Dyslipidemia can be a comorbidity of both insulin-resistance and atherosclerosis. Hypertriglyceridemia is common in hyperglycemia and is associated with hypoalphalipoproteinemia (low HDL) and with altered nucleotide or purinergic signaling. We therefore hypothesized that extracellular nucleotides may affect hepatic lipoprotein metabolism. Our studies confirm this view and show that nucleotides regulate cellular proteolytic pathways in liver cells and thereby control lipoprotein secretion and their metabolism by hepatic lipase (HL). Treatment of liver cells with the nucleotide, adenosine diphosphate (ADP), stimulates VLDL-apoB100 and apoE secretion, but blocks HDL-apoA-I and HL secretion. ADP functions like a proteasomal inhibitor to block proteasomal degradation and stimulate apoB100 secretion. Blocking the proteosome is known to activate autophagic pathways. The nucleotide consequently stimulates autophagic degradation in liver cells and increases cellular levels of the autophagic proteins, LC3 and p62. Confocal studies show that ADP increases cellular LC3 levels and promotes co-localization of LC3 and apoA-I in an autophagosomal degradation compartment. ADP acts through the G-protein coupled receptor, P2Y13, to stimulate autophagy and block both HDL and HL secretion. Overexpression of P2Y13 increases cellular LC3 levels and blocks the induction of both HDL and HL secretion, while P2Y13 siRNA reduce LC3 protein levels and cause up to a ten-fold stimulation in HDL and HL secretion. P2Y13 gene expression regulates autophagy through the insulin receptor (IR-β). A reduction in P2Y13 expression increases the phosphorylation of IR-β and protein kinase B (Akt) >3-fold, while increasing P2Y13 expression inhibits the activation of IR-β and Akt. Experiments with epitope-labeled apoA-I and HL show that activation of purinergic pathways has no effect on the internalization and degradation of extracellular apoA-I and HL, which confirms the view that nucleotides primarily impact intracellular protein transport and degradation. In conclusion, elevated blood glucose levels may promote dyslipidemia by stimulating purinergic signaling through P2Y13 and IR-β and perturbing the intracellular degradation and secretion of both HDL and VLDL.

Die chronische Herzinsuffizienz ist ein schwerwiegendes progredientes Krankheitsbild, das sich neben Dyspnoe und abnehmender Leistungsfähigkeit in einer nachgewiesenen Verschlechterung der HDL-Funktion manifestiert. In zahlreichen Studien, in denen der Einfluss von körperlichem Training auf die Progredienz der chronischen Herzinsuffizienz untersucht wurde, korrelierte dauerhaftes Ausdauertraining mit einer Verbesserung der eNOS-Aktivität und damit der HDLFunktion in Gefäßen. Ein Regulationsmechanismus von Endothelzellen besteht in der Expression von angiogenen microRNAs, die über negative Regulation die Proteinexpression beeinflussen. Ziel dieser Studie ist es nun, einen möglichen Zusammenhang zwischen der HDL-Funktionalität und der microRNA-Expression in Endothelzellen zu prüfen und damit die Funktionsänderung von HDL bei Herzinsuffizienten auf molekularer Ebene nachzuweisen. Zudem soll eine Beeinflussung der HDL-Funktion durch körperliches Training geprüft werden. Dafür wurde das HDL von gesunden und herzinsuffizienten Probanden (NYHAIII-Stadium) vor und nach einem vier- bzw. zwölfwöchigen Trainingsprogramm aus dem Plasma isoliert. Anschließend erfolgte mit dem gewonnenen HDL die 24-stündige Inkubation von HAEC-Kulturen. Nach Isolation der microRNAs aus dem gewonnenen Zelllysat konnte die Menge ausgewählter proangiogener miRs über RT-PCR quantifiziert werden. Die molekularbiologische Analyse der Proben zeigte eine, im Vergleich zu den Kontrollzellen, signifikant verringerte Menge an miR-21, -126 und -222 in den, mit HDLNYHAIII-inkubierten, Endothelzellen. Die miR-Expression der Endothelzellen zeigte nach dem Trainingsprogramm eine Annäherung an das Expressionsniveau der Kontrollen. Aus der dargelegten Studie wird so ersichtlich, dass das HDL von Herzinsuffizienten die Expression von proangiogenen microRNAs in Endothelzellen hemmt, was scheinbar in Korrelation mit der Ausbildung von endothelialen Dysfunktionen bei Herzinsuffizienz steht. Zudem konnte gezeigt werden, dass körperliches Training mit einer verbesserten Endothelfunktion über die Erhöhung der miR-Expression in Endothelzellen einhergeht.

