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Variation in human erythrocyte membrane unsaturated fatty acids: correlation with cardiovascular disease.

Erythrocyte (RBC) membrane composition varies with factors, including diet, oxidative stress, and genetic variation. (1-7) This is important because the RBC traverses the microvasculature and is subject to turbulence and deformation when the vasculature and microvasculature are altered by atherosclerotic disease. (8) Thus, factors that change RBC membrane fluidity, such as the RBC lipid composition, (9,10) have major effects on the progression of atherosclerotic disease. A number of drugs also modify membrane composition, including antiepileptic drugs (11,12) and drugs that modify cholesterol synthesis. (13)

Dietary effects that are well characterized include protective effects of fatty acids (FAs) found in fish oils that have unsaturation at the third carbon from the terminal (co3) carbon of the FA (14); mammalian polyunsaturated FAs, including arachidonic acid (cis-5,cis8,cis11,cis14--eicosatetranoic acid) are [omega]o6 FAs. Erythrocyte FA measurements have been shown to better correlate with dietary intake than serum FAs. (15) Erythrocyte membrane FA compositions change with progression of atherosclerotic disease, with the ratio of linoleic acid (cis9,cis12-octadecadienoic acid [C18:2]) to arachidonic acid (C20:4) being dependent on key desaturases FADS1 and FADS2, which have significant polymorphism. (1) Lower ratios of C18:2 to C20:4 are associated with inflammation and higher risk of atherosclerotic disease. (1) Erythrocyte membranes also change with drug treatment, including statins, for increased serum triglycerides or cholesterol, generally by increases in polyunsaturated FAs. (16)

It is also established that diets rich in trans-monounsaturated fats, as a result of partial hydrogenation (reduction of some unsaturation with conversion of cis-to-trans-un-saturation as a side effect), increase low-density lipoprotein (LDL) cholesterol (17) and may indirectly increase risk of vascular disease, (18,19) in part reflecting relationships to changes in inflammatory factors, including production of C-reactive protein. (20) However, the trans-fat issue is complicated by the occurrence of variants of linoleic acid with 1 cis and 1 trans double bond. (21) The significance of these cis, trans dienoic acids is complicated by the fact that in mammals, lipids are stored for practical purposes solely as saturated fat, and that in processing, the enoyl-coenzyme A isomerase system (22) interconverts cis, trans dienoic acids. Thus, although cis, trans dienoic acid accumulation may reflect genetic differences related to vascular diseases and other factors, it is not clear why dienoic acid variants occur in inconstant amounts in cell membranes.

Additional interesting dietary correlates of atherosclerotic risk include association of consumption of vegetable oils that contain natural polyunsaturated and singly cis-unsaturated FAs, especially olive oil, and moderate amounts of alcohol, with lower incidence of atherosclerotic disease. (23) However, although changes in diet reflecting these epidemiologic factors may ultimately reduce the overall risk of atherosclerotic disease, including myocardial infarction, in specific populations, it remains unclear how clinical testing may determine the risk status of an individual patient from FAs in vivo. To address this issue, we obtained blood samples from healthy volunteers, from unselected hospitalized patients, and from patients with newly diagnosed clinical myocardial damage as selected by cardiac troponin I screening, mostly emergency room patients. We then extracted the RBC membrane FAs and analyzed all of the major components by gas chromatography and mass spectrometry. Erythrocyte unsaturated FA composition may change significantly with, and may be a marker of, occurrence of atherosclerotic disease in individual patients. Shorter long-chain FAs (C16-C18) may correlate with increases in plasma LDL cholesterol. However, trans-monounsaturated FA differences may have wide variation between individuals.

MATERIALS AND METHODS

Clinical Samples

Blood samples were collected with institutional review board approval. Samples collected for clinical study, including from cardiac patients with elevated troponin I and random hospitalized patients or from volunteer donors, were delabeled by an honest broker--a person not involved in the study who forwarded samples with selected data but without patient identifiers. Troponin assays were performed with the Centaur cTnl Ultra Assay (Siemens Healthcare Diagnostics, Deerfield, Illinois) with a 99th percentile for the reference (normal) population equal to 0.044 ng/ mL. For the purposes of this study, samples were considered positive at levels greater than 0.5 ng/mL. Data available for analysis were age (all 40 to 60 years, except for healthy volunteers, whose average age was 41 years) and the troponin assay. For healthy volunteers, absence of known vascular disease and no treatment for atherosclerotic disease (such as statins) were criteria for inclusion, and a dietary questionnaire was completed as described in "Results." For healthy volunteers, a serum lipid profile was done and correlated with patient number but without patient identifiers.

