Cystinuria

Up to one third of primary cardiovascular and cerebrovascular thrombotic events occur in people who do not have traditional risk factors. This has led the scientific community to look for other potential reasons, i.e., predictors of these events. Numerous clinical trials have demonstrated a definite link between increased levels of homocysteine and the development of cardiovascular, cerebrovascular, and peripheral vascular disease. Hyperhomocysteinemia is caused by a disruption in normal metabolism of homocysteine by lack of nutrition, pharmaceutical agents, and by inherited or acquired disorders associated with this metabolism. Elevated levels of homocysteine promote endothelial cell damage, platelet hyperactivity, and the production of abnormal clotting factors leading to the development of thromboembolic plaques in the coronary, carotid, and peripheral vascular systems. The laboratory assessment of homocysteine has been made easier and more readily available to the clinician by the development of new enzyme immunoassay technology. Preanalytical variables, however, such as collection, processing, and storage are very important considerations for the accurate detection of elevated levels of homocysteine.

ABBREVIATIONS: CVA = cerebrovascular accidents; CVD = cardiovascular disease; EIA = enzyme immunoassay (EIA); HPLC = high-performance liquid chromatography; MTHFR = methylenetetrahydrofolate reductase enzyme.

INDEX TERMS: cerebrovascular accidents; cardiovascular disease.

Clin Lab Sci 2001:14(4);272

Many factors play a role in the development of atherosclerosis and thrombosis leading to vascular and coronary artery disease. At least 30% of the world's population will die of a thrombotic condition, and up to 25% of initial thrombotic events are fatal.1 More than 500,000 people die of cardiovascular disease (CVD) each year in the United States alone. Cerebrovascular accidents (CVA) leading to stroke related deaths occur in another 100,000. The well known risk factors for developing atherosclerosis, which can also be called predictors, are heredity, age, gender, weight, blood pressure, lipids, immobility, diabetes, increased consumption of alcohol and tobacco, as well as high levels of chronic stress. As the number of these risk factors increase, so does the risk of developing atherosclerotic and thrombotic disease. Interestingly enough, up to one-third of primary cardiovascular and cerebrovascular thromboses occur in patients who do not have these predictors. This information has lead scientists to look for other predictive criteria. Identifying other markers for increased risk would result in early detection and subsequent treatment of the disease before it becomes life threatening.

CLINICAL SIGNIFICANCE OF HOMOCYSTEINE

Numerous clinical studies have demonstrated a link between increased levels of homocysteine and the development Of cardiovascular, cerebrovascular, and peripheral vascular disease. Table 1 lists reference ranges for fasting homocysteine levels based on age and gender. Even mild elevations are considered a risk factor for the development of occlusive arterial disease. Homocysteine levels above 10 (mu)mol/L to 12 (mu)mol/ L are associated with a continuous and progressive increased risk of developing atherosclerosis and thrombotic disease.2,3

In the Physicians' Health Study, men with plasma homocysteine levels 12% above the upper limits of normal had approximately a threefold increase in the risk of myocardial infarction, as compared with those with lower levels, even after correction for other risk factors.4 In the Framingham Heart Study, a strong association was found between elevated homocysteine concentrations and occlusive vascular disease that remained even after adjustment for other conventional risk factors.4 Studies have shown that even a 5 (mu)mol/L increase in the homocysteine level elevates risk equivalent to that of a 20 mg/dL rise in serum cholesterol.5 Table 2 lists relative risk ratios for development of vascular disease based on elevations in plasma homocysteine levels.

Review of homocysteine metabolism

Homocysteine is a naturally occurring, sulfur containing amino acid formed during the metabolism of methionine, an essential amino acid derived from the diet. Dietary sources of methionine include meats, fish, vegetables, fruits, nuts, and cereal grains. Animal proteins, however, contain up to three times as much methionine as do plant proteins.6 Two pathways metabolize homocysteine: remethylation and transsulfuration. Hyperhomocysteinemia is a result of congenital or acquired defects in this metabolism.

