Scorbutic Guinea Pigs Research Articles

The regulatory role of vitamin C on the adrenal function and resistance to histamine aerosol in the scorbutic guinea-pig.

The regulatory role of vitamin C on the adrenal function and resistance to histamine aerosol in the scorbutic guinea-pig.

Capillary hemorrhage in scorbutic guinea pigs.

Capillary hemorrhage in scorbutic guinea pigs.

Glycogen Metabolism in Scorbutic Guinea Pigs

Journal Article Glycogen Metabolism in Scorbutic Guinea Pigs Get access Nutrition Reviews, Volume 8, Issue 3, March 1950, Pages 91–93, https://doi.org/10.1111/j.1753-4887.1950.tb02378.x Published: 01 March 1950

Skeletal Effects of Estrogenic Hormone in Scorbutic Guinea Pigs

Skeletal Effects of Estrogenic Hormone in Scorbutic Guinea Pigs Nutrition Reviews, Volume 7, Issue 4, April 1949, Pages 109–111, https://doi.org/10.1111/j.1753-4887.1949.tb02192.x Published: 01 April 1949

Vitamin C activity in guinea pigs of 6-O-acyl-2-O-alpha-D-glucopyranosyl-L- ascorbic acids with a branched-acyl chain.

A series of novel acylated ascorbic acid derivatives, 6-O-acyl-2-O-alpha-D-glucopyranosyl-L-ascorbic acids with a branched-acyl chain (6-bAcyl-AA-2G) were recently developed in our laboratory as stable and lipophilic ascorbate derivatives. In this study, the bioavailability of 6-bAcyl-AA-2G was investigated in guinea pigs. Various tissue homogenates from guinea pigs hydrolyzed 6-bAcyl-AA-2G to give ascorbic acid (AA), 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G), and 6-O-acyl AA. The releasing pattern of the three hydrolysates suggested that 6-bAcyl-AA-2G was hydrolyzed via 6-O-acyl AA to AA as a main pathway and via AA-2G to AA as a minor pathway. The former pathway seems to be of advantage, because 6-O-acyl AA, as well as AA, can have vitamin C activity. In addition, we found that a derivative with an acyl chain of C(12), 6-bDode-AA-2G, had a pronounced therapeutic effect in scorbutic guinea pigs by its repeated oral administrations. These results indicate that 6-bAcyl-AA-2G is a readily available source of AA in vivo, and may be a promising antioxidant for skin care and treatment of diseases associated with oxidative stress.

Bioavailability of a series of novel acylated ascorbic acid derivatives, 6-O-acyl-2-O-alpha-D-glucopyranosyl-L-ascorbic acids, as an ascorbic acid supplement in rats and guinea pigs.

The bioavailability of a series of novel acylated ascorbic acid derivatives, 6-O-acyl-2-O-alpha-D-glucopyranosyl-L-ascorbic acids (6-Acyl-AA-2G), as an ascorbic acid (AA) supplement was investigated in rats and guinea pigs. Oral administration of 6-Acyl-AA-2G to rats resulted in an increase in the plasma AA level. However, the intact form was not detectable in the plasma by high-performance liquid chromatography, indicating its hydrolysis through the process of absorption. After an intravenous injection to rats of 6-Octa-AA-2G as a representative derivative, the intact form rapidly disappeared from the plasma, being followed by a prolonged and marked elevation of the plasma AA level. Various tissue homogenates from guinea pigs were examined for their releasing activity of AA, 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G) and 6-O-acyl-AA from 6-Acyl-AA-2G. High activity was observed in the small intestine. These hydrolytic activities to AA and 6-O-acyl-AA were completely inhibited by castanospermine, an alpha-glucosidase inhibitor, and AA-2G was observed as the only resulting hydrolysate, suggesting the participation of alpha-glucosidase and esterase in the in vivo hydrolysis of 6-Acyl-AA-2G. 6-Octa-AA-2G was found to exhibit an obvious therapeutic effect in scorbutic guinea pigs from its repeated oral administration. These results indicate that 6-Acyl-AA-2G is a readily available source of AA activity in vivo, and may be useful as an effective pharmacological agent and as a promising food additive.

