Effects of hemolysis and storage condition on neuron-specific enolase (NSE) in cerebrospinal fluid and serum: implications in clinical practice.

The concentration of neuron-specific enolase (NSE) in serum and cerebrospinal fluid (CSF) has been used as a biomarker in some cancers and, more recently, in neurodegenerative diseases. Pre-analytical conditions are very important for the quality of returned results. In this study, we evaluated the effects of storage conditions (temperature and duration of storage) and hemolysis on the concentration of NSE in serum and CSF. Our results demonstrate that samples for NSE measurement may be stored at -80 degrees C for no more than 6 months in the case of CSF and 9 months in the case of serum samples. Even invisible hemolysis may increase NSE levels in samples. Consequently, an index of hemolysis should be determined before deciding whether or not to perform NSE measurement.     

Simultaneous measurement of beta-amyloid(1-42), total tau, and phosphorylated tau (Thr181) in cerebrospinal fluid by the xMAP technology

BACKGROUND: To simultaneously study several biomarkers for Alzheimer disease (AD), we used the xMAP technology to develop and evaluate a multiparametric bead-based assay for quantification of beta-amyloid((1-42)) [Abeta((1-42))], total tau (T-TAU), and hyperphosphorylated tau [P-TAU((181P))] in cerebrospinal fluid (CSF). METHODS: We compared the new multianalyte assay format with established ELISA techniques for the same proteins. We then performed a clinical study using CSF samples from patients with AD or mild cognitive impairment with progression to AD, healthy controls, and patients with other neurologic disorders. RESULTS: The INNO-BIA AlzBio3 selectively and specifically measured Abeta((1-42)), T-TAU, and P-TAU((181P)) in the CSF. The new assay format had intra- and interassay CVs <10% for all analytes, even at low concentrations. The measurement range of the new assay was 3 to 4 logs compared with 1 to 2 logs for ELISAs. By plotting the mean of the values obtained in ELISA and the xMAP technology against the difference, we found that a correction factor could be used to convert xMAP results to ELISA values. The clinical study demonstrated that the new multiparametric assay could accurately distinguish patients with AD from patients with other neurologic disorders or control patients, with the diagnostic accuracy reaching recommended consensus criteria for specificity and sensitivity. CONCLUSION: The new multiparametric method may be able to replace the corresponding ELISA methods.     

Diagnostic accuracy of ELISA and xMAP technology for analysis of amyloid beta(42) and tau proteins

BACKGROUND: Cerebrospinal fluid (CSF) concentrations of amyloid beta(42) (Abeta(42)) peptides and tau proteins may serve as biomarkers for Alzheimer disease (AD). Recently, the xMAP technology has been introduced as an alternative to ELISA for measurement of these markers. METHODS: We used xMAP assays and ELISA to analyze CSF concentrations of Abeta(42), total tau (t-tau), and tau phosphorylated at threonine 181 (p-tau(181)) in samples from 69 patients with Alzheimer disease, 26 patients with vascular dementia, and 55 controls without neurological disorders. RESULTS: High CV values (>28%) for the ratio of xMAP:ELISA were observed for each biomarker, indicating that a constant correction factor cannot be applied to recalculate xMAP results into ELISA results. When a combination of CSF markers was used, the sensitivity, specificity, and area under the ROC curves for xMAP assays and ELISAs were not significantly different in differentiating AD patients from vascular dementia patients and controls. CONCLUSIONS: A constant conversion factor cannot be used successfully to recalculate results obtained with xMAP assays to those from the ELISAs. With the use of analysis of a combination of Abeta(42), t-tau, and p-tau in CSF, however, differentiation of clinical groups is equivalent when either xMAP technology or conventional ELISA is used.     

Total tau protein, phosphorylated tau (181p) protein, beta-amyloid(1-42), and beta-amyloid(1-40) in cerebrospinal fluid of patients with dementia with Lewy bodies.

