33 Pituitary function

GLI2

FGF8

LHX3

LHX4

HESX1

SOX2

SOX3

OTX2

Prop1

POU1F1

TBX19

Growth hormone (GH)

In childhood the most prominent feature of GH deficiency is reduced growth. Measurement of IGF 1 is the most used screening test in children. For stimulation of GH release the insulin hypoglycemia test and the GHRH arginine test are used. The GHRH arginine test and the glucagon test are the usual methods for the diagnosis of GH deficiency.

Refer to Chapter 35 – Disorders of the pituitary somatotroph axis.

Adults with GH deficiency have reduced muscle mass and increased mass of fat. Functional tests are necessary for conformation of GH deficiency. The most effective test (highest sensitivity and specificity) are the insulin hypoglycemia test and the GHRH arginine test.

Gonadotropins

In premenopausal women gonadotropin deficiency is associated with menstrual irregularities including amenorrhea and disorders of conception. Low estradiol with coincidentally low FSH are the prominent results.

In men the main signs are hypo gonadotropic hypogonadism and infertility. Low morning serum testosterone level (below 10.4 nmol/L) without increased gonadotropin concentration are diagnostic results.

Prolactin

Low serum concentration of prolactin (below 100 pmol/L) is a reliable indicator of severe hypopituitarism.

Refer to Chapter 36 – Prolactin.

ACTH

Patients with chronic ACTH deficiency present with lethargy, tiredness and weight loss. Patients with a low cortisol concentration (below 80 nmol/L) at 9.00 may have ACTH deficiency. Important functional tests for ACTH deficiency are the short Synacthen test and the insulin hypoglycemia test.

Refer to Chapter 34 – Disorders of the pituitary-adrenocortical axis.

TSH

The TSH deficiency leads to central hypothyroidism. Measurement of TSH and FT4 are important. Low TSH and low FT4 confirm the diagnosis of hypopituitarism.

Refer to Chapter 30 – Thyroid function.

ADH

Central diabetes insipidus causes polyuria and polydipsia.

Refer to Chapter 8.6 – Arginine-vasopressin.

Insulin hypoglycemia test (IHT) /8/

Indication: assessment of the hypothalamic-pituitary axes function and target organs.

Principle: insulin induced hypoglycemia is a strong, nonspecific stimulus to the hypothalamic-pituitary axes. In healthy individuals, adequate hypoglycemia will result in the release of ACTH, GH, TSH, FSH, and LH. One advantage of this test is that all four axes can be assessed simultaneously. An inadequate low increase in secreting target organs is observed not only in the presence of anterior pituitary insufficiency but also in regulatory disturbances of the axes and secretory disturbances of the target organs. The main hormone determined in this test is cortisol but, depending on the clinical indication, pituitary hormones may also be determined.

Test protocol: 0.15 IU of regular (rapid acting) insulin/kg body weight are administered by intravenous bolus injection (0.05 IU/kg body weight in children). If adrenocortical insufficiency is highly likely, only 0.1 IU/kg body weight is administered. In patients with impaired glucose tolerance (Cushing’s syndrome, acromegaly, obesity), a higher dosage may be necessary (0.2 IU/kg body weight). In patients with diabetes mellitus, counter regulatory cortisol and GH secretion may have been disturbed for years due to the metabolic disturbance, thus rendering the insulin induced hypoglycemia test less valuable in these patients. Blood samples for glucose monitoring as well as the determination of ACTH, GH, TSH, FSH, LH, and cortisol are collected before as well as 15, 30, 45, 90, and 120 min. after insulin injection. Most patients will experience symptoms such as sweating, tremor, and hunger. The test should be terminated by intravenous glucose administration if neuroglycopenic symptoms such as confusion and disorientation occur. The test is contraindicated in patients with cardiovascular disease, cerebrovascular disease, and epilepsy. Patients with anterior pituitary insufficiency are subject to the risk of severe hypoglycemic reactions due to inadequate levels of anterior pituitary hormones counteracting the effects of insulin. Therefore, the performance of this test always requires close medical supervision.

