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. Author manuscript; available in PMC: 2022 Jan 31.
Published in final edited form as: J Pediatr. 2012 Nov 16;162(5):999–1003. doi: 10.1016/j.jpeds.2012.10.015

Large Neutral Amino Acid Supplementation Increases Melatonin Synthesis in Phenylketonuria: A New Biomarker

Shoji Yano 1, Kathryn Moseley 1, Colleen Azen 2
PMCID: PMC8803270  NIHMSID: NIHMS1560392  PMID: 23164313

Abstract

Objective

To determine whether levels of melatonin in blood and urine can serve as a peripheral biomarker to reflect brain serotonin synthesis in individuals with phenylketonuria (PKU).

Study design

We measured the levels of melatonin, a serotonin metabolite in the pinealocytes, in the blood and urine of individuals with PKU in a randomized double-blind placebo controlled crossover study consisting of three 3-week phases in 10 adults with PKU: phase 1 (washout), phase 2 (supplementation of large neutral amino acid [LNAA] tablets or placebo), and phase 3 (alternate supplementation). An overnight protocol to measure blood melatonin and urine 6-sulfatoxymelatonin and dopamine in first void urine specimens was conducted after each phase for subjects with PKU and once in 10 controls.

Results

Significantly lower concentrations of these neurotransmitter metabolites were observed in subjects with PKU after phase 1 compared with controls (serum melatonin P = .008, urine melatonin P = .0043, urine dopamine P < .0001), with significant increases after LNAA supplementation compared with the placebo phase (serum melatonin P = .0008, urine melatonin P = .0008, urine dopamine P = .0005). The mean tryptophan/LNAA and tyrosine/ LNAA ratios were markedly lower in subjects with PKU compared with controls, and these ratios were significantly increased in the LNAA phase compared with the placebo phase (P = .016, P = .0003, respectively). Blood phenylalanine levels in subjects with PKU were not significantly different between placebo and LNAA phases (P = .74).

Conclusion

Blood and urine melatonin levels may serve as biomarkers reflecting brain serotonin synthesis in subjects with PKU. Because this cannot be evaluated using blood phenylalanine levels, it may provide information on neurotransmitter metabolism for optimal dietary management.

The dietary treatment of phenylketonuria (PKU) with restriction of phenylalanine (Phe) to reduce blood Phe levels was first introduced by Horst Bickel in the early 1950s1 and remains the mainstay of PKU management today.2 Blood levels of other aminoacids,includingtyrosine(Tyr)andtryptophan(Trp),oftenarenotmonitoredinindividualswithPKU,despitethefact that those under poor dietary control are known to have deficiencies of neuro transmitters, including serotonin and dopamine, in the central nervous system (CNS).3–5 Neuropsychological studies of individuals with PKU who are diagnosed early and well controlled have shown a higher prevalence of characteristic deficits such as decreased executive functioning and internalizing disorders, including anxiety and depressive disorders.4,6 It is presumed that high blood Phe causes deficiencies of the precursor amino acids of the neurotransmitters in the brain by competitive inhibition at the transporter level. The large neutral amino acid (LNAA) transporter 1 (LAT1) is highly expressed in the brain capillaries and transports the LNAA including branched chain (valine, leucine, and isoleucine), aromatic (Phe, Tyr, and Trp), and other amino acids including histidine, threonine, and methionine.3 Improvement in neuropsychological symptoms and electroencephalography abnormalities in individuals with PKU have been reported after supplementation of LNAA.4,7

Supplementation of Tyr and Trp in individuals with PKU showed increases in the previously deficient cerebrospinal fluid (CSF) concentrations of homovanillic acid and 5-hydroxyindoleacetic acid, metabolites of dopamine and serotonin, respectively.8,9 It is, however, impractical to monitor these neurotransmitters in CSF in the clinical management of individuals with PKU.

Trp hydroxylase (EC 1.14.16.4) is the rate-limiting enzyme in serotonin synthesis and is unsaturated with the concentration of Trp in the brain, thus, brain Trp concentration is the most important single metabolic determinant.10 Melatonin, a serotonin metabolite in the pineal body, is released to blood during the night time and subsequently excreted into the urine as 6-sulfatoxymelatonin.11 We hypothesized that: (1) melatonin would serve as a peripheral biomarker, reflecting CNS serotonin synthesis; (2) individuals with PKU would have low blood and urine melatonin levels, suggesting low CNS serotonin synthesis; and (3) these levels would increase with LNAA supplementation. To test this hypothesis, a randomized, double-blind, placebo-controlled, crossover study with LNAA supplementation in subjects with PKU was conducted. Urine dopamine levels were also measured to evaluate any effects of LNAA supplementation in the dopamine metabolism.

