Bile Acid and Detergent Interaction with

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in plasma calcium with induced changes in plasma specific gravity, total protein, and ... fractions, plasma expanders, or other materials, charcoal will continue to ...
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R. B., Little, A. J., Williams, R. B., and Milner, J. R., In-

terpretation of serum calcium in patients teins. Br. Med. J. iv, 643 (1973).

with abnormal

serum pro-

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CLIN.CHEM.22/10, 1727-1728(1976)

Bile Acid and Detergent Interaction with Radloassays Based on Coated Charcoal Knut-Jan Andersen, Henning Schjgnsby, Dankert W. Skagen, and Gerhard von der Lippe

We tested the radioassays for cyclic AMP and human gastrin, both involving separation on coated charcoal, for interaction with bile acids and detergents, and found a concentration-dependent interaction of taurocholic and glycocholic acid as well as of the surfactant Triton X- 100 and sodium laurylsulfate in both assays. The interaction was detectable from concentrations of 0.5 mmol of bile

acid per liter or 625 mg of detergent per liter, giving rise to falsely decreased gastrin values and falsely increased or decreased values for cyclic AMP. The interactions demonstrated may be a general effect on all radioassays that are based on the use of coated charcoal in the separation of free from bound ligands.

Materials and Methods Triton X-100 (octyl phenoxypolyethoxyethanol), sodium lauryl sulfate, sodium taurocholate, and sodium glycocholate were obtained from Sigma Chemical Co., St. Louis, Mo. 63178; charcoal “carbo medicinalis” was obtained from Norit N.y., Amsterdam,

Holland.

Synthetic human gastrin-I was purchased from Imperial Chemical Industries Ltd. (ICI), Cheshire, England, and iodinated with 1m1 by a modification of the Chloramine-T method of Hunter and Greenwood (7) (spec. acty. 530 pCi of 1I per pg of gastrin). Rabbit-antiserum to albumin-synthetic human gastrin-I was a generous gift from Stadil and Rehfeld, Copenhagen. Characteristics of this antiserum (No. 2604) have been described by Rehfeld et al. (8).

Among the several adsorbents that have been used to separate free from protein-bound hormone, charcoal seems to have the widest application (1). It is well known for its adsorptive properties for many materials, and has many uses that exploits them (2). When coated with plasma, blood fractions, plasma expanders, or other materials, charcoal will continue to adsorb small molecules, but will reject molecules equal to or greater in size than those of the coating (3), giving rise to the “instant dialysis” concept where the charcoal particle acts as a “solid microsponge” and the coating as a “mo-

Our radioimmunoassay of gastrin, based upon competitive binding of ‘I-labeled synthetic human gastrin-I to antiserum followed by determination of free 125I-labeled synthetic human gastrin-I bound to dextran-covered charcoal, was according to the procedure described by Schrumpf and Sand (9),

lecular sieve” surrounding

lml.

the sponge (4).

Recently, Rimbaut et al. (5) reported a false-positive alpha-fetoprotein assay, caused by the presence of bile. We have previously shown (6) that bile acids and detergents interfere with the radioassay for vitamin B12 binders (7). It is therefore tempting to assume that bile acids and detergents may interfere with other radioassays based on the principle of “instant dialysis,” with use of coated charcoal. The radioimmunoassay for gastrin as well as the radioassay for cyclic AMP, both of which are routine methods in our laboratory, were therefore examined for such interference. Section for Clinical Research,Medical Department A, University of Bergen, 5016 Haukeland Sykehus, Bergen, Norway. Received May 17, 1976; accepted July 9, 1976.

the reproducibility of which is discussed elsewhere (10). Samples, 100 is1, containing 10 pg of gastrin were included in all the sample tubes except for those used for the standard curve, and increasing concentrations of bile acids or detergents were added before antibody. The final incubation volume was Cyclic AMP

was determined

by the protein-binding

assay

described by Tovey et al. (11), with use of The Radiochemical Centre (Amersham, U.K.) Cyclic AMP Kit. The method is based on competitive binding of tritiated cyclic AMP to a binding protein, followed by removal of free nucleotides on albumin-coated charcoal, and is valid for the concentration range 0-16 pmol of cyclic AMP, with maximum precision within the range 0.5-8 pmol. The methods recommended for use with the kit were carefully followed except that to prepare for counting we adjusted the final 200-pl sample to 1 ml with water before adding 10 ml of”Unisolve 1” (Koch-Light Laboratories Ltd., Colnbrook, Bucks, U.K.). Fifty-microliter samples containing 8 pmol of cyclic AMP and increasing concentrations of bile acids or detergents were assayed in

CLINICALCHEMISTRY. Vol. 22. No. 10, 1976 1727

0.75 ml Triton X-100, 625 mg of sodium lauryl sulfate, and 0.5 mmol glycocholic or taurocholic acid. The assay was completely distorted, the curve being reversed in direction from the standard curve (Figure 1), at concentrations of Triton X-100 or sodium lauryl sulfate exceeding 1.5 ml (g)/liter, or at concentrations of taurocholate or glycocholate exceeding 1 mmol/liter. These results are not surprising, because detergents and bile salts bind firmly to coated charcoal, tion/assay):

20400 20700 19900 19400 19000 9600 18700

17100

thereby

17400

blocking

Fig. 1. Standard curve for gastrin radioimmunoassay Values are the arithmetic mean of two determinations, each run in triplicate

the “instant

less free

10

decreased cyclic AMP

6

P

9

10

OI

12 1!J!

