Prevalence of diabetes among patients diagnosed with periodontitis: A retrospective cross-sectional study.

OBJECTIVE: The objective of this study was to assess the prevalence of diabetes among patients with periodontitis and to evaluate whether diabetes is related to extent and severity of periodontitis. METHOD: This is a retrospective study of data observed over a 10-year period in patients referred to a specialized clinic for periodontology in the Netherlands. Patients received at the intake appointment a full-mouth periodontal examination, and based on the clinical data, patients were classified with respect to extent and severity of periodontitis. In addition, the prevalence of diabetes was recorded, based on self-report. RESULTS: A total of 5375 periodontitis patients were included in the study sample (mean age of 50 years). The prevalence of diabetes in this patient sample was 3.7% (n=192). No relation between diabetes and extent or severity of periodontitis could be established. CONCLUSION: The prevalence of diabetes in a predominantly "controlled" diabetic population was not related to the extent and/or severity of periodontitis along with the finding that the prevalence was lower than the national diabetes prevalence in the Netherlands.

Plane of nutrition influences the performance, innate leukocyte responses, and resistance to an oral Salmonella enterica serotype Typhimurium challenge in Jersey calves.

Two experiments investigated how plane of nutrition influences performance, leukocyte responses, and resistance to an oral Salmonella enterica serotype Typhimurium challenge. In experiment 1, 46 (2±1 d of age) calves were randomly assigned to 2 diets: a low (LPN; n=23) and high plane of nutrition (HPN; n=23). The LPN calves were fed 409 g/d of dry matter (DM) of a 20% crude protein and 20% fat milk replacer, whereas HPN calves were fed 610 and 735 g/d of DM of a 28% crude protein and 25% fat milk replacer during wk 1 and 2 to 6, respectively. In experiment 2, 20 bull calves (LPN; n=11 and HPN; n=9) were orally challenged on d 80 with 1.5×10(7) cfu of Salmonella Typhimurium (ATCC #14028). The HPN calves had a greater incidence (87.5 vs. 45.5%) and duration of days with high fecal scores (5.5 vs. 3.5 d). The LPN calves had greater neutrophil surface expression of L-selectin on d 7, 21, and 42. Following the Salmonella Typhimurium challenge, calf starter DM intake was greater among the HPN calves. The percentage of neutrophils producing an oxidative burst was also greater among HPN calves on d 1 to 5 after the challenge. Similarly, the intensity of the oxidative burst tended to be greater among the HPN calves on d 2 and 3 postchallenge. The secretion of tumor necrosis factor-α from whole-blood cultures stimulated with lipopolysaccharide tended to be greater on d 1 and was greater on d 5 and 6 among HPN calves. The median ranks of haptoglobin concentrations were greater and plasma zinc concentrations tended to be decreased among LPN calves. These data indicate that feeding a HPN to Jersey calves improved average daily gain and feed efficiency, but increased the incidence of high fecal scores during the first few weeks of life; however, the HPN Jersey calves may be more resistant to Salmonella Typhimurium after weaning.

Group housing of Holstein calves in a poor indoor environment increases respiratory disease but does not influence performance or leukocyte responses.

The objective of the current study was to determine if group-housing Holstein heifer calves in indoor pens with poor ventilation and drainage influences performance, health, leukocytes, and behavioral responses compared with individually housed calves. Ninety colostrum-fed calves (2 ± 1 d of age) were randomly assigned to 3 treatments: individually housed (G1; n=30 calves), 2 calves per pen (G2; n=30 calves), or 3 calves per pen (G3; n=30 calves). The space allowance per calf was 2.5m(2) for all treatment groups. All calves were fed 747 and 1,010 g/d of dry matter of a 28% CP and 20% fat milk replacer during the first 2 wk and wk 3 to 6, respectively. Weaning was initiated on d 46 by removing the evening feeding, and calves were completely weaned when they consumed 800 g/d of dry matter calf starter for 2 consecutive days after d 54. Calves were randomly commingled at d 90 in groups of 5 calves per pen in outdoors pens with an attached hutch. Peripheral blood was collected during the neonatal (3, 10, 21 d), weaning (46, 48, 54 d), and commingling periods (90, 93, 98 d) and was analyzed for neutrophil oxidative burst capacity when cocultured with an Escherichia coli, neutrophil surface L-selectin protein concentration, and whole blood secretion of tumor necrosis factor-α when cocultured with lipopolysaccharide. Behavior of each calf was assessed using 2 independent tests: an approach response to a human subject and the response of the calf when placed in an isolated cage. Calf starter intake was greater for G2 and G3 during wk 8 and 9, and also at wk 11 for G3 compared with G1. No treatment × time interaction or treatment effect for average daily gain was observed. Additionally, no treatment × time or treatment effect was noted for any leukocyte or biochemical variable of biological significance throughout the entire study. Individually housed calves tended to have a reduced incidence of respiratory disease during the first 90 d of life. No other treatment differences for the other health outcomes were observed. Group-housed calves also relied more on calf-to-calf interactions than calf-to-human interactions, as evident by their reduced approach of the human observer and more frequent movement when placed in an isolated pen. Group housing in an environment with poor ventilation and drainage may increase the risk of respiratory disease.

