Preparation and metabolism of 125-sulfobromophthalein

Ciinica Chimicu Acta, 128 (1983) 321-327 Elsevier Biomedical Press

321

CCA 2431

Preparation and metabolism of 125I-sulfobromophthalein Masanori

Hirano,

Lorenz

Thei~mann, and Allan

Liver Research Center and Department

Yacov R. Stollman, W. Wolkoff

Alexander

Sosiak

of Medicine, Albert Einstein College of Medicine, Bronx, New York 10461 (USA)

(Received

July 12th; revision October

29th, 1982)

Summary Metabolism of 12sI-sulfobromophthalein (BSP) prepared by the chloramine-T method was studied in rats. ‘251-BSP is removed rapidly from the circulation. However, as compared to BSP, its plasma clearance and biliary excretion are delayed, and its accumulation in the liver is prolonged. Although BSP and ‘251-BSP show similar binding to albumin in serum, their binding properties to liver cytosohc proteins and to the liver cell plasma membrane organic anion binding protein (OABP) differ. In contrast to the X-, Y- and Z-protein binding of BSP, ‘251-BSP binds predominantly to a high molecular weight protein and only a small proportion of “‘1-BSP binds to OABP.

Sulfobromophthalein (BSP) is a compound which has been used for over 50 years to study hepatic organic anion transport and metabolism [1,2]. Availability of 3SS-BSP, which does not differ from BSP in its chemical and biological properties [3] has facilitated these studies. However, because it is a p-emitter, 35S-BSP has certain limitations (e.g. quenching of P-rays and the inability to be used as an isotope for clinical liver scans). Tubis et al (41 prepared ‘3’I-BSP by using two iodination procedures (ICL and NaNq), and studied its hepatic accumulation by liver scan. Because iodination of BSP replaces bromine at the phthalic ring with iodine, it cannot be assumed that the new molecule has the same biological properties as BSP method [7] and used it for liver [5]. Kilic [6] prepared 13’I-BSP by a chloramine-T scans in patients. The metabolism of this substance has not otherwise been de-

Address all correspondence to: Allan W. Wolkoff, MD, Liver Research Center, Ullmann Building, Room 605, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.

0~9-898~/83/~-~/$03.~

0 1983 Efsevier Biomedical

Press

322

termined. In this study, we have prepared ‘251-BSP of high specific activity by a chloramine-T method, and we have contrasted its biliary excretion and metabolism in vivo and in vitro with that of unlabelled or 35S-BSP. Materials and methods BSP was obtained from Sigma Chem. (St. Louis, MO, USA), mCi/mmol) and Na’251 (1317 mCi/mg I) from Amersham (Arlington USA); GSH-BSP was prepared by the method of Whelan et al [8].

35S-BSP (100 Heights, NJ,

Iodination of BSP The iodination mixture consisted of 20 ~1 of 0.5 mol/l phosphate buffer (pH 7.5), 20 ~1 of BSP (10 pg) in H,O, 5 ~1 of Na ‘*‘I (0.5 mCi), 20 ~1 of chloramine T (6.25 mg/ml) (Kodak, Rochester, NY, USA), 20 ~1 sodium metabisulfite (15 mg/ml) and 20 ~1 KI (100 mg/ml). Reagents were added in the above order, iodination was stopped after 5 s by adding the sodium metabisulfite and noncovalently coupled iodine displaced by addition of KI. Separation of “‘I-BSP and free iodine The iodination mixture was applied on a silica-gel thin-layer chromatography plate (Redi Plate, Fischer Scientific, Springfield, NJ, USA) and chromatographed with chloroform/acetone/methanol (4 : 2 : 1, v/v/v). BSP was visualized by exposure of the plate to NH, vapor, and was extracted with 4 x 1 ml H,O. BSP concentration was measured in a Perkin-Elmer spectrophotometer at 580 nm after alkalinization with 0.1 mol/l NaOH. Radioactivity was determined in a Packard 3002 y-counter. In vivo studies Male Sprague-Dawley rats (225-250 g) were purchased from Charles River (Wilmington, MA, USA). Chow and water were made available ad libitum. To determine the plasma clearance of ‘251-BSP, rats were anesthetized with pentobarbital and heparinized with 100 U/100 g body weight i.p. Jugular vein and femoral artery were cannulated with PElO tubing. The animals were placed in restraining cages and ‘251-BSP (1 pCi/lOO g body weight) in 0.5 ml PBS was injected into the jugular vein. Blood (50 ~1) was drawn from the femoral artery during the first 4 min. In studies of biliary excretion, the bile duct and jugular vein were cannulated under light ether anesthesia, and the rats were placed in a restraining cage. Two hours after consciousness was regained, a mixture of BSP (15 mg/lOO g body weight) and ‘251-BSP (1 pCi/lOO g body weight) was injected i.v. Bile was collected in tared vials every 10 min for 90 min. Bile volume, BSP concentration and radioactivity were measured. Accumulation of ‘251-BSP in liver was determined in seven rats from 6 to 30 min after injection. The animals were sacrificed, liver was removed, weighed and radioactivity was determined.

