Faculty of Allied Medical science

University of Nyala

The effect of feeding Gum Arabic on serum total and lipoproteins cholesterol in Hypercholesterolemic Rats

(With one table)

N. Abdelwahed; O. F. Idris*; H. I. Seri**

* Department of Biochemistry, School of Biotechnology, Al NeelainUniversity.

** College of Veterinary Medicine, SudanUniversity of Science and Technology (SUST).

تأثير إطعام الصمغ العربي علي الکولستيرول الکلي و اللايبوبروتينات في فئران عالية الکولسترول في الدم

نجم الدين عبد الواحد، عمر فضل ادريس و هشام اسماعيل سري

تماشياً مع الاهتمام المتزايد بالطب البديل (الطب الشعبي) في علاج الامراض المختلفة بما فيها ارتفاع مستوى الکوليسترول فى الدم، هدفت هذه الدراسة الي تقويم تأثير الصمغ العربى المخلوط مع الطعام على مستوى الکوليسترول الکلى وکوليسترول اللايبوبروتين (الدهن البروتيني) ذو الکثافة المنخفضة جداً واللايبوبروتين منخفض الکثافة واللايبوبروتين عالى الکثافة والجليسريدات الثلاثية فى دم الفئران المغذاه اضافياً بالکوليسترول.

أجريت التجربة علي خمسة وعشرون فأراً تم تقسيمهم  الى خمس مجموعات أ، ب، ج، د و هـ، بکل منها خمسة فئران. کانت المجموعة (أ) هى مجموعة مقارنة وأعطيت الوجبة الاساسية فقط، المجموعة (ب) أعطيت الوجبة الاساسية + 1% کوليسترول، المجموعة (ج) أعطيت وجبة المجموعة (ب) مضافاً اليها بدرة الصمغ العربى بنسبة 10%، المجموعة (د) أعطيت الوجبة الاساسية بإضافة 1% کوليسترول و بدرة الصمغ العربى بنسبة 20%، اما المجموعة الأخيرة (هـ) فأعطيت الوجبة الاساسية بإضافة 1% کوليسترول و بدرة الصمغ العربى بنسبة 30% وذلک لمدة 30 يوماً.

أخذت عينات من الدم من جميع المجموعات عند نهاية فترة الدراسة وذلک  لإجراء التحليل. خلصت النتائج  الي انخفاض مستوي الکوليسترول الکلى في المجموعات (ج)، (د) و (هـ) مقارنة بالمجموعة (ب) لکنه لم يکن معنويا. مستوى کوليسترول اللايبوبروتين منخفض الکثافة انخفض معنوياً فى المجموعات (ج)، (د)، و (هـ) مقارنة بالمجموعة (ب). مستوى کوليسترول اللايبوبروتين عالى الکثافة زاد معنوياً فى المجموعات (ج)، (د) و (هـ) مقارنة بالمجموعة (ب). انخفض مستوى الجليسريدات الثلاثية  وکوليسترول اللايبوبروتين ذو الکثافة المنخفضة جداً على نحو تدربجى فى المجموعات (ج)، (د) و (هـ) مقارنة بالمجموعة (ب)، کان هذا الانخفاض معنوياً فى المجموعتين (د) و (هـ) فقط.

مما سبق، خلصت الدراسة الي أن اضافة الصمغ العربي الي طعام الفئران أظهر تأثير ايجابي علي مستوي الکولسترول و الجليسريدات الثلاثية في الدم.

SUMMARY

Parallel with the recent increasing interest in alternative/herbal medicine for the prevention and treatment of various diseases including hypercholesterolemia, in addition to the increasing uses of Gum Arabic (GA) as one option. The objective of this study was to evaluate the effect of Gum Arabic mixed diet on serum concentration of total cholesterol (TC), very low density lipoprotein cholesterol (VLDL-c), low density lipoprotein cholesterol (LDL-c), high density lipoprotein cholesterol (HDL-c), and triacylglycerol (TAG) in rats fed diet supplemented with cholesterol.

Twenty five Wistar albino rats were divided into five groups named A, B, C, D, and E, each of five rats. Group A was fed on basal diet and served as control, group B received the basal diet mixed with 1% cholesterol powder. Gum Arabic (GA) was added to the basal diet with 1% cholesterol powder at the rate of 10%, 20% and 30% in groups C, D and E, respectively.

