Review

Homocysteine and the

pathogenesis of
atherosclerosis
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Expert Rev. Clin. Pharmacol. Early online, 1–9 (2015)

Kilmer S McCully
Pathology and Laboratory Medicine
Service, VA Boston Healthcare System,
1400 Veterans of Foreign Wars
Parkway, West Roxbury,
MA 02132, USA
and
Department of Pathology, Harvard
Medical School, Boston,
MA 02114, USA
Tel.: +1 857 203 5990
Fax: +1 857 203 5623
kilmer.mccully@va.gov
For personal use only.
The homocysteine theory of arteriosclerosis was discovered by study of arteriosclerotic
plaques occurring in homocystinuria, a disease caused by deficiencies of cystathionine
synthase, methionine synthase or methylenetetrahydrofolate reductase. According to the
homocysteine theory, metabolic and nutritional abnormalities leading to elevation of plasma
homocysteine cause atherosclerosis in the general population without these rare enzymatic
abnormalities. Through studies of metabolism of homocysteine thiolactone, the anhydride of
homocysteine, in cell cultures from homocystinuric children, the pathway for synthesis of
sulfate was found to be dependent upon thioretinamide, the amide formed from retinoic
acid and homocysteine thiolactone. Two molecules of thioretinamide form the complex
thioretinaco with cobalamin, and oxidative phosphorylation is catalyzed by reduction of
oxygen, which is bound to thioretinaco ozonide, by electrons from electron transport

particles. Atherogenesis is attributed to formation of aggregates of homocysteinylated

lipoproteins with microorganisms, which obstruct the vasa vasorum during formation of
arterial vulnerable plaques.

KEYWORDS: atherosclerosis . homocysteine . lipoprotein . microorganisms . oxidative phosphorylation . oxidative stress

. thioretinaco . thioretinamide

metabolite cystathionine is low in concentra-

Discovery & development of the
homocysteine theory of arteriosclerosis
The homocysteine theory of arteriosclerosis
was discovered in 1969 by the study of vascu-
lar pathology of children with inherited disor-
ders of homocysteine metabolism [1]. The
disease homocystinuria was first recognized in
1962 by the discovery of urinary excretion of
the sulfur amino acid homocysteine in several
subjects with mental retardation, accelerated
growth and susceptibility to arterial and
venous thrombosis [2]. The cause of homocys-
tinuria was traced to deficient function of the
enzyme cystathionine synthase, which normally
converts homocysteine to cystathionine, cata-
lyzed by the coenzyme pyridoxal phosphate [3].
Because of this enzyme deficiency, homocyste-
ine accumulates at elevated concentrations in
the tissues and fluids of subjects with homo-
cystinuria. The amino acid methionine also
accumulates at elevated concentrations because
of increased transmethylation of homocysteine
to methionine by methyl cobalamin. The
tion in tissues and fluids of subjects with
homocystinuria because of decreased conver-
sion of homocysteine to cystathionine by
cystathionine synthase.
Review of an archival case of homocystinu-
ria disclosed widespread arteriosclerotic plaques
in an 8-year-old boy who died of complica-
tions of carotid arteriosclerosis, thrombosis and
cerebral infarct [4]. The child was diagnosed
with homocystinuria because of the discovery
of homocysteine excretion in the urine of a
niece [5]. In 1968, the enzyme abnormality in
another case of homocystinuria in a 2-month-
old baby boy with pneumonia and failure to
thrive was traced to deficient function of
methionine synthase because of a derangement
in vitamin B12 metabolism, leading to homo-
cystinuria, cystathioninuria and methylmalonic
aciduria [6]. Review of the pathology of this
case, the first example of cobalamin C disease
in the literature, led to the discovery of wide-
spread, advanced arteriosclerotic plaques affect-
ing arteries to the major organs. Because

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2015 Informa UK Ltd ISSN 1751-2433 1

