**Low Blood Zinc, Iron, and Other Sociodemographic Factors Associated with Behavior Problems in Preschoolers**

**Jianghong Liu \*, Alexandra Hanlon, Chenjuan Ma, Sophie R. Zhao, Siyuan Cao and Charlene Compher** 

School of Nursing, University of Pennsylvania, 418 Curie Blvd., Philadelphia, PA 19104, USA; E-Mails: alhanlon@nursing.upenn.edu (A.H.); chenjuan@nursing.upenn.edu (C.M.); sophie.r.zhao@hotmail.com (S.R.Z.); caos@sas.upenn.edu (S.C.); compherc@nursing.upenn.edu (C.C.)

**\*** Author to whom correspondence should be addressed; E-Mail: jhliu@nursing.upenn.edu; Tel.: +1-215-573-7492; Fax: +1-215-746-3374.

*Received: 25 November 2013; in revised form: 13 January 2014 / Accepted: 20 January 2014 / Published: 27 January 2014* 

**Abstract:** Previous research supports the link among malnutrition, cognitive dysfunction, and behavioral outcomes; however, less research has focused on micronutrient deficiencies. This study investigates whether micronutrient deficiencies, specifically blood zinc and iron levels, will be associated with increased behavior problem scores, including internalizing and externalizing behaviors. 1314 Children (55% boys and 45% girls) from the Jintan Preschool Cohort in China participated in this study. Venous blood samples were collected and analyzed for zinc and iron when the children were 3–5 years old. Behavior problems were measured with the Child Behavior Checklist (CBCL), which was completed by the parents when children were in their last months of preschool (mean age 5.6 years). General linear multivariate modeling was used, with adjustment for important sociodemographic variables. The results indicate that low zinc levels alone (*p* = 0.024) and combined low zinc and iron levels (*p* = 0.022) are significantly associated with increased reports of total behavior problems. We did not find an association between low iron and behavior problems. With regards to sociodemographics, living in the suburbs is associated with increased internalizing problems, while higher mother's education and being female were associated with decreased externalizing problems. This study suggests that micronutrient deficiencies and sociodemographic facts are associated with behavior problems in preschoolers.

**Keywords:** internalizing; externalizing; total behavior; CBCL; child; micronutrient deficiency; zinc and iron

#### **1. Introduction**

The link between early nutrition deficiency and behavior outcomes has been receiving increasing attention [1–3]. At the prenatal level, Neugebauer, Hoek, and Susser [4] found that the male offspring of nutritionally-deprived pregnant women had 2.5 times the normal rate of antisocial personality disorder in adulthood. At the postnatal level, in a longitudinal study from the Mauritius birth cohort [5], it was found that children with malnutrition (protein, zinc, iron and vitamin B deficiencies) at age 3 years, compared to controls, have higher externalizing behavior problems (*i.e.*, antisocial, aggressive, and hyperactive behavior) at ages 8, 11, and 17 years [6]. In another more recent longitudinal study, Galler *et al.* [3] found that children who were malnourished at an early age showed significantly higher parent-reported levels of behavior problems, particularly aggression, and decreased executive functioning at age 9–15 and again at 11–17, independent of baseline age, sex, household standard of living, and maternal depressive symptoms. Finally, at the intervention level [7], a double-blind, placebo-controlled randomized trial from England showed that supplementation of adult prisoners' diet with vitamins, minerals, and essential fatty acids significantly reduced antisocial and violent behavior in prison. These findings have been recently replicated in young prisoners in the Netherlands [8]. This initial evidence supports the relationship between nutrition and behavioral problems; however, more research is still needed.

While increasing studies have showed the association between overall nutritional status and child behavior, few studies have specifically investigated blood zinc and iron status in relation to behavior. In developing countries, low zinc and iron levels are common [9–11]. Indeed, more than 90% of affected individuals live in developing countries, and approximately one-tenth of the worldwide population suffers from iron deficiency [12]. Furthermore, few studies have been conducted in Asian populations. In China, zinc and iron deficiency were previously very common, but over the past two to three decades, reports on zinc and iron intake have been mixed due to socioeconomic reform and rapid economic development that have taken place since 1979. The availability [13,14] and affordability [15] of foods have increased dramatically during this time, and as a result of this increased food production and access to food, the prevalence of malnutrition has decreased, while over-nutrition has increased [16,17]. Studies indicate that iron deficiency is less prevalent than zinc deficiency among Chinese children [18,19], and although the prevalence of anemia has decreased in China, it still exists among children [16,20]. Taken together, this makes the consideration of zinc and iron intake in Chinese samples a relevant issue for better understanding putative risk factors for behavioral outcomes.

Zinc and iron play important roles in children's physical and behavioral health; however, there is a relative lack of attention given to the effects of specific micronutrient (e.g., zinc and iron) deficiency on behavior problems, including internalizing and externalizing disorders. Zinc is a component of enzymes that affect growth in infancy and childhood, sexual maturation, neuromotor development, and immunity. Mental function is improved by zinc's promotion of normal brain development and physiology [21–23]. Iron similarly boosts mental functioning by serving as a co-enzyme involved in the production and release of neurotransmitters [21,22] and by influencing cognitive function [24,25] and behavioral disorders such as attention-deficit hyperactivity disorder [6,26].

