History in medicine: the story of cholesterol, lipids and cardiology

Introduction

Checking lipid values and prescribing lipid-lowering drugs is core business for cardiologists. However, they might be unaware of the interesting moments in time when cholesterol and low-density lipoprotein (LDL) receptors were discovered, of drug developments or the history of diet and lipids. Following earlier historical papers on the aortic valve and Soldier’s heart, this paper will tell you the story of lipids [1, 2] and its many Nobel prizes.

History

The word cholesterol consists of chole (bile) and stereos (solid), followed by the chemical suffix -ol for alcohol. The basic structure of this organic molecule is a sterol nucleus, synthesised from multiple molecules of acetyl-coA (Figure 1). This nucleus can be modified using various side chains to form cholesterol, cholic acid (the base of bile acids) and many steroid hormones. Cholesterol is an essential structural component of all cell membranes, and a precursor of vitamin D [3]. In vertebrates, hepatic cells produce the greatest amounts.

Atherosclerosis has been present for more than 4,000 years [4]. The relationship with lipids is much younger. In 1665, Robert Boyle (1627-1691), as in “the law of”, discovered a fat transport system in animals [5]. François Poulletier de la Salle (1719-1788) first identified solid cholesterol in gallstones in 1769 [5]. Some ten years before, he isolated crystals from cholesterol for the first time. As his work was never published, attribution and dating are known only roughly, quoted by Pierre-Joseph Macquer (1718-1784) and Felix Vicq-d'Azyr (1748-1794) [6].

Figure 1. Chemical structure of cholesterol.

It was not until 1815 that Michel Eugène Chevreul (1786-1889) rediscovered it and named the compound "cholesterine" [5, 7, 8]. Chevreul is one of the 72 famous French scientists whose names are inscribed on the Eiffel Tower in Paris. Cholesterol in human blood was first reported in 1833 by Félix-Henri Boudet (1806-1878) [5]. Then, in 1856, Rudolf Virchow (1821-1902) described the atherosclerotic plaque as a fundamental lesion of atherosclerosis [5]. The molecular structure of cholesterol (C27H46O) was described in 1888 by Friedrich Reinitzer (1857-1927) [7], followed by a case report on xanthomatosis and sudden cardiac death of an 11-year-old child by Lehzen and Knauss a year later [9].

Between 1907 and 1909, Alexander Ignatowski (1875-1955), who was involved in the laboratory of Nobel prize winner Ivan Pavlov, investigated whether an excess of protein in diet was toxic and accelerated the ageing process [10]. He fed rabbits large amounts of meat, eggs and milk. It was indeed toxic for young rabbits, while adult rabbits developed atherosclerosis. As the latter was considered a hallmark of ageing, the hypothesis was proven according to Ignatowski [10, 11].

Adolf Windaus (1876-1959) reported in 1910 that plaques in aortas from atherosclerosis patients contained 20 times more cholesterol than normal aortas [7, 10, 12]. While his PhD concerned Digitalis, he won the Nobel Prize in 1928 for his work on cholesterol [7, 8].

In 1912, Nikolai Anichkov (1885-1964) and Semen Chalatov (1884-1951) reproduced Ignatowski’s work [11], by feeding pure cholesterol to rabbits, thus eliminating proteins. This is considered to be the first experimental production of atherosclerosis [7, 10]. Anichkov, who later became a good friend of Joseph Stalin [8], laid the basis for the “Lipid Hypothesis” in 1913 [13]. However, when similar experiments in dogs and rats did not reproduce confirmatory results, his findings were rejected [10]. For the next 35 years, little work was done on the lipid hypothesis. Later, it appeared that dogs and rats, unlike rabbits, are quite efficient in converting cholesterol into bile acids [10].

