Authors' response: Poor correlation between 6β-hydroxycortisol:cortisol molar ratios and midazolam clearance as measure of hepatic CYP3A activity: a comment

We appreciate the comments of Dr Fenske [1] in the above letter. However, we disagree with his conclusions regarding the utility of the cortisol ratio once time of day and fluid intake are controlled.

First, it is incorrect to assume that a population has similar corticosteroid production patterns. To this end, morning spot urine collections have been utilized for the cortisol ratio with little success. For example, Tran et al. [2] demonstrated a high intra-individual variability (>50%) in the cortisol ratio obtained from a morning spot urine. Sleep–wake cycles affect the timing of peak cortisol concentrations [3] and episodic cortisol secretion occurs from adrenal glands in response to such varied factors as stress, nutrition and environment [4, 5]. Therefore, we disagree with Dr Fenske and believe that our collection of a 24-h urine sample for the cortisol ratio in fact assisted in normalizing for variable peak cortisol concentration times.

Although Dr Fenske is correct in stating that the variability of urinary 6β-hydroxycortisol:cortisol ratios can be influenced by fluid intake [6], urine production and elimation can also be influenced by such factors as insensible losses and fluid shifts from positioning [7]. To this end, it would be unlikely that a group of individuals given the same amount of fluid over 24 h would have the same urine flow and volume. Although Furuta et al. [8] have stated that the urinary ratio of 6β-hydroxycortisol:cortisol was valid when the renal clearance of cortisol was constant, even within the same subject there were 2.1–4.6-fold variations in the renal clearance of cortisol. It has also been demonstrated that higher fluid intake (5 l) increases the secretion of free cortisol disproportional to metabolites (17-hydroxycorticosteroids), whereas normal fluid intake does not [6]. In our study, the average urine volume was 530 ml and ranged from 375 ml to 915 ml. This is within the range of normal fluid intake and urine production. Median (range) intra-individual variability (as measured by the coefficient of variation) in urine volume was 25.5% (12.2–26.3%). Therefore, the high inter- or intra-individual variability of the urinary 6β-hydroxycortisol:cortisol ratio found in our study was probably a result of other factors than just fluid intake.

In summary, Dr Fenske has highlighted the impracticality of controlling all factors that can possibly contribute to high inter- or intra-individual variability of the urinary 6β-hydroxycortisol:cortisol ratio. Therefore, we believe Dr Fenske's comments further endorse our conclusion that the urinary 6β-hydroxycortisol:cortisol ratio is a suboptimal phenotyping tool.