ARTICLES
continued
A Rapid
Fluorometric
Method
for Determining
Bilirubin
Levels
and
Binding
in the
Blood
of
Neonates:
Comparisons
with
a Diazo
Method
and
with
2-(4’-Hydroxybenzene)azobenzoic
Acid
Dye
Binding
Audrey K. Brown, MD, Josef Elsinger, PhD, William E. Blumberg,
PhD, Jorge Flores,
MS, Gerard
Boyle,
MS
and
Angelo A. Lamola, PhD
From the Department of Pediatrics, State University of New York, Brooklyn, and Bell Laboratories, Murray Hill, New Jersey
ABSTRACT. A simple, rapid fluorometric method for determining the albumin-bound biirubin concentration,
total blood biirubin concentration, and the bilirubin
re-serve-binding capacity of albumin was clinically
evalu-ated using blood specimens from 79 neonates. This study
showed that these biirubin determinations, made by
means of the Bell Laboratories hematofluorometer,
cor-related well with plasma biirubin levels obtained by a
diazotization (Jendrassik-Grof) method. Hematofluoro-meter reserve-binding capacities correlated very well with
2-(4’-hydroxybenzene)azobenzoic acid (HABA) dye
re-serve-binding capacities for specimens of artificially
jaun-diced adult blood. For specimens of neonatal blood the HABA dye reserve capacity was, on the average, higher than the hematofluorometer reserve-binding capacity,
particularly for specimens from low-birth-weight babies (<2,000 gm). Comparison of HABA reserve capacity and hematofluorometer reserve capacity for
high-birth-weight babies (>2,000 gm) gave data very similar to those for adult blood specimens. The specific biirubin-binding capacity of albumin was found to be greater for infants whose birth weight exceeded 2,000 gm than for the lower
birth weight group. The total blood biirubin concentra-tion obtained by the hematofluorometer is shown to be
significantly higher than the concentration of biirubin
bound to albumin, an indication of other important
com-partments of biirubin in blood. Pediatrics 65:767-776, 1980; bilirubin, bilirubin binding, albumin-binding
ca-pacity, HABA reserve-binding capacity, hematofluoro-meter, kernicterus.
Received for publication Dec 4, 1978; accepted July 25, 1979. Reprint requests to (A.K.B.) Department of Pediatrics, State University of New York, Downstate Medical Center, 450
Clark-son Aye, Brooklyn, NY 11203.
PEDIATRICS (ISSN 0031 4005). Copyright © 1980 by the
American Academy of Pediatrics.
It has long been recognized that serum biirubin levels alone are poor predictors of the development of kemicterus in the neonate.’’ This is particularly true of very sick, small, premature infants in whom
kernicterus
seems
to develop
at
such low levels of biirubin that it has become difficult to apply the assignation “safe” to any degree of biirubinemia.25 Methods have been sought to measure either “un-bound” biirubin or the reserve albumin-binding capacity, which might serve as additional objective indices of the likelihood of kernicterus’”; for it is thought that only when that pool of biirubin not associated with serum albumin becomes sufficiently large do dangerous quantities of the pigment accu-mulate in the brain.’4Many of the methods presently employed to mea-sure either of these parameters are technically too cumbersome or complicated for routine laboratory use; most require too much blood and are too time consuming to allow frequent, repeated assessment. There have been very few studies of the relation between parameters obtained by these methods and the long-term outcome.’5’6
In the studies reported here, a rapid, simple, micromethod employing the Bell Laboratories bii-rubin hematofluorometer was used to study blood specimens from a group of 79 jaundiced neonates. This instrument measures total biirubin in whole blood, albumin-bound biirubin, and the reserve biirubin-binding capacity.
The objectives of the present study were:
conventional diazotization-colorimetric technique (Jendrassik-Grof) with results observed using the hematofluorometer on blood samples from an ar-bitrarily chosen group of jaundiced neonates of different birth weights.
2. To compare hematofluorometer reserve bili-rubin binding by albumin with the reserve binding
of the albumin for the dye
2-(4’-hydroxybenzene)-azobenzoic acid (HABA) in these infants; and
fur-ther, to compare these results with those obtained
using adult blood.
3. To compare the hematofluorometer values for total bilirubin-binding capacities of infants in vary-ing birth weight groups with those of adults.
These comparisons between the data obtained by
means of the hematofluorometer and those of other
assays might be helpful in ultimately evaluating the
clinical significance of reserve values obtained with the hematofluorometer, since follow-up studies are
available that relate low HABA reserve-binding
capacities to the development of kernicterus or
neurologic defects thought to be produced by bii-rubin encephalopathy. Even though a large amount
of clinical experience has been accumulated using
the dye-binding method, it must be pointed out that
no randomized, controlled study of its clinical
valid-ity has been reported.