Improved performance and heightened neutrophil responses during the neonatal and weaning periods among outdoor group-housed Holstein calves.

The objective was to determine if outdoor group housing of Holstein calves influences metabolic status, leukocyte responses, and behavior compared with individually housed calves. Forty-nine Holstein heifer calves (2 ± 1 d of age) were randomly assigned to 1 of 2 treatments: individually housed (G1; n = 22) or group housed [3 calves per pen (G3); n = 27]. The space allowances per calf were 4.8 and 7.0m(2) for G1 and G3, respectively. All calves were offered an identical plane of milk replacer nutrition (747 and 1,010 g of DM/d of a 28% CP:20% fat milk replacer from wk 1 to 2 and wk 3 to 6, respectively). Weaning was initiated during wk 7 by removing the p.m. feeding and calves were completely weaned when they consumed 900 g of calf starter/d (as fed) for 2 consecutive days after d 54. At d 90, calves were commingled into random outdoor groups of 5 calves per pen. Peripheral blood was collected during the neonatal (d 3, 10, and 21), weaning (d 46, 48, and 54), and commingling periods (d 90, 93, and 98) and was analyzed for neutrophil oxidative burst (OB) capacity when cocultured with Escherichia coli, neutrophil surface L-selectin protein expression, and whole-blood secretion of tumor necrosis factor-α when cocultured with lipopolysaccharide. Starter intake was greater for G3 during the postweaning period (wk 8 to 12). Average daily gain was greater for G3 than G1 from d 54 to 68 and tended to be greater after commingling from d 113 to 133. During the neonatal period, G3 calves had more activated neutrophils, as evidenced by increased neutrophil L-selectin protein expression and a tendency for increased percentage of neutrophils producing an OB than G1 calves. During weaning, G3 calves continued to have more activated neutrophils with increased L-selectin expression on d 46 and 48 and a greater OB intensity throughout the period. No differences were observed among leukocyte responses between treatments at d 93 and 98. Outdoor group-housed Holstein calves had improved performance and heightened neutrophil responses compared with individually housed calves.

Plane of nutrition during the preweaning period but not the grower phase influences the neutrophil activity of Holstein calves.

