Identification of a common mutation in the carnitine palmitoyltransferase II gene in familial recurrent myoglobinuria patients.

Carnitine palmitoyltransferase (CPT) II deficiency is the most common inherited disorder of lipid metabolism affecting skeletal muscle. We have identified a missense mutation (Ser113Leu) in one patient with the classical muscular symptomatology. Transfection experiments in COS cells demonstrate that the mutation drastically depresses the catalytic activity of CPT II. The mutation results in normal synthesis but a markedly reduced steady-state level of the protein, indicating decreased stability of mutant CPT II. The Ser113Leu mutation is the most frequent cause of CPT II deficiency. The mutation can be detected easily by restriction analysis enabling molecular diagnosis of most patients and identification of heterozygous carriers.

Primary carnitine deficiency: heterozygote and intrafamilial phenotypic variation.

Two boys from different families had primary carnitine deficiency: one had cardiomyopathy and myopathy, and the other had hypoglycemia and myopathy but no cardiomyopathy. Uptake of carnitine by cultured fibroblasts was negligible in both patients. Vmax for carnitine transport was reduced to 50% of controls' value in the parents and one brother (who had hypertrophic cardiomyopathy) of the first patient. A brother of the second non-cardiopathic patient died at an early age with autopsy findings of a dilated cardiomyopathy and low cardiac carnitine. Autosomal recessive primary carnitine deficiency can express a variable phenotype in different families as well as within the same family. Heterozygotes can manifest heart involvement.

Calcium and magnesium content in fetuses at risk and prenecrotic Duchenne muscular dystrophy.

We measured calcium (Ca) and magnesium (Mg) content in muscles of fetuses at risk of Duchenne muscular dystrophy (DMD) and in a premature infant who later developed typical DMD. There was a three- to six-fold increase in muscle Ca in the fetuses and in the premature infant. In contrast to our previous reports of reduced muscle Mg in DMD children, there was an 18 to 57% increase of Mg in the fetuses at risk. Opaque and Ca-positive fibers, rarely observed in normal fetuses, were numerous in fetuses at risk and in the premature infant. No necrotic fibers were detected in the fetuses or the premature infant. These findings suggest that excessive Ca accumulation precedes necrosis in DMD. Other factors related to growth and development that occur after birth may trigger the necrosis that follows muscle Ca accumulation.

Generalized lysosomal storage in Yunis Varón syndrome.

Muscle biopsy in a neonate with features of Yunis Varón syndrome revealed a vacuolar myopathy with evidence of lysosomal storage disease. Similar vacuoles were also present in heart, cartilage, central nervous system and cultured fibroblasts. Although the histologic findings in the central nervous system resembled those of infantile acid maltase deficiency, the essayed lysosomal enzymes were normal. Chromatography of urine revealed abnormal bands of unidentified oligosaccharides. This is the first report of generalized storage disease in Yunis Varón syndrome. The biochemical defect is unknown.

Lysosomal glycogen storage with normal acid maltase: a familial study with successful heart transplant.

Lysosomal glycogen storage in muscle with normal acid maltase activity is a rare inherited condition characterized by cardiomyopathy, mental retardation and mild myopathy in males, but generally only cardiomyopathy in females. Three cases (index case, his sister and her son) are described in a family with at least two other affected members. The index case underwent a successful heart transplant. The sister has cardiac involvement, myopathic changes and mental impairment--to our knowledge the first report of multisystem involvement in a female. We propose that skeletal muscle should be examined in young patients with hypertrophic cardiomyopathy. Furthermore, female relatives of males with the disease should be investigated for cardiomyopathy; they would be excellent candidates for life-saving heart transplant, since myopathy and mental retardation, if clinically evident, are mild.

Dystrophin abnormalities in Duchenne and Becker dystrophy carriers: correlation with cytoskeletal proteins and myosins.

