Deborah E. Kipp

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.

Iron overload alters iron-regulatory genes and proteins, down-regulates osteoblastic phenotype, and is associated with apoptosis in fetal rat calvaria cultures

Iron overload has been implicated in decreased bone mineral density. However, the effect of iron overload on osteoblast lineage cells remains poorly understood. The purpose of this study was to examine osteoblast differentiation, function, and apoptosis in iron-loaded cells from fetal rat calvaria. Cells were incubated with media supplemented with 0-10 microM ferrous sulfate (FeSO(4)) during differentiation (days 6-20). Intracellular iron status was assessed by measuring iron content in cell layers and changes in transferrin receptor (TrfR) and ferritin gene and protein expression. Osteoblast differentiation and function were evaluated by measuring osteoblast phenotypic gene markers and capacity of cultures to form mineralized bone nodules. Apoptotic hallmarks were evaluated by microscopy. A 2.3-fold increase in media iron concentration resulted in saturable accumulation of iron in the cell layer 20-fold higher than control (p<0.05) by mid-differentiation (day 15, D15). Iron accumulation resulted in rapid and sustained down-regulation of TrfR gene and protein levels (within 24 h) and up-regulation of light and heavy chain ferritin protein levels at late differentiation (day 20, D20). Concurrently, osteoblast phenotype gene markers were suppressed by D15 and a decreased number of mineralized nodules at D20 were observed. Apoptotic events were observed within 24 h of iron loading. These results provide evidence that iron overload alters iron metabolism and suppresses differentiation and function of cells in the osteoblast lineage associated with increased apoptosis.

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.

Plasma cortisol and adrenal ascorbic acid levels after ACTH treatment with a high intake of ascorbic acid in the guinea pig.

The influence of ascorbic acid (AA) intake on plasma cortisol (PC) and tissue AA levels after ACTH treatment was examined using guinea pigs. ACTH produced an 8- to 10-fold rise in PC levels (p less than 0.0001) over the 4-hour experimental period compared to gel-injected control animals. The magnitude of rise in PC was similar at both normal (0.50 g/kg diet) and high (10 g/kg diet) AA intakes. A 30-100% higher (p less than 0.0001) level of AA in tissues (adrenals, liver, and kidneys) and plasma was observed with the high AA diet. ACTH resulted in a 26-30% lower level of AA in the adrenals (p less than 0.0001), but not in other tissues or plasma. PC and adrenal AA responses were unrelated (r = -0.326). These results suggest that ACTH alters AA only in the adrenals, the PC response to and percent decrease in adrenal AA levels with ACTH are not related to AA status, and the absolute level of AA in the adrenals is not critical for steroidogenesis.

Iron chelator deferoxamine alters iron-regulatory genes and proteins and suppresses osteoblast phenotype in fetal rat calvaria cells.

There are few studies describing the extent to which low iron status affects osteoblastogenesis, despite evidence that iron deficiency produces adverse effects on bone density. The purpose of this study was to evaluate alterations in intracellular iron status by measuring iron-regulated gene and protein expression and to describe development of osteoblast phenotype in primary cells treated with iron chelator deferoxamine (DFOM) during differentiation. Using the well-described fetal rat calvaria model, cells were incubated with 0-8 microM DFOM throughout differentiation (confluence to day (D) 21), or only during early differentiation (confluence to D13-15) or late differentiation (D13-15 to D21). Changes in intracellular iron status were determined by measuring alterations in gene and protein expression of transferrin receptor and ferritin light chain and heavy chain. Development of osteoblast phenotype was monitored by measuring expression of genes that are known to be up-regulated during differentiation, analyzing the percentage of mineralized surface area, and counting the number of multi-layered bone nodules at the end of culture. Results indicate that treatment throughout differentiation with 8 microM DFOM alters iron-regulated genes and proteins by mid-differentiation (D13-15) in a pattern consistent with iron deficiency with concomitant down-regulation of osteoblast phenotype genes, especially osteocalcin. Additionally, alkaline phosphatase staining was lower and there was about 70% less mineralized surface area (p<0.05) by D21 in wells treated throughout differentiation with 8 microM DFOM compared to control. Down-regulation of osteocalcin and alkaline phosphatase mRNA (p<0.05) and suppressed mineralization (p<0.05) was also evident at D21 in cells treated only during early differentiation. In contrast, treatment during late differentiation did not alter osteoblastic outcomes by D21. In conclusion, it appears that iron is required for normal osteoblast phenotype development, and that early rather than late differentiation events may be more sensitive to iron availability.

