Higher platelet cytochrome oxidase specific activity in surviving than in non-surviving septic patients

Introduction In a previous study with 96 septic patients, we found that circulating platelets in 6-months surviving septic patients showed higher activity and quantity of cytochrome c oxidase (COX) normalized by citrate synthase (CS) activity at moment of severe sepsis diagnosis than non-surviving septic patients. The objective of this study was to estimate whether COX specific activity during the first week predicts 1-month sepsis survival in a larger cohort of patients. Methods Using a prospective, multicenter, observational study carried out in six Spanish intensive care units with 198 severe septic patients, we determined COX activity per proteins (COXact/Prot) in circulating platelets at day 1, 4 and 8 of the severe sepsis diagnosis. Endpoints were 1-month and 6-months mortality. Results Survivor patients (n = 130) showed higher COXact/Prot (P < 0.001) than non-survivors (n = 68) at day 1, 4 and 8 of severe sepsis diagnosis. More than a half of the 6-months survivor patients showed an increase in their COXact/Prot from day 1 to 8. However, most of the 1-month non-survivors exhibited a decrease in their COXact/Prot from day 1 to 8. Multiple logistic regression analyses showed that of platelet COXact/Prot > 0.30 mOD/min/mg at day 1 (P = 0.002), 4 (P = 0.006) and 8 (P = 0.02) was associated independently with 1-month mortality. Area under the curve of COXact/Prot at day 1, 4 and 8 to predict 30-day survival were 0.70 (95% CI = 0.63-0.76; P < 0.001), 0.71 (95% CI = 0.64-0.77; P < 0.001) and 0.71 (95% CI = 0.64-0.78; P < 0.001), respectively. Conclusions The new findings of our study, to our knowledge the largest series reporting data about mitochondrial function during follow-up in septic patients, were that septic patients that survive 1-month have a higher platelet cytochrome oxidase activity at moment of sepsis diagnosis and during the first week than non-survivors, and that platelet cytochrome oxidase activity at moment of sepsis diagnosis and during the first week could be used as biomarker to predict the clinical outcome in septic patients.


Introduction
Severe sepsis is a fatal condition associated with organ failure [1]. This organ dysfunction is related to tissue hypoxia due to cellular inability to use oxygen [2]. Most cellular oxygen is consumed by cytochrome oxidase (COX) in the electron transport chain (ETC) of the mitochondrial oxidative phosphorylation system (OXPHOS). Thus, in a previous study of 96 patients with severe sepsis, we found that 6-month survivors had higher platelet COX activity normalized by citrate synthase (CS) activity at the time of diagnosis than non-survivors [3]. Because the determination of platelet COX activity to assess ETC is a rapid, easy and not very invasive protocol, this parameter could be used as a biomarker of sepsis mortality. Moreover, patients with low COX activity could benefit from drugs that improve ETC [3].
However, despite the inclusion of 96 individuals as the largest series reported to date in which COX activity has been determined, it is still a relatively small cohort of patients. Moreover, although 6-month survival allows the analysis of long-term outcomes, this period is not the most-used end point [4] because confounder factors unrelated to the initial sepsis event would affect the results. In addition, COX activity was only determined in platelets obtained at the time of diagnosis and potential changes over time were not evaluated. To address these problems, we have now determined COX-specific activity in a larger population of septic patients, analyzed 1-month survival and obtained blood samples not only within the first 24 hours after the diagnosis of severe sepsis but also on days 4 and 8. Inclusion criteria were the diagnosis of severe sepsis according to the International Sepsis Definitions Conference criteria [5]. Exclusion criteria were: age <18 years, pregnancy, lactation, platelet transfusion, HIV, white blood cell count <1,000 cells/μl, solid or hematological tumor, or immunosuppressive, steroid or radiation therapy.

Endpoints
We assessed survival at 1 month and 6 months as the endpoints.

