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.

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.

Expression of the mutant gene for L-gulono-gamma-lactone oxidase in scurvy-prone rats.

A mutant strain of Wistar rats with L-gulono-gamma-lactone oxidase deficiency has recently been established. To investigate this deficiency by DNA and RNA blot hybridization analyses, a fragment of a previously cloned cDNA encoding rat L-gulono-gamma-lactone oxidase was used as a probe. When genomic DNA of the mutant rat was digested with several restriction enzymes, the probe hybridized to fragments of the same sizes as those produced from DNA of normal rats. Poly(A)+RNA from the liver of the mutant rat was found to contain an L-gulono-gamma-lactone oxidase-specific mRNA of a normal size at a comparable level to that of normal rats. An in vitro translation experiment revealed that the mRNA programmed the synthesis of an enzyme protein which had the same molecular weight as that of the translational product of the normal mRNA, although the amount synthesized was markedly reduced as compared with that synthesized with the normal mRNA. In accordance with this observation, a very low but definite degree of L-gulono-gamma-lactone oxidase activity was detected in the microsomes of the mutant rat by a newly developed, highly sensitive method.

A protocol for the successful long-term enzyme replacement therapy of scurvy in guinea pigs.

Gulonolactone oxidase, a key enzyme in the biosynthesis of ascorbic acid, is missing from guinea pigs and certain other scurvy-prone species. Weekly intraperitoneal injections of glutaraldehyde cross-linked immunoprecipitates of this enzyme have been shown to provide guinea pigs with the capability of synthesizing their own ascorbic acid and of surviving without an exogenous source of this vitamin. This protocol, however, was successful in only a small percentage of the animals tested. The reasons for the limited therapeutic success were investigated. Apparently, the gulonolactone oxidase-treated guinea pigs fed without ascorbic acid were receiving insufficient nutrition. By supplementing these enzyme-treated animals with vitamins A, B, D and E and selenium, we successfully maintained a high proportion of guinea pigs fed without vitamin C.

Heterologous immunoprecipitates also have potential for therapeutic use.

Potential therapeutic usefulness of administered enzymes is limited by toxicity and allergenicity. To overcome these problems we are using scurvy to test various enzyme modifications that may be suitable for therapy. L-Gulonolactone oxidase, which catalyzes the final step in ascorbic acid biosynthesis, is immunoprecipitated with specific antisera from rabbits and then cross-linked with glutaraldehyde. The modified enzyme retains activity sufficient to elicit ascorbic acid synthesis in scorbutic guinea pigs. Intraperitoneal injection of this altered enzyme to animals supplemented with L-gulonolactone increases plasma concentrations of the vitamin. Importantly, multiple doses of the complex are tolerated. Therefore, it is possible to prolong survival time of animals fed an ascorbic acid-deficient diet by this enzyme replacement therapy. This procedure can also be applied to other enzymes that have potential therapeutic use. Serum cholinesterase and asparaginase both retain activity after this modification and are tolerated in single or in weekly repeated injections. Following three or four weekly injections, an anaphylactic reaction to serum but not to enzyme can be elicited if they are injected intravascularly. We conclude that the stability of the immobilized foreign enzyme is a critical factor in lessening the toxicity to multiple injections of these foreign proteins.

Hepatic uridine-diphosphate glucuronyl transferase activity of guinea pig with scurvy.

Ascorbic acid (AsA) concentrations in plasma exert a modulating effect on the activity of liver enzymes. Since UDP-glucuronyl transferase is a liver enzyme, which is responsible for bilirubin glucuronidation, the effect of varied amounts of AsA on this enzyme activity was studied. Sixty male guinea-pigs were randomly allocated to the following six groups: controls, scorbutic and groups given 2, 5, 10 or 20 mg of ascorbic acid, respectively. All the animals with the vitamin C deficient diet presented clinical signs of scurvy at the end of the experimental period, and had lost both body and liver weight compared to all other groups. Scorbutic animals had very low levels of AsA in the liver compared with controls (0.20 +/- 0.10 and 1.65 +/- 0.45 mg/g liver, respectively) (p less than 0.001). Liver AsA levels increased as the AsA dose increased. The UDP-glucuronyl transferase activity was lower in scorbutic animals than in controls (6.20 +/- 1.95 and 23.85 +/- 4.20 mg bilirubin/g protein/h, respectively) (p less than 0.001). The other groups C, D, E and F also had higher mean levels of UDP-GT activity than the scorbutic group B. Finally, no correlation was found between UDP-glucuronyl transferase activity and ascorbic acid intake.

