Relation of Lipoprotein Subclasses as Measured by Proton Nuclear Magnetic Resonance Spectroscopy to Coronary Artery Disease

From the Division of Nutrition, Centers for Disease Control and Prevention, Atlanta, Ga (D.S.F.); the Department of Biochemistry, North Carolina State University, Raleigh (J.D.O., E.J.J.); the Milwaukee Veterans Administration Medical Center, Milwaukee, Wis (J.J.B.); and St. Luke's Hospital, Milwaukee, Wis (A.J.A., J.A.W.).

Correspondence to David S. Freedman, CDC MS–K26, 4770 Buford Hwy, Atlanta, GA 30341-3724. E-mail Dxf1{at}cdc.gov

	Abstract

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Abstract—Although each of the major lipoprotein fractions is composed of various subclasses that may differ in atherogenicity, the importance of this heterogeneity has been difficult to ascertain owing to the labor-intensive nature of subclass measurement methods. We have recently developed a procedure, using proton nuclear magnetic resonance (NMR) spectroscopy, to simultaneously quantify levels of subclasses of very low density (VLDL), low density (LDL), and high density (HDL) lipoproteins; subclass distributions determined with this method agree well with those derived by gradient gel electrophoresis. The objective of the current study of 158 men was to examine whether NMR-derived lipoprotein subclass levels improve the prediction of arteriographically documented coronary artery disease (CAD) when levels of lipids and lipoproteins are known. We found that a global measure of CAD severity was positively associated with levels of large VLDL and small HDL particles and inversely associated with intermediate size HDL particles; these associations were independent of age and standard lipid measurements. At comparable lipid and lipoprotein levels, for example, men with relatively high (higher than the median) levels of either small HDL or large VLDL particles were three to four times more likely to have extensive CAD than were the other men; the 27 men with high levels of both large VLDL and small HDL were 15 times more likely to have extensive CAD than were men with low levels. In contrast, adjustment for levels of triglycerides or HDL cholesterol greatly reduced the relation of small LDL particles to CAD. These findings suggest that large VLDL and small HDL particles may play important roles in the development of occlusive disease and that their measurement, which is not possible with routine lipid testing, may lead to more accurate risk assessment.

Key Words: nuclear magnetic resonance spectroscopy • coronary disease • angiography • lipoproteins • lipoprotein subfractions

	Introduction

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Elevated plasma levels of LDL-C and low levels of HDL-C are predictive of CHD.¹ Although there is conflicting evidence, it is also possible that high fasting or postprandial^{2 3} levels of TGs, indicative of high levels of VLDL, are also atherogenic.^{4 5} Despite the importance of these lipoproteins in the development of CHD, the extent of occlusive disease⁶ and CHD risk⁷ can vary greatly among persons with similar levels of lipids and other risk factors. Some of this variability may be due to the heterogeneity within each of the three major lipoprotein classes: each is composed of various subspecies that differ in particle diameter, density, composition, and possibly, atherogenicity.^{8 9 10 11}

Although lipoprotein subclasses have been quantified by analytical and density gradient ultracentrifugation, GGE, and chromatography,^{8 10 11 12 13} the time-consuming and labor-intensive nature of these methods has limited their widespread use. Nevertheless, several cross-sectional^{14 15 16 17 18} and cohort^{19 20 21} studies have found that normocholesterolemic persons with high levels of small, dense LDL particles are at increased risk for CHD. Furthermore, the protective effect of HDL-C has been most consistently observed for the larger HDL₂ (or HDL_2b) subfraction,^{9 22} and some evidence suggests that levels of small HDL particles (HDL_3b and HDL_3c) may be positively related to the severity of CAD.^{12 23 24}

We have developed a new procedure for quantifying plasma levels of lipoprotein subclasses by proton NMR spectroscopy.^{25 26} This method, which simultaneously provides the concentrations of VLDL, LDL, and HDL subspecies, does not require physical fractionation of the plasma and results in rapid and reproducible measurements. Furthermore, NMR-determined lipoprotein subclasses have been shown to correspond well with those obtained with established methods.^{26 27 28} The present study examines the relation of NMR-derived levels of lipoprotein subclasses to the severity of arteriographically documented CAD among 158 men.

