DETERMINATION OF ALKALINE PHOSPHATASE 445 within one hour. The optical density is measured at 650 mu, against the blank set at zero optical density. The optical densities of the phenol standards are plotted against rag. of phenol, and the phenol concentrations of the samples are deter- mined from this graph. Calculation 1. Subtract the O.D. of the nonincubated sample from that of the incubated sample. 2. From the standard curve of O.D. rs. rag. phenol, determine the rag. of phenol in the 15 mi. of incubated solution. 3. Multiply the rag. of phenol obtained in (2) by 20 and by the dilution factor used in making the "sample solution." This gives rag. phenol produced per 100 ml. of original sample (which equals K & A units of alkaline phosphatase per 100 mi.). NOTES 1. It is important to remove every last trace of bromine, and starch- iodide paper should be used to test for this in the vapors. 2. The pH will be 10.1. No substitutions may be permitted, as phos- phates, citrates, etc., interfere with the reaction rate. 3. Satisfactory readings may be obtained within a r:•nge cf about 1 to 60 units per 100 mi. The procedure includes an incubation period of fifteen minutes. The results of varying this time period are shown in Table 1, with the same data set forth graphically in Fig. 1. It can be seen that at least for the first thirty minutes the amount of phenol ester hydrolyzed is proportional to incubation time, and it is also apparent that the fifteen minute period should be timed with an accuracy of about one minute or better. 3200 2800 2400 2000 1600 1200 800 400 0 o ß 5 I0 15 20 25 MIN. TIME Figure 1.

446 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Temperature control within one degree appears to be adequate, as indicated by the results given in Table 2. Both the Folin-Ciocalteu reagent and the diluted Folin-Ciocalteu reagent were found to be unaffected by aging for at least three months. Portions of reagents were removed from the aged stock bottles and used in assays freshly prepared reagents were used to assay the same placenta extracts at the same time. Results of these experiments are shown in Tables 3 and 4. The assay methods described in some of the cited references in- volve incubation of enzyme with substrate at pH's somewhat differ- ent from that of the present proce- dure. By appropriate changes of the carbonate to bicarbonate ratio, buffers with pH's of 9.6 and 10.6 were prepared, and assays carried out using these as well as the buffer prepared according to the instruc- tions above. The results of the three assays of the same placenta extract are listed in Table 5, these TABLE 1--EvvEC'r OV INCUBATION TIME Time of Incubation Phosphatase, K & at 37øC., min. A Units/100 ml. 5 484 10 1120 13 1295 14 1290 15 1610 16 1540 17 1645 20 1635 25 2315 30 2690 TABLE 2--INCUBATION TEMPERATURE Temperature, Assay øC. Units/100 mi. 36 1838 37 1800 38 1862 results indicating that appreciable error may be introduced by improper pH control. A change in buffer concentration from 0.1 M to 0.01 M, which causes no significant pH change, introduces no assay error--this of course applies to placenta extract but may not apply to products containing suffi- cient acid or alkali to alter the pH of a dilute buffer. Table 6 shows the data from experiments with dilute buffers. It also shows the great im- portance of avoiding buffers which contain even small amounts of phos- phate. Other experiments show an inhibiting effect, resulting in low re- suits, if the reaction medium contains appreciable concentrations ofcitrate. TABLE 3--Ac,,INC, or FOLIN-CIOCALTEU TABLE 4--AGING or DILUTE-FoLIN- REAGENT CIOCALTEU REAGENT . -Assay Found----•- .... Assay Found------- Age, Aged Fresh Age, Aged Fesh Days Reagent Reagent Days Reagent Reagent 2 1826 1852 2 1823 1852 15 1815 1801 15 1774 1801 25 1807 1778 25 1752 1778 46 1764 1815 46 1802 1815 52 1769 1807 52 1810 1807 85 1826 1825 85 1841 1825