Vitamin C Deficiency in Guinea Pigs Differentially Affects the Expression of Type IV Collagen, Laminin, and Elastin in Blood Vessels

Vitamin C deficiency causes morphologic changes in the endothelial and smooth muscle compartments of guinea pig blood vessels. Endothelial cells synthesize the basement membrane components, type IV collagen and laminin, and smooth muscle cells synthesize elastin in blood vessels. Therefore, we examined the possibility that vitamin C deficiency affects the expression of these proteins. Decreased expression of types I and II collagens in other tissues of vitamin C–deficient guinea pigs is associated with weight loss and the consequent induction of insulin-like growth factor binding proteins; thus we also used food deprivation to induce weight loss. Female guinea pigs received a vitamin C–free diet, supplemented orally with ascorbate. Vitamin C–deficient guinea pigs received the same diet but no ascorbate, and the food-deprived group received no food, but were supplemented with vitamin C. Concentrations of mRNAs for basement membrane components and elastin in blood vessels were measured by Northern blotting; overall basement membrane metabolism was assessed by measuring immunoreactive laminin and type IV 7S collagen in serum. Laminin mRNA in blood vessels and serum laminin concentrations were unaffected by vitamin C deficiency. Concentrations of type IV collagen and elastin mRNAs in blood vessels were not significantly affected in moderately scorbutic guinea pigs (0–7% weight loss), but with increased weight loss, type IV collagen mRNA was 57% (P < 0.05) and elastin mRNA was 3% (P < 0.01) of normal values. In food-deprived guinea pigs, type IV collagen mRNA was 51% (P < 0.05) and elastin mRNA was 35% (P < 0.05) of normal. Serum type IV 7S collagen concentrations were 25% of normal in scorbutic guinea pigs with extensive weight loss. The lower expression of type IV collagen and elastin mRNAs in blood vessels may contribute to defects observed in blood vessels during scurvy.

Ascorbic Acid Is a Stimulatory Cofactor for Mitochondrial Glycerol-3-Phosphate Dehydrogenase

Mitochondrial glycerol 3-phosphate dehydrogenase (mGPDH) from control and scorbutic guinea pig brain, liver and skeletal muscle and from normal rat liver was stimulated several fold byl-ascorbic acid (AA). The amount of AA that gave half-maximal stimulation of guinea pig brain mGPDH was 7.1 μM. At concentrations of AA higher than 500 μM, mGPDH activity decreased to nearly the same activity as in the absence of AA. D-Ascorbic acid, erythorbic acid, was equally potent in the activation of washed mitochondrial mGPDH activity. The AA activation of mGPDH was completely inhibited by 50 μM EGTA, but could be fully restored by the sequential addition of 100 μM Ca2+. The AA activation of mGPDH was likewise completely inhibited by iron specific chelators, bathophenanthrolinedisulfonic acid, desferrioxamine, and 1,10-phenanthroline, but the activation could not be restored by the addition of excess Ca2+. In the absence of AA, mGPDH activity was not inhibited by either EGTA or the iron chelators and Ca2+addition had no effect on the activity. The iron-sulfur protein, succinic dehydrogenase (complex II), was not significantly different in brain mitochondria from control or scorbutic guinea pigs, and was not activated by the subsequent addition of AA. In the presence of AA, succinic dehydrogenase activity was not affected by either bathophenanthrolinedisulfonic acid or EGTA. The results suggest that mGPDH is a probable site of action of AA in the related glucose-coupled insulin release from pancreatic islets.

Studies on the Essential Role of Ascorbic Acid in the Energy Dependent Release of Insulin from Pancreatic Islets

Pancreatic islets from young normal and scorbutic male guinea pigs were examined for their ability to release insulin when stimulated with depolarizing levels of KCl (45 mM) and by 20 mMd-glyceraldehyde. Islets from normal guinea pigs released insulin in a K+andd-glyceraldehyde dependent manner showing a rapid initial secretion phase followed by secondary waves of insulin release during a 120 min period. Islets from scorbutic guinea pigs were able to respond to elevated K+in a manner identical to that of the control islets. In contrast, insulin release from ascorbic acid deficient islets in response to the secretagogue,d-glyceraldehyde, was significantly delayed and decreased responses were observed during the 120 min period afterd-glyceraldehyde stimulation. The results are consistent with the site of action of ascorbic acid on energy-dependent insulin release lying between the triose-phosphate level of glycolysis and the generation of ATP by oxidative phosphorylation.

