F1000Research 2022, 11:1526 Last updated: 15 DEC 2022

RESEARCH ARTICLE

Serum leucine-rich alpha-2 glycoprotein levels in rheumatoid

arthritis and spondyloarthritis: A promising biomarker
[version 1; peer review: awaiting peer review]
Rizqi Arini Siregar 1,2, Suryo Anggoro Kusumo Wibowo1, Sumariyono .1,
Aulia Rizka3, Rudy Hidayat1, Hamzah Shatri4, Sukamto Koesnoe5,
Cosphiadi Irawan6
1Division of Rheumatology, Department of Internal Medicine, Universitas Indonesia/Dr. Cipto Mangunkusumo National Central

Public Hospital, Jakarta, 10430, Indonesia

2Department of Internal Medicine, Faculty of Medicine, Universitas Sumatera Utara, Medan, 20155, Indonesia
3Division of Geriatrics, Department of Internal Medicine, Universitas Indonesia/Dr. Cipto Mangunkusumo National Central Public

Hospital,, Jakarta, 10430, Indonesia

4Division of Psychosomatic Medicine, Department of Internal Medicine, Universitas Indonesia/Dr. Cipto Mangunkusumo National

Central Public Hospital, Jakarta, 10430, Indonesia

5Division of Allergy and Immunology, Department of Internal Medicine, Universitas Indonesia/Dr. Cipto Mangunkusumo National

Central Public Hospital, Jakarta, 10430, Indonesia

6Division of Medical Hematology and Oncology, Department of Internal Medicine, Universitas Indonesia/Dr. Cipto Mangunkusumo

National Central Public Hospital, Jakarta, 10430, Indonesia

v1 First published: 15 Dec 2022, 11:1526 Open Peer Review

https://doi.org/10.12688/f1000research.128298.1
Latest published: 15 Dec 2022, 11:1526
https://doi.org/10.12688/f1000research.128298.1 Approval Status AWAITING PEER REVIEW

Any reports and responses or comments on the

Abstract article can be found at the end of the article.

Background: In the early stages of the disease, some of the signs and
symptoms of joint inflammation in rheumatoid arthritis (RA) may
resemble that of spondyloarthritis (SpA). An examination that can help
distinguish RA and SpA is warranted. One such examination is the
measurement of serum leucine-rich alpha-2 glycoprotein (LRG) levels.
This study aimed to measure serum LRG levels in RA and SpA patients
and determine the role of LRG in the diagnosis of RA and SpA.
Methods: This is a cross-sectional study consisting of 26 RA subjects
and 26 SpA subjects. The SpA subjects were further grouped into
ankylosing spondylitis (AS), psoriatic arthritis (PsA), and peripheral
SpA. Measurement of serum LRG levels were conducted using ELISA.
Difference between LRG levels of the two groups were compared
using the Mann-Whitney test.
Results: LRG levels were elevated in 76.9% and 84.6% of subjects with
RA and SpA, respectively. The median LRG levels were higher in RA
subjects (77.03 (27.16–107.73)) than SpA (68.67 (33.15–115.18)). There
was no significant difference in LRG levels in RA and SpA subjects (p =
.442). The RA and PsA group were predominated by diseases of
moderate activity, 88.5% and 58.3%, respectively. In comparison, AS
was dominated by high disease activity (85.7%). The highest median

 
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LRG levels in AR and SpA subjects were in new-onset patients (82.21

vs. 72.25 µg/dL).
Conclusions: There was no significant difference in LRG levels
between RA and SpA subjects. The role of LRG in the diagnosis of RA
and SpA remains to be determined in future studies.

Keywords
rheumatoid arthritis, spondyloarthritis, leucine-rich alpha-2
glycoprotein

Corresponding author: Rizqi Arini Siregar (arini_siregar@yahoo.com)

