J. Breath Res. 16 (2022) 026007 https://doi.org/10.

1088/1752-7163/ac57f9

Journal of Breath Research

PAPER

Levitt’s CO breath test in the differential diagnosis of chronic

OPEN ACCESS
isolated hyperbilirubinemia
RECEIVED
6 November 2021 Ling-Ling Kang1, Ze-Lin Liu2, Quan-Sheng Han3, Yuan-Wu Chen4, Ling-Wen Liu5, Xian-Hui Xie5,
REVISED Jun-Feng Luo5, Yong-Qiang Ji5, Guo-Liang Zhu5, Yong-Jian Ma5, Kun-Mei Ji6 and Hou-De Zhang1,∗
28 January 2022
1
Department of Gastroenterology, Nanshan Hospital, Guangdong Medical University, Shenzhen 518052, People’s Republic of China
ACCEPTED FOR PUBLICATION 2
23 February 2022 Department of Hematology, Nanshan Hospital, Guangdong Medical University, Shenzhen 518052, People’s Republic of China
3
Shenzhen Zhonghe Headway Bio-Sci & TechCo. Ltd, Shenzhen 518052, People’s Republic of China
PUBLISHED 4
15 March 2022
Health Center, Nanshan Hospital, Guangdong Medical University, Shenzhen 518052, People’s Republic of China
5
Institute of Breath Test Research, Shenzhen University, Shenzhen 518060, People’s Republic of China
6
Department of Biochemistry and Molecular Biology, School of Basic Medical Science, Laboratory Department of South China Hospital,
Original content from Health Science Center, Shenzhen University, Shenzhen 518060, People’s Republic of China
this work may be used ∗
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Any further distribution Keywords: isolated hyperbilirubinemia, Gilbert syndrome, hemolysis, Levitt’s CO breath test, RBC lifespan
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Abstract
A key component of the differential diagnosis of isolated hyperbilirubinemia (HB) is
distinguishing between hemolytic and non-hemolytic types. Routine hemolysis screening markers
have unsatisfactory sensitivity and specificity. Erythrocyte (RBC) lifespan shortening, the gold
standard marker of hemolysis, is seldomly measured due to the cumbersome and protracted nature
of standard methods. A new Levitt’s CO breath test method may enable simple, rapid RBC lifespan
measurement. In this pilot prospective diagnostic study, Levitt’s CO breath test was evaluated to
discriminate hemolytic from non-hemolytic HB in adults. One hundred and thirty eligible
non-smoking adult patients who were aged 18 or older, referred for chronic (>6 months) isolated
HB or had a known diagnosis of isolated HB of a rare cause, were recruited, including 77 with
non-hemolytic HB and 53 with hemolytic HB. ROC curve analysis was applied to determine the
optimal cutoff for discriminating between hemolytic and non-hemolytic HB, and the performance
was calculated. Results showed that the mean RBC lifespan in non-hemolytic HB (93 ± 26 d) was
reduced (p = 0.001 vs. normal reference value of 126 d), but longer than that in hemolytic HB
(36 ± 17 d; p = 0.001). RBC lifespans did not differ significantly between 26 patients with simple
hemolytic HB (32 ± 14 d) and 27 patients with a Gilbert syndrome comorbidity (40 ± 18 d). ROC
curve analysis revealed an optimal lifespan cutoff for discriminating between hemolytic and
non-hemolytic HB of 60 d (AUC = 0.982), with a diagnostic accuracy of 95.4%, 94.3% sensitivity
and 96.1% specificity respectively. These results indicate that Levitt’s CO breath test seems to be
very sensitive and specific for detecting hemolysis in adult patients with chronic isolated HB, and
could enable simple, rapid, and reliable differential diagnosis of isolated HB. A large-scale
validation study of the method is warranted.

