The Journal of Nutrition Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions

Rye Bran Modified with Cell Wall–Degrading Enzymes Influences the Kinetics of Plant but Not of Enterolignans in Multicatheterized Pigs

Anne K Bolvig,1 Natalja P Nørskov,1 Sophie van Vliet,1 Leslie Foldager,1,2 Mihai V Curtasu,1 Mette S Hedemann,1 Jens F Sørensen,3 Helle N Lærke,1 and Knud E Bach Knudsen1 Downloaded from https://academic.oup.com/jn/article/147/12/2220/4728058 by guest on 30 September 2021

1Department of Animal Science and 2Bioinformatics Research Centre, Faculty of Science and Technology, Aarhus University, Aarhus, Denmark; and 3DuPont Industrial Biosciences, Brabrand, Denmark

Abstract Background: Whole-grain intake is associated with a lower risk of chronic Western-style diseases, possibly brought about by the high concentration of phytochemicals, among them plant lignans (PLs), in the grains. Objective: We studied whether treatment of rye bran with cell wall–degrading enzymes changed the solubility and kinetics of PLs in multicatheterized pigs. Methods: Ten female Duroc 3 Danish Landrace 3 Yorkshire pigs (60.3 6 2.3 kg at surgery) fitted with permanent catheters were included in an incomplete crossover study. The pigs were fed 2 experimental diets for 1–7 d. The diets were rich in PLs and based on nontreated -rich [LR; lignan concentration: 20.2 mg dry matter (DM)/kg] or enzymatically treated lignan-rich (ENZLR; lignan concentration: 27.8 mg DM/kg) rye bran. Plasma concentrations of PLs and enterolignans were quantified with the use of targeted LC-tandem mass spectrometry. Data were log transformed and analyzed with mixed-effects, 1-compartment, and asymptotic regression models. Results: The availability of PLs was 38% greater in ENZLR than in LR, and the soluble fraction of PLs was 49% in ENZLR compared with 35% in LR diets. PLs appeared in the circulation 30 min after intake of both the ENZLR and LR diets. Postprandially, consumption of ENZLR resulted in a 4-times-greater (P < 0.0001) plasma PL concentration compared with LR. The area under the curve (AUC) measured 0–360 min after ENZLR intake was ;2 times higher than after LR intake. A 1-compartment model could describe the postprandial increase in plasma concentration after ENZLR intake, whereas an asymptotic regression model described the plasma concentrations after LR intake. Despite increased available and soluble PLs, ENZLR did not increase plasma enterolignans. Conclusion: The modification of rye bran with cell wall–degrading enzymes resulted in significantly greater plasma concentrations of PLs and the 4-h AUC, particularly syringaresinol, in multicatheterized pigs. JNutr2017;147:2220–7.

Keywords: lignans, cell wall–degrading enzymes, kinetics, catheterized pigs, syringaresinol

Introduction disease, and type 2 diabetes, is well established and has been The link between a high intake of whole grains and a lower risk documented in a number of epidemiological studies (1–3). One of chronic Western-style diseases, including cancer, heart of the active components that may play a role in the health- beneficial properties of whole grains and their consumption is Funding was provided by Innovation Fund Denmark (ELIN: 0603-00580B). the lignans. Lignans are diphenolic compounds found in high Author disclosures: AKB, NPN, SvV, LF, MVC, MSH, JFS, HNL, and KEBK, no conflicts of interest. concentrations in such common foods as seeds, grains, berries, Supplemental Tables 1–6 are available from the ‘‘Online Supporting Material’’ and vegetables (4–6). As concerns cereal grains, the concentra- link in the online posting of the article and from the same link in the online table of contents at http://jn.nutrition.org. tion of lignans is higher in rye than in wheat, oats, and barley (5), Address correspondence to AKB (e-mail: [email protected]). and the lignan content in grain is found mainly in the outer bran Abbreviations used: DM, dry matter; End, ; Enl, ; ENZLR, layer (pericarp or testa and aleurone) (7, 8). enzymatically treated lignan-rich diet; Hydr, 7-; I-Lari, iso-; Lari, lariciresinol; LR, lignan-rich diet; Mata, ; Med, medioresinol; Pino, ; Commonly identified plant lignans (PLs) are matairesinol

