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Blood lipids in Italy
126
Atherosclerosis, 31 (1978) 125-136 0 Elsevier/North-Holland Scientific
Publishers,
Ltd.
BLOOD LIPIDS IN ITALY * Regional Differences MENOTTI 3 GIORGIO RICCI l, INES MASI 2,** ALESSANDRO GIANCARLO URBINATI t, MARIO’MANCINI 4, EDUARDO FARINARO 4, PASQUALE ORIENTE ‘, LOREDANA POSTIGLIONE 4, SERGIO LENZI 6, GIANCARLO DESCOVICH 6, CARLA CEREDI 6, SERGIO FOREST1 6 and PAOLO MELIOTA 6
’ Istituto di Terapia Medica Sistematica dell’ Universita, Sanita, Rome, 3 Centro per le Malattie Cardiovascolari, l Istituto di Semeiotica Medica, II Facolta di Medicina, Istituto di Clinica Medico Generale e Terapia Medica, Napoli, Naples, 6 Istituto di Clinica Medica Generale e Bologna, Bologna (Italy)
Rome, * Istituto Superiore di Ospednle S. Camille, Rome, Universita di Napoli, Naples, II Facolta di Medicina, Universitci di Terapia Medica II dell’ Universita di
(Received 20 February, 1978) (Revised, received 2 June, 1978) (Accepted 6 June, 1978)
Summary Serum cholesterol and triglyceride levels have been evaluated in samples from fasting males aged 20-59 in Northern (Brisighella), Central (Rome) and Southern (Pozzuoli) Italy. Regularly performed qualit.y controls between laboratories assured comparability of data. A statistically significant difference of mean serum cholesterol and triglyceride levels was observed for most age-groups in the 3 different areas, lower values being found in the southern population as compared to the central and northern ones. These results support previous findings and the thesis that large differences in blood lipid levels may still exist even within the same country and that they at least in part may be culturally determined in connection with different dietary habits. Key words:
Dietary
habits -
Epidemiology
-Serum
cholesterol
-Serum
triglycerides
* The work in Rome has been done within the Roman Project of Coronary Heart Disease Prevention. conducted in cooperation with the Institutions to which the authors belong and the Istituto Italian0 di Medicina Sociale, Rome. ** Deceased.
Introduction Differences in levels of serum lipids and serum cholesterol in particular, between population samples or groups from the same or different countries, have attracted much attention for several decades. This is due to the large amount of evidence supporting the positive correlation between dietary habits and mean levels of serum cholesterol on the one hand and mean levels of serum cholesterol and incidence of coronary heart disease (CHD) on the other. Data collected in Italy in the sixties or before, were scanty and only seldom internally comparable. They substantially belong to either the “Seven Countries Study” or to the “WHO European Study on Prevalence of CHD”. Regional differences in serum lipids were suspected on the basis of supposedly known dietary habits, and partly confirmed. Exploratory population surveys, conducted on a small scale, showed a gradient in serum cholesterol levels in different social classes in the city of Naples and also geographically from Northern to Southern Italy [l-3]. Within the “Seven Countries Study” [4] median serum cholesterol was about 200 mg/dl in rural men from Northern and Central Italy, about 175 mg/ dl in men from Southern Italy, and about 206 mg/dl in an industrial sample from Rome and surroundings (Central Italy). All such samples included only men aged 40-59. The differences between the rural samples of Northern and Central Italy and that of Southern ltaly were accounted for by findings from the dietary surveys [ 51. The WHO study [6] showed that clerks in Milan and Naples were rather similar in terms of average serum cholesterol, but the laboratory methods were definitely not comparable. A substantial spread of interest in such problems became evident only in the early seventies when several study groups independently started field operations intended to assess the distribution of several coronary risk factors in population samples or groups. The Rome Group, represented in this article, suggested in discussions held within the “Gruppo di Studio per la Lotta alle Malattie Dismetaboliche e all’ Arteriosclerosi” that the possibility of comparing some data from the respective studies be considered since all studies included an initial screening. The idea to set up a kind of rather wide “lipid-map” of Italy had to be initially excluded but 3 areas corresponding to population samples or groups belonging to Northern, Central and Southern Italy respectively, became finally eligible for a pooling attempt. This article describes the findings concerning serum cholesterol and triglycerides. Material The 3 studies considered for comparison were as follows: (1) The Study of Brisighella (BR). This is a longitudinal population study on atherosclerosis and its complications conducted on the population of a small town of Emilia-Romagna, Northern Italy. It has a total population of about 9,500 and a typical agricultural background. Data reported here concern examination offered at entry to the males resi-
127
dent in the inner part of the town, while data on females and younger or older people have been excluded for this purpose. Only men aged 20-59 have been considered for this article. The cohort included 863 men; 739 (85%) were examined and all of them were fully available for analysis. (2) The Rome Project of Coronary Heart Disease Prevention (RO). This is the Italian Section of the “WHO European Multifactor Preventive Trial of CHD” [7 1. The aims and the structure of the study have been presented elsewhere. Here it, should be recalled only that the study is directed toward occupational groups (“factories”) allocated to either control or treatment; that entry screening examination was offered to all the subjects belonging to the treatment “factories” and to subsamples of the control “factories”. Only men aged 40--59 had been enrolled making a total of 6,027 individuals, residents of Rome, Central Italy. For the purposes of this analysis all “factories” have been pooled together. The number of men invited for screening was 3,837; those examined were 3,233 (85%); and almost all were fully eligible for the analysis. Out of the examined men about 2/3 were clerks; the remainder being engaged - on average - in moderate technical or manual work, with a negligible proportion of individuals engaged in heavy work. (3) The Olivetti Survey of Pozzuoli (PO). This was a cross-sectional study of coronary risk factors in an occupational population group in Pozzuoli, province of Naples, Southern Italy. The men were mostly engaged in light electro-mechanical work. Only men aged 20-59 have been considered for this analysis. Of the tota! number (1,900), 1,631 were examined (86%); and almost all became fully suitable for analysis, Methods The invited subjects attended the screening on work-days in the morning after a 12- to 14-h fast. Blood samples, taken from the antecubital vein without stasis, were centrifuged within 4 h and serum was kept in the refrigerator at 4°C. Chemical analysis was usually performed within the following two days. In the Bologna and Naples laboratories the same methods and apparatus were used for cholesterol and triglyceride analysis: namely the Levine and Zak extractive method for cholesterol [8] and the Kessler and Lederer method for triglycerides [9]. Both methods were adapted to the automated analyzer CLA 1510, Carlo Erba Strumentazione [lo]. In the Rome laboratory, the manual Abell-Kendall method for cholesterol [ll] and the Eggstein enzymatic method for triglycerides [12] were performed. The 3 centers participated in the lipid quality control program with the WHO Regional Lipid Reference Center of the Institute of. Clinical and Experimental Medicine, Prague. The comparability of methodologies was checked, after the screening, by periodical analysis of split samples forwarded frozen and blind from Naples to the other laboratories. Examples of the standardization procedures are given in Figs. 1 and 2. Differences between laboratories were considered to be of the same order of magnitude as the variability usually found within individual laboratories.
