1514 W. A.
BECKER
1954. A two-sample multiple decision proce-
dure for ranking means of normal populations
with common unknown variance. Biometrika,
4 1 : 170-176.
Dickerson, G. E., and H. Abplanalp, 1956. Sources
of variation in performance of entries in the
4th, 5th and 6th California Random Sample
Egg Production Test. Proc. Pacific Chicken and
Turkey Breeders' Roundtable, 9: 67-75.
Harvey, W. R., 1960. Least-squares analysis of
data with unequal subclass numbers. ARS-20-8.
USDA.
Hogsett, M. L., 1959. Effect of parent's environ-
ment upon progeny's performance. Proc. Pacific
Chicken and Turkey Breeders' Roundtable, 12:
51-57.
King, S. C , 1954. Random Sample Testing. Proc.
World's Poultry Congress, 10: 20-23.
Lush, J. L., 1945. Animal Breeding Plans, 3rd.
ed. viii, 443 pp. Collegiate Press, Ames,
Iowa.
A Simple Procedure for the Estimation of Total
Lipids in Liquid Egg White 1
O. J . COTTERILL
Poultry Husbandry, University of Missouri, Columbia, Missouri
(Received for publication January 5, 1961)
T HE estimation of small amounts of
lipids in egg white has become an im-
portant quality control test in the egg proc-
essing industry. Bergquist and Wells (1956)
adapted, to egg white, a micromethod de-
scribed by Heinemann and Rohr (1950)
for the determination of fat in skimmilk
and non-fat dry milk solids. This method
entails an ethyl alcohol, ethyl ether, petro-
leum ether extraction in an ammoniacal
system. The amount of lipid in this extract
is determined by observing the area of a
monomolecular surface film. Colburn (1960)
evaluated several factors affecting the ac-
1 equipment are required: an oxidized oil pre-
Contribution from the Missouri Agricultural
Experiment Station, Journal Series No. 2247. Ap-
proved by Director.
Nordskog, A. W., 1960. Importance of egg size
and other factors in determining net income in
Random Sample Tests. Poultry Sci. 39: 327-
338.
Nordskog, A. W., and O. Kempthorne, 1960. Im-
portance of genotype-environment interactions
in Random Sample Poultry tests. Biometrical
Genetics, pps. 159-168. Pergammon Press.
McBride, G., 1958. The environment and animal
breeding problems. Animal Breeding Abstracts,
26: 349-358.
Tukey, J. W., 1949. One degree of freedom for
non-additivity. Biometrics, 5: 232-242.
Tukey, J. W., 1955. Queries in Biometrics 11:
111.
Wilkinson, G. N., 1958. Estimation of missing
values for the analysis of incomplete data. Bio-
metrics, 14: 257-286.
Yates, F., 1936. A new method of arranging va-
riety trials involving a large number of va-
rieties. J. Agr. Sci. 26 : 424-455.
curacy of this test and devised an alternate
method to measure the surface area of the
lipid. His method gave comparable accuracy
in less time and partially corrects for film
pressure variations. Smith (1960) reported
that while increasing amounts of natural
whole egg yolk results in increasing linear
increments of film area, the individual lipid
components do not result in identical film
areas.
The monomolecular surface film tech-
nique has the advantage of being rapid, re-
quiring only about 15 minutes per observa-
tion if done routinely, and is micro in na-
ture. However, several items of specialized
pared, a standard curve constructed, and
considerable practice needed to develop the
 ESTIMATION OF LIPIDS IN EGG WHITE
skill necessary to obtain accurate and repro-
ducible results.
If the quantity of extracted fat is suffi-
ciently large to permit weighing on an ana-
lytical balance and if quick results are not
imperative, then a simplified procedure can
be used for the determination of total lipids
in egg white. The purpose of this paper is to
describe a procedure which has been used
extensively in this laboratory for the de-
termination of total lipid content of egg
white.
EXPERIMENTAL
Materials: The following reagents and
items of equipment were used:
Ammonia solution (10% NH 3 )
Ether-alcohol mixture (Containing three parts
ethyl ether and two parts of 95% ethyl al-
cohol)
Petroleum ether (ACS, b.p. 30-6O°C.)
Graduate cylinder (100 cc.)
Automatic dispensers (Two 25 cc, one 45 cc.)*
Bottle, narrow mouth, 16 oz. (Like Fisher
2-882)
Separatory funnel (500 cc.)
Funnel, glass (65 mm.)
Filter paper (Whatman # 1 2 , folded, 12.5 cm.)
Beaker, glass (250 cc.)
Hot plate or water bath
Drying oven (105°C.)
Analytical balance
With the exception of the automatic dis-
pensers, which are not necessary but do
expedite an analysis, the remaining ma-
terials are generally available in most labo-
ratories. Also, it is not mandatory that all
items of equipment be duplicated as spe-
cified above. For example, other types of
bottles would suffice.
Method: The extraction procedure was
essentially that of Bergquist and Wells
(19S6), only different proportions or con-
centrations of reagents were used. This was
found necessary for analyzing large egg
* Automatic dispensers may be obtained from
California Laboratory Equipment Co., 1717 Fifth
St., Berkeley 10, California.
ISIS
white samples. Although some laxity exists
regarding amounts of reagents, the follow-
ing practice was found to be satisfactory:
1. Place 40 gm. of egg white in a 16 oz. cork-
stoppered glass bottle.
2. Add 25 cc. of 10% ammonia solution.
Shake.
3. Add 90 cc. of the ethyl ether-alcohol mix-
ture. Shake vigorously for about one min-
ute.
4. Add 25 cc. of petroleum ether. Shake vig-
orously for about one minute. If necessary
add more of the ether-alcohol mixture to
help break the emulsion. Letting sample set
over night will permit easier decantation
of the solvent layer.
5. Decant solvent layer into a separatory fun-
nel. If the ammoniacal-aqueous phase is
liquid rather than gel, as is the case if
small samples are used, then this step per-
mits better separation of the aqueous
phase.
6. Rinse gel-like precipitate with 25 cc. of
petroleum ether. Decant into separatory
funnel.
7. Filter solvent layer through a folded filter
paper to remove any pieces of gelled pro-
tein that may be carried over in the de-
canting process. Collect extract in previ-
ously weighed glass beaker.
8. Rinse sides of separatory funnel and filter
paper with small amount of ethyl ether-
alcohol mixture.
9. Evaporate solvents using a sealed hot plate
on low heat.
10. Dry beaker containing lipid extract in an
oven at 105 °C.
11. Cool and weigh the beaker.
A recovery experiment was conducted
whereby known amounts of yolk were added
to egg white. The total lipid content of the
yolk sample was found to be 29.0%. This
determination was carried out by the acid
hydrolysis procedure (Anonymous). This
yolk sample was prepared from eggs less
than 6 hours old. All of the white was re-
moved from the yolk by rolling it on a
damp cloth. The yolk membranes were
punctured then discarded after the yolk ma-
terial had been removed. The yolk was
mixed with egg white on a magnetic mixer.
1516 O. J . COTTERILL

