This is the first longitudinal, comparative prospective study over 12 months reporting the use of CGMP-AA2 compared to conventional protein substitute L-AA in children with PKU. After 12 months of using the modified CGMP-AA2 there were no differences for Phe, Tyr, Phe:Tyr ratio and anthropometry compared to the control group using L-AA. However, in the same period within the CGMP-AA2 group, blood Phe concentrations significantly increased although this was only by 30 μmol/L. When comparing children < 12 years of age, within the CGMP-AA2 group, the same small but consistent significant increase for blood Phe concentrations were evident. Weight and BMI z scores significantly increased within the CGMP-AA2 group. Plasma and whole blood selenium improved although ferritin decreased but all nutritional measurements remained within the reference ranges. Identifying the reason for these physical and biochemical changes over 12 months in the CGMP-AA2 group is important to assess the suitability of using CGMP-AA2 as a protein substitute in children with PKU.
We have previously reported a small but significant increase in blood Phe concentrations in the first group of children recruited to the pilot study using CGMP-AA1 [11]. CGMP-AA1 was based on an AA profile that met the minimum safe levels of amino acid intake (WHO/FAO/UNU 2007) [13] for Tyr, tryptophan, leucine, and histidine. CGMP-AA1 contained 30 mg of Phe for every 20 g protein equivalent. A modified CGMP-AA formula (CGMP-AA2) was produced by making small adjustments to the AA composition of CGMP-AA1, increasing some of the LNAA (Tyr, tryptophan, leucine and histidine) in amounts similar to conventional L-AA supplements. Although in this study, we examined a different cohort of children, after using CGMP-AA2 for 12 months, blood Phe concentrations, were lower than those in the pilot study using CGMP-AA1. Median Phe concentrations at the end of the pilot study 317 μmol/L compared to CGMP-AA2 after 12 months Phe 300 μmol/L. We were also able to increase the amount of protein equivalent supplied from CGMP-AA2 to 75% of total protein equivalent, without any reduction in dietary Phe intake. However, at the end of this study period, only 14 of 29 children (48%) were able to fully transition to CGMP-AA2 as their only protein substitute, which suggests that the residual Phe concentration present in CGMP-AA2 still increases blood Phe concentrations in children.
Identifying the optimal amino acid profile for protein substitutes is challenging, although, the ratio and amount of LNAA appears important. It is suggested that LNAA supplementation competes with Phe uptake at the gut and blood brain barrier (BBB): LNAA cross the intestinal mucosa using a carrier protein similar to that at the BBB [14,15,16]. In vitro studies investigating intestinal epithelial transport of amino acids, indicate that lysine, histidine, leucine and Tyr significantly reduce Phe uptake [6]. High concentrations of LNAA compete with the transport of Phe at the gut cellular membrane and may decrease blood Phe concentrations. Additionally LNAA supplementation has been shown to reduce blood and brain Phe concentrations [17, 18] and restore some of the disturbed Phe transport across the BBB by altering monoaminergic neurotransmitter concentrations. In mice studies, mice chow with added LNAA, improved brain tryptophan, serotonin and norepinephrine concentrations [4]. There are other functional effects of protein substitutes, which indirectly affect blood Phe concentrations. These include the rate of delivery of L-AA into the systemic circulation and muscle protein anabolism. Amino acids play an important, but as yet not fully understood role in nutritional signalling and regulation of multiple cellular processes [19]. Leucine, is a potential insulin secretagogue when administered with carbohydrate and protein, acting as a pharmaconutrient AA improving muscle protein synthesis, by stimulating mRNA changes via insulin independent and dependent pathways [7, 20]. Van Loon et al. maximised endogenous insulin secretion by the combined ingestion of carbohydrate and wheat protein hydrolysate, with added leucine and Phe [21]. In vitro studies, using incubated β cells of the pancreas, have shown that arginine, leucine and phenylalanine have a strong insulinotropic effect [22]. Further studies are needed to maximize our understanding of the physiological absorption process of AA protein substitutes, with the goal, to achieve a normal absorption pattern comparable to natural intact protein.
In contrast, many L-AA are bitter tasting and unpleasant to take, particularly leucine, tryptophan and histidine [23, 24]. By adding more of these AA to CGMP-AA formulations, it potentially decreases their acceptability and palatability. It has been suggested by Van Calcar [25] that the AA profile in CGMP-AA should provide 130 to 150% of the 2002 Institute of Medicine American dietary reference intake for histidine, leucine, methionine, tryptophan and Tyr to compensate for faster absorption and degradation of AA [26].
