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Nutrition 29 (2013) 790–795

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Nutrition

journal homepage: www.nutr i t ionjrnl .com

Basic nutritional investigation

Wheat germ oil: A potential facilitator to improve lutein bioavailability in mice

Aruna Gorusupudi M.Sc., Vallikannan Baskaran M.Sc., Ph.D. *

Department of Biochemistry and Nutrition, CSIR-Central Food Technological Research Institute, Mysore, Karnataka, India

a r t i c l e i n f o

Article history:Received 24 April 2012Accepted 14 November 2012

Keywords:LuteinWheat germ oilBioavailabilityGlycolipidsLinoleic acid

The authors do not have any conflicts of interest anthe objective of the study.* Corresponding author. Tel.: þ91 8212514876; fax:

E-mail address: baskaranv@cftri.org (V. Baskaran)

0899-9007/$ - see front matter � 2013 Elsevier Inc. Ahttp://dx.doi.org/10.1016/j.nut.2012.11.003

a b s t r a c t

Objective: The aim of this study is to determine the effect of wheat germ oil (WGO) compared withgroundnut oil (GNO) and mixed micelles (control) on lutein bioavailability and bioactivity in mice.The choice of carrier lipid is critical to achieve an enhanced bioavailability of lutein.Methods: Mice were intubated with single and repeated doses (2 wk) of lutein solubilized in WGO,GNO, or control mixed micelles to study lutein bioavailability, as well as changes in the lipaseactivity, antioxidant enzymes, lipid peroxidation, and fatty-acid profile.Results: Single-dose (nmol/8 h/mL) and repeated-dose (mg/dL) studies revealed that plasma luteinlevels were higher (P > 0.05) in the WGO (88.4 � 6, 3.2 � 1) and GNO (23.36 � 4, 4.7 � 0.5) groupsthan in the control (12.4 � 1, 2.6 � 0.6) group. Liver and eye lutein levels in WGO (41% and 53%,respectively) and GNO (6% and 41%, respectively) groups also were found to be higher than thecontrol group. However, the dietary lutein response in plasma and tissues was more pronounced inthe WGO group than the GNO group. The decrease in plasma malondialdehyde (MDA) levels in theWGO (41%) and GNO (26.4%) groups compared with the control group indicates the higherbioavailability and bioactivity of absorbed lutein.Conclusion: The increased lutein bioavailability in the WGO group compared with the other twogroups may be attributed to the polar lipids and intestinal lipase activity found in this study. Theresults imply a new insight into the application of WGO for improving lutein bioavailability.

� 2013 Elsevier Inc. All rights reserved.

Introduction

Increasing evidence demonstrates that dietary carotenoidsmay delay the progression of certain degenerative and nutri-tional deficiency disorders. Specifically, lutein and zeaxanthin arethe xanthophyll carotenoids present in themacula of eyes, whichhave been implicated strongly as being protective against age-related macular degeneration (AMD) and cataracts, as well ascertain cancers and heart diseases [1]. Studies have shown thatnutritional supplements containing lutein, zeaxanthin, andmeso-zeaxanthin effectively increased the macular pigmentdensity and reduced the prevalence of AMD [2]. Christen et al. [3]studied the positive effect of vitamin E along with lutein andzeaxanthin in decreasing cataracts and AMD. Low absorption oflutein and zeaxanthin is another risk factor that increases theprevalence of AMD. Because humans cannot synthesize lutein,dietary ingestion is the only source of it; however, its intestinalabsorption depends on dietary lipids. Earlier studies [4,5]

d contributed equally for

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ll rights reserved.

reported that intestinal absorption of carotenoids depends on thenature and level of fat, its influence on the release of bile, andformation of mixed micelles in the intestine. Studies also inves-tigated the effects of vegetable oil on lutein bioavailability inrodents and in vitro [6,7]. Hu et al. [8] and Clark et al. [9] studiedthe effect of animal fats and vegetable oils on the plasmaresponse of b-carotene, lycopene, and astaxanthin in human andrats. Despite the importance of dietary lipids as carriers for lutein,little is known about their influence on lutein bioavailability.Hence, it is appropriate to determine the role of newer dietarylipids like wheat germ oil (WGO) in lutein bioavailability.

