Metabolism of bilirubin and its photoisomers in newborn infants during phototherapy

Bilirubin and its photoisomers in the biological fluids of a hyperbilirubinaemic newborn infant before and during phototherapy were analyzed by a recently improved HPLC method. In the serum, the percentages of (EZ)- and (ZE)-bilirubin in the total bilirubin concentration before phototherapy were approximately 10% and on average increased over 1.5-fold at 2 h after initiation of phototherapy. The percentage of the (EZ)-cyclobilirubin in the serum bilirubin was under 1%. In the bile, the mean concentration of (ZZ)-bilirubin, derived mainly from (ZE)-bilirubin, nearly tripled during phototherapy. The (EZ)-cyclobilirubin concentration in the bile was very low before phototherapy, increased nearly ten-fold at 3 h after initiation of phototherapy, and was 5- to 6-fold as high as that of (ZZ)-bilirubin. In the urine, upon exposure to light, the urinary concentration of (EZ)-cyclobilirubin is apparently equivalent to half of the biliary concentration of (ZZ)-bilirubin and one-fifth of that of (EZ)-cyclobilirubin. It was concluded that during phototherapy of neonatal hyperbilirubinaemia the structural photoisomer [(EZ)-cyclobilirubin] predominates considerably over the geometric photoisomer [(ZE)-bilirubin].     

Biliary and urinary excretion rates and serum concentration changes of four bilirubin photoproducts in Gunn rats during total darkness and low or high illumination.

On cycled exposure of Gunn rats to total darkness and low and high illumination, biliary excretion rates of (EZ)- and (ZE)-bilirubin and (EZ)-cyclobilirubin increased up to approx. 10-fold from the mean basal values of 1.2 and 0.2 microgram/h to the mean maximum values of 25.2 and 4.2 micrograms/h respectively, and at the same time those of (EE)-bilirubin and (EE)-cyclobilirubin also increased, but at very much lower rates than those of the first-mentioned two. During the low illumination only (EZ)- and (ZE)-bilirubin and (EZ)-cyclobilirubin appeared in the urine; during the high illumination (EE)-bilirubin and (EE)-cyclobilirubin also appeared, showing a similar excretion pattern to that observed in the bile, but the total urinary excretion rates were lower than the total biliary excretion rates. The serum bilirubin concentrations fell gradually to lower values, accompanied by an increment in (EZ)- and (ZE)-bilirubin, but (EZ)-cyclobilirubin was not detected. It is concluded that during phototherapy the predominant pathway for the removal of bilirubin from the body in the Gunn rat is by biliary excretion of the geometric photoisomers (EZ)- and (ZE)-bilirubin, derived from Z----E isomerization, and the structural photoisomer (EZ)-cyclobilirubin, formed from intramolecular endo-vinyl cyclization.     

Kinetic study of the photochemical changes of (ZZ)-bilirubin IX alpha bound to human serum albumin. Demonstration of (EZ)-bilirubin IX alpha as an intermediate in photochemical changes from (ZZ)-bilirubin IX alpha to (EZ)-cyclobilirubin IX alpha.

The present study was performed to elucidate why the photochemical reaction of (ZZ)-bilirubin bound to human serum albumin is singularly selective, and only one of the two (EZ)- and (ZE)-bilirubins, the (ZE)-isomer, is produced. In a kinetic study of the photochemical reaction in vitro, the sum of the relative rate constants of photochemical transformation of (EZ)-bilirubin into both (EZ)-cyclobilirubin and (ZZ)-bilirubin, with a significant preference for the former, was proved to be considerably larger than that of the transformation of (ZZ)-bilirubin into (EZ)-bilirubin. Therefore only one of the geometrical isomers, namely (ZE)-bilirubin, is apparently formed. It was concluded that (EZ)-bilirubin photochemically undergoes (EZ)-cyclization, i.e. structural photoisomerization, while bound to its high-affinity site on human serum albumin, and is an intermediate in the transformation of (ZZ)-bilirubin into (EZ)-cyclobilirubin.     

Wavelength-dependence of the relative rate constants for the main geometric and structural photoisomerization of bilirubin IX alpha bound to human serum albumin. Demonstration of green light at 510 nm as the most effective wavelength in photochemical changes from (ZZ)-bilirubin IX alpha to (EZ)-cyclobilirubin IX alpha via (EZ)-bilirubin.

