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Analytical Biochemistry 294, 73– 82 (2001)

doi:10.1006/abio.2001.5015, available online at on

Simultaneous Measurement of Anandamide and

2-Arachidonoylglycerol by Polymyxin B-Selective Adsorption
and Subsequent High-Performance Liquid Chromatography
Analysis: Increase in Endogenous Cannabinoids
in the Sera of Patients with Endotoxic Shock
Yin Wang,* Yan Liu,* Yasuhiko Ito,† Teruto Hashiguchi,* Isao Kitajima,* Munekazu Yamakuchi,*
Hideaki Shimizu,† Seiichi Matsuo,‡ Hitoshi Imaizumi,§ and Ikuro Maruyama* ,1
*Department of Laboratory and Molecular Medicine, Kagoshima University School of Medicine, Kagoshima, Japan;
†Chubu Rousai Hospital, Japan; ‡The Third Department of Internal Medicine, Nagoya University School of Medicine,
Nagoya, Japan; and §Intensive Care Unit, Sapporo Medical College, Sapporo, Japan

Received July 13, 2000; published online May 24, 2001

the role of the endogenous cannabinoids in the hypo-

Anandamide (ANA) and 2-arachidonoylglycerol (2- tension of human endotoxic shock. This method is
AG), two endogenous cannabinoids, can be generated rapid, sensitive, and reliable for simultaneously quan-
by activated macrophages and platelets, respectively, tifying ANA and 2-AG in biological fluids, and has po-
in the context of endotoxic shock, and are proposed to tential for clinical usage. © 2001 Academic Press
play a crucial role in the induction of the shock-re- Key Words: anandamide; 2-arachidonoylglycerol;
lated hypotension. Taking advantage of our recently polymyxin B; endotoxic shock; reverse-phase high-
discovered function of polymyxin B (PMB) binding to performance liquid chromatography.
ANA and 2-AG, we developed a new method for mea-
suring ANA and 2-AG by applying PMB-immobilized
beads to selectively adsorb them in biological fluids,
instead of organic solvent extraction. The eluate from Anandamide (ANA) 2 was initially isolated from the
beads can be directly fractionated by reverse-phase porcine brain (1) and thereafter found to exist not only
high-performance liquid chromatography (HPLC), in the mammalian brain but also in various peripheral
and the fractionations corresponding to authentic tissues (2). 2-Arachidonoylglycerol (2-AG) was initially
ANA and 2-AG are collected and derivatized with flu- isolated from canine gut (3) and, like ANA, was also
orogenic reagent and subsequently quantified by found in the central nervous system (4). These two
HPLC with fluorometric detection. The calibration molecules have been shown to exert their diverse bio-
graphs of ANA and 2-AG were linear over a range of 1 logical functions through cannabinoid receptors, CB1
to 500 pmol/ml. The limits of detection for ANA and and CB2, and are therefore defined as endogenous
2-AG were 20 and 50 fmol, respectively. Intraassay pre- cannabinoids (5, 6). The typical pharmacological effects
cision was 2.24 – 4.25 and 3.47–5.44%, and interassay of ANA and 2-AG similar to exogenous cannabinoids in
was 4.05– 6.14 and 4.92–7.28% for ANA and 2-AG, respec-
central nervous system include disruption of pain,
tively. Using this method, we first determined a 4-fold
and 3-fold higher level of ANA and 2-AG, respectively,
in the sera of patients with endotoxic shock than in 2
Abbreviations used: ANA, anandamide; 2-AG, 2-arachidonoyl-
normal serum. This finding should help in elucidating glycerol; LPS, lipopolysaccharide; PMB, polymyxin B; HPLC,
high-performance liquid chromatography; GC/MS, gas chromatogra-
phy/mass spectrometry; DBD-COCl, 4-(N-chloroformylmethyl-N-meth-
To whom correspondence should be addressed at Department of yl)amino-7-N,N-dimethylaminosulphonyl-2,1,3-benzoxadiazol; BSTFA,
Laboratory and Molecular Medicine, Kagoshima University School of bis(trimethylsilyl)trifluoroacetamide; PMSF, phenylmethylsulfonyl-
Medicine, 8-35-1 Sakuragaoka, Kagoshima City 890-8520, Japan. fluoride; TLC, thin-layer chromatography; DMEM, Dulbecco’s modified
Fax: ⫹81 (99) 275-2629. E-mail: rinken1@khosp2.kufm.kagoshima- Eagle’s medium; FBS, fetal bovine serum; RP, reverse phase; TMS, trimethysilyether.

