Download Effects of lecithin in salad dressing on the plasma appearance of fat

Graduate Theses and Dissertations
Graduate College
2014
Effects of lecithin in salad dressing on the plasma
appearance of fat-soluble micronutrients consumed
in salads: contributions of chylomicrons and large
VLDL
Yinghui Zhang
Iowa State University
Follow this and additional works at: http://lib.dr.iastate.edu/etd
Part of the Human and Clinical Nutrition Commons
Recommended Citation
Zhang, Yinghui, "Effects of lecithin in salad dressing on the plasma appearance of fat-soluble micronutrients consumed in salads:
contributions of chylomicrons and large VLDL" (2014). Graduate Theses and Dissertations. Paper 13731.
This Dissertation is brought to you for free and open access by the Graduate College at Digital Repository @ Iowa State University. It has been accepted
for inclusion in Graduate Theses and Dissertations by an authorized administrator of Digital Repository @ Iowa State University. For more
information, please contact [email protected].
Effects of lecithin in salad dressing on the plasma appearance of fat-soluble
micronutrients consumed in salads: contributions of chylomicrons and large VLDL
by
Yinghui Zhang
A dissertation submitted to the graduate faculty
in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
Major: Nutritional Sciences
Program of Study Committee:
Wendy S. White, Major Professor
Donald C. Beitz
Stephanie Clark
Matthew Rowling
Chong Wang
Iowa State University
Ames, Iowa
2014
Copyright © Yinghui Zhang, 2014. All rights reserved.
ii
TABLE OF CONTENTS
ABSTRACT
CHAPTER I. GENERAL INTRODUCTION
iii
1
CHAPTER II. LITERATURE REVIEW
Physiological Functions of Carotenoids, Phylloquinone and Tocopherols
Mechanism of Carotenoid, Phylloquinone, and Tocopherol Absorption
Distribution of Carotenoids, Tocopherols, and Phylloquinone
in Plasma Lipoproteins
Factors Influencing the Bioavailability of Carotenoids, Tocopherols,
and Phylloquinone Absorbed from Vegetables
Apolipoprotein B and Apolipoprotein B-containing Lipoproteins
References
6
6
13
0
17
0
18
26
37
CHAPTER III. EFFECTS OF LECITHIN IN SALAD DRESSING
ON THE PLASMA APPEARANCE OF FAT-SOLUBLE
MICRONUTRIENTS CONSUMED IN SALADS:
CONTRIBUTIONS OF CHYLOMICRONS AND LARGE VLDLA
Abstract
Introduction
Subjects and Methods
Results
Discussion
References
59
60
61
63
75
80
90
CHAPTER IV. CONCLUSIONS AND PERSPECTIVES
104
APPENDIX
110
ACKNOWLEDGEMENTS
113
iii
ABSTRACT
Provitamin A carotenoids, tocopherols, and phylloquinone, as the major fat-soluble
micronutrients in salad vegetables, play essential roles in maintaining various physiological
processes, such as cell differentiation and proliferation, normal organogenesis, and blood
clotting. The non-provitamin A carotenoids, particularly lutein, zeaxanthin, and lycopene are
important for maintaining ocular health and preventing chronic diseases. The intestinal
uptake of fat-soluble micronutrients involves both simple diffusion and receptor-mediated
transport. Various exogenous factors are able to affect the uptake process, e.g. food matrix
and processing, dietary fat and fiber, and nutrient species and stereoisomers. Transferring
fat-soluble micronutrients into the blood circulation depends on the normal synthesis of
apolipoproteinB (apoB) and apoB-containing lipoprotein, which is promoted by the presence
of sufficient lipids and suppressed by insulin. Overall, dietary fat is an elementary factor
regulating the absorption of fat-soluble micronutrients.
Lecithin may influence the bioavailability of fat-soluble micronutrients. The role of
the large very-low-density lipoprotein (VLDLA) plasma fraction in postprandial fat-soluble
micronutrient transport is not clearly defined. Therefore, we conducted a human study to
investigate the effects of the lecithin/oil ratio in salad dressing on the absorption of: 1)
carotenoids, phylloquinone, and tocopherols from salad vegetables; 2) retinyl palmitate
formed in the intestine from the provitamin A carotenoids. An additional objective was to
investigate the origin of plasma chylomicrons and VLDLA and their roles in the transport of
the absorbed fat-soluble micronutrients. Healthy women (n = 12) each consumed three
salads with salad dressings containing: 1) 4 g soybean oil and 0 g hydroxylated soy lecithin
iv
(Solec® 8120, Solae, St. Louis, MO); 2) 3.8 g soybean oil and 0.2 g Solec® 8120; or 3) 3.2 g
of soybean oil and 0.8 g Solec® 8120. The order in which the salads were consumed was
randomly assigned according to a Williams Latin square design; salads were separated by ≥ 2
weeks. Blood was collected at baseline and 2, 3.5, 5, 7, and 9.5 h postprandially.
Chylomicrons and VLDLA fractions were analyzed by HPLC with coulometric array
electrochemical detection. ApoB-48 and apoB-100 contents in chylomicrons and VLDLA
were determined by ELISA. There were no significant differences among the salad dressings
in the resulting AUC values of the fat-soluble micronutrients in the chylomicron and VLDLA
fractions. The exception was a decrease in the AUC value of phylloquinone in the
chylomicron fraction when the salad dressing containing 0.8 g was compared with that
containing 0 g Solec® 8120 (P < 0.02). The AUC values for α-carotene, β-carotene,
lycopene, retinyl palmitate, and phylloquinone were substantially higher in the VLDLA than
in the chylomicron fraction (P < 0.05). Both chylomicron and VLDLA fractions contained
apoB-48 and apoB-100. ApoB-48 and apoB-100 were predominantly found in the VLDLA
fraction (P < 0.0001). The AUC value of apoB-100 in VLDLA was significantly higher than
that of apoB-48 in chylomicrons (P < 0.0001). We concluded that the hydroxylated soy
lecithin in the amounts added to the salad dressing did not enhance the absorption of
carotenoids, retinyl palmitate, phylloquinone, or tocopherols. The majority of the newly
absorbed carotenoids, retinyl palmitate, and phylloquinone, as well as enterogenous (apoB48) and hepatogenous (apoB-100) lipoprotein particles, were contained within large VLDL.
Thus this lipoprotein subfraction has a major role in the transport of newly absorbed
carotenoids and fat-soluble vitamins.
1
CHAPTER I.
GENERAL INTRODUCTION
Salad consumption, as a major contributor to vegetable consumption in the United States,
is associated with a greater likelihood of meeting the recommended intakes for various nutrients,
including carotenoids and vitamin E (Su & Arab, 2006). Although it has been reported that the
bioavailability of nutrients, including provitamin A carotenoids, is low in raw vegetables (Rock
et al., 1998), the absorption of fat-soluble micronutrients from vegetables is able to be improved
by increasing the fat content of a meal (Dimitrov et al., 1988; Gijsbers et al., 1996). We
previously reported that carotenoid absorption was higher after the consumption of salads with
full-fat than with reduced-fat salad dressing, whereas no absorption of carotenoids was observed
when salads were consumed with fat-free salad dressing (Brown et al., 2004). The consumption
of 150 g avocado or 24 g avocado oil with vegetable salads resulted in similarly enhanced αcarotene, β-carotene, and lutein absorption compared with the consumption of avocado/avocado
oil-free salads (Unlu et al., 2005). More recently, we found that the absorption of carotenoids,
tocopherols, and phylloquinone increased linearly with increasing amounts of co-consumed fat
from salad dressings (Agustiana et al., 2010). However, it is not recommended to have
excessively high fat intakes to achieve high bioavailability of fat-soluble micronutrients. To
further enhance the absorption efficiency, one strategy is to find a lipid that is more efficient than
triacylglycerol in increasing the bioavailability of fat-soluble micronutrients.
2
The intestinal absorption of fat-soluble micronutrients involves the release from food
matrix, emulsification with lipid droplets in the stomach, and incorporation into mixed micelles
(Yonekura et al., 2007). Lecithin, as a structural component of mixed micelles and an excellent
emulsifier, is likely to enhance the absorption of dietary lipids and lipophilic nutrients. Soybean
lecithin was to reported to increase the lymphatic absorption of triglyceride in rats (Nishimukai
et al., 2003). Higher concentration of curcumin, a hydrophobic constituent of the spice turmeric,
was observed in rat plasma and liver when curcumin was formulated with soybean lecithin
compared with unformulated curcumin (Marczylo et al., 2007). However, few human studies
have addressed the effects of the dietary lecithin on the bioavailability of fat-soluble
micronutrients.
The utility of postprandial appearance of β-carotene and retinyl palmitate in
chylomicrons and large very low density lipoproteins (VLDLA) as an indicator of intestinal βcarotene absorption have been studied well in humans (Hu et al., 2000; Paetau et al., 1997; Li et
al., 2010). In our previous studies, after subjects consumed a β-carotene dose with a fat-rich
meal, we observed postprandial appearances of β-carotene and retinyl palmitate in the plasma
chylomicron fraction coincided in time and magnitude with their appearance in VLDLA (Hu et
al., 2000; Paetau et al., 1997). Although it is known that the VLDLA fraction is a vehicle for
assessing intestinal absorption of β-carotene and its major bioconversion product, retinyl
palmitate (Hu et al., 2000; Paetau et al., 1997; Borel et al., 1997; Li et al., 2010), there has been
little systematic study of the role of VLDLA in the transport of other newly absorbed fat-soluble
3
micronutrients, such as α-carotene, lycopene, lutein, tocopherols, and phylloquinone. The
postprandial appearance of retinyl palmitate in VLDLA was hypothesized to be attributed to
intestinal VLDL (Borel et al., 1997), but could also reflect their appearance in chylomicron
remnants (Zheng et al., 2006). However, VLDLA have long been regarded as liver-derived
lipoproteins (Karpe et al., 1994; Brodsky et al., 2008). Apolipoprotein B-48 (apoB-48) and
apolipoprotein B-100 (apoB-100) are reliable biomarkers of lipoprotein particles of intestinal and
hepatic origin, respectively (Lemieus et al., 1998; Phillips et al., 1997; Chan J, 1992). Therefore,
an investigation of apoB-48 and apoB-100 in the plasma VLDLA fraction after a single meal can
provide new insight regarding the origin of VLDLA.
The objectives of the present study were to investigate the effects of the lecithin/oil ratio
in salad dressing on the absorption of carotenoids, phylloquinone, and tocopherols from salad
vegetables and and retinyl palmitate from intestinal metabolism of the provitamin A carotenoids.
An additional objective was to investigate the origin of VLDLA and the roles of plasma
chylomicrons and VLDLA in the transport of the absorbed fat-soluble micronutrients.
REFERENCES
Agustiana A, Zhou Y, Flendrig L, White WS. The dose-response effects of the amount of oil in
salad dressing on the bioavailability of carotenoids and fat-soluble vitamins in salad
vegetables. FASEB J 2010;539.
4
Borel P, Dubois C, Mekki N, Grolier P, Partier A, Alexandre-Gouabau MC, Lairon D, AzaisBraesco V. Dietary triglycerides, up to 40 g/meal, do not affect preformed vitamin A
bioavailability in humans, Eur J Clin Nutr 1997;51:717-722.
Brodsky JL, Fisher EA. The many intersecting pathways underlying apolipoprotein B secretion
and degradation. Trends Endocrinol Metab 2008;19:254-9.
Brown MJ, Ferruzzi MG, Nguyen ML, Copper DA, Eldridge AL, Schwartz SJ, White WS.
Carotenoid bioavailability is higher from salads ingested with full-fat than with fatreduced salad dressings as measured with electrochemical detection. Am J Clin Nutr
2004;80:396-403
Chan L. Apolipoprotein B, the major protein component of triglyceride-rich and low density
lipoproteins. J Biol Chem 1992;267:25621-4.
Dimitrov NV, Meyer C, Ullrey DE, Chenoweth W, Michelakis A, Malone W, Boone C, Fink G.
Bioavailability of beta-carotene in humans. Am J Clin Nutr 1988;48:298-304.
Karpe F, Hamsten A. Determination of apolipoproteins B-48 and B-100 in triglyceride-rich
lipoproteins by analytical SDS-PAGE. J Lipid Res 1994;35:1311-7.
Lemieux S, Fontani R, Uffelman KD, Lewis GF, Steiner G. Apolipoprotein B-48 and retinyl
palmitate are not equivalent markers of postprandial intestinal lipoproteins. J Lipid Res
1998;39:1964-71.
Li S, Nugroho A, Rocheford T, White WS. Vitamin A equivalence of the b-carotene in betacarotene–biofortified maize porridge consumed by women. Am J Clin Nutr
2010;92:1105-12.
Marczylo TH, Verschoyle RD, Cooke DN, Morazzoni P, Steward WP, Gescher AJ. Comparison
of systemic availability of curcumin with that of curcumin formulated with
phosphatidylcholine. Cancer Chemother Pharmacol 2007;60:171-7.
Nishimukai M, Hara H, Aoyama Y. Enteral administration of soyabean lecithin enhanced
lymphatic absorption of triacylglycerol in rats. Br J Nutr 2003;90:565-71.
Phillips ML, Pullinger C, Kroes I, Kroes J, Hardman DA, Chen G, Curtiss LK, Gutierrez MM,
Kane JP, Schumaker VN. A single copy of apolipoprotein B-48 is present on the human
chylomicron remnant. J Lipid Res 1997;38:1170-7.
5
Rock CL, Lovalvo JL, Emenhiser C, Ruffin MT, Flatt SW, Schwartz SJ. Bioavailability of betacarotene is lower in raw than in processed carrots and spinach in women. J Nutr
1998;128:913-6.
Su LJ, Arab L. Salad and raw vegetable consumption and nutritional status in the adult US
population: Results from the Third National Health and Nutrition Examination Survey. J
Am Diet Assoc 2006;106:1394-404
Unlu NZ, Bohn T, Clinton SK, Schwartz SJ. Carotenoid absorption from salad and salsa by
humans is enhanced by the addition of avocado or avocado oil. J Nutr 2005;135:431-6.
Yonekura L, Nagao A. Intestinal absorption of dietary carotenoids. Mol Nutr Food Res
2007;51:107-15.
Zheng C, Ikewaki K, Walsh BW, Sacks FM. Metabolism of apoB lipoproteins of intestinal and
hepatic origin during constant feeding of small amounts of fat. J Lipid Res 2006;47:17719.
6
CHAPTER II.
LITERATURE REVIEW
Physiological Functions of Carotenoids, Phylloquinone and Tocopherols
Carotenoids
Carotenoids are hydrophobic pigments synthesized by photosynthetic organisms. They
provide plants with deep yellow, orange and red colors. Other non-photosynthesizing organisms
only can obtain carotenoids by dietary intake. Carotenoid accumulations in tissues can pigment
animals such as birds and fish. Carotenoids can be divided into two categories: provitamin A
carotenoids and non-provitamin A, according to whether they can be converted into provitamin
A (von Lintig et al., 2010).
The provitamin A carotenoids include α-carotene, β-carotene and β-cryptoxanthin. βCarotene can be catalyzed by β-carotene 15,15'-monooxygenase 1 (BCMO 1) to from two retinal
molecules (von Lintig et al., 2010), whereas α-carotene and β-cryptoxanthin are only able to
generate a single retinal molecule because they contain one non-hydroxylated β-ring (Davis et
al., 2008). The retinal is subsequently reduced to retinol (vitamin A) by retinal reductase, and a
minor proportion of the retinal is irreversibly oxidized to retinoic acids by retinal
dehydrogenases. Sufficient vitamin A and its derivatives (retinal and retinoic acid) are
indispensible for normal immune system, cell differentiation, normal organogenesis, and eye
7
health (Sommer et al., 2012). Vitamin A deficiency, which is a widespread health problem in the
developing world, causes more than a million cases of death and blindness every year (Sommer
et al., 2012, Sommer et al., 1981). Increasing studies indicated that high consumption of
carotenoid-rich fruits and vegetables was linked with a decreased occurrence of cancers
(Thomson et al., 2013; Reiss et al., 2012; Takata et al., 2013; Jung et al., 2013). It had been
hypothesized that β-carotene might prevent cancer development. In contrast, supplementation
trials showed that supplementation with β-carotene increased, rather than decreased, incidence of
lung cancer in cigarette smokers (The ATBC Cancer Prevention Study Group, 1994; Albanes et
al., 1995; Omenn et al., 1996). However, recently, Pham et al. (2013) reported BCMO1, which
was regulated by β-carotene uptake, had capabilities of inhibiting invasiveness of colon cancer
cells and expressions of two neoplasia-related metalloproteinases (MMP7 and MMP28). In
several animal studies, α-carotene indicated higher tumorigenesis prevention capability than βcarotene in the liver, lung, colon, and skin (Murakoshi et al., 1992, Narisawa et al., 1996). βCryptoxanthin is rich in Satsuma mandarin orange. It has been shown that Satsuma mandarin
juices have a chemopreventive effect on chemically induced tumorigenesis in rat colon and lung
(Tanaka et al., 2000, Kohno et al., 2001). Iskandar et al. (2013) recently reported that nicotineinduced emphysema and lung tumor volume and multiplicity were reduced when mice were
dosed with β-cryptoxanthin supplement. Meanwhile, β-cryptoxanthin supplementation increased
expressions of tumor suppressors including sirtuin, p53, and retinoic acid recptor (RAR)-β
(Iskandar et al., 2013). Besides suppressing oncogenesis, β-cryptoxanthin has been
8
demonstrated to play a vital role in preventing bone loss by regulating various gene expressions
that are involved in osteoblastic bone formation and osteoclastic bone resorption. The dietary
intake of β-cryptoxanthin was associated with maintaining bone homeostasis in animal models as
well as in menopausal women (Yamaguchi et al., 2012).
The non-provitamin A carotenoids, particularly lutein, zeaxanthin, and lycopene are
important for maintaining many physiological processes (Sommer et al., 2012). Lutein and
zeaxanthin are selectively accumulated in the primate retina and form the macular pigment.
They are well-known xanthophylls because of their blue light filtering and antioxidant activities.
It has been shown that lutein and zeaxanthin may play vital roles in inhibiting various ocular
diseases, including age-related macular degeneration (AMD), photophobia, and cataracts
(Kalariya et al., 2012). In addition to maintaining ocular health, increasing studies suggest that
lutein has protective effects on immune system and inflammation. Lutein can prevent various
ocular inflammation induced by endotoxin, laser, and streptozotocin. In vitro studies indicate
that lutein is able to inhibit NF kappa-B activation and iNOS (inducible nitric oxide synthase)
and COX-2 (prostaglandin-endoperoxide synthase 2) expressions (Kijlstra et al., 2012).
Lycopene, which is abundant in tomatoes and processed tomato products, contains 11 conjugated
double bonds. Therefore, as one of most powerful antioxidant, its singlet-oxygen-quenching
ability is about two times higher than that of β-carotene and 10 times higher than that of αtocopherol (Di Mascio et al., 1989). Because of the antioxidant property, lycopene was
hypothesized to be able to prevent chronic diseases. Recent studies show lycopene may exert
9
suppressive effects on various chronic diseases, including cancer and cardiovascular disease by
reducing reactive oxygen species (ROS) levels (Giovannucci et al., 1999, Rao et al., 2002,
Palozza et al., 2010a, Palozza et al., 2010b). Various redox molecules were modulated by
lycopene such as antioxidant response elements (ARE), ROS-producing enzymes, smallGTPases, mitogen-activated protein kinases (MAPK), nuclear factor-κB (NF-κB), activator
protein-1 (AP-1), as well as redox-sensitive proteins including p53, the Bcl-2 family proteins,
and the Ku proteins (Palozza et al., 2012).
Tocopherols
Vitamin E, one of major fat-soluble antioxidants, is composed of eight compounds
including α-, β-, γ-, and δ-tocopherols, as well as α-, β-, γ-, and δ-tocotrienols. α- Tocopherol, as
the most potent form of vitamin E, constitutes more than 90% of vitamin E in the human body
(Gohil et al., 2008; Brigelius-Flohe et al., 1999; Burton et al., 1990), whereas γ-tocopherol is the
predominant form of vitamin E in the diet (Rigotti et al., 2007). The α-tocopherol synthesized by
plants only have one stereoisomer, RRR, while synthetic α-tocopherol, found in supplements and
fortified food, contains equimolar concentrations of 2R-stereoisomeric and 2S-stereoisomeric
forms, i.e. RRR- , SRR- , RSR- , SSR- , RRS- , SRS- , RSS- , and SSS-α-tocopherol. The
recommended daily allowance (RDA) for vitamin E is 15 mg per day for adult males and
females, which is supposed to maintain the plasma α-tocopherol concentration as 12 μmol/L
(Johnson et al., 2003). Novotny et al. (2012) recently report that 4 mg of RRR-α-tocopherol
10
intake can maintain the plasma RRR-α-tocopherol concentrations at 23 mmol/L, which suggest
the actual vitamin E dietary requirement may be less than the current. The α-tocopherol, as a a
peroxyl radical scavenger, protects erythrocytes from oxidative damage, which prevents
peroxide-induced hemolysis. Therefore, hemolysis-induced anemia is a common symptom in
patients with vitamin E deficiency (Niki et al., 2012). Moreover, vitamin E deficiency also can
cause muscular weakness, neurological dysfunction, and reproductive failure (Burton et al.,
1990; Kayden et al., 1993; Traber et al., 1996).
α-Tocopherol is significantly associated with cell proliferation and apoptosis by affecting
cell signaling and gene expression. These activities may relate to the specific distribution of αtocopherol in plasma membrane, i.e., it tends to incorporate into lipid rafts and associate with
alterations in raft-related signaling pathways. In vascular smooth muscle cells, α-tocopherol can
inhibit protein kinase activity by dephosphorylation (Boscoboinik et al., 1991; Ricciarelli et al.,
1998). Although the mechanism details have not been clarified, protein phosphatase 2A has
been shown to be involved in this process (Boudreau et al., 2002). α-Tocopherol also can
prevent the proliferation of vascular smooth muscle cells, which is not mainly caused by its
antioxidant property because β-tocopherol did not exert this inhibition effect (Tasinato et al.,
1995). Tasinato et al. (1995) repored that the antiproliferative effect was correlated with
inhibition of protein kinase activity via gene expression alteration. Moreover, α-tocopherol has
been found to protect various cell types of the vacular wall from cholesterol oxide-induced
11
mitochondrial dysfunction and apoptosis (Vejux et al., 2009; Royer et al., 2009; Miguet-Alfonsi
et al., 2002; Lizard et al., 2000).
Phylloquinone
Phylloquinone, also named vitamin K1, is the major form of vitamin K in the diet
synthesized by plants. Another dietary form of vitamin K is menaquinones (vitamin K2) which
are synthesized by bacteria. The best sources of phylloquinone are green vegetables since
phylloquinone is correlated with photosynthetic tissues (Shearer et al., 1996). The Adequate
Intake (AI) for vitamin K is 120 μg/day for men and 90 μg/day for women, while no RDA has
been established for phylloquinone (Institute of Medicine, 2001). Phylloquinone is an essential
cofactor of γ-carboxylase, an enzyme catalyzing the γ-carboxylation of glutamate residues in
proteins. This vitamin K-dependent γ-carboxylation has been found to be a post-translational
modification process and enables the proteins, referred to as vitamin K-dependent proteins, to
bind to calcium and interact with phospholipids, which are required by blood clotting and bone
mineralization (Shearer et al., 2009).
Four vitamin K-dependent plasma proteins are indispensible for blood clotting. They are
the coagulation cascade zymogens: factor II (prothrombin), factor VII, factor IX, and factor X,
which are all synthesized in the liver. These coagulation cascade zymogens catalyze the
formation of fibrin, an insoluble fiber network essentially required in blood clotting (Shearer et
al., 2009). On the other hand, three vitamin K-dependent plasma proteins are involved in
12
anticoagulation including protein C, protein S, and protein Z (Broze et al., 2001; Furie et al.,
1992). Warfarin, an anticoagulant, can inhibit the synthesis of vitamin K by interfering with the
reducing of oxidized vitamin K to the reducted vitamin K.
Osteocalcin (bone Gla protein) was the first extrahepatic vitamin K-dependent protein
found in bone. It is secreted by osteoblasts and contains three Gla residues, which facilitate the
binding of mineral hydroxyapatites (Booth et al., 2012). Knockout mice with insufficient
osteocalcin have been found to have increased bone density (Ducy et al., 1996). Matrix Gla
protein (MGP) is another vitamin K-dependent protein secreted by osteoblasts, as well as
chondrocytes and vascular smooth muscle cells. It is related to the organic matrix and calcium
mobilization. Extensive arterial calcification has been found in MGP dicifient mice, which
suggests MGP is an important calcification inhibitor (Luo et al., 1997). The inhibition activity
may be due to its binding to bone morphogenetic protein-2 (BMP-2) . BMP-2 is a growth factor
that converts preosteoblast into bone-forming cells (Abedin et al., 2004).
