Guidelines for conducting epidemiological studies of blast injury.

Blast injuries are often caused by more than one mechanism, do not occur in isolation, and typically elicit a secondary multi-system response. Research efforts often do not separate blast injuries caused by blast waves from those caused by blunt force trauma and other mechanisms. 15 experts from nine different NATO nations developed in the HFM Research Task Group (RTG; HFM-234 (RTG)) 'Environmental Toxicology of Blast Exposures: Injury Metrics, Modelling, Methods and Standards' Guidelines for Conducting Epidemiological Studies of Blast Injury. This paper describes these guidelines, which are intended to provide blast injury researchers and clinicians with a basic set of recommendations for blast injury epidemiological study design and data collection that need to be considered and described when conducting prospective longitudinal studies of blast injury.

Quantifying physician's bias to terminate resuscitation. The TERMINATOR study.

CONTEXT: Deciding on "termination of resuscitation" (TOR) is a dilemma for any physician facing cardiac arrest. Due to the lack of evidence-based criteria and scarcity of the existing guidelines, crucial arbitration to interrupt resuscitation remains at the practitioner's discretion. AIM: Evaluate with a quantitative method the existence of a physician internal bias to terminate resuscitation. METHOD: We extracted data concerning OHCAs managed between January 2013 and September 2021 from the RéAC registry. We conducted a statistical analysis using generalized linear mixed models to model the binary TOR decision. Utstein data were used as fixed effect terms and a random effect term to model physicians personal bias towards TOR. RESULTS: 5,144 OHCAs involving 173 physicians were included. The cohort's average age was 69 (SD 18) and was composed of 62% of women. Median no-flow and low-flow times were respectively 6 (IQR [0,12]) and 18 (IQR [10,26]) minutes. Our analysis showed a significant (p < 0.001) physician effect on TOR decision. Odds ratio for the "doctor effect" was 2.48 [2.13-2.94] for a doctor one SD above the mean, lower than that of dependency for activities of daily living (41.18 [24.69-65.50]), an age of more than 85 years (38.60 [28.67-51.08]), but higher than that of oncologic, cardiovascular, respiratory disease or no-flow duration between 10 to 20 minutes (1.60 [1.26-2.00]). CONCLUSIONS: We demonstrate the existence of individual physician biases in their decision about TOR. The impact of this bias is greater than that of a no-flow duration lasting ten to twenty minutes. Our results plead in favor developing tools and guidelines to guide physicians in their decision.

Do children and adolescents with type 1 diabetes suffer from a lack of resources in France? Results from a benchmark study in the New Aquitaine region.

BACKGROUND: A benchmark study was conducted in the southwest of France, in the New Aquitaine region, to investigate metabolic outcomes and availability of resources in pediatric diabetes units. We assessed whether the level of care was in accordance with the International Society for Pediatric and Adolescent Diabetes recommendations. METHODS: Demographic and clinical data were collected, as were all HbA1c tests for the 2017 calendar year. Pediatricians specialized in diabetes care were invited to complete an online survey concerning means allocated to the management of type 1 diabetes in their centers. RESULTS: Sixteen centers provided data for 1277 patients and 3873 clinical visits. A total of 1115 children suffering from diabetes for more than 1 year were studied. Median HbA1c was 8% (7.4-8.6) for the whole region. Only 29.2% of children had good metabolic control in accordance with the <7.5% target. We identified slight but significant variation in glycemic control among centers (P=0.029). The use of an insulin pump varied greatly among centers but did not explain HbA1c differences. We did not identify a correlation between medical or paramedical time dedicated to the follow-up of diabetic patients and the mean HbA1c of each center. For 100 diabetic patients, follow-up was provided by 0.42 physicians (0.23-1.50), 0.15 nurses (0-0.56), 0.12 dietitians (0-0.48), and 0.07 psychologists (0-0.30). CONCLUSION: This study demonstrates a lack of human resources allocated to the management of type 1 diabetes in the region that is far below international recommendations. The proportion of children achieving the international glycemic target is low. There is a clear need to improve glycemic control in children, which will only be possible with improved professional practices, encouraged by benchmark studies, and by increasing the size of our multidisciplinary teams.

