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1 ENDOCRINOLOGY SYLLABUS

2 Endocrinology Syllabus Table of Contents General Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 5 Response Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 35 Charts and Data Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 67 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68 Esoterix is a wholly owned subsidiary of LabCorp, and its renowned Endocrine Sciences laboratory operates as a member of LabCorps Specialty Testing Group with a focus on endocrinology. Endocrine Sciences testing can be accessed directly from Esoterix or through LabCorp and any of its regional facilities. 1

3 GENERAL INFORMATION About Endocrine Sciences Services Endocrine Sciences is a research quality reference laboratory specializing in highly specific and sensitive endocrine testing services. By maintaining its singular focus, Endocrine Sciences provides a unique service oriented to the distinct requirements of an endocrinology practice. Endocrine Sciences is an industry leader in developing new, clinically relevant assays and establishing defined reference intervals for pediatric and adult patient populations. Since its inception in 1972, Endocrine Sciences has developed trusted relationships with clients who rely on its services daily. All laboratory testing is conducted by highly qualified professionals, specifically trained to provide knowledgeable, responsive attention to client needs. Endocrine Sciences accessible staff scientists and physicians are available to provide technical consultation and assist with clinical evaluations. Endocrine Sciences goal is to employ technology to provide useful clinical informationnot just laboratory datato affect patient care and the delivery of cost-effective medical treatment in a positive manner. Capabilities Extensive menu of laboratory assays useful in the following areas: Diabetes Bone metabolism Thyroid function Growth Hypertension Adrenal function Sexual development and reproduction Technical and clinical consultation regarding assay methods Comprehensive age-related reference intervals for accurate interpretations Strong pediatric focus Exceptionally sensitive and specific assays 2 ENDOCRINOLOGY SYLLABUS

4 GENERAL INFORMATION Hours of Operation Monday Friday 7:00 am 8:00 pm CST Saturday 8:00 am 5:00 pm CST Address: 4301 Lost Hills Road, Calabasas Hills, Calif 91301 Phone: 800-444-9111, option 1 Fax: 512-225-1253 Lab License Numbers CLIA 05D0663070 CAP 2298301 Medicare 05D0663070 CA CLF2343 FL L800004459 MD 589 NY 3072 PA021822A RILCO00265 ENDOCRINOLOGY SYLLABUS 3

5 SPECIMEN COLLECTION AND SHIPMENTS Special Specimen Collection Procedures For Endocrinology Tests The accuracy of endocrine testing depends on the quality of the specimen submitted. Endocrine specimens must be properly collected, labeled, stored, packaged, and transported. Phlebotomists and specimen managers must carefully adhere to all time limits. Following these instructions properly will assure specimen integrity and contribute to valid, consistent endocrine results. Prior to collecting the specimen, review the specimen requirements listed in the general menu section. Note the proper specimen type to be collected, amount of specimen necessary for testing, and special storage and shipping instructions. The specimen container must be properly identified with the patients full name, identification number, date and time of collection and specimen type. A properly completed test request form (TRF) must accompany each specimen. It is important that the age and/or birthdate of the patient be indicated so that age-adjusted normal values can be provided. CLIA regulations further require that we receive physician signatures on additional tests ordered or test changes not accompanying the original TRF. A complete test menu, including specimen requirements and reference intervals, can be found on the Endocrine Sciences website, www.endocrinesciences.com/test-menu. Salivary Cortisol Saliva should be collected at the precise time(s) prescribed by the physician. Saliva specimens can be collected manually by spitting into a clean sample vial, however the preferred method is to collect with the aid of a Salivette. The Salivette is a device made specifically for the purpose of collecting saliva specimens. If not using a Salivette, be sure to collect at least 1 mL of saliva. Regardless of collection technique, please observe the following guidelines: 1. Collect saliva at the times prescribed by the physician. 2. No food or fluid 30 minutes prior to collection. 3. Do not apply creams or lotions that contain steroids or use steroid inhalers for 24 hours prior to collection. (This is to avoid contamination of the Salivette or collection vial.) 4. Avoid activities that can cause your gums to bleed. This would include brushing or flossing your teeth. 5. Do not collect and submit a sample if your gums or the inside of your mouth is bleeding. Consult your physician if this is a chronic problem. 6. Carefully label the sample vial or Salivette with your name, physicians name, and the time and date of collection. 7. Return the specimen to your physician or LabCorp patient service center within one day of collection. 8. If necessary, salivation can be stimulated with sugar-free KoolAid powder. 4 ENDOCRINOLOGY SYLLABUS

6 SPECIMEN COLLECTION AND SHIPMENTS Urine, 24-Hour Collection Most urine assays require a 24-hour specimen because of diurnal variations in excretion of many hormones. Patients should receive explicit instructions for obtaining a complete 24-hour urine sample. Start the 24-hour period at exactly 7:00 am the first morning by voiding. Discard this first voiding since the urine was formed prior to the collection period. Collect all subsequent voidings until 7:00 am the next day. At exactly 7:00 am the second day, empty the bladder using this last voiding to complete the collection. Refer to specimen requirements for each assay to determine the appropriate preservative. Patients should be given two containers and instructed to avoid direct contact with collection bottles or bags containing preservatives, especially hydrochloric acid. Measure and clearly record the 24-hour volume on the TRF. Submit only the required aliquot of a well-mixed total collection. Test Menu A complete test menu can be found on the Endocrine Sciences website, www.endocrinesciences.com/test-menu. The online test menu contains test-specific information, including test numbers, CPT codes, specimen requirements, and reference intervals. ENDOCRINOLOGY SYLLABUS 5

7 ENDOCRINOLOGY RESPONSE TESTS Endocrinology Response Tests The response tests in this section represent established protocols for endocrine stimulation and suppression tests obtained from the pediatric literature. Note: Some tests may involve considerable risk to the patient. Response tests should be performed under the supervision of licensed physicians, and in some cases, only in a hospital or specialized patient center where constant medical supervision can be maintained and medications are readily available to counteract possible adverse effects. It should be noted that no test has 100% specificity and 100% sensitivity. Variability is present in patients with the same disease, and results obtained must be interpreted in conjunction with the patients history and clinical examination. Hence, interpretations of laboratory tests are guidelines and not absolutes in many cases. Contents ACTH Stimulation Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Prolonged ACTH Stimulation Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Single-dose Dexamethasone Suppression Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Standard Dexamethasone Suppression Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 DEX/CRF Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Single-dose Metyrapone Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Multiple-dose Metyrapone Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 Growth Hormone Stimulation Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Insulin-induced Hypoglycemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 16 L-Dopa/Carbidopa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 17 Clonidine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 18 Arginine Infusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 19 GHRH Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 20 GHRH - Arginine Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Glucagon Stimulation Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 TRH Test for Prolactin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 TRH Test for TSH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 rhTSH Stimulation of Thyroglobulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Gonadotropin-releasing Hormone Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 26 Combined Testing of the Anterior Pituitary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 28 hCG Stimulation Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 29 Calcitonin Stimulation Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 Calcium Infusion Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 31 Tests for Evaluating Suspected Hyperinsulinism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 Diagnostic Fast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 Calcium Infusion Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 33 Oral Glucose Tolerance Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 34 Water Deprivation Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 35 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 37 6 ENDOCRINOLOGY SYLLABUS

8 ENDOCRINOLOGY RESPONSE TESTS Adrenal Stimulation Tests ACTH Stimulation Test (Cosyntropin)1-8,13 Purpose: Evaluation of primary and secondary adrenal insufficiency and disorders of adrenal steroid biosynthesis. Rationale: Cosyntropin is a synthetic derivative of ACTH1-24 which consists of the first 24 amino acids of the N-terminal portion of the molecule. This segment comprises the biologically active region of ACTH. When given to the normal child, Cosyntropin stimulates cortisol synthesis and release from the adrenal cortex. It acts rapidly and is less allergenic than other forms of ACTH. Since the standard dose of ACTH (250 g/1.73 m2) results in supraphysiologic ACTH levels, lower doses of ACTH1-24 (0.1, 0.5, and 1.0 g/1.73 m2) which provide more physiologic stimulation, have been used. Variable results have been reported in the sensitivity of low-dose and high-dose ACTH in diagnosing secondary adrenal insufficiency (ACTH deficiency). Insulin tolerance and metyrapone tests still remain the standards for the evaluation of the hypothalamic-pituitary-adrenal axis. Procedure: The patient may have a fat-free meal prior to the test. (The test is generally performed from 8 am to 9 am.) Draw blood for baseline serum cortisol, other adrenal steroids (if indicated) and ACTH level. Inject 15 g/kg in neonates, 125 g in children 2 years and adults intravenously or intramuscularly of cosyntropin. For intravenous administration, dilute cosyntropin in 2 to 5 mL of sterile normal saline and inject over two minutes. Draw blood for serum cortisol (and other adrenal steroids) 60 minutes after ACTH injection. Specimen Requirements: Recommended: 0 .5 mL serum for each cortisol determination. (More sera will be required for determination of other steroid hormones.) Separate within one hour. Store and ship frozen in plastic vial. 1.0 mL EDTA plasma for ACTH; collect in plastic tube on ice. Separate immediately. Refrigerate during centrifugation and ship in frozen plastic tube. Minimum: 0.1 mL serum for each cortisol determination. Note: This volume does not allow repeat analysis. 0.4 mL EDTA plasma for each ACTH as above. 0.3 mL EDTA plasma for each ACTH determination. Note: This volume does not allow repeat analysis. Interpretation: Generally, cortisol levels will rise at least 7-10 g/dL, and the absolute one-hour cortisol level will exceed 18 g/dL. A normal response effectively rules out primary adrenal insufficiency, since patients with significant adrenocortical destruction cannot respond. Patients with secondary adrenal insufficiency usually show a blunted or absent response to ACTH, but occasionally may have a normal response depending on the dose of cosyntropin used and the duration of ACTH deficiency. If a subnormal response is obtained with an elevated baseline ACTH level, the patient has primary adrenal insufficiency or ACTH unresponsiveness. A subnormal response with a low baseline ACTH level suggests CRF and/or ACTH deficiency. Insulin tolerance or metyrapone stimulation tests are more definitive for evaluating the hypothalamic-pituitary-adrenal axis and should be considered. Estrogens, spironolactone, cortisone, and hydrocortisone can all interfere with the ACTH test by causing abnormally high baseline cortisol levels. The production of other adrenal steroids is also stimulated by ACTH, making it useful for assessment of biosynthetic defects in adrenocortical function. For more detailed evaluation of adrenal steroid biosynthesis and congenital adrenal hyperplasia, see the Esoterix Endocrinology publication, Adrenal Steroid Response to ACTH: Pediatrics. ENDOCRINOLOGY SYLLABUS 7

9 ENDOCRINOLOGY RESPONSE TESTS Prolonged ACTH Stimulation Test1,5,8,13 Purpose: Assessment of the secretory capacity of the adrenal cortex and differentiation of primary and secondary adrenocortical insufficiency. This test is now rarely performed, especially if one obtains a baseline ACTH level with the short ACTH stimulation test. If the ACTH level is inappropriately elevated compared to the cortisol level, the patient has primary adrenal insufficiency or ACTH unresponsiveness. This test may be useful in verifying the rare cases of ACTH unresponsiveness. Rationale: Failure to respond to a single dose of ACTH can be seen not only in primary adrenal insufficiency, but also in cases of secondary adrenal insufficiency, particularly those resulting from long-term, high-dose steroid therapy. (Those patients will have a history of steroid use.) The prolonged ACTH stimulation test is based on the premise that those patients with primary adrenal insufficiency, or a mutation in the ACTH receptor causing ACTH unresponsiveness, will show no response over successive days. Patients with secondary adrenal insufficiency will show progressive increases in serum cortisol and urinary glucocorticoid levels, thus indicating adrenal reactivation. Procedure: Patients may remain ambulatory during this test and on regular diets. Day 1: Obtain 24-hour urine for 17-hydroxycorticosteroids (17-OHCS), urine free cortisol, and creatinine beginning at 8:00 am Day 2: Repeat 24-hour urine for 17-OHCS, urine free cortisol, and creatinine. Day 3: Repeat 24-hour urine for 17-OHCS, urine free cortisol, and creatinine. Alternatively, no urine collections, basal ACTH, serum cortisol level and repeat serum cortisol level at end of ACTH infusion. Give 250 g/1.73m2 synthetic ACTH (cosyntropin) in 250 mL normal saline intravenously over 8-12 hours, beginning at 8:00 am Alternatively, 20 units/m2 ACTH gel may be given intramuscularly every 12 hours. Day 4: Repeat Day 3. Day 5: Repeat Day 3. Some investigators prefer to give dexamethasone (20 g/kg/day) to those children strongly suspected of having primary adrenal insufficiency to prevent adrenal crisis. This will not interfere with the test. Specimen Requirements: A 50 mL aliquot of a well-mixed 24-hour urine for each determination of 17-OHCS, urine free cortisol, and creatinine. Add boric acid (0.5 gm/dL) as preservative. Indicate 24-hour volume. Recommended: 0.5 mL serum for each cortisol determination. Separate within one hour. Store and ship frozen in a plastic vial. 1.0 mL EDTA plasma for ACTH; collect in plastic tube on ice. Separate immediately. Ship frozen in a plastic vial. Minimum: 0.1 mL serum for each cortisol determination. Separate within one hour. Store and ship frozen in a plastic tube. 0.3 mL EDTA plasma for each ACTH on ice. Separate immediately and store frozen in a plastic tube. Ship frozen in a plastic tube. Interpretation: In normal children, there is a 3- to 5-fold increase over baseline urinary 17-OHCS and urine free cortisol, while children with primary adrenal insufficiency do not respond or respond suboptimally. Children with secondary adrenal insufficiency generally exhibit a low baseline 17-OHCS and urine free cortisol level, which increases progressively each day after ACTH is given. Baseline plasma ACTH levels are elevated inappropriately for basal serum cortisol levels in patients with primary adrenal insufficiency and little or no rise in serum cortisol occurs. In normal patients, serum cortisol will peak after four hours of ACTH infusion at greater than 36 g/dL and no further rise is seen. In patients with secondary hypoadrenalism, there is a delayed response with a progressive increase in response on the second and third day of ACTH stimulation. 8 ENDOCRINOLOGY SYLLABUS

10 ENDOCRINOLOGY RESPONSE TESTS Single-dose Dexamethasone Suppression Test1,3,9-13 Purpose: Screening test for Cushings disease and syndrome. Rationale: The single-dose dexamethasone test is commonly used in screening patients suspected of having Cushings disease and syndrome. This test has not been used routinely in children and has not been well standardized in children. Hence, its sensitivity and specificity in children has not been established. There is up to a 30% false-positive rate in adults, especially those with chronic illness, obesity, and psychoses (Pseudo-Cushings syndrome). In normal children, administration of this synthetic glucocorticoid inhibits ACTH secretion and subsequent cortisol production. In patients with Cushings disease and syndrome, effective suppression of cortisol secretion does not occur with glucocorticoid administration. Dexamethasone is the preferred glucocorticoid for this test because it does not interfere with the measurement of cortisol or its urinary metabolites. The drug is administered in the late evening in order to block the early morning ACTH surge. This inhibits the usual morning rise in cortisol in the normal child. The single-dose dexamethasone test is valuable in screening for Cushings disease and syndrome in adolescents and adults because a normal result essentially rules out this diagnosis. Recently Gafni and coworkers at the NIH have demonstrated that a midnight salivary cortisol of greater than 0.27 g/dL identified patients with Cushings disease and syndrome.14 The diagnostic accuracy of midnight salivary cortisol and 24-hour urine free cortisol/m2 appears to be similar.12,14 In general, urine free cortisol four times the upper limit of normal is diagnostic of Cushings disease and syndrome except for patients with Pseudo-Cushings syndrome, ie, patients with psychiatric disorders, morbid obesity, poorly controlled diabetes, etc.12 A positive test requires additional studies to differentiate the causes of Cushings syndrome versus Cushings disease. Procedure: Draw 8:00 am ACTH, cortisol. Give dexamethasone orally at 11:00 pm, 20 g/kg up to 1 mg [or 0.3 mg/m2.9 Draw blood for serum ACTH and cortisol at 8:00 am the following morning prior to food ingestion. Specimen Requirements: Recommended: 0 .5 mL serum for cortisol. Separate within one hour. Store and ship frozen in plastic vial. 1.0 mL EDTA plasma on ice for ACTH. Separate immediately. Store and ship frozen in a plastic tube. Minimum: 0.1 mL serum for cortisol. Note: This volume does not allow repeat analysis. 0.3 mL EDTA plasma for ACTH on ice. Separate immediately and store frozen in a plastic tube. Ship frozen in a plastic tube. Note: This volume does not allow repeat analysis. Interpretation: Normally, serum cortisol levels are suppressed to less than 5 g/dL. False-positive tests occur in 12.5% of adults, while false-negative tests are rare (>2%). If one reduces the plasma cortisol cut-off to 3.6 g/dL or less, the sensitivity improves.10-12 False-positive tests can be seen in obese patients, in those who have had a poor nights sleep, and in patients under acute emotional or physical stress. Patients on dilantin or phenobarbital may have accelerated clearance of dexamethasone leading to a false-positive result. In these patients, the dose of dexamethasone should be doubled. Tegretol has also been shown to interfere with dexamethasone suppression. Acromegaly and Graves disease may also produce false-positive results. If a falsely positive result is suspected, the test should be repeated. If Cushings disease or syndrome is suspected, a 24-hour urine for free cortisol and/or a standard dexamethasone test should be performed to confirm the diagnosis and help differentiate the etiology. The ACTH level may be helpful, since lack of suppression of cortisol with an inappropriately elevated ACTH suggests an ACTH-dependent etiologCushings disease or ectopic ACTH syndrome. A lack of supression with a low or unmeasurable ACTH level suggests an adrenal tumor or nodular hyperplasia. ENDOCRINOLOGY SYLLABUS 9

