Dehydroepiandrosterone sulfate (DHEA-S) distribution in Spanish prepuberal children: Relationship with fasting plasma insulin concentrations and insulin resistance

Clinica Chimica Acta 366 (2006) 163 – 167 www.elsevier.com/locate/clinchim

Dehydroepiandrosterone sulfate (DHEA-S) distribution in Spanish prepuberal children: Relationship with fasting plasma insulin concentrations and insulin resistance Beatriz Cano a, Manuel de Oya a, Mercedes Benavente a, Enrique Viturro a, Iria de Oya a, Laura Lo´pez-Simo´n a, Ovidio Fernandez b, Carmen Garce´s a,* a

Lipid Unit, Fundacio´n Jime´nez Dı´az, Universidad Auto´noma de Madrid, Spain b Department of Medicine, Complejo Hospitalario de Ourense, Spain

Received 1 August 2005; received in revised form 26 September 2005; accepted 26 September 2005 Available online 26 October 2005

Abstract Background: The aim of this study was to analyze dehydroepiandrosterone sulfate (DHEA-S) levels in a population-based sample of Spanish prepuberal children and to investigate the relationship between DHEA-S and insulin. Methods: 854 (440 boys and 414 girls) randomly selected prepuberal children were included in our study after a sampling. Children were 6 to 8 years old and were classified for the analysis in half-year intervals. DHEA-S and insulin levels were measured. Results: DHEA-S levels increase significantly with age during prepuberty reaching the maximum level of DHEA-S for this period at 7.5 years old in girls and 8 years old in boys. Girls have significantly higher log DHEA-S levels than boys, except at the age of 8, where the levels are similar (median: 41.7 nmol/l girls and 41.1 nmol/l boys). DHEA-S correlates positively and significantly with weight, height, and BMI in all age intervals but the correlation between DHEA-S and insulin and HOMA is present only at the age of 6.5 in boys and 8 in girls. Conclusions: We report data about the distribution of DHEA-S in the Spanish prepuberal population. The maximum level of DHEA-S in this prepuberal period was reached before in girls than in boys, with girls having higher DHEA-S levels than boys until the end of this period. We found an important association between DHEA-S levels and weight, height and BMI but an inconsistent association of DHEA-S with insulin and HOMA. D 2005 Elsevier B.V. All rights reserved. Keywords: DHEA-S: dehydroepiandrosterone sulfate; Prepuberal children

1. Introduction Dehydroepiandrosterone sulfate (DHEA-S) is the most abundant circulating steroid hormone in humans, with a wide variety of physiological effects [1,2]. Multiple studies have analyzed the relationship of circulating DHEA-S with cardiovascular disease [3,4] and have studied its association

Abbreviations: DHEA-S, dehydroepiandrosterone sulfate; HOMA, homeostatic model assessment; BMI, body mass index. * Corresponding author. Unidad de Lı´pidos, Fundacio´n Jime´nez Dı´az, Avda. Reyes Cato´licos, 2, 28040 Madrid, Spain. Tel.: +34 91 5432880; fax: +34 91 5444100. E-mail address: [email protected] (C. Garce´s). 0009-8981/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2005.09.023

with different cardiovascular risk factors [5]. The existence of a correlation between DHEA-S and insulin has been proposed, but this correlation is controversial. While some studies in adults find that DHEA-S levels and fasting insulin levels are negatively correlated [6], other studies did not find this correlation [7,8]. The correlation seems to be ageand sex-specific [9,10]. Adrenarche is related with the onset of dehydroepiandrosterone (DHEA) and DHEA-S production, which can be detected at around 6 years of age. The phenotypic result of adrenarche is pubarche that occurs in both girls and boys at about age 8. A reversal of adrenarche appears to occur in the ageing process. Premature and exaggerated adrenarche can be indicative of the future onset of adult diseases [11], thus

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increasing the clinical relevance of adrenarche. The role of DHEA-S in this period of life is not completely understood. This article analyzes the DHEA-S throughout the prepuberal age and its relationship with insulin and insulin resistance during this period.

2. Material and methods

millimeter using a portable stadiometer, and weight was recorded to the nearest 0.1 kg using a standardized electronic digital scale. From these measurements, body mass index (BMI) (weight in kilograms divided by the square of the height in meters: kg/m2) was then computed. A questionnaire was provided along with consent forms and parents were asked to provide information regarding the child’s general health and birth weight as recorded in a legal birth certificate.

