As a prevalent, life-threatening and highly recurrent psychiatric illness, depression is characterized by a wide range of pathological changes; however, its etiology remains incompletely understood. Accumulating evidence supports that gut microbiota affects not only gastrointestinal physiology but also central nervous system (CNS) function and behavior through the microbiota-gut-brain axis. To assess the impact of gut microbiota on fecal metabolic phenotype in depressive conditions, an integrated approach of 16S rRNA gene sequencing combined with ultra high-performance liquid chromatography-mass spectrometry (UHPLC-MS) based metabolomics was performed in chronic variable stress (CVS)-induced depression rat model. Interestingly, depression led to significant gut microbiota changes, at the phylum and genus levels in rats treated with CVS compared to controls. The relative abundances of the bacterial genera Marvinbryantia, Corynebacterium, Psychrobacter, Christensenella, Lactobacillus, Peptostreptococcaceae incertae sedis, Anaerovorax, Clostridiales incertae sedis and Coprococcus were significantly decreased, whereas Candidatus Arthromitus and Oscillibacter were markedly increased in model rats compared with normal controls. Meanwhile, distinct changes in fecal metabolic phenotype of depressive rats were also found, including lower levels of amino acids, and fatty acids, and higher amounts of bile acids, hypoxanthine and stercobilins. Moreover, there were substantial associations of perturbed gut microbiota genera with the altered fecal metabolites, especially compounds involved in the metabolism of tryptophan and bile acids. These results showed that the gut microbiota was altered in association with fecal metabolism in depressive conditions. These findings suggest that the 16S rRNA gene sequencing and LC-MS based metabolomics approach can be further applied to assess pathogenesis of depression.

Using recently developed molecular-shape description algorithms, we searched the Available Chemical Directory for known compounds similar in shape to the potent HIV-1 protease inhibitor Merck L-700,417; 15 compounds most similar in shape to the inhibitor were selected for testing in vitro. Four of these inhibited the protease at 100 microM or less and the most active of the four were the naturally occurring pigments biliverdin and bilirubin. Biliverdin and bilirubin inhibited recombinant HIV-1 protease in vitro at pH 7.8 with K1 values of approx. 1 microM, and also inhibited HIV-2 and simian immunodeficiency virus proteases. The related pyrrolic pigments stercobilin, urobilin, biliverdin dimethyl ester and xanthobilirubic acid showed similar inhibitory activity at low micromolar concentrations. Biliverdin, bilirubin and xanthobilirubic acid did not inhibit viral polyprotein processing in cultured cells, but they reduced viral infectivity significantly. At 100 microM, xanthobilirubic acid affected viral assembly, resulting in a 50% decrease in the generation of infectious particles. In contrast, at the same concentrations biliverdin and bilirubin exerted little or no effect on viral assembly but blocked infection of HeLaT4 cells by 50%. These results suggest that bile pigments might be a new class of potential lead compounds for developing protease inhibitors and they raise the question of whether hyperbilirubinaemia can influence the course of HIV infection.

Bilirubin is a component of the heme catabolic pathway that is essential for liver function and has been shown to reduce hepatic fat accumulation. High plasma bilirubin levels are reflective of liver disease due to an injurious effect on hepatocytes. In healthy liver, bilirubin is conjugated and excreted to the intestine and converted by microbes to urobilinoids, which are reduced to the predominant pigment in feces, stercobilin, or reabsorbed. The function of urobilinoids in the gut or their physiological relevance of reabsorption is not well understood. In this review, we discuss the relationship of hepatic bilirubin signaling to the intestinal microbiota and its regulation of the liver-gut axis, as well as its capacity to mediate these processes.

