Recent findings indicate that aspects of high-density lipoprotein (HDL) not captured by traditionally measured HDL–cholesterol levels (HDL‑C) are likely to be cardioprotective. This review will highlight some of these studies and suggest new directions to identify the specific molecules that are responsible for the cardioprotective nature of HDL.
by Daniel S. Kim, Dr Patrick M. Hutchins and Prof. Gail P. Jarvik
Raising HDL-C does not confer cardioprotection
There is a well-established inverse association between high-density lipoprotein–cholesterol (HDL-C) levels and cardiovascular disease (CVD) in epidemiological and clinical studies [1, 2]. This robust relationship suggested that HDL-C was in the causal pathway of atheroprotection. Indeed a large number of studies have demonstrated that HDL possesses various anti-atherogenic properties, primarily the ability to accept cholesterol from macrophages in a process termed reverse cholesterol transport [3, 4].
In contrast, several high-profile studies have demonstrated that increasing levels of HDL-C does not have a significant cardioprotective effect. In a large and well-conducted clinical trial of the cholesterol ester transport protein (CETP) inhibitor, torcetrapib, there was no reduction in the incidence of CVD-related events despite significantly higher HDL-C levels [5]. A follow-up study using a different CETP inhibitor, dalcetrapib, also showed increased HDL-C levels yet there was no significant difference in CVD event rate between the treatment and placebo groups [6]. In a third randomized clinical trial that used niacin to increase HDL-C levels, there was again no reduction in cardiovascular events [7]. Finally, a large-scale Mendelian randomization study of approximately 20 000 myocardial infarction (MI) cases and 100 000 controls, showed that a genetic polymorphism which associated with approximately 10% higher HDL-C levels was not associated with decreased incidence of MI [8], again suggesting that the relationship between HDL-C and the prevention of cardiac events is not causal.
HDL particle concentration is a superior predictor of CVD
As the elevation of HDL-C was not beneficial in these studies, some have speculated that HDL itself is not cardioprotective. An alternative explanation for these negative data is that the cholesterol content of HDL – a surrogate measure of HDL – does not best reflect the anti-atherogenic properties of HDL. To resolve these issues it is critical to identify new HDL metrics that reliably reflect its cardioprotective functions.
One promising approach for assessing the role of HDL in CVD is to evaluate the individual HDL particles. HDL is a heterogeneous mixture of lipoprotein particles composed of discrete subspecies that have unique structural compositions and biological functions. As different HDL particles carry vastly different amounts of cholesterol – ranging over an order-of-magnitude [9, 10] – measuring the total HDL-C does not provide information regarding the distribution of HDL subpopulations or the number of total HDL particles.
HDL can be fractionated based on a number of physicochemical properties, most commonly size or density. Several techniques, both qualitative and quantitative, have been developed for HDL subspecies analysis. The various HDL subspecies reported by these techniques and their associated nomenclature are briefly summarized in Table 1 [see also ref. 11]. Furthermore, HDL subspecies determined by ultracentrifugation and calibrated ion mobility analysis (both are discussed in detail later) are shown in Figure 1. Many studies have demonstrated the potential clinical utility of HDL subspecies analysis, which can be achieved by techniques such as 2D gradient gel electrophoresis [12] and nuclear magnetic resonance (NMR) [13]. For example, one study (using 2D gradient gel electrophoresis) showed that very-large, cholesterol-rich α-1 HDLs were better predictors than HDL-C levels of reduced coronary heart disease (CHD) in a subset of males from the Framingham Offspring Study [14]. Another high-profile study, using NMR to assess HDL subspecies in over 2200 participants in the EPIC-Norfolk cohort, showed that higher HDL particle (HDL-P) concentrations were a predictor of reduced CHD, independent of classic CHD risk factors [15]. In more recent work from the Multi-Ethnic Study of Atherosclerosis, total HDL-P (measured by NMR) and HDL-C were evaluated at baseline for 5598 participants, who were then followed prospectively for incident CHD (n=227 events) [16]. Although both HDL-P and HDL-C were highly correlated with each other, in multivariate regression models total HDL-P concentration was the superior predictor of reduced incident CHD when compared to HDL-C. This finding indicates that although HDL-C captures a large portion of HDL-P variation, HDL-P is the better predictor of CHD.
These studies support the notion that measuring individual HDL particle subspecies provides clinically useful information beyond traditionally measured HDL-C. However, both α-1 HDLs (which are cholesterol-rich) and HDL-P measured by NMR (which relies on lipid to generate signal) are highly correlated with HDL-C. Therefore, it is possible that these observations reflect a similar inverse association observed between HDL-C and cardiovascular disease. Importantly, two recent studies (discussed below) indicate that low levels of relatively cholesterol-poor, smaller HDLs also associate with cerebrovascular disease, again suggesting that subspecies of HDL not adequately captured by measuring HDL-C may also play important roles in the pathogenesis of atherosclerotic disease.
Shifting focus: HDL-3 and medium-HDL particles
We investigated the association of the subspecies HDL-2 and HDL-3 (Table 1; Fig. 1) with carotid artery disease (CAAD) [17]. Here, HDL was sub-fractionated by ultracentrifugation and the subspecies were quantified by their cholesterol content. In a case-control cohort of 1,725 participants [part of the Carotid Lesion Epidemiology And Risk (CLEAR) cohort], stepwise linear regression was used to determine whether total HDL-C, HDL-2 cholesterol (HDL-2C), HDL-3 cholesterol (HDL-3C), or apolipoprotein A-I (apoA-I) levels were the best predictor of CAAD. In this study, the smaller HDL-3C fraction was found to be the best predictor of reduced CAAD risk. Moreover, adding HDL-3C to the model improved prediction even when HDL-C levels were also considered, demonstrating added utility of the HDL-3C measure versus HDL-C.
In a separate study using calibrated ion mobility analysis, the particle concentrations of three HDL subspecies (Table 1; Fig. 1) were measured in a subset of the same CLEAR cohort [18]. Participants with severe carotid stenosis (n=40; >80% stenosis by ultrasound in either or both internal carotid artery) had significantly lower plasma concentrations of medium-HDL particles compared with control participants (n=40; <15% stenosis by ultrasound in both carotid arteries). In this population HDL-P was a superior predictor of CAAD compared to HDL-C and this relationship was significant after controlling for HDL-C. The case-control difference in total HDL-P was driven by dramatic changes in medium-HDL particles, the next best predictor of CAAD. This medium-HDL particle inverse association also remained significant after controlling for HDL-C. Considering HDL-3 is composed of small- and medium-HDL particles (Fig. 1) and medium-HDL contributes the majority of HDL-3 cholesterol content, these results are in excellent agreement with the previous study of the CLEAR cohort. Both results support the hypothesis that relatively cholesterol-poor, smaller HDL subspecies, which are under-represented by total HDL-C, are potentially important protective factors for CVD.
Summary and future directions
Considering that increased levels of cholesterol-poor HDL subspecies – reflected by measures of HDL-3, medium size particles, and increased HDL-P – can represent superior predictors of CVD phenotypes, it is possible that pharmacologic attempts to raise HDL-C fail to affect CVD event rates because specifically elevating the cholesterol content of HDL is insufficient. The mechanism of HDL-C elevation should be considered. The agents tested thus far may have increased HDL-C by forming large, cholesterol-rich HDL particles at the expense of medium- and small-HDL particles; having an overall null effect on total particle concentration. Indeed, there is evidence from 2D-gel electrophoresis that very high HDL-C levels observed in CETP deficiency result from a shift from small- and medium-HDLs to large-HDL particles [19]. Thus, HDL directed therapies – especially CETP inhibitors – might increase HDL-C without increasing the number of total HDL-P and possibly reducing the number of potentially beneficial medium-HDL particles. Considering that medium- and total HDL particle concentrations may represent superior predictors of cardioprotection, this hypothesis could explain the failures of the CETP inhibitors and niacin to prevent CVD. We speculate that HDL directed therapies might be more effective in reducing CVD-related events if the number of circulating HDL particles was increased by therapy, especially medium-HDLs.
In light of recent research showing that certain subspecies of HDL (such as medium-HDL and HDL-3) may specifically contribute to cardioprotection, it is our opinion that the focus of research and potential therapies should shift to these promising targets. Of particular interest is the protein cargo of these HDL subspecies, which may reveal important mechanisms related to their cardioprotective properties. For instance, HDL-3 is closely associated with PON1 enzyme activity [20], which is associated with cardioprotection [21, 22]. Notably, the cardioprotective association of HDL-3 was in part independent of both PON1 activity and HDL-C, indicating that there were unmeasured predictive elements of the HDL-3 proteome; these may be apolipoproteins, or ancillary proteins that are specifically associated with HDL-3 [17].
In summary, it is our opinion that the recent failure of increased HDL-C to be cardioprotective likely reflects the fact that increasing HDL-C alone does not adequately increase the concentration or activity of cardioprotective HDL subspecies. It would be an error to say that studies of HDL-C demonstrate that HDL is not cardioprotective. Increased total HDL particle concentration, or perhaps a specific increase in medium-HDL particles, may confer greater protection against CAAD and CHD than pharmaceutically generating a preponderance of large, cholesterol-rich HDL particles. Future research should focus on narrowing down focus through computational, structural and functional studies to identify the specific molecule or molecules that are responsible for the expected cardioprotective effect of HDL.
References
1. Castelli WP. Cardiovascular disease and multifactorial risk: challenge of the 1980s. Am Heart J. 1983; 106: 1191–1200.
2. Gordon DJ, Rifkind BM. High-density lipoprotein–the clinical implications of recent studies. N Engl J Med. 1989; 321: 1311–1316.
3. Rye KA, Bursill CA, Lambert G, Tabet F, Barter PJ. The metabolism and anti-atherogenic properties of HDL. J Lipid Res. 2008; 50: S195–S200.
4. Oram JF, Heinecke JW. ATP-binding cassette transporter A1: a cell cholesterol exporter that protects against cardiovascular disease. Physiol Rev. 2005; 85: 1343–1372.
5. Barter PJ, Barter PJ, Caulfield M, Caulfield M, et al. Effects of torcetrapib in patients at high risk for coronary events. N Engl J Med. 2007; 357: 2109–2122.
6. Schwartz GG, Olsson AG, Abt M, Ballantyne CM, et al. Effects of dalcetrapib in patients with a recent acute coronary syndrome. N Engl J Med. 2012; 367: 2089–2099.
7. AIM-HIGH Investigators, Boden WE, Probstfield JL, Anderson T, et al. Niacin in patients with low HDL cholesterol levels receiving intensive statin therapy. N Engl J Med. 2011; 365: 2255–2267.
8. Voight BF, Peloso GM, Orho-Melander M, Frikke-Schmidt R, et al. Plasma HDL cholesterol and risk of myocardial infarction: a mendelian randomisation study. Lancet. 2012; 380: 572–580.
9. Shen BW, Scanu AM, Kézdy FJ. Structure of human serum lipoproteins inferred from compositional analysis. Proc Natl Acad Sci U S A. 1977; 74: 837–841.
10. Huang R, Silva RAGD, Jerome WG, Kontush A, et al. Apolipoprotein A-I structural organization in high-density lipoproteins isolated from human plasma. Nat Struct Mol Biol. 2011; 18: 416–422.
11. Rosenson RS, Brewer HB, Chapman MJ, Fazio S, et al. HDL measures, particle heterogeneity, proposed nomenclature, and relation to atherosclerotic cardiovascular events. Clin Chem. 2011; 57: 392–410.
12. Asztalos BF, Sloop CH, Wong L, Roheim PS. Two-dimensional electrophoresis of plasma lipoproteins: recognition of new apo A-I-containing subpopulations. Biochim Biophys Acta 1993; 1169: 291–300.
13. Otvos JD. Measurement of lipoprotein subclass profiles by nuclear magnetic resonance spectroscopy. Clin lab. 2002; 48: 171–180.
14. Asztalos BF, Cupples LA, Demissie S, Horvath KV, et al. High-density lipoprotein subpopulation profile and coronary heart disease prevalence in male participants of the Framingham Offspring Study. Arterioscler Thromb Vasc Biol. 2004; 24: 2181–2187.
15. Harchaoui El K, Arsenault BJ, Franssen R, Després J-P, et al. High-density lipoprotein particle size and concentration and coronary risk. Ann Intern Med. 2009; 150: 84–93.