The first pathway remethylates homocysteine to methionine. This pathway requires methionine synthase, a vitamin B12, (cobalamin)-dependent process, and a methyl group donated by 5-methyltetrahydrofolate, which requires dietary folate and the methylenetetrahydrofolate reductase enzyme (MTHFR). MTHFR is a riboflavin (vitamin B2)-dependent enzyme.7

The second pathway, present only in the liver and kidney, uses betaine as the methyl donor and is catalyzed by betaine homocysteine methyl-transferase. Homocysteine is degraded into cysteine and inorganic sulfates through a process of transsulfuration, a vitamin B6 (pyridoxine)-dependent process, initially catalyzed by cystathionine beta-synthase (Figure 1).7

A variety of factors affect plasma levels of homocysteine, including genetic disorders (enzyme deficiencies), liver and renal disease, hypothyroidism, pharmacologic agents, age, and gender (Table 1).

Genetic factors affecting homocysteine levels

Inherited genetic conditions associated with increased levels of homocysteine are cystathionine beta-synthase deficiency, methionine synthase deficiency, and methylene tetrahydrofolate reductase deficiency. Genetic defects of metabolism lead to marked elevations in the concentration of homocysteine in plasma. The most common genetic cause of severe hyperhomocysteinemia is cystathionine beta-synthase deficiency. The homozygous state of this deficiency, congenital homo-cystinuria, is associated with fasting plasma homocysteine levels of up to 40 times the upper limit of normal (400 (mu)mol/L).8 This homozygous trait is rare, occurring in only 1 in 200,000 births.8 Clinical manifestations are skeletal deformities, mental retardation, venous thrombosis, and severe, premature atherosclerosis. Children with this inborn error of metabolism develop accelerated atherosclerosis and thrombotic disease and usually die before the age of 20.(7,9) The heterozygous deficiency of this enzyme is more common and occurs in 1 in 300 births.8 Plasma homocysteine levels in these individuals are generally between 20 to 40 (mu)mol/L.8

A homozygous deficiency of the N^sup 5^,N^sup 10^- methylenetetrahydrofolate reductase enzyme (MTHFR) also leads to severe hyperhomocysteinemia. These individuals may have developmental delay, motor and gait abnormalities, seizures, and psychiatric disturbances.8 A variant of this MTHFR enzyme results in reduced activity and enhanced thermolability of the enzyme. This polymorphism is caused by a point mutation, a cytosine to thymine substitution at base 677 (C677T) in the coding region for the methylene-tetrahydrofolate (MTHF) binding site. This is the most common genetic cause of hyperhomocysteinemia. Homozygosity (genotype TT) of this mutant enzyme occurs in 9% to 17% of the population, and heterozygosity (genotype CT) can be detected in another 30% to 41%.8 People who are homozygous for this mutation have elevated plasma homocysteine levels especially in the presence of low folic acid stores. Recently, a second common mutation in the MTHFR gene, an adenine-to-cytosine substitution at base 1298 (A1298C), has been described. This mutation also results in decreased MTHFR activity but has not been associated with higher plasma homocysteine levels in either mutant genotype, AC (heterozygous) or CC (homozygous) if it is the only mutation present. Compound heterozygosity (presence of both mutant C677T genotype CT and mutant A1298C genotype AC) has been shown to increase homocysteine levels more than either genotype alone.10 Other abnormalities of the metabolic pathways associated with hyperhomocysteinemia include methionine synthase deficiency and cobalamin (vitamin B12 disorders that impair methionine synthase activity.

Acquired factors affecting homocysteine levels

Dietary deficiencies of vitamin B6, B12, and folate, also result in hyperhomo-cysteinemia. Inadequate intake of these vitamins in the diets of the general population contributes to nearly two thirds of all cases. Elevated plasma homocysteine levels are found in people who have impaired renal function, end-stage renal disease, post organ transplantation, hypothyroidism, malignant diseases, and those with hypertension. Elevations are also found in individuals with chronic alcohol or substance abuse, excessive coffee consumption, and in smokers. Hyperhomocysteinemia has been found in association with several prescription drugs also, namely, carbamazepine, phenobarbital, phenytoin, primidone, valproic acid, cyclosporine, metformin, methotrexate, nitrous oxide, and theophylline.3,8

Homocysteine's role in atherosclerosis

The vascular endothelium is sensitive to many substances, i.e., thrombin, cytokines, oxidized lipids, shear stress, and possibly infectious agents including elevated levels of homocysteine.11 In response to homocysteine-induced injury, damaged endothelial cells become prothrombotic, releasing excess tissue factor. Thrombomodulin expression becomes decreased leading to inhibition of the protein C anticoagulant pathway, thereby promoting thrombosis. Lowered levels of the natural cell surface anticoagulant heparan sulfate and impaired binding of tissue plasminogen activator also occur.