Multiple molecular targets underlie the antidiabetic effect of Nigella sativa seed extract in skeletal muscle, adipocyte and liver cells.

AIM: Nigella sativa (N. sativa) is a plant widely used in traditional medicine of North African countries. During the last decade, several studies have shown that extracts from the seeds of N. sativa have antidiabetic effects. METHODS: Our group has recently demonstrated that N. sativa seed ethanol extract (NSE) induces an important insulin-like stimulation of glucose uptake in C2C12 skeletal muscle cells and 3T3-L1 adipocytes following an 18 h treatment. The purpose of the present study was to elucidate the pathways mediating this insulin-like effect and the mechanisms through which these pathways are activated. RESULTS: Results from western immunoblot experiments indicate that in C2C12 cells as well as in H4IIE hepatocytes, but not in 3T3-L1 cells, NSE increases activity of Akt, a key mediator of the effects of insulin, and activity of AMP-activated protein kinase (AMPK), a master metabolic regulating enzyme. To test whether the activation of AMPK resulted from a disruption of mitochondrial function, the effects of NSE on oxygen consumption were assessed in isolated liver mitochondria. NSE was found to exhibit potent uncoupling activity. CONCLUSION: Finally, to provide an explanation for the effects of NSE in adipocytes, PPARgamma stimulating activity was tested using a reporter gene assay. Results indicate that NSE behaves as an agonist of PPARgamma. The data supports the ethnobotanical use of N. sativa seed oil as a treatment for diabetes, and suggests potential uses of this product, or compounds derived thereof, against obesity and the metabolic syndrome.

Antiobesity and antidiabetic effects of biotransformed blueberry juice in KKA(y) mice.

AIM: Biotransformation of blueberry juice by the Serratia vaccinii bacterium gave rise to adenosine monophosphate-activated protein kinase (AMPK) phosphorylation and glucose uptake in muscle cells and adipocytes, but inhibited adipogenesis. This study investigated the antiobesity and antidiabetic potential of biotransformed blueberry juice (BJ) in KKA(y) mice, rodent model of leptin resistance. METHODS: BJ was incorporated in drinking water of KKA(y) mice. Parameters of body weight, food intake, plasma glucose, insulin, leptin, and adiponectin were measured. Before and after therapy, animals were subjected to an oral glucose tolerance test. At the end of treatment, liver, muscle, kidney, epididymal fat pad, abdominal fat pad, and dorsal fat pad were collected and weighed. RESULTS: Incorporating BJ in drinking water protected young KKA(y) mice from hyperphagia and significantly reduced their weight gain. Moreover, BJ protected young KKA(y) mice against the development of glucose intolerance and diabetes mellitus. Chronic BJ administration in obese and diabetic KKA(y) mice reduced food intake and body weight. This effect could not fully explain the associated antidiabetic effect because BJ-treated mice still showed lower blood glucose level when compared with pair-fed controls. The adipokines pathway also seems to be involved because BJ significantly increased adiponectin levels in obese mice. CONCLUSIONS: This study shows that BJ decreases hyperglycemia in diabetic mice, at least in part by reversing adiponectin levels. BJ also protects young pre-diabetic mice from developing obesity and diabetes. Thus, BJ may represent a novel complementary therapy and a source of novel therapeutic agents against diabetes mellitus.

Management of bioburden with a burn gel that targets nociceptors.

