(2) summarized the literature in 1953 and also reported the results from repeated sampling of 42 subjects between 47 and 92 years of age. Fourteen to 20 blood ...
Proc. Natl. Acad. Sci. USA Vol. 84, pp. 6259-6261, September 1987 Medical Sciences
Individual variation in serum cholesterol levels (risk of coronary heart disease)
D. M. HEGSTED* AND ROBERT J. NICOLOSIt *New England Regional Primate Research Center, Harvard Medical School, Southboro, MA 01772; and tDepartment of Clinical Sciences, College of Health Professions, University of Lowell, Lowell, MA 01854
Contributed by D. M. Hegsted, March 30, 1987
The intraindividual variances in serum/ ABSTRACT plasma cholesterol levels from a variety of sources have been examined. It is apparent that these are very substantial with mean coefficients of variation usually between 5% and 10%, even when the diet is controlled in metabolic studies. Some subjects show extreme variability from one blood sample to the next. Thus, it is very difficult to assess the degree of risk of individuals according to the guidelines provided by the Consensus Conference on lowering blood cholesterol levels to prevent heart disease, and many individuals will be misclassified unless particular attention is paid to this problem.
The National Institutes of Health Consensus Conference on Lowering Blood Cholesterol to Prevent Heart Disease (1) proposes a goal of "reducing the blood cholesterol of our entire adult population to 200 mg/dl (180 mg/dl for those younger than 30 years).'" It has further defined moderate- and high-risk serum cholesterol levels for three age categories: 20-29 years, 200-220 mg/dl; 30-39 years, 220-240 mg/dl; and 40 years and over, 240-260 mg/dl. These levels approximate the 75th and 90th percentiles of serum cholesterol levels in the American population. Treatment is recommended for both groups, with the intensity of treatment depending on the level of risk. The Conference report also concludes that since a large percentage of the American population sees a physician at least once every year, many individuals with serum cholesterol levels above the 75th percentile would be identified in a relatively short time if serum cholesterol levels were measured at these visits. These recommendations raise important questions about the constancy of serum cholesterol levels and the reliability with which individuals at various degrees of risk can be identified. We have examined some of the data reported in the literature that provide or allow us to calculate the "intraindividual variation" in serum cholesterol levels and also present some previously unpublished data from both human subjects and nonhuman primates, which provide estimates of the variation in serum cholesterol in the same subjects from one bleeding to the next. Such data show that the serum cholesterol level is far from constant even under metabolic ward conditions where diet is not a variable. The cause of such variation is apparently unknown, but it seems clear that data obtained from occasional blood samples will, inevitably, result in misclassification of large numbers of individuals. There was substantial interest in the reliability of serum cholesterol measurements a number of years ago when methods were being developed and the significance in serum cholesterol levels was beginning to be appreciated. Watkins et al. (2) summarized the literature in 1953 and also reported The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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the results from repeated sampling of 42 subjects between 47 and 92 years of age. Fourteen to 20 blood samples were collected from each subject, and these were analyzed in duplicate so that variance due to analytical error could be subtracted from the total variance to yield the "biologic variation." These values for four age groups are summarized in Table 1 and the coefficient of variation is calculated from the data in the paper. Watkins et al. concluded that the results they obtained were reasonably consistent with other data available at that time. Table 1 also includes data from the following reports. Keys (3) measured the serum cholesterol in 38 soldiers four to six times and in 30 nonhospitalized patients who were "cautioned not to change their diets." The latter were examined four times at monthly intervals. The standard deviations and coefficients of variation in these groups have been calculated from the reported standard errors. Witchi et al. (4) reported the results of a large number of family members who were advised to lower their serum cholesterol levels. A control sample was obtained before the study began, and another was obtained several months later, after the mean cholesterol had returned to prestudy levels. The original data were available to us and the values in Table 1 have been calculated from the two samples from each subject. In a recent study (5), four blood samples were obtained from each of 31 soldiers during a 44-day field trial. These men all ate at the same mess but could choose from the foods offered. The mean cholesterol level of the group rose slightly during the trial. We have, therefore, calculated