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High Levels of Methylarginines Were Associated With Increased Mortality in Patients With Severe Sepsis

Mortensen, Karoline Myglegård; Itenov, Theis Skovsgaard; Haase, Nicolai; Müller, Rasmus Beier; Ostrowski, Sisse Rye; Johansson, Pär Ingemar; Olsen, Niels Vidiendal; Perner, Anders; Søe-Jensen, Peter; Bestle, Morten Heiberg

Author Information
doi: 10.1097/SHK.0000000000000649

Abstract

INTRODUCTION

Severe sepsis and septic shock are frequent causes of intensive care unit (ICU) admission and mortality (1). Nitric oxide (NO) has several physiological properties including potent vasodilation (2), but in sepsis the cardiovascular role of NO is debated. On the macro circulatory level, increased NO production contributes to refractory hypotension (2) with a potentially adverse outcome for the patient. However on the microcirculatory level raised NO production potentially increases microcirculatory organ perfusion (3) leading to a better outcome for the patient. Impaired organ perfusion is a hallmark of sepsis, and the balance between the effects of NO on different levels could influence the risk of irreversible organ injury and mortality. Furthermore, elevated NO levels are well documented to play a crucial role in normal leukocyte function and microbial clearance (4)—two elements also playing an important role in patient survival.

The methylated arginines—asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA)—are by-products of protein turnover. Through separate mechanisms they inhibit the production of NO. ADMA is an unselective inhibitor of the three NO synthases that produce NO from L-arginine (5); SDMA inhibits cellular uptake of arginine that is the substrate for NO production (6). Therefore, ADMA and SDMA could play a role in the pathophysiology of severe sepsis and septic shock.

So far, studies assessing ADMA in patients with sepsis have included relatively small sample sizes, and the results are conflicting regarding the effect on mortality (7–13). The role of SDMA has only been investigated in few studies, but they point to an association with mortality (11, 14).

The production of NO might be affected by co-occurring abnormal levels of both arginine and ADMA. Therefore, the ratio of arginine to ADMA (arginine/ADMA ratio)—the balance between the substrate and an inhibitor—could provide additional information (15).

We investigated if high-plasma levels of ADMA, SDMA and the arginine/ADMA ratio were associated with increased 90-day mortality in a large cohort of patients with severe sepsis or septic shock.

PATIENTS AND METHODS

Patients

In this cohort study we included patients from the Scandinavian Starch for Severe Sepsis and Septic Shock (6S) trial cohort (16). The 6S trial was a multicenter randomized clinical trial conducted from 2009 to 2011. The study compared the effect of two different resuscitation fluids (6% hydroxyethyl starch 130/0.42 and Ringer's acetate) on mortality (16). The 6S trial included adult patients admitted to ICU with severe sepsis or septic shock that needed fluid resuscitation as judged by the treating clinician. Exclusion criteria included renal replacement therapy, infusion of more than 1,000 mL synthetic colloid, and failure to obtain consent. The full list of exclusion criteria have been presented elsewhere (16). The 6S trial was approved by the Ethics Committee of the Capital Region of Denmark (protocol. no: H-A-2009-056) and the Danish Data Protection Agency (j.nr. 2007-58-0015). All patients or their legal representatives gave written informed consent. Along with the protocol (17) and the primary results from the 6S trial (16), other studies based on the 6S cohort have previously been published (18–21).

In the present study, we included all patients who were included in the 6S trial in the period of blood sample collection from December 2009 to December 2011 from four university hospitals in Denmark (Rigshospitalet, Hvidovre, Herlev and Bispebjerg Hospitals).

