Blood pressure and steroid metabolism in the first year of life in patients with congenital adrenal hyperplasia (CAH): a longitudinal study

Congenital adrenal hyperplasia (CAH) is a disorder of adrenal steroidogenesis.
In 95% of cases it is caused by 21-hydroxylase deficiency. Deficiency of
21-hydroxylase results in impaired adrenal synthesis of cortisol and often also
of aldosterone leading to increased secretion of ACTH, adrenal hyperplasia and
excessive production of adrenal precursors before the enzymatic bloc such as 17
hydroxyprogesterone. The production of adrenal androgens is not disturbed.
Therefore, adrenal hyperplasia will lead to excessive production of adrenal
androgens. Treatment with glucocorticoids and, if necessary, mineralocorticoids
prevents adrenal crises and suppresses abnormal secretion of adrenal androgens.
Usually supraphysiological doses of glucocorticoids are needed to suppress
androgen levels. In the case of illness the glucocorticoid dosage even has to
be increased.
Patients with CAH, who are treated with supraphysiological doses of
glucocorticoids, are at risk of developing signs and symptoms of Cushing*s
syndrome. As hypercortisolism is associated with hypertension, obesity with
abdominal fat accumulation and diabetes mellitus, it is not unlikely that
patients with CAH may show an adverse cardiovascular and metabolic risk
profile, possibly leading to a reduced life expectancy. Furthermore,
mineralocorticoid excess may play a role in the development of high blood
pressure. In most studies in older children with CAH a tendency towards high
blood pressure was shown. Blood pressure has not been studied in CAH patients
within the first year of life. Our pilot study showed that within the first
weeks of life blood pressure is not significantly elevated in the first year of
life. However, evaluation of the first week of life was not performed.
Not only medical treatment but also elevated concentrations of adrenal
androgens and other elevated steroid metabolites may cause an unfavorable
cardiovascular risk in CAH patients. It has been reported that in healthy
full-term newborn Japanese neonates the levels of adrenal 16 alpha-, 16 beta-,
and 15 beta-hydroxy metabolites of 3 beta-hydroxy-5-en-steroids, and 6 beta-,
18-hydroxy and 11-oxo-metabolites of corticosteroids are significantly higher
than in healthy adults. In Caucasian healthy neonates and in CAH patients the
full spectrum of adrenal steroids has not been studied with currently available
very sensitive and specific techniques.

To evaluate the blood pressure within the first year of life we will perform a
prospective longitudinal study in all newborn patients with the classical form
of CAH.
To analyze the urinary steroid profile in CAH patients and healthy controls
during the first weeks of life.

Blood pressure measurements

Newborn female CAH patients are usually hospitalized in our centre due to
ambiguous genitalia. Male newborn CAH patients will be usually hospitalized in
our center after presentation with a positive neonatal CAH screening. All blood
pressure measurements will be performed using an appropriate sized cuff on the
Dinamap Vital Signs Monitor (GE Healthcare, Finland). Before and after
initiation of treatment with hydrocortisone and fludrocortisone blood pressure
will be measured at a three hour time interval during 24-hours to evaluate the
direct effect of fludrocortisone and hydrocortisone on the blood pressure, as
ambulatory 24-hour blood pressure measurement devices are not applicable for
neonates.
During every visit to our outpatient clinic (that routinely takes place every
one to two weeks within the first two months and thereafter every 6 weeks)
blood pressure will be measured before taking the medication (08.30 a.m.). An
experienced nurse will measure all blood pressures with the parents calming the
patients, for example by feeding the child. When the child cannot be calmed the
blood pressure will be measured again at the end of the clinical visit when the
infant is calmed or sleeping. Thereafter, blood samples will be collected and
physical examination will take place according to our current follow up
protocol. Hydrocortisone replacement consists of 1 mg hydrocortisone
thrice-daily during the first eight months of life. The fludrocortisone dose
will be adjusted to serum renin levels and if blood pressure is above the 90th
percentile for age and sex.
This study will be performed within the Radboud University Nijmegen Medical
Center, department of pediatric endocrinology. No additional blood samples have
to be taken for this research project and patients will only be followed
standardized, with special attention for blood pressure measurements, during
the first year of life.
Data will be collected prospectively using a case report form. Data will be
collected by the peadiatric endocrinologists in the Radboud University Nijmegen
Medical Centre after clinical visits of the CAH patients. Data concerning blood
pressure levels will be recorded by experienced nurses, working at the
paediatric endocrinology department.

24-hour urine collection

24-hour urine will be collected in controls while they are hospitalized. In
general this will be from day 0 until day 5. 24-hour urine will be collected in
CAH patients during their hospitalization after they presented to the hospital.
Prior to every visit to our outpatient clinic parents will be asked to collect
24-hour urine nappies.
Collections can only be carried out with nappies that contain fluid-absorbing
granules such as Pampers, Huggies or similar. The same type of nappy should be
applied throughout the collection day and parents or nurses should avoid
putting creams on the baby*s bottom. A clean nappy of the same size and brand
have to be provided. This information will be used to determine the dry weight
of the specific nappy. In principle, we can*t make use of nappies that are
contaminated with faeces but only can process nappies that contain urine only.
Nurses and parents should place water-resting liners on the inner lining of the
nappies. If upon changing the nappy only contains urine, both nappy and liner
can be put in a collection bag. If the nappy contains faeces than the liner
with the faeces should be discarded while the nappy is put in the collection
bag. Odd stains and spills of faeces do not represent a problem but a
completely soiled nappy can*t be used. The collection bag with the collected
nappies will be kept in the -20 C freezer in the Radboud University Nijmegen
Medical Centre in a plastic bag that has been labeled with the name and date of
collection. The nappy bag will be sent to Birmingham on dry ice and include
the dry, clean nappy.
The urine will be extracted from the nappies and prepared for gas
chromatography-mass spectrometry (GC-MS) analysis to characterize the urine
steroid metabolome in the first weeks of life. The analysis of 24-hour urine
steroid metabolite profiles using gas chromatography/mass spectrometry is well
established as a diagnostic tool. The GC/MS set-up at the Centre for
Endocrinology, Metabolism and Diabetes at the University of Birmingham, UK,
allows for the simultaneous identification and quantification of 35 steroid
metabolites, and can be used as an integrated read-out of steroid metabolites
that specifically accumulate in CAH. Since samples are always run in scanning
mode, the traces can be further analyzed for other compounds. GC/MS has proven
its role as a pre-eminent discovery tool in clinical steroid investigations
even in the era of fast tandem mass spectrometry as it provides the opportunity
to visualize the entire steroid metabolome in one run. In the past, this
approach has defined conditions such as AME syndrome, glucocorticoid remediable
aldosteronism (GRA), P450 oxidoreductase deficiency (ORD) and apparent
cortisone reductase deficiency (ACRD). The profiles obtained from CAH patients
will be compared with urine steroid profiles from patients with other forms of
inborn primary adrenal insufficiency, which commonly present with early onset
mineralocorticoid deficiency during the first days of life.

None