Biochemical determination in platelets
Blood samples were collected from patients on day 1, 4 and 8 of diagnosis of severe sepsis for the determination of COX. Day 1 was considered as the first day that severe sepsis was diagnosed (baseline values). Day 4 was considered as the day after 72 hours had elapsed, and day 8 as the day after 7 days had elapsed since the diagnosis of severe sepsis.
Platelets were obtained according to previously described protocols [8]. We measured the COX activity (COXact) by using Mitoprofile® Human Complex IV Activity and Quantity from Mitosciences (Invitrogen) according to the manufacturer's instructions. This kit immunocaptures complex IV and activity is determined colorimetrically by following the oxidation of reduced cytochrome c as an absorbance decrease at 550 nm. Protein levels were assayed following previously described protocols [9], and were expressed in mg. A NovoStar MBG Labtech microplate instrument was used for COXact and protein determinations. COX activity corrected by proteins (COXact/Prot) was expressed as milli-optical density per minute per mg of proteins (mOD/min/mg) multiplied by 100.

Statistical analysis
Categorical variables are reported as frequencies and percentages and continuous variables are reported as medians and interquartile ranges. Comparisons of categorical variables between survivors and non-survivors were carried out with the chi-square test. Normality assumption was tested with the Komogorov-Smirnov test. Comparisons of continuous variables between survivors and non-survivors were carried out using the Wilcoxon-Mann-Whitney test. We used the Kruskal-Wallis test to compare levels of COXact/Prot between patients grouped by survival lower than 1 month, between 1 and 6 months and more than 6 month; post-hoc comparison of pairs was carried out with the Mann-Whitney test.
Multiple logistic regression analysis was used to determine the independent contribution of platelet COXact/ Prot >0.30 mOD/min/mg, lactic acid and SOFA score at day 1, 4 and 8 to the prediction of 1-and 6-month mortality. Odds ratios (ORs) and 95% confidence intervals were calculated as measures of the association. Receiver operating characteristic (ROC) analyses were carried out to determine the goodness-of-fit of the COXact/Prot at day 1, 4 and 8 to predict 1-and 6-month mortality. Analysis of survival with the Kaplan-Meier method curve and comparisons by log-rank test were carried out using COXact/Prot lower/higher than 0.30 mOD/min/mg at day 1, 4 and 8 as the independent variable and survival at 1 and 6 months as the dependent variable. A P-value less than 0.05 was considered statistically significant. Statistical analyses were performed with SPSS v. 17.0 (SPSS Inc., Chicago, IL, USA) and Med Calc v. 10.1.3.0 (Mariakerke, Belgium).

Results
In a previous study of 96 sepsis patients, we found that 6-month survivors had a higher platelet COXact/CSact ratio at the time of diagnosis than non-survivors [3]. This result suggested that OXPHOS function could be used as a biormarker of sepsis mortality. As we found a tight correlation between COXact/CSact and COXact/ Prot (Spearman rho = 0.77; P <0.001) in these patients, to confirm the association between OXPHOS function and sepsis survival and simplify clinical protocols, we have now studied COXact/Prot in a larger number of individuals (198 sepsis patients). We have observed that COXact/Prot at the time of sepsis diagnosis was significantly higher in 6-month survivors than in non-survivors among patients with sepsis (Table 1).
Although 6-month survival allows the analysis of longterm outcomes [4], confounder factors unrelated to the initial sepsis event could affect the results. Therefore, we have repeated these analyses for 1-month survival because this is a more frequently used parameter in sepsis patients. We found that 1-month survivors of sepsis (n = 130) had lower lactic acid (P <0.001), INR (P = 0.03), aPTT (P = 0.01), APACHE-II score (P <0.001) and SOFA score (P <0.001), and higher platelet count (P = 0.01) at the time of diagnosis of severe sepsis than non-survivors (n = 68). There were no differences in the other recorded variables between survivors and non-survivors of sepsis (Table 2) except in COXact/Prot. Severely septic patients who survived 1 month had significantly higher COXact/ Prot at the time of sepsis diagnosis than non-survivors (Table 1). Of 130 patients who survived 1 month, 21 died before 6 months and their COXact/Prot values were not significantly different from those of 109 patients who survived for longer than 6 months (Table 3).
In addition, we found that 1-month survivor patients had statistically significantly higher COXact/Prot at days 4 and 8 than non-survivor ones (Table 1).
We found statistically significant differences comparing the proportions of patients with increasing COXact/ Prot from days 1 to 8 between patients surviving longer than 6-months, 1 to 6 months, and less than 1 month (55.0% (60/109), 38.1% (8/21), 29.7% (11/37), respectively; P = 0.02). Comparing pairs of groups, we found an increased proportion of patients with increasing COXact/Prot from days 1 to 8 among patients surviving longer than 6 months compared with those surviving less than 1 month (P = 0.01), and we did not find statistically significant differences between the 6-month survival group and the group that survived 1 month but not 6 months (P = 0.23), or between patients who survived 1-month but not 6 months and those who did not survive 1 month (P = 0.57).
Supporting the relationship between COXact/Prot or its evolution with sepsis mortality, COXact/Prot at day 1 of 31 patients who did not survive a week (0.06 (0.05-0-18)) was significantly lower (P = 0.03) than that of 37 patients that survived more than a week but less than 1 month (0.14 (0.07-0.32)).

