Effects of exercise-induced intracellular acidosis on the phosphocreatine recovery kinetics: a 31P MRS study in three muscle groups in humans.

Little is known about the metabolic differences that exist among different muscle groups within the same subjects. Therefore, we used (31)P-magnetic resonance spectroscopy ((31)P-MRS) to investigate muscle oxidative capacity and the potential effects of pH on PCr recovery kinetics between muscles of different phenotypes (quadriceps (Q), finger (FF) and plantar flexors (PF)) in the same cohort of 16 untrained adults. The estimated muscle oxidative capacity was lower in Q (29 ± 12 mM min(-1), CV(inter-subject) = 42%) as compared with PF (46 ± 20 mM min(-1), CV(inter-subject) = 44%) and tended to be higher in FF (43 ± 35 mM min(-1), CV(inter-subject) = 80%). The coefficient of variation (CV) of oxidative capacity between muscles within the group was 59 ± 24%. PCr recovery time constant was correlated with end-exercise pH in Q (p < 0.01), FF (p < 0.05) and PF (p < 0.05) as well as proton efflux rate in FF (p < 0.01), PF (p < 0.01) and Q (p = 0.12). We also observed a steeper slope of the relationship between end-exercise acidosis and PCr recovery kinetics in FF compared with either PF or Q muscles. Overall, this study supports the concept of skeletal muscle heterogeneity by revealing a comparable inter- and intra-individual variability in oxidative capacity across three skeletal muscles in untrained individuals. These findings also indicate that the sensitivity of mitochondrial respiration to the inhibition associated with cytosolic acidosis is greater in the finger flexor muscles compared with locomotor muscles, which might be related to differences in permeability in the mitochondrial membrane and, to some extent, to proton efflux rates.

Short-term training alters the control of mitochondrial respiration rate before maximal oxidative ATP synthesis.

AIM: Short-term exercise training may induce metabolic and performance adaptations before any changes in mitochondrial enzyme potential. However, there has not been a study that has directly assessed changes in mitochondrial oxidative capacity or metabolic control as a consequence of such training in vivo. Therefore, we used (31) P-magnetic resonance spectroscopy ((31) P-MRS) to examine the effect of short-term plantar flexion exercise training on phosphocreatine (PCr) recovery kinetics and the control of respiration rate. METHOD: To this aim, we investigated 12 healthy men, experienced with this exercise modality (TRA), and 7 time-control subjects (TC). RESULTS: After 5 days of training, maximum work rate during incremental plantar flexion exercise was significantly improved (P < 0.01). During the recovery period, the maximal rate of oxidative adenosine triphosphate synthesis (PRE: 28 ± 13 mm min(-1) ; POST: 26 ± 15 mm min(-1) ) and the PCr recovery time constant (PRE: 31 ± 19 s; POST: 29 ± 16) were not significantly altered. In contrast, the Hill coefficient (nH ) describing the co-operativity between respiration rate and ADP was significantly increased in TRA (PRE: nH = 2.7 ± 1.4; POST: nH = 3.4 ± 1.9, P < 0.05). Meanwhile, there were no systematic variations in any of these variables in TC. CONCLUSION: This study reveals that 5 days of training induces rapid adaptation in the allosteric control of respiration rate by ADP before any substantial improvement in muscle oxidative capacity occurs.

In vivo and in vitro investigations of heterozygous nebulin knock-out mice disclose a mild skeletal muscle phenotype.

Nemaline myopathy is the most common congenital skeletal muscle disease, and mutations in the nebulin gene account for 50% of all cases. Recent studies suggest that the disease severity might be related to the nebulin expression levels. Considering that mutations in the nebulin gene are typically recessive, one would expect that a single functional nebulin allele would maintain nebulin protein expression which would result in preserved skeletal muscle function. We investigated skeletal muscle function of heterozygous nebulin knock-out (i.e., nebulin(+/-)) mice using a multidisciplinary approach including protein and gene expression analysis and combined in vivo and in vitro force measurements. Skeletal muscle anatomy and energy metabolism were studied strictly non-invasively using magnetic resonance imaging and 31P-magnetic resonance spectroscopy. Maximal force production was reduced by around 16% in isolated muscle of nebulin(+/-) mice while in vivo force generating capacity was preserved. Muscle weakness was associated with a shift toward a slower proteomic phenotype, but was not related to nebulin protein deficiency or to an impaired energy metabolism. Further studies would be warranted in order to determine the mechanisms leading to a mild skeletal muscle phenotype resulting from the expression of a single nebulin allele.