Methods

Ten adult patients with classic PKU and 10 adult control subjects without PKU were enrolled after obtaining informed consent. All study subjects were recruited from our outpatient population, and healthy controls were recruited from our research team and medical students. Subjects with PKU ranged in age from 20–49 years (mean ± SD: 29.1 ± 9 years), and the controls’ ages ranged from 25–67 years (mean ± SD: 44.3 ± 15.7 years). The study protocol was approved by the University of Southern California (USC) Health Science Institutional Review Board.

The subjects with PKU completed three 3-week phases. After an initial washout phase (phase 1) without use of any medical food products, subjects with PKU were randomly assigned in a double-blind manner to take either LNAA or placebo tablets during the next 3 weeks (phase 2), then crossover to the alternate supplementation for the final 3 weeks (phase 3). They consumed a regular diet but avoided high protein foods and no dietary changes were made throughout the study. LNAA or placebo tablets (PheBloc; Applied Nutrition, Cedar Knolls, New Jersey) were given following the manufacturer’s recommendation. The total number of study tablets for daily intake was determined based on the subject’s weight: body weight × 0.5 (maximum total daily intake was 45 tablets per day). The LNAA tablets provided the following amount of LNAAs (mg/kg/d): Tyr 98.4, Trp 30.6, histidine 15.6, isoleucine 15.7, leucine 15.4, methionine 24.8, threonine 16.4, valine 16, and Phe 0.

The study subjects stayed overnight at the Clinical Trials Unit at USC University Hospital at the end of each phase. The 10 control subjects received no supplementation and were studied during 1 overnight stay. All subjects were given the same protein-controlled meal during the overnight evaluation. Serum melatonin was measured every 2 hours from 7 p.m.−7 a.m., and first void urine specimens were collected at 7 a.m. to measure dopamine and 6-sulfatoxymelatonin, to which 80%−90% of melatonin is metabolized and is excreted into urine.10 Plasma amino acids were obtained before dinner at 7 p.m. Serum and urine specimens were processed and kept in a freezer (−20°C) until analyzed. Serum melatonin and urine 6-sulfatoxymelatonin were measured as described elsewhere,12 and urine dopamine and plasma amino acids were measured by a commercial laboratory. Blood specimens for serum melatonin were obtained and processed under dim light after 11 p.m. to avoid subjects’ eyes from being exposed to bright light, which potentially inhibits melatonin synthesis.

Statistical Analyses

The area under the curve (AUC) of melatonin during the overnight stay was calculated using the trapezoidal method. The ratios of Trp and Tyr to the sum of all 9 measured LNAAs, including leucine, valine, isoleucine, histidine, methionine, threonine, Phe, Tyr, and Trp, were also calculated for each subject. Comparisons between controls and subjects with PKU at the end of phase 1 and after LNAA supplementation on outcomes of interest were made with the t test or Wilcoxon rank sum test. Within-subject differences in the subjects with PKU over the 3 phases were examined using repeated measures ANOVA with post hoc contrast to compare the LNAA supplemented phase with the washout and placebo phases. Prior to analyses, a log transformation was applied to variables, as needed, to normalize distributions. Regression lines and correlation coefficient were calculated between urine 6-sulfatoxymelatonin and Trp/LNAA ratio and between urine dopamine and Tyr/LNAA ratio in controls and subjects with PKU after LNAA supplementation. Statistical tests were 2-sided with P < .05 considered statistically significant. Statistical analyses were performed using SAS v. 9.2 (SAS Institute, Cary, North Carolina).

Results

One of the control subjects (because of an accidental exposure to bright light during the overnight stay) and 1 subject with PKU (because of poor compliance) failed to complete the study. Nine subjects with PKU completed the study without experiencing any untoward effects. Dopamine and 6-sulfatoxymelatonin levels could not be measured in 1 of the 9 subjects with PKU because of an accidental loss of the urine specimens. The 9 subjects with PKU who completed the study were significantly younger than controls (P = .026) but had comparable sex distribution (PKU: 67% vs control: 44% male, P = .64).

Repeated measures ANOVA showed no differences within the PKU group between placebo and washout phases on any outcome measures. Therefore, all within group comparison reported below are for LNAA supplementation versus placebo phases.

Serum Melatonin

After the 3-week washout phase, subjects with PKU showed significantly lower nocturnal serum melatonin AUC than controls (P = .008). The subjects with PKU on LNAA supplementation showed significantly higher nocturnal melatonin AUC than after the placebo phase (P = .0008), although it did not reach the AUC of control group (P = .0169) (Figure 1).

