Minicolumn chromatography: State of the art

It was recognized in the early 1960s that a rapid screening method for aflatoxin was needed. Holaday first proposed the minicolumn chromatography method as a rapid screening method in 1968. Since that time, many improvements have been made in this method. The latest minicolumn method has a limit of detection of 5μg/kg and can be completed in 5-7 min. The minicolumn technique has been expanded to include screening commodities other than peanuts for aflatoxin, as well as for other mycotoxins including ochratoxin, zearalenone and aflatoxin M 1.     

Aflatoxin content of peanut hulls

The degree of aflatoxin contamination in peanut hulls was determined by analyzing inoculated hand-shelled hulls and hulls from peanuts known to contain aflatoxin. Hulls adjusted to 20% moisture, inoculated withAspergillus flavus, and incubated 7 days at 25 C supported growth ofA. flavus but not aflatoxin production. Peanuts from 20 selected Segregation III (visibleA. flavus) lots contained 13–353 ppb of aflatoxin. The machine-shelled hulls from these lots were analyzed and 3 lots contained no detectable aflatoxin, 13 lots contained 4–88 ppb and 4 lots contained >116 ppb. Aflatoxin concentrations of 53–87 ppb were detected in hulls when peanuts containing relatively high levels of aflatoxin (up to 26.8 ppm in damaged kernels) were carefully machine-shelled. Hulls from the same samples obtained by hand-shelling contained no detectable aflatoxin. When machine-shelled hulls were screened through successively smaller screens, the aflatoxin concentration of the smallest fraction (<3.18 mm) was always highest and indicated that small peanut kernels and peanut parts in the hulls actually contained the aflatoxin. Separating hulls over a 4.76 mm round-hole screen appeared to provide a means of removal of most aflatoxin in peanut hulls. No aflatoxin was found in hulls from uncontaminated peanuts.     

Control and removal of aflatoxin

The best approach to contain the problem of aflatoxin is prevention and enough is now known about prevention to reduce contamination drastically. Guidelines for preventing mycotoxins in farm commodities have been suggested by the U.S. Department of Agriculture. Moisture is the single most important parameter and prompt drying to safe levels is essential for control of toxigenic molds. Foreign matter and damaged seed should be removed. Provision of clean, dry, adequately cooled and ventilated storage is important and good sanitation is essential to minimize mold contamination during storage and processing: Genetic approaches which may result in resistance to elaboration of aflatoxins are under investigation. When aflatoxin is found in a sample of oilseeds the contamination generally resides in only a small proportion of the kernels, commonly less than 1%. Sorting or separation can concentrate the vast majority of aflatoxin-contaminated kernels into relatively small fractions and only a small loss is incurred as a result of their removal. Aflatoxin is frequently found deeply imbedded within individual kernels so removal by simple washing does not seem feasible. However, extraction with polar solvents such as alcohols and ketones to achieve essentially complete removal of aflatoxins appears technically feasible. Heat is relatively ineffective for destruction of aflatoxin although normal roasting, as of peanuts for the preparation of peanut butter, results in considerable reduction in aflatoxin content. Treatment withFlavobacterium aurantiacum removes aflatoxin and may be useful for beverages. Oxidizing agents readily destroy aflatoxin, and treatment with hydrogen peroxide may be useful. Treatment of defatted oilseed meals with ammonia can reduce aflatoxin content to very low or undetectable levels with only moderate damage to protein quality. One of 21 papers presented at the Symposium, “Oilseed Processors Challenged by World Protein Need,” ISF-AOCS World Congress, Chicago, September 1970. So. Market. Nutr. Res. Div., ARS, USDA.     

