key: cord-0939374-51ss01dl authors: Yan, Haixi; Liang, Xiao; Du, Juping; He, Zebao; Wang, Yu; Lyu, Mengge; Yue, Liang; Zhang, Fangfei; Xue, Zhangzhi; Xu, Luang; Ruan, Guan; Li, Jun; Zhu, Hongguo; Xu, Jiaqin; Chen, Shiyong; Zhang, Chao; Lv, Dongqing; Lin, Zongmei; Shen, Bo; Zhu, Yi; Qian, Biyun; Chen, Haixiao; Guo, Tiannan title: Proteomic and metabolomic investigation of serum lactate dehydrogenase elevation in COVID‐19 patients date: 2021-05-28 journal: Proteomics DOI: 10.1002/pmic.202100002 sha: 7756753171ffdf716b409741a245c9ccb06146fb doc_id: 939374 cord_uid: 51ss01dl Serum lactate dehydrogenase (LDH) has been established as a prognostic indicator given its differential expression in COVID‐19 patients. However, the molecular mechanisms underneath remain poorly understood. In this study, 144 COVID‐19 patients were enrolled to monitor the clinical and laboratory parameters over 3 weeks. Serum LDH was shown elevated in the COVID‐19 patients on admission and declined throughout disease course, and its ability to classify patient severity outperformed other biochemical indicators. A threshold of 247 U/L serum LDH on admission was determined for severity prognosis. Next, we classified a subset of 14 patients into high‐ and low‐risk groups based on serum LDH expression and compared their quantitative serum proteomic and metabolomic differences. The results showed that COVID‐19 patients with high serum LDH exhibited differentially expressed blood coagulation and immune responses including acute inflammatory responses, platelet degranulation, complement cascade, as well as multiple different metabolic responses including lipid metabolism, protein ubiquitination and pyruvate fermentation. Specifically, activation of hypoxia responses was highlighted in patients with high LDH expressions. Taken together, our data showed that serum LDH levels are associated with COVID‐19 severity, and that elevated serum LDH might be consequences of hypoxia and tissue injuries induced by inflammation. To better diagnose COVID-19 and monitor the disease progress, multiple molecules have been proposed as prognostic indicators [2] . Lactate dehydrogenase (LDH) is an intracellular enzyme, catalyzing pyruvate fermentation and facilitating glycolysis. LDH is released into the blood after cell death and has been reported to increase in a variety of diseases including Severe acute respiratory syndrome (SARS) [3] , diabetes [4] , and cancers [5] . Serum LDH levels in COVID-19 patients are over-expressed [2] , especially in severe and critical patients [6] [7] [8] [9] . They decrease throughout disease course [7, 10] , in correlation with viral mRNA clearance [7] . Related studies have shown that serum LDH is well correlated with respiratory failure [10] , lung injury, disease severity [11] and mortality [12] in COVID-19 patients. However, the molecular mechanisms underlying the LDH's association with the COVID-19 disease progression remains poorly understood. Most studies attribute serum LDH elevation to its release from somatic tissue and organ damage caused by either viral attack [11] or inflammation [10] . These clinical assumptions lack molecular evidence, potentially leading to biased assessments. Moreover, these explanations failed to consider the metabolic role of LDH to balance excess lactate during hypoxia. Here we have systematically explored the proteome and metabolome of sera from COVID-19 patients with low and high serum LDH, and identified the specific host responses, which shed light on the pathogenesis and convalesce of COVID-19. We collected and curated the electronic medical records of patient information in Taizhou Samples were taken throughout disease course from patient admission to discharge. More details are described in Table S1 . For laboratory tests, 404 serial blood samples from 144 patients were collected and centrifuged at 1500 × g for 10 min at room temperature. The proteomic and metabolomic data were extracted from our previous publication [13] . Briefly, serum samples from COVID-19 patients were kept at 56 • C for 30 min to inactivate potential SARS-CoV- for proteomic and metabolomic data were 10% and 5%, respectively, determined by pooled control samples in each batch, as described previously [13] . Statistical clinical data analyses were performed using SPSS soft- The patients did not exhibit significant difference between severe and non-severe groups in terms of gender unmentioned symptoms on admission, nor other medical treatments (p > 0.05) as listed in Table 1 . Based on laboratory test results, a higher percentage of severe patients have elevated levels of serum LDH that are above the upper limit of normal (ULN) value than non-severe patients (58.3% vs. 7.4%, p < 0.001). Likewise, more severe patients also showed higher level of alanine aminotransferase (ALT, p = 0.023), aspartate aminotransferase (AST, p < 0.001), urea (p = 0.004), creatinine (p = 0.029), and creatine kinase (CK, p < 0.001). We then compared their temporal changes at 7-day intervals (Table S1) . LDH, CK, and creatinine showed continuous decrease in the sera of severe patients, while only serum LDH showed a continuous decrease in non-severe patients. A Cox regression model was applied to evaluate the prognostic value of these indicators ( Table S2 ). In the Cohort 1, LDH is the only indicator with discrimination ability (p < 0.001) in the multivariate analysis, although LDH, AST, and CK were determined in the univariate analysis with statistical significance (p < 0.001). We further validated the value of serum LDH in Cohort 2 and the data showed significant discrimination (p = 0.032), suggesting that serum LDH could be a useful prognostic biomarker. We then assessed the effect of age and sex on the temporal serum LDH expression. COVID-19 patients older than 60 years showed a higher serum LDH expression throughout the disease course than that in younger patients ( Figure 1A) , probably due to ageing and underlying diseases. Males expressed higher serum LDH levels than that from females only during the initial hospitalization stage (within the first week) ( Figure 1B [14] . These data suggest anaerobic glycolysis metabolism in severe patients. To better understand the temporal dynamics of serum LDH expression over the disease course, we next monitored the serum LDH level from day 1 on admission till day 21 at a 3-day interval ( Figure 1E ). As to the non-severe patient group, the serum LDH level was slightly higher on admission while declining slowly over the hospitalization period. The serum LDH levels in the severe group comparatively were significantly higher upon admission, with a prominent variance range. They dropped significantly from 3rd to 9th day as the patients were taking medical care and by the 21st day fell below the initial serum LDH levels in the non-severe group. To establish a cohort-specific serum LDH expression threshold as a risk indicator, we took patient severity (severe vs. non-severe) as the dichotomous variable and conducted the receiver operating characteristic (ROC) analysis on the serum LDH expression levels on admission. The area under curve (AUC) was 0.864 ( Figure 1F ), confirming the discriminative power of serum LDH. The serum LDH level corresponding to the maximum Youden index was determined as 247 U/L, within the threshold to determine serum LDH abnormality from past reports (240-253.2 U/L) [12] . Fourteen characteristic patients within the cohort were selected and divided into two groups for closer inspection ( Figure 1H) . The low-risk (LR) group consists of seven non-severe patients with serum LDH expressions below 247 U/L since admission except for one exceptional detection (LR4, 5th day, 306 U/L). The high-risk (HR) group are composed of six severe patients with on admission serum LDH levels above 247 U/L, and one severe patient with serum LDH levels below the threshold throughout hospitalization (HR1). We attribute this to the relatively late first sampling timepoint (8th day), given that serum LDH levels from all the patients started at a relatively high level and declined over time. Patient HR3 and patient HR7 in particular had exceptionally high serum LDH levels (> 450 U/L) upon admission but dropped dramatically within 10 days. We inspected the detailed medical records of the 14 patients (Table 2) . COVID-19 patients with comorbidities including hypertension, chronic HBV infection, and diabetes tend to be severe patients in the HR group, in consistent with the literature [15, 16] . The serum proteomic and metabolomic datasets of the HR and LR group patients were extracted from a collateral project [13] . 78.6% (11/14) of the patient sera were sampled during the first week on admission (Table 2) , herein representing the stage when serum LDH levels exhibited sharp difference between the LR and HR groups. For the proteomic dataset, the Student's t-test highlighted 34 proteins as differentially expressed (p < 0.05) between HR and LR groups ( Figure 2A, upper panel) , 26 of which were up-regulated. Pathway enrichment analysis using Metascape [17] showed these proteins conduct three major immune-related activities including acute inflammatory responses (GO:0002526, p < 0.001), platelet degranulation (GO:0002576, p < 0.001) and regulation of complement cascade (R-HSA-977606, p < 0.001) (Figure 2A and Figure S2A ). Additional analyses using Ingenuity Pathway Analysis (IPA) nominated acute phase response signaling as the most activated immune-related pathway ( Figure S2C ) in HR patients. These findings were in consistence with the prominent immune behaviors ( Figure S1B ) as we have previously reported in COVID-19 patients with different severity [13] . Moreover, blood coagulation (GO: 007596, p < 0.001) was significantly enriched ( Figure S2A and S2C) . This pathway has been reported to be altered in COVID-19, and associated with interleukin-6 (IL-6)) [18] . Our data showed upregulation of acute phase proteins (SAA1, ORM1, AGT, and SERPINA3), complement subunits (C9, C6, and CFI), and LDH subtypes (LDHA and LDHB) in the HR group ( Figure 2B) . Thirteen of the differentiated proteins were mapped into a network wherein key regulators were focused ( Figure 2C ). Within them, pro-inflammatory cytokine IL-6 has been widely recognized as a risk factor for COVID-19 [18] [19] [20] [21] [22] and clinically observed to be positively correlated with serum LDH levels [23] . IL-6 can activate TP53, which facilitates cell apoptosis and could enhance LDHA expression in blood. Multiple COVID-19 studies involving IL-6 agree with our profiling [18, 24] . This network also includes CCAAT/enhancer-binding protein beta (CEBPB) which mediates immune and inflammatory responses [25] , and sterol regulatory element binding transcription factor 1 (SREBF1) which regulates lipid metabolisms that has been reported dysregulated in severe COVID-19 patients [13] . Taken together, the proteomic difference between LR and HR patients reflected different host responses between the non-severe and severe COVID-19 patients. Of the 34 differentially expressed metabolites listed in Figure S3A (FN1) that involves COVID-19 lung fibrosis [28] . The dysregulated metabolites further consolidate disturbed host responses in association with serum LDH increase uncovered by the proteomic data. Next, we narrowed our focus to the two patients with exceptionally high serum LDH levels on admission (HR3 and HR7, HR outliers), and compared their proteomic patterns with the other HR patients ( Figure 3A ). 38 proteins including LDHA and LDHB were differentially expressed (p < 0.05, Figure 3A For the other up-regulated proteins in HR (outliers) (Figure 3B ), CES1 is a hepatic protein and its release in blood suggests liver injuries. Protein disulfide-isomerase (P4HB) was reported to up-rise in response to hypoxia [30] . GAPDH could enhance HIF activity [31] via NF-κB induction activated in hypoxia [32] , which contributes to heat shock protein 90-alpha (HSP90AA1) upregulation [33] to form protein complexes with Hif1α. Dipeptidyl peptidase 4 (DPP4), also known as CD26, has been nominated as a potential critical marker in infection susceptibility [34] , and its inhibition has been proposed to reduce COVID-19 patient severity [35] . DPP4 is also a downstream factor to mark HIF pathway induction [36] . Especially, the down-regulated protein in HR (outliers) includes CPB2 as a basic carboxypeptidase that suppresses complement system-mediated inflammation [37] . Its deficiency could lead to accelerated acute lung injuries [38] . 84.6% (11/13) of the dysregulated metabolites were lipids ( Figure S5B ), suggesting disturbed lipid metabolism accompanied with serum LDH changes. The network analysis ( Figure S5C ) further proposed LDH elevation to be associated with HSP90AA1 and proteasomes. Taking together all the perturbed molecules as highlighted above (Figure 2B and 3B), we propose a putative working model for the serum LDH elevation in COVID-19 patients ( Figure 3C ). On the one hand, the inflammation processes triggered by the host immune system induce apoptosis of the infected cells, leading to the release of intracellular LDH into the blood. In high-risk cases, these immune activities result in over-reactive inflammation processes (like "cytokine storm") [39] , thereby releasing higher levels of serum LDH from multiple organs/tissues [10] . On the other hand, oxygen homeostasis was disturbed in severe COVID-19 patients [11] . Hypoxia reactions occur to accumulate lactate via glycolysis. LDH can balance lactate secretion via pyruvate fermentation and a series of metabolic regulation ( Figure S4A and 4C) to maintain cellular homeostasis [40] . The activated NF-κB and HIF pathways in hypoxia conditions could also induce inflammatory responses [41] . Here we systematically investigated serum LDH elevation in COVID- Taken together, we propose that elevation of serum LDH might attribute to inflammation-related tissue injuries and hypoxia-related metabolism. This study is limited by several factors. Firstly, this is a single-center study with a relatively small patient cohort due to difficulties of sample collection, therefore subject to experimental bias. Also, the standard inactivation procedures for COVID-19 serum to minimize the risk of infection may have some impact on the characterized samples. Moreover, proteome data from only 14 individuals were acquired, from which only two patients were determined as high-risk group outliers for comparative analysis. We could not obtain the samples from different patients at the identical time points. And due to the small sample size, multiple testing was not performed for molecular analyses, therefore the statistical power from the proteomic data has to be interpreted with caution. It is worth noting that serum LDH elevation is not specific to COVID-19 disease [44] . Further studies should conduct clinical validation on larger cohorts, and compare the molecular differences including control patients with other diseases with similar symptoms. Targeted approaches would also be required to validate our findings for diagnostic purposes. and Tencent Foundation. We thank Westlake University Supercomputer Center for assistance in data storage and computation. The research group of Tiannan Guo is partly supported by Tencent, Thermo Fisher Scientific, SCIEX, and Pressure Biosciences Inc. Tianna Guo and Yi Zhu are shareholders of Westlake Omics Inc. Guan Ruan is an employee of Westlake Omics Inc. The proteomics and metabolomics data that support the findings of this study could be accessed in ProteomeXchange Consortium (https: //www.iprox.org/). Project ID: IPX0002106000 and IPX0002171000. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: A descriptive study SARS: Clinical features and diagnosis Homocysteine and other biochemical parameters in Type 2 diabetes mellitus with different diabetic duration or diabetic retinopathy Elevated lactate dehydrogenase (LDH) can be a marker of immune suppression in cancer: Interplay between hematologic and solid neoplastic clones and their microenvironments Clinical features and treatment of COVID-19 patients in northeast Chongqing The correlation between viral clearance and biochemical outcomes of 94 COVID-19 infected discharged patients Discovery of a novel coronavirus associated with the recent pneumonia outbreak in humans and its potential bat origin. bioRxiv Clinical and immunological features of severe and moderate coronavirus disease 2019 Lactate dehydrogenase and C-reactive protein as predictors of respiratory failure in CoVID-19 patients Lactate dehydrogenase, an independent risk factor of severe COVID-19 patients: A retrospective and observational study Lactate dehydrogenase levels predict coronavirus disease 2019 (COVID-19) severity and mortality: A pooled analysis Proteomic and metabolomic characterization of COVID-19 patient sera Lactate dehydrogenase 5: An old friend and a new hope in the war on cancer Risk factors of critical & mortal COVID-19 cases: A systematic literature review and meta-analysis Characteristics of liver function in patients with SARS-CoV-2 and chronic HBV coinfection Metascape provides a biologist-oriented resource for the analysis of systems-level datasets Serum proteomics in COVID-19 patients: Altered coagulation and complement status as a function of IL-6 level Clinical characteristics and risk factors associated with COVID-19 disease severity in patients with cancer in Wuhan, China: A multicentre, retrospective, cohort study. The Lance Prognostic value of interleukin-6, C-reactive protein, and procalcitonin in patients with COVID-19 Interleukin-6 is a biomarker for the development of fatal severe acute respiratory syndrome coronavirus 2 pneumonia Elevated interleukin-6 and severe COVID-19: A meta-analysis The role of interleukin-6 in monitoring severe case of coronavirus disease 2019 Impaired type I interferon activity and inflammatory responses in severe COVID-19 patients A member of the C/EBP family, NF-IL6 beta, forms a heterodimer and transcriptionally synergizes with NF-IL6 Simultaneous measurement of F2-isoprostane, hydroxyoctadecadienoic acid, hydroxyeicosatetraenoic acid, and hydroxycholesterols from physiological samples Hypoxia: A key feature of COVID-19 launching activation of HIF-1 and cytokine storm SARS-CoV-2 induces transcriptional signatures in human lung epithelial cells that promote lung fibrosis New insights on the function of SCF ubiquitin E3 ligases in the lung Role of ubiquilin associated with protein-disulfide isomerase in the endoplasmic reticulum in stress-induced apoptotic cell death GAPDH enhances the aggressiveness and the vascularization of non-Hodgkin's B lymphomas via NF-kappaB-dependent induction of HIF-1alpha Crosstalk between the Akt/mTORC1 and NF-kappaB signaling pathways promotes hypoxia-induced pulmonary hypertension by increasing DPP4 expression in PASMCs Regulation and function of the human HSP90AA1 gene Assessment of risk conferred by coding and regulatory variations of TMPRSS2 and CD26 in susceptibility to SARS-CoV-2 infection in human Dipeptidyl peptidase-4 (DPP4) inhibition in COVID-19 Hypoxia-inducible factor-1 target genes as indicators of tumor vessel response to vascular endothelial growth factor inhibition Carboxypeptidase B2 and carboxypeptidase N in the crosstalk between coagulation, thrombosis, inflammation, and innate immunity Thrombin-activatable fibrinolysis inhibitor protects against acute lung injury by inhibiting the complement system COVID-19: consider cytokine storm syndromes and immunosuppression A lactate-induced response to hypoxia NF-kappaB and HIF crosstalk in immune responses Ultra-high-throughput clinical proteomics reveals classifiers of COVID-19 infection Serum proteomics in COVID-19 patients: Altered coagulation and complement status as a function of IL-6 level SARS: clinical features and diagnosis the Supporting Information section at the end of the article. How to cite this article Proteomic and metabolomic investigation of serum lactate dehydrogenase elevation in COVID-19 patients