323

Conjugation of **‘I-BSP . Bile from successive time points after injection was applied on a TLC plate. Conjugated and unconjugated ‘251-BSP were separated after development with I-propanol/acetic acid/H,0 (10 : 1 : 5, v/v/v) and identified using BSP and GSH-BSP as standards [8]. The two zones corresponding to BSP and GSH-BSP standards were scraped off, radioactivity was measured and the ratio of ‘251-BSP to unconjugated was determined. conjugated In vitro studies Binding to serum proteins. 250 pg BSP and 0.5 PCi of ‘251-BSP were mixed with 0.2 ml rat serum. This mixture containing labelled and unlabelled BSP, was charged onto a Sephadex G-200 column (Pharmacia, Fine Chemicals, Uppsala, Sweden) (1 cm x 50 cm), equilibrated with phosphate-buffered saline (20 mmol/l phosphate, 0.15 mol/l NaCl, pH 7.4) (PBS) and filtered with the same buffer at a flow rate of 5 ml/h. Fractions of 0.5 ml were collected, protein was quantitated by the absorption at 280 nm, and BSP concentration and radioactivity were determined. The same methods were used to determine binding of ‘251-BSP to plasma proteins in vivo. Five minutes after injection of ‘251-BSP (1 pCi/lOO g body weight) blood was drawn from the femoral artery and 0.2 ml of the serum was chromatographed as described above. The liver was removed Binding of BSP and ‘*-‘I-BSP to liver cytosolic proteins. from rats, weighed and perfused with ice cold PBS. A 20% homogenate was prepared in 0.25 mol/l sucrose/PBS, pH 7.4. The homogenate was centrifuged at 100 000 X g for 90 min at 4°C [9]. Supernatant cytosol was decanted and 3 ml was incubated The with 750 pg BSP and 1 PCi of ‘251-BSP for 15 min at room temperature. mixture, containing labelled and unlabelled BSP, was applied to a Sephadex G-75 column (Pharmacia, Uppsala, Sweden) (3 cm X 75 cm) equilibrated with PBS, pH 7.4, and 2-ml fractions were collected [9]. Binding to liver cell plasma membrane organic anion binding protein (OABP). OABP was purified according to Wolkoff and Chung [lo]. Five pg protein in PBS was incubated with 1 PCi ‘251-BSP and applied onto a Sephadex G-25 column (1 cm X 30 cm) and filtered with PBS. In a second experiment, the same procedure was performed using 35S-BSP (0.03 PCi) equimolar to ‘251-BSP. Radioactivity of 35S-BSP was determined in an Intertechnique ABAC-SL liquid scintillation counter (Intertechnique Instruments, Inc., Dover, NJ, USA) after addition of Hydrofluor (New England Nuclear, Boston, MA, USA). Appropriate crossover and quench corrections were made. Results Biochemical properties On TLC “‘1-BSP had an R, of 0.05 while free ‘25I had an R, of 0.55. Unlabelled BSP and 35S-BSP did not migrate in this system. The specific activity of (mean f SD). BSP and ‘251-BSP TLC purified ‘251-BSP was 1975 &-349 mCi/mmol)

324

MINUTES Fig. 1.This typical biliary excretion curve was obtained after injection of a mixture of BSP (1.Smg/ 100 g body weight) (O------O) into the jugular vein. body weight) (O0) and ‘251-BSP (1 pCi/lOOg Percentage excretion of each anion was determined in consecutive IO-min bile collections.

were extracted from silica gel with H,O. ‘251-BSP was identical to that of BSP.

