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Precursors of atherosclerosis in children: The Cittadella study. Follow-up and tracking of total serum cholesterol, triglycerides, and blood glucose
PREVENTIVE
MEDICINE
11,
381-390
(1982)
Precursors of Atherosclerosis in Children: The Cittadella Study. Follow-up and Tracking of Total Serum Cholesterol, Triglycerides, and Blood Glucose ANTONIO PAGNAN,*~’ GIOVANNI B. AMBROSIO,* MARIO VINCENZI,? PAOLO MORMINO,* PIETRO MAIOLINO,? LICIA GERIN,* ENRICO BARBIERI,~ FRANCO CAPPELLETTI,~ AND CESARE DAL PALM* *Istituto
di Medicina Clinica, tDivisione Cardiologica,
Clinica Medica II, Universita di Padova, Ospedale Civile Di Cittadella. Cittadella,
Padua, and Italy
As part of a WHO project, precursors of atherosclerosis in children, a survey was made in Cittadella, a town in Northern Italy. Four hundred and eight children (49.7% males) aged 6, 9, 12, and 15 were examined and a blood sample for total cholesterol (TC), triglycerides (TG), and blood glucose (BG) was drawn one hour after an oral glucose load of 50 g. The same procedure was performed in the same subjects 3 years later. The mean values of TC in the age groups 6,9, 12, and 15 were 155.2, 176.1, 191.5, 170.5 mg/dl, respectively. TG values were 54.4, 53.1,55.8,62.2 n&d1 and BG, 108.3,85.2,92.1,97.4 mg/dl, respectively. A marked fall of TC at reexamination was found in initial age groups of 12 and 15. BG levels fell significantly only in those subjects initially examined at age 6, while TG showed a longitudinal increase in all age groups, though more markediy in the younger ones. Different body composition, and possibly a secular trend, might explain these findings. The tracking analysis showed that serum TC tends to maintain its percentile with increasing age in contrast to serum TG and BG tracking which was less consistent, suggesting the influence of factors other than those affecting changes in TC.
INTRODUCTION The hypothesis that atherosclerosis starts in childhood was put forward more than two decades ago by Holman et al. (1958) after their studies on juvenile atherosclerosis (12). The first epidemiological survey dealing with children and families was made in Tecumseh, Michigan in 1959 (9). Since then several studies have been published, mainly in the United States, on the risk factors of atherosclerosis in children (1, 4, 7, 10, 11, 19, 21, 27, 28, 32). Recognizing the importance of this approach to the possible prevention of atherosclerosis, in 1974 the WHO held a meeting in which the plan for a collaborative study (Precursors of Atherosclerosis in Children) was drafted (29). A detailed research protocol was subsequently designed (30). In 1975, according to the WHO protocol, our Institute started a collaborative study with two hospitals in Northern Italy, Pordenone and Cittadella. The general aim of the project was to determine whether future high-risk adults could be identified by measuring the same risk factors during childhood or, the reverse1 To whom reprint requests should be addressed: Antonio Clinica, Clinica Medica II, Via Giustiniani, 2, Padua, Italy.
Pagnan, M.D.,
Istituto
di Medicina
381 0091-7435/82/040381-10$02.00/O Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
PAGNAN
382
ET AL.
TABLE AGEANDSEXDISTRIBUTIONOFTHE
1 SCHOOLPOPULATION
Age (years)
M
F
Total
6* 9 9-12 12 -+ 15 15 --f 18
51 51 50 51
52 51 51 51
103 102 101 102
whether children with high risk-factor levels do indeed develop into high-risk adults. In order to clarify the problem, the longitudinal approach, in which the tracking of risk factors is performed from childhood to early adulthood, was considered the most suitable. We describe herein the longitudinal data (3 years follow-up) on serum lipids (cholesterol and triglycerides) and blood glucose (one hour after 50 g glucose per OS)in a school population of children and adolescents from Cittadella, a town in Northern Italy. The tracking of the same variables is also investigated. MATERIALS
AND METHODS
Following the recommendations of the WHO protocol (30), we examined 444 school children (50% each sex) of four age groups: 6, 9, 12, and 15. All subjects were examined initially and reexamined after 3 years. At the 3-year follow-up, 28 (6.3%) were lost for the following reasons: parents’ refusal (18); emigration (10); and eight for whom a reliable blood sample could not be obtained, were excluded. The longitudinal analysis includes the remaining 408 subjects, 203 males and 205 females (Table 1). Subjects live in the Cittadella district, whose total population is 60,486 (49.1% males). The economy of the district is well diversified: 150 small and medium-size industries, 1,350 commercial factories, 2,228 craftsmen factories, and 2,333 farms. The population is ethnically homogeneous with few immigrants. The cooperation of school authorities (physician, presidents) and school health services was secured at the start of the study. Parents and children were informed of the aim and methods of study and written consent was obtained from the parents. Examination procedure. In the health office in each school,2 3 days a week, a physician and two nurses took a venous blood sample of approximately 10 ml from each child. The blood was taken with the child seated, one hour after ingestion of 50 g of glucose. The blood was divided into two tubes, one of which was taken to the Central Laboratory of Cittadella General Hospital for assay of blood glucose (3) and serum triglycerides (8). The other tube was centrifuged and the serum, stored at -20°C was sent within 7-10 days to the Central Laboratory of Pordenone General Hospital for serum cholesterol determination (22). Quality control for serum TC measurements was overseen by the WHO Regional Reference Center (Institute for Clinical and Experimental Medicine of Praha) which cooperates * In several schools the health offke
was a modified classroom.
