Syftet med arbetet var att försöka utveckla en metod för att isolera lipoprotein(a), ett lipoprotein som korrelerar i hög grad till kardiovaskulär sjukdom, från plasma. Det har varit problematiskt att på ett enkelt sätt separera lipoprotein(a) från LDL på grund av dess strukturella likheter. Som inspiration användes en artikel där separationen utfördes med anjonbyteskromatografi-HPLC (high performance liquid chromatography). Målet var att applicera metoden på ett FPLC-system (fast protein liquid chromatography) med en gelfiltreringskolonn och en anjonbyteskolonn där det kaotropiska saltet natriumperklorat användes som elueringsmedel under anjonbyteskromatografin. Resultaten kontrollerades med DLS (dynamic light scattering), SDS-PAGE och Western blot med antikroppar riktade mot lipoprotein(a). Resultaten visade på goda möjligheter till en bra separation vid fortsatta studier.

Biocompatibility is an important issue for the development of chromatographic stationary phases for the analysis of biomolecules (including proteins and peptides). A biocompatible stationary phase material is a material that resists nonspecific adsorption of biomolecules and does not interact with them in a way that would alter or destroy their structures or biochemical functions. The monolithic column format is a good alternative to typical spherical particle packed columns for capillary liquid chromatography of biomacromolecules. Several novel anion-exchange polymer monoliths for the analysis of proteins were synthesized for improved biocompatibility. Two novel polymeric monoliths were prepared in a single step by a simple photoinitiated copolymerization of 2-(diethylamino)ethyl methacrylate and polyethylene glycol diacrylate (PEGDA), or copolymerization of 2-(acryloyloxy)ethyl trimethylammonium chloride (AETAC) and PEGDA, in the presence of selected porogens. The resulting monoliths contained functionalities of diethylaminoethyl (DEAE) as a weak anion exchanger and quaternary amine as a strong anion exchanger, respectively. An alternative weak anion exchange monolith with DEAE functionalities was also synthesized by chemical modification after photoinitiated copolymerization of glycidyl methacrylate (GMA) and PEGDA. The dynamic binding capacities of the three monoliths were comparable or superior to values that have been reported for various other monoliths. Chromatographic performances were also similar to those provided by a modified poly(GMA-co-ethylene glycol dimethacrylate) monolith. Separations of standard proteins were achieved under gradient elution conditions using these monolithic columns. This work represents a successful attempt to prepare functionalized monoliths via direct copolymerization of monomers with desired functionalities. Compared to earlier publications, laborious surface modifications were avoided and the PEGDA crosslinker improved the biocompatibility of the monolithic backbone. Protein separations by capillary size exclusion chromatography (SEC) require a monolith that is biocompatible, has sufficient pore volume, has the appropriate pore size distribution, and is rigid. Most polymer monoliths have not possessed a biomodal pore-size distribution, i.e., especially with one distribution in the macropore region and the other in the mesopore region. Furthermore, non-specific adsorption of proteins in these stationary phases has persisted as a major unresolved problem. To overcome these difficulties, a porous poly[polyethylene glycol methyl ether acrylate (PEGMEA)-co-PEGDA] monolith which can resist adsorption of both acidic and basic proteins when using an aqueous buffer without any organic solvent additives was developed. Based on this biocompatible monolith, surfactants were introduced as porogens with the hope of significantly increasing the mesopore volume within the polymer. Two types of surfactants were studied, including poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) or PPO-PEO-PPO and Brij. Pore size distributions were examined using a well-defined molecular weight range series of proteins and peptides by inverse size exclusion chromatography, which indicated relatively large volume percentages of mesopores and micropores. The two new monoliths demonstrated different SEC behaviors, low nonspecific adsorption of proteins, and high mechanical rigidity. High density lipoprotein (HDL) is a heterogeneous class of lipoprotein particles with subspecies that differ in apolipoprotein and lipid composition, size, density, and charge. In this work, I developed a new capillary SEC method for size separation of native HDL particles from plasma using a capillary packed with BioSep-SEC-4000 particles, Three major sizes of HDL particles were separated. Additionally, capillary SEC and capillary strong anion-exchange chromatography of non-delipidated HDL were accomplished using poly(PEGMEA-co-PEGDA) and poly(AETAC-co-PEGDA) monoliths. These new LC methods using packed and monolithic stationary phases provided rapid separation of HDLs and excellent reproducibility.

