Major phenolic acids and total antioxidant activity in Mamaki leaves, Pipturus albidus.

Three phenolic acids, (+)catechins, chlorogenic acid, and rutin, were identified and quantified in Mamaki leaves using a liquid chromatograph-mass spectrometer technique. Concentrations of (+)catechins, chlorogenic acid, and rutin varied from 1.1 to 5.0 mg/g of Mamaki leaves as determined in the extract using 0.5% acetic acid in 90% aqueous methanol. This study also quantified total antioxidant capacity using the photochemiluminescence method, which was expressed in equivalents to ascorbic acid (AA). Mamaki teas brewed for 30 min contained total antioxidant activity (TAA) between 238 and 259 mg AA/g of tea. Mamaki teas brewed for 1 h and stored at 4 h, 1 d, and 3 d at 4 degrees C had available TAA 293, 271, 172, and 163 mg AA/g of tea leaves, respectively. The concentrations of (+)catechins and rutin in Mamaki leaves are compared to other types of popular teas. Mamaki teas contained relatively low amounts of TAA compared to green teas and Lipton teas.

A rapid hemolysis assay for the detection of sodium channel-specific marine toxins.

Current methods of detection for fish and shellfish biotoxins in monitoring and research purposes are either labor intensive, expensive, require specialized techniques or all of the above. This paper reports on the development of a fairly sensitive, rapid, and inexpensive assay which detects the presence of compounds that affect the sodium channel. It is based on the principles of the mouse neuroblastoma tissue culture assay for sodium channel specific-biotoxins using red blood cells (RBCs) from the red tilapia (Sarotherodon mossambicus). This assay has the potential to complement the use of live animal bioassay testing for marine toxins. Veratridine, a sodium channel activator and ouabain, an inhibitor of Na(+)/K(+) ATPase, both react with the tilapia RBCs by affecting the permeability of the cell's membrane. Saxitoxin (STX), its analogs, and tetrodotoxin (TTX) can inhibit the action of veratridine and ouabain leaving the cell morphologically normal. By sequencing the addition of veratridine and ouabain, with either the extracted samples, saxitoxin, tetrodotoxin, or ciguatoxin (CTX-a sodium channel activator) to the RBCs a sodium channel antagonist or activator can be detected. Results using pure concentrations of a sodium channel-specific toxin could be detected to inhibit hemolysis at a concentration of 0.3 microg/ml STX, 3.5 microg/ml for neo-STX, 3.0 microg/ml for GTX, and 5.0 microgl for TTX in the presence of ouabain and veratridine. CTX was detected at a concentration of 50 microg/ml. The RBCs from the red tilapia was used due to the fish's ability to osmoregulate its internal environment to survive in both fresh and saltwater. In addition, with growing opposition to live animal testing, this assay has been designed as a non-lethal means of testing for sodium channel affecting marine toxins. No test animals are sacrificed and blood may be drawn from the same fish for continued sample testing.

Formation of mutagens during the frying of Hawaiian fish: correlation with creatine and creatinine content.

Compounds mutagenic toward Salmonella typhimurium strain TA98 in the presence of rat-liver homogenates (S9) were formed when fish flesh was fried at 199 degrees C. Three species of Hawaiian fish commonly consumed in Hawaii (skipjack tuna, Katsuwonus pelamis; yellowfin tuna, Neothunnus macropterus; and milkfish, Chanos chanos) were cooked in an electric skillet, along with samples of sole (Microstomus pacificus). Organic extracts of the fish were tested in the Ames Salmonella mutagenic assay using tester strain TA98 and S9. Basic organic extracts of fried, but not raw, samples exhibited significant mutagenicity. The levels of mutagenicity were also higher among the red flesh Hawaiian fish ('ahi, aku and awa) than with the white flesh sole. Creatine and creatinine contents were highest in the Hawaiian fish and lower in the sole. Creatine levels in the fish were 50-100 times greater than the creatinine content and varied from a high of 645 mg/100 g wet weight of fish for yellowfin tuna to a low value of 251 mg/100 g for sole. Mutagen levels are only approximately related to creatine/creatinine levels suggesting that other components contained in these fish may be as important as the guanidines in determining the levels of mutagen in the cooked fish.

