280

February 1975 The Journal o f P E D I A T R I C S

Fluorescent dye method for determination of the bilirubin-binding capacity of serum albumin A simple, rapid microfluorometric method util&ing afluorescent dye, Direct Yellow 7,for quantitative measurement of serum albumin-binding capacity for bilirubin has been developed. The dye binds exclusively to serum albumin with enhancement offluorescence. The dye shares bilirubin-binding sites on albumin with up to 2 mols ofbilirubin per mol qfalbumin with differing affinities to the.first and second sites. LoweredpH reduces albumin-binding capacity and exposure to light increases binding capacity of icteric sera. The binding capacity of adult human sera is greater than that of human cord sera or purified human serum albumin compared on albumin molar basis.

Kwang-sun Lee, M.D., Lawrence M. Gartner, M.D.,* Bronx, N. Y., and nana Zarafu, M.D., Newark, N. J.

A GENERAL STATISTICAL RELATIONSHIP exists between serum unconjugated bilirubin concentrations of greater than 20 mg/dl during the newborn period and the development of kernicterus or impaired neurologic function, 1 Kernicterus has been noted in severely ill premature infants at unconjugated bilirubin concentrations as low as 8 mg/dl. 2 In contrast, older infants and children usually do not develop clinically apparent kernicterus until serum bilirubin concentrations exceed 40 mg/dl. 3 This increased susceptibility of the newborn to the development of kernicterus has been attributed to d i f f e r e n c e s in t h e " b l o o d - b r a i n b a r r i e r " a n d in albumin-binding capacity for bilirubin. 4 A l b u m i n - b i n d i n g capacity for b i l i r u b i n has b e e n s t u d i e d by various m e t h o d s . 57 A l t h o u g h neurologic outcome correlates better with albumin-binding capacity than with serum bilirubin levels, 8,9 uncertainty regarding interpretation of binding results 1~ 11 has prevented their widespread use. Serum bilirubin concentrations therefore continue to be the major indicator of the need for exchange transfusion or phototherapy to reduce the assumed risk of kernicterus. From the Department of Pediatrics and Rose F. Kennedy Center, Albert Einstein College of Medicine. *Reprint address: Department of Pediatrics and Rose F. Kennedy Center, AIbertEinstein Collegeof Medicine, 1300MorrisPark Ave., Bronx, 1~ Y. 10461.

VoL 86, No. 2, pp. 280-285

A rapid microfluorometric method for determination o f a l b u m i n - b i n d i n g capacity for b i l i r u b i n utilizing a f l u o r e s c e n t dye probe, specific for b i l i r u b i n - b i n d i n g sites on albumin, has been developed. MATERIALS

AND METHODS

C o l u m n c h r o m a t o g r a p h i c study. S e p h a d e x G - 2 0 0 (Pharmacia Fine Chemicals Co.) column chromatography of the fluorescent dye in sera was carried out in 90 x 1.5 cm column by low pressure downward flow elution with 0.1M phosphate buffer, pH 7.4, in the dark at 4 ~ The fluorescent dye, Direct Yellow 7 (DY7), a Abbreviation used DY7: Direct Yellow 7 polymer of dehydrothio-para-toluenosulfonate (Vasoflavine, Allied Chemical Co.) was added to adult human sera at concentrations from 40-300 mg/dl sera. Eluates were collected by a timed fraction collector (Buchler Instrument). Protein content of each effluent was measured by the method of Lowry and associates. 12 Conc e n t r a t i o n s o f DY7 in eluates were d e t e r m i n e d by fluorescence with a filter fluorometer, following appropriate dilutions, as described below under "filter fluorometric method." Spectrofluorometric study. Excitation and emission

Volume 86 Number 2

z o

,~ ~

Fluorescent dye method for determining bilirubin-binding capacity

281

18

1.8

18

1.6

16

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120

~ 140

i X 160

180

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EFFLUENT(mL)

60

80 1 0 0 150 200 DY 7 (mg/100ml sera)

250

d

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Fig. l. Sephadex G-20O column chromatography of adult human sera containing DY7 300 mg/dl sera (eluted with 0.1M phosphate buffer, pH 7.4, in the dark at 4~ ( ) protein content (mg/ml) and DY7 content expressed as fluorescence (__o__e~). Protein peaks from left to right; lipoproteinmacroglobulin, gamma globulin, albumin and prealbumin as a shoulder on albumin peak. 13

Fig. 2. Sephadex G-200 column chromatography of adult human sera containing increasing concentrations of DY7, from 40 to 300 mg/dl sera; hatched block = DY7 measured as fluorescence in lipoprotein-macroglobulin fraction; open block = DY7 in albumin fraction; closed block = DY7 in terminal protein-free fraction.

