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Free thyroxine results are affected by albumin concentration and nonthyroidal illness
Clinica Chin&a Acta, 130 (1983) 391-396 Elsevier
391
CCA 2529
Brief technical
note
Free thyroxine results are affected by albumin concentration and nonthyroidal illness Monika F. Bayer Division of Nuclear Medicine, Department
(Received
November
of Radiology. Stanford University School of Medicine, Stanford, CA 94305 (USA) 15th. 1982; revision February
4th, 1983)
Direct measurement of serum free thyroxine (FT,) by the AmerlexTM radioimmunoassay (RIA) is based on a iz51-T, derivative as tracer (undisclosed composition) which, according to the manufacturer, competes with serum FTd for binding sites on a high affinity anti-T4 antibody but does not bind to any significant extent to various serum proteins [I]. In-as-much as the tracer is designed not to interact with thyroxine-binding globulin (TBG), thyroxine-binding prealbumin (TBPA) and albumin, it is added directly to sera, resulting in a simple, one-step RIA procedure for FT,. However, the Amerlex RIA has been reported to produce a relatively large percentage of subnormal FT, values in clinically euthyroid patients with severe systemic nonthyroidal illnesses (NTI) [2,3] or in patients on heparin [4,5], and conversely, abnormally high values in euthyroid patients with dysalbuminic hyperthyroxinemia [2,6]. Although the actual plasma FT, concentrations in patients with NT1 are uncertain at this time, theoretical predictions and clinical findings [7,8] or FTg deter~nations by equilib~um dialysis and other commercial RIAs (9,101 tend to support FT, concentrations which are in the normal range. Stockigt et al [a] ascribed the presumed falsely low FT, results in NT1 and the falsely high values in dysalbuminemia to interactions between the Amerlex tracer and serum TBPA. This report provides data which suggest that the Amerlex FT, results may depend on albumin concentrations and on serum constituents which bind to albumin. Patients and methods
Patients Group I: 25 (15 female and 10 male) years, were selected by criteria described history, judged by retrospective review thyroid disorders, and had normal levels ~9-8981/83/$03.~
euthyroid severely ill patients, age 3 l-6 1 before [9]. Briefly, these patients had no of the charts, nor signs or symptoms of of TSH.
@ 1983 Elsevier Science Publishers
B.V.
392
Group 2:
five patients
Control group:
similar
with confirmed
20 apparently
age distribution
healthy as group 1.
hypothyroidism. individuals
(10 male and
10 female),
of
Methods
FT, was measured, unless stated otherwise, by the Amerlex FT, RIA according to the manufacturer’s protocol (Amersham Corp., Arlington Heights, IL 60005, USA). The assay is a competitive binding assay in which the standards and unknowns are incubated with an anti-T, antibody, immobilized on polymer particles, and a T,-‘251 derivative as tracer which competes with FT, for a limited number of antibody binding sites, but is said not to interact with serum proteins. Intra- and inter-assay coefficients of variation by this assay ranged from 4.7-12.1s for serum pools with values covering the hypo- to hyperthyroid range. The GammaCoat two-step FT, RIA (Clinical Assays, Cambridge, MA 02139, USA) was performed as described previously [3,9]. The dose response curves for human (HSA) and bovine serum albumin (BSA) (Fig. 2) were obtained by adding increasing amounts of crystalline HSA or BSA to T,-free serum and analysing 100 ~1 of the resulting solutions by the Amerlex FT, RIA. The BSA and HSA used did not contain any detectable FT,, or total T4, as measured by the GammaCoat FT, RIA, or a sensitive RIA for total T4. In the third experiment (Fig. 3) crystalline BSA (in increments of 25 or 50 mg/ml serum) was dissolved, immediately before the assay, in (1) ten sera from patients with NT1 and subnormal FT, by the Amerlex RIA, and (2) in ten sera from the control group. 100 ~1 of each serum (without and with added BSA) was then subjected to the Amerlex assay. Serum albumin was measured by a SMAC system (Technicon Instruments Co., Tarrytown, NY 10591, USA, Technicon method No. SG4-0030J81, Revised Sept. 1981) involving bromcresol green binding at room temperature. Total albumin concentrations were calculated from the serum albumin concentration plus the amount of BSA in the tracer solution and the BSA added. Results and discussion As described previously by Stockigt and us [2,3], approximately 40% of euthyroid patients with NT1 have FT, values in the hypothyroid range when measured by the Amerlex RIA. In contrast, FT, values obtained by the GammaCoat two-step RIA in these patients are predominantly in the euthyroid, normal range (Fig. 1) [9]. Various concentrations of human (HSA) or bovine serum albumin (BSA) in T,-free serum produced apparent FT, results directly proportional to the albumin concentration in the Amerlex assay (Fig. 2). A difference of 10% in B/B0 was observed between albumin concentrations of 30 and 55 g/l (corresponding to borderline low and borderline high serum albumin concentrations), compared to a difference of 18% for B/B, over the entire normal range for FT,. Since binding of ‘Z51-labeled albumin to the anti-T, antibody was found to be minimal, the effect of albumin appears to be caused by binding of the Amerlex tracer to albumin.
