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BIOCHEMISTRY MASTER GUIDE: Hormones, Metabolism & Diabetes

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1. STEROID HORMONE SYNTHESIS

What is a steroid hormone?

Where are they made?

The Pathway - Step by Step (from cholesterol to hormones)

1. Cholesterol is the starting material (comes from LDL uptake or synthesis from acetyl-CoA)
2. CYP11A (P450scc - side chain cleavage) - located in the inner mitochondrial membrane - cuts off 6 carbons from cholesterol's side chain → forms Pregnenolone (21 carbons)
3. 3-β-hydroxysteroid dehydrogenase (3β-HSD) converts pregnenolone → Progesterone (this is the first major branch point)
4. From progesterone, three enzymes shape the final products:
   • CYP17 (17α-hydroxylase) - produces sex steroids and cortisol precursors
   • CYP21 (21-hydroxylase) - produces cortisol and aldosterone precursors
   • CYP11B1 (11β-hydroxylase) - final step to cortisol

Cortisol Synthesis Specifically:

Cholesterol → Pregnenolone → Progesterone → 17α-OH Progesterone → 11-Deoxycortisol → CORTISOL
                                              (CYP17)              (CYP21)              (CYP11B1)

Aldosterone Synthesis:

Cholesterol → Pregnenolone → Progesterone → 11-Deoxycorticosterone → Corticosterone → Aldosterone
                                             (CYP21)                  (CYP11B1)        (CYP11B2)

Sex Steroid Synthesis:

• Progesterone → (CYP17) → DHEA → Androstenedione → Testosterone
• Testosterone → (CYP19/aromatase in ovarian granulosa cells) → Estradiol
• Testosterone → (5α-reductase in peripheral tissues) → Dihydrotestosterone (DHT) (more potent)

Enzyme Defects - Congenital Adrenal Hyperplasia (CAH):

• 21-hydroxylase deficiency (most common): no cortisol/aldosterone → excess androgens → virilization in females
• 11β-hydroxylase deficiency: no cortisol, but excess 11-deoxycorticosterone (causes hypertension)
• 17α-hydroxylase deficiency: no cortisol or sex steroids, only aldosterone

The Cytochrome P450 Enzymes - Why They Matter:

Sources: Basic Medical Biochemistry 6e, Costanzo Physiology 7th Ed

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2. THYROID HORMONE SYNTHESIS

Background - What are thyroid hormones?

Key Players:

• Thyroglobulin: a large glycoprotein (two subunits), made in thyroid cells, containing 123 tyrosine residues - only 4-8 are used for hormone synthesis. It is the "scaffold" on which hormones are built.
• Thyroid peroxidase (TPO): the enzyme that does iodination AND coupling
• Iodine: the essential raw material, absorbed as iodide (I-)
• Colloid: the space inside thyroid follicles where thyroglobulin is stored

Step-by-Step Thyroid Hormone Synthesis:

• First product: MIT (monoiodotyrosine) - one iodine added
• Then: DIT (diiodotyrosine) - second iodine added at carbon-5

• DIT + DIT → T4 (thyroxine) + alanine side chain released
• MIT + DIT → T3 (triiodothyronine) + alanine The resulting T3 and T4 remain covalently attached to thyroglobulin in the colloid. This acts as a hormone reservoir - humans can survive 2 months without iodine before circulating levels drop.

Transport in Blood:

• Only ~0.02% of T4 and ~0.2% of T3 circulate FREE (biologically active)
• Rest is bound to: TBG (thyroxine-binding globulin) - main carrier, transthyretin (prealbumin), and albumin
• TBG is increased by: pregnancy, oral contraceptives, estrogen, hepatitis
• TBG is decreased by: androgens, corticosteroids, hypoproteinemia (nephrotic syndrome, liver disease)

T4 to T3 Conversion:

• Most T3 in circulation (~80%) is made by peripheral deiodination of T4 in liver, kidney, muscles - by deiodinase type 1 & 2
• T4 can also form reverse T3 (rT3) - biologically inactive - by type 3 deiodinase
• In illness (euthyroid sick syndrome): T3 falls, rT3 rises, TSH normal/low

Regulation - The HPT Axis:

Hypothalamus → TRH → Pituitary → TSH → Thyroid → T3/T4 → (negative feedback on both)

• Cold, stress → TRH secretion
• High T3/T4 → suppress both TRH and TSH (negative feedback)

Sources: Ganong's Review of Medical Physiology 26th Ed, Quick Compendium of Clinical Pathology 5th Ed

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3. THYROID FUNCTION TESTS (TFTs)

Why test thyroid function?

The Key Tests:

A. TSH (Thyroid Stimulating Hormone)

• Single best screening test for thyroid dysfunction
• Modern third-generation assays detect as low as 0.01 mU/L
• TSH is inversely related to thyroid hormone levels (TSH goes up when T3/T4 go down)
• Normal: ~0.4-4.0 mU/L

B. Free T4 (fT4)

• Measures only the unbound, biologically active T4
• More reliable than total T4 because it is unaffected by TBG changes
• Low in hypothyroidism, high in hyperthyroidism

C. Free T3 (fT3)

• Used when T3 toxicosis is suspected (T3-predominant hyperthyroid state)
• Monitors patients on oral T3 therapy
• Checks for impaired T4→T3 conversion

D. Total T4 / Total T3 (less commonly used now)

• Includes both bound and free hormone
• Affected by TBG levels: TBG↑ → Total T4↑ even if free T4 is normal
• T3 resin uptake (T3RU): old test, now replaced by direct free hormone assays

E. Radioactive Iodine Uptake (RAIU)

• Patient given radioactive iodine dose, then scanned
• High RAIU: Graves disease, toxic nodular goitre (gland is overactive)
• Low RAIU: Thyroiditis (destruction releasing pre-formed hormone), iodine excess, exogenous T4 use

F. Thyroid Antibodies

• Anti-TPO antibodies: Hashimoto's thyroiditis (autoimmune hypothyroid)
• TSI (thyroid stimulating immunoglobulin) or anti-TSH receptor antibodies: Graves disease (autoimmune hyperthyroid)
• Anti-thyroglobulin antibodies: Hashimoto's or thyroid cancer monitoring

G. Thyroglobulin (Tg)

• Used as tumor marker to monitor differentiated thyroid cancer after total thyroidectomy
• Should be undetectable after thyroid removal

Non-Thyroidal Illness (Sick Euthyroid Syndrome):

Sources: Quick Compendium of Clinical Pathology 5th Ed, Washington Manual of Medical Therapeutics

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4. PTH AND CALCIUM METABOLISM

Why is calcium regulated so tightly?

Forms of Calcium in Blood:

• ~45% bound to albumin (inactive)
• ~10% complexed to anions like citrate, phosphate (inactive)
• ~45% ionized/free Ca2+ (biologically active - the form that matters)

The Inverse Ca-Phosphate Relationship:

• If phosphate goes up → calcium must go down
• Alkalosis promotes calcium deposition in bone → hypocalcemia
• Acidosis leaches calcium from bone → hypercalcemia

PTH (Parathyroid Hormone) - The Calcium Guardian:

• Low ionized Ca2+ is the main trigger
• Sensed by Calcium Sensing Receptor (CaSR) on parathyroid cells
• When Ca2+ falls → CaSR stops inhibiting → PTH is secreted
• High phosphate also stimulates PTH (independently)

Vitamin D (Calcitriol) - The "Sunshine Hormone":

1. Skin: UV light converts 7-dehydrocholesterol → Cholecalciferol (Vitamin D3)
2. Liver: 25-hydroxylase adds OH at C25 → 25-hydroxyvitamin D (calcidiol) - this is the storage form, measured in blood
3. Kidney: 1α-hydroxylase (stimulated by PTH, low phosphate, low Ca) adds OH at C1 → 1,25-(OH)2-D3 (calcitriol) - the active hormone