spectra of 1.6 tzg DY7/dl diluted h u m a n serum, h u m a n serum albumin (Sigma) or 0.1M phosphate buffer with and without added bilirubin equivalent to 15 mg/dl undiluted sera, were determined with an Aminco-Bowman spectrofluorometer. Filter f l u o r o m e t r i e study (standard method). T h e filter f l u o r o m e t e r (C-12, C o l e m a n Jr., primary filter 12-221, secondary filter 14-211) was calibrated with quinine sulfate solution (Fisher Scientific Co.). The zero p o i n t was set with 0.15 mg q u i n i n e sulfate/dl 0.1N sulfuric acid and the i00 point with 0.3 mg/dl. To determ i n e s a t u r a t i o n c o n c e n t r a t i o n of DY7 b i n d i n g to h u m a n adult sera, a broader calibration of the fluorometer was achieved by setting the zero point with distilled water and the 100 point with 0.5 mg/dl quinine sulfate solution. Three microliters of sera were added m a n u a l l y or by an automatic micropipetter (Brinkmann) to 10 ml of 0.1M phosphate buffer Solution, pH 7.4, Containing DY7 at a standardized concentration (see below) followed by rapid, thorough mixing. All measurements of fluorescence were done within 20 seconds. For each assay, fluorescence of DY7 solution w i t h o u t added s e r u m was d e t e r m i n e d first ( b l a n k ) . Fluorescence of the solution after addition of the serum was then read (total). The net difference between blank and total reading was designated as " e n h a n c e d fluorescence" (AF). Since the different batches of DY7 vary in

purity, the concentration of the dye was adjusted by dilution to a fluorescence of 20. In the first batch of the dye used, this degree of fluorescence was produced by 0.35 mg/dl, which resulted in maximal fluorescence in the assay system using fresh adult human sera. Solutions of the dye were prepared and kept in the dark and all procedures were p e r f o r m e d in dim light. Serum b i l i r u b i n c o n c e n t r a t i o n s above 5 mg/dl r e s u l t e d in quenching of fluorescence and corrections were made from a table established by the addition of bilirubin of varying concentrations to an aqueous solution of DY7 (Table I). Serum a l b u m i n concentrations were determined by measurement of total protein 12 and per cent a l b u m i n by 2% a g a r o s e gel e l e c t r o p h o r e s i s in a Spectrophor I ( B a u s c h a n d L o m b , Inc.) in 0.01M phosphate buffer, pH 8.5, with 300 volts applied for 90 minutes. Densitometric reading of protein fractions was carried out at 205 nm. RESULTS C o l u m n c h r o m a t o g r a p h i c study. I n c r e a s i n g conzentrations of DY7 in adult h u m a n sera demonstrated almost exclusive fluorescence in association with the albumin peak (Fig. 1). Less than 3% of total fluorescence was associated with lipoprotein-macroglobulin and prealbumin peaks. Fluorescence was also detected in the final protein-free eluates and was assumed to be

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The Journal of Pediatrics February 1975

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Fig. 3. The effect of added human sera on activation, A, and emission, B, spectra of DY7 in 0.1M phosphate buffer, ph 7.4; ( )DY7, 1.6 Fg/dl buffer; (__o_o) DY7, 1.6/~g/dl buffer with added sera (sera diluted with the buffer 3/10,000). 20

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Fig. 4. Enhanced fluorescence (AF) of DY7 following addition of 3/xl of adult human sera (albumin concentration, 3.3 gm/dl) with increasing concentrations of DY7 in 0.1M phosphate buffer, pH 7.4 (n = 4). Calibration of filter fluorometer: 0 point set with distilled water, 100 point With 0.50 mg/dl quinine sulfate solution. Table I. Quench correction values: Inhibition of fluorescence of DY7 in 0.1M phosphate buffer, pH 7.4, by addition of bilirubin Bilirubin concentrations (mg/dl) Fluorescence change (AF)

5 -1.0

l0 -1.5

15 -2.0

u n b o u n d DY7, The DY7 content of each peak, with increasing concentrations of the dye in adult h u m a n sera, was expressed by the histogram in Fig. 2. Saturation of a l b u m i n - b i n d i n g capacity for DY7 was o b s e r v e d at DY7 concentrations above 200 mg/dl of sera, with all additional dye being eluted in the protein-free fraction. Speetrofluorometrie study. DY7, when exposed to its maximum excitation wavelength, 370 n m (Fig. 3 A), yielded emission spectra of longer wavelength (fluorescence) with a m a x i m u m at 440 n m (Fig. 3 B). W h e n DY7 was added to adult h u m a n sera or albumin solutions, there was a shift of the excitation and emission peaks of DY7 to 390 and 430 nm, respectively, with