393 GAMMACOAT
2-STEP
AMERLEX
18
10 8
I
I c
2
i
c
I
5
Fig. 1. Distribution of FT, results by the GammaCoat two-step ET RIA, Clinical Assays, and by Amerlex FT, RIA, Amersham Corp., in healthy individuals (control group, C), in euthyroid severely ill patients (group l), and hypothyroid patients (group 2).
HSA (or BSAltglL) 50
25 9
100
I
200
8
I’
NORMAL
~4.3
x MEAN
NORMAL
HSA
15
I I I
95 -
20 I I
10 0.59 x MEAN NORMAL 5: 04
FT4d 1 NORMAL ITI
I
I
1.7
4.8
9.8 FT4
$ t
i
‘
20
44
89
WL)
Effect of human (HSA, 0 Fig. 2. Typical standard curve for the Amerlex FT., assay (0 -0). 0) on the binding of the Amerlex ‘2sI-Tq analog by the anti-T, or bovine serum albumin (BSA, A -A) antibody: increasing concentrations of HSA or BSA were added to T,-free serum and analysed by the AmerIex ET4 RIA. A 50% inhibition of T4 analog binding was observed at 0.59 times the mean normal FT., concentration and, for comparison, at 4.3 times the mean normal HSA concentration.
394
The Amerlex tracer solution itself contains a relatively large amount of BSA. Approximately 4.5 mg BSA are added to each 100 ~1 of serum in the course of the assay, so that the assay is performed in the presence of an albumin concentration twice that of the original serum sample. The third experiment was therefore designed to determine how exogenous BSA might affect individual serum FT, values, specifically those of euthyroid patients with NT1 who had unexplained low FT, results by the Amerlex RIA. As illustrated in Fig. 3, addition of BSA shifted all FT, values to higher levels (in agreement with the data in Fig. 2); however, this FT, increase varied. For instance, when 5 mg of BSA were added, immediately before the FT, assay, to 100 ~1 of serum from various patients and controls, the mean FT, increase (AFT, f SEM) was 8.0 f 0.8 rig/l in ten healthy individuals as opposed to only 4.2 f 0.3 rig/l (significantly different at p < 0.001) in ten patients with NTI. The differences in the FT, increases upon addition of BSA may be due to individual variations of serum constituents which bind to BSA and compete with the r25I-T, analog for binding sites.
28 26 24 22 20 18 2
16-
‘D = *
14-
t
1210 -
a642n
“25
45
50
75
100
125
150
ALBUMIN (g/L)
Fig. 3. Differential effect of exogenous BSA present in the Amerlex tracer solution on FT., results in individual sera. (Closed symbols), sera of six healthy individuals with normal FT, levels were measured (a) according to the manufacturer’s protocol which includes 45 g BSA per 1 serum (0); (b) in the presence of 70 g BSA per 1 serum (A); (c) in presence of 95 g BSA per 1 serum (m). (Open symbols), sera of six euthyroid severely ill patients with subnormal FT, by the Amerlex RIA: (a) according to the manufacturer’s protocol (0); (b) in the presence of 70 g BSA per 1 serum (A);(c)in the presence of 95 g BSA per 1 serum (0).
