Scorbutic Diet Research Articles

Clinical changes observed in Clarias gariepinus (Burchell 1822) fed varying levels of ascorbic acid supplementation

This study was undertaken to observe the clinical changes associated with the dietary ascorbic acid supplementation in the diet of African catfish, Clarias gariepinus, using the presence or absence of scorbutic (ascorbic acid deficiency) symptoms as indices. C. gariepinus fingerlings weighing 6.02±0.4 g were randomly distributed into glass tanks of 60 × 45 × 45 cm3 dimension at ten fish per tank in a triplicate treatment. Five isonitrogenous and isocalorific diets containing 40% crude protein was formulated. Ascorbic acid (AA) was supplemented in the diets as ascorbyl-2-polyphosphate (a mixture of phosphate esters of ascorbate). Each treatment had varying levels of ascorbic acid (AA) supplementation, at 0 (Control) 50, 100, 150 and 200 mg AA/kg, in Treatment 1, 2, 3, 4 and 5 respectively. Fish were fed practical diets twice daily at 900 and 1600 h GMT. Weekly weighing of fish was done and the data collected were subjected to statistical analysis. At the end of week 4, fish fed scorbutic diets (diets without ascorbic acid supplementation) had significantly lower weight than fish fed AA supplemented diets (P<0.05). After week 6 fish fed scorbutic diet began to develop clinical symptoms including lordosis (lateral curvature), broken skull, pigmentation and scoliosis (vertical curvature). Radiographs confirmed defects in the vertebral columns of fish in this treatment. Highest mortality was recorded in this treatment as 30%, followed by treatment two fed 50 mg AA/kg which had 10% mortality, fish in this treatment also showed some clinical signs like eroded fins and pigmentation. In all parameters considered, treatment 4 fed 150 mg AA/kg gave the best AA supplementation that prevented growth reduction and clinical signs of AA deficiency in this study. Key words: Clinical, ascorbic acid, Clarias gariepinus, scorbutic, supplementation.

Ascorbic acid deficiency aggravates stress-induced gastric mucosal lesions in genetically scorbutic ODS rats

We examined whether ascorbic acid (AA) deficiency aggravates water immersion restraint stress (WIRS)-induced gastric mucosal lesions in genetically scorbutic ODS rats. ODS rats received scorbutic diet with either distilled water containing AA (1 g/l) or distilled water for 2 weeks. AA-deficient rats had 12% of gastric mucosal AA content in AA-sufficient rats. AA-deficient rats showed more severe gastric mucosal lesions than AA-sufficient rats at 1, 3 or 6 h after the onset of WIRS, although AA-deficient rats had a slight decrease in gastric mucosal AA content, while AA-sufficient rats had a large decrease in that content. AA-deficient rats had more decreased gastric mucosal nonprotein SH and vitamin E contents and increased gastric mucosal lipid peroxide content than AA-sufficient rats at 1, 3 or 6 h of WIRS. These results indicate that AA deficiency aggravates WIRS-induced gastric mucosal lesions in ODS rats by enhancing oxidative damage in the gastric mucosa.

Scurvy Leads to Endoplasmic Reticulum Stress and Apoptosis in the Liver of Guinea Pigs1

Insufficient ascorbate intake causes scurvy in certain species. Beyond its known functions, it has been suggested that ascorbate participates in oxidative protein folding in the endoplasmic reticulum (ER). Because redox imbalance in this organelle might cause ER stress and apoptosis, we hypothesized that this might contribute to the pathology of scurvy. Guinea pigs were divided into 7 groups: the control group was fed a commercial guinea pig food containing 0.1 g/100 g ascorbate for 4 wk, 5 groups consumed an ascorbate-free food for 0, 1, 2, 3, or 4 wk and 1 group was fed this scorbutic diet for 2 wk and then the commercial food plus 1 g/L ascorbate in drinking water for 2 wk. TBARS generation and the expression of some ER chaperones and foldases were determined in hepatic microsomes. The apoptotic index was assessed in histological sections. Although ascorbate, measured by HPLC, was undetectable in the livers of the guinea pigs after they had consumed the scorbutic diet for 2 wk, the microsomal TBARS level was elevated relative to the initial value only at wk 4. Western blot revealed the induction of GRP78, GRP94, and protein disulfide isomerase at wk 3 and 4. Apoptosis was greater than in the control, beginning at wk 3. None of the alterations occurred in the groups fed the commercial guinea pig food or ascorbate-free food followed by ascorbate supplementation. Therefore, persistent ascorbate deficiency leads to ER stress, unfolded protein response, and apoptosis in the liver, suggesting that insufficient protein processing participates in the pathology of scurvy.

