Maternal and fetal vitamin D and their roles in mineral homeostasis and fetal bone development.

During pregnancy, female physiology adapts to meet the additional mineral demands of the developing fetus. Meanwhile, the fetus actively transports minerals across the placenta and maintains high circulating levels to mineralize the rapidly developing skeleton. Most of this mineral is accreted during the last trimester, including 30 g of calcium, 20 g of phosphate and 0.8 g of magnesium. Given the dependence of calcium homeostasis on vitamin D and calcitriol in the adult and child, it may be expected that vitamin D sufficiency would be even more critical during pregnancy and fetal development. However, the pregnant mother and fetus appear to meet their mineral needs independent of vitamin D. Adaptations in maternal mineral and bone metabolism during pregnancy appear to be invoked independent of maternal vitamin D, while fetal mineral metabolism and skeletal development appear to be protected from vitamin D deficiency and genetic disorders of vitamin D physiology. This review discusses key data from both animal models and human studies to address our current knowledge on the role of vitamin D and calcitriol during pregnancy and fetal development.

A Preliminary Study of Clinical Trial Enrollment Decisions Among People With Mild Cognitive Impairment and Their Study Partners.

OBJECTIVE: All Alzheimer disease (AD) clinical trials, including those enrolling patients with mild cognitive impairment (MCI), require dyadic participation. The purpose of this study was to elucidate how people with MCI and their study partners decide whether to enroll in clinical trials. METHODS: This was a mixed methods interview study. We interviewed patient participants with a consensus research diagnosis of MCI and their study partners. Interviews examined how dyads decide whether to enroll in a clinical trial and whether AD biomarker testing affects willingness to enroll. RESULTS: Though most MCI patients and study partners would decide in partnership whether to enroll in a clinical trial, agreement was lower among nonspousal, compared with spousal, dyads. Deterrents to enrollment included concerns about patient safety and inconvenience, especially for study partners. Motivators to enrollment included altruism, the desire to contribute to research, hope for patient benefit, and the desire to learn more about the patient's condition. When asked open-ended questions about motivators to enroll in trials, few patients cited access to biomarker testing specifically, though most expressed a desire to undergo biomarker testing when asked directly. CONCLUSION: Spousal and nonspousal MCI dyads may approach clinical trial decisions differently. Future research should investigate how AD biomarker testing affects participants' willingness to enroll in trials.

Pulmonary function in nonsmokers following exposure to sidestream cigarette smoke.

Ten healthy male and 10 healthy female, "never-smoking" subjects (ages 21-50) participated in a 5-day environmental room study to determine if an acute exposure to a high level of fresh diluted sidestream smoke (FDSS) would alter pulmonary function. On Monday, Tuesday, Thursday and Friday, the twenty subjects sat in an environmental room for 7.33 hours and were exposed to filtered and humidified air. On Wednesday, the twenty subjects were exposed in an environmental room for 7.33 hours to an average respirable suspended particle (RSP) concentration of 179 micrograms per m3 of FDSS generated by machine smoking Kentucky 1R4F reference cigarettes. This level of FDSS is 3.3 times the 95th percentile concentration of workplace environmental tobacco smoke exposure levels previously measured in the US. FVC and FEV1 decreased approximately 1.6% (p < 0.05) in both males and females after exposure. Similarly, PEF decreased approximately 1.3% (p < 0.03) following exposure. The observed decrease in pulmonary function was consistent with a "stress" related norepinephrine-induced alteration in blood flow leading to transient bronchoconstriction. Alternatively, a cholinergic reflex due to activation of bronchopulmonary C fibers may have also played a role in the transient bronchoconstriction. These small exposure-related decrements in pulmonary function were reversible.

Urinary mutagenicity in nonsmokers following exposure to fresh diluted sidestream cigarette smoke.

