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PubMed1991

Influence of phloretin and alcohols on barrier defects in the erythrocyte membrane caused by oxidative injury and electroporation.

Oxidative damage by diamide, periodate and oxygen-derived reactive species, but also exposure to electroporation induce in the erythrocyte membrane dynamic, presumably fluctuating, defects having the properties of aqueous holes with definable radii and selectivities. These leaks, which can be quantified by measuring tracer fluxes or rates of colloid-osmotic lysis, are here shown to be inhibited by phloretin and a small number of related phenol compounds (phenolphthalein, hydroxyacetophenones, nitrophenol), while a host of other 'membrane-active' agents is not effective in this respect. I50 values range from about 200 microM for phloretin and phenolphthalein to about 10 mM for 4-nitrophenol. Inhibition by phloretin is reversible, not competitive and not related in its extent to the extent of leakiness. In contrast, the enhancement of transbilayer mobility of amphiphilic lipid probes, which invariably goes along with leak formation of the type described, is not affected by phloretin. Aliphatic alcohols (hexanol, butanol) have an amplifying effect on leaks induced by oxidative damage but do not affect leaks induced by electroporation. The alcohol-amplified leaks maintain the properties of aqueous holes as indicated by a low activation energy of leak fluxes. Since both, inhibition and stimulation of leak fluxes do not go along with appreciable changes of the apparent radii of the aqueous holes, changes in the dynamics (opening and closing) of the defects are proposed to underly the effects of phloretin and alkanols. The membrane lipid domain is likely to be the site of the leaks and of their modulation.

Alcohols↗
PubMed1977

Biochemical characterization of alkaline phosphatase in guinea pig thymus.

1. Alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) in guinea pig thymus was extracted optimally in 10 mM Tris - HCl buffer at pH 8.0 containing 5 g/l Triton X-100. 2. alpha-Glycerophosphate, beta-glycerophosphate and phenolphthalein monophosphate were hydrolyzed by thymus extract with a pH optimum at 9.8-10.0, whereas p-nitrophenylphosphate and alpha-naphthylphosphate were hydrolyzed with pH optima at 10.7-10.8 and beta-naphthylphosphate at pH 11.2. P-Nitrophenylphosphate and phenolphthalein monophosphate proved to be the most suitable substrates. 3. Alkaline phosphatase was effectively inhibited by EDTA, Zn2+, histidine and urea therefore resembling the inhibition characteristics of alkaline phosphatase in the placenta and kidney, but not that in the liver and intestine, which differed markedly. 4. DEAE-cellulose chromatography and polyacrylamide disc electrophoresis revealed three enzyme peaks which did not differ in their substrate specificities and modifier characteristics. 5. Polyacrylamide disc electrophoresis of thymus, serum, placenta, kidney, liver, bone and intestine revealed no alkaline phosphatase bands definitely unique to thymus.

Alkaline Phosphatase↗
PubMed1987

Human adult hepatocytes in primary monolayer culture. Maintenance of mixed function oxidase and conjugation pathways of drug metabolism.

The stabilities of several drug oxidation and conjugation pathways in human adult hepatocytes have been investigated during 72 hr culture. Cytochrome P-450-dependent mixed function oxidase was measured by the O-dealkylations of ethoxyresorufin (EROD), pentoxyresorufin (PROD) and benzyloxyresorufin (BROD), which are probes for different isozymes of cytochrome P-450 in the rat. EROD declined to 64% of initial fresh cell values after 72 hr in culture, whereas PROD increased to 162% and BROD remained relatively constant. Addition of phenobarbitone to the culture medium selectively increased PROD to a greater extent than EROD and did not affect BROD. NADPH-cytochrome c reductase and NADH-cytochrome b5 reductase were markedly labile during culture, declining to 32% and 22% of fresh cell values respectively. Epoxide hydrolase (EH) showed a large transient increase (2-5-fold) in enzyme activity 24 hr after culture, declining to fresh cell values by 48 hr. UDP-glucuronyltransferase (GT) activity towards phenolphthalein and 1-naphthol also increased (2-3-fold) during the 72 hr of culture, the greater and more rapid increase being observed with phenolphthalein glucuronidation. Sulphotransferase activity declined rapidly within 24 hr of culture, whereas reduced glutathione (GSH) levels and GSH conjugation were maintained at fresh cell values for 72 hr.

