81
Clinica Chimica Acta, 88 (1978) 81-88 @ Elsevier/North-Holland Biomedical Press
CCA 9514
RADIOIMMUNOASSAY SERUM
MARTIN
GRUSSENDORF
FOR 3,3’-DIIODOTHYRONINE
* and MICHAEL
IN HUMAN
HUFNER
Medizinische Poliklinik, Klinikwm der Universitri’t Heidelberg, Hospitalstrasse 3, 6900 Heidelberg 1 (G.F.R.) (Received
February
21st, 1978)
Summary A specific radioimmunoassay for measurement of 3,3’diiodothyronine (T,‘) is presented. With the method described (ethanol extraction of native serum and lyophilisation of the extract) the application of 400 ~1 serum equivalent in the assay is possible. Standards and sera are treated similarly. The detection limit is 0.625 ng/dl, comparison between direct assay and dried extract assay shows good correlation. Mean normal Tz’ serum concentration in man is 7.2 ng/dl (range 3 to 11 ng/ dl), hypothyroid: below 3.0 ng/dl, hyperthyroid: 11-64 ng/dl (range). Tz’ level in cord-blood of newborns: 16.5 ng/dl. The urinary excretion of free Tz’ of normal man is 0.49 pg/24 h (mean), a relatively high excretion rate in comparison to the low serum level.
Introduction Since 3,3’,5’-triiodothyronine (reverse T3, rT3) (besides the metabolically active triiodothyronine (T3), a second deiodination product from thyroxine (T4)) has been proved to be a physiological metabolite in human serum [ 1,2], it has stimulated many clinical and experimental investigations in the last years. Clinically it could be proved that it is generated to a great extent from T4 in the periphery [ 3,4]. Furthermore, it demonstrated a behavior parallel to the known thyroid hormones T3 and Tq in thyroid dysfunction (elevated in hyper-, reduced in hypothyroidism). A reciprocal fluctuation of serum levels of T3 and rTg could be demonstrated in man in certain nonthyroidal diseases (for example in hunger, shock, severe illness [5-7]), indicating an involvement of peripheral T4 deiodination in metabolic processes. * To whom
correspondence
should
be addressed.
82
More recently, in vitro experiments with rat liver homogenate showed strong instability of rT3 (in contrary to T3) in the homogenate [8,9]. 3,3’-T2 (T2’) was found to be the product of this degradation (i.e. 5’-deiodination). Additionally, rT, was found to inhibit T4 to T3 conversion in vitro [lO,ll], therefore it was concluded that rapid degradation of rTg within the cell may be of physiological importance. In order to investigate whether these results were comparable to fluctuations in man, several authors developed a method for measurement of Tz’ in human serum [ 12-151. As very different results were obtained, a normal range has not been established until now. In this paper we describe a method using vacuum dried ethanol extracts and compare it with direct measurements, Furthermore, some clinical findings are presented. Materials and methods Materials T,‘-conjugate to bovine serum albumin (Tz’-BSA conjugate) and Tz’ standard was a gift of Fa. Henning (Berlin). Chloramine T, metabisulfate and Na,HPO, came from Fa. Merck (Darmstadt), “‘1 was obtained from Fa. Hijchst (Frankfurt), Tq, T3 and rT 3 from Henning (Berlin). Human serum albumin (HSA) came from Behringwerke (Marburg); DASP (double antibody solid-phase) from Organon, Miinchen; complete Freund’s adjuvant from Difco Laboratories (Detroit). Production of antibodies 1 mg of T,‘-BSA conjugate was dissolved in 500 addition of 500 ~1 of complete Freund’s adjuvant the administered subcutaneously to 3 rabbits (1 mg each). tered after 4 and 8 weeks and blood was taken at 9th ear vein.
~1 distilled water, after emulsified mixture was The animals were boosweek by puncture of an
Buffer solutions Buffer I (assay buffer): 0.05 M Na2HP04 + 0.9 g% NaCl + 0.1 g% HSA, pH 7.4. Buffer II (antibody dilution buffer): 0.9 g% NaCl + 0.1 g% HSA. 1251-77~1
T2’ was labelled using the chloramin T method (labelling and separation have been reported previously [2]). 1251-T2’ was diluted with antibody dilution buffer (approx. 10 000 cpm/50 ~1) and stored at -20°C. It could be used for 6 weeks without significant loss of binding. Preparation
of standards
For preparation of T2’-free serum (O-serum), pooled normal sera were extracted with charcoal (method of Mitsuma et al. [16]): 20 g of charcoal were added to 100 ml serum and stirred for 12 h at 4°C. Afterwards the mixture was centrifuged at 20 000 X g to separate the charcoal. With this method 99.18 + 0.07 (S.D.) of added T2’ is eliminated. The following T2’ standard concentrations were dissolved in this O-serum: 0.625,1.25, 2.5, 5, 10 and 20 ng/dl.
