Protein Content of Liquor Amnii in Prediction of Severity of Haemolytic Disease of Newborn

Two series of cases of Rh isoimmunization were subjected to liquor examination for bilirubin and protein level. Series 1 comprised 298 cases for the years 1962 and 1963. Series 2 comprised 179 consecutive cases for 1967 in which preliminary selection for liquor examination had been made on the basis of previous history and maternal antibody titre.Bilirubin was measured as the liquor bilirubin ratio, and protein levels were estimated in series 1 by the Folin and Ciocalteau technique and in series 2 by a modified biuret method.Correlations with severity of haemolytic disease in the foetus was made, taking into account the stage of gestation of liquor examination. Both bilirubin and protein levels correlate with severity, but bilirubin is superior to protein. Interrelation of these measurements as bilirubin/protein ratio was inferior to bilirubin level as a method of forecasting severity.     

Structural and functional differences between fetal and adult serum albumin

The binding of bilirubin with adult of fetal human serum albumin has been studied by steady-state fluorescence emission spectroscopy. The 1:1 complex between bilirubin and the two albumin samples shows very similar fluorescence properties, as well as essentially identical accessibility of the protein-bound bilirubin to fluorescence quenchers added to the aqueous medium. The intramolecular distance between bilirubin and the single tryptophyl residue can be estimated to be 2.4 +/- 0.2 nm for both proteins by singlet-singlet energy transfer. These findings suggest that fetal and adult human serum albumin have a very similar three-dimensional structure; the different binding capacity for bilirubin displayed by the two proteins is likely to be the consequence of small differences in the physico-chemical properties of some amino acid residues close to the bilirubin binding site, as indicated by pH-titration experiments of the intrinsic albumin fluorescence.     

Liquor Bilirubin Levels in Normal Pregnancy: A Reassessment of Early Prediction of Haemolytic Disease

A comparison was made between chemically and spectrophotometrically determined concentrations of total bile pigment in the liquor amnii. For the period 16 to 26 weeks'' gestation the upper limit of normal bile pigment to protein ratio was found to be 0·4. Levels above this would be required as an indication for intrauterine transfusion. In 110 cases of isoimmunization of pregnancy these criteria were applied in the diagnosis of severity of the condition. The ratio of the bile pigment to protein was used rather than a simple bile pigment measurement, and found to be valuable.Nevertheless, it is important to emphasize that fallacious high bilirubin readings may arise by using whatever method, particularly owing to bilirubin and other breakdown products of blood contaminating the liquor.     

Maternal, amniotic fluid and cord blood metabolic profile in normal pregnant and gestational diabetics during recurrent withholding of food

In order to advise regarding the religious practice of withholding food, we studied the metabolic changes after successive 15 days of recurrent fasting of 13 hours every day in maternal plasma and liquor amnii of obese normal gravids and gestational diabetics in their third trimester. There were no significant differences between those who fasted that period for one day prior to elective cesarean section (CS) and those who fasted the same period repeatedly for 15 days. The fasted gravids had significant rises in glycerol, beta-hydroxybutyrate (BOHB) and nonesterified fatty acids (NEFA) (P less than 0.0001, P less than 0.005 and P less than 0.01, respectively) in maternal plasma, compared to unfasted gravid groups and ungravid fasted group. No significant metabolic difference was found in the liquor amnii withdrawn from fasted and unfasted groups. The influence of such short term of starvation on the fetal metabolic profile was studied in the cord blood during cesarean section (CS). Glucose, glycerol and NEFA were significantly lower in arterial than in venous cord plasma (P less than 0.05, P less than 0.01 and P less than 0.01, respectively) indicating that the fetus could utilize these substrates. Positive correlation was found between the levels of BOHB in the mother and venous cord plasma on the one hand and their levels in the arterial cord plasma and liquor amnii on the other hand implying that this substrate passes unutilized through the fetus to the liquor amnii. A pregnant woman in the third trimester should not withhold food for long periods.     

