The Lipid Hypothesis and the Seven Countries Study

CHAPTER 4

The Lipid Hypothesis and the Seven Countries Study Ronan Lordan, Alexandros Tsoupras, Ioannis Zabetakis Department of Biological Sciences, University of Limerick, Limerick, Ireland

Chapter Outline 4.1 Introduction 119 4.2 The Understanding of Atherosclerosis Circa 1800–1930 120 4.3 Cholesterol 122 4.3.1 Serum Cholesterol, Serum Triglycerides, and Diet 123 4.3.2 Lipoproteins and Coronary Heart Disease 127

4.4 Lipid Hypothesis: Origin, Development, and Prelude to the Seven Countries Study 129 4.5 Ancel Keys (1904–2004) 130 4.5.1 The Seven Countries Study (1958–2000) 131 4.5.2 The Seven Countries Study: Serum Cholesterol and the Link to CVD 132

4.6 Familial Hypercholesterolemia 135 4.7 Concluding Remarks 136 References 136

4.1 Introduction There is evidence from studies of Egyptian mummies that cardiovascular diseases were as commonplace 3000 years ago as they are now (Allam et al., 2011; Thompson et al., 2013). Before the 20th century, cardiovascular diseases were believed to naturally accompany the aging process. By the beginning of the 1800s, physiologists began to examine the pathology of vascular lesions, which led to the coining of the term “arteriosclerosis” by Jean Lobstein in 1829 (Lobstein, 1833). It wasn’t until 1904 at a congress in Leipzig that the term “arteriosclerosis” was referred to pathologically by Marchand (1904), who is also credited with coining the term atherosclerosis (Kritchevsky, 1967). This chapter explores the early development of the lipid hypothesis and how it came to the fore following the “Seven Countries Study.” The Impact of Nutrition and Statins on Cardiovascular Diseases. https://doi.org/10.1016/B978-0-12-813792-5.00004-5 # 2019 Elsevier Inc. All rights reserved.

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4.2 The Understanding of Atherosclerosis Circa 1800–1930 The formation of atherosclerotic lesions had become a major interest of physiologists in the 1800s. This included Rudolf von Virchow, who observed that lipids and cholesterol were present in the arteries dissected from human cadavers. These findings were later confirmed by Windaus (1910), who found that atheromatous aortas contained 6 times more free cholesterol and 20 times more cholesterol ester than normal arteries; this was later confirmed by Sch€ onheimer (1926). Virchow had also discerned that endothelial cell injury initiates atherogenesis (Virchow, 1856), and that the cholesterol in the lesions could only have come from the blood. Virchow would later formulate the terms embolism and thrombosis from his research elucidating the mechanisms of pulmonary thromboembolism (Kumar et al., 2010). In 1908, Alexander Ignatowski was the first to carry out a nutritional investigation into experimental atherosclerosis, believing that a toxic metabolite found in animal protein may be the root cause of the disease due to the formation of nodular lesions of the aorta (Bailey, 1916). Stuckey (1910) carried out similar experiments using foods rich in animal protein such as egg whites, milk, and meat juice. However, he did not observe significant lesion development until he used the nonprotein components of foods such as egg yolk and brain. Pure neutral animal or plant-derived fats did not produce lesions. It was not until 1913 at the Pathology Institute of the Royal Military Medical Academy in St Petersburg, where colleagues Ignatowski and Stuckey studied, that experimental pathologist Nikolai Nikolajewitsch Anitschkow wanted to decipher whether cholesterol played a role in the pathology of atherosclerosis. This was in contrast to the ideas of his colleagues and the research trends of his time examining diets high in protein (Konstantinov and Jankovic, 2013). Anitschkow and his student Chalatow fed healthy rabbits a diet of cholesterol derived from egg yolk dissolved in sunflower seed oil daily for 10, 25, 26, 79, 81, and 139 days. The dose administered ranged from 3 to 82 g. Changes were observed in the subintimal layer of the aorta that resembled human atherosclerotic lesions, which were dose-dependent and increased with prolonged cholesterol consumption (Anitschkow et al., 1913). The control animals that consumed only sunflower oil exhibited no atherosclerotic lesions (Finking and Hanke, 1997). The resulting atherosclerotic lesions led to the development of a number of key concepts, as highlighted by Steinberg (2004a). These are discussed in brief. During the early development of an atheroma, cholesterol-filled cells now known as “foam cells” are visible as fatty streaks. These early lesions occur at the root of the aorta and the aortic arch and their pattern of distribution is characterized by hemodynamic factors. The consistent consumption of cholesterol led to the alteration of the fatty streak to a fibrous cap. Anitschkow found that early lesions are reversible. However, in advanced lesions, although lipids can be mobilized, some remnants of cholesterol esters, lipids, and the irreparable fibrous plaque still remain (Wolkoff, 1930). Anitschkow and Ascoff were both very aware that other factors