Adult↗
PubMed1992

Fecal osmotic gap and pH in experimental diarrhea of various causes.

Although the osmotic gap of fecal fluid is often used to distinguish osmotic diarrhea from secretory diarrhea, there has never been a scientific evaluation of the validity of this concept. Similarly, although a low fecal fluid pH value is used to indicate that diarrhea is mediated by carbohydrate malabsorption, the validity of this method is unproven. Therefore, in the present study, diarrhea was induced in normal subjects by different mechanisms and fecal fluid osmotic gap (using an assumed fecal fluid osmolality of 290 mOsm/kg) and pH were measured. In secretory diarrhea caused by phenolphthalein, the osmotic gap was always less than 50 mOsm/kg, whereas in osmotic diarrhea caused by polyethylene glycol, magnesium hydroxide, lactulose, and sorbitol, the osmotic gap always exceeded 50 mOsm/kg. In osmotic diarrhea caused by sodium sulfate, the fecal fluid osmotic gap was less than 50 mOsm/kg, but phenolphthalein-induced secretory diarrhea could be distinguished from sodium sulfate-induced osmotic diarrhea by the fecal chloride concentration. When diarrhea was caused by carbohydrate malabsorption (lactulose or sorbitol), the fecal fluid pH was always less than 5.6 and usually less than 5.3; by contrast, other causes of diarrhea rarely caused a fecal pH as low as 5.6 and never caused a pH less than 5.3. It is concluded that measurement of fecal fluid osmotic gap and pH can distinguish various mechanisms of experimental diarrhea in normal subjects. The concepts on which these tests are based are therefore verified experimentally.

Adult↗
PubMed1992

Biliary function studies during multiple time periods in freely moving rats. A useful system and set of marker solutes.

Biliary output of endogenous and exogenous compounds is altered by anesthesia, depletion of bile salts, and hydrostatic pressure. The described system for bile function studies minimizes these confounding factors by substantially modifying existing methods. Experiments were conducted in freely moving rats which eliminates effects of anesthesia or restraint-induced stress. Depletion of bile salts was prevented by intraduodenal infusion of taurocholate which maintains bile volume. Bile was collected in containers taped to the rat's back which minimizes hydrostatic forces induced by lengthy or elevated biliary cannulas. Animals were prepared for hepatobiliary function studies 1 week before experiments by placement and exteriorization of a jugular cannula and a bile duct to duodenal fistula. Experiments involved monitoring biliary outputs of marker solutes for various pathways of bile formation during three sequential time periods of 120 min, that is, a basal period in the morning and two experimental periods in the afternoon. We found similar patterns of biliary output in each time period for small i.v. doses of conventional exogenous markers [3H-taurocholate, phenolphthalein glucuronide, indocyanine green, and horseradish peroxidase] and for less commonly studied endogenous markers [glucose, inorganic phosphate (Pi), total protein, and leucine aminopeptidase]. This temporal stability indicates a lack of confounding circadian variability for these markers during the course of the biliary function study. Biliary excretion patterns of these marker solutes (e.g., rapid high recoveries of phenolphthalein glucuronide and low concentrations of Pi and glucose) demonstrated that our system for bile function studies is associated with intactness of the examined pathways of bile formation. These results validate our system and set of marker solutes for in vivo biliary function studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2006

Amino terminal domains of human UDP-glucuronosyltransferases (UGT) 2B7 and 2B15 associated with substrate selectivity and autoactivation.

