Fourteen young male baboons (Papio ursinus) were divided into two groups. All the animals received the same dietary regimen during a 2%-month adaptation period. During the next 3 months one group received 250 mg and the other 20 mg vitamin C daily. For the last 2% months of the experiment no vitamin C was given to the first group, and that of the second group was increased to 350 mg daily. Simultaneously with the switchover, 14C-4-cholesterol was administered. A classical twopool system for the kinetic behaviour of cholesterol in the body was confirmed. Vitamin C treatment did not alter the serum cholesterol levels significantly, but the production rate was repressed. It was also shown that vitamin C was depleted from the body in a typical two-pool fashion.S. Afr. Med. J., 48, 1182 (1974)

de Klerk, W.A.

Kotze, J.P.

Menne, I.V.

Weight, M.J.

Weight, M.J.A.

English

Fourteen young male baboons (Papio ursinus) were divided into two groups. All the animals received the same dietary regimen during a 2½-month adaptation period. During the next 3 months one group received 250 mg and the other 20 mg vitamin C daily. For the last 2½ months of the experiment no vitamin C was given to the first group, and that of the second group was increased to 350 mg daily. Simultaneously with the switchover, 14C-4-cholesterol was administered. A classical twopool system for the kinetic behaviour of cholesterol in the body was confirmed. Vitamin C treatment did not alter the serum cholesterol levels significantly, but the production rate was repressed. It was also shown that vitamin C was depleted from the body in a typical two-pool fashion.S. Afr. Med. J., 48, 1182 (1974)