The vitamin K-dependent protein Gas6 has been shown to be associated with cellular
growth by binding to Axl receptor, a tyrosine-protein kinase receptor. Gas6 can activate Axl
tyrosine phosphorylation and enhace cell migration and growth. Li et al.(1996) reported that γcarboxylated Gas6 has growth factor effects on Schwann cells and smooth muscle cells in the
central nervous system, and lack of γ-carboxylated Gas6 may be associated with the
pathogenesis of Alzheimer’s disease.
13
Mechanism of Carotenoid, Phylloquinone, and Tocopherol Absorption
Absorption of fat-soluble micronutrients from vegetables into the blood circulation starts
with the mechanical and enzymatic break down of plant matrices by which carotenoids and fatsoluble vitamins are transferred from the food matrix into the lipid droplets in the stomach. The
carotenoids and fat-soluble vitamins are then transferred from the lipid droplets into self
assembled intestinal dietary mixed micelles. Micelles are composed of phospholipids, bile salts,
dietary lipids, and their hydrolysis products, including monoglycerides, diglycerides, and free
fatty acids. Bile salts, the salt form of bile acids, are synthesized in the liver and secreted by the
gallbllader. Bile salts and phospholipids exhibit amphipathic properties and reversibly form the
exterior of micelles at neutral pH, and lipophilic compounds such as monoglycerides,
diglycerides, free fatty acids, cholesterols, as well as fat-soluble micronutrients are incoporated
into micelle hydrophobic interior. After incorperations into micelles, the fat-soluble
micronutrients are uptaken by the enterocytes, incorporated into chylomicrons and transferred
into the blood circulation via the lymphatic system. Although the mechanism of fat-soluble
micronutrient aborption by the enterocytes is not completely understood, it has been considered
to invlove simple diffusion and receptor-mediated transport.
Simple diffusion
Carotenoid absorption has long been thought to occur by simple diffusion. In
unanesthetized rats, β-carotene absorption rate linearly responded to its concentration in the
14
perfusate from 0.5 to 11 μmol/L (Hollander et al., 1978). Also, the β-carotene absorption was
enhanced when simple diffusion was facilitated by increasing perfusate hydrogen ion
concentration and perfusate flow rate, as well as by adding different types of fatty acids
(Hollander et al., 1978). The concentration dependence of β-carotene uptake also has been found
in the rat small intestinal cells (Scita et al., 1992) and Caco-2 cells (Garrett et al., 1999). The
former showed no saturation up to 25 μmol/L β-carotene, and the latter indicated that β-carotene
content increased when culture medium contained 2 to 27 μmol/L β-carotene. Similarly,
intestinal absorption of tocopherols is also assumed to be by simple diffusion. Intestinal
perfusion studies in rats have shown significantly higher intestinal permeability for α-tocopherol
compared with γ-tocotrienol, which mainly contributes to the higher bioavailability of αtocopherol (Abuasal et al., 2012).
Receptor-mediated transport
Although intestinal absorption of fat-soluble micronutrients has been considered for a
long time as a passive process, it has recently been revealed that several lipid transporters are
involved in mediating carotenoid and fat-soluble vitamin intestinal uptake. The first identified
transporter is the scavenger receptor class B type I (SR-BI), which is a single-chain
trasmembrane glycoprotein expressed at the brush border membrane of enterocytes from the
duodenum to the colon (Lobo et al., 2001). SR-BI is able to bind lipoproteins and facilitate the
selective uptake of lipophilic compounds including cholesterol, phospholipids, and triglyceride
15
hydrolysis products (Terpstra et al., 2000; Hauser et al., 1998; Bietrix et al., 2006). It has been
identified that SR-BI plays a role in regulating the intestinal absorption of α-carotene, β-carotene,
lycopene, lutein, zeaxanthin, and vitamin E (van Bennekum et al., 2005; During et al., 2005;
Moussa et al., 2008; Reboul et al., 2005; Reboul et al., 2006).
Cluster determinant 36 (CD36) is another scavenger receptor involved in fat-soluble
micronutrient uptake. Similar to SR-BI, CD36 is a single chain-membrane glycoprotein found in
the brush border membrane of the duodenum and jejunum (Terpstra et al., 2000). It has been
reported that CD36 plays a key role in fatty acid uptake in the intestine, adipose tissue, and
muscle. In CD36-null mice, intestinal lipid secretion and catabolism rate of chylomicrons in the
blood were reduced after a high-fat intake (Drover et al., 2005). Although lipid secretion was
decreased, the accumulation of chylomicrons in the blood masked this phenomenon and resulted
in postprandial hypertriglyceridemia. It was assumed that CD36 regulated secretion and
clearance of intestinal lipoproteins. van Bennekum et al. (2005) firstly reported that CD36 was
associated with β-carotene uptake in transfected COS-7 cells and mouse brush border membrane
vesicles. Sakudoh et al. (2010) further showed that the selective delivery of carotenoid depended
on a CD36-related protein, Cameo2, in Bomyx mori. In addition, genetic variants in CD36 are
associated with plasma lutein content in humans (Borel et al., 2011). More recently, CD36 was
shown to facilitate the uptake of lycopene and lutein in mouse adipocytes and adipose tissue
cultures (Moussa et al., 2011).
16
Niemann-Pick C1-like1 (NPC1L1) is a polytopic protein expressed in various human
tissues, such as the liver and brush border membrane of the intestine (Davies et al., 2000;
Altmann et al., 2004; Davies et al., 2005; Garcia-Calvo et al., 2005). It is the major cholesterol
and phytosterol transporter in the intestine, which contributes 60% of dietary cholesterol uptake
(Altmann et al., 2004; Davies et al., 2005; Davis et al., 2004). NPC1L1 also has been shown to
interact with α-tocopherol and γ-tocotrienol and play a role in their absorption (Narushima et al.,
2008; Abuasal et al., 2010). Further NPC1L1 kinetic studies demonstrated significant lower Vmax
and Km values for γ-tocotrienol compared to α-tocopherol. The role of NPC1L1 in carotenoid
absorption is not completely clear (During et al., 2005). Ezetimibe, an inhibitor of cholesterol
transporter, was firstly shown to have an inhibition function in absorption of α-carotene, βcarotene, β-cryptoxanthin, lycopene, lutein, and zeaxanthin in Caco-2 cells (During et al., 2005).
However, the following study demonstrated neither ezetimibe nor an anti-human NPC1L1
antibody reduced lycopene uptake in Caco-2 cells (Moussa et al., 2008). Additional studies are
thus worthwhile to investigate these discrepancies.
On the basolateral side of enterocytes, another transmembrane transporter ATP-binding
cassette-A1 (ABCA1) is expressed. It medicates basolateral efflux of tocopherols. Besides
incorporating into chylomicrons, newly absorbed tocopherols also can be secreted in nascent
HDL. This process depends on ABCA1 (Reboul et al., 2009; Anwar et al., 2007). Reboul et al.
(2009) reported that no secretion of α-tocopherol in chylomicrons was observed when lipids are
absent, and Anwar et al. (2007) suggested tocopherols were mostly incorporated into HDL in the
17
absence of oleic acid. In humans, ABCA1 also is found in the retinal pigment epithelium. Its
gene variant is associated with retinal pigment optical density level response to lutein
supplementation (Yonova-Doing et al., 2013). In chicks that have a recessive mutation in
ABCA1 resulting in a severe HDL deficiency, the plasma lutein content was only 9% of control
level (Connor et al., 2007). It is very likely that ABCA1 may also be involved in lutein efflux
through enterocytes.
Distribution of Carotenoids, Tocopherols, and Phylloquinone in
Plasma Lipoproteins
In humans, carotenoids are mostly transported in the low-density lipoproteins (LDL) and
high-density lipoproteins (HDL). The hydrocarbon carotenoids are different from the
xanthophylls in the relative distribution between LDL and HDL. The former (e.g., α- and βcarotene) are mainly detected in LDL, whereas the latter (e.g., zeaxanthin and lutein), which are
more polar, are found in both HDL and LDL (Stahl et al., 2002). Recently, increasing studies
emphasized the importance of HDL in the transport and retinal uptake of lutein (Loane et al.,
2010; Mutungi et al., 2009; Wang et al., 2007). Moreover, lutein has been reported to exchange
between human very low-density lipoprotein (VLDL) and HDL, whereas no exchanges were
observed in α-carotene, β-carotene, and lycopene (Tyssandier et al., 2002).
The major carriers of tocopherols are HDL and LDL with similar proportions, and less
than 20% of tocopherols are carried in VLDL and other lipoproteins (Perugini et al., 2000). The
18
transfer of α-tocopherol between circulating lipoproteins occurs either spontaneously or with
assistance of plasma phospholipid transfer protein (PLTP) (Kostner et al., 1995). Spontaneous
transfer is slow and only can account for little portion of the high exchange/transfer rates that
have been demonstrated in vivo. PLTP has the ability to transfer various
amphipathic/hydrophobic compounds, including phospholipids, diacylglycerides, unesterified
cholesterol, and tocopherols, between lipoproteins in the blood circulation (Tzotzas et al., 2009).
Similar to carotenoids and tocopherols, phylloquinone is mainly distributed between LDL
and HDL with approximately equally proportion, and less amount of phylloquinone is carried by
IDL (Schurgers et al., 2002; Lamon-Fava et al., 1998).
Factors Influencing the Bioavailability of Carotenoids, Tocopherols and
Phylloquinone Absorbed from Vegetables
The bioavailability of fat-soluble micronutrients is influenced by multiple factors. These
factors can be classified as host-related factors or dietary factors (Yonekura et al., 2007). The
digestion and absorption of fat-soluble micronutrients are tightly associated with the health status
of individuals, which include gut health, nutritional and physiological status, lifestyle, and
genotype. This review is mainly focused on dietary factors that affect the bioavailability of fatsoluble micronutrients in vegetables.
19
Food matrix and food processing
Vegetables, especially green leafy vegetables, are the primary sources of fat-soluble
micronutrients, including carotenoids, tocopherols, and phylloquinone in the U.S diet
(Eitenmiller et al., 1995; Murphy et al., 1990; Maras et al., 2004). However, the bioavailability
of fat-soluble micronutrients is rather low in raw vegetables. One of the reasons is that the fatsoluble micronutrients embedded in the matrix of vegetables are more difficult to be released
compared with those in supplements. To improve their bioavailability, food processing, e.g.
heating and mechanical treatments, is often applied, which facilitates the release of fat-soluble
micronutrients from the food matrix by disrupting the plant cell wall and organelles. Rock et al.
(1998) reported that β-carotene plasma concentration was higher when women consumed
thermally processed and pureed carrots and spinach versus raw carrots and spinach. Similarly,
lycopene AUC (area under the curve) value in chylomicrons was significantly higher when
tomato paste was consumed compared with fresh tomatoes (Gärtner et al., 1997). Moreover,
food processing, including boiling, cooking, and microwaving, increased the phylloquinone
content in carrots, spinach, as well as tomatoes (Damon et al., 2004).
In the matrix of vegetables, carotenoids are located either in the photosynthetic organelles
(green leafy vegetables), or as part of semi-crystalline membrane-bounded solids (carrots and
tomatoes) (Castenmiller et al., 1998; van het Hof et al., 1998). The differences in food matrix
microenvironment are also critical to carotenoid absorption. It has been demonstrated that the
20
solubilization of β-carotene from raw carrot juice to oil phase was substantially higher than that
from raw spinach (Rich et al., 2003).
Dietary fat
Besides food processing, the absorption of fat-soluble micronutrients from vegetables is
also improved by increasing the fat content of a meal (Stahl, 2002). The fat intake is able to
stimulate bile and pancreatic enzyme secretion and facilitate micelle formation. Brown et al.
(2004) reported that no absorption of carotenoids was observed when salads were consumed with
fat free salad dressing, and the caroteniod absorption was higher after the consumption of salad
with full-fat (28 g canola oil) than with reduce-fat dressing (6 g canola oil). Likewise, serum
phylloquinone concentration was higher when boiled spinach was consumed with 25 g butter
versus without added fat (Gijsbers et al., 1996). In addition, it has been demonstrated that the
consumptions of vitamin E-fortified apples with low-fat (2.4 g fat) and regular-fat (11 g fat)
breakfasts increase the maximal plasma concentration and AUC value of α-tocopherol by 2- and
3-fold, respectively (Bruno et al., 2006). However, increasing fat content seems to affect the
bioavailability of different carotenoid species to a different extent, in particular, it has stronger
influence on the highly lipophilic carotenes (α-carotene, β-carotene, and lycopene) compared
with less lipophilic xanthophylls (e.g., lutein and zeaxanthin). The β-carotene and lutein,
released from the digesta of spinach puree containing 10% corn oil, showed no significant
difference in micellerization in an in vitro digestion model (Ferruzzi et al., 2001). However, in
21
the presence of relatively low amount of fat (2-3.5%), the micellerization of lutein extremely
exceeded that of β-carotene after an in vitro simulated digestion (Garrett et al., 1999;
Chitchumroonchokchai et al., 2004; Garrett et al., 2000). It suggests that, with insufficient
dietary fat, the micellerization of the more lipophilic carotenes is limited, whereas the less
lipophilic xanthophylls seem to be more freely released from the food matrix to oil phase and
mixed micelles.
Furthermore, unprocessed vegetable consumption seems to require more fat to achieve
the optimal absorption of fat-soluble micronutrients. As mentioned above, the chylomicron
carotenoid content was higher when salad vegetables were consumed with 28 g canola oil than
with either 6 g or 0 g canola oil (Brown et al., 2004). Similarly, the addition of avocado or
avocado oil significantly increased the absorption of α-carotene, β-carotene, and lutein from
salad and salsa (Unlu et al., 2005). In contrast, when α- and β-carotene supplements were
consumed with a meal containing 3 g or 36 g dietary fat, no significant differences in plasma αand β-carotene contents were observed between the low- and high-fat groups (Roodenburg et al.,
2000).
The fat composition also plays a role in the bioavailability of fat-soluble micronutrients.
Long-chain triglycerides are able to promote chylomicron formation, whereas medium-chain
triglycerides diminish chylomicron formation. Therefore, the presence of long-chain
triglycerides in both the first and second meals significantly enhanced carotenoid absorption
compared with the presence of medium-chain triglycerides (Borel et al., 1998). Several studies
22
also have shown the degree of unsaturation in fatty acids can affect carotenoid absorption. The
absorption of β-carotene was higher when a β-carotene supplement was ingested with beef tallow
than with sunflower oil (Hu et al., 2000). Also, micellar oleic, but not linoleic acid, have been
reported to enhance β-carotene absorption and its cleavage into retinol in rats (Raju et al., 2006).
Furthermore, Huo et al. (2007) reported that the efficiency of lutein uptake by Caco-2 cells was
significantly higher from that of micelles generated during the in vitro digestion of salad in the
presence of triolein (monounsaturated fatty acid) compared with trioctanoin (saturated fatty
acid), while the uptake of β-carotene, α-carotene and lycopene were not affected by the degree of
unsaturation of the fatty acids in the added triglycerides. Few human studies have investigated
the role of the degree of unsaturation of fatty acids in the absorption of lutein, α-carotene, and
lycopene. It is likely that the degree of unsaturation in fatty acids may play a role in the
bioavailability of carotenoids. According to the in vitro study of Huo et al. (2007), lutein may
have a different response to the degree of unsaturation of fatty acids in vivo as compared with carotene, α-carotene, and lycopene.
Phospholipids and other lipids
The absorption of fat-soluble micronutrients involves gastric oil phase and dietary mixed
micelles. Phospholipids, as one of the components of mixed micelles and an emulsifier for oil,
have been studied for their bioavailability enhancing effects. They commonly exist in both fresh
and processed food matrixes, especially phosphatidylcholine, which is also the major form of
23
phospholipids in bile. Under normal conditions, most dietary and biliary phosphatidylcholine is
hydrolyzed by the action of phopholipase A2 to produce lysophosphatidylcholine. It has been
shown that lysophosphatidylcholine, a common component in soy lecithin, can enhance the
bioavailability of β-carotene, lutein, and α-tocopherol in rats, mice, and Caco-2 human intestinal
cells (Lakshminarayana, 2006; Sugawara, 2001; Baskaran, 2003; Koo, 2001). Moreover, the
acyl chain length of phospholipids is also a critical factor influencing their effects on the
bioavailability of fat-soluble micronutrients (Yonekura, 2006). Yonekura et al. (2006) reported
that medium-chain phosphatidylcholine and medium-to-long-chain lysophosphatidylcholine
increased β-carotene uptake, whereas long-chain phosphatidylcholine inhibited and short-chain
phosphatidylcholine as well as short-to-medium-chain lysophosphatidylcholine had no effects on
the β-carotene uptake.
Sucrose polyesters are often used as nonabsorbable fat replacers because they cannot be
hydrolyzed by gastric and pancreatic lipases. They are able to partition fat-soluble
micronutrients from mixed micelles and consequently decrease the nutrient uptake (Weststrate
and van het Hof, 1995, Koonvitsky et al., 1997, Schlagheck et al., 1997). However, due to the
relatively less lipophilic nature of xanthophylls, their absorption is less inhibited by sucrose
polyesters when compared to β-carotene (Schlagheck et al., 1997; Broekmans et al., 2003).
Plant sterols and stanols have been reported to lower plasma cholesterol levels in humans
(Nguyen et al., 1999). However, they can compete with fat-soluble micronutrients for the
incorporation in mixed micelles. In the presence of β-sitosterol, β-carotene absorption was
24
diminished to approximately 50% in Caco-2 cells, and both of them were completely dissolved
(Fahy et al., 2004). Also, the plasma TRL β-carotene content was reduced by 50% when human
subjects consumed a single meal containing free or esterified plant sterols (Richelle, 2004).
Dietary fiber
Dietary fiber, especially soluble fiber, has been considered to have an inhibitory role in
the absorption of lipophilic compounds, such as cholesterol (Brown et al., 1999). Similarly, as
hydrophobic bioactive food components, the absorption of carotenoids was diminished by some
types of fibers in humans (Riedl et al., 1999; Rock et al., 1992), rats (Zanutto et al., 2002), chicks
(Erdman et al., 1986), and Mongolian gerbils (Deming et al., 2000). The mechanism of the
inhibitory effects of fiber on carotenoid absorption has not been completely elucidated. Such
mechanisms might include: 1) influencing the pancreatic enzyme activity and consequently
inhibiting micelle formation; 2) slowing the diffusion of micelles to the enterocytes by increasing
the viscosity and volume of the intestinal contents; 3) diminishing second meal effects by
altering enterocyte renewal (Riedl et al., 1999). On the other hand, in one human study,
volunteers were given a supplement containing carotenoids and α-tocopherol with or without
various types of fiber, and significantly lower AUC values for carotenoids were observed in the
fiber groups, whereas no differences were found for α-tocopherol AUC values (Riedl et al.,
1999). Animal studies suggested that the effects of dietary fiber on vitamin E absorption might
25
be apparent only if the fiber intake reached an “effective level” (De Lumen et al., 1982; Schaus
et al., 1985).
Species and stereoisomers of fat-soluble micronutrients
As mentioned above, dietary lipids do not affect all carotenoid species to the same
degree. Increasing the fat content of the meal efficiently enhances the absorption of the more
lipophilic carotenes, whereas the transfer of the less lipophilic xanthophylls from the oil phase to
micelles occurs much more easily in low-fat dietary conditions (Yonekura et al., 2007). Thus,
different carotenoid species may require different amounts and/or types of lipids for optimal
absorption. Within one carotenoid species, the bioavailabilty of different stereoisomers may be
different. The cis-isomer of lycopene has been shown to be more bioavailable than all-trans
lycopene, which may be due to the shorter length of the cis-isomer which facilitates
micellerization and uptake by intestinal cells (Boileau et al., 2002; Failla et al., 2008). However,
the preferential uptake of the all-trans β-carotene, rather than the cis-isomers, was observed in
several human studies (Gaziano et al., 1995; Stahl et al., 1993; You et al., 1996). Moreover, the
α-tocopherol constitutes more than 90% of vitamin E in the human body (Gohil et al., 2008;
Brigelius-Flohe et al., 1999; Burton et al., 1990), even though γ-tocopherol is the predominant
form of vitamin E in the diet (Rigotti et al., 2007). The limited bioavailability of γ-tocopherol is
caused by its low affinity of α-tocopherol transfer protein (α-TTP), which is only 9% compared
with RRR-α-tocopherol (Hosomi et al., 1997). Likewise, the high α-TTP affinity of RRR-α-
26
tocopherol also contributes to its 2-fold higher plasma concentration after a single dose or 8
consecutive daily doses when compared to all-rac-α-tocopherol (Burton et al., 1998).
Apolipoprotein B and Apolipoprotein B-containing Lipoproteins
Apolipoprotein B (apoB) is a large plasma protein that binds to the surface of lipoproteins
and plays an essential role in the assembly of triglyceride-rich lipoproteins. ApoB occurs in two
isoforms, apolipoprotein B-48 (apoB-48) and apolipoprotein B-100 (apoB-100). They are
encoded by the same gene on chromosome 2 (Welty et al., 1999; Veniant et al., 1999). In
humans, apoB-48 is secreted by the intestine as a result of a post-transcriptional mRNA editing
catalyzed by the apoB editing complex 1 (apoBEC-1). This enzyme converts codon 2153 to a
stop codon (UAA) that truncates the polypeptide at 48% of its full length. The full length apoB,
apoB-100, is primarily synthesized by the liver and contains 4536 amino acids.
Unlike all other apolipoproteins, apoB cannot exchange between lipoproteins, and it
remains with the apoB-containing lipoprotein through its entire metabolic process. Therefore,
apoB-48 and apoB-100 are regarded as reliable markers of intestinal and hepatic origin
lipoprotein particles, respectively (Phillips et al., 1997; Karpe et al., 1994; Chan et al., 1992).
ApoB-containing lipoproteins have long been referred to as chylomicrons and VLDL (Lemieus
et al., 1998; Karpe et al., 1994). Chylomicrons are lipoprotein particles of intestinal origin,
which transport newly absorbed lipids and fat-soluble nutrients. VLDL is synthesized by the
liver, and transports endogenous lipids. VLDL can be converted to intermediate-density
27
lipoproteins (IDL) and LDL by the action of lipoprotein lipase. However, data from cell culture,
rat liver perfusion, and human studies demonstrate that the liver has the capability of producing a
wide spectrum of apoB-100 containing lipoproteins from VLDL down to LDL (Marsh et al.,
1976). Recently, apoB-48 has also been found in lipoproteins with various sizes and densities,
including VLDL, IDL, and LDL (Campos et al., 2005; Cartwright et al., 2001; Zheng et al.,
2006; Johanson et al., 2004; Mero et al., 2000; Battula et al., 2000). By labeling apoB-100 and
apoB-48 with trideuterated leucine, Zhang et al. (2006) reported that the liver and intestine were
capable of secreting apoB-100- and apoB-48-containing lipoproteins, respectively, with a range
of sizes from chylomicron-sized down to IDL-sized particles. The production rate of apoB-100
in the liver was higher than that of apoB-48 in the intestine (Zheng et al., 2006). Among the
apoB-containing lipoproteins, this review will focus on chylomicrons and VLDL.
ApoB synthesis and apoB-containing lipoprotein assembly
As mentioned above, apoB transcriptional regulation is tissue-specific. In enterocytes of
all mammals, apoB-48 is synthesized as the 48% of apoB-100 fragment due to the action of
apoBEC-1. With the absence of apoBEC-1 in human liver, only apoB-100 is assembled into
VLDL. ApoB translation is mainly regulated by 5' and 3' untranslated regions (UTRs) of apoB
mRNA. In the 5' UTR of apoB mRNA, about 76% of the bases are either guanine or cytosine.
The GC-rich regions are able to form stable secondary structures (Kozak et al., 1991). The 3'
UTR of apoB mRNA is rich in adenine and uracil, and the AU-rich regions can also form
28
secondary structures that may play a role in the regulation of mRNA stability (Day and Tuite,
1998; Pontrelli et al., 2004). Several studies have investigated the effects of 5' and 3' UTRs in
the translation of apoB by binding the UTRs with a luciferase reporter gene or apoB15 cDNA
(Pontrelli et al., 2004; Sidiropoulos et al., 2005, 2007). The results suggested that the 5' UTRs
facilitated apoB translation, but, in contrast, the 3' UTR had an adverse impact on the apoB
translation by reducing the stability of apoB mRNA (Pontrelli et al., 2004; Sidiropoulos et al.,
2005, 2007). Furthermore, insulin can also negatively affect apoB translation by the action of
insulin-sensitive factor. The 110 kDa insulin-sensitive factor is able to bind the first 64
nucleotides of the 5' UTR that has been demonstrated to be the response region of apoB mRNA
to insulin, and the binding subsequently induces reduction in apoB translation (Sidiropoulos et
al., 2005, 2007). This process is assumed to be associated with phosphoinositide 3 kinase and
mTOR pathways, because the binding of the insulin-sensitive factor to the 5' UTR and the
insulin inhibition of apoB translation were diminished when HepG2 cells were treated with
either phosphoinositide 3 kinase inhibitor (wortmannin) or mTOR inhibitor (rapamycin)
(Sidiropoulos et al., 2007).
