Combined Assessment of Gastrointestinal Hormones and Metabolomic Profiling Following Mixed-Meal Tolerance Tests in Patients at High Risk of Refeeding Syndrome: Study Protocol

1. Introduction Refeeding syndrome (RS) is a potentially life-threatening metabolic complication that occurs when nutrition is reintroduced after prolonged fasting or malnutrition, leading to electrolyte imbalances - especially low serum phosphate, magnesium and potassium - and fluid shifts that may cause organ dysfunction. Although RS has been traditionally reported in hospitalized high-risk and critical ill patients, its prevalence is probably underestimated, especially in ambulatory individuals needing artificial feeding. Actually, only a few years ago standardized criteria to diagnose and stage RS were published for the first time in the literature by the American Society for Parenteral and Enteral Nutrition updating the classic high-risk RS risk criteria described by the National Institute for Health and Clinical Excellence (NICE).

Some studies suggest that refeeding hypophosphatemia is more common in enteral than parenteral feeding in adult hospitalized patients [5]. However, in patients undergoing percutaneous endoscopic gastrostomy (PEG) for long-term enteral nutrition due to chronic disease with prolonged dysphagia, a particular high-risk subgroup, clinical and laboratory signs of RS are seldom identified. In fact, chronic starvation induces histological, ultrastructural and hormonal changes in the duodenal mucosa of patients with high-risk of RS as it was already demonstrated in two recent prospective controlled studies. These changes include shortening of the villi with or without mucosal atrophy, ultrastructural evidence of autophagy and increased tissue expression of glucagon-like peptide 1 (GLP-1) and gastric inhibitory polypeptide (GIP). GLP-1 and GIP are the two primary incretin hormones secreted from the intestine after food ingestion to stimulate insulin secretion from pancreatic beta cells. This incretin overexpression may constitute an adaptative response to maintain insulinogenic activity for essential anabolic processes during long-term low food supply. Actually, although the impaired absorption expected from the histologic and ultrastructural changes in the duodenum may probably avoid RS, the enteroendocrine adaptative response of the duodenal mucosa may paradoxically increase its risk after enteral PEG refeeding.

Gastrointestinal hormones are bioactive peptides released by the gut in response to nutrient ingestion, playing a key role in regulating digestion, absorption, appetite regulation, and systemic metabolic homeostasis. Beyond incretins, ghrelin and peptide YY (PYY) are other gastrointestinal hormones with complementary roles: ghrelin, produced mainly in the stomach, stimulates appetite, whereas PYY, secreted primary in the distal intestine, promotes satiety and slows gastric emptying, together regulating energy balance and postprandial metabolism.

Mixed Meal Tolerance Tests (MMTT) are standardized clinical procedures in which a subject ingests a mix-nutrient meal containing carbohydrates, proteins and fats, followed by serial measurements of serum glucose, insulin and other metabolic hormones over time. These tests are used to assess postprandial metabolic responses, including beta-cell function, insulin sensitivity and gastrointestinal hormone secretion.

Metabolomic analysis is the systematic study of small-molecule metabolites within biological samples, that reflects a functional snapshot of cellular metabolism and its response to physiological and pathological changes.