Protein oxidation: towards a non-invasive assessment of anabolic competence
Reckman, Gerlof
DOI:
10.33612/diss.136482233
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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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Milk protein oxidation in healthy subjects:
A preliminary study
After submitting this thesis to the Thesis Committee, the content of this chapter has been revised and thereafter published in International Dairy Journal 2020:111. Therefore, this chapter is printed as it was submitted to and approved by the Thesis Committee.
G.A.R. Reckman M. Koehorst H. Schierbeek R.J. Vonk
ABSTRACT
The role of protein oxidation in the regulation of whole body protein metabolism is unknown. Previously, it was observed that vigorous exercise led to increased protein oxidation. To further characterize
13C-milk protein oxidation in healthy subjects, the oxidation of
ingested 13C-protein after an overnight fast was measured using
a non-invasive 13C-protein breath test. This approach enables the
analysis of 13C-protein oxidation kinetics and the effect of interfering
factors. It was found that the estimated maximal 13C-milk protein
oxidation was 0.07 g min-1, corresponding to a theoretical maximal
oxidation capacity of ≈1.4 g protein/kg bw/d. No indications were found for preferential oxidation of non-essential amino acids. Combined ingestion of 30 g 13C-whey protein with 30 g glucose
resulted in a 19% decrease of 13C-whey protein oxidation. It was
concluded that exogenous 13C-whey protein oxidation is affected by
co-ingested glucose, which shows that macronutrients other than proteins affect protein oxidation.
1. INTRODUCTION
Fat and lactose form the main energy sources in both human and cow’s milk [1]. After ingestion and digestion the milk proteins are utilized for protein synthesis and thus support maintenance or increase in lean body mass (LBM). Protein forms the main source of nitrogen for the body and protein derived essential amino acids can neither be synthesized, nor stored in large quantities to meet protein synthesis demand [2]. Therefore, regular ingestion and subsequent economical utilization of ingested protein is of vital importance. Prolonged dysregulation of protein metabolism leads to loss of LBM as seen in for example malnutrition. Consequently, malnutrition is associated with increased risk of complications such as impaired wound healing, increased hospital length of stay, and higher mortality [3,4]. The loss of protein through dysregulation of protein oxidation might have medical relevance.
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It was recently observed that exogenous protein oxidation, measured non-invasively with a 13C-breath test was increased after a short
bout of vigorous aerobic exercise in healthy males [5]. In another study in healthy males, exogenous protein oxidation after a 4-day protein restricted diet was not diminished, but the response was highly variable (~0.65 g protein/kg bw/d) [6]. These results showed that the protein oxidation in healthy subjects is variable by a short change in physiological conditions. Our studies also showed that fitting the concentration model to the data resulted in a mean R2 value of >0.9. For reference, a R2 value of 1.0 means a perfect fit
between the model and the data. This is supportive evidence that the breath test captures the oxidation process well and therefore suggests that the oxidation variation found with the breath test has a biological origin.
Comparing the oxidation kinetics under different physiological circumstances can increase our knowledge about the role of the oxidation process in overall protein metabolism. This can lead to improved treatment aimed to minimize loss of LBM. In the present study we address four separate aims: The first aim was to compare the oxidation kinetics of 13C-lactose, 13C-milk protein, and 13C-milk
fat. The second aim was to characterize the dose response curve of
13C-milk protein and cumulative protein oxidation. These experiments
also enabled the estimation of the protein oxidation capacity. The third aim was to investigate whether the kinetics of protein oxidation measured in breath is comparable to the appearance kinetics of individual amino acid concentrations measured in blood plasma. In addition, it enabled to determine whether non-essential amino acids are more prone to oxidation compared to essential amino acids based upon potential differences in their the appearance kinetics. Therefore, blood samples were collected in parallel with 13C-milk
protein breath test. The fourth aim was to investigate the influence of added energy in the form of glucose on whey protein oxidation as it is known that protein synthesis is diminished when insufficient energy is available [7].
