Connective Tissue Research, 1991. Vol.

26. pp. 1-10

Reprints available directly from the publisher Photocopying permitted by license only 0 1991 Gordon and Breach Science Publishers S.A. Printed in the United States of America

THE EFFECT OF VARIOUS AVOCADO OILS ON SKIN COLLAGEN METABOLISM

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M. J. WERMAN,t S . MOKADY,t M. E. NIMNI,* and I. NEEMAN? ?Department of Food Engineering and Biotechnology, Technion-Israel Institute of Technology, Haifa, 32000, Israel $Department of Biochemistry and Medicine, Bone and Connective Tissue Research Laboratory, University of Southern California, Los Angeles, CA 90033, USA (Received October 23. 1989; in revised form July 25, 1990; accepted August 6 . 1990)

The effects of various avocado oils on collagen metabolism in skin were studied in growing rats fed diets containing 10% (w/w) of the tested oils. Rats fed the unrefined avocado oil extracted with hexane from the intact fruit, its unsaponifiables or the avocado seed oil, showed significant increases in soluble collagen content in skin, though total collagen content was not affected. The increased soluble collagen content appears to be a consequence of the inhibition of lysyl oxidase activity. The active factor was found to be present in the unrefined avocado oil and probably originated from the avocado seed, since collagen metabolism was affected only by fractions which contained lipids fraction from the seed. In comparison rats fed the refined or unrefined soybean oils showed no effects.

KEYWORDS: dietary avocado oils, skin collagen, lysyl oxidase

INTRODUCTION The special properties that enable collagen to act as the major supporting framework of the body are largely dependent on the high structural stability of the collagen fibers. Under appropriate conditions, such as those encountered in the extracellular space, the newly synthesized collagen molecules aggregate end to end, and side to side, into organized fibrils showing microscopic characteristics of mature tissue collagen. During early stages the aggregated material is presumably held together by relatively weak electrostatic forces, hydrogen bonding and hydrophobic interactions. With time, these fibers mature into highly insoluble and inert structures, characterized by an increase in the mechanical, chemical and thermal stability. This change is attributed to the formation of stable inter- and intramolecular bonds.' Covalent crosslinks have been shown to occur between lysine residues on adjacent polypeptides chains. The reaction that initiates crosslink formation is catalyzed by lysyl oxidase, EC 1.4.3.13, first demonstrated by Pinnell & Martin.2 and shown to be an extracellular, copper-requiring, enzyme.3,4. In this reaction, the E-HN, group of certain lysyl and hydroxylysyl residues are oxidatively deaminated to form the corresponding 6-semialdehydes. These then condense either with E-NH, groups of other lysyl or hydroxylysyl residues or with other semialdehydes to form Schiff base or aldol cross1inks.l Address for correspondence: Dr. 1. Neeman, Dept. of Food Engineering & Biotechnology, Technion-Israel Institute of Technology, Haifa, 32000, Israel. Fax no.: 972-4-320742 I

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A few substances are able to influence selectively the metabolism of collagen. Among these the lathyrogen P-aminopropionitrile (BAPN), produces osteolathyrism by inhibiting lysyl oxidases and penicillamine and other compounds that contain adjacent thiol and amino group, alter collagen crosslinking by directly reacting with aldehydes on the surface of the collagen molecule.6 Nutritional factors have been suggested to play a role in the crosslinking of collagen.7.8The most important trace element required for crosslinking is copper,9.10 a cofactor for lysyl oxidase, and possibly for the regulation of lysyl oxidase synthesis.11 Metals, such as cadmium or zinc, which interfere with the metabolism of copper may inhibit crosslink formation. These metals might replace copper in the molecule of lysyl oxidase, causing a decrease in enzyme activity. However, the loss of copper is not proportional to the inhibition of the enzyme activity. 12 Of the vitamins, deficiencies of ascorbic acid,13.14 a-tocopherol,ls pyridoxine16 and cholecalciferoll7 have been suggested to reduce crosslink formation. A lipidic mixture derived from avocado and soybean non-saponifiables administered orally appear to be useful in the treatment of disorders of connective tissue, such as scleroderma, as well as in wound healing. 18 Robert et al. 19-21 reported the pharmacological effects of this mixture on carrageenan-induced granuloma and skin of rats, but no single purified constituent was isolated. In this study, the influence of various avocado oils were examined and compared to soybean oils, in order to understand the mechanism by which they affect collagen metabolism in the skin of growing rats.

