ω-3 Long-Chain Polyunsaturated Fatty Acids and Fatty Acid Desaturase Activity Ratios as Eventual Endophenotypes for ADHD

Abstract

Objective: Epidemiological studies suggest that long-chain polyunsaturated fatty acids (LC-PUFAs) may be suitable as endophenotypes for ADHD. To be appropriated vulnerability traits, endophenotypes should be altered in unaffected relatives of index cases. Serum profiles of LC-PUFAs in unaffected relatives of ADHD patients remain understudied. The main objective of this study was to compare serum LC-PUFAs in ADHD patients, unaffected relatives of index cases, and general-population unaffected participants. Method: LC-PUFA profiles of 72 participants (27 ADHD patients, 27 unaffected relatives, and 18 general-population participants) were obtained by gas chromatography–mass spectrometry (GC-MS). Groups were compared by parametrical statistics. Results: Unaffected females from the general population presented lower Docosapentaenoic acid (DPA; p = .0012) and a-linolenic acid (ALA; p = .0091) levels compared with ADHD females and unaffected relatives. In addition, docosahexaenoic acid (DHA)/ALA and DHA/DPA ratios, addressing desaturase activity, were significantly lower in ADHD patients and unaffected relatives of ADHD patients in the female-subgroup (p = .022 and .04, respectively). Conclusion: DHA/ALA, DHA/DPA, serum DPA, and serum ALA may be suitable as endophenotypes for ADHD women.

Keywords

ADHD ω-3 LC-PUFAs endophenotype

Introduction

Although the genetic base of ADHD is extensively supported by twin and adoption studies, which estimate a genetic component of around 75% for the disorder (Derks et al., 2008; Faraone & Doyle, 2000; Freitag, Rohde, Lempp, & Romanos, 2010), the efforts devoted to identify specific variants conferring genetic risk to ADHD have been less fruitful than anticipated, with many studies yielding to inconsistent results and providing less potentially clinically relevant information than previously expected. In opinion of some authors, the power of genetic studies in neuropsychiatry is actually hampered by traditional nosological definitions, which may be suboptimal for genetic analysis, because they are based in expert consensus and could comprise heterogeneous phenotypes, some of them corresponding to nongenetic phenocopies (Castellanos & Tannock, 2002; Goldman & Ducci, 2007). Alternatively, it has been proposed that “intermediate phenotypes” or “endophenotypes” should replace nosological definitions as phenotypes for genetic studies, because they better represent gene action and may act as heritable vulnerability traits.

The search for potential ADHD endophenotypes has been focus on neurocognitive and electrophysiological/neuroimaging markers with some promising results, but biochemical markers have been largely ignored (Durston, de Zeeuw, & Staal, 2009; Goos, Crosbie, Payne, & Schachar, 2009; Hale et al., 2010; Loo et al., 2010; Nigg, Blaskey, Stawicki, & Sachek, 2004; Slaats-Willemse, Swaab-Barneveld, de Sonneville, van der Meulen, & Buitelaar, 2003). In general, biochemical markers may be advantageous when compared with neurocognitives and electrophysiological/neuroimagings, because they are generally easier to quantify by interlaboratory reliable, sensitive, and specific methods, allowing valid multicenter collaboration (Henriquez-Henriquez, Zamorano-Mendieta, Rothhammer-Engel, & Aboitiz, 2010; Kuntsi, Andreou, Ma, Borger, & van der Meere, 2005).

