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Absorption of Folic Acid and Ascorbic Acid fromNutrient Comparable BeveragesBrett Carter, Pablo Monsivais, and Adam Drewnowski

Abstract: One hundred percent fruit juices can help consumers increase the nutrient content of the diet since these bev-erages can be naturally rich in micronutrients. Micronutrient-fortified low-calorie beverages are an important alternativeto those wishing to minimize their calorie intakes. However, little is known about the bioavailability of nutrients fromfortified beverages relative to 100% fruit juices. The present study examined the bioavailability of ascorbic acid (AA) andfolic acid (FA) in 100% orange juice (OJ) and a low-calorie beverage fortified with these nutrients. In a within-subjects,cross-over design, 12 adult men consumed a 591 mL serving of OJ, a low-calorie beverage fortified with AA and FA,and 1% low fat milk. Participants were aged 20 to 35 y, with body mass indexes between 20 and 30 kg/m2. Blood plasmaconcentrations of AA and serum concentrations of FA were assayed by serial blood draws, made at 30 min intervals for4.5 h. Blood plasma concentration of AA was significantly greater after ingestion of the fortified beverage comparedto after OJ ingestion. However, the bioavailability of AA did not significantly differ from that of OJ. Analyses of FAindicated no significant difference between fortified beverage and OJ. Consumption of both vitamin containing beveragesled to higher concentrations of AA and FA than the milk control. This study showed that similar levels of AA and FAbioavailability can be attained through ingestion of 100% OJ and a fortified beverage.

Keywords: absorption, ascorbic acid, bioavailability, folic acid

Practical Application: A nutrient fortified beverage and a 100% OJ delivered similar amounts of folic acid and AA.However, the fortified beverage contained far fewer calories than the juice. Fortification can provide an effective way toincrease the nutrient-to-calorie ratio of the diet.

IntroductionImproving the nutrients-to-energy ratio in the American diet

is a stated goal of the 2005 Dietary Guidelines for Americans andthe 2005 USDA MyPyramid (U.S. Dept. of Agriculture, 2005;U.S. Dept. of Health and Human Services/Dept. of Agriculture,2005). One way to increase the dietary nutrient-to-energy ratio isto consume more nutrient-dense foods (Drewnowski and Fulgoni2008). Another way is to consume more nutrient-fortified foodsand beverages. Fortified beverages have been used in place of mealsfor weight loss and for optimizing nutrition in clinical settings(Winkels and others 2007).

One concern is that the bioavailability of fortified nutrients maybe different than the bioavailability of naturally occurring nutri-ents. Bioavailability, the proportion of a nutrient that is absorbedby the gut and that becomes available in the body for metabolicprocesses (Winkels and others 2007), has been shown to differbetween foods fortified with micronutrients and those that nat-urally contain equivalent levels of the same nutrient (Sauberlichand others 1987; Vinson and Bose 1988; McNulty and Pentieva2004; Winkels and others 2007).

MS 20100435 Submitted 4/21/2010, Accepted 8/6/2010. Authors are from theNutritional Sciences Program, School of Public Health and Community Medicine (BC,PM, AD), and the Dept. of Dental Public Health Sciences, School of Dentistry (AD),Univ. of Washington, Seattle, WA 98195, U.S.A. Direct inquiries to author Carter(E-mail: brettc2@u.washington.edu).

Supported by a research grant from GlaceauR©

, Inc.

Previous research has shown that ingestion of folic acid (FA)in fortified foods can result in greater bioavailability than thatof naturally occurring food folate (Sauberlich and others 1987;McNulty and Pentieva 2004; Winkels and others 2007). Poorbioavailability of natural food folate has been associated with inad-equate blood levels of FA (McNulty and Pentieva 2004). Studieson the bioavailability of ascorbic acid (AA) have been inconsis-tent (Nelson and others 1975; Vinson and Bose 1988; Mangelsand others 1993). Some studies have found no difference in thebioavailability of naturally occurring AA in fruits and vegetablescompared with supplements (Nelson and others 1975; Mangelsand others 1993). However, other studies have found that whenAA is consumed by participants in synthetic form, unaccompa-nied by other nutrients, it was associated with lower bioavailabilitythan when the AA was consumed in a natural citrus extract con-taining protein, carbohydrate, and bioflavonoids (Vinson and Bose1988).

