α-HBDH Is a Probably Higher Sensitive and Specic the Biomarkers of Poor Prognosis in Patients With COVID-19

Background: The coronavirus disease 2019 (COVID-19) that is caused by the severe acute respiratory syndrome-coronavirus2 (SARS-CoV2) has spread rapidly worldwide during the past nearly a year. SARS-CoV-2 particles spread through the respiratory mucosa and infect other cells, causing a storm of cytokines in the body 1 , producing a series of immune responses, and causing multiple organ dysfunction, including the heart. Some patients present with cardiovascular system damage, such as palpitations and shortness of breath as the rst or secondary symptoms. Previous studies suggested that LDH, α-HBDH, CK and CK-MB reect myocardial function. 2 Here, we aim to investigate whether these markers can predict poor prognosis of patients with COVID-19. Methods: We collected data from 2338 patients with laboratory-conrmed COVID-19. Patients were then screened, and we focused on 49 moderate cases, 98 severe cases and 53 critical cases (27 recovered cases, 26 deaths). We divided these patients into non-critical group (n = 49) and critical group (n = 151). Then, we also divided the length of hospitalization into ve time points, namely admission, 25%, 50%, 75% and discharge or death, according to the principle of interquartile distance. Blood was collected from patients on the above ve time points. Patients with ve blood tests were 49 moderate cases, 98 severe cases and 53 critical cases (27 recovered cases, 26 deaths). LDH, α-HBDH, CK and CK-MB of each group were collected for analysis. Results: Our research found that α-HBDH and LDH of the critical groups signicantly increased, diagnostic eciency of LDH and α-HBDH have more advantages than that of CK and CK-MB compared with the non-critical group, and patients with α-HBDH greater than 182IU/L and LDH greater than 250IU/L at admission had lower survival rates. Then, CK, LDH, α-HBDH and CK-MB were observed dynamically in the 49 moderate cases, 98 severe cases and 53 critical cases (27 recovered cases, 26 deaths). It turns out that they increased progressively in the dead patients, while they decreased regularly in


Background
The coronavirus disease 2019  that is caused by the severe acute respiratory syndrome-coronavirus2 (SARS-CoV2) has spread rapidly worldwide during the over past year 3,4 . As a novel infectious disease, COVID-19 has the ability of human-to-human transmission and can lead to acute respiratory distress syndrome (ARDS), multiple organ dysfunction and even death [5][6][7][8] . It has become a global crisis with a tremendous socio-economic impact 9 . On March 11, 2020, COVID-19 was declared by the World Health Organization (WHO) as a global public health emergency due to its pandemicity.
However, e cient indicators for predicting the poor prognosis of COVID-19 patients, therapeutic responses and disease outcome have not been fully investigated. Currently, these indicators include obesity 10,11 , liver injury 12 , chest CT 13 , alteration of taste or smell 14 , combined IL-6 and CD8+ T cell counts 15 , serum amyloid A 16 , cardiac troponin-I, homocysteine 17 , elevated N-terminal pro-brain natriuretic peptide 18 , angiopoietin-2 19 , lymphocyte-to-C-reactive protein ratio 20 , albumin 21 , peripheral lymphocyte count 22-25 , neutrophil to lymphocyte ratio 26 . However, most of these indexes are limited to speci c populations, with small sample sizes, and are di cult to detect or to reproduce on a large scale. We found that many patients with COVID-19 had abnormal level of CK, LDH, α-HBDH and CK-MB. These indicators were the most available, e cient and economic examination, as this low-cost, easily acquired biomarker is readily available even in remote areas. Their signi cance for the mortality and severity of COVID-19 are essential in this pandemic situation.
SARS-CoV-2 particles spread through the respiratory mucosa and infect other cells, causing a storm of cytokines in the body 1 , producing a series of immune responses, and causing multiple organ dysfunction, including the heart. The clinical manifestations of COVID-19 are mainly respiratory symptoms, but some patients present with cardiovascular system damage, such as palpitations and shortness of breath as the rst or secondary symptoms. In addition, some people with basic cardiovascular disease (CVD) may have an increased risk of death. Therefore, it is important to understand the potential mechanisms of SARS-CoV-2 damage to the cardiovascular system. 27 Previous studies suggested that LDH, α-HBDH, CK and CK-MB re ect myocardial function 2 . The increased number of these cardiac injury markers is related to in-hospital mortality in patients with COVID-19 28 . This study aims to retrospectively analyze the time courses of LDH, α-HBDH, CK and CK-MB of cured and dead patients with COVID-19, in order to provide timely and effective treatment and reduce mortality in these patients.

