Pitfalls in Endocrine Testing Part 3: Adrenal, Cortisol, Prolactin, and Gonadal Axis Testing High, and Gonadal Axis Notes on the Gonadal Axis

Introduction and Learning Objectives

• Presenter: Dr. Nimali Pereira, Royal Prince Alfred Hospital.

• Context: This is Part 3 of the "Pitfalls in Endocrine Testing" series, focusing on the adrenal gland, cortisol, prolactin, and the gonadal axis.

• Core Learning Objectives:

  • Recognize common causes of false endocrine test results.

  • Identify causes of increased prolactin and when to suspect macroprolactin.

  • Understand why cortisol tests often provide discordant or disagreeing results.

  • Identify key pre-analytical factors including timing, posture, medications, and collection requirements.

  • Explain the necessity of morning measurements for $\text{testosterone}$ and $\text{cortisol}$ due to diurnal variation.

Prolactin and Macroprolactin

• Physiological Role of Prolactin:

  • Produced by the lactotrophs in the pituitary gland.

  • Primary function: Stimulation of milk production.

  • Biological Effect: Suppresses the reproductive axis (hypothalamic-pituitary-gonadal axis).

• Clinical Implications of Hyperprolactinemia:

  • In females: Causes irregular periods (menstrual dysfunction) and infertility.

  • In males: Causes low $\text{testosterone}$ and sexual dysfunction.

Causes of Elevated Prolactin

• Physiological: Pregnancy is a primary cause in women of reproductive age.

• Pathological: Pituitary adenomas (microadenomas and rarely macroadenomas) that autonomously secrete prolactin.

• Iatrogenic (Medications): Prolactin is controlled by dopamine, which acts as a "brake" on secretion.

  • Dopamine blockers (antipsychotics) are classic causes.

  • Anti-nausea medications: $\text{metoclopramide}$ and $\text{domperidone}$.

• Stress and Environment: Prolactin is highly sensitive to physical and emotional stress.

  • Factors like a difficult needle draw (venepuncture pain), exercise on the morning of the test, rushing to the appointment, or waiting-room anxiety can elevate levels.

  • Stress can push levels to $\text{5}$ to $\text{10}$ times the upper limit of normal ($ULN$).

Macroprolactin (Big Big Prolactin)

• Definition: A molecular form where prolactin is bound to an antibody (IgG), creating a large complex.

• Clinical Significance:

  • It is biologically inactive because it is too large to bind to receptors.

  • Standard immunoassays often count it as "total prolactin," leading to falsely elevated results in asymptomatic patients.

  • Misinterpretation can lead to unnecessary brain $\text{MRIs}$ and medication.

• Laboratory Detection (PEG Precipitation):

  • The lab adds $\text{Polyethylene Glycol (PEG)}$ to the sample.

  • ${PEG}$ precipitates/removes the "big big" prolactin complexes.

  • The supernatant is then measured for monomeric, biologically active prolactin.

Clinical Management of High Prolactin

• Step 1: Exclude pregnancy.

• Step 2: Repeat the test on a different day.

• Conditioning: The patient must sit quietly for $\text{15}$ to $\text{30}$ minutes before blood collection to eliminate stress-induced surges.

• Next Step: Request a macroprolactin screen via the lab if levels remain high and symptoms are absent.

Cushing's Syndrome and Cortisol Testing

• Diagnosis Principle: Never rely on a single test. Diagnosis requires multiple biochemical tests interpreted in a clinical context.

• Discordance: Results from different tests (urine, saliva, blood) frequently disagree; this is considered normal and expected, not a mistake.

Standard Screening Tests

1. 1 mg Overnight Dexamethasone Suppression Test (DST): Tests if exogenous steroids suppress morning cortisol.

2. 24-Hour Urine Free Cortisol (UFC): Measures total cortisol output over a full day.

3. Late-Night Salivary Cortisol: Measures the "nadir" or lowest point of cortisol production.

Pre-analytical Factors for Cortisol and ACTH

• Diurnal Rhythm: Cortisol is highest between $\text{8:00 AM}$ and $\text{9:00 AM}$ and lowest at midnight. Collection time must always be documented.

• ACTH Handling: Highly unstable.

  • Requires $\text{EDTA}$ tubes.

  • Must be kept on ice and processed rapidly by the lab to avoid spurious low results.

Case Study: Method Variability

• Patient: $\text{62}$-year-old woman with an incidental adrenal adenoma ($lipid-rich$), hypertension, and diabetes.

• Test Results:

  • Overnight $DST$: Cortisol not suppressed (abnormal).

  • Salivary Cortisol: Elevated (abnormal).

  • $24$-hour UFC (measured by $LC-MS/MS$): Consistently normal.