Despite the important role of UDP-glucuronosyltransferases (UGT) in the metabolism of drugs, environmental chemicals and endogenous compounds, the structural features of these enzymes responsible for substrate binding and selectivity remain poorly understood. Since UGT2B7 and UGT2B15 exhibit distinct, but overlapping, substrate selectivities, UGT2B7-UGT2B15 chimeras were constructed here to identify substrate binding domains. A UGT2B7-15-7 chimera that incorporated amino acids 61-194 of UGT2B15 glucuronidated the UGT2B15 substrates testosterone and phenolphthalein, but not the UGT2B7 substrates zidovudine and 11alpha-hydroxyprogesterone. Derived apparent K(m) values for testosterone and phenolphthalein glucuronidation by UGT2B7-15((61-194))-7 were similar in magnitude to those determined for UGT2B15. Moreover, glucuronidation of the non-selective substrate 4-methylumbelliferone (4MU) by UGT2B7-15((61-194))-7 and UGT2B15 followed Michaelis-Menten and weak substrate inhibition kinetics, respectively, whereas 4MU glucuronidation by UGT2B7 exhibited sigmoidal kinetics characteristic of autoactivation. Six UGT2B7-15-7 chimeras that incorporated smaller domains of UGT2B15 were subsequently generated. Of these, UGT2B7-15((61-157))-7, UGT2B7-15((91-157))-7 and UGT2B7-15((61-91))-7 glucuronidated 4MU, but activity towards the other substrates investigated here was not detected. Like UGT2B7, the UGT2B7-15((61-157))-7, UGT2B7-15((91-157))-7 and UGT2B7-15((61-91))-7 chimeras exhibited sigmoidal 4MU glucuronidation kinetics. The sigmoidal 4MU kinetic data were well modelled using both the Hill equation and the expression for a two-site model that assumes the simultaneous binding of two substrate molecules at equivalent sites. It may be concluded that residues 61-194 of UGT2B15 are responsible for substrate binding and for conferring the unique substrate selectivity of UGT2B15, while residues 158-194 of UGT2B7 appear to facilitate the binding of multiple 4MU molecules within the active site.

Amino Acid Motifs↗
PubMed2002

A hydrogel readout for autonomous detection of ions in microchannels.

Readouts that work autonomously on colorimetric principles were fabricated inside a microchannel by immobilizing dyes in a hydrogel matrix. Two pH-sensitive dyes (congo red and phenolphthalein) were studied. The response time of the readout varied between 20 s for phenolphthalein and 45 s for congo red dyes. The robustness of the dyed gels was tested at different flow rates (0.1, 0.25 and 0.5 mL min(-1)). Combinatorial readouts consisting of gels with the different dyes were fabricated and tested. Based on the limits of human perception, the fabrication methodology and the principles of functioning; general design rules for fabricating a readout device are discussed. The simple and low cost fabrication technique combined with autonomous functioning make the readout appropriate for portable microfluidic systems.

Journal Article↗
PubMed1975

Effects of pH indicators on various activities of chromatophroes of Rhodospirillum rubrum.

1. The effects of pH indicators on activities for ATP hydrolysis in the dark and ATP-Pi exchange in the dark were examined with chromatophores from Rhodospirillum rubrum. Of thirty-one pH indicators tested, eleven (metanil yellow, 2, 4-dinitrophenol, ethyl orange, bromocresol green, resazurin, neutral red, bromthymol blue, alpha-naphtholphthalein, o-cresolphthalein, phenolphthalein, and alizarin yellow G) almost completely inhibited the activities for ATP formation and ATP-Pi exchange at concentrations of 1 mM, and were studied in detail. 2. Of the eleven pH indicators, those other than alpha-naptholphthalein, o-cresolphthalein and phenolphthalein, when assayed at appropriate concentrations, inhibited ATP-Pi exchange, but not ATP hydrolysis. In ATP-Pi exchange, these eight pH indicators at the concentrations described above were competitive against Pi, and non-competitive against ATP. The remaining three kinds of pH indicators were non-competitive against either Pi or ATP, when assayed at concentrations of the dyes that inhibited both activities. 3. The amounts of pH indicators bound with chromatophores were measured. No correlation was found between the amounts of the bound dyes and the extents of their inhibition of either ATP formation or ATP-Pi exchange. 4. Ethyl orange (pKa=4.1) and 2, 4-dinitrophenol (pKa=3.9) stimulated ATP hydrolysis to the greatest extent. The latter dye was hardly bound with chromatophores. 5. The stimulatory effects of pH indicators on ATP hydrolysis were hardly affected by extraction of quinones from chromatophores. 6. Most of the pH indicators stimulated both succinate-cytochrome c2 and NADH-cytochrome c2 reductions in the dark. 7. The mechanism of uncoupling of the electron transfer system and the phosphorylation system by pH indicators and the mechanism of the coupling are discussed.

Adenosine Triphosphate↗
PubMed1988

Phosphatase activity is a constant feature of all isolates of all major species of the family Enterobacteriaceae.

