University of Groningen
Protein oxidation: towards a non-invasive assessment of anabolic competence
Reckman, Gerlof
DOI:
10.33612/diss.136482233
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Publication date: 2020
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Reckman, G. (2020). Protein oxidation: towards a non-invasive assessment of anabolic competence. University of Groningen. https://doi.org/10.33612/diss.136482233
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CHAPTER 1
MOTIVATION
Loss of lean body mass (LBM) is an important problem in many acute and chronic clinical conditions. Whereas adequate protein intake is a main prerequisite for maintenance of LBM, however, for many patients the LBM loss is not compensated or reversed by simply adding more protein to the diet. Apparently, the added dietary proteins are not adequately utilized by the patient’s body for protein synthesis in order to maintain lean body mass. To help determine the cause(s) of underutilization of dietary proteins, better understanding of protein metabolism in a clinical context is urgently needed. However, currently available monitoring methodology is either informative but invasive [1], or invasive but non-informative on pathophysiology [2]. A simple non-invasive test to measure (specific parts of) protein metabolism, therefore, could be of great clinical value. The protein oxidation pathway is part of protein metabolism and could be measured non-invasively. However, it is unclear what the biological relevance of the protein oxidation pathway is. Therefore, protein oxidation research forms the core of this thesis.
PROTEIN METABOLISM
Proteins are essential molecules that serve many critical functions, i.e., they act as enzymes, antibodies, messengers, transporters, and provide structural integrity. The structural integrity is a result of proteins forming tissue, organs, and muscles. Proteins are composed of building blocks called amino acids. The individual amino acids do not function merely as building blocks for protein, but some individual amino acid also serves one or more specific roles. For example, amino acids play a role in cell signaling, serve as regulators of gene expression, and are part of metabolic pathways [3]. Maintenance of protein content is essential for survival. In the absence of sufficient dietary protein intake, the muscles will be used as a reservoir for amino acids in order to maintain protein metabolism in vital organs [4]. If prolonged, this process will lead to loss of LBM.
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Protein metabolism describes the biochemical pathways involved in the synthesis and degradation of both proteins and amino acids. Anabolism is the process of linking amino acids together to form proteins. Catabolism is both the process of protein degradation into amino acids and the breakdown of amino acids. Whole body net anabolism or catabolism results in changes in LBM.
Protein utilization refers to the proportion of ingested dietary protein that is used in the protein synthesis process. It can be considered as a marker of efficiency with which the body handles the ingested dietary protein. Regular and sufficient dietary protein intake is vital, as it provides the main source of nitrogen intake and enables the continuation of whole body protein metabolism (Figure 1) [5– 7]. After ingestion, the dietary proteins are digested into amino acids and absorbed, after which they become available for protein synthesis. Of note, the body does not have a dedicated organ or tissue to store and retrieve proteins and amino acids. Although proteins within muscles can be broken down into amino acids to support vital processes, muscles do not primarily serve a protein storage function. Since proteins and amino acids are not stored by the body [8,9], the body instead maintains a dynamic and tightly regulated pool of ~100 g of amino acids within cells and plasma [10]. The regulation of this pool is brought about by continuous protein degradation and protein synthesis, called protein turnover [6]. The function of the amino acid pool is to quickly respond to changes in protein and amino acid demands [6]. The main influx of amino acids into this pool is formed by, the dietary protein intake, for which the WHO recommends 0.8 g protein/kg bw/day, and the degradation of body proteins (~300-400 g/day) [11]. The absence of a protein storage organ or tissue results in the breakdown of all amino acids in excess of protein synthesis requirements (~300-400 g/day) and in excess of the amino acid pool [6].
The breakdown of amino acids starts with the deamination of the amino acids, whereby the resulting ammonia is converted into urea via the urea cycle, and is subsequently excreted in urine. The remaining
amino acid carbon skeletons are oxidized within the citric acid cycle (CAC) or enter glycolysis to release energy, which is utilized to synthesize the high-energy molecule adenosine triphosphate. After oxidation, the carbon skeletons are no longer available for protein synthesis. The process of oxidation leads to the formation of energy, water and carbon dioxide. The latter is removed from the body into the surrounding air by pulmonary exhalation. Protein oxidation is considered to be, under normal conditions, a simple pathway for the disposal of excess amino acids [6]. The biological relevance of protein oxidation as a proxy for disturbed protein metabolism is unknown.
