Lactic Acid Output Research Articles

Design and Optimization of Lactic Acid Purification Process by Reactive Distillation with Isoamyl Alcohol

The purpose of this study was to investigate a continuous process for the recovery of lactic acid from fermentation broth. A reactive distillation process to purify lactic acid by esterification with isoamyl alcohol and hydrolysis was designed and simulated by Aspen. Five columns were included in the process: esterification column, purification column, hydrolysis column, dehydration column and recovery column. Effects of process parameters (operating pressure, feed composition, feed position, number of reaction and separation stages, and reboiler duty) of the esterification column on the yield and purity of lactic ester was evaluated. Further evaluation was carried out to reveal the effects of process parameters of the hydrolysis column on the yield and purity of lactic acid. The total annual cost of the whole process was calculated. Simulation studies are carried out using Aspen Plus RADFRAC module. It was shown that under the optimized condition, the concentration of lactic acid can reach up to 82.4 wt%, and the organic acid impurities were less than 3 ppm. Compared with the traditional usage of methanol and butanol, the isoamyl alcohol process can save about 20% of the total annual cost at the same output of lactic acid. Results obtained in this work can be used for scale-up study of an industrial reactive distillation operation as an efficient and economical alternative to recover lactic acid from fermentation broth.

Dynamic analysis of lactic acid fermentation in membrane bioreactor

In this paper, a mathematical model for the lactic acid fermentation in membrane bioreactor is investigated. This novel theoretical framework could result in an objective criterion on how to control the substrate concentration in order to keep a sustainable and steady output of lactic acid. Firstly, continuous input substrate is taken. The existence and local stability of two equilibria are studied. According to Poincaré–Bendixson Theorem, we obtain the conditions for the globally asymptotical stability of the equilibrium. Secondly, impulsive input substrate is also considered. Using Floquet's theorem and small-amplitude perturbation, we obtain the biomass-free periodic solution is locally stable if some conditions are satisfied. In a certain limiting case, it is shown that a nontrivial periodic solution emerges via a supercritical bifurcation. Finally, our findings are confirmed by means of numerical simulations.

Regulation of tumor pH and the role of carbonic anhydrase 9

The high metabolic rate required for tumor growth often leads to hypoxia in poorly-perfused regions. Hypoxia activates a complex gene expression program, mediated by hypoxia inducible factor 1 (HIF1alpha). One of the consequences of HIF1alpha activation is up-regulation of glycolysis and hence the production of lactic acid. In addition to the lactic acid-output, intracellular titration of acid with bicarbonate and the engagement of the pentose phosphate shunt release CO(2) from cells. Expression of the enzyme carbonic anhydrase 9 on the tumor cell surface catalyses the extracellular trapping of acid by hydrating cell-generated CO(2) into [see text] and H(+). These mechanisms contribute towards an acidic extracellular milieu favoring tumor growth, invasion and development. The lactic acid released by tumor cells is further metabolized by the tumor stroma. Low extracellular pH may adversely affect the intracellular milieu, possibly triggering apoptosis. Therefore, primary and secondary active transporters operate in the tumor cell membrane to protect the cytosol from acidosis. We review mechanisms regulating tumor intracellular and extracellular pH, with a focus on carbonic anhydrase 9. We also review recent evidence that may suggest a role for CA9 in coordinating pH(i) among cells of large, unvascularized cell-clusters.

Maternally inherited hearing loss, ataxia and myoclonus associated with a novel point mutation in mitochondrial tRNASer(UCN) gene.

We report on a new maternally-inherited syndrome characterized by a combination of sensorineural hearing loss, ataxia and myoclonus in a large kindred from Sicily. Hearing loss was the most widespread and sometimes the only symptom found in family members. Sequence analysis of the mitochondrial DNA regions encompassing the tRNA genes revealed the presence of a heteroplasmic insertion at nucleotide position 7472. The insertion adds a seventh cytosine to a six-cytosine run that is part of the mitochondrial tRNASer(UCN) gene. Conformational analysis showed that this mutation is likely to alter the structure of the T psi C loop in the tRNASer(UCN) clover leaf secondary structure. Moreover, the degree of heteroplasmy in blood and muscle was correlated with the clinical phenotype, and homoplasmic mutant hybrids showed decreased complex I activity, low oxygen consumption and high lactic acid output, indicating faulty oxidative phosphorylation. Finally, mutation was absent in 381 unrelated maternal lineages, suggesting specific segregation with the disease. We propose that the C7472 insertion-mutation is pathogenic, and etiologically related to hearing loss and other symptoms that define a novel maternally-inherited clinical entity.