In this study we evaluated phosphatase activity in members of the family Enterobacteriaceae by conventional methods and by a novel method. The novel method is based on the formation of bright-green-strained colonies by phosphatase-positive, but not phosphatase-negative, strains in the presence of a phosphate substrate, such as phenolphthalein monophosphate or 6-benzoylnaphthyl phosphate (6-BNP), and methyl green. A total of 1,055 strains belonging to 65 different species of Enterobacteriaceae were tested for green staining of the colonies in the presence of methyl green and either phenolphthalein monophosphate or 6-BNP and for phosphatase activity by three different conventional methods. With the sole exception of one Leminorella richardii type strain, all isolates of all of the species formed green-stained colonies in the presence of the substrate 6-BNP. All strains were phosphatase positive by all of the conventional methods.

Enterobacteriaceae↗
PubMed1994

Selective interaction of chemical dyes with inositol 1,4,5-trisphosphate recognition sites.

Inositol 1,4,5-trisphosphate (InsP3) is an important second messenger that interacts with a specific intracellular receptor and triggers a release of Ca2+ from intracellular stores. InsP3 is preferentially metabolized by two enzymes. A specific 5-phosphatase (InsP3 phosphatase) produces an inactive metabolite, inositol 1,4-bisphosphate, while a specific 3-kinase (InsP3 kinase) produces an active metabolite, inositol 1,3,4,5-tetrakisphosphate. With the goal of developing selective ligands of the diverse InsP3 recognition sites, we have studied the effects of some chemical dyes on the binding of InsP3 to its receptor and on the activity of its metabolic enzymes. Although these dyes possess similar chemical structures, they showed varied selectivities towards the three recognition sites. Thymol Blue was the most potent inhibitor of InsP3 binding activity, with an IC50 of 105 microM. Phenol Red demonstrated a higher selectivity for InsP3 phosphatase inhibition, with an IC50 of 100 microM. 3',3",5',5"-Tetraiodophenolsulfonephthalein showed its most potent inhibitory effect on InsP3 kinase activity, with an IC50 of 35 microM. Tetrabromophenol Blue potently inhibited InsP3 phosphatase and InsP3 kinase activities, with respective IC50 values of 25 and 12 microM. Phenolphthalein Diphosphate and Phenolphthalein Carbinol Disulfate demonstrated weak inhibitory effects towards the three recognition sites for InsP3. These results reveal certain structural clues that should help in the development of more selective inhibitors.

Adrenal Cortex↗
PubMedDate not supplied

An evaluation of the hemizygous transgenic Tg.AC mouse for carcinogenicity testing of pharmaceuticals. I. Evidence for a confounding nonresponder phenotype.

We have completed 2 26-wk studies to evaluate the hemizygous transgenic Tg.AC mouse, which has been proposed as an alternative short term model for testing carcinogenicity. We attempted to evaluate the response to the known rodent carcinogens cyclophosphamide, phenolphthalein, and tamoxifen and to the noncarcinogen chlorpheniramine following topical application. In the first study, a weak response (2/17 animals) was observed to the positive control 12-O-tetradecanoylphorbol 13-acetate (TPA in ethanol, 1.25 micrograms), and no response was observed to cyclophosphamide, phenolphthalein, or chlorpheniramine, despite evidence for skin penetration. The second study compared 1.25 micrograms and 6.25 micrograms of TPA in ethanol and acetone solutions. Tamoxifen was also evaluated in both solvents and orally. No significant response was observed to tamoxifen by skin paint or oral routes. Over 60% of the high dose TPA-treated animals showed no (0 or 1) papilloma response, and 30% of the animals each developed more than 32 papillomas. The heterogenous response to high dose TPA may be related to variability in the responsiveness of hemizygous animals. In light of these findings, further Tg.AC studies should employ homozygous animals, and the underlying cause for heterogeneity in the tumorigenic response of Tg.AC mice should be identified and eliminated.

Administration, Topical↗
PubMed2003

Skin breakdown and blisters from senna-containing laxatives in young children.