Interpretation: the test can only be assessed, if adequate hypoglycemia occurred, i.e., a blood glucose level of less than 36 mg/dL (2.0 mmol/L) or less than 50% of the baseline glucose concentration. A rise in plasma cortisol to a maximum level of ≥ 8 μg/dL (500 nmol/L) is considered to be a normal cortisol response. If this does not occur, this indicates dysfunction of the hypothalamic-pituitary-adrenal axis and probably of other axes and further investigation is necessary. A normal response in the insulin-hypoglycemia test rules out disturbances of the hypothalamic-pituitary axes and secondary insufficiency of the target organs.

Metyrapone test /9/

Indication: secondary adrenocortical insufficiency.

Principle: metyrapone inhibits the adrenal enzyme 11β-hydroxylase, which converts 11-deoxycortisol to cortisol in the final step of adrenal steroidogenesis (Fig. 34.1-2 – Biosynthesis of adrenocortical steroids). 11-deoxycortisol does not have glucocorticoid activity and therefore does not inhibit ACTH production. When metyrapone is given to a normal individual, the decline in serum cortisol stimulates ACTH production, driving adrenal steroidogenesis proximal to the enzyme blockade and causing 11-deoxycortisol to accumulate. When metyrapone is given to a patient with adrenal insufficiency of any type, 11-deoxycortisol fails to increase.

Test protocol: 30 mg of metyrapone/kg body weight (2.5–3 g) is administered orally at 11:00 p.m. together with a snack. A blood sample for the determination of 11-deoxycortisol is collected the following morning at 8:00 a.m.

Interpretation: the metyrapone test was developed specifically as a test of pituitary reserve. The test evaluates the ability of the pituitary to react to a reduction in feedback exerted by 11-hydroxylated steroids by means of an adequate rise in ACTH secretion. The maximum serum level of 11-deoxycortisol is evaluated. A value of ≥ 70 μg/L (180 nmol/L) indicates that the entire hypothalamic-pituitary-adrenal axis is functioning normally. Clinically, the metyrapone test is as valuable as the insulin-hypoglycemia test, although for the patient, it is associated with less risk and fewer side effects.

A subnormal reaction after metyrapone administration suggests the presence of primary or secondary adrenocortical insufficiency but cannot differentiate between the two.

In cases of secondary Cushing’s syndrome, normal or excessive increases in 11-deoxycortisol are observed whereas in primary Cushing’s syndrome due to the presence of an autonomous adrenocortical tumor and in most cases of ectopic ACTH syndrome, no rise in 11-deoxycortisol occurs. In large, hormonally active adrenal tumors, increased synthesis of 11-deoxycortisol rather than cortisol may occur solely due to the effect of metyrapone; this in turn results in a rise in 11-deoxycortisol, which may mimic central Cushing’s syndrome.

Phenytoin may increase the metabolism of metyrapone, thereby reducing metyrapone level and decreasing 11β-hydroxylase blockade. For this reason, plasma cortisol should be determined in patients taking this medication in addition. A morning cortisol level of less than 10 μg/dL (280 nmol/L) is considered evidence of adequate suppression.

Since the metyrapone test may trigger acute adrenocortical insufficiency in patients with a low basal cortisol concentration, this test should be performed on an inpatient basis only.

Corticotropin-releasing hormone stimulating test (CRH test) /9/

Indication: to diagnose and localize adrenal insufficiency.

CRH is a peptide composed of 41 amino acids. CRH is synthesized in neurons of the para ventricular nucleus of the hypothalamus; from there, CRH is transported to the capillary system of the median eminence. Finally, CRH reaches the ACTH secreting cells via the portal system of the anterior pituitary and stimulates ACTH production. CRH is inhibited by cortisol, plasma levels of CRH rise in primary adrenocortical insufficiency.

Principle: the pattern of ACTH and cortisol response to CRH allows differentiation between primary, secondary and tertiary adrenal insufficiency.

Test protocol: a resting period of 2 h should precede the administration of CRH. During this time, serum cortisol decreases markedly in healthy individuals. The lower the initial level of serum cortisol, the more pronounced the CRH induced rise in ACTH and cortisol will be. 1 μg/kg of CRH is administered by slow intravenous injection. Blood samples for the determination of cortisol and ACTH are collected before as well as 15, 30, 60, 90, and 120 min. after CRH injection. A transient hot sensation with flushes occurs occasionally after the injection of CRH. The test should be performed on the awake patient since the ACTH and cortisol response to the administration of CRH may be reduced during sleep.

