T H E AMERICAN JOURNAL OF CLINICAL PATHOLOGY
Vol. 40, No. 2, pp. 113-122
August, 1963
Copyright © 1963 by The Williams & Wilkins Co.
Printed in U.S.A.
HEMATIN—STUDIES ON PROTEIN COMPLEXES AND
DETERMINATION I N HUMAN PLASMA
GEORGE Y. SHINOWARA, P H . D . , AND MARTHA I. WALTERS, M.Sc.
Department of Pathology, New York University School of Medicine and Bellevue Hospital,
New York, New York
The occurrence of hematin, ferriporphyrin
hydroxide, as a protein complex was first
demonstrated spectrophotometrically by
Heilmeyer12 on specimens obtained from patients with hemolytic anemia and on experimental solutions of this pigment in normal
serums. On the basis of spectroscopic observations of experimental protein solutions
and of fractions of plasma specimens obtained from patients with blackwater fever
and other hemolytic dyscrasias, Fairley 8,9
concluded that hematin binds exclusively
with albumin and named this complex,
methemalbumin. Miller and Ailing16 and
others, 2, " • , 8 ' 2 0 ' 2 8 however, have reported
that hematin forms complexes not only with
albumin but also with alpha-2 or beta
globulins, or both.
The binding of hematin with beta-alpha-2
globulins is also significant from the perspective that some of these proteins are
associated with hemoglobin-coupling substances, or haptoglobins, first reported by
Polonovsky and Jayle19 and by Jayle and
Conas,14 and a subject recently reviewed by
Dacie.0 Therefore, in view of (1) this relation
of 2 heme pigments to these globulins, and
(2) the occurrence of hematinemia or hemoglobinemia, or both, in certain hemolytic
states, the authors' principal objectives in
the work described in this paper were (1) to
study the binding capacity of hematin to
plasma proteins, (2) to compare hematin
protein complexes prepared experimentally
and occurring pathologically, and (3) to
attempt the quantitation of hematin in the
presence of hemoglobin and bilirubin.
Received, December 13, 1962; accepted for
publication May 1, 1963.
Dr. Shinowarti is Professor, Department of
Pathology, New York University School of Medicine, and Director of Biochemistry, Bellevue
Hospital. Miss Walters is Senior Research Assistant in Pathology.
This study was supported, in part, by Grant
No. H-4843 from the National Institutes of
Health, United States Public Health Service. A
preliminary report on a portion of the results was
presented to the American Society of Biological
Chemists, Atlantic City, 1962.
Dr. Shinowara was recipient of Investigatorship
of the Health Research Council of the City of'New
York under Contracts Nos. 1-146 and U-1083.'
113
METHODS
Collection and Processing of Blood
Approximately 10 ml. of fasting blood was
obtained with an 18-gage needle and a
syringe containing 1.0 ml. of 4 per cent
sodium citrate solution, pH 7.4. After removal of the needle, a bubble of air was
introduced and the syringe gently inverted
several times. The specimen was then transferred to a calibrated 15-ml. centrifuge tube
and centrifuged for 30 min. at 1650 X g at
1 C. The cell and total volumes were recorded, the plasma removed, and the citrate
correction factor was calculated as previously
described.23 If spectrophotometry was not
performed immediately, the plasma was
frozen for a maximum of 1 week. If tire specimen was not crystal clear, it was filtered as
follows. Approximately 5 ml. of sample were
placed in a Boerner centrifugal apparatus,
consisting of a Seitz filter disk with a porosity
of 0.1 ix, then centrifuged for 10 min. or less
at 1650 X g and 1 C. If the filtrate was still
cloudy, this treatment was repeated.
