Interestingly enough, the effect of the digitalis glycosides is inhibited by a high concentration of potassium in the incubation medium and is enhanced by the absence of potassium (Wolff, 1960).
B.

Organification of iodine

The precise mechanism for organification of iodine in the thyroid is not as yet completely understood.
However, the formation of organically bound iodine, mainly mono-iodotyrosine, can be accomplished in cell-free systems.
In the absence of additions to the homogenate, the product formed is an iodinated particulate protein (Fawcett and Kirkwood, 1953;
Taurog, Potter and Chaikoff, 1955;
Taurog, Potter, Tong, and Chaikoff, 1956;
Serif and Kirkwood, 1958;
De Groot and Carvalho, 1960).
This iodoprotein does not appear to be the same as what is normally present in the thyroid, and there is no evidence so far that thyroglobulin can be iodinated in vitro by cell-free systems.
In addition, the iodoamino acid formed in largest quantity in the intact thyroid is di-iodotyrosine.
If tyrosine and a system generating hydrogen peroxide are added to a cell-free homogenate of the thyroid, large quantities of free mono-iodotyrosine can be formed (Alexander, 1959).
It is not clear whether this system bears any resemblance to the in vivo iodinating mechanism, and a system generating peroxide has not been identified in thyroid tissue.
On chemical grounds it seems most likely that iodide is first converted to Af and then to Af as the active iodinating species.
In the thyroid gland it appears that proteins (chiefly thyroglobulin) are iodinated and that free tyrosine and thyronine are not iodinated.
Iodination of tyrosine, however, is not enough for the synthesis of hormone.
The mono- and di-iodotyrosine must be coupled to form tri-iodothyronine and thyroxine.
The mechanism of this coupling has been studied in some detail with non-enzymatic systems in vitro and can be simulated by certain di-iodotyrosine analogues (Pitt-Rivers and James, 1958).
There is so far no evidence to indicate conclusively that this coupling is under enzymatic control.

The chemical nature of the iodocompounds is discussed below (pp. 76 et seq.).
C.

Thyroglobulin synthesis

Little is known of the synthetic mechanisms for formation of thyroglobulin.
Its synthesis has not been demonstrated in cell-free systems, nor has its synthesis by systems with intact thyroid cells in vitro been unequivocally proven.
There is some reason to think that thyroglobulin synthesis may proceed independently of iodination, for in certain transplantable tumours of the rat thyroid containing essentially no iodinated thyroglobulin, a protein that appears to be thyroglobulin has been observed in ultracentrifuge experiments (Wolff, Robbins and Rall, 1959).
Similar findings have been noted in a patient with congenital absence of the organification enzymes, whose thyroid tissue could only concentrate iodide.
In addition, depending on availability of dietary iodine, thyroglobulin may contain varying quantities of iodine.
D.

Secretion

Since the circulating thyroid hormones are the amino acids thyroxine and tri-iodothyronine (cf. Section C), it is clear that some mechanism must exist in the thyroid gland for their release from proteins before secretion.
The presence of several proteases and peptidases has been demonstrated in the thyroid.
One of the proteases has pH optimum of about 3.7 and another of about 5.7 (McQuillan, Stanley and Trikojus, 1954;
Alpers, Robbins and Rall, 1955).
The finding that the concentration of one of these proteases is increased in thyroid glands from TSH-treated animals suggests that this protease may be active in vivo.
There is no conclusive evidence yet that either of the proteases has been prepared in highly purified form nor is their specificity known.
A study of their activity on thyroglobulin has shown that thyroxine is not preferentially released and that the degradation proceeds stepwise with the formation of macromolecular intermediates (Alpers, Petermann and Rall, 1956).
Besides proteolytic enzymes the thyroid possesses de-iodinating enzymes.
A microsomal de-iodinase with a pH optimum of around 8, and requiring reduced triphosphopyridine nucleotide for activity, has been identified in the thyroid (Stanbury, 1957).
This de-iodinating enzyme is effective against mono- and di-iodotyrosine, but does not de-iodinate thyroxine or tri-iodothyronine.
It is assumed that the iodine released from the iodotyrosines remains in the iodide pool of the thyroid, where it is oxidised and re-incorporated into thyroglobulin.
The thyroxine and tri-iodothyronine released by proteolysis and so escaping de-iodination presumably diffuse into the blood stream.
It has been shown that thyroglobulin binds thyroxine, but the binding does not appear to be particularly strong.
It has been suggested that the plasma thyroxine-binding proteins, which have an extremely high affinity for thyroxine, compete with thyroglobulin for thyroxine (Ingbar and Freinkel, 1957).
E.

Antithyroid drugs

Antithyroid drugs are of two general types.
One type has a small univalent anion of the thiocyanate-perchlorate-fluoro type.
This ion inhibits thyroid hormone synthesis by interfering with iodide concentration in the thyroid.
It does not appear to affect the iodinating mechanism as such.
The other group of antithyroid agents or drugs is typified by thiouracil.
These drugs have no effect on the iodide concentrating mechanism, but they inhibit organification.
The mechanism of action of these drugs has not been completely worked out, but certain of them appear to act by reducing the oxidised form of iodine before it can iodinate thyroglobulin (Astwood, 1954).
On the other hand, there are a few antithyroid drugs of this same general type, such as resorcinol, possessing no reducing activity and possibly acting through formation of a complex with molecular iodine.
Any of the antithyroid drugs, of either type, if given in large enough doses for a long period of time will cause goitre, owing to inhibition of thyroid hormone synthesis, with production of hypothyroidism.
The anterior lobe of the pituitary then responds by an increased output of TSH, causing the thyroid to enlarge.
The effect of drugs that act on the iodide-concentrating mechanism can be counteracted by addition of relatively large amounts of iodine to the diet.
The antithyroid drugs of the thiouracil type, however, are not antagonised by such means.
Besides those of the thiouracil and resorcinol types, certain antithyroid drugs have been found in naturally occurring foods.
The most conclusively identified is L-5-vinyl-2-thio-oxazolidone, which was isolated from rutabaga (Greer, 1950).
It is presumed to occur in other members of the Brassica family.
There is some evidence that naturally occurring goitrogens may play a role in the development of goitre, particularly in Tasmania and Australia (Clements and Wishart, 1956).
There it seems that the goitrogen ingested by dairy animals is itself inactive but is converted in the animal to an active goitrogen, which is then secreted in the milk.
F.