11 ENDOCRINOLOGY RESPONSE TESTS Standard Dexamethasone Suppression Test1,3,11-13,15 Purpose: Diagnosis and differentiation of Cushings disease and syndrome. Rationale: Excessive circulating glucocorticoids produce Cushings syndrome. This is most commonly seen with exogenously administered glucocorticoids. Endogenous Cushings syndrome may be due to Cushings disease (pituitary hypersecretion of ACTH), adrenal adenomas or carcinomas, ectopic ACTH production, or nodular adrenal hyperplasia. The standard dexamethasone suppression test is used to help establish the etiology of endogenous Cushings syndrome. This test is especially useful in separating Cushings disease from other causes of Cushings syndrome. Procedure: Day 1Collect a 24-hour urine for 17-hydroxycorticosteroids (17-OHCS), free cortisol, and creatinine. The creatinine helps determine the completeness of the collection. Day 2Repeat Day 1 (if collecting urines). Day 3Obtain a plasma ACTH and serum cortisol at 8:00 am, 8:30 am, 9:00 am from indwelling access site (heplock, IV, etc) and/or a 24-hour urine for 17-OHCS, free cortisol, and creatinine. At the beginning of this urine collection and after the basal ACTH and cortisols are obtained, give dexamethasone orally (5 g/kg up to 0.5 mg). Repeat dosage every six hours for 24 hours (20 g/kg/day). Total maximum dose should be 2 mg of dexamethasone daily. Day 4Repeat Day 3. Day 5Obtain 8:00 am plasma ACTH and serum cortisol and/or 24-hour urine for 17-OHCS, free cortisol, and creatinine. At the beginning of this urine collection, give dexamethasone (20 g/kg up to 2.0 mg) orally. Repeat dosage every six hours for 24 hours. (80 g/kg/day) Total maximum dose should be 8 mg dexamethasone daily. Day 6Repeat Day 5 dexamethasone dose. Plasma ACTH and serum at 8:00 am and/or 24 hour urine collection for 17-OHCS, free cortisol, and creatinine. Day 7Plasma ACTH and serum cortisol only. There is no restriction on diet or activity during the entire test period. Note: If collecting plasma ACTH and serum cortisol only, blood may be drawn on Days 3, 5, and 7 only; however, samples on Days 4 and 6 may be informative if results are equivocal on other days. Multiple samples over one hour are helpful, especially on the baseline sample, because cortisol and ACTH levels oscillate both diurnally and during a one-hour period. Specimen Requirements: A 50-mL aliquot of a well-mixed 24-hour urine for each determination of 17-OHCS, free cortisol, and creatinine. Add boric acid (0.5 gm/dL) as preservative. Indicate 24-hour volume. Recommended: 0.5 mL for each cortisol determination. Separate in one hour. Store and ship frozen in a plastic vial. 1.0 mL EDTA plasma for each ACTH determination; collect in plastic tube on ice. Separate immediately. Refrigerate during centrifugation and ship frozen in a plastic tube. Minimum: 0.1 mL serum for each cortisol determination. Separate in one hour. Store and ship frozen in a plastic tube. 0.3 mL EDTA plasma for each ACTH on ice. Separate immediately and store frozen in a plastic tube. Ship frozen in a plastic tube. Interpretation: Normal children (and obese children with elevated baseline 17-OHCS) will suppress their 17-OHCS excretion to less than 3 mg/m2/day on low-dose dexamethasone by day four (second day of low-dose dexamethasone). Plasma cortisol should suppress to less than 2 g/ dL after 48 hours (8 doses) of low-dose dexamethasone, and (similarly) urine free cortisol should suppress. Patients with Cushings disease and syndrome do not suppress to these levels and will have 17-OHCS levels that remain elevated. On high-dose dexamethasone by day six (second day high-dose dexamethasone), patients with Cushings disease will generally suppress urine 17-OHCS by a mean 10 ENDOCRINOLOGY SYLLABUS

12 ENDOCRINOLOGY RESPONSE TESTS of 64% (range 18% to 100%).15 Urine free cortisol in patients with Cushings disease suppressed to 90% (range 40% to100%) after high-dose dexamethasone in one study.15 Plasma cortisols generally suppress to greater than 50% of baseline level in patients with Cushings disease. Patients with Cushings syndrome due to adenoma, carcinoma, or ectopic ACTH production rarely suppress urinary 17-OHCS, free cortisol, or plasma cortisol levels. Patients with severe depression or those taking dilantin may not suppress after low-dose dexamethasone but generally have suppressed 17-OHCS levels after high-dose dexamethasone. The adequacy of urinary collections should be checked by a comparison of the creatinines of the baseline and postdexamethasone collections. Dexamethasone-suppressed CRF Test12,16 Purpose: Yanovski and coworkers reported that CRF administration after two days of low-dose dexamethasone suppression (2 mg/day) in adults can readily distinguish normals and pseudo-Cushings patients from those with Cushings disease.16 Procedure: (In adults) Dexamethasone 0.5 mg orally every six hours for eight doses starting at 12:00 pm x eight doses (48 hours) Administer ovine CRF 1 g/kg by IV bolus injection two hours after the patients last dose of dexamethasone. Draw blood samples for serum cortisol and plasma ACTH at -15, -10, -5, -1 minute before CRH stimulation and at 5, 15, 30, 45, and 60 minutes after CRH. Specimen Requirements: Recommended: 0 .5 mL for each cortisol determination. Separate in one hour. Store and ship frozen in a plastic vial. 1.0 mL EDTA plasma for each ACTH determination; collect in plastic tube on ice. Separate immediately. Refrigerate during centrifugation and ship frozen in a plastic tube. Minimum: 0.1 mL serum for each cortisol determination. Separate in one hour. Store and ship frozen in a plastic tube. 0.4 mL EDTA plasma for ACTH on ice. Separate immediately and store frozen in a plastic tube. Ship frozen in a plastic tube. Interpretation: A serum cortisol above 2 g/dL (38 nmol/L) at 15 minutes after CRH was found in all patients with Cushings disease and in no normal patient or patient with pseudo-Cushings. This test had 100% specificity and 100% sensitivity in this study.14,16 As noted however, there are no data in either normal children or those with Cushings disease. The CRF test has been used without dexamethasone suppression to distinguish Cushings disease from Cushings syndrome in adults.12 Flushing, tachycardia, and hypotension have all been reported after CRF infusion. ENDOCRINOLOGY SYLLABUS 11

13 ENDOCRINOLOGY RESPONSE TESTS Single-dose Metyrapone Test1,6 Purpose: Assessment of the functional integrity of the hypothalamic-pituitary-adrenal axis. Rationale: 11-deoxycortisol (compond S) is converted to cortisol by the adrenal enzyme, 11--hydroxylase, which is selectively inhibited by metyrapone. When metyrapone is given to a normal child, cortisol levels fall, stimulating ACTH production by the pituitary. The increased ACTH levels in turn stimulate the adrenal gland to produce additional 11-deoxycortisol since cortisol production is blocked. Because 11-deoxycortisol exerts no feedback control on ACTH production, ACTH continues to rise in response to metyrapone in the normal individual and results in a sharp increase in the serum 11-deoxycortisol level. The changes in 11-deoxycortisol levels can be measured most reliably in serum. The magnitude of the increase over baseline far exceeds that observed when measuring urinary metabolites. Procedure: Draw baseline ACTH, cortisol, and 11-deoxycortisol. Give metyrapone (30 mg/kg; maximum 3.0 g) orally in a single dose at midnight. Milk or a light snack should be given at the same time to decrease the nausea that often occurs with this drug. At 8:00 am the following morning, draw blood for plasma ACTH, serum cortisol, and 11-deoxycortisol. Specimen Requirements: Recommended: 0 .5 mL serum for each determination of cortisol and 11-deoxycortisol (sompound S, metyrapone test). Separate within one hour. Store and ship frozen in a plastic vial. 1.0 mL EDTA plasma for ACTH. Collect and place on ice. Centrifuge and separate plasma immediately. Store and ship frozen in plastic vial. Minimum: 0.1 mL serum for cortisol; 0.2 mL serum for 11-deoxycortisol. Note: These volumes do not allow repeat analyses. 0.3 mL EDTA plasma for ACTH on ice. Separate immediately and store frozen in a plastic tube. Ship frozen in a plastic tube. Interpretation: The normal response is an increase in serum 11-deoxycortisol to levels greater than 7 g/dL with a rise in ACTH to greater than 100 pg/mL. In patients with primary or secondary adrenocortical insufficiency, 11-deoxycortisol generally will be less than 5 g/dL. If a normal rise in 11-deoxycortisol is not observed, the ACTH and cortisol levels should be checked to confirm adequate metyrapone blockade. Cortisol levels below 5 g/dL usually indicate adequate suppression. Cortisol levels above 10 g/dL in the presence of an inadequate rise in 11-deoxycortisol may indicate ineffective suppression and the test should be repeated. An abnormal metyrapone test does not differentiate primary from secondary adrenal insufficiency. Secondary adrenal insufficiency however, is strongly suggested in those who respond to ACTH stimulation tests but not to metyrapone, and in those who do not have a rise in ACTH with adequate metyrapone blockade. Patients with primary adrenal insufficiency generally have markedly elevated baseline plasma ACTH levels as well as elevated plasma ACTH levels postmetyrapone. Patients on dilantin or other drugs that increase hepatic P450 enzyme activity generally have subnormal increases in 11-deoxycortisol levels after metyrapone administration due to increased metabolism of the drug. An abnormal response may also be seen following glucocorticoid therapy because of suppression of the hypothalamic-pituitary- adrenal axis. Precautions: Metyrapone may produce gastric irritation and consequent nausea and vomiting. Thus, it should be given with milk or food to prevent vomiting. Caution should be observed when administering metyrapone to patients suspected of having primary adrenal insufficiency since there is a risk of adrenal crisis in these cases. Patients of primary adrenal insufficiency generally have markedly elevated baseline plasma ACTH levels. All patients should be tested in the hospital and be observed closely during the test so that supportive measures can be instituted if the patient develops hypotension or vomiting. After the test, one can consider hydrocortisone coverage for the day depending on the patients clinical status. 12 ENDOCRINOLOGY SYLLABUS

14 ENDOCRINOLOGY RESPONSE TESTS Multiple-dose Metyrapone Test, Serum6,17 Purpose: Assessment of the functional integrity of the pituitary-adrenal axis. Rationale: See Single-dose Metyrapone Test. Although most investigators now use the single-dose metyrapone test, others still consider the multiple-dose test to be more reliable because of more effective enzymatic inhibition. This test may be given over one or four days. The multiple-dose metyrapone test was originally standardized on the basis of 24-hour urine 17-hydroxycorticosteroids. Because of the difficulties inherent in collecting 24-hour urines in children for four consecutive days, and the development of radioimmunoassay for plasma 11-deoxycortisol, the one-day test has superseded the four-day test. Procedure: Draw 8:00 am ACTH, cortisol, 11-deoxycortisol levels. Administer metyrapone 15 mg/kg or 300 mg/m2 orally every four hours for six doses from 8:00 am the first morning to 4:00 am the following morning (maximum total dose 3 g/24 hours). Four hours following the last metyrapone dose (at 8:00 am), draw blood for ACTH, cortisol, and 11-deoxycortisol. Specimen Requirements: Recommended: 0 .5 mL serum for each determination of cortisol and 11-deoxycortisol (Compound S, Metyrapone Test). Separate within one hour. Store and ship frozen in plastic vial. 1.0 mL EDTA plasma for ACTH. Collect and place on ice. Centrifuge separate plasma immediately. Store and ship frozen in a plastic tube. Minimum: 0.1 mL serum for cortisol; 0.2 mL serum for 11-deoxycortisol. Note: These volumes do not allow repeat analyses. mL EDTA plasma for ACTH on ice. Separate immediately and store frozen in a plastic tube. Ship frozen in a plastic tube. Interpretation: The normal response is an increase in serum 11-deoxycortisol to levels greater than 7 g/dL with an ACTH rise to greater than 100 pg/mL. In patients with primary or secondary adrenocortical insufficiency, 11-deoxycortisol generally will be less than 5 g/dL. If a normal rise in 11-deoxycortisol is not observed, the cortisol level should be checked to confirm adequate metyrapone blockade. Cortisol levels below 5 g/dL usually indicate adequate suppression. Cortisol levels above 10 g/dL in the presence of an inadequate rise in 11-deoxycortisol and ACTH indicates ineffective suppression. An abnormal metyrapone test does not differentiate primary from secondary adrenal insufficiency. Patients with primary adrenal insufficiency usually have markedly elevated baseline ACTH levels. Secondary adrenal insufficiency is strongly suggested in those who respond to ACTH stimulation tests but not to metyrapone and in those whose ACTH levels do not rise in response to metyrapone. Patients on dilantin or other drugs that increase hepatic P450 enzyme activity generally have subnormal increases in 11-deoxycortisol levels after metyrapone administration due to increased metabolism of the drug. An abnormal response may also be seen following glucocorticoid therapy. Precautions: Metyrapone may produce gastric irritation. Because this is especially common in children, the drug should be given with milk or food. Caution should be observed when administering metyrapone to patients suspected of having adrenal insufficiency since there is a risk of adrenal crisis in these cases. Patients should be tested as inpatients and be observed closely during the test so that supportive measures can be instituted if the patient develops hypotension or vomiting. After completion of the test, one can consider hydrocortisone coverage for the remainder of the day. ENDOCRINOLOGY SYLLABUS 13

15 ENDOCRINOLOGY RESPONSE TESTS Pituitary Stimulation Tests Growth Hormone Stimulation Tests1,18-23 General Comments: Stimulation tests are useful for the evaluation of a child with suspected growth hormone deficiency. They are also widely used to evaluate pituitary function and serve as indicators of destructive lesions of the pituitary or hypothalamus. Occasionally, these tests are used to evaluate the therapeutic response to treatment of acromegaly or gigantism. Stimulation tests are often required because basal growth hormone is usually low and fluctuates periodically throughout the day. Its secretion can be stimulated physiologically (during sleep or after strenuous exercise) or pharmacologically. The measurement of serum IGF-1 and/or IGFBP-3 or sleep or exercise tests are employed by clinicians as screening tests for growth hormone deficiency. It must be emphasized that growth hormone stimulation testing should be considered in patients who have auxological, clinical, and chemical criteria strongly suggesting the presence of growth hormone deficiency. Some investigators18,19,21 have suggested that in a child with no other indication of systematic or endocrine disease, the presence of low levels of IGF-1 and IGFBP-3, along with auxological data consistent with growth hormone deficiency are adequate for diagnosis or at least no worse than the results obtained by stimulation tests for growth hormone deficiency. Nevertheless, many clinicians still feel that definitive testing for growth hormone deficiency requires the use of pharmacologic agents.20,22,23 The most popular and reliable of these agents are insulin, arginine, L-dopa, and clonidine. Although all can elicit growth hormone increases in a majority of patients, a normal child may fail to respond appropriately to any one of these pharmacologic stimuli. Therefore, an inadequate response to a single stimulation test cannot be considered diagnostic of growth hormone deficiency. An inadequate response to at least two stimulation tests, along with auxologic data consistent with growth hormone deficiency, are recommended to confirm a diagnosis. Sex steroids and propranolol (not with insulin) have been found to augment growth hormone release and to decrease the incidence of false-negative responses, especially in patients with delayed adolescence.24,25 Conversely, there are some circumstances in which stimulation can evoke a normal responseeven though physiologic conditions suggest growth hormone secretion is insufficient, such as after radiation treatment for leukemia. In some cases, therefore, it may be necessary to assess endogenous growth hormone (sampling every 20 minutes for 24 hours or 12 hours at night). However, this is difficult and costly to perform. Finally, many drugs, nonpituitary illnesses, and poor nutritional states may alter either the basal level or maximal response to these tests. Obesity or hypothyroidism may cause decreased growth hormone responses to stimulatory agents. Hypothyroidism must be corrected before pituitary function can be reliably evaluated for growth hormone. A blunted response in the obese patient must be viewed with caution. Certainly, the diagnosis of growth hormone deficiency should be viewed with caution in the patient in whom no evidence of an abnormality in the hypothalamus or pituitary is seen on MRI. Insulin-induced Hypoglycemia Test1,21-24 Purpose: Assessment of growth hormone secretion and the hypothalamic-pituitary-adrenal axis. Rationale: See General Comments. Hypoglycemia can promote the release of a number of hormones, including growth hormone, ACTH, and prolactin. The administration of insulin to the normal child will lower blood glucose levels and generally, but not invariably, lead to a compensatory rise in growth hormone secretion. 14 ENDOCRINOLOGY SYLLABUS