In our study, 52% of children in the CGMP-AA2 group were prescribed a combination of CGMP-AA2 and separate Phe-free L-AA supplements, as they could not fully transition onto CGMP-AA2 in order to maintain blood Phe concentrations within the target range of 120 to 360 μmol/L. The median amount of protein substitute provided by CGMP-AA2 that could be tolerated without affecting blood Phe control was 75% of the total amount. This contrasts with other researchers findings that have reported that the Phe content of CGMP-AA has little effect on blood Phe concentration. In an uncontrolled, short term study reported in 10 children (aged 4 to 16 years) with PKU, when 50% of their total protein requirements were supplied by ‘GMP cheese’ for 9 weeks, blood Phe concentrations decreased by a median of 114 μmol/L although this was not statistically significant [27]. In a short-term randomized cross over trial in 30 patients aged 15 years or over, comparing CGMP-AA with L-amino acid supplements only, CGMP-AA was associated with a non-significant increase in Phe of 62 ± 40 μmol/L, although 10 of the 30 patients were prescribed sapropterin (likely to have improved Phe tolerance), 6 patients appeared less adherent with CGMP-AA and overall subjects were only studied over a short time and had a higher baseline blood Phe levels in comparison to our study group [28]. Furthermore, increases of between 60 to 102 μmol/L may be unacceptable in children particularly as evidence is accumulating to suggest that optimal blood Phe may be below 240 μmol/L [29].
Median nutritional blood parameters in both groups at baseline and 26 weeks were all within reference ranges, with the exception on vitamin B12 being higher than the reference range at 26 weeks in both groups. There were no biochemical signs of vitamin or mineral deficiencies in line with what was retrospectively reported in adult PKU patients [30]. There was a significant increase in whole blood and plasma selenium between the groups at 26 weeks, and within the CGMP-AA2 group from baseline to 26 weeks. The selenium content of both products was similar, a median intake of 60 g protein equivalent from Phe-free L-AA provided 87 mg of selenium compared with CGMP-AA2 providing 90 mg. It is only conjecture, but absorption of selenium in the CGMP-AA2 group may be enhanced based on its bioactive properties. It is also possible that CGMP may modulate microbiota resulting in a different absorption or bioavailability. Furthermore, whey protein is rich in the sulfhydryl amino acid cysteine, which is a precursor of glutathione, and may in part explain the higher selenium concentrations in the CGMP-AA2 group. Muniz-Naveiro [31] reported the largest percentage of selenium in cow’s milk was found in the whey phase, although unmodified CGMP is not high in cysteine or selenium. Peptides containing isoleucine, proline, lysine, glutamine, aspartic and glutamic acid have been shown to have antioxidant properties; hence peptide structure and amino acid sequencing influence biological function. The bioactive antioxidant properties of CGMP and absorption in the gut may have a selenium sparing effect compared to protein substitutes without a peptide base [32, 33].
Weight and BMI increased significantly in the CGMP-AA2 group with an increase in weight and BMI first becoming evident from 26 weeks, this may be related to some children using a protein free milk replacement to make up their powdered CGMP-AA2 protein substitute. A mean intake of 400 ml/day of protein-free milk replacement would increase energy intake by 270 kcal/day. Only 9 children were routinely adding additional milk substitute to their CGMP-AA2 but there were no significant differences evident between children using protein free milk or water to prepare the CGMP-AA 2, but this may not be apparent due to the small numbers in the group. We changed this practice when it was observed that children were gaining extra weight. Another consideration influencing growth is age; the Phe-free L-AA group were an older patient cohort and therefore, over the 12-month period it is difficult to quantify how many were actively reaching puberty and the effect this has on weight, height and BMI. A further consideration is that CGMP based on a protein source may be more efficiently utilized increasing muscle mass compared to L-AA. Longer-term observations of both study groups will hopefully answer this question with data describing both fat free and fat mass index.
There are several limitations to this study that need to be considered. We were unable to conduct a randomised controlled blind trial. CGMP-AA2 and conventional L-AA supplements are very different in taste, texture and appearance rendering any blind or randomised trial very challenging in children who may not easily accept changes to their protein substitutes [34]. In this study, 40% of children preferred to stay on their usual Phe-free L-amino acid supplement (control group) and so they were a self-selected, not age-matched group and children in the control group were older than the study group. Also in teenage children in both study groups, ensuring dietary adherence was difficult although patients were monitored closely with monthly home visits to check stock levels of protein substitute and dietary intake. A further limitation was the use of low protein milk substitute to make up the protein substitute, in addition to increasing energy intake it possibly altered CGMP-AA2 absorption, affecting amino acid kinetics.