Impaired fertility in the jaundiced female (Gunn) rat

The hyperbilirubinemic female Gunn rat has been reported to have impaired fertility. A total of 267 jaundiced (j/j) and 91 nonjaundiced (+/j) female Gunn rats were used in a series of experiments to characterize the nature of this reduced fertility. Sixteen percent of the jaundiced females mated and delivered litters which were characteristically small in number (4.5 pups). A comparison of the gross observations at necropsy of jaundiced and nonjaundiced pregnant rats indicated that the number of implantation sites and live fetuses were significantly lower in the jaundiced females. The number of fetal resorptions in these rats were significantly higher; whereas, the number of corpra lutea was similar for both genotypes. The significantly lower plasma bilirubin levels in the pregnant jaundiced rats compared to the nonpregnant suggested that the observed effect on fertility was related to the concentration of plasma bilirubin.     

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.     

Bilirubin and the risk of common non-hepatic diseases

Bilirubin is a potent antioxidant but can be toxic at high concentrations. This article critically reviews the reported relationships of plasma bilirubin levels to the severity and/or incidence of various common non-hepatic diseases. Plasma bilirubin levels are reportedly negatively related to the risk of atherosclerotic diseases, cancers, demyelinating neuropathies and seasonal affective disorder. By contrast, the incidence and severity of schizophrenia are increased by elevated bilirubin levels. The data strongly suggest that the level of plasma bilirubin should be considered as a risk factor for several common non-hepatic diseases. Additional studies are needed to clarify the mechanisms of this influence, which are thought to be related to unconjugated bilirubin counteracting the oxidative stress underlying these disorders.     

Fetal proteinuria in diagnosis of congenital nephrosis detected by raised alpha-fetoprotein in maternal serum.

High concentrations of alpha-fetoprotein (alpha-FP) were found at 14, 19, and 21 weeks gestation in the serum of a woman with a history of unexplained fetal death in her previous pregnancies. The alpha-FP concentration of the liquor also was high at 21 weeks and the pregnancy was terminated. Though the fetus was macroscopically normal, measurement of albumin, alpha-FP, IgG, and alpha2-macroglobulin in the fetal urine showed a selective proteinuria, and congenital nephrosis was diagnosed after examination of the fetal kidneys by electron microscopy. Possibly some fetuses reported to be "false-positive for neural tube defect" may have had renal lesions of this nature. Examination of fetal urine may be the simplest initial diagnostic procedure in any future case.     

Effect of Bilirubin on the Membrane Potential of Rat Brain Synaptosomes

The effect of the neurotoxic pigment bilirubin on the membrane potential of rat brain synaptosomes was studied by using the tetraphenylphosphonium ion (TTP+) technique. Bilirubin induces a rapid depolarization of synaptosomes, as reflected by an efflux of previously accumulated [3H]TTP+. This phenomenon persisted when the membrane potential across either the plasma membrane of the synaptosome or the inner membrane of the entrapped mitochondria was selectively depressed, thus indicating that both components of the synaptosomal membrane potential were affected by bilirubin. Bovine serum albumin, used at a albumin/bilirubin molar ratio of 1:1, had the capacity to completely prevent and reverse the effect of bilirubin. This fact demonstrates that the bilirubin-induced TPP+ release from synaptosomes is a reversible process that requires the presence of bilirubin interacting with the synaptosomal membranes. These results, together with the inhibition by bilirubin of [3H]TPP+ and [2-14C]acetate uptake by synaptosomal plasma membrane vesicles isolated from rat brain, suggest that bilirubin depresses the membrane potential across the synaptosomal plasma membrane by a mechanism involving alterations in ion permeability. This effect could be of relevance in the pathogenesis of bilirubin encephalopathy.     

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.     