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such as blood pressure may play a role in the development of atherosclerotic lesions (Bailey, 1916). Anitschkow later postulated that the cholesterol in the lesions had come from the blood because rabbits that develop lesions also had raised blood cholesterol levels. Initial findings did not indicate that the rabbit’s lesions had a similarity to human lesions due to the higher levels of lipid in the arteries. However, on low doses of cholesterol over 2.5 years, the arterial lesions more closely resembled human lesions. Initially, the scientific community rejected Anitschkow’s studies as a number of researchers were concerned by the use of a rabbit as a model organism to study human atherosclerosis as it was a herbivore that did not consume such high levels of cholesterol regularly (Steinberg, 2013). Anitschkow himself encountered problems with his theory when rats fed egg yolks and milk for up to 5 months failed to demonstrate macroscopic or microscopic alterations of the subintimal layer in the aorta (Truswell, 2010c). Knack (1915) compared the atherogenic effects of dietary cholesterol with diets containing eggs and milk and found the latter to be more uniformly atherogenic. Anitschkow had difficulty in repeating his studies on dogs, cats, rats, monkeys, and other carnivores. Even with a greater intake of dietary cholesterol, the animals’ blood cholesterol levels did not rise high enough to induce lesion formation (Anitschkow, 1925; Ahrens et al., 1957b). Of course, it was later found that these animals are efficient in converting cholesterol into bile acids that are excreted. Some studies have attributed cholesterol consumption to lesion development in rabbits, guinea pigs, goats, hens, parrots, and nonhuman primates (Bailey, 1916; Anitschkow, 1922, 1925; Chalatow, 1929; Clarkson, 1972; Fuster et al., 2012). The disparity between these studies is a result of various factors, including the fact that some animal models are genetically resistant to diet-induced atherosclerosis (Getz and Reardon, 2012). This particularly applies to mice and other experimental animals, where plaque rupture and thrombosis are rarely observed (Thomas and Hartroft, 1959; Plump and Lum, 2009). The studies by Stuckey, Anitschkow, and Chalatow also received criticism from Clarkson and Newburgh (1926) because they believed that “the feeding of cholesterol was not extensive enough; the cholesterol administered to the animals on diets which were abnormal and well might have let to metabolic disturbance under any condition; or the cholesterol was fed in conjunction with large amounts of protein.” Although the focus of cardiovascular research shifted from the effects of protein consumption to cholesterol consumption, protein consumption was still researched at length. Newburgh and Squier (1920) carried out a study to investigate the effect of beef consumption on the development of atherosclerosis in rabbits and found that a diet containing 36% protein mainly composed of beef protein was more atherogenic than a diet containing 27% protein that had a much lower composition of beef protein. The two diets contained 36 or 28 mg of cholesterol, respectively, levels that were too low to cause atherosclerosis. Newburgh and Clarkson (1922) attributed the atherogenic effect to the levels of protein in the diet, which they later proved by feeding rabbits 25, 113, 253, or 507 mg of cholesterol

122 Chapter 4 daily for extended periods of time. Rabbits that consumed 25 mg of cholesterol for up to 288 days were normocholesterolemic and free of atherosclerotic lesions. No elevation in serum cholesterol was observed in rabbits fed 113 mg of cholesterol for up to 302 days, and only one of 19 rabbits displayed evidence of lesion development. They found that the amount of cholesterol required to cause atherosclerosis in the rabbits was at least 10 times the amount present in the beef diets. Studies also examined the atherogenicity of plant- and vegetable-derived proteins versus animal proteins in rabbits and found that some animal proteins are atherogenic (Meeker and Kesten, 1940, 1941; Howard et al., 1965; Kritchevsky and Klurfeld, 2013). A study by Carroll and Hamilton (1975) inspired renewed interest in dietary protein by showing that rabbits fed defatted protein animal sources were more cholesterolemic than those of plant origin.

4.3 Cholesterol In 1665, Irish scientist Robert Boyle noted that the blood of animals had a milky appearance following a fatty meal, which was confirmed by Henson in 1774. In 1924, French scientists Gage and Fish determined that after a fatty meal, humans had 1 μm particles in their bloodstream, which we now recognize as chylomicrons (Olson, 1998). Cholesterol was first isolated from a gallstone in 1769 by Poulletier de la Salle and later by Antoine Franc¸ois de Fourcroy (McNamara et al., 2006). Michel Eugene Chevreul, famous for the saponification process, suggested the name “cholesterine” (from the Greek words chole: bile and stereos: solid) due its appearance in gallstones. In 1859, Berthelot proposed that the substance be renamed cholesterol due to the presence of a hydroxyl group. Cholesterol (C27H45OH) is a sterol molecule that is biosynthesized by all animal cells and is an essential component of the cell membrane that allows for fluidity and movement. The hydroxyl group on cholesterol interacts with the polar head groups of the membrane phospholipids, as opposed to the hydrophobic steroids and hydrocarbon chain, which are embedded in the membrane alongside the nonpolar fatty acid chains of the other lipids (Za´rate et al., 2016). The majority of cholesterol production is carried out by hepatocytes through a complex 37-step process, beginning with the mevalonate pathway and ending with a 19-step conversion of lanosterol to cholesterol before entering the circulatory system to be distributed by lipoproteins around the body. Cholesterol carries out vital metabolic and regulatory functions and is the precursor molecule for endogenous biosynthesis of vitamin D, bile acids, and steroid hormones such as estrogen, progesterone, and testosterone. Cholesterol is insoluble in blood and therefore is transported via lipoproteins. Lipoproteins are globular particles consisting of a hydrophobic core of triacylglycerols and cholesterol esters surrounded in a coat of apoproteins, phospholipids, and cholesterol. The apoproteins on the surface help to solubolize the lipids and determine their target tissues.

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4.3.1 Serum Cholesterol, Serum Triglycerides, and Diet In the early 1900s, the observations of pathologists such as Anitschkow and Virchow confirmed that higher serum cholesterol was associated with the severity of atherosclerosis at the time of death. However, measurements of human plasma cholesterol levels were inconsistent between studies (Elliott and Nuzum, 1936; Davis et al., 1937; Kritchevsky, 1958). The reasons for these discrepancies include the fact that the diagnosis of atherosclerosis in some studies was centered on the palpitation of the peripheral blood vessels; serum lipids were incorrectly taken and in some cases taken postmortem; the number of subjects was small and not age standardized; and genetic risk factors such as familial hypercholesterolemia (FH) were not widely known or considered, among other reasons. Since 1938, FH has been associated with CHD (M€ uller, 1938, 1939; Boas et al., 1948). By the end of the 1940s, CHD was associated with many factors, including a faulty cholesterol metabolism, local arterial strain, hypertension, infection, allergy, and heredity, but none had been proved or consistently found (Truswell, 2010d). However, no link between serum cholesterol or dietary fat had been made with CHD at the time, even though it was widely acknowledged that lipids were present within the atheroma of CHD sufferers (Oliver, 1987; Truswell, 2010d). The first acknowledgment of a possible causal link between raised blood cholesterol levels and CHD was in the book “Heart Disease” by White (1947). This was followed by London-based cardiologist Paul Wood, who had said that “consideration should be given to the possibility that raised cholesterol might result from CHD” (Wood, 1950). As aforementioned, Windaus (1910) and Sch€onheimer (1926) had discovered increased cholesterol esters in human atheroma. Sch€ onheimer found that the overall cholesterol content of the aorta increased with the severity of the atheroma and the ratio of circulating cholesterol ester to free cholesterol ester was the same in the plasma (3:1). He hypothesised that the level of cholesterol present was too high to have been synthesized in the aorta itself, therefore the lipids in the wall emanated from direct infilitration from blood in the arterial lumen (Truswell, 2010d). The composition of aortic lesions has since been comprehensively characterized and studies have identified that cholesterol ester accumulates in the atheroma as they increase in severity. It was also found that similarly to plasma cholesterol ester, linoleic acid is the most abundant fatty acid present (Truswell, 2010d). Many researchers in the early 20th century noticed that lipids present in the blood serum must be either associated with a protein or contained within an emulsion. The nature of lipoproteins was first reported by Macheboeuf (1929), who isolated a crude lipoprotein from horse serum using salt precipitation. He later succeeded in purifying and characterizing α-lipoprotein from horse serum and showed that the lipid-free protein remained soluble. In the 1930s and 1940s, methods were developed for the large-scale fractionation of human serum, including researchers at Harvard who used the fractionated material to treat wounds in World War II (Cohn et al., 1946; Oncley et al., 1947). They later discovered that serum lipids were concentrated in two major

