The most important rhythms are circadian and menstrual.
Circadian changes mean changes over the 24 hours of the day-night cycle and is best shown for the glucocorticoid cortisol axis. plasma cortisol levels measured over 24 hours-levels are highest in the early morning and lowest overnight. Additionally, cortisol release is pulsatile, following the pulsatility of pituitary ACTH. Thus ‘normal’ cortisol levels (stippled areas) vary during the day and great variations can be seen in samples take only 30 min apart. The circadian (lightdark) rhythm is seen in reverse with the pineal hormone, melatonin, which shows high levels during dark, though there is no clear clinical role for this.
The menstrual cycle is the best example of a longer (28-day) biological rhythm.
Other regulatory factors
STRESS. Though difficult to define, stress can produce rapid increases in ACTH and cortisol, growth hormone (GH), prolactin, adrenaline and noradrenaline. These can occur within seconds or minutes. SLEEP. Secretion of GH and prolactin is increased during sleep, especially the rapid eye movement (REM) phase.
Testing endocrine function
Ideally, cellular levels of hormones would be measured, but this is currently impossible. Body fluids are the normal substitute and are usually an excellent approximation, but it must be remembered that they do not always reflect the current tissue action of the relevant hormone.
Assays for all important hormones are now available. Obviously the time, day and condition of measurement may make great differences to hormone levels. The method and timing of samples will depend upon the characteristics of the endocrine system involved. BASAL LEVELS are especially useful for systems with long half-lives, e.g. T. and T3• These vary little over the short term and random samples are therefore satisfactory. BASAL SAMPLES may also be satisfactory if interpreted with respect to normal ranges for the time of day/month, diet or posture concerned. Examples are FSH, oestrogen and progesterone and aldosterone. All relevant details must be recorded or the data may prove uninterpretable.
STRESS-RELATED HORMONES (e.g. catecholamines, prolactin, GH, ACTH and cortisol) may require samples to be taken via an indwelling needle some time after venepuncture; otherwise, high levels may be artefactual.
24-Hour collections have the advantage of providing an ‘integrated mean’ of a day’s secretion but are often incomplete or wrongly timed. They also vary with sex and body size or age. Written instructions should be provided. Saliva is sometimes used for steroid estimations, especially in children.
Stimulation and suppression tests
These are valuable in instances of hormone deficiency or excess:
WHERE SECRETORY CAPACITY OF A GLAND IS DAMAGED, maximal stimulation by the trophic hormone will give a diminished output. Thus, in the Synacthen (SYNthetic-ACTH-en) test for adrenal reserve, subject A shows a normal response (stippled area); subject B with primary hypoadrenalism (Addison’s disease) demonstrates an impaired cortisol response to ACTH.
A PATIENT WITH A HORMONE-PRODUCING TUMOUR usually fails to show normal negative feedback. A patient with Cushing’s disease (excess pituitary ACTH) will thus fail to suppress ACTH and cortisol production when given a dose of synthetic steroid, as would normal subjects. the response of a normal subject (A) given 1 mg dexamethasone at midnight; cortisol is suppressed the following morning. Subject B with Cushing’s disease shows inadequate suppression.
The detailed protocol for each test must be followed exactly, since even slight differences in technique will produce variations in results. Details of commoner tests are given in the Appendix.
Measurement of hormone concentrations
Circulating levels of most hormones are very low (10-‘_ 10-12 mol litre:”) and cannot be measured by simple chemical techniques. Radioimmunoassay (RIA), previously by far the most common technique in endocrine assays, is being rapidly supplanted by immunoradiometric type assays (IRMA). These are increasingly being automated and using non-radioactive end-points such as colorimetric. Other techniques include high pressure liquid chromatography (HPLC). RIA has limitations; in particular the immunological activity of a hormone, as used in developing the antibody, may not necessarily correspond to biological activity, and the increasing stringency of Health and Safety requirementshas led to a search for methods not involving radioactivity.
RIA is, however, widely being replaced by IRMAs. These rely on highly specific antibodies (usually monoclonal) that are themselves labelled rather than labelling the hormone concerned. Usually employing a solid-phase system, the principles are otherwise similar to those of RIA, requiring incubation and separation of bound and free fractions, except that the ‘signal’ is obviously proportional to the amount of substance present (i.e. this is a saturation analysis). The label need not be a radioactive label, but may involve a fiuorimetric, colorimetric, chemiluminescent or enzymatic end-point.