Binding of bilirubin by bovine and human alpha-fetoprotein.

alpha-Fetoprotein, a fetal protein associated with certain tumors, was found to bind bilirubin. Addition of human or bovine alpha-fetoprotein to bilirubin solutions enhanced the light absorbance of bilirubin and shifted its maximum. Bovine alpha-fetoprotein caused a marked shift towards shorter wavelengths, while human alpha-fetoprotein gave a slight red shift. The spectral changes were used to study the characteristics of the binding of bilirubin by bovine alpha-fetoprotein. These studies indicated the presence of one binding site/molecule of alpha-fetoprotein with an association constant of about 1.1 . 10(6) M-1. A difference between the spectral changes brought about by alpha-fetoprotein and albumin allowed comparison of their relative affinities for bilirubin. The spectrum approximated the average between the spectra induced by the two proteins when the ratio of bovine alpha-fetoprotein to bovine albumin was 6.3 : 1, and of the human proteins 21 : 1, respectively. These results show that alpha-fetoprotein from two species binds bilirubin with an affinity somewhat lower than that of albumin. Binding of bilirubin by alpha-fetoprotein is in agreement with the recent demonstration of structural homology between alpha-fetoprotein and albumin. Whether alpha-fetoprotein plays a role in the metabolism of bilirubin or other degradation products of heme remains to be investigated.     

The physiology of bilirubin: health and disease equilibrium

Bilirubin has several physiological functions, both beneficial and harmful. In addition to reactive oxygen species-scavenging activities, bilirubin has potent immunosuppressive effects associated with long-term pathophysiological sequelae. It has been recently recognized as a hormone with endocrine actions and interconnected effects on various cellular signaling pathways. Current studies show that bilirubin also decreases adiposity and prevents metabolic and cardiovascular diseases. All in all, the physiological importance of bilirubin is only now coming to light, and strategies for increasing plasma bilirubin levels to combat chronic diseases are starting to be considered. This review discusses the beneficial effects of increasing plasma bilirubin, incorporates emerging areas of bilirubin biology, and provides key concepts to advance the field.     

Role of apolipoprotein D in the transport of bilirubin in plasma

Apolipoprotein D (apo D) is a 30-kDa glycoprotein of unknown function that is associated with high-density lipoproteins (HDL). Because unconjugated bilirubin has been shown to bind apo D with a 0. 8:1 stoichiometry, we examined the contribution of this protein to transport of bilirubin in human plasma. Density gradient centrifugation analysis using physiological concentrations of [(14)C]bilirubin reveals that 9% of unconjugated bilirubin is associated with HDL, with the remaining pigment bound primarily to serum proteins (i.e., albumin). The percentage of total plasma bilirubin bound to HDL was found to increase proportionally with bilirubin concentration. Affinity of human apo D for bilirubin was determined by steady-state fluorescence quenching, with Scatchard analysis demonstrating a single binding site for unconjugated bilirubin with an affinity constant (K(a)) of approximately 3 x 10(7) M(-1). Incorporation of apo D into phosphatidylcholine vesicles had no effect on K(a), suggesting that a lipid environment does not alter the affinity of the protein for bilirubin. Using stopped-flow techniques, the first-order rate constant for bilirubin dissociation from apo D was measured at 5.4 s(-1) (half-time = 129 ms). Our findings indicate that HDL is the principal nonalbumin carrier of bilirubin in human plasma and further support the proposition that the affinity of HDL for bilirubin is primarily the result of binding to apo D.     

Differences in the protein binding of several drugs and bilirubin in serum and heparinized plasma of rats.

The purpose of this investigation was to compare the protein binding of salicylic acid, phenytoin, warfarin and bilirubin in serum and heparinized plasma of rats. Protein binding was determined by equilibrium dialysis (drugs) or by a reaction rate method (bilirubin), using serum and plasma obtained from the same animals. The three drugs were significantly less protein bound in heparinized plasma than in serum; this difference was particularly pronounced in the case of warfarin. Addition of heparin to serum also resulted in a decrease in the protein binding of the drugs but to a lesser extent than in plasma. The protein binding of bilirubin was more extensive in plasma than in serum, irrespective of the anticoagulant used (heparin, sodium citrate, or disodium ethylenediamine-tetraacetate). It may be desirable to perform all binding studies with serum rather than plasma.     

