Serum lipocalin-2 levels positively correlate with coronary artery disease and metabolic syndrome
Serum lipocalin-2 levels positively correlate with coronary artery disease and metabolic syndrome
Jie Ni 0
Xiaojing Ma 0
Mi Zhou 0
Xiaoping Pan 0
Junling Tang 0
Yaping Hao 0
Yuqian Bao 0
Weiping Jia 0
0 Department of Endocrinology and Metabolism, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai Clinical Center for Diabetes, Shanghai Key Laboratory of Diabetes Mellitus, Shanghai Diabetes Institute , 600 Yishan Road, Shanghai, 200233 , China
Background: The lipocalin-2 (LCN2) cytokine, primarily known as a protein of the granules of human neutrophils, has been recently reported to be implicated in metabolic and inflammatory disorders. This study was designed to evaluate the relationship between serum LCN2 levels and coronary artery disease (CAD). Methods: Serum LCN2 levels of 261 in-patients who underwent coronary angiography were measured by sandwich enzyme immunoassay. Demographic (169 men and 92 postmenopausal women) and clinical (metabolic syndrome (MS), triglyceride (TG) and C-reactive protein (CRP) levels) characteristics were collected to assess independent factors of CAD (CAD: 188 and non-CAD: 73) and serum LCN2 levels by multiple logistic regression and multivariate stepwise regression analyses, respectively. Results: Serum LCN2 levels were significantly higher in men (37.5 (27.4-55.4) vs. women: 28.2 (18.7-45.9) ng/mL, p < 0.01) and men with CAD (39.2 (29.3-56.5) vs. non-CAD men: 32.7 (20.5-49.7) ng/mL, p < 0.05), and showed significant positive correlation with CAD in men (odds ratio = 2.218, 95% confidence interval: 1.017-4.839). Similarly, serum LCN2 levels were significantly higher in men with MS (40.2 (31.9-59.4) vs. non-MS: 32.0 (21.7-47.6) ng/mL, p < 0.01) and showed a significant positive correlation with the number of MS components (p for trend < 0.05). No significant differences or correlations were seen in women. TG and neutrophils (standard = 0.238 and 0.173) were independent factors of serum LCN2 levels in men, and only neutrophils (standard = 0.286) affected levels in women (all p < 0.05). Conclusions: Increased serum LCN2 levels are positively correlated with the presence of CAD and MS in a Chinese cohort.
Atherosclerosis; Coronary artery disease; Metabolic syndrome; Serum LCN2 levels
Coronary artery disease (CAD) is commonly encountered
in long-term and urgent clinical care settings, yet high
mortality and disability rates persist . The fundamental
pathological change observed in CAD patients is
atherosclerosis, and this cholesterol and lipid-based blockage has
also been implicated in metabolic disorders and chronic
The extensive research efforts put forth to elucidate
the molecular mechanisms of CAD pathogenesis have
indicated a potential role for the adipocyte-related
proinflammatory cytokine lipocalin-2 (LCN2). It has also
been detected in some organs with normal metabolism,
including bronchus, stomach, small intestine, pancreas,
kidney, prostate gland and thymus. Additionally, LCN2
can be secreted by activated neutrophils, adipocytes and
macrophages . While in metabolic and inflammatory
disorders, high LCN2 levels were noticed in adipocytes
. Therefore, LCN2 has been characterized as a potential
contributing factor to obesity, insulin resistance,
hyperglycemia, chronic inflammation [5,6], apoptosis, and impaired
renal function [7-9].
LCN2 exerts structural effects which can perturb normal
physiological functions. For example, LCN2 complexes
with matrix metalloproteinase-9 (MMP-9) and contributes
to fibrosis, and this functional interaction is recognized by
the alternative designation of LCN2 as the
neutrophilgelatinase associated lipocalin (NGAL) . In addition,
Lcn2 expression has been detected in clinical specimens
of atherosclerosis plaques (particularly in the vascular
endothelial cell, smooth muscle cell and macrophage
components) and damaged myocardium, as well as in vitro
analysis of arterial plaques [11,12]. Studies in animal models
have shown that murine atherosclerosis is accompanied by
increased levels of serum LCN2 and that conditions of
hypoxia and myocardial infarction (MI) induce Lcn2 mRNA
expression . Serum LCN2 levels have also been found
to be related to glucose metabolism and blood lipid
In recent years, the possible association of serum LCN2
levels and CAD has been addressed by clinical trials, and
studies based on Caucasian and Korean populations have
suggested positive associations between expression of this
multifunctional protein and development of this
lifethreatening disease with systemic implications [16-18].