Fatigue of mammalian skeletal muscle in situ during repetitive contractions

When the gastrocnemius-plantaris muscle group of the dog is stimulated to contract repetitively for 30 min at frequencies high enough to generate VO2 levels at or near VO2 max, VO2 and mechanical performance decline with time. This decline with time is fatigue, and it occurs during twitch and tetanic contractions that are isometric or isotonic. There is oxidation of the mitochondrial electron transport system, and net lactic acid output is transient, ending after 20 min of contractions. Energy and substrate stores and intracellular pH are only moderately changed and do not appear to be well correlated with the development of fatigue. Blood flow through the muscle is well correlated with the development of fatigue and decreases as fatigue develops in a manner that keeps the blood arteriovenous O2 difference nearly constant. Changing the blood flow alters the rate of development of fatigue as an inverse relation, and this response does not appear to be related to changes in the availability of O2 in the mitochondria. Nerve-muscle transmission of excitation does not seem to be involved in the development of fatigue. Excitation-contraction coupling is well accepted to be at least part of the genesis of the development of fatigue. Metabolic limitations and control may affect excitation-contraction coupling by one or more changes in the internal environment. Blood flow affects this system by an unknown mechanism. The role of blood flow in fatigue deserves further consideration.

On the mechanism of impaired insulin secretion in chronic renal failure.

It has been suggested that a sustained rise in resting levels of cytosolic calcium [Ca2+]i of pancreatic islets is responsible for impaired insulin secretion in chronic renal failure (CRF). Evidence for such an event is lacking and the mechanisms through which it may affect insulin secretion are not known. Studies were conducted in normal, CRF, and normocalcemic, parathyroidectomized (PTX) CRF rats to answer these questions. Resting levels of [Ca2+]i of islets from CRF rats were higher (P less than 0.01) than in control of CRF-PTX rats. [3H]2-deoxyglucose uptake and cAMP production by islets were not different in the three groups. Insulin content of, and glucose-induced insulin secretion by islets from CRF rats was lower (P less than 0.01) than in control and CRF-PTX rats. In contrast, glyceraldehyde-induced insulin release by CRF islets was normal. Basal ATP content, both glucose-stimulated ATP content and ATP/ADP ratio, net lactic acid output, Vmax of phosphofructokinase-1, and Ca2+ ATPase of islets from CRF rats were lower (P less than 0.02-less than 0.01) than in normal or CRF-PTX animals. Data show that: (a) Glucose but not glyceraldehyde-induced insulin secretion is impaired in CRF; (b) the impairment in glucose-induced insulin release in CRF is due to a defect in the metabolism of glucose; (c) this latter defect is due to reduced ATP content induced partly by high [Ca2+]i of islets; and (d) the high [Ca2+]i in islets of CRF rats is due to augmented PTH-induced calcium entry into cells and decreased calcium extrusion from the islets secondary to reduced activity of the Ca2+ ATPase.

Effects of ischemic and hypoxic hypoxia on VO2 and lactic acid output during tetanic contractions

We measured O2 uptake (VO2), CO2 output (VCO2), and net lactic acid output (L) during a 30-min period of repetitive 1/s isotonic tetanic contractions of the dog gastrocnemius-plantaris muscle group. The conditions were modest ischemic hypoxia (ischemia), hypoxia hypoxia (hypoxia), and free-flow normoxia (control). The major goal was to assess the effects of these perturbations on L during contractions. Ischemia and hypoxia were initiated just before the start of the contractions and at minute 7 of contractions in separate groups of experiments. Whenever applied, both ischemia and hypoxia reduced VO2 compared with the control values. When ischemia was initiated at the start of contractions, L was reduced transiently compared with the controls. When ischemia began at minute 7, L was increased modestly but transiently compared with the controls. When hypoxia was initiated at the start of contractions, L was increased during the entire period of contractions. The L pattern was the same as in the controls, rising to a maximal value at 3 min and declining steadily to a lower value at 30 min. When hypoxia began at minute 7, L declined initially at a slower rate than it did in the controls and was thereby elevated above the controls from 9 to 30 min. Ischemia was associated with a more rapid reduction in mechanical performance than hypoxia. The data suggest that the mechanisms of the decreased mechanical performance and VO2 are different for ischemia and hypoxia.