MATERIALS AND METHODS Avocado Fruit and Oil The avocado fruit (Persea americana) belongs to the family Lauraceae and is one of the few cultivated fruits in which oil is the main component.22The two most prominent varieties in the California and Israel avocado industry are the Fuerte, a Guatemalan-Mexican hybrid, and the Hass, which originated from the Guatemalan seedling. According to Biale and Young23 one might expect similarities in physiological and biochemical properties of fruits from different races or varieties. There are two major methods for producing of avocado oil, namely organic solvent extraction and centrifugal separation. The oil, which makes up 15-30% of the fresh weight, is extracted from hard or soft mature fruit, either cored or intact. The unrefined oil is mainly used in the cosmetic industry. Purification steps namely neutralization, bleaching, degumming and deodorization are used for the production of refined avocado oil recently introduced into the food market.

Animals and Diets Female Charles River CD rats, weighing 80-100 g obtained from the animal colony of the department of Food Engineering and Biotechnology, Technion, Haifa, Israel were used. They were randomly divided into groups of eight rats each, housed individually and maintained at 25°C with a 12-hr lighvdark cycle. Food and water were given ad libitum, except for the pair-fed group. Each group was fed for 8 weeks the basic diet, ground

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AVOCADO OILS AND

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commercial rat feed (obtained from Asia Maabarot Ltd., Israel) supplemented at a level of 10% (w/w) with either avocado oils derived from different sources or with soybean oils as follows: Group A . Centrifugal separated refined avocado oil derived from cored avocado fruit (CRAO-C), Hass variety, obtained from Avochem, Santa Paula, CA, USA. Group B . Refined soybean oil (RSO), obtained from Shemen Ltd., Haifa, Israel. Group C. Unrefined avocado oil extracted from intact avocado fruit (E-URAO-I), Fuerte variety, obtained from Miluot Ltd., Haifa, Israel. Group D. Unrefined soybean oil (URSO), obtained from Shemen Ltd., Haifa, Israel. Group E . C-RAO-C as in Group A, containing 5% unsaponifiable material, prepared according to the official method of the AOCS24 from E-URAO-I (Group C). Group E C-RAO-C, as in Group A, containing 10%avocado-seed oil (ASO), Hass variety, laboratory extracted with a mixture of chloroform-methanol (2:1 v/v), respectively. Group G . Centrifugal separated unrefined avocado oil from intact avocado fruit (CURAO-I), Fuerte variety, obtained from E. Shmueli Industries Ltd., Ashdod, Israel. Group H . Centrifugal separated unrefined avocado oil from cored avocado fruit (CURAO-C), Fuerte variety, obtained from E. Shmueli Industries Ltd., Ashdod, Israel. Group I . Laboratory unrefined avocado oil from cored avocado fruit (L-URAO-C), Fuerte variety, extracted with a mixture of chloroform-methanol (2:1), respectively. In order to exclude possible influences due to low food consumption, a matched group was pair-fed with the rats fed avocado seed oil (Group E) since this group showed the lowest food consumption (unpublished data). The pair-fed group was maintained on the diet containing C-RAO-C as in Group A. Experimental At the end of the feeding period, the rats were fasted overnight and then killed by C 0 2 asphyxiation. The dorsal skin was shaved, cleaned of adhering tissue, sliced and weighed for the determination of moisture, protein and collagen content as well as for soluble collagen fractions and lysyl oxidase activity. Moisture content. Skin slices, 2-2.5 g each, were freeze-dried for 24 hr to constant weight. Protein content. Total nitrogen content of the freeze-dried skin samples, 0.3/ 0.5 g each, was determined by the Kjeldahl procedure as described in the AOAC official method.25 Protein content was expressed as nitrogen content X 6.25. Collagen content. Total collagen content of the freeze-dried skin was determined, with some modifications, according to the method of Rompala and Jones.26 Samples, 0.05-0.1 g each, were hydrolyzed with 3 ml of 6N HCl at 115°C for 24 hr. The acid was vaporized at 60°C in the presence of solid KOH. Hydroxyproline (Hy-Pro) content, as an index of skin collagen in the hydrolyzed samples, was determined as described by Grant.27 Collagen solublefractions. Skin samples, 0.5 g each, were extracted with 20 ml of 0.5N NaCl in 0.02 Tris-HC1 buffer pH 7.4, for 24 hr in a shaker at a rate of 100 strokedmin. Following extraction, the samples were centrifuged at 10000 X g for 20 min. The supernatant was dialyzed overnight against 0.5N acetic acid, pH 3.4. All these operations were performed at 4°C.Aliquots after acid hydrolysis (6N HCl, 115"C, 24 hr) were analyzed for Hy-Pro, as an index of collagen, and neutral soluble collagen (NSC) was expressed as HyPro content in the hydrolyzate. The residues were re-extracted with 0.5N acetic acid pH 3.4,

