2010 TRI/PRINCETON CONFERENCE 241 Shampoo B. Aqua, sodium laureth sulfate, disodium cocoamphodiacetate, citric acid, so- dium chloride, sodium benzoate, proteolipid SR, salicylic acid. Proteolipid SR. All investigations were performed with proteolipid CR which consists of an alkyl chain derived from coco palm oil (C8–C18), a hydrolyzed protein present in wool keratin and a quaternized adapter group (cocodimonium hydroxypropyl hydrolyzed ker- atin). Proteolipid SR is water soluble. METHODS MULTIPLE DAILY STRESS SIMULATION The stress simulation, consisting of shampooing, drying and sun exposure, was automatically run by the RV E2 robot from Mitsubishi Electric Corporation, Tokyo (Japan). Firstly all hair samples were immersed into 160 ml of a solution of shampoo A for 3 min at a temperature of 38°C. Afterwards the hair samples were removed from the vessel and rinsed with 20 liter tap water at a temperature of 38°C for 2 min and dried with an air heater (LE 10000S, Leister Process Technologies, Kaegiswil, Switzerland) at 80 °C for 30 min. Sun exposure was simu- lated for 60 min at 764 W with an ATLAS Suntest (CPS+, Chicago, USA). The time for one cycle was approx. 2.5 h. Fifty cycles in total are run automatically within 5 days (12). ULTRA BLEACHING Hair strands were treated for 45 min with a mixture of 6% hydrogen peroxide (Hyprox 500®, Evonik) and 3% potassium persulfate (KPS-5®, Evonik) at pH 10. This mixture was spread by using a brush and washed out with tap water at approx. 30 °C for 3 min. MEDIUM BLEACHING (I) Hair strands were treated for 50 min with 7% hydrogen peroxide (Hyprox 500®, Evonik) at pH 8.8. This mixture was spread by using a brush and washed out with tap water at approx. 30 °C for 3 min. MEDIUM BLEACHING (II) Hair strands were treated for 25 min with 3% hydrogen peroxide (Hyprox 500®, Evonik) at pH 9.5. This mixture was spread by using a brush and washed out with tap water at approx. 30 °C for 3 min. QUANTIFICATION OF COVALENTLY BOUND LIPIDS The hair samples were subjected to a 16 h extraction chloroform/methanol azeotrope in a Soxhlet apparatus. Alkaline catalyzed total hydrolysis of the hair material was performed with 2M alcoholic potassium hydroxide solution and the lipids were recovered by repeated

JOURNAL OF COSMETIC SCIENCE 242 hexane extraction. Aliquots of the lipid extract were reacted with a silylation (MSHFBA, Macherey & Nagel) reagent to form their trimethylsilylester derivatives and the fatty acids, namely 18-methylicosanoic acid, were analyzed by gas-liquid chromatography/ mass-spectrometry coupling (GC-MS). Tridecanoic acid was applied as internal standard for quantitative measurements. Duplicate sub-samples of each lipid extract were prepared and analyzed by GC-MS. FLUORESCENCE MICROSCOPY The fl uorescence microscopic investigation was performed with a scanning photometer micro- scope MPM03, Zeiss, Jena, Germany). The fl uorescence labeling of the proteins was carried out with fl uorescein isothiocyanate (FITC) isomer I (Sigma, St. Louis, USA) (14,15). SCANNING FORCE MICROSCOPY Scanning force microscopy (SFM) studies were conducted using a Nanoscope llla from Dig- ital Instruments operating in the tapping mode. Standard silicon cantilevers were used (PPP-NCH from Nanosensors) with a spring constant k ≈ 42 Ν/m and an oscillation fre- quency f0 ≈ 330 kHz. Height and phase images were recorded simultaneously at a scan rate of 1 Hz. All measurements were performed at amplitude A0 of the freely oscillating canti- lever of 30–50 nm. Set-point amplitudes Asp were in the range of 0.85–0.95A0 and 0.35– 0.45A0, corresponding to light-tapping and hard-tapping conditions, respectively (16). DYNAMIC CONTACT ANGLE MEASUREMENTS The determination of the dynamic contact angles was performed by the Tensiometer K14 (Krüss GmbH, Hamburg, Germany). The hair perimeter L was determined by immers- ing the hair fi ber in n-heptan (99%), which shows total wettability (Θ=0). Advancing contact angle was measured in distilled water at 20°C. The immersion rate was 1mm/min in root-tip orientation at the maximum immersion depth of 1mm. PSEUDO-STATIC CONTACT ANGLE MEASUREMENTS AND DETERMINATION OF DROPLET RESORPTION TIME A droplet of 25 μl of distilled water was placed by using a micro-pipette on a parallel aligned and fi xed hair strand of 2 g weight and 18 cm length. The side of the droplet is recorded by digital camera (D40 F/3.5-5.6, Nikon co., Japan). Camera perspective is a perpendicular to the hair fi ber axes on level with the hair strand. RESULTS AND DISCUSSION AGING OF OUTER HAIR SURFACE After simulation of approximately 100 Middle European summer days (equivalent to 50 cycles of shampooing, blow drying and sun light exposure) signifi cant changes both in