Characterization with a panel of six antibodies revealed abnormal dystrophin expression in 6 of 20 Duchenne muscular dystrophy (DMD) carriers examined, and in 5 of 12 Becker muscular dystrophy (BMD) carriers examined. The immunocytochemistry of muscle fibres was normal with five of the antibodies in two BMD carriers, but some muscle fibres were negative to the antibody directed against a portion of the dystrophin rod domain. Mosaicism was detected with all six antibodies in the other three BMD (but in only a small number of fibres) and in all DMD carriers muscles. Spectrin, vinculin and talin were immunolocalized in the same muscle specimens in order to assess membrane cytoskeletal integrity and to correlate their expression with that of dystrophin. These proteins, including vinculin, which was previously reported to be reduced in DMD patient muscles, were normally present on the surface of all dystrophin-deficient fibres. Muscle fibre types were characterized using monoclonal antibodies against fetal myosin and adult fast and adult slow myosin heavy chains. In both the DMD and BMD carriers, a significant reduction in type 2B fibres, as well as an increase in type 2C and fetal myosin-containing fibres was found - as has also been reported in DMD patients. Altered dystrophin expression was observed more frequently in type 2 than type 1 fibres. Dystrophin deficiency was found in a high percentage of type 2C fibres as well as in all fibres expressing fetal myosin; this suggests that dystrophin-deficient fibres are more susceptible to degeneration, leading to regeneration.

Cramps: a sign of motoneurone 'bistability' in a human patient.

In a patient suffering from severe long-lasting cramps, cramps were triggered in the triceps surae by volleys in homonymous Ia afferents (elicited by electrical stimulation or by tendon taps) and were interrupted by antidromic invasion and Renshaw inhibition of triceps surae motoneurones (evoked by a single maximal stimulation of motor axons). This result suggests that the mechanisms which generate the cramps are intrinsic to alpha-motoneurone somata. A similar on-off switching of a self-sustained motor discharge has been observed in the decerebrate cat and recognized to depend on 'bistability' of the motoneuronal membrane. We propose that the same mechanism may be at the origin of the cramp discharge.

Dystrophin characterization in BMD patients: correlation of abnormal protein with clinical phenotype.

We have investigated protein expression and genotype in 59 Becker muscular dystrophy (BMD) patients. The aim was to identify possible causes of the marked variability in phenotype in patients with similar deletions/mutations. The patients were examined neurologically and functionally and underwent Manual Muscle Testing. Dystrophin expression was analysed by immunohistochemistry and western blot using antibodies against six different segments of the protein. DNA mutations were investigated by PCR amplification of 30 exons. Based on dystrophin expression at the sarcolemma, two groups of patients were identified: group A (29 patients) with the classic patchy distribution of dystrophin and group B (30 patients) with absence or reduction of one or more dystrophin portions and variable, although mostly normal, expression of the other portions of the protein. Dystrophin molecular weight was normal or slightly reduced in group A and was variably reduced, generally conspicuously so, in group B. The quantity of dystrophin expressed varied markedly in both groups. The pattern of immunohistochemical staining in group B patients correlated with milder clinical phenotype, suggesting that small dystrophin molecules lacking a portion in the N-terminus or in the rod domain, are more functional than proteins with normal or slightly reduced molecular weight that display the BMD-typical patchy distribution at the sarcolemma.

Muscle pathology in ankylosing spondylitis.

The results of histological, histochemical and electron microscopic study of the muscular involvement in eight patients with Ankylosing Spondylitis (AS) are described. Muscle tissue from all these patients showed gross abnormalities of the sacrospinalis muscle, which had a 'targetoid core' appearance common to several myopathic and neurological diseases. In our opinion, there is always constant and early involvement of this muscle in AS. Several factors associated with the disease (chronic inflammation, multiple enthesopathies, muscle stiffness and bone lesions) could be responsible for this not fully understood aspect of AS. They may lead to a non specific damage of the nervous and/or muscular components of the spine in these patients.

Neurogenic arthrogryposis multiplex congenita: clinical and MRI findings.

