Effects of Dietary Factors on the Pharmacokinetics of 58Fe-labeled Hemin After Oral Administration in Normal Rats and the Iron-deficient Rats

Hemin, iron (III) protoporphyrin chloride (IX), as a stable form of heme iron, has been used in iron absorption studies. The aim of the present study was to elucidate the influences of body iron status and three dietary factors (green tea extract, ascorbic acid, and calcium) on the pharmacokinetics of hemin using stable isotope labeling methods followed by ICP-MS measurement. In this study, a rapid, sensitive, and specific ICP-MS method for the determination of ⁵⁸Fe originating from hemin in rat plasma was developed and a rat model of iron deficiency anemia was established. It was found that hemin iron absorption increased significantly under iron deficiency anemia status, with AUC_0?t and AUC_0–∞ showing significant increase in anemic rats compared to normal ones. Green tea extract strongly inhibited hemin iron absorption in both normal rats and iron-deficient rats. In normal rats administered with green tea extract, C _max resulted significantly reduced, whereas in anemic rats administered with green tea extract both AUC_0?t and AUC_0–∞ were reduced. On the other hand, ascorbic acid significantly affected hemin iron absorption only in iron-deficient rats, in which C _max showed a significant increase. Interestingly, calcium slowed down the hemin iron absorption rate in normal rats, MRT_0–t being significantly different in calcium-treated animals compared to untreated ones. This trend also appeared in the iron-deficient group but it did not reach statistical significance. Our data suggest that the mechanism of hemin iron absorption is regulated by body iron status and dietary factors can influence hemin iron absorption to varying degrees. Moreover, these results may also have general implication in the iron deficiency treatment with iron supplements and fortification of foods.     

Effect of phytic acid,tannic acid and pectin on fasting iron bioavailability both in the presence and absence of calcium

ObjectiveTo determine the effect of phytic acid, tannic acid and pectin on fasting non-heme iron bioavailability in both the presence and absence of calcium.Research methodsTwenty-eight apparently healthy adult females participated in two iron absorption studies using radioactive iron isotopes (⁵⁹Fe and ⁵⁵Fe). One group received 5 mg of iron (as FeSO₄) alone (control), together with 10 mg of phytic acid, 100 mg of tannic acid and 250 mg of pectin (study A), on different days. The second group received the same iron doses and compounds as the other group, plus 800 mg of calcium (CaCl₂) (study B). The compounds were administered after an overnight fast, and no food or beverages were consumed for the following 3 h. Iron status and circulating radioactivity were measured in venous blood samples.ResultsThe geometric means of iron bioavailability (range ± 1SD) for iron alone, iron with phytic acid, iron with tannic acid, and iron with citrus pectin were 25.0% (11.9–52.0); 18.9% (9.9–35.8); 16.8% (8.7–32.3); and 21.1% (10.2–43.9), respectively (repeated-measures ANOVA, p < 0.02 (Dunnett's post hoc: control vs tannic acid p < 0.05). When 800 mg of calcium was added (study B), iron bioavailability was 16.7% (10.1–27.5); 13.2% (7.1–24.6); 14.8% (8.8–25.1); and 12.6% (5.5–28.8), respectively (repeated-measures ANOVA, NS).ConclusionsTannic acid decreases the fasting bioavailability of non-heme iron, however this effect did not exist in the presence of calcium. No effect was observed by phytic acid or citrus pectin on fasting non-heme iron bioavailability in both the presence and absence of calcium.     