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M . J. WERMAN

el nl

under the same conditions. Acid soluble collagen (ASC) was expressed as Hy-Pro content in the supernatant. Total soluble collagen (TSC) was determined by extraction of fresh skin samples, 0.5 g each, directly with 0.5N acetic acid pH 3.4 as described above, and the HyPro measured in the supernatant. Lysyl oxidase assay. Skin samples were cut finely with scissors and homogenized with 19 volumes of 4M urea and 0.16M NaCl in 0.05M phosphate buffer pH 7.4 at 4"C, using a Virtis homogenizer. The resultant supernatant after centrifuging at 135000 X g for 45 min, and dialyzing for 24 hr against 0.16M NaCl in 0.05M phosphate buffer, pH 7.4 to remove the urea, served as the enzyme extract. Protein content in the enzyme extract was determined according to Lowry et al.28 For preparation of collagen substrate, calvarial parietal bones from eighteen 17-day-old chick embryos were incubated at 37°C in a 50 ml Erlenmeyer flask with 10 ml of Eagle's minimum essential medium without lysine and supplemented with 4 mg of proline, 5 mg of glycine, 5 mg of BAPN, 5 mg of ascorbic acid, and 2oooO units of penicillin G per 100 ml of medium. After preincubation for 60 min to reduce the free lysine pool, the medium was changed, 250 mCi of DL-[4,VH] lysine added to each flask, and the flasks were gassed for 1 min with a mixture of 5% Co,/%% 0,. After incubation for 24 hr, the calvaria were washed twice with 10 ml of 0.15M NaCl in 0.1M phosphate buffer (PBS), pH 7.8, and four flasks were pooled and homogenized in 40 ml of 1M NaCl in 0.05M TrisHCI, pH 7.4 at 4"C, for 10 rnin with a Ystral homogenizer and then centrifuged at 2oooOxg for 10 min. The collagen in the supernatant was precipitated by the addition of solid NaCl to a final concentration of 20%. The precipitate was collected by centrifugation at 2oooO x g for 10 min, resuspended in 15 ml of PBS and then dialyzed for 72 hr against the same buffer at 4°C. The dialyzate was clarified by centrifugation at 3oooOXg for 15 min. In order to inactivate endogenous lysyl oxidase, the supernatant was incubated with 5 mM BAPN for 2 hr at 37°C. During this treatment, the collagen formed fibrils which were recovered by centrifugation. The pellet was redissolved in PBS, dialyzed against the same buffer for 24 hr at 4"C, and the dialyzate used as the substrate. Lysyl oxidase activity was determined by measuring the tritiated water formed during incubation of enzyme extracts and radioactive substrate. The composition of the standard reaction mixtures was as follows: 1.2 ml enzyme preparation, containing 2-3 mg protein per assay;29 0.3 ml of the substrate (2-4 x 105 dpm); 0.4 ml of 1M NaCl in 0.05M phosphate buffer pH 7.4, with L-lysine at a final concentration of 0.01M per assay;300.1 ml water and 1 drop of toluene to prevent microbial growth. The control contained 0.1 ml of BAPN fumarate solution (4 mg/ml) instead of water. After incubation for 20 hr at 37"C, the tubes were rapidly chilled in ice and the reaction terminated by the addition of 0.25 ml of 50% trichloroacetic acid (TCA). The resulting protein precipitate was removed by centrifugation in a table centrifuge. The tritiated water formed during the incubation was collected by ion exchange columns as described by Melet et al.31 Columns were prepared by pipetting a slurry of Dowex 50w-X8, 200-400 mesh (Serva feinbiochemica, Heidelberg, Germany) in distilled water into plugged disposable Pasteur pipettes. Immediately before use the columns were flushed with 3 ml of 5% TCA and then 0.5 ml of the TCA supernatants were passed through. The samples were eluted with 5% TCA directly into 2 ml volumetric flasks. Volumes of 1 ml of the effluent were counted in a Beckman LS-9800 liquid scintillation spectrometer. Lysyl oxidase activity was expressed as dpm of 3H20 per mg protein. Statistical analysis. All data were expressed as the mean k SD and analyzed by one-way analysis of variance (ANOVA). If the ANOVA showed a significant value ( p < 0.01), Fisher's least significant difference test (LSD) for multiple comparison, was applied.32