However, no study to date has assessed the relationship
between serum LCN2 levels and CAD in a Chinese
population. The current study was designed to investigate the
serum LCN2 levels detected in Chinese patients who
underwent coronary angiography (CAG) to address
symptoms of chest pain or/and chest tightness and determine
the independent factors of CAD and increased serum
Materials and methods
Patient selection and demographic/clinical characteristics
Between July 2008 to January 2010, the in-patient
population of the Department of Cardiology of Shanghai Jiao
Tong University Affiliated to the Sixth Peoples Hospital
was searched for patients who were admitted to undergo
CAG to address reported experience of or current
suffering from chest pain or/and chest tightness.
The identified patients were required to complete a
standardized questionnaire upon enrollment to collect data on
past and present illnesses, drug use, and smoking habits.
Subjects were identified as current smokers if they reported
regular use of inhaled tobacco products or having smoked at
least one cigarette per day at any time over the previous
6 months . Patients were denied study enrollment if they
reported MI occurrence at any time during the past
3 months, history of congestive heart failure (New York
Heart Association Class III-IV) or related treatment
(coronary bypass surgery or percutaneous coronary intervention) at
any time during the past 6 months, ongoing hepatic or renal
dysfunction, current acute infection, history of malignancy
or autoimmune diseases, or diagnosis of mental illness.
The study was carried out with pre-approval from the
local Ethics Committee of Shanghai Jiao Tong University
affiliated Sixth Peoples Hospital and complied with the
Declaration of Helsinki. All enrolled subjects provided
A total of 261 patients were enrolled in the study, including
169 men and 92 postmenopausal women ranging in age
from 39 to 86 years-old (mean age: 65.9 9.5 years). A
complete physical examination was given to each study
participant upon enrollment, and included body mass index
(BMI, calculated as kg weight/m2 height), waist
circumference (W, measured at the midpoint between the lowest rib
and the superior border of the iliac crest on the midaxillary
line), and blood pressure (measured by conventional sleeve
Blood samples were taken from all study participants after an
overnight fast (of 10 h) and stored at 80C until testing.
Fasting plasma glucose (FPG) and 2 hours postprandial
glucose (2hPG) were determined by the glucose oxidase method.
Fasting insulin was assayed by radioimmunoassay (Linco
Research, St Charles, Missouri, USA). Insulin resistance was
estimated via the homeostasis model assessment index
(HOMA-IR) . Glycated hemoglobin (HbA1c)
concentration was measured by high-pressure liquid chromatography
(Bio-Rad Inc., Hercules, CA, USA). Serum creatinine (Scr),
uric acid (UA) and lipid profiles, including triglyceride (TG),
total cholesterol (TC), high-density lipoprotein cholesterol
(HDL-c) and low-density lipoprotein cholesterol (LDL-c),
were determined by standard enzymatic procedures on an
automated bioanalyzer (7600020; Hitachi, Tokyo, Japan). The
24 h urine albumin (24hALB) concentration was measured
by the standard rate nephelometry method. Estimated
glomerular filtration rate (eGFR, mL/min/1.73 m2) was calculated
according to the equation from the Modification of Diet in
Renal Disease (MDRD) study: [186 (Scr/88.4) -1.154 (age)
-0.203 0.742 (if women)] . Serum C-reactive protein
(CRP) was measured by particle-enhanced
immunonephelometry assay (Dade Behring Inc., Newark, NJ, USA). Serum
LCN2 and adiponectin levels were measured by sandwich
enzyme immunoassays (Antibody and Immunoassay Services,
The University of Hong Kong). Concerning the assay of
LCN2, the immunoplate was pre-coated with a monoclonal
antibody specific for human LCN2. A second horseradish
peroxidase (HRP)-linked monoclonal antibody specific to
human LCN2 was co-incubated with the samples. While for
adiponectin, a mouse monoclonal antibody specific to human
adiponectin was pre-coated onto a micro-tire plate and a
second HRP-linked monoclonal antibody specific to human
adiponectin was co-incubated with the samples. The inter- and
intra-assay coefficients of variation were 6.77% and 1.84% for
LCN2 and 8.6% and 7.3% for serum adiponectin.