Author roles: Siregar RA: Conceptualization, Formal Analysis, Investigation, Methodology, Writing – Original Draft Preparation, Writing
– Review & Editing; Wibowo SAK: Formal Analysis, Investigation, Writing – Original Draft Preparation, Writing – Review & Editing; . S:
Formal Analysis, Investigation, Writing – Original Draft Preparation, Writing – Review & Editing; Rizka A: Formal Analysis, Investigation,
Writing – Original Draft Preparation, Writing – Review & Editing; Hidayat R: Formal Analysis, Investigation, Writing – Original Draft
Preparation, Writing – Review & Editing; Shatri H: Formal Analysis, Investigation, Writing – Original Draft Preparation, Writing – Review &
Editing; Koesnoe S: Formal Analysis, Investigation, Writing – Original Draft Preparation, Writing – Review & Editing; Irawan C: Formal
Analysis, Investigation, Writing – Original Draft Preparation, Writing – Review & Editing
Competing interests: No competing interests were disclosed.
Grant information: The author(s) declared that no grants were involved in supporting this work.
Copyright: © 2022 Siregar RA et al. This is an open access article distributed under the terms of the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
How to cite this article: Siregar RA, Wibowo SAK, . S et al. Serum leucine-rich alpha-2 glycoprotein levels in rheumatoid arthritis
and spondyloarthritis: A promising biomarker [version 1; peer review: awaiting peer review] F1000Research 2022, 11:1526
https://doi.org/10.12688/f1000research.128298.1
First published: 15 Dec 2022, 11:1526 https://doi.org/10.12688/f1000research.128298.1

 
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Introduction
Arthritis is an inflammatory process characterized by pain, swelling and stiffness of the joints. Chronic inflammation of
the joints can lead to joint deformity and destruction of structures in proximity to the joints. Early identification of the
etiologies of arthritis is pivotal to promote clinical improvement, prevent the development of structural and functional
joint damage, and consequently preserve patient quality of life.1 Two of the major causes of arthritis are systemic
autoimmune diseases, such as rheumatoid arthritis (RA) and spondyloarthritis (SpA).2,3

The prevalence of these two diseases is similar. The global prevalence of RA is 0.5–1%, while the exact prevalence of RA
in Indonesia is unknown. However, based on a prevalence of 0.5–1% of the total population of Indonesia, it can be
estimated that RA occurs in around 1.3 million Indonesians in 2020.4 Meanwhile, the prevalence of SpA is about 0.5–2%
of the world population.5,6

The pathogenesis of RA and SpA is complex and distinct from one another. RA is an autoimmune disease characterized
by the production of rheumatoid factor (RF) and anti-citrullinating protein antibody (ACPA).7,8 Patients with RA who are
positive for RF, ACPA, or both, which represents approximately 75–85% of all patients with RA, are classified as
seropositive RA. However, approximately 20% of patients with RA are negative for RF and/or ACPA and are
consequently classified as seronegative RA.5 The absence of antibodies in SpA renders it difficult to distinguish from
seronegative RA in some cases. SpA is a group of systemic rheumatic diseases characterized by chronic autoinflamma-
tion of the innate immune system and is associated with HLA-B27.6,7 SpA is further subdivided into psoriatic arthritis
(PsA), ankylosing spondylitis (AS), reactive arthritis, arthritis associated with inflammatory bowel disease (IBD) and
non-specific arthritis.9,10

In the early stages of the disease, some of the signs and symptoms of joint inflammation in RA may resemble that of SpA.
This can lead to difficulty in pinpointing a diagnosis, which then affects the extent of joint damage. A cohort study by
Combe et al., suggested that early diagnosis is the key to a good outcome in the management of arthritis.11 Despite this
urgency, classification criteria and clinical diagnostic standards are often only useful one or two years after disease onset.
Additionally, clinicians may not use them consistently and uniformly when classifying early-phase arthritis.11–13

The annual incidence of early inflammatory arthritis (EIA) ranges from 115 to 271 per 100,000 adults. The annual
incidence of undifferentiated arthritis (UA) ranges from 41 to 149 per 100,000 adults. Around 13–54% of UA will
progress to RA, whereas 21–87% of UA will persist. Several cohort studies report that early-phase arthritis patients who
are referred to rheumatology for further assessment had to wait for 2 to 36 months to be reclassified to a more specific
arthritis.14 These holdups can lead to delays in diagnosis and administration of therapy. Several studies have shown that
patients with RA diagnosed in more than 12 weeks since the onset of symptoms eventually exhibited more severe joint
destruction. Over a course of 10 years, 92% of patients showed decreased functional ability and about 50% required
assistance in performing daily activities.15,16

Providing a correct diagnosis will positively impact the management of these patients as some medical therapies for RA
may not be effective in SpA. For instance, the B cell targeting drug rituximab is considered effective in RA but has yet to
be proven effective in SpA. By contrast, the administration of drugs targeting the interleukin (IL)-12/IL-23 and IL-17
pathways have proven beneficial in PsA, but not in RA.17,18