Preparation of Stock Hemoglobin Solutions
Fresh normal titrated whole blood was
centrifuged, the plasma and buffy coat removed, and the red cells washed 3 times with
a buffered saline solution, pH 7.3 to 7.4, or
until the supernatant was negative for protein. To 1 volume of the middle layer of
washed packed cells, 7 volumes of distilled
water were added. The resulting solution was
filtered through a 0.1-M porosity Seitz disk
114
SHhVOWAKA AND
Vol. 40
WALTERS
as described above. The filtrate was free of
stroma as evidenced by phase microscopy at
500 X magnification. The concentration in
the final solution, approximately 3 per cent,
was obtained on a spectrophotometer that
had been standardized for hemoglobin by
iron and by determinations of oxygen capacity, and also gravimetrically after
lyophilization.24
Chemical Determination of Total Heme
(Hematin plus Hemoglobin)
Aqueous Bilirubin Solutions
RESULTS
This was prepared by a procedure previously reported,23 using bilirubin (C.P., Eastman) and the stock albumin described above.
Physical Properties of Pure Hematin
Hematin dissolved in pH 11.3 buffer (see
Methods) was added to buffer, whole plasma,
Total heme concentrations of all experimental solutions and certain pathologic
plasma specimens were performed at least in
duplicate by this laboratory's photometric
modification of the Bing and Baker procedure.4 Stock benzidine reagent was prepared from benzidine dihydrochloride (Baker
and Adamson). Hydrogen peroxide, 0.6 per
Preparation of Stock A queous
cent, was freshly prepared for each series
Hematin Solutions
from the stock 30 per cent solution. The
Stock acid acetone hematin was prepared specimens were diluted with water so that
according to a modification of the Anson and the maximal heme concentration was 1.0
Mirsky technic,3 as follows. One volume of mg. per 100 ml.; normal plasma was diluted
purified stock hemoglobin was added to 9 10-fold. (Final concentrations of total heme,
volumes of acid acetone (99.5 volumes of as well as hematin and hemoglobin, are exC.P. acetone plus 0.5 volumes of C.P. con- pressed throughout this communication as
centrated hydrochloric acid) at — 5 to — 8 C. milligrams of hemoglobin per 100 ml.) The
The mixture was stirred occasionally during total iron content 13 ' 29 was determined not
a 20-min. period, centrifuged at 1650 X g for only on the hemoglobin standards for the
15 min. at — 5 C , and the supernatant benzidine colorimetric procedure, but also on
decanted off. From 15 to 20 ml. of this the stock hematin solutions.
solution were transferred to a round bottom
flask containing a few glass beads and evapo- Spectrophotometric Determination of Hematin,
Hemoglobin, and Bilirubin
rated to dryness under reduced pressure. The
dry residue, at room temperature and under
All quantitative data were obtained, using
nitrogen, was dissolved in 5 ml. of NaOH- Beckman, model DU, spectrophotometers.
phosphate buffer (pH 11.3).23 Usually, 1 Cells with a 1-cm. light path and minimal
volume of this solution was immediately volume of 0.1 or 2.8 ml. were used throughadded to 18 volumes of protein solutions or out; the slit was less than 0.1 mm. for all
plasma, followed by 1 volume of appropriate wave lengths. The wave length calibrations
phosphate buffer so that the final pH was of all instruments were made periodically acbetween 7.3 and 7.6. With each series, a pro- cording to the procedure of the National
tein or plasma solution was similarly pre- Bureau of Standards. 10
pared without hematin, which was then used
The procedure for spectrophotometry of
as an optical reference or as a diluent.
plasma was to oxygenate the undiluted
specimen by inverting several times. Then
Stock Albumin Solutions
absorbancies were obtained at 675, 615, 575,
560,
and 450 nut, with distilled water as the
Albumin as Fraction IV, which was at
least 98 per cent pure, was prepared from optical reference. Dilution of the specimen
frozen fresh human plasma by this labora- was made when necessary with Sorensen
tory's low ionic strength, low temperature, buffer, 0.066 M, pH 7.4, to obtain at 450
ethanol fractionation system.26 Five per cent nut only an absorbancy between 0.5 and 1.2.
solutions were prepared by reconstituting The concentrations of the 3 plasma pigments
lyophilized albumin with distilled water; the were calculated from Equations 1, 3 to 9, 11,
and 13 (see Results).
final pH was usually between 6.8 and 7.2.
