四氢生物喋呤负荷试验在高苯丙氨酸血症鉴别诊断中的临床应用

目的探讨四氢生物喋呤（BH4）负荷试验在高苯丙氨酸血症（HPA）鉴别诊断中的应用价值。方法自2005年5月到2007年4月，51例HPA患儿采用口服BH4（20mg／kg）负荷试验。对其中血苯丙氨酸（Phe）浓度小于600μmol／L患儿采用口服Phe—BH4联合负荷试验，结合尿喋呤分析、血红细胞二氢喋呤还原酶（DHPR）活性测定。结果（1）在BH4负荷试验中，不同类型HPA患儿的血Phe浓度表现出各不相同的改变。51例HPA患儿中，共鉴别出5例BH4缺乏症，10例BH4反应性苯丙氨酸羟化酶（PAH）缺乏症，36例BH4无反应性苯丙氨酸羟化酶（PAH）缺乏症。（2）在17例中度苯丙酮尿症（PKU）患儿中，9例（52．9％）为BH4反应性PAH缺乏症。结论BH4负荷试验在HPA早期鉴别诊断中十分重要，部分中度PKU对BH4有反应，可使用BH4替代治疗。     

四氢生物蝶呤负荷试验在高苯丙氨酸血症鉴别诊断中的价值

目的 对高苯丙氨酸血症患者进行口服四氢生物蝶呤（tetrahydrobiopterin，BH4）负荷试验，了解该方法在高苯丙氨酸血症中的鉴别诊断价值。方法 新生儿筛查阳性以及临床诊断的高苯丙氨酸血症患儿73例（男47例、女26例），中位数年龄1．93月，进行口服BH4负荷试验（20mg／kg）。对其中血苯丙氨酸（phenylalanine Phe）浓度〈600／μmol／L者给予口服Phe—BH4联合负荷试验，同时进行尿蝶呤谱分析，红细胞二氢生物蝶啶还原酶测定。结果 在BH4负荷试验或Phe－BH4联合负荷试验中，经典型苯丙酮尿症（phenylketonuria，PKU）的血Phe对BH4无反应；苯丙氨酸羟化酶缺乏引起的中度PKU的血Phe 24 h下降32．8％；BH4缺乏症患者服BH4后血Phe表现出特征性的快速下降，4 h降至正常水平，并且维持至24h。结合其他检测结果，22例诊断为经典型PKU，39例中度PKU，12例BH4缺乏症。结论 高苯丙氨酸血症是由苯丙氨酸羟化酶缺乏或者BH4缺乏所引起，早期鉴别诊断十分重要。BH4负荷试验是快速、简便的体内诊断试验，安全可靠，有较高的鉴别诊断价值。     

宝鸡地区2007∽2013年新生儿筛查高苯丙氨酸血症结果分析

目的对宝鸡市2007年∽2013年间的204 868例新生儿筛查苯丙氨酸浓度测定,并对部分阳性患儿血、尿标本进行MS/MS、GS/MS分析,探讨我市PKU发病情况和发病率。方法采用ELISA时间分辨荧光免疫多标记免疫分析法（DELFIA）检测血苯丙氨酸浓度。结果筛查出高苯丙氨酸血症71例,其中经典型PKU17例,轻度PKU22例,轻度HPA30例,BH4缺乏症2例,高HPA检出率为1/2885（71/204868）,高出全国HPA发生率（1/11444）,BH4缺乏症为2.82%。结论开展新生儿筛查是早期发现HPA的有效方法,对HPA患儿应进一步进行鉴别诊断,排除四氢喋呤缺乏症,并进行及时治疗,避免残障儿的发生。     

