Experimental colitis induced by dextran sulphate sodium in mice: beneficial effects of sulphasalazine and olsalazine.

BACKGROUND: Animal models of inflammatory bowel disease are artificial and more or less representative of human disease. However, the dextran sulphate sodium (DSS) induced intestinal inflammation model has recently been shown to fulfil some pathological criteria for an adequate experimental model. AIM: To determine whether this form of experimental intestinal inflammation responds to established therapy used for human inflammatory bowel disease. METHODS: DSS was used to induce intestinal inflammation in conventional Balb/c mice and athymic nu/nu CD-1(BR) mice, and the well-documented 5-aminosalicylic acid (5-ASA) based anticolitis drugs sulphasalazine (SASP) and olsalazine (OLZ) were used to study therapeutic effects. Parameters which have been shown to reflect DSS-induced intestinal inflammation (body weight, colon length, spleen weight, diarrhoea, and rectal bleeding) were measured in the Balb/c mice. RESULTS: Significant amelioration was seen on these parameters after different treatment protocols. Survival in nu/nu CD-1 mice was studied, and after 16 days a death rate of 50% was noted in the DSS group. SASP (100 mg/kg/day) and OLZ (50 mg/kg/day) significantly prolonged the survival to 29 and 38 days, respectively. SASP and OLZ showed a dose-dependent effect in the range between 10 and 100 mg/kg/day, doses closely corresponding to those used in humans. CONCLUSIONS: SASP and OLZ are able to ameliorate the DSS-induced intestinal inflammation. The dose-response patterns suggested that the active therapeutic moiety for the two drugs appears to be mainly the liberated 5-ASA molecule.

Dextran sulfate sodium (DSS) induced experimental colitis in immunodeficient mice: effects in CD4(+) -cell depleted, athymic and NK-cell depleted SCID mice.

Administration of dextran sulfate to mice, given in the drinking water results in acute or subacute colonic inflammation, depending on the administration protocol. This colonic inflammation exhibits ulceration, healing and repair, and a therapeutic response that makes it valuable for the study of mechanisms that could act in the pathogenesis of human ulcerative colitis, a disease thought to have an immunologically dependent pathogenesis. To investigate if immunological mechanisms were involved in the induction of colonic inflammation in this model, mice with different degrees of immunodeficiency were used. It was shown that dextran sulfate induced colitis could be induced in Balb/c mice depleted of CD4(+) helper T cells by treatment with monoclonal antibodies preceded by adult thymectomy. The depletion of CD4(+) was verified by flow cytometric analysis. Furthermore, the colonic inflammation could equally be induced in athymic CD-1 nu/nu mice lacking thymus-derived T cells, in T and B-cell deficient SCID mice, and also in SCID mice depleted of NK cells by treatment with anti-asialo GM1 antibodies. The NK-cell depletion was verified by measuring spleen NK-cell activity. The resulting colonic inflammation in all these types of deficient mice was qualitatively comparable, as shown by clinical and histological appearance. These results indicate that the presence of functional T, B and NK cells is not crucial for the induction of dextran sulfate colitis in mice.

Changes in gastric mucosal microcirculation and purine nucleotide metabolism during hemorrhagic shock in rats.

Intravital fluorescence microscopy and morphometry were used to study the microcirculation in the rat gastric mucosa during and after hemorrhagic shock. Under control conditions the circulation appeared homogeneous and unaffected by superfusion with 0.1 N HCl. During hemorrhagic shock, scattered areas of the mucosa lost circulation. Morphometry showed that the number of perfused mucosal vessels decreased significantly in the abluminal part of the mucosa both in perfused and ischemic areas, the reduction being most pronounced in the ischemic areas, where the number of perfused luminal vessels also decreased significantly. During reperfusion, bleedings occurred from the mucosa. A key finding was that the bleedings always had their origin at the border zone between ischemic and perfused areas. After hemorrhagic shock adenosine triphosphate and energy charge levels dropped significantly in both perfused and ischemic areas but with significantly lower levels in the ischemic areas. The hypoxanthine levels increased in both perfused and ischemic areas. The experiments show that local mucosal ischemia and accelerated nucleotide degradation are of pathogenetic importance in the development of stress ulcers after hemorrhagic shock. The border zone between circulated and ischemic areas seems to be an area of special interest.

Analysis of purine nucleotides in muscle tissue by HPLC.

