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  Rice bran oil, not fiber, lowers cholesterol in humans 1–3  Marlene M Most, Richard Tulley, Silvia Morales, and Michael Lefevre ABSTRACTBackground: Thecholesterol-loweringabilitiesofricebran’sfiberand oil apart from its fatty acid composition remain unclear. Objective:  The objective of the study was to assess the effects of defatted rice bran and rice bran oil in an average American diet onblood lipids in moderately hypercholesterolemic persons. Design:  Study 1 used a parallel-arm design. Twenty-six healthyvolunteersconsumedadietwith13–22gdietaryfiber/dfor3wk,andthen 13 of the volunteers were switched to a diet with defatted ricebran to double the fiber intake for 5 wk. Study 2 was a randomized,crossover, 10-wk feeding study performed in 14 volunteers whoconsumed a diet with rice bran oil (1/3 of the total dietary fat)substituted for an oil blend that had a fatty acid composition similartothatofthericebranoil.SerumlipidsandfactorVIIweremeasuredin both studies. Results:  Defatted rice bran did not lower lipid concentrations. Instudy 2, total cholesterol was significantly lower with consumptionof the diet containing rice bran oil than with consumption of thecontrol diet. Moreover, with consumption of the rice bran oil diet,LDL cholesterol decreased by 7% ( P    0.0004), whereas HDLcholesterol was unchanged. Conclusions: Rice bran oil, not fiber, lowers cholesterol in healthy,moderately hypercholesterolemic adults. There were no substantialdifferences in the fatty acid composition of the diets; therefore, thereduction of cholesterol was due to other components present in therice bran oil, such as unsaponifiable compounds.  Am J Clin Nutr   2005;81:64–8. KEYWORDS  Ricebranfiber,ricebranoil,lipoproteins,phyt-osterols INTRODUCTION Rice bran, a coproduct of milled rice, and its oil may havecardiovascularhealthbenefits.Humanconsumptionofricebranhasbeenlimited,primarilybecauseoftherapidonsetofrancidityinricebran,butmethodstostabilizericebranandtoextractitsoilhave been developed. Interest in rice bran grew from the deter-mination that the inclusion of oat bran in the diet lowers serumcholesterol (1, 2). Studies of rice bran supplementation in humansfound similar beneficial effects on lipoproteins (3–6). In a 10-wk controlled feeding trial, rice bran was as effective as oat bran inloweringbloodcholesterolconcentrationsinmenandwomenwithmoderately high blood cholesterol concentrations (7).Rice bran contains 10–23% oil (8) and (unlike oat bran) neg-ligible amounts of water-soluble   -glucans and larger amountsof insoluble dietary fiber. Because of these differences, it isbelieved that rice bran lowers cholesterol by a mechanism dif-ferentfromthatofoatbran.Decreasesincholesterolwerefoundinhypercholesterolemicsubjectswhoreplacedtheirusualcook-ing oils with rice bran oil (9) and in middle-aged and elderlysubjects consuming a low-fat diet containing rice bran oil (10).Yetricebranoiltypicallycontains20%saturatedfattyacidsandapproximately equal amounts of oleic and linoleic fatty acids(11). Previous research showed the deleterious effects of satu-rated fatty acids on total cholesterol concentrations, and the factthat rice bran oil lowers cholesterol is contrary to these findings.Research now suggests that rice bran oil’s cholesterol-loweringproperties are explained by its unsaponifiable components morethan by its fatty acid composition (12, 13). Attention has begunto focus on the components of rice bran oil, including phytoster-ols,triterpenealcohols,tocopherols,andtocotrienols,aspossiblehypocholesterolemic agents.Weexaminedfurtherthecholesterol-loweringabilitiesofricebran’s fiber and oil apart from its fatty acid composition. Thiswas accomplished