Measurement of sugars and starches in foods by a modification of the AOAC total dietary fiber method.

A separation scheme for the determination of sugars and starch in processed food was developed. It is based on AOAC Method 985.29 for total dietary fiber with these modifications: carbohydrate starches are separated into soluble and insoluble fractions before they are hydrolyzed; acetonitrile is used instead of ethanol to separate sugars from enzyme-resistant carbohydrates, proteins, and other macromolecules; and a solid-phase extraction filter is included to remove substances that interfere with high-performance liquid chromatography (HPLC). Recovery studies indicate a > 97% sugar recovery. Twenty foods were analyzed. After enzymatic hydrolysis, fructose, glucose, sucrose, maltose, and lactose were extracted and determined by HPLC using a refractive index detector. Starch content was calculated from the increase in the amount of glucose. The results were compared with values listed on the "Nutrition Facts" panel for that food. The analyzed amounts of sugars and starches were 73-96% of declared values.

Uptake, translocation, and transformation of pentachlorophenol in soybean and spinach plants.

Soybean plants were grown for 90 days and spinach plants for 64 days in a mixture of sterilized greenhouse soil and sand containing 10 ppm pentachlorophenol. All plant parts and soil samples were extracted and separated into nonpolar and polar fractions. Major nonpolar and polar metabolites were identified by gas-liquid chromatography and mass spectrometry. Nonpolar fractions from both soybean and spinach plants were found to contain pentachlorophenol and its metabolites, 2,3,4,6-tetrachlorophenol, methoxytetrachlorophenol, 2,3,4,6-tetrachloroanisole, and pentachloroanisole. Cleavage of polar metabolites from the soybean plants by acid hydrolysis yielded organic solvent-extractable products. These products were identified as pentachlorophenol, 2,3,4,6-tetrachlorophenol, and methoxytetrachlorophenol. Cleavage of polar materials from spinach plants yielded only pentachlorophenol. The polar metabolites from the soybean plants were also subjected to enzymatic cleavage by beta-glucosidase. The conjugates consisted mostly of O-glucosides of the same metabolites released by acid hydrolysis. Failure of hydrolysis by aryl sulfatase indicated that very little or no sulfates were present. The metabolites found in the plants were not detected in soil samples obtained from pots immediately after the plants were harvested.

Screening of fresh water fish extracts for enzyme-inducing substances by an aryl hydrocarbon hydroxylase induction bioassay technique.

A sensitive biological test to detect the presence of certain contaminants, such as highly toxic halogenated dioxins, dibenzofurans, and biphenyls in foods, was applied to extracts of fresh water fish that had been prepared by a food extraction-cleanup procedure developed by the Food and Drug Administration for pesticides and industrial chemicals. Aryl hydrocarbon hydroxylase (AHH) activity in a rat hepatoma cell line was used as the biological detection system for residues that induce enzyme activity. The induction of AHH activity by the extracts was compared with a standard AHH-induction curve for the most active compound known, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and results were computed as TCDD equivalents. Several dilutions of fish extracts were used to produce AHH-induction curves from which an optimal dose-response range was determined and used to estimate TCDD equivalents. Cleaned-up extracts of fish obtained from different water bodies in the United States were examined for AHH activity. The samples which had low levels of polyhalogenated contaminants produced low biological activity, while a higher activity was obtained from fish that contained higher levels of polyhalogenated contaminants. The results suggest that the fish extracts can be screened for AHH inducers before chemical analysis.

Influence of Mycoplasma arginini infection on the induction of aryl hydrocarbon hydroxylase by TCDD in rat hepatoma cell cultures.

Mycoplasma arginini was eliminated from a rat hepatoma cell line (H-4-II-E) by plating at low cell density and treatment with chlortetracycline (250 micrograms/ml), kanamycin (250 micrograms/ml), tylosin (100 micrograms/ml), 3% M. arginini antiserum and 5% fresh guinea-pig serum. The induction of AHH activity in the cell culture was measured in response to increasing concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The ED50 values (estimated doses that produce 50% maximum enzyme induction) were calculated to be 0.256, 0.452 and 0.344 pmol TCDD/plate for original, mycoplasma-free and reinfected cells, respectively. Although the absence of M. arginini in the rat hepatoma cell line makes the cells slightly less responsive to AHH induction by TCDD, this decrease does not detract from the use of the method to screen food extracts and environmental samples for the presence of certain toxic planar organic compounds.