Short-term ascorbic acid deficiency induced oxidative stress in the retinas of young Guinea pigs.

We examined whether short-term ascorbic acid deficiency induces oxidative stress in the retinas of young guinea pigs. Four-week-old guinea pigs were given a scorbutic diet (20 g/animal/day) with and without adequate ascorbic acid (400 mg/animal/day) in drinking water for 3 weeks. The serum concentrations of the reduced form of ascorbic acid and the oxidized form of ascorbic acid in the deficient group were 14.1 and 4.1%, respectively, of those in the adequate group. The retinal contents of the reduced form of ascorbic acid and the oxidized form of ascorbic acid in the deficient group were 6.4 and 27.3%, respectively, of those in the adequate group. The retinal content of thiobarbituric acid-reactive substances, an index of lipid peroxidation, was 1.9-fold higher in the deficient group than in the adequate group. Retinal reduced glutathione and vitamin E contents in the deficient group were 70.1 and 69.4%, respectively, of those in the adequate group. This ascorbic acid deficiency did not affect serum thiobarbituric acid-reactive substances and reduced glutathione concentrations but increased serum vitamin E concentration. These results indicate that short-term ascorbic acid deficiency induces oxidative stress in the retinas of young guinea pigs without disrupting systemic antioxidant status.

Short-Term Ascorbic Acid Deficiency Induced Oxidative Stress in the Retinas of Young Guinea Pigs

We examined whether short-term ascorbic acid deficiency induces oxidative stress in the retinas of young guinea pigs. Four-week-old guinea pigs were given a scorbutic diet (20 g/animal/day) with and without adequate ascorbic acid (400 mg/animal/day) in drinking water for 3 weeks. The serum concentrations of the reduced form of ascorbic acid and the oxidized form of ascorbic acid in the deficient group were 14.1 and 4.1%, respectively, of those in the adequate group. The retinal contents of the reduced form of ascorbic acid and the oxidized form of ascorbic acid in the deficient group were 6.4 and 27.3%, respectively, of those in the adequate group. The retinal content of thiobarbituric acid-reactive substances, an index of lipid peroxidation, was 1.9-fold higher in the deficient group than in the adequate group. Retinal reduced glutathione and vitamin E contents in the deficient group were 70.1 and 69.4%, respectively, of those in the adequate group. This ascorbic acid deficiency did not affect serum thiobarbituric acid-reactive substances and reduced glutathione concentrations but increased serum vitamin E concentration. These results indicate that short-term ascorbic acid deficiency induces oxidative stress in the retinas of young guinea pigs without disrupting systemic antioxidant status.

Short-term ascorbic acid deficiency does not impair antioxidant status in lens of guinea pigs.

We examined whether short-term ascorbic acid deficiency impairs antioxidant status in the lens of guinea pigs. Male guinea pigs aged 4 wk were given a scorbutic diet (20 g/animal per day) with and without ascorbic acid (400 mg/animal per day) in drinking water for 3 wk. The ascorbic acid-deficient group showed no lens opacity. The ascorbic acid-deficient group had 14% of serum ascorbic acid concentration, 6% of aqueous humor ascorbic acid concentration, and 18% of lens ascorbic acid content in the ascorbic acid-adequate group. There were no differences in the contents of lens reduced glutathione and thiobarbituric acid reactive substances, an index of lipid peroxidation, between the ascorbic acid-deficient and adequate groups, while the deficient group had higher lens vitamin E content than the adequate group. The ascorbic acid-deficient group had higher serum vitamin E concentration than the ascorbic acid adequate group, while there were no differences in the concentrations of serum reduced glutathione and tiobarbituric acid reactive substances between the deficient and adequate groups. These results indicate that short-term ascorbic acid deficiency does not impair antioxidant status in the lens of guinea pigs despite induction of severe ascorbic acid depletion in the tissue, which may result in no cataract formation.