In some cases, RA and SpA may show clinical symptoms that are difficult to distinguish from one another. Therefore, an
examination to assist the diagnosis of RA and SpA is warranted. One such examination is the measurement of leucine-rich
alpha-2 glycoprotein (LRG) levels. LRG is a 50 kDa leucine-rich glycoprotein synthesized in hepatocytes, neutrophils,
macrophages, and epithelial cells of inflamed tissues. LRG expression is mostly stimulated by IL-6 or IL-1 but could also
be induced by IL-1β, IL-22, and NF-α. Increased pro-inflammatory cytokines, especially IL-6, has been shown to
indirectly increase LRG.19–21

Studies assessing LRG levels in the synovial fluid of RA and SpA patients have previously been conducted, with varying
results being obtained. Several studies examining LRG levels in synovial fluid, including one by Birkelund et al.,
reported that synovial fluid proteomics analysis from SpA and RA patients showed striking differences in the amount of
innate immune system proteins, especially those from neutrophil granulocytes, including LRG. This protein was found to
be more abundant in the RA patient group.22 Furthermore, there are also studies examining the utility of serum LRG
levels in differentiating EIA. McArdle et al., analyzed serum protein biomarkers from RA and PsA patients using multiple
reaction monitoring (MRM). LRG was recognized as one of the proteins that was able to distinguish RA from PsA, which
was found to be significantly increased in RA relative to PsA. This increase in LRG is associated with a relatively higher
level of IL-6 in RA compared to SpA, which in turn produced a greater increase in LRG expression.17 Nakajima et al.,
found that serum LRG levels were also elevated in patients with PsA compared to patients with psoriasis vulgaris.23
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Currently, there are no existing studies comparing LRG values in RA and SpA patients using blood samples. Therefore,
we analyzed serum LRG levels in patients with confirmed RA and SpA. The results of this study can hopefully be used as
primary data to distinguish RA and SpA in cases where they are difficult to discern and aid in better understanding the role
of LRG the diagnosis of RA and SpA.

Methods
Ethics statement
This study has received approval from the Research Ethics Committee of Faculty of Medicine Universitas Indonesia and
Dr. Cipto Mangunkusumo National Central Public Hospital (KET-197/UN2.F1/ETIK/PPM.00.02/2022) on 28 February
2022. Written informed consent for their participation in the study and publication of the patients’ details was obtained
from each of the patients. The patients were anonymized and identified through the medical record number.

Selection and recruitment of subjects

This research was a cross-sectional study conducted at the Rheumatology Polyclinic of Dr. Cipto Mangunkusumo
National Central Public Hospital (RSCM) (Jakarta, Indonesia) from March to May of 2022. This study is reported in line
with the STROBE guidelines.24 The research subjects were recruited by consecutive sampling. The inclusion criteria
included: patients aged over 18 years old; confirmed seropositive active RA patients in accordance with the disease
activity score-28 for RA (DAS28); active SpA patients in accordance with the classification criteria of each disease,
namely AS disease activity score (ASDAS) for AS and peripheral SpA and disease activity index for PsA (DAPSA) for
PsA; and (4) patients able to fill out the consent form and are willing to participate in the study. Patients with other
systemic autoimmune diseases, acute infectious diseases, liver disorders, and malignancies were excluded from the study.

This study also divided the subjects to several subgroups. C-reactive protein (CRP) levels were measured and patients
with elevated CRP, defined as greater than 5 mg/dL, were analyzed separately. The LRG levels of both RA and SpA
patients were also analyzed separately based on their disease activity and disease onset. Naïve patients, defined as patients
with no prior exposure to conventional or disease-modifying anti-rheumatic drugs (DMARD), were categorized as “new
onset”. Meanwhile patients who have received DMARD were categorized as “old onset”. Appropriate criteria cut-off
points were used according to the corresponding disease in determining disease activity. Determination of sample size can
be found as Underlying data.24

Measurement of leucine-rich alpha-2 glycoprotein and c-reactive protein levels

Blood samples collected from patients were analyzed for LRG and CRP levels in an ISO-certified laboratory at
Universitas Indonesia. To extract the serum, 6 mL of whole blood was drawn and centrifuged (Heraeus Labofuge
200, Thermo Fisher Scientific, Waltham (MA), USA) at 3,000 rpm for 15 minutes and 2,000 rpm for 5 minutes for LRG
and CRP, respectively. LRG measurement was performed with Human LRG ELISA Assay Kit (Immuno-Biological
Laboratories Co., Ltd., Fujioka, Gunma, Japan) and read with spectrophotometry at 450 nm. Meanwhile, CRP
measurement was performed with immunoturbidimetry using cobas c 311 analyzer (Roche Holding AG, Basel,
Switzerland).

