CLIN. CHEM. 28/3, 432-439 (1982)

432 CLINICALCHEMISTRY,Vol. 28, No. 3, 1982

Relationshipsof BilirubinBindingParametersRhondda Wells,1 Keith Hammond,1 Angelo A. Lamola,2 and William E. Blumberg2

The apparent unbound bilirubin concentration by the“peroxidase” method (U) and the total unconjugated bili-rubin in blood (T), albumin-bound bilirubin (B), and reservebilirubin binding capacity (A) by the bilirubin hematofluo-rometer were measured in 164 specimens from 98 neo-nates and in a series of artifactual specimens, made byadding bilirubin to the blood of a single adult donor. Linearcorrelations between U and (B/R) were found for both theprepared specimens (r = 0.99) and the clinical specimens(r = 0.87), with the slopes of the regression lines beingclose to the reciprocal of the albumin-bilirubin bindingconstant, a prediction of the mass action law. An excellentlinear correlation was observed for the prepared speci-mens (r = 0.96) between U and (1- B), the concentrationof bilirubin bound to low-affinity secondary sites (“looselybound bilirubin”). A simple model for low-affinity bindingof bilirubin in blood predicts this simple relation. A signif-icant linear correlation between U and (T - B) was foundfor the clinical specimens, although the correlation wasless good (r = 0.72), as one would expect. The demon-strated simple linear relationships between U, (B/A), and(T - B) support the hypothesis that both the hematofluo-rometer and peroxidase methods provide valid measure-ments of bilirubin binding status.

AddItional Keyphrases: hematofluorometry . newborns#{149}pediatric chemistry . enzymic methods . hem.ato-fluorometer saturation index - bound and unbound bilirubinin blood . kernicterus . liver disease - encepha-lopathy

It is widely accepted that the unconjugated bilirubin con-centration in serum, by itself, inadequately indicates the riskfor bilirubin encephalopathy in individual hyperbilirubinemicneonates (1-7). Based upon the idea that, because of its re-markable affinity for the pigment, serum albumin acts to se-quester as well as to transport bilirubin, it is argued that onlywhen the bilirubin binding capacity of the albumin ap-proaches saturation does a significant quantity of bilirubinaccumulate within cells, including those in the central nervous

system (8). More than a dozen methods for assessing the al-bumin-bilirubin binding status in blood specimens fromneonates have been reported over the past 15 or so years(9-33). Despite the development of these assays and the ap-parently wide interest in utilizing bilirubin binding assays inthe management of jaundiced infants, very few centers haveintroduced such assays into routine use. Few of the methodsare amenable to critical-care use in typical hospitals (i.e., rapidemergency assays, available on a 24-h basis), owing to thespecial requirements and (or) the time and blood-volume re-quirements of most of the protocols (34). Moreover, partlybecause most binding assays are tedious but primarily becauseof ethical considerations, large-scale studies to provide an

‘Department of Pediatrics, School of Medicine, University ofColorado, Denver, CO 80262.

2 Bell Laboratories, Murray Hill, NJ 07974. Address correspon-

dence to A.A.L. at this address.Received Aug. 26, 1981; accepted Dec. 3, 1981.

unequivocal relationship between neurological sequelae andbilirubin binding status during the neonatal period have notbeen performed. One must rely on the few studies of this kindreported over the years (35-38) and the inadvertent experi-ence with the potent competitor of bilirubin for albuminbinding, suluisoxazole (39). Finally, because of apparent dis-agreements of results from the various methods for assessingbilirubin binding status or the lack of demonstration of therelationships among the various methods, all binding assayshave become somewhat suspect and their utility has beenquestioned (40-44).

One can now perform rapid and exceedingly simple fluo-rometric assays for albumin-bound bilirubin, total blood bi-lirubin, and assess reserve bilirubin-binding capacity, usingless than 200 iL of whole blood and an instrument called thebilirubin hematofluorometer (31-33). The physicochemicalbasis of these fluorometric assays is simple and well under-stood (31). It has recently been demonstrated for clinicalspecimens that the binding capacity as determined inwhole-blood specimens with the hematofluorometer agreesvery well with the value determined by stepwise titration ofsera with bilirubin until albumin saturation is reached, asindicated by either Sephadex chromatography or peroxi-dase-catalyzed oxidation rates (33). In another study the re-lationship between reserve binding capacity by hematofluo-rometry and the HABA (hydroxybenzeneazobenzoic acid)reserve binding capacity was discerned (32).

Here we report the results of a study of the relationshipsbetween quantities determined with the hematofluorometerand the unbound bilirubin concentration as determined bythe “peroxidase” method (18, 19). These results, plus thoseof the previous studies, clearly demonstrate that the deter-mination of bilirubin binding status in blood is, in fact, wellin hand, both conceptually and in practice.

