dIN.
CHEM.
26/1,
84-88
(1980)
Lipid Interference in Steroid Radioimmunoassay
Judith M. Rash, lvanka Jerkunica, and Demetrios S. Sgoutas
To study lipid interference in steroid radioimmunoassays
in which dextran-coated charcoal is used as the separating
agent, we tested triolein and phosphatidylcholine as model
hydrophobic and amphipathic lipids, respectively. Addition
ofeithercauseddistortion
ofthestandardcurvetoan extent that was inversely related to the polarity of the steroid
molecule. Both lipids form a dispersion that entraps steroid
molecules. When we increased the charcoal concentration, the effect of phosphatidyicholine additon was eliminated for assays of both polar and nonpolar steroids. In
contrast, the effect from triacylglycerol
was not corrected,
particularly
inassaysof nonpolar steroids. We also studied
mixtu-es oflipids
mimicking the mixture oflipids
extracted
from plasma of normolipemic and hyperlipemic individuals.
The degree of lipemia that can be tolerated differs from
assay to assay, and primarily varies directly with the polarity
ofthesteroid
beingassayed.
Keyphrases:
variation, source of#{149}
effects of hydrophobic and amphipathic lipids compared. phospholipidsseparation on dextran-coated charcoal- liposome (micelle)
AdditIonal
preparation
Previous work in our laboratory
has shown that free fatty
acids in plasma interfere
in two ways with steroid radioimmunoassays
in which bound and free steroid are separated by
use of dextran-coated
charcoal
(1).First, when included in
reaction
tubes with steroid standards,
free fatty acids form
micelles and entrap 3H-labeled
steroids, causing an apparent
decrease in the binding capacity of the system. Second, free
fatty acids bind to the dextran-coated
charcoal,
thereby
blocking the adsorption
of free 3H-labeled steroids and giving
rise to an apparent, but false, enhanced binding capacity in
the system. Increasing the charcoal concentration eliminates
the effect of free fatty acids on the separation
step for assays
of polar steroids, and greatly decreases it for assays of nonpolar
steroids (1).
Here, we extend our studies of lipid interference
in steroid
radioimmunoassays
by examining
a typically
hydrophobic
lipid, triolein,
dylcholine.
termine
and a typically
amphipathic
We have also included
what degree
of lipemia
lipid, phosphati-
mixtures
of lipids,
would jeopardize
to de-
the re-
sults.
purchased
purity
from
were reagent
Steraloids,
was checked
grade.
Unlabeled
steroids
Pawling,
NY 12564, and
by melting-point
determination
were
their
and
thin-layer
chromatography,
as previously
described
(1).Tntium-Iabeled
steroids
(stated spec. acty, 90 kCi/mol)
were
purchased
from New England
Nuclear
Corp., Boston, MA
02118, and their radiochemical purity, checked by thin-layer
chromatography
(1),in all cases exceeded 98% as judged by
the absence of other radioactive spots on the chromatogram.
Department of Pathology and Laboratory Medicine,
versity School of Medicine, Atlanta, GA 30322.
Received Aug. 20, 1979; accepted Oct. 11, 1979.
84
ether-extraction
method of the “Bio-Ria [‘251]Testosterone”
kit (Bio-Ria Inc., Louisville, KY 40299).
Anti-cortisol-21-hemisuccinate/bovrne
serum albumin was
purchased
from Endocrine
Science, Tarzana, CA 91356;
anti-i la-progesterone-li
-hemisuccinate/bovine
bumin
and anti-6-ketoestradiol-1713,6-(O-carboxymethyl)-
serum
al-
oxime/bovine serum albumin were from Miles Laboratories,
Elkhart,
IN 46514; and anti-testosterone-3-(O-carboxymethyl)oxime/bovine
serum albumin was from Wien Laboratories,
without
Succasunna,
NY
07876.
All antisera
were
used
further purification.
Triolein,
egg-yolk
3-sn-phosphatidylcholine,
and other
lipids and reagents were purchased
from Sigma Chemical Co.,
St. Louis, MO 63178, and from Supelco, Inc., Bellefonte,
PA
16823. [i-14C]Triolein (spec. acty, 60 Ci/mol) was purchased
from Amersham Corp., Arlington Heights, IL 60005.
