Reserve Bilirubin Binding Capacity Research Articles

FLUOROMETRIC BILIRUBIN BINDING ASSAY (FBBA) AND TRANSCUTANEOUS BILIRUBINOMETRY (TcB) IN TERM NEWBORNS

Fifty-four white full term newborns (gestational age 40.0±1.2 wks; birth wt 3447±440 gms), not under phototherapy, were studied. FBBA including reserve bilirubin binding capacity (RBBC) and saturation index (SI) were determined, as well as the bilirubin levels by TcB and modified diazo method. There is a significant linear relationship between serum bilirubin levels and values obtained by TcB (figure). This study differs from other published works on TcB by demonstrating the RBBC and SI levels of infants who belong to Area AB (RBBC=11.75±3.18, SI=1.75±0.5) and Area CD (RBBC=13.23±1.6, SI=2.86±0.79). These FBBA values including the RBBC (12.12±1.97) and SI (3.55±1.62) of the infants outside these areas are normal, demonstrating that this group of infants are not in danger of Kernicterus regardless of the TcB and serum bilirubin values at 3 days of life.

THE EFFECT OF SLOW LIPID INFUSION AND DOSAGE ON BILIRUBIN BINDING TO ALBUMIN IN THE PREMATURE INFANT

The effect of 15-hour lipid infusions at doses of 1, 2, and 3 g/kg, administered on successive days, on unbound bilirubin and the serum free fatty acid:albumin molar ratio (F/A) was studied in 20 AGA premature infants (BW mean ± SEM - 1.43 kg ± 0.09; EGA 30.7 wks ± 0.63) with physiologic hyperbilirubinemia (7.7 mg/dl ± 0.30). Albumin-bound bilirubin (B), reserve bilirubin binding capacity (R), and total unconjugated bilirubin were measured fluorometrically pre-infusion and 15-45 minutes before end-infusion. Serum free fatty acid levels were determined pre- and at end-infusion. Serum albumin was measured on day 1. The ratio B/R was used to estimate unbound bilirubin (Wells et al, Clin Chem 28:432,1982).Infants <30 wks gest. demonstrated a significant (p<.05) rise in the F/A ratio with each increase in lipid dose while infants ≥30 wks did not. The concentration of unbound bilirubin (B/R) increased linearly with increasing F/A ratio (r=.65,p<.001). The largest increases in B/R were seen in infants with F/A> 4.0; the EGA of these infants (28.7 wks ± .47) was significantly less (p<.01) than those infants whose F/A ratio was<4.0 (31.1 wks ± .40). In 10/58 infusions there was a fall in unbound bilirubin unrelated to BW, EGA or postnatal age. In all such instances, the end-infusion F/A was≤3.0.No significant increase in unbound bilirubin was seen in any infant receiving 1 g/kg fat emulsion despite increases in F/A. At doses of 2 and 3 g/kg increases in F/A in the infants < 30 wks resulted in increased unbound bilirubin. Monitoring of the F/A ratio may identify infants at risk for increased unbound bilirubin during lipid infusion. (Supported by NIH Grant RR-00240)

BILIRUBIN ALBUMIN BINDING PARAMETERS IN SHEEP DURING EXTRACORPOREAL MEMBRANE OXYGENATION (ECMO)

Three sheep (average wt. 32 kg) were anesthetized, ventilated and cannulated for venoarterial (VA-ECMO) by-pass procedure (Trans Am Soc Artif Intern Organ 28:350, 1982). Bilirubin albumin binding parameters were determined using a Bilirubin Hematoflurometer (Pediatr Res 17:127A, 1983). Pre-VA ECMO values were: hemoglobin (Hgb) 8-10 gm/dl; plasma Hgb 1-7mg/dl; Fibrinogen (F) 110-245 mg/dl; Fibrin split products (FSP) 10 μg/ml and platelet count (Plt) 495-880×103/mm3. During VA-ECMO, F was 96-225 mg/dl, FSP over 40 μg/ml and the Plt was 94-269×103/mm3. As shown (Figure), the Hgb and bilirubin levels varied, with elevation of plasma Hgb. In spite of hemoglobinemia suggesting intravascular hemolysis, the reserve bilirubin binding capacity (RBBC) and saturation index (SI) values were normal during the study. The normal RBBC and SI values would make the development of bilirubin neurotoxicity unlikely during VAECMO.

