Preparation of high specific activity tritium-labelled leukotriene B 4 suitable for radioligand binding assay

Article abstract of DOI:10.1002/jlcr.1488

We describe a method of preparation of high specific activity tritium-labelled leukotriene (LT) B₄ from [5,6,8,9,11,12, 14,15- ³H] arachidonic acid (AA; 6.66 TBq/mmol) utilizing a LTB ₄-synthesizing enzyme system from rat basophilic leukemia (RBL-1) cells. It was shown that both cyclooxygenase inhibitor indomethacin and adenosine 5′-triphosphate induced [³H] AA transformation to [³H] LTB₄. In optimized conditions up to 15% of total radioactivity of the incubation mixture was present in [³H] LTB ₄. A separation of [³H] LTB₄ from other labelled C_20:4 products was achieved by a three-step reverse phase-high-performance liquid chromatography in methanol- and acetonitrile-based solvent systems. [³H] LTB₄ was confirmed to be identical to the naturally occurring LTB₄ by a radioligand binding assay using a culture of HF1 cells that express a BLT₁ receptor. Copyright

Full text of DOI:10.1002/jlcr.1488

Research Article
Received 18 July 2007,
Revised 7 November 2007,
Accepted 16 November 2007
Published online 21 January 2008 in Wiley Interscience
(www.interscience.wiley.com) DOI. 10.1002/jlcr.1488
Preparation of high specific activity tritium-
labelled leukotriene B4 suitable for
radioligand binding assay
Ã
Stanislav I. Schramm,a Igor Yu. Nagaev,a Alan Sabirsh,b Valeriy P.
a
Shevchenko, Anastasiya S. Arkhipova, Jesper Z. Haeggstrom, and
a
b
¨
Nikolay F. Myasoedova
We describe a method of preparation of high specific activity tritium-labelled leukotriene (LT) B4 from [5,6,8,9,11,12,
14,15-3H] arachidonic acid (AA; 6.66 TBq/mmol) utilizing a LTB4-synthesizing enzyme system from rat basophilic leukemia
(RBL-1) cells. It was shown that both cyclooxygenase inhibitor indomethacin and adenosine 50-triphosphate induced [3H] AA
transformation to [3H] LTB4. In optimized conditions up to 15% of total radioactivity of the incubation mixture was present
in [3H] LTB4. A separation of [3H] LTB4 from other labelled C20:4 products was achieved by a three-step reverse phase-high-
performance liquid chromatography in methanol- and acetonitrile-based solvent systems. [3H] LTB4 was confirmed to be
identical to the naturally occurring LTB4 by a radioligand binding assay using a culture of HF1 cells that express a BLT1
receptor.
Keywords: [3H] leukotriene B4; enzymatic synthesis; rat basophilic leukemia (RBL-1) cells; BLT1 receptor; radioligand binding assay
LTB4 is not the only product of AA metabolism in RBL-1 cells.
Exogenous AA is also converted to the products of cyclo-
Introduction
Leukotriene (LT) B4 (5S,12R-dihydroxy-6,8,10,14-ZEEZ-eicosate-
traenoic acid) is a potent mediator of inflammation, and it is
primarily synthesized by polymorphonuclear cells (PMNs).1 It
promotes the adhesion of leukocytes to the vascular endothe-
lium and plays an important role in attracting leukocytes to
inflammatory sites.2
oxygenase and 12-lipoxygenase oxygenation, and in the
presence of reduced glutathione to the peptide LTs LTC4,
LTD4, and LTE4.8,9 LTA4 (5,6-oxido-7,9,11,14-ZZEE-eicosatetrae-
noic acid) is a common precursor for all LTs. In the absence of
L-glutathione, LTA4 is converted enzymatically to LTB4 or non-
enzymatically to dihydroxy derivatives of eicosatetraenoic acid
(diHETEs): 5,6-diHETEs, 5S,12S- and 5S,12R-dihydroxy-6,8,10,14-
EEEZ-eicosatetraenoic acid (the two latter compounds are the
6-trans isomers of LTB4)9.
