*Editor's note: Adapted with permission from CL Emson et al, A pilot study demonstrating a non-invasive method for the measurement of protein turnover in skin disorders: Application to psoriasis, Clinical and Translational Medicine 2:12 (2013)
Due to the hyperproliferation and hyperkeratinization observed in psoriatic skin, substantial work examining epidermal keratinocyte and keratin dynamics has been undertaken. Abnormal epidermal turnover can impair skin barrier function and tissue repair capabilities, and keratinocyte or keratin turnover may be critical to understanding disorders of the skin as well as providing therapy.1
Methods using heavy water in human tissue have been developed to measure the kinetics of keratin,2, 3 triglycerides, fatty acids and cholesterol,4 as well as the kinetics of complex lipids, e.g., galactocerebrosides, from brain myelin.5 Specifically, protein synthesis can be measured via continuous oral heavy water administration and the rise of deuterium to the top of the stratum corneum (SC).
This approach is based on the incorporation of deuterium (2H) from heavy water (2H2O) into nonessential amino acids (NEAA) in newly synthesized proteins.5 This 2H2O technique has been used to measure the synthesis of proteins in muscle, bone, liver, lung and other tissues. Similarly, the incorporation of 2H from 2H2O into cholesterol ester, free fatty acids or triglycerides can be used to determine the synthetic rates of lipid turnover. When adhesive tape strips are used on human skin, a layer composed primarily of lipids, keratin and other epidermal constituents is removed.6 Thus, the turnover rate of epidermal keratin detected from the tape strips provides a strategy for assessing psoriatic disease activity and treatment effectiveness.
Epidermal keratin synthesis was previously measured by combining a collection of tape strips with heavy water labeling and mass spectrometric analysis.3 Researchers observed that keratin and keratinocytes had equivalent rates of fractional synthesis in a psoriatic animal model, and that keratin turnover from tape strips matched keratin turnover from tissue samples.3 In this article, the keratin synthesis method is used to measure skin protein turnover rates in involved and uninvolved skin of psoriatic individuals. This noninvasive technique should extend skin physiology knowledge regarding the effects of cosmetic ingredients and formulations on normal and abnormal skin.
Four males with dermatologist-confirmed severe plaque psoriasis participated in the present study. All subjects had no family history of disease and were concurrently receiving Goeckerman treatment, which involves exposing affected skin areas daily to ultraviolet B phototherapy and crude coal tar. Sampled areas were blocked from receiving Goeckerman treatment with the application of non-adhering dressingsa secured with surgical tapeb. All participants gave written informed consent.7
Normal Skin Model
Variability exists in an individual’s skin cell turnover rate depending on their age and genetics. Thus, the uninvolved, non-psoriatic area of each person was tape stripped and compared with the turnover rates of the cells in the involved area. Skin tape strips collected from the same individuals, but from uninvolved skin, had a significantly delayed appearance of detectable deuterium in the SC and slower skin turnover rates, as shown in Figure 1. Its appearance was detected around 10–20 days after the start of labeling. These results are consistent with published data of the keratin turnover rates from healthy, non-psoriatic individuals.3 Also, although psoriasis is considered a systemic disease, these results indicate the kinetics of uninvolved skin in psoriasis patients were comparable to normal skin and did not exhibit an increased basal turnover rate.
Samples from tape strips taken from the uninvolved areas were then analyzed for protein content with nanoscale liquid chromatography-tandem mass spectrometry (LC-MS/MS). The majority (98%) of the peptides identified from the uninvolved tape strips were derived from keratin, as determined by LC-MS/MS spectral counts, consistent with previous findings. A dramatic difference in the epidermal barrier function between psoriatic and normal skin was observed, as shown in Figure 2.
Psoriatic Skin Model
Although the detected proteins were almost completely newly synthesized, the appearance of labeled protein in psoriatic lesions was rapid. Deuterated proteins were detected in tape strips from psoriatic lesions as early as three days after starting heavy water administration (see Figure 1). The label was observed in all samples from lesions between 3–8 days after label administration. It should be noted the first time point sampled was on the third day, so initial appearance may have been even sooner. Moreover, the protein isolated was nearly 100% newly synthesized, demonstrating that the entire SC compartment in psoriatic subjects is replaced in less than five days.
