Correction: Endogenous tetrahydrobiopterin in humans: circadian rhythm, sex, race, age, and disease status
Year: 2025
Type: erratum
Abstract: 6,7, ) is an essential cofactor and natural stabilizer for multiple aromatic amino acid hydroxylation enzymes, such as phenylalanine hydroxylase (PAH), tyrosine hydroxylase (TH), and tryptophan hydroxylase (TPH), alkylglycerol monooxygenase (AGMO), and nitric oxide synthase (NOS) (Thony et al., 2004;Thony et al., 2008;Komleva et al., 2025). Multiple inborn errors of metabolism are closely related to the biosynthesis and regeneration of BH 4 ; therefore, it serves as an important drug target to treat metabolic disorders such as phenylketonuria (PKU), primary tetrahydrobiopterin deficiencies (PBD), and tyrosine hydroxylase deficiency (Werner-Felmayer et al., 2002;Werner et al., 2011;Werner, 2013;Fanet et al., 2021;Eichwald et al., 2023;Jung-Kc et al., 2024). Exogenously supplemented BH 4 (sapropterin dihydrochloride) has been approved for the treatment of hyperphenylalaninemia (HPA) due to BH 4 -responsive PKU (Blau et al., 2010;Blau, 2013). Results from recent Phase 3 studies indicated that a broader population of patients with PKU could benefit from treatment with sepiapterin, a natural precursor of BH 4 that was recently approved in the EU and US (Muntau et al., 2024;van Spronsen et al., 2025).Despite the importance of BH 4 in the pathology of BH 4 -dependent enzyme metabolic diseases, studies of natural fluctuation of endogenous BH 4 in human were rarely reported. This could be due to the lability of BH 4 , which is subject to autoxidation under physiologic conditions (Mortensen and Lykkesfeldt, 2013;Telegina et al., 2018). BH 4 represents 65%-80% of total biopterins in plasma, and large variations in biopterin concentration from 6.9 nmol/L to 23.6 nmol/L have been observed, which have been attributed to BH 4 lability (Fukushima and Nixon, 1980;Fiege et al., 2004;Fekkes and Voskuilen-Kooijman, 2007). The lability of BH 4 has been well recognized for >50 years, and it remains an active area of research today (Blair and Pearson, 1973;Buglak et al., 2021;Boulghobra et al., 2023).Determining the plasma concentration of BH 4 is not feasible without stabilization. Various antioxidants such as dithioerythritol (DTE), dithiothreitol (DTT), and ascorbic acid (Fiege et al., 2004;Fekkes and Voskuilen-Kooijman, 2007;Kaushik et al., 2024); acids to control pH, like meta-phosphoric acid, perchloric acid, and trichloroacetic acid (Mortensen and Lykkesfeldt, 2013); and chelating agents, such as ethylenediaminetetraacetic acid and diethylenetriaminepentaacetic acid, have been explored to stabilize BH 4 in the biomatrix (Guibal et al., 2017). Depending on the procedures followed and the concentrations of reagents used, very different results were observed. However, it is generally agreed that lower pH reduces the rate of autoxidation, and the half-life of BH 4 ranges from 15 min to 127 min, depending on the buffer and pH (Mortensen and Lykkesfeldt, 2013;Boulghobra et al., 2023). Accordingly, it is critical to stabilize BH 4 in blood immediately after sample collection.Additionally, the most widely used method to measure BH 4 is the indirect method introduced by Fukushima and Nixon about half a century ago (Fukushima and Nixon, 1980). The method utilizes chemical oxidation under acidic conditions to convert both BH 4 and 7,8-dihydrobiopterin (BH 2 ) to biopterin, and oxidation under alkaline conditions to convert BH 2 to biopterin and BH 4 into pterin. Biopterin is quantified using high-performance liquid chromatography (HPLC) with fluorescence detection (FD). The difference in the amount of biopterin between the two HPLC-FD runs reflects the amount of BH 4 . The indirect method is not only tedious, but also subject to variations in oxidation conversion efficiency, which has been reported to be far less than complete, approximately 47% in human plasma (Zhao et al., 2009). Additionally, there is potential interference from the matrix and variability in multiple HPLC runs. The lower limit of quantitation (LLOQ) of the indirect method for plasma BH 4 measurements is rarely reported, and it is generally assumed to lack adequate sensitivity for reliably measuring endogenous plasma BH 4 concentration. Various adjustments have been made to improve the indirect method since its introduction, such as the direct measurement of BH 4 with post-column coulometric oxidation by HPLC-FD (Hyland, 1985;Guibal et al., 2014) and HPLC mass spectrometry (HPLC-MS) (Fismen et al., 2012;Kim et al., 2012).Recently, a highly selective and sensitive HPLC-MS method to quantify BH 4 in human plasma has been developed and fully validated following Good Laboratory Practice guidance (Kaushik et al., 2024). Blood samples were placed in wet ice immediately after collection and stabilized within 10 min of collection with 10% ascorbic acid at a 100:11 (v:v) ratio to reach a final ascorbic acid concentration of 