The influence of different levels of protein diet on protein oxidation measured by 13CO2 breath test

Malnutrition is a major negative determinant for the clinical outcome of
patients. The hallmark of malnutrition is disturbed protein metabolism, i.e.
increased oxidation of amino acids and decreased incorporation of amino acids,
which leads to loss of muscle mass.
Current detection of malnutrition relies on clinical signs, such as weight
loss, or on identification of risk factors, such as poor dietary intake, and as
such provides the basis for dietary intervention. To initiate, guide and
evaluate (dietary) intervention, 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. This breath
test seems valuable for patient populations, because it elicits the possibility
to develop a simple bedside monitoring tool for quantification of amino acid
oxidation and amino acid incorporation. The underlying principle of the test
is: 13C-protein + O2 --> 13CO2 + H2O.
In our pilot experiments we have found clear differences in the oxidation
kinetics of whey and casein (both repeated 4 times within 1 person). Whey is
oxidized faster than casein. This corresponds with measurements by Boirie et
al. 1997 in which they measured the arrival of amino acids of whey and casein
in blood. A different pilot experiment showed that a 3-day very low protein
diet (repeated 4 times within 1 person) results in less oxidation of protein
compared to a habitual diet rich in protein (repeated 6 times within 1 person),
which shows that the breath test has discriminative powers.

The aim of this study is to determine whether our 13CO2 breath test is
discriminative for different protein diets in healthy young volunteers.

Crossover study.

Controlled isocaloric dietary protein intake for 4 days at different levels of
intake: 0.25 g protein/kg/day, 0.8 g protein/kg/day and 1.35 g protein/kg/day
followed on the fifth day by consumption on of 30 g of naturally low enriched
13C-milk protein, which is produced by NIZO (Dutch Institute for Dairy
Research). After consumption, breath samples will be taken every 10 minutes for
a total duration of 5.5 hours. All subjects will participate in all diet
conditions in a determined sequence separated by at least 1 week between each
diet. Dietsequence: 1.35, 0.80, 0.25 g protein/kg/day.

The subjects will keep a food diary for 4 days in order to collect data on
their habitual diet. On the fourth day, the subjects will collect 24-hour urine
in which urea and creatinine will be measured, which will serve as a measure
for muscle mass. Age will be noted. Physical parameters such as height, weight,
body mass index, waist-circumference and fat-free mass will be measured and/or
determined. Fat-free mass will be measured by bioelectrical impedance, which is
a non-invasive method. Body surface area will be calculated upon height and
weight, using the formula described by Haycock et al. Taken together, these
will serve as their baseline values. The subjects will follow a 4-day protein
isocaloric diet three times, using three levels of protein. The subjects will
collect 24-hour urine every day during each 4-day diet.

During the 4-day diets, subjects will refrain from all 13C enriched products
(e.g. maize, sugar cane and pineapple), alcohol, exercise. On the fourth day,
subjects will start fasting from 22:00 until 09:15 the following morning. The
fifth day will be the day of the breath test in which subjects 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. The planning of the diets will be in
consultation with the participant. There will be at least 1 week between each
diet in order to flush out remaining 13C enrichment and to normalize the
subjects from the effects of the 4-day diet.

The breath tests will be performed under the supervision of the coordinating
investigator. The risks of all described items are considered negligible.
There is no direct benefit for the subjects. Due to the fact that we will
recruit at the Hanzehogeschool, Wiebengacomplex the chances are that the
subjects will be enrolled in a healthcurriculum. Therefore it could be very
interesting for the subjects to get a sense of what research entails.
Especially dieting, fasting and also the breath test. We offer the subjects the
possibility to be present at a final meeting which will take place after the
results have been analyzed.