Endoplasmic reticulum thiol oxidase deficiency leads to ascorbic acid depletion and noncanonical scurvy in mice.

Endoplasmic reticulum (ER) thiol oxidases initiate a disulfide relay to oxidatively fold secreted proteins. We found that combined loss-of-function mutations in genes encoding the ER thiol oxidases ERO1α, ERO1ß, and PRDX4 compromised the extracellular matrix in mice and interfered with the intracellular maturation of procollagen. These severe abnormalities were associated with an unexpectedly modest delay in disulfide bond formation in secreted proteins but a profound, 5-fold lower procollagen 4-hydroxyproline content and enhanced cysteinyl sulfenic acid modification of ER proteins. Tissue ascorbic acid content was lower in mutant mice, and ascorbic acid supplementation improved procollagen maturation and lowered sulfenic acid content in vivo. In vitro, the presence of a sulfenic acid donor accelerated the oxidative inactivation of ascorbate by an H(2)O(2)-generating system. Compromised ER disulfide relay thus exposes protein thiols to competing oxidation to sulfenic acid, resulting in depletion of ascorbic acid, impaired procollagen proline 4-hydroxylation, and a noncanonical form of scurvy.

Ascorbic acid deficiency decreases hepatic cytochrome P-450, especially CYP2B1/2B2, and simultaneously induces heme oxygenase-1 gene expression in scurvy-prone ODS rats.

The mechanisms underlying the decrease in hepatic cytochrome P-450 (CYP) content in ascorbic acid deficiency was investigated in scurvy-prone ODS rats. First, male ODS rats were fed a diet containing sufficient ascorbic acid (control) or a diet without ascorbic acid (deficient) for 18 days, with or without the intraperitoneal injection of phenobarbital. Ascorbic acid deficiency decreased hepatic microsomal total CYP content, CYP2B1/2B2 protein, and mitochondrial cytochrome oxidase (COX) complex IV subunit I protein, and simultaneously increased heme oxygenase-1 protein in microsomes and mitochondria. Next, heme oxygenase-1 inducers, that is lipopolysaccharide and hemin, were administered to phenobaribital-treated ODS rats fed sufficient ascorbic acid. The administration of these inducers decreased hepatic microsomal total CYP content, CYP2B1/2B2 protein, and mitochondrial COX complex IV subunit I protein. These results suggested that the stimulation of hepatic heme oxygenase-1 expression by ascorbic acid deficiency caused the decrease in CYP content in liver.

Vitamin C Deficiency in the Young Brain-Findings from Experimental Animal Models.

Severe and long-term vitamin C deficiency can lead to fatal scurvy, which is fortunately considered rare today. However, a moderate state of vitamin C (vitC) deficiency (hypovitaminosis C)-defined as a plasma concentration below 23 µM-is estimated to affect up to 10% of the population in the Western world, albeit clinical hallmarks in addition to scurvy have not been linked to vitC deficiency. The brain maintains a high vitC content and uniquely high levels during deficiency, supporting vitC's importance in the brain. Actions include both antioxidant and co-factor functions, rendering vitamin C deficiency likely to affect several targets in the brain, and it could be particularly significant during development where a high cellular metabolism and an immature antioxidant system might increase sensitivity. However, investigations of a non-scorbutic state of vitC deficiency and effects on the developing young brain are scarce. This narrative review provides a comprehensive overview of the complex mechanisms that regulate vitC homeostasis in vivo and in the brain in particular. Functions of vitC in the brain and the potential consequences of deficiency during brain development are highlighted, based primarily on findings from experimental animal models. Perspectives for future investigations of vitC are outlined.

Ascorbic Acid Route to the Endoplasmic Reticulum: Function and Role in Disease.

Significance: Humans cannot synthesize ascorbic acid (AscH2) (vitamin C), so deficiencies in dietary AscH2 cause the life-threatening disease of scurvy and many other diseases. After oral ingestion, plasma AscH2 concentrations are strictly controlled by transporters, which are required for entry into the cell and into intracellular organelles. Recent Advances: Besides its general antioxidant function, AscH2 is a cofactor for endoplasmic reticulum (ER)-localized collagen hydroxylases. Its important role in ER homeostasis is also highlighted by the fact that AscH2 deficiency in auxotrophic species triggers ER stress. Critical Issues: Characterizations of the molecular basis of diseases suggest that intracellular AscH2 deficiency is due not only to limited dietary access but also to its limited intracellular transport and net loss under conditions of intracellular hyperoxidation in the ER. This essay will offer an overview of the different transporters of vitamin C regulating its intracellular concentration, its function inside the ER, and the phenotypes of the diseases that can be triggered by increased depletion of this vitamin in the ER. Future Directions: When considering the benefits of increasing dietary AscH2, it is important to consider pharmacokinetic differences in the bioavailability between orally and intravenously administered AscH2: the latter bypasses intestinal absorption and is, therefore, the only route that can lead to the high plasma concentrations that may provide some health effects, and it is this route that needs to be chosen in clinical trials for those diseases associated with a deficiency of AscH2. Antioxid. Redox Signal. 34, 845-855.