Figure 1. Schematic overview of dietary protein intake, protein turnover,
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ASSESSMENT OF PROTEIN METABOLISM
Many methods are able to measure distinct aspects of protein metabolism. These methods can be categorized into indirect measurements and direct measurements. Indirect measurements include the nitrogen balance and changes in body weight. The nitrogen balance is the difference between dietary protein intake and protein loss, whereby dietary protein intake is the main source of nitrogen intake, and urinary urea is the main source of nitrogen excretion [2]. This method is non-invasive, as it consists of measurements on the dietary protein intake and urinary urea excretion which are both collected without piercing the skin [2]. A positive nitrogen balance indicates anabolism, whereas a negative balance indicates catabolism [2]. However, measuring the nitrogen balance has some disadvantages. First, it is difficult to get a reliable measurement of dietary nitrogen intake because most dietary assessment methods, such as the food frequency questionnaire, have an inherent bias [12]. The food frequency questionnaire has an average bias towards underreporting and the reporting accuracy differs per person [12,13]. Second, sudden changes in protein intake are not immediately reflected by the amount of nitrogen excreted, as it takes three to four days for the nitrogen to reflect the new level of protein intake [14,15]. Third, the nitrogen balance does not provide insight in changes in protein synthesis and breakdown [16]. Another indirect measure of anabolism versus catabolism is the monitoring of changes in LBM, for instance by bioelectrical impedance analysis (BIA) or dual x-ray absorptiometry (DEXA) [17,18]. The disadvantage of measuring changes in LBM as an indicator of anabolism or catabolism is that changes in LBM occur over days or weeks instead of minutes or hours. The speed at which LBM changes occur is dependent on the severity of lack of dietary intake, rate of catabolism, or both. The lag between presence of a catabolic state and its detection through the measurement of loss in LBM results in losing valuable time to minimize or prevent loss of LBM. Therefore, a direct measurement of protein metabolism which
does not lag behind the actual anabolic or catabolic state could in principle be extremely beneficial for minimizing the loss of LBM in, for example, malnutrition, by allowing early detection and hence, early intervention.
Alongside indirect measurements, direct measurements to assess protein metabolism are available. Direct measurements of protein metabolism include for example measurements of protein synthesis [1,19–23], protein turnover [19], protein breakdown [1,19], and amino acid oxidation [1,22]. These measurements are conducted within the research setting and make use of isotope tracers. The most common tracer applied in protein metabolism research is the stable isotope of carbon, i.e., carbon-13 (13C). The 13C-tracer is
synthetically incorporated into the essential amino acids leucine and phenylalanine to form 13C-leucine [1,22,23] and 13C-phenylalanine
[1,19–22], respectively. However, the disadvantages of these direct measurements are that they are complex, invasive, and involve the use of synthetically enriched substrates which are more expensive than naturally enriched substrates.
PROTEIN OXIDATION BREATH TEST
Currently, no clinically feasible method to directly measure aspects of protein metabolism is available. Such a method would have to be non-invasive, less complex than the current research methods, and cost-effective, which could be achieved through the use of naturally enriched 13C-substrates. Combining existing methods has
led to the development of the protein oxidation breath test. The protein oxidation breath test requires ingestion of naturally enriched
13C-protein by the study subject. After ingestion, digestion, and
uptake into the bloodstream, the 13C-amino acids are available for
synthesis or oxidation. The 13C-proteins that are oxidized emerge as
13CO
2 in the exhaled breath. Samples of exhaled breath are collected
at regular intervals by exhaling into small glass containers through a drinking straw. Immediately after exhalation the glass container
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is closed with a screwcap, which has a rubber seal. The collected breath samples are measured for their 12CO
2:13CO2 ratio with an
isotope ratio mass spectrometer (IRMS). The IRMS compares the breath sample to a high 13C-enriched international standard, Pee
Dee Belemnite (PDB), which has an accepted absolute 13C/12C ratio
of 0.0112372. The differences (delta, δ) between the breath samples and the standard is expressed in parts per 1000 (‰). The PDB standard 13C/12C ratio is defined as 0 ‰. As a result of comparing
breath samples to the high 13C-enriched PDB, the δ-value of breath
samples are negative. The requirement for the protein oxidation breath test is that the 12CO
2:13CO2 ratio before ingestion of the
naturally enriched 13C-substrate, i.e the background value, is as low
as possible. This needs to be verified by the collection of baseline breath samples. All in all, the protein oxidation breath test is non-invasive and less complex than the current research methods, but its value for assessment of protein metabolism so far has not been evaluated.