Dietary influences

Besides the presence of goitrogens in the diet, the level of iodine itself in the diet plays a major role in governing the activity of the thyroid gland.
In the experimental animal and in man gross deficiency in dietary iodine causes thyroid hyperplasia, hypertrophy and increased thyroid activity (Money, Rall and Rawson, 1952;
Stanbury, Brownell, Riggs, Perinetti, Itoiz, and Del Castillo, 1954).
In man the normal level of iodine in the diet and the level necessary to prevent development of goitre is about 100 **ymg per day.
With lower levels, thyroid hypertrophy and increased thyroid blood-flow enable the thyroid to accumulate a larger proportion of the daily intake of iodine.
Further, the gland is able to re-use a larger fraction of the thyroid hormone de-iodinated peripherally.
In the presence of a low iodine intake, thyroglobulin labelled in vivo with Af is found to contain more mono-iodotyrosine than normal, the amounts of di-iodotyrosine and iodothyronines being correspondingly reduced.
This appears to result from both a reduced amount of the iodine substrate and a more rapid secretion of newly iodinated thyroglobulin.
If the deficiency persists long enough, it is reasonable to suppose that the Af label will reflect the Af distribution in the thyroglobulin.
Similar results might be expected from the influence of drugs or pathological conditions that limit iodide trapping, or organification, or accelerate thyroglobulin proteolysis.

B.

The thyroid-stimulating hormone

The name thyroid-stimulating hormone (TSH) has been given to a substance found in the anterior pituitary gland of all species of animal so tested for its presence.
The hormone has also been called thyrotrophin or thyrotrophic hormone.
At the present time we do not know by what biochemical mechanism TSH acts on the thyroid, but for bio-assay of the hormone there are a number of properties by which its activity may be estimated, including release of iodine from the thyroid, increase in thyroid weight, increase in mean height of the follicular cells and increase in the thyroidal uptake of Af.
Here we shall restrict discussion to those methods that appear sufficiently sensitive and precise for determining the concentration of TSH in blood.
Brown (1959) has reviewed generally the various methods of assaying TSH, and the reader is referred to her paper for further information on the subject.

1.

Chemical constitution and physical properties of pituitary tsh

As long ago as 1851 it was pointed out by Niepce (1851) that there is a connection between the pituitary and the thyroid.
This connection was clarified by Smith and Smith (1922), who showed that saline extracts of fresh bovine pituitary glands could re-activate the atrophied thyroids of hypophysectomised tadpoles.
The first attempts to isolate TSH came a decade later, when Janssen and Loeser (1931) used trichloroacetic acid to separate the soluble TSH from insoluble impurities.
After their work other investigators applied salt-fractionation techniques to the problem, as well as fractionation with organic solvents, such as acetone.
Albert (1949) has concluded that the most active preparations of TSH made during this period, from 1931 to 1945, were probably about 100 to 300 times as potent as the starting material.
Much of this work has been reviewed by White (1944) and by Albert (1949).
Developments up to about 1957 have been discussed by Sonenberg (1958).

In the last few years, the application of chromatographic and other modern techniques to the problem of isolating TSH has led to further purification (Bates and Condliffe, 1960;
Pierce, Carsten and Wynston, 1960).
The most active preparations obtained by these two groups of investigators appear to be similar in potency, composition and physical properties.

Two problems present themselves in considering any hormone in blood.
First, is the circulating form of the hormone the same as that found in the gland where it is synthesised and stored?
Second, what is its concentration in normal circumstances and in what circumstances will this concentration depart from the normal level and in which direction?
It is therefore necessary to consider the properties of pituitary TSH if the fragmentary chemical information about blood TSH is to be discussed rationally.
The importance of knowing in what chemical forms the hormone may exist is accentuated by the recent observation that there exists an abnormally long-acting TSH in blood drawn from many thyrotoxic patients (Adams, 1958).
Whether this abnormal TSH differs chemically from pituitary TSH, or is, alternatively, normal TSH with its period of effectiveness modified by some other blood constituent, cannot be decided without chemical study of the activity in the blood of these patients and a comparison of the substance responsible for the blood activity with pituitary Aj.

In evaluating data on the concentration of TSH in blood, one must examine critically the bio-assay methods used to obtain them.
The introduction of the United States Pharmacopoeia reference standard in 1952 and the redefinition and equating of the USP and international units of thyroid-stimulating activity have made it possible to compare results published by different investigators since that time.
We should like to re-emphasise the importance of stating results solely in terms of international units of TSH activity and of avoiding the re-introduction of biological units.
For the most part, this discussion will be confined to results obtained since the introduction of the reference standard.
A.

Standard preparations and units of thyroid-stimulating activity

The international unit (u.), adopted to make possible the comparison of results from different laboratories (Mussett and Perry, 1955), has been defined as the amount of activity present in 13.5 mg of the International Standard Preparation.
The international unit is equipotent with the USP unit adopted in 1952, which was defined as the amount of activity present in 20 mg of the USP reference substance.