Connrcriw Tissue Research. Vol. 37(3-4). pp. 303-31 I Reprints available directly from the publisher Photocopying permitted by license only

C> 1998 OPA (Overseas Publishers Association) N.V. Published by license under

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Acylated Ascorbate Stimulates Collagen Synthesis in Cultured Human Foreskin Fibroblasts at Lower Doses

than does Ascorbic Acid* GENNADY ROSENBLAT NATASHA PERELMAN b, ELLA KATZIR a , SISSI GAL- OR^,

AD1 JONASa, MARCEL E. NIMNI ’, NINO SORGENTEb and ISHAK NEEMANa

Department of Food Engineering and Biotechnology, Technion-Israel Institute of Technology. Haija 32000, Israel: bDepartment of Biochemislry and Molecular Biology and Surgery, Division of Surgical Research,

Children’s Hospiial Los Angeles. University of Southern Calijornia. 4650 Sunset Blvd., #35. Los Angeles. C A 90027, USA

(Received 6 June 1997; Revised 4 November 1997: Accepted I0 January 1998)

Acylated derivatives of ascorbic acid were found to be active in a number of biochemical and physiological processes. In the present study we investigated the effects of 6-0- palmitoyl ascorbate on collagen synthesis by cultured foreskin human fibroblasts. Our observations indicate a marked stimulatory effect on collagen synthesis by 6-0-palmitoyl ascorbate in the concentration range of 5-20pM, while the synthesis stimulated by ascorbic acid was maximal a t concentrations of 20-100 pM. Cells treated with 10 pM palmitoyl ascorbate for 36 h exhibited a production of collagen threefold greater than those in the presence of 10 pM ascorbic acid, and it was about the same as in cells treated with 100 pM ascorbic acid. By 48 h differences were not significant. Acylated ascorbate impaired vitality of the treated fibroblasts a t concentrations exceeding 20 pM in media supplemented with 0.5% FCS. However, most of the cytotoxic effect was neutralized by FCS at a concentration of 10%. The resistance of acylated ascorbate against oxidative degradation as well as the role of free radicals in the modulation of collagen synthesis by ascorbic acid and by its derivatives is discussed.

Ke)words: Collagen synthesis. ascorbate

INTRODUCTION for the expression and deposition of collagen in the extracellular m a t r i ~ . [ ~ ” ~ * ’ ~ ] In cultured dermal

Ascorbic acid (AA) plays an important role in human fibroblasts AA induces an increase in various functions of living organisms,[’91 particu- collagen gene transcription and in procollagen larly in collagen metabolism as a required cofactor mRNA levels.[’’ The mechanism of these effects

*Supported in part by a grant from The Michigan Applied Research Foundation “S.T.A.R.” I Corresponding author.

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304 G. ROSENBLAT el ul.

is poorly understood but allegedly involve AA- induced lipid peroxidation and stimulation of gene e x p r e ~ s i o n . [ ~ ~ ~ * ~ ] AA also serves as cofactor in a post- translational hydroxylation of specific procollagen proline residues.".'] This modification stabilizes the procollagen helix and stimulates the rate of procol- lagen secretion in cell culture systems. Based on its biological effects, AA is widely employed in cos- metics and medicine, as well as in experiments entailing the study of collagen synthesis in normal and diseased cell lines. Under normal culture conditions AA is quickly oxidized to dehydroascor- bic acid which is not effective in stimulating collagen synthesis at low concentrations. This instability of aqueous AA has promoted a number of investiga- tors to seek a more stable AA derivative. It was shown that a stable form ofascorbate such as 2-0-a- D-glucopyranosyl-L-ascorbic acidcz5] as well as ascorbyl-2-phosphate or its sodium and magnesium salts adequately stimulate collagen synthesis of human skin fibroblasts,"01 while the sodium salt of ascorbyl-Zphosphate requires at least a tenfold greater concentration to produce the same effect. 6-0-palmitoyl ascorbic acid (6PA) is an hydropho- bic derivative of AA showing surface activity. The ene-diol group in C-2-C-3 position of 6PA, causes it aggregate in a micelle-like form that probably renders it more difficult to oxidize.[18] Acylated derivatives of AA are found to be active in a number of biochemical and physiological processes. For instance, 6PA is a more potent antitumor agent than AA (Miwa and Yamazaki, 1986), probably owing to differences in production of reactive oxygen species"81 it alters DNA synthesis in tumor cells and tumor cell pr~l i ferat ion,"~~ leads to the release of membrane phospholipid^"^] and suppresses the post-transcriptional induction of ornithine decarb~xylase."~~