Homocysteine promotes the binding of Lp(a) to fibrin, impairing plasmin-induced fibrinolysis. It increases platelet synthesis of thromboxane A2 and enhances expression of the platelet adhesion molecule, selectin, thereby increasing platelet aggregation activity. Homocysteine also promotes smooth muscle cell proliferation, increasing arterial wall thickness. Endothelial cell injury, platelet hyperactivity, and the presence of abnormal clotting factors contribute directly to the development of thromboembolic plaque formations of the coronary, carotid, and peripheral arteries. These obstructions can lead to transient ischemic attacks, coronary heart disease, stroke, and peripheral arterial disease especially in the elderly.8

Treatment of hyperhomocysteinemia

Hyperhomocysteinemia is a risk factor that can generally be controlled through dietary modification and vitamin supplementation. Under medical supervision, treatment is simple and inexpensive: dietary manipulation providing adequate amounts of folic acid, vitamin B12, and vitamin B6. Ubbink studied the effect of multiple vitamins in moderately hyperhomocysteinemic men and found that folic acid supplementation given alone or in combination with B6 and B12 decreased homocysteine levels by 41.7%.12 Landgren found that in post MI patients folic acid administration decreased the homocysteine level by 27%.12 All three vitamins should be given together, however, to achieve the desired effect and to prevent possible side effects of folic acid administration alone.12

Laboratory assessment of homocysteine

Plasma levels of homocysteine can be measured by several different techniques: acid hydrolysis followed by amino acid analysis, gas chromatography-mass spectrometry, high-performance liquid chromatography (HPLC), and by enzyme immunoassay (EIA). Until the recent development of the EIA methods, the most commonly performed testing method for homocysteine was HPLC. Most methods of HPLC require: 1) treatment of the plasma sample with a reducing agent to convert the disulfide forms, homocystine and cysteine-homocysteine to homocysteine and other thiols, and an acid treatment to remove all protein-bound homocysteine; 2) a derivatization step to form a fluorescent derivative; and 3) analysis with fluorometric detection. The technical expertise, time, and equipment costs involved with HPLC methods have made them an impractical clinical laboratory test, especially with the advent of the EIA methods. Axis Biochemical ASA (Oslo Norway) has developed both a manual method, using absorbance readings from a microtiter plate (Bio-Rad, Hercules CA) and an automated fluorescence polarization immunoassay (FPIA) methodology on the IMx system (Abbott Laboratories, Abbott Park IL). Test results from these methods correlate with those obtained with HPLC at a level of at least 0.98.(6) The first step in the EIA methods is mixing the plasma sample with buffer containing the reducing agent dithiothreitol to remove protein-bound homocysteine. The first incubation phase of both assays includes the enzyme S-adenosylhomocysteine hydrolase and excess adenosine to convert free homocysteine to S-- adenosylhomocysteine (SAH). In the fluorescence polarization EIA, the SAH that is formed is incubated with a monoclonal antibody against SAH and fluorescently labeled with S-adenosylcysteine. The degree of depolarization is inversely proportional to the amount of label-bound antibody thus quantitating the amount of homocysteine in the sample. In the microtiter plate method, the enzyme-- treated sample is transferred to immunoplates coated with anti-- SAH and incubated. After removal of unbound anti-SAH antibody, a secondary antibody labeled with horseradish peroxidase is added. After substrate addition and incubation the peroxidase activity is measured spectrophotometrically. The absorbance is inversely proportional to the concentration of total homocysteine in the sample. The development of these assays has made it possible for more laboratories to perform homocysteine analyses. It should be noted that specimens from patients who have received preparations of mouse monoclonal antibodies may contain human antimouse antibodies and should not be assayed using the EIA methods. Homocysteine levels may also be affected in patients receiving S-adenosyl-methionine, methotrexate, carbamazepine, phenytoin, nitrous oxide, and 6-azauridine triacetate due to either interference in test methodology or by disruption of the metabolic pathways of homocysteine metabolism.13

One very important issue in the laboratory analysis of homocysteine is the control of pre-analytical variables. The patient should be fasting to obtain a true baseline homocysteine value; eating will increase methionine levels thereby increasing homocysteine levels. Homocysteine in whole blood is very unstable at room temperature. Artifactual elevations occur as red blood cells metabolize S-- adenosylmethionine and release homocysteine into the plasma. Plasma homocysteine values increase by 10% within one hour and up to 75% in 24 hours if whole blood is stored at room temperature after collection.1,8 Therefore blood samples collected for homocysteine analysis should be drawn on fasting subjects, kept cold, protected from light, and the plasma should be separated from the cells immediately.

