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thesis entitled

ENDOGENOUS RETINOIDS
IN EARLY JAPANESE QUAIL EHBRYO

presented by

DING DONG

has been accepted towards fulfillment
of the requirements for

MASTER OF SCIENCE degree in HUMAN mmunon

 

 

 

Date AUGUST 3, 1995

 

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ENDOGENOUS RETINOIDS IN EARLY JAPANESE QUAIL EMBRYO
BY

DING DONG

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Food Science and Human Nutrition

1995

ABSTRACT
ENDOGENOUS RETINOIDS IN EARLY JAPANESE QUAIL EMBRYO
By

DING DONG

It has been demonstrated that vitamin A-active retinoids are required in stage 5-8 quail
embryos for normal development, including cardiovascular development, but their identity is
not known This study was undertaken to isolate, identify and quantitate by HPLC methods
the endogenous retinoids in quail eggs and embryos. Yolks of eggs from quail fed normal
chow diet were found to contain retinal, 3,4-didehydroretinol, and retinyl esters. No retinoids
were detected in the yolks of eggs from quail fed vitamin A-deficient diet supplemented with
13-cis-retinoic acid. Egg albumen did not contain any retinoids. Stage 5-8 embryos from
normal eggs were found to contain all-trans-retinoic acid, 3,4-didehydroretinoic acid, all-
trans-retinol, 3,4—didehydroretinol, retinal, and retinyl esters. No retinoids were detected in
the stage 5-8 embryos from vitamin A-deficient quail eggs.

The results suggest that all-trans-retinoic acid and 3,4-didehydroretinoic acid are
biogenerated in the early avian embryo at the time when cardiovascular development is
initiated, i.e. stages 5-8. The data support the hypothesis that the absence of bioactive
retinoids in the quail embryo at stage 5-8 is linked to an abnormal cardiovascular development
and embryo death. All-trans-retinoic acid and/or 3,4—didehydroretinoic acid are most likely
the active forms of vitamin A initiating normal cardiovascular development in the quail

embryo.

ACKNOWLEDGMENTS

I am most indebted to my advisor, Professor Maija H. Zile, for her constant
encouragement and support during my graduate study at Michigan State University. I would
like to thank her for suggesting the problem and the helpfirl direction which made this work
possible.

I would like to express my thanks and appreciation to the members of my graduate
committee, Professor Wanda L. Chenoweth and Professor Maurice R Bennink for their
valuable suggestions and precious time.

I wish to express my gratitude to my family, and fellow graduate students and

researchers.

iii

TABLE OF CONTENTS

1. INTRODUCTION .................................................. 1
1.1 Historical background of vitamin A .................................. 1
1.2 Structures of retinoids ............................................ 4
1.3 Vitamin A in human nutrition ...................................... 7
1.4 Metabolism of vitamin A .......................................... 8
1.5 Retinoids in avian cardiovascular development ......................... 22
1.6 Retinoids in avian limb development ................................ 24
1.7 Rationale and objectives of this study ................................ 25

1.7.1 Retinoids in quail eggs ...................................... 25
1.7.2 Retinoids in early quail embryos ............................... 26
1.7.3 Analysis of vitamin A-defrcient embryos ......................... 27

2. MATERIALS AND METHODS ...................................... 28
2.1 Animals and diets ............................................... 28
2.2 Quail embryo model ............................................. 30
2.3 Retinoids and chemicals .......................................... 30
2.4 Protein assay .................................................. 31
2.5 Retinoids in quail eggs from hens fed different diets ..................... 31

2.5.1 Sample preparation ........................................ 31
2.5.2 Extraction of lyophilized yolks and whites ....................... 31
2.5.3 HPLC analysis of retinoids ................................... 32
2.6 Extraction and HPLC analysis of retinoids from early normal quail embryos . . . 33
2.6.1 Sample preparation ........................................ 33
2.6.2 Pilot studies to establish methods of analysis ..................... 34
2.6.3 Extraction of 2000 pooled stage 5-8 normal embryos .............. 35
2.6.4 HPLC analysis of the extract from 2000 pooled normal embryos ...... 38
2.6.5 Methylation of retinoic acid and metabolites; characterization of
HPLC elution profiles of authentic methylated retinoids ............. 38
2.6.6 Characterization of unknown retinoic acids and other metabolites

by methylation and rechromatography .......................... 39
2.6.7 Characterization of non-polar retinoids ......................... 40
2.6.8 Quantitation of retinoids .................................... 40

2.7 Extraction and HPLC analysis of retinoids from early vitamin A—deficient
quail embryos ................................................. 43
2.7.1 Sample preparation ........................................ 43

iv

2.7.2 Extraction of 2100 pooled stage 5-8 embryos from

vitamin A—deficient eggs .................................... 43
2.7.3 HPLC analysis of the extract from 2100 pooled embryos
fi'om vitamin A-deficient eggs ................................. 44
3. RESULTS ....................................................... 45

3.1 Retinoid concentration in quail eggs obtained fi'om hens fed difi‘erent diets . . . . 45
3.2 HPLC profile of retinoids from the extract of 2000 pooled

normal quail embryos ............................................ 48
3.2.1 Characterization of retinoids in polar region A .................... 53
3.2.2 Characterization of retinoids in polar region B .................... 56
3.2.3 Characterization of retinoids in polar region C .................... 56
3.2.4 Characterization of retinoids in polar region D .................... 59
3.2.5 Characterization of retinoids in nonpolar region B ................. 59
3.2.6 Characterization of retinoids in nonpolar region F ................. 60
3.2.7 Quantitation of retinoids in normal quail embryos .................. 66

3.3 HPLC profile of retinoids from the extract of 2100 pooled
vitamin A—deficient embryos ....................................... 67
4. DISCUSSION .................................................... 72
4.1 Summary and conclusions ........................................ 85
4.2 Future research ................................................ 87
LIST OF REFERENCES .............................................. 89

LIST OF TABLES

Table 1. Recommended vitamin A intakes .................................. 7
Table 2. Data for methyl retinoate standard curve ........................... 42
Table 3. Retinoids in yolks of eggs from hens fed difl‘erent diets ................ 48
Table 4. Retinoid metabolites in stage 5-8 normal quail embryo ................ 66

LIST OF FIGURES

Figure 1. Carbon skeleton and the numbering system for retinoids ............... 5
Figure 2. Structures of all-trans-retinol and 3,4-didehydroretinol ................. 5
Figure 3. Structures of some biologically important retinoic acids ................ 6
Figure 4. Flow chart of the extraction of 2012 stage 5-8 normal embryos ......... 37
Figure 5. Standard curve for methyl retinoate .............................. 42
Figure 6. Standard curve for retinyl acetate ................................ 44
Figure 7. HPLC analysis of retinoids in quail eggs ........................... 47
Figure 8. HPLC profile of the extract from 2000 pooled normal embryos ......... 50
Figure 9. HPLC profile of authentic retinoids .............................. 52

Figure 10. Chromatography of methylated polar region A from HPLC in Figure 7 . . . 54
Figure 11. Chromatography of methylated polar region A from HPLC in Figure 7 . . . 55
Figure 12. Chromatography of methylated polar region B fi'om HPLC in Figure 7 . . . 57
Figure 13. Chromatography of methylated polar region C from HPLC in Figure 7 . . . 58

Figure 14. Chromatography of methylated polar region D from HPLC in Figure 7 . . . 61

Figure 15. Rechromatography of region B from HPLC in Figure 7 ............... 62
Figure 16. Rechromatography of region B from HPLC in Figure 7 ............... 63
Figure 17. Rechromatography of region F from HPLC in Figure 7 ............... 64
Figure 18. Rechromatography of region F from HPLC in Figure 7 ............... 65

Figure 19.

Figure 20.
Figure 21.

Figure 22.

HPLC profile of the extract fi'om 2100 pooled

stage 5-8 embryos from vitamin A-deficient quail eggs ............... 69
Rechromatography of peak A from HPLC in Figure 18 ............... 70
Chromatography of methylated peak A from HPLC in Figure 18 ........ 71

Proposed pathways of endogenous vitamin A metabolism
in early Japanese quail embryo ................................. 86

1. INTRODUCTION

1.1 Historical background of vitamin A

Vitamin A was the first vitamin to be discovered. Since vitamin A was identified as
an essential, fat-soluble nutrient in 1913 by McCollum andDavis, for eighty-two years it has
been the subject of continuous and fiuitfirl research. It is a nutrient that still fascinates the
public and nutritional. scientists alike. This fascination has prompted a succession of
productive investigations that have revealed the multiple and diverse roles of this essential
nutrient in a variety of tissues. Our present information about vitamin A, which is extensive
but still far from complete, has been derived through several distinct sources, which have
merged the contributions at various points into the general stream of knowledge about this
vitamin.

The first recognition of the existence of vitamin A as a nutritional factor capable of
correcting the inability to see properly at night dates back several thousand years. Eber's
Papyrus, an ancient Egyptian medical treatise of about 1500 BC, recommended roasted ox
liver, or the liver of a black cock, as curative agents. The famous Greek philosopher
Hippocrates also prescribed ox liver, but suggested that it should be eaten in a raw state after
dipping in honey (Moore, 1957). In 1915, McCollum and Davis took the initial step towards
subdivision of the vitamins by postulating the existence of two factors, Fat-soluble A and

Water-soluble B.

2

In 1925, a considerable advance in the study of vitamin A deficiency was made by
Wolbach and Howe, who provided a detailed description of the histological changes in
epithelia associated with vitamin A deficiency. Later, Green and Mellanby (1928) associated
vitamin A deficiency with infectious disease. Subsequently, remarkable chemical research led
to elucidation of the structure of B-carotene by Karrer and associates (193 0) and of retinol
(Karrer et al. 1931).

In 1937, Holmes and Corbet (1937) were able to crystalize pure retinol from fish liver.
A decade later, Arena and van Dorp (1946), and Isler and his associates (1947) succeeded in
achieving chemical synthesis of pure retinoic acid and retinol. Shortly after, the total synthesis
of B-carotene was reported by Karrer and Eugster (1950).

Many studies were conducted in the first half of this century dealing with various
aspects of the physiology and metabolism of vitamin A Particularly noteworthy was the
identification by Wald (1934) and by Morton (1944) of the chromophore of the visual
pigment as retinal. These various studies provided considerable information about the role of
vitamin A in vision (Wald, 1968) and about the pathology and pathophysiology of vitamin A
deficiency. By 1968, the stage was set for more sophisticated forays into metabolism,
regulation, function and therapeutic applications of vitamin A

Based on evidence that retinol was associated with the protein fi'action of plasma,
Goodman's laboratory isolated and partially characterized in 1968 the first retinol-binding
protein (RBP) (Kanai et al., 1968). Five years later, Bashor et al. (1973) reported that the
cytoplasm of cells also contained a protein which specifically binds retinol, named cellular
retinal-binding protein (CRBP). This important observation was quickly followed by

identification of cellular retinoic acid-binding protein (CRABP) (Sani and Hill, 1974).

3

Because the pharrnacologic use of vitamin A compounds is limited by their
tissue accurrmlation and intrinsic toxicity, organic chemists have worked since the late 19608
(Bollag, 1983) to create analogs of retinol and retinoic acid with good biological activity and
reduced toxicity. In the late 19703 and early 1980s, as the result of a search for
anticarcinogenic compounds, the "retinoid revolution” occurred (Olson, 1994a), namely, the
synthesis of very large numbers of compounds that possessed either the biological activity and
(or) the therapeutic utility of vitamin A against certain skin disorders and some neoplasms.
"Retinoids" were defined as natural vitamin A compounds and synthetic derivatives of it, with
or without biological activity. The search still continues for retinoid compounds with
therapeutic effectiveness and little or no toxic side effects.

The mechanism of retinoid action remained unclear until 1987 when experiments in
the laboratories of Chambon (Petkovich et al., 1987) and Evans (Giguere et al., 1987) showed
that the nuclei of cells contain retinoic acid receptors (RARs) with strong homology to the
steroid hormone/thyroid hormone/vitamin D receptor family. Since then, a more distinctly
related family of "orphan receptors", the retinoid X receptors (RXRs) have been shown to
have low afi'rnity for all-trans-retinoic acid , but to function as coregulators with the RARs
(Mangelsdorf et al., 1990). A search for putative retinoid ligands of RXR has revealed 9-cis-
retinoic acid as a strong activator of this receptor family (Heyman et al., 1992). Recent
advances in understanding the actions of retinoids in the nucleus have pointed to diverse
genes that the nuclear retinoid-receptor proteins which are transcription factors, may
potentially regulate. Much of the current vitamin A research is focusing on the understanding
of the types, or possrbly groups of genes that may be responsive to retinoic acid. Of particular

interest is the understanding of the expression of the retinoic acid receptors and the genes

4

responsive to retinoic acid or other forms of vitamin A during development, normal cellular

differentiation and neoplastic transformation.

1.2 Structures of retinoids

"Vitamin A" is a generic term that designates any compounds possessing the biological
activity of retinol, while the term ”retinoids” includes both naturally occurring forms of
vitamin A and the many synthetic analogs of retinol, with or without biological activity
(IUPAC-IUB, 1982).

The natural retinoids are 20 carbon isoprenoids with a B-ionylidene ring, a side chain
with conjugated double bonds allowing for a number of isomeric configurations, and a
terminal functional group in one of three oxidation states. The carbon skeleton and commonly
used system for numbering the carbon atoms of the naturally occurring retinoids, retinol,
retimrl, and retinoic acid, as well as their derivatives is shown in Figuil (F rickel, 1984). Of
interest in the present study are two forms of retinol: all-trans-retinol and 3,4-didehydro—
retinal (EM), and five forms of retinoic acid: all-trans-retinoic acid, 3,4-didehydroretinoic
acid, 9-cis-retinoic acid, 13-cis-retinoic acid, and 4-oxo-all-trans-retinoic acid (EMS).

Retinoids are hydrophobic compounds that are unstable in the presence of oxygen and
yield a mixture of dehydrated and double-bonded rearrangement products in acids. Light
catalyzes double-bond isomerization of most retinoids. \Vrth light of higher intensity, other
photochemical reactions take place, leading to dimerization and the formation of kitol and
other polymers (Blomhofi‘, 1994). These properties require that retinoids be handled

experimentally in an inert atmosphere under dim illumination, and avoiding contact with acids.

Figure 1. Carbon skeleton and the numbering system for retinoids

' CH,0H
‘ \ \ \ \

all-trans-retinol

\ \ \ CHZOH

3,4-didehydroretinol

Figure 2. Structures of all-trans-retinol and 3,4-didehydrorctinol

\\\\

E
E

all-trans-retinoic acid

\\\\coon

3,4-didehydroretinoic acid

9-cis-retinoic acid

‘1\\\
COOH

13-cis-retinoic acid

‘l\\\\COOH

O

4-oxoretinoic acid

Figure 3. Structures of some biologically important retinoic acids

1.3 Vitamin A in human nutrition

Olson (1994b) has identified five states of vitamin A nutriture: deficient, marginal,
satisfactory, excessive, and toxic. Clinical, histological, physiological, biochemical, and dietary
indicators have been developed to assess vitamin A status (Olson, 1992; Underwood and
Olson, 1993). Children with marginal vitamin A status are generally at risk of mortality and
the incidence of severe morbidity (Sommer et al., 1983; Sommer 1994); therefore, particular
attention is currently being given to indicators that measure marginal status. Three indicators
that have been very useful in this regard are: conjunctiva] impression cytology (CIC) and the
two response assays: the relative dose response (RDR) and the modified relative dose
response (MRDR) (Underwood and Olson, 1993).

The recommended vitamin A intakes of FAQ/WHO (Food and Agriculture
Organimtion, 1988) and National Research Council (NRC) (Food and Nutrition Board, 1989)

are given in Table 1.