PRECURSORS
OF ATHEROSCLEROSIS
IN
CHILDHOOD
383
with the WHO International Lipid Standardization Laboratory at the Centers for Disease Control, Atlanta, Georgia. Further data collected are irrelevant to this paper and will not be discussed. Participation at the initial examination was 100% in all age classes. It was necessary to reach eight children at home, three aged 12 and five aged 15 years, with a staff of two physicians, two nurses, and one secretary. All clinical and laboratory data were collected in the appropriate form. An identical examination was performed 3 years later. The 80th percentile of the distribution was arbitrarily taken as a cutoff point between “normal” and “abnormal” values in the various age classes (6, 9, 12, 15 years) both at the initial and follow-up examination. Statistical methods. A paired t test was used in the comparison of the baseline and rescreening values. The “tracking” pattern, i.e., whether or not the subjects, with increasing age, have been keeping the rank within their age group, was determined by means of (a) correlation analysis (Pearson’s Y) between initial and follow-up values; (b) analysis of the distribution in various quintiles at retest of the subjects in the 5th quintile at initial examination; (c) application of the Van der Waerden scores (18) in order to evaluate any baseline-retest changes in the differences between subjects in the 5th quintile and those in the lower ones (lst-4th). RESULTS
The mean values of TC in the age groups 6,9, 12, 15, were, respectively: 155.2, 176.1, 191.5, 170.5, mg/dl. Those of TG: 54.5, 53.1, 55.8, 62.2 mg/dl and those of BG: 108.3, 85.2, 92.1, and 97.4 mg/dl. No significant differences were found between the two sexes in any age group, which were subsequently analyzed together. In the longitudinal analysis, mean values of serum TC showed a significant increase from age 6 to 9, while a significant decrease was observed from ages 12 to 15 and 15 to 18 (Table 2 and Fig. 1). Mean serum TG levels showed a steady, significant increase from ages 6 to 9, 9 to 12, and 12 to 15 years (Table 3 and Fig. 2). Mean BG levels significantly decreased from age 6 to 9, while a significant increase was observed from ages 12 to 15 and 15 to 18 (Table 4 and Fig. 3). A significant correlation was found between serum TC at initial testing and at retest in all age groups; the same was true for serum TG, except for age group 6 to 9 years (Tables 2 and 3). No significant correlation was found for BG, however, except for the triennium 9 to 12 years (Table 4). The age-specific 80th percentile values of serum TC, TG, and BG have been calculated (Table 5). At retest, the mode of distribution for subjects initially in the 5th quintile remained the same only for serum TC, while serum TG and BG showed a greater dispersion (Table 6). Comparison at retest between the mean values for serum TC of children in the top quintile at initial test and those of all other quintiles showed significant differences in all age groups, except for that of the 6- to 9-year group. For serum TG, the above differences were significant only in the age group 12 to 15 years and for BG only in that of 9 to 12 years (Table 7).
PAGNAN ET AL.
384
TABLE 2 LONGITUDINAL BEHAVIOROF SERUMTOTAL CHOLESTEROL(TC) IN mgldl Age (years)
Mean ” SD
P
155.2
9
186.0
Correlation coefficient* 69
Range
+*
21.7
95-255
22.1
139-255
P < 0.001
9 J 12
176.1 2 32.3
95-260
0.511
182.3 k 25.1
96-252
P < 0.001
12
191.5
f 28.2
135-305
0.530
15
156.8
+ 28.9
80-265
15 J 18
170.5 2 28.6
75-240
0.527
156.6 f 28.4
75-222
P < 0.001
Mean of the change c SD
0.360 +30.8
f
Range of the change
-
55
119
28.4 -49
112
- 148
40
-119
58
+ 6.2 k 28.9
i P < 0.001
-3.41
t 27.8
- 13.9 f 27.6
* r between initial and retest values.
DISCUSSION
We found no sex differences in the mean values of serum TC, TG, and BG, thus confirming the results of several studies (1,4,7, 15, 23). Other studies, however, have found higher levels in girls than in age-matched boys (10, 21). The similarity TABLE 3 LONGITUDINAL BEHAVIOROF SERUMTRIGLYCERIDES(TG)IN mg/dl Correlation coefficient* (r)
Age (years)
Mean 2 SD
6 J 9
54.4 -c 12.7
36
100
0.055
66.1 2 27.9
28
184
NS
9 L 12
53.1 2 13.8
36
100
0.199
64.9 2 23.6
35
230
12 L 15
55.8 k 16.4
43
140
0.279
63.2 “_ 24.3
23
125
P < 0.01
15 1 18
62.2 r 13.9
43
100
0.266
62.6 h 18.9
25
163
Range
* r between initial and retest values.
P < 0.05
P < 0.01
Mean of the change f SD
Range of the change -43
141
-43
173
-48
75
-54
92
-21.1 f 32.4
+11.8 k 24.8
f 1.4 f 25.2
+ 0.4 k 20.2
PRECURSORS OF ATHEROSCLEROSIS IN CHILDHOOD 200
385
serumTC Pg A)
/ 170
160 160
\
\
I
150 140 190 !