In the present study we investigated the effects of 6PA on collagen synthesis in cultured foreskin human fibroblasts as we set out to clarify the role of free radical processes in collagen synthesis. Additionally, we also examined the effect of 6PA on DNA synthesis in human foreskin fibro- blast.

MATERIALS AND METHODS

Fibroblast Culture

Fibroblasts were established in our laboratory from explants of human foreskins. Fibroblasts were grown in 75 cm2 plastic flasks (Corning Glass Works, Corning, NY) in Dulbecco's modified Eagle medium (DMEM) containing 4500 mg/l glucose supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 50 pg/ml gentamycin sulfate, 2.5 mg/ml amphoteracin B (all supple- ments were products of Biological Industries, Kibbutz Beit Haemek, Israel). The cultures were incubated at 37°C in 5% co2-95% air until confluent, subcultured by trypsinization and used between the 3rd and 18th passage.

Assay for Collagen Production by Fibroblasts

Fibroblasts were freshly trypsinized and plated in 24-well microculture plates (Corning Glass Works, Corning, New York, USA) at a density of 50.000 per/well and this in 1 ml DMEM supplemented with 10% FCS, and incubated for 4-6 days to con- fluency. The medium was removed and replaced with 1 ml of DMEM supplemented with 0.5% or 10% FCS containing 100 pg/ml beta-aminopro- pionitrile, 3 pCi or 10 pCi [3,4-3H]-proline (60 Ci/ mmol, ARC Inc., St. Louis, USA) and either 0- 100 pM AA or 6-0-palmitoyl ascorbate (Sigma Chemical Company, St. Louis, USA), following which the cultures were incubated for an addi- tional 24 h. The 3H-proline incorporation into pepsin-resistant, salt precipitated extracellular col- lagen was determined by the method of Webster and Harvey['31 with small modifications. Briefly, extracellular 'H-labeled collagen was extracted for 4 h at room temperature with 0.5 M acetic acid containing pepsin (Worthington Biochemical Cor- poration, New Jersey, USA) at a final concentra- tion of 0.5mg/ml. The extracted collagen was purified by successive salt precipitation, at acid and neutral pH. The final precipitate was solubi- lized in 0.5M acetic acid, placed in a scintillation

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PALMITOYL ASCORBATE-STIMULATED COLLAGEN SYNTHESIS 305

liquid and counted. Results were averaged from four identically treated wells and corrected for cell number. An aliquot of the cells was counted in a hemocytometer. The effect of FCS on collagen synthesis by fibroblasts was studied in identical condition described above using medium supple- mented with l00pM AA.

In the experiments with palmitic acid or with the antioxidants propyl gallate (Sigma Chemical Company, St. Louis, USA) and hydroquinone (Fluka Chemika-BioChemika, Switzerland) both compounds were evaluated in the presence of ethanol (final concentration in the media 0.1 YO)

Preparation of Palmitoyl Ascorbate Suspension

6-0-palmitoyl ascorbate suspension was prepared by either of two methods. In the first, acylated AA in final concentration of 1 mM was vigorously mixed with a Vortex with phosphate buffer (1OmM p H = 10.7) at 4°C for 5min and the pH adjusted to 6.8 with phosphoric acid. The other method entailed adding an ethanol solution of palmitoyl ascorbate to the media in a final ethanol concentration of 0.1 YO. In both cases UV spectrometry was used to assess the palmitoyl ascorbate stability in solution.