After translation of a signal peptide consisting of 24-27 amino acids, the nascent apoB
chain and ribosomes are transferred to the rough endoplasmic reticulum (ER) (Boerwinkle and
Chan, 1989). With sufficient lipid ligands, the nascent apoB is translocated into the ER lumen
via a proteinaceous channel, known as the translocon (Chen et al., 1998). The translocon is
primarily composed of the Sec61 protein and binds to ribosomes with high affinity (Alder et al.,
29
2004; Rapoport et al., 2007). Following translocation, apoB translation continues. During and
after translation, apoB is lipidated by the microsomal transfer protein (MTP) and subsequently
forms a pre-VLDL (Olofsson et al., 2000; Gordon et al., 2000; Shelness et al., 2005). MTP is a
membrane-associated heterodimer of protein disulfide isomerase (PDI) and a 97 kDa polypeptide
subunit that improves the delivery of phospholipids, cholesteryl esters, and triglyceride to apoB
(Hussain et al., 2003; Shoulders et al., 2005). Individuals with a mutation of the MTP gene have
abetalipoproteinmia, which is characterized by a deficiency of VLDL formation (Di Leo et al.,
2005). Although MTP plays an indispensable role in VLDL formation, a recent study implies
that MTP is not required for apoB translocation in McA-RH7777 cells (Dashti et al., 2007). In
addition to MTP, apoB co-translationally and post-translationally interacts with various
molecular chaperones, including cyclophilin, PDI, BiP (an Hsp70 chaperone), Grp94 (an Hsp90
chaperone), calnexin, and calreticulin (chaperone-like lectins) (Chen et al., 1998; Tatu et al.,
1997; Rashid et al., 2002; Stillemark et al., 2000; Zhang et al., 2003; Qiu et al., 2005). Most of
the molecular chaperones may be associated with apoB posttranslational modification or
degradation, or facilitate insertion of apoB into multiprotein complexes; however, the complete
mechanism of each chaperone effect on apoB is not clear (Brodsky et al., 2008).
Following the formation of pre-VLDL, the primordial lipoprotein particles are packed
and transported by coatomere Protein Ⅱ (COPⅡ) to the Golgi apparatus (Gurkan et al., 2006).
This process is stimulated by a GTPase known as Sar1. Mutations in Sar1 are responsible for
chylomicron retention disease, which is characterized by chylomicron deposition in intestinal
30
cells (Jones et al., 2003). Likewise, Sar1 also plays an essential role in apoB export from ER in
rat hepatoma McA-RH7777 cells (Gusarova et al., 2003). In the Golgi apparatus, the primordial
lipoprotein particles were further lipidated to achieve VLDL density, by which VLDL is formed.
The final VLDL lipidation is mediated by phospholipase D1 and extracellular signal regulated
kinase 2 (ERK2). They facilitate the formation of VLDL by increasing the production of
cytosolic lipid droplets that deliver additional lipids into the Golgi apparatus.
ApoB degradation
ER-associated degradation
ER-associated degradation (ERAD) is a cellular pathway that recognizes misfolded
secretory proteins in the ER and subsequently induces their ubiquitin-dependent degradation by
the proteasome (Brodsky et al., 2007). The ERAD is able to prevent the development of
cytotoxic aggregates caused by concentrated misfolded proteins. When the concentration of
misfolded proteins increases rapidly in the ER and exceeds the capability of ERAD, the unfolded
protein response (UPR) is activated by transmembrane UPR sensors (Schroder et al., 2005; Ron
et al., 2007). The UPR stimulates the enhancement of ER chaperone synthesis, ERAD
efficiency, as well as other degradation pathways. In addition to ER quality control, the ERAD
also has the ability to regulate metabolic processes. When lipid loading is limited, which may be
caused by low MTP activity or low lipid availability, apoB is targeted for ubiquitination and
31
subsequently degradation by the proteasome during translation process (Benoist et al., 1997;
Dixon et al., 1991; Zhou et al., 1998).
Polyunsaturated fatty acid-induced degradation
The apoB degradation can also happen after the ER exit of pre-VLDL, known as PERPP
(post-ER presecretory proteolysis). Dietary polyunsaturated fatty acids (PUFAs) are able to
trigger PERPP and then decrease VLDL levels (Fisher et al., 2001). In vitro and animal studies
have shown that in the presence of PUFAs, apoB is damaged by lipid peroxides, and aggregates
are formed and subsequently degraded by autophagy (Pan et al., 2004). This process most likely
occurs in the Golgi apparatus, because pre-VLDL requires further addition of lipids in the Golgi
in which VLDL may be exposed to PUFAs (Brodsky et al., 2008).
Insulin-induced degradation
Insulin, as an essential peptide hormone, is involved in lipid metabolism by inhibiting
hormone-sensitive lipase, promoting storage of triglycerides, as well as diminishing VLDL
production inthe liver. Acute increases in insulin reduce apoB synthesis and VLDL secretion in
HepG2 cells, rat and human hepatocytes, and rats (Patsch et al., 2004; Sparks et al., 1986;
Pullinger et al., 1989; Salhanick et al., 1991; Durrington et al., 1982; Chirieac et al., 2000).
These inhibitory effects of insulin primarily depend on PI3K (phosphatidylinositol 3-kinase)
activity (Phung et al., 1997). Insulin is able to activate PI3K by tyrosine phosphorylation of
32
insulin receptor substrates. The activated PI3K subsequently: 1) inactivates protein phosphatase
1B (PTP1B), which increases the expression of a cysteine protease ER60 and consequently
promotes apoB degradation; 2) suppresses the activities of FoxO1 and Foxa2, two transcription
activators of MTP, and then MTP expression is reduced; 3) decreases phosphatidylinositol
biphosphate (PIP2) that is responsible for activating phospholipase D1 and ARF-1, two factors
required in pre-VLDL formation (Vergès et al., 2010). The PUFAs-induced PERPP may also
play a role in the effects of insulin on the production of apoB-containing lipoproteins since it has
been observed that PUFA-rich diets increase insulin sensitivity (Riserus et al., 2008; Galgani et
al., 2008).
ApoB-100 synthesis in human intestine
It is universally known that human apoB-48 is primarily secreted by the intestine and
apoB-100 is the predominant isoform in human liver. However, the detection of apoB-100 in the
human intestine by using anapoB-100 cDNA probe (Knott et al., 1985) raises a question: Can the
human intestine synthesize apoB-100? Several in vitro studies reported Caco-2 cells were able
to produce apoB-100 (Hughes et al., 1987; Lee et al., 1988). A human intestinal organ culture
study indicated apoB-100 represented 16 ± 3% of the intestinal apoB mRNA, whereas apoB-100
only contributed 3-5% of newly synthesized intestinal apoB (Hoeg et al., 1990). The synthesis
of apoB-100 may be associated with age. Immunoprecipitates of 11-week fetal intestine showed
major radioactivity incorporation into apoB-100 and little incorporation into apoB-48. The
33
intestine from 16 week fetuses incorporated radioactivity into both apoB-100 and apoB-48,
whereas adult intestine immunoprecipitates only contained radioactivity in apoB-48 (Glickman
et al., 1986). Another immunoprecipitate study, in which intestinal biopsy samples were
obtained from 6 children aged 3 months to 8 years, reported radioactivity primarily in apoB-48,
but a relatively small amount was detected in apoB-100 (Levy et al., 1990). In the same study,
apoB-100 was found in thoracic duct lymph chylomicrons donated by a 5-year old boy with
severe hypertension (Levy et al., 1990). This finding was consistent with studies of Kane et al.
(1980) and Ruf et al. (1999), where apoB-100 presented in chylomicrons from thoracic duct
lymph samples obtained from five normolipidemic patients and a patient undergoing thoracic
surgery, respectively. However, hepatic lymph contributes about 25 to 50% of lymph flowing
through the thoracic duct (Ohtani et al., 2008). The detection of apoB-100 in thoracic duct
lymph might be caused by contamination of hepatic apoB-100-containing large VLDL presented
in the thoracic duct lymph and being collected with chylomicrons. To examine this possibility,
Lock et al. (1983) investigated apoB isoforms in chylomicrons isolated from a male patient with
chyluria. Chyluria is a medical disorder with symptoms of chyle urine and lymph channel
blocking. The apolipoprotein composition in urine chylomicrons was determined by using
sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with Coomassie Blue
staining. Lock et al (1983) concluded that the apoB-48 is virtually the only apoB in
chylomicrons. However, a weak band in the apoB-100 size position was observed in urine
chylomicrons isolated after an overnight fasting. Although the intestine has the ability to
34
synthesize apoB-100, it tends to choose apoB-48 to form chylomicrons. Mice with apobec-1
gene knock out, whose intestine secreted only apoB-100, had a reduction of chylomicron
secretion into the lymphatic system and triglyceride accumulation in the enterocytes after being
fed a large dose of lipids (Lo et al., 2008). The synthesis rate of apoB-100 in human intestine
still needs to be quantified, and whether it is regulated by diet and insulin is not clear.
Factors affecting apoB secretion and apoB-containing lipoprotein assembly
Dietary fat
As mentioned above, dietary PUFAs (polyunsaturated fatty acids), particularly the n-3
fatty acids enriched in fish oil, are able to reduce VLDL levels by PUFAs-induced post ER
nonproteasomal degradation. A VLDL apoB-100 kinetics study reported that triglyceride level
was decreased in 10 normal male subjects after consuming a daily fish oil supplement for four
weeks. The triglyceride reduction is due to a decreased production rate of VLDL apoB-100
(Bordin et al., 1998). In addition, dietary fat may affect VLDL particle size (Stacpoole et al.,
1991). When normal subjects consumed very low fat diets, more apoB-100 was secreted with
VLDL/IDL-like particles, whereas high fat diets increased larger VLDL particle secretion. Also,
the fractional clearance rate of VLDL/IDL was increased in both normal and familial
hypercholesterolemic subjects after they consumed a very low fat diet.
35
Aging
Dietary fat is not the only factor regulating apoB and apoB-containing lipoprotein
secretion. The magnitude of apoB-100 in VLDL, IDL, and LDL has been positively correlated
with age in normolipidemic male subjects (Millar et al., 1995). ApoB-100 kinetic analyses also
revealed that aging was linked with an increased production of VLDL and a decreased fractional
clearance rate of apoB-100 in IDL and LDL in the fed state. This is supported by an earlier
study with radiolabeled LDL apoB, which reported that the LDL apoB fractional catabolic rate
was higher in young male subjects (20-39 y) compared withold male subjects (60-80 y)
(Ericsson, 1991). Besides aging, postmenopausal status has been shown to result in greater
postprandial lipid responses in women (Cohn et al., 1988; van Beek et al., 1999; Masding et al.,
2003). In sequential dietary studies recently conducted at Reading University, postprandial data
from 98 healthy UK female adults were pooled and analyzed (Jackson et al., 2008). The
investigators reported that triglyceride absorption and maximal triglyceride concentration were
augmented in postmenopausal women, and the most evident difference in triglyceride absorption
was observed between the younger and the older premenopausal women. Normal apoB secretion
depends on the availability of lipids; therefore, it could be hypothesized that menopausal status
might affect apoB and apoB-containing lipoprotein metabolism by regulating the postprandial
lipid responses.
36
Gender
In several kinetic studies, a gender difference in lipoprotein metabolism was noted.
Women have been shown to have higher triglyceride-rich lipoprotein and LDL-apoB fractional
catabolic rates than men, whereas no significant differences are found in production rates
(Matthan et al., 2008; Watts et al., 2000). In addition, gender differences observed in younger
subjects were more significant than in older subjects (Stanhope et al., 2011; 2009). The younger
male adults exhibited greater fasting triglyceride and apoB responses after 2 weeks of sugar
consumption, whereas the responses in postprandial triglyceride and apoB were comparable
between the genders (Stanhope et al., 2011). It was suggested that the gender difference in
lipoprotein metabolism may reflect VLDL clearance rate rather than its secretion rate.
ApoB and apoB-containing lipoprotein kinetics
Kinetic studies of apoB-containing lipoproteins began with chylomicrons by labeling the
triglyceride component. Chylomicron triglyceride was found to be cleared from the blood within
minutes in healthy subjects, whereas the clearance rate was relatively slow in subjects with
coronary heart disease (Nestel et al., 1964; Grundy et al., 1976). By labeling chylomicrons with
either retinyl palmitate or iodinated protein components, subsequent studies reported the halftime of chylomicrons in blood was as short as 15-30 min (Berr et al., 1984; Cortner et al., 1987;
Karpe et al., 1997; Schaefer et al., 1978; Stalenhoef et al., 1984; Schaefer et al., 1986).
Although, in the early stages of kinetic investigations, radioiodinated apoB-48 and apoB-100
37
were shown to be depleted within 15 min and 30 min, respectively, in normal subjects
(Stalenhoef et al., 1984), the apoB-48 and apoB-100 were radiolabeled in vitro after isolation
from plasma. To avoid radiation hazards and exogenous labeling, amino acid precursors labeled
with stable isotopes are now widely used. When apoB-48 and apoB-100 were endogenously
labeled with stable isotopes, the catabolism of chylomicrons was found to be significantly slower
than it had been assumed. Several studies reported that apoB-48 had similar fractional turnover
rate to apoB-100 in triglyceride-rich lipoproteins, with an average fractional catabolic rate (FCR)
of four to six per day and an average residence time of 4-6 h (Lichtenstein et al., 1992; Welty et
al., 1999; Welty et al., 2000; Batista et al., 2004; Welty et al., 2004;). The FCR difference was
observed in the two major VLDL subfractions (large and small VLDL); i.e., the FCR of large
VLDL is significantly faster than that of small VLDL, but no consistent differences were found
in the production rate (PR) between the two subfractions (Marsh et al., 2002). Moreover, the
postprandial PR of apoB-100 in VLDL is about twice as great as that in the fasting condition
(Marsh et al., 2002).
REFERENCES
Abedin M, Tintut Y, Demer LL. Vascular calcification: mechanisms and clinical ramifications.
Arterioscler Thromb Vasc Biol 2004;24:1161-70.
Abuasal B, Sylvester PW, Kaddoumi A. Intestinal absorption of gamma-tocotrienol is mediated
by Niemann–Pick C1-like 1: in situ rat intestinal perfusion studies. Drug Metab Dispos
2010;38:939-45.
38
Abuasal BS, Qosa H, Sylvester PW, Kaddoumi A. Comparison of the intestinal absorption and
bioavailability of γ-tocotrienol and α-tocopherol: in vitro, in situ and in vivo studies.
Biopharm Drug Dispos 2012;33:246-56.
Albanes D, Heinonen OP, Huttunen JK, Taylor PR, Virtamo J, Edwards BK, Haapakoski J,
Rautalahti M, Hartman AM, Palmgren J, Greenwald P. Effects of alpha-tocopherol amid
beta-carotene supplements on cancer incidence in the Alpha-Tocopherol. Beta-Carotene
Cancer Prevention Study. Am J Clin Nutr 1995;62:l427-30.
Alder NN, Johnson AE. Cotranslational membrane protein biogenesis at the endoplasmic
reticulum. J Biol Chem 2004;279:22787-90.
Altmann SW, Davis HR Jr, Zhu LJ, Yao X, Hoos LM, Tetzloff G, Iyer SP, Maguire M, Golovko
A, Zeng M, Wang L, Murgolo N, Graziano MP. Niemann– Pick C1 Like 1 protein is
critical for intestinal cholesterol absorption. Science 2004;303:1201-4.
Anwar K, Iqbal J, Hussain MM. Mechanisms involved in vitamin E transport by primary
enterocytes and in vivo absorption. J Lipid Res 2007;48:2028-38.
Batista MC, Welty FK, Diffenderfer MR, Sarnak MJ, Schaefer EJ, Lamon-Fava S, Asztalos BF,
Dolnikowski GG, Brousseau ME, Marsh JB. Apolipoprotein A-I, B-100, and B-48
metabolism in subjects with chronic kidney disease, obesity, and the metabolic syndrome.
Metabolism 2004;53:1255-61.
Benoist F, Grand-Perret T. Co-translational degradation of apolipoprotein B100 by the
proteasome is prevented by microsomal triglyceride transfer protein. Synchronized
translation studies on HepG2 cells treated with an inhibitor of microsomal triglyceride
transfer protein. J Biol Chem 1997;272:20435-42.
Berr F, Kern F Jr. Plasma clearance of chylomicrons labeled with retinyl palmitate in healthy
human subjects. J Lipid Res 1984;25:805-12.
Bietrix F, Yan D, Nauze M, Rolland C, Bertrand-Michel J, Coméra C, Schaak S, Barbaras R,
Groen AK, Perret B, Tercé F, Collet X. Accelerated lipid absorption in mice
overexpressing intestinal SR-BI. J Biol Chem 2006;281:7214-9.
Boerwinkle E, Chan L. A three codon insertion/deletion polymorphism in the signal peptide
region of the human apolipoprotein B (APOB) gene directly typed by the polymerase
chain reaction. Nucleic Acids Res 1989;17:4003.
39
Boileau TW, Boileau AC, Erdman JW Jr. Bioavailability of all-trans and cis-isomers of
lycopene. Exp Biol Med (Maywood) 2002;227:914-9.
Bordin P, Bodamer OA, Venkatesan S, Gray RM, Bannister PFA, Halliday D. Effects of fish oil
supplementation on apolipoprotein B 100 production and lipoprotein metabolism in
normolipidaemic males. Eur J Clin Nutr 1998;52:104-9.
Borel P, de Edelenyi FS, Vincent-Baudry S, Malezet-Desmoulin C, Margotat A, Lyan B,
Gorrand JM, Meunier N, Drouault-Holowacz S, Bieuvelet S. Genetic variants in BCMO1
and CD36 are associated with plasma lutein concentrations and macular pigment optical
density in humans. Ann Med 2011;43:47-59.
Borel P, Tyssandier V, Mekki N, Grolier P, Rochette Y, Alexandre-Gouabau MC, Lairon D,
Azaïs-Braesco V. Chylomicron β-carotene and retinylpalmitate responses are
dramatically diminished when men ingest β-carotene with medium-chain rather than
long-chain triglycerides. J Nutr 1998;128:1361-7.
Boscoboinik D, Szewczyk A, Hensey C, Azzi A. Inhibition of cell proliferation by α-tocopherol.
Role of protein kinase C. J. Biol. Chem 1991;266:6188-94.
Boudreau RT, Garduno R, Lin TJ. Protein phosphatase 2A and protein kinase Ca are physically
associated and are involved in Pseudomonas aeruginosa-induced interleukin 6 production
by mast cells. J Biol Chem 2002;277:5322-9.
Brigelius-Flohé R, Traber MG. Vitamin E: function and metabolism. FASEB J 1999;13:1145-55.
Brodsky JL, Fisher EA. The many intersecting pathways underlying apolipoprotein B secretion
and degradation. Trends Endocrinol Metab 2008;19:254-9.
Brodsky JL. The protective and destructive roles played by molecular chaperones during ERAD
(ER associated degradation). Biochem. J 2007;404:353-63.
Broekmans WM, Klöpping-Ketelaars IA, Weststrate JA, Tijburg LB, van Poppel G, Vink AA,
Berendschot TT, Bots ML, Castenmiller WA, Kardinaal AF. Decreased carotenoid
concentrations due to dietary sucrose polyesters do not affect possible markers of disease
risk in humans. J Nutr 2003;133:720-6.
Brown L, Rosner B, Willett WW, Sacks FM. Cholesterol-lowering effects of dietary fiber: a
meta-analysis. Am J Clin Nutr 1999;69:30-42.
40
Brown MJ, Ferruzzi MG, Nguyen ML, Copper DA, Eldridge AL, Schwartz SJ, White WS.
Carotenoid bioavailability is higher from salads ingested with full-fat than with fatreduced salad dressings as measured with electrochemical detection. Am J Clin Nutr
2004;80:396-403
Broze GJ Jr. Protein Z-dependent regulation of coagulation. Thromb Haemost 2001;86:8-13.
Bruno RS, Leonard SW, Park SI, Zhao Y, Traber MG. Human vitamin E requirements assessed
with the use of apples fortified with deuterium-labeled alpha-tocopheryl acetate. Am J
Clin Nutr 2006;83:299-304.
Burton GW, Traber MG, Acuff RV, Walters DN, Kayden H, Hughes L, Ingold KU. Human
plasma and tissue alpha-tocopherol concentrations in response to supplementation with
deuterated natural and synthetic vitamin E. Am J Clin Nutr 1998;67:669-84.
Burton GW, Traber MG. Vitamin E: antioxidant activity, biokinetics and bioavailability. Annu
Rev Nutr 1990;10:357-382.
Burton GW, Traber MG. Vitamin E: antioxidant activity, biokinetics, and bioavailability. Annu
Rev Nutr 1990;10:357-82.
Castenmiller JJ, West CE. Bioavailability and bioconversion of carotenoids. Annu Rev Nutr
1998;18:19-38.
Chen Y, Le Cahérec F, Chuck SL. Calnexin and other factors that alter translocation affect the
rapid binding of ubiquitin to apoB in the Sec61 complex. J Biol Chem 1998;273:1188794.
Chen Y, Le Cahérec F, Chuck SL. Calnexin and other factors that alter translocation affect the
rapid binding of ubiquitin to apoB in the Sec61 complex. J Biol Chem 1998;273:1188794
Chirieac DV, Chirieac LR, Corsetti JP, Cianci J, Sparks CE, Sparks JD. Glucose-stimulated
insulin secretion suppresses hepatic triglyceride-rich lipoprotein and apoB production.
Am J Physiol Endocrinol Metab 2000;279:1003-11.
Chitchumroonchokchai C, Schwartz SJ, Failla ML. Assessment of lutein bioavailability from
meals and a supplement using simulated digestion and Caco-2 human intestinal cells. J
Nutr 2004;134:2280-6.
41
Cohn JS, McNamra JR, Cohn SD, Ordovas JM, Schaefer EJ. Postprandial plasma lipoprotein
changes in human subjects of different ages. J Lipid Res 1988;29:469-79.
Connor WE, Duell PB, Kean R, Wang Y. The prime role of HDL to transport lutein into the
retina: evidence from HDL-deficient WHAM chicks having a mutant ABCA1
transporter. Invest Ophthalmol Vis Sci 2007;48:4226-31.
Cortner JA, Coates PM, Le NA, Cryer DR, Ragni MC, Faulkner A, Langer T. Kinetics of
chylomicron remnant clearance in normal and in hyperlipoproteinemic subjects. J Lipid
Res 1987;28:195-206.
Damon M, Zhang NZ, Haytowitz DB, Booth SL. Phylloquinone (vitamin K1) content of
vegetables. J Food Comp Anal 2005;18:751-8.
Dashti N, Manchekar M, Liu Y, Sun Z, Segrest JP. Microsomal triglyceride transfer protein
activity is not required for the initiation of apolipoprotein B-containing lipoprotein
assembly in McA-RH7777 cells. J Biol Chem 2007;282:28597-608.
Davies JP, Levy B, Ioannou YA. Evidence for a Niemann–Pick C (NPC) gene family:
identification and characterization of NPC1L1. Genomics 2000;65:137-45.
Davies JP, Scott C, Oishi K, Liapis A, Ioannou YA. Inactivation of NPC1L1 causes multiple
lipid transport defects and protects against diet-induced hypercholesterolemia. J Biol
Chem 2005;280:12710-20.
Davis CR, Howe JA, Rocheford TR, Tanumihardjo SA. The xanthophyll composition of
biofortified maize (Zea mays Sp.) does not influence the bioefficacy of provitamin A
carotenoids in Mongolian gerbils (Meriones unguiculatus). J Agric Food Chem
2008;56:6745-50.
Davis HR Jr, Zhu LJ, Hoos LM, Tetzloff G, Maguire M, Liu J, Yao X, Iyer SP, Lam MH, Lund
EG, Detmers PA, Graziano MP, Altmann SW. Niemann–Pick C1 Like 1 (NPC1L1) is the
intestinal phytosterol and cholesterol transporter and a key modulator of whole-body
cholesterol homeostasis. J Biol Chem 2004;279:33586-92.
Day DA, Tuite MF. Post-transcriptional gene regulatory mechanisms in eukaryotes: an overview.
J Endocrinol 1998;157:361-71.
de Lumen, B, Lubin B, Chiu D, Reyes PS, Omaye ST. Bioavailability of vitamin E in rats fed
diets containing pectin. Nutr Res 1982;2:73-83.
42
Deming DM, Boileau AC, Lee CM, Erdman JW Jr. Amount of dietary fat and type of soluble
fiber independently modulate postabsorptive conversion of beta-carotene to vitamin A in
mongolian gerbils. J Nutr 2000;130:2789-96.
Di Leo E, Lancellotti S, Penacchioni JY, Cefalù AB, Averna M, Pisciotta L, Bertolini S,
Calandra S, Gabelli C, Tarugi P. Mutations in MTP gene in abeta and hypobetalipoproteinemia. Atherosclerosis 2005;180:311-8.