Ten healthy male and 10 healthy female 'never-smoking' subjects (ages 21-50) participated in a 5-day environmental room study to determine if an acute exposure to a high level of fresh diluted sidestream smoke (FDSS) would alter urinary mutagenicity. On Monday, Tuesday, Thursday and Friday, the 20 subjects sat in environmental rooms for 7.33h and were exposed to filtered and humidified air. On Wednesday, the 20 subjects were exposed in the environmental rooms for 7.33h to an average respirable suspended particle (RSP) concentration of 179 microg/m(3) of FDSS generated by machine smoking 1R4F Kentucky reference cigarettes. This level of FDSS is approximately three times the ETS level seen in the top 5% of US workplaces which allow smoking. A cumulative 7.33h air sample from each environmental room was collected and determined to be mutagenic by Ames Salmonella assay. Subjects' urinary mutagenicity was measured on Wednesday as compared with Tuesday or Thursday by assaying concentrates of 24h urine samples in Ames Salmonella bacterial strains TA98 and YG1024. Diet was strictly controlled on all study days, with broiled and pan-fried meat not served to minimize ingestion of mutagenic protein pyrolysis products. Although all the urinary mutagenicity values were within the range reported for minor changes in diet, the subjects experienced a small but statistically significant increase (p<0.05) in urinary mutagenicity in strain YG1024, but not in the less sensitive strain TA98 on the day of FDSS exposure.

Rule compliance and peer sociability: a study of family process, school-focused parent-child interactions, and children's classroom behavior.

This study examined the associations among family processes (cohesion, control, and conflict), school-focused parent-child interactions (support and pressure about achievement), and the child's own characteristics (assertiveness, frustration tolerance, intellectual effectiveness, and self-esteem) as correlates of rule compliance and peer sociability in the classroom. The sample consisted of 161 Grade 4 and 151 Grade 7 children. Family processes and parent-child interactions about school issues were associated with children's personal characteristics, which, in turn, predicted children's rule compliance and peer sociability. Some differences were found between the 4th- and 7th-grade samples; however, many variables consistently predicted the same outcomes across grades.

Reactions of sperm whale myoglobin with hydrogen peroxide. Effects of distal pocket mutations on the formation and stability of the ferryl intermediate.

Distal pocket mutants of sperm whale oxymyoglobin (oxy-Mb) were reacted with a 2.5-fold excess of hydrogen peroxide (HOOH) in phosphate buffer at pH 7.0, 37 degreesC. We describe a mechanism composed of three distinct steps: 1) initial oxidation of oxy- to ferryl-Mb, 2) autoreduction of the ferryl intermediate to ferric metmyoglobin (metMb), and 3) reaction of metMb with an additional HOOH molecule to regenerate the ferryl intermediate creating a pseudoperoxidase catalytic cycle. Mutation of Leu-29(B10) to Phe slows the initial oxidation reaction 3-fold but has little effect on the rate of ferryl reduction to ferric met-aquo-myoglobin. In contrast, the Val-68(E11) to Phe mutation causes a small, 60% increase in the initial oxidation reaction and a much larger 2. 5-fold increase in the rate of autoreduction. Double insertion of Phe at both the B10- and E11-positions (L29F/V68F) produces a mutant with oxidation characteristics of both single mutants, slow initial oxidation, and rapid autoreduction, but an extraordinarily high affinity for O2. Replacing His-64(E7) with Gln produces 3-4-fold increases in both processes. Combining the mutation H64Q with L29F results in a myoglobin with enhanced resistance to metMb formation in the absence of antioxidant enzymes (i.e. catalase and superoxide dismutase) due to its own high pseudoperoxidase activity, which rapidly removes any HOOH produced in the initial stages of autoxidation. This double substitution occurs naturally in the myoglobin of Asian elephants, and similar multiple replacements have been used to reduce selectively the rate of nitric oxide (NO)-induced oxidation of both recombinant MbO2 and HbO2 blood substitute prototypes without altering O2 affinity.

Cardiovascular effects of odors.

