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SEMINAR OUTLINE

Biochemical Basis of Diagnosis and Disorders of Endocrine Glands

For MD Biochemistry 2nd Year PG Students

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📋 SLIDE-BY-SLIDE OUTLINE

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SECTION I: PITUITARY DISORDERS (Slides 1–7)

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• Title: Biochemical Basis of Diagnosis of Endocrine Disorders: Pituitary, Adrenal & Thyroid
• Sub-topics listed
• Presenter name, date, institution

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• Hypothalamic-pituitary portal system
• Anterior pituitary: ACTH, TSH, GH, LH/FSH, Prolactin — each from specific cell types (corticotrophs, thyrotrophs, somatotrophs, gonadotrophs, lactotrophs)
• Posterior pituitary: ADH (Vasopressin), Oxytocin
• Triple feedback loop: long, short, ultra-short
• Key diagram: hypothalamic-releasing hormones and their targets (Table 55.1)
• 📖 Tietz 7th ed., Chapter 55, p. 2204–2207

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• Definition: Glucocorticoid excess (endogenous vs. exogenous)
• Classification:
  • ACTH-dependent (~80%): Cushing disease (pituitary adenoma), ectopic ACTH
  • ACTH-independent (~20%): adrenocortical adenoma/carcinoma
• Pathophysiology: Loss of diurnal variation of cortisol; loss of ACTH negative feedback
• Key biochemical features: ↑serum cortisol (loss of diurnal rhythm), ↑24h urinary free cortisol (UFC), ↑plasma ACTH (in ACTH-dependent)
• 📖 Tietz 7th ed., Chapter 56, p. 2283

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• Step 1 – Screening tests (any one):
  • 24h urinary free cortisol (UFC) — threshold >4× upper limit
  • Late-night salivary cortisol (×2)
  • Overnight low-dose dexamethasone suppression test (1 mg DST): cortisol >1.8 µg/dL = abnormal
• Step 2 – Confirmatory: Low-dose DST (2-day, 0.5 mg q6h): UFC not suppressed <50% = Cushing confirmed
• Step 3 – Source localization:
  • Measure plasma ACTH:
    • ACTH <10 pg/mL → adrenal tumor (ACTH-independent)
    • ACTH >50 pg/mL → pituitary or ectopic
  • High-dose DST (8 mg): suppression → Cushing disease (pituitary); no suppression → ectopic ACTH (ACTH may be >300 pg/mL)
  • MRI pituitary / Inferior petrosal sinus sampling (IPSS)
• 📖 Tietz 7th ed., Chapter 56, p. 2283–2284

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• ADH (Vasopressin/AVP): nonapeptide from posterior pituitary; t½ = 15–20 min; acts on collecting duct to promote water reabsorption via V2 receptor → aquaporin-2 insertion
• Types of DI:
  • Central DI: ↓ADH secretion (head injury, tumors, idiopathic)
  • Nephrogenic DI: end-organ resistance to ADH
• Biochemical features: Polyuria (>3L/day), polydipsia, dilute urine (osmolality <300 mOsm/kg), hypernatremia, ↑serum osmolality (>295 mOsm/kg)
• Diagnosis:
  • Water deprivation test → measure urine osmolality
  • DDAVP challenge test: central DI responds (urine osmolality rises >50%), nephrogenic DI does not
  • Copeptin measurement: newer; stable prohormone of ADH; longer half-life; replaces direct AVP measurement
• 📖 Tietz 7th ed., Chapter 31 (Block 10), p. 940

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• Definition: Excess ADH secretion relative to plasma osmolality → water retention → dilutional hyponatremia
• Causes: CNS disorders, pulmonary disease (TB, pneumonia), drugs (carbamazepine, SSRIs, vincristine), ectopic ADH (small cell lung carcinoma)
• Biochemical criteria (Bartter & Schwartz):
  1. Hyponatremia with low plasma osmolality (<275 mOsm/kg)
  2. Inappropriately concentrated urine (osmolality >100 mOsm/kg)
  3. Urine sodium >40 mmol/L
  4. Euvolemia (no edema, ascites)
  5. Normal renal, adrenal, thyroid function
• Differentiation from other hyponatremia: urine Na+ & osmolality, volume status, clinical context
• 📖 Tietz 7th ed., Chapter 31, p. 940

