New plasma LC-MS/MS assays for the quantitation of beta-amyloid peptides and identification of apolipoprotein E proteoforms for Alzheimer's disease risk assessment.

Early detection of Alzheimer's disease (AD) represents an unmet clinical need. Beta-amyloid (Aß) plays an important role in AD pathology, and the Aß42/40 peptide ratio is a good indicator for amyloid deposition. In addition, variants of the apolipoprotein E (APOE) gene are associated with variable AD risk. Here, we describe the development and validation of high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays for plasma Aß40 and Aß42 quantitation, as well as apolipoprotein E (ApoE) proteotype determination as a surrogate for APOE genotype. Aß40 and Aß42 were simultaneously immunoprecipitated from plasma, proteolytically digested, and quantitated by LC-MS/MS. ApoE proteotype status was qualitatively assessed by targeting tryptic peptides from the ApoE2, ApoE3, and ApoE4 proteoforms. Both assays were validated according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Within-run precision was 1.8%-4.2% (Aß40), 1.9%-7.2% (Aß42), and 2.6%-8.3% (Aß42/40 ratio). Between-run precision was 3.5%-5.9% (Aß40), 3.8%-8.0% (Aß42), and 3.3%-8.7% (Aß42/40 ratio). Both Aß40 and Aß42 were linear from 10 to 2500 pg/mL. Identified ApoE proteotypes had 100% concordance with APOE genotypes. We have developed a precise, accurate, and sensitive high-throughput LC-MS/MS assay for plasma Aß40, Aß42, and proteoforms of ApoE.

The Panama Aging Research Initiative Longitudinal Study.

The Panama Aging Research Initiative is a cohort study of 423 adults aged ≥65 years recruited from an outpatient geriatric department of Panama's largest public hospital, the Social Security Fund's Dr Arnulfo Arias Madrid Hospital Complex (Complejo Hospitalario Dr Arnulfo Arias Madrid de la Caja de Seguro Social). The study provides the first reports of modifiable and non-modifiable risk factors of cognitive impairment and dementia, as well as various health conditions common among older adults in Panama, including chronic illnesses, polypharmacy and rates of comorbidity. The initial study, conducted September 2012-May 2016, included a clinical interview; physical assessments of body mass index and handgrip strength; and cognitive testing, plus non-fasting blood draws for measurements of genetic (Apolipoprotein E genotype) and blood-based biological markers. Information was collected regarding limitations in activities of daily living, symptoms of depression and fall incidents. A subsample of participants provided cerebrospinal fluid to measure proteins related to Alzheimer's disease; another subsample underwent ultrasonography and electroencephalography. This report describes the general study design and highlights lessons learned and future directions. In particular, drawing on lessons learned from this clinical research, a community-based prospective cohort study is currently under way among older adults in Panama to validate a blood-based biomarker profile for detecting mild cognitive impairment and Alzheimer's disease, as well as risk factors for cognitive decline. KEYWORDS: Dementia, biomarkers, Latin America, aging, cognition, chronic disease, Panama.

Assessment of the Relationship between Carotid Intima-Media Thickening and Early-Stage Diabetic Kidney Disease Coupled with Helicobacter pylori Infection.

OBJECTIVE: This study aimed to explore the associations between carotid intima-media thickness (CIMT) and early-stage diabetic kidney disease (DKD) coupled with Helicobacter pylori (H. pylori) infection in type 2 diabetic patients. METHODS: A cross-sectional study including 180 type 2 diabetic participants was conducted to explore the associations between CIMT and early-stage DKD coupled with H. pylori infection, and a stepwise multivariate regression analysis evaluated the correlations of CIMT with clinical and serologic parameters. RESULTS: The type 2 diabetic patients with early-stage DKD coupled with H. pylori infections had the highest CIMT values. Apolipoprotein B (ApoB), urine albumin/creatinine ratio (UACR), and interleukin-6 (IL-6) were independent predictors of CIMT. CONCLUSIONS: Early-stage DKD coupled with H. pylori infection may synergistically lead to significant CIMT thickening in type 2 diabetic patients. Additionally, ApoB, UACR, and IL-6 levels were important independent risk factors for increased CIMT.

Identifying cost-effective predictive rules of amyloid-ß level by integrating neuropsychological tests and plasma-based markers.

