Baboons kept on hypovitaminotic C diets, but without clinical signs of scurvy, had significantly higher heart muscle lipoprotein lipase activity than baboons on vitamin C 34 mg/kg body mass/day. When the serum vitamin C levels were above 0,35 mg/100 ml the heart muscle lipoprotein lipase was repressed. Serum vitamin C levels below 0,35 mg/100 ml stimulated lipoprotein lipase to between 2 and 3 times the repressed value. Heart muscle lipoprotein lipase from baboons receiving dietary vitamin C was inhibited by 0,34 mM vitamin C in vitro, whereas heart muscle lipoprotein lipase from baboons on scorbutogenic diets were stimulated by addition of vitamin C in vitro.S. Afr. Med. J., 48, 511 (1974)

de Klerk, W.A.

Kotze, J.P.

Matthews, M.J.A.

English

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13 Maart 1974 S.-A. MEDIESE TYDSKRIF(Byvoegsel tot die SI/id - Afri~aal1se Mediese Tydskrif)511V 33'instant coffee', made from 100% coffee, contains 4,38%.Details of the history, mode of manufacture andchemical compositions of tea, coffee and cocoa are givenbv a number of authors, for example Sinclair andHollingsworth.' An account of the active principles inthese and other beverages is also given by Sapeika.'REFERENCESI. Horwitz, W., ed. (1970): Official Methods of Analysis of rhe Asso-ciation of Official Analytical Chemists, 11th ed. Washington, DC:Benjamin FrankJin Station.2. Sinclair, H. M. and Hollingsworth, D. F. (1969): Huechison's Foodand the Principles of Nutrition. London: Edward Arnold.3. Sapeika, N. (1969): Food Pharmacology. Springfield, Ill.: Charles C.Thomas.Effect of Ascorbic Acid on LipoproteinLipase ActivityJ. P. KOTZt, M. J. A. MATTHEWS, W. A. DE KLERKSUMMARYBaboons kept on hypovitaminotic C diets, but withoutclinical signs of scurvy, had significantly higher heartmuscle lipoprotein lipase activity than baboons onvitamin C 34 mg/kg body mass/day. When the serumvitamin C levels were above 0,35 mg/100 ml the heartmuscle lipoprotein lipase was repressed. Serum vitamin Clevels below 0,35 mg/100 ml stimulated lipoprotein lipaseto between 2 and 3 times the repressed value.Heart muscle lipoprotein lipase from baboons receivingdietary vitamin C was inhibited by 0,34 mM vitamin Cin vitro, whereas heart muscle lipoprotein lipase frombaboons on scorbutogenic diets were stimulated by addi-tion of vitamin C in vitro.S. Afr. Med. l., 48, 511 (1974).The uptake of triglyceride fatty acids from the plasma byextrahepatic tissues is facilitated through hydrolysis of thetriglycerides by the enzyme, clearing factor lipase or lipo-protein lipase. Various authors have given evidence thatNational Research Institute for Nutritional Diseases of theSouth African Medical Research Council, PretoriaJ. P. KOTZEM. J. A. MATfHEWSW. A DE KLERKDate received: 23 November 1973.the enzyme acts at the surface of the capillary endothelialcells where the triglycerides, carried in the plasma very-low-density lipoproteins or chylomicrons, are sequestered. I,'Robinson3,< also maintained that lipoprotein lipase playsa directive role in determining the pattern of triglyceridefatty acid uptake by extrahepatic tissues.Recently, various workers have presented data indica-ting that the adipose tissue and heart muscle contain lipo-protein lipases having different characteristics. l .'.'The activities of the enzyme in specific tissues havebeen shown to change in particular physiological situationsand such changes can be correlated with alterations in theuptake of triglyceride fatty acids by these tissues. In adiposetissue, for example, the activity of the enzyme is high inthe fed state, when triglyceride fatty acids are taken up,and low in the fasted state, when uptake of triglyceridefatty acids is low. In the heart muscle, on the other hand.the activity of lipoprotein lipase is low during feeding andis elevated during fasting, when the flow of triglyceridefatty acids is from the depot fat region towards utilisationof the heart muscle.Ascorbic acid has been implicated in the control ofserum