283

Clinica Chimica Acta, 93 (1979) @ Elsevier/North-Holland

283-294 Biomedical Press

CCA 10200

MECHANISMS OF INTERFERENCE OF NONESTERIFIED IN RADIOIMMUNOASSAYS OF STEROIDS

JUDITH

M. RASH,

IVANKA

JERKUNICA

and DEMETRIOS

Department of Pathology and Laboratory Medicine, Emory Atlanta, GA 30322 (U.S.A.) (Received

October

SGOUTAS

FATTY ACIDS

*

University, School of Medicine,

31st, 1978)

Summary Addition of nonesterified fatty acids caused an apparent increase in unbound steroid present in supernatants in radioimmunoassays for steroids utilizing dextran-coated charcoal. Nonesterified fatty acid interference occurred at the initial binding interaction between steroid and its antiserum, and also at the step separating bound from unbound steroid. It was determined that nonesterified fatty acids, which had been added to radioimmunoassays, formed micelles and trapped steroids. The extent of the entrapment was inversely related to the number of polar groups in the steroid molecule, that is, hydrophobic steroids more easily interacted with the nonesterified fatty acid micelles. However, if the charcoal concentration were increased, the nonesterified fatty acid effect was eliminated for assays of polar steroids and greatly reduced for non-polar steroid assays.

Introduction There are problems associated with steroid radioimmunoassays (RIAs), some of which are caused by the interference of plasma components such as proteins, heavy metals and lipids. Pratt et al. [l] studied the effect of plasma proteins on the specificity of steroid RIAs. In 1970, it was determined that heavy metals influence testosterone assays, yielding falsely increased values [ 21; the effect of plasma cholesterol was also examined in the study [ 21. Tillson et al. [ 31 investigated the effect of pH, ionic strength, and the composition of buffers upon the assays of testosterone, estradiol-170, and progesterone. Abraham [4] concluded that excess of lipids and proteins may cause negative blanks resulting in underestimations of steroid analytes in biological fluids. * TO whom correspondence should be addressed.

Other scattered but consistent reports [ 5,6] have provided data showing that plasma lipids produce spuriously low or high values read from typical standard curves. In light of the above, it was of interest to investigate more thoroughly the effect of lipids on steroid RIAs. Therefore, such interference for several assays which are routine procedures in our laboratory was examined. The effect of non-esterified fatty acids (NEFA) is dealt with in this paper. Materials and methods In all cases, solvents and reagents were of analytical grade quality. Double distilled deionized water was used, and all glassware was acid-washed and rinsed with water until free of acid. [1,2,6,7-3H]Cortisol (S.A. 100 Ci/mmol), [2,4,6,7-“Hlestradiol-170 (S.A. 100 Ci/mmol), [ 1,2,6,7-“H] testosterone (S.A. 91 Ci/mmol), [ 1,2,6,7-“HIprogesterone (S.A. 97 Ci/mmol), and [1,2,6,7-“Hldehydroepiandrosterone sulfate (DHEA-S; S.A. 85 Ci/mmol) were purchased from New England Nuclear Corporation (Boston, MA). The purity of these steroids was determined by thinlayer chromatography (TLC) using the solvent systems recommended by Stahl [7] and in all cases was better than 96% as judged by the absence of other radioactive spots on the chromatogram. The tritiated compounds were diluted with absolute ethanol to appropriate concentrations before use. Unlabeled steroids were purchased from Steraloids (Pawling, NY), and their purities were checked by melting point determinations and TLC [ 71. Bovine serum albumin (BSA) essentially free of fatty acids and bovine globulin were obtained from Sigma Chemical Co. (St. Louis, MO). Anti-cortisol-21-hemisuccinate-BSA was purchased from Endocrine Science (Tarzana, CA), anti-progesterone-lla-hemisuccinate-BSA and anti-17fl-estradiol-6-(0-carboxymethyl)oxime-BSA were from Miles Laboratories (Elkhart, IN), anti-dehydroepiandrosterone-3-hemisuccinate-BSA from Harbor General Hospital (Torrance, CA), and anti-testosterone-3-( 0-carboxymethyl)oxime-BSA from Wein Laboratories (Succasuna, NJ). All antisera were utilized without further purification. Fatty acids were purchased from Sigma Chemical Co. and Supelco, Inc. (Bellefonte, PA); they were more than 95% pure by gas-liquid chromatography. [ l-‘4C]Oleic acid was purchased from New England Nuclear, and its purity was 97% as judged by radiogas liquid chromatography. Procedures for RIAs were performed as previously described [S--12], except for modifications to optimize the assay under the applied conditions. Buffer solutions. A: Phosphate (0.01 M), pH 7.4, containing 1 g/l of BSA, 9 g/l NaCl, and 1 g/l sodium azide; B: Tris (0.05 M), pH 8.0, containing 1 g/l bovine globulin, and 1 g/l sodium azide. Buffer A was used for cortisol, DHEA-S, and testosterone assays. Buffer B was employed for estradiol and progesterone assays. Bound and unbound steroids were separated by adsorption onto active charcoal. In order to obtain reproducible results, the procedures of preparing and employing charcoal were followed precisely. Charcoal (Neutral Norit) from Fisher Scientific Co. (Atlanta, GA) was washed three times with distilled water and centrifuged each time at 700 Xg for 10 min in order to remove the fine particles. After drying in an oven at 50°C for 24 h, 100 parts of charcoal and