Scurvy results in decreased collagen synthesis and bone density in the guinea pig animal model

The effect of severe ascorbic acid deficiency on bone remodeling and collagen synthesis was evaluated in a 21 day experiment, using the scorbutic guinea pig model. Animals (n = 6-7/group) were assigned to one of three groups: scorbutic, pair-fed ascorbic acid-replete, or ad libitum ascorbic acid-replete groups. After 2 weeks, scorbutic animals started voluntarily decreasing food intake and losing weight. By day 19-21, at which time bone and tissue samples were collected and analyzed, scorbutic animals decreased food intake to 46% of usual and lost 9% body weight. Serum 25OHD3, 1,25(OH)2D3, calcium, and albumin were significantly lower (p < 0.05) in the scorbutic animals than in the other groups. Bone mineral density and bone mineral content of the proximal and central femur were significantly lower in the scorbutic group than in the other groups (p < 0.05). Morphometric analysis of tibia indicated significantly lower bone volume, fewer and thinner trabeculae, and a thinner growth plate in the scorbutic group, compared to the pair-fed and ad libitum groups (p < 0.05). Osteoclast surface was about 60% higher in the scorbutic group than in the pair-fed and ad libitum control groups (0.05 < p < 0.10). Mechanical strength of the femur and lumbar vertebral body tended to be lower when bone mass was altered in the same group. Collagen synthesis of articular cartilage and tendons was lower in the scorbutic group than in the pair-fed or ad libitum groups (p < 0.05). In conclusion, scurvy but not food restriction, per se, results in alterations in bone mass and tissue collagen synthesis.

Ascorbic acid is essential for the release of insulin from scorbutic guinea pig pancreatic islets.

Pancreatic islets from young normal and scorbutic male guinea pigs were examined for their ability to release insulin when stimulated with elevated D-glucose. Islets from normal guinea pigs released insulin in a D-glucose-dependent manner showing a rapid initial secretion phase and three secondary secretion waves during a 120-min period. Islets from scorbutic guinea pigs failed to release insulin during the immediate period, and only delayed and decreased responses were observed over the 40-60 min after D-glucose elevation. Insulin release from scorbutic islets was greatly elevated if 5 mM L-ascorbic acid 2-phosphate was supplemented in the perifusion medium during the last 60 min of perifusion. When 5 mM L-ascorbic acid 2-phosphate was added to the perifusion medium concurrently with elevation of medium D-glucose, islets from scorbutic guinea pigs released insulin as rapidly as control guinea pig islets and to a somewhat greater extent. L-Ascorbic acid 2-phosphate without elevated D-glucose had no effect on insulin release by islets from normal or scorbutic guinea pigs. The pancreas from scorbutic guinea pigs contained 2.4 times more insulin than that from control guinea pigs, suggesting that the decreased insulin release from the scorbutic islets was not due to decreased insulin synthesis but due to abnormal insulin secretion.

The ability of guinea pigs to synthesize carnitine at a normal rate from ϵ- N-trimethyllysine or γ-butyrobetaine in vivo is not compromised by experimental vitamin C deficiency

