Apo CIII Proteoforms, Plasma Lipids, and Cardiovascular Risk in MESA.

BACKGROUND: Apo CIII (apolipoprotein CIII) is an important regulator of triglyceride metabolism and was associated with cardiovascular risk in several cohorts. It is present in 4 major proteoforms, a native peptide (CIII0a), and glycosylated proteoforms with zero (CIII0b), 1 (CIII1, most abundant), or 2 (CIII2) sialic acids, which may differentially modify lipoprotein metabolism. We studied the relationships of these proteoforms with plasma lipids and cardiovascular risk. METHODS: Apo CIII proteoforms were measured by mass spectrometry immunoassay in baseline plasma samples of 5791 participants of Multi-Ethnic Study of Atherosclerosis, an observational community-based cohort. Standard plasma lipids were collected for up to 16 years and cardiovascular events (myocardial infarction, resuscitated cardiac arrest, or stroke) were adjudicated for up to 17 years. RESULTS: Apo CIII proteoform composition differed by age, sex, race and ethnicity, body mass index, and fasting glucose. Notably, CIII1 was lower in older participants, men and Black and Chinese (versus White) participants, and higher in obesity and diabetes. In contrast, CIII2 was higher in older participants, men, Black, and Chinese persons, and lower in Hispanic individuals and obesity. Higher CIII2 to CIII1 ratio (CIII2/III1) was associated with lower triglycerides and higher HDL (high-density lipoprotein) in cross-sectional and longitudinal models, independently of clinical and demographic risk factors and total apo CIII. The associations of CIII0a/III1 and CIII0b/III1 with plasma lipids were weaker and varied through cross-sectional and longitudinal analyses. Total apo CIII and CIII2/III1 were positively associated with cardiovascular disease risk (n=669 events, hazard ratios, 1.14 [95% CI, 1.04-1.25] and 1.21 [1.11-1.31], respectively); however, the associations were attenuated after adjustment for clinical and demographic characteristics (1.07 [0.98-1.16]; 1.07 [0.97-1.17]). In contrast, CIII0b/III1 was inversely associated with cardiovascular disease risk even after full adjustment including plasma lipids (0.86 [0.79-0.93]). CONCLUSIONS: Our data indicate differences in clinical and demographic relationships of apo CIII proteoforms, and highlight the importance of apo CIII proteoform composition in predicting future lipid patterns and cardiovascular disease risk.

Plasma proteoforms of apolipoproteins C-I and C-II are associated with plasma lipids in the Multi-Ethnic Study of Atherosclerosis.

Apolipoproteins (apo) C-I and C-II are key regulators of triglyceride and HDL metabolism. Both exist as full-size native and truncated (apoC-I'; apoC-II') posttranslational proteoforms. However, the determinants and the role of these proteoforms in lipid metabolism are unknown. Here, we measured apoC-I and apoC-II proteoforms by mass spectrometry immunoassay in baseline and 10-year follow-up plasma samples from the Multi-Ethnic Study of Atherosclerosis. We found that baseline total apoC-I (mean = 9.2 mg/dl) was lower in African Americans (AA), Chinese Americans (CA), and Hispanics (by 1.8; 1.0; 1.0 mg/dl vs. whites), higher in women (by 1.2 mg/dl), and positively associated with plasma triglycerides and HDL. Furthermore, we observed that the truncated-to-native apoC-I ratio (apoC-I'/C-I) was lower in CA, negatively associated with triglycerides, and positively associated with HDL. We determined that total apoC-II (8.8 mg/dl) was lower in AA (by 0.8 mg/dl) and higher in CA and Hispanics (by 0.5 and 0.4 mg/dl), positively associated with triglycerides, and negatively associated with HDL. In addition, apoC-II'/C-II was higher in AA and women, negatively associated with triglycerides, and positively associated with HDL. We showed that the change in triglycerides was positively associated with changes in total apoC-I and apoC-II and negatively associated with changes in apoC-I'/C-I and apoC-II'/C-II, whereas the change in HDL was positively associated with changes in total apoC-I and apoC-II'/C-II and negatively associated with change in total apoC-II. This study documents racial/ethnic variation in apoC-I and apoC-II plasma levels and highlights apolipoprotein posttranslational modification as a potential regulator of plasma lipids.