Collection of clinical data

A range of clinical data was collected during the 6S trial. The following data were retrieved from the study database for use in the present study: patient demographics (age and gender), comorbidities (major cardiovascular disease, diabetes, hypertension), the number of patients with shock and acute kidney injury, disease severity scores (Simplified Acute Physiology Score (SAPS) II and Sepsis-related Organ Failure Assessment (SOFA) without the cerebral component), and most extreme values of relevant biochemistry and physiology parameters within 24 h prior to inclusion (creatinine, bilirubin, platelet count, white blood cell count, partial pressure of oxygen in arterial blood (PaO2), fraction of inspired oxygen (FiO2), mechanical ventilation, mean arterial pressure, heart rate and vasopressor treatment). Days alive during follow-up censored at 90 days were retrieved as follow-up data.

Measurements

Plasma blood levels of ADMA, arginine, and SDMA were measured in duplicate by a commercially available enzyme-linked immunosorbent assay (ELISA) (EA207/92 and EA203/96, DLD Diagnostica, Hamburg, Germany). The measurements were conducted in baseline blood samples only. Outliers within the standards were excluded to draw proper standard curves. Standards with a concentration of 0 were set to 0.01 to allow for logarithmic transformation. The 75% quartile was set as cut-off between high and low levels of methylarginines. The lower limits of detection were: ADMA: 0.03 μmol/L; arginine: 6 μmol/L; and SDMA 0.03 μmol/L. Highest detection ranges of the standard curves were: ADMA: 3.0 μmol/L; arginine: 300 μmol/L; and SDMA 3.0 μmol/L.

Handling of the arginine/ADMA ratio

To differentiate between ratios consisting of high arginine/high ADMA and low arginine/low ADMA, we categorized arginine and ADMA by separating each variable into groups below or above the 50% percentile and analyzed the arginine/ADMA ratio in four groups: high arginine/low ADMA, high arginine/high ADMA, low arginine/high ADMA, and low arginine/low ADMA.

Statistical analysis

Categorical variables are presented as numbers (%) and continuous variables are presented as medians (interquartile range (IQR)) or medians (IQR, and range), and compared by chi-square test and Man–Whitney U test, respectively. The association between 90-day mortality and methylarginines was modeled with Cox regression and presented as hazard ratios (HR) with 95% confidence intervals (CI). In multivariate Cox regression analyses methylarginines were adjusted for gender, age ≥65 years, previous major cardiovascular disease, diabetes, hypertension, respiratory failure (PaO2/FiO2 <200 mm Hg and mechanical ventilation), vasopressor treatment, highest quartile of creatinine and bilirubin, and lowest quartile of platelet count. The baseline biochemistry and physiological parameters were chosen as adjustment variables before initiation of the present study, because these parameters are commonly used to assess the degree of organ dysfunction in ICU patients. The comorbidities major cardiovascular disease, diabetes, and hypertension were chosen because these are known to be associated with increased concentrations of ADMA (22–24). The effects of the intervention (hydroxyethyl starch vs. Ringer acetate) in the 6S trial and storage time of blood samples were investigated in sensitivity analyses.

ADMA, SDMA, and/or arginine measurements out of the assay range were replaced by either the lower or upper limit of detection depending on in which direction the measurement was out of range. In the regression analyses missing values in one or more of the adjusting variables or missing methylarginine concentrations were estimated by multiple imputation using the remaining variables as predictors (25). Other analyses were based upon patients with measured methylarginine concentrations only.

The mortality stratified by methylarginine levels are presented in Kaplan–Meier plots. The assumption of proportional hazards was checked using log–log plots and Schoenfeld residuals.

Statistical significance was set at P <0.05 and all analyses were done using R, version 3.2.0 (The R-project, http://www.r-project.org/) (26).

RESULTS

Patients and outcome

From a total of 267 included patients, 223 patients had a plasma blood sample available for analysis; thus, 44 patients missed measured methylarginine concentrations.

The 267 patients had a median age of 66 years (IQR, 57–74 years). A majority of the patients presented with septic shock (228, 85.4%) and received mechanical ventilation (203, 76.0%) and/or vasopressor treatment (160, 59.9%). Table 1 summarizes baseline characteristics.

