Atherogenic Effect of Homocysteine, a Biomarker of Inflammation and Its Treatment

Abstract

Hyperhomocysteinemia (HHcy) is an independent risk factor for atherosclerosis. Ischemic stroke and heart disease, coronary heart disease, and cardiovascular disease are events resulting from long-lasting and silent atherosclerosis. This paper deals with the synthesis of homocysteine (Hcy), causes of HHcy, mechanism of HHcy-induced atherosclerosis, and treatment of HHcy. Synthesis and metabolism of Hcy involves demethylation, transmethylation, and transsulfuration, and these processes require vitamin B ₆ and vitamin B ₁₂ folic acid (vitamin B ₉ ). Causes of HHcy include deficiency of vitamins B ₆ , B ₉ , and B ₁₂ , genetic defects, use of smokeless tobacco, cigarette smoking, alcohol consumption, diabetes, rheumatoid arthritis, low thyroid hormone, consumption of caffeine, folic acid antagonist, cholesterol-lowering drugs (niacin), folic acid antagonist (phenytoin), prolonged use of proton pump inhibitors, metformin, and hypertension. HHcy-induced atherosclerosis may be mediated through oxidative stress, decreased availability of nitric oxide (NO), increased expression of monocyte chemoattractant protein-1, smooth muscle cell proliferation, increased thrombogenicity, and induction of arterial connective tissue. HHcy increases the generation of atherogenic biomolecules such as nuclear factor-kappa B, proinflammatory cytokines (IL-1β, IL-6, and IL-8), cell adhesion molecules (intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selection), growth factors (IGF-1 and TGF-β), and monocyte colony-stimulating factor which lead to the development of atherosclerosis. NO which is protective against the development of atherosclerosis is reduced by HHcy. Therapy with folic acid, vitamin B ₆ , and vitamin B ₁₂ lowers the levels of Hcy, with folic acid being the most effective. Dietary sources of folic acid, vitamin B ₆ , vitamin B ₁₂ , omega-3 fatty acid, and green coffee extract reduce Hcy. Abstaining from drinking coffee and alcohol, and smoking also reduces blood levels of Hcy. In conclusion, HHcy induces atherosclerosis by generating atherogenic biomolecules, and treatment of atherosclerosis-induced diseases may be by reducing the levels of Hcy.

Homocysteine (Hcy) is associated with ischemic stroke, ischemic heart disease, cardiovascular disease, and atherosclerosis, and these pathological outcomes are events from long-lasting and silent process known as atherosclerosis. Elevation of circulating Hcy is associated with inflammation and is considered a biomarker of inflammation. Hyperhomocysteinemia (HHcy) is associated with numerous diseases including, ischemic stroke, ^{1 2} ischemic heart disease, ³ coronary artery disease (CAD), ^{4 5 6} cardiovascular disease, ^{7 8} hypertension, ^{9 10 11} chronic renal failure, ¹² atherosclerosis, ^{13 14} venous thrombosis, ¹⁵ dementia, ¹⁶ and Alzheimer disease. ^{17 18} All these pathological outcomes are events resulting from the long-lasting and silent process known as atherosclerosis. It appears that HHcy is associated with numerous diseases. This association is not limited to severe homocysteinemia, ¹⁹ but it also occurs with moderately elevated Hcy. ^{20 21 22 23} Several findings support the hypothesis that HHcy is causally related to atherosclerotic diseases such as CAD, cerebrovascular disease, and peripheral arterial disease. ^{20 21 22 23 24} The above findings suggest that HHcy is causally associated with atherosclerotic diseases. HHcy is an independent risk factor for atherosclerosclerosis. ¹³ This review deals with the synthesis of Hcy, causes of HHcy, HHcy and atherosclerosis, and mechanism of HHcy-induced atherosclerosis and its prevention and treatment.

Synthesis and Metabolism of Homocysteine

The synthesis of Hcy is given in detail by Prasad ²⁵ and Lee and Prasad. ²⁶ Hcy is a sulfur-containing amino acid derived from methionine and is essential for several biochemical processes including the metabolism of nucleic acids, fats, and high-energy bonds.