Abbreviations
HB hyperbilirubinemia CBC complete blood count
ROC Receiver operating characteristic LDH lactate dehydrogenase
AUC area under the curve RBC Erythrocyte
UGT1A1 uridine diphosphate G6PD glucose-6-phosphate dehydrogenase
glucuronosyltransferase family 1 CI confidence interval
member A1 ROC receiver operating characteristic

© 2022 The Author(s). Published by IOP Publishing Ltd

J. Breath Res. 16 (2022) 026007
1. Introduction
Isolated hyperbilirubinemia (HB) is a common clin-
ical laboratory finding of elevated serum bilirubin
levels in the context of apparently normal global liver
function and normal levels of biochemical markers of
hepatocellular injury and cholestasis [1]. Three basic
mechanisms have been found to result in isolated
HB. The first is increased bilirubin production, which
is seen in various hemolytic diseases and in indi-
viduals with hematopoiesis (hemolysis in situ). The
second mechanism is decreased hepatocellular uptake
and conjugation of unconjugated bilirubin, which is
seen mainly in Gilbert syndrome and Crigler–Najjar
syndrome. Both of these syndromes are caused by
dysfunctional hepatic uridine diphosphate glucuro-
nosyltransferase family 1 member A1 (UGT1A1),
due to mutations affecting the gene UGT1A1. The
third mechanism is decreased hepatocellular secre-
tion of conjugated bilirubin, which occurs in Dubin–
Johnson syndrome, a rare condition, as well as in the
even more rare Rotor syndrome.
Clinically, the evaluation of a patient with isol-
ated HB is quite different from that of a patient
who presents with elevated bilirubin associated with
elevated liver enzyme levels, with the latter suggest-
ing either a cholestatic or hepatocellular process [2].
Traditionally, the first step in the evaluation of a
patient presenting with isolated HB is to fractionate
a sample of bilirubin from the patient to determine
if the patient’s bilirubin is conjugated or unconjug-
ated. In the latter case, the second step is to dif-
ferentiate between a hemolytic and non-hemolytic
origin. Based on these findings, the type of isol-
ated HB can then be classified (i.e. (un)conjugated,
(non-)hemolytic) [2].
Direct bilirubin levels measured by the commonly
employed diazo reaction method often overestim-
ates the actual conjugated bilirubin level, especially
in hemolytic samples, resulting in potential misclas-
sification of isolated HB cases [2–4]. However, isol-
ated conjugated HB is extremely rare (i.e. principally
consisting of patients with Dubin–Johnson syndrome
and Rotor syndrome). Thus, in the vast majority of
isolated HB cases, unconjugated HB is observed [5],
making reliable differentiation between HB of hemo-
lytic versus non-hemolytic origin a key diagnostic
objective. Current hemolytic marker-based clinical
tests—such as a complete blood count (CBC), retic-
ulocyte count, peripheral blood smear, and lactate
dehydrogenase (LDH) assay, as well as assessments
of serum haptoglobin and plasma free hemoglobin—
are far from satisfactory with respect to sensitivity and
specificity. Many patients do not have a clear HD clas-
sification diagnosis even after the numerous etiolo-
gical tests.
Erythrocyte (RBC) lifespan refers to the aver-
age period that RBCs survive after being released
2
L-L Kang et al
into circulation from bone marrow. Typically, healthy
adults have an average RBC lifespan of 120 d [6, 7].
An abnormally short RBC lifespan is the fundamental
characteristic of hemolysis. Therefore, RBC lifespan
measurement should give a more accurate diagnosis
of hemolysis than indirect hemolytic marker assays
[8]. However, RBC lifespan measurement is seldomly
conducted due to the cumbersome and protracted
nature of classical labelling methods (e.g.51 Cr and
15
N-glycine), which can take several weeks, or even
months.
Based on the knowledge that exhaled endogen-
ous CO comes primarily from degraded hemoglobin
following RBC destruction, established mainly by
Rurnal et al [9–12], and thus the notion that mean
RBC lifespan can be estimated based on endogenous
CO production rate, Levitt’s team [13, 14] developed
a simple, rapid CO breath test to determinate RBC
lifespan. Our previous study confirmed that Levitt’s