Interpretation: maximum cortisol concentrations of 18.6–22.3 μg/dL (514–615 nmol/L) largely rule out adrenocortical insufficiency, while concentrations of less than 12.6–15.2 μg/dL (349–420 nmol/L) indicate a very high likelihood of the condition. The pattern of ACTH and cortisol response to CRH allows differentiation between primary, secondary, and tertiary adrenocortical insufficiency /9/.

• Primary adrenocortical insufficiency: high baseline ACTH levels which increase after CRH and then decrease slowly toward the baseline.
• Secondary adrenocortical insufficiency: low baseline ACTH levels which does not respond to CRH. In such cases, a CRH/lysine vasopressin test and an overnight version of the 8 mg dexamethasone test should also be performed, which increases the diagnostic sensitivity to almost 100%.
• Tertiary adrenocortical insufficiency: elevated baseline ACTH levels which do not respond to CRH. The lack of a rise in ACTH in conjunction with high basal ACTH levels suggests the presence of ectopic ACTH syndrome, but it may also be observed in pituitary macro adenoma.

Suppression of the hypothalamic-pituitary-adrenal axis by exogenous glucocorticoid therapy correlates poorly with the size of the administered glucocorticoid dose and the duration of therapy. The extent of suppression in any given patient is therefore unpredictable and should be checked, repeatedly if necessary, by means of a CRH test when glucocorticoid therapy is discontinued /10/. The ACTH stimulation test is not suited for use in this setting since suppression of endogenous cortisol production by the administration of exogenous glucocorticoids occurs primarily as a result of inhibition of pituitary ACTH secretion.

Pituitary stimulation test using several releasing hormones (global stimulation test)

Indication: evaluation of the integrity of the hypothalamic-pituitary axes and target organs. Use of the test is only justified if imaging investigations fail to produce conclusive evidence of e.g., trauma, degenerative process, Sheehan’s syndrome, empty sella syndrome, inflammatory lesion (hypophysitis, tuberculosis, syphilis, encephalitis), histiocytosis X, tumor (craniopharyngioma, pituitary adenoma, hamartoma, hypothalamic astrocytoma, ectopic pinealoma), or congenital disorders (Kallmann syndrome, Prader-Labhart-Willi syndrome, Laurence-Moon-Bardet-Biedl syndrome).

Test protocol: the test should be conducted after a 2-hour rest period, during which time cortisol secretion decreases markedly. The test is performed with the patient in a supine position.

• At 8:00 a.m., after a light breakfast, insertion of an intravenous catheter
• At 10 a.m., intravenous injection of the following releasing hormones: CRH 100 μg; GHRH 1–44 100 μg; TRH 200 μg; LHRH 25 μg (women) and 100 μg (men). The releasing hormones are injected at 30-second intervals in any order.

Blood sampling: for the determination of ACTH (usually dispensable), cortisol, LH, TSH, FSH, prolactin, and GH, blood samples are collected at the following times: –120, –60, 0, 15, 30, 45, and 60 min.; for example, at: 8:00, 9:00, 10:00, 10:15, 10:30, 10:45, and 11:00 a.m.

Serious side effects are rare; occasionally, temporary hot sensation, dizziness, urge to urinate, transient flushes.

Interpretation: the test results clearly indicate whether the baseline and stimulated anterior pituitary hormones fall within the reference interval. Slight impairments of one or more individual pituitary functions are detected as readily as cases of insufficiency affecting the entire pituitary gland. The hormonal reactions observed in the global pituitary stimulation test are not identical to those found in individual stimulation tests using each releasing hormone by itself. Accordingly, the rise in TSH is significantly higher in the global test than in the single TRH test. The global pituitary stimulation test is of little use in the diagnosis of pituitary hyper function because nonspecific effects of individual releasing hormones may occur. Thus, TRH in patients with acromegaly typically leads to stimulation of GH secretion; in hypothalamic-pituitary Cushing’s syndrome, TRH also results in stimulation of ACTH and cortisol secretion.