20 -2.5

25 -2.8

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40 -3.5

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70 -5.0

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e n h a n c e m e n t of absorbance or fluorescence (Fig. 3). The enhanced absorbance or fluorescence was proportional to the concentrations of albumin in the solutions. W h e n DY7 was added to artificialIy jaundiced sera or albumin solutions, the intensity of fluorescence was reduced. Filter fluorometrie study. Increasing concentrations of DY7 in sera resulted in a progressive e n h a n c e m e n t of fluorescence up to a DY7 concentration of 0.35 mg/ dl (Fig. 4). No further increase in fluorescence was noted above this level, indicating saturation of albuminbinding capacity for DY7. At this concentration, DY7 in aqueous solution yielded a base fluorescence (blank)

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Fluorescent dye method for determining bilirubin-binding capacity

283

60

40 AF 20

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Fig. 6. Effect on increasing concentrations of bilirubin in adult human sera (albumin concentrations, 3.3 gm/dl) on enhanced fluorescence (AF) of DY7 in 0.1M phosphate buffer, pH 7.4 (n=4). Slopes of regression lines were calculated by the method of least squares. Slope 1 is significantly different from slope !I by t test (p (0.001). Filter fluorometer calibration: 0 point set with 0.15 mgldl quinine sulfate, 100 point with 0.30 mg/dl quinine sulfate solution. of 20. The addition of 3/zl offresh adult h u m a n sera to the DY7 solution gave a mean enhanced fluorescence (hF) of 44.6 __ 1.6 (SD) (n=4). The effect Of ~emperiiture and pH changes on the fluorescence measurements were determined. Increasi n g . t h e t e m p e r a t u r e of the DY7 solution, w i t h o u t added sera, fi'om 10 to 40 ~ a small but progressive decrease in blank fluorescence was noted, reaching a m a x i m u m decrease of 5% at 40 ~ Total fluorescence was also decreased with increasing temperature in a similar m a n n e r , resulting in no net change in AF over the range of 10 to 40 ~ Decreasing the pH of the DY7 solution without added sera (blank) led to a marked

decrease in the intensity of fluorescence, b u t no appreciable change was noted above pH 7.4. Addition of sera to the buffer-dye solutions at increasing pH demonstrated a progressive reduction in albumin-binding capacity (AF) by s u b t r a c t i n g the m a t c h e d b l a n k of same pH (Fig. 5). A drop in pH from 7.4 to 7.0 caused a decrease of 25% in albumin-binding capacity. In contrast, less than a 5% increase in binding capacity was noted when the pH was raised from 7.4 to 8 (Fig. 5). The addition of increasing concentrations of unconj u g a t e d b i l i r u b i n (5-70 mg/dl) to adult h u m a n sera resulted in a progressive inhibition of AF, with complete inhibition of AF at 55 mg/dl (Fig. 6). Multiple re-

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The Journal of Pediatrics February 1975

Table II. Enhanced fluorescence (AF) of DY7 following addition of bilirubin to adult human sera (in 0.1M phosphate buffer, pH 7.4) and the exposure to room illumination for 3 and 6 hours Bilirubin (mg/dl sera)

I

DISCUSSION 0

Time of light exposure 0 hours 43.0 After 3 hours 43.5 After 6 hours 43.0

10

20

30

40

35.0 44.0 44.0

24.5 37.0 41.0

15.0 35.5 39.5

9.5 35.0 39.0

Table III. Enhanced fluorescence ( A F ) of DY7 by addition of adult human sera, cord sera and crystalline albumin solution (Sigma) (in 0.1M phosphate buffer, pH 7.4)

Specimen

Adult Cord Crystalline (3.5 gm/dl)

AF o f 27.4 _ 3.9 (SD) ( n = 4 1 ) ( T a b l e I I I ) . S e r u m bilirubin concentrations in cord sera were all less than 1.5 mg/dl.