Assay of DNA Synthesis

DNA synthesis was assayed by monitoring ['HI-thymidine incorporation into the acid-insolu- ble Confluent cells were maintained for 24 h in the presence of ascorbate or palmitoyl ascorbate in 24-multiwell culture plates in DMEM media supplemented with 10% dialyzed FCS, then pulsed for 4 h with 2 pCi/ml [methyL3H]-thyrnidine (6.7Ci/mmol, ARC Inc., St. Louis. USA). In experiments with antioxidants these compounds were added to the medium simultaneously with AA or its derivative. Supernatants were aspirated, and cells washed three times with phosphate buffered saline (PBS), solubilized for 2 h at room temperature in 1 ml of 0.1% sodium dodecyl sulfate (SDS) containing 0.1 YO bovine serum

albumin (BSA) (both Sigma Chemical Company, St. Louis, USA) as a carrier protein, and pre- cipitated with 100 p1 of 50% trichloroacetic acid (TCA). After 30min incubation at 4"C, the TCA precipitable material was pelleted by centrifuga- tion at 2000g for 15 min, redissolved in 500 pl of a mixture of 0.1% SDS with 0.3N NaOH and processed for liquid scintillation counting.

Cytotoxicity Assay

The procedure used was essentially that of Romijn et a1.[221 Briefly, confluent cells plated in 24-well plates were treated with a palmitoyl ascorbate suspension for 24 h in DMEM supplemented with 0.5% or 10% FCS. At the end of this incubation period supernatants were discarded and I ml of the same media (without palmitoyl ascorbate) added. Thirty p1 of a 5mg/ml solution of MTT 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bro- mide (Sigma Chemical Company, St. Louis, USA) in phosphate-buffered saline was added to each well. After 4 h of incubation with MTT at 37"C, the supernatant was carefully aspirated and 0.1 ml of dimethylsulfoxide (DMSO) was added to each well. The plates were shaken for 5 min and the absorbency at 5 I5 nm read spectrophotometrically.

Ascorbate Oxidase-dependent Oxidation of AA and 6-0-palmitoyl Ascorbate

The detection of ascorbate and palmitoyl ascorbate oxidation in presence of purified ascorbate oxidase (Sigma Chemical Company, St. Louis, USA) was carried out using direct ultraviolet spectrophotom- etry. The method was based on the different UV spectral characteristic of reduced and oxidized AA or 6PA. The assay was performed by mixing the samples of AA or palmitoyl ascorbate (at final concentration 100 pM) in phosphate buffer (10 mM,pH 5.5)withascorbateoxidase(O.l04units/ ml). The absorption of AA or 6PA at 277 or 266 nm respectively was measured on a Biochrom 4060 spectrophotometer (Pharmacia, LKB, Biochrom Ltd). Diminishing UV absorption dosing the course

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306 G. ROSENBLAT et al.

of the enzymatic reaction was used as an index of sample oxidation. The data were expressed as percent ofnonoxidized ascorbic acid or its derivative remaining in the mixture.

RESULTS

Comparative Effects of AA and 6-0-palmitoyl ascorbate on Collagen Synthesis in Tissue Culture

