background

Accumulation of oxidative damage to proteins and lipids is considered a major contributor to age-related skin deterioration (1). To date, we have shown that arNOX, a potent generator of superoxide, can be found in serum, saliva, sweat, urine and many cell types, including skin epidermis and dermis (2), and is subject to external modulation (3). We have shown that arNOX in both sun-exposed and sun-protected skin increases with age beginning at about the age of thirty and peaks around the age of 70 (4) and that oxidative skin damage in the elderly correlates with arNOX activity (5).

Further, we have shown that individuals who appear younger for their age have, on average, lower levels of serum arNOX activity compared to those who look older for their age (6). This work suggested that the simplest assessment tool of all, apparent age or appearance, may be related to the amount of oxidative damage dependent on superoxide-generating arNOX activity. Others have made similar observations on health and appearance leading us to believe that appearance is more than a matter of subjective beauty. And, most recently, we reported a relationship between appearance, arNOX activity and lipid oxidation (7).

Borkan et. al. in 1982 (8) using data from the Baltimore Longitudinal Study of the Gerontology Research Center, NIA, reported that the visual estimation of age could be used as an overall indicator of the rate of aging within a population. In another more recent study, examining pairs of twins, 70 years of age and older, the twin who visually appeared to be older was more likely to die within the next two years compared to the twin who appeared younger (9). Even specific facial features have been offered as useful predictors of chronic disease risk. Independent of age, gender and other risk factors, severe peri-orbital wrinkling (crow’s feet) in Korean subjects was correlated with lower estimated glomerular filtration rate and higher lipid hydroperoxide levels (10). Some have offered that the skin can be considered a mirror of the aging process (11).

objective

Demonstrate a relationship between aging of appearance and metabolic inhibitors of age-related NADH oxidase in Japanese subjects.

Methods & Materials

This was an IRB-approved, single-center clinical study designed to evaluate a possible relationship between visual appearance and metabolic inhibitors of arNOX. A diagram of the subject selection method is shown in Figure 1. Beginning with a pool of 150 Japanese women 45-60 years of age, by personal interview two study clinicians selected those who looked much younger than their chronological age and those who looked much older than their chronological age. Three photos, front, left and right side were taken of the face under controlled lighting of 35 subjects from each group. Without disclosing chronological age, photos were evaluated for skin appearance and overall age estimation by three dermatologists. Subjects were rank-ordered based on the error in age assessment by these dermatologists. Eighteen subjects from the most extreme “much younger than chronological age” and 24 subjects from the most extreme “much older than chronological age” were selected for saliva, serum and plasma collection.

Fasting blood was drawn, prepared, stored and analyzed according to a commercial metabolic profiling platform employing GC and LC methodologies (12). Metabolites within all classes (lipids, carbohydrate, energy, amino acids, etc) comprised the 471 profiled in this study. Statistical analyses were performed on (natural) log-transformed data to account for increases in data variance that occurs as the level of response increases. A statistical cut off of p≤0.05 was employed to indicate significant metabolite differences between the two groups. The data were typically displayed with box plots (Fig. 2) with the y-axis representing the median scaled value.

ArNOX activity in serum was measured as the production of superoxide using the standard method where reduction of ferricytochrome c by superoxide is monitored over 45 minutes as the increase in absorbance at 550 nm with reference at 540 nm (13). Measurement of arNOX inhibition by selected compounds was conducted using saliva from a 72 year old male. Compounds were prepared in water or ethanol and tested at the concentration given (final concentration in the reaction cuvette).

results

Serum and saliva arNOX activities were on average 1.5 and 1.6 times higher, respectively, in the group of subjects appear-ing 6.2 years older than those appearing 4.5 years younger based on age estimation by a panel of dermatologists (Table 1).

Comparing metabolic profiles of the older-appearing group to the younger-appearing group, only a limited number of metabolites achieved strong statistical significance. Most notably, metabolites related to lipid metabolism appeared to distinguish the two subject groups. Lysophosphoinositides and inositol (p=0.009) were lower in the older-appearing group of subjects (Fig. 2). Also lower in the older-appearing group was the sulfated steroid, dehydroepiandrosterone sulfate (DHEAS). Two long-chain dicarboxylic acids higher in the older-appearing group were octadecanedioate and hexadecanedioate (p= 0.0185). All other metabolites were statistically unchanged.