Materials

Solvents were high-performance liquid chromatography grade (Sigma, St Louis, Missouri). The trans9-octadecenoic (elaidic) and cis9-octadecenoic (oleic) acids were from Alltech (State College, Pennsylvania). N,O-bis-(trimethylsilyl)-acetamide-trimethylchlorosilane (Sylon BT) was from Supelco Inc (Bellefonte, Pennsylvania). Tetradeuterated long-chain FA standards ([D.sub.4]-C20, C22, C24) were from Herman J. ten Brink (Academic Hospital VU Metabolic Laboratory, Amsterdam, the Netherlands).

Lipid Profiles

Serum was separated by centrifugation, with cholesterol, triglycerides, and high-density lipoprotein cholesterol measured on a Vitros 950 instrument (Ortho Clinical, Piscataway, New Jersey). Briefly, cholesterol was disassociated by addition of polyoxyethylene octyl phenyl ether, and cholesterol esters were hydrolyzed with cholesterol ester hydrolase, with cholesterol oxidase to yield H2O2, quantified by peroxidase oxidation of triarylimidazolone dye as reflectance at 540 nm. (24) Triglycerides were measured similarly after lipase de-esterification with [H.sub.2][O.sub.2] produced by l-a-glycerophosphate oxidase. High-density lipoprotein was precipitated in 50-kDa dextran sulfate, 45 mM Mg[Cl.sub.2], (25) measuring cholesterol in the precipitate as in total cholesterol. Low-density lipoprotein cholesterol was calculated as total cholesterol less high-density lipoprotein cholesterol and very low-density lipoprotein cholesterol (estimated as triglycerides/5). (26)

RBC Membrane FA Extraction and Derivitization

A total of 250 [micro]L of packed red blood cells were washed with saline, and [D.sub.4]-C22, 24, and 26 internal standards were added, and the cells then were subjected to lipid extraction and de-esterification. Red blood cell FAs were converted to methyl esters in a 3:1 methanol to methylene chloride solution with acetyl chloride catalyst at 75[degrees]C for 90 minutes. The resulting mixture was neutralized with 7% potassium carbonate, and esterified acids were extracted with hexane as described previously. (27) The hexane layer was dried under nitrogen. The extracted material was further derivatized with 40 [micro]L of N,O-bis(trimethylsilyl)acetamidetrimethylchlorosilane for 20 minutes at 85[degrees]C, (28) and the resulting mixture was used for analysis; analysis (Figure 1, A) showed sialyated residues only in trace quantities, indicating that the methylation was essentially quantitative. Where replicates are shown, these represent separate extractions.

Chromatography and Identification of Separated Components

A Hewlett-Packard 6890 instrument (Agilent Technologies, Santa Clara, California) was used for gas chromatography/mass spectometry. The column was a DB-1ms (Agilent Technologies), 30 meters X 0.25 mm diameter, with a dimethyl polysiloxane stationary phase and a film thickness of 0.25 [micro]m. Samples were run in helium with a flow rate of 0.80 mL per minute. Initial temperature was 180[degrees]C, and this temperature was held for 3 minutes, then temperature was increased to 250[degrees]C at 5[degrees]C per minute, and after 10 minutes the heating rate increased to 10[degrees]C per minute to 300[degrees]C at 15 minutes, and then temperature was held at 300[degrees]C until the end of the run at 37 minutes. Electron impact ionization was used to monitor elution of components, scanning m/z from 50 to 550 for each eluted peak. Data were not collected during the exclusion (5 minutes); thereafter, ionizable material was recorded as a function of time, and m/z signals were used for eluant identification by comparison with Wiley275 and Nist02 libraries.

Statistics

Curve fits used least squares, with correlation coefficients (R2) indicated. Comparisons of groups used Student t test. Mean [+ or -] range, SD, or SEM are shown as indicated.