16. Mackey RH, Greenland P, Goff DC, Lloyd-Jones D, et al. High-density lipoprotein cholesterol and particle concentrations, carotid atherosclerosis, and coronary events: MESA (multi-ethnic study of atherosclerosis). J Am Coll Cardiol. 2012; 60: 508–516.
17. Kim DS, Burt AA, Rosenthal EA, Ranchalis JE, et al. HDL-3 is a superior predictor of carotid artery disease in a case-control cohort of 1725 participants. J Am Heart Assoc. 2014; 3: e000902.
18. Hutchins PM, Ronsein GE, Monette JS, Pamir N, et al. Quantification of HDL particle concentration by calibrated ion mobility analysis. Clin Chem. 2014; 60: 1393–1401.
19. Asztalos BF. Apolipoprotein composition of HDL in cholesteryl ester transfer protein deficiency. J Lipid Res. 2003; 45: 448–455.
20. Kontush A, Chantepie S, Chapman MJ. Small, dense HDL particles exert potent protection of atherogenic LDL against oxidative stress. Arterioscler Thromb Vasc Biol. 2003; 23: 1881–1888.
21. Jarvik GP, Rozek LS, Brophy VH, Hatsukami TS, et al. Paraoxonase (PON1) Phenotype Is a Better Predictor of Vascular Disease Than Is PON1192 or PON155 Genotype. Arterioscler Thromb Vasc Biol. 2000; 20: 2441–2447.
22. Kim DS, Marsillach J, Furlong CE, Jarvik GP. Pharmacogenetics of paraoxonase activity: elucidating the role of high-density lipoprotein in disease. Pharmacogenomics 2013; 14: 1495–1515.
The authors
Daniel Seung Kim1–3† BS; Patrick M. Hutchins4† PhD; Gail P. Jarvik1,2 MD, PhD
1Department of Genome Sciences, University of Washington, Seattle, WA, USA
2Division of Medical Genetics, Department of Medicine, University of Washington, Seattle, WA, USA
3Department of Biostatistics, University of Washington, Seattle, WA, USA
4Division of Metabolism, Endocrinology, and Nutrition, Department of Medicine, University of Washington, Seattle, WA, USA
†Authors contributed equally to this work
*Corresponding author
E-mail: pair@u.washington.edu
Acknowledgement
DSK is supported in part by 1F31MH101905-01 and a Markey Foundation Award. PMH is supported by a Cardiovascular Fellowship Training Grant (NIH T32HL007828). Work on the CLEAR study referenced within was supported by National Institutes of Health RO1 HL67406 and a State of Washington Life Sciences Discovery Award (265508) to the Northwest Institute of Genetic Medicine.
Possible polio resurgence and the anti-vax movement
, /in Featured Articles /by 3wmediaLast month was the 100th anniversary of the birth of Jonas Salk who developed the first effective polio vaccine. Prior to its widespread use in the West from the mid 1950s on, seasonal polio outbreaks in North America and Europe killed some children and caused life long paralysis in others. In the 1952 polio epidemic in the United States, 57,628 cases were reported with 3,145 fatalities and 21,269 cases of paralysis. Indeed those of us attending school before the advent of routine polio vaccination saw some of our fellow pupils returning after the summer break in leg braces; sadly occasionally a desk would remain empty. Now the global polio eradication effort has essentially eliminated the disease from all but three countries where it remains endemic, namely Nigeria, Afghanistan and Pakistan. In 2013 there were just 416 cases worldwide; so far this year there have been 306 cases. The aim is to totally eradicate the disease by 2018, but this goal may be thwarted because of the increased international spread of wild polio virus from endemic countries. The situation in Pakistan is causing most concern as the number of cases has more than quadrupled from 53 last year to 260 so far this year, and a major factor has been the ruthless militant violence against Pakistani teams vaccinating children against polio. More than 65 healthcare workers and supporting staff have been killed in the last two years, the latest shot dead in late November.
The anti-vax movement in the West is less immediately perilous, but is unfortunately growing, greatly facilitated by misinformed pressure groups disseminating dangerously misleading information using social media. One reason is that medical success has bred complacency: thanks to effective vaccination programmes polio is no longer endemic, and the former childhood scourges of measles, pertussis, tetanus and diphtheria are currently rare. Parents thus focus on the possible health risks of the vaccines – and most of these perceived risks have no scientific basis – rather than on the morbidity and mortality rate of the diseases themselves and the increasing danger of epidemics in non-immune populations. A common fallacy is that parental decisions have no repercussions for other families. But we know that 95% of children must be vaccinated against a disease to achieve ‘herd immunity’; this allows even hypersensitive children who cannot be vaccinated to be safe. If Western parents can’t be persuaded by means of pertinent information to protect their nation’s children from disease, it is high time for some coercion.
Metabolic syndrome: definition, grading and treatment
, /in Featured Articles /by 3wmediaMetabolic syndrome is characterized by a collection of disorders, making it difficult to diagnose and stage. This article describes the criteria used for diagnosis as well as discussing treatment strategies.
by Prof. Giuseppe Derosa and Dr Pamela Maffioli
Definition and grading
Metabolic syndrome is a combination of medical disorders that increases the risk of developing cardiovascular disease; it affects one in five people in the United States, and prevalence increases with age. There are different definitions of metabolic syndrome; according to the Adult Treatment Panel (ATP) III [1], metabolic syndrome requires the presence of at least three of the listed criteria (Table 1).
Recently insulin resistance has been cited to be associated with other metabolic risk factors and correlates with cardiovascular risk. The pro-inflammatory state has also been developed and used as a marker to predict coronary vascular diseases in metabolic syndrome: it is identified by higher C-reactive protein (CRP) levels, commonly present in people with metabolic syndrome. One cause of elevated CRP is obesity, because adipose tissue releases inflammatory cytokines that may elicit higher CRP levels. Also, the pro-thrombotic state has been recently considered for the definition of metabolic syndrome, characterized by increased plasma plasminogen activator inhibitor-1 (PAI-1) and fibrinogen. However, the ATP III panel did not find adequate evidence to recommend routine measurement of insulin-resistance, pro-inflammatory state (e.g. high-sensitivity C-reactive protein), or pro-thrombotic state (e.g. fibrinogen or PAI-1) in the diagnosis of the metabolic syndrome.
The World Health Organization (WHO) criteria, instead, emphasized insulin resistance as the major underlying risk factor and required evidence of insulin resistance for diagnosis (Table 2) [2, 3].
The International Diabetes Federation (IDF), instead, dropped the WHO requirement for insulin resistance, but made abdominal obesity necessary for the diagnosis, with particular emphasis on waist measurement as a simple screening tool [4]; the other criteria (Table 3) were essentially identical to those provided by ATP III [1].
The American Association of Clinical Endocrinologists (AACE) proposed a third set of clinical criteria for the insulin resistance syndrome [5]. These criteria appear to be a hybrid of those of the ATP III and WHO metabolic syndrome. However, no defined number of risk factors is specified and diagnosis is left to clinical judgment (Table 4).
Given that multiple definitions of the same disease can generate confusion among physicians, the major organizations made an attempt to unify the various criteria for the definition of metabolic syndrome [6]. It was agreed that there should not be an obligatory component, but that waist measurement would continue to be a useful preliminary screening tool. Three abnormal findings out of five would qualify a person for the metabolic syndrome according to the unified definition shown in Table 5.
As readers can easily understand, individuals with metabolic syndrome are at increased risk for coronary heart disease (CHD) [7]. In particular, in the absence of diabetes, the metabolic syndrome generally did not raise the 10-year risk for CHD by more than 20% [8], in particular 10-year risk generally ranged from 10% to 20% for men and did not exceed 10% for women. However, in the presence of diabetes, the risk increases. Obviously, patients fulfilling all or almost all of the metabolic syndrome potential criteria, have earlier and more serious organ damage, at both cardiac and vascular levels, than patients with only three out of five components of the metabolic syndrome definition.
Treatment
Despite the grade of metabolic syndrome, however, there are two general approaches to its treatment. The first strategy modifies root causes, overweight/obesity and physical inactivity, and their closely associated condition, insulin resistance. The second approach directly treats the metabolic risk factors such as atherogenic dyslipidemia, hypertension, the pro-thrombotic state, and underlying insulin resistance. ATP III recommended that obesity be the primary target of intervention for metabolic syndrome [9]. First-line therapy should be weight reduction; the current recommendations for the treatment of overweight and obese people include increased physical activity and reduced calorie intake [10, 11]. Pharmacological treatment with orlistat can be another option, and when it is not tolerated, bariatric surgery should be considered. However, surgery irreversibly changes the overall architecture of the digestive tract; in this regard, the endoscopic duodenal–jejunal bypass liner can be another option. It consists of a sheath that is inserted endoscopically through the mouth into the digestive tract of the obese patient creating a physical barrier between the intestinal wall and the food ingested. The device can be considered as an alternative to bariatric surgery because of the minimal adverse events and the possibility to easily remove the device when the desired weight has been achieved [12]. Weight loss is important because it lowers serum cholesterol and triglycerides, raises HDL-cholesterol, lowers blood pressure and glucose, and reduces insulin resistance. Published data further show that weight reduction can decrease serum levels of CRP and PAI-1 [13–16]. In addition, other lipid and non-lipid risk factors associated with the metabolic syndrome should be appropriately treated. Atherogenic dyslipidemia includes elevated serum triglycerides and apolipoprotein B, increased small LDL particles, and reduced level of HDL-cholesterol. The treatment strategy for atherogenic dyslipidemia in metabolic syndrome focuses on triglycerides. If triglycerides are ≥150 mg/dL and HDL-cholesterol is <40 mg/dL, a diagnosis of atherogenic dyslipidemia is made. If triglycerides are <200 mg/dL, and specific drug therapy to reduce triglyceride-rich lipoproteins is not indicated. However, if the patient has CHD or CHD risk equivalents, LDL-cholesterol goal has to be considered together with the use of a drug to raise HDL-cholesterol (fibrate). On the other hand, if triglycerides are 200–499 mg/dL, non-HDL cholesterol becomes a secondary target of therapy. Goals for non-HDL cholesterol are 30 mg/dL higher than those for LDL-cholesterol. First the LDL-cholesterol goal is attained, and if non-HDL remains elevated, additional therapy may be required to achieve the non-HDL goal. Alternative approaches for treatment of elevated non-HDL cholesterol that persists after the LDL goal has been achieved are (a) higher doses of statins, or (b) moderate doses of statins + triglyceride-lowering drug (fibrate). If triglycerides are very high (≥500 mg/dL), attention turns first to prevention of acute pancreatitis, which is more likely to occur when triglycerides are >1000 mg/dL. Triglyceride-lowering drugs (fibrate) become the first line therapy; although statins can be used to lower LDL-cholesterol to reach the LDL-cholesterol goal, in these patients it is often difficult (and unnecessary) to achieve a non-HDL cholesterol goal of only 30 mg/dL higher than for LDL-cholesterol [9].
Conclusion
In conclusion, metabolic syndrome increases cardiovascular risk; a multifactorial approach is necessary in order to prevent the development of the various components of this disease.
References
1. National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). Third report of the National Cholesterol Education Program (NCEP) expert panel on detection, evaluation, and treatment of high blood cholesterol in adults (Adult Treatment Panel III) final report. Circulation 2002; 106: 3143–3421.
2. Alberti KG, Zimmet PZ. Definition, diagnosis and classification of diabetes mellitus and its complications. Part 1: diagnosis and classification of diabetes mellitus: provisional report of a WHO consultation. Diabet Med. 1998; 15: 539–553.
3. World Health Organization. Definition, diagnosis and classification of diabetes mellitus and its complications: report of a WHO Consultation. Part 1: diagnosis and classification of diabetes mellitus. Geneva, Switzerland: World Health Organization; 1999. Available at: http:// whqlibdoc.who.int/hq/1999/WHO_NCD_NCS_99.2.pdf.
4. Alberti KG, Zimmet P, Shaw J; IDF Epidemiology Task Force Consensus Group. The metabolic syndrome: a new worldwide definition. Lancet 2005; 366: 1059–1062.
5. Einhorn D, Reaven GM, Cobin RH, Ford E, Ganda OP, Handelsman Y, Hellman R, Jellinger PS, Kendall D, Krauss RM, Neufeld ND, Petak SM, Rodbard HW, Seibel JA, Smith DA, Wilson PW. American College of Endocrinology position statement on the insulin resistance syndrome. Endocr Pract. 2003; 9: 237–252.
