ß-carotene bioavailability from differently processed carrot meals in human ileostomy volunteers

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 differently processed carrot meals in human ileostomy

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Eur J Nutr 42 : 338345 (2003)DOI 10.1007/s00394-003-0430-6 ORIGINAL CONTRIBUTIONOrly LivnyRam ReifenItzhak LevyZecharia MadarRichard FaulksSue SouthonBetty Schwartz-carotene bioavailability from differentlyprocessed carrot meals in human ileostomyvolunteersEJN 430Received: 8 January 2003Accepted: 7 May 2003O. Livny R. Reifen Z. Madar B. Schwartz, Ph.D. ()Institute of Biochemistry, Food Science and NutritionFaculty of Agricultural, Food and Environmental Quality SciencesThe Hebrew University of JerusalemRehovot 76100, IsraelTel.: +9 72-8/9 48-90 07Fax: +9 72-8/9 36-32 08E-Mail: bschwart@agri.huji.ac.ilI. LevyDept. of Surgery CSoroka Medical Center and Ben-Gurion UniversityBeer-Sheva, IsraelR. Faulks S. SouthonDept. of Nutrition, Diet, and HealthInstitute of Food ResearchNorwich Research ParkColney, Norfolk, UK Summary BackgroundCarotenoids contribute to the ben-eficial effects of fruits and vegeta-bles consumption; however, thebioavailability of these com-pounds from fresh or processedfoods is not well established. Aim ofthe study We evaluated thebioavailability of -carotene(15 mg) from a single meal com-posed of cooked, pureed carrotsand compared it to raw, choppedcarrots. Methods Test meals weregiven to overnight-fasted-ileostomy volunteers (n = 8) alongwith skimmed-milk yogurt con-taining 40 g of added sunflower oil.Blood and complete ileal effluentsamples were collected over a 24 hperiod. Samples were solvent-ex-tracted and the -carotene contentmeasured by HPLC. Results Kinet-ics of excretion of cis and trans -carotene were similar. More -carotene was absorbed from pureeas compared to raw carrots.Carotenoid mass-balance calcula-tions indicated that 65.17.4 % ofthe -carotene was absorbed fromcooked pureed carrot meals,vs. 41.47.4 % from raw, choppedcarrot meals. Gastrointestinal tran-sit parameters did not differ signif-icantly among the volunteers. Asexpected, the calculated lag phasewas five times longer for raw vs.cooked carrots. Mean t-end, t-1/2and rate of mass transit resulted insimilar values for both raw andcooked carrot meals. A moderateresponse in carotenoid plasma pro-file was observed for cooked carrottest meals. Conclusions Signifi-cantly more -carotene was ab-sorbed from meals containingcooked, pureed carrots than frommeals containing the raw vegetable.Moderate carotenoid plasma re-sponse was detected within 6 h fol-lowing the administration ofcooked processed carotenoid-con-taining single meal. Key words carotenoids -carotene ileostomy transittimeIntroductionEpidemiological studies have shown that a high veg-etable intake is associated with reduced risk of free rad-ical-mediated degenerative diseases, such as epithelialcancers [1], cardiovascular disease [2] and age-relatedeye diseases [35]. Among the major carotenoids (-carotene, -carotene, lycopene, lutein, zeaxanthin, -cryptoxanthin and -cryptoxanthin) absorbed by hu-mans and incorporated into their blood and tissues, -carotene has been studied most extensively [6].A significant positive correlation exists between theextent of vegetable intake and plasma concentration ofcarotenoids [79]. In addition, increased vegetable con-sumption results in increased plasma levels ofcarotenoids [1012].In studies in which -carotene was given as supple-O. Livny et al. 339-carotene bioavailability from carrot mealsment, or in which -carotene was a constituent part offoods, all have shown plasma alterations that are highlyvariable in magnitude and duration, and include non-responders [13] to elevated levels that are still de-tectable 10 to 20 days post-dose [13, 14].The absorption of carotenoids from test meals is re-ported to range between 2 and 90 % depending on themeal and the model used to assess absorption [15]. Thebenefit obtained from a high intake of carotenoids fromfruits and vegetables is dependent on the bioavailabilityof these molecules, which is influenced by food