Scottish soldiers from the Battle of Dunbar 1650: A prosopographical approach to a skeletal assemblage.

After the Battle Dunbar between English and Scottish forces in 1650, captured Scottish soldiers were imprisoned in Durham and many hundreds died there within a few weeks. The partial skeletal remains of 28 of these men were discovered in 2013. Building on previous osteological work, here we report wide-ranging scientific studies of the remains to address the following questions: Did they have comparable diet, health and disease throughout their lives? Did they have common histories of movement (or lack of movement) during their childhoods? Can we create a collective biography of these men? Strontium and oxygen isotope analysis of tooth enamel investigated childhood movement. Carbon and nitrogen isotope analysis of incrementally sampled dentine addressed childhood diet and nutrition. Metaproteomic analysis of dental calculus investigated oral microbiomes and food residues; this was complemented by microscopic analysis of debris in calculus from ingested materials. Selected individuals were examined for dental microwear. The extent of hydroxylation of proline in collagen was examined as a potential biomarker for scurvy. An osteobiography for each man was created using the full range of data generated about him, and these were synthesised using an approach based on the historical method for a collective biography or prosopography. The childhood residences of the men were primarily within the Midland Valley of Scotland, though some spent parts of their childhood outside the British Isles. This is concordant with the known recruitment areas of the Scottish army in 1650. Their diets included oats, brassicas and milk but little seafood, as expected for lowland rather than highland diets of the period. Childhood periods of starvation or illness were almost ubiquitous, but not simultaneous, suggesting regionally variable food shortages in the 1620s and 1630s. It is likely there was widespread low-level scurvy, ameliorating in later years of life, which suggests historically unrecorded shortages of fruit and vegetables in the early 1640s. Almost all men were exposed to burnt plant matter, probably as inhaled soot, and this may relate to the high proportion of them with of sinusitis. Interpersonal violence causing skeletal trauma was rare. Based on commonalities in their osteobiographies, we argue that these men were drawn from the same stratum of society. This study is perhaps the most extensive to date of individuals from 17th century Scotland. Combined with a precise historical context it allows the lives of these men to be investigated and compared to the historical record with unprecedented precision. It illustrates the power of archaeological science methods to confirm, challenge and complement historical evidence.

Tocopherols, tocotrienols and tocomonoenols: Many similar molecules but only one vitamin E.

The aim of this article is to correct a very general error in scientific articles, in textbooks and in the Internet that has become an accepted fact. In this literature, the term "vitamin E″ is used for several similar molecules (both tocopherols and tocotrienols) that have never been shown to have vitamin property, i.e. a protective effect against the human deficiency disease. In fact, the name "vitamin E″ should only be used to define molecules that prevent the human deficiency disease "Ataxia with Vitamin E Deficiency" (AVED). Only one such molecule is known, α-tocopherol. This error may confuse consumers as well as medical doctors, who prescribe vitamin E without realizing that the current use of the name includes molecules of unknown, if not unwanted functions.

Specifically decreased collagen biosynthesis in scurvy dissociated from an effect on proline hydroxylation and correlated with body weight loss. In vitro studies in guinea pig calvarial bones.

The question whether ascorbate regulates collagen production solely through its direct role in proline hydroxylation was investigated. Proteins in calvarial bones from control and scorbutic weanling guinea pigs were labeled in short-term cultures with radioactive proline. Proteins were digested with purified bacterial collagenase to distinguish between effects on collagen polypeptide production and hydroxyproline formation. There was a preferential decrease in the absolute rate of collagen biosynthesis beginning after 2 wk of ascorbate deficiency, and this effect was temporally dissociated from decreased proline hydroxylation. There were no significant changes in the absolute rates of collagen degradation or noncollagen protein production. In vitro inhibition of proline hydroxylation in normal bone with alpha, alpha'-dipyridyl did not affect the relative rate of collagen synthesis, further dissociating these functions. Ascorbate added to scorbutic bone cultures reversed defective proline hydroxylation but not defective collagen synthesis, suggesting that the latter was an indirect effect of scurvy. There was a linear correlation between the extent of body weight lost during the 3rd and 4th wk of scurvy and the rate of collagen synthesis in scorbutic bone. This correlation also applied to control animals receiving ascorbate, but with weight loss induced by food restriction. These studies establish for the first time that ascorbate deficiency in guinea pigs leads to a specific decrease in collagen polypeptide synthesis and suggest that this decrease results from the reduced food intake and/or weight-loss characteristic of scurvy.

