42 IRISH JOURNAL OF MEDICAL SCIENCE--SUPPLEMENT, JULY 1974

a poor response to a hypoglycemic stimulus of growth hormone release as the mechanism of failure to this group is shown in Fig. 5. With substitution therapy with Eltroxin growth hormone output increases. Finally, I should like to conclude by indicating the economic limiting factors to immunoassay. Apart from the minimum requirement of, at least, two skil led trained per- sonnel, automatic isotope counting equipment (in the region of s +) and preparatory equipment, e.g. refrigerated centrifuge (s +) , space to house the equipment and personnel is also required. This type of assay therefore is not really an undertaking to be attempted unless the personnel and equipment can be guaranteed to b3 reasonably extended at all times.

I wish to thank the Medical Research Council of Ireland for their support and Miss M. Ryan for technical assistance.

The following references will be found useful: Radio Immuno Assay Methods. 1971. Ed. by K. E. Kirkham and W. M. Hunter. Churchill

Livingstone. Protein and Polppeptide Hormones. 1969. Parts 2 and 3. Ed. by M. Margoulies. Excerpta

Medica Publications.

CHAIRMAN

Now I call on Dr. David Powell to give his communication 'Clinical Sig- nif icance of Circulating Form on Substances'. Dr. Powell has recently returned from Boston and is already a distinguished contr ibutor in the literature pertaining to parathyroid physiology and pathology.

CLINICAL SIGNIFICANCE OF CIRCULATING HORMONAL SUBSTANCES

David Powell

Federated Dublin Voluntary Hospitals.

T HE advent in recent years of methods for measuring circulat ing hormonal substances--especial ly radioimmunoassay techniques--has revolution- ized endocrinology, and medicine generally. These methods have been

applied to study diagnosis, prognosis and localization of endocrine abnor- malities. In addition, they have permitted us to learn a great deal about the normal physiology of hormones-- thei r synthesis, secretion, actions and fate, and factors influencing these events, and about the dynamic pathophysiol- ogy underlying many abnormal endocrine processes.

in relatively few instances, a hormone measurement per se is diagnostic. More often, because of the complex homeostatic system of feedback con- trols, and because our assays may measure multiple hormonal forms rather than a single active principle, it is necessary to relate the measured hor- mone level to other aspects of the clinical situation before it can be of real value: the qualitative nature of the substance measured, and the signific- ance of its quantitation must be fully considered.

RADIOIMMUNOASSAY IN CLINICAL MEDICINE 43

The hormonal substance being measured This may be the hormone itself, its precursor, or a metabolite, or a com-

bination of these. The major metabolic pathways involved in steroid bio- synthesis, secretion and metabolism are well known (Harper, 1971), and the clinical significance of metabolic alterations are appreciated (Bondy, 1969). More recently, the peptide hormones have been shown to occur in multiple forms. Some peptide hormones at least are synthesized in a pro- hormone form, are cleaved within the cells of origin to the hormonal form which is then released under normal c i r cumstances . The hormone circulates and is in turn broken down in peripheral sites to hormonal fragments and cleared from the circulation (see Fig. 1) at various rates. Evidence for

N

CEll CIRCULATION

Fig. 1.

prohormone synthes~o~ has been shown for insulin (Steiner et al., 1968) and for parathyroid hormone (Kemper et al., 1972; Habener et al., 1971). Cleavage of hormone in the circulation into biologically intact and inert fragments, each with its own half-life, has been shown to occur with ACTH and parathyroid hormone (Habener et al., 1971). Production rates of pep- tides may be altered by disease states, or an abnormal form of the hormone may be released by neoplasms into the circulation, as happens in the case of insulinoma (Melani et al., 1970) or parathyroid adenoma (Habener et al., 1971). As in the case of ectopic peptide hormones from non-endocrine tissues this may occur because neoplastic derepression permits synthesis of pro-peptide, but not co-ordinate synthesis of the cleaving enzyme for conversion of pro-hormone to hormone.

Disease may alter measured peptide hormone levels in blood not by effecting production rates, but by retarding or enhancing clearance rates of one or more peptide fragment moieties. For example the half-life of para- thyroid hormone fragments in the plasma is greatly prolonged in chronic renal failure (Potts et al., 1972). Moreover, immunochemical differences

44 IRISH JOURNAL OF MEDICAL SCIENCE--SUPPLEMENT, JULY 1974

between gland extract standard and hormone in blood may cause non- parallelism in assays (Segre et al., 1972) leading to a falsely low (but variable) quantitative result for the circulating hormone level obtained.

