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Arch Dermatol Res (2005)DOI 10.1007/s00403-005-0584-6 A. Barel Æ M. Calomme Æ A. TimchenkoK. De. Paepe Æ N. Demeester Æ V. RogiersP. Clarys Æ D. Vanden Berghe Effect of oral intake of choline-stabilized orthosilicic acid on skin, nailsand hair in women with photodamaged skin Received: 10 January 2005 / Revised: 20 April 2005Accepted: 23 June 2005 Springer-Verlag 2005 Abstract Chronic exposure of the skin to sunlight causes group but decreased in the ch-OSA group suggesting damage to the underlying connective tissue with a loss of improvement in isotropy of the skin. VAS scores for nail elasticity and firmness. Silicon (Si) was suggested to have and hair brittleness were significantly lower after an important function in the formation and maintenance 20 weeks in the ch-OSA group compared to baseline of connective tissue. Choline-stabilized orthosilicic acid scores. Oral intake of ch-OSA during the 20 weeks re- (‘‘ch-OSA'') is a bioavailable form of silicon which was sults in a significant positive effect on skin surface and found to increase the hydroxyproline concentration in skin mechanical properties, and on brittleness of hair the dermis of animals. The effect of ch-OSA on skin, nails and hair was investigated in a randomized, doubleblind, placebo-controlled study. Fifty women with Keywords Photodamaged skin Æ Silicon Æ Orthosilicic photodamaged facial skin were administered orally acid Æ Nails Æ Hair during 20 weeks, 10 mg Si/day in the form of ch-OSApellets (n=25) or a placebo (n=25). Noninvasivemethods were used to evaluate skin microrelief (fore- arm), hydration (forearm) and mechanical anisotropy(forehead). Volunteers evaluated on a virtual analog Healthy skin impedes the penetration of microorgan- scale (VAS, ‘‘none=0, severe=3'') brittleness of hair isms which can cause infections and protects against and nails. The serum Si concentration was significantly irritants. Ageing leads to several changes in the skin higher after a 20-week supplementation in subjects with and its appendages (hair, nails). These changes can be ch-OSA compared to the placebo group. Skin roughness broadly categorized as either intrinsic ageing (chrono- parameters increased in the placebo group (Rt:+8%; biological) or photoageing (actinic ageing). Intrinsic Rm: +11%; Rz: +6%) but decreased in the ch-OSA ageing results in subtle but important alterations of cutaneous function that are presumed to be due to in roughness from baseline was significantly different time alone, whereas photoageing is the result of between ch-OSA and placebo groups for Rt and Rm.
preventable chronic exposure The difference in longitudinal and lateral shear propa- radiation superimposed on intrinsic ageing. Major gation time increased after 20 weeks in the placebo changes of photoageing occur in the dermis. A markeddecrease in collagen, glycosaminoglycans and proteo- A. Barel Æ A. Timchenko Æ P. Clarys glycans is observed combined with a degeneration of Faculty of Physical Education and Physiotherapy, elastic fibers (elastosis) resulting in a rough leathery Vrije Universiteit Brussel, Brussels, Belgium skin surface with fine and coarse wrinkles. Further-more, a loss of elasticity and an increase in mechanical M. Calomme (&) Æ N. Demeester Æ D. Vanden BergheDepartment of Pharmaceutical Sciences anisotropy of the skin is observed. Premature age- Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, ing of the skin due to excessive exposure to UV light University of Antwerp, Universiteitsplein 1, either from the sun or/and from sun benches is an B-2610 Wilrijk-Antwerp, Belgium increasing problem [32].
E-mail: microfar@ua.ac.beTel.: +32-3-820-2550 Silicon (Si) is a ubiquitous element present in various Fax: +32-3-820-2544 tissues in the human body [1] and is present in 1–10 partsper million in hair [29] and nails [1]. Studies of silicon K. De. Paepe Æ V. Rogiers deprivation in growing animals indicated growth retar- Faculty of Medicine and Pharmacy, dation and marked defects of bone and connective tissue Vrije Universiteit Brussel, Brussels, Belgium [9]. Nutritional Si deficiency was found to decrease boththe collagen synthesis and the formation of glycosami- Subjects and methods noglycans in bone and cartilage [7]. In vitro, the activityof prolyl hydroxylase was reported to be dependent on the Si concentration in the medium of bone cultures,suggesting a Si-dependent pathway for collagen type I Fifty healthy Caucasian females, aged between 40 and synthesis [8]. Others have suggested a structural role of 65 years, with clear clinical signs of photo-ageing of Si in the cross-linking of glycosaminoglycans in con- facial skin were included in this study after written in- nective tissue [25].
