Benzophenone

Samples collected at visit 1 were analyzed at the National Center for Environmental Health laboratories at CDC for nine phthalate metabolites [n = 1,149; monoethylphthalate (MEP), mono-butyl phthalate, mono-iso-butyl phthalate, mono-benzyl phthalate, mono-3-carboxypropyl phthalate, mono-2-ethyl-5-carboxypentyl phthalate, mono-(2-ethyl-5-hydroxylhexyl) phthalate, mono-(2-ethyl-5-oxohexyl) phthalate, and mono-(2-ethylhexyl) phthalate (MEHP)], seven phenols (benzophenone-3, bisphenol A, 2,5-dichlorophenol, triclosan; n = 1,149; methyl-, butyl-, and propyl- parabens, n = 1,059), and three phytoestrogens (daidzein, genistein, enterolactone; n = 1,150). Parabens were not measured early in the study. At least one urinary biomarker measurement was available among 1,151 girls, 985 with breast stages. We substituted limit of detection ( for results below the LOD. Adjustment for urine dilution was accomplished using creatinine, to account for difference in sampling (spot specimens at MSSM and KPNC, early-morning samples at Cincinnati) and interindividual variation in urine dilution. We included log-creatinine in models using continuous log-biomarker variables, and we created quintile cut points from creatinine-corrected concentrations (micrograms per gram creatinine). As previously described, to reduce multiple comparisons we combined the phthalate metabolites into two groups that represent similar sources and similar biologic activity, low- (< 250 Da) and high-molecular-weight (> 250 Da) phthalate metabolites (low MWP and high MWP) [details in Supplemental Material, Table 2 (doi:10.1289/ehp.0901690)]. We expressed high MWP molar sum as MEHP (molecular weight 278) and the low MWP as MEP (molecular weight 194) so that units were the same as the other analytes (micrograms per liter). Similarly, a molar sum of the paraben metabolites was created (paraben sum) expressed as propyl paraben (molecular weight 180.2). Models with the individual phthalate and paraben metabolites were consistent with the molar sum variables. Results using di(2-ethylhexyl)phthalate (DEHP)-sum metabolites were almost identical to those for the high MWP, and they represented 75% ± 16% (mean ± SD) of the high MWP biomarkers. Therefore, only the latter models are presented.
Laboratory techniques and quality control protocols are identical to those reported previously in a pilot study (Wolff et al. 2007 (link)). Briefly, urine undergoes an automated cleanup with enzymatic deconjugation, followed by high-performance liquid chromatography-isotope dilution tandem mass spectrometry quantification (Kato et al. 2005 (link); Rybak et al. 2008 ; Ye et al. 2005 (link), 2006 ). In addition to the internal CDC quality control procedures, we incorporated approximately 10% masked quality control specimens (n = 101) from a single urine pool. The coefficients of variation (SD/mean concentration) were < 10% for 13 analytes and were between 10% and 21% for the remaining six biomarkers.