with 2 well-controlled feeding studies de-signed to evaluate the effects of using defatted rice bran and ricebran oil in an average American diet on cardiovascular diseaserisk factors in men and women. SUBJECTS AND METHODS Subjects For study 1, 27 healthy men and women were recruited in 2cohorts. One woman was taking hormone replacement therapy,and the other women were premenopausal and not taking oralcontraceptives. One man was dropped from the study after ran-dom assignment to the treatment diet because of an allergicreaction. Fourteen healthy men and premenopausal women (3takingoralcontraceptives)participatedinstudy2.Thesubjects’characteristics are shown in  Table 1 .Eligible subjects were 18–50 y old and had total serum cho-lesterol concentrations between the 25th and 90th percentilesafter adjustment for sex, age, and race (14); triacylglycerol con-centrations  90th percentile after adjustment for sex, age, and 1 From the Division of Functional Foods Research, Pennington Biomed-ical Research Center, Louisiana State University, Baton Rouge, LA. 2 Supported by the Louisiana Education Quality Support Fund–Enhancement–Private Leveraging for Excellence Program of the Loui-siana Board of Regents. 3 Reprints not available. Address correspondence to MM Most, Penning-ton Biomedical Research Center, 6400 Perkins Road, Baton Rouge, LA70808. E-mail: February 19, 2004.Accepted for publication September 28, 2004. 64  Am J Clin Nutr   2005;81:64–8. Printed in USA. © 2005 American Society for Clinical Nutrition  race;LDLconcentrations  4.91mmol/L(190mg/dL)andHDLconcentrations    0.65 mmol/L (25 mg/dL); and a body massindex (BMI; in kg/m 2 )  30. Exclusion criteria included renal,hepatic,cardiovascular,endocrine,gastrointestinal,orothersys-temic disease; hypertension; pregnancy (for women); history of drugoralcoholabuse;smokingorothertobaccouse;chronicuseof prescribed medication; extreme dietary habits such as vege-tarianism or severely low fat intakes; multiple food allergies;extreme levels of physical or athletic activities; and currentweight-loss efforts.The study protocol and consent form were approved by theLouisiana State University’s Institutional Review Board. Writ-ten informed consent was obtained from all subjects, and allsubjectsreceivedmonetarycompensationfortheirparticipation. Experimental design Study 1: defatted rice bran All subjects began with consumption of a run-in diet for 3 wk and then were randomly assigned to either the control or inter-vention diet for an additional 5 wk. Assessment of outcomemeasures occurred at the end of the run-in period and at the endof the study. Blood samples were collected in triplicate on sep-arate days to minimize the influence of biologic variability inthesemeasures.Inaddition,asinglemeasurementofbloodlipidswas taken 2 wk after the randomization. Body weight was mea-sured twice a week throughout the study, and energy was ad- justed to ensure weight stability.The study 1 diets were a low-fiber control diet (control 1) anda high-fiber intervention diet containing defatted rice bran(DRB). The control 1 diet provided 13–22 g dietary fiber/d,varying with total energy, whereas the addition of DRB (56–94g/d, varying with total energy level) to the intervention dietdoubled the fiber content. Both diets provided 37% of total en-ergy as fat. DRB was incorporated into muffins, cookies, andbreads. A 4-d menu rotation was used to maintain varietythroughout the study. The macronutrient composition of eachdiet (an average of the 4 menu plans, each at 2 different energyamounts),asdeterminedbythePenningtonCenter’sFoodAnal-ysis Laboratory, is shown in  Table 2 . Study 2: rice bran oil Study 2 used a randomized, double-blind, crossover designwith two 5-wk