Bioassay

of TSH Using Dog Thyroid Cells in Monolayer Culture 6. Rapoport

and R. J. Adams

The CAMP response to TSH stimulation in dog thyroid cells in monolayer culture was adapted as a means to assay TSH bioactivity. Of a variety of polypeptide hormones examined, only TSH and LH stimulated

thyroid

cell

stimulation

by

contamination thyroid was

G-200

gel

range

filtration, the

peak.

effect

of serum,

from

serum

achieved

response

using

void

were

TSH

as meausred TSH

variation

this

S-200

calibrated

wide

and

the

of TSH and

was

gel filtrawith

human and

by bioassay. thyroid

the

inhibitory

by radioimmunoassay of

in

a

purification

necessary,

as measured

bovine

over

volume of

Sephacryl

Columns

TSH

and, on Sephadex

partial

tion.

to

of

present

eluted

Because was

assay

was found inhibitor

inhibitor(s)

nondialyzable

between

albumin

Serum

CAMP

The

serum

TSH.

but

represented

noncompetitive

cell

stimulation.

generation,

probably

with

to be a potent. the

CAMP

LH

cell

Intra-

response 9.2%.

to a 100

TSH

serum

following

tracted

by

intra-assay

variation

was with

14 normal on

in

between

serum

noactivity, displayed

a

dence

that

serum

of

thyroidism

and

interassay was

of 25

hypothyroidism,

4 of

none

of 3 patients

replacement. was

bioactivity

and immu-

between

This study provides immunoassayable

patients

with

is not

necessarily

Al-

observed

individual

dichotomy

measurements.

19.9%;

bioactivity

correlation TSH

for 100

21

and

numerous

ex-

of

from

thyroxine

a positive

was

TSH

primary

exogenous

though

standards

serum

subjects

was

assayed

of 25 KU/ml,

34.5%.

was

to human

TSH

and

variation

standards

patients

the

TSH

with TSH

standard

added

filtration

With

demonstrable

TSH

were

which

gel

bioactivity. @/ml

lU/ml

standards

TSH primary

samples the

two

new eviin the hypo-

synonymous

with TSH bioactivity.

CAMP

D

ESPITE THE ADVENT of the radioimmunoassay for TSH which has revolutionized the clinical evaluation of thyroid function,‘.” interest still remains in the bioassay of TSH in biologic fluids. Among the reasons for this continuing interest is the recognition that human pituitary TSH”-” and serum TSH” are heterogenous. In addition, there is evidence that TSH radioimmunoactivity and biologic activity are not necessarily synonymous.5 There is, therefore, a need for a systematic study to evaluate human sera for both TSH bioactivity and radioimmunologic activity, Numerous bioassays for TSH have been employed over the past half century. The most widely used assay at present is the mouse radioiodine release assay of McKenzie,’ an adaptation of the guinea pig assay of Adams and Purves.’ This assay has provided important information, but is somewhat limited in being an in vivo assay of only moderate sensitivity and subject to many influencing factors.‘-“’ The use of a highly sensitive cytochemical assay for the measurement of TSH in

From The Department of Medicine. Veterans Admkistrarion Hospital. San Francisco. and the Department of Medicine. University of California, San Franci.sco. Calif Receivedforpuhlication November 16, 1977. Supported by the National Institute ofArthritis, Metabolic and Digestive Diseases. Bethesda, Md. (AM 19289). and the Medical Research Serviceof the Veterans Administration. Address reprint requests to B. Rapoport. Department of Medicine /I I I Fi. Veterans .3dmini.~tration Hospital, 4150 Clement Street, San Francisco, Calif: 94121. 1111978 bv Grune & Stratton, Inc. 0026~0495/78/2712-0004$I)z.1)0/0

Metabolism,

Vol. 27.

No.

