Standardized Meals with Different Macronutrient Ratios on the Postprandial Metabolome and Metabolic Flexibility

Enrolled subjects are followed every week for one month. At each visit, a questionnaire assesses daily time activity patterns relevant to energy expenditure, general health status, including infectious symptoms, and confirmation of basic social and demographic characteristics. Dietary intake is assessed by a food frequency questionnaire and a multi-step 24-hour dietary recall for quantitative analysis. If symptoms of infection are present, participants are treated with ad-hoc broad antibiotics. Anthropometrics are obtained. Subjects will then be randomized to receive the metabolic challenges in a different order. The procedures will be performed before (fasting; 8-10 hours) and after (postprandial) consumption of the metabolic challenges. Capillary blood samples (40µ) are obtained in the morning after an 8-hour fasting and after test meal consumption. To obtain the capillary blood sample, sterilize the ring finger with alcohol and allow it to dry. Then, puncture the area with a sterile 2 mm long lancet. Once the drop of blood is formed, it is placed directly into the CardioCheck Plus® cassette to determine glucose triglyceride, LDL-cholesterol, HDL-cholesterol, and total cholesterol. A second drop of capillary blood shall be placed on a filter paper (S&S 903) until a circle of filter paper is filled with blood to saturate the paper throughout its thickness. Insulin concentration shall be determined following the protocol for dried blood, which is standardized in the laboratory. Indirect fasting calorimetry is also performed. After the indirect calorimetry, the metabolic and hormonal response to the test meal is performed.

Each challenge should be consumed within 15 minutes. After 5 minutes of rest, indirect calorimetry will be postprandially, lasting 30 minutes. Capillary blood shall be obtained at the following times: 15-30-45-60-90 and 120 min after ingestion of food.

Nutrient composition of standardized meal and meal example.

• High carbohydrate challenge: Energy, 479.6 kcal; Carbohydrate, 83.6%; Lipids, 12.4% and Protein, 4.0% (70 g of hot-Cake, 100 g of mango, 270 ml of peach nectar, and 40 g of strawberry jam)
• High lipid challenge: Energy, 1043.4 kcal; Carbohydrate, 4.9%; Lipids, 86.8%, and Protein, 8.3% (60 g of manchego cheese, 25 g of egg, white, dried, 24 g of bacon, 5 ml of oil, 65 g of cream cheese, 70 g of cream, and 16 g of poblano pepper)
• High protein challenge: Energy, 441.3 kcal; Carbohydrate, 1.6%; Lipids, 5.1%, and Protein, 93.3% (2 scoop Isopure Zero Carb, 180 g of chicken breast, 20 g of lettuce and 24 g of ham turkey breast)

Determination of metabolites in dried blood To determine the concentration of metabolites (amino acids and acyl-carnitines), a circle of 3 mm diameter shall be punched out of the filter paper and placed in a 96-well plate. Add 100 µl of the acyl-carnitine and amino acid standards from the NeoBase PerkinElmer kit. Subsequently, follow the manufacturer's instructions for determining metabolites by liquid chromatography coupled to mass spectrometry (LC-MS).