-II
9-
6-9
-I
I
12
I
12-15
I
---(,Zs,
FIG. 1. Longitudinal behavior of serum total cholesterol (TC).
between girls and boys could be related to the fact that sexual development progresses during childhood and adolescence. Serum lipid values in our population are similar to those reported in several studies of young population samples in different ethnic, geographic, and environmental settings (1, 4, 5, 7, 10, 15, 20, 23). This observation, in contrast with the well-known differences observed for serum lipid levels in adult populations (13) from different geographic areas, stresses the importance of environmental factors in the modulation of serum lipid levels. Looking at the longitudinal behavior of serum TC, we observe a marked, significant fall from age 12 to I5 and, although less consistent, from age 15 to 18, around puberty and adolescence, confirming the results of many previous crosssectional and longitudinal studies (10, 11, 15, 17, 20, 21, 24). Such behavior draws attention to marked hormonal and growth changes as modulating factors of serum
,
6-9
,
,
9 -
12
,
12-15
,
,
15-
,
18&:s,
FIG. 2. Longitudinal behavior of serum triglycerides (TG).
386
PAGNAN
ET AL.
FIG. 3. Longitudinal behavior of blood glucose (BG), one hour after 50 g glucose per OS.
TC levels. In particular, besides sex hormones, especially testosterone (20), the spectacular TC fall might be related to the thyroid gland, the weight of which during puberty almost doubles (2). The longitudinal behavior of serum TG levels showed a steady increase at the rescreening, 3 years later. We have observed a similar trend in children in a different area in Northern Italy (25). Whether these changes are due to a secular trend or an age-related trend is unclear. The fact that the mean TG values in children aged 6, 9, and 15 years at initial testing were all similar as were their TABLE 4 LONGITUDINAL BEHAVIOR OF BLOOD GLUCOSE(BG), 1 HR AFTER50 g GLUCOSEPEROS, IN mg/dl Age (years)
Mean k SD
Range
Correlation coefficient* (4
108.3 f 30.2
60
218
0.179
85.2 f 18.7
58
130
NS
85.2 ? 20.2
44
144
0.252
12
96.1 k 21.9
41
175
P < 0.05
12 J 15
92.1 k 23.2
46
163
0.143
93.9 ” 24.0
50
170
NS
15 1 18
97.4 k 24.2
55
180
0.139
93.3 5 28.7
46
280
NS
9
* r between initial and retest values.
Mean of the change k SD
Range of the change -113
43
-53
111
- 88
90
- 75
190
-23.1 + 32.4
+ 10.9 + 25.7
+ 1.8230.7
- 4.1 k 34.7
PRECURSORS
OF ATHEROSCLEROSIS
IN
387
CHJLDHOOD
TABLE 5 80~~ PERCENTILE, AGE-SPECIFIC VALUES FOR SERUM TC, TG, AND BG Serum TC
Follow-up
BG (mddl)
(m&W
Serum TG (mg/dl)
175.0
71.0
127.0
200.0
80.0
101.0
203.0
57.0
100.0
12
197.0
80.0
111.0
12
210.0
71.0
110.0
1:
175.0
86.0
112.0
15
195.0
71.0
114.0
18
182.0
71.0
107.0
(years)
9
means three years later might suggest a secular trend. However, Widhalm et al. (31) reported a “significant age dependency for the mean TG values” of ll-yearold children followed for 4 years, in keeping with the change we observed between ages 12 and 15. Interestingly, for BG (after load) the highest mean values were observed in the youngest subjects, aged 6, while 3 years later it showed a significant fall. Comparison of our data with those published by other investigators (26) is difficult for technical reasons, such as different glucose load and different methods of determining BG. The interpretation of this finding is obscure, though it might be suggested that the highest glucose values observed at age 6 as compared with age 9 are related to differences in body composition, i.e., the ratio of muscle mass to adipose tissue. The tracking analysis showed that TC measurement tracks reasonably with age, confirming other recent study results (16). Such behavior, also reported by Lee et
TABLE 6 QUINTILE DISTRIBUTION, AT RETEST (3 YEARS LATER), OF THE SUBJECTS IN THE TOP QUINTILE AT INITIAL TEST Years (quintiles) 5” 4” 3” 2” 1”
TC
6+9 TG
BG
TC
7 4 6 1 3
5 4 4 2 6
3 4 8 3 2
7 6 6 1 0
9+ 12 TG BG 5 4 7 3 1
8 3 2 4 3
TC 11 5 1 3 0
12 --) 15 TG BG 6 4 3 5 2
6 1 5 4 4
TC 10 6 3 0 1
15 + 18 TG BG 3 3 3 5 6
3 8 4 2 2
TABLE I
18
179.9 + 24.7
151.0 ? 26.4
160.7 -t 21.9
210.7 k 13.1
15
L
150.1 2 23.9
181.1 ? 17.3
233.7 f 23.2
184.1 5 32.0
178.2 t 24.5
199.3 + 20.1
165.6 f 25.9
219.1 2 15.4
15
i
12
12
L
9
9
183.9 5 21.5
6
193.9 e 22.9
145.0 f 18.5
195.3 2 19.3
Follow-up
L
Subjects in the lst-4th quintile at intake
Subjects in the 5th quintile at intake
Serum cholesterol (mgldl)
to.001
to.001
NS
P
66.5 2 18.0
81.2 2 11.4
73.7 + 27.1
81.2 f 16.0
68.8 5 16.6
74.9 2 13.6
65.6 _’ 25.9
15.2 + 8.1
Subjects in the 5th quintile at intake
61.6 2 19.0
57.5 + 9.9
60.7 _t 23.1
49.5 + 8.5
63.9 k 25.0
47.8 k 6.9
66.3 k 28.5
49.1 zk 6.8
Subjects in the lst-4th quinrile at intake P