Distinct patterns of apolipoprotein C-I, C-II, and C-III isoforms are associated with markers of Alzheimer's disease.

Apolipoproteins C-I, C-II, and C-III interact with ApoE to regulate lipoprotein metabolism and contribute to Alzheimer's disease pathophysiology. In plasma, apoC-I and C-II exist as truncated isoforms, while apoC-III exhibits multiple glycoforms. This study aimed to 1) delineate apoC-I, C-II, and C-III isoform profiles in cerebrospinal fluid (CSF) and plasma in a cohort of nondemented older individuals (n = 61), and 2) examine the effect of APOE4 on these isoforms and their correlation with CSF Aß42, a surrogate of brain amyloid accumulation. The isoforms of the apoCs were immunoaffinity enriched and measured with MALDI-TOF mass spectrometry, revealing a significantly higher percentage of truncated apoC-I and apoC-II in CSF compared with matched plasma, with positive correlation between CSF and plasma. A greater percentage of monosialylated and disialylated apoC-III isoforms was detected in CSF, accompanied by a lower percentage of the two nonsialylated apoC-III isoforms, with significant linear correlations between CSF and plasma. Furthermore, a greater percentage of truncated apoC-I in CSF and apoC-II in plasma and CSF was observed in individuals carrying at least one APOE Ɛ4 allele. Increased apoC-I and apoC-II truncations were associated with lower CSF Aß42. Finally, monosialylated apoC-III was lower, and disialylated apoC-III greater in the CSF of Ɛ4 carriers. Together, these results reveal distinct patterns of the apoCs isoforms in CSF, implying CSF-specific apoCs processing. These patterns were accentuated in APOE Ɛ4 allele carriers, suggesting an association between APOE4 genotype and Alzheimer's disease pathology with apoCs processing and function in the brain.

ApoC-III Glycoforms Are Differentially Cleared by Hepatic TRL (Triglyceride-Rich Lipoprotein) Receptors.

OBJECTIVE: ApoC-III (apolipoprotein C-III) glycosylation can predict cardiovascular disease risk. Higher abundance of disialylated (apoC-III2) over monosialylated (apoC-III1) glycoforms is associated with lower plasma triglyceride levels. Yet, it remains unclear whether apoC-III glycosylation impacts TRL (triglyceride-rich lipoprotein) clearance and whether apoC-III antisense therapy (volanesorsen) affects distribution of apoC-III glycoforms. Approach and Results: To measure the abundance of human apoC-III glycoforms in plasma over time, human TRLs were injected into wild-type mice and mice lacking hepatic TRL clearance receptors, namely HSPGs (heparan sulfate proteoglycans) or both LDLR (low-density lipoprotein receptor) and LRP1 (LDLR-related protein 1). ApoC-III was more rapidly cleared in the absence of HSPG (t1/2=25.4 minutes) than in wild-type animals (t1/2=55.1 minutes). In contrast, deficiency of LDLR and LRP1 (t1/2=56.1 minutes) did not affect clearance of apoC-III. After injection, a significant increase in the relative abundance of apoC-III2 was observed in HSPG-deficient mice, whereas the opposite was observed in mice lacking LDLR and LRP1. In patients, abundance of plasma apoC-III glycoforms was assessed after placebo or volanesorsen administration. Volanesorsen treatment correlated with a statistically significant 1.4-fold increase in the relative abundance of apoC-III2 and a 15% decrease in that of apoC-III1. The decrease in relative apoC-III1 abundance was strongly correlated with decreased plasma triglyceride levels in patients. CONCLUSIONS: Our results indicate that HSPGs preferentially clear apoC-III2. In contrast, apoC-III1 is more effectively cleared by LDLR/LRP1. Clinically, the increase in the apoC-III2/apoC-III1 ratio on antisense lowering of apoC-III might reflect faster clearance of apoC-III1 because this metabolic shift associates with improved triglyceride levels.

Complexity, cost, and content - three important factors for translation of clinical protein mass spectrometry tests, and the case for apolipoprotein C-III proteoform testing.