Toxicity of Aflatoxin B1 to Helicoverpa zea and Bioactivation by Cytochrome P450 Monooxygenases

Infestation of corn (Zea mays) by corn earworm (Helicoverpa zea) predisposes the plant to infection by Aspergillus fungi and concomitant contamination with the carcinogenic mycotoxin aflatoxin B1 (AFB1). Although effects of ingesting AFB1 are well documented in livestock and humans, the effects on insects that naturally encounter this mycotoxin are not as well defined. Toxicity of AFB1 to different stages of H. zea (first, third, and fifth instars) was evaluated with artificial diets containing varying concentrations. Although not acutely toxic at low concentrations (1−20 ng/g), AFB1 had significant chronic effects, including protracted development, increased mortality, decreased pupation rate, and reduced pupal weight. Sensitivity varied with developmental stage; whereas intermediate concentrations (200 ng/g) caused complete mortality in first instars, this same concentration had no detectable adverse effects on larvae encountering AFB1 in fifth instar. Fifth instars consuming AFB1 at higher concentrations (1 μg/g), however, displayed morphological deformities at pupation. That cytochrome P450 monooxygenases (P450s) are involved in the bioactivation of aflatoxin in this species is evidenced by the effects of piperonyl butoxide (PBO), a known P450 inhibitor, on toxicity; whereas no fourth instars pupated in the presence of 1 μg/g AFB1 in the diet, the presence of 0.1% PBO increased the pupation rate to 71.7%. Pupation rates of both fourth and fifth instars on diets containing 1 μg/g AFB1 also increased significantly in the presence of PBO. Effects of phenobarbital, a P450 inducer, on AFB1 toxicity were less dramatic than those of PBO. Collectively, these findings indicate that, as in many other vertebrates and invertebrates, toxicity of AFB1 to H. zea results from P450-mediated metabolic bioactivation.An erratum to this article can be found at     

Aflatoxin formation on whole and ground cumin and anise seeds

The purpose of this study was to evaluate the potential productivity and growth ofAspergillus parasiticus (NRRL 2999) and the resulting toxin production on natural and autoclaved (cooked) cumin and anise spice seed substrates. Both whole and ground seeds were used. Mycelia and sporulation were also noted in this 17-day experiment. Cumin and anise seeds are capable of supporting mycelial growth, sporulation and toxin production when the seeds are moist and maintained at room temperature. Toxin yields were higher on ground sterile seed substrates. Of the commercial samples tested, neither the resulting cultures of natural flora nor dry whole seeds were found to contain aflatoxin or aflatoxin-like producing organisms. The anise substrates were more conducive to mycelial growth, sporulation and aflatoxin production than the cumin. Toxin levels in the various anise substrates ranged from 0.83 to 6.5μg/g total for the 4 aflatoxins, B₁, B₂, G₁ and G₂. Cumin seed substrates usually showed only B, and G, at total levels ranging from 0.23 to 0.63μg/g- Both spice seeds had mycelial growth and sporulation to occur at some time during the experimental period. Both substrates could be considered as low-level-producer substrates for aflatoxins. Anise seeds should be monitored occasionally for aflatoxin contamination when the commodities are purchased and used in large quantities.     

Theoretical investigations into the accuracy of sampling shelled peanuts for aflatoxin

Within a population of shelled peanuts, aflatoxin may be concentrated in less than 0.5% of the peanuts. Those peanuts containing aflatoxin might have concentrations up to 1,000,000 μg of aflatoxin per kilogram of peanuts. Because of the distribution pattern, sample means vary widely, and the true average level of aflatoxin in the population is difficult to estimate. The objective of this study was to determine the effect of sample size, N, on sampling accuracy. The negative binomial distribution of aflatoxin since it allowed for a high probability of zero counts along with small probabilities of large counts. Using both the Monte Carlo technique and a direct computation method, the effect of sample size on sampling accuracy was quantitatively described. Journal Paper No. 2775, North Carolina State University Agricultural Experiment Station, Raleigh, N.C.     

