The acetaminophen metabolite N-acetyl-p-benzoquinone imine (NAPQI) inhibits glutathione synthetase in vitro; a clue to the mechanism of 5-oxoprolinuric acidosis?

1. Metabolic acidosis due to accumulation of l-5-oxoproline is a rare, poorly understood, disorder associated with acetaminophen treatment in malnourished patients with chronic morbidity. l-5-Oxoprolinuria signals abnormal functioning of the γ-glutamyl cycle, which recycles and synthesises glutathione. Inhibition of glutathione synthetase (GS) by N-acetyl-p-benzoquinone imine (NAPQI) could contribute to 5-oxoprolinuric acidosis in such patients. We investigated the interaction of NAPQI with GS in vitro. 2. Peptide mapping of co-incubated NAPQI and GS using mass spectrometry demonstrated binding of NAPQI with cysteine-422 of GS, which is known to be essential for GS activity. Computational docking shows that NAPQI is properly positioned for covalent bonding with cysteine-422 via Michael addition and hence supports adduct formation. 3. Co-incubation of 0.77 µM of GS with NAPQI (25-400 µM) decreased enzyme activity by 16-89%. Inhibition correlated strongly with the concentration of NAPQI and was irreversible. 4. NAPQI binds covalently to GS causing irreversible enzyme inhibition in vitro. This is an important novel biochemical observation. It is the first indication that NAPQI may inhibit glutathione synthesis, which is pivotal in NAPQI detoxification. Further studies are required to investigate its biological significance and its role in 5-oxoprolinuric acidosis.

Salivary testosterone changes during oral glucose tolerance tests in overweight and obese men - postprandial or circadian variation?

BACKGROUND: Serum total testosterone (T) decreases postprandially. Postprandial salivary testosterone (SalT) responses, however, have not been studied. We report on the effect of glucose ingestion on fasting SalT concentrations. OBJECTIVE: To investigate the effect of oral glucose ingestion on fasting SalT. METHODS: Salivary and blood samples were collected between 09.00 and 09.30 and two hours after a 75g oral glucose load in 32 men with mean (standard deviation) age of 52 (5.7) years and body mass index of 32.6 (5.56) kg/m2. Free T and bioavailable testosterone (BAT) were calculated using the Vermeulen equation. RESULTS: Two hours following oral glucose, there was a decrease in fasting mean (standard deviation) SalT [178.2 (56.6) vs 146.0 (42.2) pmol/L; p = 0.0003], serum cortisol [332 (105.0) vs 239 (75.3) nmol/L; p = <0.0001], prolactin [193 (75.0) vs 127 (55.9) mIU/L; p = <0.0001] and TSH [1.60 (0.801) vs 1.16 (0.584) mIU/L; p = <0.0001]. Plasma glucose increased [6.2 (0.72) vs 8.1 (3.71) mmol/L; p = 0.0029]. Serum total T, SHBG, albumin, Free T, BAT, gonadotrophins and FT4 remained unchanged. CONCLUSIONS: SalT decreased postprandially. A concomitant decrease in serum cortisol, prolactin and TSH reflecting diurnal variation offers an alternative explanation for the decrease in SalT independent of food consumption. Further studies are required to determine whether morning temporal changes in SalT are related to food consumption or circadian rhythm or both.

Conflicting effects of haemolysis on plasma sodium and chloride are due to different haemolysis study protocols: A case for standardisation.

