Background:
Patients in the intensive care unit often suffer from problems such as high metabolic rate
and high tissue breakdown rate under the severe stress of critical diseases. Phenylalanine is
an amino acid in the human body and is rich in muscle tissue. However, current studies have
found that if the concentration of phenylalanine in the blood is abnormally increased, it may
indicate that the tissue is decomposing in large quantities. This phenomenon may be related
to disease severity. Our recent observational study of patients in the intensive care unit
(ICU) found that patients with elevated blood phenylalanine had a four-fold increase in
mortality within one year.
The most common cause of phenylalanine concentration elevation in the blood is a rare disease
that occurs in infants with congenital metabolic abnormalities, called congenital
phenylketonuria (PKU). The abnormally elevated phenylalanine concentration can lead to
toxicity and cause brain hypoplasia, mental retardation, consciousness disturbance, and even
seizure. These PKU patients must be treated with milk diet that contains low amount of
phenylalanine in order to lower the phenylalanine concentrations. At present, in medicine,
although there have been clear medical care strategy for infants with congenital PKU, there
is still no clear medical care strategy for adult patients.
Research purpose: To investigate whether patients with high blood phenylalanine concentration
in the intensive care unit can reduce the blood phenylalanine concentration by a diet without
phenylalanine, so as to develop personalized high-quality and specific treatments.
Methods:
The investigators aim to have complete study in 70 patients (based on our previous study, to
recruit 70 patients with high phenylalanine, the investigators need 300 ICU patients for
screening).
Intervention protocol:
Once a plasma phenylalanine level of ≥ 95 μM is noted, phenylalanine-free diet (Phenex-2®,
Abbott Nutrition, Ohio, USA; or Phenyl-Free 2®, Mead Johnson Nutrition, Minnesota, USA) is
started as the only nutrition source for enteral feeding, as per the recommendation of
dieticians, in the following 4 days. The phenylalanine-free diet is free of phenylalanine but
is enriched with protein, potassium, tyrosine, and antioxidant micronutrients. After the
completion of the study, diet will be back to normal diet for all patients.
Study end points: the 5-day study period completed.
Blood sampling and examination for Phenylalanine measurement (dry blood spot) Fasting
phenylalanine level will be measured by LC/MS-MS using the dry blood spot method (blood
sample is collected by finger sting) in all included patients in the next morning after
informed consent signed. Blood applied on filter cards is dried for at least 3 h at room
temperature. Dry blood spot cards are stored at room temperature for 0-6 days prior to
analyses. Phenylalanine concentrations in dry blood spot are measured with LC-MS/MS, using a
calibration curve in 0.1 N HCl. The report will be received one day after dry blood spot
collected. Once the result of measurement shows the concentration of phenylalanine ≥95 μM,
this patient is enrolled for formal study. All enrolled patients will take the measurement of
phenylalanine levels by dry blood spot every 12 hours for the following 4 days until study
protocol finished.
Phenylalanine measurement by UPLC:
In the meanwhile, fasting blood will be collected by venipuncture in EDTA-containing tubes on
the morning of screening, 2 and 4 days after diet intervention. The collected blood will be
used to measure phenylalanine level by UPLC (ultra-performance liquid chromatography). Plasma
samples (100μL) are precipitated with 10% sulfosalicylic acid. After protein precipitation
and centrifugation, derivatization is initiated by AQC in acetonitrile. Amino acids are then
analyzed using the ACQUITY UPLC System, consisting of a Binary Solvent Manager, a Sample
Manager, and a Tunable UV detector. The investigators use EmpowerTM 2 Software to control the
system and collect data. Separations are performed on a 2.1×100mm ACQUITY BEH C18 column at a
flow rate of 0.70mL/min. The average intra-assay coefficient of variation is 2.6% for
phenylalanine. The total coefficient of variation is 2.7% for phenylalanine. The detection
limit is 3.3μM for phenylalanine. The linear range is 25-500 μM.
APACHE II scores Disease severity is evaluated by calculating APACHE II score on the first
day of ICU admission.