PL, plant lignan; PLOther, plant lignans other than Syr; PLTotal, total plant lignans; SDG, (Mata), pinoresinol (Pino), medioresinol (Med), lariciresinol (Lari), diglycoside; Seco, secoisolariciresinol; Syr, syringaresinol; WO, washout. iso-lariciresinol (I-Lari), syringaresinol (Syr), secoisolariciresinol

ã 2017 American Society for Nutrition. 2220 Manuscript received July 18, 2017. Initial review completed August 18, 2017. Revision accepted September 12, 2017. First published online October 4, 2017; doi: https://doi.org/10.3945/jn.117.258483. (Seco), and the glycosylated form of Seco, secoisolariciresinol The pigs were transferred to individual pens (1.25 3 3.75 m) without diglycoside (SDG). Absorption of PLs has not been well charac- bedding material but with elevated plastic grids, which allowed the pigs terized; however, recent studies in rats (9) and pigs (10), along to rest and remain dry. The pigs were acclimatized for 4 d before being with studies in humans, fed a lignan-rich (LR) diet or a single surgically fitted with permanent catheters in the portal and hepatic veins dose of SDG (11, 12) have shown rapid postprandial absorption and in the mesenteric artery. A flow probe was fitted around the portal vein (17), but because of technical problems, flow probe data were not of PLs and reported peak concentrations within 0–2.5 h from included in the present study. intake. The rapid absorption implies that part of the PLs is The pigs had a 7–9-d recovery period after the surgery, at which time absorbed from the small intestine to the circulation, which is the pigs entered the experimental period. Originally, the study was in contrast to the enterolignans [enterodiol (End) and enterolactone designed as a crossover study; however, because of the malfunctioning (Enl)], which are produced by the gut microbiota and are absorbed catheters, the crossover was not completed and the experiment was mainly from the large intestine (13). terminated on day 16. The pigs were fed the WO diet on days 1–4. This The lignans in cereals are concentrated to a large extent in the was followed by intake of one of the LR diets (LR or ENZLR) on days dietary fiber–rich outer bran fraction, which also contains a high 5–11. On days 12–15, the WO was fed again, and on the last day of the concentration of other phenolic compounds. Bioprocessing with experimental period (day 16), the pigs were fed the LR diet (LR or xylanases alone or in combination with arabinofuranosidase has ENZLR) that was not fed on days 5–11. Ten pigs completed the been shown to make a substantial part of the insoluble ferulic postprandial profile conducted on days 5 and 16 on each diet, whereas Downloaded from https://academic.oup.com/jn/article/147/12/2220/4728058 by guest on 30 September 2021 5 pigs/diet completed days 5–11 during which the samples collected acid available for absorption in the small intestine (14). Further- when the pigs were feed deprived were sampled. The experiment was more, enzymatic treatment with xylanases has resulted in the terminated after the postprandial profile was completed on day 16. enrichment of PLs in soluble rye bran extract (15). The effect of The pigs were offered water ad libitum and were allowed physical enzyme modification of the lignan source has not been investi- and visual contact through the railings of the pens. The pigs were gated in vivo, however, and its influence on the kinetic param- weighed 1 time/wk, and an iron supplement (400 mg; Uniferon, eters is unknown. Pharmacosmos) was administered intramuscularly weekly and addi- We hypothesized that the treatment of rye bran with cell tionally on profile days. wall–degrading enzymes would result in the increased solubili- Blood samples were collected (0 min) from the catheters of feed- zation of PLs, which would lead to an altered absorption kinetic. deprived pigs on days 5–16. On days 5 and 16, additional postprandial The aim of the present study was to investigate the absorption blood samples were collected at 30, 60, 120, 180, 240, 300, and 360 min relative to the morning meal. On days 12–15, a single sample was and elimination kinetics of PLs upon intake of an LR diet collected 360 min after the morning meal in addition to the 0-min sample modified with a cocktail of cell wall–degrading enzymes: xylanases, taken after feed deprivation for 12 h. After sampling was completed, the b -glucanases, and cellulases. The enzyme-modified rye bran or indwelling catheters were treated as described by Ingerslev et al. (16). nontreated rye bran was incorporated into a low-lignan wheat Blood samples were collected into sodium heparin Vacutainers (Becton- flour–based diet replacing refined wheat–concentrated fiber at an Dickinson), centrifuged at 2000 3 g for 10 min at 4°C, and plasma was iso-dietary fiber level. The effect of the enzyme treatment was stored at 280°C until analysis. Hematocrit levels were measured on days assessed in terms of absorption kinetic consequences for PLs and 5, 12, and 16. The pigs were killed with an overdose of sodium enterolignans in a catheterized pig model. pentobarbital followed by exsanguination (18). The animal experiment was conducted according to the protocols approved by the Danish Animal Experiments Inspectorate and complied with the guidelines of the Ministry of Food, Agriculture, and Fisheries concerning animal Methods experiments and the care of animals under study. Diets Analytical methods Three experimental diets were produced: a washout (WO) diet low in PLs Chemical analyses of the diets were performed in duplicate on freeze- based on white wheat flour supplemented with purified wheat fiber (Vitacel dried samples. Dry matter (DM) was determined by drying at 103°Ctoa WF 600; J. Rettenmeier and So¨ hne GmbH) and 2 LR diets in which constant weight, and ash was analyzed according to the Association of 270 g/kg nontreated (LR); and enzymatically treated (ENZLR) rye brans Analytical Communities method number 942.06. Dietary nitrogen was replaced the supplemented purified wheat fiber. The diets were created to analyzed by Dumas, and protein was calculated as N 3 6.25 (19). Gross have equal amounts of protein, fat, digestible carbohydrates (sugars and energy was analyzed on the 6300 Automatic Isoperibol Calorimeter starch), dietary fiber, and gross energy but to vary in the content and system (Parr Instruments). The dietary sugars glucose, fructose, and proportion of soluble and insoluble PLs (Supplemental Table 1). DuPont sucrose were determined as described elsewhere (20). Starch and Industrial Biosciences produced the enzyme-treated rye bran (rye bran nonstarch polysaccharide were determined essentially as described by from Lantma¨nnen Cerealia, Nordmills), which was essentially prepared Knudsen (21) and modified to include measurements of low-molecular- as described by Ingerslev et al. (16). The enzyme mix used consisted of weight nondigestible carbohydrates as described by Kasprzak et al. (22). 3 cell wall matrix–degrading enzymes: xylanase from Bacillus subtilis Klason lignin was measured as the sulfuric acid–insoluble residue as (0.5 g dry enzyme product/kg bran) and glucanase and cellulase from described by Theander and A˚ man (23). Dietary lignan concentrations Trichoderma reesei (5 g liquid enzyme product/kg bran). The diet were determined through the use of a newly developed LC-MS method ingredients were mixed and cold pelleted in a feed production unit at described by Nørskov and Bach Knudsen (24). Aarhus University and stored at 220°C. During the experimental period, Plasma concentrations of Seco, Mata, Lari, I-Lari, Syr, Pino, the pigs were provided daily feed corresponding to 2.7% of the mean 7-hydroxymatairesinol (Hydr), End, and Enl were determined according body weight divided into 3 equal meals fed at 0900, 1500, and 2100. to Nørskov et al. (25). Total PL (PLTotal) concentration represents the sum Portion size was adjusted 1 time/wk according to body weight. On the of Seco, Mata, Lari, I-Lari, Syr, Pino, and Hydr, whereas Syr is the days with postprandial blood sampling, the pigs were allowed 30 min to quantification of Syr alone, and other PLs (PLOther) represent the sum of complete their ingestion of the morning meal, after which the remaining the PLs Seco, Mata, Lari, I-Lari, Pino, and Hydr. feed was removed. Calculations and statistical analysis Animals and experimental design Data were analyzed with RStudio version 0.99.489. Data were log A total of 10 female Duroc 3 Danish Landrace 3 Yorkshire pigs (60.3 6 transformed before analysis to obtain normality and to stabilize the 2.3 kg at surgery) were purchased from local farmers and raised by the variances. Estimates for feed-deprived and postprandial plasma variables Department of Animal Science, Aarhus University (Foulum, Denmark). were determined for each sampling site (arterial, portal vein, or hepatic