Furthermore, samples from kinetically assessed tape strips showed that only 72% of the identified peptides were keratins. The different makeup of the surface proteins in the involved skin reflected the disrupted profile of the plaque with detection of inflammatory and antimicrobial defense proteins (S100A8, histone H4 and SERPINB4), as well as epithelial organizational proteins (Plakoglobin). A technical point worth noting regarding kinetics, however, is the near 100% fractional replacement of protein observed in epidermal skin strips from psoriatic subjects. This finding means that the keratin present is fully and rapidly turned over regardless of the presence of contaminant proteins. Thus, the altered kinetics of the label appearance in proteins from psoriatic patients does not represent labeling of inflammatory proteins but must primarily reflect turnover of keratin itself.
Previous attempts have been made to measure cell kinetics in normal and psoriatic skin. Methods used to measure proliferative activity in the skin include bromodeoxyuridine (BrdU), DNA flow cytometry (FCM), cell cycle markers such as Ki-67 antigen, and radio-isotopic tracers. Increased DNA synthesis in cells has been demonstrated in the psoriatic epidermis using methods including tritiated, thymidine and Ki-67 staining. However, the current technique has distinct advantages over these traditional methods. Many involve the labeling of human subjects or ex vivo skin biopsies with radioactive tracers, and both BrdU and 3HdT are toxic and mutagenic, raising ethical and practical considerations that prevent their use. Stable isotopes such as deuterium (2H) have a long history of safety, and provide an effective means for measuring the synthesis of molecules in experimental animals and humans. And since deuterium is safe for human use, it is easily translated into clinical studies. In addition, by using stable isotopes, longer periods of time can be labeled, allowing the disease to be tracked over time during a treatment or intervention.
Further, many historical studies use punch biopsies but this is invasive and not suitable for already damaged lesional skin, especially since injury alone may induce further exacerbations in uninvolved skin. Thus, tape stripping is a less invasive method of sampling the skin surface that also allows researchers to take multiple samples from the same subjects over time. The safety and simplicity of tape-stripping also enables much larger scale clinical studies, giving a more accurate determination of kinetics based on a larger population size.
Using the technique applied in the present study, previous clinical studies measuring keratin synthesis in normal subjects demonstrated a transit time of about 18 days from the start of heavy water administration until label appearance at the skin surface.3 A degree of variability in keratin synthesis kinetics was also observed among normal subjects. Here, similar appearance times were demonstrated for uninvolved skin from psoriatic individuals. This implies that basal turnover rates in skin are similar in psoriatic and non-psoriatic individuals, and that abnormal keratin kinetics are only observed in skin undergoing active disease. Also observed were similar keratin turnover rates in uninvolved skin in different anatomic locations on the same individual, again suggesting consistent basal skin kinetics for these subjects except in areas of psoriatic plaques.
Most striking was the detection of labeled protein, predominantly keratin, in lesional psoriatic skin within as little as three days following heavy water administration. And as previously stated, the keratin detected appeared to be nearly 100% newly synthesized during the labeling period. Despite the small scale of this study, with limited subject numbers and sampling times, the difference in keratin turnover between involved and non-involved skin was striking and consistent. Furthermore, this approach yielded an average turnover rate for the extracted proteins.
This approach also works well for healthy epidermis, which is thought to be composed largely of layers that work their way to the surface at a fairly uniform rate. Psoriatic skin is less ordered and could conceivably vary in the turnover rates of some components. In the future, the use of advanced dynamic proteomic techniques to pull out the turnover rates of individual proteins from the labeling of peptides may be applied with the same heavy water labeling/skin strip collection methodology described here.
The administration of deuterium oxide and collection of skin samples is relatively easy, making this assay highly attractive. However, as with any new assay, feasibility and cost must be considered. Unlike other methods of assessing protein turnover, this study allows subjects to drink pre-bottled D2O at home, and tape strips to be taken from them later. In this study, tape strips were collected by a single technician to minimize any sampling variability between subjects. However, it is conceivable to imagine a kit mailed to the subjects’ home including D2O, tape strips and collection cards. Following collection, tape strips would be placed onto a laminated card and stored in the subjects’ freezer until collection. The abundance of keratin in the tape strip sample also means protein isolation could be conducted by any chemical laboratory.
