Changes in the Composition of Bilirubin‐IX Isomers During Human Prenatal Development

Abstract

We analyzed the isomeric composition of bilirubin-IX in human fetal bile using HPLC. The approximate ratio of the bilirubin-IX isomers obtained from the fetal bile at 20 weeks of gestation was IXa, 6 % ; IXP, 87 % ; IXy, 0.5 % ; and 1x6, 6 %.From 15 to 22 weeks, bilirubin-IXP was predominant and bilirubin-1x8 and bilirubin-IXa were also present in the bile as minor components. By 28 weeks, bilirubin-IXa constituted about 50% of the total bilirubin.There was a general correlation between fetal age and the proportion of bilirubin-IXa to bilirubin-IXP in the bile and the small intestinal contents of fetuses. As development proceeded from mid-gestation to near term, the isomeric composition dramatically changed, with a decrease in the IXP isomer and a subsequent increment of the IXa isomer. In contrast, the 1x6 isomer changes little.Recently, we identified four forms of biliverdin reductase including two biliverdin-IXa reductases and two biliverdin-IXP reductases in human liver cytosolic fractions [Yamaguchi, T.) Komoda, Y. & Nakajima, H. (1994) J. B i d . Chem. 269, 24343-243481. The proportion of the total activity of biliverdin-IXP reductases to that of biliverdin-IXa reductases was considerably higher in the fetal, than in the adult liver.Keywords: bilirubin-IX isomer; biliverdin-IX isomer ; bile pigment; biliverdin reductase ; heme metabolism.Protoheme-IX, undergoes enzymic ring cleavage and is converted to biliverdin via several intermediate metabolic pathways (Nakajima et al., 1963;Tenhunen et al., 1969; Maines et al., 1977;Yoshida and Kikuchi, 1978;Yoshinaga et al., 1982).To salvage and reuse the iron within heme, the sites of ring cleavage of heme are theoretically considered to be the 5(a)-, lo@-, 15(y)-, and 20(6)-methene bridges. The end products of heme degradation are biliverdin-IXa,-IXP,-IXy and -1X6, respectively. In most mammals, heme is converted by heme oxygenase at the a-methene bridge into biliverdin, and biliverdin is reduced to bilirubin by the cytosolic enzyme, biliverdin reductase (Singleton and Laster, 1965;Tenhunen et al.. 1970;Colleran and O'Carra, 1977) and excreted into the bile after conjugation.Naturally occurring bilirubin is believed to have mainly a IXa configuration due to the cleavage of heme at the S(a)-methene bridge and the subsequent reduction of the formed biliverdin. However, small amounts of other bilirubin isomers as well as several similar unidentified pigments have been found under both physiological and pathological conditions in vivo (Heirwegh et al., 1970;Gordon et al., 1977;.Several procedures for investigating the isomeric structure of biliverdin-IX and bilirubin-IX have been reported. These are alkaline permanganate degradation (Petryka et al., 1962), dichromate degradation (Rudiger, 1968), combined mass spectrometry and NMR (Nichol and Morel], 1969) and one-dimensional (Rudiger, 1968) or two-dimensional TLC (Bonnett and McDonagh, 1973). All these methods have disadvantages when applied to biological specimens since they are often obtainable only in small amount...

BLVRB redox mutation defines heme degradation in a metabolic pathway of enhanced thrombopoiesis in humans

“…All natural BVs are formed by ring cleavage of preassembled heme and not by de novo assembly from smaller units 40 ; although cleavage could occur at any of the 4 meso bridge carbons, there is striking regioselectivity of HMOX for the a-meso (IXa) carbon bridge with limited generation of BLVRB-specific IXb, IXd, and IXg isomers. 28 Interestingly, BV isomer generation appears distinct in the fetus where BV IXb may predominate, 41 although the mechanism for BV IXb generation remains enigmatic. Nonetheless, BLVRB binds promiscuously (and nonproductively) to other tetrapyrroles such as protohemin 27 or BV IXa, 28 suggesting that its redox activity could be modulated by endogenous cellular heme byproducts with resultant effects on redox coupling and lineage commitment (ie, function as a metabolically regulated cellular rheostat 34 ).…”

Studies on the Specificity of the Tetrapyrrole Substrate for Human Biliverdin-IXα Reductase and Biliverdin-IXβ Reductase

“…In previous work with ox kidney BVR-A (42) and rat kidney BVR-A (43), we have attempted to define the effect of BSA as simple sequestration of the verdin substrate; however, detailed work in our laboratory suggests that this is not the only function (41). 3 The extinction coefficient for bilirubin-IX␣ at 460 nm is increased on binding to albumin, and the free concentration of biliverdin IX␣ is also reduced by binding to BSA; however, it is clear that other factor(s) are also operative (44). By monitoring the ⌬A 660, as opposed to ⌬A 460, some of these complications are overcome, albeit at the expense of sensitivity.…”

Excretion of biliary compounds during intrauterine life

“…However, small amounts of the other three isomers are also generated depending on the position of the protoporphyrin IX that is cleaved. These include biliverdin-IXγ, biliverdin-IXδ and biliverdin-IXβ, which is the most abundant of these three pigments in humans and other mammals [35] .…”

“…Regarding bile pigments, although the IXβ isomer of bilirubin constitutes only a small fraction of the total amount produced in the fetus [33] , this more water-soluble isomer is the most abundant isomer found in fetal gallbladder bile and meconium [34,35] . The reason for this is two-fold: (1) bilirubin IXβ cannot easily cross the placenta; and (2) it can be excreted into bile without previous conjugation with glucuronic acid [36] .…”