Urinary Pterins Research Articles

Analysis of GCH1 gene variant in a consanguineous Chinese pedigree affected with tetrahydrobiopterin deficiency

To explore the genetic basis for a child featuring tetrahydrobiopterin deficiency and global developmental delay. Clinical and laboratory examinations were carried out for the child. Genomic DNA of the patient was subjected to high-throughput sequencing to identify genetic variants associated with hyperphenylalaninemia. Candidate variants were verified by Sanger sequencing. The result of blood tandem mass spectrometry showed that the Phenylalanine in the blood was 642.7 μmol/l, and the ratio of Phenylalanine/Tyrosine was 5.42. Analysis of urinary pterin: neopterin 0.09 mmol/mol Cr, biopterin 0.04 mmol/mol Cr, biopterin% 77%, which suggested tetrahydrobiopterin deficiency. The parents of the proband were first cousins. DNA sequencing revealed that the proband has harbored homozygous c.353A>T variants in exon 2 of the GCH1 gene, for which his great grandmother, grandfather, mother, uncle, father and elder brother were heterozygous carriers with normal phenotype and no clinical symptoms associated with dopa responsive dystonia. The homozygous c.353A>T variant of the GCH1 gene probably underlay the tetrahydrobiopterin deficiency in this pedigree of consanguineous marriage.

Influencing Factors on the Use of Tetrahydrobiopterin in Patients with Phenylketonuria.

Objective To explore and analyze the influencing factors of tetrahydrobiopterin therapy in patients with phenylketonuria. Methods 86 children with phenylketonuria (PKU) diagnosed and treated in our hospital from February 2019 to September 2021 were randomly enrolled. All the children underwent coenzyme hydroxybiopterin and urinary pterin spectrum analysis, and the children with deficiency received gene mutation testing. Results The results of urine pterin analysis showed that 82 patients had higher urinary N and B contents than the normal reference values, with the N/B slightly higher than the normal B% within the normal range. 4 patients had extremely high urinary N/B and B% <5% and were diagnosed as BH4 deficiency caused by 6-pyruvoyl-tetrahydropterin synthase (PTPS) deficiency, and a combined stress test was performed. The blood Phe level was (720–1200) μmol/L 3 h after Phe loading, and the blood Phe concentration decreased to (120–240) μmol/L 4–6 h after oral administration of 7.5 mg/kg BH4 tablet. After one week of treatment, the blood Phe concentration decreased significantly to 239 ± 173 μmol/L, with a decrease rate of 52.14 ± 25.28%. It shows that the application of tetrahydrobiopterin intervention therapy is effective in patients with PKU. The results of the full-length cDNA analysis of the PTPS gene showed that a total of 4 gene mutations were found. A C ⟶ T substitution occurred at the 259th base, and the 87th proline (Pro) in the coding region was converted to serine (Ser) (P87S). G ⟶ A substitution at base 286 converts aspartic acid (Asp) at position 96 of the coding region to asparagine (Asn) (D96N). A ⟶ G substitution occurs at the 155th base to convert asparagine (Asn) at position 52 of the coding region to serine (Ser) (N52S). G ⟶ C substitution occurs at the 430th base to convert glycine at position 144 (Gly) to arginine (Arg) (G144R). G144R is a new mutation type. The gene mutation types of the 4 patients were P87S/D96N, N52S/G144R, D96N/P87S, and P87S/P87S, all of which were from their parents, which conformed to the law of autosomal recessive inheritance. Conclusion PKU is caused by the defect of phenylalanine hydroxylase activity in children, which causes phenylalanine metabolism disorder, and tetrahydrobiopterin intervention therapy can affect the activity of phenylalanine hydroxylase, increase the decline rate of blood Phe, significantly reduce the level of phenylalanine in children, and promote intellectual recovery. The dose of tetrahydrobiopterin should be tailored, with small doses for mild phenotypes and long-term treatment using even smaller doses.

BH4-deficient hyperphenylalaninemia in Russia.