METHODS
The present study was performed with a
popu-lation of 79 neonates whose clinical condition,
usu-ally jaundice, prompted requests for biirubin as-says. Blood was obtained either by heel stick or by venipuncture using heparmn or
ethylenediaminetet-raacetic acid (EDTA) as an anticoagulant. A
por-tion of the blood specimen was used for standard
bilirubin assessment and the remainder for the
as-says of this study. All but three of the infants were under 2 weeks of age, and their average birth weight
was 1,991 gm (737 gm to 4,345 gm). Forty-three had
birth weights of less than 2,500 gm and 25 had birth weights of more then 2,500 gm.
Adult blood specimens were obtained by veni-puncture from three healthy male volunteers.
Bilirubin and Albumin
Direct and total diazo-reacting plasma biirubin concentrations were determined by the method of
Jendrassik and Grof.’7 The human serum albumin (HSA) concentrations were determined using brom-ocresol green.’8 Artificially jaundiced normal adult blood specimens were prepared by adding small volumes of highly concentrated (300 mg/100 ml
solutions of bilirubin, prepared in 0.02 M NaOH, to
the plasma fractions, followed by addition of
well-packed original red cells to a hematocrit of 40%. These mixtures were incubated in the dark and at room temperature for at least one hour before use.
HABA Reserve-Binding Capacity
The reserve-binding capacity was determined by HABA binding using the method of Porter and Waters.6 The amount of HABA bound by a refer-ence, pooled cord serum, containing 3.7 gm/100 ml of albumin and less than 1.0 mg/100 ml of biirubin, was measured and compared with that of the un-known serum. The reference and test sera were added to separate aliquots of the HABA dye solu-tion as well as to buffer blanks, and the optical absorbances were measured at 510 nm. The HABA binding of the test serum, as reflected in the net absorbance at 510 nm, is expressed as a percentage of that of the reference serum.
The Hematofluorometer
The bilirubin hematofluorometer employed in
this study is a dedicated, front-face, filter fluoro-meter developed as a prototype instrument for ex-perimental purposes. Although details of its design
are given elsewhere,’92’ it is necessary here to
es-tablish the operational meaning of the three param-eters that the instrument measures. Its design is based on the results of a series of experiments exploring the fluorescence and binding properties of biirubin in serum and in blood, also given in detail elsewhere,’9 and summarized below:
1. Biirubin has a single strong binding site on
human serum albumin (HSA), characterized by an association constant of at least 106 M’ at 37 C. Biirubin bound at this site fluoresces with a quan-tum yield of about 0.002. Unbound biirubin, as well
as biirubin bound to secondary HSA sites, has at
most ‘/so the fluorescence quantum yield of the
strongly bound molecules. Biirubin bound to sec-ondary albumin sites does not affect the fluores-cence of the strongly bound biirubin. Biirubin bound to other serum proteins contributes only negligibly to the observed fluorescence.
2. Biirubin bound to erythrocytes has at most
‘/54) the fluorescence quantum yield of biirubin
strongly bound to albumin and contributes
negligi-bly to the biirubin fluorescence from blood
speci-mens.
3. The detergent dodecyldimethylamine oxide
(DDAO) lyses the red blood cells and forms micelles which scavenge biirubin from all blood sites. The micelle-bound biirubin fluoresces with about one third the quantum yield of HSA-bound biirubin.
4. At a constant hematocrit, the fluorescence emission stimulated by front-face illumination of blood by blue light has an intensity that is propor-tional to the concentration of the fluorescent bili-rubin species.
readily applicable to biirubin in blood, because the wavelength oflight used to excite bilirubin (430 nm) is near that of the strong Soret absorption band of hemoglobin. Thus, the exciting light penetrates whole or hemolyzed blood only a few microns. The fluorescence of bilirubin is peaked in the green region of the spectrum (520 nm) so that there is little self-absorption within the sample. It is moni-tored by a photomultiplier after passing through an interference filter centered at 530 nm.
In practice, three drops of blood-one, plain whole blood and two others that are mixed with additives (bilirubin and DDAO as described be-low)-are deposited on a thin glass slide (no. 1 #{189} coverslip), which is then placed on a sliding carriage. When the hematofluorometer is activated, the three samples are automatically drawn into the instru-ment, where they are incubated at 37 C for 15
seconds. The fluorescence intensities from the three drops of blood are measured automatically in se-quence, along with the fluorescence intensity of a stable fluorescence glass and of a blank portion of the sample slide. The latter is used in a background
correction, and the former is used to normalize the blood fluorescence intensities.
The fluorescence intensities measured for the
three drops of blood yield numerical values for three
biirubin levels which have relevance to the man-agement of neonatal jaundice: the fluorescence from the unprocessed drop of blood gives the
concentra-tion of bilirubin strongly bound by albumin, and
its value (in units of mffligrams per 100 ml of
plasma) is computed and displayed digitally on the front of the instrument. To obtain a value for the
bilirubin reserve-binding capacity, a second drop
of blood is mixed with sufficient biirubin (50 mgI 100 ml of blood) to saturate all strong albumin-binding sites under normal conditions. The
differ-ence between its fluorescence intensity and that of
the whole blood yields the concentration of strong HSA sites available for biirubin binding. The third drop of blood, previously mixed with DDAO and therefore hemolyzed, fluoresces with an intensity
that is proportional to the total bilirubin level in
the blood; and its value, again in milligrams per 100 ml of plasma, is also displayed. The
hematofluoro-meter was calibrated by using normal adult blood specimens artificially jaundiced with known amounts of unconjugated biirubin. The definitions of the parameters determined by the hematofluo-rometer are summarized in the Table.