Quercetin Metabolites Up-Regulate the Antioxidant Response in Osteoblasts Isolated From Fetal Rat Calvaria.

Oxidative stress contributes to osteoporosis by suppressing differentiation of osteoblasts, suggesting the osteoblast antioxidant response may be a viable strategy for osteoporosis prevention. Quercetin, an antioxidant flavonol, up‐regulates the antioxidant response in many cell types, but studies are needed to understand the effects of quercetin plasma metabolites on the osteoblast antioxidant response. The first specific aim was to examine antioxidant response genes and proteins in osteoblasts exposed to plasma quercetin metabolites. The second specific aim was to identify potential signaling pathways in the osteoblast antioxidant response that mediate the effect of quercetin, specifically Nrf2, ERK1/2, and NFκB p65. Osteoblasts isolated from fetal rat calvaria were treated with doses up to 20 μM of three different quercetin metabolites found in blood plasma after consumption of quercetin‐rich foods or supplements: quercetin aglycone (QRC), isorhamnetin (ISO), or quercetin 3‐O‐glucuronide (Q3G). Alternatively, some cells received a 2:1:1 mixture of all three metabolites (10 μM Q3G: 5 μM ISO: 5 μM QRC) to evaluate synergistic effects. Antioxidant response genes and proteins known to be up‐regulated by quercetin were analyzed along with Nrf2, ERK1/2, and NFκB proteins. Both QRC and ISO, but not Q3G, up‐regulated heme oxygenase‐1 (HO‐1) and γ‐glutamate cysteine ligase catalytic subunit (GCLC) at the mRNA and protein level. Synergistic effects of metabolites were not observed. Up‐regulation of HO‐1 and GCLC was associated with suppression of phosphorylated ERK1/2 and NFκB, but no alterations in Nrf2 protein levels were observed. This study shows that the antioxidant response of osteoblasts is differentially stimulated by quercetin metabolites. J. Cell. Biochem. 116: 1857–1866, 2015.

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 nonrepair 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‐β1, and transforming growth factor‐β2 were significantly expressed in implants, but their patterns of expression did not correlate with changes in procollagen gene expression.

Tissue vitamin C levels of guinea pig offspring are influenced by maternal vitamin C intake during pregnancy

Abstract The effect of a low to excessive maternal vitamin C intake during pregnancy on the vitamin C concentration of plasma and tissues of guinea pig dams and their pups was evaluated on the day of birth of the litter. Dams were provided low (0.15 g/kg diet) ( n = 7 litters), normal (0.50 g/kg) ( n = 2 litters), or excess (10 g/kg) ( n = 8 litters) dietary vitamin C levels. Maternal weight gain during pregnancy, gestational length, and weight and crown-rump length of pups were similar among vitamin C groups. For plasma, vitamin C values were 5 fold higher for pups and dams of the excess vitamin C group, compared with pups and dams of the low vitamin C group ( P P P P P P P

Maternal PKU and Breastfeeding: Case Report of Identical Twin Mothers

were diagnosed at birth (1969) with PKU. Both women were on diet during uneventful full-term pregnancies that ended with delivery of female offspring. These twins have been followed up through the PKU Clinic at the Kansas University Medical Center since birth. Dietary phenylalanine intake was well controlled when they were small children, but owing to poor dietary compliance, dietary phenylalanine restrictions were discontinued during their adolescent period. The girls were healthy and developed normally. Both were evaluated in 1994 and were determined to be of low normal intelligence (IQ of 80 and 74, respectively). The phenylalanine-restricted diet instituted prior to conception and during pregnancy included consumption of Phenex 2 medical formula (Ross Laboratories, Columbus, Ohio) and 200 mg of phenylalanine from food.4 Subjects recorded all food and fluids consumed for 3 days prior to sample collection. Food records were analyzed for total kilocalories (kcal), protein, and

Reflections on Teaching Graduate Students How to Use and Publish Nutrition Information on the World Wide Web

Abstract This article describes an approach to teaching graduate nutrition students how to search the Internet and publish on the World wide Web (WWW). The students first learned how to search for information and then developed documents on a nutrition topic. The first WWW document was first used as a supplement to an in-class lecture and then was expanded to include links to other documents the students developed and to other locations on the WWW that were relevant to the topic. Students learned how to develop the programs using Hyper Text Markup Language (HTML) and link the documents to the WWW home page for the course that had been developed by the instructor. The outcome was very positive. Pros and cons to this educational approach are discussed.