 Review McCully
elevation of plasma homocysteine was the only metabolic
abnormality in common between these two cases with different
inherited enzyme abnormalities, the conclusion was reached
that homocysteine causes arteriosclerotic plaques by a direct
effect of the amino acid on the cells and tissues of the arter-
ies [1]. Independent confirmation of this conclusion is derived
from the observation of arteriosclerotic plaques in a child with
homocystinuria caused by methylenetetrahydrofolate reductase
deficiency, the third major enzymatic deficiency which causes
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homocystinuria [7].
Development of the homocysteine theory of arteriosclerosis
is based on observations of the effect of homocysteine on pro-
duction of arteriosclerosis in experimental animals, abnormali-
ties of extracellular matrix aggregation by sulfation in cell
cultures from subjects with homocystinuria, effects of homocys-
teine on growth in normal and hypophysectomized animals
and discovery of a biochemical pathway by which the sulfur
atom of homocysteine thiolactone is converted to sulfate [8].
According to the homocysteine theory of arteriosclerosis, meta-
bolic and nutritional factors leading to elevation of blood
homocysteine levels produce increased risk of atherosclerosis
and its manifestations, including coronary heart disease, cere-
brovascular disease and peripheral vascular disease in subjects
without these rare inherited enzyme abnormalities. Indeed, in
For personal use only.
the years since the discovery of the homocysteine theory of
arteriosclerosis in 1969, many retrospective and prospective
human observational studies have established hyperhomocystei-
nemia as a potent independent risk factor for atherosclerosis [9].
It is noteworthy that these cases of arteriosclerosis in homocys-
tinuria do not exhibit changes in blood levels of cholesterol,
lipoproteins or other traditional risk factors for atherosclerosis.
Homocysteine thiolactone metabolism in
homocystinuria & malignancy
Investigation of the cellular morphology and function of cell
cultures from the skin of subjects with homocystinuria revealed
aggregation of extracellular matrix, associated with increased
sulfation and with altered contact inhibition of growth [10].
Studies with S35-labeled homocysteine thiolactone, the cyclic
anhydride of homocysteine, revealed a new pathway for conver-
sion of the sulfur atom to sulfate, since the cultured cells from
homocystinuric subjects are deficient in cystathionine synthase,
the first step in catabolism of homocysteine to cysteine and sul-
fate by the trans-sulfuration pathway [11].
Observations with cultured malignant cells revealed a meta-
bolic blockade in the conversion of homocysteine thiolactone
to sulfate, resulting in homocysteinylation of cellular macromo-
lecules by its reaction with free amino groups of proteins,
nucleic acids and glycosaminoglycans to produce peptide-
bound homocysteine containing free sulfhydryl groups [12].
This abnormality of sulfate synthesis in malignant cells was
attributed to deficiency of an unknown substance containing
homocysteine thiolactone that is present in normal cells.
Organic synthesis of model compounds containing homocyste-
ine thiolactone revealed derivatives that inhibit the growth of
doi: 10.1586/17512433.2015.1010516
transplanted malignant neoplasms in experimental animals [13].
One of the synthetic compounds with antineoplastic activity is
thioretinamide, the amide formed from homocysteine thiolac-
tone and retinoic acid [14]. Thioretinamide combines with
cobalamin to form thioretinaco, a complex which contains two
molecules of thioretinamide bound to the cobalt atom of cobal-
amin [15]. Both thioretinamide and thioretinaco are antiathero-
genic in animals injected with homocysteine thiolactone [16].
The blockade in conversion of the sulfur atom of homocysteine
thiolactone to sulfate in malignant cells is attributed to cellular
deficiency of thioretinamide, an intermediate in the conversion
of homocysteine thiolactone to sulfate [13].
Thioretinaco ozonide & oxidative phosphorylation
According to a detailed theory of oxidative phosphorylation,
thioretinaco is activated by ozone and oxygen to form thioreti-
naco ozonide oxygen, which stereospecifically binds ATP, facili-
tating the removal of ATP from the F1F0 ATPase complex of
mitochondrial membranes [13]. ATP is released from the
F1F0 ATPase complex, concomitant with reduction of oxygen
to water by electrons from electron transport particles of the
mitochondria. Oxidative stress is caused by accumulation of
oxygen radicals because of inhibition of thioretinaco ozonide
function within the mitochondria and the endoplasmic
reticulum [17].
The active site of oxidative phosphorylation is proposed to
consist of two molecules of thioretinamide bound to cobala-
min, forming thioretinaco, complexed with ozone, oxygen, nic-
otinamide adenine dinucleotide and inorganic phosphate
(TR2CoO3O2NAD+H2PO4-) [18]. Reduction of the pyridinium
nitrogen of the nicotinamide group by an electron from elec-
tron transport complexes initiates polymerization of phosphate
with ADP yielding nicotinamide riboside and ATP bound to
thioretinaco ozonide oxygen. A second electron reduces oxygen
to hydroperoxyl radical, releasing ATP from the active site.
A proton gradient is created within F1F0 ATPase complexes of
the mitochondria by reaction of protons with reduced nicotin-
amide riboside and with hydroperoxyl radical yielding reduced
nicotinamide riboside and hydroperoxide. The hyperhomocys-
teinemia of aging and dementia is attributed to decreased syn-
thesis of adenosyl methionine by thioretinaco ozonide and
ATP, causing decreased allosteric activation of cystathionine
synthase and decreased allosteric inhibition of methylenetetra-
hydrofolate reductase and producing dysregulation of
methionine metabolism.
Excitotoxicity, oxidative stress, endothelial dysfunction
& inflammation
Depletion of thioretinaco ozonide from cellular membranes was
suggested to cause overproduction of homocysteine thiolactone
in atherogenesis and carcinogenesis [13]. Homocysteine is a potent
excitatory neurotransmitter that binds to the N-methyl-D-aspar-
tate receptor leading to oxidative stress, cytoplasmic calcium
influx, cellular apoptosis and endothelial dysfunction [17]. Endo-
thelial dysfunction is considered by many investigators to be the
Expert Rev. Clin. Pharmacol.

