Heavy Water Labeling of Keratin as a Non-Invasive Biomarker of Skin Turnover In Vivo in Rodents and Humans  Glen Lindwall, Elaine A. Hsieh, Lisa M. Misell, Christine M. Chai, Scott M. Turner, Marc K. Hellerstein  Journal of Investigative Dermatology  Volume 126, Issue 4, Pages 841-848 (April 2006) DOI: 10.1038/sj.jid.5700189 Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Purification of skin keratins: hair keratins are not extracted by this method unless reducing agents are added. MW: BioRad Precision Plus Protein All Blue molecular weight standards. a: Human keratin purified from tape strips as described. The bands lie between the 50 and 75kDa markers. b: Mouse keratin purified by the same method from whole epidermis harvested from a freshly collected skin sample. c: Mouse keratin purified by the same method from whole epidermis harvested from a previously frozen skin sample. d: Rat keratin purified from tape strips collected from a killed depilitated rat. e: Mouse hair extracted by the standard keratin extraction method. f: The same amount of mouse hair extracted by the standard keratin extraction method except that mercaptoethanol is added. Lanes (e and f) were overloaded to emphasize that no hair is extracted in lane e. (Criterion 4–20% Tris HCl PAGE, Bio-Rad Laboratories, Hercules, CA.) Journal of Investigative Dermatology 2006 126, 841-848DOI: (10.1038/sj.jid.5700189) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Skin kinetics in mouse whole epidermis. Keratin turnover is complete in about 3 weeks in normal mice but in only about 4 days in fsn mice. (a) Normal c57bl/6 mice were given an intraperitoneal bolus of 2H2O to achieve 5% 2H2O in body water. That level of 2H2O enrichment in body water was maintained by supplying the animals with 8% 2H2O in their drinking water. Animals were killed at the noted times and whole epidermis was isolated using dispase treatment. Keratin was extracted from the epidermis and analyzed for the incorporation of 2H. The level of incorporated 2H is expressed as excess fractional M+1 (EM1) as described in Materials and Methods. (b) In fsn mice, both keratin and keratinocytes approach complete turnover in about 4 days. Fsn mice were given the same 2H2O labeling regimen as the normal mice in (a) but for 2 or 4 days, at which time both keratin and keratinocytes were extracted from the epidermis. To enable comparison of the two measurements (protein from keratin and DNA from keratinocytes) 2H incorporation is expressed as fractional replacement (f). f is calculated from EM1 values by comparison with measured body water enrichments from blood samples collected at the time of killing, and is expressed as a percent of the theoretical maximum, or asymptotic EM1 value, at the measured body water (Hellerstein and Neese, 1999). Journal of Investigative Dermatology 2006 126, 841-848DOI: (10.1038/sj.jid.5700189) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Antipsoriatic treatments have little effect on epidermal kinetics. Homozygous fsn mice have much faster turnover than their heterozygous and wild-type littermates. Fsn mutants (fsn) and littermates, including heterozygous and wild-type mice (wt/het), were given no treatment, treated with cetomacrogol vehicle, or treated with an anti-psoriatic cream containing either triamcinolone or calcipotriene. 2H2O labeling was for 2 days. (a) Epidermal cell keratinocyte and (b) keratin turnover were measured and expressed as f, fractional replacement. Data are in mean±SD (n=2–11 per group). No substantial effects of any treatment were apparent for either keratin or keratinocytes. Journal of Investigative Dermatology 2006 126, 841-848DOI: (10.1038/sj.jid.5700189) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Incorporation of 2H into keratin at the skin surface in a human subject with 28 days of oral administration of 2H2O. A human subject was administered 2H2O for 28 days (5 days at a priming dose followed by 23 days at a dose designed to maintain a steady concentration of 2H2O in body water). Tape strips were collected every 2–5 days. Collection was five strips deep in each of two spots on the forearm, one washed with an isopropanol swab and one unwashed. Keratin extracted from the tape strips was analyzed for incorporation of 2H. Data from the third and fourth strips of the washed and unwashed skin are combined here. Differences between washed and unwashed or strips 3 and 4 were negligible (not shown). Journal of Investigative Dermatology 2006 126, 841-848DOI: (10.1038/sj.jid.5700189) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 2H incorporated into keratin from oral 2H2O can take several weeks to reach the skin surface. Three human subjects self-administered 2H2O for 7 days. Beginning on the eighth day, they collected tape strips once a week for 8 weeks, 20 strips deep in a single spot on the forearm that was varied each week. (a) Weekly 2H incorporation in keratin extracted from the fifth strip deep of subject #3. (b) 2H incorporation in keratin plotted by strip depth for each of the 8 weeks that strips were collected. 2H incorporation in keratin increases with depth at week 3. By week 7, peak incorporation has passed in strip 1 and the label is decreasing with depth. Traces for these 2 weeks are shown in bold. These data are from subject #2. (c) Skin keratin turnover was variable in three human subjects. The incorporation of 2H in keratin is plotted for each week for each of the three subjects. Values used are the average of six tape strips (strips 1, 5, 9, 13, 17, and 20 deep) at each week. Journal of Investigative Dermatology 2006 126, 841-848DOI: (10.1038/sj.jid.5700189) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions