Attention-Deficit/Hyperactivity Disorder and Urinary Metabolites of Organophosphate Pesticides

Study Design and Population

The National Health and Nutrition Examination Survey (NHANES) is a population-based, health survey of noninstitutionalized US residents conducted by the National Center for Health Statistics of the Centers for Disease Control and Prevention. The NHANES uses a complex, multistage, probability sampling design, with oversampling of certain subgroups. Participants completed household surveys, which included questions about demographic features and health history, and blood and urine samples were collected during physical examinations at mobile centers.¹⁶ We used data from 2000–2004, years for which ADHD was assessed in children 8 to 15 years of age; diagnoses were available for 3998 children. Urinary DAP metabolite levels were measured for a random subsample of the NHANES participants. From 2000 to 2002, the sampling rate was 50% for ages 6 to 11 years and 33% for ages 12 to 15 years. From 2003 to 2004, the sampling rate was 33% for all ages. Measurements of urinary DAP levels were available for 1481 children among those with ADHD diagnoses. The NHANES was approved by the National Center for Health Statistics institutional review board, and all participants provided written informed consent.

Children who received newborn care in an ICU or premature nursery (n = 167) and those with birth weights of <2500 g (n = 126) were excluded because these are important risk factors for developmental disorders.¹⁷ We excluded 24 children with extremely dilute urine (creatinine levels of <20 mg/dL) and 1 outlier with respect to urinary DAP concentrations. Children with missing data were excluded (poverty/income ratio [PIR], n = 43; fasting duration, n = 38; urinary creatinine level, n = 1).

ADHD Assessment

The Diagnostic Interview Schedule for Children IV (DISC-IV), a structured diagnostic interview designed for use in epidemiological studies,¹⁸ was used to assess the presence of ADHD on the basis of slightly modified criteria from the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV).¹⁹ The interview was conducted with the mother or another caretaker over the telephone by trained interviewers, 2 to 3 weeks after the physical examination. The interview was conducted by bilingual interviewers in English or Spanish. The DISC-IV has evidence of substantial validity,¹⁸ reliability for both its English¹⁸ and Spanish^20,21 versions, and successful use via telephone in DSM-IV field trials.²² The use of DISC-IV data is restricted for confidentiality reasons; therefore, we accessed the data through the National Center for Health Statistics Research Data Center.

The DISC-IV scoring algorithms determine ADHD diagnostic status for the previous year, as well as ADHD subtype, that is, predominately inattentive subtype, predominately hyperactive/impulsive subtype, or combined subtype. The diagnosis is based on the presence, during the previous 12 months, of symptoms related to inattention, hyperactivity, and impulsivity, with significant impairment in ≥2 settings (eg, at school and home).²³ The DSM-IV criterion that symptoms must not occur in conjunction with another neuropsychiatric disorder is not assessed by the DISC-IV. We chose not to use the DSM-IV criterion that symptoms must have been present before 7 years of age, because our hypothesis was that urinary DAP concentrations would be associated with increased odds of concurrent ADHD.

The DISC-IV identified 119 cases of ADHD, but 30 children did not meet the diagnostic criteria although the parent reported use of doctor-prescribed ADHD medication during the previous year. Because the diagnostic interview addresses the presence of symptoms, children whose symptoms were well controlled with medication would not meet the diagnostic criteria. Therefore, we also conducted analyses in which ADHD cases were defined as either meeting the DISC-IV diagnostic criteria or regularly taking ADHD medication during the previous year.

Measurement of Urinary Metabolites of Organophosphate Pesticides

During the physical examination, “spot” urine specimens were collected from participants, divided into aliquots, and stored cold (2–4°C) or frozen. Samples collected for DAP measurements were shipped on dry ice to the Centers for Disease Control and Prevention's National Center for Environmental Health. Six urinary DAP metabolites, resulting from the degradation of ≥28 different organophosphates, were measured in urine to provide an indicator of the body burden of common organophosphates.³ The urinary DAP metabolites measured were 3 dimethyl alkylphosphate (DMAP) molecules (dimethyl phosphate, dimethyl thiophosphate, and dimethyl dithiophosphate) and 3 diethyl alkylphosphate (DEAP) molecules (diethyl phosphate, diethyl thiophosphate, and diethyl dithiophosphate). DMAP metabolites are derived from O,O-dimethyl-substituted organophosphate pesticides such as malathion; DEAP metabolites result from the degradation of O,O-diethyl-substituted organophosphates such as chlorpyrifos. The measurements were performed through lyophilization and chemical derivatization, followed by analysis through isotope-dilution gas chromatography-tandem mass spectrometry.²⁴ Stable-isotope analogues were used as internal standards for each of the metabolites, which resulted in a high degree of accuracy and precision, with low analytical limits of detection. Concentrations below the detection limit were imputed a value corresponding to the value of the detection limit divided by √2.²⁵ Urinary creatinine concentrations were determined using an automated colorimetric method based on a modified Jaffe reaction on a Beckman Synchron AS/ASTRA clinical analyzer (Beckman Instruments, Inc., Brea, CA) at the Fairview University Medical Center, Minneapolis, Minnesota.²⁶

Other Covariates

The following variables were considered as potential confounders: gender, age (in months), child race/ethnicity (non-Hispanic white, black, Mexican American, or other/multiracial), PIR (the ratio of self-reported family income to the family's appropriate poverty threshold value, on the basis of Census data, recoded into 4 categories), BMI (in quartiles), blood lead concentrations (logarithmically transformed), maternal age at birth, maternal smoking during pregnancy (yes or no), and time since last consuming food or drink, recorded at the time of blood and urine sampling (recorded on a scale of 1 [0–2 hours] to 7 [21–24 hours]).

Data Analyses

We used the complex samples module of SPSS 17.0 (SPSS Inc, Chicago, IL) to conduct all analyses, with accounting for the multistage probability sampling design of the NHANES. Strata, primary sampling units, and sample weights were used to obtain robust linearized SEs and unbiased point estimates. The threshold for statistical significance was set at P < .05. All statistical tests were 2-sided. The DAP metabolite concentrations were divided by the respective molecular weights before being summed to yield DEAP, DMAP, and total DAP concentrations. Because DMAP and DEAP metabolites might have different relationships to the outcomes,¹² they were examined separately. Because the distributions of total DAP, DMAP, and DEAP concentrations were skewed, logarithmic transformations (base 10) were applied. Correlates of urinary DAP metabolite concentrations were examined with a general linear model. Logistic regression analyses were used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) for ADHD (any subtype) and ADHD subtypes, per 10-fold increases in total DAP, DMAP, and DEAP metabolite concentrations (in nanomoles per liter). Results are presented for crude analyses and adjusted analyses. Gender- and age-related differences were examined in subpopulation analyses and, because there were no differences in effect estimates and no significant interaction, these variables were included as covariates in the models. In addition, race/ethnicity, PIR, fasting time, and logarithmically transformed urinary creatinine concentrations (as recommended by Barr et al,²⁶ to adjust for urine dilution) were included in the models because they are important potential confounders; other covariates also were examined in sensitivity analyses. Because individual urinary DAP metabolite levels were below the analytical limit of detection for a large proportion of children (Table 1), which might bias effect estimates,²⁷ we conducted further analyses on the metabolite with the highest detection frequency, namely, dimethyl thiophosphate. Cases were categorized as below the limit of detection or lower or higher than the median of detectable concentrations adjusted for creatinine levels.