The systemic administration of local anaesthetics produces a selective depression of C-afferent fibre evoked activity in the spinal cord

An electrophysiological analysis of the antinociceptive effects of systemic lidocaine and its longer acting primary amine congener, tocainide, has been performed in the decerebrate-spinal unanaesthetised rat. Neither of these local anaesthetic drugs when administered systemically in doses of up to 10 mg/kg (lidocaine) or 100 mg/kg (tocainide), produced any evidence of a block in the conduction of action potentials in A beta, A delta or C primary afferents. The local anaesthetics also failed to reduce mustard oil induced neurogenic extravasation, a test of cutaneous C-fibre terminal function. Lidocaine produced a transient (1-2 min) depression in monosynaptic reflexes at doses of greater than or equal to 1 mg/kg while tocainide had no effect on this reflex at any dose up to a 100 mg/kg. Both drugs, however, significantly suppressed the C-fibre evoked polysynaptic reflex generated by stimulating the sural nerve. The tocainide effect was longer lasting with less action on the short latency A beta-evoked reflex than lidocaine. The reflex activity in hamstring flexor alpha-motoneurones evoked by pinching the toes of the ipsilateral hind paw was reduced by both drugs but not abolished. Thermal and noxious chemical evoked reflexes were, however, completely suppressed by the local anaesthetic drugs, again with a longer action from tocainide. These results demonstrate that the systemic administration of drugs which increase the inactivation of sodium channels can produce a selective central block of certain types of afferent evoked activity in the spinal cord. There are resemblances between the selective C-fibre suppressing actions of systemically administered local anaesthetic and the pharmacological actions of narcotic opiates which may represent a similar mechanism for the analgesic action of these quite different classes of drugs.     

The formalin test: a tonic pain model in the primate

The formalin test has been used in monkeys for assessing pain. After formalin injection in the palmar surface of the hand just proximal to the base of the fingers, the monkey's responses are rated for 1 h according to objective behavioral criteria. The present 'tonic' pain model has a fair degree of objectivity, validity, reproducibility and quantifiability. The analgesic effects of morphine and pethidine have been evaluated.     

Amino acid composition of gliadin fractions which may be toxic to individuals with coeliac disease

Fraction 9, prepared by chromatography of a peptic-tryptic-pancreatinic digest of gliadin on S.P. Sephadex C-25, was re-chromatographed on Q.A.E. Sephadex A-25 and subfractions 9-1 and 9-2 further purified on S.P. Sephadex C-25. Sub-fractions 9-1 and 9-2 and the purified sub-fractions 9-1B and 9-2B appeared to be toxic to patients with coeliac disease on the basis of causing a reduction in d-xylose absorption.Amino acid analysis of undigested residues from sub-fractions 9-1B and 9-2B obtained after ‘in vitro’ digestion with remission coeliac mucosa contained mainly glutamine/glutamic acid and proline with some serine, leucine, phenylalanine and glycine.Another fraction (fraction 3) of wheat gliadin prepared by peptic-tryptic digestion and ion-exchange chromatography on S.P. Sephadex, previously shown to produce a skin-reaction in adults with coeliac disease, has been further purified by ion exchange chromatography, isoelectric focusing and gel filtration. The sub-fractions were submitted to amino acid analysis and the results compared with those from the undigested residues above.Isoelectric focusing of fraction 3 of the P.T. digest and its sub-fractions showed the presence of peptides of pI approximately 4.8 and 5.6 with only small amounts of peptides on either side of this region.Mucosal digestion of fractions of peptic-tryptic-pancreatinic gliadin digests ‘in vitro’ appears to be a promising method for the elucidation of the primary structure of that section of the gliadin which may be responsible for the lesion in coeliac disease. The evaluation of higher molecular mass peptic-tryptic digests by intradermal skin tests could also be useful for the preliminary screening of fractions for feeding tests, but this approach seems less likely to indicate the toxic region of the gliadin molecule.     