12x
LIPID
QUALITY
CONTROL
: SERUM
CHOLESTEROL
JUNE 1975
SEPTEMBER ,975
n=15
x = 179 y = 183
r =0.982 y=
--1-----T------rtc--T------
100
100
..
P E”
1 /
,1.468+0.956x
200
300
mg/dl
NPlPLES
.a
;
200
,"
.
n = 15
;/I/
x=
+
; = 203
i
r = 0.995 100.
y = *.71g+,.o35x
??
100
___I__
200
y= n= 15
.
x=
/
v/d1
189
I/
y=-12
=
J
1
JJ r iz
i
1
‘T
y=,oo
r = 0.995
r = 0.085
y = 0.117 +1.070x
2;o
lb0
IOO-
between
y=
I
mddl
100
BOLOGNA
control
n = 15
E=133
t = 203
j///
1. Quality
I
P =100
.
Fig.
200
n =15
.
. 1_1 i’
Y
200
/
rng/dl
/
2 300
E
100
300
NAPLES
P
:
254+,.124x
200
NAPLES
;
170
r = 0.997
I_T-.--T~~.+5-100
I
100
/
_;
187
200 BOLOGNA
Centers
for serum
cholesterol.
-10.423+1.140x
I
3
300 mgpl
LIPID JUNE
QUALITY
CONTROL : TRIGLYCERIDES
1975
SEPTEMBER
1975
T----
300
200
m&l
----260100
300
1 1
*
ilil ??
LL
:
/
.*
-I
/.
3004
4
ddl
/
r
100
300 NAPLES
?I
P
mehl
NAPLES
NAPLES
2
300
200
NAPLES
??
200
P i
n= 15
z=
99
5=
100
1004
r = 0.994 y=
1
-14.678+,.161x
/
----
100
msPl BOLOGNA
Fit?. 2. Quality control
between
y=
,’
-8.106+,.0*3x
li/
100
200 BOLOGNA
Centers
for serum triglycerides.
300
msbl
P
Br-PO
2x
mmoles/l
0.89
3.96
34.17
153.00
_
0.96
4.76
37.00
184.00
311
0.87
0.85
13
5.36
5.08
32.67
32.96
86
0.95
4.93
36.66
190.30
462
1.18
5.44
45.70
210.06
71
SERUM
3om-34
OF
2 5-29
206.96
(yr)
(SD)
195.96
49
20-24
Age
DEVIATION
__~.___.
SD
’ SD
mgldl
n
rx
‘“SD
(PO)
mmolesil
1’“sD
n
STANDARD
mg/dl
AND
Pozzuoli
(Br)
Brisighella
(A)
1
(f)
TABLE
MEAN
267
1.06
4.95
41.01
191.23
0.98
4.84
37.88
187.09
1,053
1.06
5.50
5.78 1.09
40.94
212.17
310
20-39
42.23
223.35
104
35-39
(mg/dl
~~
corrected
Age-
CHOLESTEROL
mmoles/l)
’
n
n.s.
Ro-PO
1.09
5.07
Br-Po
SD
1.r
Br-Ro
.__~____~~~~.~ ~~~. .._
P
mmoles/l
42.12
195.84
4
’SD
w-0)
1.08
SD 263
5.54
(0.001
1.17
5.15
45.16
198.86
189
1.08
5.66
41.70
218.34
214.01 41.84
868
860
1.21
1.01
S
,.’ SD
6.15
5.66
237.31
218.73 46.65
132
114 38.88
45--49
(yr)
MIDDLE-AGED
4044
Age
AND
n
mgldl
n
(A)
x ‘. SD
YOUNG
mmoles/l
mgjdl
mg/dl
IN
Pozzuoli
R0ma @o)
(Br)
Brisighella
(B)
and
<0.001
ro.001
n.s.
