Study Design and Participants Selection
We conducted a retrospective longitudinal study of patients being treated for PKU and exclusively followed-up at Centro de Genética Médica, Centro Hospitalar Universitário do Porto. Patients were given CGMP-AA as their primary nitrogen source if they had difficulties with taking L-AA or if CGMP-AA was considered a suitable alternative.
The inclusion criteria were diagnosis of PKU, absence of co-existent conditions and taking CGMP-AA as part of their protein substitute prescription. Exclusion criteria were the use of sapropterin, use of large neutral amino acids, pregnancy, lack of biochemical markers or body composition analysis.
All patients followed a low-Phe diet, avoiding high protein foods, and supplemented with L-AA and special low protein foods. Phe intake was controlled using a Phe exchange system (1 exchange = 20 mg of Phe).
The present study included data on 11 patients from a previous study reported by Pinto et al.  but an extended follow-up period of 2.9 years if patients remained on CGMP-AA.
Data was collected between May 2013 and December 2018, whereas the previous study by Pinto et al.  retrieved data until February 2016. The first annual nutritional status evaluation (ANSE) was performed when taking L-AA and the last ANSE with the addition of CGMP-AA. The baseline assessment was conducted for a mean of 6 months before CGMP-AA commencement when patients were taking L-AA only as their primary nitrogen source. The last assessment was carried out when CGMP-AA had been given for a mean of 29 months. CGMP-AA either fully or partially replaced L-AA; CGMP-AA contribution to the total protein substitute intake was: 100%, n=4, 50% to < 100%, n=4, < 50%, n=3). This was according to patient’s protein substitute preference or by the nutritionist’s prescription after assessing metabolic control, nutritional status, nutritional intake, anthropometry and body composition.
The PKU classification was based on the Portuguese guidelines as follows: hyperphenylalaninaemia (HPA) [blood Phe < 360 µmol/L (6 mg/dL)]; mild PKU [blood Phe ≥ 360 µmol/L and ≤ 1200 µmol/L (≥ 6 mg/dL and ≤ 20 mg/dL)] and classical PKU [blood Phe > 1200 µmol/L (> 20 mg/dL)] .
Blood Phe and Tyr control was also evaluated over 2-time intervals as follows: i) from May 2013 until CGMP-AA introduction (13 ± 5 months) and ii) from CGMP-AA introduction until the last ANSE taking CGMP-AA (29 ± 16 months). The median number of blood Phe measurements while patients were taking L-AA was 11 (7-16) and with CGMP-AA was 40 (21-71).
The study design is presented in Figure 1.
Figure 1. Study design.
ANSE: annual nutritional status evaluation; CGMP-AA: casein glycomacropeptide supplements; HPA: hyperphenylalaninaemia; L-AA: phenylalanine-free L-amino acid supplements; PKU: phenylketonuria.
Data collection and Outcomes Measured
The following parameters were collected from patients’ records by trained research nutritionists (M.J.P. and A.P.):
Height (cm) was measured with light clothes, using a stadiometer (SECA GmbH & CO., Hamburg, Germany) (measured to the nearest millimetre) and weight (kg) was assessed with a mechanical weighing scale (SECA GmbH & CO., Hamburg, Germany) (measured to the nearest 100 g). Waist circumference (WC) (cm) was measured in the standing position, midway between the lower rib margin and the iliac crest, at the end of a normal exhalation, to the nearest millimetre and using a non-extensive metric tape. Anthropometric measures were performed by trained nutritionists (M.F.A. and J.C.R.).
Body composition analysis
Body composition was performed in the fasted state using a single-frequency (50 Hz) bioelectrical impedance analyzer, Akern, Quantum/S (RJL systems, Florence, Italy) according to described standards and measurement conditions. Total fat mass, percentage of body fat mass, percentage of lean mass and phase angle were assessed in the programme BodyGram™️ version 1.3 (Akern Bioresearch, Florence, Italy) which uses validated prediction equations . The measures were carried out by trained nutritionists (M.F.A. and J.C.R.).
Total protein intake, natural protein intake (g/kg/day), protein equivalent from the protein substitute (g/kg/day and g/day), Phe intake (mg/day) from both natural foods and CGMP-AA, Tyr (g/day) and Leu (g/day) intake from the protein substitutes were calculated using a 24-hour food recall. Dietary assessments were performed by M.F.A. and J.C.R..
Blood samples for biochemical analysis were taken after an overnight fast. Uric acid, glucose, creatinine, urea, glycated haemoglobin (HbA1c), lipid panel [total cholesterol, triglycerides, high-density lipoprotein (HDL)-cholesterol, low-density lipoprotein (LDL)-cholesterol, very low-density lipoproteins (VLDL)-cholesterol, apolipoprotein A1, apolipoprotein B], iron, transferrin, ferritin, albumin, homocysteine, prealbumin, C-reactive protein, insulin, calcium, phosphorus, selenium, zinc, vitamin B12, vitamin D and folic acid were determined. Blood urea nitrogen (BUN) was calculated from urea and homeostatic model of insulin resistance (HOMA-IR) was calculated as follows: HOMA-IR = fasting plasma glucose (mg/dL) × fasting serum insulin (μU/mL)/405 .
A Critikon Dinamap™️ vital signs monitor 8100 was used to measure resting systolic and diastolic blood pressure and heart rate with individuals seated for at least 5 minutes, according to standard techniques.
Blood Phe and Tyr were measured by fasting blood spots and analysed by tandem mass spectrometry. Patients or caregiver were trained to perform routine blood spots. Good metabolic control was defined as median blood Phe level within 120-360 µmol/L (2-6 mg/dL) ≤ 12 years or 120-480 µmol/L (2-8 mg/dL) > 12 years of age, according to the Portuguese criteria . The percentage of median blood Phe within the target range was also calculated.
Body mass index (BMI) was calculated as the ratio of weight (kg) and height (m2) and classified according to the World Health Organisation (WHO) criteria. Overweight and obesity were defined when BMI was between 25.0 and 29.9 kg/m2 or was ≥ 30.0 kg/m2, respectively . The Anthro Plus® programme version 1.0.4 was used to calculate the BMI z-scores for individuals under 19 years. Overweight and obesity were identified when the BMI z-score was between 1 and 2 standard deviations (S.D.) or above 2 S.D., respectively .
All statistical analyses were performed with SPSS® version 26.0 for Mac (IBM Company, Chicago, IL, USA). Normal distribution was checked using Shapiro-Wilk test. Categorical variables are expressed as percentage and continuous variables as mean ± S.D. or median (P25-P75) where appropriate. Paired t-test and Wilcoxon signed ranks test was used to analyse the differences when normal distribution or non-normal was found, respectively. The McNemar test was used to determine if there are differences on a dichotomous dependent variable between 2 related groups. Significance was set at the level of p-value less than 0.05.