Prolonged marginal ascorbic acid deficiency induces oxidative stress in retina of guinea pigs.

We examined whether prolonged marginal ascorbic acid deficiency induces oxidative stress in the retina of guinea pigs. Male guinea pigs aged four weeks were given a scorbutic diet (20 g/animal per day) with either marginally deficient ascorbic acid (0.5 mg/animal per day) or adequate ascorbic acid (1 g/animal per day) in drinking water for three and six months. The retinal contents of the reduced form of ascorbic acid in the deficient group at three and six months were 68.1 and 43.5%, respectively, of that in the corresponding adequate group. The retinal contents of the oxidized form of ascorbic acid in the deficient group at three and six months were 1.9- and 2.7-fold, respectively, higher than that in the corresponding adequate group. The content of retinal thiobarbituric acid reactive substances (TBARS), an index of lipid peroxidation, in the deficient group was 2.5-fold higher than that in the adequate group at six months. The retinal contents of reduced glutathione (GSH) in the deficient group at three and six months were 84.8 and 66.7%, respectively, of that in the corresponding adequate group. The deficient group had 37.5% of retinal vitamin E content of the adequate group at six months. The deficient group had higher serum vitamin E concentration than the adequate group in both experimental periods. There were no differences in serum TBARS and GSH concentrations between the groups at both periods. These results indicate that prolonged marginal ascorbic acid deficiency induces oxidative stress in the retina of guinea pigs without systemic oxidative stress.

Effect of prolonged marginal ascorbic acid deficiency on lenticular levels of antioxidants and lipid peroxide in guinea pigs.

We examined the effect of prolonged marginal ascorbic acid deficiency of the levels of antioxidants and lipid peroxide in lenses of guinea pigs in order to clarify lenticular antioxidant status under ascorbic acid deficiency. Male guinea pigs aged 4 weeks were given a scorbutic diet (20 g/animal per day) with either marginally deficient ascorbic acid (0.5 mg/animal per day) or sufficient ascorbic acid (1 g/animal per day) in drinking water for 3 and 6 months. The deficient group showed no lens opacity during the administration period. The deficient group had 62.3 and 53.9% of lenticular ascorbic acid content in the sufficient group at 3 and 6 months of ascorbic acid deficiency, respectively. There were no differences in lenticular contents of reduced glutathione and thiobarbituric acid reactive substances, an index of lipid peroxidation, between both groups at 3 and 6 months of ascorbic acid deficiency, while the deficient group tended to have higher lenticular vitamin E content than the sufficient group. The deficient group had higher serum vitamin E concentration than the sufficient group at 3 and 6 months of ascorbic acid deficiency. These results indicate that lenticular antioxidant status is maintained well in guinea pigs with prolonged marginal ascorbic acid deficiency, which may result in no cataract formation.

Vitamin C deficiency in the catfishClarias gariepinus

Regardless of the absence of spinal deformities, vitamin C deprivation in adult-sized Clarias gariepinus challenged the physical integrity and survival of fish, inducing changes that greatly resembled those of fingerlings of other species. Vitamin C was presumed to be a dietary facilitator, which when deprived in the diet of C. gariepinus, would obstruct intestinal absorption of iron and/or impair its release from the reticulo-endothelial stores. This impaired erythrocyte synthesis led into anaemia (as indicated by lowered levels of red blood cells, haemoglobin and haematocrit). Leukopenia noted in fish on a scorbutic (lacking vitamin C) diet suggested functional depression of phagocytic engulfment with increased susceptibility to the deteriorating action of pathogenic agents. Changes in histology included hypertrophy, oedematous separation and telangiectasia of gill lamellae. Shrunken glomeruli, sloughed Bowman’s capsules and tubular dilation and distortion were also shown. Shrunken hepatocytes and the occurrence of yellowish ceroid pigments were the concrete histopathological signs in liver. Vitamin C is thus suggested as a potent antioxidant that offers protection against oxidative damage to various fish tissues.