The National Health and Nutrition Examination Survey (NHANES), conducted continuously since 1999 by the CDC, assesses the health and nutritional status of the civilian noninstitutionalized U.S. population ≥ 2 months of age (CDC 2003 ). The survey includes household interviews, medical histories, standardized physical examinations, and collection of biologic specimens, some of which can be used to assess exposure to environmental chemicals. NHANES 2003–2004 included examinations of 9,282 people (CDC 2006a ). We measured BP-3 by analyzing a random one-third subset of urine samples (n = 2,517) collected from NHANES participants ≥ 6 years of age. Because this subset was randomly selected from the entire set, it maintained the representativeness of the survey. Participants provided informed written consent; parents provided informed written consent for their children.
Urine specimens were shipped on dry ice to the CDC’s National Center for Environmental Health and stored frozen at or below –20°C until analyzed. We measured total (free plus conjugated species) concentrations of BP-3 in urine by online solid-phase extraction coupled to high-performance liquid chromatography–tandem mass spectrometry described in detail elsewhere (Ye et al. 2005a (link)). Briefly, conjugated BP-3 in 100 μL of urine was hydrolyzed using β-glucuronidase/ sulfatase (Helix pomatia; Sigma Chemical Co., St. Louis, MO). After hydrolysis, samples were acidified with 0.1 M formic acid; BP-3 was preconcentrated by online solid-phase extraction, separated by reversed-phase high-performance liquid chromatography, and detected by atmospheric pressure chemical ionization–tandem mass spectrometry. Because a stable isotope-labeled BP-3 was not available, we used ¹³C₁₂-bisphenol A as internal standard (Ye et al. 2005a (link)). The limit of detection (LOD), calculated as 3S₀, where S₀ is the standard deviation as the concentration approaches zero (Taylor 1987 ), was 0.34 μg/L, and the precision ranged from 17.6% (at 18.5 μg/L) to 16.2% (at 46 μg/L). Low-concentration (~ 20 μg/L) and high-concentration (~ 45 μg/L) quality control materials, prepared from pooled human urine, were analyzed with standard, reagent blank, and NHANES samples (Ye et al. 2005a (link)).
We analyzed the data using Statistical Analysis System (version 9.1.3; SAS Institute, Inc., Cary, NC) and SUDAAN (version 9.0.1; Research Triangle Institute, Research Triangle Park, NC). SUDAAN calculates variance estimates after incorporating the sample population weights, nonresponse rates, and sample design effects. We calculated the percentage of detection and the geometric mean and distribution percentiles for both the volume-based (in micrograms per liter urine) and creatinine-corrected (in micrograms per gram creatinine) concentrations. For concentrations below the LOD, as recommended for the analysis of NHANES data (CDC 2006b ), we used a value equal to the LOD divided by the square root of 2 (Hornung and Reed 1990 ).
A composite racial/ethnic variable based on self-reported data defined three major racial/ethnic groups: non-Hispanic black, non-Hispanic white, and Mexican American. We included participants not defined by these racial/ethnic groups only in the total population estimate. Age, reported in years at the last birthday, was stratified in groups (6–11, 12–19, 20–59, and ≥ 60 years of age) for calculation of the geometric mean and the various percentiles.
We used analysis of covariance to examine the influence of several variables, selected on the basis of statistical, demographic, and biologic considerations, on the concentrations of BP-3. For the multiple regression models, we used the variables described below and all possible two-way interactions to calculate the adjusted least square geometric mean (LSGM) concentrations. LSGM concentrations provide geometric mean estimates (in micrograms per liter) after adjustment for all covariates in the model. Because the distributions of BP-3 and creatinine concentrations were skewed, these variables were log transformed. We analyzed two separate models: one for adults (≥ 20 years of age) and one for children and teenagers (≤ 19 years of age). We considered age (continuous), age squared, sex, race/ethnicity, and log-transformed crea-tinine concentration for both models. When the model included both age and age squared, we centered age by subtracting 50 from each participant’s age, thus avoiding multi-collinearity and obtaining the least weighted correlation between these two variables (Bradley and Srivastava 1979 ). Additionally, to further evaluate the relation between the log-transformed BP-3 concentration and age, we used age group (20–29, 30–39, 40–49, and ≥ 50 years of age) as a categorical variable in the model and generated a bar chart of LSGM concentrations by age group.
To reach the final reduced model, we used backward elimination with a threshold of p < 0.05 for retaining the variable in the model, using Satterwaite-adjusted F statistics. We evaluated for potential confounding by adding each of the excluded variables back into the final model one by one and examining changes in the β coefficients of the statistically significant main effects or interactions. If addition of one of these excluded variables caused a change in a β coefficient by ≥ 10%, we re-added the variable to the model.
We also conducted weighted univariate and multiple logistic regressions to examine the association of BP-3 concentrations above the 95th percentile with sex, age group, and race/ ethnicity for all ages.

In a Young’s tube, [{SiN^Dipp}MgNa]₂ (4, 21.6 mg, 0.02 mmol) was dissolved
in 0.4 mL of d₆-benzene before the addition
of benzophenone (7.2 mg, 0.04 mmol) to the bright yellow solution.
The reaction mixture instantaneously turned into a purple solution.
The reaction mixture was then left standing at room temperature overnight,
whereupon the formation of orange precipitates was observed. The purple
supernatant was then carefully decanted and examined by EPR spectroscopy.
The orange solid was collected and redissolved in toluene, and bright
yellow crystals suitable for single-crystal X-ray diffraction were
obtained by storage of the orange solution at −30 °C.
This reaction was then repeated with a modified stoichiometry of substrates:
[{SiN^Dipp}MgNa]₂ (4, 21.6 mg, 0.02
mmol) was dissolved in 0.4 mL of d₆-benzene
before the addition of benzophenone (21.6 mg, 0.12 mmol) to the bright
yellow solution. This entry also provided a purple solution with orange
solids when the reaction mixture was kept at ambient temperature overnight.
The purple supernatant provided a virtually identical EPR spectrum,
and the recrystallization of the orange solids provided compound 9 (confirmed by unit-cell screening). Yield 6.9 mg, 19%. Compound 9 can also be prepared by treatment of 2 equiv of benzophenone
(7.2 mg, 0.04 mmol) with [{SiN^Dipp}Mg] (10.4 mg, 0.02 mmol).^33a The reaction was conducted with 0.4 mL of C₆D₆ inside a J-Young’s tube, providing quantitative
conversion into 9 (verified by NMR spectroscopy), and
slow evaporation of the benzene solution provided 9 as
bright yellow crystals. Yield 15.2 mg, 86%. No meaningful result was
obtained for elemental analysis after multiple attempts. ¹H NMR (500 MHz, 298 K, benzene-d₆) δ:
7.19–7.16* (m, 8H, ArH,*overlapping with C₆D₆), 7.14–7.00 (m, 11H, ArH), 6.95–6.92 (m, 7H, ArH), 4.31 (sept, J = 6.9 Hz, 4H, CHMe₂), 1.44
(s, 4H, SiCH₂), 1.41 (d, J = 6.9 Hz, 12H, CHMe₂), 0.89 (d, J = 6.9 Hz, 12H, CHMe₂), 0.38
(s, 12H, SiMe₂). ¹³C NMR (126
MHz, 298 K, Benzene-d₆) δ: 151.6
(i-C₆H₃),
151.3 (i-C₆H₅ on OCPh₂), 145.6 (o-C₆H₃), 133.5 (o-C₆H₅ on OCPh₂), 131.3 (p-C₆H₅ on OCPh₂),
128.6 (m-C₆H₅ on OCPh₂), 123.5 (m-C₆H₃), 120.3 (p-C₆H₃), 114.1 (OCPh₂), 27.5 (CHMe₂), 25.3 (CHMe₂), 24.3 (CHMe₂), 13.4 (SiCH₂), 1.4 (SiMe₂).