cholesterol levels by a number of investigations"o'l>although Anderson et al." have questioned the serumlipid-lowering effect of ascorbic acid.Sokoloff et al." also discussed the decrease of serumlipoprotein lipase activity during severe atherosclerosis, andserum lipid values and the improvement of lesions duringlipid intake restriction and high ascorbic acid intakes.512N 34S.A. MEDICAL JOURNAL(Supplement to the SOl/th African Medical Journal)13 March 1974TABLE I. RELATIONSHIP BETWEEN SERUM ASCORBIC AClfJLEVELS AND BABOON HEART MUSCLE LIPOPROTEINLIPASENational Park." The level of ascorbic acid of 34 mg/kgday is also much higher than the requirements of 2 mg/kgday'" and 7,5 mg/kg/day reported by Shaw et al.'" fa'primates.The heart lipoprotein lipase activity decreased by afactor of 7,2, from a high value of 549 to a low value of76 (Table I).Examination of the data indicated that apparently aminimal serum ascorbic acid level of about 0,35 mg/ dlexisted, below which the lipoprotein lipase actively in-creased steeply (Fig. 1). At serum ascorbic acid valuesabove 0,35 mg/dl, however, heart lipoprotein lipase ap-peared to be repressed. This is of interest, because Taylor"reported that petechial haemorrhages around hair folliclesappeared at serum ascorbic acid levels of about 0,2 mg/dl.which corresponds to 15 fLg ascorbic acid per 10' leuco-cytes.When heart muscle homogenates of baboons with dif-ferent serum ascorbic acid levels were incubated in vitrowith 0,34 mM ascorbic acid during the assay, it was foundthat at low serum ascorbic acid levels the addition ofascorbic acid stimulated the enzyme activity (103,7%), andat high serum ascorbic acid levels inhibited it (61,3%)(Table II).The acetone powder of baboon heart muscle was de-pleted of ascorbic acid. Addition of ascorbic acid to theassay medium also effected a stimulation of the lipoproteinlipase activity to 122,2 % of the original value. Althoughstimulation of lipoprotein lipase by ascorbic acid occurred,the effect was always small. .Heart muscle vitamin C levels paralleled those of theserum.Andrews et al. H found that in the elderly human, vitaminC deficiencies often occurred and that higher intakes thanthe recommended 30 mg/day were necessary to bringleucocyte values back to those of young people. Similarly,postheparin lipase activity was often found to be deficientin old age." We therefore set out to determine the re-lationship between serum ascorbic acid levels and heartlipoprotein lipase. A preliminary report of our findingshas been published."MATERIALS AND METHODSBaboons were kept on diets as described by Kotze andKempff," and De Klerk et al. lSThe diets were supplemented with various quantities ofascorbic acid to obtain different serum levels. Bloodsamples were obtained by venepuncture under phencycli-dine hydrochloride anaesthesia (1 mg/kg body mass)(Sernylan; Parke-Davis). Blood samples were allowed toclot and the serum was separated by centrifugation. Serumascorbic acid was determined according to the method ofRoe and Kuether19 as modified by Schaffert and Kingsley."Heart muscle samples were obtained after phenobarbi-tone euthanasia and immediate autopsy. The apex of theheart muscle was excised and rinsed in Krebs-Ringer bi-carbonate buffer, blotted on filter paper, weighed, andthree times its mass of Krebs-Ringer bicarbonate bufferadded. It was then minced with scissors and homogenisedfor 1 minute with Ultra Turrax homogeniser in the cold.Heparin was added to the homogenate to yield a finalconcentration of 250 units/ml, and this was subsequentlyincubated for 30 minutes at 25°C under a constant flowof a gas mixture containing 95% 0, and 5% CO,.Lipoprotein lipase was determined according to themethod of Huttunen and Steinberg,Ol with the modifica-tion that the liberated free fatty acids were determinedaccording to N ovak 's" method. Dry mass of the homo-genate was determined by mass difference after drying analiquot for 24 hours at 105°C.Lipoprotein lipase results are always the average of atleast 6 different