Kotze, JP

Weight, MJ

de Klerk, WA

Menne, IV

Weight, MJA

AJOL - African Journals Online

1182LCM 34S.A. MEDICAL JOURNAL(Supplement·-South African Journal of Laboratory and Clinical Medicine)12 June 1974 lEffect of Ascorbic Acid on Serum CholesterolLevels and on Die-Away Curves of14C-4-Cholesterol in BaboonsJ. P. KOTZE, M. J. WEIGHT, W. A. DE KLERK, I. V. MENNE, M. J. A. WEIGHTSUMMARYFourteen young male baboons (Papio ursinus) weredivided into two groups. All the animals received thesame dietary regimen during a 2%-month adaptationperiod. During the next 3 months one group received250 mg and the other 20 mg vitamin C daily. For the last2% months of the experiment no vitamin C was given tothe first group, and that of the second group was in-creased to 350 mg daily. Simultaneously with the switch-over, I·C-4-cholesterol was administered. A classical two-pool system for the kinetic behaviour of cholesterol in thebody was confirmed. Vitamin C treatment did not alter theserum cholesterol levels significantly, but the productionrate was repressed. It was also shown that vitamin C wasdepleted from the body in a typical two-pool fashion.s. A fr. Med. J., 48, 1182 (1974).Myasnikova' was the first to show that vitamin Chadthe ability to influence ~erum cholesterol levels of patients.She observed that the intravenous administration of highdoses of vitamin C to patients with high levels of serumcholesterol resulted in a distinct decrease, whereas inpatients with low values it produced increased serumcholesterol levels.Tyapina' studied the influence of 500-mg intravenousdoses of vitamin C on the cholesterol levels of patientssuffering from hypertension and atherosclerosis. She ob-served a hypercholesterolaemic effect within a few hoursand a decrease in blood cholesterol after 10 days.Spittle' made the chance observation that she couldvary her own serum cholesterol level by means of hervitamin C intake. She reported that the serum cholesterollevels of healthy people under 25 years of age fell afterdietary administration of 1 g of vitamin C per day. TheNational Research Institute for Nutritional Diseases of theSouth African Medical Research Council, PretoriaJ. P. KOTZE (Present address: National Food Research In-stitute or the CSIR, P.O. Box 395, Pretoria- 0001)M. J. WEIGHTW. A. DE KLERKI. V. MENNEM. J. A. WEIGHTDale received: 3 December 1973.same treatment of atherosclerotic patients showed a risein serum cholesterol, which she attributed to mobilisationof arterial cholesterol by the dietary vitamin C. In anattempt to repeat these findings, Anderson et af! foundthat in 41 subjects aged 18 - 24 years, vitamin C causedan increase in serum cholesterol rather than a decrease.In a review Ginter5 discussed the normalising effectof vitamin C on serum cholesterol, but also pointed outthat too often methodological errors were committed.A common fault was the administration of vitamin Ctogether with therapeutic measures which resulted inreactions falsely ascribed to vitamin C administration.Schaffer6 argued strongly in favour of the preventativeeffect that vitamin C has on the development ofatherosclerosis. Bronte-Stewart et aC found that in vitaminC-deficient patients, fats known to elevate serum choles-terol under normal conditions failed to produce a responsein serum cholesterol levels. Administration of vitamin Ccaused an increase in serum cholesterol values. Oraladministration of vitamin C resulted in a faster increasein serum chole~terol than intramuscular administration.They also reported that vitamin C failed to influence tbelevel of serum cholesterol of healthy individuals.Chronic ascorbic acid deficiency lowered catabolismof cholesterol in guinea pigs." Cholesterol-fed guinea pigsalso had a raised vitamin C consumption.'It is known that baboons, like other primates, needdietary vitamin C, and therefore appear to be appro-priate research models in experimentation regarding vita-min C and cholesterol metabolism. Previous