Clinical and Diagnostic Laboratory Immunology, May 1998, p. 303-307, Vol. 5, No. 3
1071-412X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Development and Evaluation of a Chromatographic
Procedure for Partial Purification of Substance P with Quantitation
by an Enzyme Immunoassay
William P.
Fehder,1,
Wen-Zhe
Ho,2,3
Donald E.
Campbell,1,2,3
Wallace W.
Tourtellotte,4
Lisa
Michaels,1
Joann R.
Cutilli,2
Marina
Uvaydova,2 and
Steven D.
Douglas1,2,3,*
Division of Immunologic and Infectious
Diseases, Department of Pediatrics, University of Pennsylvania
School of Medicine,1
Joseph Stokes Jr.
Research Institute,2 and
Clinical
Immunology Laboratories, Children's Hospital of
Philadelphia,3 Philadelphia, Pennsylvania, and
Multiple Sclerosis Human Neurospecimen Bank, Veterans
Affairs Medical Center, Los Angeles, California4
Received 15 October 1997/Returned for modification 15 December
1997/Accepted 21 January 1998
 |
ABSTRACT |
We have developed a simple chromatographic procedure for the
partial purification of substance P (SP) from acidified plasma and
serum samples. We have evaluated a sensitive antigen competition enzyme
immunoassay (EIA) for the quantitation of SP. The chromatographic procedure has recovery efficiencies ranging from 94.8 to 125%. The
immunoreactivity of unknown amounts of purified SP subjected to the
preparative procedure yielded a coefficient of variance of 9.4%. The
EIA yielded reproducible standard curves having an interassay
(n = 8) correlation coefficient of 0.984. The
evaluation of normal adult control serum yielded a mean value of 51 pg/ml (range, 35 to 61 pg/ml). The evaluation of 3.33× concentrates of
serum-derived partially purified SP provided uncorrected SP values of
117 to 201 pg/ml, which fell within the midpoint of the three-decalog
standard curve. These studies indicate that both the preparative and
quantitative procedures are required for the detection of SP in plasma
or serum samples collected from patients with several clinical
disorders.
| |
INTRODUCTION |
Substance P (SP), a bioactive
undecapeptide, was discovered in 1930 (7) and is found at
varying concentrations in the central nervous system (11).
Observations by Lembeck that there is a much higher concentration of SP
in the dorsal than in the ventral roots of the spinal cord led to the
speculation that SP was associated with sensory neuron transmission
(18). Chang and Leeman (5) reported the chemical
structure of SP, which they determined to be an undecapeptide
(Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-amide), leading to its
synthesis and availability for experimentation. Recently, immunoassay
techniques have made it possible to study the distribution of SP in
different tissues (6). SP is widely distributed in the
central and peripheral nervous systems and has been implicated in
immune (14) and hematopoietic (20, 21)
modulation. We have recently demonstrated that human immune cells such
as monocytes and macrophages express SP at both mRNA and protein levels
(10). SP may have a major role in the pathogenesis of
several inflammatory diseases such as rheumatoid arthritis, bullous
pemphigus, asthma, inflammatory bowel disease (Crohn's disease), and
ulcerative colitis as well as in the transmission of pain (4,
16).
An area of intense investigation within the field of
psychoneuroimmunology centers on the elucidation of the roles played by
certain neuropeptides in the modulation of immunological responses in
various disease states. SP, one of three well-characterized neuropeptides of the tachykinin peptide family, has been extensively studied in terms of its role in physiological processes such as vasodilation, smooth-muscle contraction, and nerve conduction (3,
12, 27). More recently, SP has been shown to play an important
role in the induction of cytokines such as interleukin 1
(IL-1),
IL-6, and tumor necrosis factor alpha, which are central to the
initiation of inflammatory responses leading to modulation of both
specific and nonspecific immune responses (13, 24). With the
observation of SP receptors on both T and B lymphocytes (26), several studies have strengthened the hypothesis that SP plays a pivotal role in the trafficking of lymphocytes
(15) leading to enhanced humoral (25) as well as
cellular humoral responses (16, 17). Elevated plasma SP and
immunoglobulin A levels were noted in human immunodeficiency virus type
1 (HIV-1)-infected infants compared to their uninfected cohorts
(2, 23). Macrophages play a central role in both
inflammatory responses and antigen presentation. The finding of SP
augmentation of lipopolysaccharide (LPS)-induced cytokine
production (IL-1, IL-6, and tumor necrosis factor alpha) by
macrophages lends additional evidence for the role of SP in
the modulation of host immune function (4).