The intra vitam diagnosis of different dementias is still based on clinical grounds. So far, no technical investigations have been available to support these diagnoses. For tau protein and beta-amyloid(1-42) in cerebrospinal fluid (CSF), promising results for the diagnosis of Alzheimer's disease (AD) have been reported; however, their differential diagnostic spectrum is limited, as was recently shown for dementia with Lewy bodies (DLB) and for AD. Therefore, further marker proteins have to be established to ameliorate, support, and differentiate these clinical diagnoses. We evaluated beta-amyloid(1-40) and phosphorylated tau protein (181p), in addition to total tau protein and beta-amyloid(1-42), in 20 patients with DLB, 34 AD patients, and 20 non-demented neurological controls (NDCs). All markers could differentiate between the dementia groups (AD, DLB) and the controls. AD and DLB could be differentiated only by levels of total tau protein and by the ratio total tau protein/phosphorylated tau protein. However, values still overlapped markedly. In some cases, tau protein levels in CSF may contribute to the clinical distinction between DLB and AD, but the value of the markers is still limited, especially because of mixed pathology. We conclude that more specific markers have to be established to differentiate between these diseases.     

Cerebrospinal fluid tau and Abeta42 concentrations in healthy subjects: delineation of reference intervals and their limitations.

Many limitations and conflicting results have cast serious doubts on the validity of cerebrospinal fluid tau and Abeta42 levels for the biological diagnosis of Alzheimer's disease, particularly extreme variations of the reference limits found by unrelated groups as a consequence of different reference populations used. In this study, we addressed the issue of defining reference limits for cerebrospinal fluid tau and Abeta42 in healthy adult individuals. One hundred and five neurologically intact subjects were enrolled according to strict inclusion criteria, 10 of them with autopsy confirmation of brain integrity. All cerebrospinal fluid samples were similarly and optimally processed as were the dosage methods used and the statistical analyses performed. A robust correlation with age was demonstrated for Abeta42 but not for tau. For tau, we found that an upper cut-off value of 443 ng/l allowed 95% of the subjects to be correctly classified as normal. For Abeta42, a lower cut-off value of 90 ng/l allowed a correct classification of 90% of the subjects. However, a large variance of the reference values, partly explained by the potential contamination of the reference population with presymptomatic dementia patients, may limit the use of reference limits based on living subjects. We propose that the issue of defining reference limits for both cerebrospinal fluid tau and Abeta42 may ultimately be settled by studying large numbers of autopsy-proven neurologically intact individuals only.     

喹硫平对阿尔茨海默小鼠淀粉样β蛋白42表达的影响及机制研究

目的探索喹硫平对阿尔茨海默小鼠淀粉样β蛋白42(Aβ42)的表达的影响及其机制。方法C57BL/6小鼠20只作为对照组,APP/PS1小鼠40只分为APP/PS1组、APP/PS1+喹硫平组,各20只。APP/PS1+喹硫平组小鼠通过腹腔给予喹硫平溶液2.5 mg/(kg·d),连续给药2个月,对照组和APP/PS1组小鼠每天腹腔给予等量双蒸水。通过新事物识别实验检测三组小鼠的记忆功能;通过Western blot检测三组小鼠海马脑区Aβ42蛋白、核因子-κB(NF-κB)p65蛋白的表达水平;通过酶联免疫吸附(ELISA)检测海马脑区白细胞介素-1β(IL-1β)和肿瘤坏死因子α(TNF-α)的表达水平,免疫荧光染色计算小胶质细胞数量。结果与对照组比较,APP/PS1小鼠对新事物的探索时间显著降低(P<0.05),小胶质细胞明显激活,小胶质细胞的数量显著增加(P<0.05),IL-10和TNF-α的表达水平均显著增加(P<0.05),Aβ42、NF-κB p65蛋白表达水平显著增加(P<0.05);与APP/PS1组比较,APP/PS1+喹硫平组小鼠的探索时间显著延长(P<0.05),小胶质细胞数量显著减少(P<0.05),IL-10和TNF-α表达水平均显著降低(P<0.05),Aβ42、NF-κB p65蛋白表达水平显著降低(P<0.05)。结论喹硫平能降低Aβ42蛋白的产生,促进AD小鼠的记忆功能恢复,其发生机制可能与小胶质细胞激活和NF-κB信号通路活化相关。     