1%. Treated blood was centrifuged at 4 °C for 10 min at a centrifugal force of 2000-g within 30 min of blood sample collection. It was then stored at -80 °C and analyzed within the established stability period. During the process of blood sample collection, processing, and plasma sample analysis, samples were protected from light. The method has an LLOQ of 0.5 ng/mL for measuring BH 4 concentrations in human plasma. The sensitivity and robustness of this HPLC-MS method were demonstrated during method validation, as well as by its application in multiple clinical studies, in which the accuracy and precision of every analytical run and incurred sample reanalysis were examined. The high specificity and sensitivity of this method made it feasible to investigate endogenous BH 4 concentrations to detect small fluctuations in healthy volunteers and in patients with PKU or PBD.In this study, data were pooled together from measurements of blood samples collected at predose or placebo treatment in healthy adults and patients of all ages with PKU or PBD from eight sepiapterin clinical studies. The aim of this study was to assess the impacts of circadian rhythm, sex, age, race, and disease status on endogenous BH 4 concentrations to gain a better understanding of the molecule and its role in pharmacology.Blood samples were collected from healthy volunteers, patients with PKU, and patients with PBD (due to 6-pyruvoyltetrahydrobiopterin synthase [6-PTPS] deficiency) from eight clinical studies and plasma BH 4 concentrations were measured using a validated HPLC-MS method (Kaushik et al., 2024). The clinical studies included in this analysis are summarized in Supplementary Table S1. All clinical studies were conducted following Good Clinical Practice guidelines and in compliance with the principles of the Declaration of Helsinki. The protocols and informed consent forms were approved by local independent ethics committees or institutional review boards, and regulatory agencies, as required. Signed consent forms were provided by all participants prior to enrollment.To reflect the endogenous BH 4 concentrations, only data from samples collected prior to the first treatment or predose samples of each treatment period following a minimum washout of 4 days if there were multiple treatment periods, and samples following placebo treatments were included in the analysis.Data were binned by hour following the 24-h clock period according to the actual sampling time. For example, data from samples collected from 7:00 to 7:59 would be binned for the 7-h period. If data from multiple samples collected from the same participant in the same hour period were available, the geometric mean would be calculated and used for the analysis. Hence, a subject had only one value in any specific hour period.Following the hourly binned analysis, data were further binned by 4-h time blocks, including 7:00-10:00 (7:00 to 10:59), 11:00-14: 00, 15:00-18:00, 19:00-22:00, except for the 23:00-06:00 time block. Since there were only two data points available in the 23: 00-06:00 time block, it was not further divided. If multiple values were available from the same participant in the same time block, the geometric mean would be calculated and used for the analysis. Hence, a subject had only one value in any 4-h time block.Descriptive statistics were used to summarize data by categories. Analysis of variance (ANOVA) was used to compare the differences between categories. Sex, age, race, and disease status comparisons were conducted based on data collected during the 7-11 time block, as this time block contained the richest data; later analysis also suggested that endogenous BH 4 concentrations were relatively stable during this period.This analysis was conducted using R (version 4.3.3; R Foundation for Statistical Computing, Vienna, Austria) with R Studio (version 2023.09.1, Posit Software, PBC, Boston, MA, United States of America).Data from eight clinical studies in adult healthy volunteers and participants of all ages with PKU or PBD were included in this analysis (Supplementary Table S1). Designs and results from these clinical studies have been previously reported (Smith et al., 2019;Gao et al., 2024a;Gao et al., 2024b;Gao et al., 2024c;Muntau et al., 2024;van Spronsen et al., 2025).A total of 236 participants provided 1175 BH 4 measurements for this study (Table 1). Among them, 167 participants were healthy volunteers, with a median (minimum, maximum) age of 32.0 (18.0, 56.6) years; seven participants with PBD (all due to 6-PTPS deficiency), with a median of age 11.0 (6.00, 20.0) years; and 62 participants with PKU, with a median age of 16.0 (0.380, 61.0) years. Overall, approximately equal numbers of male (48.7%) and female participants (51.3%) were enrolled in the studies. In healthy volunteers, males and females represented 49.1% and 50.9% of the population, respectively; in participants with PKU, males and females represented 46.8% and 