	Methods

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Sample
The subjects were 240 men admitted to St. Luke's Hospital in 1989 to 1990 for diagnostic coronary arteriography performed by a cardiologist who was participating in The Milwaukee Cardiovascular Data Registry, a database in which the relation of various risk factors to CAD severity has been extensively examined.^{6 29} (Indications for coronary arteriography included severe or unstable angina pectoris, evidence of myocardial ischemia after myocardial infarction, and recurrent chest pain of unknown origin.) Men were excluded from the current study if they reported using cholesterol-lowering drugs (n=44), did not have a lipid or lipoprotein measurement (n=26), or did not have plasma available for an NMR determination (n=4), because LDL-C concentrations were estimated by the Friedewald equation [TC-HDL-C-(TG/5)],³⁰ where TC is total cholesterol; 8 men with a TG level 400* mg/dL were also excluded.

The 158 men included in the analyses ranged in age from 30 to 84 years (mean, 63 years), and Quetelet Index (weight in kilograms divided by the square of height in meters) was used as a measure of relative weight. Other characteristics, such as diabetes (n=31) or use of diuretics (n=32), were included as covariates in some analyses, but several men had missing information on the history of various medical conditions or use of antihypertensive medications. All analyses were limited to men because only 43 eligible women underwent coronary arteriography during the time frame of the study.

Chemical Analyses of Lipids and Lipoproteins
After a 12-hour fast, blood samples were drawn (prior to arteriography) into tubes containing EDTA (final EDTA concentration, 1 g/L). After centrifugation at 4°C, the separated plasma was analyzed for the concentrations of TC and TG by automated procedures³¹ in a laboratory that was standardized (and monitored) by the Centers for Disease Control and Prevention. HDL-C levels were measured after selective precipitation of apoB-containing lipoproteins with heparin/MnCl₂,³² and levels of apoB and apoA-I were measured by rate immunonephelometry on an automated system (Array, Beckman Instruments), with calibration materials supplied by the manufacturer. Levels of lipids, lipoproteins, and apolipoproteins based on these methods are referred to as "chemical determinations" throughout the text.

NMR Spectroscopy
The basis for NMR analysis of lipoprotein subclasses is that each lipoprotein particle in plasma within a given diameter range "broadcasts" a distinctive lipid NMR signal, the intensity of which is proportional to its bulk lipid mass concentration.^{25 27 28 33} The methodology used in this study to acquire and process the NMR data has been described in detail²⁶ and consisted of three steps: (1) acquisition of 250-MHz proton NMR spectra of the plasma specimens (0.5 mL, stored at 4°C for up to 5 days) at 45°C, with a Bruker WM-250 spectrometer; (2) deconvolution of the lipid methyl group signal envelope appearing in these spectra at 0.8 ppm, yielding the derived signal amplitudes broadcast by 18 modeled lipoprotein subclasses; and (3) conversion of these signal amplitudes to lipoprotein subclass concentrations by using experimentally determined factors that relate the signal amplitudes of isolated subfraction standards to their chemically measured cholesterol and TG concentrations. Levels of chylomicrons and VLDL subclasses are expressed in units of TG (mg/dL), and those of LDL and HDL subclasses in units of cholesterol (mg/dL). Close agreement has previously been demonstrated between NMR- and chemically determined LDL-C (r=0.91) and HDL-C (r=0.93) levels with this methodology.²⁶ LDL and HDL subclass distributions determined by GGE and NMR have also been shown to be closely related.²⁶

Because detailed information on the exact line shapes and chemical shifts of the lipid methyl signals of homogeneous subclass standards was not yet available at the time the NMR data from this study were acquired and processed (1989 to 1990), the deconvolution procedure employed 18 digitally shifted reference spectra to simulate the spectral characteristics of isolated subclasses.²⁶ To simplify presentation and analysis of the NMR data, the 18 modeled subclasses were combined into a smaller number (10) of particle-size groupings; classification was based on recently determined relations between isolated-subclass chemical shifts and particle-size estimates from GGE or electron microscopy measurements.^{27 28} The 10 lipoprotein subclass categories used were the following: chylomicrons (diameter >100 nm), large VLDL and remnants (60 to 100 nm), intermediate VLDL (40 to 60 nm), small VLDL (30 to 40 nm), large LDL (23 to 30 nm), intermediate LDL (20.5 to 23 nm), small LDL (18 to 20.5 nm), large HDL (10 to 13 nm, similar to HDL_2b), intermediate HDL (8.2 to 10 nm, similar to HDL_2a and HDL_3a), and small HDL (7.3 to 8.2 nm, similar to HDL_3b and HDL_3c).

