Tofogliflozin: a highly selective SGLT2 inhibitor for the treatment of type 2 diabetes.

Diabetes mellitus impacts a substantial number of people worldwide and despite numerous antidiabetic medications available, approximately half of the drugs do not attain their recommended target, glycated hemoglobin (HbA1c). Recently, the kidney and its role in glucose reabsorption through the sodium/glucose cotransporter 2 (SGLT2) has been the target for novel antidiabetic treatments. Pharmacologic inhibition of SGLT2 in patients with diabetes results in increased urinary glucose excretion and decreased blood glucose levels, decreasing HbA1c levels. Tofogliflozin is the most selective SGLT2 inhibitor, with HbA1c reductions ranging from -0.44% to -0.99% throughout clinical studies, and it is well tolerated with a low rate of drug-related adverse events. Tofogliflozin has demonstrated efficacy and safety as monotherapy or as add-on to various antidiabetic agents, and it is currently undergoing phase IV clinical studies in Japanese patients with diabetes on background insulin therapy. Tofogliflozin is currently approved in Japan for use in patients with type 2 diabetes at a dose of 20 mg orally once daily in the morning, either before or after breakfast.

Improving the damage accumulation in a biomechanical bone remodelling model.

We extend, reformulate and analyse a phenomenological model for bone remodelling. The original macrobiomechanical model (MBM), proposed by Hazelwood et al. [J Biomech 2001; 34:299-308], couples a population equation for the cellular activities of the basic multicellular units (BMUs) in the bone and a rate equation to account for microdamage and repair. We propose to account for bone failure under severe overstressing by incorporating a Paris-like power-law damage accumulation term. The extended model agrees with the Hazelwood et al. predictions when the bone is under-stressed, and allows for suitably loaded bones to fail, in agreement with other MBM and experimental data regarding damage by fatigue. We numerically solve the extended model using a convergent algorithm and show that for unchanging loads, the stationary solution captures fully the model behaviour. We compute and analyse the stationary solutions. Our analysis helps guide additional extensions to this and other BMU activity based models.

Peripheral neuropathies in Waldenström's macroglobulinaemia.

OBJECTIVE: We sought to determine the prevalence, clinical features, and laboratory characteristics of polyneuropathies in Waldenström's macroglobulinaemia (WM), a malignant bone marrow disorder with lymphocytes that produce monoclonal IgM. METHODS: We prospectively studied 119 patients with WM and 58 controls. Medical history was taken, and neurological examinations, electrodiagnostic tests, and serum studies were performed by different examiners who were blinded to results except the diagnosis of WM. RESULTS: Polyneuropathy symptoms, including discomfort and sensory loss in the legs, occurred more frequently (p<0.001) in patients with WM (47%) than in controls (9%). Patients with WM had 35% lower quantitative vibration scores, and more frequent pin loss (3.4 times) and gait disorders (5.5 times) than controls (all p<0.001). Patients with IgM binding to sulphatide (5% of WM) had sensory axon loss; those with IgM binding to myelin associated glycoprotein (MAG) (4% of WM) had sensorimotor axon loss and demyelination. Patients with WM with IgM binding to sulphatide (p<0.005) or MAG (p<0.001) had more severe sensory axon loss than other patients with WM. Demyelination occurred in 4% of patients with WM with no IgM binding to MAG. Age related reductions in vibration sense and sural SNAP amplitudes were similar ( approximately 30%) in WM and controls. CONCLUSIONS: Peripheral nerve symptoms and signs occur more frequently in patients with WM than controls, involve sensory modalities, and are often associated with gait disorders. IgM binding to MAG or sulphatide is associated with a further increase in the frequency and severity of peripheral nerve involvement. Age related changes, similar to those in controls, add to the degree of reduced nerve function in patients with WM.

Treatment of IgM antibody associated polyneuropathies using rituximab.

