263
Clinica Chimica Acta, 60 (1975) 263-266 @ Elsevier Scientific Publishing Company,
SHORT ___
Amsterdam
-
Printed
in The Netherlands
COMMUNICATION
CCA 7014
SIMPLE INEXPENSIVE SERUM THYROXINE
PROCEDURE
WILLIAM
O’KELLY
P. TAAFFE,
Psycho-Endocrine Dublin (Eire) (Received
D.A.
Centre,
December
Department
FOR THE MEASUREMENT
OF
and A. DARRAGH of Psychiatry,
St. James’s
Hospital,
P.O. Box
469,
3, 1974)
Summary A simple, rapid, reliable and inexpensive procedure for the measurement of serum thyroxine is described. Evaporation of the serum extract is not necessary and the sensitivity, precision and accuracy of the method compare well with more elaborate procedures. The cost per duplicate assay of less than ten pence is considerably less than that of any of the available kit methods. Introduction The competitive protein binding assay described by Murphy and Pattee [l] in 1964 has provided the basis of many methods subsequently developed for the measurement of thyroxine (T4 ) in serum. Most of the currently available kit procedures also depend on modifications of this technique. We have developed a reliable method for measuring serum T4 which combines the speed and simplicity of a typical kit procedure but which is considerably less expensive. Materials L-Thyroxine sodium salt (--5H, 0) was obtained from Sigma (London). Centre, Amersham, England ] ’ “11 T4 was obtained from the Radiochemical and had a specific activity of 44 pCi/pg. The resin used was Amberlite IRA-400 analytical grade from BDH (England) and was soaked overnight in 0.1 M barbital buffer pH 8.6 and dried overnight at 80°C before use. The binding reagent was diluted normal serum. Pooled serum from healthy individuals was collected every six months, divided into lo-ml portions and deep-frozen. At weekly intervals the serum was thawed at 37°C and diluted
264
to 0.8 ml/100 ml with 0.1 M barbital buffer pH 8.6 containing propylene glycol (0.1 ml/100 ml) and phenol (10 mg/lOO ml). Radioactive T, was added to a final concentration of 0.02 pCi/ml and the reagent was dispensed in 4 ml aliquots into small screw-capped glass vials and stored at 4°C until required. One litre of reagent so prepared was sufficient to carry out duplicate assays on 100 serum samples plus appropriate controls and standards. Assay procedure One ml of 95% ethanol is added to 0.5 ml serum in 10 ml polythene tubes which are then stoppered, vortex-mixed for 15 seconds and centrifuged at 2500 rpm for 5 min. Aliquots (0.3 ml) are transferred to the vials containing the binding reagent and the same volume is transferred from a series of standard solutions containing 0, 10, 20 and 40 ng/ml T4 in 66% ethanol. All vials are capped, mixed briefly by inversion and incubated at 37°C for 10 min and at 4°C for 30 min. The resin (150 mg) is transferred to the vials by means of a small polythene dispenser and the vials are mixed on a slow rotary mixer (15 rpm) for 15 min at 4°C. After transferring all vials to an ice-bath, 1 ml of each supernatant is taken for counting. Evaluation
of the method
Accuracy Thyroxine-free serum was prepared by charcoal treatment of pooled normal serum. Concentrations of 2.5, 5.0, 8.0 and 10.0 pg/lOO ml of T4 were added and the assay carried out as described above. Results are summarised in Fig. 1. Precision At a level of 9.6 pg/lOO ml T4 the intra-assay precision (n = 25), and the inter-assay precision was 27.1% (n = 46).
2
4
6 T4 Added
Fig.
1.
Measurements
estimations.
of
thyroxine
was +4.4%
10 6 (T4 pg/lOOml) added
to
thyroxine
free
sera.
