A 28-day oral toxicity evaluation of small interfering RNAs and a long double-stranded RNA targeting vacuolar ATPase in mice.

New biotechnology-derived crop traits have been developed utilizing the natural process of RNA interference (RNAi). However, plant-produced double stranded RNAs (dsRNAs) are not known to present a hazard to mammals because numerous biological barriers limit uptake and potential for activity. To evaluate this experimentally, dsRNA sequences matching the mouse vATPase gene (an established target for control of corn rootworms) were evaluated in a 28-day toxicity study with mice. Test groups were orally gavaged with escalating doses of either a pool of four 21-mer vATPase small interfering RNAs (siRNAs) or a 218-base pair vATPase dsRNA. There were no treatment-related effects on body weight, food consumption, clinical observations, clinical chemistry, hematology, gross pathology, or histopathology endpoints. The highest dose levels tested were considered to be the no observed adverse effect levels (NOAELs) for the 21-mer siRNAs (48 mg/kg/day) and the 218 bp dsRNA (64 mg/kg/day). As an additional exploratory endpoint, vATPase gene expression, was evaluated in selected gastrointestinal tract and systemic tissues. The results of this assay did not indicate treatment-related suppression of vATPase. The results of this study indicate that orally ingested dsRNAs, even those targeting a gene in the test species, do not produce adverse health effects in mammals.

Consumption of stearidonic acid-rich oil in foods increases red blood cell eicosapentaenoic acid.

BACKGROUND: Consumption of soybean oil enriched with stearidonic acid was previously shown to increase eicosapentaenoic acid (EPA) in red blood cells (RBC). OBJECTIVE: This study was designed to evaluate the effect of stearidonic acid oil used as a food ingredient in baked products and beverages on the RBC fatty acid profile. DESIGN: This was a randomized, double-blind, controlled, parallel-arm study. PARTICIPANTS: Healthy men and women 21 to 65 years of age were included. INTERVENTION: Participants consumed either negative control (1.5 g/day high-oleic sunflower ethyl ester oil softgel capsules+foods containing 7 g/day high-oleic sunflower oil), positive control (1.5 g/day EPA oil ethyl ester softgel capsules+foods containing 7 g/day high-oleic sunflower oil), or active (1.5 g/day high-oleic sunflower ethyl ester oil softgel capsules+foods containing 7 g/day stearidonic acid soybean oil) for 12 weeks. MAIN OUTCOME MEASURES: RBC membrane fatty acid profile (at weeks 0, 2, 4, 6, 8, 10, and 12); fasting serum lipids (weeks -2, 0, 6, 10, and 12); and fasting plasma glucose and insulin (weeks -2, 0, 10, and 12) were assessed. STATISTICAL ANALYSES PERFORMED: A repeated measures analysis of covariance was used for continuous variables, and pair-wise comparisons between treatments were adjusted using a step-down Bonferroni method. Fisher's exact or χ(2) tests were used for categorical data. RESULTS: RBC %EPA throughout the 12-week study were significantly different between all groups. Means at 12 weeks were 0.50%±0.03%, 2.17%±0.21%, and 0.85%±0.05% for control, EPA, and stearidonic acid, respectively. Other efficacy outcome measures were not significantly different among treatment groups. CONCLUSIONS: Consumption of stearidonic acid-enriched soybean oil incorporated into common foods increased RBC %EPA in healthy men and women. Stearidonic acid soybean oil, a sustainable and accessible source of long-chain n-3, can effectively be used to increase EPA in RBC.

Dietary intake of stearidonic acid-enriched soybean oil increases the omega-3 index: randomized, double-blind clinical study of efficacy and safety.