Comparative Effects of Bicarbonate and Dichloroacetate in Newborn Swine with Hypoxic Lactic Acidosis

Sodium bicarbonate (BC) and dichloroacetate (DCA) were studied in 7- to 14-day-old (n = 25) anesthetized swine with hypoxic acidosis. BC (base deficit X kg X 0.3, n = 10), DCA (300 mg/kg, n = 7) or saline (n = 8) was infused for 1 h. Blood lactic acid, dP/dtmax, heart rate and cardiac output increased and base excess and total arterial and carotid resistances (R) decreased with acidosis; aortic pressure, renal and mesenteric R did not change. BC induced higher pH, base excess and lactic acid. Heart rate in all and dP/dtmax with BC and DCA were restored; renal and mesenteric R and aortic pressure decreased in all. Cardiovascular responses to DCA and BC did not differ except for renal R. BC is a more effective alkalizer than DCA, which induced a greater renal vasodilation; both restored contractility.

Oxidation/reduction state of cytochrome oxidase during repetitive contractions

There is disagreement regarding whether inadequate O2 determines maximal O2 uptake (VO2max) and lactic acid output (L) during muscular activity. Direct assessment of mitochondrial cytochrome oxidase (cytochrome a-a3) oxidation/reduction (O/R) state should provide an unequivocal answer for this issue. A new near-infrared spectrophotometric method was used to measure the O/R state of cytochrome a-a3 of dog gastrocnemius-plantaris muscle in situ during repetitive isotonic twitch and tetanic contractions. Three contraction frequencies were used for each contraction type in alternating sequence to provide a wide range of VO2 up to VO2max. VO2 and L were measured after 3 and 9 min of a 10-min contraction period, and 15 min were allowed for recovery between contraction periods. VO2 increased with contraction frequency. L was variably increased with contraction frequency at 3 min and uptake usually occurred at 9 min, except at the highest tetanic frequency. The O/R span of cytochrome a-a3 was determined by respiring the animals with 100% N2 to determine the most reduced state. This was followed by respiration with 100% O2, which gave the most oxidized state transiently during recovery. Within this span in muscles at rest, cytochrome a-a3 was 50-80% oxidized. During contractions of both types at all frequencies, cytochrome a-a3 always became more oxidized by an additional 10-20%. These findings should put to rest any arguments that inadequate O2 is a determinant of VO2max or L under the conditions of these experiments: repetitive contractions with free flow in self-perfused muscles and normoxia.

A possible mechanism of toxicity by the antidepressant amoxapine based on its effects in three in vitro models

When the antidepressant amoxapine is taken in overdose, it can cause metabolic acidosis, brain damage and sometimes death. In previous studies, biochemical evidence has been presented that amoxapine disrupts reactions of membrane-associated multi-enzyme complexes, and mitochondrial energy conservation may be one of the first systems affected. Three in vitro systems were investigated to determine whether general membrane disruption or more specific mitochondrial effects of amoxapine could be responsible for toxicity and for lactic acid accumulation. In Saccharomyces cerevisiae, increased amoxapine concentrations led to decreased oxygen uptake associated with decreased survival of cells. In Chinese hamster ovary cells in culture, an initial increase in oxygen uptake was observed up to 10 μg amoxapine/ml and a decrease thereafter. At drug levels that caused an increased oxygen uptake, there was increased lactic acid output by cells, but no observable toxicity. At higher drug levels, the decreased oxygen uptake was accompanied by cell death, reduced lactic acid output and a change in the mitochondrial cristae configuration. Cell death in both of the above systems was attributed to interference with energy conservation. Isolated, beating guinea-pig hearts perfused with 5 μg amoxapine/ml stopped after 13 min, but no lactic acid accumulated. This may be explained by the membrane-stabilizing activity of the drug. In an arrested perfused heart, increased concentrations of amoxapine stimulated oxygen consumption and lactic acid production. Hence, membrane-stabilizing activity alone is not adequate to explain the action of amoxapine on isolated cells, and on the perfused arrested heart, or the clinical pattern of overdose, and disruption of energy conservation in cells is likely to be involved also.

Effect of systemic pH on pHi and lactic acid generation in exhaustive forearm exercise.

