A-111 “Activation” Of macro-ast by pyridoxal phosphate supplementation

Abstract

Abstract Background Type 1 macro-AST is a high molecular weight AST (Aspartate Aminotransferase) formed between an AST monomer and immunoglobulins in plasma, which can cause elevated AST values. It is believed that reduced clearance or excretion of macro-AST can cause elevated AST results. Here we report a case of macro-AST that produced high AST values only by the AST assay that includes pyridoxal phosphate (PLP), not by the one without PLP. Case description A 54 year-old male patient diagnosed with disseminated Bartonellosis was monitored with ‘liver function” tests on a monthly basis, with AST varying between 24 to 73 U/L (Reference interval, 13-47 U/L). The patient’s AST value suddenly jumped to 378 U/L when the laboratory switched to an AST assay supplemented with PLP (named ASTPM) on Roche Cobas c702 analyzer (c702). When the same patient sample was tested using the previous AST assay which does not include PLP, a result of 14 U/L was obtained. Macro-AST was suspected, as all other “liver function” tests were within reference interval. Methods Protein-A and Protein-G were used to remove immunoglobulins from the patient sample and another sample with matching AST as a control, respectively. Three hundred microliters of washed Protein-A or Protein-G Sepharose bead slurry was mixed with 300 microliters of each sample, respectively. Each mixture was incubated at 37oC for 3 hours on a rocker. After centrifugation, the supernatant was transferred to a new tube. AST, ASTPM, and all other tests were performed on c702, and results corrected for dilution. The recovery of enzyme activity or immunoglobulin concentration after the removal of immunoglobulins was calculated, respectively. Results In the original sample, the ratio of ASTPM to AST was 28 (389 U/L vs. 14 U/L) for the patient, and 1.1 (304 U/L vs. 275 U/L) for the control. After the complete removal of immunoglobulin G (IgG), the recovery of ASTPM for the patient was 3% and for the control was 74%. The recovery of ALTPM (supplemented with PLP) was 89% for the patient and 85% for the control, showing the expected loss of enzymatic activity by Protein-A or Protein-G treatment. Conclusions The significant reduction of ASTPM activity (recovery of 3%) after removal of IgG in the patient sample proved that the AST enzyme was associated with IgG. The slight increase of ASTPM activity compared to AST (ratio of 1.1) in the control sample was an expected effect of supplementing PLP in the AST assay, however, the huge increase of ASTPM activity (ASTPM/AST ratio of 28) in the patient sample suggests that PLP “activated” the macro-AST and produced high AST result. This is a concerning effect of supplementing PLP in AST assays because it can cause spurious AST elevations in cases of macro-AST. As more laboratories begin to employ the PLP supplemented aminotransferase assays, it is important to acknowledge the interactions between PLP and macro-AST and the role of the laboratory to identify macro-AST in order to avoid unnecessary, costly, and time-consuming testing and procedures for these patients.