Each
point
represents
the
mean
of
six
265
TABLE
1
SERUM
T4
LEVELS
OF
EUTHYROID
SUBJECTS,
INFANTS
AND
PATIENTS
WITH
THYROID
DISEASE
Mean
k SD.
n
Range
Euthyroid
8.4
* 2.2
4.5-13.0
Hypothyroid
2.5
f 1.1
0.2-
Hyperthyroid
15.9
i; 2.2
13.3-22.6
Infants
15.6
f 2.6
13.3-21.3
41 4.4
22 24 7
Sensitivity The assay as described has been optimised to provide satisfactory precision and accuracy over the range 1.5 to 25 pg/lOO ml T, . The absolute assay sensitivity can be greatly increased by further dilution of the binding reagent and decrease of the [ ’ ’ 5I] T4 concentration. The method has been applied to the measurement of Tq in species (e.g. the horse) where the levels of the hormone are considerably lower than those found in humans.
Normal range In a group of clinically euthyroid human subjects the range of T, levels in serum was 4.5 to 13.0 r.lg/lOO ml (n = 45). Table I summa&es the serum T4 levels found in euthyroid, hypothyroid and hyperthyroid subjects as well as in a group of infants less than 8 weeks old.
T4 response to intravenous
thyrotrophin
releasing hormone
(TRH)
Intravenous administration of 200 pg TRH to a group of 10 healthy subjects produced, in addition to the expected rapid rise in serum TSH, a slower, less marked but significant rise in serum T, in each case (Table II).
TABLE
II
EFFECT
OF INTRAVENOUS
IN EUTHYROID
Subject
ADMINISTRATION
OF
200
fig TRH
ON
SERUM
T4 LEVELS
SUBJECTS
Oh
lh
4h
6h
8h
J.W.
11.2
11.2
12.9
13.8
13.0
D.R.
10.9
10.5
12.8
11.7
10.9
A.C.
8.1
9.1
10.0
11.0
9.6
Z.B.
9.8
9.8
13.9
12.5
11.9 7.5
E.McW.
5.3
7.1
6.6
7.5
I.H.
6.9
6.9
6.6
6.9
5.3
S.I.
7.6
8.4
11.9
9.8
10.5
S.B.
9.7
11.7
12.4
12.0
I.C.
10.2 8.5
10.2
10.3
9.4
10.8
O.H.
7.6
8.1
9.8
11.0
9.0
7.5
24.4
23.9
17.6
Mean
%
increase
(fig/100
ml)
266
Concluding remarks Many procedures based on the method of Murphy and Pattee [l] in 1964 for the measurement of T4 in serum have been described: Ekins et al. [2] in 1969, Thorson et al [3] in 1970 and Seligson and Seligson [4] in 1972. Of these, the procedures which do not necessitate the evaporation to dryness of the serum extract are to be preferred because of the considerable saving in time involved. Several commercially available kits for T, make use of this approach but the relatively high cost of these kits renders them an uneconomic proposition for many laboratories. The procedure described above is simple, reliable and inexpensive (costing less than ten pence to carry out an assay in duplicate), and the excellent reproducibility of the method makes it possible to measure with confidence even small changes in serum T4 levels.
Acknowledgement The generous financial fully acknowledged.
support
of F. Hoffmann-La
Roche
and Co. is grate-
References 1
Murphy.
2
Ekins,
3
Thorson, 64,
4
B.E.P. R.P..
Pattee.
S.C..
E.S.
Tsujikawa.
C.J. and R..
(1964) Ellis,
J. Clin.
S. (1969)
Brown,
J.L.,
Endocrinol. Clin.
24,
Biochem.
Morrison,
R.T.
and
187 2. 253-288 McIntosh,
H.W.
(1970)
Acta
Endocrinol.
630-636
Seligson,
CONTENTS Short
and
Williams,
H. and
Seligson.
(continued
D.
(1972)
from
Clin.
Chim.
Acta
38,
199-205
cover)
Communications
The
use of o-L-iduronidase activity determinations in leucocytes for the detection of Hurler and Scheie syndromes K.O. Liem and G.J.M. Hooghwinkel (Leiden and Amsterdam, The Netherlands) (November 22,1974). . . Simple inexpensive procedure for the measurement of serum thyroxine . W.P. Taaffe, D.A. O’Kelley and A. Darragh (Dublin, Eire) (December 3, 1974)
259 263
Clinica Chimica Acta, 60 (1975) 263-266 @ Elsevier Scientific Publishing Company,
SHORT ___
Amsterdam
-
Printed
in The Netherlands
COMMUNICATION
CCA 7014
SIMPLE INEXPENSIVE SERUM THYROXINE
PROCEDURE
WILLIAM
O’KELLY
P. TAAFFE,
Psycho-Endocrine Dublin (Eire) (Received
D.A.