There is now increasing evidence of the relationship between malnutrition and childhood behavior problems [27–30], though more data are needed to address the impact of specific micronutrient deficiencies on both internalizing and externalizing problems separately [2]. The importance of zinc and iron in physiological development seems to warrant particular attention with regard to how these micronutrients relate to behavioral outcomes. Childhood behavioral problems represent an important sub-area of developmental psychopathology [31–34]. Thus, identifying early childhood behavior problems—and, perhaps more importantly, their early risk factors, including nutritional and sociodemographic factors—is important for understanding and preventing problem behaviors later in life [35,36]. The purpose of this study is to assess the association of micronutrients controlled for sociodemographic factors with behavior outcomes. We hypothesize that nutritional deficiencies, specifically zinc and iron deficiencies, will be associated with increased behavior problems.

#### **2. Experimental Section**

#### *2.1. Participants and Procedures*

The current study was part of a population-based community preschool cohort study of 1656 Chinese children (55.5% boys, 44.5% girls) initially recruited between the Fall of 2004 and the Spring of 2005 from four preschools in the city of Jintan, located in the southeastern coastal region of Mainland China. In China, preschools are called kindergartens and enroll children from ages 3–6 years, after which children enter the elementary school system; to be consistent, we use preschool to refer to our study sample. Detailed sampling and research procedures of this larger cohort study have been described elsewhere [37,38]. Briefly, all children and parents taking part in the original cohort study were invited to participate for assessment of children's behaviors while the children were in the final few months of their senior year in preschool (spring 2005 to spring 2007). At that point, some children dropped out of the study because they changed schools or because data were not fully available. Therefore, only 1385 children in the original sample were followed up in the later waves. There was no statistically significant difference between those who dropped out of the study and those who were retained [37,39].

In the last year of preschool, parents were asked to assess their children with the Chinese version of the Child Behavior Checklist (CBCL/1.5–5). Since some of the children were beyond the age limit of the CBCL/1.5–5, the current analysis only addressed the subset of the original sample that was under age 6 to adhere to the age requirement of the measure. Our final data set for analysis was thus comprised of 1314 preschoolers with a mean age of 66.6 months (SD = 5, range = 50–71), which is close to the common kindergarten age in the US. Written informed consent was obtained from parents. Institutional Review Board (IRB) approval was obtained from the University of Pennsylvania and the ethical committee for research at Jintan Hospital in China.

#### *2.2. Measures*

#### 2.2.1. Micronutrient Deficiency

Blood specimens were collected in Fall 2004 and Spring 2005 by trained pediatric nurses using a strict research protocol to avoid lead contamination. Approximately 0.5 mL of venous blood was collected in a lead-free EDTA tube for zinc and iron analysis. Samples were frozen and shipped to the Child Development Center, Nanjing Medical University, Nanjing, China, for analysis. Specimens remained frozen at í °C until analysis. Blood concentrations of zinc and iron were determined by atomic absorption spectrophotometry (BH model 5.100 manufactured by Beijing Bohu Innovative Electronic Technology Corporation), with duplicate readings taken with an integration time of 2 s. The reliability and validity of the analysis and the detailed analytic procedure have been described previously [40]. Detailed information on blood sample data collection and analysis is given in [39].

/RZ]LQFOHYHOVZHUHGHILQHGE\FRQFHQWUDWLRQȝJG/DQGORZLURQE\FRQFHQWUDWLRQȝJG/ in blood, with cutoffs determined from the middle of the normal range. Combined low zinc and iron were defined as currently low zinc and low iron concentrations, *i.e.*, children in this category have ERWK=QȝJG/DQG)HȝJG/

#### 2.2.2. Behavior Problems at Ages 5–6

Childhood behavior problems were measured with the Chinese version of the Achenbach System of Empirically Based Assessment (ASEBA) CBCL/1.5–5 [41]. The CBCL is a widely used scale for assessing behavioral and emotional problems in children. In this study, parents were asked to answer the 99 items of the CBCL instrument, which dealt with their children's behavior within the past 12 months, and give a rating from a 3-point scale (0 = not true; 1 = sometimes true, or 2 = often true) [41]. Factor analysis performed on the CBCL/1.5–5 has revealed two broadband factors: Internalizing behaviors and Externalizing behaviors [42]. Separately, factor analysis has produced four syndromes for Internalizing behaviors: Emotionally Reactive, Anxious/Depressed, Somatic Complaints, and Withdrawn; and two syndromes for Externalizing behaviors: Attention Problems and Aggressive Behavior [41,43]. These factor structures have also been validated in our previous study [44]. The internal reliabilities (coefficient alpha) for the scales in our study sample were as follows: Emotionally Reactive (0.71), Anxious/Depressed (0.64), Somatic Complaints (0.58), and Withdrawn (0.73), Attention Problems (0.64) and Aggression (0.87). The strategy we employ in this study is to use these established scales as predictors of latent construct "Internalizing behavior" and "Externalizing Behavior". The sum of the items in the scales was used.

#### 2.2.3. Sociodemographic Variables

Sociodemographic information was obtained from the questionnaire filled out by the parents, and included information on gender, parental education, home living conditions, and the age of mother when the child was born. These data were collected as control variables given their potential direct effects on child behavioral problems. As discussed in our previous publication [39], we did not ask for data on household income because it is often not the best indicator of socioeconomic status, therefore we used information on house size as a proxy for evaluating socioeconomic status. A descriptive summary of these demographic variables is presented in Table 1.


**Table 1.** Baseline characteristics of study population (*N* = 1314).
