0) x 10(-5) M was deduced. Results have been explained on the basis of Michaelis-Menton type of mechanism for the heme binding, in which heme first binds reversibly to His146 at the surface of the protein to form an intermediate complex, followed by irreversible binding to Tyr161 in the interior of the protein.

Increased body weight as a prognostic parameter for complications in the course of acute pancreatitis.

To evaluate whether obesity is a negative prognostic parameter in the course of acute pancreatitis, we examined 149 patients and divided them into four weight groups. Single (methemalbumin) and multiple (Ranson's signs) prognostic parameters were found to be independent of increased body weight in all groups, although the incidence of patients with more than six Ranson's signs or a positive methemalbumin test was highest in the most obese group. There was also no direct positive correlation between increased body weight and the incidence of mortality and late complications such as pseudocysts and abscesses. However, when compared with patients of normal weight, the obese groups showed a slight increase in the incidence of early complications such as shock and renal insufficiency and a significant increase in respiratory insufficiency necessitating artificial ventilation. Thus, increased body weight was associated with increased incidence of early extrapancreatic complications.

Quantitative determination of serum methemalbumin.

We have devised a simple, sensitive quantitative method for the determination of serum methemalbumin. The method uses a modified Allen correction to correct the alpha band of methemalbumin at 623 nm for background turbidity. The technique is robust and is more sensitive than Schumm's test.

Reductive activation of HCFC-123 by methaemalbumin.

Hydrochlorofluorocarbon 1,1-dichloro-2,2,2-trifluoroethane (HCFC-123), a close structural analogue of the hepatotoxic anaesthetic halotane and a replacement for some ozone-depleting chlorofluorocarbons, is metabolized by liver cytochrome P450 (P450), both in vitro and in vivo. P450 activates HCFC-123, both oxidatively and reductively, to reactive species which attack P450 itself and also damage other targets leading to hepatotoxicity. Previous work in our laboratory has shown that some haloalkanes, including halomethanes CCl4, CCl3Br, CHCl4 and CH2Cl2 as well as halothane, are activated by different haemoproteins to reactive metabolites resulting in the protein's suicidal inactivation. Among these is methaemalbumin (MHA), a synthetic complex of haem with human albumin often used as a model for various natural haemoproteins, such as P450. The aim of this study was to use MHA as a model to investigate the mechanism of P450 inactivation by HCFC-123. We found that MHA can reductively activate HCFC-123 to reactive species resulting in the loss of its haem group. During anaerobic incubation of MHA with 10 mM HCFC-123, a typical reduced difference spectrum was observed with a 470-nm peak that increased with time, indicating an interaction between HCFC-123 or HCFC-123 metabolites and haem. In similar anaerobic incubations, a significant loss of haem was measured using both the pyridine-haemochromogen technique and an ion-pairing reverse-phase HPLC method (37 and 30%, respectively). The loss of haem was time-, but not dose-dependent. No statistically significant loss of protoporphyrin IX, as measured by a fluorescence technique, or of the absolute haem spectrum produced in presence of CO (CO-haem complex) was observed up to 10 mM HCFC-123. Finally, a small but statistically significant inorganic fluoride production was measured in the presence of 20 mM HCFC-123 using an F(-)-specific electrode. Taken together, these results indicate that incubation of the non-enzymatic P450 model MHA with HCFC-123 under anaerobic conditions leads to reductive activation of the substrate, resulting in the modification of haem, as was previously shown to occur for halothane. The haem modification is due to interaction of the prosthetic haem group of MHA with HCFC-123 metabolites. These data confirm the results of previous work with rat liver microsomal P450 and confirm suicidal destruction of haem to be the mechanism responsible for the HCFC-123-dependent loss of the enzyme's content and catalytic function.

The role of RBC destruction in vascular regions with high turbulence on atherosclerosis.