Two different suspensions of palmitoyl ascorbate were used to study the effects of an acylated derivative of AA on collagen synthesis by foreskin fibroblasts. Unlike AA, its acylated derivative is insoluble in water. Consequently we used an ethanol solution for preparing one type of suspension, while a second type of suspension was prepared by mixing the 6PA in phosphate buffer at pH 10.7 with sub- sequent pH adjustment to neutral or weak acid level. The rational was to increase the solubility of the hydrophobic derivative of AA by transforming it into sodium salt by analogy with free fatty acids. AA, like other lactones, is unstable in basic solu- tions, oxidizing withacleavageofthelactonering.~'91 However for brief periods, palmitoyl ascorbate (at a concentration of 1 mM) proved resistant to pH degradation. The comparative effect of AA and 6- 0-palmitoyl ascorbate on collagen synthesis is shown in Fig. 1. In monolayer cultures of fibroblasts treated under nonproliferating conditions in media containing 0.5% dialyzed FCS, collagen synthesis was previously shown to be markedly stimulated by ascorbate levels greater than 20 pM and was max- imal at ascorbate concentration 50- 150 pM.[s*201 Our acylated derivative, however, markedly stimu- lated collagen synthesis already at a concentration of 5 pM. Maximal collagen synthesis was achieved at concentrations of 10-20 pM of palmitoyl ascorbate. Similar effects were observed whether cells were treated in media supplemented with either 0.5% or 10% FCS,althoughin presenceof 10% FCSthelevel of collagen synthesis was greater. In media supple- mented with FCS concentrations up to 5%, no significant differences in the effect of 6PA on

1.200 lo4

T 4 1.000 lo4 I 0.5 % FCS

0 1 2.5 5 10 20 40 60 100 concentration, pM

1.25 lo4 , I

3 1.00 104 -I 4 10 Yo FCS

0 1 2.5 5 10 20 40 60 100 concentration, pM

FIGURE 1 Comparative effect of AA and 6PA on collagen synthesis by human foreskin fibroblasts. Suspension of 6PA was prepared in phosphate buffer at pH 10.7 as described in "Materials and Methods". Confluent cultures were treated for 24 h with ascorbic acid - 8 or with 6PA in the presence of 3 pCi (3,4-3H) - proline in media supplemented with 0.5% FCS or with 10% FCS. Here and in other figures each data point repre- sents the mean of quadruplicate determination fS.E.M.

collagen synthesis by foreskin fibroblasts were observed when the suspension of 6PA prepared with phosphate buffer was replaced by a suspension containing ethanol (Fig. 2). The only difference between these two suspensions was in 10% FCS. In this concentration collagen synthesis was signifi- cantly lower in the ethanol suspension.

The Effect of Fetal Calf Serum on Collagen Synthesis

The effect of dialyzed fetal calf serum on collagen synthesis is shown in Fig. 2. The optimal FCS con- centrations in the presence of AA were different

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PALMITOYL ASCORBATE-STIMULATED COLLAGEN SYNTHESIS 307

5.0 la‘ I 3.5 1 o4 I I

4 3.0 104 -

3 2.5 1 o4 - Y) f! 4.0 104

3.0 104

In

5 2.0 104

1.0 104 5

n 0.5 2 5 10 15

% fetal calf serum

12 36 48 hours

FIGURE 2 Effect of fetal calf serum on ascorbate- or 6-0- palmitoyl ascorbate-stimulated collagen synthesis. Confluent cells were treated for 24h with IOpCi [3,4-3H]-proline in media supplemented with different concentrations of FCS in the presence of: lOpM 6PA suspension in buffer B; lOpM 6PA solution in ethanol m; 100pM ascorbic acid .

than those with its derivative. Maximal collagen biosynthesis in the presence of AA was obtained when the concentration of FCS in the media was lo%, which is consistent with studies published by Freiberger ef a/.[” In the presence of both types of 6PA suspensions, maximal collagen production occurred in media supplemented with 5% FCS.

Duration of Collagen Synthesis Stimulation in the Presence of 6PA

Duration of the stimulatory effects of AA and its derivative on collagen synthesis is shown in Fig. 3. In this experiment cells were treated with ascorbate or with 6-0-palmitoyl ascorbate for different time intervals and except for the case of the 12 h-treated cells the labeling period for all cells was invariably 24 h. As can be seen, in the 12-h cells, differences in collagen synthesis between the cells treated with AA (10 or IOOpM) and those treated with 6PA (10 pM) were insignificant. On the other hand after 36h the level of collagen synthesis level was markedly higher in cells treated with 10pM 6PA than in cells with 10pM AA and in fact it was about the same as in cells treated with 100 pM AA. No collagen stimulatory activity was detected