Kinetics of plant lignans 2221 vein) and at each time (hours after intake or days after switching the diet) constant, and t is time. The time to reach plateau is estimated as the time through use of the following mixed-effects model (package ‘‘lmer’’): it took to reach the modeled asymptotic concentrations 20.25 nmol/L. The postprandial half-life (t1/2) was calculated by Yijk ¼ ai þ yk þ 3ijk ð1Þ logð2Þ t1=2 ¼ ð4Þ where Yijk is the response, ai is the time (i = 0, 30, 60, 120, 180, 240, 300, expðlKeÞ 360 min, or i = 1, 2, 3, 4, 5, 6, 7 d after switching the diet), the variance component yk (k = pig) accounts for the repeated measurements within with the use of the Ke found by the SSfol function. each pig, and 3ijk is the normally distributed residual error component. The AUC was calculated using the trapezoidal rule estimating the A number of relevant comparisons were made, and P values were AUC obtained 0–360 min after feed intake (Figure 1A) and the AUC adjusted for multiple comparisons according to a single-step procedure estimated on days 5–11 when the pigs were feed deprived (Figure 1B). based on the joint distribution as implemented in the package ‘‘glht.’’ In Differences between groups were evaluated based on calculated CIs. all of the analyses, a significance level of 5% was chosen, whereas The elimination t1/2 found during the WO phase was determined tendency was reported when P values were between 0.05 and 0.1; the with the use of P values have been corrected for multiple comparisons. r f ðt = Þ¼ ð5Þ The variation in postprandial plasma concentrations of PLTotal and 1 2 2

