- Email: [email protected]
Race and gender differences in cord blood lipoproteins
Atherosclerosis 148 (2000) 57 – 65 www.elsevier.com/locate/atherosclerosis
Race and gender differences in cord blood lipoproteins Clodagh M. Loughrey a,*, Eric Rimm b, Gerardo Heiss c, Nader Rifai a,1 a
Department of Laboratory Medicine, Children’s Hospital, Har6ard Medical School, 300 Longwood A6e., Boston, MA 02115, USA b Department of Nutrition, Har6ard School of Public Health, Boston, MA, USA c Department of Epidemiology, School of Public Health, Uni6ersity of North Carolina, Chapel Hill, NC, USA Received 19 October 1998; received in revised form 23 April 1999; accepted 4 June 1999
Abstract Race and gender differences in lipoproteins and apolipoproteins have repeatedly been demonstrated in adults. The same disparities have been observed in children of different race and gender, implying that these differences may be influenced by genetic factors. To further explore this hypothesis, cord blood concentrations of both lipoproteins and apolipoproteins were examined in black and white neonates of both sexes. Results were controlled for the observed effects of gestational age and growth parameters. Similar patterns of lipoprotein differences to those observed in adults and children were also detected between black and white, and between male and female neonates. These findings support the concept that the aetiology of the difference in lipoprotein concentration observed between race and gender groups includes a significant genetic component. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Lipoproteins; Apolipoproteins; Lipids; Race; Gender; Newborn; Infants
1. Introduction Increased concentration of cholesterol in plasma is associated with a higher risk of atherosclerosis [1]. Low-density lipoprotein (LDL) is the major cholesterolcarrying particle in plasma. High-density lipoprotein (HDL) is responsible for transporting cholesterol back from the tissues to the liver. High blood levels of LDL-C (low-density lipoprotein cholesterol) and low concentrations of HDL-C (high-density lipoprotein cholesterol) are widely accepted as independent risk factors for coronary heart disease [2]. The protein component of LDL consists of one molecule of apolipoprotein B (apo B), and most of the apo B found in plasma circulates in LDL. Apo B is a ligand for the LDL receptor which controls cellular uptake of LDL. Similarly, most of the protein content * Corresponding author. Present address: Department of Clinical Chemistry, Belfast City Hospital, Lisburn Road, Belfast BT9 7AB, Northern Ireland, UK. Tel.: +44-1232-263628; fax: + 44-1232263969. E-mail addresses: [email protected] (C.M. Loughrey), [email protected] (N. Rifai) 1 Corresponding author also.
of HDL consists of apolipoprotein A-I (apo A-I) and most apo A-I in plasma exists in association with HDL. Apo A-1 modulates HDL metabolism: it is a ligand for HDL uptake and a cofactor for lecithin: cholesterol acyltransferase (LCAT), which esterifies cholesterol in HDL-C. These associations have led to the view that apo A-I and apo B may be useful adjuncts to more conventional lipid and lipoprotein measurements in the assessment of coronary risk [3]. Increased blood concentrations of total cholesterol, LDL-C and apo B, and decreased HDL-C and apo A-1 have been shown to correlate with increased risk of developing coronary heart disease [1,2,4]. The precise contribution of plasma triglyceride concentration to atherosclerotic risk has been difficult to elucidate and its relevance has been more widely accepted in Europe than in the US. The difficulty with resolution of this issue has been at least in part due to the association of raised plasma triglyceride levels with diminished concentrations of other lipoproteins, notably HDL-C, and in many studies the association of high plasma triglycerides with CHD risk does not withstand multivariate analysis. However the significance of hypertriglyceridemia as an independent risk factor has been demonstrated both in women [5] and, more recently, in men [6].
0021-9150/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 2 1 - 9 1 5 0 ( 9 9 ) 0 0 2 3 8 - 5
58
C.M. Loughrey et al. / Atherosclerosis 148 (2000) 57–65
Race and gender differences in lipoproteins and apolipoproteins have repeatedly been demonstrated in adults, most recently by the NHANESIII study [7]. Black men and women have lower LDL-C and apo B, and higher HDL-C and apo A-I than white men and women. Women have higher HDL-C and apo A-1 than men. These differences have also been noted in children, supporting the concept that the variance is due to genetic influences, rather than environmental factors [8]. However even among children, racial and gender differences in lipids may be attributable to environmental influences such as diet, exercise or obesity [9–11]. A considerable body of work now exists which examines lipid, lipoprotein and apolipoprotein levels and compositions in cord blood; some fetal and maternal factors are recognised to affect these [12 – 19]. If these factors are taken into consideration, cord blood measurements are an effective means of controlling as far as possible for the effects of early life environmental factors on various blood parameters. We examined cord blood lipoproteins and apolipoproteins concentrations in black and white male and female neonates to help determine whether genetic influences play a significant role in the observed differences in these groups.
reagents from Boehringer–Mannheim Diagnostics (BMD), Indianapolis, IN. HDL-C was assayed by the dextran sulfate-magnesium precipitation method as previously described [20] with enzymatic measurement of cholesterol. LDL-C was estimated by Friedewald’s equation [21]. Apolipoproteins A-I, A-II and B were determined by immunoturbidimetry using antisera from BMD and calibrators from Washington Research Foundation, Seattle, WA. In order to verify the reliability of Friedewald’s equation in cord blood, 20 cord blood samples were obtained and LDL-C concentrations were assessed in each by two methods: by modified b-quantification, which employs both ultracentrifugation and chemical precipitation steps [22], and by Friedewald’s equation. The following results were obtained: b-quantification Friedewald’s equation
Mean9 SD 0.769 0.25 mmol/l Mean9 SD 0.779 0.25 mmol/l 0.99
Slope of the linear regression curve Correlation coefficient r 0.98 There was no significant difference between the respective means and standard deviations.
2. Methods
2.1. Subjects The study population comprised a total of 256 neonates, which included 60 black males, 70 white males, 65 black females and 61 white females. These were consecutive births taken over a 6-week period (for white children) extended to 10 weeks (to recruit a comparable number of black children) at the University of North Carolina Hospitals in Chapel Hill, NC. Gestational age, head circumference, height and weight were analysed as indicators of fetal maturity. Also examined as potential covariates were time of birth, type of delivery (vaginal spontaneous, vaginal induced and Caesarean section), Apgar scores, maternal age, and occupation and income of the head of household. All information was abstracted from the hospital charts by a single observer (N.R.).
2.3. Statistical analysis Pearson product moment correlations were calculated to determine the potential effects of neonatal maturity indices and maternal parameters on cord blood lipids and lipoproteins. Factors which had negligible effect in any of the four race or gender groups were eliminated from further analysis. To analyse the differences in cord blood lipids and lipoproteins between the four groups, analysis of covariance was conducted using SAS-PC statistical software (SAS Inst., Cary, NC). Probability values of less than or equal to 0.05 were taken to be significant.
3. Results
3.1. Characteristics of the study population 2.2. Laboratory methods Blood was sampled from the umbilical cord immediately following delivery and separated after clotting for at least 45 min at room temperature. Serum was stored at 4°C for a maximum of 24 h prior to analysis. Total cholesterol and triglycerides were measured enzymatically with correction of triglycerides for endogenous glycerol on a Cobas-BIO centrifugal analyzer (Roche Analytical Instruments, Nutley, NJ) using
Basic descriptive statistics are presented by race and gender in Table 1. Gestational age was similar in males and females, but slightly longer in whites compared to blacks (39.29 2.5 vs. 38.8 93.0 weeks). Maternal age was equivalent in male and female neonates but the mothers of the white infants were older than the black mothers (25.59 6.5 vs. 23.595.7 years). All growth parameters were higher in male compared to female, and in white compared to black neonates.
C.M. Loughrey et al. / Atherosclerosis 148 (2000) 57–65
3.2. Correlations between neonatal maturity indices and lipid le6els For each gender and race classification, a Pearson correlation matrix between birth characteristics and biochemical measures of cord blood was calculated (Table 2A–D). Among white neonates, only gestational age was significantly correlated with any cord blood measurements. In white males, gestational age was significantly correlated negatively with LDL-C and positively with apo A-I and apo A-II (Table 2A); in white females there was a positive association with HDL-C and a negative association with triglycerides (Table 2B). In black newborn infants, both gestational age and neonatal growth parameters (weight, length and head circumference) were significantly related to cord blood lipoprotein concentrations. In black males, LDL-C and apo B correlated negatively, and apo A-II positively, with gestational age and with all size parameters (Table 2C). Apo A-I correlated positively with growth parameters alone. LDL-C was also positively associated with maternal age. In black females there were significant correlations between TC, HDL-C and triglycerides and both gestational age and all growth parameters (Table 2D). In view of the consistent strong associations which emerged from these analyses, we further controlled for the combined effects of gestational age and head circumference to examine differences between the four race and gender groups. Partial correlation coefficients are displayed in Table 3. The following relationships were noted. In white neonates LDL-C was positively correlated with apo A-I and apo A-II; however this pattern was not evident in black neonates. In all black neonates there was a consistent positive association between
59
LDL-C and apo B, a relation which was also apparent in white male newborns (Table 3A). Both white and black males (Table 3B) exhibited a positive correlation between LDL-C and triglycerides. In males of both racial groups, HDL-C was significantly correlated positively with both apo A-I and apo A-II, and negatively with triglycerides. Similarly, in both female racial groups, HDL-C was significantly correlated positively with apo A-I and apo A-II, although a similar trend only existed between HDL-C and triglycerides. In all four race and gender groups, apo B correlated positively with triglycerides. In black females there was an additional significant positive association between apo A-II and triglycerides.