Aug. 1963
HUMAN
PLASMA
PROTEIN
HEMATIN
115
COMPLEXES
TABLE 1
E F F E C T OF PLASMA AND I T S FRACTIONS ON THE P A P E R E L E C T R O P H O R E S I S OF
HEMATIN*
Distribution
Specimen
Concentration
of Protein
Stain
Gamma
Phi
Gm./lOO ml.
Beta
Alpha-2
Alpha-1
Albumin
6.3
5.5
per cent
Fraction 1II-A
3.1
BPBf
PBH J
Fraction IV
2.5
BPB
PBH
Whole plasma
0.5
BPB
PBH
Buffer
0.0
PBH
51.4
5.7 | 31.0
<-100->
19.0
12.4
9.0
7.5
0.1
27.3
11.7
0.9
<-39.7->
99.9
53.7
0.9
10. S
53.1
42.0
100.0
* Hematin was added to each solution to a final concentration of 58 mg. per 100 ml.; p H 7.4.
t B P B = Bromphenol blue for protein.
t P B H = Pyridine benzidine dihydrochloride for hematin.
globulins as Fraction III-A, and albumin as
Fraction IV,26 all at a final pH of 7.4. The
hematin at this concentration, 58 mg. per
100 ml., was completely soluble in all 4
solutions. Paper strip electrophoresis, at pH
8.6 and T/2, 0.05, was performed at least
in duplicate on each of these solutions with
controls consisting of the same solution
without hematin and of plasma with a known
concentration of added hemoglobin. Half of
the strips were stained by a modification of
the pyridine benzidine hydrochloride peroxide procedure of Connelly and associates6 and
the remainder with bromphenol blue. The
pigment in Fraction III-A solution migrated
exclusively in the beta-alpha-2 region,
demonstrating its capacity to bind with
globulins (Table 1). None of the hematin
migrated with albumin, which comprised
more than 5 per cent of the total protein.
This preparation represented a subfraction of
Fraction IIP 5 and contained at least 95 per
cent of the gamma globulins present in the
original plasma. On the other hand, in
Fraction IV, containing almost pure albumin, most of the hematin migrated in both
the beta and albumin regions. In 16 similar
experiments, hematin was added to normal
plasma in concentrations from 3 to 57 mg.
per 100 ml.: 44 (32 to 62) per cent of the pigment migrated in the beta-alpha-2 region;
and 49 (25 to 67), in the albumin-alpha-1
region. At higher concentrations, 60 to 236
mg. per 100 ml., the proportion found with
the phi component increased to as high as
36 per cent, with the remainder migrating
almost equally in the beta-alpha-2 and in the
alpha-1-albumin positions (12 experiments).
In 21 normal plasma specimens without
hemoglobin or added hematin, trace benzidine-positive reactions were consistently
noted in the phi and albumin positions only.
In 108 control experiments, absolutely all of
the added hemoglobin migrated in the betaalpha-2 position when the concentration was
less than 60 mg. per 100 ml. At higher concentrations, hemoglobin was also found in
the phi and albumin positions. Not more
than 50 per cent of the hematin added to
normal plasma containing up to 60 mg.
hemoglobin per 100 ml. migrated with albumin; the remainder was found together
with hemoglobin in the beta-alpha-2 position (7 experiments).
The spectrophotometric absorption curves
(Nos. 2 to 6, Fig. 1) of hematin in albumin
and globulin solutions could be superimposed
between 475 and 700 mn, manifesting specific
maxima at 500, 530, and 615 mji. From 425
to 475 my, the curves are similar, but between 400 and 425 my, there is a maximum
at which hematin in albumin solution absorbs to a greater extent than that in globulin
solution. Flematin in pH 7.4 solution not
450
500
550
WAVELENGTH
450
500
550
WAVELENGTH
600
650
(mjj)
600
650
(m»)
FIG. 1 (upper). Wave length absorption curves of hematin in protein and buffer solutions.