earliest manifestation of atherogenesis. Homocysteine thiolactone
reacts with the free amino groups of the apoB protein of LDL to
form peptide-bound homocysteine causing aggregation of LDL
and leading to foam cell formation from phagocytosis of LDL
aggregates by cultured human macrophages [19].
Homocysteine, microbes, lipoproteins & the origin of
vulnerable plaques
Lipoproteins have long been known to participate in the innate
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immune system by binding and inactivating bacteria, viruses and
their toxins by complex formation [20]. Microbes have been
implicated in the production of atherosclerotic plaques, and evi-
dence for microbial remnants of a wide variety of bacteria, viruses
and protozoans within plaques is based on the detection of
microbial ribosomal DNA by FISH [21]. Chlamydia pneumoniae,
Mycoplasma pneumoniae, Helicobacter pylori, cytomegalovirus, her-
pesvirus and other microbes have been identified in atheroscle-
rotic plaques by culture, immunohistochemistry and electron
microscopy [22]. Based on these observations, vulnerable plaques
were suggested to originate from obstruction of the vasa vasorum
by aggregates of lipoproteins complexed with microbes, exacer-
bated by aggregation of lipoproteins by homocysteine thiolac-
tone [23]. Obstruction of the circulation in the vasa vasorum,
caused by the aggregated complexes, leads to ischemia of the arte-
For personal use only.
rial wall, intramural cell death, rupture of capillaries and release
of microorganisms within the arterial intima causing hemor-
rhage, inflammation and an intimal microabscess, the vulnerable
plaque. Hyperhomocysteinemia contributes to obstruction of the
vasa vasorum because of swelling of endothelial cells during
endothelial dysfunction, impeding passage of LDL aggregates
through narrowed lumens of the vasa vasorum.
Status of homocysteine patents
Because of the antiatherogenic and antineoplastic activities of
thioretinamide and thioretinaco in experimental animals, pat-
ents were granted for these compounds by the US Patent and
Trademark Office [24,25]. An improved method for synthesis of
thioretinamide in high yield and high purity was developed,
and antiatherogenic activities of thioretinamide and thioretinaco
were demonstrated in rats injected with homocysteine thiolac-
tone [16]. Patents were granted for this method of synthesis of
thioretinamide and thioretinaco, and this intellectual property
is in the public domain [26,27]. A patent for thioretinaco ozon-
ide and the use of thioretinaco ozonide in combination with
interferon was granted [28], and a patent for liposomal thioreti-
naco ozonide and the use of a liposomal carrier was also
granted [29]. A metabolic and nutritional protocol for utilization
of thioretinamide in the treatment of degenerative disease of
aging is the basis of a current patent application [30]. None of
these patents is the subject of a clinical trial to date.
Biosynthesis of thioretinamide by cystathionine
synthase
Recent developments in the understanding of cystathionine
synthase structure and function and in the role of transthyretin
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Homocysteine & atherosclerosis Review
and retinol in protein energy malnutrition have led to a
proposed process for the biosynthesis of thioretinamide from
retinol and homocysteine thiolactone [31]. The enzyme cysta-
thionine synthase contains a hemeoxygenase group that was
demonstrated to catalyze superoxide radical generation from
dioxygen [32]. Retinol is transported to tissues by the plasma
protein transthyretin, and hyperhomocysteinemia and decreased
plasma transthyretin are associated with protein energy malnu-
trition. Hyperhomocysteinemia is caused by downregulation of
the trans-sulfuration pathway in this metabolic condition [33].
In scorbutic guinea pigs, oxidation of the sulfur atom of homo-
cysteine is inhibited, implicating ascorbate function in sulfate
synthesis from homocysteine [34]. Because of these observations,
it was proposed that biosynthesis of thioretinamide from the
retinol of plasma transthyretin is produced by oxidation of reti-
nol to retinoic acid by superoxide radical generated from the
hemeoxygenase group of cystathionine synthase, and this reac-
tion is facilitated by conversion of deoxyascorbate to semi-
dehydroascorbate radical [31]. Enzyme-bound retinoic acid is
combined with homocysteine thiolactone to produce thioretina-
mide, catalyzed by cystathionine synthase. Thioretinaco is the
complex formed by two molecules of thioretinamide with one
molecule of cobalamin, and thioretinaco is activated by ozone
and oxygen to function as the active site of oxidative phosphor-
ylation [13,18]. Recent evidence demonstrates that embryonic
and malignant cells are deficient in cystathionine synthase activ-
ity [35]. Since malignant cells are deficient in the hemeoxygenase
function of cystathionine synthase, this observation explains the
failure of oxidation of homocysteine thiolactone to sulfate
observed in malignant cells [12].
Homocysteine & the enzyme treatment of cancer
Because embryonic pancreatic enzymes facilitate the conversion
of the trophoblast to the placenta and because of the similar
biological behavior of trophoblastic cells and cancer cells,
administration of pancreatic enzymes was proposed as a treat-
ment for primary or metastatic malignancy [36]. This treatment
consists of parenteral injection of enzymes and proenzymes
extracted from porcine pancreas in patients with cancer.
According to the trophoblastic theory of the origin of cancer,
malignant cells arise from adult stem cells. The embryologist
John Beard discovered that adult stem cells are derived from
trophoblastic cells which migrate from the yolk sac into
somatic tissues of the developing embryo. The observation that
human fetal and malignant cells produce chorionic gonadotro-
pin supports the trophoblastic theory of the origin of cancer [37].
Oncolysis by pancreatic enzymes and proenzymes is attributed
to susceptibility of cancer cells to enzymatic therapy because of
their derivation from trophoblastic cells [38,39]. Accumulation of
homocysteinylated enzymes, proteins, RNA, DNA and glycosa-
minoglycans occurs by reaction of homocysteine thiolactone
with the free amino groups of these macromolecules during
aging, atherogenesis, carcinogenesis and autoimmune dis-
eases [13,40]. The active and proenzyme forms of trypsin, chy-
motrypsin, hyaluronidase, elastase, amylase, lipase, ribonuclease