一例Pah基因杂合缺失合并半合子突变病例分析

目的 对一例苯丙氨酸羟化酶(PAH)缺乏症患儿进行基因组测序,分析其Pah基因变异情况,以明确病因。方法 采用免疫荧光法对新生儿进行血苯丙氨酸(Phe)浓度检测,血Phe浓度>2.0mg/dL进行尿蝶呤谱分析以鉴别病因;进一步用基因测序技术对其静脉血进行Pah基因测序,测序数据与正常人基因组进行比对,分析基因变异情况。结果 该患儿血Phe浓度为46.88 mg/dL(2765.92μmol/L),正常新生儿Phe浓度<2.0mg/dL,苯丙氨酸/酪氨酸(Phe/Tyr)为49.65(参考范围0.1～1.5),尿蝶呤谱结果为52.08(正常新生儿：19.8～50.3);基因测序结果发现1个缺失突变和1个剪接突变,分别为：exon6杂合缺失和第6外显子c.611A>G剪接突变,其中exon6片段缺失在人类基因突变数据库(HGMD)中未报道;患儿根据治疗前最高血Phe进行分类为经典型PAH缺乏症,需低Phe饮食治疗,并定期监测血Phe浓度水平。结论 初步明确了该患儿的病因为杂合缺失合并半合子突变,Pah基因型为exon6杂合缺失/c.611A>G;并发现了1种未报道基...     

四氢生物蝶呤反应性的苯丙酮尿症研究进展

本文综述对四氢生物蝶呤（BH4）反应性的苯丙氨酸羟化酶（PAH）基因缺陷的苯丙酮尿症（PKU）的研究进展。分析BH4反应性的PAH基因突变，探讨这一现象潜在的发生机制，介绍一种新的临床分类法，并对PKU的BH4替代治疗前景进行展望。     

中国南方四氢生物蝶呤缺乏症筛查、临床及基因突变的系列研究

目的 得出我国南方高苯丙氨酸血症（hyperphenylalaninemia,HPA)者四氢生物蝶呤（tetrahydrobiopterin,BH4)缺乏症的发病率,总结BH4缺乏症者基因突变和临床转归的研究。方法 对87例HPA者做尿新蝶呤（N）和生物蝶呤（B）分析;对BH4缺乏症者进行基因突变检测及治疗随访。结果 11例诊断为6－丙酮酰四氢蝶呤合成酶（PTPS）缺乏所致BH4缺乏症,其尿N／B＞38,B％＜5％,在HPA中其发病率为12％。5例中发现4种PTPS基因突变类型,即P87S、N52S、D96N及G144R（新突变类型）。5例经BH4、L－DOPA及5－羟色氨酸治疗后体格发育良好,4例智商（IQ）70－80分。结论 对所有HPA者需进行BH4缺乏症的筛查,以降低误诊率。     

1066例高苯丙氨酸血症患者诊断及治疗随访

目的1066例高苯丙氨酸血症（HPA）患者治疗随访。方法自1984年10月到2009年12月,我院共诊治HPA患者1066例。采用高效液相色谱法进行尿喋呤分析,血红细胞二氢喋啶还原酶（DHPR）活性测定及四氢生物喋呤（BH4）负荷试验,进行四氢生物喋呤缺乏症（BH。D）的鉴别;对部分患者进行MRI、1HMRS检查;对不同类型的HPA进行治疗。结果（1）1066例中1016例为苯丙氨酸羟化酶缺乏症（PAHD）,其中51例为BH4反应性PAHD;50例为BH4Dc（2）28例筛查早治的BH4D患儿智商为（96±15）分,晚治疗的11例患儿治疗前为（46±15）分,治疗后为（69±11）分,前后有显著意义（P〈0．05）。DQ／IQ水平与治疗开始时间呈明显的负相关（r=-0．714,P〈0．01）。（3）33例BH4反应性PAHD患儿的智商为（92±18）分。（4）PAHD患者治疗中筛查组智商与非筛查组智商比较,筛查治疗理想组智商与筛查治疗不理想组智商比较,非筛查治疗理想组智商与非筛查治疗不理想组智商比较均有意义（P〈0．001）,47例非新筛并控制理想血Phe浓度PKU患儿智商从治疗前的（60．66±7．78）分,提高到治疗后的（76．62±7．55）分（P〈0．001）。（5）合并癫痫的患儿脑电图的异常率为94．3％,将血苯丙氨酸浓度控制在理想范围,可使脑电图异常有改善。（6）22例大于4个月的患儿血、脑Phe浓度与智商均呈负相关关系r血=-0．5045,r脑=-0．6471（P〈0．01）。结论对所有HPA患者都必须进行BH4D的鉴别诊断,尽早确诊和治疗效果越好。严格控制血苯丙氨酸浓度是减少智能落后的最好措施。     