Table 1. Recommended vitamin A intakes

( in pg of Retinol Equivalents, RE)

 

FAO/WHO, 1988 NRC, 1989

 

 

Category

Basal Safe RDA
Young infants 180 350 375
Adult males 300 600 1000
Adult females 270 500 800
Pregnancy +100 +100 +0

Lactation +180 +100 +500

 

8

One international unit (IU) of vitamin A is defined as 0.3 pg of all-trans-retinol. For
nutritional purpose, a better term is "retinol equivalents" (RE), which converts all dietary
sources of vitamin A and carotenoids into a single unit. Thus, 1 pg of all-trans-retinol equals
1 RE. Generally, 1 pg of retinol is assumed to be biologically equivalent to 6 pg of [3-
carotene or 12 pg of mixed dietary carotenoids (IUPAC-IUB, 1982).

The term ”vitamin A” is conventionally used to include both preformed vitamin A, i.e.
the biologically active derivatives of retinoL and provitamin A carotenoids that can serve as
precursors of vitamin A. Preformed vitamin A is found in very high concentrations in
vertebrate livers and fish liver oils and , to a moderate degree, in milk and eggs. In essence,
preformed vitamin A is largely found in foods of animal origin. In contrast, carotenoids are
present in a variety of fiuits and vegetables (Olson 1994b). A large number of other foods
contain substantial amounts of either vitamin A or carotenoids, and many of these foods are
widespread and inexpensive. However, vitamin A deficiency remains a major public health
problem among children in many countries, particularly in the less industrialized world and

also in specific disadvantaged population groups in the industrialized countries.

1.4 Metabolism of vitamin A

In most animal tissues, the predominant retinoid is retinyl palmitate, but other fatty
acid esters, such as retinyl oleate and retinyl stearate, are also found. Most of these
metabolites occur in all-trans configuration. The ll-cis aldehyde form, ll-cis-retinal, is the
chromophore in the retina of the eye, and several acid forms such as the all-trans-, 3,4-
didehydro- and 9—cis retinoic acids, are active metabolites of retinol found in most if not all

tissues. Many more metabolites of retinol are detected in difl‘erent tissues. It is now clear that

9

many cells contain one or more distinct cytoplasmic proteins that specifically bind retinol or
retinal or retinoic acid. Four of the cellular retinoid-binding proteins ( CRBP-I, CRBP-II,
CRABP-I, and CRABP-II) are well-characterized members of FABP (fatty acid binding
protein)/CRBP family of soluble, ~15 kDa, proteins that each bind a single molecule of ligand
(See Ong et al., 1994 for review).

Vitamin A exists in the plant world only in the form of precursor compounds, the
carotenoids. The most efi‘ective provitamin A is B-carotene, a member of a large class of
naturally occurring carotenoids. In the intestine, the provitamin A molecule is split and at
least one intact molecule of a vitamin A compound, i.e. retinal, or retinoic acid can be
obtained. Relatively little is known quantitatively of the efficiency of intestinal absorption of
provitamin A carotenoids (Blomhofi‘ et al., 1991a). B-carotene, and presumably the other
provitamin A carotenoids, can undergo oxidative cleavage to form retinoids by one of two
pathways. The central cleavage pathway involves attack and subsequent cleavage at the
central double bond to form at least one molecule of retinal (Olson and Hayaishi, 1965;
Goodman et al., 1966). Depending on the reduction potential of the tissue and the availability
of appropriate enzymes, this retinal can be either reduced to retinol or oxidized to retinoic
acid. The excentric pathway involves oxidative attack and subsequent cleavage at any of the
double bonds in the polyene chain of carotenoids to form apocarotenoids (Glover, 1960;
Sharma et al., 1976; Sharma et al., 1977). The apocarotenoids may be firrther processed to
retinoic acid (Krinsky et al., 1993; 1994). Several publications (Tang et al., 1991; Wang et
al., 1992; Krinsky et al., 1993) have reported that citral, an inhibitor of the oxidative
formation of retinoic acid fiom retinal, can be used to differentiate between the two pathways.

These studies have shown that B-carotene and D-apocarotenal can be converted to retinoic

10

acid without any interference by citral, and thus have provided evidence for the formation of
vitamin A-active compounds from provitamin A by excentric cleavage. Clearly, the
conversion of carotenoids to retinoids in viva should be firrther investigated.

Retinyl esters fi'om the diet are hydrolyzed in the intestinal lumen. Several enzymes,
including pancreatic lipase, pancreatic carboxyl ester lipases, and one or more retinyl ester
hydrolases associated with the brush border membranes (Harrison, 1993) have been
implicated in this hydrolysis. Although the relative roles of these enzymes in the digestion of
retinyl esters remain to be determined, it has been suggested that each may play a role
because of their ability to hydrolyze retinyl esters in difi‘erent physicochemical forms
(Harrison, 1993). Published data suggest that absorption of retinol is less than 75%, and that
it is dependent on both the quantity and quality of dietary fat (Blomhofi‘et al., 1991a). More
research is needed to determine what factors influence retinol absorption.

Almost all of the retinol absorbed into the enterocytes leaves via the lymphatics as
retinyl esters in chylomicra. Two enzymes have been identified as being important for the
esterification of retinol in enterocytes: acyl CoAzretinol acyltransferase (ARAT) (Helgerud
et al., 1982; 1983) and lecithin2retinol acyltransferase (LRAT) (Ong et al., 1987; MacDonald
and Ong, 1988). Ong and his collaborators found that retinol complexed to CRBP-II was
csterified by LRAT (MacDonald and Ong, 1988). In contrast, uncomplexed retinol in
membranes may be esterified by ARAT. It has been suggested (Blomhofi‘ et al., 1991a) that
LRAT esterifies retinol during the absorption of a normal load of retinol, and ARAT esterifies
excess retinol when large doses are absorbed and CRBP-II becomes saturated. Thus, CRBP-
Il may play a critical role in the normal carrier-mediated absorption of retinol. Free retinol,

if present in excessive amounts, can disrupt normal membrane structure and function. Thus,

11

the intracellular binding proteins play an important role in facilitating normal metabolism of
retina] and also in protecting cells fi'om free retinol incorporating into the membranes.

Almost all of the retinyl esters present in the chylomicra remain with this particle
during conversion to chylomicron remnants. Although chylomicron remnants are mainly
cleared by the liver, extrahepatic uptake of remnants may be important in the delivery of
retinol and carotenoids to extrahepatic tissues such as bone marrow, peripheral blood cells,
spleen, adipose tissue, skeletal muscle, kidney, and lung. In light of the importance of
retinoids in regulating gene expression and cellular difi‘erentiation, chylomicra may be an
important transport complex for delivering retinol and carotenoids to tissues with intensive
cell proliferation and difi‘erentiation (Blomhofi‘ et al., 1990a; Blomhofi‘ et al., 1991b).

Most of the absorbed dietary vitamin A is delivered to hepatic parenchymal cells
(hepatocytes) when chylomicron remnants are metabolized by the liver. Retinyl esters are then
hydrolyzed at the plasma membrane or in early endosomes. It is not clear which of the several
hepatic retinyl ester hydrolases is responsible for this hydrolysis, but the retinyl ester hydrolase
activity described by Harrison and Gad (1989) is probably the best candidate. Retinol is
subsequently found in endosomes with other ligands that are taken up by receptor-mediated
endocytosis (Blomhofi‘ et al., 1985a). In contrast to many other ligands that are transferred
to lysosomes after processing in endosomes, retinol is transferred to the endoplasmic
reticulum, where RBP is found in high concentration. Binding of retinol to RBP apparently
initiates a translocation of retinol-RBP from endoplasmic reticulum to the Golgi complex,
followed by secretion of retinol-RBP fiom cells (Ronne et al.,1983). The mechanism for RBP
retention in absence of retinol is not well understood. Most of the chylomicron remnant

retinyl esters taken up by hepatocytes are transferred as retinol to stellate cells in the liver

12

(Blomhoff et al, 1982). Since stellate cells can take up the RBP-retinol complex and since
hepatocytes secrete retinol bound to RBP (Blomhofi‘ et al., 1985b), it was suggested that RBP
mediates the transfer of retinol from hepatocytes to stellate cells. During an in situ perfusion
of rat livers, it was observed that labeled retinol was transferred from hepatocytes to stellate
cells. Furthermore, antibodies against RBP blocked the transfer, indicating that RBP was the
transport protein mediating transfer of retinol from hepatocytes to stellate cells (Blomhofl‘ et
al., 1988).

In mammals 50-80% of the total retinol in the body is normally present in the liver.
Under most conditions, stellate cells contain 90-95% of the liver retinol. Most (98%) of the
stellate cell vitamin A is in the form of retinyl esters packed together in cytoplasmic lipid
droplets. The normal reserve of vitamin A in stellate cells is adequate to last for several
months (Moriwaki et al, 1988; Blomhofl‘et al., 1990b; Blomhofl‘ et al., 1991a). Extrahepatic
vitamin A-storing stellate cells are found in higher vertebrates when excessive doses of
vitamin A are administered (Blomhofl‘ et al., 1991b). It is not clear at present whether these
cells also play a role in retinal metabolism under normal conditions.

The relative importance of the two enzymes, ARAT and LRAT, that have been
implicated in retinol esterification, varies between cell types. LRAT seems to be the main
intestinal enzyme esterifying retinol under normal conditions. LRAT was also recently
identified in liver stellate cells (Blaner et al., 1990). The high level of CRBP-I (Blomhofl‘ et
al., 1985c) in stellate cells points to an important role for LRAT in stellate cell retinol
esterification. Expression of both CRBP-I (Smith et al., 1991) and LRAT (Matsuura and
Ross, 1993) is induced by retinoids. When retinol is present in normal amounts, CRBP-I

directs it to LRAT for esterification in stellate cells. When the vitamin is present at high levels

13

and CRBP-I becomes saturated, ARAT may esterify the excess. The massive storage of
retinyl esters in stellate cells, and the ability of cell to control the mobilization of retinol,
ensures that the plasma retinol concentration is close to 2 pM in spite of the normal
fluctuations in daily vitamin A intake in human It is likely that the retinoid-regulated CRBP-I
and LRAT expression, and the saturation of CRBP-I and RBP by retinol, are the main
regulators of retinol uptake, storage, and mobilization by stellate cells.

Two mechanisms have been suggested for retinol mobilization fiom stellate cells.
First, it may be transferred fi'om stellate cells to hepatocytes before secretion of retinol-RBP
from the hepatocytes. This mechanism was suggested by the observation that cultured
hepatocytes synthesize and secrete RBP. It has been assumed that hepatocytes are the
exclusive site of retinol mobilization from liver (Blanner, 1989). Second, data now available
suggest that a direct mobilization of retinol fi'om stellate cells to the general circulation also
may occur, and may be the predominant pathway. One line of evidence that supports this
possibility comes from a study by Green et al. (1993), which uses a whole-body multi-
compartrnental model of retinol dynamics in rats. The model suggests that stellate cells are
the mq'or hepatic site of retinol secretion into blood. The model predicts that the retinol pool
in stellate cells responsible for the secretion is small and is rapidly tuming over; this is
compatible with the relatively small amounts of RBP observed in stellate cells.

Approximately 95% of the plasma RBP is associated with transthyretin (TTR)-retinol
(1:1, mol:mol) complex and, except in the postprandial state, essentially all of the plasma
vitamin A is bound to RBP. Complexing with 'ITR reduces the glomerular filtration of retinol.
As a result of extensive work in the laboratories of Goodman and Peterson, as well as in those

of other researchers, RBP has been well characterized (see Blaner and Olson, 1994 for

14

review). Until recently, it was thought that liver parenchymal cells were the primary, if not
the exclusive, site of RBP synthesis. Most of the RBP secreted by the liver contains retinol
in a 1:1 molar ratio; retinol binding to RBP is required for the normal release of retinol from
liver. It has now been established, mainly by work fiom Green's laboratory (1994), that retinol
recycles among plasma, liver, and extrahepatic tissues. As a consequence, a significant
fraction of the total input of retinol into plasma is from extrahepatic tissues. The extensive
recycling of retinol raises intriguing questions about the function of extrahepatic RBP
synthesis and about whole-body retinol homeostasis.

A number of retinoids other than retinol and retinyl esters are also present in plasma,
in nanomolar concentrations. These include all-trans-retinoic acid, 13-cis-retinoic acid, 13-cis-
4-oxoretinoic acid, and all-trans-retinoyl-B-glucuronide. The level of most of these retinoids
is dependent on the intake of vitamin A and will typically increase two- to four-fold after
ingestion of a large amount of vitamin A (see Blomhofi‘ et al., 1992 for review).

The mechanism by which retinol is taken up by cells is not yet firlly understood.
Retinol is transported to cells in two major forms by two principal carriers: as esterified
retinol in chylomicra secreted by intestinal enterocytes, and as retinol carried between the liver
and peripheral organs by RBP. The quantity of chylomicron retinyl ester varies directly with
dietary vitamin A intake. The concentration of retinol bound to plasma RBP is maintained
at a nearly constant level (1-3 pM) depending on age and other factors (Life Science
Research Office, 1985). The concentration of cytoplasmic CRBP may determine the capacity
of cells for retinol accumulation and thus serve to regulate cellular retinol uptake (Harrison
et al., 1987; Noy and Blaner, 1991). Enzymatic reactions by which retinol is removed would

also be expected to contribute to retinol flux within the cells. In retinal pigment epithelium

15

cells, retinol uptake has been proposed to be closely coupled to retinol esterification, which
may involve a membrane-associated form of retinol-binding protein (Ottonello et al., 1987).

Cytoplasmic all-trans-retinol occupies an important branch point fi'om which reactions
may lead to esterification, oxidation, hydroxylation, isomerization, glucuronidation or release
as the retinol-RBP complex. In vivo, the oxidation of retinal to retinoic acid is irreversible,
accounting for the inability of exogenous retinoic acid to support vision in spite of its ability
to restore growth and difi‘erentiation of most tissues in the retinol-depleted animal (Dowling
and Wald, 1960). Other irreversible oxidation reactions lead to a number of more polar
products, including the 4- and 18- hydroxylated metabolites of retinol and retinoic acid (see
Ross, 1993 for review).

Retinol and retinal are interconvertible through oxidation and reduction reactions. It
is likely that retinol dehydrogenase is important in viva (Posch et al., 1989). Multiple routes
of retinoid oxidation are indicated by the conversion of retinol to retinal in both tissue
microsome (Leo et al., 1987) and cytosol fi'actions (Kim et al., 1992). Retinal-binding
proteins have been irnplicated as important determinants of some of the redox reactions
involving retinal pigment epithelium, liver, and other tissues (Saari and Bredberg, 1982;
Kakkad and Ong, 1988; Posch et al., 1991).

Since the discovery of nuclear receptors for retinoic acid (Petkovich et al., 1987;
Giguere et al., 1987), increasing attention has focused on the processes that generate and
maintain cellular retinoic acid levels. The fasting plasma all-trans-retinoic acid level is in the
range of 4-14 nM in humans (De Leenheer et al., 1982; Eckhofl‘ and Nau, 1990) and 7.3-9
nM in rats (Cullum and Zile, 1985; Napoli et al., 1985). Plasma retinoic acid can be derived

fiorn endogenous metabolism in tissues. Circulating retinoic acid is bound to serum albumin

16

but not to RBP (Smith et al.,1973). At physiological pH, uncharged retinoic acid can cross
membranes rapidly and spontaneously (Nay, 1992). In addition to all-trans-retinoic acid, both
13-cis-retinoic acid and 13-cis-4-oxoretinoic acid are present at significant levels in normal
human plasma (Eckhofi‘ and Nau, 1990). At present, the regulation of concentration of
retinoic acid in plasma and tissues is poorly understood.