doi: 10.1586/17512433.2015.1010516
 Review McCully
and deoxyribonuclease and other potent digestive enzymes of
pancreatic extracts are capable of hydrolyzing the homocystei-
nylated proteins, nucleic acids and glycosaminoglycans in sub-
jects with degenerative diseases of aging, increasing the
catabolism of homocysteinylated macromolecules [31].
Nitrilosides, hydrogen sulfide & the homocysteine
theory
Nitrilosides are cyanogenic compounds containing nitrile
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groups which are widely distributed in grasses, roots, legumes,
fruits and berries [41]. The important plant nitriloside, amygda-
lin (mandelonitrile b-diglucoside), is found in seeds of apples
and apricots and other fruits, and the nitrilosides dhurrin,
lotaustralin and linamarin are cyanogens of tropical plants such
as cassava roots. Normal and malignant mammalian cells con-
tain glucosidases which convert the nitrile group of plant nitri-
losides to cyanide [41]. Normal cells contain rhodanese, a
sulfotransferase enzyme which catalyzes thiocyanate formation
from cyanide and thiosulfate. Thiocyanate is less toxic than
cyanide and possesses mild antithyroid properties. Malignant
cells contain insufficient rodanese to catalyze thiocyanate syn-
thesis from cyanide and thiosulfate, theoretically producing tox-
icity from increased intracellular concentrations of cyanide
which potentially inactivate thioretinaco ozonide [17] and
For personal use only.
explain the inhibitory effects of nitrilosides on growth of malig-
nant neoplasms in experimental animals [41]. Konzo is a toxic
upper motor neuron myelopathy, which is associated with pro-
tein energy malnutrition and is attributed to cyanide consump-
tion from improperly processed cassava [42]. Because of a
dietary deficiency of sulfur amino acids, conversion of cyanide
to thiocyanate is inhibited and cyanide is preferentially con-
verted to cyanate, a known neurotoxin [43].
Hydrogen sulfide is a powerful reducing agent that acts as a
gasotransmitter in sensing oxygen concentrations in tissues [44].
Hydrogen sulfide is produced from homocysteine by cystathio-
nine synthase and is produced from cystathionine by cystathio-
nase. Low levels of hydrogen sulfide decrease oxidative stress
and counteract experimental ischemia-reperfusion injury, hyper-
tension and renal failure [45]. The reducing properties of hydro-
gen sulfide inhibit reactive oxygen species induced by
homocysteine, thereby ameliorating experimental myocardial
injury [46]. Recently, hydrogen sulfide has been shown to atten-
uate neurodegeneration and neurovascular dysfunction induced
by intracerebral administration of homocysteine in mice [47].
The beneficial effects of dietary nitrilosides may be attributed
to reaction of thiosulfate with cyanide to produce thiocyanate.
Cyanide is converted to thiocyanate by rhodanese, and thiosul-
fate is produced by oxidation of hydrogen sulfide, catalyzed by
sulfide oxidase [48].
Epidemiology of coronary heart disease & the
homocysteine theory
Since 1958, the mortality rate from coronary heart disease in
the USA has steadily declined from 560 deaths per 100,000 to
180 deaths per 100,000 in 2010, an impressive decline of 68%
doi: 10.1586/17512433.2015.1010516
in mortality rate during a half century of observation. The
decline in stroke mortality during this period was even more
dramatic, falling from 180 deaths per 100,000 in 1958 to
40 deaths per 100,000 in 2010, a decline of 79% in stroke
mortality rate. The decline in coronary heart disease mortality
was already evident in 1978, when a nationwide conference of
subject matter experts was convened by the NIH to examine
the reasons for the decline [49]. Although trends in smoking
cessation, exercise, coronary care units, coronary bypass surgery,
treatment of hypertension and a 5% reduction in blood choles-
terol levels were favorable, the total fat and meat consumption
increased, and an increased incidence of obesity was observed.
The final conclusion of the conference was that ‘Although
there was general agreement that the decline in coronary heart
disease is real, the probable cause or causes could not be pre-
cisely identified.’ This conference was concluded before wide-
spread use of cholesterol-lowering drugs was introduced in
the 1980s.
The answer to the critical question concerning why US coro-
nary heart disease mortality declined during the years from
1958 to 1978 is related to adequate dietary consumption of
pyridoxal and folate. These two B vitamins are required for
normal homocysteine metabolism, and dietary deficiencies of
these nutrients produce elevated plasma homocysteine concen-
trations. Traditional methods of food processing, such as heat-
ing, chemical additives, extraction of purified carbohydrates
and oils from foods, and milling of grains, cause major losses
of these sensitive vitamins [50]. Introduction of these highly
processed foods during the early decades of the 20th century
may have contributed to the precipitous rise in mortality from
coronary heart disease in the 1940s and 1950s. A study of
elderly Framingham participants aged 67 to 96 years demon-
strated major nutritional deficiencies of pyridoxal, folate and
cobalamin in two-thirds of subjects, leading to elevated homo-
cysteine levels [51].
An explanation of the decline in coronary heart disease mor-
tality is related to increased consumption of pyridoxal and folate
from fortification and supplementation beginning in the 1950s.
The daily human consumption of synthetic pyridoxine, a pre-
cursor of pyridoxal phosphate (vitamin B6), was minimal in the
1940s and early 1950s, amounting to 0.04 mg/day in 1944 and
0.23 mg/day in 1955 [8]. By 1961, the daily human consump-
tion of synthetic pyridoxine had risen to 0.53 mg/day, and in
the 1960s and 1970s, consumption rose to 2.5–3.5 mg/day,
supplying more than the 1998 Recommended Dietary Intake of
vitamin B6, 1.7 mg/day. Synthetic folic acid, a precursor of
folate, was also added to the human diet in the form of supple-
ments beginning in the 1960s, and in 1998 the US FDA man-
dated fortification of processed grain foods with folic acid.
A study of Framingham participants demonstrated that plasma
folate levels doubled after folic acid fortification, and plasma
homocysteine levels decreased about 15%, compared to the lev-
els prior to fortification with folic acid in 1998 [52]. The conclu-
sion from this analysis is that the dramatic decline in
cardiovascular mortality in the USA since the 1950s may be
Expert Rev. Clin. Pharmacol.