The kinetics for the quantitatively important reaction: (Formula: see text) that is, the photochemical interconversion between bilirubin and its geometric and structural photoisomers bound to human serum albumin in aqueous solution when various wavelengths of monochromatic light were used, were assayed by h.p.l.c. In order to clarify the wavelength-dependence of the relative rate constants in the individual steps, a light-source with a half-bandwidth of 10 nm was used at increments of 20 nm, in the range from 410 nm to 550 nm. We describe for the first time studies on the wavelength-dependence of rate constants in geometric and structural photoisomerization reactions in vitro of (ZZ)-bilirubin or (EZ)-bilirubin bound to human serum albumin, especially the relative rate constants of cyclization of (EZ)-bilirubin into (EZ)-cyclobilirubin. Because studies in vitro have demonstrated that the wavelengths from 350 to 450 nm are mutagenic, the results obtained indicated that the safest and ideal light-source for phototherapy is green light of 510 nm, which keeps (ZE)-bilirubin concentrations as low as possible, as shown by a maximal value of k2 at 510 nm and a relatively low value of k1 at 510 nm. This light-source still ensures the substantial absorption of (ZZ)-bilirubin, which is the precursor of (EZ)-bilirubin, the intermediate in (EZ)-cyclobilirubin formation and, furthermore, as shown by the maximal value of k5 and a considerable value of k4 at 510 nm, promotes the cyclization of (EZ)-bilirubin derived from (ZZ)-bilirubin even though k3 at 510 nm also shows a peak value.     

Effect of the binding of bilirubin to either the first class or the second class of binding sites of the human serum albumin molecule on its photochemical reaction.

The kinetics of the photochemical changes of bilirubin were studied at a constant concentration of bilirubin bound either to the first class or to the second class of binding sites of the human serum albumin molecule. The more the bilirubin binds to the first class of binding sites in the human serum albumin molecule, the more readily geometric photoequilibrium to give (ZE)-bilirubin takes place. The more the bilirubin binds to the second class of binding sites or allosterically transformed binding sites induced by added SDS, the more readily structural photoisomerization, i.e. the formation of (EZ)-cyclobilirubin, takes place. When the serum bilirubin concentration is at low, safe, values bilirubin binds exclusively to the first class of binding sites and serves as an antioxidant [Onishi, Yamakawa & Ogawa (1971) Perinatology 1, 373-379]; at these concentrations human serum albumin protects bilirubin from irreversible photodegradation by only allowing readily reversible geometric photoisomerization. As the serum bilirubin concentration increases to high, and potentially dangerous, values, bilirubin binds to the second class of binding sites, and under these conditions human serum albumin seems to promote the photocyclization of bilirubin. During irradiation human serum albumin seems to act by retaining low, useful, concentrations of bilirubin while facilitating irreversible photoisomerization of excess bilirubin.     

Comparison of kinetic study of the photochemical changes of (ZZ)-bilirubin IX alpha bound to human serum albumin with that bound to rat serum albumin.

It has been stated by McDonagh, Palma & Lightner [(1982) J. Am. Chem. Soc. 104, 6867-6871] that complexing of bilirubin with serum albumin has a marked species-dependent influence on bilirubin photoisomerization in vitro and in vivo. Therefore the kinetics for the quantitatively important reaction: (Formula: see text) of the photochemical interconversion between bilirubin and its photoisomers bound to human or rat serum albumin in aqueous solution, assayed by h.p.l.c., was used to elucidate the observed species-dependent difference. The relative rate constants for bilirubin bound to human serum albumin, except for k4, the rate of interconversion from (ZZ)-bilirubin into (EZ)-bilirubin, proved to be considerably larger than those for bilirubin bound to rat serum albumin. In accordance with these rate constants, the formation of photoisomers of bilirubin bound to human serum albumin, except for (EZ)-bilirubin, is very rapid and much greater than that for bilirubin bound to rat serum albumin.     

Significance of the endo-vinyl group of bilirubin in photochemical reactions.

In photochemical experiments on bilirubin III alpha (no endo-vinyl group), IX alpha (one endo-vinyl group) and XIII alpha (two endo-vinyl groups) and in the photochemical, thermal and catalytical reversion of their photoproducts under anaerobic conditions, much more instability and complexity of photoproducts of bilirubin XIII alpha were observed than for those of bilirubin IX alpha or III alpha. On the basis of present and previous results of photochemical experiments in vitro and the fact that large amounts of (EZ)-cyclobilirubin IX alpha appear in the bile during phototherapy of neonatal hyperbilirubinaemia [Onishi, Kawade, Itoh, Isobe & Sugiyama (1980) Biochem. J. 190, 527-532], it is concluded that the endo-vinyl group plays a crucial role in the photochemical reaction of bilirubin IX alpha. On reversed-phase high-pressure liquid chromatography of photoisomers, it was found that the retention times of geometric isomers and E-cyclized structural isomers were shortened compared with those of Z-isomer and E-isomer, respectively, as precursor substances.     

