A method for the analysis of natural and synthetic folate in foods

The essentiality of dietary folates for human beings has been known for many years. Over the shorter term, biological activities associated with several human maladies and the attenuation of biomarkers for several chronic diseases also have been assigned to folates. In the United States, these observations have led to the addition of folic acid to several foods and food ingredients (food fortification) and to dietary recommendations that assign biological activity to each of the forms of folate in the food supply. There currently is unavailable a robust, instrumental procedure that will distinguish between naturally occurring food folates and synthetic folic acid as part of the routine analysis of foods. The procedure proposed in this publication is unique in that it uses "off-the-shelf" supplies and instrumentation, to the extent possible, and was developed with "normal" corporate work schedules in mind. This method takes advantage of the tri-enzyme food digestion and folate deconjugation steps but was optimized with a commercially available rat plasma as the source of conjugase. A high-capacity styrene-divinylbenzene-based solid-phase extraction column was identified, and conditions were developed for quantitative recovery of 5-methyltetrahydrofolate and folic acid (FA) with it. The various forms of food folates are separated on a C-18 high-performance liquid chromatography (HPLC) column which is resistant to degradation at low pH. As a result, the mobile phase was simplified to a gradient of low-pH phosphate buffer (pH 2.2) and acetonitrile. Although FA does not exhibit fluorescence, a UV-induced photolysis system was added, which is controlled by the HPLC system, so that an appropriate segment of the HPLC column effluent is subjected to photolytic conditions and, thereby, FA can be measured as a fluorescent product. The application of the system was verified by analyzing several certified reference materials and foods and comparing results with certified values and/or total folate values as determined by microbiological assay.     

Optimization of the trienzyme extraction for the microbiological assay of folate in vegetables

Response surface methodology was applied to optimize the trienzyme digestion for the extraction of folate from vegetables. Trienzyme extraction is a combined enzymatic digestion by protease, alpha-amylase, and conjugase (gamma-glutamyl hydrolase) to liberate the carbohydrate and protein-bound folates from food matrices for total folate analysis. It is the extraction method used in AOAC Official Method 2004.05 for assay of total folate in cereal grain products. Certified reference material (CRM) 485 mixed vegetables was used to represent the matrix of vegetables. Regression and ridge analysis were performed by statistical analysis software. The predicted second-order polynomial model was adequate (R2 = 0.947), without significant lack of fit (p > 0.1). Both protease and alpha-amylase have significant effects on the extraction. Ridge analysis gave an optimum trienzyme digestion time: Pronase, 1.5 h; alpha-amylase, 1.5 h; and conjugase, 3 h. The experimental value for CRM 485 under this optimum digestion was close to the predicted value from the model, confirming the validity and adequacy of the model. The optimized trienzyme digestion condition was applied to five vegetables and yielded higher folate levels than the trienzyme digestion parameters employed in AOAC Official Method 2004.05.     

Determination of folate concentrations in diverse potato germplasm using a trienzyme extraction and a microbiological assay

Folate deficiency is a leading cause of birth defects and is implicated in several other diseases. We are interested in how much folate concentrations vary among potato germplasm. We determined total folate concentrations of potato tubers from 67 cultivars, advanced breeding lines, or wild species. Folates were extracted by a tri-enzyme treatment and analyzed by using a Lactobacillus rhamnosus microbiological assay. Folate concentrations varied from 521 +/- 96 to 1373 +/- 230 ng/g dry weight and were genotype and location dependent. The highest folate concentrations were mostly found in color-fleshed potatoes. Variations of folate concentrations within either color- or white-fleshed tubers were similar ( approximately 2-fold). Skin contained approximately 30% higher folate concentrations than flesh. Storage of tubers for 7 months generally led to an increase in folate contents. Semiquantitative RT-PCR analyses showed that higher folate contents were correlated with lower mRNA expression of some folate genes.     

