1. Introduction

Dietary retinol esters are cleaved to retinol andfatty acids by pancreatic and mucosal esterase andhydrolase, and are absorbed via the intestinallymphatics. Cellular retinol-binding protein, type Ⅱ,i.e., CRBP (Ⅱ), present in the small intestine, mayplay an important role in the absorption and metabo-lism of retinol and β-carotene. Absorption of freeretinol is followed by re-esterification within themucosal enterocytes with long-chain saturated andunsaturated free fatty acids. The composition of newlyformed retinyl esters is independent of the fatty acidcomposition of dietary lipid during absorption and

re-esterification1). On the other hand, dietary supple-mentations of retinol and lipid resulted in higherserum levels of retinyl esters. The dominant esterhere was retinyl stearate followed by retinyl palmitateand retinyl oleate in ferrets2, 3), dogs and silver foxes4),while, in the serum of raccoon dogs4) and humans5),retinyl palmitate dominated over retinyl stearate.There were no reports on the relationship betweenthe composition of fatty acids in dietary lipids and thecomposition of retinyl esters recovered in serum.Therefore, the present study was designed to investi-gate which fatty acid is attributable to the newlyformed retinyl esters in human serum.

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1)Department of Laboratory Medicine, Toho UniversityOhashi Medical Center, 2-17-6 Ohashi, Meguro, Tokyo153-8515, Japan2)Department of Health and Nutrition, Wayo Women's

University, 2-3-1 Konodai, Ichikawa, Chiba 227-8533,JapanRecieved for Publication November 11, 2011Accepted for Publication November 21, 2011

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Metabolism of retinyl esters in humans after ingestion of retinol

Hiroshi Ihara1), Asuka Tani1), Takashi Kakinoki1), Yoshio Shino1), Yoshikazu Morita1),Nobuaki Matsumoto1), Yuji Oikawa1), Mio Koyasu1), Kiyoshi Takamiya1),

Makoto Suzuki1) and Naotaka Hashizume2)

Summary Using high-performance liquid chromatography, we determined which retinyl esters

predominately circulate in human serum after the ingestion of rapeseed oil and a few drops of

retinyl palmitate. Fatty acids, predominately as palmitic acid followed by oleic, linoleic, and stearic

acids, as well as retinyl esters, predominately as retinyl palmitate followed by retinyl stearate, oleate

and linoleate, increased in serum after the ingestion of rapeseed oil and a few drops of retinyl

palmitate. Re-esterification ratios (molar ratios between the concentrations of fatty acids and the corre-

sponding retinyl esters recovered in serum after the ingestion of rapeseed oil and a retinyl palmitate)

were markedly increased in saturated fatty acids (palmitic and stearic acids). Re-esterification

ratios of unsaturated fatty acids (oleic and linoleic acids) were low compared with saturated fatty acids.

Key words: Vitamin A, Fatty acid, Palmitic acid, Stearic acid, LDL receptor

〈Original Article〉

2. Materials and methods

1. SubjectsSeven healthy volunteers, five men (age 30 to 49

years) and two women (age 52 and 55 years), all witha normal body mass index (21 to 25), were enrolled inthis study. Venous serum specimens were collectedfrom these volunteers before and 1, 2, 3, 4, 6 and 8hours after the ingestion of 37 g of rapeseed oil and afew drops of retinyl palmitate (Chocola A, Eisai Co.,Ltd. Tokyo, Japan) equivalent to 32.7 mg of retinol(0.114 mmol). All subjects had fasted overnight for 12hours, remaining in a fasting state except for water anda light lunch (soba, i.e., buckwheat noodles) for 4hours until the end of the study. They reported noside effects other than occasional mild headaches andgastric upset. From all volunteers, informed writtenconsent for these studies was obtained, and studieswere approved by the guidelines established by theProtection of Human Subjects Committee of theJapanese Society of Nutrition and Food Science.