Centre,
December
Department
FOR THE MEASUREMENT
OF
and A. DARRAGH of Psychiatry,
St. James’s
Hospital,
P.O. Box
469,
3, 1974)
Summary A simple, rapid, reliable and inexpensive procedure for the measurement of serum thyroxine is described. Evaporation of the serum extract is not necessary and the sensitivity, precision and accuracy of the method compare well with more elaborate procedures. The cost per duplicate assay of less than ten pence is considerably less than that of any of the available kit methods. Introduction The competitive protein binding assay described by Murphy and Pattee [l] in 1964 has provided the basis of many methods subsequently developed for the measurement of thyroxine (T4 ) in serum. Most of the currently available kit procedures also depend on modifications of this technique. We have developed a reliable method for measuring serum T4 which combines the speed and simplicity of a typical kit procedure but which is considerably less expensive. Materials L-Thyroxine sodium salt (--5H, 0) was obtained from Sigma (London). Centre, Amersham, England ] ’ “11 T4 was obtained from the Radiochemical and had a specific activity of 44 pCi/pg. The resin used was Amberlite IRA-400 analytical grade from BDH (England) and was soaked overnight in 0.1 M barbital buffer pH 8.6 and dried overnight at 80°C before use. The binding reagent was diluted normal serum. Pooled serum from healthy individuals was collected every six months, divided into lo-ml portions and deep-frozen. At weekly intervals the serum was thawed at 37°C and diluted
264
to 0.8 ml/100 ml with 0.1 M barbital buffer pH 8.6 containing propylene glycol (0.1 ml/100 ml) and phenol (10 mg/lOO ml). Radioactive T, was added to a final concentration of 0.02 pCi/ml and the reagent was dispensed in 4 ml aliquots into small screw-capped glass vials and stored at 4°C until required. One litre of reagent so prepared was sufficient to carry out duplicate assays on 100 serum samples plus appropriate controls and standards. Assay procedure One ml of 95% ethanol is added to 0.5 ml serum in 10 ml polythene tubes which are then stoppered, vortex-mixed for 15 seconds and centrifuged at 2500 rpm for 5 min. Aliquots (0.3 ml) are transferred to the vials containing the binding reagent and the same volume is transferred from a series of standard solutions containing 0, 10, 20 and 40 ng/ml T4 in 66% ethanol. All vials are capped, mixed briefly by inversion and incubated at 37°C for 10 min and at 4°C for 30 min. The resin (150 mg) is transferred to the vials by means of a small polythene dispenser and the vials are mixed on a slow rotary mixer (15 rpm) for 15 min at 4°C. After transferring all vials to an ice-bath, 1 ml of each supernatant is taken for counting. Evaluation
of the method
Accuracy Thyroxine-free serum was prepared by charcoal treatment of pooled normal serum. Concentrations of 2.5, 5.0, 8.0 and 10.0 pg/lOO ml of T4 were added and the assay carried out as described above. Results are summarised in Fig. 1. Precision At a level of 9.6 pg/lOO ml T4 the intra-assay precision (n = 25), and the inter-assay precision was 27.1% (n = 46).
2
4
6 T4 Added
Fig.
1.
Measurements
estimations.
of
thyroxine
was +4.4%
10 6 (T4 pg/lOOml) added
to
thyroxine
free
sera.