Background Patterns of dyslipidemia and incidence of hypertension have been rarely reported in Asian populations with inconsistent findings. To accumulate further evidence in Asian populations, the study aimed to investigate the relationship between lipid profiles and hypertension in Chinese adult males. Methods We conducted a cross-sectional study based on the data from the DATADRYAD database. The overall population was divided into hypertensive and non-hypertensive groups based on baseline blood pressure levels. For continuous variables, Mann-Whitney test was performed between two groups, while Kruskal-Wallis and Dunn tests were used among multiple groups. The chi-square test was carried out for dichotomous variables. Spearman's correlation coefficient was employed to assess the association between systolic blood pressure (SBP), diastolic blood pressure (DBP) and lipid profiles, whereas the relationship between lipid profiles and the incidence of hypertension was evaluated using multivariate logistic regression. The Bayesian network (BN) model was adopted to investigate the relationship between clinical characteristics and hypertension, and the importance of related predictor to the incidence of hypertension was obtained to make conditional probability analysis. Results Finally, totally 62,957 participants were included in this study. In the lipid profiles, total cholesterol (TC), low-density cholesterol (LDL-c), and non- high-density lipoprotein cholesterol (non-HDL-c) were higher in the hypertensive population (p <0.001). In the fully multivariate model, for every 1 mg/dl increase in TC, LDL-c and non-HDL, the risk of hypertension increased by 0.2% [1.002 (1.001–1.003)], 0.1% [1.001 (1.000–1.002)], and 0.1% [1.001 (1.000–1.002)]. Meanwhile, HDL-c became positively associated with the incidence of hypertension (p for trend < 0.001) after adjusting for the body mass index (BMI), and 1 mg/dl increment in HDL-c increased the risk of hypertension by 0.2% [1.002 (1.000–1.002)] after fully adjusting for multiple variables. Furthermore, the BN showed that the importance of age, BMI, fasting plasma glucose (FPG), and TC to the effect of hypertension is 43.3, 27.2, 11.8, and 5.1%, respectively. Conclusion Elevated TC, LDL-c, and non-HDL-c were related to incidence of hypertension in Chinese adult males, whereas triglycerides (TG) was not significantly associated. The relationship between HDL-c and hypertension incidence shifted from no association to a positive correlation after adjusting for the BMI. Moreover, the BN model displayed that age, the BMI, FPG, and TC were strongly associated with hypertension incidence.

Background: Dyslipidemia is strongly associated with the development of hypertension. In our previous study, it was shown that elevated TC, LDL-c, and non-HDL-c were associated with the prevalence of hypertension in Chinese men, whereas the relationship between HDL-c and hypertension shifted from no association to a positive association after adjusting for the BMI. To further accumulate epidemiological evidence in Asian women, this study aimed to investigate the relationship between lipid profile and prevalence of hypertension in Chinese adult women. Methods: This is a cross-sectional study including 54,099 Chinese women aged>20 years at 32 health screening centers in 11 cities from 2010-2016. The original data were obtained from DATADRYAD database (www.datadryad.org). Besides, the overall women were classified into non-hypertensive and hypertensive groups based on baseline blood pressure levels. Differences between the two groups were examined by Man-Whitney test or Chi-square test. Spearman’s correlation coefficient was employed to evaluate the correlation between systolic blood pressure (SBP), diastolic blood pressure (DBP) and lipid profiles. Multivariate logistic regression was performed to estimate the relationship between different lipid levels and the prevalence of hypertension. Odds ratios (ORs) and 95% confidence intervals (CIs) indicated the risk of lipid and hypertension. Bayesian model (BN) model was constructed to further assess the relationship between baseline characteristics and the prevalence of hypertension, as well as the importance of each variable for the prevalence of hypertension. Results: Compared to the non-hypertensive population, the hypertensive population was older, and had the higher body mass index (BMI), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-c), serum creatinine (Scr), fasting blood glucose (FPG), blood urea nitrogen (BUN), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and non-high-density lipoprotein cholesterol (non-HDL-c), but HDL-c and the presence concerning the family history of diabetes were lower. Multivariate logistic regression analysis revealed that TC, LDL-c, and non-HDL-c showed a positive trend with hypertension risk (p for trend < 0.05) whereas TC and HDL-c were not significantly associated with hypertension prevalence. Moreover, each 1 mg/dl increase in TC, LDL, and non-HDL hypertension prevalence increased by 0.2% [1.002 (1.000-1.003)], 0.2% [1.002 (1.000- 1.004)], and 0.2% [1.002(1.001-1.004)], respectively. BN suggested that the importance of age, BMI, FPG, non-HDL-c on the prevalence of hypertension was 52.73%, 24.98%, 11.22%, and 2.34%, respectively. Conclusion: Overall, in Chinese adult women, TC, LDL-c and non-HDL-c levels were higher and HDL-c level was lower in the hypertensive population, whereas TG did not differ significantly from the non-hypertensive population. Meanwhile, TC, LDL-c, and non-HDL-c were positively associated with prevalence of hypertension, and HDL-c was negatively associated with prevalence of hypertension but became nonsignificant after full adjustment for variables. Moreover, BN model suggested that age, BMI, FPG, and non-HDL-c had a greater effect on the development of hypertension.