NS
co.05
NS
NS
Serum triglycerides (mg/dI)
99.8 k 22.0
134.4 2 19.4
97.7 ‘- 29.0
126.6 t- 17.1
106.3 e 24.3
115.0 + 13.6
88.3 -t 17.5
153.5 e 19.7
Subjects in the 5th quintile at intake
91.7 f 30.0
88.4 -e 14.6
93.0 k 22.7
83.6 -c 15.1
93.6 2 20.7
78.0 + 13.8
84.5 2 19.0
96.8 ? 19.5
Subjects in the lst-4th quintile at intake
Blood glucose (mgldl)
NS
NS
<0.05
co.oo1
NS
P
COMPARISON AT RETEST (3 YEARS LATER) BETWEEN THE MEAN VALUES (?SD) FOR SERUM TC, TG, AND BG OF CHILDREN IN THE TOP QUINTILE AT INITIAL TEST (3 YEARS BEFORE) AND THOSE OF ALL OTHERS (1s~ TO ~TH QUINTILE)
PRECURSORS
OF ATHEROSCLEROSIS
IN
CHILDHOOD
389
al. (17) and Laskarzewski et al. (14), confirms the possibility of detecting high-risk
children at an early age, and thus of laying the foundations for intervention trials at that time (7). Yet, the fact that not all the children and adolescents maintain values in the highest quintile 3 years later, clearly indicates that a single TC determination is not sufficient for an absolute diagnosis of hypercholesterolemia (25). The dispersion of the TC values at retest might be partly related to regression toward the mean (6, 29, although quintile grouping minimizes this effect. The degree of tracking for serum TG was less consistent than that of TC, since the dispersion throughout the lower quintiles was much more marked for TG than for TC. This finding, which confirms previous reports (14), suggests that serum TG might be largely influenced by the environment. Similarly, BG did not appear to track with age as well as serum TC; data of BG tracking are not available in the literature for comparison. REFERENCES 1. Askevold, R., Hostmark, A. T., Vellar, 0. D., Von Kraemer, B., and Glattre, E. Serum cholesterol and triglyceride levels in Norwegian adolescent school children. Acta. Puediutr. Stand. 67, 157-160 (1978). 2. Barnes, H. V. Symposium on adolescent medicine. Physical growth and development during puberty. Med. Clin. North Amer. 59160, 130.5-1317 (1975). 3. Binner, D. L., and Manning, J. Automated neocuproine glucose method: Critical factors and normal values, in “Automation in Clinical Chemistry,” Technicon Symp. 1, 33-39 (1966). 4. Blumenthal, S., Jesse, M. J., and Hennekens, C. H. Risk factors for coronary artery disease in children of affected families. J. Pedkztr. 87, 1187- 1192 (1975). 5. Clarke, W. R., Schrott, H. G., Leaverton, P. E., Connor, W. E., and Lauer, R. M. Tracking of blood lipids and blood pressures in school age children: The Muscatine Study. Circulation 58, 626-634 (1978). 6. Davis, C. E. The effect of the regression to the mean in epidemiological and clinical studies. Amer. J. Epidemiol.
104, 493-501
(1976).
7. Drash, A. Atherosclerosis, cholesterol and the Pediatrician. J. Pediatr. 80, 693-696 (1978). 8. Egstein, M., and Kreutz, F. H. Eine neue Bestimmung der Neutralfette in Blutserum und Gewebe. Klin. Wochenschr. 44, 267-273 (1966). 9. Epstein, F. H., Block, W. I)., Hand, E. A., and Francis, T., Jr. Familial hypercholesterolemia, xanthomatosis and coronary heart disease. Amer. J. Med. 26, 39-53 (1959). 10. Frerichs, R. R., Srinivasan, S. R., Webber, L. S., and Berenson, G. S. Serum cholesterol and triglyceride levels in 3446 children from a biracial community. Circulation 54, 302-309 (1966). 11. Hennekens, C. H., Jesse, M. J., Klein, B. E., Gourley, J. E., and Blumenthal, S. Cholesterol among children of men with myocardial infarction. Pediatrics 58, 211-217 (1976). 12. Holman, R. L., McGill, H. C., Strong, J. P., and Geer, J. C. The natural history of atherosclerosis: The early aortic lesions as seen in New Orleans in the middle of the 20th century. Amer. J. Puthol. 34, 209-220 (1958). 13. Keys, A. Coronary heart disease in seven countries. Circulation 41, (Suppl. l), 1-211 (1970). 14. Laskarzewski, P., Morrison, J. A., De Groot, I., Kelly, K. A., Mellies, M. J., et al. Lipid and lipoprotein tracking in 108 children over a four-year period. Pediatrics 64, 584-591 (1979). 15. Lauer, R. M., Connor, W. E., Leaverton, P. E., Reiter, M. A., and Clarke, W. R. Coronary heart disease risk factors in school children: The Muscatine Study. J. Pediatr. 86, 697-706 (1975). 16. Lauer, R. M., and Clarke, W. R. Tracking of coronary risk factors in children: The Muscatine Study, in “Atherosclerosis: Proceedings of the 5th International Symposium” (A. M. Gotto, L. C. Smith, and B. Allen, Eds), p. 294. Springer-Verlag, Berlin/New York, 1980. 17. Lee, V. A. Individual trends in the total serum cholesterol of children and adolescents over a 10 year period. Amer. J. Clin. Nutr. 20, 5-11 (1967). 18. Lehman, E. L. “Nonparametrics,” p. 97. Holden-Day Inc. Press, San Francisco, 1975.
