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1 From the Divisions of Gastroenterology (KEH, AKM, and RFB) and Endocrinology (XL and JMM), Department of Medicine; the Department of Pathology (TJM); and the Clinical Nutrition Research Unit (KEH, JMM, and RFB), Vanderbilt University School of Medicine, Nashville, TN.
2 Supported by NIH grants R01 AG16236 and P30 DK26657. 3 Address reprint requests to RF Burk, C2104, Medical Center North, Vanderbilt Medical Center, Nashville, TN 37221-2279. E-mail: raymond.burk{at}vanderbilt.edu.
ABSTRACT
Background: On the basis of in vitro studies, the antioxidant nutrients vitamins E and C are postulated to interact in vivo.
Objective: We developed a guinea pig model to evaluate the combined deficiency of vitamins E and C in vivo.
Design: Weanling guinea pigs were fed a control diet or a vitamin Edeficient diet for 14 d, after which one-half of each group had vitamin C removed from their diet, thus creating 4 diet groups. Some animals were observed for clinical signs. Others were killed for evaluation.
Results: Of 21 guinea pigs that were observed after being fed the diet deficient in both vitamins, 8 died 9 ± 2 d (
Key Words: Combined vitamin E and vitamin C deficiency guinea pigs limb paralysis antioxidant nutrient deficiency central nervous system dysfunction ascorbate -tocopherol
INTRODUCTION
Vitamin E, vitamin C, and selenium are essential nutrients that function as antioxidants in vivo (1). They have been postulated to protect against oxidative injury in the pathogenesis of atherosclerosis, diabetes, cancer, and other conditions. Although rat and mouse models of vitamin E deficiency and selenium deficiency are readily available, vitamin C (ascorbate) deficiency cannot be produced in most rodents. For this reason, there have been relatively few in vivo studies of the effect of vitamin C deficiency on oxidative injury.
Each nutrient has specific biochemical functions that combat oxidative injury. Vitamin E (primarily -tocopherol) is a lipid-soluble free radical scavenger that resides in membranes. Vitamin C is a redox-active molecule in the water phase. In donating an electron to a free radical, -tocopherol is converted into the relatively stable -tocopheroxyl radical. Further oxidation inactivates the vitamin, but there is evidence that it can be recycled from the -tocopheroxyl radical to -tocopherol (2, 3). In vitro studies indicate that water-soluble ascorbate can donate a hydrogen atom to the -tocopheroxyl radical, reconverting it to -tocopherol. This recycling action on -tocopherol has been postulated to account for some of the antioxidant function of vitamin C (2). Thus, in vivo studies probing the relation between vitamin E and vitamin C could provide support for this attractive recycling hypothesis.
Simultaneous deficiencies of vitamins E and C have been produced in fish. Growth and feed efficiency were adversely affected by the double deficiency, but those effects could be counteracted by relatively small amounts of vitamin C or large amounts of vitamin E (4, 5). There was a slight increase in mortality in fish deficient in both nutrients. Similar studies in a strain of rats that cannot synthesize vitamin C did not describe a clinical effect of the double deficiency (6). Thus, there is some, but not overwhelming, evidence that an in vivo interaction between vitamin E and vitamin C exists.
Like human beings, guinea pigs require all 3 of the antioxidant nutrients: vitamin E, vitamin C, and selenium. We set out to develop guinea pig models of individual and combined antioxidant nutrient deficiencies in the hope that the study of guinea pigs would improve our understanding of these nutrients in humans. Our first model involved selenium and vitamin E deficiencies, which produced severe skeletal muscle necrosis (7). In the present study, we developed a guinea pig model of combined vitamin E and vitamin C deficiency.
MATERIALS AND METHODS
Animals
Weanling male Hartley guinea pigs (101136 g) were purchased from Elm Hill Breeding Labs (Chelmsford, MA). On arrival, the guinea pigs were housed in pairs in plastic cages with aspen shavings as bedding. They were fed torula yeastbased diets formulated according to our specifications. The control diet used in the present study has been described in detail (7), and the control diet and the 3 iterations of it that were used in the present study are described in Table 1. The diets used in these experiments contained 0.5 mg Se as sodium selenate/kg. The diets were purchased from Harlan-Teklad (Madison, WI). The animals had free access to food and water. The experiments were approved by the Vanderbilt University Animal Care and Use Committee.