A "particle size index," describing the mass-weighted average size of particles within each lipoprotein class, was calculated by weighting each subclass concentration by a numerical size designation (1 to 4 for VLDL, 1 to 3 for LDL and HDL) with larger values representing larger particle subclasses. For example, a person with an LDL-C level of 124 mg/dL, distributed as 18 mg/dL in small LDL, 56 mg/dL in intermediate LDL, and 50 mg/dL in large LDL, would have an LDL particle size index of 2.26 [(1x18)+(2x56)+ (3x50)÷124], a value slightly above the mean.

Extent of CAD
Coronary arteriograms were evaluated by a radiologist and cardiologist (without knowledge of risk factor data) to determine the extent of occlusive disease. Reductions in lumen diameter due to the most serious stenosis in the left anterior descending, circumflex, and right coronary arteries were incorporated into a global occlusion score representing the severity of CAD, as suggested by Rowe et al.³⁴ We inverted the scale so that a score of 0 indicates no observed occlusion, and a score of 300 represents total occlusion of all vessels. The mean occlusion score was 162 (SD=97), the interquartile range was from 82 to 247, and 11% (n=17) of the men had no detectable occlusive disease.

Statistical Analyses
Because the distributions of several lipoprotein subclasses were skewed toward higher values, analyses included the use of logarithmically transformed values, Spearman correlations, and robust regression techniques (such as minimizing the absolute rather than the squared deviations).³⁵ (In general, the exclusion or downweighting of influential observations generally strengthened the relation of subclass levels to CAD severity.) After the age-adjusted associations between the NMR parameters and CAD severity were examined, linear and logistic regression models focussed on which, if any, NMR determinations could provide information, beyond that obtained with chemically determined lipoprotein levels, on CAD severity. In addition to hypotheses generated from the literature, we used various stepwise regression procedures (forward selection with a P value <0.05, maximum R², and the C_p statistic)³⁶ to determine the set of NMR parameters most predictive of occlusive disease. In all regression models, we used quadratic terms and natural splines to assess nonlinearity³⁷ and product terms to assess interactions.

Although CAD severity was modeled primarily as a continuous variable, some analyses treated disease status as extensive (occlusion score 200, n=61) versus minimal (occlusion score <100, n=48). In these analyses, which excluded men with occlusion scores of 100 to 199, ORs were used to summarize the magnitudes of the associations with lipoprotein subclass levels (dichotomized at approximately the median). Some results from these analyses are presented graphically, with the predicted logits transformed to probabilities and covariates set to median values.³⁷ Although data on other characteristics, such as use of diuretics, were missing for several men, these adjustments (performed among the men with no missing data) did not substantially alter the relation of the lipoprotein subclasses to CAD.

	Results

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The size index for LDL particles was moderately associated with those for HDL (r_s=0.2) and VLDL (r_s=-0.28), and the relation of these particle size indices to other characteristics are shown in Table 1. In general, the strongest associations were with TG levels: relatively high TG levels were seen among men who had smaller LDL and HDL particles and among those with larger VLDL particles. Moderate associations, but in the opposite direction, were also seen between the particle size indices and levels of HDL-C. Although men with relatively large LDL particles also had higher levels of the LDL-C/apoB ratio than did other men, several of these associations were not linear. The association between TG levels and the LDL particle size index, for example, was much stronger among the 70 men with a TG level >150 mg/dL (r_s=-0.66) than among men with lower TG levels (r_s=0.12; data not shown).

After controlling for the association between CAD severity and age (r_s=0.33), the extent of CAD was most strongly related to levels of apoB (Spearman partial [r_s] correlation=0.30), but statistically significantly associations were seen with most of the chemically determined lipid and lipoprotein levels (Table 2). Although age-adjusted levels of the LDL and HDL particle size indices were also inversely related to the extent of occlusive disease (r_s=-0.17 to -0.19), these associations were not statistically significant after further adjustment for the chemical determinations.