The Blood–Brain Barrier and Bilirubin Encephalopathy

1. The pathogenesis of bilirubin encephalopathy is multifactorial, involving the transport of bilirubin or albumin/bilirubin across the blood–brain barrier and delivering bilirubin to target neurons.2. The relative importance of the blood–brain barrier, unconjugated bilirubin levels, serum binding, and tissue susceptibility in this process is only partially understood. Even at dangerously high serum levels, bilirubin traverses the intact blood–brain barrier slowly, requiring time for encephalopathy to occur, although deposition of bilirubin can be rapid if a surge in plasma unbound bilirubin is produced by administering a drug which competes with bilirubin for binding to albumin.3. There may be maturational changes in permeability both in the fetus and postnatally which protect the brain from bilirubin.4. Disruption or partial disruption of the blood–brain barrier by disease or hypoxic ischemic injury will facilitate transport of bilirubin/albumin into brain, but the relative affinities of albumin and target neurons will determine whether the tissue bilirubin load is sufficient for toxicity to occur.     

Dietary Modulation of Plasma Bilirubin and of Hepatic Microsomal Lipid Peroxidation in the Gunn Rat

An in vitro assay for the simultaneous measurement of lipid peroxidation (LPO) and bilirubin degradation BRD) activities in rat liver microsomes has been developed; a good correlation between the 2 activities was observed (r = 0.78). In the Gunn rat a lipid free diet caused an increase in plasma bilirubin (62.4 ± 25.8%, n = 6) and a concomitant decrease in both hepatic microsomal LPO and BRD to zero. In contrast, on a 25% lipid diet there was a decrease in plasma bilirubin (46.1 ± 3.6%; n = 8) associated with an increase in LPO (1.26 ± 0.11 nmol/min/mg protein, and BRD (0.21 ± 0.6 nmol/min/mg protein). Therefore, in the absence of bilirubin glucuronidation, dietary modulation of plasma bilirubin and lipid peroxidation appear to be closely associated.     

Amperometric detection of bilirubin from a micro-sensing electrode with a synthetic bilirubin imprinted poly(MAA-co-EGDMA) film

Poly(methacrylic acid-co-ethyl glycol dimethylacrylate) (poly(MAA-co-EGDMA)) imprinted with alpha-bilirubin was shown to be able to bind alpha-bilirubin in our previous work. In this work, the corresponding imprinted polymer thin film was synthesized onto a thiol treated Au electrode by surface grafting polymerization. Bilirubin was able to be detected by an Au electrode, however, the electrode was not be able to discriminate bilirubin from the other matrix components if clinical samples were used. Therefore, the imprinted material was introduced so that the modified Au electrode could specifically detect bilirubin. Optimal potential was found to be 0.55 V and this was set for the rest of experiments. The imprinting factor of 3.16 was confirmed by comparing the signals from the MIP-Au and the NIP (non-imprinted polymer)-Au electrode. Calibration of the bilirubin concentration with respect to the current by the MIP-Au electrode was made within the range of 5mg/dl and a detection sensitivity of 0.644 microA/mg/dl (2.58 microA/cm(2)/mg/dl) was obtained. Furthermore, a linear correlation of the bilirubin concentration within 1.0mg/dl versus detection current was also achieved. Bilirubin was further detected by the MIP-Au electrode in the presence of fetal bovine serum (FBS). Repeated detection of bilirubin with at least three detection batches was performed and the reproducibility of the same piece of MIP-Au electrode was confirmed. The result was compared to those obtained from the serum and the solvent solution. The results indicated the feasibility of using the bilirubin imprinted poly(MAA-co-EGDMA) film as a sensing electrode for the clinical detection of bilirubin in serum.     

