Ferrihaemoglobin in Normal Blood 



the moment when the blood left the living circulation. In fact, for 

 certain purposes they were able actually to include the cuvette in the 

 circulation of the living, nembutalized dog. Total pigment was 

 determined in three different ways : as ferrihaemoglobin cyanide, after 

 treatment with ferricyanide and KCN ; as oxyhaemoglobin after 

 saturation with oxygen at one atmosphere pressure ; and as ferro- 

 haemoglobin after reduction. If the original specimens had contained 

 ferrihaemoglobin, the determination as oxyhaemoglobin would have 

 been lower than the other two, while these should have given identical 

 results. In point of fact the three values agreed within 0-5 per cent 

 in all three of the cases published in detail. This indicated that none 

 of these specimens could have contained as much as 0-5 per cent of 

 the total pigment in the ferric form. It is a pity that more figures are 

 not available by this technique, which might indicate a real, but small 

 difference between arterial and venous blood. It would be unwise at 

 present to suggest any such difference, especially as in all cases the 

 analytical methods are stretched very near their limits. 



The work so far described suggests strongly that although there may 

 be traces of ferrihaemoglobin in normal blood, the gasometric methods 

 give results which, for reasons which are not yet altogether clear, are 

 usually much too high ; and this in spite of the fact that the complete 

 gasometric analysis consumes an appreciable time, during which 

 ferrihaemoglobin will tend to disappear. In 1946 an invaluable 

 comparison of the gasometric method and a spectrophotometric 

 method was published by Van Slyke, Hiller, Weissiger and Cruz 25 . 

 The gasometric technique employed was a modification of earlier 

 techniques designed to eliminate as many potential sources of error 

 as possible. The pressure of CO used for all saturations was of the 

 order of 150 mm and it was found possible to reduce the time required 

 for saturation to as little as 1-5 to 2 minutes. The spectrophotometric 

 method used was that of Horecker and Brackett 26 , in which ferri- 

 haemoglobin is determined from the fall in extinction coefficient at 

 800 mji. which results from the addition of KCN. The most stringent 

 precautions against analytical errors seem to have been taken, and the 

 two methods were compared in 19 cases. In all of these the gaso- 

 metric method detected some ' inactive haemoglobin ', usually of the 

 order of 1 per cent of the total pigment. In almost all the cases the 

 spectrophotometrically determined ferrihaemoglobin was much lower 

 than this (the mean was 0-4 per cent of the total pigment), and in 

 several it was not possible to detect any. Previous observations, such 

 as the gradual disappearance of ' inactive haemoglobin ' from drawn 

 blood, were confirmed, but although the CO pressure was as high as 

 that used by Kallner 15 , this did not eliminate entirely the ' inactive 



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