ANABOLIC COMPETENCE
To integrate the modifiable factors which support and impede anabolism, and to translate fundamental knowledge about protein metabolism into a broader clinical paradigm, the concept of anabolic competence was formulated [24]. Anabolic competence is defined as “that state which optimally supports protein synthesis and lean body mass, global aspects of muscle and organ function, and immune response” [24]. Optimal protein synthesis is reached when protein synthesis rate is high enough to at least offset the rate of amino acid breakdown. For the continuation of optimal protein metabolism, sufficient intake of energy, macro- and micronutrients and in particular dietary protein intake is paramount, as protein is the nutrient from which new proteins can be synthesized and lean body mass can be maintained.
Age is a main non-modifiable factor to be considered, as protein and energy requirements respectively increase and decrease, in elderly subjects, and moreover, the risk for nutrition deficiencies is higher in the elderly [25,26]. Also, the risk for nutrient deficiencies is higher in overweight and obese older adults than in those with normal weight [27,28]. Of note, whereas overweight and obesity are intuitively associated with “too much”, in fact, obesity is associated with protein deficiency [26]. Information on actual dietary protein intake is an important starting point for strategies aimed at optimization of protein status. For design of screening strategies it would be relevant to know which populations have the highest risk of an unbalanced dietary intake, and, more specifically, inadequate protein intake. Thus, it would be important to know whether, in older adults, there are differences in protein intake between overweight and obese subjects, as compared to normal weight subjects, and to review the available literature to this purpose.
Whereas assessment of dietary protein intake is a highly relevant first step, it does not provide information on protein utilization. Utilization of proteins can be impaired by many pathophysiological conditions. These include, for example, gastrointestinal diseases that impair intestinal absorption or digestion, inflammation, hormonal disturbances, adverse effects of medication (e.g. corticosteroids) or other therapy, or lack of physical activity as an anabolic trigger [29– 32]. This underutilization of protein could in theory be compensated by higher than normal dietary intake of protein. Accordingly, for relevant clinical translation, the nutritional aspects of protein metabolism need to be integrated into the broader context of anabolic competence, integrating the domain of nutrition with other supportive and impeding factors of anabolism which are involved in the actual utilization of protein for protein synthesis. These other supportive and impeding factors are assigned to the anabolic competence domains of physical activity and internal milieu [33]. The assessment and monitoring of factors from the three domains of anabolic competence combined can help pinpoint causes of
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disturbed protein metabolism in the clinical setting. Disturbed protein metabolism is characterized by an imbalance between decreased anabolism and increased catabolism, resulting in net catabolism. The protein breath test is a possible candidate to expand the toolkit for assessing factors of anabolic competence. For the protein breath test to be of added value for both the research and the clinical setting, it should be able to distinguish different levels of protein oxidation under different physiological circumstances which influence protein metabolism.
Disease-related malnutrition (DRM) is a clinical example to illustrate the need for a non-invasive method to assess protein metabolism as disturbed protein metabolism is a hallmark of DRM. It has been shown that in DRM, amongst others, the decline of LBM results in increased risk of adverse clinical outcomes of disease such as, increased risk for complications, increased hospital length of stay, and higher mortality [34,35]. In Western countries, the prevalence of DRM in the clinical setting is approximately ~40% depending on the definition and assessment tools used [34,36,37]. Therefore, better understanding of protein metabolism, in particular the utilization of protein, is pivotal for this large population and for the operationalization of the paradigm of anabolic competence. To summarize, there is not only a demand for expanding the knowledge on protein metabolism fundamentally, there is also demand for the development towards a clinically relevant and feasible method to put the acquired knowledge into clinical practice. Such a method should provide information on severity of disturbed protein metabolism, which would guide better assessment, monitoring, and treatment of disturbed protein metabolism.
AIM OF THESIS
The aim of this thesis is to determine the value of the protein breath test to assess protein oxidation and to consider the role of protein oxidation within protein metabolism. This could contribute to form a methodological and empirical foundation for the paradigm of anabolic competence.
Therefore, the objectives of this thesis were:
a. To determine the capabilities of the non-invasive 13C-milk protein
breath test to measure protein oxidation (Chapter 2).
b. To determine the effect of various milk protein doses, the effect of added energy on protein oxidation, and explore serum amino acid enrichment in parallel with the 13C-milk protein breath test
(Chapter 3).
c. To determine the effect of a protein restricted diet on protein oxidation (Chapter 4).
d. To determine the effect of exercise on protein oxidation (Chapter 5).
e. To determine the actual differences in protein intake between normal weight, overweight, and obese older adults (Chapter 6). f. To describe the paradigm of anabolic competence in relation to
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