Among biochemical putative endophenotypes for ADHD, long-chain polyunsaturated fatty acids (LC-PUFAs) seem especially attractive due to their key role as structural components of the nervous system and as pivotal actors in neural development, neurotransmission (especially related to monoaminergic systems), synaptic plasticity, and apoptosis (Chalon, 2006; du Bois, Deng, & Huang, 2005; Kim, Akbar, & Kim, 2001; Singh, 2005; Uauy, Hoffman, Peirano, Birch, & Birch, 2001; Wainwright, 2002). However, epidemiological studies consistently report significant reductions in ω-3 LC-PUFAs in ADHD patients when compared with controls, which are not explained by dietary differences (Antalis et al., 2006; Chen, Hsu, Hsu, Hwang, & Yang, 2004; Colter, Cutler, & Meckling, 2008; Laasonen, Hokkanen, Leppamaki, Tani, & Erkkila, 2009; Mitchell, Aman, Turbott, & Manku, 1987; Young, Maharaj, & Conquer, 2004). Even more, in LC-PUFA dietary restriction animal models, deficiency of ω-3 fatty acids was associated with increased motor activity and learning abilities deficit, which revert after fatty acid supplementation (Vancassel et al., 2007; Yamamoto et al., 1988; Yamamoto, Saitoh, Moriuchi, Nomura, & Okuyama, 1987).

LC-PUFAs have 18 or more carbon units in their hydrocarbon chain and at least two double bonds. ω-6 LC-PUFAs are named as such due to the positioning of the first double carbon bond, which is located on the sixth atom from the methyl end of the acyl chain. In the same way, the first double bond in ω-3 LC-PUFAs is located on the third carbon atom. The 18-carbon precursors linoleic acid (LA; C18:2 ω-6) and a-linolenic acid (ALA; C18:2 ω-3) must be obtained from diet because mammals are unable to synthesize them. Interestingly, the same enzymatic system of elongases and desaturases leads LA and ALA to important derivates that are longer and/or present an increase in the extent of instauration of the acyl chain. The rate-limiting step in this process is mediated by the enzymes Δ5 desaturase and Δ6 desaturase (see Figure 1), encoded by the genes Fatty acid desaturase1 (FADS1) and FADS2, respectively (Bennett & Horrobin, 2000; Schuchardt, Huss, Stauss-Grabo, & Hahn, 2010; Sprecher & Chen, 1999).

Figure 1.

Biosynthetic pathways for ω-3 and ω-6 long-chain polyunsaturated fatty acids.

Interestingly, Brookes, Chen, Xu, Taylor, and Asherson, 2006, have reported a significant association between ADHD and SNP rs498793 in FADS2 (which is in linkage disequilibrium to SNPs previously associated to the regulation of LC-PUFA levels). Therefore, there is some evidence suggesting that LC-PUFAs not only are phenotypically associated to ADHD but also may share common genetic pathways.

Based on the aforementioned evidence, we suggest that LC-PUFAs may be useful endophenotypes for molecular genetic studies. Among other conditions, endophenotypes must be present in unaffected relatives of index cases at a higher rate than in the general population to be suitable as markers of genetic vulnerability (Kendler & Neale, 2010; Walters & Owen, 2007). This study was designed as an exploratory research to compare serum LC-PUFAs profiles and Δ5/Δ6 desaturase activity indices in ADHD patients, unaffected first-degree relatives, and unaffected participants from the general population. To our knowledge, this is the first study evaluating the suitability of LC-PUFAs as genetic markers for ADHD, by means of an exploration of their distribution in unaffected relatives of ADHD patients.