After 30 days, blood samples were collected from all groups for analysis. There was non-significant reduction on TC in groups C, D and E compared to group B. The level of LDL-c was found to be significantly (P<0.05) decreased in groups C, D and E compared to group B. In groups C, D and E HDL-c level was significantly (P<0.05) increased compared to group B. Serum TAG and VLDL-c were reduced in dose response manner of Gum Arabic in groups C, D and E compared to group B, although the decrease was only significant (P< 0.05) in groups D and E.

The present study concluded that the addition of Gum Arabic to rat’s diet has positive effect in lowering serum cholesterol and triacylglycerol levels.

Keywords: Gum Arabic, Hyper-cholesterolaemia, Rats.

Introduction

Coronary heart disease (CHD) is a global health problem. High intake of fats and dyslipidemia are risk factors for its development. Certain food items which are rich in dietary fiber in association with exercise may be protective (Buriro and Tayyab, 2007). The association of raised serum cholesterol with Coronary heart disease is well known; some studies suggest that the increase of serum triacylglycerol may also be a hazard (Sharma et al., 1996).

There are many chemical drugs that lower cholesterol level in the body such as: statins, fibrates, ezetimibe and nicotinic acid, but most of them are expensive and have undesirable effect (Thomas, 2003). So there are increasing interest in alternative/herbal medicine for the prevention and treatment of hypercholesterolemia.

Gum Arabic (GA) is a dried exudates obtained from the branches and stems of Acacia senegal and closely related species (FAO, 1999). It is a complex polysaccharide of high molecular weight which contains neutral sugars as rhamnose, arabinose, and galactose; acids such as glucuronic acid; minerals such as calcium, magnesium, potassium, sodium, and phosphorous (Leung, 1980). Gum Arabic has wide industrial uses as a stabilizer, thickening agent and emulsifier, mainly in the food industry also in the textile, pottery, lithography, cosmetics and pharmaceutical industries (Verbeken et al., 2003).

In folk medicine, GA has been reported to be used internally for the treatment of inflammation of the intestinal mucosa, and externally to cover inflamed surfaces (Gamal el-din et al., 2003). Some recent reports have claimed that GA possesses anti-oxidant, nephro-protectant and other effects (Rehman et al., 2001, Gamal el-din et al., 2003, Ali et al., 2008).

Clinically, GA has been tried in patients with chronic renal failure, and it was claimed that it helps to reduce urea and creatinine plasma concentrations and reduces the need for dialysis from 3 to 2 times per week (Suliman et al., 2000).

Ross et al., (1983) and Sharma (1985) reported reductions of total serum cholesterol by 6% and 10.4%, respectively when human subjects received 25 g/day and 30 g/day of GA for periods of 21 and 30 days. The decrease was confined to LDL and VLDL cholesterol only, with no effect on HDL and triacylglycerol. In contrast, consumption of GA at a dose of 15 g/day for 4 weeks by healthy human subjects or hypercholesterolemic subjects had no significant effect on plasma lipids (Haskell et al., 1992., Jensen et al., 1993).

In rats, the results were contradictory. Topping et al. (1985) showed that plasma cholesterol concentrations were unaffected by feeding GA, but plasma triacylglycerols were significantly lower than in controls. 

In another study, GA was fed to rats replacing cellulose in purified diets supplemented with cholesterol and cholic acid, no significant effects of increasing concentrations of GA were found on the concentrations of plasma cholesterol when compared to levels found in rats that consumed control diet containing cellulose alone. Plasma triacylglycerol concentrations were, however, higher in rats fed GA (Annison et al., 1995). These findings are not universally accepted and their confirmation, validity, reliability and mode of action await further studies.

The aim of this study is to evaluate the effect of feeding Gum Arabic mixed diet on the level of serum total and lipoproteins cholesterol in induced hypercholesterolemic Wistar albino rats.

Materials and Methods

Experimental animals: Twenty five Wistar albino rats obtained from the NationalCenter for Research, Khartoum were used in this study. The rats were housed identically in stainless steel cages in an air room under suitable conditions. All of the rats were initially fed a standard laboratory diet for 7days as adaptation period. Tap water was freely available.