AIM: To assess the effectiveness of RescuDerm, an amorphous, water-soluble burn gel in controlling Pseudomonas aeruginosa growth in rat full-thickness wounds contaminated with 10(3), 10(5) or 10(7) CFU/g tissue. METHOD: Wounds were treated daily for 72 hours with a placebo gel, a 5% w/w mafenide acetate gel (MAF), or with four modalities of RescuDerm application. RESULTS: All RescuDerm treatments were equally effective within 24 hours in preventing further Pseudomonas aeruginosa growth in wounds contaminated with 10(3) CFU/g tissue. Pseudomonas aeruginosa levels remained at or below this baseline count for 72 hours in all but one of the RescuDerm treatments. The bioburdens in MAF-treated wounds were negligible, averaging 0.14 +/- 0.09 log10 CFU/g tissue. While RescuDerm and MAF remained bacteriostatic in wounds contaminated with 10(5) CFU/g tissue, this property disappeared at higher bioburdens. CONCLUSION: RescuDerm can be used for the management of cutaneous injuries sustained in environments deemed marginally or moderately contaminated. Heavily contaminated wounds would require irrigation prior to application to reduce their bioburden below 10(5) CFU/g tissue.

Age-associated alterations in cardiac and skeletal muscle glucose transporters, insulin and IGF-1 receptors, and PI3-kinase protein contents in the C57BL/6 mouse.

We investigated potential age-related changes in cardiac and skeletal muscle protein contents of glut-4 and glut-1 transporters, insulin and insulin-like growth factor-1 (IGF-1) receptors, and phosphatidylinositol 3-kinase (PI3-kinase) in the C57B1/6 mouse. Myocardial glut-4 content increased four- to five-fold between mid- to late-adulthood with no further age-related changes. Increases in myocardial glut-1 preceded the increase in glut-4 and was of a much smaller magnitude (25-40%). Skeletal muscle glut-4 was also increased (38-49%) and no further changes were noted between adulthood and old age. Cardiac insulin receptor and the p85 alpha subunit of PI3-kinase both declined by about 40%, whereas the skeletal muscle content of these two proteins were unaffected by aging. Cardiac (-23 to -24%) and skeletal muscle (-40 to -62%) IGF-1 receptor levels were decreased in adult and old animals with senescence being associated with a further decrease in cardiac IGF-1 receptor levels to 20% of controls. A two- to three-fold increase in both basal and maximal in vitro autophosphorylation of the cardiac insulin and IGF-1 receptors by their respective ligands was observed with senescence. It appears that cardiac and skeletal muscle demonstrate differential responses in terms of the magnitude and temporal responses of age-associated alterations in glucose transport related protein contents in the C57B1/6 mouse.

Resistance of the aged myocardium to exercise-induced chronic changes in glucose transport related protein content.

The effects of acute exercise on myocardial content of glut-1 and glut-4 transporters, insulin and IGF-1 receptors were assessed in control and chronically exercised 24-month-old C57B1/6 mice. Myocardial glut-1, glut-4, insulin receptor (Ins R) and insulin like growth factor-1 receptor (IGF-1 R) protein levels were unaffected by 36 weeks of chronic exercise. However, myocardial protein content of glut-1, but not glut-4, was increased 12 h following an acute exercise bout in control (46%) and chronically exercised (83%) aged animals. This increased glut-1 response following acute exercise occurred despite the finding that the chronic exercise failed to increase cardiac or skeletal muscle oxidative capacity as indicated by no change in citrate synthase activity. Myocardial IGF-1 R content was unaffected by acute exercise whereas Ins R protein content was decreased 12 h following the acute exercise bout in the chronically exercised (-52%) and control (-28%) animals. The effect of acute exercise on the protein content of glut-1 and Ins R was 80 and 84% greater respectively, in the chronically exercised animals. This suggests that the amplitude of the expression of these two proteins may be increased by chronic exercise, thus constituting a form of adaptation.

NMR characterization of a diamagnetic model of unliganded alpha chains from human hemoglobin.