16 11, AMP

gastrin

function

(6), and

is bound

concentrations.

The increased values

for cyclic AMP observed at low detergent concentrations may be due to a combination of effects. Possibly the detergents and bile acids also inhibit the binding of cyclic AMP to the binding protein, and consequently less tritiated cyclic AMP will be detected in the supernatant fluid (increased Co/Cs).

E5

4

‘251-labeled

Possibly the detergents were blocking the binding between albumin-coated charcoal and free cyclic AMP (6), giving rise to increased radioactivity in the supernate (increased C1), and

A

2

dialysis”

to charcoal. Cyclic AMP assay. At concentrations greater than 10 ml/liter for the detergent or 15 mmol/liter for the bile salt, falsely decreased cyclic AMP concentrations (decreasedC0/C1 ratio) were observed, while at a concentration of 5 ml of detergent or 5 mmol of bile salt per liter, falsely increased values for cyclic AMP (increased C0/C5 ratio) were found (Figure 2). consequently

18

References B

C 12

8

8 I

E!,p.5t.d

flLAS

,.AMP

p

#{163}p,.cIod vct40

8po

c-AMP

8 E

1

0.5 .0 5 2 D.t.rge.l co-c,c1m1On

0

2.5 I%i

25 Ole

Cc! dconcpnlrat!or

50

75 mI-Il

Fig. 2. Results obtained with the Cyclic AMP Kit Top to bottom: Standardcurve (A), and the effect of including detergents (B) X-100 (x), sodium lauryl sulfate(#{149}); Or bileacids (C)-glycocholic acid (x), taurocholic acid (#{149}). in the assayof 8 pmol of cyclic AMP.Valuesare the arithmetic mean of two determinations, each run in triplicate -Triton

addition

to the standard

1. Yalow, R. S., and Berson, S. A., Introduction and generalconsiderations. In Principles of Competitive Protein-Binding Assays, W. D. Odell and W. H. Daughaday, Eds. J. B. Lippincott Co., Philadelphia, Pa., 1971, pp 1-21. 2. Hassler, J. W., ActivatedCarbon,rev. ed. Leonard Hill, London, 1967, p 278. 3. Herbert, V., Coated charcoal separation of free labelled hormone from hormone bound to antibody. In Protein and Peptide Hormones, Part 1, M. Margoulies, Ed. Excerpts Medica Foundation, Amsterdam, 1969, pp 55-60. 4. Gottlieb, C. W., Lau, K.-S., Wasserman, L. R., and Herbert, V., Rapid charcoal assay for intrinsic factor (IF), gastric juice unsaturated B,2 binding capasity, antibody to IF, and serum unsaturated B12 binding capasity. Blood 25,875 (1965). 5. Rim baut, C., Rudant, C., and Buffe, D., Biliary factor and falsepositive alpha-fetoprotein assays. Lancet ii, 934(1975).

6. Andersen, K.-J., von der Lippe, G., and Schjnsby, H., Bile and detergent interaction with the radio-assay for vitamin B12binders, using protein and dextran covered charcoal. Anal. Biochern., in press (1976).

curve. The radioactivity (cpm) of cyclic I3HJAMP bound in the absence (C0) and in the presence

7. Hunter, W. M., and Greenwood, F. C., Preparation of iodine-131

(C1) of standard or unknown unlabeled cyclic AMP was measured and the ratio C0IC, for each concentration of cyclic AMP and the unknowns were calculated. Radioactivity was determined in an automatic beta-gamma spectrometer (Nuclear Enterprise, Model 8312).

495 (1962).

Results and Discussion Detergents and bile acids interfered with both the assay for gastrin and cyclic AMP, and the effects were concentration dependent. Gastrin assay. Falsely low gastrin values were observed

from the following concentrations

(final per-liter concentra-

1728 CLINICALCHEMISTRY,Vol. 22. No. 10. 1976

labelled human growth hormone of high specific

activity.

Nature

194,

8. Rehfeld, J. F., Stadil, F., and Rubin, B., Production and evaluation of antibodies for the radioimmunoassay of gastrin. Scand. J. Clin. Lab. Invest. 30, 221 (1972).

9.

Schrumpf, E., and Sand, T., Radioimmunoassay of gastrin with activated charcoal. Scand. J. Gastroenterol. 7,683 (1972). 10. Schjnsby, H., and Willassen, Y., Renal extraction ofendogenous gastrin in patients with normal renal function. Scand. J. Gastroenterol., in press (1976). 11. Tovey, K. C., Oldham, K. G., and Wheland, J. A. M., A simple direct assay for cyclic AMP in plasma and other biological samples using an improved competitive protein binding technique. Clin. Chim. Acta 56,221(1974).