6. Alberti KG, Eckel RH, Grundy SM, Zimmet PZ, Cleeman JI, Donato KA, Fruchart JC, James WP, Loria CM, Smith SC Jr; International Diabetes Federation Task Force on Epidemiology and Prevention; Hational Heart, Lung, and Blood Institute; American Heart Association; World Heart Federation; International Atherosclerosis Society; International Association for the Study of Obesity. Harmonizing the metabolic syndrome: a joint interim statement of the International Diabetes Federation Task Force on Epidemiology and Prevention; National Heart, Lung, and Blood Institute; American Heart Association; World Heart Federation; International Atherosclerosis Society; and International Association for the Study of Obesity. Circulation 2009; 120 (16): 1640–1645.
7. Lakka HM, Laaksonen DE, Lakka TA, et al. The metabolic syndrome and total and cardiovascular disease mortality in middle-aged men. JAMA 2002; 288: 2709–2716.
8. Wilson PW, D’Agostino RB, Levy D, Belanger AM, Silbrshatz H, Kannel WB. Prediction of coronary heart disease using risk factor categories. Circulation 1998; 97: 1837–1847.
9. National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III) final report. Circulation. 2002; 106 (25): 3143–3421.
10. American Diabetes Association. Nutrition principles and recommendations in diabetes. Diabetes Care 2004; 27(S1): 36–46.
11. American Diabetes Association. Physical activity/exercise and diabetes. Diabetes Care 2004; 27(S1): 58–62.
12. Derosa G, Maffioli P. Possible therapies for obesity: focus on the available options for its treatment. Nutrition 2014; doi: 10.1016/j.nut.2014.09.005.
13. Dengel DR, Galecki AT, Hagberg JM, Pratley RE. The independent and combined effects of weight loss and aerobic exercise on blood pressure and oral glucose tolerance in older men. Am J Hypertens. 1998; 11: 1405–1412.
14. Ahmad F, Considine RV, Bauer TL, Ohannesian JP, Marco CC, Goldstein BJ. Improved sensitivity to insulin in obese subjects following weight loss is accompanied by reduced protein-tyrosine phosphatases in adipose tissue. Metabolism 1997; 46: 1140–1145.
15. Su HY, Sheu WH, Chin HM, Jeng CY, Chen YD, Reaven GM. Effect of weight loss on blood pressure and insulin resistance in normotensive and hypertensive obese individuals. Am J Hypertens. 1995; 8: 1067–1071.
16. Derosa G, Limas CP, Macías PC, Estrella A, Maffioli P. Dietary and nutraceutical approach to type 2 diabetes. Arch Med Sci. 2014; 10(2): 336–344.
The authors
Giuseppe Derosa1,2 MD, PhD, Pamela Maffioli1,3 MD
1Department of Internal Medicine and Therapeutics, University of Pavia, Fondazione IRCCS Policlinico S. Matteo, PAVIA, Italy.
2Center for the Study of Endocrine-Metabolic Pathophysiology and Clinical Research, University of Pavia, PAVIA, Italy.
3PhD School in Experimental Medicine, University of Pavia, PAVIA, Italy
E-mail: giuseppe.derosa@unipv.it
Cardiac biomarkers – new weapons against cardiovascular disease
, /in Featured Articles /by 3wmediaAlthough formally defined as recently as the early 2000s, biomarkers have quickly begun to gain acceptance in clinical practice. Many experts believe they will become an indispensable tool for the diagnosis and management of a wide variety of medical conditions in the near future.
Cardiovascular disease now a global priority
One of the priority applications for biomarkers is likely to be for cardiovascular diseases (CVD) – the leading cause of mortality and disability in the Western world. In Europe, CVD causes 1.9 million deaths a year, while the toll in the US is about 1 million.
The prevalence of CVD is also increasing rapidly in newly industrializing countries, especially among the more affluent urban populations adopting Western lifestyles. Indeed, “CVD is now more numerous in India and China than in all economically developed countries in the world added together.”
Mapping the disease progression pathway
It has, for some time, been accepted that CVD follows a relatively clear-cut pathway from subclinical to overt status. The Multi-Ethnic Study of Atherosclerosis (MESA), sponsored in the year 2000 by the US National Institutes of Health, has been seeking to assess the characteristics of subclinical CVD and means to predict its progression to clinically overt cardiovascular disease. More recently, in 2010, Spain’s Banco Santander and the Istituto de Salud Carlos III launched a similar effort in Europe called PESA (Progression of Early Subclinical Atherosclerosis).
Such efforts are targeted at providing clinicians with tools to help assess CVD and identify vulnerable, at-risk patients. In many respects, they complement the world’s most ambitious effort in the area, the Framingham Heart Study, which began in 1948 in a town in Massachusetts in the US with 5,209 adult subjects. The Study, which has now enrolled its third generation of participants, has resulted in the publication of over 1,000 medical papers. It has also provided many commonplace tools for the contemporary understanding of CVD, including the impact of smoking, diet and exercise, medications such as aspirin etc. – as well as the term ‘risk factor’.
The Framingham project: clarifying the role of biomarkers
Biomarkers began to be part of the Framingham project in the 2000s, although initial results were unclear. For instance, enthusiasm about elevated levels of the inflammation marker C-reactive protein (CRP) as an independent risk factor for future CVD events were dispelled in a 2005 study supported by the Framingham sponsors.
In September 2012, a study in the American Heart Association’s journal ‘Circulation’ pointed to one reason for such conflicting assessments, namely the “lack of cardiovascular specificity” in many of the new biomarkers. The authors sought to address such limitations by studying three key CVD biomarkers (soluble ST2, growth differentiation factor-15 and high-sensitivity troponin I) in almost 3,500 patients. The findings were conclusive: “Multiple biomarkers of cardiovascular stress,” they said “add prognostic value to standard risk factors for predicting death, overall cardiovascular events, and heart failure.”
In 2014, another study of 2,680 Framingham participants sought to associate circulating biomarkers with The American Heart Association Cardiovascular Health score (CVH score). The authors concluded there was an “inverse association” between ideal CVH and CVD incidence, and that this was partly attributable to its “favourable impact on CVD biomarker levels and subclinical disease.” The list of CVD biomarkers in the 2014 study includes natriuretic peptides (N-terminal pro-atrial and B-type natriuretic peptide), plasminogen activator inhibitor-1, aldosterone, C-reactive protein, D-dimer, fibrinogen, homocysteine and growth differentiation factor-15.
Identification of at-risk patients
One of the most promising biomarkers seems to be cardiac troponin, first identified in the early 1990s. Changes in cardiac troponin T (cTnT) levels over time appear to correlate with heart failure risk, especially in a major study of elderly subjects.
The potential of circulating cTnT may also extend beyond the heart failure setting. Some argue that circulating cTnT is representative of subclinical myocardial dysfunction. In the general population, studies show that elevated cTnT is associated with subclinical cardiac injury, and marks an increased risk for structural heart disease and all-cause mortality.
Other studies have found that myeloperoxidase (MPO) and high-sensitivity C-reactive protein (hsCRP) in apparently healthy populations can predict risk of coronary disease, allowing for early preventative treatment. Together, MPO and C-reactive protein have also shown promise in prognostic risk assessment for patients with systolic heart failure.
Enabling targeted and timely treatment
While screening the general population is bound to draw considerable attention, the more immediate application of CVD biomarkers is to enable treatment in a risk-stratified and timely fashion.
One of the biggest challenges faced by physicians is to differentiate between patients with unstable angina and acute myocardial infarction (AMI) in an emergency setting. Here too cTnT – as well as cardiac troponin I (cTnI) – have catalysed some of the greatest excitement, due to their high sensitivity and specificity for cardiac damage.
In 2007, the US National Academy of Clinical Biochemistry Laboratory Medicine Practice recommended the use of cardiac troponin as a ‘preferred’ biomarker for MI diagnosis, in conjunction with clinical evidence of myocardial ischemia. Creatine kinase-MB was positioned as an ‘acceptable alternative’. These recommendations were endorsed by the joint European Society of Cardiology/American College of Cardiology/American Heart Association/World Heart Federation task force for the definition of myocardial infarction.
Cardiac troponin ‘the best single marker’
Levels of cardiac troponin are dependent on infarct size, and directly indicate the prognosis following MI. Indeed, in recent years, some experts suggest that CTnI and CTnT have “displaced myoglobin and creatine kinase-MB as the preferred markers of myocardial injury.”
In 2013, a Health Technology Asssessment (HTA) by Britain’s National Institute for Health Research (NIHR) concurred with this view, observing that “high-sensitivity cardiac troponin is the best single marker in patients presenting with chest pain.” Additional measurements of myoglobin or creatine kinase-MB, it noted were “not clinically effective or cost-effective.”
Debate on troponin not over
Nevertheless, considerable debate remains about the utility of troponin in real world CVD management. Although patients with undetectable troponins are considered to have excellent short-term prognosis, levels may be undetectable “for six hours after the onset of myocardial cell injury,” making myoglobin “a preferred early marker” for MI. This limitation, which seems to go against the 2013 NIHR Health Technology Assessment, is also acknowledged by some proponents of troponin, who admit that although it “may be useful for risk assessment and management” in asymptomatic populations, there is no evidence that it confers “an advantage in the context of MI diagnosis.” In addition, they also note that “cTnI assays are not standardized; thus, there can be a substantial difference in values depending on the assay used.”
Defining assay sensitivity, differentiating troponin I and T
One challenge lies in the definition of a ‘high sensitivity’ assay, which can measure cTn in the single digit range of nanograms per litre. The term is used by vendors “for marketing purposes,” and there “is still no consensus” regarding its application. Making matters tougher is the fact that most manufacturers’ claims for assay precision “cannot be achieved in clinical laboratories.”
In effect, the jury on troponin is likely to be out for some time to come, accompanied by continuing uncertainties.
For instance, Britain’s respected health advisory site patient.co.uk suggests that troponin I and T “are of equal clinical value” while a 2010 guideline from NICE (National Institute for Healthcare and Clinical Excellence) advises taking a blood sample for troponin I or T as “preferred biochemical markers to diagnose acute MI.”
However, a very recent study published by the Journal of the American College of Cardiology finds that patients with neuromuscular disease can show elevated levels of troponin T but not I, thus questioning the guidelines which regard both as being “equally sensitive and specific for the diagnosis of myocardial injury.”
These may be some of the reasons why the US Food and Drug Administration (FDA) decided in June 2014 to discuss clarification of claims and protocols with vendors of troponin assays, in order to “modernize the performance evaluation and regulatory review.” In Britain, NICE is currently updating its 2010 guideline.
The role of B-type natriuretic peptide
Once acute MI is confirmed, a variety of other biomarkers are used to help make assessments.
One of the most promising of these is B-type natriuretic peptide or BNP, designated by the FDA in the year 2000 as a Class II diagnostic device.
Nevertheless, it is important to underline that only troponin has been used to direct therapeutic intervention. Though it is evident that the adoption of proven new biomarkers will increase prognostic accuracy, they have yet to be tested to alter outcomes of therapeutic intervention.
Thus, in spite of statements from reputable sources claiming that BNP is “already used to diagnose heart failure,” the truth is somewhat different, with the difference in the details. At the end of 2013, the US Agency for Healthcare Research and Quality (AHRQ), investigated BNP and the related N-terminal proBNP (NT-proBNP) for detecting heart failure (HF). The findings were guarded: “BNP and NT-proBNP had good diagnostic performance for ruling out HF but were less accurate for ruling in HF.” In addition, it found that the “therapeutic value was inconclusive.”
Other biomarkers remain valuable
In the meanwhile, clinicians in emergency settings have recourse to a variety of other established CVD risk markers, such as cholesterol. “Research is also under way on markers with strong predictive value that are not used in the clinic for cardiovascular disease risk prediction, such as fibrinogen, vitamin D, and cystatin C.” Some of these “are of special interest as these may prove to be valuable biomarkers in the future.”
To have clinical utility, however, such biomarkers will need to provide risk assessments independently of other established markers. They also require the presence of standardized assays which are specific and sensitive for the markers, with easy-to-interpret results.
In effect, biomarker-mediated approaches to CVD need to yield superior patient outcomes compared to current standard-of-care management schemes.