Mutagen formation during commercial processing of foods.

Levels of bacterial mutagenicity 3-17 times above spontaneous are generated during commercial thermal processing (canning) of foods, particularly foods high in protein. The potential for other processing operations, including pasteurization, dehydration, and concentration, to produce substances active in the Ames Salmonella assay was also examined. Two heated fish model systems, canned salmon and fried sole, were established by extracting mutagen precursors from fish tissues with water. The model system studies suggest that the limiting reactants for mutagen formation differ from one food product to another, and that Maillard type browning reactions are involved in mutagen production. Bisulfite treatment was found to inhibit mutagen formation in modal systems and whole food products. Isolation and partial characterization of the mutagens in both fried and canned pink salmon showed that at least three distinct mutagens were present. These mutagens exhibited HPLC retention time patterns on C18, cyano, and amino columns different than the major mutagens present in other cooked and grilled meats and fish.

Occurrence of mutagens in canned foods.

Mutagens are shown to be present in a variety of commercially heat-processed foods. Since these substances are not present in the unheated raw material, it appears that they are produced during processing. Canned salmon and beef broth showed the highest mutagenicity while other canned beef and fish products yielded lower but detectable levels. These findings are significant not only because of the large proportion of the food supply which is processed by canning, but also because the mutagens in these foods exhibit chemical behaviors and Salmonella strain specificity similar to mutagens in grilled foods which have been shown to be mammalian carcinogens.

Enzymatic transformation of PSP toxins in the littleneck clam (Protothaca staminea).

Stemming from investigations into the relationship between toxins produced by Gonyaulax sp. and accumulated in shellfish, we wish to report enzymatic transformations of the PSP toxins to decarbamoyl derivatives in the littleneck clam (Protothaca staminea). No toxin transformations were observed in either mussels (Mytilus edulis) or in butter clams (Saxidomus giganteus). In addition, littleneck clam samples from the natural environment contained predominantly the decarbamoyl derivatives, while other shellfish species collected from the same vicinity contained the previously reported PSP toxins.

High pressure liquid chromatographic determination of toxins associated with paralytic shellfish poisoning.

A high pressure liquid chromatographic procedure is described for assay of toxins associated with paralytic shellfish poisoning (PSP). The method is applicable to saxitoxin, neosaxitoxin, gonyautoxins I through IV, and their sulfocarbamoyl derivatives. Toxins are separated on a bonded phase cyano column and detected by fluorescence following alkaline oxidation (NH+4 and periodic acid). The utility of the HPLC procedure for research and monitoring is discussed.

Effects of benzo(a)pyrene on early development of flatfish.

The ontogenetic effects of the environmental carcinogen benzo(a)pyrene (BAP) on three species of larval flatfish were investigated using concentrations (from 0.10 to 4.2 ppb) which were comparable to levels found in polluted harbors. BAP-treated sand sole (Psettichthys melanostichus) eggs displayed a significant decline in hatching success and a significantly higher incidence of developmental anomalies than did control eggs. Flathead sole (Hippoglossoides elassodon) eggs exposed to a single dose of a water-soluble BAP-bovine serum albumin complex demonstrated evidence of toxic injury with pycnotic nuclei present in the integument and, more commonly, in ocular and neural tissues. An increased incidence of morphological anomalies in English sole (Parophyrs vetulus) eggs and larvae exposed to BAP was not detected.

Mutagen formation during the cooking of fish.