This study appraises the effect of WGO compared withgroundnut oil (GNO) andmixed micelles on lutein bioavailabilityand the activity of intestinal lipase in mice. Wheat germ andWGO are among the best sources of vitamin E, polar lipids, andlinoleic acid (the precursor for u-3 and u-9 fatty acids thatimproves b-carotene and lutein absorption) [10,11].

Materials and methods

Chemicals

Luteinwas extracted and purified (72%) frommarigold (Tagetes erecta) petalsfor animal feeding studies. Wheat germ (Triticum vulgare) was donated by Fluor

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Fig. 1. Percent difference over control in the lutein plus zeaxanthin levels in plasma(plasma AUC values), liver, eyes (#pooled samples), and intestine after single dose(A) and repeated intubation (2 wk) of lutein in WGO and GNO mixed micelles (B).

Table 1Lutein plus zeaxanthin levels in plasma, liver, eyes, and intestine of mice aftera single intubation (8 h) of lutein solubilized in either WGO, GNO, or controlmixed micelles

Groups Plasma AUC(nmol/8 h/mL)

Liver(pmol/g)

Eyes#(pmol/g)

Intestine(pmol/15 cm)

Control 12.4 � 1.9* 2.06 � 0.2* ND NDGNO 23.39 � 2z ND 21.18 0.96 � 0.1y

WGO 88.8 � 6y 7.42 � 1y 8.87 13.06 � 9.5*

Values are mean � SD (five mice per group) ND, below the detectable limit(1 pmol). Values in each column not sharing a common symbol are significantlydifferent (P < 0.05) between groups determined by ANOVA with Tukey’s test.#Pooled eye samples

A. Gorusupudi, V. Baskaran / Nutrition 29 (2013) 790–795 791

Milling, Baking and Confectionary Technology Department, CFTRI, Mysore, India.Standard lutein (99%), butylated hydroxyl toluene, monooleylglycerol (MOG),oleic acid (OLA), sodium taurocholate, glutathione reductase (GR), cytochrome C,xanthine oxidase, TMP standard (99%), Dinitro- 5-thiobenzoic acid, thiobarbituricacid, and fatty-acid standards were purchased from Sigma-Aldrich (St. Louis,USA). Analytical, high-performance liquid chromatography (HPLC)-gradesolvents and other chemicals mentioned elsewhere in this study were purchasedfrom Sisco Research Laboratories (Mumbai, India). Lipase assay kit was purchasedfrom Randox Laboratories, Mumbai, India. Rat feed pellets were purchased fromSai Durga feeds (Bangalore, India). GNO was purchased from local market inMysore, India.

Extraction and purification of lutein and WGO

Lutein was extracted and purified from marigold petals as per the methoddescribed by Lakshminarayana et al. [11] to obtain 99% pure lutein. The purity oflutein was quantified by HPLC (LC-10A; Schimadzu, Kyoto, Japan), based on thepeak area of standard lutein at 445 nm and used in this study.

WGO was extracted from wheat germ as per the method described bySugawara and Miyazawa [12]. Glycolipid, phospholipid, and neutral lipid frac-tions were separated from WGO by open-column chromatography (OCC) usingsilica gel (60–200 mesh). The neutral lipids were eluted with chloroform,followed by glycolipids with acetone and phospholipids with methanol. Thefatty-acid composition of crude WGO and GNO were analyzed by gas chroma-tography as methyl esters, as per Morrison and Smith [13].

Preparation of mixed micelles

Mixed micelles were prepared in phosphate-buffered saline (pH 7.0) con-taining MOG (2.5 mM), OLA (2.5 mM), sodium taurocholate (12 mM), cholesterol(0.5 mM), and lutein (200 mM) with WGO (300 mg/mL), GNO (300 mg/mL), andcontrol (without WGO and GNO). Chemicals and lipids were dissolved in chlo-roform and mixed well. The solvent was evaporated to dryness using nitrogen.The mixture was suspended in phosphate-buffered saline (pH 7.0), vortexed for10 min, and sonicated (PCI, Mumbai) for 30 min to obtain mixed GNO, WGO, andcontrol micelles, which are used for gavage studies.