The HDL particle: frontiers for new discovery in cardioprotection
, /in Featured Articles /by 3wmediaRecent findings indicate that aspects of high-density lipoprotein (HDL) not captured by traditionally measured HDL–cholesterol levels (HDL‑C) are likely to be cardioprotective. This review will highlight some of these studies and suggest new directions to identify the specific molecules that are responsible for the cardioprotective nature of HDL.
by Daniel S. Kim, Dr Patrick M. Hutchins and Prof. Gail P. Jarvik
Raising HDL-C does not confer cardioprotection
There is a well-established inverse association between high-density lipoprotein–cholesterol (HDL-C) levels and cardiovascular disease (CVD) in epidemiological and clinical studies [1, 2]. This robust relationship suggested that HDL-C was in the causal pathway of atheroprotection. Indeed a large number of studies have demonstrated that HDL possesses various anti-atherogenic properties, primarily the ability to accept cholesterol from macrophages in a process termed reverse cholesterol transport [3, 4].
In contrast, several high-profile studies have demonstrated that increasing levels of HDL-C does not have a significant cardioprotective effect. In a large and well-conducted clinical trial of the cholesterol ester transport protein (CETP) inhibitor, torcetrapib, there was no reduction in the incidence of CVD-related events despite significantly higher HDL-C levels [5]. A follow-up study using a different CETP inhibitor, dalcetrapib, also showed increased HDL-C levels yet there was no significant difference in CVD event rate between the treatment and placebo groups [6]. In a third randomized clinical trial that used niacin to increase HDL-C levels, there was again no reduction in cardiovascular events [7]. Finally, a large-scale Mendelian randomization study of approximately 20 000 myocardial infarction (MI) cases and 100 000 controls, showed that a genetic polymorphism which associated with approximately 10% higher HDL-C levels was not associated with decreased incidence of MI [8], again suggesting that the relationship between HDL-C and the prevention of cardiac events is not causal.
HDL particle concentration is a superior predictor of CVD
As the elevation of HDL-C was not beneficial in these studies, some have speculated that HDL itself is not cardioprotective. An alternative explanation for these negative data is that the cholesterol content of HDL – a surrogate measure of HDL – does not best reflect the anti-atherogenic properties of HDL. To resolve these issues it is critical to identify new HDL metrics that reliably reflect its cardioprotective functions.
One promising approach for assessing the role of HDL in CVD is to evaluate the individual HDL particles. HDL is a heterogeneous mixture of lipoprotein particles composed of discrete subspecies that have unique structural compositions and biological functions. As different HDL particles carry vastly different amounts of cholesterol – ranging over an order-of-magnitude [9, 10] – measuring the total HDL-C does not provide information regarding the distribution of HDL subpopulations or the number of total HDL particles.
HDL can be fractionated based on a number of physicochemical properties, most commonly size or density. Several techniques, both qualitative and quantitative, have been developed for HDL subspecies analysis. The various HDL subspecies reported by these techniques and their associated nomenclature are briefly summarized in Table 1 [see also ref. 11]. Furthermore, HDL subspecies determined by ultracentrifugation and calibrated ion mobility analysis (both are discussed in detail later) are shown in Figure 1. Many studies have demonstrated the potential clinical utility of HDL subspecies analysis, which can be achieved by techniques such as 2D gradient gel electrophoresis [12] and nuclear magnetic resonance (NMR) [13]. For example, one study (using 2D gradient gel electrophoresis) showed that very-large, cholesterol-rich α-1 HDLs were better predictors than HDL-C levels of reduced coronary heart disease (CHD) in a subset of males from the Framingham Offspring Study [14]. Another high-profile study, using NMR to assess HDL subspecies in over 2200 participants in the EPIC-Norfolk cohort, showed that higher HDL particle (HDL-P) concentrations were a predictor of reduced CHD, independent of classic CHD risk factors [15]. In more recent work from the Multi-Ethnic Study of Atherosclerosis, total HDL-P (measured by NMR) and HDL-C were evaluated at baseline for 5598 participants, who were then followed prospectively for incident CHD (n=227 events) [16]. Although both HDL-P and HDL-C were highly correlated with each other, in multivariate regression models total HDL-P concentration was the superior predictor of reduced incident CHD when compared to HDL-C. This finding indicates that although HDL-C captures a large portion of HDL-P variation, HDL-P is the better predictor of CHD.
These studies support the notion that measuring individual HDL particle subspecies provides clinically useful information beyond traditionally measured HDL-C. However, both α-1 HDLs (which are cholesterol-rich) and HDL-P measured by NMR (which relies on lipid to generate signal) are highly correlated with HDL-C. Therefore, it is possible that these observations reflect a similar inverse association observed between HDL-C and cardiovascular disease. Importantly, two recent studies (discussed below) indicate that low levels of relatively cholesterol-poor, smaller HDLs also associate with cerebrovascular disease, again suggesting that subspecies of HDL not adequately captured by measuring HDL-C may also play important roles in the pathogenesis of atherosclerotic disease.
Shifting focus: HDL-3 and medium-HDL particles
We investigated the association of the subspecies HDL-2 and HDL-3 (Table 1; Fig. 1) with carotid artery disease (CAAD) [17]. Here, HDL was sub-fractionated by ultracentrifugation and the subspecies were quantified by their cholesterol content. In a case-control cohort of 1,725 participants [part of the Carotid Lesion Epidemiology And Risk (CLEAR) cohort], stepwise linear regression was used to determine whether total HDL-C, HDL-2 cholesterol (HDL-2C), HDL-3 cholesterol (HDL-3C), or apolipoprotein A-I (apoA-I) levels were the best predictor of CAAD. In this study, the smaller HDL-3C fraction was found to be the best predictor of reduced CAAD risk. Moreover, adding HDL-3C to the model improved prediction even when HDL-C levels were also considered, demonstrating added utility of the HDL-3C measure versus HDL-C.
In a separate study using calibrated ion mobility analysis, the particle concentrations of three HDL subspecies (Table 1; Fig. 1) were measured in a subset of the same CLEAR cohort [18]. Participants with severe carotid stenosis (n=40; >80% stenosis by ultrasound in either or both internal carotid artery) had significantly lower plasma concentrations of medium-HDL particles compared with control participants (n=40; <15% stenosis by ultrasound in both carotid arteries). In this population HDL-P was a superior predictor of CAAD compared to HDL-C and this relationship was significant after controlling for HDL-C. The case-control difference in total HDL-P was driven by dramatic changes in medium-HDL particles, the next best predictor of CAAD. This medium-HDL particle inverse association also remained significant after controlling for HDL-C. Considering HDL-3 is composed of small- and medium-HDL particles (Fig. 1) and medium-HDL contributes the majority of HDL-3 cholesterol content, these results are in excellent agreement with the previous study of the CLEAR cohort. Both results support the hypothesis that relatively cholesterol-poor, smaller HDL subspecies, which are under-represented by total HDL-C, are potentially important protective factors for CVD.
Summary and future directions
Considering that increased levels of cholesterol-poor HDL subspecies – reflected by measures of HDL-3, medium size particles, and increased HDL-P – can represent superior predictors of CVD phenotypes, it is possible that pharmacologic attempts to raise HDL-C fail to affect CVD event rates because specifically elevating the cholesterol content of HDL is insufficient. The mechanism of HDL-C elevation should be considered. The agents tested thus far may have increased HDL-C by forming large, cholesterol-rich HDL particles at the expense of medium- and small-HDL particles; having an overall null effect on total particle concentration. Indeed, there is evidence from 2D-gel electrophoresis that very high HDL-C levels observed in CETP deficiency result from a shift from small- and medium-HDLs to large-HDL particles [19]. Thus, HDL directed therapies – especially CETP inhibitors – might increase HDL-C without increasing the number of total HDL-P and possibly reducing the number of potentially beneficial medium-HDL particles. Considering that medium- and total HDL particle concentrations may represent superior predictors of cardioprotection, this hypothesis could explain the failures of the CETP inhibitors and niacin to prevent CVD. We speculate that HDL directed therapies might be more effective in reducing CVD-related events if the number of circulating HDL particles was increased by therapy, especially medium-HDLs.
In light of recent research showing that certain subspecies of HDL (such as medium-HDL and HDL-3) may specifically contribute to cardioprotection, it is our opinion that the focus of research and potential therapies should shift to these promising targets. Of particular interest is the protein cargo of these HDL subspecies, which may reveal important mechanisms related to their cardioprotective properties. For instance, HDL-3 is closely associated with PON1 enzyme activity [20], which is associated with cardioprotection [21, 22]. Notably, the cardioprotective association of HDL-3 was in part independent of both PON1 activity and HDL-C, indicating that there were unmeasured predictive elements of the HDL-3 proteome; these may be apolipoproteins, or ancillary proteins that are specifically associated with HDL-3 [17].
In summary, it is our opinion that the recent failure of increased HDL-C to be cardioprotective likely reflects the fact that increasing HDL-C alone does not adequately increase the concentration or activity of cardioprotective HDL subspecies. It would be an error to say that studies of HDL-C demonstrate that HDL is not cardioprotective. Increased total HDL particle concentration, or perhaps a specific increase in medium-HDL particles, may confer greater protection against CAAD and CHD than pharmaceutically generating a preponderance of large, cholesterol-rich HDL particles. Future research should focus on narrowing down focus through computational, structural and functional studies to identify the specific molecule or molecules that are responsible for the expected cardioprotective effect of HDL.
References
1. Castelli WP. Cardiovascular disease and multifactorial risk: challenge of the 1980s. Am Heart J. 1983; 106: 1191–1200.
2. Gordon DJ, Rifkind BM. High-density lipoprotein–the clinical implications of recent studies. N Engl J Med. 1989; 321: 1311–1316.
3. Rye KA, Bursill CA, Lambert G, Tabet F, Barter PJ. The metabolism and anti-atherogenic properties of HDL. J Lipid Res. 2008; 50: S195–S200.
4. Oram JF, Heinecke JW. ATP-binding cassette transporter A1: a cell cholesterol exporter that protects against cardiovascular disease. Physiol Rev. 2005; 85: 1343–1372.
5. Barter PJ, Barter PJ, Caulfield M, Caulfield M, et al. Effects of torcetrapib in patients at high risk for coronary events. N Engl J Med. 2007; 357: 2109–2122.
6. Schwartz GG, Olsson AG, Abt M, Ballantyne CM, et al. Effects of dalcetrapib in patients with a recent acute coronary syndrome. N Engl J Med. 2012; 367: 2089–2099.
7. AIM-HIGH Investigators, Boden WE, Probstfield JL, Anderson T, et al. Niacin in patients with low HDL cholesterol levels receiving intensive statin therapy. N Engl J Med. 2011; 365: 2255–2267.
8. Voight BF, Peloso GM, Orho-Melander M, Frikke-Schmidt R, et al. Plasma HDL cholesterol and risk of myocardial infarction: a mendelian randomisation study. Lancet. 2012; 380: 572–580.
9. Shen BW, Scanu AM, Kézdy FJ. Structure of human serum lipoproteins inferred from compositional analysis. Proc Natl Acad Sci U S A. 1977; 74: 837–841.
10. Huang R, Silva RAGD, Jerome WG, Kontush A, et al. Apolipoprotein A-I structural organization in high-density lipoproteins isolated from human plasma. Nat Struct Mol Biol. 2011; 18: 416–422.
11. Rosenson RS, Brewer HB, Chapman MJ, Fazio S, et al. HDL measures, particle heterogeneity, proposed nomenclature, and relation to atherosclerotic cardiovascular events. Clin Chem. 2011; 57: 392–410.
12. Asztalos BF, Sloop CH, Wong L, Roheim PS. Two-dimensional electrophoresis of plasma lipoproteins: recognition of new apo A-I-containing subpopulations. Biochim Biophys Acta 1993; 1169: 291–300.
13. Otvos JD. Measurement of lipoprotein subclass profiles by nuclear magnetic resonance spectroscopy. Clin lab. 2002; 48: 171–180.
14. Asztalos BF, Cupples LA, Demissie S, Horvath KV, et al. High-density lipoprotein subpopulation profile and coronary heart disease prevalence in male participants of the Framingham Offspring Study. Arterioscler Thromb Vasc Biol. 2004; 24: 2181–2187.
15. Harchaoui El K, Arsenault BJ, Franssen R, Després J-P, et al. High-density lipoprotein particle size and concentration and coronary risk. Ann Intern Med. 2009; 150: 84–93.
