Flaws in the Serum Anticholinergic Activity Assay: Implications for the Study of Delirium

To the Editor: Delirium is a common adverse outcome after surgery, with significant morbidity and mortality; however, the etiology of postoperative delirium remains unclear, making advances in diagnosis and treatment difficult. Anticholinergic activity has long been suggested as a major factor in the development of postoperative delirium and has usually been quantified using the serum anticholinergic activity (SAA) assay. The current study found a serious flaw in the SAA assay that may have hampered progress in determining the contribution of anticholinergic activity to delirium. One hundred six elderly (aged 65) patients undergoing elective knee arthroplasty at Duke University Medical Center (DUMC) were recruited and tested for delirium using Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, criteria before surgery and on postoperative days 2 (POD2) and 3 (POD3). Blood samples were collected at each test. Of 106 patients, 27 developed postoperative delirium. The anticholinergic activity in preand postsurgery plasma was determined according to the SAA. This assay measures inhibition of quinuclidinyl benzilate, L-[benzilic4,40-3H] ([H]QNB) binding to rat brain muscarinic acetylcholine receptors by serum or plasma. The assays were performed in a total volume of 200 uL containing phosphate buffered saline, 0.3 nM [H]QNB (specific activity 52.0 Ci/mmol; PerkinElmer, Boston, MA) with no plasma (control) or 20 mL plasma, and approximately 13mg of rat brain membrane proteins. Inhibition of [H]QNB binding was expressed as percentage inhibition and in pmol/mL atropine equivalents. Anticholinergic activity was measured in preoperative and POD2 plasma from 48 patients without delirium and 17 with. Anticholinergic activity in presurgery plasma was 57 9% (2.55 1.2 pmol/mL atropine equivalents), increasing to 69 8% (4.56 2.2 pmol/mL atropine equivalents) in POD2 plasma (Po.001). In patients with delirium, anticholinergic activity in presurgery plasma was 53 15% (2.35 1.2 pmol/mL atropine equivalents), increasing to 62 13% (3.43 1.9 pmol/mL atropine equivalents) in POD2 plasma (P 5.003). These results indicate that plasma anticholinergic activity was not significantly different in patients with and without delirium. The precise source of the anticholinergic activity was investigated by filtering preoperative plasma from several patients using a Microcon YM-50 filtration device (Millipore, Bedford, MA). In this filtration device, molecules larger than 50 kD are retained while smaller molecules are allowed to pass into the filtrate. When this filtered plasma was used instead of whole plasma in the SAA assay, there was less than 10% inhibition of [H]QNB binding. These results indicate that high-molecular-weight plasma proteins rather than small-molecular-weight anticholinergic drugs cause anticholinergic activity seen with plasma. Plasma proteins can inhibit [H]QNB binding to muscarinic receptors by binding to the receptors or sequestering [H]QNB. The latter possibility was tested by incubating [H]QNB in the presence or absence of plasma for 2 hours at room temperature and filtering the mixture on Microcon YM-50 filters to assess the concentration of free [H]QNB. Using preoperative plasma from seven patients, the percentage of free [H]QNB in the filtrate from YM-50 filters was found to be 44.2 8.2% of the added [H]QNB. Similar results were seen with serum. These results indicate that, in the SAA, more than 50% of added [H]QNB binds to high-molecular-weight plasma and serum proteins and is thus not available to bind to muscarinic receptors. It appears that a decrease in the concentration of free [H]QNB, because of its binding to plasma proteins, causes the anticholinergic activity seen with preoperative plasma. When these experiments were repeated using POD2 plasma from the same seven patients, the concentration of free [H]QNB in the filtered plasma was found to be 33.4 10.7%Fa further decrease of 10.4% (P 5.03) from preoperative plasma. A greater decrease in the concentration of free [H]QNB in the presence of POD2 plasma explains the observed increase in plasma anticholinergic activity seen after surgery. Two previous studies demonstrate that patients with delirium in cardiac surgery and surgical intensive care settings had higher serum and plasma anticholinergic activity than those without. The current study shows no correlation between high plasma anticholinergic activity and postoperative delirium in knee arthroplasty patients. Perhaps more importantly, this study has uncovered a serious problem with this long-used assay. When the assay was developed, its developers were aware that serum inhibited [H]QNB binding to rat brain membranes but did not consider it significant, because, in their hands, the inhibition by serum was only 29%. However, the current data show that plasma and serum inhibits [H]QNB binding to rat brain membranes by more than 50%, a conclusion that other reports support. As early as 1981, it was reported that 10% plasma (an amount typically used in the SAA assay) inhibits [H]QNB binding to pig brain muscarinic receptors by 70%. This inhibition was attributed to [H]QNB’s binding to plasma proteins, because when the plasma was filtered on a Centriflo cone (Millipore) with a molecular-weight cutoff of 50 kDa, the filtered plasma showed no inhibition of [H]QNB binding to muscarinic receptors. In summary, the present study demonstrates that the frequently used serum anticholinergic assay is not an accurate representation of anticholinergic activity because of extensive binding of [H]QNB to plasma proteins. Consequently,