儿童运动及智能障碍者四氢生物蝶呤代谢病筛查及基因研究

目的探讨四氢生物蝶呤（tetrahydrobiopterin，BH4）代谢中各酶缺乏在儿童运动及智能发育障碍者中发生率及基因突变。方法对100例运动及智能障碍患者进行苯丙氨酸（phenylalanine，Phe）及BH4负荷试验、尿蝶呤谱分析、红细胞二氢蝶啶还原酶测定，并对部分患者进行多巴治疗性诊断；对诊断为多巴反应性肌张力障碍（dopa-responsive dystonia，DRD）及6-丙酮酰四氢蝶呤合成酶（6-pyruvoyl tetrahydropterin synthase，PTS）缺乏者做基因突变检测。结果100例中70例基础血Phe浓度正常，6例（6％）诊断为DRD；30例有高苯丙氨酸血症[Phe（1022±290）μmol／L]，8例（8％）诊断为VIS缺乏症，22例（22％）诊断为苯丙氨酸羟化酶缺乏症。发现2例DRD患者其三磷酸鸟苷环化酶基因（GTP cyclohydrolase 1 gene，GCHI）突变为IVS5＋3insT，8例FIS缺乏症患者存在PTS基因7种突变类型，其中259C→T，286G→A，155A→G最常见，占75％。结论一些肌张力障碍或智能障碍者是由于BH4代谢障碍所致，有必要做筛查诊断以明确诊断。     

中国北方人群四氢生物蝶呤缺乏症的研究

目的探讨中国北方人群四氢生物蝶呤(tetrahydrobiopterin,BH4)缺乏症的筛查、治疗和长期预后。方法自1992年到2005年,我院共诊治主要来自中国北方地区的高苯丙氨酸血症(hyperpheny lalaninemia,HPA)患者618例。采用尿蝶呤谱分析,红细胞二氢蝶啶还原酶(dihydropteridinereductase,DHPR)活性测定及四氢生物蝶呤负荷试验,对这些患者进行BH4缺乏症的鉴别诊断;确诊患者立即进行BH4、左旋多巴和5羟色胺三药联合治疗。定期随诊患者的精神运动和智能发育情况。结果(1)在618例HPA患者中,共鉴别出38例BH4缺乏症,而且皆表现为尿生物蝶呤显著降低,新蝶呤显著增加,红细胞DHPR活性正常,血苯丙氨酸浓度在BH4负荷后4～8小时内降至正常,即全部是6丙酮酰四氢蝶呤合成酶(6pyruvoyl tetrahydropterinsynthase,PTPS)缺乏型,被确诊时的年龄为2岁零1个月～13岁;(2)其中,只有27例患儿接受规律治疗并定期随诊。在经过3个月～10年的治疗后,患者的发育商或智商(developmentorintelligence quotient,DQ/IQ)明显提高(治疗前后分别为52±16,79±15),但智能发育各个方面并不平行;(3)通过对患儿目前DQ/IQ与治疗开始时间进行Pearson相关分析发现,二者呈明显负相关(r=-0.714,P<0.01)。结论对所有HPA患者都必须早期进行BH4缺乏症的鉴别诊断;PTPS缺乏症是中国北方导致BH4缺乏症的主要原因;PTPS缺乏症患者必须进行BH4、左旋多巴和5羟色胺三药联合治疗,而且治疗越早,效果越好。     

