key: cord-0929304-n6ad11zt
authors: Tonon, Federica; Di Bella, Stefano; Giudici, Fabiola; Zerbato, Verena; Segat, Ludovica; Koncan, Raffaella; Misin, Andrea; Toffoli, Barbara; D'Agaro, Pierlanfranco; Luzzati, Roberto; Fabris, Bruno; Bernardi, Stella
title: Discriminatory Value of Adiponectin to Leptin Ratio for COVID-19 Pneumonia
date: 2022-04-26
journal: Int J Endocrinol
DOI: 10.1155/2022/9908450
sha: e1f8cec945b12206cd8fdd4c7a2de31c078592df
doc_id: 929304
cord_uid: n6ad11zt

PURPOSE: Obesity is a risk factor for severe coronavirus disease 2019 (COVID-19). Circulating adipokines have been associated with inflammatory burden and amplified or dysregulated immune responses. This study aimed to evaluate the discriminatory ability of adipokines to identify COVID-19 pneumonia and to assess disease severity. METHODS: We conducted an observational case-control study, with a prospective design, and recruited patients with diagnosis of COVID-19 pneumonia (n = 48) and healthy controls (n = 36), who were matched by age, sex, and BMI. Leptin, adiponectin, IL-6, and TNF-α were measured by ELISA. RESULTS: Patients with COVID-19 pneumonia had higher levels of leptin, lower adiponectin/leptin (Adpn/Lep) ratio, and higher expression of IL-6. Leptin had an acceptable discriminatory accuracy for COVID-19 pneumonia in patients with BMI >30 (AUC 0.74 [0.58, 0.90]) with a cutoff of 7852 pg/mL and it was associated with maximum respiratory support. By contrast, Adpn/Lep had an excellent discriminatory accuracy for COVID-19 pneumonia in patients with BMI <25 (AUC 0.9 [0.74, 1.06]) with a cutoff of 2.23. CONCLUSION: Our data indicate that high Adpn/Lep (>2.23) in lean patients is consistent with a state of good health, which decreases in case of inflammatory states, ranging from adipose tissue dysfunction with low-grade inflammation to COVID-19 pneumonia.

e year 2019 will be remembered for the deadly outbreak of coronavirus disease 19 (COVID-19), a pandemic due to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). With the worldwide spread of SARS-CoV-2, obesity has emerged as an important determinant of COVID-19 prognosis [1] . Obesity is an independent risk factor for the development of severe COVID-19, requiring intensive care support [2, 3] , and leading to higher death rates in hospitalized patients [4, 5] . We have recently demonstrated that neck circumference predicts mortality in hospitalized COVID-19 patients [6] . e relationship between obesity and worse clinical outcomes in viral infections is not new [7] . Frydrych et al., for instance, suggested that this association could be due to an immune system dysfunction triggered by the chronic lowgrade inflammation that characterizes obesity [7, 8] . Adipose tissue is an endocrine organ capable of synthesizing a wide variety of molecules called adipokines. When it enlarges, there is a change in adipocyte size, an increase of free fatty acids and tissue hypoxia. ese changes are accompanied by a modification of adipose tissue secretory profile and macrophage infiltration, leading to a moderate but chronic release of acute-phase proteins, proinflammatory adipokines, and adhesion molecules [9] [10] [11] , which could contribute to the development of cytokine storm and cytokine release syndrome, which are life-threatening systemic inflammatory syndromes that can be triggered by various conditions/agents such as SARS-CoV-2 infection [12] .

In addition to the proimmunogenic state of adipose tissue in obesity, adipose tissue cells can be targets of multiple viruses, including SARS-CoV-2, because they express ACE2, which is the main receptor of SARS-CoV-2 [13] . Interestingly, patients with obesity exhibit an overexpression of ACE2 as well as of other alternative SARS-CoV-2 receptors (CD147, DPP4, and NRP1) in their visceral fat, increasing their susceptibility to viral infection [14] .

is could lead to prolonged viral shedding in this organ, with extended activation of local immune cells and inflammatory response [13, 14] .

