EVALUATION OF COLORIMETRIC DETERMINATIONS
OF VITAMIN A IN FOODS
by
REGINA EGBUONU, B.S.
A THESIS
IN
FOOD TECHNOLOGY
Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for
the Degree of
MASTER OF SCIENCE
T2
ACKNOWLEDGEMENTS
I wish to express my sincere appreciation to Dr. Leslie D.
Thompson and Dr. Gordon W. Davis for their assistance and criticism
during my research. I am deeply grateful to Dr. J. E. McCroskey and
Dr. Ronald M. Miller for their helpful advice and counsel during the
research. I would like to thank Dr. James R. Clark, Dr. David B.
Wester and Mr. Luke J. Celantano for their help in the statistical
analysis of the data.
Very special thanks and appreciation are extended to my parents
for their understanding and encouragement throughout this graduate
study.
n
CONTENTS
ACKNOWLEDGEMENTS ii
LIST OF TABLES iv
LIST OF FIGURES v
I. INTRODUCTION 1
II. REVIEW OF LITERATURE 5
Nomenclature 5
Chemical Properties 7
Physical Properties 8
Metabolism of Vitamin A 11
Uptake, Distribution and Storage 13
Colorimetric Determination of Vitamin A 15
III. EVALUATION OF COLORIMETRIC DETERMINATIONS
OF VITAMIN A IN FOODS 19
Summary 19
Introduction 20
Experimental Procedure 22
Colorimetric Determination 23
Color Development and Stability 24
Statistical Analyses 25
Results and Discussion 26
Conclusions 62
REFERENCES 65
m
CONTENTS
ACKNOWLEDGEMENTS ii
LIST OF TABLES iv
LIST OF FIGURES v
I. INTRODUCTION 1
II. REVIEW OF LITERATURE 5
Nomenclature 5
Chemical Properties 7
Physical Properties 8
Metabolism of Vitamin A 11
Uptake, Distribution and Storage 13
Colorimetric Determination of Vitamin A 15
III. EVALUATION OF COLORIMETRIC DETERMINATIONS
OF VITAMIN A IN FOODS 19
Summary 19
Introduction 20
Experimental Procedure 22
Colorimetric Determination 23
Color Development and Stability 24
Statistical Analyses 25
Results and Discussion 26
Conclusions 62
REFERENCES 65
111
LIST OF TABLES
1. Effect of wavelength on absorbance within chloroform and methylene chloride 37
2. Effect of wavelength on quantitation of vitamin A and recovery in liver samples 38
3. Wavelength and solvent effect on recovery of standard vitamin A from samples 39
4. Effect of solvents on mean recovery of vitamin A acetate from liver samples 41
5. Effect of solvent on the color intensity produced at different vitamin A concentrations 42
6. Effect of storage (days) on activity of TCA reagent in chloroform at 620 and 616nm 45
7. Effect of storage (days) on activity of TCA reagent in methylene chloride at 620 and 616nm 48
8. Mean vitamin A concentration in liver tissue 60
9. Vitamin A concentration in liver as determined with TCA-CH2CI2 and TCA-CHCI3 at 616nm and 620nm 61
10. Mean vitamin A and recovery of vitamin Afrom liver tissue 63
IV
LIST OF FIGURES
Figures
1. Absorption spectra of blue species produced by vitamin Aand TCA in chloroform and methylene chloride at 0 day 27
2. Absorption spectra of blue species produced by vitamin A and TCA in chloroform and methylene chloride after 2 days of storage 28
3. Absorption spectra of blue species produced by vitamin A and TCA in chloroform and methylene chloride after 4 days of reagent storage 29
4. Absorption spectra of blue-colored species produced by vitamin A and TCA in chloroform and methylene chloride after 6 days of reagent storage 30
5. Absorption curve of the violet color produced by vitamin A and 1, 3-DCP/SbCL3 at 0 day of reagent storage 32
6. Absorption curve of the violet color produced by vitamin A and 1, 3-DCP/SbCL3 after 2 days of reagent storage 33
7. Absorption curve of the violet color produced by vitamin A and 1, 3-DCP/SbCL3 after 4 days of reagent storage 34
8. Absorption curve of the violet color produced by vitamin A and 1, 3-DCP/SbCL3 after 6 days of reagent storage 35
9. Effect of storage time (days) on stability of TCA reagent in chloroform and all-trans vitamin A acetate at 620 and 616nm 44
10. Effect of storage time (days) on stability of TCA reagent in methylene chloride and all-trans vitamin A acetate at 620 and 616nm . 46
11. Absorbance at 620nm versus time (min) of blue complex formed during determination of vitamin A . . . 49
12. Absorbance at 616nm versus time (min) of blue complex formed during determination of vitamin A . . . 50
13. Absorbance at 620nm versus time (min) of blue complex formed during determination of vitamin A . . . . 51
14. Absorbance at 616nm versus time (min) of blue complex formed during determination of vitamin A . . . . 52
15. Absorbance at 550nm versus time (min) of violet color formed during determination of vitamin A 54
16. Standard calibration curves for vitamin A acetate using TCA in methylene chloride (-.-), and chloroform (-*-) as solvents at 620nm 55
17. Standard calibration curves for vitamin A acetate using TCA in methylene chloride (-.-), and chloroform (-*-) as solvents at 616nm 57
18. Standard calibration curve for vitamin A acetate using 1, 3-DCP/SbCL3 at 550nm 59
VI
CHAPTER I
INTRODUCTION
Retinoids are a group of molecules comprising retinol, retinalde-
hyde, retinoic acid and their synthetic analogs. Various synonyms of
vitamin A include retinol, axerophthol, biosterol, vitamin A,, antixer-
ophthalmic vitamin and anti-infective vitamin. The history of vitamin
A dates back to 1550 B.C. when nyctalopia (night blindness) was reversed
with topical application of fat soluble factor A to the eyes (Wolf,
1978). It wasn't until the mid thirties that the "factor" was isolated
and synthesized (Isler et al., 1947; Moore, 1957).
Physiological forms of vitamin A include retinol and esters, 3-
dehydroretinol (vit A2) and esters, retinal (retinene, vitamin A alde
hyde), 3-dehydroretinal Iretiene-2), retinoic acid, neovitamin A and
neo-b-vitamin A,. Active analogs and related compounds include a, 3,
Y-carotene, neo-b-carotene B, cryptoxanthine, myxoxanthine and torula-
harhodin (Bauernfiend, 1981). Vitamin A is stored in the liver adipose
tissue as the esters, retinyl palmitate and retinyl acetate, and
approximately 5% is stored while 95% is hydrolyzed and circulated
throughout the body (Heller, 1979). Three forms of vitamin A exist in
the free state and these are known as retinol (vitamin A alcohol),
retinal (vitamin A aldehyde), and retinoic acid (vitamin A acid). Retin
oic acid maintains growth in animals and is not stored in the liver
(Sherman et al., 1981).
Carotenoids are a group of lipid soluble compounds responsible
for many of the yellow and red colors of plants and animal products
(Isler et al., 1967). They are widespread and occur naturally in large
quantities. At present, nearly 300 naturally occurring carotenoids are
known with most of them found in the form of fucoxanthin in algae,
lutein, violaxanthin and neoxanthin in green leaves (Straub, 1971).
Any carotenoid which contains the retinol structure in the molecule
may be converted in vivo to retinol (Olson and Hayaishi, 1965). Recent
evidence suggests that, in addition to its property as vitamin A precur
sors, carotenoids may be involved in other biochemical processes in
higher animals and humans. For example, a protective effect of 3-caro-
tene against cancer has been postulated (Peto et al., 1981). This hypo
thesis, though somewhat speculative, has aroused new interest in the
fate of carotenoids in the living cell with respect to human beings.
Vitamin A is essential for the maintenance and normal function of
the eyes and epithelial tissues lining the respiratory and reproductive
tracts, and bone development in animals and humans (NRG/NAS, 1979).
Recent evidence suggests vitamin A plays a primary role in glyco-protein
synthesis (DeLuca, 1977). A deficiency may decrease resistance to
infection, cause xerophthalmia or cutaneous changes (WHO, 1982; Sauber-
lich et al., 1974; Krishnan et al., 1974). Toxicity of the vitamin has
been observed in animals and humans. Dosages in excess of 50,000 lU per
day (10 times the RDA) for extended periods in humans have been documen
ted with clinical symptoms of drowsiness, increased cranial pressure,
hypercalcemia, hair loss and hypercarotenemia in the skin, eyes and
other tissues (Jeghers and Marraro, 1958). Acute and chronic toxicity
can occur in growing pigs with doses ranging from 440,000 to 1,100,000
lU/kg feed. Clinical findings of toxicity in animals include emaciation,
nausea, joint swelling, skin inflammation, growth depression and irrit
ability (Wolbach and Maddock, 1951).