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TABLE 1 . Amounts of ascorbate and all-rac--tocopheryl acetate added to the 4 diets used in all of the studies1
Guinea pigs were exsanguinated from the portal vein while they were anesthetized with pentobarbital (65 mg/kg intraperitoneal). Blood was treated with disodium EDTA (1 mg/mL) to prevent coagulation, and plasma was separated from cells by centrifugation for 20 min at 1000 x g and 4 °C. Liver and skeletal muscle (quadriceps) samples were harvested, and the brain, spinal cord, and sciatic nerve were carefully removed. Samples of all tissues were fixed in 10% (by vol) buffered formalin for light microscopic study. Quadriceps muscle necrosis was assessed histopathologically as before (7). A spinal cord sample was fixed in 2% (by vol) glutaraldehyde for electron microscopic study. Samples of all tissues and of plasma were quick-frozen in liquid nitrogen and stored at -70 °C for biochemical analysis. At the time that the animals were killed, their hind limbs were observed for signs of scurvy. Scurvy was diagnosed when hemorrhage in the connective tissues surrounding the joints was present.
Development of the experimental protocol
Guinea pigs that are fed a vitamin Edeficient diet begin to develop muscle injury at 45 wk (7). Guinea pigs fed a vitamin Cdeficient diet develop scurvy at 3 wk (8). On the basis of this information, we carried out 2 preliminary studies in which weanling animals were fed the vitamin Edeficient diet for 1 and 2 wk, respectively, before the second deficiency (vitamin C) was imposed. In both of the preliminary studies, the guinea pigs were found dead or were noted to have paralysis of the limbs 79 d after institution of the double deficiency. These affected animals had no signs of scurvy.
On the basis of these observations, we used a standard diet protocol to carry out a study that was performed 4 times. In that protocol, weanling animals were fed the vitamin Edeficient or the control diet for 14 d. Then a subset of each group had vitamin C removed from their diet. In this way, the following 4 diet groups were formed: control, vitamin C deficient, vitamin E deficient, and doubly deficient (Table 1).
Assays
For assay of vitamin C in plasma, a 0.1-mL plasma sample was added to 0.9 mL of a 0.2-mol perchloric acid/L solution and was mixed by vortex. The sample was allowed to stand on ice for 10 min and then was centrifuged for 10 min at 6000 x g and 4 °C. The supernatant fluid was removed and assayed for ascorbate by HPLC with electrochemical detection as previously described (9), except that tetrapentyl ammonium bromide was used as the ion pair reagent.
For assay of -tocopherol in plasma, 0.1 mL plasma was treated with 0.05 mL 0.5% (wt:vol, in water) pyrogallol. The sample was treated with 0.3 mL reagent alcohol [ethanol:isopropanol (95:5, by vol)], mixed by vortex, and treated with 0.6 mL heptane. The sample was then mixed by vortex for ≥ 1 min and centrifuged for 15 s at 13 000 x g and 4 °C to separate the layers. An aliquot of the upper heptane phase was transferred to a glass tube and evaporated to dryness under nitrogen. For assay of -tocopherol by HPLC (9), the residue was dissolved in a volume of methanol:reagent alcohol (1:1, by vol) equal to the volume of the upper heptane phase.
Measurement of -tocopherol in tissue samples was carried out as described by Lang et al (10), except that heptane rather than hexane was used in the extraction step. Vitamin C in tissues was measured after homogenization of a 100-mg sample in a polytetrafluoroethylene-glass homogenizer in 1 mL of a solution containing ice-cold 80% (by vol) methanol and 2 mmol EDTA/L. The homogenate was allowed to sit on ice for 5 min and then was centrifuged for 5 min at 6000 x g and 4 °C. Aliquots of the supernatant fluid were taken for assay of vitamin C as described for plasma samples.
Tissue F2-isoprostanes were measured after Folch extraction (11), base hydrolysis, and derivatization (12). Plasma creatine phosphokinase (CPK) activity was determined by using a CPK kit (procedure number 45-UV; Sigma Chemical Co, St Louis).
Materials
HPLC solvents and perchloric acid were purchased from Fisher Scientific Co (Atlanta). Pyrogallol, tetrapentyl ammonium bromide, and the CPK kits were purchased from Sigma Chemical Co. The TUNEL (terminal deoxynucleotidyl transferasemediated X-dUTP nick end labeling) staining kit ApoTag was obtained from Serologicals Corp (Norcross, GA). The HAM-56 antibody was purchased from Chemicon (Temecula, CA).
Statistical analyses
Statistical analyses were performed by using the analysis of variance method in the STATVIEW software program, version 5.0.1 (SAS Institute Inc, Cary, NC), on a Macintosh G4 computer (Apple Computer, Cupertino, CA). Tukey-Kramer procedures were used for post hoc testing to determine the statistical significance of differences. Values are presented as means ± SDs.