Determination of volatile N-nitroso compounds in various samples of edible vegetable oils and margarine (commercially available products)

The examination of samples of various commer-cially available vegetable oils (olive oil, sunflower oil, thistle oil, linseed oil, plant germ oil, etc.) and of various samples of margarine for the presence of vola-tile N-nitroso-compounds yielded the following results. By means of the above mentioned procedure (gas liquid chromatography — AFID gas liquid chromatography — TEA), N-nitrosodimethylamine (NDMA) was found to be present in 21 of 61 dif-ferent samples of vegetable oil, in concentrations ranging from < 1 μg/kg to 23 μg/kg. 18 samples con-tained N-nitrosodiethylamine (NDEA) in concentra-tions varying between < 1μg/kg and 27.8 μg/kg. 37 out of 107 different samples of margarine were shown to contain N-nitroso compounds. N-nitroso-dimethylamine was found to be present in 15 samples. The range of concentrations determined was between < 1 μg/kg and 5.8 μg/kg. 33 samples con-tained N-nitrosodiethylamine in concentrations varying between < 1 μg/kg and 7.5 μg/kg.     

Sample preparation for aflatoxin assay: The nature of the problem and approaches to a solution

Cases have been reported of individual peanuts, cottonseeds or Brazil nuts so highly contaminated with aflatoxin that, for a 50 g portion to be representative of the whole, the sample preparation procedures should grind each unit to a large number of particles and distribute them uniformly throughout the sample. Assuming uniform contamination of the individual kernel, each 50 g sample should contain 1/100 of that kernel. Even though these extreme cases may be encountered only infrequently, the more usual situation still presents difficulties because of great variability in individual kernel contamination. However, if the extreme can be handled, one can expect to handle the more usual situation. Equipment and procedures to achieve this distribution goal are described. The equipment studied includes a food chopper (Hobart), a nut mill (Thomas Mills), a disc mill (Bauer), a hammer mill (Fitzpatrick Model D comminuting machine), a hammer mill designed specifically for peanut samples (Dicken’s subsampling mill), a Polytron homogenizer (Bronwill Scientific), a vertical cutter-mixer (Hobart), and a sample splitter (Jones riffle). Commodities examined were shelled peanuts and in-shell Brazil nuts, walnuts, pecans and almonds. Comminution and mixing effectiveness were determined by particle size analysis, by distribution of kernels made radioactive by neutron activation and by aflatoxin analysis of naturally contaminated products. From the results we conclude that the ultimate in sample uniformity can be achieved with a disc mill, solvent addition to obtain a fluid system and mixing and grinding of the fluid with a dispersion mixer-grinder. A practical uniformity can be achieved in a vertical cutter-mixer with less expenditure of time and effort for the commodities studied. Presented in part at the AOCS-AACC Joint Meeting, Washington, D.C., April 1968.     

Comparison of the observed distribution of aflatoxin in shelled peanuts to the negative binomial distribution

Suitability of the negative binomial distribution for use in estimating the probabilities associated with sampling lots of shelled peanuts for aflatoxin analysis has been studied. Large samples, called “minilots,” were drawn from 29 lots of shelled peanuts contaminated with aflatoxin. These minilots were subdivided into ca. 12 lb samples which were analyzed for aflatoxin. The mean and variance of these aflatoxin determinations for each minilot were determined. The shape parameterk and the mean aflatoxin concentrationm were estimated for each minilot. A regression analysis indicated the functional relationship betweenk andm to be:k=(2.0866+2.3898m) × 10⁻⁶. The observed distribution of sample concentrations from each of the 29 minilots was compared to the negative binomial distribution by means of the Kolmogorov-Smirnov test. The null hypothesis that each of the true unknown distribution functions was negative binomial was not rejected at the 5% significance level for all 29 comparisons. Journal Series Paper of the North Carolina State University Agricultural Experiment Station, Raleigh, N.C.     