studies haveindicated spontaneous intimal lesions in free-livingbaboons.'" We also found that during the initial stress ofcaptivity, known to increase serum cholesterol,]] oraladministration of vitamin C tended to lower serumcholesterol."It was decided to study the effect of dietary vitamin Con serum cholesterol levels and the die-away curves of"C-4-cholester01 from the serum pool of young malebaboons (Papio ursinus) in an effort to resolve thecontroversy regarding the interaction between vitamin, Cand cholesterol metabolism.MATERIALS AND METHODSIn order to vary the serum vitamin C levels and todetermine whether this might influence serum cholesterol12 Junie 1974 S.-A. MEDIESE TYDSKRIF(Byvoegsel-SlIid-Afrikaanse Tydskrif vir Laboratorillm- en Kliniekwerk)1183LKW 35levels, 14 young male baboons were kept on diets" whichvaried only in their ascorbic acid content.All baboons were first kept on 1 - 2 mg vitamin C/kgbody weight/day for 2t months to adapt to captivity(period 1).After this period 7 baboons were kept on 20 mgvitamin C/day and the other 7 received 250 mg vitaminC/day for another 3 months (period 2). Subsequently theanimals receiving 20 mg/day were given 350 mg/day,whereas the other 7 baboons receiving 250 mg vitaminC/day until then were put on a diet depleted ofvitamin C (period 3). The average body weight of thebaboons was 13,0 kg, and varied from 7 to 16,5 kg.Simultaneously with the last 2t months' treatment"C-4-cholesterol (16,8 fLCi) (Amersham, England) in aphysiological saline ethanol solution was injected intra-venously into each baboon. Blood samples obtained byvenepuncture were allowed to clot, and serum sampleswere collected after separation by centrifugation in thecold. Radioactive determinations in the serum were doneby liquid scintillation (Instagel, Packard) counting, andestimations by the tomatine method showed that morethan 95% of the radioactivity resided in cholesterol.Cholesterol was determined by the method of Zak andRessler.H Vitamin C was determined by the method ofRoe and Kuether" as modified by Schaffert and Kingsley.16The calculation of the half-life values for the choles-terol pools (tt), the pool sizes (MA) and the kineticconstants, were done according to the method of Good-man and Noble.'"RESULTS AND DISCUSSIONThe 14 baboons were kept on a standard diet" supple-mented with fruit and vegetables to give an intake ofabout 1 - 2 mg vitamin C/kg body weight/day during theperiod of adjustment to their new environment. During theinitial adjustment period of 2t months (period 1) thevitamin C requirement apparently increased, as the serumvitamin C level decreased from an average value of 1,45mg/lOO ml to 0,79 mg/lOO ml (Table I). Day" reportedthe requirement of vitamin C for another primate, theRhesus monkey, to be 2 mg/kg body mass/day.While the vitamin C requirement increased during theadjustment period, a simultaneous significant increase inserum cholesterol was observed, from an initial value of100 mg/IOO ml to 124 mg/loo ml. St Clair et al." reportedcomiderab'e increases in the serum cholesterol of squirrelmonkeys during transportation and acclimatisation.During the next 3 months 7 baboons received 250 mgvitamin C/kg/day while the control group received about1 - 2 mg vitamin C/kg/day (period 2). At the averageintake level of 19 mg/kg/day the serum vitamin C levelrose to the initial value of the free-living baboons.With the average intake of 2 mg vitamin C/kg/day of thecontrol group during period 2 the serum level eventuallydropped to 0,51 mg/IOO m!. The oral administration ofan average of 19 mg vitamin C/kg/day did not cause anysignificant change of the average serum cholesterol of the7 treated baboons when compared with the 2 mg/kg/dayof the control group (Table I, period 2).The regimens for the two groups of baboons werechanged during period 3 so that the previous controlgroup now received an average of 27 mg vitamin C/kgbody weight/day, whereas the group previously receiving19 