Assays of vitamin A in tissue extracts are often affected by the
presence of restrictive or inhibitory contaminants (Moore, 1957; Olson,
1965), phytofluene in fluorescence assays (Thompson et al., 1971) and
3-carotene in the Carr-Price assay (Neeld and Pearson, 1963). In
nutritional surveys, however, the chromatographic purification of vitamin
A before analysis is time consuming and costly while the correction form
ula method (Morton and Stubbs, 1948) is often not applicable.
Vitamin A determination in animal tissues has been the subject of
considerable attention and some criticism in past years. A solvent
extraction procedure employing acetone and light petroleum, in equal
amounts, at room temperature was described by Bayfield (1975) for determ
ination of vitamin A products in liver. The estimation of vitamin A by
methods that utilize colorimetric finish generally provide the analyst
with some problems, such as end-point detection and sensitivity of the
reagent in various solvent systems (Subramanyam and Parrish, 1976;
Rosenheim and Drummond, 1925). The use of trichloroacetic acid (TCA) as
a reagent for vitamin A determination was investigated because it is
readily available for routine laboratory analysis, inexpensive, provides
adequate sensitivity (10-20 yg), specific for most purposes and less
toxic than trifluoroacetic acid and antimony trichloride (SbCls).
The purpose of this study was to examine the use of trichloroacetic
acid (TCA) as a reagent in two solvent systems (chloroform and methylene
chloride) and investigate the effect of wavelength solvents and storage
time on the reactivity and sensitivity of TCA during colorimetric determ
ination of vitamin A in foods.
CHAPTER II
REVIEW OF LITERATURE
The recognition of the existence of vitamin A as a factor capable
of correcting defective dark vision dates back several thousand years.
Aykroyd (1944) reported how Eber's paprus, an ancient Egyptian Medical
treatise of about 1550 B.C., recommends roast ox-liver, or liver of a
black cock, as curative agents. Vitamin A as a dietary factor has been
learned by the practical experience of sufferers from the "dark adapt
ation to light" condition in parts of the world that are too remote to
reach or that do not read from the writings of ancient and recent scien
tists. The evidence of the relationship of vitamin A to dark adaptation
was reported in 1925 by Friderica and Holme (1925) who demonstrated that
the pigment "visual purple" in deficient rats could form slowly in their
retinas.
Nomenclature
The term "vitamin A'' is used generically for all 3-ionone deriva
tives, other than the provitamin A carotenoids, which exhibit the biolog
ical activity of all-trans retinol (WHO, 1982). Six isomers of retinol
(Olson, 1968) include all-trans, 13-cis, 9-cis, 11-cis, 9-13-cis and 11-
13-cis retinol. Both biological activity and physical properties of
retinol, retinal and retinoic acid are dependent on their isomeric form.
Retinoic acid has biological activity in growth and differentiation of
cells but not in vision or reproduction (Deluca, 1977; Takase et al.,
1979). The biologically active form of vitamin A in vision is U-cis
retinaldehyde. The isomerization of 11-cis retinal, the chromophore
of visual pigment was elucidated by Hubbard (1966) as being stimulated
by temperature, light and iodine.
Of the more than 400 carotenoids that have been reasonably well
characterized, only about 30 have provitamin A activity (for example,
3-carotene, cryptoxanthin and echinenone). Like retinol, carotene
exists in isomeric forms (Rosenheim and Starling, 1931; Karrer, 1931).
These include a- and 3-carotene. Physiologically, 3-carotene is more
active in promoting growth and causing the storage of vitamin A in the
liver than a-carotene. The difference in activity has been linked to
the ring structure necessary for the formation of vitamin A at both
ends of its molecule, whereas a-carotene only has the required forma
tion at one end.
Recently, the term "retinoid" has been defined to include both
the naturally occurring compounds with vitamin A activity and synthetic
analogs of retinol, with or without biological activity. In a report of
the lUPAC-IUB Joint Commission on Biochemical Nomenclature (1982), it
was stated that:
Retinoids are a class of compounds consisting of four isoprenoid units joined in a head-to-tail manner. All retinoids may be formally derived from a monocyclic parent compound containing five carbon-carbon double bonds and a functional group at the terminus of the acyclic portion.
To avoid confusion with previously used names in this field, they recom
mended, "The term vitamin A should be used as the generic descriptor
for retinoids exhibiting qualitatively the biological activity of
retinol." According to lUPAG-IUB (1982), the basic structures are
7
called retinol (1), retinaldehyde (2), and retinoic acid (3), and
these names always refer to the completely trans compounds.
Chemical Properties
Retinoid isomers undergo isomerization reaction at the side chain
positions. Cisoid retinoids have been isomerized to yield the corres-
poinding trans compounds by catalysis with iodine (Hubbard, 1966;
Reif and Grassner, 1973). The rate of isomerization increased rapidly
with temperature and in the presence of iodine (Hubbard, 1966). In the
membrane of the rod outer segment, phsophatidylethanolamine specific
ally catalyzes the isomerization of retinaldehydes in a dark reaction
at the A " double bond (Groenendijk et al., 1980a). Furthermore,
retinol isomerization has been catalyzed by flavins (Walker and Radda,
1967) in methanolic solution. The reaction was characterized by a
decrease in activation energy at 328nm upon illumination.
Retinyl acetate in the presence of trichloroacetic acid polymerizes
to a complex of three to five molecules of retinyl acetate with ill-
defined structure (Blatz and Pippert, 1966). Similarly, when retinoic
acid was treated with antimony trichloride (SbCl3) or an acid, a charac
teristic transient intermediate with a peak intensity of 573nm was
formed, which was converted to a second compound with absorption maxima
at 470nm (Katsui et al., 1966).
Pronation of retinoids with trifluoroaceticacid (Dugan et al.,
1964) and antimony trichloride (Blatz and Estrada, 1972) was reported
8
to yield colored cations which has long been used as the basis of the
Carr-Price reaction for the colorimetric determination of retinoids.
Retinol may be irreversibly oxidized by the transfer of electrons
to oxygen. Atmospheric oxidation of retinol as a colloidal suspension
in saline is stimulated by ferrous ion but inhibited by a-tocopherol,
a-tocopheryl acetate (Lucy, 1966) and other antioxidants such as butyl-
ated hydroxy toluene (BHT) with retinyl acetate and retinyl palmitate
(Bayfield, 1975).
Photo isomerization of retinaldehydes is one of the fundamental
reaction steps in the process of vision. All-trans retinaldehyde and/or
its 3, 4-didehydro derivate are chromophores in all species (Wald, 1968).
Hubbard (1954) discovered that by bonding to various lipoproteins these
aldehydes form the light-absorbing systems and that all visual pigments
contained one retinaldehyde per molecule of opsin. Rhodopsin of the
rods which absorb maximally at 487nm and three iodopsins in different
cone cells, which absorb at 420nm (blue cones), 534nm (green cones) and
563nm (red cones) are the four main kinds of vitamin A-containing photo-
pigments (Bors and Fells, 1971) for vision.
Physical Properties
Retinol and retinyl acetate are light yellow crystals (pure form)
with melting points ranging from 62-54''G and 57-58''C, respectively.
Retinyl palmitate on the other hand is a semi-solid, yellow crystal with
a melting point below 30''G. Vitamin A and its esters are soluble in
fats, oils, ether and chloroform, while solubility is < 10% in isopro-
panol (Bauernfiend and Corte, 1974).
9
Spectrophotometrically, vitamin A and its esters have different
1%
^Icm *̂" different solvent systems. The biological activities of vita
min A alcohol, vitamin A acetate and vitamin A palmitate, expressed on
the basis of vitamin A value in lU/g, are 3.33 x 10^, 1.9 x 10^, and
1.82 X 10 , respectively. One international unit is equal by defini
tion (WHO, 1950) to 0.6 yg vitamin A alcohol, 0.344 yg of vitamin A
acetate and of 0.55 yg of vitamin A palmitate.
Retinol and its esters are the only naturally occurring retinoids
that fluoresce appreciably under normal conditions. Sobotka et al.