16 ENDOCRINOLOGY RESPONSE TESTS Procedure: Perform the test in the morning after an overnight fast or, if the patient is an infant, after a four-hour fast. An indwelling intravenous (IV) line should be placed and maintained patent with normal saline or Ringers lactate solution throughout the entire test so that the test can be terminated promptly, if necessary, with IV glucose. The IV line also may be used for administering insulin and obtaining blood samples throughout the test. Draw baseline 0 time blood samples for growth hormone, glucose, ACTH, and cortisol. If baseline glucose is greater than 70 mg/dL, inject regular insulin, 0.075 or 0.1 units/kg intravenously. When panhypopituitarism is strongly suspected, use 0.05 units/kg IV. For accuracy in administering insulin, dilute insulin 1:10 with sterile normal saline prior to injection. Obtain blood samples for glucose and growth hormone at 15, 30, 45, 60, 90, and 120 minutes after insulin injection. If evaluation of ACTH reserve is desired, additional samples for ACTH and cortisol should be obtained at 0 and 60 minutes. During the test, the patient should be continuously assessed and monitored by a physician in attendance. A solution of 25% glucose in water should be available for IV administration, if needed. At the termination of the test, a meal or intravenous glucose in water should be given immediately. This test may be performed in sequence following an arginine infusion test. (See pages 18-19). Because of the high frequency of false-positive results some investigators recommend administering 40 g/m2 ethinyl estradiol/day, divided into three doses with meals x two days,24 or micronized estradiol valerate 1 mg (up to 20 kg weight), 2 mg (over 20 kg) nightly for three days prior to test25 to facilitate a growth hormone response. Priming with sex steroids is especially helpful in short patients with delayed adolescence, since these patients are functionally growth hormone deficient due to a lack of endogenous sex steroids. Sex steroid priming may be useful in general with other provocative stimuli such as arginine and L-dopa/Carbidopa. Specimen Requirements: Growth Hormone: Recommended: 1.0 mL serum for each determination. Separate within one hour. Store and ship frozen in plastic vial. Minimum: 0.4 mL serum for each determination. Note: This volume does not allow repeat analysis. Glucose: Recommended: 0.5 mL serum for each determination. Collect in a tube with sodium fluoride. Separate within one hour. Store in plastic vial with 5 mg NaF. Minimum: 0.1 mL serum for each determination. Note: This volume does not allow repeat analysis. Recommended: 0 .5 mL for each cortisol determination. Separate in one hour. Store and ship frozen in a plastic vial. ACTH 1.0 mL EDTA plasma; collect in plastic tube on ice. Refrigerate during centrifugation. Separate immediately and ship frozen in a plastic tube. Minimum: 0.1 mL for each cortisol determination. 0.4 mL EDTA plasma for ACTH on ice. Separate immediately and store frozen in a plastic tube. Ship frozen in a plastic tube. Interpretation: It is important that adequate hypoglycemia be achieved to provide the stimulus necessary for growth hormone secretion. Serum glucose levels should fall to values less than 45 mg/dL. Generally, patients will demonstrate mild symptoms of hypoglycemia during the test (nervousness, sweating, or tachycardia). At least one additional growth hormone stimulation test should be performed on those patients who fail to respond to insulin-induced hypoglycemia in order to confirm a diagnosis of growth hormone deficiency. The assessment of GH secretory capacity is complicated because of the episodic nature of GH release from the pituitary. Basal GH levels can exhibit considerable variability throughout a 24-hour period, thus limiting their clinical utility. Alternatively, measurement of GH response to various stimuli has commonly been used to improve the diagnostic assessment of GH secretion. GH response to provocative ENDOCRINOLOGY SYLLABUS 15

17 ENDOCRINOLOGY RESPONSE TESTS stimuli among normal individuals, however, is highly variable. Response values greater than 10 ng/mL have historically been considered to reflect normal GH secretory function, while values below 10 ng/mL have been considered to indicate some degree of GH deficiency. However, it should be noted that this limit is arbitrarily derived. Hence, some clinicians use 7 ng/mL as the minimal normal response in children and adolescents.22-24 A significant percentage of normal controls exhibit response values well below this 10 ng/mL limit. The clinical research literature should be consulted for a more recent detailed review of the interpretation of GH response data.22-27 Precautions: This test is relatively safe and reliable if adequate precautions are observed. Patients will usually have mild symptoms of hypoglycemia during the procedure. Indeed, the absence of any symptoms suggests an inadequate test. However, severe hypoglycemic reactions can occur. Constant medical attention is required during the entire procedure. Preparations for adverse reaction should be made prior to testing and hypoglycemia reversed immediately if confusion, hypotension, loss of consciousness, or seizures occur. This test is contraindicated in patients with a history of hypoglycemia, heart disease, or seizure disorder. L-Dopa/Carbidopa Test1,21-23,28,29 Purpose: Assessment of growth hormone secretion. Rationale: See General Comments. Levo-dopa, a dopamine precursor that crosses the blood-brain barrier, has been used to evaluate growth hormone secretory capacity. Although the mechanism of action is not entirely known, it appears that L-dopa is capable of promoting growth hormone release by either a direct effect on the pituitary, or more likely through stimulation of hypothalamic growth hormone-releasing hormone. It has also been shown to stimulate the release of ACTH and cortisol.28 The manufacture of L-dopa has been curtailed in the US, making it unavailable for growth hormone stimulation testing. However, a combination of L-dopa/carbidopa (250/25 mg) is available under the commercial name of Sinemet (Merck). Carbidopa decreases the activity of dopamine carboxylase, resulting in a longer half-life of circulating L-dopa and, hence, greater potency. This combination (along with propranolol priming) has been used for growth hormone stimulation in children,28 and the subject has recently been extensively reviewed by the Drug and Therapeutic Committee of the Lawson Wilkins Pediatric Endocrine Society.29 Procedure: Perform the test in the morning after an overnight fast. Draw a baseline blood sample for growth hormone, ACTH, and cortisol. Administer L-dopa/carbidopa orally, 125 mg/12.5 for body weight less than 30 lbs, 250 mg/25 for body weight over 30 lbs. Obtain blood samples for growth hormone at 30, 60, 90, and 120 minutes. Samples can be obtained from an indwelling intravenous line which is kept open with normal saline. Some investigators recommend pretreatment with propranolol, 0.75 mg/kg to a maximum of 40 mg, given orally one hour prior to L-dopa/carbidopa administration. This significantly reduces the incidence of false-negative responses. Propranolol should not be given to children with asthma, diminished cardiac reserve, a history of hypoglycemia, or extreme thinness. The serum glucose should be checked prior to propranolol administration, and it should not be given unless the glucose level is normalthat is about 70 mg/dL. Obtain ACTH and cortisol level at 60 minutes. 16 ENDOCRINOLOGY SYLLABUS

18 ENDOCRINOLOGY RESPONSE TESTS Specimen Requirements: Recommended: 1.0 mL serum for each growth hormone determination. Separate within one hour. Store and ship frozen in plastic vial. 0.5 mL serum for each cortisol determination. Separate within one hour. Store and ship frozen in plastic vial. 1.0 mL EDTA plasma for ACTH; collect in a plastic tube on ice. Refrigerate during centrifugation. Separate immediately and ship frozen in a plastic tube. Minimum: 0.4 mL serum for each growth hormone determination. Note: This volume does not allow repeat analysis. 0.1 mL serum for each cortisol determination. Note: This volume does not allow repeat analysis. 0.3 mL EDTA plasma for ACTH on ice. Separate immediately and store frozen in a plastic tube. Ship frozen in a plastic tube. Interpretation: The assessment of GH secretory capacity is complicated because of the episodic nature of GH release from the pituitary. Basal GH levels can exhibit considerable variability throughout a 24-hour period, thus limiting their clinical utility. Alternatively, measurement of GH response to various stimuli has commonly been used to improve the diagnostic assessment of GH secretion. GH response to provocative stimuli among normal individuals, however, is highly variable. Response values greater than 10 ng/mL have historically been considered to reflect normal GH secretory function, while values below 10 ng/mL have been considered to indicate some degree of GH deficiency. This limit is arbitrarily derived. A significant percentage of normal controls exhibit response values well below this 10 ng/mL limit. Some clinicians consider 7 ng/mL to be the minimal normal response to provocative stimuli in children and adolescents. The clinical research literature should be consulted for a more recent detailed review of the interpretation of GH response data.22-27 Patients should be tested while fasting since hyperglycemia will blunt the normal response. As with other tests of growth hormone secretory capacity, a normal patient occasionally will not respond to L-dopa/carbidopa. Therefore, an additional stimulatory test should be used to confirm the diagnosis of growth hormone deficiency. Precautions: Generally, side effects are minor when present. These include nausea or vomiting, vertigo, and mild headaches. Clonidine Test1,27,30 Purpose: Assessment of growth hormone secretion. Rationale: Clonidine, an -adrenergic stimulus, has been used in recent years to evaluate growth hormone secretory capacity. Although the mechanism of action is not entirely known, it appears that clonidine is capable of promoting growth hormone release by a direct effect on the stimulation of hypothalamic growth hormone-releasing hormone. Procedure: Perform the test in the morning after an overnight fast. Start IV and draw a baseline blood sample for growth hormone. Administer clonidine orally, 0.15 mg/m2. Obtain blood samples for growth hormone at 30, 60, 90, and 120 minutes. Samples can be obtained from an indwelling intravenous line, which is kept open with normal saline. ENDOCRINOLOGY SYLLABUS 17

19 ENDOCRINOLOGY RESPONSE TESTS Specimen Requirements: Recommended: 1.0 mL serum for each growth hormone determination. Separate within one hour. Store and ship frozen in plastic vial. Minimum: 0.4 mL serum for each determination. Note: This volume does not allow repeat analysis. Interpretation: The assessment of GH secretory capacity is complicated because of the episodic nature of GH release from the pituitary. Basal GH levels can exhibit considerable variability throughout a 24-hour period, thus limiting their clinical utility. Alternatively, measurement of GH response to various stimuli has commonly been used to improve the diagnostic assessment of GH secretion. GH response to provocative stimuli among normal individuals, however, is highly variable. Response values greater than 10 ng/mL have historically been considered to reflect normal GH secretory function, while values below 10 ng/mL have been considered to indicate some degree of GH deficiency. This limit is arbitrarily derived. A significant percentage of normal controls exhibit response values well below this 10 ng/mL limit. Some clinicians use 7 ng/mL as the lower limit of normal response in children and adolescents. The clinical research literature should be consulted for a more recent detailed review of the interpretation of GH response data.22-27 Patients should be tested while fasting since hyperglycemia will blunt the normal response. As with other tests of growth hormone secretory capacity, a normal patient occasionally will not respond to clonidine. Therefore, an additional stimulatory test should be used to confirm the diagnosis of growth hormone deficiency. Precautions: Generally, side effects are minor when present. These include hypotension and somnolence. Therefore, it is prudent to keep the patient supine during the test to prevent consequences that could result from hypotension and somnolence. Arginine Infusion Test1,18,22,27 Purpose: Assessment of growth hormone secretion. Rationale: See General Comments. It has been shown that a variety of amino acids given intravenously can stimulate growth hormone secretion. Arginine is the most commonly used of these preparations. Its mechanism of action is not entirely clear but may involve adrenergic stimulation of the hypothalamus. Procedure: Perform test in the morning after an overnight fast. Start IV and draw a baseline blood sample for growth hormone and glucose. Infuse a sterile 10% solution of arginine monohydrochloride 0.5 g/kg (maximum dose 20 g) intravenously (IV) over 30 minutes. Obtain blood samples for growth hormone at 30, 60, 90, and 120 minutes. These may be obtained from the indwelling IV line, which can be kept open with normal saline Pretreatment with estrogen two days prior to the test or propranolol at one hour prior to the test may enhance the growth hormone response and decrease the percentage of false-negative response. This test may be performed in sequence with insulin or L-dopa after the arginine test. Note: Studies using L-dopa/carbidopa stimulation after arginine infusion have not been published. Hence, the efficacy and possible side effects are not documented. Specimen Requirements: Recommended: 1.0 mL serum for each growth hormone determination. Separate within one hour. Store and ship frozen in plastic vial. Minimum: 0.4 mL serum for each determination. Note: This volume does not allow repeat analysis. 18 ENDOCRINOLOGY SYLLABUS

20 ENDOCRINOLOGY RESPONSE TESTS Interpretation: The assessment of GH secretory capacity is complicated because of the episodic nature of GH release from the pituitary. Basal GH levels can exhibit considerable variability throughout a 24-hour period, thus limiting their clinical utility. Alternatively, measurement of GH response to various stimuli has commonly been used to improve the diagnostic assessment of GH secretion. GH response to provocative stimuli among normal individuals, however, is highly variable. Response values greater than 10 ng/mL have historically been considered to reflect normal GH secretory function, while values below 10 ng/mL have been considered to indicate some degree of GH deficiency. This limit is arbitrarily derived. A significant percentage of normal controls exhibit response values well below this 10 ng/mL limit.23 Some clinicians use 7 ng/mL as their minimal normal response to provocative stimuli in children and adolescents. The clinical research literature should be consulted for a more recent detailed review of the interpretation of GH response data.22-27 Precautions: Generally, this is a safe test with few side effects. Arginine stimulates release of insulin and glucagon. Serum glucose usually remains normal but hypoglycemia can occur. Arginine should be given with caution to patients with severe hepatic or renal disease and in patients who are acidotic, since arginine monohydrochloride infusion induces acidosis. Growth Hormone-releasing Hormone (GHRH) Test1,27,31,32 Purpose: Assessment of growth hormone secretion. Rationale: See General Comments. GHRH is a potent hypothalamic hormone that stimulates growth hormone secretion. It is available for diagnostic testing as sermorelin acetate (GEREF Serono). Sermorelin is an acetate salt of a synthetic, 29-amino acid polypeptide that is the amino-terminal segment of the naturally occurring human growth hormone-releasing hormone (GHRH or GRF) consisting of 40 and 44 amino acid residues, respectively. Sermorelin increases plasma growth hormone (GH) concentrations by direct stimulation of the pituitary gland to release GH. GEREF appears to be equivalent to GRF (1-40) and (1-44) in its ability to stimulate growth hormone secretion in humans. Maximal stimulatory effect is obtained by an IV bolus dose of 1 g/kg; a dose of 0.1 g/kg is minimally effective. Peak plasma GH responses occur between 15 to 60 minutes following administration. The growth hormone-releasing hormone test may be helpful as a confirmatory diagnostic test for evaluating the ability of the somatotrophs of the pituitary gland to secrete growth hormone (GH) with a high diagnostic specificity (approximately 100%). However, most cases of idiopathic growth hormone deficiency are not due to a lack of the somatotrophs secondary to malformation, destruction, or absence of the pituitary gland but rather to a deficiency of hypothalamic GRF secretion. Therefore, a normal response to the GHRH test does not rule out growth hormone deficiency, and further evaluation of the hypothalamic-pituitary axis is necessary. Procedure: Perform test in the morning after an overnight fast. Sermorelin should be administered in a single IV dose of 1 g/kg body weight in the morning following the overnight fast. Start IV with normal saline. Venous blood samples for growth hormone determinations should be drawn 15 minutes before and immediately prior to sermorelin administration. Administer a bolus of 1 g/kg body weight sermorelin IV followed by a 3 mL normal saline flush. Draw venous blood samples for growth hormone determinations at 15, 30, 45, and 60 minutes after sermorelin administration. Specimen Requirements: Recommended: 1.0 mL serum for each growth hormone determination. Separate within one hour. Store and ship frozen in plastic vial. Minimum: 0.4 mL serum for each determination. Note: This volume does not allow repeat analysis. ENDOCRINOLOGY SYLLABUS 19

21 ENDOCRINOLOGY RESPONSE TESTS Interpretation: A peak GH level of less than 7 ng/mL is diagnostic of growth hormone deficiency. Values above that indicate that further study of the hypothalamic-pituitary unit are necessary.26,27,31,32 Precautions: The administration of sermorelin may be associated with transient warmth and/or flushing of the face, injection site pain, redness and/or swelling at injection site, nausea, headache, vomiting, strange taste in the mouth, paleness, and tightness in the chest. GHRH - Arginine Infusion Test1,26,27,31,33,34 Purpose: Assessment of growth hormone secretion. Rationale: See General Comments. Ghigo and coworkers31-34 have proposed a protocol using both GHRH and arginine to assess the maximal secretory capacity of growth hormone in children. The combination of GHRH and arginine is more potent than either alone and is more reproducible. GHRH is the most potent hypothalamic factor in the natural stimulation of growth hormone secretion and acts by a direct action on the pituitary somatotroph. Its usefulness as a test has been limited by the fact that under normal circumstances the response of the somatotroph to GHRH depends on the interplay with somatostatin and other hypothalamic factors. The use of arginine in addition to GHRH is thought to neutralize these other factors in the response of the somatotroph and lead to a maximum release of GH. Procedure: Perform test in the morning after an overnight fast. Draw a baseline blood sample for growth hormone. Start IV with normal saline. Give GHRH: 1 g/kg IV at 0 minutes followed by arginine monohydrochloride 0.5 g/kg (maximum dose 20 g) from 0 to 30 minutes.26,30,31 Obtain blood samples for growth hormone at 15, 30, 60, 90, and 120 minutes. These may be obtained from the indwelling IV line, which can be kept open with normal saline. Specimen Requirements: Recommended: 1.0 mL serum for each growth hormone determination. Separate within one hour. Store and ship frozen in plastic vial. Minimum: 0.4 mL serum for each determination. Note: This volume does not allow repeat analysis. Interpretation: This combination test attempts to assess the maximum GH secretory capacity of the pituitary. Therefore, the expected normal responses are considerably higher than those observed with traditional GH response tests. Any peak value below 19 ng/mL is considered diagnostic of impaired GH secretory capacity. Thus, the use of this test requires a change in thinking of both the physicians and the third party payers. However, it has the potential advantage that only a few, if any, normal controls (none out of 81 patients tested32,84 exhibit response values below 19 ng/m). It should be noted that this test has not been evaluated as yet in many centers, and further validation will be needed before its use becomes widespread. Precautions: Generally, this is a safe response test with few side effects. Arginine stimulates release of insulin and glucagon. Serum glucose levels usually remain normal, but hypoglycemia can occur. Arginine should be given with caution to patients with severe hepatic, renal disease, or to those who are acidotic. GHRH administration may be associated with transient warmth and/or flushing of the face, injection site pain, redness and/or swelling at injection site, nausea, headache, vomiting, strange taste in the mouth, paleness, and tightness in the chest. 20 ENDOCRINOLOGY SYLLABUS