This study used serial blood draws to compare bioavailability ofAA and FA from a fortified beverage, 100% orange juice (OJ), and1% low fat milk as a control condition.

Methods

ParticipantsTwelve men ranging in age from 20 to 35 were recruited for

the study. Participants were normal to overweight, with body massindexes ranging from 20 to 30 kg/m2. All were nonsmokers ingood health with no history of diabetes, cardiovascular disease,

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dislipidemia, food allergies, or asthma. Participants were recruitedfrom the Univ. of Washington campus and the surrounding neigh-borhood through fliers, campus newspapers, and online postings.All procedures were approved by the Institutional Review Boardof the Univ. of Washington prior to the initiation of the study.

Candidates were invited to attend 1 preliminary screening ses-sion in the lab where they completed a written consent form.This consent described the procedures of the experiment, and ex-plained the voluntary nature of participation. After the consent,each candidate’s weight and height was measured by study staff us-ing a Detecto physicians scale (Detecto, Webb City, Mo., U.S.A.)to calculate and verify reported BMI.

Potential subjects were screened through an in-person interviewto verify eligibility criteria. Subjects were excluded if their personalschedule did not permit attendance at scheduled testing sessionsor if they were currently taking vitamin or mineral supplements.Qualified individuals who agreed to be in the study were sentemail reminders about their weekly testing sessions.

ProtocolThe study followed a within-subject design, with each subject

serving as his own control. Participants came to the laboratory on3 different study dates, at least 1 wk apart, on the same day of theweek for each session. To keep variability at a minimum, subjectswere asked to keep their evening meals and activity levels on theday before the test and breakfast on the day of the test as similaras possible. Participants were asked to avoid eating cold breakfastcereals or toaster pastries (for example, Pop Tarts) for breakfast onthe morning of their testing sessions, since these foods are typicallyfortified with vitamins and minerals. Subjects were also requiredto refrain from drinking alcohol for 24 h before the test.

Participants arrived at 1145 h on testing days and reported toclinical staff at the Fred Hutchison Cancer Research Center. Par-ticipants were permitted to work quietly during the study ses-sion. On each laboratory visit, baseline blood draws were takenat 1200 h, after which participants consumed the test beverage(20 fl ounces, 591 mL) and a low-AA, low-FA snack (see below)amounting to 234 kcal. Participants remained in the laboratoryuntil 1630 h, when a final blood draw was performed. A totalof 10 blood draws were collected for each participant under eachbeverage condition.

Snack and test beveragesThe snack items served were selected to be low in AA and FA

and provide a moderate amount of energy to simulate a morenatural setting. The snack consisted of sliced turkey breast (28 g),sliced low-fat cheese (22 g), and rice crackers (39 g) amounting to234 kcal. Participants were asked to consume the entire snack andbeverage within 20 min.

Each participant was exposed to 3 beverage conditions duringthe study. The order of beverage presentation followed a LatinSquare design, so that on any given testing day, each partici-pant received a different beverage condition in a counterbalancedorder.

Beverage characteristics and compositionThe comparison of the test beverage and OJ was the primary

outcome. Low fat milk (1% fat) was also included as a controlcondition. The purpose of the control beverage was to match OJin energy density (kcal/g), but provide little AA or FA.

Table 1 shows the characteristics of the beverages used in thisstudy. In all cases, the beverage was served in a 591 mL (20 fl oz)

portion, chilled in an opaque cup with lid, and straw. All bever-ages tested in this study were obtained by the investigator fromsingle manufacturing lots through commercial outlets. The forti-fied beverage was ordered in a single, unopened case of 24 units(591 mL each). Milk (1% fat, organic) and OJ (plain, not fromconcentrate) were also obtained in unopened cases through FoodServices of America, a major food distributor. All 3 beverageswere maintained at 4 ◦C until they were presented to participantsduring the testing phase of the project, after which samples of eachbeverage were frozen for later shipment to the testing laboratory(Medallion Laboratories, Minneapolis, Minn., U.S.A.).