(1944) first developed a quantitative fluorimetric assay for retinyl
ester. The fluorescence faded rapidly and carotenoids interfered with
the assay. Retinol and its esters emit a pale green fluorescence when
excited with near-ultra violet light. Popper and Greenberg (1941)
also used this property to identify retinol in t-ssues and cells contain
ing the vitamin.
Careful application of fluorescence assays for the measurement of
retinol in plasma and liver took place in the late 1960s and 1970s
(Hansen and Warwick, 1968; 1969a,b; Drujan et al., 1968; Thompson et al.,
1971, 1972, 1973). The wave lengths for excitation and emission are
near 340 and 490nm, respectively, with reference to the type of solvent
employed during analysis. Generally, hexane, cyclohexane, xylene,
dioxane of some other nonpolar hydrocarbon solvent is used. Of various
solvents, the fluorescence of retinol is highest in dioxane followed
by cyclohexane, whereas polar solvents, such as ethanol, chloroform and
acetone, tend to quency the fluorescence (Olson and Pungpapongsa,
(1967).
10
The reliability of this assay is influenced by many factors. One
of these factors, as reported by Thompson and his workers (1971), is
the presence of other natural fluorescent compounds in the tissue
extract. Another concern has been the presence of fluorescent contamin
ants in the solvents employed (Davidson, 1979).
Despite some of these drawbacks, fluorescent measurement of retinol
is extremely useful where a spectrofluoremeter is already at hand and
the assay method has been properly standardized. Recently, scores of
studies have shown that the measurement of retinol has been automated
(Thompson and Mad^re, 1978; Giuliany, 1981).
Retinol and its esters also have the ability to absorb strongly at
325nm, a property that been utilized in direct measurement of their
absorbances using colorimeters and spectrophotometers. The principle
is based on the fact that all-trans vitamin A alcohol in isopropanol has
maximum absorption at 325nm (Pharmacopoeia of the United States, 1970).
At this wavelength, the absorbance is directly proportional to vitamin
A concentration, and the method can be used for the determination of
synthetic vitamin A esters in pharmaceutical preparations.
In the presence of interfering substances which absorb light
between 300 and 350nm, a false value of vitamin A usually is obtained.
Irrelevant absorption can be overcome, according to Morton and Stubbs
(1948), by using a mathematical correction formula. The mathematical
correction formula is based on knowledge of the absorption curve of the
vitamin in the particular solvent being used, a measurement of the
absorption at a maximum wavelength known for pure vitamin A (gross read
ing), and a measurement at two other chosen wavelengths ("fixation
11
points") one on each side of the maximum. However, a few years later,
this formula was surpassed by the use of standardized equations which
are less cumbersome and based partially on the same principles (Assoc.
Vit. Chem., 1951; Olson, 1979).
• Another property of retinol and its esters which has been utilized
in their measurements in tissue extracts is based on the principle:
under exposure with ultraviolet light, they tend to photooxidize and
polymerize to nonabsorbing products (Bessey et al., 1946). Photo oxida
tion and polymerization of retinol to non absorbing products enabled
de Araujo and Flores (1978) to reduce the work load satisfactorily to
small amount.
Retinol and its esters have the ability to mix with any Lewis acid
under anhydrous conditions yielding an intense transient blue color.
Carr and Price (1926) developed a quantitative assay for retinol using
antimony trichloride (SbCl3). Various assay methods have been developed
using a variety of Lewis acids, including TFA/TCA (Dugan et al., 1964).
The reaction involves the extraction of the hydroxyl moiety, leaving a
retinylic cation which forms a complex with SbCl3 at the C-4 or C-15
position.
Metabolism of Vitamin A
After ingestion of foods, preformed vitamin A and carotenoids are
released by the action of pepsin in the stomach and various proteolytic
enzymes in the upper intestinal tract. Many factors influence the
absorption efficiency of carotenoids and vitamin A: Digestibility of
the foodstuff, the presence of fat, protein, and antioxidants and the
integrity of the mucosal cells (Thompson, 1965; Plack, 1965).
12
Within the mucosal cells, lipids pass progressively from oil drop
lets to emulsions, micelles and even into a true solution when polar
lipids are in large amount (Hoffman and Small, 1967). Among dietary
carotenoids, xanthophylls predominate, but 3-carotene is present in
significant amounts in most plants. The absorption efficiency of
dietary vitamin A is usually 80-90%, with only a slight reduction in
efficiency at high doses while that of dietary 3-carotene is lower (40-
60%), and decreases rapidly with increasing dosage (Olson, 1978).
Transformations of vitamin A and carotenoids mostly occur in the
mucosal cells. The absorbed retinol is esterified with palmitic acid.
Coenzyme A and adenosine triphosphate (ATP) are involved in the ester-
ification reaction (Ross, 1980), incorporated into chylomicra, and
transported via the lymphatics to the general circulation. Some retinol
is oxidized to retinoic acid. 3-carotene and other provitamin A caro
tenoids such as cryptoxanthins are cleaved at the 15, 15'-double bond to
yield 2 molecules of retinaldehyde (Goodman et al., 1965). Activity of
this enzyme (15, 15'-carotenoid dioxygenase) tends to decrease with
inadequate protein intake (Stoecker and Arnich, 1973).
The absorption of some carotenoids varies with species. Conse
quently, some carotenoids may escape cleavage in the gut and appear in
the chylomicra. In some species such as the rat, pig, goat, sheep and
dog almost all of the carotene is cleaved in the intestine while signif
icant amounts of uncleaved carotene are absorbed by man, cattle and
horses (Olson, 1978).
13
The fate of carotenoids in chylomicra has been unresolved. The
tissue site and mechanisms whereby carotenoids are removed from chylom
icra and repackaged with low density lipoprotein (LDL) are not knwon.
Another phase of carotenoids metabolism that will require further
research deals with the question of "Why do some vertebrate species
(man, horses) absorb dietary carotenoids unchanged? Is there a special
mucosal receptor for carotenoids that renders them unavailable for
cleavage by intestinal enzymes in the species indicated, which might be
absent in the white fat species (pig, sheep)?" (Underwood, 1984).
Lotthammer and Ahlswede (1978). were close to finding a possible cellu
lar role of carotenoids in animals. Fertility in cows was enhanced by
dietary carotenoids. While working with humans, some workers have
cited possible cellular roles for carotenoids because of suggestive
epidemiological evidence associating carotenoid intake with risk of
certain types of tumor (Ibrahim et al., 1977; Peto et al., 1981).
Uptake, Distribution and Storage
The retinyl esters in chylomicra are taken up as micron remnants by
the liver (Cooper and Yu, 1978; Ross and Zilversmith, 1977). In ani
mals the uptake of retinyl esters has been found to.be efficient (Carney
et al., 1976). Except in severe cases of chronic hypervitaminosis A in
certain liver diseases, retinyl ester in circulation is seldomly more
than 10% of the circulating vitamin A (McKenna and Bieri, 1982). Vita
min A is ultimately stored in a special perisinusoidal cell called the
lipocyte (Wake, 1980).
The liver plays the major role in the uptake and metabolism of the
newly absorbed retinol (retinyl esters). A quantitative and detailed
14
study by Goodman and associates (1965) revealed that retinyl esters
appear to remain almost completely with the hydrophobic core of the
chylomicron during its extrahepatic conversion to remnant particle.
Recent studies also have shown that the liver not only takes up retinyl
esters from chylomicra but also from infused solutions (McKenna and
Bieri, 1982) and from circulating retinol binding protein-bound retinol
(RBP-retinol) (Lewis et al., 1981).
Hydrolysis of retinyl esters occurs in the liver during the hepa
tic uptake of dietary vitamin A and during mobilization of retinol from
its stores in the liver. Retinyl ester was reported to be hydrolized
in the "nuclear" and "mitochondria-lysosome rich" fraction of rat liver
homogenates (Mahadevan et al., 1966) after previous studies that com
pounded an inability of the liver to hydrolyze the naturally occurring,
long-chain retinyl ester in vitro (Olson, 1964). While working with
liver of several species (pig, ox, man), Betram and Krisch (1969)
reported that retinyl palmitate was hydrolyzed by purified carboxyles
terase from the livers of the mentioned species.
Although vitamin A is found in most tissues, more than 90% of the
vitamin is stored in the liver. The storage form in the liver is prob
ably a lipoglycoprotein complex in intact lipocytes and ruptured liver
cells (Olson and Gunning, 1980). In its complex form, vitamin A con
sists of 96% retinyl esters and 4% unesterified retinol (Heller, 1979).