diet periods. Assessment of outcome measuresoccurred at the end of each diet period. Blood samples werecollectedintriplicateonseparatedaystominimizetheinfluenceofbiologicvariabilityinthesemeasures.Bodyweightwasmea-sured twice a week, and energy was adjusted to ensure weightstability.To determine whether unsaponifiable components present inrice bran oil (RBO) affect lipid metabolism, the fatty acid com-position of RBO was matched with that of an oil blend that wasusedinthecontroldiet.ThefattyacidprofileoftheRBOthatwasobtained for the second feeding study was determined. Thenother oils were combined, chemically analyzed, and adjusteduntilthebestmatchoftheRBO’sfattyacidprofilewasachieved.The oil blend was composed of peanut oil, olive oil, corn oil,canola oil, palm oil, and butter. The comparison of the controlblendandRBOsforthemajorfattyacidsandforthetocopherol,tocotrienol, and oryzanol contents is shown in  Table 3 .Bothdietsweredesignedtoprovide37%oftotalenergyasfat.For the study 2 control diet (control 2), one-third of the totaldietary fat was in the form of the oil blend, and for the RBOintervention diet, the oil blend was replaced with RBO. The oilblendorricebranoilwasincorporatedintorecipesfora5-dmenurotation. Other fats were added to the diets so that the total dietwouldprovide15%,17%,and6%ofenergyassaturated,mono-unsaturated, and polyunsaturated fat, respectively. The total di-etary cholesterol was  125 mg/1000 kcal. To keep the partici-pants blinded to their diet assignment, the control 2 (oil blend) TABLE 1 Baseline characteristics at screening of subjects in study 1 and study 2 1 Study 1( n  13 F, 13 M)Study 2( n  7 F, 7 M)Age (y) 32.9  1.7 33.6  2.8BMI (kg/m 2 ) 24.3  0.6 24.8  0.7Total cholesterol (mmol/L) 5.03  0.12 5.33  0.11HDL cholesterol (mmol/L) 1.29  0.08 1.19  0.07LDL cholesterol (mmol/L) 3.33  0.15 3.65  0.12Triacylglycerols (mmol/L) 0.84  0.08 1.00  0.09 1 All values are  x       SEM of subject’s average of replicate measure-ments.Toconvertcholesterolvaluestomg/dL,divideby0.02586;toconverttriacylglycerol values to mg/dL, divide by 0.01129. TABLE 2 Chemical composition of the diets provided to subjects participating instudy 1 1 Control diet Defatted rice bran dietFat (% of energy)Total 36.6  1.0 36.5  1.2Saturated 14.6  0.4 14.5  0.6Polyunsaturated 10.6  0.4 10.6  0.4Monounsaturated 11.4  0.4 11.3  0.4Carbohydrate (% of energy) 51.3  1.4 50.6  1.3 2 Protein (% of energy) 12.1  0.6 12.9  0.5 2 Fiber (g) 16.6  1.8 33.3  4.7 31 Allvaluesare  x      SEMandreflecttheaveragevaluesof4menuplansat 2 energy levels (9205 and 12 552 kJ; 2200 and 3000 kcal). 2,3 Significantly different from control diet (two-sample  t   test):  2 P  0.05,  3 P  0.002. TABLE 3 Comparison of the fatty acid profile and of the tocopherol, tocotrienol, andoryzanol content of the control oil blend and the rice bran oil used in study2, as determined by chemical analysisControl oil blend Rice bran oil14:0 (g/100 g) 0.37 0.4016:0 (g/100 g) 12.96 14.6018:0 (g/100 g) 2.97 2.0918:1 (g/100 g) 45.43 44.5118:2 (g/100 g) 35.90 36.5918:3n (g/100 g) 0.84 0.87  -Tocopherol (  g/g) 108.4 180.0  -Tocotrienol (  g/g) 34.4 218.0   -Tocopherol (  g/g) 127.5 38.0   -Tocotrienol (  g/g) 11.7 59.0  -Tocopherol (  g/g) 2.92 —  -Tocotrienol (  g/g) — —Oryzanol (mg/g) 0.04 15.8 RICE BRAN OIL AND CHOLESTEROL  65  dietwassimilarinappearancetotheRBOinterventiondiet.Themacronutrient composition of both diets (an average of the 5menu plans, each at 2 different energy amounts), as determinedbythePenningtonCenter’sFoodAnalysisLaboratory,isshownin  Table 4 .Forbothstudies,thesubjectswereprovidedwithallfoodsforthedurationofthestudy.Onweekdays,subjectswererequiredtoconsume breakfast and dinner under supervision at the Penning-ton