AVOCADO OILS

AND SKIN COLLAGEN

5

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RESULTS Feeding weanling rats for 8 weeks with avocado oils (C-RAO-C, E-URAO-I) or soybean oils (RSO, URSO) did not affect moisture, protein and collagen contents in the dorsal skin. However, the E-URAO-I diet caused a 30% elevation in the total soluble collagen, while the refined avocado oil (C-RAO-C) and refined or unrefined soybean oils (RSO, URSO) had no effect (Table I). The effects of differently produced avocado oils on collagen content and solubility in the skin of growing rats is shown in Table 11. No changes were observed in total collagen content. However, total soluble collagen was increased in rats fed E-URAO-I, while those fed C-RAO-C and the unrefined oils obtained by centrifugal separation or by laboratory extraction had no effect. Hitherto, it seems likely that only E-URAO-I influenced collagen metabolism in the skin of rats. To locate the origin of the active factor(s) that may be present in E-URAO-I, rats were fed the unsaponifiables obtained from this oil or avocado seed oil (ASO). Total collagen content remained constant (20-21 mg Hy-Pro/g wet skin) within all the dietary groups including the pair-fed one. TSC content increased by 28.5% in rats fed E-URAO-I, by 31% in rats fed unsaponifiables and by 36% in rats fed avocado seed oil as compared with rats fed C-RAO-C and the pair-fed group (Table 111). Determination of soluble collagen fractions in the skin revealed that the contents of NSC and ASC were significantly increased by E-URAO-I, avocado unsaponifiables and ASO. In contrast, no differences in total collagen and TSC were observed between the pair-fed group and the rats fed C-RAO-C (Table 111). Compared to rats fed C-RAO-C or the pair-fed group the activity of lysyl oxidase was decreased by 32%, 48% or 56% in the skin of rats fed E-URAOI, its unsaponifiables or ASO, respectively (Table 111). Lysyl oxidase activity was significantly correlated ( p < 0.01) with total soluble collagen content in the skin (Fig. 1).

