Analyses on Heat-coagulated Blood and Serum H. The Determination of Serum Inorganic Phosphate

A method is described that does not employ chemical precipitation for the quanti-tative separation of serum inorganic phosphate from the proteins present. The separation is accomplished by heat coagulation of serum and subsequent extraction of phosphate from the intact clot with dilute sulfuric acid. Serum (0.25 ml.) is placed at the bottom of a 25 X 95-mm. shell vial and coagulated in a boiling-water bath for 2 mm. The resultant coagulum is firm, adherent to the container, and not easily disrupted; 6.0 ml. of O.3N H2S04 is then added to cover the coagulum, and the phosphate extracted. The phosphate in the extract is determined by a phospho-molybdate reduction procedure. N/Il OST PROCEDURES for the determination of serum inorganic phos-phate are based on the reduction of phosphomolybdate (1-6), and have three distinct steps. The first is the preparation of a protein-free fil-trate with trichloroacetic acid (TCA). An acid molybdate reagent is then added to an aliquot of filtrate, with the formation of phosphomo-lybdic acid. Finally the phosphomolybdic acid is reduced to a blue complex (molybdenum blue). TCA has several disadvantages as a protein precipitant. It must be used in fairly high concentrations to be effective, and requires refrigeration. On occasion the protein-free filtrate formed is not clear, and elimination of the cloudiness requires addition of chloroform or carbon tetrachloride (7). The presence of TCA is known to inhibit the formation of molybdenum blue (1). The selection of a reducing agent is important. If it is too strong, molybdic acid itself will be reduced, and the values obtained falsely elevated. Conversely, if it is too weak, there will be incomplete reduction of th