T1-5
Table 1:
Baseline characteristics

A total of 141/267 (52.8%) patients died during the 90-day follow-up. As compared with survivors, patients who died were significantly older (65 vs. 68 years, P = 0.001), had higher SAPS II and modified SOFA score (48 vs. 56, P <0.001 and 7 vs. 8, P = 0.004, respectively), and more frequently presented with shock (101 vs. 127, P = 0.034). Survivors and non-survivors were similar with regard to baseline biochemistry and physiology, and in the prevalence of previous major cardiovascular disease, diabetes, and hypertension (Table 1). Twenty-five patients had a missing value in one or more of the baseline variables used as adjusting variables in the multivariate analyses.

Values of methylarginines

The median (IQR, range) measured concentration of the methylarginines at the time of inclusion was: ADMA: 0.41 μmol/L (0.28–0.56 μmol/L, range: 0.086–2.18 μmol/L); SDMA: 0.89 μmol/L (0.61–1.42 μmol/L, range: 0.25–2.67 μmol/L); arginine: 35.7 μmol/L (25.7–49.5 μmol/L, range: 6.0–141.5 μmol/L); and arginine/ADMA ratio 91 (65–133, range: 4–373). A total of 10 patients had an ADMA, SDMA, and/or arginine measurement out of the assay range. Lactate levels (P <0.01), vasopressor treatment (P = 0.047), and use of mechanical ventilation (P = 0.026) independently predicted the ADMA concentration at baseline. The level of ADMA was independent of age (P = 0.83), gender (P = 0.83), creatinine (P = 0.98), bilirubin (P = 0.12), and platelet count (P = 0.99).

ADMA and mortality within 90 days

The measured ADMA plasma concentration (median and IQR) in survivors was 0.40 μmol/L (0.27–0.52 μmol/L) versus 0.41 μmol/L (0.30–0.61 μmol/L) in non-survivors, P = 0.11. The risk of death within 90 days was significantly increased in patients with ADMA in the highest quartile versus the three lower quartiles and independently associated with this outcome (hazard ratio 1.54; 95% CI, 1.00–2.38; P = 0.046) (Table 2 and Fig. 1).

T2-5
Table 2:
Asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), arginine/ADMA ratio, and 90 day-mortality—Cox regression, n = 267
F1-5
Fig. 1:
90-day survival stratified by asymmetric dimethylarginine (ADMA)—Kaplan–Meier curve.Q1–3 indicate patients with plasma ADMA level in the three lower quartiles; Q4, patients with plasma ADMA level in the upper quartile. The dashed lines indicate the time intervals were the effect of ADMA is further investigated (see Table 3 for results). ADMA indicates asymmetric dimethylarginine.

SDMA and mortality within 90 days

The measured SDMA plasma concentration (median and IQR) in survivors was 0.82 μmol/L (0.56–1.17 μmol/L) versus 0.99 μmol/L (0.67–1.57 μmol/L) in non-survivors, P = 0.017. As for ADMA the risk of death within 90 days was significantly increased in patients with a SDMA in the highest quartile versus the three lower quartiles and independently associated with mortality (hazard ratio 1.78; 95% CI, 1.14–2.72; P = 0.011) (Table 2 and Fig. 2).

F2-5
Fig. 2:
90-day survival stratified by symmetric dimethylarginine (SDMA)—Kaplan–Meier curve.Q1–3 indicate patients with plasma SDMA level in the three lower quartiles; Q4, patients with plasma SDMA level in the upper quartile.

Arginine/ADMA ratio and mortality within 90 days

The measured arginine/ADMA ratio (median and IQR) in survivors was 102 (65–136) versus 87 (65–126) in non-survivors, P = 0.24. Arginine/ADMA ratio was not associated with 90-day mortality in neither univariate nor multivariate analyses (Table 2).