the regression of the serum cholesterol for each man over time and report the deviation from regression. One would expect that the variation in serum cholesterol would be reduced when the diet is constant. Jacobs et al. (6) have recently examined the data collected by Keys et al. (7) in their extensive studies to define the serum cholesterol response to changes in the intake of fat. The mean intraindividual standard deviation in these studies was 17.8 mg/dl, and it ranged from 6.0 to 44.9 mg/dl. In the double-blind study of Roberts et al. (8), to evaluate the effects of dietary cholesterol on serum cholesterol, three weekly samples were obtained from each subject during each dietary period. The standard deviations shown in Table 1 were calculated from the data presented. Wolf and Grundy (9) recently reported studies on the effects of weight reduction on serum cholesterol levels of obese patients. During the base-line period of 4-5 weeks (weight maintenance period), four to nine samples were obtained while the subjects were receiving a formula diet of constant composition. The mean intraindividual standard deviation was 7.0 mg/dl, and it ranged from 3 to 14 mg/dl. Finally, we have data from cynomolgus monkeys (Macaca fascicularis) that have received semipurified diets of constant composition for several months. Eight blood samples were collected into EDTA-containing tubes for cholesterol analy-
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Table 1. Intraindividual variation in serum cholesterol from various sources Intraindividual variation in serum cholesterol Range No. of Mean Mean of SD, determinations SD, C.V., No. of per subject % mg/dl Source mg/dl subjects Little or no dietary control 17-20 14.0 6.7 5.4-29.2 Watkins et al. (2) 10 12 17.6 8.8 4.4-30.4 19-20 Watkins et al. (2) 6.2 8.6-21.2 10 14-20 12.6 Watkins et al. (2) 10 12.3 6.7 9.7-22.6 Watkins et al. (2) 19-20 NA NA 38 4-6 27.0 Keys (3) 4 NA NA 30 48.0 Keys (3) 2 5.2 1.0-27.0 210 10.1 Witchi et al. (4) 4 9.2 1.3-23.0 This report and ref. 5 31 19.1 Metabolic ward conditions 11 17.8 9.0 6.0-44.9 58 Jacobs et al. (6) Roberts et al. (8) Wolf and Grundy (9) Cynomolgus monkeys This report NA, not available.
Comments
Subjects 47-59 yr old; hospital diet Subjects 65-60 yr old; hospital diet Subjects 72-79 yr old; hospital diet Subjects 80-92 yr old; hospital diet U.S. Army soldiers Nonhospitalized coronary patients Family member examined several months apart U.S. soldiers during field trial
16 15
3 4-9
10.6 7.0
4.8 3.1
2.1-24.0 3.0-14.0
Data of Keys et al. (7) while subjects were receiving the same diet Double-blind trial on dietary cholesterol Obese subjects; formula diet
15
8
16.9
9.3
5.3-35.0
Animals receiving a constant semipurified diet
sest at weekly intervals after the animals had received the diet for at least one month. As is apparent from the table, these animals show variations in serum cholesterol as large as those reported for human subjects. The reasons for the large differences observed in the serum cholesterol level of the same subject are not known. Most papers do not provide estimates of the proportion of the variation that might be attributed to analytical error, but it is generally agreed that in well controlled laboratories the reproducibility of the values should be within 1-2% and cannot, apparently, account for the range of values observed. Whatever the source of the variation, however, the practical implications are the same. In attempting to evaluate the significance of these kinds of data one should note that the mean serum cholesterol level in American men is now -220 mg/dl (1). If one assumes a mean intraindividual standard deviation of only 5% of the mean value, then one would expect that a single sample would be likely to fall within 2 standard deviations above or below the true mean. Thus, a single sample from a man with a mean serum cholesterol level of 220 mg/dl can be expected to fall between 200 and 240 mg/dl-i.e., range from essentially no risk to high risk. Even so, this is a highly optimistic assumption since a standard deviation of 5% is only about twice the analytical error in well controlled laboratories and, obviously, many individuals will show greater variation than this. It might be noted that in any type of field trial where one or a few blood samples are obtained before and after efforts to modify serum cholesterol levels, very large numbers of subjects can be expected to "improve" or "deteriorate" from chance alone. It is also pertinent to note that a variety of methods are available to estimate serum cholesterol levels but may not yield the same absolute values. Blank et al. (11) compared two commonly used clinical laboratory instruments (Technicon SMAC and Du Pont aca) with the Lipid Research Clinics (LRC) methodology (12). They conclude that altPlasma was harvested
(1987)
at low-speed centrifugation at 1500 x
g
for
30 min at 40C. Plasma cholesterol levels were determined in duplicate by an enzymatic method (10). The coefficient of variation for duplicate samples is 0.5%. The laboratory methodology has been certified by the Lipid Standardization Program of the Center for Disease Control.
