Determination of free and total cholesterol in human bile samples using an enzyme electrode

A reliable cholesterol electrode was assembled with a Clark type oxygen electrode and cholesterol oxidase immobilised on a nylon net fixed on the electrode surface. Determination of free (unesterified) and total (esterified and unesterified) cholesterol in human bile samples was performed. Results were compared with the enzymatic-spectrophotometric procedure based on the Roeschlau method. The electrode has a usable life of more than two weeks, and more than 300 determinations could be performed. The CV values for free and total cholesterol in bile samples is 5-6%. The comparison between the proposed procedure and the spectrophotometric procedure is fair for the half of the results (delta less than 5%) and acceptable in the other half (delta less than 25%).     

An improved gas-chromatographic method for the determination of sulfated and unsulfated bile acids in serum.

This study has been aimed at improving some steps in the gas-liquid chromatographic determination of sulfated bile acids. The best conditions for the enzymatic hydrolysis with cholylglycine hydrolase of sulfolithocholylglycine and sulfolithocholyltaurine are described. Recoveries of more than 85% were obtained after prolonging the incubation to 12 h. A single-step procedure for solvolysis and methylation of bile acids was achieved by using, as a water scavenger, 2,2-dimethoxypropane added directly to the hydrolysis mixture. This procedure avoided the difficulty of the extraction of sulfated bile acids from aqueous solutions.     

Effects of cysteamine and other thiols on cystine egress from isolated lysosome-rich fractions of cystinotic and normal leucocytes

After loading isolated normal and cystinotic white blood cell lysosomes with radioactive amino acid methyl esters, egress of the corresponding amino acids was measured in the presence or absence of reducing agents. Cysteamine greatly enhanced disposal of cystine- and, to a lesser extent, cysteine-loaded lysosomes, resulting in final similar rates of radioactive clearance. Cysteamine did not alter leucine egress. Cysteamine comparably accelerated cystine egress from normal and cystinotic lysosomes. Reduced glutathione, ascorbic acid, coenzyme A and acetylcoenzyme A, which are much less effective cystine-depleting agents on intact cystinotic cells than cysteamine, failed to increase egress rates from isolated lysosomes loaded with cystine. We believe this in vitro system can help in the future in evaluating drug efficacy in reducing cystine content in cystinotics in addition to the already existing tissue culture technique.     

Homogeneous enzymoimmunoassay for anticonvulsant drugs: effects of hapten-enzyme bridge length.

The effects of the enzyme-hapten chemical bridge on the quality of the enzymatic tracer in homogeneous enzymoimmunoassay were studied, using as a model the conjugates of glucose-6-phosphate dehydrogenase with different phenobarbital and diphenylhydantoin carboxyalkyl derivatives. For both series of conjugates the maximal immunoinhibition level was found to depend critically on the bridge length, best results being obtained with carboxymethyl derivatives. A tendency to immuno re-activation with increasing degrees of hapten substitution in the enzyme structure, particularly evident for phenobarbital-carboxyethyl and carboxypropyl conjugates, was also observed. Effects of steric hindrance on the antibody interaction, in the case of short bridges, and ineffectiveness of antibody interaction towards the active enzyme site, in the case of long bridges, have been hypothesized to contribute in defining a critical bridge size for immuno inhibition processes. Instead the tendency to immunoactivation has been tentatively interpreted in terms of occurrence of "conformational" antibody interactions involving hapten residues in enzyme regions other than the catalytic site.     