Final concentration of hematin, 58 mg. per 100 ml., and pH 7.4:/—phosphate buffer; 8—
Fraction III-A; S, 4, and 5—Fraction IV (0.31, 1.25, and 2.50 Gm. of albumin per 100 ml.);
6—whole plasma.
FIG. 2 (lower). Wave length absorption curves of bilirubin, hematin, and hemoglobin in
solutions of albumin, alone and combined. The pigments and their concentrations per 100
ml. are as follows: 1—bilirubin, 1.1 mg.; 2—hematin, 16.6 mg.; 3—hemoglobin, 11.2 mg.;
4—a solution with all 3 pigments and in the same finai concentrations, pH 7.3 to 7.6.
116
Aug. 1963
HUMAN
PLASMA
PROTEIN
containing protein was characterized not
only by a lack of electrophoretic mobility,
but also by a nonspecific type of absorption
(No. 1, Fig. 1). Moreover, these solutions,
unlike those containing protein, changed in
their optical properties after 4 to 6 hr. at
room temperature.
Effect of Bilirubin and Hemoglobin on
Hematin Spectrophotometry
Inasmuch as bilirubin always appears in
plasma, and hemoglobin is present under
certain pathologic conditions, the spectrophotometric relation of these 2 pigments to
hematin was next investigated. The major
maxima of hematin, hemoglobin, and bilirubin in the blue range are 403, 415, and 450
nut, respectively (Fig. 2), in protein solutions.
The spectrum of a solution containing all 3
pigments in final concentrations as those in
the pure pigment solutions is also illustrated
in Figure 2. In the blue range there are 2
maxima at approximately 450 and 408 m/i
despite the comparatively low concentration
of bilirubin and the high concentrations of
the 2 heme pigments. It is noted that the
effect of hematin and bilirubin on the
A675 mii-Amm*, characteristic for hemoglobin,23 is not great. The most significant
observation here is that hemoglobin and
bilirubin have very little effect on the absorption of hematin in the red region of the
visual spectrum. Table 2 is a summary of the
HEMATIN
117
COMPLEXES
extinctions, i i Y i of all 3 pigments at the
following wave lengths: 450 nut, the maximum for bilirubin; 560 and 575 nut, the most
specific wave lengths for hemoglobin; and
also at 615 and 675 m/t. I t is apparent that
bilirubin at 450 nut has at least an 80-fold
greater absorption than either hematin or
hemoglobin. On the contrary, EiJ°. obtained by dividing the concentration into
the difference in absorption between 615 and
675 nn« is 0 for bilirubin and very slight for
hemoglobin. For these and other reasons,
this extinction is apparently most specific
for hematin in plasma and solutions containing the other 2 pigments.
Absorption Characteristics in the Visual Red
Spectrum of Normal Plasma
In 14 clear postabsorptive plasma specimens (hemoglobin, 0.0 mg. per 100 ml.;
bilirubin, 0.2 to 0.8 mg. per 100 ml.) the
mean A6i5 mn-A6n ^ was 0.022 ± S.D.
0.0063. The mean total heme determined
chemically was 0.69 mg. per 100 ml. (range,
0.22 to 2.10) or 0.00069 per cent. Therefore,
the average absorption owing to heme pigments could be calculated as follows:
0.00069 X 3.02 = 0.002, The latter subtracted from the mean gross Au& mn-A61s m„
of 0.022 would equal 0.020 or the average
absorption of clear normal plasma owing to
substances other than hematin, bilirubin, or
hemoglobin.
TABLE 2
E X T I N C T I O N S (E\l°m.) AT 5 W A V E L E N G T H S OF P U R E H E M A T I N , OXYHEMOGLOBIN, AND
B I L I R U B I N IN ALBUMIN SOLUTIONS AT p H 7.3 TO 7.6
Hematin
Oxyhemoglobin's
Wave* Length
E
R*
Mean
dbS.E.f
9.09
4.11
3.05
3.49
0.47
3.02
0.20
0.06
0.03
0.08
0.01
0.05
Mean
±S.E.