RESULTS

Analysis of RBC-Derived FAs

We assayed RBC FA content from 10 healthy volunteer samples, 10 samples with plasma cardiac troponin I measurements higher than 0.5 ng/mL, and 10 random hospitalized inpatient samples. The FA components were resolved by gas chromatography/mass spectometry using selected ion monitoring to identify the components (Figure 1, A). Hexadecanoic (C16:0), octadecadienoic (C18:2), octadecenoic (C18:1), octadecanoic (C18:0), and eicosatetraenoic acids (C20:4) had a mean recovery of more than 1% of membrane lipids and were compared between sample types. Three samples in which one or more components were not resolved were excluded (1 from a troponin-positive patient and 2 from random hospitalized patients). For C18:1, cis-andtrans-isomers were resolved (Figure 1, B) and were analyzed because of the interest in trans-octadecenoic acid (t-C18:1), which is not synthesized in mammals but is derived from consumption of modified fat, predominantly as partially hydrogenated vegetable oil. Trans-unsaturation also occurs to variable extents in multiple unsaturated FAs, which were not resolved on the 30-meter column used (see "Comment"). The cis-and trans-C18:1 have identical molecular weights and principal ion fragments, and so they were verified using isolated oleic (cis-C18:1) and elaidic (trans-C18:1) acids (Figure 1, B). The drawing on the right in Figure 1, B shows the structures of cis-andtrans-C18:1, which have surprisingly disparate physical properties. Oleic acid is a liquid at room temperature, whereas elaidic acid is a hard wax; this difference is the basis for conversion of liquid vegetable oil to solid partially hydrogenated oil. To validate the recovery of cis-andtrans-C18:1, which varied widely in relative quantity in clinical samples, standard solutions with varying octadecanoic acids and constant amounts of deuterated lignoceric acid (C24 saturated FA) were calculated for cis-(Figure 2, A) and trans-(Figure 2, B) octadecenoic acid. This showed that recoveries were essentially linear with quantity in the ranges used for analysis (Figure 1, A).

[FIGURE 1 OMITTED]

FAs From RBCs of Healthy Subjects

On average, 55% of the FAs from RBCs produced by methylene chloride--methanol extraction were C16 and C18 saturated acids (Figure 3). Small amounts of other saturated FAs, less than 1% of recovered material, were resolved in some cases, were not quantified, and were not included in calculations of fraction of total FAs recovered. Of the unsaturated FAs, there were approximately equal amounts--about 15% each--of C20:4, C18:1, and C18:2. Standard deviations were typically 15% of analyte value, with trans-C18:1 and C20:4 having greater variability. Although the predominant dienoic and tetraenoic acids are known to be linoleic and arachidonic acids (all cis 9,12-octadecadienoic acid and all cis 5,8,11,14-eicosatetraenoic acid), the method does not resolve other isomers.

Trans-octadecenoic Acid in RBC Membranes Was Highly Variable but Did Not Differ Between Groups

There was no relation between trans-C18:1 load and sample type. Figure 4, A shows trans-C18:1 as percentage of recovered FAs for healthy volunteers (average age, 41 years) and 9 samples from inpatients with elevated cardiac troponin I (average age, 50 years). Healthy volunteers were given a questionnaire about diet, which was remarkable only in that all subjects stated that they avoided foods with trans fats (see "Comment"). Note that the range of trans-C18:1 was approximately 10-fold in the subjects (0.5%-5%), but there was no relation to subject type. Elevated troponin samples also gave the same average and had a similar SD; random inpatient samples (data not shown) also averaged -2.5% trans-C18:1, with SDs of about 1%. This variability, despite the linearity of recovery of standards (Figure 2), suggested the possibility of variable recovery in extraction, because trans-C18:1 was the least abundant FA quantified. However, duplicates run for 12 samples showed that replicates averaged [+ or -] 10% of analyte value (Figure 4, B; see "Comment"). The lack of difference in patients with documented myocardial damage may reflect both genetic differences and differences in trans-unsaturation in polyunsaturated FAs (see "Comment").

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

Patients With Myocardial Damage Have Increased RBC Octadecadienoic Acid

Although differences in C18:1 isomers between groups were not found, specimens with elevated troponin had significantly (P = .02) increased C18:2 compared with FAs extracted from healthy controls or random inpatient samples (Figure 5, A). Other comparisons of saturated and unsaturated FA components showed no significant differences, except that overall unsaturated FAs as a proportion of total membrane FAs were significantly lower in the healthy volunteers compared with either hospitalized random samples or patients with myocardial damage (Figure 5, B). This comparison should be viewed with some caution in that the patient samples were from an older population (see "Comment"), but nevertheless, there was a highly significant increase in unsaturated FAs in sick patients, with or without myocardial infarcts (P = .01).

Short-Chain FAs, but Not Trans-octadecenoic Acid, Correlate With Serum Cholesterol

Triglycerides were, as expected, directly related to total or LDL cholesterol in healthy control samples (Figure 6, A and B). However, despite the strong relation of dietary trans-fat to increased serum cholesterol and related risk factors for atherosclerotic disease risk, RBC octadecenoic acids, cis or trans, were not related to serum cholesterol (Figure 6, C). It is possible that in a larger sample a relationship might be resolved, but clearly for an individual subject there will be no useful predictive relationship between red blood cell cis-or trans-C18:1 and lipid profile. On the other hand, total C16-C18 FAs from RBC membranes correlated significantly with LDL or total cholesterol in serum samples (Figure 6, D).