Optimal conditions for simultaneous analysis of the purine nucleotides adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), inosine monophosphate (IMP), inosine, adenosine, hypoxanthine, xanthine and uric acid in muscle samples by high-performance liquid chromatography (HPLC) were evaluated. A neutralized perchloric acid extract of freeze-dried human or rat skeletal muscle tissue was injected on to a reversed-phase silica column and eluted by a gradient composed of ammonium dihydrogen phosphate buffer and methanol. Good resolution for all the nucleotides was achieved within a retention time of about 20 min. Linearity for each of the nucleotides within the concentration intervals obtained in the samples was demonstrated. Purity of each peak was verified by use of the photodiode array technique. Reproducibility for biological samples with variation coefficients below 3.6% for ATP, ADP, AMP, inosine and hypoxanthine and 6.7% for IMP was obtained. The stability of the compounds after extraction was specifically addressed. Storing of frozen extracts at -20 degrees C for 24 h gave acceptable values, while storage for 7 days cannot be recommended. Storage of unfrozen extracts at 4 degrees C was acceptable for (up to) 7 h. This technique provides a sensitive, convenient and reliable method for simultaneous analysis of a large number of purine nucleotides in small skeletal muscle biopsies, provided that certain precautions are taken with respect to the instability of these metabolites.

Purine metabolism after in vivo ischemia and reperfusion in rat skeletal muscle.

An in vivo rat hindlimb tourniquet ischemia model was used to study the purine nucleotide metabolism in response to 2, 4, and 6 h of ischemia and to the same ischemia periods followed by 1 h of reperfusion. All purine intermediates from ATP to uric acid were determined in skeletal muscle with a high-performance liquid chromatography (HPLC) system. The major metabolic event during ischemia is to temporarily save the nucleotide pool as inosine-5'-monophosphate (IMP. On restitution of the circulation as the energy state recovers, the IMP is converted back to AMP via the purine nucleotide cycle. Six hours of ischemia is associated with irreversible damage and no recovery fo the adenine nucleotides on reperfusion. Fast-twitch muscles appear to be more susceptible than slow-twitch muscles in response to ischemia and reperfusion. A severalfold increase of intracellular hypoxanthine occurred during ischemia, whereas uric acid formation is observed only after reperfusion. These findings are discussed in relation to the proposed role of xanthine oxidase, as an enzyme generating tissue-injurious oxygen free radicals.

Renal function during reperfusion after warm ischaemia in rabbits: an experimental study on the possible protective effects of pretreatment with oxygen radical scavengers or lidoflazine.

Survival and renal function were studied after 60 min of renal ischaemia and contralateral nephrectomy in four groups of French loop rabbits. One group served as untreated control animals. The other groups were pretreated with superoxide dismutase (SOD) and catalase, lidoflazine or a buffered albumin solution containing mannitol, L-methionine and MgCl2. Six out of nine rabbits died during the 14-day follow-up period in the untreated control group, while the corresponding figure in each of the three treatment groups was one out of nine. Five of the rabbits that died exhibited azotaemia or hyperpotassaemia that could explain the death, while four died of non-renal related causes. In the surviving animals, no differences in serum creatinine, potassium and sodium concentration or urinary output of osmoles was observed between the four groups. The increase in serum creatinine of the French loop rabbits observed after 60 min of ischaemia was considerably less pronounced than the corresponding increase observed in New Zealand White rabbits, indicating that the kidneys of the former strain are more tolerant to ischaemia. A cardiomyodepressant factor could be demonstrated in the venous effluent from previously ischaemic kidneys. This release could not be prevented by pretreatment with SOD and catalase.

Improved method for quantification of tissue PMN accumulation measured by myeloperoxidase activity.

Myeloperoxidase (MPO) was used as a marker enzyme for measuring polymorphonuclear leukocyte (PMN) accumulation in tissue samples. The MPO recovery from kidney, liver, myocardium, skeletal muscle (iliopsoas), and skin was measured, and a variation of 5%-100% was found, depending on the tissue analyzed. The recovery could be improved to 100% in all tissues by incubation of the tissue samples at 60 degrees C for 2 hr. This improved method was used to measure PMN accumulation in rabbit myocardium after regional ischemia and reperfusion. The MPO activity increased fivefold in the occluded area as compared with intact myocardium. Treatment with IB4, a monoclonal antibody recognizing the leukocyte adhesion molecule Mac-1, decreased the MPO activity in the occluded area almost to the level in intact myocardium.

Dynamics of skeletal muscle energetics during ischemia and reperfusion assessed by in vivo 31P NMR.