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SECTION II: ADRENAL MEDULLA — PHEOCHROMOCYTOMA (Slides 8–11)

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• Location: inner portion of adrenal gland, ~1/10th of total gland weight
• Chromaffin cells: catecholamine storage granules (100–300 nm); chromaffin reaction (brown with K-dichromate)
• Catecholamine synthesis: Tyrosine → DOPA → Dopamine → Norepinephrine → Epinephrine (PNMT enzyme, stimulated by glucocorticoids from cortex)
• Metabolism: MAO + COMT → metanephrines (normetanephrine, metanephrine) → VMA (vanillylmandelic acid)
• Adrenal medulla produces mainly epinephrine (80%); metabolic hormone (lipolysis, glycogenolysis, ketogenesis, thermogenesis)
• 📖 Tietz 7th ed., Chapter 53, p. 2082

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• Definition (WHO): Tumor of chromaffin cells in adrenal medulla; extra-adrenal = paraganglioma
• "Rule of 10" (historical): 10% bilateral, 10% extra-adrenal, 10% malignant, 10% in children, 10% familial
• Modern update: up to 40% have germline mutations (RET, VHL, NF1, SDHB/C/D, MAX, TMEM127)
• Prevalence: 2–5 cases/million/year; 0.2–0.6% of hypertensive patients
• Clinical: Paroxysmal hypertension, headache, diaphoresis, palpitations ("5 Ps" – pressure, pain, perspiration, pallor, palpitation)
• 📖 Tietz 7th ed., Chapter 53, p. 2091–2092

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• Gold standard: Plasma free metanephrines (sensitivity ~97%, specificity ~93%) OR 24h urinary fractionated metanephrines
• Why metanephrines?: Continuously produced within chromaffin cells (independent of episodic secretion); more stable than catecholamines
• Test hierarchy:
  1. Plasma free metanephrines (best screening)
  2. 24h urine fractionated metanephrines + catecholamines
  3. Urine VMA (less sensitive, historical)
• Analytic method: LC-MS/MS (gold standard); immunoassay available
• Clonidine suppression test: Used when plasma normetanephrine mildly elevated; pheochromocytoma: no suppression; essential hypertension: suppression
• 📖 Tietz 7th ed., Chapter 53, p. 2091–2100

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• Imaging: CT/MRI abdomen (90% adrenal); MIBG scintigraphy (functional imaging)
• Malignancy (metastatic pheochromocytoma): PASS score; biochemical markers: ↑chromogranin A, ↑neuropeptide Y, ↑dopamine-predominant biochemistry suggests malignancy
• Chromogranin A: General neuroendocrine tumor marker; elevated in pheochromocytoma
• Genetic testing recommended for all patients (index of suspicion for MEN2, VHL, SDH mutations)
• 📖 Tietz 7th ed., Chapter 53, p. 2095–2098

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SECTION III: ADRENAL CORTEX DISORDERS (Slides 12–20)

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• Three zones: Zona Glomerulosa (aldosterone), Zona Fasciculata (cortisol), Zona Reticularis (androgens)
• Steroidogenesis: Cholesterol → Pregnenolone (rate-limiting: cholesterol desmolase/CYP11A1) → divergent pathways
• Key enzymes: CYP11B1 (11β-hydroxylase), CYP11B2 (aldosterone synthase), CYP21A2 (21-hydroxylase), CYP17A1 (17α-hydroxylase)
• ACTH controls glucocorticoid and androgen production; angiotensin II + K+ control aldosterone
• 📖 Tietz 7th ed., Chapter 56, p. 2271–2275