Clinical Significance When the exact hormone moiety being measured has been ascertained,

the value obtained may per se be of diagnostic help. Often however it is necessary to relate hormone measurement to "target product" concentra- tion, or to consider hormone production pattern over a time span, or to examine the hormone response to induced alteration in homeostasis.

(a) Hormone level per se (i) Presence diagnost ic : Detection in blood of hormone such as human

chorionic gonadotrophin (HCG) in the male subject (Tashjian et al., 1973) or calcitonin in appreciable amounts (Deftos et al., 1972) is indicative of carcinoma. The finding of a relatively high proportion of a hormone cir- culating in the pro-hormone state, will I feel also turn out to be indicative of neoplastic transformation.

(ii) Normal~abnormal level d iagnost ic : Most hormones have a normal range in the circulation, and disease states are diagnosed by comparing the patients' hormone level with established normal ranges. When so doing, the following points should be considered.

(i) Binding proteins. Hormones such as thyroxine or cortisol circulate both bound to protein and unbound, the protein-bound (inactive) fraction making up most of the measured hormone level. Alterations in binding pro- teins (Ingbar and Braverman, 1966; Biessel et al., 1964) can cause marked alterations in the total hormone levels in blood, although the active (un- bound) hormone fraction remains normal.

(ii) Assay Specif icity. Chemical substances may interfere with hormone assay methods (a good example is the effect of spironolactine on the fluorimetic corticol assay and on urinary 17-keto and 17-hydroxy steroid estimations. (Desper et al., 1966) ). Immunochemical interference may occur in radioimmunoassays and cross-reaction between glycoproteins (T.S.H. and the pituitary and placental gonadotropins. (McArthur and Powell, 1973) ) or bs:we~n growth hormone, prolactin and placental lactogen, in addition to the problem of multiple hormone forms already mentioned.

(iii) End-organ resistance. A hormone deficiency state may in certain instances exist despite the presence of adequate circulating levels of active hormone, b~cause the hormone's target tissues fail to respond to its action. Examples include pseudo-hypoparathyroidism (Albright et al., 1942) or the hypoparathyroidism associated with magnesium deficiency (Muldowney et al., 1970), nephrogenic diabetes insipidus, and some forms of dwarfism. Ob3sity leads to resistance to insulin action (Salans et al., 1968).

(iv) Effect of stress. Stress may cause marked alterations in hormone levels in normal subjects-- far outside 'normal' limits. Growth hormone pro- duction may be stimulated, and, in women, pituitary gonadotropin produc- tion may be suppressed. The pituitary--adrenal axis is profoundly affected by stress--a good practical rule is that one cannot, using hormone assays,

RADIOIMMUNOASSAY IN CLINICAL MEDICINE 45

establish (or exclude) a diagnosis of Cushing's disease during an episode of stress, since the stimulant effect of the stress may override the normal response to suppression tests.

(b) Hormone level relative to target product

For many hormones the concept of 'normal range' is only applicable when the target product is also within its normal range. For example the 'normal' serum insulin levels only apply when blood sugar is within normal limits. In normal subjects, serum insulin levels may well exceed the "nor- mal range" in response to induced hyperglycemia. The concept of this relationship is illustrated schematically for the parathyroid-hormone-calcium axis. Analogous relationships occur in other systems such as the aldoster- one-renin-salt axis.

(c) Hormone pattern over time

For certain tropic hormones and their target-organ hormone products, it is necessary to consider their temporal relationships. In the early morning hours plasma ACTH is elevated and the plasma cortisol low, whereas later in the forenoon the reverse holds true. The absolute and relative normal ranges for the hormones in this feed-back axis change with time of day. Similarly for the gonadotropinoestrogen-progesterone axis--normal ranges and relationships vary with the time of month.

(d) Response to altered homeostasis

Subtle hormonal disorders exist in which the measured hormone levels are normal or equivocal is the basal state, and abnormality only becomes apparent when attempts are made to stimulate or suppress hormone pro- duction.

In mild examples of Cushing's disease the morning cortisol may not be abnormal under basal conditions, but may show a very abnormal lack of response to attempts to suppress cortisol production by means of dexa- methazcne. In mild acromegaly, a lack of HGH suppression in response to a glucose load may be the only striking abnormality. Conversely, mild pit- uitary-adrenal axis deficiency state may be inapparent under basal condit- ions, but obvious under the stress of hypoglycemia during insulin tolerance testing. In attempts to differentiate between endocrine abnormalities of hypothalamic and of pituitary origin, it is only now becoming possible to assay releasing factors in plasma, and the differentiation has ha~ to be made by administering releasing factors (LHRF, TRH) to attempt to stimu- late pituitary hormone production.

