Clin. Biochem. (6) 292-294 (1980)

Clinical Relevance of the Plasma Reserve Albumin

Binding Capacity for Bilirubin (RABC) and "Free" Bilirubin Concentration

G E O R G E CHAN, ROMA I L K I W and DAVID S C H I F F

D e p a r t m e n t s of P e d i a t r i c s , Obs t e t r i c s a n d Gyneco logy , U n i v e r s i t y of A l b e r t a , E d m o n t o n , A l b e r t a , C a n a d a T6G 2G3

An analysis of 55 plasma samples from 46 jaundiced newborn infants showed that endogenous "free" bilirubin levels bear no significant correlation to reserve albumin binding capacities for bilirubin as determined by both the Sephadex G-25 gel filtration and the enzymatic peroxidation techniques (r=0.14, p>0.05). This contrasts with the significant correlation between the "free" bilirubin concentrations and the bilirubin/albumin molar ratios in the same plasma samples (r=0.75, p< 0.001). The results of this study suggest that if "free" bilirubin is the "driv- ing force" in the pathogenesis of bilirubin encephalopathy then the use of the reserve albumin binding capacity for bilirubin as the only guide in predicting the risk of kernicterus may not be adequate.

THE USE OF PLASMA RESERVE ALBUMIN BINDING

capacity for bil irubin (RABC) and/or "free" bilirubin concentrat ions( i -6) are helpful guidelines for the management of the hyperbi l i rubinemic newborn infant. Clinical and labora tory da ta suppor t the concept tha t albumin-bound biluribin is non-toxic. As long as the molar concentrat ions of albumin exceeds those of bflirubin, the level of free or neurotoxic bilirubin would be below the threshold required to cause any neurologic damage (7). The higher incidence of kernic terus in sick p remature infants compared with their heal thy full t e rm counterpar t s has been a t t r ibu ted to factors such as acidosis, hypoxia and hypoglycemia (8-9). These fac- tors presumably e i ther in terfere with bilirubin binding to albumin or increase t issue affinity for "free" bil irubin riO). Hence, as with total bilirubin, the safe plasma "free" bil irubin concentrat ions for one infant may be critical for another . The following s tudy at- t empts to de te rmine whether a relat ionship exists be- tween rese rve albumin binding capacity for bil irubin and "free" bilirubin levels in jaundiced plasma from newborns.

MATERIALS AND METHODS

Fifty five plasma samples from 46 jaundiced newborn infants were studied. The samples were obtained from newborn in- fants in the first 24 hours of life from the Neonatal Intensive Care Unit of the University of Alberta Hospital. The determination of "free" bilirubin concentrations and the RABC of plasma is a routine procedure for monitoring hyperbilirubi- nemia in our Unit. Diagnosis of the group of infants include un- complicated prematurity, birth asphyxia, respiratory distress syndrome and idiopathic hyperbilirubinemia. None of the in-

rants had either Rh or ABO blood group incompatibility. The birth weights of the infants ranged from 840 to 3856 grams (mean _+ S.D. : 2453 _+ 813 grams) and gestational age ranged be- tween 27 and 42 weeks (36 _+ 4 weeks). The samples were assayed for total bilirubin (11) and albumin(12), and free bilirubin levels (5). By enriching aliquots of plasma with graded quantities of exogenous bilirubin the RABC's for bilirubin were also obtained by both the Sephadex G-25 gel filtration (2) and the peroxidase oxidation methods (6), respectively. The RABC for bilirubin is defined as the quantity of bilirubin (mg/dl) which the plasma can tolerate before the primary bind- ing sites of albumin are saturated with bilirubin.

RESULTS

In the group of infants studied, the total plasma bilirubin concentrat ions ranged be tween 1.0 to 18.3 mg/dl and albumin concentrat ions from 1.8 to 4.7 g/dl. F igure 1 demons t ra tes a good correlat ion in rese rve albumin binding capaci ty be tween the Sephadex gel f i l t rat ion and peroxidase assays {r = 0.99, p < 0.001). F igure 2 shows the re la t ionship be tween the RABC for bil irubin obtained by the Sephadex t i t ra t ion method and "free" bil irubin concentrat ion obtained by the peroxidase method. A t r e se rve RABC's ranging be- tween 4.0 and 21.5 mg/dl, the corresponding "free" bilirubin concentrat ions ranged between 0.5 to 19.7 nmol/1 (0.03 to 1.15 ~g/dl). No significant correlat ion was found be tween "free" bilirubin levels and the RABC (r = 0.14, p > 0.05). In the range of binding capacit ies ob- ta ined i t was not possible to show whether there was an increase of endogenous plasma "free" bil irubin concen- t ra t ion when the p r imary binding si tes of albumin were sa tura ted . F igure 3 i l lus t ra tes the re la t ionship be tween p l a sma " f r e e " b i l i rub in c o n c e n t r a t i o n and the bil irubin/albumin molar rat io in the same group of in- fants. A s ta t i s t ica l ly significant correlat ion was found be tween these two pa rame te r s (r = 0.75, p < 0.001).