Measurement of plasma unbound unconjugated bilirubin

A method is described for measuring the unconjugated fraction of the unbound bilirubin concentration in plasma by combining the peroxidase method for determining unbound bilirubin with a diazo method for measuring conjugated and unconjugated bilirubin. The accuracy of the unbound bilirubin determination is improved by decreasing sample dilution, eliminating interference by conjugated bilirubin, monitoring changes in bilirubin concentration using diazo derivatives, and correcting for rate-limiting dissociation of bilirubin from albumin. The unbound unconjugated bilirubin concentration by the combined method in plasma from 20 jaundiced newborns was significantly greater than and poorly correlated with the unbound bilirubin determined by the existing peroxidase method (r = 0.7), possibly due to differences in sample dilution between the methods. The unbound unconjugated bilirubin was an unpredictable fraction of the unbound bilirubin in plasma samples from patients with similar total bilirubin concentrations but varying levels of conjugated bilirubin. A bilirubin-binding competitor was readily detected at a sample dilution typically used for the combined test but not at the dilution used for the existing peroxidase method. The combined method is ideally suited to measuring unbound unconjugated bilirubin in jaundiced human newborns or animal models of kernicterus.     

Self-association of unconjugated bilirubin-IX alpha in aqueous solution at pH 10.0 and physical-chemical interactions with bile salt monomers and micelles.

Spectrophotometric measurements of bilirubin-IX alpha in water and in aqueous/organic solvent mixtures at pH 10.0 as a function of bilirubin-IX alpha concentration (approx. 0.6--400 microM) are consistent with the formation of dimers (KD - 1.5 microM) in dilute (less than 10 microM) aqueous solution and further self-aggregation to multimers at higher concentrations. Added urea (to 10M) and increases in temperature (to 62 degrees C) obliterate the dimer-multimer transition at 10 microM, but added NaCl (to 0.30 M) promotes strong aggregation of dimers over a narrow concentration range, suggesting a 'micellization' phenomenon. Concentrations of dioxan or ethanol greater than 60% (v/v) in water were required to obtain the absorption spectrum of bilirubin-IX alpha monomers, suggesting that both hydrophobic and electrostatic (pi-orbital) interactions are involved in stabilizing the dimeric state in water. Micellar concentrations of sodium dodecyl sulphate induced spectrophotometric shifts in the dimer absorption spectrum of bilirubin-IX alpha consistent with progressive partitioning of bilirubin-IX alpha monomers into a relatively non-polar region of the micelles and allowed a deduction of the apparent critical micellar concentration that closely approximated the literature values. The pattern of bilirubin IX alpha association with bile salts is complex, since the absorption spectrum shifts hypsochromically below and bathochromically above the critical micellar concentration of the bile salts. Consistent with these observations, bilirubin IX alpha appears to bind to the polar face of bile salt monomers and to the polar perimeter of small bile salt micelles. At higher bile salt concentrations some-bilirubin-IX alpha monomers partition into the hydrophobic interior of the bile salt micelles. Our results suggest that under physiological conditions the natural conjugates of bilirubin-IX alpha may exhibit similar physical chemical properties in bile, in that dimers, highly aggregated multimers and bile salt-associated monomers may co-exist.     

Kinetics of bilirubin oxidase and modeling of an immobilized bilirubin oxidase reactor for bilirubin detoxification

The unbound bilirubin concentration and the enzymatic rate of bilirubin degradation by bilirubin oxidase in bilirubin-serum albumin solutions have been investigated experimentally and theoretically. A stoichiometric bilirubin-serum albumin binding analysis shows that the unbound bilirubin concentration depends only on the molar ratio of the total bilirubin concentration to the total serum albumin concentration. From the theoretical analysis and the measured unbound bilirubin concentrations, serum albumin may be modelled as a molecule having two binding sites, primary and secondary, with stoichiometric equilibrium constants of K(1) = 6 x 10(7)M(-1) and K(2) = 4.5 x 10(6)M(-1), respectively. The rate of total bilirubin degradation in bilirubin-serum albumin mixtures is zero order. An immobilized bilirubin oxidase reactor model, which shows good agreement with experimental bilirubin conversions, is presented. At a flow rate of 1 mL/min with a 8-mL reactor volume, a 50% bilirubin conversion per pass was observed with an inlet bilirubin concentration of 350muM and a serum albumin concentration of 500muM.     