• Gut: increases synthesis of calcium-binding proteins → increases Ca AND phosphate absorption
• Bone: promotes mineralization at normal levels; high levels can cause bone resorption
• Kidney: mild increase in Ca reabsorption
• Calcitriol feeds back to suppress PTH synthesis

Calcitonin - The Opposite of PTH:

• Made by parafollicular C-cells of the thyroid
• Released when calcium is HIGH
• Actions: inhibits osteoclasts → less bone resorption → lowers blood calcium
• Clinical significance: less important in day-to-day regulation in adults; important marker for medullary thyroid carcinoma

Summary: Ca Regulation at a Glance:

Low Ca2+ → PTH↑ → Bone resorption↑ + Renal Ca reabsorption↑ + Vitamin D activation↑
         ↓
Gut Ca absorption↑ (via Vit D) + Renal phosphate excretion↑
         ↓
Ca2+ returns to normal → CaSR activated → PTH suppressed (negative feedback)

Sources: Henry's Clinical Diagnosis - Lab Methods, Basic Medical Biochemistry 6e

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5. CALCIUM AND PHOSPHATE - CLINICAL POINTS

Normal Values:

• Calcium: 8.5-10.5 mg/dL (ionized: 4.5-5.6 mg/dL)
• Phosphate: 2.5-4.5 mg/dL (adults)

Causes of Hypocalcemia:

• Hypoparathyroidism (post-surgical, autoimmune)
• Vitamin D deficiency / malabsorption
• Renal failure (↓ 1α-hydroxylase → ↓ Vit D; ↑ phosphate)
• Hypoalbuminemia (check ionized Ca)
• Alkalosis

Causes of Hypercalcemia (mnemonic: "CHIMPANZEES"):

• Calcium supplements excess
• Hyperparathyroidism (primary: usually parathyroid adenoma)
• Iatrogenic (thiazides, vitamin D)
• Malignancy (PTH-rP secretion, bone mets)
• Paget's disease
• Addison's disease
• Neoplasm (lymphoma via calcitriol)
• Zollinger-Ellison / Zinc toxicity
• Excess Vitamin D
• Endocrine (hyperthyroidism, acromegaly)
• Sarcoidosis (activated macrophages make calcitriol)

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6. INSULIN - STRUCTURE AND RECEPTOR

What is Insulin?

Structure of Insulin:

1. Beta cells of the islets of Langerhans (in pancreas) synthesize pre-proinsulin on the rough ER
2. Signal peptide is cleaved → Proinsulin (single polypeptide of ~86 amino acids)
3. Proinsulin is folded and three disulfide bonds form:
   • Two bonds connect the A and B chains
   • One internal bond within the A chain
4. In secretory granules (Golgi apparatus), protease cleaves proinsulin into:
   • Insulin (A chain: 21 amino acids + B chain: 30 amino acids, connected by 2 disulfide bonds)
   • C-peptide (connecting peptide: 31 amino acids) - equimolar release with insulin!
5. Insulin + C-peptide are stored as zinc-insulin hexamers in secretory granules and released together by exocytosis

What triggers insulin secretion?

1. Glucose enters beta cell via GLUT2 transporter (always open, high Km)
2. Glucose is phosphorylated by glucokinase (hexokinase IV) - the "glucose sensor"
3. Glucose metabolism → ↑ ATP/ADP ratio
4. ATP closes KATP channels (ATP-sensitive K+ channels)
5. Cell depolarizes (K+ can't exit)
6. Voltage-gated Ca2+ channels open → Ca2+ influx
7. Ca2+ triggers exocytosis of secretory granules → insulin released

Insulin Receptor:

• Tetramer: 2 alpha (α) subunits + 2 beta (β) subunits linked by disulfide bonds
• α-subunits: extracellular, contain the insulin-binding domain
• β-subunits: transmembrane + intracellular tyrosine kinase domain