Materials and MethodsNeonate population. We studied specimens from 98 neo-

nates whose weights at birth ranged from 0.54 to 4 kg andwhose gestational ages ranged from 25 to 41 weeks. Blood wasobtained either by heel-stick or from the umbilical vein. Thespecimens to be used for the hematofluorometer assays werecollected into heparinized tubes, kept refrigerated, and as-sayed within 24 h. The specimens to be used for determinationof the apparent concentration of unbound bilirubin werecollected into pediatric blood-collection tubes or VacutainerTubes (both from Becton Dickinson and Co., Rutherford, NJ07070). After clotting, serum was separated and stored at -25#{176}Cuntil assay. All neonate specimens were coordinated withthe drawing of blood for routine blood chemistries or for thestate health-screening test.

A heparinized blood specimen was obtained from an ap-parently healthy adult individual after an overnight fast. Theplasma was divided into three parts, two of which were dilutedwith different amounts of 0.1 molfL phosphate-bufferedisotonic saline, pH 7.4, to provide specimens with lower con-centrations of albumin. Aliquots of a concentrated bilirubinsolution, freshly prepared by dissolving bilirubin (Sigma) in0.1 molfL NaOH, were added to provide a series of specimenswith bilirubin concentrations ranging from 12 to 345 mg/L.The plasma with added bilirubin was allowed to stand in thedark for at least 30 miii. The packed cells, after three washings

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with phosphate-buffered saline, were added to the plasmaspecimens and allowed to stand in the dark for an additionalhour, with intermittent mixing. The specimens were centri-fuged at 500 X g and some plasma was removed for determi-nation of the apparent concentration of unbound bilirubin.The cells were resuspended, providing reconstituted bloodspecimens with hematocrits ranging from 0.39 to 0.44. Thesespecimens were used for measuring the bilirubin binding pa-rameters with the hematofluorometer.

Peroxidase. Apparent concentrations of unbound bilirubinin plasma and serum specimens were determined from theinitial rate of decrease in absorbance at 460 nm, according tothe peroxidase-catalyzed oxidation method of Jacobsen andWennberg (18) as modified by Wennberg et al. (19). The assaywas performed at 37 #{176}C,with use of a recording spectropho-tometer (Model 25; Beckman Instruments, Fullerton, CA92634).

Hernatofluorometer.3 We used the bilirubun hematofluo-rometer developed by the Bell Laboratories, Murray Hill, NJ07974, to determine the following parameters: albumin-boundbilirubin (B), reserve biirubin-binding capacity (R), and totalblood bilirubin (T) as described by Cashore et al. (33) exceptthat 15 tL of dodecyldimethylamine oxide (DDAO), 100 g/L,was added to 60 tL of whole blood to prepare the samples forassay of total blood bilirubin.4

In brief, the bilirubin hematofluorometer is an automated“front-face” filter fluorometer, dedicated to the assay of bi-lirubun and bilirubin binding in specimens of whole blood.Essentially, only that bilirubin bound to the high-affinitybinding site on albumin is fluorescent in whole blood. Becausethere are no important interferences, the fluorescence signalnear 520 nm is proportional to B. Empty high-affinity sitesfor biirubin can be populated by adding an excess of bilirubinto the blood specimen. The increase in the fluorescence signalupon addition of excess bilirubin to the blood is then pro-portional to R. The nonionic detergent DDAO efficiently ex-tracts bilirubin from all binding sites in blood. Because bili-rubin is fluorescent in this detergent, the fluorescence signalin the presence of DDAO is proportional to T.

ResultsA series of 32 blood specimens with albumin concentrations

from 16 to 40 g/L, unconjugated bilirubin concentrations from7 to 360 mg/L, hematocrit 0.39 to 0.44, and with a pH of 7.4were prepared from freshly drawn blood of a normal adultvolunteer. Total blood bilirubin, albumin-bound bilirubin,and reserve bilirubin-binding capacity were run in duplicateand averaged. The precision (CV) observed for each of theparameters was (mean of duplicate determinations on 32specimens): B, 5.4%; T, 4.2%; R, 8.4%. The larger apparent CVfor R than for the other two parameters is mainly digital error,owing to the fact that R is displayed to the nearest 10 mg/Lwhile B and Tare displayed to the nearest 1 mg/L. The day-to-day CV (monitored over several months) for the readingof the fluorescent glass secondary standard in the hemato-fluorometer was 0.8%. The plasma fraction of each specimenwas assayed (single determination) for the apparent unboundbilirubin concentration by the “peroxidase” method. Thewithin-run CV was 3.4%, the day-to-day CV 12.5%.