Body mass index,iron absorption and iron status in childbearing age women

BackgroundThe prevalence of obesity has increased at an alarming rate worldwide. Some studies have observed an association between iron (Fe) deficiency (ID) and obesity, however more research is needed.ObjectiveTo assess whether body mass index (BMI) is associated with both Fe absorption and Fe status.MethodsA cross sectional sample of 318 Chilean childbearing age women was studied. The women received either a single dose of 0.5 mg of Fe (n = 137, group 1) or 3 mg of Fe plus ascorbic acid (1:2 molar ratio) (n = 181, group 2), both as FeSO₄ with labeled radioisotopes. Fe absorption was assessed through radio Fe erythrocyte incorporation. Fe status was determined by hemoglobin (Hb), mean corpuscular volume, serum Fe, total iron binding capacity, transferrin saturation, erythrocyte Zn protoporphyrin and serum ferritin (SF).Results29%, 47% and 24% of the women were classified as normal, overweight or obese, respectively. Fe absorption was significantly lower in obese women (p < 0.05). In group 1, the geometric mean and range ±1 SD of the percentage of Fe absorption for normal-weight women was 32.9% vs. 19.7% in obese. For group 2, this percentage was 36% vs. 30%, respectively (2-way ANOVA: BMI classification and Fe dose p < 0.05; interaction p = 0.34). Although Fe absorption was lower in obese women, they had higher SF (p < 0.01) and Hb (p < 0.05) concentrations.ConclusionAlthough we did not observe a relationship between BMI and Fe status, obese women displayed lower Fe absorption compared with overweight and normal weight women, possibly due to subclinical inflammation associated with obesity.     

Supramolecular interaction of 6-shogaol,a therapeutic agent of Zingiber officinale with human serum albumin as elucidated by spectroscopic,calorimetric and molecular docking methods

Background6-Shogaol, one of the main bioactive constituents of Zingiber officinale has been shown to possess various therapeutic properties. Interaction of a therapeutic compound with plasma proteins greatly affects its pharmacokinetic and pharmacodynamic properties.PurposeThe present investigation was undertaken to characterize the interaction between 6-shogaol and the main in vivo transporter, human serum albumin (HSA).MethodsVarious binding characteristics of 6-shogaol–HSA interaction were studied using fluorescence spectroscopy. Thermal stability of 6-shogaol–HSA system was determined by circular dichroism (CD) and differential scanning calorimetric (DSC) techniques. Identification of the 6-shogaol binding site on HSA was made by competitive drug displacement and molecular docking experiments.ResultsFluorescence quench titration results revealed the association constant, K_a of 6-shogaol–HSA interaction as 6.29 ± 0.33 × 10⁴ M⁻¹ at 25 ºC. Values of the enthalpy change (−11.76 kJ mol⁻¹) and the entropy change (52.52 J mol⁻¹ K⁻¹), obtained for the binding reaction suggested involvement of hydrophobic and van der Waals forces along with hydrogen bonds in the complex formation. Higher thermal stability of HSA was noticed in the presence of 6-shogaol, as revealed by DSC and thermal denaturation profiles. Competitive ligand displacement experiments along with molecular docking results suggested the binding preference of 6-shogaol for Sudlow's site I of HSA.ConclusionAll these results suggest that 6-shogaol binds to Sudlow's site I of HSA through moderate binding affinity and involves hydrophobic and van der Waals forces along with hydrogen bonds.     

2-(3,5-Dibromo-4-hydroxyphenyl)imidazo[4,5-<Emphasis Type="Italic">f</Emphasis>][1,10]phenanthrolinoruthenium(II) complexes: synthesis,characterization, cytotoxicity,apoptosis, DNA-binding and antioxidant activity

A new ligand DBHIP and its two ruthenium(II) complexes [Ru(dmb)₂(DBHIP)](ClO₄)₂ (1) and [Ru(dmp)₂(DBHIP)](ClO₄)₂ (2) have been synthesized and characterized. The cytotoxicity of DBHIP and complexes 1 and 2 has been assessed by MTT assay. The apoptosis studies were carried out with acridine orange/ethidium bromide (AO/EB) staining methods. The binding behaviors of these complexes to calf thymus DNA (CT-DNA) were studied by absorption titration, viscosity measurements, thermal denaturation and photoactivated cleavage. The DNA-binding constants of complexes 1 and 2 were determined to be 8.64 ± 0.16 × 10⁴ (s = 1.34) and 2.79 ± 0.21 × 10⁴ (s = 2.17) M⁻¹. The results suggest that these complexes interact with DNA through intercalative mode. The studies on the mechanism of photocleavage demonstrate that superoxide anion radical (O₂ ^•–) and singlet oxygen (¹O₂) may play an important role in the DNA cleavage. The experiments on antioxidant activity show that these compounds also exhibit good antioxidant activity against hydroxyl radical (OH^•).     