124 Chapter 4 fractions having α1- and β-mobility, respectively, on either free, starch, or paper electrophoresis. They estimated that 25% of the total cholesterol forms part of the α1-lipoproteins and the remainder is found in the β-lipoprotein fraction (Fredrickson et al., 1967). In 1951, groundbreaking research from Russ et al. (1951), using the same methods as Cohn and Oncley, demonstrated that different lipoprotein classes possess distinct biological functions, and proposed that specific lipoprotein patterns may be associated with CHD (Steinberg, 2004b). Around the same time, four large case-control studies published data indicating that people with CHD had higher average serum cholesterol levels than controls. These studies were much larger and more stringent and organized than previous studies. However, there was an overlap in plasma cholesterols between CHD cases and controls (Morrison et al., 1948; Gertler et al., 1950; Steiner et al., 1952; Oliver and Boyd, 1953). Surprisingly, few studies were carried out to test if changing dietary cholesterol intake affected people’s serum cholesterol levels. Indeed, it was widely assumed prior to the 1950s that the amount of cholesterol in the diet is reflected in the concentration of cholesterol in the blood serum, and eventually in the tendancy to develop atherosclerosis (Dock, 1946). Researchers had not previously thoroughly investigated dietary cholesterol, as its role as a risk factor for CHD had not been established yet (Truswell, 2010a). One study found that feeding eight eggs to three healthy subjects for 4 days had no effect on plasma cholesterol levels (Mjassnikow, 1926). A study perfomed by Keys and his collegues measured the cholesterol intake of more than 400 men and found no difference in serum cholesterols between those with low and those with high cholesterol intake (Keys et al., 1950b). Within the same study, a group of 41 middle-aged men lowered their dietary cholesterol by 50%, and it was found that their mean serum cholesterol did not change. In a further study, Keys varied the diets of 21 male diagnosed schizophrenics and found that dietary cholesterol had no significant effect on serum cholesterol, but changes in total fat intake had a substantial effect (Keys, 1952). Keys deduced that the serum cholesterol level is noticeably influenced by the amount of calories supplied by fats in the diet, that vegetable as well as animal fats have this effect, and that dietary cholesterol itself is insignificant at all levels of intake (Keys, 1952). This trend was noticed (Kempner, 1948; Chapman et al., 1950) and confirmed by other studies (Mellinkoff et al., 1950; Hildreth et al., 1951; Mayer et al., 1954). At first, Keys thought that dietary cholesterol did not significantly affect serum cholesterol levels. But, after more carefully controlled experiments in the Metabolic Unit of the Hastings State Hospital with 22 stable schizophrenic patients who had large varying amounts of cholesterol added to their already low cholesterol diet, researchers found that serum cholesterols were higher with added cholesterol. Taking into account other studies at the time that all reported an increase in serum cholesterol in individuals with increased dietary cholesterol (Beveridge et al., 1960; Connor et al., 1961; Steiner et al., 1962), Keys suggested that serum cholesterol elevations were related to the square root of the change in cholesterol intake (Grande et al., 1965). Therefore, the usual range of cholesterol intake was relatively small and easily masked by inter- and intraindividual variance in serum cholesterol as well as the effect of other dietary changes (Truswell, 2010b).

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At Harvard, a similar set of experiments also tested men with chronic schizophrenia in Danzers State Hospital (Hegsted et al., 1965). Each patient was fed an adequate diet with various fats and levels of cholesterol. It was found that across intakes up to 600 mg of dietary cholesterol, the response of serum cholesterol was linear. Many other experiment were carried out in the 1960s and 1970s that had flawed designs, low levels of participants, and a short time span, as highlighted by McGill (1979). He concluded that independent association between serum cholesterol and dietary cholesterol has not yet been observed in cross-sectional studies. But, in controlled experiments, increases of up to 600 mg cholesterol/day may increase serum cholesterol levels by 3–12 mg/100 mL/250 mg cholesterol. Increases above 600 mg/day in dietary cholesterol produce no significant rise in serum cholesterol. However, there seems to be a notable variability among individuals in response to dietary cholesterol, which may mean that some individuals are very responsive to dietary cholesterol while others are not (McGill, 1979; Truswell, 2010b). Research in this field continued to investigate the link between dietary cholesterol consumption and serum cholesterol through the 1970s. The use of isotope-labeled sterol human balance studies showed that as more cholesterol is absorbed, its excretion is increased and endogenous synthesis is decreased via suppresion of HMG CoA reductase. In the 1980s, research examined inconsistent results in relation to individual differences in the response of serum cholesterol to dietary cholesterol. It is thought that the genetic basis for cholesterol responders is the 4/4 genotype of apolipoprotein E. In the 1950s, Keys (1952) led a study that varied the type of fat used in their studies and examined how that affects serum cholesterol levels. Studies indicated that plasma cholesterol levels were decreased when a diet high in vegetable fat was substituted for a diet high in animal fat (Groen et al., 1952; Ahrens et al., 1954). Further studies showed that serum cholesterol was lower when individuals consummed corn oil versus the same intake of olive oil, coconut oil, or lard (Ahrens et al., 1955). These studies and more paved the way for further discoveries on the effects of different types of fats on serum cholesterol levels. A South African study by Bronte-Stewart et al. (1956) suggested that different effects on serum cholesterol affected by various animal- and plant-derived oils may be connected with the proportion of highly unsaturated and saturated fatty acids within the oils. Shortly after, these findings were confirmed by various studies (Ahrens et al., 1957a; Anderson et al., 1957; Malmros and Wigand, 1957; Williams and Thomas, 1957). A second succession of human metabolic studies on the effects of different fats was carried out by various groups, including Hegsted et al. (1965) at Harvard where it emerged that myristic acid (14:0) was a potent cholesterol elevator, which was later confirmed by Zock et al. (1994) and was also noticed by Sundram et al. (1994), to increase cholesterol levels when combined with lauric acid (12:0). Keys et al. (1965) concluded from their studies that stearic acid (18:0) (which can be converted to oleic acid (18:1) in the body) and saturated fatty acids containing less than 12 carbon atoms have very little effect on serim cholesterol, as was later confirmed by Bonanome and Grundy (1988). Research trends indicated that mainly lauric, myristic, and palmitic saturated fatty acids were responsible for elevating serum