Complexity, cost, and content are three important factors that can impede translation of clinical protein mass spectrometry (MS) tests at a larger scale. Complexity stems from the many components/steps involved in bottom-up protein MS workflows, making them significantly more complicated than enzymatic immunoassays (EIA) that currently dominate clinical testing. This complexity inevitably leads to increased costs, which is detrimental in the price-competitive clinical marketplace. To successfully compete, new clinical protein MS tests need to offer something new and unique that EIAs cannot - a new content of proteoform detection. The preferred method for proteoform profiling is intact protein MS analysis, in which all proteins are measured as intact species thus allowing discovery of new proteoforms. To illustrate the importance of intact proteoform testing with MS and its potential clinical implications, we discuss here recent findings from multiple studies on the distribution of apolipoprotein C-III proteoforms and their correlations with key clinical measures of dyslipidemia. Such studies are only made possible with assays that are low in cost, avoid unnecessary complexity, and are unique in providing the content of proteoforms.

Quantification of circulating Mycobacterium tuberculosis antigen peptides allows rapid diagnosis of active disease and treatment monitoring.

Tuberculosis (TB) is a major global health threat, resulting in an urgent unmet need for a rapid, non-sputum-based quantitative test to detect active Mycobacterium tuberculosis (Mtb) infections in clinically diverse populations and quickly assess Mtb treatment responses for emerging drug-resistant strains. We have identified Mtb-specific peptide fragments and developed a method to rapidly quantify their serum concentrations, using antibody-labeled and energy-focusing porous discoidal silicon nanoparticles (nanodisks) and high-throughput mass spectrometry (MS) to enhance sensitivity and specificity. NanoDisk-MS diagnosed active Mtb cases with high sensitivity and specificity in a case-control study with cohorts reflecting the complexity of clinical practice. Similar robust sensitivities were obtained for cases of culture-positive pulmonary TB (PTB; 91.3%) and extrapulmonary TB (EPTB; 92.3%), and the sensitivities obtained for culture-negative PTB (82.4%) and EPTB (75.0%) in HIV-positive patients significantly outperformed those reported for other available assays. NanoDisk-MS also exhibited high specificity (87.1-100%) in both healthy and high-risk groups. Absolute quantification of serum Mtb antigen concentration was informative in assessing responses to antimycobacterial treatment. Thus, a NanoDisk-MS assay approach could significantly improve the diagnosis and management of active TB cases, and perhaps other infectious diseases as well.

PEGylated-nanoliposomal clusterin for amyloidogenic light chain-induced endothelial dysfunction.

Light chain (AL) amyloidosis is a disease associated with significant morbidity and mortality arising from multi-organ injury induced by amyloidogenic light chain proteins (LC). There is no available treatment to reverse the toxicity of LC. We previously showed that chaperone glycoprotein clusterin (CLU) and nanoliposomes (NL), separately, restore human microvascular endothelial function impaired by LC. In this work, we aim to prepare PEGylated-nanoliposomal clusterin (NL-CLU) formulations that could allow combined benefit against LC while potentially enabling efficient delivery to microvascular tissue, and test efficacy on human arteriole endothelial function. NL-CLU was prepared by a conjugation reaction between the carboxylated surface of NL and the primary amines of the CLU protein. NL were made of phosphatidylcholine (PC), cholesterol (Chol) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (DSPE-PEG 2000 carboxylic acid) at 70:25:5 mol%. The protective effect of NL-CLU was tested by measuring the dilation response to acetylcholine and papaverine in human adipose arterioles exposed to LC. LC treatment significantly reduced the dilation response to acetylcholine and papaverine; co-treatment of LC with PEGylated-nanoliposomal CLU or free CLU restored the dilator response. NL-CLU is a feasible and promising approach to reverse LC-induced endothelial damage.

Human proteoforms as new targets for clinical mass spectrometry protein tests.