22 ENDOCRINOLOGY RESPONSE TESTS Glucagon Stimulation Test1,27 Purpose: Assessment of growth hormone secretion. Rationale: See General Comments. Glucagon has been shown to increase growth hormone release and thus has been used as a pharmacologic stimulus for growth hormone testing. It is relatively safe, especially in neonates who may have low glucose levels secondary to their multiple pituitary hormone deficiencies. Procedure: Perform test after an overnight fast or after a two- to three-hour fast in the neonate or infant with low blood sugar. Start IV with normal saline solution. Draw baseline glucose and growth hormone samples. Give glucagon 0.03 mg/kg (maximum dose 1 mg) IM or SC. Obtain blood samples for glucose and growth hormone at 15, 30, 60, 90, 120, 150, and 180 minutes. Specimen Requirements: Recommended: 1.0 mL serum for each growth hormone determination. Separate within one hour. Store and ship frozen in plastic vial. Minimum: 0.4 mL serum for each determination. Note: This volume does not allow repeat analysis. Interpretation: The assessment of GH secretion capacity is complicated because of the episodic nature of GH release from the pituitary. Basal GH levels can exhibit considerable variability throughout a 24-hour period, thus limiting their clinical utility except in neonates where levels are generally elevated above 10 ng/mL.26,27 Response values in neonates are not well documented, but it has been suggested that responses greater than 25 ng/mL are appropriate, while a response of less than 20 ng/mL is suggestive of growth hormone deficiency.26,27 In older patients, response values greater than 10 ng/mL have historically been considered to reflect normal secretory function, while values below 10 ng/mL have been considered to indicate some degree of GH deficiency. This limit is arbitrarily derived, and a significant percentage of normal control children exhibit response values below the 10 ng/mL limit. Hence, some clinicians use 7 ng/mL as their minimal normal response to provocative stimuli in children and adolescents. Growth hormone peak values are generally seen at 120 minutes after glucagon and mean values are reported to be higher than those obtained with arginine or insulin-induced hypoglycemia. Precautions: Generally, glucagon stimulation is a safe response test, as it stimulates the breakdown of glycogen in the liver to glucose and its release with a consequent rise in blood glucose. Hypoglycemia can occur after the peak glucose rise in the first 30 to 60 minutes, and, consequently, serum glucose levels should be monitored closely, especially in the neonate with presumed hypopituitarism. The test should be terminated with feeding or IV glucose if the patient becomes symptomatically neuroglycopenia or the serum glucose decreases to less than 45 mg/dL. Nausea, vomiting, and abdominal pain may also occur as a consequence of glucagon administration. ENDOCRINOLOGY SYLLABUS 21

23 ENDOCRINOLOGY RESPONSE TESTS rhTSH Stimulation of Thyroglobulin (Tg)76-78 Purpose: To detect residual normal thyroid tissue, persistent papillary, and follicular thyroid carcinoma. Rationale: Thyroglobulin (Tg) has been demonstrated to be a valuable clinical marker for periodic assessment of the posttreatment status of thyroid cancer patients. Historically, Tg has been measured with conventional polyclonal radioimmunoassay procedures. New immunometric methods for determining Tg have improved the value of this tumor marker by providing greater sensitivity and faster turnaround time. These new assays, however, are particularly subject to interference from Tg autoantibodies, which occur in a significant percentage of patients with thyroid cancer. Therefore, when evaluating thyroid cancer patients, it is critical that a highly sensitive method be used to determine the presence of antithyroglobulin autoantibodies. Current protocols for Tg testing are offered as a comprehensive thyroglobulin testing service. All samples are first screened with a highly sensitive thyroglobulin autoantibody assay. Samples with undetectable autoantibodies (

24 ENDOCRINOLOGY RESPONSE TESTS Gonadotropin-releasing Hormone (GnRH) Test1,38-40 Purpose: Assessment of pituitary gonadotropin secretion. Rationale: Gonadotropin-releasing hormone (GnRH) is a hypothalamic deca-peptide that stimulates secretion of pituitary LH and FSH. This hormone has been synthesized (Factrel) and can be used to evaluate pituitary gonadotropin reserve. The response to exogenous GnRH in children appears to be dependent on the amount of previous exposure of the pituitary to endogenous GnRH. In prepubertal children, the hypothalamic neurons that secrete GnRH are very sensitive to feedback inhibition by small amounts of gonadal sex steroids. As children approach puberty, hypothalamic sensitivity is believed to diminish gradually, resulting in increased GnRH secretion. With continued exposure to GnRH, the pituitary becomes more responsive and secretes greater amounts of gonadotropins, particularly LH. Thus, endogenous GnRH probably acts as a self-primer and augments pituitary sensitivity to exogenous GnRH as children approach puberty. This likely accounts for the increased response observed in pubertal and adult patients. Traditional GnRH testing has employed six samples collected during a two-hour period. Using two-site ICMA LH and FSH assays allows shorter overall test times, fewer samples, and reduced patient costs. A current protocol requires only a single baseline sample and one other sample collection at 30 or 40 minutes post GnRH administration to assess gonadotropic hormone secretion accurately, albeit multiple samples during one hour may have a higher degree of sensitivity. Procedure: Draw blood for baseline LH and FSH at 0 minutes. Give GnRH, Factrel, 100 g maximum, by rapid (less than five seconds) intramuscularly or subcutaneously. Draw blood for LH and FSH at 30 to 40 minutes after GnRH administration for short test and at 20, 40, and 60 minutes for the standard test. Specimen Requirements: Recommended: 1.0 mL serum for each determination of LH or FSH (2.0 mL for both). Separate within one hour. Store and ship frozen in plastic vial. Minimum: 0.4 mL serum for either LH or FSH (0.8 mL for both). Note: This volume does not allow repeat analysis. Interpretation: The data obtained using new two-site immunoassays have resulted in a major improvement in the value of this stimulation test. Using these assays, basal LH-ICMA values are very low in prepubertal children (

25 ENDOCRINOLOGY RESPONSE TESTS The GnRH test has not proved valuable in differentiating between pituitary and hypothalamic disorders. Absent responses can be seen in patients who have hypothalamic diseases, and conversely, many patients with known pituitary disorders have a normal response to exogenous GnRH. Thus, while the GnRH test can demonstrate that the pituitary is capable of gonadotropin release when stimulated, it cannot differentiate conclusively between pituitary and hypothalamic disorders. The GnRH induced LH release in children with true sexual precocity is in the pubertal range, while it is in the prepubertal range in children with precocious thelarche or adrenarche. During treatment of precocious puberty with GnRH agonists, LH-ICMA progressively decreases to very low levels and responds minimally to GnRH testing. LH-ICMA values of

26 ENDOCRINOLOGY RESPONSE TESTS Combined Testing of The Anterior Pituitary Gland1,44,45 General Comments: Although specific tests for the secretory capacity of the anterior pituitary hormones have been traditionally performed separately, sufficient evidence has now accumulated to justify simultaneous dynamic testing of several hypophyseal hormones. These tests have now been shown to be safe, reproducible, and (most significantly) to yield results identical to those obtained by testing the same pituitary-hypothalamic functions on separate days. Simultaneous challenge with three agentsinsulin, thyrotropin-releasing hormone (TRH), and gonadotropin-releasing hormone (GnRH)may provide information on the secretory capacity of growth hormone (GH), ACTH, TSH, prolactin, LH, and FSH. This procedure may be particularly helpful for evaluating pituitary reserve when the major pituitary dysfunction has been established and when either pre or postoperative assessment or regular follow-ups are sought. The value of TRH and GnRH in the evaluation of short children is questionable, since basal TSH and gonadotropin may be as informative.45,82 Since the combined test can be completed within three hours in a single session, it offers a timely and cost-effective method for hypothalamic- pituitary evaluation. Procedure: Place an indwelling catheter in a peripheral vein. Keep the line open by means of a very slow saline drip or a heparin lock. Obtain baseline samples for growth hormone, TSH, LH, FSH, prolactin, cortisol, ACTH, and glucose.. If baseline glucose is normalgreater than 70 mg/dL, inject (IV) regular insulin (0.075 or 0.100 units/kg).* If hypopituitarism is suspected, use 0.05 units/kg. TRH (5-7 g/kg to a maximum dose of 200 g), and GnRH (100 g) consecutively as close together as possible, by means of three separate syringes. Obtain blood samples at 0, 30, 60, 90, and 120 minutes. * When hypopituitarism is strongly suspected, use 0.05 units/kg IV. For accuracy in administering insulin, dilute insulin 1:10 with sterile normal saline prior to injection. Specimen Requirements: Specimen requirements for each time point may be calculated by adding the serum volume required for all hormones to be measured. Prolactin 0.1 mL serum Growth Hormone 0.3 mL serum TSH 0.2 mL serum Cortisol 0.1 mL serum FSH 0.3 mL serum LH 0.3 mL serum ACTH 0.4 mL EDTA plasma To evaluate all hormones, collect enough blood to yield 1.4 mL serum at each time point and 0.4 mL EDTA plasma for ACTH determination at each time point. Separate serum within one hour. Store and ship frozen in plastic vials. In order to avoid confusion when interpreting results, clearly mark each vial with the time of collection and the tests desired. Interpretation: Criteria for the interpretation of results obtained from the combined test are the same as indicated for the corresponding tests when carried out separately. Only two interactions have been observed between agents used in the combined assessment, neither of which affects the diagnostic usefulness of the test. Some preparations of TRH induce the release of small amounts of FSH but not LH in normal male subjects. However, the range of FSH response to GnRH is wide enough to obviate the significance of this small variation. Secondly, normal females may display an enhanced response of GH to the combined challenge compared to that observed during insulin-induced hypoglycemia alone. Since any GH peak level greater than 7-10 ng/mL is considered normal, further possible enhancement of GH by combined testing may have little effect on the interpretation. ENDOCRINOLOGY SYLLABUS 25

27 ENDOCRINOLOGY RESPONSE TESTS A summary of the suggested criteria for normal response under each separate test is provided in the table below: Normal Responses For Combined Testing Of The Anterior Pituitary Measured Insulin Provocative Agent TRH GnRH Hormone GH * S ee interpretation under insulin-induced hypoglycemia Cortisol * Peak >20 g/dL or an increase >8 g/dL or 2x normal baseline (8-12 g/dL) if not already elevated in high normal range. TSH 10-30 U/mL above baseline PRL Peak response >20 ng/mL or 3x baseline FSH Variable, depending on age and stage LH of sexual development *Interpretation of cortisol and GH is dependent on achieving an adequate degree of hypoglycemia. Serum glucose levels should fall at least 50% from baseline levels (usually to values less than 45 mg/dL). *See Precautions for Insulin-induced Hypoglycemia test on page 29. Gonadal Stimulation Tests hCG Stimulation Test1,46-49 Purpose: Assessment of gonadal Leydig cell responsiveness in males with suspected Leydig cell agenesis or hypoplasia; differentiation of anorchia and undescended testes; diagnosis of 5--reductase-2 deficiency and biosynthetic errors of testosterone synthesis. Basal anti-Mllerian hormone (AMH) and inhibin B levels are accurate discriminators of Sertoli cell integrity. They can be used to discriminate between anorchia, dysgenetic testes, and functional but undescended testes.50 Rationale: Human chorionic gonadotropin (hCG) is a glycoprotein that possesses biologic and immunologic similarities to luteinizing hormone (LH). Like LH, it can stimulate Leydig cell function in males and increase testosterone production. Low testosterone output, resulting from testicular failure, usually leads to increased LH release. Since there is no precise inverse correlation between testosterone and LH in hypogonadal males, LH levels are frequently not a reliable index of gonadal failure. FSH and inhibin B are better. Moreover, in prepubertal children between the ages of three and nine years, central neural inhibition of gonadotropin secretion can mask the lack of gonadal feedback on gonadotropin secretion so that even girls with gonadal dysgenesis and boys with anorchia may have normal LH and FSH levels at these ages. Consequently, a dynamic stimulation test is required to elucidate suspected gonadal deficiency. In the normal male, the testes respond to hCG with a pronounced increase in synthesis and release of testosterone. In females, ovarian steroid synthesis is primarily FSH-dependent, therefore, this test is not employed in them. Several hCG stimulation test protocols are available. Generally, the testosterone response to hCG increases in proportion to the daily dose and to the number of injections. The protocol outlined below, employing three hCG injections, was chosen because of its relative simplicity and the availability of reliable data on age-related normal responses. Detailed normal response data have also been established for prolonged tests consisting of seven alternate-day injections of hCG.46 26 ENDOCRINOLOGY SYLLABUS

28 ENDOCRINOLOGY RESPONSE TESTS Procedure: Draw a baseline blood sample for testosterone, FSH, LH, and (where appropriate) dihydrotestosterone (DHT) and intermediates. Inject 1500 IU hCG IM every other morning on days 1, 3, and 5. Alternatively, hCG, 5000 IU/m2 can be injected intramuscularly in a single dose. Twenty-four hours after the third injection (day 6), or 72 hours after the single injection, draw a blood sample for testosterone and DHT and intermediates if desired. Specimen Requirements: Recommended: 1.0 mL serum for each testosterone determination. Separate within one hour. Store and ship frozen in plastic vial. Minimum: 0.5 mL serum for each determination. Note: This volume does not allow repeat analysis. Interpretation: Normal males respond to hCG administration with a significant rise in serum testosterone levels; however, that response is age- dependent. Infants younger than six months of age may exhibit relatively high baseline levels of testosterone with a brisk response to hCG. Later on during the first year of life, both baseline and hCG-stimulated testosterone levels decline and increase again only during puberty. A table of age-related expected values is given below. Testosterone and DHT responses were obtained after administration of three 1500 IU doses of hCG (no normative data for intermediates with the one and three dose hCG test are available). A normal response to hCG may be seen in patients with unilateral or bilateral undescended testes, while lack of response is compatible with anorchia or Leydig cell agenesis. A reduced response is generally observed in Leydig cell hypoplasia or other causes of primary hypogonadism. The hCG test is also helpful in the differential diagnosis of male pseudohermaphroditism when 5--reductase-2 deficiency and abnormalities in testosterone synthesis are suspected. Patients with 5--reductase-2 deficiency exhibit impaired conversion of testosterone to dihydrotestosterone, but the basal unstimulated testosterone:DHT ratio is frequently not high enough to establish the diagnosis unequivocally. However, following hCG administration, testosterone increases in both normal boys and those with 5--reductase-2 deficiency, whereas dihydrotestosterone rises substantially only in normals. In normal prepubertal males, the mean T:DHT ratio following hCG stimulation is 10.7 with ranges from 3.5-14. In male infants, the stimulated ratio is somewhat lower, and is usually less than 10. The T:DHT ratio in prepubertal patients with 5--reductase-2 deficiency generally exceeds 30. In adults, the discriminatory value of the post hCG T:DHT ratio is even higher. Normals respond with ratios from 8 to 16, while patients with 5--reductase-2 deficiency exhibit T:DHT ratios from 35 to 84. Testosterone And Dht Response To 3X1500 Iu Doses Of Hcg BASELINE POST-hCG STIMULATION Normal Subjects Testosterone DHT T:DHT Testosterone DHT T:DHT (Males) (ng/dL) (ng/dL) Ratio (ng/dL) (ng/dL) Ratio Infants 1 wk - 6 mos. Mean 190 43 4.4 395 76 7.2 (N = 16) Range 4 - 530

29 ENDOCRINOLOGY RESPONSE TESTS Calcitonin Stimulation Tests General Comments51-54,80,81: Calcitonin is a 32-amino-acid peptide secreted by C-cells of the thyroid. Measurement of calcitonin has been found to be of value in detecting C-cell hyperplasia and subsequent medullary carcinoma of the thyroid. Elevated calcitonin levels are not specific for medullary carcinoma and may be found in renal failure, pregnancy, subacute thyroiditis, pernicious anemia, cystic fibrosis, various bone diseases, a variety of tumors (particularly oat cell carcinoma of the lung), and infants in the first year of life. Medullary thyroid cancer (MTC) occurs as a sporadic disease in approximately 70% of cases (1%-7% have RET mutations) and familial MTC accounts of the remaining 30%, with a very high percentage of RET mutations. The RET proto-oncogene is a receptor protein tyrosine kinase encoded on chromosome 10. Germline mutations in this gene have been shown to lead to familial MTC development in cells derived from neural crest tissue (C cells), as well as hyperplasia or tumor formation in the adrenal medulla and parathyroid glands (MEN 2A, 2B). Somatic mutations in C cells are found in sporatic forms of MCT. Calcitonin levels are elevated early in the development of MTC when premalignant C cell hyperplasia is present. Although the majority of patients with this tumor have clearly elevated calcitonin levels, a significant number of patients with the RET proto-oncogene mutations have borderline or normal calcitonin levels. In the past, stimulation tests with pentagastrin and calcium have been useful in detecting early abnormalities of calcitonin secretionoften before clinical evidence of the tumor is present. Further, because medullary carcinoma of the thyroid commonly occurs in families and has an autosomal-dominant mode of inheritance with variable penetrance, provocative tests with pentagastrin and calcium have been useful in the past for screening family members. Recent studies suggest, however, that molecular genetic analysis of the RET proto-oncogene is superior to biochemical screening and should be performed at an early age, before one year of age for children in families with RET mutations. It should be emphasized that when a familial form of MTC is suspected, all family members should be screened for a RET germ cell line mutation with analysis of exons 10-16, and, if they are negative, the remaining 15 RET exons should be examined. If no RET mutation is found, a small risk of hereditary MTC exists, 0.18%. If clinically indicated, provocative calcitonin screening can be considered in these patients; however, at present, pentagastrin is not commercially available. Calcium infusion can be used for screening, since calcium has been shown to augment calcitonin secretion. Basal and calcium- infusion-stimulated calcitonin should not be used in place of RET molecular screening. Calcium Infusion Tests51-54 Purpose: The detection of medullary thyroid carcinoma. Rationale: Calcium has been shown to increase calcitonin levels above the normal range in patients with C cell hyperplasia or early medullary carcinoma who have normal baseline calcitonin levels. The calcitonin response can also be used postoperatively to detect residual medulary thyroid carcinoma or recurrence of the tumor. Procedure: The patient is NPO overnight (shorter in young children). Start IV with normal saline and draw baseline calcitonin level. With the patient supine, be sure IV is securely in place in a large peripheral vein and that it flushes well since extravasation of calcium can result in a severe local reaction and necrosis. Infuse 2 mg/kg of elemental calcium as calcium gluconate (10% calcium gluconate = 100 mg/mL = 9 mg elemental calcium/mL). Dilute 10% calcium gluconate to 2% solution (1 part calcium gluconate to 4 parts normal saline) and infuse carefully over 60 seconds. 2 mg of elemental calcium/kg can be given as calcium chloride (27% elemental calcium) or calcium acetate (13% elemental calcium) Monitor blood pressure and pulse during infusion. Draw blood samples for calcitonin at 0, 1, 2, 5, 10, and 30 minutes. 28 ENDOCRINOLOGY SYLLABUS