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• Clinical: Central obesity, moon face, buffalo hump, purple striae, hypertension, hyperglycemia, osteoporosis, proximal myopathy, hypokalemia
• Key biochemical findings:
  • ↑Serum cortisol (loss of diurnal rhythm)
  • ↑24h UFC (>4× upper limit diagnostic)
  • ↑Late-night salivary cortisol
  • Hyperglycemia, hypokalemia, metabolic alkalosis
  • ↑ACTH if pituitary/ectopic; ↓ACTH if adrenal tumor
• Pseudo-Cushing: Alcoholism, depression, obesity — can mimic biochemically; differentiated by CRH stimulation test
• 📖 Tietz 7th ed., Chapter 56, p. 2283–2289

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• Definition: Primary adrenocortical insufficiency → ↓cortisol + ↓aldosterone
• Causes: Autoimmune (most common in developed countries; anti-21-hydroxylase antibodies; associated with APS type 2: also type 1 DM, autoimmune thyroiditis, celiac disease); TB (most common worldwide); granulomatous diseases, hemorrhage, malignancy
• Biochemical features:
  • ↓Serum cortisol (<3 µg/dL = diagnostic)
  • ↑Plasma ACTH (>300 pg/mL in primary)
  • Hyponatremia (↓aldosterone → Na+ loss)
  • Hyperkalemia, hypoglycemia, hypercalcemia
  • Eosinophilia, lymphocytosis
  • Hyperpigmentation (↑MSH due to POMC cleavage with ACTH)
• 📖 Tietz 7th ed., Chapter 56, p. 2271–2278

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• Basal cortisol (morning, 8 AM): <3 µg/dL strongly suggests adrenal insufficiency; >18 µg/dL rules it out
• Short Synacthen (Cosyntropin) Stimulation Test:
  • 250 µg ACTH IV/IM; cortisol at 0, 30, 60 min
  • Normal: peak cortisol ≥18–20 µg/dL (500–550 nmol/L); Δcortisol ≥7–10 µg/dL
  • Abnormal response = adrenal insufficiency
  • Low-dose variant (1 µg): greater sensitivity for mild adrenal insufficiency
• ACTH level: Differentiates primary (↑↑ACTH) from secondary (↓/normal ACTH)
• Aldosterone & renin: ↓Aldosterone, ↑renin in primary AI
• Autoantibodies: Anti-21-hydroxylase (CYP21A2) antibodies — confirm autoimmune etiology
• Addisonian crisis: Precipitated by stress; acute hypotension, hyponatremia, hyperkalemia, hypoglycemia
• 📖 Tietz 7th ed., Chapter 56, p. 2276–2282; cosyntropin protocol p. 2286–2289

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• Definition: Autonomous overproduction of aldosterone independent of renin-angiotensin system
• Causes: Aldosterone-producing adenoma (APA/Conn adenoma ~35%), bilateral adrenal hyperplasia (~60%), adrenal carcinoma (rare)
• Biochemical features:
  • Hypertension (often resistant)
  • Hypokalemia (↓K+), metabolic alkalosis
  • Hypernatremia (mild)
  • ↑Aldosterone (PAC), suppressed Plasma Renin Activity (PRA)
  • ↑Aldosterone-to-Renin Ratio (ARR): Screening test — ARR >30 (ng/dL)/(ng/mL/h) with PAC >15 ng/dL is positive
• Confirmatory: Oral sodium loading test / IV saline infusion test — aldosterone not suppressed
• Subtype differentiation: Adrenal CT + Adrenal Vein Sampling (AVS) — lateralization ratio >4:1 suggests APA
• 📖 Tietz 7th ed., Chapter 56, p. 2260–2270