A clinical and magnetic resonance imaging (MRI) study on a selected group of 11 children, with a diagnosis of neurogenic arthrogryposis multiplex congenita (AMC) based on clinical, electromyographic, and muscle biopsy findings, is presented to determine the extent of central nervous system involvement in AMC. Family history, pregnancy, perinatal problems, other abnormalities, and epileptic seizures were reviewed. Neurologic examination, electroencephalography, intellectual assessment, and MRI study both of spinal cord and brain were performed. The clinical and laboratory findings disclosed evidence of spinal cord lesions with involvement of anterior horn cell function in all patients, and impairment of cerebral function in 5 patients. MRI revealed spinal cord atrophy in 3 patients, diffuse atrophy in 2 patients, and involved thoraco-lumbar segments in 1 patient. Cranial MRI studies demonstrated features of developmental brain abnormalities in 3 patients, cortical frontal atrophy in 2, and was normal in 4. In neurogenic AMC patients, MRI examination of the spinal cord and brain may help to clarify the pathogenesis of the disease and is helpful for prognostic and therapeutic purposes.

Effect of total parenteral nutrition on the protein kinetics of patients with cancer cachexia.

AIMS AND BACKGROUND: The question of whether TPN is able to reverse lean body mass depletion in cachectic cancer patients and, in particular, its effect on protein kinetics is a matter of some controversy. This study investigates the impact of TPN on protein kinetics in patients with gastric cancer. METHODS: The study involved three patients with 14-30% weight loss. They were administered a TPN regimen including 33-40 kcal/kg/day and 1.4-1.7 g amino acid/kg/day. The protein metabolism was studied before and during TPN using a stable amino acid isotope. RESULTS: Whole body protein turnover and breakdown did not change during TPN, whereas whole body protein synthesis increased from 3.39 +/- 1.04 to 6.05 +/- 0.48 g/kg/day (P = 0.03). However, the net balance, which was slightly negative prior to TPN, became positive during nutritional support. In the skeletal muscle compartment the synthesis improved with TPN (from 9.38 +/- 2.6 nmol/100 mL/min to 35.95 +/- 3.4 nmol/100 mL/min; P = 0.0143), whereas breakdown did not change significantly. CONCLUSIONS: TPN triggers a positive metabolic response in cachectic cancer patients. Whether this results in a clinical benefit for the patient requires further investigation.

Motor neuron 'bistability'. A pathogenetic mechanism for cramps and myokymia.

In three patients suffering from chronic muscle cramps, spasms and myokymia, these involuntary contractions were triggered in the triceps surae, quadriceps, flexor carpi radialis or flexor digitorum by means of single or short-train stimulation of homonymous Ia afferents, elicited by electrical means or tendon taps. In some cases cramp was induced by the first afferent volleys; more often, however, continued stimulation produced stepwise recruitment of motor units (whose rhythmic firing was visible as myokymia in the muscle) until cramp developed. Cramps and myokymic discharges could usually be terminated by a single maximal stimulus to the motor axons (producing antidromic invasion and Renshaw inhibition of the motor neurons), or by short trains of volleys in inhibitory pathways from the skin. The fact that it was possible to induce myokymia and cramps by brief synaptic excitation and terminate them by antidromic invasion or synaptic inhibition, suggests that the mechanism generating these disturbances is intrinsic to alpha-motor neuron somata. Similar on-off switching of self-sustained motor discharges has been observed in the decerebrate cat and is known to depend on 'bistability' of the motor neuron membrane. We propose that a similar mechanism is responsible for discharges that produce cramp.

Partial rupture of the pectoralis major muscle in athletes.

