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Squalene and Noncholesterol Sterols in Serum and Lipoproteins of Children with and Without Familial Hypercholesterolemia

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Squalene and Noncholesterol Sterols in Serum and Lipoproteins of Children with and Without Familial Hypercholesterolemia 0031-3998/03/5304-0648 PEDIATRIC RESEARCH Vol. 53, No. 4, 2003 Copyright © 2003 International Pediatric Research Foundation, Inc. Printed in U.S.A. Squalene and Noncholesterol Sterols in Serum and Lipoproteins of Children with and without Familial Hypercholesterolemia ANNA KETOMÄKI, HELENA GYLLING, MARTTI A. SIIMES, ALPO VUORIO, AND TATU A. MIETTINEN Division of Internal Medicine, Department of Medicine and Biomedicine [A.K., A.V., T.A.M.], Hospital for Children and Adolescents [M.A.S.], University of Helsinki, Helsinki, and Department of Clinical Nutrition, University of Kuopio and Kuopio University Hospital, Kuopio [H.G.], Finland ABSTRACT Squalene and noncholesterol sterols, e.g. lathosterol and plant ilar in serum and lipoproteins of the two groups. The ratios of sterols, the respective markers of cholesterol synthesis and ab- squalene and lathosterol were higher in VLDL and intermediate sorption, are transported with cholesterol in serum lipoproteins. density lipoprotein, whereas in LDL that of lathosterol was lower Their concentrations and ratios to cholesterol in serum and than the respective serum values in both groups. Absorption lipoproteins have not been carefully compared, especially in marker sterol ratios were highest in HDL in both groups. Thus, children and in marked hypercholesterolemia. Thus, we mea- even though the ratios of noncholesterol sterols to cholesterol in sured these variables with gas-liquid chromatography in 18 serum reflect, in general, synthesis and absorption of cholesterol, children with and 29 without familial hypercholesterolemia, all their ratios in different lipoproteins could give additional infor- aged 5–17 y. Concentrations of most noncholesterol sterols were mation of cholesterol metabolism. (Pediatr Res 53: 648–653, higher in serum, LDL, and intermediate density lipoprotein in the 2003) children with than those without familial hypercholesterolemia. Despite accumulation of noncholesterol sterols mainly in LDL Abbreviations (75% in familial hypercholesterolemia and 55% in non-familial FH, familial hypercholesterolemia hypercholesterolemia, p Ͻ 0.001), their ratios were mostly sim- IDL, intermediate density lipoprotein The deranged catabolism of apo B containing LDL increases expressed as ratios to cholesterol (5) to eliminate the influence serum cholesterol levels in FH (1), even already at birth (2), of varying cholesterol concentrations between different indi- resulting in development of arterial atherosclerotic lesions viduals as well as different lipoproteins. However, it is not already at the ages of 3–19 y (3, 4). In addition to cholesterol, known how exactly the serum noncholesterol sterol to choles- other minor sterols, called noncholesterol sterols, and squalene terol ratios are related to the respective ratios in different are also carried by serum lipoproteins. The cholesterol precur- lipoproteins, especially in healthy children and those with FH. sors squalene, lanosterol and other methyl sterols, and dem- The measurement of the sterols in lipoproteins are expected to ethyl sterols (⌬8-cholestenol, desmosterol, and lathosterol) re- give more accurate information on the release of cholesterol flect cholesterol synthesis (5–7), whereas plant sterols, precursors from liver into the circulation and on the reverse traffic campesterol and sitosterol, and cholestanol, a metabolite of mechanism of sterols from tissues through HDL. Thus, the aim of cholesterol, reflect the efficiency of cholesterol absorption in the present study was to investigate serum and lipoprotein non- normal and hyperlipidemic populations (8–10), including het- cholesterol sterol concentrations in markedly hypercholester- erozygous adults with FH (11). The values of serum noncho- olemic children and in controls, and to compare the ratios of lesterol sterols and squalene are, in general, standardized and sterols to cholesterol in the different lipoproteins and relate them to the respective ratios in the serum. Accordingly, the study population was chosen to comprise children with genetically Received November 2, 2001; accepted September 12, 2002. Correspondence: Tatu A. Miettinen, M.D., Division of Internal Medicine, Department diagnosed heterozygous FH and healthy subjects of similar age. of Medicine, University of Helsinki, P.O. Box 340, FIN-00029 HUS, Finland; e-mail: tatu.a.miettinen@helsinki.fi Supported by The Finnish Medical Society Duodecim, Research Foundation of Orion METHODS Corporation, The Paavo Nurmi Foundation, The Paulo Foundation, and Helsinki Univer- sity Central Hospital. Subjects. The study population consisted of 47 children aged DOI: 10.1203/01.PDR.0000055771.28409.40 5–16 y (24 boys and 23 girls) who either had a relative with 648 NONCHOLESTEROL STEROLS IN FH