It is recycled back into methionine or converted into another amino acid, cysteine. Synthesis and metabolism of Hcy involves demethylation, transmethylation, and transsulfuration. The demethylation process converts methionine to Hcy. In the transmethylation process, Hcy is remethylated to methionine catalyzed by methionine synthase which uses vitamin B _12. ²⁷ In the transsulfuration process, Hcy is irreversibly converted to cysteine and requires vitamin B ₆ for this process. ^{27 28}

In human fasting, plasma levels of Hcy concentration is below 12 to 15 µmol/L. ²⁹ Average Hcy concentration in men is 12.6 µmol/L and in women 9.6 µmol/L and increases with age such as 4.6 to 8.1 µmol at ages 0 to 30 years, 6.3 to 11.2 µmol/L at ages of 30 to 59 years in males, 4.5 to 7.9 µmol/L in female, and 5.8 to 11.9 µmole/L at ages above 59 years. ³⁰ The plasma level of Hcy increases with age by 1 µmol/L/decade. ³¹

Causes of Hyperhomocysteinemia

The causes of HHcy include deficiency of vitamins B _6, B ₉ (folic acid), and B _12, ^{32 33 34} genetic defects, ^{35 36} smokeless tobacco, ³⁷ cigarette smoking, ³⁸ alcohol consumption, ³⁹ diabetes, ⁴⁰ rheumatoid arthritis, ⁴¹ and low thyroid hormone. ⁴² Caffeine increases the Hcy levels. ⁴³

HHcy is associated with pernicious anemias, ⁴⁴ and several types of cancer such as breast, ovary, and pancreas. ⁴⁵ Certain drugs such as folic acid antagonist (phenytoin, carbamazopin, ⁴⁶ and cholesterol-lowering drug [niacin]). ⁴⁷ Prolonged use of proton pump inhibitors causes deficiency of vitamin B ₁₂ which may cause HHcy. ⁴⁸ _. Metformin could increase the concentration of Hcy when exogenous B-group vitamins or folic acid supplementation is not given. ⁴⁹ HHcy arises from genetic defects in enzymes involved in Hcy metabolism. ⁵⁰ Increasing age, male sex, unfavorable lipid profile, high creatinine, and fatty diets are associated with HHcy. ⁵¹ Elevated Hcy is associated with a higher risk of CAD in patients with chronic renal dysfunction. ⁵² Hypertension has been positively associated with both systolic and diastolic blood pressiures. ⁵³

Hyperhomocysteinemia and Atherosclerosis

McCully in1969 had suggested that HHcy is associated with atherosclerosis. ⁵⁴ Prevalence of CAD is high in patients with HHcy. ^{54 55 56} Boushey et al reported that for every 5 µmol/L increase in Hcy there was a 70% higher risk of CAD and a 50% increase in cerebrovascular disease, and there was a strong association with peripheral arterial disease. ⁵⁷ They also reported that the risk of CAD in 10% of the population is attributed to Hcy. Eikelboom et al ⁵⁸ reported that there is a strong dose-dependent positive association between plasma levels of Hcy and the risk of cardiovascular disease.

Mechanism of Homocysteine-Induced Atherosclerosis

HHcy-induced atherosclerosis may be mediated through oxidative stress, decreased bioavailability of endothelial nitric oxide (NO), increased expression of monocyte chemoattractant protein-1 (MCP-1), smooth muscle cell proliferation, increased thrombogenicity, and induction of arterial connective tissue.

