For some of the hormones, a substance is given that would normally affect hormone production and then the level of the hormone is measured. For example, if insulin is injected, the levels of ACTH, GH and prolactin should increase. Sometimes, rather than measuring GH levels directly, another hormone, insulin like growth factor I (IGF-1) is often measured. GH is produced in bursts and its levels quickly fall, but IGF-1 levels reflect the overall daily production of GH.

A stimulation/provocation test is a test that is performed to reveal a clinical condition; suspected but not fully manifested clinically.

Any procedure in which a suspected pathophysiological abnormality is deliberately induced by manipulating conditions known to provoke the abnormality.

The test can be either stimulation for hypoactive medical problem, or suppression for hyperactive function.

In general

GH TSH ACTH

Stairs up

IndicationsConfirming suspected diagnosis.Monitoring the course of disease.Following the effect of therapy.Establishing the significance of borderline low values by

stimulation tests.Document the presence of hyperfunction by suppression

tests.Distinguish primary from secondary causes of endocrine

dysfunction.

Clinical applicationsAlmost every system disorder in clinical evaluation of disease

could use a stimulation test.It can be the only test available in certain situations like in “drug

induced diseases” by challenging the patient with the suspected drug.

Stimulation testing is commonly used in endocrinology based on the feedback system.

There are several different types of stimulation testing that can be ordered to help the doctor diagnose child’s medical condition.

All stimulation testing can take several hours to complete. The test should take from 1 to 3 hours, including registration, the test, and a snack afterward.

Most stimulation tests are “fasting,” meaning that child should not eat or drink before the test. Follow specific instructions given about eating and drinking before the test.

The child will need to have an intravenous (IV) line placed to start the testing. Blood samples will be removed from the IV line at different times throughout the test.

Stimulation testing measures the response of certain glands within the endocrine system to different types of hormones. Some of the stimulation tests that might be ordered include:

Growth Hormone Stimulation — used to find out if child’s pituitary gland is producing enough growth hormone

Lupron Stimulation — used to diagnose precocious (too early) or delayed (too late) puberty in boys and girls

Glucose Tolerance — used to rule out diabetes, hypoglycemia and insulin resistance

ACTH Stimulation — used to find out if your child’s adrenal glands are producing enough cortisol and to rule out congenital adrenal hyperplasia

PURPOSEGrowth hormone tests are ordered for the following reasons:

to identify growth deficiencies, including delayed puberty and small stature in adolescents that result from pituitary or thyroid malfunction

to aid in the diagnosis of hyperpituitarism that is evident in gigantism or acromegaly

to screen for inadequate or reduced pituitary gland functionto assist in the diagnosis of pituitary tumors or tumors related

to the hypothalamus, an area of the brainto evaluate hGH therapy

GROWTH HORMONEQuantitative GH

Fasting levelProvocative test for suspected deficiency

Hypoglycemia causes GH levels to riseGive insulin, L-dopa or arginine take samples at 0, 15, 39, 45, 60, 90, and 120 minutesSmall rise partial deficiencyNo rise inability to secrete GH

Suppression testHyperglycemia suppresses GH but not in autonomous or

elevated GHGive glucose

NORMAL RESULTS Results are reported in nanograms per milliliter (ng/ml). Normal

results may vary from laboratory to laboratory depending upon the method used for measurement, but results are usually within the following ranges.

SOMATOTROPIN (hGH): men: 5 ng/ml women: less than 10 ng/ml children: 0–10 ng/ml newborn: 10–40 ng/mlSOMATOMEDIN C: adult: 42–110 ng/ml child: 0–8 years; girls 7–110 ng/ml; boys 4–87 ng/ml 9–10 years: girls 39–186 ng/ml; boys 26–98 ng/ml 11–13 years: girls 66–215 ng/ml; boys 44–207 ng/ml 14–16 years: girls 96–256 ng/ml; boys 48–255 ng/ml

ABNORMAL RESULTSSomatotropin hormone: Excess hGH is responsible for the

syndromes of gigantism and acromegaly. Excess secretion is stimulated by anorexia nervosa, stress, hypoglycemia, and exercise. Decreased levels are seen in hGH deficiency, dwarfism, hyperglycemia, failure to thrive, and delayed sexual maturity.

