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。     

四氢生物蝶呤反应性苯丙氨酸羟化酶缺乏症在我国南北地区差异的临床初步研究

目的通过对不同类型高苯丙氨酸血症（hyperphenylalaninemia,HPA）临床特点的分析,探讨我国南、北方四氢生物蝶呤（tetrahydrobiopterin,BH4）反应性苯丙氨酸羟化酶（phenylalanine hydroxylase,PAH）缺乏症患者对BH4的反应性。方法（1）108例HPA患儿,男63例、女45例,平均年龄7.05个月。所有患者都进行口服BH4负荷试验,同时进行尿蝶呤谱分析、红细胞二氢蝶啶还原酶测定。对其中血苯丙氨酸（phenylalanine,Phe）浓度〈600μmol/L者给予口服Phe-BH4联合负荷试验。（2）根据患儿父母双方祖籍,以长江为界将诊断为BH4反应性PAH缺乏症的患儿分为南、北两组。比较南、北方组BH4反应性PAH缺乏症患儿在BH4负荷试验中血Phe浓度的变化。结果（1）HPA中诊断BH4反应性PAH缺乏症36人（33.3%）,BH4无反应性苯丙酮尿症（phenlketonuria,PKU）49人（45.4%）,四氢生物蝶呤缺乏症（BH4D）23人（21.3%）。BH4反应性PAH缺乏症血Phe浓度8h、24h时分别平均下降了49.24%和65.35%。（2）36例BH4反应性患者分为南方组23人、北方组13人。南、北方组BH4反应性患儿服药后24h时血Phe浓度均值分别为（217.02±189.03）μmol/L和（458.75±342.54）μmol/L（P〈0.05）,而两者在服药后2h、4h、8h、24h时血Phe浓度下降的百分数差异均无统计学意义（P〉0.05）。结论部分因PAH缺乏引起的PKU患儿口服BH420mg/kg后24h,血Phe浓度较服药前下降30%以上,其中绝大多数为轻、中度HPA（血Phe120～1200μmol/L）,少数为经典型PKU（血Phe〉1200μmol/L）。本研究中我国南方组BH4反应性PAH缺乏症服药24h时血Phe浓度较北方组低,但是南、北方患者对药物的总体反应性差异无统计学意义。     

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。     

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。     

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。     

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。     

高苯丙氨酸血症的抽搐机制研究

目的四氢生物喋呤缺乏症和苯丙酮尿症均可导致继发性癫痫,本研究拟探讨两类患者的临床与实验室特点。方法将两组患者出现癫痫的年龄、发作形式、脑电图特点及治疗预后进行比较。结果在391例晚治的高苯丙氨酸血症患者中,共有98例苯丙酮尿症和12例四氢生物喋呤缺乏症患者合并癫痫。98名苯丙酮尿症患者出现癫痫发作的年龄是10.7±4.6(4.5~27.1)个月,表现为多种形式,其中55例(56.1%)表现为婴儿痉挛症。经丙戊酸钠和其他抗癫痫药物治疗后,癫痫较难控制,经低苯丙氨酸饮食治疗后临床发作及脑电图均有所减轻。12例四氢生物喋呤缺乏症患儿出现癫痫的年龄为5.1±1.9(2.7~11.0)个月,主要表现为肢体铅管样扭转,哈气样症状。其中10例患者进行了脑电图检查,3例有轻度的痫样放电,7例脑电图正常。治疗后随访脑电图无特异性变化。在服用美多巴后,发作立即得到控制。结论四氢生物喋呤缺乏症和苯丙酮尿症导致癫痫的机制不同,两组患者发作开始年龄、形式、脑电图表现差异显著,治疗方法及预后完全不同。     