Lysine vasopressin test /9, 11/

Indication: differential diagnosis of Cushing’s syndrome, pituitary insufficiency.

Principle: vasopressin like CRH stimulates the secretion of ACTH, because certain vasopressin producing neurons in the para ventricular nucleus of the hypothalamus project into the median eminence. There, vasopressin is released and reaches the corticotropin cells via the portal system of the anterior pituitary.

Test protocol: 5 IU of lysine or 8-arginine vasopressin in 50 mL of 0.9% NaCl solution are infused intravenously over 60 min. Blood samples for the determination of cortisol and ACTH are collected 10 min. before, at the start, and 15, 30, 45, 60, and 90 min. after the infusion. Noteworthy side effects include: vasoconstriction with facial pallor, slight elevation in blood pressure (therefore contraindicated in patients with hypertension and with coronary heart disease), urge to defecate, generally tolerable abdominal pain.

Interpretation: lysine or 8-arginine vasopressin administration results in a definite increase in plasma ACTH and cortisol in most cases of secondary Cushing’s syndrome. On the contrary, in primary Cushing’s syndrome no rise in ACTH and cortisol is observed.

A lack of increase in ACTH following the administration of lysine or 8-arginine vasopressin in conjunction with low basal ACTH and cortisol levels indicates pituitary ACTH deficiency. However, due to extensive individual variability in the ACTH response to vasopressin administration, pituitary insufficiency can neither be confirmed nor ruled out in the case of a weak ACTH response. In such cases, an ACTH stimulation test and metyrapone test should also be performed. In primary adrenocortical insufficiency, a rise in ACTH is sometimes observed after vasopressin administration; such a rise, however, cannot be demonstrated in the insulin hypoglycemia test.

ACTH test /9, 11/

Indication: the ACTH test is a diagnostic test for patients suspected of having chronic adrenal insufficiency. Detection of heterozygous or nonclassical congenital adrenal hyperplasia due to deficiency of 21-hydroxylase, 11β-hydroxylase, or 3β-hydroxy steroid dehydrogenase (3β-HSD).

Principle: serum cortisol is measured after intravenous administration of 0.25 mg ACTH.

1-h ACTH test: 25 IE (0.25 mg) of synthetic ACTH 1–24 is administered as an intravenous bolus injection. Blood samples for the determination of serum cortisol are collected before as well as 30 and 60 min. after the injection. In order to differentiate between primary and secondary adrenocortical insufficiency, additional determination of the basal ACTH concentration may be necessary. The ACTH test is not sufficiently sensitive to diagnose secondary adrenocortical insufficiency of recent onset. The functional tests of choice in this case are the insulin hypoglycemia test and the metyrapone test.

8-h infusion test: 50 IU (0.50 mg) of synthetic ACTH 1–24 in 500 mL of 0.9% NaCl solution are administered by infusion over a period of 8 h. Blood samples for the determination of plasma cortisol are collected at baseline, 4, 6, and 8 hours after the start of the infusion. The 8-h infusion test achieves similar cortisol responses and is in fact only indicated if free urinary cortisol is also taken into account as a diagnostic criterion besides serum cortisol.

Interpretation: a peak serum cortisol of > 20 μg/dL (550 nmol/L) 60 min. after ACTH injection rules out adrenal insufficiency. The maximum cortisol concentration and not the relative increase from the initial baseline level is the essential diagnostic criterion. This implies that the test may be conducted independently from the initial baseline cortisol level and thus at any time during the day.

In the case of inadequate cortisol increases, no distinction can be made between primary and secondary adrenal insufficiency based on the cortisol level. When ACTH production is impaired by pituitary or hypothalamic disease, the adrenal gland loses the capacity to respond to exogenous stimulation. Stimulation will only take place after ACTH challenges have been repeated for several days. Differentiation is accomplished much more easily by determining the ACTH concentration before the stimulation test. In primary adrenal insufficiency, ACTH is elevated whereas in the secondary type it is decreased.

