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3 Interdisciplinary Graduate Program in Nutritional Sciences, University of Washington, Seattle, WA 98195-3410; 4 Fred Hutchinson Cancer Research Center, Seattle, WA 98109; 5 Institute for Preventive Medicine, Nutrition and Cancer, Folkhälsan Research Center and Division of Clinical Chemistry, University of Helsinki, Helsinki, Finland; and 6 Department of Epidemiology, University of Washington, Seattle, WA 98195-7236
* To whom correspondence should be addressed. E-mail: jlampe{at}fhcrc.org.
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ABSTRACT |
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 TOP ABSTRACT IntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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Introduction |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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5-n-Alkylresorcinols (AR)7 comprise one of the major groups of phenolic compounds in cereal grains (9). They are amphiphilic lipids that are present in the bran layer of several cereal grains but not in the germ or endosperm layers (9), making them candidates as biomarkers of whole-grain intake. The potential for AR to serve as a whole-grain wheat + rye biomarker has been supported by several studies conducted in Nordic countries (particularly Finland), where wheat and rye are the primary grains consumed (8). Using chromatographic analysis, AR content has been shown to be measurable in cereal grains (specifically whole-grain wheat, rye, triticale and barley in human diets) (10), stable during food processing (10,11), and detected in whole-grain wheat and rye-containing food products but not in refined-grain food products related to wheat or rye (11). Although variable AR content has been measured in cereal grains (normally ranging from 300 to 1500 µg/g) (9), it is likely that whole-grain wheat + rye food products will contain at least 200 µg/g (10). Such levels are clearly distinguishable from those of refined products. The AR are absorbed by humans (absorption estimated at 60%) (12), are measurable intact in circulation (9,13,14), and are present as polar metabolites 3-(3,5-dihydroxyphenyl)-1-propanoic acid (DHPPA) and 3,5-dihydroxybenzoic acid (DHBA) in urine samples (15,16). Feeding trials in humans have shown measurable levels of AR or their metabolites in plasma or urine samples after whole-grain wheat and rye consumption but not after refined wheat and rye grain consumption (15,17). Such investigations have shown promise for AR as a biomarker specific for whole-grain wheat and rye intake. However, no studies have examined the potential of these biomarkers to reflect whole-grain exposure in the context of observational studies in U.S. populations.
Our study aimed to examine the potential for AR to serve as a whole-grain wheat + rye biomarker by investigating specifically the relationship between whole-grain wheat + rye intake and DHPPA excretion. We also aimed to determine the utility of DHPPA as a biomarker for whole-grain intake as defined more broadly by epidemiologic studies conducted in the US (4,5,18). This also is the first study, to our knowledge, to quantify urinary AR metabolite excretion in a subgroup of the U.S. population and examine its association with self-reported whole-grain intake.
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Materials and Methods |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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Data collection. At an initial informational meeting (d 0), participants were trained by a registered dietitian on how to keep food records. Participants completed a FFQ relative to their dietary intake within the past 3 mo and a health and demographics questionnaire. The questionnaire included the following categories for race, which was self-declared by each participant: Asian, Black, Caucasian, Mixed, Native Hawaiian, Unknown, African. They completed a 3-d food record (3DFR; 3 consecutive days), which they returned at the d 4 visit when body weight and height also were measured. A single 12-h overnight urine sample was collected following this visit.
Dietary assessment. 3DFR were reviewed by a registered dietitian with the participant and analyzed by trained nutritionists using the University of Minnesota Nutrition Coordinating Center database (20). This database includes a comprehensive food product list and nutrient database (20) to estimate daily intake of nutrients. 3DFR data were available for 99 study participants. The FFQ used was developed by the Nutrition Assessment Shared Resource of FHCRC. The FFQ collected information about frequency of intake and portion size of 122 foods and food groups over the past 3 mo. Completed FFQ were analyzed for annual servings of specific foods and daily nutrient consumption using the database from the University of Minnesota Nutrition Coordinating Center database (20). Annual servings of foods were adjusted for serving size and usual frequency of consumption and then converted to number of daily servings. FFQ data were available for 95 study participants.
