New insights into tetrahydrobiopterin pharmacodynamics from Pah, a mouse model for compound heterozygous tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency

Phenylketonuria (PKU), an autosomal recessive disease with phenylalanine hydroxylase (PAH) deficiency, was recently shown to be a protein misfolding disease with loss-of-function. It can be treated by oral application of the natural PAH cofactor tetrahydrobiopterin (BH₄) that acts as a pharmacological chaperone and rescues enzyme function in vivo. Here we identified Pah^enu1/2 bearing a mild and a severe mutation (V106A/F363S) as a new mouse model for compound heterozygous mild PKU. Although BH₄ treatment has become established in clinical routine, there is substantial lack of knowledge with regard to BH₄ pharmacodynamics and the effect of the genotype on the response to treatment with the natural cofactor. To address these questions we applied an elaborate methodological setup analyzing: (i) blood phenylalanine elimination, (ii) blood phenylalanine/tyrosine ratios, and (iii) kinetics of in vivo phenylalanine oxidation using ¹³C-phenylalanine breath tests. We compared pharmacodynamics in wild-type, Pah^enu1/1, and Pah^enu1/2 mice and observed crucial differences in terms of effect size as well as effect kinetics and dose response. Results from in vivo experiments were substantiated in vitro after overexpression of wild-type, V106A, and F263S in COS-7 cells. Pharmacokinetics did not differ between Pah^enu1/1 and Pah^enu1/2 indicating that the differences in pharmacodynamics were not induced by divergent pharmacokinetic behavior of BH₄. In conclusion, our findings show a significant impact of the genotype on the response to BH₄ in PAH deficient mice. This may lead to important consequences concerning the diagnostic and therapeutic management of patients with PAH deficiency underscoring the need for individualized procedures addressing pharmacodynamic aspects.     

芪苈强心胶囊辅治老年慢性心力衰竭疗效观察

目的观察芪苈强心胶囊辅治老年慢性心力衰竭(CHF)对心功能、血浆NT-proBNP水平的影响及临床安全性。方法采用随机方法将64例老年CHF患者分为2组:对照组(32例)和治疗组(32例)。2组均给予常规西药治疗,治疗组在常规西药治疗的基础上加用芪苈强心胶囊,疗程均为4周。观察治疗前后患者体质量变化、Lee氏心力衰竭积分、6 min步行试验、血浆NT-proBNP水平,超声心动图检查左心室舒张末内径(LVEDD)、左心室射血分数(LVEF)、心输出量(CO)等疗效指标,并检测肝肾功能、电解质等安全性指标。结果 (1)治疗组与对照组体质量较治疗前均有减轻,但组间比较差异无统计学意义(P>0.05);(2)Lee氏心力衰竭积分:治疗组总有效率90.6%,对照组总有效率78.2%,差异有统计学意义(P<0.05);(3)6 min步行试验:治疗组总有效率87.5%,对照组总有效率75.0%,差异有统计学意义(P<0.05);(4)血浆NT-proBNP水平比较:治疗4周后2组NT-proBNP水平均有所下降,且治疗组较对照组下降更为明显,差异有统计学意义(P<0.05);(5)心功能超声变化:治疗4周后2组IVEDD较治疗前均有所缩小,LVEF、CO均有提高,且治疗组优于对照组,差异有统计学意义(P<0.05)。2组治疗过程中均未发现明显不良反应,治疗后复查肝肾功能、电解质、血常规未见异常。结论芪苈强心胶囊联合西药治疗中重度老年CHF,可以更有效地改善患者心功能,缓解临床症状,且药物安全性好。     

参草通脉颗粒对慢性心衰患者生活质量的影响

目的观察参草通脉颗粒对气虚血瘀型慢性心衰患者的生活质量的影响。方法将40例慢性心衰患者随机分为2组,每组20例。对照组采用西医常规治疗,治疗组在西医常规治疗的基础上,加用参草通脉颗粒,用药疗程均为3个月。比较两组患者治疗前后明尼苏达生活质量(LHFQ)评分变化、6分钟步行距离改善情况及治疗后6个月再住院率和病死率。结果两组患者LHFQ评分均降低,6分钟步行距离改善,再住院率、病死率降低,且治疗组明显优于对照组(P<0.05)。结论参草通脉颗粒可明显提高气虚血瘀型心衰患者的生活质量,改善患者的运动耐量,降低其住院率和病死率。     