Structure and thermal interconversion of cyclobilirubin IX alpha.

One of the two main photoproducts in bilirubin metabolism during phototherapy in neonatal hyperbilirubinaemia is (EZ)-cyclobilirubin. However, it has not yet been possible to come to a final conclusion as to its chemical structure, despite the fact that much effort has been expended on the problem. The present paper demonstrates that (EZ)-cyclobilirubin is formed by the intramolecular cyclization of the C-3-vinyl group with the position at C-7 rather than at C-6, without delta-lactone-ring formation. The evidence comes from 13C-n.m.r. spectra, which indicate that an oxygen-bound quaternary carbon atom is not present, and from 1H-n.m.r. spectra, which indicate that the orientation of the methyl group at C-2 is equatorial; these findings are supported by mass spectra. The existence of both an epimeric relationship at C-7 between (EE)- and (EZ)-cyclobilirubins A and B and of steric isomers of the hydrogen atom and methyl group at C-2 is supported by the fact that the methyl-group protons at C-2 and C-7 are observed as a paired signal in 1H-n.m.r. spectra, and that new signals at C-7, C-2 and C-3 beta appear in 13C-n.m.r. spectra, that mass spectra of (EZ)-cyclobilirubins A and B are extremely similar and that, furthermore, thermal interconversion between (EE)- and (EZ)-cyclobilirubins A and B is observed.     

Degradation of hemin IX and synthetic hemins to α-bilirubins and their conjugates in the isolated perfused rat liver

Hemin IX was perfused through rat liver of a normal, untreated animal. Its degradation products, collected in the bile fluid over a period of 90 min, were found to consist of the bilirubin IX-α diglucuronide (56%), the mixture of bilirubin IX-α monoglucuronides (42%), and free bilirubin IX-α (2%). When the synthetic hemin XIII $\text{2}$ was perfused with the same technique, it was found to be degraded in the same way. The bile fluid contained the diglucuronide of bilirubin XIII-α $\text{10}$ (55%), the monoglucuronide of bilirubin XIII-α $\text{9}$ (43%) and the free bilirubin XIII-α 8 (2%). Similar results were obtained when the iron 1,4-di(β-hydroxyethyl)-2,3,5,8-tetramethyl-6,7-di(β-carboxyethyl) porphyrin $\text{3}$ was perfused; the diglucuronide of the α-bilirubin $\text{11}$ comprised 65% of the excreted bile bilirubins, the monoglucuronide was 25% of the total and the free α-bilirubin $\text{11}$ 10% of the total. Perfusion of hematohemin gave 58% of the diglucuronide of α-hematobilirubin, as well as 40% of the monoglucuronides, and 2% of the free α-hematobilirubin. The simultaneous perfusion of hematohemin and of hemin IX produced an inhibition of the degradation of the hemin IX, while hematohemin was degraded as described above. It was concluded that the normal rat liver is prepared to dispose of exogenously added hemins by their oxidation to α-biliverdins, reduction of the latter to the corresponding α-bilirubin and excretion of their conjugated derivatives through the bile duct.     

Effect of cucurbitacins on bilirubin-albumin binding in human plasma

The aim of this study is to investigate the effect of three cucurbitacins (Cuc) E, D and I on the bilirubin-albumin binding, both in human serum albumin (HSA) and in plasma. Bilirubin-HSA solution and plasma free of cucurbitacins were prepared as well as others containing serial concentrations of cucurbitacins. The concentration of unbound bilirubin was determined in bilirubin-HSA solution and the direct and total bilirubin concentrations were measured in plasma (with normal or elevated bilirubinemia) by Jendrassik and Grof method. In the conditions we adopted Cuc E and D (to a lesser extent), decreased the levels of unbound bilirubin in bilirubin-HSA solution and decreased direct bilirubin concentration and total bilirubin concentration in plasma in a dose-dependent manner while Cuc I had no effect. The effect of Cuc is related to the presence of native HSA. Thus, when albumin was absent or has been denatured by heating or by urea, Cuc E did not modify bilirubin levels, suggesting that the native structure of albumin is essential for such activity. The interaction of HSA with Cuc E was investigated by fluorescence spectroscopy. Cuc E increased the intrinsic fluorescence of the protein and the magnitude of fluorescence intensity of bilirubin-albumin complex. We concluded that Cuc E and D produced a rearrangement in the structure of albumin, particularly in the domain-II, resulting in an increase in the binding of bilirubin to albumin regardless to whether it's conjugated to glucuronic acid or unconjugated.     

