Titrimetric determination of muscle buffering capacity (beta mtitr) in biopsy samples

Abstract

In vitro titration of muscle homogenates has been used to assess muscle buffering capacity (beta mtitr) in a variety of species. In the present study, factors likely to affect the estimation of beta mtitr were investigated. Also, values of beta mtitr from normal Thoroughbred horses are presented. A non-linear titration curve was obtained with addition of HCl to muscle homogenates. As a result, beta mtitr is expressed as the mumol H+ required to change the pH of 1g of dry muscle or wet muscle from 7.1 to 6.5. An effect of dilution on the initial pH was found below 40 mg wet muscle per ml homogenising reagent (10 mg dry muscle per ml) and on beta mtitr below 10 mg wet muscle. As a result, 40 mg wet muscle or 10 mg dry muscle per ml was chosen as the minimum concentration for determination of beta mtitr. Incubation of homogenates up to 60 mins did not affect beta mtitr significantly. As a mean, beta mtitr in wet muscle was approximately 25 per cent higher compared to dry muscle. The beta mtitr of dry muscle was increased by approximately 18 per cent when HCO3- was added in an amount equivalent to the calculated HCO3- content of wet muscle at rest. The homogenisation process resulted in complete loss of adenosine triphosphate and phosphocreatine with only small changes in adenosine diphosphate and adenosine monophosphate. It was concluded that the estimates of beta mtitr did not include any contribution from 'dynamic' buffering via rephosphorylation of adenosine diphosphate by phosphocreatine, and in dry muscle it was accounted for mainly through physico-chemical buffering by phosphates, proteins and dipeptides. beta mtitr determined in biopsy samples of muscle from 20 Thoroughbred horses ranged from 100.8 to 131.8 mumol H+/g dry muscle pH 7.1 to 6.5 (mean 121.2, sd +/- 7.4).