Magister Pharmaceuticae - MPharm / Cardiovascular diseases (CVDs) such as ischaemic heart diseases, heart failure and stroke remain a major cause of death globally. Various deep-rooted factors influence CVD development; these include but are not limited to elevated blood lipids, high blood pressure, obesity and diabetes. A considerable number of proteins are involved directly and indirectly in the transport, maintenance and elimination of plasma lipids, including high and low-density lipoprotein cholesterol (HDL-C and LDL-C). There are several mechanisms involved in the removal of LDL particles from systemic circulation. One such mechanism is associated with the gene that encodes proprotein convertase subtilisin/kexin type 9 (PCSK9), which has become an exciting therapeutic target for the reduction of residual risk of CVDs. Currently, statins are the mainstay treatment to reduce LDL-C, and a need exists to further develop more effective LDL-C-lowering drugs that might supplement statins. This study was aimed at contributing to the generation of knowledge regarding the effect of niacin in reducing LDL levels through PCSK9 interaction. The aims/objectives of this study were achieved by utilizing two approaches, which included animal intervention with niacin followed by genetic screening of five prioritized genes involved in cholesterol synthesis and regulation. For animal intervention, 16 vervet monkeys were divided into two groups of eight animals consisting of a control and an experimental (niacin) group. The control group was given a normal standard diet of pre-cooked maize meal throughout the study, while the experimental group received the same diet supplemented with 100 mg/kg of niacin (SR) for 12 weeks. During the niacin intervention, blood was collected at baseline, every four weeks during the treatment period and the end of the washout period. The collected blood was used for biochemical analysis (total cholesterol, triglycerides, LDL-C, and HDL-C) and downstream genetic applications. The second phase included the screening of PCSK9, LDLR, SREBP-2, CETP and APOB-100 using genotyping and gene expression. Niacin administration produced statistically significant increases in plasma HDL-C at fourtime points (T1, T2, T3 and T4), which resulted in an overall increase in plasma HDL-C. Additionally, niacin administration resulted in a slight reduction in LDL-C and total cholesterol levels. Furthermore, the genotyping analysis revealed 13 sequence variants identified in PCSK9, LDLR, SREBP-2, CETP and APOB-100 genes. Five of these variants were predicted to be disease-causing and correlated with gene expression patterns. Three identified PCSK9 variants (H177N, R148S, G635G) were categorized as LOF mutations, and this was supported by a decline in gene expression in animals harbouring these variants. The LDLR also had LOF variants that were the reason for its decreased mRNA expression. Additionally, SREBP-2 proved to be a key mediator of cholesterol pathways. Therefore, the findings of the study conclusively suggest that niacin does increase HDL-C and decrease LDL-C and total cholesterol. Moreover, an interaction between niacin administration and PCSK9 was observed which resulted in decreased gene expression.

Background: Several previous studies have reported that dyslipidemia is associated with the risk of hypertension, but these studies are mainly conducted in European and US populations, with a very few studies in the Asian population. Moreover, the effects of atherosclerotic indices, including atherogenic coefficient (AC) and atherogenic risk of plasma (AIP), on hypertension in Asians have not been well described so far. Methods: From 2010 to 2016, altogether 211,833 Chinese adults were ultimately recruited at the health centers in 11 Chinese cities (including Shanghai, Beijing, Nanjing, Suzhou, Shenzhen, Changzhou, Chengdu, Guangzhou, Hefei, Wuhan, and Nantong). Differences in continuous variables between the two groups were analyzed by the Mann–Whitney test, while those in categorical variables were examined by the Chi-squared test. Logistic regression was applied to evaluate the association between lipid profiles and the risk of hypertension. The predictive values of AC and AIP for the incidence of hypertension were analyzed using the area under the receiver operating characteristic (ROC) curve. Meanwhile, Bayesian network (BN) models were performed to further analyze the associations between the different covariates and the incidence of hypertension. Results: A total of 117,056 participants were included in the final analysis. There were significant differences in baseline characteristics between normotension and hypertension groups (p < 0.001). In multivariate logistic regression, the risk of hypertension increased by 0.2% (1.002 [1.001–1.003]), 0.2% (1.002 [1.001–1.003]), and 0.2% (1.002 [1.001–1.003]) per 1 mg/dl increase in total cholesterol (TC), low-density lipoprotein (LDL), and non-high-density lipoprotein cholesterol (non-HDL-c), respectively. However, after adjusting for body mass index (BMI), an increase in HDL level was associated with a higher risk of hypertension (p for a trend < 0.001), and the risk of hypertension increased by 0.6% per 1 mg/dl increase in HDL-c (1.006 [1.003–1.008]). In women, AC had the highest predictive value for the incidence of hypertension with an area under the curve (AUC) of 0.667 [95% confidence interval (CI): 0.659–0.674]. BN models suggested that TC and LDL were more closely related to the incidence of hypertension. Conclusions: Overall, lipid profiles were significantly abnormal in the hypertensive population than in the normotensive population. TC and LDL were strongly associated with the incidence of hypertension. TC, LDL, and non-HDL-c levels show a positive association, HDL-c shows a negative association, while TG is not significantly associated with the risk of hypertension. After adjusting for BMI, HDL-c turns out to be positively associated with the risk of hypertension. In addition, AC has a good predictive value for the incidence of hypertension in women.

