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The laboratory's role in identifying lipid and lipoprotein risk factors for CHD.

Periodic physical exams are part of a program of good health habits, and clinical laboratory tests are usually part of any periodic physical exam. Among the laboratory tests under these circumstances are those that screen for the long-term risk of coronary heart disease (CHD), which can often be prevented if physicians and patients work together when necessary in response to the laboratory results. CHD is a complex phenomenon with multiple risk factors that are either modifiable or nonmodifiable. Age, gender, and genetic predisposition are nonmodifiable risk factors. Other established risk factors, such as smoking, hypertension, obesity, and dyslipoproteinemias are generally considered to be within one's control to normalize. Abnormalities in hemostatic, oxidative, or inflammatory factors might also increase the long-term risk for CHD but are not discussed here.

CHD develops slowly and asymptomatically for many years, and the first stage of the disease is often evident at a surprisingly early age. Coronary fatty streaks can present even in the late teens and twenties, as observed in a high proportion of postmortem exams of individuals who suffered traumatic, non-CHD fatalities.[1-3] At the present time, approximately 30% of CHD-related deaths occur in individuals with no prior symptomatic manifestations of the disease, partially because more than 65% of myocardial infarctions occur in vessels with less than 50% stenosis.[4]

Despite significant declines over the past 20 years, CHD is the number one cause of death in the United States, outranking all cancers combined by approximately 2:1. Declines in CHD-related mortality can be credited to a combination of factors, including (1) increased public awareness through campaigns that focus on smoking prevention and cessation; (2) increased understanding of the contribution of hypertension, diet, exercise, and cholesterol levels to disease; and (3) increased effectiveness of various therapies. However, substantial progress is still required, and new risk factors continue to be proposed and evaluated. If shown to add significantly to the risk profile, assays for these risks may become part of future laboratory test menus, in addition to the established risk factors that already involve clinical laboratory testing.

Traditional lipoprotein profile

Approximately 30 years ago, a cholesterol level was considered high risk when it exceeded 7.8 mmol/L (300 mg/dL). Low density lipoprotein (LDL) and high density lipoprotein (HDL) cholesterol were not determined clinically, and triglycerides were rarely measured. Since the late 1970s, however, the standard lipoprotein profile has included measurements for total cholesterol, triglycerides, HDL cholesterol, and a calculated estimate of LDL cholesterol based on the equation by Friedewald et al.[5] This profile is still an important diagnostic tool for lipoprotein disorders.

It became increasingly clear over time that an elevated cholesterol level was associated with increased risk of CHD.[6-8] In 1988, the Adult Treatment Panel of the National Cholesterol Education Program (NCEP) set the cutoff for high-risk total serum cholesterol at [greater than or equal to] 6.2 mmol/L (240 mg/dL) by using data from Multiple Risk Factor Intervention Trial (MRFIT).[8] A value of [greater than or equal to]4.1 mmol/L (160 mg/dL) was selected for LDL cholesterol because it corresponds to this total cholesterol level, and both values approximate the 75th percentile for adults. In addition, patients with LDL cholesterol levels of [greater than or equal to] 4.1 mmol/L (160 mg/dL) in the Lipid Research Center Coronary Primary Prevention Trial (LRC/CPPT) benefited by lowering their LDL cholesterol values.[7] Total cholesterol of 5.2-6.2 mmol/L (200-239 mg/dL) was in the borderline category. For HDL cholesterol, a value of [less than] 0.9 mmol/L (35 mg/dL) is the NCEP low cutoff for high risk based on Framingham data, and approximates the 20th percentile in men. NCEP considered triglyceride levels of [less than] 2.25 mmol/L (200 mg/dL) to be optimal, 2.25-4.50 mmol/L (200-359 mg/dL) to be borderline, and [greater than or equal to] 4.50 mmol/L (400 mg/dL) to be a high value. However, there was a lack of consensus in these studies on the value of using triglyceride as a CHD risk factor.

Total and HDL cholesterol results are used to screen and monitor patients; triglyceride results are often used to calculate an estimate of LDL cholesterol concentration, using the formula developed by Friedewald et al: LDL cholesterol = total cholesterol HDL cholesterol - (triglycerides/5).[5]

Triglyceride measurements are also important for diagnosing and monitoring individuals with severe hypertriglyceridemia. Borderline hypertriglyceridemia (2.25-4.50 mmol/L; 200-400 mg/dL) is associated with decreased HDL cholesterol levels and is commonly found in diabetics and CHD patients. Although not associated with CHD, severe hypertriglyceridemia ([greater than] 11 mmol/L; [greater than] 1000 mg/dL) is important to diagnose and treat because it can produce pancreatitis, which is a life-threatening condition.

In the late 1980s, the NCEP Laboratory Standardization Panel first established precision (coefficient of variation) and accuracy (bias) limits at [less than or equal to] 5% for total cholesterol and later tightened them to the current limits of [less than or equal to] 3 % for each.9 Precision and accuracy limits were later set at [less than or equal to] 5% for triglycerides.[10]

Preanalytical considerations. There are additional factors separate from the actual analyses that can affect the reliability of individual lipid results. Sources of biological variation and other preanalytical variables are often difficult to control but must be minimized to whatever extent possible.[11] Although capillary blood is often used for screening purposes, results are potentially more variable than those from venous blood. However, fingerstick specimens collected from a puncture that allows an adequate blood flow without excessively squeezing the finger can provide results that are as reliable as those from venous blood.[12]

In addition to proper technique for specimen collection, the position of an individual during collection is also important because lipid values vary depending on whether the person is standing, sitting, or prone.[13,14] When standing and prone positions are compared, cholesterol concentrations are approximately 10% higher when the patient stands versus when the patient is prone; triglycerides are approximately 12% higher; and HDL cholesterol is approximately 7% higher.[14] Although complete equilibration takes about 30 minutes when changing position, sitting quietly for 5 minutes before specimen collection accounts for most of the hemodynamic changes.[14] Consequently, this time for sitting has been incorporated into "standard procedure" for specimen collection. These changes are considered in the general population but will necessarily affect results obtained from hospitalized patients who cannot sit up for blood collection. Therefore, it is generally advisable to defer lipid testing until the patient is ambulatory and preferably free living.

Patient stress during hospitalization also affects lipid concentrations. Types of stress include both physiological (e.g., during hospitalization) and psychological. Total and HDL cholesterol concentrations can be markedly reduced under these conditions.[15,16] Viral infections for both inpatients and outpatients can also substantially reduce total and HDL cholesterol concentrations. If possible, specimen collection should be postponed until after a vital infection has resolved. It is rarely necessary to determine lipid/lipoprotein concentrations for inpatients; these analytes are better determined on an outpatient basis whenever possible.