Experimental vitamin C deficiency in guinea pigs is associated with low carnitine concentrations in blood and some tissues. Ascorbic acid is a cofactor for two enzymes in the pathway of carnitine biosynthesis. The effect of experimental vitamin C deficiency on the ability of guinea pigs to synthesize carnitine was investigated in animals fed a vitamin C-deficient diet for 28 days. On days 19 to 28, supplements (0.5 mmol · kg body weight −1 · d −) of the carnitine precursors ϵ- N-trimethyllysine or γ-butyrobetaine were administered orally. Ascorbate-supplemented, ascorbate-deficient, and pair-fed (to ascorbate-deficient) animals showed an increase in the rate of carnitine biosynthesis (as estimated from measured rates of carnitine excretion) of 32 to 40 μmol · kg body weight −1 · d −1 following supplementation with ϵ- N-trimethyllysine. Likewise, animals in each experimental group showed an increase in the rate of carnitine biosynthesis of 41 to 50 μmol · kg body weight −1 · d −1 after supplementation with γ-butyrobetaine. These results indicate that scorbutic guinea pigs are able to synthesize carnitine at a normal or above-normal rate. For guinea pigs not given a carnitine precursor supplement, rates of free and total carnitine excretion for ascorbate-deficient (but not pair-fed) animals were threefold higher than for ascorbate-supplemented animals during days 19 to 28 of the feeding regimen. Thus, carnitine depletion in vitamin C deficiency likely is due to excessive urinary excretion of carnitine and not to a decreased rate of carnitine biosynthesis.

Differential regulation of collagen gene expression in granulation tissue and nonrepair connective tissues in vitamin C‐deficient guinea pigs

Poor wound healing during vitamin C deficiency is thought to be due to decreased hydroxylation of proline residues in collagen. In non-repair connective tissues of guinea pigs, however, procollagen gene expression is not decreased until weight loss occurs during the third and fourth weeks of scurvy (phase II) with only a moderate decrease in proline hydroxylation. Decreased procollagen gene expression is related to the induction of insulin-like growth factor binding proteins 1 and 2 that inhibit insulin-like growth factor-I action. We examined wound healing and granulation tissue formation during phase I of vitamin C deficiency. Synthetic sponges were implanted on day 7 of vitamin C deficiency and analyzed at 6 and 10 days after surgery, when there was no weight loss or induction of insulin-like growth factor binding proteins. Healing of incisions was almost complete at 10 days after surgery in normal controls but not in scorbutic animals. The area around the incision and implant exhibited excessive angiogenesis and hemorrhaging of vessels in the scorbutic animals at 6 and 10 days after surgery. At 10 days after surgery, collagen synthesis in the implants of scorbutic guinea pigs was 36% lower than control values, with a normal extent of proline hydroxylation. Concentrations of messenger RNAs for types I and III procollagens were slightly increased by scurvy at 6 days after surgery but were decreased by 26% and 40%, respectively, at 10 days. Fibronectin mRNA levels were unaffected by scurvy at both time points. Our results suggest that poor wound healing in phase I of scurvy may be related to defective interstitial procollagen gene expression and defective blood vessel formation, but it does not involve inhibition of proline hydroxylation or induction of insulin-like growth factor binding proteins. mRNA for insulin-like growth factor-II, transforming growth factor-beta(1), and transforming growth factor-beta(2) were significantly expressed in implants, but their patterns of expression did not correlate with changes in procollagen gene expression.

Evidence for an in vivo role of insulin-like growth factor-binding protein-1 and -2 as inhibitors of collagen gene expression in vitamin C- deficient and fasted guinea pigs

Acutely scorbutic and fasted (vitamin C-supplemented) guinea pigs exhibit decreased collagen gene expression associated with weight loss. We recently demonstrated that circulating insulin-like growth factor-binding protein-1 and -2 (IGFBP-1 and -2) are induced in these deficiencies, and that removal of IGFBP-1 and -2 from serum of such animals by specific antibodies reverses inhibition of cellular IGF-I-dependent functions, including collagen and DNA synthesis. Here we investigated the kinetics of induction of IGFBP-1 and -2 relative to suppression of collagen gene expression. Guinea pigs were fasted for 10-96 h, with 3-24% weight loss, or received an ascorbate-free diet for up to 4 weeks, with 5-28% weight loss during the third and fourth weeks (phase II of scurvy). In both deficiencies, there was noncoordinate regulation of collagen mRNA expression in tissues. Type I collagen mRNA concentrations in skin decreased rapidly after 5-10% weight loss and reached about 10% of normal levels, whereas in bone, there was a later, and not as extensive, decrease. The concentration of cartilage type II collagen mRNA decreased rapidly initially, but then remained at 40-50% of normal. Circulating IGF-I concentrations remained normal during the period when collagen gene expression was initially suppressed, although there was a later decrease. In contrast, mRNAs for IGFBP-1 and -2 and the circulating proteins were induced before or concomitantly with the suppression of collagen gene expression. The ability of fasted or scorbutic guinea pig sera to inhibit IGF-I action in cells increased in parallel with IGFBP activity ([125I]IGF-I binding), which, in turn, mainly reflected the concentration of IGFBP-1 in sera. Serum insulin may be the primary regulator of the IGFBPs. Its levels were decreased to 10-13% of normal when weight loss commenced, whereas cortisol levels, although increased, did not correlate with the induction of IGFBPs. The overall results taken together with our recent findings from cell culture experiments are compatible with circulating IGFBP-1 and -2 acting as inhibitors of collagen gene expression by blocking IGF-I action during fasting and phase II of vitamin C deficiency.