Serum dopamine β-hydroxylase (DBH) activity in acute myocardial infarction

Serial serum dopamine β-hydroxylase (DBH) activities were determined in 32 patients admitted to the Coronary Care Unit with chest pain, 9 with acute myocardial infarction and 23 with other diagnoses. The values of DBH were elevated in both groups on day one. The acute myocardial infarction group showed a slower decline and reached a baseline plateau at day five when compared to the control group whose values fell significantly by day two. The reason for the quantitative difference between the two groups is speculative and may be related to changes in plasma volume. Increases in DBH are not specific for myocardial infarction but a nonspecific response to stress.     

The toxic fraction of gliadin digests in coeliac disease. Isolation by chromatography on Biogel P-10

Improvement in the fractionation of gliadin digests and in the isolation of toxic fractions was achieved using chromatography on Biogel P-10. Fraction V, one of the 11 fractions eluted from a peptic-tryptic digest of crude gliadin extracted from Cappelle wheat, significantly affected coeliac jejunal mucosa in organ culture. BV, gamma V, omega V, the corresponding fractions V from beta-, gamma- and omega-gliadins, displayed similar toxic effects. Fraction VI containing peptides with a lower molecular mass did not show any significant cytotoxic activity and, moreover, inhibited the toxicity of fraction V. Analysis of the toxic fractions V showed that they contained peptides of 7-8,000 molecular mass, rich in proline and glutamine and poor in aromatic amino acids and carbohydrates. Among the various fractions, V, beta V from beta-gliadin appeared the less heterogeneous.     

Identification of β-aminoisobutyric acid in uremic serum

An unidentified ninhydrin-positive substance found in uremic sera but not found in normal sera was isolated by gel-filtration through Sephadex G-75 followed by high voltage paper electrophoresis (pH 3.5), and identified as β-aminoisobutyric acid using paper chromatography and automated amino acid analyzer.

The quantitative determination of β-aminoisobutyric acid in serum revealed that the level of β-aminoisobutyric acid in uremic sera was much higher than that of normal sera.

Gas chromatographic determination of the enantiomorphs of β-aminoisobutyric acid showed that uremic sera contain R- and S-isomers of the amino acid, but with the R-isomer as the dominating form.     

Spectrophotometric and fluorimetric assays of galactocerebrosidase activity,their use in the diagnosis of Krabbe's disease

Derivatives of galactocerebroside were prepared containing coloured (ω-2,4,6-trinitrophenylaminolauric acid) or fluorescent (11-(9-anthroyloxy) undecanoic acid) fatty acid moieties. These cerebrosides were used as substrates for galactocerebrosidase activity. By overcoming problems associated with the radioactively labelled substrates normally used, yet retaining good enzyme-substrate specificity, these derivatives provided useful and reliable alternative substrates for galactocerebrosidase activity. Enzyme activities in whole extracts of brain, liver, fibroblasts and cultured amniotic fluid cells were compared, using as substrates the novel cerebrosides as well as [³H] galactocerebroside. Good correlation of activities was obtained. In extracts derived from patients with Krabbe's disease marked deficiency of galactocerebrosidase activity was observed with each substrate, whereas extracts from heterozygous carriers exhibited a partial reduction in enzyme activity. The results show that these coloured and fluorescent galactocerebrosides may be used with confidence in the diagnosis and carrier detection of Krabbe's disease.     

Hepatic collagen proline hydroxylase activity in alcoholic liver disease.

The activity of hepatic collagen proline hydroxylase was examined in biopsy samples as a factor in collagen synthesis in 77 patients with alcoholic liver disease. The urinary excretion of peptide bound hydroxyproline was also measured in most of the patients, as an index of collagen degradation. The highest activities of collagen proline hydroxylase were found in the patients with alcoholic hepatitis. Enzyme activity was markedly increased in patients with non-specific changes on liver biopsy, whereas, patients with fatty infiltration had only mild elevations, and those with inactive cirrhosis had normal enzyme activity. Urinary hydroxyproline was elevated only in patients with alcoholic hepatitis and inactive cirrhosis. Follow-up determinations in 16 patients with alcoholic hepatitis, after 4 to 5 weeks, revealed a decrease in enzyme activity, but no change in urinary hydroxyproline. We conclude that among the types of alcohol-related liver diseases, alcoholic hepatitis is associated with the greatest turnover of hepatic collagen.     