Preparation of14C-labeled aflatoxins and incorporation of unlabeled aflatoxins in a blocked versicolorin-A-accumulating mutant ofaspergillus parasiticus

We have compared 2 methods for preparing radiolabeled aflatoxins from [¹⁴C] acetate for use in biosynthetic studies at the end of the aflatoxin pathway. The Salhab-Edwards method (SE) used a 3-day-old mycelium and a defined medium containing MnCl₂ with incubation at 28 C. The Lee-Bennett method (LB) used a 2-day-old mycelium and a defined medium containing low levels of Mn with incubation at 30 C. Generally, the LB method produced lower quantities of aflatoxin but the product had higher specific activities (sp act). The SE method produced 0.157μmol of aflatoxin B₁ and 0.028μmol of G₁ compared to the LB method with 0.058μmol of aflatoxin B₁ and 0.001μmol of G₁. The sp act (inμCi/μmol) for aflatoxin produced by the LB method were: B₁ = 1.379; B₂ = 0.130; G₁ = 5.0 and G₂ - 0.063. The sp act of aflatoxin produced by the SE method were: B₁ = 0.267; B₂ = 0.014; G₁ = 0.178; and G₂ =0.133. Unlabeled aflatoxins were presented to resting cell cultures of the versicolorin-A-accumulating mutant ofAspergillus parasiticus. No conversion of aflatoxin B, was noted. However, when aflatoxins B₂ or G₁ were presented low levels of aflatoxins B₁ and G₂ were recovered. Aflatoxins B₂ and G₁ were recovered when aflatoxin G₂ was presented. Similar low levels of recovery were obtained in experiments using autoclaved mycelia. Thus we conclude that these minor quantities of aflatoxins recovered were not produced enzymatically.     

A water slurry method of extracting aflatoxin from peanuts

A water slurry method in which 1100 g of comminuted peanuts was blended with 1500 ml of tap water for 3 min in a blender and the aflatoxin in a 130-g portion of the water slurry was extracted by solvent according to methods similar to those used in Method II of AOAC was compared to the method presently used by the Food Safety and Quality Service, USDA. The proposed water slurry method requires only 180 and 60 ml per sample, respectively, of methanol and hexane compared to the 1650 and 1000 ml, respectively, required by the FSQS method. Blending comminuted peanuts with water reduced the average particle size and distributed the contaminated particles throughout the slurry. Ninety-four percent of the blended particles passed a sieve with 149-μ openings compared to only 66% of the unblended product. Variance among analyses with the FSQS method did not differ significantly from the variance among analyses with the slurry method. However, analyses with the slurry method averaged 16% more aflatoxin than with the FSQS method. Paper No. 6189 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC.     

Distribution of aflatoxin-containing cottonseed within intact locks

Location of aflatoxin-containing seeds within locks ofAspergillus flavus contaminated bolls was determined. Of the 141 seeds examined from 22 intact locks, 78 exhibited bright greenish yellow fluorescence (BGYF) on the linters. Twenty-four seeds contained aflatoxins ranging from 0.231 to 151.3 μg of toxin per gram of seed. Twenty-one of these aflatoxin-positive seeds had linters exhibiting BGYF, and three had nonfluorescent linters. With one exception, aflatoxin contamination was concentrated in only one or two highly contaminated seeds in the apex half of tight locks, and the rest of the three to five seeds were negative. Explanations for this type of infection are discussed.     

Replacement of benzene as a solvent for aflatoxin standards

Cyclohexane, heptane and toluene were evaluated for possible replacement of benzene in the preparation of aflatoxin standard solutions. The results of a 6-month study showed that these solvents can be substituted for benzene, provided they are not unduly exposed to light. The superiority of benzene as a solvent for aflatoxin standards was clearly demonstrated in a test in which these solvent systems containing 5μg B₁ /mL, were exposed to daily lighting in the laboratory for 3 months. Complete photodegradation of aflatoxin B₁ occurred in the cyclohexane, heptane and toluene systems whereas only a 15% B₁ decrease occurred in the benzene system.     

