Titrimetric determination of muscle buffering capacity (βm titr ) in biopsy samples

Summary In vitro titration of muscle homogenates has been used to assess muscle buffering capacity (βm titr ) in a variety of species. In the present study, factors likely to affect the estimation of βm titr were investigated. Also, values of βm titr from normal Thoroughbred horses are presented. A non‐linear titration curve was obtained with addition of HCl to muscle homogenates. As a result, βm titr is expressed as the μmol 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 βm titr 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 βm titr . Incubation of homogenates up to 60 mins did not affect βm titr significantly. As a mean, βm titr in wet muscle was approximately 25 per cent higher compared to dry muscle. The βm titr of dry muscle was increased by approximately 18 per cent when HCO 3 − was added in an amount equivalent to the calculated HCO 3 − 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 βm titr 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. βm titr determined in biopsy samples of muscle from 20 Thoroughbred horses ranged from 100.8 to 131.8 μmol H+/g dry muscle pH 7.1 to 6.5 (mean 121.2, sd ± 7.4).