12

(December). 1978

1732

BIOASSAY

OF TSH IN MONOLAYER

1733

CULTURE

serum has been described.“,‘2 Further experience with this promising assay is awaited. We have been impressed by the sensitivity and precision of the CAMP response to TSH in dog thyroid cells in monolayer culture. I3 The present report describes the adaptation of this system as an in vitro assay to measure TSH bioactivity. Using this assay, the bioactivity of TSH in human serum was measured and compared with TSH as measured by radioimmunoassay. MATERIALS

AND METHODS

Dog thyroid cells in monolayer culture were prepared and maintained as previously described.13 For use as target cells to measure TSH bioactivity, the thyroid cells were subcultured in 35 mm diameter culture dishes (Corning). In a typical experiment, confluent cells from one 75 cm2 flask (Falcon) were subcultured in 20 dishes, providing a density of approximately IO5 thyroid cells per dish. The culture medium was Minimum Essential Medium containing 20% fetal calf serum, 2 mM glutamine and nonessential amino acids, as well as 100 U penicillin, 100 r,tg neomycin, and 2.5 rg fungizone per milliliter of medium. The cells were maintained at 37” C in a water-saturated incubator in an atmosphere of 5% CO, in air. After subculture the cells, which were not confluent, were allowed to recover for approximately 24 hr before they were used. Substances to be tested for bioactivity were dissolved in serum-free Leibovitz-I5 (L-15) medium, pH 7.4, containing 20 mM N-2-hydroxethylpiperazine-N’-2-ethanesulfonic acid (Hepes), and 0.5 mM 3-isobutyl-I-methylxanthine. Target dog thyroid cells in 35 mm dishes were incubated with these solutions for I5 min at 37°C in room air in a water bath. At the end of the incubation period, the medium was aspirated and the thyroid CAMP was extracted and assayed by the method of Steiner et al.,‘” as previously described.lJ Gel filtration of serum was performed using columns of Sephadex G-200 (90 x 2.6 cm), and Sephacryl S-200 (150 x 2.6 cm) equilibrated with 50 mM NH,HCO,, pH 7.7. For the latter, columns of 90 cm and 60 cm were used in series. The columns were eluted by reverse flow at 0.38 ml/min using a Buchler Polystaltic pump. Four milliliter samples were applied using a four-way valve (Pharmacia Fine Chemical Co., Piscataway, N.J.). Appropriate fractions were lyophilized, and the residues then were resuspended in a volume of medium equal to the original serum sample and tested for their ability to stimulate thyroid cell CAMP content as described above. Fraction optical densities (280 mr) were measured with a Beckman model 24 spectrophotometer with a return sipper. This accessory allowed the rapid assay of many samples without the loss of sample volume and hence without the loss of sample bioactivity. Radioimmunoassay of human TSH (hTSH) was performed in the laboratory of Dr. Ralph R. Cavalieri by the double-antibody method of Odell et al.2 Serum samples for testing were obtained from the storage bank maintained by the Nuclear Medicine Department, San Francisco Veterans Administration Hospital (Dr. Ralph R. Cavalieri). Sera were stored at -20°C. Samples older than 6 ma were not used, and the majority were no more than l-2 mo old. Materials were purchased from the following sources: Minimum Essential Medium, Leibovitz-15 medium, fetal calf serum, glutamine, nonessential amino acids, neomycin and fungizone, from Grand Island Biological Co., Grand Island, N.Y; antiserum for the CAMP radioimmunoassay, from Schwarz/Mann Div., Becton Dickinson & Co; collagenase-CLS (1255200 U/mg), from Worthington Biochemical Corp; Hepes, 3-isobutyl-I-methylxanthine, 0”-monosuccinyl CAMP tyrosine methyl ester and glucagon, from Sigma Chemical Co., St. Louis, MO; bovine TSH (bTSH) from Armour Pharmaceutical, Phoenix, Ariz. Prolactin (P-B,), LH(B9). FSH(B-I). hCG(CRI IS) and GH(B-II) were kindly provided by the Hormone Distribution Officer, N.I.A.M.D.D., Bethesda, Md., and hTSH (Internstional Standard, 68/38) by Dr. D. R. Bangham, Mill Hill, England. RESULTS

E#ect ofpHon

the Thyroid Cell CAMP Response to TSHStimulation

Because it was observed in preliminary experiments that solutions of different fractions of human serum varied in pH, it was important to examine the effect of medium pH on the cultured thyroid cell CAMP response to TSH stimulation.

1734

AAPOPORT

AND

ADAMS

60

I:

Fig. 1. The effect of pli on the thyroid cell CAMP response to TSH stimulation. Cells were incubated for 16 min at 37” C in L-l 5 medium containing 20 mM Hepes and 0.5 mM 3-isobutyl-I-methylxanthine. Brackets indicate the SE of cellular CAMP content in triplicate dishes of cells.