Patients and Method

Participants

We studied 72 participants belonging to one of the following categories: ADHD patients (n = 27), unaffected first-degree relatives of ADHD patients (n = 27), and unaffected participants without familiar history of ADHD (n = 18). ADHD patients and unaffected relatives were originally recruited as part of an ongoing genetic association study and were referred from general psychiatric/neurological and familiar-medicine outpatient services. ADHD diagnosis was based on Diagnostic and Statistical Manual of Mental Disorders (4th ed., text rev.; DSM-IV-TR; American Psychiatric Association, 2000) criteria in all cases. Inclusion criteria for ADHD patients were age between 5 and 18 years and average IQ or more (assessed by Wechsler Intelligence Scales for Children–Revised [WISC-R]; Wechsler, 1974). Exclusion criteria included major systemic and neurological illness and active ω-3/ω-6 supplementation. Unaffected relatives share the same inclusion/exclusion criteria, with exception of age (adult siblings and/or parents were also suitable for this group). Unaffected participants from the general population were children recruited from a medium-income school of the urban Santiago and/or adult healthy participants referred from general annual checkup. ADHD diagnosis was excluded from the unaffected groups by means of a brief clinical interview exploring DSM-IV-TR criteria. Because we were not able to asses unaffected participants by specific IQ scales, we enrolled children presenting average-to-high school performance and/or high school graduates in the case of adults (mean IQ estimation = 105 according to Crawford, et al., 1989). Because groups significantly differ in terms of age distribution, all reported results were age/sex controlled (ADHD group: M = 208 months, SD = 144 months; unaffected relatives: M = 450 months, SD = 257 months; unaffected from general population: M = 266 months, SD = 146 months; p < .0001) and sex distribution (15/27 men in ADHD group, 3/27 men in the group of unaffected relatives of ADHD patients, and 8/18 men in the group of unaffected participants from the general population; p = .006).

Method

Blood samples were obtained between 8 and 9 hr, after an 8-hr fast. Samples were centrifuged, and serum was stored at −20°C until analysis.

Long-chain polyunsaturated fatty acids assessment

Lipids were extracted and transesterified from serum samples according to Bligh and Dyer (1959), using heneicosaenoic acid (C21:0) as internal standard. Briefly, proteins were precipitated using methanol-Butylated hydroxytoluene (BHT): chloroform 2:1 v/v, and the lipid-containing organic phase was collected after a mixing/centrifugation process and evaporated to dryness with nitrogen. The fatty acids were transesterified overnight at 40°C with methanolic HCl (1 N):toluene 2:1 v/v. Finally, fatty acid methyl esters (FAMEs) were recovered by liquid/liquid extraction with sodium bicarbonate/n-hexane 2:1 v/v. FAMEs were separated and analyzed using an Agilent 6890N gas chromatography (GC) and an Agilent 5973N mass spectrometry (MS) detector (Agilent Technologies Cheadle, UK). Chromatography was performed using a DB-23 capillary column of 60 m × 0.25 mm × 0.25 mm (Crawford Scientific, Strathaven, UK) and helium as carrier gas at a flow rate of 1.0 mL/min and a constant pressure of 22.02 psi. The inlet temperature was 250°C. Volume injection was 0.2 µL in splitless mode. The oven temperature was initially held at 50°C for 1 min and then increased to 175°C at a rate of 25°C/min. Then, temperature was programmed to increase to 235°C at a rate of 4°C/min for 9 min. The MS transfer line was maintained at a temperature of 250°C. GC-MS was carried out using 70 eV. Data were evaluated using the selected ion monitoring (SIM) mode. Total run time was 30 min for identification and quantification.

To validate the GC-MS analysis for the intended use, we assessed the following metrological parameters prior to running clinical samples: limit of detection (LOD), linearity, intra-assay and inter-assay imprecision, inaccuracy, percentage of recovery, and matrix effect. Validation was according to the requirements of ISO 15189/Nch15189 for quality and competence in clinical laboratories. The obtained results are summarized in Table 1.

Table 1.

Metrological Parameters From the Prerun Validation of Our GC-MS Method for Serum LC-PUFA Detection/Quantification.

			Precision (CV%)
	LOD (µg/mL)	LOQ (µg/mL)	Intra-assay	Inter-assay	Inaccuracy (%)	Recovery (%)	Matrix effect (%)	Efficiency (%)
LA	37.37	52.08	0.84	3.46	9.94	95.64	114.29	108.67
GLA	0.17	0.34	1.41	2.08	0.46	84.47	113.49	95.85
ALA	0.16	0.29	1.27	0.46	8.90	84.41	121.92	103.46
DGLA	0.09	0.16	0.82	1.67	8.25	86.30	125.46	108.25
AA	0.34	0.50	1.81	0.92	8.95	85.59	171.11	146.97
EPA	0.24	0.51	0.85	3.22	5.45	84.33	117.73	99.26
DPA	0.23	0.48	0.83	9.15	5.26	84.56	123.00	103.92
DHA	0.24	0.45	0.80	7.05	5.49	84.46	131.36	110.86
IS	—	—	—	—	—	88.85	101.85	90.68