Animal experiment

Animal experiment was performed after due clearance from the institutionalanimal ethics committee. Thirty male albino mice (OUTB/Swiss Albino/IND/CFT[2c]) weighing 24 � 2 g, were housed in individual stainless steel cages at roomtemperature (28� 2�C) with a 12-h light/dark cycle in the institute animal housefacility. The mice received a pellet diet and had free access to water. Afteracclimatization (7 d), the mice were administrated a daily dose of lutein solu-bilized in mixed micelles (control) or WGO-mixed or GNO-mixed micelles. Dietandmixedmicelles were processed for lutein analysis to ascertain its level beforeintubating to animals.

Single-dose time-course study

Groups of mice (five per group) were gavaged a daily dose (0.2 mL permouse) of lutein solubilized in mixed micelles with WGO, GNO, or controlmicelles. In order to minimize the usage of animals, blood (100 ml) was drawnusing heparinized capillaries (Hirschmann laborgerate GmbH & Co., Germany)from the caudal vein [14] at 0, 2, and 4 h after gavage of lutein, without sacrificingthe animal. At the end of 8 h, animals were sacrificed, and blood was drawndirectly from the heart into heparinized tubes and centrifuged at 1000g for15 min at 4�C to separate plasma. The liver, intestine, and eyes were excised,washed with ice-cold isotonic saline, and stored at �80�C until analyzed.

Repeated-dose study

Mice (six per group) were administered a daily dose (0.2 mL per mouse) ofmicellar lutein (control), lutein dispersed inmixedmicelles withWGO or GNO, ormicelles with no lutein or lipids (baseline) for 2 wk. At termination of theexperiment, animals were sacrificed and bloodwas drawn directly from the heartto heparinized tubes and centrifuged at 1000g for 15 min at 4�C to separateplasma. Liver, intestine, and eyes were excised, washed with ice-cold saline, andstored at �80�C until analyzed.

Extraction of lutein from plasma, tissues, and diet and HPLC analysis

Lutein was extracted from plasma as per Lakshminarayana et al. [11]. Theextracts were pooled, evaporated to dryness using nitrogen, and redissolved in100 mL of Acetonitrile: MeOH: DCM (60:20:20, v/v/v, 0.1% ammonium acetate,mobile phase) for HPLC analysis. Liver, eyes (pooled eyes from five mice), intes-tine, feces, and diet samples were homogenized (Potter-Elvehjem homogenizer,

Remi Instruments Ltd. Mumbai, India) separately with nine parts of ice-coldisotonic saline and used (0.8 mL) for lutein extraction. In the case of the intes-tine, the mucosal layer was scraped using a cover glass, homogenized to forma uniform phase, and used (1 mL) for lutein extraction. In the case of the liver,samples were saponified separately with 2 mL of 10 M potassium hydroxide at60�C for 45min and vortexed; 2mL of ice-cold deionized water was added beforelutein extraction. Sample handling, homogenization, and extractionwere carriedout on ice under dim yellow light to minimize light-induced isomerization andoxidation of lutein.

Lutein extracted from the plasma, liver, eyes, intestine, feces, and dietsamples were quantified with an HPLC system equipped with photodiode arraydetector (SPD-M20A, Shimadzu). Lutein was separated on a Princeton SPHERC-30 (ODS) column (250 mm � 4.6 mm; 5 mm) isocratically eluting with1 mL/min of mobile phase at 445 nm (Shimadzu Class-VP version 6.14 SP1software). Because lutein and zeaxanthin are structural isomers, lutein wasquantified as lutein plus zeaxanthin. The peak identity of lutein and zeaxanthinwere confirmed by their characteristic spectrums and were quantified bycomparing their peak area with authentic standards.

Assay for antioxidant enzymes, lipase, and lipid peroxides

Activities of catalase (CAT) [15], glutathione peroxidase (GPx) [16], super-oxide dismutase (SOD) [17], glutathione S-transferase (GST) [18], and glutathione

Fig. 2. Representative chromatograms of HPLC elution of lutein in plasma (A) and eyes (B) of mice after repeated intubation (2 wk) of lutein in WGO, GNO, or control micelles.