0003-2697/01 $35.00 73
Copyright © 2001 by Academic Press
All rights of reproduction in any form reserved.

memory formation, and motor coordination, all of at high affinity (20). This observation prompted us to
which depend on the N-methyl-D-aspartate receptor- develop a rapid method using PMB-immobilized beads
mediated excitatory neurotransmission (7, 8). Besides selectively to adsorb ANA and 2-AG, instead of organic
these actions, ANA and 2-AG have recently been pro- extraction, for quantifying ANA and 2-AG. The use of
posed to exert cardiovascular actions as a contributor PMB-immobilized beads to adsorb ANA and 2-AG is
to endotoxic and hemorrhagic shock-induced hypoten- roughly analogous to antigen- or antibody-conjugated
sion (9 –11), and ANA induces vasodilation through agarose immunoprecipitation, which has been univer-
vanilloid receptors (12). The conclusive evidence con- sally and conveniently applied to molecular biochemi-
sists mainly of the findings that ANA can be generated cal investigation. This is a greatly simplified and
by the brain during pain stimulation (7) and by the highly reproducible method for the simultaneous mea-
activated macrophages during endotoxic and hemor- surement of ANA and 2-AG. The increased content in
rhagic shock, and that 2-AG is generated by activated these endogenous cannabinoids in the sera of some
platelets during endotoxic shock (9 –11). Therefore, in- patients with endotoxic shock further supported the
creasing studies aimed at clarifying the physiological notion, established on the data obtained from a rat
and pathological roles of ANA and 2-AG in vivo can be model, that ANA and 2-AG are endogenous mediators
anticipated. To this end, it is expected that a rapid, of endotoxic shock-induced hypotension.
sensitive, and reliable quantitative method to measure
ANA and 2-AG in clinical samples will be established. MATERIALS AND METHODS
Quantitative measurements of ANA and 2-AG in
biological samples are often hindered by the low con-
centrations of these mediators. At present, several ANA and lipopolysaccharide (LPS, from Esche-
methods are applicable to the quantitative measure- richia coli strain 055:B5) were purchased from
ment of ANA or 2-AG in biological samples. All of the Sigma Chemical Co. (St. Louis, MO); 2-AG from Cal-
methods for measuring ANA or 2-AG in biological tis- biochem-Novabiochem Co. (La Jolla, CA), 4-(N-
sues or fluids include organic solvent extraction at the chloroformylmethyl-N-methyl)amino-7-N,N-dimeth-
first step. The obtained extracts must be prepurified by ylaminosulphonyl-2,1,3-benzoxadiazol (DBD-COCl)
thin-layer chromatography (TLC) (1, 13), C-18 car- from Tokyo Kasei Kogyo Co., Ltd. (Tokyo, Japan);
tridges (14), silica gel column chromatography (15), or bis(trimethylsilyl)trifluoroacetamide (BSTFA), phe-
high-performance liquid chromatography (HPLC) (11) nylmethylsulfonyl fluoride (PMSF) from Wako Pure
to remove the potentially interfering substances in the Chem. Ind. (Osaka, Japan); and Affi-Prep polymyxin
extracts before further analysis by HPLC, or by liquid matrix from Bio-Rad Laboratories (Hercules, CA).
or gas chromatography/mass spectrometry (GC/MS). Silica gel K6 TLC plates (250 ␮m) were from What-
The organic solvent system used for the extraction is man (Clifton, NJ), and anhydrate benzene, acetoni-
generally a mixture of chloroform and methanol (16, trile, and water were of HPLC grade. All other re-
17), although in some modified methods these solvents agents used were of analytical grade.
are replaced by ethyl acetate or toluene (14, 18).
Schmid et al. used acetone precipitation to remove the Cell Cultures and Stimulation
proteins in analyzed samples before extraction, rather
Mouse macrophage-like RAW264.7 cells were ob-
than prepurifying the extracts; after the acetone pre-
tained from the American Type Culture Collection
cipitation, the samples could be directly subjected to
(Manassas, VA) and maintained in Dulbecco’s modified
HPLC fractionation (19). By using these methods, ANA
Eagle’s medium (DMEM) supplemented with 10% fetal
and 2-AG have been measured in the mammalian
bovine serum (FBS) and 1% each of penicillin and
brain (14, 16, 18, 20), mouse peritoneal macrophages
streptomycin. The cells were passaged in a 100-mm
and uterus (21), and rat blood (19). The recovery of
plastic uncoated dish overnight. Then, the cells were
ANA or 2-AG described in the above-noted procedures
treated with 100 ␮g/ml LPS or left untreated in 3 ml of
varied from 50 to 80%. In preparation for the present
fresh DMEM medium containing 1% FBS and 200 ␮M
experiment, we repeated some of these methods and
of PMSF. Two hours later, the medium was collected
found that it was difficult to obtain a recovery over 70%
and supplemented with 500 ␮l of 50% Affi-Prep PMB
by a procedure utilizing organic extraction, TLC pre-
suspension for the adsorption of the released ANA. The
purification, HPLC fractionation, and GC/MS analysis,
adsorption was then assayed as described below.
since this long and complicated procedure resulted in
the loss of the ANA and 2-AG to be measured.
Very recently, we demonstrated that polymyxin B PMB-Immobilized Beads Adsorption Assay
(PMB), which is formed from a cyclic heptapeptide The various amounts of ANA and 2-AG were seeded
moiety linked to a peptide side chain that terminates in different tubes with 1 ml serum containing 1 mM
with a short fatty acid residue, adsorbs ANA and 2-AG PMSF to prevent their breakdown. After diluting with

