Homocysteine vs. Cholesterol
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Competing Views, or a Unifying Explanation of Arteriosclerotic Cardiovascular Disease?
A discovery made nearly three decades ago--little noted at the time and just nowattracting serious attention from physicians--promises to drastically alter ourview of what causes heart attacks and strokes.It also may mean that the simplest dietary precautions, adopted early in life,can substantially lower the risk.
Since at least the 1950s, the dominant view of arteriosclerotic cardiovascular disease (ACVD) has been that it is mainly the outcome of a lifelong diet rich in animal fats, cholesterol in particular. This theory holds that cholesterol, mainly its low-density-lipoprotein (LDL) variety, collects in plaques that line the insides of large and small arteries. In time, the plaques themselves, or the clots they cause to form, may impede blood flow to the heart, the brain, and the arteries supplying the lower limbs. The consequences are heart attacks, strokes, and crippling peripheral vascular disease.
Most researchers and clinicians today hold to the "cholesterol theory" although it has never been rigorously proved. Even if high lipid levels do increase the risk of ACVD, that does not mean that lipids themselves cause the condition that we commonly think of as "hardening of the arteries." Exactly how they might do this remains a mystery.
First Insights
In 1968, while attending conferences in human genetics at the
Massachusetts General Hospital in Boston, Kilmer S. McCully, MD,
encountered two children with a genetic disorder called
homocystinuria. In patients with this disorder, homocysteine, an
amino acid formed from methionine, is present in the blood in
excessive amounts and excreted in the urine.
Strikingly, these children--one of them a boy only 2 months old--had an advanced stage of arteriosclerosis that, to McCully, closely resembled that seen in older adults with ACVD except for one thing: The plaques contained no lipid.(1,2) Similar changes had been seen in other homocystinuric children but, because lipid deposits were not evident, they were dismissed as a special kind of "toxic" effect of homocysteine on the arteries.
Fortuitously, autopsy tissues were available from an 8-year-old homocystinuric child who had died of a stroke 35 years earlier. The arteries looked exactly like those of elderly men with arteriosclerosis. It occurred to McCully that the primary pathology is fibrotic (and fibrocalcific) change in the inner arterial lining, which literally produces "hardened" arteries. Could this be a direct result of exposure to an elevated level of homocysteine in the circulating blood?
Genetic Clues
At least three forms of homocystinuria occur in children who
inherit enzyme deficiencies. The most common form is a lack of
cystathione-psynthase, which converts homocysteine to cystathione.
These children are very likely to develop arteriosclerosis at an
early age, and many of them die young of coronary heart disease or
stroke. Two much rarer forms of homocystinuria likewise accelerate
fibrosclerotic arterial changes.
Are carriers of homocystinuria-those with a single abnormal gene-also at risk of arterial disease? It would seem so. Studies of individuals with premature ACVD who lack the usual risk factors for arteriosclerosis show that 30% of them do have abnormally high blood homocysteine levels.(3) It is claimed that as many as one in every three persons with ACVD carries a gene that impairs homocysteine metabolisms but what causes ACVD in the other two persons?
Metabolism Gone Awry:
The Homocysteine Theory
When the essential amino acid methionine is metabolized, one
of the products formed is homocysteine, itself a sulfur-containing
amino acid. Homocysteine is further metabolized by either
remethylation, a process requiring vitamin B12 and folic acid, or
by a transsulfuration pathway that involves a vitamin B6-dependent enzyme. Normally homocysteine is rapidly metabolized
enzymatically, keeping it from building up in the circulation.
Animal proteins in meats, poultry, and dairy products, however, contain up to three times as much methionine as does plant protein, creating much more homocysteine to be dealt with. It is this excess homocysteine that causes tissue damage, either in children with homocystinuria or to the arteries in persons of any age.
Dietary studies further support a correlation between homocysteine levels and intake of micronutrients. A study of nearly 1,200 elderly persons showed that the lower the plasma levels of vitamins B6, B12 and folate, the higher the blood homocysteine level.(5) Populations with high rates of ACVD are the same ones whose diet typically includes processed foods containing little of these vital micronutrients. In addition, these vitamins are readily destroyed when foods are heated, refined, dried, or irradiated. The milling of flour from grains is a key example. Groups of people who eat mainly plant proteins and fresh, unprocessed, unrefined foods develop vascular disease much less often than those eating a "modern" diet.