Patient fasting is essential for accurate results from lipoprotein profiles; however, fasting is a relative term and can affect the accuracy of results and increase the variability among serial results. The time required to return to a true fasting state after eating depends on the amount and the type of food ingested and each individual's metabolic efficiency, which varies with body type, age, level of exercise, and lipid status. The triglyceride levels for young, fit individuals with low fasting values will often return to baseline within 8 hours, even after a high-fat, high-calorie meal. However, triglyceride levels for individuals with high fasting values and less efficient metabolism may require as many as 14 hours to return to baseline.[17,18] NCEP recommends a 9-hour fast. Based on our studies,[17,18] we recommend a 12- to 14-hour fast after a low-fat, low-calorie meal, with no intake of alcohol for 24 hours. Our recommendations differ from those of the NCEP for several reasons, one of which is that our data are based on a high-fat meal (worst case scenario), whereas NCEP recommends a low-fat meal. Most lipid labs use 12 hours.

LDL cholesterol

Listed behind only smoking and hypertension, LDL and HDL cholesterol are currently considered to be the most important modifiable risk factors indicative of CHD. Therefore, laboratory assessment of these analytes must be precise and accurate. The NCEP Adult Treatment Panel uses LDL and HDL cholesterol concentrations as the basis for national guidelines to diagnose and treat CHD.[19] Consequently, the NCEP Laboratory Standardization panels have set national guidelines for laboratory precision and accuracy when measuring lipid analytes.[9,10,20,21]

Indirect measurement. Historically, LDL cholesterol has been measured after separation of lipoprotein classes by ultracentrifugation[22,23] or estimated using a formula.[5,24] As performed at the Centers for Disease Control and Prevention, the ultracentrifugation method, also known as beta quantification, has become the reference method, even though it does not directly measure LDL cholesterol. Rather, this method determines LDL cholesterol by subtracting HDL cholesterol from the cholesterol in the 1.006 kg/L infranate. In addition to LDL and HDL, the 1.006 kg/L infranate also includes intermediate density lipoproteins (IDL) and lipoprotein(a) [Lp(a)]. After subtracting the HDL cholesterol concentration, LDL cholesterol equals the sum of the cholesterol in LDL plus IDL plus Lp(a).

For most individuals, the impact on LDL cholesterol from IDL and Lp(a) is negligible. However, for the few patients with increased levels of either IDL or Lp(a), the impact on the total LDL cholesterol concentration can be significant. Generally, IDL are considered to represent a subspecies of triglyceride-rich lipoprotein (TRL) remnants. Compared with very low-density lipoproteins (VLDL), IDL are relatively cholesterol-enriched and their accumulation is associated with type III hyperlipoproteinemia. In some studies, increased Lp(a) has been associated with increased CHD risk, and this is covered in more detail in a later section.

While measurement of LDL cholesterol based on beta quantification is indirect and influenced by "contamination" with other lipoproteins [IDL and Lp(a)], beta quantification is considered the reference procedure because it has been used to determine LDL cholesterol concentrations in many of the large studies,[23] whose results were used by the NCEP Adult Treatment Panel to determine the national guidelines. Also, beta quantification results are not subject to interference from high triglycerides or from nonfasting status. However, beta quantification is unsuitable for use in most clinical laboratories because of its many disadvantages, including expensive equipment, slow throughput, high labor intensity, and large sample volume requirements.

Most clinical laboratories have relied instead on a mathematical formula developed from beta quantification data derived from studies performed at the National Institutes of Health by Friedewald et al.[5] For accurate estimation of LDL cholesterol concentration, the Friedewald formula requires accurate measurement of total cholesterol, HDL cholesterol, and triglycerides from fasting samples. This formula is useful when (1) samples are truly fasting, (2) the 3 measured analytes are determined with very little bias, and (3) triglyceride concentrations are [less than] 2.8 mmol/L (250 mg/dL). However, reliability of the formula decreases markedly when any one of these criteria is not met, including a fasting triglyceride in the range of 2.8-4.5 mmol/L (250-400 mg/dL).[25]

Use of triglyceride/5 as an estimate of VLDL in the Friedewald equation involves 2 assumptions: (1) all serum triglyceride resides in VLDL, and (2) all VLDL contain 20% of their mass as cholesterol. While not totally correct, the first assumption is reasonable because most serum triglyceride is found in VLDL when samples have relatively normal triglyceride values and are obtained in the truly fasting state (ideally, 12-14 hours after a low-fat meal with no alcoholic intake for 24 hours). Only small amounts of triglyceride are carried in LDL and HDL. However, substantial quantities of triglyceride are carried in chylomicrons in postprandial and severely hypertriglyceridemic samples and in IDL in samples from patients with type III hyperhpoproteinemia.

The second assumption is also reliable when triglycerides are fasting and relatively low ([less than] 2.8 mmol/L or 250 mg/dL). However, when triglycerides are elevated, the presence of particles with abnormal composition is more likely. These particles have either (1) a much higher proportion of cholesterol as found in individuals with type III hyperlipoproteinemia (more than 30% of mass as cholesterol), or (2) a much lower proportion of cholesterol, as in the case of nonfasting samples where chylomicrons are present (less than 10% of mass as cholesterol). Consequently, when the composition of particles is abnormal, the Friedewald formula doesn't provide accurate results. In our opinion, a reasonable solution is to use the Friedewald equation when samples are from fasting subjects and triglycerides are below 2.8 mmol/L (250 mg/dL) but to reflexively use one of the direct LDL cholesterol assays when either of those 2 criteria are not met.

Direct measurement. Even within the confines of precision and accuracy limits set by the NCEP Laboratory Standardization Panel[9] and the Working Group on Lipoprotein Measurement[10,20,21] for total, LDL, and HDL cholesterol and triglyceride, it is possible for LDL cholesterol concentrations to be outside recommended limits when estimated by the Friedewald formula. Consequently, the NCEP Working Group on Lipoprotein Measurement recommended the development of methods for direct measurement of LDL cholesterol that would be suitable for use in clinical laboratories and would not require ultracentrifugation.[20] Subsequent methods have been developed and are commercially available.[26-29]

Manufacturers have retained Lp(a) in LDL supernates to mimic the beta quantification LDL. However, depending on their formulation, manufacturers differ in inclusion of IDL among the different direct LDL cholesterol methods. For example, immunoseparation methods that employ anti-apo E necessarily remove IDL along with VLDL because both species contain apo E. Other methods that use detergents to isolate specific lipoprotein classes have slightly different separation characteristics. Indeed, whenever methodological or environmental differences (such as density, charge, immunology, size, carbohydrate content, pH, and temperature) are the basis for isolation, the specific particles isolated will differ slightly. For certain individuals whose particles have unique properties, separation will be quite different according to the method used.

Manufacturers of total and LDL cholesterol reagents and analytical systems have access to the Cholesterol Reference Method Laboratory Network (CRMLN) certification program established by the CDC to evaluate assays and calibration, using fresh samples compared with the reference method. However, incorporating these LDL methods into clinical laboratory test menus has been slow, partly due to their limited use in large studies to date, but primarily due to reagent costs, which are not associated with a mathematical formula. Consequently, the perceived cost associated with the direct LDL cholesterol reagents is relatively high.

The NCEP Adult Treatment Panel has made LDL cholesterol the primary determinant of therapy in relation to lipoprotein risk factors. Despite these high costs, therefore, laboratories must provide physicians with the most accurate LDL cholesterol levels possible so that physicians can then provide the patient with the most accurate estimates of risk. Neither over- nor undertreatment is in the best interest of the individual patient or the healthcare industry as a whole. Laboratories must have the appropriate tests in place that can help diagnose patients and monitor therapy.