Oxidative Stress

Hcy is toxic to the endothelial cells. ^{59 60} This toxicity of Hcy could be due to the Hcy-induced generation of reactive oxygen species (ROS). ROS are known to produce endothelial cell injury. ^{24 61 62} The sulfhydryl group of Hcy acts catalytically with ferric or cupric ions to generate oxygen radicals, hydrogen peroxide, and Hcy radicals. ^{63 64} Hcy activates PAR-4 which induces ROS production by increasing NADPH oxidase and decreasing thioredoxin expression and reduces NO bioavailability in cultured cardiac microvascular endothelial cells. ⁶⁵ HHcy induces oxidative stress. ⁶⁶ Hcy can produce hydrogen peroxide (H ₂ O ₂ ) during metal-catalyzed oxidation and in the presence of NO. ⁶⁷ Autooxidation of Hcy generates superoxide anion, hydroxyl radicals (OH), and hydrogen peroxide (H ₂ O ₂ ). ⁶⁸ Hcy increases the release of superoxide anion by neutrophil. ⁶⁹ Hcy also reduces the antioxidants leading to increased levels of oxygen radicals. Hcy decreases the activity of antioxidant enzymes (superoxide dismutase (SOD), catalase, glutathione peroxidase [GSH-Px]), in plasma of methionine-induced atherosclerosis in rabbits. ⁷⁰ In welders, HHcy was associated with reduced SOD activity in plasma. ⁷¹

How Oxygen Radicals are Involved in Atherosclerosis?

The role of ROS in the development of atherosclerosis has been reported by Steinberg, ⁷² Prasad and Kalra, ⁷³ and Prasad and Mishra. ⁷⁴ ROS increases the expression of cell adhesion molecules (CAM), intercellular adhesion molecule-1 (ICAM-1), ⁷⁵ vascular cell adhesion molecule-1 (VCAM-1), ⁷⁶ and E-selectin. ⁷⁷ ROS activates nuclear factor kappa B (NF-kB) ⁷⁸ which in turn activates proinflammatory cytokines genes including, interleukin (IL)-1, IL-2, IL-6 and NF-kB. ^{79 80 81} ROS stimulates insulin-like growth factor-1 (IGF-1) in vascular smooth muscle ⁸² and transforming growth factor-β (TGF-β). ⁸³ Oxidation of low-density lipoprotein-cholesterol (LDL-C) by ROS has numerous functions in the development of atherosclerosis. ^{84 85 86 87 88} ROS oxidizes LDL-C to minimally modified LDL (MM-LDL) which is further oxidized to form maximally oxidized LDL (OX-LDL). ⁸⁹ MM-LDL activates smooth muscle cells and endothelial cells to produce MCP-1 that helps in the migration of monocytes (leukocytes) from the endothelial surface to subendothelial space. Monocytes have LDL receptors which combine with native LDL. However, the amount of native LDL is not enough to form foam cells. The function of MM-LDL is to stimulate endothelial cells to generate monocyte colony-stimulating factor which helps in the differentiation of monocytes to macrophages that develop receptors for OX-LDL. Differentiated macrophages combine with OX-LDL to form foam cells. Macrophages/foam cells generate a host of growth-regulating molecules platelet-derived growth factor (PDGF), basic fibroblast growth factor, and TGF-β. Endothelial cells produce PDGF and IGF-1. ⁹⁰ Foam cells are involved in the formation of numerous growth factors. Above-mentioned growth factors help in vascular smooth muscle cell proliferation and migration, stimulate the synthesis of connective tissue and matrix including collagens, proteoglycans, and elastic fiber proteins leading to the development and progression of atherosclerosis. Fatty streak development results in full-fledged atherosclerosis.

