Purine Nucleotide Degradation

Overview: Where It Happens

• Biochemistry, 8th ed - Lippincott Illustrated Reviews, p. 840

Stepwise Degradation to Uric Acid

Step 1 - AMP to IMP (Deamination)

• AMP deaminase removes the amino group from AMP → IMP (inosine monophosphate), releasing NH4+
• Alternatively, adenosine deaminase (ADA) deaminates adenosine → inosine directly

Step 2 - Nucleotides to Nucleosides (Dephosphorylation)

• 5'-nucleotidase removes phosphate from IMP → inosine and from GMP → guanosine

Step 3 - Nucleosides to Free Bases (Phosphorolysis)

• Purine nucleoside phosphorylase (PNP) cleaves inosine → hypoxanthine + ribose 1-phosphate
• PNP cleaves guanosine → guanine + ribose 1-phosphate
• (A mutase interconverts ribose 1-phosphate and ribose 5-phosphate)

Step 4 - Guanine Deamination

• Guanine deaminase removes the amino group from guanine → xanthine + NH4+

Step 5 - Xanthine Oxidase (the key enzyme, x2 reactions)

• Xanthine oxidase (XO) oxidizes hypoxanthine → xanthine (uses O2, produces H2O2)
• XO further oxidizes xanthine → uric acid (uses O2, produces H2O2)

• Biochemistry, 8th ed - Lippincott Illustrated Reviews, p. 841; Basic Medical Biochemistry - A Clinical Approach - 6e, p. 1434

Dietary digestion overview

Properties of Uric Acid

• pKa = 5.4 → in the body (pH ~7.4), it is largely ionized as urate
• Urate is poorly soluble in aqueous environments; plasma urate is near its solubility limit
• In non-primate mammals, urate oxidase (uricase) converts uric acid to the more soluble allantoin
• Uric acid is also a potent antioxidant - one proposed reason humans retained it

Associated Diseases

1. Gout (hyperuricemia)

• Underexcretion (>90% of cases) - primary or secondary (e.g., thiazide diuretics, lactic acidosis, lead exposure "saturnine gout")
• Overproduction - idiopathic; or from:
  • Gain-of-function mutations in PRPP synthetase (increased PRPP → more de novo purines)
  • Lesch-Nyhan syndrome (below)
  • High cell turnover (myeloproliferative disorders, chemotherapy)

2. Lesch-Nyhan Syndrome (HGPRT deficiency)

• X-linked recessive deficiency of hypoxanthine-guanine phosphoribosyltransferase (HGPRT)
• Cannot salvage hypoxanthine → IMP or guanine → GMP
• Hypoxanthine and guanine are degraded to uric acid instead → severe hyperuricemia
• Also causes self-mutilating behavior, choreoathetosis, spasticity, and intellectual disability (neurologic features dominate)
• PRPP accumulates (not consumed by salvage), driving excess de novo purine synthesis

3. Adenosine Deaminase (ADA) Deficiency

• ADA deaminates adenosine → inosine and deoxyadenosine → deoxyinosine
• Deficiency → toxic accumulation of dATP (deoxyadenosine triphosphate) in lymphocytes
• Inhibits ribonucleotide reductase → blocks DNA synthesis → lymphocyte death
• Leads to Severe Combined Immunodeficiency (SCID) - accounts for 10-20% of SCID cases
• Lymphocytes have the highest ADA activity of any tissue; they are most sensitive to its deficiency

4. Purine Nucleoside Phosphorylase (PNP) Deficiency

• Deficiency → accumulation of guanosine and inosine → toxic to T lymphocytes
• Results in T-cell immunodeficiency (predominantly T-cell SCID, B cells relatively spared)

Summary Pathway

AMP ──(AMP deaminase)──→ IMP
Adenosine ──(ADA)──→ Inosine

IMP ──(5'-nucleotidase)──→ Inosine ──(PNP)──→ Hypoxanthine + Ribose-1-P
GMP ──(5'-nucleotidase)──→ Guanosine ──(PNP)──→ Guanine + Ribose-1-P

Guanine ──(guanine deaminase)──→ Xanthine + NH4+
Hypoxanthine ──(Xanthine Oxidase)──→ Xanthine ──(Xanthine Oxidase)──→ URIC ACID
                                        ↑ [Allopurinol/Febuxostat block here]

• Biochemistry, 8th ed - Lippincott Illustrated Reviews, p. 840-848; Basic Medical Biochemistry - A Clinical Approach - 6e, p. 1434-1435

Gout

Pathogenesis

1. Hyperuricemia

2. MSU Crystal Formation & Inflammation

• Low temperature (explains predilection for peripheral, cooler joints - first MTP, ankles)
• Low pH
• Intra-articular dehydration
• Collagen and proteoglycans (promote crystal nucleation)

