Insulin and C-Peptide

Insulin is made in pancreatic beta cells as proinsulin, which is cleaved into insulin and C-peptide in equal amounts. C-peptide is cleared by the kidney, has a longer half life (30 minutes against 4 to 5 minutes for insulin), and is not extracted by the liver, so it reflects endogenous insulin secretion better than insulin itself. Both are measured by immunoassay in serum. Proinsulin and beta hydroxybutyrate are added in investigation of hypoglycaemia.

The tests are used to investigate hypoglycaemia (distinguishing insulinoma from factitious insulin or sulfonylurea use), to classify diabetes (type 1 against type 2, monogenic forms), and to assess insulin resistance and residual beta cell function.

Insulinoma is the commonest functioning pancreatic neuroendocrine tumour, with an incidence of 1 to 4 per million each year, and over 90% are benign and single. Hypoglycaemia in patients on insulin or sulfonylureas is far more common, and factitious hypoglycaemia needs exclusion in people with access to these drugs.

Indications. Spontaneous hypoglycaemia (Whipple triad: low glucose, symptoms, relief with glucose), suspected insulinoma, suspected factitious or malicious hypoglycaemia, unclear diabetes type (adult with young onset or ketosis prone), monogenic diabetes, assessment of beta cell reserve, hyperinsulinism in infancy, and polycystic ovary syndrome or metabolic syndrome in selected research or clinical contexts.

Specimen. For hypoglycaemia, a critical sample is taken during a spontaneous episode or a supervised prolonged fast, at the time of plasma glucose under 3.0 mmol/L (under 54 mg/dL): glucose, insulin, C-peptide, proinsulin, beta hydroxybutyrate and a sulfonylurea or meglitinide screen. Serum or plasma separated promptly, since haemolysis destroys insulin.

For diabetes classification, a random or stimulated C-peptide is taken at least 3 years after diagnosis and with glucose over 8 mmol/L, because low glucose suppresses C-peptide. Urinary C-peptide to creatinine ratio is stable at room temperature in boric acid.

Ranges are method specific. Conversion: insulin microIU/mL (mU/L) multiplied by 6.0 gives pmol/L, and C-peptide ng/mL multiplied by 0.33 gives nmol/L.

Insulin. Fasting: 2 to 20 mU/L (about 12 to 120 pmol/L). Raised: insulin resistance (obesity, type 2 diabetes, polycystic ovary syndrome), insulinoma, exogenous insulin, nesidioblastosis and congenital hyperinsulinism, and sulfonylurea use. Lowered: type 1 diabetes, late type 2 diabetes, pancreatic disease, and hypopituitarism.

C-peptide. Fasting: 0.3 to 0.6 nmol/L (0.8 to 3.1 ng/mL). Raised: insulinoma, sulfonylurea or meglitinide ingestion, insulin resistance and type 2 diabetes, kidney failure (reduced clearance), and insulin antibodies. Lowered: type 1 diabetes (under 0.2 nmol/L after the honeymoon), pancreatic failure, and exogenous insulin (suppresses endogenous production).

Proinsulin. Fasting under 5 pmol/L (under 20% of immunoreactive insulin). Raised in insulinoma (over 22% of insulin, or over 5 pmol/L during hypoglycaemia), type 2 diabetes and kidney failure.

Interpretation of hypoglycaemia (plasma glucose under 3.0 mmol/L).

• Insulinoma or endogenous hyperinsulinism: insulin 3 mU/L or more (18 pmol/L), C-peptide 0.6 ng/mL or more (0.2 nmol/L), proinsulin 5 pmol/L or more, beta hydroxybutyrate 2.7 mmol/L or less, no sulfonylurea in screen, and glucose rise 1.4 mmol/L or more after glucagon.
• Factitious insulin administration: high insulin, suppressed C-peptide and proinsulin, low beta hydroxybutyrate. Insulin analogues may be missed by some insulin assays.
• Sulfonylurea or meglitinide ingestion: biochemically indistinguishable from insulinoma (high insulin, C-peptide and proinsulin), with a positive drug screen.
• Insulin autoimmune syndrome: very high total insulin with high C-peptide and anti-insulin antibodies, and postprandial hypoglycaemia.
• Non-insulin-mediated hypoglycaemia: insulin and C-peptide suppressed, beta hydroxybutyrate raised (fasting, adrenal insufficiency, hypopituitarism, liver failure, sepsis), or IGF-2-mediated tumour hypoglycaemia (low insulin, low C-peptide, high IGF-2 to IGF-1 ratio over 10).

