A simple and accurate method is described for the quantitative determination of alkaline phosphatase in tissue homogenates, utilizing stable buffered phenolphthalein monophosphate as substrate. The compound, which is hydrolyzed at a rate linear to enzyme concentration and incubation time, provides a chromogen directly for spectrophotometric determination. The assay does not require the addition of a non-specific protein or Mg++ for optimal activity and is equally reactive in glass and plastic flasks. The technique affords good sensitivity by measuring enzyme activity in homogenates as dilute as 0.0005% (small intestine). Quadruplicate determinations varied <1%. Small intestine was the most reactive of the rat organs tested, followed by the kidney, lung, heart and spleen.

In intact rats, or in rats castrated on day 4 and receiving progesterone replacement, the decidual alkaline phosphatase activity was 12 times greater on day 8 of pseudopregnancy than in the contralateral nontraumatized horn ( 1-3). This pseudopregnant decidual enzyme response was entirely similar to the alkaline phosphatase reaction in day 8 implantation sites of pregnant rats (1, 3-6). No increased enzyme was elicited by the nondecidualized uteri of nontraumatized, pseudopregnant animals treated with 17p-estradiol and/or progesterone or rats ovariectomized and traumatized on day 4 and given only estrogen. Additionally, the changes in alkaline phosphatase from day 5 to 10 in the intact and hormone-treated ovariectomized pseudopregnant rats with traumatized uteri were comparable to those demonstrated by the intact pregnant animals, i.e., an increased reaction from day 5 to 8 and decreased activity from day 8 to 10 (1). These data supported the view that the requirements for the elevation of decidual alkaline phosphatase in the pseudopregnant animal were similar to those in the pregnant rat; that is, physical and/or chemical irritation (day 4) and a progesterone-sensitized endometrium ( 1, 3, 5 , 6). The results for the pseudopregnant deciduae, however, do not entirely exclude the possibility of some residual ovarian estrogen entering directly into the mechanism of alkaline phosphatase stimulation because the traumatized animals were ovariectomized and treated with hormones starting on day 4 of pseudopregnancy .The purpose of this study was to discover whether estrogen is necessary for the rise in decidual alkaline phosphatase activity and, if so, when and how much is required. In order to exlude the influence of ovarian estrogen, rats castrated on day 1 of pseudopregnancy were utilized for this investigation.Materials and Methods. The rats used were 180-200 g adult virgin albinos (KG Farms). The animals were housed in an airconditioned, controlled light-reverse room (12 hr of light, 12 hr of darkness). Standard rat chow and water were available ad lib;tum. Only animals with normal 5-day estrus cycles by vaginal smear were used.Pseudopregnancy was induced bmetween 10-1 2 AM by electric stimulation (constant voltage of 9