After intravascular red blood cell (RBC) destruction, released hemoglobin exceeding the binding capacities of haptoglobin and hemopexin would contribute, as free hemoglobin and/or hemin and/or methemalbumin, to the pathogenesis of atherosclerosis. Especially in some regions of vasculature with a high turbulence strong enough to destruct fragile or old RBCs, free hemoglobin concentrations can exceed locally the binding capacities of haptoglobin and hemopexin although their measured venous concentrations are normal. As a very simple model of this possible very local event we analysed free hemoglobin and methemalbumin levels after blood collection into evacuated tubes, and found that the increase of methemalbumin is more significant than free hemoglobin. The measurement of free hemoglobin and methemalbumin concentrations of arterial blood samples just from pre- and post-atherosclerotic lesions can likely shed new light on our understanding of the development of atherosclerosis.

Haptoglobins in some Galliformes birds.

In several groups of galliform birds (chicken, turkey, pheasant and guinea fowl) the presence and function of plasma haptoglobin (Hp) have been studied. Paper, acetate and starch-gel electrophoresis were carried out and two benzidine stain regions were observed when haemoglobin (Hb) was added to normal plasma (Hb-Hp complex and methemalbumin) besides the free Hb when the binding capacity of these proteins were surpassed. The disappearance of the administered Hb-59Fe was exponential which is evidence of the existence of two different rates. Liver, bone marrow and kidney are the main organs related to the 59Fe uptake. These results on Hp in galliforms and the metabolism of the Hb are concordant with the data in the literature on mammals. This suggests the same common physiological role for Hp.

[Methemalbuminnemia after massive hemolysis during blackwater fever]. / Methémmalbuminémie après hémolyse massive au cours d'une fièvre bilieuse hémoglobinurique.

We report a case of blackwater fever with brown plasma due to the presence of methemalbumin. The discovery of plasma with this color is a rare event at the laboratory. This compound appears during intravascular hemolysis or hemorrhagic pancreatitis when the ability of haptoglobin and hemopexin to bind free hemoglobin has been exceeded. In these cases some of heme is oxidized to hematin and taken up by serum albumin to form an albumin-hematin complex called methemalbumin. The major clinical problem is to evoke the diagnosis of methemalbuminemia and not confuse with methemoglobinemia. In our case, methemalbumin was detected and quantified using a scanning spectrophotometer. Its diagnostic and clinicals consequences are discussed.

[Voluntary poisoning by intravenous injection of caustic soda]. / Intoxication volontaire par injection intraveineuse de soude caustique.

A voluntary intoxication by injection in the left basilic vein of 10 ml of concentrated caustic soda is reported. The main effects were, besides local necrosis, haemolysis, acute renal failure with initial anuresis, intravascular coagulation and cyanosis with a normal Pao2 due to methaemalbuminaemia. This was confirmed by using the usual spectrophotometric methods as well as electrophoretic methods.

Inhibition of heme oxygenase activity in newborn mice by azalanstat.

Inhibition of heme oxygenase (HO), the rate-limiting enzyme in heme catabolism, may be an ideal strategy for preventing neonatal jaundice. Although natural and synthetic heme analogs, called metalloporphyrins (Mps), have been extensively investigated for this purpose, some Mps are phototoxic, affect the activity of other enzymes, or induce HO-1 transcription-properties that may limit their clinical use. Another class of compounds, imidazole-dioxolanes, has been shown to selectively inhibit the inducible isozyme HO-1. Therefore, we investigated the efficacy of azalanstat (AZA), an imidazole-dioxolane, towards inhibiting HO activity in 7-day-old mice. We found that a single dose of AZA at 500 micromol.kg(-1) body mass (BM) administered i.p. significantly inhibited HO activity and reduced in vivo bilirubin production. In the spleen, HO inhibition (>50%) was observed within 0.25-3 h after administration. After 24 h, however, spleen HO activity, HO-1 protein, and HO-1 mRNA levels significantly increased 1.2-, 2.4-, and 4.0-fold, respectively. We conclude that AZA effectively inhibits in vivo HO activity only at a high dose and that it also induces spleen HO-1 gene transcription. Therefore, other imidazole-dioxolanes should be evaluated to determine whether they are more potent than AZA for use in treating neonatal jaundice.

