Isobutyl alcohol

BMSC isolation and characterization was performed as previously described with minor modification [39 (link)]. Briefly, 4-week-old male Sprague Dawley rats were sacrificed by cervical dislocation, and the body was soaked in 70% ethanol alcohol for 10 min. Afterwards, the bone marrow was separated from tibias and femurs and subsequently flushed with MEM alpha containing 10% FBS. The bone marrow was cultured in MEM alpha medium within 10% FBS and 1% penicillin-streptomycin using 75-cm² tissue culture flasks. After cell growth for 24 h, the liquid suspension was replaced with new culture medium. The adherent cells were passaged at 80% confluency, and the cells from only passage three to five were used in the experiments.
Fluorescence-activated cell sorting (FACS) was applied to quantify the level of expression of mesenchymal and hematopoietic markers [3 (link), 40 (link)]. Cells were harvested at 80% confluency, centrifuged at 900 rpm for 10 min, supernatant discarded, and cell pellets were resuspended in 3 mL FACS buffer (99% PBS, 1% FBS). Thereafter, the cells at a concentration of 1 × 10⁶/mL were incubated with anti-rat CD 45 FITC, anti-Mouse/Rat CD 90.1 (Thy-1.1) FITC or anti-mouse/rat CD 29 FITC at 4 °C for 30 min, respectively. The expression level of the antigen markers was analyzed through a BD FACSCalibur (BD Biosciences, San Jose, CA). Collected data were analyzed by the software FlowJo 7.6 (Tree Star, Ashland OR, USA).
BMSCs were also identified by investigating the differentiation potential towards osteocytes and adipocytes [41 (link)]. For osteogenic differentiation, cells were seeded in 12-well plates at a density of 4 × 10⁴/mL. The culture medium was replaced with osteogenic differentiation medium consisting of DMEM, 10% FBS, 1% penicillin-streptomycin, 1% l-glutamin, 0.1 μM dexamethasone, 50 μM ascorbic acid, and 10 mM glycerol 2-phosphate (BGP) once the cells were approximately 60% confluent. The medium was changed every 3 days. After 21-day culture, cells were gently washed in PBS twice followed by fixation with 4% paraformaldehyde for 20 min at room temperature. Then, cells were stained with 2% Alizarin Red S staining solution for 25 min at room temperature to identify the Ca²⁺ deposits. For adipogenic differentiation, cells were seeded in 12-well plates at a density of 3 × 10⁴/mL. The culture medium was replaced with adipogenic differentiation medium consisting of DMEM, supplemented with 10% FBS, 1% penicillin-streptomycin, 1% l-glutamin, 1 μM dexamethasone, 0.5 mM 3-isobutyl-1-methylxantine (IBMX), 10 μg/mL insulin, and 100 μM indomethacin once the cells were approximately 80% confluent. Fresh medium was changed every 3 days. After 21-day culture, cells were washed with PBS twice and fixed with 4% paraformaldehyde for 20 min at room temperature. Four percent paraformaldehyde was washed with PBS twice followed by being stained with Oil Red O solution for 15 min at room temperature to identify the lipid droplets.

