Impact of cerebrospinal fluid matrix on the detection of Alzheimer's disease with Aß42 and influence of disease on the total-Aß42/Aß40 ratio.

The 42-amino acid fragment of amyloid ß (Aß1-42) in cerebrospinal fluid has continued to be important for detecting cerebral ß-amyloidosis in Alzheimer's disease (AD). However, there are impediments to our ability to fully understand this measurement, including matrix interference and changes linked to apolipoprotein E (APOE) ε4 genotype. This study investigated matrix interference as a contributing factor for detecting AD in APOE ε4-negative patients by comparing total extractable Aß1-42 to free Aß1-42. It also examined the ratio of total Aß1-42 to Aß1-40, since changes relative to other Aß peptides may provide a measurement of cerebral ß-amyloidosis that is neutral to changes in APP metabolism. Total Aß1-42 lost the diagnostic power for detecting AD, confirming a role for matrix in the diagnostic. However, when total Aß1-42/Aß1-40 was examined, the separation between groups was reestablished. This result was confirmed in a second sample set of unknown APOE status. These results confirmed that matrix interference in some cerebrospinal fluid samples appears to contribute to identifying AD patients and this can be compensated by using a total extracted Aß1-42/Aß1-40 ratio when matrix interference is small. It may be preferable to employ a total Aß1-42/Aß1-40 measurement, since this could minimize variability because of matrix and compensate for across patient differences. Aß1-42 measurement in CSF has provided an important tool for early detection of AD. However, it appears that most assays measure a free fraction of Aß1-42. This study examined total extracted Aß1-42, since this would provide a more accurate assessment of Aß1-42 in AD CSF. Total Aß1-42 measurements alone were not good for detecting AD but total Aß1-42/Aß1-40 performed well.

An integrated multi-study analysis of intra-subject variability in cerebrospinal fluid amyloid-ß concentrations collected by lumbar puncture and indwelling lumbar catheter.

INTRODUCTION: Amyloid-ß (Aß) has been investigated as a diagnostic biomarker and therapeutic drug target. Recent studies found that cerebrospinal fluid (CSF) Aß fluctuates over time, including as a diurnal pattern, and increases in absolute concentration with serial collection. It is currently unknown what effect differences in CSF collection methodology have on Aß variability. In this study, we sought to determine the effect of different collection methodologies on the stability of CSF Aß concentrations over time. METHODS: Grouped analysis of CSF Aß levels from multiple industry and academic groups collected by either lumbar puncture (n=83) or indwelling lumbar catheter (n=178). Participants were either placebo or untreated subjects from clinical drug trials or observational studies. Participants had CSF collected by lumbar puncture or lumbar catheter for quantitation of Aß concentration by enzyme linked immunosorbent assay. Data from all sponsors was converted to percent of the mean for Aß40 and Aß42 for comparison. Repeated measures analysis of variance was performed to assess for factors affecting the linear rise of Aß concentrations over time. RESULTS: Analysis of studies collecting CSF via lumbar catheter revealed tremendous inter-subject variability of Aß40 and Aß42 as well as an Aß diurnal pattern in all of the sponsors' studies. In contrast, Aß concentrations from CSF samples collected at two time points by lumbar puncture showed no significant differences. Repeated measures analysis of variance found that only time and draw frequency were significantly associated with the slope of linear rise in Aß40 and Aß42 concentrations during the first 6 hours of collection. CONCLUSIONS: Based on our findings, we recommend minimizing the frequency of CSF draws in studies measuring Aß levels and keeping the frequency standardized between experimental groups. The Aß diurnal pattern was noted in all sponsors' studies and was not an artifact of study design. Averaging Aß concentrations at each time point is recommended to minimize the effect of individual variability. Indwelling lumbar catheters are an invaluable research tool for following changes in CSF Aß over 24-48 hours, but factors affecting Aß concentration such as linear rise and diurnal variation need to be accounted for in planning study designs.

Characterization of novel CSF Tau and ptau biomarkers for Alzheimer's disease.

Cerebral spinal fluid (CSF) Aß42, tau and p181tau are widely accepted biomarkers of Alzheimer's disease (AD). Numerous studies show that CSF tau and p181tau levels are elevated in mild-to-moderate AD compared to age-matched controls. In addition, these increases might predict preclinical AD in cognitively normal elderly. Despite their importance as biomarkers, the molecular nature of CSF tau and ptau is not known. In the current study, reverse-phase high performance liquid chromatography was used to enrich and concentrate tau prior to western-blot analysis. Multiple N-terminal and mid-domain fragments of tau were detected in pooled CSF with apparent sizes ranging from <20 kDa to ~40 kDa. The pattern of tau fragments in AD and control samples were similar. In contrast, full-length tau and C-terminal-containing fragments were not detected. To quantify levels, five tau ELISAs and three ptau ELISAs were developed to detect different overlapping regions of the protein. The discriminatory potential of each assay was determined using 20 AD and 20 age-matched control CSF samples. Of the tau ELISAs, the two assays specific for tau containing N-terminal sequences, amino acids 9-198 (numbering based on tau 441) and 9-163, exhibited the most significant differences between AD and control samples. In contrast, CSF tau was not detected with an ELISA specific for a more C-terminal region (amino acids 159-335). Significant discrimination was also observed with ptau assays measuring amino acids 159-p181 and 159-p231. Interestingly, the discriminatory potential of p181 was reduced when measured in the context of tau species containing amino acids 9-p181. Taken together, these results demonstrate that tau in CSF occurs as a series of fragments and that discrimination of AD from control is dependent on the subset of tau species measured. These assays provide novel tools to investigate CSF tau and ptau as biomarkers for other neurodegenerative diseases.