Leptin and adiponectin are the two best known adipokines produced by the adipose tissue. In physiological conditions, circulating concentrations of leptin are around 2-8 ng/mL [15] , while those of adiponectin are around 5-30 µg/mL [16, 17] . ese values may vary, due to sex differences, with leptin and adiponectin levels being higher in women [18] , as well as due to the presence of comorbidities [19] , or to the methods used for their measurement [20, 21] . Nevertheless, leptin is directly associated with stored body fat [15] , while adiponectin is inversely related to it [19, 22] . In addition, leptin has proinflammatory actions [23] , while adiponectin has anti-inflammatory properties, such as the inhibition of macrophage activity and their production of TNF-α [24] . Consequently, these adipokines appear to be the link between obesity, unhealthy adipose tissue, low-grade inflammation, and cardio-metabolic alterations. In this setting, the adiponectin to leptin ratio (Adpn/Lep) has been recently proposed as a marker of adipose tissue dysfunction [23] . Consistent with this concept, it has been demonstrated that the adipocyte dysfunction induced by SARS-CoV-2 infection was associated with a decrease in Adpn/Lep ratio and an upregulation of factors involved in the innate immune response [25] .

Based on this background, in this study, we aimed to evaluate whether leptin, adiponectin, as well as Adpn/Lep could help discriminate the presence of COVID-19 pneumonia as well as assess disease severity.

is is an observational case-control study, with a prospective design, aiming at evaluating the role of adipokines in patients with COVID-19 of different severity as compared to healthy controls. Between October 2020 and April 2021, patients with diagnosis of COVID-19 pneumonia (cases), who were admitted to the Institute of Malattie Infettive (ASUGI), were consecutively recruited for this study, while age-and sex-matched healthy volunteers with no history of COVID-19 and/or other intercurrent diseases (controls) were recruited at the Institute of Medicina Clinica (ASUGI).

Diagnosis of COVID-19 pneumonia was based on the following criteria: (i) presence of SARS-CoV-2 nucleic acids as assessed by real-time RT-PCR from nasopharyngeal swabs and (ii) respiratory distress (respiratory rate >30 breaths/min), (iii) mean oxygen saturation ≤93%, and (iv) arterial oxygen pressure/oxygen concentration (PaO 2 /FiO 2 ) ≤300 mmHg, as well as (v) typical ground-glass pulmonary opacities. Exclusion criteria: age <18 years, refusal to participate in the study, and for controls, history or presence of COVID-19 and/or intercurrent diseases.

All participants gave informed consent to participate in this study. In patients with COVID-19 pneumonia (cases), the following parameters were collected: demographics, medical history, medication, vital signs, body mass index, PaO 2 /FiO 2 , complete white blood cell count, CRP, estimated GFR, fasting glucose, and lipid levels. During hospitalization, we recorded the medication prescribed, the maximum respiratory support given, days of noninvasive mechanical ventilation, time to negativity, and death. e maximum respiratory support given was dived into two groups: venturi mask/high-flow nasal cannula (VM-HFNC) group and continuous positive airway pressure (CPAP)/noninvasive mechanical ventilation (NIMV) group. In control patients, the following parameters were collected: demographics, vital signs, body mass index, complete white blood cell count, estimated GFR, fasting glucose, and lipid levels.

In all patients, a fasting blood sample was collected to measure adipokines the day after hospital admission.

is study was conducted in accordance with the Declaration of Helsinki, and it was approved by our Institutional Review Board and regional Ethics Committee on 27/ 09/2019 (CEUR-2019-SPER-113). All subjects gave their written informed consent before participation in the study.

Leptin (DY398-05, R&D Systems), adiponectin (DY1065-05, R&D Systems), IL-6 (DY206-05, R&D Systems), and TNF-α (DY210-05, R&D Systems) were measured by ELISA, according to manufacturer's instructions. e intra-assay coefficients of variations were 6.39% for leptin, 6.96% for adiponectin, 5.37% for IL-6, and 3.72% for TNF-α. e interassay coefficients of variations were 7.26% for leptin, 7.30% for adiponectin, 11.40% for IL-6, and 8.74% for TNF-α.

All statistical analyses were carried out in R system for statistical computing (Ver 5.0; R Development Core Team, 2018). Statistical significance was set at p < 0.05.

Shapiro-Wilk test was applied to quantitative (continuous) variables to check for distribution normality. Continuous variables were reported as median with range (min-max) or mean ± standard deviation, depending on their distribution. Categorical variables were reported as absolute frequencies and/or percentages. Continuous variables were compared by Mann-Whitney test or t-test, depending on data distribution. Linear associations were evaluated with the Pearson or Spearman coefficient. Two logistic multivariate regression models were performed: the first to evaluate factors associated with COVID-19 pneumonia and the second to identify variables related to maximum respiratory support.