a3
Extraction and assay procedure 1.5 ml of serum or standard was extracted with 3.0 ml of 98% ethanol. The mixture was centrifuged 2 times (5 min, 3000 X g) and exactly 3 ml of the clear supematant were transferred into plastic tubes (1.5 X 10 cm). The tubes were closed with a perforated cover and the extracts were dried in room temperature (21°C) in a big glass vial by means of a lyophilisator. 1200 ~1 of assay buffer were added to the dried extract and the solution was shaken strongly on a Vortex mixer. Afterwards, 500 ~1 of the turpid solution representing 400 ~1 of native serum or standards, respectively were added into the assay vials (plastic tubes 1 X 5.5 cm), 50 ~1 of antibody (final solution 1 : 100 000) and 50 ~1 labelled Tz’ were added, and the assay mixture was incubated 20 h at room temperature. Afterwards bound Tz’ was separated using the double-antibody solid-phase technique (DASP, Organon). Measurement of TBG binding of T,‘, TJ, rT3 and T4 10, 20, 50, 100 and 200 ~1 of 4 normal sera and 50 ~1 of labelled iodothyronine were incubated in the assay buffer at +4”C, after 16 h, free tracer was separated with dextran-coated charcoal (0.1 M NazHP04, pH 7.6, plus 150 mg/ dl Dextran + 1.0 g/d1 charcoal). Direct radioimmunoassay For direct measurement of blood with high Tz’ concentrations 50 ~1 of native serum were incubated with 500 ~1 assay buffer, 50 ~1 of labelled Tz’ and 50 ~1 of antibody (final dilution 1 : 100 000). After 16 h incubation at room temperature free and bound T2 was separated using DASP.’ Measurement of free urinary T,’ 50 ~1 of urine were added to incubation mixture as described above Direct radioimmunoassay). Standards were dissolved in assay buffer.
(see
Results Antibodies The BSA-T2’ conjugate raised very potent antibodies in all three rabbits. They could be used in a final solution of 1 : 100 000 (40% binding). Table I shows the cross-reactivity with important iodothyronines. The low cross-
TABLE I CROSS-REACTIVITY
OF 3,3’-T2
3,3’-Diiodothyronine (Tz’) Tyrosine 3-Monoiodotyrosine (MIT) Diiodotyrosine (DIT) 3’-Monoiodothyronine (TI) 3’,5’-Diiodothyronine (3’,5’-T2) 3,5,3’-Triiodothyronine (T3) 3,3’,5’-Triiodothyronine (rT3) Thyroxine (T4)
ANTIBODY 100%
Clinica Chimica Acta, 88 (1978) 81-88 @ Elsevier/North-Holland Biomedical Press
CCA 9514
RADIOIMMUNOASSAY SERUM
MARTIN
GRUSSENDORF
FOR 3,3’-DIIODOTHYRONINE
* and MICHAEL
IN HUMAN
HUFNER
Medizinische Poliklinik, Klinikwm der Universitri’t Heidelberg, Hospitalstrasse 3, 6900 Heidelberg 1 (G.F.R.) (Received
February
21st, 1978)
Summary A specific radioimmunoassay for measurement of 3,3’diiodothyronine (T,‘) is presented. With the method described (ethanol extraction of native serum and lyophilisation of the extract) the application of 400 ~1 serum equivalent in the assay is possible. Standards and sera are treated similarly. The detection limit is 0.625 ng/dl, comparison between direct assay and dried extract assay shows good correlation. Mean normal Tz’ serum concentration in man is 7.2 ng/dl (range 3 to 11 ng/ dl), hypothyroid: below 3.0 ng/dl, hyperthyroid: 11-64 ng/dl (range). Tz’ level in cord-blood of newborns: 16.5 ng/dl. The urinary excretion of free Tz’ of normal man is 0.49 pg/24 h (mean), a relatively high excretion rate in comparison to the low serum level.
Introduction Since 3,3’,5’-triiodothyronine (reverse T3, rT3) (besides the metabolically active triiodothyronine (T3), a second deiodination product from thyroxine (T4)) has been proved to be a physiological metabolite in human serum [ 1,2], it has stimulated many clinical and experimental investigations in the last years. Clinically it could be proved that it is generated to a great extent from T4 in the periphery [ 3,4]. Furthermore, it demonstrated a behavior parallel to the known thyroid hormones T3 and Tq in thyroid dysfunction (elevated in hyper-, reduced in hypothyroidism). A reciprocal fluctuation of serum levels of T3 and rTg could be demonstrated in man in certain nonthyroidal diseases (for example in hunger, shock, severe illness [5-7]), indicating an involvement of peripheral T4 deiodination in metabolic processes. * To whom
correspondence
should
be addressed.