Note. CV = coefficient of variation; LOD = limit of detection; LOQ = limit of quantification; LA = linoleic acid; GLA = γ-linoleic acid; ALA = a-linolenic acid; DGLA = dihomogammalinolenic acid; AA = arachidonic acid; EPA = eicosapentaenoic acid; DPA = docosapentanoic acid; DHA = docosahexaenoic acid; IS = internal standard. Herein we present precision/inaccuracy values corresponding to a tested level of 10 mg/mL, which presented the higher metrological variation.

Data analysis

Desaturase activity was estimated by means of product/precursor indices previously established (Bokor et al., 2010). In particular, Δ6 desaturase activity was estimated directly by the γ-linoleic acid (GLA)/LA ratio and indirectly by docosahexaenoic acid (DHA)/docosapentanoic acid (DPA); Δ5 desaturase, however, was estimated by the arachidonic acid (AA)/dihomogammalinolenic acid (DGLA) ratio. Aggregated desaturase activity ratios (overall desaturation activity in the metabolic pathway) were also explored: eicosapentaenoic acid (EPA)/ALA and DHA/ALA for ω-3 pathway and AA/LA for ω-6 pathway. Additionally ω 6/total, ω 3/total and ω 6/ω 3 ratios were also included in the analysis due to previous reports on their association to ADHD (see Figure 1).

Statistical analysis was performed by SAS 9.2 software (SAS Institute, Cary, North Carolina) using parametrical methods. Reported significance levels were adjusted by age and sex by means of linear regression with clinical status, age, and sex (which took values 1 for men and 0 for women) as independent variables. We adjusted two different regression models to account for “three level or stair-like distributions” where ADHD patients and unaffected participants from the general population represent the uppers/lowers levels, on one hand, and to account for “two levels or step-like distributions,” where levels from unaffected relatives of index cases and from ADHD patients do not present significant differences, on the other hand. In the first models, “clinical status” took values 1 for unaffected participants from the general population, 2 for unaffected relatives of index cases, and 3 for ADHD patients. In the latest models, “clinical status” took values 1 for unaffected participants from the general population and 2 for ADHD patients and/or unaffected relatives of index cases. We considered p < .05 to be statistically significant. Due to the pilot nature of the present study, multiple testing corrections were not applied.

Ethical issues

All the procedures performed as part of this study have been approved by the Ethics Committee of the Pontificia Universidad Católica de Chile. The study was fully explained to children and their parents, and they both agreed to participate by signing written consent forms.

Results

Levels of DPA and ALA and the Desaturase Activity Ratios DHA/DPA and DHA/ALA Follow a Typical Endophenotype-Like Distribution

Table 2 summarizes the mean LC-PUFA serum levels and desaturase activity ratios found in ADHD patients compared with unaffected relatives of index cases and unaffected participants from the general population. Unadjusted one-factor ANOVA demonstrated significant differences among the clinical groups for the following dependent variables: DPA level (p = .005), DHA level (p = .031), and total ω-3 (p = .013). Bonferroni post hoc analysis showed that levels of DPA where significantly higher in unaffected relatives of ADHD patients compared with unaffected participants from the general population (confidence interval [CI] for difference = 1.2-7.7 µg/mL), whereas they did not significantly differ among the ADHD patients and unaffected relatives of index cases. Mean total ω-3 levels were significantly higher in unaffected relatives of ADHD patients when compared with unaffected participants from the general population (CI for difference = 1.8-41.2 µg/mL) and with ADHD patients (CI for difference = 0.5-35.8 µg/mL). Only DPA differences were significant after age–sex adjustment (p = .0031). Interestingly, linear regression models accounting for step-like distributions demonstrated two additional significant findings: a positive correlation between clinical status and ω-3/total ratio (r = .3; p = .0089), with ADHD patients and unaffected relatives of index cases presenting the highest values, and a negative correlation between clinical group and DHA/DPA ratio, with ADHD patients and unaffected relatives from the general population presenting the lowest values (r = −.23; p = .041). These step-like distributions of ω-3/total and DHA/DPA ratios, in which patients and unaffected relatives may be modeled as a single population, could be considered characteristic for endophenotype.