上海部分地区新生儿苯丙酮尿症筛查14年临床总结

从１９８１年１０月至１９９５年１２月，我室对在上海部分医院出生的６０万新生儿进行了苯丙酮尿症（ＰＫＵ）的筛查，共查出３５个阳性病例，发病率为１／１７１７８（３５／６０１２１８）。３３例确诊为经典型ＰＫＵ，另２例确诊为四氢生物喋呤缺乏症（ＢＨ＿４ｄｅｆｉｃｉｅｎｃｙ）．所有ＰＫＵ患者（除１例外）均在生后３２±１６天开始接受低苯丙氨酸奶方的治疗，取得了显著的疗效．在对本组ＰＫＵ患儿的随访中发现，绝大部分患儿２岁以前的血苯丙氨酸（ｐｈｅｎｙｌａｌａｎｉｎｅ，Ｐｈｅ）浓度控制在较理想的范围内（４－１０ｍｇ／ｄｌ），智商正常；２岁以后其血苯丙氨酸浓度常常超过１０ｍｇ／ｄｌ，智商明显下降。作者对比作了分析并就完善ＰＫＵ筛查阳性病例的临床管理作了初步探讨。     

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。     

16例新生儿苯丙酮尿症筛查确诊患儿治疗效果初步分析

目的 对新生儿苯丙酮尿症（PKU）筛查确诊患儿的治疗效果进行初步分析。方法 采用化学荧光分析法或细菌抑制法进行足跟血苯丙氨酸（phe）检测，对高苯丙氨酸症者进行尿碟呤谱分析、红细胞二氢碟啶还原酶（DHPR）活性检测，以及phe加BH。联合负荷试验；PKU患儿用饮食疗法，四氢生物碟呤缺乏症（BH4D）用药物治疗。结果 我省PKU发病率为1：18447，BH4D占55％；接受饮食治疗的PKU患儿7例，其智商〉90分5例，占71．4％，智商50—69分2例，占28．6％；接受药物治疗的BH4D患儿9例，其智商≥90分2例，占22．2％、智商70—84分3例，占33．3％、智商50—69分4例，占44．4％。结论 PKU患儿早期诊疗能达到避免严重弱智的目的。     

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.     

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.     

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.     

Long-term treatment with tetrahydrobiopterin increases phenylalanine tolerance in children with severe phenotype of phenylketonuria

Hyperphenylalaninemia caused by phenylalanine hydroxylase (PAH) deficiency requires lifelong rigorous diet starting in early infancy to prevent severe neurodevelopmental handicap. In a considerable number of children with mild hyperphenylalaninemia, long-term tetrahydrobiopterin (BH4) treatment significantly improves phenylalanine (phe) tolerance, but it has never been investigated in classic phenylketonuria (PKU). We performed a BH4-loading test in 40 consecutive infants with phe serum concentrations exceeding 240 microM, who had been detected by newborn screening programs. Eighteen out of 40 infants were found to be BH4 responsive. Five of them, responding to the neonatal BH4-loading test, showed a phe tolerance of less than 20 mg/kg/day and a phe pretreatment level of >1000 microM. They were treated with BH4 (20 mg/kg/day) over a period of 24 months. All five children had a sustained response to BH4, allowing substantial easing of dietary restrictions. Before BH4 treatment daily phe tolerance was 18-19 mg/kg, increasing to 30-80 mg/kg on BH4 treatment and decreasing again to 12-17 mg/kg after termination of BH4 treatment. Mutation analysis revealed compound heterozygosity for a putative null and a variant PAH mutation in four patients and homozygosity for a variant PAH mutation in one patient. We conclude that BH4 sensitivity is not restricted to mild hyperphenylalaninemia and that long-term BH4 treatment may also improve phenylalanine tolerance in a considerable number of children with a more severe PKU phenotype.     

Tetrahydrobiopterin sensitivity in German patients with mild phenylalanine hydroxylase deficiency

We report the results of tetrahydrobiopterin (BH4) loading tests in 10 German patients with mild phenylketonuria. A significant decline of phenylalanine values after application of BH4 was observed in all but one patients. Molecular genetic analyses revealed a range of different PAH gene mutations. Re-testing of one patient previously reported as non-responsive to BH4 loading showed a moderate response with a higher dose of BH4. Nevertheless, there appear to be kinetic differences in phenylalanine hydroxylation in patients with the same genotype. Non-responsiveness to 20 mg/kg BH4 was observed only in a single patient who was compound heterozygous for the novel mutation R176P (c.527G>C) and the common null-mutation P281L. In summary, our data are in line with recent reports indicating that BH4 sensitivity is a normal feature of most mild forms of PAH deficiency but may be influenced by other factors.