Methods
Patients' involvement and data collection All hospitalized patients (n=2338) (admission date from February 10 to March 20, 2020) in Huoshenshan Hospital of Wuhan, diagnosed with COVID-19 based on their clinical symptoms (fever or respiratory symptoms) with typical changes in chest radiology and positive nucleic acid detection results, were involved in this study. Pharyngeal swab specimens of these patients were collected and used for COVID-19 viral nucleic acid detection using a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay in the designated hospitals. Patients with positive test results were admitted to hospital and included in this study. All patients involved in this study were living in Wuhan during the outbreak period of COVID-19.
COVID-19 severity was de ned according to the diagnostic and treatment guidelines for COVID-19 issued by the Chinese National Health Committee (version 7). Severe COVID-19 was designated when the patients had one of the following criteria: (a) respiratory distress with respiratory frequency ≥30/min; (b) pulse oximeter oxygen saturation ≤93% at rest; and (c) oxygenation index (artery partial pressure of oxygen/inspired oxygen fraction, PaO 2 /FiO 2 ) ≤300 mm Hg. Critical COVID-19 was designated when the patients had one of the following criteria: (a) respiratory failure with mechanical ventilation; (b) shock; and (c) combination with other organ failure; requirement of ICU for monitoring and treatment.
A total of 2338 patients diagnosed with COVID-19 were included in this study, with 2126 non-critical patients (20 mild cases and 2106 moderate cases), 212 severe and critical patients (152 severe cases and 60 critical cases) ( Figure 1). In order to facilitate the study, we divided the length of hospitalization into ve time points, namely admission, 25%, 50%, 75% and discharge or death, according to the principle of interquartile distance. Blood was collected from patients on the above ve time points. Patients with ve blood tests were 49 moderate cases, 98 severe cases and 53 critical cases (27 recovered cases, 26 deaths) (Figure 1,2). The mean age for 200 patients was 66.4 years, ranging from 24 to 91 years old, and 125 (62.5%) patients were 60-80 years old. More than half (57.5%) of cases were male (Table1). The date of disease onset, hospital admission date and time of discharge or death, as well as the severity of COVID-19, were also recorded. The onset date was de ned as the day when any symptoms were observed by the patients.

Laboratory testing
Blood testing for all patients was performed by the clinical laboratory of Huoshenshan Hospital of Wuhan. Medical laboratory results in this study, including the plasma levels of CK, LDH, α-HBDH and CK-MB, were collected for each patient. All medical laboratory data were generated by the clinical laboratory of Huoshenshan Hospital of Wuhan. As the disease progressed, the updated secondary results for laboratory ndings (including levels of LDH CK, LDH, α-HBDH and CK-MB) during the hospital stay were also collected.
Although COVID-19 infections clustered within 30 (15.0%) patients whose family members or friends were also infected with COVID-19 in this study, 170 patients (85.0%) did not have a clear history of exposure. About 74.5% of patients had one or more underlying comorbidity, the most common of which were chronic diseases, such as hypertension, diabetes and coronary heart disease. In particular, many patients concomitantly suffered from a variety of underlying comorbidities (Table 1).
Clinical symptoms of all patients at the onset of illness are shown in Table 2. The most common symptoms were cough, fever, chest tightness/dyspnea, fatigue and muscle aches. Less common symptoms were gastrointestinal symptoms, dizziness/headache, expectoration, sore throat etc. Frequently, many patients also had multiple symptoms at the same time.

Radiological and laboratory ndings at admission
Abnormalities in chest CT images were detected in all patients. Of the 200 patients, 133 (66.5%) had multiple ground glass in both lungs; 51 (25.5%) patients had multiple patchy shadows in both lungs and 13 (6.5%) patients had multiple consolidation in both lungs ( Table 2). There were signi cant differences between the moderate cases, severe cases, critical recovered cases and deaths(P 0.001).
The blood tests of patients were collected at admission. The results showed that there were differences in CK, LDH, α-HBDH and CK-MB in the non-critical groups and critical groups ( Table 3). Compared with the non-critical groups, the LDH and α-HBDH of critical groups signi cantly increased(P 0.001), while their CK and CK-MB remained almost at the same level (P = 0.355, 0.051, respectively). Further analysis found that 67.3% of non-critical groups displayed normal level of LDH, while 62.9% of critical groups displayed abnormality levels (P = 0.001). The study also found 51.0% of non-critical groups displayed normal level of α-HBDH, while 71.5% of critical groups displayed abnormality levels (P = 0.021) ( Table 4).
Correlation analysis of CK-MB and LDH or α-HBDH in the non-critical groups and critical groups There was no correlation of CK-MB and LDH or α-HBDH in the non-critical groups (r = 0.2659 and P = 0.0648, r = 0.2066 and P = 0.1543, respectively). However, there was a positive correlation of CK-MB and LDH or α-HBDH in the critical groups (r = 0.2960 and P = 0.0002, r = 0.3182 and P 0.0001, respectively). Furthermore, there was also a positive correlation of CK-MB and LDH or α-HBDH in the non-critical groups and critical groups(r = 0.3008 and P 0.0001, r = 0.3119 and P 0.0001, respectively) ( Figure 4).