BACKGROUND: Haemolysis has been reported as having a positive, negative or no effect on plasma sodium (PNa) and chloride (PCl). We investigated the haemoltytic effect of different haemolysis protocols on PNa and PCl using modelling and laboratory experiments. METHODS: In a modelling experiment, percentage change and recovery due to dilution in routinely (in vitro) haemolysed samples were compared against shear stress haemolysis and samples spiked with haemolysate from whole blood freeze-thaw, packed cells freeze-thaw and osmotic shock protocols. The results were compared against a control base pool. Additionally, for the osmotic shock method, results were compared against saline- and deionised water (DIW)-spiked controls. In a laboratory experiment, percentage change and recovery were similarly compared using haemolysate from whole blood freeze-thaw and osmotic shock protocols. PNa, PCl and H-index were measured on the Abbott Architect and haemoglobin on the Sysmex XN-9000. RESULTS: In the modelling experiment, the percentage decrease in PNa and PCl was similar in in vitro haemolysis, shear stress haemolysis, whole blood freeze-thaw haemolysis and packed cells freeze-thaw haemolysis and this was lower compared to the osmotic shock method. In the laboratory experiment, the change in PNa compared to the base pool was less (p < 0.001) per unit increase in H-index in the freeze-thaw method (-0.33 mmol, 95% CI -0.35 to -0.31) compared to the osmotic shock method (-0.65 mmol, 95% CI -0.66 to -0.64). PCl did not change with haemolysis in the freeze-thaw method and changed by -0.21 ± 0.01 mmol per unit increase in the H-index in the osmotic shock method. Recovery of PNa and PCl increased with increasing H-index in both methods. CONCLUSION: The osmotic shock protocol is inappropriate for haemolysis studies because of dilution with DIW used for cell lysis. Recovery calculations may incorrectly compensate for genuine dilution caused by haemolysis.

Utility of plasma NGAL for the diagnosis of AKI following cardiac surgery requiring cardiopulmonary bypass: a systematic review and meta-analysis.

The objective of this study was to assess the diagnostic value of plasma neutrophil gelatinase-associated lipocalin (pNGAL) for the early diagnosis of acute kidney injury (AKI) in adult patients following cardiac surgery requiring cardiopulmonary bypass (CPB). Electronic databases and other resources were systematically searched for relevant studies. Risk of bias was assessed using the Quality Assessment for Diagnostic Accuracy Studies 2 (QUADAS-2) tool. Studies were assigned to a sub-group based on the timing of the pNGAL sample in relation to the cessation of CPB. These were < 4 h, 4-8 h, 12 h or 24 h post-cessation of CPB. Summary values for sensitivity and specificity were estimated using the hierarchical summary receiver operator characteristic (ROC) curve model. A random-effects meta-analysis of each pair of sensitivity and specificity estimates from each included study was performed. In total, 3131 patients from 16 studies were included. When taken at 4-8 h following CPB, pNGAL had superior performance for the diagnosis of AKI in the defined population when compared to earlier and later time points. Prediction regions and confidence intervals, however, demonstrated significant variability in pooled estimates of sensitivity and specificity. This is likely due to population and study design heterogeneity, lack of standardisation of assays and thresholds, and inability to distinguish the different molecular forms of NGAL. In conclusion, the diagnostic utility of pNGAL in this clinical setting is inconclusive and large individual studies of representative populations of cardiac surgery patients using assays that specifically detect NGAL in its monomeric form are required.

Diagnostic performance of serum calprotectin in discriminating active from inactive ulcerative colitis in an outpatient setting.

There are limited and conflicting data on the value of serum calprotectin (sCp) in discriminating active from inactive disease activity in ulcerative colitis (UC). Faecal calprotectin (fCp), sCp, serum C-reactive protein (sCRP) and platelets were compared in patients with UC who had clinically active (n = 29) and clinically inactive (n = 42) disease. Serum calprotectin was measured with Bühlmann® (BMN sCp) and ImmunodiagnostikTM (IDK sCp) assays. Median (interquartile range) fCp was higher in active than inactive disease [1004 (466-1922) versus 151 (55-280) µg/g; p < 0.0001). BMN sCp [4534 (3387-6416) versus 4031 (2401-5414) ng/mL; p = 0.1825], IDK sCp [4531 (2920-6433) versus 3307 (2104-4789) ng/mL; p = 0.1065], sCRP [ 4 (2-8) versus 2 (1-4) mg/L; p = 0.0638) and platelets [269 (233-331) versus 280 (227-325) ×10-9/L; p = 0.8055] were similar in active and inactive disease respectively. The area under the receiver operator characteristics curves with 95% confidence limits were 0.85 (0.76-0.94) for fCp, 0.61 (0.47-0.74) for BMN sCp, 0.61 (0.48-0.75) for IDK sCp, 0.69 (0.56-0.81) for sCRP and 0.52 (0.38-0.66) for blood platelets. Faecal calprotectin is the optimum biomarker for discriminating between active and inactive UC. The diagnostic performance of sCp, irrespective of assay, and systemic biomarkers was poor; of these sCRP performed best.