腹腔镜胆总管探查胆道一期缝合与T管引流疗效的比较

目的探讨与腹腔镜胆总管T管引流相比,腹腔镜胆总管一期缝合的优点,手术操作的技术关键,手术适应证以及并发症的预防。方法回顾对照分析2008年7月至2010年7月间行腹腔镜胆总管一期缝合的24例患者以及同期行腹腔镜T管引流的24例患者的临床病历资料,分为一期缝合组和T管引流组。结果一期缝合组与T管引流组的平均手术时间分别为（54.03±8.46）、（49.83±7.25）min,术中出血量分别为（15.13±4.26）、（16.23±5.25）ml,两组差异无统计学意义（P〉0.05）。而一期缝合组与T管引流组的首次肛门排气时间分别为（1.16±0.46）与（2.02±0.19）d、术后补液量分别为（8.05±2.73）与（11.56±4.72）L、术后住院时间分别为（7.73±1.76）与（13.85±4.09）d、住院费用分别为（11 393±283）与（15 836±296）元,带管时间分别为（6.45±2.15）与（73.68±9.15）d。一期缝合组的平均带管时间较T管引流组缩短（67.23±7）d。以上观察指标,两组差异有统计学意义（P〈0.01）。一期缝合组4例术后2～3 d发生胆汁漏,经保守治疗治愈,无严重并发症,全组患者均痊愈出院,随访1个月到2年,无结石复发和胆管狭窄等并发症。结论胆总管一期缝合有利于患者快速恢复;术中确保胆道无残余结石和胆总管下端通畅是开展胆总管一期缝合的前提条件;严格掌握手术适应证,注重操作技术要点是取得良好疗效的关键。     

Western and dot immunoblotting analysis of viral antigens and antibodies: application to murine hepatitis virus

Viral proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred quantitatively to nitrocellulose by electroblotting in SDS-containing buffer. Monoclonal antibodies directed against previously defined epitopes on the viral proteins were used as probes to detect viral protein synthesis and processing, as well as expression in animal tissues. Circulating polyclonal antibodies were also probed and characterized for their polypeptide specificities. Under appropriate conditions, this Western immunoblotting technique was quantitative. Finally, a highly sensitive dot immunoblotting assay was used to analyze the sensitivity to denaturation of various epitopes on the viral proteins. This assay detected picogram quantities of viral antigens and antibodies.     

In vivo [14C]amino acid profiles in discrete hypothalamic regions during push-pull perfusion in the unrestrained rat.

In the unanesthetized rat, the differential release of [¹⁴C]amino acids into a perfusate from a push-pull cannula was analyzed for 20 circumscribed areas of the hypothalamus. To serve as an exogenous precursor for [¹⁴C]amino acid synthesis. [U-¹⁴C]glucose was injected in a vol of 0.5–1.0 μl into a discrete hypothalamic region 20 min prior to a push-pull perfusion. Then, at 15min intervals, the labeled site was perfused for 5 min with an artificial cerebrospinal fluid usually at a rate of 10μl per min. Each sample of perfusate was extracted and assayed by two-dimensional, thin-layer chromatography for the presence of eight amino acids and residual glucose.The pattern of recovery of ¹⁴C-labeled substances as well as the distribution of ¹⁴C between γ-aminobutyric acid, glutamate, aspartate, alanine, taurine and glycine depended solely upon the neuroanatomical region perfused. Near the lateral edge of the ventromedial nucleus, 67% of the amino acid activity was accounted for by [¹⁴C]GABA. Perfusion sites medial to or within this nucleus, or others near the anterior hypothalamus or paraventricular nucleus, yielded a recovery of labeled GABA that was equal to that of glutamate, together accounting for two-thirds of the total ¹⁴C recovery. Within many perfusion loci, small amounts of [¹⁴C]alanine and [¹⁴C]aspartate were detected, whereas within most sites the respective recovery of [¹⁴C]aspartate, [¹⁴C]alanine, [¹⁴C]taurine and [¹⁴C]glycine was negligible. Finally, the metabolism of [¹⁴C]amino acids in the rat's hypothalamus decreased exponentially over time with little identifiable content obtained 80 min following injection of the glucose precursor.These results demonstrate that putative amino acid transmitters can be recovered from the brain of a freely moving animal, and can be characterized in vivo within specific neuroanatomical regions.     