Is brain maturation comparable in fetuses and premature neonates at term equivalent age?

BACKGROUND AND PURPOSE: Improved knowledge of brain maturation in fetuses and premature neonates is crucial for the early detection of pathologies and would help determine whether MR data from the premature brain might be used to evaluate fetal maturation. Using diffusion-weighted MR imaging and (1)H-MR spectroscopy, we compared cerebral microstructure and metabolism in normal in utero fetuses imaged near term and premature neonates imaged at term equivalent. MATERIALS AND METHODS: Forty-eight subjects were investigated: 24 in utero fetuses (mean gestational age, 37 ± 1 weeks) and 24 premature neonates (mean postconceptional age, 37 ± 1 weeks). ADC values were measured in cerebellum, pons, white matter, brain stem, basal ganglia, and thalamus. MR spectroscopy was performed in deep white matter. RESULTS: Mean ADC values from fetuses and premature neonates were comparable except for the pons and the parietal white matter. ADC values were lower in the pons of premature neonates, whereas greater values were found in their parietal white matter compared with fetuses. Proton MR spectroscopy showed higher levels of NAA/H(2)O, Glx/H(2)O, tCr/H(2)O, and mIns/H(2)O in premature neonates compared with fetuses. CONCLUSIONS: Our study provides evidence of subtle anomalies in the parietal white matter of healthy premature neonates. In addition, the reduced ADC values in the pons together with the increased levels of NAA/H(2)O, tCr/H(2)O, and Glx/H(2)O in the centrum semiovale suggest a more advanced maturation in some white matter regions. Our results indicate that MR data from the premature brain are not appropriate for the assessment of the fetal brain maturation.

Capsiate administration results in an uncoupling protein-3 downregulation, an enhanced muscle oxidative capacity and a decreased abdominal fat content in vivo.

OBJECTIVES: The involvement of skeletal muscle mitochondrial uncoupling protein-3 (UCP3) in the control of energy expenditure in skeletal muscle and at the whole-body level is still a matter of debate. We previously reported that UCP3 downregulation is linked to an enhanced mitochondrial energy metabolism in rat skeletal muscle as a result of acute capsiate treatment. Here, we aimed at investigating noninvasively the effects of chronic capsiate ingestion on metabolic changes occurring in exercising gastrocnemius muscle and at the whole-body level. METHODS: We used an original experimental setup allowing a complete noninvasive investigation of gastrocnemius muscle function in situ using 31-phosphorus magnetic resonance spectroscopy. Whole-body fat composition was determined using magnetic resonance imaging and UCP3 gene expression was measured by quantitative real-time RT-PCR analysis. RESULTS: We found that a 14-day daily administration of capsiate (100 mg kg(-1) body weight) reduced UCP3 gene expression and increased phosphocreatine level at baseline and during the stimulation period in gastrocnemius muscle. During muscle stimulation, pH(i) showed a larger alkalosis in the capsiate group suggesting a lower glycolysis and a compensatory higher aerobic contribution to ATP production. Although the capsiate-treated rats were hyperphagic as compared to control animals, they showed a lower weight gain coupled to a decreased abdominal fat content. CONCLUSION: Overall, our data indicated that capsiate administration contributes to the enhancement of aerobic ATP production and the reduction of body fat content coupled to a UCP3 gene downregulation.

A MRS-MRI-fMRI exploration of the brain. Impact of long-lasting persistent vegetative state.

BACKGROUND: The persistent vegetative state (PVS) is a devastating medical condition characterized by preserved wakefulness contrasting with absent voluntary interaction with the environment. However, very little is known about the actual degree of perception in these patients and the extent of progressive brain injury induced by very prolonged unawareness. METHODS: The authors have conducted a 2-year longitudinal study using a multimodal MRI-MRSI-fMRI protocol in four patients in long-lasting PVS (over 3 years at inclusion) characterized by various brain injuries. RESULTS: Although one subject showed initially preserved local brain metabolism and brain activity related to primary perception suggesting the presence of potential residual brain plasticity even in this critical stage, none of the four patients recovered to consciousness during the 2 years of the protocol. Moreover, significant deterioration of parameters related to brain atrophy, metabolism and functional excitability of primary cortices was observed in all patients during the follow-up. CONCLUSIONS: Heterogeneity of brain injury, consequences of long term minimal brain activity and potential factors that prevent recovery to consciousness are discussed.

Multiparametric differentiation of posterior fossa tumors in children using diffusion-weighted imaging and short echo-time 1H-MR spectroscopy.

