Biliverdin Reductase Activity Research Articles

Dose-related increase in liver heme catabolism during rabbit aflatoxicosis

Aflatoxin B1 (AFB1) has been reported to decrease microsomal hepatic cytochrome P450 (P450) content and increase both total plasma bilirubin concentration and liver heme oxygenase activity. The purposes of this study were to determine whether liver hemoproteins contents and heme catabolizing enzymes were affected by the mycotoxin and whether these alterations were linked to hyperbilirubinemia. Male New Zealand rabbits were divided into three groups of five animals, each receiving for 5 days either arabic gum as vehicle or AFB1 at a daily oral dose of 0.05 or 0.10 mg/kg. These treatments affected neither cytochrome b5 content nor NADPH-cytochrome reductase activity. A linear dose-dependent decrease in cytochrome P450 content and increases in both heme oxygenase and biliverdin reductase activities were observed. Bilirubin UDP-glucuronyltransferase activity was dramatically decreased at both doses, whereas cholestasis occurred only at 0.10 mg/kg. An exponential dose-dependent increase in plasma bilirubin concentration was also observed. Both the simultaneous exponential increase in bilirubinemia associated to a reduced bilirubin UDP-glucuronyltransferase activity and the absence of cholestasis at 0.05 mg/kg, suggested that the hyperbilirubinemia is more probably related to an increased heme catabolism than to an altered bile duct permeability.

Mitochondrial heme oxygenase of Mastomys coucha

While studying the fate of heme generated during malaria infection, it was observed that mitochondrial preparations were highly enriched with heme compared to other subcellular particles. With this background, the present study aimed to determine the status of mitochondrial heme oxygenase and compare it with the microsomal enzyme. Mitochondrial and microsomal preparations were obtained from liver, spleen, kidney and brain of normal, inducer (cobalt chloride and hemin)-treated and Plasmodium berghei-infected Mastomys coucha. Heme oxygenase activity was determined by monitoring the formation of bilirubin. Biliverdin reductase activity was assayed by following the decrease in biliverdin content. Heme levels were measured by pyridine haemochromogen formation. Mitochondria from different tissues showed significant activity of heme oxygenase only after inducer (CoCl 2 and hemin) treatment. In contrast, cerebral mitochondria did not show any enzyme activity. Hepatic, splenic and renal mitochondria of P. berghei-infected M. coucha showed noticeable heme oxygenase and biliverdin reductase activity. The response of hepatic mitochondrial heme oxygenase towards Triton X-100, trypsin, hydrogen peroxide, temperature, freezing and thawing and hemin was distinguishable from microsomal heme oxygenase. It is concluded that the mitochondria of different tissues from Mastomys display stress (biological and chemical)-induced activity of heme oxygenase. In addition, distinct differences between microsomal and mitochondrial heme oxygenase were observed.

Heme oxygenase and related indices in chloroquine-resistant and -sensitive strains of Plasmodium berghei

Chloroquine-resistant (CQR) and -sensitive (CQS) Plasmodium berghei K173 strains possessed significant activities of heme oxygenase, biliverdin reductase and heme polymerase. Heme oxygenase and biliverdin reductase activities of CQR were significantly higher (7–10-fold) as compared to that of CQS P. berghei, whereas heme polymerase showed a reverse trend (two-fold decrease). However, a 10-fold increase in heme, three-fold increase in ferriprotoporphyrin IX and a two-fold increase in hemozoin levels were noted in CQS as compared to CQR strains of P. berghei. This study suggests the importance of heme oxygenase and related components in the biochemical regulation of malaria parasites and in understanding the mechanism of the acquisition of chloroquine resistance.

Immunohistochemical localization of biliverdin reductase in rat brain: age related expression of protein and transcript