395
Serum albumin concentrations (x f SD = 24.2 + 5.7 1 g/l) were significantly lower ( p < 0.001) in the severely ill patients with low FT, than those in the control group (44.5 k 4.58 g/l) and 7 of 10 patients with NT1 and subnormal FT, had borderline or subnormal serum albumin concentrations. If, on the other hand, FT, values were extrapolated to a mean normal HSA concentration of 45 g/l (and without any additional BSA), they were within a relatively narrow range and no longer showed vast differences between healthy individuals and euthyroid patients with NT1 (Fig. 3). The fact that all extrapolated FT, values are lower is of no clinical significance since the units for FT, are arbitrarily defined in the kit and refer to FT, standards measured also in the presence of exogenous BSA. These observations suggest that the subnormal FT, results obtained by the Amerlex assay in patients with NT1 could be an artefact of the methodology, i.e., due to (1) generally lower serum albumin concentrations in these patients, and (2) abnormal concentrations of anions with high affinity for albumin. Conceivably, certain drugs, steroids, fatty acids, urate, bilirubin and other metabolites which compete with the Amerlex tracer for binding sites on BSA could cause low FT, values. For instance, in vitro addition of non-esterified fatty acids to sera (> 2.0 mmol/l) decreases Amerlex FT, values significantly (unpublished observation) and could also explain the sometimes abnormally low FT, values by this method in heparinized patients [4,5]. Heparin causes a surge in the concentrations of nonesterified fatty acids which, in turn, may displace the labeled analog from albumin [ 111. Conversely, abnormally high concentrations of albumin binding sites in hyperdysalbuminemia, or very low levels of various ligands binding to albumin, may contribute to falsely high FT, results [2]. In conclusion, the concept of the Amerlex FT, measurement is elegant, but the T4 tracer may interact appreciably with the plasma proteins. This interaction may be diminished in NTI, perhaps due to interfering drugs, or other substances, leading to spuriously low results for FT,. Physicians should be made aware that the Amerlex FT, RIA does not differentiate well between euthyroid and hypothyroid sick patients, and erroneous results could be generated when this assay is applied to evaluation of thyroid status in a hospital setting involving patients with severe systemic illnesses. References 1 Midgley JEM, Wilkins TA. A defense of the Amerlex free thyroxine kit. Clin Chem 1982; 28: 2183-2184, (letter). 2 Stock@ JR, deGaris M, Csicsmann J, Barlow JW, White EL, Hurley DM. Limitations of a new free thyroxine assay (Amerlex free T4). Clin Endocrinol 198 1; 15 : 3 13-3 18. 3 Bayer MF, McDougall IR. The value of free thyroxine by solid phase radioimmunoassay in hospitalized patients. In: Albertini A, ed. Neuropeptides and free hormones assay. Amsterdam: Elsevier Biomedical Press, 1982: in press. 4 McDougall IR, Bayer MF, Nierenberg D, Lewis SJ. Disparate effects of heparin on free thyroxine as measured by two radioimmunoassays. J Nucl Med 1982; 23: 507-510. 5 Boss M, Kingstone D, Chan MK, Varghese Z. Contradictory findings in the measurement of free thyroxine after administration of heparin. Clin Chem 1982; 28: 1238-1239. 6 Stockigt JR. Influence of altered plasma binding on free and total thyroid hormone levels. In:
7 8 9 10 11
Albertini A, ed. Neuropeptides and free hormones assay. Amsterdam: Elsevier Biomedical Press, 1982: in press. Robbins J, Rall JE. Proteins associated with thyroid hormones. Physiol Rev 1960; 40: 415. Kaptein EM, Grieb DA, Spencer CA, Wheeler WS, Nicoloff JT. Thyroxine metabolism in the low thyroxine state of ctitical nonthyroidal illnesses. J Clin Endocrinol Metab 1981; 53: 764-771. Bayer MF, McDougall IR. Free thyroxine by solid phase radioimmunoassay: improvement in the laboratory diagnosis of thyroid status in severely ill patients. Clin Chim Acta 1982; 118: 209-218. Kaptein EM, Macintyre SS, Weiner JM, Spencer CA, Nicoloff JT. Free thyroxine estimates in nonthyroidal illness: comparison of eight methods. J Clin Endocrinol Metab 1981; 52: 1073-1077. Spector AA. Fatty acid binding to albumin. J Lipid Res 1975; 16: 165-179.