FIGURE 3 Time-dependence of the collagen stimulatory effect of palmitoyl ascorbic acid in contact with fibroblasts. Cells were seeded at near confluent density in 24-well microcult- ure plates (80 000-90000 cells/well) and at actual confluence the cells were exposed for 12, 36 or 48 h to 1OpM ascorbate , 100pM ascorbate or 10pM 6-0-palmitoyl ascorbate m; con- trol .. Media were supplemented with 10% FCS. Except for the cells treated for 12 h. the labeling period lasted 24 h.

following 48 h incubation in either 10 pM ascor- bate or 10pM 6PA.

Role of Surface Activity of 6PA on Collagen Synthesis Stimulation

In order to clarify whether it is the surface activity of 6PA which is responsible for its effect on collagen synthesis and to elucidate the role of palmitic acid, we exposed the test cells to a mixture of AA ( 5 or 20 pM) with n-octyl P-glucopyranoside (5 or 20 pM) (which is a surfactant of somewhat similar structure to 6PA) or to n-octyl P-glucopyranoside alone or to a mixture of AA with palmitic acid (5pM). We found that contrary to 6PA, all the above-men- tioned reagents affected collagen synthesis the same as did AA or else they reduced it (data not shown).

Cytotoxic Effect of 6PA

The cytotoxic action of 6PA was studied by an assay which is based on the reduction of a tetrazolium salt (MTT) to a colored formazan product by mitochon- drial enzymes. We found that incubation of the cells in media supplemented with 0.5% FCS led to reduction in MTT transformation to formazan

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308 G. ROSENBLAT e? al.

when the cells were treated with 6PA in concentra- tions exceeding 20 pM. This finding suggested impaired vitality of the treated cells, possible due to toxicity of the acylated AA at the higher concentration. Yet, no differences were detected between control and experimental cells upon treat- ment with acylated ascorbate (up to IOOpM) in media containing 10% FCS (data not shown). The effect of 6PA on DNA synthesis by cells treated in media supplemented with 10% FCS is shown in Table 1. The incorporation of [3H]-thymidine into the DNA of cells following their exposure for 4 h to 6PA up to a concentration of 40pM was not significantly inhibited compared to control cells. However when the cells were exposed for 24 h to 6PA at the same concentrations (up to 40 pM) their DNA synthesis was enhanced compared to the control, albeit at higher concentrations of the

TABLE 1 Effects of palmitoyl ascorbate on DNA synthesis by foreskin fibroblasts

Dose of 6-0-palmitoyl ascorbate (pM)

Relative incorporation of [3H] thymidine (% of control)

After 4 h After 24 h

0 5

10 20 40 60

100

100 100 87.8 f 3.5 128.9f 1.7 83.4f 3.7 134.9f 1.2 85.0 f 7.0 137.9 f 2.3 82.3 f 4.6 159.2 f 3.3

104.0 f 3.9 144.5f 1.2 101.0f 0.9 134.6 f 7.2

Confluent cells were either treated for 4h with palmitoyl ascorbate in the presence of [3H]thymidine or for 24h with palmitoyl ascorbate in which case the ['Hlthymidine was added during last 4 h.

acylated ascorbate there was again an inhibitory effect.

Effect of the Antioxidants on the Synthesis of Collagen and DNA

In the presence of antioxidants such as propyl gallate or desferoxamine B (which is also an ion chelator) at a concentration of 10pM, there was reduction in the ascorbate- and palmitoyl ascorbate-stimulated collagen synthesis. The anti- oxidants when added to fibroblasts in absence of AA, inhibited the synthesis of the DNA too (Table 11). The results from the experiments suggest that their effect on collagen synthesis may be non- specific. Hydroquinone at a concentration 10 pM, which is alleged to be a singlet oxygen scavenger, exerted no significant effect on the collagen synthesis induced by ascorbic acid although palmitoyl ascorbate-induced collagen synthesis was more susceptible to this antioxidant (Table 11). In this concentration hydroquinone did not affect the synthesis of DNA by fibroblasts.