Experimental design: The animals were divided into five groups each of five animals. These groups were named as A, B, C, D, and E. Group A was fed on basal diet and served as control, group B received the basal diet mixed with 1% cholesterol powder. Gum Arabic (GA) was added to the basal diet with 1% cholesterol powder at the rate of 10%, 20% and 30% in groups C, D and E, respectively. Blood samples were collected at the end of the experiment for the determination of lipid fractions concentration.

The composition of rat basal diet: The rats were given a basal diet which fulfilled their requirements, as follows:

Gum Arabic: Gum Arabic powder was purchased from (the Gum Arabic Company limited, Khartoum), which is pre-identified, and then added to the rats diet according to the different groups.

Cholesterol supplementation in the diet:  Cholesterol powder (Lab Tech Chemicals) was supplemented to the basal diet of the rats so as to induce hypercholesterolemia in all groups except the control one according to (Son et al., 2003).

Blood sampling: following 30 days, blood was collected from the rat's orbital plexus (1.5 ml) after an overnight fasting by capillary tubes and then transferred into test tubes; the blood was centrifuged at 5000 rpm for 10 minutes. Then the serum was placed into plain containers and used immediately.

Analytical methods:  All lipid fractions were estimated in the Research and Laboratory Unit, Khartoum Teaching Hospital, using Roche diagnostic Hitachi 902 analyzer, Germany, which is fully automated and computerized machine to report test results on various body fluid samples for wide range of analysis. It uses spectrophotometric principle.

Calculation of Very low density lipoprotein-cholesterol (VLDL-c): Very low density lipoprotein-cholesterol (VLDL-c) in samples was calculated according to the formula of (Friedewald, et al., 1972) which state that: VLDL-c= TG/5

Statistical analysis: Data are expressed as the means ± SE. The effect of Gum Arabic was determined by the one-way analysis of variance (ANOVA) procedure, using SPSS version 17 software. Differences were considered significant at P < 0.05.

Results

The induction of hypercholesterolemia: Table (1) Shows significant increase (P<0.05) in the total cholesterol level as well as LDL-c, and a significant decrease (P<0.05) in HDL-c in group B compared to group A (control).

The effect of feeding Gum Arabic mixed diet on serum total cholesterol level: Table (1) show the results of serum total cholesterol of groups A, B, C, D and E. The level of serum total cholesterol in group B is significantly (P< 0.05) higher than the level of serum total cholesterol in group A, but there is non- significant (P> 0.05) difference between groups B, C, D and E.                                                           

The effect of feeding Gum Arabic mixed diet on serum LDL-c level: Table (1) Show the results of serum LDL- c of groups A, B, C, D and E. The level of serum LDL-c in group B is significantly (P< 0.05) higher than the level of serum LDL-c in groups A, C, D and E. In group C the level of serum LDL-c is significantly (P<0.05) different compared to the level of serum LDL-c of group A. However, there is non-significant difference between groups A, D and E.  

The effect of feeding Gum Arabic mixed diet on serum HDL-c level: as we could observe in table (1),  the level of serum HDL-c in group B is significantly (P< 0.05) lower than the level of serum HDL-c in groups A, C, D and E. In group C the level of serum HDL-c is non-significantly (P>0.05) different compared to the level of serum HDL-c of group A, but the level of serum HDL-c of group D and E is significantly (P < 0.05) higher than that of group A ( the control group).

The effect of feeding Gum Arabic mixed diet on serum triacylglycerol and VLDL-c level: Table (1) Show the results of serum triacylglycerol (TAG) and VLDL-c level of groups A, B, C, D and E. Since they are dependent (VLDL-c equal 20% of TAG), the level of serum TAG and VLDL-c in group B are non-significantly higher than that of groups A and C, but significantly (P< 0.05) higher than those of groups D and E. Serum TAG and VLDL-c in group D and E are significantly (P< 0.05) lower than that of group A (control group).

Discussion

The objective of this study was to evaluate the effect of feeding Gum Arabic mixed diet on serum levels of total cholesterol (TC), VLDL-c, LDL-c, HDL-c and triacylglycerol (TAG) in induced hypercholesterolemic Wistar albino rats.