Table 2.

LC-PUFA Profiles Among the Studied Groups.

	Unaffected participants from the general population	Unaffected relatives from index cases	ADHD patients	Unadjusted p value	Sex–age adjusted p value
LA	960.92	985.03	892.24	.303	.282
GLA	14.49	15.56	14.55	.854	.693
ALA	15.44	21.10	18.00	.097	.084
DGLA	50.43	48.55	44.38	.513	.296
AA	153.51	160.70	148.49	.488	.606
EPA	14.60	15.96	11.98	.275	.221
DPA	11.80	16.27	14.35	.005*	.003*
DHA	39.98	50.04	40.89	.031*	.291
ω-6	1179.34	1209.84	1099.63	.287	.283
ω-6/total	0.93	0.92	0.96	.076	.063
ω-3	81.83	103.37	85.22	.013*	.14
ω-3/total	0.06	0.07	0.07	.142	.009*
ω-3/ω-6	0.07	0.09	0.08	.080	.105
AA/DHA	4.00	3.50	3.85	.304	.492
AA/EPA	13.46	12.20	14.27	.275	.367
AA/LA	0.16	0.17	0.17	.807	.567
EPA/ALA	1.03	0.91	0.76	.376	.148
EPA/DHA	0.36	0.32	0.30	.520	.261
DHA/ALA	2.479	2.161	2.132	.611	.241
GLA/LA	0.015	0.016	0.016	.823	.479
AA/DGLA	3.368	3.617	3.634	.725	.605
DHA/DPA	3.52	3.1	2.97	.165	.041*

Note. LC-PUFAs = long-chain polyunsaturated fatty acids; LA = linoleic acid; GLA = γ-linoleic acid; ALA = a-linolenic acid; DGLA = dihomogammalinolenic acid; AA = arachidonic acid; EPA = eicosapentaenoic acid; DPA = docosapentanoic acid; DHA = docosahexaenoic acid. Presented values correspond to mean levels and are expressed in µg/mL.

p < .05.

Intra-analysis by sex subgroups revealed that the differences in DPA levels, ω-3/total ratio, and DHA/DPA ratio were only present in the female subgroup (age-adjusted p values in females and men, respectively—for DPA: .0012 and .485; for ω-3/total ratio: .0021 and .636; for DHA/DPA: .022 and .2). In addition, ALA levels were significantly lower in unaffected females from the general population when compared with ADHD females and with unaffected females with family history of ADHD (age-adjusted p = .009), suggesting an inefficient conversion rate from ALA to longer intermediates (Table 3, Figure 2). In addition, DHA/ALA ratio, which addresses “aggregated desaturase activity,” significantly correlated with clinical status in regression models accounting for stair-like distributions (r = .3; p = .041), with ADHD females presenting the lowest ratios and unaffected females from the general population the highest. Finally, in “step-like” regression models, we observed that unaffected females from the general population presented significantly lower ω-6/total ratios (p = .015) and higher total ω-3 (p = .012) and ω-3/ω-6 ratios (p = .034) than ADHD females and unaffected relatives of index cases.

Table 3.

LC-PUFA Profiles Among the Studied Groups (Subgroup Analysis by Sex).

