Near-Infrared Spectroscopy, Acute Taurine Supplementation, and Isolated Muscular Endurance and Fatigue Resistance in Young Healthy Adults

Near-Infrared Spectroscopy-Derived Mitochondrial Oxidative Phosphorylation Capacity, Local Muscular Fatigue Resistance, and the Effects of Acute Taurine Supplementation

The goal of this clinical trial is to determine the association between near-infrared spectroscopy (NIRS)-derived mitochondrial oxidative phosphorylation (OXPHOS) capacity of the vastus lateralis muscles, and both local muscular endurance capacity and local muscular fatigability of the thigh muscles in young adults. Additionally, this trial aims to determine the effect of acute taurine supplementation on local muscular endurance capacity and local muscular fatigability when compared to a placebo condition. The main questions it aims to answer are:

1. Will there be a positive correlation between NIRS rate constant (a marker of mitochondrial function) and local muscular endurance capacity, along with a negative correlation between NIRS rate constant and local muscular fatigability?
2. Will acute taurine supplementation lead to improvements in local muscular endurance capacity and local muscular fatigability when compared to a placebo condition?

Participants will:

1. Complete a NIRS assessment to non-invasively assess the mitochondrial capacity of both vastus lateralis muscles
2. Complete two acute supplementation assessment visits (separated by 7-10 days) which involve small blood collections before and after taurine or placebo supplementation
3. Perform a single-leg extension time-to-exhaustion test to measure local muscular endurance capacity on one leg, along with a repeated maximal voluntary isometric contraction fatigue index test to measure local muscular fatigability on the opposite leg, during each acute supplementation assessment visit.

Participants will be recruited from the Louisiana State University campus and the Greater Baton Rouge area. Interested and potentially eligible participants will be identified based on the responses provided to questions on a prescreen assessment survey completed electronically via RedCap. These individuals will then be scheduled for the in-person Screening and Familiarization Visit which will include: a review of the informed consent form; a review of the inclusion/exclusion criteria; a self-report medical history and medication inventory; the collection of demographic information; an assessment of height and weight; an assessment of body composition via bioelectrical impedance analysis; an assessment of resting blood pressure and heart rate; the completion of the Physical Activity Readiness Questionnaire for Everyone (PARQ+) and the Global Physical Activity Questionnaire (GPAQ); an ultrasound assessment to determine adipose tissue thickness at the NIRS assessment sites; and familiarization with primary study assessments. If eligible according to the Screening and Familiarization Visit, participants will complete the NIRS Assessment Visit. Following an overnight fast (≥ 12 hours), with ≥ 24 hours since most recent exercise participation, a NIRS occlusion-based recovery kinetics approach will be used to non-invasively assess the mitochondrial OXPHOS capacity of the vastus lateralis muscle of each leg. On the day prior to this visit, participants will be asked to complete a 24-hour food log. Participants will then be asked to repeat this reported 24-hour food consumption on the day prior to each of the two remaining study assessment visits. Acute Supplementation Assessment Visit 1 will be completed following an overnight fast (≥ 12 hours), with ≥ 72 hours since most recent exercise participation and ≥ 48 hours following the NIRS Assessment Visit. During Acute Supplementation Assessment Visit 1, a baseline blood sample (10 ml) will be collected upon participants' arrival. Participants will then ingest either a taurine or placebo supplement depending on a randomized supplementation order. Next, participants will wait for 60 minutes following supplement ingestion before having another blood sample (10 ml) collected. Then participants will complete the submaximal, single-leg extension (SLE) isotonic contraction time-to-exhaustion (TTE) test on a randomly determined leg, followed by the 4-minute repeated, single-leg maximal voluntary isometric contraction (MVIC) fatigue index test on the contralateral leg. 7-10 days after Acute Supplementation Assessment Visit 1, following a ≥ 12 hour overnight fast and ≥ 72 hours since most recent exercise participation, participants will complete Acute Supplementation Assessment Visit 2. During this visit, all visit procedures will be conducted identically relative to Acute Supplementation Assessment Visit 1, but participants will consume the alternate supplement according to a randomized supplementation order. The 7-10 days between acute supplementation assessment visits will serve as a standard washout period. An unblinded member of the study team will be responsible for randomizing the supplementation order and preparing the supplements for all participant visits. This unblinded study team member will not be involved in any other aspects of data collection, data processing, or data analysis. All study visits will be conducted in the LSU Exercise Physiology Laboratory.