2.1. Subjects and study design 2.4. Biochemical data The population included 854 healthy school children (440 boys and 414 girls) 6 to 8 years old (mean age of 6.7 years) who participated in a voluntary survey of cardiovascular risk factors in Spain over the period 1998– 2000 in whom DHEA-S levels have been measured. Children were selected by means of random cluster-sampling in schools, and stratified by sex and type of school (i.e., public versus private). More detailed information about the design of the study is available in previous publications [12,13]. The study protocol complied with Helsinki Declaration guidelines and Spanish legal provisions governing clinical research on humans, and was approved by the Clinical Research Ethics Committee of the Fundacio´n Jime´nez Dı´az in Madrid. 2.2. Data collection and study variables The study was orally presented to the School Board of each of the schools. Following this, a letter was circulated to the parents of all children invited to participate in the study, outlining the study goals and procedures. Parents were required to sign a written consent for participation of their children in the study. All children reported by parents to be suffering from metabolic, endocrine, liver or kidney disorders were excluded to rule out any possible alteration in the values of the variables of interest. 2.3. Anthropometric variables Measurements were taken with the children lightly dressed and barefoot. Height was measured to the last

Fasting (12 h) venous blood samples were obtained by venipuncture into Vacutainer tubes. Samples were kept on ice and sent to the study’s central laboratory for analysis. Once centrifuged, the fractions were separated and frozen at 70 -C. Serum insulin concentrations were measured by RIA using a commercial kit (BI-Insulin IRMA, Bio-Rad, France). Insulin resistance was estimated using the homeostasis model assessment for insulin resistance (HOMA; fasting insulin [AU/ml]  fasting glucose [mmol/l] / 22.5) [14]. Plasma DHEA-S was determined by RIA, using a commercial kit (DHEA-S RIA, DSL, Texas, USA). Plasma glucose concentration was determinated using a method which involves the phosphorylation of glucose by hexokinase and the action of glucose-6-phosphate dehydrogenase on the product. 2.5. Statistical analysis Descriptive statistics (mean, standard deviation, median) were used to characterize our study population. Differences in anthropometric and biochemical measurements between boys and girls were evaluated using a t-test. Analysis of variance (ANOVA) was used to compare anthropometric and biochemical measurements by age group. Because DHEA-S exhibited important departures from normality, logarithmic transformations (log10) of the variable were used to compare DHEA-S concentration by gender or age. We used sample quantiles to estimate the corresponding population percentiles. Spearman correlation coefficients were used to examine the relationship between DHEA-S

Table 1 Anthropometric variables, insulin and HOMA (mean T S.D.) among Spanish school children by group of age and sex 6.5 years

Weight (kg) Height (m) BMI (kg/m2) Birth weight (kg) Glucose (mg/dl) Insulin (nmol/l) HOMA

7 years

7.5 years

8 years

Boys (n = 134)

Girls (n = 130)

Boys (n = 129)

Girls (n = 129)

Boys (n = 98)

Girls (n = 76)

Boys (n = 79)

Girls (n = 79)

24.5 T 4.1 1.22 T 0.05 16.4 T 2.1 3.5 T 0.6 90.6 T 8.1 24.6 T 20.3 0.78 T 0.68

23.8 T 3.6 1.20 T 0.05 16.3 T 2.0 3.2 T 0.5 86.9 T 9.0 23.2 T 16.6 0.68 T 0.53

26.7 T 4.6 1.25 T 0.05 17.0 T 2.4 3.4 T 0.5 91.7 T 7.7 23.9 T 15.9 0.74 T 0.52

25.9 T 4.8 1.24 T 0.06 16.9 T 2.3 3.2 T 0.5 88.2 T 7.9 23.9 T 16.6 0.73 T 0.54

28.3 T 6.4 1.28 T 0.06 17.3 T 2.9 3.5 T 0.5 93.0 T 8.4 23.9 T 15.9 0.77 T 0.55

28.5 T 5.8 1.27 T 0.06 17.7 T 3.1 3.2 T 0.5 90.4 T 8.8 25.4 T 15.9 0.79 T 0.50

29.7 T 6.0 1.29 T 0.05 17.6 T 2.6 3.5 T 0.5 95.3 T 9.4 25.4 T 20.3 0.85 T 0.78

29.2 T 5.2 1.29 T 0.06 17.5 T 2.4 3.3 T 0.5 92.2 T 7.6 31.2 T 18.1 0.98 T 0.6

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Table 2 Descriptive statistics for DHEA-S and Log DHEA-S in boys and girls by age Total Boys (n = 440) DHEA-S (nmol/l) Mean T S.D. 924 T 940 Median 575 Log DHEA-S (nmol/l) Median 36.0 pa 0.004 a

6.5 years

7 years

7.5 years

8 years

Girls (n = 414)

Boys (n = 134)

Girls (n = 130)

Boys (n = 129)

Girls (n = 129)

Boys (n = 98)

Girls (n = 76)

Boy (n = 79)

Girls (n = 79)

1022 T 1018 677

635 T 750 406

643 T 550 523

962 T 1018 559

1156 T 1243 810

953 T 817 739

1199 T 970 892

1319 T 1092 892

1256 T 1094 936

37.8

31.8 0.05

34.7

35.6 0.03

39.8

38.9 0.03

41.1

41.1 0.68

41.7

T-test analysis between genders of mean values of Log DHEA-S by age group.

and anthropometric variables. Statistical analyses were carried out using the SPSS software package, version 9.0 (SPSS Inc., Chicago, IL).