Note that
the same polymer is used in this study as in our previously published
procedure.^{33 (link)}mPEG- macroinitiator
(0.05 mmol, 100 mg), diethylaminoethyl methacrylate (DEAEMA) (10 mmol,
2 mL) and benzophenone methacrylate (BMA) (1.12 mmol, 300 mg) were
added to a Schlenk flask and dissolved in 6 mL of 2-butanone. Subsequently,
2,2′-bipyridine (0.1 mmol, ∼16 mg) was added to the
solution. The CuBr (0.1 mmol) catalyst was added, and the flask was
immediately frozen in liquid N₂ and subjected to three
freeze–pump–thaw cycles before placing it in a preheated
oil bath at 60 °C. After the reaction, the solution was diluted
with 60 mL of THF and passed through an alumina column to remove the
catalyst. The filtrate was concentrated and precipitated in cold hexane
to give the final polymer.

Leaves of four-month-old plants (10 g dry weight) were lyophilised and homogenised (M20 Universal mill; IKA, Staufen, Germany). Eight aliquots of the resulting powder were transferred to 50 mL conical reaction tubes and extracted three times with 50 mL dichloromethane. The combined extract was centrifuged, the supernatant filtrated through filter paper, and the solvent evaporated under N₂ gas. The residue (1 g) was fractionated using an Isolera One Flash Purification Chromatography system (Biotage, Uppsala, Sweden), equipped with a Chromabond Flash BT cartridge (SiOH, 40–63 µm, 40 g; Macherey-Nagel, Düren, Germany). Separation was stretched over 23 column volumes (CV), using n-hexane (A) and ethyl acetate (B) as mobile phase (gradient: 2 CV 0% B, 6 CV 0–10% B, 15 CV 10–60% B). A total of 30 fractions a 45 mL were collected, automatically controlled by detection of UV signals in the λ-all mode. After solvent evaporation under N₂ gas, the fractions were passed through a Chromabond HR-X SPE column (6 mL, 55–60 µm; Macherey-Nagel) to remove chlorophyll. Lipophilic target compounds were isolated by semi-preparative HPLC on a normal-phase EC NUCLEODUR column (100-5 silica, 4.6×250 mm, 5 µm; Macherey-Nagel) with a flow rate of 3 mL min⁻¹. The mobile phase consisting of n-hexane (A) and ethyl acetate (B) was used for isocratic elution at 3% B for 16 min. Separation of fractions 16 and 17 yielded pure nemosampsone 4 (7 mg), while fraction 18 yielded 7-epi-nemorosone 3 (6 mg), compound 4 (4 mg), and 7-epi-clusianone 5 (3 mg). More polar target compounds were purified on a reversed-phase EC NUCLEODUR column (π², 10 × 250 mm, 5 µm; Macherey-Nagel) at a flow rate of 3 mL min⁻¹ using 0.1% formic acid in water (A) and acetonitrile (B) as mobile phase (80–92% B from 0–15 min). Separation of fractions 23–28 resulted in the isolation of grandone 1 (5 mg) and kolanone 2 (2 mg). 7-epi-secohyperforin 6 (0.28 mg) was isolated on the above normal-phase HPLC system from fresh H. sampsonii roots used for RNA isolation.