Vitamin B12 Added as a Fortificant to Flour Retains High Bioavailability when Baked in Bread.

Vitamin B12 deficiency and depletion are common world-wide, particularly in populations that consume low amounts of animal source foods. WHO and the Food Fortification Initiative recommend that wheat flour be fortified with vitamin B12 in regions where intake of B12 is low. The purpose of this pilot study in five participants was to determine if fortification of flour with B12 produced a bread product with intact B12 still present and to determine if healthy elderly absorb sufficient B12 from bread fortified in this manner. High-purity crystalline 14C-B12 was dissolved in water and added to flour (2 µg B12 /100 g flour) in a bread maker and made into rolls (average 1.17 kBq (31.5 nCi) 14C-B12 in a total of 0.8 µg B12 per roll). Excess 14C first appeared in plasma 4 h after ingestion of the 14C fortified bread and plasma levels returned almost to background by 72 h. Measurement of 14C in plasma verified that the dose was absorbed into the systemic circulation. The cumulative % dose recovered in urine was 4.8-37.0% (mean = 20.1%). Most of the 14C label in the stool appeared by day 4, and the cumulative % dose recovered in stool was 24.5- 43.0% (mean = 31.8%). Bioavailability among the 5 participants, calculated by subtracting the sum of urinary and fecal 14C excretion from the administered dose, was 28.4-63.7% (mean = 48.0%). This study showed that when B12 is added as a fortificant to flour it survives the fermentation and baking processes, and retains ~ 50% bioavailability when fed in small doses to healthy subjects. The Recommended Dietary Allowance of B12 for adults is 2.4 µg/d. This recommendation assumes that usual bioavailability of low doses of the vitamin in the crystalline form is 60%, while for the same amount in foods such as meat and fish it is 50%. Our pilot study shows that B12 added to bread as a fortificant in flour was absorbed as well as it is from endogenous food sources such as meat and fish.

Maternal exposure to an environmentally relevant dose of triclocarban results in perinatal exposure and potential alterations in offspring development in the mouse model.

Triclocarban (TCC) is among the top 10 most commonly detected wastewater contaminants in both concentration and frequency. Its presence in water, as well as its propensity to bioaccumulate, has raised numerous questions about potential endocrine and developmental effects. Here, we investigated whether exposure to an environmentally relevant concentration of TCC could result in transfer from mother to offspring in CD-1 mice during gestation and lactation using accelerator mass spectrometry (AMS). 14C-TCC (100 nM) was administered to dams through drinking water up to gestation day 18, or from birth to post-natal day 10. AMS was used to quantify 14C-concentrations in offspring and dams after exposure. We demonstrated that TCC does effectively transfer from mother to offspring, both trans-placentally and via lactation. TCC-related compounds were detected in the tissues of offspring with significantly higher concentrations in the brain, heart and fat. In addition to transfer from mother to offspring, exposed offspring were heavier in weight than unexposed controls demonstrating an 11% and 8.5% increase in body weight for females and males, respectively. Quantitative real-time polymerase chain reaction (qPCR) was used to examine changes in gene expression in liver and adipose tissue in exposed offspring. qPCR suggested alterations in genes involved in lipid metabolism in exposed female offspring, which was consistent with the observed increased fat pad weights and hepatic triglycerides. This study represents the first report to quantify the transfer of an environmentally relevant concentration of TCC from mother to offspring in the mouse model and evaluate bio-distribution after exposure using AMS. Our findings suggest that early-life exposure to TCC may interfere with lipid metabolism and could have implications for human health.

DNA isolation and sample preparation for quantification of adduct levels by accelerator mass spectrometry.

Accelerator mass spectrometry (AMS) is a highly sensitive technique used for the quantification of adducts following exposure to carbon-14- or tritium-labeled chemicals, with detection limits in the range of one adduct per 10(11)-10(12) nucleotides. The protocol described in this chapter provides an optimal method for isolating and preparing DNA samples to measure isotope-labeled DNA adducts by AMS. When preparing samples, special precautions must be taken to avoid cross-contamination of isotope among samples and produce a sample that is compatible with AMS. The DNA isolation method described is based upon digestion of tissue with proteinase K, followed by extraction of DNA using Qiagen isolation columns. The extracted DNA is precipitated with isopropanol, washed repeatedly with 70 % ethanol to remove salt, and then dissolved in water. DNA samples are then converted to graphite or titanium hydride and the isotope content measured by AMS to quantify adduct levels. This method has been used to reliably generate good yields of uncontaminated, pure DNA from animal and human tissues for analysis of adduct levels.

Free flow electrophoresis separation and AMS quantitation of C-naphthalene-protein adducts.

Naphthalene is a volatile aromatic hydrocarbon to which humans are exposed from a variety of sources including mobile air sources and cigarette smoke. Naphthalene produces dose- (concentration) dependent injury to airway epithelial cells of murine lung which is observed at concentrations well below the current occupational exposure standard. Toxicity is dependent upon the cytochrome P450 mediated metabolic activation of the parent substrate to unstable metabolites which become bound covalently to tissue proteins. Nearly 70 proteins have been identified as forming adducts with reactive naphthalene metabolites using in vitro systems but very little work has been conducted in vivo because reasonably large amounts (100 µCi) of (14)C labeled parent compound must be administered to generate detectable adduct levels on storage phosphor screens following separation of labeled proteins by 2 D gel electrophoresis. The work described here was done to provide proof of concept that protein separation by free flow electrophoresis followed by AMS detection of protein fractions containing protein bound reactive metabolites would provide adducted protein profiles in animals dosed with trace quantities of labeled naphthalene. Mice were administered 200 mg/kg naphthalene intraperitoneally at a calculated specific activity of 2 DPM/nmol (1 pCi/nmol) and respiratory epithelial tissue was obtained by lysis lavage 4 hr post injection. Free flow electrophoresis (FFE) separates proteins in the liquid phase over a large pH range (2.5-11.5) using low molecular weight acids and bases to modify the pH. The apparatus separates fractions into standard 96-well plates that can be used in other protein analysis techniques. The buffers of the fractions have very high carbon content, however, and need to be dialyzed to yield buffers compatible with (14)C-AMS. We describe the processing techniques required to couple FFE to AMS for quantitation of protein adducts.

DNA isolation and sample preparation for quantification of adduct levels by accelerator mass spectrometry.

A protocol is described for the isolation of DNA and subsequent preparation of samples for the measurement of adduct levels by accelerator mass spectrometry (AMS). AMS is a highly sensitive technique used for the quantification of adducts following exposure to carbon-14- or tritium-labeled chemicals, with detection limits in the range of one adduct per 10(11)-10(12) nucleotides. However, special precautions must be taken to avoid cross-contamination of isotope between samples and to produce a sample that is compatible with AMS. The DNA isolation method described is based on digestion of tissue with proteinase K, followed by extraction of DNA using Qiagen DNA isolation columns. DNA is then precipitated with isopropanol, washed repeatedly with 70% ethanol to remove salt, and then dissolved in water. This method has been used to generate reliably good yields of uncontaminated, pure DNA from animal and human tissues for analysis of adduct levels. For quantification of adduct levels from 14C-labeled compounds, DNA samples are then converted to graphite, and the 14C content is measured by AMS.