INTRODUCTION: Only about dozen mass spectrometry (MS) protein tests have been translated into clinical laboratories since the MALDI and ESI approaches were developed thirty years ago. While the cost and complexity of these assays are important factors impeding their clinical adoption, new content generated via proteoforms detection could provide the impetus for further development and translation. Areas covered: Provided here are several examples of MS-based protein assays capable of detecting proteoforms, including those for B-type natriuretic peptide (BNP) and parathyroid hormone (PTH). The evidence suggests that the ability to detect proteoforms is not enough to drive the clinical adoption of the MS-based tests - clinical utility of those proteoforms needs to be demonstrated first. Along those lines, recent efforts to discover, clinically validate, and initiate translation of novel proteoform biomarkers such as those of apolipoprotein C-III will be discussed. Expert commentary: MS protein tests face a challenging future. Both the sample preparation steps and the MS platforms need to be simplified to bring the cost per test down, and then the new content brought by the detection of proteoforms will drive the proliferation of these MS tests - first in clinical utility studies and then for routine diagnostics.

Mass spectrometry protein tests: ready for prime time (or not).

INTRODUCTION: Mass spectrometry has played an important role in protein biomarker discovery. Yet, very few of the candidate biomarkers have been validated, and mass spectrometry-based protein tests have not made a significant inroad into clinical laboratories. Areas covered: Offered here is a unique perspective on the future of mass spectrometry protein tests, in view of the following determinants: the true demand for such clinical tests, end-users requirements, platforms and systems design, sample preparation bottlenecks, analytical and clinical validation, and regulatory approval. Expert commentary: Fresh thoughts and attitudes toward MS protein tests are required in order to move them toward clinical utilization and diagnostic use en masse, with critical emphasis on content, simplicity and cost. In its current format and state of the art, they are simply not ready for prime time.

Disialylated apolipoprotein C-III proteoform is associated with improved lipids in prediabetes and type 2 diabetes.

The apoC-III proteoform containing two sialic acid residues (apoC-III2) has different in vitro effects on lipid metabolism compared with asialylated (apoC-III0) or the most abundant monosialylated (apoC-III1) proteoforms. Cross-sectional and longitudinal associations between plasma apoC-III proteoforms (by mass spectrometric immunoassay) and plasma lipids were tested in two randomized clinical trials: ACT NOW, a study of pioglitazone in subjects with impaired glucose tolerance (n = 531), and RACED (n = 296), a study of intensive glycemic control and atherosclerosis in type 2 diabetes patients. At baseline, higher relative apoC-III2 and apoC-III2/apoC-III1 ratios were associated with lower triglycerides and total cholesterol in both cohorts, and with lower small dense LDL in the RACED. Longitudinally, changes in apoC-III2/apoC-III1 were inversely associated with changes in triglycerides in both cohorts, and with total and small dense LDL in the RACED. apoC-III2/apoC-III1 was also higher in patients treated with PPAR-γ agonists and was associated with reduced cardiovascular events in the RACED control group. Ex vivo studies of apoC-III complexes with higher apoC-III2/apoC-III1 showed attenuated inhibition of VLDL uptake by HepG2 cells and LPL-mediated lipolysis, providing possible functional explanations for the inverse association between a higher apoC-III2/apoC-III1 and hypertriglyceridemia, proatherogenic plasma lipid profiles, and cardiovascular risk.

Applications of MALDI Mass Spectrometry in Clinical Chemistry.

BACKGROUND: MALDI-TOF mass spectrometry (MS) is set to make inroads into clinical chemistry because it offers advantages over other analytical platforms. These advantages include low acquisition and operating costs, ease of use, ruggedness, and high throughput. When coupled with innovative front-end strategies and applied to important clinical problems, it can deliver rapid, sensitive, and cost-effective assays. CONTENT: This review describes the general principles of MALDI-TOF MS, highlights the unique features of the platform, and discusses some practical methods based upon it. There is substantial potential for MALDI-TOF MS to make further inroads into clinical chemistry because of the selectivity of mass detection and its ability to independently quantify proteoforms. SUMMARY: MALDI-TOF MS has already transformed the practice of clinical microbiology and this review illustrates how and why it is now set to play an increasingly important role in in vitro diagnostics in particular, and clinical chemistry in general.

The association of plasma cystatin C proteoforms with diabetic chronic kidney disease.