2. AssaysSerum concentrations of retinyl esters were

measured by high-performance liquid chromatog-raphy (HPLC) as described previously6, 7, 8) on C18column 150 x 4.6 mm inner diameter, containing 5μm particles (STR-Ⅱ, Shimadzu Techno-ResearchInc., Kyoto, Japan). The mobile phase was comprisedof 10 g/L of AgNO3 dissolved in ethanol/H2O, 97:3(v/v). The elution rate was 1.0 mL/min, and the eluentwas monitored at 325 nm. To calibrate thechromatogram, we used retinol and retinyl palmitate(from Wako Pure Chemical Industries, Ltd., Osaka,Japan). Retinyl oleate, stearate, and linoleate weresynthesized by reacting retinol with their corre-sponding acyl chlorides. Concentrations in the eluentwere calculated based on the molar absorptivity ofretinol9). This was possible because retinol and severalretinyl esters have nearly identical molar absorptivitiesat 325 nm owing to their retinol moiety10). The fattyacid compositions of rape oil and serum specimenswere determined by gas chromatography (GC) after amodified Folch extraction and transmethylation11).An incremental area under the response curve

(IAUC) was calculated by plotting the concentrationsof fatty acids, retinyl esters and their molar rations vs.time. The IAUC was the sum of the trapezoids fittingunder the curve.

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Fig. 1 Fatty acid levels in serum from zero to 8 hours after ingestion of 37 g of rapeseed oil and a fewdrops of retinyl palmitate.■, palmitic acid; ◆, linoleic acid; ▲, oleic acid;○, stearic acid.

Fig. 2 Retinyl ester levels in serum from zero to 8 hoursafter ingestion of 37 g of rapeseed oil and a fewdrops of retinyl palmitate.■, retinyl palmitate; ◆, retinyl linoleate; ▲, retinyl oleate; ○, retinyl stearate.

3. Results

1. Fatty acid composition of rape oil and serumBy GC analysis, 37 g of rapeseed oil predomi-

nantly contained 62.4 mmol (16.0 g) palmitic acid,57.4 mmol (16.2 g) oleic acid, 8.2 mmol (2.3 g)linoleic acid, and 8.1 mmol (2.3 g) stearic acid. Afew drops of retinyl palmitate contained palmitic acidas low as 0.114 mmol. In fasting serum specimens,fatty acids (mainly as linoleic acid) dominatedwith 3264±596μmol/L (Table 1), followed bypalmitic acid (2997±718μmol/L), oleic acid(2218±741μmol/L) and stearic acid (861±122μmol/L). Among these fatty acids, palmitic acid(IAUC=5639±3807μmol/L x hr) and oleic acid(IAUC=4469±3495μmol/L x hr) increased markedlyfollowing the ingestion of rapeseed oil and a fewdrops of retinyl palmitate in accord with the fattyacid composition of the rapeseed oil (Fig. 1).

2. Re-esterification of retinolIn fasting serum specimens, retinyl palmitate

dominated with 46±13 nmol/L (Table 1), followed byretinyl oleate (35±14 nmol/L), retinyl stearate (21±6 nmol/L) and retinyl linoleate (19±21 nmol/L).Serum concentrations of these four retinyl estersincreased after the ingestion of 37 g rapeseed oil and

a few drops of retinyl palmitate (Fig. 2). Compared tothe concentrations in a fasting state, retinyl palmitateincreased maximally to 57-fold (IAUC=12159±3455nmol/L x hr) in accord with the increased serumconcentration of palmitic acid. Contrary to the lower

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Time after ingestion (hr)

Fatty acids (μmol/L)

Linoleic acid

Oleic acid

Palmitic acid

Stearic acid

Retinyl esters (nmol/L)

Retinyl linoleate

Retinyl oleate

Retinyl palmitate

Retinyl stearate

Retinyl ester/Fatty acid (molar ratio, x 105)

