Suboptimal rise in awakening-induced cortisol is an accurate marker of cortisol insufficiency in patients with normal renal function (eGFR >60 mL/min).

Background The insulin tolerance test is the gold standard for diagnosis of cortisol insufficiency. However, it is cumbersome, invasive, requires supervised hospital facilities and has unpleasant side-effects. A non-invasive outpatient-based test will be useful. We hypothesized that free cortisol concentrations in multiple spot urine samples can be used to diagnose cortisol insufficiency in patients with normal renal function (eGFR > 60 mL/min). Method Patients and controls provided urine samples at bedtime (S1), and first (S2) and second (S3) void the next day. Cortisol and creatinine were measured in all three samples, and cortisol:creatinine ratio (S1, S2 and S3) was used for further analysis. The sum of S1 + S2 + S3 was used to calculate total cortisol secretion (T). Variation (V) in cortisol secretion in response to circadian rhythm was calculated as the modulus of the difference between S1 and S2 and S2 and S3. Results Samples were collected from 96 controls and 11 patients. S1 was significantly lower vs . S2 and S3 in controls ( P < 0.0001) but not in patients. S2, S3, T and V were significantly lower in patients vs . controls ( P < 0.0001). ROC curve analysis using insulin tolerance test as gold standard showed that S2, S3, T and V were all equally accurate diagnostic markers for cortisol insufficiency (AUC: 0.87, NPV: 100%). The best balance of sensitivity and specificity was achieved using T (sensitivity: 100%, specificity: 58%). Conclusion Multiple spot urine samples test is an accurate, relatively inexpensive, non-invasive, convenient outpatient-based screening test for exclusion of cortisol insufficiency.

Assessment of a semi-quantitative screening method for diagnosis of ethylene glycol poisoning.

Background Ethylene glycol poisoning remains a rare but important presentation to acute toxicology units. Guidelines recommended that ethylene glycol should be available as an 'urgent' test within 4 h, but these are difficult to deliver in practice. This study assessed a semi-quantitative enzymatic spectrophotometric assay for ethylene glycol compatible with automated platforms. Methods The ethylene glycol method was assessed in 21 samples from patients with an increased anion gap and metabolic acidosis not due to ethylene glycol ingestion, and seven samples known to contain ethylene glycol. All samples were analysed in random order in a blinded manner to their origin on a laboratory spectrophotometer. Results In this study, seven samples were known to contain ethylene glycol at concentrations >100 mg/L. The method correctly identified all seven samples as containing ethylene glycol. No false-positives were observed. Thirteen samples gave clear negative results. Ethylene glycol was present at <20 mg/L in one sample, but this sample remained within the limits of the negative control. Passing-Bablock correlation of estimates of ethylene glycol concentration against results obtained when the samples had been analysed using the quantitative method on an automated analyser showed a good correlation (R = 0.84) but with an apparent under-recovery. Conclusions A semi-quantitative assay for ethylene glycol was able to discriminate well between samples containing ethylene glycol and those with other causes of acidosis. It is a practical small-scale assay for rapid identification of cases of ethylene glycol poisoning.

The relationship between pro-resorptive inflammatory cytokines and the effect of high dose vitamin D supplementation on their circulating concentrations.

Inflammatory/pro-resorptive cytokines and chemokines form part of a complex inter-dependent network and may be influenced by vitamin D. We investigated their inter-relationship and the effect of a loading dose of vitamin D. We measured plasma concentrations of an array of cytokines including tumour necrosis factor-alpha (TNF-α), interleukin-1 (IL-1), IL-6, IL-17, IL-8, granulocyte macrophage colony stimulating factor (GM-CSF), and the chemokine monocyte chemoattractant protein-1 (MCP-1). Cytokines, 25 (OH) vitamin D, 1,25 (OH)2 vitamin D, the Wnt inhibitor, DKK1 concentrations and bone turnover markers were measured at baseline and 3 months following a bolus dose (300,000 IU) of vitamin D2 in 39 subjects with vitamin D insufficiency. We observed strong correlations between TNF-α with GM-CSF (r=0.628, p<0.001), IL-17 (r=0.7, p<0.001) and MCP-1 (r=0.5, p=0.001), between IL-1ß with IL-17 (r=0.45, p=0.004) and between the 2 chemokines, IL-8 and MCP-1 (r=0.45, p=0.004). A positive correlation was seen between DKK1 and IL-1ß (r=0.35, p=-0.029). Following vitamin D loading at 3 months, the relationships between some of the cytokines changed (TNF-α and MCP-1: r=0.38, p=0.017, IL-1ß and IL-17: r=0.3, p=0.06). 1,25 (OH)2 vitamin D increased markedly following supplementation. Significant correlations were seen between 25 (OH) vitamin D (r=0.4 p=0.016) and 1,25 (OH)2 vitamin D (r=0.39 p=0.02) with plasma CTX (marker of bone resorption) at 3 months. TNF-α and IL-1ß increased significantly at 3 months (p<0.05). The close association between several cytokines is influenced by vitamin D status. Acute increases in 1,25 (OH)2 vitamin D, achieved with loading doses of vitamin D, lead to increases in pro-resorptive cytokines.

Plasma insulin-like factor 3 (INSL3) in male patients with osteoporosis and Klinefelter's syndrome.

Insulin-like factor 3 (INSL3) is a peptide hormone produced in leydig cells of the testes. Its role in the adult male is unknown but INSL3 and its receptor RXFP2 have been linked to bone cell differentiation. It is speculated that low levels of INSL3 could be responsible for low bone mineral density in patients with primary osteoporosis and Klinefelter's Syndrome. The aim of this study was to assess plasma INSL3 in patients with osteoporosis and Klinefelter's Syndrome compared to healthy males. Fourteen healthy males, 21 males with osteoporosis (4 primary and 17 secondary) and 4 patients with Klinefelter's Syndrome were studied. Plasma INSL3, testosterone, LH, FSH and Sex hormone-binding globulin were evaluated. Plasma INSL3 concentrations were similar in osteoporosis patients compared to healthy controls (0.72 vs. 0.69 ng/mL, p = 0.26). INSL3 was significantly higher in patients with primary osteoporosis (n = 4) compared to age-matched healthy controls (n = 8) (0.845 vs. 0.665 ng/mL, p = 0.021). INSL3 levels in Klinefelter's Syndrome patients were significantly lower compared to healthy controls (0.39 vs. 0.69 ng/mL, p = 0.01). Plasma INSL3 levels were lower in Klinefelter's Syndrome reflecting testicular failure. INSL3 levels were not lower in men with osteoporosis. The relationship between INSL3, its receptor and bone metabolism requires further study.