Testing for the Presence of Insulin
The gold standard for diagnosing endogenous hyperinsulinism is a supervised 48-72 hour fasting test, measuring simultaneous insulin, C-peptide, and glucose levels when hypoglycemia occurs, with diagnostic criteria of insulin >3 mcIU/mL, C-peptide ≥0.6 ng/mL, and glucose <55 mg/dL. 1, 2
Diagnostic Testing Algorithm
First-Line: Supervised Fasting Test
The supervised fast remains the definitive diagnostic approach:
- Duration: 48-72 hours under medical supervision, continuing until hypoglycemia develops or time limit reached 1, 2
- Monitoring: Measure capillary blood glucose at regular intervals and when symptoms occur 3
- Termination criteria: Stop when blood glucose drops to <40-45 mg/dL or patient develops neuroglycopenic symptoms (confusion, lethargy, seizures) 1
At the time of hypoglycemia, obtain simultaneous measurements of:
- Plasma glucose
- Serum insulin
- Serum C-peptide
- Proinsulin levels
- Urine sulfonylureas (to exclude factitious hypoglycemia) 2
Diagnostic Thresholds for Endogenous Hyperinsulinism
The National Comprehensive Cancer Network establishes these criteria at the termination of the fast: 1
- Insulin: >3 mcIU/mL (>21 pmol/L)
- C-peptide: ≥0.6 ng/mL (≥0.2 nmol/L)
- Proinsulin: ≥5 pmol/L
- Glucose: <55 mg/dL (<3.0 mmol/L)
- Insulin-to-glucose ratio: ≥0.3
Proinsulin >5 pmol/L with glucose <2.5 mmol/l achieves 100% diagnostic specificity and sensitivity for endogenous hyperinsulinism, making it the single most reliable biochemical marker. 4
Alternative: Shortened Fasting Protocols
Recent evidence supports shorter fasting tests in select cases:
- 12-hour mini-fasting test: Performed over 3 consecutive days, this identified 100% of patients with pathologically proven endogenous hyperinsulinism when insulin ≥3 μU/ml and C-peptide ≥0.6 ng/ml occurred with symptomatic hypoglycemia 5
- 24-hour short fasting test: Diagnosed 91.7% (11/12) of insulinoma cases within 24 hours, offering a valid first-line outpatient approach 3
- 48-hour fasting test combined with glucagon stimulatory test: Achieved 93.3% sensitivity and 95.0% specificity, providing a less invasive alternative 6
However, the 72-hour fast remains the gold standard when shorter tests are negative but clinical suspicion persists. 5
Understanding C-Peptide vs. Insulin Measurements
C-peptide is superior to insulin for diagnosing endogenous hyperinsulinism because:
- Produced in equimolar quantities with insulin during proinsulin cleavage 2
- Longer half-life than insulin (30 minutes vs. 4 minutes)
- Not extracted by the liver, unlike insulin which undergoes 50% first-pass hepatic metabolism 2
- Distinguishes endogenous insulin production from exogenous insulin administration 7
Elevated C-peptide with elevated insulin confirms endogenous hyperinsulinism; elevated insulin with suppressed C-peptide indicates exogenous insulin administration. 7, 2
Clinical Context: When to Test
The American Diabetes Association states routine testing for insulin or proinsulin is NOT recommended in most people with diabetes or cardiovascular disease risk. 7
Testing is indicated when:
- Whipple's triad is present: symptomatic hypoglycemia + documented low glucose (<55 mg/dL) + symptom resolution after glucose administration 1, 2
- Differentiating type 1 from type 2 diabetes in ambiguous cases (e.g., ketoacidosis with type 2 phenotype) 7
- Investigating suspected insulinoma or other causes of endogenous hyperinsulinism 1, 2
- Evaluating nondiabetic hypoglycemia to rule out surreptitious insulin administration 7
- Diagnosing islet cell tumors when inappropriately elevated insulin persists despite low glucose 7
Critical Pitfalls to Avoid
Do not rely on random insulin or C-peptide measurements: These must be obtained during documented hypoglycemia to have diagnostic value 1, 2
Ensure proper fasting conditions: For C-peptide measurement required by payers for insulin pump coverage, measure when simultaneous fasting glucose is ≤220 mg/dL (12.5 mmol/L) 7
Consider insulin resistance confounders: Isolated C-peptide elevation without hypoglycemia reflects insulin resistance (obesity, PCOS, early type 2 diabetes), not pathological hyperinsulinism 2
Avoid premature test termination: The 48-72 hour fast should continue until true hypoglycemia occurs or the time limit is reached, not stopped at arbitrary glucose thresholds 1
Test for sulfonylureas: Always check urine for sulfonylureas during the fasting test to exclude factitious hypoglycemia from oral hypoglycemic agents 2