CLB-09236; No. of pages: 4; 4C:
Clinical Biochemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Clinical Biochemistry
journal homepage: www.elsevier.com/locate/clinbiochem
Short Communication
Analytical and clinical validation of an LC–MS/MS method for urine
leukotriene E4: A marker of systemic mastocytosis
Alan J. Lueke a, Jeffrey W. Meeusen a,⁎, Leslie J. Donato a, Amber V. Gray a, J.H. Butterfield b,c, Amy K. Saenger a
a
b
c
Department of Laboratory Medicine & Pathology, Mayo Clinic, Rochester, MN, United States
Department of Medicine, Division of Allergic Diseases, Mayo Clinic, Rochester, MN, United States
Mayo Clinic Program for Mast Cell and Eosinophil Disorders, Mayo Clinic, Rochester, MN, United States
a r t i c l e
i n f o
Article history:
Received 31 December 2015
Received in revised form 15 February 2016
Accepted 16 February 2016
Available online xxxx
Keywords:
Allergic disease
Mast cell disorder
Arachidonic acids
Prostaglandin
Histamine
Tryptase
Mass spectrometry
Method development
a b s t r a c t
Objectives: Systemic mastocytosis (SM) is a disorder characterized by the excessive accumulation of clonally
derived mast cells in various tissues. When triggered, mast cells release large amounts of histamine, prostaglandins and leukotrienes. Leukotriene E4 (LTE4) is the primary stable metabolite of total cysteinyl leukotrienes. We
hypothesized that secretion of LTE4 would be increased in SM and could be used alone or in combination with
current urinary biomarkers to optimize screening for SM.
Design and methods: LTE4 was measured by liquid chromatography followed by tandem mass spectrometry
(LC–MS/MS). Analytical assay validation was performed using residual urine specimens. LTE4 results were normalized to urine creatinine for clinical use. Reference interval was established using a healthy volunteer cohort.
Clinical sensitivity and specificity for SM detection were determined by measuring urinary biomarkers (LTE4, Nmethyl histamine [NMH] and 11β-prostaglandin F2α [BPG]) in a cohort of 409 patients referred to allergy specialists, 66 (16%) of which were diagnosed with SM.
Results: Urinary LTE4 measurement was accurate, precise and linear across a range of 31–3020 pg/mL. The
95th percentile of the reference interval population was b 104 pg/mg creatinine. Median urine LTE4 concentrations were significantly higher among patients with SM (97 pg/mg cr. vs. 50 pg/mg cr.; p b 0.01). Elevated urinary
LTE4 was 48% sensitive and 84% specific for SM. Clinical sensitivity was 53% for BPG (N 1000 ng/mL) and 71% for
NMH (N 200 ng/mL). Incorporating all three urinary metabolites improved the SM diagnostic sensitivity to 97%,
with minimal change in specificity.
Conclusions: We have developed a sensitive and precise LC–MS/MS assay for quantitation of LTE4 in urine.
Incorporating LTE4 into a panel including BPG and NMH provides a much-needed screening tool for a complicated disease with non-specific symptoms and invasive confirmatory testing.
© 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
1. Introduction
Systemic mastocytosis (SM) is a disorder characterized by the excessive accumulation of clonally derived mast cells in various tissues. When
triggered, mast cells release large amounts of tryptase, histamine, prostaglandins and leukotrienes. This release of signal molecules causes intermittent “spells” with varying symptoms that may include itching,
flushing, lightheadedness, tachycardia, gastrointestinal distress, or
even loss of consciousness. Diagnostic criteria established by the
World Health Organization (WHO) recommend a bone marrow biopsy,
specialized cytology studies or genetic testing [1].
Urine concentrations of N-methyl histamine (NMH) and 11-beta
prostaglandin F2α (BPG), which are metabolites of mast cell derived histamine and prostaglandin, have been used to aid in screening and reduce unnecessary biopsies [2–4]. Cysteinyl leukotrienes are another
⁎ Corresponding author at: 200 First St. SW, Rochester, MN 55905, United States.
E-mail address: meeusen.jeffrey@mayo.edu (J.W. Meeusen).
class of mast cell secretory molecules and potent inflammatory mediators. Leukotriene E4 (LTE4) is the primary stable metabolite of total
cysteinyl leukotrienes [5]. Concentrations of LTE4 are low in circulation
but accumulate in the urine. We hypothesized that urinary LTE4 could
be used alone or in combination with NMH and BPG to optimize screening for SM. Here we describe a novel (LC–MS/MS) assay to accurately
and precisely quantitate LTE4 in urine and outline its clinical utility in
SM screening.