Table 2Lutein plus zeaxanthin levels in plasma, liver, eyes, and feces of mice afterrepeated intubation (2 wk) of lutein solubilized in either WGO, GNO, or controlmixed micelles

Groups Plasma(mg/dL)

Liver(ng/g)

Eyes#(ng/g)

Intestine(ng/15 cm)

Feaces(mg/1 g)

Baseline 0.8 � 0.1* 1.4 � 0.2* 0.34 0.5 � 0.0* 7 � 0.2*Control 2.6 � 0.8y 23 � 4y 4.21 4.8 � 0.8z 133.8 � 3z

WGO 3.2 � 1.1y 39 � 1.1z 25.63 4.7 � 0.4z 130 � 0.5z

GNO 4.7 � 0.5z 24.6 � 5.1y 7.16 3.36 � 0.2y 124.5 � 3y

Baseline, group fed mixed micelles containing no lutein and no lipid; control,group fed lutein mixed micelles without any lipid; WGO, group fed lutein withWGO; GNO, group fed lutein with GNO#- pooled eyes (six mice per group). Values are mean � SD. Values in eachcolumn not sharing a common symbol are significantly different (P < 0.05)between groups determined by ANOVA with Tukey’s test

A. Gorusupudi, V. Baskaran / Nutrition 29 (2013) 790–795792

reductase (GR) [19] were measured using standard procedures. The glutathione(GSH) level [20], protein [21], and lipid peroxides [22] in plasma and liverhomogenates were measured using standard procedures. Lipase activity in liverand intestine were measured using lipase assay kit by monitoring the rate ofdecrease in absorption of substrate at 410 nm. Total lipids [23] and fatty-acidmethyl esters [13] were analyzed from plasma and tissues by adopting stan-dard procedures.

Statistical analysis

To quantify the postprandial plasma lutein level over 8 h, area under thecurve (AUC) was calculated by trapezoidal approximation. Data were tested forhomogeneity of variances by the Bartlett test. When homogenous variances wereconfirmed, the data were tested by analysis of variance and significant differ-ences between the groups were evaluated by Tukey’s test. Differences in meanswere considered significant at P < 0.05.

Results

The purity of lutein obtained from the marigold petal extractby OCC was 97% � 2% and their lmax was 445 and 452 nm. Thetotal lipid content of wheat germwas 8% (dry wt. basis) in whichthe levels of glycolipids, phospholipids, and neutral lipids were30%, 10%, and 50%, respectively. The major fatty acids present inWGO and GNO were linoleic acid (58%) and OLA (54%), respec-tively (Table 3a).

Single-dose study

Lutein was not detected in plasma of the 0 h samples. Aftergavage of a single dose of lutein, its levels reached a maximum at4 h (pmol/mL) in WGO (22.36 � 9), GNO (4.8 � 0.8), and control(3.8� 0.1) anddecreased significantly from4 to 8h. ThemeanAUCvalues for plasma lutein (nmol/8 h/mL) in WGO (88.4 � 6) andGNO (23.39 � 2) were higher than the control group (12.4 � 1.9)(Fig. 1a). The liver lutein levels (pmol/g) were higher in the WGOgroup (7.9 � 2) than in the GNO (ND) and control (2.02 � 0.2)groups, whereas in eyes (pmol/g) it was higher in the GNO group(21.18) than in theWGO (8.87) and control (ND) groups. Similarly,the intestinal lutein levels were higher in the WGO group(13.06 � 9) than in the GNO group (0.96 � 0.1) (Table 1).

Repeated-dose study

The lutein levels in mice plasma and eyes after repeatedintubation of lutein (2 wk) solubilized in WGO, GNO, or controlmixed micelles is shown in Figure 2. The lutein level (mg/dL) inplasma of the WGO (3.2 � 1.1) and GNO (4.7 � 0.5) groups were

higher than in the control group (2.6 � 0.8). The liver luteinlevels (mg/g) in the WGO (39 � 1.1) and GNO (24.6 � 5) groupswere also higher than the control group (23 � 4), whereas itslevel in the WGO group was higher by 37% compared with theGNO group. Similarly, lutein level in the eyes of the WGO andGNO groups were higher by 53% and 41% compared with control,whereas the WGO group was higher by 71% compared with theGNO group. The lutein level (mg/g) in feces of control group(133.8 � 4) was higher than the GNO (130 � 0.7) and WGO(124� 0.8) groups. In the case of intestine, the lutein levels werehigher in the control group, by 2% and 42% when compared withthe WGO and GNO groups, respectively (Fig. 1b and Table 2).