Myo-inositol and the mono- and diphosphorylated myo-inositols were inhibitory to arNOX (Table 2). Inhibition by myo-inositol was sterio-specific. Phosphatidylinositol was inactive as an arNOX inhibitor. However, lysophophatidylinsitol and other lysophospholipids were significant arNOX inhibitors (Table 3).

table 1.Test Parameter “Looks Younger” Group “Looks Older” GroupError in age assessment (apparent age estimation minus real age in years)

- 4.5 ± 1.8 (n=18)

+ 6.2 ± 2.4 (n=24)

Total apparent age span 10.7 years

arNOX (total ferricytochrome c reduction before SOD, nmoles/min/200 ul) Ratio

Anticipated from previous work 1.6Saliva 0.082 ± 0.033 0.120 ± 0.036 1.5Serum 0.095 ± 0.034 0.154 ± 0.045 1.6

Aggregate Data: The ratio shown is the arNOX activity of the “looks older” group divided by the activity of the “looks younger” group over the average apparent age span of 10.7 years. (*Reduction of ferricytochrome c inhibited by SOD).

table 2.Test compound Concentration Inhibition, %

Myo-inositol 1 mM 35 ± 12

Scyllo-inositol 100 μM 0 ± 1

Allo-inositol 100 μM 4 ± 4D-chiro-inositol 100 μM 3 ± 4Phosphatidylinositol 100 μM 0 ± 5.5L-alpha-glycerophospho-D-myo-inositol 4-monophosphate 10 μM 2 ± 31,2-dipalmitoyl phosphatidylinositol 4,5, diphosphate 100 μM 3 ± 12Myo-inositol 2-monophosphate 1 mM 33 ± 4D-myo-inositol 1,4-diphosphate 500 μM 50 ± 16D-myo-inositol 1,4,5-triphosphate 100 μM 32 ± 3L-myo-inositol 1,4,5-triphosphate 100 μM 0 ± 2D-myo-inositol 1,3,4-triphosphate 50 μM 33 ± 5Phytic acid (fully phosphorylated inositol) 1 mM 11 ± 11

Inhibition of age-related NOX (arNOX) of saliva by inositols and phosphosylate inositols based on cytochrome c reduction as a measure of superoxide production. Compounds were tested over the concentration range 100 μM to 1mM. Inhibitions at concen-trations resulting in maximum inhibition are reported.

table 3.Test compound Inhibition, %

Phosphatidylinositol 0 ± 5.5

Lysophosphatidylinositol 43 ± 10

Phosphatidylcholine 0 ± 3Lysophosphatidylcholine 22 ± 8Phosphatidylethanolamine 0 ± 6Lysophosphatidylethanolamine 20 ± 8Phosphatidic acid 7 ± 7Lysophosphatidic acid 33 ± 6

Inhibition of age-related NOX (arNOX) of saliva by phospholipids and lysophospholipids based on cytochrome c reduction as a measure of superoxide production. Compounds were dissolved in ethanol and tested at a final concentration of 100 μM (0.1 % ethanol). Results are averages of determinations with three different saliva collections ± standard deviations.

discussion

In Japanese women, apparent age (looking older or younger than one’s chronological age) and certain inositides and lysophospholipids appear to correlate with superoxide dismutase-inhibitable superoxide production in the blood, the bulk of which is derived from endogenous circulating arNOX activity. The principle metabolites, inositols and lysolipids, im-portant in maintaining osmotic balance, modification of enzyme activity, modulating the inflammatory response and cell signaling have now been shown to also be naturally-occurring inhibitors of an aging-related NADH oxidase (arNOX).

conclusion

This, our previous work and ongoing studies suggest that the increase in arNOX activity and reduction in arNOX inhibi-tors with age are significant contributors to increased oxidative damage to skin structural elements and appearance aging.

appearance in japanese WoMen correlates With the presence of inosotides and lysolipids, both significant

inhibitors of age-related nadh oxidase levels

D. J. Morré,1 D. M. Morré,1 D. G. Kern,2 S. M. Wood,2 H. Toyoda,3 H. E. Knaggs21NOX Technologies, Inc., West Lafayette, Indiana, 2Nu Skin Enterprises, Inc., Provo, Utah and 3Nu Skin Japan Co., Ltd., Tokyo, Japan