Für die Ätiopathogenese der Atherosklerose spielen eine Vielzahl von Mediatoren eine Rolle. Dabei werden durch das Endothel sowohl protektive als auch schädliche Mediatoren sezerniert. High Density Lipoproteine (HDL) stellen einen bedeutenden protektiven Marker für das kardiovaskuläre Risiko dar, u.a. durch die Aktivierung der endothelialen NO-Synthase (eNOS). HDL besteht zu 50 % aus Proteinen und zu 50 % aus Lipiden. Welche Komponenten des HDL für die eNOS Aktivierung verantwortlich sind, ist nicht bekannt gewesen. Im ersten Abschnitt dieser Promotionsarbeit konnte erfolgreich gezeigt werden, dass die Lysophospholipide, Sphingosin-1-Phosphat (S1P) und Sphinsosylphosphorylcholin (SPC), die strukturelle Bestandteile der Lipidfraktion von HDL darstellen, für einen Teil der HDL induzierten eNOS Aktivierung durch Stimulation des S1P3-Rezeptors verantwortlich sind. Diese eNOS Aktivierung wird durch den intrazellulären Einstrom von Calcium und durch die Aktivierung der Akt-Kinase induziert. Im zweiten Abschnitt dieser Promotionsarbeit konnte nachgewiesen werden, dass das oral verfügbare Lysophospholipid-basierte Medikament, FTY720, das ein strukturelles Analogon des S1P ist, den HDL induzierten Signaltransduktionsweg der eNOS Aktivierung in gleicher Weise induziert. Im dritten Abschnitt dieser Promotionsarbeit konnte ein neuartiges endothelabhängig sezerniertes gemischtes Dinukleosidpolyphosphat, Uridin-Adenosin-Tetraphosphat (Up4A), identifiziert werden. Up4A ist ein Agonist an den P2X- und P2Y-Purinrezeptoren. Up4A induziert bei Applikation in eine isoliert perfundierte Rattenniere hauptsächlich über die Aktivierung des P2X1-Rezeptors und des P2Y2/P2Y4-Rezeptors eine starke Vasokonstriktion im renalen Perfusionsgebiet mit einhergehender Erhöhung des mittleren renalen Perfusionsdrucks. Die direkte Infusion von Up4A in vivo in eine WKY-Ratte führt zu einer signifikanten Erhöhung des mittleren arteriellen Blutdrucks. / In the pathogenesis of atherosclerosis many mediators are included. Therefore the endothelium plays a crucial part by secreting protective but also deleterious factors. High density lipoproteins are an established protective factor in the risk profile of cardiovascular events especially by activating the endothelial NO synthase (eNOS). HDL is composed of 50 % proteins and 50 % lipids. Which component of HDL is responsible for the eNOS activation was not known. In the first part of this dissertation it could be shown, that the lysophospholipids, sphingosine-1-phosphate (S1P) and sphingosylphosphorylcholine (SPC), which are structural compounds of the lipid fraction of HDL, are responsible for a significant part of the HDL induced eNOS activation by stimulating the specific S1P3 receptor. In the signal transduction mechanism the activation of Akt kinase and an influx of calcium is involved. In the second part of this dissertation it could be shown, that the orally active lysophospholipide based drug FTY720, which is a structural analogue of S1P, is able to induce the same signal transduction mechanism activated by HDL including the stimulation of the S1P3 receptor. In the last part of this dissertation a new endothelium dependent vasoconstrictor, the dinucleoside polyphosphate uridine-adenosine-tetraphosphate (Up4A), could be for the first time identified. Up4A is a potent agonist of the P2X- and P2Y-purinoceptors. Via activating the P2X1 receptor and the P2Y2/P2Y4 receptor Up4A induce a strong vasoconstriction in the renal perfusion system in the model of the isolated perfused rat kidney with an adjacent increase of the mean perfusion pressure. By injection of Up4A in vivo in a Wistar Kyoto rat the mean arterial pressure also increase significantly.