Measurement of Aß1-42 in cerebrospinal fluid is influenced by matrix effects.

Aß1-42 measurement in CSF is an important biochemical marker for Alzheimer disease (AD). However, our understanding of why this biomarker is predictive and why it is often difficult to measure in a reproducible fashion is still lacking. To study these questions, the concentration of Aß1-42 in CSF was compared before and after denaturation with 6M guanidine and reverse-phase HPLC. Measurement of the Aß1-42 after denaturation and reverse-phase HPLC demonstrated that considerably more Aß1-42 was present in CSF than revealed when assaying non-denatured CSF. A comparison of Aß1-42 concentrations before and after HPLC in AD CSF with that in normal controls suggested that matrix interference may affect the differentiation between the diagnostic groups. A similar effect was observed with dilutions of crude CSF. Together, these results suggested that at least part of the mechanism by which low Aß1-42 concentrations in CSF function as a biomarker of AD is related to matrix components which preferentially hide a portion of the Aß1-42 from detection in AD CSF. In contrast, we show that the association of the APOEε4 allele with lower Aß1-42 concentrations in CSF is preserved even after denaturation and HPLC. A similar relationship between the presence of the APOEε4 allele and lower concentrations of Aß1-40 was also apparent, thereby generating similar ratios of Aß1-42/ Aß1-40 across the APOE genotypes. The results from the present study suggested that Aß1-42 in CSF functions as a biomarker of AD in tandem with other CSF matrix components that are increased in AD CSF. Further studies are needed to identify which matrix factors (e.g. binding of Aß to proteins) underlie the increased detection of Aß1-42 concentrations after denaturation and HPLC. The data also suggested that denaturation and HPLC of CSF may be a useful approach for studies using Aß1-42 as a pharmacodynamic marker or in other paradigms where measurement of total non-covalently bound Aß1-42 is required.

Enrichment and analysis of Alzheimer's Abeta1-42 peptide in human plasma and whole blood.

The Abeta1-42 fragment from the Amyloid Precursor Protein (APP) has presented considerable challenges from an analytical perspective. It is present at low levels in the circulation and can bind to proteins which mask its presence in assays. A number of therapeutic strategies target the lowering of this peptide, necessitating more robust and sensitive methods for its measurement. In this study, conditions for extracting and enriching Abeta1-42 using solid-phase extraction (SPE) and reverse-phase HPLC (RP-HPLC) were optimized. The new process provided reproducible recovery of Abeta1-42 of about 80% and allowed for concentration of the peptide prior to assay. Radiolabeled Abeta1-42 and ELISA for Abeta1-42 were used to determine the recovery and distribution of the peptide from whole blood collected in the presence of potassium-EDTA. Endogenous Abeta1-42 yielded a cell pellet:plasma ratio near 40:60 while exogenously added peptide distributed with a ratio of about 27:73. Additionally, the Abeta1-42 in the plasma and cell pellet fractions maintained stability over many hours. Comparing the measurement of Abeta1-42 using a commercial ELISA before and after enrichment demonstrated noticeable improvement of signal in samples enriched for the peptide. The current study also showed that conspicuous amounts of Abeta1-42 partition to the cell pellet but that this fraction can be robustly recovered and measured with SPE and HPLC. The process utilized established chromatographic techniques and is suitable for automation. It is also compatible with other detection methods including mass spectrometry.

Distribution of Abeta peptide in whole blood.

The measurement of amyloid beta peptides (Abeta) in blood and plasma is expected to be a useful biomarker as potential therapeutics designed to lower Abeta peptide enter clinical trials. Many reports have suggested that Abeta could bind to substances in blood that may influence the recovery of Abeta peptide in plasma, its detection by conventional ELISAs or the actual turnover and half-life of the peptide in blood. In this study we describe a process for analyzing total Abeta in whole blood and plasma using denaturing solid-phase extraction followed by reverse-phase HPLC linked to ELISA. Comparison of total Abeta peptide levels in whole blood and plasma from the same bleed showed that most of the Abeta peptide is captured in the plasma if the samples are first denatured. In contrast, plasma that was assayed without denaturation could show greater than 70% reduction in apparent total Abeta peptide. This suggested that there was a pool of Abeta peptide in non-denatured plasma that is occluded from detection by ELISA, perhaps by binding to plasma proteins.

Analysis of protein fragmentation inhibition by an MMP-inhibitor in an in vivo model of heart failure using automated chromatography.

Proteomic strategies have continued to demonstrate value in studying disease by exploiting new technologies that can develop significant numbers of measurements from single samples. However, using complex samples such as tissues or blood has continued to be problematic due to the presence of major interfering substances. In this study, a process is described that uses denaturing peptide extraction from whole tissue and automated chromatography in order to allow subsequent analysis of more than 1000 tissue-derived peptides per sample. The process was employed to identify cardiac proteins that were spared degradation by administration of a heart-protecting matrix metalloproteinase (MMP) inhibitor (compound SC-621) following experimental myocardial infarction (MI). HPLC peptide fingerprints were developed from rat heart left ventricles and the resultant integrated peak data was compared across experimental animals. Surprisingly, although protein fragmentation was generally increased in MI hearts, the effect of the MMP inhibitor was only observed on a few species. The results from this study demonstrated that whole-tissue sample enrichment and peptide analysis using HPLC could be linked in order to study the effects of new compounds on a disease state. The system is flexible and amenable to improvements such as incorporating detection by mass spectrometry.