PURPOSE: To assess the combined value of diffusion-weighted imaging (DWI) and proton magnetic resonance spectroscopy (1H-MRS) in differentiating medulloblastoma, ependymoma, pilocytic astrocytoma, and infiltrating glioma in a pediatric population. MATERIALS AND METHODS: A total of 17 children with untreated posterior fossa tumors (seven medulloblastoma, four infiltrating glioma, two ependymoma, and four pilocytic astrocytoma), were investigated with conventional MRI, DWI, and MRS using a single-voxel technique. Within the nonnecrotic tumor core, apparent diffusion coefficient (ADC) values using a standardized region of interest (ROI) were retrieved. Quantification of water signal and analysis of metabolite signals from MRS measurements in the same tumorous area were reviewed using multivariant linear discriminant analysis. RESULTS: Combination of ADC values and metabolites, which were normalized using water as an internal standard, allowed discrimination between the four tumor groups with a likelihood below 1 x 10(-9). Positive predictive value was 1 in all cases. Tumors could not be discriminated when using metabolite ratios or ADC values alone, nor could they be differentiated using creatine (Cr) as an internal reference even in combination with ADC values. CONCLUSION: Linear discriminant analysis using DWI and MRS using water as internal reference, fully discriminates the four most frequent posterior fossa tumors in children.

[Indications for cerebral MR proton spectroscopy in 2007]. / Spectroscopie par résonance magnétique du proton. Quelles indications neurologiques en 2007?

Magnetic resonance spectroscopy (MRS) is being increasingly performed alongside the more conventional MRI sequences in the exploration of neurological disorders. It is however important to clearly differentiate its clinical applications aiming at improving the differential diagnosis or the prognostic evaluation of the patient, from the research protocols, when MRS can contribute to a better understanding of the pathophysiology of the disease or to the evaluation of new treatments. The most important applications in clinical practice are intracranial space occupying lesions (especially the positive diagnosis of intracranial abscesses and gliomatosis cerebri and the differential diagnosis between edema and tumor infiltration), alcoholic, hepatic, and HIV-related encephalopathies and the exploration of metabolic diseases. Among the research applications, MRS is widely used in multiple sclerosis, ischemia and brain injury, epilepsy and neuro degenerative diseases.

Diffusion-weighted imaging in normal fetal brain maturation.

Diffusion-weighted imaging (DWI) provides information about tissue maturation not seen on conventional magnetic resonance imaging. The aim of this study is to analyze the evolution over time of the apparent diffusion coefficient (ADC) of normal fetal brain in utero. DWI was performed on 78 fetuses, ranging from 23 to 37 gestational weeks (GW). All children showed at follow-up a normal neurological evaluation. ADC values were obtained in the deep white matter (DWM) of the centrum semiovale, the frontal, parietal, occipital and temporal lobe, in the cerebellar hemisphere, the brainstem, the basal ganglia (BG) and the thalamus. Mean ADC values in supratentorial DWM areas (1.68 +/- 0.05 mm(2)/s) were higher compared with the cerebellar hemisphere (1.25 +/- 0.06 mm(2)/s) and lowest in the pons (1.11 +/- 0.05 mm(2)/s). Thalamus and BG showed intermediate values (1.25 +/- 0.04 mm(2)/s). Brainstem, cerebellar hemisphere and thalamus showed a linear negative correlation with gestational age. Supratentorial areas revealed an increase in ADC values, followed by a decrease after the 30th GW. This study provides a normative data set that allows insights in the normal fetal brain maturation in utero, which has not yet been observed in previous studies on premature babies.

[Brain magnetic resonance spectroscopy]. / Spectroscopie par résonance magnétique cérébrale.

MR spectroscopy (MRS) sequences allow noninvasive exploration of brain metabolism during a MRI examination. Their day-to-day use in a clinical setting has recently been improved by simple programming of sequences and automated quantification of metabolites. However, a few simple rules should be observed in the choice of sequences and the location of the voxels so as to obtain an informative, high-quality examination. The research applications of MR spectroscopy, where use of this examination seeks to better understand the pathophysiology of the disease, must be distinguished from its clinical indications, where MRS provides information that can be used directly in patient management. The most significant of the clinical uses are imaging intracranial tumors (positive and differential diagnosis, extension, treatment follow-up), diffuse brain injury, encephalopathies (especially hepatic and HIV-related), and the diagnosis of metabolic disorders.

Infratentorial pediatric brain tumors: the value of new imaging modalities.