The dynamics of rat skeletal muscle energy metabolism in response to ischemia and reperfusion have been investigated by in vivo 31P NMR spectroscopy. The time course of changes in the phosphocreatine, inorganic phosphate, ATP peaks and in intracellular pH during 2 and 4 h of tourniquet ischemia followed by up to 24 h of tissue reperfusion have been determined. Furthermore, the ATP and IMP concentrations in the soleus and tibialis muscles have been determined by high performance liquid chromatography analysis in response to ischemia and subsequent reperfusion. The results demonstrate an initial overshoot in the pH during the first minutes of ischemia. It is also shown that the muscles recover completely after 2 h of ischemia whereas the energy state of the muscle cell is not restored after 4 h of ischemia followed by up to 24 h of reperfusion. However, the soleus muscle recovers better than the tibialis. The results are discussed in terms of oxygen availability, reperfusion injury, IMP accumulation and different response between muscles with different fibre composition.

Cytochrome c oxidase and cardiolipin alterations in response to skeletal muscle ischaemia and reperfusion.

The effect of 2 and 4 h of tourniquet ischaemia followed by 1 h of reperfusion on the major mitochondrial phospholipids and on the cytochrome c oxidase kinetic parameters has been investigated in rat skeletal muscle. There was no change either in the mitochondrial phospholipid content or in the Vmax and the Km of the enzyme after 2 h of ischaemia with and without subsequent reperfusion. Four hours of ischaemia had no effect on the lecithin and the cephalin content, while the cardiolipin content decreased as well as the Vmax of the enzyme (P less than 0.05). Tissue reperfusion caused a dramatic decrease in both cardiolipin (55% of the control, P less than 0.001) and Vmax (38% of the control, P less than 0.001). The corresponding reduction in lecithin and cephalin contents was 12% and 14% respectively (P less than 0.05). The Km remained unchanged at all conditions. These findings suggest that mitochondrial dysfunction in response to ischaemia and reperfusion could be a consequence of the reperfusion itself following severe ischaemia. The results are discussed in terms of cardiolipin peroxidation and cytochrome oxidase as a functional parameter.

Biological responses of sheep treated with endotoxin-contaminated superoxide dismutase and endotoxin preparations.

The purpose of this study was to evaluate the biological response of sheep to different doses of endotoxin and endotoxin-contaminated enzyme preparations. The enzyme used in this experiment was superoxide dismutase (SOD), as it is currently being used in many different experiments and because several preparations were found to be heavily contaminated with endotoxin. A group of ewes were injected intravenously with a variety of different treatments. Peripheral blood was used to determine the total number of leukocytes, a differential cell count to find out the numbers of polymorphonucleocytes (PMN) and monocytes (M), and to measure the concentration of 15-ketodihydro-PGF2 alpha. In addition, rectal temperature was recorded. Treatments included saline (control), Pharmacia-Chiron's Cu/Zn-SOD (r-hSOD, 8 mg/kg), Sigma's bovine SOD (bSOD, 8 mg/kg), Grünenthal's bSOD (8 mg/kg), various doses of Salmonella typhimurium endotoxin (1000, 200, 100, 50, 20, 10, 5, and 1 ng/kg), and a mixture of endotoxin (200 ng/kg) plus r-hSOD (8 mg/kg). Results indicate that sheep react to endotoxin-contaminated SOD preparations with an endotoxemia which is similar to that seen in animals receiving endotoxin alone. This endotoxemia includes, among other things, a rise in rectal temperature, a peak in the PGF2 alpha metabolite, and an increased PMN/M ratio. Endotoxin administered at doses of 50 to 200 ng/kg also caused the expected signs of endotoxemia. At 1000 ng/kg endotoxin actually led to a decreased rectal temperature. This may be due to a type of endotoxemic shock, resulting in a decrease in peripheral blood circulation. Low doses of endotoxin (10, 5, and 1 ng/kg) caused a leukocytosis via increases in PMN; no greater changes in rectal temperature or the PGF2 alpha metabolite were noted. The combination of r-hSOD with 200 ng/kg of endotoxin caused an endotoxemia similar to that caused by 200 ng/kg of endotoxin alone. In conclusion, if an endotoxin-contaminated SOD-preparation was used to study the efficacy of SOD, there would be a serious risk of interaction by the endotoxins. In such a case it would be impossible to distinguish the effects of the endotoxin from those of the preparation itself. It is therefore important that researchers are alert to the problem of endotoxin contamination.

