In vitro permeability and metabolic stability of bile pigments and the effects of hydrophilic and lipophilic modification of biliverdin

Bile pigments, including bilirubin and biliverdin are tetrapyrrolic, dicarboxylic acids capable of forming conjugates at their propionic acid groups via ester or amide bonds. They possess substantial antioxidant and anti-mutagenic activities and therefore their intestinal absorption might influence the development of cardiovascular disease and cancer. The aim of this study was to investigate whether altering the physico-chemical properties of bile pigments would improve their permeability in an in vitro assay of absorption. Native and synthetically modified bile pigments were tested for gastrointestinal permeability and metabolic stability using the Caco-2 cell line. In addition, a gross measure of their toxic effects was tested in a red blood cell co-incubation assay. The apparent permeability of unconjugated bilirubin (1), bilirubin ditaurate (2) and biliverdin (3) through Caco-2 cell monolayers was determined to be 10.4+/-1.2x10(-7), 35.2+/-3.4x10(-7) and 37.0+/-1.6x10(-7) cm/s (mean+/-SD), respectively, while biliverdin diglucosamine (4), and biliverdin dioctylamine (5) were impermeable. Unconjugated bilirubin, biliverdin, bilirubin ditaurate and biliverdin diglucosamine did not decompose when incubated in Caco-2 cell homogenates, whereas biliverdin dioctylamine decomposed over time. Only unconjugated bilirubin showed toxicity towards red blood cells (> or = 1000 microM), an effect that was abolished by the addition of 40 g/L serum albumin. The data presented here suggest that bile pigments are absorbed across the Caco-2 cell monolayer and that conjugation of biliverdin to hydrophilic or lipophilic moieties decreases their absorption and can reduce their metabolic stability. In summary, exogenous bilirubin and biliverdin supplements could be absorbed across the intestinal epithelium in vivo and potentially increase circulating concentrations of these antioxidant compounds.     

Biliverdin IX delta and neobiliverdin IX delta, isolated from the ovaries of the marine snail, Turbo cornutus

The ovaries of the marine snail Turbo cornutus contain a number of pigments. So far, the presence of carotenoids and a chromoprotein with a bile pigment, called turboverdin (= 3(2)-hydroxy-mesobiliverdin IX alpha), as its prosthetic group are known. The present work describes the isolation and structure elucidation of two further bile pigments, biliverdin IX delta and neobiliverdin IX delta. This is the first report of naturally occurring bile pigments with IX delta structure.     

Tissue levels of bilirubin and biliverdin in the sea lamprey, Petromyzon marinus L., before and after biliary atresia

1. Liver, intestine, kidney, muscle and epidermis from larvae, juvenile adults and upstream migrants of the sea lamprey, Petromyzon marinus L., were assayed for the presence of biliverdin and bilirubin. Urine was also examined for these bile pigments in juveniles and upstream migrants. 2. Bilirubin concentration increased dramatically in the liver and caudal intestine following loss of larval bile ducts while biliverdin levels were highest in the liver of upstream migrants and rose sharply in the caudal intestine immediately following the atresia. 3. Small amounts of bile pigment were present in larval kidneys but high concentrations were found in this organ in upstream migrants. The urine of the latter possessed biliverdin. 4. Mucus of the epidermis may be a vehicle for transport and release of bilirubin in upstream migrants. 5. These data indicate that lampreys utilize different avenues for bile pigment storage and elimination over the course of their life cycle.     

Study of the heme catabolism of fish

• 1.1. The composition of bile pigments in the blood and bile of 39 species were studied.
• 2.2. Conjugated bilirubin (trace to 4.62 mg/100 ml) was detected in the serum of most fish, while biliverdin (trace to 2.0 mg/100ml) was detected only in Anguilla Japonica, Thalassoma lunare and Clinocottus analis.
• 3.3. Analysis showed tht there are two types of bile pigments excretion pattern in these fishes. The first pattern excretes bilirubin (most conjugate) predominantly, the other excretes mostly biliverdin with some bilirubin. However, during starvation, the excretion of conjugate bilirubin gradually shifted to unconjugated biliverdin. The rate of shifting varies with species.
• 4.4. Introduction of bilirubin into Anguilla japonica produced an initial excretion of mono-conjugates, followed by di-conjugates. Introduction of biliverdin caused an increased in the excretion of unconjugated biliverdin, but no significant increase of bilirubin in the bile was detected.
• 5.5. A binary excretion pathway of bile pigments in fish is proposed. The evolutionary characteristics of heme catabolism in terrestrial animals with respect to this pathway is discussed.