BACKGROUND: At the direction of the Food and Drug Administration, phenolphthalein was removed from all over-the-counter laxatives in 1999. Phenolphthalein was then replaced in most laxative products with the natural product senna from Cassia acutifolia Delile, which contains various anthraquinones. No data are available on the safety of senna use in children <6 years of age. OBJECTIVE: To describe the clinical outcomes of exposure to unintentional ingestion of senna-containing laxatives in young children. METHODS: All ingestion exposures of senna-containing laxatives in children <5 years of age from 6 poison centers over a 9-month period were evaluated. Inclusion criteria required 24-hour follow-up and the presence of diarrhea to confirm ingestion. Parents were told routinely that severe diaper rash was possible and to protect the perianal area with frequent cleansing and a barrier ointment if the child was wearing diapers. RESULTS: During the study period, 111 cases were reported: 19 children experienced no diarrhea, 4 were lost to follow-up, and 88 exposures were evaluated. Fifty-two children (59%) were </=2 years old. Fifty children remained in diapers, 28 children were fully toilet trained, and 10 wore diapers (pull-up pants) overnight. Twenty-nine children (33%) experienced severe diaper rash. The mean +/- SD time to recognition of the diaper rash was 15.6 +/- 8.6 hours. Ten children (11%) had blisters and skin sloughing. There was a significant increase in severe diaper rash (p < 0.05) and onset of blisters and skin breakdown (p < 0.05) in children wearing diapers versus those who were fully toilet trained. The mean time to onset of blisters was 14.5 +/- 6.8 hours. Skin burns and loss were seen primarily on the buttocks and perineum, loosely following the diaper area. CONCLUSIONS: Unintentional ingestion of senna-containing laxatives in young children may potentially cause severe diaper rash, blisters, and skin sloughing.

Blister↗
PubMed1992

A fully automated enzyme immunoassay for the measurement of cortisol in biological fluids.

This paper describes a fully automated assay on the Serono SR1 for the measurement of cortisol in serum, heparinised plasma and urine. The assay incorporates a specific polyclonal antibody to cortisol and cortisol conjugated to alkaline phosphatase as a label. Following immunoincubation, bound and free labelled cortisol are separated by magnetic sedimentation of the antibody complex. Phenolphthalein is liberated by the enzymatic hydrolysis of the substrate (phenolphthalein monophosphate) and the absorbance generated is measured as the assay end-point. All processing steps are performed by the SR1. The assay has good analytical performance with respect to precision (within and between run CVs less than 10 and 11.5% respectively), detection limit (less than 5 nmol/l) and recovery of added cortisol (100 +/- 10%). The assay agrees well with cortisol concentrations determined by ID-MS and by established immunoassays (r > 0.96). Reference ranges of normal urine samples (pretreated by solvent extraction) are in good agreement with accepted values. The study demonstrates that the SR1 Cortisol assay on the SR1 analyzer is suitable for the routine determination of cortisol in serum, heparinised plasma and urine samples.

Adult↗
PubMed1993

An enzyme-linked immunoadsorbent assay for human pituitary glycoprotein free alpha-subunit: evaluation and initial experience in patients with pituitary disease.

An enzyme-linked immunoadsorbent assay (ELISA) for measuring human pituitary glycoprotein free alpha-subunit is described. Pituitary tumours may secrete glycoprotein hormones, their free subunits (alpha, beta) or a combination of these. Non-functioning adenomas often secrete free alpha-subunit. Assays for free alpha-subunit have previously used radioimmunoassay or immunoradiometric principles. Some of these methods are time-consuming and lack specificity and sensitivity. In order to overcome these problems, we have developed a two-site ELISA for alpha-subunit which uses a monoclonal antibody to alpha-subunit as the capture antibody to provide greater specificity. An affinity-purified polyclonal anti alpha-subunit conjugated to alkaline phosphatase was the signal antibody. Detection and quantification were based on phenolphthalein monophosphate conversion to phenolphthalein. The ELISA specifically measured glycoprotein free alpha-subunit in serum or plasma, discriminating between alpha-subunit and the intact glycoprotein hormones. The assay can be completed in 4 h. The assay sensitivity was 0.03 micrograms/l, and a normal range of 0.05 to 0.22 micrograms/l was established. In a retrospective study, elevated circulating glycoprotein alpha-subunit was detected in 22% of patients with non-functioning pituitary tumours previously treated with surgery and/or radiotherapy.

Adolescent↗
PubMed1981

Inhibition of housefly oxidative detoxication by phthaleins, fluoresceins and related compounds.

1. Phenolphthalein, halogenated fluoresceins, and other triphenylmethane and diphenylmethane derivatives inhibited biphenyl hydroxylation, aldrin epoxidation and several O-dealkylations in insect abdomen homogenates. Phenolphthalein and eosin (50 muM) were 2-3 times more effective than SKF 525-A and piperonyl butoxide (50 muM) as inhibitors of biphenyl hydroxylation in vitro. 2. The phthaleins, Aurin and Aluminon, inhibited both epoxidation and hydroxylation to similar extents, but fluoresceins, Rhodamine B, Malachite Green, and basic diphenylmethane derivatives preferentially inhibited hydroxylation. 3. Tetrabromophenolphthalein ethyl ester and bis-(N-dimethyl-4-aminophenyl-methane inhibited biphenyl hydroxylation in vivo. Bis-(N-dimethyl-4-aminophenyl) methane synergized the toxic effects of 1-naphthyl N-methylcarbamate in live houseflies.