Two experiments were conducted to determine the influence of plane of nutrition during (1) the pre- and immediate postweaning periods and (2) the grower phase on the performance and leukocyte responses of Holstein calves. In experiment 1, 39 (2 ± 1 d old) colostrum-fed heifer calves were randomly assigned to 2 planes of nutrition, a low (LPN; n = 19) and a high plane of nutrition (HPN; n = 20). Calves in the LPN treatment were offered 418 g/d of dry matter (DM) of a 20% crude protein (CP)/20% fat milk replacer, whereas calves in the HPN treatment were offered 747 and 1,010 g/d of DM of a 28% CP/20% fat milk replacer during wk 1 and wk 2 to 6, respectively. Calves were offered ad libitum access to a calf starter until the end of the study. Peripheral blood samples were collected on d 3, 10, 21, 45, 47, 53, and 91 for many ex vivo leukocyte responses and biochemical analyses. A nutrition × time interaction was present for average daily gain and feed efficiency (feed:gain ratio). A nutrition × time interaction existed for plasma glucose concentration. Neutrophil L-selectin expression was greater in calves fed the LPN than HPN on d 3 and 21. The percentage of neutrophils producing an oxidative burst (OB) when cocultured with Escherichia coli tended to be greater and was greater in calves fed the LPN than HPN on d 10 and 21, respectively. In addition, neutrophils from calves fed the LPN had greater OB intensity throughout the neonatal period (0 to 21 d). However, plasma haptoglobin was not different between the 2 planes of nutrition throughout the study. In experiment 2, 50 heifer calves that were all previously fed a HPN similar to that described for experiment 1 were randomly assigned to 2 planes of nutrition (HPN and LPN) during the grower phase (5 pens/treatment; 5 calves/pen). Calves fed the HPN during the grower phase were fed 4.1 kg of concentrate DM (pellets; 22.4% CP, DM basis) per head per day, whereas calves on the LPN during the grower phase were fed 1.6 kg of DM of the same concentrate per head per day. All calves were fed alfalfa hay (16.2% CP; DM basis) ad libitum. Overall, average daily gain was greater in HPN calves than LPN calves. No differences were noticed for concentrations of plasma urea nitrogen, glucose, neutrophil L-selectin expression, percentage of neutrophils producing OB, and plasma haptoglobin concentration between the 2 planes of nutrition. In summary, intake and performance were improved in calves fed the HPN than calves fed the LPN in both experiments. The neutrophil responses of calves fed an LPN were more active during the preweaning period than calves fed an HPN; however, this response was not observed during the immediate postweaning period or the grower phase.

Interproximal cervical lesions caused by incorrect flossing technique.

UNLABELLED: This case report describes an interproximal cervical lesion caused by the incorrect use of dental floss. A 58-year-old man who was asymptomatic, presented with unusual notch-like cervical lesions. After clinical and radiographical examinations, it was concluded that the aetiology of these lesions was an incorrect flossing technique. The treatment plan included extraction of maxillary 3rd molars and re-education of the patient in oral hygiene technique. CONCLUSION: These lesions are irreversible and often go undiagnosed; therefore, it is important for the clinician to be familiar with the clinical presentation and aetiology.

The effects of early weaning on innate immune responses of Holstein calves.

The objectives of this study were to compare innate immune responses of calves weaned early (EW; n=23; weaned at 23.7 ± 2.3 d of age) with those of conventionally weaned calves (CW; n=22; weaned at 44.7 ± 2.3 d of age). All calves were fed 3.8L of colostrum within 12h of birth and were subsequently fed milk replacer twice daily. The weaning process began by withdrawal of the afternoon milk-replacer feeding. Milk was fully withdrawn, and the calf was considered completely weaned when it consumed 900 g of calf starter as-fed for 2 consecutive days. Blood samples were collected from all calves at 24, 27, 31, 45, 48, 52, and 66 ± 2.3 d of age. Early weaned calves took a variable amount of time to completely wean from milk replacer; therefore, data were also analyzed by comparing calves grouped by latency to completely weaned (fast=1 to 5 d; intermediate=6 to 8 d; slow=15 to 17 d). Slow-EW calves weighed less than either the fast- or intermediate-EW calves before initiating weaning. At 27 d of age, circulating neutrophils were greater among EW calves than CW calves. Moreover, fast-EW calves had lower neutrophil:mononuclear cell ratios at 45 d of age than other EW calves. Slow-EW calves had lower TNF-α concentrations from whole blood stimulated with endotoxin at 27 and 31 d of age compared with fast- and intermediate-EW calves. All EW calves had decreased neutrophil L-selectin at d 27 and increased neutrophil L-selectin at 31 d of age. At 31 d of age, neutrophil ß(2)-integrin was the greatest among the fast-EW calves. All EW calves had decreased neutrophil oxidative burst at 27 and 31 d of age. Three days after CW calves were weaned they had higher neutrophils, hematocrit percentages, and circulating cortisol than EW calves. In addition, 3 d after CW calves were weaned they had decreased neutrophil oxidative burst responses to Escherichia coli. Weaning, irrespective of age, suppressed many innate immune responses. In addition, early weaning transiently suppressed L-selectin expression on neutrophils; however, the immunological significance in the context of the resistance to disease is unknown because EW calves likely had greater protection from passively derived immunoglobulins when they were weaned. Finally, calves with lower BW around 24 d of age may not be suitable for early weaning programs as evident in the suppressed secretion of TNF-α from whole blood cultures during the week following the initiation of weaning.