Discussion
Platelets contain small numbers of fully functional mitochondria. Their abundance in blood as well as the relative ease to obtain them with minimally invasive protocols has made platelets an attractive source of human mitochondria. Platelet OXPHOS function has frequently been used to study OXPHOS dysfunction as a biomarker in human disease [10]. COXact/Prot can be used as a surrogate of OXPHOS function. Because COX subunits are lower in Table 3 Platelet cytochrome c oxidase activity per proteins (COXact/Prot) of 6-month survivors, 1-month but non-6-month survivors and 1-month non-survivors    platelets than in other blood cells, the detection of a pathology associated with decreased COXact/Prot will be most readily revealed in platelets [11]. Some commercial kits are able to determine COXact/Prot using a microplate reader, an instrument frequently found in the clinical chemistry laboratories from most of the hospitals. Therefore, to verify our previous observation that platelet OXPHOS function can be used as a biomarker of sepsis mortality [3] and simplify clinical protocols, we have now studied the COXact/Prot in a larger number of sepsis patients using a fast and easy test.
We have confirmed that platelet COXact/Prot at the time of diagnosis of sepsis is significantly higher in 1-month survivors than in non-survivors. It would be possible that some individuals have a lower basal OXPHOS capacity and, for them, the sepsis insult may represent an unbearable stress. In fact, we have already observed that there are OXPHOS differences between transmitochondrial cell lines with distinct mitochondrial DNA (mtDNA) genetic backgrounds (haplogroups) obtained from normal individuals [12] and that patients from mtDNA haplogroup JT have a higher platelet OXPHOS function and 1-month survival [13,14].
It has been reported that OXPHOS inhibition can affect the platelet prothrombotic function [15,16]. However, on the contrary, activation of coagulation frequently occurs in sepsis [17]. Therefore, platelet OXPHOS dysfunction observed in sepsis patients probably is not enough to alter platelet function and increase mortality. However, as previously discussed, sepsis provokes mitochondrial dysfunction in different tissues from different species [3]. Thus, the lower platelet COXact/Prot in non-surviving patients probably mirrors a general situation in other tissues and organs and this general lower OXPHOS function would be one of the reasons for death.
It is possible that independently of their basal COXact/ Prot, non-surviving patients have an impaired response to the septic insult. COXact/Prot changes along the time. Thus, a higher percentage of 6-month survivors have increased COXact/Prot along the first week compared to patients who do not survive 1 month. An interesting aspect is that the patients who died after 1 month but within 6 months of the sepsis event had an initial COXact/Prot similar to that of those who survived more than 6 months; however, the percentage of patients with increased COXact/Prot along the first week was lower among patients who survived 1 month but died within 6 months of the sepsis compared to those who survived 6 months. If this was the case, early determinations of platelet COXact/Prot along the first week might help to predict the survival probabilities and, for those patients with higher risk of death, the administration of different compounds that increase COXact/Prot may improve their survival chances [18][19][20].

Conclusions
Septic patients who survive 1 month have higher platelet cytochrome oxidase-specific activity at the time of diagnosis than non survivors. A decrease in the platelet cytochrome oxidase specific activity along the first week after the sepsis diagnosis is a factor for poor prognosis. Therefore, platelet cytochrome oxidase-specific activity can be used as a biomarker of outcome in septic patients.

Key messages
Septic patients who survive 1 month have a higher platelet cytochrome oxidase-specific activity at the time of diagnosis than non survivors. A decrease in the platelet cytochrome oxidase-specific activity along the first week after the sepsis diagnosis a factor for poor prognosis. Platelet cytochrome oxidase-specific activity can be used as a biomarker of outcome in septic patients.