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).
Determination of the respiratory quotient (RQ) To determine the respiratory quotient (RQ=
VmaxCO2/ VmaxO2) and lipid and carbohydrate oxidation examinations, the Cardio Coach CO2
Vmax Encore 29 System calorimeter software (Korr, Inc, UT, USA) will be used according to
the instructions of the supplier. Examinations are invariably performed in the morning
(8:00-9:00 am) in a thermoneutral environment with controlled pressure, humidity, and
temperature, with the patient supine but awake. The investigators examine a maximum of
two subjects per day. Oxygen consumption and carbon dioxide production were obtained
using a canopy and were monitored continuously for 30 minutes. The initial 10 minutes of
the measurement are discarded for the calculation to ensure greater data homogeneity. O2
consumption and CO2 production will be recorded continuously for 30 min. VO2 and VCO2
values will be used in the equation proposed by Weir (Energy Expenditure = [3.941(VO2) +
1.11(VCO2)] x 1440 min/day), considered as the standard method [34]. Additionally, one
day before the test, subjects are instructed to fast for 8 hours and not engage in
physical activity or consume caffeine the day before the exam. All participants will be
asked to eat the same dinner the night before each test. Dinner provides 15% of the daily
energy intake; 20 g of protein, 7 g of lipids, and 34 g of carbohydrates)