Retinyl linoleate/Lioleic acid

Retinyl oleate/Oleic acid

Retinyl palmitate/Palmitic acid

Retinyl stearate/Stearic acid

　　0

3264±596

2218±741

2997±718

861±122

19±21

35±14

46±13

21±6

0.6±0.8

1.8±1.2

1.6±0.6

2.5±0.9

　　1

3353±626

2276±788

3082±777

887±145

　22±16

　36±12

107±59

　36±15

0.6±0.5

1.8±0.8

3.5±1.7

4.2±1.9

　　2

3592±797

2894±1154

3834±1214

1013±218

　60±21

114±45

952±394

263±128

1.7±0.6

4.4±2.4

26.3±12.4

26.3±12.3

　　3　

3747±800

3388±1593

4397±1661

1102±258

94±53

217±114

1968±965

508±258

　2.4±1.1

　6.6±2.5

44.4±14.2

44.8±16.8

　　4

3577±729

2963±1056

3932±1182

1020±203

111±53

264±109

2424±935

592±222

　3.1±1.2

　9.4±4.1

62.2±18.9

58.4±21.3

　　6

3477±780

2945±1450

3906±1566

984±237

116±31

296±104

2627±1209

607±206

　3.4±0.8

11.0±3.5

67.5±20.5

62.3±20.6

　　8

3129±573

2064±761

2943±782

864±183

　47±13

　94±12

603±129

164±28

　1.5±0.3

　5.0±1.6

21.0±4.1

19.3±3.7

　 IAUC

1694±2065

4469±3495

5639±3807

930±800

474±219

1188±428

12159±3455

2917±717

　 13±7

　 40±10

311±44

288±61

The values are means±SD for seven healthy subjects. IAUC: incremental area under response curve.

Table 1 Concentrations of fatty acids and retinyl esters, and their molar ratios before and after ingestion of rapeseed oil and retinyl palmitate

Fig. 3 Re-esterification ratios (molar ratios) between concentrations of fatty acids and the correspondingretinyl esters recovered in serum from zero to 8 hours after ingestion of 37 g of rapeseed oil anda few drops of retinyl palmitate.■, retinyl palmitate/palmitic acid; ◆, retinyl linoleate/linoleic acid; ▲, retinyl oleate/oleic acid; ○, retinyl stearate/stearic acid.

increase of stearic acid (IAUC= 930±800μmol/L xhr) and the higher increase of oleic acid (IAUC=4469±3495μmol/L x hr), the corresponding increaseof retinyl ester was high in retinyl stearate (maximally,29-fold: IAUC= 2917±717 nmol/L x hr) and lower inretinyl oleate (maximally, 9-fold: IAUC=1188±428nmol/L x hr), respectively. The increase of retinyllinoleate was as low as 6-fold (IAUC=474±219nmol/L x hr).We calculated the re-esterification ratios (molar

ratios) between concentrations of fatty acids and theircorresponding retinyl esters recovered in serum afterthe ingestion of rapeseed oil and a retinyl palmitate(Table 1). A marked increase was observed in there-esterification ratios of retinyl palmitate to palmiticacid, and retinyl stearate to stearic acid (Fig. 3). Theratios of retinyl stearate to stearic acid were identicalnear to the same ratios of retinyl palmitate to palmiticacid. The re-esterification of fatty acids with retinolwas revealed to be low in both oleic and linoleicacids when compared with those of palmitic acid andstearic acid.

4. Discussion

In humans, the dietary sources of retinol wereretinyl esters and carotenoids, i.e., β-carotene. Retinylesters were hydrolyzed to retinol, which was re-ester-ified and packaged in the core of chylomicrons asretinyl ester (i.e., retinyl palmitate and retinyl stearate).Some of theβ-carotene was cleaved to form twomoles of retinal, which when formed was reduced toretinol, that was then taken up by chylomicrons also asretinyl esters12).It is natural to consider why saturated fatty acids

rather than unsaturated fatty acids were preferentiallyesterified with retinol. We hypothesize that retinylesters together with esterified cholesterol present in thecore of chylomicron remnants would be released intothe liver cells via the LDL receptor, wherein saturatedfatty acids regulate the LDL receptor pathway largelyat the mRNA level13). Once the cell has satisfied itsrequirement for retinol, saturated fatty acids inhibitreceptor activity, so that the receptor is no longerable to internalize retinyl esters. When the liver cells

need more retinol, the receptors again come intoplay14, 15). In response to cellular requirements, theliver releases retinol in the form of a retinol-RBP4complex bound to the transthyretin in circulation. Intarget cells, a cell-surface receptor for retinol-RBP4removes retinol from RBP4. Thus, we conclude thatsufficient intake of saturated fatty acids is required tomaintain the nutritional status of retinol. Althoughunsaturated fatty acids, i.e., oleic acid and linoleicacid (essential fatty acid) are needed by the humanbody, they may provide only a minor poor contributionto retinol absorption. While among animal speciesother than humans, retinyl stearate as a dominatingform of retinyl ester in circulation is unknown, but itappears to be among the essential physiological condi-tions in these animals.

AcknowledgmentsWe thank Dr. Yoshifumi Kurosaki, Department of

Clinical Chemistry, School of Allied Health Science,Kitasato University, for his helpful suggestions onthis manuscript.

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