No description available.

No description available.

No description available.

No description available.

The liver regulates inflammation during sepsis, and most liver functions are carried out by hepatocytes. Bacterial lipids, including lipopolysaccharide (LPS) and lipoteichoic acid (LTA), can be cleared by hepatocytes, but the underlying mechanisms are uncertain. Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates uptake of LPS by hepatocytes, but it is unknown whether LTA uptake is similarly regulated. Therefore, our objectives were to characterize the PCSK9-regulated pathway of bacterial lipid uptake by hepatocytes by identifying whether low-density lipoprotein (LDL) receptor (LDLR) and LDLR-related protein 1 (LRP1) are the target receptors, and by determining which lipoproteins are involved. To study this pathway, we assessed the uptake of fluorescently-labeled LPS or LTA by human HepG2 hepatocytes using flow cytometry. We pre-treated HepG2 cells with PCSK9, alone or in combination with anti-LDLR or anti-LRP1 antibodies, in order to identify the PCSK9-regulated receptors that are involved, and utilized media containing normal serum or lipoprotein-deficient serum to investigate the lipoprotein- dependence of this pathway. We also determined the roles of LDL and HDL in bacterial lipid uptake through a series of add-back experiments to lipoprotein-deficient serum, and blocked LDLR to confirm that LDLR mediates LDL-dependent uptake. The HepG2 cell response to variable degrees of bacterial lipid uptake was also assessed in a subset of experiments by measuring several cytokines and extracellular alanine aminotransferase (ALT) activity in the cell culture supernatant. We found that PCSK9 regulates LDLR-mediated uptake of both LPS and LTA through an LDL-dependent mechanism, while LRP1 is not involved. Increased bacterial lipid uptake did not result in any hepatocellular injury or cytokine production, as measured by ALT activity and interleukin (IL)-6, IL-8, IL-10, and IL-17 concentrations. In conclusion, we completed our objective of characterizing the PCSK9-regulated pathway of bacterial lipid uptake, and provide supporting evidence for targeting PCSK9 as a novel therapeutic avenue in sepsis. / Thesis / Master of Science (MSc) / Bacterial compounds stimulate inflammation that can be overwhelming during sepsis. Understanding the processes behind uptake and clearance of these compounds may lead to better sepsis treatments. Therefore, our goal was to understand how uptake of two bacterial compounds, lipopolysaccharide and lipoteichoic acid, occurs by liver cells called hepatocytes. Hepatocytes are naturally equipped to clear foreign compounds, so understanding their role in clearing bacterial compounds is important. Another goal was to identify the role of the protein PCSK9 in this uptake process, as treatments targeting PCSK9 could be applied to sepsis once we understand its role in this disease. Our research demonstrates the negative role of PCSK9 in regulating uptake of lipopolysaccharide and lipoteichoic acid through a lipoprotein receptor called LDLR, and identifies the role of lipoproteins in this process. These findings further our understanding of the hepatocyte response to bacterial compounds in relation to sepsis, and identify PCSK9 as a potential target for new sepsis therapies.

Extracellular vesicles (EV) originate from various cell types and reflect the contents of the originating cells. EVs are ubiquitous in nearly all body fluids, including blood plasma, and exhibit significant potential as biomarkers in disease diagnostics. However, isolating EVs from blood plasma remains challenging due to the lack of a standardised method. This study aimed to compare and optimize a density gradient ultracentrifugation workflow (DUC) against size exclusion chromatography-cation exchange chromatography (SEC-CEC) and evaluate SEC versus SEC-CEC. Common contaminants during isolation include lipoproteins (LP); previous studies have shown that lowering the pH of blood plasma can precipitate LP, enhancing isolation efficiency. Acidified blood plasma was compared with neutral plasma for EV isolation using all above mentioned methods. To assess the ability of the isolation methods to purify contaminants while retaining maximal EV yield, samples were analysed using multiple techniques, including particle quantity, free proteins, LP-associated apolipoprotein B, purity index (μg protein/particle), and EV-associated surface markers. The results indicate potential for DUC, but further optimization is necessary to improve the method and its isolation of EV. SEC-CEC emerged as an effective method, reducing contaminants by 71% (SEC) to 99% (SEC-CEC), increasing purity by 80%, and yielding positive signals from EV markers (SEC-CEC). The effect of acidification was ambiguous, it reduced apolipoprotein-B levels in plasma pre-isolation. However, post- isolation, neutral plasma exhibited significantly lower contaminations, albeit at the expense of total particle content and risking EV loss. The study underscored several advantages of SEC-CEC but indicated that acidification did not optimise isolation efficiency.