Article

High-Density Lipoprotein-Associated Paraoxonase-

1 (PON-1) and Scavenger Receptor Class B Type 1
(SRB-1) in Coronary
Artery Disease: Correlation with Disease Severity
Manish Kumar 1, Wahid Ali 1,*, Kusum Yadav 2, Swati Kaumri 1, Sridhar Mishra 2, Paolo Nardi
3
,
Ferdinando Iellamo 4, Sergio Bernardini 5, Akshyaya Pradhan 6 and Marco Alfonso Perrone 4

1
Department of Pathology, King George Medical University, Lucknow 226003, Uttar Pradesh,
India;
mankrshukla95@[Link] (M.K.); swatijaiswal621@[Link] (S.K.)
2
Department of Pathology, Dr. Ram Manohar Lohia Institute of Medical Sciences, Lucknow
226003,
Uttar Pradesh, India; kusummedico9453@[Link] (K.Y.); shridhar.mishra17@[Link]
(S.M.)
3
Department of Surgical Sciences, University of Rome Tor Vergata, 00133 Rome, Italy;
[Link]@[Link]
4
Division of Cardiology and Cardio Lab, Department of Clinical Sciences and Translational
Medicine,
University of Rome Tor Vergata, 00133 Rome, Italy; iellamo@[Link] (F.I.);
[Link]@[Link] (M.A.P.)
5
Department of Experimental Medicine, University of Rome Tor Vergata, 00133 Rome, Italy;
bernardini@[Link]
6
Lari Cardiology Center, King George Medical University, Lucknow 226003, Uttar Pradesh,
India; akshyaya33@[Link]

J. Clin. Med. 2024, 13, x. [Link] [Link]/journal/jcm

J. Clin. Med. 2024, 13, x FOR PEER REVIEW 2 of 22

Citation: Kumar, M.; Ali, W.;

Yadav, K.; Kaumri, S.; Mishra,
S.; Nardi, P.; Iellamo, F.;
Bernardini, S.; Pradhan, A.;
Perrone, M.A. High-Density
Lipoprotein-Associated
Paraoxonase-1 (PON-1) and
Scavenger Receptor Class B
Type 1 (SRB-1) in Coronary
Artery Disease: Correlation
with Disease Severity. J. Clin.
Med. 2024, 13, x.
[Link]

Academic Editor: Dinesh K.

Kalra

Received: 16 August 2024

Revised: 4 September 2024
Accepted: 11 September 2024
Published: date

Copyright: © 2024 by the

authors. Submitted for possible
open access publication under
the terms and conditions of the
Creative Commons Attribution
(CC BY) license
([Link]
censes/by/4.0/).
* Correspondence: aliwahid78@[Link]; Tel.: +91-522-2257542; Fax: +91-522-2257539

Abstract: Background: Coronary artery disease (CAD) is the leading cause of death
worldwide. High- Ddensity lipoprotein (HDL) is a well-established marker associated
with CAD. The current re research goes beyond the conventional HDL-C
measurement in previous studies and dives into the functional intricacies of HDL. By
understanding how HDL works, rather than just how much of it exists, we can better
tailor diagnostic and therapeutic strategies for CAD and related conditions. Hence,
the current study quantifies the serum levels of two novel, HDL- associated markers,
Paraoxonase- 1 (PON-1) and Scavenger Receptor Class B Type 1 (SRB-1), in CAD
cases vs. controls. Methods: A total of 92 subjects, including 69 CAD and 23 healthy
controls, were included, based on the prevalence of the disease. Further, based on
J. Clin. Med. 2024, 13, x FOR PEER REVIEW 3 of 22

the severity of the diseases, CAD cases were subcategorized in as CAD-I, -II, and -III.
Serum PON-1 and SRB-1 levels were measured and compared between patient and
control groups. Results: The lLevels of PON-1 and SRB-1 (32.6 ng/mL & and 12.49
ng/mL) were significantly lowered in CAD patient’s vs. the healthy control, at 60.36
ng/mL and 15.85 ng/mL, respectively (p < 0.000). A fFurther intergroup comparison
showed a statistically significant difference between the CAT-I [Link] -III for PON-1
(p < 0.025), the CAT-I [Link] -III, and CAT-II [Link] -III for SRB-1 (p < 0.000). The
rReceiver operating characteristics (ROC) curve showed a cut off values of 48.20
ng/mL and 14.90 ng/mL for PON-1 and, SRB-1. Conclusions: The current study
showed found that serum levels of HDL- associated PON-1 and SRB-1 are
significantly lower in CAD cases, and were also inversely related to the increasing
severity of coronary artery disease. This inference implies that serum PON-1 and
SRB-1 could be used for as non-invasive tools for the identification of coronary
atherosclerosis and risk assessment in CAD cases.