BILIRUBIN ALBUMIN BINDING (BAB) ASSAY IN NEWBORN INFANTS BEFORE, DURING AND AFTER EXTRACORPOREAL MEMBRANE OXYGENATION (ECMO)

Red blood cell destruction during ECMO may increase the risk for hyperbilirubinemia and bilirubin neurotoxicity. For this reason we performed bilirubin binding studies on 12 newborn infants (mean ± SD, gestational age 37.6±3.6 wks, birth wt 2889±706 gms) managed with ECMO for respiratory failure. The mean duration of ECMO was 91.1±37.2 hrs. Bilirubin binding studies including reserve bilirubin binding capacity (RBBC), and saturation index (SI) were performed using a bilirubin fluorometer. No significant changes pre, on, or post ECMO were noted on the hemoglobin and fibrinogen levels. The bilirubin levels were not significantly different pre and on ECMO and were lower post ECMO. As shown in the table, there were significant changes pre, on, and post ECMO plasma hemoglobin values. Significant changes between pre and on ECMO values only were noted on the infant's platelet, fibrin split products and SGOT levels. Thus, the hemolysis that occurs during ECMO does not adversely effect the RBBC and SI from any alterations in the bilirubin load or in the BAB sites. ECMO would not predispose an infant to bilirubin neurotoxicity because of the normal BAB values pre, on, and post ECMO.

Alterations in Reserve Bilirubin Binding Capacity of Albumin by Free Fatty Acids. II. In Vitro and In Vivo Studies Using Difference Spectroscopy

SummaryThe effects of free fatty acids (FFA) on reserve bilirubin binding capacity of solutions of human serum albumin (Alb) were determined in vitro using difference spectroscopy. Additionally, sera from 15 infants requiring parenteral nutrition were obtained before, during, and after the administration of a 1.0 g/kg intravenous dose of safflower oil emulsion, and determinations of FFA/Alb molar ratios and reserve bilirobin binding capacities were made for in vivo confirmation. The in vitro experiments demonstrated that increasing the linoleic acid/Alb molar ratio from 0 to 3.5 produced a 37% increase in binding capacity, but further increases to 9 ultimately resulted in a 32% decrease. Similar results were obtained using Alb at pH 7.4 and using defatted whole human serum. Using salicylate to block secondary binding sites demonstrated that linoleic acid enhanced binding at primary sites. In contrast to linoleic acid, octanoic acid did not alter binding at primary sites but lowered total binding capacity when its concentration exceeded 2.5 times that of the Alb in the sample. The in vivo measurements demonstrated that the total reserve bilirubin binding capacity dropped to 30% below base line only when the FFA/Alb molar ratio was greater than 7:1. Increases in total reserve bilirubin binding capacity above 30% over base line occurred in the range of FFA/Alb molar ratios of 1.6:4.0. Binding at primary sites was enhanced in a like manner at moderate FFA concentrations and did not fall below base line even at very high concentrations. These data support the concept of a positive cooperative binding effect between fatty acids and bilirubin at moderate fatty acid/Alb molar ratios and reconfirm the negative effect of fatty acids on bilirubin binding at high molar ratios. These data suggest that hyperbilirubinemic infants may be offered the nutritional benefits of intravenous safflower oil emulsion without additional risk of kernicterus as long as the FFA/Alb molar ratio is kept below 4 or the reserve bilirubin binding capacity is not decreased.

Reserve Bilirubin Binding Capacity Determined by Difference Spectroscopy. I. Modifications and Performance of the Difference Spectroscopy Assay

SummaryThe difference spectroscopy assay for reserve bilirubin binding capacity, modified to improve its performance, was used to study solutions of human serum albumin and bilirubin. The results indicate that the assay is performed simply and rapidly: that it uses very small sample volumes, making it suitable for use in premature human neonates; that it measures reserve binding capacity and not free bilirubin resulting from exceeding binding capacity; that it measures binding at two sites, which can be discriminated by blocking the low‐affinity site with concentrated salicylate: and that it can be performed at varying pH depending upon the experimental need.