At present there are on-going studies to find new inhibitors of
LTB4 synthesizing enzymes and antagonists of LTB4 receptors as
potential drugs.3 A radiolabelled LTB4 with high specific activity
is an essential tool for this work. The simplest method of
preparation of such substances is a selective hydrogenation of
14, 15-acetylene precursors with gaseous tritium.4 However, in
practice, the specific radioactivity of LTs prepared by this way
does not exceed 1.5 GBq/mol. Another method is to employ an
enzymatic transformation of tritium-labelled arachidonic acid
(AA) to produce the desired substances.5,6 This approach results
in products with considerably higher specific radioactivity.
Moreover, a number of other labelled compounds can be made
from [3H] AA using the same enzyme.5 An enzymatic method for
the preparation of [3H] LTB4 has not been described in the
literature to date.
Results and discussion
In RBL-1 cells, the activities of 5-LO and LTA4-hydrolase have
been found in 10 000g supernatant fraction derived from cellular
homogenates.9,10 Therefore, we utilized this cellular fraction for
the preparation of tritium-labelled LTB4.
aDepartment of Chemistry of Physiologically Active Compounds, Institute of
Molecular Genetics of the Russian Academy of Sciences, Kurchatov Sq. 2,
Moscow 123182, Russia
This paper reports a synthesis of high specific radioactivity
tritium-labelled LTB4 from [3H8] AA using an enzymatic system
from cultured rat basophilic leukemia (RBL-1) cells as well as
receptor-binding properties of the compound.
bDepartment of Medical Biochemistry and Biophysics, Karolinska Institutet,
S-171 77 Stockholm, Sweden
The synthesis of LTB4 from AA in RBL-1 cells comprises three
consecutive enzymatic reactions catalyzed by two enzymes: a
5-lipoxygenase (5-LO) and a LTA4-hydrolase (Scheme 1).7,8,9
*Correspondence to: Stanislav I. Schramm, Department of Chemistry, Institute of
Molecular Genetics, Kurchatov Sq. 2, Moscow 123182, Russia.
E-mail: shram@img.ras.ru
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S. I. Schramm et al.
reaches 15% of the total radioactivity. This is two or three times
as much as in the procedures described elsewhere.
An analysis of kinetics of the LTB4 synthesis showed that the
metabolism of AA and the production of 5-HETE, LTB4, and
6-trans-LTB4 isomers occurred concurrently (Figure 2). This fact
suggests that the conversion of AA to 5S-hydroperoxy-
eicosatetraenoic acid (5-HpETE) is the rate-limiting step of
LTB4 synthesis. Once synthesized 5-HpETE is then rapidly
converted to either LTA4 (enzymatically) or 5-HETE (enzymati-
cally or non-enzymatically). The high content of non-enzymatic
products of 5-HpETE and LTA4 suggests that the LTA-synthase
and LTA-hydrolase activities were insufficient in the enzyme
preparation used. Similar results have been reported early by
Jakschik and Kuo.10
HPLC analysis data on C20:4 products derived from exogenous
AA in polymorphonuclear leukocytes, RBL-1 cells, and lung
homogenate have been well described in literature.12,13
According to these publications, LTB4 and its two 6-trans
isomers can easily be separated using reverse phase (RP)-HPLC
in methanol-based solvent system, resulting in a chromato-
graphic triple peak that is typical for this reaction. In our
experiments, an enzymatically prepared tritium-labelled LTB4
and an unlabelled standard behaved in the same way in all
variants of RP-HPLC used.
It has been demonstrated that in RBL-1 cell homogenates AA
can undergo double oxygenation by 5- and 12-lipooxy-
genases.10 One of the main products of these reactions is a
5S,12S-dihydroxy-6,8,10,14-EZEZ-eicosatetraenoic acid (5,12-di-
HETE). During HPLC analysis in the methanol-based system, this
compound co-eluted with LTB4. To reveal the 5,12-diHETE
content in LTB4 fraction obtained after the first HPLC,
we analyzed it once more using an acetonitrile-based system
(Figure 3). This analysis showed that the content of 5,12-diHETE
in LTB4 fraction did not exceed 15% of the total radioactivity.
