Eleven men from our ongoing study of the relationship between environmental agents and male reproductive health agreed to participate in the phthalate variability study. Participant recruitment into the environmental agents and male reproductive health study has been previously described (Hauser et al. 2003 (link)). Briefly, men who were the partner in couples seeking fertility evaluation for inability to conceive were recruited to participate. The study site was the Massachusetts General Hospital (MGH) Andrology Laboratory, so most men resided in the New England area. At the clinic visit, each man was asked to produce a single semen sample and to collect a single spot urine sample.
For each of the 11 men in the phthalate temporal variability study, up to nine additional spot urine samples were collected during three cycles over a 92-day period. Ten of these 11 men each contributed a total of 10 urine samples (nine for the variability study and one for the male reproductive study), whereas one of the men provided a total of seven samples (including six for the variability study). Nested within each of the three cycles were three urine samples, collected on the first 3 consecutive days of each cycle. The first cycle began upon enrollment into the phthalate temporal variability study, and urine samples were collected on days 0, 1, and 2. Cycles 2 and 3 began 30 days and 90 days after cycle 1, respectively. Therefore, the nine urine samples were collected on days 0, 1, and 2 (cycle 1); days 30, 31, and 32 (cycle 2); and days 90, 91, and 92 (cycle 3).
All the urine samples were collected in a sterile specimen cup. The urine sample on day 0 was collected at the MGH Andrology laboratory. All other samples were collected at the subject’s home and frozen before overnight shipment to the Harvard School of Public Health (HSPH) on blue ice. All urine samples were then shipped frozen on dry ice from HSPH to CDC. Eight phthalate monoesters—MBzP, MBP, MEP, MEHP, monomethyl phthalate (MMP), mono-n-octyl phthalate (MOP), mono-3-methyl-5-dimethylhexyl phthalate (MINP), and monocyclohexyl phthalate (MCHP)—were measured in each spot urine sample using an analytical approach developed at the CDC (Silva et al. 2003 ). Briefly, the determination of phthalate metabolites in urine involved enzymatic deconjugation of the glucuronidated metabolites, solid-phase extraction, separation with high-performance liquid chromatography, and detection by tandem mass spectrometry. Detection limits were in the low micrograms per liter range. Reagent blanks and 13C4-labeled internal standards were used along with conjugated internal standards to increase the precision of the measurements. One method blank, two quality control samples (human urine spiked with phthalates), and two sets of standards were analyzed along with every 21 unknown urine samples. Analysts at the CDC were blind to all information concerning subjects.
Several methods adjust for urine volume (Boeniger et al. 1993 (link); Teass et al. 1998 ). Although creatinine is a frequently used form of adjustment, if a compound is excreted primarily by tubular secretion it is not appropriate to adjust for creatinine level (Teass et al. 1998 ). Although the methods of excretion of the phthalate monoesters measured in this study are unknown, terephthalic acid was found to be actively secreted by renal tubules and actively reabsorbed by the kidney (Tremaine and Quebbemann 1985 (link)). Furthermore, because organic compounds that are glucuronidated in the liver, like the phthalates, are eliminated by active tubular secretion (Boeniger et al. 1993 (link)), creatinine adjustment may not be appropriate for phthalates. Additionally, creatinine levels may be confounded by muscularity, physical activity, urine flow, time of day, diet, and disease states (Boeniger et al. 1993 (link); Teass et al. 1998 ). For these reasons, specific gravity, rather than creatinine, was used to normalize phthalate levels.
Urinary phthalate levels were normalized for dilution by specific gravity adjustment using the formula Pc = P × [(1.024 – 1)/(SG – 1)], where Pc is the specific-gravity–corrected phthalate concentration (micrograms per liter), P is the observed phthalate concentration (micrograms per liter), and SG is the specific gravity of the urine sample (Boeniger et al. 1993 (link); Teass et al. 1998 ). Specific gravity was measured using a handheld refractometer (National Instrument Company, Inc., Baltimore, MD), which was calibrated with deionized water before each measurement.
For each of the 11 men in the phthalate temporal variability study, up to nine additional spot urine samples were collected during three cycles over a 92-day period. Ten of these 11 men each contributed a total of 10 urine samples (nine for the variability study and one for the male reproductive study), whereas one of the men provided a total of seven samples (including six for the variability study). Nested within each of the three cycles were three urine samples, collected on the first 3 consecutive days of each cycle. The first cycle began upon enrollment into the phthalate temporal variability study, and urine samples were collected on days 0, 1, and 2. Cycles 2 and 3 began 30 days and 90 days after cycle 1, respectively. Therefore, the nine urine samples were collected on days 0, 1, and 2 (cycle 1); days 30, 31, and 32 (cycle 2); and days 90, 91, and 92 (cycle 3).
All the urine samples were collected in a sterile specimen cup. The urine sample on day 0 was collected at the MGH Andrology laboratory. All other samples were collected at the subject’s home and frozen before overnight shipment to the Harvard School of Public Health (HSPH) on blue ice. All urine samples were then shipped frozen on dry ice from HSPH to CDC. Eight phthalate monoesters—MBzP, MBP, MEP, MEHP, monomethyl phthalate (MMP), mono-n-octyl phthalate (MOP), mono-3-methyl-5-dimethylhexyl phthalate (MINP), and monocyclohexyl phthalate (MCHP)—were measured in each spot urine sample using an analytical approach developed at the CDC (Silva et al. 2003 ). Briefly, the determination of phthalate metabolites in urine involved enzymatic deconjugation of the glucuronidated metabolites, solid-phase extraction, separation with high-performance liquid chromatography, and detection by tandem mass spectrometry. Detection limits were in the low micrograms per liter range. Reagent blanks and 13C4-labeled internal standards were used along with conjugated internal standards to increase the precision of the measurements. One method blank, two quality control samples (human urine spiked with phthalates), and two sets of standards were analyzed along with every 21 unknown urine samples. Analysts at the CDC were blind to all information concerning subjects.
Several methods adjust for urine volume (Boeniger et al. 1993 (link); Teass et al. 1998 ). Although creatinine is a frequently used form of adjustment, if a compound is excreted primarily by tubular secretion it is not appropriate to adjust for creatinine level (Teass et al. 1998 ). Although the methods of excretion of the phthalate monoesters measured in this study are unknown, terephthalic acid was found to be actively secreted by renal tubules and actively reabsorbed by the kidney (Tremaine and Quebbemann 1985 (link)). Furthermore, because organic compounds that are glucuronidated in the liver, like the phthalates, are eliminated by active tubular secretion (Boeniger et al. 1993 (link)), creatinine adjustment may not be appropriate for phthalates. Additionally, creatinine levels may be confounded by muscularity, physical activity, urine flow, time of day, diet, and disease states (Boeniger et al. 1993 (link); Teass et al. 1998 ). For these reasons, specific gravity, rather than creatinine, was used to normalize phthalate levels.
Urinary phthalate levels were normalized for dilution by specific gravity adjustment using the formula Pc = P × [(1.024 – 1)/(SG – 1)], where Pc is the specific-gravity–corrected phthalate concentration (micrograms per liter), P is the observed phthalate concentration (micrograms per liter), and SG is the specific gravity of the urine sample (Boeniger et al. 1993 (link); Teass et al. 1998 ). Specific gravity was measured using a handheld refractometer (National Instrument Company, Inc., Baltimore, MD), which was calibrated with deionized water before each measurement.
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