3.3. Lipoprotein differences between race and gender groups When controlling for the combined effects of gestational age, head circumference and gender, black children had significantly lower levels of LDL-C, mean difference 106.7 mmol/l, PB 0.05, and apo B, mean difference 68.6 mg/l, PB 0.0001, than white children (Fig. 1). There were also distinct trends to lower TC and TG, and higher HDL-C, apo A-1 and apo A-II in black neonates, although these associations did not reach significance. When controlling for the combined effects of gestational age, head circumference and race, female neonates had significantly lower triglyceride concentrations than males, with a mean M-F difference of 103 mmol/l (P B0.001) (Fig. 2). There were also tendencies to higher concentrations of LDL-C, HDL-C, apo A-I and apo A-II in females, none of which were statistically significant.
Table 1 Descriptive and lipid characteristics of the study population, by race and gendera
Gestational age (weeks) Head circumference (cm) Length (cm) Weight (kg) Maternal age (years) Total cholesterol (mmol/l) HDL-C (mmol/l) LDL-C (mmol/l) Triglycerides (mmol/l) Apo A-I (g/l) Apo A-II (g/l) Apo B (g/l) Apo A-I/Apo B HDL/TChol a
Females Mean (SD) n= 126
Males Mean (SD) n=130
Blacks Mean (SD) n =125
Whites Mean (SD) n = 131
39.0 33.5 48.9 3.11 24.3 1.73 0.73 0.88 0.35 0.83 0.22 0.32 2.6 0.42
39.0 34.2 49.6 3.21 24.8 1.67 0.67 0.85 0.46 0.80 0.21 0.33 2.4 0.40
38.8 33.4 49.0 3.10 23.5 1.69 0.72 0.83 0.38 0.79 0.22 0.30 2.6 0.43
39.2 34.3 49.6 3.22 25.5 1.72 0.67 0.89 0.43 0.84 0.21 0.36 2.3 0.39
(2.7) (2.2) (3.6) (0.68) (6.5) (0.47) (0.21) (0.40) (0.20) (0.23) (0.05) (0.11) (1.2) (0.11)
(2.8) (2.1) (3.4) (0.72) (5.9) (0.53) (0.22) (0.47) (0.30) (0.24) (0.06) (0.14) (1.3) (0.13)
(3.0) (2.5) (3.8) (0.75) (5.7) (0.50) (0.21) (0.45) (0.24) (0.20) (0.05) (0.12) (1.3) (0.11)
(2.5) (1.7) (3.2) (0.66) (6.5) (0.50) (0.22) (0.43) (0.27) (0.26) (0.05) (0.13) (1.2) (0.12)
LDL-C, low density lipoprotein cholesterol; HDL-C, high density lipoprotein cholesterol; Apo, apolipoprotein; TChol, total cholesterol.
60
Table 2 Correlations between maturity indices and maternal characteristics, and lipids, lipoproteins and apolipoproteins, by race and sex (A) White males (n= 69) TCholb
LDL-C
HDL-C
−0.13
0.17
0.10
−0.04
−0.28 B0.02 −0.06
0.07
−0.04
0.01
−0.04
0.10
−0.15
−0.21
−0.04
−0.09
Trigs
Apo A-I
Apo A-II
Apo B
TChol
LDL-C
HDL-C
Trigs
0.36 B0.002 0.15
0.24 B0.047 0.12
−0.08
0.07
0.23
0.09
−0.04
0.07
0.03
0.06
−0.04
−0.04
−0.05
−0.10
0.34 B0.008 0.12
−0.27 B0.035 −0.11
0.24
−0.03
0.01
−0.49
0.08
0.20
0.12
−0.08
−0.06
−0.13
0.17
0.03
−0.18
0.09
0.06
(C) Black males (n= 59)
Gestational age P Head circumference P Height P Weight P Maternal age P a b
Apo A-I
Apo A-II
Apo B
0.08
0.10
−0.05
−0.13
0.00
−0.16
0.03
0.05
0.24
0.01
0.20
−0.18
0.00
0.11
−0.08
0.09
0.06
−0.14
−0.07
0.24
Apo A-I
Apo A-II
Apo B
0.03
0.13
−0.23
−0.06
0.06
−0.19
0.11
0.08
0.05
−0.14
0.09
0.04
0.00
−0.12
0.12
−0.02
−0.11
0.04
(D) Black females (n= 64)
TCholb
LDL-C
HDL-C
−0.53 B0.0001 −0.42 B0.0012 −0.39 B0.0021 −0.3 B0.022 0.33 B0.012
−0.62 B0.0001 −0.46 B0.0002 −0.5 B0.0001 −0.36 B0.005 0.28 B0.035
0.14
0.04
0.24
0.08
0.01
0.25
−0.05
0.13
0.03
0.16
0.08
0.27 B0.038 0.31 B0.016 0.29 B0.026 0.19
Trigs
Apo A-I
Apo A-II
Apo B
TChol
LDL-C
HDL-C
Trigs
0.33 B0.009 0.43 B0.001 0.32 B0.14 0.38 B0.003 0.08
−0.4 B0.002 −0.35 B0.007 −0.44 B0.001 −0.26 B0.043 0.23
−0.55
−0.55 B0.0001 −0.54 B0.0001 −0.49 B0.0001 −0.40 B0.0011 0.15
−0.31 B0.0001 −0.36 B0.0037 −0.31 B0.0123 −0.25 B0.47 −0.14
0.18 B0.0119 0.08
−0.59 B0.0001 −0.52 B0.0001 −0.42 B0.0006 0.07
Pearson product moment correlation: P value insignificant if not stated. TChol, total cholesterol; LDL-C, low density lipoprotein cholesterol; HDL-C, high density lipoprotein cholesterol; Trigs, triglycerides; Apo, apolipoprotein.
C.M. Loughrey et al. / Atherosclerosis 148 (2000) 57–65
Gestational age Pa Head circumference P Height P Weight P Maternal age P
(B) White females (n= 58)
C.M. Loughrey et al. / Atherosclerosis 148 (2000) 57–65
61
Table 3 Correlations between maturity indices and maternal characteristics, and lipids, lipoproteins and apolipoproteins, by race and sex (A) White males (n= 66) LDL-Cb
HDL-C
Trigs
Tchol 0.92 0.44 0.38 Pa B0.001 B0.05 B0.01 LDL-C 0.10 0.35 P B0.01 HDL-C −0.27 P B0.05 Trigs P Apo A-I P Apo A-II P (C) Black males (n=57) LDL-Cb TChol 0.89 P B0.001 LDL-C P HDL-C P Trigs P Apo A-I P Apo A-II P
(B) White females (n = 58) Apo A-I
Apo A-II Apo B
LDL-C
HDL-C
0.46 B0.001 0.34 B0.01 0.33 B0.01 0.23
0.52 B0.001 0.40 B0.01 0.35 B0.01 0.24
0.90 B0.001
0.50 B0.001 0.12
0.37 B0.01
0.53 B0.001 0.61 B0.001 −0.24
Trigs 0.01 −0.09 −0.19
0.67 B0.001 0.10
Apo A-I
Apo A-II Apo B
0.37 B0.01 0.26 B0.05 0.31 B0.05 0.04
0.36 B0.01 0.29 B0.05 0.31 B0.05 −0.11
0.16
0.10 0.19 −0.23 0.28 B0.05 0.06 0.08
(D) Black females (n =62)
HDL-C
Trigs
Apo A-I
0.34 B0.05 −0.06
0.31 B0.05 0.28 B0.05 −0.30 B0.05
0.24
0.25
−0.05
0.09
0.56 B0.001 0.18
Apo A-II Apo B
0.37 B0.01 0.05 0.38 B0.01
0.60 B0.001 0.62 B0.001 −0.22 0.69 B0.001 0.10 0.12
LDL-C
HDL-C
0.86 B0.001
0.47 B0.001 0.00
Trigs 0.23 0.12 −0.10
Apo A-I
Apo A-II Apo B
0.42 B0.001 0.18
0.33 B0.05 0.08
0.52 B0.001 0.10
0.42 B0.001 0.35 B0.01 0.38 B0.01
0.51 B0.001 0.49 B0.001 −0.04 0.68 B0.001 0.13 0.28 B0.05
a
Pearson product moment correlation: P value insignificant if not stated. TChol, total cholesterol; LDL-C, low density lipoprotein cholesterol; HDL-C, high density lipoprotein cholesterol; Trigs, triglycerides; Apo, apolipoprotein. b
4. Discussion Previous studies have repeatedly established differences in lipoprotein patterns between race and gender groups in adults [7,23 – 25] which are also evident in children [26,27]. Our results demonstrate that these features are already apparent at birth. We have also confirmed in newborn infants associations between lipoproteins and apolipoproteins which have been well documented in adults. These correlations are already strongly established at birth, despite the very different lipid and lipoprotein profiles which exist in newborns compared to those typically seen in adults. Blood total cholesterol at birth is much lower than in adults and is distributed in an approx. 40:60 ratio between LDL-C and HDL-C [12]; by contrast, in adults, approx. 65% of blood cholesterol is carried in LDL-C, with much of the rest being transported by HDL-C. Thus neonatal LDL concentrations are strikingly lower than in adults, whilst HDL levels are decreased to a lesser extent [13–15]. Triglycerides are also considerably lower in the neonate. In addition, there are lipoprotein compositional changes in neonates, HDL having a higher apolipoprotein content
[17] and a characteristic size distribution profile [17], which is dependent on lipid levels [18]; LDL also exhibits considerable heterogeneity [17]. Lipids and apolipoprotein levels at birth have been shown to be influenced by gestational age and birthweight, although not by method of delivery [19] and the effects of these factors were confirmed and controlled for in our study. Lipoprotein profiles change dramatically in the first week of life, total cholesterol, LDL-C and triglyceride levels rising sharply to 80% of adult values by day 4 [28]. These parameters continue to rise until they approach adult levels sometime in the second year of life [29], after which concentrations remain relatively stable until puberty, when there is a transient fall in total cholesterol, LDL and HDL [28]. Subsequently gender differences become more apparent under the influence of endogenous sex hormones [30]: in adolescent males there are significant decreases in HDL-C and apo A-I post-puberty, which continue to decline over the next decade, and then remain stable until at least age 55. By contrast, in females, HDL and apo A-I increase gradually from menarche until menopause. Male/female differences in lipoprotein composition have also been reported [31].