Statistical analysis
Statistical analyses of the collected data were processed using IBM SPSS Statistics (RRID:SCR_016479) 24.0 for Mac
program (IBM Corp., Armonk (NY), USA). Comparison of LRG levels were analyzed using the parametric independent
samples t-test or Mann-Whitney non-parametric test depending on the distribution of the data, which was determined via
the Shapiro-Wilk test.

Results
Ultimately, 26 patients with RA and 26 patients with SpA were included as research subjects, totaling at 52 patients. A
flow diagram of subject inclusion can be found as Underlying data.24 Characteristics of the research subjects are shown in
Table 1.24

LRG levels of the SpA and RA patients were analyzed with the Mann-Whitney test because the subjects obtained were not
normally distributed. The test showed no statistical difference between the LRG levels of patients in the RA group (mean
rank = 28.12) and those in the SpA group (mean rank = 24.88); Mann-Whitney U = 296, p = .442 two-tailed.

In this study, patient LRG levels were further grouped based on disease activity, onset, comorbidity, and treatment
(Table 2). Meanwhile, Table 3 shows the serum LRG levels of RA and SpA patients based on disease onset. Additional
analyses were also conducted to determine the difference in LRG levels between the RA group and the SpA group based
on CRP levels and disease activity.

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Table 1. Characteristics of included research subjects.

Characteristics RA (n = 26) SpA (n = 26)

Sex (n [%])
 Male 2 (7.7%) 8 (30.8%)
 Female 24 (92.3%) 18 (69.2%)
Age (years) (mean  SD) 49  12 41  10
Diagnosis (n [%])
 Peripheral SpA - 1 (3.8%)
 Ankylosing spondylitis - 13 (50%)
 Psoriatic arthritis - 12 (46.2%)
DAS28 (n [%])
 Moderate (> 3.2–5.1) 22 (88.5%) -
 Severe (> 5.1) 4 (11.5%) -
DAPSA (n [%])
 Moderate (15–23, 25–29) - 7 (58.3%)
 Severe (> 28) - 5 (41.7%)
ASDAS (n [%])
 High (≥ 2.1–3.5) - 12 (85.7%)
 Very high (≥ 3.5) - 2 (14.3%)
Rheumatoid factor positive (n [%]) 26 (100%) -
LRG (n [%])
 Normal (21–50 μg/dL) 6 (23.1%) 4 (15.4%)
 Increased (> 50 μg/dL) 20 (76.9%) 22 (84.6%)
LRG level (μg/dL) (median [range]) 77.03 (27.16–107.73) 68.9  21.5*
CRP level (mg/dL) (median [range]) 13.9 (1.2–45.5) 9.45 (0.6–54.2)
Treatment (n [%])
 Non-DMARD 5 (19.2%) 2 (7.7%)
 DMARD monotherapy 12 (46.2%) 18 (69.2%)
 Combination of 2 DMARD 7 (26.9%) 5 (19.2%)
 Combination of 3 DMARD 2 (7.7%) 1 (3.9%)
Glucocorticoid use (n [%])
 Did not use 6 (23.1%) 6 (23.1%)
 Methylprednisolone ≤ 4 mg 11 (42.3%) 3 (11.5%)
 Methylprednisolone > 4 mg 9 (34.6%) -
 NSAID - 17 (65.4%)
Disease onset (n [%])
 New onset 5 (19.2%) 1 (3.85%)
 Old onset 21 (80.8%) 25 (96.15%)
Comorbidities (n [%])
 No comorbidity 6 (23%) 10 (38.5%)
 One comorbidity 10 (38.5%) 11 (42.3%)
 More than one comorbidity 10 (38.5%) 5 (19.2%)
*
Mean was used as the data was normally distributed; RA: rheumatoid arthritis; SpA: spondyloarthritis; DAS28: disease activity score-28 for
rheumatoid arthritis; DAPSA: disease activity index for psoriatic arthritis; ASDAS: ankylosing spondylitis disease activity score; LRG: leucine-
rich alpha-2 glycoprotein; CRP: C-reactive protein; DMARD: disease-modifying anti-rheumatic drugs; NSAID: non-steroidal anti-
inflammatory drugs
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Table 2. LRG levels based on disease activity, onset, comorbidities, and treatment in RA and SpA subjects.