The correct assessment of the four most frequent infratentorial brain tumors in children (medulloblastoma, ependymoma, pilocytic astrocytoma and infiltrating glioma) has always been problematic. They are known to often resemble one another on conventional magnetic resonance (MR) imaging. We tested the hypothesis whether the combined strength of diffusion-weighted imaging (DWI) and proton MR spectroscopy (MRS) could help differentiate these tumors. Seventeen children with untreated posterior fossa tumors were investigated between January 2005 and January 2006 with conventional MR imaging and combined DWI and MR spectroscopy using a single-voxel technique at short and long echo time (TE) of 30 ms and 135 ms respectively. Apparent diffusion coefficient (ADC) values were retrieved after regions of interest were manually positioned within non necrotic tumor core. Water signal was quantified and metabolite signals were compared and analyzed using linear discriminant analysis. When a combination of ADC values and normalized metabolites was used, all tumors could be discriminated against one other. This could only be achieved when metabolites were normalized using water as an internal standard. They could not be discriminated when using metabolite ratios or ADC values alone, nor could they be differentiated using creatine (Cr) as an internal reference even in combination with ADC values. In conclusion, linear discriminant analysis and multiparametric combination of DWI and MRS, although not replacing histology, fully discriminates the four most frequent posterior fossa tumors in children, but metabolites have to be normalized using water and not Cr signal as an internal reference.

In vivo and in vitro characterization of skeletal muscle metabolism in patients with statin-induced adverse effects.

OBJECTIVE: Statins (3-hydroxymethylglutaryl-coenzyme A reductase inhibitor) are widely used to treat hypercholesterolemia. They are generally well tolerated, but myotoxic effects have been reported and the corresponding mechanisms are still a matter of debate. The aim of the present study was to determine whether impairment of calcium homeostasis and/or mitochondrial impairment could account for the adverse effects of statins in skeletal muscle. METHODS: Eleven patients with increased creatine kinase levels and myalgias after statin treatment were evaluated using in vitro contracture tests (IVCTs), histology, and 31P magnetic resonance spectroscopy (31P-MRS). RESULTS: IVCT results were abnormal in 7 of the 9 patients, indicating an impaired calcium homeostasis. The 31P-MRS investigation disclosed no anomaly at rest, and the aerobic function assessed during the postexercise recovery period was normal. On the contrary, the pH recovery kinetics was significantly slowed down as indicated by a reduced proton efflux, which could be ultimately linked to a failure of calcium homeostasis. Overall, our observations indicate a normal mitochondrial function and raise the possibility that statins may unmask a latent pathology involving an impairment of calcium homeostasis such as malignant hyperthermia (MH). CONCLUSION: In case of susceptibility to MH, statins treatment must be administered with caution, and signs of adverse effects should be checked.

[MR spectroscopy of brain tumors]. / Spectroscopie par résonance magnétique des tumeurs cérébrales.

MR spectroscopy (MRS) can complement MRI in the evaluation of intracranial tumors. Before treatment, MRS can contribute to the differential diagnosis between tumor and non tumoral lesion (especially intracranial abscesses), to assess the aggressiveness of a glial tumor or to determine its extension to better delineate the surgical removal or the target volume of radiotherapy. During treatment follow-up, MRS helps differentiate recurrent tumor from radionecrosis or physiological post-surgical contrast enhancement. The current studies are trying to determine if the indications of MRS, alone or in association with other MR sequences can further be extended in the study of brain tumors, in particular the follow-up of lesions undergoing chemo or radiotherapy.

1H-MRS imaging in intractable frontal lobe epilepsies characterized by depth electrode recording.

Presurgical evaluation of frontal lobe epilepsy (FLE) remains a challenging issue and frequently requires invasive depth electrode recording. In this study, we aimed at evaluating the potential usefulness of a non-invasive technique such as proton magnetic resonance spectroscopic imaging ((1)H-MRSI) in the presurgical evaluation of FLE and at investigating the potential electrophysiological correlates of the metabolic disturbances as defined by (1)H-MRSI. We compared the distribution of (1)H-MRSI abnormalities with the electrophysiological abnormalities defined by stereo-electroencephalography (SEEG) recording in 12 patients presenting with several subtypes of FLE. We also used 12 control subjects in order to obtain normative (1)H-MRSI data. We used a multilevel (1)H-MRSI protocol to better sample the principal regions of the frontal lobe. We also applied a metabolic mapping technique allowing a visual display of metabolic data. A significant decrease of both N-acetyl-aspartate/phosphocreatine-creatine and N-acetyl-aspartate/(choline-compounds + phosphocreatine-creatine) ratios was observed in regions involved in the epileptogenic zone (EZ) and/or the irritative zone (IZ) compared to regions without electrical abnormalities in the same patients (P = 0.044 and P = 0.018, respectively), and also compared to controls (P = 0.004 and P = 0.0001, respectively). No significant differences in metabolic ratios were observed between those regions involved in the EZ and those involved in the IZ only. Our results suggest a link between the relative decrease of N-acetyl-aspartate and the EZ as well as the IZ in FLE. Thus, multilevel (1)H-MRSI protocol may add pertinent information during the non-invasive presurgical evaluation of FLE.