62
C.M. Loughrey et al. / Atherosclerosis 148 (2000) 57–65
Racial differences in lipoprotein patterns are more evident in pre-pubertal children than gender differences. Black children have noticeably lower LDL and apo-B, and higher HDL and apo A-1 than white children. They also exhibit a higher LDL cholesterol:apo-B ratio, suggesting a larger, less dense LDL particle in black children compared to whites [32,33]. This more favourable lipoprotein profile in blacks persists into adulthood. It has been documented in the Bogalusa Heart Study [34], the Muscatine Study [35] and others [36] that childhood lipoprotein levels track reasonably well into adulthood. Some inconsistency noted in children identified as hypercholesterolaemic in the Muscatine study, who did not remain so in adulthood, may conceivably be due to dietary and other lifestyle modifications which were not controlled for in this work. It has also been reported that there is a reasonably good correlation between infant lipid profiles and subsequent childhood patterns [14], although this is thought to be best after age 6 months [37] and in those on a homogeneous diet such as exclusive breast-feeding [38]. The differences observed in previous studies of children and adults between race and gender groups concur with those observed in our work in newborn black and white neonates. The lower triglycerides noted in infant females compared to males are consistent with the higher HDL-C concentrations observed in adult females. Most previous studies of cord blood lipoproteins have been
unable to demonstrate any racial or gender differences [16,39], although the results of one study were in agreement with our findings [40]. However the consistency of our results suggests that cord blood lipoprotein patterns are likely to reflect and be predictive of the different profiles seen in later life. It is conceivable that maternal diet may play a role in determining fetal blood lipoproteins and apolipoproteins, although no data exists regarding this issue and dietary information was not examined in this study. No correlation has been observed between maternal and neonatal levels of total cholesterol or apo B; however HDL-C and apo A in mothers have been shown to be weakly associated with cord blood levels [41]. Another potentially confounding factor is the effect of maternal smoking, which has been shown to be associated with low umbilical cord HDL and apo A-I concentrations [42], and was not controlled for in this study. However, given that direct exposure to conventional environmental factors which can influence lipid parameters is minimal in utero, and age and body mass are controlled for, our findings imply a major heritable influence on the different patterns of lipoproteins observed between race and gender groups. This conclusion is supported by a recent paper describing analysis of lipoprotein concentrations in 790 sibling pairs, in which lipoprotein patterns appeared to be influenced by obesity in black much more so than in white children. The authors concluded that there is a major genetic
Fig. 1. White/black differences in lipids and lipoproteins. Mean differences are derived from a multiple regression model, controlling for head circumference, gestational age and gender. Values for whites are used as a baseline. TChol, total cholesterol; LDL-C, low density lipoprotein cholesterol; HDL-C, high density lipoprotein cholesterol; Trigs, triglycerides; Apo, apolipoprotein.
C.M. Loughrey et al. / Atherosclerosis 148 (2000) 57–65
63
Fig. 2. Female/male differences in lipids and lipoproteins. Mean differences are derived from a multiple regression model, controlling for head circumference, gestational age and gender. Values for females are used as a baseline. TChol, total cholesterol; LDL-C, low density lipoprotein cholesterol; HDL-C, high density lipoprotein cholesterol; Trigs, triglycerides; Apo, apolipoprotein.
component determining lipoprotein concentrations in white children which is less apparent in black children [43]. Lipoprotein(a) (Lp(a)) concentration in blood is recognized to be an independent risk factor for coronary artery disease and is thought to be strongly influenced by hereditary factors. Black adults are known to have considerably higher levels of Lp(a) than white adults and this disparity has also been demonstrated by the Bogalusa Heart Study in black compared to white children [44]. However in a previous study we were unable to establish any difference in Lp(a) concentration between black and white neonates [45]. Lp(a) concentrations in cord blood samples were shown to be extremely low, relative to typical adult concentrations, in all four race and gender groups. The absence of any detectable difference between racial groups is conceivably due to the processes by which Lp(a) is manufactured remaining immature or unchallenged at this stage of life. In this work, we were able to show that the lipoprotein/apolipoprotein associations characteristic of adult phenotypes are already apparent in neonates, implying that the pathways responsible for the synthesis of LDL and HDL are already well established. Thus any differences in concentrations of these lipoprotein due to hereditary influences are likely to be evident at birth.
On the basis of this work, we propose that the racial and gender differences in lipoprotein profiles which have been demonstrated repeatedly in previous studies are feasibly due primarily to genetic influences, subject to subsequent modification by environmental factors. Our findings also provide support for the concept that the adverse lipid profile found more frequently in whites compared to blacks is present from birth. This would imply that exposure to the atherogenic risk attributable to lipoproteins is more prolonged than that due to risk factors which develop later in life and is thus probably of greater health impact in the white than the black population. Given the higher frequency of occurrence in the black population of hypertension [46], obesity in women [23] and diabetes mellitus [47], it would be particularly pertinent to determine whether these risk factors are also established early in life. The Bogalusa Heart Study recently described postmortem studies of children and young adults and indicated strong correlation between the number of ante-mortem cardiovascular risk factors and the extent of atherosclerotic involvement of the aorta and coronary arteries [48]. These findings are consistent with a similar autopsy study of young men and women in which LDL plus VLDL cholesterol levels correlated positively, and HDL cholesterol negatively, with extent of aortic and coronary arterial fatty streaks and fibrous plaques [49]. These studies lend weight to the issue of
64
C.M. Loughrey et al. / Atherosclerosis 148 (2000) 57–65
targeted screening of populations of children at higher genetic risk of adverse lipid profiles, particularly in view of the fact that risk factors were noted to cluster in the Bogalusa autopsy study. Although the benefits of pharmacological intervention in children with dyslipidaemia have yet to be proven, focused efforts should be made at least to educate these children from an early age on dietary and other lifestyle measures which might modify an inherited risk of cardiovascular disease.
Acknowledgements We would like to acknowledge the assistance of Genesis Farrow, Department of Laboratory Medicine, Children’s Hospital, Boston, for measurement of cord blood lipids in the validation of the Friedewald equation in cord blood.
References [1] LaRosa JC, Hunninghake D, Bush D, et al. The cholesterol facts. A summary of the evidence relating dietary fats, serum cholesterol, and coronary heart disease. A joint statement by the American Heart Association and the National Heart, Lung, and Blood Institute. The Task Force on Cholesterol Issues, American Heart Association. Circulation 1990;81(5):1721–33. [2] Wilson PW. High-density lipoprotein, low-density lipoprotein and coronary artery disease. Am J Cardiol 1990;66(6):7A – 10. [3] Bachorik PS, Kwiterovich PO. Apolipoprotein measurements in clinical biochemistry and their utility vis-a`-vis conventional assays. Clin Chim Acta 1988;178(1):1–34. [4] Wald NJ, Law M, Watt HC, et al. Apolipoproteins and ischaemic heart disease: implications for screening. Lancet 1994;343(8889):75– 9. [5] Austin MA, Hokanson JE, Edwards KL. Hypertriglyceridemia as a cardiovascular risk factor. Am J Cardiol 1988;81(4A):7B– 12. [6] Stampfer MJ, Krauss RM, Ma J, Blanche PJ, Holl LG, Sacks FM, Hennekens CH. A prospective study of triglyceride level, low-density lipoprotein particle diameter, and risk of myocardial infarction. J Am Med Assoc 1996;276(11):882–8. [7] Bachorik PS, Lovejoy KL, Carroll MD, Johnson CL. Apolipoprotein B and AI distributions in the United States, 1988 – 1991: results of the National Health and Nutrition Examination Survey III (NHANES III). Clin Chem 1997;43(12):2364–78. [8] Srinivasan SR, Berenson GS. Childhood lipoprotein profiles and implications for adult coronary artery disease: the Bogalusa Heart Study. Am J Med Sci 1995;310(Suppl 1):S62–7. [9] Durant RH, Linder CW, Harkess JW, Gray RG. The relationship between physical activity and serum lipids and lipoproteins in black children and adolescents. J Adolesc Health Care 1983;4(1):55 – 60. [10] Connor SL, Connor WE, Sexton G, Calvin L, Bacon S. The effects of age, body wight and family relationships on plasma lipoproteins and lipids in men, women and children of randomly selected families. Circulation 1982;65(7):1290–8. [11] Berenson GS, Srinivasan SR, Frank GC, Webber LS. Serum lipid and lipoprotein in infants and children and their relationship with diet. Prog Clin Biol Res 1981;61:73–94.