82
More recently, in vitro experiments with rat liver homogenate showed strong instability of rT3 (in contrary to T3) in the homogenate [8,9]. 3,3’-T2 (T2’) was found to be the product of this degradation (i.e. 5’-deiodination). Additionally, rT, was found to inhibit T4 to T3 conversion in vitro [lO,ll], therefore it was concluded that rapid degradation of rTg within the cell may be of physiological importance. In order to investigate whether these results were comparable to fluctuations in man, several authors developed a method for measurement of Tz’ in human serum [ 12-151. As very different results were obtained, a normal range has not been established until now. In this paper we describe a method using vacuum dried ethanol extracts and compare it with direct measurements, Furthermore, some clinical findings are presented. Materials and methods Materials T,‘-conjugate to bovine serum albumin (Tz’-BSA conjugate) and Tz’ standard was a gift of Fa. Henning (Berlin). Chloramine T, metabisulfate and Na,HPO, came from Fa. Merck (Darmstadt), “‘1 was obtained from Fa. Hijchst (Frankfurt), Tq, T3 and rT 3 from Henning (Berlin). Human serum albumin (HSA) came from Behringwerke (Marburg); DASP (double antibody solid-phase) from Organon, Miinchen; complete Freund’s adjuvant from Difco Laboratories (Detroit). Production of antibodies 1 mg of T,‘-BSA conjugate was dissolved in 500 addition of 500 ~1 of complete Freund’s adjuvant the administered subcutaneously to 3 rabbits (1 mg each). tered after 4 and 8 weeks and blood was taken at 9th ear vein.
~1 distilled water, after emulsified mixture was The animals were boosweek by puncture of an
Buffer solutions Buffer I (assay buffer): 0.05 M Na2HP04 + 0.9 g% NaCl + 0.1 g% HSA, pH 7.4. Buffer II (antibody dilution buffer): 0.9 g% NaCl + 0.1 g% HSA. 1251-77~1
T2’ was labelled using the chloramin T method (labelling and separation have been reported previously [2]). 1251-T2’ was diluted with antibody dilution buffer (approx. 10 000 cpm/50 ~1) and stored at -20°C. It could be used for 6 weeks without significant loss of binding. Preparation
of standards
For preparation of T2’-free serum (O-serum), pooled normal sera were extracted with charcoal (method of Mitsuma et al. [16]): 20 g of charcoal were added to 100 ml serum and stirred for 12 h at 4°C. Afterwards the mixture was centrifuged at 20 000 X g to separate the charcoal. With this method 99.18 + 0.07 (S.D.) of added T2’ is eliminated. The following T2’ standard concentrations were dissolved in this O-serum: 0.625,1.25, 2.5, 5, 10 and 20 ng/dl.
a3
Extraction and assay procedure 1.5 ml of serum or standard was extracted with 3.0 ml of 98% ethanol. The mixture was centrifuged 2 times (5 min, 3000 X g) and exactly 3 ml of the clear supematant were transferred into plastic tubes (1.5 X 10 cm). The tubes were closed with a perforated cover and the extracts were dried in room temperature (21°C) in a big glass vial by means of a lyophilisator. 1200 ~1 of assay buffer were added to the dried extract and the solution was shaken strongly on a Vortex mixer. Afterwards, 500 ~1 of the turpid solution representing 400 ~1 of native serum or standards, respectively were added into the assay vials (plastic tubes 1 X 5.5 cm), 50 ~1 of antibody (final solution 1 : 100 000) and 50 ~1 labelled Tz’ were added, and the assay mixture was incubated 20 h at room temperature. Afterwards bound Tz’ was separated using the double-antibody solid-phase technique (DASP, Organon). Measurement of TBG binding of T,‘, TJ, rT3 and T4 10, 20, 50, 100 and 200 ~1 of 4 normal sera and 50 ~1 of labelled iodothyronine were incubated in the assay buffer at +4”C, after 16 h, free tracer was separated with dextran-coated charcoal (0.1 M NazHP04, pH 7.6, plus 150 mg/ dl Dextran + 1.0 g/d1 charcoal). Direct radioimmunoassay For direct measurement of blood with high Tz’ concentrations 50 ~1 of native serum were incubated with 500 ~1 assay buffer, 50 ~1 of labelled Tz’ and 50 ~1 of antibody (final dilution 1 : 100 000). After 16 h incubation at room temperature free and bound T2 was separated using DASP.’ Measurement of free urinary T,’ 50 ~1 of urine were added to incubation mixture as described above Direct radioimmunoassay). Standards were dissolved in assay buffer.
(see
Results Antibodies The BSA-T2’ conjugate raised very potent antibodies in all three rabbits. They could be used in a final solution of 1 : 100 000 (40% binding). Table I shows the cross-reactivity with important iodothyronines. The low cross-
TABLE I CROSS-REACTIVITY
OF 3,3’-T2
3,3’-Diiodothyronine (Tz’) Tyrosine 3-Monoiodotyrosine (MIT) Diiodotyrosine (DIT) 3’-Monoiodothyronine (TI) 3’,5’-Diiodothyronine (3’,5’-T2) 3,5,3’-Triiodothyronine (T3) 3,3’,5’-Triiodothyronine (rT3) Thyroxine (T4)
ANTIBODY 100%