We synthesized
radiolabeled
phosphatidylcholine
by a
modification (8)of the biosynthetic
procedure
of Robertson
and Lands (9). [l-14C]Linoleic acid (stated spec. acty 40 Ci!
mol; New
England
Nuclear)
was incorporated
into
the 2-
position of i-monoacylglycerophosphorylcholine,
and the
radiolabeled product was purified by thin-layer chromatography (8).
A solution of lipids in chloroform was added to the reaction
tubes and the organic solvent was evaporated
under nitrogen.
Alternatively,
preformed
liposomes were added to the reaction
mixture simultaneously
with the 3H-labeled steroid, and the
volume of the control sample was adjusted with buffer to
compensate for the difference in volume.
We investigated the adsorption of free steroid by charcoal
in a system containing 0.1 mL of diluted isotope, about 50 pg,
in 10 mmol!L phosphate-buffered
saline, pH 7.4, containing
1 g each of bovine albumin and NaN3 per liter. After incubating
the system
for 10 mm at 4 #{176}C,
we added
0.5 mL of
dextran-coated
charcoal solution, re-incubated
the mixture
for 10 mm at 4#{176}C,
and then centrifuged at 1700 X g for 10 min.
To check whether the charcoal could adsorb lipids, we incubated the dextran-coated
charcoal (0.5 mL of a 12.5 g/L suspension of charcoal in buffer) with increasing concentration
Materials and Methods
All solvents
Radioimmunoassays
were performed
as previously
described (2-7), except for modifications to optimize the assays
under the applied conditions
(1).Bound and unbound steroids
were separated by adsorption onto active charcoal (2-7) under
conditions described in our previous publication (1). In addition to our routine assay (4) we used the double-antibody
CLINICAL CHEMISTRY, Vol. 26, No. 1, 1980
Emory Uni-
of radiolabeled lipid.
Sephadex G-25 (coarse) was purchased from Pharmacia
Fine Chemicals, Piscataway, NJ 08854. The assays were performed in vials containing 300mg of Sephadex G-25, 1.8 mL
of phosphate buffer (50 mmol/L, pH 7.4) containing 1 g of
NaN3
per
liter,
and
defined
quantities
of 3H-labeled
steroids
and antibody. After addition of the sample in 0.2 mL of buffer,
the vials were capped and placed in a rotating
mixer for 30
mm; then they were removed from the mixer, the Sephadex
was allowed to settle, and a 0.5-mL aliquot of the supernate
was removed for counting.
Equilibrium
dialysis experiments
were carried out with use
of Visking bags (size 8; Union Carbide Corp., Chicago, IL
60638) containing 1 mL of a solution of antiserum, labeled
steroid, phosphate buffer (100 mmol/L,
pH 7.4), and the in-
lipid. In preliminary
experiments, we incubated the
dialysis bags for 16 h with radiolabeled lipid. More than 95%
terfering
of the radioactivity
was recovered,
indicating
that the tested
lipids did not bind to the dialysis tubing. The dialysis tubing
was soaked in distilled
water overnight
to remove glycerol.
Duplicate bags were
20 mL of phosphate
bath at 20 #{176}C
for 16
and 0.1-mL samples
placed in Erlenmeyer flasks containing
buffer. All flasks were shaken in a water
h. Bags were removed
of their contents
at various
times,
were taken for liquid
scintillation
counting.
The radioactivity
of 0.1 mL of the solution inside each bag
was then counted to determine the total steroid concentration
within the bag. Samples of the outside solution were counted
to determine the concentration of the unbound steroid outside
the bag. Duplicate 0.1-mL samples of the inside solutions were
counted
before
equilibrium
dialysis
to determine
the recovery
of radioactivity.
Sampling error was less than 2%; the analytical recovery of radioactivity exceeded 90%.
Egg-yolk
dylcholine
shaken
phosphatidyicholine
and sphingomyelin
or vortex-mixed
contained
as impurities.
in aqueous
solutions,
tidylcholmne
dispersed
(liposomes).