1. MSU crystals are phagocytosed by resident synovial macrophages
2. Crystals activate the NLRP3 inflammasome (NOD-like receptor protein 3)
3. Inflammasome activates caspase-1 → cleaves pro-IL-1β → mature IL-1β
4. IL-1β (and IL-6, IL-8, TNF-α) drives massive neutrophil recruitment
5. Neutrophils phagocytose crystals → respiratory burst → lysosomal enzyme release, superoxide, leukotriene B4
6. Crystals also physically damage phagolysosomal membranes → enzyme leakage
7. Neutrophil mediators lower local pH → further urate precipitation (vicious cycle)

3. Chronic Tophaceous Gout

Clinical Stages

Risk Factors

• Older age, male sex
• Obesity, metabolic syndrome, insulin resistance
• Diet: red meat, shellfish/seafood, beer, spirits, high-fructose foods
• Medications: thiazide diuretics, loop diuretics, low-dose aspirin, pyrazinamide, ciclosporin
• Comorbidities: hypertension, renal disease, diabetes, hyperlipidemia, heart failure
• Genetic factors: HGPRT deficiency (partial = gout without neurology; complete = Lesch-Nyhan), PRPP synthetase superactivity, ABCG2/URAT1/SLC22A12 variants

Diagnosis

• Musculoskeletal ultrasound: "Double-contour sign" over articular cartilage (MSU deposition) - useful early
• Dual-energy CT (DECT): Identifies MSU based on chemical composition - highly specific
• Plain X-ray: In advanced disease - well-defined erosions with sclerotic margins and overhanging edges ("rat-bite" erosions), soft tissue tophi calcifications; typically absent early

Treatment

Acute Gout Flare

Urate-Lowering Therapy (ULT) - Chronic Management

Xanthine Oxidase Inhibitors (XOI) - reduce urate synthesis

• Structural analog of hypoxanthine; oxidized by XO to oxypurinol, a long-lived (t½ ~18-30h) competitive inhibitor of XO
• Blocks both: hypoxanthine → xanthine AND xanthine → uric acid
• Accumulating hypoxanthine is salvaged by HGPRT → reduces PRPP → decreases de novo purine synthesis (bonus effect)
• Starting dose: 100 mg/day, titrate by 100 mg increments weekly; most patients: 300 mg/day
• Reduce dose in renal impairment
• Key drug interaction: Inhibits XO-mediated inactivation of azathioprine and 6-mercaptopurine → must reduce those doses to 25-33% when co-prescribed
• Adverse effects: Hypersensitivity rash (1-5%), rarely Stevens-Johnson syndrome / toxic epidermal necrolysis. Risk is strongly associated with HLA-B*5801 allele (screen in Korean, Thai, Han Chinese, Sardinian patients before initiating)
• Also inhibits warfarin metabolism (monitor INR)

• Non-purine XO inhibitor; inhibits both oxidized and reduced forms of XO (more complete inhibition than oxypurinol)
• Used when allopurinol is not tolerated or contraindicated
• Cardiovascular signal: higher rates of MI and stroke vs. allopurinol in cardiovascular-risk patients (CARES trial) - use with caution in existing CVD; prefer allopurinol when possible

Uricosuric Agents - increase renal urate excretion

Uricase Agents - enzymatic urate degradation

Complications

Differential Diagnosis

• Pseudogout (CPPD) - calcium pyrophosphate crystals, weakly positive birefringence, rhomboid shape; often affects knees and wrists
• Septic arthritis - always culture synovial fluid (infection can coexist with MSU crystals)
• Cellulitis - gout over first MTP can mimic cellulitis ("pseudocellulitis")
• Reactive arthritis, psoriatic arthritis - can overlap

Lifestyle & Dietary Advice

• Avoid purine-rich foods: organ meats, red meat, shellfish (especially mussels, scallops, anchovies)
• Avoid beer and spirits; moderate wine consumption is less strongly associated with gout
• Avoid high-fructose corn syrup (fructose promotes purine synthesis and reduces urate excretion)
• Low-fat dairy products are associated with decreased gout risk
• Maintain high fluid intake (>2L/day)
• Weight loss, treat metabolic syndrome, discontinue offending drugs if possible
• Harrison's Principles of Internal Medicine 22E, p. 3000-3003; Robbins & Kumar Basic Pathology, p. 840-846; Goodman & Gilman's Pharmacological Basis of Therapeutics, p. 867-875

Pyrimidine Synthesis

Key Distinction from Purine Synthesis

Atomic Origins of the Pyrimidine Ring

• N-1 and C-2: from carbamoyl phosphate (derived from glutamine's amide nitrogen + CO₂)
• N-3, C-4, C-5, C-6: from the entire aspartate molecule

Stage 1: De Novo Synthesis of the Pyrimidine Ring (Steps 1-3)

Step 1 - Carbamoyl phosphate synthesis (RATE-LIMITING STEP)