390
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19. Long, M., Dunlop, J. R., and Holland, W. W. Blood pressure recording in children. Arch. Dis. Child. 46, 636-640 (1971). 20. Morrison, J. A., Laskarzewski, P. M., Rauh, J. L., Brookman, R., Mellies, M. J., et al. Lipids, lipoproteins, and sexual maturation during adolescence: The Princeton Maturation Study. Metabolism 28, 641-649 (1979). 21. Morrison, J. A., De Groot, I., Edwards, B. K., Kelly, K. A., Rauh, J. L., et al. Plasma cholesterol and triglycerides levels in 6775 school children, ages 6- 17. Metabolism 26, 1199- 1211 (1977). 22. Ness, A. T., Pastewko, J. V., and Peacock, A. C. Evaluation of a recently reported stable Lieberman Burchard reagent and its use for the direct determination of serum total cholesterol. Chin. Chim. Acta 10, 229-233 (1964). 23. Pagnan, A., Antonini Canterin, A., Donadon, V., Menin, A., Targa. S., ef a/. Precursori dell’arteriosclerosi nell’infanzia. Studio di Pordenone: Primi risultati concementi il comportamento “trasversale” e “longitudinale” della pressione arteriosa in un gruppo di bambini ed adolescenti. G. Ital. Cardiol. 9, 472-479 (1979). 24. Pagnan, A., Vincenzi, M., Barbieri, E., Zanetti, G., Guarini, P., and Dal Palh, C. Lipidlipoprotein composition in hypercholesterolemic children and their parents. Atherosclerosis 32, 121-129 (1979). 25. Pagnan, A., Antonini Canterin, A., Donadon, V., Menin, A., Targa, S., et al. Precursori dell’arteriosclerosi nell’infanzia. Studio di Pordenone: Comportamento “longitudinale” (two years follow-up) de1colesterolo e dei trigliceridi in una casistica scolare di bambini ed adolescenti. G. Ital. Cardiol. 10, 821-825 (1980). 26. Pickens, J. M., Burkeholder, J. N., and Womak, W. N. Oral glucose tolerance test in normal children. Diabetes 16, 11-14 (1962). 27. Srinivasan, S. R., Frerichs, R. R., and Berenson, G. S. Serum lipid and lipoprotein profile in school children from a rural community. C/in. Chim. Acta 60, 293-302 (1975). 28. Uppal, S. C., De Haas, .I. H., and Amtzenius, A. C. Westland school children survey. A preliminary report on risk factors for CHD. Hart Bull. 5, 95-102 (1974). 29. World Health Organization. “Study of Atherosclerosis Precursors in Children. Report of a W.H.O. Consultation on Prevention of Adult Cardiovascular Diseases in Childhood.” Geneva, February 4-6, 1974. 30. World Health Organization. “Study of the Precursors of Atherosclerosis. Protocol of a W.H.O. Collaborative Project” (Appendix). Geneva, October 12- 14, 1977. 31. Widhalm, K., Strobl, W., and Westphal, G. Age dependency and tracking of serum lipids and lipoproteins in healthy children aged 11 to 14 years. Atherosclerosis 38, 189-196 (1981). 32. Willmore, J., and McNamara, J. Prevalence of coronary heart disease risk factors in boys 8 to 12 years of age. .I. Pediatr. 84, 527-533 (1974).
MEDICINE
11,
381-390
(1982)
Precursors of Atherosclerosis in Children: The Cittadella Study. Follow-up and Tracking of Total Serum Cholesterol, Triglycerides, and Blood Glucose ANTONIO PAGNAN,*~’ GIOVANNI B. AMBROSIO,* MARIO VINCENZI,? PAOLO MORMINO,* PIETRO MAIOLINO,? LICIA GERIN,* ENRICO BARBIERI,~ FRANCO CAPPELLETTI,~ AND CESARE DAL PALM* *Istituto
di Medicina Clinica, tDivisione Cardiologica,
Clinica Medica II, Universita di Padova, Ospedale Civile Di Cittadella. Cittadella,
Padua, and Italy
As part of a WHO project, precursors of atherosclerosis in children, a survey was made in Cittadella, a town in Northern Italy. Four hundred and eight children (49.7% males) aged 6, 9, 12, and 15 were examined and a blood sample for total cholesterol (TC), triglycerides (TG), and blood glucose (BG) was drawn one hour after an oral glucose load of 50 g. The same procedure was performed in the same subjects 3 years later. The mean values of TC in the age groups 6,9, 12, and 15 were 155.2, 176.1, 191.5, 170.5 mg/dl, respectively. TG values were 54.4, 53.1,55.8,62.2 n&d1 and BG, 108.3,85.2,92.1,97.4 mg/dl, respectively. A marked fall of TC at reexamination was found in initial age groups of 12 and 15. BG levels fell significantly only in those subjects initially examined at age 6, while TG showed a longitudinal increase in all age groups, though more markediy in the younger ones. Different body composition, and possibly a secular trend, might explain these findings. The tracking analysis showed that serum TC tends to maintain its percentile with increasing age in contrast to serum TG and BG tracking which was less consistent, suggesting the influence of factors other than those affecting changes in TC.
INTRODUCTION The hypothesis that atherosclerosis starts in childhood was put forward more than two decades ago by Holman et al. (1958) after their studies on juvenile atherosclerosis (12). The first epidemiological survey dealing with children and families was made in Tecumseh, Michigan in 1959 (9). Since then several studies have been published, mainly in the United States, on the risk factors of atherosclerosis in children (1, 4, 7, 10, 11, 19, 21, 27, 28, 32). Recognizing the importance of this approach to the possible prevention of atherosclerosis, in 1974 the WHO held a meeting in which the plan for a collaborative study (Precursors of Atherosclerosis in Children) was drafted (29). A detailed research protocol was subsequently designed (30). In 1975, according to the WHO protocol, our Institute started a collaborative study with two hospitals in Northern Italy, Pordenone and Cittadella. The general aim of the project was to determine whether future high-risk adults could be identified by measuring the same risk factors during childhood or, the reverse1 To whom reprint requests should be addressed: Antonio Clinica, Clinica Medica II, Via Giustiniani, 2, Padua, Italy.
