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1 Background

Critical illness is associated with loss of lean body mass. In particular sarcopenia is a characteristic feature of long-standing critical illness [1–3]. This is often obvious at bedside, but in addition it has been extensively documented in terms of negative nitrogen balance and loss of muscle tissue [4–6]. The loss of both muscle proteins and of muscle mass is in rough terms proportional to the severity of critical illness [2]. When it comes to nutrition support, the major target of this intervention may be defined as to minimize the loss of lean body mass.

A loss of body proteins corresponds to an imbalance between synthesis and degradation of proteins. On the whole body level, this is reflected by a negative whole body nitrogen balance. This balance, however, is extremely unevenly distributed between organs and tissues and even between singular proteins in a given tissue. This means that protein metabolism in the critically ill may be discussed on a whole body level, on an organ level, or on the level of individual proteins.

Besides the loss of lean body mass, the most characteristic feature of protein metabolism in the critically ill is the elevated whole body protein turnover as illustrated in Fig. 9.1 [7]. As compared to healthy individuals, both de novo synthesis rate and degradation rate are doubled. The net protein balance is, as illustrated, not different from zero in the fed state of critical illness, but as pointed out above this is very different between tissues. For contractile proteins in skeletal muscle, there is clearly a negative balance [8].

Fig. 9.1
figure 1

Whole body protein turnover in healthy individuals in the postabsorptive state and in the IV-fed state, together with IV-fed critically ill patients. All kinetic measurements made by isotopic labeled phenylalanine. Open bars represent protein synthesis, hatched bars protein degradation, filled bars protein balance, and finally cross-hatched bars protein oxidation. For the healthy subjects, statistical comparisons of paired measurements were made as indicated, where ** represents P < 0.001 and *** P < 0.0001. Between healthy subjects and patients, no formal comparisons were made, but the higher levels of both synthesis rate and degradation rate are obvious. In addition the protein balance for the patients is not different from zero (Combined data from [7, 13] with permission)

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2 Techniques of Assessment

2.1 Nitrogen Balance

The classic technique to evaluate protein metabolism is by whole body nitrogen balance. In healthy subjects this may be done by monitoring intake and collecting and analyzing excretions. In addition there is a small insensible nitrogen loss. In a weight stable subject adapted to a standardized nutrition intake (in terms of caloric and protein intake), the nitrogen balance technique allows for comparisons between different isocaloric or isonitrogenous products. Comparisons between nutrition products differing in protein and caloric content is much more complicated and must be accompanied by sufficient time periods for adaptation to the different intake levels [9]. Furthermore, studying malnourished or catabolic subjects add concerns over comparability between groups of subjects.

2.2 Imaging

Recent developments of imaging techniques have made ultrasound and radiology techniques more useful in estimating muscle mass and lean body mass [10]. Single-slice CT scans on the level L3 for estimation of muscle mass are reported to give good prediction of outcomes in critical illness [11]. Simultaneously temporal patterns of thigh muscle thickness estimated by transcutaneous ultrasound give information over development of sarcopenia and have illustrated the severity of disease as a determinant of degree of sarcopenia [2, 3]. Possible limitations lie in reproducibility of measurements and sensitivity when different treatments are compared in the critically ill. Neutron activation analysis is often regarded as the gold standard to follow changes in lean body mass in critical illness. A number of studies from New Zeeland have been instrumental in expanding our knowledge of the temporal development of depletion associated with critical illness, including the effects of feeding and overfeeding [4, 6]. The obvious limitation of the technique is its sparse availability and logistic challenge in applying the technique for body mass diagnostics only in critically ill subjects.