2-Polyunsaturated Acyl Lysophosphatidylethanolamine Attenuates Inflammatory Response in Zymosan A-Induced Peritonitis in Mice

In the present study, the anti-inflammatory action of lysophosphatidylethanolamine (lysoPtdEtn), orally administered, in zymosan A-induced peritonitis was examined. Oral administration of 2-DHA-lysoPtdEtn (ED₅₀, ~111 μg/kg) or 2-ARA-lysoPtdEtn (ED₅₀, 221 μg/kg) was found to inhibit the plasma leakage in mice treated with zymosan A. In support of this, 2-polyunsaturated acyl-lysoPtdEtn diminished the formation of LTC₄, a lipid mediator responsible for vascular permeability. Next, 2-DHA-lysoPtdEtn (ED₅₀, 110 μg/kg) or 2-ARA-lysoPtdEtn (ED₅₀, 123 μg/kg) effectively inhibited the leukocyte extravasation into the peritoneum. Consistent with this, each polyunsaturated-lysoPtdEtn diminished the formation of LTB₄ and 12-HETE, potent chemotactic factors. Additionally, the level of pro-inflammatory mediator (IL-1 β, IL-6, TNF-α or NO) was lowered remarkably in contrast to the augmentation of anti-inflammatory interleukin IL-10. Furthermore, 2-(15-HETE)-lysoPtdEtn and 2-(17-HDHE)-lysoPtdEtn, 15-lipoxygenation product of 2-ARA-lysoPtdEtn and 2-DHA-lysoPtdEtn, respectively, were more potent than corresponding lysoPtdEtn, suggesting the action of 2-acyl-lysoPtdEtn might be expressed through 15-lipoxygenation. In support of this, the formation of 15-HETE and LXA₄ was upgraded in accordance with an increasing dose of 2-ARA-lysoPtdEtn. Separately, anti-inflammatory actions, 2-polyunsaturated acyl-lysoPtdEtns also drastically diminished leukocyte infiltration in a later phase of zymosan A-induced peritonitis, indicating that these lipids also possess pro-resolving activity. Taken together, it is suggested that polyunsaturated lysoPtdEtns and their lipoxygenation derivatives, could be classified as potent anti-inflammatory lipids.     

Determination of aflatoxins in individual peanuts and peanut sections

Subsamples of a given lot of peanuts may vary greatly in aflatoxin content due to extreme variability in the degree of contamination of individual kernels. A micro method, adapted from the aqueous acetone procedure recently proposed by Pons and Goldblatt for the determination of aflatoxins in cottonseed products, was developed to permit accurate determination of aflatoxins in individual kernels and kernel sections. Use of this procedure permitted the topographic distribution of aflatoxins within single kernels to be mapped and indicated that the toxins are not uniformly distributed within contaminated kernels, even when the kernel contains a high level of aflatoxins. Although wrinkling or discoloration sometimes indicated that a kernel was contaminated, this type of physical damage was not found to be a reliable indication of aflatoxin content. Also it was noted that a few apparently sound and mature kernels contained high levels of aflatoxins. Presented at the AOCS Meeting, Houston, April 1965. Honorable Mention Bond Award Competition. So. Utiliz. Res. Dev. Div., ARS, USDA.     

Soybean phytoalexin,glyceollin, prevents accumulation of aflatoxin B1 in cultures ofAspergillus flavus

The soybean phytoalexin, glyceollin, suppresses the accumulation of aflatoxin B₁ in cultures ofAspergillus flavus. At concentrations of 6.25g/ ml and 62.5g/ml, glyceollin causes 70% and 95% decreases in the maximum observed levels of aflatoxin B₁, respectively. In contrast to the dramatic effect on aflatoxin B₁ levels, these concentrations have little effect on fungal growth. For example, at 62.5g/ml in liquid culture, glyceollin causes a barely discernible lag in the beginning of growth and a 11.5% decrease in maximum fungal mass. When the same concentration of glyceollin is added to the colony margin on semisolid medium, an inhibition zone is formed and then overgrown in one day. Glyceollin appears to act by inhibiting aflatoxin B₁ synthesis, since the rate of aflatoxin B₁ breakdown is not increased in fungal cultures that have been grown in the presence of glyceollin. Glyceollin does accumulate in viable soybean seeds that have been infected withAspergillus flavus. Such seeds accumulate aflatoxin B₁ at one-third the rate of non-glyceollin-producing, nonviable seeds. These results suggest that the synthesis of glyceollin in infected seeds may explain, at least in part, why aflatoxin contamination of soybeans is not a common problem.     