Quantification of whole-grain intake. The primary exposure variable, whole-grain intake, was estimated in several ways. For the 3DFR data, literature estimates of AR-containing foods were used to identify food record line items including whole-grain wheat- and rye-containing foods. A categorical variable was created from this data to classify participants as consuming any or none of these foods (any consumption was >0.0 g dry flour or >0.0 g fresh food item). For the FFQ data, whole-grain intake was estimated from food group intakes as defined by line items. Whole-grain intake (continuous) was estimated in 2 ways: 1) in accordance with the 3DFR data methods, estimating particularly whole-grain rye and wheat intake [summation of intakes of 2 line items, "high-fiber or bran cereals" (1-cup (45 g) servings) and "dark breads" (medium slice servings)]; and 2) representing whole-grain intake as defined by epidemiologic studies (summation of intakes of 7 line items; Table 1), following as closely as possible classifications employed by Jacobs et al. (4), Liu et al. (5), and Mellen et al. (18).
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Statistical analysis.
Whole-grain intake was analyzed as a categorical variable for the 3DFR data, because amounts of AR in each food item have not been determined consistently and whole-grain wheat and rye intake as determined by 3DFR was low in this population. For the FFQ data, frequency of intake of whole grains was used as a continuous variable without further weighting by AR content or concentration of whole-grain. DHPPA excretion [µmol/12 h and µmol/mol creatinine] was analyzed as a continuous variable and, because of its positively skewed distribution, was log-transformed to normalize (data presented as back-transformed values unless otherwise noted). For part of the analysis, urinary DHPPA was adjusted for creatinine to adjust for variable dilutions in collected urine samples (21). Nutrient data were examined and FFQ or 3DFR data were excluded if daily energy intake was outside reasonable ranges [1675–16,748 kJ for females; 2512–20,935 kJ for males; a modification of ranges from (22)]. Nutrient intakes were adjusted for energy intake. Whole-grain intake and DHPPA excretion were adjusted for demographic and nutrient intake variables that exhibited at least 1 of 2 criteria for confounding: 1) a significant association with both the exposure and outcome variables; and/or 2) change in the partial β-coefficient of the main effect by >10%. We used linear and multiple regression, chi-square tests, t tests, and pair-wise Pearson correlation tests. Effect estimates of change in log (excretion) were back-transformed into relative change per unit change in independent variable, expressed as a ratio. A priori, adjustments for BMI were made to account for the possibility that AR may be stored in adipose tissue (23). All the tests were 2-sided and at 
level of 0.05. P-values between 0.05 and 0.10 were considered indicative of a trend toward significance. All statistical analyses were performed using Stata (version 9.0; StataCorp LP).
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Results |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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Urinary DHPPA excretion. 12-h urinary DHPPA excretion (n = 100) ranged from 1.3 to 99.4 µmol/12 h and concentrations ranged from 0.8 to 166 µmol/L (226 – 15,950 µmol/mol creatinine). Although between-group differences in mean 12-h DHPPA excretion were not significant for sex, age, and BMI (Table 2), DHPPA excretion tended to be higher in non-Asians than in Asians (P = 0.08). After adjusting for whole-grain intake, race was no longer associated with DHPPA excretion (3DFR, P = 0.28; FFQ wheat + rye, P = 0.6; FFQ all grains, P = 0.7).
Based on 3DFR, energy intake was significantly associated with DHPPA excretion and most fiber-related variables, adjusted for energy, were significantly associated with DHPPA excretion (Table 3). In contrast, protein and fat intakes were not significantly associated with DHPPA excretion. Similar relationships were observed between DHPPA excretion and nutrient measures as estimated by FFQ (data not shown). Adjusting for BMI, energy, fiber, and whole-grain wheat + rye intake, the variances in DHPPA excretion explained by the data were 31% (R2 = 0.31; P = 0.003) for the 3DFR and 25% (R2 = 0.25; P = 0.003) for the FFQ. Adjusting for BMI, energy, fiber, and overall whole-grain intake, the FFQ data explained 26% of the variance in DHPPA excretion (R2 = 0.26; P < 0.0001).
