Elsevier

Clinica Chimica Acta

Volume 359, Issues 1–2, September 2005, Pages 189-194
Clinica Chimica Acta

Plasma free cortisol fraction reflects levels of functioning corticosteroid-binding globulin

https://doi.org/10.1016/j.cccn.2005.03.044Get rights and content

Abstract

Background

In normal plasma free cortisol accounts for less than 6% of the total with 80–90% bound to corticosteroid-binding globulin (CBG) and the remainder associated albumin. However little is known about the distribution of free cortisol in plasma where CBG is inactivated or in congenital CBG deficiency.

Methods and results

Here we describe ligand binding experiments revealing that while free cortisol in unstressed individuals is less than 6% of total cortisol this rises markedly to 25% when CBG is totally inactivated by heat. Similar elevations of the free cortisol fraction were noted in a patient with a rare genetically determined complete lack of CBG (mean 32% on frequent circadian sampling). Following heat inactivation of CBG or in the congenital absence of CBG, there is a shift in cortisol binding from CBG to albumin. That this shift occurs is further supported by experiments adding [3H]-cortisol to physiological human serum albumin solutions, where 25% of cortisol remained in the free fraction.

Conclusion

Taken together the data provide strong evidence that when CBG is inactivated or congenitally absent then more than 25% of the total cortisol appears in the free fraction with the remainder associated with albumin. The proportion of free cortisol measured in plasma thus reflects a simple measure of functional corticosteroid-binding globulin.

Introduction

Human plasma corticosteroid-binding globulin (CBG) is a 52 kDa glycoprotein, which is reported, based on theoretical considerations of albumin and CBG binding affinities, to bind 80–90% of circulating cortisol with high affinity [1], [2], [3]. Albumin bound cortisol is reported to represent 14% and free cortisol 6% of total plasma cortisol under basal conditions [4]. We recently found a patient with a genetically determined complete deficiency of circulating CBG, providing a unique opportunity to examine the distribution of cortisol in plasma [5]. Our aim was to determine the true quantitative role of CBG in binding circulating cortisol and to this end we studied healthy subjects' plasma with ligand competition with and without heat treatment to inactivate CBG. We also measured circadian total and free cortisol levels in a CBG-deficient subject and a control individual. We found that CBG binds more than 80% of circulating cortisol in normal intact plasma. However when CBG is inactivated by heat or absent then the majority of cortisol becomes associated with albumin (75%) with the percentage free cortisol rising markedly to 25% or more.

Section snippets

Measurements of free and total cortisol

Plasma was collected from normal subjects in EDTA-containing tubes between 0800 and 1000 h, unless otherwise stated, and from subjects undergoing an ACTH 1–24 (cosyntropin 250 μg i.m.) with cortisol levels at 0 and 30 min post-dose. Plasma was stored frozen until required. Free cortisol was measured by an ultrafiltration/ligand binding method [6] validated in our laboratory. Briefly, plasma (0.5 mL) was equilibrated with [3H]-cortisol (0.1 μCi, Amersham TRK 407) for 30 min at 37 °C followed by

Results

Free cortisol fractions in plasma and albumin, expressed as a percentage (free cortisol/total cortisol × 100) are shown in Table 1. Untreated normal plasma showed expected free cortisol fractions of less than 6% which is similar to normal subjects (Table 2). Heat treatment of plasma led to undetectable CBG levels by ELISA and unchanged total cortisol levels. Either heat-treatment of plasma or adding an excess of unlabelled cortisol or both led to free cortisol fractions of 20–26%. Human albumin

Discussion

Our data indicate that while most cortisol is bound to CBG under normal conditions and less than 6% is free this increases markedly to between 25% and 35% in CBG-deficient individuals and when plasma CBG is heat inactivated. This is also in contrast to patients with abnormally high total basal cortisol levels (1500–2000 nmol/L, data not shown) or following cosyntropin where the free cortisol normally rises but only to a maximum of 14% (Table 3) when CBG would be expected to be saturated. The

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