Keywords: coronary artery disease; atherosclerosis; Paraoxonase- 1; Scavenger

Receptor Class B Type 1; hHigh- dDensity lLipoprotein
J. Clin. Med. 2024, 13, x FOR PEER REVIEW 4 of 22

1. Introduction
Cardiovascular disease (CVD) is the leading cause of death worldwide.
Approximately 17.7 million people worldwide suffer from various types of
cardiovascular diseases each year [1]. Ischemic heart disease (IHD) and
stroke are the leading causes of cardiovascular disease-related deaths in
India (~83%) [2]. The protective response of Hhigh-Ddensity Llipoprotein
(HDL), and the atherogenic effect of Llow-Ddensity Llipoprotein (LDL), are
well-established risk factors for coronary artery disease (CAD) development.
HDL has numerous effects on the function and integrity of endothelial cells,
e.g., endothelial anti- and prothrombotic activity, modulation of nitric oxide
(NO) production, tissue repair, apoptosis, and adhesion of molecules. The
aforementioned functions are mediated by Sscavenger Rreceptor Cclass B
Ttype 1 (SRB-1) and S1P3 receptor-dependent signaling pathways [3,4].
HDL stimulates the production of NO via the endothelial nitric oxide
synthase (eNOS) enzyme, and it binds via the SRB-1 receptor. NO plays a
crucial role in the protective response, including vasodilation, anti-
inflammatory, and anti-adhesion effects [5]. Much emphasis has been placed
on the HDL value, not only on the numerical values, but also on the faulty
HDL’s functioning; for example,, e.g., B. Functional HDL deficiency is the
underlying cause of the development of atherosclerosis [5]. Experiments
with knockout mice have shown that the antioxidant property of HDL is
largely due to the Paraoxonase-1 (PON-1) enzyme that is found on it [6,7].
PON-1 is an enzyme primarily associated with HDL particles. It has
antioxidant properties and plays a role in protecting against oxidative stress.
PON-1 enhances cholesterol efflux from macrophages in atherosclerotic
plaques. This process helps prevent plaque from progressing and breaking
down. Inflammation is a major trigger of atherosclerosis, and PON-1 has
been found to reduce inflammation by inhibiting the oxidation of Llow-
Ddensity Llipoprotein (LDL) cholesterol. In addition to this, PON-1 can
detoxify certain organophosphate compounds, which may indirectly affect
CAD risk [8]. Low serum PON-1 activity is significantly reduced in patients
with myocardial infarction (MI), which may be the reason for the
development of CAD [8]. Research has shown that the genetic polymorphism
in PON-1 leads to a reduction in LDL peroxidation prevention activity and
the development of atherosclerosis [8]. The potential assaying of PON-1 may
be more effective than conventional serum HDL levels in predicting
atherosclerosis in patients with type II diabetes mellitus [9].
SRB-1 is an important component of reverse cholesterol transport
because it binds to HDL and facilitates the selective transfer of lipids
[10,11]. It is critical for cholesterol sensing and facilitates bidirectional
cellular cholesterol flow [11]. The role of SRB-1 in knockout mice was
investigated, and it showed was found that knockout mice lacking SRB-1
reduce cholesterol reverse transport and increase the risk of developing
atherosclerosis [11,12]. On the other hand, SRB-1 overexpression in the liver
reduces serum HDL levels and reduces the risk of atherosclerosis,
J. Clin. Med. 2024, 13, x FOR PEER REVIEW 5 of 22

suggesting that SRB-1 overexpression in the liver may promote cholesterol

efflux through reverse cholesterol pathways [13]. It has been postulated that
PON-1 and SRB-1 are part of the complex interplay between HDL particles,
cholesterol metabolism, and inflammation. Various studies have shown that
the balance between cholesterol uptake (via SRB-1) and cholesterol efflux
(augmented by PON-1) is crucial. Dysregulation can lead to lipid
accumulation and plaque formation. Variability in SRB-1 expression levels
may impact cholesterol management and CAD risk. Understanding these
mechanisms could guide therapeutic strategies. For example, increasing
PON-1 activity or promoting SRB-1 expression could be beneficial. Genetic
variations in PON-1 and SRB-1 may explain why some individuals are more
susceptible to CAD, despite similar HDL-C levels [11–13].
The studies have reported inconsistencies in PON-1 polymorphism
associations. While many studies have examined the association of PON-1
polymorphisms with CAD, results have not been consistent across the
literature. Our study aims to provide clarity by analyzing the serum
association of PON-1 levels with CAD severity. In addition, we address the
role of SRB-1 in CAD. The existing literature provides some insights, but
gaps remain. Our research aims to address these gaps and provide a
comprehensive understanding of the relevance of SRB-1 to CAD
pathophysiology. Hence, the current study was conducted to measure and
correlate SRB-1 and PON-1 levels with the severity of coronary obstruction
in patients with CAD, compared with to the normal control group.