Motivation Alcohol consumption is one of the major risk factors of cardiovascular disease (CVD). Excessive alcohol drinking is the fifth leading cause of death worldwide and the prevalence of alcohol abuse continues to increase especially in low-income areas of sub-Saharan Africa. The alarming rate of urbanisation seems to be the driving force for excessive alcohol intake in the developing world. In addition to its influence on CVD, heavy drinking also results in a number of non-cardiovascular consequences that include injury, risky sexual behaviour, violent crime and family dysfunction among black South Africans, contributing to high mortality. Moreover, the highest number of individuals with human immunodeficiency virus (HIV) infection in South Africa is partly attributable to high intake of alcohol. HIV remains a major concern in South Africa with significant funding diverted to address the pandemic. The continued increases in mortality from preventable outcomes such as stroke, myocardial infarction and renal failure are largely due to urbanisation, poverty and dysfunctional health systems working with limited budgets. These are some of the factors requiring in-depth study of the scientific aspects of alcohol intake in South Africa. Although there is enough evidence that links excessive drinking with hypertension and CVD, the markers of alcohol intake – self reporting of alcohol, gamma-glutamyltransferase (GGT) and carbohydrate deficient transferrin – are still not specific enough to isolate other confounding factors in the association of alcohol intake with CVD. The markers of alcohol that independently predict CVD and mortality need to be explored. Finally, the severe lack of longitudinal investigations on alcohol-related hypertension development and total mortality in black South Africans has compromised the early identification of risk factors associated with these outcomes. This study will therefore attempt to address the limited availability of longitudinal studies and stimulate interest for continued investigation. Aim The aim of this study was to investigate whether alcohol intake of black South Africans is related to specific measures of cardiovascular function (change in blood pressure (BP), hypertension development) and mortality over a period of 5 years. Methodology This study was based on the international Prospective Urban and Rural Epidemiology (PURE) study which includes 26 countries, investigating the cause and development of cardiovascular risk factors in low, middle and high income countries. This South African leg of the PURE study started in 2005 in which the baseline data was collected from 2021 black South Africans from rural and urban areas in Ikageng, Ganyesa and Tlakgameng in the North West Province. Eleven participants presented with missing data, leaving 2010 participants with complete datasets at baseline. However, data from these 11 participants was useful, especially for Chapter 4. All participants gave informed consent and the Ethics committee of the North-West University (Potchefstroom Campus) approved the study. The follow-up data collection was done in 2010. General health questionnaires, anthropometric measurements, lipid profiles and cardiovascular measurements were taken both at baseline and follow-up using appropriate methods. We also collected blood samples and performed biochemical analyses for lipid markers, liver enzymes, inflammatory markers and percentage carbohydrate deficient transferrin (%CDT). Finally, we obtained data on cardiovascular and non-cardiovascular mortality through verbal autopsy and death certificates. We made use of analysis of variance (ANOVA) and Chi-square tests to compare means and proportions, respectively. We used dependent t-tests and the McNemar test to compare baseline and follow-up variables. Furthermore, we employed single and partial linear regression analyses to correlate alcohol markers with each other and with the cardiovascular measures. Multiple regression analyses were used to correlate dependent variables in the study with various independent variables as required. Finally, we employed multivariable-adjusted Cox regression analyses to assess the association of the selected alcohol markers with mortality while adjusting for several independent variables. Results and Conclusions of each manuscript - With the first research article (Chapter 4), we aimed to compare self-reported alcohol intake estimates with GGT and %CDT, considering their relationship with percentage change in brachial blood pressure (BP) and central systolic blood pressure (cSBP) over 5 years. The results indicated that only self-reported alcohol intake independently predicted % change in brachial BP and cSBP. This was not found for the biochemical markers GGT and %CDT. Self-reported alcohol intake seems to be an important measure to implement by health systems in low income areas of sub-Saharan Africa, where honest reporting is expected. - Given the likely presence of high GGT levels in both alcohol consumption and non-alcoholic fatty liver disease (NAFLD), the second manuscript (Chapter 5) aimed to compare the cardiovascular