Abdomen↗
PubMed1981

Substrate specificity of human UDP-glucuronyltransferase in cultured lymphocytes.

1. This study establishes the presence of UDP-glucuronyltransferase activity for non-steroidal as well as steroidal substrates, in cultured human B-lymphocytes. Glucuronidation of alpha-naphthol and testosterone was demonstrated in homogenates of two cell lines, SN1006 and RPMI-1788, and that of phenolphthalein, 4-methylumbelliferone, p-nitrophenol and estradiol in the cell line with the higher glucuronyltransferase activity, SN1006. 2. Kinetic studies of testosterone glucuronidation in homogenates of both cell lines revealed a similarity in the behaviour of glucuronyltransferase of these cells. Thus, comparable apparent Km values for UDPGA (0.63 mM) and for testosterone (14 microgram) were observed, although apparent maximal velocities, Vmax, differed several-fold (3.0 versus 0.55 pmol/10(6) cells per min, in SN1006 and RPMI-1788 cells, respectively). 3. Kinetic studies of glucuronidation of testosterone, estradiol, phenolphthalein, alpha-naphthol, 4-methylumbelliferone, and p-nitrophenol yielded comparable apparent Km values for UDPGA (0.56-0.67 mM), suggesting that the same, or similar, glucuronyltransferase(s) catalyse(s) glucuronidation of this wide range of substrates in lymphocytes. This was reinforced by the observation of competitive inhibition of testosterone glucuronidation by alpha-naphthol (Ki 0.25mM), 4-methylumbelliferone (Ki 0.8mM) and p-nitrophenol (Ki 0.8 mM). Thus, lymphocyte glucuronyltransferase activity with a broad substrate specificity, for steroidal and non-steroidal aglycones, is indicated.

Cell Line↗
PubMed1993

Extent and implications of interspecies differences in the intestinal hydrolysis of certain glucuronide conjugates.

1. Interspecies differences in the intestinal hydrolysis of glucuronide conjugates (phenolphthalein glucuronide, 4-methylumbelliferone glucuronide, morphine-3-glucuronide) were evaluated in mouse, rat and rabbit small intestine and caecum. 2. beta-Glucuronidase activity in the caecum was 50-200-fold higher than in the small intestine for all species and substrates studied. 3. There was evidence of a similarity between species in the capacity of the gut contents cultures from the proximal and distal small intestine to hydrolyse phenolphthalein glucuronide and 4-methylumbelliferone glucuronide. 4. Morphine was not liberated from morphine-3-glucuronide in detectable amounts in the proximal and distal small intestine. 5. Species- and substrate-specific differences were identified in the capacity of the caecal microbiota to hydrolyse the glucuronide conjugates studied. 6. The capacity of the rabbit caecal microbiota to hydrolyse all three glucuronides was significantly lower than those of both rat and mouse. 7. Morphine was metabolized to codeine in low, but detectable levels in all three species.

Animals↗
PubMedDate not supplied

Colorimetric determination of alkaline phosphatase as indicator of mammalian feces in corn meal: collaborative study.

In the official method for rodent filth in corn meal, filth and corn meal are separated in organic solvents, and particles are identified by the presence of hair and a mucous coating. The solvents are toxic, poor separation yields low recoveries, and fecal characteristics are rarely present on all fragments, especially on small particles. The official AOAC alkaline phosphatase test for mammalian feces, 44.181-44.184, has therefore been adapted to determine the presence of mammalian feces in corn meal. The enzyme cleaves phosphate radicals from a test indicator/substrate, phenolphthalein diphosphate. As free phenolphthalein accumulates, a pink-to-red color develops in the gelled test agar medium. In a collaborative study conducted to compare the proposed method with the official method for corn meal, 44.049, the proposed method yielded 45.5% higher recoveries than the official method. Repeatability and reproducibility for the official method were roughly 1.8 times more variable than for the proposed method. The method has been adopted official first action.

Alkaline Phosphatase↗