Hemoglobin
Am
Control: For use, the
control must be diluted 10- to 15-fold
with the hemolyzing
actual dilution required
reagent.
(The
varies with lot
be determined
of control,
and must
empirically
for each lot.)
Benzidine
working solution (prepare
immediately
before use): 5 ml of benzidine stock, 2 g/liter;
10 ml of dilute
Buffer and blotted
Three
dry.
such cellulose
acetate
plates
may be electrophoresed
simultaneously
on each zip-zone electrophoresis
chamber. Several chambers can be run simultaneously
from the same power supply.
Electrophoreses
for 15 mm at 400 V.
Immediately
after
place the plates
in
TCA
electrophoresis
(50 g/liter)
for 10
acetic acid (5 ml + 95 ml of water);
1 ml of sodium
nitroferricyanide,
10
g/liter;
1 ml of hydrogen peroxide, 300
mm, followed by two 3-mm washes in
the dilute acetic acid. Transfer
to freshly
prepared working benzidine stain for 5
g/liter
mm.
(the “3%”
solution
available
at
any pharmacy).
Trichloroacetic acid (TcA): 50 g/liter.
Dilute acetic acid: 5 ml of glacial acetic
acid and plus 95 ml of water.
The procedure
is as follows: Whole
is collected in
Follow
with
two 3-mm
washes
in
the dilute acetic acid. Allow to air dry
and read, or densitometric quantitation
may be carried out either directly or
after the clearing by conventional technique.
blood from a finger-stick
10-l, heparinized
capillary
tubes. Prepare racks of small test tubes containing
0.340ml of hemolyzing
reagent in each
tube (an automatic
dispenser
may be
used). Add the contents
of one blood-
collecting capillary
(10 l) to each tube.
Mix by twirling. Place 2 l of hemolysate
into each of the first seven wells of a
“zip-zone
sample plate.”
Place 2
of
Reference
1. Nalbandian,
cellulose
viously
to a 1 inch
acetate
been
X 3 inch Titan
plate
soaked
that
in
III
has preSupreHeme
et al., Automated
A. P.
diluted control in the eight well. With
the zip-zone applicator, apply the eight
samples
R. M.,
dithionite
test for rapid, inexpensive detection
of hemoglobin
S and non-S
sickling hemoglobinopathies.
CLIN. CHEM.
17,1033 (1971).
ROSENBERG
NORMA
BYRNES
LEONA
SEXTON
Biochemistry
Associates Inter,zational
American Biomedical Corp.
Miami, Fla. 38136
490 CLINICAL CHEMISTRY, Vol. 18, No. 5, 1972
RapidTLCSeparationand Detection
of Lecithin and Sphlngomyelin in
AmnioticFluid
To the Editor:
In recent years much interest has been
focused on the relative concentrations
of
the phospholipids
lecithin and sphingomyelin in amniotic fluid. These phospholipids are believed to originate principally from the fetal lung, and the ratio
of lecithin to sphingomyelin
in the amniotic fluid can be correlated
with fetal
pulmonary
maturity
( 2). This communication
describes
a rapid and convenient
thin-layer
chromatographic
method for separation
and visualization
of these two phospholipids.
Studies on
the evaluation
of lecithin/sphingomyelin
ratios in amniotic
fluid and correlation
of these results with the clinical fetal
state will be described elsewhere.
The procedure
for extraction
of phospholipids
from amniotic
fluid is essentially the same as that of ‘Gluck et al.
(1). About 5 ml of amniotic fluid is cen-
trifuged to remove any cells present. An
equal
volume
of absolute
methanol
and
twice the volume of chloroform is added,
the mixture is shaken for 5 mm, then
centrifuged
to separate
the layers.
The
phase is carefully removed and discarded; the chloroform
aqueous
(upper)
(lower)
phase, containing
lipids, is filtered through
the phospho-
Whatman
No.
1 paper. The filtrate is transferred
to an
evaporating
dish and concentrated
to
‘about 0.5 ml. Thms concentrate
is placed
in a 13 X 100 mm tube, the dish rinsed
with an additional
1 to 2 ml of chloroform, and the risings
are added to the
contents of the tube. The material in the
tube is then evaporated further to about
40to50zl.
Alloftheconcentrated
chloro-
form extract
is used for the thin-layer
chromatographic
separation.
Silica
gel-impregnated
glass fiber
sheets (Gelman Instrument
Co.), 20 X
20 cm, are used for chromatography.
The
following purified phospholmpids, purchased from General Biochemicals, are
used as standards
(5 mg/mi of chloroform): lecithin (dipalmitoyl),
sphingomyelin, phosphatidyl
inositide, phosphatidyl serine, and phosphatidyl
ethanolamine; the latter three are included
because they have been reported to be
present in fetal lung extracts (3). Eleven
light pencil marks are made 2.5 cm from
the bottom edge of the sheet with the
help of the Gelman spotting guide (cat.
