Is serum angiotensin-converting enzyme a suitable biomarker for differentiating idiopathic and demyelinating optic neuritis?

Abstract

PURPOSE:

The purpose of the study was to measure serum angiotensin-converting enzyme (ACE) in patients diagnosed with idiopathic and demyelinating optic neuritis (ON) as well as heathy individuals and assess its viability as a biomarker.

METHODS:

This clinical case–control study was conducted on patients diagnosed with acute ON as the case group and healthy controls aged between 18 and 50 years. Based on findings from systemic and neurological evaluation, the study group was divided into: Idiopathic ON and demyelinating ON (including multiple sclerosis [MS], neuromyelitis optica [NMO], and clinically isolated syndrome [CIS]) subgroups. Serum ACE levels were measured using enzyme-linked immunosorbent assay and compared between groups.

RESULTS:

Fifty-one patients with acute ON (78.4% female, mean age 29.8 ± 8.4 years) and 51 controls (78.4% female, mean age 29.9 ± 6.9 years) were enrolled in this study. In the case group, 29 patients were diagnosed with idiopathic ON (56.9%), and 22 patients with demyelinating types of ON (43.1%) (MS-ON, n = 16; NMO-ON, n = 3; and CIS, n = 3). The median serum ACE levels were 33.50 U/L (range: 29.40–46.20), and 34.25 U/L (range: 29.30–46.10) for patients diagnosed with idiopathic ON and demyelinating ON, respectively (P > 0.99). The mean serum ACE level for healthy individuals was 26.2 U/L (range: 24.80–20.50), and compared to other groups, it was significantly different (P < 0.001). Abnormal levels of serum ACE (defined as a serum ACE >65 U/L) were measured in 3 (10.3%) patients with idiopathic ON and 2 (9.1%) patients with the demyelinating ON (P > 0.99).

CONCLUSION:

Our findings did not exhibit a significant difference in terms of serum ACE level between patients diagnosed with idiopathic ON and demyelinating ON. However, serum ACE level in both groups is significantly higher compared to healthy subjects.

INTRODUCTION

Optic neuritis (ON), as an initial ocular manifestation in multiple sclerosis (MS), is reported in up to one-third of the MS cases or may occur in up to 70% of patients during the clinical course of MS.[1,2] Typical ON, associated with MS, usually presents with subacute monocular vision loss accompanied by painful eye movements and dyschromatopsia, predominantly in young adult women.[3,4]

ON is identified by inflammatory neuropathy triggered by (1) a demyelinating process (e.g., MS-associated ON, ON associated with neuromyelitis optica (NMO-ON)), (2) systemic disorders (e.g., infections, connective tissue diseases, and granulomatous disease), or (3) other idiopathic causes with no systemic involvement (e.g., recurrent or solitary isolated ON, and chronic relapsing inflammatory optic neuropathy).[5,6,7,8,9]

Regardless of the traditional role of the renin–angiotensin system (RAS) in the regulation of cardiovascular homeostasis, many components of RAS have been identified in the central nervous system.[10,11] It has been implied by several studies that angiotensin II (Ang II) modulates various neurophysiological functions throughout two distinct axes, in addition to its vasoconstriction role. Signaling through angiotensin type-1 receptor (AT1R) causes aberrant neural damage.[12,13] However, AT2R activation provides a neuroprotective effect by counteracting the Ang II/AT1R axis.[14,15,16,17]

In an animal model of MS, oral administration of candesartan (angiotensin receptor blocker) drastically decreased demyelination of the optic nerve and retinal ganglion cell loss.[13] In addition to that, Ang II upregulation in astrocytes plays an important role in deteriorating the inflammatory pathogenesis of MS.[14,18,19] A study has shown that angiotensin-converting enzyme (ACE) level was higher in patients diagnosed with MS compared to healthy subjects.[20] Moreover, our previous study demonstrated that the mean serum ACE level was remarkably increased in idiopathic acute ON patients compared to the control group.[21] This sheds light on the clinical implications of serum levels of ACE, as a component of the risk profile, in terms of reflecting the ON prognosis.[22,23,24,25] This study aimed to compare serum ACE levels in patients diagnosed with idiopathic ON to ones diagnosed with demyelinating ON.