BASAL

I:-xc-I:-=-=

0

I

I

I

I

I

6.0

6.5

7.0

75

80

PH

Over a range of pH 6.0 to pH 8.0, both basal and TSH stimulation CAMP values increased progressively with increasing pH (Fig. 1). Maximal stimulation was observed at pH 7.5-8.0. A wider pH range was not examined because of the likelihood of severe cell damage at extreme pH values. Similar results were obtained whether 20 mM Hepes or 20 mM phosphate was used as a buffer. Because of the marked effect of pH on cellular CAMP generation, care was taken in all subsequent experiments to ensure uniformity in pH. Serum fraction residues were resuspended in medium containing 20 mA4 Hepes, and, if necessary, the pH was adjusted further with minimal volumes of NaOH or HCI.

Specijcity

of the CAMP Response to TSH

The effect of a variety of polypeptide hormones on dog thyroid CAMP content was examined (Table 1). Of the hormones tested, only bovine TSH and bovine LH had stimulatory activity. The extent of stimulation with LH was surprising but probably is explained by contamination of the LH preparation with TSH.15 This Table 1. Effect of Polypeptide Hormones on Dog Thyroid Cell CAMP Content COllCWltG3tlOtl

Hormone

Cyclic AMP

ng/ml’

pmole/Dlsht

Control

1.29

f

07

100

4.14

*

.31

LH-B9

100

2.48

f

.19

FSH-Bl

100

1.28

f

.22

HCG-CR115

100

0.98

f

09

GH-B18

100

1.42

I

100

152i

TSH

(Thytropar)

Prolactin

P-B3

Glucagon ‘The mated

concentrations specific

activity

1000 indicated of 30

are of impure

U/mg

TSH.

the

preparations absolute

f

SE of values

obtamed

in tnplicate

dishes

the exception

concentration

pU/ml). t Mean

1.10 with

of cells

of TSH

11 17

f

of glucagon IS approxunately

.04 Based

on an estl-

3 ng/ml

(100

BIOASSAY

OF TSH IN MONOLAYER

CULTURE

1735

explanation is supported by further observations in a separate experiment in which highly purified porcine LH (kindly provided by Dr. Harold Papkoff) had considerably less bioactivity than the relatively impure bovine LH preparation. Thus, 1000 ng/ml of this porcine preparation has the same stimulatory effect on thyroid cell CAMP generation (370% of basal values) as 3 ng/ml of pure bTSH. That is, the TSH was approximately 300 times more potent than the LH on a weight basis. Inhibitory Effect ofSerum on the CAMP Response to TSH Potent inhibitors of the thyroid CAMP response to TSH have been demonstrated previously in serum.‘“.” The presence in culture medium of 20% human serum reduced the thyroid cell CAMP response to TSH stimulation by approximately 40% (Fig. 2). Predialysis of the serum did not alter its inhibitory effect on CAMP generation. A double-reciprocal plot of TSH concentration versus CAMP response indicated that the inhibitory effect of serum was noncompetitive in nature (Fig. 2, inset). In order to quantitate TSH bioactivity in serum using the cultured dog thyroid cell system, it was, therefore, necessary to remove first the inhibitory substances(s) from the serum sample to be assayed. Filtration of human serum on Sephadex G-200 suggested that there was no single inhibitor present (Fig. 3). Thus, inhibitory activity was distributed across a wide range of fractions. Most inhibition, however, occurred with the third protein peak corresponding to albumin. No inhibitory activity was observed with fractions eluting after the albumin peak. Since purified bovine serum albumin does not have inhibitory activity at a concentration of 1.5%, and since fetal calf serum and human serum are equipotent

0.4

NP

L #’

0.3

C

,’

0.2

0

,*d

,’

+ 0.1 $0//

Fig. 2. The effect of serum on the dog thyroid CAMP response to TSH stimulation. Cells were incubated for 16 min et 37°C in L-15 medium, pH 7.4, containing 20 mM Hepes and 0.5 mM 3-isobutyl-I-methylxanthine. Each point represents the mean of CAMP values obtained in triplicate dishes of cells. The solid line represents data obtained in serum-free medium. and the overlapping dashed lines represent data obtained with medium containing either untreated or dialyzed human serum. A double reciprocal plot of these data is shown in the inset: e-e serum-free medium; e--e 20% human serum.