30 ENDOCRINOLOGY RESPONSE TESTS Specimen Requirements: Recommended: 1.0 mL serum for each calcitonin determination. Separate within one hour. Store and ship frozen in plastic vial. Minimum: 0.5 mL serum for each determination. Note: This volume does not allow repeat analysis. Interpretation: Calcitonin measurements are method and laboratory dependent. Thus, the results must be interpreted with this caveat in mind. Normal calcitonin levels in adults are less than 10 pg/mL using two-site immunometric assays. Calcitonin levels are lower in females than males. At present, there are no published data for calcium-stimulated calcitonin levels in children. No data using two-site assays have been published. Adult normals demonstrated a two-fold to five-fold increase in calcitonin when measured by RIA.84 Gender-related differences were not examined. Precautions: Infiltration of calcium into subcutaneous tissues will cause a severe local reaction and may result in necrosis at the site. Calcium infusion may cause flushing, a feeling of warmth, an urge to urinate, and a sensation of gastric fullness lasting 1 to 5 minutes. Tests For Evaluating Suspected Hyperinsulinism General Comments1,55-59: Hyperinsulinism is the most common cause of hypoglycemia in infants and children after the first day of life. Recent studies have defined the pathophysiology of basic genetic forms of persistent hypoglycemia due to hyperinsulinism.55-58 These include mutations in the sulfonylurea receptor, the inward-rectifying 6.2 kDA potassium channel, glucokinase and glutamate dehydrogenase gene.57,58 Patients with mutations in the glutamate dehydrogenase gene are leucine-sensitive and have elevated ammonia levels.57,58 Hyperinsulinism should be suspected in a child who develops hypoglycemia a few hours after a meal and is not acidotic (increased lactate) or ketotic (increased -hydroxybutyrate or aceto-acetate levels). Mid-line defects, such as cleft lip and/or palate, optic hypolasia, and micropenis are seen in patients with hypopituitarism. Critical for the diagnosis of any form of hyperinsulinism is the demonstration of a measurable insulin level greater than 2 U/mL in the face of hypoglycemia (a serum glucose below 50mg/dL). In addition, hyperinsulinism is associated with low ketone bodies and free fatty acids at the time of hypoglycemia, as well as a glycemic response of greater than 30 mg/dL to glucagon stimulation when hypoglycemic. Patients with hyperinsulinism usually require greater than normal amounts of glucose (5-10 mg/kg/minute in the neonate) to maintain their blood sugars in the normal range. At the time of hypoglycemia it is essential to obtain a critical sample of blood for intermediate metabolites and hormones in order to clarify the etiology of the hypoglycemia. However, rarely are the appropriate tests done at the time of the first spontaneous hypoglycemic episode. Therefore, it may be incumbent on the physician to do a careful, closely observed diagnostic fast in order to obtain these samples. The diagnostic fast to obtain critical samples lasts as long as necessary to achieve hypoglycemia (serum glucose less than 50mg/dL) and may in adults entail a 48- to 72-hour fast. Since there are questions about the safety of diagnostic fasts in patients with -oxidation defects, total and free carnitine and acetylcarnitine profile levels should be ascertained in any child with nonketotic hypoglycemic epidsodes or with a suspected fatty acid oxidation defect before a fast is attempted. ENDOCRINOLOGY SYLLABUS 29

31 ENDOCRINOLOGY RESPONSE TESTS Diagnostic Fast55-59 Procedure: NPO: In infants and children it is necessary to have an IV access site available in case symptomatic hypoglycemia occurs. Monitor glucose every 30 to 120 minutes depending on age, starting serum glucose and clinical history until glucose decreases to less than 50 mg/dL. At the time of hypoglycemia, draw critical sample for glucose, lactate, pyruvate, -hydroxybutyrate, free fatty acids, ammonia, insulin, C peptide, cortisol and growth hormone. Obtain urine for ketones, reducing substances, amino and organic acids. Thereafter give glucagon: neonates 0.3 mg/kg, children 0.1 mg/kg (maximum dose 1 mg) IV Obtain serum glucose at 10, 20 and 30 minutes. In the event of symptomatic hypoglycemia (ie, a seizure), administer 2 mL/kg 10% glucose (0.2 g/kg) of glucose IV over 1 minute and then 5mL/kg/hr (8 mg/kg/minute) of 10% glucose or more IV as needed to maintain the glucose in the normal range. Specimen Requirements: Recommended: Insulin: 1.0 mL for each insulin determination. Separate within one hour. Store and ship frozen in a plastic vial. Cortisol: 0.5 mL for each determination. Separate within one hour. Store and ship frozen in a plastic vial. Growth hormone: 1.0 mL for each determination. Separate within one hour. Store and ship frozen in a plastic vial. C-peptide: 1 mL EDTA plasma. Separate within one hour. Store and ship frozen in a plastic vial. Minimum: Insulin: 0.2 mL serum Cortisol: 0.1 mL serum Growth hormone: 0.4 mL serum C-peptide: 0.1 mL EDTA plasma Note: These volumes do not allow repeat analysis. Interpretation: The criteria for diagnosing hyperinsulinism using the critical sample when the glucose is less than 50 mg/dL is (1) an insulin level greater than 2 U/mL, (2) decreased free fatty acids

32 ENDOCRINOLOGY RESPONSE TESTS Specimen Requirements: Recommended: 0.5 mL serum for each determination. Separate within one hour. Store and ship frozen in plastic vial. Minimum: 0.2 mL serum for each determination. Note: This volume does not allow repeat analysis. Interpretation: Patients with diffuse hyperinsulinism caused by SUR mutation or KIR6 mutations have significant increase in insulin with calcium infusion (8/9 patients).60 Patients with focal disease and glutamate dehydrogenase deficiency have a slight rise or fall in insulin. This test, along with intra-arterial infusion with selective pancreatic venous sampling may help to differentiate focal from diffuse hyperplasia of the pancreas in the infant with profound and persistent hypoglycemia who is unresponsive to medical therapy and thus a candidate for surgical resection of the pancreas. Tolbutamide has also been used as a diagnostic test in infants to distinguish focal from diffuse disease. However, IV tolbutamide is not available at present.62 No data using two-site assays have been published. Adult normals demonstrated a two-fold to five-fold increase in calcitonin when measured by RIA.84 Gender-related differences were not examined. Precautions: Infiltration of calcium into subcutaneous tissue will cause a severe reaction and may result in necrosis at the site. Calcium infusion may cause flushing, a feeling of warmth, an urge to urinate, and a sensation of gastric fullness lasting 1 to 5 minutes. Oral Glucose Tolerance Test (OGTT)63-69 Purpose: Diagnosis of impaired glucose tolerance (IGT). Rationale: Following a glucose load in normal subjects, blood glucose rises leading to a prompt increase in circulating insulin. This in turn enhances glucose entry into cells, which promotes glycolytic metabolism and limits the extent and duration of hyperglycemia. In diabetic patients, however, the insulin response may be delayed, insufficient or completely blunted allowing blood glucose to rise above the normal range. In type 1 diabetes where clinical symptoms are usually manifest, there is little indication for performing an OGTT. The ADA diagnostic criteria63,64 for diabetes are: a fasting blood glucose of greater than 126 mg/dL (7.0 nM/L) and a two-hour postprandial blood sugar of greater than 200 mg/dL (11.1 nM/L). The World Health Organization criteria65 are identical. In patients with obesity or other factors predisposing to type 2 diabetes, OGTT with concomitant measurement of plasma insulin may help identify those patients with secretory abnormalities of insulin, as well as those with resistance and covert type 2 diabetes. The oral glucose tolerance test is the most sensitive method of detecting early diabetes. In a recent study, impaired glucose tolerance was detected in 25% of 55 obese children and 21% of 112 obese adolescents. In addition, silent diabetes was diagnosed in four adolescents (4%). There is no question that insulin resistance is a transitional phase in the development of type 2 diabetes. There is rarely an indication in childhood to do an OGTT to rule out hypoglycemia, since reactive hypoglycemia rarely occurs in childhood and flat glucose tolerance tests are common. Procedure: The test should be delayed for at least two weeks following any acute illness. Many drugs are known to interfere with either the laboratory test for serum glucose or are associated with impaired glucose tolerance and should thus be avoided whenever possible. Physical activity should be encouraged in the days preceding the test. Children should be maintained on a diet in which 50% of the calories are consumed as carbohydrates or a minimum of 150 grams of carbohydrates daily. Quiet activity is permitted during the test. Following an overnight or 12-hour fast, a baseline blood sample for glucose, insulin, proinsulin, and lipid profile is obtained. Glucose, 1.75 gm/kg of ideal body weight up to 75 grams, is given orally. Blood samples for glucose and insulin are collected at 15, 30, 60, 90, 120, 150, 180, 240, and 300 minutes. ENDOCRINOLOGY SYLLABUS 31

33 ENDOCRINOLOGY RESPONSE TESTS Specimen Requirements: Insulin: Recommended: 0.5 mL serum for each determination. Separate within one hour. Store and ship frozen in plastic vial. Minimum: 0.2 mL serum for each determination. Note: This volume does not allow repeat analysis. Glucose: Recommended: 0.5 mL serum for each determination. Store in plastic vial with 5 mg NaF. Minimum: 0.1 mL serum for each determination. Note: This volume does not allow repeat analysis. Proinsulin: Recommended: 1 mL EDTA plasma only. Separate within one hour. Store and ship frozen in a plastic vial. Minimum: 0.5 mL EDTA plasma only Interpretation: Diabetes mellitus in children Either of the following is considered diagnostic of diabetes: Presence of the classic symptoms, such as polyuria, polydipsia, ketonuria, and rapid weight loss, with a random plasma glucose of 200 mg/dL (11.1 nM/mL) or greater, Hg AIC >6.5% or diabetic retinopathy. A glucose tolerance test is not indicated in these individuals. In asymptomatic individuals, both an elevated fasting glucose concentration greater than 126 mg/dL (7.0 nM/mL) and a sustained elevated glucose level during the OGTT with a glucose over 200 mg/dL at 2 hours. Impaired glucose tolerance (IGT) in children (insulin resistance) Two criteria must be met: The fasting glucose concentration must be below the value that is diagnostic of diabetes (126 mg/dL). The glucose concentration two hours after an oral glucose challenge must be elevated (140-200 mg/dL). The insulin response to OGTT is somewhat age-dependent. Obese children display an enhanced response, which plays a role in their long-term prognosis with regard to the evolution of diabetes mellitus. Children with type 2 diabetes, at least initially, have a delayed, though often enhanced insulin response to an oral glucose load. The simplest method of determining insulin resistance (although not as sensitive as the OGTT) is the determination of the fasting glucose:insulin ratio or simply the fasting insulin level. A glucose:insulin ratio of less than 7 connotes some degree of insulin resistance. Other indexes of insulin resistance such as the HOMA or Quicki can be calculated from the fasting glucose:insulin ratio. Water Deprivation Test70-73 Purpose: Evaluation of patients suspected of having diabetes insipidus. Rationale: Water balance and plasma osmolality are tightly controlled in humans by thirst, antidiuretic hormone (ADH) secretion from the posterior pituitary and the kidneys. A small change either upward or downward in plasma osmolality elicits either increased or decreased release of ADH from the posterior pituitary gland with resultant antidiuresis or diuresis at the renal level. ADH acts on the kidney tubules by binding to the V2 receptors affecting the aquaporins, which mediate the transport of free water from the nephrons into the circulation. Maximum antidiuresis occurs at plasma ADH concentrations of 2-4 pg/mL. Patients with diabetes insipidus (either central or nephrogenic) have polyuria and urinate large volumes (>2 liters/m2/24 hours in older children and adults) of dilute urine. Before undertaking the water deprivation test to determine the patients ability to concentrate their urine and respond to desmopressin (DDAVP), it is prudent to exclude osmotic and metabolic causes of polyuria, as well as to document the 24-hour volume of urine. Serum electrolytes, glucose, calcium, creatinine, plasma osmolality, and simultaneous first-morning urine osmolality should be obtained. A diagnosis of diabetes insipidus can sometimes be made simply from the first am urine osmolality and concurrent plasma osmolality.74 32 ENDOCRINOLOGY SYLLABUS

34 ENDOCRINOLOGY RESPONSE TESTS Procedure: Water deprivation tests should never be done at home. They are performed in the hospital or outpatient setting under very close medical supervision during the day. Patients must be observed closely and their free access to water prevented. The patient may be allowed fluids overnight but no solid breakfast. NPO at 8:00 am Record weight and vital signs. Draw blood for electrolytes, plasma osmolality, and plasma ADH level. Collect urine for urinalysis, specific gravity, and urine osmolality. Collect all urine specimens for osmolality. Draw serum electrolytes, creatinine, and plasma osmolality every two hours. Weigh patient and record vital signs every two hours. Stop test before seven to eight hours if weight decreases >3% to 5% from baseline, plasma osmolality exceeds 300 mOsm/kg, or the patient becomes hypotensive and/or tachycardic. At the end of seven to eight hours of water deprivation, collect urine for specific gravity, osmolality and plasma for electrolytes, osmolality, and ADH level. A urine osmolality above 750 mOsm/kg makes central diabetes insipidus unlikely, and the test may be terminated, albeit it is important to draw a plasma ADH level at the end of the test. Note: In some patients who have not lost 3% to 5% of body weight and have normal vital signs, and appear to be well hydrated, the test sometimes has been prolonged until urine osmolality becomes fixed; that is, the osmolality does not change more than 10% between consecutive voids. A further increase in urine osmolality after desmopressin administration in the patient suggests central diabetes insipidus, while no change indicates maximal concentration ability has been achieved, and the patient may have nephrogenic diabetes insipidus or primary polydypsia with loss on the counter current concentrating mechanism. The plasma ADH level achieved before the desmopressin is given may clarify the diagnosis. If results suggest diabetes insipidus, urine osmolality less than 450 mOsm/kg, and/or plasma osmolality greater than 300, give desmopressin 0.025 g/kg subcutaneous (1 g maximum) or 0.25 g/kg intranasally (10 g maximum). Continue to monitor input and output, urine volume, specific gravity, and osmolality with each void. Restrict patients intake of fluids to the amount of urine excreted during the test plus that voided until the antidiuretic effect of the desmopressin injection has waned (ie, urine specific gravity 1.005 or lower, urine osmolality less than 200). Solids may be eaten ad libitum. (Caveat! Solids contain water.) At 8:00 am the next morning (after the desmopressin injection) collect blood for electrolytes, creatinine, and plasma osmolality, as well as urine for osmolality. Specimen Requirements: ADH levels Recommended: Adult - 2.0 mL of EDTA plasma only. Centrifuge cold (2C to 8C) within 30 minutes of collection, separate plasma immediately. Store and ship frozen in a plastic vial. Pediatric - 1.0 mL of EDTA plasma only. Centrifuge cold (2C to 8C) within 30 minutes of collection, separate plasma immediately. Store and ship frozen in a plastic vial. Minimum: Pediatric - 1.0 mL of EDTA plasma only. Note: this volume does not allow for repeat analysis. Interpretation: The interpretation of the water deprivation test is complicated by several factors that include (1) the fact that polyuria may compromise the ability of the kidney to respond to ADH, (2) cortisol deficiency may mask central diabetes insipidus, and (3) the criteria for a response to seven to eight hours of water deprivation in children differs widely depending on the report cited. Certainly, using stringent criteria, patients who concentrate their urine to 750 mOsm/kg, maintain their plasma osmolality below 300, and have measurable plasma ADH levels do not have diabetes insipidus.71 Frasier et al proposed less stringent criteria and suggested that a urine osmolality greater than 450 mOsm/kg with a serum osmolality less than 300 rules out central diabetes insipidus.70 Patients with nephrogenic diabetes insipidus generally do not concentrate their urine to greater than 300 mOsm/kg after water deprivation, do not respond to multiple physiologic doses of desmopressin over several days, and have ADH levels that are elevated. Further, patients with central diabetes insipidus may sometimes concentrate adequately on one day, however, may be unable to concentrate when deprived of water on a subsequent day. Although rarely performed, it has been suggested that in children older than five years of age, a definitive diagnosis of central versus nephrogenic versus dipsogenic diabetes insipidus can be made using hypertonic saline infusion and the measurement of plasma ADH levels.71,75 ENDOCRINOLOGY SYLLABUS 33