CO breath test gives similar results to those obtained
with classical labelling methods [15]. The aim of this
pilot prospective diagnostic study was to evaluate
Levitt’s CO breath test to discriminate hemolytic from
non-hemolytic HB in adults.
2. Patients and methods
2.1. Study design and participants
This pilot prospective diagnostic study was under-
taken at Nanshan Hospital, Guangdong Medical Uni-
versity in Shenzhen, China. We recruited newly
referred patients with isolated HB. We also conduc-
ted a search of medical record archives to find patients
with rare causes of HB and invited them to par-
ticipate in the study. The inclusion criteria were:
being ⩾18 years old; having had a disease course of
>6 months; and having a confirmed cause of HB. The
exclusion criteria were: severe chronic cardiopulmon-
ary disease; acute disease or emergent medical needs;
a blood transfusion within 3 weeks of the study; and
active smoking, severe passive smoking, or other sim-
ilar air contamination exposure within 24 h before the
test; inability to complete sampling; and unwilling-
ness to sign the informed consent.
The diagnostic criteria of isolated HB was that a
general liver function test shows an isolated elevation
of the serum total bilirubin level ⩾ 21.0 µmol L −1
(diazo reaction method) concomitant with normal
serum albumin, normal levels of biochemical mark-
ers of hepatocellular injury (alanine transaminase and
aspartate transaminase), and cholestasis (gamma-
glutamyl transferase and alkaline phosphatase activit-
ies). Etiological diagnosis was based on comprehens-
ive clinical and laboratory evaluation.
All patients underwent a panel of routine laborat-
ory examinations, encompassing genetic tests, hemo-
lysis screening tests, and biochemical tests. First,
Sanger sequencing of UGT1A1 was conducted to
J. Breath Res. 16 (2022) 026007
identify genetic causes of HB, namely Gilbert syn-
drome and Crigler–Najjar syndrome (Beijing Gen-
omics Institute, Beijing, China). Second, to screen
for hemolysis, hemolytic markers were assessed
with a CBC, reticulocyte count, peripheral blood
smear, LDH assay, and routine chemical urinalysis.
Third, two hemolytic etiology diagnostic tests were
performed, including glucose-6-phosphate dehydro-
genase (G6PD) activity measurement for detection of
G6PD deficiency and Coomb’s test for the diagnosis
of autoimmune hemolytic anemia. Other hemolytic
etiology tests were carried out according to specific
case indications. For example, hemoglobin electro-
phoresis was conducted for definitive diagnosis of
thalassemia, flow cytometry of blood CD55/CD59
RBCs was conducted for definitive diagnosis of par-
oxysmal nocturnal hemoglobinuria, and we meas-
ured serum levels of foliate (a.k.a. vitamin B9), vit-
amin B12 (a.k.a. cobalamin), and homocysteine to
diagnose megaloblastic anemia. Patients with clin-
ical and/or laboratory evidence of hemolysis that
remained unexplained after these diagnostic investig-
ations were submitted to diagnostic next-generation
sequencing with a commercial HO-41 sequencing
panel (MyGenotics, Beijing, China) that covered 208
genes linked to hereditary erythroid related diseases.
Those patients who had no history or signs
of hemolysis, were negative for hemolytic mark-
ers, had normal G6PD activity, and had a negative
Coomb’s test result formed the non-hemolytic HB
cohort. Those for whom a definitive hemolytic cause
was established were included in the hemolytic HB
cohort, regardless of other associated diseases.
Nanshan Hospital Ethics Committee approved
the study protocol. Written informed consent was
obtained from all study participants. This trial is
registered with www.chictr.org.cn number, CHiC-
TRDDD17011592.
2.2. Levitt’s CO breath test
The principle of Levitt’s CO breath test is that endo-
genous CO in the breath originates mainly (∼70%)
from heme oxidation during hemoglobin degrad-
ation following RBC rupture, such that the total
capacity of CO from hemoglobin divided by the
CO quantity released per day equates to mean RBC
lifespan [10]. Given that the lungs mediate the only
mechanism available to the body for CO excre-
tion and the fact that alveolar ventilation volume
(mL min−1 ) is roughly equal to total blood volume