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• Definition: Autosomal recessive disorder of adrenocortical steroid biosynthesis
• Enzyme deficiencies in order of frequency:
  1. 21-Hydroxylase (CYP21A2): 95% of cases
  2. 11β-Hydroxylase (CYP11B1): ~5% of cases
  3. Others: 17α-hydroxylase, 3β-HSD, StAR protein deficiency (rare)
• Pathophysiology (21-OHD): ↓Cortisol → ↑ACTH (negative feedback loss) → adrenal hyperplasia + ↑androgen precursors (17-OHP, androstenedione) → virilization
• Clinical forms:
  • Salt-wasting: ↓cortisol + ↓aldosterone; neonatal crisis within 2 weeks of birth
  • Simple virilizing: virilization, normal mineralocorticoid activity
  • Non-classical (late-onset): mild, presents as PCOS-like in females
• 📖 Tietz 7th ed., Chapter 56, p. 2292–2297; Chapter 14, p. 1259

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• 21-Hydroxylase deficiency:
  • ↑17-OHP (>1000 ng/dL; typically 5–10× normal; gold standard marker)
  • ↑Androstenedione (5–10× normal)
  • ↓11-DOC, ↓11-DC (distinguishes from 11β-OHD where both are elevated)
  • Neonatal screening: Heel-prick blood spot, immunoassay for 17-OHP; LC-MS/MS for confirmation
  • Stimulated 17-OHP (after ACTH stimulation) used for non-classical form diagnosis
• 11β-Hydroxylase deficiency:
  • ↑11-Deoxycortisol (11-DC), ↑11-DOC → ↑mineralocorticoid activity → hypokalemic hypertension
  • ↓Cortisol, virilization
  • No salt-wasting (aldosterone pathway intact)
• Prenatal diagnosis: CYP21A2 gene mutation analysis (amniocentesis/CVS); dexamethasone to mother to suppress fetal ACTH
• 📖 Tietz 7th ed., Chapter 56, p. 2293–2298

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SECTION IV: THYROID DISORDERS (Slides 20–28)

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• Thyroid hormones: T4 (thyroxine, 3,5,3',5'-tetraiodothyronine) and T3 (3,5,3'-triiodothyronine)
• HPT axis: Hypothalamus (TRH) → Anterior pituitary (TSH) → Thyroid (T3/T4) → Negative feedback primarily on pituitary (THRB-2 receptor)
• TSH actions on thyroid: ↑Iodide uptake (NIS), organification, coupling, thyroglobulin proteolysis
• Circulating forms: 99.97% T4 bound (TBG 70%, transthyretin 10–15%, albumin 15–20%); only free fraction biologically active
• T4 → T3 conversion: Peripheral deiodination (5'-deiodinase types I, II, III); T3 is the active hormone (3–4× more potent than T4); rT3 is inactive
• Functions: ↑BMR, calorigenesis, cardiac stimulation, protein synthesis, neural development, lipid metabolism
• 📖 Tietz 7th ed., Chapter 57, p. 2304; Chapter 55, p. 2241

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• Step 1: Iodide trapping — NIS (Na+/I− symporter) at basolateral membrane; TSH-driven
• Step 2: Oxidation of I− to I0/I+ (thyroid peroxidase, TPO; H2O2 generated by DUOX2)
• Step 3: Organification — iodination of tyrosyl residues on thyroglobulin → MIT (monoiodotyrosine), DIT (diiodotyrosine)
• Step 4: Coupling reaction (TPO) — DIT + DIT → T4; MIT + DIT → T3
• Step 5: Secretion — TSH triggers pinocytosis of colloid, lysosomal digestion of thyroglobulin, release of T4/T3
• Wolff-Chaikoff effect: High iodide transiently inhibits organification (mechanism of iodide therapy in thyroid storm)
• Key enzymes: TPO (target of anti-TPO antibodies in autoimmune disease)
• 📖 Tietz 7th ed., Chapter 57, p. 2304–2307