High-performance liquid chromatographic determination of naturally occurring folates during tempe preparation

A trienzyme treatment (protease, alpha-amylase, and human plasma conjugase), followed by purification using SPE with SAX cartridges and reversed-phase HPLC with UV-PDA detection, was performed for determination of the distribution of various folate forms and content at various stages of tempe preparation. The major folate form in soybean identified was 5-formyl tetrahydrofolate (5-CHO-H4folate), followed by 10-formyl tetrahydrofolate (10-CHO-PGA), and 5-methyl tetrahydrofolate (5-CH3-H4folate), whereas folic acid was not detected and tetrahydrofolic acid (H4folate) was not detectable. The most predominant form in tempe was also 5-CHO-H4folate, followed by 10-CHO-PGA, whereas the quantities of 5-CH3-H4folate and folic acid were negligible. Quantities and retention of folate significantly decreased during the first boiling, dehulling, soaking, and second boiling procedures, yielding folate retention of 32%. A remarkable increase in folate content was found after fermentation, 5.2-fold higher than that of the boiled soybean. This may be due to de novo formation of folate by Rhizopus oligosporus, the principal mold in tempe fermentation. HPLC results were approximately 38-55% lower than the values obtained from the microbiological assay using Lactobacillus casei.     

Analysis for total sulfite in foods by using rapid distillation followed by redox titration

A rapid and accurate analysis for total sulfite as sulfur dioxide has been developed for foods and food products. The method, which combines a selective distillation cleanup procedure with the selective redox titration of sulfite ion by iodine, has been applied to a variety of foods and food products over a period of time with no significant interference encountered in any matrixes other than garlic and leeks. For the foods analyzed, the method typically shows a detection limit of 10 ppm, a relative standard deviation of 7.5% (compared with 10.4% for similar matrixes by the Monier-Williams method), and recoveries of 97.9 +/- 6.4%. Comparison of results for this method with those obtained using the Monier-Williams method showed a mean value for the distillation/titration method of 241 ppm compared with 242 ppm for the Monier-Williams method. A correlation of 0.991 and odds of a difference between methods of 10.7% (Student's paired t-test (1-alpha) X 100) were obtained for those matrixes where no interferences were encountered with either method.     

Effects of preparation and cooking of folic acid-fortified foods on the availability of folic acid in a folate depletion/repletion rat model

The practice of food fortification with folic acid offers the potential to increase the folate intake of the general population. To fully exploit the potential of fortification for raising folate nutriture, appropriate food vehicles need to be selected. Selection should involve determination of the availability of folic acid as affected by characteristics of the carrier food, food matrix, food preparation, and cooking. The present study investigated the effects of preparation and cooking of a range of folic acid-fortified foods on the folate status of folate-deficient rats. Fifty-six weanling male rats (Wistar strain) were fed a folate-deficient diet containing 1% succinyl sulfathiazole for 28 days. Following depletion, six rats were randomly assigned to each of eight repletion diets containing cooked or uncooked meringue mix, quick bread mix, brownie mix, or pizza base mix. The test foods were fortified with 1400 microg of folic acid/kg of food and incorporated as 19% of the repletion diets. Each of the first four groups was pair-fed a diet containing a cooked fortified food with another group fed the corresponding uncooked fortified food. After a further 28 days, plasma, liver, and kidney folate concentrations were determined by microbiological assay. Mean plasma and liver folate concentrations of rats fed diets containing cooked fortified foods were similar to those of rats fed uncooked fortified foods. Preparation and cooking did not affect the availability of folic acid from the selected cereal-based convenience foods in this rat model system, suggesting that these foods are appropriate vehicles for fortification with folic acid.     

Protocol for the production of concentrated extracts of food folate for use in human bioavailability studies

To provide a tool to study folate bioavailability under controlled conditions, a methodology was developed to produce extracts representative of natural food folates but removed from their matrix and sufficiently concentrated so as to elicit a response in biomarkers of folate status without distorting usual dietary intake patterns. Egg, spinach, and yeast were selected to represent the wide range in extent of folate conjugation found in foods (0, 60, and 100% polyglutamyl folate, respectively). The protocol, which was based on extracting food folates using only reagents safe for human consumption, was optimized in the laboratory (thermal extraction for 10 min in a 2% ascorbate solution at pH 5) and then adapted for industrial scale production in a food-processing facility. Results showed that the extracts were 2.3-12 times more concentrated in folate compared with their corresponding food sources. Neither the mono- to polyglutamate ratio nor the distribution of the main folate derivatives was altered during processing, making these extracts suitable for use in human bioavailability studies.     