Compounds mutagenic toward Salmonella typhimurium strains sensitive to frameshift mutation (1537, 1538 and TA98) were formed when fish flesh was fried at 190 degrees c. Four species of marine fish commonly consumed in the United States were cooked in an electric skillet and broiled beneath the elements of an electric oven. Organic extracts of the fish were tested in the Salmonella mutagenic assay using strains 1535, 1537, 1538, TA98 and TA100. Basic organic extracts of fried but not raw or broiled samples exhibited significant mutagenicity with metabolic activation. Mutagenic activity ratios ranging from 3.3 to 15.7 for the extract from 20 g of fish were observed. The mutagenicity produced during the frying of fish was dependent on time. Frying times of less than 6 min produced no mutagenic activity, while at 6 min or greater substantial mutagenicity was generated.

Uptake of benzo[a]pyrene by gonadal tissue of flatfish (family Pleuronectidae) and its effects on subsequent egg development.

Accumulation of benzo[a]pyrene (BaP) by sexually mature flatfish gonad, its transfer to developing gametes, and its subsequent effects on developing embryos were studied. Thin-layer chromatography revealed both unmetabolized BaP and polar metabolites in the ovary, wolffian ducts, oocytes, and semen of English sole 24 h after ip injection with 200 microCi [3H]BaP. Concentrations of BaP and its metabolites were 3-11 times higher in oocytes and semen than in gonadal tissue. Fertilized eggs from flathead sole that had been fed 4.0 mg BaP 5 h before spawning demonstrated a significantly lower (p less than 0.001) hatching success (11.9%) than eggs from control fish (56.6%). Morphological abnormalities were found in only 1.6% of control embryos but in 5.6% of embryos from treated females.

Effect of pH and ammonium ions on mutagenic activity in cooked beef.

Controversy surrounding the extraction procedure commonly used for isolating and concentrating mutagens from foods has resulted in a need for the re-examination of the reported mutagenicity in fried hamburgers. Using a procedure in which Na2SO4 and NaOH are substituted for (NH4)2SO4 and NH4OH respectively, mutagenic activity in extracts of hamburgers fried for 5 min appeared to be unchanged. However, when organic extractions are performed at pH conditions more moderate than those generally employed to isolate mutagens from foods, a 30-50% decrease in mutagenicity is observed.

A source of error in mutagen testing of foods.

Mutagenic compounds reported to be present in foods may be forming during the extraction process rather than during cooking or baking. In this study, the formation of mutagenic substances in biscuits was examined using in the extraction procedure either ammonium sulfate and ammonium hydroxide or sodium sulfate and sodium hydroxide. Compounds producing high mutagenic activity in Salmonella strains 1538 and TA 98 obtained from aqueous biscuit extracts containing ammonium ions. No mutagenic activity was observed in extracts from aqueous biscuit extracts containing sodium ions until some ammonium ions (NH4OH) were added. We suggest that ammonium sulfate and ammonium hydroxide not be used in the extraction procedure of food when studying mutagen formation.

Nutritional effects of the cyclic monomers of methyl linolenate in the rat.

Low levels (0.0075, 0.0225, and 0.15%) of cyclic fatty acid methyl esters (greater than 98% pure) were incorporated into diets of weanling rats fed different levels of protein. Animals on low protein diets (8-10% casein) exhibited decreased wt gains and feed consumption with increasing levels of cyclic esters in their diets after 6 weeks. Liver enlargements due to a significant (P less than 0.01) accumulation of liver lipid were noted in animals receiving 0.15% cyclic fatty acid esters in their diets.

Chromatographic separation of conformers of substituted asymmetric nitrosamines.

The syn and anti conformers of N-nitrosoproline, N-nitrososarcosine and N-nitroso-2-(ethylamino)-ethanol, have been separated by liquid chromatography. These conformers result from hindered rotation about the N-N bond. Separation was achieved using adsorption, reversed-phase, and ion-exchange modes. For the nitroso-amino acids, a shift in the equilibrium conformer concentration was observed with changes in pH.