One major advantage of both reference and direct assays that measure LDL cholesterol is the ability to assay nonfasting samples[26,28] because LDL cholesterol testing can then be included in random screening panels. However, regardless of the method used, LDL and HDL cholesterol concentrations measured in the postprandial state will, on average, be slightly lower than their fasting counterparts because of actual physiological changes rather than a miscalculation.[17] The decreased concentration for both LDL cholesterol and HDL cholesterol content is due to transfer of cholesterol out of these particles and into newly synthesized chylomicrons and VLDL. The decreases in LDL cholesterol are moderate, generally [less than] 3-5% but as high as approximately 8%, when postprandial triglyceridemia peaks after a very high-fat meal. The level of decrease varies with the meal's size and fat content and with the length of time between the meal and specimen collection. While the concentration decreases are relatively small, they should be considered when interpreting borderline results.

HDL cholesterol

HDL cholesterol is another well established CHD risk factor,[30-32] and the NCEP Adult Treatment Panel recommends that HDL cholesterol be assayed along with total cholesterol as part of a screening program to assess a patient's risk for CHD.[19] Concentrations [less than] 0.90 mmol/L (3 5 mg/dL) are considered to confer increased risk, and concentrations [greater than] 1.55 mmol/L (60 mg/dL) are generally considered protective.[19] These tests can be performed in the nonfasting state, although HDL cholesterol levels will be slightly lower postprandially, as discussed earlier.[17] Individuals whose HDL cholesterol concentrations are [less than] 0.90 mmol/L (35 mg/dL) at screening should then have the test repeated in the fasting state for a complete lipoprotein profile.

While high levels of HDL cholesterol are known to be protective against CHD, until recently it was not known if patients with low HDL cholesterol levels could decrease their risk for CHD by raising their HDL cholesterol levels. The 7-year Veterans Administration HDL Intervention Trial (VA-HIT), a secondary prevention study of 2,500 men,[33] found that when compared to men taking a placebo, men who raised their HDL cholesterol concentrations while taking gem-fibrozil experienced a significant reduction in primary (combined incidence of and nonfatal MI and CHD death) and secondary (stroke, transient ischemia, death by any cause, revascularization, carotid endoarterectomy, and hospitalization for unstable angina or congestive heart failure) endpoints.[34] This study enrolled men with previously diagnosed coronary disease who had relatively normal LDL cholesterol and triglyceride levels, i.e., LDL cholesterol [less than] 3.6 mmol/L (140 mg/dL) and triglycerides [less than] 3.4 mmol/L (300 mg/dL) but who had low HDL cholesterol levels ([less than] 40 mg/dL). It is difficult to raise HDL cholesterol levels, and in the HIT study, the magnitude of change associated with benefit was small but significant; a 22% decrease in coronary events was associated with a 7.5% increase in HDL cholesterol and a 24.5% decrease in triglycerides. These changes were not related to changes in LDL cholesterol, which were not different between placebo and gemfibrozil groups.

The precision and accuracy of HDL cholesterol assays are very important because both the range of HDL cholesterol concentration in the population and the therapeutic changes associated with therapy are small and individuals at greatest CHD risk have the lowest HDL cholesterol concentrations. Manufacturers can evaluate the calibration of their HDL cholesterol assay systems through the CRMLN HDL cholesterol certification program. Laboratories should monitor precision closely and check accuracy through enrollment in an approved proficiency testing program. In 1998, NCEP recommended that acceptable limits for bias and precision be tightened.[21] The bias limit was changed to [+ or -] 5 % or less and precision limits were [less than or equal to] 4% coefficient of variation (CV) for concentrations [greater than or equal to] 1.09 mmol/L (42 mg/dL) and [greater than or equal to] 0.04 mmol/L (1.7 mg/dL) standard deviation for concentrations [less than] 1.09 mmol/L (42 mg/dL).

For clinical analysis of HDL cholesterol, interference from high levels of triglyceride in the sample is a problem, particularly in nonfasting and previously frozen samples, where the interference is generally more evident at lower triglyceride levels than in fasting samples. The CDC developed and uses the reference method for HDL cholesterol, which consists of a heparin-manganese precipitation from a 1.006 kg/L infranate. By using the 1.006 kg/L infranate, any interference from triglyceride is eliminated, but all the other disadvantages of ultracentrifugation are still present. Unfortunately, triglyceride interference has not been totally eliminated by the newer direct nonprecipitation-based assays for HDL cholesterol.[35] Dilution, filtration, and (for frozen samples) a modification of the dextran sulfate-magnesium method[36] can each be used to eliminate triglyceride-rich particles from supernates when using precipitation methods. For the direct nonprecipitation-based assays, dilution is probably the option. However, precision data reported for the assays have been tighter than those typically obtained using precipitation methods.[35,37]

Research assays for possible future use in the clinical laboratory

Lipoprotein (a). Based primarily on case-control studies, elevated serum levels of Lp(a) are thought to confer increased risk of CHD,[38-43] but prospective studies have been inconsistent in confirming the association.[41,44,45] Lp(a) particles are synthesized in the liver and consist of LDL particles with an extra apolipoprotein [apo(a)] linked to apo B-100 by 2 disulfide bonds.[46] The length of apo(a) is heterogeneous because of varying numbers of repeating peptide sequences called kringles. Molecular weights range from approximately 185 kda to 650 kda, and protein length is genetically determined.

Lp(a) by electrophoresis. The plasma concentration of Lp(a) is inversely related to the apo(a) isoform length, that is, the longer the peptide chain, the lower the plasma concentration. Lp(a) has a density range of approximately 1.05-1.15 kg/L, which overlaps the LDL and HDL density ranges. On agarose gel electrophoresis, Lp(a) migrate in the pre-beta range and were once called sinking pre-beta lipoprotein.[47,48] If whole plasma is applied to the gel, visualization of Lp(a) is obscured by the presence of VLDL, which also has pre-beta mobility. Consequently, to observe Lp(a) by agarose electrophoresis, ultracentrifugation must first be performed to isolate the 1.006 kg/L infranate. Apo(a) also has more than 75% homology with plasminogen,[49] and is thought to compete with plasminogen for fibrin binding sites, thereby interfering with fibrinolysis.[50-52]

Quantitative assays. Lp(a) can be measured either (1) by determining apo(a) protein concentration, which is then converted to an estimate of total Lp(a) particle mass (sum of the molar concentrations of the protein, cholesterol, triglyceride, and phospholipid that comprise the particles)[53] or (2) by measuring Lp(a) cholesterol concentration,[43,54] as is done to assay LDL and HDL concentration. Protein and cholesterol each account for approximately 30% of Lp(a) mass. Mass assays involving immunologic separation of apo(a) must use specific epitopes to avoid regions homologous to plasminogen and regions subject to kringle IV, type 2 sequences, which are the peptides that account for size heterogeneity. The protein is then quantitated by ELISA, EIA, or nephelometric or turbidometric assays. For the Lp(a) cholesterol assay, Lp(a) are bound to lectin, taking advantage of the high degree of glycosylation found in Lp(a). After eluting Lp(a) off the column, the cholesterol is measured. Standardization procedures and materials are currently being evaluated through the International Federation of Clinical Chemistry and the National Institutes of Health. Reference materials selected by the IFCC committee will be used by manufacturers to set calibrator concentrations in the same way that reference materials developed for apos A-I and B are used. Evaluation of assays with respect to isoform size, currently being conducted under an NIH contract, will help assure that the antibodies employed identify all peptides equally.