Biliverdin reductase regulates heme oxygenase activity by removing the inhibitory product of the oxygenase activity, biliverdin; and reducing it to bilirubin. The other products of the oxygenase are carbon monoxide and Fe. To date, biliverdin reductase remains unique among all enzymes described by using 2 different cofactors (NADPH and NADH) at different pH ranges. The present study reports on the developmentally regulated changes in the patter of protein expression and the level of biliverdin reductase transcript in rat brain. Biliverdin reductase activity of the brain cytosol with both NADPH (pH 8.7) and NADH (pH 6.7) exhibited developmental changes with the activity increasing after birth, reaching an adult level by day 28 postpartum. When analyzed by Western blotting the immunoreactive protein detected increased as the animal matured (day 1 to 28 postparturition). Northern blot hybridization of RNA isolated from rat brain revealed the presence of ∼ 1.5 kb biliverdin reductase transcript at all stages of development ranging from 1 day post partum to 20 months. The level of the transcript was developmentally regulated and a gradual increase (≈ 4-fold) was observed from day 1 after birth to adulthood and was maintained in 20 month old animals. Cellular localization, using immunohistochemical technique, revealed age-related pattern of expression of the reductase in select regions sich as the cortex, substantia nigra, hippocampus and in the cerebellum; the changes, however, did not follow the same pattern. To elaborate, in the cortex, the reductase expression increased when 7-day-old animals were compared with young adults (2 months old) and then declined in the 20-month-old animals. In the substantia nigra the level of reductase expression progressively declined with age when 7-day-old neonate, 2- and 20-month-old animals were compared. In the hippocampus, a distinct reductase-expressing cell population residing between CA1 and the dentate gyrus was observed in the 7-day-old animals; these cells were not detected in the adults (2 or 20 months old). In the cerebellum, the expression of the reductase reflected the developmental organization of this region. We postulate that age-dependent increase of the brain reductase at the transcript and protein levels in the course of maturation serves to control heme oxygenase activity which also displays a developmental pattern in the organ. As such, the reductase modulates generation of biologically active heme degradation products; bilirubin, carbon monoxide and Fe.

Biliverdin Reductase Is Heat Resistant and Coexpressed with Constitutive and Heat Shock Forms of Heme Oxygenase in Brain

Two heme oxygenase (HO) isozymes--HO-1, which is a heat shock protein (HSP32), and HO-2--catalyze the isomer-specific production of biliverdin IX alpha and carbon monoxide. The latter has the potential of functioning as a neurotransmitter, whereas the reduced form of biliverdin, bilirubin, has potent antioxidant activity. Formation of bilirubin is catalyzed by biliverdin reductase (BVR). The reductase is a unique enzyme in being dual pyridine nucleotide and dual pH dependent. Here, we show that the reductase is resistant to thermal stress at both the protein and message level. We further demonstrate that the reductase is coexpressed in cells that display HO-1 and/or HO-2 under normal conditions, as well as in regions and cell types that have the potential to express heat shock-inducible HO-1 protein. Exposure of male rats to 42 degrees C for 20 min did not decrease brain BVR activity, but caused a slight increase in NADPH- and NADH-dependent activities at 1 and 6 h following hyperthermia. High levels of the approximately 1.5-kb BVR mRNA were detected in control brain; it too displayed thermal tolerance. Similarly, the pattern of multiplicity of net charge variants of the enzyme purified from brain of heat-shocked rats did not differ from the control pattern. Immunochemical localization of BVR protein in normal brain correlated well with the presence of HO-1 and/or HO-2 throughout the forebrain, diencephalon, cerebellum, and brainstem regions. There were select neuronal and nonneuronal cells in the substantia nigra and cerebellum that did express the reductase under normal conditions, wherein no HO isozymes could be detected.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of heme oxygenase in intestinal epithelial cells: studies in Caco-2 cell cultures.

Enterally administered, heme is a good source of iron in humans and other animals, but the metabolism of heme by enterocytes has not been fully characterized. Caco-2 cells in culture provide a useful model for studying cells that resemble small intestinal epithelium, both morphologically and functionally. In this paper we show that heme oxygenase, the rate-controlling enzyme of heme catabolism, is present in abundance in Caco-2 cells, and that levels of its mRNA and activity can be increased by exposure of the cells to heme or metal ions (cadmium, cobalt). Caco-2 cells also contain biliverdin reductase activity which, in the basal state, is similar to that of heme oxygenase (approximately 40 pmole of product per mg protein per minute); however, when heme oxygenase is induced, biliverdin reductase may become rate-limiting for bilirubin production.

Biliverdin reductase activity in relation to bilirubin.

Biliverdin reductase activity in relation to bilirubin.