(mL), Levitt’s team [13, 14] deduced that RBC
lifespan can be calculated from exhaled endogen-
ous alveolar CO concentration (ppm) and peripheral
blood hemoglobin concentration (g mL−1 ) according
to the following formula:
RBC survival (d)
= (1380) × ([hemoglobin]) /
endogenous alveolar CO concentration.
3
L-L Kang et al
We conducted the breath test with an automated
instrument, namely the ELS Tester (Seekya Biotec.
Co., Ltd, Shenzhen, China). The test involved three
steps. (1) For breath sample collection, in the morn-
ing (8:00–11:30) without a fasting requirement, each
subject inhaled deeply, held their breath for 10 s, and
then exhaled into a tri-channel collection system that
discards the first 300 ml, presumed to be dead space
air, and then directs subsequent alveolar air into a foil
bag (700 ml). If needed, the procedure was repeated
until the collected air sample reached the collection
bag capacity. The filled bag was detached and sealed.
An atmospheric sample was collected just after breath
sampling. Alveolar air and atmospheric samples were
stored at room temperature and analyzed within 2 d
of collection. (2) To measure hemoglobin concentra-
tion, a CBC was carried out on the breath sampling
day. (3) Finally, for RBC lifespan determination, an
automated ELS tester was used to measure endo-
genous alveolar CO concentration by nondispersive
infrared spectroscopy of the paired alveolar and envir-
onmental air samples. The ELS tester used these data,
together with an inputted hemoglobin concentration
value, to calculate RBC lifespan according to Levitt’s
formula (above). Briefly, for step 3, the alveolar and
environmental air sample bags were connected to
inlet ports and each patient’s hemoglobin concentra-
tion datum was inputted; then, automatic measure-
ment by the ELS TESTER was initiated by pushing
the start button on the machine, which reported each
participant’s RBC lifespan within 15 min.
Previously, we showed that the mean normal RBC
lifespan determined Levitt’s CO breath test was 126 d
(range, 75–177 d), a value similar to that obtained by
the standard biotin-labelling technique (mean, 115 d;
range, 70–140 d) [7, 15].
2.3. Outcomes
The primary outcome of this study was the accuracy
of Levitt’s CO breath test for detecting hemolysis in
adults with chronic isolated HB.
2.4. Statistics
Power analysis indicated that we would need a sample
size of 70 to achieve a power of ⩾90%and thus show
a diagnostic accuracy of ⩾90% (lower level of two-
sided 95% confidence interval (CI)) to discriminate
between patients with hemolytic HB from those with
non-hemolytic HB. Normally distributed data are
reported as means with standard deviations. Abnor-
mally distributed data are reported as medians with
inter-quartile ranges. Enumeration data are expressed
as percentages.
Student’s t-test and Wilcoxon rank-sum test or
chi-square tests were applied to analyze differences
in measurement data and enumerated data, respect-
ively. For hemolytic HB diagnosis, we used receiver
operating characteristic (ROC) curves to establish
an optimal discrimination value. Diagnosis accuracy
J. Breath Res. 16 (2022) 026007
rate was calculated as the number of correctly dia-
gnosed cases divided by the total number of HB cases.
All data analyses were conducted in SPSS for Win-
dows, version 22 (IBM, Chicago, IL), with a signific-
ance criterion of p < 0.05.
3. Results
3.1. Patient characteristics
Patients were recruited from 1 May 2018 to 1 Novem-
ber 2020. Non-hemolytic HB patients were gathered
quickly owing to the relatively high prevalence of
Gilbert syndrome in the population. Recruitment
of patients with hemolytic HB, which is already
relatively rare, was quite slow and further slowed
by the discovery that some patients with hemo-
lytic HB had a comorbidity of Gilbert syndrome.
To ensure there were enough cases of simple isol-
ated hemolytic HB in the sample, we determined
to enlarge the sample size by 50% to 105%. Ulti-
mately, 137 patients were recruited, including 128
newly referred patients and 2 with rare diagnoses,
including 1 patient with Crigler-Najjar syndrome
type II and 1 patient with Dubin-Johnson syndrome.
