Sunday, May 18, 2014

ITT: What are the hormones involved?

     Have you ever had a feeling of anxiety, loss of emotional control, fatigue, decreased libido, gain of weight, easy bruising and having purple stretch marks on the abdomen? Have yourself checked now. You might have the symptoms of some Pituitary diseases like Cushing's Syndrome and Hypopituitarism.

What to know?

     One test that will detect the pituitary problem is through stimulation tests, particularly Insulin tolerance test. What comes into your mind when you first hear the word "Insulin"? Hmm. All about Diabetes right? Yes it can be. But to inform you, there are deeper things you should know about Insulin.

     Insulin Tolerance Test is the most sensitive and most specific test in determining the function of Growth Hormone (GH) and Cortisol which are both hormones. 

Growth Hormone

Growth hormone is a protein-based peptide hormone. It is responsible for the growth and cell production and regeneration in the body. It is stimulated by the Growth Hormone Releasing Hormone (GHRH) and is produced by the Pituitary gland.


Cortisol is a glucocorticoid hormone that is produced by the zona fasciculata of the adrenal cortex. It is responsible in response to fear and stress as part of the fight and flight mechanism. It is stimulated by Adenocorticotropin Hormone (ACTH). These hormones are produced when the blood glucose levels are lowered.


        Insulin is a peptide hormone which is being produced by the Islets of Langerhans in the pancreas. It is important in utilizing energy and for metabolism. The role of Insulin in the test is to lower down the levels of glucose in the body so that the Growth Hormone and Cortisol will be produced.

Brook, Charles, et. al, Brook’s Clinical Pediatric Endocrinology, 6th edition, page 141
Pagana, Kathleen D., et. al., Mosby’s Diagnostic and Laboratory Test Reference, page 495
Warrel, David A., et. al., Oxford Textbook of Medicine, 4th edition, page 195

ITT: How is the test done?

What to avoid?

There are certain situations wherein the test is not applicable for it could cause harm to their health or it may produce an erroneous result. These are:
  •      Persons who have epilepsy, heart problems, or untreated hypothyroidism
  •      Avoid vigorous exercise for this may produce an erroneous result
  •      Patient should fast for 8 hours
Before the test, the patient should have the knowledge on the procedure being done. The patient should be warn on the possible side effects that may happen. Since hypoglycemia will happen, the patient must be observed for the tendency of fainting. After the test, the patient must be properly taken cared of by observing the punctured site for bleeding and giving something to eat to compensate for the induced hypoglycemia.

What to expect during the test?

    1.        The patient should be lying on the bed.
    2.        On the vein of the arm, the clinician will place a small tube used for collecting blood samples (cannula) and measure the growth hormone, cortisol and glucose concentration. 

    3.          Patients must be hypoglycemic. So, clinician would induced hypoglycemia by giving insulin to the patient. The insulin will cause stress to the body that will stimulate the production of growth hormone and cortisol.
4.           The blood glucose level should be <2.2 mmol/L. If the insulin produced no effect on the blood glucose level, a second dose of insulin may be given.
5.            The blood glucose level should be monitored throughout the test.
6.         After 1-2 hours, the clinician should take blood to know the concentration of growth hormone and cortisol.
7.          The clinician will monitor you for possible side effects and give something to eat to compensate for the induced hypoglycemia.

What to know more?

     When there is a decrease level of glucose in the blood (hypoglycemia), the Hypothalamus will stimulate the production of Cortisol Releasing Hormone (CRH), then the CRH will stimulate the release of Adrenocorticotropin Hormone (ACTH) then the adrenl cortex will release Cortisol. In this test, we are measuring the levels of the Cortisol that is responding in our body during decrease levels of glucose.

    Same as with the Growth hormone. If the body is put under stress (i.e hypoglycemia), the Hypothalamus will stimulate the production of Growth Hormone Releasing Hormone (GHRH) to stimulate the release of Growth hormone by the Anterior Pituitary. 

     A normal cortisol response will rise to 580 nmol/ L or more while a normal growth hormone response must increase to more than 20 mU/L. Results under and over the normal values indicate abnormal production or function of Adenocorticotropin hormone (ACTH) and/or Growth hormone.

These abnormal findings may indicate:
  • Cushing's Syndrome

         Also known as hypercortisolism, a disease wherein there is elevated cortisol levels in the patient’s result. It is caused by taking long-term medications in conditions like Asthma, Rheumatoid Arthritis, and other inflammation containing the hormone (Cortisol).