The role of albumin in the hepatic transport of bilirubin: studies in mutant analbuminemic rats

Bilirubin and other cholephilic organic anions are bound to albumin in the circulation; their hepatic uptake involves a carrier-mediated process. To investigate the possible role of serum albumin in the transhepatic transport of a cholephilic ligand, plasma clearance of radioactive bilirubin and its biliary excretion as well as its interaction with plasma proteins were compared between normal and mutant analbuminemic rats (NAR). With a tracer amount of 3H-labeled bilirubin, its plasma clearance and biliary excretion were comparable in both animal groups. However, the plasma clearance of a loading dose of the ligand was significantly increased and its biliary recovery was low in NAR as compared with normal animals. In accord with these findings in vivo, gel permeation chromatographic analysis revealed that the bilirubin binding capacity of serum proteins was significantly lower in NAR than in control animals. When bilirubin was administered to NAR as a mixture with equimolar albumin, its plasma disappearance was considerably decreased and its biliary recovery was increased. Similar effects were observed when albumin was replaced by an equimolar amount of glutathione S-transferases (ligandins). These observations indicate that, although ligand-protein interaction in the circulation is important for directing bilirubin to the plasma membranes of the hepatocyte, this mechanism is not specific for albumin.     

Degradation of plasma bilirubin by a bilirubin oxidase derivative which has a relatively long half-life in the circulation

To enhance degradation of unconjugated bilirubin in hyperbilirubinemic subjects, we synthesized a bilirubin oxidase (EC 1.3.3.5) (BO) derivative (PEGBO) by covalently linking (2,4-bis[O-methoxy(polyethyleneglycol)]-6-chloro-s-triazine) (PEG) to the enzyme. Intravenously injected BO in rats disappeared from the circulation with a half-life of 2.5 min; the half-life of PEGBO was 190 min. Intravenously injected BO minimally and transiently decreased plasma bilirubin levels in jaundiced Gunn rats and in bile-duct-ligated jaundiced rats. In contrast, PEGBO rapidly and substantially decreased plasma bilirubin levels and the effect persisted for longer than 3 h. Renal dysfunction often occurs in patients with liver diseases. To study the role of bilirubin toxicity for the kidney, functions of transtubular transport for organic anions was measured in bile-duct-ligated jaundiced animals before and after treatment with PEGBO. Bile duct ligation decreased urinary excretion of phenolsulfophthalein (PSP), an organic anion used for renal function test. Treatment of the jaundiced animals with PEGBO increased the rate of PSP disappearance from the circulation and normalized its urinary excretion. Thus, PEGBO might be useful for the study of bilirubin toxicity in jaundiced animals.     

Binding of bilirubin with albumin-coupled liposomes: implications in the treatment of jaundice

In the present study, we demonstrated the suitability of liposomes as a method of removing plasma bilirubin in hyperbilirubinemic rats. The liposomes have innate tendency to bind with bilirubin through hydrophobic interaction. Among different types of liposomes, the positively charged liposomes were found to have maximum affinity to free bilirubin. However, the entrapment or coupling of serum albumin on the surface of egg phosphatidylcholine liposomes can render a several-fold increase in their bilirubin binding capacity. The proteoliposomes were able to preferentially bind with bilirubin even in the presence of erythrocytes. Interestingly, these liposomes were found to displace bilirubin bound on the surface of erythrocytes as well. The results of the present study further demonstrate that albumin-bearing liposomes were equally effective in removing plasma bilirubin in experimental jaundiced animals. These observations indicate that liposome-mediated selective homing of excess plasma bilirubin to the liver cells (cf. hepatocytes) may help in the development of safer strategy for the treatment of hyperbilirubinemic conditions in the model animals.