type,mode of preparation and individual response [16].Plasma concentrations have been erroneously usedin the vast majority of the bioavailability nutritionalstudies to indicate the proportion of carotenoids that areavailable to the body. In addition, determination of ab-sorption of carotenoids has been carried out in humanvolunteers using a large number of methodologies, suchas, oral-fecal mass balance [17], chronic dosing [6,1820], acute dosing [16, 21], triglyceride-rich lipopro-tein carotenoid from acute dosing [22, 23], radioactivetracers [24, 25], stable-isotope tracers [2628], total gas-trointestinal washout (mass balance) method [29], andmore recently, the ileostomy mass-balance model [30].This last methodology provides a system to assess theextent of carotenoid absorption and has been recentlyused to determine the extent of carotenoid intake fromcooked spinach meals; however, it has not been tested onadditional vegetables.Mass-balance techniques in ileostomy patients [30]have been shown to provide useful absorption datawhile avoiding the confounding influence of the largeintestinal microflora. Even though some residual micro-bial activity remains (colonization of the terminal ileumin ileostomy patients), this activity is not thought tocause any significant loss during transit.The purpose of this study was to characterize the ab-sorption and disposal of -carotene in ileostomy sub-jects fed a single portion (equivalent to 15 mgcarotenoids) of raw carrots compared to a single portionof cooked carrots.Subjects and methods EthicsThe study protocol was approved by the Ethics Commit-tee for human studies of the Israeli Ministry of Health,and the experimental work was carried out in accor-dance with the Declaration of Helsinki (1989). Informedwritten consent was obtained from each volunteer. VolunteersEight volunteers (3875 years) were recruited for thisstudy, which was conducted in Soroka Medical Center.The weight range of the volunteers was 52 to 77 kg and amean BMI of 20.5 kg/m2 (range 18.525.2). The subjectshad undergone ileostomy surgery at least 2.5 years pre-vious to the experiment. They were free of intestinal dis-ease and otherwise healthy, as determined by medicalhistories, blood chemistry tests, lipidogram and com-plete blood count and urine analyses, which were allwithin normal limits. Study protocolThe volunteers avoided consumption of food itemsknown to contain large amounts of carotenoids (a listwas provided) and recorded their habitual diet for 3 daysprior to the test day. The volunteers fasted from 19:00 hthe day before the test but were free to drink water. At08:00 h on the test day (fasted, t = 0) blood samples weretaken,participants then changed the stomal effluent col-lection bag and ate a breakfast containing fat-free yogurtto which 40 g of sunflower oil was added, homogenizedin a low speed blender, and the carotenoid-containingtest meal in the form of ground raw carrots or cookedpureed carrot. Each test meal contained an equivalent of15 mg trans -carotene. Volunteers were provided withcarotenoid-free midday (12:0012:30 h) consisting offat-free chicken breast meat or tofu-based meal accom-panied by potato puree and light refreshments. Theevening meals consisted of sandwich with low fat cot-tage cheese, three slices of bread and tea or coffee. Ve-nous blood samples were collected via antecubital can-nula into 10-ml heparin tubes every 2 h up to 12 h andthe last sample was collected after 24 h. Plasma was sep-arated by centrifugation (5 min, 5000 x g) and frozen at80 C. Stomal effluents were collected into pre-weighedbags every 2 h up to 12 h and the last sample was col-lected after 24 h. Analytical methodsThe carotenoid extraction method from effluent andserum samples was evaluated by introducing the inter-nal standard -apo-8-carotenal into the effluent orserum. This internal standard was prepared by dissolv-ing -apo-8-carotenal to a concentration of 0.7 g/ml inhexane. One ml of the internal standard was added to ef-fluent or serum before extraction. The mean percent ofrecovery was 91 % (range 8794).Plasma samples (500 l) were treated with sodiumdodecyl sulfate (0.5 ml, 10 mmol/l) and ethanol (1 ml) toprecipitate plasma proteins. The carotenoids were ex-340 European Journal of Nutrition, Vol. 42, Number 6 (2003) Steinkopff Verlag 2003tracted three times by the addition of hexane (2 ml), andthe pooled hexane fraction was dried with a stream ofnitrogen gas. The dry residue was dissolved indichloromethane (100 l) before adding 400 l of ace-tonitrile/methanol (79:21). The carotenoids were mea-sured by HPLC [30]. Effluent samples (2 g) were placedin 50-ml screw-top glass centrifuge tubes and 20 ml ofacetone added. The effluent was thoroughly dispersedusing a small ultra-turrax, the mixture was centrifuged(800 x g, 10 min) to pellet the solids and the supernatantwas transferred to a 100-ml volumetric flask. The pelletwas resuspended in 20 ml acetone and the process re-peated 3 more times. The acetone extract was brought to100 ml, thoroughly mixed and a filtered sub-sample(20 ml) stored at 20 C. A sub-sample (1 ml) of the fil-tered acetone extract was dried under N2, resuspendedin HPLC mobile phase, diluted if needed, and assayed byHPLC as above. HPLC analysisSamples were injected by using a JASCO Autosampler(model AS-95010; JASCO, Tokyo, Japan) onto a C18, RP(Vydac 201 TP 45) column fitted with a C18 guard col-umn and biocompatible frits. Samples were eluted iso-cratically in the HPLC mobile phase at a flow rate of1.2 ml/min with a model LC-1150 pump (GBC, ScientificEquipment, Victoria, Australia), a multiwave program-mable detector (model MD 910, JASCO) and a BorwinPDA version 1.50 system controller (JASCO).Carotenoids were identified and quantified at 450 nmusing known standards. Data analysesCis and trans -carotene was calculated as the subtrac-tion of cis and betacarotene released into the effluentfrom the ingested concentration found in raw and pureecarrot meals for each time point.The kinetics was drawnaccordingly.The normalized cumulative collection (percent) ofthe carrot meals was plotted against time to provide thetransit profile of the carrot, and then the data were fittedusing a logistics model to calculate t-1/2 of gastroin-testinal (GI) residue time. The portion of the curve thatappeared to be steeply increasing with time was fittedwith a regression model. The slope was equivalent to theeffluent production rate and the intercept (x at y = 0) wasused to calculate the lag time. The normalized amount(percentage) of the original carrot meal collected at eachtime point was calculated from the total recovered -carotene and the amount of -carotene in each collec-tion. This calculation took into consideration constantpercentage absorption of -carotene at all time points. Calculation and statisticsValues are given as means SD or SEM, as indicated.One or two-tailed t-tests were used to compare absorp-tion, excretion and transit-time values between testmeals in each volunteer.Results Carotenoid composition of carrot mealsThe average composition of the raw carrot meal con-sisted of 54.8 g/g trans -carotene, 3.02 g/g cis -carotene, 25.2 g/g trans -carotene, 0.78 g/g cis -carotene, 1.42 g/g lutein (total) and 0.08 g/gzeaxanthin. We aimed at receiving 15 mg of trans -carotene in each test meal; therefore the volunteers re-ceived 275 g of raw carrots in each test meal. The aver-age composition of the cooked carrot puree mealconsisted of 62.5 g/g trans -carotene, 2.52 g/g cis -carotene, 23.6 g/g trans -carotene, 1.18 g/g cis -carotene, 1.37 g/g lutein (total) and 0.05 g/g zeaxan-thin. We aimed at receiving 15 mg of trans -carotene ineach test meal; therefore the volunteers received 240 g ofpureed carrots in each test meal. Excretion kinetics of cis and trans -caroteneThe concentrations of all-trans -carotene (Fig. 1a) andcis--carotene (Fig. 1b) isomers apparently absorbed(calculated as secreted quantity subtracted from themeasured amount present in the test meal) from rawand cooked carrots were determined. The kinetics forexcretion of both isomers and the two meals was simi-lar; however the final amounts absorbed differed. Fortrans--carotene the excretion of the carotenoid follow-ing consumption of the raw carrot meal was signifi-cantly higher starting 