BACKGROUND: Cystatin C (CysC) is an endogenous cysteine protease inhibitor that can be used to assess the progression of kidney function. Recent studies demonstrate that CysC is a more specific indicator of glomerular filtration rate (GFR) than creatinine. CysC in plasma exists in multiple proteoforms. The goal of this study was to clarify the association of native CysC, CysC missing N-terminal Serine (CysC des-S), and CysC without three N-terminal residues (CysC des-SSP) with diabetic chronic kidney disease (CKD). RESULTS: Using mass spectrometric immunoassay, the plasma concentrations of native CysC and the two CysC truncation proteoforms were examined in 111 individuals from three groups: 33 non-diabetic controls, 34 participants with type 2 diabetes (DM) and without CKD and 44 participants with diabetic CKD. Native CysC concentrations were 1.4 fold greater in CKD compared to DM group (p = 0.02) and 1.5 fold greater in CKD compared to the control group (p = 0.001). CysC des-S concentrations were 1.55 fold greater in CKD compared to the DM group (p = 0.002) and 1.9 fold greater in CKD compared to the control group (p = 0.0002). CysC des-SSP concentrations were 1.8 fold greater in CKD compared to the DM group (p = 0.008) and 1.52 fold greater in CKD compared to the control group (p = 0.002). In addition, the concentrations of CysC proteoforms were greater in the setting of albuminuria. The truncated CysC proteoform concentrations were associated with estimated GFR independent of native CysC concentrations. CONCLUSION: Our findings demonstrate a greater amount of CysC proteoforms in diabetic CKD. We therefore suggest assessing the role of cystatin C proteoforms in the progression of CKD.

Development of quantitative mass spectrometric immunoassay for serum amyloid A.

OBJECTIVE: Proteins can exist as multiple proteoforms in vivo that can have important roles in physiological and pathological states. METHODS: We present the development and characterization of mass spectrometric immunoassay (MSIA) for quantitative determination of serum amyloid A (SAA) proteoforms. RESULTS: Intra- and inter-day precision revealed CVs <10%. Against existing SAA ELISA, the developed MSIA showed good correlation according to the Altman-Bland plot. Individual concentrations of the SAA proteoforms across a cohort of 170 samples revealed 7 diverse SAA polymorphic types and 12 different proteoforms. CONCLUSION: The new SAA MSIA enables parallel analysis of SAA polymorphisms and quantification of all expressed SAA proteoforms, in a high-throughput and time-efficient manner.

Development of multiplex mass spectrometric immunoassay for detection and quantification of apolipoproteins C-I, C-II, C-III and their proteoforms.

The impetus for discovery and evaluation of protein biomarkers has been accelerated by recent development of advanced technologies for rapid and broad proteome analyses. Mass spectrometry (MS)-based protein assays hold great potential for in vitro biomarker studies. Described here is the development of a multiplex mass spectrometric immunoassay (MSIA) for quantification of apolipoprotein C-I (apoC-I), apolipoprotein C-II (apoC-II), apolipoprotein C-III (apoC-III) and their proteoforms. The multiplex MSIA assay was fast (∼ 40 min) and high-throughput (96 samples at a time). The assay was applied to a small cohort of human plasma samples, revealing the existence of multiple proteoforms for each apolipoprotein C. The quantitative aspect of the assay enabled determination of the concentration for each proteoform individually. Low-abundance proteoforms, such as fucosylated apoC-III, were detected in less than 20% of the samples. The distribution of apoC-III proteoforms varied among samples with similar total apoC-III concentrations. The multiplex analysis of the three apolipoproteins C and their proteoforms using quantitative MSIA represents a significant step forward toward better understanding of their physiological roles in health and disease.

An automated, high-throughput method for targeted quantification of intact insulin and its therapeutic analogs in human serum or plasma coupling mass spectrometric immunoassay with high resolution and accurate mass detection (MSIA-HR/AM).

The detection and quantification of insulin and its therapeutic analogs is important for medical, sports doping, and forensic applications. Synthetic variants contain slight sequence variations to affect bioavailability. To reduce sample handling bias, a universal extraction method is required for simultaneous extraction of endogenous and variant insulins with subsequent targeted quantification by LC-MS. A mass spectrometric immunoassay (MSIA), a multiplexed assay for intact insulin and its analogues that couples immunoenrichment with high resolution and accurate mass (HR/AM) spectrometric detection across the clinical range is presented in this report. The assay is sensitive, selective, semi-automated and can potentially be applied to detect new insulin isoforms allowing their further incorporation into second or third generation assays.