Role of Nitric Oxide in Hyperhomocysteinemia-Induced Atherosclerosis

NO is protective against atherosclerosis. ⁹¹ It serves as an antiatherosclerotic factor in the endothelium. ⁹² NO interferes with the adhesion of monocytes and leukocytes to the endothelium in the process of the development of atherosclerosis. ^{93 94 95} Hcy damages the endothelial cells. Endothelial NO inhibits endothelial cell activation and macrophage infiltration, ⁹⁶ platelet aggregation, ⁹⁷ and thrombosis, ⁹⁸ which are involved in the formation of atherosclerosis. Hcy inhibits the synthesis of NO. ⁹⁹ Hcy combines with NO in the presence of oxygen resulting in S-nitroso- Hcy. ⁶⁴ Hcy inhibits NO synthase enzyme. ⁹⁹ Hcy promotes lipid peroxidation that may decrease the expression of endothelial NO synthase and directly degrade NO. ^{100 101 102} NO interacts with superoxide anion to form peroxynitrite, ¹⁰³ which can cause vascular pathology by various mechanisms including, cell death, upregulation of CAM in endothelial cells, enhancing neutrophil adhesion, and atherosclerosis. ¹⁰⁴ Hcy suppresses the expression of cellular GSH-Px by endothelial cells that may promote lipid peroxidation by ROS generated by Hcy. ¹⁰⁵ Hcy decreases the bioavailability of NO by a mechanism involving GSH-Px. ¹⁰² Bioproduct of oxidation of Hcy gives Hcy thiolactone which combines with LDL to form foam cells. ¹⁰⁶ Hcy-induced in oxidative metabolism results in the overproduction of ROS which induce endothelial injury. Resulting in the development of atherosclerosis. ¹⁰¹

In summary, NO is protective against the development of atherosclerosis. Hcy decreases the bioavailability of NO by decreasing the synthesis of NO that helps in the development of atherosclerosis.

Role of Homocysteine in the Generation of Atherogenic Biomolecules

Hcy stimulates MCP-1 in endothelial cells ¹⁰⁷ which helps in the migration of monocytes into the endothelial cell of the artery. Hcy increases the expression of MCP-1 and IL-8 ^{108 109} that assist in the development of atherosclerosis. Hcy-induced superoxide anion production may play a role in NF-kB activation. ¹¹⁰ Hcy induces MCP-1 expression by activating NF-kB in THP-macrophage-derived macrophages. ¹¹¹ HHcy increases the secretion of proinflammatory cytokines and IL-1β and IL-6 secreted by peripheral blood mononuclear cells. ¹¹² Hcy increases monocyte recruitment into developing atherosclerosis by upregulating MCP-1 and IL-8 expression in vascular smooth muscle cells. ¹¹³ Hcy increases the expression of IL-1β, IL-6, IL-8, and IL-12 in human monocytes in vitro. ¹¹⁴ Silverman et al ¹¹⁵ have reported that HHcy increases VCAM-1 expression and monocyte adhesion in cultured human aortic endothelial cells. It has been shown that Hcy significantly increases the expression of ICAM-1 and E-selectin. ¹¹⁶ HHcy is associated with increased expression of TGF-β ₁ in the rat model. ¹¹⁷ Hcy induces the expression of CRP, ¹¹⁸ stimulates the proliferation of vascular smooth muscle cells, ¹¹⁹ and induces alteration in arterial connective tissue. ¹²⁰ From above, it appears that HHcy increases the expression of numerous biomolecules which could induce atherosclerosis.

In summary, Hcy increases the generation of atherogenic biomolecules such as ROS, NF-kB, CAM (ICAM-1, VCAM-1, Eselectin-1), proinflammatory cytokines (IL-1β, IL-6, IL-8), MCP-1, and growth factors (IGF-1, TGF-β) which leads to the development of atherosclerosis.

Effects of Homocysteine on Vascular Cell Proliferation

Hcy increases the proliferation of vascular smooth muscle cells ^{121 122 123} which assists the development of atherosclerosis.

Is There Any Evidence That Hyperhomocysteinemia Induces/Accelerates the Development of Atherosclerosis?

Hofmann et al ¹²⁴ and Zhou et al ¹²⁵ have reported that apoE null mice fed on a hyperhomocysteinemic diet for 8 weeks developed atherosclerotic lesions in the aorta that was greater in size and complexity than those fed a normal chow diet. Hofmann et al ¹²⁴ used low-fat diet enriched with methionine and deficient in folate, vitamin B ₆ , and vitamin B ₁₂ , while Zhou et al ¹²⁵ used high-fat diet plus Hcy or excess methionine. These studies show that HHcy accelerates the development of atherosclerosis.