Induction of aryl hydrocarbon hydroxylase activity in cell cultures by aroclors, residues from yusho oil samples, and polychlorinated biphenyl residues from fish samples.

Four Aroclor reference materials, cleaned-up extracts of 2 yusho rice oil samples, and cleaned-up extracts of 3 fish samples containing polychlorinated biphenyl (PCB) residues were tested for their ability to induce aryl hydrocarbon hydroxylase (AHH) activity in a rat hepatoma cell line. Before the AHH bioassay, the samples were fractionated by a Florisil column chromatographic method. All samples contained about 1000 micrograms PCBs before Florisil column chromatography. The first Florisil eluate contains about 95% of the PCBs in a typical Aroclor, and the second contains the more polar or adsorbent PCB congeners. In this study, the first eluate for all samples produced no quantifiable AHH activity. The second Florisil eluates of both Aroclors 1242 and 1248 induced AHH activity, whereas these eluates of both Aroclors 1254 and 1260 did not. This difference may be due to the presence in Aroclors 1242 and 1248 of 3,3',4,4'-tetrachlorobiphenyl, which has not been detected in Aroclors 1254 and 1260. The second Florisil eluates of the fish samples induced somewhat less AHH activity than did Aroclor 1242 or 1248. The second Florisil eluates of the PCB residues from yusho rice oil samples induced significantly greater AHH activity than these eluates of either Aroclor 1242 or 1248, perhaps because yusho rice oil contains a greater amount of polychlorinated dibenzofurans than PCB commercial mixtures on a PCB equivalent basis.

Cadmium-binding components in soybean plants.

Soybean (Glycine max L.) plants exposed to (109)Cd readily absorb the element. Differential centrifugation of leaf, stem, and root homogenates followed by radioassay showed that Cd was associated primarily with the 105,000g supernatant. Separation of this fraction by gel chromatography and subsequent analysis by radioassays revealed that (109)Cd was bound to macromolecules of >50,000, 13,800, and 2,280 molecular weights. The >50,000 and 2,280 molecular weight fractions probably are nonspecific binding of Cd to normal cell components. The 13,800 molecular weight (109)Cd-bound component was found to be inducible by cadmium. It had a high ultraviolet absorbance at 254 nm and a low absorbance at 280 nm at pH 8.6.

Induction of enzyme activity in cell culture: a rapid screen for detection of planar polychlorinated organic compounds.

Induction of aryl hydrocarbon hydroxylase (AHH) activity in rat hepatoma cell line serves as a simple and rapid method to detect minute (pg) amounts of certain classes of compounds, e.g., dibenzo-p-dioxins, dibenzofurans, and biphenyls. This method may provide a quick screen for such substances in extracts from foods prior to chemical identification. AHH activity is measured by conversion of benzo[a]pyrene (BP) to 3-hydroxy BP in homogenized cell extracts from control and treated cultures and is reported as pmol product formed/mg protein/min. Substances screened by this method include polyhalogenated analogs of dibenzo-p-dioxin (24 compounds), dibenzofuran (11 compounds), biphenyl (7 compounds), and extracts from several food sources. Response of the most reactive compound, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was used to prepare a standard curve, and the AHH activity induced by mole doses of test substance is reported as an ED50 response (the estimated dose needed to produce 50% maximum enzyme induction). The AHH activity induced by food extracts is equated to the standard curve and reported as TCDD equivalents. A potent ED50 response in cell culture appears to correlate well with known toxic responses in other mammalian and avian systems for certain test substances. This correlation suggests that the cell culture enzyme induction method is a useful model for screening food extracts that are suspected to be contaminated with polychlorinated planar substances. A collaborative study would demonstrate the reproducibility of the method.

The distribution and binding of cadmium in oyster, soybean, and rat liver and kidney.

Exposure to Cd and/or 109Cd has shown that the element was efficiently accumulated in oysters and soybeans as well as in rats. Differential centrifugation of oyster, soybean, rat liver, and rat kidney homogenates followed by analysis showed that Cd was associated primarily with proteins in the 105,000 X g supernatants. Separation of these proteins by Sephadex chromatography and subsequent analysis by atomic absorption spectroscopy or by radioactivity measurements revealed that Cd in oysters and rat organs was principally bound to proteins of 9,200 to 13,800 molecular weight. A significant amount of Cd in oysters was also associated with fractions of greater than 50,000 and less than 3,000 molecular weights. Almost all of the Cd in soybeans was found to be bound to molecules of greater than 50,000 molecular weight.