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Urinary DHPPA adjusted for creatinine. Given that spot urines, rather than 12- or 24-h urines, are often more commonly available for large-scale observational studies, we also examined dietary intake data relative to urinary DHPPA excretion adjusted for creatinine. Demographics (sex, age, race, and BMI) did not differ for DHPPA excretion expressed as µmol DHPPA/mol creatinine. Whole-grain wheat + rye intake was associated with DHPPA/mol creatinine using both 3DFR and FFQ data. DHPPA/mol creatinine was significantly associated with fiber-related variables but not with total energy, protein, or fat intakes (data not shown). 3DFR data showed that consumers of whole-grain wheat + rye had a 75% higher DHPPA/mg creatinine excretion than nonconsumers and a 42% higher DHPPA/mol creatinine excretion than nonconsumers before and after adjusting for BMI, energy, and fiber intake [ratio of excretion (95% CI) = 1.75 (1.26, 2.45); P = 0.001 unadjusted; ratio of excretion (95% CI) = 1.42 (1.05, 1.93); P = 0.025 adjusted]. Similarly, DHPPA/mol creatinine increased per serving increase in whole-grain wheat + rye as estimated by FFQ: ratio of excretion (95% CI) = 2.12 (1.58, 2.84), P < 0.001 unadjusted; ratio of excretion (95% CI) = 1.90 (1.36, 2.67); P < 0.001 adjusted. Overall whole-grain intake estimated by FFQ was also associated with DHPPA/mol creatinine [ratio of excretion (95% CI) = 1.67 (1.32, 2.10); P < 0.001 unadjusted; ratio of excretion (95% CI) = 1.55 (1.21, 2.00); P = 0.001 adjusted].
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Discussion |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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Findings pertaining to demographics indicated that whole-grain intake was fairly consistent across sex, age, and BMI groups, although our small sample may have reduced the likelihood of detecting differences. A significant difference in the consumption of whole grains across race was not unexpected, as many Asian cultures consume rice as a staple grain (compared with wheat + rye or other grains) (24). The observation that race was significantly associated with DHPPA excretion prior to, but not after adjusting for, whole-grain intake indicates that race differences in DHPPA excretion are likely attributable to differences in whole-grain intake rather than race itself. Our finding of a lack of significant difference in DHPPA excretion by sex was consistent with other published work on plasma AR (13).
The significant association between energy intake and DHPPA excretion is aligned with results of other studies, indicating that a higher energy consumption commonly yields higher nutrient/intake marker levels (25). The positive associations of fiber- and grain-related variables (e.g. phytic acid and vegetable protein) and the inverse associations of protein and fat intakes with DHPPA excretion also support the hypothesis that DHPPA excretion reflects primarily whole-grain intake.
Overall, whole-grain wheat + rye intake (3DFR and FFQ) and whole-grain intake (FFQ) were significantly associated with AR excretion. The association for the FFQ data were slightly diluted by the extension of whole grains to include oat and corn products (i.e. a serving increase in whole-grain intake increased DHPPA by 67% rather than 94%), but the precision of the estimate was improved as shown by the narrower CI and lower P-values (Table 4). Intake of the 5 items representing whole-grain oat and corn did not differ between the whole-grain wheat + rye consumers and nonconsumers, suggesting that whole-grain wheat and rye, and not other grains, were contributing to DHPPA excretion. Further, based on the 3DFR, only 12% of participants reported consuming whole-grain rye, suggesting that, in our study population, whole-grain wheat is the predominant source of AR. Whether the observations from our study [i.e. wheat + rye consumers were not more likely to be the primary consumers of other whole grains (e.g. oats and corn) and consumption of whole-grains was dominated by wheat] is generalizable to a broader segment of the U.S. population remains to be determined.
Our findings also support the utility of DHPPA concentration adjusted for creatinine (µmol DHPPA/mol creatinine) as a potential biomarker for whole-grain wheat + rye. This could extend the use of urinary DHPPA to large, population-based studies. Further studies should examine the associations between whole-grain intake and DHPPA adjusted for creatinine from a spot urine sample rather than from a defined 12-h urine sample to determine whether these findings hold.