53.2% of the population, respectively; and in participants with PBD, males and females represented 57.1% and 42.9% of the population, respectively. The majority of participants were White (67.4%), followed by Asian (16.1%) and Black or African American (9.3%). Participants with unidentified race or identified with multiple races of different were as healthy participants were adults In participants with PBD, there were adult participants and and years. In participants with PKU, there were adults each from age and years, and years, and years, and of age (Table BH 4 measurements for participants with PBD were all reported the limit of Table 1). were as during the analysis. It is that this reflects the value in patients with PBD, as suggested by the of the This is also 6-PTPS deficiency in patients with PBD BH 4 biosynthesis et al., there were BH 4 measurements from healthy volunteers and measurements from participants with PKU reported as represented and of the data from healthy volunteers and participants with PKU, (Table both were of the total measurements of each data were as and were from the analysis. Since these data of of total of these data had a on the of the analysis et al., BH 4 measurements were from 167 adult healthy volunteers (Table 1). hourly there were BH 4 from 167 healthy BH 4 data points were collected between 7:00 to was only one data each at and The geometric mean of BH 4 was ng/mL with and of of and with the mean geometric mean of median with and of for each period were calculated and are summarized in Supplementary Table The data and the of median BH 4 concentration with time is in was a relatively of endogenous BH 4 ; the was approximately the median BH 4 a with circadian BH 4 concentration was the in the and relatively stable between and then during the the concentration was between and 1). were only two data points available between and and there were data available between and (Supplementary Table was conducted after data were binned into the 4-h period time blocks, except for data in the 23:00-06:00 time block which had only two BH 4 measurements and was to have data for the (Table In the 7:00-10:00 time block, the geometric mean BH 4 concentration was ng/mL and to concentrations of ng/mL in the between and with the 7:00-10:00 time block, BH 4 was for the of in the and time BH 4 further in the in the time block, and the was with the 11:00-14: and time (Table from the 7:00-10:00 time block were used to investigate the potential differences in endogenous BH 4 between and This period contained the richest and endogenous BH 4 was relatively which potential interference from from of 167 healthy participants were available in this time block for the were approximately equal numbers of male and female adult healthy volunteers included in this BH 4 concentrations between male and female healthy volunteers the geometric mean concentration was in male participants than in female participants Table the majority of the participants available for this were White there were Asian and Black or African American healthy volunteers to of endogenous BH 4 between races Asian participants had of endogenous BH 4 to White were between White and Black or African American participants Table same data from the available healthy volunteers in the time block were to the of age on BH 4 concentrations, which included participants from ages years, with a median age of in there was in endogenous BH 4 concentration with age in both male and female The of for male and female healthy volunteers were within ng/mL of analysis was conducted to endogenous BH 4 concentration with The for male and female participants were both within the was between and and placebo treatment samples were collected from participants with PBD in a Phase study and from participants with PKU in a Phase 3 study and an Phase 3 study (Supplementary Table S1). were data a time period to assess the circadian of BH 4 concentrations in participants with PBD or However, it was that the would that in healthy data collected in the 7:00-10:00 time block were for the following analysis.Data were available from a total of seven participants with PBD (all with 6-PTPS deficiency) with one BH 4 measurement from each the endogenous BH 4 data were all 0.5 Table 6-PTPS deficiency results in of BH 4 biosynthesis et al., and placebo treatment samples were collected from 62 participants with PKU (Table and data in the time block were available from participants with PKU for the age and analysis. The in that there is an with a BH 4 concentration of which was than the BH 4 concentration and was from the there was a difference in endogenous BH 4 concentration between male and female healthy volunteers, the difference was explored for participants with The analysis was to adult participants with PKU, as only this age had an adequate of participants for such analysis. difference in endogenous BH 4 concentration was for participants with PKU, and the ranges of