Protein oxidation in stable renal transplant patients measured with a breath test

The human body has two options to utilize protein-derived amino acids. Ingested
protein is used for synthesizing new proteins until requirements are met, and
all surplus protein will be oxidized for energy. Disturbed protein metabolism,
i.e. increased oxidation of amino acids and/or decreased incorporation of amino
acids, is a major negative determinant for the clinical outcome of patients.
The role of disturbed protein metabolism, which can lead to loss of muscle
mass, has not been fully elucidated. To initiate, guide and evaluate
interventions to minimize or prevent loss of muscle mass, it would be highly
useful to be able to monitor the derangements in protein oxidation directly,
but until recently a bedside tool to assess protein oxidation was not available.
A recently developed non-invasive low naturally enriched 13C-protein 13CO2
breath test has shown to be able to quantify the oxidation rate of ingested low
naturally 13C-enriched milk-proteins derived amino acids directly and therefore
elicits the possibility to develop a simple bedside monitoring tool for
quantification of amino acid oxidation. The underlying principle of the test
is: 13C-protein + O2 * 13CO2 + H2O.
Generally, there are two major fates for the amino acids derived from protein
ingestion: 1) uptake into amino acid pool thereby being available for protein
synthesis and 2) oxidation of excess amino acids. All amino acids that are
oxidized, are not incorporated [6].
For the healthy adult population, a protein intake of 0.8 g/kg bw/day is
recommended. However, stable renal transplant patients benefit from a higher
protein intake of *1.1 g protein/kg bw/day, suggesting higher protein
requirements. Possible reasons for higher protein requirement could be the
chronic use of corticosteroids in renal transplant patients, a low grade
inflammatory state, or other. Corticosteroids are used to prevent graft
failure, but also have side effects, such as muscle wasting/protein catabolism.
Whether protein catabolism is altered in renal transplant recipients however,
is unknown.
The question is whether in stable renal transplant patients higher levels of
protein catabolism (oxidation) can be measured with the 13C-milk protein breath
test, as compared to age-controlled healthy subjects.

To study whether stable renal transplant patients show higher protein oxidation
measured with the 13C-breath test as compared to healthy kidney donor subjects.

Case control study

The following data will be already collected as part of the standard medical
dossier: age, height, weight, Body Mass Index, waist-circumference and fat-free
mass. Fat-free mass will be measured by bioelectrical impedance analysis, which
is a non-invasive method. Body surface area will be calculated upon height and
weight, using the formula described by Haycock et al. [4] which is necessary
for the calculation of 13CO2 produced (mmol/hour) by the subject. 24-hour urine
collection is also part of the standard medical dossier. From the 24-hour urine
collection, urea and creatinine are measured, which will serve as a measure for
protein intake (estimated by Maroni formula) and lean body mass, respectively
[5].
Resting metabolic rate will be measured by indirect calorimetry, which will be
used as a covariate.
Before the breath test, all subjects will keep a food diary for three days in
order to collect data on their habitual diet (main interest: energy and protein
intake).

Two days before the breath test, all subjects will be asked to refrain from all
13C-enriched products (e.g. maize, sugar cane and pineapple), alcohol, and
exercise in order to keep the 13C content in each subjects as low as possible.
On the day of the breath test, the subjects will give breath samples by
exhalation through a drinking straw into a glass (12 ml) container. The test
drink, which is part of the breath test, will consist of 30 g of milk protein
dissolved in 500 ml water (t=0). The planning of the experiment will be in
consultation with the subjects.

Blood sampling will performed at three time points on the breath test day. At
three time points, t=-10, t=120 and t=240 minutes blood will be drawn, unless
described otherwise. The blood samples will be analysed for:
- Glucose
- Insulin
- C-reactive protein (only at t=-10)
- Insulin-like growth factor 1 (only at t=-10)