高苯丙氨酸血症患儿尿蝶呤谱分析及正常人群尿蝶呤谱参考区间的建立

目的探讨尿蝶呤谱分析、口服四氢生物蝶呤(tetrahydrobiopterin,BH_4)药物负荷试验及酸学分析在高苯丙氨酸血症(hyperphenylalaninemia,HPA)诊断与分型中的意义,同时建立本地区正常人群尿蝶呤谱参考区间,为我国正常人群尿蝶呤谱参考区间的制订提供可靠数据。方法 2014年1月至2017年6月本中心正常健康人群及无先天性遗传代谢缺陷的人群859例为正常对照组,确诊的137例高苯丙氨酸血症患者为研究组,运用串联质谱(LC-MS/MS)测定血苯丙氨酸(Phe)、高效液相色谱(HPLC)对尿液蝶呤谱分析,同时根据BH_4药物负荷试验,红细胞二氢生物蝶啶还原酶(DHPR)进行分组研究,运用统计学进行差异性分析及正常人群各年龄段参考区间建立。结果 137例HPA中,BH_4反应型HPA22例,占16.05%(22/137),无反应型HPA79例,占57.66%(79/137),BH_4缺乏症36例,占26.27%(36/137),其中6-丙酮酰四氢蝶呤合成酶(6-pyruvoyl tetrahydropterin synthase deficiency,PTPS)34例,占BH_4缺乏症94.44%(34/36)例,二氢生物蝶啶还原酶(dilydropterindine reductase deficiency,DHPR)2例,占5.56%(2/36)。尿蝶呤谱PTPS缺乏组年龄差异无统计学意义(P0.05),新蝶呤(neopterin,N)高于正常对照组和无反应型HPA组,生物蝶呤(biopterin,B)及生物蝶呤百分比(B/N+B,B%)明显低于各组,差异有统计学意义(P0.05);BH_4反应型HPA和无反应型HPA组N、B均高于正常对照组,差异有统计学意义(P0.05),而B%差异无统计学意义(P0.05);BH_4反应型HPA与无反应型HPA两组差异无统计学意义(P0.05)。口服BH_4药物负荷试验PTPS组血Phe浓度下降最快并在4h内降至正常,无反应型HPA组24h变化不明显,BH_4反应型HPA和DHPR缺乏组下降速度和幅度相似,但DHPR缺乏组酶活性低于正常。结论尿蝶呤谱分析对BH_4缺乏症分型及鉴别BH_4反应型HPA、无反应型HPA有辅助作用。口服BH_4药物负荷试验能快速判断血Phe对BH_4反应程度、血Phe的变化趋势,为可为HPA患者药物治疗提供依据,同时结合本地区正常人群尿蝶呤谱参考区间,为提高BH_4缺乏症诊断率及制定我国尿蝶呤谱参考值提供可参考依据。     

Extended tetrahydrobiopterin loading test in the diagnosis of cofactor-responsive phenylketonuria: a pilot study

Patients with tetrahydrobiopterin (BH4)-responsive phenylalanine hydroxylase (PAH) deficiency may benefit from BH4 therapy instead or in addition to the low-phenylalanine diet. Different loading test protocols are currently used to detect these patients. As a consequence, data on the rate of BH4-responsiveness within patients with mild phenylketonuria (PKU) and/or more severe phenotypes show high variation and a more sensitive and standardised BH4 loading test protocol needs to be defined. We modified the current standard BH4 loading test (20 mg/kg) to a second administration of 20 mg/kg after 24 h and extended blood sampling to 48 h in 24 patients with PAH deficiency. Using this extended loading test (2 x 20 mg BH4/kg), the rate of BH4-responsiveness was calculated at 8, 24, and 48 h after BH4 administration. We defined three groups of patients: "rapid responders" in 10/24 patients (4 mild HPA, 2 mild PKU, 2 moderate PKU, and 2 classic PKU), "moderate responders" in 4/24 patients (4 classic PKU), and "slow responder" in 4/24 patients (4 mild PKU). Six out of 24 patients (1 mild HPA, 1 moderate PKU, and 4 classic PKU) were found to be "non-responder." Individual phenylalanine profiles show variations in responsiveness at different time points and sampling over 48 h was more informative than over 24h in patients with mild and moderate PKU compared to mild HPA. Analysis of BH4 loading tests in 209 patients with the standard BH4 loading test protocol confirms only minor importance of the 24 h response: the rate of responsiveness to BH4 after 24 h was shown to be equal to or even lower than after 8h among most phenotypes. However, extension of the BH4 loading test to 48 h and repeated BH4 administration seems to be useful to detect BH4-responsiveness in more severe phenotypes and allows detecting "slow responders" who may benefit from BH4 therapy.     