391
CCA 2529
Brief technical
note
Free thyroxine results are affected by albumin concentration and nonthyroidal illness Monika F. Bayer Division of Nuclear Medicine, Department
(Received
November
of Radiology. Stanford University School of Medicine, Stanford, CA 94305 (USA) 15th. 1982; revision February
4th, 1983)
Direct measurement of serum free thyroxine (FT,) by the AmerlexTM radioimmunoassay (RIA) is based on a iz51-T, derivative as tracer (undisclosed composition) which, according to the manufacturer, competes with serum FTd for binding sites on a high affinity anti-T4 antibody but does not bind to any significant extent to various serum proteins [I]. In-as-much as the tracer is designed not to interact with thyroxine-binding globulin (TBG), thyroxine-binding prealbumin (TBPA) and albumin, it is added directly to sera, resulting in a simple, one-step RIA procedure for FT,. However, the Amerlex RIA has been reported to produce a relatively large percentage of subnormal FT, values in clinically euthyroid patients with severe systemic nonthyroidal illnesses (NTI) [2,3] or in patients on heparin [4,5], and conversely, abnormally high values in euthyroid patients with dysalbuminic hyperthyroxinemia [2,6]. Although the actual plasma FT, concentrations in patients with NT1 are uncertain at this time, theoretical predictions and clinical findings [7,8] or FTg deter~nations by equilib~um dialysis and other commercial RIAs (9,101 tend to support FT, concentrations which are in the normal range. Stockigt et al [a] ascribed the presumed falsely low FT, results in NT1 and the falsely high values in dysalbuminemia to interactions between the Amerlex tracer and serum TBPA. This report provides data which suggest that the Amerlex FT, results may depend on albumin concentrations and on serum constituents which bind to albumin. Patients and methods
Patients Group I: 25 (15 female and 10 male) years, were selected by criteria described history, judged by retrospective review thyroid disorders, and had normal levels ~9-8981/83/$03.~
euthyroid severely ill patients, age 3 l-6 1 before [9]. Briefly, these patients had no of the charts, nor signs or symptoms of of TSH.
@ 1983 Elsevier Science Publishers
B.V.
392
Group 2:
five patients
Control group:
similar
with confirmed
20 apparently
age distribution
healthy as group 1.
hypothyroidism. individuals
(10 male and
10 female),
of
Methods
FT, was measured, unless stated otherwise, by the Amerlex FT, RIA according to the manufacturer’s protocol (Amersham Corp., Arlington Heights, IL 60005, USA). The assay is a competitive binding assay in which the standards and unknowns are incubated with an anti-T, antibody, immobilized on polymer particles, and a T,-‘251 derivative as tracer which competes with FT, for a limited number of antibody binding sites, but is said not to interact with serum proteins. Intra- and inter-assay coefficients of variation by this assay ranged from 4.7-12.1s for serum pools with values covering the hypo- to hyperthyroid range. The GammaCoat two-step FT, RIA (Clinical Assays, Cambridge, MA 02139, USA) was performed as described previously [3,9]. The dose response curves for human (HSA) and bovine serum albumin (BSA) (Fig. 2) were obtained by adding increasing amounts of crystalline HSA or BSA to T,-free serum and analysing 100 ~1 of the resulting solutions by the Amerlex FT, RIA. The BSA and HSA used did not contain any detectable FT,, or total T4, as measured by the GammaCoat FT, RIA, or a sensitive RIA for total T4. In the third experiment (Fig. 3) crystalline BSA (in increments of 25 or 50 mg/ml serum) was dissolved, immediately before the assay, in (1) ten sera from patients with NT1 and subnormal FT, by the Amerlex RIA, and (2) in ten sera from the control group. 100 ~1 of each serum (without and with added BSA) was then subjected to the Amerlex assay. Serum albumin was measured by a SMAC system (Technicon Instruments Co., Tarrytown, NY 10591, USA, Technicon method No. SG4-0030J81, Revised Sept. 1981) involving bromcresol green binding at room temperature. Total albumin concentrations were calculated from the serum albumin concentration plus the amount of BSA in the tracer solution and the BSA added. Results and discussion As described previously by Stockigt and us [2,3], approximately 40% of euthyroid patients with NT1 have FT, values in the hypothyroid range when measured by the Amerlex RIA. In contrast, FT, values obtained by the GammaCoat two-step RIA in these patients are predominantly in the euthyroid, normal range (Fig. 1) [9]. Various concentrations of human (HSA) or bovine serum albumin (BSA) in T,-free serum produced apparent FT, results directly proportional to the albumin concentration in the Amerlex assay (Fig. 2). A difference of 10% in B/B0 was observed between albumin concentrations of 30 and 55 g/l (corresponding to borderline low and borderline high serum albumin concentrations), compared to a difference of 18% for B/B, over the entire normal range for FT,. Since binding of ‘Z51-labeled albumin to the anti-T, antibody was found to be minimal, the effect of albumin appears to be caused by binding of the Amerlex tracer to albumin.