METHODS

Study design and patients

In this case–control study, patients diagnosed with acute ON were evaluated for findings consistent with demyelination disorders at the Isfahan Eye Research Center, a referral ophthalmology center affiliated with Isfahan University of Medical Sciences, Isfahan, Iran. After thorough systemic and neurological evaluation, the study group was divided into two subgroups: Idiopathic ON and demyelinating ON; the latter including MS-ON, NMO-ON, and clinically isolated syndrome (CIS). The healthy controls were selected from candidates for refractive surgery with similar age and gender distribution as the case group.

The ethics committee of the Isfahan University of Medical Sciences approved the study protocol (Registration number: IR.MUI.MED.REC.1399.1120). This study abides by the guidelines of the Helsinki Declaration, and subjects were provided with written informed consent to participate in the study.

Patients aged between 18 and 50 years, regardless of gender, diagnosed with acute ON of one eye in the setting of clinical examinations and magnetic resonance imaging (MRI); in the absence of a known diagnosis of demyelination disorder, they were recruited.

The excluding criteria were defined as (1) any progressive known neurological disorder, medical condition, or limiting psychiatric disease that may impair the subject’s capacity to participate and adhere to study; (2) other causes of optic neuropathy (e.g., intrinsic or extrinsic compression anywhere along the optic nerve, infections, ischemia, toxic and nutritional neuropathies); (3) medical comorbidities that may alter serum ACE level (e.g., hypertension, diabetes mellitus, renal parenchymal diseases, sarcoidosis, chronic liver diseases, moderate or severe cardiopulmonary disorders, and taking any medicines that may affect the RAS function); (4) inability to acquire MRI from the subject; and (5) contraindications to MRI such as glomerular filtration rates <30 mL/min/1.73 m², cardiac pacemaker, metal implants, and claustrophobia. This clinical report follows the Strengthening the Reporting of Observational Studies in Epidemiology checklist for case–control studies. This study is meant to extend and complete the findings from our previous study conducted on serum ACE levels of patients diagnosed with ON.[21]

Ocular examination

The diagnosis of ON was clinically determined for each eligible patient by a detailed history of periorbital pain with eye movement, subacute monocular vision loss developed over several days, and confirmed by dyschromatopsia and relative afferent pupillary defect findings upon examination, accompanied by any pattern of visual field loss with or without disc edema.

All patients had a thorough ophthalmological examination, including assessment of the best-corrected visual acuity (BCVA), pupillary response to light stimuli, ishihara color blindness test, extraocular muscle function testing, ocular tonometry to define intraocular pressure, and dilated-pupil fundus examination using 78D lens and binocular slit-lamp examination of anterior segments. BCVA was converted to a logarithm of the minimum angle of resolution for statistical analysis.

Neurological examination

All the subjects underwent brain and orbital MRI, and a lumbar puncture was performed on any suspected individual. Cerebrospinal fluid (CSF) samples were collected to assess for MS or Neuromyelitis Optica Spectrum Disorder. AQP4-IgG was measured by a live cell-based assay method. MS diagnosis was established according to the 2017 Mcdonald’s criteria.[26]

Sample storage and biomarker assessment

Venous blood samples of all participants were obtained from the cubital vein to measure the serum ACE level. Coagulated samples were centrifuged, and the sera were then frozen at 20°C. A standardized enzyme-linked immunosorbent assay method for ACE was used to quantify ACE concentration in the blood (R and D Systems, Minneapolis, Minnesota, USA). Healthy controls had their serum ACE levels measured using the same method as the ON subjects. The normal ACE reference range was considered 8–65 units.

Statistical analysis

The Statistical Package for the Social Sciences (SPSS, version 26; IBM Corp., Armonk, NY, USA) was used to analyze the data. Means, standard deviations, medians, interquartile ranges, and box plots were utilized to explain and illustrate the numerical variables. Qualitative characteristics were quantified using numbers and percentages. Where appropriate, the Shapiro–Wilk test was performed to determine the normality of the distribution of numerical variables. Due to nonnormal distribution of variables, comparisons between groups were conducted using the Mann–Whitney U and Kruskal–Wallis tests. Pairwise post hoc testing with Bonferroni correction was used to make comparisons. The adjusted associations were assessed using a multinominal logistic regression model and reported as adjusted odds ratios (OR). P < 0.05 was considered significant.