Effect of ADMA on mortality across time intervals

The Kaplan–Meier curve and proportional hazards test revealed a time-varying effect of ADMA on mortality (Fig. 1). An ADMA concentration in the upper quartile versus the three lower quartiles was significantly associated with increased mortality within the first 7 days (Table 3). For patients alive at day 7 and day 21 the risks of dying days 8 to 21 and 22 to 90 were not associated with the baseline concentration of ADMA (Table 3).

T3-5
Table 3:
Effect of asymmetric dimethylarginine (ADMA) on mortality across time intervals—Cox regression, n = 276

Sensitivity analysis

We found no effect of the intervention in the 6S trial or the storage time of blood samples on the association between the methylarginines and mortality, neither for analyses of mortality within 90 days nor mortality across time intervals (see Tables 4a, 4b, 4c, and 4d in Supplemental Digital Content 1, at https://links.lww.com/SHK/A399).

DISCUSSION

We investigated if ADMA, SDMA, and arginine/ADMA ratio were associated with increased risk of mortality within 90 days in a large cohort of patients with severe sepsis or septic shock. We found that high levels of plasma ADMA and SDMA were associated with increased mortality independently of other factors known to be associated with mortality. The arginine/ADMA ratio was not associated with mortality. Baseline plasma ADMA were highly associated with death within the first week, but less so in the following time periods. These results could imply that ADMA is involved in the acute circulatory collapse seen in severe sepsis and septic shock rather than more subtle organ damage that could influence the risk of death on a long term. On ICU admission the level of ADMA was higher in patients with signs of hypoperfusion (elevated lactate) and shock (need for vasopressor treatment), whereas there was no direct association to other manifestations of organ failures, except mechanical ventilation that may also be an indication of insufficient circulation. This supports the hypothesis that ADMA is closely associated with the development of insufficient micro- and macrocirculation.

There are conflicting data on the association between ADMA, SDMA, and mortality (7, 10, 11, 13, 14, 27, 28). Most studies have included small numbers of septic patients (10, 11, 13) except two studies that included 255 and 247 patients, respectively (7, 14); however in these studies the population was mixed ICU patients and of these severe sepsis and septic shock comprised only 64% and 65%, respectively. In one study the researchers found that baseline SDMA, but not ADMA, was associated with ICU mortality (11); however, the cut-off levels used for SDMA and ADMA are unclear.

We also found a significant difference in age of survivors versus non-survivors. This raises the question whether levels of methylated arginines are related to severity of illness or to age. The ADMA level has been shown to be positively correlated with age in healthy individuals (29). To account for the age difference between groups all our multivariate analyses were adjusted for age.

The association between the methylarginines and mortality could be attributed to inhibition of the NO system. The pathophysiology of SDMA is less investigated than ADMA, but the association between SDMA and mortality could be explained by SDMA-induced inhibition of arginine uptake leading to decreased NO production. An in vitro study of cultured endothelial cells found that SDMA dose dependently inhibited NO synthesis (30).

The role of NO in sepsis is still not fully elucidated. In a macrocirculatory perspective high levels of NO contribute to hypotension through vasodilation (2), making pharmacological lowering of NO a potential intervention. However in a randomized trial of 797 patients with septic shock increased mortality was observed with unselective pharmacological inhibition of NO synthases (31).

In a microcirculatory perspective, endothelial cells produce NO to regulate blood flow in microvessels (32). Microcirculation is decreased in patients with severe sepsis and septic shock and even more pronounced in patients that die (33, 34). Intravenous infusion of nitroglycerin, causing levels of NO to increase, seems to improve microcirculatory organ perfusion (3), which could explain why treatment with NO donors in septic rats and mice results in decreased mortality (35). However, a more recent study of nitroglycerin administration in humans indicated increased mortality, though without reaching statistical significance (36).

