After obtaining institutional review board approval and subject consent, human hair was cut from non-pregnant and pregnant females, and from males. All subjects denied any type of chemical processing of their hair in the past year. Subject demographics included age, gender, race/ethnicity, medication and tobacco use and hair hygiene (frequency of shampooing, use of heat treatments).
Hair was cut from the posterior vertex region as close to the scalp as possible using scissors or thinning shears. Hair length was recorded and proximal ends of the hair samples were marked. Hair samples were stored in aluminum foil for protection as recommended [18 (link)]. All hair samples were divided into a minimum of two clusters to a maximum of six clusters of full-length hair. If hair was longer than 5 cm, the strands were divided at 5 cm from the scalp and only the proximal segment used for analysis. For experiment 1, clusters of hair were exposed to various common products used for chemical processing. One cluster was set aside from each subject and used as the “native” hair cluster, to which all other clusters were compared. The other clusters were exposed to one or more of seven hair treatments (depending on the number of available hair clusters): potassium persulfate (bleach), <2% peroxide (H2O2), (demi-perm or semi-permanent colour), 6% H2O2, 9% H2O2 or 12% H2O2. All peroxide treatments also contained ammonia. A licensed cosmetologist (AG) instructed the first author (CH) on standard salon procedures using Wella Koleston Perfect© and Wella Color Touch© (Wella Professionals, Procter & Gamble, USA) products prior to processing hair samples.
For Experiment 2, a variety of common commercially available shampoos, to represent the assortment of shampoos by cost, hair texture, and popularity used commonly by our subjects, were used to wash hair samples from untreated clusters described previously. Hair samples were placed in individual 15 ml conical centrifuge tubes with shampoo diluted 5-fold. Each tube was vigorously shaken for 5-10 seconds. The hair was removed from the tube and placed under a running stream of deionized water for 5 seconds to rinse the hair after each wash step. Hair samples were washed either 15 or 30 times with the same hair product for comparison to native hair.
Following either chemical treatment or shampoo, each cluster from Experiments 1 and 2 was washed three times in isopropanol and dried as previously described [3 (link)]. After washing, drying, and weighing, the hair was ground in 2 ml cryovials (Wheaton, Millville, NJ, USA) using a ball mill (Retsch, Haan, Germany) that held the cryovial and sample as well as a 3/16 inch stainless steel ball bearing in specially milled aluminum cassettes holding three cryovials. The cassette containing the cryotubes was immersed in liquid nitrogen to snap freeze the hair prior to grinding to facilitate the process. Samples were ground for approximately 5 min. The powdered hair (5-10 mg) was extracted in the same cryovial in HPLC grade methanol at room temperature for 24 hours with slow shaking. Following extraction, the cryovial was spun for 120 s in a microcentrifuge at 20000 g and 200 μl supernatant was removed, placed into a second microcentrifuge tube and dried for 24 hr under a hood. The dried extract was then reconstituted in 133 μl of assay buffer and cortisol levels determined using a commercial high sensitivity EIA kit (Salimetrics LLC, State College, PA, USA) according to manufacturer's directions. A pooled control consisting of previously ground hair was extracted and run on each EIA plate for determination of inter-assay coefficients of variation. Inter-assay coefficient of variation (CV) for the control hair pool was 11.6% and intra-assay CV was 1.9%.
Statistical analyses applied paired t-tests comparing native to chemically processed and shampooed hair from the same individual. Pearson correlations evaluated unprocessed hair cortisol levels versus the matching chemically processed hair cortisol levels. Age, sex, race/ethnicity and tobacco status were also assessed using ANOVA. All analyses were performed using Prism (GraphPad Software, Inc., San Diego, CA).
Hair was cut from the posterior vertex region as close to the scalp as possible using scissors or thinning shears. Hair length was recorded and proximal ends of the hair samples were marked. Hair samples were stored in aluminum foil for protection as recommended [18 (link)]. All hair samples were divided into a minimum of two clusters to a maximum of six clusters of full-length hair. If hair was longer than 5 cm, the strands were divided at 5 cm from the scalp and only the proximal segment used for analysis. For experiment 1, clusters of hair were exposed to various common products used for chemical processing. One cluster was set aside from each subject and used as the “native” hair cluster, to which all other clusters were compared. The other clusters were exposed to one or more of seven hair treatments (depending on the number of available hair clusters): potassium persulfate (bleach), <2% peroxide (H2O2), (demi-perm or semi-permanent colour), 6% H2O2, 9% H2O2 or 12% H2O2. All peroxide treatments also contained ammonia. A licensed cosmetologist (AG) instructed the first author (CH) on standard salon procedures using Wella Koleston Perfect© and Wella Color Touch© (Wella Professionals, Procter & Gamble, USA) products prior to processing hair samples.
For Experiment 2, a variety of common commercially available shampoos, to represent the assortment of shampoos by cost, hair texture, and popularity used commonly by our subjects, were used to wash hair samples from untreated clusters described previously. Hair samples were placed in individual 15 ml conical centrifuge tubes with shampoo diluted 5-fold. Each tube was vigorously shaken for 5-10 seconds. The hair was removed from the tube and placed under a running stream of deionized water for 5 seconds to rinse the hair after each wash step. Hair samples were washed either 15 or 30 times with the same hair product for comparison to native hair.
Following either chemical treatment or shampoo, each cluster from Experiments 1 and 2 was washed three times in isopropanol and dried as previously described [3 (link)]. After washing, drying, and weighing, the hair was ground in 2 ml cryovials (Wheaton, Millville, NJ, USA) using a ball mill (Retsch, Haan, Germany) that held the cryovial and sample as well as a 3/16 inch stainless steel ball bearing in specially milled aluminum cassettes holding three cryovials. The cassette containing the cryotubes was immersed in liquid nitrogen to snap freeze the hair prior to grinding to facilitate the process. Samples were ground for approximately 5 min. The powdered hair (5-10 mg) was extracted in the same cryovial in HPLC grade methanol at room temperature for 24 hours with slow shaking. Following extraction, the cryovial was spun for 120 s in a microcentrifuge at 20000 g and 200 μl supernatant was removed, placed into a second microcentrifuge tube and dried for 24 hr under a hood. The dried extract was then reconstituted in 133 μl of assay buffer and cortisol levels determined using a commercial high sensitivity EIA kit (Salimetrics LLC, State College, PA, USA) according to manufacturer's directions. A pooled control consisting of previously ground hair was extracted and run on each EIA plate for determination of inter-assay coefficients of variation. Inter-assay coefficient of variation (CV) for the control hair pool was 11.6% and intra-assay CV was 1.9%.
Statistical analyses applied paired t-tests comparing native to chemically processed and shampooed hair from the same individual. Pearson correlations evaluated unprocessed hair cortisol levels versus the matching chemically processed hair cortisol levels. Age, sex, race/ethnicity and tobacco status were also assessed using ANOVA. All analyses were performed using Prism (GraphPad Software, Inc., San Diego, CA).