Receiver operating characteristic (ROC) curves were constructed to evaluate the sensitivity and specificity of leptin and Adpn/Lep in predicting COVID-19 pneumonia. e area under the curve (AUC) was calculated, with higher values indicating better discriminatory ability. Sensitivity, specificity, and positive and negative predictive values with 95% confidence interval (CI) were then calculated. Areas under the curve (AUCs) for Adpn/Lep respect to BMI were compared using DeLong's test implemented in the R package pROC.

A total of 48 patients with COVID-19 pneumonia and 36 healthy controls were recruited. Groups did not differ in terms of age, sex, BMI, and presence of metabolic syndrome, as assessed based on the International Diabetes Federation criteria [20] , as shown in Table 1 . Patients with COVID-19 pneumonia were on average 64 years old; they were predominantly men (71%) and overweight, as their mean BMI was 29. On admission, their median CRP was 70 mg/L (4-220). e PaO 2 /FiO 2 was less than 100 in 35.4% of them, 100 to 200 in 35.4%, 200 to 300 in 29.2%, and greater than 300 only in 4.2% of them. All these patients required oxygen support during hospitalization; the maximum respiratory support given was recorded and a total of 32/48 patients (66.67%) received VM/HFNC (Venturi mask/high-flow nasal cannula), while a total of 16/ 48 patients (33.33%) received NIMV (continuous positive airway pressure/noninvasive mechanical ventilation) in the sub-ICU/ICU for 8.5 days. During hospitalization, patients received preventive (24/48; 50%) or therapeutic antithrombotic therapy (24/48; 50%), corticosteroids (47/48; 97.9%), and/or remdesivir (9/48; 18.75%). e median duration of hospital stay was 10 days (3-49). 

Adiponectin was not associated with sex, age, and BMI. It did not change between healthy subjects and patients with COVID-19 pneumonia, apart from the subgroup of patients with BMI <25 (Figure 1 ). We also looked at the ratio adiponectin/leptin (Adpn/Lep) [26] . Adpn/Lep did not correlate with age, but it was associated with sex, being higher in male subjects (p � 0.03) and inversely related to BMI (rho � −0.46; p < 0.0001). In our study, Adpn/Lep was 1.65 (0. 13-12.9 ) in the control group and it decreased significantly to 0.98 (0.26-3.98) in patients with COVID-19 pneumonia (p � 0.025). Nevertheless, when we stratified our population based on BMI, it was only in the subgroup of patients with BMI <25 that we observed a significant change of this ratio in case of COVID-19 pneumonia (Figure 2 ).

e levels of IL-6 and TNF-α were not always detectable, so we looked at the percentage of patients with detectable levels (vs those with undetectable levels). e percentage of patients with COVID-19 pneumonia who expressed detectable levels of IL-6 (but not TNFα) was significantly higher than that of controls (p � 0.0055), as shown in Figure 1 . Patients with detectable levels of IL-6 were older (p � 0.04), and they were more often males (47% of males expressed it as compared to 29% of females; p � 0.12), as previously reported [27] . 

Pneumonia. Consistent with our previous data, also in the subgroup of patients with COVID-19 pneumonia, leptin was directly associated to BMI (rho � 0.592; p < 0.0001), and Adpn/Lep was inversely related to it (rho � −0.354; p � 0.014). When we looked at the immune response of patients with COVID-19 pneumonia, the number of leukocytes and lymphocytes was inversely related to IL-6 and TNF-α (Table 3 ). When we looked at the maximum respiratory support given, which we divided into VM/HFNC and NIMV, only BMI and leptin were related to it, being significantly higher in the NIMV group ( Figure 3 ). BMI was also associated with the days of NIMV, while age and BMI were associated with the days of hospital stay. Logistic multivariate regression models suggested that age and BMI, but not leptin, were independently associated with the maximum respiratory support given in patients with COVID-19 pneumonia (Table 4 ). Consequently, we did not look for the discriminatory cutoffs of leptin and Adpn/Lep for outcomes of COVID-19 pneumonia.

is study shows for the first time that Adpn/Lep had an excellent discriminatory accuracy for the presence of COVID-19 pneumonia in lean people. Adpn/Lep is a ratio that was initially proposed as a marker of adipose tissue dysfunction, increased cardio-metabolic risk [26, 28] , and low-grade inflammation [29] . Obesity [15, 22] , insulin resistance [19] , and inflammation [30] [31] [32] are associated with a decrease of adiponectin and an increase of circulating leptin. Consistent with this, Adpn/Lep decreases with the increasing number of metabolic risk factors [28] and it is negatively correlated with CRP levels and systemic inflammation [29] . For these reasons, it has been suggested that Adpn/Lep might discriminate between the patients that are "metabolically healthy" and those that are "metabolically unhealthy" [28] .