126 Chapter 4 cholesterol levels. Several other studies were conducted to show that polyunsaturated fatty acids (PUFA) seemed to lower serum cholesterol. It was well-recognized that hydrogenation may convert some fats into cholesterol elevators (Bronte-Stewart et al., 1956), and fats with an intermediate degree of saturaton had little or no effect on serum cholesterol levels (Ahrens et al., 1957a). It was suggested that the reduction of serum cholesterol levels by PUFA was a correction of essential fatty acid deficiency (Sinclair, 1956). However, this was refuted in time as the vast majority of people who saw a reduction in their cholesterol level after an increased intake of dietary PUFA, typically from marine sources, never demonstrated signs that they had an essential fatty acid deficiency (Truswell, 2010a). The mechanisms behind how certain lipids affect serum cholesterol remained elusive for some time, and many thoeries surrounding excretion, increased fecal bile, and lipid absorption were develped (Gordon et al., 1957; Spritz et al., 1965; Truswell, 2010a). It was hypothesized by Spritz and Mishkel (1969) that as unsaturated fatty acids occupy more space due to their bent shape, this may alter the spatial configuration of the lipids so that fewer lipid molecules can be accommodated by the apoprotein of low-density lipoprotein (LDL). However, the mechanim wasn’t elucidated until after the discovery of the low-density lipoprotein receptor (LDLR) by Brown and Goldstein (1986), for which they were awarded the Noble Prize for Medicine in 1985. It has been revealed that if triglycerides containing myristate or palmitate are circulating in high amounts after feeding, then the LDLR activity is suppressed and less plasma LDL is taken up by the cells (Dietschy et al., 1993). If triglycerides containing predominantly unsaturated fatty acids are circulating, then the opposite effect occurs. It has been shown that saturated fatty acids reduce the size of the intracellular cholesterol ester pool whereas unsaturated fatty acids increase it. The size of the pool seems to regulate the activity of the hepatic LDLR (Dietschy et al., 1993; Truswell, 2010a). Another risk factor for CHD was proposed by Albrink et al. (1961) in the United States from a case-control (not perspective) study of 115 pateints who had a myocardial infarction. They proposed that increased fasting serum triglyceride levels was a better risk factor for CHD than serum cholesterol. Indeed, high levels of triglycerides are associated with a high total cholesterol/high-density lipoprotein (HDL) cholesterol ratio, which is an indicator of CHD risk (Castelli, 1986). Furthermore, men and women who have high triglyceride levels (>1.7 mmol/L) and a low high-density lipoprotein level (<1.03 mmol/L) have a significantly higher rate of CVD development (Castelli, 1992). The time consumption of food has little effect on serum cholesterol concentration. However, serum triglyceride level measurements require blood to be taken after an overnight fasting period, but before breakfast (Truswell, 2010e). These are endogeous triglycerides or very low-density lipoportiens (VLDL), which are typically induced by the overconsumption of carbohydrates (Ahrens et al., 1961). Many studies have tried to demonstrate that triglyceride levels are a risk factor for CVD development. The first propective study to examine triglyceride levels

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indicated that raised triglyceride seemed to be a risk factor for CHD events along with, and independent of, raised cholesterol at the 9-year follow up (Carlson and B€ottiger, 1972). Later, at the 14.5-year follow up, serum triglycerides were more significantly associated with deaths than cholesterol (B€ ottiger and Carlson, 1980). However, there was a strong correlation between cholesterol and triglyceride (Truswell, 2010e). Research in the area of serum triglycerides has been reviewed by Hulley et al. (1980), who found that, after multivariate adjustment for major risk variables such as total cholesterol, highdensity lipoprotein, and obesity, triglycerides are not a suitable independent risk factor for CHD (Truswell, 2010e). Hulley et al. (1980) suggested that the screening and treatment of persons with elevated triglyceride levels should cease until further, more conclusive evidence is available. As result, triglyceride measurements have not ceased as many still value triglycerides as a risk factor (Hokanson and Austin, 1996). However, triglyceride levels are now in the second row of CHD risk factors for several reasons: they show wider variation between and within individuals than cholesterol; they are reduced by exercise; triglycerides also steadily increase as age and weight increase; use of oral contraceptives among women elevates triglycerides; and other lifestyle factors such as high alcohol intake increases triglycerides (Altekruse and Wilmore, 1973; Cullinane et al., 1982; Castelli, 1986; Truswell, 2010e). However, some studies such as the Framingham studies indicate that the triglyceride level may be an independent risk factor for CHD in women, but not men. Therefore, it should be considered in patient treatment options (Castelli, 1986).

