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Reduced Apolipoprotein E-Rich High-Density Lipoprotein Level at Birth Is Restored to the Normal Range in Patients with Familial Hypercholesterolemia in the First Year of Life

Division of Metabolism (H.N., T.T., M.T.), Chiba Children’s Hospital, Chiba 266-0007, Japan; Division of Clinical Preventive Medicine (T.M.), Niigata University, Graduate School of Medical and Dental Sciences, Niigata 951-8510, Japan; Department of Cardiovascular Medicine (K.H., A.O.), Osaka University, Graduate School of Medicine, and Department of Pediatrics (T.Y.), Kyoto University, Graduate School of Medicine, Kyoto 606-8507, Japan; Department of Pediatrics (H.T.), Fukui University, Graduate School of Medicine, Fukui 910-1193, Japan; Departments of Clinical Laboratory Medicine (H.C., S.-P.H.), Hokkaido University Hospital, and Department of Pediatrics (K.K.), Hokkaido University, Graduate School of Medicine, Sapporo 060-8638, Japan

Address all correspondence and requests for reprints to: Hironori Nagasaka, M.D., Chiba Children’s Hospital, 579-1 Heta Cho, Midori-Ku, Chiba, Japan. E-mail: nagasa-hirono{at}k2.dion.ne.jp; or Takashi Miida, M.D., Ph.D., Niigata University, Graduate School of Medical and Dental Sciences, Niigata 951-8510, Japan, Department of Cardiovascular Medicine, Asahimachi 1-754, Chuo-ku, Niigata, Niigata 951-8520, Japan.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Background: High-density lipoprotein (HDL) consists of apolipoprotein E (apoE)-rich and apoE-poor HDL particles. ApoE-rich HDL level is high at birth but decreases after birth with reciprocal elevation in low-density lipoprotein (LDL)-cholesterol.

Objectives: The objective of the study was to clarify whether apoE-rich HDL decreases after birth in children with familial hypercholesterolemia (FH), a disorder caused by impaired LDL clearance.

Methods: We measured apoE-rich HDL-cholesterol and LDL-cholesterol during the first year of life in 10 FH children (one homozygote and nine heterozygotes), 12 non-FH siblings, and 75 healthy controls.

Results: At birth, apoE-rich HDL-cholesterol was undetectable in a homozygous FH child and lower in heterozygous FH children than non-FH siblings and controls (4 ± 2 vs. 12 ± 4 and 11 ± 4 mg/dl, P < 0.001). At 3–4 months, apoE-rich HDL-cholesterol increased in homozygous and heterozygous FH children and decreased in non-FH siblings and controls. At 12 months, apoE-rich HDL-cholesterol levels were similar among these four groups (6–7 mg/dl). In contrast, LDL-cholesterol concentration was always twice as high in heterozygous FH children as non-FH siblings and controls (at birth, 50 ± 15 vs. 25 ± 7 and 25 ± 5 mg/dl, P < 0.001; at 3–4 months of age, 159 ± 29 vs. 71 ± 16 and 73 ± 15 mg/dl, P < 0.001; at 12 months of age, 156 ± 29 vs. 75 ± 18 and 76 ± 17 mg/dl, P < 0.001).

Conclusion: ApoE-rich HDL level is low at birth in FH children and increases to the normal level in the first year of life, opposite to the change in normal children.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
The lipoprotein profile in fetuses is quite different from that in adults (1, 2, 3, 4) because lipoprotein synthesis is low due to immature hepatic function and the absence of intestinal lipid absorption. The major plasma lipoprotein in fetuses is high-density lipoprotein (HDL), whereas in adults it is low-density lipoprotein (LDL) (1, 2, 3, 4). HDL particles can be classified into two subpopulations based on apolipoprotein composition: apolipoprotein (apo)-E-rich HDL and apoE-poor HDL. In the cord plasma, apoE-rich HDL is 2-fold higher than in adult plasma (1). In contrast, LDL-cholesterol (C) concentration is extremely low in cord plasma (20–40 mg/dl) (1, 2) a level similar to that in nonprimate mammals. Once milk is given to newborn infants enterally, however, their lipoprotein profiles change dramatically within a few months from an HDL-dominant to a LDL-dominant pattern (3, 4, 5). Such changes are likely to be closely related to the increased synthesis of lipoproteins in the small intestine as well as liver.