Each
point
represents
the
mean
of
six
265
TABLE
1
SERUM
T4
LEVELS
OF
EUTHYROID
SUBJECTS,
INFANTS
AND
PATIENTS
WITH
THYROID
DISEASE
Mean
k SD.
n
Range
Euthyroid
8.4
* 2.2
4.5-13.0
Hypothyroid
2.5
f 1.1
0.2-
Hyperthyroid
15.9
i; 2.2
13.3-22.6
Infants
15.6
f 2.6
13.3-21.3
41 4.4
22 24 7
Sensitivity The assay as described has been optimised to provide satisfactory precision and accuracy over the range 1.5 to 25 pg/lOO ml T, . The absolute assay sensitivity can be greatly increased by further dilution of the binding reagent and decrease of the [ ’ ’ 5I] T4 concentration. The method has been applied to the measurement of Tq in species (e.g. the horse) where the levels of the hormone are considerably lower than those found in humans.
Normal range In a group of clinically euthyroid human subjects the range of T, levels in serum was 4.5 to 13.0 r.lg/lOO ml (n = 45). Table I summa&es the serum T4 levels found in euthyroid, hypothyroid and hyperthyroid subjects as well as in a group of infants less than 8 weeks old.
T4 response to intravenous
thyrotrophin
releasing hormone
(TRH)
Intravenous administration of 200 pg TRH to a group of 10 healthy subjects produced, in addition to the expected rapid rise in serum TSH, a slower, less marked but significant rise in serum T, in each case (Table II).
TABLE
II
EFFECT
OF INTRAVENOUS
IN EUTHYROID
Subject
ADMINISTRATION
OF
200
fig TRH
ON
SERUM
T4 LEVELS
SUBJECTS
Oh
lh
4h
6h
8h
J.W.
11.2
11.2
12.9
13.8
13.0
D.R.
10.9
10.5
12.8
11.7
10.9
A.C.
8.1
9.1
10.0
11.0
9.6
Z.B.
9.8
9.8
13.9
12.5
11.9 7.5
E.McW.
5.3
7.1
6.6
7.5
I.H.
6.9
6.9
6.6
6.9
5.3
S.I.
7.6
8.4
11.9
9.8
10.5
S.B.
9.7
11.7
12.4
12.0
I.C.
10.2 8.5
10.2
10.3
9.4
10.8
O.H.
7.6
8.1
9.8
11.0
9.0
7.5
24.4
23.9
17.6
Mean
%
increase
(fig/100
ml)
266
Concluding remarks Many procedures based on the method of Murphy and Pattee [l] in 1964 for the measurement of T4 in serum have been described: Ekins et al. [2] in 1969, Thorson et al [3] in 1970 and Seligson and Seligson [4] in 1972. Of these, the procedures which do not necessitate the evaporation to dryness of the serum extract are to be preferred because of the considerable saving in time involved. Several commercially available kits for T, make use of this approach but the relatively high cost of these kits renders them an uneconomic proposition for many laboratories. The procedure described above is simple, reliable and inexpensive (costing less than ten pence to carry out an assay in duplicate), and the excellent reproducibility of the method makes it possible to measure with confidence even small changes in serum T4 levels.
Acknowledgement The generous financial fully acknowledged.
support
of F. Hoffmann-La
Roche
and Co. is grate-
References 1
Murphy.
2
Ekins,
3
Thorson, 64,
4
B.E.P. R.P..
Pattee.
S.C..
E.S.
Tsujikawa.
C.J. and R..
(1964) Ellis,
J. Clin.
S. (1969)
Brown,
J.L.,
Endocrinol. Clin.
24,
Biochem.
Morrison,
R.T.
and
187 2. 253-288 McIntosh,
H.W.
(1970)
Acta
Endocrinol.
630-636
Seligson,
CONTENTS Short
and
Williams,
H. and
Seligson.
(continued
D.
(1972)
from
Clin.
Chim.
Acta
38,
199-205
cover)
Communications
The
use of o-L-iduronidase activity determinations in leucocytes for the detection of Hurler and Scheie syndromes K.O. Liem and G.J.M. Hooghwinkel (Leiden and Amsterdam, The Netherlands) (November 22,1974). . . Simple inexpensive procedure for the measurement of serum thyroxine . W.P. Taaffe, D.A. O’Kelley and A. Darragh (Dublin, Eire) (December 3, 1974)
259 263