Each globule is composed
ular membrane
into
spheres
microscopic
(multilamellar
lysophosphatiWhen hand-
the phospha-
spherical
globules
of a series of bimolecdispersions),
packed
one inside the other, each membrane separated from the next
by a water layer; when further dispersed by ultrasonication,
single-compartment
spheres with a one-membrane
wall
(unilamellar
vesicles)
Liposomes
are produced.
were prepared
by dissolving
110 mg of phos-
phatidylcholine in chloroform in a beaker and evaporating
the
solvent to leave a thin film of lipid. After 5 mL of buffer was
added,
the mixture
was either
vortex-mixed
(multilamellar
preparation),
or immersed in an ice bath, and exposed to the
maximal output of a 100-W ultrasonic
probe (Model W14OD;
Heat Systems-Ultrasonics,
Inc., Plainview,
Long Island, NY
11803) for five 90-s periods interrupted
by 20-s cooling periods. The resulting
dispersion,
in either case, was centrifuged
at 74 000 X g for 60 mm and the clear supernate, containing
the liposomes, was removed. Radiolabeled
steroids were added
simultaneously
with the phosphatidylcholine
or, in some ex-
periments,
after the formation
of liposomes.
To study the entrapment
of steroids within liposomes,
we
used Sephadex
G-50 columns
(the barrels of 1-mL plastic
syringes) according
to the method of Fry et al. (10). Excess
fluid was removed from the Sephadex beads by centrifuging
at 1000 X g for 3 mm. The mixture of the micellarly entrapped
3H-labeled
steroid
and free 3H-labeled
steroid was applied to
was repeated
at 50 X g for 10
mm, followed by 1000 X g for 3 mm, forcing the micellar material through the column into a test tube while the free steroid
was quantitatively
retained.
Radioactivity
was determined
with a liquid-scintillation
the column bed. Centrifugation
counter
(Beckman
Instruments,
Inc., Fullerton,
CA 92634)
by adding aliquots to 10 mL of scintillation
fluid per vial (8
g of 2,5-diphenyloxazole,
0.4 g of 1,4-bis[2-(5-phenyloxazol-
steroid. At low concentrations
of ligand, triolein and phosphatidylcholine
appeared to reduce the steroid binding to its
antiserum,
as seen by the downward displacement
of the
standard curve. The distortion of the standard curve was
greater
terone>
with hydrophobic
steroids:
estradiol
> cortisol.
our preparations
either lipid class yielded a turbid dispersion.
Figure
1D also shows the effect upon the standard
curve for
testosterone
of a lipid extract
of testosterone. An extract of an artificial mixture consisting
of triolein and phosphatidylcholine,
4.8 and 4.56 g/L, respectively,
gave similar
counting error below 2%.
We assayed serum lipids by usual methods
the
(11-13).
Results
Figure
standard
curves obtained
or absence of triolein
tidylcholmne.
Addition
of either lipid distorted
dose-response
curve, which was a function
of
centration,
and the amount and the structure
methods
1 shows
in the presence
by the usual
and phosphathe “pure”
the lipid conof the assayed
From the experiments
shown
In the testosterone
and progesterone
assays,
we examined
the effect of an artificial mixture of lipids that resembled
plasma lipids in composition (Table 1). The effect was studied
with use of three different serum pools (a: low testosterone;
b; high testosterone;
C: normal
progesterone)
supplemented
with lipid to give several different
concentrations.
Addition
of 10 zmol of lipid per milliliter of plasma caused a significant
increase in assay values (p <0.01). Addition of larger amounts
of lipid (<20 mol/mL)
increased assay values by 30 to 100%.
The three serum pools tested (Table 1) were normolipemic,
with a total lipid content of about 10 mmol/L of plasma.
The data in Table 2 show that the percentages of radiolabeled cortisol, estradiol-17fl, and progesterone not adsorbed
by charcoal were a function of the concentration
of the charcoal, either in the absence or presence
of interfering
phophatidylcholine
or triolein. In these experiments
we were investigating
the effect of those lipids upon the efficiency
of
charcoal separation.
It has already been shown that separation
with charcoal is influenced by several nonspecific substances,
including lipids (14-16). In the absence of interfering lipid,
all (0.3 mol/L) but 5 to 10% of [3Hlcortisol,
[3H]estradiol-17f3,
and [3Hprogesterone
was precipitated
at a suboptimal
charcoal concentration
(2.5 g per liter of assay mixture). In the
presence of 2 zmol of phosphatidylcholine,
approximately
37,
30, and 26%, respectively,
of the above-named
labeled steroids
remained in the supernate.