A timely detection of patients with tetrahydrobiopterin (BH4) -deficient types of hyperphenylalaninemia (HPABH4) is important for assignment of correct therapy, allowing to avoid complications. Often HPABH4 patients receive the same therapy as phenylalanine hydroxylase (PAH) -deficiency (phenylketonuria) patients—dietary treatment—and do not receive substitutive BH4 therapy until the diagnosis is confirmed by molecular genetic means. In this study, we present a cohort of 30 Russian patients with HPABH4 with detected variants in genes causing different types of HPA. Family diagnostics and biochemical urinary pterin spectrum analyses were carried out. HPABH4A is shown to be the prevalent type, 83.3% of all HPABH4 cases. The mutation spectrum for the PTS gene was defined, the most common variants in Russia were p.Thr106Met—32%, p.Asn72Lys—20%, p.Arg9His—8%, p.Ser32Gly—6%. We also detected 7 novel PTS variants and 3 novel QDPR variants. HPABH4 prevalence was estimated to be 0.5–0.9% of all HPA cases in Russia, which is significantly lower than in European countries on average, China, and Saudi Arabia. The results of this research show the necessity of introducing differential diagnostics for HPABH4 into neonatal screening practice.

Determination of Urinary Pterins by Capillary Electrophoresis Coupled with LED-Induced Fluorescence Detector.

Urinary pterins have been found as potential biomarkers in many pathophysiological conditions including inflammation, viral infections, and cancer. However, pterins determination in biological samples is difficult due to their degradation under exposure to air, light, and heat. Besides, they occur at shallow concentration levels, and thus, standard UV detectors cannot be used without additional sample preconcentration. On the other hand, ultra-sensitive laser-induced fluorescence (LIF) detection can be used since pterins exhibit native fluorescence. The main factor that limits an everyday use of LIF detectors is its high price. Here, an alternative detector, i.e., light-emitted diode induced fluorescence (LEDIF) detector, was evaluated for the determination of pterins in urine samples after capillary electrophoresis (CE) separation. An optimized method was validated in terms of linearity range, limit of detection (LOD), limit of quantification (LOQ), intra- and interday precision and accuracy, sample stability in the autosampler, and sample stability during the freezing/thawing cycle. The obtained LOD (0.1 µM) and LOQ (0.3 µM) values were three-order of magnitude lower compared to UV detector, and two orders of magnitude higher compared to previously reported house-built LIF detector. The applicability of the validated method was demonstrated in the analysis of urine samples from healthy individuals and cancer patients.

Application of isoxanthopterin as a new pterin marker in the differential diagnosis of hyperphenylalaninemia.

This study aimed to explore the value of applying a new pterin marker (isoxanthopterin) to the traditional urine pterin analysis to reduce the rate of mis-diagnosis of 6-pyruvoyltetrahydropterin synthase deficiency (PTPSD) and improve the accuracy of diagnosis. We compared the urine neopterin (N), biopterin (B), isoxanthopterin (Iso), B% and Iso% levels between patients with phenylalanine hydroxylase deficiency and those with PTPSD, and found the most specific pterin biomarkers by ROC analysis. A positive cut-off value of urine pterins was determined. The effect of combined Iso% + B + B% in reducing PTPSD mis-diagnosis was evaluated, and the different urine pterin levels in PTPSD and false PTPSD (FPTPSD) were compared. The concordance of PTPSD diagnosis by the new pterin scheme and gene mutation analysis was determined. (1) Urinary B, B%, Iso and Iso% were significantly lower in PTPSD than those in phenylalanine hydroxylase-deficiency group (P < 0.01); (2) Iso%, B%, and B were the most specific markers; (3) The positive cut-off values of B, B%, Iso% for PTPSD were < 0.17 mmoL/moLCr, < 5.0%, and < 9.5%, respectively; (4) urinary B + B% + Iso% scheme significantly reduced the false-positive rate of PTPSD compared to traditional ones. The Iso% levels in FPTPSD group were higher than the ones in PTPSD group; (5) an accuracy of diagnosis for PTPSD was increased by 9-19% when Iso% was introduced to urinary pterin scheme. Iso% is helpful to reduce the rate of misdiagnosis of PTPSD in the diagnosis by urinary pterin analysis for hyperphenylalaninemias and improve the accuracy of diagnosis. This approach is worthy of further development and increased utilization.

