Arginine metabolism is markedly impaired in polymicrobial infected mice

Since sepsis has been identified as an arginine deficiency state, there is an ongoing controversial debate about arginine supplementation in the treatment of patients with sepsis. Little is known, however, about the different catabolic and anabolic downstream and upstream products or byproducts in arginine metabolism in the course of sepsis. 
 
Using a clinical chemistry and mass spectrometric approach we analyzed metabolites related to arginine metabolism from plasma samples of polymicrobial infected mice at 6 hours and 24 hours post sepsis induction in comparison with healthy animals. Following ethical approval by the responsible animal welfare committee (Thueringer Landesamt fuer Lebensmittelsicherheit und Verbraucherschutz; TVA 02-10/10), animals (male C57BL/6N mice) were randomized into three groups: healthy controls without any intervention, mice 6 hours post sepsis induction, and mice 24 hours post sepsis induction. Peritonitis in mice was induced as described in more detail for a rat model [1] by injection of stool suspension into the right lower quadrant of the abdomen with a 21-gauge cannula. Blood samples were drawn using direct needle puncture of the right ventricle. EDTA-anticoagulated blood was immediately centrifuged, and plasma was collected and snap-frozen in liquid nitrogen and stored at -80°C until use. Mass spectrometric analysis was performed using the API4000™ LC/MS/MS system (AB SCIEX, Foster City, CA, USA) equipped with an electrospray ionization source. All animals received a standard pellet rodent diet (ssniff, Soest, Germany) and water ad libitum. The pellets contain 33% proteins, 58% carbohydrates and 9% fats. In our translational approach we did not focus on an additional nutritional support. To avoid the influence of sepsis-induced anorexia, however, we limited the observational period to 24 hours. 
 
Our data clearly show that the arginine plasma concentration is substantially decreased in sepsis, which is not caused by lower availability of amino acids glutamine or citrulline (Table ​(Table1).1). The data support the concept that the arginine catabolic pathway outweighs the anabolic pathway, producing for example proline. Increased arginase activity synthesizes ornithine and urea, the latter significantly enhanced in the late phase during sepsis. The increased urea plasma concentration might reflect not only enhanced arginase activity but also a developing state of acute renal injury. Furthermore, ornithine might be catalyzed rapidly by ornithine de carboxy lase to synthesize putrescine, which is the com mitted reaction in the synthesis of polyamines. Further explanation for the observed decrease in concentration of ornithine, however, cannot be given from our present data and remains unclear. 
 
 
 
Table 1 
 
Metabolites from plasma samples of healthy and polymicrobial infected mice 
 
 
 
Interestingly, whilst the putrescine plasma concentration was significantly increased, the concentration of spermidine decreased after 24 hours. The increasing putrescine pool might indicate a restricted activity of spermidine synthase resulting in an insufficient converted concentration of spermidine. This finding might be interesting because polyamines exert pleiotropic biological activities, including modulation of cell signaling, cell growth as well as cell apoptosis [2]. Furthermore, it is important to note that administration of polyamines can function as either protective or harmful in lethal experimental sepsis [3]. 
 
Proline plays various roles in cell metabolism and physiology; for example, in cell growth, wound healing, scavenging oxidants as well as in polyamine and protein syntheses [4]. In our model, the plasma concentration of proline was significantly decreased during sepsis compared with healthy controls. One could speculate that, beside the reduced arginine availability, increasing hydroxy lation of proline to hydroxyproline - an essential step to provide collagen for tissue repair, remodeling or wound healing - contributes to the substantially reducedproline plasma concentration during sepsis even in the early phase of the disease [5]. Consequently, targeting arginine deficiency might be essential in patients withsepsis. Owing to the complexity of arginine metabolism that underlies multiple biochemical and physiologic processes (Figure ​(Figure1),1), however, the potential benefits and risks of arginine supplementation have to be carefully evaluated in further studies by a multiparameter approach measuring additional pathway-related products such as polyamines and ornithine, but also the activity of enzymes. 
 
 
 
Figure 1 
 
Scheme of the arginine metabolism and pathway-related products. Simplified overview presenting the L-arginine pathway, including major metabolites and their related pathways and functions. NO, nitric oxide; NOS, nitric oxide synthase; ODC, ornithine decarboxylase; ...

