Comparison of particulate 3,3',5,5'-tetramethylbenzidine and 3,3'-diaminobenzidine as chromogenic substrates for immunoblot.

In horseradish peroxidase (EC: 1.11.1.7)-dependent immunoblot assays, particulate 3,3',5,5'-tetramethylbenzidine (TMB) is shown to be a more efficient immunoblot substrate than the standard substrate 3,3'-diaminobenzidine (DAB), because TMB is easily prepared, stable, and less carcinogenic than is DAB. Assays of antibody in a serially diluted human immunodeficiency virus (HIV) control serum (CDC reference CAT# VS2151) have the same sensitivity limits with both DAB and TMB (1:312,500). Complete, working substrate solutions of H2O2/TMB/enhancer and of H2O2/DAB were stored at room temperatures and at 48 degrees C respectively. Periodic tests showed the TMB substrate system to be functional after four weeks at 48 degrees C and after eight weeks at room temperature, while the DAB system was functional after one week at 48 degrees C and after four weeks at room temperature. The stability, safety, and convenience of the commercially available TMB kits make this substrate ideal for immunoblot tests.

Evidence of sympathetic augmentation mediated increase in the blood and the plasma volume following vagotomy.

Cervical vagotomy was performed bilaterally in three series, i.e. normal (untreated), atropine treated and propranolol treated animals. The classical effect of the nerve section on heart rate, arterial blood pressure and respiration respectively were verified in the three series to confirm the reproducibility of the findings. The pre- and post-vagotomy plasma volume and haematocrit were estimated and thus the blood volume was calculated in each animal. The investigation indicated that: The interruption of vagal sensory information reflexively expanded the plasma and blood volume. This information was normally tonically active and inhibitory in nature. The volume expansion after vagotomy was brought about by stimulation of autonomic nerves, most probably the sympathetic nerves. These three conclusions suggested that expansion of vascular capacity as well as blood and plasma volume was controlled through vagal sensory information.

Intensification of the base deficit induced by vagotomy after atropine treatment in dogs.

The effects of atropine treatment on the heart rate, the arterial blood pressure, the respiratory rate, the acid-base profile and the oxygenation of arterial blood were investigated in chloralose anaesthetized dogs. As the I.V. atropine sulphate (1mg/kg) in saline (9% w/v) did not induce base deficit or alter the acid-base profile, the section of vagal parasympathetic (motor) fibres in normal (untreated) ones could not be held responsible for the observed base deficit after their vagotomy. Such observed base deficit was most likely engendered reflexively due to the section of vagal sensory fibres; as only general activation, not the withdrawal (section) of vagal sympathetic fibres might induce such an effect. Moreover, the bilateral vagotomy in these atropinized dogs induced a larger base deficit, which mostly contributed to the increase of H+ion concentration therein. This comparative enhancement of base deficit suggests that the opening of the negative feed back loop of the (aortic chemoreceptor) sensory fibres points to the blocking effect of atropine on the cholinergic link in the central (medulla) chemosensitive mechanism. The base excess observed after vagotomy in propranolol treated animals suggested that the base deficit vagal sensory fibres was mediated through reflexive activation of sympatho-adrenal axis.

An increase in metabolic acidosis induced by chloralose anaesthesia in dogs after sino-vagal denervation.

Chloralose anaesthesia in dogs increased the H+ ion concentration significantly from its reference values. The findings favoured that it was most probably engendered through anaesthetic depression of neural centre regulating H+ ion concentration of blood. Such increase was largely contributed by a significant increase in its metabolic fraction. A further increase of metabolic fraction after separate and joint section of carotid sinus nerves and vagi indicated their holding effect. The section of carotid sinus nerve induced greater increase in this fraction than that of vagi. It indicated differences between the two nerves in their metabolic fraction controlling influence. Hyperpnoea after vagi section decreased the carbonic acid fraction, whereas marginally reduced ventilation after carotid sinus nerve section increased the carbonic acid fraction. Moreover, the overall changes in H+ ion concentration followed the changes in carbonic acid fraction. The present study suggested that the depressive effect of chloralose anaesthesia on H+ ion controlling neural mechanism could be largely determined by degree of increase in its metabolic fraction.

Intensification of the metabolic acidosis induced under alpha-chloralose anaesthesia with graded increase in the dose.

Surgical anaesthesia between planes 2-3 with alpha-chloralose in dogs induced simultaneous depression of ventilation viz., low oxygen (PaO2) tension, high carbon dioxide (PaCO2) tension and H+ion homeostasis (high base deficit). The anaesthesia on the aforesaid planes could be achieved only by doses not below 100 mg/kg. The progressive increase in the administered dose between 100 and 200 mg/kg did not alter the acid-base profile. Further increase beyond 200 mg/kg dose was followed by a steep increase in the base deficit enhancing H+ion (cH+) concentration. However, the steep fall in PaO2 accompanied by steep rise in PaCO2 was observed only when the dose was above 250 mg/kg. The findings in the present study indicated that depression of H+ion homeostasis under chloralose anaesthesia was induced by neural mechanism independent of respiratory centres.

The effect of polyethylene glycol-200 on metabolic acidosis induced by chloralose anaesthesia.

Chloralose may be used in a 10% solution as an anaesthetic in dogs. The solubility of chloralose was found to be much higher in polyethylene glycol-200 (PEG-200) than in either warm (body temperature) or cold saline (0.9% NaCl). The intravenous (i.v.) administration of chloralose in warm saline solution induced acidosis as a result of the increase in the level of metabolic acids. The acidity generated by chloralose in almost neutral saline was probably the result of increase in the base deficit in the animal. The infusion of PEG-200 (almost neutral) significantly reduced the base deficit without disturbing the PaO2 or PaCO2 in the arterial blood. The base deficit value was significantly lower after administration of chloralose solution in PEG-200 (almost neutral) than after administration in saline. The use of PEG-200 as a solvent for chloralose was advantageous in two ways. Firstly, it prevented the production of acids in anaesthetic solution and neutralized the blood metabolic acids generated by chloralose administration in saline. Secondly, the solubility of chloralose (10% w/v solution) in PEG-200 was very much higher than in warm or cold saline.

Acid-base profile of healthy mongrel Indian familaris canis determined by different techniques under two non-volatile anaesthetics.

The acid-base status of healthy mongrel Indian dogs was compared under pentobarbital sodium and alpha-chloralose anaesthesia. The findings showed similarity of canine acid-base profile under the two types of anaesthesia. Comparison among various techniques of determination of acid-base profile indicated accuracy of findings. However, a slight metabolic acidosis was persistently observed in the majority of dogs under both types of anaesthesia.