2. Material and Methods

Study participants were recruited between December 2021 and June
2022 from the Lari Cardiology Center, Department of Cardiology, King
George Medical University, Lucknow, UP, India. Coronary angiograms were
evaluated by a clinician who performed a visual assessment of luminal
narrowing in multiple segments using the AHA/ACC classification of the
coronary tree [14].
A total of 92 subjects were included in the study, 69 of whom had
angiographically proven CAD and varying degrees of vascular blockage
severity. The diagnosis of coronary artery disease was made by a
cardiologist based on the patient’s history, clinical examination, and relevant
investigations, including angiography (whereby >50% stenosis in one or
more coronary arteries was used as a diagnostic criterion). The degree of
blockage and severity of each case were evaluated by an experienced
cardiologist and divided into three groups, namely Category 1, Category 2,
and Category 3, based on the presence of angiographically proven single-,
double-, and three-vessel blockages, respectively.
The healthy control group consisted of 23 subjects without detectable
coronary stenosis or atherosclerotic vascular disease. Patients were
interviewed to record their medical history and lifestyle habits. All patients
who met the inclusion criteria were recruited for the study. Written informed
consent was obtained from all patients. The research related to human use
J. Clin. Med. 2024, 13, x FOR PEER REVIEW 6 of 22

was conducted in accordance with all relevant national regulations and

institutional guidelines, and in accordance with the principles of the
Declaration of Helsinki. The study was approved by the authors’ institutional
ethics committee of King George Medical University, Lucknow, before the
start of the study. The exclusion criteria included cases with recent acute
coronary syndrome (within three months), diabetes mellitus type I and type
II, chronic kidney and liver diseases, active systemic infections, morbid
obesity, and connective tissue diseases. All demographic data were collected
from the OPD- and IPD- enrolled patients whose angiographic had already
been performed.

2.1. Sample Collection and Serum Isolation

A total of 5.0 mL of peripheral blood was collected from 92 subjects in
plain EDTA vials (NOVAC, POLYMED, Poly Medicure Ltd., India). The
sSerum was separated by centrifugation at 1900× g for 10 min, followed by
a 10 min high-speed centrifugation at 16,000× g, and stored at 80 °C until
further processing.

2.2. Biochemical Examination

Biochemical parameters, including Total Cholesterol (TC), (mg/dL),
Triglyceride (TG), (mg/dL), High-Ddensity Llipoproteins (HDL-C) (mg/dL),
Low-Ddensity Llipoproteins (LDL-C) (mg/dL), and Very- Llow-Ddensity
Llipoproteins (VLDL-C) (mg/dL), were recorded. All biochemical parameters
were measured using a fully automated biochemical analyzer (ARCHITECT
i2000SR, Abbott Diagnostic & Selectra ProXL, ELITech Group).

2.3. Enzyme-Linked Immunosorbent Assay for PON-1 and SRB-1

Serum PON-1 and SRB-1 levels were determined using an ELISA kit
(Elabbiosciences) based on the sandwich ELISA principle. After performing
several steps, the stop solution was added, and the optical density (OD) was
immediately measured spectrophotometrically at a wavelength of 450 nm ±
2 nm. Finally, based on this OD, the serum concentrations of PON-1 and
SRB-1 were measured in ng/dL. To minimize intra-assay variability, the
coefficient of variation (CV), which is the standard deviation divided by the
mean (expressed as a percentage), was calculated. Intra-assay CV values
less than 10% were found, and the results were considered reliable. Similar
to the intra-assay CV, the inter-assay CV was calculated from the standard
deviation and the mean of measurements over different assay runs, and it
was less than 12%. All samples were routinely analyzed in triplicate by
ELISA, and the results were averaged to minimize measurement errors and
calculate the final concentration in the samples.

2.4. Statistical Analysis

Statistical analysis was performed using SPSS (Statistical Package for
Social Sciences) Version 21.0 Sstatistical Analysis Software. Normally
J. Clin. Med. 2024, 13, x FOR PEER REVIEW 7 of 22

distributed continuous variables are presented as mMean ± SD (sstandard

dDeviation), and in number (%). The ANOVA test, followed by pPost hoc
tTests (Tukey-HSD), was used to compare the within-group and between-
group variances. A receiver operating characteristic curve (ROC) was
developed to evaluate the diagnostic performance of SRB-1 and PON-1. A 2
2 table was built to categorize tests as positive or negative based on
individual cutoffs obtained from ROC curves. A p value < 0.05 (two- tailed)
was considered significant.

3. Results
3.1. Demographic and Baseline Characteristics of the Patients and Controls
The demographic and clinical parameters of the patients and controls
are presented in Table 1. When baseline characteristics were compared
between the CAD cases and control groups, older age was observed in the
CAD cases (p < 0.000). Male predominance was observed in the
development of coronary heart disease (p < 0.000), along with a significantly
higher number of subjects who were smokers, alcohol consumers, and non-
vegetarians who developed coronary heart disease (p < 0.012, p< 0.052,
and p < 0.048). Obesity, BMI, and hypertension showed no significant
difference between the CAD cases and control groups, as shown in Table 1.
The subject who attended the OPD, underwent angiography, and had no
significant CAD and was considered a normal control (non-significant CAD).

Table 1. Basic and clinical characteristics of the study subjects.