Figure 1.

Figure 1.

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Melatonin over time. P values are from Wilcoxon rank sum test comparing PKU after washout phase with controls.

Urine 6-Sulfatoxymelatonin

First void urine 6-sulfatoxymelatonin levels were statistically lower in the subjects with PKU than in controls (P = .0043). Supplementation with LNAA increased urine 6-sulfatoxymelatonin levels (P = .0008), although those levels were still significantly lower than the levels in controls (P = .021) (Figure 2).

Figure 2.

Figure 2.

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Urine 6-sulfatoxymelatonin and dopamine in controls and subjects with PKU. Column heights and error bars represent the mean and SEs, respectively. PKU-W, PKU-washout phase; PKU-P, PKU-placebo phase; PKU-A, PKU-LNAA supplemented phase.

Urine Dopamine

First void urine dopamine was statistically lower in the subjects with PKU than in controls (P < .0001). Supplementation with LNAA increased urine dopamine levels in patients with PKU (P = .0005), although those levels were still significantly lower than the levels in controls (P = .0072) (Figure 2).

Urine 6-Sulfatoxymelatonin versus Plasma Trp/LNAA

The mean Trp/LNAA ratio was markedly lower in the subjects with PKU than in controls, with no overlap between the groups (Figure 3). The ratios in the control group were all above 0.045 whereas the ratios in the PKU group were between 0.01 and 0.04. The mean Trp/LNAA ratio was significantly higher in the subjects with PKU in LNAA phase (0.025 ± 0.008) than in placebo phase (0.018 ± 0.003: P = .016). Urine melatonin was positively correlated with Trp/LNAA ratio (R = 0.59, P = .013).

Figure 3.

Figure 3.

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Urine 6-sulfatoxymelatonin and Trp/LNAA ratio. Trp/LNAA is the ratio of Trp to the LNAAs (Trp, Tyr, Phe, histidine, threonine, valine, isoleucine, leucine, methionine).

Urine Dopamine versus Plasma Tyr/LNAA

The plasma Tyr/LNAA ratios in the control group were above 0.07, and the ratios in the PKU group were between 0.015 and 0.035 after the washout and placebo phases, and between 0.035 and 0.09 after 3 weeks of LNAA supplementation (Figure 4). The mean Tyr/LNAA ratio was significantly higher in the subjects with PKU in LNAA phase (0.053 ± 0.022) than in placebo phase (0.025 ± 0.004: P = .0003). Urine dopamine was positively correlated with Tyr/LNAA ratio (R = 0.69, P = .0021).

Figure 4.

Figure 4.

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Urine dopamine and Tyr/LNAA ratio. Tyr/LNAA is the ratio of Tye to the LNAAs (Trp, Tyr, Phe, histidine, threonine, valine, isoleucine, leucine, methionine).

Plasma Phe

Blood Phe levels in the subjects with PKU did not change significantly from the placebo phase after supplementation with LNAA (1574 ± 239 vs 1548 ± 250 uM, respectively, P = .74).

Discussion

It is widely accepted that dietary management of individuals with PKU should be lifelong. High blood Phe is known to inhibit brain protein synthesis.13 This may explain why infants and children with PKU are more susceptible to intellectual disability and microcephaly, and adults with PKU tend to develop neuropsychological disturbances, such as anxiety or depressive disorders, which are probably due to secondary chronic neurotransmitter deficiencies. Individuals with PKU who are under poor dietary control are reported to have brain serotonin and dopamine deficiencies, as evidenced by low levels of 5-hydroxyindoleacetic acid and homovanillic acid in CSF.8,9 The results of this study, showing low levels of blood melatonin and urine 6-sulfatoxymelatonin in individuals with PKU indicating decreased serotonin synthesis in the brain, are consistent with the previous reports.8,9

The precursor of serotonin is Trp, which is transported through LAT1 into the brain, hydroxylated to 5-hydroxytryptophan by tryptophan hydroxylase, and subsequently decarboxylated by aromatic L-amino acid decarboxylase (EC 4.1.1.28) forming 5-hydroxytryptamine (serotonin). LAT1 is expressed abundantly at the blood brain barrier in the brain tissues. The kinetics of LNAA in LAT1 have been studied, showing that LAT1 has the highest affinity for Phe.14 Because tryptophan hydroxylase is believed unsaturated with regard to Trp, brain serotonin content is thought to be dependent on blood Trp levels.10 N-acetylation of serotonin, followed by O-methylation in the pinealocytes, forms melatonin. Nocturnal melatonin secretion is a unique phenomenon, which occurs only in the pineal gland, and reduced blood Trp concentration decreases night secretion of melatonin in humans.15 Trp uptake from blood into the pinealocytes is thought to be achieved through monocarboxylate transporter 10 or T-type amino acid transporter 1 (TAT1).16 Although expression studies of human TAT1 in Xenopus laevis oocytes demonstrated the Na+ independent transport of Trp, Tyr, Phe, and L-Dopa, characteristics of this transporter in human pinealocytes have not been well studied.17 Because the pinealocyte is believed to use TAT1 and the other brain tissues to use LAT1 to transport Trp into the cells, concentrations of Trp in these tissues are likely to be different. Thus, the evaluation of blood melatonin and urine 6-sulfatoxymelatonin levels may serve as important biologic markers to evaluate CNS serotonin metabolism that cannot be evaluated by the measurement of blood Phe levels in individuals with PKU.