BACKGROUND: The benefits of omega-3 (n-3) long-chain polyunsaturated fatty acids to heart health are well established. Stearidonic acid (SDA, 18:4n-3) may contribute to these benefits. OBJECTIVE: The objective was to evaluate the ability of SDA-containing soybean oil to increase the omega-3 index [erythrocyte eicosapentaenoic acid (EPA) + docosahexaenoic acid, as a percentage of total fatty acids] and to affect other cardiovascular disease risk markers compared with EPA and regular soy oil (control). DESIGN: This was a randomized, placebo-controlled, double-blind multicenter study in which 252 overweight subjects were randomly assigned to 1 of 3 treatments for 12 wk: 1 g encapsulated soybean oil/d plus 14.7 g liquid soybean oil/d to be mixed in food (control group), 1 g encapsulated EPA/d plus 14.7 g liquid soybean oil/d (EPA group), and 1 g encapsulated soybean oil/d plus 14.7 g liquid SDA-enriched soybean oil/d, providing 4.2 g SDA (SDA group). Subjects consumed treatment oils in exchange for other oils in their diet. RESULTS: The mean (±SE) baseline omega-3 index was similar between treatments, but after 12 wk of treatment values for this index were 4.15 ± 0.12%, 4.84 ± 0.13%, and 4.69 ± 0.15% for control, EPA, and SDA groups, respectively. Values for the EPA and SDA groups were greater than those for control subjects in the intent-to-treat population (P < 0.001 and P = 0.006, respectively). No adverse treatment-related effects of SDA-enriched soybean oil were reported. CONCLUSIONS: SDA-enriched soybean oil increased the omega-3 index by raising erythrocyte EPA concentrations. SDA-enriched soybean oil is a land-based n-3 fatty acid that is a sustainable approach to increasing tissue concentrations of long-chain polyunsaturated n-3 fatty acids.

Stearidonic acid-enriched soybean oil increased the omega-3 index, an emerging cardiovascular risk marker.

A plant source of omega-3 fatty acid (FA) that can raise tissue eicosapentaenoic acid (EPA) and/or docosahexaenoic acid (DHA) is needed. A soybean oil (SBO) containing approximately 20% stearidonic acid [SDA; the delta-6 desaturase product of alpha-linolenic acid (ALA)] derived from genetically modified soybeans is under development. This study compared the effects of EPA to SDA-SBO on erythrocyte EPA+DHA levels (the omega-3 index). Overweight healthy volunteers (n=45) were randomized to SDA-SBO (24 ml/day providing approximately 3.7 g SDA) or to regular SBO (control group) without or with EPA ethyl esters (approximately 1 g/day) for 16 weeks. Serum lipids, blood pressure, heart rate, platelet function and safety laboratory tests were measured along with the omega-3 index. A per-protocol analysis was conducted on 33 subjects (11 per group). Compared to baseline, average omega-3 index levels increased 19.5% in the SDA group and 25.4% in the EPA group (p<0.05 for both, vs. control). DHA did not change in any group. Relative to EPA, SDA increased RBC EPA with about 17% efficiency. No other clinical endpoints were affected by SDA or EPA treatment (vs. control). In conclusion, SDA-enriched SBO significantly raised the omega-3 index. Since EPA supplementation has been shown to raise the omega-3 index and to lower risk for cardiac events, SDA-SBO may be a viable plant-based alternative for providing meaningful intakes of cardioprotective omega-3 FAs.

Effect of substitution of high stearic low linolenic acid soybean oil for hydrogenated soybean oil on fatty acid intake.

High stearic, low alpha-linolenic acid soybean oil (HSLL) has been developed via traditional breeding to serve as a substitute for partially hydrogenated soybean oils used in food manufacturing. The purpose of this study was to estimate the impact on fatty acid intake in the United States if HSLL were substituted for partially hydrogenated soybean oils used in several food categories, including baked goods, shortenings, fried foods, and margarines. Using National Health and Nutrition Examination Survey (NHANES) data (1999-2002), baseline intakes of five fatty acids and trans fatty acids (TFA) were determined at the mean and 90th percentile of fat consumption. Then intakes of these fatty acids were determined after HSLL was substituted for 100% of the partially hydrogenated soybean oils used in these four food categories. The results show that baseline intake of stearic acid is 3.0% energy at the mean and 3.3% energy at the 90th percentile. Use of HSLL could increase stearic acid intake to about 4-5% energy. Mean intakes of TFA could decrease from 2.5 to 0.9% energy, and intake of palmitic acid would remain unchanged. Use of HSLL as a substitute for partially hydrogenated soybean oils would result in changes in the fatty acid composition of the US diet consistent with current dietary recommendations.

Absorption and retinol equivalence of beta-carotene in humans is influenced by dietary vitamin A intake.