Phenylketonuria and Hirschsprung Disease—A Report of an Unusual Neonatal Presentation

We describe a term born boy of non-consanguineous Swiss parents with tetrahydrobiopterine (BH4)-responsive Phenylketonuria (PKU) and Hirschsprung disease with unusual neonatal presentation. The child presented with floppiness, irritability, recurrent bilious vomiting and failure to pass meconium until 32 hours after birth, resulting in the clinical suspicion of an intoxication-type metabolic disease such as maple syrup urine disease (MSUD). Although the slightly elevated branched-chain amino acids in newborn screening on the fourth day of life initially supported the clinical suspicion of MSUD, the elevated Phenylalanine (Phe) of 650 µmol/L, low Tyrosine (Tyr) of 30 µmol/L, and a Phe/Tyr ratio of 22, led to the diagnosis of PKU. BH4-testing resulted in a significant decrease of Phe from 1011 to 437 µmol/L within 24 h. Urinary pterins and dihydropteridine reductase (DHPR) activity were normal, supporting the diagnosis of BH4-responsive PKU. Dietary restriction of Phe was initiated immediately, but oral feeding turned out to be difficult because of gastrointestinal symptoms. Intestinal motility disorder was suspected due to distended abdomen, obstructive symptoms and radiological findings with dilated intestinal loops and lack of intestinal gas in the anorectal region. Hirschsprung disease was confirmed by rectal suction biopsies and treated by a laparoscopically-assisted transanal pull-through (de la Torre) procedure. The boy is additionally compound heterozygous for two mutations in the phenylalanine hydroxylase (PAH) gene, which confirmed BH4-responsive PKU. It is the first case to be described in the literature of the comorbidity of PKU and Hirschsprung disease.

PTPS gene analysis and prenatal diagnosis in patients with 6-pyruvoyl-tetra hydropterin synthase deficiency

Objective: To analyze the variations of PTPS gene in patients with suspected 6-pyruvoyl-tetra hydropterin synthase deficiency (PTPSD) and to make prenatal diagnosis in high-risk families. Methods: Chemiluminescence was used for phenylalanine detection in blood or dried blood spots.Patients with phenylalanine concentration over 120 μmol/L were detected by urine pterin analysis, and the activity of dihydropteridine reductase (DHPR) was detected. tetrahydrobiopterin loading tests were performed in suspected patients with abnormal urinary pterin profiles. PTPS gene variation analysis was performed by direct Sanger sequencing based on PCR amplification. Prenatal diagnosis in 7 high-risk families was performed by chorionic villus sampling when the genotype was identified. Results: In 656 patients with hyperphenylalanine, 22 cases were diagnosed as PTPSD clinically. 16 variations were detected in the 22 PTPSD cases. The 5 variations, p.Lys77Arg, p.Ile84Phe, c.315-2A>G, c.244-2A>T, c.187-1G>T, were identified as novel variations. Two fetuses carried the same mutation with the proband and therefore were thought to be PTPSD fetuses. Three fetuses carried only one mutant allele and thus were thought to be PTPSD carriers. The other 2 fetuses carried no mutations and were presumed normal. Conclusions: PTPS gene variation analysis is necessary to confirm the diagnosis. Prenatal diagnosis could help avoiding the defect birth in PTPSD families.

Analysis of neonatal hyperphenylalaninemia screening and therapeutic effect in Xuzhou from 2003 to 2015