285

10 parts of Dextran T-70 (Pharmacia Fine Chemicals; Piscataway, NJ) were suspended in the buffer solutions at appropriate dilutions. Assessment of the effects of various fatty acids on the assays and other experiments was performed by introduction of the fatty acid along with the 3H-labeled steroid to the reaction mixture. In all instances, the volume of the control samples was adjusted with buffer to compensate for the difference in volume. Fatty acids (1 mmol) were dissolved in 5 ml of 25% ethanol and converted to the respective potassium salts by adding 1 mmol of KOH. The suspensions in buffers were prepared by vigorously shaking 200 mg of dry material in 5 ml water and then diluting to final strength with buffer. Binding parameters were determined from Scatchard plot analyses [13] as described earlier [14]. Aliquots of 3H-labeled steroid were incubated in the presence and absence of varying amounts of unlabeled hormone. Unbound steroid was removed with dextran-coated charcoal. The influence of NEFA on the binding was examined after controlled addition of NEFA into the assay tubes. The adsorption of free steroid by charcoal was investigated in a system containing 0.1 ml of isotope solution (0.1 pmol in buffer A) and 0.4 ml buffer A which was incubated for 10 min at 4°C. Dextran-coated charcoal solution (1 ml) was added, the mixture incubated for 20 min at 4°C and then centrifuged at 700 X g for 10 min. To test whether charcoal was able to adsorb NEFA, the dextran-coated charcoal used in an assay (0.5 ml of the following: 1.25 g charcoal and 100 ml of buffer B) was incubated with increasing concentrations of oleic acid containing 0.05 PCi of [ 1-14C] oleic acid. To test whether NEFA bind to antisera under our experimental conditions, progesterone antiserum was diluted in buffer B and incubated with varying amounts of cold oleic acid and a constant amount of [1-14C]oleic acid (0.05 &i) overnight at 4°C. One-ml aliquots of dextran-coated charcoal, prepared in buffer B were added to the samples. The tubes were shaken for 15 min at 4°C and left for 15 min before centrifugation at 700 X g for 15 min. Equilibrium dialysis experiments were carried out using Visking bags (Union Carbide Corp., Chicago, IL) containing 1 ml of a solution containing antiserum, labeled steroid, buffer B, and the interfering lipid. Bags were previously incubated for 16 h with [1-14C]oleic acid. Greater than 95% of the radioactivity was recovered, indicating that NEFA did not bind to the dialysis tubing and that the tubing was suitable for equilibrium dialysis experiments. Surgical gloves were worn throughout the handling of the dialysis tubing. Size 8 dialysis tubing was soaked in distilled water overnight to remove glycerol from the tubing. Each bag was placed in an Erlenmeyer flask containing 20 ml of assay buffer. Duplicate flasks were employed for each steroid concentration and all flasks were shaken in a water bath at 4°C. It was determined in a preliminary experiment that equilibrium was reached in 6 to 8 h, however, all equilibrium dialysis experiments were run for 16 h. The radioactivity of. the inside solution of each was then determined in triplicate samples (0.1 ml) to obtain the total steroid concentration within the bag. Six l-ml samples of the outside solution were counted to obtain the concentration of the unbound steroid outside the bag. Duplicate O.l-ml samples of the inside solutions before equilibrium dialysis