Somatomedin C: Increased levels contribute to the syndromes of gigantism and acromegaly. Stress, major surgery, hypoglycemia, starvation, and exercise stimulate hGH secretion, which in turn stimulates somatomedin C.

Growth hormone stimulation: Decreased levels are seen in pituitary deficiency and hGH deficiency. Diseases of the pituitary can result in failure of the pituitary to secrete hGH and/or all the pituitary hormones. As a result, the hGH stimulation test will fail to stimulate hGH secretion.

PitfallsFalse negative or positive results if the test is not

conducted properly.Failure to provide safety measures during a challenge

test may harm the patients.Inadequate preparation following the exact procedure

for the test can provide misleading results.Lab errors may confuse the results (wrong sample

labeling, wrong time, poor calibration).

Thyroid hormone production is under the control of pituitary TSH and in turn hypothalamic TRH. Plasma TSH levels normally increase rapidly (2 to 5 minutes) after an IV bolus of TRH, with a subsequent more gradual increase in T3 secretion from the thyroid. The TRH test thus allows the integrity of the thyroid axis to be tested.

Abnormalities of the prolactin response to TRH may occur in pituitary tumors (especially GH or prolactin producing) or in pituitary stalk pathology. Responses are not diagnostically pathognomonic, but indicate axis disruption or dysregulation.

In GH excess states (gigantism/acromegaly), TRH may cause an elevation of GH, but not in normal subjects.

Indications1.To assess the response of pituitary TSH secretion and thyroid

hormone production to stimulation. The main indication is in suspected secondary (pituitary) or tertiary (hypothalamic) hypothyroidism. Less frequently may be of assistance in mild primary hypothyroidism.

2.To assess the response of prolactin to stimulation3.Less commonly used in investigation of gigantism/acromegaly.

Often performed as part of a combined pituitary function test (triple test).

Dose:

200 micrograms/m2 BSA by slow intravenous injection over 1 min.

Sample:

0 min, 15min, 30min, 45min, 60min, 90min and 120min

Analysis:

TSH, Free T3, Free T4, Prolactin

InterpretationA normal response is a rapid rise in TSH, peaking between 10-

30 mU/l at 20-30 minutes, then gradually declining to reach baseline after 2 to 3 hrs. T3 values show a rise, but do not peak until 3-4 hrs (30-70% rise from baseline).

Prolactin levels are age dependent. Above 1 year age, mean basal levels are approximately 240 mU/l, rising to approximately 725 mU/l with TRH stimulation (i.e. a 2-3 fold rise).

In hyperthyroidism (Grave's disease), T3 and T4 are elevated and TSH levels are suppressed and unresponsive to TRH stimulation.

In primary hypothyroidism basal T3 and T4 are low, with elevated basal TSH and an exaggerated TSH response (usually to a peak > 30 mU/l at 30-40 min).

Conti……In secondary (pituitary) hypothyroidism T3 and T4 levels are

likely to be low and there is a poor TSH response and poor T3 response.

In tertiary (hypothalamic) hypothyroidism, an exaggerated and prolonged TSH peak may be seen, and a T3 response occurs. While prolactin responses are variable, an exaggerated prolactin response suggests hypothalamic disease or stalk disruption, owing to loss of inhibitory effects of dopamine.

A poor prolactin response suggests pituitary disease.In normal subjects TRH induces no rise in GH levels, but it

may do so in pituitary gigantism / acromegaly.

Adverse effectsNausea, flushing, dizziness, urinary urgency, unusual taste

in mouth, occasionally headaches. Increases in BP and pulse rate frequently observedCaution in heart failure, myocardial ischemia and asthma.Caution in severe hypopituitarism - risk of hypoglycemiaCertain drugs may diminish response.

Cortisol Cortisol Cortisol

ACTH Fasting Glucose

RationaleACTH is the primary regulator of glucocorticoid production,

and also plays some role in adrenal androgen production.

Synacthen is a synthetic form of ACTH, is used to assess the stimulated cortisol response of the adrenal cortex and is valuable in diagnosing suspected primary adrenal insufficiency.

The test is also useful in suspected secondary or tertiary

adrenal insufficiency since chronic CRH/ACTH deficiency or dysregulation results in temporary quiescence of the adrenal cortex and inability to respond acutely.