Pagnan, M.D.,
Istituto
di Medicina
381 0091-7435/82/040381-10$02.00/O Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
PAGNAN
382
ET AL.
TABLE AGEANDSEXDISTRIBUTIONOFTHE
1 SCHOOLPOPULATION
Age (years)
M
F
Total
6* 9 9-12 12 -+ 15 15 --f 18
51 51 50 51
52 51 51 51
103 102 101 102
whether children with high risk-factor levels do indeed develop into high-risk adults. In order to clarify the problem, the longitudinal approach, in which the tracking of risk factors is performed from childhood to early adulthood, was considered the most suitable. We describe herein the longitudinal data (3 years follow-up) on serum lipids (cholesterol and triglycerides) and blood glucose (one hour after 50 g glucose per OS)in a school population of children and adolescents from Cittadella, a town in Northern Italy. The tracking of the same variables is also investigated. MATERIALS
AND METHODS
Following the recommendations of the WHO protocol (30), we examined 444 school children (50% each sex) of four age groups: 6, 9, 12, and 15. All subjects were examined initially and reexamined after 3 years. At the 3-year follow-up, 28 (6.3%) were lost for the following reasons: parents’ refusal (18); emigration (10); and eight for whom a reliable blood sample could not be obtained, were excluded. The longitudinal analysis includes the remaining 408 subjects, 203 males and 205 females (Table 1). Subjects live in the Cittadella district, whose total population is 60,486 (49.1% males). The economy of the district is well diversified: 150 small and medium-size industries, 1,350 commercial factories, 2,228 craftsmen factories, and 2,333 farms. The population is ethnically homogeneous with few immigrants. The cooperation of school authorities (physician, presidents) and school health services was secured at the start of the study. Parents and children were informed of the aim and methods of study and written consent was obtained from the parents. Examination procedure. In the health office in each school,2 3 days a week, a physician and two nurses took a venous blood sample of approximately 10 ml from each child. The blood was taken with the child seated, one hour after ingestion of 50 g of glucose. The blood was divided into two tubes, one of which was taken to the Central Laboratory of Cittadella General Hospital for assay of blood glucose (3) and serum triglycerides (8). The other tube was centrifuged and the serum, stored at -20°C was sent within 7-10 days to the Central Laboratory of Pordenone General Hospital for serum cholesterol determination (22). Quality control for serum TC measurements was overseen by the WHO Regional Reference Center (Institute for Clinical and Experimental Medicine of Praha) which cooperates * In several schools the health offke
was a modified classroom.
PRECURSORS
OF ATHEROSCLEROSIS
IN
CHILDHOOD
383
with the WHO International Lipid Standardization Laboratory at the Centers for Disease Control, Atlanta, Georgia. Further data collected are irrelevant to this paper and will not be discussed. Participation at the initial examination was 100% in all age classes. It was necessary to reach eight children at home, three aged 12 and five aged 15 years, with a staff of two physicians, two nurses, and one secretary. All clinical and laboratory data were collected in the appropriate form. An identical examination was performed 3 years later. The 80th percentile of the distribution was arbitrarily taken as a cutoff point between “normal” and “abnormal” values in the various age classes (6, 9, 12, 15 years) both at the initial and follow-up examination. Statistical methods. A paired t test was used in the comparison of the baseline and rescreening values. The “tracking” pattern, i.e., whether or not the subjects, with increasing age, have been keeping the rank within their age group, was determined by means of (a) correlation analysis (Pearson’s Y) between initial and follow-up values; (b) analysis of the distribution in various quintiles at retest of the subjects in the 5th quintile at initial examination; (c) application of the Van der Waerden scores (18) in order to evaluate any baseline-retest changes in the differences between subjects in the 5th quintile and those in the lower ones (lst-4th). RESULTS
The mean values of TC in the age groups 6,9, 12, 15, were, respectively: 155.2, 176.1, 191.5, 170.5, mg/dl. Those of TG: 54.5, 53.1, 55.8, 62.2 mg/dl and those of BG: 108.3, 85.2, 92.1, and 97.4 mg/dl. No significant differences were found between the two sexes in any age group, which were subsequently analyzed together. In the longitudinal analysis, mean values of serum TC showed a significant increase from age 6 to 9, while a significant decrease was observed from ages 12 to 15 and 15 to 18 (Table 2 and Fig. 1). Mean serum TG levels showed a steady, significant increase from ages 6 to 9, 9 to 12, and 12 to 15 years (Table 3 and Fig. 2). Mean BG levels significantly decreased from age 6 to 9, while a significant increase was observed from ages 12 to 15 and 15 to 18 (Table 4 and Fig. 3). A significant correlation was found between serum TC at initial testing and at retest in all age groups; the same was true for serum TG, except for age group 6 to 9 years (Tables 2 and 3). No significant correlation was found for BG, however, except for the triennium 9 to 12 years (Table 4). The age-specific 80th percentile values of serum TC, TG, and BG have been calculated (Table 5). At retest, the mode of distribution for subjects initially in the 5th quintile remained the same only for serum TC, while serum TG and BG showed a greater dispersion (Table 6). Comparison at retest between the mean values for serum TC of children in the top quintile at initial test and those of all other quintiles showed significant differences in all age groups, except for that of the 6- to 9-year group. For serum TG, the above differences were significant only in the age group 12 to 15 years and for BG only in that of 9 to 12 years (Table 7).