286

were counted to determine the recovery of radioactivity. Sampling error was less than 2%; the recovery of radioactivity was greater than 90%. Radioactivity was measured in a Beckman liquid scintillation counter (Fullerton, CA) by addition of aliquots to 10 ml of scintillation fluid (Aquasol; New England Nuclear) and counted with an external standard for correction of quenching. Counting was conducted at a level which maintained the error below 2%. Samples containing the radioisotopes 3H and 14C were acidified and extracted with diethyl ether; the solvent was evaporated, and the radioactive compounds dissolved in 10 ml of toluene containing 3 g/l of 2,5-diphenyloxazole and 0.1 g/l of 1,4-di-[ 2( 5-phenyloxazolyl)]-benzene. These samples were counted with split channel settings, the lower channel registering 14C counts only. Corrections were made for background and quenching. The discriminator ratio method was used for calculating the 3H/‘4C ratio in the same sample. Sufficient counts were accumulated so that the errors of the ratios were less than 6%. All calculations were performed with the aid of a UNIVAC series 70/7 computer. In order to study the entrapment of steroids with fatty acid micelles, minicolumns of Sephadex G-50 (Pharmacia Fine Chemicals) were made from the barrels of l-ml plastic syringes according to the method of Fry et al. [15]. Excess fluid was removed from the Sephadex beads by centrifuging at 1000 X g for 3 min. The mixture of micellarly entrapped 3H-steroid and free 3H-steroid was applied to the column bed. Centrifugation was repeated at 50 X g for 10 min followed by 1000 X g for 3 min, forcing the micellar material through the column into a test tube (glass, 12 X 75 mm) while the free steroid was quantitatively retained. Turbidity was measured in a l-cm cell at 300 nm, using a Gilford 240 spectrophotometer. Specific turbidity is defined as the turbidity per PM of fatty acid. The observed specific turbidity is constant when the vesicle size distribution is homogeneous; when it becomes variable as in micelle formation, the specific turbidity is non-linear. Results Fig. 1 shows the effect of oleic acid on the shape of standard curves in several assays. Oleic acid was selected as the representative fatty acid because it is the most common fatty acid in plasma lipids [16], and is neither saturated nor polyunsaturated. The addition of oleic acid distorted the “pure” doseresponse curves by causing an apparent decrease in the concentration of bound 3H-steroid. The distortion was a downward displacement resulting in decreased sensitivity (Table I); the extent of the distortion was a function of oleic acid concentration, and also depended on the steroid assay. That is, with a steroid concentration of 3.0 X lo-” M and an antiserum concentration of 1.2 X lo-” M, a 106-fold excess of oleic acid reduced maximum binding of the cortisol and DHEA-S assays by 5--lo%, of the testosterone assay by 15-20%, and of the progesterone assay by 50-60% (Fig. 1). To evaluate the role of the fatty acid structure, various fatty acids were tested for interference in the estradiol assay. At the tested concentration (0.5 pmol), the chain length of the fatty acid increased the effect progressively

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WYs at various wmcentrations of oleic acid. Percent bound/total counts B: DHEA-S; C: testosterone: D: progesterone. Oieic acid concentrations: .

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288

TABLE

I

ANTISERUM

SENSlTIVITY

IN VARIOUS Fatty

Assav

Cortisol * * DHEA-S ** Testosterone ** Progesterone ** Estradiol * * * Androstenedione

***

STEROID

ASSAYS

acid concentration

*

(pmol)

0.00

0.50

2.00

0.18 0.04 0.03 0.04 0.02 0.10

0.30 0.05 0.04 0.12 0.03 0.15

0.36 0.06 0.05 >0.20 0.06 0.22

* The detection limit is approximately equal to the mean blank result + 2 S.D. (ng of ligand). * * Calculated from a mean of 8 determinations. *** Calculated from a mean of 3 determinations.