4.3.2 Lipoproteins and Coronary Heart Disease In his early academic career, John W. Gofman went to medical school, earned a PhD in physics, and worked under two Nobel laureates on the Manhattan Project. Gofman was convinced that blood cholesterol was a major contributor to the development of CHD. Gofman adapted a technique developed by Swedish chemist Theodore Svedberg known as analytical ultracentrifugation. A colleague of Svedberg’s, Pedersen had studied proteins in human serum but encountered an artifact he could not understand or rationalize, which he called “X-protein.” It was found in varying concentrations in different samples and seemed to change concentration as the analysis went on (Pedersen, 1947). He deduced that the X-protein may in fact be a lipoprotein, which was later confirmed by Gofman et al. (1949) to be LDL and other lipoproteins. Gofman and his team went on to develop accurate and reproducible techniques to isolate plasma lipoproteins into subclasses and measure their concentration. Gofman’s new technique gave rise to breakthrough pioneering research. Gofman even postulated that certain lipoprotein fractions were more atherogenic than others (Gofman et al., 1950) when he found that most of the 104 male patients with proven myocardial infarction had cholesterol supporting low-density lipoproteins of the Sf 10–20 fraction, whereas only half the controls presented with these lipid profiles. Gofman suggested that the controls who carried the Sf 10–20 fraction were developing atherosclerosis that would manifest later

128 Chapter 4 in life (Truswell, 2010d). Gofman had discerned that, of all the lipoproteins, LDL was associated with CHD. In a later case control study of 50 cases versus 50 controls, Oliver and Boyd (1955) discerned that β-lipoprotein was increased in CHD cases, but α-lipoprotein was lower in comparison to the controls. This revolutionary research was the first of its kind to acknowledge the presence of low-density lipoproteins (LDL, then known as β-lipoproteins) and high-density lipoproteins (HDL, then known as α-lipoproteins), which were divided based on their densities (LDL: below 1.05 g/mL; HDL: above 1.05 g/mL) and floatation rates (Gofman et al., 1949). Later, Gofman developed his “atherogenic index” that divided different subclasses according to their supposed atherogenicity (Gofman, 1956). Much of Gofman’s research was considered controversial at the time, compounded by the fact that Ancel Keys had said that the use of lipid classes could not discriminate between healthy individuals and those with atherosclerosis (Keys, 1951). Gofman’s theory was not widely recognized, partly due to his background in physics and not the conventional medical community (Reynolds and Tansey, 2006). Gofman went on to propose that using a scale such as his atherogenic index or a similar profile that accounts for the size and concentration of lipoproteins should possess the capability to predict coronary risk better than just total cholesterol level. However, as highlighted by Steinberg (2004b), Gofman never went on to do a large prospective study due to financial constraints. Instead Gofman was funded by the National Institutes of Health (NIH) to expand his research and form a collaboration between the University of California at Berkeley, the Cleveland Clinic, the University of Pittsburgh, and the Harvard School of Public Health. In total, the collaboration analyzed almost 5000 healthy men between 40 and 59 years old who were followed for 3 years. Of these, 82 participants suffered a cardiac event. The overall study had its flaws but the final report made a number of key points; it was clear that the lipoprotein pattern was similar to total serum cholesterol levels as an indicator of CHD risk (Gofman et al., 1956). Also in 1956, Gofman and his colleagues examined the lipoproteins of 351 patients who had previously suffered a myocardial infarction in a blind study over a 5-year period. At the end of the study, 52 patients had passed away. However, Gofman had observed that the 299 patients who survived were characterized by lower atherogenic index values versus those who died (Lyon et al., 1956). In the same report, Gofman analyzed 280 patients who suffered a myocardial infarction and were prescribed a low-fat and low-cholesterol diet. The study reported that those who adhered to the diet maintained a significantly lower atherogenic index value. In patients who had not adhered to that diet, the recurrence of myocardial infarction and death rate was four times as high as those patients who said they adhered to the diet (Lyon et al., 1956). The study was flawed in its design, as these deductions were made using a written questionnaire and a relative completed the questionnaire for those who died. However, for the time it was very forward thinking and innovative. Gofman’s work publicized the complexity of human plasma lipoproteins and introduced the notion that lipoproteins were associated with atherogenic indices and that diet can alter these

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indices and the relative risk of CHD. Between 1955 and 1965, diet was firmly in the crosshairs of many physicians as the cause and treatment target for CHD (Keys et al., 1955; Keys, 1956; Ahrens et al., 1957b; Balch et al., 1958; Stamler, 1958; Central Committee for Medical and Community Program of the American Heart Association, 1961; Stamler et al., 1963).

4.4 Lipid Hypothesis: Origin, Development, and Prelude to the Seven Countries Study The concept that cholesterol plays a causal role in atherosclerosis and cardiovascular disease has been the subject of controversy since the 1950s (Thompson, 2009). The relationship between dietary fat, SFA, serum cholesterol, and CHD has been referred to as the “diet-heart hypothesis,” “cholesterol hypothesis,” or “lipid hypothesis.” This hypothesis has been refuted by many (Mann, 1977; Ahrens, 1979; McMichael, 1979; Oliver, 1981) and so the term “cholesterol controversy” emerged (Steinberg, 2006). The studies of Gofman and the four large case-control studies of Morrison et al. (1948), Gertler et al. (1950), Steiner et al. (1952), and Oliver and Boyd (1953) played a major role in establishing a link between diet and cholesterol levels. The idea that hypercholesterolemia was a causative factor for CHD began to take hold among many physicians, one of whom was Dr. Ancel Keys. Already an accomplished physiologist by 1947, Keys suggested that studies are required in humans to discern the effects of diet on blood lipids, as he was convinced that serum cholesterol was affected by the type and amount of fat in the diet (Keys et al., 1955). Keys hypothesized that if serum cholesterol was a major determinant of CHD, then populations who had fat-rich diets should have higher serum cholesterol levels leading to higher heart attack rates in comparison to countries who had lower serum levels of cholesterol due to a diet lower in fat (Steinberg, 2005). Keys and his colleagues devised a systematic cohort study examining risk factors implicated in the incidence of CHD in business men in Minnesota (Keys et al., 1963). Keys was perplexed by the fact that seemingly healthy middle-aged (45–55 years) business and professional men were frequently dying across America due to CHD. Keys had many questions and wanted to differentiate whether preexisting characteristics or risk factors between individuals would provide information on the causes for preventative means against CHD. Keys recruited 281 male executives who received an annual physical examination over a 15-year period and a further examination in a follow-up study in 1983. Keys analyzed participant’s blood pressure, weight, and urine, followed by an ECG and a blood sample. Serum cholesterol levels were estimated and skinfold thickness measurements were taken between 1948 and 1954. Over the course of 15 years, 32 participants developed CHD, where 22 men suffered a myocardial infarction and 17 deaths were recorded. Participants were followed for 35 years thereafter; in that period, 183 men died, 110 survived, and 1 was never found. In 1951, Keys was asked to speak at a FAO congress in Rome, where he had said that in the United States, 50% of males from the age of 39–59 were condemned to die of a heart attack and