An HPLC analysis of the radioactive AA products derived from
the reaction mixture revealed
a number of unidentified
products amounting to 50% of the total radioactivity. The
larger portion of these products consists of polar substances
with shorter retention times (1.5–5.0 min) when using RP-HPLC
and a methanol-based solvent system. The proportion of these
products increased when a cellular homogenate was used
instead of 10 000g supernatants and decreased when the
incubation temperature was reduced from 37 to 301C. This
agrees well with previously published data by Jakschik et al.14
showing that the proportion of unidentified polar
products produced following the incubation of AA with
Scheme 1
Only small amounts of 5S-hydroxy-eicosatetraenoic acid calcium-free RBL-1 cell enzyme preparations reaches 55%. The
(5-HETE) were detected when enzyme preparations from RBL-1 addition of Ca21 reduced the proportion of these products
cells were incubated with labelled AA under standard conditions notably. The authors believe that these polar products may be a
(Table 1). But when cyclooxygenase inhibitor indomethacin was mixture of different phospholipids as well as unmetabolized
added to the standard reaction mixture, the total amount of radiolabelled AA.
5-LO products notably increased (Table 1). The high-perfor-
A scaled-up synthesis of [3H] LTB4 was carried out under the
mance liquid chromatography (HPLC) analysis showed that LTB4, optimal conditions determined above. [5,6,8,9,11,12,14,15-3H]
6-trans isomers of LTB4, 5,6-diHETEs, and 5-HETE are the main AA (6.66 TBq/mmol, 2.59 GBq) was used as a parent substance.
radioactive products detected (Figure 1). LTB4 content was The content of labelled LTB4 in extracts after incubation was
higher when incubations were carried out at 301C.
about 15% of the total radioactivity. The first two steps of
Since it is known that adenosine-50-triphosphate (ATP) purification (RP-HPLC in methanol-based solvent system)
stimulates the 5-LO,7,11 we also investigated the effect of this isolated LTB4 and its two 6-trans isomers. And a third RP-HPLC
compound on the LTB4 synthesis. Table 1 shows that ATP is able (in an acetonitrile-based solvent system) was used to separate
to promote the synthesis of LTB4 even in the absence of LTB4 from 5,12-diHETE. One hundred and thirty MBq of LTB4
indomethacin but ATP had a larger effect when given together with a radiochemical purity of 97% and specific radioactivity of
with a cyclooxygenase inhibitor. The yield of LTB4 in this case 6.29 TBq/mmol was obtained after the final purification. The
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S. I. Schramm et al.
Table 1. Effect of indomethacin and ATP on the content of radioactive C20:4 products formed following the incubation of [3H8]
AA with 10 000g supernatant of RBL-1 cell homogenates
Yield (%)
Ã
Additives
T (1C)
LTB4
6-trans-isomers of LTB4
5,6-diHETEs
5-HETE
AA
No additives
30
37
o0.5
o0.5
o0.5
o0.5
o0.5
o0.5
5.9
3.8
34.7
47.1
Indomethacin – 25 mM
30
37
5.8
1.9
3.7
6.3
3.7
5.1
14.1
14.5
8.3
10.2
ATP – 1 mM
30
30
2.8
12.0
8.9
4.4
7.3
12.4
6.5
17.6
11.1
Indomethacin – 25 mM
ATP – 1 mM
14.4
Ã
As it was shown in this work (see the text), the LTB4 peak obtained after the analytical reverse phase-HPLC in methanol-based
solvent system corresponds not only to the LTB4 but also to the 5,12-diHETE. The content of latter did not exceed 15% of the
total radioactivity in this fraction.
Figure 3. Separation of tritium-labelled LTB4 and 5,12-diHETE by RP-HPLC in an
acetonitrile-based solvent system. The peaks on the chromatogram correspond to:
1 – LTB4; 2 – 5,12-diHETE. RP-column – Inertsil (4.6 ꢀ 125 mm, 5 mm).
Figure 1. RP-HPLC analysis of the radioactive C20:4 products in methanol-based
solvent system. The peaks on the chromatogram are corresponded to: 1 and 2–
6-trans isomers of LTB4 ; 3 – LTB4 ; 4–5,6-diHETEs; 5–5-HETE; 6–AA. RP-column –
Kromasil 100 C18 (4 ꢀ 150 mm, 6 mm).
for [3H] LTB4 were determined to be 1.270.2 nM and
221720 fmol/mg membrane protein (Figure 4). There was a
linear correlation between the amount of a radioligand bound
and the amount of a membrane protein added. No specific [3H]
LTB4 binding to sham-transfected HF1 cells was observed. Under
similar conditions using membranes obtained from human
PMNs, [3H] LTB4 bound to LTB4 membrane binding sites with a
Kd = 1.770.7 nM, and the calculated Bmax value was 1972 fmol/
mg membrane protein (data not shown).