Microheterogeneity of biliverdin reductase in rat liver and spleen: Selective suppression of enzyme variants in liver by bromobenzene

Recently we have reported the detection of multiple net-charge and molecular mass variants of biliverdin reductase in the rat liver. We now report an apparent selective change in the electrophoretic profile of the reductase variants in the liver by in vivo bromobenzene treatment (2 mmol/kg, sc, 24 h). Using two-dimensional electrophoresis and isoelectric focusing, one molecular mass species of the reductase ( M r 30,400) appeared to be selectively suppressed by bromobenzene treatment. This molecular mass species was the main component of two isoelectric focusing bands with p I 6.23 and 5.91. The effect in vivo of bromobenzene could not be duplicated by in vitro experiments involving treatment of purified enzyme with bromobenzene in the presence of a NADPH-dependent microsomal drug metabolizing system. The phenomenon of multiplicity of the reductase was not limited to the liver. Multiplicity of the enzyme was detected also in the spleen; however, the pattern of composition of the reductase variants vastly differed from that of the liver. In the spleen, variants with p I 5.76, 5.61, and 5.48 were the prevalent forms; the variant with p I 6.23 was absent, and p I 5.91 was present in a minute amount. Further, bromobenzene did not affect the composition pattern of net-charge variants in this organ. Also, the splenic biliverdin reductase activity was refractory to in vivo bromobenzene treatment, whereas the liver reductase activity with both NADH and NADPH was altered by the treatment. The possible significance of the presence of multiple variants of biliverdin reductase and the change in their composition caused by bromobenzene is discussed.

Biliverdin reductase activity in cattle, sheep, rabbits and rats

1. 1. Biliverdin reductase (BVR) activity was measured in post-microsomal supernatants of livers of cattle, sheep, rabbits and rats. BVR activities in bovine and ovine livers were 4.7 and 5.0%, respectively, of rat liver activity. 2. 2. The finding of BVR activity in ruminants is in contrast to a previous report and may be due to the use of a different assay system. 3. 3. Lapine liver had the lowest BVR activity of only 0.37% of rat liver activity. 4. 4. Increasing the available heme by phenylhydrazine administration did not induce increased hepatic or splenic BVR activity in rabbits. 5. 5. Maximal BVR activities were attained using NADPH as cofactor at pH 8.7 in sheep and rabbits and at pH 8.4 in cattle. 6. 6. Differing concentrations of bovine or human albumins enhanced or inhibited BVR activity quite differently in the various species. 7. 7. The finding of a very low, but measurable BVR activity in lapine liver and spleen may explain, in part, why rabbits, unlike rats, cattle and sheep, excrete primarily biliverdin (70%) into bile.

Tin-protoporphyrin suppression of hyperbilirubinemia in the jaundiced Gunn rat.

Suppression of hyperbilirubinemia was studied in jaundiced adult and neonatal Gunn rats following treatment with tin-protoporphyrin. The effects of tin-protoporphyrin treatment on heme oxygenase, NADPH cytochrome c reductase, and biliverdin reductase activities were studied in adult jaundiced Gunn rat renal, hepatic and splenic tissues. Hepatic heme oxygenase activity was studied in nonjaundiced and jaundiced Gunn neonates. Significant decreases in plasma bilirubin levels were observed for both adult and neonatal rats treated with 50 or 100 mumol tin-protoporphyrin/kg body weight. Tin-protoporphyrin-treated rats had significantly lower hepatic and renal heme oxygenase activities, splenic and renal cytochrome c reductase activities, and a significantly higher splenic biliverdin reductase activity. Hepatic heme oxygenase activity was also significantly reduced in the neonatal rats.

Bile pigment formation and excretion in the rabbit

1. 1. Bile and plasma levels of biliverdin and bilirubin, together with the hepatic biliverdin reductase and bilirubin UDP-glucuronosyl transferase activities, were studied in the rabbit. 2. 2. No biliverdin could be detected in the blood plasma. The bilirubin concentration in blood was 7.81 ± 0.79 μmol/1. 3. 3. Biliverdin was the predominant pigment in bile (63%). 4. 4. Hepatic biliverdin reductase activity was 0.086 ±0.016 nmol/mg protein/hr. The synthesis of bilirubin was apparently limited by the enzyme activity. 5. 5. Most of the bilirubin in bile was conjugated (90%) with monoconjugates predominating (75%). 6. 6. Hepatic UDP-glucuronosyl transferase activity was 2.65 ± 0.18 and 1.14 ± 0.16 μmol/mg protein/hr with and without activation, respectively.

Prevention of neonatal hyperbilirubinaemia in non-human primates by Zn-protoporphyrin.