Controls (n CAD Cases (n = p-

Variables
= 23) 69) Value
Age (Yrs.) 49.08 ± 14.09 57.55 ± 8.59 <0.000
Sex (Male/Female) (14/9) (65/4) <0.000
Alcohol Cconsumer (N) 2 (8.6%) 25 (36.23%) <0.012
Family History 2 (8.6%) 18 (20.0%) 0.486
Dietary Habits * (13/10) (23/46) 0.048
Nature of Work # (5/15/3) (8/51/10) 0.480
Smoker (N) 6 (26%) 34 (49.27%) <0.052
Hypertensive (N) 4 (17.39%) 24 (34.78%) <0.116
BMI (kg/m2) 23.34 ± 3.43 24.22 ± 3.80 <0.325
Obesity $ 4 (17.3%) 21 (30.4%) <0.223
Healthy Controls 23 (25%) -
Single- Vvessel Bblockage
23 (25%)
(CAD-I)
Double- Vvessels Bblockage
23 (25%)
(CAD-II)
Triple- Vvessels Bblockage 23 (25%
J. Clin. Med. 2024, 13, x FOR PEER REVIEW 8 of 22

(CAD-III)
#
* Dietary habits (Vegetarian/Non- Vvegetarian); Nature of Wwork
(Hard/Moderate/Sedentary);, $ >30 BMI is considered as obese.

3.2. Lipid Parameters and PON-1 and SRB-1 in Cases and Controls
The lipid parameters and PON-1 and SRB-1 levels are shown in Table 2.
Lower HDL levels were observed in CAD patients (44.76 ± 8.15 mg/dL)
compared with to controls (50.65 ± 12.43 mg/dL, p = 0.011), but no
significant differences were found in LDL, VLDL, and TG levels, as shown in
Table 2. A low level of PON-1 level was observed in CAD cases (32.65 ±
14.67 ng/dL) compared to controls (60.36 ± 12.63 ng, p = 0.000).
Furthermore, a significantly lower SRB-1 level was observed in CAD cases
(12.49 ± 3.34 ng/dL) compared to controls (15.85 ± 2.76, p = < 0.000), as
presented in Table 2).

Table 2. Lipid parameters and PON-1 & and SRB-1 levels in CAD cases and controls.

CAD Cases (n =
Variables Controls (n = 23) p Value
69)
Total Cholesterol (mg/dL) 142.4 ± 41.5 142.2 ± 40.1 0.983
High- Density Lipoprotein (HDL) (mg/dL) 50.65 ± 12.43 44.76 ± 8.15 0.011
Low- Density Lipoprotein (LDL) (mg/dL) 63.41 ± 31.96 67.29 ± 35.49 0.731
Very- Low- Density Lipoprotein (VLDL)
33.03 ± 11.81 33.84 ± 17.90 0.840
(mg/dL)
Triglyceride (mg/dL) 166.67 ± 89.47 166.34 ± 88.07 0.942
PON-1 (ng/dL) 60.36 ± 12.63 32.65 ± 14.67 0.000
SRB-1 (ng/dL) 15.85 ± 2.76 12.49 ± 3.34 0.000
CAD-I CAD-II CAD-III
27.05 ±
SRB-1 (ng/dL) 38.74 ± 19.41 32.16 ± 12.45
7.86
10.06 ±
PON-1 (ng/dL) 14.15 ± 2.20 14.25 ± 2.02
3.97

3.3. Intergroup Comparison of PON-1 and SRB-1 with Different Categories

of CAD and Normal Control
PON-1 concentration was found to be inversely correlated with
increasing severity of vascular obstruction, i.e., from a single- vessel
obstruction to a triple- vessel obstruction (CAD-I to CAD-III), as shown in
Table 3 and Figure 1. All categories (CAD -I, -II, and -III) show a statically
significant difference compared to the controls (p =< 0.000). Intergroup
comparisons were performed in CAD patients, and a statistically significant
difference was found between CAD-I (38.74 ± 19.41 ng/dL) and CAD-III
(27.05 ± 7.86 ng/dL, p = 0.025). However, no statistically significant
difference was found between CAD-I (38.74 ± 19.41 ng/dL) [Link] CAD-II
J. Clin. Med. 2024, 13, x FOR PEER REVIEW 9 of 22

(13.25 ± 2.03 ng/dL), and between CAD-II vs. and CAD-III (27.05 ± 7.86
ng/dL), as shown in Table 3. The maximum SRB-1 level was found in the
control subject (20.43 ng/dL), while the minimum level (4.01 ng/mL) was
found in CAD-III cases. It was observed that 17 out of 69 (24.63%) CAD
patients had decreased SRB-1 levels, despite higher-than-normal HDL levels
(mean 53.81, maximum 66.6 mg/dL) (mean 10.82, minimum 4.9 ng/dL).
Similarly, SRB-1 levels were also found to be inversely related to increasing
severity of vascular obstruction. CAD-II and CAD-III showed a statically
significant difference compared to healthy controls (p = 0.014 and p =
0.000, respectively), as shown in Table 3 and Figure 2. An intergroup
comparison was performed, showing a statistically significant difference
between CAD-I (14.15 ± 2.20 ng/dL) andvs. CAD-III (10.06 ± 3.98 ng/dL, p =
0.014), and CAD-II (13.25 ± 2.03 ng/dL) [Link] CAD-III (10.06 ± 3.98 ng/dL,
p = 0.001);, however, no significant difference was observed between CAD-I
(14.15 ± 2.20 ng/dL) and CAD-II (13.25 ± 2.03 ng/dL).
J. Clin. Med. 2024, 13, x FOR PEER REVIEW 10 of 22

Table 3. Intergroup comparison for PON-1 and SRB-1 in controls and cases with
different categories of disease severity.