5

1

10I

TW TSH (mu/ml)

No Serum

RAPOPORT AND ADAMS

1736

Fig. 3. Sephadex G-200 gel filtration of human serum demonstrating the distribution of the inhibitor on the thyroid cell CAMP response to TSH stimulation (50 mU/mll. The column (90 x 2.6 cm) was equilibrated and eluted with 50 mM NH,HCO,. pH 7.7. The sample size was 4 ml. Six ml collected. fractions were lyophilized, resuspended in 4 ml L-15 medium, pH 7.4, containing 20 mM Hepes and 0.5 mM 3 isobutyl - I - methylxanthine, and aliquots incubated for 15 min at 37°C with duplicate dishes of dog thyroid cells.

60 50

I 40 30 20 10

f 0 Fraction

Numbel

in their inhibitory effect on CAMP generation, is not albumin itself. Separation

of TSH Bioactivity

From Inhibitory

‘I it seems likely that the inhibitor(s)

Factorfs 1in Serum

Adequate separation of the inhibitory activity in serum from serum TSH bioactivity was achieved by reverse flow filtration through Sephacryl S-200 (Fig. 4). In separate experiments, it was found that the inhibitory activity in serum became negligible when the OD (280 mp) reading following a 4-ml serum application fell below 0.1. Consequently, for the assay of unknown samples for TSH bioactivity, fractions were pooled for lyophilization beginning with the first fraction with an OD less than 0. I. This excluded less than 10% of the TSH bioactivity recovered from the column (Fig. 4). Good agreement was observed between the elution patterns of bTSH bioactivity and hTSH by radioimmunoassay (Fig. 4).

Fig. 4. Filtration of human serum with added TSH on Sephacryl S-200. Two columns in series (SO x 2.6 cm and 60 x 2.6 cm) were equilibrated and eluted with 50 mM NH,HCO,, pH 7.7. Nine milliliter fractions were collected. In the upper panel 200 plJ of bTSH in 4 ml serum was applied, the fractions lyophilized, resuspended in 4 ml Leibovitz-15 medium, pH 7.4, containing 20 mM Hepes and 0.5 mM 3-isobutyl-1-methylxanthine, and tested for their ability to stimulate dog thyroid cell CAMP content in duplicate dishes of cells. In the lower panel, 200 PU of hTSH (68/38) in 2 ml serum was applied, the fractions lyophilired, resuspended in 2 ml of dog serum, and hT8H measured by radioimmunoassay using hTSH standards in dog serum. A total of 160 pLJ hTSH was recovered (80%).

14 12 10

8 6 4 2 0

10

20

30

Fraction

40 Number

50

60

BIOASSAY

OF TSH IN MONOLAYER

Table 2.

CULTURE

1737

Recovery of a Physiologic Concentration

of TSH From Serum as Measured

by Bioassay

Thyrold Cell CAMP Content pmole/Dlsh’ Basal

*Mean serum from

f

(no TSH)

TSH

in serum-free

TSH

extracted

SE

of tnplicate

and serum-free the

from

serum

*

.05

3.23

+

.lO

serum

3.81

f

.14

values.

medium.

by filtration

1.87 medium

The

Solutions former

was

on Sephacryl-$200.

of bTSH

(50

maintained

pU/ml)

were

m 4” C untll

Bioactivity

in the

two

simultaneously completion

prepared

in whole

of the extraction

preparations

was

then

of TSH

measured

SI-

multaneously.

Because the foregoing calibration experiments utilized pharmacologic concentrations of TSH, it was important to examine the recovery, as measured by bioassay, of a physiologic concentration of TSH. Solutions of bTSH were prepared simultaneously in whole serum and serum-free medium at a concentration of 50 pU/ml. The TSH in the serum-free medium was maintained at 4°C until completion of the extraction of TSH from serum by gel filtration, after which the bioactivity in the two samples was compared. Similar activity was observed in both samples (Table 2). Bioassay of TSH in Human Serum