The apparent unbound biirubin concentrations were found

3The hematofluorometer is neither manufactured nor sold by BellLaboratories or any other company in the Bell System. Bell Labora-tories and Western Electric Co. have made the technology of hema-tofluorometry available to interested instrument manufacturers.

“Nonstandard abbreviations: B, albumin-bound bilirubin; R, re-serve bilirubin-binding capacity; T, total blood bilirubin; U, unboundbilirubin; DDAO, dodecyldimethylamine oxide; HABA, hydroxy-benzeneazobenzoic acid, SI, hematofluorometer saturation index.

Fig. 1. A plot of the apparent unbound bilirubin concentrationas determined by the “peroxidase” method (U) vs the ratio ofalbumin-bound bilirubin to reserve bilirubin binding capacity asdetermined with the hematofluorometer (B/R) for a series ofspecimens prepared by addition of bilirubin to blood obtainedfrom a single adult donor

to increase linearly with an increase in the ratio between al-bumin-bound bilirubun and reserve binding capacity, B/R. Aplot of U vs (B/R) (Figure 1) gave the regression line: U =

-0.14 + 7.4 (B/R), withr = 0.996, and = 0.96 nmol/L.Values of U for 164 specimens of serum obtained from

neonates were assayed (single determinations) and hemato-fluorometer parameters were determined (duplicate deter-minations) on whole-blood specimens drawn at the same time.The plot of U vs (B/R) (Figure 2) gave the regression line: U= -0.7 + 10.1 (B/R), with r = 0.87 and S,,1 = 3.8 nmol/L. Ofthe 164 specimens from neonates, 44 were from babies notbeing treated by phototherapy. The plot of U vs (B/R) forthese specimens gave the regression line: U = 0.6 + 6.9 (B/R),with r = 0.73 and = 2.1 nmol/L. However, analysis showedthat the difference between the slopes of the regression linesfor the data from infants receiving and not receiving pho-totherapy was statistically insignificant.

In a previous smaller study, values of U by the peroxidasemethod and hematofluorometer values were obtained butwere not compared (33). A plot of U vs (B/R) for these data(Figure 3) gave the regression line: U = 0.4 + 10.5 (B/R), withr = 0.80 and S,,1 = 3.2 nmol/L (n = 14; one outlying pointdeleted from the regression analysis).

A plot of U vs (T - B), the difference between the totalblood bilirubin and albumin-bound bilirubin by hematoflu-orometry, for the adult blood specimens “spiked” (i.e., sup-plemented) with bilirubin also showed a good linear correla-tion (Figure 4). The regression line obtained was U = 0.5 +0.63 (T - B), with r 0.96 and S1 = 3 nmol/L. A similar plotfor the neonate specimens (Figure 5) gave U = 0.5 + 0.29 (T- B) (mg/L), with r = 0.72 and Syl,: = 5.6 nmol/L. Because theapparent value of (T - B) is perturbed by the presence ofphotobilirubin (45), we made a similar plot (not shown) ofdata from infants not receiving phototherapy. The regressionline obtained-U = 0.7 + 0.27 (T - B), with r = 0.70 and= 5 nmol/L-was not significantly different.

Plots of (B/R) vs (T - B), not shown, for both the adult andneonate specimens were found to have linear regressions withsimilar correlation coefficients as those for the U vs (T - B)plots. This is, of course, expected because of the good linearrelationship between U and (B/R) described above.

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Discussion

Relationship between U and B/R

Albumin possesses one high-affinity site for bilirubin andat least one low-affinity site (8, 31, 46). Independent of thelow-affinity site or of any other binding sites for bilirubin inserum or blood, at equilibrium

K0 = [bilirubin-HSA]/[bilirubin] [HSA]

where K0 is the affinity constant for the high-affinity site,[bilirubin-HSAJ is the concentration of albumin having abilirubin molecule bound to the high-affinity site, [bilirubin]is the concentration of unbound5 or free bilirubin, and [HSAJrepresents the concentration of albumin with empty high-af-finity sites or the reserve albumin binding capacity for strongbinding of bilirubin.

The initial rate of oxidation of bilirubin by peroxides cat-alyzed by peroxidase is proportional to the unbound bilirubinconcentration (U) (18). The fluorometric method embodiedin the hematofluorometer directly measures the albumin-bound bilirubin concentration (31). The operational definitionof reserve binding capacity by the hematofluorometer methodis the increase in the measured value of albumin-bound bili-rubin upon addition of a specific and excess quantity of bili-rubin to the specimen. In the absence of competitors for the

We use the term “unbound bilirubin” for that bilirubin which is“free,” that is, for which the chemical activity is not significantlydifferent from monodispersed bilirubin in an aqueous medium. Theterm “unbound” represents an accurate description of such “free”bilirubin and should not also be used, as it has been (23,42), to rep-resent bilirubin bound to low-affinity sites (“loosely bound bili-rubin”).