Non-human primates were used as a model of human neonatal hyperbilirubinaemia and its chemotherapeutic suppression. High levels of haem oxygenase activity were detected in the liver and the spleen of neonatal rhesus (Macaca mulatta) and cynomolgus (Macaca irus) monkeys. When 1-day-old neonatal animals were given a single injection of Zn-protoporphyrin (40 mumol/kg, subcutaneously), serum bilirubin levels declined to nearly normal adult levels within 24 h and remained suppressed throughout the postnatal period (12 days). This treatment inhibited the activities of haem oxygenase and biliverdin reductase in the liver and the spleen, without affecting that of the brain. Zn-protoporphyrin treatment did not alter the activity of brain biliverdin reductase or increase brain bilirubin levels. The biological disposition of Zn-protoporphyrin was examined by measuring the biliary and urinary excretion of the metalloporphyrin complex, as well as its uptake and deposition in blood cells and tissues. Biliary excretion of the metalloporphyrin was minimal (0.12% over a 28 h period), and no evidence was detected for the urinary excretion of Zn-protoporphyrin. However, the concentration of metalloporphyrin in erythrocytes increased over the duration of the experiment (11 days) to such an extent that 46% of the administered compound was taken up by the cells. It appeared that the molecular basis for the sustained suppression of haem oxygenase activity and bilirubin production by Zn-protoporphyrin involved the release of the metalloporphyrin in the normal process of the degradation of fetal erythrocytes. The scope of the biological activity of Zn-protoporphyrin to alter haem-dependent processes appeared limited in nature, insofar as the microsomal contents of cytochrome P-450 and b5, as well as the aniline hydroxylase, were similar to those of the control animals. Also, the concentration of glutathione in the liver was unchanged. These findings suggest the potential usefulness of Zn-protoporphyrin in experimental and perhaps clinical conditions in which hyperbilirubinaemia occurs.

Hemoglobin-degrading enzymes in experimental subcutaneous hematomas.

The activities of heme oxygenase and biliverdin reductase were determined in subcutaneous (s.c.) hematomas of rats after different periods of vital time. The postmortem stabilities of the heme-degrading enzymes were also studied by keeping the rats with vital hematomas for 1-5 days at temperatures of +4 degrees C and +22 degrees C. A tenfold increase of heme oxygenase activity over the starting level was observed in 2-9-day-old vital hematomas, when the specimens were taken immediately after death. Biliverdin reductase showed only negligible changes. Postmortally, heme oxygenase activity started to decrease in hematomas immediately at +22 degrees C and from day 2 on at +4 degrees C.

Hepatic heme metabolism: Possible role of biliverdin in the regulation of heme oxygenase activity

Treatment of rats with biliverdin (48 h) resulted in an increase in microsomal heme oxygenase activity in the liver. This was accompanied by decreases in the microsomal heme and cytochrome P-450 contents. In these respects cellular responses elicited by biliverdin resembled those produced by hematin (48 h). When rats were treated with biliverdin for a short interval (3 h) an inhibition of the activities of heme oxygenase and biliverdin reductase, concomitant with an increase in microsomal heme and cytochrome P-450 contents, were observed. Hematin was ineffective in altering these parameters under similar conditions. Biliverdin, in a concentration-dependent manner, inhibited the activities of purified heme oxygenase and biliverdin reductase. The activity of purified rat liver heme oxygenase was refractory to bilirubin, whereas that of purified biliverdin reductase was severely inhibited. It is suggested that biliverdin regulates cellular heme degradation processes by occupying the heme binding site on heme oxygenase, thus hindering the access of the substrate to the catalytic site of the enzyme.

Isolation from rat liver of a peroxisomal enzyme which converts molecular form 1 of biliverdin reductase into molecular form 3

Cobaltous chloride induced in rat liver an enzyme which converted biliverdin reductase molecular form 1 into the molecular form 3. This conversion involves the oxidation of two sulfhydryl groups of form 1 giving rise to a disulfide bond in form 3. The converting enzyme was isolated from the liver peroxisomal fraction (which was devoid of biliverdin reductase activity), and was absent in liver peroxisomes of control rats. The enzyme was solubilized by treatment of the peroxisomes with 0.1% sodium deoxycholate, and partially purified by DEAE-cellulose and Sephadex G-100 filtration. It is a NAD + dependent enzyme which was inactivated by trypsing and heat treatments. It did not oxidize either reduced glutathione or cysteine. The converting enzyme had a molecular weight of about 54,000 daltons. The oxidation of biliverdin reductase molecular form 1 mediated by the converting enzyme did not affect the latter's molecular weight or activity.

Biliverdin reductase-like activity in buffalo mammary tissues

Lactating buffalo mammary tissues exhibited biliverdin reductase activity. The supernatant fraction obtained from the tissue possessed the enzyme activity. Enzyme activity could not be detected in non-lactating mammary tissues, and was absent in lactating and non-lactating mammary tissues from cow and goat.