Detection of polycyclic aromatic hydrocarbons in commonly consumed edible oils and their likely intake in the Indian population

Edible oils such as coconut, groundnut, hydrogenated vegetable, linseed, mustard, olive, palm, refined vegetable, rice bran, safflower, sesame, soybean, and sunflower were analyzed for the presence of light and heavy polycyclic aromatic hydrocarbon (PAH) residues using liquid-liquid extraction, cleanup on a silica gel column, and resolution and determination by HPLC using a fluorescence detector. Ten PAH viz. acenaphthene, anthracene, benzo(a)pyrene, benzo(e)pyrene, benz(ghi)perylene, chrysene, coronene, cyclopenta(def)phenanthrene, phenanthrene, and pyrene were monitored. Analysis of 296 oil samples showed that 88.5% (262) samples were contaminated with different PAH. Of 262 contaminated edible oil samples, 66.4% of the samples showed PAH content of more than the 25 μg/kg recommended by the German Society for Fat Science. The total PAH content was highest in virgin olive oil (624 μg/kg) and lowest in refined vegetable oils (40.2 μg/kg). The maximum content (265 μg/kg) of heavy PAH was found in olive oil and the minimum (4.6 μg/kg) in rice bran oil. Phenanthrene was present in 58.3% of the oil samples analyzed, followed by anthracene (53%). Among the heavy PAH, benzo(e)pyrene was observed in 31.2% of the samples followed by benzo(a)pyrene (25.5%). The intake of PAH was highest through olive oil (20.8 μg/day) followed by soybean oil (5.0 μg/day) and lowest through refined vegetable oil (1.3 μg/day). Based on these monitoring studies, international and national guidelines for permissible levels of PAH can be prepared so as to restrict the intake of these toxic contaminants.     

Studies of long term administration of aflatoxin to rats as a natural food contaminant

The effect of feeding aflatoxin, as a natural food contaminant, to rats over long periods of time was studied using multigeneration and longevity tests. The test animals in the multigeneration study consisted of three groups of rats fed diets containing 0, 1 and 10 ppb of aflatoxin (predominantly B₁) continued over four generations, with animals of the first and fourth generation fed the diets for 104 weeks. These diets were in proper nutritional balance and included 35% ground roasted peanut products; the ration with 0 ppb aflatoxin excluded the peanuts usually discarded; the one with 1 ppb had the roasted discards returned, while the ration with 10 ppb included the discards in amount 10 times that which had been initially removed. Another longevity study was also performed in which rats were fed diets containing aflatoxin at a level of 80 ppb. In this case, the test peanuts, also fed as a simulated peanut butter at 35% concentration, consisted entirely of roasted peanut discards. Control diets provided no peanut components. Animals fed the low levels of aflatoxin grew as well and actually had a higher percentage survival at 104 weeks than did the animals on the control, aflatoxinfree diets. Organ weights, liver total lipid and cholesterol levels were comparable in all groups. Pathological abnormalities, e.g., hemorrhagic and opaque spots and mottling in some of the livers, were attributed to the aging process since the abnormalities appeared in the control as well as the experimental groups. In the animals fed the aflatoxin at 80 ppb, which has been reported by several investigators to produce well-defined hepatomas in rats, there was liver involvement and some biochemical changes occurred that were not noted in the controls. However, no hepatomas were observed in these animals even after 21 months on this diet. The liver lesions, indicative of a toxic effect, have not been associated with the development of hepatomas. It is possible that some components of the diet used in these experiments may have protected the animal against hepatoma formation. Our studies indicate that there may be a tolerance for aflatoxin as judged by results in one species of rats when whole ground roasted peanuts provide the natural contaminant. Presented in part at the AOCS Meeting, New York, October 1968.     