Induction of hypercholesterolemia: The results showed that the serum total cholesterol and LDL-c levels were significantly (P<0.05) increased  following administration of 1% cholesterol powder mixed with the basal diet in group B compared to group A (control), but serum HDL-c level was significantly (P< 0.05) decreased. These results were in agreement with the study of Son et al. (2003), who reported that administration of 1% cholesterol powder resulted in an increase in TC and LDL-c, but the HDL-c was reported to be decreased. Also the results were in line with the results obtained from other studies that administration of cholesterol powder or other high fat diet resulted in an increase in TC as well as LDL-c, but decreased HDL-c level (Chithra and leelaman, 1997; Hwang et al., 2001)

The effect of feeding Gum Arabic on serum total cholesterol level:    The level of serum total cholesterol was decreased, but non-significantly after the administration of 10%, 20% and 30% Gum Arabic compared to the hypercholestrolemic group (group B). This result was in line with the result obtained by Ross et al. (1983) who reported that, administration of Gum Arabic to men for 3 weeks result in modest fall in serum cholesterol, and he reported that the mechanism of the decrease in cholesterol with GA is not by adsorption of cholesterol metabolites as the fecal bile acids and neutral sterols did not increase. The findings in the present study are also consistent with those reported by Kelley and Tsai (1978), they noticed that Gum Arabic reduces serum cholesterol in rats, suggesting that gum interferes with dietary cholesterol absorption. In chicken, El-kheir et al. (2009) reported that Gum Arabic in the basal laying hen diet significantly reduced serum cholesterol in a gradual manner.

            On the other hand, these results disagreed with Topping et al. (1985) who showed that plasma cholesterol concentration was unaffected by feeding GA, and Annison et al. (1995) who reported that GA fed to rats replacing cellulose in purified diets supplemented with cholesterol and cholic acid, has no significant affect on plasma cholesterol when compared to levels found in rats that consumed control diet containing cellulose alone. It is also not in agreement with the work of Tageldin et al. (2006) who reported increase on cholesterol level in rabbits fed GA, and that GA associated with an increase in total cholesterol biosynthesis.

           Al-Othman et al. (1998) reported that, the soluble fraction of various dietary fiber sources, as found in oat bran and barley, seem to have the potential for lowering plasma total and LDL cholesterol levels, and Gel-forming soluble fibers, such as pectin and guar gum, have also been observed to be effective hypocholesterolemic agents, on the other hand, insoluble fibers, such as cellulose or wheat bran, have to be relatively ineffective in lowering serum cholesterol levels.

           Gum Arabic (soluble dietary fiber) was observed to be effective in lowering the total plasma cholesterol level compared with insoluble fiber (cellulose) when rats were fed diets supplemented with 1% cholesterol (Al-Othman et al. 1998).

           According to the above mentioned results the hypocholesterolaemic effect of GA may be due to its interference with dietary cholesterol absorption as suggested by Kelley and Tsai (1978).

The effect of feeding Gum Arabic on serumLDL-c: The level of Plasma LDL-c decreased significantly (P<0.05) after the administration of 10%, 20% and 30% Gum Arabic. These results were in line with the results stated by Ross et al., (1983) and Sharma (1985) who reported reduction of serum cholesterol when human subjects received GA, and that the decrease was confined to LDL and VLDL cholesterol. The results were also in agreement with that obtained by Al-Othman et al. (1998), but it disagreed with Jensen et al. (1993) who found that administration of GA in usual beverages of hypercholesterolemic males daily for 4 weeks showed no change in any plasma lipid parameters. 

Hwang et al. (2001), reported that feeding of 5% Allium sativum whole fruits to rats after high fat diet decreased LDL-c level significantly, and that was due to the fiber content of the fruit, that increases LDL-c receptor activity.

The effect of feeding Gum Arabic on serumHDL-c: In the present study addition of 10%, 20% and 30% Gum Arabic to the basal diet of induced hypercholesterolemic Wistar albino rats resulted in a significant increase of serum HDL-c. A finding that is in consistent with that reported by Al-Othman et al. (1998), who stated that supplementing Gum Arabic to diet with 1% cholesterol resulted in a significantly higher HDL-c content.

Hannan et al. (2003) reported that the administration of the soluble dietary fiber (SDF) fraction of Trigonella foenum graecum to rats orally twice a day for 28 days increased the serum HDL-c level. The present study is not in agreement with Ross et al., (1983) and Sharma (1985) who reported no effect on HDL when human subjects received 25 g/day and 30 g/day of GA for periods of 21 and 30 days, respectively, it is also disagreed with that of Davidson et al. (1998) who stated that soluble dietary fiber supplement containing Gum Arabic and Pectin in apple juice did not change HDL-c in hypercholestrolemic men and women.