and metabolic characteristics of excessive alcohol users and individuals with suspected NAFLD (confirmed with self-report, GGT and %CDT). We found that different sex and cardiometabolic profiles characterised excessive alcohol users and individuals suspected with NAFLD. Lean body mass and male sex were the dominant characteristics in excessive alcohol use while the NAFLD group had a dysmetabolic profile with obese women making up the higher proportion of this group. In excessive alcohol users systolic blood pressure and pulse pressure were independently associated with high-density lipoprotein cholesterol. Diastolic blood pressure showed a significant correlation with waist circumference. These disparate profiles may guide healthcare practitioners in primary healthcare clinics to identify individuals with elevated GGT levels who may suffer from NAFLD or alcohol overuse. These results emphasise the importance of modifiable risk factors as the main contributors to CVD and that lifestyle change should be the main focus in developing countries such as South Africa. - The third manuscript (Chapter 6) aimed to determine the measure of alcohol intake (selfreported alcohol intake, GGT and %CDT) that related best with hypertension development, cardiovascular and all-cause mortality over 5 years in the same population of black South Africans. We found that GGT was the only independent predictor of hypertension development, cardiovascular as well as all-cause mortality. Moreover, self-reporting of alcohol intake predicted incident hypertension, confirming our findings from Chapter 4. The third marker, %CDT, a highly specific marker of alcohol intake, was not related with any outcome variable, perhaps due to its low sensitivity. Although self-reported alcohol intake is useful in low-resource primary healthcare settings, measurement of GGT is encouraged due to its predictive value for hypertension and mortality. GGT represents alcohol intake, non-alcoholic steatohepatitis and obesity - all known to have severe cardiovascular consequences. Discussion and Conclusions Excessive alcohol intake remains a major concern in the development of hypertension, CVD and premature death in sub-Saharan Africa. Despite their weaknesses such as bias and nonspecificity, self-reporting of alcohol consumption and GGT emerged as reliable alcohol markers that independently predicted 5-year change in BP, hypertension development and total mortality in this population. Serum %CDT did not show any association with the mentioned cardiovascular markers. Finally, we were also able to show that black South Africans with suspected NAFLD (i.e. with high GGT levels who do not consume alcohol) are typically obese women, whereas lean men were more likely to have high alcohol consumption. Further prospective investigations are encouraged regarding (a) these mentioned associations, as well as (b) other self-reporting estimates such as quantity and frequency of drinking and (c) the use of %CDT as a highly specific marker of alcohol intake. The simultaneous presence of HIV infection in alcohol abuse in this population also warrants further investigation. / PhD (Physiology), North-West University, Potchefstroom Campus, 2015

Motivation Alcohol consumption is one of the major risk factors of cardiovascular disease (CVD). Excessive alcohol drinking is the fifth leading cause of death worldwide and the prevalence of alcohol abuse continues to increase especially in low-income areas of sub-Saharan Africa. The alarming rate of urbanisation seems to be the driving force for excessive alcohol intake in the developing world. In addition to its influence on CVD, heavy drinking also results in a number of non-cardiovascular consequences that include injury, risky sexual behaviour, violent crime and family dysfunction among black South Africans, contributing to high mortality. Moreover, the highest number of individuals with human immunodeficiency virus (HIV) infection in South Africa is partly attributable to high intake of alcohol. HIV remains a major concern in South Africa with significant funding diverted to address the pandemic. The continued increases in mortality from preventable outcomes such as stroke, myocardial infarction and renal failure are largely due to urbanisation, poverty and dysfunctional health systems working with limited budgets. These are some of the factors requiring in-depth study of the scientific aspects of alcohol intake in South Africa. Although there is enough evidence that links excessive drinking with hypertension and CVD, the markers of alcohol intake – self reporting of alcohol, gamma-glutamyltransferase (GGT) and carbohydrate deficient transferrin – are still not specific enough to isolate other confounding factors in the association of alcohol intake with CVD. The markers of alcohol that independently predict CVD and mortality need to be explored. Finally, the severe lack of longitudinal investigations on alcohol-related hypertension development and total mortality in black South Africans has compromised the early identification of risk factors associated with these outcomes. This study will