mg vitamin C/kg body weight/day now received novitamin C. As could be expected, the serum vitamin Clevel of the 7 baboons receiving 27 mg vitamin C/kg/dayrose dramatically to 1,22 mg/lOO m!' The withdrawal ofvitamin C caused a sharp drop in serum vitamin Clevels. If a semilogarithmic plot is drawn of the serumvitamin C levels against time, our data comply with aclassical two-pool system for vitamin C in the body.Since we did not use radio-isotope tracers, calculationof all the parameters of a two-pool system is notpermissible. We can, however, calculate the tt values ofthe two pools. The tt value of the first fast turning-overpool is 7 days, with the tt value of the second slowerturning-over pool 156 days. Burns'o reported tt valuesof 15 and 3 days for man and guinea pigs, respectively.The tt value of 7 days for the pool falls well withinTABLE I. SERUM CHOLESTEROL AND VITAMIN C LEVELS OF 14 YOUNG MALE BABOONS TREATED WITHDIFFERENT DIETARY LEVELS OF VITAMIN CVitamin C treatmentPeriod 1 (1 - 2 mg/day) Period 2 (250 mg/day) Period 3 (0 mg/day)Date 27/2 14/3 29/3 10/4 24/4 8/5 22/5 7/6 19/6 3/7 17/7 1/8 10/8 30/8 11/9 26/9 11/10 23/10jl Aver. 1,46 0,58 0,72 0,67 0,73 0,78 1,35 1,12 1,26 1,28 1,48 1,48 1,37 0,43 0,27 0,26 0,24 0,18SD 0,30 0,17 0,20 0,18 0,10 0,13 0,13 0,16 0,19 0,16 0,12 0,16 0,12 0,08 0,02 0,03 0,02 0,051 - 2 mg/day 20 mg/day 350 mg/dayAver. 1,44 0,73 0,73 0,54 0,76 0,79 0,56 0,55 0,55 0,64 0,69 0,57 0,51 1,22 1,22 1,29 1,35 1,27SD 0,21 0,18 0,15 0,19 0,12 0,18 0,12 0,16 0,10 0,21 0,19 0,19 0,07 0,14 0,14 0,20 0,30 0,101 - 2 mg/day 250 mg/day o mg/day11Aver. 94 108 112 117 107 116 125 119 117 129 119 128 147 130 128 119 96 85SD 13 15 21 12 8 11 16 15 5 23 10 8 20 14 11 17 13 121 - 2 mg/day 20 mg/day 350 mg/dayAver. 106 119 125 127 121 131 126 133 127 128 139 125 141 131 132 129 109 96SD 27 11 12 21 12 6 10 14 18 36 26 9 25 12 30 17 7 91184LCM 36S.A. MEDICAL JOURNAL(S/lpplemelll-So/lth African ]ollrn[ll of Laboratory and Clinical Medicine)12 June 1974RAk = 1,44.H = turnover rate.RArJ.{3PRA = rJ. C ~ fJ C = production rate of cholesterol in pool A.a . ATABLE 11. AVERAGE KINETIC PARAMETERS CALCULATEDAFTER INTRAVENOUS INJECTION OF "C-4-CHOLESTEROLINTO 7 YOUNG MALE BABOONS WITH DIETARY DEPLETIONOF VITAMIN C AND 7 YOUNG MALE BABOONS RECEIVING27 MG VITAMIN C/KG/DAYCONCLUSIONSREFERENCESwith 9,5 days for baboons treated with vitamin C. The sizeof the first pool, viA, can be calculated with a reasonabledegree of certainty. It was indicated that the size of thefirst pool MA of the vitamin C-treated animals was slightlylarger than that of the baboons receiving no vitamin C,n2mely 10,61 g versus 9,95 g. The first pool normallycomprises the free cholesterol of serum, cholesterol estersof serum, red blood cell cholesterol and liver cholesterol."''''It is also likely that cholesterol from the spleen, kidney,lung and intestines equilibrates sufficiently fast to comprisepart of pool MA."Our findings on the tt values and size of the first poolMA for the two groups of baboons indicate that vitaminC treatment increases the turnover of this pool. Ourestimates for the pool size of 9,95 - 10,61 g for the firstpool MA, per baboon of 13,2 kg body weight, was higherthan 6,7 g reported by Wilson'·l for baboons (23,2 kg), andalso higher than the 0,309 - 1,843 g of the much smallersquirrel monkeys."The tt values of the second pool were significantlyhigher for the vitamin C-treated baboons (85,5 days) thanfor the vitamin C-deficient animals (69,0 days). Thesevalues were also considerably higher than the 23 - 37 daysreported for baboons by Wilson," but were more withinthe range of the 35 - 67 days reported by Eggen et al.'"Vitamin C apparently depressed the production rate(PRA) of cholesterol of the first pool, A, from 300 mg/dayof the control group to 172 mg/day for the vitamin C-treated group. Wilson found the PRA of baboon cholesterolto be 629 mg/day, which was more than twice as highas the values reported here. The turnover