Apart from genetic disorders and a lack of sufficient micronutrients in the diet, the following factors also may contribute to hyperhomocysteinemia:
- Being male or elderly
- Impaired renal function
- Hypothyroidism
- Such drugs as methotrexate and phenytoin, which alter folate metabolism; and theophylline, which affects the synthesis of pyridoxal phosphate (and therefore the vitamin B6 level)
Summing Up
McCully, now a staff pathologist at the Veterans Affairs Medical
Center in Providence, RI, is amused to note that, after 15 years
of being almost alone in taking the homocysteine theory seriously,
at present there is virtually a "new paper every day" on
the subject. Elevated homocysteine, he says, "is likely to be
of extreme importance in the pathogenesis of arteriosclerosis,
whether related to dietary deficiencies of vitamin B6, folic acid,
or vitamin B12; genetic defects; toxic agents; hormonal changes; aging;
or other general factors."
After publishing The Homocysteine Revolution in 1997, McCully heard of a Czechoslovakian pathologist who, when World War II ended, supervised more than 10,000 autopsies of starved death camp victims. Despite a total lack of dietary fat, most of them had severe arteriosclerotic changes-much like homocystinuric children. After the fat reserves of these starving prisoners were exhausted, their bodies metabolized muscle tissue and even tendon and bone, all of which contain proteins rich in methionine. At the same time, their daily rations were grossly deficient in B6 and folate, says McCully. Anecdotal, certainly, but this episode hints at much of what homocysteine can tell us about arteriosclerosis that the lipid theory cannot.
Joseph A. Knight, MD, professor of pathology at the University of Utah School of Medicine in Salt Lake City, acknowledges McCully's "beautiful work" and goes on to say that "the nice thing about [homocysteine] is that it's manipulable in most individuals, because if they increase their intake of folic acid, B12 and B6 as well, this will decrease the level and remove, for most people, that risk factor."
Testing and Screening
Without any doubt, the demand for plasma homocysteine testing will
increase in the coming months and years, perhaps explosively. What
has long been the standard procedure for quantifying plasma
homocysteine is soon to be replaced by an equally accurate but
much more efficient and less costly enzyme immunoassay (see
"Today's Test/ Tomorrow's Test").
McCully says that, at present, it is reasonable to screen for elevated homocysteine in two groups-those who have:
- A strong family history of atherosclerotic disease
- Early (before age 50) symptoms of coronary heart disease, cerebrovascular disease, or peripheral vascular disease
What Homocysteine Does to Arteries
Experiments on arterial segments and on live animals as well as
observations of hyperhomocysteinemic patients have shown that
homocysteine can contribute to several processes that lead to
atherosclerosis:
- Homocysteine generates superoxide and hydrogen peroxide, both of which have been linked to damage of the endothelial lining of arterial vessels.
- It changes coagulation factor levels so as to encourage blood clot formation. It prevents small arteries from dilating, thereby making them more vulnerable to obstruction by clot or plaque.(6)
- It causes the smooth muscle cells that support the arterial wall to multiply-part of the atherogenic process.(7)
- In its reactive form, homocysteine thiolactone, it causes platelets to aggregate-part of the clotting process.
- When infused into baboon arteries, homocysteine causes the endothelial lining cells to actually slough off. The resulting lesions look very much like human atheromas.(8)
The Clinical Evidence Mounts
Numerous studies reported in the past 15 years provide
impressive support for the homocysteine theory of
arteriosclerosis. When nearly 15,000 male physicians were followed
for 5 years, those with the highest homocysteine levels were three
times more likely to suffer a heart attack, even when taking other
risk factors into account.' In another prospective study, focusing
on about 600 Norwegian patients with coronary artery disease,
heart attacks, and deaths rose in straight-line relation to the
plasma homocysteine level.(10) An international study of healthy
middle-aged men from 11 countries convincingly shows that the
risk of dying from ACVD increases with the average homocysteine
level in a given nation (see "Homocysteine Levels and
Cardiovascular Mortality"). Finally, a prospective study
reported early this year shows that, for each 5-[Lmol/L increase
in blood homocysteine content, the risk of middle-aged men dying
of ischemic heart disease rises by one third.(12)
A 1995 report analyzing results from no fewer than 27 studies concluded that the risk of arteriosclerotic vascular disease does in fact increase with the homocysteine level, regardless of whether cholesterol is normal or elevated. The authors conclude that 10% of the overall risk of coronary artery disease can be blamed on excessive homocysteine.(13)
But not all studies support this association. A recent look at about 700 men at moderately high risk of coronary heart disease failed to show any substantial difference in homocysteine levels between those who developed heart disease and those who did not, although mean levels for both groups were near the upper limit of normal (15µmol/L).(14)
In time, it may be necessary to lower the upper limit of normal for homocysteine levels. Many studies have found a gradation in risk with increasing levels even within the accepted normal range. The forthcoming enzyme immunoassay for homocysteine (see "Tomorrow's Test") should make much more data available and give us a more precise idea of what is "normal."