highly the value was in male participants with PKU (Table endogenous BH 4 concentrations were not between male and female participants with PKU, and there were approximately equal numbers of male and female the endogenous BH 4 variations with age in participants with PKU were explored using male and female data collected from participants with PKU was not the age the age were 3 for age and for with ages years. was a of participants for any for years. BH 4 concentrations in participants with PKU were lower than in years. The geometric mean of endogenous BH 4 for PKU patients of age and were and (Supplementary Table BH 4 concentration was the endogenous BH 4 was the age that endogenous of the the between endogenous BH 4 concentration and age The and values were ng/mL and years, respectively. was in of endogenous BH 4 concentration in participants with PKU years. This was with the analysis for data in the years, which a of and a of (Supplementary only data from participants with PKU were included to assess the population difference in with different disease The endogenous BH 4 concentration was in participants with PKU than in healthy volunteers (Table It was that there was of participants with PBD with healthy volunteers or participants with PKU, and of participants with PKU or healthy healthy volunteers, a circadian of BH 4 concentrations was BH 4 concentration was the in the between 7:00 and mean and the at between and this was to the variability ng/mL for the in the 7:00-10:00 time BH 4 was relatively stable in the 7:00-10:00 time block, and this it a period to endogenous BH 4 concentrations according to different participant including age, sex, race, and disease healthy adult volunteers, there was a difference in endogenous plasma BH 4 concentration between male and female participants However, this difference clinical as it the a difference between Asian and White was this difference not into clinical to BH 4 concentrations were in participants with PKU ng/mL for to healthy adults BH 4 concentrations in both participants with PKU and healthy volunteers were than the concentration in participants with PBD.In participants with PKU with age 2 years, the majority of data were collected between 7:00 and and the were collected between and was data for a analysis to detect the in participants with However, an analysis indicated that the would be from to to the if there were any (Supplementary The difference in endogenous plasma BH 4 between male and female participants with PKU was not this was attributed to the which was than that in healthy Since endogenous BH 4 concentrations were in participants with PBD, with circadian or was seven participants with PBD were by an deficiency in and endogenous BH 4 concentrations were 0.5 PBD from in for the biosynthesis and regeneration of BH 4 , including sepiapterin and et al., and are important for biosynthesis of BH 4 , and are for BH 4 regeneration et al., enzyme of analysis of BH 4 , geometric of healthy PBD, primary tetrahydrobiopterin PKU, in could biosynthesis of BH 4 , to lower plasma BH 4 , which was as in this It has been that BH 4 deficiency results from the enzyme are by the of during and following analysis biopterin, and in a blood or analysis. Additionally, patients with PBD in such as and BH 4 concentration has not been used as a Results from this study that plasma BH 4 concentrations in participants with 6-PTPS deficiency be from in healthy volunteers and with such as is a metabolic by a or deficiency in due to in the than and have been identified et al., the hydroxylation of phenylalanine to deficiency in results in blood concentration as et al., Spronsen et al., is a enzyme that in as a between and et al., by to the enzyme and the from to et al., BH 4 as an essential cofactor for et al., BH 4 to the of to block the and stabilize the enzyme et al., et al., 2013). During BH 4 is two to the enzyme the as et al., blood concentration BH 4 concentration to the et al., et al., 2013). it is to endogenous BH 4 concentrations in patients with PKU to healthy The BH 4 concentrations to of and in the of its role in BH 4 as a cofactor for aromatic amino acid including and endogenous BH 4 concentration is with these BH 4 is for the of which and nitric oxide in BH 4 could to a in and an in which are to diseases, such as and and et al., BH 4 in the could the enzyme in and has been with disease et al., 2023). deficiency in BH 4 is to diseases, such as and et al., 2023). a for plasma BH 4 concentrations could in healthy adult volunteers and participants with PKU of age, endogenous BH 4 concentrations had with However, lower plasma BH 4 concentrations were in participants with PKU years. The between this and the pathology of PKU in this age is not fully further research is to the a recent endogenous BH 4 concentrations from volunteers and patients were summarized et al., 2024). The mean BH 4 concentration calculated as a . . . . and to the of and mean BH 4 reported