determinations. The effect of ascorbic acidon lipoprotein lipase activity was done by addition ofascorbic acid to the assay medium to a final concentra-tion of 0,34 mM (6 mg/lOO ml).Acetone extracts of heart muscle homogenates wereprepared as described by Garfinkel and Schotz."RESULTSDeterminations of serum ascorbic acid levels and the lipo-protein lipase activities of baboon heart muscle homo-genates indicate a definite inverse relationship betweenthese entities. The serum ascorbic acid level varied 12,7-fold from a low value of 0,11 mg/dl to 1,41 mg/dl. It is ofinterest because after 7 young baboons had been kept onascorbic acid 34 mg/kg body mass/day for at least 3months, 1,41 mg / dl was the highest serum level obtained,which was still lower than the average value of 1,56 mg/dlfound in more than 200 free-living baboons in the KrugerSerum ascorbic acid(mg/l00 ml)0,110,120,160,160,180,210,240,250,290,300,340,451,091,251,261,261,261,361,41Lipoprotein lipase actiVityn.e.q FFA released per mgdry mass per minute38454949549244432024934527114226920118676209231217222125I3 MOlart 1974d55lIII :1z>j~> IIIi= .1IltJ (( :!III >3001III lI: III~ C I-~ Cl :J2501z :! ziii lI: ~~ III lI:200[0 a. IIIlI: C a.a. III0 IIIa. (~ III 150.JIII III.J lI:tJ~III 100:J 11.soL:!~lI:(III%0S.-A. MEDIESE TYDSKRIF(Byvoegsel lOt die SlIid - AfrJkaill1Se Mediesc T.'·dskrif)•• •••••I , I I I I0,6 0,7 0,8 0,9 1,0 1,1 1,2 1,3 1,4 1,5513V 35SERUM ASCORBIC ACIDMG/100MLFig. 1. The relationship between serum ascorbic acid levels and baboon heartmuscie lipoprotein lipase.TABLE 11. EFFECT OF ADDED ASCORBIC ACID TOIN VITRO LIPOPROTEIN LIPASE ACTIVITIES OF BABOONHEART MUSCLE HOMOGENATES (ASCORBIC ACID INASSAY WAS 0,34 mM)Lipoprotein Iif=aseactivity as % Heart(control 100%) Serum muscleAA level AA level- Asc. + Asc. (mg/100 9 (mg/100 9acid acid wet mass) wet mass100 35,0 0,60 9,1100 56,8 1,27 14,8100 84,2 0,76 9,3100 39,5 0,41 2,2100 91,0 1,29 15,6Average 61,3 0,87 10,2SD -+-25,46 -+-0,40 -+- 5,39I100 105,6 0,31 5,2100 106,0 0,29 2,7100 107,0 0,30 5,7100 103,7 0,09 1,4100 107,0 0,22 4,5100 93,1 0,18 4,6Average 103,7 0,27 4,0SO -+- 2546 -+-0,10 -+- 1,14Acetonepowder 100 122,2DISCUSSIONThere is experimental evidence that during fasting lipo-protein lipase from heart and skeletal muscle increases:·7 ,.Borenstajn et al.' found that insulin could not preyentthe increase of heart lipoprotein lipase elicited by starva-tion. It thus appears that there are other factors besidesinsulin that control heart lipoprotein lipase activity.It is not clear how the feeding of ascorbic acid canhave a repressive effect on lipoprotein lipase synthesis;because such a mechanism, if any, is unknown.According to recent reports the lipoprotein lipase fromheart muscle is not a single entity. Schotz and Garfinkel'described two molecular species of rat heart lipoproteinlipase, one, LPL.. was stimulated by heparin, whereas theother, LPLb , was inhibited by heparin, and concluded thatboth species were increased during fasting.Sokoloff et al." suggested that ascorbic acid at doses of150 mg / kg body mass / day could counteract the decreaseof lipoprotein lipase of rabbit and rat serums caused bycholesterol feeding. This finding is, however, not directlyrelevant, since these animals synthesise their daily ascorbicacid requirements from glucose, whereas primates, like thebaboon and man, do not synthesise their own ascorbicacid but must receive it from external sources.The fact that adipose tissue and heart muscle lipoproteinlipase react differently to physiological stimuli like feedingand fasting, suggests that whereas an ascorbic acid de-ficiency caused a rise in heart lipoprotein lipase activity,a concomitant decrease of adipose tissue lipoprotein lipaseshould occur. However, high serum ascorbic acid levelsshould increase adipose tissue lipoprotein lipase and, there-fore, tend to lower serum triglyceride fatty acid levels.Sokoloff et al." maintained that large doses of ascorbicacid (2 - 3 g/ day) and restriction of lipid intake loweredthe