Pediatric reference intervals for lipids and apolipoproteins on the VITROS 5,1 FS Chemistry System

Clinical Biochemistry 39 (2006) 978 – 983

Pediatric reference intervals for lipids and apolipoproteins on the VITROS 5,1 FS Chemistry System Paul M. Yip a,b,1 , Man Khun Chan a , Joanna Nelken a , Nathalie Lepage c , George Brotea d , Khosrow Adeli a,b,⁎ b

a Division of Clinical Biochemistry, The Hospital for Sick Children, Toronto, ON, Canada M5G 1X8 Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada M5G 1L5 c Division of Biochemistry, Children's Hospital of Eastern Ontario, Ottawa, ON, Canada K1H 8L1 d Ortho-Clinical Diagnostics, Rochester, NY, USA

Received 10 March 2006; received in revised form 2 June 2006; accepted 26 June 2006 Available online 22 July 2006

Abstract Objectives: Lipid biomarkers are integral in the assessment of dyslipidemia and cardiovascular risk, conditions that have become increasingly prevalent in pediatric populations. A comprehensive set of pediatric reference intervals for traditional or recently established lipid analytes is not currently available. Design and methods: 525 outpatient samples from a pediatric population were categorized into five age groups ranging from 0 to 20 years of age. Groups were further partitioned by gender. Serum or plasma samples were analyzed on the VITROS 5,1 FS Chemistry System for cholesterol and triglycerides by dry-film methods, direct HDL-C and LDL-C by selective detergent elimination, and apolipoproteins AI and B by immunoturbidimetry. Reference intervals were established by non-parametric methods at the 2.5th and 97.5th percentiles. Results: Lipid levels show age- and gender-related differences, particularly during the first year of life and in young adults following puberty. Concentrations of total cholesterol, LDL-C, and apo B were lowest in the 12 months after birth and remained relatively constant throughout childhood, but decreased for males in early adulthood. Triglyceride levels increased gradually throughout childhood and adolescence, and along with cholesterol, the upper limits of these intervals exceeded the recommended concentrations of lipid levels in children. For HDL-C and apo AI, no age- or sex-related differences were found until after puberty when values for males decreased slightly. Conclusions: Our current reference intervals in children and adolescents provide an important update for lipid markers and suggest earlier incidence of hypercholesterolemia when compared to previous ranges. Increased profiling of lipids is anticipated, and these will aid in the early assessment of cardiovascular risk in pediatric populations. © 2006 The Canadian Society of Clinical Chemists. All rights reserved. Keywords: Pediatric chemistry; Newborns; Adolescents; Cholesterol; Triglycerides; HDL cholesterol; LDL cholesterol; Apolipoprotein; Cardiovascular risk factor; Metabolic syndrome

Introduction In adults, the utility of lipid biomarkers has evolved from traditional reference intervals to defined dyslipidemic states with ⁎ Corresponding author. Department of Paediatric Laboratory Medicine, Hospital for Sick Children, 555 University Avenue, Rm. 3652, Toronto, ON, Canada M5G 1X8. Fax: +1 416 813 6257. E-mail addresses: [email protected] (P.M. Yip), [email protected] (K. Adeli). 1 Current address: Toronto Medical Laboratories, University Health Network, 200 Elizabeth Street, 3ES-412, Toronto, ON, Canada M5G 2C4. Fax: +1 416 586 1426.

emphasis placed on risk reduction of cardiovascular events, diabetes, and their co-morbidities. Reference to established marker levels like cholesterol, triglycerides, HDL-cholesterol (HDL-C), and LDL-cholesterol (LDL-C) is frequently made in major clinical practice guidelines and position statements [1–3]. Despite the recent and widespread recognition that changes in these lipid-related risk factors begin in childhood, our understanding of normal and abnormal values in the pediatric population remains limited. Although the National Cholesterol Education Program (NCEP) made recommendations on the cholesterol levels in children and adolescents [4], screening of youth remains controversial and reference intervals are needed