150JapaneseWomen45–60years

PersonalInterview

Much

YoungerYounger Same Older Much

Older

35MuchYounger

35MuchOlder

PhotosandBlinding

3Dermatologists:AgeEsImaIon

Ranking

ErrorinAgeAssessmentYounger Older

Saliva,serum,plasma

BiochemicalAssay

Figure 1. Selection of study subjects.

references

Figure 2. Inositol (top) and lysoglycerophosphoinositol (bottom) levels were lower in “older” group.

myo-inositol

younger older

0.0

0.5

1.0

1.5

1-arachidonylglycerophosphoinositol

younger older

0.0

0.5

1.0

1.5

2.0

1-palmitoylglycerophosphoinositol

younger older

0.0

0.5

1.0

1.5

2.0

2.5

1-stearoylglycerophosphoinositol

younger older

0.0

0.5

1.0

1.5

2.0

2.5

1. Makrantonaki E, Zouboulis CC. William J. Cunliffe Scientific Awards. Characteristics and pathomechanisms of endog-enously aged skin. Dermatology 2007;214(4):352–60.

2. Kern D, Draelos Z, Morré DM, Morré DJ. Age-related NADH oxidase (arNOX) activity of epidermal punch biopsies correlate with subject age and arNOX activities of serum and saliva. Society for Investigative Dermatology Meeting, Kyoto, Japan, May 2008.

3. Morré DM, Meadows C, Morré DJ. arNOX: Generator of reactive oxygen species in the skin and sera of aging individu-als subject to external modulation. Rejuvenation Res. 2010; March 26 [Epub ahead of print].

4. Kern DG, Draelos ZD, Meadows C, Morré DM, Morré DJ. Controlling reactive oxygen species in skin at their source to reduce skin aging. Rejuvenation Res. 2009 Dec 2 [Epub ahead of print]

5. Morré DM, Meadows C, Hostetler B, Weston N, Kern D, Draelos Z, Morré DJ. Age-related ENOX protein (arNOX) activity correlated with oxidative skin damage in the elderly. Biofactors 2009;34(3):237–44.

6. Rehmus WE, Kern DG, Janiqua R, Knaggs HE, Morré DM, Morré DJ. A randomized pilot study of the relationship between arNOX levels and appearance of skin aging in healthy women. Congress of the International Federation of the Societies of Cosmetic Chemists, Barcelona, Spain, September 2008.

7. Morré DJ, Morré DM, Kern DG, Wood SM, Toyoda H, Knaggs HE. Appearance in Japanese women correlates with age-related NADH oxidase levels and blood markers of lipid oxidation. Society for Investigative Dermatology Meeting, Atlanta, United States, May 2010.

8. Borkan GA, Bachman SS, Norris AH. Comparison of visually estimated age with physiologically predicted age as indica-tors of rates of aging. Soc. Sci. & Med. 1982;16:197–204.

9. Christensen K, Iachina M, Rexbye H, Tomassini C, Frederiksen H, McGue M, Vaupel JW. “Looking Old for Your Age”: Genetics and Mortality. Epidemiology 2004;15(2):251–252.

10. Park BH, Lee S, Park JW, Kim KA, Kim HU, Lee JH, Koh DH, Youm JH, Yoo N, Park SK, Kwon KS. Facial wrinkles as a predictor of decreased renal function. Nephrology 2008;13(6):522–7.

11. Makrantonaki E, Zouboulis CC. The skin as a mirror of the aging process in the human organism—state of the art and results of the aging research in the German National Genome Research Network 2 (NGFN-2). Exp. Gerontol. 2007;42(9);879–86.

12. Lawton KA, Beebe K, Berger A, Guo L, Rose D, Roulston A, Tsutsui N, Ryals JA, Milburn MV. Technology and applica-tions of metabolomics: Comprehensive biochemical profiling to solve complex biological problems. Computational and Systems Biology. Methods and Applications, 2009: 115–140, ISBYH: 978-81-308-0298-5.

13. Butler J, Koppenol WH, Margoliash E. Kinetics and mechanism of the reduction of ferricytochrome c by the superoxide anion. J. Biol. Chem. 1982;257:10747–10750.

004021_ESDR_2010poster.indd 1 8/23/10 2:08 PM