triglyceride and cholesterol levels of hyperlipidaemicpatients.It is also of interest that a serum ascorbic acid level of514N 36S.A. MEDICAL JOUR AL(5l/pplemem [0 [he 501/[h African Medical }ollmQ/)13 March 1974about 0,35 mg/ dl was found to be sufficient to result in alow heart muscle lipoprotein lipase activity and probablyto ensure an efficient level of adipose tissue lipoproteinlipase activity. This serum ascorbic acid level is just abovethe 0,2 mg/ dl serum ascorbic acid level reported to causepetechial haemorrhage of hair follicles."In vitro incubation of heart lipoprotein lipase with addi-tional 0,34 mM ascorbic acid caused a stimulation of theenzyme activity if the homogenate originated from a baboonwith a low serum ascorbic acid level, and inhibited thelipoprotein lipase when the homogenate was from ababoon with a high serum ascorbic acid level. This findingsuggests that we may be dealing with three phenomena.Firstly, the feeding of ascorbic acid controls the level oflipoprotein lipase synthesis, or only one of the LPL. orLPLh species as reported by Garfinkel and Schotz.,"J Se-condly, that in addition to this effect, which probably in-volves protein synthesis, we have a more direct effect ofallosteric enzyme activity modulation by different ascorbicacid concentrations in vitro. A third possibility exists inthat the two species LPLa and LPL;) react differently toascorbic acid, as was reported for the reaction withheparin.'It would seem, therefore, that ascorbic acid definitelyexerts a controlling effect on lipoprotein lipase activityand consequently on serum lipid levels.At present our knowledge about the intricacies of theprocess is still lacking and further investigation is war-ranted.REFERE 'CES1. Robinson. D. S. and Wing, D. R. ill Jeanrenaud, B. and Hepp, D..eds (1970): Regulation and Metabolic Functions, p. 41. Stuttgart:Georg Thieme Verlag.2. Blanchene-Mackie. E. J. and Scow, R. O. (1971): J. Cell Bio!., 51.1.3. Robinson, D. S. (1963): Advanc. Lipid Res., I, 134.4. Idem (1970): Biochemistry, 18, 51.5. Borenstajn, H., Otway, S. and Robinson, D. S. (1970): 1. Lipid Res ..11, 102.6. Borenstajn, J. and Robinson, D. S. (1970): Ibid., 11, 11.7. Borenstaln, J., Samols, D. R. and Rubenstein, A. H. (1972): Amer.J. Physiol., 223, 1271.8. Schotz. M. C. and Garfinkel, A. D. (1972): Biochem. biophys. Acta.270, 472.9. Kotze, J. P. and Steinberg, D. (1972): Unpublished data.10. Spittle, C. R. (197 I): Lancet, 2, 1280.I!. Sokoloff, B., Hon, M., Saelhof, C., McConnelI, B. and Imai, T.(1967): J. utr., 91, 107.12. Gimer, L., Cerven, l., Nemec, R. and Mikus, L. (1971): Amer. l.C1in. Nutr., 24, 1238.13. Anderson, T. W., Reid, D. B. w. and Beaton, G. H. (1972): Lancet.2, 876.14. Andrews, J., Letcher, M. and Brook. M. (1969): Brit. Med. l., 2.416.15. Brodows, R. G. and CampbelI, R. G. (1972): New Engl. J. Med.,287, 969.16. Kotze, J. P., Matthews, M. J. A. and De Klerk, W. A. (1973):Lancet, I, 610.17. Kotze, 1. P. and Kempff, P. G. (1972): S. Afr. Assoc. Food. Sci.Techn. Symposium, Pretor,a, 4 - 5 October 1972, p. 185.18. De Klerk, W. A., Kotze, J. P., Weight, M. l., Menne, 1. V..Matthews, M. l. A. and McDonald, 1. (1973): S. Afr. Med. J., 47,1503.19. Roe, J. H. and Kuether, C. A. (1943): 1. BioI. Chem., 147, 399.20. Schaffert, R. R. and Kingsley, G. R. (1955): Ibid., 212, 59.21. Huttunen, J. K. and Steinberg, D. (1971): Biochim. biopys. Acta.239, 411.22. 'ovak, M. (1965): J. Lipid Res., 6, 431.23. Garfinkel, S. A. and Schotz, A. G. (1972): Ibid., 13, 63.24. Kotze, J. P., De Klerk, W. A., Weight, M. l., Menne, I. V..Matthews, M. J. A., Laubscher, N. F. and Horn, L. P. (1973):S. Afr. Med. J. (in press).25. Day, P. L. cited by Kerr, G. R. «(1972): Physiol. Rev., 52, 41;.26. Shaw, J. A., PhilIips, P. A. and Elvehjem, C. A. (1945): J. Nutr..29, 365.

Effect of ascorbic acid on lipoprotein lipase activity

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Effect of ascorbic acid on lipoprotein lipase activity

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