The patient with Dubin-Johnson syndrome was the
only patient in this study suffering from conjugated
HB (serum bilirubin 70.5 mmol L−1 , direct biliru-
bin 44.9 mmol L−1 ). Seven patients were excluded
because of cigarette smoking (N = 3), polycythemia
with unexplained HB (N = 3 cases), and pregnancy
with unexplained mildly elevated reticulocyte count
(N = 1). Therefore, 130 patients were eligible and
enrolled into the study. The de-identified, indi-
vidual participant-level data that underlie the res-
ults reported in this article are freely accessible at
https://figshare.com/(DOI:10.6084/m9.figshare.1413
0209.
The 130 eligible patients included 77 with non-
hemolytic HB (75 with Gilbert syndrome, 1 with
Crigler-Najjar syndrome type II, and 1 with Dubin-
Johnson syndrome). The remaining 53 patients were
diagnosed with hemolytic HB (26 with simple hemo-
lysis and 27 with hemolysis and Gilbert syndrome).
Of the 26 patients with simple hemolysis; 9 had
thalassemia; 7 had megaloblastic anemia; 2 had
hypersplenism of liver cirrhosis; 2 had autoim-
mune hemolytic anemia; and 1 had each of the
following: hereditary non-spherical polycythemia,
primary myelofibrosis, hemophagocytic syndrome,
paroxysmal nocturnal hemoglobinuria, Tha with
megaloblastic anemia, and Tha with G6PD defi-
ciency. Of the 27 patients who had hemolysis
coexisting with Gilbert syndrome, 8 had heredit-
ary spherocytosis; 7 had a G6PD deficiency; 5 had
thalassemia; and 1 had each of the following: megalo-
blastic anemia, Evans syndrome, cardiac mechanical
valve induced hemolysis, drug-induced hemolysis,
4
L-L Kang et al
thalassemia with G6PD deficiency, megaloblastic
anemia with G6PD deficiency, and thalassemia with
hereditary spherocytosis.
The demographic characteristics, clinical charac-
teristics, and laboratory data of the enrolled patients
are in summarized in table 1. The non-hemolytic HB
and hemolytic HB groups were similar with respect to
gender and age. Splenomegaly was detected in hemo-
lytic HB patients. While there was no difference in
total serum bilirubin level or direct bilirubin frac-
tion between the two groups, other laboratory signs of
hemolysis were found only in patients with hemolytic
HB. In the hemolytic group, patients with comorbid
hemolysis and Gilbert syndrome had higher bilirubin
levels but fewer severe hemoglobin reductions than
patients with simple hemolysis.
3.2. RBC lifespans
The RBC lifespans obtained for each of the 130 sub-
jects are shown in figure 1. The mean RBC lifespan of
the 77 patients with non-hemolytic HB was 93 ± 26 d,
which was significantly shorter than the normal mean
reference value (126 d; t = −11.254, p = 0.001), but
much longer than that of the 53 patients with hemo-
lytic HB (36 ± 17 d; t = 14.2, p = 0.001). RBC lifespan
did not differ significantly between the 26 patients
with simple hemolytic HB and the 27 patients with
comorbid hemolysis and Gilbert syndrome (32 ± 14 d
vs. 40 ± 18 d; t = − 1.983, p = 0.053).
3.3. Diagnostic performance
All 53 (100%) patients with hemolytic HB and 21/77
(27.3%) patients with non-hemolytic HB had RBC
lifespans below normal range (lower limit of normal
by Levitt’s breath test = 75 d). ROC analysis indic-
ated that a cutoff RBC lifespan of 60 d was optimal
for group separation (figure 2). With this, we reached
an accuracy of 95.4% (95%CI 90.0–98.1) with 94.3%
sensitivity (95%CI 84.0–98.7) and 96.1% specificity
(95%CI 88.7–99.1). The area under for the ROC
curve (AUC) was 0.982 (95%CI 0.965–0.999).
4. Discussion
Of 130 eligible adult patients with chronic isolated
HB enrolled over a 2 year period, only one patient
(0.08%), who was found by a medical record search,
had conjugated HB (table 1), confirming that the
vast majority of cases of isolated HB are unconjug-
ated, and thus that the key objective of differential
diagnosis for isolated HB is to determine whether
there is hemolysis. Levitt’s CO breath test was con-
firmed to have very high sensitivity for detection
of hemolysis, even mild hemolysis, in adult patients
with chronic isolated HB. Using an RBC lifespan
cutoffof 60 d, the diagnostic performance of the
breath test for hemolytic HB was excellent with
J. Breath Res. 16 (2022) 026007 L-L Kang et al