2. Patients and methods
2.1. Study populations
All patient data were accessed in compliance with the Institutional
Review Board. A normal reference population of 128 apparently healthy
donors (64 men and 64 women) with a mean age of 43 (SD 14) years
was recruited. Patients were self-reported as not having allergic disease
or immunologic disorders and had not taken any antihistamines, non-
http://dx.doi.org/10.1016/j.clinbiochem.2016.02.007
0009-9120/© 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Please cite this article as: A.J. Lueke, et al., Analytical and clinical validation of an LC–MS/MS method for urine leukotriene E4: A marker of systemic
mastocytosis, Clin Biochem (2016), http://dx.doi.org/10.1016/j.clinbiochem.2016.02.007
2
A.J. Lueke et al. / Clinical Biochemistry xxx (2016) xxx–xxx
steroidal anti-inflammatories, cyclooxygenase inhibitors, or 5lipoxygenase inhibitors within 2 weeks.
Clinical performance was determined among a retrospective cohort
of consecutive patients with clinically ordered 24-hour urine NMH measurement. Patients b 18 years of age, solid organ transplant recipients or
currently pregnant were excluded. A total of 409 patients were enrolled
between April 26th, 2012 and March 21st, 2013. An allergy specialist
evaluated all patients and final diagnoses were adjudicated by a chart
review. The WHO criteria for the diagnosis of SM include the presence
of the major criterion (the presence of multifocal dense infiltrates of
mast cells (MC) in tryptase-stained biopsy sections of the bone marrow
or of another extracutaneous organ) plus one minor criterion (more
than 25% of MC show abnormal morphology; the presence of KIT
Asp816Val mutation, abnormal mast cell phenotype indicated by the
presence of CD25 on MC; serum total tryptase N20 ng/mL). Alternatively, the presence of three minor criteria will satisfy the requirements for
the diagnosis [6]. Detailed diagnoses and medication usage are included
in Supplemental Table 1.
2.2. LTE4 method
D5-labeled internal standard (D5-LTE4) was added to waste urine
specimens, controls, and standards, followed by the addition of acetonitrile (ACN) to precipitate any excess salts. Precipitate was removed
using positive pressure filtration through a 0.2 micron 96 well PTFE filter plate prior to injection of 45 μL via a CTC Pal autosampler and onto a
Turboflow MAX mixed-mode anion exchange column (0.5 × 50 mm,
Thermo Fisher Scientific). Following elution with Methanol(MeOH)/
ammonium hydroxide, LTE4 was further chromatographically separated
on a C8 2.5-μm analytical column (Waters Xbridge, 2.1 × 50 mm, 60 °C)
using a H2O/MeOH mobile phase with ammonium hydroxide as the
modifier. LTE4 was monitored in negative MRM mode (AB Sciex API
5000 MS/MS). Detailed parameters are listed in Supplemental Table 2.
The total analysis time was 10.3 min. All LTE4 concentrations were normalized to creatinine (enzymatic method, Roche Diagnostics). SI unit
conversions are 0.25 pg LTE4/mg creatinine = 1.0 pmol LTE4/mmol creatinine, 0.90 ng NMH/mg creatinine = 1.0 nmol/mmol creatinine, and
2.82 ng BPG/24 h = 1.0 pmol BPG/24 h.
2.3. NMH and BPG analytical methods
NMH was isolated from urine specimens by solid phase extraction and measured by LC–MS/MS using a stable isotope labeled internal D3-N-methyl histamine. BPG was measured by competitive ELISA
(Cayman Chemicals, Ann Arbor, MI). The method uses an
acetylcholinesterase-linked BPG tracer, which competes with patient BPG for a limited number of rabbit-antibody binding sites. The
unbound BPG is then washed away, and the remaining BPG tracer
signal is inversely proportional to the concentration of BPG in the
urine sample.
2.4. Method validation
Accuracy of the clinical LC–MS/MS method for LTE4 was determined
by spiking pooled urine with known amounts of purified LTE4 between
100 to 2000 pg/mL. Average recovery was 111% (range 99–120%). Assay
precision was determined by repeat analysis of three urine pools with
average LTE4 concentrations of 44, 445 and 1378 pg/mL over 20 days.
