Metabolic Pathways

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O. Rooyackers and J. Wernerman

Introduction

With a better understanding of sepsis induced organ dysfunctions, metabolic ori- ented treatments as well as the metabolic effects of currently available treatments are coming into focus. Good examples of this are tight glucose control with feeding and insulin treatment, as well as intravenous glutamine supplementation [1, 2].

Understanding of metabolic pathways is therefore crucial in optimizing treatment in sepsis. In all phases of sepsis this is obvious – resuscitation, low ﬂow phases as well as high ﬂow phases, multiple organ failure (MOF), and during recovery. How- ever, timing and relation to nutrition are important, and, therefore, insights into metabolic derangements in relation to nutrition may help to improve outcomes.

Energy metabolism in sepsis is mainly characterized by an increase in insulin resistance. In addition, lipid oxidation is increased, and in protein metabolism it is the increase in net protein degradation that dominates. This overview will focus on protein metabolism, glutamine metabolism, glutathione metabolism, and ﬁnally mitochondrial metabolism.

Protein Metabolism

In sepsis, a large proportion of the whole body energy expenditure is used on protein metabolism [3, 4]. This is particularly true in patients with MOF when several organ functions are more or less managed by external medical support, such as ventilators, dialysis machines and blood transfusions. Nevertheless, whole body expenditure is often elevated above the expected level of basal energy expenditure.

Also, when techniques are used to estimate the whole body turnover of a labeled amino acid (usually leucine or phenylalanine) in order to estimate whole body protein turnover, values above what are found in healthy individuals are seen. In the basal state, humans are able to adapt to under-nutrition or starvation by decreasing energy expenditure, which mainly reﬂects a decrease in protein metabolism. This is, unfortunately, not an option for the septic patient. If the septic patient is under- nourished, no such adaptation is seen. Therefore an under-nourished patient is rapidly energy depleted and the incidence and seriousness of complications that ensue are related to this energy deﬁcit [5–7].

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312 O. Rooyackers, J. Wernerman

So far there is no direct evidence that failure of protein metabolism is related to such under-nutrition. It is more likely that prioritized protein synthesis is maintained at the expense of endogenous protein mainly in skeletal muscle, which continues as long as there are any endogenous reserves. On the other hand, there is some evidence that over-nutrition is harmful. Over-nutrition is a situation where calories are given in excess of the actual energy expenditure, and/or when large quantities of proteins or amino acids are administered. Employing enteral feeding usually eliminates the danger of over-feeding; however the concept of enteral feeding does not eliminate the risk of under-feeding. These aspects are related to the general success rate of enteral feeding, which is typically around 60% of the prescribed amount.

Protein depletion is a strong predictor of outcome in sepsis. Attenuation of ongoing protein depletion in septic states is, therefore, a cornerstone in treatment.

In general terms, during sepsis this protein depletion is the result of increased protein synthesis in combination with an even greater increased protein degradation at the whole body level [8]. This is perhaps not the case during the initial low flow phase, a period not very well characterized in clinical studies. However, later on during the high flow phase and MOF this mechanism is definitely true. It is also well known that these changes in protein metabolism are not equally dis- tributed between organs and tissues, and that in individual organs or tissues the de novo synthesis and degradation of individual proteins is altered in a non-uniform way [9]. The complexity of these changes has encouraged modern techniques to be used in this field. Genomics and proteomics provide information regarding genetic regulation of protein metabolism as well as the presence or absence of individual proteins. This information gives insight into how these processes are regulated at a tissue level as well as a cellular level. However, these measures should ideally be combined with quantification of the related metabolic pathways. Quantitative estimates are possible at the whole body level, at the tissue level and at the level of individual proteins.