BACKGROUND: Detecting participants who are positive for amyloid-ß (Aß) pathology is germane in designing prevention trials by enriching for those cases that are more likely to be amyloid positive. Existing brain amyloid measurement techniques, such as the Pittsburgh Compound B-positron emission tomography and cerebrospinal fluid, are not reasonable first-line approaches limited by either feasibility or cost. OBJECTIVE: We aimed to identify simple and cost-effective rules that can predict brain Aß level by integrating both neuropsychological measurements and blood-based markers. METHOD: Several decision tree models were built for extracting the predictive rules based on the Alzheimer's Disease Neuroimaging Initiative cohort. RESULTS: We successfully extracted predictive rules of Aß level. For cognitive function variables, cases above the 45th percentile in total cognitive score (TOTALMOD), above the 52nd percentile of delayed word recall, and above the 70th percentile in orientation resulted in a group that was highly enriched for amyloid negative cases. Conversely scoring below the 15th percentile of TOTALMOD resulted in a group highly enriched for amyloid positive cases. For blood protein markers, scoring below the 57th percentile for apolipoprotein E (ApoE) levels (irrespective of genotype) enriched two fold for the risk of being amyloid positive. In the high ApoE cases, scoring above the 60th percentile for transthyretin resulted in a group that was >90% amyloid negative. A third decision tree using both cognitive and blood-marker data slightly improved the classification of cases. CONCLUSION: Our study demonstrated that the integration of the neuropsychological measurements and blood-based markers significantly improved prediction accuracy. The prediction model has led to several simple rules, which have a great potential of being naturally translated into clinical settings such as enrichment screening for AD prevention trials of anti-amyloid treatments.

How proteomic ApoE serotyping could impact Alzheimer's disease risk assessment: genetic testing by proteomics.

Humans have three major apolipoprotein E (ApoE) alleles (APOE; ε2, ε3 and ε4) that produce three ApoE protein isoforms. The ε2 allele encodes the ApoE2 isoform (Cys112, Cys158), whereas ε3 encodes the wild-type ApoE3 isoform (Cys112, Arg158) and ε4 encodes the ApoE4 isoform (Arg112, Arg158). Because the type of ApoE expressed is related to sporadic Alzheimer's disease risk and familial hyperlipidemia, many clinical studies have utilized ApoE typing in recent years. ApoE serotyping is based on the correlation between ApoE genotype and isoform; it is therefore possible to determine the genotype from the blood ApoE isoform combination. Serotyping ApoE using mass spectrometry promises highly accurate results while requiring minimal amounts of blood and reagents, resulting in lower costs, which suggest that proteomic-based ApoE serotyping may eventually become a routine clinical laboratory test. Not limited to ApoE, proteomic analysis of human samples could be used to intentionally determine - and perhaps unintentionally reveal - personal genetic information.

Post-heparin LPL activity measurement using VLDL as a substrate: a new robust method for routine assessment of plasma triglyceride lipolysis defects.

BACKGROUND: Determination of lipoprotein lipase (LPL) activity is important for hyperchylomicronemia diagnosis, but remains both unreliable and cumbersome with current methods. Consequently by using human VLDL as substrate we developed a new LPL assay which does not require sonication, radioactive or fluorescent particles. METHODS: Post-heparin plasma was added to the VLDL substrate prepared by ultracentrifugation of heat inactivated normolipidemic human serums, diluted in buffer, pH 8.15. Following incubation at 37°c, the NEFA (non esterified fatty acids) produced were assayed hourly for 4 hours. LPL activity was expressed as µmol/l/min after subtraction of hepatic lipase (HL) activity, obtained following LPL inhibition with NaCl 1.5 mmol/l. Molecular analysis of LPL, GPIHBP1, APOA5, APOC2, APOE genes was available for 62 patients. RESULTS: Our method was reproducible (coefficient of variation (CV): intra-assay 5.6%, inter-assay 7.1%), and tightly correlated with the conventional radiolabelled triolein emulsion method (n = 26, r = 0.88). Normal values were established at 34.8 ± 12.8 µmol/l/min (mean ± SD) from 20 control subjects. LPL activities obtained from 71 patients with documented history of major hypertriglyceridemia showed a trimodal distribution. Among the 11 patients with a very low LPL activity (<10 µmol/l/min), 5 were homozygous or compound heterozygous for LPL or GPIHBP1 deleterious mutations, 3 were compound heterozygous for APOA5 deleterious mutations and the p.S19W APOA5 susceptibility variant, and 2 were free of any mutations in the usual candidate genes. No homozygous gene alteration in LPL, GPIHBP1 and APOC2 genes was found in any of the patients with LPL activity >10 µmol/l/min. CONCLUSION: This new reproducible method is a valuable tool for routine diagnosis and reliably identifies LPL activity defects.

Increased apolipoprotein E gene expression and protein concentration in lung cancer tissue do not contribute to the clinical assessment of non-small cell lung cancer patients.