Diagnosis of metabolic syndrome
Metabolic syndrome (MS) was assessed among the study
population according to the criteria published by the
2007 Chinese Joint Committee for Developing Chinese
Guidelines on Prevention and Treatment of
Dyslipidemia in Adults. Three of the following five
components were required for MS diagnosis: 1) central obesity
(W >90 cm for men and >85 cm for women); 2) fasting
TG of 1.7 mmol/L; 3) fasting HDL-c of <1.04 mmol/L; 4)
systolic blood pressure (SBP) of 130 mmHg and/or
diastolic blood pressure (DBP) of 85 mmHg, or known
treatment for hypertension; 5) FPG of 6.1 mmol/L and/
or 2hPG of 7.8 mmol/L, or receipt of hypoglycemic
therapy for diabetes .
CAG and coronary stenosis index
Selective CAG was carried out with standard
Judkins techniques  and all major coronary arteries
were imaged in more than two orthogonal views.
The analysis of angiographic images was carried out
by two experienced cardiologists, who were blinded
to the patients clinical information. CAD was
diagnosed when 50% diameter lumen stenosis was
detected in a major coronary artery, including the
left main coronary artery, left anterior descending
artery or its first diagonal branch, left circumflex
artery or its first obtuse marginal branch, and right
coronary artery. The severity of CAD was assessed
by calculating the coronary stenosis index (CSI) as
the sum of the following scores of stenosis for each
lesion: none = 0; <25% = 1; 25-49% = 2; 50-74% = 3;
75-100% = 4 .
Serum adiponectin (ug/mL)
eGFR (mL/min/1.73 m2)
CAD family history, n (%)
Hypoglycemic therapy, n (%)
Anti-hypertensive therapy, n (%)
Lipid-lowering therapy, n (%) 11 (15.1) 6 (15.8) 5 (14.3) 62 (33.0)aa 43 (32.8)b 19 (33.3)c
CSI: Coronary stenosis index; BMI: Body mass index; W: Waist circumference; SBP: Systolic blood pressure; DBP: Diastolic blood pressure; FPG: Fasting plasma glucose;
2hPG: 2 h postprandial glucose; HbA1c: Glycated hemoglobin A1c; HOMA-IR: Homeostasis model assessment index; TC: Total cholesterol; TG: Triglyceride; UA: Uric acid;
Scr: Serum creatinine; 24hALB: 24 h urine albumin; eGFR: Estimated glomerular filtration rate; CRP: C-reactive protein; Neutrophils: number of neutrophils.
For the total subjects: ap < 0.05, CAD vs. non-CAD; aap < 0.01, CAD vs. non-CAD; for the men subgroup: bp < 0.05, CAD vs. non-CAD, bbp < 0.01, CAD vs. non-CAD;
for the women subgroup: c p < 0.05, CAD vs. non-CAD, cc p < 0.01, CAD vs. non-CAD. Data are presented as mean SD or median (inter-quartile range).
All statistical analyses were carried out with the SPSS
statistical software (version 16.0; Chicago, IL, USA).
Variables with normal distribution are presented as mean
standard deviation and variables with skewed
distribution are expressed as median with inter-quartile range.
Students t-test (for normally distributed variables) and
Wilcoxon rank-sum test (for skewed variables) were used
to assess the significance of differences found between the
CAD group and the non-CAD group. Chi-squared test was
used to assess between-group differences for dichotomous
or categorical variables. Kruskal-Wallis rank-sum test was
used to compare serum LCN2 levels in groups according to
the number of MS components, and Spearmans
correlation was used to evaluate the relation between serum
LCN2 levels and other clinical parameters. Multiple logistic
regression analysis was performed to identify factors that
were independently correlated with CAD, and multivariate
stepwise regression analysis was used to further assess the
independent correlated clinical parameters of serum LCN2
levels. The skewed variables had been transformed by
natural logarithm before regression analyses. The threshold for
statistical significance was a two-sided p-value of <0.05.