The effects of non-painful transcutaneous electrical nerve stimulation on cutaneous pain threshold and muscular reflexes in normal men and in subjects with chronic pain

In healthy subjects and in subjects with chronic myofascial pain of one lower limb, the following was measured in both lower limbs: (i) sequential Hoffman (H) reflex, (ii) sequential Achilles tendon (T) reflex, (iii) cutaneous pain threshold determined with electrical stimuli, before, during and after transcutaneous electrical nerve stimulation (TENS).In healthy subjects no significant differences were observed between the pain thresholds of the two limbs. During and after TENS, changes of the reflexes were related to the pain thresholds.In the pathological subjects a significant difference of pain threshold was present between the affected limb and the contralateral one. An important difference between healthy and pathological subjects is not the quality but the quantity of the changes induced by TENS, in the sense that the levels of inhibition and facilitation of the reflexes are more evident in patients with pain. Indeed, TENS induces a reset of sensory and of motor system and a parallel long lasting effect both on sensory and on muscular function, with concomitant pain relief in the pathological subjects.     

Plasma lipids, lipoproteins and apoproteins in a case of apo C-II deficiency

A new case of apo C-II deficiency is described. The patient had plasma triglyceride levels ranging from 10.2–30.5 mmol/1. Apo C-II deficiency was confirmed by gel electrophoresis, isoelectric focusing and immunochemistry.

In this patient plasma lipoproteins were mainly chylomicrons and very low density lipoproteins, LDL and HDL levels being very low. Infusion of normal plasma effectively reduced plasma triglycerides and enhanced low density and high density lipoproteins cholesterol levels. These data suggest that in vivo a precursorproduct relationship exists between triglyceride rich lipoproteins and LDL and HDL, and further stress the role of the lipoprotein lipase-apo C-II system in modulating these metabolic interconversions.     

Sphingomyelinase activities of various human tissues in control subjects and in Niemann-Pick disease — development and evaluation of a microprocedure

A micromethod was elaborated for the assay of sphingomyelinase activities with native labelled substrate in leukocytes, cultivated skin fibroblasts, liver tissue and cultivated amniotic fluid cells. The optimal assay conditions and specific activities in control samples were investigated for each enzyme source. No significant difference was found between results obtained either with the micromethod or with our previous procedure. Findings obtained in pathological material from 62 patients with the various forms of Niemann-Pick disease and 21 obligate heterozygotes by one or another method are reported. A generalized severe sphingomyelinase deficiency was observed in all cases with Niemann-Pick disease type A or B, while in Niemann-Pick disease type C, sphingomyelinase activities were normal in leukocytes, elevated in liver tissue and partially deficient in cultivated skin fibroblasts. Six pregnancies at risk were monitored.     

A simple and reliable method for the purification of human alphafetoprotein (AFP) from amniotic fluid and fetal livers.

Highly purified human alphafetoprotein has been isolated from amniotic fluid and fetal livers by a combination of salting-out, and gel filtration, ion exchange, and concanavalin-A affinity chromatography. They yield ranged from 25 to 37%, and purity was demonstrated by radioimmunodiffusion, crossed radioimmunoelectrophoresis, polyacrylamide gel electrophoresis, and comparison with other preparations by radioimmunoassay. Immunochemical potency by weight of alphafetoprotein purified from amniotic fluid was similar to that from fetal liver, with molecular weights of 70 000 and 68 500 respectively. The amino acid and carbohydrate composition is also reported. This physicochemical method is relatively simple and inexpensive and is well suited for large scale production.     

Respiratory mucous secretions in patients with cystic fibrosis: relationship between levels of highly sulfated mucin component and severity of the disease

The tracheobronchial secretions from cystic fibrosis patients contained higher levels of protein, DNA and sialic acid than the tracheobronchial secretions from healthy donors. In contrast, the neutral hexose content in CF secretions was strikingly lower than in secretions from normal subjects. The levels of neutral hexose and sialic acid in the CF secretions were found to increase with increasing severity of the disease. The alterations in the levels of these chemical parameters in the secretions of patients with increased disease severity are as a result of increased levels of the mucin content of the secretions, especially of the highly sulfated mucin component. Since mucins are considered, to a large extent, responsible for the viscoelastic properties of the secretions, the enhanced levels of the highly sulfated mucin component in the secretions of the patients with increased disease severity, may contribute to altered rheological properties and hence decreased mucociliary transport of the secretions.