In order to use the thyroid cell CAMP response to measure TSH bioactivity in serum, studies were done to estimate the precision and reproducibil$y of this assay. When the CAMP response to 100 PI-J/ml bTSH in serum-free medium was measured in 10 replicate dishes of cultured thyroid cells the coefficient of variation,180r the standard deviation expressed as a percentage of the mean, was 9.15% (Table 3). After gel filtration, hTSH bioactivity in sera was assayed in triplicate dishes of thyroid cells. Bovine TSH standards of 0, 25, 50, 100 and occasionally 150 pU/ml were used. In each assay, a group of dishes without thyroid cells was similarly processed, and the mean of these values was subtracted from values obtained with dishes containing cells. This blank value was typically 20%-30% of basal thyroid cell CAMP values. In a representative standard curve (Fig. 5), thyroid cell CAMP content increased three to fourfold at a TSH concentration of 100 pU/ml. As previously described, the limit of sensitivity of the assay was approximately 10 pU/m1.13 In this study, it was also noted that the net CAMP response to TSH stimulation resembled Michaelis-Menten enzyme kinetics.13 Thus, the standard curve was more linear when the CAMP response was plotted against the arithmetic-TSH dose rather than the log-TSH dose. Even so, the standard curve Table 3. Precision of the CAMP Response to TSH Measured Replicate

TSH

Number

WJ/ml~

Ten replicate pH

7.4,

dishes

contaming

0.5

of thyroid mM

cells were

3.isobutyl-1

bTSH. ‘Calculated

as described

in Results.J7.20

incubated

-methylxanthtne.

9.15

for 15 min at 37” C in the same 20

mM

Hepes

and

of

Llm!ts’

339.3-386.7

zklO.5

363.0

Coefflclent

95% Confidence

+SEM

Mean

100

10

CAMP Reponse

% Increase

the

assay.

Indicated

in L-l 5 medium. concentration

of

RAPOPORT

1738

AND

ADAMS

15

? a \ 7

10

E 0. :

5

50

100

150

TSH (_.uU/ml)

Fig. 5. Representative standard curva of TSH bioassay. Bovine TSH in L-15 medium containing 20 mM Hepas and 0.5 mM 3-isobutyl-1-methylxanthine, was added to dishes of dog thyroid cells for 15 min at 37” C. The brackets indicate the mean S.E. of CAMP values obtained in triplicate (0 TSH) or duplicate (25, 50.100, and 15OlU TSH/ml) dishes of cells.

was not always precisely linear and in different assays slight curvatures in either direction *were noted. For this reason the standard curves were drawn visually to obtain the best fit for the standard points. Estimates of TSH concentrations in unknown samples were then made by visual substitution in the standard curve of the mean CAMP response produced by the unknown sample in triplicate determinations. In order to obtain a statistical analysis of the precision of the assay, the X coefficientlY of the standard curves were determined in 20 separate bioassays. Although this measurement is best suited to a standard curve which is linear when the response is plotted against the log-dose, as pointed out by Feldman and Rodbard,*O a curved standard plot can be approximated by a straight line in an appropriately small region of the curve. Thus, the portion of the standard curve between 25 VU/ml and 100 kU/ml TSH was taken as a representative section to determine the X coefficient (Table 4). As calculated by McKenzie,6 using the method minimum error of the assay was 14%. described by Gaddum, I9 the approximate This is based on the calculation that the minimum variance (V (M)) of the logarithm of the result of the assay equals 4 X2/N, where N is the total number of culture dishes in an assay, e.g., 17. To assess further the intra-assay variation, normal human serum (TSH undetectable by radioimmunoassay) was “spiked” with bTSH to a concentration of 100 pU/ml. Aliquots were frozen and were then individually processed by gel filtration. Assay of bioactivity in five replicate serum samples resulted in an intra-assay Table 4. Number of

Pomts on

Precision of TSH Bioassay Standard Curves his/b)’

Number of

Standard Curve

Assays

Meal-S’

Mean b’

4-5

20

29.7

366.6

‘Calculated

as described

m Results.eJBJg

Mean 0.119

SD

Range

0.079

0.019-0.284

BIOASSAY OF TSH IN MONOLAYER CULTURE

1739

Table 5. Precision of the Bioassay of TSH in Serum Number of Dishes of

Measured

Estimated TSH Concentrationt

CAMP Response

Sample

Cells Assayed

Mean 96 Increase + SEM

Mean i

1

3

436.4 & 33.9

2 3

3 3

410.1

f 21.8

446.9

f 42.3

105 f

4

3

435.8

i

14.4

101*5

5

3

599.1 i

11.3

148zt

Mean

12

(63-l

23)

(53-l

57)

(82-120) 3

1133-162)

110

Llz33.9

*lo (82-137)

Coefficient of variation*

(59-143)