Multiple Comparisons (Tukey-HSD)

95% Confidence In-
Depen-
Mean Difference Std. Er- Sig. terval
dent Vari- (I) GROUP (J) GROUP
(I-J) ror * Lower Upper
able
Bound Bound
0.00
CATEGORY-1 21.62 * 4.04 11.02 32.22
0
0.00
CONTROL CATEGORY-2 28.20 * 4.04 17.60 38.80
0
0.00
CATEGORY-3 33.30 * 4.04 22.71 43.90
0
0.36
CATEGORY-2 6.58 4.04 −4.01 17.18
CATE- 9
GORY-1 0.02
PON-1 CATEGORY-3 11.68 * 4.04 1.08 22.28
5
0.36
CATEGORY-1 −6.58 4.04 −17.18 4.01
CATE- 9
GORY-2 0.59
CATEGORY-3 5.10 4.04 −5.49 15.70
0
0.02
CATEGORY-1 −11.68 * 4.04 −22.28 −1.08
CATE- 5
GORY-3 0.59
CATEGORY-2 −5.10 4.04 −15.70 5.49
0
0.19
CATEGORY-1 1.69 0.83 −0.50 3.89
0
0.01
CONTROL CATEGORY-2 2.59 * 0.83 0.39 4.79
4
0.00
CATEGORY-3 5.78 * 0.83 3.59 7.98
0
0.70
CATEGORY-2 0.90 0.83 −1.29 3.09
CATE- 7
GORY-1 0.00
SRB-1 CATEGORY-3 4.09 * 0.83 1.89 6.29
0
0.70
CATEGORY-1 0.90 0.83 −3.09 1.29
CATE- 7
GORY-2 0.00
CATEGORY-3 3.19 * 0.83 0.99 5.39
1
0.00
CATEGORY-1 −4.09 * 0.83 −6.29 −1.89
CATE- 0
GORY-3 0.00
CATEGORY-2 −3.19 * 0.83 −5.39 −0.99
1
J. Clin. Med. 2024, 13, x FOR PEER REVIEW 11 of 22

* The mean difference is significant at the 0.05 level. One- way ANOVA with a Tukey-
HSD post -hock test was applied to see the statically significant difference.

Figure 1. Scatter plot showing the level of PON-1 in cases and controls, as well as
different categories of the CAD severity.
J. Clin. Med. 2024, 13, x FOR PEER REVIEW 12 of 22

Figure 2. Scatter plot showing the level of SRB-1 in cases and controls, as well as
different categories of the CAD severity.

3.4. Association of PON-1 and SRB-1 Level with Demographic

Characteristics
We have also analyzed the level of PON-1 and SRB-1 in CAD cases and
controls with various other demographic and clinical parameters that has
have been established as a risk factors for the development of coronary
artery disease, including tobacco uses, alcohol consumption, family history,
obesity, and hypertension; these findings are and is presented in Table 4.
The PON-1 level was significantly different, and higher, in cases that were
presented with hypertension, compared to cases with no hypertension (p =
0.002). Furthermore, tthe PON-1 level was higher inn cases with obesity
compared to non-obese cases;, however, the difference was not significant (p =
0.330).

Table 4. Association of PON-1 and SRB-1 level with demographic characteristics.

Cases Controls
J. Clin. Med. 2024, 13, x FOR PEER REVIEW 13 of 22

PON-1 SRB-1 PON-1 SRB-1

p p p- p
n (%) Mean ± Mean ± n (%) Mean ± Mean ±
Value Value Value Value
SD SD SD SD
46 32.76 ± 12.37 ± 61.96 ± 15.31 ±
Yes 13 (56.5)
(66.67) 15.85 3.26 11.73 2.82
Tobacco use 0.932 0.942 0.584 0.654
32.43 ± 12.73 ± 58.81 ± 16.40 ±
No 23 (33.3) 10(43.5)
12.05 3.48 13.42 2.45
27.14 ± 13.47 ± 59.75 ± 14.19 ±
Yes 08 (11.6) 2(8.7)
10.56 3.53 2.25 0.09
Alcohol use 0.26 0.823 0.513 0.587
33.37 ± 12.35 ± 60.77 ± 15.91 ±
No 61 (88.4) 21 (91.3)
14.86 3.26 12.75 2.81
32.29 ± 12.55 ± 74.75 ± 14.99 ±
Yes 04 (5.8) 2(8.7)
14.47 3.39 3.95 1.69
Family history 0.419 0.523 0.105 0.432
38.45 ± 11.36 ± 59.27 ± 15.83 ±
No 65 (94.2) 21(91.3)
14.91 1.15 12.24 2.80
36.86 ± 12.30 ± 66.47 ± 17.30 ±
Yes 10 (14.5) 5(21.7)
10.81 2.56 12.05 2.70
Obesity 0.330 0.895 0.329 0.422
31.93 ± 12.51 ± 59.38 ± 15.41 ±
No 59 (85.5) 18(78.3)
14.99 3.42 12.29 2.61
38.97 ± 12.73 ± 57.52 ± 18.49 ±
Yes 30 (43.5) 5(21.7)
16.08 3.37 13.27 1.48
Hypertension 0.002 0.622 0.545 0.983
28.34 ± 12.32 ± 61.60 ± 14.95 ±
No 39 (56.5) 18()
11.33 3.29 12.17 2.48
J. Clin. Med. 2024, 13, x FOR PEER REVIEW 14 of 22