Mean
±S.E.
10.10
5.60
9.50
0.33
0.00
0.24
0.06
0.04
0.11
0.02
0.01
0.01
800.80
17.50
12.40
1.06J
l.OOt
5.07
0.94
O.SO
0.12
0.12
inn
450
560
575
015
075
015 and 075
3.01
1.36
1.21
1.00
o.oot
-
* R = ratios of E for hematin at 450, 560, and 575 mn to E = (Acis mn Ans mjO/concentration.
t S.E. = s t a n d a r d error.
t Nine specimens, 4.3 to 19.2 mg. per 100 ml.
118
SHhVOWARA
Quantitative Optical Relation of Pyrolle
Pigments in Plasma
The preceding results in the visual spectrum demonstrate that the difference in the
absorbancies at 015 m/z and 675 m/z is the
most specific evidence for hematin in the
presence of bilirubin and hemoglobin, and
that normal human plasma has a mean net
absorbancy at these wave lengths of 0.02 not
the result of any of these pigments. Therefore, the net difference, A t, owing to hematin
can be expressed as follows:
At = (,L„615m/T"-*l 67 5111/1,.) - 0.02
(1)
Moreover, the net absorbancies (A,,) owing
to bilirubin and hemoglobin can be found by
subtracting from the absorbancies found
(A/) at 450, 560, and 575 m^j the absorbancy
of hematin:
An
= As -
RA,
(2)
where R is the ratio of the extinction of
hematin at each of these wave lengths to that
obtained at 615 m/i-G75 m/u. Therefore, substituting the appropriate R in Table 2, the
following are obtained:
—
-'t,l450mji = A/450mv
3.01 (At/D)
-'ln560ni(i = A.fwomit — 1.36 At
-'l«576mji = -'I / 575in^ — 1.21 At
(3)
(4)
(5)
The foregoing corrected absorbancies can
now be substituted in the previously reported23 Equations 6 and 8 for calculating
the concentration of total bilirubin (C&):
AND
Vol. Ifi
WALTERS
Cb = 1.27 A,n50™„ - 1.35 (AnmmJD)
Mg. bilirubin per 100 ml.
= (CV/)-citi'ate correction) — 0.44
(0)
(7)
In Equations 3, 4, and 5, D refers to the
-fold dilution, if any, of the specimen necessary for compliance to tlie Lambert-Beer
law at 450 m/z only, i.e., A/m m« between 0.5
and 1.2. The calculation of hemoglobin concentration is similarly performed by substituting the hematin-conected absorbancies
(AH) in tlie previously reported 'Equations 7
and 9.23
Ch = 256(v , l„575 m „ — -'l„5G0m)<) H" 1.31 C'(,
(S)
Mg. hemoglobin per 100 ml.
= CV citrate correction
(9)
The i?6i5 mji-676 m/i for hematin, hemoglobin,
and bilirubin listed in Table 2 are 3.02, 0.24,
and 0, respectively. These facts indicate that
100 mg. of hemoglobin per 100 ml. would
result in an apparent false hematin concentration of less than 8 mg. Nevertheless, this
slight hemoglobin effect at the critical
hematin wave lengths can be negated:
At - (0.24-6V10-3)
(10)
-3
where 10 is to convert per cent to milligrams per 100 ml. Simplifying Equation 10,
the following is obtained:
At -
(11)
(0.00024 Ch)
Finally, the hematin concentration (Ct) can
be calculated:
Ct = A ,„• 1073.02
(12)
TABLE 3
SPECTROFIIOTOMETRIC R E S U L T S ON H U M A N PLASMA CONTAINING ADDED H E M A T I N ,
H E M O G L O B I N , AND B I L I R U B I N
Concentration* Ranges of Pigments:
Category
Hematin
»t
Hematin
Hemoglobin
Bilirubin
Mean
concentration*
0
5 to 25
0 to 9
0 to 92
<1
<1
8 to 10
0 to 10
23
19
11
20
78.1
74.0
S7.0
0.4
Bilirubin
Mean
concentration*
Mean
concentration*
r\
ft
Known
I
(i to 240
II
0 to 151
III 37 to 14G
IV 0 to 2
Hemoglobin
Found
Known
Found
77.9 0.99
70.0 0.99
91. G 0.99
0.7
0.0
13.3
4.1
22.1
0.0
13.3
3.5
22.3
0.94
0.75
0.99
Known
Found
0.5
0.5
12.1
5.2
0.5
0.5
11.5
5.1
't
0.99
0.99
* Expressed as milligrams per 100 ml.