The computation of these three biirubin levels, as well as the automatic operation of the instru-ment, are controlled by a microprocessor. The com-putations involve the hematocrit of the blood used, which must be determined by conventional meth-ods and supplied to the instrument. The automatic calibration accompanying each biirubin assay
as-sures long-term reproducibility. The volumes of the
three drops of blood are not critical as long as each drop covers the optical aperture.
The following protocol was used in this study.
Approximately 150 tl of blood was collected in
heparinized tubes, and the hematocrit was deter-mined after good mixing. Five microliters of 30% DDAO (Onyx Chemical Co, Jersey City, NJ) was placed at the bottom of a small (3x30 mm) test tube, and 50 tl of well-mixed blood was added and mixed with the detergent. The lysed blood was set aside for five to ten minutes. Another 50 tl of blood was added to a similar tube containing a premea-sured deposit of sodium biirubinate and was mixed well. Aliquots of 10 to 20 tl from this tube, the DDAO test tube, and unprocessed blood were placed on the appropriate sites on the glass slide in the sample carriage. Each drop was mixed again just prior to the activation of the instrument to begin the incubation, measurement, computation, and display cycle.
RESULTS
Bilirubin Levels
Fig 1 (top) shows a high correlation between the total blood bilirubin levels obtained by the hema-tofluorometer (single determinations) and the total plasma biirubin levels obtained by the Jendrassik-Grof method (single determinations) in specimens from 68 of the neonates in the population studied. This relationship is characterized by a correlation coefficient of r = .89 and a slope of 0.91. The
HSA-TABLE. Hematofluorometer Terminology*
Instrument Notation Operational Definition Biochemical Definition Albumin bound biirubin Fluorescence from whole blood due to
biirubin
Biirubin strongly bound to primary
al-bumin site
Total blood biirubin Fluorescence from whole blood due to
biirubin after addition of detergent
(DDAO)
All biirubin in specimen ends up in de-tergent micelles and is assayed
Albumin reserve-binding Incremental biin,bin fluorescence from Incremental biirubin which binds
capacity whole blood after adding biirubin (50 mg/100 ml)
strongly to the primary albumin site after adding bilirubin (50 mg/l00 ml)
E
I
I
z
I C I
-J
I
-J
0
Furthermore, the hematofluorometer readout does not distinguish between direct and indirect biiru-bin. However, when the indirect biirubin values, as determined by the Jendrassik-Grof method, were subtracted from the bound or total biirubin values, obtained with the hematofluorometer, there were excellent correlations (r .9) between these differ-ences and the direct biirubin values (see Fig 2, top and bottom). The slopes of both regression lines were about 2.5. This means that each 1 mg/100 ml of direct biirubin in the specimen contributes 2.5 mg/100 ml to the hematofluorometer readings for either bound or total blood biirubin.
Reserve Bilirubin-Binding Capacity
The reserve biirubin-binding capacity of albu-mm, as determined by the hematofluorometer, was
DIAZO BILIRUBIN (mg/dt PLASMA)
V
E
0
NJ
0
U L’j
0
z
-I
4
0
U. I
DIRECT BILIRUBIN (mg/dl PLASMA)
DIAZO BIURUBIN (mg/dt PLASMA)
Fig 1. Comparisons between the plasma biirubin levels of 68 neonates, as determined by the Jendrassik-Grof
diazotization method and the total blood biirubin level (top) and the albumin bound biirubin level obtained by means of the hematofluorometer (bottom). The straight
lines through the individual data points fitted by least squares criteria are shown. The corresponding correlation
coefficients are 0.89 and 0.88.
bound biirubin levels obtained by the hematofluo-rometer are equally well correlated to the Jendras-sik-Grof total plasma bilirubin levels (Fig 1, bottom) with r = .88 and a slope of 0.83. All the samples
used to determine these correlations contained less than 0.5 mg/100 ml of direct biirubin.
Each of the specimens examined by
hematofluo-rometry exhibited a value of total blood biirubin
equal to or greater than the value for albumin-bound biirubin. On the average the total blood bilirubin level was 9% higher than that of the al-bumin-bound biirubin, as reflected in the difference in the slopes of the plots of Fig 1.
When samples with higher levels of direct biiru-bin were used, both total and bound biirubin values from the hematofluorometer were erroneously high.
V
E
0 NJ
4
0
I-U
I
0 z
z
C
0
1.