When 2 tmol of triolein was added
to each tube, the effect was quite pronounced
in the estradiol
assay, more so in the progesterone
assay. Increasing
the concentration
of charcoal overcame
this effect of phosphatidylcholine, but not that of triolein.
To determine
whether the phosphatidylcholine
and triolein
were actually adsorbed
onto the charcoal, we incubated
the
dextran
as to keep
results.
in Figure 1D, carried out with a double-antibody
technique,
we concluded that the effect was independent
of the method
of separating bound from unbound ligand.
quenching;
was such
from plasma from a hyperli-
pemic woman (triacyiglycerols
4.78 g/L, total cholesterol 4.48
g/L, and phospholipids
2.28 g/L) with very low concentrations
same amount
of counts
> testos-
Phosphatidylcholine
and triolein were tested at concentrations approximating
the concentrations
of phospholipids
and triacylglycerols
found in plasma, 2.5 g/L and 2 g/L, respectively. In blood, however, phospholipids
and triacylglycerols in the form of lipoproteins
are in solution, whereas in
one)]benzene,
1 L of Triton X-lOO, 2 L of toluene). The samples were counted with an external standard for correction of
the number
progesterone
assay
of dextran-coated
(0.5 mL of a suspension
charcoal
as that used in an
of 10 g of charcoal
and 1 g of
per liter of phosphate buffer, 10 mmol/L, pH 4) with
increasing
concentrations
of phosphatidylcholine
containing
0.05 zCi of [14C]phosphatidylcholine.
Radioactivity
was detected only in the supernates
of the assays; evidently,
adsorption
of phosphatidylcholine
by charcoal was negligible.
Radiolabeled
triolein gave similar results, which led to the
same conclusion.
To assess the influence of phosphatidylcholine
and triolein
at the primary binding step, we examined
by nondissociating
separation the binding of labeled steroid to its antiserum.
CLINICAL CHEMISTRY,
Vol. 26, No. 1, 1980
85
60
A
B
60
50
50
40
40
30
30
20
21
a
!
I
10
.31
1.25
.62
2.5
5.0
0.003
10.0
0.006
0.0125
0.025
0.05
0.1
ngitube
ngl lube
60
C
D
50
- 40
a
‘a
C
0
I
30
20
10
.01
.025
.05
.1
.2
.4
0.025
.$
0.05
ng!tube
0.1
0.25
0.5
1.0
ng!tubs
Fig. 1. Dose-response
curves for charcoal assays of cortisol (A), estradiol (B), and progesterone (C) in the absence (0-0)
or
presence of triolein ((>-G, 0.2 ftmol; U-U,
2 tmol) or phosphatidylcholine
1 tmol;
A-A.
2 fLmol); (L, testosterone
assay in the absence (0-0)
or presence of 1 ,umol of phosphatidylcholine
(#{149}-S), lipid extract from lipemic serum (#{149}-),
and artificial lipid mixture (0-0)
Percent bound/total count vs concentrations (ng/tube) of unlabeledhormone. A, B, C meansof four determinatIons; D means of two determInatIons
Table 1. Effect of Total Lipid on Assay Values8
Testosterone,
PrOge$t.rOnO.
Lipid.
pmoib
-
POOl b
Pool C
(0.06)
1.43 (0.25)
2.04(0.24)
8.30 (0.54)
10.39 (0.58)
11.99(0.17)
0.17 (0.01)
2.87 (0.24)
15.82 (0.97)
POOl
A
0.89
10
20
50
0.73(0.02)
0.92 (0.07)
1.07 (0.05)
3H-Labeied
steroid plus
lIpid added per assay8
[3H] Cortisol (no addition)
(SD) of four determinations.
Artificial lipidaddedto 1 mL of pooled plasma had the following composItion:
-
Charcoal concn, g/L
of final
2.5
assay
10
mixture
20
% of total radioactivity”
9
2
1
a Average
Phosphatidylcholine (1 imol)
33
4
2
b
Phosphatidylcholine (2 ,umol)
37
5
2
Trioleln (0.2 lLmol)
14
2
1
Triolein (2 imol)
13
2
1
5
4
2
19
30
5
5
4
7
5
4
13
12
8
4
3
2
cholesteryloleate.36.4%; cholesterol,29.0%
phosphatidylcholine.
triacylglycerols.