This paper reports the reconstitution and spectroscopic characterization of a complex between alpha globin from human adult hemoglobin and protoporphyrin IX-Zn(II). Optical and proton one-dimensional (1-D) NMR spectra indicate that the prosthetic group binds in a 1:1 stoichiometry to the apoglobin in a single conformation. Using 2-D proton NMR techniques we assigned resonances corresponding to the majority of porphyrin substituents and to several side chains of amino acids in contact with the porphyrin. Analysis of nuclear Overhauser enhancement interactions between identified protons indicated that the complex contains only one rotation isomer of the prosthetic group. The diamagnetic Zn(II) ion is coordinated to the proximal histidine (His87) and does not bind O2 or CO as a sixth ligand. The ring current effects on protons from the distal valine (Val62) are considerably higher than in the liganded form providing strong evidence for a more compact ligand binding pocket relative to the carbon monoxy state. Therefore, protoporphyrin-Zn(II)/alpha globin complex is a suitable diamagnetic model for unliganded alpha chains and will be used for structure determination by NMR and modeling methods.

A sustained release bacterial inoculum infusion model of intra-abdominal infection in conscious rats: bacteriology, metabolism, and histopathology.

This report describes the development of a rat peritonitis model that simulates a slow, sustained bacterial release from the gut. Septic animals (SEP) received an intraperitoneal infusion of a bacterial inoculum (6.5 x 10(8) colony forming units Escherichia coli) over 12 h, while control rats (CON) received a sterile inoculum. This model yielded a 52% mortality over 7 days in SEP, with deaths usually occurring 24-48 h after the onset of infusion. Septic rats showed greater febrile responses and body weight losses than those of CON, as well as mild hyperlactacidemia, hypoglycemia, and episodic bacteremia. Maximum bacterial counts in peritoneal fluid and several organs of SEP were observed at 36 h, with bacterial counts progressively decreasing by 7 days to levels similar to those observed at 12 h. Lung and spleen wet weights increased by 17% at 36 h and 7 days post-infection in SEP. Histological evaluation of random organ samples revealed mild to moderate morphological changes in SEP while CON showed no or minimal changes in the parameters measured during the study. This new model of chronic peritonitis in the rat reproduces many of the clinical features observed in human sepsis, and thus should prove to be a useful tool in further studies of the pathophysiology of peritonitis.

Muscle glycogen utilization during shivering thermogenesis in humans.

The purpose of the present study was to clarify the importance of skeletal muscle glycogen as a fuel for shivering thermogenesis in humans during cold-water immersion. Fourteen seminude subjects were immersed to the shoulders in 18 degrees C water for 90 min or until rectal temperature (Tre) decreased to 35.5 degrees C. Biopsies from the vastus lateralis muscle and venous blood samples were obtained before and immediately after the immersion. Metabolic rate increased during the immersion to 3.5 +/- 0.3 (SE) times resting values, whereas Tre decreased by 0.9 degrees C to approximately 35.8 degrees C at the end of the immersion. Intramuscular glycogen concentration in the vastus lateralis decreased from 410 +/- 15 to 332 +/- 18 mmol glucose/kg dry muscle, with each subject showing a decrease (P less than 0.001). Plasma volume decreased (P less than 0.001) markedly during the immersion (-24 +/- 1%). After correcting for this decrease, blood lactate and plasma glycerol levels increased by 60 (P less than 0.05) and 38% (P less than 0.01), respectively, whereas plasma glucose levels were reduced by 20% after the immersion (P less than 0.001). The mean expiratory exchange ratio showed a biphasic pattern, increasing initially during the first 30 min of the immersion from 0.80 +/- 0.06 to 0.85 +/- 0.05 (P less than 0.01) and decreasing thereafter toward basal values. The results demonstrate clearly that intramuscular glycogen reserves are used as a metabolic substrate to fuel intensive thermogenic shivering activity of human skeletal muscle.

Free fatty acid availability and temperature regulation in cold water.