attributed in part to voluntary fortification of the food supply
with pyridoxine and folic acid [53]. Some direct evidence for this
conclusion is the observation of an accelerated decline in stroke
mortality in the USA and Canada following mandated fortifica-
tion of processed grain foods with folic acid in 1998 [54].
Interventional trials of B vitamin therapy in
atherosclerosis
Several large-scale interventional trials commenced in the 1990s
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to determine whether subjects with established vascular disease
are benefited by high-dose vitamin therapy with vitamin B6,
vitamin B9 and vitamin B12 [53]. Studies of subjects with cere-
brovascular disease in the VISP trial [55], cardiovascular disease
in the NORVIT and HOPE2 trials [56,57], and vascular disease
from chronic renal failure in the HOST trial [58] showed no
reduction in stroke or heart attack or improvement in mortality
from B vitamin intervention. In the HOPE2 trial, a significant
24% reduction in stroke was observed from B vitamin ther-
apy [57], and a subgroup analysis of VISP participants without
renal failure or malabsorption of B12 had a significant 21%
reduction in adverse events [59]. These interventional studies
show little benefit of high-dose B vitamins in subjects with
advanced vascular disease who are consuming adequate dietary
B vitamins.
For personal use only.
The apparent lack of efficacy of B vitamin therapy in the
large interventional trials was traced to failure to correct for
reduced renal function and metabolic deficiency of vitamin
B12, both of which are common in elderly vascular patients [60].
Consequently, a subgroup analysis which corrects for these fac-
tors helps to explain the complexity of response of participants
in these interventional trials [61]. High-dose folic acid and cya-
nocobalamin increased adverse vascular events in patients with
diabetic nephropathy [62,63], accounting for the benefit of the
efficacy analysis of the VISP trial [59] and explaining the lack of
benefit in the main VISP trial [55]. High-dose folic acid may
increase the levels of asymmetric dimethylarginine, a nitric
oxide antagonist [64]. Nitric oxide reacts with thiols, including
homocysteine, and with the thiol groups of proteins to produce
S-nitroso derivatives with endothelium relaxing factor activities,
including vasodilation and platelet inhibition [65]. The adverse
vascular effects of homocysteine are modulated by the release
of endothelium relaxing factor and formation of S-nitroso-
homocysteine [66]. Another possible explanation is accumulation
of cyanide from high-dose cyanocobalamin in dialysis patients,
since methylcobalamin reduced the levels of both plasma
homocysteine and asymmetric dimethylarginine [67,68]. Accumu-
lation of cyanide may deplete hydrogen sulfide formation from
homocysteine by synthesis of thiocyanate, decreasing the antia-
therogenic effect of hydrogen sulfide, as demonstrated in apoli-
poprotein E knockout mice [69]. A recent commentary
regarding the evidence for the causal effect of homocysteine in
coronary heart disease, based on analysis of 107 methylenetetra-
hydrofolate reductase polymorphism studies, concludes that ‘a
role for homocysteine in causing ischemic heart disease remains
open [70].’
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Homocysteine & atherosclerosis Review
Homocysteine & diseases of aging
Since the introduction of the homocysteine theory of arterio-
sclerosis in 1969, many diseases and conditions correlated with
aging have been found to be associated with elevation of plasma
homocysteine. Some examples are chronic renal failure, hypo-
thyroidism, metastatic cancer, autoimmune diseases, dementia,
macular degeneration and the aging process itself. A recent
book, ‘Homocysteine: Biosynthesis and Health Implications,’
summarizes the current status of knowledge in several diseases
associated with hyperhomocysteinemia [71]. The topics include
cystathionine synthase function, experimental models of hyper-
homocysteinemia and their relation to dietary antioxidants,
the relation of homocysteine to neurodegenerative diseases and
antiepileptic drugs, uremia, bone disease, vascular disease, HIV
disease, eye diseases, bariatric surgery, celiac disease and
psoriasis.
Homocysteine & protein energy malnutrition
The WHO estimates that more than half of the 17 million deaths
worldwide from cardiovascular disease occur in populations of
countries where vegetarian diets of starches and legumes have
prevailed for millennia. Protein energy malnutrition was studied
in one of these countries, Chad in central Africa, where the popu-
lation subsists on cassava, groundnuts, sweet potatoes, beans and
millets [33]. This semiarid area with poor soils leads to poorly
nourished livestock, few dairy foods and rare consumption of
meat, except for some poultry and eggs. In this study, decreased
transthyretin and hyperhomocysteinemia were correlated with
downregulation of cystathionine synthase and the trans-
sulfuration pathway, explaining the increased susceptibility to
vascular disease in this population, despite adequate dietary
folate, pyridoxal, normal serum lipids and a lack of consumption
of saturated fats. The concept of dietary sulfur deficiency is a
plausible explanation of the nutritional and metabolic abnormali-
ties observed in protein energy malnutrition [33].
Aftermath of the homocysteine theory
When the homocysteine theory of arteriosclerosis was first for-
mulated and published in 1969 [1], plasma cholesterol and lip-
ids were placed in a secondary role to the primary effect of
homocysteine in producing atherosclerotic plaques. As
described in my recently published scientific memoir, ‘Pioneer
of the Homocysteine Theory,’ I was able to develop the homo-
cysteine theory for a decade at Massachusetts General Hospital
and Harvard Medical School [72]. I was unprepared, however,
for the violent rejection of the homocysteine theory by col-
leagues at Massachusetts Institute of Technology and the Fra-
mingham Study. Thereafter, the scientific leaders of Harvard
medicine denied me promotions and tenure, blackballed me,
and forced me into exile at the Veterans Affairs Medical Center
in Providence, where I was able to continue my homocysteine
research under much reduced conditions.
Some colleagues suggested that rejection of my homocysteine
research by the Harvard elders was occasioned by criticism
from the ‘cholesterol cartel.’ However, I was never aware of
doi: 10.1586/17512433.2015.1010516
 Review McCully
any direct criticism from the pharmaceutical industry, but my