Partial rupture of the pectoralis major muscle is quite a rare event in sports traumatology and information about its treatment is lacking in literature. In this paper the long term effect of conservative non-surgical treatment in two body-builders and one shot-putter is discussed, who reported the partial rupture while performing bench lifts with barbells. Functional recovery was evaluated a few years after the injury (from 4 to 7) with an isokinetic dynamometer (Cybex II, Lumex INC N.Y.), measuring maximal shoulder adduction-abduction torques at different angular speeds (60, 180, and 300 degrees/s). The results were compared with those of five healthy athletes practicing either body-building or weight-lifting. From the present study we conclude the following: the non-invasive treatment of a partial rupture of the pectoralis major muscle may produce almost complete functional recovery; in normal subjects adduction muscles are advantageous in comparison to the abduction ones.

Myoadenylate deaminase deficiency in a 5-year-old boy with intermittent muscle pain.

A 5-year-old boy with occasional pain of brief duration at the right or the left leg during exercise since the age of 4 years is reported. There was no weakness nor any other abnormality at the neurological examination. The serum creatine phosphokinase activity was elevated three times out of four. The electromyogram showed myopathic abnormalities in the biceps, it was normal in quadriceps and anterior tibial muscles. A quadriceps muscle biopsy was performed. There were no histological nor histochemical abnormalities at the routine techniques. However, a completely negative reaction with myoadenylate deaminase ( MADA ) stain was found. The MADA enzymatic activity in the muscle was very low. It is likely that the clinical syndrome is related to MADA deficiency.

Direct determination of leucine metabolism and protein breakdown in humans using L-[1-13C, 15N]-leucine and the forearm model.

We have used the forearm model to study protein metabolism in six normal healthy subjects in the fed state using L-[1-13C, 15N]-leucine as the substrate tracer. Deep venous and arterialized venous blood samples from the forearm were collected at 10-min intervals 2.5 h into a primed-continuous infusion of the dilabelled tracer. Arterialized venous blood was obtained using a 'hot-box' technique and forearm blood flow was measured by mercury strain-gauge plethysmography. The concentration and isotope enrichment of leucine and its metabolites, alpha-ketoisocaproic acid and CO2, in deep venous and arterialized venous blood were measured by gas chromatography-mass spectrometry and isotope ratio-mass spectrometry. The rates of leucine deamination and reamination were 388 +/- 24 (mean +/- SEM) and 330 +/- 23 nmol (100 ml)-1 min-1 respectively, whilst protein synthesis and breakdown rates were 127 +/- 11 and 87 +/- 10 nmol (100 ml)-1 min-1 respectively across the forearm in the fed state. We have demonstrated that the use of doubly labelled leucine as tracer and application of the mathematical model developed in this study, permits the comprehensive quantification of leucine kinetics including protein breakdown.

Influence of fasting on leucine and muscle protein metabolism across the human forearm determined using L-[1-13C,15N]leucine as the tracer.

1. We have used L-[1-13C,15N]leucine as the substrate tracer to study leucine and muscle protein metabolism across the forearm of eight normal fasting adults. 2. The rates of protein synthesis and breakdown, de- and re-amination of leucine, and the oxidative decarboxylation of its keto acid were calculated directly from the arteriovenous metabolite balances and isotope dilutions as described by the metabolic model. 3. The results were compared with those obtained previously when subjects were fed. The effects of fasting on protein and leucine metabolism were a significant decrease in protein synthesis from 127 (SEM 11; n = 6) to 70 (SEM 6; n = 12) nmol of leucine min-1 100 ml-1 of forearm tissue (P less than 0.001) and a marked decrease in leucine catabolism in the forearm muscle. 4. This model has demonstrated that each subject was in negative protein balance across the forearm during fasting while positive during feeding, the mean values being -29(SEM 5; n = 12) and +39(SEM 9; n = 6) nmol of leucine min-1 100 ml-1 of forearm tissue respectively. 5. These results are sufficiently encouraging to suggest a role for this model in future studies on muscle protein metabolism.

Effects of cachexia due to cancer on whole body and skeletal muscle protein turnover.