from CB to the Cl2 member, which approached the effect of oleic acid, and then decreased through the Czz member (Fig. 2, panel A). On the other hand, unsaturated fatty acids were more effective than saturated fatty acids, and the effectiveness increased with a greater number of double bonds (Fig. 2, panel B). Binding parameters were measured by plotting data in the form of Scatchard plots. Non-linear curves were obtained for controls and samples containing fatty acids. A decrease in slope in the linear steep portion of the curve for mixtures containing fatty acidswas observed (Fig. 3). The linear portion of the curve represents high affinity, saturable binding, and on extrapolation is used to determine the equilibrium dissociation constant (&) for the steroid-antiserum complex, as well as the molar number of binding sites (n). Fig. 3 and Table II show that the presence of oleic acid caused an apparent increase in the & and an apparent decrease in antiserum binding sites for estradiol,(Fig. 3A) and progesterone (Fig. 3B) assays. The results in percent of [3H]estradiol-17fl unadsorbed against weight of charcoal are shown in Fig. 4. In the absence of oleic acid, all but 2% of [ 3H]estradiol-170 (at 3 X lo-” M) was precipitated at a charcoal concentration of 0.5 g/dl. With the addition of 2pmol oleic acid approximately 25% of the A

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y

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R

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60

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OF DOUBLE

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4

BONDS

Fig. 2. The effect of various fatty acids (0.5 pmol) on maximum binding in the estradiol assay (mean of 3 determinations). Panel A: Percent binding versus chain length of saturated fatty acid. Panel B: Percent binding versus degree of saturation of CI 8 or C!z 2 fatty acid.

289

BOUND

IO-Ismoles

BOUND

IO-‘5 moles

Fig. 3. Scatchard plots for estradiol (A) and progesterone (B) assays. 0.25 Clmol; e------o, 0.5 /.lmol; Ah. acid additions (o -o, means of 3 experiments.

without (0 --C) and with oleic 1.0 pmol). Calculated from the

steroid was recovered in the supernatant; however, by increasing the charcoal concentration less steroid remained in the supernatant. The effect was a function of the structure of the steroid. At a low concentration of charcoal (0.25%) more progesterone than cortisol was left in the supernatant; this effect, however, was abolished by increasing the amount of charcoal, as shown in Table III. With the use of [1-‘4C]oleic acid, it was determined that the oleic acid was actually adsorbed onto the charcoal (Table IV). Charcoal (0.25 g/dl) became saturated with 1 mM or greater concentration of oleic acid. To determine if increasing concentrations of estradiol compete with the binding of NEFA to dextran-coated charcoal, 1 mM of 1 mM of [l-14C]oleic acid (0.054Ji) was incubated with increasing concentrations of estradiol and the appropriate

TABLE

II

SCATCHARD ANALYSES NEFA ADDITION Figures presented Oleic acid added (I.tmol/tube)

0.0 0.3 0.4 0.5 1.0 2.0

OF ESTRADIOL

AND

PROGESTERONE

ASSAYS

are means of 3 determinations. Estradiol

Progesterone

Kd *

.**

Kd *

n **

2.0

90

13.2 13.9

135 100

27.8

82 25.0 35.7

85 75

75.0

58

* Equilibrium dissociation constant expressed ** Number of binding sites expressed as lo-l5

as lo-lo M.

M.

WITH

AND WITHOUT

290

c 0i CHARCOAL Fig,

4.

Percent

centrations, A-,

without 2.00

g/d1

non-specific

binding ) and

(*+

@molf.

Calculated

of with

from

[‘Hlestradiol aleic

acid

remaining additions

in supernatant

(Cm,

0.25

at various

pmol;

a------

charcoal e, 0.50

con~mol:

4 experiments.