Some effects of gram-negative bacterial endotoxin and its importance as a contaminator of biological preparations.

The purpose of this study was to establish a model which can be used to examine the biological response to Salmonella typhimurium endotoxin in both anaesthetized and unanaesthetized rabbits, and then compare this response to that of rabbits injected with an endotoxin-contaminated biological preparation. The parameters used to evaluate the biological response included total white blood cell and differential counts, 15-ketodihydro-PGF2 alpha concentration, and rectal temperature. Unanaesthetized groups of rabbits received 1000, 100, 10, or 1 ng/kg of the endotoxin via intravenous injection (i.v.); the anaesthetized group of rabbits received 100 ng/kg endotoxin i.v. (anaesthesia induced with Hypnorm). In addition, groups of rabbits were treated under anaesthesia with Pharmacia-Chiron's recombinant human Cu/Zn superoxide dismutase (SOD) (10 mg/kg body weight = 1.6 endotoxin units (EU)/kg) or Grünenthal's bovine SOD (two doses: 10 mg/kg = 400 EU/kg, or 50 mg/kg = 2000 EU/kg). Results demonstrated that at the lower doses of endotoxin (10 and 1 ng/kg) and r-hSOD (10 mg/kg), no leukopenia was observed. There was however a slight shift in the leukocyte population so that polymorphonucleocytes increased and monocytes decreased in number. Rabbits treated with higher doses of endotoxin (1000 and 100 ng/kg) showed many of the common signs of endotoxemia, including leukopenia, increased prostaglandin metabolite levels, and increased body temperature, as did the rabbits treated with endotoxin-contaminated bSOD. There was a definite dose-dependency, with the higher dose of bSOD giving a more marked rise in all parameters. These findings indicate that use of this or other endotoxin-contaminated biological preparations in live-animal experiments could produce erratic, and therefore unreliable, results.

1H-n.m.r. evaluation of the ferricytochrome c-cardiolipin interaction. Effect of superoxide radicals.

The interaction between ferricytochrome c and cardiolipin was investigated by 1H n.m.r. at 270 MHz. From the phospholipid-induced changes of the protein spectral features it is concluded that the first 2 equivalents of cardiolipin cause a conformational change at the lower part of the solvent-exposed haem edge, involving a rearrangement of the hydrogen-bond interactions of propionate 6, thus partly accounting for the lowered redox potential of cytochrome c in the presence of cardiolipin. The increased value for the pK of the alkaline isomerization of ferricytochrome c shows that cardiolipin stabilizes the native structure of the protein, indicating that the oxidized form assumes ferrocytochrome c-like properties. Peroxidation of cardiolipin by superoxide radical ions drastically decreases the protein binding to this phospholipid. The implications of this finding, and the likelihood of the ternary cytochrome c-cardiolipin-cytochrome c oxidase complex, for the binding of cytochrome c to cytochrome c oxidase in vivo, are discussed in relation to peroxidative damage following ischaemia and reperfusion.

The hydroxylamine OXANOH and its reaction product, the nitroxide OXANO., act as complementary inhibitors of lipid peroxidation.

The effects of the nitroxide 2-ethyl-2,5,5-trimethyl-3-oxazolidinoxyl (OXANO.) and the corresponding hydroxylamine 2-ethyl-1-hydroxy-2,5,5-trimethyl-3-oxazolidine (OXANOH) on in vitro lipid peroxidation in rat liver microsomes and reconstituted lipid vesicles were investigated, and compared with those of some commonly used spin trapping agents. OXANO. and OXANOH (10-100 microM) inhibited iron-dependent lipid peroxidation, as did the spin trapping agents (10-100 mM). OXANO. mainly inhibited the rate of peroxidation, but caused only a small delay in the time of onset. OXANOH exerted its effect by delaying the onset of peroxidation in an antioxidant fashion, and also by inhibiting the rate. Higher concentrations of both substances were required to inhibit t-butylhydroperoxide-dependent lipid peroxidation. OXANO. was found to oxidize the ferrous-ADP complex required for initiation of peroxidation, and this is probably the basis of the inhibitory effect of this compound. Since the reaction of OXANO. tends to produce OXANOH and vice versa, either one could inhibit all reactions of lipid peroxidation.

Muscle enzyme adaptation in patients with peripheral arterial insufficiency: spontaneous adaptation, effect of different treatments and consequences on walking performance.