Using unlabelled LTB4, [3H] LTB4 could also be displaced from
HF1pBLT1-membrane binding sites with Ki = 1.4 nM (95% con-
fidence interval – from 0.9 to 2.2 nM) (Figure 5). A BLT1-binding
monoclonal antibody that has previously been shown to
15
competitively inhibit ligand binding to BLT1 could also be
used to displace [3H] LTB4 with Ki = 6.8 nM (95% confidence
interval – from 4.8 to 9.6 nM). No [3H] LTB4 displacement was
observed when using the chemically related LTs LTC4 and LTD4
or a 7B1 isotype control antibody even at concentrations up to
10 mM (data not shown).
Figure 2. Kinetics of radioactive C20:4 product formation during the incubation of
[3H8] AA with 10 000g supernatant of RBL-1 cell homogenates.
Thus, the affinity of the prepared [3H] LTB4 to BLT1 receptor
purified radioactive product had an UV spectrum typical of LTB4 agrees well with the previously published work that uses [3H]
with an absorbance maximum at 270 nm.
LTB4 from other sources16 as well as with the data obtained
Using cell membranes prepared from HF1 cells expressing using fluorescence polarisation.17 Specific binding required the
BLT1 receptors (HF1pBLT1), the Kd and Bmax (both mean7SEM) presence of BLT1 receptors and this binding could be
J. Label Compd. Radiopharm 2008, 51 101–105
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S. I. Schramm et al.
was obtained from ‘BIOMOL Res. Lab.’ (Plymouth Meeting, USA).
A BLT1 monoclonal antibody (clone 7B1) was obtained from
‘Serotec’ (Hamar, Norway). Tissue culture media and reagents
were from ‘Biolot’ (St.-Petersburg, Russia) and ‘Life Technologies’
¨
3
(Taby, Sweden). [5,6,8,9,11,12,14,15- H] AA (6.66 TBq/mmol) was
prepared as previously described.18
Cell cultures: Culture of RBL-1 cells was obtained from the
Russian Collection of Cellular Cultures (St.-Petersburg, Russia)
and cultivated as described by Jakschik et al.8 Cultures of HeLa
HF1 luciferase reporter cells were created and maintained
according to Kotarsky et al.19 HF1pSham cells and HF1pBLT1 cell
lines expressing wild-type untagged BLT1 receptors were
created by transfecting parent HF1 cells with plasmid DNAs
using Lipofectamine PLUS (‘Invitrogen’, Carlsbad, USA) as
described by the manufacturer, and stable receptor-expressing
clones were selected using puromycin.
Human PMNs were isolated from buffy coats, following
red cell sedimentation with 0.6% dextran, Lymphoprep density
gradient centrifugation (‘Axis Shield’, Olsa, Norway) and
washing.
Enzyme preparation for LTB4 synthesis: A 10 000g supernatant
of RBL-1 cell homogenates was prepared as described by
Jaskchik et al.14
Figure 4. Saturation binding of [3H] LTB4 to membranes prepared from HF1pBLT1.
Data are shown as the mean value (7SEM) from three experiments using duplicate
wells. The error bars are hidden by symbols in some cases.
Enzyme incubations and analysis of C20:4 products derived
from AA
Standard procedures for measuring the enzymatic conversion of
AA to LTB4 under different incubation conditions were as
follows. Aliquots (200 ml) of 10 000g supernatant were placed in
1.5 ml plastic microcentrifuge tubes and mixed with 250 ml of
`
reagent containing indomethacin (12.5 nmol), Ca2(NO)3 (1 mmol),
and ATP (0.5 mmol). After pre-incubation (3 min at 30oC), tritium-
labelled AA (37 MBq, 5.6 nmol) in 50 ml of a 50 mM sodium-
phosphate buffer pH 7.4, containing Na2 ꢁ EDTA (1 mM), and
gelatine (0.1%) were added to the reaction tubes and the
samples were incubated for 15 min at 301C at continuous
stirring. To stop the reaction, 1 ml of cooled methanol contain-
ing 0.1% acetic acid was added to the reaction mixture and the
samples were incubated for 5 min in an ice bath. The resulting
solutions were centrifuged and radioactive products were
extracted with 3 ml of chloroform. The organic phase was
collected and evaporated to dryness under nitrogen atmo-
sphere. The residue was dissolved in 100 ml of methanol, and
C20:4 products were analyzed by HPLC using a Kromasil 100 C18
(4 ꢀ 150 mm, 6 mm) RP column. The column was eluted with
methanol/water mobile phase containing 0.1% acetic acid at a
flow rate of 1 ml/min. The percentage of methanol in eluent
was increased linearly from 70 to 100% over 30 min. Labelled
Figure 5. [3H] LTB4 (0.5 nM) displacement from specific binding sites on HF1pBLT1
cell membranes by LTB4 and BLT1 monoclonal antibody. Data are shown as the
mean value (7SEM) from three experiments using duplicate wells.
