Randomized Crossover Trial

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To determine the effect of post-exercise protein-leucine coingestion with CHO-lipid on subsequent high-intensity endurance performance and to investigate candidate mechanisms using stable isotope methods and metabolomics.

Well-trained male cyclists. 

• Failed to pass a health screening
• Recently donated blood
• Recently consumed caffeine, alcohol, medications or drugs
• Smoked during the experimentally controlled period.

Recruitment

Not discussed.

Design

• This is a double-blind, randomized crossover study during a six-day block of controlled high-intensity training and diet
• Before the first experimental block, participants recorded their habitual training and diet for seven and three days, respectively. Training was tapered in the three days before the experimental block, including a rest day before starting.
• To standardize lead-in fatigue and minimize diet variation, this training and dietary regimen was repeated preceding Block Two
• Each block comprised four intermittent high-intensity rides
  • Day One (three hours of cycling) consisted of a warm-up (15 minuted at 30%, 10 minutes at 40% and six minutes at 50% W_max), followed by loading intervals: Three blocks of 10  two-minute intervals at 90/80/70% W_max (two minutes at 50% W_max between intervals and six minutes at 50% W_max between blocks) and a cool down of 11 minutes at 30% W_max.
  • Day Two (2.5 hours of cycling) comprised a warm-up (10 minutes at 30%, 10 minutes at 40%, five minutes at 50% W_max), followed by two blocks of longer loading intervals (four five-minute intervals at 70% W_max interspersed with three of five minutes at 50% W_max and three of four minutes at 70%, interspersed with three of four minutes at 50% W_max) and separated by three of one minute at 90% and three of one minute at 80% W_max (interspersed by two minutes at 50% W_max)
  • Days Four and Six comprised 90 minutes at 50% of W_max followed by the repeated sprint performance test
  • Rides on Days Three and Five comprised 60 minutes at 30% of W_max.
• All rides were conducted at the same time of day for a given participant.

Blinding Used
Double-blind.

Intervention 

• Isocaloric supplements fed one to three hours after exercise during a six-day training block (intense intervals, recovery, repeated-sprint performance rides). Daily protein intake was clamped at 1.9g per kg^-1 per day^-1 (LEUPRO) and 1.5g per kg^-1 per day^-1 (control)
• LEUPRO: Leucine/protein/CHO/fat supplement (7.5g/20g/89g/22g per hour^-1)
• Control: CHO/fat (119g/22g per hour^-1).

Statistical Analysis

• The effect of treatment on outcomes was estimated with mixed modeling. Most outcome variables were 100x log-transformed before modeling to reduce non-uniformity of error and to express outcomes as percentages, with the exception of data sets with negative values (nitrogen balance).
• Most outcomes and comparisons were generated from fixed-effects models based on the interaction between the respective levels of treatment, test day and order of treatment
• For the analysis of sprint mean power, sprint number was a numeric effect (as in linear regression)
• Appropriate random-effect models for each parameter included all or some of between-athlete variation, additional treatment associated variation and additional variation associated with moving between test days
• Variability between blocks at baseline in blood measures was identified a priori as a potential confounder; value at baseline was hence included as a covariate within the appropriate model
• All covariates were first normalized to and expressed as a proportion of the within-subject SD for the covariate
• For performance, we used 0.93% (0.3x3.1%) as the threshold and for a mechanistic outcome, we used the standardized difference (effect size, ES).

Stable isotope infusions [1-(13)C-leucine and 6,6-(2)H2-glucose], mass spectrometry-based metabolomics and nitrogen balance methods were used to determine the effects of LEUPRO on whole-body branched-chain amino acid (BCAA) and glucose metabolism and protein turnover.

Timing of Measurements

• Whole-body leucine turnover was measured using a primed constant 1-13C-leucine infusion during the three-hour post-exercise recovery period on Day One and at rest (pre-exercise) and during steady-state exercise on Day Six
• Expired breath samples for indirect calorimetry were directed through a five-L mixing chamber attached to a Douglas bag for 10- to 12-minute gas collections at rest and three to five minutes during exercise
• Blood samples for additional parameters were collected on Days One and Six at sampling times, as described for leucine/glucose infusions, as well as after exercise on Day Six. Pre-exercise and post-exercise samples were taken on Days Two and Four by venipuncture.
• Throughout the six-day block, 24-hour urine was collected for quantification of nitrogen excretion and urinary metabolite analysis
• Sweat was collected during exercise on Day Two and during the 90-minute steady-state ride on Day Six
• Net nitrogen balance was calculated during five approximately 24-hour collections (Day One being only approximately 12 hours) to a total of 108 hours
• Measured nitrogen outputs were urinary urea and creatinine, with additional estimated nitrogen losses from sweat at rest and during exercise on Days Two and Six.