2 ml saline, 500 ␮l of 50% Affi-Prep PMB suspension v/v) at a flow rate of 1 ml/min. Absorbance was moni-
was added for the adsorption process and shaken for tored at 204 nm. The dried eluate from the PMB-
1 h at 4°C. To measure the recovery of ANA in DMEM immobilized beads column was dissolved in absolute
medium containing 10% FBS, 500 ␮l of the PMB sus- ethanol and fractionated by reverse-phase HPLC (RP-
pension was added to 3 ml of this medium. After ad- HPLC). The fractions corresponding to the elution time
sorption the PMB-immobilized beads were pelleted by of authentic ANA (6.95 min) and authentic 2-AG (8.01
centrifuge, washed twice with 2 ml of saline, trans- min) were pooled and dried under nitrogen for deriva-
ferred to a minichromatography column (0.5 ⫻ 3 cm), tization.
and centrifuged a final time to remove the remaining
saline on the beads. ANA and 2-AG on the beads were Derivatization and HPLC Analysis with
eluted twice with 200 ␮l of absolute ethanol. The elu- Fluorometric Detection
ates were dried in a centrifugal concentrator and re-
constituted in absolute ethanol for HPLC fraction- The fractions corresponding to the elution time of
ation. authentic ANA and 2-AG were dissolved in anhydrous
benzene. ANA and 2-AG were derivatized according to
the method of Koga et al. (22) with some modifications.
Adsorption of ANA and 2-AG in Serum Samples Briefly, 3 ␮l of saturated DBD-COCl in anhydrous ben-
The serum (3 ml) from the patients with endotoxic zene was added to 30 ␮l of ANA-anhydrous benzene
shock was collected, and PMSF was added to reach the solution. The mixture was then heated at 60°C for 40
concentration of 1 mM for the inhibition of the hydro- min (Fig. 1). The reaction was stopped by addition of 30
lysis of ANA and 2-AG. Following the dilution of the ␮l of benzene containing 1% acetic acid. The resulting
serum with 2 vol of saline, 1.5 ml of 50% Affi-Prep solution was dried by a centrifugal concentrator and
Polymyxin B was added for the adsorption process, reconstituted in 40 ␮l of acetonitrile, and then a 5-␮l
which was performed under shaking for 1 h at 4°C. The aliquot of the solution was subjected to RP-HPLC anal-
following steps are the same as those in the above- ysis. HPLC with fluorometric detection was carried out
described procedure. using the same system as described above for fraction-
ation of RP-HPLC, but with a S-3370 fluorescence de-
Extraction and TLC Purification of ANA tector (Soma Optics, Ltd., Tokyo, Japan). The separa-
in Cell Culture Medium and Serum tion was performed isocratically with a mobile phase of
acetonitrile: water (8:2, v/v) at a flow rate of 1.0 ml/min.
The various amounts of ANA were seeded in differ- The fluorometric detection was performed at 560 nm
ent tubes containing 3 ml of DMEM medium with 10% with excitation at 450 nm (22). The concentrations of
FBS or 3 ml of saline with 30% of human serum; PMSF ANA and 2-AG in biological fluids were quantified by
was immediately added to each solution to prevent the comparison with calibration curves constructed using
breakdown of ANA. Chloroform and methanol were synthetic ANA and 2-AG.
directly added to achieve an extraction system with a
chloroform:methanol:water ratio of 8:4:3, v/v/v. The or-
ganic phases were recovered, evaporated to dryness Calibration Curve
under nitrogen gas, reconstituted in 50 ␮l of chloro- Varied amounts of synthetic ANA and 2-AG were
form:methanol (85:15, v/v), and applied to a TLC plate, added to individual normal human serum samples.
which was then developed under a chloroform:hexane: Each sample was then adsorbed with PMB-immobi-
methanol ratio of 40:20:5, v/v/v. The area correspond- lized beads, purified by RP-HPLC, and converted to the
ing to authentic ANA was scraped off the TLC plate, DBD-COCl derivative. To each sample in the standard
eluted with chloroform:methanol (85:15, v/v), dried un- curve set, varied amounts of derivatives of ANA (1, 3.6,
der nitrogen gas, and fractionated by HPLC using the 18, 36, 180, 360, 500 pmol) and 2-AG (1, 3.3, 16.5, 33,
same procedure as that used for fractionating the dried 165, 330, 500 pmol) were subjected to HPLC with flu-
eluate from the PMB-immobilized beads. orometric detection, respectively.