rate, k, forthe baboons receiving vitamin C was also 19% higherthan that of the control group.Kritchevsky" reported half-life values for babooncholesterol between 31 and 51 days, which is shorterthan the values reported for the second pool, B, in thisarticle. They calculated the daily synthesis rate of choles-terol as 47 mg/kg/day, which would be equivalent to 611mg/day/baboon in our experiment, which would be morethan twice as high as the values reported here.Our results seem to indicate that vitamin C repres~es thein vivo cholesterol synthesis and increases the turnoverrate of the fast miscible pool, but decreases the rate ofremoval from the slow miscible pooLHigh oral doses of vitamin C did not significantly lowerthe serum cholesterol l~vels in baboons. Withdrawal ofall dietary vitamin C did not result in a concurrent risein serum cholesteroLBody cholesterol of baboons is metabolised accordingto a two-pool system, as in man. A similar two·poolsystem was found for body vitamin C.Vitamin C repressed the production rate (PRA) ofcholesterol in vivo and increased the turnover rate, k, ofthe fast miscible pool, but decreased the rate of removalfrom the slow miscible pool.+ Vitamin8,5 days85,5 days1752 dpm/mg1730 dpm/mg0,08150,0081110,61 9172 mg/day-0,045-0,0450,869-Vitamin C9,5 days69,0 days2413 dpm/mg1310 dpm/mg0,07930,01059,95 9300 mg/day-0,055-0,0350,728Parameterst1f2At%BCACsClthis range. If only one kinetic vitamin C pool werepresent in the body, our young baboons with an averagevitamin C level of about 1,4 mg/dl would have a serumlevel of less than 0, I mg/dl after 28 days of no vitaminC intake. At this level clinical scurvy would normallyarise. In another experiment, however, we observed thatbaboons receiving no vitamin C for 8 months did stillnot show any clinical signs of scurvy. Our observationstherefore substantiate the existence of a second slowt::rturning-over pool for vitamin C.The average serum cholesterol of the 7 baboonsreceiving no dietary vitamin C decreased faster (62 mg/lOOml total) in 74 days (period 3) than that of the 7 baboonsreceiving 27 mg/kg/day (45 mg/lOO ml total) during thesame period, although the difference did not seem to besignificant.Cholesterol labelled with uc in position 4 was injectedintravenously during the last dietary switchover of thevitamin C regimen (period 3). By assuming a two-poolsystem for cholesterol the average values of the specificradioactivity of cholesterol were plotted semilogarithmi-cally against time. The data obtained conformed with atwo-pool system for body cholesterol." From these plotsall the relevant parameters were calculated (Table 11).The tt values of the first pool of the two groups ofbaboons differed only slightly. A tt value of 8,5 days wasfound for baboons receiving no vitamin C, comparedHA ~ half-life of first pool A; Ha ~ half-life of second pool B; CA andC t t N 0,693 P 0,693 M h . RAB=consans;c<.=--, ~--; =t eSlzeofpooIA=---HA Ha A CA + Ca-rJ. MA CA - PMA CakAA ~ R ; ka• = -(IX + P+ kAA) ~ total rate of removalAof cholesterol from pool B.RA = amount of isotope injected into pool A. 1. Myasnikova, I. A. (1947): Transactions of the Academy of Militaryand 'aval Medicine, Leningrad, 8, 140.-12 Junie 1974 S.-A. MEDIESE TYDSKRIF(Byvoegsel-SlIid-Afrikaanse Tydskrif vir Laboratorillm- en Kliniekwerk)1185LKW 372. Tyapina, L. A. (1952): Hypertensive Disease, p. 108. Moscow.3. SpIttle, C. (1971): Lancet, I, 1280.4. Anderson, T. W., Reid, D. B. W. and Beaton, G. H. (1972):Ibid., I, 876.5. Ginter, E. (1968): Csek fysio!., 17, 423.6. Schaffer, C. F. (1970): AIDer. J. Clin. Nutr., 23, 27.7. Bronte-Stewart, B., Robetts, B. and Wells, V. M. (1963): Brit. J.NUlr., 17. 