Coronavirus disease 2019 (COVID-19) is the new viral respiratory disease that targets the lungs and causes severe pneumonia and acute respiratory distress syndrome (ARDS). It has been demonstrated in recent studies that similar to the severe acute respiratory syndrome coronavirus (SARS-CoV), the SARS-CoV-2 also uses angiotensin-converting enzyme (ACE) 2 as a functional receptor to infect the host cell (1–5). After binding to the ACE2 via the S-protein, the virus transfers its RNA to human cells that undergo translation to produce new viral particles. It is assumed that as a result of binding of the viral S-protein with the ACE2 on the cell, ACE2 activity is decreased, and an imbalance occurs in the renin-angiotensin system (6–8).
Under normal conditions, the ACE2 enzyme converts angiotensin I (1–10) to angiotensin (1–9) and converts angiotensin II (1–8) to angiotensin (1–7) (9). When ACE2 level decreases due to viral blockage, angiotensin (1–9) and angiotensin (1–7) levels also decrease. Conversely, angiotensin II (1–8) is expected to increase. Angiotensin (1–9) and angiotensin (1–7) have anti-inflammatory properties, whereas angiotensin II (1–8) has proinflammatory properties (9–12). In addition, since the virus presumably does not affect ACE1, it is assumed that conversion of angiotensin I to angiotensin II continues (9). Angiotensin II causes vasoconstriction, cell proliferation, inflammatory responses, blood coagulation, and remodeling of the extracellular matrix due to its binding to the angiotensin type 1 receptor (AT1R). The angiotensin II also binds to another receptor, namely angiotensin type 2 receptor, which counteracts the effects mediated by AT1R (6,8,13–17).
The serum angiotensin II levels were thought to be associated with lung injury in viral pneumonias. Higher concentrations of angiotensin II (1–8) in circulation in influenza A (H7N9) pneumonia were associated with higher mortality rates (18). In a study conducted during the COVID-19 pandemic, it was reported that the angiotensin II serum levels were positively associated with both SARS-CoV-2 viral load and lung injury (19).
There is no clear evidence of serum angiotensin II levels in COVID-19 infection. Based on these hypothesis put forth with regard to the angiotensin II levels in COVID-19 infection, we aimed to determine the serum angiotensin II levels in COVID-19 patients with pneumonia in the emergency department and to investigate the effect of these levels on the prognosis of the disease.
This study was conducted in accordance with the research rules following the principles of the “World Medical Association Declaration of Helsinki” after obtaining the approval of the Clinical Research Ethics Committee of Istanbul University-Cerrahpasa, Cerrahpasa Faculty of Medicine (Number: A-25, Date: June 06, 2020).
In this study, we included patients above 18 years old who were diagnosed with COVID-19 infection in the emergency department and had a positive polymerase chain reaction (PCR) test result and had findings of pneumonia in tomography.
We excluded the following patients from our study: patients having pneumonia and a negative PCR test result, patients whose second and/or third blood samples could not be obtained, patients who used ACE inhibitors and angiotensin receptor blockers (ARBs) due to their chronic disease, and pregnant women.
We collected the first blood samples for serum angiotensin II levels from the patients at the time of admission to the emergency department. We collected the second blood samples at either of the following stages: when the patients’ symptoms progressed, laboratory values deteriorated, clinical findings of ARDS developed, or indications for admission to the ICU developed. The second blood samples were taken on the median days of hospitalization of patients whose clinics did not deteriorate. The third blood samples were collected before recovery/discharge/exitus. We recorded the patients’ initial symptoms, vital signs, chronic diseases, routine blood variables, PCR results, and tomography results. The second and third blood samples of the patients were collected in the department or the ICU. We followed the patients during their hospital stay and recorded their mortality or discharge status.
The control group consisted of healthy volunteers, and a blood sample was collected from this group for serum angiotensin II level only once.
Power analysis for the sample size was 0.92 (Type 1 error = 0.05). The need for ICU, development of ARDS, and in-hospital mortality were accepted as primary endpoints.
