A semi‐automated method for 5′‐ectonucleotidase measurement in lymphocytes

Abstract

Summary We describe a simple, non-radioactive, semi-automated method for measuring lymphocyte ccto-5'-nucleotidase activity in isolated cell homogenates. The method which uses inosine 5'-monophosphate(IMP)asa subslrate was optimized and requires a total of 4x 10*' lympbocytes. The reference range obtained (38 7-180 0 nmol/h per mg protein; .v= 106-6) agrees with other isotopic methods which use ''*C-IMP. The simplicity, sensitivity (lower limit 10, upper limit 400 nmol/h per mL) and precision (at 114 nmol/h per mL, interday4-2%s.d.,M = 20; intraday 5-8%, n * 10) makes this method suitable for routine eliniea! laboratory use.

Relationship between psychological stress and lymphocytic 5′-ectonucleotidase

“…However, if this were so one would have expected their stress levels plus drug intake to further reduce NT below the stress group not on drugs (Study 1). The degree of reduction of NT in stressed groups was similar to that found in a previous study for persons who were well but antibody positive to HIV and, in retrospect, suggested that psychological stress in these HIV-positive patients may have been contributing to their low NT activities (Chalmers et at., 1990). NT appears to be a reliable marker for depressed patients, and as shown by the control group (Fig.…”

“…The effects seen with NT are not a generalized effect of other ectoproteins on the cell and appear to be specific to NT. For example, the activity of dipeptidyl peptidase IV, another lymphocyte ectoenzyme also related to immunity, was unaffected in both HIV-positive and depressed patients (Chalmers et aL, 1990). This is not to imply, however, that other ectoproteins important to lymphocyte function and immunity are not affected by psychological stress and contribute to the immune suppression seen in depressed patients.…”

“…Apparatus for the NT assay is described in Chalmers and Hare (1990). The following psychological tests were administered: the State Trait Anxiety Inventory-trait (STAl-trait), a measure of dispositional or trait anxiety (Spielberger et al, 1983), and the POMS, a measure of mood disturbance (McNair et al, 1992).…”

“…NT was stable in blood maintained at room temperature (23~ for up to 24 hours after collection. NT activities were estimated spectrophotometrically as previously described (Chalmers & Hare, 1990). The study was done as a blind trial with the analyst unaware of the subject's grouping.…”

“…Moreover, a 50% reduction of NT activity has been reported in recently diagnosed (and otherwise healthy) HIV-positive patients who were not immunocompromised (Chalmers, Hare, Wooley, & Frazer, 1990). It is possible that the psychological stress associated with a positive HIV status may account for the low levels of this enzyme reported by Chalmers et al (1990). Finally, in addition to the medical relevance outlined above, NT can easily be measured using an automated spectrophotometric method (Chalmers & Hare, 1990), thereby avoiding complex procedures such as cell culturing or use of radioisotopes which can complicate data interpretation.…”