Serum angiotensin-converting enzyme levels indicating early sarcoidosis diagnosis and immunosuppressive therapy efficacy

Subjects

We retrospectively examined 3781 patients (≥20 years old) in whom ACE was measured for any reason including suspected sarcoidosis between 2009 and 2020 at Fujita Health University Hospital.

Data collection

The presence of hypertension, diabetes mellitus, dyslipidaemia, chronic kidney disease, and other co-existing diseases that can affect the ACE level were collected from the medical records. Hypertension was defined as blood pressure ≥140/90 mmHg or current use of antihypertensive medication. Dyslipidaemia was defined as a low-density lipoprotein cholesterol level ≥140 mg/dL, a high-density lipoprotein cholesterol level <40 mg/dL, a triglyceride level ≥150 mg/dL, or treatment with lipid-lowering medication. Diabetes mellitus was defined as a fasting blood glucose ≥126 mg/dL or haemoglobin A1c (National Glycohemoglobin Standardisation Programme) ≥ 6.5% or treatment with hypoglycaemic medication. Similarly, medical agents including ACE inhibitor (ACEi), steroids, or immunosuppressive agents were also examined. The diagnosis of sarcoidosis was determined by diagnostic standards and guidelines for sarcoidosis^{6, 7} and JCS Guideline on Diagnosis and Treatment of Cardiac Sarcoidosis.⁵ For patients diagnosed with sarcoidosis, ACE levels were measured after the initial diagnosis.

The study protocol was approved by the Institutional Review Board and ethics committees of Fujita Health University. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a prior approval by the institution's human research committee. We applied an opt-out method to obtain consent for this study on the website of our department.

Angiotensin-converting enzyme measurement

Peripheral blood samples for the measurements of biomarkers were collected at the time of clinical evaluation. Serum ACE levels were measured by a colorimetric method using p-hydroxyhip-puryl-l-histidyl-l-leucine as the substrate: Kasahara method (BML, Inc.).⁸ The normal range was ≤21.4 IU/L. In patients diagnosed with sarcoidosis, the lowest points of ACE levels after the initial diagnosis were also recorded to compare ACE levels between patients having sarcoidosis with and without immunosuppression initiation.⁶

Statistical analysis

The Shapiro–Wilk test was used to assess the normality of continuous data. Variables with a normal distribution are expressed as mean values ± standard deviation, and asymmetrically distributed data are given as the median and interquartile range. Categorical variables were presented as frequency (percentage). Differences between the two groups were evaluated by the Mann–Whitney test or Student's t-test for continuous variables and by the chi-squared test for categorical variables. Differences among the four groups were evaluated using the Kruskal–Wallis test for continuous variables in asymmetrically distributed data. A probability value of <0.05 was considered significant. Receiver operating characteristics (ROC) analysis was performed to calculate the sensitivity, specificity, area under the ROC curve, and optimal cut-off with 95% confidence limits. Two-way repeated-measures analysis of variance was used to compare the change in ACE levels between patients having sarcoidosis with and without immunosuppression initiation after ACE measurement. All statistical analyses were conducted using JMP version 13 (SAS Institute, Cary, NC, USA).

Subject characteristics

Of 3781 patients (51.1% men, 60.1 ± 17.0 years old), 293 were diagnosed with sarcoidosis. Of them, 197 (5.2%) and 109 (2.9%) patients were taking ACEi and steroid/immunosuppressive agents, respectively (Table 1). Of the 293 patients with sarcoidosis, 101, 212, 84, and 88 were diagnosed with sarcoidosis in the heart, lung, skin, and eyes, respectively (Table 2). First, we compared serum ACE levels among patients with ACEi, angiotensin II receptor blockers (ARB), both and none, because ACEi has been reported to lead to low ACE levels.^{9, 10} Serum ACE levels of patients taking ACEi or both were lower than those of patients with neither (6.9 vs. 11.1, P < 0.001, 4.3 vs. 11.1, P < 0.001), although that of patients taking ARB was similar (11.0 vs. 11.1, P = 0.627) (Figure 1). Therefore, we analysed 3487 patients (including 223 with sarcoidosis) after excluding 294 taking with ACEi and/or steroid/immunosuppressive agents (category I). Furthermore, 3304 patients (215 with sarcoidosis) after excluding 183 with other diseases that can affect serum ACE levels were analysed in category II (Figure 2).