Analytical sensitivity was determined by repeat injection (n = 20) of
a charcoal stripped urine pool and a mobile phase blank. Linearity was
assessed by mixing urine specimens with high and low LTE4 concentrations. Three mixing studies were performed on three different days for a
total of nine unique experiments (Fig. 1). Specimen stability was determined by measuring 10 specimens immediately after collection and at
1, 3, 7, 14, 30, and 90 days stored ambient, refrigerated, or frozen
Fig. 1. Analytical performance of a novel liquid chromatography tandem mass
spectrometry (LC–MS/MS) method for urinary leukotriene E4 measurement. A) Linearity
was demonstrated by mixing urine samples with high and low concentrations of LTE4
(slope 0.9929; R2 0.9985). B) LTE4 values were measured in 128 healthy donors (67
males, closed circles; 61 females, open circles); the 95th percentile value was 104 pg/mg
cr. (dotted line).
(−20 °C). Analytical specificity was assessed by spiking a urine sample
with 11-trans LTE4.
2.5. Statistical analyses
Statistical analyses were performed using JMP software (SAS Inc.;
Cary, NC). Relationships of the appropriate percentiles with age and
sex were evaluated using quantile regression by minimizing an asymmetrically weighted sum of absolute errors; 95th percentile was
established using a smoothed empirical likelihood quantile regression.
Receiver operator characteristic (ROC) curve analysis was used to identify the optimal diagnostic cutoff and the discrimination c-statistic for
each biomarker. Linearity was assessed by linear regression and Passing–Bablok regression. Significant relationships were defined as a pvalue b0.05.
Please cite this article as: A.J. Lueke, et al., Analytical and clinical validation of an LC–MS/MS method for urine leukotriene E4: A marker of systemic
mastocytosis, Clin Biochem (2016), http://dx.doi.org/10.1016/j.clinbiochem.2016.02.007
A.J. Lueke et al. / Clinical Biochemistry xxx (2016) xxx–xxx
3
3. Results
3.3. LTE4 as a clinical biomarker of SM
3.1. LTE4 analytical method accuracy, precision, linearity, and stability
The clinical utility of urinary LTE4 as a biomarker of SM was
determined in a cohort of 409 patients referred for allergic disease evaluations. The median LTE4 concentration in the entire cohort was
55 pg/mg creatinine (IQR 33–90). Chart review identified 66 patients
(16%) as being positive for SM. The most common alternate diagnoses
included spells of unknown etiology (21%), idiopathic angioedema
(11%), chronic urticaria (9%), idiopathic anaphylaxis (7%), reaction to
an identified allergen (6%), irritable bowel syndrome (4%), and eosinophilia (3%; Supplemental Table 1). The median urine LTE4 among patients with SM was 97 pg/mg creatinine, significantly higher than
patients without SM (50 pg/mg creatinine; p b 0.01; Table 2). The
only other diagnoses with elevated median LTE4 were eosinophilia
(119 pg/mg cr.; n = 12), monoclonal mast cell activation syndrome
(119 pg/mg cr.; n = 3), and aleukemic mast cell leukemia (630 pg/mg
cr.; n = 2); however the small number of patients with these diagnoses
prevented statistical comparisons. NMH and BPG were also significantly
elevated among SM patients (Table 2).
Receiver-operating-characteristic (ROC) analysis identified an optimal urine LTE4 cutoff of 84 pg/mg creatinine with an area under the
curve (AUC) of 0.705 (Table 2). ROC curve analysis for NMH and BPG
in this population identified AUCs of 0.853 and 0.649, respectively. The
reference interval cutoff of 104 pg/mg creatinine for LTE4 was 48% sensitive and 84% specific for a diagnosis of SM. Clinical sensitivity was 45%
for BPG (N 1000 ng/mL) and 73% for NMH (N 200 ng/mL). Sensitivity improved to 86% with a specificity of 68% when BPG and NMH were both
considered. Adding LTE4 improved the SM diagnostic sensitivity to 97%
with a specificity of 61%.
The method was accurate and linear between 31 and 3154 pg/mL
LTE4 (slope 0.9929; R2 0.9985), and imprecision was b 10% (Table 1).