Di Mascio P, Kaiser S, Sies H. Lycopene as the most efficient biological carotenoid singlet
oxygen quencher. Arch Biochem Biophys 1989;274:532-8.
Dixon JL, Furukawa S, Ginsberg HN. Oleate stimulates secretion of apolipoprotein Bcontaining
lipoproteins from HepG2 cells by inhibiting early intracellular degradation of
apolipoprotein B. J Biol Chem 1991;266:5080-6.
Drover VA, Ajmal M, Nassir F, Davidson NO, Nauli AM, Sahoo D, Tso P, Abumrad NA. CD36
deficiency impairs intestinal lipid secretion and clearance of chylomicrons from the
blood. J Clin Invest 2005;115:1290-7.
Ducy P, Desbois C, Boyce B, Pinero G, Story B, Dunstan C, Smith E, Bonadio J, Goldstein S,
Gundberg C, Bradley A, Karsenty G. Increased bone formation in osteocalcin-deficient
mice. Nature 1996;382:448-52.
During A, Dawson HD, Harrison EH. Carotenoid transport is decreased, expression of the lipid
transporters SR-BI, NPC1L1, and ABCA1 is downregulated in Caco-2 cells treated with
ezetimibe. J Nutr 2005;135:2305-12.
Durrington PN, Newton RS, Weinstein DB, Steinberg D. Effects of insulin and glucose on very
low density lipoprotein triglyceride secretion by cultured rat hepatocytes. J Clin Invest
1982;70:63-73.
Eitenmiller RR, Lee J. Vitamin E. Food chemistry, composition and analysis. New York, NY:
Marcel Dekker, Inc, 2004.
Erdman JW Jr, Fahey GC Jr, White CB. Effects of purified dietary fiber sources on beta-carotene
utilization by the chick. J Nutr 1986;116:2415-23.
Ericsson S, Eriksson M, Vitols S, Einarsson K, Berglund L, Angelin B. Influence of age on the
metabolism of plasma low density lipoproteins in healthy males. J Clin Invest
1991;87:591-6.
43
Fahy DM, O'Callaghan YC, O'Brien NM. Phytosterols: lack of cytotoxicity but interference with
beta-carotene uptake in Caco-2 cells in culture. Food Addit Contam 2004;21:42-51.
Failla ML, Huo T, Thakkar SK. In vitro screening of relative bioaccessibility of carotenoids from
foods. Asian Pacific J Clin Nutr 2008;17:200-3.
Ferruzzi MG, Failla ML, Schwartz SJ. Assessment of degradation and intestinal cell uptake of
carotenoids and chlorophyll derivatives from spinach puree using an in vitro digestion
and Caco-2 human cell model. J Agric Food Chem 2001;49:2082-9.
Fisher EA, Pan M, Chen X, Wu X, Wang H, Jamil H, Sparks JD, Williams KJ. The triple threat
to nascent apolipoprotein B. Evidence for multiple, distinct degradative pathways. J Biol
Chem 2001;276:27855-63.
Fu X, Moreines J, Booth SL. Vitamin K supplementation does not prevent bone loss in
ovariectomized Norway rats. Nutr Metab (Lond) 2012;20:9-12.
Furie B, Furie BC. Molecular and cellular biology of blood coagulation. N Engl J Med
1992;326:800-6.
Galgani JE, Uauy RD, Aguirre CA, Díaz EO. Effect of the dietary fat quality on insulin
sensitivity. Br. J Nutr 2008;100:471-9.
Garcia-Calvo M, Lisnock J, Bull HG, Hawes BE, Burnett DA, Braun MP, Crona JH, Davis HR
Jr, Dean DC, Detmers PA, Graziano MP, Hughes M, Macintyre DE, Ogawa A, O'neill
KA, Iyer SP, Shevell DE, Smith MM, Tang YS, Makarewicz AM, Ujjainwalla F,
Altmann SW, Chapman KT, Thornberry NA. The target of ezetimibe is Niemann–Pick
C1-Like 1 (NPC1L1). Proc Natl Acad Sci USA 2005;102:8132-7.
Garrett DA, Failla ML, Sarama RJ, Craft N. Accumulation and retention of micellar betacarotene and lutein by Caco-2 human intestinal cells. J Nutr Biochem 1999;10:573-81.
Garrett DA, Failla ML, Sarama RJ. Development of an in vitro digestion method to assess
carotenoid bioavailability from meals. J Agric Food Chem 1999;47:4301-9.
Garrett DA, Failla ML, Sarama RJ. Estimation of carotenoid bioavailability from fresh stir-fried
vegetables using an in vitro digestion/Caco-2 cell culture model. J Nutr Biochem
2000;11:574-80.
44
Gärtner C, Stahl W, Sies H. Lycopene is more bioavailable from tomato paste than from fresh
tomatoes. Am J Clin Nutr 1997;66:116-22.
Gaziano JM, Johnson EJ, Russell RM, Manson JE, Stampfer MJ, Ridker PM, Frei B, Hennekens
CH, Krinsky NI. Discrimination in absorption or transport of beta-carotene isomers after
oral supplementation with either all-trans- or 9-cis-beta-carotene. Am J Clin Nutr
1995;61:1248-1252.
Gijsbers BL, Jie KS, Vermeer C. Effect of food composition on vitamin K absorption in human
volunteers. Br J Nutr 1996;76:223-9.
Giovannucci E. Tomatoes, tomato-based products, lycopene, and cancer: review of the
epidemiologic literature. J Natl Cancer Inst 1999;91:317-31.
Glickman RM, Rogers M, Glickman JN. Apolipoprotein B synthesis by human liver and
intestine in vitro. Proc Natl Acad Sci 1986;83:5296-300.
Gohil K, Oommen S, Quach HT, Vasu VT, Aung HH, Schock B, Cross CE, Vatassery GT. Mice
lacking alpha-tocopherol transfer protein gene have severe alpha-tocopherol deficiency in
multiple regions of the central nervous system. Brain Res 2008;1201:167-76.
Gordon DA, Jamil H. Progress towards understanding the role of microsomal triglyceride
transfer protein in apolipoprotein-B lipoprotein assembly. Biochim Biophys Acta
2000;1486:72-83.
Grundy SM, Mok HY. Chylomicron clearance in normal and hyperlipidemic man. Metabolism
1976;25:1225-39.
Gürkan C, Stagg SM, Lapointe P, Balch WE. The COPII cage: unifying principles of vesicle
coat assembly. Nat Rev Mol Cell Biol 2006;7:727-38.
Gusarova V, Brodsky JL, Fisher EA. (2003) Apolipoprotein B100 exit from the endoplasmic
reticulum (ER) is COPII-dependent, and its lipidation to very low density lipoprotein
occurs post-ER. J Biol Chem 2003;278:48051-8.
Hauser H, Dyer JH, Nandy A, Vega MA, Werder M, Bieliauskaite E, Weber FE, Compassi S,
Gemperli A, Boffelli D, Wehrli E, Schulthess G, Phillips MC. Identification of a receptor
mediating absorption of dietary cholesterol in the intestine. Biochemistry 1998;37:1784350.
45
Hoeg JM, Sviridov DD, Tennyson GE, Demosky SJ Jr, Meng MS, Bojanovski D, Safonova IG,
Repin VS, Kuberger MB, Smirnov VN, Higuchi K, Gregg RE, Brewer HB Jr. Both
apolipoproteins B-48 and B-100 are synthesized and secreted by the human intestine J
Lipid Res 1990; 31:1761-9.
Hollander D, Ruble PE Jr. beta-carotene intestinal absorption: bile, fatty acid, pH, and flow rate
effects on transport. Am J Physiol 1978;235:686-91.
Hosomi A, Arita M, Sato Y, Kiyose C, Ueda T, Igarashi O, Arai H, Inoue K. Affinity for alphatocopherol transfer protein as a determinant of the biological activities of vitamin E
analogs. FEBS Lett 1997;409:105–8.
Hu X, Jandacek RJ, White WS. Intestinal absorption of beta-carotene ingested with a meal rich
in sunflower oil or beef tallow: postprandial appearance in triacylglycerol-rich
lipoproteins in women. Am J Clin Nutr 2000;71:1170-80.
Hughes TE, Sasak WV, Ordovas JM, Forte TM, Lamon-Fava S, Schaefer EJ. A novel cell line
(Caco-2) for the study of intestinal lipoprotein synthesis. J Biol Chem 1987;262:3762-7.
Huo T, Ferruzzi MG, Schwartz SJ, Failla ML. Impact of fatty acyl composition and quantity of
triglycerides on bioaccessibility of dietary carotenoids. J Agric Food Chem.
2007;55:8950-7.
Hussain MM, Shi J, Dreizen P. Microsomal triglyceride transfer protein and its role in apoBlipoprotein assembly. J Lipid Res 2003;44:22-32
Iskandar AR, Liu C, Smith DE, Hu KQ, Choi SW, Ausman LM, Wang XD. β-cryptoxanthin
restores nicotine-reduced lung SIRT1 to normal levels and inhibits nicotine-promoted
lung tumorigenesis and emphysema in A/J mice. Cancer Prev Res (Phila) 2013;6:309-20.
Jackson KG, Abraham EC, Smith AM, Murray P, O’Malley B, WilliamsCM, MinihaneAM.
Impact of age and menopausal status on the postprandial triacylglycerol response in
healthy women. Atherosclerosis 2010;208:246-52.
Johnson LJ1, Meacham SL, Kruskall LJ. The antioxidants--vitamin C,vitamin E, selenium, and
carotenoids. J Agromedicine 2003;9:65-82.
Jones B, Jones EL, Bonney SA, Patel HN, Mensenkamp AR, Eichenbaum-Voline S, Rudling M,
Myrdal U, Annesi G, Naik S, Meadows N, Quattrone A, Islam SA, Naoumova RP,
Angelin B, Infante R, Levy E, Roy CC, Freemont PS, Scott J, Shoulders CC. Mutations
46
in a Sar1 GTPase of COPII vesicles are associated with lipid absorption disorders. Nat.
Genet 2003;34:29-31.
Jung S, Wu K, Giovannucci E, Spiegelman D, Willett WC, Smith-Warner SA. Carotenoid intake
and risk of colorectal adenomas in a cohort of male health professionals. Cancer Causes
Control 2013;24:705-17.
Kane JP, Hardman DA, Paulus HE. Heterogeneity of apolipoprotein B: Isolation of a new
species from human chylomicrons. Proc Natl Acad Sci 1980;77:2465-9.
Karpe F, Olivecrona T, Hamsten A, Hultin M. Chylomicron/chylomicron remnant turnover in
humans: evidence for margination of chylomicrons and poor conversion of larger to
smaller chylomicron remnants. J Lipid Res 1997;38:949-61.
Kayden HJ, Traber MG. Absorption, lipoprotein transport, and regulation of plasma
concentrations of vitamin E in humans. J Lipid Res 1993;34:343-58.
Kijlstra A, Tian Y, Kelly ER, Berendschot TT. Lutein: more than just a filter for blue light. Prog
Retin Eye Res 2012;31:303-15.
Knott TJ, Rall SC Jr, Innerarity TL, Jacobson SF, Urdea MS, Levy-Wilson B, Powell LM, Pease
RJ, Eddy R, Nakai H. Human apoliopoprotein B: structure of carboxylterminal domains,
sites of gene expression, and chromosomal localization. Science 1985;230:37-43.
Kohno H, Taima M, Sumida T, Azuma Y, Ogawa H, Tanaka T. Inhibitory effect of mandarin
juice rich in β-cryptoxanthin and hesperidin on 4-(methylnitrosamino)-1-(3-pyridyl)- 1butanone-induced pulmonary tumorigenesis in mice. Cancer Lett 2001;174:141-50.
Koonvitsky BP, Berry DA, Jones MB, Lin PYT, Cooper DA,Jones DY, Jackson JE. Olestra
affects serum concentrations ofa-tocopherol and carotenoids but not vitamin D or vitamin
K status in free-livingsubjects. J Nutr 1997;127:1636-45.
Kostner GM, Oettl K, Jauhiainen M, Ehnholm C, Esterbauer H, Dieplinger H. Human plasma
phospholipid transfer protein accelerates exchange/transfer of alpha-tocopherol between
lipoproteins and cells. Biochem J 1995;305:65-–67.
Kozak M. Structural features in eukaryotic mRNAs that modulate the initiation of translation. J
Biol Chem. 1991;266:19867-70.
47
Lamon-Fava S, Sadowski JA, Davidson KW, O’Brien ME, McNamara JR, Schaefer EJ. Plasma
lipoproteins as carriers of phylloquinone (vitamin K1) in humans. Am J Clin Nutr
1998;67:1226-31.
Lee DM, Dashti N, Mok T. Apolipoprotein B-100 is the major form of this apolipoprotein
secreted by human intestinal Caco-2 cells. Biochem Biophys Res Commun
1988;156:581-7.
Levy E, Rochette C, Londono I, Roy CC, Milne RW, Marcel YL, Bendayan M. Apolipoprotein
B-100: immunolocalization and synthesis in human intestinal mucosa. J Lipid Res
1990;31:1937-46.
Li R, Chen J, Hammonds G, Phillips H, Armanini M, Wood P, Bunge R, Godowski PJ,
Sliwkowski MX, Mather JP. Identification of Gas6 as a growth factor for human
Schwann cells. J Neurosci 1996;16:2012-9.
Lichtenstein AH, Hachey DL, Millar JS, Jenner JL, Booth L, Ordovas J, Schaefer EJ.
Measurement of human apolipoprotein B-48 and B-100 kinetics in triglyceride-rich
lipoproteins using [5,5,5-2H3]leucine. J Lipid Res 1992;33:907-14.
Lizard G, Miguet C, Besséde G, Monier S, Gueldry S, Neel D, Gambert P. Impairment with
various antioxydants of the loss of mitochondrial transmembrane potential and of the
cytosolic release of cytochrome c occurring during 7-ketocholesterolinduced apoptosis.
Free Radic Biol Med 2000;28:743-53.
Lo CM, Nordskog BK, Nauli AM, Zheng S, Vonlehmden SB, Yang Q, Lee D, Swift LL,
Davidson NO, Tso P. Why does the gut choose apolipoprotein B48 but not B100 for
chylomicron formation? Am J Physiol Gastrointest Liver Physiol 2008;294:344-52.
Loane E, Nolan JM, Beatty S. The respective relationships between lipoprotein profile, macular
pigment optical density and serum concentrations of lutein and zeaxanthin. Invest
Ophthalmol Vis Sci 2010;51:5897-905.
Lobo MV, Huerta L, Ruiz-Velasco N, Teixeiro E, de la Cueva P, Celdrán A, Martín-Hidalgo A,
Vega MA, Bragado R. Localization of the lipid receptors CD36 and CLA-1/SR-BI in the
human gastrointestinal tract: Towards the identification of receptors mediating the
intestinal absorption of dietary lipids. J. Histochem. Cytochem 2001;49:1253-60.
48
Lock DR, Hockenberry D, Cunningham J, Hahm KS, Kantor O, Schonfeld G. Apolipoprotein B
subspecies in chylomicrons isolated from a patient with chyluria. Am J Med
1983;75:360-4.
Luo G, Ducy P, McKee MD, Pinero GJ, Loyer E, Behringer RR, Karsenty G. Spontaneous
calcification of arteries and cartilage in mice lacking matrix GLA protein. Nature
1997;386:78-81.
Maras JE, Bermudez OI, Qiao N, Bakun PJ, Boody-Alter EL, Tucker KL. Intake of alphatocopherol is limited among US adults. J Am Diet Assoc 2004;104:567-575.
Marsh JB, Welty FK, Lichtenstein AH, Lamon-Fava S, Schaefer EJ. Apolipoprotein B
metabolism in humans: studies with stable isotope-labeled amino acid precursors.
Atherosclerosis 2002;162:227-44.
Marsh JB. Apoproteins of the lipoproteins in a nonrecirculating perfusate of rat liver. J Lipid Res
1976;17:35-90.
Masding MG, Stears AJ, Burdge GC,Wooton SA, Sandeman DD. Premenopausal advantage in
postprandial lipid metabolism are lost in women with type 2 diabetes. Diabetes Care
2003;26:3243-9.
Matthan NR, Jalbert SM, Barrett PH, Dolnikowski GG, Schaefer EJ, Lichtenstein AH. Genderspecific differences in the kinetics of nonfasting TRL, IDL, and LDL apolipoprotein B100 in men and premenopausal women. Arterioscler Thromb Vasc Biol 2008;28:183843.
Miguet-Alfonsi C, Prunet C, Monier S, Bessède G, Lemaire-Ewing S, Berthier A, Ménétrier F,
Néel D, Gambert P, Lizard G. Analysis of oxidative processes and of myelin Figs
formation before and after the loss of mitochondrial transmembrane potential during 7beta-hydroxycholesterol and 7-ketocholesterol-induced apoptosis: comparison with
various pro-apoptotic chemicals. Biochem. Pharmacol 2002;64: 527-41.
Millar JS, Lichtenstein AH, Cuchel M, Dolnikowski GG, Hachel DL, Cohn JS, Schaefer EJ.
Impact of age on the metabolism of VLDL, IDL, and LDL apolipoprotein B-100 in men.
J Lipid Res 1995;36:1155-67.
Moussa M, Gouranton E, Gleize B, Yazidi CE, Niot I, Besnard P, Borel P, Landrier JF. CD36 is
involved in lycopene and lutein uptake by adipocytes and adipose tissue cultures. Mol
Nutr Food Res 2011;55:578-84.
49
Moussa M, Landrier JF, Reboul E, Ghiringhelli O, Coméra C, Collet X, Fröhlich K, Böhm V,
Borel P. Lycopene absorption in human intestinal cells and in mice involves scavenger
receptor class B type I but not Niemann-Pick C1-like 1. J Nutr 2008;138:1432-6.
Murakoshi M, Nishino H, Satomi Y, Takayasu J, Hasegawa T, Tokuda H, Iwashima A, Okuzumi
J, Okabe H, Kitano H, Iwasaki R. Potent preventive action of alpha-carotene against
carcinogenesis: Spontaneous liver carcinogenesis and promoting stage of lung and skin
carcinogenesis in mice are suppressed more effectively by alpha-carotene than by betacarotene. Cancer Res 1992;52:6583-7.
Murphy SP, Subar AF, Block G. Vitamin E intakes and sources in the United States. Am J Clin
Nutr 1990;52:361-7.
Mutungi G, Waters D, Ratliff J, Puglisi M, Clark RM, Volek JS, Fernandez ML. Eggs distinctly
modulate plasma carotenoid and lipoprotein subclasses in adult men following a
carbohydrate-restricted diet. J Nutr Biochem 2009;21:261-7.
Narisawa T, Fukaura Y, Hasebe M, Ito M, Aizawa R, Murakoshi M, Uemura S, Khachik F,
Nishino H. Inhibitory effects of natural carotenoids, alpha-carotene, beta-carotene,
lycopene and lutein, on colonic aberrant crypt foci formation in rats. Cancer Lett
1996;107: 137-42.
Narushima K, Takada T, Yamanashi Y, Suzuki H. Niemann–Pick C1-like 1 mediates alphatocopherol transport. Mol Pharmacol 2008;74:42–9.
Nestel PJ. Relationship between plasma triglycerides and removal of chylomicrons. J Clin Invest
1964;43:943-9.
Nguyen TT. The cholesterol-lowering action of plant stanol esters. J Nutr 1999;129:2109-12.
Niki E, Traber MG. A history of vitamin E. Ann Nutr Metab 2012;61:207-12.
Nilesh M. Kalariya, Kota V. Ramana, Frederik J.G.M. vanKuijk. Focus on Molecules: Lutein
Experimental Eye Res 2012;102:107-8.
Novotny JA, Fadel JG, Holstege DM, Furr HC, Clifford AJ. This kinetic, bioavailability, and
metabolism study of RRR-α-tocopherol in healthy adults suggests lower intake
requirements than previous estimates. J Nutr 2012;142:2105-11.
Ohtani O, Ohtani Y. Lymph circulation in the liver. Anat Rec (Hoboken) 2008; 291:643-52.
50
Olofsson SO, Stillemark-Billton P, Asp L. Intracellular assembly of VLDL: two major steps in
separate cell compartments. Trends Cardiovasc Med 2000;10:338-45.
Omenn GS, Goodman GE, Thornquist MD, Balmes J, Cullen MR, Glass A, Keogh JP, Meyskens
FL, Valanis B, Williams JH, Barnhart S, Hammar S. Effects of the combination of betacarotene and vitamin A on lung cancer incidence, total mnortality, and cardiovascular
mortality in smokers and asbestos-exposed workers. N Engl J Med 1996;334:1150-5.
Palozza P, Catalano A, Simone R, Cittadini A. Lycopene as a guardian of redox signalling. Acta
Biochim Po 2012;59:21-5.
Palozza P, Parrone N, Simone R, Catalano A. Role of lycopene in the control of ROS-mediated
cell growth: implications in cancer prevention. Curr Med Chem 2011a;18: 1846-60.
Palozza P, Simone R, Catalano A, Monego G, Barini A, Mele MC, Parrone N, Trombino S, Picci
N, Ranelletti FO. Lycopene prevention of oxysterol-induced proinflammatory cytokine
cascade in human macrophages: inhibition of NF-κB nuclear binding and increase in
PPARγ expression. J Nutr Biochem 2011b;22:259-68.
Pan M, Cederbaum AI, Zhang YL, Ginsberg HN, Williams KJ, Fisher EA. Lipid peroxidation
and oxidant stress regulate hepatic apolipoprotein B degradation and VLDL production. J
Clin Invest 2004;113:1277-87.
Patsch W, Franz S, Schonfeld G. Role of insulin in lipoprotein secretion by cultured rat
hepatocytes. J Clin Invest 1983;71:1161-74.
Perugini C, Bagnati M, Cau C, Bordone R, Paffoni P, Re R, Zoppis E, Albano E, Bellomo G.
Distribution of lipid-soluble antioxidants in lipoproteins from healthy subjects. Effects of
in vivo supplementation with α-tocopherol. Pharmacol Res 2000;41:65-72.
Pham DN, Leclerc D, Lévesque N, Deng L, Rozen R. β,β-Carotene 15,15'-monooxygenase and
its substrate β-carotene modulate migration and invasion in colorectal carcinoma cells.
Am J Clin Nutr 2013;98:413-22.
Phung TL, Roncone A, Jensen KL, Sparks CE, Sparks JD. Phosphoinositide 3-kinase activity is
necessary for insulin-dependent inhibition of apolipoprotein B secretion by rat
hepatocytes and localizes to the endoplasmic reticulum. J Biol Chem 1997;272:30693702.
51
Pontrelli, L, Sidiropoulos, K.G., Adeli, K. Translational control of apolipoprotein B mRNA:
regulation via cis elements in the 5’ and 3’ untranslated regions. Biochemistry
2004;43:6734-44.
Pullinger CR, North JD, Teng BB, Rifici VA, Ronhild de Brito AE, Scott J. The apolipoprotein
B gene is constitutively expressed in HepG2 cells: regulation of secretion by oleic acid,
albumin, and insulin, and measurement of the mRNA half-life. J Lipid Res
1989;30:1065-77.
Qiu W, Kohen-Avramoglu R, Mhapsekar S, Tsai J, Austin RC, Adeli K. Glucosamine-induced
endoplasmic reticulum stress promotes apoB100 degradation: evidence for Grp78mediated targeting to proteasomal degradation. Arterioscler Thromb Vasc Biol
2005;25:571-7.
Raju M, Lakshminarayana R, Krishnakantha TP, Baskaran V. Micellar oleic and
eicosapentaenoic acid but not linoleic acid influences the beta-carotene uptake and its
cleavage into retinol in rats. Mol Cell Biochem 2006;288:7-15.
Rao AV. Lycopene, tomatoes, and the prevention of coronary heart disease. Exp Biol Med
(Maywood) 2002;227:908-13.
Rapoport TA. Protein translocation across the eukaryotic endoplasmic reticulum and bacterial
plasma membranes. Nature 2007;450:663-9.
Rashid KA, Hevi S, Chen Y, Le Cahérec F, Chuck SL. A proteomic approach identifies proteins
in hepatocytes that bind nascent apolipoprotein B. J Biol Chem 2002;277:22010-7.
Reboul E, Abou L, Mikail C, Ghiringhelli O, André M, Portugal H, Jourdheuil-Rahmani D,
Amiot MJ, Lairon D, Borel P. Lutein transport by Caco-2 TC-7 cells occurs partly by a
facilitated process involving the scavenger receptor class B type I (SR-BI). Biochem J
2005;387:455-61.
Reboul E, Klein A, Bietrix F, Gleize B, Malezet-Desmoulins C, Schneider M, Margotat A,
Lagrost L, Collet X, Borel P. Scavenger receptor class B type I (SR-BI) is involved in
vitamin E transport across the enterocyte. J Biol Chem 2006;281:4739-45.