To investigate whether changes in systemic pH affect intracellular pH (pHi), energy-rich phosphates, and lactic acid generation in muscle, eight normal volunteers performed exhaustive forearm exercise with arterial blood flow occluded for 2 min on three occasions. Subjects ingested 4 mmol/kg NH4Cl (acidosis; A) or NaHCO3 (alkalosis; B) or nothing (control; C) 3 h before the exercise. Muscle pHi and phosphocreatine (PCr) content were measured with 31P-nuclear magnetic resonance (31P-NMR) spectroscopy during exercise and recovery. Lactate output during 0.5-7 min of recovery was calculated as deep venous-arterial concentration differences times forearm blood flow. Before exercise, blood pH and bicarbonate were lower in acidosis (7.303 +/- 0.009, 18.6 +/- 0.5 meq/l) than alkalosis (7.457 +/- 0.010, 32.2 +/- 0.7 meq/l) and intermediate in control (7.389 +/- 0.007, 25.3 +/- 0.6 meq/l). Lactic acid output during recovery was less with A (245 +/- 39 mumol/100 ml) than B (340 +/- 55 mumol/100 ml) (P less than 0.05) and intermediate in C (293 +/- 31 mumol/100 ml). PCr utilization and resynthesis were not affected by extracellular pH changes. pHi did not differ before exercise (A, 7.04 +/- 0.01; B, 7.09 +/- 0.01; C, 7.06 +/- 0.01) or at its end (A, 6.28 +/- 0.07; B, 6.28 +/- 0.11; C, 6.31 +/- 0.09). Hence systemic acidosis inhibited and alkalosis stimulated lactic acid output. These findings suggest that systemic pH regulates cellular acid production, protecting muscle pH, at the expense of energy availability.

Effects of adrenergic agonists and antagonists on muscle O2 uptake and lactate metabolism.

To investigate adrenergic receptor-mediated responses in dog gastrocnemius-plantaris muscle, several catecholamine agonists, isoproterenol, epinephrine, norepinephrine, and phenylephrine, and two antagonists, propranolol and phenoxybenzamine, were given during repetitive, isotonic, tetanic contractions. The response variables that were measured were muscle blood flow, shortening during constant load contractions, and arterial and venous O2 and lactate concentrations. The calculated variables were O2 uptake (VO2), net lactic acid output (L), and power output. In the control experiments, the contractions increased VO2 to approximately 50 times rest by 2 min. Thereafter, shortening, work, and VO2 declined together by 17% at 30 min, indicating muscle fatigue. L increased rapidly to nearly 0.8 mumol X g-1 X min-1 by 2 min, declined to 0.3-0.4 mumol X g-1 X min-1 by 7 min, and was like rest at 15, 22.5, and 30 min. The arterial lactate concentration rose steadily from rest to 30 min of contractions. Epinephrine infusion stopped the decline of VO2 during the contractions, but this effect was not observed with the other agonists. Propranolol decreased VO2 compared with controls at 22.5 and 30 min of contractions. Phenoxybenzamine decreased VO2 compared with controls at all times during contraction, and the decline with time was present. Coinfusion of epinephrine with propranolol reduced the decline in VO2 observed with propranolol alone. Both epinephrine and isoproterenol increased L compared with controls. This epinephrine response was antagonized by propranolol but enhanced by phenoxybenzamine. Both isoproterenol and epinephrine infusions increased arterial lactate concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolic and secretory response of tumoral-insulin producing cells to D-fructose and D-galactose

At variance with normal islet cells, tumoral insulin-producing cells of the RINm5F line were found to display a positive secretory response not solely to D-glucose and D-mannose, but also to D-fructose and D-galactose. All hexoses increased the ATP/ADP ratio, exerted a sparing action upon the oxidation of endogenous nutrients in cells prelabelled with either L-[U-14C]glutamine or [U-14C]palmitate, increased the output of lactic acid and, as judged from data collected in the presence of D-[U-14C]hexoses, underwent oxidation in the RINm5F cells. The secretory response to these four hexoses appeared commensurate with the extent of their metabolic effects.