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P.M. Yip et al. / Clinical Biochemistry 39 (2006) 978–983

in the interpretation of laboratory results. Furthermore, several new and emerging markers have moved from the research setting into the clinical laboratories. For example apo AI and apo B have demonstrated utility in major studies which may enhance and potentially exceed the value of traditional markers [5]. Childhood dyslipidemia has become a highly important health concern due to its association with increased risk for cardiovascular disease and the metabolic syndrome with consequent occurrence of cardiovascular mortality and type 2 diabetes later on in adulthood, respectively. The existence of an obesity epidemic in children and adolescents is now well appreciated [6–8], and childhood obesity is commonly associated with dyslipidemia [9]. From large population studies such as the Bogalusa Heart Study and others, cardiovascular risk factors traditionally applied to the adult population are apparent in children and young adults which are strongly associated with the development of atherosclerotic disease [10–12]. Moreover, dyslipidemia is among various factors that cluster with obesity and insulin resistance states in the criteria that define the metabolic syndrome [13], and epidemiological studies indicate that the biochemical changes in the lipid profile and glycemic control originate during childhood [14]. In a recent review by Mansoub et al. [15], a gap analysis of reference intervals for the pediatric population for several established and emerging biomarkers was presented. In the present study, we have established reference intervals on a new clinical chemistry platform (VITROS 5,1 FS) for five age groups covering 0–20 years of age from an outpatient population for six biochemical lipid markers (cholesterol, triglycerides, HDL-C, LDL-C, apo AI and apo B) for the assessment of these levels in children and young adults.

Materials and methods Subject selection and sample collection Serum or plasma was obtained from leftover specimens from an outpatient population deemed to lack the presence of a metabolic illness. For children and adolescents, this included dentistry, orthopedic, and plastic surgery patients and patients undergoing elective surgeries, while neonatal and infant samples were from elective surgeries, orthopedic, dermatology, and infectious disease clinics. Serum and heparin plasma samples were collected from plastic tubes or syringes without gel over a six-month period in 2005. All samples were stored frozen at − 80°C prior to analysis and subjected to only one freeze thaw cycle. Samples were anonymized prior to analysis and data collection as required by our institutional ethics review board. Analytical method and performance All analyses were performed on the VITROS 5,1 FS Chemistry System (Ortho-Clinical Diagnostics, Raritan, NJ) using the manufacturer's reagents and calibrators. For the

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cholesterol and triglycerides lipids, 5.5 μL of sample was measured by multilayer film dry-slide chemistry with colorimetric detection. Lipoprotein analysis of HDL-C and LDL-C was measured in 2.7 μL samples in a two-step reaction sequence whereby free cholesterol and the undesired cholesterol esters are eliminated in the first reaction. The addition of specific surfactants in the second reaction then dissociates the desired lipoprotein particle for total cholesterol measurement by a colorimetric reaction. Specific protein analysis of apolipoproteins AI and B was performed by immunoturbidimetric analysis of light scattering at 340 nm in an end-point reaction. Briefly, a 6.7 μL sample is diluted with reagent containing surfactant and polymer and subsequently reacted with specific polyclonal antisera to generate immune complexes which increase the turbidity of the solution. The total imprecision of the lipid and lipoprotein assays was <3% and <4%, respectively, for two levels of quality control over 84 days and a single calibration. These methods are traceable to their respective national reference methods [16–20]. For the apo AI and apo B, imprecision was <4% and <5%, respectively, based on one level of quality control over 82 days and a single calibration. Statistical analysis A preliminary screening procedure to assess the quality of data was performed before subsequent software-based computation. Outliers were identified by applying Chauvenet's criteria, and in a few cases, samples with outliers for multiple analytes were completely removed from the sample set. For each analyte, the data were partitioned by gender, and the central 95% interval was determined by non-parametric rank number analysis. The Z test for equality of means and the Ratio Test for equality of variance were used to compare groups after partitioning by gender. The direct LDL-C result was compared against the calculated LDL-C as performed with the Friedewald equation for SI units [LDL-C = TC − HDL-C − (TG / 2.2)].

Results and discussion Reference intervals for serum (or plasma) lipid biomarkers determined on the VITROS 5,1 FS Chemistry System are summarized in Table 1 and Fig. 1. The number of samples for each reference interval depended on the availability of leftover laboratory specimens from presumptive healthy outpatients for each analyte. No statistical differences in the mean or variance were found between males and females for the age groups spanning 1–14 years. While differences in the mean existed for some analytes in the 0–12 months and 15–20 years age groups, the limited sample availability, especially from males, may be a contributing reason. Regardless, non-parametric methods were used in all cases. Until large population-based studies are performed that include the early years, hospital-based studies using outpatient samples are a practical approach and should provide useful clinical information. Each analyte is discussed separately below.