The role of diet in cholesterol was described as early as 1916 [9]. Cornelis de Langen (1887-1967) reported much lower cholesterol levels in natives of Indonesia than in Dutch colonists. In 1922, he performed probably the first controlled study of dietary effect on cholesterol. Indonesians, put on a “Dutch diet” (rich in eggs and meat) for 3 months, presented an average cholesterol rise of 27%. Indonesians who had migrated to Amsterdam had the same high cholesterol levels as the Dutch. In 1924, Simon Henry Gage (1851-1944) and Pierre Augustine Fish (1865-1931) showed that after a fatty meal human blood contained tiny particles (1 µm), chylomicrons [5]. Heinrich Wieland’s (1877-1957) important work on bile acids and sterols earned him the Nobel prize in 1927 [7, 8].

Lipoproteins

In 1929, Michel Macheboeuf (1900-1953) suggested that circulating lipids exist in complexes with proteins: this was the discovery of lipoproteins [10, 13]. Later it was shown to be an α-globulin, nowadays called high-density lipoprotein (HDL) [5]. Ultracentrifuges were only invented in 1925 by Theodor Svedberg (1884-1971) (Nobel prize 1926), an important asset in further research. By 1932, Wieland showed the correct structure of cholesterol [8]. In 1938, Siegfried Thannhauser (1885-1962) and Heinz Magendantz (1899-1972) were among the first to see the relationship between atherosclerosis, xanthomas and high cholesterol [5]. The genetic connection between cholesterol and heart attacks was reviewed in 1939 by Carl Müller (1886-1983) [7]. He saw clinical pictures fitting the work of Francis Harbitz (1867-1950). Familiar hypercholesterolaemia (FH) was at first called Müller-Harbitz disease [9, 12, 14]. Müller was already advising that FH patients should be put on a “poor in cholesterol diet”, although there were no empirical data at that time: this was the “diet-heart hypothesis” [15].

In 1941, using plasma gel electrophoresis, Gunnar Blix (1894-1981) observed that significant amounts of lipids were associated with α- and ß-globulins [5, 13]. In 1947, Kai Pedersen, student of Svedberg, found a labile lipid protein, which was considered an artefact in Schlieren profiles. Therefore, he concluded that serum was not suitable to study due to interference with “protein X” [10, 13]. Nobel prize winner (1964) Dorothy Crawford Hodgkin (1910-1994), the first to use X-ray diffraction, established unambiguously the cholesterol structure in 1945 (Figure 2) [8].

Figure 2. Dorothy Crawford Hodgkin (1910-1994).

During and after World War II (WWII), Edwin Cohn (1892-1953) was working on isolating proteins in human plasma (“Blood fractionating project”) for use in war casualties (1946) [5]. As cholesterol is the starting material for preparing steroids, everybody was eager to synthesise cholesterol during WWII. In Oxford, Robert Robinson (1886-1975) (Nobel prize 1947) with his student John Cornforth (1917-2013) (Nobel prize 1975), and in Harvard Robert Burns Woodward (1917-1979) (Nobel prize 1965) almost simultaneously performed the first total syntheses of cholesterol in 1951 [8].

John Gofman (1918-2007), later crowned as “the father of clinical lipidology” [16], was responsible for the major breakthrough in ultracentrifuge research on lipoproteins [13]. He was a scientist who started medical school, but before finishing it he went to work on the Manhattan Project [10]. The Manhattan Project, a research and development project during World War II that produced the first nuclear weapons, was led by the USA with the support of the United Kingdom and Canada. Nuclear physicist Robert Oppenheimer was the director of the Los Alamos Laboratory that designed the actual bombs. Gofman with his student Frank Lindgren (1924-2007) solved the problem of the anomaly of “protein X” in 1949, which appeared to be LDL, by using one of the few ultracentrifuges in the world [10, 13].

In the meantime, inspired by the death of American President Franklin D. Roosevelt in 1945 due to cerebrovascular disease [17], the American National Heart Institute (NHI) was established in 1948.