A Physiological Level of Ascorbate Inhibits Galactose Cataract in Guinea Pigs by Decreasing Polyol Accumulation in the Lens Epithelium: a Dehydroascorbate-linked Mechanism

It was reported previously that dietary ascorbate (ASC) delays the development of galactose-induced cataract in guinea pigs compared to the rate which is observed in ASC-deficient animals. Experiments were conducted to explore the possible mechanism of this phenomenon. Guinea pigs were fed for a period of up to 4 weeks either a normal diet (1 g ASC/kg diet) or a scorbutic diet (< 0·04 g ASC/kg diet) combined with 10% galactose in the drinking water. After 2 weeks, levels of ASC in animals on the scorbutic diet decreased by 95% in the aqueous humor and by 78% in the lens. Slit lamp examination showed that galactose-induced vacuoles in the lens equator formed at a significantly faster rate in the scorbutic animals. However, examination of biochemical parameters in whole lenses of the two groups of animals after 2 weeks showed no significant differences with regard to accumulation of galactose and galactitol, decreases in the levels of myoinositol, taurine and GSH or changes in cation concentrations. In order to examine possible regional changes in the lenses, various parameters were studied in the lens capsule-epithelium. On day 4, the capsule epithelia of scorbutic animals on a galactose diet had a content of galactitol two-and-a-half times higher than that of normal galactose-fed animals. Scorbutic conditions also intensified the loss of Na +-K + ATPase activity in the lens capsule-epithelium caused by galactose feeding. Oxidized glutathione was not detectable in the lens capsule epithelia of any of the animals studied. Hexose monophosphate shunt activity was elevated in lenses of normal galactose-fed animals during the first hour of culture after death whereas lenses of scorbutic galaclose-fed animals were not. Consistent with the in vivo findings, galactitol accumulation in dog lens epithelial cells exposed to 30 mM galactose was significantly inhibited by the presence of either ASC or dehydroascorbate (DHA) in the medium. Hexose monophosphate shunt activity in the cells was stimulated to two-and-a-half times its initial level by either 1 mM DHA or 30 mu galactose and slightly more than three-fold by a combination of the two challenges. The results suggest that decreased polyol accumulation in the lens epithelium of the normal galactose-fed guinea pig, which has a high level of ASC in the aqueous humor, accounts for the delay in onset of cataract compared to that for the ASC-deficient animal. It appears that when the lens epithelium is exposed to both high sugar and GSH-oxidants such as DHA and H 2O 2 (both of which are present in normal guinea pig aqueous humor as autoxidatlon products of ASC), NADPH is utilized to a greater extent by glutathione reductase for the reduction of GSSG compared to aldose reductase for the synthesis of polyol. This phenomenon is presumably a result of the relatively high K m of galactose for aldose reductase compared to the K m of GSSG for glutathione reductase. No evidence was found to indicate that oxidative stress contributed to the formation of epithelial vacuoles observed in the early stage of galactose cataract. While ASC inhibited the development of sugar cataract, it was by a pro-oxidant rather than an antioxidant mechanism, and involved a preferential consumption of NADPH associated with the reduction of DHA and H 2O 2.

The effect of a scorbutic diet on ferritin-hemosiderin iron stores in the liver and spleen of female guinea pigs

Ascorbic acid deprivation for 21 days in female guinea pigs was associated with an increase in splenic hemosiderin and total iron, but no change in splenic ferritin iron. Decreases in hepatic ferritin, hemosiderin, and total iron were also produced. Upon refeeding ascorbic acid for only 3 days, elevated splenic total iron returned to control levels while the diminished hepatic iron stores remained low.

Tratment of scleroderma patients with a scorbutic diet. Negative result

Scleroderma is characterized by hyperplasia of collagen, a substance which may require vitamin C for its biosynthesis. Eleven patients were treated with a vitamin C-deficient diet in order to determine whether lack of this substance would impair collagen synthesis and consequently improve the lesions of scleroderma. None of the signs or symptoms of scleroderma improved in any of the patients. Impairment of collagen synthesis profound enough to improve the lesions of scleroderma may require vitamin C deficiency of greater duration and severity than reported here.