Heme-mediated binding of α-casein to ferritin: evidence for preferential α-casein binding to ferrous iron

Bovine milk α-casein was identified as a ferritin-binding protein, and ferritin is known to be a heme-binding protein. In this study, we found that the binding of α-casein to bovine spleen ferritin in vitro was blocked by hemin, but not by iron-free hemin (protoporphyrin IX) or zinc-protoporphyrin IX, suggesting that the presence of iron in heme play a key role in this interaction. Indeed, the binding of α-casein to ferritin and biotinylated hemin was inhibited by adding excess ferrous ammonium sulfate (FAS). To further elucidate the binding mechanism of α-casein to biotinylated hemin, Ferrozine and nitrilotriacetic acid (NTA) were used as ferrous and ferric iron chelators, respectively. FAS-mediated inhibition of α-casein to biotinylated hemin was neutralized with Ferrozine, but not NTA, while FAS- as well as ferric chloride-mediated inhibition in their interaction was neutralized by NTA. The following ions also inhibited α-casein-biotinylated hemin binding in order of potency of inhibition: FAS (Fe²⁺) ≪ ferric chloride (Fe³⁺) < copper sulfate (Cu²⁺) < zinc sulfate (Zn²⁺) < manganese chloride (Mn²⁺) < calcium chloride (Ca²⁺) < magnesium sulfate (Mg²⁺). These results suggests that the binding of α-casein to ferritin is heme-mediated through direct binding of α-casein to iron in the heme on the surface of ferritin molecule, and that α-casein preferentially binds Fe²⁺ compared with any other metal ions, including Fe³⁺.     

Isotope Ratio Measurements of Iron in Blood Samples by Multi-collector ICP-MS to Support Nutritional Investigations in Humans

With the perspective of embarking on a human study using a double iron (Fe) stable isotope tracer protocol to assess iron bioavailability, investigations were conducted on Fe isotope ratios in blood samples using a VG Axiom Multi-collector ICP-MS. The factors affecting the precision and accuracy of Fe isotopic ratios, such as spectral- and matrix-induced interferences and Fe recoveries from sample preparation, have been identified and optimized. Major polyatomic interferences (e.g., Ar-O, Ar-OH, and FeH) were significantly reduced by using an Aridus nebulizer and desolvating system. Isobaric metal (e.g., ⁵⁴Cr⁺ on ⁵⁴Fe⁺ and ⁵⁸Ni⁺ on ⁵⁸Fe⁺) interferences and Ca-oxides and hydroxides were quantitatively removed during chemical purification of blood samples and selective isolation of Fe by anion-exchange resin, after mineralization of the blood samples by microwave digestion. Quantitative recoveries of Fe from different steps of sample preparation were verified using whole blood reference material. Fe isotopic compositions of the samples were corrected for instrumental mass bias by the standard-sample bracketing method using the certified reference standard IRMM-014. External precisions on the order of 0.008–0.05 (% RSD), 0.007–0.015 (% RSD), and 0.03–0.09 (% RSD) were obtained for ⁵⁴Fe/⁵⁶Fe, ⁵⁷Fe/⁵⁶Fe, and ⁵⁸Fe/⁵⁶Fe, respectively, in the blood for three replicate measurements. The level of precision obtained in this work enables the detection of low enrichments of Fe in blood, which is highly desired in nutrition tracer studies.     