0.97
5.26
37.44
203.08
91
1.20
5.75
46.33
Age-
1.09
CO.001 ,: O.fJ
1.08
5.16
41.90
199.36
570
.5.65
42.18
218.02
3.216
1.06
5.90
41.09
227.91
429
40-59
corrected
n.s. 1
1.10
5.19
42.41
200.56
27
1.11
5.65
42.89
574 218.29
221.97
1.05
5.84
914
0.97
5.95
40.53
37.34
79
55-59
225.44
MEN
229.58
104
50-54
(B)
Levels of serum cholesterol and triglycerides have been expressed both in mg/dl and mmoles/l in order to follow the recently recommended rules for units of measurement. Data have been locally processed on the basis of standard and pre-agreed computer programs, and then pooled together and compared at the Rome Center. Since the Rome population group did not include men aged less than 40, two different and independent comparisons have been set up: that concerning men aged 20~-39 (young) and including only BR and PO; and another concerning men aged 40.---59 including also RO. In both cases yuinquennial subgroups and whole (young plus middle-aged) groups have been considered for analysis. For comparison of whole groups (i.e. young = age 20-39; or middle-aged = age 40-59) the means have been corrected by direct standardization, taking as the reference population the quinquennial age distribution of the pool of all groups. The age distribution of the men examined is given in each table. Results Mean levels of serum cholesterol are given in Table 1. They are significantly different (t-test) in 17 out of 20 comparisons involving two populations of young men, and three populations of middle-aged men and a total of 8 age subgroups and 2 age-pools. All the 4 comparisons made on the corrected means have a chance explanation of less than 0.001. The trend is typical - for Italy going downward from North, to Center, to South. The histograms concerning the distributions of data, broken down into arbitrary classes of 20 mg/dl are, as expected, slightly positively skewed and suggest that the modal class tend to present lower and lower levels going from BR to RO to PO (Fig. 3). Such distributions, as the next for triglycerides, are limited as examples, to a single quinquennial age class owing to the non-comparability if data on all ages were presented. Data on triglycerides are given in Table 2 and Fig. 4. Again the highest mean level belongs to BR, the lowest to PO, RO being in between. Such differences are significant, (t-test) in 13 out of 20 possible comparisons. The trend is particularly evident when examining the corrected mean levels. The distribution of triglycerides is even more skewed to the right than that of serum cholesterol and again the modal class is different in the 3 populations, where referred to a sub-class of the middle-aged men. But in this particular case the median classes in BR and PO are similar, while that in RO has a lower value. The overall results, in terms of inter-group comparisons, are somewhat different, a larger number of comparisons being statistically significant. Looking back to the means it appears that the age trend for serum cholesterol - as shown by quinquennial groups - is rather typical in all the 3 cohorts showing a clear increase from age 20-24 to age 50--54 and then a steady-state or a slight decline. Serum triglycerides on the other hand tend to decline after the age-group 40-44 in PO, to be steady in RO, whereas they show a curious peak at the age 50--54 in BR.
132
SERUM
CHOLESTEROL
n = 132
BRISIGHELLA
/ i I
l-1
i
I
I I
‘1 i
1
n =
ROME
868
-
I=POZZUOLl
1
"Cl89
9
3
: 140
Fig. pies.
3. Frquency
distribution
lb0
of
serum
cholesterol
values
in men
aged
50-54.
in the
3 PoPulation
s%‘n-
133
SERUM BRISIGHELLA
n :
I
_
ROME
TRIGLYCERIDE5
132
r
1 i
i, = 868
400
Fig. PIES.
4.
Frequency
distribution
of
serum
triglyceride
values
in men
aged
50-54,
in the
440
3 population
mg/dl
s%n-
P
&-PO
e-0 )
Pozzucli
(Br)
Brisighda
(A)
MEAN
TABLE2
(f)AND
‘SD x
n
JX
mmoles/l
w!/dl
SD
Iy
'SD _
jsI
n
mmoles/l { SD
wc/dl
(yr)
~.O.OOl
~.O.OOl
448 132.31 67.36 1.50 0.76
303 119.02 67.92 1.34 0.77
9 94.78 34.37 1.07 0.39
n.s.
86 163.92 101.19 1.85 1.14
30-34
71 153.51 80.43 1.73 0.91
25-29
128.86 62.36 1.46 0.10
49
20-24
Age
310 159.53 92.21 1.80 1.04
:0.001
P
Br-Ro Br-PO Ro-PO
(PO)
Pozzuoli
(Ro)
ROITlll
(Br)
Brisighella
(B)
SD
mm&s/1
mgldl
;" SD
SD
n
SD
SD
_ :x
mmoles/1
x
n
SD
x
:
;
n x
mg/dl
mmo1esp
m&U
(m~/dlandmmoles/l)INYOUNG
Agecorrected 20-39
267 1,027 137.95 128.53 75.60 68.86 1.56 1.45 0.85 0.78
104 164.08 93.28 1.85 1.05
35-39
STANDARDDEVIATION(SD)OFSERUMTRIGLYCERIDES
(yr)
<0.05 0.01 n.s.
260 142.25 69.68 1.61 0.79
860 149.19 92.63 1.68 1.05
114 169.96 95.30 1.92 1.08
40-44
Age
D.S. '0.01 ,-0.01
185 135.91 70.83 1.54 0.80
868 160.43 103.15 1.81 1.16
132 160.74 85.10 1.82 0.96
45-49
(A)ANDMIDDLE-AGED
0.01 0.01 n.s.
90 136.94 81.81 1.55 0.92
914 153.67 96.42 1.74 1.09
429 169.43 107.68 1.91 1.22
Agecorrected 40-59
0.001 0.001 0.001
ll.S.
562 136.58 79.78 1.54 0.90
ll.s. n.s.
21 126.94 105.09 1.43 1.19
573 3,215 157.82 154.05 142.89 105.99 1.74 1.78 1.20 1.61
79 160.80 110.60 1.82 1.25
55-59
-~~-~ -
104 183.43 137.43 2.07 1.55
-50-54
-.