Alterations in the activities of intestinal enzymes in vitamin-C-deficient guinea pigs.

The effect of vitamin C deficiency on various enzymes of the intestinal epithelium has been studied in guinea pigs. Brush border sucrase and alkaline phosphatase activities were considerably enhanced (p less than 0.001), but leucine aminopeptidase levels were reduced in scorbutic animals compared to the control group. There was essentially no change in the activity of maltase under these conditions. Kinetic studies with sucrase and alkaline phosphatase in control and scorbutic animals revealed that augmentation of the enzyme activities in scurvy is due to enhanced enzyme contents. Lactate dehydrogenase, succinate dehydrogenase, glucose-6-phosphatase and Mg+2 ATPase also exhibited reduced activities in the intestine of vitamin-C-deficient animals. Observed alterations in the activities of intestinal enzymes in scurvy were restored to control levels upon feeding of vitamin C to scorbutic guinea pigs.

Activation of prolyl hydroxylase in tissue homogenates of scorbutic guinea pigs.

Prolyl hydroxylase activity was measured in a variety of tissues from scorbutic and control guinea pigs. The activity of the enzyme was lower in skin, induced granuloma, aorta, arteries and lung of scorbutic animals. However, in liver and kidney the enzyme levels were the same or higher than in control animals. The low prolyl hydroxylase activity in the former tissues could be increased by incubating the homogenates with all the cofactors of prolyl hydroxylase such as ascorbate, ferrous ions and alpha-ketoglutarate, prior to the assay. An incubation time of 2-3 hours at 30 degrees was needed for optimal activation. The low enzyme activity in scorbutic animals was not due to the competition of underhydroxylated collagen present in the homogenate with the radioactive substrateused in the assay. Moreover, unlike in the control animals, the enzyme derived from granuloma of scorbutic animals was highly resistant to dithiothreitol inactivation. These data suggest that some tissues of scorbutic guinea pigs contain a prolyl hydroxylase which is an activatable form.

Immunologic evidence that the gene for L-gulono-gamma-lactone oxidase is not expressed in animals subject to scurvy.

L-Gulono-gamma-lactone oxidase (L-gulono-gamma-lactone:oxygen 2-oxidoreductase, EC 1.1.3.8) is the enzyme that catalyzes the terminal step of L-ascorbic acid biosynthesis in mammalian liver. The absence of the oxidase activity in primates and guinea pigs is the reason why these animals are subject to scurvy, which must be considered an inborn error of metabolism. Attempts were made to determine if a protein immunologically crossreactive with L-gulono-gamma-lactone oxidase is present in these animals. Detergent-solubilized microsomal preparations from guinea pig and African green monkey liver did not precipitate the antisera directed to either rat or goat enzyme, nor did any of the other cell fractions obtained from guinea pig liver react with either antiserum. No crossreactive protein was detectable in guinea pig microsomes even with the sensitive procedure or micro-complement fixation. On the other hand, extracts of all 10 other mammalian (4 orders) liver microsomes tested were shown to contain L-gulono-gamma-lactone oxidase activity that did crossreact with antibodies to the rat and goat enzymes. One explanation of these findings is that, in the guinea pig, and perhaps in primates too, the structural gene for L-gulono-gamma-lactone oxidase is not expressed.

Disruption of healed scars in scurvy -- the result of a disequilibrium in collagen metabolism.

Old scars break open in scorbutic patients because (1) the rate of collagen degradation is greater in an old scar than it is in normal skin, and (2) the rate of collagen synthesis is diminished throughout the body in ascorbate deficiency.