Lactic acid: New roles in a new millennium.

T study of lactic acid (HLa) and muscular contraction has a long history, beginning perhaps as early as 1807 when Berzelius found HLa in muscular fluid and thought that ‘‘the amount of free lactic acid in a muscle [was] proportional to the extent to which the muscle had previously been exercised’’ (cited in ref. 1). Several subsequent studies in the 19th century established the view that HLa was a byproduct of metabolism under conditions of O2 limitation. For example, in 1891, Araki (cited in ref. 2) reported elevated HLa levels in the blood and urine of a variety of animals subjected to hypoxia. In the early part of the 20th century, Fletcher and Hopkins (3) found an accumulation of HLa in anoxia as well as after prolonged stimulation to fatigue in amphibian muscle in vitro. Subsequently, based on the work of Fletcher and Hopkins (3) as well as his own studies, Hill (and colleagues; ref. 4) postulated that HLa increased during muscular exercise because of a lack of O2 for the energy requirements of the contracting muscles. These studies laid the groundwork for the anaerobic threshold concept, which was introduced and detailed by Wasserman and colleagues in the 1960s and early 1970s (5–7). The basic anaerobic threshold paradigm is that elevated HLa production and concentration during muscular contractions or exercise are the result of cellular hypoxia. Table 1 summarizes the essential components of the anaerobic threshold concept. However, several studies during the past '30 years have presented evidence questioning the idea that O2 limitation is a prerequisite for HLa production and accumulation in muscle and blood. Jöbsis and Stainsby (8) stimulated the canine gastrocnemius in situ at a rate known to elicit peak twitch oxygen uptake (V̇O2) and high net HLa output. They (8) reasoned that if the HLA output was caused by O2-limited oxidative phosphorylation, then there should be an accompanying reduction of members of the respiratory chain, including the NADHyNAD1 pair. Instead, muscle surface fluorometry indicated NADHyNAD1 oxidation in comparison to the resting condition. Later, Connett and colleagues (9–11), by using myoglobin cryomicrospectroscopy in small volumes of dog gracilis muscle, were unable to find loci with a PO2 less than the critical PO2 for maximal mitochondrial ox idative phosphorylation (0.1– 0.5 mmHg) during muscle contractions resulting in HLa output and an increase in muscle HLa concentration. More recently, Richardson and colleagues (12) used proton magnetic resonance spectroscopy to determine myoglobin saturation (and thereby an estimate of intramuscular PO2) during progressive exercise in humans. They found that HLa efflux was unrelated to muscle cytoplasmic PO2 during normoxia. Although there are legitimate criticisms of these studies, they and many others of a related nature have led to alternative explanations for HLa production that do not involve O2 limitation. In the present issue of PNAS, two papers (13, 14) illustrate the dichotomous relationship between lactic acid and oxygen. First, Kemper and colleagues (13) add further evidence against O2 as the key regulator of HLa production. They (13) used a unique model, the rattlesnake tailshaker muscle complex, to study intracellular glycolysis during ischemia in comparison to HLa efflux during free flow conditions; in both protocols, the muscle complex was active and producing rattling. In their first experiment, rattling was induced for 29 s during ischemia resulting from blood pressure cuff inflation between the cloaca and tailshaker muscle complex. In a second experiment, measures were taken during 108 s of rattling with normal, spontaneous blood flow. In both experiments, 31P magnetic resonance spectroscopy permitted measurement of changes in muscle levels of PCr, ATP, Pi, and pH before, during, and after rattling. Based on previous methods established in their laboratory, Kemper and colleagues (13) estimated glycolytic f lux during the ischemic and aerobic rattling protocols. The result was that total glycolytic f lux was the same under both conditions! Kemper and colleagues (13) conclude that HLa generation does not necessarily ref lect O2 limitation. To be fair, there are potential limitations to the excellent paper by Kemper and colleagues (13). First, and most importantly, they studied muscle metabolism in the transition from rest to rattling (29 s during ischemia and 108 s during free flow). Some investigators argue that oxidative phosphorylation is limited by O2 delivery to the exercising muscles during this nonsteady-state transition even with spontaneous blood flow (for review, see ref. 15). This remains a matter of debate, and the role of O2 in the transition from rest to contractions may depend on the intensity of contractions (16, 17). Of course, it is possible that the role of O2 in the transition to rattling may be tempered by the high volume density of mitochondria and the high blood supply to this unique muscle complex (13, 18). Second, there could be significant early lactate production within the first seconds of the transition (19). Third, it would have been helpful to have measurements of intramuscular lactate and glycogen concentra-