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37 ENDOCRINOLOGY ADRENAL STEROID RESPONSE Charts And Data Tables ADRENAL STEROID RESPONSE TO ACTH: PEDIATRICS Adrenal Steroid Response To ACTH MINERALOCORTICOID GLUCOCORTICOID ANDROGEN PATHWAY PATHWAY PATHWAY Cholesterol CH3 CH3 C=0 C=0 20.22- 17,20- 0 17-OH OH Des Des HO HO HO 5 Pregnenolone 17aHydroxypregnenolone Dehydroepiandrosterone (DHEA) CH3 CH3 3b-HSD C=0 3b-HSD C=0 3b-HSD 0 17-OH OH 17,20- Des O O O Progesterone 17a-Hydroxyprogesterone 4 Androstenedione CH2OH CH2OH 21-OH C=0 21-OH 17b-HSD C=0 0H OH O O O 11-Deoxycorticosterone (DOC) 11-Deoxycortisol (Cpd S) Testosterone CH2OH CH2OH 11-OH 11-OH 5a-Reduct C=0 C=0 0H HO HO OH O O O H Corticosterone Cortisol Dihydrotestosterone (DHT) 18-OH CH2OH HO CH2 C=0 HO Esoterix Endocrinologys Pediatric Steroid Profiles for the O evaluation of defects in steroid biosynthesis. 18-Hydroxycorticosterone (18-OH-B) 18-HSD CH2OH Adrenal Androgen Profile CAH Profile 5 (17,20 Des. Def.) C=0 5-Alpha-Reductase Profile CAH Profile 6 (Comprehensive) HCO HO CAH Profile 1 (21-OH Deficiency) CAH Profile 7 (Treatment Profile) CAH Profile 2 (11-OH Deficiency) Mineralcorticoid Profile O CAH Profile 3 (17-OH Deficiency) Premature Adrenarche Profile I Aldosterone CAH Profile 4 (3bHSD Deficiency) Premature Adrenarche Profile II 36 ENDOCRINOLOGY SYLLABUS

38 ENDOCRINOLOGY ADRENAL STEROID RESPONSE Adrenal Steroid Response To ACTH: Pediatrics Introduction The data presented in this booklet summarize the normal studies performed at Esoterix Endocrinology over the past several years on adrenal steroid response to ACTH in children. Through collaborative efforts with several pediatric institutions, we have been able to establish normal basal and response values for nearly all age groups. These comprehensive data now extend from premature infants through infancy, early childhood, pubertal ages, and adults. To our knowledge, they are the most extensive data currently reported on ACTH stimulation in children. Since the ACTH response of many adrenal steroids varies dramatically with age, the availability of these age-related normal data has proven to be very useful in the interpretation of laboratory results and the evaluation of adrenal function in pediatrics. Test Protocol And Conditions Stimulation tests were performed on ambulatory subjects (when applicable) using a standard ACTH dose of 25 units (0.25 mg Cortrosyn) given as an IV bolus. Blood samples were drawn at 0 and 60 minutes. No attempt was made to control posture in older subjects, or dietary intake of electrolytes. In some studies involving older children and adults, additional samples were drawn at 30 minutes after ACTH. These data have been omitted from the table because of space limitations, and also because our results do not support the need for additional poststimulation values. The amount of ACTH used for the short 60-minute test does not appear to be important, since all commonly used doses are pharmacological. Comparable response values have been observed with doses ranging from 0.05 - 0.5 U/kg. Data Tables Data are presented as the range and mean for baseline and 60-minute samples and for the increase above baseline or delta () value. Precursor:product ratios were determined separately for each subject and are not computed from the means and ranges of the respective steroids. The number of control subjects is given in the discussion section for each age group. Assay Procedures The steroids in these studies were determined with research quality procedures that have been used in the laboratory at Esoterix Endocrinology for many years. With the exception of cortisol and DHEA-S, which are assayed directly on aliquots of diluted serum, all of the methods involve some combination of solvent extraction and chromatographic purification. High-pressure liquid, column, and paper chromatography systems are all used routinely for sample purification. Recovery is monitored on each sample with a 3H steroid tracer. Steroid-free samples (blanks), known standards, multiple-level control pools, and randomly repeated samples are included in every assay run. An involved quality control system serves to prevent assay blanks by insuring that nitrogen drying systems are free of organic material, that solvents are redistilled, and that all glassware, chromatography media, and other materials used in the assays are solvent-washed to eliminate possible interference from nonvolatile organic residues. This program is costly and time-consuming, but it is essential for reliable pediatric endocrine testing on a continuous daily basis. Assays are performed in the laboratory by well-trained graduate-level biochemists and technologists who have demonstrated the ability to handle this kind of methodology reliably. The laboratory is supervised by PhD-level chemists who have been involved with the development of steroid assays and laboratory management for many years. Assays are reviewed by these section supervisors before any results are reported. All the antisera used in the assays were developed at Esoterix Endocrinology and were selected because their high sensitivity and low cross-reactivity enable us to obtain specific results on small volumes of pediatric samples. Since our steroid laboratory uses more complex chromatography procedures, the specificity of our methods is not entirely dependent on antibody cross-reactivity as it is with many commonly used diagnostic kits and other shortcut assays. Instead, specificity is achieved by using a high-quality antiserum in combination with a chromatography procedure that will provide specific assay results in a wide variety of abnormal and normal specimens. ENDOCRINOLOGY SYLLABUS 37

39 ENDOCRINOLOGY ADRENAL STEROID RESPONSE The reliability of our routine steroid methods has been examined during development and in numerous studies over the years. These evaluation procedures have included combinations or, in some cases, all of the following verification checks: 1. Cross-reaction of selected antisera with a large number of related steroids. 2. Comparison of assay results with those obtained using different antisera. 3. Evaluation of dose-response linearity obtained with multiple volumes of sample extracts and/or chromatography eluates. 4. Comparison of the values obtained with alternate chromatography procedures. 5. Comparison of values after a second or additional chromatography procedure is added. 6. Evaluation of chromatogram peak homogeneity by determining the specific activity of the steroid in multiple sections of the steroid peak. 7. Evaluation of results in abnormal sera known to contain high levels of precursor steroids. 8. Evaluation of the steroid response to physiological maneuvers, primarily stimulation and suppression tests. 9. Comparison of normal and abnormal values with those obtained using established assays. 10. Use of special chemical alterations or derivatives in situations where assay specificity requires additional confirmation. Interpretation of Results Some caution is indicated in the interpretation of ACTH response tests in children. In most cases, laboratory results are clear because they either fall within the normal reference interval or stimulate well beyond the upper limit of normal in those patients with biosynthetic defects. In other situations, however, the interpretation of laboratory data is not apparent, and a number of variables, including those outlined below, should be considered. 1. Age-Related ChangesThe ACTH response of many clinically relevant steroids changes substantially with age; therefore, meaningful interpretation of results requires valid normal data for the age group in question. 2. Newborns and Premature InfantsIt is recommended that newborn samples be taken on day two or three of life when steroid levels have become more stabilized and normal reference data are available. Values of many steroids in premature infants including 17-OH-progesterone are much higher than in full-term infants. Other steroids, in addition to 17-OH-progesterone, should be ordered to evaluate possible defects in these cases. 3. MethodologyThe steroid values obtained in various laboratories are often not comparable because of methodological differences. Comparing results from one laboratory with either normal reference intervals or results from another laboratory may be misleading. In many situations, it is important to have normal data that are specific for the methods being used in the laboratory performing the test. 4. Variability of ACTH Response in ChildrenThe results from large numbers of ACTH response tests processed in our laboratory over the past few years clearly demonstrate that a large variation exists in the response patterns of different steroids in children. Unusual response patterns are sometimes seen which are unlikely to be associated with any abnormality in steroid biosynthesis. 5. Use of Statistical DataThe clinical evaluation of test results based on strict application of the statistical data published by Esoterix Endocrinology or other laboratories is questionable because of the number of factors that can influence test results. While the ACTH response test has been useful for amplifying defects in steroid biosynthesis and identifying cases of late-onset congenital adrenal hyperplasia (CAH), care should be taken to avoid interpreting data too narrowly. It should be recognized that defects capable of causing clinical problems are usually associated with dramatic elevations in precursor steroids and usually become obvious after stimulation with ACTH. 6. Variability of Steroid Responsiveness to ACTHThe ACTH responses of different adrenal steroids vary substantially and are frequently not well correlated. This situation results in wide variability in both the absolute levels and the ratios of different steroids. In addition, those steroids that are very sensitive to ACTH, such as 17-OH-pregnenolone and corticosterone, may have more pronounced fluctuations during the day. Unstimulated values may change substantially. This kind of variation must be considered when evaluating laboratory data. 7. Precursor/Product RatiosPrecursor:product ratios are useful, but they should not be rigidly interpreted and must be evaluated relative to the actual stimulated levels of the steroids involved. In baseline samples, the use of precursor:product ratios is not meaningful unless the steroid levels are highly elevated. 38 ENDOCRINOLOGY SYLLABUS

40 ENDOCRINOLOGY ADRENAL STEROID RESPONSE 8. Increases Above BaselineEvaluating results by computing the response as a percentage or multiple increase above baseline is also useful, but again must be interpreted relative to the actual stimulated levels of the steroids in question. The serum levels in baseline samples can vary substantially as a result of diurnal variation, episodic secretion, stress, or other conditions. Thus, the percentage increase can vary significantly depending on the time of day and conditions under which the test was performed. For example, if response tests are performed in the afternoon, some steroids, which have a large diurnal variation, may have an abnormal response when expressed as a multiple above baseline even though the stimulated serum levels never exceed the normal range. Adrenal Steroid Response To ACTH In Normal Pediatric Subjects Premature Infants Premature Infants Infants Steroid 26-28 Weeks (N=21) 34-36 Weeks (N=12) 1-6 Months (N=15) Data Presented as Mean and Range 0 Min 60 Min Delta 0 Min 60 Min Delta 0 Min 60 Min Delta 192 320 128 178 316 148 27 77 50 Aldosterone (ng/dL) 5-635 13-1046 8-517 12-736 42-1365 28-629 2-71 4.8-166 2.7-123 Androstenendione 239 607 329 323 731 369 24 (ng/dL) 63-935 121-1323

41 ENDOCRINOLOGY ADRENAL STEROID RESPONSE Adrenal Steroid Response To ACTH In Normal Pediatric Subjects Premature Infants Premature Infants Infants Steroid 26-28 Weeks (N=21) 34-36 Weeks (N=12) 1-6 Months (N=15) Data Presented as Mean and Range 0 Min 60 Min Delta 0 Min 60 Min Delta 0 Min 60 Min Delta DHEA Sulfate (g/dL) + + + + + + + + + 47 105 58 44 59 17 20 75 55 DOC (ng/dL) 20-105 44-320 17-215 28-78 28-95 1-67 7-48 40-158 13-144 793 1879 1086 585 1218 686 60 220 160 Pregnenolone (ng/dL) 260-2104 962-3179 70-2673 203-1024 637-1888 162-1685 10-150 100-359 20-282 1402 5176 3775 1242 3803 2561 320 1465 1144 17-OH-Pregnenolone (ng/dL) 375-3559 2331-11,440 1219-9799 559-2906 831-9760 346-8911 52-828 633-3286 229-3104 471 774 302 306 562 256 47 172 125 17-OH-Progesterone (ng/dL) 124-841 285-1310 50-596 186-472 334-1725 18-1253 13-173 85-250 52-193 91 156 Testosterone (M) (ng/dL) * * * * * * * 59-125 2-501 11 4.4 Testosterone (F) (ng/dL) * * * * * * * 5-16 2-8 Premature Infants Results obtained with different 17-OH-pregenolone antisera are also basically equivalent. Similarly, values obtained with methods using column, high pressure liquid, or paper chromatography are not different. Oxidation of samples with permanganate followed by chromatography also does not lower results. Finally, peaks from paper chromatograms are homogeneous and show a constant specific activity after elution and RIA of multiple fractions. Infants 1-12 Months Stimulation studies were performed on 27 normal infants (16 males and 11 females) between the ages of 1 and 12 months. The results have been segregated into two groups, 1-6 months (N=15) and 6-12 months (N=12), in order to provide reference ranges for more restricted age groups and also because the levels of some steroids change significantly during this time period. The ACTH response values in this age group are significantly lower than those reported above for premature infants. The response of many r-4 adrenal steroids (progesterone, 17-OH-progesterone, cortisol, corticosterone, and 11-desoxycortisol) are similar to those observed in children and adults. 40 ENDOCRINOLOGY SYLLABUS

42 ENDOCRINOLOGY ADRENAL STEROID RESPONSE Adrenal Steroid Response To ACTH In Normal Pediatric Subjects Premature Infants Premature Infants Infants Steroid Precursor: 26-28 Weeks (N=21) 34-36 Weeks (N=12) 1-6 Months (N=15) Product Ratios Data Presented as Mean and Range 0 Min 60 Min Delta 0 Min 60 Min Delta 0 Min 60 Min Delta Pregnenolone 0.56 0.36 0.29 0.41 0.50 0.27 0.25 0.15 0.13 17-OH-Pregnenolone 0.25-0.70 0.25-5.0 0.17-0.73 0.17-1.3 0.1-0.7 0.03-0.25 17-OH-Pregnenolone 2.9 6.7 3.8 3.9 6.7 7.0 6.1 8.5 9.1 17-OH-Progesterone 1.1-5.2 3.6-11 1.8-6.5 3.6-12 2-22 3-20 17-OH-Progesterone 0.95 1.17 1.03 2.1 2.8 4.5 1.2 1.1 1.0 Compound S 0.35-2.4 0.25-2.1 0.9-4.8 0.8-4.2 0.4-3.1 0.5-2.0 17-OH-Progesterone (ng/dL) 78 41 23 28 18 17 5.0 4.6 4.6 Cortisol (g/dL) 18-220 15-170 9.0-71 6.2-51 0.8-12 0.2-6.9 Compound S (ng/dL) 89 44 22 22 7.5 3.8 4.4 4.7 4.8 Cortisol (g/dL) 25-300 10-189 2.9-115 3.1-26 0.8-10 2.4-10 DOC X 100 8.3 2.5 1.6 2.8 0.7 0.29 10 2.0 1.7 Corticosterone 2.3-25 1.2-7.0 1.1-5.5 0.4-0.9 0.5-30 0.7-4.9 Corticosterone 2.6 9.1 15 4.8 8.9 11.0 8.9 16 15 18-OH-Corticosterone 1.6-43 3.9-58 2.9-11 4.4-28 3-26 7-30 18-OH-Corticosterone 1.2 1.4 1.8 1.8 2.6 3.8 2.7 4.9 4.0 Aldosterone 1.0-4.5 0.8-2.6 1.1-10 1.2-11 1.3-5.0 2-13 Corticosterone (ng/dL) 93 217 275 108 210 279 42 100 120 Cortisol (g/dL) 35-198 34-800 42-242 140-375 8-120 37-146 +DHEA-S does not respond acutely to ACTH. For baseline values please refer to age-related reference ranges. *Testosterone levels are not signifcantly changed by low dose ACTH stimulation. Baseline data are presented for reference purposes only. ENDOCRINOLOGY SYLLABUS 41

43 ENDOCRINOLOGY ADRENAL STEROID RESPONSE Adrenal Steroid Response To ACTH In Normal Pediatric Subjects Steroid Infants 6-12 Months (N=12) Infants 1-2 Years (N=11) Children 2-6 Years (N=15) Data Presented as Mean and Range 0 Min 60 Min Delta 0 Min 60 Min Delta 0 Min 60 Min Delta 16 54 38 18 44 21 11 29 18 Aldosterone (ng/dL) 2-39 5-94 3-55 5-43 28-85 7-53 2-22 13-50 6-41 Androstenendione (ng/dL)

44 ENDOCRINOLOGY ADRENAL STEROID RESPONSE Adrenal Steroid Response To ACTH In Normal Pediatric Subjects Infants 6-12 Months (N=12) Infants 1-2 Years (N=11) Children 2-6 Years (N=15) Steroid Data Presented as Mean and Range 0 Min 60 Min Delta 0 Min 60 Min Delta 0 Min 60 Min Delta DHEA Sulfate (g/dL) + + + + + + + + + 22 73 52 18 85 67 12 69 57 DOC (ng/dL) 9-57 46-149 5-91 5-42 41-152 34-134 4-49 26-139 16-128 43 159 116 33 89 55 35 69 35 Pregnenolone (ng/dL) 12-137 75-294 43-282 10-93 44-135 4-113 17-50 34-99 15-75 124 937 813 42 299 257 41 172 131 17-OH-Pregnenolone (ng/dL) 14-647 257-2173 221-1981 14-207 55-732 35-712 10-103 45-347 16-276 25 170 145 30 179 154 34 135 110 17-OH-Progesterone (ng/dL) 11-106 102-267 48-247 4-105 65-353 40-341 7-114 50-269 16-176 4.5 4.0 4.0 Testosterone (M) (ng/dL) * * * * * * 2-8 2-8 2-8 3.1 Testosterone (F) (ng/dL) * * * * * * * * 2-8 +DHEA-S does not respond acutely to ACTH. For baseline values please refer to age-related reference ranges. *Testosterone levels are not signifcantly changed by low dose ACTH stimulation. Baseline data are presented for reference purposes only. ENDOCRINOLOGY SYLLABUS 43