Objective To investigate the prevalence, clinical classification, treatment and prognosis of neonatal hyperphenylalaninemia(HPA) in Xuzhou area, China. Methods Infants born between July 1, 2003 and July 1, 2015 in Xuzhou area were investigated. Heel blood samples of neonates were collected at 72 hours after birth, and the concentration of blood phenylalanine(Phe) was determined by fluorescent quantitative method in Xuzhou Maternity and Child Health Care Station Neonatal Disease Screening Center. Differential diagnosis was performed in all 265 cases diagnosed as HPA by urinary pterin analysis and dihydropteridine reductase activity determination. The blood Phe concentration and mental development were followed up regularly in infants with HPA. Mutations of phenylalanine hydroxylase (PAH) gene were analyzed by gene sequencing. The relationship between blood Phe concentration and mental development was analyzed by Bivariate correlation analysis. Results (1) The prevalence of HPA in neonates in Xuzhou was l/4 635. Among the 265 confirmed HPA cases, 260 cases(98.11%) had PAH deficiency, including 90(33.96%) classical phenylketonuria(PKU), 84(31.70%) mild PKU and 86(32.45%) mild HPA. The other five patients(1.89%) diagnosed with tetrahydrobiopterin (BH4) deficiency all had 6-pyruvoyl tetrahydropteirn synthase(PTPS) deficiency. (2) Among the 265 HPA cases, 26 cases refused any treatment, including five cases of PTPS deficiency and 21 cases of PKU. Of the five patients with PTPS deficiency, two died and the other three had normal mental and physical development. Twenty-one PKU patients who refused treatment had mental retardation of various degrees. Among 153 PKU patients who received medical treatment, three died and 12 were lost to follow-up. (3) For 138 PKU patients who received dietary treatment and follow-up, the ages at the last visit were two months to 12 years, 116 of them had normal mental development, the remaining 22 patients had mental retardation, and a negative correlation was observed between mental development and the average Phe concentration. (4) Thirty-five patients with PAH deficiency underwent gene sequencing, and 22 kinds of mutations of PAH gene were detected. Conclusions The prevalence of HPA in Xuzhou area is higher than the average national level. With early diagnosis and standard treatment, most of PKU neonates can have normal mental development. Phe level control is an important factor for mental development. Key words: Phenylketonurias; Phenylalanine hydroxylase; Biopterin; Neonatal screening; Intelligence; Treatment outcome

Differences in pteridine urinary levels in patients with malignant and benign ovarian tumors in comparison with healthy individuals

Pteridines belong to a class of fluorescent metabolites that are excreted by humans in urine and their concentrations can reflect various pathophysiological states. We quantified the differences in urinary pteridine levels in patients with malignant and benign ovarian tumors and in healthy individuals. Urine samples were centrifuged and supernatants were oxidized by MnO2 before analysis. Levels of neopterin, biopterin, and pterin were assessed by fluorescence analysis of human urine after HPLC separation.We have revealed that the median neopterin levels were higher in urine samples from patients with malignant (0.226μmol/mmol creatinine) and benign ovarian tumors (0.150μmol/mmol creatinine) than in healthy subjects (0.056μmol/mmol creatinine). The median neopterin levels of patients with malignant tumors were higher (1.5-times) than in patients with benign tumors. The median biopterin level in urine of patients with benign ovarian tumors (0.268μmol/mmol creatinine) was found to be very close to the level in patients with malignant ovarian tumors (0.239μmol/mmol creatinine), and both were higher than in healthy samples (0.096μmol/mmol creatinine). The levels of urine pterin followed a pattern similar to neopterin levels for both ovarian tumors, but their concentrations were about three times lower than neopterin levels.

Impact‐induced muscle damage and urinary pterins in professional rugby: 7,8‐dihydroneopterin oxidation by myoglobin

Muscle damage caused through impacts in rugby union is known to increase oxidative stress and inflammation. Pterins have been used clinically as markers of oxidative stress, inflammation, and neurotransmitter synthesis. This study investigates the release of myoglobin from muscle tissue due to force-related impacts and how it is related to the subsequent oxidation of 7,8-dihydroneopterin to specific pterins. Effects of iron and myoglobin on 7,8-dihydroneopterin oxidation were examined in vitro via strong cation-exchange high-performance liquid chromatography (SCX-HPLC) analysis of neopterin, xanthopterin, and 7,8-dihydroxanthopterin. Urine samples were collected from 25 professional rugby players pre and post four games and analyzed for myoglobin by enzyme-linked immunosorbent assay, and 7,8-dihydroneopterin oxidation products by HPLC. Iron and myoglobin oxidized 7,8-dihydroneopterin to neopterin, xanthopterin, and 7,8-dihydroxanthopterin at concentrations at or above 10 μM and 50 μg/mL, respectively. All four games showed significant increases in myoglobin, neopterin, total neopterin, biopterin, and total biopterin, which correlated between each variable (P < 0.05). Myoglobin and iron facilitate 7,8-dihydroneopterin oxidation to neopterin and xanthopterin. In vivo delocalization of myoglobin due to muscle damage may contribute to oxidative stress and inflammation after rugby. Increased concentrations of biopterin and total biopterin may indicate production of nitric oxide and monoamine neurotransmitters in response to the physical stress.