2. MATERIALS AND METHODS
2.1 Subjects
Healthy subjects, mean age 22 years, participated in the experiments. Exclusion criteria were: having a diagnosed disease and/or being medically treated (e.g. diabetes mellitus and heart condition), milk protein allergy or intolerance. Mean height and weight of the males (n = 15) was 1.86 m ± 0.09 and 80.5 kg ± 10.8, respectively. Mean height and weight of the females (n = 27) was 1.72 m ± 0.07 and 63.2 kg ± 7.6, respectively. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of the University Medical Center Groningen (2016.144).
2.2
13C-substrates
The following naturally enriched 13C-substrates were used: total 13C-milk proteins (consisting of 20% whey protein and 80% casein), 13C-whey protein, 13C-milk fat, 13C-lactose and 13C-glucose. The
characteristics of the 13C-enriched milk components are described
elsewhere [8]. The 13C-enrichment of the 13C-milk protein was
1.0956116%. The 13C-glucose (maize sugar) was bought at a local
supermarket and the 13C-enrichment was 1.0988139%. Each test
drink consisted of dissolving the 13C-substrate in 500 ml water. The
standard dose dissolved was 30 g, as this is the amount of protein responsible for maximal protein synthesis response [9,10].
2.3 STUDY PROTOCOL
Prior to each breath test the subjects were instructed to minimize the consumption of naturally enriched 13C-products, such as corn
and sugarcane, for at least one day prior the breath test to keep baseline 13C-enrichment low. The subjects were also instructed to
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arrived at the test day after an overnight fast starting from 22:00 PM onwards. Drinking water, tea or coffee without sugar of milk was allowed during the overnight fast.
Before the consumption of the test drink, basal breath samples were collected in threefold. The test drink ingestion started at 09:15 AM and was completed within 5 min and was followed by 10 min interval collection of breath samples, from 09:25 AM until 14:45 PM (5.5 h). Subjects remained seated upright at a desk for the duration of the breath test and were allowed, for example, to read, to write, and to work on their laptop.
In the first experiment the oxidation rate and cumulative oxidation of 13C-lactose, 13C-milk protein, and 13C-milk fat was measured in
one male subject. This subject repeated the ingestion of 30 g of lactose, 30 g of milk protein, and 30 g of milk fat, 4, 6, and 4 times respectively.
In the second experiment we investigated the cumulative oxidation of 13C-milk protein with different doses of 13C-milk protein in 10 male
and 16 female subjects. Dose of 10 g was ingested by 3 males and 4 females. One of the males repeated the 10 g dose 4 times, which brings the total datapoints to 10. Dose of 30 g was ingested by 4 males and 4 females. One of the males repeated the 30 g dose 6 times, which brings the total datapoints to 13. Dose of 50 g was ingested by 3 males and 4 females. One of the males repeated the 50 g dose 4 times, which brings the total datapoints to 10. Dose of 60 g was ingested by one male subject and was repeated 4 times. Dose of 70 g was ingested by 2 males and 4 females. One of the males repeated the 70 g dose 4 times, which brings the total datapoints to 9.
The third experiment which investigated the amino acid accumulation in blood plasma after the ingestion of 30 g of 13C-milk protein was
measured in one male subject with 3 analytical analyses. Blood was collected at t=0, 60, 120, and 180 min.
The fourth experiment was aimed to determine if the protein oxidation was influenced by administration of other energy components. Therefore, glucose and whey protein were administered simultaneously to the test drink prior to the breath test, using both the combination of 13C-whey protein + glucose and whey protein
+ 13C-glucose. The oxidation of 30 g 13C-whey protein with and
without added 30 g glucose was measured in 5 male and 11 female subjects. The ingestion of 30 g 13C-glucose was performed by 1 male
and 2 females (n = 3). The ingestion of 30 g 13C-whey protein was
performed by 1 male and 3 females (n = 4). The ingestion of 30 g 13C-glucose + 30 g whey protein was performed by 1 male and
3 females (n = 4). The ingestion of 30 g 13C-whey protein + 30 g
glucose was performed by 2 males and 3 females (n = 5).
