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

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.

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.

AimsTo 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.MethodsIn 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).ResultsThe 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.ConclusionWe 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.