Since sepsis has been identifi ed as an arginine defi ciency state, there is an ongoing controversial debate about arginine supplementation in the treatment of patients with sepsis. Little is known, however, about the diff erent cata bolic and anabolic downstream and upstream products or byproducts in arginine metabolism in the course of sepsis.
Using a clinical chemistry and mass spectrometric approach we analyzed metabolites related to arginine metabolism from plasma samples of polymicrobial infected mice at 6 hours and 24 hours post sepsis induction in comparison with healthy animals. Following ethical approval by the responsible animal welfare committee (Th ueringer Landesamt fuer Lebensmittelsicher heit und Verbraucherschutz; TVA 02-10/10), animals (male C57BL/6N mice) were randomized into three groups: healthy controls without any intervention, mice 6 hours post sepsis induction, and mice 24 hours post sepsis induction. Peritonitis in mice was induced as described in more detail for a rat model [1] by injection of stool suspension into the right lower quadrant of the abdomen with a 21-gauge cannula. Blood samples were drawn using direct needle puncture of the right ventricle. EDTA-anticoagulated blood was immediately centri fuged, and plasma was collected and snap-frozen in liquid nitrogen and stored at -80°C until use. Mass spectrometric analysis was performed using the API4000™ LC/MS/MS system (AB SCIEX, Foster City, CA, USA) equipped with an electrospray ionization source. All animals received a standard pellet rodent diet (ssniff , Soest, Germany) and water ad libitum. Th e pellets contain 33% proteins, 58% carbohydrates and 9% fats. In our translational approach we did not focus on an additional nutritional support. To avoid the infl uence of sepsis-induced anorexia, however, we limited the observational period to 24 hours.
Our data clearly show that the arginine plasma concentration is substantially decreased in sepsis, which is not caused by lower availability of amino acids glutamine or citrulline (Table 1). Th e data support the concept that the arginine catabolic pathway outweighs the anabolic pathway, producing for example proline. Increased arginase activity synthesizes ornithine and urea, the latter signifi cantly enhanced in the late phase during sepsis. Th e increased urea plasma concentration might refl ect not only enhanced arginase activity but also a developing state of acute renal injury. Furthermore, ornithine might be catalyzed rapidly by ornithine de carboxy lase to synthesize putrescine, which is the com mitted reaction in the synthesis of polyamines. Further explanation for the observed decrease in concentration of ornithine, however, cannot be given from our present data and remains unclear.
Interestingly, whilst the putrescine plasma concentration was signifi cantly increased, the concentration of spermidine decreased after 24 hours. Th e increasing putrescine pool might indicate a restricted activity of spermidine synthase resulting in an insuffi cient converted concentration of spermidine. Th is fi nding might be interest ing because polyamines exert pleiotropic biological activities, including modulation of cell signaling, cell growth as well as cell apoptosis [2]. Furthermore, it is important to note that administration of polyamines can function as either protective or harmful in lethal experimental sepsis [3].
Proline plays various roles in cell metabolism and physiology; for example, in cell growth, wound healing, scavenging oxidants as well as in polyamine and protein syntheses [4]. In our model, the plasma concentration of proline was signifi cantly decreased during sepsis compared with healthy controls. One could speculate that, beside the reduced arginine availability, increasing hydroxy lation of proline to hydroxyproline -an essential step to provide collagen for tissue repair, remodeling or wound healing -contributes to the substantially reduced proline plasma concentration during sepsis even in the early phase of the disease [5]. Consequently, targeting arginine defi ci ency might be essential in patients with sepsis. Owing to the complexity of arginine metabolism that underlies multiple biochemical and physiologic processes (Figure 1), however, the potential benefi ts and risks of arginine supplementation have to be carefully evalu ated in further studies by a multiparameter approach measuring additional pathway-related products such as polyamines and ornithine, but also the activity of enzymes. Data presented as mean ± standard error of the mean. Investigated metabolites from plasma samples of healthy controls (n = 10) and of polymicrobial infected mice 6 hours (n = 10) and 24 hours (n = 10) post sepsis induction. Statistical analysis by one-way analysis of variance (ANOVA) and post-hoc tests using the Scheff é procedure. *Statistically signifi cant diff erence compared with healthy controls (P ≤0.05; ANOVA). † Statistically signifi cant diff erence compared with healthy controls (P ≤0.001; ANOVA). ‡ Statistically signifi cant diff erence between 6 hours and 24 hours post sepsis induction (P ≤0.05; ANOVA). Signifi cant data are indicated in bold.