It is generally recognized that liver protein synthesis is enhanced in septic patients. This enhancement is usually attributed to the so-called ‘acute phase’

reaction, involving proteins that appear in increased concentrations in the serum, which are synthesized in increased amounts, and which have functions in the acute inﬂammatory reaction. In contrast, the plasma concentration of albumin decreases. This decrease is, however, not a reﬂection of low albumin synthesis (Fig. 1). On the contrary, albumin synthesis is elevated and there are even indices that albumin synthesis is increased to a maximum in these situations [10, 11]. The low plasma concentration of albumin is rather related to capillary leakage and to the degradation of albumin. The latter is a process not very well characterized and its assessment carries considerable methodological problems. In addition, increased protein synthesis rates are seen in circulating immune cells in septic patients (Fig. 1) [12, 13]. This is no surprise, as these cells are supposed to be activated, to produce export proteins and to undergo cell division.

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Fig. 1. Fractional synthesis rate (%/24 hours) in skeletal muscle proteins [16, 51], albumin [11, 52, 53], and proteins of circulating leukocytes [13,54] in sepsis (open bars), healthy controls (hatched bars), and during cholecystitis (ﬁlled bars). In muscle there is no change, while in albumin and leukocyte proteins a considerable increase is seen. For albumin the increase is much larger in sepsis than that seen in an acute inﬂammatory state such as cholecystitis. Values are given as means± SD

Muscle tissue undergoes considerable wasting in septic patients. This is related to physical inactivity, but also to metabolism, which places muscle as a provider and net exporter of free amino acids. This is particularly true for alanine and glutamine, which will be elaborated on below. The amino acids are produced from a net degradation of muscle proteins. Muscle tissue decreases rapidly in the septic patient related to this export of amino acids, even in the fully fed state [14]. This is quite different from the situation in healthy individuals, who have a net uptake of amino acids in the fed state. In septic patients, protein synthesis is unaltered and degradation of muscle is increased [12, 15–20]. The unaltered rate of muscle protein synthesis is a bit unexpected, as the protein synthesis rate in muscle decreases following elective surgery trauma [21]. However, it seems that with in- creasing size of trauma, protein synthesis in muscle moves from being decreased to being unaltered [22]. In septic patients, however, it remains unaltered (Fig. 1), and in some individuals it is actually markedly increased [23]. Protein degradation, on the other hand is generally increased. Due to methodological difficulties, quantification of this increase in degradation is still problematic, but qualitative assays clearly indicate that proteasome activity is increased, that the synthesis of proteasome subunits increased, and that the de novo synthesis and gene expression of ubiquitin is increased [15,17–20]. The few quantitative estimates on muscle protein degradation in septic patients further confirm this pattern [24]. Techniques used to investigate protein degradation quantitatively, involve the incorporation

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and/or balance of labeled amino acids across tissues. This opens the possibility of evaluating the natural course of sepsis as well as the effects of treatments in the septic patient. The initial phase of sepsis may be studied in man by the use of an endotoxic challenge in healthy volunteers. During the initial 4 hours following endotoxemia, protein metabolism mainly displays a low ﬂow phase with decreases rather than increases in muscle protein synthesis and protein degradation [25]. This is a pattern that does not correspond to what is seen later on during MOF.

Glutamine Metabolism

Among the amino acids, glutamine has a special role. It is a non-essential amino acid, but there are indices that endogenous production is insufﬁcient during critical illness and also that exogenous intravenous supplementation has beneﬁcial effects on outcome. In general terms, glutamine has a central role in amino acid metabolism and in the interface between amino acids and carbohydrate metabolism. It is a main transporter of carbon skeletons as well as amino groups between skeletal muscle and the ‘central’ organs in the splanchnic area. The carbon skeleton of glutamine,α-ketoglutarate, is also a constituent of Kreb’s cycle and con- sequently is involved in energy production. In addition, glutamine is the precursor for nucleotide synthesis, and the availability of glutamine is crucial when cell division is prioritized. Cells that are rapidly dividing commonly utilize glutamine as an energy substrate, such as cells in the intestinal mucosa and immune cells.