(B)MEN
Comment The indications given by these data suggest that in Italy a rather wide range of mean levels of the main serum lipids is still common, on the basis of a geographical and generally speaking “cultural” background. In these particular groups a kind of self-selection due to engagement at work might have some influence on men in RO and mainly in PO (the first being civil servants or employees in public utility services; the latter being employees in private industry). In any case the geographical trend is rather impressive and in line with previous studies. Roughly, such a trend is similar to those of age-corrected coronary death rates observed in the Italian regions [ 131. The mean age-corrected level of serum cholesterol for middle-aged men in BR is about 28 mg/dl higher than that observed in Crevalcore, another rural area of the same region (Emilia-Romagna) located about 100 km distant, studied in 1960 within the “Seven Countries Study” [4]. But it is also known that in this area (Crevalcore) men reaching the same age 10 years later (in 1970) had a serum cholesterol of about 20-25 mg/dl higher than those belonging to the previous generations [14]. This suggests not only that the mean serum cholesterol of middle-aged men has probably increased with time in Italy, but also that some of the previous regional differences have been maintained. While the comparison of data collected in 1960 and 1970 in Crevalcore is guaranteed by the use of the same method in a laboratory standardized with the Atlanta Reference Center, that between the 1960 data in Crevalcore and the 1975 data in BR is supposedly acceptable since the BR laboratory is indirectly standardized with the same Center through the WHO Prague Lipid Reference Center. If - as may logically be assumed - such changes are at least partly attributable to a changing pattern in dietary habits, this should have occurred in a rather parallel way in the different latitudes of the country. We were aware that the extrapolation from 3 non-representative samples to whole populations of the respective region is not entirely justified, but once again, at least by chance the traditional geographical trend has been found. Unfortunately there is no basis for a suitable comparison between the 3 groups in terms of dietary habits. In fact while in BR a rather complete dietary survey has been made on all subjects, a simpler survey has been conducted only in sub-samples of the RO groups, and no dietary study has been planned in PO. However, a very rough comparison can probably be attempted assuming that some other data collected in Naples on other groups can be extended to the PO population. The total estimated kcalories per day was 3,795 in BR and 2,700 in RO; the total fats (as % of total kcalories) was 34 in both areas; the saturated fatty acids (as % of total kcalories) were 14 in BR and 10 in RO. No data are available for PO, where presumably the diet may be similar to that of the other areas of Southern Italy, i.e. relatively poor in total kcalories, total fats and saturated fatty acids. This picture, if true, would justify the difference found in terms of serum lipids, and serum cholesterol in particular. It is clear that more surveys are still needed in order to have a kind of “lipid-
136
map” of Italy, also considering the likelihood of a net time trend. The studies pooled here, on the other hand, were not designed for this purpose, and have, only by a lucky coincidence, made a small contribution to this problem. References 1 Keys, A., Fidanza, F., Scardi. V., Bergami, G., Keys. H.M. and Di Lorenzo, F.. Studies on serum cholesterol and other characteristics on clinically healthy men in Naples, Arch. Intern. Med., 93 (1954) 328. 2 Keys, A., Fidanza. F. and Keys. H.M., Further studies on serum cholesterol of clinically healthy men in Italy, Voeding. 16 (1955) 492. B., Fidanza, F., Keys, 3 Keys, A., Anderson, J.T., Aresu, M., Biork, G., Brock, J.F., Bronte-Stewart, H.M., Malmros, H., Poppi, A., Posteli, T., Swahn, B. and Del Vecchio, A., Physical activity and the diet in populations differing in serum cholesterol, J. Clin. Invest., 35 (1956) 1173. 4 Keys, A., Aravanis, C., Blackburn, H.W., Van Buchem. F.S.P., Buzina, R., Djordievi& B.S., Dontas, A.S., Fidanza. F., Karvonen, M.J., Kimura, N., Lekos, D., Monti, M., Puddu, V. and Taylor, H.L.,
5
6
‘7 8
Epidemiological studies related to coronary heart disease -Characteristics of men aged 40--59 in seven countries, Acta Med. Stand., 460, Suppl. 180 (1967) 1. Fidanza, F., Fidanza-Alberti, A., Ferro-Luzzi, G. and Proja, M., Dietary surveys in connection with the epidemiology of heart disease: results in Italy. In: C. Den Hartog, R. Buzina. F. Fidanza, A. Keys and P. Roine (Eds.). Dietary Studies and Epidemiology of Heart Disease, Stichting tot Wetenschappelijke Voorlichting op Voedingsgebied, The Hague, 1968. Rose, G.A., Ahmeteli. M., Checcacci, L., Fidanza, F.. Glazunov, I., De Haas, J., Horstman. P., Kornitzer. M.D.. Meloni, C.. Menotti, A., Van der Sande. D., De Soto-Hartgrink, M.K., Pi&, Z. and Thomsen, B., Ischaemic heart disease in middle-aged men - Prevalence comparison in Europe, Bull. Wld Hlth Org.. 38 (1968) 885. WHO European Collaborative Group: An international controlled trial in the multifactorial prevention of coronary heart disease. Int. J. Epidemiol., 3 (1974) 219. Levine, J.B. and Zak. B., Automated determination of serum total cholesterol, Clin. Chim. Acta, 10
(1964) 381. 9 Kessler. G. and Lederer, H., Fluorimetric measurement of triglycerides. In: Automation in Analytical Chemistry, Medical Inc., New York, NY, 1966, PP. 314-344. 10 Oriente, P., Farinaro. E., Marotta, G., Mastranzo, P. and Di Marina. I,., Determinazione simultanea di colesterolo e trigliceridi de1 siero con l’analizzatore automatico CLA 1510. In: Atti 3O Congresso Nazionale di Biochimica Clinlca, Bari. 1974. 11 Abell, L.L., Levy, G.B., Brodie. B.B. and Kendall, F.E., A simplified method for the estimation of total cholesterol in serum and demonstration of its specificity, J. Biol. Chem., 195 (19521 357. 12 Eggstein, M. and Kreutz, K.H., Eine new Bestimmung der Neutralfette im Blutserum und Gewebr. I. Mitteilung (Prinzip. Durchfilhrung und Besprechung der Methode), Klin. Wschr., 44 (1966) 262. 13 Puddu, V. and Menotti, A., Ischemic heart disease and cerebrovascular accident mortality trends in the Italian regions - Their relationship to some socio-economic indexes, Acta Cardiol. (Brux.1. 21 14
(1966) 655. Fidanza, F. and Puddu,
V., Final Report,
Grant no. HE-O4672-USPHS
(19701.