61.8. Ginter, E., Cerven, 1., Nemec, J. and Mikus, L. (1971): AIDer. J.Clin. Nutr., 24, 1238.9. G,nter, E. and Zloch, Z. (1972): Int. J. Vitam. Nutr. Res., 42, 72.10. Van der Watt, J. J., Kotze, J. P., Kempff, P. G., Du Plessis, J. P.and Laubscher, N. F. (1973): J. Med. Primatol., 2, 25.11. Kotze, J. P. and Kempff, P. G. (1972): S. Afr. Assoc. for FoodSci. and Tech. Symposium. p. 185.12 De KJerk, W. A., Kotze, J. P., Weight, M. J., Menoe, I. V.,Matthews, M. J. A. and MacDonaId, T. (1973): S. Afr. Med. J.,47, 1503.13. Kabara, J. H., McLaugWin, J. T. and Riegel, C. A. (1961). Ana!.Chem., 33, 305.14. Zak, B. and Ressler, N. (1955): Amer. J. Clin. Path., 25, 433.15. Roe, J. M. and Kuether, C. A (1943): J. BioI. Chem., 147, 399.16. Schaffert, R. R. and Kingsley, G. R. (1955): Ibid., 212, 59.17. Goodman, D. S. and oble, R. P. (1968): J. Clin. lnvest., 47, 231.18. Day, P. L. (1962): Nat!. Acad. Sci. Natl. Res. Council Pub!. 990.19. St Clair, R W., MacNintch, J. E., Middleton, C. C., Clarkson,T. B. and Lofland, H. B. (1967): Lab. Invest., 6, 828.20. Burns, J. J. (1973): Nature (Land.), 244, 540.21. Avigan, J., Steinberg, D. and Berman, M. (1962): J. Lipid. Res., 3,216.22. Chobanian, A. V. and Hollander, W. (1962): J. Clin. Invest., 41,1732.23. Goodman, D. S. (1965): Physio!. Rev., 45, 747.24. Wilson, J. D. (1970): J. Clin. lnvest., 49, 655.25. Lofland, H. B., Clark, T. B. and Bullock, B C. (1970): Exp. Mo!.Patho!., 13, 1.26. Eggen, D. A" Newman. W. P. and Slrong, 1. P. ill Vagtborg, H.ed' (1967): The Baboon ill Medical Research, vo!. 2, p. 559. Austin:University of Texas Press.27. Kritchevsky, D. (1970): Trans. '. Y. Acad. Sci., 33, 821Transferable Resistance to Carbenicillin andGentamicin in Pseudomonas aeruginosaA. J. VAN RENSBURGSUMMARYAntibiograms on 482 routine isolates of Pseudomonasaeruginosa yielded 64 (13,3%) carbenicillin-resistantstrains. In 40 of these strains resistance could be ascribedto R factors. R factors accounted for resistance in 3 outof 11 gentamicin-resistant strains. The significance ofresistance in Ps. aeruginosa is discussed.S. Afr. Med. J., 48, 1185 (1974).Gentamicin' and carbenicillin' are the drugs of choicein the treatment of infection with Pseudomonas aemginosa.However, this regimen of treatment has been accompaniedby the emergenc:: of drug resistance, particularly tocarbenicillin."; Since the establishment of intensive careunits at the Bloemfontein National and Pelonomi Hospitalswe have noted an exceptional increase in the number ofPs. aeruginosa strains isolated, as well as in its incidenceof resistance to gentamicin and carbenicillin.This article reports on the occurrence of transmissibledrug resistance in Ps. aeruginosa strains, isolated in ourlaboratory.Department of Microbiology, University of the Orange FreeState, Bloemfontein, OFSA. J. VAN RENSBURGDate received: 7 January 1974.MATERIALS AND MEmODSAntibiotic Sensitivity TestsMast sensitivity discs were used to screen isolates ofPseudomonas aemginosa for resistance to carbenicillinand gentamicin. Resistance was confirmed by plating onMacCcnkey agar containing the antibiotics at a concen-tration of 1 000 ,ug/ml for carbenicillin or 25 !-'g/ml forgentamicin.Minimum inhibitory concentrations (MIC) of the anti-biotics were determined by spotting small drops (0,1 ml)of diluted cultures (ID' bacteria/ml) on Welco sensitivitytest agar plates containing doubling dilutions of carbeni-cillin or gentamicin. The MIC was taken as the lowestconcentration of antibiotic that prevented growth.Transfer of Carbenicillin and Gentamicin ResistanceTransfer of R factors was done as previously described.'"The recipient was Escherichia coli Kl2 strain J53."RESULTSCarbenicillin Resistance in Pseudomonas aernginosaOut of 482 routine isolates of Ps. aeruginosa 64 (13,3 %)showed high levels of resistance to carbenicillin (>2500

Effect of Ascorbic Acid on Serum Cholesterol Levels and on Die-Away Curves of 14C-4-Cholesterol in Baboons

Effect of ascorbic acid on serum cholesterol levels and on die-away curves of 14C-4-cholesterol in baboons

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Effect of ascorbic acid on serum cholesterol levels and on die-away curves of 14C-4-cholesterol in baboons

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