The Berlin criteria were used for the diagnosis of ARDS in patients.
The indications for intensive care admission in the patients were determined according to the Ministry of Health COVID-19 patient management guidelines. According to this guideline, the indications for intensive care hospitalization are as follows:
- Respiratory rate greater than or equal to 30/min.
- Dyspnea and signs of respiratory distress.
- Cases with oxygen saturation below 90% and partial oxygen pressure below 70 mm Hg despite nasal oxygen support of 5 L/min and above.
- Pao2/Fio2 less than 300 mm Hg.
- Lactate greater than 4 mmol/L.
Written consents were obtained from the patients who expressed their desire to participate in the study.
According to the Ministry of Health COVID-19 patient management guidelines, all patients were treated with hydroxychloroquine for a minimum of 5 and a maximum of 10 days and with favipiravir for 5 days. Tocilizumab was administered to patients who had increased C-reactive protein (CRP), ferritin, d-dimer values and/or decreased lymphocyte, fibrinogen, and platelet counts on consecutive evaluations.
Blood samples collected from the patients were centrifuged at 3,500 rpm for 10 minutes, and serum samples were portioned and stored in the freezer at –80°C until the angiotensin II analysis was performed.
Serum Angiotensin II Measurement
The human angiotensin II enzyme-linked immunosorbent assay (ELISA) kit (Elabscience Biotechnology Catalog No: E-EL-H0326, Houston, TX) was used for the determination of serum angiotensin II levels. This ELISA kit uses the competitive-ELISA principle. The assay was performed according to manufacturer’s instructions. The micro ELISA plate provided in this kit has been precoated with human angiotensin II. 50 μL of standard samples were incubated in ELISA plates precoated with the specific antibody. During the reaction, human angiotensin II in the sample or standard competes with a fixed amount of human angiotensin II on the solid phase supporter for sites on the biotinylated detection antibody specific to human angiotensin II. Excess conjugate and unbound samples or standards are washed from the plate, and avidin conjugated to horseradish peroxidase are added to each microplate well and incubated for 30 minutes at 37°C. Fluid aspirated and plate washed for five times with wash buffer. Then, 90 μL of tetramethylbenzidine substrate solution is added to each well and incubated for 15 minutes at 37°C. The enzyme-substrate reaction is terminated by the addition of 50 μL of stop solution. The color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm, immediately. The concentration of human angiotensin II in the samples is then determined by comparing the optical density of the samples to the standard curve. Inter- and intraassay coefficients of variability are 10% and 9.5%, respectively. Results were given as pg/mL, and the sensitivity of the angiotensin II kit was 18.75 pg/mL.
Statistical analyses of the data obtained were performed with the SPSS 24.0 program (Statistics Program for Social Scientists, SPSS, Chicago, IL). The Kolmogorov-Smirnov test was used for normality analysis of the data. The Student t test was used to compare two independent groups with normal distribution, whereas the Mann-Whitney U test was used for those who did not show normal distribution. For comparing three dependent variables, the Friedman test was used for those who did not show normal distribution. The post hoc analysis of the groups with statistical significance was made with the Wilcoxon signed-rank test. The Kruskal Wallis test was used to compare three independent groups. The Mann-Whitney U test was used for post hoc analysis. The chi-square test was used to compare the frequency data of independent groups. Spearman test was used for correlation. p value of less than 0.05 was considered statistically significant.
A total of 112 patients who met the study criteria were included in the study, of which 63.4% of the patients were men. The mean age of the patients was 58.6 ± 15.9 years (minimum 20, maximum 94). The healthy control group consisted of 15 women and 12 men with a mean age of 57.2 ± 14.5 years.
The in-hospital mortality rate was found to be 29.5% (n = 33). There was no significant gender difference in terms of mortality. The mean age of the patients who died was significantly higher than those who survived. In 70.5% of the patients, there was comorbidity accompanying the clinical picture. The rate of chronic obstructive pulmonary disease (COPD), chronic renal failure (CRF), heart failure (HF), and malignancy was statistically significantly higher in the patients who died (p < 0.05) (Table 1).
