Intracellular oxygen (O 2 ) availability and the impact of ambient hypoxia have far reaching ramifications in terms of cell signalling and homeostasis; however, in vivo cellular oxygenation has been an elusive variable to assess. Within skeletal muscle the extent to which myoglobin desaturates (deoxy-Mb) and the extent of this desaturation in relation to O 2 availability provide an endogenous probe for intracellular O 2 partial pressure (P iO 2 ). By combining proton nuclear magnetic resonance spectroscopy ( 1 H NMRS) at a high field strength (4 T), assessing a large muscle volume in a highly efficient coil, and extended signal averaging (30 min) we assessed the level of skeletal muscle deoxy-Mb in 10 healthy men (30 ± 4 years) at rest in both normoxia and hypoxia (10% O 2 ). In normoxia there was an average deoxy-Mb signal of 9 ± 1%, which, when converted to P iO 2 using an O 2 /Mb half-saturation (P 50 ) of 3.2 mmHg, revealed an P iO 2 of 34 ± 6 mmHg. In ambient hypoxia the deoxy-Mb signal rose to 13 ± 3% (P iO 2 = 23 ± 6 mmHg). However, intersubject variation in the defence of arterial oxygenation (S aO 2 ) in hypoxia (S aO 2 range: 86-67%) revealed a significant relationship between the changes in S aO 2 and P iO 2 (r 2 = 0.5). These data are the first to document resting intracellular oxygenation in human skeletal muscle, highlighting the relatively high P iO 2 values that contrast markedly with those previously recorded during exercise (∼2-5 mmHg). Additionally, the impact of ambient hypoxia on P iO 2 and the relationship between changes in S aO 2 and P iO 2 stress the importance of the O 2 cascade from air to cell that ultimately effects O 2 availability and O 2 sensing at the cellular level.

T 1 -based determination of perfusion was performed with the high temporal and spatial resolution that monitoring of exercise physiology requires. As no data were available on the validation of this approach in human muscles, T 1 -based NMRI of perfusion was compared to standard strain-gauge venous occlusion plethysmography performed simultaneously within a 4 T magnet. Two different situations were investigated in 21 healthy young volunteers: 1) a 5-min ischemia of the leg, or 2) a 2-3 min ischemic exercise consisting of a plantar flexion on an amagnetic ergometer. Leg perfusion was monitored over 5-15 min of the recovery phase, after the air-cuff arterial occlusion had been released. The interesting features of the sequence were the use of a saturation-recovery module for the introduction of a T 1 modulation and of single-shot spin echo for imaging. Spatial resolution was 1.7 ؋ 2.0 mm and temporal resolution was 2 s. For data analysis, ROIs were traced on different muscles and perfusion was calculated from the differences in muscle signal intensity in successive images. To allow comparison with the global measurement of perfusion by plethysmography, the T 1 -based NMR measurements in exercising muscles were rescaled to the leg cross-section.

Skeletal muscle voluntary contractions (VC) and electrical stimulations (ES) were compared in eight healthy men. High-energy phosphates and myoglobin oxygenation were simultaneously monitored in the quadriceps by interleaved (1)H- and (31)P-NMR spectroscopy. For the VC protocol, subjects performed five or six bouts of 5 min with a workload increment of 10% of maximal voluntary torque (MVT) at each step. The ES protocol consisted of a 13-min exercise with a load corresponding to 10% MVT. For both protocols, exercise consisted of 6-s isometric contractions and 6-s rest cycles. For an identical mechanical level (10% MVT), ES induced larger changes than VC in the P(i)-to-phosphocreatine ratio [1.38 +/- 1.14 (ES) vs. 0.13 +/- 0.04 (VC)], pH [6.69 +/- 0.11 (ES) vs. 7.04 +/- 0.07 (VC)] and myoglobin desaturation [43 +/- 15.9 (ES) vs. 6.1 +/- 4.6% (VC)]. ES activated the muscle facing the NMR coil to a greater extent than did VCs when evaluated under identical technical conditions. This metabolic pattern can be interpreted in terms of specific temporal and spatial muscle cell recruitment. Furthermore, at identical levels of energy charge, the muscle was more acidotic and cytoplasm appeared more oxygenated during ES than during VC. These results are in accordance with a preferential recruitment of type II fibers and a relative muscle hyperperfusion during ES.

Mechanisms generating BOLD contrast are complex and depend on parameters that are prone to large variations, in particular in skeletal muscle. Here, we simultaneously measured perfusion by ASL, and BOLD response in the calf muscle of 6 healthy volunteers during post-ischemic reactive hyperemia. We tested whether the relation between the two was altered for varying degrees of leg vascular replenishment induced by prior positioning of the leg at different heights relative to the heart. We found that the BOLD response depended on perfusion, but also on the degree of repletion of leg blood vessels. We conclude that simultaneous determination of perfusion by ASL is important to identify the mechanisms underlying BOLD contrast in the skeletal muscle.

Together, these preliminary data provide evidence of an age-related decline in tissue perfusion and increased "metabolic stress" during exercise but demonstrate that overall oxidative capacity in the elderly does not appear negatively affected by this relatively hypoperfused state.

We previously identified a systemic metabolic defect associated with early weight loss in patients with Huntington's disease (HD), suggesting a lack of substrates for the Krebs cycle. Dietary anaplerotic therapy with triheptanoin is used in clinical trials to promote energy production in patients with peripheral and brain Krebs cycle deficit, as its metabolites -C5 ketone bodiescross the blood-brain barrier. We conducted a short-term clinical trial in six HD patients (UHDRS (Unified Huntington Disease Rating Scale)¼33±13, 15-49) to monitor the tolerability of triheptanoin. We also assessed peripheral markers of short-term efficacy that were shown to be altered in the early stages of HD, that is, low serum IGF1 and 31 P-NMR spectroscopy (NMRS) in muscle. At baseline, 31 P-NMRS displayed two patients with end-exercise muscle acidosis despite a low work output. On day 2, the introduction of triheptanoin was well tolerated in all patients, and in particular, there was no evidence of mitochondrial overload from triheptanoin-derived metabolites. After 4 days of triheptanoin-enriched diet, muscle pH regulation was normalized in the two patients with pretreatment metabolic abnormalities. A significant increase in serum IGF1 was also observed in all patients (205±60 ng/ml versus 246±68 ng/ml, P¼0.010). This study provides a rationale for extending our anaplerotic approach with triheptanoin in HD.

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