Diagnosis, treatment and follow-up of patients with tetrahydrobiopterin deficiency in Shandong province, China.

To summarize the clinical and biochemical data, mutation analysis, treatment, outcome and the follow-up data of patients with BH4 deficiency from 2004 to 2012 in Shandong province, China. We analyzed the clinical, biochemical and treatment data of 40 patients with BH4 deficiency. Urinary neopterin and biopterin were analyzed. Further BH4 loading tests were performed in suspected patients with abnormal urinary pterin profiles. The patients with BH4 deficiency were treated with BH4 and neurotransmitter after diagnosis. Blood phenylalanine level, clinical symptoms and mental development were followed up. 40 cases with BH4 deficiency were identified and all classified as PTPS deficiency between 2004 and 2012 in Shandong province, China. They were diagnosed at the age of 20d - 41m and most patients received treatment with BH4, l-dopa and 5-HTP after diagnosis. Seven different mutations (P87S, K91R, T106M, D96N, N52S, S21R, and L127F) were detected in 11 patients. But outcome assessments were not always available. We obtained 19 records of DQ/IQ assessment. In 9 patients (7 early and 2 late diagnosed) no development delay is observed, while in 10 patients (8 early and 2 late diagnosed) development was delayed. Our study emphasized that screening for BH4 deficiency should be carried out in all patients with HPA in order to minimize misdiagnosis. Although the outcomes of BH4 deficiency are highly variable, early diagnosis and treatment is essential for good outcomes.

Deficiencias de tetrahidrobiopterina (BH4): diagnóstico y tratamiento

Tetrahydrobiopterin (BH4) is an enzyme cofactor of phenylalanine hydroxylase, tyrosine hydroxylase and tryptophan hydroxylase. Three different enzymes affect BH4 synthesis: guanosine triphosphate cyclohydrolase I (GTP CH 1), 6-piruvoil tetrahydropterin synthase (PTPS), sepiapterin reductase (SR) and dihydropteridine reductase (DHPR) affects regeneration. BH4 deficiency causes defects in monoaminergic neurotransmitters synthesis and can be or not associated with hyperphenylalaninemia (HPA). Symptoms often begin in the first months of life with mental retardation, dystonic movements and posture, temperature control disorders, seizures, abnormal posture and tone, salivation and swallowing difficulties. Diagnosis is based on the following tests: pterins in urine, neurotransmitters and pterins in cerebrospinal fluid, reduced enzyme activity (in blood or fibroblasts) and mutational analysis. Treatment should be directed to normalize blood Phe levels and brain neurotransmitters with the administration of BH4 10 mg / kg / d, L-DOPA 10 mg/kg/day and 5-OH-tryptophan, 5 to 6 mg/kg/day. LDOPA should be given with carbidopa in order to inhibit the destruction of the L-dopa by the carboxylase. We describe 3 patients with BH4 deficiency treated at the Ramon y Cajal Hospital. All of them had a good neurological outcome with almost normal mental development.

The complexity of newborn screening follow-up in phenylketonuria.