HPLC Fractionation Precision and Accuracy

The method employed for HPLC fractionation was The sample measurement was validated by spiking
modified from that of Lang et al. (21). Briefly, a Gilson serum samples with a known mixture of the analyzed
System (Gilson Inc., Middleton, WI) equipped with a compounds, ANA and 2-AG. The particular sample of
305 pump and a 118 UV/Vis detector was used. Sepa- sera used for this purpose had previously been shown
ration was carried out on a TSK gel ODS 80TM (50 ⫻ not to contain any of these analytes. The spiked sera
4.6-mm i.d. ⫻ 5 ␮m) column (TOSOH, Tokyo, Japan) samples were measured according to the above-de-
with an elution solution of acetonitrile and water (8:2, scribed procedures. The accuracy was determined by

FIG. 1. Structure and derivatization scheme for ANA and 2-AG. ANA and 2-AG were derivatized with DBD-COCl in dehydrated benzene
at 60°C for 40 min to form the DBD-CO-ANA and DBD-CO-2-AG derivatives, respectively.

comparing the results from analyses of spiked serum the beads into a 10% FBS medium or diluted human
samples with results from analyses of standard sam- serum containing various amounts of ANA or 2-AG.
ples of known composition. The precision of this system After washing with saline, the beads were eluted with
was evaluated by analyzing the same serum samples absolute ethanol. The eluate was subjected to RP-
four consecutive times on 4 successive days. HPLC for fractionation. In our analytic system, the
retention times for standard ANA and 2-AG were 6.95
GC/MS Analysis and 8.01 min, respectively (Fig. 2A). The selective ad-
sorption of the PMB-immobilized beads to ANA and
The fractions from fractionating HPLC correspond- 2-AG from the diluted serum allowed them to avoid
ing to the elution time of authentic ANA or 2-AG were intense interference by the large amounts of other lip-
dried and derivatized with BSTFA for 1 h at 70°C. The
trimethylsilylether (TMS) derivatives were dried
again, reconstituted in n-hexane, and injected in the
splitless mode into a gas chromatograph (GC; Hewlett-
Packard 5890A) equipped with an HP-5 capillary col-
umn (30 m ⫻ 0.25 mm ⫻ 0.33 ␮m) and interfaced with
a mass spectrometer (Hewlett-Packard 5988A). One
minute after injection, the oven temperature was in-
creased from 150° to 280°C at a rate of 8°C/min. He-
lium was used as a carrier gas. The injector and detec-
tor temperatures were maintained at 250°C and the
source temperature was maintained at 230°C. The ion
energy was 70 eV. Under these conditions, the TMS
derivatives of ANA and 2-AG were eluted from the GC
column at around 17 min after injection.