Treatment of Homocysteine-Induced Atherosclerosis and Cardiovascular Diseases

If low levels of folic acid (vitamin B ₉ ), vitamin B ₆ (pyridoxine), and vitamin B ₁₂ (cyanocobalamin) induce HHcy, then the treatment of atherosclerosis should be to provide the above vitamins. Therapy with folate, vitamin B ₆ , and vitamin B ₁₂ prevents stroke especially ischemic stroke. ¹²⁶ Folic acid in the dose of 400 µg or more daily has been reported to reduce the levels of Hcy by 30 to 42%. ¹²⁷ Combination of folic acid (0.65 mg/d), vitamin B ₆ (10 mg/d), and vitamin B ₁₂ (o.4 mg/d) for 6 weeks reduced the levels of Hcy by 49.8% which was similar to folic acid. ¹²⁶ However, vitamin B ₆ in the dose of 1,200 mg/d for 12 weeks decreased Hcy from 14.2 ±  3. 4 to 11.8 ± 2.0 µmole/L. ¹²⁸ Doses lower than this do not produce sustained reduction in Hcy. ¹²⁸ Several other studies have shown that 0.65 to 10 mg/d of folic acid alone or in combination with B ₁₂ and/or B ₆ reduce fasting and postmethionine loading Hcy by 25 to 50% in healthy, hyperhomocysteinemia subjects and in patients with vascular disease. ^{126 129 130} Treatment with folic acid alone reduces Hcy by 40 to 50% within 6 weeks. The doses were graded with the initial dose of >5 mg/d, then 3 mg, and 1 mg daily. Doses as low as 0.65 mg/d have been shown to be effective; however, the recommended dose is 1 to 2 mg/d. ¹²⁶ Vitamin B ₁₂ reduces the plasma Hcy to a maximum of 10 to 15%. ¹²⁶ A 1 mg/d dose of vitamin B ₁₂ is recommended. Vitamin B12 supplementation does a less dramatic reduction in total Hcy. ¹²⁶ Vitamin B ₆ in the dose of 250 mg/d normalized Hcy in 56% of cases of HHcy. ^{130 131} Vitamin B ₁₂ in the dose of 1,000 µg daily results in the absorption of 10 µg by passive diffusion. ¹³²

Dietary Sources of Folic Acid, Vitamin B ₆ , and Vitamin B ₁₂

Overweight and obesity impair folate metabolism causing HHcy. ¹³³ Dietary folate intake lowers HHcy. ¹³⁴ High consumption of meat and dairy products and excess protein intake increase Hcy levels. ¹³⁵ Vegetarians get inadequate vitamin B ₁₂ and have high Hcy. ^{136 137} The Mediterranean diet which balances a high intake of olive oil, fruits, vegetables, whole grains, plant proteins with modest consumption of dairy products, and sea food has healthy Hcy levels. ¹³⁸ Fruits, vegetables, potato, cassava, cornmeal, fish, and chicken are associated with lower Hcy. ¹³⁹

Folic Acid

Good dietary sources of natural folic acid include leafy green vegetables, asparagus, broccoli, cauliflower, strawberries, avocado, beef-like poultry, and egg yolk. ¹⁴⁰ Other sources are many breakfast cereals, fortified grain products, lentils, and most beans.

Vitamin B ₆

Foods that are rich in vitamin B ₆ include fortified breakfast cereals, potatoes, bananas, garbanzo beans (chickpeas), and chicken.

Vitamin B ₁₂

Good sources of vitamin B ₁₂ include dairy products, liver, beef, and some types of fish. ¹⁴¹ The U.S. Food and Drug Administration mandated fortification of flour and cereal products with 140 µg of folic acid/100 gm. This intervention is expected to have a population effect of lowering total Hcy levels by an average of 3 µmole/L and may potentially prevent 17,000 due to atrioventricular septal defect each year.