In the United States, and most developed nations, the newborn screening (NBS) panel covers many primary disorders of metabolism, including phenylketonuria (PKU). When an elevated phenylalanine level is identified, the infant is evaluated for PKU and should also be tested for tetrahydrobiopterin (BH4) deficiency. A neonate presented with a phenylalanine level of 254μmol/L (reference range <138μmol/L) on newborn screening. The infant's confirmatory phenylalanine was 118μmol/L (reference range <77μmol/L). Her urine pterin profile was normal, and initially she had no measurable activity of red blood cell (RBC) dihydropteridine reductase (DHPR). Subsequent study revealed normal levels of CSF tetrahydrobiopterin and neurotransmitter metabolites, and by 18 months of age, her RBC DHPR activity was detectable at 0.5nmol/min/mgHgb (reference range 0.8-3.9). Sequencing of the QDPR gene for DHPR revealed c.1A>T nucleotide substitution in exon 3 expressed as "p.MET1?" Phenylalanine hydroxylase (PAH) gene sequencing revealed compound heterozygosity for L249F and A300S. Although initial testing suggested the child was affected with DHPR deficiency, further analysis, finding increasing levels of DHPR activity and PAH compound mutant heterozygosity, indicated that the primary disorder is mild hyperphenylalaninemia with carrier status for DHPR deficiency. This is an example of newborn screening results leading to confusing findings requiring extensive biochemical studies and genotyping in order to arrive at the appropriate diagnosis.

Determination of pterins in urine by HPLC with UV and fluorescent detection using different types of chromatographic stationary phases (HILIC, RP C8, RP C18)

Pterins are a class of potential cancer biomarkers. New methods involving hydrophilic interaction liquid chromatography (HILIC) and reversed phase (RP) high-performance liquid chromatography have been developed for analysis of eight pterin compounds: 6,7-dimethylpterin, pterin, 6-OH-methylpterin, biopterin, isoxanthopterin, neopterin, xanthopterin, and pterin-6-carboxylic acid. The effect of mobile phase composition, buffer type, pH and concentration on retention using HILIC, C8 and C18 RP stationary phases were examined. Separation of pterins on RP and HILIC stationary phase was performed and optimized. Eight pterins were successfully separated on HILIC Luna diol-bonded phases, Aquasil C18 RP column and LiChrospher C8 RP column. Determination and separation of the pterins from urine samples were performed on HILIC Luna and LiChrospher C8 RP columns which were chosen as the most appropriate ones. Finally, LiChrospher C8 RP column with fluorescence detection was selected for further validation of the method. The optimum chromatographic condition was mobile phase methanol (A)/phosphoric buffer pH 7, 10mM (B), isocratic elution 0–15min 5% A flow=0.5ml/min 15–17min. 5% A, flow=0.5–1ml/min the linearity (R2>0.997) and retention time repeatability (RSD%<1) were at satisfactory level. The precision of peak areas expressed as RSD in % was between 0.55 and 14. Pterins detection limits varied from 0.041ng/ml to 2.9ng/ml. Finally, HPLC method was used for the analysis of pterins in urine samples with two different oxidation procedures. Concentration levels of pterin compounds in bladder cancer patients and healthy subjects were compared.

The proportion of tetrahydrobiopterin deficiency and PAH gene deficiency variants among cases with hyperphenyalaninemia in Western Iran

BACKGROUND:Defects either in phenylalanine hydroxylase (PheOH) or in the production and recycling of its cofactor (tetrahydrobiopterin [BH4]) are the causes of primary hyperphenylalaninemia (HPA). The aim of our study was to investigate the current status of different variants of HPA Kurdish patients in Kermanshah province, Iran.MATERIALS AND METHODS:From 33 cases enrolled in our study, 32 were identified as HPA patients. Reassessing of pre-treatment phenylalanine concentrations and the analysis of urinary pterins was done by high-performance liquid chromatography method.RESULTS:A total of 30 patients showed PAH deficiency and two patients were diagnosed with BH4 deficiency (BH4/HPA ratio = 6.25%). Both of these two BH4-deficient patients were assigned to severe variant of dihydropteridine reductase (DHPR) deficiency. More than 75% of patients with PAH deficiency classified as classic phenylketonuria (PKU) according their levels of pre-treatment phenylalanine concentrations.CONCLUSION:Based on the performed study, we think that the frequency of milder forms of PKU is higher than those was estimated before and/or our findings here. Furthermore, the frequency of DHPR deficiency seems to be relatively high in our province. Since the clinical symptoms of DHPR deficiency are confusingly similar to that of classic PKU and its prognosis are much worse than classical PKU and cannot be solely treated with the PKU regime, our pilot study support that it is crucial to set up screening for BH4 deficiency, along with PAH deficiency, among all HPA patients diagnosed with HPA.