COVID-19 seroprevalence after the first UK wave of the pandemic and its association with the physical and mental wellbeing of secondary care healthcare workers.

Objectives: To determine the seroprevalence of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) antibody status amongst healthcare workers (HCWs) working through the first wave of the Coronavirus (COVID-19) pandemic in 2020. To examine the association of seroprevalence and self-reported COVID-19 symptoms with occupation, sex, and ethnicity; and how these factors were associated with physical and mental wellbeing. Design: Single-centre cohort study. Setting: Large public hospital in the United Kingdom. Intervention: All HCWs who had been tested for anti-SARS-CoV-2 immunoglobulin (Ig) G nucleocapsid antibody in summer 2020 were asked to complete an electronic survey focusing on their physical and mental health in Winter 2020-21. This survey was comprised of the Short Form 12v2, Physical Component Summary (PCS), Mental Component Summary (MCS), and Generalised Anxiety Disorder 7-item (GAD-7) questionnaires. Results: 7604/9781 (77.7%) HCWs were antibody tested, of which 1082 completed the full survey. Antibody testing was conducted between 17/06/20-30/07/20, during which time our seroprevalence rate was 28% (299/1082). Of those self-reporting COVID-19 symptoms, 51% (201/395) were antibody positive. Antibody-positive participants had lower PCS scores (p = 0.016), indicating poorer physical health. Lower PCS scores were also found in those deemed high risk for COVID-19 by their GP (p = 0.001), and those aged >44 years (p = 0.009). Antibody-negative participants had lower MCS scores (p = 0.044), indicating poorer mental health. Those who self-reported COVID-19 symptoms had lower PCS scores (p=<0.001) than those with no symptoms. Lower MCS scores were found in women (p = 0.001), Caucasians (p = 0.018), non-clinicians (p = 0.001), and those aged <44 years (p = 0.009). Significantly higher GAD-7 anxiety scores were evident in staff aged <44 years (p = 0.023), and those with self-reported COVID symptoms (p = 0.031). Doctors had lower GAD-7 anxiety scores (p = 0.009). Conclusion: Self-reported symptoms did not correlate with seroprevalence; data surrounding this can be useful for future workforce planning. Interventions are needed to reduce the mental and physical burden of the pandemic on HCWs. Further work is needed to identify which particular HCWs may require further support, to ensure well-being and effective patient care. Trial registration: Sponsor Protocol number - 2020COV112, Clinicaltrials.gov number -NCT04527432.

Samples spiked with pituitary-derived thyroid-stimulating hormone may disguise the extent of differences between thyroid-stimulating hormone assays.