In addition to lipoprotein production, receptor-mediated clearance is another important determinant for plasma lipoprotein profiles. In general, lipoproteins deliver cholesterol to the peripheral tissues in which cholesterol is taken up as a component of lipoproteins by lipoprotein receptors. LDL receptor recognizes apoB and apoE, whereas scavenger receptor B-I recognizes apoAI. Several additional lipoprotein receptors also recognize apoE. Because fetuses have less LDL and more HDL (especially apoE-rich HDL) than adults, the extent to which these receptors contribute to cholesterol delivery in fetuses remains to be determined.

Familial hypercholesterolemia (FH) is an autosomal dominant disorder caused by mutations in the LDL receptor gene (6, 7, 8). The impaired uptake of LDL by the liver results in high LDL-C levels in plasma (6, 7, 8). Although reduced delivery of LDL-derived cholesterol may be compensated by other delivery systems, we have little information on the detailed lipoprotein profiles or the consequences of reduced cholesterol delivery from LDL in FH children, especially in the first year of life (6, 9, 10, 11).

To clarify whether apoE-rich HDL decreases after birth in FH children as it does in normal children, we determined apoE-rich HDL-C and LDL-C concentrations in FH children, and compared them with those of their non-FH siblings and healthy control children during the first year of life.

	Subjects and Methods

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Subjects

We examined 10 children with FH, and their 12 nonaffected siblings (non-FH siblings) in 10 Japanese kindreds. FH children consisted of one male homozygote and nine heterozygotes. In all the kindreds, each FH child had the same mutation of LDL receptor gene as either parent. The affected parents met the clinical criteria for heterozygous FH of Japan Atherosclerosis Society (12). We also obtained control data from healthy children with normal birth history (1): 75 neonates at birth and 30 infants (15 boys, and 15 girls) at the ages of 3–12 months. All subjects were fed with mother’s milk in the first 2 months and artificial milk thereafter. The protocol was approved by the ethical committees of participating institutions, and written informed consent was obtained from the parents of all subjects.

Study design and laboratory measurements

Blood samples were obtained from cord veins at birth or cubitus veins at the ages of 3–4 and12 months in the fasting state. We separated serum by centrifugation and measured total cholesterol (TC) and triglyceride (TG) concentrations by enzymatic methods using commercial kits (Kyowa Medex, Tokyo, Japan). LDL-C concentrations in cord blood were determined by Friedwald’s formula (TC – HDL-C – TG/5) and in cubitus venous blood by a homogeneous assay using a commercial kit (Choletest LDL-C; Daiichi Pure Chemicals, Tokyo, Japan). apoA-I, apoA-II, apoB, and apoE levels were measured by turbidimetric immunoassays with commercial kits (Daiichi Pure Chemicals). The protein mass of cholesteryl ester transfer protein (CETP), one of the strong determinants for HDL-C, was measured by sandwich enzyme immunoassay according to the method of Kiyohara et al. (13).

Determination of total, apoE-poor, and apoE-rich HDL-C

We quantified HDL fractions using two precipitating reagents for HDL-C measurements [13% polyethylene glycol (PEG) (PEG 6000; Wako Pure Chemicals, Osaka, Japan); dextran sulfate-sodium phosphotungstate-MgCl₂ (DS-PT-Mg) (HDL-C Daiichi; Daiichi Pure Chemicals)] (1, 14). PEG precipitates neither apoE-poor nor apoE-rich HDL, whereas DS-PT-Mg precipitates apoE-rich HDL together with apoB-containing lipoproteins. Total HDL-C and apoE-poor HDL-C were defined as cholesterol concentration in the supernatant after precipitation with PEG and DS-PT-Mg. apoE-rich HDL was calculated by subtraction of apoE-poor HDL-C from total HDL-C.