Determination of Six Pterins in Urine By Lc–MS/MS

The present work describes an analytical method for urinary pterins by LC-MS/MS, with emphasis on the separation of 6- and 7-positional isomers of bio- and neopterins. Urine sample preparation consisted of oxidation by MnO(2), filtration and direct dilution in the mobile phase. The method was validated in urine spiked at five concentration levels with true triplicates of each level. Separation of the pterins, including the positional isomers, was achieved by employing a LUNA amino column. Six pterins were quantified (pterin, isoxanthopterin, 6-biopterin, 7-biopterin, 6-neopterin, 7-neopterin) and a linear behavior was observed; LOD varied from 7 to 360 pg/ml and correlation coefficients above 0.98 were obtained for all pterins. In addition, pterin levels were evaluated in 41 urine samples of healthy subjects, in ten urine samples of patients with classical phenylketonuria (PKU) and in one with atypical PKU. The proposed method allowed to identify, separate and quantify six pterins in urine, using a simple and rapid sample preparation. The atypical PKU was unequivocally differentiated from the classical form, demonstrating that this method could be very useful for characterization and follow-up of diseases.

Analysis of Pterins in Urine by HILIC

Pterins are a class of compounds excreted in urine. Levels of the pterins are found to be significantly elevated in a variety of diseases. A new method involving hydrophilic interaction chromatography with fluorescence detection has been developed for analysis of neopterin, biopterin, and isoxanthopterin in urine samples. Separation of these pterins on an aminopropyl hydrophilic interaction column was achieved by isocratic elution. The effects of the organic modifier content, ionic strength, and pH of the mobile phase on the hydrophilic behavior of the pterins were studied and the mechanism of their separation was also investigated. Under the optimum chromatographic conditions the linearity (r ≥ 0.9995) and repeatability (relative standard deviation < 4.0%) of the method are good. Compared with reversed-phase high-performance liquid chromatography, the method is simple and convenient. The method was applied to the analysis of pterins in urine samples with satisfactory results.

The investigation of differential diagnostic development and incidence of tetrahydrobiopterin deficiency

To investigate the development of differential diagnosis of tetrahydrobiopterin (BH4) deficiency among patients with hyperphenylalaninemia (HPA) in provinces or cities of China and to investigate the incidence of BH4 deficiency. Of the thirteen hundreds and ninety-two patients with HPA received, the differential diagnosis for BH4 deficiency during 1993 - 2007 were enrolled in this study. Of which, 591 patients came from outpatient and 801 patients' samples from other provinces or cities were sent to author's laboratory to investigate the case number of differential diagnosis for BH4 deficiency in provinces or cities of China according to the data from both outpatient case histories and laboratory as to investigating the development of differential diagnosis in the whole country. To discuss the diagnostic criteria for BH4 deficiency was according to the results of urinary pterin analysis, determination of dihydropteridine reductase (DHPR) activity and the tetrahydrobiopterin loading test as well as to get the incidence of BH4 deficiency and find some provinces or cities with higher incidence of BH4 deficiency in China. (1) The number of HPA patients, who were performed by urinary pterin analysis and the determination of DHPR activity, were remarkably increased in last three years (2005 - 2007). The patient numbers of both urinary pterin analysis and DHPR activity determination were 217 and 198 respectively in 2005. And in 2007 they increased to 511 and 458, which was about 2.3 times than that in 2005. The patients came from 29 provinces or cities in 2007. (2) The urinary biopterin and biopterin percent were key marks for diagnosis of 6-pyruvoyl tetrahydropterin synthase (PTPS) deficiency. The less than 5% [(1.41 +/- 1.10)%] biopterin percent and very low biopterin level [(0.14 +/- 0.17) mmol/mol Cr] were found in 96.83% (61/63) patients with PTPS deficiency in this study. The blood phenylalanine level was remarkably decreased to normal range at 2 - 6 hours after BH4 loading test. The very low DHPR activity was a final diagnostic mark for DHPR deficiency. The very low DHPR activities of 0.27 nmol/(min x 5 mm disc) (6.11% - 7.00% of normal controls) were found in two patients with DHPR deficiency in this study. (3) The incidences of PTPS deficiency and DHPR deficiency among 1392 patients with hyperphenylalaninemia were 8.41% (117/1392) and 0.18% (2/1108) respectively. About 67.23% (80/119) patients with BH4 deficiency came from the south of Yangtze liver. The 80% (8/10) provinces or cities with higher incidence of BH4 deficiency are located in eastern and southern China. The incidence of PTPS deficiency among patients with HPA and normal newborns was 10.81% (8/74) and 0.007 per thousand (8/1,121,429) respectively in Shanghai, China according to data from neonatal screening. The awareness of differential diagnosis for BH4 deficiency from clinic pediatricians has been increased in most provinces or cities of China in last three years, but it should be more strengthened.