amount of dextran-coated charcoal. Up to 2 mM e&radio1 did not give rise to any additional 14C activity displacement in the supernatant. To assess the interference of NEFA at the binding step, the binding of [ 3H] progesterone to its antiserum in the presence and absence of NEFA was examined by non~issociating methods. Two series of dialysis experiments were carried out. In the first series, the effect of various oleic acid concentrations on the rate of dialysis of progesterone in the presence of constant concentrations of antiserum and [3H]progesterone were measured. In Fig. 5, curve A shows the [3H]progesterone-antiserum interaction in the absence of oleic acid. Curves B, C, D, and E show [3H]progesterone binding to its antiserum at the same progesterone and ~tise~rn concentra~ons as for curve A, but with oleic acid added at the indicated concentrations. There were significant alterations (greater than ?2 S.D.) in the apparent (3H]progesterone binding after the addition of 0.5 mM or greater oleic acid concentration. The second series of experiments were similar to the first, but measurements were taken after equilibrium was reached (18 h). The [3H]progesterone fraction bound to antiserum in the presence of increasing concentrations of oleic acid is shown in Fig. 6. With increasing concentrations of oleic acid, less r3H]TABLE

III

PERCENT

3H-STEROID

DEXTRAN-COATED Figures

presented

Assay

UNBOUND

are means

of

Charcoal 0.25

Cortisol

IN

SUPERNATANTS

CHARCOAL 3 determinations. concentration 0.50

(g/dI) 1.00

12.7%

4.5%

2.2%

Estradiol

6.1%

3.5%

2.9%

Progesterone

5.6%

4.2%

3.6%

AT

VARIOUS

CONCENTRATIONS

OF

291 TABLE

IV

ADSORPTION

OF [1-14CIOLEIC

ACID ONTO

DEXTRAN-COATED

CHARCOAL

Results expressed as pmol/ml and represent the mean of 3 experiments [l-14C101eic

acid added

Charcoal concentration (g/dl)

(umoilml)

0.6 1.25 2.50 5.00

0.25

0.50

1.25

0.06 0.12 0.74 0.81

0.07 0.16 0.81 1.92

0.08 0.18 1.15 2.87

progesterone interacted with the antiserum and at 2 mM oleic acid no binding between [3H]progesterone and antiserum occurred. Results were identical in experiments repeated with different concentrations of [‘HI progesterone. To test whether or not [ l-14C] oleic acid was able to react with progesterone antiserum, increasing amounts of [l-14C]oleic acid at three different levels of antiserum concentration were incubated as described in Materials and Methods. Only insignificant amounts of [ 14C]oleic acid were found in the supernatant bound to the antiserum. We further investigated the interaction between oleic acid and [3H]progesterone as well as other steroids by employing Sephadex G-50 minicolumns as described in Materials and Methods. This procedure separates low molecular

60

0123456 TIME

(hrs)

mM

oleote

Fig. 5. Concentration (lo-12 mol) of [3Hlprogesterone bound within dialysis tubing over a 6-h period from 4 separate experiments. Individual bags contained antiserum and tracer without (A: 0--0) and with oleic acid addition (B: l-, 0.25 pmol; C: q0, 0.50 umol; D: A-, 1.0 pmol; E: n-, 2.0 umol). Fig. 6. Percent [3Hlprogesterone bound within dialysis tubing following equilibrium. as a function of oleic acid concentration (mM). The curves represent means of 3 individual experiments. l -n, maximum binding bags containing antiserum and tracer; o-----o , non-specific binding with tracer alone; A-. maximum binding minus non-specific binding.

OLEIC

ACID

immoies)

Fig. 7. The entrapment of steroids in fatty acid micelles, expressed as 9%interacted versus mM oleic acid (mean of 3 determinations). Sephadex G-50 minicolumns were utilized as described in Materials and estradiol; r----4, cortisol. A-. testosterone: n ----m. progesterone; Methods. o----3,