3. Results For our study we classified prepuberal children by halfyear age intervals. Anthropometric variables, insulin and HOMA in boys and girls in these groups are shown in Table 1. Understandably, weight and height increased gradually and significantly with age in both genders. The boys had significantly higher mean height ( p < 0.05) than girls at the 6.5-year age group. Insulin and HOMA increased gradually only in girls, with these values significantly higher in the 8year-old girls than in the 6.5- and 7-year-old girls. In boys, mean DHEA-S levels were 924 T 940 nmol/l, with a median value of 575 nmol/l; in girls, mean concentration were 1022 T 1018 nmol/l, with a median value of 677 nmol/l (Table 2). These high standard deviations and these discrepancies between mean and median occurred in all age intervals. This data shows that the distribution for this variable exhibited departures from normality in this population. Because of this, logarithmic transformations (log10) were used to compare levels between groups. Using the One-Sample Kolmogorov – Smirnov Test, we observed that log transformed DHEA-S levels present a normal distribution. When looking at log DHEA-S levels by gender and age group (Table 2), we observed that, in both sexes, log DHEA-S levels increased gradually with age. In an ANOVA we observed that, in boys, this increase was significantly different between each age interval, except between the 7- and 7.5-year-old age groups. In girls, log

DHEA-S levels increased gradually reaching a maximum in the 7.5-year-old age group, which was equivalent to the levels of the girls in the 8-year-old age interval and equal to the levels of the boys at 8 years (median, 41.1nmol/l). In girls, there were significant differences between DHEA-S levels in the 6.5-year-old age group and all the other age groups. Analyzing log DHEA-S by gender we observed that girls have significantly higher overall median levels than boys. When compared by age group we see that, although the girls have significantly higher levels in every other age group, by age 8 the levels are the same between genders. Both girls and boys in this interval of age (6 to 8 years) reach the same place, but girls get there earlier. The percentile values of plasma DHEA-S concentrations by sex and age group are shown in Table 3. Girls had significantly higher DHEA-S concentrations than boys for all the percentiles except at the 90th percentile. The correlations between DHEA-S and anthropometric values, insulin measurements and HOMA for boys and girls were examined (Table 4). DHEA-S correlated positively and significantly with weight, height and BMI except at 7 for BMI, where correlation values were small (r = 0.074 in boys; r = 0.058 in girls). DHEA-S correlated negatively with birth weight except at 7.5 years in boys and 8 years in girls where correlation values were also small. Insulin and HOMA significantly correlated with DHEA-S in boys only in the 6.5-year age group and in girls only in the 8-year age interval.

4. Discussion There is evidence that the production of dehydroepiandrosterone sulfate (DHEA-S) increases before puberty,

Table 3 Percentile distribution of DHEA-S (nmol/l) concentrations in boys and girls Percentile (95% CI)

Total (n = 854) Boys (n = 440) Girls (n = 414)

P 10

P 25

P 50

P 75

P 90

152 (144 – 160) 133 (122 – 146) 184 (171 – 200)

230 (219 – 241) 198 (187 – 211) 271 (255 – 287)

496 (485 – 507) 453 (439 – 469) 539 (523 – 553)

862 (843 – 886) 805 (779 – 835) 929 (894 – 962)

1780 (1729 – 1832) 1745 (1650 – 1837) 1799 (1762 – 1856)

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Table 4 Spearman correlation coefficients for DHEA-S with anthropometric and biochemical variables in boys and girls by age group DHEA-S 6.5 years Boys (n = 134) Weight Height BMI Birth weight Insulin HOMA

0.189 0.182 0.142 0.240* 0.230* 0.207*

7 years Girls (n = 130) 0.202* 0.181 0.245* 0.296** 0.134 0.124

Boys (n = 129)

7.5 years Girls (n = 129)