Pancreatitis causes brownish-black peritoneal dialysate due to the presence of methaemalbumin.

Two patients treated with continuous ambulatory peritoneal dialysis had striking brownish-black coloured peritoneal dialysate concomitant with underlying pancreatitis. Examination of the second patient's dialysate by recording spectrophotometry showed the discoloration to be due to methaemalbumin. Haemorrhagic pancreatitis, by releasing proteolytic enzymes and perhaps by using the lesser sac as an anatomical cul-de-sac for chemical reaction, provides a suitable milieu for the production of methaemalbumin from red blood cells in dialysate.

[Methemalbumin--a biocatalyst of aromatic amine oxidation by hydrogen peroxide]. / Metgemal'bumin--biokatalizator okisleniia aromaticheskikh aminov perekis'iu vodoroda.

The kinetics of hydrogen peroxide-dependent oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) and o-phenylenediamine catalyzed by hemin and hemin-bovine serum albumin (BSA) complex (methemalbumin) was studied in buffered physiological solution containing dimethylformamide (40%) and dimethyl sulfoxide (2%), respectively. The formation of hemin-BSA complex enhanced hemin catalytic activity in oxidation of both amines. The reaction follows first-order kinetics in biocatalyst concentrations, H2O2, and H+ ions from pH 6.5 to 9.0. The catalytic constants, Michaelis constants, and kcat/K(m) ratios for both substrates in hemin- and methemalbumin-catalyzed reactions were calculated using double reciprocal plots of the effect of the initial TMB and PDA concentrations on the initial reaction rates. Mechanism of radical oxidation of amines in hemin-H2O2 and hemin-BSA-H2O2 systems is discussed. Both systems can effectively initiate radicals free radicals formation; their activity is similar to the previously studied ferritin-H2O2 system.

Spectroscopic studies on human serum albumin and methemalbumin: optical, steady-state, and picosecond time-resolved fluorescence studies, and kinetics of substrate oxidation by methemalbumin.

The nature of the heme environment in methemalbumin, the Fe(III) protoporphyrin IX (heme)-human serum albumin (HSA) complex, was investigated by optical spectroscopy. Comparison of the optical spectra of methemalbumin, ferro-hemalbumin in the absence and presence of 2-methylimidazole, and their carbon monoxide derivatives with horseradish peroxidase (HRP) and its corresponding derivatives indicates that histidine is not present in the first coordination sphere of heme in methemalbumin and that the protein is devoid of a well-defined heme cavity. The complex exhibits peroxidase activity by catalyzing oxidation of 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) by hydrogen peroxide. Its activity ( K(M)=433 microM, molar catalytic activity=0.33 s(-1)), however, is considerably lower compared to HRP, indicating differences in the heme environments. Fluorescence intensity decays of Trp214 in HSA and methemalbumin, best fitted to a three-exponential model, gave the lifetimes 7.03 ns (30%), 3.17 ns (38%), and 0.68 ns (32%) for HSA and 8.04 ns (1.7%), 2.42 ns (19.7%), and 0.64 ns (78.6%) for methemalbumin. These lifetime values were further confirmed by a model-independent maximum entropy method. Similarity in the lifetimes and variations in the amplitudes suggest that while conformational heterogeneity of HSA is unperturbed on heme binding, redistribution of the populations of the three conformations occurs and the sub-state associated with the shortest lifetime dominates the total population by approximately 80%. Decay associated spectra (DAS) indicate that the observed lifetime variation with wavelength is predominantly due to ground state heterogeneity, though solvent dipolar relaxation also contributes. Time-resolved fluorescence anisotropy measurements of the Trp214 residue yielded information on motion within the protein together with the whole protein molecule. The binding of heme did not affect the rotational correlation time of the albumin molecule (approximately 20 ns). However, the motion of tryptophan within the protein matrix increased by a factor of approximately 3 (0.46 ns to 0.15 ns). This indicates that while the overall hydrodynamic volume of the albumin molecule remained the same, tryptophan underwent a more rapid internal rotation because of the efficient energy transfer to the bound heme. Optical studies, analysis of lifetime measurements, DAS, and anisotropy measurements together suggest that heme binds to a surface residue. The rapid internal motion of Trp214 during its excited state lifetime for the approximately 80% populated conformer of methemalbumin allows the orientation factor, kappa(2), to approach the average value of 2/3. From the time-resolved fluorescence measurements and the energy transfer calculations on methemalbumin, a Trp214-heme distance of 22 A was deduced.