LRG levels RA (n = 26) SpA (n = 26)

AS (n = 13) PsA (n = 12) Peripheral
SpA (n = 1)
Total LRG level (μg/dL) 77.03 (27.16–107.73) 58.6 (41.6–92.4) 75.3 (33.15–115.18) 55.07
(median [range])
LRG level based on disease activity (μg/dL) (median [range])
Moderate 82.62 (27.16–107.73) - 68.7 (33.15–115.18) -
High 59.45 (34.85–97.68) 72.25 (41.6–92.4) 92.9 (89.4–103.5) -
Very high - 59.5 (56.2–62.7) - 55.07
LRG level based on disease onset (μg/dL) (median [range])
New onset 82.21 (54.26–107.73) 72.25 - -
Old onset 72 (27.16–104.12) 57.4 (41.6–92.4) 75.3 (33.2–115.2) 55.07
LRG level based on presence of comorbidities (median [range])
No comorbidity 86.92 (41.33–104.02) 64.3 (44.4–79.7) 85.9 (68.2–115.2) 55.07
One comorbidity 66.48 (47.6 –104.12) 60.6 (41.5–92.4) 80.7 (70–92.6) -
More than one 71.26 (27.16–107.73) 52.5 70.9 (36.9–93.4) -
comorbidity
LRG level based on treatment (median [range])
Have not received DMARD 82.21 (54.26–107.73) 64.25 (56.24-72.25) - -
DMARD monotherapy 59.56 (27.16–104.12) 56.12 (41.63–92.41) 80.7 (33.15–103.53) 55.07
Combination of 2 DMARD 90.81 (51.43–104.02) 79.7 60.37 (36.87–103.53) -
Combination of 3 DMARD 73.98 (47.6–100.35) - 89.44 -
LRG: leucine-rich alpha-2 glycoprotein; RA: rheumatoid arthritis; SpA: spondyloarthritis; AS: ankylosing spondylitis; PsA: psoriatic arthritis;
DMARD: disease-modifying anti-rheumatic drugs.

Table 3. Serum LRG levels based on disease onset.

LRG levels (n [%]) New onset Old Onset

RA SpA RA SpA
Normal (21–50 μg/dL) - - 6 (28.6%) 4 (16%)
Increased (> 50 μg/dL) 5 (100%) 1 (100%) 15 (71.4%) 21 (84%)
LRG: leucine-rich alpha-2 glycoprotein; RA: rheumatoid arthritis; SpA: spondyloarthritis.

Patients with elevated CRP were analyzed separately as means to control the potential subjectivity in the assessment of
disease activity. Elevated CRP was observed in 21 RA patients and 20 SpA patients. As the data were not normally
distributed, the Mann-Whitney test was used. The test showed no statistical difference between the LRG levels of patients
with RA (mean rank = 23.24) and those with SpA (mean rank = 18.65); Mann-Whitney U = 163, p = .220 two-tailed.

Comparison of LRG levels between RA subjects with moderate disease activity (n = 22) and SpA with high-very high
disease activity (n = 19) was also conducted. This additional analysis was performed to further clarify the difference in
LRG levels between the two largest groups, which was composed mostly of the RA and SpA group. As the data were not
normally distributed, the Mann-Whitney test was used. The test showed no statistical difference between the LRG levels
of patients with RA of moderate activity (mean rank = 21.82) and those with SpA of high-very high activity (mean rank =
20.05); Mann-Whitney U = 191, n1 = 22 n2 = 19, p = .144 two-tailed.

Discussion
Leucine-rich alpha-2 glycoprotein levels in rheumatoid arthritis and spondyloarthritis based on age,
sex, and onset
This study recruited 26 RA patients and 26 SpA patients who visited the RSCM Rheumatology Polyclinic from March to
of May 2022 and met the inclusion and exclusion criteria of the study. The sex of the RA subjects was predominantly
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female, aligning with the theory that women have a 2–3 times higher risk of developing RA than men. However, it was
reported that no meaningful difference exists in RA incidence between men and women men over 70 years old.5 There is
strong evidence that autoimmunity is influenced by genetics and sexual chromosomes. For instance, estrogen has been
shown to increase the secretion of pro-inflammatory cytokines and exert anti-apoptotic activity and its receptors are found
on various immune cells. Hence, estrogen is associated with the development of RA.25