2.4 BREATH GAS ANALYSIS
The isotope ratio mass spectrometer (IRMS) (Delta XL, Thermo Fisher Scientific, Bremen, Germany) measures a reference sample twice before each run and twice after every tenth sample and data are expressed as delta value per breath sample. The gas chromatgraph (GC) used was an Agilent 6890 series (Agilent Technologies, Amstelveen, The Netherlands). Calibration of the IRMS was performed with known reference gases against δ 13C-Vienna Pee
Dee Belemnite. Pee Dee Belemnite has a defined 13C:12C ratio of
0.01123720 and a δ13C value of zero. Comparison of breath samples 13C-enrichment against this highly 13C-enriched Pee Dee Belemnite
standard results in a negative delta value for breath sample. Further details of the IRMS and GC equipment, the protocols and sampling methods used are described in detail previously [8].
2.5 CALCULATIONS
The acquired delta values of the breath samples were used to calculate the percentage of substrate oxidized per h. Details are described elsewhere [6].
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2.6 ANALYSIS OF ENRICHMENT OF PLASMA
AMINO ACIDS
Blood samples were collected in heparin tubes at t=0, 60, 120, and 180 min. Within one h the blood samples were centrifuged at 3000 rpm for 10 min. Thereafter, the blood plasma was collected and stored at -20 °C. Sample preparation: To 50 µL of plasma was added 20 µL hydrochloride (HCl; pH<3), 20 µL internal standards mixture, and 200 µL prewashed Dowex solution (Ag 50W-X8 H+, 200-400 mesh), and the samples were thoroughly shaken and centrifuged at 4000 rpm for one minute. The supernatant was discarded, and the pellet was washed twice with 1.0 mL H2O. After
another centrifugation step, the amino acids were extracted from the remaining pellet using 0.5 mL ammonia 6N and transferred to a new vial. The original vial was rinsed with 0.2 mL ammonia 6N, and 0.2 mL of the supernatant was added to the new vial and evaporated with a speedvac (GeneVac miVac, GeneVac Ltd., Ipswich, England). The samples were redissolved in 200 µL H2O and derivatized with
ethyl chloroformate (ECF) by adding 140 µL ethanol/pyridine (4:1) and 20 µL ECF. The samples were left at room temperature for 5 minutes before extraction with 400 µL hexane/dichloromethane/ ECF (50:50:1). After centrifugation, 200 µL of the supernatant was transferred to a vial. The extraction step was repeated, and the second time 400 µL of the supernatant was added to the first portion. The combined solutions were evaporated under a stream of N2 at room temperature, then redissolved in 50 µL ethylacetate and
analyzed in triplicate [11].
The 13C-enrichment of the following amino acids was determined with
IRMS in triplo; alanine, glycine, valine, leucine, isoleucine, proline, phenylalanine, lysine, and tyrosine. 13C amino acid enrichments
in plasma were measured by a Delta-XP IRMS which was coupled online with a trace GC and a combustion interface type 3 (Thermo Fisher Scientific, Bremen, Germany). Aliquots of 1 µL of the ethyl acetate suspension containing the amino acid derivatives were introduced into the GC system by a CTC PAL autosampler (CTC,
Basel, Switzerland). Chromatographic conditions used were the same as described previously. After separation using capillary GC, amino acids were combusted online at 940°C and introduced as CO2
into the isotope ratio MS, where the [13C/12C] ratio was measured for
each amino acid [11].
2.7 STATISTICS
In these exploratory experiments with relatively small sample sizes, possible differences were tested for with the Student’s t-test with Microsoft Excel (2013). Differences were considered statistically significant at p < 0.05. All data are represented as mean ± standard deviation.