High cerebral scyllo-inositol: a new marker of brain metabolism disturbances induced by chronic alcoholism.

Cerebral metabolic changes that concur to motor and/or cognitive disorders in actively drinking alcoholics are not well established. We tested the hypothesis that chronic alcoholics exhibit profound alterations in the cerebral metabolism of scyllo-inositol. Brain metabolism was explored in nine actively drinking and 11 recently detoxified chronic alcoholics by in vivo brain (1)H-MRS and in vitro(1)H-MRS of blood serum and cerebrospinal fluid. The cohort was composed of individuals with acute, subacute or chronic encephalopathy or without any clinical encephalopathy. Chronic alcoholism is associated with a hitherto unrecognized accumulation of brain scyllo-inositol. Our results suggest that scyllo-inositol is produced within the central nervous system and shows a diffuse but heterogenous distribution in brain where it can persist several weeks after detoxification. Its highest levels were observed in subjects with a clinically symptomatic alcohol-related encephalopathy. When detected, brain scyllo-inositol takes part in a metabolic encephalopathy since it is associated with reduced N-acetylaspartate and increased creatine. High levels of cerebral scyllo-inositol are correlated with altered glial and neuronal metabolism. Our findings suggest that the accumulation of scyllo-inositol may precede and take part in the development of symptomatic alcoholic metabolic encephalopathy.

MRI/MRS of corpus callosum in patients with clinically isolated syndrome suggestive of multiple sclerosis.

Atrophy of corpus callosum (CC) related to axonal loss has previously been observed in patients at the early stage of clinically definite multiple sclerosis (CDMS). Atrophy increases with the progression of the disease. Nevertheless, no data concerning the onset of atrophy of CC are currently available. The purpose of this study is to determine if damage in callosal tissue was present at the earliest stage of MS, in a subgroup of patients presenting with a clinically isolated syndrome suggestive of MS (CISSMS), fulfilling the dissemination in space criteria according to McDonald. Magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) techniques were applied to measure CC volume, magnetization transfer ratio (MTR), mean diffusivity (MD), N-acetyl aspartate/choline-containing compounds (NAA/Cho) ratio, N-acetyl aspartate/total creatine (NAA/Cr) ratio and Cho/Cr ratio inside the CC of 46 CISSMS patients and 24 sex- and age-matched controls. No atrophy of CC was observed in the CISSMS group. CC of patients was characterized by decreased MTR and increased MD. No change in the NAA/Cr ratio was observed while the NAA/Cho ratio decreased and Cho/Cr ratio increased in the splenium and the central anterior part of CC. These abnormalities were present in patients with, but also without, macroscopic lesions inside the CC. Our results indicate that diffuse structural and metabolic changes, which may be interpreted as representing predominantly myelin pathology, occur in the CC at the earliest stage of MS before any atrophy is detected.

Multimodal magnetic resonance imaging of the central nervous system.

The physiological and biochemical properties of the diseased brain that can be explored with magnetic resonance imaging (MRI) are increasing. Progress in MR-based technology affords a large panel of MRI sequences that explore different phenomena and, thus, provide complementary informations. The diagnostic accuracy of MRI is improved by the combination of all MR modalities. However, this abundance of data requires an efficient multiparametric analysis to fully achieve the goal of the multimodal strategy. We will discuss the potential impact of this advanced MRI analysis in the clinical management and the therapeutical strategies of the most common brain pathologies (intracranial tumors, multiple sclerosis, stroke, epilepsy and dementia). This non-invasive approach is of utmost importance since it already improves the diagnosis and the therapeutic choice in the management of several central nervous system diseases.

Combined in situ analysis of metabolic and myoelectrical changes associated with electrically induced fatigue.