formed consent. The subjects were assigned to two Recent animal studies confirm the involvement of Si groups which were matched on the basis of photo type, in bone metabolism both in young animals [26, 27] and age and actinic ageing. Subjects were randomly supple- in models for postmenopausal osteoporosis [15, 24].
mented with ch-OSA or a placebo in each group. Wo- Subcutaneously implanted sponges of Si-deprived rats men, using silicon supplements less than 3 months were found to contain less hydroxyproline compared to before the start of the trial or any food supplement other rats on a normal diet indicating that Si deprivation de- than the study medication during the trial, were ex- creases collagen formation which is associated with cluded. In addition, subjects following any dermato- wound healing [28]. In addition, the activity of liver logical or cosmetical antiageing or antiwrinkle therapy ornithine aminotransferase, an important enzyme in the including collagen, hyaluronic or botox injections, pathway of collagen formation, was lower in Si-deprived chemical and laser peelings, retinoic and alpha hydroxy rats compared to Si-adequate rats [28].
acid treatment during the trial, were excluded. Fur- Soluble Si is present as orthosilicic acid (OSA) in thermore, exposure to sun benches or sunlight was beverages and water. It is stable in dilute concentrations prohibited during the trial. The subjects provided a de- (<10 4 M) but polymerizes at higher concentrations tailed list of all cosmetic products that they use daily.
around neutral pH into a range of silica species.
Subjects agreed not to change this daily regimen during Absorption studies indicated that only OSA is bio- the trial. On the day that noninvasive tests took place, available, whereas its polymers are not absorbed [16].
subjects were instructed to refrain from using lotions, Dietary silicates undergo hydrolysis, forming OSA creams or other products on face and forearms. The trial which is readily absorbed in the gastrointestinal tract.
was started in the autumn of 2003 and was completed in Physiological concentrations of OSA stimulate skin fi- the spring of 2004.
broblasts to secrete collagen type I [23].
Ethical approval was obtained from the regional A stabilized form of OSA, choline-stabilized OSA Ethics Committee (Academic Hospital, Vrije Universi- (‘‘ch-OSA''), was found to have a high bioavailability in teit Brussel, Brussels, Belgium, protocol number 03/4 humans compared to other Si supplements that contain entitled antiageing effect of ch-OSA on photodamaged polymerized forms of OSA [3, 33]. Supplementation of skin in healthy volunteers). The study was carried out in animals with low doses of ch-OSA resulted in a higher accordance to the Declaration of Helsinki (1964) chan- collagen concentration in the skin [5] and in an increased ged by the 29th World Medical Assembly at Tokyo femoral bone density [4, 6].
Choline, the stabilizing agent in ch-OSA, is classified by the Food and Nutrition Board as an essential nutri- ent [12]. Although humans can synthesize it in smallamounts, dietary sources are needed to maintain normal All patients were supplemented during 20 weeks with health [2]. Choline is important for the structural two capsules daily containing either the excipiens (mi- integrity of cellular membranes since it is the precursor crocrystalline cellulose pellets, Pharmatrans Sanaq AG, of phospholipids (phosphatidylcholine and sphingomy- Switzerland) or 10 mg of silicon in the form of ch-OSA elin) which are essential components of biological pellets (Bio Minerals n.v., Belgium). Subjects were in- membranes. One of its metabolites, betaine, participates structed to take one capsule in the morning and another in the methylation of homocysteine to form methionine.
in the evening with a glass of water or juice. Placebo and Betaine is also known as an essential intracellular osm- ch-OSA capsules were identical in color, taste, odor and olyte [35]. Choline directly affects nerve signaling (as a packaging and their content was blinded to the subjects precursor of the neurotransmitter acetylcholine), cell and investigator.
signaling (as a precursor for intracellular messengerssuch as diacylglycerol or ceramide, platelet-activatingfactor and sphingosylphosphorylcholine) and lipid transport/metabolism (required in the biosynthesis ofvery low-density lipoproteins).