Plasma levels of vitamin C and serum levels of folic acidRepeated blood samples were made from an in-dwelling

catheter that was inserted into each participant’s arm and moni-tored by trained staff of the Prevention Center at the Fred Hutchin-son Cancer Research Center. After clearing the catheter tube, eachblood draw was approximately 4 mL in volume. Samples were col-lected at 30 min intervals and aliquoted into cryovials (SST “tiger-top”) containing a clotting factor and serum separator. Aliquotsdestined for AA assays were aspirated into the precipitating agentMPA/DTT, used for stabilization prior to freezing. Plasma AA andserum FA assays were performed by staff of the Dept. of Labora-tory Medicine at the Univ. of Washington. Analysis of plasma AAlevels was conducted using a spectrophotometric method (Liu andothers 1982). Serum FA was analyzed using a chemiluminesencemethod (Song and Zhou 2001).

Statistical analysesAll statistical analyses were conducted in SPSS version 11. Prior

to analyses, intrasubject variation in baseline nutrient concentra-tions (from each day of testing) was addressed by setting baselinemeasurements to zero and expressing the nutrient levels as change(�) from baseline (level at 1200 h).

The dependent variables were blood plasma concentrations ofAA and blood serum concentrations for FA. Independent vari-ables were time and type of beverage ingested. Repeated measuresanalysis of variance (ANOVA) was used for analysis. In addition,differences in the overall serum levels and the change in serumlevels from baseline were tested. A 3rd analysis was conducted onthe area-under-the-curve (AUC) for the change in concentrationprofiles. The AUC was computed according to Kamp using thefollowing formula (Kamp and others 2003):

Table 1–Description of beverage conditions by nutrient and en-ergy composition.

Fortified beverage Orange juice 1% milk

Volume (mL) 591 591 591Energy (kcal) 125 274 256Energy density (kcal/g) 0.20 0.44 0.42Ascorbic acid (mg) 284 68 <3Folate (μg) 245 407 30Proteina (g) 0 4 21Fata (g) 0 <1 6Carbohydratea (g) 13 67 30Sugarsa (g) 13 52 30Vitamin Aa (mg) 1.5 <1 <1Calciuma (mg) 40 56 724

Nutrient data for all beverages based on Univ. of Washington commissioned independentanalyses.aThe values were calculated using standard items from Food Processor 8.1 software (ESHAResearch, Salem, Oreg., U.S.A.).

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AUC = 0.5�(�Ci−1 + �Ci ) × (ti − ti−1)

where �Ci and �Ci−1 are the increment in plasma micronutrientconcentration at time points ti and ti−1. Positive AUC measuresindicated a rise in nutrient level from baseline while negative AUCmeasures indicated a fall in nutrient level from baseline.

Bioavailability of nutrients from the 3 beverage conditions wasexpressed as the ratio between the AUC and the concentrations ofeach nutrient present in the beverage. Beverage concentrations ofFA and AA were assayed at the Medallion Laboratories. Concen-trations of FA and AA were assayed by high-performance liquidchromatography (HPLC).

Repeated-measures ANOVA was conducted on data trans-formed using the method described above. The purpose of theANOVA was to detect the possible differences in the 3 beverageconditions with each subject serving as his own control. Whenthe overall ANOVA was significant, Bonferroni-corrected pair-wise comparisons were made to identify which pairs of beverageconditions were significantly different from each other.

Results and Discussion

Participant characteristicsMale participants had a mean age of 26 y (range 20 to 35 y)

with mean BMI of 24.7 (range 20 to 30).

Ascorbic acid (AA)Analysis of the serial blood draws revealed that plasma AA con-

centrations rose to the highest levels after consumption of the

Figure 1–Temporal profiles of mean baseline change in blood serum ofascorbic acid as a function of preload condition (n = 12).