Reboul E, Trompier D, Moussa M, Klein A, Landrier JF, Chimini G, Borel P. ATP-binding
cassette transporter A1 is significantly involved in the intestinal absorption of alpha- and
gamma-tocopherol but not in that of retinyl palmitate in mice. Am J Clin Nutr
2009;89:177-84.
52
Reiss R, Johnston J, Tucker K, DeSesso JM, Keen CL. Estimation of cancer risks and benefits
associated with a potential increased consumption of fruits and vegetables. Food Chem
Toxicol 2012;50:4421-7.
Ricciarelli R, Tasinato A, Clément S, Ozer NK, Boscoboinik D, Azzi A. Alpha-tocopherol
specifically inactivates cellular protein kinase C alpha by changing its phosphorylation
state. Biochem J 1998;334:243-9.
Rich GT, Faulks RM, Wickham MS, Fillery-Travis A. Solubilization of carotenoids from carrot
juice and spinach in lipid phases: II. Modeling the duodenal environment. Lipids
2003;38:947-56.
Richelle M, Enslen M, Hager C, Groux M, Tavazzi I, Godin JP, Berger A, Métairon S, Quaile S,
Piguet-Welsch C, Sagalowicz L, Green H, Fay LB. Both free and esterified plant sterols
reduce cholesterol absorption and the bioavailability of beta-carotene and alphatocopherol in normocholesterolemic humans. Am J Clin Nutr 2004;80:171-7.
Riedl J, Linseisen J, Hoffmann J, Wolfram G. Some dietary fibers reduce the absorption of
carotenoids in women. J Nutr 1999;129:2170-6.
Rigotti A. Absorption, transport, and tissue delivery of vitamin E. Mol Aspects Med
2007;28:423-36.
Riserus U. Fatty acids and insulin sensitivity. Curr. Opin. Clin. Nutr. Metab. Care 2008;11:1005.
Rock CL, Lovalvo JL, Emenhiser C, Ruffin MT, Flatt SW, Schwartz SJ. Bioavailability of betacarotene is lower in raw than in processed carrots and spinach in women. J Nutr
1998;128:913-6.
Rock CL, Swendseid ME. Plasma beta-carotene response in humans after meals supplemented
with dietary pectin. Am J Clin Nutr 1992;55:96-9.
Ron D, Walter P. Signal integration in the endoplasmic reticulum unfolded protein response. Nat
Rev Mol Cell Biol 2007;8:519-29.
Roodenburg AJ, Leenen R, van het Hof KH, Weststrate JA, Tijburg LB. Amount of fat in the
diet affects bioavailability of lutein esters but not of alpha-carotene, beta-carotene, and
vitamin E in humans. Am J Clin Nutr 2000;71:1187-93.
53
Royer MC, Lemaire-Ewing S, Desrumaux C, Monier S, Pais de Barros JP, Athias A, Néel D,
Lagrost L. 7-ketocholesterol incorporation into sphingolipid/ cholesterol-enriched (lipid
raft) domains is impaired by Vitamin E: a specific role for alpha-tocopherol with
consequences on cell death. J Biol Chem 2009;284:15826-34.
Ruf H, Gould BJ. Size distributions of chylomicrons from human lymph from dynamic light
scattering measurements. Eur Biophys J 1999;28:1-11.
Sakudoh T, Iizuka T, Narukawa J, Sezutsu H, Kobayashi I, Kuwazaki S, Banno Y, Kitamura A,
Sugiyama H, Takada N, Fujimoto H, Kadono-Okuda K, Mita K, Tamura T, Yamamoto
K, Tsuchida K. A CD36-related transmembrane protein is coordinated with an
intracellular lipid-binding protein in selective carotenoid transport for cocoon coloration.
J Biol Chem 2010;285:7739-51.
Salhanick AI, Schwartz SI, Amatruda JM. Insulin inhibits apolipoprotein B secretion in isolated
human hepatocytes. Metabolism 1991;40:275-9.
Schaefer EJ, Gregg RE, Ghiselli G, Forte TM, Ordovas JM, Zech LA, Brewer HB Jr. Familial
apolipoprotein E deficiency. J Clin Invest 1986;78:1206-19.
Schaefer EJ, Jenkins LL, Brewer HB Jr. Human chylomicron apolipoprotein metabolism.
Biochem. Biophys. Res. Commun 1978;80:405–12.
Schaus EE, de Lumen BO, Chow FI, Reyes P, Omaye ST. Bioavailability of vitamin E in rats fed
graded levels of pectin. J Nutr 1985;115:263-70.
Schlagheck TG, Riccardi KA, Zoric NL, Torri SA, Dugan LD, Peters JC. Olestra dose response
on fat-soluble and water-solublenutrients in humans. J Nutr 1997;127:1646-65.
Schroder M, Kaufman RJ. The mammalian unfolded protein response. Annu Rev Biochem
2005;74:739-89.
Schurgers LJ, Vermeer C. Differential lipoprotein transport pathways of K-vitamins in healthy
subjects. Biochim Biophys Acta 2002;1570:27-32.
Scita G, Aponte GW, Wolf G. Uptake and cleavage of beta-carotene by cultures of rat small
intestinal cells and human lung fibroblasts. Methods Enzymol 1993;214:21-32.
Shearer MJ, Bach A, Kohlmeier M. Chemistry, nutritional sources, tissue distribution and
metabolism of vitamin K with special reference to bone health. J Nutr 1996;126:1181-6.
54
Shearer MJ. Vitamin K in parenteral nutrition. Gastroenterology 2009;137:105-18.
Shelness GS, Ledford AS. Evolution and mechanism of apolipoprotein Bcontaining lipoprotein
assembly. Curr Opin Lipidol 2005;16:325-32.
Shoulders CC, Shelness GS. Current biology of MTP: implications for selective inhibition. Curr
Top Med Chem 2005;5:283-300
Sidiropoulos KG, Meshkani R, Avramoglu-Kohen R, Adeli K. Insulin inhibition of
apolipoprotein B mRNA translation is mediated via the PI-3 kinase/mTOR signaling
cascade but does not involve internal ribosomal entry site (IRES) initiation. Arch
Biochem Biophys 2007;465:380-8.
Sidiropoulos KG, Pontrelli L, Adeli K. Insulinmediated suppression of apolipoprotein B mRNA
translation requires the 5’ UTR and is characterized by decreased binding of an insulinsensitive 110-kDa 5’ UTR RNA-binding protein. Biochemistry 2005;44:12572-81.
Sommer A, Tarwotjo I, Hussaini G, Susanto D, Soegiharto T. Incidence, prevalence, and scale of
blinding malnutrition. Lancet 1981;317:1407-8.
Sommer A, Vyas KS. A global clinical view on vitamin A and carotenoids. Am J Clin Nutr
2012;96:1204-6.
Sparks CE, Sparks JD, Bolognino M, Salhanick A, Strumph PS, Amatruda JM. Insulin effects on
apolipoprotein B lipoprotein synthesis and secretion by primary cultures of rat
hepatocytes. Metabolism 1986;35:1128-36.
Stacpoole PW, Von Bergmann K, Kilgore LL, Zech LA, Fisher WR. Nutritional regulation of
cholesterol synthesis and apolipoprotein B kinetics: studies in patients with familial
hypercholesterolemia and normal subjects treated with a high carbohydrate, low fat diet. J
Lipid Res 1991;32:1837-48.
Stahl W, Schwarz W, Sies H. Human serum concentrations of all-trans beta- and alpha-carotene
but not 9-cis beta-carotene increase upon ingestion of a natural isomer mixture obtained
from Dunaliella salina (Betatene). J Nutr 1993;123,847-851.
Stahl W, Sies H. Uptake of lycopene and its geometrical isomer is greater from heat-processed
than from unprocessed tomato juice in humans. J Nutr 1992;122:2161-6.
55
Stahl W, van den Berg H, Arthur J, Bast A, Dainty J, Faulks RM, Gärtner C, Haenen G, Hollman
P, Holst B, Kelly FJ, Polidori MC, Rice-Evans C, Southon S, van Vliet T, Viña-Ribes J,
Williamson G, Astley SB. Bioavailability and metabolism. Mol Aspects Med
2002;23:39-100.
Stalenhoef AF, Malloy MJ, Kane JP, Havel RJ. Metabolism of apolipoproteins B-48 and B-100
of triglyceride-rich lipoproteins in normal and lipoprotein lipase-deficient humans. Proc
Natl Acad Sci USA 1984;81:1839-43.
Stanhope KL, Bremer AA, Medici V, Nakajima K, Ito Y, Nakano T, Chen G, Fong TH, Lee V,
Menorca RI, Keim NL, Havel PJ. Consumption of fructose and high fructose corn syrup
increase postprandial triglycerides, LDL-cholesterol, and apolipoprotein-B in young men
and women. J Clin Endocrinol Metab 2011;96:1596-605.
Stanhope KL, Schwarz JM, Keim NL, Griffen SC, Bremer AA, Graham JL, Hatcher B, Cox CL,
Dyachenko A, Zhang W, McGahan JP, Seibert A, Krauss RM, Chiu S, Schaefer EJ, Ai
M, Otokozawa S, Nakajima K, Nakano T, Beysen C, Hellerstein MK, Berglund L, Havel
PJ. Consuming fructose-sweetened, not glucose-sweetened, beverages increases visceral
adiposity and lipids and decreases insulin sensitivity in overweight/obese humans. J Clin
Invest 2009;119:1322-34.
Stillemark P, Borén J, Andersson M, Larsson T, Rustaeus S, Karlsson KA, Olofsson SO. The
assembly and secretion of apolipoprotein B-48-containing very low density lipoproteins
in McA-RH7777 cells. J Biol Chem 2000;275:10506-13.
Takata Y, Xiang Y-B, Yang G, Li H, Gao J, Cai H, Gao Y-T, Zheng W, Shu X-O. Intakes of
fruits, vegetables, and related vitamins and lung cancer risk: results from the Shanghai
Men’s Health Study (2002-2009). Nutr Cancer 2013;65:51-61.
Tanaka T, Kohno H, Murakami M, Shimada R, Kagami S, Sumida T, Azuma Y, Ogawa H.
Suppression of azoxymethane-induced colon carcinogenesis in male F344 rats by
mandarin juices rich in β-cryptoxanthin and hesperidin. Int J Cancer 2000;88:146-50.
Tasinato A, Boscoboinik D, Bartoli GM, Maroni P, Azzi A. d-α-Tocopherol inhibition of
vascular smooth muscle cell proliferation occurs at physiological concentrations,
correlates with protein kinase C inhibition, and is independent of its antioxidant
properties. Proc Natl Acad Sci USA 1995;92:12190-4.
56
Tatu U, Helenius A. Interactions between newly synthesized glycoproteins, calnexin and a
network of resident chaperones in the endoplasmic reticulum. J Cell Biol 1997;136:55565.
Terpstra V, van Amersfoort ES, van Velzen AG, Kuiper J, van Berkel TJ. Hepatic and
extrahepatic scavenger receptors: Function in relation to disease. Arterioscler Thromb
Vasc Biol 2000;20:1860-72.
Terpstra, V.; van Amersfoort, E.S.; van Velzen, A.G.; Kuiper, J.; van Berkel, T.J. Hepatic and
extrahepatic scavenger receptors: Function in relation to disease. Arterioscler Thromb
Vasc Biol 2000;20:1860-72.
The ATBC Cancer Prevention Study Group. The effect of vitamin E and beta-carotene on the
incidence of lung cancer and other cancers in male smokers. N EngI J Med
1994;330:1029-35.
Thomson CA, Thomson PA. Fruit and vegetable intake and breast cancer risk: a case for
subtype-specific risk? J Natl Cancer Inst 2013;105:164-5.
Traber MG, Sies H. Vitamin E in humans: demand and delivery. Annu Rev Nutr 1996;16:32147.
Tyssandier V1, Choubert G, Grolier P, Borel P. Carotenoids, mostly the xanthophylls, exchange
between plasma lipoproteins. Int J Vitam Nutr Res 2002;72:300-8.
Tzotzas, T., Desrumaux, C., Lagrost, L., Plasma phospholipid transfer protein (PLTP): review of
an emerging cardiometabolic risk factor. Obes Rev 2009;10:403-11.
Unlu NZ, Bohn T, Clinton SK, Schwartz SJ. Carotenoid absorption from salad and salsa by
humans is enhanced by the addition of avocado or avocado oil. J Nutr 2005;135:431-6.
van Beek AP, de Ruijter-Heijstek FC, Erkelens DW, de Bruin TWA. Menopause is associated
with reduced protection from postprandial lipaemia. Arterioscler Thromb Vasc Biol
1999;19:2737–41.
van Bennekum A, Werder M, Thuahnai ST, Han CH, Duong P, Williams DL, Wettstein P,
Schulthess G, Phillips MC, Hauser H. Class B scavenger receptor-mediated intestinal
absorption of dietary beta-carotene and cholesterol. Biochemistry 2005;44:4517-25.
57
van het Hof KH, Gärtner C, West CE, Tijburg LB. Potential of vegetable processing to increase
the delivery of carotenoids to man. Int J Vitam Nutr Res 1998;68:366-70.
Vejux A, Guyot S, Montange T, Riedinger JM, Kahn E, Lizard G. Phospholipidosis and downregulation of the PI3-K/PDK-1/ Akt signalling pathway are vitamin E inhibitable events
associated with 7-ketocholesterol-induced apoptosis. J. Nutr. Biochem 2009;20:45-61.
Veniant MM, Kim E, McCormick S, Boren J, Nielsen LB, Raabe M, Young SG. Insights into
apolipoprotein B biology from transgenic and gene-targeted mice. J Nutr 1999;129:4515.
Vergès B. Abnormal hepatic apolipoprotein B metabolism in type 2 diabetes. Atherosclerosis
2010;211:353-60.
von Lintig, J. Colors with functions: Elucidating the biochemical and molecular basis of
carotenoid metabolism. Annual Review of Nutrition 2010;30:35-56.
Wang Y, Connor SL, Wang W, Johnson EJ, Connor WE. The selective retention of lutein, mesozeaxanthin and zeaxanthin in the retina of chicks fed a xanthophyll-free diet.
Experimental Eye Res 2007;84:591-8.
Watts GF, Moroz P, Barrett PH. Kinetics of very-low-density lipoprotein apolipoprotein B-100
in normolipidemic subjects:pooled analysis of stable-isotope studies. Metabolism
2000;49:1204-10.
Welty FK, Lichtenstein AH, Barrett PH, Dolnikowski GG, Schaefer EJ. Human apolipoprotein
(apo) B-48 and apoB-100 kinetics with stable isotopes. Arterioscler Thromb Vasc Biol
1999;19:2966-74.
Welty FK, Lichtenstein AH, Barrett PH, Dolnikowski GG, Schaefer EJ. Interrelationships
between human apolipoprotein A-I and apolipoproteins B-48 and B-100 kinetics using
stable isotopes. Arterioscler Thromb Vasc Biol 2004;24:1703-7.
Welty FK, Lichtenstein AH, Barrett PH, Jenner JL, Dolnikowski GG, Schaefer EJ. Effects of
apoE genotype on apoB-48 and apoB-100 kinetics with stable isotopes in humans.
Arterioscler Thromb Vasc Biol 2000;20:1807-10.
Weststrate JA, van het Hof KH. Sucrose polyester and plasma carotenoid concentrations in
healthy subjects. Am J Clin Nutr 1995;62:591-7.
58
Yamaguchi M. Role of carotenoid β-cryptoxanthin in bone homeostasis. J Biomed Sci
2012;19:36
Yonekura L, Nagao A. Intestinal absorption of dietary carotenoids. Mol Nutr Food Res
2007;51:107-15.
Yonekura L, Tsuzuki W, Nagao A. Acyl moieties modulate the effects of phospholipids on betacarotene uptake by Caco-2 cells. Lipids 2006;41:629-36.
Yonova-Doing E, Hysi PG, Venturini C, Williams KM, Nag A, Beatty S, Liew SH, Gilbert CE,
Hammond CJ. Candidate gene study of macular response to supplemental lutein and
zeaxanthin. Exp Eye Res 2013;115:172-7.
You CS, Parker RS, Goodman KJ, Swanson JE, Corso TN. Evidence of cis–trans isomerization
of 9-cis-beta-carotene during absorption in humans. Am J Clin Nutr 1996;64:177-183.
Zanutto ME, Jordão Júnior AA, Meirelles MS, Fávaro RM, Vannucchi H. Effect of citric pectin
on beta-carotene bioavailability in rats. Int J Vitam Nutr Res 2002;72:199-203.
Zhang, J, Herscovitz H. Nascent lipidated apolipoprotein B is transported to the Golgi as an
incompletely folded intermediate as probed by its association with network of
endoplasmic reticulum molecular chaperones, GRP94, ERp72, BiP, calreticulin, and
cyclophilin B. J Biol Chem 2003;278:7459-68.
Zhou M, Fisher EA, Ginsberg HN. Regulated co-translational ubiquitination of apolipoprotein
B100. A new paradigm for proteasomal degradation of a secretory protein. J Biol Chem
1998;273:24649-53.
59
CHAPTER III.
EFFECTS OF LECITHIN IN SALAD DRESSING ON THE PLASMA APPEARANCE
OF FAT-SOLUBLE MICRONUTRIENTS CONSUMED IN SALADS:
CONTRIBUTIONS OF CHYLOMICRONS AND LARGE VLDL
A paper to be submitted to The American Journal of Clinical Nutrition
Yinghui Zhang, Yang Zhou, Leonard Flendrig, Michiel Gribnau, and Wendy S. White1-3
1
Author affiliations at the time the work was completed: Department of Food Science and
Human Nutrition, Iowa State University, Ames, IA (YZ, YZ, WSW); Unilever R&D,
Vlaardingen, The Netherlands (LF, MG).
2
Supported by Unilever R&D, Vlaardingen, The Netherlands.
3
Corresponding author: WS White, Department of Food Science and Human Nutrition, 1111
Human Nutritional Sciences Building, Iowa State University, Ames, IA 50011-1120. Tel: 515
294-3347. FAX: 515 294-6193. Email: [email protected]. Reprints will not be available
from the author.
Authors last name: Zhang, Zhou, Flendrig, Gribnau,White
Running head: PLASMA APPEARANCE OF FAT-SOLUBLE MICRONUTRIENTS
Abbreviations used: VLDLA, large very-low-density lipoprotein subfraction; ApoB-48,
apolipoprotein B-48; ApoB-100, apolipoprotein B-10
60
ABSTRACT
Background: Lecithin may influence the bioavailability of fat-soluble micronutrients. The role
of the large very-low-density lipoprotein (VLDLA) plasma fraction in postprandial fat-soluble
micronutrient transport is not clearly defined.
Objectives: This study investigated the: 1) effects of the lecithin/oil ratio in salad dressing on the
absorption of carotenoids, phylloquinone, and tocopherols from salad vegetables and retinyl
palmitate from intestinal metabolism of the provitamin A carotenoids; 2) roles of the plasma
chylomicron and VLDLA fractions in the transport of the absorbed fat-soluble micronutrients.
Design: Healthy women (n = 12) each consumed three salads with salad dressings containing: 1)
4 g soybean oil and 0 g hydroxylated soy lecithin (Solec® 8120, Solae, St. Louis, MO); 2) 3.8 g
soybean oil and 0.2 g Solec® 8120; or 3) 3.2 g of soybean oil and 0.8 g Solec® 8120. The
salads were separated by ≥ 2 wk. Blood was collected at baseline and 2, 3.5, 5, 7, and 9.5 h
postprandially. Fat-soluble micronutrients in the lipoprotein fractions were analyzed by HPLC
with coulometric array electrochemical detection. Apolipoprotein (apo) B-48 and apo B-100
contents in chylomicrons and VLDLA were determined by ELISA.
Results: There were no differences among the salad dressings in the area under the curve (AUC)
values for the fat-soluble micronutrients in the chylomicron or VLDLA fractions. The exception
was a decrease in the AUC for phylloquinone in the chylomicron fraction when the salad
dressing containing 0.8 g was compared with that containing 0 g Solec® 8120 (P < 0.02). The
AUC values for α-carotene, β-carotene, lycopene, retinyl palmitate, and phylloquinone were
61
substantially higher in the VLDLA than in the chylomicron fraction (P < 0.05). ApoB-48 and
apoB-100 contents were higher in the VLDLA fraction than in the chylomicron fraction (P <
0.0001).
Conclusions: The hydroxylated soy lecithin in the amounts added to the salad dressing did not
enhance the absorption of fat-soluble micronutrients. The majority of the absorbed fat-soluble
micronutrients and enterogenous (apoB-48) and hepatogenous (apoB-100) lipoprotein particles
were contained within the large VLDL fraction. Thus this VLDL subfraction has a major role in
the transport of newly absorbed carotenoids and fat-soluble vitamins.
INTRODUCTION
Salad consumption is a major contributor to vegetable consumption in the United States
(1). Although the bioavailability of carotenoids and fat-soluble micronutrients is low in raw
vegetables (2-5), the absorption is able to be improved by increasing the fat content of a meal (6,
7). Because high fat intakes are not recommended, one strategy is to find a lipid that is more
efficient than triacylglycerol in increasing the bioavailability of fat-soluble micronutrients.
Lecithin, as a structural component of mixed micelles and an excellent emulsifier, is likely to
enhance the absorption of dietary lipids and lipophilic nutrients. Soybean lecithin was reported
to increase the lymphatic absorption of triglyceride in rats (8). Also, after 10 days of lutein
consumption with either phosphatidylcholine or lysophosphatidylcholine, the lutein contents in
rat plasma, liver, and eyes were significantly higher than those in which lutein was consumed
62
without phospholipids (9). However, it has not been addressed the effects of dietary lecithin on
the bioavailability of carotenoids and fat-soluble vitamins in humans. The exception was one
study which showed that multiple doses of an oral preparation of phylloquinone containing
lecithin and glycocholic acid maintained phylloquinone status in infants at least equal to that of
an intramuscular injection (10).
In our previous studies, after the consumption of a β-carotene dose with a fat-rich meal, we
observed that the postprandial appearance of β-carotene and retinyl palmitate in the plasma
chylomicron fraction coincided in time and magnitude with their appearance in large very low
density lipoproteins (VLDLA) (11, 12). Although the VLDLA subfraction has been used as a
vehicle for assessing the intestinal absorption of β-carotene and its major bioconversion product,
retinyl palmitate (11-14); there has been little systematic study of the role of VLDLA in the
transport of other newly absorbed fat-soluble micronutrients. The postprandial appearance of
retinyl palmitate in VLDLA was hypothesized to be attributed to intestinal VLDL (13), but could
also reflect the appearance of retinyl palmitate in chylomicron remnants (15). However,
VLDLA have long been regarded as liver-derived lipoproteins (16, 17). Apolipoprotein B-48
(apoB-48) and apolipoprotein B-100 (apoB-100) are reliable biomarkers of lipoprotein particles
of intestinal and hepatic origin, respectively (18-20). Therefore, an investigation of apoB-48 and
apoB-100 in the plasma VLDLA fraction after a single meal could provide new insight regarding
the origin of VLDLA.
63
The objectives of the present study were to investigate the effects of the lecithin/oil ratio in
salad dressing on the absorption of fat-soluble micronutrients from salad vegetables. An
additional objective was to investigate the origin of VLDLA and the roles of the plasma
chylomicron and VLDLA fractions in the transport of the absorbed fat-soluble micronutrients.
SUBJECTS AND METHODS
Subjects
Thirteen healthy, nonsmoking, normolipidemic women, aged 18-40 y, were enrolled in this
study. One subject dropped out before completing the first study period due to a gastrointestinal
problem that prevented her from consuming the test salad. The mean (± SD) age of the
remaining 12 subjects was 27 ± 6.9 y; the mean (± SD) body mass index (BMI; in kg/m2) was
20.7 ± 3.0. The subjects were screened initially by using a standardized questionnaire that
addressed health and lifestyle factors. At the time of interview, the applicants’ body weights and
heights were measured and recorded. Those who met the eligibility criteria donated a fasting
blood sample for a blood biochemistry profile, blood lipid profile, and complete blood count.
The exclusion criteria included frequent consumption of alcoholic beverages (> 1 drink/d),
current or recent (previous 12 mo) cigarette smoking, pregnant or planning to become pregnant
during the study, current or recent (previous 4 mo) use of hormonal contraceptives, use of dietary
supplements in the past 1 mo, current or recent (previous 1 mo) ingestion of plant sterols and/or
medications known to affect lipid metabolism, body mass index (BMI) ≤ 18 and ≥ 30, known
food allergy, and vegetarianism. The SCOFF questionnaire was used to access whether
64
applicants had a history of restrictive eating (21). Subjects who had 2 or more “yes” responses
on the written questionnaire were excluded. All procedures involving human subjects were
approved by the Iowa State University Institutional Review Board.