C20:4 products were monitored in eluate with a flow radioactivity
detector.
displaced in a predictable way using either unlabelled LTB4 or an
antibody that has been previously shown to interfere with LTB4
binding to BLT1.15 Taken together these results demonstrate
that the [3H] LTB4 prepared is useful for labelling LTB4 receptors
obtained from both recombinant and ex vivo cell cultures.
Preparation of tritium-labelled LTB4
An amount of 2.59 GBq of [5,6,8,9,11,12,14,15-3H] AA in
methanol was placed into a round-bottom flask. After the
solvent was removed, 10 ml of H2O and 3 ml of 50 mM sodium-
phosphate buffer, pH 7.4, containing 1 mM Na2 ꢁ EDTA and 0.1%
gelatine, were added to the flask. The solution was thoroughly
mixed and pre-incubated for 5 min at 301C. Then water solutions
Experimental
General
Chemicals and reagents: Indomethacin, Na2 ꢁ EDTA, ATP, and 15- of ATP (10 mM, 350 ml), Ca(NO3)2 (100 mM, 700 ml), indomethacin
HETE were purchased from ‘Sigma-Aldrich’ (St. Louis, USA). LT B4 (10 mM, 88 ml), and 17 ml of 10 000g supernatant of RBL-1 cell
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S. I. Schramm et al.
homogenates were added. The resulting reaction mixture was
incubated for 15 min at 301C with constant stirring. The reaction
was stopped by adding two volumes of cooled methanol.
Undissolved components were removed by centrifugation,
and the reaction products were extracted using two volumes
of chloroform.
Acknowledgement
The authors are thankful to Dr Alexander Susan (IICH Inc.) for his
helpful discussions and valuable remarks concerning this work.
References
The chloroform–methanol solution was evaporated under
vacuum. The remaining dry substance was dissolved in 1 ml of
starting solvent for the first HPLC, and the sample was injected
into the preparative Kromasil 100 C18 (8 ꢀ 150 mm, 7 mm) RP
column. The column was eluted using a methanol/water mobile
phase containing 0.1% acetic acid at a flow rate of 2 ml/min. The
percentage of methanol in the eluent was increased linearly
from 70 to 100% over 30 min. The fractions with [3H] LTB4 were
combined before undergoing the second HPLC purification on
an analytical Kromasil 100 C18 (4 ꢀ 150 mm, 6 mm) RP column as
defined above. To separate tritium-labelled LTB4 from 5,12-
diHETE, the final purification of [3H] LTB4 was performed using
an Inertsil (4.6 ꢀ 125 mm, 5 mm) RP column, which was eluted
with acetonitrile/water mobile phase containing 0.1% acetic acid
at a flow rate of 1 ml/min. A 30 min linear gradient of acetonitrile
(70–100%) was used.
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Radioligand binding assays
Membranes from the sham-transfected HF1 cells, the HF1pBLT1
cell line, and from purified human PMNs were prepared as
previously described.16 Radioligand binding assays were per-
formed as follows. Radiolabelled LTB4 was added to 1.0 mg
resuspended cell membranes to a final concentration of 0.5 nM,
and incubated for 1 h in opaque white 96-well filter plates
MAFC-NOB with FC glass fiber filters (‘Millipore’, Bedford, USA).
Unlabelled LTB4 was added as necessary to determine the non-
specific binding. The reaction was terminated by rapid filtration
and the filters were then washed, dried, and 25 ml of Microscint-
O (‘Packard Instrument Co.’, Meriden, USA) was added to each
well. Measurement of radioactivity in plates was evaluated by
using a Micro Beta scintillation counter (‘EG&G Wallac’, Turku,
Finland). Statistical analysis was performed using Prism, by
GraphPad Software.
J. Label Compd. Radiopharm 2008, 51 101–105
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