Cyanmethemoglobin method

Total plasma protein concentration was determined by the buiret method (16) after correction of plasma hemoglobin which was determined by the cyanmethemoglobin method (15). Plasma osmolality was measured by a Wescor model 5100 vapor pressure osmometer. 

Preparation of Hemolysate. Washed red cells were lysed in distilled water (20% HCT) for 15 min. The stroma was extracted in carbon tetrachloride, and the aqueous layer containing the hemolysate was recovered. This solution was diluted appropriately with PBS to a specified hemoglobin concentration, and labeled fibrinogen was added. The final solution was adjusted to isotonicity and pH 7.35. Hemoglobin concentrations were determined by the cyanmethemoglobin method (18). 

Total hemoglobin was measured as cyanmethemoglobin (22). Trace concentration of hemoglobin, such as that in the extracellular fluid of suspension A, was measured using a modified benzidine method (23). 

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