PMB-Immobilized Beads Selectively Adsorbed ANA
and 2-AG in Normal Individual and
FIG. 2. Chromatogram of prepurification of ANA and 2-AG in
Shock-Patients Sera eluent from a PMB-immobilized beads column. (A) Chromatogram
We have recently shown that PMB-immobilized showing baseline separation and retention times for authentic ANA
beads efficiently adsorb ANA and 2-AG (20). This ob- (6.95 min) and 2-AG (8.01 min). (B) Representative chromatograms
of the eluate from PMB-immobilized beads that were employed to
servation raised the possibility that PMB-immobilized adsorb ANA and 2-AG in the sera of patients with endotoxic shock.
beads might be utilized for the measurement of ANA Chromatographic conditions and the sample preparation are given
and 2-AG. To verify this hypothesis, we directly added under Materials and Methods.

50% serum; sharp declination in that containing more

than 70% serum; and the lowest point of the declina-
tion (about 50% recovery at 1.44 nmol/ml of ANA
added) in a full serum adsorption system. Even then, if
we dilute serum with 2 vol of saline, a more than 70%
recovery of ANA could still be obtained in an adsorp-
tion system containing 1.44 nmol/ml of ANA (Fig. 3).
Therefore, the adsorption procedure of PMB-immobi-
lized beads to ANA exercised in practical measurement
is better performed in an adsorption system containing
less than 40% serum. The lower adsorption efficiency of
PMB-immobilized beads to ANA in full serum seemed
attributable to nonselective adsorption of PMB beads
FIG. 3. The effect of human serum on the capacity of PMB-immo-
bilized beads to adsorb ANA. PMB-immobilized beads were added to to undesired substances in full serum. Thus the diluted
ANA solution with increasing levels of human serum as indicated. serum naturally attenuated the nonselective adsorp-
After shaking for 1 h at 4°C, the beads were washed by saline. ANA tion and enhanced the selective adsorption of PMB
adsorbed on the beads was eluted twice with 200 ␮l of absolute beads to ANA.
ethanol and total contents in the collected eluates were determined
by the following HPLC procedure. The recovery of ANA was ex-
pressed as the ratio of total ANA in the eluate of absolute ethanol to Comparison of the ANA Recovery Measured by Two
the content of ANA prior to loading. Methods, the Adsorption Method and the
Conventional Extraction Method
Most cells are commonly cultured in a medium con-
ids and proteins in the serum, resulting in a clear taining 10% FBS. To extend the application of this
separation between eluates by HPLC (data not shown). adsorption procedure to the measurement of ANA re-
We next examined the adsorption action of PMB- leased by elicited cells into culture medium, we exam-
immobilized beads to endogenous ANA or 2-AG. It has ined and compared the recovery of exogenous ANA in
been reported that LPS-activated macrophages and 10% FBS of DMEM medium or in diluted human se-
platelets generated ANA and 2-AG in vitro, respec- rum by two methods, the adsorption method and the
tively, indicating the possibility of their generation in conventional extraction-TLC method. In terms of the
vivo during endotoxemia. To investigate this possibil- DMEM medium containing 10% FBS, the adsorption
ity, blood was collected from patients with endotoxic procedure rendered higher recovery of ANA and lower
shock, and the obtained serum was subjected to anal- mean deviation of measurement than the extraction-
ysis by the adsorption procedure. The results demon- TLC method (Table 1). Although the high concentra-
strated that the selective adsorption of endogeneous tion serum present in the adsorption system affected
cannabinoids was reproducible in the sera of patients,
and that it resulted in a clear separation between ANA
and 2-AG (Fig. 2B). TABLE 1
Comparison of the Recovery of ANA between the
Effect of Serum Concentration on the Adsorption Extraction-TLC and Adsorption Methods
Efficiency of PMB-Immobilized Beads to ANA and
2-AG in the Adsorption Assay Method Recovery of ANA