Mass Spectrometric Immunoassay for the qualitative and quantitative analysis of the cytokine Macrophage Migration Inhibitory Factor (MIF).

BACKGROUND: The cytokine MIF (Macrophage Migration Inhibitory Factor) has diverse physiological roles and is present at elevated concentrations in numerous disease states. However, its molecular heterogeneity has not been previously investigated in biological samples. Mass Spectrometric Immunoassay (MSIA) may help elucidate MIF post-translational modifications existing in vivo and provide additional clarity regarding its relationship to diverse pathologies. RESULTS: In this work, we have developed and validated a fully quantitative MSIA assay for MIF, and used it in the discovery and quantification of different proteoforms of MIF in serum samples, including cysteinylated and glycated MIF. The MSIA assay had a linear range of 1.56-50 ng/mL, and exhibited good precision, linearity, and recovery characteristics. The new assay was applied to a small cohort of human serum samples, and benchmarked against an MIF ELISA assay. CONCLUSIONS: The quantitative MIF MSIA assay provides a sensitive, precise and high throughput method to delineate and quantify MIF proteoforms in biological samples.

Relationship of apolipoprotein C-III proteoform composition with ankle-brachial index and peripheral artery disease in the Multi-Ethnic Study of Atherosclerosis (MESA).

BACKGROUND AND AIMS: Apolipoprotein C-III (apoC-III) proteoform composition shows distinct relationships with plasma lipids and cardiovascular risk. The present study tested whether apoC-III proteoforms are associated with risk of peripheral artery disease (PAD). METHODS: ApoC-III proteoforms, i.e., native (C-III0a), and glycosylated with zero (C-III0b), one (C-III1) or two (C-III2) sialic acids, were measured by mass spectrometry immunoassay on 5,734 Multi-Ethnic Study of Atherosclerosis participants who were subsequently followed for clinical PAD over 17 years. Ankle-brachial index (ABI) was also assessed at baseline and then 3 and 10 years later in 4,830 participants. RESULTS: Higher baseline C-III0b/C-III1 and lower baseline C-III2/C-III1 were associated with slower decline in ABI (follow-up adjusted for baseline) over time, independently of cardiometabolic risk factors, and plasma triglycerides and HDL cholesterol levels (estimated difference per 1 SD was 0.31 % for both, p < 0.01). The associations between C-III2/C-III1 and changes in ABI were stronger in men (-1.21 % vs. -0.27 % in women), and in Black and Chinese participants (-0.83 % and -0.86 % vs. 0.12 % in White). Higher C-III0b/C-III1 was associated with a trend for lower risk of PAD (HR = 0.84 [95%CI: 0.67-1.04]) that became stronger after excluding participants on lipid-lowering medications (0.73 [95%CI: 0.57-0.94]). Neither change in ABI nor clinical PAD was related to total apoC-III levels. CONCLUSIONS: We found associations of apoC-III proteoform composition with changes in ABI that were independent of other risk factors, including plasma lipids. Our data further support unique properties of apoC-III proteoforms in modulating vascular health that go beyond total apoC-III levels.

Association of apolipoproteins C-I and C-II truncations with coronary heart disease and progression of coronary artery calcium: Multi-Ethnic Study of Atherosclerosis.

