An in vitro comparison of microdialysis relative recovery of Met- and Leu-enkephalin using cyclodextrins and antibodies as affinity agents

Abstract

Cyclodextrins and antibodies have been used as affinity agents to improve relative recovery during microdialysis sampling. Two neuropeptides, methionine-enkephalin (ME) and leucine-enkephalin (LE), were chosen to compare the use of cyclodextrins and antibodies as possible affinity agents for improving their relative recovery across polycarbonate and polyethersulfone membranes during in vitro sampling. Cyclodextrins (CD) including β-CD, 2-hydroxypropyl-β-cyclodextrin (2HPβ-CD), and γ-CD gave improvements of relative recovery for both peptides of less than 2-fold as compared to controls. Comparisons of relative recovery between tyrosine-glycine-glycine, tyrosine, and phenylalanine using different cyclodextrins in the perfusion fluid were also obtained. Inclusion of an antibody against met-enkephalin in the microdialysis perfusion fluid resulted in relative recovery increases of up to 2.5-fold. These results show that using antibodies as affinity agents during microdialysis sampling may be more effective agents to improve the relative recovery of these opioid neuropeptides.Keywords microdialysis sampling; neuropeptides; affinity agents; cyclodextrins; antibodies IntroductionNeuropeptides are a class of neurotransmitters that are the most structurally diverse group of neuromodulators [1]. Enkephalins are endogenous opioid neuropeptides that have morphinelike action or pain relief in the body and are readily associated with the neurochemistry of addiction [2]. Enkephalins also play significant roles in mediating body temperature control, food intake, reward mechanisms, and stress [3].Microdialysis sampling has been used for over thirty years for chemical sampling applications in neuroscience, pharmacokinetics, and drug metabolism [4][5][6]. It is by far the most commonly used method for the collection of low molecular weight hydrophilic neurotransmitters, such as acetylcholine, dopamine, and glutamate from mammalian brain with high relative recovery Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptAnal Chim Acta. Author manuscript; available in PMC 2009 July 14. Published in final edited form as:Anal Chim Acta. 2008 July 14; 620(1-2): 170-175. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript [7][8][9]. Despite the successful applications of microdialysis sampling, the main limitations for sampling large neuropeptides are a combination of their low relative recoveries and low basal concentrations. A neuropeptide with a high molecular weight will exhibit a smaller aqueous diffusion coefficient, which directly aff...

Analytical Considerations for Microdialysis Sampling

“…Enhancements varied from 1.5 -to almost 10 -fold using a polycarbonate membrane and depended on the structure of the tricyclic compound investigated. The use of cyclodextrins as an additive to increase the recovery of enkephalins has also been investigated [37] . However, in this case, the recoveries were increased by less than a factor of 2.…”

“…Use of a perfusate containing antibodies immobilized on microspheres was evaluated as an approach to improve recovery of cytokines [38] , neuropeptides [37] , and endocrine hormones in vitro using a 100 -kDa MWCO polysulfone membrane. Although the antibodies were found to enhance the recovery of these peptides 3 -to 20 -fold in vitro, one potential drawback of this method in vivo is the possible saturation by the endogenous peptides of all the available binding sites on the microspheres, which would limit the dynamic range of the assay.…”

Electroosmotic perfusion of tissue: sampling the extracellular space and quantitative assessment of membrane-bound enzyme activity in organotypic hippocampal slice cultures

“…The perfusion fluid’s velocity affects the mass transport by its effect on the concentration gradient during diffusion [12], although with high-molecular-weight (MW)-cutoff membranes, flow through the membrane into the tissue can occur [13]. Recent improvements include Stenken’s use of affinity-based recovery enhancement via molecular receptors including cyclodextrins (for nonspecific binding to low-MW molecules, for example YGGFL and YGGFM), antibody-immobilized beads (for binding to cytokines and endocrine hormones), heparin (for binding to human cytokines), and bovine-serum-albumin–heparin conjugates (for cytokine and tumor necrosis factor-α recovery) [14–16]. Membranes with larger MW cutoffs are available for detection of large molecules, for example some peptides and proteins [14, 15, 17, 18].…”

Rapid Preconcentration for Liquid Chromatography–Mass Spectrometry Assay of Trace Level Neuropeptides

“…Measurement of neuropeptides is challenging due to their low extracellular concentration (usually pM) and tendency to degrade during storage [9]. The difficulty is further exacerbated by low recovery by microdialysis sampling [10]; although recent developments have illustrated routes to improved recovery [11–13]. Measurement is also hampered by slow analysis times.…”