The slight disadvantage in terms of moles ATP per mole substrate in comparison with glucose is easily counterbalanced by the opportunity of having a substrate ﬂow which can be turned easily into nucleotide synthesis when necessary. It is well documented that in various stressed states, cells are more likely to use glutamine as an energy substrate. In the basal state, endogenous glutamine production is 50–

70 grams per 24 hours, mainly taking place in muscle tissue [26, 27]. Glutamine is constantly exported from muscle and taken up in the splanchnic area, preferably as an energy substrate where the amino groups are transaminated to other amino acids or turned into urea in the liver. In sepsis, this turnover of glutamine is not changed [28–30]. However, it does not increase either, although the demand for glutamine may be elevated. This is the situation where availability of glutamine may be a limiting factor.

In the early phase of sepsis, as reproduced by an endotoxin challenge in healthy volunteers, glutamine plasma concentration decreases, free glutamine concentration in muscle decreases, and the export of glutamine from muscle increases [31].

Later on during MOF, the export of glutamine from muscle is relatively constant, while the concentrations of glutamine in plasma and in muscle tissue stay low [30,32]. The uptake of glutamine across the splanchnic area is related to plasma concentration. Plasma glutamine concentration on admission to the ICU is a prognostic factor [33]; a low value indicates a much worse prognosis. This factor is

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unrelated to the prognostic elements summarized in the APACHE II score. Intra- venous glutamine supplementation can normalize plasma glutamine level in all patients [16]. An exogenous supply of amino acids does not increase the endogenous production of glutamine [29, 34]. On the other hand, an exogenous supply of intravenous glutamine does not suppress de novo production but makes more glu- tamine available [28]. For muscle tissue, a short term exogenous glutamine supply does not restore the intracellular glutamine depletion and the efflux of glutamine from muscle is also unaltered [16, 29, 35]. Exogenous glutamine supplementation, therefore, does not per se save muscle protein, but makes more glutamine available for other organs. On a long term perspective, glutamine may also be beneficial in terms of saving muscle protein, but this remains to be demonstrated in septic patients. The clinical evidence for a beneficial effect of glutamine on mortality is so far limited to studies of intravenous supplementation of glutamine to patients with MOF [36, 37]. The effects of enteral supplementation during earlier phases of sepsis are less well documented, but there are several reports of beneficial effects on morbidity [1]. There are also animal studies showing beneficial effects of glutamine supplementation during resuscitation [38]. Furthermore, the expression and production of heat shock proteins are enhanced in response to exogenous glutamine supplementation [38, 39]. The clinical relevance of these findings is still not settled, however.

To evaluate the need for glutamine supplementation, as well as the optimal dose and the best route of administration, it is necessary to be able to measure endogenous glutamine production. This is best done as a measurement of the rate of appearance of glutamine. The conventional technique is to give a primed constant infusion of isotopically labeled glutamine and estimate the rate of appearance at steady state [27, 34]. An alternative approach is to use the decay curve, which can also be obtained after a single injection of glutamine [26, 29, 40]. This latter approach avoids the problem related to the large distribution volume of glutamine, and also makes it possible to do estimates when a steady state is not possible. This technique has enabled us to estimate the rate of appearance of glutamine, and will also make it possible to evaluate glutamine supplementation by the entral route.

Glutathione Metabolism

Glutathione is a tri-peptide (glutamyl–cysteinyl–glycine) that is synthesized in all cells. As a thiol it appears in a reduced and an oxidized form. The oxidized form is a dimer, with the residues are connected via sulfhydryl bonds. Glutathione has many functions, but the most important is attributed to its ability to remove reactive oxygen species (ROS) as an antioxidant. In this capacity, glutathione is quantitatively the most important antioxidant in man. In plasma, the glutathione concentration is very low, but intracellularly it appears in mmole concentrations in all tissues. The reduced form of glutathione is the dominant form representing

> 80% of the glutathione in most tissues.