I
DIRECT BILIRUBIN (mg/dR PLASMA)
Fig 2. Top, Direct reacting biirubin (diazo) levels in 79
specimens (of which 1 1 had direct biirubin > 0.5 mg/100 ml plotted vs the difference between the total blood bilirubin (determined by the hematofluorometer) and the indirect bilirubin level (by diazotization). Note that the
data points are well correlated (r = .95) and that the fitted line (least squares criterion) has a slope of 2.6.
Bottom, Direct plasma biirubin level plotted vs the
dii-ference between the albumin-bound biirubin level
(he-matofluorometer) and the indirect biirubin level for the
same group of neonates. Note that as in the top figure the correlation is good (r = .89) and the slope is similar (2.4).
E
I
I
z
I
I C
z
0
...-E
w > a: Li C,) Li
a:
I
30
25
20
‘5
)0
5
0
0 20 40 60 80 )00
V
a,
E
Li
>
a:
Li
C,)
Li
a:
IL.
I
HABA RESERVE (%)
Fig 3. Comparison of the hematofluorometer albumin
reserve-binding capacities and the HABA reserve values
expressed as a percentage of the binding capacity of the
reference plasma for a series of 17 artificially jaundiced
adult blood specimens. The curve is a quadratic fitted by
the least squares criterion.
0
compared with values obtained by the HABA dye-binding method, which has been used in several clinical studies of neonatal jaundice.
The reserve-binding capacities obtained by these two methods for a series of artificially jaundiced adult blood specimens are plotted against each other in Fig 3. The data were obtained using
spec-imens with biirubin concentrations ranging from
0.2 to 20 mg/100 ml of plasma and albumin concen-trations adjusted to range from 1.4 to 2.7 gm/100
ml of plasma. The HABA reference sample used in
this study was pooled cord serum with 3.7 mg/100
nil of albumin.
A quadratic curve passing through the origin was found to provide a better fit to the data of Fig 3 than does a straight line (a = 1.2 and 1.4 mg/100 ml, respectively), and the correlation coefficient is
0.97. (By the correlation coefficient between two variables, x and y, which are fitted by curve y = f
(x) , we mean the multiple correlation coefficient against the hypothesis that x and y are correlated
by y = f(x).) The quadratic term has significance at
the P < .03 level.
Fig 4 shows a similar plot, using blood specimens from a randomly selected group of 36 jaundiced neonates. In keeping with the results obtained with the artificially jaundiced adult blood, these data were also fitted with a quadratic (not shown)
through the origin and led to a multiple correlation
coefficient of 0.73, corresponding to P < i0. It is clear from Fig 4 that, compared with the hematofluorometer, the HABA method yields a
HABA RESERVE (%)
Fig 4. The hematofluorometer albumin reserve-bind-ing capacity for biirubin as a function of the HABA
reserve values for blood specimens from a group of
neo-nates. The open and solid symbols correspond to speci-mens from babies with birth weights greater and smaller than 2,000 gm, respectively. The solid curves are the fitted quadratics (least squares criterion) for data corre-sponding to these two classes ofbirth weights. The dashed line is the quadratic obtained by fitting similar data for adult blood specimens, the same as the curve shown in Fig 3.
larger value of the reserve-binding capacity in neo-nates than in adults. In order to understand this observation better, the specimens were divided into two groups, those coming from infants whose birth weights were greater than 2,000 gm and those from infants whose birth weights were smaller than 2,000 gm. The values obtained for the hematofluorometer and HABA reserves are shown plotted separately in Fig 4. It is clear that, while the data points for the heavier weight group (>2,000 gm) on the aver-age fall closest to the curve obtained by fitting data for artificially jaundiced adult blood (Fig 4), most
of the low-birth-weight points yield HABA reserves which are approximately twice as great as those
corresponding to the same hematofluorometer re-serve in adult serum.
V a,
V
a,
E
.,
a,
E
0.5 - 1.0 1.0 - 1.5 1.5 -2.5 >2.5
BIRTH WEIGHT (kg)
e
. S
.
0 Li
I-C-)
Li a.
x
Li
4
4 I ..,, 0 Li >
Li C,)
0
4
4 I
:.:.b
.
. S
S #{149} a
a
.
and 0.30, their difference is found to have statistical
significance, with P < .001.
Bilirubin-Binding Capacity of Albumin
The sum of the hematofluorometer bound bii-rubin level and the hematofluorometer reserve-binding capacity is a measure of the bilirubin-bind-ing capacity of the albumin in a specimen.
The bilirubin-binding capacities of the neonatal blood specimens studied here and determined in
this way ranged from 9 to 38 mg/100 ml of plasma and are shown averaged for several birth weight
ranges in Fig 6. In consonance with the gestational
age dependence of binding capacity previously
re-ported using other methods,2224 the binding
capac-ity was found to depend upon birth weight. The
very small infants weighing less than 1,000 gm had a mean capacity of 15 mg/100 ml of plasma, while those born weighing more than 2,500 gm had a
mean capacity of 28 mg/100, which approaches the
value for a series of male adults (33 mg/100 ml). The lower capacities at lower birth weights is only partially accounted for by the well-known
obser-vation that the neonates of lower birth weight or
lower gestational age tend to have lower plasma albumin concentrations.