16. Mackey RH, Greenland P, Goff DC, Lloyd-Jones D, et al. High-density lipoprotein cholesterol and particle concentrations, carotid atherosclerosis, and coronary events: MESA (multi-ethnic study of atherosclerosis). J Am Coll Cardiol. 2012; 60: 508–516.
17. Kim DS, Burt AA, Rosenthal EA, Ranchalis JE, et al. HDL-3 is a superior predictor of carotid artery disease in a case-control cohort of 1725 participants. J Am Heart Assoc. 2014; 3: e000902.
18. Hutchins PM, Ronsein GE, Monette JS, Pamir N, et al. Quantification of HDL particle concentration by calibrated ion mobility analysis. Clin Chem. 2014; 60: 1393–1401.
19. Asztalos BF. Apolipoprotein composition of HDL in cholesteryl ester transfer protein deficiency. J Lipid Res. 2003; 45: 448–455.
20. Kontush A, Chantepie S, Chapman MJ. Small, dense HDL particles exert potent protection of atherogenic LDL against oxidative stress. Arterioscler Thromb Vasc Biol. 2003; 23: 1881–1888.
21. Jarvik GP, Rozek LS, Brophy VH, Hatsukami TS, et al. Paraoxonase (PON1) Phenotype Is a Better Predictor of Vascular Disease Than Is PON1192 or PON155 Genotype. Arterioscler Thromb Vasc Biol. 2000; 20: 2441–2447.
22. Kim DS, Marsillach J, Furlong CE, Jarvik GP. Pharmacogenetics of paraoxonase activity: elucidating the role of high-density lipoprotein in disease. Pharmacogenomics 2013; 14: 1495–1515.
The authors
Daniel Seung Kim1–3† BS; Patrick M. Hutchins4† PhD; Gail P. Jarvik1,2 MD, PhD
1Department of Genome Sciences, University of Washington, Seattle, WA, USA
2Division of Medical Genetics, Department of Medicine, University of Washington, Seattle, WA, USA
3Department of Biostatistics, University of Washington, Seattle, WA, USA
4Division of Metabolism, Endocrinology, and Nutrition, Department of Medicine, University of Washington, Seattle, WA, USA
†Authors contributed equally to this work
*Corresponding author
E-mail: pair@u.washington.edu
Acknowledgement
DSK is supported in part by 1F31MH101905-01 and a Markey Foundation Award. PMH is supported by a Cardiovascular Fellowship Training Grant (NIH T32HL007828). Work on the CLEAR study referenced within was supported by National Institutes of Health RO1 HL67406 and a State of Washington Life Sciences Discovery Award (265508) to the Northwest Institute of Genetic Medicine.
Latest studies confirm TnI assay performance in early rule-out protocols for AMI
, /in Featured Articles /by 3wmediaThe burden of cardiovascular disease continues to take its toll in terms of diminished quality of life, reduced life expectancy, and the direct and indirect medical costs of treating and caring for patients. This is in spite of downward trends in mortality in the US and Western Europe, especially in the two decades leading up to the year 2000.
by Bernard Cook, PhD
This decline could, in part, be attributed to the success of patient educational campaigns to reduce smoking, lower levels of cholesterol and blood pressure, and encourage lifestyle changes in exercise and diet. Alongside the wider use of effective medications such as cholesterol-lowering statins, these steps are said to have contributed to an observed decline in coronary heart disease (CHD) deaths [1].
However, the rate of improvement is being affected by other, conflicting trends, such as increasing extreme obesity, sedentary lifestyles, the prevalence of hypertension and the rise in type 2 diabetes mellitus even in children.
The World Health Organization first defined myocardial infarction (MI) in 1979 based on clinical presentation, ECG results and elevated blood enzymes such as total CK and the CK-MB isoform. However, there were no standardized definitions, with clinicians defining MI differently, even within the same hospital. The arrival of cardiac troponin assays in the 1990s opened the door for a much needed reappraisal. It was discovered that a greater sensitivity and specificity for MI diagnosis could be achieved by using troponin (a more cardiac-specific marker than CK-MB) and lower cut-offs, presenting the possibility of using the rise and fall of troponin levels to establish ‘early rule-out protocols’.
Highly sensitive troponin is biomarker of choice
Performance expectations have risen as the sensitivity of troponin assays has improved. Nowadays, the modern, highly sensitive troponin assay has become the gold standard biomarker for the early diagnosis of an acute MI, with standards benchmarked by recent international guidelines.
The first steps towards international conformity were taken in 2000 when the European Society of Cardiology (ESC) and the American College of Cardiology (ACC) collaborated on a redefinition of MI. For the first time this gave a central role to the use of biomarkers such as troponin [2]. By 2007, this collaboration had expanded to include the American Heart Association (AHA) and the World Heart Federation (WHF) and saw the publication of the first universal definition of MI. This further established troponin as the preferred biomarker when diagnosing a heart attack [3].
Five years later, in 2012, the Third Universal Definition of Myocardial Infarction [4] laid down the criteria for the contemporary use of troponin assays by today’s clinicians to reduce the time it takes to rule out acute myocardial infarction (AMI). Crucially, this defined an increased cTn concentration as a value exceeding the 99th percentile of a normal reference population apparently free from heart disease [5, 6].
Establishing upper reference limits
The current definition states that the 99th percentile limit should be determined using a healthy population [5, 7]. It is acceptable to confirm this cut-off from information published in peer-reviewed literature or in the assay manufacturer’s own product literature. Further, troponin assays should demonstrate optimal precision with a coefficient of variation of 10% or less at the 99th percentile value and high sensitivity assays should be able to detect troponin in at least 50% of the reference population [7, 8, 9]. Troponin assays with an imprecision greater than 20% CV at the 99th percentile do not fit the criteria for contemporary use.
In today’s current clinical practice, when patients present to an emergency department with chest pain and acute coronary syndrome is suspected, the requirement for the highly sensitive troponin (Tn) assays, such as Beckman Coulter’s AccuTnI+3 assay, is to rule out non-ST-segment-elevation myocardial infarction (NSTEMI) as quickly as possible. Further, distinguishing acute from chronic c-Tn elevations requires serial measurements to detect significant changes [5].
There are conflicting positions about how to best establish a 99th percentile upper reference limit (URL) for troponin. The first maintains that the URL study should include younger subjects, and should not include subjects with potential cardiovascular disease or cardiac risk factors. The second contends the study should include subjects from a population that represents the intended use for troponin: patients whose demographics reflect those of subjects presenting to the emergency department, including older individuals without known cardiovascular disease. Additionally, other methods for selecting a cardiac-healthy population have been employed, such as samples collected from apparently healthy blood donors.
In a study by Moretti et al to establish that the AccuTnI +3 assay demonstrates a coefficient of variation of 10%, the authors found that 62% of the apparently 330 healthy group of blood donors used in the study had measurable values of troponin between the Limit of Detection (LoD) and 99th percentile values [10]. There were no significant differences related to gender and no correlation between cTnI and age.
Predictive accuracy at early observation times
Storrow et al conducted a multicentre, prospective study, involving more than 1,900 patients, to investigate and compare clinical performance at pre-defined serial sampling intervals: on admission/at 1-3 hours/3-6 hours and 6-9 hours. Patients selected from 14 centres were those presenting with chest pain or equivalent ischemic symptoms suggestive of acute coronary syndromes [11]. Results from this study reinforced current clinical practice that troponin testing provides a high degree of accuracy at early observation times, on admission and three hours later, only needing to be repeated after six hours when clinical suspicion remains high.
The findings, published late 2014 in Clinical Biochemistry, compared emergency department TnI serial sampling intervals to determine optimal diagnostic thresholds, and reported on representative diagnostic performance characteristics for early rule-in and rule-out of MI [11]. Diagnosis was adjudicated by an independent central committee of cardiologists. Study samples were tested using Beckman Coulter’s AccuTnI+3 assay at four independent testing facilities.
Specific results from Storrow showed that TnI ≥0.03 ng/mL provided 96.0% sensitivity and 89.4% specificity at 1-3 hours after admission, and 94.9% sensitivity and 86.7% specificity at 3–6 hours. When troponin levels were <0.03 ng/mL, being able to give a negative predictive value (NPV) depended on knowing the time symptoms started. If it was determined that symptoms started approximately eight hours before admission or examination, the NPV was 99.1%. Testing at 1-3 hours gave a NPV of 99.5%; and 99.0% at 3-6 hours when TnI is >0.03 ng/mL. However, Storrow noted that 50–58% of patients with troponin levels of ≥0.03 ng/mL were diagnosed with MI, depending on the time symptoms started or admission.
Positive predictive values emphasize the importance of taking serial samples and observing rising or falling patterns of the delta TnI when low cut-offs are used. Storrow noted that even a single elevated TnI value increased the risk of MI. As TnI values rose, the probability of MI increased, with values ≥0.20 ng/mL associated with an almost 90% probability [11].
Importance of establishing absolute delta values
A change in serial troponin values (delta) can be reported as a percentage or absolute concentrations between the repeat measurements. However, serial measurements must be calculated with values from the same cTn assay [12]. The larger the value set for the delta, the higher the specificity (and the lower the sensitivity) for acute cardiac injury including AMI [13], and the smaller the value set, the higher the sensitivity.
In a separate report using the AccuTnI+3 assay, Storrow confirmed that absolute delta performed significantly better than relative delta at each time interval; for example, at 1-3 hours (AUC were 0.84 vs 0.69), 3-6 hours (0.85 vs 0.73), and 6-9 hours (0.91 vs 0.79) [14]. Current recommendations propose ≥20% delta within 3-6 hours; however, in this study, results were optimized using an absolute delta of 0.01 or 0.02 ng/mL.
Being able to determine the degree of serial change in high sensitive troponin assay concentrations seems to be the most accurate way of differentiating between those patients suffering AMI and those with more chronic heart conditions. Currently, there is no official consensus on a way of establishing or confirming delta values. Until this is in place, the US Task Force in Clinical Applications of Cardiac Bio-markers recommends that institutions agree on a delta value based on available peer-reviewed data for individual assays and then modify based on empirical findings and feedback [15].
Another study of 874 patients, published in the International Journal of Cardiology, used the Beckman Coulter AccuTnI assay to demonstrate that an algorithm incorporating cTnI concentration and delta cTn values with this assay could allow accurate diagnosis of AMI within two hours from presentation and an earlier rule-out of AMI in the majority of patients.
Cullen et al assessed the accuracy of delta cTn at two and six hours compared to the cTn concentration above the 99th percentile reference value for AMI in a prospective study of adult patients presenting with symptoms suggestive of possible acute coronary syndrome [16]. The area under the ROC curve for diagnosing AMI at two hours was 0.89 [95%CI, 0.84–0.95] for absolute delta cTn versus 0.79 [95%CI 0.73–0.85] for the relative change. Specificity and PPV at two hours were optimized using a delta cTnI ≥0.03 μg/L (95.8% [95%CI 94.1–97.0] and 61.4% [95%CI 50.9–70.9] respectively). Sensitivity and NPV for AMI were optimized using the 99th percentile with the addition of a delta of 0.03 μg/L (97.1% [95%CI 90.2–99.2] and 99.7% [95%CI 99–99.9] respectively).
Labs to adopt lower high sensitive assay cut-offs
With the newer highly sensitive troponin assays, the resulting shift to lower cTn cutoffs will increase the number of patients that are monitored for MI, and will also identify patients with elevated cTn due to other conditions. Published guidance now recommends that all manufacturers demonstrate the true clinical performance of their cTn assays in the contemporary clinical setting through an appropriately designed clinical study [17]. Out-of-date clinical cut-offs and diagnostic criteria may not accurately diagnose MI in some instances. It may take time for laboratories to adopt lower cut-offs, but doing so will ultimately improve patient care [18].
References
1. Ford ES, Capewell S. Coronary heart disease mortality among young adults in the U.S. from 1980 through 2002: concealed leveling of mortality rates. J Am Coll Cardiol. 2007 Nov 27; 50(22):2128-32. Epub 2007 Nov 13.
2. Myocardial Infarction Redefined – a consensus document of the Joint European Society of Cardiology/American College of Cardiology Committee for the redefinition of myocardial infarction. Eur. Heart J 2000; 21: 1502-1513. J Am Coll Cardiol 2000; 36: 959-969.
3. Thygesen K, Alpert JS, White HD; Joint ESC/ACC/AHA/WHF Task Force for the Redefinition of Myocardial Infarction. Universal definition of myocardial infarction. Eur Heart J 2007; 28: 2525-38.