The measurement of SP levels in various body fluids may serve as an
important surrogate marker in monitoring host immune responses in a
variety of disease states. Therefore, development of techniques to
accurately measure SP is of obvious importance. Historically, attempts
to measure neuropeptides in various body fluids, particularly serum or
plasma, have been difficult due to the nonspecific binding of
neuropeptides to other plasma or serum proteins (1, 22). Thus, various liquid organic extraction methods have been described for
the partial purification of neuropeptides including SP with varying
levels of success (9, 22). Further, until recently, labor-intensive radioimmunoassays (RIA) were the only methods available
for the quantitation of SP after partial purification (9).
The RIA technique, however, is less than satisfactory in terms of
safety, ease of handling, and sensitivity.
In an effort to improve upon existing procedures for both the
preparation and quantitation of SP in serum or plasma, the present paper describes the development of a simple chromatographic procedure for the partial purification of SP and describes the evaluation of a
sensitive enzyme immunoassay (EIA) for the determination of SP level in
serum and plasma samples from patients with several clinical disorders.
| |
MATERIALS AND METHODS |
Patient populations.
The 305 samples were from adults
undergoing a diagnostic procedure three times, twice in the hospital
and once at home (n = 23 [69 samples]), children
hospitalized with sickle-cell disease (n = 51), women
with human papillomavirus (HPV) infection (n = 11),
patients infected with HIV (n = 43), patients with
chronic inflammatory demyelinating polyneuropathy (n = 3), patients with multiple sclerosis (n = 23), healthy adult
volunteers (n = 19), umbilical cord blood from
HIV-positive mothers (n = 4), cultured monocytes from
umbilical cord blood treated with cytokines (n = 77),
and a cultured monocyte cell line from umbilical cord blood (n = 5). The choice of these diverse groups was based
on sample availability as well as the desire to test the procedures on
a wide range of patients.
Specimen collection.
Blood was collected in serum separation
tubes and centrifuged at 1,500 × g for 20 min. The
blood was processed in the laboratory approximately 2 h after the
specimen was drawn. The serum was collected, aprotinin (5 U/ml), a
protease inhibitor (Sigma Chemical Co., St. Louis, Mo.), was added to
each specimen, and the specimen was stored at 
70°C until analysis
was performed. The mean storage time prior to analysis was 8 days. The
samples were dried under nitrogen gas and reconstituted with deionized
water to 1/3 of the initial volume and analyzed with the EIA as
described below.
Extraction of SP from plasma and sera.
The method used for
the extraction of SP from acidified plasma or serum samples is a
modification of a previously described procedure in which plasma
samples were extracted with acetone followed by ether. The extracts
were then air dried and reconstituted with assay buffer (9).
Rissler (22) reviews the problems associated with
liquid-liquid extraction of peptides such as SP from body fluids and
advocates solid-phase extraction of samples on small disposable
cartridges such as the ones used in this study.