1. The activities of phosphofructokinase (PFK), citrate synthetase (CS), lactate dehydrogenase (LDH), 3-hydroxyacyl-CoA dehydrogenase (ACDH) and cytochrome-c oxidase(Cyt-ox) in the calf muscle tissue were compared in subjects with intermittent claudication (n = 38) and controls (n = 20). The activities of CS, ACDH and Cyt-ox were increased and the activity of Cytox was positively correlated to the maximal walking distance (MWD) in the patients. 2. Thirty-three patients with intermittent claudication were randomized to three treatment groups: (1) operative surgery, (2) operative surgery supplemented with physical training and (3) physical training alone. Before and after 6-12 months of treatment, symptom-free walking distance (SFWD), MWD, ankle-brachial blood pressure quotient (ankle index), maximal plethysmographic calf blood flow (MPBF) and the activities of PFK, CS, LDH, ACDH and Cyt-ox were measured. 3. SFWD and MWD increased in all three groups. Ankle index and MPBF increased in groups 1 and 2, but were unchanged in group 3. The activities of Cyt-ox and CS decreased with operation, but the activity of Cyt-ox was further augmented with training in group 3. Overall, the change in ankle index explained 80-90% of the variability in walking performance. In a separate analysis, the increased activity of Cyt-ox in group 3 was positively correlated to, and explained 31% of the variability in, the improvement in SFWD. 4. These findings indicate that both physical activity and a reduced calf blood flow are necessary conditions for the enzymatic adaptation to take place. A causal relationship between metabolic adaptation in the muscle tissue and walking performance is suggested.(ABSTRACT TRUNCATED AT 250 WORDS)

Radical production during in vivo intestinal ischemia and reperfusion in the cat.

Free radical formation was studied with electron spin resonance during 2 h of intestinal ischemia in the cat, at a blood flow less than 5 ml.min-1.100 g-1, followed by 30-min reperfusion. A modification of the spin trapping technique was used to stabilize highly reactive free radicals. The rate of secondary radical formation was 0.32 +/- 0.06 mumol.min-1.100 g intestine-1 before ischemia and increased to a maximum of 0.66 +/- 0.09 mumol.min-1.100 g-1 during the first minutes of reperfusion (mean +/- SE, n = 5). This could be prevented either by maintaining intestinal blood flow at 8-15 ml.min-1.100 g-1, by administering allopurinol before and during ischemia, or by perfusing the intestinal lumen with an O2-saturated buffer solution during ischemia, resulting in maximum rates of radical production during reperfusion of 0.37 +/- 0.04 (n = 6), 0.33 +/- 0.04 (n = 5), and 0.39 +/- 0.13 mumol.min-1.100 g-1 (n = 5), respectively. The results demonstrate that free radicals are produced in the intestine during reperfusion after a period of reduced blood flow below a certain critical level, and that inhibition of xanthine oxidase and prevention of hypoxia will eliminate this radical production.

Inhibition of lipid peroxidation by spin labels. Relationships between structure and function.

Inhibition of lipid peroxidation by nitroxide radicals and their corresponding hydroxylamines was investigated. The nitroxides were either oxazolidines or piperidines, differing in substitution of the backbone of the molecule (a five or six-membered ring structure, respectively). Concentration requirements for 50% inhibition of microsomal lipid peroxidation varied from 340 to 6 microM for the nitroxides, and from 120 to 3 microM for the hydroxylamines, correlating with lipophilicity and chemical structure. Intramembrane concentrations required for 50% inhibition was independent of lipophilicity when peroxidation was initiated with ADP-Fe2+ but increased with lipophilicity when peroxidation was initiated with t-butylhydroperoxide. During studies of the kinetics of the inhibition, two modes were seen: a delay or a decreased rate of the process. The former mode was seen with the more lipophilic inhibitors. The mechanism of inhibition was similar for all nitroxides and consisted of the following three major components: blocking of primary initiation, prevention of secondary (peroxide-dependent) initiation, and scavenging of various lipoid radicals in the membrane, the major mode of action of the hydroxylamines. Inhibitory efficiency was interpreted in terms of steric hindrance, diffusibility, regeneration of inhibitor, and ability to interact with hydrophilic sites in a hydrophobic environment.

Kinetic parameters of cytochrome c oxidase in rat skeletal muscle: effect of endurance training.