Biliverdin reductase: Characterization in the rat kidney and the inhibition of activity by mercuric chloride

The effects of metal ions on the activities of biliverdin reductase in the rat kidney and liver were examined; the pH optimum and the cofactor requirement for the enzyme activity in the kidney were also studied. The reduction of biliverdin IXα by biliverdin reductase in the rat kidney cytosol fraction could be supported by NADPH and NADH. The activity was optimal around pH 8.7 when NADPH was the cofactor. The activity with NADH was undetectable at this pH. NADH-dependent biliverdin reductase was optimal at pH 7.0, where the NADPH-dependent activity was negligible. Biliverdin reductase activity was not inducible in the kidney or liver in response to treatment of rats with metal ions—Co 2−, Ni 2−, Pb 2+, Sn 2−, Zn 2−, Cd 2+, and Cu 2- or sodium selenite. Rather, both NADPH- and NADH-dependent activities in the kidney were decreased markedly in a time- and dose-related manner following the adminstration of HgCl 2 (10–30 μmoles/kg, 24 hr). The pretreatment of rats (30 min) with sodium selenite (5 μmoles/kg) effectively blocked the Hg 2+ (20 μmoles/kg. 24 hr) inhibition of the kidney cytosol biliverdin reductase activity. Similarly, in vitro Hg 2+ was an effective inhibitor of the kidney biliverdin reductase. In addition, highly purified biliverdin reductase also was extremely sensitive to Hg 2+ and the thiol reagent, 5, 5'-dithiobis-(2-nitrobenzoic acid). The inhibition of purified reductase by 5,5'-dithiobis-(2-nitrobenzoic acid), but not by Hg 2-, could be reversed by dithiothreitol.

Electrophoretic characterization and genetics of human biliverdin reductase (BLVR; EC 1.3.1.24); assignment of BLVR to the p14 → cen region of human chromosome 7 in mouse-human somatic cell hybrids

A simple chromogenic staining procedure is described for the specific identification of biliverdin reductase (BLVR) after gel electrophoresis. Using this procedure a gene for BLVR was assigned to the p14 leads to cen region of human chromosome 7 in human-mouse somatic cell hybrids. Incidentally, the study indicated that the NADH-as well as the NADPH-dependent BLVR activities are due to one and the same enzyme and that, most probably, only one gene in the human genome codes for BLVR and the BLVR is a monomer in its functional configuration.

Raised serum biliverdin in hepatic necrosis

Serum biliverdin has been found to be very high in patients with paracetamol-induced hepatic necrosis who die but only slightly raised in those who survive. The rise in nontoxic biliverdin is proportionately greater than that of potentially toxic bilirubin. Studies using rats show that paracetamol or galactosamine induced necrosis or long-standing biliary obstruction cause a reduction in biliverdin reductase activity in the liver. In man, biliverdin accumulation in obstructive jaundice is proportional to the accumulation of bilirubin and this might be accounted for by the accompanying increase in bile acids which impair the reductase activity.

Purification and characterization of biliverdin reductase from rat liver.

Biliverdin reductase in a stable form was purified to homogeneity from rat liver cytosol. The purified enzyme showed 3700-fold increase in specific activity when compared with the crude preparation, and the extent of recovery was 30-35%. The molecular weight was estimated at 34,000-36,000. The amino acid analysis of the purified preparation revealed the presence of 3 cysteine residues/mol of enzyme. The reductase utilized NADPH and NADH as electron donors. The NADPH-dependent biliverdin reductase activity was extremely sensitive to SH reagents, including 5,5'-dithiobis(2-nitrobenzoic acid), N-ethylmaleimide, p-chloromercuribenzoic acid, and iodoacetamide. However, the pretreatment of the enzyme with NADPH and biliverdin fully protected the reductase from inactivation by these reagents. The enzyme activity was irreversibly inhibited by HgCl2. The addition of dithiothreitol to the enzyme inhibited by 5,5'-dithiobis(2-nitrobenzoic acid) promoted the full reversal of inhibition. The enzyme exhibited different pH optima for activity with NADPH (pH 8.7) and NADH (pH 7.0). The apparent Km for biliverdin was established to be 5.0 microM with NADH and 3.0 microM with NADPH. The apparent Km for NADPH was 3.0 microM, while that of NADH was 270 microM. The enzyme activity was inhibited by the substrate when the concentration exceeded 4.0-5.0 microM. The product, bilirubin, inhibited the enzyme activity in a competitive manner. In addition, the reductase was inhibited by hematin and zinc-protoporphyrin. Dilution produced instability in the enzyme, but the presence of exogenous proteins, such as serum albumin, beta-lactoglobulin, and lysozyme, stabilized the enzyme protein.