2.3 Isotopic Tracer Techniques

The use of isotopic studies to assess and monitor protein turnover opens up opportunities to study protein de novo synthesis and protein degradation simultaneously. Applications may be on the whole body level as well as on the tissue level or for individual proteins [12]. Just as for nitrogen balance studies, the presence of metabolic as well as isotopic steady states is a necessary prerequisite for reliable results. In contrast to nitrogen balance, the time patterns of steady states may be much shorter and therefore more practical. Whole body measurements, as illustrated in Fig. 9.1, may be performed when steady state periods of just 3–4 h exist. Results of comparisons between such short periods may be difficult to interpret, which calls for caution. Then it is advisable to predefine criteria for comparability between time-points of interest. In addition to quantification of synthesis rate and degradation rate, measurement of whole body turnover rate also enables determination of amino acid oxidation [7]. As proteins may not be stored in the human body in the way adipose tissue may store a surplus of energy intake, a possibility to measure the amount of protein oxidized will reflect the fraction of protein intake that is not utilized in protein turnover, but has to be eliminated as an increase in oxidation, which may be interpreted as a metabolic burden of overnutrition. Figure 9.2 illustrates how an increase of protein intake from 0.53 g/kg/24 h to 1.06 g/kg/24 h increase whole body net protein balance in head trauma patients without increasing protein oxidation [13].

Fig. 9.2
figure 2

Whole body protein turnover in IV-fed critically ill patients (n = 17) with traumatic brain injury or subarachnoid bleedings. All kinetic measurements made by isotopic labeled phenylalanine. Open bars represent protein synthesis, hatched bars protein degradation, filled bars protein balance, and finally cross-hatched bars protein oxidation. Hypocaloric feeding combined with low protein intake gave a lower protein synthesis rate and a less favorable protein balance. It is noteworthy that the protein oxidation does not increase by doubling protein intake in this level of intake (Data from [13] with permission)

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The demand for a metabolic steady state also includes a steady state of nutrition intake. When parenteral nutrition is given, this can usually be controlled, but as the majority of critically ill patients are fed enterally, both a steady state of enteral intake and a steady state of first pass elimination through the splanchnic organs have to be documented. At measurements of whole body turnover, sampling is done in the central compartment (plasma), which necessitates documentation of the constancy of this first pass elimination during the measurement period. This may be done by adding an isotopic tracer to the enteral feeding given and assess its appearance in plasma [14].

3 Tissues and Individual Proteins

The overview of techniques above reflects the whole body situation, but it is in principle also applicable to individual tissues or proteins. Balance techniques may be used for individual organs or tissues. For example, urinary excretion of 3-methyl-histidine has been widely used to study degradation of contractile proteins [15]. If quantified, this may be interpreted as the degradation rate of skeletal muscle proteins. The background is the irreversible (in man) posttranslational modification of histidine in contractile proteins. Furthermore, the arteriovenous balance of 3-methyl-histidine may also be analyzed across muscle tissue [16]. A limb, most often the leg, is then postulated to represent muscle tissue. The use of 3-methyl-histidine is, however, associated with a number of underlying assumptions, and also the fact that only degradation is reflected is another limitation.

Arteriovenous balances also including measurements of blood flow may be used to study balances of a wide variety of substances across organs or tissues. In the area of protein metabolism, amino acid balances are frequently applied [17]. Besides skeletal muscle, the liver, kidney, brain, and lungs are examples of tissues where this approach has been used. A necessary prerequisite is that the tissue studied is supplied and drained by accessible blood vessels, and if concentration differences are small, a high precision in concentration determinations is necessary. The obvious limitation in plain balances is that it is not possible to discriminate between what is attributable to synthesis and degradation, respectively.

A further development of arteriovenous balance techniques is to also include isotopic labels [18]. This carries the possibility to quantitatively asses both synthesis and degradation at the same time. With a constant infusion of an isotopically labeled amino acid, the rate of appearance from a given tissue will correspond to the degradation rate, while the rate of disappearance will correspond to the synthesis rate. A critical assumption is the isotopic enrichment of the labeled amino acid in the immediate precursor pool for incorporation of amino acids into protein. A snapshot of the intracellular pool may be obtained by adding tissue biopsies. If biopsies are included, a 3-pool model is established, otherwise a 2-pool model [8].

Isotopically labeled amino acids are also widely used to assess de novo protein synthesis, when the incorporation rate of the labeled amino acid into proteins is measured. This may be applied to mixed tissue proteins or fraction of proteins or individual proteins [19, 20]. If proteins are in a solid organ, tissue biopsies are required. If it is circulating proteins or proteins in circulating cells, blood sampling will be sufficient [21, 22]. Just as for the calculation of synthesis rate from the rate of disappearance requires an estimate of the isotopic enrichment in the precursor pool for protein synthesis, correct estimation of the precursor pool in these calculated synthesis rates is critical. In particular when metabolic steady states are not obvious, which, for example, may be the case after feeding, erroneous precursor enrichment estimates may introduce considerable uncertainty.