Conti…..Furthermore, the test is not reliable in assessing secondary or

tertiary insufficiency within 2 weeks of surgery to the hypothalamic-pituitary region or a major alteration in any glucocorticoid therapy.

In congenital adrenal hyperplasia, Synacthen test is useful in diagnosing milder or rare enzyme blocks by examining ratios of various adrenal steroids to their precursor compounds. The commonest ratio examined is that of 17-hydroxyprogesterone / cortisol in suspected non-classical or simple virilizing CAH or the heterozygote state.

Standard doze of synacthenOver 1 yr: Single IM or IV injection of 250 micrograms.

Under 1 yr: Single IM or IV dose of 125 micrograms.

Alternatively a dose of 250 micrograms/m2 BSA may be used.

Blood samplingSamples are collected at 0 min, 30 min and 60 min.

Cortisol, 17-OHProgesterone, ACTH and Androgens (DHEAS, Androstendione & Testosterone).

InterpretationSerum cortisol rise of > 280 nmol/l with maximal level >

600 nmol/l.

Normal ratio of 17-OHP to cortisol at 30 mins < 0.023.

Ratios up to 0.08 suggest heterozygosity for 21-hydroxylase deficiency.

ratios > 0.1 suggest CAH (21-hydroxylase deficiency).

Adverse effectsHypersensitivity or anaphylactic reaction - rare, but full

resuscitation facilities and drugs must be available.

GnRH (gonadotropin releasing hormone) is a decapeptide secreted by the hypothalamus which stimulates the production and secretion of LH and FSH by the anterior pituitary.

The GnRH stimulation test evaluates the ability of gonadotropes to secrete LH and FSH after exposure to the natural hypothalamic releasing hormone, GnRH, or an analog.

The GnRH test is extensively evaluated for the discrimination of disorders of precocious or delayed puberty and amenorrhea.

The GnRH test is useful to monitor adequate treatment of gonadotropin-dependent precocious puberty.

MethodsThis test may be done with either recombinant GnRH or a GnRH

agonist, such as leuprolide. Several different methods have been used:

Administer 100 micrograms of GnRH (or GnRH analogue) IV x 1 at time 0. Measure serum LH at time -15 minutes, 0, +15, +30, +45, and +60 minutes after GnRH administration.

Administer 25 micrograms/M2 of GnRH IV at 8am after an overnight fast. Measure serum LH and FSH at time 0, 15 min, 30 min, 60 min, and 90 min after GnRH injection.

Administer leuprolide 20 mcg/kg x 1, measure LH and FSH at baseline and after 30 and 60 minutes.

Assay requirementsIn general, assays for LH and sex hormones (estradiol and

testosterone) must be sensitive enough to detect pre-pubertal levels. Prepubertal LH is less than 0.1 IU/L and pre-pubertal E2 is <1 pg/L (undetectable with most available assays).

Immunochemiluminometric assays are much more sensitive than radioimmunoassay for gonadotropin levels in the pre-pubertal, thus these should be used, especially when measuring basal gonadotropin levels.

Interpretation In general, in gonadotropin dependent precocious puberty

(GDPP), basal LH is elevated (pubertal range) and increases further with GnRH stimulation (peak LH >5-8 IU/L suggests GDPP), and in gonadotropin independent precocious puberty (GIPP), basal LH is low (in the pre-pubertal range) and does not increase with GnRH stimulation. In addition, stimulated LH/FSH ratio may be help distinguish GDPP (higher LH:FSH) from nonprogressive PP, which does not require treatment with exogenous GnRH. Otherwise, the FSH values (basal or stimulated) are generally not useful.

With either GnRH or GnRHa tests, the ratio of peak to basal LH is also used to define precocious puberty. In general, a ratio of LH (peak): LH (basal) <3.0 is considered normal and a ratio of LH (peak): LH (basal) >3.0 is consistent with precocious puberty.

Side effectsThe patient may react to the side effect of GnRH, transient

thirst may occur. Abdominal or stomach discomfort, flushing (lasting only a short time), headaches, lightheadedness, nausea may also occur but less common.

Hypersensitivity reactions to GnRH, such as itching, redness or swelling of skin at place of injection, skin rash, breathing difficulty etc may occur but they are rare.

Rarely, GnRH injection has been associated with pituitary apoplexy.