Transferrin receptors,iron transport and ferritin metabolism in friend erythroleukemia cells

Four aspects of iron metabolism were studied in cultured Friend erythroleukemia cells before and after induction of erythroid differentiation by dimethyl sulfoxide. (1) The binding of ¹²⁵I-labeled transferrin was determined over a range of transferrin concentrations from 0.5 to 15 μM. Scatchard analysis of the binding curves demonstrated equivalent numbers of transferrin binding sites per cell: 7.78 ± 2.41 · 10⁵ in non-induced cells and 9.28 ± 1.57 · 10⁵ after 4 days of exposure to dimethyl sulfoxide. (2) The rate of iron transport was determined by measuring iron uptake from ⁵⁹Fe-labeled transferrin. Iron uptake in non-induced cells was approx. 17 000 molecules of iron/cell per min; 24 h after addition of dimethyl sulfoxide it increased to 38 000, and it rose to maximal levels of approx. 130 000 at 72 h. (3) Heme synthesis, assayed qualitatively by benzidine staining and measured quantitatively by incorporation of ⁵⁹Fe or [2-¹⁴C]glycine into cyclohexanone-extracted or crystallized heme, was not detected until 3 days after addition of dimethyl sulfoxide, when 12% of the cells were stained by benzidine and 6 pmol ⁵⁹Fe and 32 pmol [2-¹⁴C]glycine were incorporated into heme per 10⁸ cells/h. After 4 days, 60% of the cells were benzidine positive and 34 pmol ⁵⁹Fe and 90 pmol [2-¹⁴C]glycine were incorporated into heme per 10⁸ cells/h. (4) The rate of incorporation of ⁵⁹Fe into ferritin, measured by immunoprecipitation of ferritin by specific antimouse ferritin immunoglobulin G, rose from 4.4 ± 0.6 cells to 18.4 ± 1.3 pmol ⁵⁹Fe/h per 10⁸ cells 3 days after addition of dimethyl sulfoxide, and then fell to 11.6 ± 3.1 pmol 4 days after dimethyl sulfoxide when heme synthesis was maximal. These studies indicate that one or more steps in cellular iron transport distal to transferrin binding is induced early by dimethyl sulfoxide and that ferritin may play an active role in iron delivery for heme synthesis.     

Non-Heme Iron Absorption and Utilization from Typical Whole Chinese Diets in Young Chinese Urban Men Measured by a Double-Labeled Stable Isotope Technique

BackgroundThis study was to observe the non-heme iron absorption and biological utilization from typical whole Chinese diets in young Chinese healthy urban men, and to observe if the iron absorption and utilization could be affected by the staple food patterns of Southern and Northern China.ResultsNon-heme iron intake values overall, and in the rice and steamed buns groups were 12.8 ±2.1, 11.3±1.3 and 14.3±1.5 mg, respectively; the mean ⁵⁷Fe absorption rates were 11±7%, 13±7%, and 8±4%, respectively; and the mean infused ⁵⁸Fe utilization rates were 85±8%, 84±6%, and 85±10%, respectively. There was no significantly difference in the iron intakes, and ⁵⁷Fe absorption and infused ⁵⁸Fe utilization rates between rice and steamed buns groups (all P>0.05).ConclusionWe present the non-heme iron absorption and utilization rates from typical whole Chinese diets among young Chinese healthy urban men, which was not affected by the representative staple food patterns of Southern and Northern China. This study will provide a basis for the setting of Chinese iron DRIs.     

Rates of intercalator-driven platination of DNA determined by a restriction enzyme cleavage inhibition assay

A restriction enzyme cleavage inhibition assay was designed to determine the rates of DNA platination by four non-cross-linking platinum–acridine agents represented by the formula [Pt(am₂)LCl](NO₃)₂, where am is a diamine nonleaving group and L is an acridine derived from the intercalator 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea (ACRAMTU). The formation of monofunctional adducts in the target sequence 5′-CGA was studied in a 40-base-pair probe containing the EcoRI restriction site GAATTC. The time dependence of endonuclease inhibition was quantitatively analyzed by polyacrylamide gel electrophoresis. The formation of monoadducts is approximately 3 times faster with double-stranded DNA than with simple nucleic acid fragments. Compound 1 (am₂ is ethane-1,2-diamine, L is ACRAMTU) reacts with a first-order rate constant of k _obs = 1.4 ± 0.37 × 10⁻⁴ s⁻¹ (t _1/2 = 83 ± 22 min). Replacement of the thiourea group in ACRAMTU with an amidine group (compound 2) accelerates the rate by fourfold (k _obs = 5.7 ± 0.58 × 10⁻⁴ s⁻¹, t _1/2 = 21 ± 2 min), and introduction of a propane-1,3-diamine nonleaving group results in a 1.5-fold enhancement in reactivity (compound 3, k _obs = 2.1 ± 0.40 × 10⁻⁴ s⁻¹, t _1/2 = 55 ± 10 min) compared with the prototype. Derivative 4, containing a 4,9-disubstituted acridine threading intercalator, was the least reactive compound in the series (k _obs = 1.1 ± 0.40 × 10⁻⁴ s⁻¹, t _1/2 = 104 ± 38 min). The data suggest a correlation may exist between the binding rates and the biological activity of the compounds. Potential pharmacological advantages of rapid formation of cytotoxic monofunctional adducts over the common purine–purine cross-links are discussed.     

