We purchased mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), mono(2-ethyl-5-oxohexyl) phthalate (MEOHP), mono(2-ethylhexyl) phthalate (MEHP), mono-3-methyl-5-dimethylhexyl phthalate (MINP), ¹³C₄-MEHP, ¹³C₄-MEHHP, ¹³C₄-MEOHP, ¹³C₄-MINP, and ¹³C₄-4-methylumbel-liferone (¹³C₄-MeUmb) from Cambridge Isotopes Laboratories Inc. (Andover, MA). Mono(2-ethyl-5-carboxypentyl) phthalate (MECPP) and D₄ -MECPP were gifts from J. Angerer (University of Erlangen, Nuremberg, Germany). HPLC-grade acetonitrile and water were purchased from Tedia (Fairfield, OH), and MeUmb and its glucuronide (MeUmb-glu) were purchased from Sigma Chemical Co. (St. Louis, MO). β-Glucuronidase (Escherichia coli-K12) was purchased from Roche Biomedical (Mannheim, Germany). Stock solutions of standards (MEHP, MEOHP, MEHHP, and MeUmb) and internal standards (¹³C₄-MEHP, ¹³C₄-MEHHP, ¹³C₄-MEOHP, and ¹³C₄-MeUmb) were prepared in acetonitrile. ¹³C₄-MEOHP was used as the internal standard for MOINP, D₄-MECPP was used as the internal standard for MCIOP, and ¹³C₄-MEHHP was used as the internal standard for MHINP.

Urine samples were collected by each study participant directly into a phthalate-free prescreened urine cup. The analytical method for measuring DINP oxidative metabolites in urine was adapted from previously published methods (Blount et al. 2000; Silva et al. 2003, 2004b). Briefly, the urine samples (1 mL) were spiked with an internal standard solution containing ¹³C₄-MEHP, ¹³C₄-MEOHP, ¹³C₄-MEHHP, D₄-MECPP, ¹³C₄-MINP, and 4-MeUmb. MeUmb-glu was added to evaluate the completion of the deglucuronidation reaction with β-glucuronidase. Phthalate monoester metabolites were extracted by automated solid-phase extraction (SPE) using a commercial SPE system (Zymark Corp., Hopkinton, MA) after enzymatic hydrolysis. The metabolites in the urine extract were chromatographically resolved by high-performance liquid chromatography (HPLC) using a Surveyor HPLC system (ThermoFinnigan, San Jose, CA) equipped with a Betasil phenyl HPLC column (3 μm, 100 mm × 2.1 mm; ThermoHypersil-Keystone, Bellefonte, PA) using a nonlinear water:acetonitrile solvent gradient. The metabolites were detected by negative ion electrospray ionization tandem mass spectrometry using a ThermoFinnigan TSQ Quantum triple quadrupole mass spectrometer (ThermoFinnigan). For the measurement of the unconjugated metabolites, we eliminated treatment with β-glucuronidase. Under our experimental conditions, the isomeric metabolites of DINP were not chromatographically resolved and eluted as broad peaks. The entire area under the peak encompassing all isomers was integrated for quantification. The limits of detection (LODs) were 0.25 ng/mL for MOINP, MHINP, and MCIOP. The LOD for MINP was 0.36 ng/mL. Oxidative metabolism is an enzymatically mediated reaction. Therefore, oxidative metabolites cannot result from potential contamination with DINP during sampling, storage, or analysis.

Statistical analysis of the data was performed using the Statistical Analysis System (SAS) software (SAS Institute Inc., Cary, NC). Samples with values below the LOD were assigned a concentration equal to the LOD divided by the square root of 2 for the statistical analyses (Hornung and Reed 1990). Statistical significance was set at p < 0.05.

Although DINP is a less potent inducer of peroxisomal proliferation than DEHP (McKee et al. 2000), DINP exerts antiandrogenic effects similar to that of DEHP in DINP-dosed rats (Gray et al. 2000). The effects of DINP exposure in humans are not currently known.

Phthalates with long alkyl side chains, such as DEHP and DnOP, metabolize extensively before being excreted in urine both in rodents and humans (Albro 1986; Albro and Moore 1974; Koch et al. 2004, 2005b; Silva et al. 2005). Similarly, in rats administered DINP, some MINP was excreted in urine, but oxidative metabolites of MINP, MHINP, MCIOP, and MOINP were excreted as the major urinary metabolites (Silva et al. 2006a).