The effect of two intravenous fat emulsions and their components on bilirubin binding to albumin

The effect of two intravenous lipid emulsions on the binding of bilirubin to albumin was investigated in vitro. Various concentrations of a soybean (Intralipid) or a safflower (Liposyn) oil emulsion in 0.48 mM albumin were assayed for changed in bilirubin displacement using Sephadex G-25 gel filtration and for alternations of albumin reserve binding capacity by a novel difference spectroscopy technique. Two major components of the emulsions, glycerol and egg phosphatides, were also assayed by difference spectroscopy. A significant enhancement of the total reserve bilirubin binding capacity of albumin was noted with concentrations of 200 to 500 mg/100 ml lipid emulsion using difference spectroscopy. Using salicylate to block secondary albumin binding sites for bilirubin, reserve bilirubin binding capacity at nonsalicylate competitive sites was shown to have increased (maximum 61%) over the range of 50 to 1000 mg/100 ml lipid emulsion. Only changes in nonsalicylate competitive binding sites could be detected at concentrations normally achieved in vivo. Egg phosphatides had little effect and glycerol decreased reserve bilirubin binding capacity. Sephadex studies were unable to detect any significant change in bilirubin retained on the column with 50 or 500 mg/100 ml lipid emulsion at any bilirubin/albumin ratio. The enhancement of reserve bilirubin binding capacity caused by lipid emulsions is mediated through changes in nonsalicylate competitive albumin binding sites. Product differences demonstrated in vitro point out the complexity of their effects on bilirubin-albumin binding and the necessity for in vivo investigations with structurally different nutritional products designed for use in newborns.

Relationships of bilirubin binding parameters.

The apparent unbound bilirubin concentration by the "peroxidase" method (U) and the total unconjugated bilirubin in blood (T), albumin-bound bilirubin (B), and reserve bilirubin binding capacity (R) by the bilirubin hematofluorometer were measured in 164 specimens from 98 neonates and in a series of artifactual specimens, made by adding bilirubin to the blood of a single adult donor. Linear correlations between U and (B/R) were found for both the prepared specimens (r = 0.99) and the clinical specimens (r = 0.87), with the slopes of the regression lines being close to the reciprocal of the albumin-bilirubin binding constant, a prediction of the mass action law. An excellent linear correlation was observed for the prepared specimens (r = 0.96) between U and (T--B), the concentration of bilirubin bound to low-affinity secondary sites ("loosely bound bilirubin"). A simple model for low-affinity binding of bilirubin in blood predicts this simple relation. A significant linear correlation between U and (T--B) was found for the clinical specimens, although the correlation was less good (r = 0.72), as one would expect. The demonstrated simple linear relationships between U, (B/R), and (T--B) support the hypothesis that both the hematofluorometer and peroxidase methods provide valid measurements of bilirubin binding status.

220 RESERVE ALBUMIN CAPACITY FOR BILIRUBIN MEASURED BY SEPHADEX ADSORPTION (SA) AND BY ABSORBANCE DEVIATION (AD) AT SATURATION OF PRIMARY ALBUMIN-BINDING SITES

The Reserve Bilirubin Binding Capacity (RBBC) of albumin is an indicator of the chances for brain damage due to kernicterus in the neonate. This study compares the RBBC of the neonatal serum by SA (Clin. Chem. 18(1):43, 1972) to that of AD (described elsewhere in this Program Issue, APS-SPR). The two methods were correlated in 54 pairs of measurements done on 25 neonates. Data shows that the results obtained by both methods are related (p < .001) with a correlation of +0.5. By altering pH and ionic strength from those in our previous work, RBBC results obtained by AD more closely resemble the values of SA. Determination of RBBC by AD is more time-efficient, uses a smaller quantity of serum, and could be performed with equipment usually available in most laboratories.

Reserve bilirubin binding capacity assessed by difference spectroscopy: Assay statistics and results on newborn sera

Bilirubin binding properties of newborn sera and assay parameters have been investigated using a difference spectroscopy procedure [9]. Reserve bilirubin binding capacity, serum bilirubin and the total bilirubin binding capacity can be determined using only 40 μl of serum. The measured total binding capacities agreed with the theoretical binding capacities calculated from serum albumin concentrations assuming a 1:1 molar binding ratio of bilirubin to albumin; in 102 assays on newborn sera, the ratio of experimental to theoretical total binding capacity was 1.04. Bilirubin binding capacity measurements were linear over the range 0–600 mg/1. Day to day precision of binding capacity determinations on 6 albumin controls yielded coefficients of variation between 4.1 and 7.2%. Recovery for the reserve bilirubin binding capacity determinations was 99.6%. In a study of 22 newborns, reserve bilirubin binding capacities showed an inverse relationship with the changes in serum bilirubin concentrations. None of the newborns included in our study appeared to be in danger of bilirubin encephalopathy.