Supervised 72-hour fast. Patient fasts, with glucose checked every 4 to 6 hours and every hour once under 3.3 mmol/L, until glucose under 3.0 mmol/L with symptoms or 72 hours. About 65% of insulinomas show hypoglycaemia in 24 hours and 95% in 48 hours. The critical sample is drawn at the end, followed by a glucagon response.

Diabetes classification.

• C-peptide under 0.2 nmol/L (under 0.6 ng/mL) at glucose over 8 mmol/L after 3 or more years indicates insulin deficiency (type 1 diabetes). Values over 0.6 nmol/L indicate preserved secretion (type 2 diabetes or monogenic forms).
• Urinary C-peptide to creatinine ratio under 0.2 nmol/mmol on two samples confirms insulin deficiency.
• Islet autoantibodies (glutamic acid decarboxylase, IA-2, ZnT8) support type 1 diabetes and latent autoimmune diabetes.
• In a young adult with preserved C-peptide and no autoantibodies, genetic testing for maturity onset diabetes of the young is considered.

Insulin resistance. Fasting insulin and glucose give the HOMA index. The clinical use is limited, and the test is not required for the diagnosis of metabolic syndrome or polycystic ovary syndrome.

Critical values and findings needing immediate action.

• Glucose under 3.0 mmol/L with symptoms, or under 2.2 mmol/L, which needs immediate glucose and a critical sample before treatment if this does not delay it.
• Hypoglycaemia with confusion, seizures or coma.
• Hypoglycaemia in a non-diabetic patient with raised insulin and C-peptide, needing imaging and specialist referral.
• Suspected factitious hypoglycaemia with high insulin and low C-peptide in a vulnerable patient.
• Neonatal hypoglycaemia with hyperinsulinism, needing urgent specialist treatment to avoid brain injury.

Interferences and pitfalls.

• Samples drawn when glucose is not low are not informative for hypoglycaemia workup.
• Haemolysis and delayed separation degrade insulin and give falsely low results.
• Insulin antibodies cause false insulin and C-peptide results.
• Exogenous insulin analogues (glargine, detemir, lispro, aspart) can be detected poorly or not at all by insulin immunoassays.
• Kidney failure raises C-peptide, and eGFR must be considered.
• C-peptide is suppressed by hypoglycaemia and so a random C-peptide in a patient with low glucose may mislead for diabetes classification.
• Sulfonylurea screening needs a specific assay.
• Reference ranges vary greatly between assays.

Special groups.

• Neonates and infants: persistent hyperinsulinaemic hypoglycaemia needs a critical sample and genetic testing (ABCC8, KCNJ11, GCK).
• Children: ketotic hypoglycaemia is common, with ketones high and insulin suppressed.
• Pregnancy: insulin resistance rises in the second and third trimesters, and gestational diabetes is diagnosed by glucose tests, not insulin.
• Older adults: sulfonylurea hypoglycaemia is common, and renal impairment prolongs it.
• Regions: access to C-peptide and autoantibody testing is limited in many settings, and type classification relies on clinical features.

Follow up tests. Plasma glucose series, proinsulin and beta hydroxybutyrate, sulfonylurea and meglitinide screen, insulin antibodies, IGF-1 and IGF-2, cortisol, pituitary and liver function, CT or MRI of the pancreas, endoscopic ultrasound, gallium-68 DOTATATE or exendin PET, calcium and parathyroid hormone and genetic testing for MEN1, islet autoantibodies, and genetic testing for monogenic diabetes.