Clinical characteristics of study participants
The serum LCN2 levels measured in the entire study
population ranged from 5.8 to 221.1 ng/mL, with an average
level of 34.9 (23.4-51.2) ng/mL. The average level in men
was significantly higher than that in women (37.5
(27.455.4) vs. 28.2 (18.7-45.9) ng/mL, p < 0.01).
Compared with non-CAD subjects, the patients with
CAD showed significantly higher age, CSI, and proportion
of lipid-lowering therapy. Stratification analysis by gender
showed that men with CAD had significantly lower uric
acid levels than those without CAD and that women with
CAD had significantly lower BMI but significantly higher
proportion of hypoglycemic therapy than their non-CAD
counterparts (all p < 0.05, Table 1).
Relationship between serum LCN2 levels and CAD
In general, the patients with CAD had significantly higher
serum LCN2 levels than non-CAD subjects (37.6
(25.256.3) vs. 31.1 (19.9-45.0) ng/mL, p < 0.01). Men with CAD
had significantly higher serum LCN2 levels than their
counterparts without CAD (39.2 (29.3-56.5) vs. 32.7
(20.549.7) ng/mL, p < 0.05); however, the CAD-related
significant trend was not found among the women (29.0
(18.5-51.9) vs. 27.0 (19.2-40.9) ng/mL, p > 0.05, Figure 1).
The gender-related significant findings prompted a focus
of the multiple logistic regression analysis to identify
factors that were independently correlated with CAD to be
performed for the men cohort only. When CAD was set as
the dependent variable, and age, BMI, HOMA-IR, LDL-c,
Figure 1 Comparison of serum LCN2 levels between subjects
with and without CAD. White bars, non-CAD subgroup; grey bars,
CAD subgroup. ** p < 0.01, CAD vs. non-CAD in total; * p < 0.05, CAD
vs. non-CAD in men. Data are presented as median (inter-quartile range).
serum adiponectin, eGFR, CRP, number of neutrophils,
serum LCN2, smoking, CAD family history, hypoglycemic
therapy, anti-hypertensive therapy, lipid-lowering therapy,
MS and its components (central obesity, hypertension,
hyperglycemia, hypertriglyceridemia, and low HDL-c) were
set as the independent variables , serum LCN2 levels (odds
ratio (OR) = 2.218, 95% confidence interval (CI):
1.0174.839) and lipid-lowering therapy (OR = 3.428, 95% CI:
1.192-9.864) were identified as independent risk factors for
CAD, while anti-hypertensive (OR = 0.360, 95% CI:
0.1380.938) therapy was identified as an independent protective
factor for CAD (all p < 0.05, Table 2).
Relationship between serum LCN2 levels and MS in men
Similar to the CAD findings, men with MS showed
significantly higher serum LCN2 levels than their non-MS
counterparts (40.2 (31.9-59.4) vs. 32.0 (21.7-47.6) ng/mL,
p < 0.01, Figure 2A). To further evaluate this relationship,
the contribution of the extent of MS was evaluated by
stratification analysis according to the number of MS
components, using the following sub-groups: 0 ~ 1 component,
n = 23; 2 components, n = 41; 3 components, n = 37; 4
components, n = 40; and 5 components, n = 28. The
Table 2 Multivariate logistic regression analysis showing
factors independently associated with CAD in men
0.398 0.045 2.218 1.017-4.839
Anti-hypertensive therapy 1.022 0.489 0.036 0.360 0.138-0.938
0.539 0.022 3.428 1.192-9.864
Variables of the original model included: age, BMI, HOMA-IR, LDL-c, serum
adiponectin, eGFR, CRP, Neutrophils, serum LCN2, smoking, CAD family history,
hypoglycemic therapy, anti-hypertensive therapy, lipid-lowering therapy, MS, central
obesity, hypertension, hyperglycemia, hypertriglyceridemia, and low HDL-c.