The average specific albumin capacity for biiru-bin, that is, milligrams of bound biirubin per gram of HSA, was also found to decrease with decreasing birth weight (Fig 6). The differences between the
specific albumin capacity of the group >2,500 gm
.
3.
2.
--1 #{149}
C I
0 1000 2000 3000 4000
BIRTH WEIGHT (9)
Fig 5. The ratio of HABA reserve values obtained for specimens from neonates and the HABA reserve expected for specimens from adults with the same
hematofluoro-meter reserve values (cf Fig 3), as a function ofthe babies’
birth weights. The average values for this ratio are shown as horizontal lines for the two groups with birth weights
less than and greater than 2,000 gm, with the stippled areas corresponding to the standard deviations from these
averages (1.98 and 1.33, respectively).
Fig 6. The bars show the albumin levels, the total
albumin-binding capacities for biirubin and the specific albumin-binding capacities of blood specimens from
neo-nates according to their birth weights and the same
parameters for a group of specimens from adults. The
error bars represent the standard errors.
and any of the lower weight groups was found to be statistically significant to the level P < .04.
DISCUSSION
Bilirubin Assays
The hematofluorometer used in this study was calibrated using adult blood, jaundiced artificially with unconjugated bilirubin. To avoid possible com-plications arising from the presence of conjugated biirubin in the specimens, this discussion of biiru-bin assays will be confined to specimens that con-tam virtually no bilirubin conjugates (<0.5 mg di-rect diazo-reacting biirubin per 100 ml of plasma).
bound biirubin by the hematofluorometer. In such a case the hematofluorometer bound bilirubin would be lower than the plasma biirubin level. There may also be a significant quantity of bilirubin associated with erythrocytes. This erythrocyte bil-irubin is, of course, not assayed by plasma biirubin methods. Thus, the hematofluorometer reading for
total blood biirubin may be higher than the plasma
bilirubin level.
In this connection it should be noted that the hematofluorometer assay for total blood bilirubin has a coefficient of variation of about 8% (W. J. Cashore, W. Oh, W. E. Blumberg, et al, unpublished data, 1979), about the same as that of the
Jendras-sik-Grof method for determining plasma biirubin
levels. The correlations between the
hematofluo-rometer bilirubin levels and the plasma biirubin
levels (Fig 1, top and bottom) appear somewhat
poorer (r = .9) than would be expected on the basis
of reproducibiities of these assays. (The correlation coefficient which would be expected for two differ-ent measurement methods, each having a standard
deviation a and taken over a range R, would be r = cos (ira/R). For a = 2 mg/100 ml and a measure-ment range of 0 to 20 mg/100 ml, this would give an expected r of 0.95.) This, at least in part, probably
reflects disparities between albumin-bound biliru-bin and total blood biirubin on the one hand and albumin-bound bilirubin and plasma biirubin on the other.
It should be noted that the hematofluorometer and the diazo method employed were calibrated
separately in different laboratories, using different
sets of calibration specimens. Error in calibration of one or both of the methods could explain some of the differences observed.
The relationships and differences between the bilirubin values obtained by the Jendrassik-Grof diazotization technique and by the hematofluoro-meter should be considered, in terms of both the
potential use of the hematofluorometer in clinical
situations and the provision of new information not
at present available by conventional methods alone. We have not found the differences between either the bound or the total hematofluorometer bilirubin levels and the plasma bilirubin levels to be sufficient
to preclude the use of the hematofluorometer in
clinical practice as a replacement for plasma
bili-rubin assays. At the same time, these differences should not be ignored: not only are the differences between plasma bilirubin levels and albumin-bound bilirubin levels real (reference 19 and Cashore et al,
unpublished data, 1979), but the
hematofluorome-ter makes it possible to assess the biirubin bound to all blood sites, including red cells. In other words, the potential exists for gaining new insights into the
pattern of biirubin distribution in individuals and in populations characterized by birth weight, ges-tational age and various states of health, under a variety of circumstances.
In particular, it wifi be of great interest to see whether the difference between the hematofluoro-meter total blood and HSA-bound biirubin levels is correlated with other blood parameters and din-ical observations of the infant’s health or the in-fant’s medical history or prognosis. Some studies attempting to explore the relationships between this difference and reserve-binding capacity, total binding capacity, albumin level, and erythrocyte biirubin are under way.
Bilirubin Conjugates
Biirubin conjugates and biirubin have virtually identical fluorescence spectra. It follows therefore that the hematofluorometer, as constituted for this
study, is sensitive to both conjugated and
uncon-jugated biirubin. The analysis of data obtained with specimens containing elevated direct biirubin
shows that their direct bilirubin fractions evoke a
consistent response from the hematofluorometer. However, the response is 2.5 times greater for con-jugated than for unconjugated bilirubin. That is, 1 mg/100 ml of direct biirubin contributes 2.5 mgI 100 ml to the hematofluorometer reading for bound as well as total biirubin. In terms of fluorescence characteristics, this suggests that, both when pres-ent in whole blood and when present in detergent micelles, the direct-reacting biirubin in these spec-imens from neonates has an effective fluorescence quantum yield about 2.5 times greater than that of indirect-reacting biirubin under the same condi-tions, when present in whole blood and when pres-ent in detergent micelles.