93 f 7

1371.5-559.7)

Five replicate samples of serum containing

(95% Confidence)’

101 zt 10

456.6

SEM 95% confidence’

SEM

16.6%

19.9%

100 ~rU/ml bTSH were extracted for TSH and the TSH

bioassayed as described in Materials and Methods. ‘Calculated as deswbed in Results.17Jo t@

TSH/ml. TSH concentrations estimated by substitution of the measured CAMP response in a standard

curve obtamed using bTSH in serum-free medium.

coefficient of variation of 19.9% (Table 5). The 95% confidence limits were determined as described by Snedecor and Cochran. 21 The interassay coefficient of variation, measured by assaying five serum samples “spiked” with 25 pU/ml bTSH was 34.5 %. Sera were assayed from 25 patients with primary hypothyroidism (TSH of > 8 pU/ml by radioimmunoassay in conjunction with a subnormal serum thyroxine concentration), 14 euthyroid control subjects, and 3 patients on exogenous thyroxine replacement therapy. TSH bioactivity was demonstrable in 21 of the hypothyroid patients, 4 of the euthyroid subjects and none of the patients taking exogenous thyroxine (Fig. 6). While there was significant correlation between

Fig. 6. Comparison of hTSH immunoassayable and bioassayable activitY in sera from normal subjects and patients with primary hypothyroidism. The shaded areas indicate the limit of sensitivity of the assays used. The diagonal line is not derived from the data but indicates concordance between bioassayable and radioimmunoassayable TSH activity. This line does not intercept the origin because the different sensitivities of the two assays necessitated different scales for each assay. The point at the lower left indicating undatectabla TSH by both radioimmuno- and bioassay represents 13 separate determinations from 10 normal euthyroid subjects and 3 patients on exogenous thyroid hormone replacement.

10

20 hTSH

40 Bioassay

100 blJ/ml)

200

1740

RAPOPORT

samples with bioassayable and radioimmunoassayable p < O.OOl), there were a number of individual samples observed between the two measurements.

AND ADAMS

TSH activity, (r = 0.754, in which a dichotomy was

DISCUSSION

There is need at present for a sensitive, convenient, in vitro bioassay for TSH. The present data indicate that cultured dog thyroid cells can serve this need very well if the TSH to be assayed is in dilute (< 10%) serum or in serum-free medium. Possible applications for the assay under these conditions include the bioassay of TSH in pituitary extracts and the monitoring of bioactivity during the purification of TSH. Under these circumstances, this assay is clearly more convenient, sensitive, and precise than the in vivo mouse bioassay system” that is presently in general use for this purpose. Thus, the threshhold of sensitivity in the cultured thyroid cell system is 5-IOpU TSH/ml. This is 5-10 times more sensitive than the mouse bioassay system. The excellent precision of the thyroid cell CAMP response to TSH stimulation (9.2% coefficient of variation) reduces the need for large numbers of replicate determinations for each sample. In addition, the index of precision (X) in our assay was 0.119 when measured at TSH concentrations of between 25 and lOO~U/ml. This contrasts with the lesser precision (X of 0.240) in the mouse bioassay system determined at higher TSH concentrations (2551600 pU/ml).6 The tissue culture system is more convenient than keeping and preparing laboratory animals. The cells used are from a single pool and the biologic variability among animals in a single experiment is therefore avoided. In contrast to the bioassay of TSH in serum-free medium, the presence in serum of a potent inhibitor of the thyroid cell CAMP response makes the application of our assay for the measurement of TSH bioactivity in serum more difficult. This difficulty was overcome, in part, by prior gel filtration of serum to remove this inhibitor. The price paid for this maneuver, however, was a decrease in the convenience and precision of the assay. Thus, the intra-assay coefficient of variation for replicate samples of 100 MU/ml was increased from 9.2% to 19.9% by the gel filtration procedure. Despite this disadvantage, the excellent sensitivity of the cultured thyroid cell assay made it feasible to measure TSH bioactivity in the majority of sera from patients with primary hypothyroidism. The lesser sensitivity and precision of the mouse bioassay system makes this assay less attractive than the cultured thyroid cell assay for TSH bioactivity in serum, as is attested to by the paucity of data accumulated over 20 yr with the former assay.6,2” To our knowledge, other than a preliminary report using the cytochemical TSH bioassay technique, I2 there has not been a systematic study to compare TSH radioimmunoactivity and bioactivity in human serum. As anticipated, considering the known sensitivity of our assay,‘” we were unable to detect TSH bioactivity in serum in the majority of euthyroid individuals. Surprisingly, however, TSH bioactivity was detected in sera from 4 of 14 euthyroid subjects. In contrast, only 4 of 25 patients with primary hypothyroidism had no detectable TSH bioactivity in their serum. Of potential significance was the observation that although there was a positive correlation between bioassayable and immunoassayable TSH in hypothyroid serum, many individual samples revealed discrepancies between these two measurements. The question arises, however, whether these discrepancies reflect imprecision in the assay or true differences between bio- and immunoassayable TSH concentra-