1. Insulin binds to α-subunit
2. Conformational change activates the β-subunit's intrinsic tyrosine kinase
3. β-subunits transphosphorylate each other (autophosphorylation)
4. Activated receptor phosphorylates IRS proteins (Insulin Receptor Substrate 1, 2)
5. IRS activates PI3-kinase → PIP3 → PDK1 → Akt (PKB)
6. Akt phosphorylates many downstream targets:
   • GLUT4 translocation to cell surface (muscle, fat) → glucose uptake↑
   • Glycogen synthase activation → glycogen synthesis↑
   • Inhibition of FOXO1 → gluconeogenesis↓
   • Protein synthesis↑ (via mTOR)
   • Lipogenesis↑ (fatty acid synthesis in liver)
   • Lipolysis↓ (inhibits hormone-sensitive lipase in fat cells)

Sources: Guyton & Hall Textbook of Medical Physiology, Medical Physiology (Boron & Boulpaep)

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7. GLUCAGON

What is Glucagon?

What Triggers Glucagon Secretion?

• Low blood glucose (hypoglycemia) - main stimulus
• Amino acids (especially alanine, arginine) - same meal stimulus as insulin
• Sympathetic stimulation (exercise, stress) via β-adrenergic receptors
• ACh (vagus nerve)

What Inhibits Glucagon?

• High blood glucose (fed state)
• Insulin (paracrine - from adjacent beta cells through portal blood)
• Somatostatin (from delta cells)
• Free fatty acids and ketone bodies (high levels)

Glucagon Receptors and Signal Transduction:

• GPCR (Gs-coupled) - different from insulin's RTK!
• Glucagon binds → Gs protein activates → Adenylyl cyclase → ↑cAMP → PKA (protein kinase A) activation
• PKA phosphorylates:
  • Phosphorylase kinase → activates glycogen phosphorylase → glycogenolysis (breaks down liver glycogen)
  • Inhibits glycogen synthase → no new glycogen stored
  • Activates PEPCK and fructose-1,6-bisphosphatase → gluconeogenesis
  • Activates hormone-sensitive lipase → lipolysis in adipocytes → free fatty acids released → ketogenesis in liver

Actions of Glucagon (mainly in LIVER - liver has most glucagon receptors):

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8. SOMATOSTATIN

What is Somatostatin?

• SS-14 (14 amino acids) - in pancreas (delta cells), gut, neurons
• SS-28 (28 amino acids) - mainly in the gut (intestinal L-cells and D-cells)

What Triggers Somatostatin Release?

• High blood glucose
• High amino acids (especially arginine)
• High fatty acids
• GI hormones (gastrin, secretin, CCK, GIP)
• ACh

Somatostatin Receptors (SSTRs):

• 5 receptor subtypes: SSTR1-5
• All are Gi-coupled GPCRs (inhibitory G protein)
• Signal: ↓cAMP, ↑K+ outflow (hyperpolarization), ↓Ca2+ influx → inhibition of secretion

Actions of Somatostatin:

1. Pancreas (local/paracrine): Inhibits BOTH insulin AND glucagon secretion from β and α cells
2. Stomach: Decreases gastric acid secretion (inhibits parietal cells), slows gastric motility
3. Intestine: Decreases intestinal motility and absorption, inhibits secretin and CCK
4. Gallbladder: Decreases contraction (less bile release)
5. Hypothalamus/Pituitary: Inhibits GH (growth hormone) secretion and TSH secretion
6. Liver: Inhibits IGF-1 release

Somatostatin Metabolism:

• Rapidly degraded by serum and tissue peptidases (half-life ~3 minutes)
• Cleared mainly in the liver and kidneys
• Due to rapid clearance, it mainly acts in a paracrine/autocrine fashion locally

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9. GLUT TRANSPORTERS

What are GLUT Transporters?