Atherosclerosis, 31 (1978) 125-136 0 Elsevier/North-Holland Scientific
Publishers,
Ltd.
BLOOD LIPIDS IN ITALY * Regional Differences MENOTTI 3 GIORGIO RICCI l, INES MASI 2,** ALESSANDRO GIANCARLO URBINATI t, MARIO’MANCINI 4, EDUARDO FARINARO 4, PASQUALE ORIENTE ‘, LOREDANA POSTIGLIONE 4, SERGIO LENZI 6, GIANCARLO DESCOVICH 6, CARLA CEREDI 6, SERGIO FOREST1 6 and PAOLO MELIOTA 6
’ Istituto di Terapia Medica Sistematica dell’ Universita, Sanita, Rome, 3 Centro per le Malattie Cardiovascolari, l Istituto di Semeiotica Medica, II Facolta di Medicina, Istituto di Clinica Medico Generale e Terapia Medica, Napoli, Naples, 6 Istituto di Clinica Medica Generale e Bologna, Bologna (Italy)
Rome, * Istituto Superiore di Ospednle S. Camille, Rome, Universita di Napoli, Naples, II Facolta di Medicina, Universitci di Terapia Medica II dell’ Universita di
(Received 20 February, 1978) (Revised, received 2 June, 1978) (Accepted 6 June, 1978)
Summary Serum cholesterol and triglyceride levels have been evaluated in samples from fasting males aged 20-59 in Northern (Brisighella), Central (Rome) and Southern (Pozzuoli) Italy. Regularly performed qualit.y controls between laboratories assured comparability of data. A statistically significant difference of mean serum cholesterol and triglyceride levels was observed for most age-groups in the 3 different areas, lower values being found in the southern population as compared to the central and northern ones. These results support previous findings and the thesis that large differences in blood lipid levels may still exist even within the same country and that they at least in part may be culturally determined in connection with different dietary habits. Key words:
Dietary
habits -
Epidemiology
-Serum
cholesterol
-Serum
triglycerides
* The work in Rome has been done within the Roman Project of Coronary Heart Disease Prevention. conducted in cooperation with the Institutions to which the authors belong and the Istituto Italian0 di Medicina Sociale, Rome. ** Deceased.
Introduction Differences in levels of serum lipids and serum cholesterol in particular, between population samples or groups from the same or different countries, have attracted much attention for several decades. This is due to the large amount of evidence supporting the positive correlation between dietary habits and mean levels of serum cholesterol on the one hand and mean levels of serum cholesterol and incidence of coronary heart disease (CHD) on the other. Data collected in Italy in the sixties or before, were scanty and only seldom internally comparable. They substantially belong to either the “Seven Countries Study” or to the “WHO European Study on Prevalence of CHD”. Regional differences in serum lipids were suspected on the basis of supposedly known dietary habits, and partly confirmed. Exploratory population surveys, conducted on a small scale, showed a gradient in serum cholesterol levels in different social classes in the city of Naples and also geographically from Northern to Southern Italy [l-3]. Within the “Seven Countries Study” [4] median serum cholesterol was about 200 mg/dl in rural men from Northern and Central Italy, about 175 mg/ dl in men from Southern Italy, and about 206 mg/dl in an industrial sample from Rome and surroundings (Central Italy). All such samples included only men aged 40-59. The differences between the rural samples of Northern and Central Italy and that of Southern ltaly were accounted for by findings from the dietary surveys [ 51. The WHO study [6] showed that clerks in Milan and Naples were rather similar in terms of average serum cholesterol, but the laboratory methods were definitely not comparable. A substantial spread of interest in such problems became evident only in the early seventies when several study groups independently started field operations intended to assess the distribution of several coronary risk factors in population samples or groups. The Rome Group, represented in this article, suggested in discussions held within the “Gruppo di Studio per la Lotta alle Malattie Dismetaboliche e all’ Arteriosclerosi” that the possibility of comparing some data from the respective studies be considered since all studies included an initial screening. The idea to set up a kind of rather wide “lipid-map” of Italy had to be initially excluded but 3 areas corresponding to population samples or groups belonging to Northern, Central and Southern Italy respectively, became finally eligible for a pooling attempt. This article describes the findings concerning serum cholesterol and triglycerides. Material The 3 studies considered for comparison were as follows: (1) The Study of Brisighella (BR). This is a longitudinal population study on atherosclerosis and its complications conducted on the population of a small town of Emilia-Romagna, Northern Italy. It has a total population of about 9,500 and a typical agricultural background. Data reported here concern examination offered at entry to the males resi-
127
dent in the inner part of the town, while data on females and younger or older people have been excluded for this purpose. Only men aged 20-59 have been considered for this article. The cohort included 863 men; 739 (85%) were examined and all of them were fully available for analysis. (2) The Rome Project of Coronary Heart Disease Prevention (RO). This is the Italian Section of the “WHO European Multifactor Preventive Trial of CHD” [7 1. The aims and the structure of the study have been presented elsewhere. Here it, should be recalled only that the study is directed toward occupational groups (“factories”) allocated to either control or treatment; that entry screening examination was offered to all the subjects belonging to the treatment “factories” and to subsamples of the control “factories”. Only men aged 40--59 had been enrolled making a total of 6,027 individuals, residents of Rome, Central Italy. For the purposes of this analysis all “factories” have been pooled together. The number of men invited for screening was 3,837; those examined were 3,233 (85%); and almost all were fully eligible for the analysis. Out of the examined men about 2/3 were clerks; the remainder being engaged - on average - in moderate technical or manual work, with a negligible proportion of individuals engaged in heavy work. (3) The Olivetti Survey of Pozzuoli (PO). This was a cross-sectional study of coronary risk factors in an occupational population group in Pozzuoli, province of Naples, Southern Italy. The men were mostly engaged in light electro-mechanical work. Only men aged 20-59 have been considered for this analysis. Of the tota! number (1,900), 1,631 were examined (86%); and almost all became fully suitable for analysis, Methods The invited subjects attended the screening on work-days in the morning after a 12- to 14-h fast. Blood samples, taken from the antecubital vein without stasis, were centrifuged within 4 h and serum was kept in the refrigerator at 4°C. Chemical analysis was usually performed within the following two days. In the Bologna and Naples laboratories the same methods and apparatus were used for cholesterol and triglyceride analysis: namely the Levine and Zak extractive method for cholesterol [8] and the Kessler and Lederer method for triglycerides [9]. Both methods were adapted to the automated analyzer CLA 1510, Carlo Erba Strumentazione [lo]. In the Rome laboratory, the manual Abell-Kendall method for cholesterol [ll] and the Eggstein enzymatic method for triglycerides [12] were performed. The 3 centers participated in the lipid quality control program with the WHO Regional Lipid Reference Center of the Institute of. Clinical and Experimental Medicine, Prague. The comparability of methodologies was checked, after the screening, by periodical analysis of split samples forwarded frozen and blind from Naples to the other laboratories. Examples of the standardization procedures are given in Figs. 1 and 2. Differences between laboratories were considered to be of the same order of magnitude as the variability usually found within individual laboratories.