Blood samples were collected from fasting subjects at In the present study we investigated the effect of oral baseline and after a 20-week supplementation, using Si- intake of ch-OSA on skin, hair and nails in a random- free polypropylene syringes (Sarstedt, Germany) and ized, placebo-controlled double-blind study in subjects needles (Microlance, Becton Dickinson, Spain). Imme- with photo-aged facial skin.
diately after the sample was taken, the blood was transferred into Si-free polypropylene tubes without longitudinal and lateral shear wave propagation time anticoagulant (Sarstedt, Germany).
[14]. Microrelief (roughness) of the skin was measured Si concentration in serum was analyzed in one batch with the skin visiometer SV 600 (Courage-Khazaka, by electrothermal atomic absorption spectrometry with Colgne, Germany). Investigated roughness parameters inverse longitudinal Zeeman background correction were Rt (depth of roughness), Rm (maximum rough- (AAnalyst 800, Perkin Elmer, Bodenseewerk, Germany).
ness) and Rz (mean depth of roughness) [11].
Pyrolytic-coated graphite tubes were used. The hollowcathode lamp settings were respectively 30 mA lamp Evaluation of hair and nail brittleness current, 251.6 nm spectral line and 0.2 nm band width.
The injected sample volume was 20 ll and signals were Subjects were evaluated at baseline and after 20 weeks measured in the peak-area mode. Serum samples were of supplementation; the degree of brittleness of hair and measured in duplicate by standard addition. Standards nails on a 4 point scale with ‘‘0'' no brittle hair/nails, ‘‘1'' and serum dilutions were prepared in matrix modifier slight, ‘‘2'' moderate and ‘‘3'' severe.
solution containing 72 mg/l CaCl2 (Aldrich, Belgium),1.508 g/l NH4H2PO4 (Merck, Belgium) and 0.5 g/lNa4EDTA (Aldrich, Belgium) in ultrapure water (con- Statistical analysis ductance £ 0.08 lS). The sensitivity determined as theamount of silicon yielding a 0.0044 Abs.s signal was Differences between groups were evaluated with a 90 pg. A pool of serum obtained from fasting healthy Mann–Whitney U test and differences within groups subjects was analyzed on several days to determine the were analyzed with a Wilcoxon-matched pairs signed inter-assay c.v. and was found to be 8.7% for a mean Si rank test. P<0.05 was considered to be significant.
concentration of 109.09 lg/l (n=16).
To evaluate the safety of oral treatment with ch-OSA, serum concentrations of urea, creatinine, uric acid, total protein, cholesterol, HDL-cholesterol, LDL-cholesteroland direct bilirubin, glutamic-oxalacetic transaminase The mean age (±SD) in the placebo and the ch-OSA (GOT), glutamic-pyruvic transaminase (GPT), gamma- group was 49.2±4.7 years and 51.8±6.0 years, respec- glutamyltransferase (gamma-GT), cholinesterase, crea- tively. The mean body mass index was not significantly tine kinase (CK), amylase and lipase were determined.
different between both groups (placebo: 24.1±4.4, ch- Other parameters analyzed were sodium, calcium, OSA: 26.3±5.7).
phosphorus and zinc. All parameters were measured in Mean baseline values of total cholesterol, LDL cho- serum at baseline and after 20 weeks of supplementation.
lesterol and bilirubin were higher than the upper limit ofthe normal range in both the placebo and the ch-OSAgroup. All remaining parameters were within the normal Noninvasive methods range at baseline and after the 20-week supplementationin both groups. Twenty-four subjects in both the placebo All measurements were performed under standardized and the ch-OSA group completed the study. In neither of conditions, i.e., room temperature of 19±2C and a the two treatment groups there were adverse effects re- relative humidity level of 45–55%. An acclimatization ported that were related to the study medication.
time of at least 30 min was respected before measure- The mean serum Si concentration was comparable ments started. Hydration and microrelief of the skin for both groups at baseline but was significantly in- were evaluated on the forearm, whereas visco-elastic creased after 20 weeks of ch-OSA supplementation properties were measured on the forehead, at baseline (P<0.0001 vs T0 and P=0.0005 vs 20 weeks placebo, and after 20 weeks of supplementation, respectively, Table 1), whereas no differences were observed in the with the following noninvasive methods.
placebo group.
