False Positive Very Long-Chain Fatty Acids in a Case of Autoimmune Adrenal Insufficiency

Abstract

Background:

X-linked adrenoleukodystrophy (ALD) affects up to 25% of boys diagnosed with adrenal insufficiency in childhood. Because early identification of these individuals can be lifesaving, all boys with new-onset primary adrenal insufficiency should be tested for ALD with a plasma very long-chain fatty acid (VLCFA) level. While plasma VLCFA is a diagnostic test with high sensitivity and specificity, false positive results have been reported in individuals on a ketogenic diet.

Case presentation:

We present a case of an 11-year-old boy with new-onset primary adrenal insufficiency due to autoimmune adrenalitis who was initially found to have elevated VLCFA levels, suggestive of ALD, that normalized on repeat testing.

Conclusions:

As advances in gene therapy and newborn screening for ALD expand, VLCFA testing may increase, and clinicians should be aware that testing during the initial presentation of primary adrenal insufficiency may lead to false positive results and associated psychosocial distress.

Background

Primary adrenal insufficiency is a life-threatening disorder caused by deficient production of cortisol and aldosterone by the adrenal cortex. In a school-aged child with new-onset adrenal insufficiency, a common etiology is autoimmune adrenalitis, commonly referred to as Addison’s disease [1]. Primary adrenal insufficiency in boys can also be the initial manifestation of X-linked adrenoleukodystrophy (ALD), a devasting disorder of peroxisomal oxidation that can lead to rapid neurological deterioration, disability, and death in the one-third of boys with the cerebral phenotype [2]. Because treatment with hematopoietic stem cell transplantation (HSCT) can modify the disease course if performed during the early stages of cerebral disease, all boys with new-onset primary adrenal insufficiency should be tested for ALD by measuring the plasma very long-chain fatty acid (VLCFA) level. While the diagnostic VLCFA test has high sensitivity and specificity, false positive results have been reported in individuals on a ketogenic diet, with liver insufficiency, and in one case of diabetic ketoacidosis [3, 4]. Because of the possibility of false positives, a positive VLCFA test should be repeated to establish the diagnosis of ALD, and genetic testing is recommended when possible to confirm the diagnosis in boys [2].

We report a case of an 11-year-old boy with new-onset primary adrenal insufficiency caused by autoimmune adrenalitis, who was initially found to have elevated VLCFA levels, suggestive of ALD, that normalized on repeat testing. This case illustrates the limitations of the VLCFA test for the diagnosis of ALD during the initial clinical presentation of autoimmune adrenal insufficiency.

Case Presentation

An 11-year-old boy with a history of cyclic vomiting presented to an emergency department with vomiting and abdominal pain after several weeks of fatigue, anorexia, headache, and a 7-lb weight loss. On examination, his blood pressure was 72/43 mmHg, and he had a thin body habitus and diffuse hyperpigmentation. Laboratory evaluation showed a plasma glucose of 43 mg/dL; serum sodium 135 and potassium 3.7 mmol/L; BUN 36 and creatinine 0.8 mg/dL; serum creatine kinase 960 U/L. His complete blood count showed a hemoglobulin of 12.8 g/dL and hematocrit 37.4. His liver function tests were unremarkable, and his urinalysis showed 2+ ketonuria. He received several dextrose-containing intravenous saline boluses and was started on dextrose-containing intravenous saline prior to transfer to our hospital, a quaternary care facility. On arrival to our hospital, he was started on D10-based intravenous saline at a rate of 1.5 times maintenance for persistent hypoglycemia and was admitted to the floor.

His past medical history was notable for five years of recurrent episodes of vomiting and abdominal pain associated with elevations in creatine kinase to ~1000 U/L that required hospitalization approximately three times per year. Nine months before his presentation, he had a severe episode associated with acute renal failure that improved with supportive therapy. He had no prior episodes of hypoglycemia or muscle pain, and the episodes were not precipitated by strenuous physical activity. Family history revealed that his mother had hypothyroidism and rheumatoid arthritis, and his maternal uncle had type 1 diabetes. There was no history of genetic or metabolic disorders.

The endocrinology service was consulted for the evaluation of adrenal insufficiency, which revealed an undetectable serum cortisol level at baseline that remained undetectable 60 minutes after receiving intravenous high-dose (250 μg) Cosyntropin. Several days later, his remaining laboratory evaluation resulted and showed a markedly elevated plasma ACTH level of 1380 pg/mL (normal: ≤46 pg/mL), elevated plasma renin activity of 9.0 ng/mL/hr (reference range: 0.3 to 3.0 supine), and an undetectable aldosterone level. He was diagnosed with primary adrenal insufficiency. He responded well to dextrose-containing intravenous fluids, and by day 3 of hospitalization, he appeared clinically well and was started on maintenance glucocorticoid replacement with hydrocortisone 5 mg three times per day (14 mg/m²/day) and stress-dose steroids for significant physiologic stress, as needed, in addition to mineralocorticoid replacement as fludrocortisone 0.1 mg daily.