Plasma biopterin levels and tetrahydrobiopterin responsiveness

Tetrahydrobiopterin (BH4) responsive hyperphenylalaninemia (HPA) with a mutant phenylalanine hydroxylase (PAH) gene was found during neonatal screening for PKU. This study determined blood BH4 and phenylalanine in two patients with hyperphenylalaninemia following oral load with BH4 10 mg/kg. Our patients underwent neonatal screening for PKU, had normal biopterin metabolism and their PAH mutations were determined. Peak plasma biopterin levels in Case 1, which were reached at between 2 and 4h after loading, were 612, 297, and 178 nmol/L at age 30 days, 55 days, and 19 months, respectively, and the maximum phenylalanine decreasing rates, which were found at 24h, were 54, 16, and 4%, respectively. In Case 2, peak plasma biopterin levels were 747 and 327 nmol/L at age 20 and 55 days, respectively, and the maximum phenylalanine decreasing rates were 39 and 32%, respectively. In the BH4 loading test, the peaks of BH4 in both patients lowered ( approximately 50%), on the same dose schedule of BH4, as patients got older.     

Response of patients with phenylketonuria in the US to tetrahydrobiopterin

Tetrahydrobiopterin (BH4) responsive forms of phenylketonuria (PKU) have been recognized since 1999. Subsequent studies have shown that patients with PKU, especially those with mild mutations, respond with lower blood phenylalanine (Phe) concentrations following oral administration of 6-R-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4). To determine the incidence of BH4 responding PKU patients in the United States and characterize their phenylalanine hydroxylase (PAH) mutations, a study was undertaken at UTMB in Galveston and the Children's Hospital of Los Angeles on 38 patients with PKU. Patients were screened by a single oral dose of BH4, 10 mg/kg and blood Phe and tyrosine were determined at 0, 4, 8, and 24 h. Twenty-two individuals (58%) responded with marked decrease in blood Phe (>30%) at 24h. Some of the patients that responded favourably were clinically described as having Classical PKU. Blood tyrosine concentrations did not change significantly. Twenty subjects with PKU, responsive and non-responsive to BH4, were enrolled in a second study to evaluate blood Phe response to ascending single doses of BH4 with 10, 20, and 40 mg/kg and to evaluate multiple daily doses, for 7 days each, with 10 and 20 mg/kg BH4. The 7-day trial showed a sustained decrease in blood Phe in 14 of 20 patients taking 20 mg/kg BH4 (70%). Of these 14 patients, 10 (71%) responded with a significant decrease in blood Phe following 10 mg/kg BH4 daily. To understand the mechanism of response to BH4, the kinetics and stability of mutant PAH were studied. We found that mutant PAH responds with increase in the residual enzyme activity following BH4 administration. The increase in activity is multi-factorial caused by increased stability, chaperone effect, and correction of the mutant Km. These studies indicate that BH4 can be of help to patients with PKU, including some considered to have Classical PKU. The PKU population in US is heterogeneous and mutations can be varied so mutations need to be characterized and response to BH4 tested. It is more likely that mutations with residual activity should respond to BH4, therefore the clinical definition of "Classical PKU" should be reconciled with the residual activity of PAH mutations.     