According to the above mentioned findings the elevated level of HDL-c may be attributed to the soluble fiber nature of GA which led to decrease LDL-c and increase HDL-c production.

The effect of feeding Gum Arabic on serum triacylglycerol and VLDL-c: In the present study addition of 20% and 30% Gum Arabic to the basal diet of induced hypercholesterolemic Wistar albino rats resulted in a significant decrease in serum triacylglycerol level, but the level of 10% GA resulted in non-significant decrease. These findings were in line with the result obtained by Ross et al. (1983) and Sharma (1985). These results are also in agreement with the results obtained by Topping et al. (1985) who showed that, in rats fed GA plasma triacylglycerols were significantly lower than in controls. On the same line, McNaughton (1978) observed that plasma triacylglycerol decreased as dietary fiber level increased in diet fed to laying hens.

 However, these results disagree with Jensen et al. (1993) who reported that consumption of GA for 4 weeks by hypercholesterolemic subjects had no significant effect on plasma lipids, and Annison et al. (1995) who reported that GA fed to rats replacing cellulose in purified diets supplemented with cholesterol and cholic acid resulted in higher triacylglycerol.

CONCLUSION

Soluble fraction of various dietary fiber sources seem to have the potential for lowering plasma total and LDL cholesterol levels, and observed to be effective hypocholesterolemic agents.

From the present results and previous findings, mixing of Gum Arabicwith diet has diverse effects on atherogenic lipids among animal species including human, with most studies concluded that Gum Arabic has positive effect in lowering serum cholesterol.

Accordingly further studies are needed to specify the effective dose and the treatment period of GA that exerts protective and/or curative response to human. Further studies are also needed to target other lipid classes, and other biochemical parameters, with more focusing on how Gum Arabic exert its mechanism in lowering or increasing certain biochemical parameters. 

References

Ali, A.A.; Ali, K.E.; Fadlalla, A.; and Khalid, K.E. (2008): The effects of G.A. oral treatment on the metabolic profile of chronic renal failure patients under regular haemodialysis in Central Sudan. Nat. Prod. Res. 22: 12-21.

Al-Othman, A.A.; Al-Shagrawi, R.A.; Hewedy, F.M. and Hamdi, M. (1998): Plasma total, lipoprotein cholesterol, organs cholesterol and growth performance in rats fed dietary Gum Arabic. Food Chem. 62: 69-72.

Annison, G.; Trimble, R.P. and Topping, D.L. (1995): Feeding Australian Acacia gums and gum arabic leads to non-starch polysaccharide accumulation in the cecum of rats. J. Nutr. 125: 283- 292.

Buriro, M.A. and Tayyab, M. (2007): Effect of Nigella sativa on lipid profile in albino rats. Gomal J. of Med. Sci., 5 (1): 28 -31.  

Chithra, V. and Leelaman, S. (1997): Hypolipidemic effect of Coriander seeds (Coriandrum sativum): Mechanism of action. Plant Foods for Hum. Nutr. 51 (2): 167-172.

Davidson, M.H.; Dugan, L.D.; Stocki, J.; Dicklin, M.R.; Maki, K.C.; Coletta, F.; Cotter, R.; McLeod, M. and Hoersten, R. (1998): A low viscosity soluble fiber juice supplement fails to lower cholesterol in hypercholesterolemic men and women. J. Nutr. 128: 1927- 1932.   

El-kheir, M.K. S.; Ishag, K.E.A.; Yagoub. A.A. and Abu Baker, A.A. (2009): Supplementation Laying Hen Diet with Gum Arabic (Acacia senegal): Effect on Egg Production, shell Thickness and Yolk Content of Cholesterol, Calcium, and Phosphorus. Asian J. Poultry Sci., 3 (1): 9-14.

FAO (1999): Gum Arabic. Food and Nutrition Paper, 52, addendum 7. 

Friedewald, W.T.; Levy, R.I. and Fredrickson, D.S. (1972): Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clinical Chem. 18 (6): 499-502.