Fig. 3. Unbound bilirubin as measured by the “peroxidase” vshematofluorometer value (B/R) for 14 blood specimens fromdifferent neonates.An outlying point (U = 14 nmol/L; (B/A) = 5.1) was deleted from the series

albumin high-affinity site, the value of R by hematofluo-rometry is an accurate measure of the concentration of albu-min with empty high-affinity sites for bilirubin (31).

Thus, the equilibrium equation given above may be re-written in terms of the experimental parameters:

and rearranged to

K0’ =B/(U.R)

U = (K0’) (B/R).

If K0’ is constant, this equation predicts a linear relationshipbetween U and (B/R). Furthermore, because (B/R) is a di-

Fig. 4. Apparent unbound bilirubin concentration as determinedby the “peroxidaso” method (U) vs the difference between thetotal unconjugated bilirubin concentration and the albumin-boundbilirubin concentration as determined in whole blood with thehematofluorometer (T - B) for a series of specimens preparedby addition of bilirubin to blood obtained from a single adultdonor

40 60(1-B) (mq/L)

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FIg. 5. A histogram of the average values found by the “per-oxidase” method for unbound bilirubin found at various valuesof hematofluorometer (1 - B) for the neonatal specimensThe regession parameters were determined from the Individual data points

mensionless parameter, the slope, (K0’Y, of the plot of U vs(B/R) should, if the system is well behaved, reflect the valuefor the albumin-bilirubin binding constant associated withthe “peroxidase” method.

The plot of U vs (B/R) for the specimens prepared fromblood of a single adult (Figure 1) demonstrates, at least for thisseries of specimens, the validity of both the “peroxidase”method for U and the hematofluorometer assays for B and R.Not only is U precisely proportional to (B/R) over the entirerange of albumin binding saturation tested, 2 to 83%, but thereciprocal of the slope, 1.4 X 108 L/mol, was found to beidentical to the value of the albumin-bilirubin binding con-stant associated with the “peroxidase” method originally re-ported by Jacobsen (47) and within the range of values re-ported by others (0.5 to 2.5) X 10 L/mol (8,46).

The linear correlation between U and (BfR) for the neonatal

specimens (Figure 2) is not expected to be as good as that forthe specimens made from the blood of one individual, for atleast two reasons. Variability was introduced by the necessityto collect the data over a period of several months, and per-turbations introduced by phototherapy are expected. Thesetwo factors alone may well account for the magnitude of thestandard error observed. However, one must also consider thepossibility of variability in the relation between U and (B/R)among the individual neonates, that is, the effective value ofK0’ may vary somewhat from individual to individual. Ja-cobsen and Wennberg have reported lower effective bindingof bilirubin in neonatal sera as compared with an adult serumby the peroxidase method (18). Wennberg et al. reportedfurther examples (46). They ascribe this to the presence ofcompetitors for bilirubin binding rather than to an inherentdifference in the albumin.

A biased difference in the effective albumin binding of bi-lirubin would exhibit itself as a difference in the slope of theU vs (B/R) plot for the neonatal specimens as compared withspecimens prepared from the blood of the adult. The slope ofthe regression line for the neonates was, in fact, found to beabout 1.4 times greater than that for the adult specimens,indicative of an average effective binding constant 1.4 timessmaller for the neonates. However, the difference is muchsmaller than the apparent difference reported by other in-vestigators (18,46). The largest U to (B/R) ratios found amongthe neonate data are about 19, larger by 2.5 times than thatof the adult. Given the day-to-day variability in the peroxidaseand hematofluorometer results, no statistically significantdifference between the adult and neonatal slopes can be

claimed. It was satisfying to observe that the slope of the Uvs (B/R) plot (Figure 3) obtained for data accumulated by adifferent worker (W. J. Cashore) in another location was vir-tually identical to that obtained for the clinical specimens inthe present study.

Because the serum specimen is diluted by a factor of 40 inthe determination of U, the effect of competitors that re-versibly bind albumin at the high-affinity bilirubin site maybe greatly reduced and the apparent U value may be lowerthan the true value. The methodology used in determining Rwith the hematofluorometer involves use of undiluted bloodbut may also underestimate the effect of a relatively weakreversible competitor (31). However, it is expected, and shownfor the case of oleate anion as a competitor, that for thequantity of excess bilirubin used in the assay of R, the valueobtained is minimally overestimated (31).

We suggest that the observed correlation between U and(B/R) indicates that reversible competition by adventitiousmetabolites is, in general, not important. This conclusion isalso supported by the observed agreement of the R values byhematofluorometry with those obtained by serial titration ofsera with bilirubin by use of Sephadex chromatography andperoxidase oxidation rates as endpoint indicators (33).