Angiotensin-converting enzyme levels among patients with each type of sarcoidosis and those without

In category I, the serum ACE level was 19.9 [interquartile range (IQR), 15.3–31.7] IU/L in patients with sarcoidosis and 10.8 [8.4–16.8] in those without it (P < 0.01). In the ROC curve analysis of ACE for the diagnosis of sarcoidosis, the cut-off point associated with sarcoidosis was 14.7 IU/L. The area under the ROC curve was 0.864 [95% confidence interval (CI): 0.836–0.891, P < 0.001]. Similarly, in category II, the serum ACE levels were 19.6 [IQR, 15.1–31.5] IU/L in 215 patients with sarcoidosis and 10.7 [8.4–16.5] IU/L in 3089 patients without it (P < 0.01). The area under the ROC curve was 0.865 (95% CI: 0.837–0.893, P < 0.001) (Figure 3). In the current cut-off value (≥21.4), the sensitivity, specificity, positive predictive value, negative predictive value, and accuracy were 42.3, 98.6, 68.4, 96.1, and 95.0, respectively. When we used the new cut-off value (≥14.7), the sensitivity, specificity, positive predictive value, negative predictive value, and accuracy were 78.1, 81.7, 23.0, 98.2 and 81.5%, respectively (Table 3). Finally, the 3304 patients were divided into four groups: group A, CS (+) extra CS (eCS) (+); group B, CS (−) eCS (+); group C, CS (+) eCS (−); and group D, CS (−) eCS (−)]. The ACE levels in groups A, B, C, and D were 17.9 (15.6–21.9), 20.9 (15.0–24.9), 18.6 (11.0–21.9), and 10.7 (8.4–13.5), respectively. Although the ACE levels in patients with isolated CS showed a decreasing trend, the difference was not significant. The ACE level in group D was significantly lower than that of groups A, B, or C (Figure 4).

Change in angiotensin-converting enzyme levels after the initiation of immunosuppressive agents or not for patients with sarcoidosis

In 223 patients with sarcoidosis in category I, an immunosuppressive therapy was initiated for 56 and not for 167; repeated ACE data were obtained in 48 of 56 and 120 of 167 patients, respectively. The pattern of change in ACE levels was different between the two groups (P for interaction <0.001). In both groups, the ACE levels decreased from 20.6 to 10.3 (P < 0.001) in patients with immunosuppression therapy and from 18.5 to 13.9 (P < 0.001) in those without; the reduction was more remarkable in those with immunosuppression therapy (P < 0.001) (Figure 5).

Previous studies

Approximately 30–80% of patients with sarcoidosis have increased ACE levels, and sensitivity ranges between 22% and 86% and specificity between 54% and 95%.^{11, 12} Given the variabilities in ACE levels, its value as a diagnostic tool remains controversial.

First, ACE levels are reported to be influenced by ACE gene polymorphisms. Allele I (presence of a nonsense DNA fragment) is associated with low ACE activity.¹³ Tomita et al. examined the distribution of genotypes in Japanese patients and the serum ACE levels of each genotype in both patients with sarcoidosis and normal controls.¹⁴ In normal controls, the average ACE levels of II, deletion/insertion (DI) and deletion/deletion (DD) genotype were 10.8 ± 3.1, 13.8 ± 4.3, and 17.2 ± 4.0 IU/L, respectively. On the contrary, the corresponding values in patients with sarcoidosis were 21.4 ± 7.9, 23.9 ± 7.2, and 27.3 ± 7.5 IU/L, respectively. The prevalence of DD was comparable in patients with sarcoidosis and normal controls (14.0% vs. 12.4%). Differences in ACE levels between Caucasians and Japanese may be attributed to gene polymorphisms. Compared with Caucasians, the prevalence of allele I is high in Japanese, resulting in a low ACE activity. Second, ACE levels correlate well with the estimated total bulk of granulomata in the body.¹⁵ Compared with systemic sarcoidosis, patients with only a single organ involved, such as isolated CS, may have lower ACE levels. Finally, the assay of serum ACE may be influenced; the variously reported sensitivities are related to the assay method.

Clinical implications

We examined the new cut-off value of serum ACE, which is useful for the diagnosis of sarcoidosis in the clinical setting, after excluding patients with diseases that can affect ACE levels or were taking ACEi or immunosuppressive agents. Serum ACE serves as a gatekeeper for detecting patients with sarcoidosis, and further examinations are needed for patients with ACE positive. If the sensitivity is low, some patients with ACE false negative may not undergo imaging or pathological examinations. Specifically, the presence of CS is associated with the prognosis of the patients. Immunosuppression therapy, usually with corticosteroids, is suggested for the treatment of clinically manifest CS. Compared with those with moderately reduced left ventricular ejection fraction (30–54%), immunosuppressive therapy does not improve the left ventricular function of patients with severely reduced left ventricular ejection fraction (≤30%) containing a large scar burden.¹⁶ The identification of patients at the early stage of CS is essential. ACE is expected to serve as a screening tool for the diagnosis of sarcoidosis.

Limitations

This study has several limitations. First, the study was retrospectively conducted in a single hospital, although many serial subjects were included. Second, in patients with sarcoidosis, all the involved organs might be not completely examined, although the diagnosis of sarcoidosis was confirmed. Finally, we did not examine the polymorphism of ACE in the patients. The study of ACE levels for each genotype is a great force in this study. The judgement based on the new normal range for each genotype improved the accuracy of diagnosing sarcoidosis.¹⁴

Conclusions

In the current standard value of serum ACE, the sensitivity for detecting sarcoidosis is comparatively low. Further examinations are needed for patients suspected with sarcoidosis and have relatively high ACE levels in the normal range (>14.7 IU/L). In patients with sarcoidosis, ACE levels decreased more in those with immunosuppression therapy than in those without it.