No. o f specimens

4 41 4

AF

. AF/gm albumin

44.6 _+ 1.7 (SD) 27.4 +_ 3.9 (SD) 25.0 +_ 2.9 (SD)

13.4 9.3 7.1

gression analysis by the method of least squares yielded the best fit of the data, as shown in Fig. 6, to be two significantly different slopes. The first line extended from 0 to 27 mg/dl, and the second line, from 27 to 55 mg/dl. The mean concentration of serum albumin for the four subjects was 3.3 gm/dl. Based on this, a bilirubin concentration of 27 mg/dl corresponded to a bilirubin to a l b u m i n molar ratio o f 0.97, while 55 m g / d l corresponded to a ratio of 1.97. Enhanced fluorescence of DY7 resulted entirely from binding of the dye to the two bilirubin-binding sites on albumin, since AF was c o m p l e t e l y i n h i b i t e d by the a d d i t i o n of 2 mols of bilirubin per mole of albumin. The exposure of DY7 solution without sera to the incident light of the fluorometer or our ordinary laboratory illumination for varying p e r i o d s of time increased f l u o r e s c e n c e i n t e n s i t y . A q u e o u s b i l i r u b i n in 0.1M phosphate buffer, pH 7.4, did not produce any fluorescence, either initially or following the exposure of the solution to light for 6 hours. A more pronounced increase was noted in icteric sera (after blank correction) compared to its original reading before the exposure to light (Table II). By the present method, freshly obtained human adult sera had a mean binding capacity, AF of 44.6 ___ 1.6 (SD) (n=4); the mean AF of human serum albumin was 25.0 • 2.9 (SD) (n:--4); and cord sera had a mean

In 1960, Bethei114 d e s c r i b e d a m i c r o f l u o r o m e t r i c method for the measurement of serum albumin utilizing the fluorescent dye, Direct Yellow 7. He noted that icteric sera from patients with hepatic cirrhosis produced falsely low albumin concentrations) 5This observation suggested that DY7 might be useful for the estimation of albumin-binding capacity for bilirubin. A s originally r e p o r t e d , I4 DY7 is b o u n d to s e r u m albumin, and binding to other serum proteins is insignificant. Direct Yellow 7 shares bilirubin-binding sites on a l b u m i n in adult human sera up to a bilirubin concentration equivalent to a bilirubin to albumin molar ratio of 1.97. The regression lines indicate that the dye distinguishes two different binding sites with different affinities. Molar binding capacity of DY7 to albumin, as well as binding constants for both binding sites, could not be calculated because the polymeric character of DY7 prevented determination of its molecular weight. Direct Yellow 7 appears to be a precise fluorescent probe for the two bilirubin-binding sites on albumin. It c o u l d t h u s r e a s o n a b l y be e x p e c t e d to e s t i m a t e the reserve albumin-binding capacity for bilirubin. Lowered blood pH in neonates has been implicated as a risk factor in the development of kernicterus by d e c r e a s i n g a l b u m i n - b i n d i n g capacity for bilirubin. 16 The reduction of pH in our assay below 7.4 also resulted in a reduced albumin-binding capacity for DY7. This pH dependency makes it imperative that the pH of the buffer used in this assay is the same as the blood pH of the patient whose albumin-binding capacity is being studied. Marked differences in binding capacities were noted among pooled cord sera, adult human sera, and purified h u m a n serum albumin (4 gm/dl). The binding capacity of purified human serum albumin was approximately one-half that of the adult human sera on a molar basis while that of cord sera was intermediate, confirming a previous report. 17 Although the time required for the assay in a previously c a l i b r a t e d filter f l u o r o m e t e r is less than 1 minute, it is possible that exposure of icteric sera to room illumination or inadvertently to the incident light of the fluorometer for a prolonged time may occur. Bilirubin is highly susceptible to photo-oxidation and further increase in photo-oxidation may result from the exposure to the emission spectra of DY7 (350-550 n m