No. 51333). Five microliters of each
standard
is applied in the following
three combinations:
lecithin + phosphatidyl inositide + phosphatidyl
serine
sphingomyein
+ phosphatidyl ethanol-
lecithin + sphingomyelin; these
amine,
combinations are applied in spots number 1, 2, and 5, respectively, and again
in spots number 7, 8, and 11, respectively. Two concentrated amniotic fluid
extracts can be placed on the same sheet,
the first at spots 3 and 9 and the second
at spots 4 and 10; the extracts are applied 5 d at a time so that the spot size
will be no larger than 4-5 mm. The reason
for applying
extracts
may
both
in duplicate
be halved
standards
is that
by dividmng
and
the sheet
lengthwise
(along spot number 6) after development
phospholipids. These spots are outlined
lightly in pencil because the color fades
soon after the section is removed from
the
chamber.
The
second
sheet is sprayed thoroughly
half
of the
with a modi-
fled bismuth
spray (4) consisting
ml of a solution of 1.7 g of bismuth
of 20
sub-
nitrate in 100 ml of dilute (20 ml/100
ml) acetic acid, 5 ml of a solution of 40
g of potassium iodide in 100 ml water,
and 70 ml of dilute (20 mi/lOOm)) acetic
acid. This solution
is stable for about
one week. The sprayed
portion
is allowed to air dry for several
minutes,
in the solvent for the purpose of detection by two different visualization
techniques. The chromatogram
is developed
then dipped into a pan containing dilute
with the Eastman
Kodak chromatography kit (cat. No. 6071), with which
the sandwich plate technique is used,
this technique,
only the choline-containing phospholipids,
lecithin and sphingomyelin, appear as bright orange spots
thereby
eliminating
the need for presaturation
of the chamber.
The solvent
system used is chioroform/methanol/
concentrated
ammonium
hydroxide
while the other three phospholipids
not made visible. The lecithin
(170:20:3,
gradually
reappears.
Visual inspection
of the chromatogram
enables
one to
estimate
the relative
proportions
of
lecithin/sphingomyelin
present. With the
solvent
system
described
above,
the
by
vol).
Separations
are
allowed to proceed 20 mm (solvent front
moves about 15 cm) and then the chromatogram
is removed
and air dried.
The dried sheet is now divided in half
length-wise.
One section is placed within
a
closed
crystals,
either
chamber
the vapor
containing
from which
yellow or brown
iodine
produces
spots with all
(20 ml/100
ml)
background
color is bleached
sphingomyelin
acetic
acid,
until
spots are outlined
with pencil before the background
Rf’s
of the
various
the
out. With
standards
are
and
lightly
color
were:
lecithin 0.56, sphingomyelin
0.45, phosphotidyl
ethanolamine
0.67, phosphatidyl inositide
0.21, and phosphatidyl
CLINICAL CHEMISTRY, Vol. 18, No. 5, 1972 491
serine
0.17.
cluding
The
sample
entire
procedure-in-
extraction,
chromatography,
and
quires about 90 mm.
thin-layer
detection-re-
References
1. Gluck, L., Kulovich, M., Borer, R. C.,
Brenner, P. H., Anderson, G. A., and
Spellacy, W., Diagnosis of the respiratory
distress syndrome by amniocentesis.
Amer.
J. Obstet. Gynecol. 109, 440 (1971).
2. Gluck, L., Pulmonary
surfactant
and
Jr.,
neonatal
respiratory
distress.
Hospital
Practice 6, 1971, p 45.
3. Gluck, L., Kulovich, M., and Brody,
S., Rapid quantitative
measurement
of
lung tissue phospholipids.
J. Lipid Res.
7,570 (1966).
4. Haer, Frederick,
C., An Introduction
to Chromatography
on Impregnated
Glass
Fiber,
Ann
Arbor-Humphrey
Science
Publ., Ann Arbor, Mich., 1969, p 73.
EMILY
H.
Sigma, rather than obtained from an
outside source. The bottle did not have
the usual PFS symbol on its label, which
was the sole customer notification of a
change in source. This lot was subsequently
withdrawn
by a letter to
purchasers,
and replacement offered.
Considering
the above,
COCH
GERALD KESSLER
Medicine
The Jewish Hospital of St. Louis
occasion, a solution being
readied
for calcium analysis by atomic
absorption was filtered through What-
man No. 1 filter paper to remove a
protein filtrate. Nonlinearity of standard
with the recommended
unease reagent
concentration,
and adjust upwards if
necessary.
We would also recommend,
as a gen-
revealed
amount
a variable
but significant
of calcium eluted from the
eral note, that clinical chemists voice
their objections vigorously to manufacturers
when they recemve unannounced
substitutions
of materials.
