Quantitative Spectrophotometric Microplate Assay for Angiotensin-converting Enzyme in Cerebrospinal Fluid

[摘要] Angiotensin-converting enzyme (ACE; EC 3.4.15.1) catalyzes the formation of angiotensin II by cleaving the C-terminal histidylleucine dipeptide from angiotensin I (1). Indications are that ACE is affiliated with an autonomous renin-angiotensin system of the brain that participates in physiologic processes inside the brain (2)(3). In addition, studies suggest that changes in ACE concentrations in brain tissue, caused by various neurologic disorders, are reflected by alterations in ACE activity in cerebrospinal fluid (CSF) (4). For example, increased ACE concentrations in CSF are associated with neurosarcoidosis (4)(5)(6)(7), with affected patients generally having activities approximately twofold or more higher than those of healthy individuals (4)(6)(7). Increased CSF ACE has also been implicated in neurologic diseases, such as bacterial and viral meningitis and Behcet disease (4)(5)(6)(7). Decreased concentrations have been reported in patients with Alzheimer disease, Parkinson disease, and progressive supranuclear palsy (8)(9).The spectrophotometric assays customarily used for serum ACE lack the sensitivity for measuring the concentrations typically found in CSF. Consequently, more sensitive and costly methodologies are routinely used, such as fluorometric assays or HPLC (4).To provide a more economic assay to measure CSF ACE for the screening of neurosarcoidosis, several attempts were made at modifying commercially available reagents and protocols used specifically for spectrophotometric measurement of serum ACE. A successful assay was eventually established that utilizes the ability of ACE to hydrolyze the tripeptide N -[3-(2-furyl)acryloyl]-l-phenylalanylglycylglycine to furylacryloylphenylalanine and glycylglycine (10). However, the sample volume (0.6 mL) required to achieve acceptable sensitivity was impractical. The assay was therefore reformatted for a 96-well microplate. This dramatically decreased sample and reagent volumes and increased accuracy by making duplicate measurements viable for practically all specimens received in our laboratory. …

[学科分类] 过敏症与临床免疫学