Enzymatic Oxidation of AA and 6PA by Ascorbate Oxidase

6PA was more resistant against ascorbate oxidase- stimulated oxidation. As can be seen in Fig. 4, 100 pM AA was completely oxidized after 7 min of enzymatic reaction, although only 16% palmitoyl ascorbate was oxidized under similar conditions. Moreover even after 22 h of enzymatic reaction a residual amount of nonoxidised palmitoyl ascor- bate was detected in the reaction mixture.

TABLE I 1 lagen synthesis and on DNA synthesis by foreskin fibroblasts

Comparative effects of antioxidants on ascorbate- and 6-0-palmitoyl ascorbate-stimulated col-

Treatment Collagen synthesis Relative [3H]thymidine incorporation (YO of control) into DNA (YO of control)

Ascorbic acid, Palmitoyl ascorbic acid, 100 pM 10pM

Control (without antioxidant) 100 I00 100 10 pM hydroquinone 83.2 51.7 113.8 10 pM propylgallate 62.0 71.2 58.7 10 pM desferoxamine B 85.0 59.2 26.8

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PALMITOYL ASCORBATE-STIMULATED COLLAGEN SYNTHESIS 309

20 -

0-

8

I2O < \

4 0 2 4 6 8 1 0 1 2

min

FIGURE 4 ascorbate oxidase. 0 AA; A 6PA.

Enzymatic oxidation of AA and of 6PA by

DISCUSSION

The hydrophobic derivative of AA employed by us, namely, 6PA, was originally developed for the use as an antitumor agent. Its effects on normal (non- malignant) cells has yet to be established. Our present observations indicate a marked stimulatory effect on collagen synthesis by 6PA concentration in the range of 5-20 pM, with no toxic effects on the cultured foreskin fibroblasts. It also stimulated DNA synthesis in the range of concentrations effective on collagen synthesis. I t has been shown that the stimulatory effect of AA on collagen synthesis is essentially diminished when its concen- tration exceeds 200 pM and this in a dose response manner.l51 In terms of concentrations effecting collagen synthesis, both AA and 6PA behave similarly, except that the latter achieves it in smaller concentration. An interesting finding of the present study is that the fibroblasts, during their initial 12 h of exposure to 10 or 100 pM AA or to 10 pM 6PA, displayed about the same level of collagen synthesis (Fig. 3). This suggests that 10 pM (or probably less) of ascorbic acid in the medium suffices to maintain an essential level of ascorbate concentration in the cells during a short period. The higher ascorbate concentrations (about 100-200 pM) which are usually administered to maintain essential levels of this reagent, are actually necessary during pro- longed exposure (of about 24 h, which is optimal

for collagen determination) probably because AA is unstable and may fully oxidize within its first 10 h in the culture medium even in the absence of cells."91 Our speculation is in agreement with Kipp and Schwarz[I6] who assumed that minimum levels of ascorbate needed by the cells to maintain high procollagen production are much lower than would be predicated by extrapolation from steady state tissue levels in vitro. Another finding of interest was that in cells treated with 10 pM 6PA for 36 h, the production ofcollagen was 3-fold greater than in the presence 10pM of AA. This enhanced efficacy of palmitoyl ascorbate in stimulating collagen synth- esis over longer time intervals might be explained by the resistance of the ascorb ate derivative to oxidation. This assumption of ours is in agreement both with the present observation about the rela- tively high resistance of palmitoyl ascorbic acid against enzymatic oxidation, and with the results of Miwa et aZ."81 who demonstrated the resistance of palmitoyl ascorbate to autoxidation. We also may conjecture that the hydrophobicity of 6PA enables it to incorporate efficiently into cells. Both of the mentioned factors make it possible to achieve higher and more prolonged levels of 6PA steady-state concentrations in cells than that does AA. That said, one cannot exclude that 6PA may stimulate collagen synthesis through a different mechanism, in light of its effects on other metabolic reactions in cells. In this connection, it is pertinent that Gillery et uf.["] found the optimal concentration of AA for collagen synthesis in fibroblasts cultured in fibrin lattices to be 10 pM (which is 5- 10 fold smaller than the optimum for monolayared cells). These data suggest that it may be possible to render fibroblasts more responsive to smaller ascorbate concentra- tions. In any case, the suggested structural changes in plasma membranes due to the surfactant effect of our acylated ascorbate derivative is probably not responsible for its stimulatory effect since other surface active compounds such as n-octyl 0-glucopyranoside or palmitic acid, when mixed with AA, did not stimulate collagen synthesis. The different prooxidant effects of ascorbate and its acylated derivative may account for their different