Effects of changing milk replacer feedings from twice to once daily on Holstein calf innate immune responses before and after weaning.

The objectives of this study were to determine the effects of switching Holstein calves to once-daily feeding during the fourth week of life (24 ± 2.3 d of age; once-fed n=22; twice-fed n=22) on innate immune responses, and to evaluate whether carry-over effects occurred when the calves were weaned during the seventh week of life. Peripheral blood samples were taken immediately before the change in feeding strategy (24 d of age) and at 27, 31, 45, 48, 52, and 66 d of age and were analyzed for circulating cortisol, haptoglobin, total leukocyte counts, neutrophil:mononuclear cells, and hematocrit percentage. Heparinized whole blood was also stimulated with lipopolysaccharide (LPS) for 24h and the concentration of tumor necrosis factor-α (TNF-α) in the supernatant was analyzed. Neutrophil L-selectin and ß(2)-integrin expression were analyzed by flow cytometry. Simultaneous neutrophil phagocytic and oxidative burst responses to a heat-killed Escherichia coli were quantified by dual-color flow-cytometry. Treatment (once-daily or twice daily feeding) had no effect on pre- or postweaning performance. Once-fed calves tended to have more circulating neutrophils at 27 d of age, greater expression of L-selectin on neutrophils at 31 and 45 d of age, and greater intensity of phagocytosis at 45 d of age. Once-fed calves secreted less TNF-α in LPS-stimulated whole blood cultures at 45 d of age compared with twice-fed calves and this tended to persist through the immediate postweaning period. None of the other immune parameters differed after weaning between the preweaning feeding strategies. Consolidating calf milk replacer into one feeding during the fourth week of life was likely a mild and acute stressor, as evidenced by transient neutrophilia in the absence of suppressed functional capacities of neutrophils. Future research should address the mechanism and immunological significance of the persistent decreased TNF-α response in once-fed calves.

Evaluating the acute effects of oral, non-combustible potential reduced exposure products marketed to smokers.

BACKGROUND: Non-combustible potential reduced exposure products (PREPs; eg, Star Scientific's Ariva; a variety of other smokeless tobacco products) are marketed to reduce the harm associated with smoking. This marketing occurs despite an absence of objective data concerning the toxicant exposure and effects of these PREPs. Methods used to examine combustible PREPs were adapted to assess the acute effects of non-combustible PREPs for smokers. METHODS: 28 overnight abstinent cigarette smokers (17 men, 14 non-white) each completed seven, Latin-squared ordered, approximately 2.5 h laboratory sessions that differed by product administered: Ariva, Marlboro Snus (Philip Morris, USA), Camel Snus (RJ Reynolds, Winston-Salem, North Carolina, USA), Commit nicotine lozenge (GlaxoSmithKline; 2 mg), own brand cigarettes, Quest cigarettes (Vector Tobacco; delivers very low levels of nicotine) and sham smoking (ie, puffing on an unlit cigarette). In each session, the product was administered twice (separated by 60 min), and plasma nicotine levels, expired air CO and subjective effects were assessed regularly. RESULTS: Non-combustible products delivered less nicotine than own brand cigarettes, did not expose smokers to CO and failed to suppress tobacco abstinence symptoms as effectively as combustible products. CONCLUSIONS: While decreased toxicant exposure is a potential indicator of harm reduction potential, a failure to suppress abstinence symptoms suggests that currently marketed non-combustible PREPs may not be a viable harm reduction strategy for US smokers. This study demonstrates how clinical laboratory methods can be used to evaluate the short-term effects of non-combustible PREPs for smokers.

Antidiuretic action of angiotensin II in the river lamprey Lampetra fluviatilis: evidence for endocrine control of kidney function in cyclostomes.