393 GAMMACOAT
2-STEP
AMERLEX
18
10 8
I
I c
2
i
c
I
5
Fig. 1. Distribution of FT, results by the GammaCoat two-step ET RIA, Clinical Assays, and by Amerlex FT, RIA, Amersham Corp., in healthy individuals (control group, C), in euthyroid severely ill patients (group l), and hypothyroid patients (group 2).
HSA (or BSAltglL) 50
25 9
100
I
200
8
I’
NORMAL
~4.3
x MEAN
NORMAL
HSA
15
I I I
95 -
20 I I
10 0.59 x MEAN NORMAL 5: 04
FT4d 1 NORMAL ITI
I
I
1.7
4.8
9.8 FT4
$ t
i
‘
20
44
89
WL)
Effect of human (HSA, 0 Fig. 2. Typical standard curve for the Amerlex FT., assay (0 -0). 0) on the binding of the Amerlex ‘2sI-Tq analog by the anti-T, or bovine serum albumin (BSA, A -A) antibody: increasing concentrations of HSA or BSA were added to T,-free serum and analysed by the AmerIex ET4 RIA. A 50% inhibition of T4 analog binding was observed at 0.59 times the mean normal FT., concentration and, for comparison, at 4.3 times the mean normal HSA concentration.
394
The Amerlex tracer solution itself contains a relatively large amount of BSA. Approximately 4.5 mg BSA are added to each 100 ~1 of serum in the course of the assay, so that the assay is performed in the presence of an albumin concentration twice that of the original serum sample. The third experiment was therefore designed to determine how exogenous BSA might affect individual serum FT, values, specifically those of euthyroid patients with NT1 who had unexplained low FT, results by the Amerlex RIA. As illustrated in Fig. 3, addition of BSA shifted all FT, values to higher levels (in agreement with the data in Fig. 2); however, this FT, increase varied. For instance, when 5 mg of BSA were added, immediately before the FT, assay, to 100 ~1 of serum from various patients and controls, the mean FT, increase (AFT, f SEM) was 8.0 f 0.8 rig/l in ten healthy individuals as opposed to only 4.2 f 0.3 rig/l (significantly different at p < 0.001) in ten patients with NTI. The differences in the FT, increases upon addition of BSA may be due to individual variations of serum constituents which bind to BSA and compete with the r25I-T, analog for binding sites.
28 26 24 22 20 18 2
16-
‘D = *
14-
t
1210 -
a642n
“25
45
50
75
100
125
150
ALBUMIN (g/L)
Fig. 3. Differential effect of exogenous BSA present in the Amerlex tracer solution on FT., results in individual sera. (Closed symbols), sera of six healthy individuals with normal FT, levels were measured (a) according to the manufacturer’s protocol which includes 45 g BSA per 1 serum (0); (b) in the presence of 70 g BSA per 1 serum (A); (c) in presence of 95 g BSA per 1 serum (m). (Open symbols), sera of six euthyroid severely ill patients with subnormal FT, by the Amerlex RIA: (a) according to the manufacturer’s protocol (0); (b) in the presence of 70 g BSA per 1 serum (A);(c)in the presence of 95 g BSA per 1 serum (0).