130 Chapter 4 nobody could explain why. Keys wrote in his private memoirs, Adventure of a Medical Scientist, that he had encountered Professor Gino Bergami, a medical doctor from the University Hospital of Naples. Professor Bergami had stated that cardiovascular diseases were not present in his hospital and he did not know why, which may be useful to investigate. Keys returned to England where he was spending a year on sabbatical as a Senior Fulbright Scholar at Magdalen College Oxford from 1951 to 1952. However, Keys was fascinated by Bergami’s claim that people in Naples had no cardiovascular diseases (Moro, 2016). Keys kept correspondence with Bergami through telegram and arranged to visit Naples to see for himself. By invitation of Bergami and Professor Flaminio Fidanza, who also claimed heart attacks among workers in Naples were rare, Keys bought a car and he and his wife Margaret Haney Keys traveled for four days before reaching Naples (Moro, 2016). Margaret Keys was a biochemist and played a role in collecting blood samples from the Neapolitan steelworkers and locals. Keys noticed that, in contrast to, the blood samples obtained from business men in the Minnesota, the Neapolitan population had much lower cholesterol levels. Keys also reported that total cholesterol levels in men from England and Minnesota were similar but levels in Naples were lower, and considerably lower again in working-class citizens from poor socioeconomic backgrounds in Madrid (Keys, 1952). He theorized that cholesterol may cause cardiovascular disease. Keys’ intuition led him to believe that diet may be a significant difference between these cohorts, so they gathered data on the types of food the locals ate. They found that the Neapolitan diet included vegetables, legumes, broccoli, all kinds of fruits, unrefined cereals, and dairy products, some fish, but very little meat (Moro, 2016), a diet that is promoted to this day as the Mediterranean diet (Stamler, 2013). On sabbatical, Keys recruited volunteers from laboring and executive classes who provided informal surveys to Keys and his new colleagues, who then collected blood, queried the volunteer’s diet, and surveyed local hospitals for heart attack victims. Keys noticed that serum cholesterol levels tended to be low in working populations and higher in executive groups and heart attack victims (Keys et al., 1954a,b). From 1954 to 1956, Keys ran a series of informal assessments in contrasting populations with the participation of cardiologist Paul Dudley White in Boston. They found collaborators in Finland, Japan, South Africa, and Spain whose surveys suggested that CHD rates were high in the United States, Finland, and in people of European descent in South Africa, but were low in the Mediterranean countries, Japan, and the native populations of South Africa. These studies served as a prelude to the formal Seven Countries Study.

4.5 Ancel Keys (1904–2004) Keys was born in Colorado Springs, Colorado, in 1904. His family moved to San Francisco in 1906 where he first began his academic career, initially studying chemistry. After taking time off to work, he returned and studied for a bachelors in economics and political science.

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An M.Sc. in Zoology and a PhD in oceanography and biology at the University of California, Berkeley, shortly followed. On completion of his PhD, he was awarded a fellowship under the supervision of August Krogh where he published numerous papers on aquatic physiology. Once his fellowship was complete, Keys established himself at Cambridge before taking time off to teach at Harvard University. He then returned to Cambridge to receive another PhD, this time in physiology. From there, Keys was involved in a number of famous physiological studies investigating the effects of elevation and starvation on human physiology (Keys et al., 1950a). Keys was also involved in the development of the K rations that were used by the US military in World War II, which were supposedly named after him (Schemmel et al., 2001; Cheng, 2005). When Keys was director of the Laboratory of Physiological Hygiene at the University of Minnesota, he conceived the “Seven Countries Study” which aimed to elucidate the lifestyles and mass phenomena that determine high and low population rates of heart attacks (Blackburn, 1995).

Reprinted with permission from Elsevier (The Lancet, 2004, 364 (9452), 2174 pp).

4.5.1 The Seven Countries Study (1958–2000) The Seven Countries Study (SCS) was a collaborative project initiated and developed by Keys. The focus of the study was to determine associations between the intake of food groups and 25-year mortality rates caused by coronary heart disease (CHD). This study was the first attempt to show strong relationships between the eating habits of contrasting populations and their long-term incidence of CHD. The objective was to explore in detail the associations of diet, other risk factors, and disease rates between populations and among individuals within populations, using standard measures by trained survey teams with blindfolded coding and analysis of data (The Seven Countries Study, 2017a). Baseline measurements were carried out between 1958 and 1964. Several individual characteristics were measured

132 Chapter 4 in 12,763 middle-aged men belonging to 16 cohorts in seven countries. The participating countries were the United States, Finland, the former Yugoslavia, the Netherlands, Italy, Greece, and Japan (Menotti et al., 1999). On examination, participants provided information on their background, lifestyle, and medical history, followed by a physical examination, laboratory testing, and a measurement of dietary data. The physical examination included measurements of height, weight, skinfolds, mid-arm circumference, blood pressure, heart and lung auscultation, peripheral pulses, electrocardiogram, resting heart rate, and a fundus examination. Laboratory tests included the examination of respiratory function, resting and postexercise electrocardiograms, and finally the collection of blood for biochemical tests and the evaluation of serum lipids. Dietary information was gathered from 20 to 50 men using the weighed record method in random subsamples of the 16 cohorts of men aged 40–59. An effort was made to standardize all methods and almost all principal investigators had been trained at the Laboratory of Physiological Hygiene at the University of Minnesota (The Seven Countries Study, 2017b). Follow-up surveys and reexaminations of all survivors were conducted in all cohorts after 5 and 10 years, following the same procedures as in the baseline examination. A systematic monitoring of nonfatal cardiovascular events took place during the first 10 years of follow-up, with periodic collection of interim information on illness and death. Mortality data collection continued in all cohorts for a minimum of 25 years. Using repeat surveys, cardiovascular risk factors of survivors in nine European cohorts after 25, 30, 35, and 40 years were also reexamined. Mortality data was collected up to 40 years in 13 of the 16 cohorts and is due to be collected again on the 50th anniversary of the start of the study (The Seven Countries Study, 2017b).