Pharmacokinetic disposition and protein binding of furosemide in newborn infants

Using a one-compartment model, the pharmacokinetic disposition of furosemide was studied in eight premature and term neonates with fluid overload. Following a single intravenous injection of furosemide (1 to 1.5 mg/kg), multiple blood samples were obtained from a heelstick or an arterial catheter and analyzed for furosemide by gas liquid chromatography. The mean (+/- SE) apparent volume of distribution was 829.2 +/- 118.9 ml/kg; t1/2 was 7.7 +/- 1.0 hour; elimination rate constant was 0.102 +/- 0.013/hour and plasma clearance was 81.61 +/- 14.98 ml/kg/hour. Compared to the disposition of furosemide in normal adults. AVd is almost fourfold greater in the neonate with an eightfold prolongation in plasma t1/2, an eightfold decrease in Ke1, and a twofold decrease in plasma clearance. Neither gestational and postnatal age nor birth weight correlated with the pharmacokinetic variables. No significant change in reserve bilirubin-binding capacity after an intravenous dose of furosemide was noted. Slow elimination of furosemide may partly explain the prolonged diuretic and saluretic effect of furosemide in the neonate.

1045 PROTEIN BINDING AND PHARMACOKINETIC DISPOSITION OF FUROSEMIDE (F) IN NEWBORN INFANTS

The pharmacokinetic disposition of F was studied in 8 premature and full-term neonates with fluid overload using a one compartment model. Mean (±S.E.) birth weight was 2391.3 ± 289.9 g; gestational age was 35.0 ± 1.8 wks and postnatal age was 11.5 ± 5.9 days. BUN was 10.4 ± 1.5 mg/d1 and serum creatinine was 0.9 mg/d1. Following a single IV dose of F, (1-1.5 mg/kg) blood samples (0.2 ml/sample) were obtained from a heelstick or an arterial catheter at times 0, 0.5, 1,2,4,6,9,12 and 24 hrs and analyzed for F, measured by gas liquid chromatography. The mean (±S.E) volume of distribution was 829.2 ±118.9 ml.kg-1; T½ was 7.7 ± 1.0 h; elimination rate constant (Kel) was 0.102 ± 0.013h-1 and plasma clearance was 81.61 ± 14.98 ml.kg-1.h-1. Compared to the disposition of F in normal adults, aVd is almost 4-fold greater in the neonate with an 8-fold prolongation in plasma T½, an 8-fold decrease in Kel and a 2-fold decrease in plasma clearance. Neither gestational and postnatal age nor birth weight correlated with the pharmacokinetic variables. F had no effect on the reserve bilirubin binding capacity (RBBC) measured by sephadexgel filtration in 6 neonates (age 2.1 ± 0.3 d; weight 2329.9 ± 339.1 g). Mean RBBC 5 min before and 30-60 min after F were 6.2±0.2 and 6.8 ± 0.5 mg/d1 respectively. Slow F elimination may partly explain the prolonged diuretic and saluretic effect of F in the neonate. This must be taken into account whenever repetitive or chronic administration of F is used in the newborn infant.

Faster estimation of reserve bilirubin binding capacity of serum from the neonate by thin-layer chromatography on sephadex.

Knowledge of the reserve bilirubin-binding capacity is useful in management of hyperbilirubinemia in the newborn. We describe thin-layer gel chromatography that permits measurement of this variable, with a 250-mul sample of serum, within 50 min. Single samples from four different infants can be analyzed on the same plate. The capacity of 40 sera from jaundiced and of 19 sera from nonjaundiced infants was measured. Results by thin-layer Sephadex gel chromatography and column Sephadex chromatography correlated well. Dithiothreitol, added to the standard bilirubin solution, protected bilirubin from oxidation for at least six days, which obviated the time consuming preparation of standard solution before such determination.

“SINGLE-POINT” SEPHADEX GEL FILTRATION ASSAY FOR ESTIMATION OF RESERVE BILIRUBIN BINDING CAPACITY

The sephadex G-25 elution method for assay of reserve bilirubin binding capacity has proven to be a valid and useful technique for evaluating the danger level of hyperbilirubinemia in the newborn. A major disadvantage of the method is the relatively large volume of serum required from very small newborns who usually have high hematocrits. A modification of the method has been developed which requires only 0.5 ml total serum. A concentration of bilirubin is chosen to add to half the sample such that the binding capacity will be closely approached but not exceeded, and the optical density of this eluted sample is compared to that of the patient's own serum blank. The bilirubin binding capacity is thus expressed as “greater than” or “less than” the concentration of bilirubin added. Results comparing the same sera using the “single-point” method and the classical 3 or 4 point assay show excellent agreement, thus confirming the “single-point” assay as a valid and useful alternate method when sample size is limited.