Syr after intake of ENZLR (days 5 and 16) was described by the Downloaded from https://academic.oup.com/jn/article/147/12/2220/4728058 by guest on 30 September 2021 nonlinear mixed-effects model (package ‘‘nlme,’’ version 3.1–117, where RStudio version 0.99.489) with the first-order compartment model function (SSfol) (26) fðtÞ ¼ a þ ðr2aÞ exp½2expðlÞ t ð6Þ

gðtÞ where a is the asymptote, r is the intercept, and l is a numeric parameter Dose expðlKe þ lKa2lClÞfexp½2expðlKeÞ t2exp½2expðlKaÞtg ¼ representing the natural logarithm of the rate constant. expðlKaÞ2expðlKeÞ ð2Þ Results where dose is a numeric value representing the initial dose; t is time (h); and lKe, lKa, and lCl are numeric parameters representing the natural Dietary composition, animal performance, and effect of logarithm of the rates of elimination, absorption, and clearance, cell wall–degrading enzymes respectively. The diets were created to contain equal amounts of energy, fat, When pigs were fed the LR diet, the corresponding postprandial protein, digestible carbohydrates (sugars and starch), and dietary plasma concentrations were modeled with the use of the asymptotic fiber but to have varying concentrations of soluble and insoluble regression function SSasymp (27) from the RStudio package nlme: PLs, which was achieved (Supplemental Table 2). gðtÞ¼a þ ðb2aÞ exp½2expðgÞ t ð3Þ The lignan concentrations in LR and ENZLR were 13 and 19 times higher, respectively, than they were in WO. Processing of the where a is a numeric parameter representing the horizontal asymptote, rye bran with cell wall–degrading enzymes converted part of the b is a numeric parameter representing the response when time is 0, g is insoluble noncellulosic polysaccharides into low-molecular-weight a numeric parameter representing the natural logarithm of the rate nondigestible carbohydrates, and the concentrations of detectable

FIGURE 1 AUCs of PLTotal, Syr, and PLOther measured in the mesenteric artery, portal vein, and hepatic vein in pigs 0–360 min after intake of LR or ENZLR, n = 10/diet (A) or 0–7 d after intake of LR or ENZLR, n =5/diet(B). Values are means 6 SDs. *Different from LR at that time. ENZLR, enzymat- ically treated lignan-rich diet; LR, lignan- rich diet; PLOther, plant lignans other than Syr; PLTotal, total plant lignans; Syr, syringaresinol.