RESULTS

Fifty-one patients with ON were enrolled as the study group (78.4% female, mean age 29.8 ± 8.4 years) and 51 healthy participants in the control group (78.4% female, mean age: 29.9 ± 6.9 years). The study group was comprised 29 patients (28.4%) diagnosed with idiopathic ON, 16 patients (15.7%) with MS-ON, three patients (2.9%) with NMO-ON, and three patients (2.9%) with CIS. The three preceding categories were considered to have a demyelinating disorder (n = 22). The baseline demographics and clinical characteristics of the study group are summarized in Table 1.

Table 1.

Data characteristics of the study group with acute optic neuritis

Variables	Idiopathic ON (n=29)	Demyelinating ON (n=22)	P*
Sex, n (%)
Male	8 (27.6)	3 (13.6)	0.31
Female	21 (72.4)	19 (86.4)
Age, (years)
Mean±SD	30.9±8.93	28.86±6.83	0.38
Median (minimum–maximum)	30 (18–49)	27 (18–42)
Eye, n (%)
Right	14 (48.3)	9 (40.9)	0.60
Left	15 (51.7)	13 (59.1)
RAPD, n (%)
1+	1 (3.4)	0	0.64
2+	9 (31.0)	8 (36.4)
3+	19 (65.6)	14 (63.6)
Visual acuity, logMAR
Mean±SD	1.69±1.20	1.61±1.27	0.82
Median (minimum–maximum)	1.4 (0–3.7)	1.5 (0.18–3.7)
Disc swelling, n (%)
Yes	6 (20.7)	2 (9.1)	0.44
No	23 (79.3)	20 (90.9)
ACE level, n (%)
Abnormal (>65)	3 (10.3)	2 (9.1)	>0.99
Normal (≤65)	26 (89.7)	20 (90.9)

*Mann–Whitney U-test. ON: Optic neuritis, logMAR: Logarithm of the minimum angle of resolution, SD: Standard deviation, RAPD: Relative afferent pupillary defect, ACE: Angiotensin-converting enzyme

The median serum ACE levels were 33.50 U/L (range: 29.40–46.20), 34.25 U/L (range: 29.30–46.10), and 26.2 U/L (range: 24.80–20.50) for patients suffering from idiopathic ON, demyelinating ON and control group, respectively (P < 0.001) [Table 2].

Table 2.

Serum angiotensin-converting enzyme level in patients with idiopathic optic neuritis, demyelinating optic neuritis, and control groups

	Serum ACE level, U/L
	Mean±SD	Median	Quantile 1	Quantile 3
Study groups
Idiopathic ON	66.47±102.91	33.50	29.40	46.20
Demyelinating ON	44.37±27.72	34.25	29.30	46.10
Control group	30.87±11.61	26.2	24.80	29.50

P-Value (Kruskal–Wallis): <0.001. ACE: Angiotensin-converting enzyme, ON: Optic neuritis, SD: Standard deviation

The median serum ACE level was higher in patients with idiopathic and demyelinating ON compared to the control group (P < 0.001 and P < 0.001, respectively), but there was no significant difference in serum ACE level between idiopathic and demyelinating ON groups (P > 0.99) [Figure 1].

Figure 1.

Boxplot of the serum angiotensin-converting enzyme level in patients with Idiopathic and demyelinating optic neuritis and the control group. Outlier data points are not depicted. ACE: Angiotensin-converting enzyme, ON: Optic neuritis

Also, abnormal levels of serum ACE (defined as a serum ACE >65 U/L) was present in 3 (10.3%) patients with idiopathic ON (ACE level: 540, 220 and 216) and 2 (9.1%) patients with the demyelinating ON (ACE level: 140 and 108) (P > 0.99) [Table 1]. It is important to note that these outlier data points are not depicted in Figure 1. Furthermore, patients diagnosed with idiopathic ON whom had abnormal levels of ACE underwent further workup for diseases such as sarcoidosis. They were found healthy, and no autoimmune diagnoses were established for them.

According to multinominal logistic regression analysis, patients with idiopathic (OR: 1.057, 95% confidence interval [CI]: 1.013–1.104) and demyelinating ON (OR: 1.054, 95% CI: 1.009–1.100) had a greater chance of having increased serum ACE when compared to the control group (P = 0.01 and P = 0.02, respectively) [Table 3].

Table 3.