Hydration level of the skin surface was measured Skin hydration decreased significantly after supple- with the Corneometer CM 825 (Courage-Khazaka, mentation in both groups but no differences were found Colgne, Germany) [10] and visco-elastic properties of between the placebo and the ch-OSA group (Fig. 1).
the skin were measured with the Reviscometer MPA 5 Skin roughness parameters increased in the placebo (Courage-Khazaka, Colgne, Germany). The measuring group (Rt: +8%; Rm: +11%; Rz: +6%) but decreased principle of the Reviscometer is based on resonance in the ch-OSA group (Rt: running time. The time to propagate from transmitter to 8%). The change in roughness from baseline was sig- receiver is measured (shear wave propagation time) and nificantly different between ch-OSA and placebo groups is expressed in arbitrary units. This parameter is for Rt ( 0.12 vs +0.02 mm, P<0.05) and Rm ( 0.13 vs depending on the direction of the collagen fibers.
+0.05 mm, P<0.05, Fig. 2).
Therefore, two measurements are made in a different The difference in longitudinal and lateral shear propagation time increased after 20 weeks in the placebo Mechanical anisotropy is an indicator of skin photo- group but decreased in the ch-OSA group (P<0.05, ageing and was evaluated by the difference between


Table 1 Serum concentrations of silicon and safety parameters at baseline and after 20 weeks of supplementation (T20) with placebo andcholine-stabilized orthosilicic acid (ch-OSA) Uric acid(lmol/l) Total proteins(g/dl) HDL cholesterol(mmol/l) LDL cholesterol(mmol/l) Bilirubin direct(lmol/l) Fig. 1 Skin hydration(Corneometer CM 825)measured at the forearm atbaseline and after 20 weeks ofsupplementation (T20) withplacebo (n=24) and choline-stabilized orthosilicic acid (ch-OSA, n=24). * P<0.05 vsbaseline, Wilcoxon-matchedpairs signed rank test. Meanvalues ± SE are given VAS scores for nail and hair brittleness were signifi- Few studies investigating the effects of oral supple- cantly lower after the 20-week supplementation with ch- mentation of minerals on aged skin have been published.
OSA (P<0.05) compared to baseline scores (Fig. 4), Combined oral and topical treatment with colloidal si- whereas no significant differences were observed in the licic acid was found to have a positive effect on hair and placebo group.
nail brittleness in an open study [18]. However, no evi-dence was presented that the colloidal silica was ab-sorbed in the gastrointestinal tract. In fact, polymerized forms of OSA such as colloidal silica are known to havea very low bioavailability compared to OSA [22]. Oral Several studies have illustrated the beneficial effects of intake of extracts, derived from marine fish cartilage, topical treatment with tretinoin [20] or alpha hydroxy was reported to have a repairing effect on photodam- acid [31] containing creams in cutaneous ageing.
aged skin [19, 17]. However, it must be emphasized that



Fig. 2 Change in skinmicrorelief parameters(Visiometer SV 600) frombaseline, measured at theforearm, after supplementationwith placebo (n=24) or ch-OSA(n=24). Rt, depth of roughness;Rm, maximum roughness; Rz,mean depth of roughness. *P<0.05 vs placebo, Mann–Whitney U test. Mean values ±SE are given Fig. 3 Change in mechanicalskin anisotropy (ReviscometerMPA 5) from baseline,measured at the forehead, aftersupplementation with placebo(n=24) or ch-OSA (n=24).
Mechanical anisotropy wascalculated as the differencebetween longitudinal andlateral shear propagation time.
* P<0.05 vs placebo, Mann–Whitney U test. Mean values ±SE are given none of these studies were double blind nor placebo- with more than 90% compared to the baseline level controlled. Consequently, the obtained results could which confirms the high bioavailability demonstrated in have been biased by seasonal influence and subjective clinical [3, 33] and animal studies [5, 4]. In a compara- tive, bioavailability study [3], the silicon absorption from The present study is the first randomized, double a single dose of ch-OSA (20 mg Si) was compared with blind and placebo-controlled study that illustrates a colloidal silicic acid and phytolytic silica in healthy positive effect of an oral supplement on skin microrelief volunteers. Total silicon absorption was evaluated as the and skin anisotropy in women with photoaged skin. The area under the time curve (AUC, serum Si concentra- dose of ch-OSA supplementation (10 mg Si/day) was tion) and was found to be significantly higher for ch- low compared to the average daily Si intake of 20–50 mg OSA compared to the other silicon supplements and a reported previously by Pennington [21]. The major die- placebo. In another study [33], the bioavailability of ch- tary sources of Si are cereal/grain-based products and OSA was compared with a silicon-rich diet and phyto- vegetables but modern food processing, including lytic silica. After a 4-day intake of ch-OSA (10 mg Si/ refining, is likely to reduce the dietary Si intake as it was day), both the serum Si concentration and the urinary Si shown that fibers contribute the most to the silicon excretion increased, whereas no increase was found after content in plant-based foods [30]. After ch-OSA sup- the intake of a Si-rich diet (45 mg Si/day, 31 days) and a plementation the serum silicon concentration increased normal diet (14 mg Si/day, 4 days).