t n = number of items; r = positive correlation coefficient of known and found values, with each
coefficient being more t h a n twice the s t a n d a r d error.
Aug. 1963
119
HUMAN PLASMA PROTEIN HEMATIN COMPLEXES
TABLE 4
E F F E C T S OP T U R B I D I T Y AND S E I T Z F I L T R A T I O N ON T H E SPECTROIMIOTOMETRIC D E T E R M I N A T I O N
OF P Y R O L L E P I G M E N T S IN PLASMA
Concentrations*
Number
of
Specimen
!
Seitz
Filtration
Appearance
Hematin
Hemoglobin
Known
Found
Known
Bilirubin
Found
Known
Found
1
Before
After
Cloudy
Clear
0.5
0.5
12.0
0.3
0.0
0.0
0.0
0.0
0.5
0.5
0.5
0.4
2
Before
After
Cloudy
Clear
0.5
0.5
11.6
1.3
20.0
20.0
10.6
20.0
0.5
0.5
0.5
0.4
3
Before
After
Cloudy
Clear
16.5
16.5
29.8
17.2
0.0
0.0
0.0
0.0
0.5
0.5
0.9
0.7
Expressed as milligrams per 100 ml
or,
Mg. hematin per 100 ml.
= Am-331 -citrate correction
(13)
Recovery Ex-periments
In order to evaluate critically the quantitative relation formulated, spectrophotometry studies were made on 73 plasma specimens of known hematin, hemoglobin, and
bilirubin concentrations. These have been
classified according to the relative concentrations of the 3 pigments which might be found
in various pathologic states, and are listed in
Table 3. The upper and lower concentrations
of bilirubin known were identical to those
found, in Categories I and II. In Category I,
all 23 specimens had the expected results for
hemoglobin. I t is apparent, in Categories I,
II, and III, that the hematin yields in
pathologic concentrations are excellent despite the presence of the other 2 pigments.
The hemoglobin and bilirubin recoveries in
specimens (Category IV) containing exceedingly low concentrations of hematin confirm
the findings previously reported.23 In the experiments described above, it was demonstrated that the A 6i5 ,,^-A 675 nm hi clear
normal plasma was 0.022 =fc S.D. 0.0063.
This comparatively large fluctuation would
indicate that in most instances this alone
could account for a theoretical error in ± 2 . 1
mg. of hematin per 100 ml. This inherent insensitivity in hematin determinations in the
range of physiologic concentration was
demonstrated empirically in Category IV
specimens.
On nonfasting normal plasma an /l6i5 nviA675 nip severalfold higher than the usual
findings was observed. Therefore, turbid
normal specimens and the cleai'ing effect of
Seitz filtration (see Methods) were studied.
The data in Table 4 demonstrate not only
the false hematin effect of turbidity but also
the removal of this artifact by Seitz filtration
without affecting the concentrations of
hematin, hemoglobin, or bilirubin.
Plasma Hematin in Vivo
In all experiments described thus far the
hematin was prepared from human adult
hemoglobin by hydrochloric acid hydrolysis.
Experiments were next performed to determine the relation between pathologically
occurring and experimentally prepared
hematin. The plasma specimen obtained
from an 18-year-old woman in sickle cell disease crisis contained the following pigments,
as determined spectrophotometrically, per
100 ml.: hemoglobin 2.8 mg., hematin 68.9
mg., and bilirubin 4.7 mg. The total heme
level in this specimen was 71.7 mg. per 100
ml., obtained as the sum of the hemoglobin
and hematin concentrations, and 68.0 mg.,
by means of direct chemical determination.
