Scorbutic Animals Research Articles

The Role of Physiological Vitamin C Concentrations on Key Functions of Neutrophils Isolated from Healthy Individuals.

Vitamin C (ascorbate) is important for neutrophil function and immune health. Studies showing improved immune function have primarily used cells from scorbutic animals or from individuals with infectious conditions or immune cell disorders. Few studies have focused on the requirements of neutrophils from healthy adults. Therefore, we have investigated the role of vitamin C, at concentrations equivalent to those obtained in plasma from oral intakes (i.e., 50–200 µmol/L), on key functions of neutrophils isolated from healthy individuals. Cells were either pre-loaded with dehydroascorbic acid, which is rapidly reduced intracellularly to ascorbate, or the cells were activated in the presence of extracellular ascorbate. We measured the effects of enhanced ascorbate uptake on the essential functions of chemotaxis, oxidant production, programmed cell death and neutrophil extracellular trap (NET) formation. We found that neutrophils isolated from healthy individuals already had replete ascorbate status (0.35 nmol/106 cells), therefore they did not uptake additional ascorbate. However, they readily took up dehydroascorbic acid, thus significantly increasing their intracellular ascorbate concentrations, although this was found to have no additional effect on superoxide production or chemotaxis. Interestingly, extracellular ascorbate appeared to enhance directional mobilityin the presence of the chemoattractant formyl-methionyl-leucyl-phenylalanine (fMLP). Stimulation of the cells in the presence of ascorbate significantly increased intracellular ascorbate concentrations and, although this exhibited a non-significant increase in phosphatidylserine exposure, NET formation was significantly attenuated. Our findings demonstrate the ability of neutrophils to regulate their uptake of ascorbate from the plasma of healthy humans to maintain an optimal level within the cell for proper functioning. Higher oral intakes, however, may help reduce tissue damage and inflammatory pathologies associated with NET formation.

Regulation and Properties of Bone Alkaline Phosphatase during Vitamin C Deficiency in Guinea Pigs

The precise physiological role of alkaline phosphatase is unknown, although evidence suggests it is involved in bone mineralization. Previous studies showed that serum and bone alkaline phosphatase activity is decreased during vitamin C deficiency. Some effects of scurvy, such as inhibition of collagen synthesis, are related to weight loss and subsequent induction of insulin-like growth factor binding proteins and they can be duplicated in fasted guinea pigs receiving vitamin C. We found that decreased alkaline phosphatase activity in bone and serum during scurvy was not completely due to the “fasting effect” and that the decrease in serum was due to loss of bone isoenzyme activity. There also was a decrease in immunoreactive enzyme protein and alkaline phosphatase mRNA concentrations in bone of scorbutic animals, indicating that synthesis of the enzyme was inhibited. Sialylation and addition of the glycosylphosphatidylinositol anchor to the enzyme in bone tissue were not affected by scurvy. The concentration of mRNA for osteocalcin, a bone-specific marker, also fell during scurvy and to a much greater extent than either alkaline phosphatase or type I collagen mRNAs, while osteopontin mRNA concentrations increased. These results differ from the reported role of ascorbic acid on the pattern of expression of these proteins during differentiation of osteoblasts in culture. The decreased expression of collagen, alkaline phosphatase, and osteocalcin could explain the defects in bone caused by scurvy.

Growth and bone remodelling in a scorbutic rat model

This study used the scorbutic Osteogenic Disorder Shionogi rat model and a specific diet to reliably induce a state of sub-scurvy scorbutus. Under these conditions overall somatic growth was assessed, as well as that of the caudal vertebrae, as an example of scorbutic bone growth. Tail loops were then used to mechanically stress mature caudal vertebrae under scorbutic conditions, and the vertebrae's adaptation to these applied forces was assessed, using measurements of bend deformation and histologic analysis of osteogenesis. Scorbutic animals exhibited significant somatic growth retardation (p < 0.05), and abnormal reductions in osteogenesis and periosteal responsiveness to growth. Scorbutic vertebrae also showed greater bend angles of deformation (p < 0.05), and a marked reduction in cortical osseous remodelling and periosteal differentiation. It appeared that the sub-scurvy scorbutic bones were smaller, weaker and less able to adapt to physical stresses: this pattern was reflected at the histologic level.

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.