2 h following the meal and con-tinued in a steeper rate onwards. Plateau was reached at12 h. Fifty percent more trans--carotene was absorbedfrom cooked puree carrots than from raw carrots. Ap-proximately 100 % cis--carotene was secreted into theeffluent 24 h following the test meal consumption con-taining raw carrots (Fig. 1b). In contrast, volunteers con-suming the cooked puree carrot meal secreted only 73 %of cis--carotene. For both differently processed testmeals the kinetics of excretion was similar; most of the-carotene was secreted within 12 hours following con-sumption. During the last 12 hours the excretion ratereached a plateau.An average group calculation of -carotene excretionto the effluent is shown in Fig. 2. The average concentra-tion of -carotene in the effluent showed that theamount of -carotene secreted by volunteers fed cookedO. Livny et al. 341-carotene bioavailability from carrot mealscarrots was higher than for the volunteers fed raw car-rots for the first 4-h time periods. However, at later timeperiods the trend was exchanged and showed an oppo-site pattern, i. e. the excretion of -carotene in the efflu-ent was more pronounced in volunteers fed cookedpureed carrots and less evident in effluents from volun-teers fed raw carrots. Gatrointestinal transit parametersThe results for the normalized excretion of -carotenefrom the carrot meals to the ileal effluent are shown inFig. 3 for the raw and processed meals. For cookedpureed carrots, 90 % of -carotene was secreted within6h while in the effluent of the volunteers fed raw carrotsit took 12h to secrete a similar amount of the carotenoid.Additional meal-behavior parameters for variouslyprocessed carrot test meals along the GI tract are sum-marized in Table 1. The calculated mean lag phase of theinitial appearance of effluent from the cooked carrotmeal (0.660.3 h) was shorter than that calculated fromthe raw carrot meal (2.550.7 h). The mean half-life (t-1/2) of the cooked carrot meal in the GI tract (4.20.3 h)was shorter than that for the raw carrot meal (6.550.6h). The mean rate of mass transit through the GI tract asestimated from the slope of the cumulative collection ofileal effluent was similar for the cooked carrot meal(12.42.3 h) as for the raw carrot meal (12.71.0 h).TheFig. 1 a Kinetics of trans -carotene excretion into the ileal effluent. The concen-tration (calculated as secreted quantity subtracted from the measured amountpresent in the test meal) of all-trans -carotene isomer absorbed from raw andcooked carrots was drawn as a function of time. * P < 0.001 puree vs raw carrot. bKinetics of cis -carotene excretion into the ileal effluent. The concentration SD(calculated as secreted quantity subtracted from the measured amount present inthe test meal) of cis -carotene isomer apparently absorbed from raw and cookedcarrots was drawn as a function of time. * P < 0.001 puree vs raw carrotFig. 2 The amount of trans -carotene in effluent samples collected at 2-hour in-tervals was calculated. The calculations are mean SD for 8 volunteers. * P < 0.05Fig. 3 Percent excretion of -carotene in effluents collected for 24 h at intervalsof 2 hours. The plot represents the calculations obtained from 8 volunteers fed rawcarrots and the second plot that obtained from 8 volunteers following feedingcooked carrots. * P < 0.01342 European Journal of Nutrition, Vol. 42, Number 6 (2003) Steinkopff Verlag 2003mean t-end or time that the meal was completely voidedof the two carrot meals in the GI tract was similar(10.60.3 h for raw, 9.81.5 h for cooked). -carotene absorptionAll the meals contained 15 mg -carotene. The absorp-tion of trans--carotene was calculated as the differencebetween the level of trans--carotene in samples fromraw carrot meals and the level obtained in the ileal efflu-ent. Fig. 4 shows that the absorption of trans--carotenein volunteers who were fed raw carrots was 41.4+7.4 %,whereas volunteers fed cooked carrots, absorption of -carotene reached 65.1+7.4 %. Plasma b-carotene responseSignificant plasma -carotene response was observedwhen the volunteers consumed 15 mg -carotene fromprocessed carrot test meals, mainly 