BACKGROUND AND AIMS: Higher truncated-to-native proteoform ratios of apolipoproteins (apo) C-I (C-I'/C-I) and C-II (C-II'/C-II) are associated with less atherogenic lipid profiles. We examined prospective relationships of C-I'/C-II and C-II'/C-II with coronary heart disease (CHD) and coronary artery calcium (CAC). METHODS: ApoC-I and apoC-II proteoforms were measured by mass spectrometry immunoassay in 5790 MESA baseline plasma samples. CHD events (myocardial infarction, resuscitated cardiac arrest, fatal CHD, n = 434) were evaluated for up to 17 years. CAC was measured 1-4 times over 10 years for incident CAC (if baseline CAC = 0), and changes (follow-up adjusted for baseline) in CAC score and density (if baseline CAC>0). RESULTS: C-II'/C-II was inversely associated with CHD (n = 434 events) after adjusting for non-lipid cardiovascular risk factors (Hazard ratio: 0.89 [95% CI: 0.81-0.98] per SD), however, the association was attenuated after further adjustment for HDL levels (0.93 [0.83-1.03]). There was no association between C-I'/C-I and CHD (0.98 [0.88-1.08]). C-II'/C-II was positively associated with changes in CAC score (3.4% [95%CI: 0.6, 6.3]) and density (6.3% [0.3, 4.2]), while C-I'/C-I was inversely associated with incident CAC (Risk ratio: 0.89 [95% CI: 0.81, 0.98]) in fully adjusted models that included plasma lipids. Total apoC-I and apoC-II concentrations were not associated with CHD, incident CAC or change in CAC score. CONCLUSIONS: Increased apoC-II truncation was associated with reduced CHD, possibly explained by differences in lipid metabolism. Increased apoC-I and apoC-II truncations were also associated with less CAC progression and/or development of denser coronary plaques.

An association of CSF apolipoprotein E glycosylation and amyloid-beta 42 in individuals who carry the APOE4 allele.

Carrying the apolipoprotein E (ApoE) Ɛ4 allele is associated with an increased risk of cerebral amyloidosis and late-onset Alzheimer's disease, but the degree to which apoE glycosylation affects its development is not clear. In a previous pilot study, we identified distinct total and secondary isoform-specific cerebral spinal fluid (CSF) apoE glycosylation profiles, with the E4 isoform having the lowest glycosylation percentage (E2 > E3 > E4). In this work, we extend the analysis to a larger cohort of individuals (n = 106), utilizing matched plasma and CSF samples with clinical measures of AD biomarkers. The results confirm the isoform-specific glycosylation of apoE in CSF, resulting from secondary CSF apoE glycosylation patterns. CSF apoE glycosylation percentages positively correlated with CSF Aß42 levels (r = 0.53, p < 0.0001). These correlations were not observed for plasma apoE glycosylation. CSF total and secondary apoE glycosylation percentages also correlated with the concentration of CSF small high-density lipoprotein particles (s-HDL-P), which we have previously shown to be correlated with CSF Aß42 levels and measures of cognitive function. Desialylation of apoE purified from CSF showed reduced Aß42 degradation in microglia with E4 > E3 and increased binding affinity to heparin. These results indicate that apoE glycosylation has a new and important role in influencing brain Aß metabolism and can be a potential target of treatment.

A novel substitution of proline (P32L) destabilises ß2-microglobulin inducing hereditary systemic amyloidosis.

BACKGROUND: ß2-microglobulin amyloidosis was first described in the 1980s as a protein deposition disease associated with long-term haemodialysis. More recently, two inherited forms resulting from separate point mutations in the ß2-microglobulin gene have been identified. In this report, we detail a novel ß2M variant, P32L, caused by a unique dinucleotide mutation that is linked to systemic hereditary ß2-microglobulin amyloidosis. METHODS: Three family members from a Portuguese kinship featured cardiomyopathy, requiring organ transplantation in one case, along with soft tissue involvement; other involvements included gastrointestinal, neuropathic and sicca syndrome. In vitro studies with recombinant P32L, P32G, D76N and wild-type ß2-microglobulin were undertaken to compare the biophysical properties of the proteins. RESULTS: The P32L variant was caused by the unique heterozygous dinucleotide mutation c.154_155delinsTT. Amyloid disease featured lowered serum ß2-microglobulin levels with near equal amounts of circulating P32L and wild-type proteins; amyloid deposits were composed exclusively of P32L variant protein. In vitro studies of P32L demonstrated thermodynamic and chemical instability and enhanced susceptibility to proteolysis with rapid formation of pre-fibrillar oligomeric structures by N- and C-terminally truncated species under physiological conditions. CONCLUSIONS: This work provides both clinical and experimental evidence supporting the critical role of P32 residue replacement in ß2M amyloid fibrillogenesis.