4. Joint ESC/ACCF/AHA/WHF Task Force for the Universal Definition of Myocardial Infarction. Third universal definition of myocardial infarction. European Heart Journal 2012; 33: 2551-2567. Available online at: http://www.escardio.org/guidelines.
5. Thygesen K, Alpert JS, Jaffe AS, et al. Third universal definition of myocardial infarction. Eur Heart J 2012; 33: 2251–2267.
6. Morrow DA, Cannon CP, Jesse RL, et al. National Academy of Clinical Biochemistry laboratory medicine practice guidelines: clinical characteristics and utilization of biochemical markers in acute coronary syndromes. Clin Chem 2007; 53: 552–574.
7. Collinson PO, Heung YM, Gaze D, Boa F, Senior R, Christenson R, et al. Influence of population selection on the 99th percentile reference value for cardiac troponin assays. Clin Chem 2012; 58:219-25.
8. Apple FS, Collinson PO, and for the IFCC Task Force on Clinical Applications of Cardiac Biomarkers: analytical characteristics of high-sensitivity cardiac troponin assays. Clin Chem 2012; 58:54-61.
9. Apple FS. Ler R, Murakami MM. Determination of 19 cardiac troponin I and T assay 99th percentile values from a common, presumably healthy, population. Clin Chem 2012; 58:1574-81.
10. Moretti M et al. Analytical performance and clinical decision limit of a new release for cardiac troponin I assay. Ann Clin Biochem, April 7, 2014.
11. Storrow AB, et al. Diagnostic performance of cardiac Troponin I for early rule-in and rule-out of acute myocardial infarction: Results of a prospective multicenter trial. Clinical Biochemistry 2014; e-pub ahead of print.
12. Keller T, Zeller T, Ojeda F, Tzikas S, Lillpopp L, Sinning C, et al. Serial changes in highly sensitive troponin I assay and early diagnosis of myocardial infarction. JAMA 2011; 306:2684-93.
13. Korley FK, Jaffe ASJ. Preparing the United States for high-sensitivity cardiac troponin assays. J Am Coll Cardiol 2013; 61:1753-8.
14. Storrow AB, et al. Absolute and relative changes (delta) in troponin I for early diagnosis of myocardial infarction: Results of a prospective multicentre trial. Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.09.012.
15. Task Force On Clinical Applications of Cardiac Bio-Markers. Using High Sensitivity Cardiac Troponin Assays in Practice. The International Federation of Clinical Chemistry and Laboratory Medicine (IFCC). 2014. http://www.ifcc.org/media/259738/201405_TF_CB_IFCC_practice%20Summary.pdf.
16. Cullen L, Parsonage WA, Greenslade J, Lamanna A, Hammett CJ, Than M, et al. Delta troponin for the early diagnosis of AMI in emergency patients with chest pain. Int J Cardiol 2013;168:2602–8.
17. Letter to Manufacturers of Troponin Assays Listed with the FDA. Available online at: http://www.fda.gov/MedicalDevices/ProductsandMedicalProcedures/InVitroDiagnostics/ucm230118.htm.
18. Mills NL, Lee KK, McAllister DA, Churchhouse AMD, MacLeod M, Stoddart M, Walker S, Denvir MA, Fox KAA, Newby DE. Implications of lowering threshold of plasma troponin concentration in diagnosis of myocardial infarction: cohort study. BMJ 2012; 344: e1533.
The author
Dr Bernard Cook is Senior Scientific Manager, Beckman Coulter Diagnostics. He has co-authored several scientific papers and is actively involved in the diagnostics industry, which includes being the former chairman of the industry division of the American Association for Clinical Chemistry.
Anti-DFS70 antibodies: detection and significance
, /in Autoimmunity & Allergy, Featured Articles /by 3wmediaby Professor M. Herold Systemic autoimmune rheumatic diseases (SARD) are rare and difficult to diagnose. Antinuclear antibodies (ANAs) are one of the most important serological features in the diagnostic work-up of patients with SARD. However, up to 30% of healthy individuals tested are also positive for ANAs and in about 50% of these cases ANA […]
G protein-coupled receptors, accessory proteins and signalling
, /in Featured Articles /by 3wmediaMolecular diagnostics is increasingly embracing pharmacogenomics. Here we discuss the role of G protein-coupled receptors and their accessory proteins in disease, drawing on our experience addressing the role of the calcium-sensing receptor polymorphisms/variation in familial hypocalciuric hypercalcemia and autosomal dominant hypocalcemia in order to highlight the role that pharmacogenomics may play in personalized treatment.
by Dr M. D. Thompson, Dr D. E. C. Cole and Dr G. N. Hendy
Introduction
The identification and characterization of gene families encoding G protein-coupled receptors (GPCRs) and the proteins necessary for the processes of ligand binding, GPCR activation, inactivation and receptor trafficking facilitates the study of drug response in the context of human genetic disease. Thompson et al. reviewed these topics in Volume 1175 of Methods in Molecular Biology in 2014 [1–3].
With the advance of genomic technologies, there has been a substantial increase in the inventory of naturally occurring rare and common GPCR variants [2, 3]. In addition to functional GPCR variants, genetic variation has been found in a variety of G protein subunits and accessory proteins that normally modify or organize heterotrimeric G protein coupling. These include variants of the regulator of G protein signalling (RGS) protein associated with hypertension; variants of the activator of G protein signalling (AGS) proteins associated with various phenotypes (such as the type III AGS8 variant to hypoxia); variants in of the G protein-coupled receptor kinase (GRK) proteins, such as GRK4, associated with disorders such as hypertension [1]. Variation in GPCR, G protein and accessory protein structure and function provides the basis for examining the pharmacogenomics of GPCRs and the genetics of related monogenic disorders [1–3].
GPCR variants and variant G protein subunits associated with human disease
Diseases caused by the genetic disruption of GPCR functions may be selectively targeted by drugs that rescue altered receptors. The identification of variants in these receptors provides genetic reagents useful in drug screens. Examples of drugs developed as a result of targeting GPCRs mutated in disease include: the calcimimetics and calcilytics, drugs targeting melanocortin receptors in obesity and interventions that alter gonadotropin-releasing hormone receptor (GNRHR) loss from the cell surface in idiopathic hypogonadotropic hypogonadism [2, 3].
Inactive, overactive and constitutively active receptors
Genetic variations in GPCR genes disrupt GPCR function in a variety of human genetic diseases. In vitro studies and animal models have been used to identify the molecular pathologies underlying these GPCR mutations. Inactive, overactive, or constitutively active receptors have been identified. These receptor variants alter ligand binding, G protein coupling, receptor desensitization, or receptor recycling. Variant GPCRs disrupted in disease include rhodopsin, thyrotropin, parathyroid hormone (PTH), melanocortin, follicle-stimulating hormone (FSH), luteinizing hormone, GNRHR, adrenocorticotropic hormone, vasopressin, endothelin-β, purinergic, and the G protein associated with asthma [GPRA or neuropeptide S receptor 1 (NPSR1)] [2]. Data on the role of activating and inactivating calcium-sensing receptor (CASR) mutations provide examples that will be discussed in detail with respect to familial hypocalciuric hypercalcemia (FHH) and autosomal dominant hypocalcemia (ADH) [4].
Calcium-sensing receptor mutations and hypercalcemia/hypocalcemia
The CASR functions as an extracellular calcium sensor for the parathyroid gland and the kidney. The CASR itself is a plasma membrane GPCR that is abundantly expressed in the PTH secreting cells of the parathyroid gland and the cells lining the renal tubule lumen [2, 4]. The activity and/or expression levels of the CASR dictate the calcium set-point at which PTH is secreted from the parathyroid gland [2]. CASR gene variants may influence many physiological processes by contributing to individual differences in calcium metabolism [2].
Inherited abnormalities of the CASR gene give rise to a variety of disorders of mineral ion homeostasis [5]. Heterozygous loss-of-function mutations cause familial (benign) hypocalciuric hypercalcemia (FHH) in which the lifelong mild hypercalcemia is generally asymptomatic. Homozygous inactivating mutations give rise to neonatal severe hyperparathyroidism (NSHPT) with extreme hypercalcemia and marked skeletal changes [5–7]. Heterozygous activating mutations of the CASR cause ADH that may be asymptomatic or present with seizures in the neonatal period or childhood or later in life [2].
Familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism
The syndrome known as familial hypocalciuric hypercalcemia (FHH), or familial benign hypercalcemia, results in mild primary hyperparathyroidism and relatively normal serum concentrations of PTH [8]. A key feature of FHH is the unusually high renal tubular reabsorption of calcium and magnesium in the face of hypercalcemia. However, some FHH families have affected members in which calcium excretion is increased and this may reflect the particular CASR mutation involved [2].
NSHPT involves multiglandular parathyroid hyperplasia. Children under the age of 6 months develop severe, symptomatic hypercalcemia with bony changes of hyperparathyroidism. Delay in treatment can lead to a devastating neurodevelopmental disorder. Some forms of neonatal hyperparathyroidism, involving either a de novo or paternal inheritance of a mutated CASR allele, present with milder symptoms [2].
Upwards of 200 unique inactivating, FHH/NSHPT type mutations in the CASR have been identified [2], as shown in Figure 1 (http://www.casrdb.mcgill.ca/). Although FHH is inherited in an autosomal dominant manner with almost 100% penetrance and variable expressivity, the population prevalence is not well defined. The FHH trait was initially mapped to chromosome 3q21, the locus of the CASR gene: two-thirds of FHH cases are due to mutations in the CASR gene and the disorder is FHH type 1 [2].
In some kindreds, however, the FHH trait maps to either chromosome 19p13.3 (FHH type 2) or 19q13.3 (FHH type 3). FHH2 is due to heterozygous loss-of-function mutations in GNA11, the gene encoding the alpha subunit of G11 that couples the activated CASR to intracellular signalling pathways [9]. FHH3 is due to inactivating mutations in the AP2S1 gene that encodes the sigma subunit of adaptor protein complex 2 critical for clathrin-mediated endocytosis of a variety of cell surface proteins including GPCRs such as the CASR [2].
Hypocalcemia, hypoparathyroidism, and hypocalcemic hypercalciuria
Gain-of-function mutations in the CASR gene have been identified in several families previously diagnosed with ADH, autosomal dominant hypoparathyroidism, and hypocalcemic hypercalciuria [2]. In the parathyroid gland, the activated CASR suppresses PTH secretion and in the kidney, it induces hypercalciuria [4]. De novo mutations are common [2]. Mosaicism for de novo mutation in an otherwise healthy parent has been described and this has important implications for counselling parents about the risk of recurrence [2].
In a subset of ADH families, CASR gain-of-function mutations have been associated with the onset of tonic–clonic seizures. In ADH, brain calcifications – sometimes accompanied by seizures – suggest that activating mutations may alter calcium homeostasis in the brain. The abnormal set-point of calcium regulation complicates treatment with calcitriol and dietary calcium supplementation because the CASR expressed in the kidney may override other regulators of calcium excretion. The constitutively activated CASR mutant induces hypercalciuria, which may exacerbate the hypocalcemia [2, 10].
More than 100 activating mutations (virtually all missense) have been identified and appear almost equally divided between the amino-terminal third of the extracellular domain (ECD) and the transmembrane domain shown in Figure 1 (http://www.casrdb.mcgill.ca/).
GPCR pharmacogenomics
Pharmacogenetics investigates the influence of genetic variants on physiological phenotypes related to drug response and disease, while pharmacogenomics takes a genome-wide approach to advancing this knowledge. Both play an important role in identifying responders and non-responders to medication, avoiding adverse drug reactions, and optimizing drug dose for the individual.
The CASR provides an example of GPCR variability in the population. While CASR variants contribute to monogenic disorders such as FHH and ADH, common CASR polymorphisms also account for some of the population variation in calcium response that is a risk factor for a variety of disease susceptibilities. CASR single nucleotide polymorphisms (SNPs) have been associated with a number of complex phenotypes. For example, the Ala986Ser variant may contribute to bone mineral density, primary hyperparathyroidism, and Paget disease [11].
The cluster of missense polymorphisms located in the cytoplasmic tail of the receptor is associated with inter-individual population differences in Ca2+ metabolism [12]. Different haplotypes are associated with primary hyperparathyroidism and the frequency of kidney stones. More recent genome-wide association studies in ~33,000 individuals of European and Indian Asian ancestry confirmed that the blood calcium concentration associated most significantly with SNPs in the CASR gene [13].