Enfuvirtide cerebrospinal fluid (CSF) pharmacokinetics and potential use in defining CSF HIV-1 origin

BACKGROUND: Enfuvirtide is a potent inhibitor of systemic HIV-1 replication, but its penetration into the human central nervous system (CNS) has not been analysed. Here, we define cerebrospinal fluid (CSF) enfuvirtide pharmacokinetics and present a case illustrating the use of enfuvirtide as a probe to trace the origins of CSF HIV-1 quasispecies. METHODS: Enfuvirtide CSF pharmacokinetics were assessed in 18 CSF and plasma sample pairs from four HIV-1-infected individuals. Enfuvirtide levels were measured by liquid chromatography tandem mass spectrometry using known standards and controls that included spiked CSF samples from untreated, HIV-negative individuals. A segment of the gp41 coding region encompassing the heptad repeat HR-1 and HR-2 domains was amplified from selected CSF and plasma samples and independent clones sequenced to assess resistance-associated mutations. RESULTS: CSF and plasma samples obtained between 2 and 20 h after enfuvirtide injection showed plasma concentrations similar to previous reports (mean 3.687 SD +/- 1.828 mg/ml) with prolonged decay. By contrast, enfuvirtide in all CSF samples was below the assay detection limit of 0.025 mg/ml. In one individual, who developed a transient increase in CSF HIV-1 RNA, seven of seven CSF and plasma clones had identical enfuvirtide resistance-associated V38A mutations, suggesting that the CSF quasispecies derived from that of blood. CONCLUSIONS: Enfuvirtide penetration into CSF is negligible; thus, in clinical settings, where direct CNS drug exposure is crucial, this drug Is not likely to directly contribute to the local therapeutic effect. Enfuvirtide can be used as a tool to dissect the origin of the CNS virus.     

脑梗死并糖尿病认知障碍患者脑脊液中tau蛋白和淀粉样β蛋白42水平的测定

目的:探讨脑梗死并2型糖尿病认知障碍患者脑脊液中tau蛋白和淀粉样β蛋白42(Aβ42)水平测定的意义,以及2型糖尿病对脑梗死认知障碍患者脑脊液中tau蛋白和Aβ42水平的影响。方法:所有病例为1996-05/2003-06沈阳医学院附属中心医院收治患者,病例组为入选的脑梗死并2型糖尿病组认知障碍患者21例,对照组1为脑梗死并糖尿病无认知障碍组21例,对照组2为脑梗死认知障碍无糖尿病患者21例。认知功能测定选用韦氏成人记忆量表(WMS)。用酶联免疫吸附法(ELISA)对各组患者的脑脊液中tau蛋白和Aβ42水平进行检测。结果:病例组脑脊液中tau蛋白水平明显升高,为(532.59±323.15)ng/L,Aβ42水平明显下降,为(289.00±70.91)ng/L,与对照组1犤(200.59±147.63),(493.80±83.29)ng/L犦和对照组2犤(312.11±289.56),(409.33±76.35)ng/L犦比较,差异有显著性意义(P<0.05)。结论:检测脑脊液中tau蛋白,Aβ42水平有助于脑梗死并糖尿病认知障碍临床诊断,而且2型糖尿病进一步促进脑梗死认知障碍患者脑脊液中tau蛋白进一步释放,Aβ42水平进一步下降。     

Human serum amyloid A (SAA) protein: a prominent acute-phase reactant for clinical practice

