Recent studies have demonstrated that carbon-oxygen (CH···O) hydrogen bonds have important roles in S-adenosylmethionine (AdoMet) recognition and catalysis in methyltransferases. Here, we investigate noncovalent interactions that occur between the AdoMet sulfur cation and oxygen atoms in methyltransferase active sites. These interactions represent sulfur-oxygen (S···O) chalcogen bonds in which the oxygen atom donates a lone pair of electrons to the σ antibonding orbital of the AdoMet sulfur atom. Structural, biochemical, and computational analyses of an asparagine mutation in the lysine methyltransferase SET7/9 that abolishes AdoMet S···O chalcogen bonding reveal that this interaction enhances substrate binding affinity relative to the product S-adenosylhomocysteine. Corroborative quantum mechanical calculations demonstrate that sulfonium systems form strong S···O chalcogen bonds relative to their neutral thioether counterparts. An inspection of high-resolution crystal structures reveals the presence of AdoMet S···O chalcogen bonding in different classes of methyltransferases, illustrating that these interactions are not limited to SET domain methyltransferases. Together, these results demonstrate that S···O chalcogen bonds contribute to AdoMet recognition and can enable methyltransferases to distinguish between substrate and product.

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Introduction Serum androstenedione (ASD) is a useful biomarker in the diagnostic workup of hyperandrogenism, congenital adrenal hyperplasia, premature adrenarche, and polycystic ovary syndrome (PCOS). Recently, the Elecsys ASD assay (Roche Diagnostics), a competitive electrochemiluminescence immunoassay, became available in the US. Herein, the analytical and clinical performance of the Elecsys ASD assay was tested and compared with the Immulite assay (our current assay) and an LC-MS/MS assay (the gold standard) using deidentified residual patient specimens. Method In this study, the linearity, analytical measuring range (AMR), precision, and accuracy of the Elecsys ASD assay (cobas e602 analyzer) were tested. ASD from 40 deidentified residual serum/plasma samples was measured and compared between the Elecsys assay, the Immulite assay, and an LC-MS/MS assay. The reference intervals (RIs) provided by Roche for healthy male (0.280-1.52 ng/mL), healthy female (0.490-1.31 ng/mL), postmenopausal female (0.187-1.07 ng/mL), healthy children (<0.519 ng/mL), and patients with PCOS (0.645-3.47 ng/mL) were tested with at least 20 specimens, according to CLSI C28A3. Statistical analysis was performed using EP evaluator and R program. Result and conclusion The assay had a linear response across the AMR (0.3-10.0 ng/mL). The inter- and intra-assay coefficients of variation measured at multiple concentrations were less than 4.5% and 2.0%, respectively. The Elecsys ASD assay had an excellent correlation with the LC-MS/MS assay with a mean bias of -0.0542 ng/mL (-2%). The Immulite assay had a mean bias of 1.15 ng/mL (44%) and 1.22 ng/mL (32%) compared to the LC-MS/MS and Elecsys ASD assays, respectively. The Roche recommended RIs for healthy males and postmenopausal females were successfully verified in our patient population. However, the ASD concentrations for the healthy children were outside of the suggested RI, with concentrations up to 1.41 ng/mL. Therefore, the RIs for healthy children were adopted from RIs established using the same LC-MS/MS method used for method comparison. RI verification for the healthy female group also failed since many low ASD values were observed. Instead, a RI of < 1.30 ng/mL was established using 60 deidentified residual serum/plasma specimens. Finally, a separate RI for the PCOS group was not established since it may not provide useful clinical information due to the heterogeneity of the group. Unlike some published studies, hormone therapies such as oral contraceptive pills did not cause a significant decrease in ASD in patient specimens (p=0.4967). Overall, the Elecsys ASD assay has superior comparability to the LC-MS/MS assay than the Immulite assay. We were unable to verify the applicability of the RIs recommended by Roche for healthy females and children, warranting the need to establish or transfer them. When RI verification is challenging due to limited qualified specimens, transferring from an LC-MS/MS established RI is possible as long as the methods are comparable.