This method would be most applicable to interpreting clinical trials rather than for routine disease management of patients. In this scenario, access to mass spectrometry instruments and a chemical lab is not limiting. Additionally, new mass spectrometry methods such as multiple reaction monitoring are high throughput and have been used to translate biomarker approaches from discovery to clinical trials. These methods can be used to measure a targeted protein of interest from a complex biological sample, and could be adapted to this platform.
As well as analyzing keratin turnover as described above, other skin constituents can be obtained from the tape strip. Methods previously been have developed to measure the kinetics of lipids4, 5 and cell proliferation8, 9 using heavy water in human tissue. The adaptation of this tape strip technique to enable kinetic assessment of specific skin cells, lipids or proteins may yield further insights into the mechanisms behind this complex disorder. Methods have recently been devised to measure multiple protein kinetics simultaneously,10-12 which could further expand the utility of this method in looking at the pathology of psoriasis or response to treatment.
In summary, this study has noninvasively verified that keratin turnover is dramatically accelerated in psoriatic lesions, while uninvolved psoriatic skin has keratin turnover rates similar to non-psoriatic individuals.
Although it is only a pilot study, it suggests keratin synthesis kinetics may be a sensitive biomarker of psoriasis that could be used to assess individual disease activity or to provide a quantitative measure of response to treatment in clinical trials. This kinetic method of assessing skin activity or skin turnover is noninvasive and safe. Application of this technology could be used as a quantitative means to standardize either basic science or clinical studies of psoriasis, or of other skin conditions such as icthyosis or aging skin. Applications to objective quantification in cosmetics, skin, hair and nail biology appears obvious.
- MA Lowes, AM Bowcock and JG Krueger, Pathogenesis and therapy of psoriasis, Nature 445 866–873 (2007)
- EA Hsieh, CM Chai, BO De Lumen , RA Neese and MK Hellerstein, Dynamics of keratinocytes in vivo using HO labeling: A sensitive marker of epidermal proliferation state, J Invest Dermatol 123 530–536 (2004)
- G Lindwall, EA Hsieh, LM Misell, CM Chai, SM Turner and MK Hellerstein, Heavy water labeling of keratin as a noninvasive biomarker of skin turnover in vivo in rodents and humans, J Invest Dermatol 126 841–848 (2006)
- SM Turner, S Roy, HS Sul, RA Neese, EJ Murphy, W Samandi, DJ Roohk and MK Hellerstein, Dissociation between adipose tissue fluxes and lipogenic gene expression in ob/ob mice, Am J Physiol 292 E1101–E1109 (2007)
- MK Hellerstein, In vivo measurement of fluxes through metabolic pathways: The missing link in functional genomics and pharmaceutical research, Annu Rev Nutr 23 379–402 (2003)
- AMH Alikhan, Biology of the stratum corneum: Tape stripping and protein quantification, JSBR 10 2–9 (2008)
- CL Emson, S Fitzmaurice, G Lindwall, KW Li, MK Hellerstein, HI Maibach, W Liao and SM Turner, A pilot study demonstrating a noninvasive method for the measurement of protein turnover in skin disorders: Application to psoriasis, Clinical and Translational Medicine 2:12 (2013)
- R Busch, RA Neese, M Awada, GM Hayes and MK Hellerstein, Measurement of cell proliferation by heavy water labeling, Nat Protoc 2 3045–3057 (2007)
- RA Neese et al, Measurement in vivo of proliferation rates of slow turnover cells by 2H2O labeling of the deoxyribose moiety of DNA, Proc Natl Acad Sci USA 99:15345–15350 (2002)
- JC Price, WE Holmes, KW Li, NA Floreani, RA Neese, SM Turner and MK Hellerstein, Measurement of human plasma proteome dynamics with (2)H(2)O and liquid chromatography tandem mass spectrometry, Anal Biochem 420 73–83 (2012)
- JC Price et al, The effect of long term calorie restriction on in vivo hepatic proteostatis: A novel combination of dynamic and quantitative proteomics, Mol Cell Proteomics 11 1801–1814 (2012)
- P Fanara et al, Cerebrospinal fluid-based kinetic biomarkers of axonal transport in monitoring neurodegeneration, J Clin Invest 122 3159–3169 (2012)