Statistical analyses

All pair-wise comparisons were performed with the two-sided Student’s t test. The correlation analyses were examined by Pearson’s correlation test. The regression line and its 95% confident interval were drawn by StatView (version 5.0J; Hulinks, Tokyo, Japan). P < 0.05 was considered significant.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
At birth, the heterozygous FH subjects had twice as high LDL-C and apoB concentrations as non-FH siblings and controls (Table 1). In the three groups, both LDL-C and apoB levels doubled at 3–4 months of age with little change at 12 months. In the homozygous FH subject, LDL-C and apoB concentrations at birth were nearly double those in heterozygous FH subjects and further increased at 12 months of age.

To our surprise, apoE-rich HDL-C in the FH children changed in the opposite direction, compared with changes in non-FH siblings and control children. At birth, apoE-rich HDL-C in heterozygous FH subjects was about one third of that in non-FH siblings and controls. At 3–4 months of age, apoE-rich HDL-C increased by 50% in heterozygous FH subjects, whereas it decreased by 33 and 18% in non-FH siblings and controls, respectively. At 12 months, apoE-rich HDL-C levels were virtually the same among the three groups.

Unlike apoE-rich HDL, apoE-poor HDL concentrations at birth and at 3–4 months were nearly identical among the groups except homozygous FH (Table 1). The apoE-poor HDL concentrations at 12 months were significantly lower in FH children than non-FH siblings and controls.

Scatter plot analysis showed that the relationship between apoE-rich HDL-C and LDL-C concentrations changed dramatically in the first year of life in control children. At birth, apoE-rich HDL-C was positively correlated with LDL-C in control neonates (Fig. 1A). This relationship gradually inverted, resulting in significant negative correlation between them at 12 months (Fig. 1, B and C). In contrast, heterozygous FH had significant inverse correlations between apoE-rich HDL and HDL-C, even at birth, and this relationship did not change throughout the year. Interestingly, apoE-rich HDL and LDL-C levels at birth were positively correlated with birth weight (r = 0.712, P < 0.05) and head circumference at birth (r = 0.689, P < 0.05) in control children but not heterozygous FH children. These associations disappeared after 3–4 months of age in the control children.

View larger version (30K): [in this window] [in a new window]   FIG. 1. The correlations between LDL-C and apoE-rich HDL-C in heterozygous FH children and non-FH siblings. LDL-C and apoE-rich HDL-C were measured in nine heterozygous FH children (closed circles) and 12 non-FH siblings (open circles) at birth (A), 3–4 months (B), and 12 months of age (C) as described in Subjects and Methods. Correlation lines (bold solid lines) and 95% confidence intervals for slopes (thin solid lines) and intercepts (dotted lines) were drawn using the data of control children with the aid of StatView software (Hulinks).

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
The present study indicates that apoE-rich HDL is quite low at birth in FH children and increases to a normal level in the first year of life, which is the opposite change in normal children. We found that the mean apoE-rich HDL-C concentration at birth in heterozygous FH children was one third of those in the non-FH siblings and healthy controls (Table 1). After birth, apoE-rich HDL-C increased in the former but decreased in the latter, resulting in no significant difference among these groups at 12 months of age. A homozygous FH child exhibited the same trend as heterozygous FH children, although apoE-rich HDL-C was undetectable level at birth.

Because the transfer of cholesteryl ester (CE) among lipoproteins depends greatly on the amounts of acceptor and donor lipoproteins, elevated LDL levels may partly explain low apoE-rich HDL levels in FH neonates. In the present study, CETP mass levels were similar among the all groups but 1.5 times higher than levels in adults (1). Several lines of studies have provided evidence that the in vivo CETP activity is intensively influenced not by CETP mass but the concentrations of CE acceptor lipoproteins involving LDL (15, 16). For example, statins reduce not only LDL-C but also CE transfer from HDL to LDL via CETP but do not change CETP protein mass in FH patients (15, 17). In both FH and healthy neonates, cord plasma has little very low-density lipoprotein (1, 2, 3, 4), the major CE acceptor in the adults. On the other hand, LDL-C concentration in FH neonates is twice as high as in non-FH neonates. Therefore, all FH neonates have more CE acceptor than non-FH neonates. The strong inverse correlation between LDL-C and apoE-rich HDL-C in heterozygous FH children at birth supports our hypothesis (Fig. 1A).