[12] Parker CR, Carr BR, Simpson ER, MacDonald PC. Decline in the concentration of low-density lipoprotein-cholesterol in human fetal plasma near term. Metabolism 1983;32 (9):919–23. [13] McConathy WJ, Lane DM. Studies on the apolipoproteins and lipoproteins of cord serum. Pediatr Res 1980;14(5):757–61. [14] Tsang RC, Fallat RW, Glueck CJ. Cholesterol at birth and age 1: comparison of normal and hypercholesterolemic neonates. Pediatrics 1974;53(4):458– 70. [15] Dolphin PJ, Breckenridge WC, Dolphin MA, Tan MH. The lipoproteins of human umbilical cord blood apolipoprotein and lipid levels. Atherosclerosis 1984;51(1):109– 22. [16] Parker CR Jr., Fortunato SJ, Carr BR, Owen J, Hankins GD, Hauth JC. Apolipoprotein A-1 in umbilical cord blood of newborn infants: relation to gestational age and high-density lipoprotein cholesterol. Pediatr Res 1988;23(4):348– 51. [17] Davis PA, Forte TM, Kane JP, Hardman DA, Krauss RM, Blum CB. Apolipoprotein and size heterogeneity in human umbilical cord blood low density lipoproteins. Biochim Biophys Acta 1983;753(2):186– 94. [18] Genzel-Boroviczeny O, Forte TM, Austin MA. High-density lipoprotein subclass distribution and human cord blood lipid levels. Pediatr Res 1986;20(6):487– 91. [19] Lane DM, McConathy WJ. Factors affecting the lipid and apolipoprotein levels of cord sera. Pediatr Res 1983;17(2):83–91. [20] Warnick GR, Benderson J, Albers JJ. Dextran sulfate-Mg2 + precipitation procedure for quantitation of high-density-lipoprotein cholesterol. Clin Chem 1982;28(6):1379– 88. [21] Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low density lipoprotein cholesterol in plasma without the use of preparative ultracentrifuge. Clin Chem 1974;20:470. [22] Rifai N, Iannotti E, DeAngelis K, Law T. Analytical and clinical performance of a homogenous enzymatic LDL-cholesterol assay compared with the ultracentrifugation-dextran sulfate Mg2 + method. Clin Chem 1998;44(6):1242– 50. [23] Hutchinson RG, Watson RL, Davis CE, et al. Racial differences in risk factors for atherosclerosis. The ARIC Study. Atherosclerosis Risk in Communities. Angiology 1997;48(4):279–90. [24] Tyroler HA, Heiss G, Schonfeld G, Cooper G, Heyden S, Hames CG. Apolipoprotein A-I, A-II and C-II in black and white residents of Evans County. Circulation 1980;62(2):249–54. [25] Morrison JA, deGroot I, Kelly KA, et al. Black-white differences in plasma lipids and lipoproteins in adults: the Cincinnati Lipid Research Clinic population study. Prev Med 1979;8(1):34– 9. [26] Srinivasan SR, Frerichs RR, Webber LS, Berenson GS. Serum lipoprotein profile in children from a biracial community: the Bogalusa Heart Study. Circulation 1976;54(2):309– 18. [27] Morrison JA, deGroot I, Kelly KA, et al. Black-white differences in plasma lipoproteins in Cincinnati schoolchildren (oneto-one pair matched by total plasma cholesterol, sex, and age). Metabolism 1979;28(3):241– 5. [28] Strobl W, Widhalm K. The natural history of serum lipids and lipoproteins during childhood. Prog Clin Biol Res 1985;188:101– 21. [29] Berenson GS, Srinivasan SR, Frerichs RR, Webber LS. Serum high density lipoprotein and its relationship to cardiovascular disease risk factor variables in children — the Bogalusa heart study. Lipids 1979;14(1):91 – 8. [30] Srinivasan SR, Sundaram GS, Williamson GD, Webber LS, Berenson GS. Serum lipoproteins and endogenous sex hormones in early life: observations in children with different lipoprotein profiles. Metabolism 1985;34(9):861– 7. [31] McNamara JR, Campos H, Ordovas JM, Peterson J, Wilson PW, Schaefer EJ. Effect of gender, age, and lipid status on low density lipoprotein subfraction distribution. Results from the Framingham Offspring Study. Arteriosclerosis 1987;7(5):483–90.
C.M. Loughrey et al. / Atherosclerosis 148 (2000) 57–65 [32] Srinivasan SR, Wattigney W, Webber LS, Berenson GS. Relation of cholesterol to apolipoprotein B in low density lipoproteins of children. The Bogalusa Heart Study. Arteriosclerosis 1989;9(4):493 – 500. [33] Islam S, Gutin B, Manos T, Smith C, Treiber F. Low density lipoprotein cholesterol/apolipoprotein B-100 ratio: interaction of family history of premature atherosclerotic coronary artery disease with race and gender in 7 to 11-year-olds. Pediatrics 1994;94(4 Pt 1):494 –9. [34] Bao W, Srinivasan SR, Wattigney WA, Bao W, Berenson GS. Usefulness of childhood low-density lipoprotein cholesterol level in predicting adult dyslipidemia and other cardiovascular risks. The Bogalusa Heart Study. Arch Int Med 1996;156(12):1315– 20. [35] Lauer RM, Lee J, Clarke WR. Factors affecting the relationship between childhood and adult cholesterol levels: the Muscatine Study. Pediatrics 1988;82(3):309–18. [36] Guo S, Beckett L, Chumlea WC, Roche AF, Siervogel RM. Serial analysis of plasma lipids and lipoproteins from individuals 9– 21 years of age. Am J Clin Nutr 1993;58(1):61–7. [37] Freedman DS, Srinivasan SR, Cresanta JL, Webber LS, Berenson GS. Cardiovascular risk factors from birth to 7 years of age: the Bogalusa Heart Study. Serum lipids and lipoproteins. Pediatrics 1987;80 (5 Pt. 2):789–96. [38] Kallio MJ, Salmenpera L, Siimes MA, Perheentupa J, Miettinen TA. Tracking of serum cholesterol and lipoprotein levels from the first year of life. Pediatrics 1993;91(5):949–54. [39] Glueck CJ, Gartside PS, Tsang RC, Mellies M, Steiner PM. Black-white similarities in cord blood lipids and lipoproteins. Metabolism 1977;26(4):347–50. [40] Frerichs RR, Srinivasan SR, Webber LS, Rieth MC, Berenson GS. Serum lipids and lipoproteins at birth in a biracial population: the Bogalusa heart study. Pediatr Res 1978;12(8):858– 63.
.
65
[41] Pocovi M, Ordovas JM, Grande F. Lecithin:cholesterol acyltransferase, lipids and lipoproteins in maternal and umbilical cord plasma. Artery 1983;11(4):264– 72. [42] Iscan A, Yigitoglu MR, Ece A, Ari Z, Akyildiz M. The effect of cigarette smoking during pregnancy on cord blood lipid, lipoprotein and apolipoprotein levels. Jpn Heart J 1997;38(4):497– 502. [43] Chen W, Srinivasan SR, Bao W, Wattigney WA, Berenson GS. Sibling aggregation of low- and high-density lipoprotein cholesterol and apolipoproteins B and A-I levels in black and white children: the Bogalusa Heart Study. Ethn Dis 1997;7(3):241–9. [44] Srinivasan SR, Dahlen GH, Jarpa RA, Webber LS, Berenson GS. Racial (black – white) differences in serum lipoprotein (a) distribution and its relation to parental myocardial infarction in children. Bogalusa Heart Study. Circulation 1991;84(1):160–7. [45] Rifai N, Heiss G, Doetsch K. Lipoprotein(a) at birth, in blacks and whites. Atherosclerosis 1992;92(2 – 3):123 – 9. [46] Arnett DK, Tyroler HA, Burke G, Hutchinson R, Howard G, Heiss G. Hypertension and subclinical carotid artery atherosclerosis in blacks and whites. The Atherosclerosis Risk in Communities Study. ARIC Investigators. Arch Int Med 1996;156(17):1983– 9. [47] Miller JM, Miller JM. Diabetes mellitus and hypertension in black and white populations. South Med J 1986;79(10):1229. [48] Berenson GS, Sathanur R, Srinivasan SR, Weihang Bao B, Newman WPI, Tracy RE, Wattigney WA. Association between multiple cardiovascular risk factors and atherosclerosis in children and young adults. New Engl J Med 1998;338:1650–6. [49] McGill HC Jr., McMahan CA, Malcom GT, Oalmann MC, Strong JP. Effects of serum lipoproteins and smoking on atherosclerosis in young men and women. The PDAY Research Group. Pathobiological Determinants of Atherosclerosis in Youth. Arterioscler Thromb Vasc Biol 1997;17(1):95 – 106.