	Unaffected participants from the general population		Unaffected participants from index cases		ADHD
	Female	Male	Female	Male	Female	Male
LA	940.696	986.195	1003.610	842.533	938.820	854.984
GLA	13.170	16.130	15.144	18.713	13.583	15.327
ALA	12.435**	19.195*	21.964**	14.353*	20.642**	15.877*
DGLA	48.328	53.058	49.514	41.200	43.930	44.681
AA	155.002	151.636	161.702	153.037	144.057	152.033
EPA	11.274	18.763	16.654	10.640	9.133	14.264
DPA	10.172***	13.845	16.485***	14.607	13.278***	15.205
DHA	38.595	41.720	52.483	31.320	40.562	41.146
ω-6	1157.200	1207.020	1229.97	1055.48	1140.39	1067.02
ω-6/total	0.940*	0.927	0.918*	0.975	0.932*	0.923
ω-3	72.476*	93.522	107.586*	70.920	83.616*	86.493
ω-3/total	0.0600***	0.066	0.077***	0.040	0.067***	0.070
ω-3/ω-6	0.064*	0.080	0.090*	0.067	0.073*	0.083
AA/DHA	4.095	3.877	3.287	5.142	3.882	3.831
AA/EPA	15.559	10.830	11.891	14.482	16.273	12.668
AA/LA	0.161	0.160	0.166	0.181	0.157	0.181
EPA/ALA	1.015	1.014	0.936	0.744	0.562	0.921
EPA/DHA	0.301	0.433	0.317	0.351	0.242	0.350
DHA/ALA	2.921*	2.085	2.183*	2.012	1.770*	3.395
GLA/LA	0.014	0.016	0.015	0.022	0.014	0.018
AA/DGLA	3.443	3.274	3.595	3.795	3.474	3.761
DHA/DPA	3.867*	3.090	3.222*	2.116	3.103*	2.875

p < .05. **p < .01. ***p < .005. ****p < .001.

Figure 2.

Box-plot graphics representing the distribution of DPA levels, ALA levels, and DHA/ALA ratio among ADHD patients, unaffected relatives of index cases, and participants from the general population in the female subgroup. (A) Serum levels of ALA, (B) serum levels of DPA, and (C) DHA/ALA ratio (addressing aggregated desaturase activity).

Contrary to females, ADHD males and unaffected males with family history of ADHD presented significantly lower ALA levels when compared with unaffected males from the general population (age-adjusted p = .039), which may be explained by differences in ALA consumption.

Discussion

Our results suggest that DPA levels and the desaturase activity indices DHA/DPA and DHA/ALA may be suitable as endophenotypes for ADHD. In addition, subgroup analysis suggests that the aforementioned markers may be related to a pathogenic pathway predominantly expressed in females. Regarding this last point, there is consistent evidence suggesting that LC-PUFAs may be differentially regulated in men and women, with increased conversion of ALA to DHA in females (Bakewell, Burdge, & Calder, 2006; G. Burdge, 2004; G. C. Burdge, 2006; G. C. Burdge & Wootton, 2003; Childs, Romeu-Nadal, Burdge, & Calder, 2008). Based on this evidence and our results, it is possible to hypothesize that both ADHD females and female relatives of ADHD patients may present an alteration in the mechanisms involved in sex-related regulation of LC-PUFA metabolism.

Even when it is impossible to rule out to be in front of diet-induced alterations (not to endophenotypes), the co-occurrence in ADHD patients and unaffected relatives of high levels of ALA and DPA (substrates for Δ6 desaturase)—on one hand—and of low desaturase activity ratios—on the other hand—is highly concordant with the presence of decreased enzymatic activity and seems improbable that diet differences could reproduce such an alteration pattern.

Interestingly, there is consistent evidence demonstrating that serum/tissue LC-PUFA levels (including ALA, DPA, and DHA in the ω-3 pathway) and desaturase activity (assessed by the same ratios included in this analysis) are genetically regulated by variants of FADS1 and FADS, common in the European and Canadian population (Bokor et al., 2010; Glaser, Heinrich, & Koletzko, 2009; Lattka, Illig, Koletzko, & Heinrich, 2010). Even more, previous reports of a putative association between FADS2 variants and ADHD strongly suggest a pathogenic role for endogenous variations in the LC-PUFAs metabolism in ADHD, at least in a subset of patients.