Fig. 4 Brittleness of hair andnails at baseline and after20 weeks of supplementation(T20) with placebo (n=24) andch-OSA (n=24). Brittleness wasevaluated on a 4-point scalewith ‘‘0'' no brittle hair/nails,‘‘1'' slight, ‘‘2'' moderate and‘‘3'' severe. Plot (a) nails; Plot(b) hair. * P<0.05 vs baseline,Wilcoxon- matched pairs signedrank test. Mean values ± SEare given The intake of 10 mg Si in the form of ch-OSA is safe major constituents of skin, the improvement in skin as no adverse effects related to the study medication parameters after ch-OSA supplementation points to po- were reported. Serum safety parameters remained within tential regeneration or de novo synthesis of collagen fi- the normal range. Total cholesterol, LDL cholesterol bers. Silicon was also reported to be involved in the and bilirubin levels were already increased at baseline synthesis of glycosaminoglycans [25] and was suggested which is most likely due to the consumption of a diet to have a structural role as a cross-linking agent in con- high in cholesterol and saturated fats [13].
nective tissue. Accordingly, treatment with ch-OSA Both the ch-OSA group and the placebo group might improve the glycosaminoglycan structure in the showed a small increase in hydration after 20 weeks of dermis and the keratin structure in hair and nails. Fur- supplementation compared to baseline. Since all subjects thermore, the choline compound present in ch-OSA started the study in October and finished the study in might have a synergistic effect with OSA since it is well March, these changes are likely to be the result of a sea- known that choline is involved in several basic biological sonal shift in temperature and relative humidity and are processes [2] including the fact that choline is a precursor not related to the study medication. After 20 weeks of ch- of phospholipids such as phosphatidyl choline which is an essential component of cellular membranes. The physio- mechanical properties improved. We previously demon- logical significance of choline is substantiated by the fact strated that oral intake of low doses of ch-OSA (5% in- that intentional deprivation of choline disrupts cell crease of total dietary Si intake) during 24 weeks in calves growth and division [34].
resulted in a significant higher hydroxyproline content in To our knowledge, the present study is the first ran- the dermis compared to placebo and found a significant domized, double blind placebo-controlled trial that correlation between the serum Si concentration and the illustrates the positive effect of an oral mineral supple- hydroxyproline content in cartilage [5]. Reffitt et al.
ment on skin surface and mechanical properties and on found that low levels of OSA (typical serum concentra- hair and nails brittleness, respectively.
tions) stimulate the synthesis of collagen type I in culturesof human osteoblasts and skin fibroblasts [23]. The OSA- Acknowledgements The authors thank Dr. Andre´ Moreels and thetechnical staff of The Medical Centre, Vrije Universiteit Brussel, for dependent stimulation of collagen synthesis was abol- blood sampling of the study subjects. ch-OSA was developed by ished in the presence of prolyl hydroxylase inhibitors. As Dirk Vanden Berghe for Bio Minerals n.v. This study was sup- type I collagen and its monomer hydroxyproline are ported by a grant of Bio Minerals n.v.
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Characterizing new fluorescent tools for studying 5-ht3 receptor pharmacology

Contents lists available at Characterizing new fluorescent tools for studying 5-HT3 receptorpharmacology Thomas Jack , Jonathan Simonin Marc-David Ruepp Andrew J. Thompson , Jürg Gertsch , Martin Lochner * a Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, 3012 Bern, Switzerlandb Institute of Biochemistry and Molecular Medicine, University of Bern, Bühlstrasse 28, 3012 Bern, Switzerland