Specimens were stable 24 h at room temperature, 7 days at 4 °C, and
30 days at −20 °C. Samples were unaffected by use of toluene, acetic
acid, boric acid or sodium carbonate 24 hour collection preservatives.
The limit of detection was determined to be 2 pg/mL and the limit of
quantitation was 8 pg/mL. Spiking with 2000 pg/mL 11-trans-LTE4 increased measured LTE4 concentrations by 1730 pg/mL.
3.2. Urine LTE4 values in a healthy population
The distribution of urinary LTE4 concentrations was compared between 46 paired 24-h and random urine collections. No significant difference in LTE4 concentration was observed between the random
(mean ± SD; 99 ± 61 pg/mL) and 24-h (109 ± 67 pg/mL) urine samples. LTE4 values observed in a healthy population (n = 128) were determined using a random urine collection and values normalized to
creatinine. Urinary LTE4 concentrations were not significantly altered
by age or gender (Fig. 1). The median LTE4 value was 42 pg/mg creatinine (IQR 31–62) and the 95th percentile was 104 pg/mg creatinine
(95CI 63–145). The 95th percentile values for NMH and BPG in the
same cohort were 194 ng/mg creatinine (95CI 164–224) and
1118 pg/mg creatinine (95CI 892–1342), respectively.
4. Discussion
Table 1
Analytical performance characteristics of the LTE4 LC–MS/MS method.
Precision
Mean (pg/mL)/SD/%CV
Within run
Pool 1
Pool 2
Pool 3
41
631
1452
Accuracy
Between run
2.0
21
38
5.0%
3.4%
2.6%
44
445
1380
3.3
31
112
7.6%
6.9%
8.2%
Recovery; mean (SD)
Range
Slope; R2
111% (9%)
100–2000 pg/mL
1.128; 0.994
2
Linearity
Range (pg/mL)
Slope; R
% difference; mean (SD)
Mixing study 1
Mixing study 2
Mixing study 3
33–3154
53–2630
31–3020
0.979; 0.999
1.006; 0.998
0.999; 0.999
−1.2% (2.9%)
−2.6% (4.4%)
−1.2% (4.5%)
Analytical sensitivity
Mean
SD
%CV
Limit of quantitation
8.2
0.9
11%
Stability; % difference
(mean ± SD)
1 day
3 days
7 days
90 days
Ambient
Refrigerate
Frozen
−3.7 ± 11%
−0.8 ± 7.6%
−2.6 ± 12%
−15 ± 18%
4.0 ± 15%
8.4 ± 17%
−38 ± 17%
0.5 ± 14.7%
7.0 ± 19%
n.p.
n.p.
−1.8 ± 17%
24 h collection
preservative
1 day
Slope; R2 (mean difference)
7 days
Slope; R2 (mean difference)
Toluene
Acetic acid
Boric acid
Sodium carbonate
0.926; 0.962 (−3.3 pg/mL)
0.959; 0.984 (−7.1 pg/mL)
1.069; 0.982 (−0.7 pg/mL)
1.066; 0.984 (1.7 pg/mL)
0.911; 0.952 (−2.9 pg/mL)
0.962; 0.988 (−6.2 pg/mL)
0.914; 0.978 (−2.0 pg/mL)
1.011; 0.988 (1.5 pg/mL)
Reference interval
N
Median (95CI)
95th percentile (95CI)
Overall
Female
Male
128
61
67
42 pg/mg cr. (38–48)
47 pg/mg cr. (41–57)
39 pg/mg cr. (34–45)
104 pg/mg cr. (85–145)
109 pg/mg cr. (86–158)
102 pg/mg cr. (74–153)
To convert LTE4 from pg/mL to pmol/L multiply by 2.27; to convert from pg/mg cr. to
pmol/mmol cr. multiply by 0.25.
We have developed a high-throughput LC–MS/MS method to measure urinary LTE4 with suitable accuracy, precision, and stability for clinical applications. Using a population of apparently healthy normal
volunteers and normalizing the analyte concentration to concentration
of creatinine we established a urinary LTE4 reference value of 104 pg/mg
creatinine and confirmed previously established reference ranges of
200 ng/mg creatinine (NMH) and 1000 pg/24 h (BPG). Furthermore,
in a cohort of N400 patients presenting with suspicion of mast cell disease, elevated urinary LTE4 was highly specific at identifying patients
with SM.