Intravenous infusion of angiotensin II ([Asn¹ Val5]-Ang II) at 10⁻9 mol min⁻¹ kg⁻¹ body mass produced a significant antidiuresis in river lamprey Lampetra fluviatilis, captured during upstream migration and maintained in fresh water. Although the renin-angiotensin hormonal system (RAS) is now recognized in jawless fishes, until this study, the role of homologous Ang II in L. fluviatilis kidney function had not been examined. This study provides the first evidence for an antidiuretic action of Ang II in cyclostomes and, in evolutionary terms, suggests a renal function for the RAS in early vertebrates.

Septic tank additive impacts on microbial populations.

Environmental health specialists, other onsite wastewater professionals, scientists, and homeowners have questioned the effectiveness of septic tank additives. This paper describes an independent, third-party, field scale, research study of the effects of three liquid bacterial septic tank additives and a control (no additive) on septic tank microbial populations. Microbial populations were measured quarterly in a field study for 12 months in 48 full-size, functioning septic tanks. Bacterial populations in the 48 septic tanks were statistically analyzed with a mixed linear model. Additive effects were assessed for three septic tank maintenance levels (low, intermediate, and high). Dunnett's t-test for tank bacteria (alpha = .05) indicated that none of the treatments were significantly different, overall, from the control at the statistical level tested. In addition, the additives had no significant effects on septic tank bacterial populations at any of the septic tank maintenance levels. Additional controlled, field-based research iswarranted, however, to address additional additives and experimental conditions.

Can placing lead-rubber inferolateral to the light beam diaphragm limit ionising radiation to multiple radiosensitive organs?

INTRODUCTION: This article investigates a practical method of reducing the impact of scattered radiation during a lateral radiographic projection of the elbow. The light beam diaphragm (LBD) is generally accepted to limit ionising radiation using horizontal and longitudinal lead shutters, yet this article evidences further dose limitation by placing lead-rubber inferolateral to the LBD device. METHODS: Using an anthropomorphic phantom and arm construction scattered radiation was recorded at multiple radiosensitive organs. A 15 cc ionisation chamber (model 10100 AT TRIAD) was placed on each radiosensitive organ (eye, thyroid, breast, testes, spleen and ovaries) measuring exposure rate (µGy/s). Dose readings were recorded before and after the placement of lead-rubber inferolateral to the LBD. A paired two sample t-test was undertaken affirming how likely dose limitation was attributable to chance (p < 0.05). RESULTS: Descriptive and inferential statistics demonstrate dose reduction to radiosensitive organs (right eye 53%, right breast 53%, left eye 39%, thyroid 13%, left ovary 9%, testes 6%, left breast 3% and spleen 2%) upon placement of the lead-rubber inferolateral to the LBD. The paired two sample t-test demonstrated statistically significant dose limitation (t = 2.04, df = 7, p = 0.04) thus significant for radiographic practice. CONCLUSION: Placement of lead-rubber inferolateral to the LBD limits dose to multiple radiosensitive organs. Right (53%) and left (39%) eye lens, right breast (53%), thyroid (13%), left ovary (9%), testes (6%), left breast (3%) and spleen (2%) statistically demonstrate dose limiting opportunities to patients.

Manure nutrient excretion by lactating cows fed exogenous phytase and cellulase.

The effect of an exogenous phytase and cellulase-containing enzyme formulation on nutrient digestibility and excretion was evaluated in 24 Holstein cows. Cows were fed corn silage- and alfalfa silage-based diets with or without a cellulase-phytase blend for 31 d in a continuous random design. Treatment groups were balanced for parity, days in milk, and mature-equivalent projected milk yield. Diets contained 37% forage, 18.3% crude protein, 35.4% neutral detergent fiber, 18% acid detergent fiber, and 0.42% P (no supplemental P). Cows were fed once daily in Calan doors and milked 2 times daily. Body weight and milk yield were recorded at each milking. Milk samples were collected on d 28 to 31 at 8 consecutive milkings. On d 28 to 31, fecal grab samples were collected every 8 h, with sampling times advanced by 2 h each day. Feces samples were pooled by cow. Feed and feces samples were analyzed for acid detergent lignin (used as an internal marker) and for N, P, neutral detergent fiber, and acid detergent fiber. Days in milk were similar between treatments, and body weight and milk yield were unaffected by treatment. Cows fed the enzyme formulation had reduced fecal dry matter, neutral detergent fiber, and acid detergent fiber excretion and reduced fecal excretion of N and P. Apparent digestibility of dry matter, acid detergent fiber, neutral detergent fiber, and N tended to increase with the enzyme formulation. Addition of an exogenous phytase and cellulase enzyme formulation to diets for lactating cows reduced fecal nutrient excretion.