RESULTS
Clinical observations
Four study replications were carried out by using the diet protocol designed to produce simultaneous deficiencies of vitamins E and C. In each replication, after ≥ 1 animal in the doubly deficient group had died or developed paralysis, 3 or 4 animals from each diet group were killed for biochemical and histologic study. In the 4 replications, the groups of animals were killed at 8, 9, 13, and 16 d, respectively, after vitamin C had been removed from some of the diets (ie, after the end of the 14-d period in which the animals consumed either the control or the vitamin Edeficient diet). All other animals were observed for clinical signs of the double deficiency.
There were 21 animals in the doubly deficient diet group that were observed for development of clinical signs. Eight of them were found dead at 9 ± 2 d after the doubly deficient diet had been instituted, and 8 of them were observed to have paralysis of their limbs and were euthanized at 11 ± 3 d after the doubly deficient diet had been instituted. Only one of the animals with paralysis showed signs of scurvy, and that animal had been fed the doubly deficient diet for 13 d. The remaining 5 animals in the doubly deficient diet group survived to 2122 d without paralysis and were euthanized. All of them had signs of scurvy. No animals in the other diet groups died or developed paralysis, although those in the vitamin Cdeficient diet group developed signs of scurvy after 2 wk.
The regimen of simultaneous vitamin E and vitamin C deficiencies produced a striking and characteristic clinical picture that was different from the scurvy observed in vitamin Cdeficient animals and the muscle injury observed in vitamin Edeficient animals. The guinea pigs in the doubly deficient diet group appeared healthy for 1 wk after the double deficiency had been imposed. Then some were found dead and some were observed to have limb paralysis. No signs of pain or distress were observed. The plasma CPK activities in the animals with paralysis ranged from 140 to 2600 IU/L, with a mean of 1000 ± 870 IU/L (n = 8). Muscle (quadriceps) histology with hematoxylin and eosin staining was assessed in 4 of the animals with paralysis. Degeneration of muscle fibers was rare (< 1 in 50) in 3 of those animals and mild (1 in 15) in the other animal. This indicates that the muscle injury was mild (compared with that observed in reference 7) and could not have accounted for the paralysis.
The paralysis was characteristic. It began in the hind limbs and progressed to paralysis of all 4 limbs within hours. The rate of progression was somewhat variable, but the animals usually became completely paralyzed over a few hours, to the point of developing respiratory difficulty. The deaths were probably caused by paralysis that occurred during the night, while the animals were not being observed. This clinical picture would be most easily explained by dysfunction of the central nervous system, especially of the spinal cord.
We carried out biochemical and histologic assessment of animals in all 4 trials to seek the cause of the paralysis. The histopathologic results presented for the central nervous system are mostly from a single trial, but individual animals that developed paralysis in the other trials were also examined. The biochemical results presented are from a different trial because we could not obtain enough tissue from the spinal cord to carry out both histologic and biochemical analyses of it in the same trial. The plasma CPK activities of the guinea pigs used for the biochemical analysis were as follows: control diet group, 170 ± 59 IU/L; vitamin Cdeficient diet group, 190 ± 62 IU/L; vitamin Edeficient diet group, 320 ± 210 IU/L; and doubly deficient diet group, 1600 ± 880 IU/L. Histopathologic examination of the quadriceps muscles of these same animals revealed normal muscle in the control animals and vitamin Cdeficient animals, mild necrosis in 1 of the 3 vitamin Edeficient animals, and mild necrosis in all 3 of the animals in the doubly deficient diet group. These results indicate that muscle injury was mild in the vitamin Edeficient and doubly deficient animals. Clinical weakness was observed in a previous study only when CPK activity exceeded 30 000 IU/L and histopathologic necrosis was severe (7).
Ascorbate and -tocopherol concentrations
Ascorbate concentrations in the plasma and tissues of the guinea pigs in the 4 diet groups are shown in Table 2. Sixteen days after the initiation of vitamin C deficiency, the animals in both groups receiving deficient diets had essentially undetectable amounts of ascorbate in plasma and liver, which is consistent with severe vitamin C deficiency. Brain ascorbate concentrations in the animals in these groups were much better preserved, at 70% of the concentrations in the animals that received vitamin C in their diet. In addition, the animals in the vitamin Cdeficient and doubly deficient diet groups had spinal cord ascorbate concentrations that were 40% of those in the animals in the control diet group. These results confirm that the central nervous system retains ascorbate better than do plasma and liver under deficiency conditions. They also suggest that spinal cord ascorbate decreases more than does brain ascorbate under deficiency conditions.