Survival analysis of patients with COVID-19 on the admission
A total of 200 patients with COVID-19 were divided into a 200IU group (180 cases) and a 200IU group (20 cases), a 250IU group (90 cases) and a 250IU group(110 cases), a 182IU group (68 cases) and a 182IU group (132 cases), a 24IU group(186 cases) and a 24IU group(14 cases), according to normal level of CK, LDH, α-HBDH and CK-MB, respectively. Analyzing survival, we found that there was no signi cant difference between the two groups according to level of CK and CK-MB (P = 0.5962 and 0.9676, respectively), while there was signi cant difference between the two groups according to level of LDH and α-HBDH (P 0.0001 and P = 0.0075, respectively) ( Figure 5).
Dynamic changes of CK, LDH, α-HBDH and CK-MB in patients with COVID-19 The blood tests of patients during their hospitalization were collected at admission, 25%, 50%, 75% and discharge or death. The results showed that there were differences in CK, LDH, α-HBDH and CK-MB at almost every time point in the four groups (Table 5). Compared with the time of admission, the CK, LDH, α-HBDH and CK-MB of dead patients increased gradually, while the CK, LDH, α-HBDH and CK-MB of other patients were decreasing gradually (Table5 and Figure 6). Further analysis found that the CK, LDH, α-HBDH and CK-MB of death patients were signi cantly higher than that of other patients(P 0.001). Through the longitudinal comparison, we also found that CK, LDH and α-HBDH of them were signi cantly changed in 200 patients, except CK-MB(P 0.001). Further subgroup analysis revealed that LDH and α-HBDH of them gradually returned to normal in the severe case and the critical cases(recovered) (P 0.001) , while LDH and α-HBDH did not recover or even worsened in the dead patients (Table5).