The enzyme, retinyl palmitate hydrolate, hydrolizes the complex,
releasing and transferring the retinol (all-trans) to the aporetinol
binding protein (RBP). Smith and Goodman (1979) suggested that the
resultant holo-RBP enzyme is further processed through the Golgi
apparatus and finally secreted into the plasma.
15
The specificity of apo-RBP for retinol in the liver was determined
by Tosukhowong and Olson (1978) who reported that of all the various
processes involving vitamin A, the combination of all-trans retinol with
RBP was the most specific. Also the synthetic analogs of vitamin A
which include 15-methyl and 15-dimethyl retinol were apparently being
transported by RBP in vivo in the absence of retinol.
Cellular retinol binding protein (CRBP), cellular retinoic acid
binding protein (CRABP) and cellular retinaldehyde binding protein
(CRALBP) have been implicated in vitamin A metabolism. CRPP has been
isolated from livers of different species including rats (Ong and Chytil,
1978) and humans (Fex and Johanneson, 1982). Unlike RBP, CRBP has no
binding affinity for prealbumin and has absorption peaks at 277 and
345nm. On entering the target cell, retinol was quickly bound to CRBP
which has been isolated in the brain, testes, liver, kidney and lungs.
CRAB is specific for all-trans retinoic acid while CRALBP is specific
for retinal and is found only in the eye (Stubbs et al., 1979).
Colorimetric Determination of Vitamin A
When retinol or its ester is mixed with a Lewis acid under anhy
drous conditions, an intense transient blue color forms. Carr and
Price (1926) developed a quantitative assay for retinol using antimony
trichloride and anhydrous chloroform as the solvent. Presently it is
the AOAC official method (AOAC, 1975). A large number of assay methods
have subsequently been developed using a variety of Lewis acids includ
ing TFA and TCA (Dugan et al., 1964).
Several disadvantages have been cited for the original Carr-Price
reaction in chloroform: developed color which fades rapidly.
16
Interference of moisture with the determination, use of corrosive and
toxic chemicals, and the discovery that carotenoids tend to react with
antimony trichloride (SbCl3) which must be removed or corrections made
(AOAC, 1975).
Of other blue-color developing reagents being used, TFA has been
claimed to be more sensitive, free from moisture interference and safer
(Dugan et al., 1964; Neeld and Pearson, 1963). Bayfield (1971) and Grys
(1975) cited similar advantates plus lower cost and availability for
TCA. The stability of the color complex formed with various blue-
color reagents in different solvents was studied by Subramanyam and
Parrish (1976) who recommended that one or more of the new reagents
should be studied collaboratively for possible adoption as an alterna
tive to SbCl3 in chloroform. TFA in methylene chloride (CH2CI2) was
found to be more stable than the others.
A second colorimetric procedure was introduced by Budowski and
Bondi (1957). It involves the conversion of vitamin A to anhydrovitamin
A with anhydrous ethanolic-HCL or p-toluene-sulfuric acid. The reaction
is very specific for vitamin A but has the drawback of giving low results
in the presence of small traces of water.
Sobel and Werbin (1945) described a third colorimetric method.
They showed that the reaction of "activated" 1, 3-dichloro-2-propanoT
(1, 3-DCP) with retinoids produces a pink colored complex with maximum
absorption at 555nm and which was stable for 2 to 10 minutes. One of the
disadvantages associated with this method is that the activating procedure
is unreproducible and that the Carr-Price method is more sensitive. The
17
activation process was accomplished by vacuum distillation of 1, 3-DCP
from a 2.0% solution of SbCl3 in chloroform.
A few years later, Penketh (1948) suggested that the activating
technique involved hydrogen ion and that the reagent could be made
sufficiently sensitive to replace Carr-Price reagent only at the
expense of the advantage which it claims over it, namely, stability of
the chromophor. The addition of small quantities of acids to 1, 3-DCP
was investigated by Allen and Fox (1950) together with vacuum distilla
tion methods of Sobel and Werbin (1945). They reported that activation
of glycerol dichlorohydrin (1, 3-DCP) by vacuum distillation with 1%
SbCl3 produced a reagent with high activity and was more stable than
that produced by the acid-activated reagents.
Oliver and Bolz (1976), observed poor reproducibility of results
from Sobel-Snow assay was due to preparations of 1, 3-DCP containing
peroxides which are known scavengers of double bonds (Gordon and Ford,
1972). The 2, 3-DCP was purified by passing it through Woelm aluminum
oxide (basic activity grade) and collecting the distilled fraction
boiling at 70-75*'C. Activation was accomplished by the addition of
0.32M of acetyl chloride.
Blake and Moran (1976) developed procedures using 2, 3-DCP prepared
from allyl alcohol which appeared to contain few if any impurities and
was activated reproducibily by direct addition of SbCl3. A relatively
stable complex was formed which absorbed maximally at 555nm in the pres
ence of both SbCl3 and 2, 3-DCP.
The interaction of Lewis acids with retinol initially involves the
extraction of the hydroxyl moiety, leaving a retinylic cation
18
(x 586nm), which forms a complex with SbCl3 at the C-4 position
(x 619nm) or at the C-15 position (x ^„ 586nm). Blatz and Estrada max ^ max
(1972) elucidated that deprotonation of the retinylic cation or disso
ciation of the latter two complexes yielded anhydroretinol. In the
presence of DCP, C-15 Lewis acid complexes interacts further with the
anhydrovitamin A to form a n-complex (x^^ 555nm) with the conjugated md A
system (Blake and Moran, 1976).
CHAPTER III
EVALUATION OF COLORIMETRIC DETERMINATIONS
OF VITAMIN A IN FOODS
Summary
Selected chemical and physical characteristics of vitamin A in TCA
reagent along with varying effects of solvents and wavelengths used dur
ing colorimetric determination was investigated. The main effects were
solvent (chloroform and methylene chloride) and wavelength (616 and
620nm) as measured in a Beckman Spectrophotometer (Model 35 DB).
The absorption maxima of TCA in chloroform and methylene chloride
over a six day period was 616nm which could be attributed to the retinod
rather than the solvent or reagent employed. With chloroform as solv
ent, wavelength of determination was found to have an effect on the
absorbance. Thus, indicating the importance of wavelength selection
prior to analysis. Abosrbance was higher at 620nm than at 616nm. The
difference could be due to dissociation of blue-colored complex at
shorter wavelength when chloroform is used during reactions involving
retinoids and acids. Generally, the intensity of the blue-color formed
by TCA and methylene chloride was greater than that by TCA in chloroform
during preliminary and application investigation. The dielectric con
stants (s) of each solvent could influence the intensity of the color
complex formed by increasing or decreasing the attraction between com
plex formed. During colorimetric determination of vitamin A in liver
samples, wavelength interacted with solvent to affect percent recovery
19
20
of vitamin A from samples between solvents. Recoveries were higher
with methylene chloride at both wavelengths.
Activity of TCA reagent seemed to increase with both solvents from
0 days to approximately 4 days. This suggests that fresh preparations
of TCA or Lewis acids in either chloroform or methylene chloride could be
stored for complete equilibration of the reagent mixture that could
optimize results during analysis. The time course fading of blue-color
reaction products of vitamin A with TCA in methylene chloride reflected
a less steeper slope than in chloroform. Stability of the colored com
plex was maintained for a longer period of time (approximately 6 sec.)
in methylene chloride than in chloroform. This could be due to radiation
intensity in the photometer. -
Introduction
Retinoids are a group of molecules comprised of retinol (vitamin
A), retinaldehyde (vitamin A aldehyde), retinoic acid (vitamin A acid)
and their synthetic analogs. Vitamin A is essential for the maintenance
and normal functioning of epithelial tissues lining the respiratory and
reproductive tracts, and bone development in animals and humans (NRC/NAS,
1979). A possible role in glycoprotein synthesis was reported by
Deluca (1977). Toxicity of the vitamin was observed in animals (Wolbach
and Maddock, 1951) and humans (Jeghers and Marraro, 1958).
Assays of vitamin A in animal tissues have been the subject of con
siderable attention and criticism in past years. When retinol and its
esters are mixed with Lewis acids under anhydrous condition an intense
blue-color is formed. Carr and Price (1926) developed a quantitative
assay for retinol using antimony trichloride (SbCl3) and anhydrous
21
chloroform as the solvent. Various assay methods have been developed
using a variety of Lewis acids including TCA and TFA (Dugan et al.,
1964).