Partners in Crime: Unifying the Homocysteine and
Cholesterol Theories
One need not choose between the two major views of how
arteriosclerotic disease develops; there is plenty of "pathogenetic
room" to accommodate both of them. McCully has claimed from
the outset that excess homocysteine was chiefly responsible for
the early fibrotic and calcific changes in the arterial wall. He
believed that lipid deposits developed later-after years of eating
saturated fats-to produce the classic atheromas of what today is
usually called atherosclerosis. (The term athero refers to
the porridge-like appearance of lipid deposits in the arterial
wall.)
It now appears that, beyond being merely additive risk factors, homocysteine and cholesterol, specifically its LDL fraction, combine to damage the arterial endothelial lining cells - the prelude to full-blown ACVD.
Homocysteine thiolactone, a chemically reactive intermediate that forms when homocysteine is being metabolized, combines with the apolipoprotein B component of LDL cholesterol. When their sulffiydryl groups are oxidized, the LDL becomes more dense, aggregates, and precipitates spontaneously. This material is taken up by phagocytic cells in the arterial wall (the "foam cells") and, when it breaks down, cholesterol, and other lipids are deposited in the arterial wall. The cycle is completed when homocysteine released from degraded LDL auto-oxidizes to form superoxide and hydrogen peroxide, which in turn oxidize LDL and stimulate the arterial smooth muscle cells to proliferate.(15,16)
This "unitary" theory shows how two outstanding risk factors for arteriosclerosis combine to initiate the disease. Much of its appeal lies in its ability to explain the mystery of why some persons live to a ripe old age despite having very high cholesterol levels, while others who eat a healthy diet and have normal cholesterol nevertheless develop debilitating peripheral vascular disease or die of a stroke or heart attack.
As McCully says, "You know that there's something else going on [besides high cholesterol], and I'm suggesting that it's homocysteine." Knight agrees that current research points to a final common pathway through which these risk factors produce the earliest changes of ACVD. But he cautions that numerous other risk factors must not be overlooked. They include altered levels of certain coagulation factors, such as factor Vll; decreased estrogen levels in postmenopausal women; and possibly even elevated iron. Maintaining homocysteine within the normal range will not in itself be a "magic bullet."
Homocysteine and Cancer
High blood levels of homocysteine also have been found in
patients with deep venous thrombosis and infants born with neural
tube defects such as spina bifida (V. Ricchiuti, PhD, March 1998,
written communication). The same maybe the case for rheumatoid
arthritis. Perhaps most importantly, modestly elevated serum
homocysteine levels were detected in patients with various
carcinomas.
James Wu, PhD, professor of pathology at the University of Utah in Salt Lake City, is looking closely at this association and believes it to be most evident in patients with cancers of the breast, ovary, colon, and possibly the prostate. In many patients, the homocysteine concentration correlates with levels of serum tumor markers in their serial specimens and reliably declines when the tumor regresses during treatment. Laboratory studies show that as breast cancer cells grow in tissue culture, they secrete increasing amounts of homocysteine. Wu believes that elevated homocysteine probably is a result of the carcinoma, not its cause. It may someday be used clinically as a marker for certain epithelial cancers. If these associations are confirmed and others emerge, requests for homocysteine testing will expand beyond the realm of vascular disease.
Remedies for Cardiovascular Disease?