3.5. Diagnostic Value of PON-1 and SRB-1 in Discriminating Cases from

Controls
To find out diagnostic accuracy (sensitivity and specificity) of markers
(PON-1 and SRB-1) in predicting CAD, both of the markers were subjected to
ROC curve analysis, and they were summarized in Table 5 and. also depicted
in Figure 3. In the discrimination of CAD cases from controls, the area under
the curve for the PON-1 was 0.93. Furthermore, at a cutoff point of ≤39.10
ng/dL (PON-1), the sensitivity and specificity were 75.36% and 100.00,
respectively. Moreover, we further calculated the diagnostic potential of
PON-1 in the discrimination of CAD cases based on blockage level, viz., CAD-
I, CAD-II, AND and CAD-III, which were selected from the normal control.
The AUC was highest for the discrimination of CAD-III from normal controls
(AUC = 0.99), with a sensitivity and specificity of 95.65% and 100.00%,
respectively. The AUCs for the discrimination of CAD-I and CAD-II was were
0.86 and 0.94, respectively.

Table 5. Ddiagnostic value of PON-1 and SRB-1 in differentiation of CAD from

controls.

Cut oOff Sensitivity Specificity

Marker AUC p Value
Value (95% CI) (95% CI)
PON-1
75.36 100.0
Case vs. Control <39.10 0.93 <0.0001 (64.04– (85.13–
84.01) 100.0)
90.91
73.91
Category 1 vs. Ccontrol <44.90 0.86 <0.0001 (72.19–
53.53–87.45
98.38)
73.91 100.0
Category 2 vs. Ccontrol <38.15 0.94 <0.0001 (53.53– (85.13–
87.45) 100.0)
95.65 100.0
Category 3 vs. Ccontrol <39.10 0.99 <0.0001 (79.01– (85.13–
99.78) 100.0)
SRB-1
72.73
57.97
Case vs. Control <13.63 0.75 0.0005 51.85–
46.21–68.89
86.85
54.55
60.87
Category 1 vs. Ccontrol <14.94 0.62 0.112 34.66–
40.79–77.84
73.08
Category 2 vs. Ccontrol <13.63 0.72 0.008 56.52 72.73
(36.81– (51.85–
J. Clin. Med. 2024, 13, x FOR PEER REVIEW 15 of 22

74.37) 86.85)
78.26 90.91
Category 3 vs. Ccontrol <12.75 0.88 <0.001 (58.10– (72.19–
90.34) 98.38)

Figure 3. ROC curve showing the sensitivity and specificity of PON-1 and SRB-1 in
cases and controls and with different categories of the CAD severity.

3.6. Diagnostic Value of SRB-1

When distinguishing CAD cases from controls, the area under the curve
for SERB-1 was 0.75. Furthermore, the sensitivity and specificity at a cutoff
value of 13.63 ng/dL (SRB-1) were 57.97% and 72.73%, respectively. In
addition, we further calculated the diagnostic potential of SRB-1 in
distinguishing CAD cases based on the degree of blockade, viz., CAD-I, CAD-
II, AND and CAD-III, from the normal control. The AUC was highest for
distinguishing CAD-III from normal controls (AUC = 0.88), with a sensitivity
and specificity of 78.26% and 90.91%, respectively. The AUCs for
distinguishing CAD-I and CAD-II for SRB-1 was were 0.62 and 0.72,
respectively, as depicted in Table 5 and Figure 3.

4. Discussion
Asian Indians are more susceptible to developing coronary heart disease
a decade earlier than the Caucasian population [15]. Furthermore, a recent
J. Clin. Med. 2024, 13, x FOR PEER REVIEW 16 of 22