The majority of SpA subjects in this study were female. This predilection was also observed in the SpA subgroups,
namely AS and PsA. Previous study by Baumberger et al., had shown that the majority of AS patients are male, with a
ratio of 57:1 in 1980 and 1.03:1 in 2016.26 This is also supported by a study conducted by Tsui et al., which had shown
that the presence of the tissue non-specific alkaline phosphatase (TNAP) haplotype, which interacts with the progressive
ankylosis protein homolog (ANKH) gene (a gene that is involved in osteogenesis and ankylosis in AS), is associated with
AS in men.27 Although AS is more dominant in men than women. Another subtype of SpA, namely PsA, affects men and
women equally.28–30 Sex distribution is related to disease presentation because men tend to have axial involvement,
whereas peripheral joint involvement is more commonly seen in women. Kennedy et al., reported that the ratio of male to
female sex in PsA subjects is 2.1–4.8:1. However, it should be noted that the study took many samples with axial
involvement, which is more commonly seen in men.31 Meanwhile, Nishina et al., reported a different male-dominated
result in peripheral SpA patients with a ratio of 2.3:1.6.32

The mean age of RA subjects in this study was 49 years old. Other studies show that the highest incidence of RA cases is in
the 50–54 year age group.33 The incidence of RA tends to increase with age. The SpA subjects had a mean age of 41 years,
which was consistent with the mean age of the SpA subjects reported by Kennedy et al., i.e., under 50 years old.31 The
mean age of the AS subjects in this study was 38 years old. In one study, 92% of patients with AS were less than 45 years
old at disease onset.34 The mean age of the PsA subjects in this study was 41 years old. Research by Deike et al., showed
that most cases of PsA began at 50–59 years old. The incidence of PsA increases with age, peaks before the age of 60 and
declines aftewards.35 The age of the sole female peripheral SpA patient in this study was 48 years old, consistent with the
findings of de Winter et al., which stated that the median age of patients with peripheral SpA was 48 (36–55) years old.36

Increased LRG levels were observed in the majority of both RA and SpA subjects. The highest median LRG levels were
found in the RA group and the median CRP level was also higher in patients with RA. However, normal LRG results were
found in six RA subjects (23.1%) and four SpA subjects (15.4%). The elevated LRG levels is consistent with findings by
Birkelund et al., wherein joint fluid analysis in RA and SpA subjects showed striking differences in the number of
proteins from the innate immune system, especially those from neutrophil granulocytes, including LRG. This protein was
found in higher concentration in the RA patient group.22 Moreover, Nakajima et al., reported that LRG is able to
differentiate RA from PsA, as shown by the significant increase in LRG levels in patients with RA compared to those with
PsA. The increase in LRG concentration may be associated with higher levels of IL-6 in RA, thus increasing the activity
of LRG expression.23

LRG is a protein secreted during the inflammatory process, which like CRP, is also an acute phase protein. In patients with
RA, serum LRG levels were correlated with DAS28-ESR score and CRP level.19 From the six RA subjects who had
normal LRG levels in this study, two had normal CRP levels. In addition, all RA patients with normal LRG levels were
old onset patients who have previously received DMARD. Methotrexate is a DMARD that lowers IL-6, an important
cytokine in the formation of LRG.32 The same is true for SpA subjects; from the four subjects with normal LRG levels,
there was one subject with normal CRP levels. All subjects classified as old onset had received DMARD therapy in the
form of methotrexate and sulfasalazine. Hence, the observed normal CRP levels and the effect of DMARD treatment may
explain the presence of subjects with normal LRG levels in this study.

Based on disease onset, five RA subjects were classified as new onset patients, while 21 subjects were classified as old
onset patients. LRG levels showed higher results in RA subjects classified as new onset patients. As for the SpA subjects,
one subject was categorized as new onset and 25 other subjects were categorized as old onset. Aside from being untreated,
the higher level of LRG observed in subjects with new onset disease may be related to IL-6 expression in the early stages
of the disease. As suggested by animal studies, synovial tissue samples of animals with antigen-induced arthritis (AIA)
and adjuvant-induced arthritis (AA), revealed that the expression of IL-6 peaked in the early stages of the disease and
decreased in the later stages of the disease.37 Another study also found that IL-6 is expressed in the sacroiliac joints AS
subjects of new disease onset.38

The effect of comorbidity and treatment on leucine-rich alpha-2 glycoprotein levels

In this study, 17 RA subjects had comorbidities, among them were type 2 diabetes mellitus (T2DM), osteoarthritis (OA),
fibromyalgia, controlled hypertension, and pregnancy of eight weeks. The highest concentrations of LRG were found in

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RA patients who also had OA and fibromyalgia. Of all the subjects with SpA, 14 had comorbidities, including controlled
hypertension, IBD, dyslipidemia, T2DM, osteoporosis, OA, and hyper-IgE syndrome. The highest LRG levels were
obtained in subjects without comorbidities as they happen to be new onset patients.