Embryonal disposition of salicylate: in vivo-in vitro comparisons

The distribution of salicylate to embryonal compartments for in situ and in vitro rat embryos under equivalent exposure conditions, and salicylate disposition in the in vivo mid-gestation embryo and late gestation fetus, were compared. Pregnant Sprague-Dawley CD rats were exposed to steady-state blood levels of salicylate by infusing 14C-salicylic acid iv for a 24 hour period from gestation day 11.5 to 12.5. Cultured Sprague-Dawley rat embryos (in medium consisting of 100% male rat serum) were exposed to the steady-state 14C-salicylate concentration achieved in maternal serum in vivo for the same 24 hour developmental period. At the end of the exposure period radioactivity in visceral yolk sac, extra-embryonic fluid and embryos, and in maternal tissues, was measured. The distribution of salicylate to embryonal tissues was statistically comparable in vivo and in vitro, although the embryos in vitro accumulated slightly (but not significantly) less of the chemical. There was considerable binding of salicylate by maternal serum and culture medium proteins: less than 20% of the chemical was free at the 40 micrograms/ml concentration used in this experiment. Consequently, the salicylate concentration in embryonal compartments appeared to be quite low when compared to the surrounding serum/medium, but was actually equal to or greater than the concentration of unbound salicylate in serum or culture medium. The proportion of free salicylate in serum increased at concentrations higher than 40 micrograms/ml, resulting in somewhat higher concentrations of salicylate in in vitro embryos and extraembryonic fluid (as compared to medium) when cultured in the presence of 200 or 400 micrograms/ml salicylate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multidrug resistance associated protein 1 protects against bilirubin-induced cytotoxicity

We have shown that multidrug resistance associated protein 1 (MRP1) mediates ATP-dependent extrusion of bilirubin, possibly limiting its potentially toxic accumulation in cells. To determine directly if Mrp1 protects cells against unconjugated bilirubin (UCB) toxicity, mouse embryo fibroblasts (MEF) were isolated from Mrp1 knockout (-/-) mice and their wild type (WT) (+/+) littermates. Compared to WT cells, cultured MEF (-/-) cells exposed to 40-140 nM unbound [H3]-bilirubin accumulated twice as much [H3]-bilirubin (P<0.01). This was associated with greater, dose-related cytotoxicity, assessed by the methylthiazoletetrazolium test, lactate dehydrogenase release and cellular ATP content. The data confirm that Mrp1 limits intracellular accumulation of UCB and thus decreases its cytotoxicity.     

Possible antagonism of (Z)‐2‐hexenyl (E)‐3‐hexenoate against the attractiveness of (E)‐2‐hexenyl (Z)‐3‐hexenoate to Ooencyrtus nezarae (Hymenoptera: Encyrtidae)

Ooencyrtus nezarae (Hymenoptera: Encyrtidae) is an egg parasitoid of bean bug Riptortus pedestris (Hemiptera: Alydidae) which is a major pest of beans. Females of O. nezarae are attracted to (E)‐2‐hexenyl (Z)‐3‐hexenoate (EZ), one of the components of aggregation pheromone of R. pedestris. Effects of three isomers (ZE, EE and ZZ) of EZ on the attractiveness of O. nezarae were tested using electroantennography (EAG) and field bioassays. EAG analyses revealed that the response of O. nezarae to ZE was significantly higher than those to air, hexane and two other isomers, even though the response was lower than that to EZ. ZE affected the attractiveness of EZ dose‐dependently in the field. Addition of ZE (100 mg) to EZ (10 mg) caused a significant reduction in the catches of O. nezarae females. Single or binary addition of two other isomers (EE and ZZ) to EZ could not decrease or increase significantly the number of O. nezarae catches of EZ. Even though addition of ZZ (10, 50 or 100 mg) to EZ (10 mg) caused dose‐dependent reduction in the number of O. nezarae female catches, the reductions were not significantly different from that of EZ. EZ and its three isomers were not attractive to O. nezarae males at all.     