Genetic distribution. Lp(a) concentrations are unevenly distributed in Caucasian populations, with most individuals having very low concentrations. For example, in the third offspring exam of the Framingham Heart Study, the mean total mass concentration was approximately 15 mg/dL, whereas the median value was only 8 mg/dL.[55] Concentrations in some individuals can exceed 100 mg/dL and can occasionally be as high as 200-300 mg/dL. African-Americans demonstrate a more Gaussian distribution of Lp(a) values than Caucasians but with a more elevated median level. In the CARDIA study, the median value for African-Americans was 3 times higher than that for Caucasians.[56] It is not yet clear whether the more elevated levels observed in the African-American population confer greater CHD risk.[57-59] However, regardless of race, an Lp(a) total mass concentration [greater than] 30 mg/dL is considered to be elevated; the corresponding Lp(a) cholesterol cutpoint is 10 mg/dL.[40]

Despite the similarity between Lp(a) and LDL, most LDL-lowering drugs have little or no effect on Lp(a) concentrations. Only niacin and the estrogen replacement preparations for postmenopausal women have substantially decreased Lp(a) levels. Prospective studies generally confirm Lp(a)'s atherogenicity.[41,44,45] Treatment with niacin in such patients with CHD can be recommended because niacin has been shown to reduce secondary CHD events in unselected CHD patients.[61] An elevated Lp(a) level can also indicate the need to be assertive in normalizing other modifiable CHD risk factors.

TRL remnants

Triglyceride-rich lipoproteins (TRLs) include chylomicrons, chylomicron remnants, VLDL, and VLDL remnants. Both chylomicron and VLDL remnants may be atherogenic.[62-66] Chylomicrons synthesized in the intestine after a meal rapidly undergo lipolysis via the action of the enzyme, lipoprotein lipase (LPL), causing a loss of triglyceride from the core, which is partially replaced by a transfer of cholesteryl esters from LDL and HDL. Importantly, these remnant particles are now relatively enriched in apo E. Chylomicron remnants[62] are eventually removed by the liver through a receptor-mediated process.

VLDL constitute another species of TRL but are synthesized in the liver rather than the intestine. As with chylomicrons, VLDLs have a hydrated density of [less than] 1.006 kg/L and are heterogeneous in size. The main compositional difference between VLDL remnants and chylomicron remnants is the presence of apo B 100 as the major protein of VLDL remnants, rather than apo B48, and the lower relative triglyceride concentration. VLDL remnants can be removed from circulation directly by the liver or can undergo further degradation to become IDL (density 1.006-1.019 kg/L) and then LDL (density 1.019-1.063 kg/L). IDL are another species of TRL remnants that are even more cholesterol- and apo E-enriched and triglyceride-poor.

While many patients with CHD present with moderately elevated triglycerides, independent associations between triglycerides and CHD have been inconsistent.[67-70] Lack of association in many studies is probably related to biologic variability and to the compositional heterogeneity within TRL that also causes difficulty with the Friedewald calculation. Proposed mechanisms of action of TRL remnants include interference with vasorelaxation[63,71] and subendothelial accumulation of chylomicron remnants.[64]

Separation of remnant lipoproteins has always been difficult because of overlapping size, density, and composition among the native and remnant forms of TRL. Traditionally, remnants have been separated using ultracentrifugation and column separation techniques, but these are not suitable for large-scale studies. Nonetheless, the ability to separate remnants could possibly allow direct quantitation of the specific atherogenic subspecies within TRL without analyzing newly formed native TRL, which may not be atherogenic. Studies have shown that remnants can circulate for extended periods of time postprandially, even in individuals with normal fasting triglyceride concentrations.[17,18,64,72-74]

A recently developed immunologic assay isolates a subset of TRL that are enriched in apo E and display classic remnant-like characteristics.[75-77] These unbound remnants remain in the supernate and have been identified as remnant-like particles (RLP). Reference ranges have been generated in 2 populations,[78,79] and initial case control and prevalence studies examining the relationship between RLP and CHD indicate an association,[80-82] and the first prospective study has yet to be completed.

The RLP assay may also eventually be used in clinical laboratories to assess risk in diabetes[84] and to diagnose individuals with type III hyperlipoproteinemia or dysbetalipoproteinemia[85,86] who are also at high risk for developing CHD. Diagnosis of type III hyperlipoproteinemia currently requires (1) ultracentifugation to calculate the ratio of VLDL cholesterol to total triglyceride ([greater than] 0.3 in type III patients) and to document the presence of beta VLDLs on agarose electrophoresis and (2) apo E pheno-typing or genotyping to document an apo E 2/2 phenotype. Sophisticated techniques are used to fulfill these requirements, and consequently, many type III patients are not identified and are categorized as having combined hypeflipoproteinemia. Use of the ratio for RLP cholesterol to total triglyceride could replace the ratio to VLDL cholesterol to triglyceride, thereby eliminating the need for ultracentrifugation. Type III patients generally have elevated RLP cholesterol concentrations (at or above the 75th percentile of the population) and RLP cholesterol to triglyceride ratios of more than 0.10.[85,87] In our view, elevated RLP cholesterol concentrations can be effectively lowered with some inhibitors of 3-hydroxy 3-methylglutaryl coenzyme A (HMG CoA) reductase such as atorvastatin.[87,88]

Homocysteine

Homocysteinurias are a class of inborn errors of metabolism that affect metabolism of sulfur-containing amino acids and result in extremely elevated plasma and urinary levels of homocysteine. When untreated, individuals with this defect frequently develop CHD by their teens and as early as infancy. It was thus hypothesized that individuals with more moderate increases in plasma homocysteine levels might also be at some increased risk for CHD, and most studies have confirmed this hypothesis.[89-96] The metabolic pathway involved in the synthesis and catabolism of homocysteine contains several enzymes that, if defective or inactive, can interfere with homocysteine metabolism and result in its elevation. These enzymes include cystathionine beta-synthase, methylenetetrahydrofolate reductase, and N-5-methylenetetrahydrofolate:homocysteine methyltransferase.[97] Elevated plasma homocysteine levels have been associated with oxidative cell damage, vasoconstriction, vascular smooth muscle proliferation, and hypercoagulability,[98-100] all of which are potential mechanisms for homocysteine's positive association with CHD risk.