The purpose of this study was to investigate whether a reduced availability of plasma free fatty acids (FFA) would impair human temperature regulation during cold exposure. Seven seminude male subjects were immersed on two occasions in 18 degrees C water for 90 min or until their rectal temperature (Tre) decreased to 35.5 degrees C. The immersion occurred after 2 h of intermittent oral ingestion of either nicotinic acid (NIC) or a placebo (PLAC). Plasma FFA levels immediately before the immersion were significantly lower in NIC (87 +/- 15 mumol/l) than in PLAC (655 +/- 116 mumol/l, P less than 0.05). Although FFA levels increased by 73% in NIC during the immersion (P less than 0.05), they remained significantly lower than in PLAC (151 +/- 19 vs. 716 +/- 74 mumol/l, P less than 0.05) throughout the immersion. Muscle glycogen concentrations in the vastus lateralis decreased after cold water immersion in both trials (P less than 0.05), but the rate of glycogen utilization was similar, averaging 1.00 +/- 0.27 mmol glucose unit.kg dry muscle-1.min-1). Plasma glucose levels were significantly reduced after immersion in both trials (P less than 0.05), this decrease being greater in NIC (1.3 +/- 0.2 mmol/l) than in PLAC (0.7 +/- 0.1 mmol/l, P less than 0.05). O2 uptake increased to 3.8 +/- 0.3 times preimmersion values in both trials (P less than 0.05). Mean respiratory exchange ratio (RER) immediately before the immersion was greater in NIC (0.87 +/- 0.02) than in PLAC (0.77 +/- 0.01, P less than 0.05). Cold exposure increased RER in PLAC but not in NIC.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of muscle glycogen and plasma FFA availability on human metabolic responses in cold water.

The purpose of this study was to investigate whether simultaneous alterations in the availability of plasma free fatty acids and muscle glycogen would impair the maintenance of thermal balance during cold water immersion in humans. Eight seminude subjects were immersed on two occasions in 18 degrees C water for 90 min or until rectal temperature (Tre) decreased to 35.5 degrees C. Each immersion followed 2.5 days of a specific dietary and exercise regimen designed to elicit low (LOW) or high glycogen levels (HIGH) in large skeletal muscle groups. Nicotinic acid (1.6 mg/kg) was administered for 2 h before and during immersion to inhibit white adipose tissue lipolysis. Biopsies from the vastus lateralis showed that the glycogen concentration before the immersion was significantly lower in LOW than in HIGH (223 +/- 19 vs. 473 +/- 24 mmol glucose units/kg dry muscle). However, the mean rates of glycogen utilization were not significantly different between trials (LOW 0.62 +/- 0.14 vs. HIGH 0.88 +/- 0.15 mmol glucose units.kg-1.min-1). Nicotinic acid dramatically reduced plasma free fatty acid levels in both trials, averaging 127 +/- 21 mumol/l immediately before the immersion. Cold water immersion did not significantly alter those levels. Plasma glucose levels were significantly reduced after cold water immersion to a similar extent in both trials (18 +/- 4%). Mean respiratory exchange ratio at rest and during immersion was greater in HIGH than LOW, whereas there were no intertrial differences in O2 uptake. The calculated average metabolic heat production during immersion tended to be lower (P = 0.054) in LOW than in HIGH (15.3 +/- 1.9 vs. 17.5 +/- 1.9 kJ/min).(ABSTRACT TRUNCATED AT 250 WORDS)

Muscle glycogen availability and temperature regulation in humans.

The effects of intramuscular glycogen availability on human temperature regulation were studied in eight seminude subjects immersed in 18 degrees C water for 90 min or until rectal temperature (Tre) decreased to 35.5 degrees C. Each subject was immersed three times over a 3-wk period. Each immersion followed 2.5 days of a specific dietary and/or exercise regimen designed to elicit low (L), normal (N), or high (H) glycogen levels in large skeletal muscle groups. Muscle glycogen concentration was determined in biopsies taken from the vastus lateralis muscle before and after each immersion. Intramuscular glycogen concentration before the immersion was significantly different among the L, N, and H trials (P less than 0.01), averaging 247 +/- 15, 406 +/- 23, and 548 +/- 42 (SE) mmol glucose units.kg dry muscle-1, respectively. The calculated metabolic heat production during the first 30 min of immersion was significantly lower during L compared with N or H (P less than 0.05). The rate at which Tre decreased was more rapid during the L immersion than either N or H (P less than 0.05), and the time during the immersion at which Tre first began to decrease also appeared sooner during L than N or H. The results suggest that low skeletal muscle glycogen levels are associated with more rapid body cooling during water immersion in humans. Higher than normal muscle glycogen levels, however, do not increase cold tolerance.