The kinetic properties of cytochrome c oxidase (EC 1.9.3.1) in skeletal muscle tissue of sedentary and endurance-trained rats were studied. The initial velocity of the cytochrome c oxidase reaction was determined polarographically over a large range of cytochrome c concentrations and the maximal velocity (Vmax) and the Michaelis constant (Km) were calculated. The catalytic activity of cytochrome c oxidase in isolated mitochondria was also investigated. The training programme consisted of treadmill running for 2 h a day, 6 days a week, at a speed of 30 m min-1 and 30 degrees elevation, for 4 weeks. Vmax of cytochrome oxidase with respect to cytochrome c increased significantly from 254 to 310 mumol O2 min-1 g-1 protein in response to training (P less than 0.001), whereas Km remained unchanged (18.9 and 18.7 microM). The turnover number (TN) increased from 11.1 S-1 in sedentary rats to 16.6 S-1 in trained rats (P less than 0.001). The results suggest a qualitative change in the enzyme molecule in addition to a true Vmax change of cytochrome c oxidase in response to endurance training.

Detection of oxygen radicals during reperfusion of intestinal cells in vitro.

The nitroxide OXANO. (2-Ethyl-2,5,5-trimethyl-3-oxazolidinoxyl) which in its reduced form, OXANOH (2-Ethyl-1-hydroxy-2,5,5-trimethyl-3-oxazolidine), is capable of reacting with short-lived radicals, forming a secondary stable radical, was used for ESR-detection of radical production in isolated cells. The properties of OXANO. and OXANOH in terms of stability in cellular and subcellular systems, membrane permeability and effects on cellular viability were evaluated. Ischemia and reperfusion was simulated in vitro in a preparation of cells from rat intestinal mucosa by incubation at high density (4 X 10(8) cells/ml) under an atmosphere of nitrogen for 25 min and resuspended with fresh oxygenated buffer containing 5 mM OXANOH. A significant increase in radical formation during the 15 min reperfusion period studied was obtained in cells exposed to ischemia compared to control cells incubated at normal density under an atmosphere of oxygen. The addition of 5 microM of the scavenging enzyme superoxide dismutase reduced the radical formation by 50%. The time sequence of the superoxide formation was calculated as the difference in radical production in the presence and absence of superoxide dismutase.

Enzymatic activities in heart and skeletal muscle of children with cyanotic and noncyanotic congenital heart disease.

The activity of phosphofructokinase (PFK), citrate synthetase (CS), lactate dehydrogenase (LDH), 3-OH-CoA dehydrogenase (ACDH) and cytochrome-c-oxidase (cyt-ox) was measured in right atrial auricle and abdominal rectal muscle biopsies from 24 children, aged 3-12 years, with congenital heart malformations. Twelve children had cyanotic conditions (tetralogy of Fallot or truncus malformations) and 14 were noncyanotic (septal defects or vascular lesions). The cyt-ox activity was significantly higher in the cyanotic subgroup than in the noncyanotic (skeletal muscle: 55.71 +/- 10.4 vs 19.48 +/- 2.6 mmol/g protein/min, p less than 0.01; auricle: 93.1 +/- 11.8 vs 65.58 +/- 7.5, p less than 0.05). There were no significant differences between the activities of PFK, LDH, CS or ACDH in the cyanotic and noncyanotic groups. Within the normal range of hemoglobin and hematocrit, there was no correlation between these parameters and cyt-ox. On the other hand, above the normal range of hemoglobin and hematocrit a correlation coefficient of 0.89 (p less than 0.01) was observed which suggests the higher cyt-ox activity to be an adaptive phenomenon triggered by reduced availability of oxygen.

Substrate exchange in human limb muscle during exercise at reduced blood flow.

The substrate exchange of the calf muscles during leg exercise was compared in patients with chronically reduced blood flow and in matched controls. The arteriovenous differences of glucose, lactate, pyruvate, free fatty acids, glycerol, acetoacetate, beta-OH-butyrate, oxygen, and carbon dioxide were analyzed at rest, at the end of two exercise periods at various work loads, and after 10 min of recovery. Calf blood flow was measured with an electrocardiogram-triggered, computerized, strain gauge, venous occlusion plethysmograph. The results indicate that there was increased extraction of oxygen and ketone bodies in patients with reduced blood flow during exercise, whereas the glucose extraction tended to be lower than in controls. The leg respiratory quotient was lower in the patients even at the point of claudicating pain, suggesting oxidation of endogenous fat. The simultaneously elevated lactate release can be explained by local hypoxia in some muscle fiber populations. The findings are discussed in relation to the enzymatic adaptations known to occur in the calf muscle tissue of these patients.