BIOASSAY

OF TSH IN MONOLAYER

CULTURE

1741

tions. At a serum TSH concentration of 25 pU/ml (close to the median value observed in those sera in which TSH bioactivity was measureable) the inter-assay coefficient of variation was 34.5%. Despite this relatively large potential error, discrepancies of even greater magnitude exist between TSH bioactivity and immunoactivity in some sera with bioactivity values above the median. Obviously, such differences are less easy to interpret at a TSH bioactivity of below 25pU/ml, at which level the precision of the assay is very likely reduced. Evidence in support of our data suggesting that there may be a discrepancy between serum bioactivity and immunoactivity in some individuals is the previous observation of similar differences in extracts of human pituitaries from patients with primary hypothyroidism.” In contrast to these data, however, a close correlation between serum TSH bioactivity and immunoactivity was found using the cytochemical assay.12 The reasons for a possible discrepancy between TSH bioactivity and immunoactivity are unclear. One consideration is the apparent heterogeneity of TSH.“-5 Another possibility is the presence of biologically inactive free CYand @ chain subunits of TSH in serum of patients with hypothyroidism,23.24 of which the latter may cross-react in the hTSH radioimmunoassay. It is also possible, but unlikely, that a non-TSH thyroid stimulator is present in the serum of patients with hypothyroidism that coelutes with TSH on gel filtration. Finally, although we found that three rapid freeze-thaw cycles did not affect TSH bioactivity, we cannot exclude that storage for relatively long periods of time may have altered TSH bioactivity in serum. Another consideration in interpreting the present data is that we used a heterologous system, measuring human TSH bioactivity against bovine TSH standards. Bovine TSH was chosen after preliminary experiments showed hTSH (International Standard 68/38) to have approximately 10% of the bioactivity of bTSH (Thytropar) when tested with dog thyroid cells. The same potency ratio between bTSH and hTSH was found when these agents were tested with cultured human thyroid cells. Interestingly, the dose-response effect of bTSH on CAMP generation was identical in human and dog thyroid cells. This suggested that the lesser response with hTSH in both dog and human cells did not occur because of a species difference, but rather because of diminished hTSH bioactivity. Because we used bTSH as standards in the hTSH bioassay, it is clear that the results cannot be directly compared to immunoassayable hTSH measured with the use of hTSH standards. It is important to note, however, that any difference in bioactivity between hTSH and bTSH would be constant throughout the study and would, therefore, not contribute towards the discrepancy between TSH bioactivity and immunoactivity observed in some hypothyroid sera. Inhibition by serum of thyroid adenylate cyclase activation by TSH has been described previously, but very little is known about the inhibitor other than it is not albumin.16*‘7The present report confirms that this inhibitor is nondialyzable and that the inhibition is noncompetitive in nature. I6 New information is provided on the molecular size of this inhibitor and chromatographic separation of the inhibitor from TSH was achieved. The physiologic role of such a powerful inhibitor of thyroid function is of interest. How is TSH in serum able to influence thyroid function in vivo in the presence of this inhibitor? It has been shown previously that the thyroid cell CAMP response to physiological concentrations of TSH is obliterated in the presence of 100% serum.” One possibility is that because of its

1742

RAPOPORT

AND

ADAMS

large molecular size the inhibitor is primarily within the intravascular space, with a relatively low concentration in the interstitial fluid bathing the thyroid cells. In contrast to an in vivo assay for TSH, there is no endothelial barrier between medium and thyroid cells in tissue culture. ACKNOWLEDGMENT We thank Dr. Ralph R. Cavalieri for generously providing the human study as well as for his helpful comments during the course of the study.

serum

samples

used in this

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