In our opinion, these first findings warrant the need of future studies to overcome the restrictions inherent to pilot research. Notwithstanding, these results may be suggesting the presence of an understudied pathogenic pathway for ADHD in females, related to ω-3 LC-PUFA processing.

Footnotes

Acknowledgements

The authors would like to thank all children and families who participated in this research and physicians and technical staffs from Centro de Medicina Familiar Madre Teresa de Calcuta, Puente Alto, Santiago, for their valuable collaboration in this study.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by funds from School of Medicine, Pontificia Universidad Católica de Chile.

Author Biographies

Marcela Henríquez-Henríquez finished her MD studies at School of Medicine, Pontificia Universidad Católica de Chile, in 2003 and obtained her PhD in Medical Sciences in the same institution in 2008. Her doctoral thesis was based on the search of new endophenotypes for ADHD. Currently, she is finishing her clinical specialization in Clinical Pathology and Laboratory Medicine at the School of Medicine of Pontificia Universidad Católica de Chile.

Sandra Solari has obtained her MD degree in 1996 at Pontificia Universidad Católica de Chile where she completed the residency in Clinical Pathology and Laboratory Medicine, followed by a post doctoral fellowship in Therapeutic Drug Monitoring and Clinical Toxicology in Dr. Leslie Shaw laboratory at University of Pennsylvania, USA. Actually she is Assistant Professor at the Faculty of Medicine of Pontificia Universidad Católica de Chile and director of the Clinical Pharmacology and Toxicology Laboratory of the Catholic University Health System.

Gisela Várgas has obtained her Bachelor in Biochemistry at Pontificia Universidad Católica de Chile in 2012. Her thesis work was based on the implementation and validation of a measurement methodology for LC-PUFAs analysis by GC/MS. Currently she is working as research assistance at the Toxicology and Pharmacology Laboratory of the Clinical Laboratory Department, School of Medicine. Pontificia Universidad Católica de Chile.

Luis Vásquez has obtained his Bachelor in Biochemistry in 1997at Universidad de Concepción (Chile) and his Master in Chemistry in the same institution by 2000. Currently he is working as technical and research assistant at the Toxicology and Pharmacology Laboratory of the Clinical Laboratories Department, School of Medicine, Pontificia Universidad Católica de Chile.

Fidel Allende has obtained his Bachelor in Chemistry in 2005 at Universidad Tecnológica Metropolitana (Chile) and his Master in Toxicology by Colegio de Químicos de Sevilla, Spain in 2009. Since 2006, he works as technical and research assistant at the Clinical Laboratories Department, School of Medicine, Pontificia Universidad Católica de Chile.

Carla Castañón S. finished her MD studies at School of Medicine, Pontificia Universidad Católica de Chile, in 2000, and obtained her clinical specialization in Family Medicine in the same institution in 2004. Currently, she is finishing a Master in Epidemiology at the School of Medicine of Pontificia Universidad Católica de Chile.

Marcela Tenorio finished her studies in Psychology at Universidad de los Andes (Bogotá-Colombia) in 2001 and obtained her PhD in Psychology in Pontificia Universidad Católica de Chile in 2011. Her research is based on the study of neuropsychology development across life-span with special interest in the development of new methods for cognitive assessment. Currently, she is begging a post-doctoral research in Centro de Desarrollo de Tecnologías de Inclusión (CEDETi UC).

Teresa Quiroga Gutiérrez finished her MD studies at School of Medicine, Pontificia Universidad Católica de Chile, in 1980 and finished her clinical specialization in Clinical Pathology and Laboratory Medicine at the School of Medicine of Pontificia Universidad Católica de Chile, in 1985. She became Professor at the Department of Clinical Laboratory, Faculty of Medicine, since 1986 at the same University. Currently, she is Chief Director of the Clinical Laboratories Department of the School of Medicine.

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