Biomedical Research Center dining facility, and weekdaylunches and snacks and all weekend meals were packaged fortake-out. Subjects were initially assigned a total energy level tomaintain body weight, and energy adjustments were made asneededtoattempttomaintainweightwithin2kgofeachperson’sinitial value. They were not allowed to take vitamin or mineralsupplementsduringthestudy.Eachparticipantcompletedadailyfood diary to assist with compliance assessment; in this diary,they recorded study foods not eaten, nonstudy foods eaten, andbeverages consumed. Laboratory analyses Total cholesterol and triacylglycerol concentrations weremeasured on a Synchron CX5 automated chemistry analyzer(Beckman, Brea, CA). HDL cholesterol was measured as cho-lesterolontheCX5afterprecipitationoflower-densitylipidsbyusing dextran sulfate (DML, Dallas). LDL cholesterol was cal-culated by using the Friedewald equation (15) and by assumingthat triacylglycerols were within normal limits. Serum apoli-poproteinsA1andBweremeasuredonanArrayanalyzer(Beck-man) employing reagents supplied by the manufacturer. FactorVII activity was measured by using an ACL 3000  coagulationinstrument (Instrument Laboratory, Lexington, MA). The inter-assay CVs for these assays are  7%. Statistical analysis For study 1, lipids, apolipoproteins, and factor VII were ana-lyzedwiththehelpofamixedmodel.Sex,diet,andtimeandtheirinteractions were treated as fixed effects to compare the effectson the outcome variables of DRB with those of the control diet.The measurements at the end of the run-in period and BMI wereincluded as covariates. The covariance matrix included a vari-ance component for subject and one for subject  time to takeinto account the repeated nature of the observations in a subject.Toallowforcomparisoinoftheresponseundertreatmentandtherun-in measurements, the difference between the treatment re-sponse and the run-in measurement was analyzed in the samemanner as described above.Similarly, in study 2, sex and diet and their interactions wereconsideredfixedeffectsinthetwo-waymixed-modelanalysisof the primary endpoints. In this study, the covariance matrix wasmodeled by using the random effects subject and subject  dietto accommodate the repeated measurements. BMI was includedas a covariate.Because the triacylglycerol data were not normally distrib-uted, statistical analysis was performed on log-transformed val-ues.Alltestswerenondirectionalortwo-sidedandwerebasedonadjusted means predicted by the model (least-squares means).Statements concerning the significance of tests were based on atype I error rate of 0.05. All statistical analyses were performedby using SAS software (version 8.2; SAS Institute, Cary, NC). RESULTS Study 1: defatted rice bran Ofthe27subjectsenrolled,26completedtheentirestudy.Theone subject who dropped out had only run-in period data, whichwas not taken into account in the analysis. The mean (  SEM)age of the subjects was 32.9  1.7 y, and their mean BMI was24.3  0.6 (Table 1). Body weight did not change during thestudy (data not shown). All participants consumed the controldiet during run-in and thus are sampled from one population.Theysubsequentlywererandomlyassignedtotheseparatetreat-ment groups—control or DRB. Thus, the fixed-effects modelincludedtheend-of-run-inlevelofresponsevariableasacovari-ate to account for any possible differences that might have ex-isted. On the basis of this model, lipid and lipoprotein concen-trationsandfactorVIIactivityinresponsetothedietsareshownin  Table 5 . Unexpectedly, after 5 wk, LDL cholesterol andapolipoproteinBwerehigherinthesubjectsconsumingtheDRBthan in those consuming the control diet. Despite the change inLDLcholesterol,totalcholesterolwasnotsignificantlychangedby