Key words
urinary cortisol - adrenal insufficiency - hydrocortisone replacement therapy
Introduction
Patients with adrenal insufficiency (AI) require life-long cortisol replacement therapy
usually consisting of 2 or 3 hydrocortisone doses, the last dose in mid-afternoon
or early evening. These treatment regimens, however, fail to mimic the circadian rhythm
of cortisol secretion. Due to the short half-life of hydrocortisone, cortisol levels
during the night are invariably low, whereas they are found to be supraphysiological
2 h after ingestion of hydrocortisone [1]
[2]
[3]. The consequences of nocturnal cortisol deficiency as well as those of temporary
hypercortisolism are yet unknown [4]; however, inadequate treatment with short-acting glucocorticoids may result in adrenal
crisis [5]. It has also been suggested that inadequate levels of cortisol in hydrocortisone
replacement therapy (HRT) lead to a higher prevalence of cardiovascular disease and
diabetes mellitus and affect quality of life [6]
[7]. Therefore, surveillance of cortisol replacement therapy is essential to avoid serious
side effects. Due to the pharmacokinetic structure of oral hydrocortisone, biochemical
analyses of serum cortisol are of no use and surveillance should be achieved through
clinical monitoring [8]. Recently, several studies used urinary corticosteroid profiles to monitor replacement
doses in patients with adrenal hypofunction [9]
[10]. However, a possible difference in the metabolism of cortisol between healthy subjects
and patients with primary or secondary AI may confound interpretation. In fact, little
information is available whether urinary excretion of cortisol is a useful alternative
to assess the quality of HRT. Aim of the present study was to perform a quantitative
analysis of urinary cortisol excretion of affected AI patients compared to healthy
subjects.
Materials and Methods
Fourteen patients with primary or secondary AI (11 women, 3 men, age 29–70 years)
received hydrocortisone infusion between midnight and 08:00 AM at a dose according
to Al-Shoumer et al. [11], in order to mimic the physiological rise in healthy subjects ([Table 1] for hydrocortisone infusion rates) and to identify possible differences in cortisol
metabolism between AI patients and controls. Blood for serum cortisol analysis was
drawn at 08:00 AM. The study was approved by the Ethics committee at the University
of Kiel (AZ D 424/03) and informed consent was obtained from each subject.
Table 1 Hydrocortisone infusion rate.
|
Time
|
Hydrocortisone dose (μg/kg/min)
|
|
00:00–01:00 AM
|
0.15
|
|
01:00–02:00 AM
|
0.15
|
|
02:00–03:00 AM
|
0.10
|
|
03:00–04:00 AM
|
0.20
|
|
04:00–05:00 AM
|
0.20
|
|
05:00–06:00 AM
|
0.80
|
|
06:00–07:00 AM
|
0.90
|
|
07:00–08:00 AM
|
0.63
|
Urinary samples were analysed for cortisol excretion levels by using a commercial
immunoassay (Immulite 2000, Siemens Healthcare Diagnostics, Deerfield, IL, USA). Healthy
volunteers without clinical signs of hypercortisolism collected their urine from 00:00
to 08:00 AM to serve as controls. A history of pathological renal or liver test results
was an exclusion criterion, and all participants of the control group were reported
to have no prior existing endocrine disorder. The volunteers were divided into 3 subgroups
depending on age: 20–34 years, 35–49 years, and at least 50 years old. In AI patients,
values urinary and blood cortisol were comparatively analysed. Values of urinary cortisol
excretion are depicted as mean±standard deviation as well as range per group, and
were compared between groups using student’s t-test. A p-value<0.05 was considered significant. Fisher’s exact test was used to
compare categorical data. To exclude a potential role of renal function on urinary
cortisol excretion, serum creatinine values of AI patients were measured with subsequent
calculation of the glomerular filtration rate (eGFR) using the CKD-Epi formula that
incorporates age and sex. Pearson’s correlation coefficient was determined to assess
the relationship between serum and urinary cortisol levels in AI patients as well
as between age or glomerular function and urinary cortisol excretion.
Results
A total of 14 AI patients and 53 healthy volunteers were included in the study. Both
groups were comparable with respect to age (51.1±12.3 vs. 43.1±14.3 years), body weight
(74.8±13.3 vs. 73.3±15.0 kg), and BMI (26.5±4.9 vs. 24.2±3.7 kg/m²). Median age of
primary and secondary AI patients was 54 vs. 43 years (p=0.27); body weight was not
significantly different (78.5 vs. 73.7 kg; p=0.77).