METHODS

1. Blood Sample Collections. 10 ml of blood will be collected from an antecubital vein both before and 60 minutes after supplement ingestion during each of the two acute supplementation assessment visits. The whole blood will be processed to plasma, frozen at -80⁰ C and subsequently used to assess plasma taurine levels. This will allow the researchers to directly account for plasma taurine levels in response to acute supplement ingestion.

2. NIRS Assessment to Measure Muscle Mitochondrial OXPHOS. A portable NIRS sensor (Train.Red Plus) will be placed on the vastus lateralis muscle at ~ 30-40% of the distance from the lateral epicondyle to the greater trochanter of the femur of each leg. An Easi-Fit Tourniquet Cuff specifically adapted to work with the Hokanson Rapid Cuff Inflation System will be placed on the upper thigh. After a 5-min rest period, an ischemic calibration procedure will be performed to account for individual differences in adipose tissue thickness. Following a brief recovery (3-5 minutes), the mitochondrial OXPHOS capacity will be measured using a modified mito-6 protocol. In brief, after 2 minutes of cycle ergometry, a series of 8 arterial occlusions will be used to measure the recovery rate of muscle oxygen consumption (mVO2) in a semi-recumbent position. The first 4 arterial occlusions will be 3-5 seconds "on," and 3-5 seconds "off," and the last 4 arterial occlusions will be 5-7 seconds "on" and 5 seconds "off." After a brief rest, the modified mito-6 protocol will be repeated 3 additional times. Performing the modified mito-6 protocol four total times will increase the reliability of the measurement while also affording the exclusion of outlier bouts. Following the last modified mito-6 bout, the participants will rest quietly for 5-10 minutes before one to two final 30-40 second arterial occlusions. These occlusions provide resting mvo2 slopes against which the mito-6 slopes can be compared during analysis. Vastus lateralis stimulation will be achieved using cycle ergometry at approximately 1 watt per kilogram body weight, which may be adjusted slightly up or down based on participant fitness. The goal is to achieve moderate intensity muscle contractions that optimally stimulate the vastus lateralis prior to arterial occlusions. This test procedure will be performed on the vastus lateralis of each leg, preferably simultaneously. The post-exercise mVO2 data will be fitted with a mono-exponential curve to determine the rate constant (k). This data analysis will be conducted in IRXstudio. For each leg, the k-values from the four modified mito-6 bouts will be used to calculate a median. All k-values that are greater than 30% different from this median will be excluded as outliers. All k-values not deemed outliers will be averaged, and these averaged k-values will be the main outcome variables derived from the NIRS assessment. Immediately following the NIRS assessment, adipose tissue thickness and muscular characteristics will be measured using B-mode ultrasound (LOGICe, GE) at the locations of the NIRS sensors. Ultrasound images will be taken in the transverse plane of the vastus lateralis muscles with frequency at 10 MHz, gain at 45%, and depth at 6 cm. These adipose tissue and muscle measures will provide valuable context to the NIRS assessment and may serve as covariates in subsequent analyses.

3. Submaximal Single-Leg Extension Isotonic Contraction Time-to-Exhaustion Test. A multimodal dynamometer (Humac NORM) will be used to perform this assessment on the quadriceps muscles/knee extensors. First, maximal voluntary isometric contraction (MVIC) will be recorded as the highest torque produced during three attempts in which MVIC is held for five consecutive seconds followed by 30 seconds of rest. Following the establishment of MVIC, there will be a five-minute rest period to allow full recovery prior to initiating the TTE test. For the TTE test, the torque for the isotonic contractions will be set to 20% of MVIC. This low relative load requires low muscle force production and thus will allow isotonic contractions to be sustained for an extended duration (≥ 2 minutes) to test muscular endurance capacity. All isotonic contractions will be performed at 60⁰ range of motion (ROM) (100-160⁰, with 180⁰ indicating a straight leg). Participants will perform one isotonic contraction every two seconds to the cadence of a metronome. A timer will be started at initiation of the TTE test, and participants will continue performing isotonic contractions until either of the following criteria are met: a) the participant stops due to volitional exhaustion; b) the participant cannot maintain the pace of the metronome for ≥ 5 seconds; or c) the participant cannot perform the full ROM for > 2 consecutive attempts. The time in seconds, from the initiation of the first isotonic contraction until any criterion for stopping is met, will be recorded as the TTE and serve as the main outcome variable from this test. A Train.Red NIRS probe and surface EMG probes will be placed on the vastus lateralis muscle to assess muscle oxygen saturation and excitation during the test. The TTE test will be performed first during both acute supplementation assessment visits. The leg on which the TTE test will be performed will be randomly determined for each participant and kept consistent for both acute supplementation assessment visits. This approach will allow the contralateral leg to be designated for the four-minute repeated, single-leg MVIC fatigue index test. The two tests will be performed on separate legs to prevent the risk of fatigue from one test influencing the other test. Randomizing which leg performs the TTE test will prevent systematic bias (e.g., always choosing the dominant leg).