Test salads
The test salads consisted of 48 g romaine lettuce (Classic Romaine® Salad Mix, Dole Food
Company, Thousand Oaks, CA), 48 g fresh spinach leaves (Spinach, Dole Food Company), 66 g
fresh, shredded carrots (Shredded Carrots, Dole Food Company), and 85 g fresh cherry tomatoes
(NatureSweet, San Antonio, TX). The total weight (247 g) equaled the 90th percentile of the
quantity of salad (lettuce and other vegetables) that was eaten by adults per eating occasion in the
1994-1996 Continuing Survey of Food Intakes by Individuals (CSFII) (22). The prepackaged
salad vegetables were special ordered to minimize and standardize the time spent in shipment.
The cherry tomatoes, romaine lettuce, and spinach leaves were manually sorted to achieve
uniform color intensity from week-to-week, and thereby minimize variation in the carotenoid/fatsoluble vitamin contents in the salads. The green color of the romaine and spinach leaves was
used as an indicator of chlorophyll and, by extrapolation, carotenoid contents (23).
Representative samples of the spinach, romaine lettuce, cherry tomatoes, and carrots from the
test salads consumed during each of the 6 weeks of the study were stored at -70°C. The salad
dressings were supplied by Unilever R&D (Vlaardingen, The Netherlands). The salad dressings
were prepared by combining 4 g of the lipid phase with 56 g of an aqueous salad dressing base
formulation. The lipid phase (4 g total) in the form of phylloquinone- and tocopherol-stripped
65
soybean oil contained either: 1) 0 g; 2) 0.2 g; or 3) 0.8 g hydroxylated soy lecithin (Solec® 8120,
Solae, St. Louis, MO). The phylloquinone and tocopherols were stripped from the soybean oil at
Unilever R&D by using a standard short path distillation procedure. The total phospholipid
contents of the lipid phases were analyzed by Spectral Service (Cologne, Germany). The 60 g
serving of the salad dressing was chosen to simulate the typical intake of salad dressing for a
person eating a salad as all or most of a meal. This amount of salad dressing was chosen to
correspond with the amount of salad vegetables; thus, 60 g was the 90th percentile of the quantity
of low-calorie and full-fat salad dressings reported by adults eating salad components at an
eating occasion in the 1994–1996 CSFII (22). Each salad dressing was weighed directly onto the
salad and tossed with the salad vegetables to ensure even distribution.
Before consuming the test salad, the subjects first drank 250 mL of water and then waited
15 min to allow for emptying of the stomach. Then, the test salads were consumed together with
another 250 mL of water within a 30 min-period. Subjects were instructed to consume the entire
salad and then to scrape the residual salad dressing from the bowl with a spatula to ensure that all
of the test salad was consumed.
Experimental design
Each subject consumed three test salads that had identical vegetable composition and
identical aqueous base phase in the salad dressing, but different composition of the lipid phase in
the salad dressing: 4 g of soybean oil containing 0 g of Solec® 8120, 3.8 g of soybean oil
containing 0.2 g of Solec® 8120, or 3.2 g of soybean oil containing 0.8 g of Solec® 8120. The
66
subjects consumed each of the three test salads according to the treatment orders of a Williams
Latin square design (24). The 12 subjects were each randomly assigned to one of the 6 treatment
orders, so that each of the 6 treatment orders was represented twice. The washout period
between the test salads was ≥ 2 weeks.
Blood samples were collected from a forearm vein by a licensed medical technician using a
butterfly needle blood collection set (BD Safety-Lok TM Blood Collection Set, Becton,
Dickinson, and Co, Franklin Lakes, NJ). For days 1-3 of each study period, the subjects were
instructed to avoid foods containing high amounts of carotenoids, phylloquinone, tocopherols,
and vitamin A. A list of foods to avoid was provided. On day 4, the subjects consumed a
weighed, standardized diet containing only minor amounts of carotenoids, tocopherols,
phylloquinone, and vitamin A. On day 4, breakfast and dinner were consumed under supervision
in the Iowa State University Nutrition and Wellness Research Center; the lunch and snacks were
carried out. The nutrient contents of the diet were analyzed by Nutritionist Pro nutrition analysis
software (Axxya Systems, Stafford, TX). The daily diet provided 2249 Kcals, 71.57 g protein,
48.10 g fat, and 380.53 g carbohydrate. The calculated carotenoid and fat-soluble vitamin
contents of the daily diet were 11.23 μg β-carotene, 0.18 μg lutein (plus zeaxanthin), 0.05 βcryptoxanthin, 2.97 mg vitamin E, 15.44 μg vitamin K, 56.96 μg RAE vitamin A. On day 5,
subjects again reported to the Nutrition and Wellness Research Center. A 10-mL blood sample
was collected at baseline after an overnight fast. The subjects consumed the test salad, and
additional blood samples were collected at 2, 3.5, 5, 7 and 9.5 h after the test salad consumption.
67
Blood samples were transferred to 10-mL vacutainer tubes containing spray-dried K2 EDTA as
anticoagulant (BD Vacutainer®, Becton, Dickinson, and Co). Blood samples were immediately
placed on ice and then centrifuged (700 × g, 4° C, 30 min) to separate plasma. During these
procedures, blood samples were protected from light. After the 5-h blood sample collection, the
subjects consumed a low-fat lunch that was also low in carotenoids, tocopherols, phylloquinone,
and vitamin A. On the basis of analysis with the Nutritionist Pro software, the morning snack
and lunch were estimated to contain 0.32 g and 1.50 g fat, respectively.
Triglyceride-rich lipoprotein (TRL) isolation
Chylomicrons and large VLDL (VLDLA) were isolated from plasma by cumulative rate
ultracentrifugation (25, 26). To form the density gradient, salt solutions were prepared,
including a 1.006 g/mL sodium chloride stock solution at pH 7.0, and 1.020 g/mL and 1.065
g/mL potassium bromide (KBr) solutions prepared from the 1.006 g/mL sodium chloride stock
solution. The densities were confirmed by using a digital density meter (DMA-48; Anton-Paar
USA, Ashland, VA). Four milliliters of plasma were transferred to a centrifuge tube (Ultra Clear,
Beckman Instruments, Inc, Spinco Division, Palo Alto, CA), and 0.14 g KBr/mL was added to
adjust the density to 1.10 g/mL. The prepared salt solutions were overlaid on the plasma in order
of highest to lowest density: 1) 3 mL of 1.0065 g/mL density solution; 2) 3 mL of 1.020 g/mL
density solution; 3) 2 mL of 1.006 g/mL density solution. Because there were only six places in
each Beckman SW 40i swinging bucket rotor, two ultracentrifuges (L8-70M, L-90K; Beckman
Instruments Inc,) were used to centrifuge the plasma samples at 28,300 rpm (101,136 × g) for 43
68
min. The chylomicron fraction was removed and the tube was refilled with 1.006 g/mL density
solution. The tube then underwent a second ultracentrifugation at 40,000 rpm (142,948 × g) for
67 min. The VLDLA fraction was then removed from the top of the centrifuge tube. The
chylomicron and VLDLA fractions were stored at -70°C until analyzed. All procedures were
performed under yellow light.
ELISA assays of apolipoproteinB-48 and total apolipoprotein B
The contents of apolipoprotein B-48 (apoB-48) and total apolipoprotein B (total apoB) in
the chylomicron and VLDLA fractions were analyzed in triplicate by using commercial apoB-48
(Human ApoB-48 ELISA kit, Shibayagi, Gunma, Japan) and total apoB (Total Human
Apolipoprotein B ELISA assay, ALerCHECK, Inc. Portland, Maine) ELISA kits. A 175-L
aliquot of each chylomicron fraction and a 60-µL aliquot of each VLDLA fraction were analyzed
by ELISA. The remainder of each lipoprotein fraction was used for fat-soluble micronutrient
analyses by HPLC with coulometric array electrochemical detection (HPLC-ECD). The
postprandial apoB-100 contents were obtained by subtracting the apoB-48 content from the total
apoB content within each chylomicron and VLDLA sample. The calculated apoB-100 contents
were accurate because the apoB-48 ELISA has no cross reactivity with apoB-100 (27, 28), and
the total apoB standard was calibrated against an international reference material (29).
To evaluate the precision of the ELISA assays, we used a pooled plasma chylomicron and
VLDLA fraction as a quality-control material. Excess plasma collected during the study was
69
combined and used to isolate the chylomicron and VLDLA fractions. The two fractions were
then pooled, divided into multiple vials, and stored at -70°C. Three replicates of the qualitycontrol material were routinely analyzed with each ELISA assay. The mean (± SD) apoB-48 and
total apoB contents in the quality-control material were 0.945 ± 0.0386 mg/L and 7.59 ± 0.264
mg/L, respectively. For the apoB-48 ELISA assay, the inter-assay CVs were 5.89% and 7.07%
in the chylomicron and VLDLA sample sets, respectively. For the total apoB ELISA assay, the
inter-assay CVs were 7.16% and 6.36% in the chylomicron and VLDLA sample sets,
respectively.
HPLC-ECD analyses of carotenoids and fat-soluble vitamins in the plasma triacylglycerolrich lipoprotein (TRL) fractions
The contents of the α- and β-carotene, lutein, lycopene, retinyl palmitate, phylloquinone,
and α- and γ-tocopherol in the plasma triacylglycerol-rich lipoprotein fractions were analyzed by
HPLC with coulometric array electrochemical detection (HPLC‒ECD). The retinyl palmitate
was analyzed as the major bioconversion product of the α- and -carotene consumed in the salad
vegetables. Each chylomicron/VLDLA sample (~2 mL) was deproteinated by addition of an
equal volume of methanol (Fisher Scientific, Chicago, IL), followed by two extractions with 4
mL of hexane (EMD Chemicals Inc. Gibbstown, NJ) containing 1 g/L butylated hydroxytoluene
(BHT) (Fisher Scientific, Chicago, IL) to extract carotenoids, retinyl palmitate, phylloquinone,
and tocopherols. The combined hexane layers were dried by using a speed vacuum evaporator
(Model SPD 131 DDA, Thermo Electron Corporation, Milford, MA) with a universal vacuum
70
system (UVS 800 DDA, Thermo Electron Corporation), and redissolved in 30 μL methanol
(EMD Chemicals Inc. Gibbstown, NJ) and 30 μL methyl-tert-butyl ether (MTBE) (Sigma, St.
Louis, MO). A 25 μL aliquot was injected into the HPLC−ECD system. The carotenoids,
retinyl palmitate, phylloquinone, and tocopherols were separated on a C30 Carotenoid Column
(Waters, Milford, MA) (30). The mobile phase consisted of methanol (EMD Chemicals Inc.,
Gibbstown, NJ), MTBE (Sigma, St. Louis, MO) and aqueous ammonium acetate buffer (1.0 M,
pH 4.6) (Sigma, St. Louis MO). The proportions in mobile phases A and B of
methanol:MTBE:ammonium acetate were 95:3:2 (by vol) and 25:73:2 (by vol), respectively.
The following gradient was used: 0-1 min, 100% mobile phase A; 1-4 min, linear gradient to
75% mobile phase B; 40-60 min, linear gradient to 100% mobile phase B. The flow rate was 1.0
mL/min. An ESA CoulArray system (Chelmsford, MA) was operated by using CoulArray
software (CoulArrayWin 2.0) and consisted of a 542 auto sampler set at 4°C, two 582 solvent
delivery modules, CoulArray thermal organizer, and a 16-channel 5600A CoulArray detector.
The applied detector cell potentials included the predominant response potential for carotenoids
(450 mV), retinyl palmitate (750 mV), and tocopherols (300 mV). For the simultaneous analysis
of phylloquinone, reductive potentials (-800 mV) were applied to two detector channels, which
were followed by two oxidative potentials (50 mV, 200 mV) on the subsequent two detector
channels. The 50 mV channel was used for the quantification of phylloquinone. The detection
limits for α-carotene, β-carotene, lycopene, lutein, phylloquinone, α-tocopherol, and γ-tocopherol
were 0.010 g/L, 0.008 g/L, 0.046 g/L, 0.046 g/L, 0.027 g/L, 3.872 g/L, and 0.272 g/L,
71
respectively. Trans-lycopene was the only lycopene isomer detected in the samples. All sample
preparations and extractions were performed under yellow light.
HPLC-ECD analyses of carotenoids and fat-soluble vitamins in the salad vegetables
Representative samples of the spinach, romaine lettuce, cherry tomatoes, and carrots from
the test salads consumed during each of the 6 weeks of the study were stored at -70°C and
analyzed in duplicate. A modification of the method of Granado et al. (30) was used to extract
and analyze carotenoids and tocopherols. Frozen vegetable samples were thawed at room
temperature before being processed in a food processor (Handy Chopper Plus HC3000; Black &
Decker Corp, Towson, MD). Briefly, carotenoids and tocopherols were extracted from 1 g of the
vegetable homogenate with 12 mL of methanol containing 1 g/L BHT (Fisher Scientific,
Chicago, IL) and 12 mL of tetrahydrofuran (THF) containing 1 g/L BHT (VWR, Boston, MA).
An 0.5 mL aliquot of the methanol/THF extract was transferred to a screw-capped test tube and
combined with 1.0 mL of 0.4 g/mL potassium hydroxide in methanol containing 0.1 M
pyrogallol. Each tube was flushed with argon and tightly capped before vortexing for 3 minutes.
Two mL of ultrapure water were added and the tube was vortexed again for 30 sec. Four mL of
hexane/methylene chloride (5:1 by volume) was added and thoroughly mixed by vortexing for
60 sec. The sample was then centrifuged to separate the organic phase. The organic phase was
evaporated to dryness under vacuum. The dried lipid extract was reconstituted with 1000 µL
each of MTBE and methanol; a 25 µL aliquot was injected into the HPLC-ECD system. All
procedures were carried out under yellow light.
72
The procedure for extracting phylloquinone from the vegetables was a modification of a
method of Koivu et al. (31). Ten milliliters of 2-propanol were added to 1 g of homogenized
vegetable sample. After vortexing for 2 min, 5 mL of hexane were added and the sample was
vortexed for 2 min. The sample was then re-extracted using another 5 mL of hexane. Five mL
of ultrapure water were added followed by vigorous shaking. Each sample was centrifuged for 5
min at 700  g to separate the organic and water phases. For romaine lettuce and spinach, the
total volume of the hexane layer was recorded and a 1 mL aliquot was evaporated to dryness
under vacuum. Thus, the phylloquinone content in 1 g of lettuce or spinach was obtained by
multiplying the content in 1 mL aliquot by the recorded total hexane volume. Because of the low
contents of phylloquinone in cherry tomatoes and carrots, the entire hexane layer (about 10 mL)
was dried down under vacuum. Each dried extract was reconstituted with 100 µL of MTBE and
100 µL of methanol. A 25 µL aliquot was injected into the HPLC-ECD system as described
above.
Statistical analyses
Statistical analyses were performed by using SAS (version 9.2; SAS Institute Inc., Cary,
NC). The outcome variables were the 0–9.5 h area under the curve (AUC) values in the plasma
chylomicron and VLDLA fractions for carotenoids, phylloquinone, retinyl palmitate,
tocopherols, apoB-48, apoB-100 and total apoB. The AUC values were calculated by the
trapezoidal method using analyte content (expressed as the content in the chylomicron or
VLDLA fractions isolated from 1 L plasma) as the y axis and time (h) as the x axis.
73
ANCOVA was used to estimate the differences among the salad dressings in the AUC
values of the fat-soluble micronutrients in the chylomicron and VLDLA fractions. Visual
inspection of the QQ plots of the residuals showed that log transformation of the AUC values
eliminated unwanted curvature. Therefore, the AUC values were log transformed to achieve
normal distributions and analyzed by mixed model analysis of covariance (ANCOVA) using
treatment and study period as fixed factors, subject as random factor, and the log of the baseline
(0 h) analyte content [log(baseline)] as a covariate. Log(baseline) was not a significant covariate
for phylloquinone in chylomicron and VLDLA fractions. Therefore, these outcome variables
were analyzed by a mixed model analysis of variance (ANOVA) with AUC as the dependent
variable and treatment and study period as fixed factors and subject as random factor. The need
for inclusion of treatment × study period and log(baseline) × treatment interaction terms was
evaluated by using a custom model that included treatment and covariate as main effects and also
these interaction terms. The log(baseline) × treatment interaction was not significant for any of
the analytes in either lipoprotein fraction (chylomicron or VLDLA) and thus this interaction term
was excluded from the model. There was a significant treatment × study period interaction for αcarotene and β-carotene in VLDLA; thus this interaction term was retained as an additional fixed
effect in the model for these outcome variables. Posthoc multiple comparisons of least squares
means were calculated by paired Student’s t-test using Tukey’s adjustment. P values < 0.05
were considered significant
74
The baseline (0 h) contents of carotenoids, retinyl palmitate, and tocopherols in the plasma
chylomicron fraction were compared with those in the VLDLA fraction by using paired
Student’s t-tests, which indicated significant baseline differences between the two fractions (P <
0.001). Due to these baseline imbalances, ANCOVA with baseline as covariate became
problematic. If log baseline values were used as a covariate, AUC values for each analyte in
each of the two lipoprotein fractions were modeled as least squares means. To adjust for
differences in baseline analyte content across subjects, the least squares mean for the AUC
values with log baseline as a covariate was calculated at the average baseline value for that
analyte across all subjects. However, this approach cannot adjust for the baseline differences
between fractions. Thus, the incremental AUC (iAUC) values were calculated and used to
compare the postprandial responses for each carotenoid or fat-soluble vitamin in the plasma
chylomicron versus VLDLA fractions. The iAUC, which was defined as the area under the
curve and above the baseline (0 h) value, was calculated by trapezoidal approximation. If the
postprandial content of a carotenoid or fat-soluble vitamin in a lipoprotein fraction at a specific
time point was lower than the baseline fasting content, then that area below the baseline was
included as a negative contribution toward the total calculated iAUC value. The iAUC values in
chylomicron and VLDLA fractions were compared by ANOVA with fraction and treatment as
main factors and treatment × fraction interaction as random factor.
Repeated-measures ANOVA was used to compare differences between the baseline (0 h)
and postprandial contents of carotenoids, fat soluble vitamins, apoB-48, and apoB-100 in the
75
plasma lipoprotein fractions at the individual time points, 2 h, 3.5 h, 5 h, 7 h, and 9.5 h. The
repeated-measures ANOVA involved treatment and time as fixed factors and subject and subject
× treatment as random factors. The least squares means were compared by post-hoc t-tests by
using Tukey’s adjustment.
For the analytes that had significant postprandial increments, the times to reach maximal
contents in the plasma lipoprotein fractions (tmax) were determined from visual examination of
the data. The tmax values of each analyte were compared between fractions by ANOVA with
fraction, treatment and treatment × fraction interaction as fixed factors. The least squares means
were compared by post-hoc t-tests.
The AUC values of apoB-48 and apoB-100 were compared between fractions by ANOVA
with fraction and treatment as main factors and treatment × fraction as random factor. The
apoB-48 AUC value and apoB-100 AUC values were compared within each fraction by
ANOVA with analyte and treatment as main factors and treatment × analyte as random factor. P
values < 0.05 were considered significant.
RESULTS
Carotenoid, phylloquinone, and tocopherol contents in the test salads
The carotenoid, phylloquinone, and tocopherol contents in the test salads are presented in
Table 1. Across the 6 wk of the study, the mean contents (± SEs) of total carotenoids (αcarotene, β-carotene, lutein, zeaxanthin, and lycopene), total tocopherols (α- and γ-tocopherols),
and phylloquinone in the 247-g test salads were 30.660 ± 0.430 mg, 4.786 ± 0.202 mg, and 0.153
76
± 0.006 mg, respectively. Thus, the contents of the bioactive components of interest in the raw
vegetables were remarkably consistent. The contents of α- and γ- tocopherol varied the most
among all of the analytes in the salad vegetables. Tocopherol contents in vegetables are
dependent on growing conditions, e.g., growing season, sunlight intensity, and soil state (32-34).
Postprandial changes in carotenoid and fat-soluble vitamin contents in the plasma
chylomicron and VLDLA fractions
There were no significant differences among the three salad dressings in the AUC values for
the carotenoids or fat-soluble vitamins in the postprandial chylomicron or VLDLA fractions.
The exception was a decrease in the AUC value of phylloquinone in the plasma chylomicron
fraction when the salad dressing containing 3.2 g soybean oil and 0.8 g Solec® 8120 was
consumed compared with the salad dressing containing 4 g soybean oil and 0 g Solec® 8120 (P
< 0.02, Figure 1). In contrast, the AUC values for phylloquinone in the VLDLA fraction were
not different between the three salad dressings. No significant differences were observed among
the salad dressings in the AUC values for apoB-48 and apoB-100 in the postprandial
chylomicron and VLDLA fractions.
The iAUC values for each analyte between chylomicron and VLDLA fractions were
compared. In this statistical model, the effects of the salad dressing treatments were not
significant for any of the analytes, including phylloquinone. In contrast, in the previous
statistical model (above), the chylomicron AUC value for phylloquinone was significantly lower
when salad vegetables were consumed with the salad dressing containing 0.8 g Solec® 8120
77
compared with the salad dressing containing 0 g Solec® 8120. This discrepancy reflects the
overwhelming predominance of the appearance of phylloquinone in the VLDLA fraction, no
matter which of the three salad dressings was consumed. In other words, the postprandial
distribution of phylloquinone between the chylomicron and VLDLA fractions was not affected
by the addition of Solec® 8120 hydroxylated soy lecithin to salad dressing in the amounts used
in the current study. The iAUC values for α-carotene, β-carotene, lycopene, retinyl palmitate,
and phylloquinone were substantially higher in the VLDLA than in the chylomicron fraction (P
< 0.05, Table 2). For example, β-Carotene, the major pro-vitamin A carotenoid in the salad
vegetables, was primarily accumulated in the VLDLA fraction with mean (± SE) iAUC value of
58.805 ± 8.608 nmol•h/L, whereas its mean (± SE) iAUC value in the chylomicron fraction was
18.515 ± 2.055 nmol•h/L. In addition, significant increases from baseline in the α-carotene, βcarotene, lycopene, retinyl palmitate, and phylloquinone contents were observed in both the
chylomicron and VLDLA fractions as early as 2 h after salad consumption (Figures 2 and 3).
The mean (± SE) difference for tmax values of α-carotene, β-carotene, lycopene, retinyl
palmitate, and phylloquinone in chylomicron fraction versus VLDLA fraction was -0.986 ±
0.505, -0.792 ± 0.492, -1.361 ± 0.868, -2.153 ± 0.592, -1.000 ± 0.6931, respectively. The tmax
values of α-carotene, β-carotene, lycopene, retinyl palmitate, and phylloquinone were
significantly lower in the chylomicron fraction than in the VLDLA fraction (P < 0.01), which
means the maximal contents of these fat-soluble micronutrients were reached earlier in the
chylomicron fraction compared with the VLDLA fraction (Figures 2 and 3).
78
In contrast with the other carotenoids, the mean iAUC value for lutein in the plasma
VLDLA fraction was negative (-5.086 ± 2.918 nmol•h/L), even though there was a substantial
amount of lutein in the salad vegetables (Table 1). The VLDLA lutein contents at 2, 3.5, 5, 7, or
9.5 h were not significantly different from the baseline (0 h) content (Figure 4). However, the
mean iAUC value for lutein in the plasma chylomicron fraction was positive (2.608 ± 0.465
nmol•h/L), and the plasma chylomicron lutein contents at 3.5 h and 5 h were significantly higher
than the baseline (0 h) content (P < 0.05). Thus, our data suggest that, unlike the other
carotenoids, newly absorbed lutein was primarily transported by the plasma chylomicron
fraction with little or no accumulation in the VLDLA fraction.
Under our study conditions, there were no postprandial increments in α- or γ-tocopherols in
either the plasma chylomicron or VLDLA fractions. The α- and γ-tocopherol contents in the
chylomicron fraction did not significantly change until 7 h; then significant decreases from
baseline were observed at both 7 h and 9.5 h (P < 0.01) (Figure 4). In the VLDLA fraction,
significant decreases in the α- and γ-tocopherol contents were observed as early as 3.5 h (P <
0.01) (Figure 4).
Overall, after consumption of the test salads with salad dressings containing only 4 g of
total lipids, we observed notable differences among the fat-soluble micronutrients in their
postprandial appearances in the plasma lipoprotein fractions.