Activation of fibroblast procollagenase by mast cell proteases.

Proteases capable of activating procollagenase from gingiva and from fibroblast and macrophage monolayer cultures were harvested from homogenates of canine tumor mast cells. The mast cell proteases lysed casein and Azocoll but not native collagen. In low salt concentrations the enzymes existed at high molecular weight complexes, which were dissociated by increasing the salt concentration above 1.0 M (NaCl, KCl). Gel filtration in 1.4 M KCl separated the protease activity into three peaks, all of which activated procollagenase. Two of the enzymes showed substrate specificities (hydrolysis of p-tosyl-L-arginine methyl ester and benzoyl-tyrosine ethyl ester) and reactive center reactivities similar to pancreatic trypsin and chymotrypsin. Based on gel filtration, apparent molecular weights of 160 000 (p-tosyl-L-arginine methyl ester esterase), 90 000 (main procollagenase activator) and 36 000 benzoyl-tyrosine ethyl ester esterase) were determined. Activation of procollagenase resulted in a 18-20 000 decrease of the molecular weight. The activation was directly related to the amount of activator added within certain limits. Further addition of activator resulted in proteolytic inactivation of collagenase.

Procollagenase from bovine gingiva.

1. Collagenase (EC 3.4.24.3) is released from bovine gingival explants in vitro as a zymogen. The zymogen does not hydrolyze collagen and does not form a complex with alpha2-macroglobulin (alpha2-M). It elutes in gel filtration with an apparent molecular weight of approx. 80 000. 2. Incubation of the zymogen with trypsin results in a 15 000-20 000 dalton decrease in molecular weight and imparts to the enzyme the ability to hydrolyze collagen and to form a complex with alppha2-M. 3. The zymogen can be completely separated from the active enzyme to alpha2-M. Likewise, the zymogen can be harvested from cultures supplemented with serum.

Acute myocardial infarction in sickle cell anemia.

Typical acute myocardial infarction in a well-documented case of sickle cell disease, to our knowledge, has not been reported. A patient with hemoglobin SS proved by electrophoresis with typical clinical manifestation and diagnostic ECG changes died suddenly. Autopsy findings were confirmatory, but no atherogenic lesions were present. Possible mechanisms for acute infarction are discussed, including sludging of sickle cells, coronary spasm, thrombus formation, and severe anemia associated with any or all of the aforementioned.

Recycling of the ascorbate free radical by human erythrocyte membranes.

Reduction of the ascorbate free radical (AFR) at the plasma membrane provides an efficient mechanism to preserve the vitamin in a location where it can recycle alpha-tocopherol and thus prevent lipid peroxidation. Erythrocyte ghost membranes have been shown to oxidize NADH in the presence of the AFR. We report that this activity derives from an AFR reductase because it spares ascorbate from oxidation by ascorbate oxidase, and because ghost membranes decrease steady-state concentrations of the AFR in a protein- and NADH-dependent manner. The AFR reductase has a high apparent affinity for both NADH and the AFR (< 2 microM). When measured in open ghosts, the reductase is comprised of an inner membrane activity (both substrate sites on the cytosolic membrane face) and a trans-membrane activity that mediates extracellular AFR reduction using intracellular NADH. However, the trans-membrane activity constitutes only about 12% of the total measured in ghosts. Ghost AFR reductase activity can also be differentiated from NADH-dependent ferricyanide reductase(s) by its sensitivity to the detergent Triton X-100 and insensitivity to enzymatic digestion with cathepsin D. This NADH-dependent AFR reductase could serve to recycle ascorbic acid at a crucial site on the inner face of the plasma membrane.

Accessibility and reactivity of ascorbate 6-palmitate bound to erythrocyte membranes.

