Absorption
Measurements
and
Reduced
Range
Absorbance
range
mental
relative
S
measurements
0.6-1.88
Microns
Jr., and
are made
methods
and reduced
hemoglobin
of dye-dilution
studies.
in the
Experi-
,.
PECTROPHOTOMETRIC
reduced
of hemolyzed
human
reveals
characteristic
ANALYSIS
near-infrared
various
article,
regions
types
of hemoglobin
the infrared
absorbance
hemoglobin
(Hb)
was
(1).
Hb,
are
If for
relatively
determined
for
in
conjunction
permit
curves.
which
absorbs
utilized.
the
with
a more
In the
present
at 805
Since
805
a known
reliable
isosbestic
method
described
(O2Hb)
and
purpose
l0
point
for
reflection
by whole
blood
saturation
(O.S.)
changes.
is isosbestic
at this
the
From
the American
Optical
Company,
Received
for publication
Jan.
23, 1961.
O2Hb
for
wavelength
Therefore,
at 805
recording
67
and
m,
Center,
point
of
per mole,
this
point
of dye-dilution
Hb,
curves,
mole
a dye
(2) is
transmission
or
per
is independent
a dye
Research
of uncover-
an isosbestic
sq. cm.
method
for recording
dye-dilution
mj, where
#{128}
=
0.2 X 10#{176}sq. cm.
m
in
of
of tile two hemoglobins.
An
point
is defined
as a wave#{128}
of two media,
say (LHh
and
equal.
O2Hb and Hb there
exists
in the infrared
low absorption
such
that
e < 0.5 X
would
blood
in the visiabsorbailce
curves
In the experiment
of oxyhemoglobin
ing any furti1er
isosbestic
characteristics
isosbestic
characteristic
or an isosbestic
length
at which
the extinction
coefficients
then
L. Polanyi
Michael
for oxygenated
to new
in the
results show that an isosbestic region exists between
1.3 and 1.88 p., with the
extinction
coefficients
of O2Hb and Hb equaling zero at 1.3, 1.67, and 1.77
ble and
for
this
0.6-1.88
for application
Oxygenated
Hemoglobin
B. Barlow,
Robert
for
of
which
al)sorbs
Southhridge,
Mass.
oxygen
at 805
68
& POLANYI
BARLOW
is isosbestic
not
only
for
be independent
hon
0.5.
changes,
and
of conditions
of hemoglobin,
utilization
of one
805
m
permit
would
due
other
the
altering
or effects
the
detection
shape
to flow
isosbestic
detection
Clinical
of the
of red
in whole
of dye
dye
cells,
blood
wavelength
Chemistry
would
concentra-
(3,4).
Clearly,
in conjunction
independently
with
of these
condi-
tions.
It has
ance
been
of the
reduced
assumed
blood,
and
that
only
that
all
or oxygenated
O2Hb
the
Oxygenation
of heparinized
(Na0S0O4).
For
are
with
then
The
1-cm.
parallel
percentage
termined
eter
from
spectrophotometer
cording
(distilled
ous
H20
absorbin the
cuvettes
spacers,
from
0.84
to 1.9
on the
Cary
and
Perkin-Elmer
100
per
above
relative
to blood),
cent,
giving
0.9 X
path
of 0.1 cm.
to distilled
recording
Special
1120
permit
instruments.
cuvettes
a mirror
is de-
spectrophotom(Model
21) infrared
adapters
the
are
samples
containing
an optical
relative
bloods
blood
(Beckman)
of blood
(blood
with
pure
dithionite
reduced
prepared
leaving
.
of oxalated
contact
of sodium
and
The
(Model
14) automatic
and on the Perkin-Elmer
transmissions
and
oxygenated
quartz
transmission
tubes
is either
prolonged
granules
solut.ion.*
vycor
on the Cary
0.6 to 1.9
of Beckman
the
saponin
into
plane
to the
present
drawn)
through
with a few
hemolysis
a dilute
placed
0.9-cm.
contribute
Procedure
(freshly
human
blood
is achieved
reduction
is achieved
treated
Hb
form.
Experimental
bank)
oxygen;
and
hemoglobin
are
image
the
use
For
re-
reversed
of the
previ-
step.
