Human Protein Turnover Project

Skeletal muscle maintenance is determined by the balance between muscle protein
synthesis and breakdown rates, with temporary changes in either protein
synthesis or breakdown allowing net muscle protein accretion or loss. A
routinely applied method to study skeletal muscle protein metabolism in vivo in
humans is continuous intravenous infusions of stable isotope labelled amino
acids. This creates the possibility to *trace* the labelled amino acids at
different points in their metabolism using mass spectrometry. The interesting
advantage of this method is that it allows for a steady delivery of the amino
acid tracer in the blood stream. Hence, the subsequent amino acid tracer
incorporation into the skeletal muscle will provide more insight in muscle
tissue protein turnover rates. This contemporary stable isotope methodology has
been applied for several decades to show that skeletal muscle tissue turns over
at a rate of approximately 1-2% per day. This implies that skeletal muscle
tissue is largely rebuilt within a 2-3 month period.

Because of its visibility and accessibility muscle protein has been the main
interest of most stable isotope studies in humans. However, a recent bovine
study from our lab suggests that organ protein turnover is much higher than
muscle protein turnover, indicating that these tissues show rapid
reconditioning. To the authors* knowledge, nevertheless, no studies have been
performed to employ contemporary stable isotope methodology to allow a
comprehensive in vivo assessment of protein synthesis rates in various organ
tissues in humans. Furthermore, we are interested in neoplasm protein synthesis
rates in tumour tissue. Some studies on tumour metabolism found a higher
protein synthesis rate in colonic and various gastrointestinal malignancies.
However, these studies used the *flooding dose* tracer technique which has
limitations compared with the *constant infusion* tracer technique. Therefore,
besides evaluating organ-specific protein tissue protein turnover, neoplasm
protein turnover rates will be assessed using continuous intravenous infusion
of labelled amino acids.

In conclusion, the present study will provide unique insight in the dynamics of
organ- and neoplasm specific tissue protein turnover rates by assessing typical
turnover rates in skin, bone, heart, lung, kidney, oesophagus, stomach, colon
and brain. In addition to data collected in a separate study (METC 13-3-068) on
protein turnover rates in small intestine, liver, pancreas and subcutaneous and
visceral fat tissue, the current study will evaluate tissue-specific turnover
rates in the perspective of skeletal muscle protein turnover in a wide variety
of clinical settings. This basic knowledge on human tissue turnover is of
important clinical relevance for many if not all organ diseases as well as
pressure ulcers, cachexia and protein depletion and will provide knowledge to
better understand the process of organ (re)-growth and the development of
neoplasms.

The aim of this study is to investigate the differences in organ-specific
protein turnover rates by means of stable isotope labelled amino acid infusion
in patients undergoing surgery.

Primary objective:
To compare protein synthesis rates, expressed as fractional synthetic rate
(FSR) [%/h] and enrichments [mole percent excess, MPE] of skin, bone, heart,
lung, kidney, oesophagus, stomach, colon and brain tissue with the FSR and MPE
in vastus lateralis muscle as reference tissue in patients undergoing surgery.

Secondary objective:
To compare protein synthesis rates and enrichments in tumour tissue with those
in organ tissue in which the specific tumour resides.

The following hypotheses will be investigated:
1. Organ-specific protein synthesis rates and MPE will be higher than mixed
muscle protein synthesis rates.
2. Tumour-specific protein synthesis rates and MPE will be higher than
organ-specific protein synthesis rates.

Multicentre cross-sectional study conducted at Atrium Medical Centre Heerlen,
Maastricht University Medical Centre and Catharina Hospital Eindhoven.

The outline of the study protocol will be the same irrespective of the studied
organ. A separate overview of specific characteristics per studied organ is
provided in the attached protocol. The test day will start at the day of
surgery and the patients will be already admitted at the hospital.

Two and a half hours before start of surgery (t=-150 min), a polyethylene
catheter will be inserted in the dorsal hand vein for blood sampling. A
background plasma and serum sample of 10 mL of blood each will be taken for
measurement of haemoglobin, leukocyte count, HbA1c, fasting insulin, fasting
glucose, C-reactive protein (CRP), interleukin-6 and TNF- * and for
albumin-bound phenylalanine enrichment / background phenylalanine labelling. At
t=-150 min patients receive a single intravenous priming dose of
L-[ring-13C6]-phenylalanine and L-[3,5-2H2]-tyrosine labelled amino acids, and
thereafter a continuous infusion of both tracers at a rate of 0.05 µmol/kg body
weight/min. Subsequent plasma samples (10 mL) will be drawn at regular time
points, i.e. at t (min) = -120, -90, -60, and -30 min. For most surgical
procedures, as part of standard care, the anaesthetist will place a
polyethylene catheter in the radial artery for per-operative blood testing.
This catheter, or else an extra hand vein catheter, will be used for future
blood sampling (5 mL), i.e. at t= -15, 0, 15, 30, 60, 90, 120, 150 min etc.
until the muscle biopsy and tissue samples are taken. Following adequate
exposure of the organs, the organ-specific samples and m. vastus lateralis
biopsy will be taken. Thereafter tracer infusion will be stopped. A schematic
overview of the study is shown in Figure 1; time of exposure will be different
per organ/tissue (t = x min). Since all measurements and data collection are
done during hospital stay and surgery, there will be no extra time-investment
of the patient for this study day.

There are possible risks involved when participating in this study. Insertion
of the catheters in an arm vein or hand vein is comparable to a normal
venipuncture and the only risk is a small local hematoma. The same counts for
the muscle biopsy. The incision made for obtaining the muscle biopsy will be
done by an experienced physician and will heal completely, leaving a minimal
scar of 0.5-1 cm. Within our research group we have extensive experience with
taking muscle biopsies. During the follow up, several days after taking the
biopsy, no complications have been reported. The samples of the other tissues
will be taken from parts that will be resected during surgery, thus preventing
the risk for permanent complications. Potential perioperative bleeding of the
tissue will be electrocoagulated by the surgeon. All surgical procedures will
be performed by surgeons specialized in the particular organ/type of surgery.

The labelled amino acid tracers applied in this experiment are not radioactive
but stable isotopes and are completely safe. The production of the tracers for
intravenous administration will occur in a sterile environment according to GMP
guidelines in a compounding pharmacy (see Attachment K5.1: investigational
medicinal product dossier (IMPD)).