12x
LIPID
QUALITY
CONTROL
: SERUM
CHOLESTEROL
JUNE 1975
SEPTEMBER ,975
n=15
x = 179 y = 183
r =0.982 y=
--1-----T------rtc--T------
100
100
..
P E”
1 /
,1.468+0.956x
200
300
mg/dl
NPlPLES
.a
;
200
,"
.
n = 15
;/I/
x=
+
; = 203
i
r = 0.995 100.
y = *.71g+,.o35x
??
100
___I__
200
y= n= 15
.
x=
/
v/d1
189
I/
y=-12
=
J
1
JJ r iz
i
1
‘T
y=,oo
r = 0.995
r = 0.085
y = 0.117 +1.070x
2;o
lb0
IOO-
between
y=
I
mddl
100
BOLOGNA
control
n = 15
E=133
t = 203
j///
1. Quality
I
P =100
.
Fig.
200
n =15
.
. 1_1 i’
Y
200
/
rng/dl
/
2 300
E
100
300
NAPLES
P
:
254+,.124x
200
NAPLES
;
170
r = 0.997
I_T-.--T~~.+5-100
I
100
/
_;
187
200 BOLOGNA
Centers
for serum
cholesterol.
-10.423+1.140x
I
3
300 mgpl
LIPID JUNE
QUALITY
CONTROL : TRIGLYCERIDES
1975
SEPTEMBER
1975
T----
300
200
m&l
----260100
300
1 1
*
ilil ??
LL
:
/
.*
-I
/.
3004
4
ddl
/
r
100
300 NAPLES
?I
P
mehl
NAPLES
NAPLES
2
300
200
NAPLES
??
200
P i
n= 15
z=
99
5=
100
1004
r = 0.994 y=
1
-14.678+,.161x
/
----
100
msPl BOLOGNA
Fit?. 2. Quality control
between
y=
,’
-8.106+,.0*3x
li/
100
200 BOLOGNA
Centers
for serum triglycerides.
300
msbl
P
Br-PO
2x
mmoles/l
0.89
3.96
34.17
153.00
_
0.96
4.76
37.00
184.00
311
0.87
0.85
13
5.36
5.08
32.67
32.96
86
0.95
4.93
36.66
190.30
462
1.18
5.44
45.70
210.06
71
SERUM
3om-34
OF
2 5-29
206.96
(yr)
(SD)
195.96
49
20-24
Age
DEVIATION
__~.___.
SD
’ SD
mgldl
n
rx
‘“SD
(PO)
mmolesil
1’“sD
n
STANDARD
mg/dl
AND
Pozzuoli
(Br)
Brisighella
(A)
1
(f)
TABLE
MEAN
267
1.06
4.95
41.01
191.23
0.98
4.84
37.88
187.09
1,053
1.06
5.50
5.78 1.09
40.94
212.17
310
20-39
42.23
223.35
104
35-39
(mg/dl
~~
corrected
Age-
CHOLESTEROL
mmoles/l)
’
n
n.s.
Ro-PO
1.09
5.07
Br-Po
SD
1.r
Br-Ro
.__~____~~~~.~ ~~~. .._
P
mmoles/l
42.12
195.84
4
’SD
w-0)
1.08
SD 263
5.54
(0.001
1.17
5.15
45.16
198.86
189
1.08
5.66
41.70
218.34
214.01 41.84
868
860
1.21
1.01
S
,.’ SD
6.15
5.66
237.31
218.73 46.65
132
114 38.88
45--49
(yr)
MIDDLE-AGED
4044
Age
AND
n
mgldl
n
(A)
x ‘. SD
YOUNG
mmoles/l
mgjdl
mg/dl
IN
Pozzuoli
R0ma @o)
(Br)
Brisighella
(B)
and
<0.001
ro.001
n.s.