Gene Expression of Iron-Related Proteins during Iron Deficiency Caused by Scurvy in Guinea Pigs

The regulation of expression of hepatic iron-related proteins was examined during iron deficiency caused by scurvy in guinea pigs. Previous studies showed that some effects of scurvy, such as suppression of collagen gene expression, result from events associated with weight loss. During the initial phase of scurvy when vitamin C is depleted but animals grow normally, serum iron levels decreased to 50% of normal. During the second phase of scurvy when animals lose weight, there was a further decrease in iron levels to 10–15% of normal. Serum transferrin levels increased during scurvy, but this increase was related neither to the rate of weight loss nor to hepatic transferrin mRNA expression, which decreased. Serum ferritin levels diminished early in scurvy with a preferential loss of the L subunit. In liver, however, both ferritin subunits were almost undetectable even in scorbutic animals gaining weight. Ferritin gene expression during vitamin C deficiency was correlated with serum ferritin levels in that the level of mRNA for the H subunit remained relatively constant while that of the L subunit decreased early. Transferrin receptor mRNA expression in liver was induced as soon as iron levels decreased early in scurvy, which is similar to results reported for iron-depleted cultured cells. In contrast to results in cell culture, expression of iron regulatory protein 1 mRNA was decreased to approximately 50% of normal early in scurvy with a concomitant decrease in hepatic cytosolic aconitase activity. Our data indicate that iron deficiency occurs early during vitamin C deficiency and leads to changes in expression of iron-related proteins that differ in some aspects from regulation by iron in cell culture. Other events associated with weight loss in late scurvy may play a further role in this regulation.

Renal handling of carnitine in experimental vitamin C deficiency

Experimental vitamin C deficiency is associated with low carnitine concentrations in blood and some tissues, but is not due to a decreased ability of scorbutic animals to synthesize carnitine. The effect of experimental vitamin C deficiency on urinary carnitine excretion in vivo and carnitine transport into renal cortical brush-border membrane vesicles in vitro was investigated in guinea pigs fed normal and vitamin C-deficient diets for 24 days. Excretion of free and total carnitine was approximately fourfold greater in scorbutic animals as compared with normal guinea pigs during the last 6 days of the experimental regiment. The rate of carnitine transport into renal cortical brush-border membrane vesicles prepared from scorbutic animals was approximately 36% lower than the corresponding rate for vesicles prepared from normal animals. However, this effect was not specific, since rates of sodium gradient-dependent transport of glucose, lysine, and taurine (but not alanine) were also lower vesicles prepared from scorbutic animals, although the magnitude of the decrease was less than for carnitine. results are consistent with the hypothesis that carnitine depletion in vitamin C deficiency is due to decreased efficiency of carnitine reabsorption.

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.

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.

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.

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.

Effects of methamphetamine on central monoaminergic systems in normal and ascorbic acid-deficient guinea pigs

Repeated injections (s.c.) of methamphetamine (METH) were administered to normal and ascorbic acid-deficient (scorbutic) guinea pigs to assess a potential role for ascorbic acid in the METH-induced effects in central monoaminergic systems. The ascorbic acid-deficient condition differentially influenced the METH-induced responses of dopaminergic and serotonergic variables in the striatum: drug-induced changes in dopaminergic variables were identical in normal and scorbutic animals; METH-induced decreases in serotonergic variables [tryptophan hydroxylase activity, serotonin (5-HT) and 5-hydroxyindoleacetic acid concentrations], however, were prevented in scorbutic animals. The scorbutic condition did not alter significantly the distribution of METH in the brain, nor were striatal concentrations of dopamine (DA) or 5-HT affected. In vitro, ascorbic acid increased significantly DA-mediated [ 3H]5-HT release from striatal slices, thus suggesting a potential role for ascorbate in DA-mediated actions of METH on serotonergic systems. Although supplemental ascorbate failed to restore the METH-induced serotonergic effects in scorbutic guinea pigs, these data suggest that, in a normal animal, the effects of multiple injections of METH, on serotonergic systems, involve ascorbic acid.

Decreased extracellular matrix production in scurvy involves a humoral factor other than ascorbate

Our recent studies suggested that decreased collagen synthesis in bone and cartilage of scorbutic guinea pigs was not related to ascorbate-dependent proline hydroxylation. The decrease paralleled scurvy-induced weight loss and reduced proteoglycan synthesis. Those results led us to propose that the effects of ascorbate deficiency on extracellular matrix synthesis were caused by changes in humoral factors similar to those that occur in fasting. Here we present evidence for this proposal. Exposure of chick embryo chondrocytes to scorbutic guinea pig serum, in the presence of ascorbate, led to effects on extracellular matrix synthesis similar to those seen in scorbutic animals. The rates of collagen and proteoglycan synthesis were reduced to approximately 30-50% of the levels in cells cultured in normal guinea pig serum plus ascorbate, but proline hydroxylation and procollagen secretion were unaffected. Similar results were obtained with serum from fasted guinea pigs supplemented in vivo with ascorbate. The growth rate of the chondrocytes was not significantly affected by scorbutic guinea pig serum.