11.9%;
and
22.7%.
Figure 2 shows results from assays with Sephadex
G-25 as
the separating
agent. The influence
of the added lipids was
pronounced
in the progesterone
assay. In the presence
of 2
zmol of either triolein or phosphatidylcholine,
nonspecific
binding values approached
total binding, which suggests that
the lipid entrapped
the radioactive
ligand and inhibited
the
steroid from penetrating
into the matrix of the gel. It thus
appeared that the triolein and phosphatidylcholine
formation of steroid-antibody
complexes.
Additional
information
from equilibrium
regarding
dialysis experiments.
inhibited
[3HJEstradiol- 17/3 (no
addition)
Phosphatidylcholine (1 tmol)
Phosphatidylcholine
Triolein (0.2 mol)
(2 moI)
Triolein (2 ftmol)
[3H] Progesterone (no
addition)
4
was obtained
Phosphatidylcholine
(1 mol)
15
3
2
Vesicles and liposomes
Phosphatidylcholine
(2 mol)
26
11
33
3
6
24
3
6
24
this effect
are much too large to penetrate
Visking dialysis membranes,
whereas free steroid can pass through easily. This difference
was used to estimate,
in suspensions
of vesicles or liposomes,
or both, the proportion
of the total steroid that was present
in free solution.
The hormone
was allowed to partition
between two compartments
separated
by a dialysis membrane;
86
Table 2. Unadsorbed Radioactivity In the
Presence and Absence of Interfering Lipids as a
Function of Charcoal Concentration
CLINICAL CHEMISTRY, Vol. 26, No. 1, 1980
Triolein (0.2 imol)
Triolein (2 Lmol)
Assay volume,
0.5 mL throughout, corresponding to 0.1 mL of extracted
plasma.
Mean of four determInatIons.
1000
40
A
000
z
E
600
400
0
C
30
200
.31
.62
1.25
2.5
ag/tube
5.0
10.0
‘
500
Z
20
E
B
C.
1.,
400
L
300
Ia
1
200
‘
100
10
0.015
0.03
0.062
0.025
0.5
0.1
nt/tube
2
4
6
8
12
10
14
Hours
C
Fig. 3. [3H]Estradiol-17/3 inside dialysis tubing, without (A-a)
or with addition of phosphatidylcholine (0-0,
1 mol; and
0-0,2
imol)
z
Data from two experiments
300
Ia
a
.8
200
100
0.0125
0.05
0.2
0.0
nt/tube
Fig. 2. Dose-response
curves of Sephadex
assays of cortisol
(A), estradiol (B), and progesterone (C) in the absence (0-0)
and presenceof triolein (S-#{149},0.2 Lmol; 0-0,
2 imol) or
phosphatidylcholine
(A-s,
1 tmoI; A-A, 2 tmol)
Counts per mInute bound minus counts per mInute nonspecifIc binding (NSB)
vs concentration (ng/tube) of unlabeled hormone (means of two determina-
tions)
phospholipids
were present
in only one compartment
at the
indicated concentration
in 0.1 mol/L phosphate buffer.
Figure 3 shows results from such experiments. The effects
of various phosphatidyicholine
concentrations
on the rate of
dialysis of [3H]estradiol-17f3
in the presence of constant
concentrations
of antiserum
and [3H]estradiol-17/3
were
measured.
Curve A shows the [3Hjestradiol-1713
binding to
its antiserum
concentration
as for curve A, but with phosphatidylcholine
added at the indicated concentrations. There
were profound alterations in the apparent [3H]estradiol-17/3
binding at 0.5 mmol/L or greater phosphatidylcholine
concentration.
We then confirmed
that the effect was due to the interaction
between lipids and radiolabeled steroid by using several liposomal preparations
and Sephadex G-50 minicolumns
as
described in Materials
and Methods.
Table 3 shows data
obtained
with liposomes
prepared
by vortex-mixing
(multilamellar)
or sonication (unilamellar).