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Table 1 Summary of pediatric reference intervals for serum/plasma lipid biomarkers (2.5th to 97.5th percentiles) for males and females obtained in this study Age

Cholesterol (mmol/L)

Cholesterol (mg/dL)

n

Male

n

Female

n

0–12 months 1–5 years 6–10 years 11–14 years 15–20 years

28 64 53 44 43

2.20–4.21 2.57–5.46 2.82–5.27 3.11–5.21 2.81–4.89

40 48 64 62 71

2.75–4.81 3.08–5.22 2.95–5.57 3.20–5.48 2.69–5.88

28 85–163 40 106–186 64 99–211 48 119–202 53 109–204 64 114–215 44 120–201 62 124–212 43 109–189 71 104–227

Age

Triglycerides (mmol/L) n

Male

n

Female

n

0–12 months 1–5 years 6–10 years 11–14 years 15–20 years

27 64 50 43 39

0.56–2.28 0.50–1.77 0.50–2.12 0.51–2.12 0.56–2.07

39 52 63 55 67

0.73–2.64 0.47–1.75 0.50–2.19 0.57–2.36 0.60–2.35

27 64 50 43 39

Age

HDL-C (mmol/L)

Male

n

Female

Triglycerides (mg/dL) Male 50–202 44–157 44–188 45–188 50–183

n 39 52 63 55 67

Female 65–234 42–155 44–194 50–209 53–208

HDL-C (mg/dL)

n

Male

n

Female

n

0–12 months 1–5 years 6–10 years 11–14 years 15–20 years

27 65 52 43 43

0.74–2.09 0.67–1.65 0.65–1.92 0.65–1.81 0.60–1.43

42 52 63 61 69

0.61–2.18 0.75–1.86 0.78–1.72 0.69–1.81 0.74–1.78

27 65 52 43 43

Age

LDL-C (mmol/L)

Male 29–81 26–64 25–74 25–70 23–55

n 42 52 63 61 69

Female 24–84 29–72 30–67 27–70 29–69

LDL-C (mg/dL)

n

Male

n

Female

n

Male

0–12 months 1–5 years 6–10 years 11–14 years 15–20 years

27 62 52 42 42

0.64–2.31 1.03–3.31 1.27–3.35 1.32–3.39 1.24–3.02

41 47 62 60 70

0.88–2.87 1.61–3.32 1.51–3.34 1.58–3.38 1.18–3.62

27 62 52 42 42

Age

Apolipoprotein AI (g/L)

Apolipoprotein AI (mg/dL)

n

Male

n

Female

n

0–12 months 1–5 years 6–10 years 11–14 years 15–20 years

28 63 53 43 41

1.01–1.93 0.97–1.71 1.02–1.84 0.83–1.79 0.85–1.60

41 50 63 60 68

0.90–1.75 0.90–1.67 0.97–1.78 0.97–1.76 0.96–1.89

28 101–193 41 63 97–171 50 53 102–184 63 43 83–179 60 41 85–160 68

Age

Apolipoprotein B (g/L) n

Male

n

Female

n

0–12 months 1–5 years 6–10 years 11–14 years 15–20 years

27 65 54 42 43

0.35–0.77 0.46–1.01 0.42–0.95 0.50–1.00 0.47–1.02

41 51 60 60 69

0.39–0.90 0.44–0.98 0.46–1.01 0.47–1.04 0.45–1.17

27 65 54 42 43

25–89 40–128 49–130 51–131 48–117

Male

n 41 47 62 60 70

n

Female 34–111 62–128 58–129 61–131 46–140

Female 90–175 90–167 97–178 97–176 96–189

Apolipoprotein B (mg/dL) Male 35–77 46–101 42–95 50–100 47–102

n 41 51 60 60 69

Female 39–90 44–98 46–101 47–104 45–117

Cholesterol and triglycerides Widely available as part of lipid profiles in general chemistry, cholesterol and triglycerides are routinely ordered to assess individual risk for cardiovascular disease, diabetes, or dyslipidemia. Our data provide an update for pediatric reference intervals in a North American population for these two analytes since the calculation from previous sample groups dates back from over a decade ago [21,22]. For total cholesterol, values