Briefly, 1.0-ml C18 reverse-phase columns (Bond Elute;
Varton, Harbor City, Calif.) were activated by first rinsing with 5 ml
of high-performance liquid chromatography grade methanol followed by a
final rinse with 5 ml of distilled water. Plasma samples (0.5 ml) were
diluted 1:4 with 4% (vol/vol) acetic acid. Initially, acidified plasma
samples were spiked with known amounts of radiolabeled SP to evaluate
SP recovery from the C18 reverse-phase columns. Further,
known amounts of purified SP were processed through the column to
evaluate the influence of preparation on immunoreactivity of the eluted
peptide. The acidified samples were then added to the activated
reverse-phase columns and allowed to pass through the columns by
gravity. The columns were then washed five times with 2 ml of 4%
acetic acid to remove all unbound material. Three buffer systems were
evaluated for efficiency of elution of SP bound to C18
reverse-phase columns. The buffer systems tried were (i) 90% (vol/vol)
ethanol, 10% (vol/vol) water, 0.4% (vol/vol) acetic acid; (ii) 80%
(vol/vol) acetonitrile, 1% (vol/vol) trifluoroacetic acid (prepared in
distilled water); and (iii) 60% (vol/vol) acetonitrile, 1% (vol/vol)
trifluoroacetic acid (prepared in distilled water). The evaluation of
the elution buffers was accomplished by binding known amounts of
125I-radiolabeled SP (125 pg/ml) added to acidified plasma
samples. A gamma counter was used to compare the eluates to an
appropriately diluted 125I-radiolabeled SP standard which
was not processed through the column. The buffer which reproducibly
yielded more than 90% recovery was made up of 60% (vol/vol)
acetonitrile prepared in 1% (vol/vol) trifluoroacetic acid (prepared
in distilled water). Specimens to be evaluated were processed through
the column in the manner described above. The bound SP was then eluted
from the columns by the addition of 1 ml of the chosen buffer system.
The eluted samples were then dried at 45°C under a constant flow of
N2 gas, followed by reconstitution in 0.15 ml of distilled
water. Total resolubilization was achieved by incubation of the sample
for 30 min at 45°C. SP was then quantified by an antigen competition EIA described below.
SP EIA.
The quantification of SP in plasma and serum
samples, prepared as described above, was accomplished by an antigen
competition EIA (Caymen Chemical Co., Ann Arbor, Mich.). The assay
employs SP conjugated to acetylcholinesterase with acetylthiocholine as the substrate which is hydrolyzed to thiocholine and in turn reacts with 5,5'-dithio-bis-2-nitrobenzoic acid, producing the product 5-thio-2-nitrobenzoic acid, which has a maximum absorbance at 412 nm.
Acetylcholinesterase exhibits several advantages as the tracer tag over
other commonly used enzymes such as horseradish peroxidase in that it
does not autoinactivate during turnover, allowing for multiple
development of the assay. Additionally, acetylcholinesterase is highly
stable under assay conditions with a wide pH range (pH 5 to 10) and is
not inhibited by common buffer salts and preservatives. The assay was
calibrated by the use of an SP standard over a range of 7 to 1,000 pg/ml. The reported limit of detection of this assay is 17.2 pg/ml.
Briefly, 0.05 ml of an appropriately diluted unknown sample or a known
SP standard was added to each well of the microtiter plate precoated
with monoclonal antibody specific for rabbit immunoglobulin G, followed by adding 0.05 ml of acetylcholinesterase-conjugated SP to each well.
Finally, 0.05 ml of rabbit anti-SP was added to all wells except
nonspecific binding wells, which received 0.05 ml of buffer. The plates
were incubated at 4°C for 18 h, followed by five washes with
wash buffer. Enzyme substrate (0.2 ml) was added to each well, followed
by 2-h incubation at room temperature in the dark with rotation. The
absorbance (412 nm) of each well was then measured with a Labrepco Elx
800 microplate reader (Bio-Tek Instrument, Inc., Horsham, Pa.). A
computer program was developed which converts net optical density (OD)
values to B/Bo values (net OD of sample/net OD of maximum binding),
which were plotted versus the concentration of SP standards. From this
standard curve, sample B/Bo values were then used to determine
unknown-sample SP concentrations in picograms per milliliter. An
accuracy control (125 pg of SP per ml) was included with all
evaluations to monitor assay variability. Any assay in which the value
for the accuracy control was determined to be outside the 95%
confidence limit was repeated.
| |
RESULTS |
Recovery of SP from C18 reverse-phase columns.