The depletion effects of chlorpromazine, reserpine and ascorbic acid on tissue histamine of guinea-pigs

1. 1. 30 mg kg −1 chlorpromazine (CPZ) depleted more than half of the tissue histamine from lungs, stomach, ileum and skin of the normal guinea-pigs. However, the drug increased the tissue histamine content in scorbutic animals. 2. 2. In contrast, reserpine depleted histamine from the four tested tissues in both normal and scorbutic animals, except those in the lungs of the control animals. 3. 3. Ascorbic acid only depleted histamine from the stomach and ileum. 4. 4. A 24 hr period was the time limit for CPZ to deplete the histamine in all the four tested tissues. 5. 5. Histamine partially or completely recovered in the tissues after the next 24 hr.

Effect of Ascorbic Acid on Guinea Pig Adrenal Adenylate Cyclase Activity and Plasma Cortisol

Intra-adrenal ascorbic acid (AsA) concentrations exert a braking or modulating effect upon steroid release. Because changes in steroidogenesis are mediated through alterations in adenylate cyclase activity (ACL), the effect of varied AsA plasma concentrations on guinea pig adrenal ACL activity and plasma cortisol was studied. Forty-two male guinea pigs were randomly allocated to the following seven groups: controls, scorbutic, and groups given 0.1, 5, 10, 20 or 100 mg ascorbic acid/100 g body weight, respectively. Scorbutic animals had very low levels of AsA in comparison to control animals. Plasma AsA levels increased as AsA dose increased. The levels of AsA in the group given 0.1 mg AsA were higher than in controls. Basal adenylate cyclase activity did not vary significantly among animal groups. In contrast, values for NaF-stimulated ACL activity showed a progressive decrease with increasing AsA doses. A highly significant correlation was found between decreasing ACL activity and increasing plasma AsA concentrations. On the other hand, NaF-responsive ACL activity was higher in scorbutic animals than in any other group. Higher mean cortisol values were found in the scorbutic group than in the controls, correlating with high levels of NAF-stimulated ACL activity. Higher mean cortisol values were also found in the group given 0.1 mg AsA although ACL activity in this group was not affected. This finding, coupled with reduced ACL activity in these groups, is consistent with the inhibitory effect of a megadose of AsA on production of cortisol from the adrenal. The above data may suggest that differing plasma concentrations of AsA regulate in vivo steroidogenesis by altering the activity of the membrane-bound enzyme adenylate cyclase.

The fate of intravenously administered hepatic ferritin in normal, phenylhydrazine-treated and scorbutic guinea-pigs.

When highly purified hepatic 59Fe-ferritin was injected intravenously into normal guinea-pigs more than half of it was taken up by red cell precursors and the iron was used for haem formation. This was studied in more detail in animals in which a reticulocytosis had been induced either by phenylhydrazine or by repeated venescetions. 55% of the injected ferritin iron was found in reticulocytes at 1 h. Experiments using ferritin doubly labelled with 59Fe and 125I indicated that the whole molecule was taken up, with two-thirds of the radioactivity being associated with the membrane at 1 h and one third being already within the cell. There was a progressive loss of 125I activity over the ensuing hours, while most of the 59Fe was slowly internalized and incorporated into haem between 1 and 24 h. In contrast, 90% of the activity taken up by red cell precursors from 59Fe-transferrin was present as haem at all times. The liver and spleen were the two other major sites of 59Fe-ferritin uptake in phenylhydrazine treated animals. While there was an early uptake of 59Fe into haem in these organs, some redistribution occurred with time, since most of the 59Fe was in a non-haem fraction by 24 h. In a final experiment the distribution and fate of 59Fe-ferritin was studied in scorbutic animals treated with phenylhydrazine. The findings were similar to those in normals similarly treated, which suggests that ferritin iron was being effectively mobilized for haem formation despite the ascorbic acid depletion.

Ascorbic acid-deficient condition alters central effects of methamphetamine

Effects of multiple doses of methamphetamine (METH) were examined in normal and ascorbic acid-deficient (scorbutic) guinea pigs. METH-induced decreases of striatal serotonin concentrations were completely antagonized in scorbutic animals. Elevations of nigral substance P-like immunoreactivity also differed significantly in METH-treated scorbutic compared to METH-treated normal animals. Various lines of evidence indicate that dopamine is an essential mediator of METH-induced effects in both serotonergic and substance P systems in the brain areas examined; however, results from the present study indicate that, along with dopamine, ascorbic acid also plays a role in mediating the effects of METH in the central nervous system.