0.171 0.244* 0.074 0.211* 0.162 0.131

0.205* 0.220* 0.058 0.100 0.073 0.066

Boys (n = 98) 0.523** 0.418** 0.398** 0.064 0.159 0.133

8 years Girls (n = 76) 0.207 0.241* 0.078 0.198 0.051 0.104

Boys (n = 79) 0.347** 0.440** 0.186 0.225 0.043 0.030

Girls (n = 79) 0.292* 0.161 0.278* 0.090 0.273* 0.272*

**p < 0.01, *p < 0.05.

because of the maturation of the adrenal cortex at this time [15]. Little is known about the distribution of this hormone at this prepuberal age in our population and how it is related to important factors such as insulin and insulin resistance. Thus, we proposed the analysis of plasma DHEA-S in a population-based sample of prepuberal Spanish children. Serum DHEA-S concentrations are very high in the fetus, decreasing at 6 months, because of the involution of several fetal adrenal areas, and remaining low between the ages of 1 and 6. Before the start of puberty, at the ages of 6 to 8, changes, known as adrenarche, take place in the activity of the suprarenal gland, and androgen production increases, especially that of D5 steroids (DHEA and DHEA-S). The concentration of DHEA-S continues to grow reaching adult values shortly after the end of puberty, and decreasing again later in life in both sexes [16,17]. In our study of prepuberal children, when adrenarche is taking place, we observed a progressive increase in DHEA-S levels in both sexes with a maximum value towards the end of the studied age interval, reached at 7.5 years old in girls but at 8 years old in boys, an age when DHEA-S levels are equal in both sexes. With the exception of this age, girls have significantly higher DHEA-S levels than boys. We can talk of chronological differences in the maturation status in boys and girls. This data agrees with data from other studies. For example Sizonenko, established that the maturation of the androgenic area of the adrenal cortex takes places at 7 in girls and at 8 in boys [15], a period when the DHEA-S levels increase. Sulcova´ et al., analyzing DHEA-S levels from birth to the elderly, found that, for all age groups, men had higher levels of the hormone than women with the exception of children between 6 and 10 years old, when girls had higher levels [18]. However they did not separately analyze children between 8 and 10. This study also analyzes percentile values for the distribution of DHEA-S in our Spanish prepuberal children. The possibility of establishing percentile values may be useful to analyze the population in terms of a variable for which little data has been published. Again, for all percentiles examined, except the 90th, corresponding to the percentile of the highest levels, we found consistent differences between values in boys and girls, with girls having significantly higher concentrations.

We have not found data about DHEA-S levels in other Spanish prepubertal populations, but the values in our study seem to be higher than those reported in other populations of prepubertal children. However, there are few studies describing DHEA-S levels for this concrete age interval and they include a small number of children [18,19]. DHEA-S concentration in children seems to be related with fetal growth and this can be a modulator of adrenarche [20]. In our study we have observed that DHEA-S levels negatively and significantly correlate with birth weight in almost all the age intervals in both gender. There are multiple studies that associate low birth weight with high DHEA-S levels in children [21,22], thus a restriction in fetal growth can result in a prominent adrenarche. In our prepuberal children we have found important correlations between DHEA-S and weight, height and BMI. A longitudinal study has shown that an increase in BMI has a strong correlation with an increase in androgen secretion during adrenarche [23]. The association between DHEA-S and weight and height has also been found in children between 10 and 14 years old [24]. In adults, this association varies depending on sex and age [25 –28]. The reason for these discrepancies remains unclear. During puberty, the increase in sexual hormones has been associated with a state of apparent insulin resistance [29]. Negative correlations have been found between insulin sensitivity and DHEA-S levels when analyzing prepuberal and puberal children of both genders together [30]. This correlation has not been found when analyzing only prepuberal boys [31]. In our prepuberal girls the correlations between DHEA-S and insulin and HOMA were significant only at 8 years of age. In boys the correlations were significant only at the age of 6.5. Our data establishes differences between gender within the concrete prepuberal period. The relationship between adrenal androgens and insulin already present at the end of the prepuberal period for girls [32] seems to be also present in other moments of the prepuberal period studied. The presence of a correlation in boys at the beginning of the prepuberal period that is lost at the end of the period is difficult to understand and raises interesting questions. In summary, from this study in prepuberal children, we can say that in this adrenarchic period DHEA-S levels

B. Cano et al. / Clinica Chimica Acta 366 (2006) 163 – 167

increase significantly with age in both sexes. Girls reach the maximum value of DHEA-S in this period at 7.5 years while boys do not get it until 8. The association of DHEA-S levels with weight, height and BMI is clear, but the association with insulin is evident only at concrete moments of the prepuberal period studied in each gender. Further studies are needed to evaluate the reasons for these results.

Acknowledgements This study was supported by grants from the Fondo de Investigacio´n Sanitaria (FIS02/3104, FIS 020994) and Comunidad de Madrid (08.4/0006/1997, 08.4/0012.1/ 2003). B. Cano, I. de Oya, and L. Lo´pez-Simo´n are fellows of the Conchita Ra´bago Foundation. We thank Alexander G. Borun, M.D. for manuscript revision.

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