VOLUME OF BLOOD 



37 



Schiirer's foreign protein precipitation procedure, in- 

 troduced in 191 1, was also carried out on plasma, but 

 his concern for measuring blood volume as an entity 

 was such that he neglected to report data from which 

 plasma volume may be computed (209). This is true 

 of most of the earlier work, which introduced a 

 variety of plasma labels, including those mentioned 

 above, and others such as antitoxins and gum acacia. 

 Erlanger (64) has written a critical review of the 

 earlier methods, and Gregersen & Rawson {95) have 

 published a synopsis of events in the history of 

 methodology. 



The introduction of a dye label for plasma by 

 Keith et al. (127) in 1915 made it technically impera- 

 tive to measure concentrations in plasma. Since 

 plasma volume was calculated as a first step toward 

 the calculation of blood volume, it was usually 

 reported separately, along with the final value for 

 blood volume. The dye chosen for the original 

 studies, known as vital red, was nonto.xic in doses 

 adequate for visual colorimetry, and disappeared 

 from circulating plasma, after the mixing period, 

 at rates less than 20 per cent per hour. In order to 

 avoid an overestimation of plasma volume due to 

 dye loss (methods for correction had not yet been 

 proposed), plasma samples were taken at the earliest 

 moment after mixing was thougiit to be complete. 

 Usually the average concentration for two samples, 

 drawn at 3 and 6 min, was used in the calculation. 

 The mean plasma volume found in a group of 42 

 normal human males was 48,1 ml per kg (127), 

 which is in fairly good agreement with the value of 

 46.5 ml per kg obtained by current methods with 

 T-1824 (93). 



Since both the supply and the characteristics of 

 the original dye were unreliable, Dawson et al. (50) 

 in 1920 examined a large number of additional dyes 

 for their potentialities as plasma labels. Twenty-nine 

 were found with slow disappearance rates compara- 

 ble to that of vital red. All of them gave about the 

 same value for plasma volume in a group of dogs, 

 when calculations were made from the 4-minute 

 plasma dye concentration. A blue azo dye, known as 

 Evans blue, was selected as somewhat superior to 

 the rest because of its slow disappearance rate, and 

 because it could readily be distinguished from hemo- 

 globin in tlie event of hemolysis. The synthetic 

 structure of this dye is indicated by the designation 

 T-1824, which means that it is synthesized by com- 

 bining orthotolidine with 2 moles of 1 ,8-amido- 

 naphthol 2 ,4-disulphonic acid (6). 



Evans Blue Dye, T-1824. 



Systematic studies of this dye were started in 1935 

 by Gibson & Evans {77) and Gregersen and collabo- 

 rators (92). They have resulted in a more complete 

 investigation of this material as a plasma label than 

 of any other, and many of the techniques which have 

 become standard practice for T-1824 have been 

 carried over for other labels. 



The dye, with a molecular weight of 960, is readily 

 diffusible in aqueous solution, but is made non- 

 diffusible through collodion by the addition of 

 plasma. When it has been added to plasma, and its 

 electrophoretic and ultracentrifugal behavior are 

 observed, it becomes apparent that it is bound to 

 plasma albumin (187). The exact nature of its 

 attachment to protein is poorly understood, as is true 

 of biological stains in general (18). Since it is an 

 anionic or acid dye, it is thought to combine with 

 available basic groups in the albumin molecule, such 

 as lysine, arginine, and histidine (129). The absorp- 

 tion spectrum of the dye, however, differs in the 

 plasma of different species, suggesting that there are 

 several different types of binding (5). Crystalline 

 bovine albumin has 1 1 demonstrable binding sites, 

 with dissociation constants for the reaction at the 

 first site which indicate firm binding (6). Its binding 

 is not so firm, however, as to resist removal of con- 

 siderable amounts of dye by ion-e.xchange resins 



(144)- 



The kinetics of the dye-albumin interaction has 

 been studied by measuring the time required for 

 optical density to stabilize after the two are mixed. 

 At 37 °C this requires 40 to 50 sec (19). Although the 

 possibility exists that unbound, and hence diffusible, 

 dye may reach capillary beds under some conditions, 

 there is no published evidence for dye loss before 

 binding has occurred. If plasma is predyed before 

 injection, its distribution volume within the circulation 

 is the same as that of injected unbound dye (142). 

 Furthermore, plasma volume in the dog is the same 

 when it is measured by intra-arterial and by intra- 

 venous injections of T-1824 (Lawson, unpublished 

 data). Although the percentage of uncombined dye 

 increases as the molar ratio dye: albumin increases, 

 a one-hundredfold increase in the dye dosage does 

 not change the value obtained for plasma \'olume 

 in the dog (4). 



In the postmixing period, plasma dye concentra- 

 tion usually declines at the rate of 4 to 15 per cent for 

 the first 2 hours in man (77) and in the dog (7, 191, 