COMMENT

Our data indicate that increased C18:2 may be a useful indicator of risk of atherosclerotic disease, particularly myocardial infarction, even if isomers of C18:1 (including cis-andtrans-isomers) are not separately analyzed. This is in keeping with the work of Lemaitre et al, (21) who selected patients with cardiac arrest relative to age-matched controls and found increases in both linolenic acid and trans-C18:2 isomers. In that work as well as in ours, C18:1 isomers were not related to heart attack incidence, and in other studies, serum levels of trans-C18:1 above the 20th percentile (29) and higher adipose tissue trans-C18:130 were actually associated with lower risk of sudden cardiac death. A large, multinational European study found a small, statistically insignificant increased risk of myocardial infarctions in the upper quartiles of adipose transC18:1. Harris et al (31) found no association between red blood cell trans-C18:1 and acute coronary syndromes, whereas Block et al (32) showed that both trans-18:1 and trans-18:2 were associated with increased risk of acute coronary syndromes. Clearly, more research is needed to clarify this association, but it is difficult to escape the conclusion that despite the very well-established correlation of trans-fat consumption and elevated serum cholesterol and risk for cardiac disease, (17,18,33) red blood cell membrane trans-fat accumulation does not represent a direct linkage to dietary risk. On the other hand, red blood cell lipid profiles, and in particular C18:2 content, may be a useful indicator of risk, even when relatively simple FA analysis is done, without resolution of all isomers.

[FIGURE 4 OMITTED]

[FIGURE 5 OMITTED]

Several studies point to a clear association between trans-C18:2 and cardiac risk. Lemaitre et al (21) showed that the odds ratios for sudden cardiac death were 3 times higher in patients with elevated red blood cell transC18:2 compared with age- and sex-matched controls. A prospective study in female nurses showed that red blood cell trans-C18:2 content was more strongly associated with cardiovascular morbidity than trans-C18:1. (34) A study in Costa Rica, which has a higher intake of trans-C18:2 than the United States, (21) demonstrated an association between adipose tissue trans-fats and coronary heart disease, attributed mainly to trans-C18:2. (33) This is particularly interesting in that dietary trans-fat is almost entirely consumed as singly unsaturated C18 FAs, whereas C18:2 isomers are, at least to some extent, interconverted by enoyl isomerases, (22) and thus may reflect both nondietary and dietary constituents of the cell membrane FA content. Although we could not resolve isomers of C18:2, our data showing increased C18:2 are consistent with these reports, and together they suggest that C18:2 accumulation may result from either higher trans-fat intake or mechanisms affecting both RBC FA metabolism and cardiovascular risk.

[FIGURE 6 OMITTED]

In a large population study of RBC trans-FAs and risk of coronary heart disease, Sun et al (34) found a clear correlation between C18:1 trans-FA isomers and risk, as well as a very strong correlation between total trans-FA load with heart disease. These data are not inconsistent with our study in that the difference ([+ or -] SD) between cases and controls in trans-C18:1 (1.25 [+ or -] 0.35 vs 1.16 [+ or -] 0.35) would not be resolved in a small sample, such as ours. Note also that the trans-C18:1 as percentage of total FAs is smaller than that found in our study (averaging 2.5%; Figure 3), probably reflecting differences in hexane/isopropanol extraction and in assay standardization, (33) although the conclusions are qualitatively similar. The point here is that an individual determination of the trans-C18:1 is of no value whereas, surprisingly, in patients with heart attacks there is a much larger difference in total C18:2 relative to controls, suggesting that this may be a useful marker of risk in individual subjects. This hypothesis will require a larger and prospective analysis. This is despite the clear difference in odds ratios of coronary disease when trans-fat analysis is divided into quartiles (34) in that the significance of the differences depends on population differences, whereas the statistic variation makes a prediction for an individual so uncertain as to be of little potential clinical use.