Lactic acid output of cat gastrocnemius-plantaris during repetitive twitch contractions

Because fatigable, white (FF) muscle fibers have been reported to have a greater glycolytic capacity and a lower oxidative capacity than fatigue-resistant, red (FR and SR) muscle fibers, it is generally supposed that FF muscle fibers produce and therefore release more lactic acid into the blood during contractions than FR and SR muscle fibers. To test this supposition, the net lactic acid output, L, and O2 uptake, VO2, were measured for the cat gastrocnemius-plantaris muscle during repetitive isometric contractions. The results obtained from this low metabolic capacity (FF, FR and SR muscles) were compared to results obtained previously in the high metabolic capacity (FR and SR muscles) of the same muscle group in the dog during similar contractions. Preliminary studies established that 1 twitch X 2 s-1 provided a similar VO2 pattern during the contractions of cat muscle as 4 twitches X s-1 produced in the dog muscle. The decline in VO2 over a 30-min period of contractions was 12 to 18%, as developed tension declined with fatigue. Thus, the contractions of the cat muscles were matched with the dog muscles in terms of the relative aerobic capacity and development of fatigue. During the 1 twitch X 2 s-1 twitches, the VO2 reached 24.4 +/- 1.41 (SE) microliter X g-1 X min-1 at 10 min of contractions. The VO2 declined to 21.3 +/- 2.3 microliter X g-1 X min-1 by 30 min. The VO2 and tension developed changed parallel to each other. The net L reached 0.21 +/- 0.06 mumol X g-1 X min-1 at 10 min and fell to 0.13 +/- 0.05 mumol X g-1 X min by 30 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of catecholamines on lactic acid output during progressive working contractions.

Epinephrine and norepinephrine together (E + NE) and epinephrine (E) alone were infused intravenously in stepwise increasing doses during progressive isotonic tetanic contractions. The goal was to mimic, for in situ dog skeletal muscle, the concentrations of these catecholamines in the blood and the contractions during progressive exercise. The concentrations of lactate and O2 in arterial and muscle venous blood, the arterial plasma concentration of E and NE, PO2 in arterial and muscle venous blood, and the venous outflow were measured. The infusions caused a rise in plasma E and NE like those seen in progressive exercise. Compared with no-infusion controls, the E + NE infusions and the E alone infusion resulted in significant increases in maximal lactic acid output by the muscles during the contractions from 0.24 mumol X g-1 X min-1 in the controls to 0.44 and 0.54 mumol X g-1 X min-1 during E + NE and E alone infusions, respectively. The venous O2 concentrations and partial pressures were not reduced by the infusions. Both infusions resulted in a rise of arterial lactate concentration that could not be accounted for by the lactic acid output of the contracting muscles. The E alone infusions were associated with a rise in maximal O2 uptake during the contractions. Since the effects of the E + NE and E alone infusions were similar, it was suggested that E is more active than NE. It was suggested that E also increased lactic acid production in tissues other than the working muscles.

Ultrastructural differences between control and nitrosomethylurea-transformed cells of rat hepatocyte origin

Ultrastructural comparisons identify significant differences between a control cell, TRL 12–13, and a nitrosomethylurea-transformed cell line, NMU-3, which secrets lactic acid in vitro and produces carcinomas in vivo. Both cell lines were established from rat hepatocytes. The NMU-3 cells show a marked reduction in mitochondria, both at confluence and at 48 h post-confluence. This deficiency supports previous biochemical findings of altered bioenergetic pathways in the NMU-3 cells, leading to increased output of lactic acid. In addition to deficiencies in mitochondria, the NMU-3 cells show a marked reduction in other membranous structures, including rough endoplasmic reticulum, Golgi apparatus, pinocytotic and coated vesicles. Polyribosome distribution at confluence and 48 h later suggests that the transformed cells continue to synthesize intracellular protein, whereas the control cells appear to secrete protein for export at this time.

The stimulus-secretion coupling of glucose-induced insulin release. Metabolism of glucose in K+-deprived islets.

1. When pancreatic islets were exposed to a K(+)-free medium, the intracellular concentration of K(+) was decreased and that of Na(+) increased. 2. In the K(+)-deprived islets, the utilization of [5-(3)H]glucose, output of lactic acid and oxidation of [U-(14)C]-glucose were decreased by about 30-40% below the control values found at normal extracellular K(+) concentration (5.0mm). However, the oxidation of [U-(14)C]pyruvate was unaffected. 3. The omission of extracellular K(+) little affected the production of (14)CO(2) from islets prelabelled with [U-(14)C]palmitate and incubated in the absence of glucose, despite the fact that K(+) deprivation significantly increased the ATP concentration and ATP/ADP concentration ratio in the glucose-deprived islets. 4. At normal K(+) concentration, glucose increased the concentrations of phosphoenolpyruvate, NAD(P)H and ATP in the islets. In the glucose-stimulated islets, the concentration of phosphoenolpyruvate, but not that of either NAD(P)H or ATP, was higher in the absence than in the presence of extracellular K(+). In islet homogenates, the activity of pyruvate kinase (EC 2.7.1.40) was stimulated by K(+) (optimal activity at 100-150mm-K(+)) and inhibited by Na(+) (except at very low K(+) concentrations). 5. K(+) could be replaced by NH(4) (+), Rb(+), Cs(+) or Na(+) to maintain, at least to some extent, pyruvate kinase activity in islet homogenates. Addition of Rb(+) or Cs(+), but not NH(4) (+), to K(+)-deprived media also increased [U-(14)C]glucose oxidation by intact islets. 6. The omission of K(+) did not cause any obvious anomaly in the apparent dependency of (45)Ca(2+) net uptake on NAD(P)H concentration in the islets. 7. These data suggest that the coupling between metabolic and ionic events in the islet cells involves feedback mechanisms through which glucose oxidation may be modulated by cationic factors.

