Validation of the tracer-balance concept with reference to leucine: 24-h intravenous tracer studies with L-[1-13C]leucine and [15N-15N]urea

Abstract

The validity of tracer-derived estimates of whole-body leucine balance was investigated. Seven healthy young adult subjects received an adequate protein diet for 6 d; at 1800 on the last day, L-[1-13C]leucine and [15N-15N]urea were given as primed, continuous intravenous infusions for 24 h. Subjects were in a fasting state for the first 12 h and at 0600 on day 7 they then received hourly 10 equal meals to achieve a fed state. Total leucine intake (diet plus tracer) was 89.4 mg.kg-1.d-1. Mean daily leucine oxidation was equivalent to 89.5 +/- 3.3 mg leucine/kg. The predicted daily oxidation rate, from measurements made during the last hour of the fast and the fifth hour of the fed period, was 91.2 +/- 5.8 mg/kg (P = 0.25 from measured). Measured and predicted whole-body leucine balances were 0.76 +/- 2.99 and -0.98 +/- 5.54 mg/kg, respectively (P = 0.25). Urea production exceeded urea excretion by 20%; daily protein oxidation was the same when estimated from leucine oxidation or nitrogen excretion. Thus, the tracer-balance concept is valid, and reliable predictions of total daily leucine oxidation and whole-body leucine balance can be obtained from short-term measurements of leucine oxidation during fasted and fed states.

Adaptation to a long term (4weeks) arginine- and precursor (glutamate, proline and aspartate)-free diet

“…The major energy source was provided in the form of protein-free wheat starch cookies, as previously described. 8 The energy intake was maintained constant within each individual and ranged between 35 and 55 kcal kg −1 day −1 , according to diet history and an approximation of the subject's physical activity level. Energy was provided as 35-40% fat and 55-60% as beet sucrose and wheat starch carbohydrates.…”

“…9 Multi-mineral and choline supplements were also provided. 8 The total daily intake before the tracer infusion studies was consumed as three separate meals at 08:00, 12:00 and 17 00 h. At least two of these three meals were eaten in the CRC under the supervision of the dietary staff.…”

“…The procedures followed have been described previously. 8,10 Each infusion study period lasted a total of 24 h. During the first 12 h (0-720 min) the subjects received their daily protein and energy intake, provided as small, isonitrogenous, isocaloric meals every hour. For the following 12 h (720-1440 min) the subjects remained fasting.…”

“…Blood was collected in chilled, heparin-containing tubes and was centrifuged immediately at 3000 × g for 15 min at 4 °C; the plasma was stored at −20 °C until analyzed. Breath samples were collected as described previously, 8 and stored at room temperature until analyzed by isotope ratio mass spectrometry (MAT Delta E; Finnigan, Bremmen, Germany). Total carbon dioxide production was measured by indirect calorimetry (Deltatrack; Sensormedics, Anaheim, CA) on four occasions throughout the 24-h period.…”

“…8,12 The leucine flux for the present study was determined from the enrichment of plasma 13 C-leucine rather than from α-keto-[1-13 C]isocaproic acid.…”