in study . . for adult healthy volunteers was ng/mL from studies in the from which included et al., 2024). The mean BH 4 was ng/mL after healthy study healthy reported a in plasma BH 4 with age, from ng/mL at to ng/mL at with the mean BH 4 ng/mL et al., 2018). In the study, blood samples were stabilized with (v:v) immediately after collection. BH 4 was measured using HPLC-MS after with following to the sensitivity The of endogenous BH 4 with age in adults was during from to which the role of BH 4 as a cofactor for multiple essential and the of stable BH 4 for study from the same used an HPLC-MS based on et with followed by for min at to measure endogenous BH 4 from healthy to was the age was as a the age to was et al., 2024). to the first study reported from the et al., mean plasma BH 4 concentration from this recent study was at However, the of BH 4 concentration with age was age to of age and which were far from of the mean BH 4 from based on the reported data from to a of for an age of from to In study, mean BH 4 from adult healthy volunteers in the between 7:00 and was and it was independent of This value is lower than the mean value from studies and lower than the values reported for et al., et al., 2024). The be results from this study indicated that BH 4 concentrations were lower in the and in the and It is the time of blood samples were collected in studies. there is an difference in endogenous BH 4 with the mean concentration in Asian and lower in The be endogenous BH 4 between there is between and in et that of immediately blood collection is critical for measurement of plasma BH 4 . and ascorbic acid are used reagents to stabilize BH 4 (Fiege et al., 2004;Fekkes and Voskuilen-Kooijman, 2007;Kaushik et al., 2024). In this study, blood samples were stabilized with ascorbic acid within 15 min of blood collection and the from after in were under in approximately min at °C (Kaushik et al., 2024). The variations in reagents used, and of the as well as the following sample procedures are not fully at this time. The method used in this study has been used in 10 clinical to measure plasma BH 4 concentrations in blood samples from All runs have the validated and the 10% incurred sample reanalysis the as The of of this validated method the of results from this Additionally, there were large variability for BH 4 . sample is to a mean value from the This in the two studies conducted in healthy age BH 4 was from et study, which included participants and blood sample The age was in the recent study reported by et which included In this study, data were from 167 participants and blood samples in the between 7:00 and The value from this study and mean value from studies in healthy volunteers and the between this study and multiple prior patients with PKU ages years, with the of data from this study, that are 4 concentration remains stable in healthy adults of The geometric mean endogenous BH 4 concentration of healthy adults is endogenous BH 4 concentrations in participants with PKU were not reported in studies, the value could be from the BH 4 concentrations of population The BH 4 concentrations were ng/mL for participants with PKU et al., ng/mL for participants with PKU (Muntau et al., and ng/mL for participants with PKU participants were et al., with of a mean BH 4 concentration of 11.0 for participants with PKU and geometric mean of one a in BH 4 concentration in with age was reported for years, followed by relatively stable concentrations from ages in participants with disease et al., was reported for BH 4 in from participants with disorders from to in which BH 4 concentrations were stable at ages (Guibal et al., In both studies, samples were immediately at -80 °C after collection without of any The indirect chemical oxidation method with HPLC-FD was used to BH 4 The stability of BH 4 in samples was not reported in lower than adult endogenous BH 4 concentrations were in PKU patients with age in study, it be that this is not the small sample of participants with PKU within this age circadian of BH 4 concentrations was in healthy with the concentration in the and the to the concentration in the Additionally, endogenous BH 4 concentrations were in male than in female and in than in races in healthy However, the of all such fluctuations was small and to variability a lack of clinical was between endogenous BH 4 and age in healthy plasma BH 4 concentrations were to be relatively stable between 7:00 and a time for BH 4 sample collection to measurement due to endogenous plasma BH 4 concentration ranges between participants with PBD, participants with PKU, and healthy This difference could be as a
Source: Frontiers in Pharmacology
Authors: Lan Gao, Neil Smith, Ronald Kong
Institution: PTC Therapeutics (United States)
Language: English
Subfield: Clinical Biochemistry
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $3,801
OpenAlex ID: W4417490362
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