SUMMARY

It has been shown that hyperhomocysteinemia is a risk factor in the development of atherosclerosis leading to thrombosis and subsequent death. Homocysteine levels may be used to predict future thrombotic events independently or in conjunction with other risk factors. Evaluation of homocysteine should therefore, be used in vascular disease risk assessment and in its management. Elevated homocysteine levels are an easily treated risk factor through dietary manipulation and vitamin supplementation, unlike several other known risk factors. The new enzyme immunoassays have made it possible for the physician to evaluate and monitor homocysteine levels quickly and routinely.

The Focus section seeks to publish relevant and timely continuing education for clinical laboratory practitioners. Section editors, topics, and authors are selected in advance to cover current areas of interest in each discipline. Readers can obtain continuing education credit (CE) through P.A.C.E.(R) by completing the tearout form/examination questions included in each issue of CLS and mailing it with the appropriate fee to the address designated on the form. Suggestions for future Focus topics and authors, and manuscripts appropriate for CE credit are encouraged. Direct all inquiries to Carol McCoy PhD, CLS Continuing Education Editor, Department of Clinical Sciences, 343 Cowley Hall, University of Wisconsin, La Crosse WI 54601; (608) 785-6968. cmccoy@mail.uwlax.edu

REFERENCES

1. Fritsma G. Thrombosis risk testing. In Rodak B, editor. Hematology:clinical principles and applications, 2nd ed. Philadelphia PA: WB Saunders Co; (in press).

2. CAP Web Site. Sandrick K. Getting to the heart of homocysteine testing. Dec 2000. Available at http://www.cap.org/htmi/publications/feature.html. Accessed 01/05/01.

3. Malinow MR. Approaches to management of the hyperhomocysteinemic patient. Clin Insights Abbott Laboratories 1999; 1:7.

4. Welch GN, Loscalzo J. Homocysteine and atherothrombosis. N Engl J Med 1998;338:1042-50.

5. Genest J. Homocysteine as a risk factor for coronary heart disease, cerebrovascular disease and stroke. Clin Insights Abbott Laboratories 1999; 1:4.

6. Cramer DA. Homocysteine vs cholesterol: competing views, or a unifying explanation of arteriosclerotic cardiovascular disease Lab Med 1998;29(7)410-7.

7. Rozen R. Enzymology of hyperhomocysteinemia. Clin Insights Abbott Laboratories. 1999;1:5.

8. Williams RH. Hyperhomocysteinemia: pathogenesis, clinical significance, laboratory assessment, and treatment. Lab Med 1999; 30(7):468-74.

9. Abbott Diagnostics Web Site. Medical conditions: heart disease: homocysteine: eat your heart out. Available at http://www.abbottdiagnostics.com/. Accessed February 2001.

10. Lachmeijer AMA, and others. Mutations in the gene for methylenetetrahydrofolate reductase, homocysteine levels, and vitamin status in women with a history of preeclampsia. Am J Obstet Gynecol. 2001;184:394-402.

11. Jacobsen DW, and others. Potential mechanisms of disease. Clin Insights Abbott Laboratories 1999; 1:6.

12. Boers GH. Trials and treatment of hyperhomocysteinemia. Clin Insights Abbott Laboratories 1999;1:8.

13. Package Insert. Homocysteine:IMx system. Axis Biochemicals ASA/Abbott Laboratories, Oslo, Norway. September 1998.

14. Anderson JL, and others. Plasma homocysteine predicts mortality independently of traditional risk factors and C-reactive protein in patients with angiographically defined coronary artery disease. Circulation. 2000;102:1227-32.

Paige A Macy is the Diagnostic Support Specialist, Esoterix Coagulation, Aurora CO.

Address for correspondence: Paige A Macy, Esoterix Coagulation, 3177 South Parker Road, Aurora CO 80014. (800) 288-6222, (303) 399-3338 (fax). paige.macy@esoterix.com

George Fritsma MS is the Focus: Cardiovascular Disease Risk Testing guest editor.

Focus Continuing Education Credit: seepages 279 to 281 for learning objectives, test questions, and application form.

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