Stability of picrotoxin during yogurt manufacture and storage.

Picrotoxin is a neurotoxin found in the berries of Anamirta cocculus, a plant native to Southeast Asia. Picrotoxin has potential for being used as a biological weapon since the toxin is relatively easy to isolate and purify. Limited information exists on the stability and detection of picrotoxin added to foods before or after processing. The objective of this study was to determine the stability of picrotoxin during yogurt manufacture and storage. Direct, cup-set yogurt was produced by using methods that mimic the conditions used in full-scale production of yogurt. Milk (full-fat or low-fat) was pasteurized at 85 degrees C for 30 min, and then cooled to 43 degrees C. Yogurt starter culture (thermophilic culture or thermophilic + probiotic culture) and picrotoxin (200 mug/mL milk) were added. Samples of yogurt during fermentation (5 to 6 h, 43 degrees C) and during 30 d refrigerated (4 to 6 degrees C) storage were analyzed for pH, titratable acidity, and picrototoxin levels. Regardless of starter culture used or fat content of milk, there were no significant differences in the pH and titratable acidities of the picrotoxin-spiked yogurt and the control yogurt (no added picrotoxin) during fermentation and up to 4 wk of refrigerated storage. The color or texture of the yogurt was not affected by addition of picrotoxin. Levels of picrotoxinin and picrotin (components of picrotoxin) in yogurt, as measured by LC/MS (APCI(+)/SIR) did not change significantly during fermentation and storage. A separate experiment determined that addition of picrotoxin to milk before pasteurization (85 degrees C, 30 min) did not affect picrotoxin stability. These results indicate that picrotoxin is stable in yogurt during manufacture and storage.

Effect of processing on Fusarium mycotoxins.

Mycotoxins are secondary metabolites produced by a wide variety of fungal species that contaminate food or feed. Fumonisins (FUM), deoxynivalenol (DON) and zearalenone (ZEN) are examples of common mycotoxins in grains that have been shown to affect human and/or animal health. Physical, chemical and biological methods have been used for decontaminating grains containing these toxins. Some treatments reduce the concentration of mycotoxins while others are ineffective. For example, removal of damaged grain by density segregation can reduce DON and ZEN concentrations in corn and wheat. In contrast, thermal processing is usually ineffective for reducing the FUM and ZEN content of foods. More work is needed to identify effective methods for detoxifying mycotoxin contaminated food.

Effect of thermal processing on the stability of fumonisins.

Fumonisins, a group of mycotoxins produced by Fusarium moniliforme in corn, have been implicated in several animal and human diseases. F. moniliforme and the fumonisins are an area of incresing concern for corn producers and consumers. Consequently, there is interest in reducing human and animal exposure to these fungal toxins. Studies of the effects of biological, chemical, and physical treatments on the reduction of fumonisin levels in food have shown variable results. Work was conducted at the U.S. Food and Drug Administration, National Center for Food Safety and Technology, to determine the effects of thermal processing on fumonisins B1 (FB1) and B2 (FB2) in an aqueous buffer. Parameters that were studied included processing time (0-60 min), processing temperature (100-235 degrees C), and buffer pH (4, 7, and 10). The rate and extent of fumonisin decomposition increased with processing temperature. Less than 27% of FB1 and less than 20% of FB2 were lost when processing temperatures were less than or equal to 125 degrees C for 60 min. After 60 min at 150 degrees C, losses of FB1 and FB2 were 80-90% at pH 4, 18-30% at pH 7, and 40-52% at pH 10. At temperatures greater than or equal to 175 degrees C, more than 80% of FB1 and FB2 was lost after 60 min. These results indicate that foods reaching temperatures greater than 150 degrees C during processing may have lower fumonisin levels. More work is needed to quantitate the effects of different processing operations (baking, extrusion, frying) on the fumonisin content of corn-based foods.

Establishing the cut-off concentration for the detection of etorphine in horse urine.