BACKGROUND: A large discordance in the diagnosis and potential management of hypothyroidism using Abbott and Roche thyroid assays has been reported recently. The difference in Abbott and Roche thyroid-stimulating hormone (TSH) results in these studies was larger than anticipated from the external quality assessment (EQA) reports. METHODS: Abbott and Roche TSH method means in UK NEQAS for thyroid hormones distributions 430 to 454 were compared against the amount of TSH spiked. A TSH deplete serum pool was spiked with various concentrations of pooled high TSH serum and 3rd WHO International Standard for TSH (WHO-IS). Four serum pools with TSH close to clinical decision limits were spiked with two concentrations of WHO-IS. RESULTS: On review of EQA data, median (IQR) Roche: Abbott TSH ratio was lower (p < 0.001) in 48 pools spiked with TSH (1.11 (1.07-1.16)) compared to 41 pools not spiked (1.29 (1.25-1.31)) and the decrease was proportionate to the contribution of spiked TSH to total TSH in the samples (ρ=-0.908, p < 0.001). In spiking experiments, the relationship of Roche and Abbott TSH was different in TSH deplete pool spiked with WHO-IS (RocheTSH=1.13*AbbottTSH-0.52) and high TSH serum (RocheTSH=1.43*AbbottTSH-0.50), respectively. The Roche: Abbott TSH ratio decreased and the method agreement improved on spiking serum pools with WHO-IS. CONCLUSION: Abbott and Roche TSH assays are not in harmony in human serum samples but the agreement was better in samples spiked with WHO-IS which contains pituitary-derived TSH. Use of pituitary-derived TSH spiked samples, such as provided by EQA schemes, may mask clinically significant between-assay differences.

High-sensitivity cardiac troponin I: is ethnicity relevant?

AIMS: To evaluate 99th percentile upper reference limits (URLs) and investigate ethnic differences for the Abbott Architect high-sensitivity cardiac troponin I (hs-cTnI) in a middle-aged to elderly cosmopolitan population. METHODS: In subjects without cardiovascular disease and after outlier exclusion, data on hs-cTnI from 149 white men, 150 white women, 150 South Asian (SA) men and 150 SA women in their sixth, seventh and eight decades were analysed. Each ethnicity-gender-decade subgroup consisted of 50 patients except white men in their sixth decade (n=49). RESULTS: The overall, women and men hs-cTnI 99th percentile URLs were 22.1, 17.9 and 24.8 ng/L, respectively. Median (IQR) hs-cTnI was higher in men (2.7 (1.8-4.1) ng/L) than in women (1.9 (1.1-3.2) ng/L; p<0.001). White men (3.2 (2.2-4.4) ng/L) had higher hs-cTnI than SA men (2.5 (1.6-3.6) ng/L; p<0.001), white women (2.1 (1.3-3.3) ng/L; p<0.001) and SA women (1.6 (1.0-3.0) ng/L; p<0.001). Hs-cTnI in white women was similar to SA women (p=0.07) and SA men (p=0.07). Patients in the eighth decade had higher hs-cTnI (p<0.05) than those in sixth decade within each ethnicity-gender subgroup. Of significant associations, age had the greatest impact on hs-cTnI followed by gender and then ethnicity. CONCLUSION: We report white-SA differences in hs-cTnI in men and a similar trend in women. We confirm age and gender differences in hs-cTnI, irrespective of ethnicity. Further studies are required to determine whether ethnicity-specific age and gender 99th percentile URLs improve detection or exclusion of myocardial injury.

Hypothalamic-pituitary-adrenal axis suppression - The value of salivary cortisol and cortisone in assessing hypothalamic-pituitary-adrenal recovery.