CHILDREN 649 clinically or genetically documented FH, or were themselves triglycerides: GPO-PAP, Roche, Basel, Switzerland; phospho- patients at the Outpatient Lipid Clinics of the Hospital for lipids:phospholipase D choline-oxidase PAP, Wako Chemi- Children and Adolescents, University of Helsinki (Table 1). cals, Neuss, Germany; free cholesterol: CHOD-PAP, Boeh- The subjects were divided into FH (n ϭ 18) and non-FH (n ϭ ringer-Mannheim, Mannheim, Germany). Serum TSH was an- 29) by DNA analysis characterizing the four most frequent alyzed by our hospital laboratory with a chemiluminometric Finnish LDL receptor mutations; FH-Helsinki (FH-Hki; n ϭ method. Blood glucose was measured with a commercial kit 11), FH-North-Karelia (FH-NK; n ϭ 5), FH-Turku (n ϭ 2), using glucose dehydrogenase method (Roche), serum insulin and none with FH-Pori (12–14). Serum total cholesterol values by RIA (Pharmacia & Upjohn, Uppsala, Sweden), and sex were Ͻ5.0 mM in the non-FH group. Diabetes and hypothy- hormone binding globulin by commercial kits (Wallac, Turku, roidism were excluded by analyzing blood glucose and TSH Finland). levels. The possible use of plant stanol or sterol ester spread Cholesterol and cholesterol precursors, squalene, ⌬8- was ceased at least 3 wk before entry to the study. During the cholestenol, desmosterol, and lathosterol (precursor sterols called study, the subjects continued their normal habitual diet without synthesis markers of cholesterol), and cholestanol and plant ste- any hypolipidemic treatment including plant stanol and sterol rols, campesterol and sitosterol (three sterols called absorption ester spreads. One girl was a smoker and another one occa- markers of cholesterol) were determined from serum and lipopro- sionally used antihistamines. Informed consent was obtained teins by gas-liquid chromatography on a 50-m-long capillary from the parents and also from the children older than 12 y of column (Ultra-1; Hewlett-Packard, Wilmington, DE, U.S.A.) (17, age. The study protocol was approved by the Ethics Committee 18). The values are expressed as concentrations (micrograms per of the Children’s Hospital, University of Helsinki. deciliter) or as ratios defined as the concentration of particular Study design. The subjects visited the outpatient clinic once, sterol (in millimoles ϫ 100) to the concentration of cholesterol in and after fasting for at least 4 h, a blood sample was drawn moles to eliminate the effects of different transport protein con- (5–15 mL, according to the patient’s age). A routine medical centrations on the noncholesterol sterol value. No recovery cor- examination, including puberty staging (15), automatic blood rections were made for ultracentrifuge fractions. The lipoprotein pressure measurement, and clinical examination of the Achilles squalene values were obtained only from 14 FH and 24 non-FH tendons, was performed in 16 children with FH and in all children. control subjects. The apo E phenotype was determined electrophoretically by Methods. DNA was isolated from the venous blood, and the isoelectric focusing from serum samples (19). Subjects with analyses for FH-Hki, FH-NK, FH-Turku, and FH-Pori were phenotypes E2/2, E3/2, and E4/2 were combined and called carried out using the PCR technique (14, 16). Lipoproteins apo E-2 group, those with E3/3 were called apo E-3, and those were separated by ultracentrifugation into the following den- with E4/3 or E4/4 as apo E-4. The mean serum lipid and sterol sity fractions: VLDL, d Ͻ 1.006 g/mL; IDL, d ϭ 1.006–1.019 values were calculated for apo E-4 versus the other apo E g/mL; LDL, d ϭ 1.019–1.063 g/mL; and HDL, d ϭ 1.063– phenotypes. 1.210 g/mL. Total and free cholesterol, phospholipids, and Relative height was expressed as the deviation from the serum triglycerides were measured by enzymatic color reac- mean height of Finnish children of the same age, and relative tions with commercial kits (total cholesterol: CHOD-PAP, weight was expressed as a percentage of the weight of children of the same height. Table 1. Characteristics of the study population* Statistical significances between the groups were tested with 2 FH† Non-FH‡ one-way ANOVA, two-sided t test, and the ␹ test. The Variables (n ϭ 18) (n ϭ 29) differences between the lipoprotein sterols were tested with the Age (y) 11.1 Ϯ 0.8 11.2 Ϯ 0.6 paired t test. Correlation coefficients were calculated with Sex (M/F) 10/8 14/15 Pearson’s product moment correlation. When the distribution Height (cm) 144.7 Ϯ 4.3 (n ϭ 16) 146.0 Ϯ 3.3 of a variable was skewed, logarithmic transformations were Ϯ ϭ Ϯ Weight (kg) 39.0 3.7 (n 16) 40.4 3.0 performed. A p value Ͻ 0.05 was considered significant, and 2 Ϯ ϭ Ϯ Body mass index (kg/m ) 17.9 0.9 (n 16) 18.1 0.7 Ϯ Relative height (SD) 0.3 Ϯ 0.2 (n ϭ 16) 0.2 Ϯ 0.2 data are reported as mean SEM. Height weight (%) 102.8 Ϯ 4.4 (n ϭ 16) 102.2 Ϯ 2.9 Ϯ Ϯ ϭ Ϯ Ϯ Blood pressure (mm Hg) 121 4/64 2(n 16) 118 3/66 2 RESULTS Heart rate (beats/min) 76 Ϯ 4(n ϭ 16) 76 Ϯ 2 Ϯ Ϯ Blood glucose (mM) 5.2 0.1 5.2 0.1 Characteristics of the study population.
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