Regulation of collagen synthesis and mRNA levels in articular cartilage of scorbutic guinea pigs

Previous studies suggested that decreased type I collagen synthesis in calvaria of ascorbate-deficient guinea pigs was correlated with weight loss rather than defective proline hydroxylation. The generality of this correlation was examined in articular cartilage, which synthesizes mainly type II collagen, by measuring collagen synthesis and proline hydroxylation in vitro in tissue from ascorbate-supplemented and scorbutic guinea pigs. Ascorbate concentrations in tissues were almost completely depleted after 1 week of deficiency, but proline hydroxylation remained normal until after approximately 3 weeks, when it had decreased only by 10%. At that point collagen synthesis had decreased to about 50% of the control value. There was little additional effect on proline hydroxylation but collagen synthesis decreased further to 20% of normal. Procollagen mRNA levels in cartilage, as measured by dot-blot hybridization with a type II-specific cDNA probe, were unchanged after 2 weeks of scurvy, which correlated with the lack of effect on collagen synthesis during that period. Thereafter, during the period when collagen synthesis decreased, procollagen mRNA levels decreased to 20% of control values. Refeeding ascorbate to acutely scorbutic animals led to reversal of defective proline hydroxylation within 24 h with a slower increase in collagen synthesis and mRNA levels. Collagen synthesis returned to the normal level after 4 days with no further increase, while mRNA levels continued to increase to 2.7 times the control values after 7 days. Thus the major mechanism for regulation of collagen synthesis in articular cartilage during scurvy and ascorbate repletion occurs independently of the effect on proline hydroxylation and is associated with changes in mRNA levels. The lack of precise coordination between collagen synthesis and mRNA levels during repletion, however, suggests that there may be additional regulation through post-transcriptional mechanisms.

Carrageenan colitis in the guinea pig: pathological changes and the importance of ascorbic acid deficiency in disease induction.

In guinea pigs deprived of ascorbic acid, the oral administration of degraded E. spinosum carrageenan induced mild to moderate colitis, while E. cottonii consistently induced severe colitis. A variable degree of colitis was observed in scorbutic animals not receiving carrageenan. By contrast, in animals given large daily supplements of ascorbic acid, neither type of carrageenan consistently induced colitis and severe disease was rarely observed. The severe colitis induced by E. cottonii in scorbutic animals markedly affected the mid and distal colon and showed histological changes similar to human ulcerative colitis.

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) does not destroy nigrostriatal neurons in the scorbutic guinea pig

Guinea pigs were injected subcutaneously with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in maximal tolerated doses (8 mg/kg, once daily) for 10 or 15 days. No neurological effects were noted, other than sedation and hypotonia lasting a few hours after each injection, either in animals maintained on normal diet or in animals fed an ascorbate-deficient diet and rendered severely scorbutic. Subsequent chemical analyses of the striatum showed no evidence of lasting damage to nigrostriatal dopaminergic neurons in MPTPtreated guinea pigs on normal diet, and minimal evidence of permanent damage to these neurons in scorbutic animals. MPTP was undetectable in the urine of MPTP-treated animals, although a metabolite, presumably 1-methyl-4-phenylpyridinium ion (MPP +) was regularly present in urine. The relative lack of neurotoxicity of MPTP in the guinea pig remains unexplained. This species clearly is not a suitable small animal for MPTP-induced parkinsonism.

β-Hydroxyaspartic acid in vitamin K-dependent plasma proteins from scorbutic and warfarin-treated guinea pigs

β-Hydroxyaspartic acid is a rare amino acid, present in all vitamin K-dependent plasma proteins except prothrombin, and is formed by a post-translational hydroxylation of aspartic acid. We have now investigated whether this hydroxylation, like that of proline in collagen, is vitamin C-dependent. The vitamin K-dependent plasma proteins were isolated from normal and scorbutic guinea pig plasma by barium citrate adsorption and the β-hydroxyaspartic acid content was determined. Compared with normal animals, scorbutic animals showed no significant reduction of β-hydroxyaspartic acid content. In warfarin-treated animals there was a decreased content of both β-hydroxyaspartic acid and γ-carboxyglutamic acid in the barium citrate adsorbed fraction. It was concluded that the post-translational hydroxylation of aspartic acid is unlikely to be vitamin C-dependent.