study found that the risk of developing cardiovascular diseases is higher

among North Indians compared to people from other parts of India. It is
important to emphasize that the traditional risk factors have sometimes
failed to fully justify the excess risk of coronary heart disease in Indians,
raising the possibility of genetic susceptibility [16].
Our study showed that PON-1 levels decreased significantly in
Ccategory III (>90%), which included CAD cases with luminal stenosis,
regardless of the number of affected coronary arteries. One study found a
positive association between low PON-1 activity and the severity of coronary
artery disease, particularly in smokers and diabetic patients [17]. We also
found a significant association between PON-1 and hypertension in CAD
cases. Therefore, serum PON-1 may have a prognostic value in determining
the severity of CAD.
Oxidative damage plays an important role in chronic inflammatory
diseases, such as CAD. PON-1 is atheroprotective due to its antioxidant and
anti-inflammatory roles in the body [18,19];, indeed, it prevents LDL
oxidation, thereby reducing the risk of atherosclerosis [20]. The diverse
genetic variations in the PON-1 gene may influence its functional role, and
thus its ability to protect against CAD. Our results showed that PON-1 levels
were significantly lower in CAD patients compared to controls. Our data also
showed that individuals with lower PON-1 levels had a higher risk of
developing CAD. In addition, the serum PON-1 level was significantly lower
in the CAD-III category compared to CAD-I and CAD-II, as well as the control
group.
Several studies have been conducted to investigate the association
between PON-1 polymorphism and serum PON-1 activity in CAD patients.
Mackness MI et al. investigated that the PON-1 R allele polymorphism is
associated with the reduced PON-1 concentration and an increased risk of
developing coronary artery disease [6]. Fortunato G. et al. examined 310
middle-aged women for intimal thickness and PON-1 polymorphism, which
are related to premature atherosclerosis, and found that PON-1 (LL/ML)
seems to be positively correlated related to intimal thickness and carotid
atherosclerosis [21]. Coombes et al. examined the association between PON-
1 serum activity and concluded that an increase in PON-1 activity is
associated with a reduction in the risk of atherosclerosis [22]. Bhaskar et al.
conducted a cohort study in an Indian cardiovascular disease population
with and without diabetes mellitus and found that the frequency of the PON-
1-192RR genotype and R allele both were both was increased in both CAD
and T2DM patients, compared to controls [23].
PON-1 inhibits LDL-C oxidation and lipid peroxide accumulation in
macrophages. Reduced uptake of oxidized LDL is likely to be mediated by
PON-1 interaction with Sscavenger Rreceptor Cclass B Ttype 1 (SRB-1) on
the macrophage surface, leading to the suppression of macrophage
proinflammatory responses [24]. PON-1 also reduces monocyte chemotaxis
and adhesion to endothelial cells, thereby preventing endothelial damage
and atherosclerosis [25]. Research also shows that incubation of PON-1 with
HDL results in a reduction in the expression of intercellular adhesion
J. Clin. Med. 2024, 13, x FOR PEER REVIEW 17 of 22

molecule (ICAM)-1 on endothelial cells, which helps to reduce the

progression of inflammation in the endothelium. PON-1 also protects against
the proinflammatory effects of oxidized phospholipids and
lipopolysaccharides. Furthermore, PON-1 reduces cholesterol biosynthesis
by macrophages, and increases cholesterol efflux from LDL-C [26]. Some
research has shown that in carriers of the RR genotype, PON-1 enzyme
activity activities decreases, increasing oxidative stress and inflammation;
furthermore,, and all of these events may be related to the occurrence of
atherosclerosis and CAD [27].
The most important role of SRB-1 is the clearance of plasma cholesterol
via the reverse cholesterol transport pathway (RCT), in which mature HDL
particles transport cholesterol from peripheral tissues to the liver for
excretion [28,29]. Lipid-poor HDL dissociates from SRB-1 and re-enters the
circulation to continue participating in the RCT. Therefore, an efficient
interaction between SRB-1 and HDL is required to maintain cholesterol
homeostasis and protect against plaque accumulation that occurs in
atherosclerotic cardiovascular disease (ASCVD). The importance of SRB-1 in
atheroprotection is well- illustrated by in vivo studies [28–31]. When SRB-1
is genetically deleted in wild-type mice, HDL cholesterol (HDL-C) levels
double, and atherosclerotic plaques rapidly develop [30,31]. SRB-1 deletion
in atherosclerotic ApoE knockout mice results in myocardial infarction and
early death [32]. Similarly, SRB-1 deletion in Llow-Ddensity Llipoprotein
receptor (LDL) knockout mice fed a high-fat Western diet also shows early
atherosclerosis [33,34]. Alternatively, SRB-1 overexpression in wild-type
mice results in reduced HDL-C levels [35–38] and less atherosclerotic plaque
formation [39]. In addition, all nine identified human variants of SRB-1
resulted in increased plasma HDL-C levels, with two variants (P376L and
G319V-SRB-1) directly increasing the risk of ASCVD. Combining these
clinical observations with mouse studies, the importance of SRB-1 in
maintaining adequate HDL-C levels and mitigating ASCVD risk becomes
increasingly clear [40,41].
Various animal model studies (knockout mice) have shown that mice
lacking SRB-1 have an increased risk of developing atherosclerosis [11,12].
The present study also suggests that a similar mechanism is involved in the
reverse cholesterol transport of patients with CAD, as there is a minimal
SRB-1 level in patients with a triple- vessel blockade. Huang L. et al. tested
the hypothesis that SRB-1 is a receptor for HDL and LDL, and can thus
promote atherosclerosis in the aorta and other atherogenic areas of mice
[42]. They concluded that therapeutic interventions that inhibit the
endothelial delivery of LDL into the arterial vials mediated by SRB-1 and
DOCK 4 may be a potential remedy against the development of
atherosclerosis and cardiovascular disease. Wang et al. investigated the
function of a novel miR-24 on SRB-1 expression, HDL uptake, and lipid
metabolism in different types of cells extracted from mice fed a high-fat diet,
and found that miR-24 directly targets SRB-1 and decreases SRB-1 levels
and HDL uptake [43]. Similarly, Shen et al. reviewed various research
papers on the regulation and functional significance of SRB-1 in mediating
J. Clin. Med. 2024, 13, x FOR PEER REVIEW 18 of 22