To assess the etiology of his primary adrenal insufficiency, a plasma sample obtained on day five of the admission was sent to the Kennedy Krieger Institute for measurement of VLCFA and showed an elevated C26:0 of 0.698 μg/mL (normal: 0.22 [mean] ± 0.08 [SD] μg/mL) and an elevated C26:0/C22:0 ratio of 0.045 (normal: 0.01 ± 0.01) that were suggestive of a defect in peroxisomal fatty acid oxidation, such as ALD. At the time of his initial presentation in the year 2000, commercial genetic testing of the ABCD1 gene for ALD was not available. Thus, repeat plasma VLCFA samples were obtained for confirmation two weeks and six months after the initial presentation, which showed normal VLCFA profiles with a C26:0 level of 0.255 μg/mL and 0.175 and a C26/C22 ratio of 0.011 and 0.015, respectively. To assess for an autoimmune etiology of primary adrenal insufficiency, an adrenal antibody test was obtained, which showed detection of antibodies at 1:8 (normal <1:4). A follow-up 21-hydroxylase antibody level was markedly elevated at 29 U/mL (normal: <1 U/mL), which further confirmed the diagnosis of Addison’s disease.

Given his history of elevations in creatinine kinase, he was referred to the metabolism service for a consultation to evaluate the possibility of an underlying myopathy. Extensive laboratory testing, including assessment of acylcarnitines, acylglycines, carnitine, free carnitine, lactate, pyruvate, ammonia, serum amino acids, and urine organic acid levels, did not identify a metabolic defect. His serum creatinine kinase levels normalized after discharge, and he had no further episodes of emesis and dehydration requiring hospitalization or documented elevations in creatine kinase over a 15-year follow-up period.

Discussion

In this report, we describe a case of a false positive result for the diagnostic VLCFA test for ALD in a boy who was ultimately diagnosed with autoimmune primary adrenal insufficiency. Our report highlights a limitation of VLCFA testing during the acute presentation of primary adrenal insufficiency.

Among boys with primary adrenal insufficiency, ALD accounts for approximately one-third of cases not caused by Addison’s disease [5]. In addition, nearly half of boys with ALD develop adrenal insufficiency by 10 years of age [6]. As early identification of ALD can be lifesaving, all boys with new-onset primary adrenal insufficiency should be tested with a plasma VLCFA level. The diagnosis of ALD made by an elevated VLCFA level has significant implications and is followed by urgent referrals to pediatric subspecialists to facilitate genetic testing and counseling, brain MRI and, in some cases, consideration for HSCT. Thus, false positive VLCFA results can lead to substantial psychosocial distress for the patient and family while awaiting confirmatory test results.

Although non-fasting or hemolyzed samples may cause false positive VLCFA results, false positives are rare and have been most commonly reported in individuals on a ketogenic diet for the treatment of epilepsy, after acute ingestion of peanut butter, and with liver insufficiency [3, 4, 7-9]. Although the exact mechanism of VLCFA elevation in these cases are unknown, VLCFA elevations been proposed to be secondary to an imbalance of exogeneous fatty acid intake (excess in ketogenic diet and peanut ingestion) and the liver’s peroxisomal oxidation capacity (reduced in liver insufficiency) [3, 4]. In states of acute metabolic stress associated with dehydration and starvation, such as a previously reported case of diabetic ketoacidosis and possibly the current case of an adrenal crisis, fatty acids are mobilized to form ketones as an alternative fuel source to glucose [3]. Thus, it is possible that increased mobilization of fatty acids in states of physiologic stress could overwhelm the oxidation capacity of the peroxisome and result in transient elevations of VLCFA. Regardless of the mechanism, alterations in diet, liver function, and/or metabolic state can cause false elevations in VLCFA levels in individuals without a peroxisomal disorder. Thus, any positive VLCFA test should be repeated in the fasting state at the individual’s baseline clinical status.

Recent advancements in gene therapy and newborn screening for ALD are expected to increase the number of individuals tested for ALD by plasma VLCFA. A phase 3 clinical trial for ALD gene therapy was recently opened (January 2019, NCT03852498) following promising results of the initial phase 2/3 Starbeam study, which provides additional rationale for the early identification and treatment of affected boys [10]. More than a dozen U.S. states are currently screening for ALD in newborns, the number of which is expected to double in the year 2020 [11, 12]. Confirmatory plasma VLCFA testing for both male and female infants identified through newborn screening and at-risk family members will likely be a part of the multi-disciplinary evaluation process, which may vary by state [13-15]. As VLCFA testing expands with these efforts, clinicians should be aware of the limitations of this test, particularly false positive results during and in the days following the acute presentation of primary adrenal insufficiency. Thus, we recommend that clinicians aim to obtain the VLCFA level after complete resolution of an adrenal crisis (i.e., during an outpatient follow-up visit) to reduce the potential for false positive results.

Learning Points

1. ALD should be considered in any boy presenting with new-onset adrenal insufficiency, and all such boys should be tested with measurement of plasma VLCFA levels.

2. During and immediately following the presentation of acute adrenal insufficiency (not caused by ALD), plasma VLCFA may be falsely elevated.

3. When ALD is suspected, clinicians should avoid obtaining a plasma VLCFA level at the time of the initial diagnosis of primary adrenal insufficiency, which may lead to a false positive result for ALD.

What is new?

1. A false positive VLCFA result for ALD was observed in a boy who presented with new-onset primary adrenal insufficiency due to autoimmune adrenalitis.

2. During the initial presentation of primary adrenal insufficiency not due to ALD, VLCFA can be transiently elevated and falsely suggestive of ALD.

3. Although the plasma VLCFA level is considered a diagnostic test for ALD, any positive VLCFA test should be repeated in the fasting state at the individual’s baseline clinical status.

Abbreviations:

ALD

X-linked adrenoleukodystrophy

HSCT

hematopoietic stem cell transplantation

VLCFA

very long-chain fatty acid