Transitioning from intrathecal bupivacaine to mepivacaine for same-day discharge total joint arthroplasty: a quality improvement study.

INTRODUCTION: Same-day discharge total knee and hip arthroplasty is becoming more common. Anesthetic approaches that optimize readiness for discharge are important. Based on an institutional change from low-dose bupivacaine to mepivacaine, we aimed to assess the impact on postanesthesia care unit (PACU) recovery in a quaternary care, academic medical center. METHODS: In this quality improvement retrospective study, a single surgeon performed 96 combined total knee and hip arthroplasties booked as same-day discharge from September 20, 2021 to December 20, 2021. Starting on November 15, 2021 the subarachnoid block was performed with isobaric mepivacaine 37.5-45 mg instead of hyperbaric bupivacaine 9-10.5 mg. We compare these cohorts for time to discharge from PACU, perioperative oral morphine milligram equivalent (OMME) administration, PACU pain scores, conversion to general anesthesia (GA), and overnight admission. RESULTS: We found the use of isobaric mepivacaine as compared with hyperbaric bupivacaine for intrathecal block in same-day discharge total joint arthroplasty was associated with decreased length of PACU stay at our academic center (median 4.03 vs 5.33 hours; p=0.008), increased perioperative OMME (mean 22.5 vs 11.4 mg; p<0.001), increased PACU pain scores (mean 6.29 vs 3.41; p<0.01) and no difference in conversion to GA or overnight admission. CONCLUSIONS: Intrathecal mepivacaine was associated with increased perioperative OMME consumption and PACU pain scores, but still realized a decreased PACU length of stay.

Investigating the kinetic mechanism of inhibition of elongation factor 2 kinase by NH125: evidence of a common in vitro artifact.

Evidence that elongation factor 2 kinase (eEF-2K) has potential as a target for anticancer therapy and possibly for the treatment of depression is emerging. Here the steady-state kinetic mechanism of eEF-2K is presented using a peptide substrate and is shown to conform to an ordered sequential mechanism with ATP binding first. Substrate inhibition by the peptide was observed and revealed to be competitive with ATP, explaining the observed ordered mechanism. Several small molecules are reported to inhibit eEF-2K activity with the most notable being the histidine kinase inhibitor NH125, which has been used in a number of studies to characterize eEF-2K activity in cells. While NH125 was previously reported to inhibit eEF-2K in vitro with an IC(50) of 60 nM, its mechanism of action was not established. Using the same kinetic assay, the ability of an authentic sample of NH125 to inhibit eEF-2K was assessed over a range of substrate and inhibitor concentrations. A typical dose-response curve for the inhibition of eEF-2K by NH125 is best fit to an IC(50) of 18 ± 0.25 µM and a Hill coefficient of 3.7 ± 0.14, suggesting that NH125 is a weak inhibitor of eEF-2K under the experimental conditions of a standard in vitro kinase assay. To test the possibility that NH125 is a potent inhibitor of eEF2 phosphorylation, we assessed its ability to inhibit the phosphorylation of eEF2. Under standard kinase assay conditions, NH125 exhibits a similar weak ability to inhibit the phosphorylation of eEF2 by eEF-2K. Notably, the activity of NH125 is severely abrogated by the addition of 0.1% Triton to the kinase assay through a process that can be reversed upon dilution. These studies suggest that NH125 is a nonspecific colloidal aggregator in vitro, a notion further supported by the observation that NH125 inhibits other protein kinases, such as ERK2 and TRPM7 in a manner similar to that of eEF-2K. As NH125 is reported to inhibit eEF-2K in a cellular environment, its ability to inhibit eEF2 phosphorylation was assessed in MDA-MB-231 breast cancer, A549 lung cancer, and HEK-293T cell lines using a Western blot approach. No sign of a decrease in the level of eEF2 phosphorylation was observed up to 12 h following addition of NH125 to the media. Furthermore, contrary to the previously reported literatures, NH125 induced the phosphorylation of eEF-2.

Calcium/calmodulin stimulates the autophosphorylation of elongation factor 2 kinase on Thr-348 and Ser-500 to regulate its activity and calcium dependence.

Eukaryotic elongation factor 2 kinase (eEF-2K) is an atypical protein kinase regulated by Ca(2+) and calmodulin (CaM). Its only known substrate is eukaryotic elongation factor 2 (eEF-2), whose phosphorylation by eEF-2K impedes global protein synthesis. To date, the mechanism of eEF-2K autophosphorylation has not been fully elucidated. To investigate the mechanism of autophosphorylation, human eEF-2K was coexpressed with λ-phosphatase and purified from bacteria in a three-step protocol using a CaM affinity column. Purified eEF-2K was induced to autophosphorylate by incubation with Ca(2+)/CaM in the presence of MgATP. Analyzing tryptic or chymotryptic peptides by mass spectrometry monitored the autophosphorylation over 0-180 min. The following five major autophosphorylation sites were identified: Thr-348, Thr-353, Ser-445, Ser-474, and Ser-500. In the presence of Ca(2+)/CaM, robust phosphorylation of Thr-348 occurs within seconds of addition of MgATP. Mutagenesis studies suggest that phosphorylation of Thr-348 is required for substrate (eEF-2 or a peptide substrate) phosphorylation, but not self-phosphorylation. Phosphorylation of Ser-500 lags behind the phosphorylation of Thr-348 and is associated with the Ca(2+)-independent activity of eEF-2K. Mutation of Ser-500 to Asp, but not Ala, renders eEF-2K Ca(2+)-independent. Surprisingly, this Ca(2+)-independent activity requires the presence of CaM.