4.5.2 The Seven Countries Study: Serum Cholesterol and the Link to CVD By the 1950s and early 1960s, a remarkable series of epidemiologic studies had begun, some of which continue to the present day. The ultimate goal of these studies was to identify factors that could explain either differences in rates of occurrence of CHD between populations or differences in risks of coronary events among individual members of a population, of which the SCS was one (Pitsavos et al., 2003). In the mid-1950s to the 1960s, Keys and coworkers enrolled and examined more than 12,000 men. They performed an epidemiological study aiming to explore the prevalence and lifestyle/clinical determinants of the development of CHD (Menotti et al., 1996, 1999, 2007). Within these studies, the mortality rates of 16 populations from the seven countries revealed that the rates of CHD were lower in the Mediterranean region than Northern European countries, the United States, and Japan. It is notable that especially the residents of Corfu and Crete had a marked decrease in cardiovascular death rates among all the investigated cohorts. Such a low prevalence of CHD was attributed to their lifestyle and especially to their dietary habits, which are now revered as the traditional Mediterranean diet (Papandreou and Tuomilehto, 2014). Previous analyses have shown that the adoption of a dietary pattern rich in fruits, vegetables, and olive oil explains, in part, the large differences in the 25-year CHD mortality of the

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Mediterranean cohorts of Italy and Greece compared to cohorts from northern Europe, the United States, and even those of neighboring inland Mediterranean countries such as Croatia and Serbia (former Yugoslavia) (Pitsavos et al., 2003). Forty-year follow-up studies examining CHD mortality of the seven countries identified a slight decline in the United States, Finnish, Dutch, and Japanese cohorts, a moderate increase in Italy, and an exponential increase in the cohorts of Serbia and Greece (Menotti et al., 2007). The reasons for the decline in CHD death rates are unclear and possible explanations involve the development of primary and secondary prevention strategies due to modern healthcare, including emergency services and coronary care units. Recently, it was shown that many countries in the Mediterranean basin and especially Greece are drifting away from the Mediterranean dietary pattern, whereas countries in Northern Europe and some other countries around the world are taking on a Mediterranean-like dietary pattern (da Silva et al., 2009). However, an adherence to the traditional Mediterranean diet among adults living on the Greek islands such as Crete and the Ionian Islands was observed. People living in the Greek regions appear to better maintain these traditional dietary habits and this could have favorable effects on CHD mortality rates in these regions (Rees et al., 2013). The above deductions contrast with the adults living in the big cities that have adopted a Westernized lifestyle, thus a maladaptive diet and lifestyle. Besides these observations at a local level, a continuous increase in CHD mortality over the past few decades has also been observed at a national level in these Mediterranean countries, in particular Greece and Serbia. Previous results from the Seven Countries Study showed that serum cholesterol was strongly related to CHD mortality, both at the population and the individual levels (Menotti et al., 1996, 1999). The strength of the association between serum cholesterol and CHD mortality was similar in different cultures. However, the absolute risks differed substantially. Kromhout reported that at a serum cholesterol level of 200 mg/dL, the 25-year CHD mortality rate was five times higher in Northern Europe compared to Southern Europe and the Mediterranean (Kromhout, 1999). According to Kromhout, it can be concluded that the relations between diet, serum cholesterol, and CHD are more complex than originally thought because it is not just dietary cholesterol but other lipids and antioxidants that may play a role in the genesis or prevention of atherosclerosis (Kromhout, 1999). Thus, it is very important to emphasize that limitations exist in epidemiological studies and statistical correlations between risk factors and CHD frequently lack biochemical or biological mechanistic evidence. As was discussed in Chapter 3, even though there is a clear correlation between hyperlipidaemia and atherogenesis, a growing body of literature indicates that soluble and cellular immune factors associated with chronic inflammation can also promote atherogenesis, independent of hyperlipidaemia (Angelovich et al., 2015; Tsoupras et al., 2018). Also, several recent observational epidemiological studies also support the hypothesis that inflammation is the underlying cause of atherosclerosis and CVD (Herder et al., 2017; Welsh et al., 2017), either during hyperlipidaemia or independently of hyperlipidaemia (Angelovich et al., 2015).

134 Chapter 4 In addition, differences observed between population samples (i.e., like those observed between local and national adults in several countries) can also provide limitations in the criteria of choosing those populations in the Seven Countries studies and other similar epidemiological studies. For example, even from the early 1990s, the French paradox was established, pointing out that low coronary heart disease (CHD) death rates were observed in several populations in France despite their high intake of dietary cholesterol and saturated fat (Renaud and de Lorgeril, 1992). Furthermore, in the Ionian Islands, Crete, and Japan, the adherence to the healthy Mediterranean-type diets could partly explain the low percentage of CHD mortality. The inverse association between the Mediterranean diet and CHD can be attributed to its protective effects to lower BMI, blood pressure, and lipid levels as well as possible improvement in endothelial function, decreases in inflammation and oxidation, and reduced insulin resistance (Pitsavos et al., 2003; Papandreou and Tuomilehto, 2014). Unfortunately, based on the results of the Seven Countries studies and other similar epidemiological studies that correlate the levels of cholesterol with CHD, general dietary and medical practice guidelines shifted toward trying to reduce cholesterol levels as the best way to prevent CVD without taking into consideration all the other previously mentioned parameters, risk factors, and beneficial dietary patterns and lifestyles. Nevertheless, the medical community recommended the use of statin therapies in order to decrease the levels of cholesterol by inhibiting its synthesis. However, it is debated whether or not the reported benefits of statins outweigh the potential side effects they may induce, as will be discussed in Chapter 6. For example, the 2013 American College of Cardiology (ACC) and American Heart Association’s (AHA) cholesterol guidelines departed significantly from the paradigm of simply treating patients by targeting LDL cholesterol and instead shifted treatment toward differentiating between primary versus secondary CVD and the overall risk of developing CVD (Stone et al., 2013). In subjects that do not meet the criteria in the guidelines for LDL levels, other risk factors including genetic hyperlipidaemia, family history, elevated high-sensitivity C-reactive protein (CRP), coronary artery calcium score, ankle-brachial index <0.9, and elevated lifetime risk for atherosclerotic cardiovascular disease can be used to refine treatment decisions (Hong et al., 2017). Increasingly, clinical trials and guidelines are using CVD risk to guide treatment strategies. Thus, according to these guidelines, the first clinical variable affecting the initiation of statin therapy is the presence of CVD. In the absence of CVD, LDL cholesterol, diabetes, and atherosclerotic cardiovascular disease risks make up the criteria for starting statins for the primary prevention of CVD (Hong et al., 2017). Prior to the current AHA/ACC 2013 guidelines, treatments had focused on cholesterol levels for determining the initiation of statins and other nonstatin lipid-modifying therapies while the new guidelines ushered in a paradigm shift that used CVD risk, instead of LDL-C levels, as a guide to treatment. However, this change in guidelines significantly expanded the prescription of statins to approximately 56 million individuals in the United States, a 10 million patient increase due to the establishment of these new criteria (Hong et al., 2017).