Structural characteristic,pH and thermal stabilities of apo and holo forms of caprine and bovine lactoferrins

Apo and holo forms of lactoferrin (LF) from caprine and bovine species have been characterized and compared with regard to the structural stability determined by thermal denaturation temperature values (T _m), at pH 2.0–8.0. The bovine lactoferrin (bLF) showed highest thermal stability with a T _m of 90 ± 1°C at pH 7.0 whereas caprine lactoferrin (cLF) showed a lower T _m value 68 ± 1°C. The holo form was much more stable than the apo form for the bLF as compared to cLF. When pH was gradually reduced to 3.0, the T _m values of both holo bLF and holo cLF were reduced showing T _m values of 49 ± 1 and 40 ± 1°C, respectively. Both apo and holo forms of cLF and bLF were found to be most stable at pH 7.0. A significant loss in the iron content of both holo and apo forms of the cLF and bLF was observed when pH was decreased from 7.0 to 2.0. At the same time a gradual unfolding of the apo and holo forms of both cLF and bLF was shown by maximum exposure of hydrophobic regions at pH 3.0. This was supported with a loss in α-helix structure together with an increase in the content of unordered (aperiodic) structure, while β structure seemed unchanged at all pH values. Since LF is used today as fortifier in many products, like infant formulas and exerts many biological functions in human, the structural changes, iron binding and release affected by pH and thermal denaturation temperature are important factors to be clarified for more than the bovine species.     

Energy-dispersive X-ray fluorescence spectrometry as a tool for zinc, iron and selenium analysis in whole grain wheat

Background and aims

Crop biofortification programs require fast, accurate and inexpensive methods of identifying nutrient dense genotypes. This study investigated energy-dispersive X-ray fluorescence spectrometry (EDXRF) for the measurement of zinc (Zn), iron (Fe) and selenium (Se) concentrations in whole grain wheat.

Methods

Grain samples were obtained from existing biofortification programs. Reference Zn, Fe and Se concentrations were obtained using inductively coupled plasma optical emission spectrometry (ICP-OES) and/or inductively coupled plasma mass spectrometry (ICP-MS). One set of 25 samples was used to calibrate for Zn (19–60?mg?kg^–1) and Fe (26–41?mg?kg^–1), with 25 further samples used to calibrate for Se (2–31?mg?kg^–1 ). Calibrations were validated using an additional 40–50 wheat samples.

Results

EDXRF limits of quantification (LOQ) were estimated as 7, 3 and 2?mg?kg^–1 for Zn, Fe, and Se, respectively. EDXRF results were highly correlated with ICP-OES or -MS values. Standard errors of EDXRF predictions were ±2.2?mg Zn kg^–1, ±2.6?mg Fe kg^–1, and ±1.5?mg Se kg^–1.

Conclusion

EDXRF offers a fast and economical method for the assessment of Zn, Fe and Se concentration in wheat biofortification programs.     