Evidence for an in vivo role of insulin-like growth factor-binding protein-1 and -2 as inhibitors of collagen gene expression in vitamin C-deficient and fasted guinea pigs.

Acutely scorbutic and fasted (vitamin C-supplemented) guinea pigs exhibit decreased collagen gene expression associated with weight loss. We recently demonstrated that circulating insulin-like growth factor-binding protein-1 and -2 (IGFBP-1 and -2) are induced in these deficiencies, and that removal of IGFBP-1 and -2 from serum of such animals by specific antibodies reverses inhibition of cellular IGF-I-dependent functions, including collagen and DNA synthesis. Here we investigated the kinetics of induction of IGFBP-1 and -2 relative to suppression of collagen gene expression. Guinea pigs were fasted for 10-96 h, with 3-24% weight loss, or received an ascorbate-free diet for up to 4 weeks, with 5-28% weight loss during the third and fourth weeks (phase II of scurvy). In both deficiencies, there was noncoordinate regulation of collagen mRNA expression in tissues. Type I collagen mRNA concentrations in skin decreased rapidly after 5-10% weight loss and reached about 10% of normal levels, whereas in bone, there was a later, and not as extensive, decrease. The concentration of cartilage type II collagen mRNA decreased rapidly initially, but then remained at 40-50% of normal. Circulating IGF-I concentrations remained normal during the period when collagen gene expression was initially suppressed, although there was a later decrease. In contrast, mRNAs for IGFBP-1 and -2 and the circulating proteins were induced before or concomitantly with the suppression of collagen gene expression. The ability of fasted or scorbutic guinea pig sera to inhibit IGF-I action in cells increased in parallel with IGFBP activity ([125I]IGF-I binding), which, in turn, mainly reflected the concentration of IGFBP-1 in sera. Serum insulin may be the primary regulator of the IGFBPs. Its levels were decreased to 10-13% of normal when weight loss commenced, whereas cortisol levels, although increased, did not correlate with the induction of IGFBPs. The overall results taken together with our recent findings from cell culture experiments are compatible with circulating IGFBP-1 and -2 acting as inhibitors of collagen gene expression by blocking IGF-I action during fasting and phase II of vitamin C deficiency.

Circulating insulin-like growth factor binding proteins (IGFBPs) 1 and 2 induced in vitamin C-deficient or fasted guinea pigs inhibit IGF-I action in cultured cells.

Collagen gene expression and proteoglycan synthesis are decreased in vitamin C-deficient guinea pigs losing weight and in fasted guinea pigs receiving ascorbate. Sera from such guinea pigs contain an insulin-like growth factor (IGF)-I-reversible inhibitor of collagen, proteoglycan and DNA synthesis and elevated levels of 29 and 35-kDa IGF binding proteins (IGFBPs). We now have identified the induced proteins as IGFBPs 1 and 2 and investigated their role as inhibitors. Guinea pig sera were treated with antibodies to IGFBPs 1 and 2 and antibody-IGFBP complexes were removed by passage through a Protein A-Sepharose column. Inhibitor content of fasted and scorbutic sera, and Protein A pass-through fractions derived from them, was assessed by their level of stimulation of DNA and collagen synthesis in 3T3 cells, compared to analogously treated normal guinea pig serum. Removal of IGFBP-1 from scorbutic serum reversed inhibition of collagen and DNA synthesis by more than half but removal of IGFBP-2 was less effective. Removal of both IGFBPs reversed inhibition almost completely. Similar results were obtained with fasted guinea pig serum. Conversely, purified rat IGFBPs 1 and 2 inhibited DNA and collagen synthesis in cells cultured in normal guinea pig serum or IGF-I-stimulated DNA synthesis, with IGFBP-1 being more potent. Thus, IGFBP-1 and, to a lesser extent IGFBP-2, cause inhibition of IGF-I action by sera from fasted and scorbutic guinea pigs and may inhibit collagen gene expression in vivo.