Flavonoid glycosides and naphthodianthrones in the sawfly Tenthredo zonula and its host-plants, Hypericum perforatum and H. hirsutum

Larvae of the sawfly Tenthredo zonula are specialized on Hypericum. Whether the sawfly is able to sequester plant metabolites was unknown. Aerial materials of Hypericum perforatum and H. hirsutum, as well as dissected larvae and prepupae of T. zonula, were analyzed by HPLC to determine the presence and content of flavonoid glycosides (rutin, hyperoside, isoquercitrin, and quercitrin) and naphthodianthrones (pseudohypericin and hypericin). All flavonoid glycosides were detected in both Hypericum species, with hyperoside as major compound in H. perforatum (ca. 1.7 μmol/g fresh weight, FW) and isoquercitrin in H. hirsutum (0.7 μmol/g FW). Naphthodianthrones were present at low concentrations (0.02 μmol/g FW) in the former, and almost undetected in the latter species. In the body parts (i.e., hemolymph, digestive tract, salivary glands, or miscellaneous organs) of T. zonula, the surveyed compounds were detected more frequently in prepupae than in larvae. The compounds were not present in every sample, and flavonoid glycosides especially occurred in highly variable amounts, with maximal concentrations of 41 μg rutin/prepupa in salivary glands, 8 μg hyperoside/prepupa in hemolymph (= 0.36 μmol/g FW), 32 μg isoquercitrin/prepupa in salivary glands, and 63 μg quercitrin/larva in miscellaneous organs (mainly composed of the integument). We conclude that flavonoid glycosides are sequestered since they were detected in organs other than the digestive tract of larvae, and because prepupae are a non-feeding stage. The naphthodianthrone pseudohypericin, but not hypericin, occurred generally in the digestive tract (up to 0.25 μg/larva). Both naphthodianthrones and related unidentified compounds, but not flavonoid glycosides, were found in the larval excrement. The highly variable distributions of flavonoid glycosides and naphthodianthrones in T. zonula larvae and prepupae make it difficult to determine the ecological significance of these metabolites.     

Low concentration of oxidized low density lipoprotein suppresses platelet reactivity <Emphasis Type="Italic">in vitro</Emphasis>: An intracellular study

The intracellular mechanisms underlying oxidized low density lipoprotein (oxLDL)-signaling pathways in platelets remain obscure and findings have been controversial. Therefore, we examined the influence of oxLDL in washed human platelets. In this study oxLDL concentration-dependently (20–100 μg/mL) inhibited platelet aggregation in human platelets stimulated by collagen (1 μg/mL) and arachidonic acid (60 μM), but not by thrombin (0.02 U/mL). The activity of oxLDL was greater at 24 h in inhibiting platelet aggregation than at 12 h. At 24 h, oxLDL concentration-dependently inhibited intracellular Ca²⁺ mobilization and thromboxane B₂ formation in human platelets stimulated by collagen. In addition, at 24 h oxLDL (40 and 80 μg/mL) significantly increased the formation of cyclic AMP, but not cyclic GMP or nitrate. In an ESR study, 24 h-oxLDL (40 μg/mL) markedly reduced the ESR signal intensity of hydroxyl radicals (OH⁻) in both collagen (2 μg/mL)-activated platelets and Fenton reaction (H₂O₂+Fe²⁺). The inhibitory effect of oxLDL may induce radical-radical termination reactions by oxLDL-derived lipid radical interactions with free radicals (such as hydroxyl radicals) released from activated platelets, with a resultant lowering of intracellular Ca²⁺ mobilization followed by inhibition of thromboxane A₂ formation, thereby leading to increased cyclic AMP formation and finally inhibited platelet aggregation. This study provides new insights concerning the effect of oxLDL in platelet aggregation.