In
order to determine the efficiency and efficacy of using the
chromatographic extraction procedure on clinical specimens, 24 specimens were processed in duplicate both with and without the
C18 reverse-phase columns. The paired specimens were plated and EIA readings were obtained at the same times. Values were obtained
for these duplicate specimens from B/Bo plots and were compared
statistically. The mean value for SP obtained for the duplicate
specimens without the column extraction was 6.87 with a standard
deviation (SD) of 6.49. By comparison, the mean value of SP obtained
from the duplicates with the column was 18.63 with an SD of 8.91 (Fig.
1). The paired coefficient of correlation between values obtained with and without the column was 0.98 (P < 0.001). The changes in the SP concentrations for
the individual specimens based on the extraction procedure are
graphically presented in Fig. 2. In
addition to eliminating the interference with EIA measurement due to
the nonspecific binding of SP to plasma or serum proteins, the
purification procedure provides an additional advantage. Since the mean
values for the specimens subjected to the chromatographic procedure
were consistently three times the values for specimens not treated, and
the intertreatment coefficient of correlation was very high, the
purification procedure represents a worthwhile step in measuring SP
levels since it also serves to place the values closer to the center of
the EIA measurement scale.
View larger version (19K):
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FIG. 1.
Comparison of mean SP values obtained with and without
the extraction procedure (error bars represent 1 SD).
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FIG. 2.
Comparison of SP concentrations in individual specimens
treated with and without the extraction procedure.
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|
Determination of immunoreactivity of eluted SP.
In order to
evaluate the influence of sample preparation on the immunoreactivity of
SP, known amounts of synthetic SP (0.5 ml of 125 pg/ml) provided with
the EIA kits were subjected to the same preparative procedure as that
performed on unknown plasma and serum samples and then quantified by
EIA. The results of eight individual experiments yielded a mean value
of 124.5 pg/ml with an SD of 21.4 pg/ml and a coefficient of variance
of 9.4%. The 95% confidence limits (mean ± 2 SD) were 81.7 to
167.4 pg/ml. We therefore used those limits for acceptance or rejection
of individual evaluations of unknown samples. Figure
3 illustrates the mean B/Bo values ± SD for each SP standard used to produce reference standard curves
for eight individual EIA experiments and represents the typical
calibration curve for this EIA of SP. A linear displacement of
acetylcholinesterase-linked SP by synthetic SP standard concentrations
was obtained, when plotted as a semilogarithmic function from 7.8 to
1,000 pg of SP per ml. The coefficient of variation for each curve was
greater than 0.98, while the coefficient of variation for the composite
curve was 0.97. These data demonstrate an acceptable level of
interassay precision for the EIA formatted assay, particularly when the
coefficient of variance (9.4%) obtained with the multiple evaluations
of the accuracy control mentioned above is taken into consideration.
Evaluation of SP level in normal control specimens.
The limit
of detection of SP in plasma and serum samples by the EIA evaluated in
the present study was stated by the manufacturer to be 17.5 pg/ml,
which places the limit of detection near the level of normal ranges
reported elsewhere for control subjects (21.94 ± 18 pg/ml)
(9). We, therefore, elected to reconstitute eluted and dried
samples in a volume (0.15 ml) which represented a 3.33× concentrate of
the original sample volume (0.5 ml), which was applied to the
C18 reverse-phase columns. This was found to yield SP
values near the middle of the three-decalog standard curve or about 100 pg/ml. The evaluation of 19 control sera yielded an uncorrected mean
value of 126 pg of SP per ml (range, 17 to 382 pg/ml), with corrected
values having a mean of 38 pg of SP per ml (range, 5 to 115 pg/ml),
which is well within the quantitation range of the assay. The
correction used was for the 3.33× concentration effect of the
purification procedure. These conditions allow for the evaluation of
pathological samples which might have both higher and lower levels of
SP compared to a normal cohort.
Evaluation of SP levels among patient populations.
