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Shaper AG, Pocock SJ, Ashby D, Walker M, Whitehead TP. Biochemical ... Jose G. Dorea,1 Edgar Merchan-Hamann,' Douglas E. Ryan,3 and JIrI Holzbech&.

CLIN. CHEM. 36/4, 677-680 (1990)

Biological Factors Affecting Concentrations of Serum LpAI Lipoprotein Particles in Serum, and Determination of Reference Limits JoslaneStelnmetz, Ala Choukalfe,SophlaVievIkis,JosephHenny,and GerardSleet We used an electroimmunoassay to measure LpAI lipoprotein particles (Iipoproteins containing apolipoprotein Al but

not apolipoprotein All) in serum of a presumably healthy population of about 1000 subjects, noting sex- and agerelated variations for the age interval four to 70 years. Results were higher for women than men. For males, the value for the 50th percentile of the distribution was highest in

the 10- to 14-year subgroup, 0.69 g/L, decreasing to 0.60 g/L in adults. For females,

the values increased

regularly, from

0.59 g/L at ages four to 10 years to 0.79 g/L after age 55 years. The influence of puberty, menopause,

oral contraceptives, alcohol consumption, and morphometnc charactenstics was studied. Only being overweight by more than 20% statistically influenced LpAI values in men and in women. We used these results to select a reference


and to

establish reference limits of LpAI at ages 25 to 35 years: 0.40-0.95 g/L for men and 0.46-1.05 g/L for women.

AdditionalKeyphrases:sex- and age-relatedeffects source of

heart disease . “kit”methods



assay It is now well established that, in human serum, apelipeprotein (ape) Al is distributed within high-density lipoproteins (HDL) between different types of lipoprotein particles, one of them containing both apo Al and ape All (LpAI:All particles), the other containing no ape All (LpAI particles) (16).1 Atmeh et al. (4) suggested that these two populations of particles are metabolically distinct and that ape Al is not completely equilibrated between these two lipoprotein species. Thus, these lipeprotein subpepulations, which differ with respect to their



may have a

different origin, metabolic pathway, and function that could be clinically important. Both lipoproteins are rich in cholesterol and phospholipid, have an alpha electrophoretic mobility, and have the same size and density as highdensity lipoproteins (HDL) (2,3). LpAI seems to be present in all major lipoprotein density classes, but predominantly in HDL2 (3, 6, 7). Moreover, LpAI is composed of two distinct particle sizes: (LpAI)1, with a Stokes diameter of

10.8 nm, and (LpAI)2, with a Stokes diameter of 8.5 nm (5). Recently,Puchoiset al. (8) suggestedthatLpAI particles might represent the “anti-atherogenic” fraction of HDL, their concentrations in plasma being significantly lower in subjects with an angiographically defined coronary artery diseasethan in healthy patients. For preventing cardiovas-

Laboratoire du Centre de M#{233}decine Preventive et Centre du M#{233}dicament, URA CNRS 597, 2 Avenue du Doyen Jacques Pansot, 54500 Vandoeuvre-les-Nancy, France. ‘Nonstandard abbreviations: HDL, high-density lipoproteins; apo, apolipoprotein; LpAI:All, lipoprotein particles with ape A! and ape All; and LpAI, lipoprotein particles with apeATbut no ape All. Received September 1, 1989; accepted December 29, 1989.

cular diseases, quantification of this particle might offer significant advantages to better definethe risk associated with lipid abnormalities. Thus, we considered it important to study biological factors influencing the concentration of LpAl in the population, and to establish reference values for it. We report here the effect of different factors that could cause variation in a homogeneous group of about 1000 presumably healthy persons, ages four to 70 years. We used these results to select a reference population and to establish reference limits for LpAI lipoprotein particles with the use of a newly commercialized electroimmunoassay kit.