Solvent extraction procedures employing acetone and light petrol
eum in equal amounts, at room temperature was described by Bayfield
(1975) for determination of vitamin A products in liver tissue. Fur
thermore, some methods employed extraction with ethanol/hexane, drying
under nitrogen and dissolution in chloroform or mobile phase when high
pressure liquid chromotography was employed (Miller and Yang, 1985).
During extraction, hexane and cyclohexane were reported to yield similar
results to those of chloroform (Subramanyam and Parrish, 1976). Cyclo-
hexene gave low initial color, followed by a rapid secondary color
development with an unpleasant odor.
Reports for SbCl3, TCA and other Lewis acids (Bayfield, 1971;
Olson, 1979) indicated using a wavelength of 620nm during colorimetric
determination of vitamin A. However, a study by Dugan et al. (1964)
utilized 616nm in a Gary recording spectrophotometer with TFA. The
absorption maxima of the blue-colored species produced was shown to be
characteristic of the Lewis acid utilized (Subramanyam and Parrish,
1976). Trichloroacetic acid (TCA) as a reagent for vitamin A determina
tion was investigated because of its availability for routine laboratory
analysis, inexpensiveness, less moisture interference and less toxicity
(Neeld and Pearson, 1963), than other colorimetric reagents available.
Furthermore, the use of TCA has the potential for future development as a
sample kit for field tests (Dustin et al., 1978).
22
The purpose of this study was investigate the use of TCA as a
reagent in chloroform and methylene chloride and examine the effects
of wavelength, solvents and storage time (days) on the reactivity and
sensitivity of TCA during colorimetric determination of vitamin A in
foods.
Experimental Procedure
Experimental Design
A 2 X 2 factorial complete block design in which every sample was
subjected to every treatment was used to observe differences in vitamin
A concentration in liver (pig) and percent recovery. The main effects
were solvent (chloroform and methylene chloride) and wavelength
(616 and 620nm) as measured using a Beckman Model 35 DB spectrophoto
meter. Experimental variables included vitamin A concentration, per
cent recovery and absorbance. Two replicate samples were selected and
tested for every type of analysis.
Equipment
Absorbance measurements were made using a Beckman Model 35 DB
spectrophotometer (Beckman Instruments, Inc., Irvine, CA), utilizing
1 cm quartz cell (Fisher Scientific, Fair Lawn, NJ). Slit width was
selected automatically by switching to "normal" position for high-
resolution work. The spectrophotometer was operated under a double
beam (DB) mode. Automatic repipet dispensers (Lab Industries, Inc.,
Westburg, NY) were utilized for micro sampling.
23
Standard Vitamin A Solution
About 4 to 5 mg of all-trans vitamin A acetate were weighed from
a sealed vial containing 5 g of the vitamin and dissolved in 100 ml of
chloroform (A.C.S. grade, Fisher Scientific, Fair Lawn, NJ) and stored
in an amber colored bottle at -10**C.
Trichloroacetic Acid Reagent
A 30% TCA (A.G.S. grade) solution was prepared by dissolving in
chloroform (GHC13) and methylene chloride (CH2C12) and stored in a
glass stoppered amber bottle at refrigeration temperature. Before use,
the reagent was warmed to room temperature (24 ± 1°G) and an appreciable
amount transfered to a repipet dispenser bottle (low actinic).
Dichloro-2-Propanol (1, 3-DCP)
Two batches of 1, 3-DCP activated with 1-2% antimony trichloride
(SbCl3) were purchased from Eastman Kodak (Rochester, NY). The reagent
was stored at room temperature in an amber bottle. Before use the
reagent was warmed to 25°G.
Colorimetric Determination
Wavelength Experiment
The wavelength (nm) of maximum absorption for the reagents 1, 3-
DCP and (TCA) in either chloroform or methylene chloride was investi
gated over a period of 4 days using known concentrations of vitamin A
acetate in chloroform stored at -20''C. The relationship between con
centration and absorbance at 616 and 620nm were noted and further
studied during later quantitative and qualitative experiments.
24
Use of TCA Reagent
Serial dilutions of all-trans vitamin A acetate ranging from 2.5
to 10 yg/ml were prepared from stock solution on each day of analysis
using chloroform (reagent grade). To 1 ml of known concentration of
vitamin A acetate in a 1 cm quartz cell, 1 ml of TCA in either chloro
form or methylene chloride was added from a fast delivery pipette.
Absorbance at full scale deflection was recorded (within 5-7 seconds)
at 620 or 616nm. This was determined over a period of 6 days.
Use of 1, 3-DCP Reagent
Known concentrations of all-trans vitamin A acetate in 0.5 ml
chloroform (5-10 yg/ml) were pipetted into 5 ml screw top glass test
tube with micro digital pipette. 1 ml of 1, 3-DGP was added to the
tube stoppered, and mixed using a vortex mixer. The mixture was then
warmed to 25''C in a water bath (within 2 minutes), poured into 1 cm
quartz cell and absorption read at 550nm.
Color Development and Stability
With TCA-GHCI3 and TGA-CH2GI2, colorimetric determinations were
carried out at room temperature with serial dilutions of all-trans
vitamin A acetate respectively. To 1 ml of vitamin A in a cell was
added 1 ml of TCA reagent from a fast delivery pipette. Absorbance at
full scale deflection and the stability of the blue-colored complex
over a period of time (min) at 620 and 616nm was recorded. For the
stability test, absorbance as a function of time (min) was plotted for
each reagent-solvent complex stored from 0 to 6 days (0, 2, 4 and 6 days).
25
Sample Analysis
After separation an aliquot (1.0 ml) of the solvent extract was
carefully pipetted with an autopipet into a 1-cm quartz cell. To this
was added 1 ml of TCA in either chloroform or methylene chloride from
a fast delivery pipette with maximum absorbance recorded at full scale
deflection (usually within 5-7 seconds) at 620 and 616nm at different
analysis times (within each day of analysis). All samples were run in
duplicates within each day of analysis. Furthermore, 1, 3-dichlor-2-
propanol (1, 3-DCP) activated with SbCl3 practical grade was utilized
in sample analysis.
Recovery Tests
Recoveries were carried out by adding a known concentration of
vitamin A acetate standard from concentrates dissolved in chloroform,
to 1 ml of liver tissue extract, analyzed before and after the addition
of vitamin A. Samples were run in duplicates using TCA in chloroform
or methylene chloride. The percent recovery was calculated as the
ratio of actual to theoretical expressed as percent.
Calculation of Results
Quantitative results were calculated from standard calibration
graphs with reference to the reagent-solvent in question. Standard
calibration graphs were made for each day before quantitative analyses
were carried out.
Statistical Analyses
Analysis of variance and Duncan's New Multiple Range test were con
ducted by the method of Steel and Torrie (1980). Data were analyzed
26
using a 2 x 2 factorial complete block design for the main effects of
solvent (chloroform and methylene chloride) and wavelength (620 and
616nm) by testing for difference in experimental variables (absorbance)
within varying solvents (S.A.S., 1982).
During preliminary investigation with standard solution of vitamin
A, one trial was carried out before applying it to liver samples.
Data from analysis of variance tests with interactions were fur
ther tested using Duncan's Multiple Range test at five and one percent
level of significance.
Results and Discussion
Absorption Spectra
The absorption spectra of TCA in chloroform and methylene chloride
at different concentrations (2.5 and 5 yg/ml of vitamin A acetate
respectively) over a period of 6 days are shown in Figures 1-4. Absorp
tion maxima for TCA-GHCI3 and TGA-CH2CI3 with vitamin A was found to be
616nm. However, a wavelength maxima of 616 and 620nm was utilized
during the experiment to investigate relative differences in absorption
during qualitative and quantitative analysis in routine laboratory
analysis.
Reports for SbCl3, TFA and other Lewis acids (Bayfield, 1971; Olson,
1979) indicated using a wavelength of 620nm during colorimetric deter
mination of vitamin A in liver. Dugan et al. (1964) utilized a Gary
recording spectrophotometer at 616nm with TFA. The wavelength of
maximum absorption of blue-colored species produced by the reaction
between TCA and vitamin A was similar to that reported for SbGl3, TFA,
27
o c
i-O to
JD
570 590 610
Wavelength (nm)
630 650
Fig. 1-- Absorption spectra of blue species nroduced by vitamin A and TCA in chloroform (1); methylene chloride (2) at 0 day. ( 1 = 5 yg/ml vitamin A in GHGl3» 2 = 2.5 yg/ml vitamin A in GHC13).