Contrast medium causes the apparent increase in β-endorphin levels in human cerebrospinal fluid following brain stimulation

Levels of β-endorphin immunoreactivity in cerebrospinal fluid were measured in 12 chronic pain patients undergoing the surgical implantation of an electrode into the periventricular gray matter. Cerebrospinal fluid fractions were collected following placement of a cannula into the third ventricle, following injection of metrizamide contrast medium into the ventricles, following implantation of the electrode, and following electrical stimulation. A second set of samples was collected on a non-surgical day before and after stimulation.

Levels of β-endorphin immunoreactivity increased significantly from baseline levels to post-electrode implantation in one group of patients, but no significant change was seen following the onset of stimulation. Immunoreactivity increased significantly following metrizamide injection in a second group and was still elevated, in comparison to baseline, following electrode placement, but no increase was seen following the onset of stimulation. Levels of immunoreactive β-endorphin did not increase in either group after stimulation on a post-surgical day, despite consistent reports of pain relief. Addition of metrizamide or a related contrast medium, iothalamate meglumine (Conray) to the β-endorphin radioimmunoassay revealed that both compounds interfered with antigen-antibody binding and also quenched the gamma radiation emitted by iodinated peptide ligands. Due to these combined effects, the contrast media alone produced results similar to those of the β-endorphin standard. Moreover, similar observations were made when contrast media were incorporated into radioimmunoassays for met-enkephalin, dynorphin and cholecystokinin octapeptide.

These findings indicate that increased levels of β-endorphin in cerebrospinal fluid are not directly associated with patient report of pain relief following periventricular gray stimulation. This study also suggests that the previously reported association between periventricular gray stimulation and elevated levels of β-endorphin immunoreactivity in ventricular cerebrospinal fluid are artifactual due to the interaction of contrast media with the radioimmunoassay.     

In vitro studies upon the release of γ-glutamyltransferase from human liver

Samples of human liver have been incubated in different fluids for up to 48 h and the released γ-glutamyltransferase studied by gel chromatography on Sephacryl S-300 and polyacrylamide gradient gel electrophoresis. When human liver is incubated in serum, most of the released enzyme is of high M_r (greater than 1 000 000). Incubation in hepatic bile, or in a solution of glycochenodeoxycholate, results in the release of enzyme that is hydrophobic in nature and which reaggregates to a varying extent after the removal of bile salts. In contrast, incubation in saline, or in a solution of human albumin results in the release of a hydrophilic low M_r (about 120 000) form of the enzyme. These observations are discussed in relation to possible mechanisms for the release of these multiple forms.     

The assessment of pain and plasma beta-endorphin immunoactivity in burned children

The need for better analgesia during burn dressing changes (BDCs) in acutely burned children led us to assess pain during BDC with a large 0-10 thermometer-like scale which was well accepted and appeared to reflect the varying degrees of pain that patients experienced. Pain scores were obtained at least once each minute throughout 33 BDCs in 15 patients of 8-17 years. Plasma levels of beta-endorphin immunoactivity (iB-EP) were measured at 5 intervals before and after BDC; mean values (+/- S.E.M.) ranged from 30.5 +/- 4.63 pg/ml (before BDC and analgesic) to 19.2 +/- 3.02 pg/ml (immediately following BDC). The mean pain score (MPS) for each BDC was inversely related to the iB-EP levels of that day (P less than 0.001 with 4 of the 5 iB-EP determinations). The MPS varied directly with the extent of burn injury and inversely with weight; the 2 variables together predicted MPS as well as the iB-EP alone (r2 = 43 and 36% respectively).     

On the composition of capillary and venous blood serum

The concentrations of various clinical-chemical substances in the capillary and venous blood serum of apparently healthy adults (20–30 yr) were examined in the fasting state. Total protein, bilirubin, calcium, sodium and chloride concentrations were significantly lower (? 5%) in capillary than in venous serum. In non-hemolytic capillary sera, the concentration of potassium was nearly the same as in venous samples. Inorganic phosphorus and urea concentrations were identical in both specimens. There was a tendency for glucose concentrations to be higher in capillary than in venous serum. It is concluded that capillary and venous blood serum can be used interchangeably only for certain purposes. It should be emphasized that these results are valid for apparently healthy adults in the fasting state; other population samples remain to be investigated.     