A review from 2010 states that currently there is no research to support a benefit in microcirculation by increasing mean arterial pressure neither from vasopressors or inotropes (37). Identifying ways to balance the effect of NO on macro and microcirculation could be a key in ensuring a sufficient systemic blood pressure AND sufficient microcirculation to maintain organ perfusion in septic patients. Unavoidably, such balance would also have to take other NO functions in sepsis into account, i.e., microbial clearance. The methylated arginines could be interesting pharmacological targets for achieving such balance, though effects of the methylarginines besides that of the NO system have not yet been elucidated. As an example the ADMA level could be altered through manipulation of the enzymes that metabolizes ADMA, dimethylarginine dimethylaminohydrolase 1 and 2 (DDAH1 and 2). Manipulation of DDAH1 and 2 has shown promising results in recent animal studies (38, 39).

STRENGTHS AND LIMITATIONS

We found a lower baseline ADMA concentration than reported in other studies of septic patients (7, 9–12). This might be due to the use of citrate plasma as opposed to EDTA plasma or serum. The use of citrate plasma dilutes the samples slightly (40), which might cause our values to be shifted. A systematic shift in concentration will not interfere with the association with mortality; however, it is a limitation that the concentrations are not directly comparable to previous studies. Further, we only analyzed methylarginines in baseline blood samples, and not as continuous measurements on consecutive days. Our results are therefore limited to account for baseline methylarginine measurements. To further support the hypothesis that the effect of methylarginines on mortality was mediated through NO we could have included plasma measurements of stable NO metabolites as nitrate and nitrite. Unfortunately, the amount of plasma in the bio bank restricted the number of laboratory analyses. Finally, 44 patients were missed during the blood sample collection period. To account for the potential bias of excluding these patients they were assigned methylarginine concentrations by multiple imputation and thereby included in the regression analyses.

To the best of our knowledge, the present study is the largest so far to investigate the association between ADMA, SDMA, and mortality in patients with severe sepsis or septic shock, but since this study design is observational, the associations are only suggestive. Sensitivity analyses of 6S trial intervention and storage time showed no effects on the associations between methylarginines and mortality, which is strength of the present study.

Future perspectives

The hypothesis that the effect of methylarginines on mortality is due to inhibition of the NO system needs to be further elucidated. The central role of NO in the pathophysiology of sepsis makes it likely that the methylarginines work through this pathway. However, this observational study does not investigate the causal connection and therefore the link between ADMA, SDMA, and NO production remains speculative. Future research should investigate the hypothesis through observational and experimental studies of methylarginines, NO concentrations, organ failures, microcirculation, and mortality. If the hypothesis is strengthened, lowering ADMA and SDMA may represent a novel therapeutic target in patients with severe sepsis or septic shock.

CONCLUSIONS

High levels of ADMA and SDMA in plasma are associated with increased 90-day mortality in patients with severe sepsis or septic shock, independently of other factors associated with death in this population. One could hypothesize a pathway of actions from methylarginines to inhibition of the NO system, leading to failed microcirculation, organ failure, and ultimately death, though this observational study does not investigate the link of actions. If the hypothesis is strengthened through further studies, lowering ADMA and SDMA may represent a novel therapeutic target in patients with severe sepsis or septic shock.

Acknowledgments

The authors thank 6S trial investigators and staff at the ICUs at Rigshospitalet, Herlev, Hvidovre and Bispebjerg Hospitals.

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Keywords:

Critical illness; endothelium; intensive care; nitric oxide; nitric oxide synthase; 6S trial; Scandinavian Starch for Severe Sepsis and Septic Shock trial; ADMA; asymmetric dimethylarginine; CI; confidence interval; CVD; cardiovascular disease; DDAH; dimethylarginine dimethylaminohydrolase; ELISA; enzyme-linked immunosorbent assay; FiO2; fraction of inspired oxygen; HR; hazard ratio; ICU; intensive care unit; IQR; interquartile range; NO; nitric oxide; PaO2; partial pressure of oxygen in arterial blood; Q1–3; the three lower quartiles; Q4; upper quartile; SAPS; simplified acute physiology score; SDMA; symmetric dimethylarginine; SOFA; sepsis-related organ failure assessment score

Supplemental Digital Content

© 2016 by the Shock Society