Lipid profile

The plasma fatty-acid profile of WGO, GNO, and controlgroups show that OLA content in theWGO groupwas 30% higherthan control group; the GNO group was 24% lower than control(Table 3b). The liver fatty-acid profile, after repeated gavage oflutein solubilized in WGO, GNO, or control, showed that OLAlevels in the WGO and GNO groups were higher by 16% and 35%compared with control (Table 3c). The arachidonic acid (20:4)content increased in the WGO group by 36% more than in thecontrol group.

Antioxidant enzymes

The activity of antioxidant enzymes after repeated gavage oflutein solubilized in WGO, GNO, or control mixed micelles werecompared with a baseline group. CAT activity in plasma was

Table 3Fatty acid profile of (a) WGO and GNO, (b) plasma, and (c) liver of mice afterrepeated dose (2 wk) of lutein solubilized in either WGO, GNO, or control mixedmicelles

Fatty acids (%) GNO WGO

16:0 17 18.518:0 5.6 1.218:1 54.2 21.718:2 16.5 57.720:0 1.9 0.722:0 3.6 0.2

Fatty acids (%) Baseline Control GNO WGO

16:0 32.3 � 4.5* 30.4 � 3.5* 27.69 � 0.5y 23 � 6y

16:1 3.13 � 0.0* 2.52 � 0.4y 2.32 � 0.3y 1.01 � 0.2z

18:0 12.83 � 0.8* 12.43 � 1.8* 13.05 � 0.2* 15 � 1.2y

18:1 21.44 � 1.22* 27.96 � 1.4y 21.51 � 0.6* 30.93 � 2y,z

18:2 21.16 � 0.94* 19.77 � 0.7y 19.98 � 0.7y 18.66 � 2y

20:0 ND 0.4 � 0.1* 1.1 � 0.13y 1.38 � 0.4y

20:4 9 � 3.5* 7.57 � 0.8* 17.62 � 1.33y 8.88 � 1*

Fatty acids (%) Baseline Control GNO WGO

16:0 37.42 � 1.5* 35.8 � 3.5* 33.68 � 3.0* 31 � 5.0*18:0 20.86 � 0.8* 20.77 � 1.8* 11.16 � 0.2y 17.7 � 1.2z

18:1 22.28 � 1.2* 26.62 � 1.4y 41.04 � 0.6z 31.7 � 2x

18:2 17.09 � 1.4* 13.68 � 0.7y 9.72 � 0.7z 13.8 � 0.2y

20:4 2.28 � 0.13* 3.4 � 0.8y 1.28 � 0.4z 5.37 � 1x

ND, not detectedValues are mean � SD, (six mice per group). Values in rows not sharinga common symbol are significantly different (P < 0.05) between groups deter-mined by ANOVA with Tukey’s test. Refer to Table 2 for details of groups

A. Gorusupudi, V. Baskaran / Nutrition 29 (2013) 790–795 793

higher by 44% in the WGO group; it was lower by 13.5% in theGNO group than in control. Plasma GSH levels in the WGO andGNO groups increased by 19% and 5.1% over the control group(Table 4 and Fig. 3a). SOD activity in liver was higher in the WGO(55%) and GNO (16%) groups compared with control. In the GNOgroup, GST activity was lower by 19% compared with control. GRactivity was higher by 35% and 7% in WGO and GNO groups,respectively, compared with control. Liver GSH (mmol/g) in WGO(98� 2) was higher than in GNO (82� 1.2) and control (84� 3.2)groups (Table 4 and Fig. 3b).

No significant difference was observed in plasma and liverGPx. The CATactivity and GSH levels in plasma of theWGO groupwas higher by 51% and 14%, respectively, comparedwith the GNOgroup. In the liver, GPx, SOD, GST, GR, and GSH levels in theWGOgroupwere higher by 14%, 47%, 22%, 30%, and 16.3%, respectively,than in the GNO group.