Among all
306 specimens analyzed, the mean value for SP was 32 pg/ml, with
corrected values ranging from a low of 2 pg/ml to a high of 242 pg/ml
(SD, 39 pg/ml). The mean values for each group of subjects are
presented in Table 1. Statistical
analysis of the mean SP values using analysis of variance revealed
significant differences among the groups (F(9,312) = 3.7;
P < 0.01). The post hoc Tukey-B test revealed that the
SP values obtained from the cord blood from mothers infected with HIV-1
were significantly higher than those in the diagnostic-procedure group,
the children hospitalized with sickle-cell disease, the women with HPV,
patients infected with HIV-1, and patients with multiple sclerosis. The concentrations of SP released by monocyte cell cultures in serum-free medium were similar to those obtained from the plasma or serum samples
presented in Table 1 (18 ± 31.5 pg/ml for monocytes treated with
cytokines [n = 77] and 35 ± 52.1 pg/ml for
untreated monocytes [n = 5]; the value for
lymphocytes reported in reference 10 is 32 pg/ml
[n = 3]).
| |
DISCUSSION |
This study illustrates the usefulness of the procedures in
obtaining valid measures of SP from a variety of clinical settings. The
mean values we obtained compare favorably with those obtained by means
of RIA from plasma samples from healthy blood bank donors (20 to 151 pg/ml) reported by Powell et al. (19). Similar results were
also reported by Fernandez-Rodriguez et al. (9), who used an
RIA technique to measure SP and found mean values ranging from 65 to
128 pg/ml in a comparison between normal control volunteers and
patients with cirrhosis. In the experiments designed to study the
relationship between SP and anxiety we found that they are significantly correlated and that EIA is sufficiently sensitive to
detect changes within individual subjects over time (8). Interestingly, umbilical cord plasma from HIV-positive mothers had
significantly higher SP levels than the other groups studied. Although
we had only four samples of cord blood, these data support the results
obtained by Azzari and coworkers (2, 23), who found that the
SP levels of HIV-seropositive children born to HIV-seropositive mothers
were significantly higher than the SP levels of HIV-seronegative
children born to HIV-seropositive mothers. Elevated SP levels in the
HIV-seropositive children might be associated with the imbalance
between T-helper lymphocyte subsets and the cytokines they secrete.
The variability of SP concentration within certain groups such as
children with sickle-cell disease (SD = 51.1) and umbilical cord
blood from HIV-positive mothers (SD = 68.5) might be due to the
heterogeneous factors within the groups. The children with sickle-cell
disease may have had different levels of pain or have been at different
stages of the disease, resulting in the high variability exhibited by
the group. Similarly, the umbilical cord blood from HIV-positive
mothers might or might not have been infected with HIV, possibly
causing the high variability demonstrated. Thus, the variability of SP
values seen within the diagnostic groups might be a further
demonstration of the usefulness of the techniques described in this
paper.
We have demonstrated that the described procedures for purifying and
quantifying SP are reliable and useful. The SP measures obtained from
diverse sources were comparable to the published results obtained by
other researchers using different techniques. The use of the
chromatographic procedure to partially purify SP in plasma and serum
samples can be omitted when cell culture medium is serum free.
| |
ACKNOWLEDGMENTS |
This study was supported in part by NIH grant R01 MH 49981 and a
grant from the Xi Chapter of Sigma Theta Tau, Inc. (International Honor
Society of Nursing).
Some tissue and fluid specimens were obtained from the National
Neurological Research Specimen Bank, VAMC, Los Angeles, Calif., which
is sponsored by NINDS/NIMH, National Multiple Sclerosis Society,
Hereditary Disease Foundation, Comprehensive Epilepsy Program,
Tourette's Syndrome Association, Dystonia Medical Research Foundation,
and Veterans Health Services and Research Administration, Department of
Veterans Affairs.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Division of
Infectious Diseases and Immunology, Children's Hospital of
Philadelphia, 34th St. and Civic Center Blvd., Philadelphia, PA 19104. Phone: (215) 590-2353. Fax: (215) 590-3044. E-mail:
douglas{at}email.chop.edu.
Present address: School of Nursing, Allegheny University of The
Health Sciences, Philadelphia, Pa.
| |
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Clinical and Diagnostic Laboratory Immunology, May 1998, p. 303-307, Vol. 5, No. 3
1071-412X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