We have observed large discrepancies between U and (B/R)values for specimens for only two infants, the outlier in thedata set of Figure 3 and a recent case in the present ongoingstudy that appeared after analysis of the data presented here.In both cases very large values of (B/R), >5, were accompaniedby values of U of only 10 to 15 nmol/L. We speculate that thesespecimens may have contained competitors for bilirubinbinding whose presence may have escaped detection by theperoxidase method.

Relationship between U and (T - B)In the absence of photobilirubin, the parameter (T - B) by

hematofluorometry is an accurate measure of the bilirubinthat is not bound to the high-affinity site of albumin (31). Thisincludes bilirubin bound at the low-affinity site(s) of albumin,that bound to other plasma proteins, that bound to erythro-cytes, and the unbound bilirubin. The magnitude of (T - B)is of the order of 20 mg/L in adult blood when the high-affmitybinding sites of the albumin are about 50% occupied. If onetakes the tight binding affinity of bilirubin and albumin to be108 L/mol, the unbound bilirubin concentration is of the orderof 0.005 mg/L, that is, more than 1000 times smaller than (T- B). Thus, almost all of the bilirubin not bound to high-af-finity albumin sites is bound to secondary sites and is not“free.” A simple demonstration (A. A. Lamola, unpublishedresults) of this fact is afforded by ultrafiltration. When 600mg of bilirubin per liter, or about twice the molar concentra-tion of albumin, is added to a plasma specimen from an adultand the clear solution subsequently passed through an ul-trafilter (Worthington Diagnostics, Freehold, NJ) designedto retain plasma protein, the concentration of bilirubin in thefiltrate is found to be only about 5 mg/L. Furthermore, thebilirubin in the filtrate is not a true measure of the free orunbound bilirubin, because some leakage of protein cannotbe avoided.

Except for binding to hemopexin (48), affinity constantsfor binding of bilirubin to secondary sites in blood are two tothree orders of magnitude smaller than that for the high-af-finity albumin site. Relative capacities and affinities are suchthat when the hematocrit is about 0.5 the bilirubin not boundto high-affinity albumin sites is apportioned roughly equallybetween sites on plasma proteins and sites in erythrocytes(31). Thus (T - B) is related to two quantities that have beenpreviously introduced as measures of bilirubin.binding status,namely, serum or plasma “loosely bound bilirubin” (23,49,50) and erythrocyte bilirubin (28,51-53).

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Sephadex chromatography has been used to assess bilirubinbinding in three different ways: (a) as a test for saturation ofhigh-affinity sites (54), (b) as an endpoint indicator for ti-trations of reserve high-affinity sites (25),and (c) to obtaina measure of the so-called loosely bound bilirubin (23,49,50).In Sephadex chromatography, binding and entrainment ofbilirubin by the Sephadex competes with binding of bilirubinby serum (or plasma) components. Because of the very largedifference in binding constants, the Sephadex competes muchmore successfully with the secondary plasma binding sites ascompared with the high-affinity albumin sites. Thus bilirubinthat remains on the column after elution of the protein is re-lated to the “loosely bound bilirubin,” that is, biirubin boundto low-affinity secondary sites. However, it is not expectedthat this determination of “loosely bound bilirubin” is a veryquantitative one. For example, the concentration of “looselybound bilirubin” indicated by Sephadex chromatography doesnot often exceed 1 mg/L, whereas (T - B), which is a measure

of all plasma bilirubin bound at low-affinity sites, can be ashigh as 50 times that concentration. While it is expected thatthe Sephadex “loosely bound bilirubin” should be roughlyproportional to (T - B), an experimental test of this conjec-ture has not been carried out.

Bratlid reasoned that increased red cell bilirubin should beindicative of a reduced reserve capacity for binding of bilirubinto albumin, and he and others have reported erythrocyteconcentrations of bilirubin in jaundiced neonates (28,51-53).Bratlid (28) demonstrated that erythrocyte bilirubin con-centrations can easily be as high as 60 mg/L in clinical speci-mens. As stated above for blood from normal adults, roughlyhalf of the observed (T - B) is due to erythrocyte-bound bi-lirubin. No comparisons of (T - B) and erythrocyte bilirubinhave yet been made for clinical specimens.