Of the individual lipoprotein subclasses, levels of large VLDL, small LDL, intermediate HDL, and small HDL particles were significantly associated with CAD severity after adjustment for age (Table 3). For example, as the number of diseased vessels increased, the (geometric) mean level of large VLDL increased from 5 to 12 mg/dL and of small LDL from 10 to 21 mg/dL. Further adjustment for the chemical determinations (final column) reduced the magnitude of the association with levels of small LDL by 50%, but other associations remained statistically significant. Because of the differing associations of the HDL subclasses with CAD severity (positive with small particles versus inverse with larger particles), a derived variable (HDL_diff) was calculated as [(large HDL+intermediate HDL)-small HDL]. Levels of HDL_diff were strongly related to CAD severity, and all 7 men with a negative value of HDL_diff had an (age-adjusted) occlusion score that was equal to or above the median occlusion score among the other 151 men (data not shown).

Associations with extensive (versus minimal) disease were then examined (Table 4). Two thirds (40/60) of the men with a level of large VLDL above the median had extensive CAD compared with 43% of the other men. The resulting OR [(40:20)÷(21:28)] indicates that men with a high level of this subspecies were 2.7 times as likely to have extensive disease as were other men. As estimated from a logistic regression model containing age and various chemical determinations as covariates, adjusted ORs for large VLDL ranged from 3 to 5 (P<0.05 for each). Adjusted ORs with extensive CAD were also statistically significant for levels of small HDL (ORs >3) and HDL_diff (OR=0.3). Furthermore, 22 of the 27 men with high levels of both small HDL and large VLDL had extensive CAD (versus 9 of the 26 men with low levels of both subclasses), yielding an adjusted OR of 15. (Levels of small HDL and large VLDL showed little correlation [r_s=-0.05].) In contrast to these associations with CAD, levels of small LDL-C were not consistently related to disease after adjustment for lipid and lipoprotein levels, and an adjusted OR of 0.6 (P>0.05) was seen for chemically determined levels of HDL-C (final column).

Several of these associations are shown in the Figure, in which the vertical distances between the roughly parallel lines reflect the additional information provided by the lipoprotein subclasses in predicting CAD. (Various interactions between the NMR data and chemical determinations were examined, and none were found to be statistically significant; P>0.25 in all cases.) For example, at a TG level of 150 mg/dL (upper right panel), the probability of extensive CAD for a 63-year-old subject was estimated to be either 0.44 or 0.75, depending on the level of large VLDL particles. In contrast, if the level of large VLDL was known, the TG level provide little additional information on CAD severity: the estimated probability of extensive CAD varied from only 0.71 to 0.82 between the 10th and 90th percentiles of TG among men with high levels of large VLDL. Furthermore, if HDL_diff was relatively high (lower right panel), even men with an HDL-C level <35 mg/dL were not at greatly increased risk for occlusive disease. Knowledge of each subspecies in the Figure significantly improved CAD prediction beyond that obtained with the corresponding chemical determination, whereas if NMR-determined subclass levels were known, the only chemical determination that was significantly related to occlusive disease was the LDL-C level.

View larger version (25K): [in this window] [in a new window]   Figure 1. Relation of levels of NMR- and chemically determined lipoproteins to the probability of extensive CAD; prevalence of extensive CAD among 109 men was 62%. Extensive CAD was defined as an occlusion score >200, and each panel shows the predicted probability of extensive CAD (versus a score 100) according to chemical determination (x axis) and high versus low levels of the NMR-determined subclass; solid and dashed lines represent predicted estimates derived from logistic regression models in which the chemical determination value was treated as a continuous variable; age has been adjusted to the median (63 years). Circles (low levels of specified subclass) and triangles (high levels) represent predicted prevalence of extensive CAD within 6 groups, based on specified subclass level and lipid/lipoprotein concentration tertile (rather than as continuous variables for predicted estimates). There was only 1 man (79-year-old with a TG level of 231 mg/dL who did not have extensive disease) in the group defined by a high TG level and a low level of large VLDL.

