A Simple Method for the Separation and Quantitation of Radiolabeled Thyroid Hormones in Thyroxine Clearance Studies

Sco-rr

J.

GROSSMAN

A method labeled injection lated

was developed

thyroxine

of radioactive

from

plasma

Cq8 sorbent

bed.

pair reversed

to facilitate

the separation

in plasma for thyroxine thyroxine,

protein The

phase

metabolites

thyroid

chromatography

and quantitation

studies.

the radiolabeled

and polar

individual

clearance

Following

thyroid

hormones

by solid phase

hormones

were

and sequentially

of radiointravenous

then

eluted

were

extraction

separated through

isoon a

by iona UV de-

and radiochromatographic detector. The radioactivity of individual radiolabeled thyroid hormones was corrected for recovery of carrier as determined from UV absorbance. The recoveries of thyroxine and 3,5,3’-triiodothyronine (T,) tector

were

96% and lOl%,

Key Words: phase

Thyroid

respectively. hormones;

Thyroxine;

Thyroxine

clearance;

Plasma,

Solid-

in rodents

after

extraction

INTRODUCTION Thyroid

hypertrophy

and hyperplasia

are frequently

encountered

chronic exposure to xenobiotics (Japundzic, 1969; Comer et al., 1985; Brown et al., 1987). This response has often been attributed to an increased clearance of thyroxine (Oppenheimer et al., 1968; Comer et al., 1985; Brown et al., 1987) that subsequently leads to a transient decrease in plasma thyroxine levels. As a result, there may be a partial

loss of feedback

inhibition

to the hypothalamus

and a compensatory

hy-

persecretion of thyroid-stimulating hormone (TSH) to stimulate the thyroid and increase thyroxine secretion. Chronic hyperstimulation of the thyroid over the life of the animal can result in thyroid adenoma portant to be able to distinguish compounds of thyroid roid.

hormone

metabolism

(McClain et al., 1988). Thus, it is imthat induce tumors due to alteration

from those that are directly

genotoxic

to the thy-

Following the injection of 12’1-thyroxine, a variety of other radiolabeled species are found circulating in plasma in addition to thyroxine, including 3,5,3’-triiodothyronine (T3), 3,3’,5’-triiodothyronine (rT,), iododinated protein, and free iodide (Bianchi

et al., 1983;

Rudolph

et al., 1978; Schwartz

et al., 1971). Thus, simple

mea-

From the Department of Safety Assessment, Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania. Address reprint requests to: Dr. S. J. Grossman, Department of Safety Assessment WP45-324, Merck Sharp & Dohme Research Laboratories, West Point, PA 19486. Received January 1990; revised and accepted April 1990. 183 Journal of Pharmacological

Methods

0 1990 Elsevier Science Publishing

24, 183-188 (1990) Co., Inc., 655 Avenue of the Americas, New York, NY 10010

160-5402190/$3.50

184

S. I. Grossman 125l in the plasma after ‘251-thyroxine administration could be misleading. In order to measure ‘251-thyroxine content specifically in the large number of plasma samples generated in thyroxine clearance studies, a rapid,

surement

simple volves

of undifferentiated

method has been developed to facilitate this procedure. ion-paired solid phase extraction of the thyroid hormones

versed phase ion-pair

high-pressure

liquid chromatography

The method infollowed by re-

(HPLC).

The radioactive

peaks of interest are then quantitated with the use of a radiochromatography detector. This assay offers the advantages of speed, high reproducible recovery (>95%),

and specificity.

surement

The utility

of 1251-thyroxine

of this method

clearance

is demonstrated

with

the mea-

in rats.

METHODS

lz51-Thyroxine Administration ‘251-Thyroxine saline

(specific

containing

10%

of approximately

activity 2200 Ci/mmol,

rat plasma

60 tKi/mL.

and sodium

This solution

DuPont/NEN) iodide

was dissolved

(IO mg/mL)

was administered

in 0.9%

to a final activity

to rats via the tail vein

at a dose of 160 $i/kg.

Plasma Collection Rats were

lightly anesthetized

into ethylenediaminetetraacetateafter 125I-thyroxine administration. ferred

to a glass tube containing

of 0.1 M sodium quired

hydroxide.

with ether and bled from the orbital

sinus (0.5 mL)

(EDTA) treated tubes at 5, IO, 24, 34, and 48 hr After centrifugation, 0.2 mL of plasma was trans50 kg each of carrier

The tubes were

sealed

thyroxine

and stored

and T3 in 0.05 mL at -20°C

until

re-

for analysis.

Solid Phase Extraction A 100 mg C18 solid phase extraction column (Analytichem) was conditioned with approximately 2 mL of methanol followed by 2 mL of ion-pair buffer (0.05 M potassium phosphate, sample was mixed

0.005 M sodium hexanesulfonate, pH 2.5). The thawed plasma with 1.8 mL of ion-pair buffer containing 10% methanol and

applied to the extraction column. The column was washed with 0.5 mL of ion-pair buffer followed by 0.5 mL of water and allowed to air dry. Thyroxine and T3 were eluted dryness

with

three

under

volumes

a gentle

(0.5 mL) of methanol.

stream

of nitrogen

This eluate

in a 35°C water

was evaporated

to

bath.