@Data were natural logarithm transformed before analysis and only significant
variables were presented.
Figure 2 Relationship between serum LCN2 levels and MS in men. (A) Serum LCN2 levels in men with and without MS; (B) p < 0.05 for
trend of increased serum LCN2 levels in men with increasing numbers of MS components. The bars represent the median, and 25th and 75th
percentile of serum LCN2 levels, respectively.
analysis showed a positive trend between increasing serum
LCN2 levels and increasing number of MS components
(p for trend <0.05, Figure 2B).
Influencing factors of increased serum LCN2 levels
Both the men and women groups were examined to
evaluate the correlation between clinical indicators and serum
LCN2 levels. In men, serum LCN2 levels were found to be
positively correlated with BMI (r = 0.195, p = 0.011), W (r =
0.164, p = 0.033), TG (r = 0.215, p = 0.005), CRP (r = 0.234,
p = 0.003) and neutrophils (r = 0.166, p = 0.032). In women,
CRP (r = 0.254, p = 0.017) and neutrophils (r = 0.249, p =
0.017) were correlated (all p < 0.05).
To further assess the independently correlated clinical
parameters of serum LCN2 levels, multivariate stepwise
regression analysis was performed in both the men and
women groups, respectively. When serum LCN2 levels
were set as the dependent variable, and age, BMI, W, SBP,
DBP, FPG, 2hPG, HbA1c, HOMA-IR, TC, TG, HDL-c,
LDL-c, serum adiponectin, UA, Scr, eGFR, CRP, number
of neutrophils, hypoglycemic therapy, anti-hypertensive
therapy and lipid-lowering therapy were set as the
independent variables, TG (Standard = 0.238, p = 0.003) and
neutrophils (Standard = 0.173, p = 0.031) were found to
be independent factors of serum LCN2 levels in men while
only neutrophils (Standard = 0.286, p = 0.009) remained
significant in women (all p < 0.05, Table 3).
Lcn2 expression is closely related to metabolism and
inflammation, both of which contribute to the pathogenic
process of atherosclerosis. In addition, serum LCN2 levels
are positively associated with the subsequent development
of CAD in patients with coronary artery atherosclerosis.
Furthermore, this relationship has been described in CAD
patients of Caucasian and Asian (Korean) ethnic origin,
with the former showing the positive association between
CAD severity in patients with myocardial infarction (vs.
those with stable angina) [16-18]. Increased serum LCN2
levels were also shown to be gender- (in men vs. women)
and weight- (in obese vs. normal weight) related . In
the present study, obvious gender-related differences
were observed as well (in agreement with the previous
studies) which led to the study design including gender
stratification analysis (with a men focus in the
regression analysis). Ultimately, the Chinese men in our study
who had CAD also had significantly higher serum LCN2
levels than either the women or their non-CAD men
Studies to elucidate the pathogenic molecular
mechanism of Lcn2 in atherosclerosis and CAD have shown that
the Lcn2/MMP-9 complex acts to destabilize the artery
plaque . This complex has been detected in clinical
specimens of plaques on the side facing the lumen area
and in lipid centers, suggesting an involvement in vascular
inflammation and reconstruction in myocardial ischemia
. MMP-9 alone, however, has been suggested to
promote atherosclerosis by degrading the vascular basement
Table 3 Multivariate stepwise regression analysis of
serum LCN2 levels
Women (N = 92)
Variables of the original model included: age, BMI, W, SBP, DBP, FPG, 2hPG,
HbA1c, HOMA-IR, TC, TG, HDL-c, LDL-c, serum adiponectin, UA, Scr, eGFR, CRP,
Neutrophils, hypoglycemic therapy, anti-hypertensive therapy, and
@Data were natural logarithm transformed before analysis and only significant
variables were presented.
membrane, thereby increasing endothelial permeability and
allowing more white blood cells and inflammatory
cytokines to infiltrate the intima. Indeed, the conditions of
plaque bleeding and hypoxia are accompanied by increased
levels of the Lcn2/MMP-9 complex; since the complexed
form also acts to protect the MMP-9 protein from
degradation, it is more capable of exerting a further effect (both in
its beneficial and detrimental manners) on the surrounding
tissue. The differential effects of MMP-9 have been seen in
different stages of CAD. It is believed that the
uncomplexed proteins degradation might be initially beneficial
to vascular repair. However, the constant inflammatory
stimulation and up-regulated Lcn2 may lead to increased
levels of the more stable Lcn2/MMP-9 complex, thereby
destabilizing the plaques and prompting the acute
cardiovascular event .