46 h following theconsumption of the test meals. Thereafter, the plasmalevels reached a plateau. Plasma -carotene levels in vol-unteers fed raw carrot test meals differed from thosemeasured in volunteers fed the cooked carrot meals; nosignificant increases were detected throughout the ex-periment. Some expected variability in the carotenoidplasma levels was observed among the different volun-teers during the course of the experiment (Fig. 5).DiscussionThe mass-balance technique using the ileostomy model[30] was shown previously to produce reliable data onabsorption and excretion of vegetable carotenoids,while avoiding the confounding influence of the activityin the large intestinal microflora.The present study demonstrates that the rate of -carotene absorbed from a single portion of cooked car-rots is significantly higher (two-tailed t-test) than therate values obtained by ileostomy volunteers eating rawcarrot meals (P = 0.048). Absorption of an oral dose of15 mg -carotene provided in a raw carrot meal was41.47.4 %, whereas absorption of -carotene from acooked carrot meal reached 65.17.4 %. These amountsare generally greater than previously published values[30] for -carotene in oil, where the mass balance wastaken as the difference between intake and fecal excre-tion over a non-specified period.Cis -carotene was completely excreted into the ef-fluent 12 hours following ingestion of the raw carrot testmeal. The volunteers apparently absorbed 21 % of cis -carotene when they consumed the puree cooked carrottest meal. For trans -carotene the excretion kineticswere similar for both raw and cooked carrot meals; how-ever, more trans -carotene was secreted from the rawcarrot test meals. For both test meals the contribution ofcis -carotene to the general -carotene profile was mar-ginal; therefore, additional calculations relate only totrans -carotene.GI-tract transit time parameter calculations indicateTable 1 Comparison of transit-time on ileal effluents from patients fed two dif-ferent carrot test mealsTransit-time parameters Raw carrots Puree carrotslag phase (h) 2.50.7* 0.660.3t (1/2) (h) 6.50.6** 4.20.7t (end) (h) 10.60.7 9.81.5slope (%/h) 12.71.0 12.42.3Data are expressed as Means SD of 8 volunteers.lag phase (h) refers to the time taken for the test meal to appear at the terminalileum; t (1/2) time (h) at which half the meal has been voided; t (end) time (h) atwhich the meal has been completely voided; slope (%h) rate expressing meal efflu-ent accumulation (%/h)* p < 0.01 raw compared to puree carrot meal, Students t test** p < 0.07 raw compared to puree carrot meal, Students t testFig. 4 Absorption SD of trans -carotene from raw compared to cooked carrottest meals. * P = 0.048 two-tail t-test comparing both test mealsFig. 5 Plasma -carotene response recorded for 24 h period. The plots representthe results of plasma trans -carotene response (ng/ml S. E. M.) of 8 different vol-unteers fed both raw and then processed carrots. * P = 0.01O. Livny et al. 343-carotene bioavailability from carrot mealssome clear differences in values between raw andprocessed carrots: the lag phase, i. e. the initial appear-ance of -carotene in the effluent was longer for the rawcarrot meal than for cooked carrots (physical change ofthe vegetable from raw to cooked stage resulted in aquicker passage through the GI tract). This differencebecame less significant after t-1/2 (the mean half life)and disappeared towards the t-end (see Fig. 3). Themean rate of mass transit through the GI tract as esti-mated from the slope of the cumulative collection of -carotene in the ileal effluent was the same for bothmeals. The cooked carrot meal passed through the GItract at 12.4 g/h, the same rate as the raw carrot meal(12.7 g/h).A study using a computerized alimentary tract modeldemonstrated that -carotene absorption falls in therange of 13 to 18 % without a meal whereas it rises to 40to 65 % with a meal of 4184 kJ, containing 40 % dietaryfat [31], results that correlate well with our findings. Thevalues found in the present mass-balance study, how-ever, may still be an overestimation of absorption, sincedestruction of