OBJECTIVES: Polyneuropathies with associated serum IgM antibodies are often difficult to treat. Rituximab is a monoclonal antibody directed against the B cell surface membrane marker CD20. Rituximab eliminates B cells from the circulation, and, over time, could reduce cells producing autoantibodies. This study tested the ability of rituximab to produce changes in serum antibody titres, and improvement in strength, in patients with neuromuscular disorders and IgM autoantibodies. METHODS: Over a period of two years, the authors evaluated changes in strength, measured by quantitative dynamometry, and concentrations of several types of serum antibodies in patients with polyneuropathies and serum IgM autoantibodies. Twenty one patients treated with rituximab were compared with 13 untreated controls. RESULTS: Treatment with rituximab was followed by improved strength (an increase of mean (SEM) 23% (2%)of normal levels of strength), a reduction in serum IgM autoantibodies (to 43% (4%) of initial values), and a reduction in total levels of IgM (to 55% (4%) of initial values). There was no change in levels of serum IgG antibodies. There were no major side effects, even though B cells were virtually eliminated from the circulation for periods up to two years. CONCLUSIONS: In patients with IgM autoantibody associated peripheral neuropathies, rituximab treatment is followed by reduced serum concentrations of IgM, but not IgG, antibodies, and by improvement in strength. Additional studies, with placebo controls and blinded outcome measures, are warranted to further test the efficacy of rituximab treatment of IgM associated polyneuropathies.

Anti-myelin associated glycoprotein antibodies: variability in patterns of IgM binding to peripheral nerve.

We previously found that serums with anti-sulfatide antibodies have several different patterns of binding to neural tissue. In this study, we asked whether serums with anti-myelin associated glycoprotein (MAG) antibodies also have similar variations in patterns of tissue binding. We examined binding to peripheral nerve in 49 serums with IgM anti-MAG antibodies and 13 serums with IgM anti-sulfoglucuronyl paragloboside (SGPG) antibodies but no MAG binding. We correlated patterns of binding with titers of IgM binding to MAG and SGPG measured by ELISA methods. Our results show that IgM in most anti-MAG serums stained areas of non-compact myelin, including the periaxonal and outer myelin membranes and Schmidt-Lanterman incisures. However, other patterns included IgM binding to areas of compact myelin and to non-myelin structures including axons and endoneurial macrophages. IgM in anti-SGPG serums bound to axons or macrophages, but rarely to myelin-related structures. A total of 11/62 (18%) of serums had IgM binding to axons, six with anti-MAG antibodies and five with anti-SGPG antibodies. The majority of these serums (73%) had SGPG titers greater than MAG titers when measured by ELISA. We conclude that anti-MAG serums have several different binding patterns to neural tissue, including axonal binding, especially when anti-MAG antibodies cross-react with SGPG. These different binding patterns may relate to the ability of anti-MAG serum IgM to bind both MAG and SGPG or to other molecules with a sulfated glucuronic acid epitope that are present in peripheral nerve.

Serum antibodies to heparan sulfate glycosaminoglycans in Guillain-Barré syndrome and other demyelinating polyneuropathies.

We tested for serum antibodies to glycosaminoglycans (GAGs), including heparan sulfate, in patients with Guillain-Barré syndrome (GBS) and other disorders. We used ELISA methods that optimize immunoglobulin binding to carbohydrate antigens to measure serum antibodies to heparan sulfate GAGs in GBS, and control neuromuscular and immune disorders. We found serum IgM or IgG antibodies to heparan sulfate GAGs in 34% of patients with GBS. Serum IgM binding to heparan sulfate GAGs was also found in some chronic demyelinating polyneuropathies, with the highest frequency (33%) in patients with IgM anti-MAG M-proteins. Antibodies to heparan sulfate GAGs were rare (1%) in control serums from patients with other disorders. This result is the first demonstration of high titer serum antibodies to a specific antigen in a substantial group of, and with some specificity for, patients with the classically described GBS syndrome of acute-onset, motor-sensory polyneuropathy with demyelinating features.

IgM anti-sulfatide autoantibodies: patterns of binding to cerebellum, dorsal root ganglion and peripheral nerve.

Anti-sulfatide antibodies are associated with polyneuropathies having a prominent sensory component, but with variable degrees of motor and sensory loss, gait dysfunction and demyelination. In this study, we asked whether patterns of IgM binding to neural tissue in anti-sulfatide serums also demonstrated heterogeneity. We used immunocytochemical methods to examine IgM binding to peripheral nerve, dorsal root ganglion, and cerebellum in 41 serums with high titers of IgM anti-sulfatide antibodies. Our results showed that there were several different patterns of IgM binding to neural tissues in anti-sulfatide serums. In peripheral nerve the most common targets of IgM were axons, resident macrophages or Schwann cell cytoplasm. In the cerebellum, IgM bound to neuronal nuclei, white matter, or neuropil in molecular and granule cell layers. There was little binding of IgM to structures in the dorsal root ganglion. Patterns of IgM binding to peripheral nerve and cerebellum were related. Binding to neuronal nuclei in the cerebellum was usually found in serums that recognized peripheral nerve axons or macrophages. Serums with binding of IgM to cerebellar white matter usually recognized Schwann cell cytoplasm. We conclude that IgM anti-sulfatide antibodies may have several different tissue binding patterns in the peripheral and central nervous systems. These differences may be related to the variation in clinical neuropathy syndromes associated with apparently similar anti-sulfatide antibodies.