Healthy volunteers showed a urinary cortisol excretion rate of 14.0±7.8 μg/8 h with
a wide inter-individual range (0.24–35.4). There was no significant difference in
cortisol excretion rates between the healthy subgroups of subjects ([Table 2]). In the subgroup of healthy volunteers between 20 and 34 years (n=19, 13 females),
the mean cortisol urinary excretion rate was 13.1±6.4 μg/8 h; in the subgroup of volunteers
between 35 and 49 years (n=16, 9 females) 16.2±9.1 μg/8 h and in the group >50 years
(n=18, 9 females), the mean rate was 12.8±7.5 μg/8 h. Although the fraction of women
in the AI group was higher than in all controls, the difference was not significant
(58 vs. 79%, p=0.32). In the AI patients, age was comparable (median 51 vs. 51 years,
p=0.65), with 3 male participants in the secondary AI group. AI patients receiving
hydrocortisone infusions showed significantly higher urinary cortisol excretion rates
than healthy controls (51.6±27.8 μg/8 h; range: 17.1–120.0; p<0.0001; [Table 2]). Of note, patients with secondary AI (n=8) showed significantly higher serum cortisol
levels and urinary cortisol excretion rates after hydrocortisone infusion than the
group with primary AI (n=6; p<0.05; [Table 2]). Urinary values clearly correlated with serum cortisol levels in patients with
both primary and secondary AI (r2=0.98, [Fig. 1a]). Urinary cortisol excretion rates in both groups of healthy volunteers and AI patients
showed no significant correlation regarding age ([Fig. 1b]) and body weight (r2=0.20, data not shown). Similarly, renal function in the AI patients (mean creatinine
0.78±0.15 mg/dl, range: 0.6–1.06) did not correlate with urinary cortisol excretion
([Fig. 1c]). There was no significant difference in eGFR between both groups with primary and
secondary AI (100.5±8.7 ml/min/1.73 m2 vs. 90.3±18.9 ml/min/1.73 m2, p=0.28).
Fig. 1 a Patients with adrenal insufficiency (AI) receiving hydrocortisone infusions showed
urinary cortisol values that clearly correlated with serum cortisol levels. b In both groups, patients with AI as well as healthy volunteers, there was no significant
correlation between urinary excretion of cortisol and age. c Urinary cortisol values in AI patients were independent from renal excretory function,
depicted as estimated glomerular filtration rate that was determined by the CKD-Epi
formula (eGFR-CKD-Epi). (Color figure available online only).
Table 2 Urinary cortisol excretion rates (8 h) and serum cortisol levels in healthy subjects
and patients with primary or secondary adrenal insufficiency.
|
Healthy subjects
|
Adrenal insufficiency
|
|
Urinary cortisol excretion rate [μg/8 h]
… under normal conditions
|
Urinary cortisol excretion rate [μg/8 h]
… following HC infusion
|
Serum cortisol level[μg/dl]
|
|
Subject
|
20–34 years
|
35–50 years
|
>50 years
|
Primary
|
Secondary
|
Primary
|
Secondary
|
|
1
|
2.6
|
0.24
|
3.3
|
17.1
|
25.8
|
6.7
|
9.9
|
|
2
|
3.4
|
4.3
|
4.9
|
21.7
|
39.0
|
10.3
|
17.7
|
|
3
|
6.1
|
6.7
|
6.0
|
31.8
|
42.0
|
12.3
|
19.4
|
|
4
|
6.5
|
8.6
|
6.8
|
35.7
|
53.4
|
14.8
|
20.6
|
|
5
|
8.0
|
10.8
|
7.4
|
42.6
|
64.8
|
17.0
|
21.4
|
|
6
|
9.4
|
10.8
|
7.6
|
64.0
|
70.4
|
17.9
|
22.0
|
|
7
|
9.6
|
11.5
|
9.1
|
93.6
|
29.5
|
|
8
|
10.0
|
14.7
|
11.1
|
120.0
|
40.3
|
|
9
|
10.2
|
18.2
|
11.5
|
|
10
|
12.6
|
19.2
|
12.0
|
|
11
|
15.5
|
19.3
|
12.6
|
|
12
|
15.5
|
19.8
|
13.8
|
|
13
|
15.7
|
25.0
|
15.0
|
|
14
|
17.0
|
28.6
|
15.0
|
|
15
|
18.4
|
30.7
|
16.1
|
|
16
|
21.0
|
31.0
|
17.9
|
|
17
|
21.5
|
25.2
|
|
18
|
22.5
|
35.4
|
|
19
|
24.0
|
|
Mean
|
13.1
|
16.2
|
12.8
|
35.5
|
63.6
|
13.2
|
22.6
|
|
SD
|
6.4
|
9.1
|
7.5
|
15.3
|
29.0
|
3.9
|
8.4
|
|
Mean
|
14.0
|
51.6
|
18.6
|
|
SD
|
7.8
|
27.8
|
8.3
|
HC: Hydrocortisone
Discussion
Urinary cortisol excretion over time has been considered a potential tool in diagnosing
and monitoring AI patients with HRT. Since little is known under normal conditions,
we determined the excretory rate in healthy subjects over the first 8 h of a normal
work day. In addition, we used a continuous hydrocortisone infusion in patients with
AI in order to mimic the physiological biorhythm during this timeframe, as previously
described [11]. Furthermore, we intended to determine possible differences in cortisol metabolism
between healthy subjects and in patients with primary and secondary AI. Jung et al.