This study was the first examination, to our knowledge, of urinary DHPPA excretion and the utility of AR as a whole-grain biomarker in a U.S. population. Strengths included the use of more than 1 dietary assessment tool, the use of a highly sensitive assay for urinary DHPPA analysis, and findings of DHPPA excretion consistent with published data. There are several limitations. This pilot study relied on a small, convenience sample in a relatively homogeneous population, which limits generalizability to the greater U.S. population. In addition, a narrow range in the age of participants also limits our potential findings. Only DHPPA was available for analysis (DHBA eluted closely after the huge acetaminophen peak, making impossible the quantification of DHBA). Although we consider this a limitation, data from Koskela et al. (16) would suggest that DHPPA suffices as a marker; DHPPA and DHBA are highly correlated and recoveries of both from urine are similar. In addition, our observation that acetaminophen, a commonly used drug, overlapped with DHBA suggests that with the available analytic methodologies, this AR metabolite might be of less utility in populations in which over-the-counter medication use is common.
Another limitation in the study design was that the timing of the urine sampling in relation to the 3DFR varied between participants (days to weeks for most, and even over a month for several) and thus use of 3DFR as a measure of "recent" intake may have been more applicable for some than others. Despite this variability, it is possible that consumption of whole grains may be more routine in pattern than sporadic (i.e. one consistently either does or does not consume whole grains) and, if so, the 3DFR in this case may adequately reflect "usual" rather than only "recent" intake. The FFQ was limited in its ability to comprehensively capture whole grains. It did not specify rye or wheat and it was not framed to comprehensively capture whole-grain intake (i.e. brown rice, wheat germ, and whole-grain crackers among other items were not included). However, U.S. whole-grain intake data show that two-thirds of whole-grain intake come from breakfast cereals and yeast breads (26); thus, it is likely that the FFQ (specifying high-fiber cereal and dark bread intakes) captured a good portion of whole-grain consumption. Another limitation is that AR may be more widespread in foods than originally believed (9), possibly making them not specific enough to whole grains (or whole-grain wheat + rye) as a biomarker. Finally, AR storage in adipose tissue or cell membranes (23) may reflect longer-term intake and will require that results from plasma and urine AR analysis reflect that consideration.
Despite these uncertainties, the results of this study provide support for the application of AR and their metabolites as biomarkers of whole-grain intake in epidemiologic studies. Further work is necessary to determine the scope of AR presence in foods and whether any other compounds (aside from absorbed AR) can be transformed or metabolized into DHPPA or DHBA. Discussion of the latter is presented in (16). Biological considerations include clarification of percent absorption of AR and determination of the extent to which habitual whole-grain intake may affect bioaccumulation of AR in tissues (and thus the potential for measurable AR upon release from storage to skew assessments of dietary intake). In addition, the half-life of AR is estimated at 
5 h (27). As a metabolite of AR, DHPPA may have a longer half-life but still likely reflects recent dietary intake of whole grains. Future studies should determine the extent to which urinary DHPPA excretion is suitable as a whole-grain biomarker in the broader U.S. population and additional controlled feeding studies are needed to calibrate the dose-response relationship between AR-containing whole grains and urinary DHPPA excretion.
In conclusion, in this U.S. population, urinary DHPPA excretion per 12 h was associated with the intake of whole-grain wheat + rye, as well as with that of whole grains in general. Further, urinary DHPPA per mol creatinine also was strongly associated with the whole-grain parameters, suggesting the possible application to spot urine collections.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Author disclosures: L. A. Guyman, H. Adlercreutz, A. Koskela, L. Li, S. A. A. Beresford, and J. W. Lampe, no conflicts of interest.
7 Abbreviations used: AR, alkylresorcinol; 3DFR, 3-day food record; DHBA, 3,5-dihydroxybenzoic acid; DHPPA, 3-(3,5-dihydroxyphenyl)-1-propanoic acid; FHCRC, Fred Hutchinson Cancer Research Center.
Manuscript received 6 May 2008. Initial review completed 18 June 2008. Revision accepted 30 July 2008.
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LITERATURE CITED |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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