Materials and Methods Population Serum samples were obtained from 1005 healthy subfrom the population who came to the Center for Preventive Medicine at Vandoeuvre-l#{232}s-Nancy between May 1988 and January 1989 for health screening. Subgroups were as follows: 158 boys and 154 girls, ages four to 14 years; an adolescent group (14-18 years) consisting of 49 boys and 47 girls; and the adult (>18 y) population, 277 men and 320 women. The subjects fasted overnight before serum was sampled, were receiving no drug treatment except for oral contraceptives, and did not smoke. Using a health questionnaire and information derived during functional exploratory tests, we obtained some information on alcohol consumption (whether more or less than 44 g of alcohol per day for men, and more or less than 22 g per day for women), use of oral contraceptives, morphometric characteristics, height (130-139 cm, 140-149 cm, 150-159 cm at ages 10-14 years, and 150-159 and 160-169 cm at ages 14-18 years), overweight (according to the Lorenz formula, by more or less than 20%), puberty in girls (based on the presence or absence of menstruation), and menopause in women. Thus we could intercompare subgroups of population with respect to those characteristics.

jects selected


of LpAI

LpAI was measured with kits (Sebia,23 rue MaximilienRobespierre, 92130 Issy-les-Moulineaux, France) that contained hydrated agarose gel and antibodiesmonospecific for ape-Al and ape All. Anti-ape All antibody, incorporated in sufficient quantities, blocked the LpAl:Afl particle family while letting LpAl particles migrate. The antibody to ape A! reacts with ape Al to generate an immunoprecipitation “rocket,” the height of which is proportional to the LpAI concentration. Two rockets are formed on the same gel. The smaller, more intensely colored peak corresponds to the particles containing ape All, the higher, less-colored one corresponds to particles containing only LpAI. A standardserum obtained from a pool of human sera,stabilized and lyophilized, is used as reference material for the quantification of these particles. CLINICAL CHEMISTRY, Vol. 36, No. 4, 1990


In brief, the procedure is as follows. Five microliters of samples diluted in isotonic saline solution (2:100, by vol) or 5 L of diluted standard is placed in the wells of the gel. After migration (4 h, 25 mA) the gel is washed, dried, and stained with Coomassie Blue. The calibration curve is drawn as a function of the heights of the rockets corresponding to various dilutions of the standard, and patients’ results are directly deduced from the calibration curve. This procedure will be described in detail elsewhere (9). We evaluated the repeatability of the technique by measuring the concentration of LpAI in a serum pool 24 times the same day. The mean value was 0.58 g/L; the coefficient of variation was 2.4%. Day-to-day reproducibility was estimated by using two different lyophilized sera (mean LpAI concentrations: 0.38 and 0.68 g/L) measured 80 times and 30 times, respectively. The respective coefficients of variation were 5.6% and 5.8%. Statistical Tests The normality of the distributions was verified by test. To assessthe significance of differences between means, we used Student’s t-test and variance analysis. Results We assessedthe effect of five potential causes of variation on the concentration of LpAI. Age and s. Figure 1 shows the variations in values for LpAI with age for 5th, 50th, and 95th percentiles of the distribution. LpAI concentration varied slightly with age in males. The 50th percentile increased from 0.62 g/L between ages four to 10 to 0.69 g/L between ages 10 to 14 years. We (gA)


1,0 0,8





verified that this variation was not significantly related to height. After age 14, the median values remained relatively constant between 0.56 and 0.62 g/L. Results for the 95th percentile varied in the same way. In females, LpAI concentrations (for the 50th percentile) increased steadily with age from 0.59 g/L in the four- to 10-year-old subgroup to 0.79 g/L after age 55. Differences in values for girls of ages 10 to 14 who were or were not menstruating were not significant. In the group between ages 45 to 55 years, we found an increase of 12% of LpAl concentrations in pest-menopausal (n = 17, median value: 0.72 g/L) as compared with premenopausal women (n = 38, median value: 0.64 g/L). Considering the small number of subjects, this difference

was judged not to be significant. The concentration of LpAI in serum was slightly higher for boys than for girls younger than 14 years old (Figure 1). Results for women older than this were higher: the difference was more consistent and increased to 0.19 g/L after age 35 years. The variations with age and sex were significant (P

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