Insight into skeletal muscle mechanotransduction: MAPK activation is quantitatively related to tension.

The mechanism by which mechanical forces acting through skeletal muscle cells generate intracellular signaling, known as mechanotransduction, and the details of how gene expression and cell size are regulated by this signaling are poorly understood. Mitogen-activated protein kinases (MAPKs) are known to be involved in mechanically induced signaling in various cell types, including skeletal muscle where MAPK activation has been reported in response to contraction and passive stretch. Therefore, the investigation of MAPK activation in response to mechanical stress in skeletal muscle may yield important information about the mechanotransduction process. With the use of a rat plantaris in situ preparation, a wide range of peak tensions was generated through passive stretch and concentric, isometric, and eccentric contractile protocols, and the resulting phosphorylation of c-Jun NH(2)-terminal kinase (JNK), extracellular regulated kinase (ERK), and p38 MAPKs was assessed. Isoforms of JNK and ERK MAPKs were found to be phosphorylated in a tension-dependent manner, such that eccentric > isometric > concentric > passive stretch. Peak tension was found to be a better predictor of MAPK phosphorylation than time-tension integral or rate of tension development. Differences in maximal response amplitude and sensitivity between JNK and ERK MAPKs suggest different roles for these two kinase families in mechanically induced signaling. A strong linear relationship between p54 JNK phosphorylation and peak tension over a 15-fold range in tension (r(2) = 0.89, n = 32) was observed, supporting the fact that contraction-type differences can be explained in terms of tension and demonstrating that MAPK activation is a quantitative reflection of the magnitude of mechanical stress applied to muscle. Thus the measurement of MAPK activation, as an assay of skeletal muscle mechanotransduction, may help elucidate mechanically induced hypertrophy.

Comparison of thermoregulatory responses between men and women immersed in cold water.

Eleven women (age = 24.4 +/- 6.3 yr, mass = 65.0 +/- 7.8 kg, height = 167 +/- 8 cm, body fatness = 22.4 +/- 5.9%, mean +/- SD) were immersed to neck level in 18 degrees C water for up to 90 min for comparison of their thermal responses with those of men (n = 14) in a previous similarly conducted protocol. Metabolic rate increased about three times resting levels in men and women, whereas the rate of rectal temperature cooling (DeltaT(re)/Deltat) in women (0.47 degrees C/h) was about one-half that in men. With use of all data, DeltaT(re)/Deltat correlates with the ratio of body surface area to size and the metabolic rate of shivering correlates inversely to the square root of body fatness. No significant gender differences in total metabolic heat production normalized for body mass or surface area were found among subjects who completed 90 min of immersion (9 women and 7 men). Nor was there a gender difference in the overall percent contribution ( approximately 60%) of fat oxidation to total heat production. Blood concentrations of free fatty acids, glycerol, beta-hydroxybutyrate, and lactate increased significantly during the 90-min immersion, whereas muscle glycogen sampled from the right quadriceps femoris vastus lateralis decreased (free fatty acids, glycerol, and beta-hydroxybutyrate were higher in women). When the subjects were subgrouped according to similar body fatness and 60 min of immersion (6 women and 5 men), no significant gender differences emerged in DeltaT(re)/Deltat, energy metabolism, and percent fat oxidation. These findings suggest that no gender adjustments are necessary for prediction models of cold response if body fatness and the ratio of body surface area to size are taken into account and that a potential gender advantage with regard to carbohydrate sparing during cold water immersion is not supported.

Clenbuterol, a beta 2-adrenergic agonist, reverses muscle wasting due to scald injury in the rat.

