4·1 Demographic Characteristics: Comparison with global patient profile
In this study, the mean age of the sepsis group was 44·3 (± 12·5) years when compared to the mean age of 60 years in the previous studies reported in the literature 7,10. Male had more predispositions to sepsis, which is consistent with the earlier studies, which has been ascribed to variety of factors including differing inherent predisposition to infection and hormonal regulation of cytokine responses (11–13). The age and the percentage of male were matched in both the groups of survivor and non-survivors, thus avoiding potential confounding factors. The source of infection in majority of the cases was pulmonary (64%) followed by CNS infections (9%). 95% of patients had radiological evidence of infection whereas microbiological cultures was positive in 33% of the recruited cases with a blood culture positivity rate of 9%.
Mean SOFA scores were 5·2 (± 2·4) and 14·9 (± 1·9) in survivor and non-survivor groups, respectively. APACHE had a perfect linear correlation (r2=·92) with SOFA as a predictor of outcome of sepsis. Sepsis survivors had a mean APACHE of 8·1 ± 3·57 (8% predicted mortality) while it was 34·5 ± 2·82 for non-survivors (73% mortality).
4.2 Metabolomics profile of sepsis and healthy controls
Metabolic changes in sepsis are highly complex and vary between individuals14. In the present study, four amino acids namely, phenylalanine, histidine, valine and isoleucine were found to differentiate between sepsis from healthy controls. Phenylalanine increased to a significant level in sepsis patients when compared to healthy controls. Muscle breakdown coupled with reduced conversion of phenylalanine to tyrosine in liver in sepsis contributes to higher levels of phenylalanine. This observation is consistent with the finding of previous studies15,16. Phenylalanine is also markedly increased in HIV17, trauma, burns, malignancy18 and SIRS19. These reflect that phenylalanine elevation is a sign of immune activation rather than infection20. Phenylalanine is converted into fumarate, which is then shunted into Kreb’s cycle for energy production under severe anorexic condition. Increased level of phenylalanine was associated with poor outcome in sepsis patients21.
Histidine concentration was lower in sepsis group when compared to controls. The trend of decreased histidine in sepsis is consistent with the results of the study by Jauria et al.22. Histidine has antioxidant and anti-inflammatory activity and low plasma histidine is associated with oxidative stress, inflammation and mortality in CKD (Chronic Kidney Disease) patient23. Histidine rich glycoprotein, a key regulator in immunity and vascular biology24 is decreased in sepsis and is being exploited as a potential prognostic marker in sepsis25.Concentration of branched chain amino acids (BCAA) namely isoleucine and valine were significantly higher in sepsis patients than in healthy controls, which agrees with the study by Mickwickz et al.26,16.Increased oxidation of BCAA by skeletal muscle coupled with conversion into glutamine decreases the BCAA levels in sepsis 27.
Levels of choline was seen to be decreased significantly in sepsis patients compared to healthy controls and it increased in sepsis non-survivors compared to survivors. Choline is an endogenous molecule that has antioxidant, anti-apoptotic properties and attenuates the pro inflammatory cytokines (IL-6,IL-1ß,TNF-ɑ) and protects the cell membrane from oxidant injury during various pathological conditions including sepsis28.Choline supplementation in sepsis has improved outcomes in mammalian model29. Among the metabolites, isoleucine has the highest diagnostic performance with AUC of 0·898 (0·781-0·973) followed by phenylalanine 0·858 (0·683- 0·976) in predicting sepsis (Fig. 4).These results are in concordance with the study of Zhicheng et al., where phenylalanine had a AUC of 0·84 in differentiating sepsis from control16.
4.3 Metabolomics profile of Sepsis survivor from non- survivors
In the present study, the concentration of phosphocreatine and creatine was significantly higher among sepsis non-survivors. Phosphocreatine-creatine system acts as a reservoir of energy which is available instantaneously .The ATP (Adenosine Tri Phosphate) combines with creatine to form high energy Phosphocreatine catalysed by Creatine kinase. In hypermetabolic state, the reaction reverses to yield ATP and creatine. This system is of particular importance in skeletal muscle and cardiac myocytes for rapid replenishment of ATP .Sepsis profoundly affects mitochondrial functions resulting in instability of membrane potential. There is autophagy induction, suppression of biogenesis inciting myocyte death with a leak of cytoplasmic creatine and phosphocreatine into serum30. Multiple studies have confirmed the loss of phosphocreatine in muscle fibres of sepsis patient with increased mortality 31,32.
Further, the serum levels of tyrosine, histidine and phenylalanine were found to be significantly increased in the sepsis non-survivor group. The AUC (Fig. 5) is highest for phosphocreatine (0·89) followed by creatine (0·83), choline (0·76) and tyrosine (0·75) for differentiating sepsis non-survivors from survivors. Elevation of phenylalanine in non-survivors has been consistently observed in many studies in the past33,16. Level of histidine and tyrosine in non-survivors are lower in non-survivors than survivors in the above studies. In the present study, the higher levels of these metabolites in non-survivors may be due to the inability to metabolise the amino acids reflecting the metabolic shutdown in patients who did not survive. A similar trend was seen with all the metabolites listed in Table 3 including choline. This is similar to the observation by Hirose et al33, i.e., the trends of metabolites in serum of an individual with sepsis varies greatly with time and severity of sepsis.