2222 Bolvig et al. TABLE 1 Dietary fiber, PLTotal, Syr, and PLOther of WO, LR, and concentration was 45.8 nmol/L for Syr, which was reached ENZLR diets1 48 min after feeding ENZLR. Intake of LR and ENZLR in cases of PLOther translated into a gradual buildup of PLs in the Diets, chemical composition units/kg DM circulation, which could be described by an asymptotic regres- WO LR ENZLR sion model with plateau concentrations at 17.7 and 9.2 nmol/L obtained after ;164 and 89 min for PLTotal and Syr, respectively. Dietary fiber, g 150 151 147 For PLOther, plateau levels at 11.2 and 8.9 nmol/L were obtained PLTotal, mg 1.4 (0.4) 20.2 (7.1) 27.8 (13.9) ;118 and 266 min after intake of ENZLR and LR, respectively. Syr, mg 0.8 (0.2) 16.0 (5.9) 23.9 (12.5) The AUCs measured 0–360 min after intake of ENZLR or LR PLOther, mg 0.6 (0.2) 4.2 (1.2) 3.9 (1.4) were affected markedly by the enzymatic treatment, as indicated 1 Values in parentheses denote the soluble fraction. DM, dry matter; ENZLR, in Figure 1A, with the AUC of PLTotal after ENZLR intake being enzymatically treated lignan-rich diet; LR, lignan-rich diet; PLOther, plant lignans other 2 times higher than that of LR (264 and 91 h 3 nmol/L, than Syr; PLTotal, total plant lignans; Syr, syringaresinol; WO, washout. respectively). Correspondingly, the AUC of Syr was 3 times higher after ENZLR intake (203 3 nmol/L and 48 h 3 nmol/L, respec- tively), whereas the AUC of ENZLR for the PLOther was 0.4 times lignans in ENZLR were 38% higher than those in LR (27.8 and Downloaded from https://academic.oup.com/jn/article/147/12/2220/4728058 by guest on 30 September 2021 20.2 mg/kg, respectively) (Table 1). Furthermore, the fraction of higher than LR, with no statistical difference between the diets 3 soluble lignans was higher in ENZLR than in LR, with 49% in (61 and 43 h nmol/L, respectively). ENZLR compared with 35% in LR. The dominant PL for both Levels of lignans in feed-deprived animals diets was Syr, which constituted $80% of the PLTotal. PLs. Increasing plasma concentrations of PLTotal, Syr, and PLOther Postprandial absorption of lignans measured when the pigs were deprived of feed were observed 24 h PLs. Low plasma concentrations of PLs were measured before from intake of ENZLR and LR (Table 3 and Figure 3A–C). This the morning meal on day 5. The concentrations, however, was followed by a slight decrease on the following 6 d when increased rapidly within 30 min from intake of LR or ENZLR, measured in the mesenteric artery (P < 0.05) and for Syr in the as presented in Table 2 and Figure 2. Enzymatic treatment of portal vein (P = 0.030) but not in the hepatic vein (Supplemental the rye bran in ENZLR gave rise to substantially higher plasma Table 5). There was a tendency toward higher AUCs after PL concentrations compared with LR, regardless of measuring ENZLR intake for PLTotal and Syr when measured in feed- site (Supplemental Table 3). For PLTotal, and specifically for deprived pigs 0–7 d after switching to the LR diet (Figure 1B). For Syr, the diet-induced differences were most pronounced PLOther, the 2 diets did not give rise to differences in the feed- 30 min after the morning meal. The concentrations were ;4 deprived plasma concentrations or in the AUCs. and6timeshigher,respectively,inENZLR-fedthaninLR-fed pigs, whereas the concentration differences measured 360 min Enterolignans. The plasma concentrations of Enl but not End after the morning meal were only 1–2 times higher. For increased gradually over time after switching from WO to LR or PLOther, the plasma concentrations after ENZLR intake were a ENZLR diets (Figures 3D, E). For Enl, the plasma concentrations maximum of 0.6 times higher compared with those after LR could be described by an asymptotic regression model, with intake, with the largest differences observed close to lignan estimated asymptotes of 144 6 28.2 and 125 6 24.6 nmol/L for intake. ENZLR and LR, respectively. The enterolignan plasma concen- trations stabilized around the modeled asymptote ;48 h after the Kinetic models. The rapid increase in the plasma concentration first LR meal was served. The diet did not affect the AUC of either of PLTotal and Syr after feeding the pigs with ENZLR could be End or Enl (data not shown). described by a 1-compartment model, whereas it was not possible to adapt the same plasma concentration model for Clearance of lignans PLOther or after feeding LR. The kinetic analyses showed that the Clearance of the PLs from the circulation was measured both maximum concentration for PLTotal was 56.3 nmol/L, which was postprandially on the first day after switching from the WO diet reached after 56 min (Supplemental Table 4); the maximum to LR or ENZLR diets (days 5 and 16) (Supplemental Table 4)