Liver and serum butyrylcholinesterase activity in scorbutic guinea pigs

The present study reports the effects of an ascorbic acid-deficient diet on liver and plasma butyrylcholinesterase activity. The activity of this enzyme decreased significantly in the plasma and liver subcellular fractions. The levels of plasma corticosterone were also significantly elevated in scurvy. The results are discussed in light of these observations.

Reduced bactericidal activity in neutrophils from scorbutic animals and the effect of ascorbic acid on these target bacteria in vivo and in vitro

Actinomycetes, involved in oral and periodontal diseases, cause serious infections in immunocompromised hosts. Severely scorbutic guinea pig leukocytes killed only 12% of phagocytosed actinomycetes, had distorted nuclear morphology, had 16 times less ascorbate, and had no chemotactic responses in vitro. Ascorbate reversed these indices and also prevented nitrosamine formation by oral organisms. Degranulating leukocytes release lactoferrin and ascorbate that chelate iron, essential for microorganisms. Ascorbic acid, 2,2'-bipyridine and 1,10-phenanthroline were bactericidal to several bacterial pathogens at millimolar concentrations. Iron alone reversed this effect. In in vivo experiments an Actinomyces viscosus monoflora was implanted in rhesus monkeys. Plaque and serum samples showed decreased (by six orders of magnitude) bacterial counts and decreased actinomycete antibody titers in animals given 1 g ascorbate/d. Removing ascorbate returned counts and titers to preascorbate concentrations. Fifteen marmosets, receiving twice daily topical applications of ascorbate or water, had comparatively lower gingival, calculus, and plaque indices and only slightly lowered actinomycete counts.

Ascorbic acid and carnitine biosynthesis

It has been suggested that early features of scurvy (fatigue and weakness) may be attributed to carnitine deficiency. Ascorbate is a cofactor for two alpha-ketoglutarate-requiring dioxygenase reactions (epsilon-N-trimethyllysine hydroxylase and gamma-butyrobetaine hydroxylase) in the pathway of carnitine biosynthesis. Carnitine concentrations are variably low in some tissues of scorbutic guinea pigs. Ascorbic acid deficiency in guinea pigs resulted in decreased activity of hepatic gamma-butyrobetaine hydroxylase and renal but not hepatic epsilon-N-trimethyllsine hydroxylase when exogenous substrates were provided. It remains unclear whether vitamin C deficiency has a significant impact on the overall rate of carnitine synthesis from endogenous substrates. Nevertheless, results of studies of enzyme preparations and perfused liver in vitro and of scorbutic guinea pigs in vivo provide compelling evidence for participation of ascorbic acid in carnitine biosynthesis.

Bio-elimination and organ retention profile of benzanthrone in scorbutic and non-scorbutic guinea pigs

The retention and bio-elimination of benzanthrone (BA) in scorbutic and non-scorbutic guinea pigs was investigated to understand the protective role of ascorbic acid. Oral intubation of 14C-BA to scorbutic and non-scorbutic guinea pigs showed a total recovery of around 91% radioactivity through urine, faeces and tissues. Recovery of radiolabelled BA through urine (28%) and faeces (22%) up to 96 hrs averaged 50%, whereas residual radioactivity in liver and testis experienced a recovery of 29% in scorbutic animals. In non-scorbutic animals there was an increased recovery of radioactivity through urine (37%) and faeces (31%) with a decrease in retention (10%) in liver and testis. These results suggest that ascorbic acid facilitates the mobilization and bio-elimination of BA and thereby can decrease the toxicity of the compound.