PAGNAN ET AL.
384
TABLE 2 LONGITUDINAL BEHAVIOROF SERUMTOTAL CHOLESTEROL(TC) IN mgldl Age (years)
Mean ” SD
P
155.2
9
186.0
Correlation coefficient* 69
Range
+*
21.7
95-255
22.1
139-255
P < 0.001
9 J 12
176.1 2 32.3
95-260
0.511
182.3 k 25.1
96-252
P < 0.001
12
191.5
f 28.2
135-305
0.530
15
156.8
+ 28.9
80-265
15 J 18
170.5 2 28.6
75-240
0.527
156.6 f 28.4
75-222
P < 0.001
Mean of the change c SD
0.360 +30.8
f
Range of the change
-
55
119
28.4 -49
112
- 148
40
-119
58
+ 6.2 k 28.9
i P < 0.001
-3.41
t 27.8
- 13.9 f 27.6
* r between initial and retest values.
DISCUSSION
We found no sex differences in the mean values of serum TC, TG, and BG, thus confirming the results of several studies (1,4,7, 15, 23). Other studies, however, have found higher levels in girls than in age-matched boys (10, 21). The similarity TABLE 3 LONGITUDINAL BEHAVIOROF SERUMTRIGLYCERIDES(TG)IN mg/dl Correlation coefficient* (r)
Age (years)
Mean 2 SD
6 J 9
54.4 -c 12.7
36
100
0.055
66.1 2 27.9
28
184
NS
9 L 12
53.1 2 13.8
36
100
0.199
64.9 2 23.6
35
230
12 L 15
55.8 k 16.4
43
140
0.279
63.2 “_ 24.3
23
125
P < 0.01
15 1 18
62.2 r 13.9
43
100
0.266
62.6 h 18.9
25
163
Range
* r between initial and retest values.
P < 0.05
P < 0.01
Mean of the change f SD
Range of the change -43
141
-43
173
-48
75
-54
92
-21.1 f 32.4
+11.8 k 24.8
f 1.4 f 25.2
+ 0.4 k 20.2
PRECURSORS OF ATHEROSCLEROSIS IN CHILDHOOD 200
385
serumTC Pg A)
/ 170
160 160
\
\
I
150 140 190 !
-II
9-
6-9
-I
I
12
I
12-15
I
---(,Zs,
FIG. 1. Longitudinal behavior of serum total cholesterol (TC).
between girls and boys could be related to the fact that sexual development progresses during childhood and adolescence. Serum lipid values in our population are similar to those reported in several studies of young population samples in different ethnic, geographic, and environmental settings (1, 4, 5, 7, 10, 15, 20, 23). This observation, in contrast with the well-known differences observed for serum lipid levels in adult populations (13) from different geographic areas, stresses the importance of environmental factors in the modulation of serum lipid levels. Looking at the longitudinal behavior of serum TC, we observe a marked, significant fall from age 12 to I5 and, although less consistent, from age 15 to 18, around puberty and adolescence, confirming the results of many previous crosssectional and longitudinal studies (10, 11, 15, 17, 20, 21, 24). Such behavior draws attention to marked hormonal and growth changes as modulating factors of serum
,
6-9
,
,
9 -
12
,
12-15
,
,
15-
,
18&:s,
FIG. 2. Longitudinal behavior of serum triglycerides (TG).
386
PAGNAN
ET AL.
FIG. 3. Longitudinal behavior of blood glucose (BG), one hour after 50 g glucose per OS.
TC levels. In particular, besides sex hormones, especially testosterone (20), the spectacular TC fall might be related to the thyroid gland, the weight of which during puberty almost doubles (2). The longitudinal behavior of serum TG levels showed a steady increase at the rescreening, 3 years later. We have observed a similar trend in children in a different area in Northern Italy (25). Whether these changes are due to a secular trend or an age-related trend is unclear. The fact that the mean TG values in children aged 6, 9, and 15 years at initial testing were all similar as were their TABLE 4 LONGITUDINAL BEHAVIOR OF BLOOD GLUCOSE(BG), 1 HR AFTER50 g GLUCOSEPEROS, IN mg/dl Age (years)
Mean k SD
Range
Correlation coefficient* (4
108.3 f 30.2
60
218
0.179
85.2 f 18.7
58
130
NS
85.2 ? 20.2
44
144
0.252
12
96.1 k 21.9
41
175
P < 0.05
12 J 15
92.1 k 23.2
46
163
0.143
93.9 ” 24.0
50
170
NS
15 1 18
97.4 k 24.2
55
180
0.139
93.3 5 28.7
46
280
NS
9
* r between initial and retest values.