Results
It has
been
therefore,
at
805
m,
oxygenation
plete,
ing
established
to make
that
the
it is necessary
and/or
it is necessary
805
m,
transmission
to
reduction
is isosbestic
curves
equate
hemoglobin
of a hemolyzed
to correct
the
of Hb
for
and
Hb
and
O2Hb
0211b;
intersect
concentrations.
blood
experimental
sample
values
If
is not
by
the
com-
follow-
procedure.
Since
hemolyzed
blood
obeys
The
diluted
saponin
solution
is prepared
to 3 gm. of powdered
saponin
in a 20-cc,
for
mixing.
The
volume
is then
incrensed
eliminate
dissolved
oxygen).
Beer’s
by
syringe
to
law,
oxygen
saturation
adding
1 ml. of iN sodium
containing
approximately
10 cc. with
distilled
water
hydroxide
0.5 ml.
(freshly
can
of
be
(NaOH)
mercury
boiled
to
Vol.
8, No.
I,
1962
determined
OXYGENATED
for
os
each
curve
(A1)
(A1)
r
-
AND
from
s
-
REDUCED
the
(Ar)
linear
(A2)
(A1)
r r
-
69
HEMOGLOBIN
relationship
x
(A1)
-
(5),
OD
OD
(A1)
(A2)
where
#{128},,
and Er are the extinctions
coefficients
of O2Hb
and Hb at the
wavelengths
) and X2, equalillg
660 m
and 805 m
for the reasons
stated
previously;
and
OD (x) is the absorbance
of the hemolyzed
sample
at wavelength
When
interpreted
from
the reported
extinc.
tion
coefficients
(1),
Equation
O.S.
if
on the
pci’
cent
curves
the
same
for
are
1.138
=
run,
O2Hb
the
solution
).
X
calculated
is not
to read
simultaneous
0.284
-
0.S.
and/or
corrected
1 becomes
using
0 per
100
cent
per
of Beer’s
Equation
for
cent
(2)
Hb,
O2Hb
2 is not
then
and
either
0 per
100
or both
cent
Hb
by
equations:
I
(A)
=
To
10-
(C0
0r
+
C0
c,)
I’
(A)
=
10
10-
(C,
e0
+
C0
e,)wL,
wL,
and
I()
where
sample
is the
T, is the
concentrations
+
C0 =
the
total
of Hb
and
C’,. and
L is the
optical
and
duced
the
matter
of
water
air
same
values
within
and
interest,
(Fig.
3)
with
i’or
similarities
HO
the
and
blood
reported
there
are
however,
are
between
sample,
the
present
for
the
per
in the
same
unit
the
the
oxygenated
reduced
sample,
and
are
#{128}
Cr and
the
the
C0 are
oxygenated
sample
(Cr
for the reduced
sample;
volume
of the hemolyzed
that
the
the
range
the
w is
sam-
each
,
of
of the
three
experimental
curves
and
1.55
and
(6).
From
1.3 to 1.85
of blood
the
The
instruments.
beyond
relative
1.0
for
pro-
As
distilled
both
1120
runs
error.
for
on
transmission
in Fig.
from
0.6 to
coefficients
1.35
of others
of distilled
water
(in
indicated
region
extinction
transmission
between
the
coefficients,
1.0 to 1.9
determined
reports
between
extinction
values
in
no reported
fronl
hands
al)sorptioll
in accord
for
Hh;
technic
by
same
path.
values
1 and
2, check
with
(1).
Beyond
1.0 ,
transmitted
is the
light
(taken
as 1.00)
O2Hb,
respectively;
O,Hb
C’0 are
of hemoglobin
experimental
0911b
and
and
weight
1),
The
vs.
light
of
I’ (x)
and
,
original
intensity
of the
coefficients
of Hb and
extinction
ple;
intensity
of wavelength
distinct
1.85
are
,
the
to
(list illed
relative
to air suggest
serum)
is displaced
by
a
H20
mir-
that
ill
the con-
70
BARLOW
& POLANYI
Clinical
Chemistry
1’
)
20
be
z
/
0
z’ ;
80
Fig.