therefore attempt to address the limited availability of longitudinal studies and stimulate interest for continued investigation. Aim The aim of this study was to investigate whether alcohol intake of black South Africans is related to specific measures of cardiovascular function (change in blood pressure (BP), hypertension development) and mortality over a period of 5 years. Methodology This study was based on the international Prospective Urban and Rural Epidemiology (PURE) study which includes 26 countries, investigating the cause and development of cardiovascular risk factors in low, middle and high income countries. This South African leg of the PURE study started in 2005 in which the baseline data was collected from 2021 black South Africans from rural and urban areas in Ikageng, Ganyesa and Tlakgameng in the North West Province. Eleven participants presented with missing data, leaving 2010 participants with complete datasets at baseline. However, data from these 11 participants was useful, especially for Chapter 4. All participants gave informed consent and the Ethics committee of the North-West University (Potchefstroom Campus) approved the study. The follow-up data collection was done in 2010. General health questionnaires, anthropometric measurements, lipid profiles and cardiovascular measurements were taken both at baseline and follow-up using appropriate methods. We also collected blood samples and performed biochemical analyses for lipid markers, liver enzymes, inflammatory markers and percentage carbohydrate deficient transferrin (%CDT). Finally, we obtained data on cardiovascular and non-cardiovascular mortality through verbal autopsy and death certificates. We made use of analysis of variance (ANOVA) and Chi-square tests to compare means and proportions, respectively. We used dependent t-tests and the McNemar test to compare baseline and follow-up variables. Furthermore, we employed single and partial linear regression analyses to correlate alcohol markers with each other and with the cardiovascular measures. Multiple regression analyses were used to correlate dependent variables in the study with various independent variables as required. Finally, we employed multivariable-adjusted Cox regression analyses to assess the association of the selected alcohol markers with mortality while adjusting for several independent variables. Results and Conclusions of each manuscript - With the first research article (Chapter 4), we aimed to compare self-reported alcohol intake estimates with GGT and %CDT, considering their relationship with percentage change in brachial blood pressure (BP) and central systolic blood pressure (cSBP) over 5 years. The results indicated that only self-reported alcohol intake independently predicted % change in brachial BP and cSBP. This was not found for the biochemical markers GGT and %CDT. Self-reported alcohol intake seems to be an important measure to implement by health systems in low income areas of sub-Saharan Africa, where honest reporting is expected. - Given the likely presence of high GGT levels in both alcohol consumption and non-alcoholic fatty liver disease (NAFLD), the second manuscript (Chapter 5) aimed to compare the cardiovascular and metabolic characteristics of excessive alcohol users and individuals with suspected NAFLD (confirmed with self-report, GGT and %CDT). We found that different sex and cardiometabolic profiles characterised excessive alcohol users and individuals suspected with NAFLD. Lean body mass and male sex were the dominant characteristics in excessive alcohol use while the NAFLD group had a dysmetabolic profile with obese women making up the higher proportion of this group. In excessive alcohol users systolic blood pressure and pulse pressure were independently associated with high-density lipoprotein cholesterol. Diastolic blood pressure showed a significant correlation with waist circumference. These disparate profiles may guide healthcare practitioners in primary healthcare clinics to identify individuals with elevated GGT levels who may suffer from NAFLD or alcohol overuse. These results emphasise the importance of modifiable risk factors as the main contributors to CVD and that lifestyle change should be the main focus in developing countries such as South Africa. - The third manuscript (Chapter 6) aimed to determine the measure of alcohol intake (selfreported alcohol intake, GGT and %CDT) that related best with hypertension development, cardiovascular and all-cause mortality over 5 years in the same population of black South Africans. We found that GGT was the only independent predictor of hypertension development, cardiovascular as well as all-cause mortality. Moreover, self-reporting of alcohol intake predicted incident hypertension, confirming our findings from Chapter 4. The third marker, %CDT, a highly specific marker of alcohol intake, was not related with any outcome variable, perhaps due to its low sensitivity. Although self-reported alcohol intake is useful in low-resource primary healthcare settings, measurement of GGT is encouraged due to its predictive value for hypertension and