either diet. All other cardiovascular disease risk factors thatwe measured were unchanged. Study 2: rice bran oil Atenrollment,theaverageageofthesubjectwas33.6  2.8y,andtheaverageBMIwas24.8  0.7(Table1).Atenrollment,thesubjects’ total cholesterol was 5.33  0.11 mmol/L, HDL was1.19  0.07, LDL was 3.65  0.12 mmol/L, and triacylglycerolwas 1.00  0.09 mmol/L. All subjects enrolled completed thestudy.Lipid and apolipoprotein concentrations and factor VII activ-ity in response to the 2 diets are shown in  Table 6 . There was asignificant effect of diet on total cholesterol, LDL cholesterol,and apolipoprotein B. Total cholesterol was lowest on the dietcontaining RBO, because of the lower concentration of LDLcholesterol; HDL cholesterol did not change. Triacylglyceroland factor VII were unchanged. DISCUSSION Evidence from these 2 well-controlled feeding studies showsthat it is the RBO, and not the fiber, that lowers blood lipids inmenandwomenwithborderlinehightotalcholesterol.Ricebran TABLE 4 Chemical composition of the diets provided to subjects participating instudy 2 1 Control oil blend diet Rice bran oil dietFat (% of energy)Total 38.4  0.5 37.8  0.3Saturated 15.6  0.2 15.7  0.2Polyunsaturated 7.0  0.2 6.7  0.1Monounsaturated 15.8  0.2 15.7  0.2Carbohydrate (% of energy) 47.1  0.4 47.5  0.5Protein (% of energy) 14.5  0.3 14.8  0.3Cholesterol (mg) 320.9  14.6 366.4  20.0 21 Allvaluesare  x      SEMandreflecttheaveragevaluesof5menuplansat 2 energy levels (9205 and 12 552 kJ; 2200 and 3000 kcal). 2 Significantly different from control diet,  P    0.002 (two-sample  t  test). 66  MOST ET AL  fiber and oil did not affect triacylglycerols or factor VII activity.During the first study, 26 men and women completed a parallel-armfeedingstudycomparingacontroldietwithadietcontainingDRB. Between the run-in diet and the RBO, there were no de-creases in total, LDL, or HDL cholesterol. During study 2, 14men and women completed a 10-wk crossover study evaluatingthe isoenergetic substitution of RBO for an oil blend in a refer-encedietdesignedtoprovide37%ofenergyasfat.Theoilblendor RBO provided one-third of the total fat. The oil blend wasdesigned to have a fatty acid profile similar to that of the RBO.Predictive equations (16) show that the variations in fatty acidsbetweenthecontroloilblendandtheRBOwouldhaveaminimaleffect on blood lipids. Total, LDL, and HDL cholesterol wouldincrease by only 0.01, 0.007, and 0.003 mmol/L, respectively.Instead, the RBO diet lowered total and LDL cholesterol signif-icantlymorethandidthecontroldiet,buttheeffectofthe2dietson HDL cholesterol was similar. Changes in apolipoprotein Bfollowed changes in LDL. Because there were no substantialdifferences in the fatty acid composition of the diets, the reduc-tionincholesterolwaslikelyduetoothercomponentspresentinthe RBO.Anotherapproachtoexaminingthecholesterol-loweringabil-itiesofRBOsterolsseparatefromthefattyacidcompositionwasreportedbyLichtensteinetal(10)inhumansandbyWilsonetal(13) in cynomologus monkeys. Moderately hypercholester-olemic men and women consumed diets enriched in rice bran,canola, corn, and olive oils at 20% of total energy. The subjectshad similar plasma total and LDL-cholesterol concentrationsafter the consumption of the diets containing rice bran, canola,and corn oils, even though the fatty acid distributions differed.The RBO diet was richer in sterols, tocotrienols, and oryzanolthanthe3otherdiets.Inthestudyofcynomologusmonkeys,thefatty acid compositions of experimental oil blends used in thediets were similar except for higher total saturates in an RBOblend, higher total monounsaturates in a canola oil blend, andhigher total polyunsaturates in a corn oil blend. Despite the dif-ferencesinfattyacids,thedietcontainingtheRBOblendreducedserum