Molecular genetics of tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency

Mutations in the phenylalanine hydroxylase (PAH) gene result in phenylketonuria (PKU). Tetrahydrobiopterin (BH(4))-responsive hyperphenylalaninemia has been recently described as a variant of PAH deficiency caused by specific mutations in the PAH gene. It has been suggested that BH(4)-responsiveness may be predicted from the corresponding genotypes. Data from BH(4) loading tests indicated an incidence of BH(4)-responsiveness of >40% in the general PKU population and >80% in mild PKU patients. The current project entailed genotype analysis of 315 BH(4)-responsive patients tabulated in the BIOPKUdb database and comparison with the data from the PAHdb locus-specific knowledgebase, as well as with previously published PAH mutations for several European countries, Northern China, and South Korea. We identified 57 mutations, presenting with a substantial residual PAH activity (average approximately 47%), presumed to be associated with BH(4)-responsiveness. More than 89% of patients are found to be compound heterozygotes. The three most common mutations found in >5% of BH(4)-responsive patients are p.A403 V, p.R261Q, and p.Y414C. Using the Hardy-Weinberg formula the predicted average frequency of BH(4)-responsiveness in European populations was calculated to be 55% (range 17-79%, lowest in Baltic countries and Poland and highest in Spain), 57% in Northern China, and 55% for South Korea. The genotype-predicted prevalence of BH(4)-responsiveness was higher than prevalence data obtained from BH(4) loading tests. Inconsistent results were observed for mutations p.L48S, p.I65 T, p.R158Q, p.R261Q, and p.Y414C. Our data suggest that BH(4)-responsiveness may be more common than assumed and to some extent may be predicted or excluded from the patient's genotype.     

Positive effect of a simplified diet on blood phenylalanine control in different phenylketonuria variants, characterized by newborn BH4 loading test and PAH analysis

Until today, the mainstay of phenylketonuria (PKU) treatment is a phenylalanine (Phe)-restricted diet. Strict dietary treatment decreases flexibility and autonomy and still has a major impact on patients and their families. Compliance is often poor, particularly in adolescence. The aim of this study was to investigate the effect of the intake of fruits and vegetables containing Phe less than 100 mg/100g ('simplified diet'), as recommended by WHO for all individuals, instead of classical totally restricted diet on the course and treatment control of the disease in a well-characterized PKU cohort (n=80). All individual blood Phe measurements of each patient (1992-2009) were statistically analyzed before and after diet switch. Epidemiological data, age at diagnosis, PAH mutations, BH(4) responsiveness, as well as Phe control measurements and detailed diet information were tabulated in a local database. 62.5% had BH4 loading test and 40% had PAH analysis; 50/80 switched from classical to simplified diet, including 26 classical PKU, 13 moderate PKU, 7 mild PKU and 4 mild hyperphenylalaninemia (HPA). Median Phe levels on a simplified diet did not differ significantly to the median Phe levels on classical diet in all disease groups. Our results indicate that a simplified diet has no negative effect on blood Phe control in patients with hyperphenylalaninemia, independent of severity of the phenotype or the age at diet switch, over the period of 3 years. Thus, a simpler approach to dietary treatment of PKU available to all HPA patients is more likely to be accepted and adhered by patients and might also increase quality of life.     

Tetrahydrobiopterin,its Mode of Action on Phenylalanine Hydroxylase,and Importance of Genotypes for Pharmacological Therapy of Phenylketonuria

In about 20%–30% of phenylketonuria (PKU) patients (all phenotypes of PAH deficiency), Phe levels may be controlled through phenylalanine hydroxylase cofactor tetrahydrobiopterin therapy. These patients can be diagnosed by an oral tetrahydrobiopterin challenge and are characterized by mutations coding for proteins with substantial residual PAH activity. They can be treated with a commercially available synthetic form of tetrahydrobiopterin, either as a monotherapy or as adjunct to the diet. This review article summarizes molecular and metabolic bases of PKU and the importance of the tetrahydrobiopterin loading test used for PKU patients. On the basis of in vitro residual PAH activity, more than 1,200 genotypes from patients challenged with tetrahydrobiopterin were categorized as predictive for tetrahydrobiopterin responsiveness or non‐responsiveness and correlated with the loading test, phenotype, and residual in vitro PAH activity. The coexpression of two distinct PAH mutant alleles revealed possible dominance effects (positive or negative) by one of the mutations on residual activity as result of interallelic complementation. The treatment of the transfected cells with tetrahydrobiopterin showed an increase in residual PAH activity with several mutations coexpressed.     