TABLE 1. -
The Comparison of the Demographic and Clinical Features of the Cases for Mortality
|Demographic and Clinical Features
||All Patients (n = 112)
||Survivors (n = 79)
||Nonsurvivors (n = 33)
|Age, yr, mean ± sd
||58.6 ± 15.9
||55.1 ± 14.6
||67.1 ± 15.9
|Gender, % (n)
|Comorbidity, % (n)
| Diabetes mellitus
| Chronic obstructive pulmonary disease
| Heart failure
| Coronary artery disease
| Cerebrovascular disease
| Chronic renal failure
|Admission symptoms, % (n)
| Loss of smell and taste
|ARDS, % (n)
| Mild ARDS
| Moderate ARDS
| Severe ARDS
|ICU need, % (n)
|Mechanical ventilatory support, % (n)
|Length of stay, d, median (interquartile range)
ARDS = acute respiratory distress syndrome.
The median of the serum angiotensin II levels in the patients with COVID-19 was 433.61 pg/mL (IQR, 15.00–2,955.67 pg/mL). The median value of the serum angiotensin II levels of healthy controls was 774.75 (IQR, 348.79–4,775.5). The serum angiotensin II levels were statistically significantly lower in the patients with COVID-19 compared with the healthy control group (p < 0.001) (Fig. 1).
There was no statistical significance between the serum angiotensin II levels measured at three different times. However, although not statistically significant, the angiotensin II levels in the patients’ second blood samples (407.92 pg/mL; IQR, 10.00–2,062.60 pg/mL) were found lower than the angiotensin II levels in the first (433.61 pg/mL; IQR, 15.00–2,955.67 pg/mL) and the third blood samples (446.09 pg/mL; IQR, 14.61–5,320.79 pg/mL) (Fig. 1).
No statistically significant difference was found between the angiotensin II levels in the blood samples of the patients with and without comorbid disease, which were taken at the time of admission to the emergency department (Table 2). When the diseases were examined individually, no statistically significant difference was found (p > 0.05).
TABLE 2. -
Admission Serum Angiotensin II Levels According to Comorbidities
||Comorbidity (+) Angiotensin II Level, pg/mL, Median (Minimum–Maximum)
||Comorbidity (–) Angiotensin II Level, pg/mL, Median (Minimum–Maximum)
|All comorbidities (n = 79)
|Hypertension (n = 40)
|Diabetes mellitus (n = 22)
|Chronic obstructive pulmonary disease (n = 7)
|Heart failure (n = 14)
|Coronary artery disease (n = 11)
|Cerebrovascular disease (n = 3)
|Chronic renal failure (n = 4)
|Malignancy (n = 21)
No significant correlation was found between the ages of patients with COVID-19 and the serum angiotensin II levels in the first blood samples (rho = –0.06; p = 0.54).
The serum angiotensin II levels in the female patients (median, 483.08 pg/mL; IQR, 21.65–2,955.67 pg/mL) were higher than the male patients (median, 424.93 pg/mL; IQR, 15.00–2,725.83 pg/mL), but the difference was not statistically significant (p = 0.22).
The rate of the patients who developed ARDS was found to be 39.3% (n = 44). The serum angiotensin II levels of the patients with ARDS were found to be statistically significantly lower than those without ARDS in three samples collected at different clinical periods (p < 0.05) (Fig. 2).
Admission to the ICU was required in 42% of the patients (n = 47). The angiotensin II levels of the patients who required admission to the ICU at all three times of blood sample collection were found to be statistically significantly lower than those who did not (p < 0.05) (Fig. 2).
Although the serum angiotensin II levels of the patients who died were low, there was no statistically significant difference in mortality at all three times (Fig. 2). The second angiotensin II levels in particular were significantly lower in the patients who died compared with the patients who survived. However, this reduction in angiotensin II levels did not create a statistically significant difference (p = 0.05).