Study population. The NHS was established in 1976 when 121,700 female registered nurses 30–55 years of age were enrolled, whereas in 1989 the younger counterpart NHSII cohort was initiated among a total of 116,430 female registered nurses 25–42 years of age. A total of 18,743 NHS participants 53–79 years of age provided blood and urine samples in 2000–2002. In 1996–2001, blood and urine samples were collected from 29,611 NHSII participants 32–52 years of age. Urine samples were collected without preservative in a polypropylene container and returned to a central biorepository via overnight courier with an icepack, where they were processed immediately upon arrival and aliquoted into polypropylene cryovials, which were stored in the vapor phase of liquid nitrogen freezers at ≤ –130°C. In both cohorts, a high follow-up rate of > 90% was maintained among participants who provided urine samples.
Assessment of covariates. NHS participants responded to a questionnaire inquiring about body weight, height, demographic and lifestyle information, and medical history at study baseline. Similar follow-up questionnaires have been administered biennially since baseline to update these variables. In 1984, 1986, and every 4 years thereafter, a validated food frequency questionnaire (FFQ) has been used to assess participants’ usual diet. In the NHSII, questionnaires similar to those used in the NHS are sent biennially to update lifestyle and health-related characteristics. The FFQ was first administered in 1991 and is updated every 4 years in the NHSII. Based on the FFQ, we derived a score of the Alternative Healthy Eating Index (AHEI), an indicator of adherence to healthy eating behavior (McCullough et al. 2002 (link)). Of note, the FFQs did not inquire about any food packaging information. Information on cigarette smoking, physical activity, family history of diabetes, menopausal status, oral contraceptive use, hormone replacement therapy, and the history of hypertension or hypercholesterolemia were also assessed in the questionnaires at baseline and during follow-up. In the questionnaires, participants were asked about the average time per week in the past year spent on leisure-time physical activities. Based on this information, we calculated energy expenditure in metabolic equivalent tasks (METs) measured in hours per week.
Ascertainment of T2D. At baseline and on all biennial follow-up questionnaires, participants were asked whether they had received a physician-diagnosis of diabetes. Those reporting a diabetes diagnosis were sent a supplementary questionnaire (Manson et al. 1991 (link)) regarding any symptoms, diagnostic tests, and treatment. We used one of the following American Diabetes Association 1998 criteria to confirm self-reported T2D diagnosis: a) an elevated glucose concentration (fasting plasma glucose ≥ 7.0 mmol/L, random plasma glucose ≥ 11.1 mmol/L, or plasma glucose ≥ 11.1 mmol/L after an oral glucose load) and at least one symptom (excessive thirst, polyuria, weight loss, or hunger) related to diabetes; b) no symptoms, but elevated glucose concentrations on two separate occasions; or c) treatment with insulin or oral hypoglycemic medication. The accuracy of self-reported diagnosis of T2D has been demonstrated in a validation study (Manson et al. 1991 (link)), in which a blinded endocrinologist confirmed the diagnosis of diabetes by reviewing medical records of 61 of 62 NHS participants who responded to the supplementary questionnaire. Only confirmed T2D cases were included in the present study in order to minimize possible misclassification.
Nested case–control study. We prospectively identified and confirmed 971 T2D cases (NHS, 394; NHSII, 577) through June 2008 (NHS) or June 2007 (NHSII) among participants who provided first morning urine samples and were free of T2D, cardiovascular disease, and major cancers except nonmelanoma skin cancer at sample collection. Using the risk–set sampling scheme, we randomly selected one control for each case from among the women who remained free of T2D at the case’s date of diagnosis (Prentice and Breslow 1978 ). We matched cases and controls for age at urine sample collection (± 1 year), date (± 3 months)/time (first morning or not) of sample collection, ethnicity (white or other), fasting status when blood was drawn (≥ 8 hr or not), and menopausal status (yes, no) and hormone replacement therapy use (yes, no) at sample collection (NHSII only). T2D cases diagnosed within the first year since urine sample collection were excluded from selection in order to reduce the potential for reverse causation bias.
The study protocol was approved by the institutional review board of the Brigham and Women’s Hospital and the Human Subjects Committee Review Board of Harvard School of Public Health. Informed consent was provided by all participants involved in this research.
Laboratory measurements. Urinary concentrations of BPA and eight phthalate metabolites [mono-(2-ethylhexyl) phthalate (MEHP), mono-(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), mono(2-ethyl-5-carboxypentyl) phthalate (MECPP), mono-(2-ethyl-5-oxohexyl) phthalate (MEOHP), monobutyl phthalate (MBP), mono-isobutyl phthalate (MiBP), monobenzyl phthalate (MBzP), monoethyl phthalate (MEP), and phthalic acid] were measured using established methods with modifications (Fox et al. 2011 (link); Kato et al. 2005 (link)) (see Supplemental Material, “Laboratory measurements,” pp. 2–3, for complete details). Briefly, samples were mixed with isotopically-labeled phthalate metabolites and BPA and treated with β-glucuronidase and sulfatase. Urinary concentrations of phthalate metabolites were measured by orbitrap-liquid chromatography–mass spectrometry (LCMS) (model Exactive; Thermo Electron, Waltham, MA), and BPA concentrations were measured by tandem-LCMS (model TSQ Ultra; Thermo Electron) at A. Franke’s laboratory at the University of Hawaii Cancer Center in 2012. Of note in the NHS only, because of technical reasons, concentrations of phthalic acid were not available for 144 case–control pairs, and only combined concentrations of MBP and MiBP were available. We also measured urinary creatinine levels using a Roche-Cobas MiraPlus clinical chemistry autoanalyzer (Roche Diagnostics, Indianapolis, IN) with a kit from Randox Laboratories (Crumlin, UK). Lastly, In the NHS we measured two liver enzymes [alanine transaminase (ALT) and γ-glutamyl transpeptidase (GGT)] using a direct enzymatic colorimetric assay, performed on the Roche P Modular system (Roche Diagnostics). In addition, fetuin-A levels were measured by an enzyme immunoassay from R&D Systems (Minneapolis, MN) in the NHS (Sun et al. 2013 (link)).
Quality control procedures. Each pair of matched case–control urine samples was shipped in the same batch and analyzed in the same run. Within each batch, samples were assayed by the same technician in a random sequence under identical conditions. Duplicates of blinded quality control samples (n = 82 for NHS; 116 for NHSII) were run along with the case–control samples to monitor the quality of these assays. We calculated intra-assay coefficients of variation (CVs) based on the measurements of these samples. The average CVs were < 10% for most metabolites (including creatinine), except for MEHP (NHS 11.4%, NHSII 10.0%) and BPA (NHS 11.5%, NHSII 13.0%).
Within-person stability of metabolites. We measured BPA and phthalates in two urine samples collected 1–3 years apart from a separate sample of 120 participants to evaluate the within-person reproducibility (Townsend et al. 2013 (link)). The creatinine-adjusted intraclass correlation coefficients (ICCs) between the two measurements were ≥ 0.30 for all metabolites [ranging from 0.30 for MiBP to 0.53 for MBP, except for MEHP and BPA (0.14 for both)].
Statistical methods. We summed molar concentrations of MEHP, MEHHP, MECPP, and MEOHP to represent total metabolites of di(2-ethylhexyl) phthalate (DEHP). Likewise, we summed molar concentrations of MBP and MiBP. We calculated total phthalate concentrations (in nanomoles per liter) as the summed values of MEHP, MEHHP, MECPP, MEOHP, MBP, MiBP, MBzP, and MEP to facilitate comparisons with previous studies. We calculated Spearman correlation coefficients (r_S) among controls to evaluate the intercorrelation among urinary metabolites.
Study participants were categorized into quartiles according to the cohort-specific distribution of metabolite concentrations among controls. We used conditional logistic regression to model the associations under investigation. We adjusted for body mass index (BMI; < 25.0 kg/m², 25.0–27.4 kg/m², 27.5–29.9 kg/m², 30.0–32.4 kg/m², ≥ 32.5 kg/m², missing category), smoking status (current smoker, past smoker, nonsmoker), oral contraceptive use (never used, past user, current user; NHSII only), hormone replacement therapy (yes, no; NHS only), physical activity (METs-hr/week), alcohol use (abstainer, < 5.0 g/day, 5.0–14.9 g/day, ≥ 15.0 g/day), family history of diabetes (yes, no), history of hypercholesterolemia or hypertension (yes, no), AHEI score, and urinary creatinine (mg/dL). In the current analysis we used measurements of covariates derived from the questionnaire administered in 2000 (1998 for AHEI and alcohol use) in NHS or 1995 in NHSII. p-Values for linear trend were calculated by modeling the median value of each quartile as a continuous variable. We pooled cohort-specific estimates for the NHS and NHSII using a random-effects meta-analysis. Heterogeneity of odds ratios (ORs) between the two cohorts was evaluated by the Cochrane Q statistic and the I² statistic.
Restricted cubic spline regressions with 3 knots were used to model potential dose–response relations between the metabolites and diabetes (Durrleman and Simon 1989 (link)). In this analysis, to maximize statistical power, we pooled data for case–control pairs from the two cohorts and then performed statistical analyses using conditional logistic regression. In addition, participants in the top 5% of metabolite concentrations were excluded to minimize the potential impact of extreme outliers. We used likelihood ratio tests (LRTs) to examine nonlinearity, comparing the model with the linear term only to the model with the linear plus the cubic spline terms.
LRTs were also used to examine effect modification of associations between metabolites and T2D by testing the significance of multiplicative interaction terms between metabolite quartiles modeled as ordinal variables and each potential modifier.
As secondary analyses, we calculated Spearman partial correlation coefficients between the chemicals and plasma levels of liver enzymes and fetuin-A, as well as their interactions on incident T2D, in the NHS to evaluate the potential adverse effects of the pollutants on liver function as suggested by animal experiments and human observational studies (Desvergne et al. 2009 (link); Lang et al. 2008 (link)).
All p-values were two-sided and p < 0.05 was considered statistical significance. Data were analyzed with SAS, version 9.2 (SAS Institute Inc., Cary, NC).