Does the 48-hour BH4 loading test miss responsive PKU patients?

BackgroundPhenylketonuria (PKU) is an inborn error of phenylalanine (Phe) metabolism. Besides dietary treatment, some patients are responsive to and treated with tetrahydrobiopterin (BH4). Our primary objective was to examine whether the 48-hour BH4 loading test misses BH4-responsive PKU patients. Secondary, we assessed if it would be beneficial to 1) use a cut-off value of 20% Phe reduction instead of commonly used 30%, and 2) extend the loading test to 7 days.Methods24 patients with a 20–30% decrease of blood Phe levels during their initial 48-hour BH4 loading test or at least one mutation associated with long-term BH4 responsiveness, were invited to participate. 22 of them underwent the 7-day BH4 loading test. During the BH4 loading test, BH4 was administered orally once daily for 7 days (20 mg/kg/day). Blood samples on filter paper were collected at 13 time points. Potential BH4 responders (≥20% decrease in blood Phe concentrations at ≥1 moment within the first 48 h or ≥30% at ≥1 moment during the entire test) underwent a treatment trial to assess true long-term responsiveness (≥30% decrease of Phe levels compared to baseline and/or ≥50% increase in natural protein tolerance in accordance with the Dutch guidelines before 2017). The duration of the treatment trial varied from 2 to 18 months.ResultsOf the 22 patients who completed the 7-day BH4 loading test, 2 were excluded, 8 had negative tests and 12 were considered to be potential BH4 responders. Of these 12 potential BH4-responsive PKU patients, 5 turned out to be false positive, 6 true-responder and 1 was withdrawn.ConclusionEven though the 48-hour BH4 loading test has proven its efficacy in the past, a full week may be necessary to detect all responders. So, if blood Phe concentrations during the 48-hour BH4 test shows a clear tendency, but not sufficient decrease, a full week (with only measurements each 24 h) could be offered. A threshold of ≥20% decrease within 48 h is not useful for predicting true BH4 responsiveness.     

Assessment of tetrahydrobiopterin (BH(4))-responsiveness and spontaneous phenylalanine reduction in a phenylalanine hydroxylase deficiency population

A BH(4) loading test was performed in 36 patients from 34 unrelated families. The patients had 29 different genotypes, and previous data on only eight of them were found in the BIOPKU database. Thirteen patients were classified as classic PKU (35.1%), 14 as mild PKU (37.8%) and 9 as MHP (27.0%). Blood Phe levels were shown to reach a plateau after three full days of increased natural protein ingestion. Measuring the 24-hour blood Phe levels (T(-24), T(-16), T(0)) on the fourth day of increased protein ingestion before BH(4) administration showed that within 24h Phe on average increased by 2.4% in MHP patients, decreased by 2.7% in mild PKU patients and increased by 9.7% in classic PKU patients (NS for all comparisons); Phe only slightly decreased in responders by 0.2% but increased in non-responders by 7.8% (P>0.05). Altogether, 16 of 36 (44.4%) patients represented by 12 of 29 (41.4%) different genotypes were proven to be BH(4) responders, and four (10.8%) were slow-responders. Responders were 6/9 (66.7%) MHP patients, 10/14 (71.4%) mild PKU patients and 0/13 classic PKU patients. Twenty of the 29 (68.9%) genotypes harbored at least one mutation with a known PRA of 10% or more but only 11 (55%) of them were BH(4)-responsive. Spontaneous reduction of blood Phe levels within 24h on the fourth day of natural protein loading was observed only in mild PKU patients and was shown not to be an important part of the BH(4)-response. 73.3% of genotypes containing at least one allele with a PRA of at least 30% were found to be BH(4) responsive; a PRA of at least 15.5% was needed for the responder genotype in our population.