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Taking into consideration all the above, it is astonishing how the major concluding remarks of the main epidemiological studies (including those of the Seven Countries and the French Paradox) concerning the benefits of a healthy lifestyle and diet toward CVD were shifted to the increased need by guidelines to reduce cholesterol levels by introducing specific drug therapies along with their observed side effects. All the above will be further analyzed in the following chapters of this book.

4.6 Familial Hypercholesterolemia Before the Seven Countries Study, one of the first suggestions that there was a link between cholesterol levels and CHD developed from a condition called xanthomatosis. Xanthomas are large benign cutaneous manifestations of cholesterol that usually develop attached to a tendon or on the back of the hands and ankle. Pinkus and Pick (1908) were the first to note that xanthomas were composed of cholesterol esters originating from the blood. Lehzen and Knauss (1889) reported that a 3-year-old boy developed xanthomatosis and suddenly died at age 11 due to large xanthomatas present in the aorta and coronary arteries. His sister also developed this condition, and we now know that these children had homozygous FH (Steinberg, 2005), which is an autosomal codominant genetic disorder characterized by increased levels of total cholesterol and LDL cholesterol within the bloodstream (Civeira, 2004). Homozygous individuals can develop chronic CHD at a young age. The genetic causes of FH have been examined since the early 1900s and it was recognized that people with FH were also at risk of developing CHD (M€ uller, 1938). The FH clinical phenotype is associated with an increased risk of CHD and premature death. FH is mainly caused by mutations of the LDLR genes, the low-density lipoprotein receptor genes (LDLR), Apo-B, or proprotein convertase subtilisin/kexin type 9 (PCSK9) (Brown and Goldstein, 1986). However, other genetic variances do exist (Austin et al., 2004). The ratio of heterozygous FH individuals is 1:500 on average; a higher incidence is present in certain populations such as French Canadians, black South Africans, and Christian Lebanese. More recent estimates from the Netherlands, which has researched FH heavily, suggest that the incidence is much higher at 1:200 and it is estimated that 34 million people are affected by FH worldwide (Nordestgaard et al., 2013). The incidence of clinical homozygous FH has been estimated at 1:1,000,000 (Vladimirova-Kitova and Kitov, 2016). Heterozygous FH individuals are at risk of developing CHD prematurely between the ages of 30–40. Gofman had confirmed that it was the LDL and IDL fractions that were elevated in FH (McGinley et al., 1952). FH is often cited as proof that cholesterol causes CHD. However, patients with FH have exceptionally high blood cholesterol levels. Those who are heterozygous FH tend to have cholesterol levels in the range of 300–400 mg/dL, but homozygous FH individuals can have as high as 1000 mg/dL. Thus, it is difficult to extrapolate meaningful comparisons to the general population. However, the belief that this is proof that cholesterol causes CHD persists among many researchers. This is partly due to the fact it is difficult to deduce what are acceptable or “normal” levels of serum cholesterol, considering that these normal values were deduced

136 Chapter 4 in affluent western countries whose cholesterol levels may not be optimal or healthy (Truswell, 2010d). Of further interest is a report published by the Simon Broome Register Group (1991), which treats FH with cholesterol-lowering drugs known as statins. They found that a subgroup of FH sufferers over 60 with cholesterol levels over 300–350 mg/dL had no increased risk of CHD (Neil et al., 2008). However, these were thought to be a highly selective group who may have been less susceptible to the atherogenic effects of LDL-C, or may have had favorable genetic polymorphisms such as an inferior functioning variant of the PCSK9 gene, which is associated with a lower risk of CHD in the general population (Cohen et al., 2006). Although FH sufferers are at greater risk of developing CVD, no associations can be drawn between high LDL levels in these individuals and CVD, as these cholesterol levels do not correspond with general cholesterol levels within populations.

4.7 Concluding Remarks The dawn of the lipid hypothesis stemmed from the early works of great scientists such as Anitschkow and Chalatow. Their research progressed, and it was clear that lipoproteins play a role in atherosclerosis. The work of Gofman identified the different types of lipoproteins, and Keys developed his theories linking CHD with certain diets that raised cholesterol levels, leading to the development of the lipid hypothesis and the global fixation to reduce cholesterol levels. Although great strides toward elucidating the causation mechanisms of CVD have been made, a unifying theory remains elusive. We have discussed in Chapters 1–3 that further research needs to focus on the role of inflammation, how the environment, genetics, diet, and lifestyle are interconnected, and how they affect our cardiovascular health over time. As will be discussed further in this book, the lipid hypothesis has raised many questions but has not provided all the answers yet. The previous notion that dietary cholesterol may increase your risk of developing CVD is not supported in the literature. However, there is a link between saturated fat intake and changes in serum cholesterol. It is not yet clear whether reducing saturated fat intake can lower the rates of CVD mortality. The lipid hypothesis has been the target of modern primary prevention of CVD to some effect. However, as will be discussed further, it is clear that a multifactorial approach is required for a multifaceted disease, while several studies have reported positive results concerning the benefits of dietary and lifestyle modifications, which will be explored throughout this book.

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