     

The excretion pattern of biliverdin and bilirubin in bile of the small skate (Raja erinacea)

Summary Bile pigment composition (biliverdin, bilirubin and their conjugates) was analyzed in stored gallbladder bile and newly synthesized hepatic bile from the small skate (Raja erinacea). During a five day period of captivity, gallbladder volume remained relatively constant while bilirubin and biliverdin content increased two to three fold. Biliverdin which accounted for 50% of the pigments did not increase as a percentage of tetrapyrroles during this period. The relative proportion of bilirubin and its conjugates also remained constant, averaging 65% for bilirubin monoglucuronide, 30% for bilirubin diglucuronide and 5% for unconjugated bilirubin as measured by HPLC methods. Intravenous administration of biliverdin resulted in significant increases in the biliary excretion of both biliverdin and all bilirubin tetrapyrroles. Insignificant quantities of³H-biliverdin were detected in hepatic bile following the intravenous administration of³H-bilirubin. These studies indicate that the small skate excreted both biliverdin and bilirubin conjugates in bile and that the biliverdin was not produced by in vitro oxidation of bilirubin or its metabolites.     

Action spectra for light-controlled pupal pigmentation in Pieris brassicae: Melanization and level of bile pigment

Pupal pigmentation in Pieris brassicae is controlled by light during a sensitive period before pupation. Action spectra for melanization and level of biliverdin-IX_λ were determined in the range of 383 to 765 nm at equal quantum flux densities. Darkness and light of 500, 728, and 765 nm result in intermediate melanization and a low level of bile pigment. Wavelengths of <500 nm promote, while those of >500 nm inhibit cuticular melanization, but the level of bile pigment is raised in both. Infrared light (748 nm) is shown to have a significant effect, but only the bile pigment is influenced. In both spectra, maximal effects are observed at identical wavelengths: 404, 577, and 661 nm; a minimum occurs at 640 nm. The degree of melanization and the level of biliverdin are correlated positively for λ<500 nm and negatively for λ>500 nm. It is argued that the action spectra may not reflect the absorption of the photoreceptor(s), but rather the action of mechanisms in the central nervous system, which influence both pigments in separate and different ways. Light does not stimulate the synthesis of bile pigment in the larva before pupation, but does inhibit its degradation in the pupa. The results are discussed in the light of the endocrine mechanisms involved, and in respect of the biological significance of colour adaptation in Pieris pupae.     

Serum biliverdin as source of colouration upon sexual maturation in male blue‐throated wrasse Notolabrus tetricus

The blue colour observed in the serum of female blue‐throated wrasse Notolabrus tetricus was related to the presence of biliverdin. The biliverdin is probably stored in the female's serum until deposited in the skin upon transition to the male reproductive status, resulting in the brilliant blue colouration typical of males of this protogynous labrid species.     

FRACTIONATION OF THE EYE PIGMENTS OF DROSOPHILA MELANOGASTER

Eye pigments of normal and mutant types of D. melanogaster have been extracted with water and fractionated by chromatographic adsorption on powdered talc. Spectra of all the fractions obtainable in solution have been measured and the general chemical behavior of the pigments is described. Two chemically distinct groups of pigments are found, to be identified with the earlier designated red and brown components. The red component in the wild-type eye contains three well defined pigments, two of them capable of further subdivision so that the total number of fractions obtained is five. There is also present a brown component pigment which could not be treated quantitatively by the methods employed. All members of the wild-type red component are found in cinnabar eyes, unaccompanied by the brown component. Conversely, brown eyes contain a pigment indistinguishable from the wild-type brown component, virtually alone. In sepia eyes, one red component and two brown component pigments can be distinguished, all three pigments differing from those of wild-type eyes. Pigments apparently identical with those found in wild-type melanogaster eyes have also been found in D. virilis.     

Mechanism of action of heme oxygenase. A study of heme degradation to bile pigment by 18O labeling

The formation of bile pigment from heme by a reconstituted heme oxygenase system containing purified bovine spleen heme oxygenase, NADPH-cytochrome P-450 reductase, and biliverdin reductase was studied under an atmosphere containing 18,18O2. The product, bilirubin, was isolated and subjected to mass spectrometry, which revealed incorporation of 18O consistent with a two-molecule mechanism, whereby the product bile pigment contains oxygen atoms derived from two different oxygen molecules.     