Summary Interpretation of hormone assay results requires :

1. Appreciation of the qualitative limitations and advantages of the assay methods used.

2. Knowledge of the complex homeostatic feedback mechanisms which operate, and of the factors which can affect quantitative hormone levels, including target product concentrations, temporal patterns of hormone production, or natural or induced alterations in homeostasis.

46 IRISH JOURNAL OF MEDICAL SCIENCE--SUPPLEMENT, JULY 1974

Re'lerences

Albright, F., Burnett, C. H., Smith, P. H. and Parsons, W. 1942. Pseudohypoparathyroidism. Endocrinology 30, 922.

Biesel, Wr., Di Raimonod, V. C. and Forsham, P. H. 1964. Cortisol transport and clearance. Ann. Int. Med. 60,641.

Bondy, P. K. 1969. Diseases of Metabolism. Ed. by P. K. Bondy. W. B. Saunders Co. p. 827. Deftos, J. T., Murray, T. M., Powe[I, D., Habener, J. F., Singer, F. R., Mayer, G. P. and Potts,

J. T., Jr. 1972. Calcium, Parathyroid Hormone and the Calcitonins. Ed by R. Talmage and P. Munson. Amsterdam. Excerpta Medica p. 161.

Desper, P. C., Flink, E. B. and Jones, J. E. 1966. Interference of spironolactone in 17- hydroxycorticoid and 17-ketosteroid determinations. Metabolism 15, 823.

Habener, J. F., Powell, D., Murray, T. M., Mayer, G. P. and Ports, J. T., Jr. 1971. Parathy- roid hormone: Secretion and metabolism in vivo. Proc. Nat. Acad. Sci. USA. 68, 2986-

Harper, H. A. 1971. Review of physiological chemistry. Ed. by H. All Harper. Lange Medi- cal Publications. p. 441.

lngbar, S. H. and Braverman, L. E. 1966. Methods for assessing the binding of thyroid hormones by plasma proteins. Endocrin. Exper. 2, 235.

Kemper, B., Habener, J. F., Potts, J.T. Jr. and Rich, A, 1972. Pro-parathyroid hormone. Proc. Nat. Acad Sci. USA. 69, 643.

McArthur, J. W., Powell, D. Gonadotropin assays in Hormones in Human Plasma. Ed. by H. N. Antonaides. Vol. 1 (2nd Edn.). Excerpta Medica. (In press).

Melani, F., Ryan, W. G., Rubenstein, A. H. and Steiner, D. F. 1970. Proinsulin secretion by a pancreatic /~-cell adenoma. New Eng. J. Med. 283, 713.

Muldowney, F. P., McKenna, T. J., Kyle, L. H., Freaney, R. and Swan, M. 1970. Parathor- mone effect of magnesium replenishment in steatorrhoea. New Eng. J. Med. 281, 61.

Potts, J. T. Jr., Murray, T. M., Peacock, M., Niall, H. D., Tregear, G. W., Keutmann, H.D., Powell, D. and Deftos, L. J. 1971. Parathyroid hormone: sequence, synthesis, immuno- assay studies. Ann. J. Meal. 50, 639.

Salans, L. B., Knittle, J. L. and Hirsch, I. 1968. The role of adipose cell size and adipose insulin sensitivity in the carbohydrate intolerance of human obesity. J. Clin. Invest. 47, 153.

Segre, G. V., Habener, J. F., Powell, D., Tregear, G. W. and Potts, J. T. Jr. 1972. Parathy- roid hormone in human plasma. J. Clin. Invest. 51, 3163.

Steiner, D. F., Cunningham, D., Spigelman, L. and Aten, B. 1967. Insulin biosynthesis. Science 157, 697.

Tashjian, A. H., Weintraub, B. D., Barowsky, N. J., Rabson, A. S. and Rosen, S. W. 1973. Subunitsof human chorionicgonadotropin. Proc. Nat. Acad. Sol. USA. 70, 1419.

CHAIRMAN Dr. Michael Cullen will open the panel discussion.

THE DIAGNOSTIC VALUE OF RADIOIMMUNOASSAY IN CLINICAL MEDICINE

Michael J. Cullen

Department of Clinical Medicine, Trinity College, and Meath Hospital, Dublin 8.

Mr. Chairman, Dr. Devlin, Dr. Powell, ladies and gentlemen, it is my pleasure to open the discussion on this "Moving Points in Medicine" ses- sion. We have heard Dr. Devlin speak about the clinical scope of radio- immunoassay and something of the clinical significance of circulating hormone substances from Dr. Powell. I will emphasise some points regard-