Multinominal logistic regression analysis to show the association between angiotensin-converting enzyme blood level and the diagnosis of each type of optic neuritis in comparison with the normal group by matching the effect of age and sex

Disease	Adjusted OR*	95% CI		P
		Lower bound	Upper bound
Idiopathic ON
Age (year)	1.016	0.95	1.08	0.64
ACE level	1.057	1.013	1.104	0.01
Gender (male)	1.078	0.34	3.43	0.89
Intercept	-	-	-	0.02
Demyelinating ON
Age (year)	0.989	0.92	1.06	0.75
ACE level	1.054	1.009	1.100	0.02
Gender (male)	0.505	0.12	2.15	0.35
Intercept	-	-	-	0.10

*The reference category is the normal group. OR: Odds ratio, CI: Confidence interval, ACE: Angiotensin-converting enzyme, ON: Optic neuritis

DISCUSSION

The present study aimed to evaluate the utility of serum ACE levels as a differential biomarker between idiopathic ON and ON secondary to demyelinating disorders. The main finding was that while serum ACE levels were significantly elevated in both idiopathic and demyelinating ON patient cohorts relative to healthy controls; however, no statistically significant difference was observed in ACE concentrations between these two ON subgroups. Clinically, this limits its diagnostic utility as a standalone biomarker for differentiating ON etiologies. Previous clinical studies have reported mixed results regarding ACE in MS and ON. One study surmised that variations in serum ACE activity are correlated with the total plaque volume in the brains of MS patients measured by brain MRI.[20] They also found a substantial difference in cerebral fluid ACE levels between patients diagnosed with MS and healthy controls. Kawajiri et al.[27] also found that CSF ACE levels of patients diagnosed with MS were significantly higher than healthy individuals. These findings suggest that ACE may be involved in the pathophysiology of ON. However, Razazian et al.[20] reported no statistically significant difference in serum ACE levels between 30 MS patients and 30 healthy controls in their investigation. On the other hand, previous data from idiopathic acute ON patients suggest that RAS may play a role in ON.[21]

As far as we are aware, this is the first study to quantify and compare the serum ACE levels of idiopathic and demyelinating ON patients. Our findings did suggest a significantly elevated serum ACE level in MS patients (included in demyelinating subgroup) compared to healthy individuals; however, elevations were also observed in the idiopathic ON subgroup. Considering the nonsignificant differences noted in serum ACE levels of idiopathic and demyelinating ON, it seems that serum ACE level elevation is not directly associated with demyelination.

The central RAS, through Ang II signaling, exerts opposing effects via AT1R and AT2R, which are two components of this system that are most closely associated with neurodegenerative disorders. Previous studies have outlined the role of AT1R signaling, which results in aberrant neurological damage.[12,13] Underlying neurodegeneration mechanisms include the production of excessive reactive oxygen species, neuro-inflammation due to altered blood–brain barrier permeability, and distorted regulation of neurotrophic factors. On the other hand, activation of the AT2R exerts neuroprotective effects by acting as a counterbalance to the Ang II/AT1R axis.[12,13,15,16,17]

RAS dysregulation in the brain significantly contributes to aging-related alterations and neurodegeneration by intensifying neuroinflammation. Therefore, serum ACE, which converts Ang I to Ang II, or any other components of RAS can be targeted through pharmacological methods in acute ON (e.g., blocking or deactivating enzymes or receptors).[16]

As with any study, this study has several limitations to be considered. In our study, all individuals were recruited from a particular ethnic group from a single center, which limits the generalizability of the results. Moreover, future studies should also prioritize other components of the RAS such as renin, angiotensin, and aldosterone as well as a multicenter study design with a larger sample size.

Continued investigation, especially a prospective cohort study of long-term data, remains critical for determining the correlation between serum ACE level and either development of demyelinating disorders or the conversion of idiopathic types to demyelinating disease.

CONCLUSION

We did not find serum ACE level a suitable biomarker for differentiating idiopathic ON from demyelinating ON. Improved understanding of RAS in the brain would enable the development of novel strategies, unique methodologies, and risk assessment tools for predicting a patient’s individual MS risk after the onset of acute ON. Validation of serum or CSF biomarkers used to estimate the risk of developing a demyelinating disorder, requires additional research with large, well-designed cohorts that include various components of RAS, and other associated risk factors with a more extended period of follow-ups.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.