SM is a rare and complicated disease that manifests with a variety of
non-specific symptoms and sequelae depending on the site of mast cell
accumulation [7,8]. Furthermore, SM signs and symptoms may be indolent or intermittent depending on the activity of the aberrant mast cells
[9,10]. A weakness of the current study is lack of serum tryptase data.
Mast cells store histamine and tryptase within intracellular granules
that are released on activation. Conversely, arachidonic acid metabolites
such as leukotrienes and prostaglandins are synthesized de novo by
mast cells. Thus, urine NMH and serum tryptase may be indicators of activation, while LTE4 and BPG may allow for better estimation of overall
mast-cell burden.
Elevations of urinary leukotrienes among patients with SM have
been previously reported in small patient cohorts (n = 9 to 25) using
immunoassays. Leukotriene E5, 11-trans-LTE4, and N-acetyl-LTE4 have
all been reported as interfering compounds that cross-react to varying
degrees with antibodies used in these methods [11,12]. Of these, only
11-trans-LTE4 has a mass identical to LTE4, and therefore is measured
as LTE4 by LC–MS/MS. However, it has been shown that the biological
activities of these two LTE4 isoforms are similar [13].
In previous reports, and in our study, there was an overlap of urine
LTE4 concentrations between cases and controls. A reference interval
has not been previously described and it is worth noting that the median LTE4 concentration among patients was within the reference interval
despite exclusion of patients taking 5-lipoxygenase inhibitors.
Please cite this article as: A.J. Lueke, et al., Analytical and clinical validation of an LC–MS/MS method for urine leukotriene E4: A marker of systemic
mastocytosis, Clin Biochem (2016), http://dx.doi.org/10.1016/j.clinbiochem.2016.02.007
4
A.J. Lueke et al. / Clinical Biochemistry xxx (2016) xxx–xxx
Table 2
Clinical performance of urinary LTE4 and other urine biomarkers among 409 allergy patients presenting with symptoms suggestive of systemic mastocytosis (SM).
Systemic mastocytosis
Biomarker
No (n = 343)
Yes (n = 66)
p-Value
Sensitivity, % (95CI)
Specificity, % (95CI)
PPV, % (95CI)
NPV, % (95CI)
c-Statistic
LTE4, pg/mg cr.; median (IQR)
BPG, pg/24 h; median (IQR)
NMH, ng/mg cr.; median (IQR)
NMH or BPG
Any marker
50 (30–80)
624 (358–987)
124 (100–163)
N/A
N/A
97 (53–177)
1046 (469–2711)
290 (175–527)
N/A
N/A
b0.01
b0.01
b0.01
N/A
N/A
48 (37–60)
45 (32–55)
73 (59–80)
86 (76–93)
97 (89–99)
84 (80–87)
79 (73–82)
88 (86–92)
68 (63–73)
61 (56–66)
36 (27–47)
29 (22–38)
59 (53–74)
34 (28–42)
32 (26–39)
87 (85–92)
88 (85–92)
94 (91–96)
96 (93–98)
99 (97–99.9)
0.705
0.649
0.853
0.857
0.901
Clinical sensitivity, specificity, PPV, and NPV were determined using established normal 95th percentile cutoffs: LTE4 104 pg/mg creatinine (26 pmol/mmol creatinine), BPG 1000 pg/24 h
(2.82 pmol/24 h), NMH 200 ng/mg creatinine (180 nmol/mmol creatinine). PPV = positive predictive value; NPV = negative predictive value; c-statistic = ROC curve area under the
curve.
A growing number of reports on the efficacy of leukotriene inhibitors, such as zileuton, add support to the pathological role of LTE4 in
SM [14–16]. Clinical data on the presence of elevated urinary LTE4
may not only identify a screening marker for SM but may also justify
therapies beyond anti-histamines in afflicted patients.
5. Conclusions
In conclusion, we have developed a sensitive and precise LC–MS/MS
assay for quantitation of urinary LTE4. Incorporating LTE4 into a panel including BPG and NMH provides a powerful screening tool for systemic
mastocytosis, a disease of protean symptoms that is commonly
overlooked due to its rarity.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.clinbiochem.2016.02.007.
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Please cite this article as: A.J. Lueke, et al., Analytical and clinical validation of an LC–MS/MS method for urine leukotriene E4: A marker of systemic
mastocytosis, Clin Biochem (2016), http://dx.doi.org/10.1016/j.clinbiochem.2016.02.007