Hydroxyprolinemia III. The origin of free hydroxyproline in hydroxyprolinemia. Collagen turnover. Evidence for a biosynthetic pathway in man

1. 1. A patient with hydroxyproline oxidase deficiency offered a unique opportunity to investigate the origin of plasma and urine hydroxyproline, since most of the free hydroxyproline produced in this patient is accumulated in blood and excreted unchanged in the urine. Dietary origin had been ruled out previously. 2. 2. The hydroxyproline-containing peptides in the urine were not different from normal. There was no evidence of abnormal collagen breakdown. Results indicate that the free hydroxyproline derives from a normal collagen turnover of about 2 g/day. 3. 3. Trial of a scorbutic diet unexpectedly resulted in a striking increase in excretion of free and peptide-bound hydroxyproline in the urine, and a further increase in plasma hydroxyproline concentration. This is in contrast to the decreased urinary hydroxyproline observed in scorbutic guinea pigs. Administration of ascorbic acid after the period of depletion resulted in further increase in excretion of free hydroxyproline. This suggests that ascorbic acid depletion in man increases turnover of mature collagen or of partially hydroxylated collagen precursors. 4. 4. When labeled glyoxylate was administered, a significant though small amount of urinary free hydroxyproline became labeled, indicating a slight degree of biosynthesis from glyoxylate in man, as has been described in rat livers.

Nature of the Cyst of Trichinella spiralis

Studies were carried out on cysts mechanically isolated from golden hamsters after 50 to 60 days of infection with T. spiralis. It was found that the outer hyaline wall of the cyst was digested by clostridial collagenase prepared as a 1% solution in phosphate buffer (pH 7.6; 37 C). The time required for digestion (15 to 25 min) was similar to that observed with pepsin. Powdered hide and fresh hamster Achilles tendon were also dissolved under the same conditions; muscle proteins were resistant to the crude collagenase. Heat-denatured cysts were more slowly digested than the native structure. Buffer controls and heat-inactivated enzyme showed no activity against cysts. Trypsin, pan- creatin, and hyaluronidase had no significant action on native cyst walls during a 6-hr period of incu- bation. Trypsin was effective in destroying the cyst wall after 18 to 24 hr of exposure at 37 C. Trypsin was rapidly effective (35 min) in digesting the hyaline walls of heat-denatured cysts. The outer cyst wall of trichinella was inadequately formed in guinea pigs on a scorbutic diet. Infected guinea pigs which were pair-fed with the same diet were able to form normal cyst walls when they received daily supplements of ascorbic acid or orange juice. The walls of isolated whole cysts were birefringent to polarized light. Native cyst walls and tendon collagens from the same host shrank at 62 C which is the thermal shrinkage temperature of mammalian collagen. Cyst walls were resistant to mineral acid and urea; they gave positive reactions for protein with several standard spot test reagents. A 1% solution of sodium hypochlorite caused the immediate dissolution of the outer walls of whole cysts. Reaction with collagen stains, sensitivity to dietary ascorbic acid, resistance to trypsin, and collagenase susceptibility are regarded as evidence that the principal structural element of the cyst wall of trich- inella is collagen.

INFLUENCE OF GLUCOSE CYCLOACETOACETATE AND ITS HYDROLYZED PRODUCT ON CAPILLARY RESISTANCE IN SCORBUTIC GUINEA PIGS.

Summary1. Daily intraperitoneal administration of GCA and GCAh to guinea pigs maintained on scorbutic diet delayed and decreased the severity of the reduction of vascular resistance. 2. Neither of ...

Suppression of tuberculin reaction in the scorbutic guinea pig.