DISCUSSION Extracts of mixtures of avocado and soybean oils are used in the treatment of scleroderma, a disease characterized by abnormalities in collagen metabolism and connective tissue disorders in various organs. 18.35 In spite of this, few studies concerning the influence of the lipid components of avocado or soybean oils on collagen metabolism have been published. 18-21.3638 In the above studies it was not clarified which oils were responsible for the observed effects nor how the oils were extracted and their components separated. Robert et a1.21 observed that feeding mixtures composed of two-thirds unsaponifiables from soybean oil and one-third from unsaponifiables from avocado oil to rats caused increased soluble collagen content in a carrageenan-induced granuloma. The present study demonstrates that refined or unrefined soybean oils as well as refined avocado oil (Table I), have no effects on skin collagen metabolism, while rats fed the unrefined avocado oil showed increased skin collagen solubility. It is now clear that only avocado oil has a unique effect on collagen solubility, while soybean unsaponifiables, although they have no direct influence on collagen,21 probably have a synergistic effect when provided with avocado unsaponifiables. Avocado oils are commercially produced by organic solvent extraction which utilizes mature, but hard, intact fruit or by centrifugal separation which utilizes mature, hard or soft, intact or cored fruits.33 In addition to the differences associated with extraction, one should consider the toxic factors known to be present in the avocado seed.34.39 Indeed, of all the different avocado oils tested, only E-URAO-I, which contained the seed during oil extrac-

M. J. WERMAN ef al.

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TABLE I

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The effect of dietary refined and unrefined avocado or soybean oils on body weight and some skin characteristics in growing rats.+

Initial body weight (g) Body weight gain (9) Skin Moisture (%) Protein (%) Total collagen, Hy-Pro: (mgk WT) TSC, Hy-Pro: (mg/g WT)

URSO

C-ROA-C

E-URAO-I

93.4 2 8.5 147.1 2 13.9.

93.4 2 7 . 9 92.4 2 8.2b

94.1 2 8.2 151.8 2 10.98

93.7 2 7.8 156.0 -+ 16.6.

62.04 2 2.08 21.22 2 1.17 21.50 2 2.13

62.09 2 2.56 21.39 2 1.73 19.65 2 1.35

63.09 2 2.56 21.92 2 1.23 20.65 2 1.78

59.93 2 3.01 22.93 2 2.28 21.95 2 2.22

7.15 2 0.620

9.31

rt

0.57b

RSO

7.51 -+ 0.53.

7.45

-+

0.748

C-RAO-C: centrifugal separated refined avocado oil from cored fruit; E-URAO-I: extracted unrefined avocado oil from intact fruit; RSO: refined soybean oil; URSO: unrefined soybean oil; TSC: total soluble collagen; Hy-Pro: hydroxyproline; WT: wet tissue. 'Values given are means -+ SD for 8 rats. Values with different superscripts differ significantly, p < 0.01 (LSD multiple comparison test).

tion, influenced collagen solubility (Table 11). In spite of the seed being present during centrifugal oil production rats fed C-URAO-I showed no effects on skin collagen. This may be due to low extraction efficiency of the oil during centrifugal separation.40 Avocado unsaponifiables or avocado seed oil demonstrated similar effects on rat's skin collagen as observed by E-URAO-I feeding. It seems likely that the active factor present in E-URAO-I originated in the lipid fraction of the seed, extracted during oil production, was within the unsaponifiable components of the oil (Table 111). Introduction of a pair-fed group demonstrated that skin soluble collagen was not affected by the low food consumption. The low food consumption shown in the group fed ASO, might be due to an unpleasant taste or odor of the seed oil, or due to a toxic factor present in the avocado seed which decreases appetite in a manner not as yet understood. Unrefined avocado oil, its unsaponifiables as well as avocado seed oil, caused an increase in total collagen solubility, characterized by an elevation in NSC and ASC fractions. Impaired collagen crosslinking could explain the increased collagen solubility in neutral salt and the diluted acid solutions, observed in this work. Crosslinking takes place in the extracellular space, and the initial stage in the formation of the intermolecular bonds is the enzymatic oxidative deamination of specific lysine or hydroxylysine residues, by lysyl oxidase, and continues as the collagen molecules are packed into fibrils.41 The fact that lipid fractions of avocado influenced only collagen solubility, but not total collagen content, coupled with the observed inhibition of lysyl oxidase activity suggest that these lipids exert their effects only on crosslinking and not on collagen synthesis or degradation. Indeed, we demonstrated that E-URAO-I, its unsaponifiables and avocado seed oil inhibited lysyl oxidase activity by 30-56% in comparison with C-RAO-C (Table 111). Furthermore, a significant inverse relationship was found between skin lysyl oxidase activity and total soluble collagen content (Fig. 1). Since this enzyme may be a major factor in regulating collagen accumulation in some connective tissue disorders, decreasing cross-link formation by lysyl oxidase inactivation and therefore increasing the rate at which collagen fibers degrade in vivo, might serve as a therapeutic tool.42 In conclusion, avocado oil (E-URAO-I) produced by organic solvent extraction from intact fruit seemed to contain some factor(s) that inhibited lysyl oxidase activity. These

