One-year follow-up of B vitamin and Iron status in patients with phenylketonuria provided tetrahydrobiopterin (BH4)

The study protocol and schematic has been described elsewhere in detail [9, 19]. Briefly, 58 patients with PKU, 4 years of age and older, were recruited between October 2008 and September 2009 through Emory University Department of Human Genetics Healthcare Clinic (Atlanta, GA). Inclusion criteria included patients with PKU naïve to BH4 treatment, not involved in other clinical trials, and age 4 years and above. Exclusion criteria included pregnancy or planning to become pregnant, breastfeeding, not diagnosed with PKU, and inability to provide informed consent. All participants were given 20 mg/kg/day of sapropterin dihydrochloride (Kuvan®, Biomarin Pharmaceutical Inc) for 1 month. Patients were assigned as preliminary responders if they experienced a decrease in plasma phe during the initial treatment month of at least 15%, and if they continued sapropterin treatment throughout the study period. Non-responders were removed from BH4 after the one-month trial and returned to their standard treatment plan. A dietary phe challenge to adjust for BH4-dependent changes in phe tolerance [20] set apart definitive responders (improved blood phe control combined with increased dietary phe tolerance) from provisional BH4 responders who experienced an initial lowering of plasma phe but no substantial long-term change in phe tolerance, MF requirement, or blood phe control [9, 19]. Patient PAH genotypes are presented in a prior publication with the assigned value (AV) sum provided [21].

Each patient was followed for up to 1 year. Data were collected at baseline and at three consecutive study visits spaced evenly throughout the year, with final data collection completed at 1 year. Three day diet records were completed at each study visit and analyzed by a registered dietitian. Average energy, phe (mg), protein (g), amino acids (g), fatty acids, and macro- and micronutrient intake were determined using the Nutrition Data System for Research (NDSR, University of Minnesota) diet analysis software, and reported as units per day. A blood draw was also performed at each visit for laboratory analysis of plasma amino acids (Emory Genetics Lab, Amino Acid Analyzer) and micronutrient biomarkers (Quest Diagnostics). Table 1 lists specific blood biomarkers for each micronutrient investigated.

To compare laboratory nutrient values in our sample with a standard population, two age and gender matched controls were selected for each patient from the most recent NHANES collection with available B12, B6, folate, and iron blood measurements (NHANES 2005–2006). NHANES serum and plasma based laboratory measurements were performed following Center for Disease Control and Prevention (CDC) guidelines [22].

Distributions for all dependent variables were tested for normality in each age strata using graphical interpretation and the Kolmogorov–Smirnov test. Several baseline or endpoint values deviated substantially from normality; given our small sample size, non-parametric two-tailed tests were chosen for analyses to maintain consistency. For all procedures, significance was tested at an alpha level of 0.05.

Correlations between laboratory values and reported dietary intake were determined using Spearman rho correlations. Significant changes in laboratory and/or dietary nutrient values were assessed using the Wilcoxon Signed Rank Test, with average values used for ties. Differences between BH4 response groups at baseline and final visit were evaluated via the Wilcoxon-Mann-Whitney test (NPAR1WAY procedure in SAS) for each age group. The Kruskal-Wallis test was conducted to compare baseline and end point values in each response group with NHANES controls.

The percentage of nutrients obtained from MF and intact food was calculated from diet record data at baseline and final visit. MF intake included primary and secondary formula, if prescribed. Differences in the percentage of micronutrients obtained from intact foods versus MF were tested using the Wilcoxon-Mann-Whitney and Signed Rank tests.

In total, 48 of the 58 recruited patients completed the one-year study. The final sample for analysis included 33 subjects, 15 non-responsive to sapropterin therapy and 18 confirmed to be responsive, henceforth referred to simply as “responders”. Exclusions are described in Fig. 1.

In the final analysis cohort, the mean age was 18.8 years, with 19 (57.5%) < 18 years of age and 14 (42.4%) ≥18 years of age. The majority of participants were female (51.5%) (Table 2). At baseline gender, anthropometrics and MF adherence, defined as amount of MF consumed below prescribed, were not significantly different between response groups. Responders were younger on average than non-responders (16.6 years, SD = 10.3 v. 21.5 years, SD = 14.2). Approximately 60% of participants reported taking the full amount of prescribed MF in both groups.

After 1 year of follow-up, median plasma phe decreased among BH4 responders while phe obtained from intact foods increased slightly (Table 3), indicating improved phe tolerance. These long term changes did not occur among non-responders. The percent of calories obtained from MF and the amount of prescribed MF was greatly reduced among responders by the end of study (Table 3, Fig. 1).

The result of greater phe tolerance among responders in this study was liberalization from a diet where intact food is restricted with measured amounts of fruits, vegetables, and specialty low-protein foods to a diet with more liberal intake of grains, legumes, nuts, fruits, vegetables, and limited amounts of animal protein, as seen in other studies [11, 20, 23].

All BH4 responders had lower dietary and serum-assessed iron, folate, and B12 after 1 year, compared to baseline (Figs. 2, 3, 4 and 5, Additional file 1: Table S1 and S2), though plasma B6 increased over the same period in both age groups. When stratified by age, the 1 year decline observed in dietary folate and iron remained statistically significant among responders < 18 years of age (Fig. 2). Furthermore, those within the lower quartile of intake fell below DRI ranges after 1 year. Serum B12 also dropped significantly among responders in the younger age group over the 1 year study period (Fig. 3). While pediatric responders had comparable serum micronutrient levels to NHANES controls at baseline, at the end of study B12 fell significantly below NHANES control values (p = 0.03) (Fig. 3). Overall serum iron also declined among young responders over the study period, driven by significant declines in serum transferrin, though total levels remained higher than NHANES controls.

Among BH4 non-responders, dietary micronutrients remained consistent from baseline to end of study (Additional file 1: Table S1). Serum-measured micronutrients increased on average among non-responders from baseline to end of study in both age groups (Additional file 1: Table S2).

Responders in both age groups experienced a decline in proportion of caloric and nutrient intake derived from MF over the one-year period (Table 4). This decline was statistically significant for all nutrients considered among those < 18 years of age (p = 0.02). Overall declines in micronutrients may be attributed to the lowered MF intake, especially given comparison with non-responders and older responders who did not see significant declines in percentage MF intake or laboratory and dietary measured micronutrients over time. AV sum [21] was also investigated as a potential source of micronutrient change, though it was not a significant factor in either laboratory or dietary micronutrient outcomes.

Median micronutrient values remained above DRI and laboratory reference ranges among responders despite noted declines over the study period. In addition, the number of responders who did not meet DRI values increased from start to end of study for all micronutrients (Table 5). For instance, only one responder reported dietary B12 below their DRI level at the start of the study, whereas six responders reported insufficient dietary B12 at the end of the study. Most responders had micronutrient values that fell within laboratory reference ranges, with no shifts outside of acceptable ranges seen from start to end of study (Table 5).

At baseline, there were no significant differences in dietary nutrient values between response groups within either age group. After 1 year, responders in both age groups had lower dietary levels compared to non-responders of all assessed micronutrients (Figs. 2 and 4). In the younger age group, there were significant differences between responder and non-responder dietary intakes at endpoint for iron (p = 0.02), folate (p = 0.004), and B6 (p = 0.04). Laboratory assessed micronutrients appeared similar between both groups at individual time points (Figs. 3 and 5), though iron levels among responders did drop below non-responders in those under 18 years (Fig. 3).