Expression of rat gene for L-gulono-gamma-lactone oxidase, the key enzyme of L-ascorbic acid biosynthesis, in guinea pig cells and in teleost fish rainbow trout (Oncorhynchus mykiss).

The ability of rainbow trout liver and kidney preparations to produce L-ascorbic acid with an added source of L-gulono-gamma-lactone oxidase (GLO) and the absence of their own GLO activity suggested that the reason for the absence of L-ascorbic acid biosynthesis in fish and in guinea pig, a scurvy-prone mammal, can be similar. Nevertheless, results of rat GLO cDNA expression in guinea pig cells and in rainbow trout proved different. In guinea pig cells, rat GLO was expressed in a functional form. Regardless of recombinant GLO transcripts detected in rainbow trout embryos, alevins and in juvenile fish, neither GLO protein nor GLO activity were found. Furthermore, production of L-ascorbic acid in transgenic rainbow trout was not revealed in feeding tests with vitamin C-free diets or after direct administration of L-gulono-gamma-lactone. These results indicate that conditions required for translation or stability of rat GLO are absent in rainbow trout tissues.

Impaired alpha-carboxyamidation of gastrin in vitamin C-deficient guinea pigs.

The biosynthetic activation of many hormonal peptides requires enzymatic alpha-carboxyamidation via glycine-extended intermediate forms. By specific radioimmunochemical measurement on extracts of guinea pig antra, we found that vitamin C deficiency induced a 30-fold increase in glycine-extended (i.e. inactive) gastrins. The results indicate that ascorbic acid is necessary for alpha-carboxyamidation in vivo, and thus, that the biosynthetic activation of hormonal peptides can be modified by simple dietary means.

Ascorbate requirement for hydroxylation and secretion of procollagen: relationship to inhibition of collagen synthesis in scurvy.

Vitamin C deficiency is associated with defective connective tissue, particularly in wound healing. Ascorbate is required for hydroxylation of proline residues in procollagen and hydroxyproline stabilizes the collagen triple helical structure. Consequently, ascorbate stimulates procollagen secretion. However, collagen synthesis in ascorbate-deficient guinea pigs is decreased with only moderate effects on proline hydroxylation. Proteoglycan synthesis, which does not require ascorbate, also is decreased and both effects are correlated with the extent of weight loss during scurvy. Fasting, with ascorbate supplementation, produces similar effects. Both functions are inhibited in cells cultured in sera from either scorbutic or starved guinea pigs and inhibition is reversed with insulin-like growth factor (IGF)-I. The inhibitor appears to consist of two IGF-binding proteins induced during vitamin C deficiency and starving and may be responsible for in vivo inhibition of collagen and proteoglycan synthesis.

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.

Aortic endothelial mitosis and Evans blue uptake in cholesterol-fed subscorbutic guinea-pigs.

SUMMARY: Groups of ovalbumin sensitised, subscorbutic guinea-pigs were subjected to various regimens for 8-10 weeks. Group 1 were given 0.25 mg/100 g body wt. cholesterol by mouth daily and were stressed with intraperitoneal injections of ovalbumin twice weekly. Group 2 received the cholesterol only and Group 3 were stressed only. Group 4 on the low vitamin C diet only acted as controls. Evans blue uptake tests and tritiated thymidine autoradiographs of Häutchen preparations of aortic endothelium were made on each animal. Endothelial mitosis was greatest in those areas of the aorta that had taken up the Evans blue most avidly. This was most apparent in Group 1, though to a lesser extent in Groups 2 and 3 also. Group 4 showed no dye uptake and the mitotic index was not raised. Blood ascorbic acid and plasma cholesterol levels are given for the various groups.

Reduced and oxidized ascorbates in guinea pig retina under normal and light-exposed conditions.

Both reduced and oxidized ascorbates were measured in aqueous, neural retina, and pigment epithelium-choroid complex (PE-C) of pigmented guinea pigs. Normal values for total ascorbate of 16 mg/dl in aqueous, 22 mg/dl in neural retina, and 7 mg/dl in PE-C were found. After mild photic damage caused by varying lengths of exposure of 10,000 to 20,000 lux of fluorescent lighting, reduced ascorbate concentrations generally decreased in the neural retina, while oxidized ascorbate generally increased in PE-C. In both normal and light-exposed retinas, reduced ascorbate was predominant in the neural retina, and oxidized ascorbate was predominant in the PE-C. Histochemical localization of reduced ascorbate occurred in the Müller cell fibers and at the apices of the retinal pigment epithelium.