In this study we have observed a strictly linear relationshipbetween (T - B) and either the “peroxidase” unbound bili-rubin concentration or (B/H) for the series of specimens pre-pared from the blood of a single donor (Figure 4). That is, overthe range examined, the unbound biirubin was proportionalto the “loosely bound bilirubin.” Is this observation expectedon the basis of our knowledge of the binding sites, capacities,and affinities? We used the following model for the system ofbilirubin in blood to answer this question. The high-affinityalbumin binding affinity (K0) was taken to be iO - 108 L/moland the capacity (A0) to range from 20 to 70 imol/L. All thelow-affinity binding sites were lumped and described by acapacity (Co) of 150 to 500 zmolfL (three to 10 times the ca-pacity of the primary albumin sites) and an effective affinity(Kr) of the order of 10 - 106 L/mol. At equilibrium, the modelsystem is described by the equations:

B = K0 . U . R

CB = K . U . C

in which A0 = B + H and C0 = CB + C, where CB is the con-centration of biirubin bound to low affinity sites and C is thereserve low-affinity binding capacity. These equations weresolved simultaneously, and computer-generated plots of U vsCB, taken to be synonymous with (T - B), were obtained forvarious values of A0, Co, K0, and K. Some of these plots areshown in Figure 6. We observed that when K0 >> K, Co >>A0,and C >>C8, the plots of the calculated values of U and (T -

B) were strictly linear over the ranges of values examinedexperimentally. That is, the linear relationship obtains whenthe capacity for secondary binding is large and far from sat-uration.

As can be seen in Figure 6, the data from Figure 4 can beclosely fit by using our model with K0 = 1 X 108, K = 1 x 106,

A0 = 50 mol/L, and C0 = 300 molIL. Of course, this choiceof parameters does not represent a unique fit.

Fig. 6. Plots of calculated values of the unbound bilirubin con-centration vs calculated values of (1 - B) taking: (1) A0 = 50imol/L, C0 = 400 jmol/L, = 5 X 10 L/mol, K0 = 5 X i0L/mol; (2) A0 = 40 tmol/L, C0 = 300 mol/L, = 1 X 108L/mol, K0= 1 X 106 L/mol; (3) A0 = 40 Lmol/L, C0 = 400LrnOl/L, Ka = 1 X 108 L/mol, K, = 1.5 X 106L/mol; (4) A0 =

4Ojzmol/L, C0 = 400 moI/L, K = 1 X 108 L/mol, K,, = 2 X10 L/molThe brolcen lines labeled A and N are the regression lines from Figs. 4 and 5,respectively

On the basis of these results it was justified to look for alinear relationship between U and (T - B) for the clinicalspecimens. Taking into account the various factors that wouldcomplicate the relationship between these parameters in sucha mixed population, the linear correlation coefficient (0.7)observed (Figure 5) may be considered to be telling. Expectedcomplicating factors include the variations in the capacity forsecondary binding (C0) within the neonate populations, i.e.,different plasma protein concentrations and hematocrits. Adifferent mix of secondary sites in individual infants wouldpresent different apparent affinity constants (Kr). In addition,phototherapy seriously affects the (T - B) value as a resultof the presence of photobilirubin, which is detected in theassay for total bilirubin (T) but not for albumin-bound bili-rubin (B) (45). The resulting relatively small percentage errorin B leads to an unfortunately large apparent increase in (T- B). However, the slopes of the regression lines and thestandard errors obtained without inclusion of infants underphototherapy were quite similar to those obtained for theentire population. This may have been due to an increase inthe apparent value of U in proportion to the increased ap-parent (T - B) since photobilirubin binds less well to albuminthan does bilirubin (55).

The much smaller slope for the regression line of the U vs(T - B) plot for the neonates as compared with that for theseries of prepared specimens is interesting, but until bloodspecimens from a number of adults are examined it cannot beconcluded that this difference represents a general distinctionbetween adults and neonates.

Our observations of the relationships between U or (B/H)and (T - B) demonstrate that the unbound bilirubin con-centration is proportional to the concentration of “looselybound bilirubin.” This simple linear relation between thesetwo quantities, both previously suggested to be useful indi-cators of bilirubin binding status, can be explained on the basisof a reasonable model for secondary binding sites for bilirubinin blood that is supported by other data. Our model, however,

predicts a somewhat different slope for the U vs (T - B) plot(generated by varying the bilirubin concentration) for dif-ferent neonates, depending upon the capacity and averageaffinity of the secondary binding sites. This would suggest that

CLINICAL CHEMISTRY, Vol. 28, No. 3, 1982 437

the hematofluoronieter (T - B) or other assays for “looselybound bilirubin” indicators of bilirubin binding status maynot be as robust as are U, (B/H), or H. Nonetheless, a largevalue (>40 mg/L) for (T - B), in the absence of phototherapy,would appear to be a significant finding, especially in con-junction with other indicators of low reserve capacity ofhigh-affinity albumin sites.

Hematofluorometer Saturation Index (SI)We suggest that the ratio of the hematofluorometer albu-

min-bound bilirubin to reserve albumin binding capacity,B/H, is a particularly good parameter for assessment of thebilirubin binding status in a blood specimen. Operationally,(B/H) is a robust parameter in that its accuracy is independentof errors or drift in the absolute calibration of the instrumentand should be very “forgiving” of volumetric inaccuracies.Except for rare circumstances, (B/H) is expected to be accu-rately proportional to the unbound bilirubin concentrationand, for blood specimens from infants not receiving pho-totherapy, also to be proportional to the “loosely bound” bi-lirubin concentration.