Background The psychoactive component of cannabis, tetrahydrocannabinol (THC), is one of many cannabinoids present in the plant. Since cannabinoids have extensive structural similarity, it is important to be aware of potential cross-reactivity with immunoassays designed to detect THC metabolite. This is especially important as cannabinoid products are increasingly marketed as legal supplements. The objective of this study was to assess the cross-reactivity of 2 commercial immunoassays designed to detect THC metabolite with 4 cannabinoids: cannabidiol, cannabinol, cannabichromene, and cannabigerol. Methods Deidentified residual patient urine samples that tested negative for THC metabolite on initial testing were pooled and fortified with the above compounds to detect cross-reactivity. We next tested a range of CBN concentrations to determine what concentration of CBN was required to trigger a positive immunoassay result. Finally, we tested whether CBN has an additive effect with THC in the immunoassay by adding CBN to 21 samples weakly positive for THC by a mass spectrometry method but negative by the EMIT II Plus immunoassay. Results Both the EMIT II Plus assay and the Microgenics MultiGent assay demonstrated cross-reactivity with CBN. For the EMIT II Plus assay, about 5-fold more CBN than THC metabolite was required to produce an assay signal equivalent to the cutoff concentration, and CBN displayed an additive effect with THC metabolite. For the Microgenics assay, 20-fold more CBN than THC metabolite was required to cross the cutoff concentration. Conclusions These data may help guide the need for confirmatory testing when results of THC metabolite testing by immunoassay are inconsistent with expectations.

Neuron specific enolase (NSE) is a serum soluble tumor marker for certain neuroendocrine tumors and a marker of some types of brain trauma. Red blood cells (RBC) also contain substantial NSE concentrations, making even slight hemolysis a source of potential false positives and reason for sample rejection. Unfortunately, hemolysis can be common in specimens from patients with in vivo hemolysis due to trauma or medical intervention, or in vitro hemolysis due to difficult draws (e.g., pediatrics). While laboratories are appropriately reluctant to report false elevations of NSE, specimen rejection due to any evidence of hemolysis underserves these patients. Correction of NSE results in hemolyzed serum was previously reported as a possible solution; however, broad inter-individual differences in RBC NSE concentrations require measuring NSE in RBCs from a whole blood specimen submitted concurrently with a hemolyzed serum sample. Unfortunately, logistical challenges impede the effective implementation of this strategy. Our objective was to establish a reference distribution of NSE concentrations in RBCs (as a ratio of NSE/Hgb (ng/mg)) that would facilitate the derivation of a cutoff to distinguish true NSE elevations from those concentrations arising from in vivo or in vitro hemolysis. Serum NSE was measured by the Fujirebio NSE enzyme immunoassay kit, and H-index was measured using the Roche cobas c501 (previously shown to demonstrate good agreement with spectrophotometrically measured hemoglobin, Hgb). Purposely hemolyzed samples (n=80) were diluted to an H-index of ~100, and NSE was measured to establish the distribution of NSE/Hgb ratios. De-identified NSE results (n=5,441) previously analyzed in our laboratory were extracted from our data warehouse to determine the distribution of NSE values in non-hemolyzed clinical samples. R statistical software was used for linear regressions, power transform analysis, and subsequent inverse square root transformation to enable appropriate normalization of each distribution for curve fitting purposes. Median NSE/Hgb ratios in the hemolyzed and non-hemolyzed distributions were 21 and 125, respectively. Curve fitting of the transformed hemolyzed distribution provides an upper 99% value of 46 NSE/Hgb. To determine whether that cut-off is appropriate at different levels of hemolysis, NSE was measured in samples (n=60) from subjects (n=15) prepared at four levels of hemolysis (H-index ~25 to 400). The mean NSE/Hgb ratio did not change significantly across the range of H-index values: regression analysis documented a mean of 23 NSE/Hgb and an upper 99% prediction value of 41 NSE/Hgb. Additionally, the increase in NSE was related to the H-index as follows, NSE=-715.3 + 21.42*H-index. The slope (representing NSE/Hgb) was again in agreement with the mean ratio of 21 NSE/Hgb observed in the initial 80 hemolyzed samples. Overall, the derived reference distribution of NSE/Hgb facilitates the interpretation of clinically relevant NSE increases from elevated NSE concentrations due to in vivo or in vitro hemolysis.