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• Classification:
  • Primary: ↓T3/T4 → ↑↑TSH (most common)
  • Secondary (pituitary): ↓TSH → ↓T3/T4
  • Tertiary (hypothalamic): ↓TRH → ↓TSH → ↓T3/T4
• Causes of primary hypothyroidism: Hashimoto's thyroiditis (anti-TPO, anti-Tg antibodies), iodine deficiency, post-radioiodine, post-thyroidectomy, drugs (amiodarone, lithium)
• Biochemical features:
  • ↑TSH (most sensitive marker; first to change)
  • ↓Free T4 (fT4)
  • ↑Total cholesterol/LDL (↓LDL receptor expression)
  • ↑CK (muscle involvement)
  • Hyponatremia (↓free water excretion)
  • Normochromic normocytic or macrocytic anaemia
• Subclinical hypothyroidism: ↑TSH, normal fT4 (progression to overt ~2–5%/year if anti-TPO positive)
• TRH stimulation test: Differentiates secondary vs. tertiary hypothyroidism (delayed TSH rise in tertiary)
• 📖 Tietz 7th ed., Chapter 46/Block 20, p. 912; Chapter 55, p. 2241

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• Causes: Graves' disease (TSI/TRAb — TSH receptor stimulating antibodies), toxic multinodular goitre, toxic adenoma, subacute thyroiditis, iodine-induced, TSH-secreting pituitary adenoma, factitious
• Graves' disease mechanism: IgG autoantibodies (TRAb/TSI) bind TSHR → constitutive activation → ↑T3/T4 + thyroid hyperplasia (goitre)
• Biochemical features:
  • ↓TSH (<0.01 mIU/L with 3rd generation assays)
  • ↑Free T4, ↑Free T3 (T3-toxicosis possible with normal fT4)
  • ↑SHBG (sensitive marker of thyroid hormone action)
  • Hypercalcemia (↑bone turnover)
  • Hyperglycemia, ↑ALP
  • ↓Total cholesterol
• Key Tests: TSH (screening), fT4 and fT3 (confirmation), TRAb (Graves' specific), radioiodine uptake scan
• Subclinical hyperthyroidism: Suppressed TSH, normal fT3/fT4
• 📖 Tietz 7th ed., Chapter 57, p. 2304–2315

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• TSH assay: 3rd/4th generation immunometric assay; detection limit ≤0.01 mIU/L; reference range 0.4–4.0 mIU/L; TSH is the single best screening test for thyroid disorders
• Free T4: Equilibrium dialysis (reference method); immunoassay (routine); index methods (FTI = T4 × T3U)
• Free T3: Assayed similarly; clinically relevant for T3 toxicosis and monitoring
• Thyroid antibodies:
  • Anti-TPO (thyroid peroxidase antibody): most sensitive for Hashimoto's
  • Anti-Tg (thyroglobulin antibody): less specific
  • TRAb (TSH receptor antibody): specific for Graves' disease
• Thyroglobulin (Tg): Tumor marker for differentiated thyroid cancer follow-up (post-thyroidectomy)
• Reverse T3 (rT3): Elevated in non-thyroidal illness syndrome; not routinely measured
• 📖 Tietz 7th ed., Chapter 55, p. 2241–2243; Chapter 57, p. 2308–2320

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• Definition: Enlargement of thyroid gland (diffuse or nodular)
• Classification:
  • Euthyroid goitre: Iodine deficiency (endemic goitre — most common cause globally; iodine <50 µg/day)
  • Toxic goitre: Graves' (diffuse), Toxic MNG, toxic adenoma
  • Non-toxic MNG (simple/colloid goitre)
  • Thyroiditis-related (Hashimoto's, subacute de Quervain's)
• Pathogenesis of endemic goitre: Iodine deficiency → ↑TSH → thyroid hyperplasia
• Biochemical assessment:
  • TSH, fT4, fT3 (euthyroid vs. toxic)
  • Urine iodine excretion (<100 µg/g creatinine = iodine deficiency)
  • Anti-TPO/TRAb (autoimmune etiology)
  • Thyroglobulin (elevated in goitre, iodine deficiency)
• 📖 Tietz 7th ed., Chapter 57