Race and gender differences in cord blood lipoproteins Clodagh M. Loughrey a,*, Eric Rimm b, Gerardo Heiss c, Nader Rifai a,1 a
Department of Laboratory Medicine, Children’s Hospital, Har6ard Medical School, 300 Longwood A6e., Boston, MA 02115, USA b Department of Nutrition, Har6ard School of Public Health, Boston, MA, USA c Department of Epidemiology, School of Public Health, Uni6ersity of North Carolina, Chapel Hill, NC, USA Received 19 October 1998; received in revised form 23 April 1999; accepted 4 June 1999
Abstract Race and gender differences in lipoproteins and apolipoproteins have repeatedly been demonstrated in adults. The same disparities have been observed in children of different race and gender, implying that these differences may be influenced by genetic factors. To further explore this hypothesis, cord blood concentrations of both lipoproteins and apolipoproteins were examined in black and white neonates of both sexes. Results were controlled for the observed effects of gestational age and growth parameters. Similar patterns of lipoprotein differences to those observed in adults and children were also detected between black and white, and between male and female neonates. These findings support the concept that the aetiology of the difference in lipoprotein concentration observed between race and gender groups includes a significant genetic component. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Lipoproteins; Apolipoproteins; Lipids; Race; Gender; Newborn; Infants
1. Introduction Increased concentration of cholesterol in plasma is associated with a higher risk of atherosclerosis [1]. Low-density lipoprotein (LDL) is the major cholesterolcarrying particle in plasma. High-density lipoprotein (HDL) is responsible for transporting cholesterol back from the tissues to the liver. High blood levels of LDL-C (low-density lipoprotein cholesterol) and low concentrations of HDL-C (high-density lipoprotein cholesterol) are widely accepted as independent risk factors for coronary heart disease [2]. The protein component of LDL consists of one molecule of apolipoprotein B (apo B), and most of the apo B found in plasma circulates in LDL. Apo B is a ligand for the LDL receptor which controls cellular uptake of LDL. Similarly, most of the protein content * Corresponding author. Present address: Department of Clinical Chemistry, Belfast City Hospital, Lisburn Road, Belfast BT9 7AB, Northern Ireland, UK. Tel.: +44-1232-263628; fax: + 44-1232263969. E-mail addresses: [email protected] (C.M. Loughrey), [email protected] (N. Rifai) 1 Corresponding author also.
of HDL consists of apolipoprotein A-I (apo A-I) and most apo A-I in plasma exists in association with HDL. Apo A-1 modulates HDL metabolism: it is a ligand for HDL uptake and a cofactor for lecithin: cholesterol acyltransferase (LCAT), which esterifies cholesterol in HDL-C. These associations have led to the view that apo A-I and apo B may be useful adjuncts to more conventional lipid and lipoprotein measurements in the assessment of coronary risk [3]. Increased blood concentrations of total cholesterol, LDL-C and apo B, and decreased HDL-C and apo A-1 have been shown to correlate with increased risk of developing coronary heart disease [1,2,4]. The precise contribution of plasma triglyceride concentration to atherosclerotic risk has been difficult to elucidate and its relevance has been more widely accepted in Europe than in the US. The difficulty with resolution of this issue has been at least in part due to the association of raised plasma triglyceride levels with diminished concentrations of other lipoproteins, notably HDL-C, and in many studies the association of high plasma triglycerides with CHD risk does not withstand multivariate analysis. However the significance of hypertriglyceridemia as an independent risk factor has been demonstrated both in women [5] and, more recently, in men [6].
0021-9150/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 2 1 - 9 1 5 0 ( 9 9 ) 0 0 2 3 8 - 5
58
C.M. Loughrey et al. / Atherosclerosis 148 (2000) 57–65
Race and gender differences in lipoproteins and apolipoproteins have repeatedly been demonstrated in adults, most recently by the NHANESIII study [7]. Black men and women have lower LDL-C and apo B, and higher HDL-C and apo A-I than white men and women. Women have higher HDL-C and apo A-1 than men. These differences have also been noted in children, supporting the concept that the variance is due to genetic influences, rather than environmental factors [8]. However even among children, racial and gender differences in lipids may be attributable to environmental influences such as diet, exercise or obesity [9–11]. A considerable body of work now exists which examines lipid, lipoprotein and apolipoprotein levels and compositions in cord blood; some fetal and maternal factors are recognised to affect these [12 – 19]. If these factors are taken into consideration, cord blood measurements are an effective means of controlling as far as possible for the effects of early life environmental factors on various blood parameters. We examined cord blood lipoproteins and apolipoproteins concentrations in black and white male and female neonates to help determine whether genetic influences play a significant role in the observed differences in these groups.
reagents from Boehringer–Mannheim Diagnostics (BMD), Indianapolis, IN. HDL-C was assayed by the dextran sulfate-magnesium precipitation method as previously described [20] with enzymatic measurement of cholesterol. LDL-C was estimated by Friedewald’s equation [21]. Apolipoproteins A-I, A-II and B were determined by immunoturbidimetry using antisera from BMD and calibrators from Washington Research Foundation, Seattle, WA. In order to verify the reliability of Friedewald’s equation in cord blood, 20 cord blood samples were obtained and LDL-C concentrations were assessed in each by two methods: by modified b-quantification, which employs both ultracentrifugation and chemical precipitation steps [22], and by Friedewald’s equation. The following results were obtained: b-quantification Friedewald’s equation
Mean9 SD 0.769 0.25 mmol/l Mean9 SD 0.779 0.25 mmol/l 0.99
Slope of the linear regression curve Correlation coefficient r 0.98 There was no significant difference between the respective means and standard deviations.
2. Methods
2.1. Subjects The study population comprised a total of 256 neonates, which included 60 black males, 70 white males, 65 black females and 61 white females. These were consecutive births taken over a 6-week period (for white children) extended to 10 weeks (to recruit a comparable number of black children) at the University of North Carolina Hospitals in Chapel Hill, NC. Gestational age, head circumference, height and weight were analysed as indicators of fetal maturity. Also examined as potential covariates were time of birth, type of delivery (vaginal spontaneous, vaginal induced and Caesarean section), Apgar scores, maternal age, and occupation and income of the head of household. All information was abstracted from the hospital charts by a single observer (N.R.).
2.3. Statistical analysis Pearson product moment correlations were calculated to determine the potential effects of neonatal maturity indices and maternal parameters on cord blood lipids and lipoproteins. Factors which had negligible effect in any of the four race or gender groups were eliminated from further analysis. To analyse the differences in cord blood lipids and lipoproteins between the four groups, analysis of covariance was conducted using SAS-PC statistical software (SAS Inst., Cary, NC). Probability values of less than or equal to 0.05 were taken to be significant.
3. Results
3.1. Characteristics of the study population 2.2. Laboratory methods Blood was sampled from the umbilical cord immediately following delivery and separated after clotting for at least 45 min at room temperature. Serum was stored at 4°C for a maximum of 24 h prior to analysis. Total cholesterol and triglycerides were measured enzymatically with correction of triglycerides for endogenous glycerol on a Cobas-BIO centrifugal analyzer (Roche Analytical Instruments, Nutley, NJ) using
Basic descriptive statistics are presented by race and gender in Table 1. Gestational age was similar in males and females, but slightly longer in whites compared to blacks (39.29 2.5 vs. 38.8 93.0 weeks). Maternal age was equivalent in male and female neonates but the mothers of the white infants were older than the black mothers (25.59 6.5 vs. 23.595.7 years). All growth parameters were higher in male compared to female, and in white compared to black neonates.
C.M. Loughrey et al. / Atherosclerosis 148 (2000) 57–65
3.2. Correlations between neonatal maturity indices and lipid le6els For each gender and race classification, a Pearson correlation matrix between birth characteristics and biochemical measures of cord blood was calculated (Table 2A–D). Among white neonates, only gestational age was significantly correlated with any cord blood measurements. In white males, gestational age was significantly correlated negatively with LDL-C and positively with apo A-I and apo A-II (Table 2A); in white females there was a positive association with HDL-C and a negative association with triglycerides (Table 2B). In black newborn infants, both gestational age and neonatal growth parameters (weight, length and head circumference) were significantly related to cord blood lipoprotein concentrations. In black males, LDL-C and apo B correlated negatively, and apo A-II positively, with gestational age and with all size parameters (Table 2C). Apo A-I correlated positively with growth parameters alone. LDL-C was also positively associated with maternal age. In black females there were significant correlations between TC, HDL-C and triglycerides and both gestational age and all growth parameters (Table 2D). In view of the consistent strong associations which emerged from these analyses, we further controlled for the combined effects of gestational age and head circumference to examine differences between the four race and gender groups. Partial correlation coefficients are displayed in Table 3. The following relationships were noted. In white neonates LDL-C was positively correlated with apo A-I and apo A-II; however this pattern was not evident in black neonates. In all black neonates there was a consistent positive association between
59
LDL-C and apo B, a relation which was also apparent in white male newborns (Table 3A). Both white and black males (Table 3B) exhibited a positive correlation between LDL-C and triglycerides. In males of both racial groups, HDL-C was significantly correlated positively with both apo A-I and apo A-II, and negatively with triglycerides. Similarly, in both female racial groups, HDL-C was significantly correlated positively with apo A-I and apo A-II, although a similar trend only existed between HDL-C and triglycerides. In all four race and gender groups, apo B correlated positively with triglycerides. In black females there was an additional significant positive association between apo A-II and triglycerides.