Several occupational and residential settings can expose both normal and sensitive human subjects to odors and irritants. These settings include intensive agricultural operations housing swine and poultry, cigarette-smoke-filled bars, landfills and manufacturing processes. The literature suggests that adverse sensory reactions to strong odors and irritants may lead to the release of catecholamines and stress hormones. Physiological and biochemical measurements related to cardiovascular risk, e.g., blood pressure, heart rate, high-density lipoprotein (HDL) cholesterol level and serum triglyceride level, may be altered as a result of exposure to odor and irritant-induced release of catecholamines. Further work in the form of field studies and chamber exposure protocols is required to determine whether the physiological and biochemical changes observed to date represent an increase in cardiovascular risk, or are reversible changes within the normal homeostatic range.

Acellular hemoglobin-mediated oxidative stress toward endothelium: a role for ferryl iron.

We tested the hypothesis that chemical modifications used to produce stable, oxygen-carrying, Hb-based blood substitutes can induce cytotoxicity in endothelial cells in culture because of altered redox activity. We examined the interaction of hydrogen peroxide with nonmodified hemoglobin (HbA0) and two chemically modified hemoglobins, alpha-cross-linked hemoglobin (alpha-DBBF) and its polymerized form (poly-alpha-DBBF). Hydrogen peroxide-induced cell death (as assessed by lactate dehydrogenase release) in bovine aortic endothelial cells (BAEC) was completely inhibited by all three hemoglobin preparations, consistent with their known pseudoperoxidase activity [hemoglobin consumes peroxide as it cycles between ferric (Fe3+) and ferryl (Fe4+) hemes]. However, reaction of the modified hemoglobins, but not HbA0, with hydrogen peroxide induced apoptotic cell death (as assessed by morphological changes and DNA fragmentation) that correlated with the formation of a long-lived ferrylhemoglobin. A preparation of ferryl-alpha-DBBF free of residual peroxide rapidly induced morphological changes and DNA fragmentation in BAEC, indicative of apoptotic cell death. Redox cycling of chemically modified hemoglobins by peroxide yielded a persistent ferryl iron that was cytotoxic to endothelial cells.

Hemoglobin A0 and alpha-crosslinked hemoglobin (alpha-DBBF) potentiate agonist-induced platelet aggregation through the platelet thromboxane receptor.

Chemically modified hemoglobins are potential oxygen-carrying blood substitutes, but their in vivo administration has been associated with a variety of unexpected side events, including increased platelet reactivity. We studied the effects of hemoglobin A0 (HbA0) and alpha-crosslinked hemoglobin (alpha-DBBF) on platelets in vitro. Neither hemoglobin A0 nor alpha-DBBF activated platelets when added alone, but both proteins potentiated submaximal agonist-induced platelet aggregation without increasing other markers of platelet activation such as serotonin secretion. Only agonists that are known to cause release of platelet arachidonic acid (AA) were potentiated while aggregation induced by ADP, which does not release AA, was not potentiated. Blockade of the thromboxane receptor with SQ-29,548 prevented the HbA0-induced and the alpha-DBBF-induced potentiation suggesting that the AA/thromboxane signaling pathway mediates the interaction of platelets with hemoglobin.

Peroxynitrite-mediated heme oxidation and protein modification of native and chemically modified hemoglobins.

Peroxynitrite (ONOO-) has been shown to play a critical role in tissue reperfusion injury. We have studied the reactions of ONOO- with native and two chemically modified hemoglobins that are being developed as oxygen-carrying reperfusion agents for use in a variety of clinical conditions. Reactions of native and chemically modified oxyhemoglobins (oxyHb) at 7.4 with ONOO- lead to a rapid oxidation of the heme iron to ferric (HbFe3+) form. Addition of excess molar ratios of ONOO- to the ferryl (HbFe4+) heme protein induced a spectral change indicative of the reduction of HbFe4+ to the HbFe3+ oxidation state. No major spectral changes were noted when ONOO- was added to methemoglobin (HbFe3+) or cyanomethemoglobin (Hb3+CN-), whereas the carbonmonoxy derivative of ferrous hemoglobin (HbCO) underwent an immediate spectral change suggesting the displacement of the CO ligand and oxidation of the heme iron. Rapid mixing of ONOO- with oxyHb in the stopped-flow spectrophotometer yielded biphasic kinetic plots for the oxidation of the ferrous iron (Fe2+). Replots of the apparent rate constants for native, cross-linked and polymerized, cross-linked hemoglobins as a function of ONOO- concentration were linear, yielding a single second-order rate for all hemoglobins of between 2 to 3 x 10(4) M-1 s-1, independent of the oxygen affinities and molecular sizes of the proteins. Oxidative modifications of the protein by ONOO-, occurring primarily at the beta subunits, were observed in reaction mixtures of oxyHb and ONOO- using reverse-phase HPLC. The immuno-detection method confirms that nitration of tyrosine residues by ONOO- occurs on the hemoglobin molecule and contributes to the modifications observed. We postulate that the presence of hemoglobin in close proximity to ONOO- production sites in the vasculature can contribute to possible in vivo toxicity by a two-step mechanism involving (i) direct oxidation of the heme iron and (ii) nitration of the tyrosine residues on the molecule, leading to subsequent instability and heme loss from the protein.