TABLE 2 Plasma concentrations of PLTotal, Syr, and PLOther measured in the mesenteric artery of pigs 0–360 min after being fed either LR or ENZLR1

PLTotal Syr PLOther Diet Diet Diet Time, min LR, nmol/L ENZLR, nmol/LP LR, nmol/L ENZLR, nmol/LP LR, nmol/L ENZLR, nmol/L P

0 1.4 (1.1, 1.6) 1.6 (1.3, 2.0) 0.89 0.4 (0.2, 1.6) 0.5 (0.2, 1.9) 0.99 1 (0.8, 1.2) 1 (0.9, 1.3) 1.00 30 8.4 (7.0, 10.2) 43.2 (34.8, 53.7) ,0.0001 5.1 (5.1, 7.3) 37.8 (31.1, 48.2) ,0.0001 3.3 (2.7, 4.0) 5.3 (4.4, 6.4) ,0.0001 60 10.6 (8.8, 12.8) 48 (38.6, 59.7) ,0.0001 6.5 (6.3, 8.9) 41.4 (34.1, 52.6) ,0.0001 4 (3.3, 4.9) 6.6 (5.5, 8.0) ,0.0001 120 11.8 (9.8, 14.3) 41.6 (33.5, 51.7) ,0.0001 6.9 (6.6, 9.4) 34 (28.2, 43.5) ,0.0001 4.9 (4.0, 6.0) 7.5 (6.2, 9.0) ,0.0001 180 12.9 (10.7, 15.6) 37.6 (30.3, 46.7) ,0.0001 7 (6.8, 9.6) 29.7 (24.7, 38.1) ,0.0001 5.8 (4.7, 7.1) 7.9 (6.6, 9.5) 0.02 240 13.9 (11.6, 16.8) 35.6 (28.7, 44.2) ,0.0001 7.5 (7.1, 10.0) 27.2 (22.7, 35.0) ,0.0001 6.3 (5.2, 7.7) 8.4 (7.0, 10.1) 0.04 300 14.3 (11.9, 17.3) 33.1 (26.6, 41.1) ,0.0001 7.4 (7.1, 10.0) 24.7 (20.7, 31.9) ,0.0001 6.7 (5.5, 8.2) 8.4 (7.0, 10.0) 0.24 360 14.1 (11.7,17.0) 29.9 (24.1, 37.1) ,0.0001 7.3 (7.0, 9.9) 21.6 (18.2, 28.1) ,0.0001 6.6 (5.4, 8.0) 8.2 (6.9, 9.9) 0.21

1 Values are modeled estimates (95% CIs), n = 10/diet. ENZLR, enzymatically treated lignan-rich diet; LR, lignan-rich diet; PLOther, plant lignans other than Syr; PLTotal, total plant lignans; Syr, syringaresinol.

Kinetics of plant lignans 2223 FIGURE 2 Plasma concentrations of

PLTotal (A), Syr (B), and PLOther (C) collected from the mesenteric artery of pigs 0–360 min after a single dose of LR or ENZLR. Values are modeled estimates (95% CIs), n = 10/diet. *Different from LR at that time, P , 0.05. ENZLR, enzymatically trea- ted lignan-rich diet; LR, lignan-rich diet;