It has been described that the adsorbing capacity of Added ANA (nmol/ml) Extraction-TLC Adsorption
LPS by PMB is markedly reduced in the presence of
serum because LPS also binds to LPS-binding protein. DMEM with 10% FBS
0.144 52.6 ⫾ 15.2 68.5 ⫾ 5.8
We therefore examined the effect of serum on the ad- 0.576 55.6 ⫾ 14.7 73.3 ⫾ 6.8
sorption efficiency of PMB-immobilized beads to ANA 1.44 57.8 ⫾ 18.6 78.9 ⫾ 8.2
and 2-AG. As shown in Fig. 3, when less than 30%
30% human serum
serum was used in the constituted adsorption system 0.144 48.6 ⫾ 11.5 62.3 ⫾ 9.0
of PMB-immobilized beads with ANA, the selective 0.576 50.3 ⫾ 12.5 68.6 ⫾ 10.4
adsorption was slightly decreased, based on the mea- 1.44 54.8 ⫾ 15.2 71.2 ⫾ 11.5
surement of the cumulative recovery. However, the
adsorption efficiency rate declined with increasing per- Note. The indicated amounts of ANA were added to a 10% FBS
DMEM medium or to saline containing 30% human serum. The
centage of serum. Slow declination of the efficiency, to recovery of ANA was determined as described under Materials and
higher amount of ANA (more than 0.576 nmol/ml), is Methods. The results are shown as mean ⫾ SE of three separate
observed in a adsorption system containing less than experiments.

can be directly subjected to HPLC for fractionation.

The fraction corresponding to authentic ANA and 2-AG
can be directly derivatized with DBD-COCl. The peaks
of the two derivatives were clearly separated from each
other and from the interfering peaks derived from the
other lipids or proteins. The fraction around the elution
time of ANA and 2-AG pooled from fractionating HPLC
of the sample from the serum of patient with endotoxic
shock (Fig. 2B) was dried, derivatized with DBD-COCl,
and separated by the HPLC with fluorometric detec-
tion Fig. 4B.

Calibration Curve, Accuracy, and Precision

of the Method
A good linear relationship of fluoresence intensity to
concentration of ANA or 2-AG was obtained over a
concentration range of 1 to 500 pmol. For ANA, the
FIG. 4. HPLC analysis of ANA and 2-AG in the sera of patients regression line was Y ⫽ 6733.209x ⫺ 3297.755 with a
with endotoxic shock with fluorometric detection. (A) Chromatogram correlation coefficient of 1.000 and for 2-AG, the regres-
showing baseline separation and retention times for authentic ANA sion line was Y ⫽ 3817.948x ⫺ 1989.450 with a corre-
(11.94 min) and 2-AG (16.05 min) derivatized with DBD-COCl. The
peak corresponds to 5 pmol of each compound. (B) Representative lation coefficient of 1.00, respectively. The detection
chromatogram of the sample from sera of patients with endotoxic limits for ANA and 2-AG were 20 and 50 fmol, respec-
shock. The fractions corresponding to the elution time of authentic tively.
ANA and 2-AG were derivatized with DBD-COCl and subjected to The precision and accuracy of the assays were as-
HPLC analysis with a fluorometric detector. Chromatographic con-
ditions and methods for the derivatization are given under Materials sessed by the intraassay and interassay variability and
and Methods. the percentage deviation from the nominal concentra-
tions, respectively. The intraassay variabilities for
ANA, assessed on 4 consecutive working days, ranged
the selective adsorption of PMB-immobilized beads to from 2.24 to 4.25%, and for 2-AG ranged from 3.47 to
ANA (Fig. 3), the adsorption procedure still imparted a 5.44% (Table 2). The interassay variabilities for ANA,
higher recovery of ANA than the extraction-TLC pro- assessed on 4 consecutive working days, ranged from
cedure when 30% human serum was added to the con- 4.05 to 6.14%, and for 2-AG ranged from 4.92 to 7.28%
stituted system (Table 1). In this case, moreover, the (Table 2). The mean percentage deviations from the
mean deviation of ANA was also lower than that of the nominal concentration were ⫺0.12 to ⫺1.5% for ANA,
extraction-TLC procedure. The recovery of 2-AG was and ⫺0.18 to ⫺0.84% for 2-AG, respectively.
similar to that of ANA (data not shown). These results
demonstrated that PMB-immobilized beads efficiently
and selectively adsorbed ANA and 2-AG in culture GC/MS Analysis
medium containing 10% FBS, and that the adsorption GC/MS analysis was also used to identify ANA and
method can be applied for the measurement of ANA 2-AG present in the conditioned medium from LPS-
and 2-AG released by elicited cells into culture me- stimulated RAW264.7 cells and in human serum from
dium. normal or endotoxin-shock blood. Figure 5 shows a
representative GC/MS chromatogram of TMS deriva-
Derivatization of ANA and 2-AG and HPLC Analysis tives of ANA and 2-AG derived from human serum of
with Fluorometric Detection endotoxin-shock blood. Figure 5A is a representative
It has been reported that ANA can be sensitively ion recording of ANA and 2-AG of this sample after
determined by HPLC with fluorometric detection after capillary GC separation. We observed components that
derivatization of the hydroxyl group with DBD-COCl were eluted from the GC at the retention times ex-
(22). We found that 2-AG can also be easily derivatized pected for ANA and 2-AG (Fig. 5A and data not shown).
with DBD-COCl. Each derivative afforded a single Diagnostic fragments of ANA were found in the high
peak, and DBD-CO-ANA and DBD-CO-2-AG were mass range in Fig. 5B. They included molecular ions
eluted at 11.95 and 16.04 min, respectively (Fig. 4A). ([M] ⫹) as well as ions produced by the loss of one
The eluate of samples from PMB-immobilized beads methyl group ([M–15] ⫹). Additional informative and