Multifocal motor neuropathy. Serum IgM anti-GM1 ganglioside antibodies in most patients detected using covalent linkage of GM1 to ELISA plates.

IgM anti-GM1 antibodies occur with increased frequency in the serum of patients with multifocal motor neuropathy (MMN). We tested the ability of serum IgM from patients with MMN to bind to GM1 ganglioside covalently bound to secondary amino groups on ELISA plates (Co-GM1). The Co-GM1 technique detected high titer (> 1,800), selective, serum IgM binding to GM1 ganglioside in 85% of our MMN patients (23/27), a significantly greater frequency compared with figures of 37% and 52% found using our previous testing methods. Selective IgM anti-GM1 antibodies showed disease specificity. The only other patients with selective, high-titer IgM anti-GM1 antibodies had either chronic motor neuropathy without conduction block or acute immune neuropathy in China. No patient from the amyotrophic lateral sclerosis, chronic inflammatory demyelinating polyneuropathy, Guillain-Barré, or systemic immune disorder control groups had selective IgM anti-GM1 antibodies at titers greater than 1,800 detected using Co-GM1 ganglioside as ELISA antigen. Titers of IgM anti-GM1 antibodies in MMN (averaging 31,000 +/- 15,000) were more than fourfold higher with Co-GM1 than with previous anti-GM1 assay methods, using conventional ELISA plates with GM-1 antigen alone (7,200 +/- 4,400) or in a lipid environment (3,600 +/- 1,300). We conclude that using ELISA testing with Co-GM1 antigen, serum anti-GM1 autoantibodies are a useful marker for MMN, because they are present in 85% of MMN patients and, at titers greater than 1,800, have strong specificity for immune-mediated motor neuropathies.

A skeletal muscle-specific form of decorin is a target antigen for a serum IgM M-protein in a patient with a proximal myopathy.

We described a myopathy in a patient, B.J., with Waldenström's macroglobulinemia and a serum IgM M-protein that binds to a glycoprotein located in skeletal muscle endomysium. The objective of this study was to identify and characterize the endomysial antigen. The antigenic protein was purified using sequential differential solubility steps, size exclusion chromatography, and anion exchange chromatography. We subjected the deglycosylated protein and several proteolytic fragments to sequence analysis. We used immunohistochemistry, Western blot, and ELISA to study the binding of the IgM monoclonal and the anti-decorin core protein antibodies to the muscle antigen before and after deglycosylation. Sequence analysis revealed amino acid residues with 100% homology to human decorin. The anti-decorin core protein antibody bound to the purified antigen by ELISA and Western blot methods and bound to skeletal muscle endomysium in a histologic pattern similar to the human IgM M-protein from the patient B.J. Deglycosylation experiments revealed that the human IgM M-protein from B.J. serum recognized a carbohydrate epitope on decorin, probably containing chondroitin sulfate. A skeletal muscle-specific form of the proteoglycan decorin, with a chondroitin sulfate-like epitope, is a target antigen for the IgM M-protein in our patient with Waldenström's macroglobulinemia and a myopathy. These results are the first demonstration of the binding of a human IgM M-protein to an endomysial antigen. The anti-decorin IgM may be relevant to disease pathogenesis because the patient studied had a myopathy with IgM monoclonal antibodies deposited in the muscle endomysium.

Multifocal motor neuropathy: serum IgM binding to a GM1 ganglioside-containing lipid mixture but not to GM1 alone.

IgM anti-GM1 antibodies are associated with motor neuropathy syndromes, including multifocal motor neuropathy (MMN). We compared the ability of serum IgM from patients with multifocal motor neuropathy to bind to GM1 ganglioside alone and to GM1 as a component of a lipid mixture that also contained galactocerebroside and cholesterol (GGC). Our results showed that high-titer selective serum IgM binding to GGC has strong specificity for MMN. Further, over 40% more serums from patients with MMN have high-titer serum IgM binding to GGC than to GM1 alone. The specific composition and structure of the lipid mixture altered the ability of serum IgM to bind to GM1 ganglioside. Substitutions of other lipids for galactocerebroside or cholesterol could completely inhibit the antibody binding. We conclude that serum IgM anti-GGC autoantibodies have specificity for MMN and their binding is strongly influenced by the lipid environment of GM1 ganglioside.