3. RESULTS
3.1 Oxidation of various milk components
In table 1 it is illustrated that the various milk components, 13C-lactose, 13C-milk protein, and 13C-milk fat are oxidized to a considerable
amount over 120 min after ingestion. 13C-Lactose showed both the
highest oxidation rate and highest cumulative oxidation, followed by
13C-milk protein, and 13C-milk fat.
Table 1. The oxidation rate and cumulative oxidation at t=120 min of,
13C-lactose (50 g; n = 4), 13C-milk protein (30 g; n = 6), and 13C-milk fat
(30 g; n = 4).
Oxidation rate
(% oxidation per h) Cumulative oxidation(% of given dose)
13C-lactose 8.1 ± 1.7 9.7 ± 1.4
13C-milk protein 7.2 ± 1.5 7.6 ± 1.4
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3.2 RELATIONSHIP BETWEEN MILK
PROTEIN DOSE AND PROTEIN OXIDATION
CAPACITY
The mean cumulative amount of milk protein oxidized over 240 min increased with increasing dose of ingested milk protein. This positive relationship weakened towards the 70 g dose as there was no significant difference in cumulative oxidation between the 60 and 70 g dose (p = 0.161). The differences were significant between 10 and 30 g (p < 0.001), 30 and 50 g (p < 0.001), and 50 and 60 g (p = 0.019). The maximal rate of exogenous oxidation was found to be 0.07 g/min with the ingestion of 60 g of 13C-milk protein.
Figure 1. Cumulative oxidation of different doses of 13C-milk protein over
4 h. Cumulative 13C-protein oxidation expressed in g as measured with the
breath test performed with different doses of 13C-milk protein (10, 30, 50,
60 and 70 g). The total number of measurements per dose were: 10 g (n = 10), 30 g (n = 13), 50 g (n = 10), 60 g (n = 4) and 70 g (n = 9). X-axis shows the ingested dose (g) used in the breath test, y-axis shows the amount of cumulative protein oxidized (g) over 4 h.
3.3 BLOOD PLASMA AMINO ACID
CONCENTRATIONS OVER TIME
To investigate whether non-essential amino acids were oxidized faster than essential amino acids, the 13C-enrichment of nine different
amino acids in blood plasma over 180 min after the ingestion of 30 g of 13C-milk protein was analyzed. It was assumed that the decrease
in enrichment of blood plasma amino acids could also represent breath amino acid oxidation. The 13C-enrichment is expressed as
delta values and it shows that the plasma amino acid enrichment ranged between -29 to -21 δ13C. The maximum enrichment of eight
out of nine amino acids was found at 60 or 120 min and started to decrease towards 180 min.
Figure 2.Delta values of nine different amino acids in blood plasma over 180 min after the ingestion of 30 g of 13C-milk protein, expressed as delta
values. Ala = alanine, Gly = glycine, Val = valine, Leu = leucine, Ile = isoleucine, Pro = proline, Phe = phenylalanine, Lys = lysine, Tyr = tyrosine.
3.4 EFFECT OF GLUCOSE
To determine if the protein oxidation is influenced by administration of other energy components, glucose and whey protein were
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ingested simultaneously prior to the breath test. The cumulative oxidation of solely ingesting 13C-glucose was 5 g, whereas solely
ingesting 13C-whey resulted in 10 g cumulative oxidation (p < 0.001).
Cumulative oxidation of 13C-glucose increased with concomitant
ingestion of unlabeled whey protein (p = 0.011). Cumulative oxidation of 13C-whey protein decreased with concomitant ingestion
of unlabeled glucose (p = 0.010).
Figure 3. Total oxidation over 255 min in g, from left to right, of 13C-glucose
(n = 3), 13C-whey protein (n = 4), 13C-glucose + whey protein (n = 4), and 13C-whey protein + glucose (n = 5), respectively.