The effects of clenbuterol, a beta 2-adrenergic agonist, on body weight, tissue masses, and protein and RNA contents were studied following scald injury (30 per cent TBSA) in the rat. While the masses of heart, liver and epididymal fat pads remained unaffected, significant reductions in gastrocnemius, plantaris and soleus muscle masses (approximately 11 per cent; P < 0.01) were observed following injury, none of which were mimicked by pair-feeding or could be attributed to dehydration. This muscle wasting was accompanied by significant reductions in protein and/or RNA content. Oral administration of clenbuterol (4 mg/kg diet) had no anabolic effects, either in the scalded animals or their pair-fed controls. While clenbuterol (12 mg/kg diet) did not affect the masses of heart and fat pads, increases in the wet weights (approximately 20 per cent), RNA (approximately 30 per cent) and protein content (approximately 20 per cent) of the gastrocnemius and plantaris muscles were observed in all animals; the magnitude of these effects was greater (P < 0.05) in the scalded animals than in their pair-fed controls. Clenbuterol had no effect on body weight but increased (P < 0.001) carcass water content. These data indicate that there is a selective mobilization of muscle protein and sparing of fat in the early phase following burn injury, and that beta 2-adrenergic agonists, such as clenbuterol, may be of therapeutic value in inhibiting or reversing muscle atrophy associated with thermal injury.

Anion deposition into ferritin.

When the iron core of equine spleen ferritin is reduced, anions in solution cross the protein shell and enter the ferritin interior as part of a charge balancing reaction. Anion sequestration inside ferritin during iron core reduction was monitored using ion selective electrodes, inductively coupled plasma emission, and energy-dispersive X-ray spectroscopy. The requirement for anion translocation to the ferritin interior occurs because upon iron core reduction, two OH(-) ions per iron are released or neutralized inside ferritin leaving a net positive charge. Halides and oxoanions were tested as anionic substrates for this reaction. A general trend for the halides showed that the smaller halides accumulated inside ferritin in greater abundance than larger halides, presumably because the protein channels restrict the transfer of the larger anionic species. In contrast, oxoanion accumulation inside ferritin did not show selectivity based on size or charge. Vanadate and molybdate accumulated to the highest concentrations and nitrate, phosphate and tungstate showed poor accumulation inside ferritin. Fe(II) remains stably sequestered inside ferritin, as shown by electron microscopy and by column chromatography. Upon oxidation of the iron core, the anions are expelled from ferritin, and OH(-) ions coordinate to the Fe(III) to form the original Fe(O)OH mineral. Anion transport across the ferritin protein shell represents an important mechanism by which ferritin maintains proper charge balance inside the protein cavity.

Ferritin iron mineralization proceeds by different mechanisms in MOPS and imidazole buffers.

The buffer used during horse spleen ferritin iron loading significantly influences the mineralization process and the quantity of iron deposited in ferritin. Ferritin iron loading in imidazole shows a rapid hyperbolic curve in contrast to iron loading in 3-(N-morpholino)propanesulfonic acid (MOPS), which displays a slower sigmoidal curve. Ferritin iron loading in an equimolar mixture of imidazole and MOPS produces an iron-loading curve that is intermediate between the imidazole and MOPS curves indicating that one buffer does not dominate the reaction mechanism. The UV-visible spectrum of the ferritin mineral has a higher absorbance from 250 to 450 nm when prepared in imidazole buffer than in MOPS buffer. These results suggest that different mineral phases form in ferritin by different loading mechanisms in imidazole and MOPS buffered reactions. Samples of 1500 Fe/ferritin were prepared in MOPS or imidazole buffer and were analyzed for crystallinity and using the electron diffraction capabilities of the electron microscope. The sample prepared in imidazole was significantly more crystalline than the sample prepared in MOPS. X-ray powder diffraction studies showed that small cores (~500 Fe/ferritin) prepared in MOPS or imidazole possess a 2-line ferrihydrite spectrum. As the core size increases the mineral phase begins to change from 2-line to 6-line ferrihydrite with the imidazole sample favoring the 6-line ferrihydrite phase. Taken together, these results suggest that the iron deposition mechanism in ferritin can be controlled by properties of the buffer with samples prepared in imidazole forming a larger, more ordered crystalline mineral than samples prepared in MOPS.