though the correlations between values measured by the different methods are high, both of the clinical instruments have a positive bias and that "using the LRC 75th percentile as a cutoff, 50% of the population would be considered hypercholesterolemic with the Technicon SMAC and 60% with the Du Pont aca." Assigning risk is, thus, a matter of the methodology used until such differences can be eliminated. The reliability of any estimate improves, of course, as more samples are obtained, but even this is not very assuring. If four samples were analyzed in the above example, one would expect the mean value to fall between 210 and 230 mg/dl. It is apparent that defining the degree of risk in a population where most people are at some risk is difficult and large numbers of individuals will be misclassified from analysis of occasional blood samples. There appears to be no satisfactory solution to the problem, but it should be appreciated that several samples are needed to define risk. We thank Colonel David D. Schnakenberg, Army Research Institute of Environmental Medicine (Natick, MA) for providing access to the data on the military subjects. Corn oil used in the animal studies was kindly provided by Best Foods, CDC North America (Englewood Cliffs, NJ). This work was supported by grants from the National Institutes of Health (HL 36101, HL 36200, and RR00168) and from the Alcoholic Beverages Medical Research Foundation (Baltimore, MD). The animals used in these studies were maintained in accordance with the guidelines of the Committee on Animals of the Harvard Medical School and those prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Resources, National Research Council [DHEW publ. no. (NIH) 85-23, revised 1985]. 1. National Heart, Lung, and Blood Institute and the Office of Medical Applications of Research (1985) J. Am. Med. Assoc. 253, 2080-2086. 2. Watkins, D. M., Lawry, E. Y., Mann, G. V. & Halperin, M. (1954) J. Clin. Invest. 33, 874-883. 3. Keys, A. (1979) in Nutrition, Lipids and Coronary Heart Disease, eds. Levy, R., Rifkind, B., Dennis, B. & Ernst, E. (Raven, New York), pp. 1-23. 4. Witschi, J. C., Singer, M., Wu-Lee, M. & Stare, F. J. (1978) J. Am. Diet. Assoc. 72, 384-389. 5. U.S. Army Research Institute and U.S. Army Combat Developments Experimentation Center (1986) Combat Field Feeding System-Force Development Test and Experimentation, Volume 1, Basic Report (U.S. Army Combat Developments Experimentation Center, Attn: ATEC-PLS, Fort Ord, CA).
Medical Sciences: Hegsted and Nicolosi 6. Jacobs, D. R., Anderson, J. T., Hannan, P., Keys, A. & Blackburn, H. (1983) Arteriosclerosis 3, 349-356. 7. Keys, A., Anderson, J. T. & Grande, F. (1957) Lancet ii, 959-966. 8. Roberts, S. L., McMurray, M. P. & Connor, W. E. (1981) Am. J. Clin. Nutr. 34, 2092-2099. 9. Wolf, R. N. & Grundy, S. M. (1983) Arteriosclerosis 3, 160169.
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10. Allain, C. C., Poon, L. S., Chan, C. S., Richmond, W. & Fu, P. C. (1974) Clin. Chem. 19, 470-475. 11. Blank, D. W., Hoeg, J. M., Kroll, M. H. & Ruddel, M. E. (1986) J. Am. Med. Assoc. 256, 2867-2870. 12. National Institutes of Health (1974) Lipid and Lipoproiein Analysis in Manual of Laboratory Operations, LRC Programs (U.S. Dept. of Health, Education, and Welfare, Washington, DC), Report (NIH) 75-628, Vol. 1.