We were somewhat surprised to see very poor correlation between red blood cell trans-fat and cholesterol or LDL cholesterol, whereas large population studies, such as Sun et al (34) found clear statistic linkage. This undoubtedly also reflects the high degree of individual variation. On the other hand, we did find a clear correlation between total C16-C18 FA content and LDL cholesterol or total cholesterol (Figure 6), with a strong enough correlation to be of potential use in individual samples. Interestingly, patients (regardless of cardiovascular risk) had a statistically significant increase in total unsaturated C18 (C18:2 and C18:1) compared with healthy volunteers. Although these findings can be attributed to an older median age, another study found a positive correlation between serum levels of total unsaturated FAs (C18:1, C18:2, and C20:4) and coronary risk factors. (35)

The very high degree of variation in trans-C18:1 is particularly remarkable in the control healthy subjects. The healthy volunteers were given a questionnaire about diet, and all subjects stated that they avoided trans-fats. This may reflect that the volunteers were from a university hospital employee population. Nevertheless, the very large range in trans-C18:1, from about 0.5% to about 5%, suggests that in some but not all subjects, dietary monounsaturated trans-fat may accumulate out of proportion to dietary exposure. This, alternatively, could indicate that subjects' beliefs about their trans-fat intake did not correspond with the amount of trans-fat actually found in their RBCs. Although this will require further study, an interesting possibility is underlying genetic variation in efficiency of degrading dietary trans-FAs. Consistent with this hypothesis, there was no clear correlation in trans-C18:1 range in the subpopulations studied, including troponin-positive subjects. Given the prevalence of atherosclerotic disease in the United States, it is highly likely that some of the random hospitalized patients also have significant vascular disease, and early disease in some of the healthy controls probably exists. This may be the basis for the relatively high variability in all groups (note that Figure 5 shows standard errors, not SDs). Further, this preliminary study only indicates the association between troponin-positive samples and higher red blood cell unsaturated FA content; prospective study will be needed to determine whether higher unsaturated FA content reflects increased risk of clinical atherosclerotic disease.

Our method is adapted from routine determination of very long-chain FAs in serum and uses instrumentation, columns, and reagents commonly available in clinical laboratories performing toxicologic or biochemical genetics analyses. The relatively short column provides sufficient resolution for the separation of C18:2 and cis-and transC18:1 isomers while maintaining a short analysis time (less than 40 minutes), compared with other gas chromatography/mass spectometry methods for FA analysis. (7,36,37) However, to be useful clinically it will be important to determine whether changes in RBC FA content may detect potential vascular disease at stages where markers of disease that are simple to assay by automated chemistry are negative.

This is a small study; therefore, the lack of statistically significant associations is not proof of their absence (possible type II error). Furthermore, the observational nature of the study does not allow the conclusion of a causal relationship between the observed changes, such as increased C18:2, and the cardiovascular risk. Finally, the de-identified nature of the samples did not allow us to gather further clinical data. Nevertheless, this study suggests that RBC unsaturated FA composition changes significantly with myocardial damage and describes a relatively simple method to assess it as a useful marker of subclinical atherosclerotic disease. In addition, we found that shorter-chain FAs (C16-C18) correlated with increases in plasma cholesterol or LDL cholesterol. Importantly, this study shows that trans-monounsaturated FA differences are not sufficiently consistent to predict individual patient risk. Further, RBC membrane unsaturated trans-FA isomer composition is highly variable and may reflect genetic polymorphism in degradation of trans-FAs in general, a subject which is not well understood and deserves further investigation.

This study was supported in part by the US Department of Veterans Affairs and by the National Institutes of Health.

Accepted for publication March 30, 2009.

Reprints: Harry C. Blair, MD, Department of Pathology, University of Pittsburgh and Veterans Affairs Medical Center, 705 Scaife Hall, Pittsburgh, PA 1 5261 (e-mail: hcblair@imap.pitt.edu).

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Jorge L. Sepulveda, MD, PhD; Yvette C. Tanhehco, MD, PhD; Monica Frey, BS; Lida Guo, MD; Lorna J. Cropcho, BS; K. Michael Gibson, PhD; Harry C. Blair, MD

From the Department of Pathology & Laboratory Medicine, University of Pennsylvania School of Medicine (Drs Sepulveda and Tanhehco), and the Veterans Affairs Medical Center (Dr Sepulveda), Philadelphia, Pennsylvania; and the Department of Pathology, University of Pittsburgh (Mss Frey and Cropcho and Drs Guo, Gibson, and Blair), the Children's Hospital (Ms Cropcho and Dr Gibson), and the Veterans Affairs Medical Center (Dr Blair), Pittsburgh, Pennsylvania. Dr Gibson is now with the Department of Biological Sciences, Michigan Technological University, Hougton, Michigan.

The authors have no relevant financial interest in the products or companies described in this article.
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Title Annotation:Original Articles
Author:Sepulveda, Jorge L.; Tanhehco, Yvette C.; Frey, Monica; Guo, Lida; Cropcho, Lorna J.; Gibson, K. Mic
Publication:Archives of Pathology & Laboratory Medicine
Article Type:Report
Geographic Code:1USA
Date:Jan 1, 2010
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