Gamal el-din, A. M.; Mostafa, A.M.; Al-Shabanah, O.A., Al-Bekairi, A.M. and Nagi, M.N. (2003): Protective effect of arabic gum against acetaminophen-induced hepatotoxicity in mice. Pharma. Res. 48: 631-35.

Hannan, J. M.; Rokeya, B.; Faruque, O.; Nahar, N.;  Mosihuzzaman, M.; Azad, A.K. and Ali, L. (2003): Effects of soluble dietary fibre fraction of Trigonella foenum graecum on glycemic, insulinemic, lipidemic and platelet aggregation status of Type 2 diabetic model rats J. Ethno. 88: 73–77.

Haskell, W.L.; Spiller, G.A.; Jensen, C.D.; Ellis, B.K. and Gates, J.E. (1992): Role of water soluble dietary fiber in the management of elevated plasma cholesterol in healthy subjects. Am. J. Cardiol. 69: 433- 439.

Hwang, G.; Heo, Y. and Lee, H., (2001): Effect of Coriandrum sativum on lipid metabolism in rats with hypertriglyceridemic diet. J. of Nutri. Sci. 4 (1): 13-19.

Jensen, C.D.; Spiller, G.A.; Gates, J.E.; Miller, A.F. and Whittam, J.H. (1993): The effect of acacia gum and a water-soluble dietary fiber mixture on blood lipids in humans. J. Coll. Nutr. 12: 147- 154.

Kelley, J.J. and Tsai, A.C. (1978): Effect of pectin, Gum Arabic and agar on cholesterol absorption, synthesis and turnover in rats. J. Nutr. 108: 630-639.

Leung. A.Y. (1980): Encyclopedia of common natural ingredients in food, drugs, and comsmetics. John Wiley and Sons. New York.

McNaughton, J.L. (1978): Effect of dietary fiber on egg yolk, liver, and plasma cholesterol concentrations of laying hen. J. Nutr. 108: 1842-1848.

Rehman, K.; Wingertzahn, M.A.; Harper, R.G. and Wapnir, R.A. (2001): Proabsorptive action of G.A.: regulation of nitric oxide metabolism in the basolateral potassium channel of the small intestine. J. Pediatr. Gastroenterol. Nutr. 32: 529- 533.

Ross, A.H.; Eastwood, M.A.; Brydon, W.G.; Anderson, J.R. and Anderson, D. M. (1983):A study of the effects of dietary gum arabic in humans. Am. J. Clin. Nutr. 37: 368– 375.

Sharma, R. D. (1985): Hypocholesterolaemic effect of gum acacia in men. Nutr. Res. 5(12): 1321-1326.

 Sharma, R.D.; Sarkar, A.; Hazra, D.K.; Misra, B.; Singh, J.B., Maheshwari, B.B. and Sharma, S.K. (1996):  Hypolipidaemic Effect of Fenugreek Seeds: a Chronic Study in Non-insulin Dependent Diabetic Patients. PHYTOTHER. RES., 10:332-334.

Son, C.G.; Choi W.J.; Shim, J.W.; Han, S. H.; Cho, J.H.; Song, K.C. and Cho, C.K. (2003):Effect of Gamichunggantang on hyperlipidemia. J. Acta. Pharmacol. 24(2): 133-139. 

Suliman, S.M.; Hamdouk, M.I. and Elfaki, M.B. (2000): G.A. fiber as a supplement to low protein diet in chronic renal failure patients. In: Sudan Association of Physicians, 17th Conference, Friendship Hall, Khartoum, Sudan, 21- 23 March.

Tageldin, S., ElKhalifa, K.F. and Abass, K. (2006): The effect of Gum Arabic on body weight and some blood elements in New Zealand California and baladi rabbits. Pakistan J.  Bio.  Sci. 9 (1):  96-98.

Thomas, S. (2003): Medications that lower cholesterol. J. Lipid Res 33(2): 79-82.

Topping, D.; Illman, R.J. and Trimble, R.P. (1985): Volatile fatty acid concentrations in rats fed diets containing gum Arabic and cellulose separately and a mixture. Nutr.Rep. Int. 32: 809- 814.

Verbeken, D.; Dierckx, S. and Dewettinck, K. (2003): Exudate gums: occurrence, production, and applications. Appl. Microbiol. Biotechnol. 63: 10- 21.