45 ENDOCRINOLOGY ADRENAL STEROID RESPONSE Adrenal Steroid Response To ACTH In Normal Pediatric Subjects Steroid Precursor: Infants 6-12 Months (N=12) Infants 1-2 Years (N=11) Children 2-6 Years (N=15) Product Ratios Data Presented as Mean and Range 0 Min 60 Min Delta 0 Min 60 Min Delta 0 Min 60 Min Delta Pregnenolone 0.53 0.2 0.14 0.9 0.5 0.20 1.1 0.5 0.3 17-OH-Pregnenolone 0.2-2.9 0.05-0.50 0.3-2.4 0.2-1.5 0.3-3.6 0.3-1.4 17-OH-Pregnenolone 4.0 5.3 5.6 1.1 1.6 1.7 1.3 1.4 1.2 17-OH-Progesterone 1.5-10 1.6-12 0.4-2.5 0.5-3.3 0.3-3.0 0.45-2.6 17-OH-Progesterone 0.8 0.9 1.1 0.7 0.8 0.9 1.1 0.7 0.7 Compound S 0.5-1.9 0.5-2.0 0.4-1.5 0.5-1.6 0.3-2.1 0.5-1.4 17-OH-Progesterone (ng/dL) 1.7 4.0 5.3 1.9 5.5 7.7 3.1 5.2 6.9 Cortisol (g/dL) 1.2-4.6 2.0-6.0 1.0-6.5 1.8-10 1.2-8.0 3.1-10 Compound S (ng/dL) 2.3 4.6 5.7 4.0 7.2 8.6 4.6 7.9 10 Cortisol (g/dL) 1.0-5.3 3.5-8.0 1.0-6.8 5-10 1.8-6.0 3.8-11 DOC X 100 7.5 1.7 1.5 3.5 2.3 1.8 3.2 1.6 1.4 Corticosterone 3-18 1.2-2.6 2.0-7.0 1.2-4.5 1.2-5.6 0.9-3.3 Corticosterone 8.7 18 18 10 19 25 19 24 25 18-OH-Corticosterone 4-20 10-33 2-32 12-27 5-36 14-50 18-OH-Corticosterone 2.7 5.3 5.0 3.6 4.9 6.3 3.8 7.1 8.5 Aldosterone 1.4-3.9 3-12 1.3-5.0 3.3-7.0 1.2-6.0 1.9-15 Corticosterone (ng/dL) 29 94 127 40 124 165 36 150 223 Cortisol (g/dL) 10-137 45-146 15-157 99-150 17-46 71-259 44 ENDOCRINOLOGY SYLLABUS

46 ENDOCRINOLOGY ADRENAL STEROID RESPONSE Adrenal Steroid Response To ACTH In Normal Pediatric Subjects Children 6-10 Years (N=18) Early Puberty Males (N=14) Late Puberty Males (N=15) Steroid Data Presented as Mean and Range 0 Min 60 Min Delta 0 Min 60 Min Delta 0 Min 60 Min Delta 9 25 16 7 23 16 7 22 15 Aldosterone (ng/dL) 4-21 14-42 5-35 2-14 10-33 7-22 3-14 13-32 7-25 19 12 30 63 112 12 Androstenendione (ng/dL)

47 ENDOCRINOLOGY ADRENAL STEROID RESPONSE Adrenal Steroid Response To ACTH In Normal Pediatric Subjects Children 6-10 Years (N=18) Early Puberty Males (N=14) Late Puberty Males (N=15) Steroid Data Presented as Mean and Range 0 Min 60 Min Delta 0 Min 60 Min Delta 0 Min 60 Min Delta DHEA Sulfate (g/dL) + + + + + + + + + 10 48 38 9 38 29 8 30 23 DOC (ng/dL) 4-17 22-120 16-70 2-15 12-74 7-44 5-13 19-46 7-36 33 82 48 28 90 62 30 92 61 Pregnenolone (ng/dL) 15-63 39-130 15-115 10-55 58-116 41-101 11-50 37-149 8-99 76 329 253 95 390 295 123 572 449 17-OH-Pregnenolone (ng/dL) 10-186 70-656 59-515 20-363 88-675 66-655 32-297 220-966 165-842 36 158 122 45 154 109 100 172 72 17-OH-Progesterone (ng/dL) 7-100 85-280 51-240 12-131 69-313 7-281 51-191 105-264 12-134 5 161 501 Testosterone (M) (ng/dL) * * * * * * 2-19 20-310 278-702 5 Testosterone (F) (ng/dL) * * 2-19 Pubertal Children (Continued) Pubertal stages were assigned on the basis of breast development and pubic hair in females and genitalia and pubic hair in males. Results on female controls in the luteal phase of their cycle (as indicated by serum progesterone levels) were excluded from the data summary. The response of D-4 steroids in pubertal age groups is similar to prepubertal children and adults. The D-5 steroids which begin changing at adrenarche continue to increase throughout puberty. The ACTH response of DHEA and17-OH- pregnenolone in late puberty is considerably higher than the early pubertal ages. This increase is reflected in significant changes of the D5/D4 steroid ratios. The shift in D-5 steroid secretion is more pronounced in females. The stimulated levels of 17-OH pregnenolone and DHEA are significantly higher in late pubertal and adult females than in age equivalent male controls. +DHEA-S does not respond acutely to ACTH. For baseline values please refer to age-related reference ranges. *Testosterone levels are not signifcantly changed by low dose ACTH stimulation. Baseline data are presented for reference purposes only. 46 ENDOCRINOLOGY SYLLABUS

48 ENDOCRINOLOGY ADRENAL STEROID RESPONSE Adrenal Steroid Response To ACTH In Normal Pediatric Subjects Steroid Precursor: Children 6-10 Years (N=18) Early Puberty Males (N=14) Late Puberty Males (N=15) Product Ratios Data Presented as Mean and Range 0 Min 60 Min Delta 0 Min 60 Min Delta 0 Min 60 Min Delta Pregnenolone 1.0 0.3 0.2 0.5 0.3 0.2 0.4 0.2 0.13 17-OH-Pregnenolone 0.2-2.8 0.16-0.85 0.1-1.7 0.15-1.0 0.1-1.1 0.1-0.3 17-OH-Pregnenolone 1.8 2.4 2.0 1.9 2.8 2.7 1.2 3.4 6.8 17-OH-Progesterone 0.5-6.0 0.3-5.3 0.5-3.3 0.5-6.3 0.4-3.4 1.8-5.2 17-OH-Progesterone 0.7 0.9 0.9 1.0 0.88 0.9 1.5 1.2 0.5 Compound S 0.2-2.1 0.5-1.6 0.5-2.0 0.4-2.1 0.8-3.7 0.6-2.7 17-OH-Progesterone (ng/dL) 2.8 5.4 8.1 5.7 7.7 9.1 9.5 7.4 5.0 Cortisol (g/dL) 0.8-5.0 2.8-9.0 2.0-13 3.6-16 5.0-19 4.0-11 Compound S (ng/dL) 4.8 6.4 9.0 6.0 6.5 8.4 5.4 6.8 8.0 Cortisol (g/dL) 1.2-9.0 2.8-9.0 2-11 4.0-11 1.8-12 3.4-11 DOC X 100 2.4 1.2 1.0 2.6 1.3 1.0 2.5 1.2 1.0 Corticosterone 1.0-4.0 0.6-2.1 1.3-5.1 0.7-2.0 1.3-4.9 0.6-2.2 Corticosterone 15 21 24 12 21 18 11 22 28 18-OH-Corticosterone 8-27 16-28 3-28 11-33 7-21 16-30 18-OH-Corticosterone 3.8 6.9 8.7 3.6 7.2 7.9 4.2 5.6 6.2 Aldosterone 2.6-7.1 5-12 2.0-5.7 3.4-13 3.0-5.6 3.9-9.6 Corticosterone (ng/dL) 45 150 228 34 159 238 32 137 207 Cortisol (g/dL) 15-91 100-225 18-95 64-232 13-90 70-213 ENDOCRINOLOGY SYLLABUS 47

49 ENDOCRINOLOGY ADRENAL STEROID RESPONSE Adrenal Steroid Response To ACTH In Normal Pediatric Subjects Children 6-10 Years (N = 18) Early Puberty Females (N = 12) Late Puberty Females (N = 15) Steroid Data Presented as Mean and Range 0 Min 60 Min Delta 0 Min 60 Min Delta 0 Min 60 Min Delta 9 25 16 10 22 12 7 17 10 Aldosterone (ng/dL) 4-21 14-42 5-35 2-20 12-31 3-21 3-15 10-34 2-26 19 39 71 93 130 Androstenendione (ng/dL)

50 ENDOCRINOLOGY ADRENAL STEROID RESPONSE Adrenal Steroid Response To ACTH In Normal Pediatric Subjects Children 6-10 Years (N = 18) Early Puberty Females (N = 12) Late Puberty Females (N = 15) Steroid Data Presented as Mean and Range 0 Min 60 Min Delta 0 Min 60 Min Delta 0 Min 60 Min Delta DHEA Sulfate (g/dL) + + + + + + + + + 10 48 38 7 37 30 8 29 22 DOC (ng/dL) 4-34 28-85 16-70 2-16 13-63 9-51 2-13 15-50 12-45 33 82 48 39 89 50 41 146 105 Pregnenolone (ng/dL) 15-63 39-170 15-115 15-84 33-139 5-95 19-87 76-218 38-191 76 329 253 132 527 395 208 1014 806 17-OH-Pregnenolone (ng/dL) 10-186 70-656 59-515 33-451 251-756 108-683 44-542 502-1402 438-1280 36 158 122 48 182 134 55 151 96 17-OH-Progesterone (ng/dL) 7-100 85-280 51-240 14-120 75-353 18-287 12-121 71-226 9-164 5 Testosterone (M) (ng/dL) * * * * * * * * 2-19 5 18 32 Testosterone (F) (ng/dL) * * * * * * 2-19 8-36 12-59 Pubertal Children (Continued) Puberal stages were assigned on the bases of the breast development and pubic hair in females and genitalia and pubic hair in males. Results on female controls in the luteal phase of their cycle (as excluded from the data summary. The response of -4 steroids in pubertal age groups is similar to prepubertal children and adults. The -5 steroids which begin changing at adrenarche continue to increase throughout puberty. The ACTH response of DHEA and 17-OH-pregnenolone in late puberty is considerably higher than the early pubertal ages. This increase is reflected in significant changes of the -5:-4 steroid ratios. The shift in -5 steroid secretion is more pronounced in females. The stimulated levels of 17-OH-pregnenolone and DHEA are significantly higher in late pubertal and adult females than in age equivalent male controls. +DHEA-S does not respond acutely to ACTH. For baseline values please refer to age-related reference ranges. *Testosterone levels are not signifcantly changed by low dose ACTH stimulation. Baseline data are presented for reference purposes only. ENDOCRINOLOGY SYLLABUS 49

51 ENDOCRINOLOGY ADRENAL STEROID RESPONSE Adrenal Steroid Response To ACTH In Normal Pediatric Subjects Steroid Precursor: Children 6-10 Years (N=18) Early Puberty Females (N=12) Late Puberty Females (N=15) Product Ratios Data Presented as Mean and Range 0 Min 60 Min Delta 0 Min 60 Min Delta 0 Min 60 Min Delta Pregnenolone 1.0 0.3 0.2 0.43 0.2 0.12 0.3 0.15 0.13 17-OH-Pregnenolone 0.2-2.8 0.16-0.85 0.2-0.9 0.1-0.3 0.1-1.1 0.08-0.27 17-OH-Pregnenolone 1.8 2.4 2.0 2.9 3.5 3.0 4.3 7.7 10 17-OH-Progesterone 0.5-6.0 0.3-5.3 0.7-8.0 1.5-7.0 0.8-10 3.0-17 17-OH-Progesterone 0.7 0.9 0.9 0.9 1.1 1.1 1.8 1.0 1.0 Compound S 0.2-2.1 0.5-1.6 0.5-4.0 0.4-1.6 0.7-4.8 0.4-1.8 17-OH-Progesterone (ng/dL) 2.8 5.4 8.1 5.2 7.7 10 6.1 5.9 5.6 Cortisol (g/dL) 0.8-5.0 2.8-9.0 2.3-14 4.4-15 1.2-18 2.8-9.0 Compound S (ng/dL) 4.8 6.4 9.0 5.8 8.1 9.0 5.5 5.5 5.6 Cortisol (g/dL) 1.2-9.0 2.8-9.0 2.4-9.0 5.0-11.0 1.1-12 3.6-8.1 DOC X 100 2.4 1.2 1.0 2.5 1.3 1.1 2.4 1.1 0.8 Corticosterone 1.0-4.0 0.6-2.1 1.7-3.8 0.3-2.0 1.8-3.8 0.5-2.2 Corticosterone 15 21 24 11 22 25 11 20 25 18-OH-Corticosterone 8-27 16-28 3-26 12-35 4-29 11-40 18-OH-Corticosterone 3.8 6.9 8.7 3.9 7.2 9.1 3.7 7.8 10 Aldosterone 2.6-7.1 5-12 1.9-5.3 5.7-12 1.7-8.0 5-13 Corticosterone (ng/dL) 45 150 228 33 142 208 28 114 169 Cortisol (g/dL) 15-91 100-225 15-65 77-236 12-43 70-145 50 ENDOCRINOLOGY SYLLABUS

52 ENDOCRINOLOGY ADRENAL STEROID RESPONSE Adrenal Steroid Response To ACTH In Normal Adult Subjects Adult Females Dex. Supp. (N=19) Adult Females (N=19) Adult Males (N=12) Steroid Data Presented as Mean and Range 0 Min 60 Min Delta 0 Min 60 Min Delta 0 Min 60 Min Delta 6.6 20 13 8.4 20 12 7 17 10 Aldosterone (ng/dL) 1.2-21 7-33 3-22 2.4-25 6-28 2-19 3-11 7-26 0.3-18 81 207 88 125 194 20 86 159 32 Androstenendione (ng/dL) 37-148 79-289

53 ENDOCRINOLOGY ADRENAL STEROID RESPONSE Adrenal Steroid Response To ACTH In Normal Adult Subjects Adult Females Dex. Supp. (N = 19) Adult Females (N = 19) Adult Males (N = 12) Steroid Data Presented as Mean and Range 0 Min 60 Min Delta 0 Min 60 Min Delta 0 Min 60 Min Delta DHEA Sulfate (g/dL) + + + + + + + + + 4.6 33 28 7 29 22 6 26 20 DOC (ng/dL) 2-15 9-87 6-78 3-19 12-90 5-70 3-13 14-38 2-32 25 144 119 65 150 95 41 99 68 Pregnenolone (ng/dL) 10-60 55-200 59-235 46-150 70-220 40-210 10-85 20-200 10-144 20 768 748 138 871 733 100 558 461 17-OH-Pregnenolone (ng/dL) 10-24 240-1255 230-1255 48-320 290-1382 175-1300 20-187 240-1000 160-850 45 124 79 58 140 82 66 168 102 17-OH-Progesterone (ng/dL) 17-135 59-247 42-202 22-140 65-250 31-190 35-150 45-258 24-139 24 35 543 Testosterone (ng/dL) * * * * * * 10-47 15-54 430-675 Steroid Values In Patients With Adrenal Hyperplasia (Continued) Because of these wide fluctuations, levels of key marker steroids may fall within the normal range when samples are drawn during the afternoon. Confusion with laboratory results or misdiagnosis in these cases can be avoided by using ACTH stimulation. 21-OH Deficiency Serum 17-OH progesterone (17-OHP) is the standard laboratory marker for this disorder. In classical 21-OH deficiency, the values of 17-OHP are strikingly elevated (up to 2000 times normal) and there is little need for stimulation tests. In mild or late onset cases, ACTH stimulation is recommended to amplify defects. If ACTH is not used, samples should be drawn during the morning hours, since late afternoon 17-OHP values may fall into the normal range. 11-OH Deficiency Serum 11-Desoxycortisol is highly elevated in patients with adrenal hyperplasia due to 11-OH deficiency and provides the most useful marker for this disorder. Stimulation tests are usually not required in young infants, but may be useful in evaluating older patients who have either a late onset or were not diagnosed until later in life. Nearly half of the cases summarized on page 113 were identified during evaluation for premature adrenarche. +DHEA-S does not respond acutely to ACTH. For baseline values please refer to age-related reference ranges. *Testosterone levels are not signifcantly changed by low dose ACTH stimulation. Baseline data are presented for reference purposes only. 52 ENDOCRINOLOGY SYLLABUS

54 ENDOCRINOLOGY ADRENAL STEROID RESPONSE Steroid Precursor To Product Ratios In Normal Adult Subjects Steroid Precursor: Adult Females Dex. Supp. (N=19) Adult Females (N=19) Adult Males (N=12) Product Ratios Data Presented as Mean and Range 0 Min 60 Min Delta 0 Min 60 Min Delta 0 Min 60 Min Delta Pregnenolone 2.0 0.20 0.16 0.5 0.2 0.14 0.48 0.14 0.15 17-OH-Pregnenolone 1.3-3.0 0.1-0.3 0.25-1.2 0.1-0.3 0.2-2.1 0.05-0.41 17-OH-Pregnenolone 0.44 6.2 9.4 2.6 6.2 7.7 1.8 3.8 4.5 17-OH-Progesterone 0.2-0.8 3.7-11 1.7-6.0 3.7-11 0.5-5.8 1.6-7.0 17-OH-Progesterone 2.5 1.0 0.8 0.9 1.0 1.3 1.9 1.2 1.1 Compound S 2.0-14 0.8-2.4 0.7-5.0 0.9-2.4 0.8-4.9 0.6-1.7 17-OH-Progesterone (ng/dL) 23 5.4 3.8 4.2 5.4 6.8 6.1 6.6 8.0 Cortisol (g/dL) 10-60 2.8-9.1 1.9-9.3 2.8-9.1 2-13 2-9 Compound S (ng/dL) 9.5 5.4 5.1 5.0 5.4 5.3 3.5 5.5 7.2 Cortisol (g/dL) 2.5-34 2.3-70 0.7-6.2 2.3-7.0 1.4-5.1 3.4-7.4 DOC X 100 8 1.2 1.0 1.0 1.2 1.0 1.6 1.2 1.0 Corticosterone 4-20 0.5-1.7 1.2-3.6 0.5-1.7 0.5-3.2 0.5-5.8 Corticosterone 3.1 21 21 18 21 18 16 24 26 18-OH-Corticosterone 1.2-7.1 16-33 10-30 16-33 10-28 17-32 18-OH-Corticosterone 3.0 7.7 10 2.1 7.7 10 2.9 6.3 11 Aldosterone 2.3-4.6 3.8-10 1.2-5.0 3.8-10 1.2-6.0 3-11 Corticosterone (ng/dL) 32 119 127 39 119 162 38 131 219 Cortisol (g/dL) 19-88 80-169 14-45 80-169 8-128 22-253 ENDOCRINOLOGY SYLLABUS 53