The prediction of occlusive disease, treated as a continuous variable, by various combinations of the chemical and NMR determinations was then examined (Table 5). Whereas levels of TC and TG (in addition to age) accounted for 22% of the variability in the occlusion score (model 1), information on levels of LDL-C and HDL-C significantly improved disease prediction, with a multiple R² of 0.25 (model 2). (No further improvements were achieved by adding apolipoprotein levels to model 2.) Further increases in the multiple R² (P=0.002) were obtained by using information on levels of large VLDL and HDL_diff (model 3), and the predictive ability was similar (R²=0.30, model 4) if levels of HDL-C and TG were not considered. (None of the other 11 other NMR parameters, described in Tables 1 through 3, further improved disease prediction.) Furthermore, adding information on diabetes, previous myocardial infarction, and use of diuretics did not alter the relation of large VLDL and HDL_diff levels to CAD. Interestingly, although the improvement in disease prediction obtained by using NMR-determined subclasses was relatively small (R²=0.06, model 3 versus 2), it was larger than that achieved by using levels of LDL-C and HDL-C rather than TC (R²=0.03, model 2 versus 1).

	Discussion

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Our results, based on data from 158 men, indicate that several lipoprotein subfractions provide information on arteriographically documented CAD that is not available through routine lipid measurements. Independently of age and levels of lipids and lipoproteins, we found that occlusive disease was associated positively with levels of large VLDL particles and small HDL particles and inversely with levels of intermediate HDL particles. Although levels of small LDL particles were also related to CAD severity in bivariate analyses, adjustment for either TG or HDL-C levels substantially reduced the magnitude of this association. These findings suggest that information on levels of lipoprotein subclasses may improve the prediction of CHD risk, a situation analogous to the realization in the 1950s and 1960s³⁸ that information on lipoprotein cholesterol levels more accurately predicted disease risk than did the TC level alone.

The principal advantage of NMR lipoprotein analysis is the elimination of the lengthy, labor-intensive step(s) needed to physically separate the subclasses of interest. Because the lipids in each lipoprotein subclass broadcast a distinguishable signal with an intensity proportional to particle concentration and lipid mass,^{27 28} quantification proceeds directly from a short (1 minute) acquisition time of the proton NMR spectrum of unfractionated plasma. Rather than arising from differences in chemical composition, the distinct properties of lipid NMR signals of various subspecies are based on particle size–dependent differences in magnetic susceptibility; these differences are induced by the orientation order of the phospholipids in the shell of lipoprotein particles that surround the neutral lipid core.³³ Because NMR lipoprotein analysis is based on differences in particle size (specifically, the phospholipid shell diameter), it cannot distinguish between VLDL particles and chylomicron remnants of similar size or between LDL and Lp(a) particles; furthermore, IDLs are included in the quantification of large LDL. However, NMR lipoprotein analysis can simultaneously quantify VLDL, LDL, and HDL subclasses and thus provide information that would otherwise be very difficult and time consuming to obtain.

Despite the differences between NMR and other methods used to quantify lipoprotein subspecies, studies of split samples have shown close agreement between LDL and HDL particle sizes measured by NMR and GGE.²⁶ Several of the cross-sectional findings in the current study (Table 1) provide additional evidence concerning the validity of NMR lipoprotein analysis. In agreement with previous reports based on GGE or ultracentrifugation,^{39 40 41 42} we found that men with a preponderance of small LDL particles also had relatively high levels of TG, large VLDL, and small HDL particles, along with low levels of HDL-C and the LDL-C/apoB ratio. We also found, as have others,^{13 43} that there is a relative excess of small HDL particles among men who are overweight or who have high TG levels.

Possible differences in the atherogenicity of lipoprotein subspecies have focussed primarily on LDL subclasses. Results of several case-control studies, including two nested within a cohort design,^{19 20} have suggested that persons with small, dense LDL particles ("pattern B") are more likely to have CAD or its clinical complications.^{14 15 16 17 18} However, because of the association of pattern B with adverse levels of TG and HDL-C, it is difficult to determine whether the size of LDL particles per se is responsible for the increased risk. We found, as have other investigators,^{14 15 16 17} that the importance of small LDL particles is substantially reduced if comparisons are made at similar levels of TG and HDL-C. However, some reports have indicated that levels of small LDL may provide some independent information concerning disease risk,^{18 21} particularly among normotriglyceridemic men.⁴⁴ Knowledge of the LDL particle-size distribution may also be useful in the choice of lipid-lowering medications.⁴⁵