An 125I radioimmunoassay to determine the pattern of urinary excretion of etorphine (a semisynthetic opiate agonist) after its administration to horses is described. Three thoroughbred horses were each given 5, 15, 30 and 100 micrograms of etorphine intramuscularly. Urine was collected for up to 72 after administration. The maximum etorphine concentration after administration of a dose of 5 micrograms was 711 pg ml-1 (concentrations were greater than 100 pg ml-1 after 23 h in all three horses); a 15 micrograms gave 2661 pg ml-1 (levels remained above 100 pg ml-1 for more than 44 h in each horse); a 30 micrograms dose gave a maximum of 3344 pg ml-1 (levels were above 100 pg ml-1 for 24, 72 and 72 h); and 100 micrograms gave in excess of 10,000 pg ml-1 (levels were greater than 300 pg ml-1 for up to 70 h). Forty-eight urine samples from horses not given etorphine all had levels of etorphine less than 100 pg ml-1. There was no increase in apparent etorphine concentrations after hydrolysis of samples with beta-glucuronidase and aryl sulfatase. The half-lives of etorphine equivalents (calculated with a mono-exponential equation after the 100 micrograms dose) in the urine of the three horses were 569, 803 and 821 min, respectively. We conclude that radioimmunoassay can provide a useful first line screening procedure for the assessment of etorphine use in racing horses.

Development of an ELISA using a universal method of enzyme-labelling drug-specific antibodies. Part I: Detection of dexamethasone in equine urine.

The development, validation, and application of an ELISA for dexamethasone in equine urine is described. The drug-protein conjugate was immobilised in microtitre plate wells and antiserum raised against the same drug-protein conjugate was allowed to compete with sample or standard drug and the immobilised drug-protein conjugate. The proportion of antiserum binding to the immobilised drug-protein conjugate was detected using a biotinylated protein G/extravidin-alkaline phosphatase complex in situ and measurement of the substrate product. The method was used to detect the presence of drug-derived material in unextracted diluted urine after the administration of a single i.m. dose of dexamethasone at approximately 0.04 mg/kg to a thoroughbred horse. Validation of the method was carried out against a radioimmunoassay and GC/MS analysis.

Heterocyclic aromatic amine content of selected beef flavors.

Previous work has shown that meat extracts contain potent mutagenic and/or carcinogenic heterocyclic aromatic amines (HAAs). Because meat extracts and some beef flavors are produced from similar precursors and processing steps, the beef flavors may also contain HAAs. This study analyzed 24 commercial beef flavors and 2 food-grade beef extracts for creatine and creatinine concentrations, mutagenic activity and HAA concentrations (IQ, MeIQ, MeIQx, DiMeIQx, Glu-P-1, Glu-P-2 and PhIP). The creatine and creatinine levels of the flavors ranged from 0 to 73 and from 0 to 21 mg/g (dry wt.), respectively. The mutagenic activities of the flavors ranged from 0 to 3200 Salmonella typhimurium TA98 revertants/g (dry wt.). No direct relationship was found between creatine and/or creatinine concentrations and mutagenic activities. However, flavors with high creatine (> 1.5 mg/g) or creatinine (> 2 mg/g) levels exhibited higher mutagenic activities than did flavors with low levels of these compounds. Flavors with high mutagenic activities (> 1500 revertants/g) contained measurable amounts of HAAs. Three flavors contained MeIQx (7.2-21.2 ng/g [dry wt.]) and one contained DiMeIQx (4.2 ng/g [dry wt.]).

The effect of dairy products on iron availability.

Many researchers report substantial reductions in iron availability when dairy products are consumed with solutions of iron. Yet other studies indicate that dairy products have little effect on iron availability when added to complex meals. The conflicting data may be due to differences in the technique used to measure availability, species of animal used, form of iron in the diet, and meal composition. Human studies show superior bioavailability of iron in human milk when compared with cow's milk. Definitive causes for the differences between human and cow's milk have not been identified. Human milk contains lower amounts of casein, phosphate, and calcium, components thought to inhibit iron absorption. More work is needed to identify the factors that influence iron-dairy interactions. The nutritional benefits provided by dairy products outweigh the slight inhibitory effect they may have on iron availability.

Evaluation of antisera to 5-(2-bromo-E-ethenyl)-2'-deoxyuridine for the radioimmunoassay of a potent antiviral agent.