28
u c n3
S-o to
550 570 590 610
Wavelength (nm)
Fiq 2 - Absorption spectra of blue species produced by vitamin A and TCA in chloroform (1), methylene chloride (2) after 2 days of storage (1 = 5 yg/ml vitamin A in CHGI3, 2 = 2 yg/ml vitamin A in GHGI3).
29
o c:
s-o t/)
550 570 590 610
Wavelendth (nm)
Fiq 3-Absorpt ion spectra of blue species produced by vitamin A and TCA*in chloroform (1) , methylene chloride (2) af ter 4 days of reagent storage ( 1 = 5 yg/ml vitamin A in GHGI3, 2 = 5 yg/ml vitamin A in
ige GHC13).
30
o c s_ o
530 550 570 590 610
Wavelength (nm)
630 650
Fig. 4—Absorption spectra of blue-colored species produced by vitamin A in chloroform (1); methylene chloride (2) after 6 days of reagent storage. ( 1 = 5 yg/ml vitamin A in GHCI3, 2.5 = yg/ml vitamin A in GHGI3).
31
and other Lewis acids (Dugan et al., 1964; Olson, 1979, and Bayfield
1975). The absorption maxima of the species produced has been shown
to be characteristic of the retinoid but independent of the Lewis acids
(Subramanyam and Parrish, 1976). For strong solutions of vitamin A,
the color of the reaction mixture was a deep blue which faded rapidly
through violet to light pink. Maximum blue-color was attained within
5 seconds after TCA addition.
The rapidly fading color might result from the cation formed cycliz-
ing to a tetraenylic species, which undergoes a slow polymerization,
spreading absorbance over ultra violet, visible, and near-infrared
regions of the spectrum (Blatz and Pippert, 1966; Dugan et al., 1964).
Therefore, the wide range (616-620nm) observed in the study could have
been due to the coupling and dismutation reaction.
Dichloro-propanol (1, 3-DGP) was found to have a sharper absorption
peak of 550nm at the end of 2 minutes over a period of 6 days (Figures
5-8). Other researchers (Oliver and Boltz, 1976; Sobel and Werbin,
1945) concur with the wavelength maxima of 550nm of the present study.
For 1, 3-DCP, a violet colored species (secondary) which absorbed
maximally at 550nm was noted (Figures 5-8). Upon the addition of this
reagent to a solution of vitamin A in chloroform, an immediate blue-
color appeared which rapidly changed into a more stable violet color
(2-10 min). The blue-color formed with 1, 3-DGP appears to follow Beer's
Law up to 2 yg/ml of vitamin A. Nevertheless, a complete study was not
made of this point in view of the stability of the second color formed.
Although differences in choice of wavelength during vitamin A
determination depend on the type of colorimeter and reagent
32
1.0 --
o
o to -Q
.8 --
6 -
4 --
.2 -
0
470 490 510 530 550 570
Wavelength (nm)
Fig 5 --Absorption curve of thev io le t color produced by vitamin A a n d ' l , 3-DGP/SbGl3 at 0 day. (5 yg vitamin A in 2.5 ml so lu t ion) .
33
1.2
1.0 . .
(U (J c
JQ s-o to
.Q
.8 - -
.4 - -
.2 --
0
490 510 550 590
Wavelength (nm)
630
Fig. 6 —Absorption curve of the violet color produced by vitamin A and 1, 3-DGP/SbGl3 after 2 days of reagent storage. (5 yg vitamin A in 2.5 ml solution).
34
o
J-o to
.8 --
.6 --
.4 ._
.2 -̂
0 __^ ^ ^ 550
Wavelength (nm)
Fig. 7--Absorption wave of the violet color produced by vitamin A and 1, 3-DGP/SbCl3 after 4 days of reagent storage (5 yg vitamin A in 2.5 ml solution).
35
510
Wavelength (nm)
Fig. 8-- Absorption curve of the violet color produced by vitamin A and 1, 3-DGP/SbCl3 after 6 days of reagent storage (5 yg vitamin A in 2.5 ml solution).
36
available, Dugan et al. (1964) reported a wavelength maxima of 616nm
for most Lewis acids in a Gary spectrophotometer.
Effects of Wavelength
Within solvent (chloroform), wavelength (616 and 620nm) had an
effect (P < 0.05) Absorbance values during preliminary study (Table 1).
Differences in absorbance were observed between the two wavelengths
which could have been due to the solvent employed. At present, no such
difference has been cited in literature as to whether a difference exists
when measurements are determined at 620 or 616nm.
An application of the technique to liver sample, difference
(P < 0.01) between wavelengths was reflected on percent recovery and
vitamin A (yg/g) in liver samples (Table 2) which implies that wave
length could have an effect during vitamin A analysis in spite of the
solvent employed.
Furthermore, wavelength interacted with solvent to effect (P < 0.01)
percent recovery between solvents (Table 3). Wavelength and solvent
effect could possibly be contributing to unknown side reaction during
analysis. The intensity of radiation in the spectrophotometer was
reported to affect the product of reaction during colorimetric determ
ination of vitamin A (Caldwell and Parris, 1945), the result implies
during routine laboratory analysis, accuracy of results could be
affected by solvent and wavelength interaction.
37
Table 1--Effect of wavelength on absorbance within chloroform and methylene chloride
Wavelength Absorbance (nm) N TGA-GI3 TCA-GH2GI2
620 10 0.442^ 0.764^
616 10 0.433^ 0.763^
^'^Means within a column with different superscripts are different (P < 0.05).
38
Table 2--Effect of wavelength on quantitation of vitamin A and recovery in liver samples^
Wavelength Vitamin A Recovery (nm) ^ (yg/g) (%)
620 20 20.00^ 93.4^
616 20 41.30^ 94.3^
^Recovery was estimated using 1 ml extract from each liver sample by adding 5 yg/ml of vitamin A acetate dissolved in chloroform at 25''G.
'Seans in the same column with different superscripts are different (P < 0.01).
II
39
Table 3--Wavelength and solvent effect on recovery of standard vitamin A from samples^
Recovery (%) Solvent N 616nm 620nm
Chloroform 10 91.410.12^^ 93.5±0.38^
Methylene Chloride 10 95.4±0.47^ 95.3±0.35^
^0.5 ml aliquot of liver extract ( in chloroform) was added to 0.5 ml of standard vitamin A acetate (5 yg/ml) and reacted with 1 ml of TCA reagent in a Beckman Spectrophotometer (Model 35) at 25**C.
^'^Means in the same column with different superscripts are different (P < 0.01).
40
Effect of Solvent
A 30% stock solution of TCA in either chloroform or methylene
chloride was prepared, stored and used during analysis. Quantitative
analyses indicate differences (P< 0.01) In recovery data when both
solvents were used (Table 4). On the other hand, differences in mean
vitamin A concentration (yg/g) were not observed between the solvents
(chloroform and methylene chloride). The absence of solvent effect on
mean concentration could be due to the combined effect of wavelengths
(616 and 620nm). Statistically, solvent effect should be expected since
a significant effect was observed on percent recovery. Generally, the
intensity of the blue-color formed by TCA in chloroform was slightly
less than that by TCA in methylene chloride as indicated by absorbance
values at 616 and 620nm (Table 5).
As noted by Carr and Price (1926) and Olson (1979), the concentra
tion of TCA in solvent was not critical. Bayfield (1975) reported
identical color yields with 22-35% TCA in chloroform. Statistically,
differences in percent recovery between chloroform and methylene chlor
ide suggests the need for chemical stabilization of solvents used
during vitamin A analysis in foods. Such methodology will ensure
reliability of results, especially in nutritional studies.
The absorption spectra of some organic solutes vary to some extent
in different solvents, even when no evidence of equilibria between
isomeric forms exists (Gilliam, 1935). Preparation of TCA in light
petroleum as solvent, was reported to have an adverse effect during
quantitative analysis (Rosenheim and Drummond, 1925). Compared to the
color obtained with chloroform, a loss in intensity of about 60% was
observed.