BACKGROUND: We tested the hypothesis that apolipoprotein E (apo E) gene expression and protein concentration are increased in resectable non-small cell lung cancer tissue and that these apo E tissue estimations may be beneficially used in clinical assessment of non-small cell lung cancer patients. METHODS: Paired samples of lung cancer and adjacent, apparently healthy, non-cancer lung tissue were collected from 42 patients with resectable non-small cell lung cancer. Apo E gene expression in tissue was measured by quantitative PCR. Apo E protein in tissue and serum was quantified by a nephelometric method. Patients were followed for 3 years. RESULTS: Apo E gene expression and protein concentration were 1.6 and 4.1-fold higher in the cancer tissue than in the adjacent non-cancer tissue (p<0.0001 in both cases). Increase of apo E protein concentration in the cancer tissue (relative to the non-cancer tissue) correlated with the decrease of apo E protein concentration in the serum (p=0.021). However, none of these apo E estimations related to stage of cancer or histological type of tumor and do not predict patient survival. CONCLUSIONS: Our preliminary study shows that despite the distinct increase of apo E gene expression and protein concentration in the cancer tissue and the concurrent decrease of apo E protein concentration in the serum, the measured apo E values have limited usefulness in clinical assessment of patients with resectable non-small cell lung cancer.

Genetic susceptibility testing for Alzheimer disease: motivation to obtain information and control as precursors to coping with increased risk.

OBJECTIVE: This study investigated appraisals, including motivation, and coping preferences for undergoing Apolipoprotein E (APOE) susceptibility testing for Alzheimer disease (AD). METHODS: Participants were 60 adult children of individuals affected with AD enrolled in a trial investigating use and impact of APOE susceptibility testing. An exploratory qualitative study was undertaken in which participants were interviewed about their testing experience. RESULTS: Most participants viewed genetic testing as providing valuable information that could help direct future health care decisions and meet their emotional concerns about living at increased risk. Participants related their motivation for genetic testing to their worries about developing AD, preference to seek information about health threats, and need to feel in control of their health. CONCLUSION: Even without prevention or treatment options, genetic testing may be a useful coping strategy for some at-risk individuals. PRACTICE IMPLICATIONS: Once testing becomes clinically available, practitioners need to address the value and limitations of testing as well as appraisals and efforts to cope.

Comparative morphometric and immunohistological assessment of the development of restenosis after arterial injury and a cholesterol-rich diet in apolipoprotein E -/-mice and C57BL/6 control mice.

OBJECTIVE: Animal models of restenosis have been a cornerstone of testing potential therapies and have improved the understanding of the underlying mechanisms. The aim of this study was to provide an in-depth comparison of the progression of restenotic lesion formation after arterial injury in apolipoprotein E -/- and C57BL/6 control mice. METHODS: In this study, we investigated the difference in lesion formation of apolipoprotein E -/- and C57BL/6 controls on a high-cholesterol, high-fat diet after arterial injury. One week prior to arterial injury of the left femoral artery, mice were started on a high-cholesterol, high-fat diet. Diets were continued after arterial injury until euthanization. At five consecutive time points (2, 5, 10, 15, and 21 days), the intimal hyperplasia in the injured arteries was analyzed. RESULTS: In the C57BL/6 control mice, a continuously increasing lesion formation, consisting primarily of alpha-smooth muscle actin-positive cells, was observed. Lesion formation in apolipoprotein E -/- mice was significantly more pronounced, resulting in complete occlusion of the arteries in four out of five vessels after 21 days. Lesions in apolipoprotein E -/- mice consisted predominantly of lipid-loaded foam cells and alpha-smooth muscle actin-positive cells. Further histological evaluation demonstrated cholesterol crystals in the lesions and neovascularizsation in cases of occlusion. CONCLUSIONS: Thus, apoE -/- mice on a high-cholesterol, high-fat diet provide a more valid model for the characterization of the development of restenotic lesions after mechanical irritation such as angioplasty than C57BL/6 mice.

Comparison of a new method for the direct and simultaneous assessment of LDL- and HDL-cholesterol with ultracentrifugation and established methods.