Redox reactivity of modified hemoglobins with hydrogen peroxide and nitric oxide: toxicological implications.

The rapid unloading of oxygen to tissue and the prevention of subunit dissociation have been the main concerns in the search for an effective hemoglobin-based red cell substitute. The presence of redox active iron however, raises some questions about its potential to enter into reactions that mediate the formation of cytotoxic oxygen free radicals. We tested the propensity of modified hemoglobins to undergo oxidative damage by peroxide (H2O2). We found differences in their susceptibility to oxidative modification and in their ability to form the highly cytotoxic ferryl species. This protein-associated oxidant may be a physiologically important contributor to reperfusion injury. Another potential mechanism of toxicity involves the reaction of cell-free hemoglobin with endothelium derived nitric oxide (NO). Marked hypertensive responses in intact animals infused with some of these hemoglobins were reported. Cell-free hemoglobin has the potential to bind the endothelial generated NO yielding methemoglobin and nitrate, an extremely rapid reaction in vivo. We describe subsequent redox reactions between NO and methemoglobin which may further deplete NO as a biological transducer, leading to greater effects on the extent of endothelial-dependent responses. The consequences of a potential linkage between oxidative toxicity of cell-free hemoglobin and its interaction with NO is addressed.

Oxidation reactions of human, opossum (Didelphis virginiana) and spot (Leiostomus xanthurus) hemoglobins: a search for a correlation with some structural-functional properties.

1. Relative to human HbA, opossum (Didelphis virginiana) hemoglobin was found to be more susceptible to autoxidation. While the initial rate of autoxidation of spot (Leiostomus xanthurus) hemoglobin is close to that of HbA, complete oxidation occurs in 50 hr. 2. Direct addition of hydrogen peroxide (H2O2) induced oxidation of hemoglobins in a definite order: spot Hb > HbA > opossum Hb. Excess H2O2 led to heme degradation and precipitation that occurred much faster for spot Hb than the case with other proteins. 3. Exposure of hemoglobins to a continuous flux of H2O2, generated by the glucose/glucose oxidase system, induced the formation of heterogeneous protein-associated oxidation products. 4. Differential reactivity among these hemoglobins under the same or different oxidative conditions, with respect to methemoglobin formation and stability of the ferric form, may reflect the differences in the local heme environment of these proteins.

Hemoglobin-based oxygen carriers (HBOCs): structural alterations that affect free radical generation.

We examined how changes in oxygen affinity brought about by different chemical modifications of hemoglobins affect their oxidation-reduction reactions. The three modified hemoglobins studied were HbA-FMDA, HbBv-FMDA, produced by the reaction of human or bovine oxyHb with fumaryl mono-dibromoaspirin; and HbA-DBBF, produced by the reaction of human deoxyHb with bis(3,5-dibromosalicyl) fumarate. Exposure of oxyHb to H2O2 causes generation of free radicals capable of cleaving dimethylsulfoxide (Me2SO) to produce formaldehyde (HCHO). Relative to the reaction rate for HbAo (630 +/- 130 M/min) the rates of HCHO formation were roughly 70% for HbA-DBBF, 50% for HbA-FMDA and 16% for HbBv-FMDA. Exposure to H2O2 also caused spectral changes at varied rates for the HBOCs analyzed. Although these rates were not directly correlated with the rates of free radical formation, addition of mannitol or thiourea slowed both the rate of spectral changes and HCHO formation. The relative ability of the ferric derivatives of the HBOCs to participate in free radical reactions was monitored by assays of non-enzymatic NADPH oxidation and aniline hydroxylation. HbBv-FMDA showed significantly slower rates than the other HBOCs in both assays. The observed differences between HBOCs in these assays indicate differences in their ability to generate or interact with free radicals.