Mean of the change k SD
Range of the change -113
43
-53
111
- 88
90
- 75
190
-23.1 + 32.4
+ 10.9 + 25.7
+ 1.8230.7
- 4.1 k 34.7
PRECURSORS
OF ATHEROSCLEROSIS
IN
387
CHJLDHOOD
TABLE 5 80~~ PERCENTILE, AGE-SPECIFIC VALUES FOR SERUM TC, TG, AND BG Serum TC
Follow-up
BG (mddl)
(m&W
Serum TG (mg/dl)
175.0
71.0
127.0
200.0
80.0
101.0
203.0
57.0
100.0
12
197.0
80.0
111.0
12
210.0
71.0
110.0
1:
175.0
86.0
112.0
15
195.0
71.0
114.0
18
182.0
71.0
107.0
(years)
9
means three years later might suggest a secular trend. However, Widhalm et al. (31) reported a “significant age dependency for the mean TG values” of ll-yearold children followed for 4 years, in keeping with the change we observed between ages 12 and 15. Interestingly, for BG (after load) the highest mean values were observed in the youngest subjects, aged 6, while 3 years later it showed a significant fall. Comparison of our data with those published by other investigators (26) is difficult for technical reasons, such as different glucose load and different methods of determining BG. The interpretation of this finding is obscure, though it might be suggested that the highest glucose values observed at age 6 as compared with age 9 are related to differences in body composition, i.e., the ratio of muscle mass to adipose tissue. The tracking analysis showed that TC measurement tracks reasonably with age, confirming other recent study results (16). Such behavior, also reported by Lee et
TABLE 6 QUINTILE DISTRIBUTION, AT RETEST (3 YEARS LATER), OF THE SUBJECTS IN THE TOP QUINTILE AT INITIAL TEST Years (quintiles) 5” 4” 3” 2” 1”
TC
6+9 TG
BG
TC
7 4 6 1 3
5 4 4 2 6
3 4 8 3 2
7 6 6 1 0
9+ 12 TG BG 5 4 7 3 1
8 3 2 4 3
TC 11 5 1 3 0
12 --) 15 TG BG 6 4 3 5 2
6 1 5 4 4
TC 10 6 3 0 1
15 + 18 TG BG 3 3 3 5 6
3 8 4 2 2
TABLE I
18
179.9 + 24.7
151.0 ? 26.4
160.7 -t 21.9
210.7 k 13.1
15
L
150.1 2 23.9
181.1 ? 17.3
233.7 f 23.2
184.1 5 32.0
178.2 t 24.5
199.3 + 20.1
165.6 f 25.9
219.1 2 15.4
15
i
12
12
L
9
9
183.9 5 21.5
6
193.9 e 22.9
145.0 f 18.5
195.3 2 19.3
Follow-up
L
Subjects in the lst-4th quintile at intake
Subjects in the 5th quintile at intake
Serum cholesterol (mgldl)
to.001
to.001
NS
P
66.5 2 18.0
81.2 2 11.4
73.7 + 27.1
81.2 f 16.0
68.8 5 16.6
74.9 2 13.6
65.6 _’ 25.9
15.2 + 8.1
Subjects in the 5th quintile at intake
61.6 2 19.0
57.5 + 9.9
60.7 _t 23.1
49.5 + 8.5
63.9 k 25.0
47.8 k 6.9
66.3 k 28.5
49.1 zk 6.8
Subjects in the lst-4th quinrile at intake P
NS
co.05
NS
NS
Serum triglycerides (mg/dI)
99.8 k 22.0
134.4 2 19.4
97.7 ‘- 29.0
126.6 t- 17.1
106.3 e 24.3
115.0 + 13.6
88.3 -t 17.5
153.5 e 19.7
Subjects in the 5th quintile at intake
91.7 f 30.0
88.4 -e 14.6
93.0 k 22.7
83.6 -c 15.1
93.6 2 20.7
78.0 + 13.8
84.5 2 19.0
96.8 ? 19.5
Subjects in the lst-4th quintile at intake
Blood glucose (mgldl)
NS
NS
<0.05
co.oo1
NS
P
COMPARISON AT RETEST (3 YEARS LATER) BETWEEN THE MEAN VALUES (?SD) FOR SERUM TC, TG, AND BG OF CHILDREN IN THE TOP QUINTILE AT INITIAL TEST (3 YEARS BEFORE) AND THOSE OF ALL OTHERS (1s~ TO ~TH QUINTILE)
PRECURSORS
OF ATHEROSCLEROSIS
IN
CHILDHOOD
389
al. (17) and Laskarzewski et al. (14), confirms the possibility of detecting high-risk
children at an early age, and thus of laying the foundations for intervention trials at that time (7). Yet, the fact that not all the children and adolescents maintain values in the highest quintile 3 years later, clearly indicates that a single TC determination is not sufficient for an absolute diagnosis of hypercholesterolemia (25). The dispersion of the TC values at retest might be partly related to regression toward the mean (6, 29, although quintile grouping minimizes this effect. The degree of tracking for serum TG was less consistent than that of TC, since the dispersion throughout the lower quintiles was much more marked for TG than for TC. This finding, which confirms previous reports (14), suggests that serum TG might be largely influenced by the environment. Similarly, BG did not appear to track with age as well as serum TC; data of BG tracking are not available in the literature for comparison. REFERENCES 1. Askevold, R., Hostmark, A. T., Vellar, 0. D., Von Kraemer, B., and Glattre, E. Serum cholesterol and triglyceride levels in Norwegian adolescent school children. Acta. Puediutr. Stand. 67, 157-160 (1978). 2. Barnes, H. V. Symposium on adolescent medicine. Physical growth and development during puberty. Med. Clin. North Amer. 59160, 130.5-1317 (1975). 3. Binner, D. L., and Manning, J. Automated neocuproine glucose method: Critical factors and normal values, in “Automation in Clinical Chemistry,” Technicon Symp. 1, 33-39 (1966). 4. Blumenthal, S., Jesse, M. J., and Hennekens, C. H. Risk factors for coronary artery disease in children of affected families. J. Pedkztr. 87, 1187- 1192 (1975). 5. Clarke, W. R., Schrott, H. G., Leaverton, P. E., Connor, W. E., and Lauer, R. M. Tracking of blood lipids and blood pressures in school age children: The Muscatine Study. Circulation 58, 626-634 (1978). 6. Davis, C. E. The effect of the regression to the mean in epidemiological and clinical studies. Amer. J. Epidemiol.
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