BACKGROUND: Automated electrophoresis combined with enzymatic cholesterol staining might improve routine assessment of LDL- and HDL-cholesterol (LDLC and HDLC), as an alternative to the Friedewald equation and precipitation. A new method (Hydrasys; SEBIA) that adapts the cholesterol esterase/cholesterol oxidase reaction within urea-free gels was evaluated. METHODS: Fresh sera from 725 subjects (512 dyslipidemics) were analyzed by electrophoresis, in parallel with sequential ultracentrifugation, beta-quantification, calculation, and precipitation. RESULTS: Electrophoresis was linear up to 4 g/L cholesterol, with a detection limit of 0.042 g/L cholesterol/band. Within-run, between-run, between-batch, and between-operator imprecision (CVs) were 1.6%, 2.0%, 1.5%, and 2.7% for LDLC, and 3.9%, 4.3%, 5.5%, and 4.9% for HDLC, and remained unchanged up to 6.3 g/L plasma triglycerides (TGs). Precision decreased with very low HDLC (<0.25 g/L). Serum storage for 3-7 days at +4 or -80 degrees C did not interfere significantly with the assay. Agreement with beta-quantification was stable for LDLC up to 5.07 g/L (r = 0.94), even at TG concentrations >4 g/L (r = 0.91). Bias (2.88% +/- 12%) and total error (7.84%) were unchanged at TG concentrations up to 18.5 g/L. Electrophoresis predicted National Cholesterol Education Program cut-points with <0.04 g/L error, exactly and appropriately classified 79% and 96% of the subjects, and divided by 2.4 (all subjects) and 5.8 (TGs >1.5 g/L) the percentage of subjects underestimated by calculation. One-half of the patients with TGs >4 g/L had LDLC >1.30 g/L. For HDLC, correlation was better with precipitation (r = 0.87) than ultracentrifugation (r = 0.76). Error (-0.10% +/- 26%) increased when HDLC decreased (<0.35 g/L). Direct assessment of the LDLC/HDLC ratio detected 45% more high-risk subjects than the calculation/precipitation combination. CONCLUSIONS: Electrophoresis provides reliable quantification of LDLC, improving precision, accuracy, and concordance over calculation, particularly with increasing plasma TGs. Implementation of methods to detect low cholesterol concentrations could extend the applications for HDLC assessment.

Determinants of resting energy expenditure in obese non-diabetic caucasian women.

OBJECTIVE: To investigate the determinants of resting energy expenditure (REE) in obese non-diabetic Caucasian women. DESIGN: A cross-sectional survey study before the beginning of a weight-reduction program. SUBJECTS: 141 obese, non-diabetic women of Caucasian origin (BMI 34.8 +/- 3.7 kg/m2, age 43.2 +/- 8.0 y, mean +/- s.d.). MEASUREMENTS: Resting energy expenditure (an indirect calorimetry), body composition (a bioelectrical impedance), fat distribution (anthropometry) and heart rate (ECG) were determined after 12 h overnight fast and apolipoprotein E phenotype was examined. RESULTS: In a linear multiple regression analysis fat-free mass, fat mass and age together with heart rate and waist-hip ratio emerged as significant determinants of REE. In the other regression model, also serum insulin emerged as a significant determinant of REE, in addition to fat-free mass, fat mass and age. There was no significant differences in REE among the different apolipoprotein E phenotype groups. CONCLUSION: Besides fat-free mass, also fat mass, age, heart rate, waist-hip-ratio, and serum insulin level make a significant contribution to REE in obese women.

Assessment of postprandial lipemia: nutritional influences.

Recent reports have contributed new information on the influence of the size and spacing of fat-containing meals on processes related to fat digestion and absorption, lymphatic transport, and postprandial responses of plasma lipoproteins. The postabsorptive concentrations of plasma triglycerides and common polymorphisms of apolipoprotein B and apolipoprotein E exert important influences on postprandial lipoprotein responses. Attention to these characteristics of individuals and groups can facilitate interpretation of responses to meals containing different fatty acids and varying amounts of cholesterol. Additional evidence is accumulating that points to disease related abnormalities of postprandial lipoprotein metabolism related to clearance mechanisms for triglyceride-rich lipoproteins and processes related to reverse cholesterol transport.

Quantitative assessment of aortic atherosclerosis in APOE*3 Leiden transgenic mice and its relationship to serum cholesterol exposure.

Transgenic mice overexpressing the human dysfunctional apolipoprotein E variant, APOE*3 Leiden, develop hyperlipidemia and are highly susceptible to diet-induced atherosclerosis. In the present study, we investigated the effects of diet composition and feeding period on serum cholesterol exposure and the amount of atherosclerosis in the aortic sinus in these mice, using quantitative image analysis. On each of the three diets tested--a low-fat diet, a high-saturated-fat/cholesterol diet, and a high saturated-fat/high-cholesterol/0.5%-cholate diet--transgenic animals showed a marked hyperlipidemia compared with nontransgenic littermates. Measurement of the atherosclerotic lesion areas in cross sections of the aortic sinus in animals exposed to these three diets for up to 6 months showed a 5 to 10 times greater lesion area in transgenic mice compared with nontransgenic controls. Highly significant positive correlations were found between the log-transformed data on lesion area and serum cholesterol exposure (r = .82 to .85 for the 1-, 2-, and 3-month treatment groups), indicating that the hyperlipidemia is likely to be a major determinant in lesion formation. On the basis of these findings, we suggest that the APOE*3 Leiden mouse represents a promising model for intervention studies with hypolipidemic and antiatherosclerotic drugs.