4. DISCUSSION
The breath test demonstrated that the ingestion of 13C-lactose, 13C-milk protein, and 13C-milk fat led to distinctive oxidation kinetics
and cumulative oxidation in healthy subjects. The distinction in oxidation rate of the different substrates is likely the result of how each of the different substrates interacts with organs at several levels for instance with the stomach, which subsequently affects the stomach emptying rate. The difference in stomach emptying rate
is a known relevant factor when comparing the oxidation kinetics between different substrates [12]. For example, whey protein and casein have been described in the literature as fast and slow proteins respectively based on their 13C-leucine derived appearance rate in
blood [12]. Additionally, it is known that the intestinal absorption capacity of the different macronutrients is not uniform as the capacity is 86.9% for protein, 87.3% for carbohydrates, and 92.5% for fat [13]. However, it is important to realize that the process of stomach emptying rate is not a significant variable when comparing the oxidation kinetics of theparticular ingested substrate under different physiological circumstances.
To analyze the capacity of the protein oxidation pathway, the ingestion of different doses of 13C-milk protein was investigated.
With increasing doses there was increasing protein oxidation. With the exception from the ingestion of 60 g of 13C-protein compared to
70 g dose, there was no further increase in cumulative exogenous
13C-protein oxidation. This could be due to the time frame over which
the oxidation was measured. The shape of the oxidation curve of the 70 g dose formed a plateau from 120 min onwards, whereas the curves of the other doses formed a peak between roughly 120 and 180 min after which the oxidation rate gradually decreased. The absolute standard deviation range was between 1.20 and 2.00 g for the different doses. However, for the 10 g dose the absolute standard deviation was 1.73 g, which results in a relative standard deviation of 17.3%, which was the highest relative standard deviation of all the doses. This was probably the result of the low absolute 13C-content
in this low 10 g dose. A possible explanation for our finding that increasing dose leads to increasing protein oxidation could be that there is a limited capacity or demand for protein synthesis in healthy subjects. This suggests that the consumption of more than 20 to 30 g of protein per meal does not provide any extra metabolic benefits. All protein ingested beyond the demand will be directed towards oxidation. A study whereby subjects after resistance exercise ingested different doses of whole egg protein found that 20 g of
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protein resulted in the maximal protein synthesis response [14]. All dietary protein in excess stimulated protein oxidation [14].
Based on the exogenous oxidation kinetics of various doses of 13C-milk proteins from 10 to 70 g it was found that after the
ingestion of the 60 g dose that the theoretical maximal capacity of exogenous oxidation was about 0.07 g/min. Thus, if the body would sustain this maximal oxidation rate for 24 h, the amount of protein, which can be theoretically be eliminated by the body, based on the measured maximal oxidation rate, is roughly 98 g protein · d-1. The
maximal oxidation results, which were measured in an adult male of 70 kg, suggest that ≈1.4 g protein/kg bw/dcan be eliminated by oxidation if the maximal oxidation rate would be sustained. In comparison, the recommended dietary protein intake by the World Health Organization is currently 0.8 g protein/kg bw/d [15] is in the same order of magnitude as the theoretical maximal oxidation rate of 1.4 g protein/kg bw/dwhich was observed with the breath test. In a weight stable individual the dietary protein intake must be in balance over time with protein loss. The finding that the estimated amount of maximal protein oxidation per day is higher and in the same range as dietary protein intake per day is therefore expected in individuals who have stable lean body mass. In our calculation, it is assumed that 100% of the protein is absorbed in the small intestine. However, literature data indicate that the mean intestinal absorption capacity is 86.9% for protein [13]. Furthermore, first it was assumed that all the administered carbon atoms ultimately will be converted into CO2 and do not remain trapped in the bicarbonate
pool [16]. Second, the CO2 production was estimated instead of measured, which also leads to the quantitative oxidation being an estimation.