PLOther, plant lignans other than Syr; PLTotal, total plant lignans; Syr, syrin- garesinol. Downloaded from https://academic.oup.com/jn/article/147/12/2220/4728058 by guest on 30 September 2021 and during intake of the WO diet (days 12–15) (Supplemental pigs fed ENZLR had peak concentrations <1 h after ingestion. In Table 6). Based on the 1-compartment model fitted to the plasma addition, the postprandial body exposure to the PLs, measured concentrations of the pigs fed the ENZLR diet, the half-lives of as the AUC, was ;2 times higher after enzymatic treatment PLTotal and Syr were estimated to be 11.4 and 7.5 h, respectively compared with an untreated diet. To our knowledge, our study is (Supplemental Table 4). For the LR diet and for PLOther, it was the first in which the kinetics of PLs in cereals (rye bran) after not possible to estimate the half-life because the variation in enzyme treatment with cell wall–degrading enzymes have been plasma PL concentrations postprandially could not be modeled investigated in vivo. It is interesting to note that enzyme treat- by a 1-compartment model. ment did not result in increased Enl plasma concentrations, When the pigs were switched to the WO diet after being fed the despite increased circulating PL concentrations. LR and ENZLR diets for 1 wk, the half-lives for PLTotal and Syr Syr has been identified as the major PL in rye (28), were estimated to be 2.5 and 2.2 h, respectively, when fed constituting $50% of the total PL concentration (29). In the ENZLR, and 9.1 and 7.2 h, respectively, when fed LR (Supple- present study, the proportion of Syr was even higher, probably mental Table 6). For PLOther, the half-life was slightly longer than because the cell walls of the bran fraction (aleurone and for Syr, being 2.8 h after ENZLR intake and 4.2 h after LR intake. pericarp or testa) have a relatively higher Syr concentration than the remaining cell walls of the whole grain (28). Among Enterolignans. The half-life of Enl was not affected by the type the PLs, at least Pino and Lari are incorporated in the lignin of diet served before the WO phase and was ;12–13 h in both complexes in the cell walls, and the present study points to Syr cases. For End, there was a tendency toward a difference as an important structural component of the cell walls in rye. between the 2 diets because the half-life was ;4 h for ENZLR Syr was the PL that was most strongly affected by the cell wall– compared with ;8 h for LR (overlapping CIs). degrading enzymes, leading to an approximate doubling in solubilized Syr and a much more rapid and higher absorption in vivo. Our study thus confirms the findings of Aura et al. (15), Discussion who found that xylanase treatment doubled the PL concen- tration in a soluble rye bran extract as compared with the The main findings of the present study were that enzymatic untreated, extruded fraction. modification of rye bran with cell wall–degrading enzymes The enzymatic treatment resulted in a higher concentration resulted in a substantial increase in the plasma concentrations of of soluble PLs than the untreated lignan diet and a greater PLs. The PLs appeared in the plasma 30 min after intake, and the concentration of PLTotal in the diet. The latter increase, however,

TABLE 3 Plasma concentrations of PLTotal, Syr, and PLOther measured in the mesenteric artery of pigs after being fed either LR or ENZLR for 0–7 d1

PLTotal Syr PLOther Diet Diet Diet Time, d LR, nmol/L ENZLR, nmol/LP LR, nmol/L ENZLR, nmol/LP LR, nmol/L ENZLR, nmol/L P

0 1.5 (1.0, 2.2) 1.5 (0.8, 2.6) 1.00 0.3 (0.1, 1.9) 0.4 (0.7, 2.9) 1.00 1.2 (0.8, 1.7) 1.1 (0.7, 1.7) 1.00 1 12.1 (8,1, 17.9) 18.5 (10.4, 33.1) 0.75 4.1 (3.4, 7.4) 9.3 (5.0, 21.1) 0.24 7.6 (5.0, 11.4) 9 (5.6, 14.4) 1.00 2 10.2 (6.9, 15.1) 17.5 (9.8, 31.3) 0.48 3.3 (2.9, 6.3) 7.7 (4.3, 17.9) 0.23 6.6 (4.4, 10.0) 8.5 (5.3, 13.6) 0.98 3 8.3 (5.6, 12.2) 17.5 (9.8, 31.2) 0.14 1.8 (1.9, 4.1) 7.9 (4.4, 18.2) 0.011 6.3 (4.1, 9.5) 8 (5.0, 12.8) 0.99 4 8.6 (5.8, 12.8) 12.1 (6.8, 21.6) 0.91 2.8 (2.6, 5.5) 6.7 (3.8, 15.8) 0.23 5.7 (3.7, 8.6) 5.1 (3.2, 8.2) 1.00 5 8.1 (5.5, 12.0) 13.3 (7.5, 23.8) 0.60 2.3 (2.2, 4.8) 7.1 (3.8, 15.8) 0.08 5.7 (3.8, 8.7) 5.9 (3.7, 9.5) 1.00 6 5.4 (3.6, 8.0) 13.2 (7.4, 23.5) 0.048 1.4 (1.6, 3.5) 6 (3.4, 14.4) 0.021 4 (2.7, 6.1) 6.4 (4.0, 10.3) 0.62 7 6.3 (4.3, 9.4) 10.1 (5.6, 18.0) 0.67 1.9 (2.0, 4.2) 4.3 (2.6, 10.9) 0.38 4.3 (2.8, 6.5) 5.5 (3.4, 8.8) 0.99

1 Values are modeled estimates (95% CIs), n = 5/diet. Samples were collected 12 h after last meal was served. ENZLR, enzymatically

treated lignan-rich diet; LR, lignan-rich diet; PLOther, plant lignans other than Syr; PLTotal, total plant lignans; Syr, syringaresinol.