In practical terms this means that since the he-matofluorometer senses both direct and indirect biirubin, at least one determination of the direct bilirubin level in a neonate’s blood should be made for proper clinical evaluation and to permit the hematofluorometer parameters to be evaluated ap-propriately. It should be pointed out that existing rapid total plasma assays for biirubin that are based on plasma absorbance also fail to differentiate between direct and indirect plasma bilirubin.
Binding Capacity
The total biirubin-binding capacity of a blood specimen as determined in this study is proportional
#{149}PED4O429iH
pediat-4
4291
added bilirubin is sufficient to saturate primary
binding by HSA.
We have checked a number of plasma specimens from low-birth-weight neonates to show that the strong binding of bilirubin by the albumin as deter-mined by the hematofluorometer was indeed “sat-urated.” These detailed titrations studies will be reported elsewhere.
Our observations confirm those of other studies, which have found a similar dependence of total binding capacity and specific binding capacity of albumin on gestational maturity among “well” neo-nates.2224 One study showed that “sick” neonates did not exhibit an increased specific capacity with
maturity. The population of neonates in our study
was considered to be predominantly “well.” The very real difference between the specific binding capacities of the albumin of low-birth-weight, premature infants compared to higher birth weight, more mature infants suggests the presence
of an albumin fraction with low binding potential.
The hematofluorometer reserve-binding capacity was compared with the reserve HABA-binding
ca-pacity for two reasons. First and most obviously, it
was of interest to test for a correlation between the two methods. Second, although the clinical validity
of the meaning of “low” HABA reserve-binding
values has never been established by a randomized,
controlled study, there are long-term follow-up data on children with known reserve HABA values in
the neonatal period. It was, therefore, of interest to
determine the relationship between
hematofluoro-meter reserve capacity values and HABA values
previously associated with both poor and good
out-comes. In previous studies by one of us (A.B.) it
was observed that values of HABA reserve above
38% were not associated with kernicterus.23 (The
reserve HABA capacity is the amount of dye bound
by an aliquot of the specimen plasma expressed as a percentage of the dye bound by a reference plasma (or HSA solution). Since the various laboratories that employ the HABA method use various differ-ent reference solutions, the scales of HABA values
may differ among them, and HABA values may not
be directly comparable. The same reference, pooled cord plasma with total biirubin less than 1.0 mgI 100 ml, was used throughout this study.)
Because of possible complexities in the neonate
specimens, it was decided first to compare the
he-matofluorometer and HABA reserves for a series of artificially jaundiced, but otherwise normal, adult blood specimens. It had already been shown’9 that hematofluorometer reserve values were perfectly linear with the expected reserve values for artifi-cially jaundiced adult blood specimens of known bilirubin and HSA content. The excellent fit (r =
.97) to quadratic function (y = ax + bx2) with zero
intercept was expected for the plot (Fig 3) of he-matofluorometer reserve vs HABA reserve on the basis of the original report of Porter and Waters on the HABA method. These authors showed that the HABA reserve values exhibited a curvilinear rela-tionship to the expected reserve values in a series of artificially jaundiced specimens. Although the binding parameters of HABA with albumin have not been determined, it is expected from simple considerations of binding competition that the HABA reserve value should not be linearly related
to the true reserve, and indeed our fitted function has significant curvature. The HABA method
mea-sures the amount of HABA dye that can be bound
by the albumin in the specimen in competition with
the biirubin (and other anions) present. It is
ex-pected that the HABA value depends not only on the albumin and the biirubin levels but also on the relative binding affinities of albumin for biirubin and HABA and upon the concentration of HABA used. The hematofluorometer gives a direct mea-sure of the incremental biirubin that can be bound
by the albumin in the specimen when excess bii-rubin is added and, in the absence of strong endog-enous competitors, is expected to reflect the true reserve.
The relationship found between hematofluoro-meter reserve and HABA reserve for the adult specimens indicates that there exists a direct cor-respondence between hematofluorometer and HABA values. For example, for the particular HABA method used in this study it appears that, for adult blood, HABA reserves of 50% and 38% correlate with hematofluorometer reserves of 11 and 8 mg/100 ml, respectively.
The greatly increased scatter in the plot (Fig 4) of hematofluorometer reserve vs HABA reserve for the neonate specimens compared to the adult spec-imens is striking. However, two observations con-cerning these data suggest a basis for what scatter exists and imply that HABA reserve values should be employed clinically with more caution than was thought necessary until now. The first observation
is that almost all the neonate data fall below the “adult curve”; that is, the HABA value is more
“adult curve.” Statistical analysis indicates that the present data allows differentiation ofthe population into only two groups. Additional data should allow the observation of a continuously increasing devia-tion (towards a lower “slope”) from the “adult curve” with decreasing birth weight. While the scat-ter in the data is smaller in each of the two birth weight groups considered in the analysis than for the neonate population as a whole, it remains much larger than the scatter in the data for adult speci-mens, indicating heterogeneity in binding within
each group.