A mass spectrometric validated high-performance liquid chromatography procedure for the determination of folates in foods

A series of five food reference materials (RM) that had certified values of folate concentrations and five frozen food samples were analyzed for 5-methyltetrahydrofolic acid (5-MTHFA) and folic acid (FA) using a high-performance liquid chromatography (HPLC) method with fluorescence detection that was validated using an HPLC mass spectrometry (MS) method with electrospray ionization. Identical sample specimens were extracted and analyzed in triplicate using both instrumental methods, and a comparison was made of the mean values of 5-MTHFA and FA resulting from these determinations. The analytes were isolated on either a high capacity strong anion exchange solid phase extraction column (HPLC method) or a phenyl Bond Elut column (MS method) prior to analyses. For quantification of the analytes by MS, (13)C-labeled 5-MTHFA and FA were added to samples as internal standards prior to enzymatic digestion and conversion of the polyglutamate forms of 5-MTHFA to the monoglutamic acid. Quantification of FA and 5-MTHFA using the HPLC analysis was carried out using external standards. With the exception of one RM (pig liver), the values established for 5-MTHFA using these methods were highly comparable. In determining the variance associated with these two procedures, it was observed that the mean relative standard error for 5-MTHFA was 12 (range, 2-27%) and 11% (range, 5-25%) for the HPLC and MS methods, respectively. FA was detected in only three of the samples, and the values obtained for it by either method were similar. This is the first paper that describes a mass spectrometric method used in the validation of an HPLC determination of food folates across a wide range of sample matrixes. The comparable values for 5-MTHFA and FA suggest that HPLC analysis with fluorescent detection may be used to accurately quantify folates present in a variety of food matrixes.     

Awareness and consumption of folate-fortified foods by women of childbearing age in Western Australia

OBJECTIVES: The introduction of voluntary fortification of some foods with folic acid in Australia has been implemented since evidence of the prevention of neural tube defects with periconceptional folic acid was published. Our objectives were to determine how many women were aware of folate and when they became aware, what was the awareness of labels on foods that mentioned folate, and how much folate-fortified food women ate. METHODS: To address these objectives we collected data by self-administered questionnaire from a random sample of 578 recently pregnant women in Western Australia between September 1997 and March 2000. RESULTS: Overall, 89% of women had heard, seen or read anything about the link between folate and birth defects such as spina bifida, 62% first became aware of the folate message before their recent pregnancy and 42% of women noticed any labels on foods that mention folate before or during their recent pregnancy. Overall, 53% of women were aware of foods that have folate added to them and 33% usually or always read the labels on food packaging. The folate-fortified foods most often consumed by women were cereals (69%), breads (34%) and milk (15%). Of the women who consumed folate-fortified foods (78%), the earlier they became aware of the folate message and noticed labels on food, the more fortified foods they consumed. CONCLUSIONS: These results indicate that staple foods fortified with folate are consumed by almost 80% of women in the population. Therefore, mandatory fortification of staple foods may reach most women, providing improved opportunity for the prevention of neural tube defects in Australia.     

Comparison of paired-ion liquid chromatographic method with AOAC fluorometric and microbiological methods for riboflavin determination in selected foods

A paired-ion liquid chromatographic (LC) technique coupled with fluorometric detection to determine riboflavin in various food matrices is described. Chromatograms of many foods showed 2 peaks of interest due to presence of riboflavin and flavin mononucleotide (FMN). Relatively high levels of FMN were found in raw beef, corned beef, chicken liver, and canned mushrooms. When riboflavin and FMN contents were summed, LC values were comparable to those obtained by the AOAC standard procedures. The LC technique was sensitive, rapid, and simple, yielding a mean standard deviation of 3.1% which was comparable to the AOAC fluorometric method (3.0%) and better than the AOAC microbiological assay (9.6%). Mean spike recoveries were 91.8% for LC compared to 90.5% and 89.6% for the AOAC fluorometric and microbiological methods, respectively.     