Incorporation
of estradiol and progesterone
into liposomes
prepared
by sonication
was several-fold
greater than incorporation
into vortex-mixed
liposomes. Table 3 also shows that the order in which the
steroid and phospholipid
were mixed or sonicated was im-
portant. More estradiol and progesterone were incorporated
into liposomes when steroid and phosphatidylcholine
were
added simultaneously
and liposomes were then prepared,
than
when the radiolabeled steroid was incubated with preformed
liposomes.
In all cases, however, the data in Table 3 indicate
that liposomes
entrapped
the radiolabeled
steroid and that
the extent was inversely
proportionaPto
the polarity
of the
steroid molecule.
Discussion
Our data show that both triolein, a representative
hydrophobic lipid, and phosphatidylcholine,
an amphipathic
lipid,
interfere with the binding between steroid and its antiserum,
by interacting
with the steroid. When sufficient
charcoal
is
added as the separating
agent, phospholipids
only insignificantly inhibit binding of free steroid to charcoal. This finding
Table 3. Relationship between the Incorporation
into Liposomes and the Lipophilic Character of
3H-Labeled Steroids under Various Conditions8
Relative % cpmb
Muitliamellar
preparation
Steroid added c
[3H] Cortisol
[3H]Estradiol
3H] Progesterone
Before
After
0.4
0.2
3.0
1.5
11
9.3
Uniiamellar
preparation
Before
0.9
44
76
After
0.9
13
27.6
a Mean of duplicate determinations.
#{176}(cpm
recovered in liposomes X 100)/cpm applied to Sephadex 0-50
column.
C Steroid added to the lipid material before or after the preparatIon of Ilposomes.
CLINICAL CHEMISTRY, Vol. 26, No. 1, 1980
87
contrasts
with data
obtained
with nonesterified
fatty
acids,
bile acids, and commercial detergents
(14-16). In contrast,
triolein impairs the efficiency of the separation step whatever
the concentration
of charcoal, particularly
in assays of nonpolar steroids.
Phospholipids
and other amphipathic lipids form micelles
(liposomes) in aqueous solution. Nonpolar steroids are more
easily entrapped in liposomes than are polar steroids, and the
hydrophobicity
of the side chain determines the extent of
incorporation of steroids into liposomes (17). Our results show
that the efficiency of entrapment
is reversibly related to the
size of liposomes: multilamellar
liposomes trapped significantly less steroid than unilamellar liposomes. Our results also
suggest that the order of addition is important:
preformed
liposomes incorporate less radiolabeled steroid.
We have previously
shown that charcoal breaks down micelles, frees the unbound
steroid, and enables charcoal
adsorption of free ligand (1). An adequate charcoal concentration
reduces the increased nonspecific binding of amphipathic
lipids to control values, although it cannot correct the distortion of the standard curve related to steroid entrapment
by liposomes
during
complex
formation.
Triolein alone in aqueous solution does not form lipo8omes.
Neither the interference
of triolein with the initial complex
formation
nor its effect on charcoal separation
can be corrected by increasing the concentration
of charcoaL However,
in the presence of amphipathic
lipids, triolein and other hydrophobic
lipids form liposomes
(18). It is noteworthy
that
emulsions with a phosphatidycholine/hydrophobic
lipid ratio
exceeding
0.4 are very stable. The stability
of these types of
lipid dispersions
towards charcoal treatment
was not tested,
however. In future experiments, we will attempt to answer this
References
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The present studies have practical
implications.
Determination of serum steroids by radioimmunoassay
requires extraction
of the steroids
with an organic solvent, which also
results
in removal
of serum lipids in the extract. Serum lipids
are a mixture
of phospholipids,
free fatty acids, triacylgly-
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cerols, cholesterol,
and cholesterol
esters, a mixture
similar
in nature to the type described above. Consequently,
extracts
of lipemic serum samples may produce spurious
values when
read from a typical standard
curve. The question
to be answered concerns the magnitude
of the lipemia that would affect the accuracy of steroid determination.
Our results (Table
1) clearly show that for testosterone
and progesterone
a twofold increase in lipid content would bias the determination.
The fact that this effect varies at different
concentrations
of
ligand means that use of a sample blank throughout
the assay
does not sufficiently
correct the effect.
Our results confirm the impression gained in earlier dis(19-21)
that defatting of lipemic plasma extracts is
cussions
necessary
if one
is to have
specific
and precise
of steroids by radioimmunoassay.
88
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in, biological
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by differential
solu-
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