Hematin, hemoglobin, and bilirubin were
then added to normal plasma so that their
120
SHINOWARA AND WALTERS
final concentrations were equivalent to those
found in the pathologic specimen. The spectrums of the pathologic and experimental
specimens were almost identical in the visual
range. At significant wave lengths, the absorbancies for the patient's plasma and the
simulated specimen were, respectively, as
follows: Am mm, 4.34 and 4.30; Aiis mir^675 ,,,„ , 0.237 and 0.233. In this and other
pathologic plasma specimens in which similar
findings were observed, the identity of the
pigment causing the absorption band in the
red region was proved to be hematin and not
methemoglobin by the persistence of the
615-nui band when the pH of the specimen
was increased to 9.0; and by the 526-m/i and
558-imi bands resulting from the alkaline
hemochromogen reaction.21 In the pathologic
specimens investigated thus far, the electrophoretic distributions of hematin were as
follows: albumin, 22 to 72 per cent; betaalpha-2, 22 to 65 per cent. These results
demonstrate primarily that hematin does
not occur exclusively as an albumin complex.
DISCUSSION
Hematin forms complexes not only with
albumin but also with various globulins.
With a highly purified plasma albumin
fraction, a significant amount of the added
hematin migrated with the trace beta globulin present. In a fraction composed of 95
per cent globulins and 5 per cent albumin, all
of the hematin migrated in the beta-alpha-2
region. Aber and Rowe,2 in studies on serum
fractions, also found that beta globulins
have a high hematin-binding capacity. In
plasma containing less than 60 mg. of
hematin per 100 ml., however, the pigment
migrated almost equally in the beta-alpha-2
and the alpha-1-albumin regions. At higher
concentrations, hematin was also found in
the starting or phi positions, or both. That
the latter represents "free" hematin is unlikely, inasmuch as quantitative recovery of
hematin in concentrations as high as 240 mg.
per 100 ml. was demonstrated spectrophotometrically (Table 3). The spectrums
in the 475 to 700 m/z range of globulin and
albumin complexes of hematin are identical,
but distinctly different from that of the free
pigment; Schwerd22 came to a similar con-
Vol. 40
clusion. The failure of globulins to couple
with hematin in Fairley's additive experiments 8 could be explained by the fact that
the ammonium sulfate procedure for serum
fractionation used by him resulted in alphabeta globulin denaturation or loss into the
albumin fraction. I t is now well known that
albumin obtained by high-salt fractionation
of serum proteins contains at least significant
traces of alpha and beta globulins. 7 ' ll
The specific determination of heme
pigments in plasma is important in the
investigations of hemoglobin catabolism
and transport, but has been exceedingly
difficult particularly in specimens in which
there is concomitant hematinemia and hemoglobinemia. Thus, the present results and
those of other investigators2- 1G-18' 2 0 ' 2 8 demonstrate the decided limitations in the electrophoretic differentiation of these heme
pigments.
Lathem and Worley,15 on the other hand,
concluded that hematin and hemoglobin can
be differentiated electrophoretically. This
conclusion was based primarily on the migration of pure hemoglobin and hematinalbumin in aqueous solutions not containing
globulins. In the present study, it was found
that hemoglobin in high concentrations in
plasma migrates not only in the betaalpha-2 region but also with albumin, confirming the findings of Neale and associates.17
These investigators also found that hemoglobin reduces the sensitivity of the
Schumm's reaction21 for the detection of
hematin. Moreover, experiments under way
in this laboratory have demonstrated that
even a slight hyperbilirubinemia is accompanied by a marked decrease in the specificity of this qualitative test. Attempts to
differentiate heme pigments in whole plasma
spectroscopically or spectrophotometrically
have been possible only at exceedingly high
concentrations and have been approximations, at best. 1 , 9 , 1 2 This laboratory has
demonstrated that the classic Vierordt27 computations are satisfactory for bilirubin but
not for hemoglobin, because the absorption
constants for the former are much higher at
all wave lengths significant for both pigments. The problem of quantitative identification as low concentrations was successfully
Aug. 1963
HUMAN PLASMA PROTEIN HEMATIN COMPLEXES
resolved for 1 heme by the demonstration
that the absorption difference A 575 mil —
Am inn is the most specific parameter of
hemoglobin concentration in a complex
solution such as plasma.23 The present studies
demonstrate that this concept of difference
in absorbancies at 2 proximal significant
wave lengths can also be applied to hematin,
as evidenced by the excellent recoveries of
this pigment in experimental and pathologic
plasma specimens containing bilirubin and
hemoglobin.