Congenital muscular dystrophy syndromes distinguished by alkaline and acid phosphatase, merosin, and dystrophin staining.

Congenital muscular dystrophy syndromes are characterized by congenital weakness, contractures, and dystrophic features on muscle biopsy. However, these syndromes are often difficult to diagnose precisely because their clinical and pathologic characteristics are not specific and resemble changes in other myopathies. We examined muscle biopsies from 20 children with a congenital muscular dystrophy syndrome. Disease controls with dystrophies or other myopathies (n=19) and normal individuals (n=15) were studied for comparison. In each biopsy we determined (1) numbers of muscle fibers with alkaline phosphatase (AlkP) staining, (2) numbers of acid phosphatase-(AcP) positive cells, (3) dystrophin levels by immunocytochemistry, and (4) the distribution of merosin and laminin-A staining. A ratio of AcP:AlkP staining was calculated for each biopsy. In nine patients with congenital muscular dystrophy (younger than 4 years of age) with normal dystrophin, the AcP:AlkP ratio was low (0.09 +/- 0.03). In contrast, in Duchenne muscular dystrophy, the AcP:AlkP ratio was 15 times higher (1.6 +/- 0.04, p=0.001). The three children with congetial muscular dystrophy syndromes and reduced dystrophin and one child with facioscapulohumeral dystrophy had AcP:AlkP ratios in the range of Duchenne muscular dystrophy patients (2.4 +/- 1.4). Low Ac:AlkP ratios were related to relative absence of AcP-positive cells. Merosin staining was absent in 5 of the 17 congenital muscular dystrophy biopsies tested. None of the 5 children with merosin-negative but all 12 with merosin-positive stains walked (p=0.0002). We conclude that a pattern of few AcP-positive cells in the setting of numerous AlkP staining muscle fibers has specificity for congenital muscular dystrophy syndromes and provides histopathologic support for the diagnosis. Reduced merosin in muscle predicts more severe weakness and long-term disability.

Treatable gait disorder and polyneuropathy associated with high titer serum IgM binding to antigens that copurify with myelin-associated glycoprotein.

We studied clinical and electrodiagnostic features of 9 patients with very high titers (> 1:10,000) of serum IgM binding to a CNS myelin antigen (CMA) preparation that copurified with myelin-associated glycoprotein (MAG). We found that 8 of the 9 patients had a combined syndrome of gait ataxia and polyneuropathy (GAPN) with late-age onset (mean = 70 years of age). In the 8 GAPN patients progressive difficulty with ambulation led to significant functional disability and frequent falling. Examination showed a wide-based unsteady gait, especially when standing still or turning. There was mild-to-moderate distal sensory loss with involvement of joint position sense only in the toes. Motor changes, when present, were mild and mainly involved distal leg musculature. Treatment of 5 GAPN patients resulted in clear improvement of 2 after intravenous human immunoglobulin and of 3 others after other immunodulating agents. Immune-mediated GAPN syndromes with high titers of serum IgM binding to CMA appear to be treatable causes of gait disorders in older patients.

Nerve-dependent recovery of metabolic pathways in regenerating soleus muscles.

The metabolic recovery potential of muscle was studied in regenerating soleus muscles of young adult rats. Degeneration was induced by subfascial injection of a myotoxic snake venom. After regeneration for selected periods up to 2 weeks, samples of whole muscle were analysed for hexokinase (EC 2.7.1.1), phosphofructokinase (EC 2.7.1.11), lactate dehydrogenase (EC 1.1.11.27), adenylokinase (EC 2.7.4.3), creatine kinase (EC 2.7.3.2), malate dehydrogenase (EC 1.1.11.37), citrate synthase (EC 4.1.3.7) and beta-hydroxyacyl CoA dehydrogenase (EC 1.1.1.35). Lactate dehydrogenase, adenylokinase, malate dehydrogenase and beta-hydroxyacyl CoA dehydrogenase were also measured in individual fibres of muscle regenerating up to 4 weeks. We found that in the presence of nerve there was complete recovery of muscle metabolic capacity. However, there were differences in the rate of recovery of the activity of enzymes belonging to different energy-generating pathways. Lactate dehydrogenase, an enzyme representing glycolytic metabolism, reached normal activity immediately upon myofibre formation, only 3 days after venom injection, while oxidative enzymes required a week or more to reach normal activity levels. The delay in oxidative enzyme recovery coincided with physiological parameters of reinnervation. Therefore, to further test the role of nerve on the metabolic recovery process, muscle regeneration was studied following venom-induced degeneration coupled with denervation. In the absence of innervation, most enzymes failed to recover to normal activity levels. Lactate dehydrogenase was the only enzyme to achieve normal levels, and it did so as rapidly as in innervated-regenerating soleus muscles. The remainder of the glycolytic enzymes and the high energy phosphate enzymes recovered only partially. Oxidative enzymes showed no recovery and were severely reduced in the absence of reinnervation.(ABSTRACT TRUNCATED AT 250 WORDS)