In the past few years, reports of several treatment trials
indicate that B-vitamin supplements reduced risk for
cardiovascular disease and stroke:
- Patients with homocysteine levels higher than 14 µmol/L and
expanding plaques in their carotid arteries had an actual
decrease in plaque area after they started daily supplements
of folic acid, B6 and B12. (17)
- Women followed up for 14 years who had the highest intake of
folate and B6 were only about half as likely as those with the
lowest intakes to have a heart attack, fatal or otherwise.(18)
(McCully feels that this study in particular lends strong
support to the homocysteine theory.(1))
- Stroke survivors given vitamins maintained lower levels of both homocysteine and thrombomodulin-a marker of endothelial-cell damage-than did control patients, and this after only 3 months on supplements.(19)
At just what point the diet needs to be supplemented by extra micronutrients remains a matter of debate, likewise the optimal doses. Clearly, however, current recommended dietary allowances (RDAS) of vitamins B6, B12, and folic acid are subject to change. Perhaps it is best to err on the high side. There is little or no risk, and the potential health benefits of controlling blood homocysteine are immense. Consensus is forming that:
- Everyone's daily diet should be rich in fresh fruits,
vegetables, grains, and cereals. (The US Department of
Agriculture recommends 2 to 4 servings of fruit, 3 to 5
servings of vegetables, and 6 to 11 servings of breads,
cereals, rice, or pasta daily.)
- Processed and refined foods should be kept to a minimum.
- Antioxidants such as vitamins C and E may prove helpful when
blood homocysteine levels are elevated (J. A. Knight, MD,
March 1998, verbal communication).
- A daily multivitamin, preferably one without iron, is a convenient source of both vitamins and minerals.
A fairly representative supplemental regimen, used in a study of stroke survivors,19 provided 5 mg of folic acid, I mg of vitamin B12, and 100 mg of vitamin B6 in addition to a daily multivitamin.
Prospective trials are needed in which patients with high homocysteine levels are randomized, to receive either micronutrient supplements or a placebo, and followed up for several years to learn whether regaining a normal level actually does retard the progress of arteriosclerotic vascular disease. Testing for homocysteine will become an integral part of what undoubtedly will be a massive public health effort to reduce heart disease and strokes. Testing will be vital at all levels: research, screening, clinical assessment, and monitoring the effects of treatment.
David A. Cramer is a Chicago-based medical writer.
Reprinted from LABORATORY MEDICINE
Vol. 29, No. 7, July 1998. Copyright © 1998
by American Society of Clinical Pathologists, Inc.
Homocysteine Glossary | Today's Test | Tomorrow's Test
Acknowledgments
The author thanks Ivanka Jerkunica, SC(ASCP); Joseph A. Knight, MD; Kilmer S. McCully, MD; Vincent Ricchiuti, PhD; and James Wu, PhD, for their invaluable assistance in preparing this manuscript.
References
1. McCully KS. Homocysteine, folate, vitamin B diovascular disease. JAMA. 1998;279:392-393.
2. McCully KS. Vascular pathology of homocysteinemia: implications for the pathogenesis of arteriosclerosis. Am J Pathol. 1969;56:111-128.
3. McCully KS. Homocysteine and vascular disease. Clinical Chemistry Check Sample, CC-94-5. Chicago, III: ASCP; 1994;34:65-77.
4. Ignarro Lj, Lippton H, Edwards JC, et al. Mechanisms of vascular smooth muscle relaxation by organic nitrates, nitrites, nitroprusside and nitric oxide: evidence for the involvement of S-nitrosothiol as active intermediates. J Pharmacol Exp Ther. 198 i;218:739-749.
5. Selhub J, Jacques PF, Wilson PWF, et al. Vitamin status and intake as primary determinants of homocysteinemia in an elderly population. JAMA. 1993;270:2693-2698.
6. Wu KS, Chook P, Lolin YI, et al. Hyperhomocyst(e)inemia is a risk factor for arterial endothelial dysfunction in humans. Circulation. 1997;96:2542-2544.
7. Tsai JC, Perrella MA, Yoshizumi M, et al. Promotion of vascular smooth muscle cell growth by homocysteine: a link to atherosclerosis. Proc Natl Acad Sci U S A. 1994;91:6369-6373.