Figure 2–Mean area under the curve (AUC) for ascorbic acid as a functionof preload condition (n = 12); ∗ Indicates significantly different at P <0.01.

fortified beverage. The mean (s.e.m.) baseline (at 1200 h) AAconcentration for all subjects on all days was 1.02 (0.098) mg/dL.Repeated-measures ANOVA showed a significant effect of timeand beverage (P = 0.03 and P < 0.001, respectively). Analysis ofthe pair-wise comparisons of the serum AA among the 3 bever-ages showed that the AA response to the fortified beverage wassignificantly different than that to either OJ or 1% fat milk (P =0.02 and P < 0.001, respectively). The response to OJ was alsosignificantly different than that to milk (P < 0.001).

Plasma AA concentrations rose most sharply in response tothe fortified beverage, increasing to a peak of approximately 0.6mg/dL relative to baseline levels. By comparison, OJ only eliciteda peak change approximately 0.2 mg/dL relative to baseline, whileAA levels declined relative to baseline with milk. Figure 1 showsthe temporal profiles of change from baseline from serum AAconcentration for each beverage condition. Each curve representsthe mean of all 12 subjects after the change from baseline wascomputed.

AUC of serum AA concentration was also highest in responseto the fortified beverage, with a mean AUC of 1.84 units, com-pared to 0.24 AUC units with OJ and −0.43 AUC units withmilk. Repeated-measures ANOVA showed that the AUC’s dif-fered significantly in relation to beverage (P < 0.001). Analysis ofthe pair-wise comparisons of the blood plasma showed that theAUC for the fortified beverage was significantly different from theAUC for OJ (P < 0.001), which in turn was significantly differentfrom the AUC for milk (P = 0.004). The mean AUCs for all 3beverage conditions are shown in Figure 2.

While the absolute levels of AA were highest in response tothe fortified beverage, the bioavailability of AA post ingestion ofthe fortified beverage was not significantly different from that ofOJ. Table 2 shows that mean bioavailability following the for-tified beverage was approximately the same as that for OJ. Therepeated measures ANOVA conducted on the corrected measure-ments found no statistical difference between these 2 conditionsalthough both were significantly different from milk (P = 0.01and P = 0.02, respectively).

Folic acidSerum FA concentrations rose to the highest levels after con-

sumption of the fortified beverage. The mean (s.e.m.) baseline (at1200 h) serum FA concentration for all subjects on all days was18.4 (2) ng/mL. Repeated-measures ANOVA showed a significanteffect of time and beverage (P < 0.001). Analysis of the pair-wisecomparisons among the 3 beverages showed that the response tothe fortified beverage was not significantly different than the re-sponse to OJ. However, both the fortified beverage and OJ weresignificantly different from milk (P < 0.001).

Serum FA concentrations rose similarly for the fortified bever-age and OJ following beverage ingestion but the response to thefortified beverage was slightly larger and showed a longer “tail”than with OJ. Figure 3 shows the temporal profiles of serum FAconcentration for each beverage condition. In response to thefortified beverage, FA increased by almost 4 ng/mL relative tobaseline levels. By comparison, OJ elicited a peak increase of ap-proximately 3 ng/mL above baseline. In the milk condition, FAlevels declined over time. Again, analyses indicated that the for-tified beverage and OJ were not significantly different from eachother but both were significantly different from milk.

The AUC of serum FA concentration was highest in responseto the fortified beverage, with a mean AUC of nearly 9 units,compared to 4.6 AUC units with OJ and −9.9 AUC units with

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milk. Repeated-measures ANOVA showed that while AUC dif-fered significantly in relation to beverage (P < 0.001), the AUCfor the fortified beverage and OJ were not significantly differentfrom each other. The AUCs of the fortified beverage and OJ wereboth significantly different from the AUC for milk. The meanAUCs for all 3 beverage conditions are shown in Figure 4.

