VOL. 4 (1950) BODY SIZE AND TISSUE RESPIRATION 253 



Specific dynamic action, they are insignificant for the conditions of basal metabolism 

 because the concentration of these substances in plasma is too low except during the 

 period of absorption from the intestine. 



The above list of 'relevant' substances can be simplified because lactate and 

 pyruvate have very similar effects which are not additive, and only one of the two 

 therefore needs to be added. Of the two, pyruvate has the advantage over L-lactate of 

 being more readily available. Furthermore, all the acids of the tricarboxylic acid cycle 

 have very similar effects^^, as may be expected from their interconvertibility. Thus the 

 addition of one of the acids should be sufficient. As for the choice, only three of the eight 

 main acids of the cycle are readily available: citrate, succinate, and fumarate. Citrate 

 has the disadvantage that it forms complexes with calcium and magnesium ions and 

 thereby upsets the ionic balance of the medium. Succinate occupies a rather special 

 position in that the first stage of its oxidation, the formation of fumarate, may proceed 

 much more rapidly than the other stages of the cycle^^; it may cause a brief period of 

 rapid oxygen consumption followed by a steady rate at a lower level. There remains 

 fumarate as the most suitable representative of the cycle. 



From the point of view of tissue respiration the list of relevant metabolites can thus 

 be reduced to four: glucose, pyruvate or lactate, fumarate, glutamate. x^s regards the 

 concentrations to be used, experiments on kidney and brain cortex show that increasing 

 the concentrations of pyruvate, lactate, fumarate or glutamate above 0.005 ^ makes 

 no difference to the rate of respiration, except in very prolonged experiments. Glucose 

 is usually not a limiting factor when its concentration is above 0.2%. 



It is therefore suggested that serum be supplemented by adding isotonic substrate 

 solutions in the following proportions : 



100 parts of serum 



3 parts of 0.16 M Na-pyruvate (or Na-L-lactate) 

 6 parts of o.i M Na- fumarate 

 3 parts of 0.16 M Na-L-glutamate 

 5 parts of 0.3 M glucose 



The mixture must be in equilibrium with a gas mixture containing about 5% COo. 

 The additions cause a dilution of the serum of about 15%. It is not possible when making 

 additions to maintain both isotonicity and concentrations, and preference is given to 

 the former. 



The blood from which the serum is prepared should be cooled immediately after 

 collection, otherwise the glycolytic activity of the blood cells will reduce the concen- 

 trations of glucose and bicarbonate and increase that of lactate. The bicarbonate content 

 of the serum should be determined and if below 0.025 M it should be adjusted to that 

 level by the addition of 1.3% NaHCOg solution. It is advisable to sterilize the medium 

 by passing it through a Seitz-filter. 



Saline serum substitute [Medium I). Serum contains unknown and variable, and 

 thus uncontrolled, constituents. It is furthermore difficult to obtain in sufficient quan- 

 tities in the case of small animals, and heterologous serum may contain inhibitory 

 antibodies. There is therefore a case for a serum substitute which can be easily prepared 

 and whose composition is exactly known. 



As a rule serum does not preserve the metabolic activities of isolated tissues more 

 effectively than do saline media supplemented with substrates. The rates of the metabo- 

 Re/erences p. 2()'j-26g. 