3.3. Lipoprotein differences between race and gender groups When controlling for the combined effects of gestational age, head circumference and gender, black children had significantly lower levels of LDL-C, mean difference 106.7 mmol/l, PB 0.05, and apo B, mean difference 68.6 mg/l, PB 0.0001, than white children (Fig. 1). There were also distinct trends to lower TC and TG, and higher HDL-C, apo A-1 and apo A-II in black neonates, although these associations did not reach significance. When controlling for the combined effects of gestational age, head circumference and race, female neonates had significantly lower triglyceride concentrations than males, with a mean M-F difference of 103 mmol/l (P B0.001) (Fig. 2). There were also tendencies to higher concentrations of LDL-C, HDL-C, apo A-I and apo A-II in females, none of which were statistically significant.
Table 1 Descriptive and lipid characteristics of the study population, by race and gendera
Gestational age (weeks) Head circumference (cm) Length (cm) Weight (kg) Maternal age (years) Total cholesterol (mmol/l) HDL-C (mmol/l) LDL-C (mmol/l) Triglycerides (mmol/l) Apo A-I (g/l) Apo A-II (g/l) Apo B (g/l) Apo A-I/Apo B HDL/TChol a
Females Mean (SD) n= 126
Males Mean (SD) n=130
Blacks Mean (SD) n =125
Whites Mean (SD) n = 131
39.0 33.5 48.9 3.11 24.3 1.73 0.73 0.88 0.35 0.83 0.22 0.32 2.6 0.42
39.0 34.2 49.6 3.21 24.8 1.67 0.67 0.85 0.46 0.80 0.21 0.33 2.4 0.40
38.8 33.4 49.0 3.10 23.5 1.69 0.72 0.83 0.38 0.79 0.22 0.30 2.6 0.43
39.2 34.3 49.6 3.22 25.5 1.72 0.67 0.89 0.43 0.84 0.21 0.36 2.3 0.39
(2.7) (2.2) (3.6) (0.68) (6.5) (0.47) (0.21) (0.40) (0.20) (0.23) (0.05) (0.11) (1.2) (0.11)
(2.8) (2.1) (3.4) (0.72) (5.9) (0.53) (0.22) (0.47) (0.30) (0.24) (0.06) (0.14) (1.3) (0.13)
(3.0) (2.5) (3.8) (0.75) (5.7) (0.50) (0.21) (0.45) (0.24) (0.20) (0.05) (0.12) (1.3) (0.11)
(2.5) (1.7) (3.2) (0.66) (6.5) (0.50) (0.22) (0.43) (0.27) (0.26) (0.05) (0.13) (1.2) (0.12)
LDL-C, low density lipoprotein cholesterol; HDL-C, high density lipoprotein cholesterol; Apo, apolipoprotein; TChol, total cholesterol.
60
Table 2 Correlations between maturity indices and maternal characteristics, and lipids, lipoproteins and apolipoproteins, by race and sex (A) White males (n= 69) TCholb
LDL-C
HDL-C
−0.13
0.17
0.10
−0.04
−0.28 B0.02 −0.06
0.07
−0.04
0.01
−0.04
0.10
−0.15
−0.21
−0.04
−0.09
Trigs
Apo A-I
Apo A-II
Apo B
TChol
LDL-C
HDL-C
Trigs
0.36 B0.002 0.15
0.24 B0.047 0.12
−0.08
0.07
0.23
0.09
−0.04
0.07
0.03
0.06
−0.04
−0.04
−0.05
−0.10
0.34 B0.008 0.12
−0.27 B0.035 −0.11
0.24
−0.03
0.01
−0.49
0.08
0.20
0.12
−0.08
−0.06
−0.13
0.17
0.03
−0.18
0.09
0.06
(C) Black males (n= 59)
Gestational age P Head circumference P Height P Weight P Maternal age P a b
Apo A-I
Apo A-II
Apo B
0.08
0.10
−0.05
−0.13
0.00
−0.16
0.03
0.05
0.24
0.01
0.20
−0.18
0.00
0.11
−0.08
0.09
0.06
−0.14
−0.07
0.24
Apo A-I
Apo A-II
Apo B
0.03
0.13
−0.23
−0.06
0.06
−0.19
0.11
0.08
0.05
−0.14
0.09
0.04
0.00
−0.12
0.12
−0.02
−0.11
0.04
(D) Black females (n= 64)
TCholb
LDL-C
HDL-C
−0.53 B0.0001 −0.42 B0.0012 −0.39 B0.0021 −0.3 B0.022 0.33 B0.012
−0.62 B0.0001 −0.46 B0.0002 −0.5 B0.0001 −0.36 B0.005 0.28 B0.035
0.14
0.04
0.24
0.08
0.01
0.25
−0.05
0.13
0.03
0.16
0.08
0.27 B0.038 0.31 B0.016 0.29 B0.026 0.19
Trigs
Apo A-I
Apo A-II
Apo B
TChol
LDL-C
HDL-C
Trigs
0.33 B0.009 0.43 B0.001 0.32 B0.14 0.38 B0.003 0.08
−0.4 B0.002 −0.35 B0.007 −0.44 B0.001 −0.26 B0.043 0.23
−0.55
−0.55 B0.0001 −0.54 B0.0001 −0.49 B0.0001 −0.40 B0.0011 0.15
−0.31 B0.0001 −0.36 B0.0037 −0.31 B0.0123 −0.25 B0.47 −0.14
0.18 B0.0119 0.08
−0.59 B0.0001 −0.52 B0.0001 −0.42 B0.0006 0.07
Pearson product moment correlation: P value insignificant if not stated. TChol, total cholesterol; LDL-C, low density lipoprotein cholesterol; HDL-C, high density lipoprotein cholesterol; Trigs, triglycerides; Apo, apolipoprotein.
C.M. Loughrey et al. / Atherosclerosis 148 (2000) 57–65
Gestational age Pa Head circumference P Height P Weight P Maternal age P
(B) White females (n= 58)
C.M. Loughrey et al. / Atherosclerosis 148 (2000) 57–65
61
Table 3 Correlations between maturity indices and maternal characteristics, and lipids, lipoproteins and apolipoproteins, by race and sex (A) White males (n= 66) LDL-Cb
HDL-C
Trigs
Tchol 0.92 0.44 0.38 Pa B0.001 B0.05 B0.01 LDL-C 0.10 0.35 P B0.01 HDL-C −0.27 P B0.05 Trigs P Apo A-I P Apo A-II P (C) Black males (n=57) LDL-Cb TChol 0.89 P B0.001 LDL-C P HDL-C P Trigs P Apo A-I P Apo A-II P
(B) White females (n = 58) Apo A-I
Apo A-II Apo B
LDL-C
HDL-C
0.46 B0.001 0.34 B0.01 0.33 B0.01 0.23
0.52 B0.001 0.40 B0.01 0.35 B0.01 0.24
0.90 B0.001
0.50 B0.001 0.12
0.37 B0.01
0.53 B0.001 0.61 B0.001 −0.24
Trigs 0.01 −0.09 −0.19
0.67 B0.001 0.10
Apo A-I
Apo A-II Apo B
0.37 B0.01 0.26 B0.05 0.31 B0.05 0.04
0.36 B0.01 0.29 B0.05 0.31 B0.05 −0.11
0.16
0.10 0.19 −0.23 0.28 B0.05 0.06 0.08
(D) Black females (n =62)
HDL-C
Trigs
Apo A-I
0.34 B0.05 −0.06
0.31 B0.05 0.28 B0.05 −0.30 B0.05
0.24
0.25
−0.05
0.09
0.56 B0.001 0.18
Apo A-II Apo B
0.37 B0.01 0.05 0.38 B0.01
0.60 B0.001 0.62 B0.001 −0.22 0.69 B0.001 0.10 0.12
LDL-C
HDL-C
0.86 B0.001
0.47 B0.001 0.00
Trigs 0.23 0.12 −0.10
Apo A-I
Apo A-II Apo B
0.42 B0.001 0.18
0.33 B0.05 0.08
0.52 B0.001 0.10
0.42 B0.001 0.35 B0.01 0.38 B0.01
0.51 B0.001 0.49 B0.001 −0.04 0.68 B0.001 0.13 0.28 B0.05
a
Pearson product moment correlation: P value insignificant if not stated. TChol, total cholesterol; LDL-C, low density lipoprotein cholesterol; HDL-C, high density lipoprotein cholesterol; Trigs, triglycerides; Apo, apolipoprotein. b
4. Discussion Previous studies have repeatedly established differences in lipoprotein patterns between race and gender groups in adults [7,23 – 25] which are also evident in children [26,27]. Our results demonstrate that these features are already apparent at birth. We have also confirmed in newborn infants associations between lipoproteins and apolipoproteins which have been well documented in adults. These correlations are already strongly established at birth, despite the very different lipid and lipoprotein profiles which exist in newborns compared to those typically seen in adults. Blood total cholesterol at birth is much lower than in adults and is distributed in an approx. 40:60 ratio between LDL-C and HDL-C [12]; by contrast, in adults, approx. 65% of blood cholesterol is carried in LDL-C, with much of the rest being transported by HDL-C. Thus neonatal LDL concentrations are strikingly lower than in adults, whilst HDL levels are decreased to a lesser extent [13–15]. Triglycerides are also considerably lower in the neonate. In addition, there are lipoprotein compositional changes in neonates, HDL having a higher apolipoprotein content
[17] and a characteristic size distribution profile [17], which is dependent on lipid levels [18]; LDL also exhibits considerable heterogeneity [17]. Lipids and apolipoprotein levels at birth have been shown to be influenced by gestational age and birthweight, although not by method of delivery [19] and the effects of these factors were confirmed and controlled for in our study. Lipoprotein profiles change dramatically in the first week of life, total cholesterol, LDL-C and triglyceride levels rising sharply to 80% of adult values by day 4 [28]. These parameters continue to rise until they approach adult levels sometime in the second year of life [29], after which concentrations remain relatively stable until puberty, when there is a transient fall in total cholesterol, LDL and HDL [28]. Subsequently gender differences become more apparent under the influence of endogenous sex hormones [30]: in adolescent males there are significant decreases in HDL-C and apo A-I post-puberty, which continue to decline over the next decade, and then remain stable until at least age 55. By contrast, in females, HDL and apo A-I increase gradually from menarche until menopause. Male/female differences in lipoprotein composition have also been reported [31].
