A high-recovery extraction procedure for quantitative analysis of
substance P and opioid peptides in human cerebrospinal fluid
Zhurong Liu
a
, Magnus Welin
b,c
, Bjo¨rn Bragee
b
, Fred Nyberg
a,
*
a
Department of Pharmaceutical Biosciences, Divison of Biological Research on Drug Dependence, Uppsala University, P.O. Box 591, S-751 24
Uppsala, Sweden
b
Sma¨rtkliniken Kronan, Stockholm, Sweden
c
Department of Medical Pharmacology, University of Uppsala, Uppsala, Sweden
Received 2 March 1999; accepted 20 March 2000
Abstract
This study reports an improved approach for the determination of neuropeptide levels in human cerebrospinal fluid (CSF). The method
is based on sample acidification followed by liquid–liquid extraction (LLE) combined with radioimmunoassay. It was applied to study the
recovery and level of some opioid peptides (Met-enkephalin-Arg
6
-Phe
7
and Leu-enkephalin-Arg
6
), substance P and the substance P
1–7
fragment, which are all compounds known to be present in human CSF. The results indicated that the use of LLE highly improved the
recovery of these peptides compared to current liquid–solid-phase extraction methods by using silica gel cartridges or mini-columns for
ion-exchange chromatography. Peptides added to CSF in concentrations down to 10 fmol/ml were recovered in yields exceeding 80%. The
mean recovery of synthetic peptides as recorded by radioimmunoassay in the LLE procedure was significantly improved when HCl was
added to the sample. In contrast, when the
125
I-labeled analogues of the peptides were added to CSF samples, the mean recovery of the four
labeled peptides using the LLE procedure was markedly reduced in acidified samples. We also found that the inclusion of HCl effectively
improved the removal of proteins present in the samples. As an application the levels of substance P and Met-enkephalin-Arg
6
-Phe
7
in CSF
samples from patients with chronic pain (fibromyalgia syndrome) were measured using the new procedure. It was possible to confirm a
significant difference in the CSF levels of both peptides when comparing patients and controls. © 2000 Elsevier Science Inc. All rights
reserved.
Keywords: Analysis; Cerebrospinal fluid; Enkephalins; Opioid peptides; Radioimmunoassay; Substance P
1. Introduction
The access of highly sensitive and reproducible methods
for the analysis of peptides in body fluids and tissue samples
is essential in many areas of biomedical research. In clinical
studies of neurologic diseases there is a need for reliable
techniques for the assessment of neuropeptide levels in the
cerebrospinal fluid (CSF). This fluid is in direct contact with
the extracellular fluid of the central nervous system (CNS)
and peptide concentrations in CSF are considered to reflect
neuronal activity, which might be correlated to particular
CNS disorders [19]. Regarding the opioid peptides, during
the past two decades most studies on their CSF levels have
utilised radioimmunoassay (RIA) [8–10,12–14]. Indeed,
RIA is a highly sensitive technique for peptide analysis,
although it has some limitations.
The major advantage with the RIA is that it allows
detection of peptides at very low levels and that it provides
high specificity for a given peptide to be analysed. How-
ever, due to the fact that peptides with similar structures
may cross-react with the specific antibodies and that the
presence of proteins and salts in biologic samples, such as
plasma and CSF, may interfere in the assay the RIA is often
combined with a preseparation or pre-extraction technique.
Many studies on the CSF levels of neuropeptides have used
procedures with reverse-phase silica gel cartridges (e.g.,
SepPak) or ion-exchange chromatography on minicolumns
for the preseparation step [13]. The disadvantage with these
preseparation or pre-extraction techniques is that they are
often connected with low recovery. Runs of samples with
peptide concentrations at the lower fmol level often result in
substantial losses of peptide activity. Because the CSF level
* Corresponding author. Tel.: ϩ46-18-174166; fax: ϩ46-18-501920.
E-mail address: fred.nyberg@farmbio.uu.se (F. Nyberg).
Peptides 21 (2000) 853–860
0196-9781/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved.
PII: S0196-9781(00)00219-9