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310 G. ROSENBLAT et al.

effects on collagen synthesis. We are still in the dark as to how ascorbate-stimulated collagen synthesis is induced. One suggested mechanism for AA induc- tion of collagen synthesis to based on the ability of AA to induce lipid peroxidation in cells at a con- centration which stimulates collagen s y n t h e ~ i s . [ ~ ~ ~ . ~ ] This hypothesis has both supported and opposed arguments.[”] The support for this conjecture was obtained from the observation that lipid peroxida- tion inhibitors also inhibit collagen synthesis in cultured cells. Both AA and its derivative produce in an aqueous environment H202 as well as super- oxide radicals and other reactive oxygen species (ROS) secondarily generated by the Haber-Weiss reaction.[241 At the same time the production of ROS by acylated ascorbic acid in aqueous media has been shown to be other than that by ascorbic acid.[’81 Such alteration of ROS produc- tion gives the edge to 6PA over AA in terms of its enhanced toxic activity against tumor cells. The cytotoxicity of 6PA was appreciably attenuated by singlet oxygen scavengers, like sodium aside or hydroquinone, at a concentration of 1 pM, but not by other radical scavengers.[18] In our study, hydro- quinone, at a concentration up to 10pM proved different in diminishing collagen synthesis in the presence of AA or its acylated derivative. The inhibition of palmitoyl ascorbate-stimulated col- lagen synthesis exceeded that of the AA-stimulated collagen synthesis by about 30%. Therefore we suggest the participation of singlet oxygen in the stimulation of collagen synthesis by the acylated derivative of ascorbic acid. Nevertheless, any differ- ences in ROS production previously reported to play a role in tumor cells cytotoxicity by the two compounds under discussion appear to be less relevant to collagen production.

In summary, we demonstrated that acylated ascorbic acid stimulated collagen synthesis in cultured foreskin fibroblasts at lower doses than does ascorbic acid. One possible explanation of this phenomenon is the resistance of palmitoyl ascorbate against oxidation. Relatively smaller amounts of palmitoyl ascorbic acid seem to be sufficient to maintain the inter- and intracellular

concentration of the reduced acylated ascorbate at a level needed for high collagen production. The results imply that free radicals may be responsible for the differences in stimulation of collagen synthesis by AA and its derivative.

References

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[2] Blanck, T.J.J. and Peterkofsky, B. (1975). The stimulation of collagen secretion by ascorbate as a result of increased proline hydroxylation in chick embryo fibroblasts. Arch. Biochem. Biophys., 171, 259-267.

[3] Chan, D., Lamande, S.R., Cole, W.G. and Batemenn, J.F. (1990). Regulation of procollagen synthesis and processing during ascorbate-induced extracellular matrix accumula- tion in vitro. Biochem. J. , 269, 175-181.

[4] Chojkier, M., Houglum, K.P., Solis-Herruzo, J. and Brenner, D.A. (1989). Stimulation of collagen gene expression by ascorbic acid in cultured human fibroblasts. A role for lipid peroxidation. J. Biol. Chem., 264, 16957- 16962.

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