Aldehydes, acetal, methanol, esters and higher alcohols (also known as fusel alcohols) were quantified by GC-FID. Twenty-one compounds were determined following two different procedures. In both cases, the equipment used was an Agilent 7890B Gas Chromatograph (Agilent Technologies, Santa Clara, CA, USA) coupled with Flame Ionization Detector.
For the analysis of acetaldehyde, acetaldehyde—diethyl acetal, methanol, ethyl acetate, n-propyl alcohol, 2-butyl alcohol, isobutyl alcohol, n-butyl alcohol, 2-methyl-1-butanol and 3-methyl-1-butanol, the samples were injected in a split mode (split 1:46, 250 °C) into a DB-624 (30 m × 250 µm × 1.4 µm, Agilent Technologies, Santa Clara, CA, USA) column. The oven temperature for the analysis was programmed as follows: 30 °C (30 min), then 6 °C/min to 100 °C (0 min). Temperatures of the injector and the detector were 250 °C and 300 °C, respectively. Nitrogen was used as a carrier at flow of 1.0 mL/min. Data acquisition and analyses were performed using OpenLAB CDS Chemstation (Agilent Technologies, Santa Clara, CA, USA) software.
For the analysis of n-hexanol, 2-phenylethyl alcohol, ethyl lactate, ethyl succinate, ethyl caproate, ethyl caprylate, ethyl caprate, ethyl laureate, ethyl myristate and ethyl palmitate, samples were injected in a splitless mode (1 min, 250 °C) onto CP-WAX 57 CB (25 m × 250 µm × 0.2 µm, Agilent Technologies, Santa Clara, CA, USA) column. The oven temperature program during analysis was as follows: 45 °C (20 min), then 3 °C/min to 170 °C (20 min). Temperatures of the injector and the detector were 250 °C and 300 °C respectively. Nitrogen was used as the carrier gas at a flow of 1.3 mL/min. The data acquisition and analyses were performed using OpenLAB CDS Chemstation (Agilent Technologies, Santa Clara, CA, USA) software.
Standards were made in an ethanol/ultrapure water solution at 40%vol. The linear standard curve of 3-methyl-1-butanol ranges from 1 to 250 mg/100 mL of 100% vol. alcohol. The linear standard curve of methanol, ethyl acetate, n-propyl alcohol, isobutyl alcohol and 2-methyl-1-butanol ranges from 1 to 100 mg/100 mL of 100% vol. alcohol. The linear standard curve of acetaldehyde and acetaldehyde—diethyl acetal ranges from 1 to 50 mg/100 mL of 100% vol. alcohol. The linear standard curve of ethyl lactate ranges from 0.5 to 25 mg/100 mL of 100% vol. alcohol. The linear standard curve of 2-butyl alcohol, n-butyl alcohol, n-hexanol, 2-phenylethyl alcohol, ethyl succinate, ethyl caproate, ethyl caprylate, ethyl caprate, ethyl laureate, ethyl myristate, and ethyl palmitate ranges from 0.1 to 5 mg/100 mL of 100% vol. alcohol. The samples were diluted at 40%vol. with ultrapure water and injected in duplicate. The results were expressed in mg of compound per 100 mL of 100% vol. alcohol.