There was a significant difference between the initial admission angiotensin II, lymphocyte, leukocyte, CRP, d-dimer, and ferritin levels of patients with and without ARDS (p < 0.05). In the receiver operating characteristic analysis, the sensitivity of the serum angiotensin II levels in detecting ARDS development was found to be 81.8% (Table 3).
TABLE 3. -
Three-Time Changes and Receiver Operating Characteristic Analysis of Laboratory Parameters
||First Blood Sample
||Second Blood Sample
||Third Blood Sample
|Angiotensin II (pg/mL)
|Platelet (μL), ×103
|C-reactive protein (mg/L)
|Lactate dehydrogenase (IU/L)
|Aspartate transaminase (IU/L)
|Alanine transaminase (IU/L)
Area Under the ROC Curve
|For acute respiratory distress syndrome development risk
| Angiotensin II (pg/mL)
| Leukocyte (μL)
| Lymphocyte (μL)
| C-reactive protein (mg/L)
| Ferritin (ng/mL)
|For ICU need
| Angiotensin II (pg/mL)
| Leukocyte (μL)
| Lymphocyte (μL)
| C-reactive protein (mg/L)
| Ferritin (ng/mL)
ROC = receiver operating characteristic.
aShows statistically significant difference with the first blood sample.
bShows statistically significant difference with the second blood sample.
cShows statistically significant difference with the third blood sample.
There was no statistical difference in the serum angiotensin II levels between patients who received and did not receive tocilizumab (p > 0.05).
Considering the correlation of angiotensin II with other laboratory values, a significant correlation was found with lymphocyte (rho = 0.309; p = 0.001), CRP (rho = –0.300; p = 0.001), and ferritin (rho = –0.336; p < 0.001) levels. There was no significant correlation between angiotensin II levels and d-dimer (rho = –0.065; p = 0.499) levels. There was no significant correlation between angiotensin II levels and fibrinogen (rho = –0.075; p = 0.434) levels.
In this study, we share concrete data by measuring serum angiotensin II levels in COVID-19 patients with pneumonia. In our study, the serum angiotensin II levels of the patients with COVID-19 were found to be statistically significantly lower than the healthy control group. The angiotensin II levels in patients with COVID-19 were first investigated by Liu et al (19). They investigated the angiotensin II levels in 12 patients with COVID-19 and eight healthy people and found that the serum angiotensin II levels were significantly higher in patients with COVID-19 as compared to healthy people (19). Henry et al (20) determined the median of the plasma angiotensin II levels in patients with COVID-19 as 71.4 pg/mL, and the median value of the healthy control group as 71.5 pg/mL. Thus, they reported that there was no significant difference between the two groups. They also reported that they did not find a significant difference between the patients who required ICU admission and those who did not (20). However, in our study, the serum angiotensin II levels measured at all three times were found to be statistically significantly lower in the patients who were admitted to the ICU. The conflicting results in the literature may be due to the heterogeneity in the patient profiles and the number of patients included in the studies. Similar to our study, pneumonia was present in all patients in the study by Liu et al (19). However, this detail was not shared in the study of Henry et al (16). In the study by Liu et al (19), 50% of the patients developed ARDS, which is slightly higher than the result of our study. In the other study, this detail was not included. The difficulty of measuring angiotensin II levels due to protein degradation may have been a contributing factor to the varied results.
Reddy et al (8) in their study, which was published before the pandemic in 2019, had observed that the plasma angiotensin II (1–8) levels in the patients with ARDS were 0.76 ng/mL (0.07–2.22 ng/mL) in the patients who survived and 0.38 ng/mL (0.12–0.63 ng/mL) in those who died, and they did not report a statistically significant difference. They also did not detect a significant difference between the two groups (patients who survived/patients who died) at any point in time in the three sampling that they performed at the 24th, 48th, and 72nd hours in terms of angiotensin II (1–8) levels (8). In our study, in the blood samples examined at all three times, the angiotensin II levels of the patients who died were lower than those who survived. However, this decrease was not statistically significant. The results of our study were consistent with the results of the study conducted by Reddy et al (8) in terms of the angiotensin II levels. ACE1 and ACE2 are cell-associated enzymes released from the lung endothelial and epithelial cells. It has been reported that cell-associated ACE has a significantly higher catalytic activity compared with ACE in circulation (21). In patients with severe ARDS, the endothelial and epithelial damage is higher in the lung, resulting in the ACE1 and ACE2 level being affected in relation to the cell. A decrease not only in ACE2 but also in ACE1 will decrease angiotensin II levels. Consistent with this, in our study, the angiotensin II levels of patients who developed ARDS were statistically significantly decreased compared with those without ARDS. These results show that serum angiotensin II levels can be used in the prediction of ARDS development and in determining the need for mechanical ventilation.