We define the parameter 10 (B/H), rounded to the nearestinteger, as the hematofluorometer saturation index (SI). TheSI, in the common well-behaved (i.e., uncomplicated) speci-men will be close to the numerical value of the apparent un-bound bilirubin concentration (in units of nanomoles per liter)as determined by the peroxidase method. (Recall that theslopes of the U vs (B/H) plots of Figures 2 and 3 are just about10 nmol/L.) The whole-number scale for SI is appropriate tothe resolution and accuracy of the hematofluorometer de-termination of (B/H).

Clinical Significance of Hematofluorometer Assays- Longitudinal studies of the correlations among bilirubin

parameters assayed with the hematofluorometer, clinicalstatus, and bilirubin encephalopathy have not yet beencompleted, but it is possible to suggest ranges of values in-dicative of significant risk based on the evident relationshipsof hematofluorometer assays to other assays of bilirubinbinding that have been directly compared with clinical out-come. Before making these suggestions, two points should bementioned. First, because of the apparent continuum ofpathologies between minimal encephalopathy and lethalcentral nervous system damage associated with neonatal hy-perbilirubinemia and the likelihood that bilirubin bindingstatus is not the sole determinant of the rate of neurologicdamage, it does not appear sensible to attempt to set exactlimits for values of binding parameters that define specifictherapeutic actions. Second, independent of the details of themechanism of bilirubin-induced brain damage, effectivebinding of unconjugated bilirubin by extracellular albuminshould always afford protection as compared with less-effec-tive binding (8,46). Thus, even in the absence of a one-to-onecorrespondence between neurological outcome and bilirubinbinding status, knowledge of the latter should be consideredas important as knowledge of the total bilirubin concentrationin the management of jaundice of the newborn.

Wennberg et al. (56) found no kernicterus among a largegroup of jaundiced neonates with peroxidase-determinedvalues for unbound bilirubin that remained <20 nmol/L andtwo cases among a much smaller number of infants with values�20 nmolfL. No studies of the neurological outcome forchildren for whom peroxidase values were determined duringthe neonatal period have yet been published.

Comparisons of data on HABA-dye-binding capacity fromthe various literature reports are made somewhat difficult bythe different and ill-defined “reference” sera that were used.Furthermore, there is considerable suspicion that HABA doesnot bind at the bilirubin binding site of albumin exclusively

but may also bind at additional sites (32, 57). Nevertheless,the studies correlating HABA dye binding with neurologicalsequelae cannot be ignored (36, 53, 58, 59). These studiessuggest that observable neurological deficiency is significantlyincreased when the reserve dye binding capacity decreasesbelow 60% of the reference value. Making use of the directcomparison between hematofluorometer reserve binding ca-pacity and HABA reserve, capacity (32), it appears that aHABA reserve of 60% corresponds to a hematofluorometerSI value of between 10 and 15. A value of 38% for the HABAreserve capacity has been noted in one study (53) as that valueabove which no cases of kernicterus were observed. A 38%HABA reserve corresponds to a hematofluorometer SI valueof between 20 and 30.

Because of the considerable uncertainty concerning themolecular basis for the salicylate saturation index, it was notpossible for us to propose a theoretical relationship betweenthe hematofluorometer saturation index and the salicylatesaturation index.

Thus, on the basis of comparisons of hematofluorometerparameters with other binding assays for which correlationswith adverse neurological outcome have been determined, wesuggest, at this point, that hematofluorometer SI values higherthan about 10 comprise significant findings with respect torisk for bilirubin encephalopathy. We also suggest that valuesof SI higher than about 20 indicate a high risk. Hematofluo-rometer SI values of 10 and 20 respectively represent 50 and67% saturation of the effective albumin high-affinity sites forbilirubin.

While no direct comparisons of (T - B) with erythrocyte

bilirubin concentrations or with “loosely bound bilirubin” asindicated by Sephadex chromatography have been made forclinical specimens, it is clear that, in the absence of phototh-erapy, (T - B) accurately represents that bilirubin presentthat is not bound at the high-affinity albumin site. Within thecontext of the model for secondary site binding presentedabove, it would appear that a large value (>40 mg/L) of (T -

B) combined with a large value for hematofluorometer SI isa certain indication of deficient high-affinity binding of bili-rubin by albumin.