Lipemia and methemoglobinemia are also
known to affect the red range of the spectrum.12 I t was demonstrated in the present
work that chylomicrons but not heme-protein pigments nor bilirubin can be removed
by Seitz filtration. Experiments under way
in this laboratory confirm Heilmeyer's impression that methemoglobin can arise from
the in vitro spontaneous oxidation of the
oxy- or reduced hemoglobin originally
present in serum.12 Thus far, in all fresh
specimens obtained from patients who had
not received recent transfusions of bank
blood, the absorption in the red spectrum
has proved to be owing to hematin.
Heme pigment, occurring physiologically
in plasma, has a concentration less than 2.1
mg. per 100 ml., reacts positively with peroxide and benzidine dihydrochloride, and
migrates electrophoretically in the phi and
albumin positions. This benzidine-positive
material can not be hemoglobin, inasmuch as
the latter migrates at low concentrations exclusively in the beta-alpha-2 position and
was found to be absent spectrophotometrically in normal plasma.
SUMMARY
Hematin added to normal human plasma
and its fractions migrates primarily not only
with albumin, but also with certain globulins
in the beta-alpha-2 region. At concentrations
greater than 60 mg. per 100 ml., hematin
added to normal plasma also was found in the
starting or phi positions, or both. That the
latter is not necessarily "free" hematin was
demonstrated spectrophotometrically, inasmuch as the absorption spectrums of
hematin-protein were evident quantitatively
to at least 240 mg. of the pigment per 100
121
ml. normal plasma. Hematin added to normal plasma and that occurring pathologically
have almost identical spectrophotometric
and similar electrophoretic characteristics.
The difference in absorbancies at 615 mu and
675 m/i is highly specific for hematin-protein
and complies with the Lambert-Beer law.
Computations and their empirical evaluation
are presented for the quantitative determination of hematin in the presence of hemoglobin and bilirubin by direct visual spectrophotometry. Normal plasma contains less
than 2.1 mg. total heme per 100 ml., none of
which is hemoglobin.
SUMMARIO I N I N T E R L I N G U A
Hematina addite a normal plasma human
o a su fractiones migra primarimente non
solo con albumina sed etiam con certe
globulinas in le region de beta-alpha-2. A
concentrationes de plus que 60 mg per 100
ml, hematina addite a plasma normal
esseva etiam trovate in le position initial o le
position phi o in ambes. Que iste ultime non
es necessarimente hematina "libere" esseva
demonstrate spectrophotometricamente in
tanto que le spectros absorptionic de hematina-proteina esseva quantitativemente evidente usque a al minus 24.0 mg del pigmento
per 100 ml de plasma normal. Hematina
addite a plasma normal e illo que occurre
pathologicamente ha quasi identic characteristicas spectrophotometric e simile characteristicas electrophoretic. Le differentia in
absorbantias a 615 imt e 675 rajj es altemente specific pro hematina-proteina e es de
accordo con le lege de Lambert-Beer. Computationes e lor evalutation empiric es
presentate pro le determination quantitative
de hematina in le presentia de hemoglobina
e de bilirubina per directe spectrophotometria visual. Plasma normal confine minus
que 2.1 mg de liemo total per 100 ml, e
nulle parte de illo es hemoglobina.
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