CASR variants are known to alter the sensitivity of the CASR and result in altered extracellular calcium-concentration set points in tissues. Web sites such as http://www.casrdb.mcgill.ca/ document a number of SNPs scattered across the more than 100 kb region of genomic DNA that encompasses the CASR gene. Common missense SNPs (Ala986Ser and Arg990Gly) are clustered in the DNA region encoding the cytoplasmic tail of CASR. The most common of these, the Ala986Ser variant, is predictive of the unbound, extracellular calcium levels [11]. The Ala986Ser variant is thus a mild inactivating variant that may predispose to hypercalcemia without being fully predictive of hypocalciuria. By contrast, the Arg990Gly variant (activating) results in the increased calcium excretion that characterizes idiopathic hypercalciuria and is predictive of nephrolithiasis [2].
Conclusion
GPCRs are the primary target of therapeutic drugs and have been the focus of these studies. These variants include SNPs and insertion/deletions that have potential to alter GPCR expression of function. In vivo and in vitro studies have determined functional roles for many GPCR variants, but genetic association studies that define the physiological impact of the majority of these common variants are still limited. Despite the breadth of pharmacogenetic data available, GPCR variants have not been included in drug labelling and are only occasionally considered in optimizing clinical use of GPCR targeted agents. As the extent of GPCR pharmacogenomic data increases, the opportunity for routine assessment of GPCR variants to predict disease risk, drug response and potential adverse drug effects will no doubt become more commonplace.
References
1. Thompson MD, Cole DE, Jose PA, et al. G protein-coupled receptor accessory proteins and signaling: pharmacogenomic insights. Methods Mol Biol. 2014; 1175: 121-52.
2. Thompson MD, Hendy GN, Percy ME, et al. G protein-coupled receptor mutations and human genetic disease. Methods Mol Biol. 2014; 1175: 153-87.
3. Thompson MD, Cole DE, Capra V, et al. Pharmacogenetics of the G protein-coupled receptors. Methods Mol Biol. 2014; 1175: 189-242.
4. Zhang C, Zhuo Y, Moniz HA, et al. Direct determination of multiple ligand interactions with the extracellular domain of the calcium sensing receptor. J Biol Chem. 2014 Oct 10. pii: jbc.M114.604652.
5. Thim SB, Birkebaek NH, et al. Activating calcium-sensing receptor gene variants in children: a case study of infant hypocalcaemia and literature review. Acta Paediatr. 2014 Jul 10. doi: 10.1111/apa.12743.
6. Toka HR, Pollak MR.The role of the calcium-sensing receptor in disorders of abnormal calcium handling and cardiovascular disease. Curr Opin Nephrol Hypertens. 2014; 23: 494-501.
7. Grzegorzewska AE, Ostromecki G. Gene polymorphism of the vitamin D receptor, vitamin D-binding protein and calcium-sensing receptor in respect of calcium-phosphate disturbances in chronic dialysis patients. Przegl Lek. 2013; 70: 735-8.
8. Jakobsen NF, Rolighed L, Nissen PH, et al. Muscle function and quality of life are not impaired in familial hypocalciuric hypercalcemia: a cross- sectional study on physiological effects of inactivating variants in the calcium-sensing receptor gene (CASR). Eur J Endocrinol. 2013; 169: 349-57.
9. Nesbit MA, Hannan FM, Howles SA, et al. Mutations affecting G-protein subunit α11 in hypercalcemia and hypocalcemia. N Engl J Med. 2013; 368: 2476-86.
10. Ranieri M, Tamma G, Di Mise A, et al. Excessive signal transduction of gain-of-function variants of the calcium-sensing receptor (CaSR) are associated with increased ER to cytosol calcium gradient. PLoS One. 2013; 8: e79113.
11. Han G, Wang O, Nie M, et al. Clinical phenotypes of Chinese primary hyperparathyroidism patients are associated with the calcium-sensing receptor gene R990G polymorphism. Eur J Endocrinol. 2013; 169: 629-38.
12. Scillitani A, Guarnieri V, Battista C, et al. Primary hyperparathyroidism and the presence of kidney stones are associated with different haplotypes of the calcium-sensing receptor. J Clin Endocrinol Metab. 2007; 92: 277-83.
13. Kapur K1, Johnson T, Beckmann ND, et al. Genome-wide meta-analysis for serum calcium identifies significantly associated SNPs near the calcium-sensing receptor (CASR) gene. PLoS Genet. 2010; 6: e1001035.
The authors
Miles D. Thompson1* PhD; David E. C. Cole2 MD, PhD; Geoffrey N. Hendy3 PhD
1Department of Pharmacology and Toxicology, Medical Sciences Building, University of Toronto, Toronto, ON, Canada. M5S 1A8.
2Departments of Laboratory Medicine and Pathobiology, Medicine and Genetics, University of Toronto, ON, Canada. M4N 3M5.
3Departments of Medicine, Physiology, and Human Genetics, McGill University, and Calcium Research Laboratory and Hormones and Cancer Unit, Royal Victoria Hospital, Montreal, QC, Canada. H3A 1A1.
*Corresponding author
E-mail: miles.thompson@utoronto.ca
Myocardial infarction outcomes: redressing sex
, /in Featured Articles /by 3wmediaIn spite of major medical advances in diagnosis and treatment, cardiovascular disease (CVD) is still the leading cause of mortality in the Western world accounting for 51 percent of female and 42 percent of male deaths. Around half of these deaths are due to coronary heart disease, and it has been recognized for more than two decades that the outcome for women with acute coronary disease (ACD) is worse than it is for men. Quite apart from the fact that surveys show older women are less aware of their risk of myocardial infarction (MI) than men, women presenting with MI are less likely to be appropriately diagnosed.
Various explanations have been given for this disparity. Clinical symptoms of ACD in women may not be the ‘typical’ sudden severe chest pain; physicians have even attributed female symptoms of more diffuse pain, dyspnea and fatigue to falling levels of estrogen and progesterone. And although sex differences in electrocardiography (ECG) were first reported around 90 years ago, with recent studies emphasizing that normal values of the adult ECG should be both age- and sex-specific, the use of sex-specific diagnostic criteria is still not routine in many hospitals. In addition clinical research into ACD was biased towards men in the past, resulting in predictive values for analytes that are not necessarily appropriate for women; results of diagnostic tests should of course take sex-related differences into consideration. Given that the diagnosis of MI relies on a combination of clinical examination, suggestive ECG abnormalities and a rise and fall of key cardiac biomarkers, it is not surprising that myocardial infarction in women is still under-diagnosed.
However, the results of a recently published study in the BMJ should be a step towards more effective diagnosis. The troponin I level of 1126 consecutive patients presenting at a regional cardiac centre with suspected MI, 46% of whom were women, was measured using a high sensitivity assay and sex-specific diagnostic thresholds (men 34 ng/L, women 16 ng/L) in place of the current recommended threshold of 50 ng/L for both sexes. There was a significant increase in the number of women diagnosed with MI (from 11% to 22%) but the increase in men (19% to 21%) was not significant. Although studies continue to show that even when diagnosed women are less likely to undergo percutaneous coronary interventions or bypass surgery, receive prompt thrombolytic therapy or even be prescribed statins on discharge from hospital, more accurate diagnosis should go a long way towards redressing sex inequalities.
LC-MS/MS-based H-type determination of Escherichia coli
, /in Featured Articles /by 3wmediaA comparative analysis of serotyping and mass spectrometry (MS) methods for the determination of the flagellar type (H type) of clinically isolated Escherichia coli has been performed. In this analysis, it was shown that determination of the correct H type of a clinical E. coli strain was better achieved by MS than serotyping. Whole genome sequencing was used for the validation of this analysis.
by M. Chan, Dr H. Chui, D. Hernandez, Dr G. Wang and Dr K. Cheng
Background
During outbreaks of disease caused by pathogenic Escherichia coli, it is important to be able to identify the precise E. coli strain involved in order to track and prevent the spread of infection. Typically, the identification of E. coli strains has been based on the serotype of the cell surface antigens such as the lipopolysaccharide O antigen and the flagellar H antigen. Even though serotyping is viewed as the ‘gold standard’ for O- and H-type determination, this technique does have its downfalls. These conventional serotyping methods are based on antisera, which makes procedures costly and laborious to perform because of the variable quality of antibody preparations and the number of antibody agglutination reactions needed to assign a final classification [1, 2, 3]. Also, when bacterial cells do not generate lipopolysaccharide on the surface, the cultured colonies become ‘rough strains’. This makes both O- and H-antigen identification by antibody-based agglutination problematic despite the cellular motility and presence of the flagella H-antigen structure [1, 3, 4]. In addition, flagellar serotyping needs the induction of motility, which can take up to two weeks, and so does not result in the fast identification required in an E. coli outbreak situation [5].
A promising technique for E. coli H-type identification is the use of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, termed ‘MS-H’. In our own analytical assays, we harvest, enrich and digest the flagella proteins. LC-MS/MS uses liquid chromatography to separate flagellar fragments after trypsin digestion, and mass spectrometry to analyse and determine the protein sequences of these fragments. The MS results are then analysed against a curated database of H antigen variants to determine the H type. Compared with serotyping, MS-H has a high throughput, requires less labour, needs less time to perform, and provides sequence-level information.
Comparative H typing of E. coli with serotyping and MS-H
Using reference strains of all the different forms of E. coli flagella, it was shown that all 53 different types of H antigen can be determined through MS-H. Table 1 details the comparisons between H serotyping and MS-H [1]. It is important to note that both methods can reach 100% sensitivity and specificity for H-type determination. However, MS-H also provides sequence-level identification of the H type. This is important because this provides more reliable results, while antisera serotyping only provides a visual conformation, making there more of a chance for subjective and inaccurate determination of the H type.
Flagella motility induction is commonly used in serotyping. This increases the time-length of the procedure and requires more hands-on involvement [5]. In comparison, MS-H uses motility induction much less frequently, which makes MS-H identification of E. coli H type quicker and less labour-intensive.
Clinically isolated strains of E. coli that had already had their H types determined by serotyping were supplied from three provinces within Canada. A preliminary H-type analysis of the same E. coli isolates was performed by MS-H [5]. The workflow that was completed to analyse the H type of these strains is detailed in Figure 1.
Things to note within the workflow are that vortexing the suspended culture in Step 2 was to shear the flagella away from the E. coli. Also in Step 2, the flagella were trapped onto a filter membrane. This serves as an isolation step to separate the flagella from the supernatant as well as an enrichment process to concentrate the flagella. We then compared the flagellar sequence obtained by MS to a curated database formed from NCBI flagella protein sequence entries. A curated database was necessary because a public database can be problematic because of size, specificity and inconsistent annotation of the data for the specific flagellar protein entries. The curated database focuses on the flagellar proteins with fit-for-purpose annotations, thus allowing differentiation between the 53 different H types better than the public database [6].
On comparison of the H types identified by serotyping and MS-H, the majority of the results agreed with one another. However, there were some discrepancies between the two methods. In these cases, whole genome sequencing and polymerase chain reaction (PCR) detection was used to identify the correct H type [5] and these results predominantly agreed with those obtained by MS-H.
Even though PCR as a form of detection for the flagella gene is popular, the accuracy for determining all forms of flagella types through PCR is low and the use a many different primers had to be implemented, making it not ideal for the detection of an unknown H type [5]. Whereas whole genome sequencing may be more expensive then PCR detection, it was much more accurate at determining the correct H-type allele than PCR detection. Thus moving into the confirmational assays for the comparative analysis of MS-H determination and H serotyping, whole genome sequencing was only used to resolve disputed results between the two methods as it is not exclusively representative of a host’s flagella phenotype. On the other hand serotyping and MS-H is representative of the bacteria’s H type. Whole genome sequencing isn’t ideal for identifying clinical strains of E. coli for it is laborious and a long process compared to MS-H which is faster and contains less labour-intensive.
MS-H in clinical laboratories
Having shown that MS-H determined E. coli H type with high accuracy and in a short time frame, the application of H typing through MS-H could become very useful for the identification of unknown E. coli strains in a clinical laboratory. This is especially useful in the identification of an E. coli strain during a disease outbreak [7]. Faster identification of the pathogenic strain of E. coli would in turn also help combat the pathogenic E. coli more quickly.
Without the use of antibodies for agglutination and procedures for motility induction that are required in serotyping, MS-H is much less laborious in comparison. This would greatly benefit the professionals in the medical microbiology and public health laboratories who perform H typing.