found that total plasma cortisol correlates significantly with urinary cortisol after
intravenous hydrocortisone application [10]. In the present study, we could confirm these findings in AI patients receiving
hydrocortisone infusions. Interestingly, there was a strong inter-individual variability
of cortisol excretion levels despite using a uniform weight-adapted hydrocortisone
infusion rate. This may point to a limited diagnostic value of urinary cortisol measurement
despite its wide use in clinical practice. Recently, Raff and colleagues addressed
this variability in urine cortisol levels and discussed the controversies of using
urine free cortisol in the diagnosis of Cushing’s syndrome [12]. The most intriguing finding in the present study, however, is a difference between
the AI subgroups: Values for serum and urinary cortisol were significantly higher
in patients with secondary compared to primary AI. Interestingly, the infusion doses
used seemed to be excessive in almost half of the patients with secondary AI. It is
possible that endogenous secretion of cortisol in the AI patients may have an effect
on the observed variants of urinary and serum cortisol levels. Thus, urinary cortisol
levels despite their variability may help to identify residual adrenal function in
AI. In addition, these data suggest that AI patients may require an individualised
dose of hydrocortisone. Normally, the majority of circulating cortisol is bound to
CBG and albumin with less than 5% of circulating cortisol as the physiologically active
being free. This unbound cortisol is filtered by the renal glomerulus and hereafter
excreted in the urine. CBG is mostly produced by the liver, upregulated by estrogens,
and suppressed by steroids with high levels observed in pregnancy and low levels in
patients with cirrhosis [13]
[14]. Since we only included healthy patients as controls, liver function was not considered
to have an effect on cortisol levels. Although also oral contraceptives may have had
an effect on urinary cortisol levels in the AI patients, this is unlikely because
age was comparable between both groups and the 3 males were in the secondary AI where
urinary cortisol levels were significantly higher.
While the present data point to a possible weak relationship between urinary cortisol
and age, Ragnarsson and co-workers recently demonstrated in a cross-sectional study
including a population sample of 348 persons aged 38–77 years that urinary free cortisol
rather decreased in the elderly [15]. The number and sort of individuals included in our study is different though so
that the results may not be comparable. Previous studies have shown that glomerular
filtration rate and the amount of fluid intake may have an effect on urinary cortisol
excretion rates [16]
[17]. However, we did not see a statistically significant correlation between renal function
and urinary cortisol excretion rates in our AI patients receiving hydrocortisone infusions.
There were also no differences with respect to glomerular filtration rate when patients
with primary and secondary AI were compared. This may be due to the fact that renal
function in AI patients was not seriously impaired as demonstrated by normal or only
slightly elevated serum creatinine levels but may be distinct in patients with more
pronounced renal insufficiency.
It has been shown that urinary cortisol excretion is dependent on the corticosteroid-binding
globulin capacity. Intravenous and oral hydrocortisone substitution exceeds the binding
capacity of corticosteroid globulin, temporarily leading to higher cortisol excretion
rates in patients receiving hydrocortisone replacement therapy. A possible residual
adrenal function in patients with secondary AI may lead to a faster saturation of
CBG capacity resulting in significantly higher urinary cortisol excretion rates in
these patients. This has also been described in patients suffering from Cushing’s
disease, where supraphysiological levels of cortisol are a result of the pathophysiological
mechanism [18]. Although measuring urinary cortisol excretion allows to determine endogenous cortisol
production, its use as a monitoring instrument may not be conceivable. Highly variable
levels of serum cortisol in hydrocortisone replacement therapy render the monitoring
of AI difficult. Other factors like liver disease, nephrotic syndrome, and oral estrogens
must also be taken into account since they influence the amount of corticosteroid
binding globulin [19]
[20]. Finally, different types of assays are used to assess urinary cortisol excretion.
These assays can be antibody-based or structurally-based, leading to varying reference
values and different levels of specificity and sensitivity [21].
The present study harbors some strengths and limitations related to the study design.
The healthy control group was large enough to determine physiological urinary cortisol
excretion. In addition, the setting of hydrocortisone infusions in AI patients within
the first 8 h of a day is ideal to mimic the physiological peak of endogenous cortisol
production in the morning. Contrary to Al-Shoumer et al. [11], whose study design was limited to hypopituitary adults, the group of AI patients
investigated here was somewhat heterogeneous with regard to the underlying diseases;
for example, patients with pituitary diseases often have an ACTH-independent residual
secretion of cortisol, which may have influenced some results. However, there was
a wide range of values possibly in part due to the small sample size in this pilot
study.
In conclusion, we showed that: (i) urinary cortisol excretion rates vary strongly
in healthy subjects and patients with AI; (ii) cortisol metabolism in AI patients
is different compared to controls; (iii) urinary cortisol correlates well with serum
cortisol in patients with AI; and (iv) patients with secondary AI demonstrate significantly
higher urinary and serum cortisol levels following HC infusions than those with primary
AI. Presumably, urinary cortisol may be helpful in the assessment of residual adrenal
function in patients with AI rather than to be useful as a monitoring parameter for
HRT.