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In sepsis, glutathione depletion is seen in erythrocytes as well as in muscle tissue (Fig. 2) [41–43]. In addition to a low concentration, the redox status of glutathione is altered into a more oxidized state. In erythrocytes, these changes are constant over time during MOF [42]. In acute sepsis, as represented by an endotoxin challenge to healthy volunteers, there is no immediate change in glutathione concentration or glutathione redox status in the erythrocytes. In muscle, glutathione is also left unaffected by an endotoxin challenge in the acute phase, but on admission to the ICU, septic patients show reduced glutathione concentrations and an altered redox status with a more oxidized state. The extent of this depletion is associated with short-term mortality [44]. Those surviving the acute phase of sepsis and developing MOF, show a gradual normalization of glutathione concentration in muscle over subsequent weeks (Fig. 2) [43]. The more oxidized state of glutathione, however, remains throughout the ICU stay. These are descrip- tive ﬁndings, which cannot be explained by a shortage of the constituent amino acids, in particular not by a shortage of cysteine [43, 45]. If anything, glutathione depletion is statistically correlated to simultaneous glutamate depletion. On the other hand, although glutamate concentrations are low, it is still quite abundant.

Following elective surgery, a similar decrease in glutathione is seen on the ﬁrst and third post-operative days [46]. This ﬁnding applies to the reduced form of glutathione only, with the redox status being unaffected. In addition, the decrease in glutathione concentration is statistically related to a decrease in free glutamate.

Supplementation with intravenous glutamine counteracts the glutathione deple-

Fig. 2. Reduced glutathione concentrations in muscle (circles) [43] and in whole blood (squares) [42] over time in septic patients during their ICU stay (open symbols). Values are compared to those of controls (closed symbols). Initially, a decrease is seen in muscle as well as in erythrocytes. During the late course of sepsis a normalization is seen in muscle but not in erythrocytes. Values are given as means± SD

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tion to some degree following surgical trauma [47]. When patients with MOF in the ICU are given extra glutamine, the low glutathione concentration seen in muscle is not restored in response to this supplementation [16].

In addition to the obvious anti-oxidative effects of glutathione, a role in the overall regulation of protein metabolism has been suggested. In particular there is evidence from animal studies concerning the level of protein degradation [48].

However, the exact function of glutathione in this setting remains to be established.

Mitochondrial Metabolism

Mitochondrial metabolism is mainly energy production, although active protein synthesis also occurs in the mitochondrion. The majority of proteins in the mitochondrion, however, are synthesized within the cellular cytoplasm encoded for by the cellular genome and not by mitochondrial DNA. These proteins are actively transported into the mitochondrion, and they are, for example, necessary for protein degradation in the mitochondrion. In sepsis, reduced mitochondrial function is described in the acute phase as well as during MOF [44,49]. In the acute phase, it is potentially difﬁcult to distinguish between a shortage of oxygen at the tissue level or at the mitochondrial level and functional impairment of the mitochondrion. In man, these pathways have been most extensively studied in skeletal muscle. In the acute phase, failure in energy production has been attributed to reduced function of complexes I and IV within the respiratory chain [44]. The reduced function is usually expressed as in vitro activity of complexes I and IV related to citrate synthase activity as an index of mitochondrial mass. If this functional depression is not reversed, energy collapse ensues and mortality is very high. Septic patients who develop MOF have reduced mitochondrial content in terms of a low citrate synthase activity related to DNA or protein content [49]. This decrease progresses with time [50]. In this situation, in the phase of MOF, the activity of the mitochondrial complexes I and IV as related to citrate synthase activity is not different from that in healthy controls. This is true for thigh muscle as well as for intercostal muscle. It can be hypothesized that this reduction in mitochondrial metabolism and the consequent decrease in energy-rich phosphates during MOF is related to muscle fatigue [49]. Hence, the underlying mechanism of reduced mitochondrial function in terms of energy production seems to be different in the acute phase of sepsis and in the later phase involving MOF.

Conclusion

The understanding of sepsis-induced organ dysfunction has advanced consid- erably over the last few years. The interaction between signaling systems and metabolic pathways is a particularly ‘hot’ area of ongoing research. Energy production and the availability of substrates in the acute phase seem to have a crucial

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impact on the course of sepsis and on mortality and morbidity. Later on, during MOF, the same metabolic pathways determine outcome, but the mechanisms and clinical picture are different. Knowledge of these mechanisms is, therefore, necessary for optimal clinical practice.

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