The antioxidant properties of protoporphyrin IX and related tetrapyrroles are poorly characterized. Therefore, eight tetrapyrroles, five of which are produced in vivo, were tested to assess their antioxidant capacities in the Salmonella reverse mutation, TEAC, FRAP and ORAC assays. Tertiary-butyl hydroperoxide (tert-BOOH) in the presence or absence of metabolic activation (±S9) was added to Salmonella strain TA102 together with the test compounds. In the absence of metabolic activation, the order of effectiveness was protoporphyrin>> biliverdin>> bilirubin ditaurate>> bilirubin>> biliverdin dimethyl ester>> stercobilin>> bilirubin dimethyl ester>> urobilin. In the presence of S9, the effectiveness was reversed, with urobilin>> biliverdin dimethyl ester>> bilirubin dimethyl ester>> stercobilin>> biliverdin>> bilirubin>> bilirubin ditaurate>> protoporphyrin. In the antioxidant capacity assays FRAP, TEAC and ORAC, mainly bilirubin, bilirubin ditaurate, biliverdin and protoporphyrin showed antioxidant activity. This study reports that previously untested tetrapyrroles of related structure prevent oxidatively induced genotoxicity, and for some, novel underlying mechanisms of antioxidant action were revealed. These results support the physiological importance and biological relevance of tetrapyrroles including protoporphyrin that might act as antioxidants, protecting from oxidatively induced DNA damage, particularly in the tissues/organs where they accumulate.

Urobilinoids belong to the heterogenous group of degradation products of bilirubin formed in the gastrointestinal tract by intestinal microflora. Among them urobilinogen and stercobilinogen with their respective oxidation products, urobilin and stercobilin, are the most important compounds. The aim of present study was to analyze the products of bacterial reduction of bilirubin in more detail. The strain of Clostridium perfringens isolated from neonatal stools, capable of reducing bilirubin, was used in the study. Bacteria were incubated under anaerobic conditions with various native as well as synthetic bile pigments, including radiolabeled unconjugated bilirubin (UCB). Their reduction products were extracted from media and separated following thin layer chromatography. Pigments isolated were analyzed by spectrophotometry, spectrofluorometry and mass spectrometry. In a special set of experiments, bilirubin diglucuronide was incubated with either bacterial lysate or partially purified bilirubin reductase and beta-glucuronidase to reveal whether bilirubin glucuronides may be directly reduced onto conjugated urobilinoids. A broad substrate activity was detected in the investigated strain of C. perfringens and a series of bilirubin reduction products was identified. These products were separated in the form of their respective chromogens and further oxidized. Based on their physical-chemical properties, as well as mass spectra, end-catabolic bilirubin products were identified to belong to urobilinogen species. The reduction process, catalyzed enzymatically by the studied bacterial strain, does not proceed to stercobilinogen. Bilirubin diglucuronide is not reduced onto urobilinoid conjugates, glucuronide hydrolysis must precede double bond reduction and thus UCB is reduced much faster.

Epidemiological studies report a negative association between circulating bilirubin concentrations and the risk for cancer and cardiovascular disease. Structurally related tetrapyrroles also possess in vitro anti-genotoxic activity and may prevent mutation prior to malignancy. Furthermore, few data suggest that tetrapyrroles exert anti-carcinogenic effects via induction of cell cycle arrest and apoptosis. To further investigate whether tetrapyrroles provoke DNA-damage in human cancer cells, they were tested in the single cell gel electrophoresis assay (SCGE). Eight tetrapyrroles (unconjugated bilirubin, bilirubin ditaurate, biliverdin, biliverdin-/bilirubin dimethyl ester, urobilin, stercobilin and protoporphyrin) were added to cultured Caco2 and HepG2 cells and their effects on comet formation (% tail DNA) were assessed. Flow cytometric assessment (apoptosis/necrosis, cell cycle, intracellular radical species generation) assisted in revealing underlying mechanisms of intracellular action. Cells were incubated with tetrapyrroles at concentrations of 0.5, 5 and 17μM for 24h. Addition of 300μM tertiary-butyl hydroperoxide to cells served as a positive control. Tetrapyrrole incubation mostly resulted in increased DNA-damage (comet formation) in Caco2 and HepG2 cells. Tetrapyrroles that are concentrated within the intestine, including protoporphyrin, urobilin and stercobilin, led to significant comet formation in both cell lines, implicating the compounds in inducing DNA-damage and apoptosis in cancer cells found within organs of the digestive system.