The biochemical process by which retinoic acid is enzymatically formed within tissues
fiom the oxidation of retinal has not been unequivocally established. The currently prevailing
hypothesis is that retinal is first oxidized to retinal, which is oxidized to retinoic acid. Thus,
the oxidation of retinal to retinoic acid is thought to be analogous to the oxidation of ethanol
to acetaldehyde, which is in turn oxidized to acetic acid. It has long been known that the
relatively non-specific alcohol dehydrogenase (ADI-I) of liver can catalyze the oxidation of
retinal to retinal (Zachman and Olson, 1961), and that aldehyde oxidase can convert retinal
to retinoic acid (F rolik, 1984). Retinal formation is likely to be rate-limiting in the pathway
to retinoic acid formation (Napoli, 1986; Posch et al., 1989). The current understanding is
that multiple enzymatic activities, such as ADH, aldehyde dehydrogenase, and aldehyde
oxidase, are involved in the conversion of retinal to retinoic acid. To which extent individual
enzymes are importantly involved, is presently unclear. The oxidation of retinal to retinal is
most likely catalyzed by a microsomal enzyme or enzymes that use retinal bound to CRBP
as a substrate. Whether the oxidation of retinal to retinoic acid is also CRBP dependent, or
is catalyzed by a soluble aldehyde dehydrogenase or aldehyde oxidase or both, remains
uncertain (Blaner and Olson, 1994).

Metabolites of all-trans-retinoic acid generated in viva include 13-cis-retinoic acid

(Cullum and Zile, 1985; Eckhofi‘et al.,1991; Tang and Russell, 1990a, b, 1991), 9-cis-retinoic

17

acid (Heyman et al., 1992; Levin et al.,1992), retinoyl-B-glucuronide (Dunagin et al., 1966;
Zile et al., 1980b; Swanson et al., 1981a; Zile et al., 1982a, b; Eckhofi‘et al.,1991; Barua et
al., 1991), 5,6-epoxyretinoic acid (McCormick et al., 1978), 4-hydroxyretinoic acid (Roberts
et al., 1980), 4-oxoretinoic acid (Eckhofi‘ et al., 1991; Tang and Russell, 1991), and 3,4-
didehydroretinoic acid (Thaller and Eichele, 1990). Some of these metabolites are active in
mediating retinoic acid function, whereas others are probably catabolic products. The
cytochrome P-450 system of the hamster is active in metabolizing all-trans-retinoic acid
(Roberts et al.,1979; Leo et al., 1984a). The cytochrome P-450 isozyme P4SOIIC8 of human
liver microsomes was shown to be responsible for oxidizing retinoic acid to 4-hydroxyretinoic
acid and 4-oxoretinoic acid (Leo et al., 1984b). A direct role for CRABP in the oxidative
metabolism of retinoic acid has been proposed by Fiorella and Napoli (1991). When all-trans-
retinoic acid is bound to CRABP, microsomal enzymes of rat testes catalyze the conversion
of retinoic acid to 3,4-didehydro-, 4-hydroxy-, 4-oxo-, 16-hydroxy-4-oxo-, and 18-
hydroxyretinoic acids. Thus, CRABP may play a direct role in the oxidative metabolism of
all-trans-retinoic acid.

Cullum and Zile (1985) using radioactive tracers demonstrated that 13-cis-retinoic
acid is an endogenous retinoid present in the intestinal mucosa, intestinal muscle, and plasma
of normal rats under steady state conditions. When a physiological dose of all-trans-retinoic
acid was administered by intrajugular injection into vitamin A-depleted rats, l3-cis-retinoic
acid appeared in the plasma and small intestine within two minutes afier dosing demonstrating
that circulating retinoic acid is rapidly taken up by tissues, metabolized and the metabolites
released into circulation. The endogenous plasma concentrations of all-trans- and 13-cis-

retinoic acids in vitamin A-depleted rats were reported to be 9.7 and 3.0 nM. Napoli et al.

18

(1985) similarly demonstrated that 13-cis-retinoic acid is a naturally occurring form of retinoic
acid. Bhat and Jetten (1987) demonstrated that cultures of rabbit tracheal epithelial cells can
convert all-trans-retinoic acid to 13-cis-retinoic acid. Tang and Russell (1990a) and Eckhofi‘
et al. (1991) showed that 13-cis-retinoic acid is an endogenous component of human serum.
Fasting serum levels of all-trans- and l3-cis-retinoic acids determined in 26 volunteers ranged
fiom 3.7 to 6.3 nM and fi'om 3.7 to 7.2 nM, respectively (Tang and Russell, 1990a). They
also reported that homogenates of human intestinal mucosa can isomerize all-trans-retinoic
acid to l3-cis-retinoic acid. The administration of an oral dose of retinyl palmitate to human
volunteers elevated their plasma levels of 13-cis-retinoic acid (Eckhofl‘ et al., 1991).

Levin et al. (1992) and Heyman et al. (1992) reported the existence of 9-cis-retinoic
acid in cells in culture, and demonstrated that it was an endogenous component of liver. Levin
et al.(1992) demonstrated that this stereoisomer is an activating ligand for RXR-a in COS-1
cells. Heyman et al. (1992) similarly reported that 9-cis-retinoic acid is a ligand for the human
RXR-a. Mangelsdorfet al. (1992) showed that 9-cis-retinoic acid can activate mouse RXR-
a, 43, and -v. This retinoid was found to be a 40-fold more potent ligand for these three
miclear receptors than all-trans-retinoic acid and 3,4-didehydroretinoic acid (Mangelsdorf et
al.1992). Creech Kraft et al. (1994a) reported the presence of 9-cis-retinoic acid in Xenopus
embryo, and demonstrated that it is a ligand for RXR Thus the formation of 9-cis-retinoic
acid, either by isomerization of its all-trans-isomer or by cleavage of 9-cis-carotenoids (N agao
and Olson, 1994), is a crucial step in mediating retinoid biological fimctions.

Thaller and Eichele (1990) found endogenous all-trans-3,4-didehydroretinoic acid
in the chicken limb bud. This retinoid like all-trans-retinoic acid, can induce pattern

duplications in the developing limb bud. They found that 3,4-didehydroretinoic acid was

l9

generated in situ fiom all-trans-retinol, through 3,4-didehydroretinol intermediate. The
mechanism of dehydrogenation has not yet been examined.

Roberts et al. (1980) studied the formation of 4-hydroxy- and 4-oxo- retinoic acids
fiom retinoic acid by hamster liver microsome preparations. In this process, retinoic acid is
first converted to 4-hydroxyretinoic acid, which is in turn oxidized to 4-oxoretinoic acid. The
formation of 4-hydroxyretinoic acid was found to require NADPH, whereas the subsequent
formation of 4-oxoretinoic acid was reported to be NAD*-dependent. Leo et al. (1984b,
1989) and Roberts et al. (1992) demonstrated that cytochrome P-450 isoforms present in rat
and human liver preparations promote the conversion of retinoic acid to its 4-hydroxy form;
consequently, the cytochrome P-450 system seems to play a role in the physiologic formation
of these 4—oxidized retinoids. Eckhoff et al. (1991) reported that both all-trans-4-oxoretinoic
acid and 13-cis-4-oxoretinoic acid can be detected in the plasma of human volunteers given
an oral dose ofretinyl palmitate for a period of20 days. Barua et al. (1991) found that in rats
given large oral doses of retinoic acid, significant amounts of both 4-hydroxy- and 4-oxo-
retinoic acids can be detected in the serum, stomach, small intestine, liver, and kidney. In early
Xenopus embryos, 4-oxoretinoic acid is available (Pijnappel et al., 1993), and is a highly
active metabolite which can modulate positional specification. This retinoid binds avidly to
and activates RARl}, and is more active than all-trans-retinoic acid in causing microcephaly
in Xenopus embryos (Pijnappel et al., 1993).

A McCormick et al (197 8) found that when vitamin A-deficient rats were given a dose
of [’H]retinoic acid, the intestinal mucosa formed 5,6-epoxyretinoic acid. Napoli et al. (1982)
reported that 5,6-epoxyretinoic acid was present in significant concentrations in the liver,

small intestinal mucosa, and intestinal contents, but not in the kidney of retinoic acid treated

20

vitamin A-deficient rats. Barua et al. (1991) showed that 5,6-epoxyretinoic acid could be
detected in the serum, small intestine, liver, and kidney of rats given a large oral dose of
retinoic acid. It is not known whether this retinoid represents retinoid catabolism or whether
it has a physiological role in vitamin A action. It was found to have no biological activity in
the rat growth assay (Zile et al., 1980a) and reproduction (see Zile et al., 1980a for review).

When all-trans-retinoic acid was orally administered to rats, all-trans-retinoyl-B-
glucuronide was excreted into bile in significant amounts (Dunagin et al., 1966). Retinoyl B-
glucuronide can be synthesized fi‘om retinoic acid and uridine diphosphoglucuronic acid in the
liver, intestine, kidney, and other tissues by a typical microsomal glucuronyl transferase
(Lippel and Olson, 1968; Frolik, 1984). It has been demonstrated that all-trans- and 13-cis-
retinoyl glucuronides are in viva metabolites of all-trans-retinoic acid and were found in the
bile alter a physiological as well as after a pharmacological dose of all-trans-retinoic acid (Zile
et al., 1982a). In late 1960's, it was suggested that retinoyl glucuronide represented the major
(90%), or perhaps the only metabolite of retinoic acid in bile (Lippel and Olson, 1968), and
that this metabolic pathway was the major elimination route for the breakdown products of
vitamin A metabolism; this concept was widely accepted. However, later in 1980, Zile et
al. using improved separation methods concluded that this metabolite accounts for only 12%
of the metabolites of retinoic acid in bile. Of various tissues, the intestinal mucosa seems to
be the most active in synthesizing and retaining retinoyl B-glucuronide (Zile et al., 1982b;
Cullum and Zile, 1985). When 13-cis-retinoic acid is administered, all-trans-retinoyl-p-
glucuronide is a major metabolite in rat tissues in viva (McCormick et al., 1983). Retinoyl-B-
glucuronide is present in fasting human plasma at a concentration of 5-17 nM (Barua and

Olson, 1986). In pregnant females of most, retinoyl-B-glucuronide becomes a major

21

metabolite after the administration of retinoic acid (Creech Kraft et al., 1987, 1991; Eckhofi‘
et al, 1989; Eckhofi‘ and Nau, 1990). In the pregnant mouse treated with 13-cis-retinoic acid,
13-cis-retinoyl-B-glucuranide is the most abundant plasma metabolite (Creech Krafi et al.,
1991b).

The interest in retinoyl glucuronide stems from its relatively low toxicity, a property
that may be usefirl therapeutically. The lack of teratogenicity of retinayl- B-glucuronide, when
administered orally to pregnant rats at very high doses, seems to result from its relatively slow
absorption fi'om the intestine, its slow hydrolysis to retinoic acid, its relatively inefi'rcient
transfer across the placenta, and its inherently low toxicity (Gunning et al., 1993). The
enzyme responsible for the hydrolysis of retinoyl glucuronide to the flue (and more toxic)
retinoic acid is B-glucuronidase which is present in most tissues, mainly compartmentalized
in lysosomes. Thus retinayl-B-glucuranide that is injected or fed is hydrolyzed to retinoic acid
at a slow rate in viva (Barua and Olson, 1989) and very slowly if at all in cultured cells in
vitra (Zile et al., 1987; Gallup et al.,1987; Janick-Buckner et al., 1991). Although serving as
a good inducer of cellular differentiation in vitra and thus being a potential anticarcinogen
(Zile et al., 1987; Gallup et al.,1987; Janick-Buckner et al., 1991), retinayl-B-glucuronide
dose not bind to cellular retinoid-binding proteins or to nuclear retinoid receptors (Mehta et
al.,1992; Sani et al., 1992), and thereforeits mechanism of action is dificult to explain.

Retinal can also be conjugated with glucuronic acid in viva and in vitra to farm
retinyl-B-glucuronide (Dunagin et al.,1966; Lippel and OlsOn, 1968); this conjugate is also
present in human plasma (Barua et al., 1989). Other retinoids, such as 5,6-epoxyretinoic acid,
hydroxyphenyl retinarnide, and 4-oxaretinoic acid, also form D-glucuranides in viva

(Swanson et al., 1981a; Fralik et al., 1981; Napoli et al., 1982). Approximately one-third of

22

the retinoid-B-glucuronides excreted in the bile of rats are recycled back to the liver, thereby
forming an enteroheptic circulation (Zachman et al.,1966; Swanson et al., 1981a).
Further studies on retinoid metabolism will most likely focus on the regulation,

particularly with regard to the function of vitamin A active forms in viva.

1.5 Retinoids in avian cardiovascular development

Thompson and ca-warkers (1969) have shown that retinoids are essential for
embryonal development in domestic fowl. In the absence of retinoids the large blood vessels
linking heart to extra embryonal membranes of the embryo fail to develop and the embryo
eventually disintegrates (Thompson et al., 1969). Hens maintained an retinoid- and
carotenoid-deficient diet, supplemented only with small amounts of retinoic acid methyl ester,
grew well and their fertility was high after artificial insemination. However, the eggs fiam
these hens invariably failed to hatch. This was probably in part due to a failure of retinoic acid
to be transferred to the egg; however, the eggs were not analyzed in these studies.

Heine et al. (1985) using the above model confirmed these results in the quail embryo
and concluded that retinoid deficiency blocks mainly the development of heart and vascular
system. The earliest observable anatomical defects due to lack of vitamin A during avian
embryonic development have been described in detail by Heine et al. (1985). Administration
of retinal or methyl retinoate to the vitamin A-deprived embryo during early arganogenesis
results in normal embryonic development, including a firnctional circulatory system
(Thompson, 1969; Heine et al. 1985). The work of these researchers indicated that the

majority of retinoid-deficient embryos developed normally during the first 24 hr of incubation

23

and that a vitamin A-dependent process in cardiovascular development took place between
24 and 48 hr of development.

Dersch and Zile (1993) used vitamin A-deficient quail supplemented with retinoic acid
to examine the biological activity of various natural retinoids and the time "window" when
vitamin A activity is required for the initiation of a normal cardiovascular development in the
quail embryo. The studies suggested that all-trans-retinoic acid is the biologically active form
of vitamin A required for normal cardiovascular development. There is a critical time point
within the first 22-28 hour of embryogenesis when all-trans-retinoic acid initiates events that
lead to normal cardiovascular development (Dersch and Zile, 1993). Twal et al. (1995) used
a monoclonal antibody specific against all-trans-retinoic acid (Zhou et al.,1991) to block
normal avian cardiovascular development, to produce vitamin A-deficiency-like syndrome,
and to localize all-trans-retinoic acid in quail embryo during early development. It was
concluded that all-trans-retinoic acid or a closely related metabolite is the physiological form
of vitamin A required for normal cardiovascular development and for other very early
developmental events in quail embryo. Using this quail embryo model, it was established that
the expression of RARa and y in the early quail embryo is independent of vitamin A status,
while the expression of RARB and CRABP-I is developmentally regulated and the expression
of RARB is vitamin A dependent in quail embryo. The 3.2 kb RARB isoforrn is regulated
by vitamin A status during early avian development and thus plays a key role in early avian

development (Kostetskii and Zile, 1993; Kostetskii et al., 1995).

24

1.6 Retinoids in avian limb development

Retinoic acid has been implicated as a morphogen in formation of the digit pattern in
the chicken limb bud. The posterior region which contains the zone of polarizing activity
(ZPA) of the bud, when transplanted to the anterior portion of a host wing bud, causes digit
pattern duplication in a mirror image of the posterior digits normally expressed in the bud
(Saunders and Gasseling, 1968; Tickle et al., 1975). One way to interpret these findings is to
assume that the ZPA releases a difiirsible molecule (a so-called morphogen) that sets up a
diffusion gradient across the wing bud (Wolpert, 1969). In such a gradient, cells along the
anteropasterior axis are exposed to difl‘erent concentrations of the signaling molecule. Such
quantitative concentration differentials could be the basis for the formation of qualitatively
different structures, the digits. Retinoic acid mimics the action of the ZPA (Tickle et al., 1982;
Summerbell, 1983; Tickle and Eichele, 1985), causing digit pattern duplication. It was
demonstrated (W edden et al., 1990) that retinoic acid forms a shallow gradient on the limb
bud showing the highest concentration in the ZPA. An inverse gradient distribution of the
cellular retinoic acid binding protein (CRABP) also exists in the chick limb bud, leading to
suggestion that this protein may render the retinoic acid gradient steeper on the bud (Maden
et al., 1988).