Certainly more studies will have to be made correlatinghematofluorometer parameters with clinical status andlong-term neurological outcome before more definitiveguidelines can be set for use of hematofluorometer assays inmaking therapeutic decisions. It is still essentially unproventhat estimates of bilirubin binding from (e.g.) reserve bindingcapacity or hematofluorometer SI are more useful in assessingrisk for bilirubin encephalopathy than is the total bilirubinconcentration or the bilirubin/albumin ratio. However, itshould be pointed out that a valid assay of bilirubin bindingstatus that can be performed rapidly and that requires a smallquantity of blood should be of interest to those neonatologistswho already use birth-weight, gestational age, complicatingfactors, etc., to modulate the value for total bilirubin at whichthey opt for therapy. These considerations find a basis inobservations such as the decrease in average total bindingcapacity with decreasing gestational age (60). The point is thatinstead of relying on such statistical findings, the actual ef-fective binding capacity (which may be quite different fromthat expected from the albumin concentration) would be de-termined for the individual infant.

Hematofluorometer Parameters: ComprehensiveSet of Binding Assays

Table 1 lists the indicators of bilirubin binding that thusfar have been suggested in the literature. The equivalenthematofluorometer parameter or combination of parametersis listed on the right side of the table. Methods with which thehematofluorometer results have been directly compared on

Assay

438 CLINICAL CHEMISTRY, Vol. 28, No. 3. 1982

Table 1. Hematofluorometer Equivalents ofBilirubin Binding Assaysa

Hematofluorometerequivalent

Total and (or) reserve binding capacity (R + B); AAlbumin or total serum proteindye-binding competition

HABA(10) (32)b

Bromphenolblue(11)Directyellow7 (12)

Titration with bilirubin, endpoint by:Sephadex column (22, 25) (33)5Sephadex thin layer (24)

Peroxidase oxidation rate (25) (33)5

Paper chromatography (27)

Paper chromatoelectrophoresis (26)

Electron spin resonance methods (13)MADDSmethod (14)

Tryptophan fluorescence quenching (29)

“High-performance”liquid

chromatography (15)Fluorescence-labeledalbumin(16, 17)

‘Loosely bound blllrubln” (T - B)Sephadex methods (21, 23)

Paper chromatoelectrophoresls (26)

Erythrocyte bilirubin (T - B)

Unbound blllrubin by “peroxidase” method B/R”

(18, 19, 46)

Salicylate saturation index (9) B/R; Ra Ntsrierals are references. b Hematofluorometry results and those from other

assay have been compared for a series of clinical specimens and shown to beequivalent.

clinical specimens are indicated, along with the literaturereference.

The parameters that can be determined with the hemato-fluorometer-B, T, R, (T - B), and (B/H)-are a compre-hensive set of bilirubin binding assays and, concomitant withtheir redundancy, offer the opportunity to judge validity fora particular specimen by assessing the consistency amongthem.

SummaryThe bilirubin hematofluorometer can be used to determine

the total concentration of unconjugated bilirubin in wholeblood (T), the concentration of bilirubin bound to the high-affinity site of albumin (B), and the reserve albumin bindingcapacity for bilirubin (R). The law of mass action demandsthat (B/H) equal the product of the albumin-bilirubin affinityconstant and the unbound bilirubin concentration. We foundthat plots of U, the apparent unbound bilirubin concentrationin serum determined by the “peroxidase” method, vs (B/H)for clinical specimens give good linear regressions, with slopesvery close to the value expected from the measured value ofthe binding constant These observations support the validityof both the “peroxidase” assay for unbound bilirubin and theassays for B and R by hematofluorometry.

We introduce the parameter 10 (B/H), which we call the“hematofluorometer saturation index” (SI).

For the reasonable model of blood in which the capacity for

low-affinity binding of bilirubin is large compared with thatfor high-affinity binding and the condition in which the low-affinity capacity is far from saturation, the simple equilibriumequations predict, for constant plasma protein and constanthematocrit, a linear relationship between the unbound bili-rubin concentration and the concentration of biirubin boundto low-affinity sites (“loosely bound bilirubin”). We found anexcellent linear correlation between U and (T - B) for a seriesof specimens made by addition of increasing amounts of bi-lirubin to the blood from a single donor. A good linear corre-lation between U and (T - B) was also found for neonatalclinical specimens.

Our observations demonstrate that unbound bilirubin,reserve binding capacity, and “loosely bound bilirubin” arerelated to each other in simple ways, a conclusion previouslyprecluded by methodological deficiencies.

The bilirubin hematofluorometer can provide a compre-hensive and accurate set of bilirubin parameters-B, T, R, (T- B), and (B/H)-within a few minutes with less than 200 Lof whole blood. The instrument is simple to use and, becauseit can provide 24-h emergency-basis assays, offers a practicalapproach for widespread introduction of bilirubin bindingassays into the management of neonatal hyperbilirubi-nemia.

We thank F. H. Doleiden for technical assistance and A. L. Simons

for writing a computer program.

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