Plasma MDA levels (nmol/mg protein) in WGO (8.9 � 1.1) andGNO (11.5 � 0.6) groups were lower than in the control group(15.1 � 1.2). Plasma MDA in WGO and GNO groups was 41% and26.4% lower, respectively, than in the control group. The decrease

Table 4Activity of antioxidant enzymes/molecule in plasma and liver of mice after repeated int

Parameters Groups

Baseline

PlasmaGlutathione peroxidase (mmol/min/mg protein) 4.45 � 1*Catalase (mmol/min/mg protein) 19 � 8*Glutathione reduced (mmol/dL) 45 � 1*

LiverGlutathione peroxidase (mol/min/mg protein) 3.93 � 0.7*Superoxide dismutase (U/min/mg protein) 10 � 0.6*Glutathione S-transferase (mmol/min/mg protein) 1.9 � 0.4*Glutathione reductase (mmol/min/mg protein) 4.0 � 0.8*Glutathione reduced (mmol/g) 58 � 1*

Values are mean � SD, (six mice per group). Values in rows not sharing a common symwith Tukey’s test. Refer to Table 2 for details of groups

in liver MDA in WGO and GNO mice over control was 17% and31%, respectively. Plasma and liver MDA levels in theWGO groupdecreased by 22.6% and 16%, respectively, compared with theGNO group (Fig. 3).

Lipase activity

Lipase activity in livers of the WGO group was higher by 13%and 15% than in the GNO and control groups, respectively. In theintestine, the lipaseactivityof theWGOandGNOgroupsdecreasedby 11% and 20% than the control group, while in WGO group theactivity decreased by 11% comparedwith the GNO group (Table 5).

Discussion

The intestinal absorption of dietary carotenoids is governedby various exogenous and endogenous factors [5]. Dietary factorsthat play a major role in lutein absorption are the nature, type,and level of fat present in food. Brown et al. [24] showed that theuse of fat-free or reduced-fat salad dressings limits the absorp-tion of b-carotene and lutein in humans. Earlier studies [6,11]showed that micellar phospholipids and olive oil increaseplasma response of lutein in rats, demonstrating that specificlipids or fatty acids may modulate the intestinal uptake of lutein.The present study demonstrate thatWGO rich in glycolipid (30%)and linoleic acid (56%, 18:2 [n-6]), compared with GNO, whichcontains mostly neutral lipids (99%) and OLA (55%, 18:1 [n-9])and mixed micelles (control) on plasma, liver and eye responseof lutein in mice. Additionally, the effects of repeated doses oflutein solubilized in those lipids on the plasma and liverfatty-acid profile, antioxidant enzymes, intestinal and liver lipaseactivity also was investigated.

The results show that lutein absorption from WGO mixedmicelles is higher (88.4 � 6 nmol/8 h/mL) than from GNO(23.4 � 2 nmol/8 h/mL) mixed micelles, demonstrating that WGOimproves the intestinal lutein uptake (Table 1). Furthermore, theresults suggest thatWGO rich in linoleic acid (56%) and glycolipids(30%) alsomay influence the intestinal uptake of lutein by favoringmicelle formation [10]. The present findings suggest a positivecorrelation between linoleic acid levels and lutein bioavailability,which is similar to OLA levels as reported in earlier studies [6,25].Despite lower OLA levels, WGO still could facilitate luteinbioavailabilitymore than GNO, possibly due tominor componentssuch as glycolipids and vitamin E, which are present in WGO.

To our knowledge, this is the first study to demonstrate thesignificant role of WGO in lutein absorption in mice. The higherlevel of plasma lutein in theWGO group may be attributed to the

ubation (2wk) of lutein solubilized in eitherWGO, GNO, or control mixedmicelles

Control WGO GNO

9.3 � 2.4y 9.4 � 2.6y 9.8 � 1.9y

37 � 2y 66 � 6z 32 � 11y

47 � 2* 58 � 1y 49.5 � 2*

4.81 � 0.5* 4.77 � 0.5* 4.11 � 0.15*12 � 0.8y 27 � 0.9x 14.3 � 0.8z

2.6 � 0.1y 2.7 � 0.16y 2.1 � 0.1*3.9 � 1* 6 � 0.3y 4.2 � 1.2*84 � 3.2y 98 � 2z 82 � 1.2y

bol are significantly different (P < 0.05) between groups determined by ANOVA

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Fig. 3. Percent difference over control in the activity of antioxidant enzymes andlipid peroxidation in plasma (A) and liver (B) after repeated intubation (2 wk) oflutein in WGO and GNO micelles. CAT, catalase; Glu, reduced glutathione; GPx,glutathione peroxidase; GR, glutathione reductase; GST, glutathione S-transferase;SOD, superoxide dismutase.