However, the hypothesis that retinoic acid is the putative morphogen for chick limb
bud development has been seriously challenged (Wanek et al.,1991; Colbert et al., 1993) and
recently disproved by the demonstration that the product of Sonic hedghag (shh) expression
is the long-hypothesized morphogen, and that retinoic acid may firnction indirectly via
induction of Shh (Riddle et al.,1993; Smith, 1994; Niswander et al., 1994). Chen et al. (1995)

found that Henson's node, the organizer center in chick embryo, from vitamin A-deficient

25
quail embryo induces limb duplication in the host chick embryo, similar to that induced by the

node from vitamin A-sufiicient control embryos. The expression of shh is not afi‘ected by the
vitamin A status of the embryo (Chen et al., 1995). These studies also support the idea that
retinoic acid may not be a direct morphogen for limb bud duplication.

In addition to retinoic acid, 3,4-didehydroretinoic acid and 9-cis-retinoic acid were
found to cause digit duplication in chick embryo (Thaller and Eichele, 1990; Thaller et al.,
1993). Retinoic acid and 3,4-didehydroretinoic acid were found to be equipotent in evoking
digit duplication (Thaller and Eichele, 1990). An in-depth study of the expression of retinoic
acid receptors at various stages of development has been conducted and indicates that precise
and specific expression patterns may be responsible for control of expression of Hox genes

and ultimately pattern formation (Dolle et al., 1990).

1.7 Rationale and objectives of this study
1.7 ,1 Retinoids in gug‘l eggs

The Japanese quail embryo model has been established as a model for the studies of
the function of vitamin A in avian embryonic development, specifically cardiovascular
development (Heine et al., 1985; Dersch and Zile, 1993). Before studying the endogenous
retinoid content in the embryos during the time when cardiovascular system is developed, it
is important to know what the vitamin A and other retinoid content are in the egg, as the yolk
is the only source of vitamin A for the embryo during the entire length of development, until
hatching. However, there is no complete information on the retinoid content in normal quail
eggs. Although the vitamin A-deficient avian model has been established, nobody has

analyzed the eggs from which the vitamin A-deficient embryos are obtained. The objective

26

of this experiment was to analyze the eggs obtained from quail fed different diets and to

determine the retinoid content and metabolite profile baseline data for fertilized eggs at the

beginning of incubation.

1.7.2 Rgingids in early guail gmbgos
The development of the avian cardiovascular system has been shown to be vitamin A

dependent. A previous study (Dersch and Zile, 1993) suggested that all-trans-retinoic acid,
the form of vitamin A generally linked to the molecular function of this vitamin, must be
present in the quail embryo during specific critical stages in development, i.e., stage 5-8 (22-
26 hr incubation), to ensure normal cardiovascular development. It was also reported (Twal
et al., 1995) that using a specific monoclonal antibody against all-trans-retinoic acid, all-trans-
retinoic acid was localized in normal quail embryos during early development. The embryo
developed abnormal cardiovascular system when the development was blocked with this
antibody. However, recently three other closely related retinoids, 9-cis, 4-oxo-, and 3,4-
didehydroretinoic acids have also been suggested as active participants in the retinoid signal
transduction system (Thaller and Eichele, 1990; Thaller et al., 1993; Pijnappel et al, 1993;
Creech Kraft et al., 1994a). These retinoids may have also a role in embryonic development.
In addition, 3,4-didehydroretinoic acid has been shown to induce normal cardiovascular
development in vitamin A-deficient embryo in ava and in culture (Dersch and Zile, 1993;
Kostetskaia et al., 1995). The objective of the present study was to obtain direct evidence for.
the hypothesis that all-trans-retinoic acid or other closely related retinoid, is the vitamin A-

active form required for normal avian cardiovascular development. If all-trans-retinoic acid

27

does have a role in avian cardiovascular development, then it should be present endogenously
during the stages when cardiovascular development is initiated, i.e., stages 5-8.

Nobody has directly analyzed retinoids in early avian embryos because of the
requirement for a large amount of tissues. This problem has been circumvented recently by
the development of molecular bioassays that utilize cell lines, such as F9 cells, transfected
with a reporter construct consisting of a retinoic acid response element (RARE) upstream of
the B-galactosidase gene. A retinoid source, when placed upon a confluent layer of
transfected cells, activates transcription at the RARE. Using this transfection assay with
pooled extracts fi'om quail embryos, Chen et al. (1995) found that stage 6-7 normal quail
embryo contains about 3.4 pg of active retinoic acids, while no retinoid activity could be
detected in the vitamin A-deficient embryos. However, this assay system is not capable of
determining which isomer or metabolite of retinoic acid is responsible for activating
transcription. Therefore, direct biochemical methods of analysis such as high-pressure liquid
chromatography (HPLC) are required. This direct method will be used in the studies

described here.

1,7,3 Msis of vitamin A-deficient embryos

Vitamin A-deficient quail embryos develop an abnormal cardiovascular system. The
purpose of the analysis of the vitamin A-deficient embryos is to show the absence of active
vitamin A metabolites in these embryos, in order to support the hypothesis that all-trans-
retinoic acid or a closely related active retinoid is required for normal avian cardiovascular
development, and thus provide indirect evidence that the absence of these retinoids causes the

abnormalities.

2. MATERIALS AND METHODS

2.1 Animals and diets

The quail (Coiumix catumix japanica) were housed on the Poultry Research and
Teaching Farm at Michigan State University. Normal eggs were obtained fi'om hens fed a
game bird chow ration (Purina Mills Inc, St. Louis, MO). Vitamin A—deficient eggs were
obtained fi'om hens fed a semi-purified diet (Teklad, Madison, WI) adequate in all nutrients
but with 10 mg of 13-cis-retinoic acid added per kg of diet as the only source of vitamin A.
The lB-cis-retinoic acid was used as the source of all-trans-retinoic acid, because in tissues
it is in equilibrium with all-trans-retinoic acid; furthermore this form of retinoic acid was
available in gelatin beadlets which protect retinoic acid from degradation. The young birds
prior to maturity were fed a semi-purified diet containing methyl retinoate (methyl retinoate
is more stable in the diet than retinoic acid ). After receiving the diets, a sample fiam every
new barrel of vitamin A-deficient diet was extracted with methanol and hexane, and analyzed
on HPLC to verify the content of retinoids.

The ingredients of the serni-purified diets are given below. Starterzg‘gower Diet
(g/kg): soybean meal (47.5%), 505.0; DL-methionine, 3.5; L-lysine HCl, 2.5; dextrose
(monohydrate), 383.4144; soybean oil, 40.0; fiber (cellulose), 10.0; mineral mix (see below),
44.856; calcium phosphate (dibasic), 4.5; calcium carbonate, 1.5; manganese sulfate, 0.123;

choline dihydrogen citrate, 4.2; biotin, 0.0006; vitamin B12 (0.1% trituration in mannitol),

28

29

0.03; vitamin D3 in oil (100,000 U/g), 0.03; folic acid, 0.005; menadione sodium bisulfite
complex, 0.016; niacin, 0.08; calcium pantothenate, 0.03; pyridoxine HCl, 0.015; methyl
retinoate, 0.01; riboflavin, 0.015; thiarnin HCl, 0.025; DL-alpha-tocopheryl acetate (100 U/g),
0.05; and butylated hydroxytoluene (BHT), 0.1. Breeder Diet (g/kg): the composition is the
same as for Starter/ Grower Diet except for the following: soybean meal (47 .5%), 464.0; L-
lysine HCl, 1.0; dextrose (monohydrate), 380.3344; fiber (cellulose), 0; calcium phosphate
(dibasic), 13.9; calcium carbonate, 48.7; choline dihydrogen citrate, 3.2; and niacin, 0.6.
Mineral Mix (g/kg): calcium phosphate (dibasic), 557.339; calcium carbonate, 142.6788;
sodium chloride, 156.0549; potassium citrate (monohydrate), 60.1926; magnesium sulfate,
55.0651; ferric citrate, 13.3761; manganese sulfate, 7.5798; zinc carbonate, 5.3505; cupric
sulfate, 1.4045; chromium potassium sulfate, 0.6465; potassium iodate, 0.2007; sodium
malybdate, 0.0557; cobalt chloride, 0.0446; and sodium selenite, 0.0112.

An initial group of quail were hatched from normal eggs and were divided into 2
groups of 25-30 chicks each. The normal group was feed Purina starter/grower diet until the
hms began to lay eggs (6-7 weeks of age); the feed was then changed to Purina breeder diet.
The vitamin A-deficient quail were fed the vitamin A-deficient starter/ grower diet containing
methyl retinoate beginning at hatching until the hens began to lay eggs; the feed was then
changed to the vitamin A-deficient breeder diet containing 13-cis-retinoic acid. Methyl
retinoate was not used as vitamin A-active supplement for hens, because it has been
demonstrated earlier (Dersch, 1992) that this form of retinoic acid was transferred to the egg.
At this time the birds were divided into groups of 30 birds with the ratio of females to males

2:1.

30

2.2 Quail embryo model

In vitamin A-deficient embryo, the earliest gross developmental alterations are visible
during formation of the cardiovascular system and are manifested by an abnormal
development of the heart and an absence of a vascular link between the primitive embryonic
heart and the extraembryonic blood pools (Thompson, 1969; Heine et al., 1985; Dersch and
Zile, 1993; Twal et al., 1995).

Quail eggs were collected daily, stored at 13°C in a cold room, and used within one
week. Eggs were placed in an incubator at 385°C, with 99.5% humidity and with a 2-hour
rotation cycle. Embryos obtained from eggs of hens fed normal chow diet are named normal
embryos. Embryos fiom eggs of hens fed the vitamin A-deficient diet containing 13-cis-
retinoic acid are referred to as vitamin A-deficient embryos.

Eggs were incubated for 24-26 hr, at which time they are in the developmental stages
5-8 (Hamburger and Hamilton, 1951). Quail embryos were dissected from eggs in ice cold
phosphate-buffered saline (PBS), staged according to Hamburger and Hamilton (1951),

washed twice with ice cold PBS, flown on dry ice, and stored at -80°C.

2.3 Retinoids and chemicals

All-trans-3,4-didehydroretinoic acid and 4-oxoretinoic acid were gifts fiom Dr.
Y.F.Shealy, Southern Research Institute (Birmingham, AL); 9-cis-retinoic acid was a gift
fiom Hofinann-LaRoche (Nutley, NJ); methyl retinoate was generously provided by Drs. M.
Spam and A. Roberts, N.I.H; all-trans-3,4-didehydroretinol was a gift fi'om Dr. B]. Burri,
Western Human Nutrition research Center (San Francisco, CA); [11,12-3H]-all-trans-retinol

and [11,12-3Ifl-all-trans-retinoic acid were purchased from New England Nuclear-DuPont

31

(Boston, MA). Other retinoids and chemicals were reagent grade and were purchased from
Sigma Chemical Company (St. Louis, MO). Retinoids were checked for purity by HPLC, by

coelution with authentic retinoids, and by UV spectroscopy.

2.4 Protein assay
The protein content of embryos was measured by a modified method of Lowry

(Markwell, 1981) using bovine serum albumin as standard.

2.5 Retinoids in quail eggs from hens fed different diets
2.5.1 Sm]; preparation

Eggs were obtained fi'om hens fed the normal chow diet and eggs fi'om hens fed the
vitamin A-deficient diet supplemented with 13—cis-retinoic acid. Eggs were stored at 13°C and
used within 7 days. Yolks and whites were separated, keeping each yolk or white as a single
sample. There was no cross-contamination of egg yolk and white. Samples were

homogenized, lyophilized and stored at -20°C.

2.5.2 Emgion of lyophilized yolks gd whites
Samples (100 mg) were extracted with 3 ml of methanol, containing 0.001% butylated

hydroxytoluene (BHT), vortexed for 1 min, placed on a rotary shaker for 20 min, centrifuged
at 3000 x g for 10 min and the supematant transferred to a separate tube. The extraction was
repeated with 1.5 ml of methanol and 1.5 ml of hexane, then with 3 ml of hexane. The

supernatants were pooled, evaporated with a stream of nitrogen, and the residue redissolved

32

in 300 pl of methanol; 100 pl of this extract was used for retinoid analysis. Recovery of

retinoids by this extraction procedure was >70%.

2.5.3 HPLC analysis of retinoids

Reversed-phase HPLC methods (Cullum and Zile, 1985; 1986; Salyers et al., 1993)
were applied to establish the vitamin A concentration in the yolks and whites. The HPLC
system consisted of model 501 solvent delivery system (Waters, Milford, MA), model 7125
syringe loading sample injector (Rheadyne, Cotati, CA), model 440 UV absorbance detector
(Waters, Milford, MA), HP 3393A computing integrator (Hewlett-Packard, Avondale, PA),
model LC-200 linear fiaction collector (Haake Buchler, Saddle Brook, NJ) and Zenith
Personal Computer (Zenith Radio Corp., Chicago, IL). Aliquots of extracts were applied to
a C1,, 10 p Partisil-ODS-III analytical column, 4.6 mm x 25 cm (Whatman, Clifton, NJ) with
Guard-Pak precolumn module containing pBondapak C1, precolumn cartridge (Waters,
Millipore, MA). Retinoids were eluted at a flow rate of 2 ml/min by a step-gradient method
using as mobile phase methanol: water (70:30) (v/v) containing 0.01 M ammonium acetate,
20 min; methanol: water (88:12) (v/v), 15 min; and methanol: chloroform (85:15) (v/v),
10 min. Between each sample run the column was washed with 100% methanol and
reequilibrated in methanol: water (70:30) (v/v). The system described above did not resolve
all-trans-retinol from all-trans-retinal. Fractions corresponding to the retinal area were
collected, pooled, evaporated to dryness under a stream of nitrogen and redissolved in
methanol. The resolution of all-trans-retinol fi'om all-trans-retinal was accomplished on
Zorbax ODS column, 4.6 mm x 25 cm (Du-Pant, Wilmington, DE) and Exacalibar

Spherisorb ODS 5 p, 4.6 mm x 25 cm column (Milton Roy, State College, PA), using the

33
solvent sequence of methanol: water (65:35) (v/v), 17 min; methanol: water (88: 12) (v/v),
12 min; and methanol: chloroform (85: 15) (WV), 8 min. UV absorbance was monitored at
340 nm Identity of retinoids was established by comparing the elution patterns with authentic
retinoids in two stepwise elution systems or by coelution with authentic retinoid standards.
Peaks corresponding to less than 2 ng of retinoids could not to be detected.

Retinoids were quantitated from the integration peak areas at 340 nm UV absorbance,
using retinyl acetate as internal standard. The following equation was applied to calculate
retinoid amount in pg, when internal standard was in pg (unpublished lab data):

Amountx= (AreaJAreamo) x RF, x Amountmn

Where, RFx is the reference factor for retinoids.

1&me ........... 0.298
RPM .............. 0.891
mm,“,,,,,,,,,e ......... 1.000
REM,” ........... 2.465

2.6 Extraction and HPLC analysis of retinoids from early normal quail embryos
2.6.1 Sample preparation 9
Eggs of hens fed normal chow diets were incubated for 24 hr. Embryos were dissected
fiom developmental stages 5 to 8. The characteristics to differentiate each stage are as follows
(Hamburger and Hamilton, 1951):
' Sggej. Head-process: The notochord or head process is visible as a rod of condense

mesoderm extending forward fiom the anterior edge of Hensen's node. The head-fold has not

yet appeared.

34

Stege__6. Head fold: A definite fold of the blastoderm anterior to notochord marks the
anterior end of the embryo proper. No somites have yet appeared in the mesoderm lateral to
the notochord.

fiagel One somite: One to two pairs of somites are visible. Neural folds are visible
in the region of the head.

Stage8. Four somites: Three to five pairs of somites are visible. Neural folds meet at
level of midbrain. Blood islands are present in posterior halfof blastoderm.