Suppliers should be kept aware of their
responsibility
to the clinical laboratory,
and it is important once again to emphasize the need for continuous monitoring of every step in a quantitative
and
of the
unnecessary
chaos
materials
The absolute
purity
cannot
be taken
for
analysis to prevent the
undesirable
contaminants,
very ones under study.
of filter
granted,
introduction of
often
the
caused
produce
runs or erroneous results.
aborted
paper.
papers
the entire profiltration
step,
Reference
1. Baer, D. M., Amer.
44, 114 (1965).
Reference
1. Gochman,
N., and Schmitz,
J. M.,
Automated
determination
of uric acid,
with use of a unicase-peroxidase
syatem.
CLIN. CHEM. 17, 1154 (1971).
NATHAN
GOCHMAN
JOAN
M. Scrwrrz
J. Clin. Pathol.
MARTIN
Sxy&oux
MARCUS
KLEINBERG
Fordham Hospital
Bronx,N.
V.10458
NucleotideSeparationon Alumina
To the Editor:
We recently reported
procedure
for serum
a new automated
uric acid deter-
mination in which unease is used as one
of the reagents (1). The recommended
product was Type IV uricase (cat. No.
U-8500, Sigma Chemical Co., St. Louis,
Mo.), which we have been using for
over one year. On two occasions, however, material
of lower activity
was
substituted
without
tion or explanation.
sufficient
notifica-
In December, 1970, we received mateas containing
12% of the
specific activity
of the usual product,
0.3 unit/mg
vs. 2.5 units/mg.
This
resulted
in nonlinear
data for absor-
bance
phase-
taken through
including
the
National Institutes of Health
Bethe8da, Md. 20014
To the Editor:
On another
that it
if
solutions
cedure,
published method
St. Louis, Mo. 63110
“Caveat Emptor”-Uricase
it
it is recommended
be established
that it is absent
separating
paper is used.
to warn potential users of the
to check for linearity
desirable
when “equivalent”
Division of Biochemistry
Department of Pathology and Laboratory
rial labeled
we thought
components,
vs. concentration,
not be corrected
uricase.
In December,
which
by addition
could
of more
1971, we received
un-
case with the same label assay, 2.5
units/mg, as usual, but which produced
Filter Paper Contamination:
Sn and Ca
Merck
To the Editor:
of nucleotides.
Analysis
fluids
of barbiturates
in biological
by the method
of Baer (1) in-
cludes filtration
to absorb
the aqueous
phase,
and use of Whatman
No. 31
filter paper is recommended.
We substituted
Whatman
phase-separating
paper PS-i, and instead of a pink to
colorless
reagent
blank
we observed
deep-purple
solutions
when diphenylcarbazone
(1 g/liter)
was added. Gas
chromatographically
pure chloroform
filtered through the same kind of paper
yielded intense colors when the same
reagent was added to the filtrate.
Conversations with H. Reeve Angel &
Co., Inc. revealed that tin is used as a
catalyst
in manufacture
of the
paper;
nonlinear
results with our aqueous uric
acid standards.
Approximately
1.5 times
diphenylcarbazone
reagent may also be
used in the determination
of tin. Ap-
the usual concentration
parently
was needed to
some
lots
of paper
may
contaminated
telephone inquiry we were informed
that this lot was manufactured
by
metric analysis and may interfere
with
biochemical
studies
of isolated
tissue
Inasmuch
492 CLINICAL CHEMISTRY, Vol. 18, No. 5, 1972
with tin.
as
tin
may
react
aluminum
oxide, grade
with
organic reagents in colori-
Recently we encountered
some difficulty with a batch of alumina
in an attempt
to separate cyclic adenosine monophosphate
(cAMP) from other
nucleotides
in tissue homogenates,
as
well as a very low recovery
of added
cAMP
as measured
isotopically.
We
were about to abandon
this approach
when we discovered that the alumina
was
with
1 g
and
and
acid. After washing
the alumina
ammonium
hydroxide
(0.5 mi/dl),
of alumina
per 1 ml of ammonia,
with distilled water until “neutral,”
finally drying at 100#{176}
C overnight,
the product gave a 90 ± 5% recovery
of cAMP.
Our experience
appears
to
demonstrate
that the pH of chroma-
tographic
for
packings
granted,
neutrality,
be
obtain linearity
with the new material.
In addition,
the diluted
reagent
was
less stable than previously
noted. Upon
numerous
neutral
activity
1 (neutral
alumina), has been
used for the chromatographic
separation
should not be taken
especially
when acidity,
or alkalinity is a criterion.
GREGORY
The Thomas
S. DUBOFF
Henry Simpson Memorial
Inst itute for Medical Research
The University of Michigan
Ann Arbor, Mich. 48104