The production and evaluation of antibodies specific for 5-(2-bromo-E-ethenyl)-2'-deoxyuridine (BVDU) is described. Antibodies were raised against 5-(2-carboxy-E-ethenyl)-2'-deoxyuridine bovine albumin conjugates (CVDU20-BA and CVDU54-BA) and 5'-carboxy-propionyl-BVDU7-BA. The molar substitution ratio of the conjugates was determined by differential ultraviolet absorption or isotope dilution and compared to a newer method based upon the determination of unsubstituted protein residues. Usable antibody titers were obtained from all the New Zealand White rabbits immunized, but only one-third of the half-lop rabbits responded. The antisera were highly specific for BVDU and did not significantly cross-react with naturally occurring nucleosides, pyrimidines, or sugars. However, the lowest cross-reaction observed (0.21%) for the major metabolite bromovinyl uracil using antisera raised to CVDU-BA could not ensure adequate assay specificity under all conditions of use. An alternative approach using 5'-carboxy-propionyl-BVDU7-BA proved unsuccessful because the antisera could not discriminate between BVDU and bromovinyl uracil.

The evaluation and application of a radioimmunoassay for the measurement of diphenoxylic acid, the major metabolite of diphenoxylate hydrochloride (Lomotil), in human plasma.

Antibodies to diphenoxylic acid, the pharmacologically active metabolite of Lomotil, were successfully used to develop a precise and specific radioimmunoassay for the measurement of diphenoxylic acid in human plasma. The observed cross-reaction of the antiserum with Lomotil (23.5%) and p-hydroxy diphenoxylic acid (2.9%) was not considered to affect significantly the accuracy of the direct determination of diphenoxylic acid in plasma from human volunteers after ingestion of Lomotil tablets. Within-day and between-day coefficients of variation were better than 3 and 6%, respectively, over the concentration range of 3.4 to 255 ng ml-1. Comparable precision could be achieved at 2 ng ml-1 by doubling the volume of sample analyzed. The assay was used to measure plasma concentrations of diphenoxylic acid in 12 human volunteers for up to 24 hr after ingestion of Lomotil (10mg) tablets. Plasma diphenoxyllic acid levels rose to a mean (SE) maximum level of 87.8 (2.7) ng ml-1 3.3 (0.3) hr after dosing. By 24 hr after dosing plasma levls had decreased to 14.26 (1.67) ng ml-1. The appearance and elimination of plasma diphenoxylic acid could be described by a biexponential function. The appearance half-life was calculated to be 0.82 (0.09) hr, and the elimination half-life was 7.24 (0.73) hr.

Manganese protoporphyrin IX. A potential intravenous paramagnetic NMR contrast agent: preliminary communication.

An endogenous, biological chelating substance--protoporphyrin--has been studied in vitro and in vivo for potential usefulness as an MRI contrast agent. In vitro data show that manganese protoporphyrin IX (Mn PP) maintains strong paramagnetic properties. Limited in vivo studies suggest that Mn PP causes marked reduction of T1 in the liver, with less pronounced effects on other body tissues.

Haloperidol pharmacokinetics: a preliminary study in rhesus monkeys using a new radioimmunoassay procedure.

The development, validation, and application of a new radioimmunoassay for haloperidol in biological fluids is described. The antiserum, raised against N-amino-butyl chlorophenyl piperidine bovine albumin conjugate, could not distinguish between haloperidol and its reduced metabolite, but it could discriminate against chlorophenyl piperidine (cross-reaction 2.6%). The fluorophenyl metabolites of haloperidol were not recognized by the antiserum. Haloperidol determinations were made on less than 100 microliter aliquots of human and rhesus monkey plasma or diluted urine without prior extraction of the sample. The radioimmunoassay was applied to the study of the pharmacokinetics of intravenous haloperidol administration to two male rhesus monkeys. Salient features of the results are as follows. As with man, the plasma concentration versus time curve could be resolved into three compartments, but there were differences in the distribution of haloperidol between the compartments. The apparent volume of distribution for the two monkeys examined was 5.87 L kg-1 and 7.37 L kg-1, considerably smaller than in man, a difference almost entirely due to a much smaller tissue compartment. The biological half-life of 15.97 hr and 7.56 hr was similar to man. The mean hepatic extraction ratio was calculated to be 0.032 and 0.056, and the data suggested that hepatic metabolism of haloperidol may be of lesser importance in rhesus monkey than in man. An insignificant proportion (0.01%) of the administered dose was excreted as haloperidol in the urine.