Anti-MAG antibodies: major effects of antigen purity and antibody cross-reactivity on ELISA results and clinical correlation.

There is controversy regarding the relationship of polyneuropathy syndromes to the presence of serum antibody binding to myelin-associated glycoprotein (MAG). Using standard ELISA methodology, we identified 74 sera that appeared to have high titers of IgM binding to MAG and found that only 34% of these sera stained MAG using Western blot methodology. Follow-up studies showed that two factors greatly influence concordance between ELISA and Western blot testing for anti-MAG antibodies. Sera with high titers of binding to both MAG and histone H3 identified by ELISA rarely stain MAG on Western blot. In addition, sera analyzed by ELISA often bind to impurities in the semipure MAG that is frequently used in ELISA assays. Further purifications to separate MAG from other contaminants improved concordance between ELISA and Western blot results to 85% to 90% in a retrospective analysis, as well as in a prospective study of 49 additional sera. Patients with a polyneuropathy and serum IgM binding to MAG preparations by ELISA but not by Western blot methodology had several different clinical syndromes, including gait disorders and asymmetric motor neuropathies. Patients with IgM binding to MAG by both assay methods usually had a distal, sensory-motor, symmetric polyneuropathy with some features of demyelination on electrodiagnostic testing.

Whole-body energy metabolism and skeletal muscle biochemical characteristics.

A low metabolic rate for a given body size and a low fat versus carbohydrate oxidation ratio are known risk factors for body weight gain, but the underlying biological mechanisms are poorly understood. Twenty-four-hour energy expenditure (24EE), sleeping metabolic rate (SMR), 24-hour respiratory quotient (24RQ), and forearm oxygen uptake were compared with respect to the proportion of skeletal muscle fiber types and the enzyme activities of the vastus lateralis in 14 subjects (seven men and seven women aged 30 +/- 6 years [mean +/- SD], 79.1 +/- 17.3 kg, 22% +/- 7% body fat). The following enzymes were chosen to represent the major energy-generating pathways: lactate dehydrogenase (LDH) and phosphofructokinase (PFK) for glycolysis; citrate synthase (CS) and beta-hydroxyacl-coenzyme A dehydrogenase (beta-OAC) for oxidation; and creatine kinase (CK) and adenylokinase (AK) for high-energy phosphate metabolism. Forearm resting oxygen uptake adjusted for muscle size correlated positively with the proportion of fast-twitch muscle fibers (IIa: r = .55, P = .04; IIb: r = .51, P = .06) and inversely with the proportion of slow oxidative fibers (I: r = -.77, P = .001). 24EE and SMR adjusted for differences in fat-free mass, fat mass, sex, and age correlated with PFK activity (r = .56, P = .04 and r = .69, P = .007, respectively). 24RQ correlated negatively with beta-OAC activity (r = -.75, P = .002). Our findings suggest that differences in muscle biochemistry account for part of the interindividual variability in muscle oxygen uptake and whole-body energy metabolism, ie, metabolic rate and substrate oxidation.

Effects of microgravity and tail suspension on enzymes of individual soleus and tibialis anterior fibers.

