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Phosphous Method

Dr SubbaRow enrolled himself at the Harvard Medical School Department of Biochemistry in the summer of 1924.

Professor Otto Folin, head of HMS Department of Biochemistry, was then directing the talents of all in his department to development of methods for analysing small quantities of blood, urine and tissue. One such method developed in the department was for estimation of phosphorus in tissues. It was however unsatisfactory in many ways, and Folin assigned SubbaRow to assist Cyrus Hartwell Fiske, associate professor, in removing the difficulties with the phosphorus method.

The task that Fiske gave SubbaRow was to search for a chemical agent that would, in small concentrations, completely convert phosphorus in the prepared tissue into a blue substance within the shortest possible time. The agent should not be affected by other tissue substances or by trichloro-acetic acid used for removing protein from the tissue and should not require prior digestion of phosphorus with sulphuric acid.

It was a challenge that held SubbaRow's interest right away. The first satisfactory agent that SubbaRow found gave some 20 per cent more colour than hydroquinone previously used in phosphorus estimation. But it required 30 minutes to produce the maximum colour. Fiske said the ideal agent would be one which did the job in just five minutes.

SubbaRow continued his search. An old sample of 1,2,6-amino-naphthol-sulphonic acid gave very encouraging results. A purified sample of this turned out to be 50 times more active than hydroquinone. It produced within a fraction of a minute more intense colour than hydroquinone did in 30 minutes. But it was a difficult chemical to prepare. Fortunately it had an equally good isomer that could be prepared and purified easily. This was 1,2,4-amino-naphthol-sulphonic acid which differed only in the way the groups of atoms were arranged in the molecule. It could also be readily bought in the market as it was in demand from dyestuff makers who called it the 1,2,4-acid.

SubbaRow worked the next four months day and night to perfect the phosphorus estimation procedure to the satisfaction of Fiske. The 1,2,4-acid stood every test. It did not require any big change in the procedure for preparing the analytical material for colorimetric estimation. It gave accurate readings even in the presence of ten times more colour-inhibiting material than was permissible with hydroquinone.

The "rapid colorimetric method for the estimation of inorganic phosphorus, organic phosphorus, organic phosphates and lipoid phosphorus in blood and urine", SubbaRow recorded proudly, "is correct to 1/100,000th of a grain". (click for review article)

Fiske and SubbaRow were invited by the American Society of Biological Chemists to demonstrate the new phosphorus method before its annual meeting on December 29, 1924. Folin promptly asked SubbaRow to a celebration dinner at his home and promised him a bigger fellowship the following year.

The phosphorus procedure was presented to the Society as "Fiske-SubbaRow Method of Estimation of Phosphorus". (click here for A Classic Paper) SubbaRow had worked under the supervision of Fiske. Besides, the association of a biochemist of Fiske's reputation would help its ready acceptance by the profession. SubbaRow was after all a novice, unknown to the scientific world until then. The method is so accurate and suitable for analysis of all kinds of biological and clinical material that it was adopted immediately by biochemical laboratories all over the world. It is used to this day in common analytical practice in many fields of biochemistry. It is one of the first procedures biochemistry students everywhere learn.