Glucagon

Plasma glucagon is a 29-amino-acid peptide secreted by the alpha cells of the pancreatic islets in response to hypoglycaemia, amino acids and sympathetic stimulation. It raises blood glucose by stimulating hepatic glycogenolysis and gluconeogenesis, and it is suppressed by hyperglycaemia, insulin and somatostatin.

Measurement is requested to diagnose glucagonoma, a rare pancreatic neuroendocrine tumour that presents with necrolytic migratory erythema, diabetes, weight loss, anaemia and venous thrombosis. A very high fasting glucagon (over 500 pg/mL) is virtually diagnostic. The assay is technically difficult, and results depend on sample handling and assay specificity.

Glucagonoma has an incidence of about 1 in 20 million a year, and most tumours are large (over 3 cm) and metastatic at diagnosis (50 to 80%). Raised glucagon without a tumour occurs in cirrhosis, renal failure, sepsis, trauma, burns, Cushing syndrome, acromegaly and prolonged fasting. Glucagon is also measured in research on diabetes and hypoglycaemia, and in alpha cell hyperplasia (glucagon cell adenomatosis).

Indications. Necrolytic migratory erythema (a spreading, erythematous, blistering rash of the perineum, groin and limbs, with a central healing zone), unexplained weight loss with new diabetes, normocytic anaemia, glossitis, stomatitis, deep vein thrombosis, depression and a pancreatic mass. Pancreatic neuroendocrine tumour workup (multiple endocrine neoplasia type 1). Follow up of resected glucagonoma.

Specimen. Fasting venous blood (overnight 8 to 12 hours) collected into a chilled EDTA tube containing aprotinin (a protease inhibitor, 500 KIU/mL) or other degradation inhibitors, placed on ice at once, centrifuged cold within 15 to 30 minutes and the plasma frozen at minus 20 °C or lower. Delay or warm storage degrades glucagon. Collection should not follow glucose infusion, glucagon therapy or extreme exercise.

Reference values (fasting plasma, assay dependent).

• Adults: 50 to 150 pg/mL (14 to 43 pmol/L). Some laboratories use 20 to 100 pg/mL.
• Glucagonoma: typically over 500 pg/mL (over 143 pmol/L), often 1000 to 10 000 pg/mL.
• Intermediate rise (150 to 500 pg/mL): non-specific, seen with non-tumour causes and some tumours.

Interpretation.

• Fasting glucagon over 500 pg/mL with a pancreatic mass and the typical syndrome: glucagonoma.
• Fasting glucagon 150 to 500 pg/mL: causes include renal failure (reduced clearance), cirrhosis, sepsis, trauma, burns, severe stress, diabetic ketoacidosis, prolonged fasting, Cushing syndrome, acromegaly, pheochromocytoma, familial hyperglucagonaemia, and drugs (danazol, glucocorticoids, catecholamines). Pancreatic tumours other than glucagonoma, including VIPomas and insulinomas, may show mild rises.
• Pancreatic polypeptide, chromogranin A, insulin and gastrin are added for other neuroendocrine tumours.
• Very low glucagon: chronic pancreatitis, pancreatectomy, and congenital glucagon deficiency (rare).
• Glucagon secreting tumours often secrete other peptides (pancreatic polypeptide, gastrin, insulin), and 3 to 17% occur in multiple endocrine neoplasia type 1.

Associated findings. Anaemia (normochromic normocytic, 60 to 90%), low amino acids (hypoaminoacidaemia, especially glycine, proline, serine), low zinc and fatty acid deficiency, raised glucose (mild diabetes in 75 to 95%), and hypocholesterolaemia. Imaging: contrast CT or MRI shows a large (average 5 to 6 cm) pancreatic tail mass with liver metastases in 50 to 70%. DOTATATE PET and endoscopic ultrasound confirm.

Critical values and findings needing immediate action.

• Glucagon over 500 pg/mL with pancreatic mass: urgent endocrine, hepatobiliary and oncology referral, imaging and biopsy.
• Deep vein thrombosis or pulmonary embolism in a glucagonoma patient: anticoagulation and prophylaxis (thromboembolism affects 30% of patients).
• Severe necrolytic migratory erythema with secondary infection: skin care and amino acid, zinc and fatty acid replacement.
• Rapid weight loss and cachexia: nutritional support, with somatostatin analogue (octreotide or lanreotide) to control symptoms and the rash.
• Hyperglycaemia: insulin may be needed if diabetes is severe.

Interferences and pitfalls.

• Pre-analytical degradation is the main cause of low results, and a sample without protease inhibitor or kept warm gives falsely low values.
• Immunoassays cross-react with proglucagon fragments (glicentin, oxyntomodulin), and radioimmunoassays using C-terminal antibodies detect these. A glucagon specific sandwich ELISA (or mass spectrometry) is more accurate.
• Renal failure, cirrhosis and other illnesses raise the level and produce false positives. A level over 500 pg/mL in these conditions is unusual.
• A normal fasting glucagon does not exclude a small glucagonoma, but the clinical syndrome and imaging are needed.
• Necrolytic migratory erythema also occurs without glucagonoma (pseudoglucagonoma syndrome) in cirrhosis, coeliac disease, malabsorption, inflammatory bowel disease and zinc deficiency, with normal glucagon.
• Samples from patients receiving glucagon injection or somatostatin analogue give altered results.
• Glucagon stimulation testing (for GH or cortisol reserve) is unrelated and uses exogenous glucagon.

Special groups.

• Multiple endocrine neoplasia type 1 carriers: yearly screening of pancreatic hormones in some programmes.
• Children: very rare, and interpretation by a specialist laboratory.
• Pregnancy: no change in normal values.
• Regions: availability of glucagon assays is limited, and samples are sent to reference laboratories on ice.

Follow up tests. Contrast CT or MRI of the pancreas and liver, endoscopic ultrasound with biopsy, 68Ga-DOTATATE PET, chromogranin A, pancreatic polypeptide, insulin, gastrin, VIP, calcium, PTH and prolactin (multiple endocrine neoplasia type 1), MEN1 gene testing, full blood count, glucose and HbA1c, zinc, amino acid profile, coagulation tests, and skin biopsy of the rash.