4 Protein Synthesis Rates

The elevation in whole body protein synthesis rate seen in the critically ill, as illustrated in Fig. 9.1, is mainly attributable to high synthesis rates in the liver and in immune cells [21, 22]. Also for export proteins such as albumin, often referred to as a negative acute phase reactant due to its low plasma concentration, there is an increase in synthesis rate [23]. In parallel to the increase in synthesis rate of proteins in the liver and in immune cells, remaining tissues are found to have synthesis rates not different from what is seen in healthy subjects. This is particularly true for skeletal muscle, which related to the large size of the tissue make up a substantial portion of whole body protein synthesis [8]. Also when muscle proteins are subgrouped into contractile and mitochondrial proteins, for all groups a similar level of synthesis rates is seen both for the critically ill and for healthy subjects [24]. As critically ill patients are a very heterogeneous group of individuals, there is a large interindividual scatter, but in principle there are no tissues so far studied that expose a decrease in protein synthesis rate in critical illness.

5 Protein Degradation Rates

Breakdown of proteins is less well studied, related to the methodological difficulties described above. The whole body increase in degradation rate is to a large extent attributable to the elevated rate of protein degradation in skeletal muscle [25]. This is also in accord with the negative protein balance seen in muscle and the pronounced sarcopenia. Just as for synthesis rates, the degradation rates are similar in between different subgroups of proteins in muscle. From small case series of critically ill subjects, muscle protein degradation rate may be threefold higher than normal [8]. This may not cover the entire doubling of whole body degradation rate reported, but at least a major portion thereof. Very little information over degradation rates on the tissue level is available from other organs, and therefore it is hard to know whether or not degradation rates in the rest of the body also increase or stay unaltered. Indirectly the balance equation of, for example, albumin with a low, but constant over time, plasma albumin concentration combined with an elevated synthesis rate indicates an increase also in degradation rate. However, this has so far not been objectively measured.

A challenging question is the role of autophagy in critical illness. In protein degradation there are three principally different tracks for degradation: (i) cytoplasmic free enzymes, (ii) the proteasome system, and (iii) the lysosomal system. The latter system is also named autophagy. All systems are stimulated in the critically ill, but in particular the lysosomal system [8, 26]. Efforts to decrease or to attenuate the development of sarcopenia by preventing degradation may result in a decrease of lysosomal activity or in other words a decrease in autophagia. Simultaneously it is hypothesized that autophagia is a necessary mechanism to overcome critical illness and to recover [27]. If so, to attenuate autophagia may not at all be a good thing. Further studies in this field will be necessary to fully comprehend these mechanisms.

6 Interfering with Protein Metabolism

The close connection between lean body mass and outcomes has fostered hypothesis that actions to attenuate the loss of lean body mass may be a good strategy to improve outcomes. There are at least four strategies to try to obtain this: (i) source control, (ii) nutrition, (iii) pharmacology, and (iv) mobilization.

6.1 Source Control

This is clearly the least controversial part. Source control may be to stop bleeding, to control infections, to stabilize fractures, to control pain, etc. This is also the main strategy in any critically ill patient. Although it is not evidence based that the clinical improvement associated with source control also is accompanied by an increased lean body mass, there is at least a temporal coincidence.

6.2 Nutrition

In malnourished but otherwise healthy subjects, adequate nutrition is necessary (but not sufficient) to increase lean body mass. In non-malnourished and healthy adults, an increase in lean body mass can only be achieved by simultaneous physical activity and adequate nutrition in combination. This is, however, not a constant finding. Subjects respond very differently in terms of lean body mass to an increase in physical activity. Also the type of physical activity may be of importance. In critically ill subjects, the response to feeding in terms of alterations in lean body mass is not sufficiently explored. Although all critically ill subjects are catabolic in the sense that they develop sarcopenia, differences in nutrition status may be crucial for the response to feeding. In this context the temporal aspect is very important. The response to nutrition may differ in the early and late phases of critical illness. The autophagy hypothesis alluded to above may be an example of such a temporal factor.