The rate of endogenous carbon monoxide production ( Vco), determined by the closed rebreathing system technique, was elevated above the normal range in four of five patients studied with ineffective erythropoiesis (four patients with primary refractory anemia, one with thalassemia). The mean molar ratio of Vco to Vheme (rate of circulating heme catabolism, determined from (51)Cr red cell survival curves) was 3.0 +/- 0.6 (SE), indicating that most of the CO originated from sources other than circulating erythrocyte hemoglobin, in contrast to previous findings in patients with hemolytic anemia, where Vco paralleled Vheme closely.After administration of glycine-2-(14)C to these patients, endogenous CO was isolated by washout of body CO stores at high pO(2) or by reacting peripheral venous blood samples with ferricyanide. The CO was then oxidized to CO(2) by palladium chloride and trapped for counting in a liquid scintillation spectrometer. "Early labeled" peaks of (14)CO were demonstrated which paralleled "early labeled" peaks of stercobilin and preceded maximal labeling of circulating heme. Production of "early labeled" (14)CO in patients with ineffective erythropoiesis was greatly increased, up to 14 times that found in a normal subject. The increased Vco and "early (14)CO" production shown by these patients are presumably related mainly to heme catabolism in the marrow. The possibility exists that hepatic heme and porphyrin compounds may also contribute significantly to Vco, as suggested by the finding of a high Vco in an additional patient with porphyria cutanea tarda.

After the simultaneous intravenous administration of unconjugated bilirubin-(3)H and delta-aminolevulinic acid-4-(14)C, the plasma disappearance curves of unconjugated bilirubin-(3)H and the plasma appearance curves of biosynthesized unconjugated bilirubin-(14)C have been defined in seven patients, three of whom had acute intermittent porphyria (AIP). The incorporation of (14)C into plasma unconjugated bilirubin, derived by an analysis which involves deconvolution of the two plasma curves, varied between 13.1 and 23.5% (mean 19.3%) of the injected dose in the nonporphyric patients and between 5.4 and 13.6% (mean 8.3%) of the injected dose in the porphyric patients. In five of the patients, the stercobilin-(14)C specific activity in a pooled specimen of feces was measured, enabling the following further values to be calculated: (a) the total (14)C radioactivity incorporated into bilirubin (21.0 and 25.3% [mean 23.2%] of the injected dose in two of the nonporphyric patients and between 8.5 and 25.3% [mean 14.2%] of the injected dose in the porphyric patients), and (b) the proportion of hepatic synthesized bilirubin delivered directly to plasma in the unconjugated form (between 0.520 and 0.904; mean for nonporphyric patients 0.712; mean for porphyric patients 0.614). The results demonstrate that a large proportion of bilirubin derived from hepatic hemes passes through the plasma in the unconjugated form before conjugation and secretion into bile.

Faecal metabolome contains information on the metabolites found in the intestine, from which knowledge about the metabolic function of the gut microbiota can be obtained. Changes in the metabolomic profile of faeces reflect, among others, changes in the composition and activity of the intestinal microorganisms. In an effort to improve our understanding of the biological effects that phenolic compounds (including red wine polyphenols) exert at the gut level, in this foodomic study we have undertaken a metabolome characterization of human faeces after moderate consumption of red wine by healthy subjects for 4 weeks. Namely, a nontargeted metabolomic approach based on the use of UHPLC-TOF MS was developed to achieve the maximum metabolite information on 82 human faecal samples. After data processing and statistical analysis, 37 metabolites were related to wine intake, from which 20 could be tentatively or completely identified, including the following: (A) wine compounds, (B) microbial-derived metabolites of wine polyphenols, and (C) endogenous metabolites and/or others derived from other nutrient pathways. After wine consumption, faecal metabolome was fortified in flavan-3-ols metabolites. Also, of relevance was the down regulation of xanthine and bilirubin-derived metabolites such as urobilinogen and stercobilin after moderate wine consumption. As far as we know, this is the first study of the faecal metabolome after wine intake.