Distribution of homocysteine levels in the population varies by age, gender, and ethnicity.[101,102] The elderly tend to have a lower dietary intake of B vitamins and thus have higher homocysteine concentrations.[103-105] Other populations at increased risk of having hyperhomocysteinemia are diabetics[93,106] and patients with renal disease because the kidney is the site of transsulfuration enzymes.[107,108]

Many individuals with increased concentrations of homocysteine are easily treated with supplementary intake of folate and vitamins [B.sub.6] and [B.sub.12].[106,109] Recently, the U.S. grain supply has been fortified with folate to reduce the incidence of neural tube defect in fetuses. This fortification will secondarily reduce homocysteine levels in the general population.[106] Whether this reduction will translate into a reduction in cardiac and vascular events awaits future evaluation. Existing evidence gained from studies where homocysteine levels have been reduced would lead to the prediction that there will be a reduction in CHD events.[94]

Generally, laboratory analysis of homocysteine has been conducted using HPLC methodology, which is not performed in most clinical laboratories. However, with the positive association found in studies evaluating homocysteine as a CHD risk factor, new methods such as immunoassays have been developed that are more useful in a routine clinical chemistry laboratory,[110,111] Regardless of the method, a fasting specimen should be collected in EDTA or heparin, and the plasma should be removed and analyzed immediately or stored at -70 [degrees] C, where it is stable for extended periods of time.

However, issues important to clinical laboratory measurement of homocysteine involve more than just assay technology. For example, assays aren't standardized, making it potentially difficult to compare results among methods,[112] While no standardization currently exists, a homocysteine proficiency testing program has recently been initiated through the Wadsworth Center of the New York State Department of Health to assess the variability among methods and laboratories. Initial comparisons showed substantial variability, indicating the need for such a program. Regular proficiency testing for homocysteine is now being offered by the New York program.

Laboratorians also question whether homocysteine levels will be requested frequently enough to add a homocysteine assay to the test menu. Because vitamin supplementation easily decreases homocysteine levels in most individuals, many physicians may elect to begin supplementation without assaying homocysteine. However, 2 problems with that approach include (1) that supplementation with folate alone will mask, but not resolve, symptoms of pernicious anemia[105] and (2) that defects in the thermolabile methylenetetrahydrofolate reductase enzyme will require greater levels of folate supplementation than would be required to normalize most increases in homocysteine.[113] This defect is a common mutation found in individuals with elevated homocysteine concentrations, indicating that both initial and follow-up homocysteine concentrations should be measured.

Early detection of risk factors for CHD is essential to reduce the incidence of this number-one killer, and detection of many of these risk factors involves laboratory determinations. Although specific assays and assay parameters have changed and improved in the last 30 years, the lipoprotein profile is still essential to help determine a patient's risk level for CHD. Prevention is essential because only 50% of myocardial infarctions occur in people who have already been diagnosed with CHD. As the prevalence of CHD remains high, identification of new risk factors will also be important. Current data indicate that CHD events can be reduced by approximately 30% when LDL cholesterol levels are reduced with diet and medication. Identification and treatment of individuals with decreased levels of HDL cholesterol levels and increased levels of Lp(a) cholesterol or particle mass, RLP cholesterol, and homocysteine should further reduce CHD risk. In addition, identification, validation, and incorporation of other new risk factors into new clinical laboratory assays should eventually reduce the risk even further.

Table

Preanalytical variables that can affect results for lipoprotein profiles

Proper specimen collection, especially for fingerstick specimens

Patient position during specimen collection

* standing

* sitting

* recumbent

Patient stress during hospitalization

* physiological (from interventional procedures and/or illness)

* psychological (from worries about health status, impending procedures, and/or pain)

Viral infections

Compliance with a true fasting status

* duration of the fast

* limitation of alcohol

* amount and the type of food ingested

* metabolic efficiency of each individual

References

1. Stary HC. Evolution and progression of atherosclerotic lesions in coronary arteries of children and young adults. Arteriosclerosis. 1989;9(Suppl I):

1-19-32.

2. Pathobiological Determinants of Atherosclerosis in Youth (P-DAY) Research Group. Natural history of aortic and coronary atherosclerotic lesions in youth. Findings of the P-DAY Study. Arterioscler Thromb. 1993; 13: 1291-1298.

3. Strong JP, Malcolm GT, McMahan CA, et al. Prevalence and extent of atherosclerosis in adolescents and young adults: implications for prevention from the Pathobiological Determinants of Atherosclerosis in Youth Study. JAMA. 1999;281:727-735.

4. Falk E, Fuster V. Angina pectoris and disease progression. Circulation. 1995;92:2033-2035.

5. Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem. 1972;18:499-502.

6. Kannel WB, Castelli WP, Gordon T. Cholesterol in the prediction of atherosclerotic disease. New perspectives based on the Framingham Study. Ann Intern Med. 1979;90:85-91.

7. Lipid Research Clinics Program. The Lipid Research Clinics Coronary Program Prevention Trial Results I and II. JAMA. 1984;251:351-374.

8. Stamler J, Wentworth D, Neaton JD. Is the relationship between serum cholesterol and risk of premature death from coronary heart disease continuous and graded? Findings in 356,222 primary screenees of the Multiple Risk Factor Intervention Trial (MRFIT). JAMA. 1986;256:2823-2828.

9. Laboratory Standardization Panel. National Cholesterol Education Program. Current status of blood cholesterol measurement in clinical laboratories in the United States: A report from the laboratory standardization panel of the National Cholesterol Education Program. Clin Chem. 1988;34:193-201.

10. Stein EA, Myers GL, for the National Cholesterol Education Program Working Group on Lipoprotein Measurement. National Cholesterol Education Program recommendations for triglyceride measurement: Executive summary. Clin Chem. 1995;41:1421-1426.

11. Cooper GR, Myers GL, Smith SJ, Schlant RC. Blood lipid measurements. JAMA. 1992;267:1652-1660.

12. Warnick GR, Leary ET, Ammirati EB, Allen MP. Cholesterol in fingerstick capillary specimens can be equivalent to conventional venous measurements. Arch Pathol Lab Med. 1994;118:1110-1114.

13. RD, Upton SJV, Avakian EV, Grundy S. Increases in serum lipid and lipoprotein levels with movement from the supine to standing position in adult men and women. Prey Med. 1986;15:18-27.

14. Tan MH, Wilmshurst EG, Gleason RE, Soldner JS. Effect of posture on serum lipids. N Engl J Med. 1973;289:416-419.

15. Genest JJ, Corbett H, McNamara JR, et al. Effect of hospitalization on high-density lipoprotein cholesterol in patients undergoing elective coronary angiography. Amer J Cardiol. 1988;61:998-1000.

16. Genest JJ, McNamara JR, Ordovas JM, et al. Effect of elective hospitalization on plasma lipoprotein cholesterol and apolipoproteins A-I, B and Lp(a). Am J Cardiol. 1990;65:677-679.

17. Cohn JS, McNamara JR, Cohn SD, et al. Postprandial plasma lipoprotein changes in human subjects of different ages. J Lipid Res. 1988; 29:469-479.

18. Cohn JS, McNamara JR, Krasinski SD, et al. Role of triglyceride-rich lipoproteins from the liver and intestine in the etiology of postprandial peaks in plasma triglyceride concentration. Metabolism. 1989;38:484-490.