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TABLE 2 . Ascorbate concentrations in plasma and tissues of guinea pigs in the 4 diet groups1
-Tocopherol concentrations in the plasma and tissues of the guinea pigs in the 4 diet groups are shown in Table 3. The vitamin Edeficient diets had been fed for 30 d. Plasma and liver -tocopherol concentrations in the animals fed a vitamin Edeficient diet were 1625% of those in the animals fed the control diet. However, brain and spinal cord -tocopherol concentrations were much less affected. The only significant effect of a deficient diet on -tocopherol concentrations in the central nervous system was a mean brain concentration that was 68% of that in the animals fed the control diet. The doubly deficient diet did not have a significant effect on brain -tocopherol. Thus, the dietary effects on -tocopherol concentrations in the central nervous system were much less than those on -tocopherol concentrations in plasma and liver.
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TABLE 3 . -Tocopherol concentrations in plasma and tissues of guinea pigs in the 4 diet groups1
F2-isoprostane concentrations
F2-isoprostanes are markers of lipid peroxidation (13). As shown in Table 4, the liver F2-isoprostane concentrations in the vitamin Cdeficient and doubly deficient diet groups were significantly higher than those in the control diet group; however, the concentrations in the vitamin Edeficient diet group were not significantly higher than those in the control diet group. Neither single deficiency affected F2-isoprostanes in the central nervous system, but combined deficiencies of the vitamins caused F2-isoprostane concentrations in the brain to be significantly higher than those in the animals fed the control diet. Compared with the control diet, the doubly deficient diet appeared to cause higher F2-isoprostane concentrations in the spinal cord, but the only groups that were significantly different from one another were the vitamin Cdeficient group and the doubly deficient group. This result suggests that the combined deficiency of vitamins E and C causes an increase in lipid peroxidation in the brain and probably in the spinal cord.
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TABLE 4 . F2-isoprostane concentrations in liver, brain, and spinal cord of guinea pigs in the 4 diet groups1
Histopathologic evaluation of the brain, spinal cord, and sciatic nerve
In the trial used for histopathologic evaluation, one animal became paralyzed 7 d after the vitamin C deficiency had been initiated, and another animal was found dead at 8 d. Consequently, we studied 3 animals from each group at 9 d. We also studied 3 of the animals that became paralyzed. Thus, we performed both a direct examination of animals that were affected by the doubly deficient diet and a systematic evaluation of all diet groups, which allowed comparison between them. Brain, spinal cord, and sciatic nerve were harvested for examination.
Multiple axial whole-mount sections of brain were stained with hematoxylin and eosin. The cerebral cortex, basal ganglia, thalamus, brainstem, and cerebellum were examined. One animal that was from the doubly deficient diet group and had become paralyzed had a small destructive lesion with a macrophage infiltrate and surrounding astrogliosis in the base of the pons. Macrophage infiltration in this lesion was confirmed by using HAM-56 immunohistochemistry. However, similar lesions were not found in the other animals that had become paralyzed or in any of the animals from the other groups. No other structural lesions were identified in any of the animals studied.
Axial sections of caudal spinal cord were evaluated both histochemically with hematoxylin and eosin and with luxol fast blueperiodic acid Schiffs reagent and immunohistochemically with antiglial fibrillary acidic protein antiserum. Neither a destructive or degenerative lesion nor astrogliosis was identified in the gray matter or white matter. TUNEL-stained sections of the spinal cords were examined, and no difference in nuclear staining was observed between the 4 diet groups.
Two caudal spinal cord sections from animals from the control diet group and 2 sections from animals from the doubly deficient diet group were evaluated ultrastructurally. Anterior horn cell cytoplasm, nuclei, and mitochondria had normal morphology in all 4 animals. Axons had normal axoplasm, mitochondria, and vesicles. The ultrastructure of myelin also was normal in all 4 animals. No abnormal inclusions were identified.
Peripheral (sciatic) nerve was evaluated by toluidine blue staining of cross sections. The number of myelinated fibers did not differ between the 4 diet groups, and no abnormal variation in myelin thickness was observed in any of the 4 groups. As expected for rodents, the animals in the control diet group showed 12 degenerating axons per nerve; a similar frequency of degenerating fibers was present in the animals fed the deficient diets.
In summary, brain, spinal cord, and peripheral nerve were evaluated by several histopathologic techniques. No consistent evidence of cell death or injury was found, even in the animals that had developed limb paralysis.
DISCUSSION
A distinct clinical syndrome of rapidly progressive paralysis is caused by combined vitamin E and vitamin C deficiency in guinea pigs. This syndrome, described here for the first time, is different from the conditions brought on by the deficiency of either nutrient alone. The occurrence of this distinct clinical syndrome indicates that these 2 nutrients interact in some way in vivo.