Several studies support the prevalence of DM in patients with RA. TNF-α and IL-6 are associated with the pathogenesis of
DM, insulin resistance and RA. Long-term exposure to elevated levels of IL-6 triggers insulin resistance, which may play
a role in the incidence of DM in patients with RA.39 Plasma LRG levels can predict the progression of kidney disease and
the incidence of albuminuria in patients with DM. LRG levels in subjects with albuminuria were significantly higher than
subjects with stable albuminuria or those in regression. LRG levels in DM subjects with chronic kidney disease (CKD)
progression were also much higher than subjects with normal kidney function.40 In another study, LRG levels were twice
as high in subjects with end-stage renal disease (ESRD) who had been treated with hemodialysis than in patients with
stage 2/3 CKD.41 Prospective study in Southeast Asia in subjects with T2DM stated that high plasma LRG levels were
associated with an increased risk of heart failure events.42 LRG is a novel factor in the pathogenesis of heart failure in
patients with T2DM. Elevated serum LRG is also associated with peripheral arterial disease and several risk factors for
cardiovascular diseases, such as arterial stiffness and endothelial dysfunction.43 Furthermore, LRG levels were found to
be elevated in subjects with IBD, as shown by the elevated levels of IL-6 in the colonic mucosal tissue of patients with
IBD.44

LRG levels are also elevated in other inflammatory diseases such as OA. In a murine model of OA, increased LRG
expression was found in the subchondral bone and articular cartilage.45 The increase in LRG is also associated with IL-6
as it is one of the pro-inflammatory cytokines that is elevated in OA synovial fluid samples.46 In addition, LRG is also a
biomarker of fibromyalgia.23,47,48 As serum IL-6 levels are elevated in subjects with fibromyalgia, it is suggested that
fibromyalgia may affect the assessment of inflammatory activity in patients with RA.49 This is in line with the results of
this study, which showed that RA subjects with OA and fibromyalgia had the highest concentration of serum LRG.

The highest levels of LRG in RA subjects were found in subjects who had not received DMARD therapy, aligning with
previous studies that showed that DMARD therapy reduced IL-6 concentration in patients with RA.50 As for AS subjects,
the highest LRG levels were found in subjects with a combination of DMARD and steroid treatment, while the lowest was
in the combination of DMARD and paracetamol. In PsA subjects, the highest LRG levels were found in the DMARD
monotherapy group and the lowest in the DMARD and steroid combination group. It can be inferred that DMARDs and
steroids play a role in regulating IL-6 and blocking NF-κB as promoters of LRG synthesis.32,50,51

Leucine-rich alpha-2 glycoprotein levels in rheumatoid arthritis

LRG levels were found to be increased in 76.9% of subjects with RA, with the highest median LRG level observed in
subjects with RA (77.03 vs. 68.67 μg/mL). These results align with the studies of Fujimoto et al., Naka et al., and Serada
et al., which reported the elevation of serum LRG in various autoimmune diseases, including RA.19,52,53 Ha et al., also
showed that LRG can differentiate RA patients from normal controls as serum LRG concentrations were significantly
increased in patients from the RA group compared with patients in the control group.54 LRG stimulation is influenced by
pro-inflammatory cytokines, one of which is IL-6, an inflammatory cytokine that is critically involved in the pathogenesis
of RA and significantly correlated with serum LRG levels.55 Serum and synovial IL-6 levels are elevated in RA patients
and are associated with disease activity.56 Previous study conducted by Madhok et al., showed that RA patients had
higher levels of IL-6 (55 (28–139) IU/mL) compared with the control group (10 (7–12) IU/mL).57

Leucine-rich alpha-2 glycoprotein levels in spondyloarthritis

Increased LRG levels were seen in 84.6% of subjects with SpA, with a median of 68.67 (33.15–115.18) μg/ml. Nakajima
et al., previously reported that LRG levels were increased in patients with PsA (51.1  32.9 μg/mL) and psoriasis vulgaris
(37.8  25.1 μg/mL) compared with controls (25.5  11.5 μg/mL).23 A multicenter retrospective observational study
showed that serum levels of IL-6 are elevated in AS and PsA subjects.38 A previous study also reported that the mean IL-6
level was significantly higher in AS subjects compared with controls.38 The highest median LRG levels in SpA subjects
were found in the PsA subset in the present study.