From the practical point of view translation be-tween hematofluorometer and HABA reserve val-ues for neonates must take into account birth weight or some other index of maturity. Thus, on the average, for the group >2,000 gm, HABA re-serves of 50% and 38% correspond to hematofluo-rometer reserves of 11, and 8 mg/100 ml, respec-tively. For the group <2,000 gm, HABA reserves of
50% and 38% correspond to hematofluorometer
re-serves of 7 and 4 mg/100 ml, respectively. As stated
above, a large data pool would allow differentiation into narrower weight groups.
In light of our observations one may ask whether it is the HABA method or the hematofluorometer method for reserve (or both) for which birth weight (or birth weight-related factors) must be taken into account if one wishes to obtain a uniform scale useful in managing neonatal jaundice. We suggest that this should be done for the HABA method, which, in comparison with the hematofluorometer method, does not “sense” binding deficiencies in the specimens from small premature neonates as compared with larger, more gestationally mature infants. This view is based upon the fact that the hematofluorometer measures a reserve binding ca-pacity by loading albumin sites with biirubin, while the HABA method measures the binding of an exogenous dye. Furthermore, in the HABA method the pH is buffered, and the plasma is diluted 30-fold, which may have the effect of minimizing al-bumin binding “deficiencies,” due to such factors as acidosis and the presence of endogenous competi-tors. The hematofluorometer uses essentially unal-tered whole blood at 37 C so that binding equilibria are not significantly perturbed. Blood gas equilibria are not maintained. Thus, the specimen becomes
fully oxygenated in the hematofluorometer proto-col. Loss of CO2 would also occur. Although the latter would cause an increase in the pH of the specimen, the change would be small. A reduction in a Pco2 from 50 torr to 0 torr alters the pH by 0.5 unit25; an original pH of 7.3 would rise to 7.8. Neither the fluorescence of biirubin nor its binding to al-bumin is significantly altered over the pH range
from 6.8 to 7.8.’ The addition of sodium bilirubi-nate to the specimens also leads to an increase in the pH. It has been determined’9 that the increase amounts to not more than 0.1 unit. The fact that, for the same hematofluorometer reserve value, the HABA reserve was observed to be higher for small, premature infants than for larger, more gestation-ally mature infants and adults strongly suggests that the HABA method does not respond to certain binding deficiencies to which the hematofluorome-ter does respond. Finally, another study (Cashore et al, unpublished data, 1979) has shown that the hematofluorometer reserve values agreed very closely with those obtained by serial titration of plasma specimens using a saturation end point based upon either gel filtration (Sephadex G-25) or peroxidase-catalyzed oxidation rates as indicators of unbound biirubin. The neonate population of this latter study was similar to the population of the present study.
It has been observed by Chan et al26 that, as the concentration of plasma-free fatty acids increases, the HABA dye-binding capacity is reduced signifi-cantly before a reduction is noted in the biirubin-binding capacity, which is measured using the gel ifitration (Sephadex G-25) end point. From these observations it was suggested that HABA is not specific for the primary biirubin-binding site on albumin. Such a conclusion is supported by our observations. It should also be noted that a com-parison of the observations reported herein with those of Chan et al26 suggests that if there were important competitors for biirubin binding present in the neonatal specimens we studied, they were not fatty acids. This suggestion is based on the following arguments. The hematofluorometer re-serve-binding capacity refers specifically to the pri-mary albumin-binding site. In many of the speci-mens examined in this study the binding capacity measured with the hematofluorometer was signifi-cantly lower than would be expected from the al-bumin concentration, but the HABA reserve capac-ity was greater than the hematofluorometer reserve capacity. Further support derives from the good agreement found between Sephadex reserve values and hematofluorometer reserve values in another study (Cashore et al, unpublished data, 1979).
CONCLUSION
Thus the assay provokes minimal perturbations of the partition of bilirubin in blood. Furthermore saturation of the strong HSA sites is performed by bilirubin addition rather than by addition of a dye as in the HABA method. It is therefore possible that the hematofluorometer parameters may be
better indicators and predictors of the risk for ex-ceeding the capacity of the blood to retain biirubin.
Our observations using the hematofluorometer to measure blood biirubin parameters in a random population of jaundiced neonates indicate that the hematofluorometer can be used to screen neonates for biirubinemia and may be useful in the manage-ment of neonatal jaundice. The hematofluorometer can rapidly identify infants with very low reserve-binding capacities despite low, seemingly safe2’3 bil-irubin levels. The hematofluorometer can supply a binding capacity assessment on a smaller specimen (<100 tl) of blood than that required by the much more tedious HABA dye method.
The work presented here suggests that, as clinical experience with the hematofluorometer is accumu-lated, a better understanding of bilirubinemia and the binding and distribution of biirubin may result.