Many studies showed that vitamin E and vitamin C have antioxidant effects in cell and membrane systems (14, 15). It has also been shown in vitro that ascorbate can regenerate -tocopherol after -tocopherol has scavenged a free radical (2). Thus, the in vivo relation between these vitamins that is implied by the occurrence of limb paralysis in animals fed a doubly deficient diet might be a consequence of separate biochemical effects that are additive or of a cooperative mechanism between the vitamins, such as recycling of -tocopherol by ascorbate. This study does not allow discrimination between these 2 possibilities.
Guinea pigs develop muscle injury in vitamin E deficiency, and we considered the possibility that the paralysis we observed in the animals in the doubly deficient diet group was caused by muscle weakness. The vitamin Edeficient diet that we used in the present study was also fed to guinea pigs in our previous study (7) for 135 d, but the animals in that study did not become paralyzed. The animals in the present study were fed the vitamin Edeficient diet for only 30 d, and we evaluated muscle injury by measuring plasma CPK activity and examining histologic sections. We concluded that muscle injury was mild in the animals fed the doubly deficient diet, including the animals that developed paralysis, and that therefore muscle injury could not account for their loss of limb function.
Our clinical observations of the progressive paralysis syndrome led us to postulate that it was caused by an injury to the nervous system. However, despite extensive histopathologic examination of the brain, spinal cord, and sciatic nerve, we found no evidence of an injury that could explain the paralysis. We took note of the axonal abnormalities described in the spinal cords of rats fed vitamin Edeficient diets for > 1 y (16) and used electron microscopy to search for similar lesions in the spinal cords of guinea pigs. However, we did not find such lesions or other abnormalities. Thus, we cannot ascribe the paralysis to a specific nervous system lesion, despite our conclusion that injury to the nervous system is likely to underlie the paralysis.
Biochemical analyses of plasma and liver showed that deficiencies of vitamins E and C were produced by the diets (Tables 2 and 3). Despite the depletion in those tissues, brain and spinal cord maintained these vitamins at much higher percentages of control values. Almost no decrease in -tocopherol concentrations was detected. Ascorbate concentrations decreased significantly, with concentrations in the spinal cord decreasing more than those in the brain. These values were obtained by using large pieces of tissue that included all regions of the organ. It seems likely that different regions of the organ and different compartments in cells could be depleted of these vitamins to different extents. Thus, there might be compartments in the nervous system that are much more vitamin E and C deficient than is the whole tissue. Such compartments might be injured and cause the dysfunction we observed.
Because vitamins E and C are antioxidant nutrients, we measured F2-isoprostane concentrations in tissues. F2-isoprostanes occur in phospholipids after they have undergone lipid peroxidation and serve as a marker of oxidative injury (13). The vitamin E deficiency produced in the guinea pigs in the present study was not severe because it did not elevate F2-isoprostane concentrations in the liver (Table 4). However, the vitamin Cdeficient animals all had elevated F2-isoprostane concentrations in the liver, indicating that the vitamin C deficiency caused lipid peroxidation in the liver. F2-isoprostane concentrations in the brain were elevated only in the doubly deficient diet group. This indicates that when both vitamins were lacking, lipid peroxidation occurred in the brain. The values in the spinal cord were similar to those in the brain, although significant differences between the diet groups were not observed in all cases. Thus, individual deficiencies of the vitamins did not cause detectable lipid peroxidation, but simultaneous deficiencies of vitamins E and C did cause lipid peroxidation in the central nervous system. We conclude that there is biochemical evidence of a relation between these vitamins in preventing lipid peroxidation in the central nervous system.
We speculate that the progressive paralysis that occurs in guinea pigs with combined vitamin E and vitamin C deficiency is caused by oxidative injury in the central nervous system, probably in the spinal cord. The injury would have to be of rapid onset so that it would cause dysfunction leading to paralysis with little or no histopathologic evidence of the injury.
On the basis of our conclusions, we can speculate that humans with vitamin E deficiency might be highly susceptible to injury if they become vitamin C deficient. Thus, maintenance of normal vitamin C status in vitamin Edeficient patients appears to be prudent.
ACKNOWLEDGMENTS
KEH contributed to the study design, statistical analysis, and manuscript preparation. TJM carried out the neuropathologic analysis. AKM contributed to the design and execution of the study. XL carried out the biochemical analyses. JMM contributed to the study design and data interpretation. RFB contributed to the study design and manuscript preparation. None of the authors had any conflicts of interest.
REFERENCES