More research into lipids, lifestyle and atherosclerosis

In the early 1950s, it became clearer (for some) that there was an association between lipoproteins and cardiovascular disease (CVD) [13]. Gofman introduced the “atherogenic index”, i.e., the association of different types of lipoprotein with coronary artery disease (CAD) risk [13]. The relationship with food pattern was also appreciated. Gofman’s wife Helen co-authored probably the first low fat, low cholesterol “diet-heart” cookbook in 1951, for which John wrote the introduction [10]. Also, in 1951 it was found that premenopausal women had higher levels of α-lipoproteins than men [10].

Epidemiological studies

In those exciting times, large population studies were born, and epidemiological evidence was gathered. In 1950, the Framingham Heart Study was started by the NHI [9]. Thomas Dawber (1913-2005) and William Kannel (1923-2011) published a landmark article in 1957 showing that higher cholesterol levels were proportionally correlated to increased CAD [8]. Another important achievement was the Seven Countries Study (Japan, Finland, the Netherlands, Yugoslavia, Italy, Greece, USA) by Ancel Keys (1904-2004), which started in 1954-1956 [9]. Keys was convinced that cholesterol levels were determined by diet, suggesting that populations using fat-rich diets should have higher cholesterol levels and more heart attacks. Around that same time, Edward Korn described an enzyme, naming it lipoprotein lipase [17].

Dietary interventions

When epidemiological evidence presented an association between diet and congenital heart disease (CHD), the first dietary intervention studies were initiated. Probably the very first was the Lester Morrison study. Morrison (1908-1991) was a cardiologist in Los Angeles, USA, who stated as early as 1946 that lowering cholesterol in diet was beneficial. In 1951 and 1955 positive results were published, but his intervention was not double-blinded [9]. In 1959, John Yudkin (1910-1995) advocated that “evidence points to a multifactorial aetiology of cardiac infarction”, including diet, mental stress, obesity, sedentary lifestyle and smoking [15]. As early as 1961 (!) the American Heart Association (AHA) recommended reducing dietary fat, calorie intake and substituting saturated by polyunsaturated fats [9]. In the late 1960s, several well designed dietary intervention studies were performed, such as the Paul Leren Oslo study (1966), Wadsworth Veterans Administration Hospital study (Los Angeles) (1969) and the Finnish Mental Hospitals Study (1968) [9]. These were NOT low-fat studies, but had decreased saturated fat and increased polyunsaturated fat, leading to a substantial drop in cholesterol levels (between 12% and 18%) [9].

As for laboratory research, in 1955 Richard Havel (1925-2016) adapted the Lindgren method to separate lipoproteins, thus enabling broad implementation and use in clinical investigations [13]. Another important researcher entering the lipid field was Donald Fredrickson (1924-2002). In 1959, he was the first to describe (apart from apolipoprotein A and apolipoprotein B) apolipoprotein C [13]. Bernard and Virgie Shore (1928-2014) did a lot of work on apolipoproteins and were contributors to the discovery of ApoE, which was published by Gerd Utermann (b. 1939) in 1975 [13].

Currently, the mouse model is most frequently used in atherosclerotic research; however, the first mouse model was described by Robert Wissler (1917-2006) around 1960 [18].

In 1964, Konrad Bloch (1912-2000) received the Nobel prize for his work on the pathway of cholesterol biosynthesis [12]. In the same year, Kåre Berg (1932-2009) described Lp(a) [13]. Years later this was shown to follow an autosomal dominant inheritance pattern and was associated with an increased risk for CHD [13].

In the meantime, in 1967 Fredrickson discovered that lipoprotein patterns could be phenotypically classified into five types [17], replacing the previous categorisation into α- and β-lipoproteins [13]. Fredrickson’s system was adapted by the World Health Organization (WHO), stimulating clinicians to use it in practice [17]. Mechanisms and genetics were still unknown. In 1969, the chairman of the AHA Arteriosclerosis Council made a groundbreaking remark promoting treatment of hyperlipoproteinaemia, and thereby preventing disease [9]. It would, however, take decades to reach this practice [9]. Another physicians’ guide on how to calculate lipoproteins was published in 1972. This one contained the famous Friedewald formula (estimation of LDL) [19].