REFERENCES
I. Harris RC, Lucey JF, MacLean JR: Kernicterus in
prema-ture infants associated with low concentrations of biirubin
in plasma. Pediatrics 21:875, 1958
2. Gartner LM, Snyder RM, Chabon RS, et al: Kernicterus:
High incidence in premature infants with low serum biirubin concentrations. Pediatrics 45:906, 1970
3_ Kim MH, Yoon JJ, Sher J, et al: Serum biirubin: A poor predictor of kernicterus. Pediatr Res, Program Issue, APS-SPR, April 1978, No. 1134
4_ Keenan
Wj,
Perlstein PH, Light IJ, et al: Kernicterus in small sick infants receiving phototherapy. Pediatrics 49:652, 19725. Stern L, Denton RL: Kernicterus in small premature infants. Pediatrics 35:483, 1965
6. Porter EG, Waters WJ: A rapid micromethod for measuring the reserve albumin binding capacity in serum from newborn infants with hyperbiirubinemia. J Lab Clin Med 67:660,
1966
7. Jirsov#{224}V, Jirs#{224}M, Heringov#{224} A: The use and possible
diagnostic significance of Sephadex gel filtration of serum from icteric newborn. Biol Neonate 11:204, 1967
8. Kaufmann NA, Kapitulnik J, Blondheim SH: The absorp-tion of bilirubin by Sephadex and its relationship to the criteria for exchange transfusion. Pediatrics 44:543,1969
9. Nakamura H, Lardinois R: Unbound bilirubin in icteric
newborn. Biol Neonate 21:400, 1972
10. Kaufmann NA, Simcha AJ, Blondheim SH: The uptake of bilirubin by blood cells from plasma and its relationship to the criteria for exchange transfusion. Gun Sci 33:201, 1967
1 1. Odell GB, Cohen SN, Kelly PC: Studies in kernicterus. II.
The determination of the saturation of serum albumin with
bilirubin. J Pediatr 74:214, 1969
12. Bratlid D: Biirubin binding by human erythrocytes. Scand
J Clin Lab Invest 29:91, 1972
13. Bergsma D, Blondheim H (eds): Bilirubin Metabolism in
the Newborn. II. Birth Defects Original Article Series, Vol
12, No. 2, 1976. New York, The National Foundation-March of Dimes
14. Diamond I, Schmid R: Experimental biirubin encephalop-athy: The mode of entry of biirubin-’4C into the central nervous system. J Clin Invest 45:678, 1966
15. Johnson L, Boggs TR: Biirubin-dependent brain damage:
Incidence and indicators for treatment, in Odell GR, Schaef-fer R, Simopoulos AP (eds): Phototherapy in the Newborn.
Washington, DC, National Academy of Sciences, 1974, pp 122-149
16. Odell GB, Storey GNB, Rosenberg LA: Studies in kernicte-rus. III. The saturation of serum proteins with biirubin during neonatal life and its relationship to brain damage at five years. J Pediatr 76:12, 1970
17. Jendrassik L, Grof P: Vereinfachte photometrische Meth-oden zur Bestimmung des Blutbilirubin. Biochem Ztschr 297:81, 1938
18. Doumas BT, Watson WA, Biggs HG: Albumin standards and the measurement of serum albumin with bromocresol
green. Clin Chim Acta 31:87, 1971
19. Lamola AA, Eisinger J, Blumberg WE, et al: Fluorometric study of the partition of biirubin among blood components: Basis for rapid microassays of biirubin and biirubin binding capacity in whole blood. Anal Biochem 100:25, 1980 20. Blumberg WE, Eisinger J, Lamola AA, et al: Zinc
protopor-phyrin level in blood determined by a portable
hematofluo-rometer: A screening device for lead poisoning. J Lab Clin
Med 89:712, 1977
21. Blumberg WE, Zuckerman DM, Eisinger J, et al: The bili-rubin hematofluorometer. J Lab Clin Med, in press 1980 22. Kapitulnik J, Horners-Mibashan R, Blondheim SH, et al:
Increase in biirubin-binding affinity of serum with age of
infant. J Pediatr 86:442, 1975
23. Brown AK, Mody MC Assessing diffusibiity of biirubin in jaundiced infants, in Bergsma D, Blondheim S (eds)
Biliru-bin Metabolism in the Newborn II. Birth Defects Original
Articles Series, Vol. 12, No. 2, 1976. New York, The National Foundation-March of Dimes, pp 205-214
24. Cashore WJ, Herwick A, Karoficin EH, et al: Influence of gestational age and clin,cal status on biirubin-binding
Ca-pacity in newborn infants. Am J Dis Child 131:898, 1977 25. Levin RM: Pediatric Respiratory Intensive Care
Hand-book. Flushing, NY, Medical Examination Publishing Co, mc, 1976
26. Chan G, Schiff D, Stem L: Competitive binding of free fatty
acids and biirubin to albumin: Differences in HBABA dye
versus Sephadex G-25 interpretation of results. Clin