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• Classification:

  Type	Cell of Origin	Differentiation	Incidence
  Papillary	Follicular cell	Well differentiated	~80%
  Follicular	Follicular cell	Well differentiated	~10%
  Medullary	Parafollicular C-cell	—	~5%
  Anaplastic	Follicular cell	Undifferentiated	~2%

• Molecular/biochemical markers:
  • Thyroglobulin (Tg): Tumor marker for papillary & follicular CA post-thyroidectomy; undetectable Tg after complete ablation = remission; rising Tg = recurrence
  • Anti-Tg antibodies: Can interfere with Tg measurement (false-low immunoassay); must be co-measured
  • Calcitonin: Specific marker for medullary thyroid carcinoma (MTC); parafollicular C-cells; also used for screening in MEN2 families (pentagastrin stimulation)
  • CEA: Also elevated in MTC; useful for monitoring
  • BRAF mutation (V600E): Most common mutation in papillary TC; molecular marker with diagnostic/prognostic value
  • RET proto-oncogene: MTC; germ-line mutation = MEN2A/MEN2B; sporadic MTC = somatic mutation
• 📖 Tietz 7th ed., Chapter 33 (Block 11), p. 2545

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SECTION V: CLOSING SLIDES (Slides 28–30)

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• Tier 1 (Screening): TSH (thyroid), cortisol AM + ACTH (adrenal axis), plasma metanephrines (pheochromocytoma), aldosterone:renin ratio (Conn's), sodium/osmolality (DI/SIADH)
• Tier 2 (Confirmatory/Dynamic): DST variants, cosyntropin stimulation, saline suppression, water deprivation/DDAVP test
• Tier 3 (Source localization): IPSS, AVS, imaging (MRI, CT, MIBG, radioiodine scan)
• Tier 4 (Molecular): Gene panels (RET, CYP21A2, MEN1), autoantibodies, tumor markers

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• Chapter 14 — Endocrine Function: Reproductive Disorders — CAH, p. 1259
• Chapter 31 (Block 10) — Vasopressin, Diabetes Insipidus, SIADH, p. 940
• Chapter 53 — The Adrenal Medulla & Pheochromocytoma, p. 2082–2098
• Chapter 55 — Pituitary Embryology, Anterior & Posterior Pituitary, TSH, p. 2204–2243
• Chapter 56 — Adrenal Cortex: Addison Disease, Cushing Syndrome, CAH, Conn's Syndrome, p. 2260–2298
• Chapter 57 — Thyroid Hormones, Thyroid Function Tests, Thyroid Disease, p. 2304–2320
• Chapter 33 (Block 11) — Thyroid Cancer & Tumor Markers, p. 2545

• Endocrine Society Clinical Practice Guidelines (Pheochromocytoma, Cushing's, Primary Hyperaldosteronism)
• WHO Classification of Endocrine Tumors, 2022

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1. TSH is the single most sensitive screening test for all thyroid disorders
2. Plasma free metanephrines are the first-line biochemical test for pheochromocytoma
3. The aldosterone:renin ratio is the best screening test for Conn's syndrome
4. 17-OHP is the biochemical hallmark of 21-hydroxylase deficiency (most common CAH)
5. Urine/serum osmolality + DDAVP test differentiates central DI from nephrogenic DI
6. Copeptin is emerging as a practical surrogate for ADH measurement
7. Late-night salivary cortisol and 24h UFC are the most reliable tests for Cushing screening
8. Thyroglobulin is the tumor marker of choice for follow-up of differentiated thyroid cancer; calcitonin for medullary thyroid cancer
9. Autoantibodies (anti-TPO, anti-21OHase, TRAb) establish the autoimmune etiology of thyroid and adrenal diseases

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📚 COMPLETE REFERENCE LIST FROM TIETZ (7th Edition)

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📐 SLIDE COUNT BREAKDOWN

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