Electrical muscle stimulation (Mstim) at a low or high frequency is associated with failure of force production, but the exact mechanisms leading to fatigue in this model are still poorly understood. Using 31P magnetic resonance spectroscopy (31PMRS), we investigated the metabolic changes in rabbit tibialis anterior muscle associated with the force decline during Mstim at low (10 Hz) and high (100 Hz) frequency. We also simultaneously recorded the compound muscle mass action potential (M-wave) evoked by direct muscle stimulation, and we analyzed its post-Mstim variations. The 100-Hz Mstim elicited marked M-wave alterations and induced mild metabolic changes at the onset of stimulation followed by a paradoxical recovery of phosphocreatine (PCr) and pH during the stimulation period. On the contrary, the 10-Hz Mstim produced significant PCr consumption and intracellular acidosis with no paradoxical recovery phenomenon and no significant changes in M-wave characteristics. In addition, the force depression was linearly linked to the stimulation-induced acidosis and PCr breakdown. These results led us to conclude that force failure during 100-Hz Mstim only results from an impaired propagation of muscle action potentials with no metabolic involvement. On the contrary, fatigue induced by 10-Hz Mstim is closely associated with metabolic changes with no alteration of the membrane excitability, thereby underlining the central role of muscle energetics in force depression when muscle is stimulated at low frequency. Finally, our results further indicate a reduction of energy cost of contraction when stimulation frequency is increased from 10 to 100 Hz.

A new approach to examine the relationships between sensory and gas chromatography-olfactometry data using Generalized Procrustes analysis applied to six French Chardonnay wines.

Six French Chardonnay wines were submitted to both sensory and combined headspace/gas chromatography-olfactometry analyses. The detection frequencies allowed five hierarchical levels to be distinguished: P25, the odorant areas (OAs) having a detection frequency > or =25% (the complete olfactogram without the odor noise); P40, > or =40%; P55, > or =55%; P70, > or =70%; and P85, > or =85%. Moreover, the detection frequencies were analyzed to distinguish 21 discriminative OAs. Wines tested by sensory analysis and the headspace samples analyzed by gas chromatography-olfactometry (GC-O) were described by a heterogeneous vocabulary distributed into nine overall classes of descriptors. The new statistical treatment to examine hierarchical or discriminative OA categories with respect to sensory data used Generalized Procrustes analysis (GPA) from coordinate tables provided by correspondence analysis (CA). The successive data sets supplied by CA were subjected to GPA to yield consensus method maps. The more selective levels of detection frequency (P70 and P85) were responsible for incomplete or distorted information with respect to sensory data. The most appropriate segmentation of the OA distribution (olfactogram) to represent the sensory profile of the six samples would correspond to the intermediate pattern (P40 and P55). The other interest was to study the reasons of distortion due to the dynamic headspace extraction. The highest proportions of the variance were at all times related to the same classes: spicy, herbaceous, and, to a lesser degree, microbiological. This would indicate that the dynamic headspace analysis induces a distortion with respect to sensory data, which systematically affected the perception of both spicy and herbaceous characters of wines.

Metabolic determinants of the onset of acidosis in exercising human muscle: a 31P-MRS study.

Onset of intracellular acidosis during muscular exercise has been generally attributed to activation or hyperactivation of nonoxidative ATP production but has not been analyzed quantitatively in terms of H(+) balance, i.e., production and removal mechanisms. To address this issue, we have analyzed the relation of intracellular acidosis to H(+) balance during exercise bouts in seven healthy subjects. Each subject performed a 6-min ramp rhythmic exercise (finger flexions) at low frequency (LF, 0.47 Hz), leading to slight acidosis, and at high frequency (HF, 0.85 Hz), inducing a larger acidosis. Metabolic changes were recorded using (31)P-magnetic resonance spectroscopy. Onset of intracellular acidosis was statistically identified after 3 and 4 min of exercise for HF and LF protocols, respectively. A detailed investigation of H(+) balance indicated that, for both protocols, nonoxidative ATP production preceded a change in pH. For HF and LF protocols, H(+) consumption through the creatine kinase equilibrium was constant in the face of increasing H(+) generation and efflux. For both protocols, changes in pH were not recorded as long as sources and sinks for H(+) approximately balanced. In contrast, a significant acidosis occurred after 4 min of LF exercise and 3 min of HF exercise, whereas the rise in H(+) generation exceeded the rise in H(+) efflux at a nearly constant H(+) uptake associated with phosphocreatine breakdown. We have clearly demonstrated that intracellular acidosis in exercising muscle does not occur exclusively as a result of nonoxidative ATP production but, rather, reflects changes in overall H(+) balance.