Serum amyloid A (SAA) proteins comprise a family of apolipoproteins synthesized in response to cytokines released by activated monocytes/macrophages. Acute-phase protein concentrations have been advocated as objective biochemical indices of disease activity in a number of different inflammatory processes. Clinical studies in large groups of patients with a variety of disorders confirmed the rapid production and exceptionally wide dynamic range of the SAA response. It is as sensitive a marker for the acute-phase as C-reactive protein (CRP). Recent studies indicate that SAA is the most sensitive non-invasive biochemical marker for allograft rejection. Further studies comparing the measurement of SAA to CRP could reveal other indications for its specific use. These studies are now more feasible given newer assays to measure this acute-phase reactant. Observations that the acute-phase response is tightly coupled to lipoprotein abnormalities and the fact that acute-SAA proteins are mainly associated with plasma lipoproteins of the high density range suggested a possible role of this apolipoprotein (apo SAA) in the development of atherosclerosis. The expression of SAA mRNA in human atherosclerotic lesions and the induction of acute-phase SAA by oxidized low-density lipoproteins strengthen the hypothesis that SAA might play a role in vascular injury and atherogenesis.     

淀粉样β蛋白（1-40）诱导血管平滑肌细胞损伤与利福平的保护效应

目的:采用淀粉样β蛋白诱导种植在人工基底胶上的血管平滑肌细胞损伤,观察利福平对血管平滑肌细胞的保护作用。方法:实验于2005-09/2006-09在武汉协和医院神经科实验室进行。①实验材料:100～150g清洁级SD大鼠;淀粉样β蛋白(1-40)(北京博奥森生物工程公司);利福平(华北制药厂)。②实验干预及分组:体外培养大鼠颈动脉平滑肌细胞;实验前所有的细胞培养板孔底经人工基底胶预处理。实验分为2组,实验组细胞培养板孔底沉积淀粉样β蛋白(1-40),对照组细胞培养板孔底无淀粉样β蛋白(1-40)沉积,实验组和对照组根据利福平终浓度不同分别分为7组(0,0.1,1,2,5,10,20g/L)。③实验评估:在显微镜下观察给药前后平滑肌细胞的形态学变化,用四甲基偶氮唑盐比色实验检测平滑肌细胞活性,依据乳酸脱氢酶漏出率检测平滑肌细胞膜的损伤程度。结果:①血管平滑肌细胞形态学变化:对照组内不同浓度利福平下细胞形态均基本正常,实验组细胞形态均异常,但经利福平处理后均有改善,并成剂量依赖关系。②细胞活性:对照组内四甲基偶氮唑盐染色吸光值无明显变化,实验组四甲基偶氮唑盐染色吸光值下降但经利福平处理也呈剂量依赖性上升。③乳酸脱氢酶漏出率:对照组中乳酸脱氢酶漏出率在利福平低浓度时无改变,高浓度时有所增加;实验组中乳酸脱氢酶漏出率均增加,在利福平浓度≤2g/L时,随利福平浓度增加,成剂量依赖关系的下降;在利福平浓度>2g/L时,乳酸脱氢酶漏出率又开始增加。结论:利福平在一定浓度范围内对淀粉样β蛋白(1-40)诱导的大鼠血管平滑肌细胞损伤有保护作用。     