  • Hypopituitarism
         It is a disease caused by the under production or failure production of the hormones of pituitary gland. The pituitary gland has short supply of pituitary hormones thus affecting the body's routine function such as blood pressure, growth and reproduction. Symptoms may include fatigue, weight loss, decreased sex drive, decreased appetite, facial puffiness and      short stature in children.

ITT: Assays for the hormones

To know the levels of the hormones, we check or test them through:

Chemiluminescence Immunoassay for Growth Hormone

Chemiluminescence Immunoaasay has been considered the diagnostic assay for Growth hormone because it has been shown to be more sensitive than the conventional colorimetric methods, and does not require long incubations or the addition of stopping reagents, as is the case in some colorimetric assays.

·        Microtiter wells (Solid phase)—Monoclonal anti-HGH Antibody
·  Conjugate solution-- Monoclonal anti-HGH Antibody in the antibody enzyme (Horseradish peroxidase)
·        Serum specimen—HGH (Antigen)

If there is the presence of HGH in the serum, after the addition of the Conjugate enzyme solution, it will bind to the antibody on the well and the conjugate enzyme solution that will result in Sandwiching of the 2 antibodies and the antigen. To remove unbound labelled antibodies, the microtiter wells are being washed after the 1 hour incubation. Then, a solution of chemiluminescent substrate is then added.
     The intensity of the emitting light is directly proportional to the amount of enzyme present and to the antigen present in the sample.

1.       In a microtiter well, dispense a 50 uL of the specimen. Mix thoroughly for 10 seconds.
2.      Dispense 100 uL of the conjugate enzyme solution. Mix thoroughly for 30 seconds.
3.      Incubate for 1 hour.
4.     Remove the incubation mixture by emptying the plate content into a waste container.
5.      Rinse and flick the wells 5 times with washing buffer.
6.     Dispense 100 uL Chemiluminescence Substrate solution into the well. Mix for 5 seconds.
7.      Read wells with a chemiluminescence microwell reader 5 minutes later.

    0.5 ng/ml

Normal Values
     In most adult subjects at rest, after an overnight fast, the HGH level in serum is 7 ng/ml or less.

Cortisol ELISA

This test is used for the quantitative determination of cortisol in serum.

·        Microwell titers—Rabbit anti-cortisol antibody coated
·        Conjugate Solution— Antigen Cortisol with Horseradish peroxidise (enzyme)
·        Substrate
·        Serum specimen—Unlabeled antigen (Cortisol)

This test employs competitive binding assay. Competition occurs between an unlabeled antigen and the enzyme-labelled antigen which is the conjugate. If the Cortisol is present in the specimen, during the 45 minutes incubation, it will compete with the enzyme-labelled antigen for the binding to the antibody on the mirowell. After the washing step, a substrate is being added. The absorbance is measured on the microtiter plate reader. The intensity of the color formed is inversely proportional to the levels of Cortisol in the serum.

1.       Dispense 20 uL of the sample in the microwell plate.
2.      Add 100 uL of the conjugate solution.
3.      Incubate for 45 minutes on room temperature.
4.     Wash the wells 3 times with 300 uL of diluted wash buffer and tap the plate firmly against absorbent paper.
5.      Add 150 uL of substrate.
6.     Incubate for 15-20 minutes.
7.      Read the plate on the reader for 450 nm filter.

    0.4 ug/dL

Normal Values;
    3.95-27.23 ug/dL



Tuesday, May 13, 2014

ITT Quiz


Select the correct answer.

  1. What are the two (2) hormones that are being tested in Insulin Tolerance Test?

  2. Growth hormone and Cortisol
    Cortisol and Luteinizing Hormone
    Follicle Stimulating Hormone and Growth Hormone

  3. What particular hormone is responsible in response to fear and stress as part of fight and flig

  4. Growth Hormone

  5. The following are conditions wherein you can't test ITT, EXCEPT:

  6. SLE
    Heart Disease

  7. What should be the blood glucose level upon injecting Insulin in the patient?

  8. <2 data-blogger-escaped-.2="" data-blogger-escaped-br="" data-blogger-escaped-dl="" data-blogger-escaped-mmol=""> 2.5 mmol/L
    <2 data-blogger-escaped-.2="" data-blogger-escaped-br="" data-blogger-escaped-mmol="">
  9. What is the most specific test in diagnosing Cushing's Syndrome?

  10. Corticotropin-Releasing Hormone Stimulation Test
    24-hr Free Cortisol Test
    Dexamethasone Suppression Test

Thank you! :)