Lipophilic derivatives of ascorbic acid may protect lipid bilayers and micelles against lipid peroxidation. In this work the binding, accessibility, and reducing capacity of ascorbate 6-palmitate (A6P) were studied in human erythrocyte membranes. In contrast to less lipophilic carbon-6-modified ascorbate derivatives, A6P bound to erythrocyte membranes in a concentration-dependent manner. This binding was preserved following centrifugation washes, but was largely reversed by extraction with bovine serum albumin. Most of the ascorbyl groups of membrane-bound A6P were readily accessible to oxidation by water-soluble oxidants. Ferricyanide quantitatively oxidized membrane-bound A6P, but the latter spared endogenous tocopherols from destruction. In EPR studies, A6P was much more effective than ascorbate in reducing nitroxide spin labels positioned at either carbon-5 or carbon-16 of membrane-bound stearic acid in both intact cells and in membranes. A6P, thus, appears to intercalate into the erythrocyte membrane with the ascorbyl group located superficially, but with access to the hydrophobic membrane interior, and with the ability to recycle endogenous alpha-tocopherol during oxidant stress.

Ascorbate recycling in human erythrocytes: role of GSH in reducing dehydroascorbate.

Human erythrocytes regenerate ascorbate from its oxidized product, dehydroascorbate. The extent to which such ascorbate recycling occurs by a GSH-dependent mechanism was investigated. In the presence of glucose, erythrocytes took up over 90% of extracellular [14C]dehydroascorbate and rapidly converted it to [14C]ascorbate, which was trapped within the cells. Dehydroascorbate uptake and reduction was not associated with generation of a monoascorbyl free radical intermediate. Uptake and reduction of dehydroascorbate by glucose-depleted erythrocytes coordinately decreased GSH and raised GSSG concentrations in erythrocytes. This effect was reversed by D-glucose, but not by L-lactate. Conversely, depletion of cellular GSH decreased the ability of cells to recycle dehydroascorbate to ascorbate, as reflected in the extent to which cells were able to reduce extracellular ferricyanide. Monoascorbyl free radical was formed during the reduction of extracellular ferricyanide, indicating that one electron transfer steps were involved in this process. In GSH-depleted cells, addition of L-lactate as an energy source for glycolysis-dependent NADH regeneration did cause a partial recovery of the ability of cells to reduce ferricyanide. However, in resealed erythrocyte ghosts containing either 4 mM GSH or 400 mu M NADH, only the GSH-containing ghosts supported regeneration of ascorbate from added dehydroascorbate. These results suggest that in human erythrocytes ascorbate regeneration from dehydroascorbate is largely GSH dependent, and that it occurs through either enzymatic or nonenzymatic reactions not involving the monoascorbyl free radical.

Ascorbate 6-palmitate protects human erythrocytes from oxidative damage.

Lipid-soluble antioxidants, such as alpha-tocopherol, protect cell membranes from oxidant damage. In this work we sought to determine whether the amphipathic derivative of ascorbate, ascorbate 6-palmitate, is retained in the cell membrane of intact erythrocytes, and whether it helps to protect the cells against peroxidative damage. We found that ascorbate 6-palmitate binding to erythrocytes was dose-dependent, and that the derivative was retained during the multiple wash steps required for preparation of ghost membranes. Ascorbate 6-palmitate remained on the extracellular surface of the cells, because it was susceptible to oxidation or removal by several cell-impermeant agents. When bound to the surface of erythrocytes, ascorbate 6-palmitate reduced ferricyanide, an effect that was associated with generation of an ascorbyl free radical signal on EPR spectroscopy. Erythrocyte-bound ascorbate 6-palmitate protected membrane alpha-tocopherol from oxidation by both ferricyanide and a water-soluble free radical initiator, suggesting that the derivative either reacted directly with the exogenously added oxidant, or that it was able to recycle the alpha-tocopheroxyl radical to alpha-tocopherol in the cell membrane. Ascorbate 6-palmitate also partially protected cis-parinaric acid from oxidation when this fluorescent fatty acid was intercalated into the membrane of intact cells. These results show that an amphipathic ascorbate derivative is retained on the exterior cell surface of human erythrocytes, where it helps to protect the membrane from oxidant damage originating outside the cells.