Miscellaneous

Exercise

Maroto-Sánchez et al ¹⁴² have reviewed in detail the effects of exercise on blood levels of Hcy. After acute exercise, such as triathlon competition, Hcy levels in the blood are higher at 1 and 24 hours postexercise. However, no consensus exists regarding the training effect due to a large variety of exercise intervention due to large variety of exercise interventions with different intensities, durations, and modes of exercises. However, there is consensus that there is an increase in total vitamin B ₁₂ and vitamin B ₆ .

Choline and Betaine

Choline is present in many foods such as egg yolks, dairy products, meat, peanuts, cruciferous vegetables, nuts and seeds, whole grains, and soybeans. Choline makes betaine which is a cofactor in the remethylation of Hcy into methionine. ¹⁴³ Betaine occurs in foods like seafood, wheat germ, and bran, beets, and spinach. ¹⁴⁴

Omega-3 Fatty Acids

Omega-3 fatty acids and fish oil reduce Hcy levels. ¹⁴⁵ Controlled clinical trials have shown that 3 grams of omega-3 fatty acids daily for 2 months significantly reduced the levels of Hcy. ¹⁴⁶

Green Coffee Extract

The green coffee extract significantly lowers the levels of Hcy. ¹⁴⁷

Magnesium

Studies suggest that low magnesium status may exacerbate the intracellular magnesium loss triggered by Hcy. ¹⁴⁸ A study on blood vessel cells cultured in a laboratory found Hcy increased production of compounds that trigger structural changes associated with plaque formation but adding magnesium to the cells' environment mitigated this atherogenic effect of Hcy. ¹⁴⁹ Magnesium has also been shown to reverse the adverse effects of Hcy on cardiac rhythm in laboratory mice. ¹⁵⁰

Abstain from Alcohol

Alcohol drinking should be curtailed because alcohol significantly reduces vitamin B12 and folate levels and increases Hcy levels. ¹⁵¹

Abstain from Smoking

Smoking should be stopped because smoking and exposure to second-hand increases Hcy levels. ^{37 152}

Conclusion

Hcy is derived from methionine and its metabolism involves remethylation, transsulfuration, and demethylation which requires vitamins B ₆ , B ₉ , and B ₁₂ . HHcy occurs with a deficiency of vitamins B _6, B ₉ , and B ₁₂ , genetic defects, smoking, consumption of alcohol and caffeinated coffee, and use of certain drugs. HHcy-induced atherosclerosis may be mediated through oxidative stress, decreased bioavailability of NO, increased expression of MCP-1, smooth muscle cell proliferation, increased thrombogenicity, and induction of arterial connective tissue. Hcy produces endothelial injury through the generation of oxidative stress and the generation of atherogenic biomolecules and decreased bioavailability of NO.

The question arises if there is proof that Hcy induces atherosclerosis. It has been reported that Hcy induces aortic atherosclerosis in apoE null mice. ^{124 125} There is some evidence of HHcy in human stroke. Clinically, there is elevated Hcy in plasma of human stroke ¹⁵³ and stroke recurrence and in the predicted mortality, especially in stroke patients with large vessel atherosclerosis. ¹⁵⁴ Lehotský et al ¹ in a review has reported that Hcy alone in combination with ischemic preconditioning affects ischemia-induced neurogenerative changes. In a general population of elderly men, a high Hcy level is common and is strongly associated with the prevalence of coronary heart disease and cerebrovascular disease. ⁴ Knekt et al ¹⁵⁵ have demonstrated that there is an association between elevated Hcy and both incidence and progression of coronary and extracoronary vascular calcification. Case control studies support the association between HHcy and CAD. ⁵⁵ Gregory et al have reported that HHcy is an independent risk factor for CAD. ¹⁴

Treatment modalities of HHcy-induced atherosclerosis and atherosclerosis-mediated diseases include Hcy lowering agents, such as vitamins B _6, B ₉ , and B ₁₂ and dietary sources of these vitamins. Abstention from drinking caffeinated coffee and alcohol, and smoking cigarettes would also reduce blood levels of Hcy. Lowering blood levels of Hcy could help in prevention, regression, and slowing the progression of Hcy-induced diseases.