We measured the urinary concentrations of MINP, MHINP, MCIOP, and MOINP in 129 human adults. We observed a wide range of exposures to DINP (Table 1). MHINP was present in all samples tested at concentrations ranging from 1.4 to 202.7 ng/mL, with 5% of the samples having > 43.7 ng/mL (Table 1). Similarly, MCIOP was detected in 97% of the samples tested at levels ranging from < LOD to 310.8 ng/mL. MOINP was detected in 87% of the samples at levels ranging from < LOD to 201.7 ng/mL (Table 1). The geometric mean concentrations of MHINP, MCIOP, and MOINP were 11.4, 8.6, and 1.2 ng/mL, respectively. Interestingly, we did not detect MINP in any of the samples analyzed.

In rats dosed with DINP, the major metabolite excreted in urine was MCIOP (Silva et al. 2006a). By contrast, in this study population, MHINP was excreted as the major urinary metabolite (Table 1). Although three oxidative metabolites of DINP were present in all samples analyzed, the urinary concentrations of these metabolites were lower than the structurally related DEHP metabolites MEHHP, MEOHP, and MECPP, also measured in this population (Silva et al. 2006b) (Figure 2). In humans, the urinary concentrations of MEHHP, MEOHP, MECPP, and MEHP represent about 75% of the DEHP dose (Koch et al. 2004, 2005b). The fraction of DINP excreted in urine as MHINP, MOINP, MCIOP, and MINP is not presently known. Based on similar physicochemical properties and metabolism between DINP and DEHP (Koch et al. 2004, 2005b; Silva et al. 2006b, 2006c), the lower concentrations of DINP oxidative metabolites than of DEHP oxidative metabolites in this group of adults suggest that environmental exposures to DINP may be lower than the exposure to DEHP. However, because DINP is a mixture of isomers, it is also possible that the prevalence of exposure to DINP is underestimated by measuring only these three oxidative metabolites. Furthermore, the elimination half-life of the oxidative metabolites of DINP is presently unknown. Therefore, the differences in urinary concentrations observed among DINP oxidative metabolites and their DEHP counterparts may also reflect differences in toxicokinetic parameters.

Because all three DINP metabolites result from the same parent compound, their urinary concentrations were highly correlated with each other, with correlation coefficients varying from 0.73 to 0.83 (p < 0.001; Figure 3), similar to previous findings regarding DEHP metabolites (Barr et al. 2003; Kato et al. 2004; Koch et al. 2004, 2005b).

Glucuronidation not only facilitates urinary excretion of phthalate metabolites but may also reduce their potential biological activity if the putative biologically active species is the free metabolite. We measured both total and free urinary concentrations of MHINP, MOINP, and MCIOP and found that although MCIOP mostly excreted in its free form, MOINP excreted mostly glucuronidated. The percentage of MHINP excreted either as a conjugate or free form was similar (Figure 4). Furthermore, the concentration of the glucuronidated form of the metabolites increased with increasing levels of the total metabolite concentrations, indicating the absence of enzyme saturation at environmental exposure levels (Figure 5).

In summary, we measured the urinary concentrations of three oxidative metabolites of DINP (MCIOP, MHINP, and MOINP) and the hydrolytic metabolite MINP in 129 anonymous adults. The oxidative metabolites were present in all samples tested, and their urinary concentrations were highly correlated with each other. By contrast, the hydrolytic monoester MINP was not detected in any of the samples. The most abundant DINP urinary metabolites were the ω and ω⁻¹ oxidative metabolites, MCIOP and MHINP, respectively. MCIOP was excreted in urine predominantly in its free form, whereas MOINP was excreted glucuronidated. The significantly higher frequency of detection and urinary levels of oxidative metabolites than of MINP confirm the validity of these oxidative metabolites as biomarkers for DINP exposure assessment. More important, these data suggest that exposure to DINP is widespread and that it has been underestimated by using MINP as the sole DINP urinary biomarker.

In Figure 5, values for urinary MOINP glucuronide on the y-axis have been modified from the original manuscript published online. The corrected values are 0.1, 1, 10, 100, and 1,000. The original values were 0.1, 1, 10, and 100.