Advantages and disadvantages of MS-H
It is significant that MS-H better determined the H type compared to conventional serotyping. As MS-H is faster, less laborious, provides sequence-level identification and has a higher throughput than serotyping, MS-H can be very useful in rapid and accurate identification of E. coli flagellar antigens. This may be a little over-idealistic as LC-MS/MS machines are very expensive and serotyping has been around for a very long time. It might be difficult currently for clinical laboratories to afford a LC-MS/MS machine or to change their workflow to incorporate MS-H as their method of H determination. However, mass spectrometer platforms are becoming more common in clinical laboratories. The uses of matrix-assisted laser desorption/ionization (MALDI) mass spectrometers have shown varying successes in microbiological identification. Also the use of MS isn’t just limited to H typing. MS could extend to the determination of the lipopolysaccharides (O antigen) of E. coli, toxins, and other relevant molecules within the spectrum of the mass spectrometer. This would not only determine the surface antigens of pathogenic E. coli, it would also give a broader profile of a pathogenic E. coli.
Conclusion and future directions
MS has potential to determine the H antigen type of E. coli better than serotyping, as shown in a comparative study between MS-H and serotyping through the use of a mass LC-MS/MS platform. MS-H provides sequence level identification of the flagellum type, whereas serotyping only provides visual agglutination assays for positive results and is therefore more prone to errors such as false positives and misidentification. Also, without the need for antisera, MS-H is less laborious, requires less time, and has a high throughput.
With the completion of the preliminary assay results of the comparative study between MS-H and serotyping in the determination of the flagella type, we are currently working on a country-wide thorough validation of the platform.
References
1. Cheng K, Drebot M, McCrea J, Peterson L, Lee D, McCorrister S, Nickel R, Gerbasi A, Sloan A, Janella D, Van Domselaar G, Beniac D, Booth T, Chui L, Tabor H, Westmacott G, Gilmour M, Wang G. MS-H: A novel proteomic approach to isolate and type the E. Coli H antigen using membrane filtration and liquid chromatography-tandem mass spectrometry (LC-MS/MS). PLoS One 2013; 8(2): 1–12.
2. Tenover FC, Arbeit RD, Goering RV. How to select and interpret molecular strain typing methods for epidemiological studies of bacterial infections: A review for healthcare epidemiologists. Molecular Typing Working Group of the Society for Healthcare Epidemiology of America. Infect Control Hosp Epidemiol. 1997; 18(6): 426–439.
3. Machado J, Grimont F, Grimont PA. Identification of Escherichia coli flagellar types by restriction of the amplified fliC gene. Res Microbiol 2000; 151(7): 535–546.
4. Edwards PR, Ewing WH. Edwards and Ewing’s Identification of Enterobacteriaceae, p536. Elsevier 1986. ISBN 978-0444009817.
5. Cheng K, Sloan A, Peterson L, McCorrister S, Robinson A, Walker M, Drew T, McCrea J, Chui L, Wylie J, Bekal S, Reimer A, Westmacott G, Drebot M, Nadon C. Knox D, Wang G. Comparative study of traditional flagellum serotyping and liquid chromatography-tandem mass spectrometry-based flagellum typing with clinical Escherichia coli isolates. J Clin Microbiol. 2014; 52(6): 2275-2278.
6. Cheng K, Sloan A, McCorrister S, Babiuk S, Bowden TR, Wang G, Knox D. Fit-for-purpose curated database application in mass spectrometry-based targeted protein identification and validation. BMC Res Notes 2014; 7: 444.
7. Cheng K, Sloan A, McCorrister S, Peterson L, Chui H, Drebot M, Nadon Celine, Knox D, Wang G. Quality evaluation of LC-MS/MS-based E. coli H antigen typing (MS-H) through label-free quantitative data analysis in a clinical sample setup. Proteomics Clin Appl. 2014; 8: 963–970.
The authors
Michael Chan*1,2, Huixia Chui1,3 MD, Drexler Hernandez1,2, Gehua Wang*1 MD and Keding Cheng*1,4 MD
1National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, MB, Canada
2University of Manitoba, Winipeg, MB, Canada
3Centre of Disease Control and Prevention, Henan Province, PR China
4Department of Human Anatomy and Cell Sciences, Faculty of Medicine, University of Manitoba, Winnipeg, MB, Canada
*Corresponding authors
E-mail: M. Chan, umchanm@myumanitoba.ca; G. Wang, gehua.wang@phac-aspc.gc.ca; K. Cheng, keding.cheng@phac-aspc.gc.ca.
Using HCV core antigen testing to improve diagnosis of acute infection
, /in Featured Articles /by 3wmediaMany people become infected with hepatitis C virus (HCV) every year and these infections often have no symptoms. A significant number of patients will go on to develop chronic liver disease and potentially hepatocellular carcinoma. Early detection of HCV infection is of great importance, but remains challenging. This article describes the advantages and limitations of methods of HCV diagnosis.
by Dr O. Blach, Dr D. Lawrence, Dr F. Cresswell and Prof. M. Fisher
Hepatitis C virus infection
It is estimated that 3% of the world’s population has hepatitis C virus (HCV), with a further 3–4 million people becoming newly infected every year [1]. Early detection of HCV infection is of great importance, as prompt diagnosis enables contact tracing, partner notification, health promotion advice to reduce the risk of onward transmission and disease progression, and the opportunity for early treatment, which may offer the best opportunity for cure [2].
However, diagnosing acute hepatitis C remains challenging. Most patients with acute infections are asymptomatic, and even when symptoms are present, they are often non-specific, not severe, and may not present in the same way as those with other hepatitis viruses (such as A, B and E). Approximately 10–20% of patients clear the virus spontaneously during acute infection; the remainder progress to chronic infection which, if unrecognized, will progress in a significant proportion to chronic liver disease, cirrhosis, end-stage liver disease and hepatocellular carcinoma [2]. Although newer directly acting antiviral drugs (DAAs) against HCV will transform management, for many individuals pegylated interferon and ribavirin may remain standard of care for some time until these can be afforded. Therefore early diagnosis for many will offer the best opportunity for cure at the present time.
Established diagnostic tests
The diagnosis of acute hepatitis C is usually made after detection of abnormal liver function tests or on routine screening in specific populations, such as those with HIV infection, on hemodialysis for end-stage renal failure, or accessing services for injecting drug users. Traditionally, seroconversion from anti-HCV antibody (anti-HCV) negative to positive, a process which takes places around 12 weeks after infection, is detected by enzyme-linked immunosorbent assays (ELISA, EIA) or chemiluminescence immunoassay (CIA) [3].
However, although the presence of anti-HCV indicates infection with HCV at some point, it does not determine whether it is acute, chronic or resolved. Furthermore, anti-HCV may not be detectable during this aforementioned 12-week ‘window period’, or if the patient is immunocompromised and therefore has an impaired ability to produce antibodies [4], with delayed seroconversion up to 18 months being reported [5].
Detection of viremia in the setting of a negative anti-HCV (during the seronegative ‘window period’), and therefore verification of active HCV infection has historically been done using nucleic acid amplification test (NAAT) for HCV RNA by quantitative reverse transcription polymerase chain reaction (qRT-PCR), which can detect HCV RNA in serum 1–3 weeks after infection [6–8]. Although the ‘gold standard’ for diagnosing acute HCV infection, HCV qRT-PCR has several shortcomings: it is costly, labour-intensive, time-consuming and requires advanced technical skills, separate facilities (separate platform) and equipment [9], which make it particularly impractical in a resource-poor setting. As a consequence, HCV core antigen (Ag) quantification as a surrogate marker of HCV replication has been suggested as an alternative assay for initial testing of acute hepatitis [10].
HCV core antigen
HCV core Ag is part of the HCV capsid formed by core protein polymerization, and as such, is one of the best ‘conserved’ products of viral genome [11]. Using HCV core Ag, acute infection with HCV can be detected in the serum earlier than with the current anti-HCV screening assays [12], and only 1–2 days later than with HCV RNA NAAT tests [13].
Since the development of the first HCV core Ag tests around 2000, newer assays, which are up to 25 times more sensitive, have become available and are licensed in several countries. A recent meta-analysis of 25 studies conducted by Gu et al. [14] compared the diagnostic accuracy of HCV core Ag (index reference) with HCV RNA (‘gold standard’) and showed good pooled sensitivity of 0.84 (95% CI, 0.83–0.85), with excellent pooled specificity of 0.98 (95% CI, 0.97–0.98) for HCV core Ag assays. HCV core Ag can therefore be used as a marker of viraemia [7] with the lower limit of detection corresponding to HCV RNA load of 700–1100 IU/mL [15]. Positive and negative predictive values reported in the literature for HCV core Ag assays are also high, with one study reporting PPV of 100% and NPV of 97% [16]. However, re-testing samples with low positive Ag values (<35fM) has been recommended after one study by Shepherd et al. [17] reported 37% false positive rates with such results. Another study by Cresswell et al. [7] recorded two false-indeterminate results, one of which was false positive on re-testing.
Furthermore, HCV core Ag levels closely track those of HCV RNA with multiple studies identifying a strong non-linear correlation between the two, thus potentially also allowing clinical monitoring of a patient’s therapy, independently of HCV genotype [15]. This is mainly the case in samples with HCV RNA levels above 20 000 IU/ml, thereby limiting their use in practice [18].
Given its slightly lower sensitivity compared with HCV RNA PCR, the utility of HCV core Ag testing in a diagnostic algorithm for acute hepatitis C is dependent on the practicalities of testing in a given population setting and the potential cost savings [19]. One appealing advantage of HCV core Ag assays lies in the potential for reflex HCV core Ag testing in anti-HCV positive samples using the same testing platform and the same sample [20], thus providing physicians with clinically meaningful results of both anti-HCV and HCV core Ag within an hour.
HCV core Ag could also prove to be more stable than HCV RNA in situations where testing cannot be done on a fresh sample or where a sample needs to be transported to another laboratory [21]. As such, HCV antigen detection could be a viable next step following a positive anti-HCV test, and additional HCV RNA testing would only be necessary with negative or low positive HCV core Ag values.
Furthermore, besides a faster processing time compared to traditional molecular tests, HCV core Ag assays are cheaper [22] and thereby especially attractive in low-resource settings or where PCR may be unavailable [9]. Cresswell et al. estimated potential cost savings of $18 275 in equipment and $6964 in manpower per year in an HIV cohort of 2200 people, had HCV core Ag been used in place of HCV RNA PCR [7].
Special populations
The usefulness of HCV core Ag as a screening investigation in the immunocompromised cohort has attracted considerable attention recently, given their impaired antibody production and the well-recognized delay in HCV antibody seroconversion [5]. High sensitivities (100%) and specificities (97.9 and 97.7%) were reported by Cresswell et al. [7] and Carney et al. [23] in diagnosing acute hepatitis C in HIV-infected individuals by HCV core Ag. Another study of dialysis patients by Moini et al. found only one HCV RNA positive patient to be HCV core Ag negative (note a low HCV viral load of <100 IU/mL) [24]. Finally, in the context of blood transfusion or organ transplantation, the modern HCV RNA assays remain the most sensitive and preferred option [25], but in a resource-limited blood bank setting, testing with HCV core Ag might be superior to no testing for HCV viremia at all. Further research is needed to determine the role of HCV core Ag testing in monitoring of both the untreated patients and those undergoing therapy, as well as in predicting the histological chances and disease progression. Looking to the future
In conclusion, given the inadequacies of HCV antibody testing in acute infection and the time and financial constraints of HCV RNA PCR, HCV core Ag detection offers a new, cheaper and effective way of testing for acute hepatitis C, and is a promising confirmatory test for anti-HCV positive patients. Given the emerging evidence on the constantly improving HCV core Ag assays, we believe that national guidelines should now begin to consider HCV core Ag testing as an integral part of the HCV screening algorithm for acute HCV infection, as illustrated in Figure 1.
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The authors
Ola Blach*1 MBChB, BSc; David Lawrence1 MBChB, MSc; Fiona Cresswell1 MD, MBBS; and Martin Fisher1,2 FRCP, MBBS, BSc
1Lawson Unit, Department of HIV and Sexual Health, Royal Sussex County Hospital, Brighton, UK
2Brighton and Sussex Medical School, Brighton, UK
*Corresponding author
E-mail: ola.blach@doctors.org.uk