Our study shows direct evidence that LNAA therapy improved serotonin production in the CNS with no significant effect on blood Phe levels. The improved serotonin synthesis with LNAA supplementation did not reach the control levels, thus, it may be necessary to increase supplementation to achieve further improvement.

We also measured urine dopamine in the present study. In contrast to 6-sulfatoxymelatonin, urine dopamine levels likely reflect neuronal cell metabolism not only in the CNS but also in the other organs including the kidneys and the gastrointestinal tract. Because decreased ratios of the precursor amino acids to Phe are not tissue specific, deficiencies of the precursor amino acids may cause similar effects on all systemic neuronal cells, including the CNS.18 Urinary dopamine levels may represent whole body dopamine metabolism including the CNS. This was supported by the findings that urine dopamine levels showed very similar profiles to one of the urine 6-sulfatoxymelatonin levels in the present study.

Our results support the mechanism of competitive inhibition of Trp and Tyr transport by showing the upward trends in urine 6-sulfatoxymelatonin and dopamine levels versus plasma Trp/LNAA and Tyr/LNAA, respectively. As reported previously, individuals with PKU who are under good dietary control can have neurotransmitter deficiencies.4,5 This is particularly true for individuals who are under strict protein restriction without sufficient supplementation of medical food products that provide LNAA. It is likely that chronic deficiencies of the neurotransmitters in individuals with PKU could well be one of the major contributing factors responsible for decreased executive function in this population. Measurement of 6-sulfatoxymelatonin and dopamine in first void urine specimens may provide invaluable clinical information about the CNS serotonin and dopamine status. Our studies suggest that management of individuals with PKU should not solely depend on blood Phe levels. Evaluation of CNS neurotransmitter metabolism including serotonin and dopamine as well as evaluation of plasma Trp/LNAA and Tyr/LNAA ratios may be essential for optimal dietary management.

It is well known that there are individuals with PKU who do well, although they are not on dietary therapy, and their blood Phe levels are chronically above the recommended range.19 Some of these individuals were identified with PKU after their babies were diagnosed with specific anomalies consistent with maternal PKU syndrome.20 If these individuals have normal or close to normal 6-sulfatoxymelatonin and dopamine levels in first void urine despite high blood Phe levels, it may establish the possible relationship between serotonin and dopamine deficiencies and neuropsychological disorders.

In summary, noninvasive monitoring of 6-sulfatoxymelatonin and dopamine in first void urine specimens may significantly affect the concept of dietary therapy for individuals with PKU. Optimizing dietary therapy based on these new biologic markers, along with blood Phe, and Trp/LNAA and Tyr/LNAA ratios, may prevent individuals with PKU from developing neuropsychological disorders and lower executive function. Further studies are indicated to establish the relationship between neuropsychological disorders in individuals with PKU and serotonin and dopamine metabolism in the CNS, which may be monitored by the 2 urine biomarkers. More experience with these biomarkers is absolutely essential to determine how to incorporate them in clinical management of PKU.

Acknowledgments

Funded by Applied Nutrition and National Institutes of Health/National Center for Research Resources Southern California-Clinical and Translational Science Institute (UL1 RR031986). The contents of this article are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.

Glossary

AUC

Area under the curve

CNS

Central nervous system

CSF

Cerebrospinal fluid

LAT1

Large neutral amino acid transporter 1

LNAA

Large neutral amino acid

Phe

Phenylalanine

PKU

Phenylketonuria

TAT1

T-type amino acid transporter 1

Trp

Tryptophan

Tyr

Tyrosine

USC

University of Southern California

Footnotes

The authors declare no conflicts of interest.

The authors appreciate Dr Frank Stanczyk (Obstetrics/Gynecology, Keck School of Medicine, USC) for performing laboratory analysis and Carolina Coleman for her assistance in the preparation of this manuscript.

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