62
C.M. Loughrey et al. / Atherosclerosis 148 (2000) 57–65
Racial differences in lipoprotein patterns are more evident in pre-pubertal children than gender differences. Black children have noticeably lower LDL and apo-B, and higher HDL and apo A-1 than white children. They also exhibit a higher LDL cholesterol:apo-B ratio, suggesting a larger, less dense LDL particle in black children compared to whites [32,33]. This more favourable lipoprotein profile in blacks persists into adulthood. It has been documented in the Bogalusa Heart Study [34], the Muscatine Study [35] and others [36] that childhood lipoprotein levels track reasonably well into adulthood. Some inconsistency noted in children identified as hypercholesterolaemic in the Muscatine study, who did not remain so in adulthood, may conceivably be due to dietary and other lifestyle modifications which were not controlled for in this work. It has also been reported that there is a reasonably good correlation between infant lipid profiles and subsequent childhood patterns [14], although this is thought to be best after age 6 months [37] and in those on a homogeneous diet such as exclusive breast-feeding [38]. The differences observed in previous studies of children and adults between race and gender groups concur with those observed in our work in newborn black and white neonates. The lower triglycerides noted in infant females compared to males are consistent with the higher HDL-C concentrations observed in adult females. Most previous studies of cord blood lipoproteins have been
unable to demonstrate any racial or gender differences [16,39], although the results of one study were in agreement with our findings [40]. However the consistency of our results suggests that cord blood lipoprotein patterns are likely to reflect and be predictive of the different profiles seen in later life. It is conceivable that maternal diet may play a role in determining fetal blood lipoproteins and apolipoproteins, although no data exists regarding this issue and dietary information was not examined in this study. No correlation has been observed between maternal and neonatal levels of total cholesterol or apo B; however HDL-C and apo A in mothers have been shown to be weakly associated with cord blood levels [41]. Another potentially confounding factor is the effect of maternal smoking, which has been shown to be associated with low umbilical cord HDL and apo A-I concentrations [42], and was not controlled for in this study. However, given that direct exposure to conventional environmental factors which can influence lipid parameters is minimal in utero, and age and body mass are controlled for, our findings imply a major heritable influence on the different patterns of lipoproteins observed between race and gender groups. This conclusion is supported by a recent paper describing analysis of lipoprotein concentrations in 790 sibling pairs, in which lipoprotein patterns appeared to be influenced by obesity in black much more so than in white children. The authors concluded that there is a major genetic
Fig. 1. White/black differences in lipids and lipoproteins. Mean differences are derived from a multiple regression model, controlling for head circumference, gestational age and gender. Values for whites are used as a baseline. TChol, total cholesterol; LDL-C, low density lipoprotein cholesterol; HDL-C, high density lipoprotein cholesterol; Trigs, triglycerides; Apo, apolipoprotein.
C.M. Loughrey et al. / Atherosclerosis 148 (2000) 57–65
63
Fig. 2. Female/male differences in lipids and lipoproteins. Mean differences are derived from a multiple regression model, controlling for head circumference, gestational age and gender. Values for females are used as a baseline. TChol, total cholesterol; LDL-C, low density lipoprotein cholesterol; HDL-C, high density lipoprotein cholesterol; Trigs, triglycerides; Apo, apolipoprotein.
component determining lipoprotein concentrations in white children which is less apparent in black children [43]. Lipoprotein(a) (Lp(a)) concentration in blood is recognized to be an independent risk factor for coronary artery disease and is thought to be strongly influenced by hereditary factors. Black adults are known to have considerably higher levels of Lp(a) than white adults and this disparity has also been demonstrated by the Bogalusa Heart Study in black compared to white children [44]. However in a previous study we were unable to establish any difference in Lp(a) concentration between black and white neonates [45]. Lp(a) concentrations in cord blood samples were shown to be extremely low, relative to typical adult concentrations, in all four race and gender groups. The absence of any detectable difference between racial groups is conceivably due to the processes by which Lp(a) is manufactured remaining immature or unchallenged at this stage of life. In this work, we were able to show that the lipoprotein/apolipoprotein associations characteristic of adult phenotypes are already apparent in neonates, implying that the pathways responsible for the synthesis of LDL and HDL are already well established. Thus any differences in concentrations of these lipoprotein due to hereditary influences are likely to be evident at birth.
On the basis of this work, we propose that the racial and gender differences in lipoprotein profiles which have been demonstrated repeatedly in previous studies are feasibly due primarily to genetic influences, subject to subsequent modification by environmental factors. Our findings also provide support for the concept that the adverse lipid profile found more frequently in whites compared to blacks is present from birth. This would imply that exposure to the atherogenic risk attributable to lipoproteins is more prolonged than that due to risk factors which develop later in life and is thus probably of greater health impact in the white than the black population. Given the higher frequency of occurrence in the black population of hypertension [46], obesity in women [23] and diabetes mellitus [47], it would be particularly pertinent to determine whether these risk factors are also established early in life. The Bogalusa Heart Study recently described postmortem studies of children and young adults and indicated strong correlation between the number of ante-mortem cardiovascular risk factors and the extent of atherosclerotic involvement of the aorta and coronary arteries [48]. These findings are consistent with a similar autopsy study of young men and women in which LDL plus VLDL cholesterol levels correlated positively, and HDL cholesterol negatively, with extent of aortic and coronary arterial fatty streaks and fibrous plaques [49]. These studies lend weight to the issue of
64
C.M. Loughrey et al. / Atherosclerosis 148 (2000) 57–65
targeted screening of populations of children at higher genetic risk of adverse lipid profiles, particularly in view of the fact that risk factors were noted to cluster in the Bogalusa autopsy study. Although the benefits of pharmacological intervention in children with dyslipidaemia have yet to be proven, focused efforts should be made at least to educate these children from an early age on dietary and other lifestyle measures which might modify an inherited risk of cardiovascular disease.
Acknowledgements We would like to acknowledge the assistance of Genesis Farrow, Department of Laboratory Medicine, Children’s Hospital, Boston, for measurement of cord blood lipids in the validation of the Friedewald equation in cord blood.
References [1] LaRosa JC, Hunninghake D, Bush D, et al. The cholesterol facts. A summary of the evidence relating dietary fats, serum cholesterol, and coronary heart disease. A joint statement by the American Heart Association and the National Heart, Lung, and Blood Institute. The Task Force on Cholesterol Issues, American Heart Association. Circulation 1990;81(5):1721–33. [2] Wilson PW. High-density lipoprotein, low-density lipoprotein and coronary artery disease. Am J Cardiol 1990;66(6):7A – 10. [3] Bachorik PS, Kwiterovich PO. Apolipoprotein measurements in clinical biochemistry and their utility vis-a`-vis conventional assays. Clin Chim Acta 1988;178(1):1–34. [4] Wald NJ, Law M, Watt HC, et al. Apolipoproteins and ischaemic heart disease: implications for screening. Lancet 1994;343(8889):75– 9. [5] Austin MA, Hokanson JE, Edwards KL. Hypertriglyceridemia as a cardiovascular risk factor. Am J Cardiol 1988;81(4A):7B– 12. [6] Stampfer MJ, Krauss RM, Ma J, Blanche PJ, Holl LG, Sacks FM, Hennekens CH. A prospective study of triglyceride level, low-density lipoprotein particle diameter, and risk of myocardial infarction. J Am Med Assoc 1996;276(11):882–8. [7] Bachorik PS, Lovejoy KL, Carroll MD, Johnson CL. Apolipoprotein B and AI distributions in the United States, 1988 – 1991: results of the National Health and Nutrition Examination Survey III (NHANES III). Clin Chem 1997;43(12):2364–78. [8] Srinivasan SR, Berenson GS. Childhood lipoprotein profiles and implications for adult coronary artery disease: the Bogalusa Heart Study. Am J Med Sci 1995;310(Suppl 1):S62–7. [9] Durant RH, Linder CW, Harkess JW, Gray RG. The relationship between physical activity and serum lipids and lipoproteins in black children and adolescents. J Adolesc Health Care 1983;4(1):55 – 60. [10] Connor SL, Connor WE, Sexton G, Calvin L, Bacon S. The effects of age, body wight and family relationships on plasma lipoproteins and lipids in men, women and children of randomly selected families. Circulation 1982;65(7):1290–8. [11] Berenson GS, Srinivasan SR, Frank GC, Webber LS. Serum lipid and lipoprotein in infants and children and their relationship with diet. Prog Clin Biol Res 1981;61:73–94.