Table 1. Baseline characteristics of patients with isolated hyperbilirubinemia (HB).

Hemolytic HB (N = 53)
Non-hemolytic
Characteristic HB (N = 77) Simple (n = 26) +GS (n = 27) Total (n = 53)

Demographic
Age, years 29.0 (26.0, 37.5) 35.0 (23.0, 50.8) 29.0 (25.0, 42.0) 33.0 (25.0, 46.5)
Sex
Male 57 (74.0%) 12 (46.2%) 23 (85.2%) 35 (66.0%)
Female 20 (26.0%) 14 (53.8%) 4 (14.8%) 18 (34.0%)
Clinical
∗∗ ∗∗ ∗∗
/#
Splenomegaly 0 14 (51%) 7 (27%) 21 (40%)
Laboratory
Serum bilirubin
∗
TBil, µmol L−1 40.8 (39.0, 49.5) 37.0 (29.2, 45.2) 53.9 /# (33.3,78.8) 41.0 (30.5, 59.8)
DBil, µmol L−1 10.8 (8.4, 12.9) 11.2 (8.58, 14.2) 9.0 (7.7, 11.8) 9.7 (8.2, 12.9)
∗
IBil, µmol L−1 28.1 (20.4, 35.8) 24.7 (14.9, 32.3) 45.1 (23.1, 69.1) /## 29.2 (19.5, 52.7)
∗∗ ∗∗
UGT1A1 mutation 76 (98.7%)a 0 27 (100%)## 27 (50.9%)
∗∗ ∗∗ ∗∗
LDH > 250 IU/L 0 13 (56.5%) 7 (25.9%) /## 20 (43.4%)
∗∗ ∗∗ ∗∗
Hemoglobin, g/dl 15.4 (14.5, 16.2) 7.3 (6.6, 9.2) 13.3 (10.8, 15.0) /## 9.5 (7.1, 13.5)
∗∗ ∗∗ ∗∗
Anemia 0 25 (94.3%) 16 (59.3%) /## 41 (77.4%)
MCV
∗∗ ∗∗ ∗∗
<80, fL 0 10 (38.5%) 5 (18.5%) 15 (28.3%)
∗∗ ∗∗
>100, fL. 0 10 (38.5%) 1 (0.4%) ## 11 (20.8%)
∗∗ ∗∗ ∗∗
Reticulocytes > 2.0% 0 12 (42.6) 20 (74.1%) /# 32 (60.4%)
∗∗ ∗∗ ∗∗
Abnor blood smear 0 18 (69.2%) 11 (40.7%) 29 (54.7%)
Hemoglobinuria 0 1 (3.8%) 0 1 (1.9%)
Hemosiderinuria 0 1 (3.8%) 0 1 (1.9%)
Coomb’s test + 0 2 (7.7%) 1 (3.7%) 3 (5.7%)
∗∗ ∗∗
G6PD deficiency 0 0 7 (25.9%) /# 7 (13.2%)
Data are reported as mean (SD), median (IQR) or n (%); a the single negative patient had Dubin-Johnson syndrome; ∗ p < 0.05,
∗∗ p < 0.01 vs. non-hemolytic; # p < 0.05, ## p < 0.01 vs. simple hemolysis; GS, Gilbert syndrome; TBil, total bilirubin; DBil, direct

bilirubin; IBil, indirect bilirubin; LDH, lactate dehydrogenase Hb, hemoglobin; MCV, mean corpuscular volume; Abnor, abnormal;
G6PD, glucose-6-phosphate dehydrogenase.