carotenoid by the intestinal microfloracannot be calculated.We have previously shown levels of90 % -carotene absorption (range 74.397 %) in mass-balance studies carried out in fasted ileostomy subjectsgiven an oral dose of -carotene extracted fromDunaliella salina and administered dispersed in veg-etable oil [30]. The physical form of carotenoids may bean important factor in the absorption process. Crys-talline -carotene was shown to be less available thandissolved -carotene [3234]. In carrots, -carotene ispresent as crystals and this may also explain the lowavailability of -carotene from carrots as compared toextracted -carotene previously dissolved in oil [19, 33].Processing, such as mechanical homogenization or heattreatment, can enhance the bioavailability ofcarotenoids from vegetables [35, 36]. Pureeing vegeta-bles results in smaller particle sizes and also mechani-cally disrupts the plant cells, so that the carotenoids arepresumably more available in the intestinal lumen forabsorption [33, 3638]. Heat treatment is suggested toenhance the bioavailability of carotenoids by looseningtheir binding to proteins [39].Mass-balance technique using this model allowed usto demonstrate that when a cooked carrot meal contain-ing 15 mg -carotene is consumed it is possible to detectplasma -carotene response, whereas consumption of15 mg -carotene from a raw carrot meal does not allowdetection of the plasma -carotene response. Van hetHof et al. [6] showed a very significant plasma responseto carotenoid meals 4 days after consumption of palmoil carotenoids, which could explain the lower levels wefound in plasma collected after a single serving. It seemsthat 24 h after the consumption of a single processedmeal is sufficient to induce a new steady state of plasmacarotenoid levels. However, a similar single dose of rawcarrot meal is not sufficient to induce a significantserum response. There is less variability in the plasmaresponse to continuous supplementation of dietarycarotenoids than after administration of a single dose[16, 19]. In addition, the amount of -carotene in themeal (15 mg) was lower relative to the dose (2060 mg)given in similar studies, which have resulted in signifi-cant changes in plasma carotenoid concentration [16,18, 19]. Nevertheless, a short intervention period as usedhere has several advantages: It mirrors a more realisticstage of the carotenoid changes in a subject and compli-ance is probably better during a short period.It has been suggested that carotenoids are absorbedby enterocytes in the chylomicrons as are fatty acids,andthen slowly released into the plasma [13]. Small changesin plasma -carotene following a single raw carrot mealmay represent poor absorption due to matrix present inthe raw carrots that interferes with free transfer of thecarotenoid crystals to the enterocytes [37]. In theprocessed cooked carrots newly absorbed carotenoidsare measured in plasma chylomicrons. Resecretion ofthe carotenoid from the liver packed in VLDL particlesmay be evident much later and at lower peak concentra-tions in LDL or HDL fractions.The extent of carotenoid absorption varies among in-dividuals. In healthy, well-nourished populations, largevariability of the response of plasma or serum -carotene concentrations to supplementation was re-ported [16, 40]. Some volunteers identified as so-callednon-responders: people who show no, or only a verysmall increase in -carotene concentration in chylomi-crons or plasma, following -carotene supplementation[13, 41]. The mechanism underlying this variation maynot only be related to differences in carotenoid absorp-tion but also to differences in lipoprotein metabolismamong individuals [42].The processing of foods involves changes in thestructural integrity of the matrix and this produces bothnegative (loss of carotenoids due to oxidation) and pos-itive (increased bioavailability) effects. The ileostomymodel used herein is proved to be a good method to per-form bioavailability studies of feeding meals for shorttime periods with low carotenoid levels. Acknowledgement R. Reifen, R. Faulks, S. 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