1.
sion
curves
0
RI
-
A
B
CURVE
CURVE
water
so
li.65
I,
100
path,
Il
20
C-
ii
80
0.6
1.0
1.2
WAVELENGTH
1.4
1.6
(0)
A
and
hemoglobin
and
hemoglobin
4#{176}
transmisdistilled
to
oxygenated
(it)
Curves
Ii’
a.
for
reduced
fV
Percentage
relative
B
with
representing
concentrations
gm./iOO
cc.,
0.1
cc.
of
and
8.45
respectively.
cm.
gm./
Optical
LB
(MICRONS)
‘4
Fig.
2.
coefficients
and
reduced
WAVELENGTH
centratioii
the
of
two
and
three
which
two
extinction
(0)
oxygenated
(it),
as
pairs
are
obtained
of
runs,
shown
on
of
Fig.
1.
(MICRONS)
hemoglobin,
cuvettes)
from
Average
of
resulting
in
in transmission
unequal
above
water
100
per
volumes
(in
cent.
Discussion
The results
in Fig. 2 show
that
not one but many
isosbestic
points,
or an isosbestic
region,
is found
to exist
for Rb and O2Hb between
1.3
and 1.88
Beyond
1.88 , the optical
path
length
of 0.1 cm. is too large
for water
absorption,
making
it necessary
to use smaller
path
lengths
.
(on the order
of 0.01 cm.) if this region
is to be investigated.
The small
deviations
noted
in Fig.
2 may
be due to experimental
error,
leaving
reason
to suspect
that Rb and O2Hb
absorb
equally
beyond
1.3
.
The
experimental
dye-dilution
curves.
results
The
suggest
first
method
two
methods
utilizes
for
the
the
isosbestic
recording
point
of
at
Vol.
8, No.
Fig.
of
I,
3.
OXYGENATED
Infrarcd
distilled
Optical
962
REDUCED
71
HEMOGLOBIN
transmission
water
path,
AND
relative
0.1
to air.
cm.
w
0
z
c-I
cr
a.
WAVELENGTH
805
in
two
curves
point
the
throughout
choose
the
one other
isosbestic
should
not actually
alid
as
extinction
to the
region
1.45 ,
isosbestic
dye
such
as
Green,”
which
from
Rb
and
of
coefficient
of
method
nonabsorbent,
i.e.,
0211b
Rb
commercially
and not at 1.45
altering
oxygen
of hemoglobin,
0211b and Rb equal
1.3, 1.67, and 1.77
zero.
The
meet these
experimental
requirements;
less
region
which
in the
lengths
to
the
first
depends
for
and
and
be detectable
method,
on
system
0.S.
visible
would
only
dye
which
one
to flow
at which
extinction
absorbs
independent
utilizes
wavelength,
detection
and
results
thus,
at
of these
two
thus
enables
in ratio
Thus,
a
.
as “Cardiobe detectable
shape
due
utilizes
a wavelength
at which
the relative
one
known
p, would
saturation,
or effects
If the
every
it would
be wise to
if any,
between
would
be negligible
and 0211b at 1.45
of
green,
at 0.805
conditions
cells, concentration
The
second
1.3 and 1.88
hypothesized-at
1.3 to 1.88 , then
a small
difference,
where
extinction
indocyanine
absorbs
independent
point
between
coincide-as
coefficients
average
)MICRONS)
of
red
in whole
blood.
hemoglobin
is
coefficients
of
in Fig. 2 show
that
a dye which
is coloreither
of these
conditions.
isosbestic
points,
permitting
waveSuperior
this
method
a completely
simultaneous
static
measurement
of
of dye-dilutions.
References
1.
2.
Horeeker,
B. L., J. Biol.
Chem.
148,
178
Fox,
I. J., et at., Staff
Meet.
Mayo
Clinic
3.
4.
Kramer,
K., et at., Am.
J. Physiol.
165,
229 (1951).
Wood,
E. H., Sutterer,
W. F., and
Cronin,
L., Medical
Yr. Bk. Pub.,
Chicago,
1960,
pp. 416, 429.
Millikan,
G. A., Rev.
Sci. Instr.
13, 434 (1942).
Curcio,
J. A., and Petty,
C. C., J. Opt.
Soc.
Am.
41, 304
5.
6.
(1943).
32, 480
(1957).
Physics,
(1951).
edited
by
Otto
Glaser.