HPLC Validation Characteristics of ANA and 2-AG in Human Sera

Variability (%)
Nominal Observed Deviation
(pmol/ml) (pmol/ml) (%) Intraassay Interassay n

1 0.985 ⫺1.5 4.25 6.14 4
20 19.85 ⫺0.75 3.61 4.05 4
150 146.3 ⫺0.25 4.18 5.01 4
350 345.8 ⫺0.12 2.24 4.22 4
1 0.948 ⫺0.52 5.44 7.28 4
20 18.32 ⫺0.84 4.66 5.91 4
150 145.1 ⫺0.33 4.5 6.63 4
350 343.7 ⫺0.18 3.47 4.92 4

prominent fragments were [M– 43] ⫹ and [M–91] ⫹, solvent extraction with prepurification. In other words,
which may be produced by the loss of one propyl group the selective adsorption process of PMB-immobilized
and TMSOH group, respectively. The electron impact beads to ANA and 2-AG simultaneously provides a
mass spectra of TMS derivatives of 2-AG are shown in clean-up step, which is necessary for HPLC or GC/MS
Fig. 5C (m/z 522 for [M] ⫹, molecular ion; m/z 507 for analyses. However, the selective adsorption action of
[M–15] ⫹, loss of methyl group; m/z 451 for [M–71] ⫹, PMB-immobilized beads to ANA and 2-AG is affected
loss of pentyl group; m/z 432 for [M–90] ⫹, loss of trim- by the presence of a high concentration of serum (more
ethylsilenol; m/z 218 for [M–RCOOH] ⫹). than 50%) (Fig. 3). We diluted the serum with 2 vol of
saline for the adsorption action, resulting in a higher
DISCUSSION recovery rate of ANA and higher reproducibility of results
than by conventional extraction methods (Table 1).
The first step in most previous methods for measur- After the selective adsorption of PMB-immobilized
ing ANA and 2-AG includes lipid extration with or-
beads to ANA and 2-AG, the eluate from the PMB-
ganic solvent. The organic solvent extraction is neither
immobilized beads column can be directly subjected to
directly subjected to HPLC analysis nor directly deri-
HPLC for fractionation. The fraction around the reten-
vatized for GC/MS analysis, since there are many po-
tion time of authentic ANA and 2-AG can be directly
tential contaminants, including large amounts of un-
derivatized for further analyses by HPLC or GC/MS. In
desired lipids in the extracts. Thus, additional
the HPLC fractionation procedure, we modified the
prepurification, such as that by TLC, C-18 catridge,
silica gel column chromatography, or HPLC is indis- eluents from acetonitrile and 8.5% aqueous phosphoric
pensable. The total recovery of ANA or 2-AG by the acid in a ratio of 9:1, v/v, to acetonitrile and water in a
chloroform/methanol extraction plus TLC prepurifica- ratio of 8:2, v/v. This modification unified the elution
tion is around 50%, as assessed from the extraction system of fractionated HPLC with that of analyzed
recovery (83.2 ⫾ 7.8%) discounted by the prepurifica- HPLC with fluorometric detection. More importantly,
tion recovery (50 –70%) (13). In addition to their low this modification eliminated the interference of phos-
recovery rates and poor reproducibility, these proce- phoric acid with the derivatization for ANA and 2-AG,
dures are costly in terms of time and reagents. Taking and allowed the collected fraction to be directly deri-
advantage of our recent observation that PMB-immo- vatized by either DBD-COCl for HPLC analysis or
bilized beads efficiently and selectively adsorb ANA TMS for GC/MS analysis. The derivatizing reaction of
and 2-AG in saline or serum, we applied PMB-immo- ANA and 2-AG by DBD-COCl dye in the original
bilized beads to selectively adsorb ANA and 2-AG from method was performed in a reaction system to which a
a cell-cultured medium or from diluted serum rather largely excess amount of the dye was added for a com-
than using organic solvent extraction. By greatly sim- plete derivatization of ANA and 2-AG. However, the
plifying the processing of samples, this substitution led excess free dye intensely interfered with the fraction-
to a high rate of recovery and reproducibility of mea- ation of ANA and 2-AG by the HPLC. The fluorescent
surement of ANA and 2-AG. The selective adsorption derivatives must be diluted at a proportion of 1:1000,
process of PMB-immobilized beads to ANA and 2-AG and only one one-thousandth of the sample was in-
virtually corresponds to the combination of the first jected for the HPLC analysis. We reduced the amount
two steps in the conventional procedure, i.e., organic of reactive dye to an extent to which the labeling reac-