19. Expert Panel. National Cholesterol Education Program. Second report of the expert panel on detection, evaluation, and treatment of high blood cholesterol in adults. (Adult Treatment Panel II). Circulation. 1994;89:1333-1445.

20. Bachorik PS, Ross JW, for the National Cholesterol Education Program Working Group on Lipoprotein Measurement. National Cholesterol Education Program recommendations for measurement of low-density lipoprotein cholesterol: Executive summary. Clin Chem. 1995; 41:1414-1420.

21. Warnick GR, Wood PD, for the National Cholesterol Education Program Working Group on Lipoprotein Measurement. National Cholesterol Education Program recommendations for measurement of high-density lipoprotein cholesterol: Executive summary. Clin Chem. 1995; 41:1427-1433.

22. Havel RJ, Eder HA, Bragdon JH. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J Clin Invest. 1955;34:1345-1353.

23. Manual of laboratory operations, Lipid Research Clinics program, lipid and lipoprotein analysis (revised 1982). Washington, DC: NIH, US Dept of Health and Human Services.

24. DeLong DM, DeLong ER, Wood PD, et al. A comparison of methods for the estimation of plasma low- and very low-density lipoprotein cholesterol: The Lipid Research Clinics Prevalence Study. JAMA. 1986;256:2372-2377.

25. McNamara JR, Cohn JS, Wilson PWF, Schaefer EJ. Calculated values for low-density lipoprotein cholesterol in the assessment of lipid abnormalities and coronary disease risk. Clin Chem. 1990;36:36-42.

26. McNamara JR, Cole TG, Contois JH, et al. Immunoseparation method for measuring low-density lipoprotein cholesterol directly from serum evaluated. Clin Chem. 1995;41:232-240.

27. Jialal I, Hirany SV, Devaraj S, Sherwood TA. Comparison of an immunoseparation method for direct measurement of LDL cholesterol with beta-quantification (ultracentrifugation). Am J Clin Path. 1995; 104:76-81.

28. Pisani T, Gebski CP, Leary ET, et al. Accurate direct determination of low-density lipoprotein cholesterol using an immunoseparation reagent and enzymatic cholesterol assay. Arch Pathol Lab Med. 1995;119:1127-1135.

29. Rifai N, Iannotti E, DeAngelis K, Law T. Analytical and clinical performance of a homogeneous enzymatic LDL-cholesterol assay compared with the ultracentrifugation-dextran sulfate-[Mg.sup.2+] method. Clin Chem. 1998;44:1242-1250.

30. NIH Consensus Conference. Triglyceride, HDL cholesterol and coronary heart disease. JAMA. 1993;269:505.

31. Castelli WP, Garrison RJ, Wilson PWF, et al. Incidence of coronary heart disease and lipoprotein cholesterol levels: The Framingham Study. JAMA. 1986;256:2835-2838.

32. Genest JJ, McNamara JR, Salem DN, Schaefer EJ. Prevalence of risk factors in men with premature coronary artery disease. Am J Cardiol. 1991;67:1185-1189.

33. Rubins HB, Robins SJ, Iwane MK, et al. Rationale and design of the Department of Veterans Affairs High-Density Lipoprotein Cholesterol Intervention Trial (HIT) for secondary prevention of coronary artery disease in men with low high-density lipoprotein cholesterol and desirable low-density lipoprotein cholesterol. Am J Cardiol. 1993;71:45-52.

34. Rubins HB, Robins SJ, Collins D, et al, for the Veterans Affairs High-Density Lipoprotein Cholesterol Intervention Trial Study Group. Gemfibrozil for the secondary prevention of coronary heart disease in men with low levels of high-density lipoprotein cholesterol. N Eng J Med. 1999;341:410-418.

35. Rifai N, Cole TG, Iannotti E, et al. Assessment of interlaboratory performance in external proficiency testing programs with a direct HDL-cholesterol assay. Clin Chem. 1998;44: 1452-1458.

36. McNamara JR, Huang C, Massov T, et al. Modification of the dextran-[Mg.sup.2+] HDL cholesterol precipitation method for use with previously frozen plasma. Clin Chem. 1994;40:233-239.

37. Nauck M, Marz W, Jarausch J, et al. Multicenter evaluation of a homogeneous assay for HDL-cholesterol without sample pretreatment. Clin Chem. 1997;43:1622-1629.

38. Hoefler G, Harnoncourt F, Faschke E, et al. Lipoprotein Lp(a). A risk factor for myocardial infarction. Arteriosclerosis. 1988;8:398-401.

39. Dahlen GH, Guyton JR, Attar M, et al. Association of levels of lipoprotein Lp(a), plasma lipids, and other lipoproteins with coronary artery disease documented by angiography. Circulation 1986;74:758-765.

40. Genest J Jr, Jenner JL, McNamara JR, et al. Prevalence of lipoprotein (a) [Lp(a)] excess in coronary artery disease. Am J Cardiol. 1991; 67:1039-1045.

41. Schaefer EJ, Lamon-Fava S, Jenner JL, et al. Lipoprotein(a) levels and risk of coronary heart disease in men. The Lipid Research Clinics Coronary Primary Prevention Trial. JAMA. 1994;271:999-1003.

42. Dahlen GH, Stenlund H. Lp(a) lipoprotein is a major risk factor for cardiovascular disease: Pathogenic mechanisms and clinical significance. Clin Genet. 1997;52:272-280.

43. Seman LJ, DeLuca C, Jenner JL, et al. Lipoprotein(a)-cholesterol and coronary heart disease in the Framingham Heart Study. Clin Chem. 1999;45:1039-1046.

44. Ridker PM, Hennekens CH, Stampfer MJ. A prospective study of lipoprotein(a) and the risk of myocardial infarction. JAMA. 1993;270: 2195-2199.

45. Berg K, Dahlen G, Christophersen B, et al. Lp(a) lipoprotein level predicts survival and major coronary events in the Scandinavian Simvastatin Survival Study. Clin Genet. 1997;52: 254-261.

46. Marcovina SM, Koschinsky ML. Lipoprotein (a): Structure, measurement, and clinical significance. In: Rifai N, Warnick GR, eds. Handbook of Lipoprotein Testing. Washington, DC: AACC Press; 1997:283-313.

47. Bostom AG, Gagnon DR, Cupples LA, et al. A prospective investigation of elevated lipoprotein(a) detected by electrophoresis and cardiovascular disease in women. The Framingham Heart Study. Circulation. 1994;90:1688-1695.

48. Bostom AG, Cupples LA, Jenner JL, et al. Elevated plasma Lp(a) and coronary heart disease in men aged 55 years and younger. A prospective study. JAMA. 1996;276:544-548.

49. McLean JW, Tomlinson JE, Kuang WJ, et al. cDNA sequence of human apolipoprotein(a) is homologous to plasminogen. Nature. 1987;330: 132-137.

50. Hajjar KA, Gavish D, Breslow JL, Nachman RL. Lipoprotein(a) modulation of endothelial cell surface fibrinolysis and its potential role in atherosclerosis. Nature. 1989;339: 303-305.