The effect of vitamin A supplements on metabolic behavior of an oral tracer dose of [14C]beta-carotene was investigated in a longitudinal test-retest design in two adults. For the test, each subject ingested 1 nmol of [14C]beta-carotene (100 nCi) in an emulsified olive oil-banana drink. Total urine and stool were collected for up to 30 days; concentration-time patterns of [14C]beta-carotene, [14C]retinyl esters, and [14C]retinol were determined for 46 days. On Day 53, the subjects were placed on a daily vitamin A supplement (10000 IU/day), and a second dose of [14C]beta-carotene (retest) was given on Day 74. All 14C determinations were made using accelerator mass spectrometry. In both subjects, the vitamin A supplementation was associated with three main effects: 1). increased apparent absorption: test versus retest values rose from 57% to 74% (Subject 1) and from 52% to 75% (Subject 2); 2). an approximately 10-fold reduction in urinary excretion; and 3). a lower ratio of labeled retinyl ester/beta-carotene concentrations in the absorptive phase. The molar vitamin A value of the dose for the test was 0.62 mol (Subject 1) and 0.54 mol (Subject 2) vitamin A to 1 mol beta-carotene. Respective values for the retest were 0.85 and 0.74. These results show that while less cleavage of beta-carotene occurred due to vitamin A supplementation, higher absorption resulted in larger molar vitamin A values.

Dual isotope test for assessing beta-carotene cleavage to vitamin A in humans.

BACKGROUND: The ability of beta-carotene to deliver bioactive retinoids to tissues is highly variable. A clearer understanding of the environmental and genetic factors that modulate the vitamin A potential of beta-carotene is needed. AIM OF STUDY: Assess the vitamin A value of orally administered beta-carotene relative to a co-administered reference dose of preformed vitamin A. METHODS: Equimolar doses (30 micromol) of hexadeuterated D6 beta-carotene and D6 retinyl acetate were orally co-administered in an emulsified formulation to a male subject. The plasma concentration time courses of D6 retinol (derived from D6 retinyl acetate) and bioderived D3 retinol (from D(6) beta-carotene) were determined for 554 h postdosing using gas chromatography/mass spectrometry. Intact D6 beta-carotene plasma concentrations were determined by high-pressure liquid chromatography. The ratio of the two forms of vitamin A, D6 retinol/D3 retinol, at any single time point is postulated to reflect the quantity of vitamin A derived from beta-carotene relative to preformed vitamin A. Additionally, a minute amount of 14C beta-carotene (50 nCi; 0.27 microg) was included in the oral dose and cumulative 24-h stool and urine samples were collected for two weeks to follow absorption and excretion of the b-carotene. The 14C nuclide was detected using accelerator mass spectrometry (AMS). Results During the absorption/distribution phase (3-11 h) the D6/D3 ratio of the two retinols was not stable and ranged between a value of 3 and 16. Between 11 and 98 h postdosing the ratio was relatively stable with a mean value of 8.5 (95 % CI: 7.5, 8.7). These data suggest that in this subject and under these conditions, 8.5 moles of beta-carotene would provide a vitamin A quantity equivalent to 1 mole of preformed vitamin A. On a mass basis, 15.9 microg of beta-carotene was equivalent to 1 microg of retinol. The total administered beta-carotene was found to be 55 % absorbed by AMS analysis of cumulative stool. CONCLUSION: The co-administration of D6 beta-carotene and D6 retinyl acetate provides a technique for assessing individual ability to process beta-carotene to vitamin A. The results indicate that a single time point taken between 11-98 h after dose administration may provide a reliable value for the relative ratio of the two forms of vitamin A. However, results from more subjects are needed to assess the general utility of this method.

Characterization of clay-based enterosorbents for the prevention of aflatoxicosis.

Appropriate chemical interventions that can block, retard, or significantly diminish foodborne exposure to aflatoxins are high priorities. A practical and effective approach to the aflatoxin problem has been the dietary inclusion of a processed calcium montmorillonite clay (HSCAS). HSCAS acts as an enterosorbent that rapidly and preferentially binds aflatoxins in the gastrointestinal tract resulting in decreased aflatoxin uptake and bioavailability. In mechanistic studies, we have shown that the intact dicarbonyl system in aflatoxin is essential for optimal sorption by HSCAS. Evidence also suggests that aflatoxins react at multiple sites on HSCAS clay surfaces (especially those within the interlayer region). Due to conceivable risks associated with the dietary inclusion of nonspecific binding agents, all aflatoxin enterosorbents should be tested in sensitive animal models for efficacy, safety, and the potential for nutrient interactions.