Postprandial changes in apolipoprotein B contents in the plasma chylomicron and VLDLA
fractions
79
The plasma chylomicron and VLDLA fractions each contained both apoB-48 and apoB100, which is consistent with other studies (15, 29, 35-38). The majority of the postprandial
apoB-48 and apoB-100 was found in the VLDLA fraction (Figure 5). The mean (± SE) absolute
AUC values for apoB-48 in the chylomicron and VLDLA fractions were 0.535 ± 0.076 mg•h/L
and 7.518 ± 0.918 mg•h/L, respectively. The apoB-48 absolute AUC value in the VLDLA
fraction was significantly higher than that in the chylomicron fraction (P < 0.0001). Also, the
mean (± SE) apoB-48 content in the VLDLA faction at baseline (0 h) (0.778 ± 0.115 mg/L) was
about 10 times higher than the maximal postprandial mean (± SE) content of apoB-48 in the
chylomicron fraction (0.080 ± 0.015 mg/L, at 2 h) (Figure 5). Significant decreases in the apoB100 content from baseline were observed at 7 h in the chylomicron fraction (P < 0.01) and at 5 h,
7 h, and 9.5 h in the VLDLA fraction (P < 0.01), and significant increases in the apoB-48 content
from baseline were observed at 2 h and 3.5 h in the chylomicron fraction (P < 0.01) whereas no
significant changes apoB-48 content were found in the VLDLA fraction (Figure 5). One
lipoprotein particle only contains one type of apoB, and apoB-48 and apoB-100 are reliable
biomarkers for intestine- and liver-derived lipoproteins, respectly (18-20). Thus, the changes in
the apoB-48 and apoB-100 contents reflect the particle number changes in intestine- and liverderived lipoproteins, which means, in the postprandial state, the particle number of chylomicronsized enterogenous lipoproteins increased, and the particle number of chylomicron- and
VLDLA-sized hepatogenous lipoproteins decreased. The mean (± SE) absolute AUC values for
apoB-100 in the chylomicron and VLDLA fractions were 1.110 ± 0.130 mg•h/L and 60.705 ±
80
5.748 mg•h/L, respectively. As expected, the apoB-100 AUC value in the VLDLA fraction was
significantly higher as compared with the chylomicron fraction (P < 0.0001). Moreover, the
apoB-100 absolute AUC value was substantially higher than the apoB-48 AUC value in both the
plasma chylomicron and VLDLA fractions (P < 0.0001). Thus, apoB-100 had greater
postprandial appearance in both the chylomicron and VLDLA fractions than did apoB-48 after
salad consumption.
DISCUSSION
In the current study, the apparent intestinal absorption of fat-soluble micronutrients from
salad vegetables was measured based upon their postprandial appearances in the plasma
chylomicron and VLDLA fractions. Chylomicrons have long been regarded as the major
lipoproteins that transport newly absorbed fat-soluble vitamins and other fat-soluble bioactive
food components. However, recently our group and others reported that measuring the
appearance of retinyl esters in the VLDLA fraction is necessary to avoid underestimating total
vitamin A absorption (13, 14). Retinyl esters are formed in the enterocytes from provitamin A
carotenoids, incorporated into chylomicrons, and subsequently cleared into liver hepatocytes in
which they are hydrolyzed to retinol (39). The liver secretes vitamin A back into the plasma
primarily in the form of retinol bound to retinol-binding protein rather than in the form of retinyl
esters with hepatogenous lipoproteins (40). Thus, in the VLDLA fraction, the great increment in
retinyl palmitate content in the VLDLA fraction most likely reflected the quantity of newly
absorbed retinyl palmitate that was formed in the enterocytes from the α- and β-carotene
81
consumed in the salad vegetables. Moreover, it has been shown that the appearance of βcarotene in plasma after a single oral dose is biphasic (41, 42). The early peak at 5-6 h post
dosing represents the newly absorbed dietary β-carotene (43, 44), and the late and larger peak at
24-48 h post dosing represents hepatic secretion of β-carotene in liver-derived lipoproteins (12).
In the current study, we observed that the contents of α- and β-carotene, lycopene, retinyl
palmitate, and phylloquinone peaked at 3.5 h and 5 h in the chylomicron and VLDLA fractions,
respectively (Figure 2 and 3). Thus, the increments in the carotenoid and phylloquinone
contents in both lipoprotein fractions should reflect the quantities that were newly absorbed.
However, their tmax values in the two fractions were not completely coincident; the later tmax
within the VLDLA fraction than within the chylomicron fraction may reflect a precursor product
relation.
Based upon the AUC values, the postprandial appearance of α-carotene, β-carotene,
lycopene, retinyl palmitate, and phylloquinone in the VLDLA fraction greatly exceeded their
appearance in the chylomicron fraction (Table 2; Figures 2 and 3) (P < 0.05). Thus, the
majority of the newly absorbed fat-soluble micronutrients from the salad vegetables were
contained within the large VLDL fraction in the context of this study in which the co-consumed
salad dressing contained only 4 g of lipids. In our earlier study, when a large β-carotene dose (25
mg) was consumed with a 33-g fat load, Paetau et al. (12) reported that β-carotene was primarily
accumulated within the plasma VLDLA fraction and there was a somewhat lower accumulation
in chylomicrons. In a subsequent study from our group, Hu et al. (11) showed that postprandial
82
accumulations of β-carotene and its major bioconversion product, retinyl palmitate, were of
similar magnitude in the chylomicron and VLDLA fractions after a large (25 mg) dose of βcarotene was consumed with 60 g of either beef tallow or sunflower oil. These findings suggest
that the distributions of β-carotene and retinyl palmitate in the chylomicron and VLDLA
fractions may depend on the amount of co-consumed fat. When the meal is rich in fat, newly
absorbed β-carotene and retinyl palmitate are incorporated into chylomicron and VLDLA
fractions in similar proportion. When fat is limited, e.g. 4 g of total lipids in the current study,
the VLDLA fraction contains the major transport particles for newly absorbed β-carotene and
retinyl palmitate. Under the current study conditions, newly absorbed α-carotene, lycopene, and
phylloquinone were primarily accumulated in the VLDLA fraction instead of the chylomicron
fraction. This finding highlights the need for additional human studies regarding the effects of
the fat content of the test meal on the distributions of these fat-soluble micronutrients in plasma
lipoproteins, especially in chylomicrons and VLDLA particles. Our study highlights the
importance of measuring fat-soluble micronutrients in the postprandial VLDLA fraction to avoid
underestimating carotenoid (-carotene, -carotene, and lycopene), retinyl palmitate, and
phylloquinone absorption.
In contrast to the other carotenoids, newly absorbed lutein from the salad vegetables was
primarily incorporated into the plasma chylomicron fraction (Figure 4). Paetau et al. (12)
reported a rapid accumulation of canthaxanthin in chylomicron, VLDL and LDL fractions that
began as early as 2 h post-dosing. Canthaxanthin, which was used as a model xanthophyll
83
carotenoid, has plasma kinetics similar to those of lutein (45, 46). The xanthophyll distribution
difference in postprandial lipoproteins might be caused by differences in fat content. In our
current study, only 4 g of total lipids were consumed, whereas 33 g of fat were consumed in our
previous study by Paetau et al. These findings seem to suggest potential effects of fat content on
the lipoprotein distribution of xanthophyll carotenoids, and the fat effects may be different from
those on the distributions of other carotenoids, such as β-carotene.
There were no postprandial increments in α- or γ-tocopherols in either the plasma
chylomicron or VLDLA fractions when the salad was consumed with the test salad dressings
containing a total of only 4 g of soybean lipids (Figure 4). Reboul et al. (47) reported that αtocopherol is not secreted in chylomicrons when lipids are absent. Similarly, Anwar et al. (48)
showed that tocopherols were mostly incorporated into high density lipoproteins (HDL) in the
absence of oleic acid. In our previous study (49), subjects consumed test salads containing
amounts of salad vegetables that matched those in the current study. Significant increments in αand γ-tocopherol contents in chylomicrons were observed only when the test salads were
consumed with salad dressings containing 8 g or 32 g soybean oil. Therefore, in the current
study, the absence of newly absorbed α- and γ-tocopherols in chylomicron and VLDLA fractions
may be caused by the limited amount of co-consumed soybean oil and a resulting incorporation
into HDL.
In humans, apoB-48 is secreted by the intestine and is a reliable marker of lipoprotein
particles of intestinal origin (18-20). ApoB-100 is primarily synthesized by the liver and is
84
regarded as a marker of lipoprotein particles of hepatic origin (17, 20). Chylomicrons and
VLDLA are usually referred to as apoB-48- and apoB-100-containing lipoproteins, respectively
(16). However, increasing evidence, including our current study, indicates that the chylomicron
and VLDLA fractions each contain both apoB-48 and apoB-100 (15, 29, 35-38). The evidence
suggests that chylomicon and VLDLA fractions as isolated from human plasma each consist of
both enterogenous and hepatogenous lipoprotein particles. The chylomicrons and VLDLAs were
isolated based upon their particle densities and sizes. Thus, the presence of apoB-48 and apoB100 in both fractions shows that some postprandial apoB-48-containing lipoproteins have similar
particle sizes with apoB-100-containing lipoproteins, as hypothesized by Nakano et al. (50). The
AUC value of apoB-48 was substantially higher in the VLDLA fraction than in the chylomicron
fraction (P < 0.0001). This finding implies that more enterogenous lipoprotein particles were
contained in the VLDLA fraction, which can, at least partially, explain the greater postprandial
appearance of α-carotene, β-carotene, lycopene, retinyl palmitate, and phylloquinone in the
VLDLA fractions as compared with the chylomicron fraction. Likewise, a higher AUC value for
apoB-100 was observed in the VLDLA fraction than in the chylomicron fraction (P < 0.0001),
which means that, as expected, more particles of hepatic origin were accumulated in the VLDLA
fraction. We observed higher baseline contents of carotenoids, retinyl palmitate, and tocopherols
in the VLDLA fraction. These higher baseline contents may be explained by the fact that plasma
hepatogenous lipoprotein particles transport fat-soluble micronutrients, except retinyl palmitate,
out of the liver (4).
85
The greater postprandial appearance of apoB-48 in the VLDLA fraction compared with the
chylomicron fraction raises the question of the origin of the apoB-48-containing particles in
VLDLA. Two hypotheses may explain their origin: 1) they are the remnants of chylomicrons; 2)
chylomicrons are not the sole precursor for VLDLA and a significant proportion of apoB-48containing VLDLA particles are directly secreted by the intestine. The first hypothesis is based
on the metabolism of fat-soluble micronutrients (51-54). Fat-soluble micronutrients are taken up
by the enterocytes, incorporated into chylomicrons, and transferred into the blood circulation via
the lymphatic system (55). In the bloodstream, the chylomicron triacylglycerols are hydrolyzed
by the action of lipoprotein lipase, yielding chylomicron remnants (56, 57). This hydrolysis
process has been examined by labeling the triacylglycerol component of chylomicrons, and
kinetic analyses showed it occurs within minutes (51, 52). The second hypothesis was obtained
from a lipoprotein kinetics study by Zheng et al. (15) in which apoB-48 and apoB-100 were
labeled by stable isotopes. The study indicated that the human intestine and liver are able to
release various sizes of apoB-48 lipoproteins and apoB-100 lipoproteins, respectively, which
range in size from chylomicrons- and VLDLs-, down to IDL-sized lipoproteins. This finding
implies that intestinal and hepatic lipoproteins do overlap in particle size, which has been
supported by several recent studies (50, 58). This hypothesis is also applicable to explain the
origin of the apoB-100-containing particles in the chylomicron fraction in our study, i.e., they are
secreted by the liver. We also observed that the contents of α- and β-carotene, lycopene, retinyl
palmitate, and phylloquinone peaked significantly earlier in the chylomicron fraction compared
86
with the VLDLA fraction (P < 0.01). This finding can be explained by the first hypothesis, i.e.
the tmax delay in the VLDLA fraction was attributed to the lipolysis of chylomicron
triacylglycerols. However, we only had 5 postprandial blood collection points. Thus, it is
possible that we missed the actual tmax for both the fractions, and they might overlap each other.
Because a limited number of time points were applied in the current study, we cannot conclude
with certainty which hypothesis is more applicable to our data.
The apoB-100 AUC value was higher than the apoB-48 AUC value in both the chylomicron
and VLDLA fractions (P < 0.0001). Thus, the overall production of apoB-100 appears to be
much higher than that of apoB-48, which has been shown by other studies (15, 36). On the other
hand, the high content of apoB-100 may be caused by the delayed lipolysis of apoB-100containing lipoproteins that must compete for lipoprotein lipase active sites with apoB-48containing lipoproteins (51-54, 59). With the markedly higher apoB-100 content compared with
apoB-48, we may conclude that the major postprandial lipoprotein is apoB-100-containing
lipoprotein. We also observed significant decreases of apoB-100 content from baseline in both
the chylomicron and VLDLA fractions (P < 0.01), which is consistent with the finding in the
apoB-48/100 kinetic study by Zheng et al. (15) that the postprandial apoB-100 mass decreased in
the chylomicron and light VLDL fractions. The reduction in apoB-100 content suggests that the
number of hepatogenous lipoproteins decreases. Recently, it was reported that the major
increase in postprandial triacylglycerol was contained in apoB-100-containing lipoproteins
instead of in apoB-48-containing lipoproteins (59). Therefore, the decrease of apoB-100-
87
containing particle number may be compensated by the increase of particle size by which apoB100-containing lipoproteins contain more fat-soluble micronutrients than apoB-48-containing
lipoproteins. In the current study, the significant increase of apoB-48 content was observed only
in the chylomicron fraction, whereas Campos et al. (29) reported a increase in both chylomicron
and VLDL fractions but a decrease in LDL fraction after a high-fat (104 g) breakfast
consumption. One explanation is that a highfat load stimulates the intestine to secrete more large
VLDL-sized apoB-48 containing particles but less small LDL-sized apoB-48 containing particles
in order to adjust the absorbed dietary fat (29). The apoB-48/100 postprandial content depends
on its production rate and clearance rate both of which were not clear in the current study due to
no stable isotope tracers were applied (15, 60).
In the current study, the addition of Solec® 8120, a commercial hydroxylated soy lecithin to
salad dressing did not enhance the intestinal absorption of fat-soluble micronutrients from salad
vegetables. The AUC value for phylloquinone in the plasma chylomicron fraction was decreased
when the salad dressing containing 3.2 g soybean oil and 0.8 g Solec® 8120 was consumed
compared with the salad dressing containing 4 g soybean oil and 0 g Solec® 8120 (P < 0.02,
Figure 1). The decrease in the AUC value of phylloquinone may be attributed to the low
postprandial contents of phylloquinone in the chylomicron fraction. In the chylomicron fraction,
the maximal postprandial content of phylloquinone (0.23 ± 0.09 nmol/L) was about 20 and 5
times lower than those of β-Carotene (4.58 ± 1.27 nmol/L) and α-carotene (1.15 ± 0.36 nmol/L),
respectively, when 3.2 g soybean oil and 0.8 g Solec® 8120 were consumed. The bioavailability
88
of phylloquinone varies not only with the consumed fat content but also with the consumed fat
composition (5, 7). Jones et al. (61) reported that a polyunsaturated fatty acid-rich meal resulted
in lower phylloquinone absorption compared with the other two meals that contained 2-fold
lower polyunsaturated fatty acid. But no systematic studies have addressed the effects of lecithin
composition and/or content on the absorption of phylloquinone. Moreover, in our study,
phylloquinone was consumed from vegetables, whereas 20 μg of free 13C-labelled phylloquinone
supplement were consumed with a fat-rich meal in the study by Jones et al (61). Further
investigations are thus needed to thoroughly assess the roles of lecithin and polyunsaturated fatty
acid  food matrix interaction in phylloquinone absorption. Lysophosphatidylcholine, a
common component in commercial soy lecithins, was shown to enhance the bioavailability of βcarotene, lutein, and α-tocopherol in rats, mice, and Caco-2 human intestinal cells (9, 62-64).
The acyl chain length of phospholipids is also a critical factor influencing the bioavailability of
fat-soluble micronutrients (65). Yonekura and colleagues (65) reported that both medium-chain
phosphatidylcholine and long-chain lysophosphatidylcholine increased β-carotene uptake. In our
study, we may have observed enhanced bioavailability of the fat-soluble micronutrients in the
salad vegetables if homogeneous long-chain lysophosphatidylcholine had been added to the
salad dressings. However, pure lysophosphatidylcholine leads to lower quality salad dressings.
Among the commercially-available lecithins, the hydroxylated soy lecithin in Solec® 8120 was
determined to have the best product properties and taste profile. It also improved acid stability.
89
In the current study, the apoB-48 content in both fractions was analyzed by apoB ELISA
assay. The assay uses a newly available apoB-48-specific monoclonal antibody that was raised
against the apoB-48 C-terminal decapeptide (KLSQLQTYMI) and calibrated by using
recombinant apoB-48 antigen (27). The recovery of apoB-48 exceeds 90% (27). The ELISA
assay values are highly correlated with values obtained by gel scanning, which has often been
used to quantify apoB-48 (28, 16). However, compared with the ELISA assay values, the apoB48 values are underestimated by 50% with the gel scanning method (28). In the current study,
the intra- and inter-assay precision of the apoB-48 ELISA was evaluated by using a qualitycontrol material. The inter-assay CVs and mean intra-assay CV were excellent (< 8%) in both
the chylomicron and VLDLA samples. The high-recovery and sensitivity of the apoB-48 ELISA
confirm that it is a reliable method to quantify apoB-48 content in plasma samples.
In conclusion, the addition of Solec® 8120 hydroxylated soy lecithin to salad dressing in
the amounts used did not enhance the intestinal absorption of fat-soluble micronutrients from
salad vegetables. This study showed that the plasma VLDLA fraction contained the major
transport particles for α-carotene, β-carotene, lycopene, retinyl palmitate, and phylloquinone
over a 9.5-h period after consuming these bioactive components in salad vegetables with only 4 g
of total lipid in salad dressing. We showed that the plasma VLDLA fraction contains more
enterogenous (apoB-48) and hepatogenous (apoB-100) lipoprotein particles as compared with
the chylomicron fraction. In this study, the fat-soluble micronutrients were consumed as they
90
naturally occur within salad vegetables. Thus, our experimental conditions were similar to real
diets and the outcomes have more realistic and physiological meanings.
REFERENCES
1. Su LJ, Arab L. Salad and raw vegetable consumption and nutritional status in the adult US
population: Results from the third national health and nutrition examination survey. J Am
Diet Assoc 2006;106:1394-404.
2. Rock CL, Lovalo JL, Emenhiser C, Ruffink MT, Schwartz SJ. Bioavailability of β-carotene
is lower in raw than in processed carrots and spinach in women. J Nutr 1998;128:913-6.
3. Gärtner C, Stahl W, Sies H. Lycopene is more bioavailable from tomato paste than from fresh
tomatoes. Am J Clin Nutr 1997;66:116-22.
4. Stahl W, van den Berg H, Arthur J, Bast A, Dainty J, Faulks RM, Gärtner C, Haenen G,
Hollman P, Holst B, Kelly FJ, Polidori MC, Rice-Evans C, Southon S, van Vliet T, ViñaRibes J, Williamson G, Astley SB. Bioavailability and metabolism. Mol Aspects Med
2002;23:39-100.
5. Booth SL, Lichtenstein AH, Dallal GE. Phylloquinone absorption from phylloquinonefortified oil is greater than from a vegetable in younger and older men and women. J Nutr
2002;132:2609-12.
6. Dimitrov NV, Meyer C, Ullrey DE, Chenoweth W, Michelakis A, Malone W, Boone C, Fink
G. Bioavailability of beta-carotene in humans. Am J Clin Nutr 1988;48:298-304.
7. Gijsbers BL, Jie KS, Vermeer C. Effect of food composition on vitamin K absorption in
human volunteers. Br J Nutr 1996;76:223-9.
8. Nishimukai M, Hara H, Aoyama Y. Enteral administration of soyabean lecithin enhanced
lymphatic absorption of triacylglycerol in rats. Br J Nutr 2003;90:565-71.
9. Lakshminarayana R, Raju M, Krishnakantha TP, Baskaran V. Enhanced lutein bioavailability
by lyso-phosphatidylcholine in rats. Mol Cell Biochem 2006;281:103-10.
91
10. Greer FR, Marshall SP, Severson RR, Smith DA, Shearer MJ, Pace DG, Joubert PH. A new
mixed micellar preparation for oral vitamin K prophylaxis: randomised controlled
comparison with an intramuscular formulation in breast fed infants. Arch Dis Child
1998;79:300-5.
11. Hu X, Jandacek RJ, White WS. Intestinal absorption of beta-carotene ingested with a meal
rich in sunflower oil or beef tallow: postprandial appearance in triacylglycerol-rich
lipoproteins in women. Am J Clin Nutr 2000;71:1170-80.
12. Paetau I, Chen H, Goh NM, White WS. Interactions in the postprandial appearance of betacarotene and canthaxanthin in plasma triacylglycerol-rich lipoproteins in humans. Am J Clin
Nutr 1997;66:1133-43.
13. Borel P, Dubois C, Mekki N, Grolier P, Partier A, Alexandre-Gouabau MC, Lairon D, AzaisBraesco V. Dietary triglycerides, up to 40 g/meal, do not affect preformed vitamin A
bioavailability in humans. Eur J Clin Nutr 1997;51:717-722.
14. Li S, Nugroho A, Rocheford T, White WS. Vitamin A equivalence of the -carotene in betacarotene–biofortified maize porridge consumed by women. Am J Clin Nutr 2010;92:1105-12.
15. Zheng C, Ikewaki K, Walsh BW, Sacks FM. Metabolism of apoB lipoproteins of intestinal
and hepatic origin during constant feeding of small amounts of fat. J Lipid Res
2006;47:1771-9.
16. Karpe F, Hamsten A. Determination of apolipoproteins B-48 and B-100 in triglyceride-rich
lipoproteins by analytical SDS-PAGE. J Lipid Res 1994;35:1311-7.
17. Brodsky JL, Fisher EA. The many intersecting pathways underlying apolipoprotein B
secretion and degradation. Trends Endocrinol Metab 2008;19:254-9.
18. Lemieux S, Fontani R, Uffelman KD, Lewis GF, Steiner G. Apolipoprotein B-48 and retinyl
palmitate are not equivalent markers of postprandial intestinal lipoproteins. J Lipid Res
1998;39:1964-71.
19. Phillips ML, Pullinger C, Kroes I, Kroes J, Hardman DA, Chen G, Curtiss LK, Gutierrez MM,
Kane JP, Schumaker VN. A single copy of apolipoprotein B-48 is present on the human
chylomicron remnant. J Lipid Res 1997;38:1170-7.
20. Chan L. Apolipoprotein B, the major protein component of triglyceride-rich and low density
lipoproteins. J BiolChem 1992;267:25621-4.
92
21. Hill LS, Reid F, Morgan JF, Lacey JH. SCOFF, the development of an eating disorder
screening questionnaire. Int J Eat Disord 2010;43:344-51.
22. U.S. Department of Health and Human Services. Advance Data from Vital and Health
Statistics. Dietary Intake of Selected Vitamins for the United States Population: 1999-2000.
Centers for Disease Control and Prevention. National Center for Health Statistics. Number
339, 2004.
23. Rabbani S, Beyer P, von Lintig J, Hugueney P, Kleinig H. Induced beta-carotene synthesis
driven by triacylglycerol deposition in the unicellularalga Dunaliellabardawil. Plant Physiol
1998;116:1239-48.
24. Williams EJ. Experimental designs balanced for the estimation of residual effects of
treatment. Austr J Sci Res, Ser A 1949;2:149-68.
25. Redgrave TG, Carlson LA. Changes in plasma very low density and low density lipoprotein
content, composition, and size after a fatty meal in normo- and hypertriglyceridemic man. J.
Lipid Res1979;20:217-29.
26. Lindgren FT. Preparative ultracentrifugal laboratory procedures and suggestions for
lipoprotein analysis. In: Perkins EG, ed. Analysis of lipids and lipoproteins. Champaign, IL:
American Oil Chemists’ Society, 1975:204-24.
27. Kinoshita M, Kojima M, Matsushima T, Teramoto T. Determination of apolipoprotein B-48
in serum by a sandwich ELISA. Clin Chim Acta 2005;351:115-20.
28. Otokozawa S, Ai M, Diffenderfer MR, Asztalos BF, Tanaka A, Lamon-Fava S, Schaefer EJ.
Fasting and postprandial apolipoprotein B-48 levels in healthy, obese, and hyperlipidemic
subjects. Metabolism 2009;58:1536-42.
29. Campos H, Khoo C, Sacks FM. Diurnal and acute patterns of postprandial apolipoprotein B48 in VLDL, IDL, and LDL from normolipidemic humans. Atherosclerosis 2005;181:345-51.
30. Granado F, Olmedilla B, Herrero C, Perez-Sacristan B, Blanco I, Blazquez S. Bioavailability
of carotenoids and tocopherols from broccoli: in vivo and in vitro assessment. Exp Biol Med
2006;231:1733–8.
31. Koivu TJ, Piironen VI, Henttonen SK, Mattila PH. Determination of phylloquinone in
vegetables, fruits,and berries by high-performance liquid chromatography with
electrochemical detection. J Agric Food Chem 1997;45:4644-9.
93
32. Munné-Bosch S. The role of alpha-tocopherol in plant stress tolerance. J Plant Physiol
2005;162:743-8.
33. Hormaetxe K, Esteban R, BecerrilJM, Garcia-Plazaola JI. Dynamics of the alpha-tocopherol
pool as affected by external (environmental) and internal (leaf age) factors in
Buxussempervirensleaves. PhysiolPlant 2005;125:333-44.