Pigment limitation and female reproductive characteristics influence egg shell spottiness and ground colour variation in the house sparrow (Passer domesticus)

One of the hypotheses to explain egg colour variation in birds lays in the context of sexual selection, where egg colour may signal the female’s physical condition and antioxidant capacity. We tested one of the assumptions following from this hypothesis, that eggshell pigment deposition should be limited for females. The study was conducted in a captive house sparrow (Passer domesticus) population over several years under constant environmental conditions. This multi-brooded species lays eggs which vary in ground colour (biliverdin pigment) and in the intensity and distribution of brownish-red spots (protoporphyrin pigment). Spot darkness, spread and ground colour diminished along the laying sequence, suggesting that the deposition of both pigments was limiting for females over the short term. Also, the proportion of eggs with biliverdin diminished in consecutive clutches laid by the same female over the breeding season, suggesting a long-term cost of biliverdin deposition. On the other hand, spots were darker at the end of the breeding season, indicating that protoporphyrin deposition was probably not limited over the long term. This result could indicate a lower capacity for calcium deposition over the long term, which was compensated for by darker spots. Female age also significantly affected the proportion of bluish eggs and spot patterns. Egg pigmentation decreased with age, indicating that senescing passerine females lay less pigmented eggs. Clutch size was positively related to the proportion of bluish eggs and to spot patterns (darker and more evenly spotted). These results are in accordance with assumptions for the sexual selection hypothesis.     

Eggshell biliverdin concentration does not sufficiently predict eggshell coloration

Avian eggshell coloration may have arisen due to selection on the biological, chemical, or physical properties of the pigments embedded within the eggshell, or due to selection on the coloration that emerges due to pigment deposition. Within both hypothetical frameworks, pigment‐based eggshell coloration would be related to metrics of egg quality; however, no one has evaluated the relative strength of coloration and pigment concentration in predicting egg quality. Here, we examined 66 European starling Sturnus vulgaris eggs and quantified eggshell biliverdin concentration (an antioxidant that produces the eggshell's blue coloration) and used 28 different coloration metrics derived from both photographic and spectrophotometric data. We also measured egg size, eggshell thickness, concentration of carotenoids in the yolk, and concentration of lysozyme in the albumen to capture variation in egg quality. We found that throughout the laying sequence, biliverdin concentration increased while eggshell thickness, yolk carotenoid concentration, and lysozyme concentration in the albumen all decreased, but this variation was not captured by any eggshell coloration metric. Both eggshell coloration and biliverdin concentration were negatively associated with yolk carotenoid concentration, but only eggshell biliverdin concentration was negatively associated with yolk mass. Lastly, biliverdin concentration explained, at most, only 46% of the variation for all eggshell coloration metrics. Our results suggest biliverdin concentration is a better predictor of egg quality than egg coloration in European starlings, supporting the hypothesis that eggshell pigment concentration per se may be the target of selection.     

UDP-glucuronosyltransferase activity and bilirubin conjugation in the bullfrog.

Bile pigments of bile and serum of Rana catesbeiana were investigated by means of high-pressure liquid chromatography. The major pigment in both bile and serum was bilirubin IX alpha. Bilirubin UDP-glucuronosyltransferase activity was found in the livers of all animals examined, but no conjugated bilirubin was detectable in the bile. Frog bile was found to contain large amounts of beta-glucuronidase. When the beta-glucuronidase inhibitor saccharo-1,4-lactone was introduced into the gall bladder followed by an exogenous bilirubin load, bilirubin glucuronide appeared in the bile.     

Purification and properties of two blue biliproteins from the larval hemolymph and integument of Rhodinia fugax (Lepidoptera: Saturniidae)

Blue biliproteins (BPs) are found in the hemolymph and integument of the fifth instar larvae of the saturniid silkworm, Rhodinia fugax. An efficient method of isolating BPs from the hemolymph, epidermis and cuticle using hydrophobic interaction chromatography and ion-exchange chromatography was devised. The BPs from the hemolymph, epidermis and cuticle have molecular weights of approximately 24 000, 48 000 and 23 000 Da by gel-filtration, respectively. Using matrix-assisted laser desorption ionization–time of flight/mass spectrometry (MALDI–TOF/MS), the respective molecular masses were determined to be 22 641, 22 908 and 22 737 Da. Based on these results, BP molecules from the hemolymph and cuticle are assumed to be monomers, whereas the epidermal BP is a dimer. The amino acid composition and N-terminal amino acid sequences of the BPs from the hemolymph and cuticle (BP-I) are very similar, but the BP from the epidermis (BP-II) is quite different. The N-terminal amino acid sequences of these BPs share approximately 50% identity with the biliproteins from other lepidopteran insects. The blue color of BP is due to the presence of bile pigments, which are non-covalently bound to the apoprotein. The absorbance spectrum of BP-I from the hemolymph revealed maxima at 280 and 669 nm, while that of BP-II showed maxima at 280, 385 and 663 nm. The pigment dimethyl esters were extracted from BP-I and BP-II with acidic methanol and dichloromethane. The results of these analyses suggest that the blue pigments of BP-I and BP-II are different; BP-I contains a phorcabilin-like pigment while BP-II contains biliverdin IXγ. In an immunoblot analysis, anti-BP-I antibodies, produced against hemolymph BP-I, reacted with immunoreactive proteins in the hemolymph and cuticle of R. fugax. These anti-BP-I antibodies did not react with BP-II and only cross-reacted weakly with Samia cynthia ricini biliverdin-binding protein (BBP)-II.     