The relation between lean body mass and outcomes does not immediately imply that preservation or attenuation of lean body mass will affect outcomes. So far it is reasonably well evidenced that caloric substitution will give a lower whole body protein loss as compared to no caloric substitution, but it is not evidenced that it will affect outcomes. If the caloric substitution turns into an over-substitution, this is associated with harm. The possible association with protein metabolism is not clear, but an attenuation of autophagy may be a suggestion. Lately the provision of protein nutrition has attained a large interest. The recommendation of a protein intake of 0.8 g/kg/24 h (together with a normocaloric energy intake) given for healthy subjects has been hypothesized to be far too low for the critically ill [28, 29]. The evidence for this hypothesis, however, is not very strong. No prospective randomized studies with outcome parameters but merely observational case series exist [9]. Nevertheless, recommendations from nutritional societies suggest a higher intake as compared to the WHO recommendations for the healthy [30, 31], 1.2–1.5 g/kg/24 h, together with a normocaloric intake is recommended by both ESPEN and ASPEN [32–34].

The use of isotopic labeling to assess whole body protein turnover offers the possibility to assess the short-term effect of an increased protein intake on protein balance and on protein oxidation. Figure 9.3 depicts data from such short-term studies in the early phase of critical illness, illustrating a clear relationship between protein intake and protein balance [14]. This must, however, be interpreted with caution. It is a short-term observation, very few observations above an intake of 1.8 g/kg/24 h are included, and studied subjects are all in the early phase of critical illness. Beyond that the relations between a positive protein balance, lean body mass, and outcome all remain to be established.

Fig. 9.3
figure 3

Observations (n = 112) of whole body protein balance in relation to protein intake for critically ill patients (n = 39) given variable amounts of energy and protein (amino acids). Whole body protein balance was assessed by measurements of isotopic labeled phenylalanine. The statistical correlation seen is suggestive of a positive whole protein balance on the protein intake level suggested by the ESPEN guidelines (Combined data from [7, 13, 14] with permission)

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6.3 Pharmacology

On the side of the rather confusing hypotheses around pharmaco-nutrition, there have been efforts to interfere with protein metabolism by pharmacological agents. The well-known example of growth hormone supplementation illustrated that although a stimulating effect on skeletal muscle combined with protein synthesis rate could be demonstrated in critically ill patients [35], a prospective randomized blinded trial showed an increase in mortality in the growth hormone-treated group of patients [36]. This experience clearly illustrates the necessity to understand the underlying mechanisms before starting large-scale trials. In addition there are small studies exploring high insulin doses [37], testosterone [38], anabolic steroids [39], as well as beta-blockers [40]. The experience of multi-supplemented formulas under the heading of pharmaco-nutrition has so far not contributed to a better outcome and should preferably be better investigated before being recommended.

6.4 Mobilization

There has been a discussion over the relative contributions to sarcopenia from critical illness itself (general inflammation) and the immobilization that also accompanies critical illness. Studies of immobilized healthy subjects indicate an initial delay in the development of sarcopenia [41]. Long-term immobilization on the other hand is associated with considerable loss of muscle mass [42]. In critically ill patients, efforts to prevent muscle wasting by activity have attained a lot of interest [43]. Presented studies are most often case series, and the absence of blinding is common. In addition selection of patients by extensive exclusion criteria is also common. In the context of protein metabolism, there are no reports over physical mobilization or training and protein turnover, although this is an area where reports of beneficial effects from muscle contractions during critical illness are emerging [44].

7 Conclusions

Protein metabolism is a core part of metabolism in particular in critical illness. Overall the critically ill subject has an elevated protein turnover to meet the demand associated with critical illness. Regulating mechanisms are incompletely understood, which make recommendations on nutrition support or other therapeutic efforts difficult. The alterations of protein metabolism in critical illness are not uniform between individual tissues, which make global nutrition protocols difficult to evaluate. Recent advances in isotopic techniques to assess protein turnovers in the whole body as well as in individual tissues or even proteins together with advances in imaging will give opportunities to better understand the mechanisms and consequently give more evidence-based recommendation over optimal care.