In 1973, Arno Motulsky (1923-2018, the father of pharmacogenomics) and Joseph Goldstein (b. 1940) laid down the basis for the first genetics-based classification of hyperlipidaemias [17]. A year later, Russell Ross (1929-1999) and John Glomset (1928-2015) made the pivotal discovery of the platelet-derived growth factor (PDGF), leading to their “Response-to-Injury hypothesis”, i.e., that mechanical injury to endothelium can lead to platelet aggregation and intimal thickening [17]. The impact of their findings on the pathogenesis of atherosclerosis was huge. About the same time, Earl Benditt (1916-1996) and his son John developed the “monoclonal hypothesis”, suggesting that the atheroma was a sort of leiomyoma. Strangely, these, at the time leading, hypotheses on atherosclerosis barely mentioned lipoproteins, although the evidence for hypercholesterolaemia as a primary causative factor was overwhelming [17].

Cholesterol wars

The “cholesterol wars” started in the 1950s, peaked in the 1970s, and subsided in the 1990s [20]. The battlefield was mostly Great Britain. In 1953, Michael Oliver (1925-2015) published that cholesterol levels in CAD patients were higher than in controls [20]. The battle started by the suggestion of Paul Wood (1907-1962 after an MI) that higher levels were a result of CHD, rather than the cause [20]. Sir John McMichael’s (1904-1993) anti-cholesterol campaign was harsh and long [20]. Years after his retirement, his reputation and previous fame enabled him to get his many critical and incorrect opinions published in leading journals [20]. Oliver was also influenced by critical remarks of McMichael and started to doubt. However, after the 4S study in 1994 he completely changed his mind and became a promoter [20].

However, now back to another very important discovery in lipidology - the LDL receptor by Joseph Goldstein (b. 1940) and Michael Brown (b. 1941) in 1974. They demonstrated that human cultured fibroblasts bind LDL and inhibit activity of HMG-CoA reductase [5] and established that cellular uptake of LDL requires the LDL receptor [17]. They met during their internships in the late 1960s and were intrigued by FH [17]. Their lifelong partnership also extended to the bridge table, and both received the Nobel prize in 1985 [17].

Figure 3. Michael Brown and Joseph Goldstein with their Nobel Prize.

With permission from Eur Heart J. 2019 Volume 40, Issue 42, 7 November 2019: 3447–3449. https://doi.org/10.1093/eurheartj/ehz723

In 1975, GJ Miller and NE Miller firmly established that HDL is a negative factor in atherosclerosis [17]. Work in 1979 from Henriksen in Oslo as well as Hessler in Cleveland formed the basis for the “oxidative modification hypothesis”: via the “endothelial cell-modified LDL”, the content of cholesterol in the macrophage is increased, and oxidised LDL is chemotactic for circulating monocytes [17]. In the same year, the PROCAM study started, showing that TG and Lp(a) are sensitive indicators of increased risk for coronary events [21].

In 1984, Bethesda (USA) housed the important “NHI Consensus Development Conference on lowering blood cholesterol to prevent coronary heart disease”. The resulting 1988 guidelines became the gold standard on how and who to treat [22].

In 1992, the first ApoE knock-out mouse model was introduced [19]. Two gene mutations had already been identified as the cause of FH, LDL receptor and Apolipoprotein B. In 2003, mutations were found in proprotein convertase subtilisin kexin-9 (PCSK9) as a cause for autosomal dominant hypercholesterolaemia [23].

History of lipid-lowering drugs

Before reaching the lifechanging era of cholesterol-lowering drugs in the 1970s, other options were tested.

Nicotinic acid

Nicotinic acid (Niacin) was first synthesised by Albert Ladenburg (1842-1911) in 1897 [8]. In 1973, Conrad Elvehjem (1901-1962) described the vitamins’ properties, calling it “Vitamine B3” [8]. Rudolf Altschul finally discovered cholesterol lowering properties in 1955 [8]. This lasted until 2001 when Anna Lorenzen described the mechanism - via the nicotinic acid receptor [8].