41
Table 4 — Effect of solvents on mean recovery of vitamin A acetate from liver samples^
Solvent N Recovery (%)
TGA-GHGL3 20 95.45^
TGA-GH2GL2 20 95.34^
G.V. 0.32
Recovery was estimated using 1 ml extract from each liver sample by adding 5 yg/ml of vitamin A acetate dissolved in chloroform.
Solvents used were CHGI3 (chloroform) and GH2GI2 (methylene chloride.
'̂ Means in the same column with different superscripts are different (P < 0.01).
42
Table 5--Effect of solvent on the color intensity produced at different vitamin A concentrations
Vitamin A Absorbance Reagent (yg/ml) 620nm 616nm
TGA-CHGI3 10-0 0-594 0.742
5.0 0.318 0.345
2.5 0.181 0.166
TGA-CH2GI2 10-0 0.979 0.984
5.0 0.412 0.487
2.5 0.222 0.230
43
Interference from carotenoids and other substances with Carr-
Price reagent was reported by Avampato and Eaton (1953).- However,
during this study, interference from carotenoids were not encountered
because of its absence in the liver tissues analyzed.
Effects of Storage Time
Within chloroform (Fig. 9) an interaction existed between wave
length and time (days). A surface lenear response in absorbance was
observed at 620nm from 0 to 4 days which decreased on the 6th day.
Conversely, this was the case at 616nm. A linear response could be
predicted from 2 to 6 days. The activity of TCA in chloroform was
found to be the same at both wavelengths after 2 and 4 days of storage
(Table 6). Activity of TCA at 620nm was observed after storage for 4
days. Activity at 616nm indicated most activity at 6 days although
this was not different (P < D.Ol) from that on the 4th day. This
suggests that storage of TCA reagent in chloroform in low actinic glass
wares) could enhance activity after four days.
Because TCA solutions deteriorate rapidly in light (Subramanyam
and Parrish, 1975) and lose 10-20 percent of their chromogenic capa
bility per day, preparations of fresh TCA solutions in CHCI3 were sug
gested by Olson (1979). Normally, during quantitative analysis of
compounds in samples, optimum activity of chemicals and reagents are
expected.
Similarly, within methylene chloride as solvent for TCA prepara
tion, there was a significant (P < 0.01) cubic effect on absorbance
due to wavelength and time interaction (Fig. 20). Within 620 and
616nm, the activity of TCA reagent differed (P < 0.01) between each
44
O)
(J
fO
S-
o to
1.0 T
.9 ••
.8--
.7 -
.6 ..
.5 -•
.4..
.3--
LEGEND
620 NM
• 616 NM
. 2 -
.I--
0
0 2 4
Storage (days)
8
Fig. 9 --Effect of storage time (days) on stability of TCA reagent in chloroform and all-trans vitamin A acetate at 620 and 616nm. (1 nl of reagent + 1 ml of vitamin A acetate (14.5 yg) at 24 ± IC.)
46
e
s-o to X2 <:
1.0^
.9 -.
.8..
.7..
.6 -.
5 ..
4 ••
3 ••
2 ..
. 1 ••
0
616 NM
0 2 4
Storage (days) 8
Fig. 10 --Effect of storage time (days) on stability of TCA rea methylene chloride and all-trans vitamin A acetate at 620 and 6 (1 ml TCA-CHCI3 + 1 ml vitamin A acetate (14.5 yg) at 24 ± IC )
47
day from 0 to 6 days. Most activity of TCA was noted after 4 days at
616nm, while at 620nm the most activity was after 6 days of storage
(0.800 and 0.823 absorban:ceunits, respectively) (Table 7).
Overall, absorbance was significantly higher (P 0.01) at 616nm
than at 620nm within each day of storage. This implies that chloro
form and methylene chloride as solvents for TCA preparations would
respond favorably at wavelength of 616nm than qt 620nm. From the
results obtained and discussed above, differences between wavelengths
depends on which time of storage in question and vice versa. Hence,
the maximum activity of TCA in either of these solvents should be
investigated further for optimization of results during field and
routine laboratory trials.
Color Stability
With chloroform, optimal absorption at both wavelengths occurred
at approximately 8 sec and was stable for about 2 sec before fading
(Figs. 11-12). Maximal absorption of blue-colored complex in methylene
chloride was maintained for a longer period of time (approximately 21
sec) over a period of four days at 620nm. On attainment, the color
was stable for about 4 sec (Figs. 13-14). The increase in activity of
the reagent over the six day period was also reflected during this
experiment.
The time-course fading of the blue-color reaction products of 3.33
yg vitamin A with TCA in methylene chloride stored for 2 days reflected
less steeper slope in curve than at 4th and 6th day (Figs. 13-14).
Subramanyam and Parrish (1976) reported similar fading curve for TCA
in chloroform. Caldwell and Parrish (1945) also reported that the
48
Table 7 --Effect of storage (days).on activity of TCA reagent in methylene chloride at 620 and 616nm.
Time Wavelength, nm
0
2
4
6
N
10
10
10
10
620
0.737^'^
0.711^'^
0.785^'^
0.823^'^
616
0.723^'^
0.742^'^
0.800^'^
0.786^'^
a,b,c,dj^g^i^^ within a column followed by different superscript are different (P < 0.01).
^'^Means within a row followed by different superscript are different (P < 0.01).
49
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>> 03
Q
SZ +J CD
1 •
CXD
CO
LO
• ^
CO
•- CM
<U
E
aouequosqv
4-> rtJ c:
• r ^
E S-cu
4-> (U
"O
cr e
•r— S-13
TD
XJ CU E i -o
M-
X CU
c— CL E O CJ
CU :3
f —
JD
H -O
CU E
• r -
+-> to 3
• to >> 03
"O
CO
S-o
*:*• W\
C\J
S-o
4 -
TD <U S-o +-> to
CM r ^ CJ CM : r CJ
1 c::
to CJ $-CU > E c
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+J 03
CU CJ c 03
X3 S-o
h-
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•r—
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r—
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ro •
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53
fading of the blue-color was being affected by the intensity of radia
tion in the photometer. Caldwell and Parrish (1945) suggested that
photometers for determining vitamin Aby Carr-Price reaction should
employ low intensity of incident light to reduce fading of the blue-
color to a minimum and make possible more precise determinations.
Results obtained with 1, 3-DCP/SbCl3 portrayed it as a more stable
reagent for vitamin A determination. During reaction with vitamin A
acetate, a transient blue-color was initially formed (£ 30 sec) that
rapidly changed to violet color (x„^„ at 550nm). This was stable for
max
about 90 seconds. A s imi lar time (minutes) fading curve, was noted at
0 and 2nd day of analysis (Fig. 15).
In the presence of 1, 3-DCP, the Lewis acid complex can covalently
in teract with 1, 3-DCP to y ie ld a conjugated pentaenic product (x
550nm) which is very stable (Blake and Moran, 1976).
The s l i gh t increase in in tens i ty and s t a b i l i t y of the color com
plex formed with methylene chloride as solvent indicates that the l a t t e r
is more re l iab le than chloroform as solvent for determining vitamin A
during routine laboratory analysis. In addi t ion, the use of 1, 3-DCP/
SbCl3 should be investigated in spi te of i t s costs as a reagent during
indepth vitamin A c l i n i ca l studies.
Cal ibrat ion Curves
Serial d i lu t ions of a l l trans-vitamin A acetate ranging from 2.5
to 10 yg/ml were prepared from stock solut ion on each day of analysis.
Figure 16 shows two typical curves of TCA in methylene chloride and
chloroform at 620nm. L inear i ty of curve was established with correla
t ion coef f i c ien t of 0.96 and 0.99 respectively. Slopes calculated
M i
Q
>> 03
Q
XJ e
CM
03 Q
4 J
Q
CO
LU CD
0 0
- - . CO
LO
CU
r -:̂ ̂
• - CO
- - '.-CM
X. , O
0 0 CO CM
aouequosqv
03
«+-o e o
• ^ +-> 03 C
*r—
E S-CU
+-> CU
T 3
cr c
• r -
s-
• to >> 03
:3 T3 -o -a CU E S-
CD
S-
o
o ^ M-
s-o
r^
o o
+ J
€S
CM
«S
CD
S-
o CU M -
^•^ o
• 1 —
>
M-O
CU E
•r—
+-> to Z3 C/1 S-
cu > E c:
CD ir> LO
+-> 03
CU
o -E 03
J 2 s-o to
• o CU
s-o +-> to
CO n—
o J D LO
a. C J Q
1 CO
Wl
1—1
CD
c n -to 13
'E
en ^
Xi un
1 1
54
CO
en
55
1.2
1.0 •
CU o zz OJ
s-o to
X3
.8-
.6 .