94.3% sensitivity and 96.1% specificity (figure 2).
However, among patients with hemolytic HB, the
test could not distinguish between simple hemolytic
HB and hemolysis coexisting with Gilbert syndrome
(figure 1).
Gilbert syndrome is a well-known phenotypic-
ally heterogenous hereditary condition characterized
by mild, chronic unconjugated HB in the absence
of liver disease or overt hemolysis. Reported incid-
ence rates for Gilbert syndrome range from 6% to
12% [2]. The pathogenesis of Gilbert syndrome has
been linked to congenital mutations in UGT1A1 and
consequent disruption of UGT1A1, the enzyme that
conjugates glucuronic acid to bilirubin in hepato-
cytes and then converts it into an excretable molecule.
Traditionally, unconjugated HB resulting from Gil-
bert syndrome has been attributed completely to
this non-hemolytic mechanism because commonly
used clinical biomarkers of hemolysis (e.g. serum
LDH, haptoglobin, and reticulocyte count) are not
found in patients with Gilbert syndrome. However,
several early studies employing classical 51 Cr-labeled
RBC re-transfusion measurement techniques sug-
gested that some 30% ∼ 80% of people with Gil-
bert syndrome have mild hemolysis indicated by a
slightly shortened RBC lifespan [16–19]. Therefore,
in addition to being a bilirubin conjugating disorder,
bilirubin overproduction with mild hemolysis can be
considered a diagnostic marker of Gilbert syndrome
[17, 19, 20]. Consistent with these previous reports,
our non-hemolytic group, which consisted mostly
of patients with Gilbert syndrome, had a slightly
reduced mean RBC lifespan (92 d vs. normal mean
of 126 d). Indeed 38% of the patients in the non-
hemolytic group had an RBC lifespan shorter than
75 d, which is the lower limit of the normal range.
With respect to diagnosis, the present results suggest
strongly that simple rapid Levitt’s CO breath testing
would be as sensitive as classical standard labelling
methods for detecting hemolysis in the context of
isolated HB.
We found that if 75 d, the lower limit of the nor-
mal RBC lifespan range, were used as the cutoff-
for hemolytic HB diagnosis, we would reach 100%

5
J. Breath Res. 16 (2022) 026007 L-L Kang et al

Figure 1. Scatter plot of RBC lifespan data observed in 130 adults with chronic isolated HB. CN2, Crigler-Najjar syndrome type
II; DJS, Dubin-Johnson syndrome; GS, Gilbert syndrome.

Figure 2. ROC curve for differentiation between patients with hemolytic HB and patients with non-hemolytic HB.