Metabolism of ascorbic acid and ascorbic-2-sulfate in man and the subhuman primate.

Man does not catabolize ascorbate to CO2, whereas the monkey does catabolize ascorbate and ascorbate sulfate to CO2 when these compounds are given orally. However, it takes the same length of time to produce frank scurvy in both man and the monkey, thus indicating that the comparative storage, rate of use, and mode of metabolism of ascorbate is similar in both species. Preliminary feeding and isotope studies conducted on monkeys are in agreement with the fact that only a small amount of labeled ascorbate or ascorbate sulfate equilibrated with body stores. These results are in agreement with published ascorbic acid requirements of 10 mg/kg body weight. In our experiments, 250 mg/day had to be fed to a 10-kg monkey to completely clear all signs of scurvy and return blood ascorbate levels to normal. Ascorbic acid administered intravenously to monkeys appears to equilibrate completely with the ascorbate pool(s). Ascorbate sulfate was found to be a urinary metabolite of both ascorbic-1-14C acid and ascorbic-6-14C acid fed orally to monkeys.

Metabolism of L-ascorbic acid in the prostate of normal and scorbutic guinea pigs.

Guinea pig prostate was found to actively participate in the biosynthesis and catabolism of ascorbic acid. The key ascorbic acid biosynthetic enzyme L-gulono-gamma-lactone oxidase and the other two lactonases were found to be present in guinea pig prostate. The activities of dehydroascorbatase and diketogulonate decarboxylase, the enzymes for ascorbic acid degradation, were also detected in guinea pig prostate. Male guinea pigs kept under scorbutic condition for 7, 14, 21 and 28 days, were examined for prostatic metabolism of ascorbic acid. A significant but gradual decrease in the concentration of L-ascorbic acid was observed in prostate, total blood and leukocytes with the progression of scorbutic condition. There was an appreciable decrease in the rate of lipid peroxidation under the scorbutic condition. In the tissue fraction of scorbutic guinea pigs, the activities of biosynthesizing enzymes, measured in vitro, under optimum conditions were found to be higher with no significant alterations in the catabolizing enzymes. The implications of these findings are discussed in this paper.

Comparison of the anti-scorbutic activity of L-ascorbic acid and Ester C in the non-ascorbate synthesizing Osteogenic Disorder Shionogi (ODS) rat.

The Osteogenic Disorder Shionogi (ODS) rat, Clea Inc., Tokyo, Japan lacks the ability to synthesize L-ascorbic acid (AA). As with man, monkey and the guinea pig, this rat lacks L-gulonolactone oxidase necessary for the synthesis of AA from glucose. This study shows this animal to be an alternative to the guinea pig in AA studies. The anti-scorbutic potency of Ester C (EC), a calcium ascorbate and calcium threonate mixture, was compared with an AA dose of equal ascorbate activity equivalents (AAE) for anti-scorbutic activity in the ODS rat. The minimal anti-scorbutic dose of EC was determined to be 0.44 mg/kg/day (AAE), while an AA dose of 0.51 mg/kg/day (AAE) was not anti-scorbutic in a 24 day study. At 24 days EC rats gained 125% of initial body weight (BW) and the AA rats only 45% BW. Scorbutic signs at 24 days were scored on a 0 (min) to 3 (max) scale. The EC/AA ratio scores were: hemorrhage 0/1.4, behavior change 0/2.0, piloerection 0/2.2, mobility 0.4/2.2, dysbasia 0.6/2.8 and ataxia 0.4/1.0. Pearson's correlation coefficient for BW versus AAE was r = .34 for the AA group and r = .90 for the EC group. The morbidity index for EC was 0/5 and for the AA group 2/5. The AAE dose of AA which was 16% higher/day than the EC AAE dose was not anti-scorbutic, while the EC dose was anti-scorbutic. EC rats had 3.5X greater weight gain, a sensitive indicator of scurvy, than the AA rats. EC rats had 3-4 times less, if any, scorbutic signs than AA rats. The results clearly show that, based on ascorbate activity equivalents, EC has more available ascorbate activity/potency than AA. The mechanism of this increased potency is believed to be due to the facilitated transport of AAE into the cell by the threonate (a normal in vivo metabolite of AA) present in the EC product. In addition, previous studies have shown EC (AAE) to be higher in plasma and excreted less rapidly than the AAE derived from AA administered orally.