Crystalline white powder of GW501516 was received from Wuhan Shu Mai Technology Co. (Wuhan, China) and solvents from Sigma-Aldrich and Merck, Taufkirchen, Germany. To obtain new polymorphs, a screening was undertaken in a variety of solvents and solvent mixtures. GW501516 (10 mg) with a purity of 99% was dissolved in a wide range of solvents (1 mL) after which the solutions were heated to approximately 40 °C and were allowed to cool to 3 °C. The vials were covered with parafilm in which very small holes were made; the solvents were left to evaporate slowly for several weeks.
The complete list of solvents used is the following: tetrahydrofuran, 2,2,2-trifluoroethanol, 1-butanol, diisobutyl ketone, dimethylformamide, ethyl acetate, 1-propanol, chloroform, 2-ethoxyethanol, 1,4-dioxane, methanol, acetone, nitromethane, ethanol, dichloromethane, dimethylacetamide, dimethylformamide, pentane, acetic acid, methyl isobutyl ketone, diisopropyl ether, and isopropyl alcohol.
The solvent mixtures used for polymorph screening were 1:1 volumetric ratios of 2,2,2-trifluoroethanol with tetrahydrofuran, methanol with tetrahydrofouran, ethyl acetate with dimethylformamide, chloroform with isopropyl alcohol, ethyl acetate with acetone, diisopropyl ether with acetic acid, and pentane with acetone.
In this way, the polymorphs were obtained as follows:(i)

GW-1 (starting polymorph) from acetonitrile;

(ii)

GW-2 from mixture of pentane and acetone;

(iii)

GW-3 in isopropyl alcohol;

(iv)

GW-5 in methyl isobutyl ketone;

(v)

GW-4 was found in the bulk of the starting sample as single crystals but was not further reproduced.

All the reagents were HPLC grade. Ethanol (99.5% purity), acetonitrile (ACN, 99.8%), acetone (99.8%), ethyl acetate (99.0%), and triEthanolamine (99.0%) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Acetaldehyde (99.0%) mEthanol (99.9%), and butanol (99.0%) were purchased from Merck (Darmstald, Germany). Isoamyl alcohol (98.5%) and isobutyl alcohol ([ISO]; 99.0%) were obtained from Labsynth (Diadema, Brazil). Benzene (99.0%) and toluene (99.5%) were purchased from Reagen (Colombo, Brazil). Deionized water was obtained using a Milli-DI water purification system from Merck. Ultrapure water was used in the blank gel matrix^{37 (link)}. [Carbopol 940] was obtained from Vetec (São Paulo, Brazil).