In our study, although it was not statistically significant, the second serum angiotensin II levels of the patients were lower than the first angiotensin II levels collected at the time of admission to the emergency department. The second blood samples were collected at a stage when the symptoms of the patients had exacerbated, blood variables had further deteriorated, and/or clinical signs of ARDS had developed. These results, in our study, show that the angiotensin II levels decrease when the severity of the disease begins to increase. This decrease was more pronounced especially in the second serum angiotensin II levels of the patients who died. We think that measuring the serum angiotensin II level in COVID-19 patients, especially when hypoxia develops, may guide the development of ARDS and prognosis.
It has been reported that thrombosis and microclotting play an important role in the pathogenesis of COVID-19. It has been shown that d-dimer elevation in COVID-19 patients is associated with thrombi formation. The mechanism of thrombi formation in the pathogenesis of the disease has not yet become fully clear (21,22). In our study, the serum angiotensin II levels did not correlate with d-dimer and fibrinogen levels. Therefore, we could not find a relationship between the decrease in angiotensin II levels and microclotting or formation of thrombosis.
At the beginning of the pandemic, many hypotheses related to chronic diseases, and the angiotensin II levels were put forward. It was stated that in patients who had a prior history of hypertension and who used ACE inhibitors and ARBs, the angiotensin II levels increased, and consequently, the severity of the disease increased (24). In the clinical studies conducted later, it was declared that these drugs had no effect on the severity of the disease (25–27). In our study, there was no significant difference observed between the angiotensin II levels of the patients with and without chronic disease. The angiotensin II levels were low in the patients with COPD, HF, and CRF, although this difference was not significant.
The relationship between the decrease in serum angiotensin II levels and the use of tocilizumab was not determined. Since hydroxychloroquine and favipiravir were given to all patients, it is not possible to give information about the effects of these drugs on the serum angiotensin II levels.
ACE1 and ACE2 are cell-associated enzymes released from the lung endothelial and epithelial cells. However, studies have shown that the circulating ACE2 levels are very low, and some effects of the ACE2 are local (28). This may explain the different angiotensin II levels. Another reason for the varied and conflicting results among the literature may be due to the difference in the methods that were employed to measure angiotensin II. There are several ELISA kits produced with different sensitivities. It is important that our results are in compliance with the results of the study by Reddy et al (8), who metabolomically measured the angiotensin II level.
The first patients admitted to the hospital at the onset of the pandemic were included in this study. During this period, the disease was not fully understood, and treatment protocols were planned according to the data reported in the literature. Therefore, the effect of favipiravir and hydroxychloroquine drugs on the serum angiotensin II levels could not be studied. Another limitation is that serum ACE 1 and ACE2 levels are not studied.
Although SARS-Cov-2 uses the ACE2 receptor to invade the cell, subsequent lung damage may cause a decrease in the activities of both the ACE1 and ACE2 enzymes. As a result, the angiotensin II levels may decrease. In most of the patients included in our study, the presence of diffuse lung involvement may be responsible for the low angiotensin II levels. The fact that the angiotensin II levels in patients with ARDS were statistically significantly lower compared with those without ARDS also corroborates this.
As a result, serum angiotensin II levels decrease significantly in patients with COVID-19, and this decrease is proportional to lung damage. Serum angiotensin II levels can be used to determine the risk of developing ARDS and the need for mechanical ventilation support in COVID-19 patients.
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