The GLUT4 Story (Most Clinically Important):

• Fasting state: GLUT4 vesicles sit inside the cell (endosomes). Very little glucose entry.
• Fed state (insulin present): Insulin → IRS → PI3K → Akt → phosphorylates AS160 → GLUT4 vesicles fuse with plasma membrane → GLUT4 translocates to surface → massive increase in glucose uptake

GLUT2 as Glucose Sensor in Beta Cells:

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10. ROLE OF C-PEPTIDE

What is C-Peptide?

Why Does C-Peptide Matter Clinically?

1. Measure of Endogenous Insulin Secretion

• Exogenously injected insulin does NOT contain C-peptide
• C-peptide is not removed by the liver (unlike insulin, which is ~50% cleared in first pass through liver)
• C-peptide has a longer half-life (~30 min vs ~6 min for insulin)
• Therefore, C-peptide is a much better marker of how much insulin your own pancreas is making

2. Detecting Insulinoma vs Factitious Hypoglycemia:

3. Monitoring Residual Beta Cell Function:

• In Type 1 DM: C-peptide is used to detect "honeymoon period" (remaining beta cell function)
• Helps decide insulin dosing in some patients

4. Possible Direct Physiological Role:

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11. LABORATORY DIAGNOSIS OF DIABETES MELLITUS

What is Diabetes Mellitus?

Diagnostic Criteria (WHO/ADA):

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12. ORAL GLUCOSE TOLERANCE TEST (OGTT)

What is OGTT?

Preparation:

• Patient must fast for 8-12 hours (overnight)
• Must have been on a normal diet (≥150g carbs/day) for 3 days before the test
• No smoking, caffeine, exercise during test

Procedure:

1. Fasting blood glucose drawn (baseline)
2. Patient drinks 75g anhydrous glucose dissolved in 250-300 mL water over 5 minutes
3. Blood glucose measured at 2 hours (standard test)
4. Additional timepoints (30, 60, 90 min) may be drawn for gestational diabetes

Interpretation (75g OGTT, 2-hour value):

Gestational Diabetes (GDM):

1. Step 1: 50g glucose challenge test (non-fasting) at 24-28 weeks
   • If 1-hr glucose ≥130-140 mg/dL → proceed to step 2
2. Step 2: 100g OGTT (fasting), measure at 1, 2, 3 hours (Carpenter-Coustan criteria)
   • Diagnose GDM if ≥2 values exceed: Fasting ≥95, 1-hr ≥180, 2-hr ≥155, 3-hr ≥140 mg/dL

Factors that Falsely Lower OGTT Values:

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13. HbA1c (GLYCATED HEMOGLOBIN)

What is HbA1c?

The Chemistry:

1. Glucose in blood reacts with the N-terminal valine of the beta chain of hemoglobin in a slow, non-enzymatic reaction
2. First forms a reversible Schiff base (aldimine)
3. Then undergoes Amadori rearrangement → stable ketoamine (HbA1c)
4. This is irreversible and stays for the life of the red blood cell

Why Does This Reflect Glucose Control Over Time?

• Red blood cells live for approximately 120 days (3 months)
• The higher the blood glucose, the more HbA1c forms
• Therefore HbA1c reflects the average blood glucose over the past 2-3 months (not just today)
• It is NOT affected by short-term glucose swings (eating, fasting just before the blood draw)

HbA1c to Average Blood Glucose Conversion:

Diagnostic Use:

• ≥6.5%: Diagnosis of diabetes (confirmed on repeat)
• 5.7-6.4%: Pre-diabetes
• Target in treated DM: Generally <7% for most patients (individualized)

Limitations of HbA1c:

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14. GLUCAGON RECEPTORS (DETAILED)

• Coupled to Gs protein (stimulatory)
• Signal transduction: Glucagon binds → Gs activation → Adenylyl cyclase → ↑cAMP → PKA (cAMP-dependent protein kinase A)