Association of alterations in energy metabolism with lead-induced delays in rat cerebral cortical development

Fifteen day-old rat pups taken from dams exposed to 200 mg of lead (Pb) in drinking water were observed to have reduced synaptic densities in the cerebral cortex as determined by ethanol-phosphotungstic acid staining. Synaptic figures of animals in the Pb groups were also observed to possess a less mature synaptic profile. Cerebral cortical slices taken from pups of Pb-treated dams exhibited enhanced respiratory responses to a 3 to 30 mM increase in K + concentration. This response was associated with a higher rate of glucose uptake by tissues of Pb-treated pups unaccompanied by a change in lactic acid output. Cerebral cortical concentrations of amino acids were not altered by Pb-treatment. Serum thyroid hormone levels were identical in 15-day old animals from the control and Pb-treated groups. These data further substantiate delayed cerebral cortical development as a result of low-level Pb exposures, and suggest that such delays may be associated with a direct effect of Pb on cerebral energy metabolism.

Effects of HCO 3−/CO 2 on the cytochrome redox potential and metabolic responses of isolated rat cerebral cortex

The importance of HCO 3 −/CO 2 to the maintenance of stable metabolic conditions in rat cerebral cortex slices has been investigated. Replacement of bicarbonate buffered media with glycylglycine or phosphate buffered salines resulted in an increased lability of the cytochrome redox potential of brain slices as measured by dual wavelength spectroscopy. Depletion of reduced cytochrome in the electron transport chain was associated with changes in the metabolic responses of tissues to electrical stimulation or elevated concentrations of potassium. This appears primarily as a loss of the late reductive responses of the tissue nicotinamide adenine dinucleotides NAD(P)H to these stimuli and decreased lactic acid output of the tissues. The effects could be largely reversed in a combined glycylglycine and HCO 3 −/CO 2 buffered media. It is suggested that although the reduction of NAD(P) in response to stimulation may be substantially located in the cytosol, it is also modulated by the mitochondrial redox potential. It is suggested that the lability of the redox potential in the absence of HCO 3 −/CO 2 may be related to depletion of TCA cycle intermediates normally replaced by CO 2 fixation.

The effect of long-term stimulation of fast muscles on their blood flow, metabolism and ability to withstand fatigue.

Chronic stimulation of fast rabbit muscles (tibialis anterior, extensor digitorum longus and the peroneal muscle group) at a frequency naturally occurring in nerves to slow muscles increased their ability to withstand fatigue. Isometric tension decreased during a 10-min period of contractions at 4 Hz by 75% in control muscles, but only 55% in muscles chronically stimulated for 4 days, and 23% in muscles stimulated for 28 days. Chronic stimulation had little effect on resting blood flow, oxygen or glucose consumption. The output or consumption of lactate and free fatty acids (FFA) at rest were also unaffected. The glycogen content was regularly increased, and was apparent after only 2 days of stimulation. The activity of fatty acid activating enzyme was increased after 28 days. During a 10-min period of isometric contractions at 4 Hz, there was a markedly greater increase in blood flow and oxygen consumption in muscles stimulated for 14-28 days than in control muscles; lactic acid output was lower in muscles stimulated for 28 days, and the uptake of FFA was significantly higher. It is therefore suggested that muscles chronically stimulated for 14-28 days use fats as the main source of energy during isometric contractions. The predominantly oxidative metabolism is probably facilitated by the higher density of capillaries. The latter also enables more efficient delivery of oxygen, and therefore smaller fatiguability, already after 4 days of chronic stimulation.