Cross-sectional studies that have examined HDL subfractions have typically found that CAD was most strongly related (inversely) to levels of the larger HDL_2a and HDL_2b subclasses.^{12 22 24} In further agreement with our results, elevated levels of small HDL particles have been found among persons with CAD^{23 24 46} and among myocardial infarction survivors.¹² Although several cohort studies^{20 47 48 49} found that information on levels of HDL₂ and HDL₃ did not improve disease prediction, the laboratory methodology used may be important, because the proportion of HDL-C in the HDL₂ subfraction has been reported to range from 10%²⁰ to 66%.⁴⁷ (The coefficient of variation for HDL subfraction determinations is frequently >10%.⁴⁹ ) It has also been suggested¹³ that levels of HDL₃, as determined by differential precipitation, are more strongly correlated with levels of HDL_2a and HDL_3a than with smaller HDL particles. Our finding that HDL_diff is related to CAD severity, independently of the HDL-C level, emphasizes the importance of considering differences in the atherogenicity of HDL subspecies when assessing CAD risk.

Because several reports have found that smaller TG-rich lipoproteins may be particularly atherogenic,^{4 5 50 51} it is interesting that we found levels of large VLDL particles (and/or chylomicron remnants) to be positively related to CAD severity. However, the lack of evidence concerning the atherogenicity of large VLDL subclasses may in part be methodological: no simple analytical procedures are available for VLDL subfractionation. It has been speculated^{51 52} that large VLDL particles may play a role in the development of CHD through either their enhanced uptake by macrophages or induction of a procoagulant state promoting thrombosis. Because fasting levels of large VLDL (S_f 60 to 400) are also correlated with delayed chylomicron clearance,⁵³ which is related to CAD severity,^{2 3} the observed association may reflect the atherogenicity of postprandial lipemia.

Several limitations of the current study should be considered. Although coronary arteriography is useful for studying factors associated with atherosclerosis, the cross-sectional design precluded the study of men who died from an initial myocardial infarction and those with asymptomatic disease. Furthermore, lipoprotein (and subclass) levels measured at arteriography may not reflect levels present during lesion development,⁵⁴ and there can also be substantial misclassification of coronary arteriograms. These biases, however, would be expected to decrease the magnitudes of the associations with both the chemical and the NMR determinations. Several statistical considerations, including our limited ability in this small study to assesses whether the relation of subclass levels to CAD varied by age or level of the corresponding chemical determination, should also be considered. It is also very difficult to select a single "best" regression model when independent variables are correlated, but we found that NMR-determined levels of VLDL and HDL subclasses were consistently included in regression models that fit the data well. It should also be emphasized that the methodology for NMR lipoprotein analysis has evolved considerably since the current study was conducted, and it is now possible to reliably quantify 6 VLDL, 3 LDL, and 5 HDL subclasses as well as IDLs.^{27 28}

Despite these limitations, our results suggest that levels of lipoprotein subfractions provide information on CHD that cannot be obtained with the routine clinical measurement of lipid and lipoprotein levels. Because the current guidelines from the National Cholesterol Education Program have relatively low sensitivity and positive predictive value,⁷ this additional information may lead to improved diagnostic accuracy. For example, our results suggest that if the HDL size distribution is weighted toward larger particles, an HDL-C level <35 mg/dL may confer little, if any, increased risk; in contrast, a high level of large VLDL particles, irrespective of the TG level, may be of concern. The advantages of lipoprotein subclass analyses through NMR, particularly its rapid and simultaneous measurement of VLDL, LDL, and HDL subspecies, make it feasible to examine the importance of various subspecies in large cohort studies.

	Selected Abbreviations and Acronyms

 

-C = cholesterol CAD = coronary artery disease CHD = coronary heart disease GGE = gradient gel electrophoresis NMR = nuclear magnetic resonance OR = odds ratio TC = total cholesterol TG = triglyceride

	Acknowledgments

 
This study was supported by National Institutes of Health grants RO1-HL43230, RO1-HL29011, and RO1-HL28692 and by the Medical Research Service of the Veterans Administration. We would like to thank Dr Gerald L Cooper for his interest in our laboratory methods and his encouragement during this project.

Received September 23, 1997; accepted January 13, 1998.

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