Table 5Activity of lipase in liver and intestine of mice after repeated intubation (2 wk) oflutein solubilized in either WGO, GNO, or control mixed micelles

Groups Liver lipase activity(EU/mg protein/g of liver)

Lipase in intestine(EU/mg protein/15 cm)

Control 19.51 � 1.2* 6.8 � 1.4*GNO 19.9 � 3.3* 8.6 � 1.*WGO 22.4 � 6.1* 7.67 � 1*Baseline 19.0 � 2.13* 5.28 � 0.8y

Values are mean � SD, (six mice per group). Values in columns not sharinga common symbol are significantly different (P < 0.05) between groups deter-mined by ANOVA with Tukey’s test. Refer to Table 2 for details of groups

A. Gorusupudi, V. Baskaran / Nutrition 29 (2013) 790–795794

synergistic effect of glycolipids, phospholipids, and neutral lipidspresent in it. In the case of GNO, the major lipid class is neutrallipids. Furthermore, difference in plasma lutein responsebetween the GNO and WGO groups may be attributed to thevariation in the chemical structure of major lipids present withrespect to fatty acids and functional groups [26]. GNO with non-polar lipids may be less soluble in water compared with WGO(combination of phospholipids, glycolipids, and neutral lipids).This may be a reason for lower plasma lutein levels in the GNO-fed group. Polar lipids (phospholipids and glycolipids) formsmaller micelles compared with non-polar lipids (neutral lipids),which may result in a difference in plasma lutein in GNO andWGO [27]. This is not evidenced in the case of repeated-dosestudy in which the plasma lutein levels were higher in theGNO than in the WGO group, which may be due to the highturnover rate of lutein and lower intestinal lipase activity, asreported by Raju et al. [28].

Lipolysis or hydrolysis of triglycerides (TGs) to release fattyacids for micelle formation in the intestinal tract by lipase arevital for the efficient uptake of carotenoids by the enterocytes[29]. WGO and GNO differ in their fatty-acid profiles, thus they

may act differently at various stages of absorption, including theactivity of lipase, which may be one of the reasons for slightlyenhanced activity of liver lipase, yet no difference in the intes-tinal lipase between the WGO and GNO groups. Because carot-enoids are transported in association with lipoproteins, plasmaTG and cholesterol concentrations usually correlate with circu-lating carotenoid concentrations [30]. In the present study,plasma and liver TGs increased after a repeated dose of luteinwith WGO and GNO compared with control (data not shown),which indicates their involvement in the transport of newlyabsorbed lutein to target tissues such as the eyes and liver.Elevated lutein levels in the eyes and liver of WGO group ratherthan in the GNO group further supports the result that TG isa carrier molecule for lutein.

Although specific fatty acids [6] play a vital role in luteinbioavailability, they are found to be involved in the regulation ofoxidative mechanisms. The present results demonstrate anenhanced protective effect of lutein ingested with WGO fromlipid peroxidation compared with GNO and control. This may bethe reason for higher plasma MDA levels in the GNO group(22%) than in the WGO group. Lutein ingestion along with WGOon lipid composition and the activity of CAT, GPx, and GSH inplasma and SOD, GPx, and GR in liver were comparatively morepronounced than in the GNO and control groups. Changes in theplasma and liver fatty-acid composition after single andrepeated doses of lutein with WGO may be due to the unsatu-rated lipids in WGO, which in turn may enhance the efficiencyof SOD and GPx [31]. The present study is in agreement withearlier studies [3,32] in which increases in plasma lutein levelsand vitamin E are reported to decrease oxidative damage,thereby protecting against degenerative diseases like AMD andcataracts.

Conclusions

The influence of lipids on lutein bioavailability in mice was inthe order ofWGO>GNO> control (mixedmicelles). The relativebioavailability of lutein could be improved with WGO, and inturn may help to modulate the tissue fatty-acid profile andantioxidant molecules. This finding may imply a new insight forthe dietary recommendations of lutein with WGO for improvedlutein bioavailability.

Acknowledgments

Aruna Gorusupudi acknowledges the University GrantsCommission, government of India, for grant of senior researchfellowship. The authors acknowledge Dr. T. R. Ramaprasad,scientist, Lipid Science and Technology Department, CFTRI,Mysore, India, for his help in editing and reviewing thismanuscript.

A. Gorusupudi, V. Baskaran / Nutrition 29 (2013) 790–795 795

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