Different stage embryos were pooled separately in Eppendorf tubes and stored at
-80°C. In 18 months, 2012 embryos were collected, they were as follows: 134 stage 5
embryos (6.7% of total); 858 stage 6 embryos (42.6% of total); 630 stage 7 embryos (31.3%
of total); and 390 stage 8 embryos (19.4% of total). The average wet weight of a quail
embryo between stage 5 to 8 was calculated fi'om the pooled sample of 2012 embryos and
was found to be 0.9 mg. The average wet volume is 1.0 pl. The protein content is estimated

to be 82 pg per embryo.

2.6,2 Pilot etpdies to established methods of analysis

Pilot studies were done first to compare difl‘erent extraction methods and HPLC
analyms systems by using 6-day normal embryos with a sample volume estimated to be that
of 2000 stage 5-8 embryos. Retinoic acid standard was added before extraction to estimate
recovery. The different extraction methods (pilot methods) for retinoids from embryonic
tissue tested are described below.

Embryos were extracted with methanol and dichloromethane two times, and the liquid

phase of the extracted mixed with 0.9% NaCl. The organic layer was evaporated, dissolved

35

in 1:3 ammonium acetate, 60 mM and methanol (v/v). The extracts were either injected
directly on HPLC (pilot method No. 1), or prepurified by SEP-Pak C13 cartridge (pilot
method No.2) or by C2 cartridges (Waters, Milford, MA) (pilot method No.3), then applied
on HPLC. Recoveries of retinoic acid and impurities in solvent front were compared in the
above three methods. It was found that using a cartridge to prepurify the sample did not
improve the results, thus in subsequent analysis the cartridges were not utilized.

Retinoid standards were used to select the best suited HPLC column, the optimum
solvent system and flow rate for resolution of retinoids, particularly 3,4-didehydro-, 13-cis-,
9-cis-, and all-trans-retinoic acids, since these retinoids have been shown to be biologically
active in other developmental models. Extracts of quail embryo samples fiom the pilot studies
were analyzed by HPLC using several solvent systems so as to select the condition to be used
for the analysis of the pooled 2000-embryo sample. Those pilot studies are not described here.
After selecting the best suited extraction procedure and HPLC system, the method for
retinoid quantitation was developed. Standard curves were calibrated for the quantitation of
retinoids by using the area ratio of specific retinoid over internal standard (retinyl acetate) vs
the corresponding amount of retinoid standard. In our HPLC system, peaks corresponding
to less than 1 ng of retinoid could not to be detected. The recoveries for retinoids were found
to be >70%. In carefully conducted control experiments, no artifacts were produced using

these extraction, isolation, and quantitation procedures.

2.6.3 Extragipn pf 2000 ppoled stage 5-8 no_rrp;a_.l quail embggos

The collected 2012 frozen embryos were placed as one sample into a 15 ml centrifuge

tube, containing 1 mg of ascorbic acid, then thawed, and centrifirged to remove PBS. After

36

the weight and volume were determined, the embryos were transferred with PBS to a 10 ml
homogenizing tube and briefly homogenized. The total homogenate was 6.5 ml; 38.8 pl of
the homogenate was set aside for measurement of protein content; this amount was calculated
to be equivalent of 12 embryos. [11, 12-3H]-all-trans-retinoic acid, 9.92><10s DPM (specific
activity , 52.5 Ci/mmol), and [11,12-3I-I]-all-trans-retinol, 8.1XIO‘ DPM (specific activity,
41.3 Ci/mmol), were added as internal standards to allow measurement of the eficiency of
extraction and to facilitate retinoic acid and retinal peak identification. One ml of methanol
with 0.001% BHT and dichloromethane (1 ml) were added, and the mixture was flushed with
nitrogen, vortexed for 2 min and sonicated for 5 min. The residue was removed by
centrifugation for 20 min, dissolved in a mixture of methanol (2 ml) and dichloromethane
(6 ml), and re-extracted as above. Both of the liquid phases were combined, 1.75 ml of 0.9%
NaCl was added, and the mixture was vortexed for 2 min and sonicated for 10 min. The
mixture was allowed to settle, and the upper (aqueous) layer was removed. The bottom layer
(organic) was evaporated by a stream of nitrogen, and the components, containing retinoids
were dissolved in 70 pl of methanol: water (75:25) (v/v) containing 40 mM ammonium
acetate. The tube was washed with 70 pl of the same solvent, the wash added to the sample
and this mixture was transferred to a 2.5 ml Eppendorf tube, centrifuged for 5 min,

and 130 pl of the supernatant injected on HPLC. The flow chart of the extraction is shown

in Figrge 4.

37

2012 embryos

 

2000 embrIyos 12 enlrbryos
WHIROH. [3mm
J 1ml MeOH, lml CH2C12 PM“ my

 

i

Residue Liquid phase
2ml MeOH, 6m] CH2C12 I 1.75m] of 0.9% NaCl

 

 

 

Y Upper layer Bottom layer
A evaporate solvent

Worn MeOH x2
Sample for HPLC

Fiflg 4. Flow chart of the extraction of 2012 stage 5-8 normal embryos

38
2.6.4 HPLC Mysis of the extract from 2000 pooled stage 5-8 norm_al embryos

The HPLC system was the same as that used in the analysis of retinoids in eggs,

 

except that a smaller particle size analytical column, PariSphere C1, 3 p, 4.6 mm X 25 cm
(Whatman, Clifton, NJ) was used . The retinoids were eluted with a flow rate of 1 ml/min by
a step-gradient method using as mobile phase methanol: water (7 5:25) (v/v) containing
0.04 M ammonium acetate, 35 min; methanol: water (90:10) (v/v), 25 min; methanol:
chloroform (85: 15) (v/v), 20 min. The eluent was collected in 0.5 ml fiactions using a
fraction collector (0.5 min/fiaction). A 20 pl aliquot fiom each fiaction was mixed with 5 ml
of Safety-Solv high flash point cocktail (Research Products International Corp., Mount
Prospect, IL) and counted for radioactivity in a Model 4430 Liquid Scintillation Counter
(Packard Instrument Co., Downers Grove, IL). Recovery of retinoic acid fiam the entire
isolation procedure was 78% and of retinal, 29%. All the eluents collected in minivials were
flushed with nitrogen, placed into sealed bags, and stored at -80°C for firrther

characterization.

2.6.5 Methylatipn of retinoic acid erg metabolites; characterizatipn of HPLC elutipn
prpfiles of authentic methylated retinoids

 

In order to identify the retinoic acids and other retinoid carboxylic acids in the embryo
extract, eluted by the above HPLC, they will be methylated, rechromatographed in another
solvent system, and compared with methylated authentic retinoic acids. The authentic
retinoids had to be first methylated and their elution patterns established. Standard solutions
of4-oxo-, 4-hydroxy, 4-oxo-13-cis-, 5,6-epoxy-, 3,4-didehydro-, 13-cis-, 9-cis-, and all-trans-
retinoic acids were first methylated, then analyzed for completeness of methylation and

recovery.

39

The methylation condition finally selected was as follows: 2-10 ng of retinoic acid is
dissolved in 20 pl of methanol and treated with 30 pg of diazomethane in an ethereal solution
for 1 hr at room temperature on a platform shaker. The solvent is then evaporated with a
stream of nitrogen and the residue is redissolved in 20 pl of methanol for firrther analysis. The
yield of methylation in the presence of excess diazomethane was 100%.

The ethereal diazomethane solution was prepared as follows (V ogel, 1978): dissolve
2.14 g of Diazald" (99% N-methyl-N-nitroso-p-toluenesulfonamide, m.p. 61-62°C, Aldrich,
Milwaukee, WI) in 30 ml of diethyl ether in a 100 ml distillation flask connected to a
condenser. Add a solution of 0.4 g of potassium hydroxide in 10 ml of 96% ethanol. After 5
minutes, distil the ethereal diazomethane solution fi'om a 60-65°C water bath into a receiving
flask kept cold on ice. The ethereal solution contains 0.32-0.35 g of diazomethane per 30 ml.
Diazomethane (CHZNZ), a liquid in room temperature, is explosive and has a green-yellow
color when dissolved in diethyl ether.

Next, the authentic methylated retinoic acid derivatives were chromatographed in
various HPLC solvent systems (not shown) to select a system that was capable of resolving

most of the compounds.

2,6.6 QMerizatipn of unknown retinoic acids end other metabolites by methyletipn Ed
rechromatography

 

Collected fi'actions corresponding to the elution areas of 4-oxoretinoic acid, 3,4-
didehydroretinoic acid, 13-cis-retinoic acid and 9-cis-retinoic acid, and all-trans-retinoic acid
were pooled separately, to each was added 2.19 ng of internal standard (retinyl acetate), and

the solvent was evaporated with a stream of nitrogen. The residue was dissolved in 20 pl of

40

methanol and treated with 30 pg of diazomethane in ethereal solution for 1 hr at room
temperature on a platform shaker. The solvent was then evaporated with a stream of nitrogen
and the residue redissolved in 20 pl of methanol. The methylated retinoids were
chromatographed on PariSphere 3 p C" analytical column and eluted with methanol: water

(90:10) (v/v), 30 min .

2.6.7 Chmprizatipn of non-mlar retinoids

Retinal area, including 3,4-didehydroretinol and all-trans-retinal, was pooled, dried
and redissolved in methanol. Aliquots were rechromatographed in two difl‘erent solvent
systems, i.e. methanol: water (90: 10), and methanol: water (85:15). Retinyl ester area was
pooled, dried and redissolved in methanol. Aliquots were rechromatographed in two different
solvent systems, i.e. methanol: chloroform (85:15) and methanol: chloroform (88:12).
Samples were coeluted with authentic standards, or their elution patterns were compared with

authentic compounds.

2.6,8 mutation of retinoids

Retinoid concentrations were calculated from peak areas with reference to standard
curves. To get the standard curve for a retinoid, known amounts of the authentic retinoid are
analyzed in the presence of the internal standard. Difl‘erent known amounts of the retinoid will
correspond to the different peak areas, fiam which one can calculate the ratio of the peak
area of this retinoid to the peak area of the internal standard. It was not possible to obtain
reproducible peak areas for 0-5 ng amounts of the retinoic acid. Therefore, these retinoids

were quantitated after methylation, using standard curves obtained for methyl retinoate.

41

Table 2 shows the data for the calculation of the standard curve of methyl retinoate using,
2.19 ng of retinyl acetate as internal standard. Since the regression of the area ratio on the
amount of the retinoid is linear (Cullum and Zile, 1986), using the method of least squares
(Milton, 1993), an equation for the line can be calculated. Fm; shows the standard line
of methyl retinoate, using the data in Table 2. The equation for the line is:
Y = 0.09 + 0.58 X (p<0.01),

where, Y is the ratio of peak area of methyl retinoate to internal standard (retinyl acetate), X
is the amount of methyl retinoate. When the peak area is obtained from HPLC analysis, the
amount of this retinoid can be calculated from the equation.

The equations for other retinoids are as follows:

all-trans-retinol ................ Y = 0.03 + 0.18 X (P<0.01)
3,4-didehydroretinol ............ Y = 0.02 + 0.12 X (P<0,01)
all-trans-retinal ................. Y = 0.05 + 0.21 X (P<0.01)

Retinoid concentrations calculated from the above equations were corrected for the
recovery ratio (retinoic acid, 78%; retinal 29%). In the case of radioactive retinoids that were
added to the embryo to serve as markers for all-trans-retinoic acid and for all-trans-retinol,
the mass contributed by the radioactive makers was subtracted from the total amount of the
retinoid contained in the detected peak.

The equation for calculating retinyl esters was adopted from the previous studies in
the laboratory (unpublished), and was as follows:

Amount = (Area/AreamD) >< Amountmn x 2.465 .

42

Table 2. Data for methyl retinoate standard curve

 

Amount...” m Aream mJArea 1m,“

 

 

0.000 0.00

1.255 0.76

2.510 1.77

3.765 2.31

5.020 2.88
'Mean of two analysis

Area (Moth Retinoate
Area

 

 

 

0’ I I I . _I I I ’ X
0 1 2 3 4 5 6
Amount (Methyl Retinoate), ng

Figpre 5. Standard curve for methyl retinoate.

43

2.7 Extraction and HPLC analysis of retinoids from early vitamin A-deficient quail
embryos

2.7.1 Sample preparation

Eggs from hens fed vitamin A-deficient diets containing 13-cis-retinoic acid were
incubated for 24 hr. The embryos from stage 5 to 8 were dissected out in ice cold PBS,
washed twice with ice cold PBS, collected in Eppendorf tubes, frozen on dry ice, and stored
at -80"C. In 2 years, 2111 vitamin A-deficient embryos were collected; they were as follows:
314 stage 5 embryos (6.7% of total); 890 stage 6 embryos (42.6% of total); 656 stage 7
embryos (31.3% of total); and 224 stage 8 embryos (19.4% of total). The average wet weight
of these quail embryos was calculated from the pooled sample of 2111 embryos and was
found to be 0.9 mg; the average wet volume was 1.0 pl per embryo. Protein content was
estimated to be 87 pg per embryo. At these stages of development there are no morphological

difi‘erences to distinguish the vitamin A-deficient quail embryo fiom that of the normal.

 

2.7 .2 Extrac_tion pf 2100 pooled stage 5-8 embryos fi'om vitamin A-deficient eggs

The pooled 2111 fiozen embryos were placed as one sample into a 15 ml centrifirge
tube. The extraction procedure was the same as that for normal embryos, except that retinyl
acetate was added as internal standard to allow for the measurement of eficiency of
extraction (recovery); an aliquot of the homogenate equivalent to the amount of 11 embryos

was set aside for measurement of protein content before extraction.

44

27,3 HPLC ngais of the extract fi'om 2100 popled embryos from vitamin A-deficient

98$

 

The HPLC analysis system was the same as that used in the analysis of the normal
embryo extract. The peak area of internal standard (retinyl acetate) was compared to the

standard curve of a known amount of retinyl acetate vs the corresponding integration peak

area (EM) to calculate recovery. The recovery of the extraction was 82%.

 
   

 

 

Y
4x105j
A Y=64773X-14000
2 <
g ”(105- p 0.01
<
. '5.
E
3 2x105-
I
.29
1x105-
OL I I I I I I, X

0 1 2 3 4 5 6
Amount (Retinyl Acetate), ng

Figug 6. Standard curve for retinyl acetate.

3. RESULTS

3.1 Retinoid concentration in quail eggs obtained from hens fed different diets

The egg whites (albumen) fiom eggs of hens fed difl‘erent diets did not contain any
retinoids (not shown). The HPLC analysis of retinoids in yolk fi'om quail eggs is shown in
Ligand.

The HPLC profile of normal quail yolk is shown in Figpre 7A. Yolks fi'om eggs
obtained from hers fed normal chow diet contained 3,4-didehydroretinol, all-trans-retinol and
retinyl esters. Retinyl palmitate was the major form of retinyl esters. All-trans-retinol was the
predominant form of vitamin A in the eggs. No retinal was detected. The molar percent of
retinal in total retinoids detected was 70-80%. In addition, in the yolks of eggs fiom hens fed
the normal chow diet, peaks of carotenoids were detected.

Figare 7 B represents the HPLC analysis of yolk extract fi'om vitamin A-deficient quail
eggs. The yolks of eggs from hens fed the vitamin A-deficient diet containing 13-cis-retinoic
acid contained no retinoids.

The results of the retinoid concentration in the yolks of quail eggs from hens fed
different diets are summarized in Table 3. Since there were no vitamin A compounds in any

of the egg whites, measurements on the yolk apply to the whole eg.

45

46

FigI_1re 7. HPLC analysis of retinoids in quail eggs.

HPLC column: Partisil ODS-III, 10p, C". Solvent system, methanol: water (70:30)
containing 0.01 M ammonium acetate, 20 min; methanol: water (88:12), 15 min; and
methanol: chloroform (85:15), 10 min. Flow rate, 2 ml/min.

A, Representative HPLC profile of yolk extract from quail eggs obtained fi'om hens fed a
normal quail diet. Inset shows chromatography of combined peaks 7 and 8 in an HPLC
system that resolves retinal and retinal.