Energetic constraints on expression of carotenoid-based plumage coloration

Carotenoid pigments are used by many bird species as feather colorants, creating brilliant yellow, orange, and red plumage displays. Such carotenoid-based plumage coloration has been shown to function as an honest signal that is used in female mate choice. Despite recent interest in carotenoid-based ornamental traits, the basis for individual variation in expression of carotenoid-based plumage coloration remains incompletely understood. I tested the hypothesis that, independent of carotenoid access, food stress during molt would cause reduced expression of carotenoid pigmentation. I fed molting male House Finches Carpodacus mexicanus seed diets supplemented with either the red carotenoid pigment canthaxanthin or the yellow/orange carotenoid pigment β-cryptoxanthin (in the form of tangerine juice). Within each diet treatment, one group of males was given restricted food access and the other group was given unrestricted food access. Carotenoid supplements were placed in water so carotenoid access was controlled independent of food access. The results indicated a strong effect of both carotenoid access and food access on color display. Some males in the β-cryptoxanthin-supplemented group grew red plumage, suggesting that they can metabolically modify yellow pigments into red pigments, but no bird supplemented with β-cryptoxanthin grew plumage as red as birds supplemented with canthaxanthin. Males in the unrestricted food groups grew redder and more intensely pigmented plumage than males in the restricted food groups. These observations provide the best evidence to date of an energetic cost of carotenoid utilization in the generation of colorful plumage.     

Excretion of fetal biliverdin by the rat placenta-maternal liver tandem

Fetal liver immaturity is accompanied by active heme catabolism. Thus fetal biliary pigments must be excreted toward the mother by the placenta. To investigate biliverdin handling by the placenta-maternal liver tandem, biliverdin-IXalpha was administered to 21-day pregnant rats through the jugular vein or the umbilical artery of an in situ perfused placenta. Jugular administration resulted in the secretion into maternal bile of both bilirubin and biliverdin (3:1). However, when biliverdin was administered to the placenta, most of it was transformed into bilirubin before being transferred to the maternal blood. Injecting Xenopus laevis oocytes with mRNA from rat liver or placenta enhanced their ability to take up biliverdin, which was inhibited by estradiol 17beta-d-glucuronide. The expression of three OATP isoforms in this system revealed that they have a varying degrees of ability to transport biliverdin (Oatp1/1a1 > Oatp2/1a4 > Oatp4/1b2). The abundance of their mRNA in rat trophoblast was Oatp1/1a1 > Oatp4/1b2 > Oatp2/1a4. The expression of biliverdin-IXalpha reductase in rat placenta was detected by RT-PCR/sequencing and Western blot analysis. The relative abundance of biliverdin-IXalpha reductase mRNA (determined by real-time quantitative RT-PCR) was fetal liver > placenta > maternal liver. Common bile duct ligation in the last week of pregnancy induced an upregulation of biliverdin-IXalpha reductase in maternal liver but had no effect on fetal liver and placenta. In conclusion, several members of the OATP family may contribute to the uptake of fetal biliverdin by the rat placenta. Before being transferred to the mother, biliverdin is extensively converted into bilirubin by biliverdin-IXalpha reductase, whose expression is maintained even though bilirubin excretion into maternal bile is impaired.     

Isolation and identification of the urinary pigment uroerythrin.