Glutamine + CO₂ + 2 ATP → Carbamoyl phosphate + Glutamate + 2 ADP + Pi

Step 2 - Carbamoylaspartate formation

Carbamoyl phosphate + Aspartate → Carbamoylaspartate + Pi

Step 3 - Ring closure

Carbamoylaspartate → Dihydroorotate + H₂O

Stage 2: Orotate Formation and Attachment to Ribose (Steps 4-6)

Step 4 - Oxidation to orotate

Dihydroorotate + FMN → Orotate + FMNH₂

Step 5 - Attachment to ribose (PRPP donation)

Orotate + PRPP → Orotidine 5'-monophosphate (OMP) + PPi

Step 6 - Decarboxylation to UMP

OMP → Uridine monophosphate (UMP) + CO₂

Full De Novo Pathway Diagram

Stage 3: UMP → All Other Pyrimidine Nucleotides

UMP → (UMP kinase) → UDP → (NDPK) → UTP

CTP Synthesis

UTP + Glutamine + ATP → CTP + Glutamate + ADP + Pi

• Glutamine provides the amino group added at C-4 of uracil to form cytosine
• Note: This amination can only occur at the triphosphate level (not at UMP or UDP)
• CTP is a feedback inhibitor of CTP synthetase (end-product inhibition)

Deoxyribonucleotides

• UDP and CDP are substrates for ribonucleotide reductase → dUDP and dCDP
• dCDP → dCTP (for DNA) or dephosphorylated → dCMP → deaminated → dUMP
• dUDP → phosphorylated → dUTP → rapidly hydrolyzed by dUTPase to dUMP (prevents erroneous U incorporation into DNA)

dTMP Synthesis (Critical for DNA)

dUMP + N⁵,N¹⁰-methylene-THF → dTMP + Dihydrofolate (DHF)

Drug targets at this step:

Regulation of Pyrimidine Synthesis

• Inhibited by UTP (end product feedback inhibition)
• Activated by PRPP (signals need for more nucleotides)

• As cells approach S-phase: CPS II becomes more sensitive to PRPP activation and less sensitive to UTP inhibition (phosphorylation by MAP kinase)
• After S-phase: inhibition by UTP is enhanced, PRPP activation reduced (phosphorylation by cAMP-dependent protein kinase)

Pyrimidine Salvage

1. Pyrimidine base + ribose 1-phosphate → nucleoside (pyrimidine nucleoside phosphorylase - reverse direction)
2. Nucleoside + ATP → nucleotide (nucleoside kinase)

Pyrimidine Degradation

• UMP/CMP → cytosine → uracil → β-alanine + NH₃ + CO₂
• TMP → thymine → β-aminoisobutyrate + NH₃ + CO₂

Clinical Diseases

Hereditary Orotic Aciduria

• Deficiency of UMP synthase (one or both activities: orotate phosphoribosyltransferase and/or OMP decarboxylase)
• Cannot convert orotate → OMP → UMP
• Results in: massive orotic acid in urine, failure to thrive, megaloblastic anemia (from pyrimidine deficiency - cells cannot divide; DNA synthesis impaired)
• Treatment: Oral uridine - bypasses the block and is phosphorylated to UMP, from which CTP and dTMP can be produced. Exogenous UMP also provides feedback inhibition of CPS II, reducing orotate accumulation.

OTC Deficiency (urea cycle) causing secondary orotic aciduria

• Carbamoyl phosphate normally used by OTC (ornithine transcarbamylase) in mitochondria accumulates and leaks into the cytosol
• Excess cytosolic carbamoyl phosphate bypasses the regulated CPS II step → excess pyrimidine synthesis → elevated urinary orotate
• Differentiates from hereditary orotic aciduria: in OTC deficiency there is hyperammonemia and the orotic aciduria is secondary

Summary Pathway

Glutamine + CO₂ + 2ATP ──(CPS II [regulated])──→ Carbamoyl phosphate
                                                           │
                                          + Aspartate (ATCase)
                                                           ↓
                                               Carbamoylaspartate
                                                           │ (Dihydroorotase)
                                                           ↓
                                                 Dihydroorotate
                                                           │ (DHODH, mitochondria, FMN)
                                                           ↓
                                                       Orotate
                                                           │ + PRPP (orotate PRTase → UMP synthase)
                                                           ↓
                                                       OMP
                                                           │ - CO₂ (OMP decarboxylase → UMP synthase)
                                                           ↓
                                     UMP → UDP → UTP ──(CTP synthetase + Gln)──→ CTP
                                                │
                                           (ribonucleotide reductase)
                                                │
                                      dUDP → dUMP ──(thymidylate synthase + N⁵,N¹⁰-CH₂-THF)──→ dTMP → dTDP → dTTP

• Biochemistry, 8th ed - Lippincott Illustrated Reviews, p. 848-854; Basic Medical Biochemistry - A Clinical Approach - 6e, p. 1425-1432