4. Four-Minute Repeated, Single-Leg Maximal Voluntary Isometric Contraction Fatigue Index Test. A multimodal dynamometer (Humac NORM) will be used to perform this assessment on the quadriceps muscles/knee extensors. Participants will be set up on the device with the knee fixed at a 60° angle relative to full knee extension ROM. Participants will perform 5-second MVICs interspersed by 5-second rest intervals for four consecutive minutes (for a total of 24 repeated MVICs). The measured MVIC torques will be used to calculate fatigue index as the main outcome variable from this test. To calculate fatigue index, the investigators will first take the highest torque achieved from the first three MVICs (i.e., kicks 1-3) and the highest torque achieved from the final three MVICs (i.e., kicks 22-24), then use the following equation:

   ((Peak torque from first 3 MVICs - Peak torque from final 3 MVICs)/(Peak torque from first 3 MVICs)) x 100. A Train.Red NIRS probe and surface EMG probes will be placed on the vastus lateralis to assess muscle oxygen saturation and excitation during the test.

5. Statistical Analyses. The participant characteristics will be summarized using mean and standard deviation for continuous variables and frequencies/percentages for categorical data. Correlation analyses will be used to determine the association between NIRS rate constant (k) and time-to-exhaustion in the placebo condition, and the association between NIRS k and fatigue index in the placebo condition. Paired t-tests will be used to assess the within-participant differences between time-to-exhaustion and fatigue index in the taurine and placebo conditions. A power analysis was conducted using G*Power for a paired t-test (two-tailed) with an assumed moderate effect of 0.50 for time-to-exhaustion performance with acute taurine supplementation, alpha of 0.05, and power of 0.80. The effect size was established by considering previous findings in the literature but taking a more conservative approach. The analysis indicated a required sample size of at least 34 participants.

Inclusion Criteria:

• Capable and willing to give written informed consent
• Capable of understanding inclusion and exclusion criteria
• No medical condition that would limit participation in supervised exercise
• No current prescription medications known to impact mitochondrial function
• Sedentary or recreationally active (self-report average MVPA < 300 mins/week)
• Willing to allow researchers to use data for research purposes

Exclusion Criteria:

• Self-report history of diabetes mellitus
• Self-report history of cardiovascular, peripheral vascular, cerebral vascular, pulmonary, or renal disease
• Self-report history of uncontrolled hypertension (resting systolic blood pressure > 180 mmHg or diastolic blood pressure > 100 mmHg)
• Self-report history of blood clotting disorders
• Self-report history of sickle cell trait
• Self-report history of myopathy leading to muscle loss, weakness, severe cramps, or myalgia
• Self-report history of musculoskeletal disorders
• Self-report history of lower limb musculoskeletal injuries that may cause significant differences in the functional capacities of the right and left legs
• Self-report history of neurological disorders
• Active tobacco, E-cigarette, or nicotine use
• Recent (within previous 2 weeks) consumption of energy drinks or supplements containing ≥ 1 gram of taurine on > 2 days/week
• Actively taking medications to treat blood-clotting disorders
• Any condition that, in the judgement of the Principal Investigator, may interfere with study participation and adherence to the protocol
• Participants may be excluded if measured adipose tissue thickness is > 2.0 cm at the site of NIRS assessment or if the accessor is unable to obtain high-quality NIRS signals