Chromatography The residue was dissolved

in 0.15 mL of methanol

and mixed with 0.1 mL of water.

An aliquot (50-150 ~.LL)was analyzed by chromatography on C,, column (Apex, 4.6 x 150 mm, 5 pm, Jones Chromatography) eluted with 57% methanol in an ion-pair buffer at 1.2 mUmin. The column effluent was passed sequentially through a Perkin Elmer LC-85 UV detector (254 nm) followed by a Beckman 171 radiochromatography detector equipped with a 0.3 mL solid scintillator cell. The integrated peak areas were recorded with a Spectra Physics 4270 dual channel integrator. Peak areas of

measurement

of Radiolabeled Thyroid Hormones

UV-absorbing carrier or radioactivity were calculated using standard curves constructed from injection of known amounts of carrier or radioactive thyroid hormones. Data Analysis

Recovery was determined by comparison of the amount of carrier present after extraction to standards not subjected to extraction. The amount of radioactivity in samples was normalized for recovery of the carrier in each sample. The elimination rate constant (k,,), half-life, volume of distribution (Vd), and systemic clearance (Cls) were calculated assuming a one-compartment model over the time course studied (Notari, 1980). RESULTS

The recovery of cold-carrier thyroxine from the plasma was 96 AI 2% (n = 240) in the course of routine analyses. Triiodothyronine recovery was also high (101 2 0.5%). The recovery of spiked 7251-thyroxine was identical to cold carrier when determined. The chromatographic resolution of the four thyroid hormones is shown in Figure 1. These compounds were completely resolved from each other and from more polar material. The total HPLC analysis time was about ‘I5 min. The quantitation of the radioactive compound of interest was most easily accomplished with a radiochromatographic detector in series with a UV detector. In this manner, the analysis was automated and capable of simultaneous measurement of recovery WV) and 1251-level. Figure 2 illustrates typical chromatograms obtained by this method. There were no interfering UV peaks that affected calculation of re-

t 0

’

L

L

J

10

5

15

Time (minutes)

FIGURE 1. Separation of thyroid hormone standards: (1) 3,5-diiodothyroni~e reverse T3, (4) 3,5,3’,5’-tetraiodothyronine (thyroxine) (T4).

(T2), (2) TS, (3)

185

186

S. J. Grossman

I

-----.A; 10

15

0

5

10

15

Time (minutes)

Time (minutes)

FIGURE 2. Representative chromatograms of carrier thyroid hormones T3 and T4 (left panel) and radioactivity (right panel) in a plasma sample from a control rat 34 hr after the intravenous administration of ‘%thyroxine. The radioactive peak represents approximately 20,000 cpm ‘%thyroxine chromatographed.

covery. The sensitivity of this configuration was routinely capable of detecting greater than 2000 dpm of 1251-injected onto the column. The clearance of thyroxine is described by a one-compartment model with the time points chosen (Figure 3). More rapid sampling at earlier time points may resolve at least three separate exponential phases. Regardless, the estimates of terminal elimination rate constant, half-life, volume of distribution, and clearance are similar to those obtained from a three-compartment model (Table 1).

3 a

0.5 0

I

I

I

I

12

24

36

48

Hours Post ’ 25 I -Thyroxine Dose FIGURE 3. Plasma ‘251-thyroxine clearance in control rats (0) or rats treated with phenobarbital (m), 80 mg/kg/day for 12 weeks. Points represent mean -I- SEM (n = 6).

Measurement

of Radiolabeled Thyroid Hormones

TABLE 1 Comparison of Experimental ‘%Thyroxine Clearance Parameters in Control Rats THREE COMPARTMEN?

ONE COMPARTMENT

PARAMETER

K,I (hr-‘)

-0.048

Half-Life

(hr)

Vdb (mL)

- 0.057

14.72

12.16

23.6

19.2

Clsb (mL/hr)

1.13

a Parameters

taken

b Normalized

from

1.08

DiStefano

et al., (1982).

to 100 g body weight.

DISCUSSION Numerous clearance

other

methods

in laboratory

exhaustive

plasma

have been

animals.

solvent

extraction

The use of immunoprecipitation

described

for the estimation

The most commonly

applied

methods

or immunoprecipitation

requires

of thyroxine involve either

(Aickin

specific antisera often followed

et al., 1977). by column

chromatography. Because thyroxine is zwitterionic and highly protein-bound, solvent extraction often leads to somewhat lower recoveries despite repeated extractions.

Previous

traction number 1981;

techniques

describing

either

solvent

extraction

or solid phase

ex-

gave low recoveries or were too cumbersome to be applicable to the large of plasma samples involved in thryoxine clearance studies (Lankmayr et al.,

Burman

A method

et al., 1981; was desired

teins reproducibly out. Solid phase C,, cartridges

Hay et al, 1981; Bianchi that would

separate

hormones

from

plasma

pro-

with high recovery and that was capable of high sample throughadsorption of the ion-paired thyroid hormones onto disposable

proved to be fast, efficient,

of processing

et al., 1984).

thyroid

many

samples

gave excellent

simultaneously

recoveries,

(typically,

eight

and was capable

samples

at a time).