B, Representative HPLC profile of yolk extracts fi'om quail eggs obtained from hens fed the
synthetic vitamin A—deficient diet containing l3-cis-retinoic acid. Note the absence of
endogenous retinoids.

Arrows indicate elution positions of authentic retinoids: 4-oxoretinoic acid (1); 3,4-
didehydroretinoic acid (2); 13-cis-retinoic acid (3); 9-cis-retinoic acid (4); all-trans-retinoic
acid (5); 3,4-didehydroretinol (6); all-trans-retinol (7); all-trans-retinal (8); retinyl acetate (9),
internal standard; retinyl palmitate (10); solvent front (SF). Peaks without designation do
not contain any of the authentic retinoids used.

 

47

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48

Table 3. Retinoids in yolks of eggs from hens fed different diets

 

 

 

Retinoids ‘ (pg / yolk)
Diet n
dd ROH at-ROH RE
Normal 11" 328321.04“ 39.10i8.27 l 1.25:1: 3.33
chow diet
Vitamin A- 11” nd" nd nd
deficient diet

 

" dd-ROH, 3,4-didehydroretinol; ROH, all-trans-retinol; RE, retinyl esters
" Number of individual eggs analyzed.

‘ Values are mean :t: SEM; n, number of samples

" nd, not detected.

3.2 HPLC profile of retinoids from the extract of 2000 pooled normal quail embryos

Ligare§ shows the HPLC profile of the extract of pooled 2000 stage 5-8 embryos
from eggs of quail hens fed the normal chow diet. The polar region (A), where 4-oxo-all-
trans-retinoic acid and other polar metabolites elute (see W for HPLC profile of
authentic retinoids), contained many impurities and was off the scale for detection. The
retention times of the peaks in the subsequent regions indicated the presence of all-trans-3,4-
didehydroretinoic acid (Area B), 13-cis-retinoic acid or 9-cis-retinoic acid(Area C), and all-
trans-retinoic acid (Area D). The elution area of retinols and retinal (Area E) contained many
impurities that obscured the detection of any retinoids. The elution area of retinyl esters (Area
F) contained several partially resolved peaks, indicating the presence of retinyl esters.

The characterization of the retinoids in the various eluted fi'actions is described in the

subsequent sections.

49

Figpge 8. HPLC profile of the extract from 2000 pooled normal embryos

HPLC column: PariSphere, 3p, C1,. Solvent, methanol: water (75:25) containing 0.04 M
ammonium acetate, 35 min; methanol: water (90: 10), 25 min; methanol: chloroform ( 85:15),
20 min. Flow rate, 1 ml/min.

Arrows indicate the elution positions of authentic retinoids: 4-oxoretinoic acid (1); 3,4-
didebydroretinoic acid (2); 13-cis-retinoic acid (3); 9-cis-retinoic acid (4); all-trans-retinoic
acid (5); 3,4-didelrydroretinol(6); all-trans-retinol(7); all-trans-retinal (8); retinyl linolenate
(9); retinyl linoleate (10); retinyl palmitate (11); retinyl stearate (12); solvent front (SF).
Solid lines show the UV absorbance at 340 nm; dotted lines indicate DPM of purified [’11]-
all-trans-retinoic acid and [’I-I]-all-trans-retinol added to the pooled extract as markers. Data
are plotted as DPM per pl aliquot from a 0.5 ml fi'action.

 

50

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51

Figug 9. HPLC profile of authentic retinoids

HPLC column: PariSphere, 3 p, C“. Solvent system: A: methanol: water (75:25)
containing 0.04 M ammonium acetate, 35 min; methanol: water (90:10), 25 min; methanol:
chloroform (85:15), 20 min. B: methanol: water (90:10). Flow rate, 1 mein.

Numbers indicate authentic retinoids: 4-oxoretinoic acid (1); 3,4-didehydroretinoic acid (2);
13-cis-retinoic acid (3); 9-cis-retinoic acid (4); all-trans-retinoic acid (5); 4-oxo-methyl-
retinoate (6); 3,4-didehydroretinol (7); all-trans-retinol (8); all-trans-retinal (9); 3,4-
didehydro-methylretinoate (10); 13-cis-methylretinoate (11); 9-cis-methylretinoate (12);
. methyl retinoate (13); retinyl linolenate (14); retinyl linoleate (15); retinyl palmitate (l6);
retinyl stearate (16); solvent fi'ont (SF). .

52

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F _ _ _ _ — _ _ —
am
-83
S
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t 2 2
o. i
m. .53.

 

uru op: 12 eaueqrosqv

53

3 .2.1 Characterization of retinoids in polar region A

The known bioactive polar retinoids that elute in this region are retinoic acid
metabolites with an intact carboxylic group. The strategy used to characterize them was to
methylate the eluted material and then to rechromatograph the methylated derivatives in
another solvent system and to compare the elution pattern and/or coelute them with authentic
methylated retinoic acids and metabolites.

The fiactions of region A in Mei were pooled, and internal standard (retinyl
acetate) was added. The solvent was evaporated under a stream of nitrogen to completed
dryness, and the residue was dissolved in 50 pl of methanol and treated with an ethereal
solution of diazomethane for methylation for one hour in room temperature. The methylated
samples were rechromatographed in solvent system consisting of methanol: water
(90: 10) (Figu_re 10). A major peak eluted at 5.9 min and two other peaks eluted at 8.0 min
and 9.0 min, respectively. The peak at 5.9 min had the retention time of authentic methylated
4-oxo-retinoic acid, but was not pure. The peaks eluting at 8.0 min and 9.0 min because of
their small size, could not be identified. In order to firrther resolve the methylated retinoids
in the fiactions within the peak at 5.9 min, they were pooled, internal standard added, and the
sample rechromatographed in a more polar system of methanol: water (85:15) (Figure 11).
Using this solvent system it was demonstrated that only a small amount (<1 ng) of retinoid
eluted at the retention time of authentic methylated all-trans-4-oxoretinoic acid (10.0 min).
The major peak at 15.5 min retention time requires further identification. These results
suggest that the embryos may contain some (<0.5 pg) all-trans-4-oxoretinoic acid, and

possibly also a derivative of it.

54

0.001 - i-

«.4

 

4—M

 

 

0.000 e

Absorbance at 340 nm

 

 

I l l I

0 5 10 15 20
Retention time (min)

Figpm 10. Chromatography of methylated polar region A from HPLC in Fiflge 8

HPLC colunm: PariSphere, 3 p, C". Solvent methanol: water ( 90:10), 25 min. Flow rate,
lml/min.

Arrows indicate the elution positions of authentic retinoids: 4-oxo-methylretinoate (l) ;
retinyl acetate (internal standard) (2); SF, solvent front (SF). Peaks without designation
could not be identified.

55

 

 

E
g 0.001- SF 2
T i
(’0
*5 1
a l
C
(U
.D
‘-
8 0.000—
.D
<
I I I fifk'
0 5 10 15 40

Retention time (min)

Figpge ll. Chromatography of methylated polar region A from HPLC in Figur_'e 8

HPLC column, PariSphere, 3 p, C1,. Solvent, methanol: water (85:15), 40 min. Flow rate,
1 ml/min.

Arrows indicate the elution positions of authentic retinoids: 4-oxo-methylretinoate (1);

retinyl acetate (2), internal standard; solvent front (SF). Peaks without designation could not
be identified. ‘

56

3.2.2 Chgaeterization of retinoids in polar region B

 

Area B in the HPLC profile shown in M corresponds to the elution area of
all-trans-3,4-didehydroretinoic acid. The fi'actions fi'om this area were pooled and internal
standard (retinyl acetate) added, then the sample was treated with diazomethane for
methylation and chromatographed in a system that separates retinoic acids and methylated
retinoic acids. Figure 12 shows the chromatograrn of methylated area B. After methylation,
peak B elution shifted from the polar region, [solvent methanol: water (7 5:25), 17.8 min] to
the nonpolar region, [solvent methanol: water (90: 10)], and a peak eluted in the peak area
of authentic methylated 3,4-didehydroretinoic acid (peak 2). This finding confirmed the

presence of 3,4-didehydroretinoic acid in stage 5-8 normal quail embryos.

3.2.3 Characterization of retinoids in polar region C

Region C in BM corresponds to the elution area of 13-cis-retinoic acid and 9-
cis-retinoic acid. Fractions eluted in this peak were treated with diazomethane for
methylation. When chromatographed with the internal standard 13-cis-N-ethylretinamide in
the solvent system of methanol: water (75:25), 30 min, and methanol: water (90: 10) 30 min,
the original peak and the peak corresponding to the methylated acids could not be detected
(Figpre 13 ). This may be because of the small amount of this compound and the losses
during transferring and methylation. Thus, if present, 9-cis- and/or 13-cis- retinoic acids

represent only a very minute fraction of the retinoids in the normal quail embryo.

57

0.001 - W H

 

‘——l
M
*—

 

 

0.000 ”’1

 

 

 

Absorbance at 340 nm

I I I I I

l
0 10 20 30 40 50 60
Retention time (min)

Figpge 12. Chromatography of methylated peak B from HPLC in Figr_rre 8

HPLC column: PariSphere, 3 p, C". Solvent, methanol: water (75:25) containing 0.04 M
ammonium acetate, 30 min; methanol: water (90: 10), 25 min. Flow rate, 1 ml/min.

Arrows indicate the elution positions of authentic retinoids: 3,4-didehydroretinoic acid (1);
3,4-didehydro-methylretinoate (2); retinyl acetate (3), added as internal standard; solvent
fi'ont (SF).

58

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59

3.2.4 Chgacterization of retinoids in polar region D

Region D in HPLC profile shown in HM corresponds to the elution area of all-
trans-retinoic acid. Furthermore, the major peak in region D coeluted with [3H]-all-trans-
retinoic acid which was added as an internal standard before tissue extraction(see ms,
dotted lines). Therefore it was very likely that this peak was all-trans-retinoic acid. To further
identify this compound, the fractions that were contained in peak D were treated with
diazomethane for methylation. When the methylated compounds were chromatographed, both
the unknown compound and radioactive all-trans-retinoic acid standard, now in the form of
[3H]-all-trans-methyl retinoate, eluted as a single peak in the nonpolar region [using the
solvent methanol: water (90: 10)]; the elution position was that of authentic methyl retinoate
(peak 3) (Figure 14). This confirmed the identity of all-trans-retinoic acid as a major

bioactive vitamin A metabolite in normal stage 5-8 quail embryos.

3.2,5 Charactm’ tion of retinoids in nonpolar region B
The nonpolar region E in the HPLC profile in Figure 8, contained many compounds

which eluted with solvent fiont; the peak was out of scale of detection. The elution of all-
trans-retinol, all-trans-retinal, and 3,4-didehydroretinol corresponds to this region. To
determine if any of these retinoids were present in the embryo, one fifth of the collected
fraction of this region plus internal standard (retinyl acetate) was rechromatographed in
methanol: water (75:25), 25 min; and methanol: water (90: 10), 30 min (Figure 15). Another
aliquot of one fifth was rechromatographed in methanol: water (85:15), 45 min (Eigrge 16).
In both solvent systems, 3 major peaks (E1, E2 and E3) were detected, eluting in the

positions of authentic 3,4-didehydroretinol (peak 1), all-trans-retinol (peak 2), all-trans-retinal

60

(peak 3), respectively. The identity of all-trans-retinol (E2) was firrther confirmed by its
coelution with [3H]-all-trans-retinol, added as an internal standard before tissue extraction.
These results established the presence of all-trans-retinol, 3,4-didehydroretinol and all-trans-

retinal in the stage 5-8 quail embryo.

3.2.6 CMaerization of retinoids in nonpolar region F

Region F in HPLC profile shown in Iiigrm is the region where retinyl esters elute.
To identify the retinyl esters present in normal embryos, halfof the collected fractions of this
region plus internal standard (retinyl acetate) were chromatographed in methanol: water
(90:10), 30 min; and methanol: chloroform (85:15), 30 min (Figure 17); the other half of the
sample was rechromatographed in a second solvent system consisting of methanol: water
(90:10), 30 min; and methanol: chloroform (88:12), 30 min (Eiguru 18). In both
chromatographic systems, retinyl palmitate (peak 4) and retinyl stearate (peak 5) were
identified as the major retinyl esters present in the embryos; they eluted at the retention times

of their respective authentic standards.

61

          

 

 

 

E 3

= l

8 SF SF

on 0.001- 2 :

a 1 l I":

a l 3

a SE

a "

.D 3:

h 0

9, 0.000-

.0 000.00"'00000....oso.o.0..00.oOOoooo.00.o°0.00°‘oo...0: ‘00....
< I l l l l I

o 10 ab 30 40 so 60 70
Retention time (min)

Figug l4. Chromatography of methylated peak D from HPLC in Figure 8

HPLC column: PariSphere, 3 u, C”. Solvent, methanol: water (75:25) containing 0.04 M
ammonium acetate, 30 min; methanol: Water (90: 10), 25 min. Flow rate, 1 ml/min.

Arrows indicate the elution positions of authentic retinoids: all-trans-retinoic acid (1); retinyl
acetate (2), added as internal standard; methyl retinoate (3); solvent front (SF). Solid lines,
absorbance at 340 nm; dotted lines, radioactivity (DPM).

62

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0.001 4 1

0.000 --]

   
 

m
ooooooo"'0. <— N

"IN 4—0)
~4—A

 

 

   

Absorbance at 340 nm

 

 

I l l I

O 10 20 3O 40
Retention time (min)

Figugg 16. Rechromatography of region E from HPLC in Figure 8

HPLC column: PariSphere, 3 u, C". Solvent, methanol: water (85:15) , 50 min. Flow rate,
1 ml/min. ‘

Arrows indicate the elution positions of authentic retinoids: 3,4-didehydroretinol (1); all-
trans-retinol (2); all-trans-retinal (3); .retinyl acetate (4), added as internal standard; solvent
fi'ont (SF). Solid lines, absorbance at 340 nm; dotted lines, radioactivity (DPM).

 

 

 

 

 

 

 

 

E 1 23 4 5
5 SF i H i i
v 0.001“ U .
(‘0
‘5
0
g 0.000-J
'2 SF
1 I l i I r l
o .10 20 30 40 so 60 70

Retention time (min)

Figug l7. Rechromatography of region F from HPLC in Figure 8

HPLC column: PariSphere, 3 u, C1,. Solvent, methanol: water (90:10), 30 nrin; methanol:
chloroform (85:15), 30 min. Flow rate, 1 ml/min. '

Arrows indicate the elution positions of authentic retinoids: retinyl acetate (1), added as
internal standard; retinyl linolenate (2); retinyl linoleate (3); retinyl palmitate (4); retinyl
stearate (5); solvent front (SF). Peaks without designation could not be identified.

65

 

 

 

 

E 1 23 4 5

C

3 3.: l u l r

m 0.001-

‘65

0

O

C

('0

e .

9, 0.000-«J

.0

< SF
l I l j r I 1
10 20 30 4o 50 60 70

Retention time (min)

Figugg l8. Rechromatography of region F from HPLC in Figure 8

HPLC column: PariSphere, 3 u, C". Solvent, methanol: water (90: 10) , 30 min; methanol:
chloroform (88:12), 30 min. Flow rate, 1 ml/min.

Arrows indicate the elution positions of authentic retinoids: retinyl acetate (1), added as
internal standard; retinyl linolenate (2); retinyl linoleate (3); retinyl palmitate (4); retinyl
stearate (5); solvent fi'ont (SF). Peaks without designation could not be identified.

66

3.2.7 Qyntitatiun of retinoids in normal guail embryos

Table 4 shows the amounts of retinoid metabolites in stage 5 to 8 normal quail

 

 

 

embryos.
Table 4. Retinoid metabolites in stage 5-8 normal quail embryos
Amount of retinoids‘
Retinoids
pg/embryo nM pg/mg of protein
3,4-didehydroretinoic acid 1 4 15
all-trans-retinoic acid 2 5 l9
3,4-didehydroretinol 53 185 641
all-trans-retinol 3 1 109 3 81
all-trans-retinal 24 85 294
retinyl palmitate 42 79 506
retinyl stearate 17 3 1 210

 

‘ Retinoids were quantitated from the elution peak areas using standard curves as
described in MATERIALS AND METHODS.