Resins

The Dow Chemical Company developed cholestyramine as a water softener. It became available in 1957. In 1965, Sami Hashim and Theodore Van Itallie reported a clinical study showing cholesterol lowering, which caused great excitement in the field of cardiology [8]. As its smell and taste were awful, Robert Fuson (1932-2012) added orange flavour to cholestyramine in 1967 for Mead Johnson Laboratories [8]. The rights for cholestyramine were with Merck and, after selling it to Bristol Myers Squibb, it was marketed as Questran [8]. Upjohn, in the meantime, had developed a similar product, colestipol (Colestid) [8].

Fibrates

In 1954, Imperial Chemical Industries (ICI) discovered that some plant hormones decreased blood cholesterol levels [8]. They discovered clofibrate (not a plant sterol) and marketed this under the name Atromid-S in 1958 [8]. Gemfibrozil was patented in 1968 and came into medical use in 1982 under the name Lopid.

Other substances

In the 1950s, by serendipity, it was found that, whilst removing a part of the thyroid as anti-anginal therapy (!), cholesterol levels in blood rose. Hence, it was deduced that supplying dextrothyroxine should lower cholesterol [8]. In 1962, several cardiologists reported seeing good results in lowering cholesterol by using equine oestrogens (Premarin) [8].

In 1973, the Coronary Drug Project’s study method and preliminary results were published (using equine oestrogens, clofibrate, dextrothyroxine, nicotinic acid, lactose placebo) [24]. During enrolment, dextrothyroxine as well as oestrogens were stopped.

Statins

In 1959, “MER-29” or triparanol, an inhibitor of cholesterol biosynthesis (inhibiting the conversion of desmosterol into cholesterol), was tested in rats and dogs by the Merrell company [12]. Just before FDA evaluation, it appeared that serious side effects in animals (cataract, hair loss) were not reported; in 1963, a lawsuit was the end of this scandal [12].

It was more than a decade before new developments were reported. In 1976, Akira Endo (b. 1933), working at Sankyo, published his discovery of ML-236B (compactin), produced by Penicillinium citrinum, showing reduction in cholesterol synthesis in rats [23]. Compactin was a powerful inhibitor of HMG-CoA reductase [7]. At the same time, Beecham Pharmaceuticals also discovered this from Penicillium brevicompactum [7]. The development of compactin as a drug by Sankyo was, however, stopped in 1980, due to claims that it caused lymphomas in dogs (in fact these dogs received 200 times the dose patients would have received) [7]. The search went on for other statins. Merck in agreement with Sankyo, obtained samples of compactin in 1976. In 1979, they isolated mevinolin from Aspergillus terreus [7]. Endo, who had left Sankyo, also discovered monacolin K from Monascus ruber in 1979. The substances appeared to be the same (later renamed lovastatin). In 1980, after five months of development, lovastatin clinical trials were stopped by Merck due to rumours that this also caused cancer in dogs [7]. As clinicians kept showing impressive results of lowering cholesterol using statins, Merck was persuaded to restart large clinical lovastatin trials in 1984. This led to FDA approval of lovastatin (Mevacor) as the first commercial statin at the end of 1987 [7, 8]. Simvastatin followed, and Sankyo developed and launched pravastatin in 1989. By 2010, two semi-synthetic statins, simvastatin (Zocor, Merck, 1991) and pravastatin (Pravachol, BMS, 1991), and four synthetic statins, fluvastatin (Lescol, Sandoz, 1994), atorvastatin (Lipitor, Parke-Davis, 1996), rosuvastatin (Crestor, AstraZeneca, 2003) and pitavastatin, were introduced to the market [7, 8].