IN a recent publication from this laboratory it was noted that vitamin C deficiency in guinea pigs prevented induction of experimental allergic encephalomyelitis as well as development of the tuberculin reaction1. Replacement of the scorbutic diet with a normal stock diet four weeks after deprivation began and three weeks after inoculation of the challenge vaccine containing brain and killed Mycobacterium butyricum in water-in-oil emulsion permitted subsequent development of tuberculin sensitivity although the guinea pigs failed to develop clinical or histological signs of experimental allergic encephalomyelitis. It was important to determine the exact basis for the animals' failure to respond to tuberculin by development of a skin reaction because of the bearing this information might have on the induction of experimental allergic encephalomyelitis. Did absence of vitamin C prevent the end organ response (development of a positive skin reaction to tuberculin or, in the case of experimental allergic encephalomyelitis, the inflammatory lesions in the brain), or was the vitamin essential to the primary immunological response to the injected antigen(s)?

Production of experimental scurvy in a bird species.

IT has been generally supposed that all species of animals except the primates and the guinea pig are able to synthesize ascorbic acid according to their requirements. In a previous communication1, using our technique of incubating liver and kidney tissues of different species of animals with D-glucuronolactone as substrate in presence of cyanide, it was shown that in some species the kidney could effect the synthesis and in some the liver. In the evolutionary ascent the mechanism for the synthesis of ascorbic acid appeared to pass from the kidney to the liver and then to disappear altogether. It was also unexpectedly observed that neither the liver nor the kidney tissue of the red-vented bulbul (Pycnonotus cafer, Linn.) and of the Indian fruit-bat (Pteropus medius) could effect the synthesis. Thus at least one avian species, the red-vented bulbul, and one mammalian species, the Indian fruit-bat, were apparently incapable of synthesizing ascorbic acid. It was suggested, therefore, that the cyanide technique might offer a simple means for determining the capacity of different species for the synthesis of ascorbic acid. This supposition has now been proved to be correct by actually placing some red-vented bulbuls on a scorbutic diet and producing in them ascorbic acid deficiency symptoms, which could be cured by ascorbic acid.

Potentiation of pteroylglutamic acid by ascorbic acid in anemia of scurvy.

SummaryTwo men with scurvy and associated anemia, while maintained on a scorbutic diet, displayed hematologic responses to 125 and 250 μg of PGA, respectively, given intramuscularly daily. A potentiation of this hematologic effect of PGA occurred on subsequent administration of 1 g of ascorbic acid daily in addition to the PGA.

Effect of hemopoietic and other agents on the hydroxyphenyluria of premature infants.

IN THE year 1939 it was found that premature infants excreted the deaminated residues of tyrosine and phenylalanine when fed high protein diets low in vitamin C.1Full-term infants behaved similarly when their high-protein diets were supplemented by tyrosine or phenylalanine.2The administration of ascorbic acid led to consistent eradication of the hydroxyphenyluria (tyrosyluria). Liver extract decreased tyrosyluria in two of four premature infants and eliminated it in one full-term infant. Independently, Sealock and Silberstein3found a similar metabolic disorder in guinea pigs fed a scorbutic diet with supplements ofl-tyrosine. The response of this hydroxyphenyluria to the administration of ascorbic acid, an anti-pernicious-anemia preparation (liver extract),4or a variety of acidic substances5suggested that many factors contribute to complete oxidation of tyrosine and phenylalanine. Studies of the tyrosyluria of patients with pernicious anemia6and of scorbutic individuals7have shown that the

A scorbutic diet in a nile cataract community

1. (1) A Nile community living in an arid area, and its restricted food economy, are described. It was reputed to be malnourished. 2. (2) The daily food intake was assessed by weighing an estimated day's food for each of eight families and averaging the man-day values for seven of them which appeared to be characteristic of the village. 3. (3) The diet was considered to be deficient in ascorbin, as the only foods contributing this were probably cooked for over an hour from the cold state and with an alkaline cooking soda. 4. (4) The villagers were lean, and in every mouth looked at there were caries or the gaps denoting missing teeth. An old case of scurvy was seen, active cases of scurvy were found in the village, and the people described a local seasonal peak of “bleeding teeth” and swellings in the limbs. Cases of scurvy were also found in a nearby hospital in persons deriving from the same or similar local environments. 5. (5) It is suggested that in such circumstances resettlement is indicated, but that as palliatives, certain trees yielding useful food or economic products may be introduced.