• Phosphorylates glycogen phosphorylase kinase → activates glycogen phosphorylase → glycogenolysis
• Phosphorylates glycogen synthase → inactivates it → no glycogen storage
• Increases expression of gluconeogenic enzymes (PEPCK, G6Pase) via CREB transcription factor
• Activates FBPase-2 / inactivates PFK-2 → lowers fructose-2,6-bisphosphate → inhibits glycolysis and activates gluconeogenesis

• Glucagon receptor antagonists (under investigation for T2DM)
• GLP-1 agonists (exenatide, liraglutide) work in part by suppressing glucagon

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15. METABOLISM OF INSULIN, GLUCAGON, AND SOMATOSTATIN

Insulin Metabolism:

• Liver is the main site - removes 40-60% in the first pass via the portal vein (which drains from the pancreas directly to the liver!)
• Mechanism: Cells take up insulin-receptor complexes by receptor-mediated endocytosis → Insulin-Degrading Enzyme (IDE, also called insulysin) degrades it
• Kidney is the second major site (filters and degrades insulin, especially important when liver disease is present)
• Peripheral tissues (muscle, fat) also degrade some insulin after receptor binding

• C-peptide is not cleared by the liver (no IDE action on C-peptide)
• C-peptide is cleared by the kidney with half-life of ~30 minutes
• This is why C-peptide blood levels are higher and more stable than insulin

Glucagon Metabolism:

• Liver is the primary site (~30-40% degraded in first pass - but less than insulin)
• Kidney is another important site
• Mechanism: receptor-mediated endocytosis and lysosomal/protease degradation
• Plasma proteases also contribute

Somatostatin Metabolism:

• Mainly by serum proteases (endopeptidases) acting on the cyclic peptide structure
• Also degraded in liver, kidney, gut wall
• The very short half-life means somatostatin acts almost entirely in a paracrine fashion (locally on neighboring cells)

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Quick Summary Mind-Map

PANCREAS
├── Alpha cells → GLUCAGON (low glucose → cAMP/PKA → glycogenolysis + gluconeogenesis)
├── Beta cells → INSULIN (high glucose → KATP closes → Ca2+ → exocytosis)
│              → C-PEPTIDE (equimolar with insulin, longer half-life, no hepatic clearance)
└── Delta cells → SOMATOSTATIN (inhibits BOTH, extends digestion, Gi-GPCR)

GLUCOSE TRANSPORT
├── GLUT1: brain, RBC, always on (low Km)
├── GLUT2: liver, beta cell, sensor (high Km)
├── GLUT3: neurons (highest affinity)
├── GLUT4: muscle + fat, insulin-dependent (stored in vesicles, Akt-triggered)
└── GLUT5: intestine, fructose transporter

CALCIUM
├── PTH (parathyroid): bone resorption↑, renal Ca↑, renal PO4↓, Vit D activation↑
├── Calcitriol (Vit D): gut Ca + PO4 absorption↑, bone mineralization
└── Calcitonin (thyroid C-cells): bone resorption↓ (opposes PTH)

STEROID SYNTHESIS
Cholesterol → (CYP11A) → Pregnenolone → (3β-HSD) → Progesterone
→ Cortisol (zona fasciculata, via CYP17/CYP21/CYP11B1)
→ Aldosterone (zona glomerulosa, via CYP21/CYP11B2, NO CYP17)
→ Testosterone (gonads, via CYP17)
→ Estradiol (ovary, via CYP19 aromatase from testosterone)

THYROID
Iodide → (NIS) → Thyrocyte → (TPO) → MIT → DIT → T4 (DIT+DIT) / T3 (MIT+DIT)
→ Stored in thyroglobulin in colloid → secreted by endocytosis/lysosomal cleavage
→ T4 is prohormone → peripheral deiodination → T3 (active) or rT3 (inactive)

DIABETES DIAGNOSIS
├── FPG ≥126 mg/dL (×2)
├── 2-hr OGTT ≥200 mg/dL
├── HbA1c ≥6.5%
└── Random glucose ≥200 + symptoms

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