6
J. Breath Res. 16 (2022) 026007
diagnostic sensitivity, but with a false positive rate as
high as 27.3%. The high false positive rate was appar-
ently the consequence of mild hemolysis in Gilbert
syndrome being detected. Thus, the adjusted cutoffis
critical for differential diagnosis of isolated HB. Based
on our ROC analysis, we recommend an optimal RBC
lifespan cutoff of 60 d to maximize both sensitivity
and specificity.
Generally, Gilbert syndrome coexisting with
hemolytic disease is considered to be uncommon,
albeit with isolated single case reports [21–25]. We
were surprised to find that more than half of our
enrolled patients with hemolytic HB (37/53) had
Gilbert syndrome, indicating that this phenomenon
maybe much more common than is appreciated. Per-
haps many such cases have been missed due to a lack
of awareness and available techniques. The preval-
ence of Gilbert syndrome in the general population,
at 6% ∼ 12%, indicates that a similar prevalence
may be found among hemolytic patients by chance.
Thus, the incidence of Gilbert syndrome among
our sample of hemolytic HB patients far exceed-
ing this range is noteworthy. Here, we were able to
find undiagnosed Gilbert syndrome in a large num-
ber of patients because all of the participants in our
study underwent Sanger sequencing of UGT1A1 and
detailed hemolysis tests. Although RBC lifespan data
were not useful for distinguishing between patients
with simple hemolytic HB and those with comorbid
hemolysis and Gilbert syndrome, it is evident from
our baseline clinical characteristic data (table 1) that
the latter patient subgroup tended to have higher
hemoglobin and bilirubin levels than patients with
simple hemolytic HB, consistent with previous single
case reports [21–25]. Thus, discordance between sig-
nificant unconjugated HB and less severe hemolysis
may be a valuable clue that a patient may have hemo-
lytic disease comorbid with Gilbert syndrome. Fur-
ther studies are needed to clarify the clinical signific-
ance of this pattern of findings.
Our study had several limitations. First, we did
not enroll patients with drug-induced non-hemolytic
HB, a condition that needs to be considered in
the differential diagnosis of isolated HB. Second,
although non-hemolytic HB was diagnosed accord-
ing to an established clinical rubric, it is possible that
a few patients might have been misclassified. Third,
because the study was initially designed as a proof-of-
concept study, the number of patients was not large
enough to be divided into a development set and
a validation set, especially for patients with simple
hemolytic HB. Therefore, further prospective testing
of the recommended optimal cutoff value’s perform-
ance should be done.
In summary, the present results show that Levitt’s
CO breath test is a highly sensitive method for
detecting hemolysis in adult patients with chronic
7
L-L Kang et al
isolated HB. RBC lifespan measurements with this
method can discriminate between patients with non-
hemolytic HB and patients with hemolytic HB with
high diagnostic accuracy. Thus, Levitt’s CO breath
test represents a new simple and rapid diagnostic
approach to differentiating among different chronic
isolated HB pathologies. The validity demonstrated
here should be confirmed in a large-scale valida-
tion study. Finally, it should be noted that because
smokers absorb large amounts of CO from tobacco
smoke, this breath test may not be appropriate for
smoking patients, at least not with the presently used
formula for calculating RBC lifespan.
Data availability statement
All authors had access to the study data and reviewed
and approved the final manuscript.
The data that support the findings of this study
are openly available at the following URL/DOI:
https://figshare.com/(DOI:10.6084/m9.figshare.1413
0209).
Author contributions
Hou-De Zhang and Ling-Ling Kang designed the
study, developed the study protocol, recruited sub-
jects, collected breath samples, analyzed the data, and
wrote the manuscript. Ze-Lin Liu edited the protocol,
supervised all experimental steps, and contributed
to hemolysis diagnoses. Quan-Sheng Han, Yuan-Wu
Chen, Ling-Wen Liu and Xian-Hui Xie contributed
to subject enrollment and data collection. Jun-Feng
Luo was responsible to breath samples measurement.
Yong-Qiang Ji and Guo-Liang Zhu contributed to
the statistical analysis and mapping. Yong-Jian Ma
and Kun-Mei Ji analyzed the data and revised the
manuscript.
Conflict of interest
Hou-De Zhang, Yong-Qiang Ji, Guo-Liang Zhu, and
Yong-Jian Ma were members of research and devel-
opment team of the ELS TESTER. The other authors
have no competing interests to declare.
Clinical trial registration
This trial is registered with www.chictr.org.cn num-
ber, CHiCTRDDD17011592.
Patient consent statement
Written informed consent was obtained from all
study participants.
J. Breath Res. 16 (2022) 026007
ORCID iDs
Yong-Jian Ma  https://orcid.org/0000-0002-4932-
4562
Hou-De Zhang  https://orcid.org/0000-0001-
8680-0073
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