Liquid chromatographic determination of polydextrose in food matrixes

A liquid chromatographic (LC) method has been developed to determine the content of polydextrose, a water-soluble 1 calorie/g bulking agent, in food matrixes such as cookies, cakes, fruit spreads, and chocolate toppings. This analysis, which requires use of a blank matrix, provides a feasible means to control the manufacture of foods containing this additive and provides a component for the accurate determination of the caloric value of a particular food product. The method involves aqueous extraction of the polydextrose from the food matrix followed by separation on a carbohydrate analysis column. The LC system uses a mobile phase of 0.005M CaSO4.2H2O and a refractive index detector for quantitation. Polydextrose recoveries from the food matrixes varied from 91.5 to 100.9% with assay precision, expressed as coefficient of variation, ranging from 0.7 to 4.3%. Each error estimate was derived from 5 parallel determinations. The present methodology is precise and selective in contrast to the modified classical phenol-sulfuric acid colorimetric method for assaying carbohydrates, which had been used for polydextrose determination in food matrixes in the past. Because the coefficient of variation frequently exceeded 10%, replicate analyses were necessary to achieve quantitation.     

Adaptation and evaluation of the National Cancer Institute's Diet History Questionnaire and nutrient database for Canadian populations

BACKGROUND AND OBJECTIVE: Despite assumed similarities in Canadian and US dietary habits, some differences in food availability and nutrient fortification exist. Food-frequency questionnaires designed for the USA may therefore not provide the most accurate estimates of dietary intake in Canadian populations. Hence, we undertook to evaluate and modify the National Cancer Institute's Diet History Questionnaire (DHQ) and nutrient database. METHODS: Of the foods queried on the DHQ, those most likely to differ in nutrient composition were identified. Where possible these foods were matched to comparable foods in the Canadian Nutrient File. Nutrient values were examined and modified to reflect the Canadian content of minerals (calcium, iron, zinc) and vitamins (A, C, D, thiamin, riboflavin, niacin, B6, folate and B12). DHQs completed by 13 181 Alberta Cohort Study participants aged 35-69 years were analysed to estimate nutrient intakes using the original US and modified versions of the DHQ databases. Misclassification of intake for meeting the Dietary Reference Intake (DRI) was determined following analysis with the US nutrient database. RESULTS: Twenty-five per cent of 2411 foods deemed most likely to differ in nutrient profile were subsequently modified for folate, 11% for vitamin D, 10% for calcium and riboflavin, and between 7 and 10% for the remaining nutrients of interest. Misclassification with respect to meeting the DRI varied but was highest for folate (7%) and vitamin A (7%) among men, and for vitamin D (7%) among women over 50 years of age. CONCLUSIOn: Errors in nutrient intake estimates owing to differences in food fortification between the USA and Canada can be reduced in Canadian populations by using nutrient databases that reflect Canadian fortification practices.     

Difference in folate content of green and red sweet peppers (Capsicum annuum) determined by liquid chromatography-mass spectrometry

Folic acid (pteroylmonoglutamic acid) is used in enriched foods; however, very little folic acid occurs naturally in fruits and vegetables. For the U.S. Department of Agriculture's National Food and Nutrient Analysis Program, a number of fruits and vegetables have been assayed for endogenous folates, by a liquid chromatography-mass spectrometry method, to evaluate the accuracy of existing data for total folate determined by standard microbiological analysis. Folate in red and green sweet peppers (Capsicum annuum) differed notably (70.2 and 20.7 microg/100 g, respectively) and exceeded existing values determined by microbiological assay (18 and 11 microg/100 g, respectively). 5-Methyltetrahydrofolate was the predominant vitamer, but a significant amount of 5-formyltetrahydrolfolate and some 10-formylfolate were present. These findings may assist in making dietary recommendations or developing research diets related to folate. The data from this study have been used to update the folate values in release 19 of the USDA Nutrient Database for Standard Reference.     