Frühbeck et al. have recently proposed that Adpn/Lep should have a value >1 in normal conditions, corresponding to a low cardio-metabolic risk, while values between >0.5 and <1 should correspond to a moderate risk, and values <0.5 should correspond to a high cardio-metabolic risk [33] . Interestingly, in this work by Frühbeck et al., lean subjects (mean BMI of 22), with mean arterial blood pressure of 123/ 71 mmHg, mean glycemia of 91 mg/dL, and lipids levels within reference targets, had a mean value of Adpn/Lep of 2.83 ± 2.16 [33] . In line with these findings, in our study, 2.23 was the optimal cutoff value discriminating between lean patients that were in good health and those with COVID-19 pneumonia, allowing to identify correctly 88% of the patients with a sensitivity of 90% and a specificity of 83%. In other words, a high value of Adpn/Lep is consistent with a state of good health in lean patients and it could rule out any low-grade or acute systemic inflammation, including the state triggered by SARS-CoV-2 infection.

Our study shows that the Adpn/Lep could be used not only to identify patients with adipose tissue dysfunction and high cardio-metabolic risk but also to rule out the presence of acute inflammatory diseases, such as COVID-19 pneumonia. is is consistent with other authors' recent findings [34] . e issue that remains to be solved is the heterogeneity of methods to measure Adpn/Lep, mostly due to the variety of methods available to measure adiponectin [20, 21] . Adiponectin can be measured with different techniques, such as radioimmunoassays, enzymelinked immunosorbent assays, chemiluminescent immunoassays, and immunoturbidimetric methods, in both manual or fully automated ways [21] . Consequently, a standardization of adiponectin assays, as well as a clear definition of normal and pathological reference ranges is still lacking [20] .

It can be speculated that the decrease of Adpn/Lep, which is seen in obese patients and patients with COVID-19 pneumonia, might contribute to development of severe form of disease, due to the anti-inflammatory actions of Adpn/Lep is for adiponectin to leptin ratio; BMI is for body mass index; CRP is for C-reactive protein; IL-6 is for interleukin-6; NIMV is for noninvasive mechanical ventilation; TNF-α is for tumor-necrosis factor-α. Spearman coefficient. 6 International Journal of Endocrinology adiponectin and the proinflammatory effects of leptin. Several studies demonstrate that obesity is a risk factor for developing a severe form of COVID-19 pneumonia [1] , requiring intensive care unit admission, intubation, and mechanical ventilation [3, [35] [36] [37] . Last year, we demonstrated that also other anthropometric indexes, such as neck circumference, might predict the need of mechanical ventilation support [38] . Consistent with the literature, in this study, we found that BMI, as well as age, was independently associated with the need of noninvasive mechanical ventilation (NIMV), the days of NIMV, and the days of hospitalization. Nevertheless, we did not find any significant association between the levels of circulating adipokines and these outcomes, possibly due to the small number of patients. e limitations of this study include the small number of patients recruited, as well as the measurement of the adipokines with manual enzyme-linked immunosorbent assays. On the other hand, it has to be taken into account that even in our small cohort, Adpn/Lep had an excellent discriminatory accuracy in identifying COVID-19 pneumonia in lean subjects.

In conclusion, our data indicate that patients with COVID-19 pneumonia had higher levels of leptin and lower Adpn/Lep. Leptin had an acceptable discriminatory accuracy for COVID-19 pneumonia in patients with BMI >30, while Adpn/Lep had an excellent discriminatory accuracy for COVID-19 pneumonia in patients with BMI <25, with a cutoff of 2.23. Our data indicate that high Adpn/Lep (>2.23) in lean patients is consistent with a state of good health, which decreases in case of inflammatory states, ranging from adipose tissue dysfunction with low-grade inflammation to COVID-19 pneumonia.