[12] Parker CR, Carr BR, Simpson ER, MacDonald PC. Decline in the concentration of low-density lipoprotein-cholesterol in human fetal plasma near term. Metabolism 1983;32 (9):919–23. [13] McConathy WJ, Lane DM. Studies on the apolipoproteins and lipoproteins of cord serum. Pediatr Res 1980;14(5):757–61. [14] Tsang RC, Fallat RW, Glueck CJ. Cholesterol at birth and age 1: comparison of normal and hypercholesterolemic neonates. Pediatrics 1974;53(4):458– 70. [15] Dolphin PJ, Breckenridge WC, Dolphin MA, Tan MH. The lipoproteins of human umbilical cord blood apolipoprotein and lipid levels. Atherosclerosis 1984;51(1):109– 22. [16] Parker CR Jr., Fortunato SJ, Carr BR, Owen J, Hankins GD, Hauth JC. Apolipoprotein A-1 in umbilical cord blood of newborn infants: relation to gestational age and high-density lipoprotein cholesterol. Pediatr Res 1988;23(4):348– 51. [17] Davis PA, Forte TM, Kane JP, Hardman DA, Krauss RM, Blum CB. Apolipoprotein and size heterogeneity in human umbilical cord blood low density lipoproteins. Biochim Biophys Acta 1983;753(2):186– 94. [18] Genzel-Boroviczeny O, Forte TM, Austin MA. High-density lipoprotein subclass distribution and human cord blood lipid levels. Pediatr Res 1986;20(6):487– 91. [19] Lane DM, McConathy WJ. Factors affecting the lipid and apolipoprotein levels of cord sera. Pediatr Res 1983;17(2):83–91. [20] Warnick GR, Benderson J, Albers JJ. Dextran sulfate-Mg2 + precipitation procedure for quantitation of high-density-lipoprotein cholesterol. Clin Chem 1982;28(6):1379– 88. [21] Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low density lipoprotein cholesterol in plasma without the use of preparative ultracentrifuge. Clin Chem 1974;20:470. [22] Rifai N, Iannotti E, DeAngelis K, Law T. Analytical and clinical performance of a homogenous enzymatic LDL-cholesterol assay compared with the ultracentrifugation-dextran sulfate Mg2 + method. Clin Chem 1998;44(6):1242– 50. [23] Hutchinson RG, Watson RL, Davis CE, et al. Racial differences in risk factors for atherosclerosis. The ARIC Study. Atherosclerosis Risk in Communities. Angiology 1997;48(4):279–90. [24] Tyroler HA, Heiss G, Schonfeld G, Cooper G, Heyden S, Hames CG. Apolipoprotein A-I, A-II and C-II in black and white residents of Evans County. Circulation 1980;62(2):249–54. [25] Morrison JA, deGroot I, Kelly KA, et al. Black-white differences in plasma lipids and lipoproteins in adults: the Cincinnati Lipid Research Clinic population study. Prev Med 1979;8(1):34– 9. [26] Srinivasan SR, Frerichs RR, Webber LS, Berenson GS. Serum lipoprotein profile in children from a biracial community: the Bogalusa Heart Study. Circulation 1976;54(2):309– 18. [27] Morrison JA, deGroot I, Kelly KA, et al. Black-white differences in plasma lipoproteins in Cincinnati schoolchildren (oneto-one pair matched by total plasma cholesterol, sex, and age). Metabolism 1979;28(3):241– 5. [28] Strobl W, Widhalm K. The natural history of serum lipids and lipoproteins during childhood. Prog Clin Biol Res 1985;188:101– 21. [29] Berenson GS, Srinivasan SR, Frerichs RR, Webber LS. Serum high density lipoprotein and its relationship to cardiovascular disease risk factor variables in children — the Bogalusa heart study. Lipids 1979;14(1):91 – 8. [30] Srinivasan SR, Sundaram GS, Williamson GD, Webber LS, Berenson GS. Serum lipoproteins and endogenous sex hormones in early life: observations in children with different lipoprotein profiles. Metabolism 1985;34(9):861– 7. [31] McNamara JR, Campos H, Ordovas JM, Peterson J, Wilson PW, Schaefer EJ. Effect of gender, age, and lipid status on low density lipoprotein subfraction distribution. Results from the Framingham Offspring Study. Arteriosclerosis 1987;7(5):483–90.
C.M. Loughrey et al. / Atherosclerosis 148 (2000) 57–65 [32] Srinivasan SR, Wattigney W, Webber LS, Berenson GS. Relation of cholesterol to apolipoprotein B in low density lipoproteins of children. The Bogalusa Heart Study. Arteriosclerosis 1989;9(4):493 – 500. [33] Islam S, Gutin B, Manos T, Smith C, Treiber F. Low density lipoprotein cholesterol/apolipoprotein B-100 ratio: interaction of family history of premature atherosclerotic coronary artery disease with race and gender in 7 to 11-year-olds. Pediatrics 1994;94(4 Pt 1):494 –9. [34] Bao W, Srinivasan SR, Wattigney WA, Bao W, Berenson GS. Usefulness of childhood low-density lipoprotein cholesterol level in predicting adult dyslipidemia and other cardiovascular risks. The Bogalusa Heart Study. Arch Int Med 1996;156(12):1315– 20. [35] Lauer RM, Lee J, Clarke WR. Factors affecting the relationship between childhood and adult cholesterol levels: the Muscatine Study. Pediatrics 1988;82(3):309–18. [36] Guo S, Beckett L, Chumlea WC, Roche AF, Siervogel RM. Serial analysis of plasma lipids and lipoproteins from individuals 9– 21 years of age. Am J Clin Nutr 1993;58(1):61–7. [37] Freedman DS, Srinivasan SR, Cresanta JL, Webber LS, Berenson GS. Cardiovascular risk factors from birth to 7 years of age: the Bogalusa Heart Study. Serum lipids and lipoproteins. Pediatrics 1987;80 (5 Pt. 2):789–96. [38] Kallio MJ, Salmenpera L, Siimes MA, Perheentupa J, Miettinen TA. Tracking of serum cholesterol and lipoprotein levels from the first year of life. Pediatrics 1993;91(5):949–54. [39] Glueck CJ, Gartside PS, Tsang RC, Mellies M, Steiner PM. Black-white similarities in cord blood lipids and lipoproteins. Metabolism 1977;26(4):347–50. [40] Frerichs RR, Srinivasan SR, Webber LS, Rieth MC, Berenson GS. Serum lipids and lipoproteins at birth in a biracial population: the Bogalusa heart study. Pediatr Res 1978;12(8):858– 63.
.
65
[41] Pocovi M, Ordovas JM, Grande F. Lecithin:cholesterol acyltransferase, lipids and lipoproteins in maternal and umbilical cord plasma. Artery 1983;11(4):264– 72. [42] Iscan A, Yigitoglu MR, Ece A, Ari Z, Akyildiz M. The effect of cigarette smoking during pregnancy on cord blood lipid, lipoprotein and apolipoprotein levels. Jpn Heart J 1997;38(4):497– 502. [43] Chen W, Srinivasan SR, Bao W, Wattigney WA, Berenson GS. Sibling aggregation of low- and high-density lipoprotein cholesterol and apolipoproteins B and A-I levels in black and white children: the Bogalusa Heart Study. Ethn Dis 1997;7(3):241–9. [44] Srinivasan SR, Dahlen GH, Jarpa RA, Webber LS, Berenson GS. Racial (black – white) differences in serum lipoprotein (a) distribution and its relation to parental myocardial infarction in children. Bogalusa Heart Study. Circulation 1991;84(1):160–7. [45] Rifai N, Heiss G, Doetsch K. Lipoprotein(a) at birth, in blacks and whites. Atherosclerosis 1992;92(2 – 3):123 – 9. [46] Arnett DK, Tyroler HA, Burke G, Hutchinson R, Howard G, Heiss G. Hypertension and subclinical carotid artery atherosclerosis in blacks and whites. The Atherosclerosis Risk in Communities Study. ARIC Investigators. Arch Int Med 1996;156(17):1983– 9. [47] Miller JM, Miller JM. Diabetes mellitus and hypertension in black and white populations. South Med J 1986;79(10):1229. [48] Berenson GS, Sathanur R, Srinivasan SR, Weihang Bao B, Newman WPI, Tracy RE, Wattigney WA. Association between multiple cardiovascular risk factors and atherosclerosis in children and young adults. New Engl J Med 1998;338:1650–6. [49] McGill HC Jr., McMahan CA, Malcom GT, Oalmann MC, Strong JP. Effects of serum lipoproteins and smoking on atherosclerosis in young men and women. The PDAY Research Group. Pathobiological Determinants of Atherosclerosis in Youth. Arterioscler Thromb Vasc Biol 1997;17(1):95 – 106.