51. Loscalzo J, Weinfield M, Fless GM, Scanu AM. Lipoprotein(a), fibrin binding, and plasminogen activation. Arteriosclerosis. 1990; 10:240-245.

52. Miles LA, Fless GM, Levin EG, et al. A potential basis for the thrombotic risks associated with lipoprotein(a). Nature. 1989;339:301-303.

53. Albers JJ, Adolphson JL, Hazzard WR. Radioimmunoassay of human plasma Lp(a) lipoprotein. J Lipid Res. 1977; 18:331-338.

54. Seman LJ, Jenner JL, McNamara JR, Schaefer EJ. Quantitation of plasma lipoprotein (a) by cholesterol assay of lectinbound lipoprotein (a). Clin Chem. 1994;40:400-403.

55. Jenner JL, Ordovas JM, Lamon-Fava S, et al. Effects of age, sex, and menopausal status on plasma lipoprotein (a) levels. The Framingham Offspring Study. Circulation. 1993;87:1135-1141.

56. Marcovina SM, Albers JJ, Jacobs DR Jr, et al. Lipoprotein(a) concentrations and apolipoprotein(a) phenotypes in Caucasians and African Americans. The CARDIA Study. Arterioscler Thromb. 1993;13:1037-1045.

57. Moliterno DJ, Jokinen EV, Miserez AR, et al. No association between plasma lipoprotein(a) concentrations and the presence or absence of coronary atherosclerosis in African-Americans. Arterioscler Thromb Vasc Biol. 1995; 15:850-855.

58. Schreiner PJ, Heiss G, Tyroler HA, et al. Race and gender differences in the association of Lp(a) with carotid artery wall thickness. The Atherosclerosis Risk in Communities (ARIC) Study. Arterioscler Thromb Vasc Biol. 1996;16: 471-478.

59. Weiss SR, Bachorik PS, Becker LC, et al. Lipoprotein(a) and coronary heart disease factors in a racially mixed population: the Johns Hopkins Sibling Study. Ethn Dis. 1998;8:60-72.

60. Seman LJ, McNamara JR, Schaefer EJ. Lipoprotein(a), homocysteine and remnant-like particles: Emerging risk factors. Curr Opin Cardiol. 1999;14:186-191.

61. Canner PL, Berge KG, Wenger NK, et al. Fifteen-year mortality in Coronary Drug Project patients: Long-term benefit with niacin. J Am Coll Cardiol. 1986;8:1245-1255.

62. Havel RJ. Triglyceride-rich lipoproteins. In: Rifai N, Warnick GR, and Dominiczak MH (Eds), Handbook of Lipoprotein Testing, 3rd edition, Washington, DC: AACC Press; 1997:451-464.

63. Doi H, Kugiyama K, Ohgushi M, et al. Remnants of chylomicron and very low density lipoprotein impair endothelium-dependent vasorelaxation. Atherosclerosis. 1998;137:341-349.

64. Ooi TC, Ooi DS. The atherogenic significance of an elevated plasma triglyceride level. Crit Rev Clin Lab Sci. 1998;35:489-516.

65. Zilversmit DB. Atherogenesis: A postprandial phenomenon. Circulation 1979;60:473-485.

66. Cohn JS. Postprandial lipemia: emerging evidence for atherogenicity of remnant lipoproteins. Can J Cardiol. 1998; 14[Suppl B]: 18B-27B.

67. Cambien F, Jacqueson A, Richard JL, et al. Is the level of serum triglyceride a significant predictor of coronary death in normocholesterolemic subjects? The Paris Prospective Study. Am J Epidemiol. 1986; 124:624-632.

68. Criqui MH, Heiss G, Cohn R, et al. Plasma triglyceride level and mortality from coronary heart disease. New Engl J Med. 1993;328:1220-1225.

69. Hokanson JE, Austin MA. Plasma triglyceride level as a risk factor for cardiovascular disease independent of high-density lipoprotein cholesterol level: A meta-analysis of population-based prospective studies. J Cardiovasc Risk. 1996;3: 213-219.

70. Miller M, Seidler A, Moalemi A, Pearson TA. Normal triglyceride levels and coronary artery disease events: the Baltimore Coronary Observational Long-Term Study. J Am Coll Cardiol. 1998;31:1252-1257.

71. Doi H, Kugiyama K, Ohgushi M, et al. Membrane active lipids in remnant lipoproteins cause impairment of endothelium-dependent vasorelaxation. Arterioscler Thromb Vasc Biol. 1999;19:1918-1924.

72. Cohn JS, McNamara JR, Cohn SD, et al. Plasma apolipoprotein changes in the triglyceride-rich lipoprotein fraction of human subjects fed a fat-rich meal. J Lipid Res. 1988;29:925-936.

73. Cohn JS, Johnson EJ, Millar JS, et al. Contribution of apoB-48 and apoB-100 triglyceride-rich lipoproteins (TRL) to postprandial increases in the plasma concentration of TRL triglycerides and retinyl esters. J Lipid Res. 1993;34:2033-2040.

74. Karpe F, Hellenius ML, Hamsten A. Differences in postprandial concentrations of very-low-density lipoprotein and chylomicron remnants between normotriglyceridemic and hypertriglyceridemic men with and without coronary heart disease. Metabolism. 1999;48:301-307.

75. Campos E, Nakajima K, Tanaka A, Havel RJ. Properties of an apolipoprotein E-enriched fraction of triglyceride-rich lipoproteins isolated from human blood plasma with a monoclonal antibody to apolipoprotein B-100. J Lipid Res. 1992;33:369-380.

76. Nakajima K, Saito T, Tamura A, et al. Cholesterol in remnant-like lipoproteins in human serum using monoclonal anti apo B-100 and anti apo A-I immunoaffinity mixed gels. Clin Chim Acta. 1993;223:53-71.

77. Nakajima K, Okazaki M, Tanaka A, et al. Separation and determination of remnant-like particles in human serum using monoclonal antibodies to apo B-100 and apo A-I. J Clin Ligand Assay. 1996;19:177-183.

78. McNamara JR, Shah PK, Nakajima K, et al. Remnant lipoprotein cholesterol and triglyceride reference ranges from the Framingham Heart Study. Clin Chem. 1998;44:1224-1232.

79. Leary ET, Wang T, Baker DJ, et al. Evaluation of an immunoseparation method for quantitative measurement of remnant-like particle-cholesterol in serum and plasma. Clin Chem. 1998;44:2490-2498.

80. McNamara JR, Shah PK, Nelson SM, et al. Lipoprotein remnant cholesterol and triglyceride values in coronary cases and Framingham controls. [Abs] Circulation. 1996;94:I-94.

81. Takeichi S, Nakajima Y, Osawa M, et al. The possible role of remnant-like particles as a risk factor for sudden cardiac death. Int J Legal Med. 1997;110:213-219.

82. Takeichi S, Yukawa N, Nakajima Y, et al. Association of plasma triglyceride-rich lipoprotein remnants with coronary atherosclerosis in cases of sudden cardiac death. Atherosclerosis. 1999; 142:309-315.

83. Kugiyama K, Doi H, Takazoe K, et al. Remnant lipoprotein levels in fasting serum predict coronary events in patients with coronary artery disease. Circulation. 1999;99(22):2858-2860.