54.53 t 3.12 7.53 t 0.61a

C-ROA-C 52.56 2 3.14 9.67 2 0.42b

E-URAO-I 53.78 2 2.17 7.97 f 0.698

C-URAO-I 52.76 2 2.79 7.27 2 0.548

C-URAO-C

53.39 2 3.08 6.97 t 0.391

L-URAO-C

3560

2

20.45 7.47

2

-

5

2

2.1

415c

0.93 0.64.

0.2b 21.65 7.20 1.57 5.67 3700 2.34 0.17’

c 342cd

2 0.3@

2

c 0.51a

t

3.1 2 0 . 3 ~

C-RAO-C

21.66 t 1.67 9.81 ? 0.56b 2.78 2 0.16b 7.15 2 0.52c 1624 ? 2641

1.5 c 0.28

AS0

20.45 2 2.34 9.25 5 0.83b 2.32 2 0.22b 6.29 t 0.62k 2532 2 456b

2.1 t 0.3b

E-URAO-I

2.93 0.75b 0.41b 0.68” 1909 2 465ab

21.92 t 9.46 -t 2.86 ? 6.58 t

2 . 0 t 0.3b

USAPO

C-RAO-C: centrifugal separated refined avocado oil from cored fruit; ASO: avocado seed oil; E-URAO-I: extracted unrefined avocado oil from intact fruit; USAPO: unsaponifiable material from E-URAO-I; Hy-Pro: hydroxyproline; TSC: total soluble collagen; NSC: neutral soluble collagen; ASC: acid soluble collagen; WT: wet tissue. Walues given are means 2 SD for 8 rats. Values with different superscripts differ significantly, p < 0.01 (LSD multiple comparison test).

Body weight gain (g/day) Skin collagen, Hy-Pro: (mg/g WT) Total Total soluble collagen Neutral soluble collagen Acid soluble collagen Lysyl oxidase activity ( d p d m g protein)

Pair-fed

The effect of dietary avocado seed oil, unrefined avocado oil and its unsaponifiables compared with refined avocado oil and pair-fed rats on the content of collagen fractions (TSC, NSC, ASC) and on lysyl oxidase activity in the skin of growing rats.’

TABLE III

C-RAO-C: centrifugal separated refined avocado oil from cored fruit; E-URAO-I: extracted unrefined avocado oil from intact fruit; C-URAO-I: centrifugal separated unrefined avocado oil from intact fruit; C-URAO-C: centrifugal separated unrefined avocado oil from cored fruit; L-URAO-C: laboratory unrefined avocado oil from cored avocado fruit; TSC: total soluble collagen; Hy-Pro: hydroxyproline; DT: dry tissue; WT: wet tissue. Values given are means 2 SD for 8 rats. Values with different superscripts differ significantly, p < 0.05 (LSD multiple comparison test).

Skin Collagen, Hy-Pro: (mglg DT) TSC, Hy-Pro: (mg/g WT)

The effect of differently produced avocado oils on skin collagen content and solubility in growing rats.?

TABLE I1

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The effect of various avocado oils on skin collagen metabolism.

The effects of various avocado oils on collagen metabolism in skin were studied in growing rats fed diets containing 10% (w/w) of the tested oils. Rat...
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