67

3.3 HPLC profile of retinoids from the extract of 2100 pooled vitamin A-deficient
embryos

Figgue 19 shows the HPLC profile of the extract of 2100 pooled stage 5-8 vitamin
A-deficient quail embryos. Peak A is close to the retention time of 4-hydroxy-all-trans-
retinoic acid and 4-oxo—l3-cis-retinoic acid. Peaks B, C and D, and peaks between 80-90 min
(region F) do not correspond to any known naturally occurring retinoids.

Collected fractions containing peak A were pooled and rechromatographed in a more
polar system, methanol: water (65:35) (Figure 20). In this system, the retention time of the
unknown compound was again close to that of 4-hydroxy-all-trans-retinoic acid and 4-oxo-
13-cis-retinoic acid; however the unknown compound did not coelute with these retinoids.
To further characterize this unknown compound and to determine if it has a carboxyl group,
the HPLC fi'actions were'pooled, methylated with diazomethane, and chromatographed using
the solvent system, methanol: water (65: 35), 15 min; and methanol: water (85: 15), 50 min,
which separates the polar methylated retinoids (Figure 21). After methylation, the unknown
peak shifted to the nonpolar region, and had a retention time of 38.6 min, which was very
different fi'om the retention times of 4-oxo-13-cis-methyl-retinoate (32.2 min) and 4-hydroxy-
all-trans-methyl-retinoate (32.6 min). It was concluded that this unknown compound has a
free carboxyl group, i.e. is a organic acid, but is not a known retinoic acid or a known
metabolite of retinoic acid. This compound, as well as, other peaks observed in the
chromatogram, needs further identification.

We conclude that the extract of 2100 pooled stage 5-8 quail embryos, obtained fiom
eggs of hens fed a vitamin A-deficient diet containing 13-cis-retinoic acid, does not contain

any known retinoids. Thus, retinoids that were detected in stage 5-8 normal quail embryos

68

and other authentic retinoids that were used in this work could not be detected in the same

stage vitamin A-deficient quail embryos.

69

 

 

1
2 3456 789 10 1213

E l W m l rruum

g 0.001JSFA SF F

g o E

g B C

4'3 J

o 0.000-

E?

< SF

 

 

I l I I I I I I 'I

o 10 20 30 40 so 60 7o 80 90 Too
Retention time (min)

Figug l9. HPLC profile of the extract from 2100 pooled stage 5-8 embryos from
vitamin A-deficient quail eggs

HPLC column, PariSphere, 3 u, C". Solvent, methanol: water (75:25) containing 0.04 M
ammonium acetate, 40 min; methanol: water (90: 10), 25 min; methanol: chloroform (85:15),
20 min. Flow rate, 1 ml/min.

Arrows indicate the elution positions of authentic retinoids: 4—oxoretinoic acid (1); 4-
hydroxy—all-trans-retinoic acid and 4-oxo-13-cis-retinoic acid coelute (2); 3,4-
didehydroretinoic acid (3); 13-cis-retinoic acid (4); 9-cis-retinoic acid (5); all-trans-retinoic
acid (6); 3,4-didehydroretinol (7); all-trans-retinol (8); all-trans—retinal (9); retinyl acetate
(10), added as internal standard; retinyl linolenate (l l); retinyl linoleate (12); retinyl
palmitate (13); retinyl stearate (l4); solvent front (SF).

7O

 

 

 

 

o 001 SF
0 .7 n
E
S 12
g i
‘5 A
0)
O
C
N
.D
h
8 0.000—«d
.0
<
l I I
5 10 15

Retention time (min)

Figug 20. Rechromatography of peak A from HPLC in Figuue l9

HPLC column, PariSphere, 3 u, C“. Solvent, methanol: water (65:35) containing 0.04 M
ammonium acetate, 20 min. Flow rate, 1 ml/min.

Arrows indicate the elution position of authentic retinoids: 4-oxo-l3-cis-retinoic acid (1);
4-hydroxy-all-trans-retinoic acid (2).

71

 

 

 

 

0001 SF SF
E ' 3
c 1 j
° 2
" l
°° l
‘6
d)
3 r
g 0.000-
I-
O
U)
.D
< ' I
I I I I 7 I
0 10 20 30 40 60

Retention time (min)

Figuue 21. Chromatography of methylated peak A from HPLC in Figure 19

HPLC colurm, PariSphere, 3 u, C". Solvent, methanol: water ( 65:35) containing 0.04 M
ammonium acetate, 15 min; methanol: water (85:15) , 50 min. Flow rate, 1 ml/min.

Arrows indicate the elution position of authentic retinoids: 4-oxo-13-cis-methylretinoate (l);
4-hydroxy—all-trans-methylretinoate (2); retinyl acetate (3), added as internal standard.

4. DISCUSSION

It has been established that vitamin A-active retinoids exert their actions through
their nuclear receptors to alter gene expression at the level of transcription (Giguere et al.,
1987; Petkovich et al., 1987; Chambon et al., 1991). To study the mechanism of retinoids in
embryonic. development, one needs to determine what retinoids are present in embryo, what
receptor types are expressed, and what genes are responding. Once all these components are
defined, it will be possrble to have a clear picture about the mechanism of action of retinoids
in embryonic development. It is therefore very important to understand the metabolism of
vitamin A

In order to study the metabolism of retinoids during avian embryonic development,
it is necessary to know the forms and concentrations of vitamin A compounds in the egg,
since the embryo is anatomically separated from the hen and the entire vitamin A complement
must be obtained from stores already in the eggs. In 1978, Sreekn'shna et al. analyzed the
yolks of duck and found 40 i 4 pg of retinol/yolk and 6.2 :1: 2 pg of retinyl esters/yolk . In
1980, Heaf et al. applied thin layer chromatography to analyze the yolks fi’Om Japanese quail
and found that the amount of retinol was 120 d: 48 nmol per yolk, equivalent to 34.37 a: 13.75
ug/yolk Retinyl esters and other retinoids were not assessed in these studies. In 1989, Spear
et al. found retinol and retinyl esters in the egg yolk of ring dove. In addition to retinol and

retinyl esters, retinal was also reported in the egg yolk of chicken (Plack, 1960, 1963a, b;

72

73

Plack et al., 1964). In the egg yolks of chicken, the amount of retinol ranged fi'om 49.3 - 122
ug/yolk, the amount of retinal ranged from 11.4 - 21.1 ug/yolk, and the amount of retinyl
esters ranged fi'om 18.8 - 25.5 ug/yolk. Joshi et al. (1973) stated that there are no vitamin A
compounds in the chicken egg white, but no data were given. Quail egg yolk was found
to contain retinol-binding protein (RBP) and transthyretin (TTR) (Heaf et al., 1980). Yolk
RBP concentration was below halfthat of plasma, whereas TTR concentration was about the
same in both fluids. Less than 4% of yolk retinol was reported to be attached to RBP
(holoRBP) and it was concluded that the holoprotein accounts for about half the total
RBP. From the above information it was suggested that a possible fimction for the small
amount of RBP and TTR present in the yolk may be to prime the retinol transport
system in the early stages of embryonic development until a supply of protein from
the embryonic liver becomes available.

The great importance of retinoids in the development of the avian embryo was first
shown by Thompson and coworkers (Thompson, 1969; Thompson et al., 1969), who
succeeded in raising vitamin A-deficient hens; the eggs fi'om these hens were unable to
generate normal embryos. This was confirmed in subsequent studies by Sporn et al. (1985),
Heine et al. (1985), and Dersch and Zile (1993). In the avian embryo, retinoid deprivation
most dramatically prevents the formation of the cardiovascular system. The earliest
observable developmental defects in the embryos fi'om eggs of retinoid-deficient hens are
localized in the heart region (Heine et al., 1985). After early addition of retinoids to
the egg during incubation, the embryos are capable of developing normally (Thompson
et al., 1969; Spom et al., 1985; Heine et al., 1985; Dersch and Zile, 1993). Such a system

could be an excellent model for the study of the molecular mechanisms of retinoid

74

action in embryonic development and in the control of expression of a wide variety
of gene products leading to altered phenotypic expression. Other advantages (Dersch,
1992) of the avian embryo model include :

1) the egg provides an isolated, self-contained system where embryonic development

takes places independently of maternal influences;
2) embryos are available without any surgical or invasive procedures to the hen;
3) avian heart development during the early period of organogenesis is similar to that
of mammals, making the avian embryo an appropriate model for application to
humans and other mammals (Thompson and Fitzharris, 1979a, 1979b, 1985;
Thompson et al., 1983; Thompson et al., 1985);

4) it is possrble to produce avian eggs which do not contain vitamin A, thus providing
a vitamin A-deficient environment for embryonic development.

5) in addition, the quail is a more convenient species than the domestic hen for the
generation of vitamin A-deficient eggs, because it takes 6 months for maturation
of the domestic hen but only 6 weeks in the quail (Thompson, 1969).

To obtain vitamin A-deficient embryos, it is essential to insure the complete absence
of retinoids from eggs. In order to obtain vitamin A-deficient eggs, the quail are reared from
the time they are hatched, on vitamin A- and carotenoid- fi'ee diets containing retinoic
acid, a form of vitamin A that supports the growth and maintenance functions as well as the
reproductive fimctions of the quail, but is not transferred to the egg. The eggs obtained
from these quails do not support normal embryonic development. In their work, Thompson

et al. (1969) and Heine et al. (1985), who used methyl retinoate as the vitamin A supplement

in these diets, implied that the eggs must be devoid of vitamin A compounds to produce the

75

abnormal embryo. However, they never analyzed these eggs for vitamin A content. Thus, it
is not accurate to refer to these eggs as vitamin A-deficient. When methyl retinoate was used
as the supplement in our laboratory, it was found that this form of retinoic acid was
transferred fiom the hen to the yolk; there were 5-15 u g of methyl retinoate per yolk (Dersch
and Zile, 1993). Therefore, in this study it was important to examine the vitamin A content
of the yolks of the eggs obtained from quail fed the vitamin A-deficient diet supplemented
with l3-cis-retinoic acid. The present study shows that there are no retinoids in the extract
from yolks fi'om hens fed the 13-cis-retinoic acid supplemented vitamin A-deficient diet.
However, the HPLC system used can not detect retinoids below 1-2 ng per injected sample.
Thus it is possible, that picogram amounts of retinoids, if present in the yolks, may remain
undetected. However, since the embryos from these eggs do not develop normally, it is very
unlikely that the yolks fi'om these eggs can supply any retinoids to the embryo.

It has been shown that the majority of the vitamin A-deficient quail embryos develop
normally during the first 24 hours of incubation (Heine et al., 1985; Thompson et al., 1969).
However, vitamin A-active molecules must be present in the quail embryo during specific
critical stages in development, i.e. stage 5-8, to ensure the subsequent normal development
(Dersch and Zile, 1993). Dersch and Zile (1993) suggested that all-trans-retinoic acid is most
likely the active form present in the embryo at that time to induce the subsequent normal
development. Indirect evidence for this was provided by the study of Twal et al. (1995) who
used a specific monoclonal antibody against all-trans-retinoic acid (Zhou et al., 1991) to block
normal cardiovascular development and to localize all-trans-retinoic acid in quail embryo

during early development. However, until now no direct evidence is available to support this

hypothesis.

76

The purpose of the present work was to obtain information about the endogenous
vitamin A compounds present in the avian embryo at the time during development,
i.e. stage 5-8, when the vitamin A-active form is required to induce subsequent normal
development, and to identify the retinoid(s) that may be the active form(s) in inducing normal
development. Furthermore, it is also important to demonstrate that it is the absence of
vitamin A-active forms in the vitamin A-deficient embryo that prevents the normal
development of the embryo.

The egg yolk is the only source of vitamin A for the embryo, therefore the metabolism
of vitamin A in the yolk is directly linked to that of the embryo. However, the information
available on vitamin A compounds in avian eggs is limited. It was therefore important to
address also this aspect of avian vitamin A metabolism in the present study. The present study
demonstrates that normal quail egg yolk contains all-trans-retinol, 3,4-didehydroretinol and
retinyl esters, and that all-trans-retinol is the predominant form of vitamin A in the yolk. This
agrees with the early work of Heaf (1980) who found in the egg of Japanese quail 120t48
nmol of retinol per yolk, equivalent to 34.37ztl3.75 rig/yolk; these findings are comparable
to those in other species, including the eggs of duck (Sreekrishna et al. 1978), ring dove
(Spear et al., 1989), and chicken (Plack, 1960; 1963a; 1963b; 1964). However , while Plack
(1963a) found retinal in chicken egg yolk, in the present study no retinal was detected in the
yolks of normal quail eggs. The difi‘erences in the results may be due to species difi‘erences
as well as due to the difl‘erent methods of analysis. .

HPLC methods are generally used to separate and directly determine retinoid
metabolites in embryonic and other biological systems. However, because of the low

level (pg) of retinoids available from embryonic tissues, the detection limit of the

77

conventional HPLC systems makes their determination in the early embryos very dimcult,
as large amounts of pooled tissue are required to generate detectable quantities of retinoids.
Thus, until now, no data have been available on the metabolite profile of endogenous vitamin
A compounds in the early avian embryo. The present study provides for the first time the
metabolite profiles and concentrations of endogenous retinoids in the avian embryo during
gastrulation.

Several laboratories have addressed the question of bioactive retinoids during
embryogenesis. Chen et al. (1992) applied a molecular bioassay to determine retinoid activity
(Wagner et al., 1992) that utilizes F9 cell lines, transfected with a reporter construct
consisting of a retinoic acid response element (RARE) upstream of the B-galactosidase gene.
They demonstrated that the early chicken embryo contains endogenous retinoids that can
activate RARE. Stage 4 embryo was found to contain activity equivalent to 0.41-0.61 pg of
retinoic acid and stage 6 embryo was found to contain activity equivalent to 1.37-2.37 pg of
retinoic acid. In subsequent studies Chen et al. (1995) found that stage 6-7 quail embryo
contains 3 .4 pg of active retinoids. However, the above assay system is not capable of
determining which isomer or metabolite of retinoic acid is present. Recent work has
suggested that not only all-trans-retinoic acid, but also 9-cis-retinoic acid, 4-oxoretinoic
acid, and 3,4-didehydroretinoic acid may be active participants in the retinoid signal
transduction system (Leid et al., 1993; Eichele, 1993; Pijnappel et al., 1993). Similarly to all-
trans-retinoic acid, digit duplication can be produced by 3,4-didehydroretinoic acid and 9-cis-
retinoic acid (Thaller and Eichele, 1990; Thaller et al., 1993). Both 9-cis-retinoic acid and
4-oxoretinoic acid have been identified in the Xenopus embryo and can cause

dysmorphogenesis of the embryo (Pijnappel et al., 1993; Creech Kraft et al., 1994a).

78

In the work reported here, all-trans—retinoic acid, 2 pg, and 3,4-didehydroretinoic
acid , 1 pg, were detected in the normal stage 5-8 quail embryo. This concentration range is
compatible with the retinoid activity obtained using reporter assay (Chen et al. 1992; Chen
et al., 1995). However, it was impossible in the present study to accurately determine the
concentration and identity of other active retinoids, for example, 4-oxoretinoic acid, 9-cis-
retinoic acid, l3-cis-retinoic acid, or other unknown compounds.