8. Harker LA, Slichter Sj, Scott CR, et al. Homocystinuria: vascular injury and arterial thrombosis. New Engl J Med. 1974;291:537-543.
9. Stampfer Mj, Malinow MR, Willett WC, et al. A prospective study of plasma homocyst(e)ine and risk of myocardial infarction in US physicians. JAMA. 1992;268:877-88 1.
10. Nyggrd 0, Nordrehaug JE, Refsum H, et al. Plasma homocysteine levels and mortality in patients with coronary artery disease. N Engl I Med. 1997;337:230-236.
11. Alfthan G, Aro A, Gey KF. Plasma homocysteine and cardiovascular disease mortality. Lancet. 1997;349:397.
12. Wald NJ, Watt HC, Law MR, et al. Homocysteine and ischemic heart disease: results of a prospective study with implications regarding prevention. Arch Intern Med. 1998;158:862-867.
13. Boushey Cj, Berestord, SSA, Omenn GS, et al. A metaanalysis of plasma homocysteine as a risk factor for arteriosclerosis vascular disease and the potential preventive role of folic acid. In: International Congress on Homocysteine Metabolism: From Basic Science to Clinical Medicine. Norwell, Mass: Kluwer Academic, 1995.
14. Evans RW, Shaten Bj, Hempel JD, et al. Homocyst(e)ine and risk of cardiovascular disease in the multiple risk factor intervention trial. Arterioscler Thromb Vasc Biol. 1997;17:1947-1953.
15. McCully KS. Chemical pathology of homocysteine: 1. Atherogenesis. Ann Clin Lab Sci. 1993;23:477-493.
16. Naruszewica M, Mirkiewicz E, Olszewski Aj, et al. Thiolation of low-density lipoprotein causes increased aggregation and interaction with cultured macrophages. Nutr Metab Cardiovasc Dis. In press.
17. Peterson JC, Spence JD. Vitamins and progression of atherosclereosis in hyper-homocyt(e)inaemia. Lancet. 1998;351:263.
18. Rimm EB, Willett WC, Hu FB, et al. Folate and vitamin B6 from diet and supplements in relation to risk of coronary heart disease among women. JAMA. 1998;279:359-364.
19. Macko RF. B-vitamins lower homocysteine levels in the blood; may reduce vessel damage in stroke patients. Press release at 23rd International joint Conference on Stroke and Cerebral Circulation; February 1998; Orlando, Fla,
Additional Readings
Graham 1, Refsum H, Rosenberg IH, et al, eds. Homocysteine Metabolism: From Basic Science to Clinical Medicine. Norwell, Mass: Kluwer Academic Publishers; 1997.
McCully KS. 7he Homocysteine Revolution: Medicine for the New Millennium. New Canaan. Conn: Keats Publishing; 1997.
Glossary
- Cystathione P-synthase
- a catalyst for homocysteine metabolism by the transsulfuration pathway. Its deficiency creates the most common form of homocystinuria.
- Homocysteine (homo-sis-TAY-een)
- a sulfur-containing amino acid formed during methionine metabolism; 70% to 80% of homocysteine forms disulfide bonds with sulffiydryl groups of plasma proteins and thus becomes a protein-bound fraction@
- Homocysteine thiolacetone
- a product of the demethylation of methionine, which is thought to damage the endothelial lining cells of arteries
- Homocystine
- the disulfide form of homocysteine, which itself is a monomer
- Methionine
- an essential amino acid derived from dietary protein, whose metabolism is a source of methyl groups and sulfur
- Remethylation
- a means of "salvaging" homocysteine by reforming methionine. Reaction is catalyzed by methionine synthase, with vitamin B12 an essential cofactor. The methyl donor is 5,10-methyltetrahydrofolate.
- Transsulfuration
- an alternative metabolic pathway used when there is excessive methionine or when cysteine synthesis is required. Reaction of homocysteine with serine is catalyzed by cystathionine-b-synthase, a vitamin B6-dependent enzyme, to form cystathione, which then is hydrolyzed to form cysteine.
Today's Test
High-performance liquid chromatography (HPLC)
Overview - The HPLC test, used for more than 10 years, is a reproducible and useful way to measure plasma homocysteine. 1% Results may be directly compared between laboratories. The time and cost involved, however, preclude the use of HPLC as a practical clinical chemistry test.