Selected enzymes of energy metabolism were measured in random individual fibers of soleus and tibialis anterior (TA) muscles from rats exposed for 2 wk to spaceflight (F) aboard COSMOS 2044 or tail suspension (T) and from synchronous controls. Average size of soleus fibers (dry weight per unit length) was reduced 37% in F and T fibers; there was little change in TA fibers. Enzyme changes were more pronounced in soleus than in TA fibers. Three enzymes characteristic of fast-twitch muscles, pyruvate kinase, glycerol-3-phosphate dehydrogenase, and 1-phosphofructokinase, were elevated in F and T soleus fibers, but changes in phosphofructokinase were not statistically significant. 3-Ketoacid-CoA transferase, characteristic of slow-twitch muscles, did not change significantly in either F or T fibers. Hexokinase, usually moderately higher in slow- than in fast-twitch muscles, increased markedly in both F and T fibers. In TA fibers analyzed for hexokinase, malate dehydrogenase, phosphohexoisomerase, and pyruvate kinase, only hexokinase and malate dehydrogenase showed significant changes. Hexokinase increased 83% in one of two T muscles. Enzyme data for TA fibers typed by myosin adenosinetriphosphatase were more informative: phosphofructokinase, phosphorylase, and glycerol-3-phosphate dehydrogenase were increased in type IIb fibers of either F or T muscles or both. Malate dehydrogenase was not changed in fibers of any type in either F or T muscle.

Metabolic transitions in rat jaw muscles during postnatal development.

The program of acquisition of adult metabolic phenotypes was studied in three jaw muscles in order to determine the link between muscle metabolism and functional development. During early postnatal stages, there were similar transitions in the masseter, anterior digastric, and internal pterygoid muscles with respect to fiber growth, fiber type composition, and whole muscle energy metabolism. Oxidative capacity, as judged by the activities of the enzymes succinate dehydrogenase (SDH), malate dehydrogenase (MDH), and beta-hydroxyacyl CoA dehydrogenase (beta OAC), rose sharply after birth to reach near maximal levels by 3 weeks. The capacities for glycolytic metabolism represented by lactate dehydrogenase (LDH), and for high-energy phosphate metabolism represented by adenylokinase (AK) and creatine kinase (CK) activities, rose gradually, not reaching peak values until 6 weeks or later. Thus, the maturation of oxidative metabolism preceded that of glycolytic metabolism in the developing jaw muscles. This was documented for individual fibers in the masseter muscle. Differential metabolic maturation among the jaw muscles was evident beyond 3 weeks. All three jaw muscles attained their specific adult fiber-type profile by about 6 weeks. This maturation program differed from that of hindlimb muscles [Nemeth et al., J Neurosci 9:2336-2343, 1989] and diaphragm muscle [Kelly et al., J Neurosci 11:1231-1242, 1991], reflecting their differential energy demands for contractile performance.

Metabolic response to a high-fat diet in neonatal and adult rat muscle.

Neonatal rats were exposed to a high-fat low-carbohydrate diet to determine how substrate availability might affect the metabolic phenotype of muscle. Mixed-fiber homogenates of extensor digitorum longus, soleus, and diaphragm muscles were assayed for beta-hydroxyacyl-CoA dehydrogenase (beta-OAC), succinate dehydrogenase, malate dehydrogenase, lactate dehydrogenase, phosphofructokinase (PFK), adenylokinase, and creatine kinase. The three muscles showed significant increases in enzyme activity for fatty acid oxidation (beta-OAC) in weaned neonatal rats maintained on the high-fat diet compared with normal weaned controls. This effect persisted for 6 wk of the diet. The other consistent metabolic change was a decrease in PFK. Adult animals subjected to the same diet had similar increases in fatty acid oxidation and a fall in PFK after 1 wk, with most of these changes persisting for the 4 wk of the diet. Examination of individual fibers revealed enzyme changes in fibers of all types, but with the largest effect in type IIb fibers. The data indicate that both adult and neonatal muscles are similarly capable of adjusting their energy metabolism in response to dietary factors.

IMP response: an indicator of metabolic stress in working muscle.

An in vitro system for the combined biochemical and physiologic analysis of striated muscle has been developed. It is hoped that its eventual application will be to the evaluation of human muscle disease. To test the method's usefulness in evaluating metabolic defects, we used iodoacetate to induce a glycolytic defect in animal muscle. This produced the expected effects of precipitous fatigue on repetitive stimulation, increased inosine monophosphate (IMP) and decreased adenosine triphosphate (ATP) and phosphocreatine; in addition, the severity of the glycolytic block was directly related to the amount of IMP produced per gram twitch tension.

Inosine monophosphate production is proportional to muscle force in vitro.

An in vitro system of muscle stimulation has been developed that combines physiologic measurements with biochemical evaluation. In the rat epitrochlearis muscle, 2 minutes of stimulation at 4 Hz results in a marked elevation of inosine monophosphate (IMP) levels. The amount of IMP generated is proportional to the amount of force developed. Depriving the muscle of oxygen results in an increased amount of IMP generation. This model provides direct confirmation of similar findings in the human forearm exercise test.