DISCUSSION

The measurement of the r e se rve albumin binding capaci ty for bil irubin by t i t ra t ion of p lasma with graded quanti t ies of bil irubin is an a t t e mp t to s imulate an an- t ic ipated r ise of bil irubin in vivo. I t e s t imates the con- centra t ion of bi l i rubin tha t serum albumin can bind before i ts p r imary binding s i tes a re sa tura ted . The good

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correlation using two different approaches, that is, Sephadex G-25 gel filtration and peroxidase oxidation shows that both methods measure the same end point beyond which "free" bilirubin levels will dramatically increase. The main objective of this study was to deter- mine, under physiological conditions, whether the level of "free" bilirubin is related to the reserve albumin binding capacity for bilirubin. The poor correlation be- tween these two parameters in the plasma of infants with adequate binding reserve is not unexpected. Theoretically, the binding of bilirubin to albumin obeys the law of mass action. The concentration of "free" bilirubin is therefore a function not only of the binding capacity of albumin, but also of the binding affinity and the bflirubin and albumin concentrations. Although a good correlation was found between the plasma bill- rub]n/albumin molar ratio and "free" bilirubin concen- tration (r = 0.75), the less than ideal fit is in all likelihood the results of differences in binding characteristics in individual infants. Saturation of the primary albumin binding site in newborn infants, especially the sick premature infants, are often achieved before the bili-

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Fig. 2 -- The endogenous "free" bilirubin concentrations as measured by the peroxidase enzymatic method are compared to the reserve albumin binding capacity for bilirubin (RABC). When the R A B C ranged from 4 to ~ mg/dl bilirubin, no sig- nificant relationship with ''free "bilirubin was found (r = 0.1~, p > 0.05).

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CLINICAL RELEVANCE OF THE PLASMA RESERVE ALBUMIN BINDING CAPACITY 293

Fig. 3 -- The relationship between plasma "free" bilirubin con- centration and bilirubin/albumin molar ratio is shown. A statistically significant correlation between these two para- meters is obtained {r = 0.075, p < 0.001).

294 CHAN, ILKIW AND SCHIFF

rubirdalbumin molar ratio of 1 (adult level) is reached (2). Hence, the molar relationship of bilirubin to albumin cannot be considered to be a specific indicator in assessing the risk of the development of bilirubin encephalopathy. Clinically diagnosed cases of ker- nicterus are consistent with elevated plasma total and "free" bilirubin concentrations and low binding ca- pacities (13, 14). The margin of safety between poten- tially "toxic" and "safe" levels of "free" bilirubin and the binding reserve of "at risk" infants, especially those associated with low bilirubin kernicterus, is unknown.

In the blood, bilirubin is bound almost exclusively to serum albumin (15). The labile fraction ("free" bilirubin) is in a constant state of flux, distributed rapidly to reach a new state of equilibrium with tissues such as liver (16) red blood cells (17) spinal fluid (18, 19, 20), and possibly the central nervous system, implicating the tissue- binding involvement of "free" bilirubin. The patho- genesis of bilirubin encephalopathy is not fully under- stood and may well be the result of a multiplicity of dif- ferent processes. In isolated mitochondriai systems, the rate of cellular respiration and uncoupling of oxidative phosphorylation is dependent upon the concentration of bil irubin(22). A different composition of brain ganglioside between the newborn and adult has been cited as a possible reason for the difference in bilirubin toxicity during different stages of development (23). A recent abstract report ing on the variations in toxic response to bilirubin in human tissue culture systems also support the hypothesis that the distribution of bilirubin is a function of the tissue composition and hence its affinity for bilirubin (24). While these studies contribute to our overall unders tanding of the cytotoxic effects of bilirubin, no conclusions could be drawn as to neurotoxic concentrations of "free" bilirubin under dif- ferent clinical conditions in the human newborn infant. The results of the present study concur with current views on bilirubin-binding tests that plasma "free" bilirubin concentrations and the reserve albumin bin- ding capacity for bilirubin are helpful guidelines, but cannot be considered to be definitive biochemical assays for the management of neonatal hyperbil irubinemia (25, 26, 27).

ACKNOWLEDGEMENT

The authors express their appreciation to all the nurses of the Neonatal Intensive Care Unit of the Universi ty of Alberta Hospital for their assistance, Mrs. Kathy Merrills and Miss Leslie Hart for labora- tory assistance, and Mrs. Dianna Tetreaul t for assist- ance in the preparation of the manuscript.

REFERENCES

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