LDL-cholesterol concentrations significantly more thandid the diets containing the other oil blends. In both studies, thechangesintotalandLDLcholesterolobservedwithconsumptionof the RBO diet represented a greater improvement than waspredicted on the basis of the fatty acid composition of the diets.HDL-cholesterolconcentrationswerenotdecreasedbytheRBO.These observations parallel the results reported in the currentstudy. Together, the results from the 2 studies provide evidencethat the cholesterol-lowering capabilities of RBO can be attrib-utedtothecompoundsitcontainswhoseeffectssurpassthoseof its fatty acid composition.Otherinvestigatorsofricebranhaveimplicatedtheunsaponi-fiable compounds as being responsible for its cholesterol-lowering properties. The amounts present in commercial RBOare dependent on the refining process (17). The most notablecompound is    -oryzanol, a ferulate ester of triterpene alcohols(12). Major components of the triterpene alcohols are cycloarte-noland24-methylenecycloartanol.Alsonotablearethephytos-terolscampesteroland  -sitosterol,whicharefoundatrelativelyhigh amounts in RBO. When the plant sterols from RBO wereincorporated into margarine and provided at 2.1 g/d to normo-lipidemicmenandwomen,totalcholesteroldecreasedby5%andLDL cholesterol decreased by 9% (18). The investigators pos-tulated that the effect was due to the   -sitosterol and other4-desmethylsterols and not to the 4,4'-dimethylsterols, such ascycloartenol and 24-methylene cycloartanol. The   -sitosterolstructure is more similar to that of cholesterol than is that of the4,4'-dimethylsterols, and it may be more effective than the 4,4'-dimethylsterols in inhibiting cholesterol absorption in the smallintestine.ThisisfurthersupportedbyWeststrateandMeijer(19), TABLE 5 Lipid and apolipoprotein concentrations and factor VII activity in study 1 1 Run-in diet( n  26)Control diet( n  13)Defatted rice bran diet( n  13)Total cholesterol (mmol/L) 5.01  0.12 4.84  0.17 5.21  0.26HDL cholesterol (mmol/L) 1.32  0.08 1.44  0.14 1.22  0.09LDL cholesterol (mmol/L) 3.25  0.13 3.04  0.16 3.53  0.25 2 Triacylglycerols (mmol/L) 3 0.93  0.08 0.76  0.09 0.99  0.12Apolipoprotein A-1 (g/L) 1.36  0.05 1.43  0.11 1.32  0.05Apolipoprotein B (g/L) 0.95  0.04 0.88  0.05 1.00  0.07 4 Factor VII (%) 81.69  3.31 80.92  5.58 85.49  5.23 1 Allvaluesare  x      SEMofeachsubject’saverageofreplicatemeasurementstakenatendofdietperiods.Toconvertcholesterolvaluestomg/dL,divideby 0.02586; to convert triacylglycerol values to mg/dL, divide by 0.01129. 2,4 Significantly different from control diet (two-sided  t   tests for pairwise comparison):  2 P  0.0204,  4 P  0.0299. 3 Statistical analyses performed on log-transformed values; no significant difference was found. TABLE 6 Lipid and apolipoprotein concentrations and factor VII activity at the endof study 2 1 Control oil blend diet( n  14)Rice bran oil diet( n  14)Total cholesterol (mmol/L) 5.22  0.15 4.95  0.14 2 HDL cholesterol (mmol/L) 1.22  0.06 1.22  0.07LDL cholesterol (mmol/L) 3.57  0.15 3.30  0.14 3 Triacylglycerols (mmol/L) 4 0.93  0.11 0.93  0.08Apolipoprotein A-1 (g/L) 1.32  0.05 1.34  0.05Apolipoprotein B (g/L) 1.03  0.05 0.97  0.04 5 Factor VII (%) 103.48  3.66 101.48  4.38 1 All values are  x       SEM of each subject’s average of replicate mea-surements. To convert cholesterol values to mg/dL, divide by 0.02586; toconvert triacylglycerol values to mg/dL, divide by 0.01129. 2,3,5 Significantly different from oil blend diet ( F   tests using a mixedmodel):  2 P  0.0036,  3 P  0.0004,  5 P  0.0054. 4 Statistical analyses performed on log-transformed values. RICE BRAN OIL AND CHOLESTEROL  67
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