Our data reinforce the notion that high Adpn/Lep is an indicator of good metabolic health, as Adpn/Lep decreases in case of inflammatory states, ranging from the low-grade inflammation associated with metabolic syndrome to acute inflammatory states as in COVID-19 pneumonia.

Some or all datasets generated and/or analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request.

is study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Institutional Review Board and the Ethics Committee of FVG (date 27/09/2019; No. CEUR-2019-SPER-113).

All participants provided informed consent for publication.

An early version of this manuscript has been presented as a preprint according to the following link: https://www. researchsquare.com/article/rs-1254160/v1 [39] .

e authors declare that there are no conflicts of interest regarding the publication of this article.

Global pandemics interconnected -obesity, impaired metabolic health and COVID-19

Obesity and COVID-19 severity in a designated hospital in shenzhen, China

High prevalence of obesity in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) requiring invasive mechanical ventilation

Severe obesity, increasing age and male sex are independently associated with worse in-hospital outcomes, and higher in-hospital mortality

Presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with COVID-19 in the New York city area

Neck circumference predicts mortality in hospitalized COVID-19 patients

Obesity and COVID-19: immune and metabolic derangement as a possible link to adverse clinical outcomes

Obesity and type 2 diabetes mellitus drive immune dysfunction, infection development, and sepsis mortality

Elevated levels of interleukin 6 are reduced in serum and subcutaneous adipose tissue of obese women after weight loss *

Effect of weight loss and lifestyle changes on vascular inflammatory markers in obese women

Osteoprotegerin increases in metabolic syndrome and promotes adipose tissue proinflammatory changes

Cytokine storm

Is adipose tissue a reservoir for viral spread, immune activation, and cytokine amplification in coronavirus disease

FNDC4 and FNDC5 reduce SARS-CoV-2 entry points and spike glycoprotein S1-induced pyroptosis, apoptosis, and necroptosis in human adipocytes

Serum immunoreactive-leptin concentrations in normal-weight and obese humans

cDNA cloning and expression of a novel adipose specific collagen-like factor, apM1 (AdiposeMost abundant gene transcript 1)

Isolation and characterization of GBP28, a novel gelatin-binding protein purified from human plasma

Sex differences in body fat distribution are related to sex differences in serum leptin and adiponectin

Adiponectin and adiponectin receptors in insulin resistance, diabetes, and the metabolic syndrome

A methodological reappraisal of total and high molecular weight adiponectin determination in human peripheral circulation: comparison of four immunometric assays

e adiponectin to leptin ratio, a still unrecognized biomarker of insulin resistance and cardiometabolic risk

Paradoxical decrease of an adipose-specific protein, adiponectin, in obesity

COVID-19 severity in obesity: leptin and inflammatory cytokine interplay in the link between high morbidity and mortality

Adiponectin suppression of high-glucose-induced reactive oxygen species in vascular endothelial cells

Hyperglycemia in acute COVID-19 is characterized by insulin resistance and adipose tissue infectivity by SARS-CoV-2

Adiponectin-leptin ratio: a promising index to estimate adipose tissue dysfunction. Relation with obesityassociated cardiometabolic risk

Sex differences in proatherogenic cytokine levels

Metabolic risk susceptibility in men is partially related to adiponectin/leptin ratio

Involvement of the leptin-adiponectin axis in inflammation and oxidative stress in the metabolic syndrome

Low serum adiponectin is associated with high circulating oxidized low-density lipoprotein in patients with type 2 diabetes mellitus and coronary artery disease

Multiple cytokines and acute inflammation raise mouse leptin levels: potential role in inflammatory anorexia

Elevated serum leptin concentrations induced by experimental acute inflammation

Adiponectinleptin ratio is a functional biomarker of adipose tissue inflammation

Adiponectin to leptin ratio reflects inflammatory burden and survival in COVID-19

Body mass index and risk for intubation or death in SARS-CoV-2 infection

Clinical outcomes in young US adults hospitalized with COVID-19

BMI as a risk factor for clinical outcomes in patients hospitalized with COVID-19 in New York

Neck circumference as reliable predictor of mechanical ventilation support in adult inpatients with COVID-19: a multicentric prospective evaluation

Discriminatory value of adiponectin to leptin ratio for covid-19 pneumonia

All authors contributed to the study conception and design. Federica Tonon Acknowledgments is work was supported by the University of Trieste (grant number: microgrant #2020 to SDB).