theories and discoveries were only ignored. Naturally, after
leaving Harvard medicine under a cloud of criticism, I was
never able to secure independent grant funding for homocyste-
ine research, relying only on collaborations with sympathetic
colleagues. My new ideas did not appear to me to be a threat
to the multi-billion dollar pharmaceutical industry, the food
and nutrition industry or the cancer industry. My expulsion
from Harvard medicine, however, suggested to some of my col-
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leagues that my theories were perceived as a threat to these
powerful financial interests.
The homocysteine theory & prevention of
atherosclerosis
After the First International Conference on Homocysteine
Metabolism was held in Ireland in 1995 [73], articles about the
homocysteine theory of arteriosclerosis were published in major
newspapers, magazines and newsletters. A description of my
discovery of the homocysteine theory and the circumstances
surrounding my exile from Harvard medicine was published in
the New York Times Magazine in August 1997 [74]. During
this period, I was encouraged to write a book describing how
the homocysteine theory was discovered. The result was ‘The
Homocysteine Revolution, Medicine for the New Millennium,’
For personal use only.
a book published in 1997 by Keats Publishing Company [75].
Because of growing interest in the media and in scientific and
medical publications in the late 1990s, I was encouraged to
write an expanded version of this account for the general
reader, ‘The Heart Revolution. The Extraordinary Discovery
that Finally Laid the Cholesterol Myth to Rest,’ a book pub-
lished in 1999 by HarperCollins [76].
The ‘Homocysteine Revolution’ describes in detail the cir-
cumstances surrounding the discovery of the homocysteine the-
ory of arteriosclerosis in terms designed for the scientific or
medical reader [75]. In the book, the traditional cholesterol/fat
hypothesis is described and related to the discovery of the
homocysteine theory and why the cholesterol hypothesis is the
favorite of the medical–pharmaceutical establishment. In regard
to the cholesterol hypothesis, ‘it is significant that the 80-year
history of the cholesterol/fat approach has yet to provide a
coherent and comprehensive scientific theory which explains
how cholesterol, a normal constituent of the body, or excess
dietary fat in the diet of susceptible populations produces arte-
riosclerotic plaques.’ The book explains in detail how food
processing removes essential nutrients, especially vitamins
B6 (pyridoxal) and B9 (folate), leading to elevation of blood
homocysteine levels in the population and causing
vascular disease.
In the ‘Heart Revolution,’ the essentials of the homocysteine
theory of arteriosclerosis are presented in a more conversational
and appealing style, emphasizing how the general reader can
benefit personally from the theory [76]. Appendix I of the book
presents a 6-week plan for making the necessary lifestyle
changes needed fully to implement the principles of the homo-
cysteine theory. Appendix II of the book presents recipes for
doi: 10.1586/17512433.2015.1010516
healthy eating based on an appetizing diet consisting of whole
nutritious foods without highly processed ingredients.
In the early years after discovery of the homocysteine theory,
the cholesterol hypothesis concerning the origin of arteriosclero-
sis held sway in medical and scientific opinion. There was no
clear connection between the conventional wisdom about cho-
lesterol and saturated fats in the diet and homocysteine as a
cause of arterial plaques. The discovery of aggregation of LDL
by homocysteine thiolactone and production of foam cells
from cultured human macrophages helped to clarify the con-
nection between homocysteine and the cholesterol of LDL [19].
Introduction of the concept of obstruction of the vasa vasorum
by LDL aggregates with microbes and homocysteinylated lipo-
proteins explains the origin of vulnerable plaques [23]. In this
view, cholesterol, as an important constituent of LDL, partici-
pates in atherogenesis by binding to the lipid constituents of
microorganisms, forming aggregates which are trapped in the
vasa vasorum of the arterial adventitia. Homocysteine exacer-
bates the process of trapping of LDL aggregates by narrowing
of arterial lumens of the vasa vasorum by causing endothelial
dysfunction, further facilitating aggregation of LDL by forming
homocysteinyl groups attached to LDL, and by antibody for-
mation to homocysteinylated LDL and to oxidized LDL
(oxLDL), impeding the passage of LDL aggregates through the
vasa vasorum. Through these pathological processes occurring
in the cells and tissues of arteries, homocysteine and the choles-
terol of LDL contribute to the pathogenesis of atherosclerosis.
Expert commentary
Homocysteine research has expanded steadily in the half century
since the disease homocystinuria was discovered in 1962 and the
homocysteine theory of arteriosclerosis was introduced in 1969.
In addition to coronary heart disease, elevated homocysteine lev-
els have been demonstrated in subjects with generalized arterio-
sclerosis, arterial and venous thrombosis, metastatic cancer,
neuropsychiatric disorders, hypothyroidism, renal failure, osteo-
porotic fractures, Alzheimer’s dementia, macular degeneration
and numerous other age-related diseases and conditions. Studies
of altered homocysteine metabolism in these diseases are cur-
rently being pursued in laboratories, clinics and hospitals world-
wide, providing new and effective strategies for successful
prevention or treatment of these diseases. The current database
for homocysteine research is approximately 17,500 articles, as
listed in PubMed. Although clinical trials with high doses of pyri-
doxine, folic acid and vitamin B12 have failed to reverse estab-
lished vascular disease, mounting evidence for primary
prevention and amelioration of cerebrovascular disease by B vita-
mins is currently being evaluated and reported by investigators
worldwide. Recent advances in understanding of the biochemical
processes involved in biosynthesis of thioretinamide by cystathio-
nine synthase and the active site of oxidative phosphorylation by
thioretinaco ozonide, oxygen, nicotinamide adenine dinucleotide
and phosphate have led to a new proposal for treatment of dis-
eases of aging by a nutritional and metabolic protocol utilizing
thioretinamide, B vitamins, nicotinamide riboside, pancreatic
Expert Rev. Clin. Pharmacol.
 Homocysteine & atherosclerosis Review