RESULTS AND DISCUSSION
The results of a recovery experiment are
shown in Figure 1. The amount of yolk lipid
recovered showed a linear relation with the
amount of yolk added. In this test the re-
gression coefficient for recovered yolk on
added yolk was .90 with a standard devia-
tion of .025. This means that between 85
and 95% of the added yolk lipid would be
recovered unless these data are more abnor-
mal than would be expected to occur by
chance in 95% of the repeated trials. On
the basis of this experiment, observed values
of recovered yolk would be equal to about
90% of the total lipid present. Therefore,
the concentration of yolk found in an egg
white sample may be calculated as follows:
Lipids observed less blank (gm.)XlO 8
Egg white sample (gm.) XLipids in yolk Yo) X Recovery correction (%)
For example:
.032 X10 6
— = . 3 0 7 % yolk (2)
40X29.0X90
Hence, equation (1) and (2) can be re-
duced to the following form:
Lipids observed less blank (gm.)
X 9.58 = % yolk (3)
The constant, 9.58, in equation (3) is
true only if a 40 gm. egg white sample is
used and if the contaminating yolk has a
lipid content of 29.0%. The experience has
been that egg yolk prepared as described
the amount of lipid added and the quantity
recovered was not determined. This devia-
tion from expected value was consistent
over the range of yolk concentration tested.
Two possibilities might be considered re-
garding this lack of agreement: (1) The
ammoniacal treatment may be less efficient
than the acid hydrolysis, hence some lipoid
component may have been incompletely ex-
tracted; (2) A mechanical loss of lipid may
have occurred during the extraction process.
Where conditions permit its use this
method may be used in place of the surface
film test outlined by Bergquist and Wells
(1956). The quantity of extracted fat and
time needed to conduct an analysis are the
= % yolk in white (1)
limiting factors. This simplified procedure is
not satisfactory for yolk concentrations less
than 0.05%. Bergquist (1960) commented
that most commercial egg white is found to
be below 0.1% yolk. Above 0.05% yolk is
— 1 1 1 1 I
/ /
// / /
Yolk /
/ /. / /