血管性痴呆患者脑脊液中tau蛋白、淀粉样β蛋白42及血清中转化生长因子α和淀粉样β蛋白42的相关性分析

目的:测定脑脊液中tau蛋白及淀粉样β蛋白42和血清中转化生长因子α和淀粉样β蛋白42表达水平对血管性痴呆患者的评估价值。方法:所有实验对象均来自沈阳医学院附属中心医院。选择2000-01/2004-10神经内科门诊和住院的包括血管性痴呆患者31例为血管性痴呆组,无痴呆脑梗死患者31例为无痴呆脑梗死组及同期健康体检者31名为健康对照组。均知情同意。运用成人韦氏记忆量表,Hachinski缺血量表和社会功能活动调查评定患者认知功能。采集所有实验对象的脑脊液及血清,用酶联免疫吸附实验测定脑脊液中tau蛋白和淀粉样β蛋白42的含量,采用放射免疫法测定血清中转化生长因子α、淀粉样β蛋白42的含量。结果:所有实验对象均采集到脑脊液及血清,测定值全部进入结果分析。①脑脊液中tau蛋白检测结果:血管性痴呆组比无痴呆脑梗死组明显升高[(528.49±296.35),(208.48±136.49)ng/L,q=4.72,P<0.05],比健康对照组明显升高[196.32±125.29)ng/L,q=4.82,P<0.05],无痴呆脑梗死组与健康对照组无差异(q=1.91,P>0.05)。②脑脊液中淀粉样β蛋白42含量:血管性痴呆组比无痴呆脑梗死组明显下降[(278.21±69.25),(496.45±81.13)ng/L,q=4.64,P<0.05],比健康对照组明显升高[(504.25±79.81)ng/L,q=4.69,P<0.05],无痴呆脑梗死组与健康     

beta(1)-adrenoceptor polymorphisms: deviation of clinical outcome with use of beta-blockers in ventricular dysfunction

While the debate continues regarding the role of polymorphisms of beta(1)-adrenoceptors on the clinical outcomes and beneficial effects of beta-blockers in patients with heart failure, we need to step back and examine the evidence in the peer-reviewed literature more closely.     

Pharmacokinetics and cerebrospinal fluid (CSF) concentrations of vancomycin in pediatric patients undergoing CSF shunt placement

Staphylococcus epidermidis has been established as the common pathogen causing cerebrospinal fluid shunt infections. In addition, clinical isolates of S. epidermidis from infected shunts are typically resistant to methicillin. Vancomycin is often used for neurosurgical prophylaxis due to its excellent in vitro activity against methicillin-resistant staphylococci. Limited data are available about the pharmacokinetics and cerebrospinal fluid concentrations of vancomycin in pediatric patients intraoperatively. The objectives of this study were to characterize the pharmacokinetics and determine the cerebrospinal fluid concentrations of vancomycin. Eight patients (mean age 8.3 +/- 7.0 years) received three doses of intravenous vancomycin, 15 mg/kg every 6 h. The first dose was administered 1 h prior to surgery. Blood samples were collected at 0, 0.5, 1, 2, 4, and 5 h after the end of the infusion. A cerebrospinal fluid sample was collected at the time of shunt insertion. Urine samples were collected over a 24-hour period. Vancomycin was measured with a fluorescence polarization immunoassay. The peak serum concentrations ranged from 15.6 to 33.7 micrograms/ml; cerebrospinal fluid concentrations ranged from less than 0.6 to 0.8 microgram/ml. The mean total clearance, renal clearance, apparent volume of distribution, and elimination half-life were 0.11 +/- 0.05 l/h/kg, 0.07 +/- 0.02 l/h/kg, 0.54 +/- 0.15 l/kg, and 4.8 +/- 4.0 h, respectively. Approximately 70% of total vancomycin dose was excreted in the urine. A 2- to 5-fold variation in total clearance and a 2.5-fold variability in renal clearance were observed. Low cerebrospinal fluid concentrations of vancomycin were present at the time of shunt insertion in these pediatric patients.(ABSTRACT TRUNCATED AT 250 WORDS)     

Establishment of a reference interval for beta 2-microglobulin in cerebrospinal fluid with use of two commercial assays

Increased concentrations of beta 2-microglobulin in cerebrospinal fluid have been used to detect central nervous system involvement with metastatic cancer and with neurological complications of AIDS. However, no adequate reference interval study has been reported for beta 2-microglobulin in cerebrospinal fluid. We established a reference interval with both the Abbott IMx microparticle enzyme-linked immunoassay (EIA) (0.6-2.0 mg/L) and the Pharmacia beta 2-micro EIA 96 method (0.8-2.2 mg/L) for beta 2-microglobulin in cerebrospinal fluid. The two methods correlate well, with the latter method giving slightly higher values. beta 2-Microglobulin increases with age in adults by about 0.1 mg/L every 7.5 years, with no significant difference between genders.