Volume 86 Number 2

Fluorescent dye method for determining bilirubin-binding capacity

with peak at 430 rim). The studies reported here indicate that these photo-oxidation products of bilirubin appear to have less affinity than DY7 or bilirubin for albumin-binding sites. Prolonged exposure to light may therefore result in a falsely high estimation of albuminbinding capacity for bilirubin. Various methods have been developed to assess the risk of kernicterus occurring in the newborn period and to serve as a guide to clinical management of the infa nt. 5-7 T h e H B A B A [ 2 - ( 4 ' - h y d r o x y b e n z e n e a z o ) benzoic acid] method is based on the premise that the d y e is c o m p e t i n g w i t h b i l i r u b i n f o r t h e p r i m a r y bilirubin-binding sites on albumin. It has been suggested that the dye measures not only bilirubin-binding sites but also other sites on albumin. 1~ There is also paradoxical effect from pH changes with an estimated increase in binding from decrease in pH below 7.4 and an estimated decrease in binding from elevated pH a b o v e 7.4, contrary to th e k n o w n effect o f pH on bilirubin-albumin binding. 5 The saturation index method is based on the shift of loosely bound bilirubin from albumin to an unbound state by the addition of a competitive anion, salicylate, to icteric sera. 6 It requires highly sophisticated spectrophotometric equipment and great precision in the performance of the test. 6 In the s e p h a d e x G-25 c o l u m n c h r o m a t o g r a p h i c method, the presence of loosely bound or free bilirubin in sera is determined by the binding of bilirubin to gel. 7 In this method the estimation of binding capacity requires performance of a more complex series of steps in which increasing amounts of unconjugated bilirubin are added to the specimen in a step-wise fashion. The fluorescent dye-binding technique described in the present study is simple, extremely rapid; and requires only 3/xl of sera. The dye appears to behave in many ways similar to bilirubin (pH effect, two binding sites on albumin, and specificity of binding sites) and may permit accurate estimation of the reserve capacity of the two primary bilirubin-binding sites on albumin. Evaluation of the dye has been limited at present, to examination of adult h u m a n and cord sera, but clinical studies are in progress to evaluate the method in neonates. It is apparent that only long-term s t u d y of a population of infants at risk will be able to determine the value of the method for prediction of neurologic outcome. We wish to thank Drs. Joseph Betheil, Irwin Arias, and Nathan Rudolph for their counsel and encouragement.

285

REFERENCES

1. Hsia DY, Allen FH Jr, Gellis SS, and Diamond LK: Erythroblastosis fetalis. VII1. Studies of serum bilirubin in relation to kernicterus, N Engl J Med 247:668, 1952. 2. Gartner LM, Snyder RN, Chabon RA, and Bernstein J: Kernicterus: High incidence in prematures with low serum bilirubin concentrations, Pediatrics 45:906, 1970. 3. Arias IM, Gartner LM, Cohen MI, Ben Ezzer J, and Levi J: Chronic non-hemolytic unconjugated hyperbilirubinemia with hepatic glucuronyl transferase deficiency: Clinical, biochemical, pharmacologic evidence for heterogeneity, Am J Med 47:395, 1969. 4. Diamond I: Bilirubin binding and kernicterus, in Schulman, I., editor: Advances in pediatrics, Vol 16, Chicago, I11., 1969, Year Book Medical Publishers, Inc., p 99. 5. Porter EG, and Waters WJ: A rapid micro method for measuring the reserve albumin binding capacity in serum from newborn infants with hyperbilirubinemia, J Lab Clin Med 67:660, 1966. 6. Odell GB: Studies in kernicterus. I. The protein binding of bilirubin, J Clin Invest 38:823, 1959. 7. Jirsova V, Jirsa M, Heringova A, Koldovsky O, and Weirichova J: The use and possible diagnostic significance of sephadex gel filtration of serum from icteric newborn, Biol Neonate 11:204, 1967. 8. Odell GB, Storey GNB, and Rosenberg LA: Studies in kernicterus III. The saturation of serum proteins with bilirubin during the neonatal life and its relationship to brain damage at five years, J PEDIATR76:12, 1970. 9. Johnson LH, and Boggs TR: Failure of exchange transfusion to prevent minimal cerebral damage when employed so as to maintain serum bilirubin concentrations under 18 and 20 mg/100 ml, Pediatr Res 4:481, 1970 (abst.). 10. Chan G, Schiff D, and Stern L: Competitive binding of free fatty acids and bilirubin to albumin: Difference in HBABA dye versus sephadex G-25 interpretation of results, Clin Biochem 4:208, 1971. 11. Wooley PV III; and Hunter MJ: Binding and circular dichroism data on bilirubin-albumin in the presence of oleate and salicylate, Arch Biochem Biophys 140:197, 1970. 12. Lowry OH, Rosebrough NJ, Farr AL, and Randall RJ: Protein measurement with the Folin phenol reagent, J Biol Chem 193:265, 1951. 13. Fireman P, Vannier WE, and Goodman HC: Immunochemical studies of human serum fractionated by get filtration with sephadex G-200, Proc Soc Exp Biol Med 115:845, 1964. t4. Betheil JJ: Fluorometric microdetermination of human serum albumin, Anal Chem 32:560, 1960. 15. Betheil JJ, and Hanok A: Determination of albumin in Cirrhotic sera by vasoflavine fluorescence enhancement procedure, Fed Proc 17:190, 1958. 16: Martin NH: Preparation and properties of serum and plasma proteins. XXI. Interactions with bilirubin, J Am Chem Soc 71:1230, 1949. 17. Kapitulnik J, Blondheim SH, and Kaufmann NA: Sephadex adsorption of bilirubin from neonatal and adult serum, Clin Chem 18:43, 1972.