BACKGROUND: The 0.25 mg short synacthen test is used to assess recovery from hypothalamic-pituitary-adrenal suppression due to chronic glucocorticoid administration. We assessed the potential role of salivary cortisol and cortisone in predicting hypothalamic-pituitary-adrenal function using the short synacthen test as the gold standard test. METHOD: Between 09:00 and 10:30, salivary and blood samples were collected just prior to a short synacthen test to assess hypothalamic-pituitary-adrenal axis recovery in patients previously treated with oral glucocorticoids. The cut-off for a normal short synacthen test was a 30-min cortisol ≥450 nmol/L. RESULTS: Fifty-six short synacthen tests were performed on 47 patients. Of these, 15 were normal. The area under receiver operating characteristic curves for serum cortisol, salivary cortisone and salivary cortisol were 0.772, 0.785 and 0.770, respectively. From the receiver operating characteristic analysis, the cut-offs for baseline serum cortisol (≥365 nmol/L) and salivary cortisone (≥37.2 nmol) predicted hypothalamic-pituitary-adrenal axis recovery with 100% specificity in 26.7% of pass short synacthen tests, whereas salivary cortisol predicted none. Baseline serum cortisol (≤170 nmol/L), salivary cortisone (≤9.42 nmol/L) and salivary cortisol (≤1.92 nmol/L) predicted hypothalamic-pituitary-adrenal suppression with 100% sensitivity in 58.5%, 53.7% and 51.2% of failed short synacthen tests, respectively. Using these cut-offs, baseline serum cortisol, salivary cortisone and salivary cortisol could reduce the need for short synacthen tests by 50%, 46% and 37%, respectively. CONCLUSION: Although marginally inferior to early morning serum cortisol, early morning salivary cortisone may be used as a first-line test for assessing hypothalamic-pituitary-adrenal function. We plan to incorporate salivary cortisone into a home-based patient pathway to identify patients with hypothalamic-pituitary-adrenal recovery, continuing hypothalamic-pituitary-adrenal suppression and those who require a short synacthen test.

Multicenter Evaluation of a High-Sensitivity Troponin I Assay and Verification of an Early Rule-Out Algorithm.

BACKGROUND: The objectives of this study were to independently evaluate the analytical performance of the STAT high-sensitivity troponin I (hs-cTnI) assay on a recently launched and CE-marked integrated chemistry and immunoassay system, confirm acceptable performance of the assay in line with The Third Global MI Task Force recommendations, and confirm suitability of the assay for continued use of an early rule-out algorithm for acute coronary syndrome at our Trust. METHODS: A multicenter evaluation of the analytical performance characteristics of the hs-cTnI assay on the Abbott Alinity ci series was performed in 5 clinical laboratories across Europe. Comparison studies were performed vs the existing Abbott ARCHITECT hs-cTnI assay. RESULTS: Passing and Bablok regression analysis revealed a slope of 0.99 [95% confidence interval (CI), 0.98-1.00] and an intercept of -0.09 ng/L (95% CI, -0.21-0.11). Intermediate imprecision ranged from 3.7% to 5.4%, 2.6% to 4.5%, and 2.0% to 6.1% at concentrations of 19.1-21.1 ng/L, 196.6-205.5 ng/L, and 15229-16265 ng/L, respectively. There was good concordance between the 2 assays at the early rule-out cutoff. CONCLUSION: Comparable analytical performance of the hs-cTnI assay on new Abbott Alinity ci series supports the continued use of the early rule out algorithm for patients with suspected ACS at our Trust.

Analytical Performance of 10 High-Volume Clinical Chemistry Assays on the Alinity c System.

BACKGROUND: Early access for routine testing with the Alinity c clinical chemistry system (Abbot Laboratories) presented the opportunity to characterize the analytical performance of multiple analytes across clinical laboratories in Europe. METHODS: A total of 8 laboratories from 7 European countries evaluated 10 high-volume chemistry assays on the Alinity c system for imprecision, linearity, and accuracy by method comparison to the routine ARCHITECT (Abbott Laboratories) method. RESULTS: Within-run precision was less than 4% coefficient of variation (CV), with total imprecision less than 5.6% CV for 5- and 20-day evaluations. Linearity met expectations, and method comparison showed strong correlation between the Alinity and ARCHITECT methods, with overall linear correlation coefficient between 0.980 to 1.000 and slopes of the regression line between 0.963 and 1.034. Mean percentage difference between the results of assays run on the ARCHITECT and the Alinity ranged between -1.7% and 2.15%. CONCLUSIONS: Our results demonstrated acceptable key analytical performance across all assays tested at each participating laboratory.