Effects of hypertonic saline (7.5%)/dextran 70 on human red cell typing, lysis, and metabolism in vitro.

The introduction of a 7.5% hypertonic saline/6% dextran 70 (HSD) solution into clinical trials for the treatment of hypovolemic states, and the past concerns regarding the possible interference of dextran with blood serology, prompted us to investigate the effects of HSD on human red-cell typing and stability. HSD was evaluated with fresh and 35-day stored CPDA-1 red cells from 12 healthy donors. A 1:5 mixture of HSD to blood in vitro had no effect on ABO, Rh, and MN typing in both fresh and stored blood. HSD produced no significant lysis with fresh cells and a minimal level with stored blood. No evidence of metabolic or morphologic changes was seen after HSD treatment. The results of this study suggest that the clinical use of HSD for the treatment of hemorrhagic shock will not affect blood group determinations or red-cell stability from stored blood which may be infused after the HSD-treated patient is transported to a hospital.

Dextran concentrations in plasma and urine following administration of 6% dextran-70/7.5% NaCl to hemorrhaged and euvolemic conscious swine.

Dextran metabolism was investigated in ten hemorrhaged and seven euvolemic conscious swine. Chronically instrumented, splenectomized swine were subjected to a progressive fixed-volume hemorrhage (27 ml/kg over 45 min). Resuscitation with 4 ml/kg of a 7.5% NaCl/6% dextran 70 (HSD) solution was begun 5 min later. Blood and urine samples were drawn before and during hemorrhage, and at 15, 60, and 120 min following intravenous HSD infusion. Hemorrhage significantly reduced cardiac output (CO) and mean arterial pressure (MAP) and eliminated urinary flow. HSD administration to hemorrhaged pigs returned CO and MAP to control values and improved urinary flow. Creatinine clearance returned to prehemorrhage values. These parameters were not affected by HSD in nonhemorrhaged animals. Plasma dextran concentrations were 20-30% higher in hemorrhaged pigs compared with euvolemic swine. In additional studies, plasma t1/2 for dextran was 9.4 hr in hemorrhaged pigs (n = 3) compared to 10.8 hr in euvolemic animals (n = 2). These data show that HSD ameliorates the effects of hemorrhage on cardiovascular and renal function and suggest that plasma clearance of dextran may be affected by hemorrhage.

A growth-limiting, mild zinc-deficiency syndrome in some southern Ontario boys with low height percentiles.

A double-blind, pair-matched 12-mo study examined the effects of a zinc supplement (10 mg Zn/d as ZnSO4) on linear growth, taste acuity, attention span, biochemical indices, and energy intakes of 60 boys (aged 5-7 y) with height less than or equal to 15th and midparent height greater than 25th percentiles. Boys with initial hair Zn less than 1.68 mumol/g (n = 16) had a lower mean (+/- SD) weight-for-age Z score (-0.44 +/- 0.59 vs -0.08 +/- 0.84), and a higher median recognition threshold for salt (15 vs 7.5 mmol; p = 0.02) than those with hair Zn greater than 1.68 mumol/g. Only boys with hair Zn less than 1.68 mumol/g responded to the Zn supplement with a higher mean change in height-for-age Z score (p less than 0.05); taste acuity, energy intakes, and attention span were unaffected. A growth-limiting Zn deficiency syndrome exists in boys with low height percentiles, hair Zn levels less than 1.68 mumol/g, and impaired taste acuity.