The interaction of protoporphyrin compounds of human origin with the major bee venom component melittin (26 a.a., Z +6) and its hybrid derivative (CM15, 15 a.a., Z +6) were studied by a combination of various spectroscopic methods. Throughout a two-state, concentration-dependent process, hemin and its metabolites (biliverdin, bilirubin, bilirubin ditaurate) increase the parallel β-sheet content of the natively unfolded melittin, suggesting the oligomerization of the peptide chains. In contrast, α-helix promoting effect was observed with the also disordered but more cationic CM15. According to fluorescence quenching experiments, the sole Trp residue of melittin is the key player during the binding, in the vicinity of which the first pigment molecule is accommodated presumably making indole-porphyrin π-π stacking interaction. As circular dichroism titration data suggest, cooperative association of additional ligands subsequently occurs, resulting in multimeric complexes with an apparent dissociation constant ranged from 20 to 65 μM. Spectroscopic measurements conducted with the bilirubin catabolite urobilin and stercobilin refer to the requirement of intact dipyrrinone moieties for inducing secondary structure transformations. The binding topography of porphyrin rings on a model parallel β-sheet motif was evaluated by absorption spectroscopy and computational modeling showing a slipped-cofacial binding mode responsible for the red shift and hypochromism of the Soret band. Our results may aid to recognize porphyrin-responsive binding motifs of biologically relevant, intrinsically disordered peptides and proteins, where transient conformations play a vital role in their functions.

A new method for the direct measurement in vivo of the synthetic rate of bilirubin from hepatic hemes is proposed. This method depends on the application of the labeled precursor-product relationship to the hepatic pool of porphobilinogen, which is a common precursor of both urinary porphobilinogen and hepatic-synthesized bilirubin. The hepatic pool of porphobilinogen is labeled by means of an intravenous injection of delta-aminolevulinic acid-4-(14)C. The proportion of total bilirubin production which is derived from hepatic hemes is calculated from the ratio of the mean (14)C specific activities of stercobilin and porphobilinogen estimated in pooled specimens of feces and urine, respectively. The method can be most readily applied to patients with acute intermittent porphyria, as the appreciable quantities of prophobilinogen in the urine of these patients greatly facilitate the measurement of porphobilinogen-(14)C specific activity. In three patients with acute intermittent porphyria, values obtained for the synthetic rate of bilirubin from hepatic hemes were 20.7, 15.8, and 13.3% of total bilirubin production.

Conventional (CONV) rats were fed by stomach tube for five days with either benzylpenicillin, ampicillin, tetracykline, oxitetracykline, neomycin, bacitracin + neomycin, kanamycin or colistin. On the 2nd-3rd day all the animals developed one or several of the following symptoms or characteristics typical for germfree (GF) rats: no coprostanol formation, no stercobilin production, a GF pattern after gel electrophoresis of fecal supernatant and proteolytic activity in the feces. Under the same conditions succinylsulfathiazole or metronidazole had much less pronounced effects than the antibiotics. When clofibrate, acetylsalicylic acid or ferrous sulphate were administered the effects were none or negligible. The GF characteristics persisted for several weeks after the end of the administration of the drugs. In some instances this was the case up to 7 weeks, when the animals were contaminated by anal route with a suspension of the cecum contents from intact CONV animals. On the 2nd day after this treatment the GF characteristics had disappeared.