55 ENDOCRINOLOGY ADRENAL STEROID RESPONSE Steroid Hormone Levels In Congenital Adrenal Hyperplasia 21-OH-Deficiency Classical 21-OH-Deficiency Late Onset Steroid 11-OH-Deficiency (N=20) (N=30) (N=20) 0 min 2-90 2-20 1-18 Aldosterone (ng/dL) 60 min * 8-41 1-40 0 min 10-520 15-45 570-26,000 Compound S (ng/dL) 60 min * 35-150 1200-50,000 0 min 45-1730 105-1110 70-1350 Corticosterone (ng/dL) 60 min * 1470-4800 101-5800 0 min 2-145 25-70 2-45 18-OH-Corticosterone (ng/dL) 60 min * 65-310 6-70 0 min 1-18 3-12 1-26 Cortisol (g/dL) 60 min * 10-34 1-45 0 min * * * DHEA (ng/dL) 60 min * * * 0 min 2-70 5-35 20-800 DOC (ng/dL) 60 min * 20-75 225-2900 0 min 190-3400 20-185 35-740 Pregnenolone (ng/dL) 60 min * 75-760 180-970 0 min 600-22,000 20-510 125-1500 17-OH-Pregnenolone (ng/dL) 60 min * 500-2890 430-3650 0 min 3000-120,000 90-2078 30-1250 17-OH-Progesterone (ng/dL) 60 min * 1400-11,500 220-2360 *Not determined 17-OH Deficiency Corticosterone and to a lesser extent DOC are highly elevated in these patients, and provide useful laboratory markers. The data on the 5 patients summarized on page 114 are relatively consistent. Cortisol, other 17-hydroxylated steroids and androgens are consistently very low. Steroids not hydroxylated at the 17-position are elevated, but the most pronounced increase appears to be in corticosterone where values may be 100 times normal. 3--HSD Deficiency Unlike most forms of CAH, the interpretation of laboratory data in late onset or mild 3-Beta-HSD deficiency is not clear, and there is considerable disagreement regarding the incidence and diagnostic criteria for this disorder. The ACTH stimulated levels of 17-OH-pregnenolone and DHEA which are key marker steroids for this disorder are widely scattered. There is no readily apparent separation of 3-HSD deficient patients from those who have elevated levels of adrenal steroids for other reasons. 54 ENDOCRINOLOGY SYLLABUS

56 ENDOCRINOLOGY ADRENAL STEROID RESPONSE Steroid Hormone Levels In Congenital Adrenal Hyperplasia Steroid 3-B-HSD Deficiency (N=6) 17-OH-Deficiency (N=5) CMO-II Deficiency (N=5) 0 min 4-200 0.7-4.0 1-12 Aldosterone (ng/dL) 60 min * 2.3-5.0 1-18 0 min 260-1800 15-65 10-180 Compound S (ng/dL) 60 min * 25-94 145-360 0 min 80-5700 9000-40,000 700-9000 Corticosterone (ng/dL) 60 min * 20,000-57,000 3400-13,000 0 min 16-380 100-300 85-2600 18-OH-Corticosterone (ng/dL) 60 min * 300-550 130-4120 0 min 1-15 0.5-3.0 3-16 Cortisol (g/dL) 60 min * 2.0-5.0 15-37 0 min * * * DHEA (ng/dL) 60 min * * * 0 min 2-53 100-250 35-250 DOC (ng/dL) 60 min * 200-480 60-550 0 min 650-5900 300-800 * Pregnenolone (ng/dL) 60 min * 600-2400 * 0 min 2200-48,000 20-50 * 17-OH-Pregnenolone (ng/dL) 60 min * 20-50 * 0 min 335-4100 10-25 80-235 17-OH-Progesterone (ng/dL) 60 min * 25-50 140-490 ENDOCRINOLOGY SYLLABUS 55

57 PAGE 22 ENDOCRINOLOGY ADRENAL STEROID RESPONSE ALDOSTERONE BASELINE/O MIN ALDOSTERONE ACTH STIMULATION/6O MIN 56 ENDOCRINOLOGY SYLLABUS

58 ENDOCRINOLOGY ADRENAL STEROID RESPONSE CORTICOSTERONE BASELINE/O MIN CORTICOSTERONE ACTH STIMULATION/6O MIN ENDOCRINOLOGY SYLLABUS 57

59 PAGE 25 ENDOCRINOLOGY ADRENAL STEROID RESPONSE 18-OH-CORTICOSTERONE BASELINE/O MIN 18-OH-CORTICOSTERONE ACTH STIMULATION/6O MIN 58 ENDOCRINOLOGY SYLLABUS

60 ENDOCRINOLOGY ADRENAL STEROID RESPONSE CORTISOL BASELINE/O MIN ug/dl CORTISOL ACTH STIMULATION/6O MIN ug/dl ENDOCRINOLOGY SYLLABUS 59

61 ENDOCRINOLOGY ADRENAL STEROID RESPONSE 11-DESOXYCORTISOL BASELINE/O MIN 11-DESOXYCORTISOL ACTH STIMULATION/6O MIN 60 ENDOCRINOLOGY SYLLABUS

62 ENDOCRINOLOGY ADRENAL STEROID RESPONSE DEOXYCORTICOSTERONE BASELINE/O MIN DEOXYCORTICOSTERONE ACTH STIMULATION/6O MIN ENDOCRINOLOGY SYLLABUS 61

63 ENDOCRINOLOGY ADRENAL STEROID RESPONSE PREGNENOLONE BASELINE/O MIN PREGNENOLONE ACTH STIMULATION/6O MIN 62 ENDOCRINOLOGY SYLLABUS

64 PAGE 31 ENDOCRINOLOGY ADRENAL STEROID RESPONSE 17-OH-PREGNENOLONE BASELINE/O MIN 17-OH-PREGNENOLONE ACTH STIMULATION/6O MIN ENDOCRINOLOGY SYLLABUS 63

65 ENDOCRINOLOGY ADRENAL STEROID RESPONSE 17-OH-PROGESTERONE BASELINE/O MIN 17-OH-PROGESTERONE ACTH STIMULATION/6O MIN 64 ENDOCRINOLOGY SYLLABUS

66 ENDOCRINOLOGY ADRENAL STEROID RESPONSE DHEA-SULFATE BASELINE/O MIN 300 250 200 ug/dl 150 100 50 0 1 Mo 6 Mo 1 2 4 6 8 10 12 14 16 AGE (Years) REFERENCES 8. Absence of Nonclassical Congenital Adrenal Hyperplasia in Patients with I. Adrenal Response to ACTH in Premature Infants. Marco Danon, Jesus Cruz, Precocious Adrenarche. Alan Morris, Edward Reiter, Mitchell Geffner, Darrel Mayes, Gail Kletter, Inese Beitin. Program Society Ped. Research. Barbara Lippe, Robert Itami and Darrel Mayes. J. Clin. Endocrinol. Metab. 430,1990. 69:709, 1989. 2. Transient 11 B-Hydroxylase Deficiency in Preterm Infants. Robert Hingre, 9. Adrenal Steroidogenic Function in a Black and Hispanic Population with Kristina Hingre, Steven Gross, Darrel Mayes and Robert Richmond. Program Precocious Pubarche. Sharon Oberfield, Darrel Mayes, Lenore Levine. J. Clin. Society Ped. Research. 447, 1990. Endocrinol. Metab. 70: 76, 1990. 3. Progressive Decrease in 3B-Hydroxysteroid Dehydrogenase Activity in 10. The Hypothalmic-Pituitary-Adrenal Axis in Partial (Late- Onset) 21- Low Birth Weight Preterm Infants. R. Hinare. K. Hinare, S. Gross, D. Mayes, R. Hydroxylase Deficiency. P. Feuillan, S. Pang, T. Schurmeyer, P.C. Avgerinos and Richman. Program of the 72nd Meeting of Endocrine Society. 841,1990. G.P. Chrousos. J. Clin. Endocrinol. Metab. 67: 154, 1988. 4. Elevated Serum 17-OH Pregnenolone in Premature Infants with High 11. Late-onset Steroid 21-Hydroxylase Deficiency: A Variant of Classical Serum 17-Hydroxyprogesterone Levels. L.R. Garibaldi, W. Keenan, C. Bayu, D. Congenital Adrenal Hyperplasia. B. Kohn, et al. J. Clin. Endocrinol. Metab. Mayes. Society for Pediatric Research. 443,1990. 55:817-27, 1982. 5 Normative Data for Adrenal Steroidogenesis: Age and Sex Related 12. Late-onset adrenal steroid 3B-Hydroxvsteroid Dehvdroaenase Changes after ACTH Stimulation. P. Saenger, G. Lashansky, K. Fishman, D. Deficiency l: A Cause of Hirsutism in Pubertal and Postpubertal Women. S. Mayes, T. Gautier, E. Reiter. Program of the 72nd Meeting of the Endocrine Pang, et al. J. Clin. Endocrinol. Metab. 60: 428-39, 1985. Society. 820,1990. 13. High Frequency of Nonclassical Steroid 21-Hydroxylase Deficiency. P.W. 6. Serum Adrenal Steroid Levels in Healthy Full-term Three Day Old Infants. Speiser, et al. Am. J. Hum. Genet. 37: 650-67, 1985. Dona Wiener, Jeffrey Smith, Steven Dahlem, Gregory Berg, Thomas 14. Attenuated Forms of Congenital Adrenal Hyperplasia due to 21- Moshang. Journal of Pediatrics. 110, (1) 122- 124,1987. Hvdroxylase Deficiency. Peter Lee, Zev Rosenwaks Maria Urban, Claude 7. Adrenal Steroid Levels in Premature-lnfants. Mary Lee, Lakshimv Migeon and Wilma Bias. J. Clin. Endocrinol. Metab. 55: 866, 1982. Rajagopalan, Gregory Berg, Thomas Moshang. J. Clin. Endocrinol. Metab. 1133, 1989 . ENDOCRINOLOGY SYLLABUS 65

67 ENDOCRINOLOGY CONVERSION TABLE S.I. Unit Conversion Table When When Multiply Multiply Hormone You To Find Hormone You To Find By By Know Know ACTH (Corticotropin) pg/mL 0.222 pmol/L Dehydroepiandrosterone-Sulfate g/dL 27.211 nmol/L (DHEA-S) Antidiuretic Hormone (ADH) pg/mL 0.923 pmol/L 18-Hydroxy-Deoxycorticosterone Albumin g/dL 10.0 g/L ng/dL 28.86 pmol/L (18-OH DOC) Aldosterone, Serum ng/dL 27.747 pmol/L 11-Desoxycortisol (Compound S) ng/dL 28.868 pmol/L Aldosterone, Urine g/24 h 2.775 nmol/d 21-Desoxycortisol ng/dL 0.0289 nmol/L Aldosterone:Creatinine g/g 0.314 nmol:mmol Desoxycorticosterone (DOC) ng/dL 30.2572 pmol/L Androstanediol ng/dL 344.3 nmol/L Dexamethasone ng/dL 25.478 pmol/L Androstanediol Glucuronide ng/dL 344.3 nmol/L Dihydrotestosterone ng/dL 34.435 pmol/L Androstenedione ng/dL 0.035 nmol/L Dopamine, Plasma pg/mL 6.528 pmol/L Angiotensin I pg/mL 0.772 pmol/L Dopamine, Urine g/24 h 6.53 nmol/d Angiotensin II pg/mL 0.957 pmol/L Dopamine:Creatinine g/g 0.739 nmol:mmol Angiotensin I-converting Enzyme U/L 0.017 ukat/L Endorphin, Beta pg/mL 1.0 ng/L Atrial Natriuretic Peptide (ANP) pg/mL 0.325 pmol/L Epinephrine, Plasma pg/mL 5.459 pmol/L C-Peptide ng/mL 0.331 nmol/L Epinephrine, Urine g/24 h 5.459 nmol/d C-Peptide, Urine ng/mL 0.331 nmol/L Epinephrine:Creatinine g/g 0.618 mol:mmol Calcitonin pg/mL 0.292 pmol/L Estradiol pg/mL 3.671 pmol/L Calcium mg/dL 0.25 mmol/L Estriol ng/mL 3.467 nmol/L Calcium, Urine mg/24 h 0.025 mmol/d Estrogens, Serum pg/mL 1.0 ng/L Catecholamines, Urine g/24 h 5.911 nmol/d Estrone pg/mL 3.698 pmol/L Catecholamines:Creatinine g/g 0.669 nmol:mmol Estrone Sulfate ng/dL 0.273 pmol/L Corticosterone ng/dL 28.864 pmol/L Folic Acid ng/mL 2.266 nmol/L 18-Hydroxy Cortisterone ng/dL 27.594 pmol/L Follicle-stimulating Hormone mlU/mL 1.0 lU/L (FSH) Cortisol, Serum g/dL 27.588 nmol/L Follicle-stimulating Hormone, Cortisol, Urine g/24 h 2.759 nmol/d IU/24 h 1.0 lU/d Urine Cortisol:Creatinine g/dL 0.3121 nmol:mmol FSH:Creatinine lU/g 0.113 lU:mol Cortisone g/dL 0.278 nmol/L Gastrin pg/mL 0.481 pmol/L Creatinine, Urine mg/24 h 884 mmol/d Glucagon pg/mL 1.0 ng/L nmol/ Growth Hormone ng/mL 1.0 g/L Cyclic AMP, Urine 1.0 mol/L mL Human Chorionic Gonadotrophin nmol/ mlU/mL 1.0 lU/L Cyclic AMP:Creatinine 1.131 mol:mol (hCG) mg hCG, Urine mlU/mL 1.0 lU/L Dehydroepiandrosterone (DHEA) ng/dL 34.674 pmol/L 66 ENDOCRINOLOGY SYLLABUS

68 ENDOCRINOLOGY CONVERSION TABLE S.I. Unit Conversion Table When When Multiply Multiply Hormone You To Find Hormone You To Find By By Know Know 5-Hydroxy-Indoleacetic Acid Somatostatin-14 pg/mL 0.611 pmol/L mg/24 h 5.236 mol/d (5-HIAA), Urine Somatostatin-28 pg/mL 0.305 pmol/L 5-HIAA:Creatinine mg/g 0.592 mol:mmol Sex Hormone-binding Globulin nmol/L 1.0 nmol/L Homovanillic Acid (HVA), Urine mg/24 h 5.489 mol/d (SHBG) (Binding Capacity) HVA:Creatinine g/g 0.621 mol:mmol Testosterone ng/dL 0.035 nmol/L IGF I (Somatomedin C) ng/mL 0.131 nmol/L Testosterone, Free pg/mL 3.467 pmol/L IGF II ng/mL 0.133 nmol/L Testosterone, Urine g/24 h 3.467 nmol/d Inhibin U/mL 1.0 arb units/L Testosterone:Creatinine g/g 0.392 nmol:mmol Insulin IU/mL 6.945 pmol/L Thyroglobulin ng/mL 1.0 g/L Luteinizing Hormone (LH) mlU/mL 1.0 lU/L Thyroid-stimulating Hormone IU/mL 1.0 IU/mL (TSH) Metanephrine, Urine g/24 h 0.005 mol/d Thyroxine (T4) g/dL 12.871 nmol/L Metanephrines, Total Urine g/24 h 5.258 nmol/d Thyroxine-binding Globulin g/mL 17.094 nmol/L Metanephrines, Total:Creatinine g/mg 0.574 mmol:mol Thyrotropin-releasing Hormone Methoxytyramine, Urine g/24 h 5.981 nmol/d pg/mL 2.762 pmol/L (TRH) Norepinephrine, Plasma pg/mL 5.914 pmol/L Triiodothyronine (T3) ng/dL 0.015 nmol/L Norepinephrine, Urine g/24 h 5.914 nmol/d Vanillylmandelic Acid (VMA), mg/24 h 5.05 mol/d Urine Norepinephrine:Creatinine g/g 0.669 nmol:mmol VMA:Creatinine mg/g 0.571 mmol:mmol Normetanephrine, Urine g/24 h 0.005 mol/d Vitamin B-12 pg/mL 0.738 pmol/L Normetanephrine:Creatinine g/g 0.618 mol:mol 25-Hydroxy-Vitamin D ng/mL 2.521 nmol/L Osteocalcin ng/mL 0.171 nmol/L 1,25-Dihydroxy-Vitamin D pg/mL 2.402 pmol/L Parathyroid Hormone (PTH) pg/mL 0.106 pmol/L Prednisolone ng/dL 27.739 pmol/L Prednisone ng/dL 27.902 pmol/L Pregnenolone ng/dL 0.032 nmol/L 17-Hydroxy-Pregnenolone ng/dL 0.03 nmol/L Progesterone ng/mL 3.18 nmol/L 17-Hydroxy-Progesterone ng/dL 0.03 nmol/L Prolactin ng/mL 43.478 pmol/L Renin (Plasma Renin Activity) ng/mL/h 0.278 ng/L/s Reverse T3 ng/dL 0.015 nmol/L Secretin pg/mL 0.327 pmol/L ENDOCRINOLOGY SYLLABUS 67

69 ENDOCRINOLOGY NOTES 68 ENDOCRINOLOGY SYLLABUS

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