Resonance Raman spectroscopy of bilirubins: band assignments and application to bilirubin/lipid complexation

Resonance Raman spectra of bilirubins IX alpha, III alpha, and XIII alpha and mesobilirubin XIII alpha in alkaline aqueous and chloroform solutions are reported. Partial band assignments of bilirubin IX alpha are proposed. The model compounds confirm assignments of bands of the Raman spectrum of bilirubin IX alpha to each of the two different pyrromethenones. Resonance Raman spectra of mesobilirubin IV alpha, vinylneoxanthobilirubinic acid, and vinylisoneoxanthobilirubinic acid in alkaline aqueous solution and of the tetra-n-butylammonium salt of bilirubin IX alpha are used to define markers for the presence or absence of internal hydrogen bonds. Interaction of bilirubin dianion and sphingomyelin liposomes is studied. The Raman evidence suggests that in the bilirubin dianion/liposome complex the intramolecular hydrogen bonds between the propionate groups and the lactam NH/CO are ruptured. It is proposed that in the complex the bilirubin propionates form ion pairs with the quaternary ammonium ion of the choline moiety of sphingomyelin.     

Preparation and properties of crystalline biliverdin IX alpha. Simple methods for preparing isomerically homogeneous biliverdin and [14C[biliverdin by using 2,3-dichloro-5,6-dicyanobenzoquinone.

Amorphous isomerically pure biliverdin IX alpha is readily prepared in more than 70% yield by dehydrogenation of bilirubin with 2,3-dichloro-5,6-dicyanobenzoquinone in dimethyl sulphoxide under carefully controlled conditions. Crystalline biliverdin IX alpha and amorphous [14C]biliverdin can be obtained similarly in more than 40+ yield. The pure crystalline pigment was characterized by elemental analysis, methylation, chemical and enzymic reduction to bilirubin, i.r.- and u.v.-visible-absorption spectroscopy, n.m.r. spectroscopy and field-desorption mass spectrometry, and its solubility was determined. Under certain conditions, dehydrogenation, gave biliverdin contaminated with III alpha and XIII alpha isomers as a result of disproporationation of bilirubin. Formation of non-IX alpha isomers depends on the concentrations of the reagents and the order in which they are mixed, and occurs under neutral anaerobic conditions. Free-radical reactions probably are responsible, suggesting that the first step in the deydrogenation of bilirubin with 2,3-dichloro-5,6-dicyanobenzoquinone in dimethyl sulphoxide is formation of a bilirubin cation radical, rather than hydride ion abstraction.     

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.     

Blood levels of corticosteroid-binding globulin, total cortisol and unbound cortisol in patients undergoing coronary artery bypass grafting surgery with cardiopulmonary bypass

Previous studies have demonstrated a persistent rise in serum cortisol concentrations after cardiac surgery. To further investigate this finding and to evaluate the effect of hemodilution that occurs with the onset of cardiopulmonary bypass (CPB), concentrations of cortisol-binding globulin (CBG), total and unbound cortisol, and packed cell volume (PCV) were studied in 28 patients undergoing coronary artery bypass graft surgery. All patients received a standardized general anesthetic using a balanced technique with sufentanil, isoflurane, and midazolam. Blood was collected preoperatively, intraoperatively during CPB, and postoperatively in the evenings on the day of surgery and on the first and second postoperative day. Cortisol and CBG concentrations were measured by radioimmunoassay and were used to calculate the fraction of unbound cortisol. Serum CBG and cortisol concentrations corrected for hemodilution were significantly higher than non-corrected values. Perioperatively, CBG measurements were significantly intercorrelated. Intraoperatively, total and unbound cortisol concentrations were not significantly increased compared to preoperative values. Postoperatively up to the end of the study period serum concentrations of total and unbound cortisol were significantly increased compared to baseline values. Our results suggest that hemodilution occurs in all patients during cardiac surgery and continues up to the second postoperative day. This may lead to an underestimation of serum cortisol and CBG concentrations in patients undergoing heart surgery with CPB. Intraoperatively, concentrations of total and unbound cortisol were not significantly elevated. The postoperative rise in serum total cortisol concentration was accompanied by an increase in unbound cortisol concentration. The postoperative increase of unbound cortisol concentrations in patients undergoing cardiac surgery with CPB was largely due to an increase in cortisol secretion.