Abnormal heme-protein patterns in hemorrhagic shock.

Allegedly toxic heme pigments have been described in the serum of animals bled to hemorrhagic shock (5,7). In addition, Sears et al. (9), and Braun et al. (1) have shown that the products derived from the degradation of hemoglobin following intravascular hemolysis were toxic (heme carried by hemopexin and albumin). The accumulation of metabolites, caused by the impeded circulation, degrades free hemoglobin into heme pigments and their concentration then reaches a level which exceeds the normal heme-carrying capacity of serum proteins. We have already demonstrated the presence of abnormal heme pigments in clinical cases of shock using scanning spectrophotometry (3). We have endeavored to identify these pigments by serum electrophoresis, and to relate the appearance of some of these compounds to mortality rates. There were no deaths associated with the presence of haptoglobin-hemoglobin alone in serum. As shock deepened and mortality rose, hemopexin-heme and methemalbumin appeared. The highest mortality rate (4 out of 5 cases) was found when both hemopexin-heme and methemalbumin were present. It appears, therefore, that the administration of serum proteins in shock may reduce the toxicity of the products of degradation formed in low-flow states.

The atomic structure of human methemalbumin at 1.9 A.

The high resolution structure of hemalbumin was determined by single crystal X-ray diffraction to a resolution of 1.9 A. The structure revealed the protoporphyrin IX bound to a single site within a hydrophobic cavity in subdomain IB, one of the principal binding sites for long chain fatty acid. The iron is penta coordinated with the fifth ligand comprised of the hydroxyl oxygen of Tyr-161 (phenolic oxygen to heme plane distance: 2.73 A) in an otherwise completely hydrophobic pocket. The heme propionic acid residues form salt bridges with His-142 and Lys-190, which together with a series of hydrophobic interactions, enclose and secure the heme within the IB helical motif. A detailed discussion of the structure together with its implications for the development of potential blood substitutes is presented.

The degradation of haem by carbon tetrachloride: metabolic activation requires a free axial coordination site on the haem iron and electron donation.

1. The ability of haem to catalyse the reductive activation of carbon tetrachloride (CCl4) in vitro has been investigated under anaerobic conditions, using methaemalbumin (MHA) and either sodium dithionite or NADPH together with NADPH-cytochrome P-450 reductase (EC 1.6.2.4) as the reducing agents. 2. In the non-enzymic system protohaem and other non-physiological haem analogues underwent rapid and extensive CCl4-dependent degradation, due to irreversible modification of their porphyrin tetrapyrrolic structure. 3. This mechanism of non-enzymic activation of CCl4 by protohaem mimics that catalyzed by cytochrome P-450 in that it requires a free, reduced haem iron and electron donation and it is largely prevented by carbon monoxide. 4. H.p.l.c. analysis of 14C-haem after anaerobic incubation with CCl4 and sodium dithionite gave radioactive products which eluted before and after haem, and exhibited significantly lower absorbance at 400 nm compared with authentic haem. When the products of CCl4-dependent haem degradation were methylated and applied to silica for t.l.c., two non-fluorescent pigments were isolated, purified and partially characterized. 5. On incubation of haem with 14CCl4 and sodium dithionite a 1:1 stoichiometry could be calculated for haem loss and 14CCl4-derived adduct formation, indicating that, as with microsomes, the loss of haem may be the result of a typical 'suicidal' inactivation reaction where the same haem moiety is both the site of CCl4 activation and the target of CCl4 reactive metabolites.