rise in the first year after birth and plateau soon thereafter as noted previously [21,22]. We observed lower levels in the total cholesterol levels in our 15–20 years male group compared to females (p < 0.001). While sexual maturation is reported to decrease levels of various serum lipids which often are more pronounced in boys than in girls [23], others have reported that male cholesterol levels rise sooner in early adulthood [24]. However, a striking difference from these previous studies is the increase in the upper limit of the reference intervals in the 1–5 years age group while other age groups in our study population remained relatively unchanged. According to the recommendations of the NCEP expert panel on blood cholesterol levels in children and adolescents [4], more individuals at younger ages would be considered to have a high level of cholesterol (i.e. > 5.2 mmol/L). In the 1–5 years, 6–10 years, and 11–14 years age groups, the proportion of individuals considered to have high cholesterol levels was approximately 5% and slightly higher for females. However, 11% of the individuals in the 15–20 years age group had high cholesterol, with most being female. Thus, our current reference intervals for cholesterol may reflect the increased prevalence of hypercholesterolemia in the pediatric population. The triglyceride reference interval was higher during the first year of life, presumably due to milk-rich feeding. Following the first year, the values decreased to their lowest levels and then gradually increased with age in agreement with other studies which indicated a similar trend in regard to age [24,25]. Furthermore, no difference between gender was observed. We were unable, however, to determine the fasting state at the time of sample collection, and thus these intervals for triglycerides would be better suited under non-fasting circumstances. Currently, fasting triglyceride levels in adults exceeding 1.7 mmol/L is a proposed criterion in national and international definitions of metabolic syndrome [2,26], while recent nutritional guidelines by the American Heart Association and endorsed by the American Academy of Pediatrics consider values above 2.26 mmol/L to be abnormal [27]. However, a proper fasting lipid profile may be difficult to obtain especially from young children and non-fasting ranges may be more practical for health assessment. Indeed, recent evidence suggests that elevated non-fasting triglycerides is associated with increased risk for coronary heart disease in adults [28]. Reference intervals in such cases remain limited [29], and improved ease of collection should prompt future studies to determine its usefulness. HDL-C and LDL-C HDL-C and LDL-C have more specific roles in the risk assessment and directing therapy, whereas cholesterol and triglycerides have primary roles as screening tests. The accessibility of HDL-C and LDL-C assays has greatly improved through the development of automated homogeneous methods that have enabled non-reference laboratories to perform routine measurements without the need for manual preparative separation (i.e. for HDL-C) or providing calculated values (i.e. for LDL-C). On the VITROS 5,1 FS, these are direct assays and do

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Fig. 1. Box plot distributions showing (A) total cholesterol, (B) triglycerides, (C) HDL-C, (D) LDL-C, (E) apo AI, and (F) apo B values (5th, 25th, 50th, 75th, and 95th percentiles) across the age groups for males (dark box) and females (white box).

not require any sample pre-treatment, and the precision of assays was acceptable by the NCEP goals for analytical performance [30]. Finally, the calculated LDL-C value from the Friedewald calculation based on the levels of non-LDL lipids showed excellent agreement with the direct LDL-C assay performed from the same sample (y = 0.999x + 0.005, R = 0.999, N = 478). The reference intervals for HDL-C were approximately the same from the first year of life to early adolescent 11–14 years age group without difference between genders. Other studies suggest that these values increase with age beginning from the newborn stage [31,32], but our wider age group of 0–12 months likely obscures this observation. In the oldest age group, the

intervals remained relatively unchanged for females while values decreased somewhat for males (p < 0.001). This pattern over childhood development is consistent with previous findings from other studies including a large cross-sectional group [24,25]. Direct measurement of LDL-C on auto-analyzers has simplified analysis and is likely to replace the calculated values used for earlier intervals [31]. Using a direct LDL-C method, our reference interval was lowest during the first year of life and then rose to a steady level that persisted over 1–14 years without difference between gender. In the 15–20 years age group, female values were higher than male values (p < 0.005).

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A recent study by Soldin et al. [33] showed a similar pattern to intervals obtained from hospitalized patients. While their use of the Hoffman technique in which Gaussian fitting and extrapolation to a limit are applied is reported to have statistical limitations in the estimation of reference intervals [34], the concordance of our interpretation for LDL-C intervals in children supports its use in situations where normal patient samples are difficult to obtain. The recent consensus guidelines for children and adolescents consider fasting LDL-C levels ≥ 2.6 mmol/L (100 mg/dL) as borderline and levels ≥ 3.4 mmol/L (130 mg/dL) as abnormal [27]. As with triglycerides, the intervals presented in this study should be applied to non-fasting situations, and the identification and treatment of dyslipidemia need to be viewed from the assessment of several lipid biomarkers and not by a single elevated result. Finally, LDL particle size is another relatively new lipid biomarker that is associated with increased risk for CVD and is characteristic of insulin resistance syndrome, and a large population study in children and adolescents has been recently published [35].