The

spectrophotometric

absorption

of

Metabolic studies ‘251-BSP shows a delay in its plasma disappearance with t: = 2.1 _+0.003 min (mean * SD) (n = 3) compared to BSP with tf = 1.48 k 0.015 (n = 2). A typical bile excretion pattern is shown in Fig. 1. The highest biliary concentration of BSP is found lo-20 min after injection. ‘251-BSP reaches the peak of its excretion at 20-30 min. Bile excretion of ‘251-BSP when injected alone is shown in Table I. The ratio of ‘251-BSP in bile was determined in one study during the conjugated to unconjugated first 40 min and averaged 0.170. The peak was 0.2 and was reached during the 20-30 min period. Six minutes after i.v. administration, 68% f 7% (mean _+ SD) (n = 3) of injected ‘251-BSP was present in liver homogenate. Fifty percent (average of 2 experiments) TABLE

I

BILIARY

EXCRETION

OF ‘251-BSP AFTER

INJECTION

INTO

RATS

Time (mitt) O-10 Bile flow (pl.min -I .1OOg-’ bodyweight)

5.9*0.4

Bile excretion of ‘251-BSP (% dose)

3.5 * 1.3

Conj./unconj. ratio

0.06 1

IO-20

20-30

6.2kO.3

6.3 + 0.3

12.Ok3.2

13.6k 1.2

0.158

Four rats were studied and all results are expressed was 52.9% f 5.0% of that injected.

0.197

30-40

40-50

50-60

55

5.5 * 0.2

5.2 + 0.4

8.1 +0.8

5.7+0.6

0.188

0.142

*0.2

10.1 kO.5

0.190

as mean f SEM. Total excretion

of ‘251-BSP at

1h

325 15

I0

t

0 *

4P

5

0” 5

1.0 0 t 0”

2-

0.5

FRACTION NUMBER

Fig. 2. Binding of BSP and ‘251-BSP to serum proteins. 250 ng BSP and 0.5 pCi of ‘ZSI-BSP were mixed with 0.2 ml rat serum and applied on a Sephadex G-200 column. In each fraction, protein concentration was measured at OD 280 nm (Ol), BSP at OD 580 nm (0 0) and “s1-BSP radioactivity (O---- -4) was determined. Both ligands were predominantly associated with a peak in which albumin eluted

was present at 60 min and 30% (average of 2 experiments) at 30 min. The binding patterns of BSP and ‘*‘I-BSP to serum protein is shown in Fig. 2 and reveal that BSP and ‘251-BSP both bind primarily to albumin [ 11,121. In contrast, gel chromatography of cytosol (Fig. 3) reveals that ‘251-BSP is predominantly associated with only one cytosolic protein peak which precedes the Y-peak to which BSP is primarily associated [9]. This peak has previously been described as binding small amounts of BSP and bilirubin, and its protein composition has not been well characterized [9]. Gel chromatography of OABP revealed that BSP was avidly bound, but only approximately 6% of ‘251-BSP bound to OABP. 12

0.6

0.4 a6 *

3

I

2

Q 4

L 20

25

30

35

40

45

50

FRACTION NUMBER

Fig. 3. Binding of BSP and ‘251-BSP to liver cytosolic proteins. Three ml of liver cytosol were incubated with 750 ng of BSP and 1 pCi ‘251-BSP for 15 min at 37°C and the mixture was applied on a Sephadex G-75 column. Protein concentration was determined at OD 280 nm (a0). BSP at OD 580 0) and I25I-BSP concentration (o- - - - - -0) was determined by measuring the radioactivity. (0 -

326

Discussion Using the chloramine-T iodination method, we prepared ‘251-BSP of a high specific activity ( - 2000 mCi/nmol). Unlike BSP, this compound migrates on TLC using chloroform/acetone/methanol (4 : 2 : 1, v/v/v) as developer, suggesting that “‘1-BSP may be more lipophilic. Our studies revealed several biological differences between the two compounds. Plasma disappearance of ‘2SI-BSP is 50% slower than BSP and less than 20% of ‘251-BSP is excreted as the GSH-conjugate as compared to 80-90% of BSP [3]. Although these compounds have similar binding patterns to plasma proteins, there is little binding of ‘251-BSP to cytosolic Y- and Z-proteins [9]. In addition, ‘2fI-BSP does not bind to OABP, a liver cell plasma membrane protein, which binds BSP avidly [lo]. by the liver, it Thus, although “‘1-BSP is rapidly removed from the circulation may not be used as a tracer for BSP [ 131. However, because of its high specific activity and its localization nearly exclusively to the liver, this compound may be useful in studies of the cellular and subcellular metabolism of organic anions. There have been several reports in which iodinated BSP has been used as a liver scanning agent in man [ 13-151. Its relatively delayed biliary excretion as compared to that of other oxganic anions may limit its usefulness in studies of the patency of the biliary tree. Acknowledgement This work AM- 17702.

was supported

in part

by NIH

grants

AM-23026,

AM-02036

and

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