A different method to measure protein oxidation, the indicator amino acid oxidation (IAAO) method, is able with the use of a L-[1-13C]phenylalanine tracer to measure the requirement of one
indispensable amino acid at a time [17]. With the IAAO method, the oxidation limit of acute leucine supplementation in five healthy men
has been measured [18]. The maximal oxidation capacity of leucine was determined to be between 550 and 700 mg/kg bw/d, which corresponds to a protein intake between 3.7 and 4.7 g protein/kg bw/d [18]. Based upon the IAAO method the requirement of protein intake is determined to be 1.2 g protein/kg body weight/d, which is close to the ≈1.4 g exogenous protein oxidation/kg bw/dwhich was found with the estimated maximal exogenous protein oxidation rate. Exhalation of oxidized protein is the endpoint of the processes of digestion, uptake in the bloodstream, and uptake by the cells of various organs. It is therefore conceivable that the protein oxidation results obtained from the breathgas samples are related to measurements of amino acids enrichment in blood plasma. To explore this, blood samples were collected in parallel with the collected breathgas samples after the 30 g 13C-milk protein administration. It was found
that the baseline 13C-enrichment of the nine measured amino acids
in blood plasma had a range of 13C-enrichment between -28.7 ±
0.2 to -22.1 ± 0.2. Even though, each of the nine amino acids had different 13C-enrichments at baseline, their mean enrichment of
-25.83 ± 2.055 at baseline was quite similar to the mean value of the triplicate baseline breath samples which was -25.80 ± 0.086. It seems logical that the average 13C-enrichment of the 9 amino
acids was comparable to the 13C-enrichment of the 13CO
2 exhaled,
because the 13CO
2 signal consists of all amino acids oxidized. It
was assumed that what is measured in breath is the oxidation of almost exclusively 13C non-essential amino acids. However, based
on these preliminary results similar enrichment patterns were found over time in blood plasma for both essential and also some non-essential amino acids with respect to the breath 13C(O
2) enrichment
over time, which suggests that there is no distinction in oxidation of essential and non-essential amino acids. However, there are two exceptions. Both alanine (non-essential) and glycine (non-essential) show minimal enrichment changes over time. Here two possible explanations for these findings are provided. First, alanine and glycine were preferably extracted by the splanchnic bed (first pass
3
effect) and were therefore immediately oxidized to 13CO
2 and exhaled
before reaching the systemic circulation. However, no evidence was found in the literature which confirms this explanation. Second, the concentrations of glycine and alanine were lower than needed for protein synthesis relative to the other amino acids. In other words, glycine and alanine were rate limiting for protein synthesis to occur. So far, the discussion describes experiments in which only one macronutrient at a time was ingested. In these type of experiments the body has no dietary choice other than to utilize or to break down the ingested protein. Therefore, experiments were performed to determine the effect of adding 30 g of glucose to 30 g of whey protein. Separately, both substrates have comparable oxidation kinetics which consists of a fast and high oxidation peak compared to milk protein oxidation kinetics. It was found that cumulative oxidation of 13C-glucose increased with concomitant ingestion of
unlabeled whey protein. Moreover, cumulative oxidation of 13C-whey
protein decreased with concomitant ingestion of unlabeled glucose. This suggests that the ingestion of carbohydrates in addition to the ingestion of protein enables a more economical utilization of protein. Detailed understanding of how, for example, carbohydrates effect the utilization of protein is of great importance for both individuals with and without disease with the aim to maintain or gain lean body mass.
5. CONCLUSION
In the current study, it was demonstrated that the process of exogenous protein oxidation can be very efficient and has a high capacity, which indicates that the protein oxidation pathway easily adapts to the ingestion of acute high protein doses. The observation that exogenous whey protein oxidation is affected by co-ingested glucose shows that ingested macronutrients other than protein affect protein oxidation.
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