Although not specifically examined,this extraction step should be easily adapted to all four physiologically relevant thyroid hormones. We have found that the radiochemical

purity of thyroxine

after the solid phase extraction

step is typically

greater

than 95% thyroxine in plasma samples from untreated rats. Depending on the nature of the study, this degree of purity may prove to be sufficient. For increased specificity, a technique

for the separation

and quantitation

hormones is necessary. Ion-suppression reversed efficient method for achieving the desired thyroid al., 1984). With a much shorter

of isolated

individual

thyroid

phase HPLC appeared to be an hormone separation (Bianchi et

the technique of ion-pair reversed phase HPLC used in this study, analysis time was achieved with an equivalent degree of resolution.

The chromatographic step makes and, with the use of simultaneous

the assay specific for the compound monitoring of a nonlabeled internal

of interest standard by

a UV detector, it provides recovery for the individual samples. As used in this laboratory, the chromatographic resolution and radiochemical and UV quantitation are completely automated. Over 600 samples have been analyzed with this method in ‘251-thyroxine clearance

187

188

S. J. Grossman

studies. Typically, 60 samples are processed per day using the automated radiochromatographic procedure. Using this method in the protocol described, increases in 1251-thyroxine clearance as small as 14% were easily discernible (S. J. Grossman, unpublished observations).

REFERENCES Aickin CM, Fraser S, Cooper E, Hall C, Burke CW (1977) Thyroid hormone kinetics: Improved method for quantitative separation and measurement of the various radioiodinated species in serum after radioiodothyronine injection. C/in fncfocrinol

7:469-479.

Bianchi R, Mariani G, Molea N, Vitek F, Cazzuola F, Carpi A, Mazzuca N, Toni MC (1983) Peripheral metabolism of thyroid hormones in man. I. Direct measurement of the conversion rate of thy roxine to 3,5,3’-triiodothyronine (T3) and determination of the peripheral and thyroidal production of Ts. 1 C/in Fnndocrinol Metab 56:1152-1163. Bianchi R, Molea N, Cazzulo F, Fusani L, Lotti M, Bertelli P, Ferdeghini M, Mariani F (1984) Highperformance liquid chromatographic separation of iodoamino acids for tracer turnover studies of thyroid hormones in viva.] Chromatogr297:393398.

Brown CC, Harland RF, Atterwill CK (1987) Increased thyroxine clearance in rats treated with high doses of a histamine Hz-Antagonist, SKF 93479. Arch Toxicol11:253-256. Burman KD, Bongiovanni R, Garis RK, Wartofsky L, Boehm TM (1981) Measurement of serum T4 concentration by high performance liquid chromatography. / C/in Endocrinol Metab 53:909-912. Comer CP, Chengelis CP, Levin S, Kotsonis FN (1985) Changes in thyroidal function and liver UDPglucuronosyltransferase activity in rats following administration of a novel imidazole (SC37211). Toxicol Appl Pharmacol 80:427-436. DiStefano JJ, Malone TK, Jang M (1982) Comprehensive kinetics of thyroxine distribution and metabolism in blood and tissue pools of the rat from only six blood samples: Dominance of

large, slowly exchanging tissue pools. Endocrinology

111:108-117.

Hay ID, Annesley TM, hang NS, Corman CA (1981) Simultaneous determination of D- and L-thyroxine in human serum by liquid chromatography with electrochemical detection. / Chromatogr 226:383-390.

Japundzic MM (1969) The goitrogenic effect of PhenobarbitaCNa on the rat thyroid. Acfa Anat 74:88-96.

Lankmayr EP, Maichin B, Knapp C (1981) Catalytic detection principle for high-performance liquid chromatography: Determination of enantiomerit iodinated thyronines in blood serum. / Chromatogr 224 :239-248. McClain RM, Posch RC, Bosakowski T, Armstrong JW (1988) Studies on the mode of action for thyroid gland tumor promotion in rats by phenobarbital. Toxicol Appl Pharmaco/94:254-265. Notari Robert E (1980) Biopharmaceutics and C/inical Pharmacokinetics. New York: Marcel Dekker, pp. 48-63. Oppenheimer JH, Bernstein G, Surks MI (1968) Increased thyroxine turnover and thyroidal function after stimulation of hepatocellular binding of thyroxine by phenobarbital. / C/in Invest 47:1399-1406.

Rudolph M, Sakurada T, Fang SL, Vagenakis AK, Braverman LE, lngbar SH (1978) Appearance of labeled metabolites in the serum of man after the administration of labeled thyroxine, triiodothyronine (TX) and reverse triiodothyronine (rT3). / Clin Endocrinol

Metab 46:923-928.

Schwartz HL, Surks Ml, Oppenheimer JH (1971) Quantitation of extrathyroidal conversion of Lthyroxine to 3,5,3’-triiodothyronine in the rat. / C/in Invest 50:1124-1130.