0.97
5.26
37.44
203.08
91
1.20
5.75
46.33
Age-
1.09
CO.001 ,: O.fJ
1.08
5.16
41.90
199.36
570
.5.65
42.18
218.02
3.216
1.06
5.90
41.09
227.91
429
40-59
corrected
n.s. 1
1.10
5.19
42.41
200.56
27
1.11
5.65
42.89
574 218.29
221.97
1.05
5.84
914
0.97
5.95
40.53
37.34
79
55-59
225.44
MEN
229.58
104
50-54
(B)
Levels of serum cholesterol and triglycerides have been expressed both in mg/dl and mmoles/l in order to follow the recently recommended rules for units of measurement. Data have been locally processed on the basis of standard and pre-agreed computer programs, and then pooled together and compared at the Rome Center. Since the Rome population group did not include men aged less than 40, two different and independent comparisons have been set up: that concerning men aged 20~-39 (young) and including only BR and PO; and another concerning men aged 40.---59 including also RO. In both cases yuinquennial subgroups and whole (young plus middle-aged) groups have been considered for analysis. For comparison of whole groups (i.e. young = age 20-39; or middle-aged = age 40-59) the means have been corrected by direct standardization, taking as the reference population the quinquennial age distribution of the pool of all groups. The age distribution of the men examined is given in each table. Results Mean levels of serum cholesterol are given in Table 1. They are significantly different (t-test) in 17 out of 20 comparisons involving two populations of young men, and three populations of middle-aged men and a total of 8 age subgroups and 2 age-pools. All the 4 comparisons made on the corrected means have a chance explanation of less than 0.001. The trend is typical - for Italy going downward from North, to Center, to South. The histograms concerning the distributions of data, broken down into arbitrary classes of 20 mg/dl are, as expected, slightly positively skewed and suggest that the modal class tend to present lower and lower levels going from BR to RO to PO (Fig. 3). Such distributions, as the next for triglycerides, are limited as examples, to a single quinquennial age class owing to the non-comparability if data on all ages were presented. Data on triglycerides are given in Table 2 and Fig. 4. Again the highest mean level belongs to BR, the lowest to PO, RO being in between. Such differences are significant, (t-test) in 13 out of 20 possible comparisons. The trend is particularly evident when examining the corrected mean levels. The distribution of triglycerides is even more skewed to the right than that of serum cholesterol and again the modal class is different in the 3 populations, where referred to a sub-class of the middle-aged men. But in this particular case the median classes in BR and PO are similar, while that in RO has a lower value. The overall results, in terms of inter-group comparisons, are somewhat different, a larger number of comparisons being statistically significant. Looking back to the means it appears that the age trend for serum cholesterol - as shown by quinquennial groups - is rather typical in all the 3 cohorts showing a clear increase from age 20-24 to age 50--54 and then a steady-state or a slight decline. Serum triglycerides on the other hand tend to decline after the age-group 40-44 in PO, to be steady in RO, whereas they show a curious peak at the age 50--54 in BR.
132
SERUM
CHOLESTEROL
n = 132
BRISIGHELLA
/ i I
l-1
i
I
I I
‘1 i
1
n =
ROME
868
-
I=POZZUOLl
1
"Cl89
9
3
: 140
Fig. pies.
3. Frquency
distribution
lb0
of
serum
cholesterol
values
in men
aged
50-54.
in the
3 PoPulation
s%‘n-
133
SERUM BRISIGHELLA
n :
I
_
ROME
TRIGLYCERIDE5
132
r
1 i
i, = 868
400
Fig. PIES.
4.
Frequency
distribution
of
serum
triglyceride
values
in men
aged
50-54,
in the
440
3 population
mg/dl
s%n-
P
&-PO
e-0 )
Pozzucli
(Br)
Brisighda
(A)
MEAN
TABLE2
(f)AND
‘SD x
n
JX
mmoles/l
w!/dl
SD
Iy
'SD _
jsI
n
mmoles/l { SD
wc/dl
(yr)
~.O.OOl
~.O.OOl
448 132.31 67.36 1.50 0.76
303 119.02 67.92 1.34 0.77
9 94.78 34.37 1.07 0.39
n.s.
86 163.92 101.19 1.85 1.14
30-34
71 153.51 80.43 1.73 0.91
25-29
128.86 62.36 1.46 0.10
49
20-24
Age
310 159.53 92.21 1.80 1.04
:0.001
P
Br-Ro Br-PO Ro-PO
(PO)
Pozzuoli
(Ro)
ROITlll
(Br)
Brisighella
(B)
SD
mm&s/1
mgldl
;" SD
SD
n
SD
SD
_ :x
mmoles/1
x
n
SD
x
:
;
n x
mg/dl
mmo1esp
m&U
(m~/dlandmmoles/l)INYOUNG
Agecorrected 20-39
267 1,027 137.95 128.53 75.60 68.86 1.56 1.45 0.85 0.78
104 164.08 93.28 1.85 1.05
35-39
STANDARDDEVIATION(SD)OFSERUMTRIGLYCERIDES
(yr)
<0.05 0.01 n.s.
260 142.25 69.68 1.61 0.79
860 149.19 92.63 1.68 1.05
114 169.96 95.30 1.92 1.08
40-44
Age
D.S. '0.01 ,-0.01
185 135.91 70.83 1.54 0.80
868 160.43 103.15 1.81 1.16
132 160.74 85.10 1.82 0.96
45-49
(A)ANDMIDDLE-AGED
0.01 0.01 n.s.
90 136.94 81.81 1.55 0.92
914 153.67 96.42 1.74 1.09
429 169.43 107.68 1.91 1.22
Agecorrected 40-59
0.001 0.001 0.001
ll.S.
562 136.58 79.78 1.54 0.90
ll.s. n.s.
21 126.94 105.09 1.43 1.19
573 3,215 157.82 154.05 142.89 105.99 1.74 1.78 1.20 1.61
79 160.80 110.60 1.82 1.25
55-59
-~~-~ -
104 183.43 137.43 2.07 1.55
-50-54
-.