Discussion
We report here a retrospective analysis on 2338 pneumonia patients with laboratory-con rmed 2019-nCoV infection, and reviewed clinical records, nursing records, laboratory ndings, and CT scans from the data of 200 patients with 5 blood tests. The important changes were presented in the CK, LDH, α-HBDH and CK-MB of patients with SARS-CoV-2 infection of different clinical types and at different follow-up time points in China. It was highly signi cant to study the level of α-HBDH of peripheral blood in patients with COVID-19 for accurate typing, illness evaluation, precise treatment, delaying the progression of the disease, and reducing mortality.
A very important characteristic is that the α-HBDH and LDH of the critical groups signi cantly increased, while their CK and CK-MB remained almost at the same level. Our research also found that 51.0% of non-critical groups displayed normal level of α-HBDH, while 71.5% of critical groups displayed abnormality levels; 67.3% of non-critical groups displayed normal level of LDH, while 62.9% of critical groups displayed abnormality levels. Therefore, compared with non-critical patients, LDH and α-HBDH in critically ill patients are signi cantly abnormal, which are more sensitive than CK and CK-MB. Previous studies suggested that LDH, α-HBDH, CK and CK-MB re ect myocardial function 2 . In order to study the four of the biomarkers indicating myocardial injury, ROC curve was used to analyze, the results showed that diagnostic e ciency of LDH and α-HBDH have more advantages than that of CK and CK-MB ( Figure   3).
Another very important feature is that there is a very slight correlation of CK-MB and LDH or α-HBDH in critical cases ( Figure 4). This suggests that LHD and α-HBDH have similar clinical signi cance to CK-MB in critically ill patients. According to the study, LDH is a glycolytic enzyme which catalyzes the oxidation of lactic acid to pyruvate. It has ve isozymes, LD1, LD2, LD3, LD4 and LD5. LD1 and LD2 mainly come from myocardium (the sum of LD1 and LD2 is α HBDH); LD3 mainly comes from lung and spleen; LD4 and LD5 mainly come from liver and skeletal muscle (Figure 7). Therefore, it can be said that α-HBDH is a higher sensitive and speci c the biomarkers of myocardial injury in patients with COVID-19. And in a previous study also showed that most of COVID-19 patients (75%) but COVID-19 patients (20%) had an abnormal α-HBDH 29 . The results suggested that 2019-nCoV infected patient may result into cardiac injury.
The third feature is that α-HBDH and LDH can be used as a marker for poor prognosis in COVID-19, especially in critical patients. LDH and α-HBDH in dead cases with COVID-19 remained at a high level on admission. Through survival analysis, we found that patients with α-HBDH greater than 182IU/L and LDH greater than 250IU/L at admission had lower survival rates ( Figure 5), which may indicate poor clinical prognosis. Therefore, CK, LDH, α-HBDH and CK-MB were observed dynamically ( Figure 6). It turns out that they increased progressively in the dead patients, while they decreased regularly in the severe case and the critical cases(recovered). Previous studies have shown that LDH and α -HBDH are common indicators of cardiotoxicity 30 . The evidence of myocardial damage during SARS-CoV-2 infection was evaluated by CK-MB, LDH and α -HBDH 28 .
The cardiac injury related to SARS-CoV-2 infection can be explained by several mechanisms. The rst mechanism is the systemic in ammatory response syndrome, such as the cytokine storm, which may contribute to the myocardial injury 31 . Novel coronavirus pneumonia is a systemic disease. Its mortality was caused mainly by a respiratory failure due to severe acute pneumonia, but systemic in ammatory response syndrome, including can lead to myocardial damage, such as acute fulminant myocarditis with arrhythmic manifestations, even to a sudden cardiac death [32][33][34][35][36] . In the heart, in ammatory changes with lymphohistiocytic in ltration were observed. The ndings of Puntmann and colleagues, 29 describing myocyte injury post-COVID-19 on MRI, suggest a clinical substrate for COVID-19-induced cardiac damage 37 . Ruan Q et al. reported that 40% of deaths were associated with circulatory failure due to cardiac injury 4,38 . The cardiac injury related to SARSCoV2 infection is considered as an important issue 39 . They could relate to either virally induced in ammation, myocardial stress, ischaemia, drugs, micro vascular thrombotic occlusion, or combinations of these factors 40 . The second one is that the serum myocardial enzyme spectrum increased abnormally after myocardial cell injury. Myocardial enzymes mainly exist in the heart tissues in good condition. When myocardial cells are damaged, it will cause the myocardial enzymes to over ow from the myocardial cells to the serum, which makes the content of serum central muscle enzymes increase abnormally. Therefore, the changes of myocardial enzymes can be used to re ect the degree of myocardial injury and the size of the lesion, especially CK-MB and α-HBDH, which is considered as the standard diagnostic parameter for various forms of cardiac injury 41 . Accordingly, it is comprehensible that the patients with an increased number of cardiac injury markers have a higher incidence of mortality.
Our study has several limitations. Firstly, the lack of a randomized control group means that we cannot draw de nitive conclusions.
Second, this is a retrospective observational study with a limited patient population. Although the different time points in this analysis were used for the LDH and α-HBDH -related survival analysis, which have strengthened our ndings; however, the sample size available was not of a su cient size to conduct such an analysis. Further validation of α-HBDH in a larger patient population is necessary. Despite this, the present results con rm the prognostic ability of α-HBDH in COVID-19. Therefore, we believe that the predictors demonstrated here are meaningful. Greater effort should be made to tackle these issues in future studies.

Conclusions
In conclusion, α-HBDH is a probably higher sensitive and speci c the biomarkers of poor prognosis in adults with COVID-19. This study revealed a feasible quantitative tool as a prognostic indicator for COVID-19. It may help clinicians identify patients with a poor prognosis and may be useful for guiding a physician for the strategy of treatment in SARS-CoV-2 infection. All data generated or analysed during this study are included in this manuscript and its tables and gures.

Funding:
This research received no external funding.

Compliance with ethical standards:
Clinical and laboratory information was collected approved by Huoshenshan Hospital of Wuhan. Moreover, when the information is obtained, the patient's name, ID number, work unit, home address, contact person and telephone information are hidden, and there is no patient privacy exposure. Thirdly, Huoshenshan Hospital of Wuhan has stopped running before the article is completed. Fourth, this is a descriptive retrospective study. Therefore, this study has not been reviewed by the hospital ethics committee.

Transparency declarations:
None to declare.

Con icts of Interest:
The authors declare no con ict of interest. Study ow chart Correlation analysis of CK-MB and LDH or α-HBDH in non-critical groups and critical groups Distribution of LDH isozymes LD1, LD2, LD3, LD4 and LD5 in human body