395
Serum albumin concentrations (x f SD = 24.2 + 5.7 1 g/l) were significantly lower ( p < 0.001) in the severely ill patients with low FT, than those in the control group (44.5 k 4.58 g/l) and 7 of 10 patients with NT1 and subnormal FT, had borderline or subnormal serum albumin concentrations. If, on the other hand, FT, values were extrapolated to a mean normal HSA concentration of 45 g/l (and without any additional BSA), they were within a relatively narrow range and no longer showed vast differences between healthy individuals and euthyroid patients with NT1 (Fig. 3). The fact that all extrapolated FT, values are lower is of no clinical significance since the units for FT, are arbitrarily defined in the kit and refer to FT, standards measured also in the presence of exogenous BSA. These observations suggest that the subnormal FT, results obtained by the Amerlex assay in patients with NT1 could be an artefact of the methodology, i.e., due to (1) generally lower serum albumin concentrations in these patients, and (2) abnormal concentrations of anions with high affinity for albumin. Conceivably, certain drugs, steroids, fatty acids, urate, bilirubin and other metabolites which compete with the Amerlex tracer for binding sites on BSA could cause low FT, values. For instance, in vitro addition of non-esterified fatty acids to sera (> 2.0 mmol/l) decreases Amerlex FT, values significantly (unpublished observation) and could also explain the sometimes abnormally low FT, values by this method in heparinized patients [4,5]. Heparin causes a surge in the concentrations of nonesterified fatty acids which, in turn, may displace the labeled analog from albumin [ 111. Conversely, abnormally high concentrations of albumin binding sites in hyperdysalbuminemia, or very low levels of various ligands binding to albumin, may contribute to falsely high FT, results [2]. In conclusion, the concept of the Amerlex FT, measurement is elegant, but the T4 tracer may interact appreciably with the plasma proteins. This interaction may be diminished in NTI, perhaps due to interfering drugs, or other substances, leading to spuriously low results for FT,. Physicians should be made aware that the Amerlex FT, RIA does not differentiate well between euthyroid and hypothyroid sick patients, and erroneous results could be generated when this assay is applied to evaluation of thyroid status in a hospital setting involving patients with severe systemic illnesses. References 1 Midgley JEM, Wilkins TA. A defense of the Amerlex free thyroxine kit. Clin Chem 1982; 28: 2183-2184, (letter). 2 Stock@ JR, deGaris M, Csicsmann J, Barlow JW, White EL, Hurley DM. Limitations of a new free thyroxine assay (Amerlex free T4). Clin Endocrinol 198 1; 15 : 3 13-3 18. 3 Bayer MF, McDougall IR. The value of free thyroxine by solid phase radioimmunoassay in hospitalized patients. In: Albertini A, ed. Neuropeptides and free hormones assay. Amsterdam: Elsevier Biomedical Press, 1982: in press. 4 McDougall IR, Bayer MF, Nierenberg D, Lewis SJ. Disparate effects of heparin on free thyroxine as measured by two radioimmunoassays. J Nucl Med 1982; 23: 507-510. 5 Boss M, Kingstone D, Chan MK, Varghese Z. Contradictory findings in the measurement of free thyroxine after administration of heparin. Clin Chem 1982; 28: 1238-1239. 6 Stockigt JR. Influence of altered plasma binding on free and total thyroid hormone levels. In:
7 8 9 10 11
Albertini A, ed. Neuropeptides and free hormones assay. Amsterdam: Elsevier Biomedical Press, 1982: in press. Robbins J, Rall JE. Proteins associated with thyroid hormones. Physiol Rev 1960; 40: 415. Kaptein EM, Grieb DA, Spencer CA, Wheeler WS, Nicoloff JT. Thyroxine metabolism in the low thyroxine state of ctitical nonthyroidal illnesses. J Clin Endocrinol Metab 1981; 53: 764-771. Bayer MF, McDougall IR. Free thyroxine by solid phase radioimmunoassay: improvement in the laboratory diagnosis of thyroid status in severely ill patients. Clin Chim Acta 1982; 118: 209-218. Kaptein EM, Macintyre SS, Weiner JM, Spencer CA, Nicoloff JT. Free thyroxine estimates in nonthyroidal illness: comparison of eight methods. J Clin Endocrinol Metab 1981; 52: 1073-1077. Spector AA. Fatty acid binding to albumin. J Lipid Res 1975; 16: 165-179.