a series of tests was developed ad hoc to demonstrate the effectiveness of pyruvate in inhibiting free - radical generation . clofibrate is a pharmaceutical known to generate free - radicals in the liver . the clofibrate causes the patient &# 39 ; s beta - peroxidase system to develop peroxisomes and to increase the organelles of the liver . within the peroxisomes there is beta - oxidation activity which is an enzyme phenomenon whose end products include hydrogen peroxide , which is a free - radical metabolite . the patient &# 39 ; s body has a normal defense to free - radicals called catalase . the available catalase cannot neutralize the abnormal generated quality of free - radicals . the surplus free - radicals break down lipid membranes ( lipid peroxidation ), resulting in cell abnormalities . with the addition of pyruvate , there is less generation of free - radicals . peroxisomes do not proliferate when pyruvate is present ; beta - oxidation does not increase . in comparison testing , vitamin e exhibited its known property of scavenging free - radicals , but did not inhibit generation of free - radicals . thirty - two male sprague - dawley rats weighing approximately 210 grams each were divided into 7 groups and fed a liquid diet ( dyets 710 , 027 , dyets , inc ., bethlehem pa .) for 22 to 26 days . the 7 groups were : pyruvate was added to the diet as sodium pyruvate ( 20 grams / liter ) and calcium pyruvate ( 17 . 9 grams / liter ). the pyruvate dosage was 10 % of the energy content of the diet of those rats receiving pyruvate . all diets were made isoenergetic by adding polyglucose ( polycosel ®, ross laboratories . columbus ohio ) to the diets not containing pyruvate . the sodium and calcium content of the diets were made similar by adding sodium citrate and calcium carbonate to those diets not containing pyruvate . the final composition of all diets was : all diets were established to be iso - energetic by calorimetric analysis . prior to grouping the animals for the experiment , in order to familiarize the animals with the liquid diet , all animals were fed the placebo liquid diet for three days during which time the mean intake was 60 ml / day . clofibrate was added to the diet to a final concentration of 0 . 018 gram / liter which is an expected dosage of 0 . 5 gram / kilogram of initial body weight . vitamin e was added to the diet to a final concentration of 2 ml / liter , corresponding to a dosage of 30 iu ( 20 mg / kg ) of the initial body weight . a modified pair technique was employed to assure equal energy intake among the groups . the intake of that group with the smallest intake for a given day ( rate - limiting group ) was fed to the other groups of animals on the following day and the rate - limiting group intake was fed the next day , et cetera . in most instances the rate - limiting group was clpy -- clofibrate plus pyruvate . the animals for all groups were fed the mean dietary intake for 22 to 26 days ( the feeding period ). during the feeding period , total intake of clofibrate ( table i ) and vitamin e ( table ii ) between the groups was similar . table i______________________________________clofibrate intakegroup clofibrate intake ( grams ) ______________________________________cl 3 . 3 ± 0 . 1cle 3 . 3 ± 0 . 1clpy 3 . 1 ± 0 . 1clepy 3 . 1 ± 0 . 1______________________________________ p = ns among these groups table ii______________________________________vitamin e intake vitamin e intake * ( internationalgroup units ) ______________________________________e 184 ± 5cle 184 ± 5clepy 173 ± 4______________________________________ p = ns among these groups after 22 days of feeding , one animal from each group was selected for an overnight fast of 16 hours . the selected animals of each group were euthanatized by decapitation ( without anaesthesia ). for the next 4 days , one animal from each group was selected , fasted , and euthanatized . the liver was obtained from each euthanatized rat for immediate biochemical analysis and prepared for light and electron inicroscope analysis . blood was obtained from the cervical stump and frozen at - 20 ° c . for later analysis . a portion of the liver was homogenized in sucrose buffer and analyzed for palmitoyl coa oxidase and catalase activity and lipofuscin - like products . differences between groups were evaluated with a 2 - tailed t test . differences were considered significant with p & gt ; 0 . 