2224 Bolvig et al. FIGURE 3 Plasma concentrations of

PLTotal (A), Syr (B), PLOther (C), End (D), or Enl (E) collected from the mesen- teric artery of feed-deprived pigs 0–7 d after switching to LR or ENZLR. Values are modeled estimates (95% CIs), Downloaded from https://academic.oup.com/jn/article/147/12/2220/4728058 by guest on 30 September 2021 n = 5/diet. *Different from LR at that time, P , 0.05. End, enterodiol; Enl, enterolactone; ENZLR, enzymatically treated lignan-rich diet; LR, lignan-rich

diet; PLOther, plant lignans other than Syr; PLTotal, total plant lignans; Syr, syringaresinol.

is caused mainly by Syr and not the other PLs Seco, Mata, Lari, first day that the LR diets were served. During this period, the I-Lari, Pino, and Hydr. The rye bran used as ingredient for LR increase was mainly driven by Syr, which showed a 3-times and ENZLR was derived from the same batch. As such, we higher AUC after enzymatic treatment compared with LR, assumed that the PLTotal concentration in the diets was identical, whereas the change was only 0.4 times for PLOther.Asimilar but that the enzymatic treatment improved the extractability of pattern, although not substantial between the 2 LR diets, was Syr, as previously found by Smeds et al. (5). seenfortheAUCmeasuredafter12-hfeeddeprivation0–7d ENZLR resulted in a rapid postprandial uptake of Syr, with after switching to the LR diets. As such, enzymatic treatment peak levels reached at 49 min postprandially. For PLOther,the increased the effective uptake of PLs, in particular Syr, from the uptake also was higher after ENZLR intake, but the plateau diet, resulting in increased exposure to PLs in the postprandial concentrations were not obtained until 118 min postconsump- phase. tion. A rapid increase in the plasma concentrations of Syr and The half-lives measured postprandially (days 5 and 16) were PLTotal also occurred in LR, but not nearly as quickly as occurred longer than those measured during the WO phase (days 12–15). in ENZLR. The results with ENZLR are comparable to kinetic For the former, it most likely reflects an ongoing absorption of studies with SDG and , in which increased concentrations PLs from the gut in the postprandial phase. Syr accounts for of PLs in human plasma were detected within hours from intake 77% of the PLTotal in the postprandial phase; as such, we were (11, 12). In a separate study, we found that the absorption of PLs surprised to find that the half-lives of PLTotal and Syr differed by from nonenzyme-treated cereal-based diets took place in an ;4 h (11.4 and 7.5 h, respectively). The difference is most likely interval of 0–2.5 h after feeding (10), thus supporting the data on driven by some of the other PLs in the diet; Lari and I-Lari have the LR diet. Collectively, these data suggested that PL absorption, been reported to have maximum plasma concentrations in irrespective of the source, takes place in the small intestine rather humans 5.5 h after intake of sesame seeds (30). Although Syr is than in the colon. This finding was reflected in a high plasma being rapidly absorbed and eliminated, Lari and I-Lari may lignan concentration in the portal vein, an intermediate con- contribute to a longer PLTotal half-life. This assumption is centration in the hepatic vein, and a low concentration in the supported further by the half-life measurements in the WO mesenteric artery. period, in which the half-lives of PLTotal and Syr were ;2.6 times Although the onset of initial absorption of PLs is similar longer when feeding LR than when feeding ENZLR before the regardless of enzymatic treatment, the AUCs, the time to reach WO phase, whereas the half-life of PLOther was only 0.5 times maximum concentrations and peak concentrations, differed longer. Enzymatic modification of rye bran therefore gave rise to markedly according to enzymatic treatment in the postprandial the rapid absorption and elimination of Syr. In contrast to phase. The AUC of PLTotal was ;2 times higher after ENZLR the PLs, the half-lives of End or Enl did not differ according intake compared with LR ingestion when measured during the to the type of diet ingested before the WO phase. Furthermore,

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