Activity of some proteolytic enzymes in normal and dystrophic human muscle.

1. The following proteolytic enzymes were measured in muscles of control subjects and patients with muscular dystrophies and related neuromuscular diseases: an elastase-like enzyme, carboxypeptidase A, carboxypeptidase B and pyroglutamyl peptidase. 2. Elastase-like enzyme and carboxypeptidase B did not show significant alterations in various disease conditions that were examined. 3. Carboxypeptidase A was moderately elevated in dystrophic as well as other diseased muscles. 4. Pyroglutamyl peptidase was not markedly altered in any disease condition except that is was slightly lower in dystrophic muscles.     

Assay of ornithine carbamoyltransferase activity in human liver using carbon-labeled ornithine and thin-layer chromatography

Ornithine carbamoyltransferase (EC 2.1.3.3) activity in human liver homogenates has been measured using 14C-labeled ornithine and unlabeled carbamoyl phosphate. A thin-layer chromatographic (TLC) procedure is used to separate the radioactive substrate and product, ornithine and citrulline, respectively, and the regions of the chromatogram corresponding to ornithine and citrulline are cut out and counted in a liquid scintillation spectrophotometer. The method has the following advantages: (1) the radioactive substrate ornithine is more stable in solution than carbamoyl phosphate, (2) 14C-labeled ornithine is available in higher specific activity than carbamoyl phosphate, (3) all radioactivity may be accounted for by using the TLC system, (4) the developed thin-layer chromatogram is stable indefinitely, (5) in contrast to colorimetric assays, other compounds in the raction mixture do not interfere with the citrulline determination, and (6) most importantly, the rate of the enzyme reaction at various time intervals can be determined by taking aliquots from the same incubation tube.     

Radioenzymatic assay of platelet serotonin, dopamine and norepinephrine in subjects with normal and increased serotonin production

Platelet serotonin, dopamine and norepinephrine concentrations were determined with sensitive and specific radioenzymatic methods in normal volunteers and in sick subjects with both normal and increased serotonin production. In the assays, a 3H-labeled methyl group is transferred from [3H]S-adenosyl methionine to the monoamine under investigation by a semi-purified enzyme. The platelet serotonin concentration (expressed as pmol serotonin/mg platelet protein) was significantly higher in the patients with increased serotonin production from carcinoid tumors (5400 +/-0 500) than in the normal volunteers (900 +/- 100) or the sick subjects with normal serotonin production (900 +/- 100). Although eight of the subjects with increased serotonin production had symptoms of the carcinoid syndrome such as flushing and diarrhea, two of the subjects with increased serotonin production did not have these symptoms. The radioenzymatic method of platelet serotonin measurement was as effective as a conventional fluorometric technique for serum serotonin in detecting patients with serotonin overproduction from carcinoid tumors, and had the advantages of being more sensitive and specific. The great sensitivity and specificity of the radioenzymatic techniques suggest that it might be useful in evaluating disease states in which platelet serotonin is decreased. Using these techniques we found that dopamine and norepinephrine were also present in platelets, although in much lower amounts than serotonin. Despite the markedly increased serotonin concentration in the platelets of subjects with serotonin overproduction from carcinoid tumors, there was no alteration in the dopamine or norepinephrine concentration of their platelets.     

A radioenzymatic assay for free and conjugated normetanephrine and octopamine excretion in man

A radioenzymatic method for the measurement of free and conjugated normetanephrine (NMN) and octopamine (OCT) is described for human urine. The assay is based on the conversion of NMN or OCT to radiolabeled metanephrine (MN) or synephrine (SYN) by phenylethanolamine-N-methyl transferase (PNMT), using tritium-labeled S-adenosyl-methionine [³H]SAM as methyl donor. Thin-layer Chromatographie separation yields an assay of high specificity. The sensitivity of the assay is 5 and 2.5 pg of NMN or OCT, respectively.The normal value of free and conjugated NMN was found to be 23 ± 13 and 102 ± 49 ng/mg creatinine. Four patients with pheochromocytoma had highly increased levels of free and conjugated NMN. The urinary excretions of free and total octopamine were 5.7 ± 2.8 and 34.8 ± 16.6 ng/mg of creatinine, respectively, in normal patients.     

Glyoxalase enzyme system in human muscular dystrophy.

Glyoxalase I and glyoxalase II activities were determined in skeletal muscle of control subjects and of patients with Duchenne dystrophy, other major forms of muscular dystrophies and certain neuromuscular disorders. The glyoxalase I activity was normal in all diseases examined except in Duchenne and limb girdle types of muscular dystrophy, where it showed a significant moderate decrease. The glyoxalase II activity in normal human muscle was very low, and the activity was unaltered in muscle of patients with Duchenne and other major forms of muscular dystrophies and spinal muscular atrophy. The selective decrease of glyoxalase I activity in recessively inherited muscular dystrophies may have some relevance to some phases of these disease processes.     

Long term results of peripheral conditioning stimulation as an analgesic measure in chronic pain.

In the present study 123 patients with chronic pain, consecutively referred for symptomatic pain treatment, were given peripheral conditioning stimulation as an analgesic measure and were followed for 2 years or till they terminated the treatment. The stimulation was either conventional transcutaneous nerve stimulation (TNS) [35] of mainly cutaneous afferents with high frequency (10-100 Hz) or acupuncture-like TNS [11] where muscle nerves are activated at a low repetition rate (1-4 Hz) with small trains of stimuli. The follow-up showed that 55, 41 and 31% of the patients continued the treatment after 3, 12 and 24 months, respectively. About 30% of the patients had to use acupuncture-like TNS to get useful analgesia, defined as a desire of the patient to continue stimulation treatment. Three-quarters of the successfully relieved patients reported more than 50% pain relief as measured from visual analogue scales and half of these reported an increased social activity and a decrease of analgesic drug intake by more than 50%. Psychogenic and visceral pains were less suitable for TNS treatment. It is concluded that peripheral conditioning stimulation is a valuable therapy in cases of chronic pain and that both conventional and acupuncture-like TNS should be tried before considering implantable devices or destructive surgery.     

A solid state fixed wavelength colorimeter for continuous-flow analyses

A completely solid state colorimeter has been built using a subminiature light emitting diode (LED) as the fixed wavelength light source and a PIN silicon photodiode as the photodetector. An LED has a very stable, narrow bandwidth light emission, thereby eliminating the need for a reference circuit and a monochromator. The LED used has an estimated useful life greater than 30 years, is not fragile, consumes little power, emits scant heat and is of low cost. The photodetector is a photodiode whose output current drives an FET input operational amplifier coupled to a logarithmic operator whose output voltage is rectilinear with sample absorbance. The output can drive a three lead (Technicon) or a two lead (Heathkit) strip chart recorder. The sample cuvette can be either a now cell for continuous flow methods or a discrete sample cuvette. The application of this colorimeter to automated serum uric acid analyses exemplifies the proof of principle. With currently available LED wavelengths it should be also possible to measure phosphate, hemoglobin, urea, carbon dioxide, cholesterol, total protein, calcium, amylase and triglycerides.