05 . data are presented as mean ± sem ( standard error of the mean ) for the animals in each group . sections of the rat liver were processed for routine histologic analysis by light microscopy and ultrastructural electron microscope evaluation . as shown in table iii , the animals fed clofibrate alone had a 42 % increase in liver size , compared to the placebo . when pyruvate was added to the clofibrate diet ( clpy and clepy ) the effects of the clofibrate were reduced , i . e ., liver size increased 14 %, about one - third of the clofibrate alone . clofibrate plus vitamin e aggravated the liver size increase to 46 %, and vitamin e along reduced liver weight by 11 %. table iii______________________________________hepatic parameters liver protein liver weight / ( mg / ggroup weight ( g ) body weight liver ) ( mg total ) ______________________________________pl 7 . 1 ± 0 . 3 0 . 024 ± 0 . 001 160 ± 6 1130 ± 67cl 10 . 1 ± 0 . 4 0 . 041 ± 0 . 002 211 ± 11 2140 ± 160clpy 8 . 1 ± 0 . 2 0 . 032 ± 0 . 001 182 ± 8 1480 ± 69cle 10 . 4 ± 0 . 3 0 . 041 ± 0 . 001 204 ± 9 2124 ± 141clepy 7 . 5 ± 0 . 3 0 . 029 ± 0 . 001 172 ± 6 1298 ± 81e 6 . 3 ± 0 . 1 0 . 024 ± 0 . 001 199 ± 10 1254 ± 78py 7 . 4 ± 0 . 5 0 . 025 ± 0 . 001 170 ± 21 1261 ± 166______________________________________ pl = animals fed placebo diet cl = diet supplemented with clofibrate clpy = diet supplemented with clofibrate + pyruvate cle = diet supplemented with clofibrate + vitamin e clepy = diet supplemented with clofibrate + pyruvate + vitamin e e = diet supplemented with vitamin e py = diet supplemented with pyruvate the protein content of the liver was significantly increased by clofibrate ( from 1130 grams to 2140 grams ) and almost completely normalized by pyruvate ( from 1130 grams to 1480 grams ). interestingly the vitamin e supplementation of the clofibrate diet had no effect on the clofibrate - induced liver size and protein change ( compare 2140 grams and 2124 grams ). similarly adding vitamin e to the clofibrate and pyruvate group did not significantly alter the results ( compare 1298 grams and 1480 grams ), indicating that the vitamin e supplementation did not appreciably enhance the normalizing effect of the pyruvate . fig1 shows the hepatic peroxisomal beta - oxidation activity ( nmol / min / mg protein ). the clofibrate induced a five - fold increase in activity of the peroxisomal enzyme system , indicating significant free - radical generation . pyruvate reduced the clofibrate - enhanced increases in beta - oxidation by 75 %, indicating substantial inhibition of free - radical generation . vitamin e did not inhibit the effects of clofibrate on the enzyme system . vitamin e ( clepy ) did not enhance the normalizing effects of pyruvate ( clpy ). note : in fig1 , 3 , the lower - case letters a , b , c indicate fig2 illustrates that the catalase activity increased nearly three - fold with clofibrate , indicating free - radical generation . addition of vitamin e ( cle ) did not significantly reduce the catalase activity . the animals fed pyruvate in addition to clofibrate ( clpy and clepy ) increased the activity by one - half or less , indicating inhibition of the free - radical generation . the relevant fluorescent intensity of lipofuscin - like - products is shown in fig3 . clofibrate induced a three - fold increase in lipofuscin - like products . the increase of lipofuscin - like products was retarded completely by the addition of pyruvate or vitamin e to the clofibrate diets . thus the vitamin e effectively scavenged free - radicals which would have caused lipofuscin - like - product . the pyruvate likewise retarded free - radical presences , partly by inhibiting generations and partly by scavenging the free - radicals that were generated . the significance of fig3 is that limofuscin - like - products are present and measurable if the body contains free - radicals . the examples illustrate the effectiveness of dyruvate to inhibit free - radical production . the clofibrate - fed rats will chronically generate free - radicals . clofibrate - feeding induced hepatomegaly with an increase in hepatic protein content . peroxisomal proliferation by electron microscopic evaluation , had a five - fold increase in peroxisomal beta - oxidation activity , an indicator of peroxisomal proliferation and hydrogen peroxide production . the auto - generated hepatic catalase , which detoxifies hydrogen peroxide , increased two - to three - fold . lipofuscin - like products , i . e ., the end - products of free - radical - induced lipid peroxidation , increased by three - fold in the clofibrate - fed animals . these findings suggest free - radical production and hydrogen peroxide production are in agreement with previous investigators . the rat tests herein reported establish that pyruvate inhibits free - radical generation . clofibrate - feeding manifests a chronic , free - radical production . clofibrate - feeding induces hepatomegaly with an increase in hematic protein content , peroxisomal proliferation by electron microscope evaluation and five - fold increase in peroxisomal beta - oxidation activity , a marker of peroxisomal proliferation and hydrogen peroxide production . the total hepatic catalase , which detoxifies hydrogen peroxide , increased by two - to - three fold . lipofuscin - like products , which are end products from free - radical - induced livid peroxidation , increased by three - fold in the clofibrate - fed animals . these findings , suggestive of hydrogen peroxide production and toxic , free - radical metabolic effects , are in agreement with previous investigations . in contrast , supplementing the clofibrate - fed animals with vitamin e had no effect on hepatic size , protein content , peroxisomal proliferation or beta - oxidation activity . these findings indicate that free - radical production continued unabated with clofibrate - feeding despite supplementation with vitamin e . however the antioxidant vitamin e , as expected , did reduce the effects of free - radicals on lipid peroxidation ; i . e ., vitamin e appeared to be an effective scavenger of free - radicals . the data suggest that pyruvate has reproducible effects on free - radical generation and subsequent lipid peroxidation . pyruvate seems to offer promise in the control of the metabolic effects of free - radicals , as a known inhibitor of free - radical production . vitamin e did not influence free - radical generation . pyruvate supplementation of a diet inhibits peroxisomal proliferation and free - radical generation induced by clofibrate . the pyruvate supplementation also inhibits free - radical - induced lipid peroxidation and enhances metabolism of nitric oxide . superoxides ( o 2 0 - ) can be detected by lucigenin - enhanced chemiluminescence ( caraceni , p . et al , j . am . physiol . 266 , g799 - g808 , 1994 ). superoxides themselves are free - radicals and are known to be a precursor of hydrogen peroxide which is a precursor of other free - radicals . for example : ## str1 ## both the superoxide and the hydroxyl are free - radicals , known to be harmful . pyruvate is known as a scavenger for hydroxyl free - radicals . it is here reported that pyruvate also inhibits the generation of superoxide which thereby lowers the proliferation of objectionable hydroxyl free - radicals . in the reported experiments , hepatocytes were recovered from adult male sprague - dawley rats and centrifuged in appropriate solutions for ten minutes at 1 , 200 g . cell viability ranged from 90 % to 97 %. cells were embedded in agarose gel threads , 0 . 5 mm diameter . all of the agarose gel threads were perfused for one hour in khb ( standard krebs - henseleit bicarbonate buffer ) at 37 ° c . thereafter the hepatocytes were segregated into three groups : 2 . pyruvate ( 1 )-- same as control containing in addition 1 . 0 mm pyruvate . 3 . pyruvate ( 5 )-- same as control containing in addition 5 . 0 mm pyruvate . each of the groups ( 1 ), ( 2 ), ( 3 ) was rendered anoxic for two hours in anoxic conditions nitrogen by volume and 5 % carbon dioxide by volume . after two hours of anoxia , the nitrogen was replaced with oxygen air restored ( reoxygenation ) while all cells were perfused with khb solution . the generation of superoxides from the three groups is illustrated in fig4 where the superoxide generation is indicated by the measured lucigenin chemiluminescence in na . during the two hours of anoxia , lucigenin chemiluminescence ceased for all groups , indicating no superoxide generation . non - generation of superoxides is expected during anoxia , i . e ., the absence of oxygen . reoxygenation started at 150 minutes ( fig4 ) and the control group ( black circles ) generated lucigenin chemiluminescence at more than 200 na . the lucigenin chemiluminescence for the two pyruvate groups ( white circles and black triangles ) was significantly lower during the reoxygenation period from 150 to 270 minutes . there were six control specimens , six specimens with five mm pyruvate and five specimens with one mm pyruvate . these data indicate that pyruvate inhibits formation of superoxide ( o 2 0 - ) and hence inhibits free - radical generation .