34. Lizarazo K, Fernández-Marín B, Becerril JM, García-Plazaola JI. Ageing and irradiance
enhance vitamin E content in green edible tissues from crop plants. J Sci Food Agric
2010;90:1994-9.
35. Cartwright IJ, Higgins JA. Direct evidence for a two step assembly of apoB48-containing
lipoproteins in the lumen of the smooth endoplasmic reticulum of rabbit enterocytes. J Biol
Chem 2001;276:48048-57.
36. Johanson EH, Jansson PA, Gustafson B, Lönn L, Smith U, Taskinen MR, Axelsen M. Early
alterations in the postprandial VLDL1 apoB-100 and apoB-48 metabolism in men with
strong heredity for type 2 diabetes. J Intern Med 2004;255:273-9.
37. Mero N, Malmström R, Steiner G, Taskinen MR, Syvänne M. Postprandial metabolism of
apolipoprotein B-48- and B-100-containing particles in type 2 diabetes mellitus: relations to
angiographically verified severity of coronary artery disease. Atherosclerosis 2000;150:16777.
38. Battula SB, Fitzsimons O, Moreno S, Owens D, Collins P, Johnson A, Tomkin GH.
Postprandial apolipoprotein B48-and B100-containing lipoproteins in type 2 diabetes: do
statins have a specific effect on triglyceride metabolism? Metabolism 2000;49:1049-54.
39. Cifelli CJ, Green JB, Green MH. Use of model-based compartmental analysis to study
vitamin A kinetics and metabolism. Vitam Horm 2007;75:161-95.
40. Ross AC, Zolfaghari R. Regulation of hepatic retinol metabolism: perspectives from studies
on vitamin A status. J Nutr. 2004;134:269-75.
41. White WS, Stacewicz-Sapuntzakis M, Erdman JW Jr, Bowen PE. Pharmacokinetics of βcaroteneandcanthaxanthin after ingestion of individual and combined doses by human
subjects. J Am Coll Nutr 1994;13:665-71.
42. Swanson JE, Wang YY, Goodman KJ, Parker RS. Experimental approaches to the study of βcarotene metabolism: potential of a tracer approach to modeling 13-carotenekinetics in
94
humans. Adv Food Nutr Res 1996;40:55-79.
43. Cornwell DG, Kruger FA, Robinson HB. Studies on the absorption of β-carotene and the
distribution of total carotenoid in human serum lipoproteins after oral administration. J Lipid
Res l962;3:65-70.
44. Johnson El, Russell RM. Distribution of orally administered β-carotene among lipoproteins
in healthy men. Am J Clin Nutr l992;56:128.
45. White WS, Stacewicz-Sapuntzakis M, Erdman 1W Jr, Bowen PE. Pharmacokinetics of βcarotene and canthaxanthin after ingestion of individual and combined doses by human
subjects. J Am Coll Nutr 1994;13:665-71.
46. Kostic D, White WS, Olson JA. Intestinal absorption, serum clearance, and interactions
between lutein and beta-carotene when administered to human adults in separate or combined
oral doses. Am J Clin Nutr 1995;62:604-10.
47. Reboul E, Trompier D, Moussa M, Klein A, LandrierJF, Chimini G, Borel P. ATP-binding
cassette transporter A1 is significantly involved in the intestinal absorption of alpha- and
gamma-tocopherol but not in that of retinyl palmitate in mice. Am J Clin Nutr 2009;89:17784.
48. Anwar K, Iqbal J, Hussain MM. Mechanisms involved in vitamin E transport by primary
enterocytes and in vivo absorption. J. Lipid Res 2007;48:2028-38.
49. Agustiana A, Zhou Y, Flendrig L, White WS. The dose-response effects of the amount of oil
in salad dressing on the bioavailability of carotenoids and fat-soluble vitamins in salad
vegetables. FASEB J 2010;539.
50. Nakano T, Tanaka A, Okazaki M, Tokita Y, Nagamine T, Nakajima K. Particle size of apoB48 carrying lipoproteins in remnant lipoproteins isolated from postprandial plasma. Ann Clin
Biochem 2011;48:57-64.
51. Bjorkegren J, Packard CJ, Hamsten A, Bedford D, Caslake M, Foster L, Shepherd J, Stewart
P, Karpe F. Accumulation of large very low density lipoproteins in plasma during intravenous
infusion of a chylomicron-like triglyceride emulsion reflects competition for a common
lipolytic pathway. J Lipid Res 1996;37:76-86.
52. Karpe F, Hultin M. Endogenous triglyceride-rich lipoproteins accumulate in rat plasma when
competing with a chylomicron-like triglyceride emulsion for a common lipolytic pathway. J
95
Lipid Res 1995;36:1557-66.
53. Karpe F, HelleÂnius M-L, Hamsten A. Differences in postprandial concentrations of very
low density lipoprotein and chylomicron remnants between normotriglyceridemic and
hypertriglyceridemic men with and without coronary artery disease. Metabolism
1999;48:301-7.
54. Karpe F. Postprandial lipoprotein metabolism and atherosclerosis. J Intern Med
1999;246:341-55.
55. Yonekura L, Nagao A. Intestinal absorption of dietary carotenoids. Mol Nutr Food Res
2007;51:107-15.
56. Nestel, PJ. Relationship between plasma triglycerides and removal of chylomicrons. J Clin
Invest 1964;43: 943-9.
57. Grundy, S. M., and H. Y. Mok. Chylomicron clearance in normal and hyperlipidemic man.
Metabolism 1976;25:1225-39.
58. Sun F, Stolinski M, Shojaee-Moradie F, Lou S, Ma Y, Hovorka R, Umpleby AM. A novel
method for measuring intestinal and hepatic triacylglycerol kinetics. Am J Physiol
Endocrinol Metab 2013;305:1041-7.
59. Nakajima K, Nakano T, Tokita Y, Nagamine T, Yatsuzuka S, Shimomura Y, Tanaka A,
Sumino H, Nara M, Machida T, Murakami M. The characteristics of remnant lipoproteins in
the fasting and postprandial plasma. Clin Chim Acta 2012;413:1077-86.
60. Welty FK, Lichtenstein AH, Barrett PHR, Dolnikowski GG, Schaefer EJ. Human
apolipoprotein (apo) B-48 and apoB-100 kinetics with stable isotopes. Arterioscler Thromb
Vasc Biol 1999;19:2966-74.
61. Jones KS, Bluck LJ, Wang LY, Stephen AM, Prynne CJ, Coward WA. The effect of different
meals on the absorption of stable isotope-labelled phylloquinone. Br J Nutr 2009;102:1195202.
62. Sugawara T, Kushiro M, Zhang H, Nara E, Ono H, Nagao A. Lysophosphatidylcholine
enhances carotenoid uptake from mixed micelles by Caco-2 human intestinal cells. J Nutr
2001;131:2921-7.
63. Baskaran V, Sugawara T, Nagao A. Phospholipids affect the intestinal absorption of
96
carotenoids in mice. Lipids 2003;38:705-11.
64. Koo SI, Noh SK. Phosphatidylcholine inhibits and lysophosphatidylcholine enhances the
lymphatic absorption of alpha-tocopherol in adult rats. J Nutr 2001;131:717-22.
65. Yonekura L, Tsuzuki W, Nagao A.Acyl moieties modulate the effects of phospholipids on
beta-carotene uptake by Caco-2 cells. Lipids 2006;41:629-36.
TABLE 1
Carotenoid, tocopherol, and phylloquinone contents in the vegetables in the test salads1
Component
Weight
α-Carotene
β-Carotene
Lutein
Lycopene
g
α-Tocopherol γ-Tocopherol Phylloquinone
mg
7.709 ±
0.211 ±
0.3182
0.372
0.022
1.394 ±
1.603 ±
0.139
0.121
2.172 ±
3.572 ±
0.078
0.171
1.200 ±
0.187 ±
0.055
5.407 ±
0.318
66
Lettuce, romaine
48
‒
Spinach, leaf
48
‒
Tomato cherry
85
‒
Total
247
0.424 ±
0.056 ±
0.019
0.001
0.281 ±
0.277 ±
0.023
0.021
1.576 ±
0.148 ±
0.135
0.008
7.068 ±
1.479 ±
0.544 ±
0.014
0.306
0.065
0.060
12.475 ±
5.574 ±
7.068 ±
3.760 ±
1.026 ±
0.566
0.292
0.306
0.128
0.041
‒3
‒
‒
1
n = 6. A representative sample of each vegetable from each of the 6 weeks of the study was analyzed in duplicate.
2
All values are means ± SEs.
3
‒ Not detected.
0.002 ± 0.000
0.075 ± 0.006
97
5.407 ±
Carrot, grated
0.071 ± 0.005
0.004 ± 0.000
0.152 ± 0.008
TABLE 2
Incremental area under the response curve (iAUC) values for carotenoids, retinyl palmitate, and phylloquinone in plasma chylomicron
and VLDLA fractions after consuming the test salads1
α-Carotene
β-Carotene
Lutein
Lycopene
Retinyl palmitate
Phylloquinone
0.913 ± 0.363
27.853 ± 3.982
1.482 ± 0.212
4.634 ± 1.1474
113.382 ± 14.7845
3.340 ± 0.5044
nmol · h/L
4.507 ± 0.6552
18.515 ± 2.055
VLDLA
22.162 ± 3.5245
58.805 ± 8.6085 -5.086 ± 2.9183
1
2.608 ± 0.465
n = 12 women; There were no significant treatment differences and data were combined across the three salad dressing treatments.
The iAUC values in chylomicron and VLDLA fractions were compared by ANOVA with fraction and treatment as main factors
and treatment × fraction interaction as a random factor.
2
All values are means ± SEs.
3,4,5
Significantly different from chylomicrons,3P < 0.05, 4P < 0.01, 5P < 0.001.
98
Chylomicrons
99
FIGURE 1.Mean (± SE) changes from baseline (fasting) in the content of phylloquinone in the plasma chylomicron fraction after 12
women each consumed test salads with salad dressing containing 0 g, 0.2 g or 0.8 g Solec® 8120 hydroxylated soy lecithin. A
significant decrease (P < 0.02) in the chylomicron phylloquinone AUC values was observed when the salad dressing containing 0.8 g
Solec® 8120 lecithin was consumed compared with the salad dressing containing 0 g Solec® 8120.
100
FIGURE 2. Mean (± SE) changes from baseline in the contents of α-carotene, β-carotene, and lycopene in the plasma chylomicron
(A) and VLDLA (B) fractions. Repeated-measures ANOVA with Tukey’s adjustment was used to compare differences between the
baseline and postprandial values of α- and β-carotene and lycopene at 2 h, 3.5 h, 5 h, 7 h, and 9.5 h, respectively. Treatment and time
were included in the model as fixed factors and subject and subject × treatment interactions as random factors.*P < 0.05, **P < 0.01,
***
P < 0.001.
101
FIGURE 3. Mean (± SE) changes from baseline in the contents of retinyl palmitate and phylloquinone in the plasma chylomicron (A)
and VLDLA (B) fractions. Repeated-measures ANOVA with Tukey’s adjustment was used to compare differences between the
baseline and postprandial values of retinyl palmitate and phylloquinone at 2 h, 3.5 h, 5 h, 7 h, and 9.5 h, respectively. Treatment and
time were included in the model as fixed factors and subject and subject × treatment interactions as random factors.
***
P < 0.001
102
FIGURE 4. Mean (± SE) changes from baseline in the contents of lutein, α-tocopherol and γ-tocopherol in the plasma chylomicron
(A) and VLDLA (B) fractions. Repeated-measures ANOVA with Tukey’s adjustment was used to compare differences between the
baseline and postprandial values of lutein and tocopherols at 2 h, 3.5 h, 5 h, 7 h, and 9.5 h, respectively. Treatment and time were
included in the model as fixed factors and subject and subject × treatment interactions as random factors.*P < 0.05, **P < 0.01, ***P <
0.001
VLDLA (B) fractions. Repeated-measures ANOVA with Tukey’s adjustment was used to compare differences between the baseline
and postprandial values of lutein and tocopherols at 2 h, 3.5 h, 5 h, 7 h, and 9.5 h, respectively. Treatment and time were included in
the model as fixed factors and subject and subject × treatment interactions as random factors. **P < 0.01 The AUC values of apoB-48
and apoB-100 were compared between fractions by ANOVA analysis with fraction and treatment as fixed factors and treatment ×
fraction as random factor. The apoB-48 AUC value and apoB-100 AUC value were compared within each fraction by ANOVA
analysis with analyte and treatment as main factors and treatment × analyte as a random factor. The apoB-48/apoB-100 AUC value in
the VLDLA fraction was significantly higher than that in the chylomicron fraction (P < 0.0001). The apoB-100 AUC value was
substantially higher than the apoB-48 AUC value in both the plasma chylomicron and VLDLA fractions (P < 0.0001).
103
FIGURE 5. Mean (± SE) changes from baseline in the contents of apoB-48 and apoB-100 in the plasma chylomicron (A) and
104
CHAPTER IV
CONCLUSIONS AND PERSPECTIVES
Salad consumption is a major contributor to vegetable consumption in the United States.
Although it is associated with a greater likelihood of meeting the recommended intakes for
various nutrients, the bioavailability of fat-soluble micronutrients, including carotenoids,
tocopherols and phylloquinone, is low in raw vegetables, especially when co-consumed fat is
limited. To achieve high absorption of fat soluble micronutrients and meanwhile to avoid
having excessively high fat intakes, we were looking for a lipid that is more efficient in
increasing the bioavailability of fat-soluble micronutrients compared with triacylglycerol.
Lecithin was a potential candidate, because it is a structural component of mixed micelles
and an excellent emulsifier, and soybean lecithin was reported to enhance the absorption of
diet triacylglycerol and curcumin, a lipophilic constituent of the spiceturmeric.
Chylomicrons have long been regarded as the major transport particles for newly
absorbed fat-soluble micronutrients. Therefore, their postprandial appearance in
chylomicrons has been utilized as an indicator of intestinal absorption. However, in previous
studies, we observed that postprandial appearances of β-carotene and retinyl palmitate in the
plasma VLDLA fraction coincided with that in the chylomicron fraction in time and
magnitude, which implied the importance of VLDLA as a vehicle for assessing intestinal
absorption of β-carotene and its major bioconversion product, retinyl palmitate. Few human
studies have addressed the origin of VLDLA and the role of VLDLA in the transport of other
newly absorbed fat-soluble micronutrients, such as α-carotene, lycopene, lutein, tocopherols,
and phylloquinone.
105
We conducted a human study to investigate the effects of the lecithin/oil ratio in salad
dressing on the absorption of fat-soluble micronutrients, as well as the roles of plasma
chylomicrons and VLDLA in the transport of the absorbed fat-soluble micronutrients. There
were no significant differences among the salad dressings in the resulting AUC values of the
fat-soluble micronutrients in the chylomicron and VLDLA fractions. The exception was a
decrease in the AUC value of phylloquinone in the chylomicron fraction when the salad
dressing containing 0.8 mg hydroxylated soy lecithin (Solec® 8120) was consumed
compared with the salad dressing containing 0 g Solec® 8120 (P < 0.02). The AUC values
for α-carotene, β-carotene, lycopene, retinyl palmitate, and phylloquinone were substantially
higher in the VLDLA than in the chylomicron fraction (P < 0.05). Both chylomicron and
VLDLA fractions contained apoB-48 and apoB-100. ApoB-48 and apoB-100 were mainly
found in the VLDLA fraction (P < 0.0001). The AUC value of apoB-100 was significantly
higher than that of apoB-48 within each of the two fractions (P < 0.0001).
We concluded that the hydroxylated soy lecithin in the amounts added to the salad
dressing did not enhance the absorption of carotenoids, retinyl palmitate, phylloquinone, or
tocopherols. Over a 9.5-h period after consuming these bioactive components in salad
vegetables with only 4 g of salad dressing, the majority of the newly absorbed α-carotene, βcarotene, lycopene, retinyl palmitate, phylloquinone, as well as enterogenous (apoB-48) and
hepatogenous (apoB-100) lipoprotein particles, were contained within large VLDL. Thus
this lipoprotein subfraction has a major role in the transport of newly absorbed carotenoids
and fat-soluble vitamins.
In this study, the fat-soluble micronutrients were consumed as they naturally occur
within salad vegetables. Thus, our experimental conditions were similar to real diets, and the
106
outcomes may reflect the actual distribution of fat-soluble micronutrients in postprandial
triacylglyride-rich lipoproteins, i.e., the majority of newly absorbed fat-soluble
micronutrients is contained in the VLDLA fraction instead of the chylomicron fraction. This
finding highlights the importance of measuring fat-soluble micronutrients in the postprandial
VLDLA fraction to avoid underestimating carotenoid (-carotene, -carotene, and lycopene),
retinyl palmitate, and phylloquinone bioavailibility. Meanwhile, the findings of apoB-48 and
apoB-100 in both the chylomicron and VLDLA fractions further support the hypothesis that
apoB-48 and apoB-100 containing lipoproteins overlap in density. Although the study
design does not allow us to conclude with certainty the origin of VLDLA-sized apoB-48
containing lipoproteins, our findings indicated the chylomicrons and VLDLA can not be
simply regarded as enterogenous (apoB-48) and hepatogenous (apoB-100) lipoprotein
particles, respectively. In contrast, they are the mixtures of intestine- and liver-derived
particles. Thus, hepatic and intestinal lipoproteins should be separated by apoB isoform
(apoB-48 or apoB-100) instead of particle size/density.
Besides these major findings, we also observed some interesting phenomena that may
inspire future studies and provide new insights regarding the mechanism of fat-soluble
micronutrient absorption:
1) Under the current study conditions, newly absorbed β-carotene and its major
bioconversion product, retinyl palmitate, were primarily contained into the VLDLA fraction
instead of the chylomicron fraction. In contrast, our previous studies reported that the βcarotene was primarily secreted into the VLDLA fraction followed by a slightly lower
accumulation in chylomicron fraction when β-carotene dose (25 mg) was consumed with a
meal containing 33 g of fat, and postprandial appearances of β-carotene and retinyl palmitate
107
in VLDLA fraction were of similar magnitude of those in chylomicron fraction after
consumption of β-carotene (25 mg) with a fat-rich (60 g) meal. These findings suggest the
distributions of β-carotene and retinyl palmitate in chylomicron and VLDLA fractions may
depend on the fat content in the co-consumed meal. When the meal is rich in fat, newly
absorbed β-carotene and retinyl palmitate are incorporated into chylomicron and VLDLA
fractions with similar proportion. When the fat is limited, VLDLAs become the major
transport particles of newly absorbed β-carotene and retinyl palmitate. This hypothesis
requires further human studies in which fats with different amounts are consumed with
identical meals and the distributions of fat-soluble micronutrients in postprandial lipoproteins
are simultaneously investigated. These studies can also provide an insight regarding the
effect of fat content on the distributions of other fat-soluble micronutrients in the
postprandial lipoproteins.
2) In contrast to the other carotenoids, newly absorbed lutein was mainly incorporated
into the chylomicron fraction after the salad consumption, whereas no increments were
observed in the VLDLA fraction. This finding highlights the difference of intestinal
absorption between lutein and hydrocarbon carotenoids (e.g. α-carotene, β-carotene,
lycopene). Canthaxanthin has similar kinetics to those of lutein and has been shown to
accumulate in chylomicron, VLDL and LDL fractions as early as 2 h post-dosing after the
consumption of fat-rich (33 g) meal. It seems to suggest potential effects of fat content on
the lipoprotein distribution of lutein, and the effects may be different from those on the
distributions of other carotenoid, such as β-carotene. Moreover, ABCA1, a transmembrane
transporter mediating nascent HDL secretion from the intestine, is associated with plasma
lutein content and retinal pigment optical density. When co-consumed lipids are limited,
108
tocopherols are mostly incorporated into nascent HDL through ABCA1. Whether the
analogous pathway exists in lutein intestinal absorption could be an interesting topic. Further
investigations into the lutein appearance in the whole spectrum of lipoproteins with various
fat contents would be worthwhile as it may help to clarify the absorption and transport
mechanism of lutein.
3) Lutein had the strongest correlation with apoB-48 in the both chylomicron and
VLDLA fractions compared with the other fat-soluble micronutrients (Table 3). The
correlation coefficients between the relative contents of apoB-48 and each fat-soluble
micronutrient were calculated, where the relative content is defined as the value after
baseline correction (posprandial content - baseline). It is not clear that whether lutein is
tightly associated with apoB-48 in molecular level. But it has been reported that lutein is
able to activate ERK1/2 (extracellular-signal-regulated kinases) that facilitate the formation
of apoB-48 containing lipoproteins. More in vitro and in vivo studies are needed to further
explore the potential linkage between apoB-48 and lutein.
4) We found both chylomicron and VLDLA fractions contain apoB-48 and apoB-100.
Additional kinetics analysis is required to conclude which of the following hypotheses is
more applicable to our data: 1) apoB-48 containing VLDLAs are the remnants of
chylomicrons; 2) chylomicrons are not the sole precursor for VLDLAs, and a significant
proportion of apoB-48 containing VLDLA are directly secreted by the intestine. Two models
can be designed: 1) Model 1 provides for the direct secretion of apoB-48 and apoB-100 into
chylomicron and VLDLA fractions and allows apoB-48 and apoB-100 to leave the plasma at
any compartment; 2) Model 2 used chylomicrons as the sole precursor for VLDLA.
Simulation, Analysis, and Modeling Software (SAAM) can be used to find the best fit model.
109
TABLE 3. Correlation coefficients between the analytes and apoB-48 responses in
chylomicron and VLDLA fractions1, 2
Correlation coefficient R
In Chylomicrons
In VLDLA
Lutein
0.734
0.534
α-Carotene
0.493
0.295
β-Carotene
0.523
0.262
Lycopene
0.589
0.240
Retinyl palmitate
0.614
0.350
α-Tocopherol
0.452
0.407
γ-Tocopherol
0.464
0.468
Phylloquinone
0.591
0.264
1
Data were combined across the 3 salad dressing treatments.
2
All analytes were significantly correlated with apoB-48 in chylomicron and VLDLA
fractions, P < 0.001.
110
APPENDIX
TABLE 4. Hydroxylated soy lecithin (Solec® 8120) composition:
Phospholipid
Weight-%
Mol-%
PC
15.14
24.81
1-LPC
/*
/*
2-LPC
3.74
9.17
PI
8.19
12.37
LPI
2.19
4.84
PS-Na
/*
/*
LPS
/*
/*
SPH
/*
/*
PE
0.66
1.15
LPE
/*
/*
APE
12.83
16.35
PG
1.02
1.7
DPG
0.69
1.27
PA
5.55
10.23
LPA
1.11
3.25
Other
9.07
14.86
Sum
60.19
100
111
TABLE 5. Salad dressing aqueous base formulation:
Salad Dressing base formulation
wt%
Ultra pure reversed osmosis water
71.1458
Sugar
10.0000
Salt, refined
2.0000
Keltrol 521 CP kelco
0.5000
Waxy corn starch, E1422, Cluster S4R4
1.5000
Spirit vinegar 12%
8.0000
Citric acid monohydrate E330
0.1800
EDTA dissolvine
0.0075
112
TABLE 6. Amount of lipid and the salad dressing (SD) base on the salad
Hydroxylated
Soybean oil
Treatment
soy lecithin (g)
(g)
SD base (g)
Total (g)
1
0.00
4.00
56.00 g
60.00
2
0.20
3.80
56.00 g
60.00
3
0.80
3.20
56.00 g
60.00
113
ACKNOWLEDGEMENTS
I would like to take this opportunity to express my thanks to those who helped and
supported me in the past four and a half years. First and foremost, I’d like to express my
deep appreciation to my major professor Dr. Wendy White, who instructed, helped and
supported me throughout the entire research. Her insights and passions on research always
inspired me to pursue my dreams in the research field.
I am also grateful to my committee members: Dr. Don Beitz, Dr. Stephanie Clark, Dr.
Matthew Rowling, and Dr. Chong Wang. Thank them for providing valuable guidance and
thoughts in my POS meeting.
I am particularly indebited to my husband, Dr. Huaxun Ye. Thank him for being
together with me in the past twelve years. Whatever happened, he is always on my side to
support, encourage and help me. I appreciate him so much.
I would like to thank my parents who give the greatest love and support to me. I also
would like to thank my parents-in-law and my grandparents, especially my grandfather
Shiyao Wei, who passed away on January 3rd, 2014. Thank him for doing many things for
me and always consider everything on my side.
My appreciation also extends to my current and former lab members in Dr. White’ s
research team, including Dr. Yang Zhou, Agatha Agustiana, Hong You, Huanyi Jiang. Thank
them for all the help they gave me. It is also my duty to record my thankfulness to my
friends and collaborators: GarYee Koh, Nidhi Shah, Haili Dong, Yan Deng, Yong Zhu, Mei
Pan, Zhe Li, Liyan Chen.
Finally, I would like to thank Iowa State University for giving me an opportunity to be a
graduate student in such a great university and so beautiful small town.