Apolipoproteins AI and B Apo AI is the major structural protein of HDL, while apo B is mostly associated with LDL but is also a component of other lipoproteins including chylomicrons, VLDL, IDL, and lipoprotein(a). Measurement of apo AI and apo B is becoming widely available on automated immunoassay platforms, and their increased use in the future is predicated upon demonstrated utility in predicting CVD risk through major prospective studies [5]. By analogy to their HDL and LDL counterparts, low levels of apo AI and high levels of apo B are present in patients with coronary heart disease [36,37]. Another advantage of these assays over traditional lipid testing is that fasting is not required. Between the two, more attention has focused on apo B because serum concentrations reflect the total number of atherogenic particles rather than LDL particles alone. Furthermore, adults with increased levels of both triglycerides and apo B are more prevalent in patients with metabolic syndrome and have higher risk for CVD than those with high triglycerides but normal apo B levels [13]. To date, limited reference intervals across a broad pediatric population have been available for apo AI and B. In parallel with our observations and interpretation, the reference intervals for apo AI and apo B follow similar patterns across age groups and between genders as with HDL-C and LDL-C, respectively. Apo AI levels appear to level during the first year of life and remain fairly constant until after puberty when levels in males fall relative to females (p < 0.001), a trend which has been observed previously although others have reported lowest levels to be in neonates [31,32]. In the NHANES III study (1988–1991), the first large population study of healthy individuals with standardized apo AI and B measurements, similar levels of apo AI occur in children followed by a decrease in males during early adulthood while levels tended to increase in females [38]. The reference intervals from our study are comparable to those in the NHANES III

study, although the latter study found higher apo AI levels in black children than in white children. In our study, apo B levels rise during the first year after birth and remained steady thereafter through puberty. As reported previously, concentrations are lowest at birth and quickly rise to childhood and adolescent levels during the first year of life [31,32], and this was reflected in our analysis. No difference in gender was noted until after puberty when female values were higher than males, although this may be a result of a limited sample size for males in the 15–20 years age group. In the NHANES III study, apo B levels were steady from 4 to 19 years with no difference between the sexes then abruptly increased after 20 years of age and continued to rise throughout adulthood for both sexes until ∼ 60 years of age [38]. However, other studies including the Framingham Offspring Study suggest that more prominent age-related changes occur in females [39]. Lastly, ethnicity was not evaluated as part of our study, but data from the NHANES III indicate that differences between black and whites exist but only in adults [38]. Therefore, reference intervals for the young adult group (15–20 years) may be easily influenced by demographics of the local population and should be interpreted with caution due to rapid rises occurring in the third decade of life. Acknowledgments P.M.Y. is supported by the Sanford Jackson Fellowship at The Hospital for Sick Children. This work was supported by a research grant from Ortho-Clinical Diagnostics, USA. References [1] Executive summary of the third report of the national cholesterol education program (NCEP) expert panel on detection, evaluation, and treatment of high blood cholesterol in adults (Adult Treatment Panel III). JAMA 2001;285:2486–97. [2] Genest J, Frohlich J, Fodor G, McPherson R. Recommendations for the management of dyslipidemia and the prevention of cardiovascular disease: summary of the 2003 update. CMAJ 2003;169:921–4. [3] Grundy SM, Cleeman JI, Merz CN, Brewer Jr HB, Clark LT, Hunninghake DB, et al. Implications of recent clinical trials for the National Cholesterol Education Program Adult Treatment Panel III guidelines. Circulation 2004;110:227–39. [4] American Academy of Pediatrics. National cholesterol education program: report of the expert panel on blood cholesterol levels in children and adolescents. Pediatrics 1992;89:525–84. [5] Sniderman AD, Furberg CD, Keech A, Roeters van Lennep JE, Frohlich J, Jungner I, et al. Apolipoproteins versus lipids as indices of coronary risk and as targets for statin treatment. Lancet 2003;361:777–80. [6] Troiano RP, Flegal KM. Overweight children and adolescents: description, epidemiology, and demographics. Pediatrics 1998;101:497–504. [7] Fagot-Campagna A, Pettitt DJ, Engelgau MM, Burrows NR, Geiss LS, Valdez R, et al. Type 2 diabetes among North American children and adolescents: an epidemiologic review and a public health perspective. J Pediatr 2000;136:664–72. [8] Daniels SR, Arnett DK, Eckel RH, Gidding SS, Hayman LL, Kumanyika S, et al. Overweight in children and adolescents: pathophysiology, consequences, prevention, and treatment. Circulation 2005;111: 1999–2012. [9] Goran MI, Gower BA. Abdominal obesity and cardiovascular risk in children. Coron Artery Dis 1998;9:483–7. [10] Berenson GS, Srinivasan SR, Bao W, Newman III WP, Tracy RE, Wattigney WA. Association between multiple cardiovascular risk factors

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