In 1997, Bayer marketed cerivastatin (Baycol/Lipobay), another pure synthetic statin, and 100 times more powerful than Mevacor. However, it had to be taken off the market in 2001, because of substantial side effects: the prevalence of rhabdomyolyse was 15-60 times higher, and 100 deaths were reported [8].

Newest-generation medication

In 2002, ezetimibe was introduced (Schering-Plough), an intestinal cholesterol absorption inhibitor. Nabil Seidah (b. 1949) discovered PCSK-9 in 2003. Alirocumab and evolocumab, fully human anti-PCSK-9 antibodies, were both FDA approved in 2015 [23]. Large PCSK-9 studies included the evolocumab FOURIER trial (2017) and the alirocumab ODYSSEY trial (2015).

Important pharmaceutical trials

The first results of the Coronary Primary Prevention Trial which started in 1973 were published in 1984 [22]. The intervention group took high doses of cholestyramine, and both groups (placebo and intervention) followed a mild diet. The LDL decrease was a disappointing 20% in the intervention group, but the number of cardiovascular events was nevertheless 19% lower in the intervention group (p<0.05) [22]. The Scandinavian Simvastatin Survival Study (4S), published in 1994, showed for the first time a significant decrease in all-cause mortality [12]. The British Heart Protection Study, using simvastatin, was the first to demonstrate that subjects with “normal” LDL levels (<100 mg/dl) also benefited from additional lowering [12]. This finding led to new guidelines which advised lowering LDL levels even further. In 2001 the American NHI advised levels below 70 mg/dL for high-risk populations. The discussion was also influenced by the fact that Bill Clinton experienced his heart attack after stopping simvastatin [8].

Many studies followed, using pravastatin, lovastatin, atorvastatin, rosuvastatin, fluvastatin, ezetimibe, and simvastatin+ezetimibe, bococizumab, evolocumab and alirocumab [26]. Of the 13 randomised controlled trials (RCTs) meeting the LDL-C reduction target, only one reported mortality benefit (ODYSSEY Outcomes, 2018) and five reported a reduction in CV events. Of the 22 RCTs that did not meet their target, four reported mortality benefit (ACAPS 2010, 4S study 1994, LIPID 1998, GREACE 2002) and 14 a reduction in CV events [26].

At this moment, developments and discoveries go faster than ever before when compared to their historical counterparts. In 2019, alternatives for anti-PCSK-9 antibodies were being tested, e.g., inhibiting translation of PCSK-9 by interfering with messenger RNA by “short interfering RNA” (siRNA), such as inclisiran [26]. Another alternative is targeting the step before HMG-coA reductase by inhibiting ATP-citrate lyase (Bempedoic acid) (2019) [26]. For FH, evinacumab, a monoclonal antibody against angiopoetin-like 3 (ANGPTL3), is currently being tested [27].

Conclusion

Every cloud has a silver lining. Wars once again played an important role in new developments, in this case lipoproteins and cholesterol in WWII, and for example presenting evidence that 75% of 300 corpses from men average 22 years of age had lesions in their arteries in the Korean War (1951) [1].

The scientific work on cholesterol and lipids led to at least 11 Nobel prizes. With hindsight, Akira Endo and John Gofman were also “Nobel prize material”.

Although the mortality benefits are much smaller than assumed, cholesterol-lowering therapy is widely accepted and is beyond any doubt a preventive means in vascular diseases. Developments in medication advance much faster nowadays than history could ever have predicted, and they quickly result in new guidelines.

For almost a century, evidence has been overwhelming that lipids and diet are related and have a negative impact on CVD. It is also clear that lipids, and especially LDL, play a crucial role in atherosclerosis. However, groups of “non-believers” decelerated developments and clinical progress, sometimes for decades.

Importantly, we should never forget that patients are individuals and LDL is not the holy grail. As early as 1959, diet, mental stress, obesity, a sedentary lifestyle and smoking were mentioned as risk factors besides lipids. Here as well, there are still “non-believers”. However, as Sir William Osler said: “The good physician treats the disease; the great physician treats the patient who has the disease”.