The bioavailability of FA post ingestion of the fortified bev-erage was not significantly different than that post OJ ingestion.Table 2 shows that the mean bioavailability for the fortified bev-erage was higher than that for OJ. Both OJ and the fortifiedbeverage produced significantly different FA bioavailability thanthe milk control (P = 0.001 and P = 0.002, respectively).

This study compared the bioavailability of AA and FA by mea-suring the blood plasma levels of AA and blood serum levels ofFA following the consumption of 591 mL (20 fl oz) servings of areduced calorie fortified beverage, 100% OJ, and milk (1% fat) asa control. This study found no significant differences in terms ofthe bioavailability of 100% OJ and a low calorie beverage fortifiedwith FA and AA.

Figure 3–Temporal profiles of mean baseline change in blood serum of folicacid as a function of preload condition (n = 12).

Figure 4–Mean area under the curve (AUC) for folate as a function ofpreload condition (n = 12); ∗ indicates significantly different at P < 0.01.

Table 2–Bioavailability of ascorbic acid (AA) and folic acid (FA).

Fortified beverage Orange juice (OJ)

AA bioavailability 0.646 (±0.17) 0.625 (±1.22)FA bioavailability 3.66 × 10−3 1.33 × 10−3

(±3.61 × 10−3) (±1.84 × 10−3)

Values shown are means (SD). Bioavailability was defined for both nutrients as the ratiobetween the mean serum area under the curve (AUC) and the nutrient content of eachbeverage. Mean bioavailability for milk was not computed since the AUC for this beveragewas negative. The fortified beverage and OJ were not significantly different from eachother.

Independent laboratory analysis did show that the fortified bev-erage contained over 4 times the AA as the OJ (Table 1). This dif-ference was not unexpected because previous research has showncomparable levels of AA in OJ (Johnston and Bowling 2002).Assays showed that the blood plasma AA concentration was sig-nificantly greater following the ingestion of the fortified beveragein comparison to OJ. However, that observation was likely due todifference in the concentration of AA between the 2 beverages.When using bioavailability as the dependent measure instead ofabsolute blood plasma levels, such a difference did not exist

Several studies have found no differences in the relative bioavail-ability of AA found naturally in foods and that added to foods orbeverages through fortification (Nelson and others 1975; Mangelsand others 1993; Kamp and others 2003). However, in studieswhere bioavailability differences have been reported, the differ-ences might have been due to study design features or the specificfoods or beverages being tested. For example, 1 study that foundhigher bioavailability of natural AA (Vinson and Bose 1988) wasconducted on fasting participants, while the present study wasconducted on participants who had eaten breakfast prior to bever-age consumption and who ingested a moderate amount of energywith the beverage.

Blood serum FA levels and FA bioavailability were not foundto differ significantly between OJ and the fortified beverage. Thefindings on FA reported here are consistent with some prior studies(Tamura and Stokstad 1973; Nelson and others 1975; Pietrzik andRemer 1989). Other studies have reported higher bioavailabilityof synthetic FA than natural food folate (Sauberlich and others1987; McNulty and Pentieva 2004; Winkels and others 2007).Indeed, we did find that the synthetic FA led to a higher peak ofserum levels of FA and a longer tail of FA levels. However, thisdifference was not significant. If the bioavailability of synthetic FAis actually higher than that of natural food folate, the differencemight have been minimized by the experimental design used in thepresent study. Bioavailability of both synthetic FA or food folate isenhanced when presented in liquid vehicles and/or accompaniedby macronutrients (Castenmiller and others 2000; McNulty andPentieva 2004), perhaps leading to a “ceiling effect.” The lack ofa difference may also have been due to the short duration of thetesting session (that is, 4.5 h) (Perry and Chanarin 1970; Brownand others 1973), or the fact that there were other food productsin the intestinal tract of participants when the beverages wereconsumed (McNulty and Pentieva 2004).

ConclusionsThe results indicate that beverages fortified with AA and FA

can provide the same nutrient bioavailability as OJ, but with fewercalories and sugars per serving.

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