The current study included a cohort of patients at high
risk of metabolic diseases, especially MS. The results
suggested that serum LCN2 levels were 1.25-times higher in
the patients with MS than those without MS; furthermore,
the levels of LCN2 increased along with increases in the
number of MS components in the men patients. Patients
with metabolic disorders have been previously shown to
have a higher rate of cardiovascular diseases . MS, in
particular, and its components (which are characterized
as conventional risk factors of cardiovascular diseases)
is known to cause endothelial damage leading to
formation of the atherosclerotic plaque and facilitating lipid
infiltration that contributes to atherosclerosis
progression [29,30]. In agreement with this pathogenic process,
serum HDL-c has been previously demonstrated as
negatively correlated with serum LCN2 levels ; likewise, in
the current study serum TG was identified as a positive
influencing factor of serum LCN2 levels, indicating that
the related pathogenic mechanism of atherosclerosis may
involve disruption of lipid metabolism.
Chronic inflammation is another well-recognized
feature of CAD development. Previous studies have shown
that patients with acute coronary syndrome also have
increased levels of serum CRP [31,32], which are positively
associated with serum LCN2 levels even after adjusting for
several confounders . In this study, the number of
neutrophils, generally known to be an index of inflammation
status, was found to be positively correlated with serum
LCN2 levels. Similar results were obtained in the
current study of Chinese cohort, suggesting that LCN2
might prompt the inflammatory process that induces
When interpreting the results from the current study,
three inherent limitations to the study design must be
remembered. Firstly, the sample size was relatively small.
Secondly, the cross-sectional nature of the study
precluded the ability to confirm whether LCN2 itself
was a predictor of CAD. Finally, additional factors that
may influence in the outcome of our population were
not measured, including MMP-9 , retinol binding
protein 4 and resistin . Subsequent investigations
should consider each of these features to provide further
insights into this pathogenesis topic.
We conclude that the presence of increased serum
LCN2 levels is closely associated with CAD and MS in a
Chinese cohort. However, further large population-based
prospective studies encompassing also other ethnicities
are needed to demonstrate the potential of serum LCN2
as a sensitive predictor for CAD.
BMI: Body mass index; CAD: Coronary artery disease; CAG: Coronary
angiography; CRP: C-reactive protein; CSI: Coronary stenosis index;
DBP: Diastolic blood pressure; eGFR: Estimated glomerular filtration rate;
FPG: Fasting plasma glucose; HbA1c: Glycated hemoglobin A1c;
HOMAIR: Homeostasis model assessment index; LCN2: Lipocalin-2; MMP-9: Matrix
metalloproteinases-9; MS: Metabolic syndrome; Neutrophils: Number of
neutrophils; NGAL: Neutrophil-gelatinase associated lipocalin; SBP: Systolic
blood pressure; Scr: Serum creatinine; TC: Total cholesterol; TG: Triglyceride;
UA: Uric acid; W: Waist circumference; 2hPG: 2 h postprandial glucose;
24hALB: 24 h urine albumin.
We declare that there is no conflict of interest that could be perceived as
prejudicing the impartiality of the research reported.
YB and WJ designed the study. MZ, YH, JT and MG collected data. JN
analyzed data and wrote the draft. XP measured serum LCN2. ZL did the
angiographic analysis. XM, YB, and WJ revised the paper and contributed to
the discussion. All authors read and approved the final manuscript.
This work was funded by 973 Program of China (2013CB530606), National
Key Technology R&D Program of China (2012BAI02B03), Project of National
Natural Science Foundation of China (81100563), Key Discipline of Public
Health of Shanghai (Epidemiology) (12GWZX0104), and Drug Innovation
Program of National Science and Technology Project (2011ZX09307-001-02).
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