Screening for tetrahydrobiopterin deficiencies using dried blood spots on filter paper

Tetrahydrobiopterin (BH 4) deficiency among newborns with hyperphenylalaninemia must be rapidly diagnosed and distinguished from classical phenylketonuria (PKU) to initiate immediately specific treatment and to prevent irreversible neurological damage. The characteristic pattern of urinary pterins makes it possible to differentiate between PKU and BH 4 deficiencies, and to identify different variants of BH 4 deficiency. However, collection, storage, and shipment of urine samples for pterin analysis is cumbersome. A method for the measurement of different pterins (neopterin, biopterin, and pterin) in blood collected on filter paper was developed as a potential alternative to the screening for BH 4 deficiencies in urine and for the monitoring of BH 4 pharmacokinetics. Pterins pattern in blood spots was comparable with those in plasma and urine. We thus established reference values for pterins in blood spots in patients with hyperphenylalaninemia and identified new patients with GTP cyclohydrolase I deficiency, 6-pyruvoyl-tetrahydropterin synthase deficiency, and dihydropteridine reductase deficiency using dried blood spots on filter paper.

Incidence of BH 4-responsiveness in phenylalanine-hydroxylase-deficient Italian patients

Background Hyperphenylalaninemia (HPA) is an inherited metabolic disorder due to deficiency of the enzyme phenylalanine hydroxylase (PAH) or its cofactor tetrahydrobiopterin (BH 4). BH 4-responsiveness in PAH-deficient HPA is a recently described characteristic of most milder phenotypes. BH 4-responsive patients show reduction of plasma phenylalanine (phe) levels after oral administration of BH 4. Aim Determination of the incidence of BH 4-responsiveness among a non-selected, cohort population of PAH-deficient hyperphenylalaninemic patients and evaluation of phenotype–genotype correlations. Patients and methods All patients born in Lombardy (Italy) between January 2000 and December 2004, and affected by HPA (107 patients) were classified after BH 4 loading test, analysis of urinary pterins, and determination of DHPR activity in blood, and investigated for BH 4-responsiveness. 6R-BH 4 (20 mg/kg) was administered orally as a single dose and plasma samples were obtained at time-points 0, 4, 8, and 24 h after BH 4 administration. In patients with basal plasma phe levels ⩽360 mmol/L a combined phe (100 mg phe/kg) and BH 4 (20 mg/kg) loading test was performed. Patients were defined “responsive to BH 4” when plasma phe levels decreased by 30% 8 h after oral BH 4 administration. Results BH 4 significantly lowered blood phe levels in 91 (85%) of 107 patients affected by PAH-deficient HPA. Most responsive patients were affected by mild HPA (77%), a smaller percentage by mild (7%) and moderate (7%) phenylketonuria (PKU). One patient with classical PKU was responsive to BH 4. Eighteen mutations were found to be associated to the BH 4-responsive phenotype. Conclusions BH 4-responsiveness is shown by a consistent number of PAH-deficient hyperphenylalaninemic patients and seems to be common in milder phenotypes. Genotype is not the only factor determining BH 4-responsiveness.