Endogenous retinoids are vitamin A compounds and therefore are not synthesized
de novo, but derived from precursor substances in the forms of retinyl esters, retinol and
carotenoids. However, at this time, it is not known what is the source of the bioactive
retinoids in the embryo: are they generated from the vitamin A compounds in the yolk and
then transferred to the embryo or does the embryo obtain retinol and retinyl esters fi'om the
yolk and then metabolize these forms to active forms in situ. This study shows that the early
quail embryos contains all-trans-retinol, 3,4-didehydroretinol, all-trans-retinal and retinyl
esters as well as two active retinoic acids. The concentrations of retinol and retinyl esters are
219 nM, i.e. 40 times greater than that of all-trans-retinoic acid (5 nM). This finding is
consistent with the hypothesis that retinol serves as an in situ precursor to retinoic acid in the
embryo. In addition, the embryo was found to contain all-trans-retinal. This firrther supports
the postulated pathway of retinoic acid biosynthesis fiom retinol since retinal is an obligatory
intermediate (see reviews by Wolf , 1984; Blaner and Olson, 1994). The presence of this
metabolic pathway in the early embryo is also supported by the work of Thaller and Eichele
(1988) who studied retinoid metabolism in the wing bud of stage .20 chicken embryos and
found that locally applied [’H]-retinol is converted primarily to [3H]-retinal and [3H]-retinoic

acid. When [3H]-retinal was locally applied, it was either oxidized to [3H]-retinoic acid or

79

reduced to [3H]-retinol. In contrast, delivery of retinoic acid to the site of the bud did not
yield retinal or retinol. The data showed that retinoid metabolism can take place in situ in the
limb bud of the chick embryo when exogenous retinoids are provided.

Further support for in situ metabolism of retinoids in the present study with the early
quail embryo comes from the finding that neither retinal nor retinoic acids were detected in
the normal quail egg yolks from where the embryo obtains its retinoids. Thus, the in situ
metabolism could provide a simple mechanism to generate in a controlled fashion the
picogram amount of the biologically active retinoic acid from its abundant (nanogram
amount) biosynthetic precursor retinol. In addition, the existence of an in situ metabolism
supports the hypothesis that retinoids are local chemical mediators involved in embryonic
development (Thaller and Eichele, 1988; Hogan et al., 1992).

Another major observation in this study is that the normal embryo contains 3,4-
didehydroretinoic acid and its amount is in the same concentration range as that of all-trans-
retinoic acid. This finding suggests a firnction for 3,4-didehydroretinoic acid in early
development. The embryo also has 3,4-didehydroretinol, the precursor to this retinoid. It was
not possible to determine if the aldehyde intermediate is a metabolite in the embryos, since
the 3,4-didehydroretinal standard is not available. This aldehyde has been found in the
Xenopus embryos (Creech Kraft et al., 1994b). Several small peaks were observed in the
region where 3,4-didehydroretinol and retinal eluted, but they remained unidentified. The 3,4-
didehydroretinol and 3,4-didehydroretinoic acid have also been isolated from 3-4-day-old
chicken embryos (Thaller and Eichele, 1990). These researchers showed that 3,4-
didehydroretinoic acid and all-trans-retinoic acid are equipotent in evoking digit duplication

and that the morphogenic activity of 3,4-didehydroretinoic acid does not depend on its

80

conversion to all-trans-retinoic acid. It was found that 3,4-didehydroretinoic acid is generated
in situ from all-trans-retinol involving several steps. They hypothesized that retinol is first
dehydrogenated at the 3,4 bond of the ionylidene ring to 3,4-didehydroretinol.
Dehydrogenation is followed by two sequential oxidations at carbon 15, which convert the
hydroxyl group of 3,4-didehydroretinol, through an aldehyde intermediate, into the carboxyl
group of 3,4-didehydroretinoic acid (Thaller and Eichele, 1990). The conversion of retinol
to 3,4-didehydroretinol is probably a rate limiting step in the formation of 3,4-
didehydroretinoic acid (Andersson et al., 1994). The ability of the early quail embryo to
gerraate 3,4-didehydroretinol in situ has also been demonstrated in our laboratory. When a
single dose of [’H]-retinol was administrated into the vitamin A-deficient quail embryo, 3,4-
didehydroretinol was identified as a major metabolite of retinol in the embryo (Dersch, 1992).
The generation of 3,4-didehydroretinol from all-trans-retinol has been also demonstrated in
several other embryonic tissues. Neural tissue from chicken embryos can readily convert
retinol into 3,4-didehydroretinol (Wagner et al., 1990). Xenopus eggs and embryos contain
3,4-didehydroretinol and 3,4-didehydroretinal (Azuma et al., 1990; Creech Kraft et al.,
1994b).

Vitamin A2 or 3,4-didehydroretinol has been long recognized as a major form of
vitamin A in fish and arnphibia (Moore, 1957) and was thought to be specific to these species.
However, recently it has also been identified in human and other mammalian tissues. Human
keratinocytes cultured in serum-supplemented medium contain variable high concentrations
of 3,4-didehydroretinol, and when supplemented with [’H]-retinol, rapidly convert this tracer
into [3H]-3,4-didehydroretinol (Andersson et al., 1994; Rollman et al.,1993; Randolph and

Simon, 1993). HeLa cells and melanoma calls contain low concentrations of 3,4-

81

didehydroretinol and are able to produce this metabolite when supplemented with all-trans-
retinol in vitro (Andersson et al., 1994). In contrast, terotocarcinoma F9 cells and monkey
kidney epithelium (CV-l) cells neither contain endogenous 3,4-didehydroretinol nor produce
[’H]-3,4-didehydroretinol when incubated with [3H]-retinol (Andersson et al., 1994). Thus,
it was suggested that the expression of the 3,4-didehydro metabolic pathway is cell- and
tissue- specific (Andersson et al., 1994). The present studies suggest that this pathway may
also be important in early avian development. However, didehydroretinoids could not be
detected in the mouse embryo (Horton and Maden, 1995). The metabolism of
didehydroretinoids in different species needs firrther study.

In the present study, the egg yolk of hens fed normal chow diet was found to contain
about 10 times more all-trans-retinol than 3,4-didehydroretinol, while in the stage 5-8 embryo
3,4-didehydroretinol is the predominant form of retinol. The amount of all-trans-retinol in egg
yolk is more than a thousand times greater than that in the embryo. The amount of 3,4-
didehydroretinol in egg yolk is only about 60 times greater than that in the embryo. This
suggests that the 3,4-didehydroretinol in embryo is most likely the product of the in-embryo
metabolism of all-transpretinol, instead of this retinoid having been transported from the yolk
to the embryo. This agrees with the previous findings that 3,4-didehydroretinol can be
generated by the limb bud (Thaller and Eichele, 1990). However, it is also possible that some
of the 3,4-didehydroretinol found in the early quail embryo in the present study may have
been transported fi'om the yolk, which was found to also contain 3,4-didehydroretinol. The
limited information available on the binding characteristics of didehydroretinol to the all-

trans-retinol transport protein, RBP, suggests that 3,4-didehydroretinol can bind to this

 

if.

82

transport protein (Taumihardjo et al., 1987); RBP has been shown to be present in chicken
yolk (Heaf, et al., 1980).

It has been reported that esterification is an obligatory pathway for 3,4-
didehydroretinol formation (Randalph and Sirnrnon, 1993) and that epidermal retinol and
3,4-didehydroretinol are to a large extent esterified (Torma and Vahquist 1985; 1990). In the
present study, a relatively large amount of retinyl esters were found. However, because there
were no standards available, 3,4-didehydroretinyl esters could not be identified in the embryos
and yolks.

All-trans-retinoic acid has been suggested as the most active form of vitamin A (Wolf,
1984; Gudas, 1992; Gudas et al., 1994). It was also demonstrated to be the physiological
active form of vitamin A to ensure normal cardiovascular development both in ovo (Dersch
and Zile, 1993) and in cultured vitamin A-deficient quail embryos (Kostetskaia et al., 1995).
With the anti-all-trans-retinoic acid monoclonal antibody, the localization of all-trans-retinoic
acid (Twal et al., 1995) in early normal embryo established that, in addition to cardiovascular
development, other very early development events, including axial organization, regression
of the primitive streak, and the development of head, may also be regulated by all-trans-
retinoic acid. Therefore, it is very likely that in the normal embryo endogenous all-trans-
retinoic acid is biosynthesized in target cells as required for all-trans-retinoic acid-mediated
gene regulation and that these autocrine events are developmentally regulated (Twal et al.,
1995; Kostetskii et al., 1995). Retinoic acid binds to nuclear receptors that function as
ligand-regulated transcription factors (Petkovich et al., 1987; Giguere et al., 1987; Zelent et
al., 1989). It has been established that the expression of RARa and y in the early quail

embryo is independent of vitamin A status, while the expression of RARBZ isoforrn is

83
regulated by vitamin A status during early avian development (Kostetskii and Zile, 1993;
Kostetskii et al., 1995). Furthermore, the expression of RARB2 in vitamin A-deficient embryo
can be induced by all-trans-retinoic acid (Kostetskii et al., 1995).

The two parallel biosynthetic pathways for all-trans—retinoic acid and 3,4-
didehydroretinoic acid initiate from the same precursor retinol and generate two equipotent
signaling molecules. The 3,4-didehydroretinoic acid can also bind to RAR (Torma et al.,
1994). X-ray structure analysis revealed that a 3,4 double bond will force the ionylidene ring
into an almost planar conformation (Simmons et al., 1986), a structural change that could
selectively affect its aflinity for one of the several retinoic acid receptors or to the cellular
retinoic acid binding protein (Daly and Redfem, 1988; Thaller and Eichele, 1990). This
suggests that 3,4-didehydroretinoic acid may be involved in signaling a pathway difi‘erent
from that of all-trans-retinoic acid. In addition, the presence of a 3,4 double bond may
influence the half life of the molecule, as degradation of retinoic acid is initiated by
hydroxylation at the 4 position (Thaller and Eichele, 1990) .

The presence of 3,4-didehydroretinol during embryonic development and in
keratinizing or malignant human epithelial cells suggests that this metabolic pathway is in
some yet unknown way coupled to the difl‘erentiation process. This is easiest explained by
3 ,4-didehydroretinol being the precursor of 3,4-didehydroretinoic acid and other specific
metabolites with high affinity for the nuclear retinoid receptor (Torma et al., 1994). Whether
3,4—didehydroretinol itself also regulates gene expression by a direct interaction with nuclear
retinoid receptors remains to be elucidated. By analogy, a recent report suggested that retinol

has a weak aflinity for RAR and thus cannot be ruled out as a RAR ligand under physiological

 

84

conditions when retinol heavily predominates over retinoic acid (Repa et al., 1993). The
regulation of the biosynthesis and degradation of 3,4-didehydroretinol is still an open issue.

It has been suggested that 3,4-didehydroretinoic acid can also participate in the
cardiovascular development of early quail embryo. The study of Dersch and Zile (1993)
demonstrated that this retinoid has some biological activity (50% of all-trans-retinoic acid)
in the in ovo induction of normal cardiovascular development in vitamin A-deficient quail
embryos. However, in cultured embryos it is as potent as all-trans-retinoic acid in inducing
normal cardiovascular development in vitamin A-deficient embryo (Kostetskaia et al., 1995).
The demonstration of 3,4-didehydroretinoic acid in normal stage 5-8 embryos in the present
work strongly supports the hypothesis that 3,4-didehydroretinoic acid is also involved in early
embryonic development, especially, cardiovascular development. Further support comes from
study of the blocking of embryonic development with the anti-all-trans-retinoic acid
monoclonal antibody (Twal et al., 1995). Since this antibody also cross reacts (80%) with 3,4-
didehydroretinoic acid, the blocking of normal cardiovascular development with this antibody
may also indicate the blocking of the firnction of 3,4-didehydroretinoic acid.

The 9-cis isomer of retinoic acid was shown to be completely inactive in the induction
of normal cardiovascular development in vitamin A-deficient quail embryos in ovo (Dersch
and Zile, 1993); similarly 4-oxoretinoic acid was also inactive for the in ovo induction of
normal cardiovascular development in vitamin A-deficient quail embryos (unpublished
results). In contrast, when tested in the cultured vitamin A-deficient embryos, these retinoids
showed the same biopotency as all-trans-retinoic acid (Kostetskaia et al., 1995). However,
in the metabolic study reported here, the presence of these two retinoids could not be firmly

established in the stage 5-8 normal embryos, i.e. the level of these retinoids was very low

85

compared to all-trans-retinoic acid or 3,4-didehydroretinoic acid. Thus, it is unlikely that 9-
cis-retinoic acid and 4-oxoretinoic acid are biosynthsized in situ and involved in the
endogenous regulation of cardiovascular development during these early stages. Their
biological activity in culture may be due to the fact, that when provided exogenously in the
media they may be able to bind to the retinoid receptors and thus substitute for the lacking
endogenously active retinoids.

The proposed pathways of endogenous vitamin A metabolism in stage 5-8 normal

quail embryo are shown in Figure 22.

4.1 Summary and Conclusions

1. The studies described here provide for the first time direct evidence for the
presence of vitamin A-active forms in the early avian embryo at the time in embryogenesis
when critical developmental events are initiated by retinoids.

2. The stage 5-8 quail embryos obtained fi'om hens fed normal chow diets
contained all-trans—retinoic acid (5><10'9 M), 3,4—didehydroretinoic acid (4x 10" M), all-trans-
retinol (1.09><10'7 M), 3,4-didehydroretinol (1.85 ><10‘7 M), all-trans-retinal (8.5 x 10" M),
retinyl palmitate (7.9><10"M), and retinyl stearate (3.1><10" M). The metabolite pattern in the
embryos suggests that the embryo is able to metabolize the vitamin A provided by the yolk
and to biosynthesize vitamin A-active form(s) required for development. The eggs fiom
which these embryos were obtained contained in their yolk all-trans-retinol (11.86-68.86
pg), 3,4-didehydroretinol (1.09-4.93 pg), and retinyl esters (389-2846 pg). No retinoids

were detected in the albumen.

86

   

M2. Proposed pathways of endogenous vitamin A metabolism in early Japanese
quail embryo

87

3. The stage 5-8 quail embryos obtained from hens fed a vitamin A-deficient diet
supplemented with l3-cis-retinoic acid did not have any identifiable active or inactive vitamin
A compounds. The eggs from which these embryos were obtained contained no retinoids in
their yolks and albumen.

4. The sensitivity for the detection and quantitation of the retinoids by the HPLC
method used in these studies is 1 ng. It is therefore possible that retinoids present in the
pooled extract from 2000 embryos at levels below the detection limit have not been
detected.

5. The results fiom this work support the hypothesis that all-trans-retinoic acid and/or
3,4-didehydroretinoic acid are the vitamin A-active forms required for normal cardiovascular
development to be initiated in the stage 5-8 quail embryo.

6. The results fiom this work support the hypothesis that the embryos from eggs of
hens fed vitamin A-deficient diet supplemented with 13-cis-retinoic acid are vitamin A-
deficient as the pooled extract from 2100 vitamin A-deficient embryos did not contain any
detectable (more than 1 ng ) vitamin A-active retinoids. The inability of these embryos to
develop normally supports the conclusion that these embryos are vitamin A-deficient or

contain an amount of retinoids that is insuflicient to support normal embryonic development.

4.2 Future research
Retinoid metabolism in avian embryonic development is an exciting area of research.
There are still many aspects to be investigated. In future research in retinoid metabolism it

will be necessary to determine:

88

1) at what time point during development the normal embryo becomes competent to
generate the vitamin A-active forms, e.g. retinoic acids;

2) if the early vitamin A-deficient quail embryo is competent to generate vitamin A-
active forms, i.e. retinoic acids from administered retinol;

3) what is the in situ enzymatic pathways of metabolism of endogenous retinoids. The
study should include the effects of known inhibitors on the conversion of retinol to retinoic
acid, using inhibitors such as citral, ethanol and other potential retinol inhibitors.

4) what is the temporal and spatial distribution pattern of retinoids in embryonic
tissues; and the distribution of binding proteins; what are the mechanisms whereby the
patterns of retinoid distribution are created and maintained during quail embryonic
development;

5) what are the interactions of retinoid metabolites with cellular binding proteins and
nuclear receptors;

6) how do teratogenic doses of retinoic acid afl‘ect retinoid distribution pattern and
metabolism during embryonic development.

These studies will provide a more complete and detailed information about retinoid
function and the transduction mechanisms regulated by vitamin A in avian embryonic

development.

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