FIG. 5. GC/MS analysis of ANA and 2-AG in the samples of sera from patients with endotoxic shock. (A) Representative ion recording of
the samples of sera of patients with endotoxic shock showing the presence of components with the chromatographic properties of ANA or
2-AG in samples, corresponding to the ones of authentic standard (data not shown). Results are from one experiment, representative of five.
(B) Electron-impact mass spectra of TMS derivatives of ANA obtained from the samples of the sera from patients with endotoxic shock. (C)
Those of TMS derivatives of 2-AG.

tion is complete enough, as compared with the original whether for fractionation of HPLC or analysis of HPLC
method (data not shown). As a result, one-eighth of the with fluorometric detection, were unified. Thus the
total sample in our labeling method was injected for method presented here provides a very convenient so-
HPLC analysis. These changes in the labeling reaction lution, i.e., simply switching the UV detector to the
resulted in a 125-fold increase in the amount of sam- fluorometric detector. It is worthy of note that the
ples for subjects, in addition to increasing the sensitiv- measurement for ANA and 2-AG can be performed
ity for detection of ANA and 2-AG. simultaneously in all procedures.
Each derivative of ANA and 2-AG afforded a clear GC/MS analysis can best provide unambiguous iden-
single peak. DBD-CO-ANA and DBD-CO-2-AG were tification of ANA and 2-AG in biological samples. We
eluted at 11.95 and 16.04 min, respectively. The peaks carried out GC/MS analysis of ANA and 2-AG by de-
of the derivatives were clearly separated from each rivatizing the fraction collected from fractionating
other and from the interfering peaks derived from the HPLC with BSTFA. By GC/MS, we measured the con-
other lipids and contaminants. Moreover, the column tent of ANA in the culture medium of LPS-stimulated
conditions and elution systems in our procedure, RAW264.7 cells and the contents of ANA and 2-AG in

respectively. In contrast, approximately 4.0 pmol/ml of

ANA and 10 pmol/ml of 2-AG were detected in normal
human serum (Fig. 6). The increased amounts of ANA
and 2-AG in the sera of the patients with endotoxic
shock further supported the notion, established on
data obtained from a rat model, that ANA and 2-AG
are endogenous mediators of shock-induced hypoten-
sion. Therefore, the removal of endogenous ANA from
the circulation of shock patients by selective adsorption
of PMB-immobilized fiber hemoperfusion column may
contribute to the improvement of endotoxin-induced
hypotension. All of these investigations imply the im-
portance of our rapid-detection method for assaying
ANA and 2-AG in blood, cerebral fluid, synovial fluid,
FIG. 6. Contents of ANA and 2-AG in the sera from health indi-
and cell-culture media in both clinical and scientific
viduals (n ⫽ 5) and from the patients with endotoxic shock (n ⫽ 5), research.
and in conditional medium of LPS-stimulated RAW264.7 cells.
RAW264.7 cells were passaged overnight and then switched to a 3 ml ACKNOWLEDGMENT
of fresh medium containing 10% FBS before the treatment with 100
␮g/ml of LPS for 2 h. PMB-immobilized beads were added to the sera We thank Toray Chemical Company for supplying the polymyxin
from normal donors or patients with endotoxic shock after dilution B-immobilized beads.
those sera with 2 vol of saline, or to the 3 ml of collected medium of
LPS-stimulated RAW264.7 cells. The endogenous ANA and 2-AG
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