cholesterol movement, and concluded that SRB-1 is a multiligand of

membrane receptors that acts as a receptor for the selective delivery of HDL
to cells, and that SRB-1 may serve as a potential marker for diagnostic and
therapeutic purposes for the selective HDL uptake pathway in individuals
with risk factors [44]. The current study also provides similar results and
underlines the importance of SRB-1 in high-risk patients. In addition, Jiang
C. et al. conducted an experiment on mice fed a high-fat and low-fat diet and
concluded that SRB-1 plays an important role in adipogenesis in high-fat diet
mice by regulating the increase in SRB-1 expression increased [45].
Zanoni et al. showed in their study that P376L carriers have an
increased risk of CHD, despite their increased HDL level, and proved that
steady-state concentrations of HDL-C do not provide causal protection
against CHD and HDL function, and that cholesterol flux may be more
important than the absolute values [46]. Zhang, Y. et al. examined the
hepatic expression of SRB-1 and plasma HDL levels in mice and concluded
that SRB-1-deficient mice have higher plasma HDL levels than mice with
hepatic SRB-1 overexpression [13]. Zanoni et al. emphasized that people
who are carriers of P376L have elevated HDL levels, but are at a higher risk
of developing coronary artery disease [46]. This can be explained by
defective expression of SRB-1 in the liver, which promotes the compensatory
increase in absolute HDL levels;, but however, this increased HDL value
does not protect against atherosclerosis, which is consistent with the
experiment of Zhang, Y. et al. on mice [13]. Similar results were observed in
our study. Our data showed that 17 of 69 (24.63%) CAD patients had varying
degrees of coronary obstruction despite elevated HDL levels and reduced
serum SRB-1 levels. The current study is an extension of previous animal model
testing hypotheses, and demonstrates the association between atherogenesis
and the role of SRB-1 in patients with CAD.
Our results support the inverse association between PON-1 and SRB-1
in patients with CAD. High-severity CAD cases have low PON-1 and SRB-1
levels compared to normal controls. Low levels of PON-1 and SRB-1 may be
responsible for a change in the functionality of the PON-1 enzyme, leading to
an increased risk of CAD. Furthermore, because CAD is a multifactorial
disease due to the interaction of various environmental and genetic
influences, there may also be other modulators that alter serum PON-1
levels in these individuals. Furthermore, PON-1 may have prognostic
significance in severe coronary artery stenosis [47–49]. The limitation of our
study was the relatively small sample size, representing only a fraction of
patients with CAD admitted to a tertiary care hospital during the given
period. However, our study shows the importance of additional risk factors,
including hypertension, and valuable markers such as PON-1 and SRB-1
levels, which may be useful for predicting CAD in the Indian population.
Prospective studies with a larger population are needed to gain a better
understanding of the role of PON-1 in increased CAD susceptibility. PON-1
and SRB-1, as potential therapeutic candidates, are also interesting points
for further study. Exploring PON-1 and SRB-1 in low-risk individuals could
improve our understanding of early CVD mechanisms. Although there are
J. Clin. Med. 2024, 13, x FOR PEER REVIEW 19 of 22

challenges, this research can serve as a guide for personalized prevention

strategies.

5. Conclusions
The present study was conducted with the primary aim of determining
the serum levels of PON-1 and SRB-1, and their association with the severity
of vascular blockage in patients with CAD. Compared to healthy controls,
sSignificantly lower levels of PON-1 and SRB-1 were observed compared
with healthy controls, with lower values in those angiographically diagnosed
with the maximum severity of vascular blockage. This knowledge can be
used to assess the functional aspect of HDL, risk assessment of
atherosclerosis in high-risk groups, and severity of coronary stenosis in
patients with CAD. Based on the above data, a non-invasive, cost-effective
serological diagnostic test can be developed. More studies will be needed to
confirm these data. Although PON-1 and SRB-1 show promise as CAD
biomarkers, their successful integration into routine practice requires
overcoming technical, clinical, and logistical challenges. Clinicians,
researchers, and policymakers must work together to manage this
complexity and maximize the clinical impact of these biomarkers.

Author Contributions: Conceptualization, M.K. and W.A.; investigation, M.K., W.A.,

K.Y., S.K. and S.M.; methodology, W.A., A.P. and M.A.P.; supervision, W.A., A.P. and
M.A.P.; validation, P.N., S.B., F.I. and A.P.; writing—review and editing, M.K., W.A. and
M.A.P. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: The study was conducted in accordance
with the Declaration of Helsinki, and was approved by the Institutional Ethics
Committee (dDocument code: -72nd ECM-II-B-Thesis/P25, approved on 21 October
2021).
Informed Consent Statement: Informed consent was obtained from all subjects
involved in the study.
Data Availability Statement: The data presented in this study are available upon
request from the corresponding author.
Conflicts of Interest: The authors declare no conflicts of interest.

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