What happens - Plasma is treated with a strong reducing agent such as n-butyl phosphine to reduce the homocystine to homocysteine, and perchloric acid is added to remove all disulfide-bound homocysteine from protein. A fluorographic reagent is added, chromatography is carried out, and total homocysteine is quantified by fluorescence detector, using a standard curve prepared with known amounts of homocysteine in plasma, and run simultaneously. The two incubation periods and processing together take 2 1 hours.
Sample preparation - Plasma is collected into a tube with heparin or EDTA. It should be frozen by dry ice and processed within 4 hours so that the red and white blood cells will not synthesize appreciable additional homocysteine from methionine. If not refrigerated, plasma must be separated within 1 hour of collection.
Staff time - 20 to 30 minutes to set up the test; 20 minutes to process and add dye; 10 minutes to set up the HPLC
Availability of results - The same day for a morning sample, or the following morning
Normal range - About 4 to 15 µmol/L
Analytic sensitivity - About 3 µmol/L
Interfering substances - None known
Quality control - Internal only; no standards available
Cost - About $80
Where performed - Large hospitals and reference laboratories
Source: Details on test procedures were provided by Ivanka Jerkunica, SC(ASCP), of Emory Medical Laboratories, Emory University Hospital, Atlanta.
Tomorrow's Test
A competitive enzyme immunoassay (EIA)
[editor's note: assay is FPIA, not EIA]
Overview - Test results correlate with those of high-performance liquid chromatography (HPLC) at a level of at least 0.98. Axis Biochemical ASA (Oslo, Norway) has developed a fluorescence polarization FPIA for Abbott Laboratories IMx System (Abbott Park, Ill.) and a microtiter FPIA using absorbance readings for Bio-Rad (Hercules, Calif). The Food and Drug Administration is reviewing both tests for marketing approval within the United States. They are already available in Europe and elsewhere. [Since publication of this article, both tests have been cleared for marketing in the United States.]
Procedure - Plasma samples are mixed with buffer containing the reducing agent dithiothreitol. The first incubation includes the enzyme S-adenosyl-L-homocysteine hydrolase and adenosine.
For the fluorescence polarization FPIA, the S-adenosyl -L-homocysteine that forms is incubated with a monoclonal antibody raised against S-adenosyl-L-homocysteine and fluorescently labeled S-adenosylcysteine. On passing polarized light through the samples, the degree of depolarization is inversely proportional to the amount of label bound antibody. Results are read automatically and printed out.
For the microtiter plate, the enzyme-treated sample is transferred to immunoplates coated with anti-S-adenosyl-L-homocysteine. The S-adenosyl-L-homocysteine is determined in a competitive immunoassay using monoclonal anti-S-adenosyl-L-homocysteine and horseradish peroxidase-conjugated antibodies.
Sample preparation - Same precautions as for
HPLC.
Staff time: For the fluorescence polarization FPIA, about 10
minutes to load samples into the carousel. For the microliter
plate, 45 minutes if the method is performed manually, 15 minutes
for the automated method.
Availability of results - For the fluorescence polarization FPIA, about an hour. For the microtiter plate, less than 3 hours for the manual method; less than 2 hours for the automated method.
Normal range - Typically 4 to 12 µmol/L for the fluorescence polarization FPIA; 5 to 15 µmol/L for the microtiter plate, but each laboratory will determine its own range.
Analytic sensitivity - Less than 0.5 µmol/L
Interfering substances - No cross-reactions extensive enough to cause problems for the fluorescence polarization FPIA. Bio-Rad reports that concentrations of greater than 10 µmol/L of S-adenosyl-L-methionine will falsely elevate values in the microliter assay. [Note: Abbott reports S-adenosyl-L-methionine at 0.5mM can yield 12.9% cross reactivity]
Quality control - Six calibrators and three controls.
Estimated cost - Not yet determined for the fluorescence polarization FPIA; $13 to $18 for each microtiter test.
Where performed - At first, the fluorescence polarization FPIA probably will be limited to larger hospitals and reference laboratories. Mid-sized hospitals may later adopt it if they get enough requests. Microplate format can be used in laboratories of all sizes.
Sources: Abbott Laboratories. Abbott Park, IL: Bio-Rad. Hercules, Calif