(B)MEN
Comment The indications given by these data suggest that in Italy a rather wide range of mean levels of the main serum lipids is still common, on the basis of a geographical and generally speaking “cultural” background. In these particular groups a kind of self-selection due to engagement at work might have some influence on men in RO and mainly in PO (the first being civil servants or employees in public utility services; the latter being employees in private industry). In any case the geographical trend is rather impressive and in line with previous studies. Roughly, such a trend is similar to those of age-corrected coronary death rates observed in the Italian regions [ 131. The mean age-corrected level of serum cholesterol for middle-aged men in BR is about 28 mg/dl higher than that observed in Crevalcore, another rural area of the same region (Emilia-Romagna) located about 100 km distant, studied in 1960 within the “Seven Countries Study” [4]. But it is also known that in this area (Crevalcore) men reaching the same age 10 years later (in 1970) had a serum cholesterol of about 20-25 mg/dl higher than those belonging to the previous generations [14]. This suggests not only that the mean serum cholesterol of middle-aged men has probably increased with time in Italy, but also that some of the previous regional differences have been maintained. While the comparison of data collected in 1960 and 1970 in Crevalcore is guaranteed by the use of the same method in a laboratory standardized with the Atlanta Reference Center, that between the 1960 data in Crevalcore and the 1975 data in BR is supposedly acceptable since the BR laboratory is indirectly standardized with the same Center through the WHO Prague Lipid Reference Center. If - as may logically be assumed - such changes are at least partly attributable to a changing pattern in dietary habits, this should have occurred in a rather parallel way in the different latitudes of the country. We were aware that the extrapolation from 3 non-representative samples to whole populations of the respective region is not entirely justified, but once again, at least by chance the traditional geographical trend has been found. Unfortunately there is no basis for a suitable comparison between the 3 groups in terms of dietary habits. In fact while in BR a rather complete dietary survey has been made on all subjects, a simpler survey has been conducted only in sub-samples of the RO groups, and no dietary study has been planned in PO. However, a very rough comparison can probably be attempted assuming that some other data collected in Naples on other groups can be extended to the PO population. The total estimated kcalories per day was 3,795 in BR and 2,700 in RO; the total fats (as % of total kcalories) was 34 in both areas; the saturated fatty acids (as % of total kcalories) were 14 in BR and 10 in RO. No data are available for PO, where presumably the diet may be similar to that of the other areas of Southern Italy, i.e. relatively poor in total kcalories, total fats and saturated fatty acids. This picture, if true, would justify the difference found in terms of serum lipids, and serum cholesterol in particular. It is clear that more surveys are still needed in order to have a kind of “lipid-
136
map” of Italy, also considering the likelihood of a net time trend. The studies pooled here, on the other hand, were not designed for this purpose, and have, only by a lucky coincidence, made a small contribution to this problem. References 1 Keys, A., Fidanza, F., Scardi. V., Bergami, G., Keys. H.M. and Di Lorenzo, F.. Studies on serum cholesterol and other characteristics on clinically healthy men in Naples, Arch. Intern. Med., 93 (1954) 328. 2 Keys, A., Fidanza. F. and Keys. H.M., Further studies on serum cholesterol of clinically healthy men in Italy, Voeding. 16 (1955) 492. B., Fidanza, F., Keys, 3 Keys, A., Anderson, J.T., Aresu, M., Biork, G., Brock, J.F., Bronte-Stewart, H.M., Malmros, H., Poppi, A., Posteli, T., Swahn, B. and Del Vecchio, A., Physical activity and the diet in populations differing in serum cholesterol, J. Clin. Invest., 35 (1956) 1173. 4 Keys, A., Aravanis, C., Blackburn, H.W., Van Buchem. F.S.P., Buzina, R., Djordievi& B.S., Dontas, A.S., Fidanza. F., Karvonen, M.J., Kimura, N., Lekos, D., Monti, M., Puddu, V. and Taylor, H.L.,
5
6
‘7 8
Epidemiological studies related to coronary heart disease -Characteristics of men aged 40--59 in seven countries, Acta Med. Stand., 460, Suppl. 180 (1967) 1. Fidanza, F., Fidanza-Alberti, A., Ferro-Luzzi, G. and Proja, M., Dietary surveys in connection with the epidemiology of heart disease: results in Italy. In: C. Den Hartog, R. Buzina. F. Fidanza, A. Keys and P. Roine (Eds.). Dietary Studies and Epidemiology of Heart Disease, Stichting tot Wetenschappelijke Voorlichting op Voedingsgebied, The Hague, 1968. Rose, G.A., Ahmeteli. M., Checcacci, L., Fidanza, F.. Glazunov, I., De Haas, J., Horstman. P., Kornitzer. M.D.. Meloni, C.. Menotti, A., Van der Sande. D., De Soto-Hartgrink, M.K., Pi&, Z. and Thomsen, B., Ischaemic heart disease in middle-aged men - Prevalence comparison in Europe, Bull. Wld Hlth Org.. 38 (1968) 885. WHO European Collaborative Group: An international controlled trial in the multifactorial prevention of coronary heart disease. Int. J. Epidemiol., 3 (1974) 219. Levine, J.B. and Zak. B., Automated determination of serum total cholesterol, Clin. Chim. Acta, 10
(1964) 381. 9 Kessler. G. and Lederer, H., Fluorimetric measurement of triglycerides. In: Automation in Analytical Chemistry, Medical Inc., New York, NY, 1966, PP. 314-344. 10 Oriente, P., Farinaro. E., Marotta, G., Mastranzo, P. and Di Marina. I,., Determinazione simultanea di colesterolo e trigliceridi de1 siero con l’analizzatore automatico CLA 1510. In: Atti 3O Congresso Nazionale di Biochimica Clinlca, Bari. 1974. 11 Abell, L.L., Levy, G.B., Brodie. B.B. and Kendall, F.E., A simplified method for the estimation of total cholesterol in serum and demonstration of its specificity, J. Biol. Chem., 195 (19521 357. 12 Eggstein, M. and Kreutz, K.H., Eine new Bestimmung der Neutralfette im Blutserum und Gewebr. I. Mitteilung (Prinzip. Durchfilhrung und Besprechung der Methode), Klin. Wschr., 44 (1966) 262. 13 Puddu, V. and Menotti, A., Ischemic heart disease and cerebrovascular accident mortality trends in the Italian regions - Their relationship to some socio-economic indexes, Acta Cardiol. (Brux.1. 21 14
(1966) 655. Fidanza, F. and Puddu,
V., Final Report,
Grant no. HE-O4672-USPHS
(19701.