enzymes, ascorbic acid, retinol, menoquinone, vitamin D3 and
adenosyl methionine. Clinical trials of this protocol are
awaiting funding.
Five-year view
With sufficient financial support, new strategies for the treatment
and prevention of arteriosclerosis, cancer, osteoporosis, dementia,
macular degeneration and other age-related diseases will be intro-
duced through clinical trials of the homocysteine theory in sus-
Expert Review of Clinical Pharmacology Downloaded from informahealthcare.com by Chinese University of Hong Kong on 02/12/15
oxidative phosphorylation by declining concentrations of thiore-
tinaco ozonide of mitochondrial membranes. Development of
direct assays for thioretinamide, thioretinaco and thioretinaco
ozonide will contribute to new understanding of conditions and
diseases characterized by hyperhomocysteinemia.
Financial & competing interests disclosure
The preparation of this article was supported by the US Department of
Veterans Affairs solely by salary. The author has no other relevant affilia-

ceptible populations. Advances in understanding of the aging tions or financial involvement with any organization or entity with a
process will be correlated with concepts of mitochondrial dys- financial interest in or financial conflict with the subject matter or materi-
function, related to depletion of nicotinamide adenine dinucleo- als discussed in the manuscript apart from those disclosed.
tide, flavin adenine dinucleotide and decreased control of No writing assistance was utilized in the production of this manuscript.