Dependent Variables

• Performance
• Plasma essential and total amino acid concentration
• Plasma and urinary concentrations of substrates and metabolites relating to the branched-chain amino acids, the urea cycle, the metabolism of alanine and aspartate, the degradation of lysine or the metabolism of arginine and proline
• Plasma concentrations of glucose and insulin
• Metabolomics
• CK concentration
• Leucine turnover
• Nitrogen balance
• Amino acid metabolomics
• RER
• Whole-body leucine and glucose kinetics. Whole-body leucine breakdown, non-oxidative disposal, oxidation and balance immediately after exercise (recovery, zero minutes) and during recovery (60 minutes to 180 minutes) on Day One and at rest and during steady-state exercise on Day Six.

Independent Variables

A leucine/protein/CHO/fat supplement (LEUPRO 7.5g/20g/89g/22g per hour^-1, respectively) or isocaloric CHO/fat control (119g/22g per hour^-1).

Control Variables

• Standardized diet and training regimen prior to testing
• Controlled diet during experimental blocks (all food provided).

• Initial N: 12 males
• Attrition (final N): 12
• Age (mean±SD): 35±10 years
• Ethnicity: NA

Other Relevant Demographics

• Maximal oxygen uptake (VO_2max): 64.8±6.8ml per kg^-1
• Peak power output (W_max): 355±36 W
• Cyclists had 9±4 years of training history, with a recent weekly training volume of 10±1 hours.

Anthropometrics

• Height: 182±5cm
• Body mass: 76.9±6.5kg
• Location: New Zealand.

Key Findings

• After exercise, LEUPRO increased BCAA levels in plasma (2.6-fold; 90% confidence limits, ×/÷1.1) and urine (2.8-fold; ×/÷1.2) and increased products of BCAA metabolism plasma acylcarnitine C5 (3.0-fold; ×/÷0.9) and urinary leucine (3.6-fold; ×/÷1.3) and beta-aminoisobutyrate (3.4-fold; ×/÷1.4), indicating that ingesting approximately 10g leucine per hour during recovery exceeds the capacity to metabolize BCAA
• LEUPRO increased leucine oxidation (5.6-fold; ×/÷1.1) and non-oxidative disposal (4.8-fold; ×/÷1.1) and left leucine balance positive relative to control
• With the exception of Day One (LEUPRO, 17±20mg N per kg^-1; control, -90±44mg N per kg^-1), subsequent (Days Two to Five) nitrogen balance was positive for both conditions (LEUPRO, 130±110mg N per kg^-1, control, 111±86mg N per kg^-1)
• Compared with control feeding, LEUPRO lowered the serum creatine kinase concentration by 21% to 25% (90% confidence limits, ±14%), but the effect on sprint power was trivial (Day Four, 0.4±1.0%; Day Six, -0.3±1.0%)
• There were no clear effects of treatment on the RER (range of means, 0.86 to 0.90) during recovery from exercise on Day One and at rest or during exercise on Days Four and Six
• Overall mean plasma glucose and insulin concentrations during the three-hour recovery period after exercise on Day One in the control were 6.9±1.6mmol per L^-1 and 89±118mcIU per ml^-1; the LEUPRO supplement led to a 12% reduction (±7%; ES, -0.80±0.51; P=7e-3) and a possible increase of 17% (±34%; ES, 19±34, P=0.23), respectively
• Nitrogen balance was positive on all of the five 24-hour collection periods with the LEUPRO supplement; with control, nitrogen balance was negative from the completion of exercise on Day One through to the morning of Day Two
• On Day One in the control, the total plasma amino acid concentration declined with time from the start of recovery
• LEUPRO supplementation led to an extremely large increase in plasma leucine (3.5-fold) and a very large increase in essential (2.2-fold) and total amino acid (1.7-fold) concentrations, relative to the control
• LEUPRO ingestion during recovery increased the concentration of plasma and urinary metabolic intermediates of BCAA degradation, indicative of protein and leucine intake that exceeded the whole-body capacity to metabolize BCAA
• LEUPRO led to small to moderate reductions of plasma CK concentrations before and after exercise on Days Four and Six.

• Post-exercise supplementation with LEUPRO resulted in a positive whole-body net leucine balance, reduced plasma CK and led to the accumulation of plasma and urinary amino acids and metabolites during the recovery period and resulted in positive leucine and nitrogen balance during the immediate hours after an intense endurance exercise
• However, the supplement provided no clear benefit to subsequent performance.

Industry:

Nestec Ltd., Vevey,Switzerland Pharmaceutical/Dietary Supplement Company:

• Results are interesting, however it is a small sample size
• Further studies on large sample, different ethnic groups and increase in duration of the study is required.