Differences in levels of leucine-rich alpha-2 glycoprotein in rheumatoid arthritis and spondyloarthritis

This study found no significant difference between LRG levels in subjects with RA and SpA. This result is different from
a previous study by Lee et al., LRG was also previously identified as a protein that can differentiate RA from PsA as
shown by the significantly increased LRG levels in MRM analysis of patients with RA.58 In another study of 32 synovial
fluid samples from RA patients and 24 synovial fluid controls, it was found that LRG in RA synovial fluid and SpA
correlated significantly with synovial fluid circulating free DNA (cfDNA), and it was known that LRG was closely
related to plasma CRP.59

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 F1000Research 2022, 11:1526 Last updated: 15 DEC 2022

The insignificant difference in this study may be due to other factors affecting LRG levels, including disease onset and
treatment received. However, the effects of DMARD treatment on LRG levels in RA and SpA subjects have not yet been
studied. Old onset subjects predominated this study. Disease onset is related to IL-6 concentration. IL-6, a known
precursor of LRG, increases in both mild and severe inflammatory conditions. Higher LRG levels in subjects with new-
onset may be related to IL-6 expression in the early stages of the disease, as suggested in a study by Ferraccioli et al.,
which showed that IL-6 is a pro-inflammatory cytokine that is expressed in the early stages of the disease and plays a role
in pathogenesis.37 Expression of IL-6 in sacroiliac joint biopsies of AS patients is also associated with new disease
onset.38 Besides IL-6 activity, disease onset is also related to the patient's initiation of the treatment regimen. Old onset
patients in the present study were defined as patients who had been diagnosed before the time of sampling and had
received DMARD. Studies have shown that RA subjects receiving DMARD therapy have decreased serum concentra-
tions of IL-6, a known precursor for LRG formation.60 Most of the research subjects also received DMARD treatment in
the form of sulfasalazine; from the results of previous studies, it was known that sulfasalazine caused a decrease in LRG
levels in four female albino rats suffering from ulcerative colitis.51 The effect on subjects with SpA and RA is still
unknown.

Because this study initially did not consider the level of disease activity, there was a possibility for the patients’ disease
activity to be distributed unevenly. The results of this study found differences in the predominant disease activity between
the two groups, wherein the RA group was predominated by moderate disease activity, while the SpA group was
predominated by high to very high disease activity. The insignificant difference in LRG levels between the two groups in
this study may be due to this distribution of disease activity. The difference in LRG levels between RA and SpA subjects
may be more apparent if the disease activity between these two groups were controlled. A previous study conducted by Ha
et al., only compared LRG levels between active and inactive RA patients, where serum LRG concentrations were found
to be significantly higher in patients with active RA compared with patients with RA in remission.54 No previous study
has compared LRG levels in moderate and high disease activity; the said study did not distinguish between disease
activity groups. Meanwhile, we found different levels of LRG in the moderate and high to very high activity groups.

Leucine-rich alpha-2 glycoprotein levels in rheumatoid arthritis and spondyloarthritis subjects with
increased c-reactive protein levels
Median LRG levels based on increased CRP levels showed higher results in RA subjects than in SpA. The Mann-Whitney
test results showed no significant difference in LRG levels in RA and SpA subjects with increased CRP levels. Previous
studies have stated that LRG is correlated with CRP levels, as such a positive correlation between the two variables were
expected.55 Future research should confirm this finding.

Research strengths and limitations

To the knowledge of the researchers, this is the first study to compare LRG levels of patients with RA and generalized
SpA. The limitation of this study was the different predominant disease activity in the two groups. Future studies with a
larger subject population along with control of DMARD status and disease activity is warranted.

Conclusions
The median LRG levels of the RA and SpA subjects were 77.03 (27.16–107.73) μg/dL and 68.67 (33.15–115.18) μg/dL,
respectively. There was no significant difference in LRG levels between RA and SpA subjects. The role of LRG in the
diagnosis of RA and SpA remains to be determined in future studies.

Data availability
Underlying data
Mendeley Data: Underlying data for ‘Serum Leucine-Rich Alpha-2 Glycoprotein Levels in Rheumatoid Arthritis and
Spondyloarthritis: A Promising Biomarker’. https://www.doi.org/10.17632/xp52nbs8r3.1.24

The project contains the following underlying data:

- Data file 1: Dataset of the study subjects.xlsx

- Supplementary 1: Flow diagram of subject inclusion.docx

- Supplementary 2: Determination of sample size.docx

- Supplementary 3: STROBE Checklist.docx

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0)
Page 9 of 12

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