weight solutes such as steroids trapped in aggregates from nonentrapped solute. Salts of fatty acids are amphiphiles which form molecular solutions in water until a fatty acid concentration designated the “critical micellar concentration” (CMC) is reached. Above the CMC excess fatty acid aggregates in the form of micelles. The results of an experiment in which several amount of oleic acid were mixed with a constant amount of 3H-steroid (1 X lo-” mol) in buffer A are shown in Fig. 7. The preparations were first checked for turbidity as described in Materials and Methods. Specific turbidity depended on the solvent system; in distilled water specific turbidity was 0.1 mM, in either buffer it was 0.3 mM, in other media it can be 1 mM or higher [ 171. After having determined the presence of micelles in preparations of 0.3 mM or greater concentration of oleic acid, these mixtures were applied to Sephadex G-50 mini~olumns. A concentration of 1.5 mM oleic acid or greater effectively entrapped the 3H-steroids. When several steroids of increasing polarity were tested, it was found that less polar > compounds were more easily entrapped, that is, progesterone > testosterone estradiol > cortisol (Fig. 7). Discussion Addition of fatty acids decreased the sensitivity ofstandard curves obtained with several steroid- ass-ays, due to a reduction in the number of binding sites, and an increase in the equilibrium dissociation constant as compared to control curves. Simultaneous variations in these parameters are usually attributed to a range of factors including interference at the initial binding step of the assay and perturbation at the separation step. Interference at the binding step can occur in two ways: (1) at the interaction of NEFA with antiserum, and/or (2) at the interaction of NEFA with ligand. The former prevails in several hormone binders in blood, where interference of NEFA with ligand binding to these proteins has been recognized [ 18-223. Contrary to this, our results strongly suggest that only insignificant amounts of

293

oleic acid bind to antisera, and only in the monomer form of the NEFA. Above the CMC there is no increase in binding of NEFA to immunoglobulins, and there is no evidence in the literature of such an infraction. One may speculate that the binding sites of immunoglobulins, which are visualized as hydrophobic crevices on the surface of protein molecules, are too small to contain long-chain fatty acids. It would, therefore, be of interest to investigate the effects of shortchain fatty acids added in similar test situations. The most important finding of this study is that oleic acid interferes with the binding between steroid and its antiserum by interacting with the steroid. This is shown in Fig. 6 as an apparent increase in nonspecific binding of steroid. Elucidation of the mechanism of the NEFA-s~roid interaction was obtained from the experiments with Sephadex minicolumns (Fig. 7). NEFA-steroid interactions were pronounced at oleic acid concentrations of 2 mM or higher, suggesting that NEFA micelles had formed and effectively entrapped free steroids. At the separation step, NEFA inhibited steroid binding to charcoal, which resulted in an apparent increase in unbound 3H-steroids in the supematants. This phenomenon accounts for the negative blanks reported by Abraham [4] and for similar interference of bile acids and commercial detergents reported with the RfAs of B-12, gas&in, and cyclic AMP [5,6]. However, adequate amounts of charcoal corrected the NEFA effect by increasing the number of effective charcoal binding sites to adsorb both NEFA and unbound steroid. Furthermore, adequate amounts of charcoal were effective in breaking down the micelles of NEFA with entrapped steroid, and NEFA and steroid were then individually adsorbed by the charcoal. These findings are of practical importance in the laboratory which determines steroid hormones by RIA. Extraction of plasma or serum by organic solvents yields fractions which are rich in lipids including steroid hormones. The majority of the latter are present in concentrations of 10W9g/ml plasma, while plasma lipids occur in concentrations of 5-10 X 10e3 g/ml. The range of concentrations of NEFA in serum is 0.5-3.5 X 10V3 g/ml. Although the fatty acid concentrations examined in this report were high in relation to the concentrations of antigen and antiserum, in several situations such as starvation and postprandial heparin injections, NEFA as high as 5 mM have been reported [ 23 J. Thus, the amount of NEFA in aliquots of serum (0.1-1.0 ml) employed for extractions in many RIA steroid procedures would approach the levels of NEFA employed in the present study. In that case, and according to our results, NEFA may interfere significantly in non-polar steroid RIA determinations. A full evaluation of the effect of NEFA and other plasma lipids in groups of patients receiving heparin after a fat meal is now in progress. References 1 Pratt, J.J., Koops. W., Woldring. M.G. and Wiegman, T. (1976) Eur. J. Nut. Med. 1.3748 2 Dessypris, A.G. 11970) J. Steroid Biochem. 1.185-193 3 Tillson, S.A., Thorneycroft, I.H., Abraham, G.E.. Searamuzzi, R.J. and Caldwell, B.V. (19’70) in Immunologic Methods in Steroid Determination (Peron. F.G. and Caldweli, B.V.. eds.). PP. 127-147, Appleton-Century-Crofts, New York 4 Abraham. G.E. (1975) J. Steroid Biochem. 6,261-270

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