Linear programming can help identify practical solutions to improve the nutritional quality of food aid

OBJECTIVES: To assess the nutritional quality of food aid delivered by food banks in France and to identify practical modifications to improve it. DESIGN: National-level data were collected for all food aid distributed by French food banks in 2004, and its nutrient content per 2000 kcal was estimated and compared with French recommendations for adults. Starting with the actual donation and allowing new foods into the food aid donation, linear programming was used to identify the minimum changes required in the actual donation to achieve the French recommendations. RESULTS: French food-bank-delivered food aid does not achieve the French recommendations for dietary fibre, ascorbic acid, vitamin D, folate, magnesium, docosahexaenoic acid, alpha-linolenic acid and the percentage of energy from saturated fatty acids. Linear programming analysis showed that these recommendations are achievable if more fruits, vegetables, legumes and fish were collected and less cheese, refined cereals and foods rich in fat, sugar and/or salt. In addition, new foods not previously collected are needed, particularly nuts, wholemeal bread and rapeseed oil. These changes increased the total edible weight (42%) and economic value (55%) of the food aid donation, with one-third of its edible weight coming from fruits and vegetables, one-third from staples, one-quarter from dairy products and approximately a tenth from meat/fish/eggs. CONCLUSIONS: Important changes in the types and amounts of food collected will improve the nutritional quality of food-bank-delivered food aid in France. Such changes are recommended to improve the diets of deprived French populations.     

Development of an in vitro digestion method to assess carotenoid bioavailability from meals.

The objective of this study was to develop a model for assessing the bioavailability of carotenoids from meals using an in vitro digestion procedure. A meal was prepared using baby food carrots, spinach, and a meat, plus tomato paste. The aqueous fraction was isolated from digesta to determine the quantity of carotenoids transferred from the food to micelles. The micellarization of lutein (25-40%) exceeded (p < 0.01) that of alpha- and beta-carotene (12-18%) and lycopene (<0.5%). Micellarization of carotenoids was not affected by elimination of the gastric phase of the digestive process. The absence of bile extract prevented the transfer of carotenoids from foods to micelles, whereas omission of pancreatin only reduced the micellarization of the carotenes. Differentiated cultures of Caco-2 human intestinal cells accumulated 28-46% of micellarized carotenoids from the medium after 6 h. These results support the usefulness of the in vitro digestion process as a rapid and cost-effective model for screening the bioavailability of carotenoids from meals.     

Issues in assessing the validity of nutrient data obtained from a food-frequency questionnaire: folate and vitamin B12 examples

OBJECTIVE: To compare methods used to assess the validity of nutrient intake data obtained from a food-frequency questionnaire (FFQ), using folate and vitamin B12 as nutrient examples. DESIGN: Cross-sectional sample from a population cohort. SETTING: Two postcode areas west of Sydney, Australia. SUBJECTS: In total, 2895 people aged 49 years and older provided dietary data using a semi-quantitative FFQ (79% of 3654 subjects examined). The validity of the FFQ was assessed against three 4-day weighed food records (WFRs) completed by 78 people (mean age 70 years). RESULTS: Folate and vitamin B12 validity data were assessed using different methods. The Spearman ranked correlations (energy-adjusted) were 0.66 for folate and 0.38 for vitamin B12. Using the Bland-Altman method, following loge transformation, no linear trend existed between the differences and means for folate and vitamin B12. Large differences existed between the FFQ and WFR in individual cases, particularly for vitamin B12. Finally, data were divided into quintile categories for the test and reference method: 79% classified folate within one quintile, 65% classified vitamin B12 within one quintile; there was no gross misclassification for folate and only 3% misclassification for vitamin B12. CONCLUSIONS: Different methods of analysis provided different information about the validity of the FFQ. Correlation coefficients should not be used alone to assess the validity of nutrient data, but should be used in conjunction with Bland-Altman analyses. Depending on the use of the data, additional assessment of classification categories is recommended. This worked example demonstrates that absolute intakes of folate and vitamin B12 should be used with caution.     