• Resolution: The sample was re-tested using an Immunoassay method, which showed elevated levels consistent with Cushing's.

• Key Distinction: Mass Spec ($LC-MS/MS$) vs. Immunoassay:

  • Mass Spec: Highly specific. Measures only true cortisol ($C_{21}H_{30}O_{5}$). It may miss "mild" cases or forget to count precursors and metabolites secreted by a tumor.

  • Immunoassay: Less specific but more sensitive in this context. It "cross-reacts" with cortisol precursors and metabolites, providing a higher total reading that may be more clinically useful for screening.

  • Rule: More specific does not always mean more sensitive.

Pitfalls in Dexamethasone Suppression

• False Positives (Failure to Suppress):

  • CYP3A4 Inducers: Medications like $\text{carbamazepine}$, $\text{phenytoin}$, and $\text{rifampicin}$ speed up the metabolism of dexamethasone.

  • Result: Dexamethasone is cleared before it can suppress the brain, leading to a false high morning cortisol.

  • Absorption: Poor absorption of the dexamethasone pill.

• Validation: Measuring the blood dexamethasone level alongside the morning cortisol confirms the drug was taken, absorbed, and not metabolized too quickly.

Aldosterone and Renin Testing

• Usage: Evaluation of Primary Hyperaldosteronism in patients with hypertension, hypokalemia, or adrenal adenomas.

• Pre-analytical Preparation (Extremely Strict):

  • Medication Stoppage: Must stop $\text{Spironolactone}$ (Aldactone) and other Mineralocorticoid Receptor Antagonists ($MRAs$) for $\text{4}$ to $\text{6}$ weeks.

  • Interfering Meds: $\text{ACE inhibitors}$, $\text{ARBs}$, and $\text{Beta-blockers}$ interfere with results.

  • Safe Alternatives: $\text{Verapamil}$, $\text{Prazosin}$, and $\text{Hydralazine}$.

  • Diet/Electrolytes: Patient needs normal salt intake and corrected potassium levels.

  • Posture: Gravity affects the renin-angiotensin-aldosterone system. Patients should be upright/walking for $\text{2}$ hours, then seated for $\text{5}$ to $\text{15}$ minutes before the draw.

Other Endocrine Tests and Pitfalls

• Plasma Metanephrines (Pheochromocytoma):

  • Requires patient to be fasting and supine (lying flat) for at least $\text{30}$ minutes in a quiet room.

  • Stress and upright posture increase sympathetic tone, causing false-positive elevations in normetanephrines.

• Growth Hormone ($GH$) vs. IGF-1:

  • Growth Hormone: Secreted in pulses; a single random measurement is useless.

  • IGF-1: Secreted by the liver in response to $GH$; stable and acts as an integrated marker of $GH$ status.

• Gonadal Axis (HPG Axis):

  • Testosterone must be measured in the morning ($\text{7:00 AM} - \text{10:00 AM}$) because levels can drop by $\text{20}$ to $\text{30}$ percent by the afternoon.

  • Screen for use of androgenic supplements, exogenous testosterone, or oral contraceptives.

The Master Diagnostic Framework

Before acting on any abnormal endocrine result, evaluate these four categories:

1. Drug/Supplement Interference: Are they taking heparin, carbamazepine, rifampicin, biotin, spironolactone, or antipsychotics?

2. Collection Problems: Was it the wrong time of day, the wrong tube, or delayed processing? Was the sample chilled/iced?

3. Patient Preparation: Did they follow fasting/dietary requirements? Did they stop interfering medications? Was their posture correct?

4. Laboratory Issues: Is the assay too specific? Is there cross-reactivity? Is there platform-to-platform variability or a transcription error?

Review Questions and Discussion

• Scenario 1: A $\text{32}$-year-old woman with prolactin of $\text{1,400}$ ($ULN \text{ is } 500$), no symptoms, and normal $MRI$.

  • Correct Step: Exclude pregnancy and repeat the test after rest; then test for macroprolactin if still high.

• Scenario 2: Patient has abnormal $DST$ (no suppression) while on $\text{carbamazepine}$, but normal 24h UFC via $LC-MS/MS$.

  • Explanations: Accelerated dexamethasone metabolism ($CYP3A4$ induction) caused the false positive $DST$. The normal UFC could be due to the high specificity of $LC-MS/MS$ or an under-collected urine sample (checked via creatinine levels).

• Scenario 3: Aldosterone-renin ratio testing.

  • Invalidating Factor: Taking spironolactone is a major interference. Posture (standing) and salt intake also invalidate results.

Final Rule: If in doubt, question the test and the collection process, not the patient. Use the laboratory test catalogue or call the lab for guidance.