BACKGROUND: Data available in the literature regarding whole body protein (WBP) kinetics in patients with cachexia due to cancer are conflicting. Some authors have reported an increase of WBP synthesis and breakdown, whereas others have not found any significant changes; only a few researchers have investigated more compartments simultaneously. The main purpose of this study was to investigate WBP and skeletal muscle protein (SMP) turnover simultaneously in cachectic patients to understand better the mechanisms underlying the general wasting of the host present in cancer cachexia. METHODS: WBP and SMP synthesis and breakdown were studied in malnourished patients with advanced gastric carcinoma and in healthy volunteers. Protein turnover was evaluated in a postabsorptive state, using a model based on a primed constant infusion of L-[(2)H5] phenylalanine and L-[(2)H4]tyrosine, and by determining the isotopic enrichment and concentration in plasma during a plateau phase by gas chromatography and mass spectrometry. RESULTS: Rates of WBP synthesis and breakdown did not differ significantly between the two groups (whole body synthesis [WBS] of 4.35 +/- 0.2 g/kg/day and whole body breakdown [WBB] of 4.77 +/- 0.2 g/kg/day in the control group and WBS of 3.34 +/- 0.7 g/kg/day and WBB of 4.5 +/- 0.4 g/kg/day in the patient group). The skeletal muscle compartment of the patients showed a significantly lower synthesis compared with controls (patients, 9.6 +/- 1.8 nmol/100 mL/minute and control, 25.9 +/- 7.6 nmol/100 mL/minute; P < 0.05), whereas the breakdown was similar in the two groups. Such reduction in SMP synthesis in the gastric carcinoma patients resulted in a more negative net balance. CONCLUSIONS: Conflicting data in the literature may be accounted for by the different selection of patients and controls. Furthermore, WBP kinetics is the result of the metabolism of at least two compartments, the muscle and the nonmuscle compartments (including the tumor), which can change in opposite ways. In patients with cachexia due to cancer, the skeletal compartment appears to be the more compromised, with a significant decrease in SMP synthesis.

Functional evaluation of Duchenne muscular dystrophy: proposal for a protocol.

A protocol for the evaluation of functional activities in subjects with Duchenne muscular dystrophy (DMD) was designed. The aim of our study was to define objective clinical criteria for the evaluation both of the clinical status of the patient and of the natural history of the illness itself. A protocol with such criteria is particularly necessary when testing the efficacy of treatment. 43 still-ambulant children with DMD between the ages of 3.10 yr and 10.4 yr were examined. Of this number 19 children were evaluated every 4 months over a period of 12 months; of these 14 formed part of a randomized double blind trial with L-carnitine (1.2-1.8 g/day) versus placebo.

Needle biopsy for muscle diagnosis and research: an Italian experience.

We report our experience of needle biopsy over the past two and a half, viz. 395 cases. We find that the technique meets the needs of diagnosis and research very satisfactorily. We describe its advantages over open biopsy and recommend it in preference to the latter, provided that the necessary technical skill for processing the sample is available.

Rate of protein synthesis in skeletal muscle of normal man and patients with muscular dystrophy: a reassessment.

1. Quadriceps muscle protein synthetic rate has been determined in healthy subjects in the post-absorptive (n = 18) and fed (n = 10) states and in patients with a variety of myopathies, by analysis of the enrichment of serial muscle biopsies taken during primed continuous infusion of L-[1-13C]leucine. 2. Quadriceps protein synthetic rates in normal subjects were (mean +/- SD) 0.046 +/- 0.012 and 0.075 +/- 0.014%/h in the post-absorptive and fed states respectively. These results are significantly lower than we previously reported (M. J. Rennie et al., Clinical Science, 1982, 63, 519-523 [1]) but show the same relative differences of direction and magnitude, confirming the effects of feeding previously reported. In patients with muscular dystrophy, muscle protein synthetic rate was, as previously reported [1], much lower in the fed state than in normal subjects. A new finding is that for patients with myotonic dystrophy the rate is also depressed in the post-absorptive state. 3. We suggest that the present estimates in post-absorptive and fed normal subjects be used as reference values for quadriceps mixed muscle protein synthetic rate.