Bilirubin exhibits antioxidant and antimutagenic effects in vitro. Additional tetrapyrroles that are naturally abundant were tested for antigenotoxicity in Salmonella. Un-/conjugated bilirubin (1 and 2), biliverdin (4), bilirubin and biliverdin dimethyl esters (3 and 5), stercobilin (6), urobilin (7), and protoporphyrin (8) were evaluated at physiological concentrations (0.01-2 μmol/plate; 3.5-714 μM) against the metabolically activated food-borne mutagens aflatoxin B1 (9) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (10). Compound 8 most effectively inhibited the mutagenic effects of 9 in strain TA102 and 10 in TA98. Compound 7 inhibited 9-induced mutagenesis in strain TA98 most effectively, while 1 and 4 were promutagenic in this strain. This is likely due to their competition with mutagens for phase-II detoxification. Mechanistic investigations into antimutagenesis demonstrate that tetrapyrroles react efficiently with a model epoxide of 9, styrene epoxide (11), to form covalent adducts. This reaction is significantly faster than that of 11 with guanine. Hence, the evaluated tetrapyrroles inhibited genotoxicity induced by poly-/heterocyclic amines found in foods, and novel evidence obtained in the present investigation suggests this may occur via chemical scavenging of genotoxic metabolites of the mutagens investigated. This may have important ramifications for maintaining health, especially with regard to cancer prevention.

i-Urobilin and 1-stercobilin were separated by high-performance liquid chromatography on a reversed-phase octadecylsilane-bonded column and detected fluorimetrically through formation of phosphor with zinc ions in the eluent. The separation and the intensity of the fluorescence response were affected by concentrations of zinc acetate and sodium borate buffer, pH and methanol content in the eluent. The optimal eluent used consisted of 0.1% zinc acetate in 75 mM boric acid buffer (pH 6.0)-methanol (25:75). The detection limit was 0.2 microgram/l for both i-urobilin and 1-stercobilin (signal-to-noise ratio 2), which makes the method 250-2500 times more sensitive than conventional methods.

The origins and the kinetics of unconjugated bilirubin were assessed in 25 healthy dogs. Bilirubin kinetics were measured by the analysis of the plasma disappearance of [3H]bilirubin in a two-compartment model. The relative bilirubin productions from erythrocyte haem degradation and early labeled bilirubin were determined by measuring the incorporation of [14C]glycine in erythrocyte haem and in fecal stercobilin. The incorporation of this relation into the model permitted the quantitation of the bilirubin production from erythrocyte destruction, ineffective erythropoiesis and the catabolism of hepatic haemoproteins. The contribution of the three bilirubin sources to the plasma concentration was derived from the calculated fraction reflux into the plasma of bilirubin produced in the liver from hepatic haemoproteins. Other calculated model-dependent and -independent parameters were plasma bilirubin clearance, hepatic bilirubin extraction efficiency, pool sizes, and the fractional transfer rates which reflect the hepatic uptake process, reflux from liver to the plasma, and the conjugating enzyme activity. In plasma of healthy dogs only unconjugated bilirubin was detected. It averaged 0.68 mumol/l, which is far below levels in man. This is probably due to the 20-fold higher hepatic clearance rate in dogs (median 32.2; range 21.6-43.9 ml/kg per min). In addition, fasting hyperbilirubinaemia could not be documented in the dog. The total bilirubin turnover was 14.9 (12.6-17.1) mumol/kg (median and 95% range), with 67 (60-70)% derived from erythrocyte degradation, 5.3 (4.7-5.5)% from ineffective erythropoiesis and 27.7 (24.5-35.3)% from hepatic haemoproteins. The figures for the plasma bilirubin turnover were 12.3 (10.3-14.2) mumol/kg per day, 79 (75-84)%, 6.3 (6.0-6.6)% and 14.8 (9.2-18.9)%, respectively. The presented model permits the simultaneous quantitation of both the origins and the kinetics of bilirubin. The application of this approach in pathological conditions is expected to provide better insight in the pathophysiology of acquired hyperbilirubinaemia.