Bradycardia evoked by hypothalamic stimulation in the rabbit: Dependence upon the arterial blood pressure

The spinal cord was transected in adult New Zealand White rabbits anaesthetized with urethane plus chloralose. The level of transection was in the mid-cervical region. The animals were then ventilated mechanically and the arterial blood pressure was maintained with an intravenous infusion of noradrenaline solution. Stimulation of the hypothalamus 1–2 mm lateral to the third ventricle and 1.5–3 mm dorsal to the mammillary nuclei, in a region known to evoke pressor responses and bradycardia in normal anaesthetized rabbits, never evoked pressor responses in the spinally transected rabbits. Bradycardia was evoked only when the mean arterial blood pressure was maintained above 44–49 mmHg. At higher pressures stimulation evoked a greater bradycardia and the relationship between bradycardia and pressure was approximately linear over much of the range of pressures tested (up to 116 mmHg).Because the threshold mean arterial blood pressure at which hypothalamic stimulation evoked bradycardia was similar to the threshold pressures reported in the literature for baroreceptor activation in the rabbit and because the curve of bradycardia:pressure was similar to published curves of baroreceptor and baroreflex activity against blood pressure, it is concluded that the bradycardia evoked by hypothalamic stimulation in the rabbit is mediated by a neural pathway in the hypothalamus that can increase the gain of the cardio-inhibitory baroreceptor reflex.     

Loss of radioactive 2-deoxy-d-glucose-6-phosphate from brains of conscious rats: Implications for quantitative autoradiographic determination of regional glucose utilization

Radioactive glucose and 2-deoxy-d-glucose (deoxyglucose) were compared as tracers for estimating the rate of rat brain glucose utilization after an intravenous injection. The [brain] : [blood] ratio of deoxyglucose content was twice as large as that of glucose at 5 min, 3 times at 45 min, and 13 times at 240 min. While [2-¹⁴C]glucose accounted for about 20% of total brain¹⁴C (acid soluble) at 10 min, labeled deoxyglucose took 45 min to fall to comparable levels. Labeled 2-deoxy-d-glucose-6-phosphate (deoxyglucose phosphate) did not accumulate after 10 min despite the fact that deoxyglucose was available continuously for phosphorylation. This resulted from loss of deoxyglucose phosphate, which was in proportion to its concentration. The disappearance of deoxyglucose phosphate was probably catalyzed by glucose-6-phosphatase, which reacts with deoxyglucose phosphate and which is present in brain.Since the [brain]: [blood] ratio of labeled deoxyglucose increases as time passes and since deoxyglucose phosphate is lost from brain at an appreciable rate, its use for quantitative determination of the rate of rat brain glucose utilization is much more complex than previously realized. Approximately 45 min are necessary to reduce background contamination to acceeptable levels for autoradiography during which time substantial amounts of deoxyglucose phosphate may be lost. The primary reason for using deoxyglucose to measure glucose utilization by the brain is the assumption that deoxyglucose phosphate is lost very slowly from the brain. In view of the fact that loss of deoxyglucose phosphate cannot be ignored, the advantage of labeled deoxyglucose over labeled glucose is open to question.     

Oxygen desaturation during a 6 min walk test is a sign of nocturnal hypoxemia

BACKGROUND/OBJECTIVES:

Patients with chronic obstructive pulmonary disease (COPD) may experience sleep disordered breathing with nocturnal desaturation. An exploratory study was performed to determine whether any commonly measured clinical parameters were useful in predicting nocturnal desaturation in patients with COPD. A validation study was subsequently performed to confirm the utility of the parameter identified in the exploratory study as most useful in this regard.

METHODS:

A total of 103 (exploratory cohort) and 200 (validation cohort) consecutive patients with COPD admitted for pulmonary rehabilitation were evaluated. Standard outcome measures including nocturnal oximetry and the 6 min walk test (6MWT) on room air with continuous pulse oximetry were assessed. Patients with sleep apnea or those undergoing long-term oxygen therapy were excluded.

RESULTS:

In the exploratory study, the mean (± SD) patient age was 70±9.9 years, with forced expiratory volume in 1 s of 0.76±0.34 L, which was 36±16% of predicted. Body mass index, arterial oxygen tension, oxygen saturation by pulse oximetry at rest and during the 6MWT all demonstrated significant correlations with percentage of time spent with a saturation <90%. When the lowest pulse oximetry during the 6MWT was ≤88%, 10 of 21 patients demonstrated a saturation <90% for at least 30% of sleep time. This measure yielded a positive likelihood ratio of 3.77 (95% CI 1.87 to 7.62) compared with those who did not reach this threshold value. The validation study confirmed similar detection characteristics.

CONCLUSIONS:

Results from the present study suggest that monitoring oxygen saturation changes during a 6MWT is useful in helping to identify COPD patients who may experience significant nocturnal desaturation.