Key issues
. The homocysteine theory was discovered by demonstration of arteriosclerotic plaques in children with homocystinuria caused by
deficiencies of cystathionine synthase, methionine synthase or methylenetetrahydrofolate reductase.
. According to the homocysteine theory, metabolic and nutritional factors leading to hyperhomocysteinemia produce an increased risk of
atherosclerosis, including coronary heart disease, stroke and peripheral vascular disease.
. Human observational studies have established hyperhomocysteinemia as a potent, independent risk factor for atherosclerosis in the
general population.
. Studies with homocysteine thiolactone, the reactive cyclic anhydride of homocysteine, in cell cultures from children with cystathionine
For personal use only.

synthase deficiency and homocystinuria demonstrated a metabolic pathway for conversion of the sulfur atom of homocysteine
to sulfate.
. Studies with cultured malignant cells revealed a metabolic blockade in conversion of the sulfur atom of homocysteine thiolactone to
sulfate, resulting in homocysteinylation of free amino groups of proteins, nucleic acids and glycosaminoglycans, and causing aerobic gly-
colysis, altered genetic expression, diffuse membrane abnormalities and other phenotypic abnormalities of malignant cells.
. The blockade in synthesis of sulfate from homocysteine thiolactone is attributed to cellular deficiency of thioretinamide, the amide
formed from retinoic acid and homocysteine thiolactone, and deficiency of thioretinaco ozonide, the complex formed from cobalamin,
ozone, oxygen and thioretinamide which is responsible for ATP synthesis during oxidative phosphorylation, catalyzed by reduction of
oxygen to water by electron transport.
. The active site of oxidative phosphorylation is proposed to comprise thioretinaco ozonide, oxygen, nicotinamide adenine dinucleotide
and phosphate, which yields ATP, nicotinamide riboside and hydroperoxide upon reduction by electrons from electron transport com-
plexes and a proton gradient across the F1Fl0 ATPase complexes of mitochondrial cristae.
. Homocysteine is a potent excitatory neurotransmitter that binds to the N-methyl-D-aspartate receptor, leading to oxidative stress,
cytoplasmic calcium influx, apoptosis and endothelial dysfunction, the earliest manifestation of atherogenesis.
. The origin of arteriosclerotic vulnerable plaques is attributed to obstruction of the vasa vasorum by aggregates of homocysteinylated
lipoproteins with microorganisms, leading to ischemia of arterial wall muscle cells, intramural cell death, hemorrhage, inflammation and
an intimal microabscess, the vulnerable plaque.
. The declining US mortality rate from coronary heart disease and stroke since 1958 is partly attributed to fortification of processed foods by
pyridoxine and folic acid, precursors of the coenzymes which activate homocysteine metabolism and prevent hyperhomocysteinemia.
. A metabolic and nutritional protocol consisting of whole nutritious foods, thioretinamide, B vitamins, ascorbic acid, vitamin D3,
menoquinone, retinol, nicotinamide riboside, adenosyl methionine and pancreatic enzymes is suggested for therapy and prevention of
diseases of aging associated with hyperhomocysteinemia, including arteriosclerosis, cancer, dementia, osteoporosis, neurodegenerative
diseases, autoimmune diseases and the aging process.

informahealthcare.com doi: 10.1586/17512433.2015.1010516

 Review McCully
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