In the case of borderline cortisol increases, partial pituitary insufficiency may be present. In this setting, residual ACTH secretion is sufficient to prevent adrenal atrophy. Thus, a subnormal increase in cortisol can take place after ACTH stimulation. These patients are exposed to the risk of insufficient pituitary ACTH secretory reserve and adequate rise in cortisol in response to stress. In such cases, further stimulation tests such as the insulin hypoglycemia test or metyrapone test should be conducted.

Simultaneous determination of the plasma aldosterone concentration may serve as an additional diagnostic criterion. In primary adrenal insufficiency, the adrenal cortex is destroyed, with the result that the aldosterone concentration does not increase after ACTH stimulation. In contrast, in secondary adrenal insufficiency, aldosterone secretion by the renin-angiotensin system remains intact; the aldosterone concentration rises to at least > 50 ng/L (133 pmol/L).

GHRH-arginine test /14/

Indication: suspected growth hormone (GH) deficiency. Clinically this test is considered the best alternative to the insulin hypoglycemia test.

Principle: growth hormone releasing hormone (GHRH) stimulates the release of GH by the pituitary and arginine inhibits the secretion of somatostatin, a physiological inhibitor of GH secretion.

Test protocol: intravenous application over a period of < 30 sec. of 0.1 μg of GHRH/kg body weight, followed by 0.5 g of arginine/kg body weight (maximum 30 g) in 500 mL of 0.9% NaCl solution over 30 min. Temporary flushing is reported as a side effect in about 20% of patients, most commonly older children. The patient must be fasting.

Blood sampling: blood samples are collected for GH determination before as well as 15, 30, 45, 60, and 90 min. after the administration of GHRH.

Interpretation: the levels of GH achieved in GHRH-arginine test stimulation are greater than those in other functional tests. It is to be expected that following the prolonged absence of hypothalamic stimulation by GHRH, an intact pituitary will not be able to secrete sufficient GH in a single test. For this reason, it is advisable to perform the test before and after repeated administration of GHRH (e.g. 1 μg/kg body weight by subcutaneous injection each day for 5 days). In a study /14/ the degree of the GH response in adults to GHRH and arginine was correlated with GH, the age and plasma IGF-I levels. There was a correlation between peak GH after GHRH and arginine and body mass index as well as abdominal circumference.

GnRH test

Indication: to assess the severity of progestin negative amenorrhea in premenopausal women. To differentiate between hypothalamic and pituitary hypogonadism in men.

Principle: Gonadotropin releasing hormone (GnRH) stimulates the pituitary to release LH and FSH.

Test protocol women: a blood sample is collected while 25 μg GnRH is administered simultaneously as an intravenous bolus injection. Another blood sample is collected 30 minutes later. FSH and LH are determined from both blood samples.

Test protocol in men: a blood sample is collected while 100 μg GnRH is administered simultaneously as an intravenous bolus injection. Further blood samples are collected 25 and 45 min. later. FSH and LH are determined from all three blood samples.

Interpretation (women): during reproductive years an increase of 2–8 times the baseline value of LH and 2–3 times the baseline value of FSH is considered normal. An absent or inadequate increase suggests hypothalamic-pituitary dysfunction. In pubertal children, an increase of twice the baseline level of the respective hormone is considered normal.

Interpretation (men): an at least 3-fold increase in LH and an at least 1.5-fold increase in FSH with respect to the baseline level suggests the presence of central hypogonadism.

In general the GnRH test is well suited:

• To differentiate forms of hypogonadism due to hypothalamic causes from those of due to pituitary causes; in the latter cases, no rise in LH and FSH is detectable /15/
• To distinguish between constitutional delays in sexual development (associated with a detectable rise in FSH and LH) and hypo gonadotropic hypogonadism (no detectable rise in FSH and LH) /16/
• To diagnose hyperandrogenemic ovarian failure; excessive LH rises are noted in conjunction with levels already elevated at baseline
• To investigate children with precocious puberty i.e., the appearance of sexual characteristics primary to the age of 8 years in girls and prior to the age of 9 years in boys. In girls with centrally induced precocious puberty, both the basal and stimulated LH levels are higher than normal whereas only the basal levels of FSH are elevated; the stimulated FSH levels, in contrast show the same pattern as in prepubertal girls /17/.