Determination of 5-methyltetrahydrofolic acid and folic acid in citrus juices using stable isotope dilution-mass spectrometry

A stable isotope liquid chromatography-mass spectrometry (LC-MS) method was developed for the quantitative determination of 5-methyltetrahydrofolic acid (5-MTHFA) and folic acid in a variety of commercial citrus juices. Folates were extracted from juices, and the polyglutamyl side chain of 5-MTHFA was cleaved to the monoglutamate form using rat plasma conjugase. The folates were purified on a Bond-Elut column and analyzed by LC-MS with electrospray ionization. The analytes were quantified using the (13)C(5) analogues of 5-MTHFA and folic acid as internal standards. The relative standard error of the method was 3.35% based on replicate analyses (n = 4). This method was then applied to the determination of 5-MTHFA and folic acid in a variety of citrus juices obtained from local supermarkets. It was observed that although both "store" brands and "national" brands of fresh (nonfrozen) juices contained similar concentrations of 5-MTHFA, the "store" brands of fresh juices had on average >5-fold the amount of folic acid compared to the "national" brands. In addition, the "total" folate concentrations were generally below values listed on the food label.     

Confirmation of the allergenic peanut protein, Ara h 1, in a model food matrix using liquid chromatography/tandem mass spectrometry (LC/MS/MS)

Enzymatic digestion of total protein along with liquid chromatography/tandem mass spectrometry (LC/MS/MS) was used to confirm the presence of a major peanut allergen in food. Several peptides obtained from the enzymatic digestion of the most abundant peanut allergen, Ara h 1, were identified as specific peptide biomarkers for peanut protein. Using ice cream as a model food matrix, a method was developed for the detection of the allergen peptide biomarkers. A key component of the method was the use of molecular mass cutoff filters to enrich the Ara h 1 in the protein extracts. By applying the method to ice cream samples containing various levels of peanut protein, levels as low as 10 mg/kg of Ara h 1 could routinely be detected. This method provides an unambiguous means of confirming the presence of the peanut allergen, Ara h 1, in foods and can easily be modified to detect other food allergens.     

Traditional food diversity predicts dietary quality for the Awajún in the Peruvian Amazon

OBJECTIVE: Our goal was to assess the potential for evaluating strengths of the Awajún traditional food system using dietary assessment, a traditional food diversity score and ranking of local foods. DESIGN: The method was used for dietary data obtained from mothers and children in the Awajún culture of the Peruvian Amazon where >90% of the dietary energy is derived from local, traditional food. Traditional food diversity scores were calculated from repeat 24-hour recalls. Group mean intakes of energy, fat, protein, iron, vitamin A and vitamin C from each food item were used to rank foods by nutrient contribution. SETTING: The study took place in six remote communities along the lower Cenepa River in the Amazonas District of Peru, South America. SUBJECTS: Dietary data were collected from 49 Awajún mothers and 34 children aged 3-6 years, representative of the six communities. RESULTS: Higher traditional food diversity was associated with greater protein, fibre, vitamin and mineral intakes when controlling for energy (partial correlations = 0.37 to 0.64). Unique sources for iron, total vitamin A and vitamin C were found in the Awajún traditional food system. CONCLUSIONS: A traditional food diversity score was a useful tool for predicting nutrient adequacy for the Awajún. Promotion of the Awajún traditional food system should focus on dietary diversity and unique nutrient-dense local foods.     

Quantitative sandwich ELISA for the determination of lupine (Lupinus spp.) in foods

The use of lupine in foods has increased considerably during the past decade, reflected by a corresponding increase in reported lupine-induced allergic incidents. Lupine allergy may arise either by primary sensitization or by clinical cross-reactivity in peanut-allergic persons. Detection of lupine proteins in food has previously been based on the use of patient serum. A novel sandwich enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of lupine in processed foods was developed, using a polyclonal rabbit antilupine capture antibody and a biotinylated conjugate of the same antibody for detection. The antibody was highly specific for lupine, apart from minor cross-reactivities to other legumes. The assay had a detection limit of 1 mug/g and was successfully used to quantify lupine protein in various food matrixes. Recoveries ranged from 60 to 116%, while the intra-and interassay coefficients of variation were <6% and <21%, respectively.