84. Shimizu H, Mori M, Saito T. An increase of serum remnant-like particles in non-insulin-dependent diabetic patients with microalbuminuria. Clin Chim Acta. 1993;221:191-196.

85. Nakajima K, Saito T, Tamura A, et al. A new approach for the detection of Type III hyper-lipoproteinemia by RLP-cholesterol assay. J Atheroscler Thromb. 1994; 1:30-36.

86. Devaraj S, Vega G, Lange R, et al. Remnant-like particle cholesterol levels in patients with dysbetalipoproteinemia or coronary artery disease. Am J Med. 1998; 104:445-50.

87. McNamara JR, Shah PK, Tayler TD, et al. Statin therapy reduces concentrations of remnant-like particles. [Abs] Atherosclerosis. 1997; 134:346.

88. McNamara JR, Shah PK, Tayler TD, Schaefer EJ. Postprandial response of remnant-like particles in coronary heart disease patients and controls. [Abs] Circulation. 1998:98:I-584.

89. Malinow MR, Kang SS, Taylor LM, et al. Prevalence of hyperhomocyst(e)inemia in patients with peripheral arterial occlusive disease. Circulation. 1989;79:1180-1188.

90. Genest JJ Jr, McNamara JR, Salem DN, et al. Plasma homocyst(e)ine levels in men with premature coronary artery disease. J Am Coil Cardiol. 1990;16:11141119.

91. Stampfer MJ, Mallinow MR, Willett WC, et al. A prospective study of plasma homocyst(e)ine and risk of myocardial infarction in US physicians. JAMA. 1991;268:877-881.

92. Boushey CJ, Beresford SA, Omenn GS, Motulsky AG. A quantitative assessment of plasma homocysteine as a risk factor for vascular disease. Probable benefits of increasing folic acid intakes. JAMA. 1995;274:1049-1057.

93. Hoogeveen EK, Kostense PJ, Beks PJ, et al. Hyperhomocysteinemia is associated with an increased risk of cardiovascular disease, especially in non-insulin-dependent diabetes mellitus: a population-based study. Arterioscler Thromb Vasc Biol. 1998;18:133-138.

94. Donner MG, Klein GK, Mathes PB, et al. Plasma total homocysteine levels in patients with early-onset coronary heart disease and a low cardiovascular risk profile. Metabolism. 1998;47:273-279.

95. Folsom AR, Nieto FJ, McGovern PG, et al. Prospective study of coronary heart disease incidence in relation to fasting total homocysteine, related genetic polymorphisms, and B vitamins: the Atherosclerosis Risk in Communities (ARIC) study. Circulation. 1998; 98:204-210.

96. Taylor LM Jr, Moneta GL, Sexton GJ, et al. Prospective blinded study of the relationship between plasma homocysteine and progression of symptomatic peripheral arterial disease. J Vasc Surg. 1999;29:8-19.

97. Selhub J, Miller JW. The pathogenesis of homocysteinemia: Interruption of the coordinate regulation by S-adenosylmethionine of the remethylation and transsulfuration of homocysteine. Am J Clin Nutr. 1992;55:131-138.

98. Lentz SR. Homocysteine and vascular dysfunction. Life Sci. 1997;61:1205-1215.

99. Tyagi SC, Smiley LM, Mujumar VS, et al. Reduction-oxidation (Redox) and vascular tissue level of homocyst(e)ine in human coronary atherosclerotic lesions and role in extracellular matrix remodeling and vascular tone. Mol Cell Biochem. 1998; 181:107-116.

100. Genest J Jr. Hyperhomocyst(e)inemia determining factors and treatment. Can J Cardiol. 1999;15(Suppl B):35B-38B.

101. Jacques PF, Rosenberg IH, Rogers G, et al. Serum total homocysteine concentrations in adolescent and adult Americans: Results from the third National Health and Nutrition Examination Survey. Am J Clin Nutr. 1999;69:482-489.

102. Osganian SK, Stampfer MJ, Spiegelman D, et al. Distribution of and factors associated with serum homocysteine levels in children. Child and Adolescent Trial for Cardiovascular Health. JAMA. 1999;281:1189-1196.

103. Selhub J, Jacques PF, Wilson PW, et al. Vitamin status and intake as primary determinants of homocysteinemia in an elderly population. JAMA. 1993;270:2693-2698.

104. Stehouwer CD, Weijenberg MP, van den Berg M, et al. Serum homocysteine and risk of coronary heart disease and cerebrovascular disease in elderly men: A 10-year follow-up. Arterioscler Thromb Vasc Biol. 1998;18:1895-1901.

105. Tucker KL, Mahnken B, Wilson PWF, et al. Folic acid fortification of the food supply. Potential benefits and risks for the elderly population. JAMA. 1996;276:1879-1885.

106. Chico A, Perez A, Cordoba A, et al. Plasma homocysteine is related to albumin excretion rate in patients with diabetes mellitus: A new link between diabetic nephropathy and cardiovascular disease? Diabetologia. 1998;41:684-693.

107. Bostom AG, Shemin D, Lapane KL, et al. Folate status is the major determinant of fasting total plasma homocysteine levels in maintenance dialysis patients. Atherosclerosis. 1996;123: 193-202.

108. Hong SY, Yang DH, Chang SK. The relationship between plasma homocysteine and amino acid concentrations in patients with end-stage renal disease. J Ren Nutr. 1998;8:34-39.

109. Malinow MR, Duell PB, Hess DL, et al. Reduction of plasma homocyst(e)ine levels by breakfast cereal fortified with folic acid in patients with coronary heart disease. N Engl J Med. 1998;338:1009-1015.

110. Shipchandler MT, Moore EG. Rapid, fully automated measurement of plasma homocyst(e)ine with the Abbott IMx analyzer. Clin Chem. 1995;41:991-994.

111. Frantzen F, Faaren AL, Alfheim I, Nordhei AK. Enzyme conversion immunoassay for determining total homocysteine in plasma or serum. Clin Chem. 1998;44:311-316.

112. Ubbink JB, Delport R, Riezler R, Vermaak WJ. Comparison of three different plasma homocysteine assays with gas chromatography-mass spectrometry. Clin Chem. 1999;45:670-675.

113. Jacques PF, Bostom AG, Williams RR, et al. Relation between folate status, a common mutation in methylenetetrahydrofolate reductase, and plasma homoeysteine concentrations. Circulation. 1996;93:7-9.

Judith R. McNamara is supervisor, Leo J. Seman is research scientist, and Ernst J. Schaefer is chief at the Lipid Metabolism Laboratory, Jean Mayer USDA Human Nutrition Research Center on Aging, Tufts University, Boston, MA. The authors are also affiliated with the Lipid Research Laboratory, Division of Endocrinology, Diabetes, Metabolism, & Molecular Medicine, at the New England Medical Center, Boston, MA.
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Title Annotation:coronary heart disease
Author:McNamara, Judith R.; Seman, Leo J.; Schaefer, Ernst J.
Publication:Medical Laboratory Observer
Date:Oct 1, 1999
Words:8939
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