. 4 '
. 2 -
0
0 4 6 8
Concentration (mg/g)
10 12
Fig. 16-- Standard calibration curves for vitamin A acetate using TCA in methylene chloride (-.-)-^nd chloroform (-*-) as solvents at 620nm.
56
from the curves were 0.083 and 0.069 for TCA-CH2CL2 and TCA-CHCL3
respectively. Similar curves were used as references to calculate the
vitamin A concentration (yg/ml) in samples. Subramanyam and Parrish
(1976) suggested using slopes calculated from such curves as factors
to calculate the concentrations of vitamin A in various extracts. The
curves indicated an obedience to Beer's Law to concentration of 10
yg/ml. Bayfield (1971) reported a concentration range of 0.7 yg/ml.
However, compared to absorbance (color intensity) obtained with TCA in
methylene chloride at similar concentrations, chloroform reflected
about 21% loss in color intensity.
At 616nm, TCA in a methylene chloride curve suggests a greater
correlation (0.99) amongst calibration points than chloroform (0.92)
(Figure 17). A loss in color intensity produced when TCA was dissolved
in chloroform was greater (32% loss) when compared to TCA in methylene
chloride. Similarly, the ability of TCA in methylene chloride to pro
duce a deeper color was affected at 616nm. An increase in absorption
was noted at 5 yg/ml concentration of vitamin A acetate over that at
620nm with TCA in methylene chloride preparation (8% increase).
Statistically, serial dilutions of vitamin A prepared from stock
was shown to have a cubic effect (P < 0.01) on absorption within each
solvent system. Practically, it could be due to the minimum variation
that developed by going back to the same stock on each day of analysis.
Secondly, it might be as a result of the radiation intensity effect in
photometer (Caldwell and Parrish, 1945).
With 1, 3-DCP/SbCl3 at 550nm, however, the relative absorbance at
same concentration was much lower when compared to TCA prepared in
57
1.2 ^
1.0 .
CU
o E 03
X3 S-
o j Q
.8 .
6 •
4 .-
. 2 •
0
0
/
4 6 8
Concentration" (mg/g) 10 12
Fig. 17--Standard calibration curves for vitamin A acetate using TCA in methylene chloride (- . - ) , and chloroform (- * -) as solvents at 616nm.
58
either methylene chloride or chloroform. Colorimetrically, 1, 3-DCP/
SbCL3 was reported by Sobel and Werbin (1945) as obeying Beer's Law up
to a concentration range of 5 I.U. of vitamin A (1.5 yg/ml). From
Figure 18, the calculated slope was 0.008. This seemed to be much
lower than that calculated from TCA preparations in either methylene
chloride or chloroform. The difference in slope might be due to dif
ferent batches of reagent obtained from the supplier (Eastman Kodak
Company, NY) that activated the reagent with 1-2% SbCL3 (antimony)
trichloride). Variations in activity between different batches of 1,
3-DCP from companies was reported by Sobel and Werbin (1945).
Liver Vitamin A Content
Mean vitamin A concentration of five liver samples from barrows
weighing approximately 104 kg was 30.65 yg/g (C.V. = 20.65) (see Table 3)
Moore and Payne (1942) reported an overall mean of 23.4 yg/g vitamin A
for all pigs (6-36 months) of age. For young pigs (6-8 months) an aver
age of 23.6 yg of vitamin A per gram of liver was reported during winter.
Lack of green forage to graze on in addition to poor conversion of
carotene to vitamin A in swine species could be the contributing factor
to low vitamin A reserves.
In spite of the fact that most of the investigations on liver vita
min A concentrations were performed with the use of rather primitive
analytical methods, the information provided is nevertheless useful with
regard to the correlation between age and level of vitamin A in liver
samples.
Results from Table 9 indicate variations exist in liver reserves
of vitamin A within and between lobes of liver. Quantitation with TCA
59
T CM
O LO LO
+J
03
CO
CJ oo Q_ CJ Q I
CO
CD 00
CO
^
CM
CM
00 CO CM
Bouequosqv
en E
•r— to
^̂ ^ en ^
^—'
E O
+->
ntr
a
CU CJ E o CJ)
CU +-> 03
-M CU CJ 03
<c
am in
+-> >
u. o
l i
eu > s-Z3 o
03
X I
03 O
-o OJ
•o E 03
•4-> oo I I
CO
CD
60
Table 8 --Mean vitamin A concentration in liver tissue
Sample Vitamin A I.D. N (yg/g)
1 8 34.35
2 8 32.00
4 8 31.25
6 8 28.50
8 8 27.25
C.V. - 20.65
61
Table 9--Vitamin A concentration in liver as determined with TCA-CH2CJ2 and TCA-CHCI3 at 616nm and 620nm
Wavelendth (nm)
620
616
N
10
10
Vitamin TCA-CHC13
19.2
20.8
A (ug /g ) TCA-CH2CI2
42.2^
40.4^
C.V.
10.06
4.46
^' 'Means within a column followed by different superscripts are different (P < 0.01).
62
in CH2CI2 and TCA-CHCT3 at 620 and 161nm showed variations of 4.46 and
20.06 respectively. Similarly, coefficient of variation within-run in
the proximity of 7.1 was reported by Miller and Yang (1985) using an
HPLC procedure.
Variation between lobes of liver in the distribution of vitamin A
was cited in literature by Hoppner et al. (1968) and McLaren et al.
(1979). Variation was found to be greater in humans than in animal
livers (Olson, 1979).
Nevertheless, the effect of solvent and wavelength should not be
ruled out in contributions to observed variations.
Recovery of Vitamin A
Percent recovery of vitamin A from pig liver is given in Table 10
where the recoveries of 93-94% were obtained when 5 yg/ml of vitamin A
acetate was added to 1 ml of extract from each extract. Mean recovery
was 93.89 [G.V. = 0.32] with TCA in methylene chloride and chloroform,
combined.
Within solvents, TGA-GH2GL2 seemed to portray a slightly higher
recovery percent (95.27) than TCA-GHGL3 (93.52) at 616nm. The same
trend was followed at 620nm when TCA in both reagents were evaluated
(see Table 11).
Bayfield (1975) reported recoveries of vitamin A acetate to be
between 97-101% using sheep liver in the presence and absence of
a-tophercol (vitamin E) as an antioxidant in the extraction solvent.
Conclusions
Based on results from this study the wavelength of maximum absorp
tion for vitamin A acetate with TCA in methylene chloride or chloroform
63
Table 10--Mean vitamin A and recovery of vitamin A from liver tissue
Mean^ Vitamin A
Pig N yg/g % Recovery
1 8
2 8
3 8
4 8
5 8
C.V.
^Mean recovery was determined by addition of 6 yg of vitamin A to 1 ml of liver extract.
24.35
32.00
31.25
28.50
27.25
20.65
93.50
93.92
93.97
93.91
93.91
0.32
64
was 616nm. The vitamin rather than the solvent or TCA could be the
attributable factor. With chloroform, the wavelength of determination
was found to have an effect on absorbance, thus indicating the impor
tance of wavelength selection during analysis. Absorbance was higher at
620nm. Variation in values could be due to end product breakdown at
shorter wavelength (616nm).
There was an interaction between sorage time (days) of the reagent
and wavelength on the absorbance values with standard vitamin A solution
in both solvents (chloroform and methylene chloride). Apparently there
was a linear time effect on absorbance within both solvents. This sug
gests that storage of fresh TCA preparation in low actinic glassware
for equilibration of reagent could enhance activity and thus eliminate
the possibility of inaccuracy.
Furthermore, there was a wavelength and solvent interaction
effect on recovery of pure vitamin A from liver samples between solv
ents and within wavelengths. In as much as differences were observed,
variations between wavelengths depends on solvent in question and vice
versa.
The time course fading curve of blue-color reaction products of
vitamin A with TCA in methylene chloride indicated a more stable complex
at 616nm (slope of -0.54 after 2 d of reagent storage) than TCA in
chloroform at 616 and 620nm (slope was -0.68 and -0.67 respectively).
Therefore, it could be concluded that wavelength and solvent used
during colorimetric analysis of vitamin A in foods affect the accuracy
of results obtained. However, further research is necessary to deter
mine the activity of TCA in methylene chloride for possible adoption
as solvent during colorimetric determination of vitamin A.
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