mortality. GGT represents alcohol intake, non-alcoholic steatohepatitis and obesity - all known to have severe cardiovascular consequences. Discussion and Conclusions Excessive alcohol intake remains a major concern in the development of hypertension, CVD and premature death in sub-Saharan Africa. Despite their weaknesses such as bias and nonspecificity, self-reporting of alcohol consumption and GGT emerged as reliable alcohol markers that independently predicted 5-year change in BP, hypertension development and total mortality in this population. Serum %CDT did not show any association with the mentioned cardiovascular markers. Finally, we were also able to show that black South Africans with suspected NAFLD (i.e. with high GGT levels who do not consume alcohol) are typically obese women, whereas lean men were more likely to have high alcohol consumption. Further prospective investigations are encouraged regarding (a) these mentioned associations, as well as (b) other self-reporting estimates such as quantity and frequency of drinking and (c) the use of %CDT as a highly specific marker of alcohol intake. The simultaneous presence of HIV infection in alcohol abuse in this population also warrants further investigation. / PhD (Physiology), North-West University, Potchefstroom Campus, 2015

Introdução: estudos recentes demonstram que o aumento do colesterol na lipoproteína de alta densidade (HDL-C), induzido por medicamentos ou mutações genéticas, não é associado à redução de eventos coronarianos. A lipoproteína de alta densidade (HDL) apresenta aspectos funcionais distintos em relação ao seu papel cardioprotetor. Objetivo: desenvolver um escore de funcionalidade da HDL (EFH) e avaliar a sua associação com algoritmos de predição de risco cardiovascular e aterosclerose subclínica em indivíduos brasileiros. Metodologia: trata-se de um estudo transversal composto por duas etapas. Na 1ª etapa, o EFH preditor de risco cardiovascular (EFH-RCV) foi desenvolvimento e validado a partir de uma subamostra do estudo CARDIONUTRI (n=354). Na 2ª etapa, o EFH preditor de aterosclerose subclínica (EFH-AS) foi desenvolvido e validado com dados de uma subamostra do estudo ELSA-Brasil (n=4549). No estudo CARDIONUTRI foram avaliadas a atividade da paraoxonase 1 (PON1) e da proteína de transferência de ésteres de colesterol (CETP), a concentração da apolipoproteína AI (APOAI), a capacidade antioxidante da HDL (lag time) e as subfrações da HDL pelo método Lipoprint®. O estudo ELSA-Brasil avaliou as subfrações da HDL pelo método Vertical Auto Profile (VAP) e Ressonância Magnética Nuclear (RMN), e a aterosclerose subclínica por tomografia computadorizada, quantificação da calcificação da artéria coronária (CAC) e calculo do escore da CAC. Resultados: no desenvolvimento do EFH-RCV, a HDL grande apresentou maior força de associação com o risco cardiovascular no modelo múltiplo final (OR = 0,797; p <0,001). O EFH-RCV demonstrou bom desempenho em relação ao escore de risco de Framingham (AUC = 0,899; p <0,001), escore de risco de Reynolds (AUC = 0,722; p <0,001) e Adult Treatment Panel III/2013 (AUC = 0,864; p <0,001). Além disso, apresentou boa reprodutibilidade e correlação com aterosclerose subclínica, quando testado na amostra do estudo ELSA-Brasil, utilizando medidas da HDL grande derivadas do método VAP (AUC = 0,864; p <0,001 e r = 0,252 p <0,001) ou do método de RMN (AUC = 0,876; p <0,001 e r = 0,277; p <0,001). O EFH-AS foi desenvolvido a partir do tamanho da HDL (nm), que apresentou a associação mais forte com aterosclerose subclínica no modelo múltiplo final (OR = 0,549; p <0,001) e demonstrou bom desempenho (AUC = 0,769; p <0,001). Conclusão: o EFH apresentou associações mais fortes com o risco cardiovascular e a aterosclerose subclínica, independente do HDL-C, com destaque para a HDL grande. Os resultados controversos entre as subfrações da HDL e o risco cardiovascular parecem manter relação com as metodologias distintas utilizadas nas análises. Portanto, a validação dos métodos e a inclusão do tamanho da HDL como marcador de risco cardiovascular revela um futuro promissor como adjuvante na estimativa do risco cardiovascular, manejo de medicamentos e tomada de decisões na prática clínica. / Introduction: current studies have not presented association between high density-lipoprotein cholesterol (HDL-C) increase, induced by drugs or genetic mutations, and coronary events reduction. HDL plays different functional cardioprotective role. Objective: to develop a HDL functionality score (HFS) and to assessment its association with predictive cardiovascular risk algorithms and subclinical atherosclerosis outcomes in Brazilian subjects. Methods: cross-sectional study based in two steps. In the first step, the HFS predictor of cardiovascular risk disease (HFS-CVR) was developed and validated on CARDIONUTRI study subsample (n=354). In second step the HFS predictor of subclinical atherosclerosis (HFS-SA) was developed and validated on ELSA-Brasil study subsample (n=4549). CARDIONUTRI study evaluated paraoxonase 1(PON1) and cholesterol ester transfer protein (CETP) activity, apolipoprotein AI (APOAI) concentration, HDL antioxidant capacity, and HDL subfractions by standard Lipoprint® method. ELSA-Brasil study evaluated the size of HDL and subfractions by Vertical Auto Profile (VAP) and Nuclear Magnetic Resonance (NMR) method, and the diagnosis of subclinical atherosclerosis by computed tomography, quantifying coronary artery calcification (CAC) and CAC score. Results: in the development of HFS-CVR, the large HDL presented greater strength of association with cardiovascular risk in the multiple final model (OR = 0.797; p <0.001). The HFS-CVR showed satisfactory performance by Framingham risk score (AUC = 0.899; p <0.001), Reynolds risk score (AUC = 0.722; p <0.001) and Adult Treatment Panel III/2013 guidelines (AUC = 0.864; p <0.001). In addition, HFS-CVR presented satisfactory reproducibility and was associated with subclinical atherosclerosis on ELSA-Brasil sample using large HDL measurements derived from the VAP method (AUC = 0.864; p <0.001 and r = 0,252; p <0,001) or the NMR method (AUC = 0.876; p <0.001 and r = 0.277; p <0,001). HFS-AS was developed from the HDL size (nm), because presented greater association with subclinical atherosclerosis in the final multiple model (OR = 0.549; p <0.001). HFS-AS demonstrated satisfactory performance (AUC = 0.769; p <0.001). Conclusion: the HFS demonstrates strong association with cardiovascular risk and subclinical atherosclerosis, independent of HDL-C, with emphasis on large HDL. Controversial results, between HDL subfractions and cardiovascular irsk seem to maintain a relation with the different methodologies used in analysis. Therefore, the validation of the methods and the inclusion of the HDL size as a cardiovascular risk marker reveal a promising future as an adjunct in the estimation of cardiovascular risk, drug management and decision making in clinical practice.

Introduction: Type 2 diabetes continues to be one of the most common non-communicable diseases worldwide and complications due to type 2 diabetes, such as cardiovascular disease (CVD) can cause severe disability and even death. Despite advances in the development and validation of cardiovascular risk scores, those used in clinical practice perform inadequately for people with type 2 diabetes. Research has suggested that particular non-traditional biomarkers and novel omics data may provide additional value to risk scores over-and-above traditional predictors. Aims: To determine whether a small panel of non-traditional biomarkers improve prediction models based on a current cardiovascular risk score (QRISK2), either individually or in combination, in people with type 2 diabetes. Furthermore, to investigate a set of 228 metabolites and their associations with CVD, independent of well-established cardiovascular risk factors, in order to identify potential new predictors of CVD for future research. Methods: Analyses used the Edinburgh Type 2 Diabetes Study (ET2DS), a prospective cohort of 1066 men and women with type 2 diabetes aged 60-75 years at baseline. Participants were followed for eight years, during which time 205 had a cardiovascular event. Additionally, for omics analyses, four cohorts from the UCL-LSHTM-Edinburgh-Bristol (UCLEB) consortium were combined with the ET2DS. Across all studies, 1005 (44.73%) participants had CVD at baseline or experienced a cardiovascular event during follow-up. Results: In the ET2DS, higher levels of high sensitivity cardiac troponin (hs-cTnT) and N-terminal pro-brain natriuretic peptide (NT-proBNP) and lower levels of ankle brachial pressure index (ABI) were associated with incident cardiovascular events, independent of QRISK2 and pre-existing cardiovascular disease (odds ratios per one SD increase in biomarker 1.35 (95% CI: 1.13, 1.61), 1.23 (1.02, 1.49) and 0.86 (0.73, 1.00) respectively). The addition of each biomarker to a model including just QRISK2 variables improved the c-statistic, with the biggest increase for hs-cTnT (from 0.722 (0.681, 0.763) to 0.732 (0.690, 0.774)). When multiple biomarkers were considered in combination, the greatest c-statistic was found for a model which included ABI, hs-cTnT and gamma-glutamyl transpeptidase (0.740 (0.699, 0.781)). In the combined cohorts from the UCLEB consortium, a small number of high-density lipoprotein (HDL) particles were found to be significantly associated with CVD: concentration of medium HDL particles, total lipids in medium HDL, phospholipids in medium HDL and phospholipids in small HDL. These associations persisted after adjustment for a range of traditional cardiovascular risk factors including age, sex, blood pressure, smoking and HDL to total cholesterol ratio. Conclusions: In older people with type 2 diabetes, a range of non-traditional biomarkers increased predictive ability for cardiovascular events over-and-above the commonly used QRISK2 score, and a combination of biomarkers may provide the best improvement. Furthermore, a small number of novel omics biomarkers were identified which may further improve risk scores or provide better prediction than traditional lipid measurements such as HDL cholesterol.

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