In heterozygous FH children at birth, a marked reduction was observed in apoE-rich HDL but not apoE-poor HDL, probably because apoE-rich HDL is a more favorable CE donor for CETP-mediated lipid exchange than apoE-poor HDL. We previously measured apoE-rich and apoE-poor HDL-C concentrations in 10 homozygotes with the intron 14A nonsense mutation (I14A) and six compound heterozygotes with the I14A and an exon 15 missense mutation (G442G) (14). I14A homozygotes had no CETP activity, whereas compound heterozygotes had a little CETP activity. Although apoE-poor HDL-C concentrations were almost the same between the two groups, the apoE-rich HDL concentration was 44% higher in I14A homozygotes than compound heterozygotes. This finding indicates that apoE-rich HDL is a better substrate for CETP activity than apoE-poor HDL. Because apoE-rich HDL in fetuses may differ in apoA-I and apoA-II content from that in normal subjects and CETP-deficient patients (1, 18), CE transfer should be directly measured in the future studies.

Another possible mechanism for low apoE-rich HDL-C in FH neonates is enhanced uptake of apoE-rich HDL via lipoprotein receptors recognizing apoE (19). Given the low LDL concentration in fetuses, impaired cholesterol delivery by LDL to systemic tissues must be compensated for by another carrier system(s). The ubiquitous distribution of apoE and apoE receptors strongly suggests that apoE plays a crucial role in the growth and development of various organs including the central nervous system in fetuses (20, 21, 22, 23). This may explain why the homozygous FH subject displayed neither mental retardation nor underdevelopment.

It remains unclear why the relationship between apoE-rich HDL-C and LDL-C was opposite between the value at birth and 12 months of age in control children (Fig. 1). As discussed above, CE of apoE-rich HDL is preferentially transferred to LDL, which is removed from the circulation by the LDL receptor pathway. Because the liver of neonates has low cholesterol content due to limited cholesterol biosynthesis, plasma LDL must be immediately cleared by LDL receptors, resulting in considerably low plasma LDL concentrations. Therefore, the CE transfer from apoE-rich HDL to LDL is likely to be reduced due to low acceptor lipoproteins (i.e. LDL), resulting in high plasma apoE-rich HDL concentrations. In contrast, the liver of infants at 12 months of age has greater cholesterol content and exhibits less receptor-mediated LDL uptake than that of the neonates. In cultured fibroblasts, LDL receptors are already down-regulated at the serum LDL levels of 12-month-old infants (24). Therefore, CE is continuously transferred from apoE-rich HDL to LDL that stays in the circulation due to reduced clearance by the liver. This situation is similar to that in heterozygous FH children.

We conclude that apoE-rich HDL is low at birth in FH children and increases to a normal level in the first year of life, which is opposite to what happens in apoE-rich HDL in normal children. Experimental study is required to clarify the underlying mechanism and physiological significance for the dramatic change in apoE-rich HDL in the first year of life.

	Footnotes

 
Disclosure Statement: The authors have nothing to disclose.

First Published Online January 8, 2008

Abbreviations: apo, Apolipoprotein; C, cholesterol; CE, cholesteryl ester; DS-PT-Mg, dextran sulfate-sodium phosphotungstate-MgCl₂; FH, familial hypercholesterolemia; HDL, high-density lipoprotein; I14A, intron 14A nonsense mutation; LDL, low-density lipoprotein; PEG, polyethylene glycol; TC, total cholesterol; TG, triglyceride.

Received July 20, 2007.

Accepted December 27, 2007.

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This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Nagasaka, H. Articles by Chiba, H. Search for Related Content PubMed PubMed Citation Articles by Nagasaka, H. Articles by Chiba, H. Related Collections Pediatric Endocrinology Lipid