odded\^/. s
previously will have a lipid content ranging
between 28.0 and 30.0%. Since the lipid in /
/
/A
/ • Yolk
? observed
these samples was from a yolk source and / /
//'/' '
the yolk lipoid components were present in
/ //*// /' -
their original natural proportions, it is pos- // '
sible to convert the observed lipoid values to o.i ////
//
% concentration of yolk. However, if this ///"
assumption could not be made, then it would ,"•
j ?

be erroneous to express the total lipids pres- 0.2 0.3 0.4

ent as egg yolk. Yolk added (%)

The reason for the discrepancy between FIG. 1. Recovery of added yolk from egg white.
 ESTIMATION OF LIPIDS IN EGG WHITE
considered to be high in yolk concentration
as far as some uses are concerned. Likewise,
in industrial processing operations, it is
often essential that a result be obtained in
much less time than is possible with the
procedure outlined in this paper. However,
during the course of our investigations on
the functional performance of yolk con-
taminated egg white, it has been desirable
to use concentrations as high as 0.4% yolk.
In the concentration range of 0.05% to
0.4% added yolk this procedure was found
to give satisfactory results. It has the ad-
vantage that only miscellaneous reagents
and equipment, generally found in most re-
search and quality control laboratories, are
needed to carry out an analysis. It would
appear to be a good indication of total lipids
present and can be conducted with only a
minimum of preparation or standardization
of materials and technique.
SUMMARY
A simple procedure for the estimation of
total lipid in egg white is described. The
ethyl alcohol, ethyl ether, and petroleum
ether extraction technique has been modi-
A Study of the Ataxias of Vitamin A and Vitamin E
Deficiencies in the Chick
M. L. SCOTT AND G. S. STOEWSAND
Department of Poultry Husbandry, Cornell University, Ithaca, New York
(Received for publication January 6, 1961)
D EFICIENCY of either vitamin A or
vitamin E in chicks may produce
severe ataxia. Coggeshall and Bieri (1960),
reporting on the pathology of the brains of
ataxic chicks suffering from single and
mixed deficiencies of vitamins A and E, ob-
served pinpoint lesions in the frontal lobes
of the brain in chicks with combined de-
ficiencies of vitamins A and E, similar to
those described by Adamstone (1947) as
1517
fled to accommodate a larger egg white
sample. Subsequently, the extracted fat is
weighed on an analytical balance. This pro-
cedure was found to recover 90% of the
lipids from a yolk contaminated egg white
in a concentration range of 0.05 to 0.4%
added yolk.
REFERENCES
Anonymous. Chemical and Bacteriological Meth-
ods for Examination of Eggs and Egg Prod-
ucts. Institute of American Poultry Industries,
Chicago, Illinois.
Bergquist, D. H., 1960. Personal communica-
tion.
Bergquist, D. C , and F. Wells, 1956. The mono-
molecular surface film method for determining
small quantities of yolk or fat in egg albumen.
Food Technol. 10: 48-50.
Colburn, J. T., 1960. A modified test for micro-
quantities of lipids. Masters' Thesis, Univer-
sity of Missouri.
Heinemann, B., and M. R. Rohr, 1950. A micro-
method for routine determination of fat in
skim milk and nonfat dry milk solids. J. Dairy
Sci. 33: 703-709.
Smith, C. F., C. K. Stearns and E. Kolen, 1960.
Determination, in egg white, of lipids and their
effects upon performance. Food Technol. 14 ( 4 ) :
26.
being characteristic of vitamin A deficiency.
In the chicks with vitamin A deficiency
only, they found a limited number of lesions
in the central nervous system, which were
similar to the neuronal changes previously
described by Rigdon (1952) in vitamin A
deficient ducklings. Coggeshall and Bieri
(1960) also reported that the Purkinje cell
layer of the cerebellum of vitamin A de-
ficient chicks occasionally showed pyknosis.