An easy procedure for determination of molar activity (or specific activity) of bile acids in bile

An easy procedure is described for determination of the molar activity of the major bile acids, labelled with 14C, in bile. The procedure involves initial enzymatic hydrolysis, by which the amino acid moieties are removed from the glycine and taurine conjugated bile acids by means of choloylglycine hydrolase, followed by thin-layer chromatographic separation of the unconjugated bile acids, cholic acid, chenodeoxycholic acid, and deoxycholic acid. Then the bile acids are eluted from the individual silica gel spots concerned. Finally, determination of the radioactivity by liquid scintillation counting and of the amount of substance by an enzymatic method using 3 alpha-hydroxysteroid dehydrogenase is performed in the eluates. The method requires only a small volume of sample and allows of separate determination of the molar activity of cholic acid and chenodeoxycholic acid, labelled with the same isotope, in the same sample.     

A new method for the assay of aminoacylase: elaboration of a fixed-incubation method for routine measurements.

A simple method suitable for routine determinations of aminoacylase (EC 3.5.1.14) in serum and tissue homogenates is described. It is based on the formation of red charge-transfer complexes from p-benzoquinone and amino acids liberated by cleavage of acylamino acids. Optimal substrates are trifluoroacetyl-L-methionine and chloroacetyl-L-methionine, the first being rapidly hydrolyzed by pancreatic tissue, the latter by liver and kidney homogenates. Sera preferentially split the chloroacetyl compound. Optimal conditions for the assay of serum activity are: 18-20 mm9l/l substrate concentration, 50 mmol/l phosphate buffer pH 6.0, no additives.     

Plasma protein profiling: the diagnostic evaluation of disorders in plasma protein composition by a new immunoelectrophoretic method.

A new electroimmunoprecipitation technique is presented by means of which a great variety of antigens e.g. plasma proteins can be simultaneously and quantitatively determined with a single-step electrophoretic separation. The essential features of the new technique are: (a) subdivision of the antibody gel into gel strips containing monospecific antibodies to individual plasma proteins. (b) sample application as a "sample gel" filling a trough over the width of the immunoplate. Quantitation is based on the fact that the distance an antigen can migrate within a gel containing a defined amount of specific antibody directed against the antigen is determined by the concentration of the appropriate antigen within the sample. The area where the antigen is finally completely consumed by immunoprecipitation and antibody present in excess is sharply delineated. The applicability of the method in simultaneous quantitative determination of 15 plasma proteins is demonstrated with plasma from healthy blood donors and patients with various diseases. The advantage of the new technique as compared to commonly used clinical acetate folia electrophoresis is the high degree of specificity for the determination of a great number of individual, diagnostically meaningful plasma proteins. The advantage over common quantitative two-dimensional immunoelectrophoresis is its uncomplicated way of evaluation. The potential clinical application of the new quantitative immunoelectrophoretic technique in diagnostic screening and differential diagnosis is discussed.     

The heterotopic effects of visceral pain: behavioural and electrophysiological approaches in the rat

The heterotopic effects of peritoneo-visceral pain were investigated in behavioural and electrophysiological experiments performed on rats. The intraperitoneal administration of acetic acid (i.p. AA), an algesic agent commonly used to induce writhing behaviour in rodents, was used as a conditioning stimulus in two parallel series of experiments involving 3 behavioural tests and recordings of dorsal horn convergent neurones. The responses to a nociceptive stimulus applied to the tail or paws were lowered by i.p. AA but these effects depended on the behavioural test used: in the tail-flick test, AA produced a transient low magnitude increase in latencies; the threshold for vocalization induced by electrical stimulation of the tail was clearly (25%) and sustainedly (full recovery taking up to 1 h) increased; the jump latency in hot plate was markedly increased (100% at 15 min). Intraperitoneal AA strongly depressed the C fibre evoked responses of coccygeal convergent neurones to suprathreshold transcutaneous electrical stimulation applied on their tail excitatory receptive fields. The time course of these inhibitory effects roughly paralleled the behavioural hypoalgesic effect observed in the vocalization test. These results are discussed with reference to diffuse noxious inhibitory controls (DNIC). Analogies with counter-irritation phenomena are emphasized.