The red pigment uroerythrin, a chromophore known to be adsorbed by the amorphous urate sediments (sedimentum lateritium), has been isolated from human urine and further purified as its trimethyl derivative. Urine was applied to a column of Amberlite XAD-2 resin on which uroerythrin and other pigments were adsorbed. The pigments were eluted with methanol and uroerythrin was further purified by extraction with ether at pH 4.0, repeated chromatography on lipophilic Sephadex LH-20 and thin-layer chromatography on silica gel. For optimal purification uroerythrin was converted into the trimethyl derivative and chromatographed on silical gel thin-layer plates. The structure of the pigment has been studied by chromate degradation followed by identification of the imide products by thin-layer chromatography. From these results and from infrared, mass spectral and nuclear magnetic resonance data a tripyrrole structure for uroerythrin is concluded. The proposed structure for the chromophore is related to that of the bile pigment biliverdin consisting, however, only of the rings A, B and C.     

不产氧光合细菌光合色素的光氧调控机制

[目的]为不产氧光合细菌光合色素研究提供可行的较系统规范的研究方法和数据,揭示固氮红细菌(Rhodobacter azotoformans 134K20)光合色素光氧适应性机制.[方法]采用光谱法和色谱法对光和氧调控下的类胡萝卜素和细菌叶绿素合成代谢进行了研究.[结果]134K20菌株光照好氧时细胞得率最高.光照厌氧时主要合成3黄、1红、1紫、2绿、2蓝9种色素,黄色素大量表达.有氧时红色素大量表达,且启动2种新的红色素和1种新的紫色素表达,而黄色和蓝绿色素则受氧抑制.黑暗好氧主要合成2黄、3红、2紫、1绿、1蓝9种色素,但不同于光照厌氧.光照好氧时黄色素减少到1种,紫色素含量增加,其余同黑暗好氧.[结论]固氮红细菌(Rhodobacter azotoformans 134K20)是通过PpsR调节途径来调节光合基因表达的.黄色和红色素属于类胡萝卜素.黄色素1属于球形烯系列,其余两种黄色素是新的类胡萝卜素组分.红色素为新的球形烯酮组分,3种红色素极性、峰形和峰位差别显著,正己烷能显示其精细结构.紫色为极性较大的细菌脱镁叶绿素,绿色和蓝色为4种极性不同的细菌叶绿素a中间产物.乙醚甲醇法适合类胡萝卜素的提取,丙酮甲醇冰冻研磨法能快速有效完全提取光合色素.溶剂效应可有效鉴别细菌叶绿素a中间产物.     

Why are birds' eggs colourful? Eggshell pigments co‐vary with life‐history and nesting ecology among British breeding non‐passerine birds

The colourful appearance of bird eggshells has long fascinated biologists and considerable research effort has focused on the structure and biochemistry of the avian eggshell matrix. The presence of tetrapyrrole pigments was identified nearly a century ago. Surprisingly, how the concentrations of avian eggshell pigments vary among related species, and whether this variability is associated with either eggshell appearance and/or species life‐history traits, remains poorly understood. We quantified the concentrations of the two key eggshell pigments, protoporphyrin IX and biliverdin, from a diverse sample of eggshells stored at the Natural History Museum, Tring, UK. We explicitly tested how these two pigments are associated with physical measures of eggshell coloration and whether the pigment concentrations and colour diversity co‐vary with phylogenetic affiliations among species. We also tested a series of comparative hypotheses regarding the association between the concentrations of the two pigments and specific life‐history and breeding ecology traits. Across species, the average concentrations of protoporphyrin and biliverdin were positively correlated, and both strongly co‐varied with phylogenetic relatedness. Controlling for phylogeny, protoporphyrin concentration was associated with a higher likelihood of cavity nesting and ground nesting, whereas biliverdin concentration was associated with a higher likelihood of non‐cavity nesting habit and bi‐parental provisioning. Although unlikely to be explained by a single function, the breeding ecology and life history‐dependence of eggshell pigment concentrations in these comparative analyses implies that related species share pigment strategies, and that those strategies relate to broad adaptive roles in the evolution of variation in avian eggshell coloration and its underlying mechanisms. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 106 , 657–672.     

Green-blood pigmentation in lizards

The green pigment in the plasma of the scincid lizard genus Prasinohaema is identified as the bile pigment biliverdin. Concentrations of biliverdin in the plasma of P. flavipes, P. prehensicauda and P. virens are 714 ± 79 μmol/1 (mean ± one standard deviation), 1020 ± 624 μmol/1 and 819 ± 89 μmol/1, respectively. These values represent the highest concentration of plasma biliverdin measured for any organism and are the first report of non-pathological biliverdin accumulation in amniotes. We review the literature for fish species with high concentrations of plasma biliverdin and pathological biliverdin accumulation in humans; we find that Prasinohaema species have plasma biliverdin concentrations approximately 1.5–30 times greater than fish species with green blood plasma and 40 times greater than humans with green jaundice.