Near real-time magnetic particle imaging for visual assessment of vascular stenosis in a phantom model

PurposeThis study aimed to investigate the potential of magnetic particle imaging (MPI) to quantify artificial stenoses in vessel phantoms in near real-time.MethodsCustom-made stenosis phantoms with different degrees of stenosis (0%, 25%, 50%, 75%, and 100%; length 40 mm, inner diameter 8 mm, Polyoxymethylene) were filled with diluted Ferucarbotran (superparamagnetic iron-oxide nanoparticle (SPION) tracer agent, 500 mmol (Fe)/l). A traveling wave MPI scanner (spatial resolution ~ 2 mm, gradient strength ~ 1.5 T/m, field of view: 65 mm length and 29 mm diameter, frequencies f₁ = 1050 Hz and f₂ = 12150 Hz) was used to acquire images of the phantoms (200 ms total acquisition time per image, 10 averages). Standardized grey scaling was used for comparability. All measured stenoses (n = 80) were graded manually using a dedicated software tool.ResultsMPI allowed for accurate visualization of stenoses at a frame rate of 5 frames per second. Less severe stenoses were detected more precisely than higher-grade stenoses and came with smaller standard deviations. In particular, the 0%, 25%, 50%, 75%, and 100% stenosis phantom were measured as 3.7 ± 2.7% (mean ± standard deviation), 18.6 ± 1.8%, 52.8 ± 3.7%, 77.8 ± 14.8% and 100 ± 0%. Geometrical distortions occurred around the center of the high-grade stenosis and led to higher standard deviations compared to lower grade stenoses. In the frame of this study the MPI signal depended linearly on the SPION concentration down to 0.05 mmol (Fe)/l.ConclusionNear real-time MPI accurately visualized and quantified different stenosis grades in vascular phantoms.     

β2-Adrenoceptor Agonist-Induced Down-Regulation After Short-Term Exposure

Abstract

We examined the effect of duration of β₂-adrenergic receptor (β₂AR) occupancy by isoproterenol on specific binding of ¹²⁵l-lodocyanopindolol (¹²⁵I-ICYP) in membranes from rat L6 myoblasts. Ten minute exposure caused a time- and concentration-dependent maximal decrease in ¹²⁵-?YP binding 24 hours after exposure equal to that following continuous exposure (p < 0.05). Low temperature, concanavalin A, H89 and ICI 118,551 blocked the decline in ¹²⁵I-ICYP binding during the first hour following exposure probably representing receptor sequestration to a compartment or change to a form incapable of ligand binding. Compared to controls, receptor binding 4 and 24 hours following exposure was reduced 56 ± 8.7% and 72 ± 8.8%, respectively (p < 0.05), and was blocked by ICI 118,551 but not CGP12177. Isoproterenol-induced, but not forskolinstimulated, cAMP accumulation was reduced 35% 24 hours following exposure (p < 0.05). ¹²⁵I-ICYP binding in intact L6 cells 4 and 24 hours after exposure were respectively 56 ± 8.9 and 61 ± 13% of controls (p < 0.05). Following agonist exposure, CHO cell membranes expressing human β₂ARs exhibited ¹²⁵I-ICYP binding 85 ± 2.0% and 6 ± 2.8% of control values 4 and 24 hours, respectively (p < 0.05). A model predicting that full occupation of the β₂AR activates receptor degradation explains our results that agonist-induced down-regulation of β₂AR does not require continuous presence of the agonist.     

Erythrocyte permeability to urea and water: comparative study in rodents,ruminants, carnivores,humans, and birds

Mammalian erythrocytes exhibit high urea permeability (P _urea) due to UT-B expression in their cytoplasmic membrane. This high P _urea allows fast equilibration of urea in erythrocytes during their transit in the hyperosmotic renal medulla. It also allows more urea (in addition to that in plasma) to participate in counter-current exchange between ascending and descending vasa recta, thus improving the trapping of urea in the medulla and improving urine concentrating ability. To determine if P _urea in erythrocytes is related to diet and urine concentrating ability, we measured P _urea in erythrocytes from 11 different mammals and 5 birds using stopped-flow light scattering. Carnivores (dog, fox, cat) exhibited high P _urea (in ×10⁻⁵ cm/s, 5.3 ± 0.6, 3.8 ± 0.5 and 2.8 ± 0.7, respectively). In contrast, herbivores (cow, donkey, sheep) showed much lower P _urea (0.8 ± 0.2, 0.7 ± 0.2, 1.0 ± 0.1, respectively). Erythrocyte P _urea in human (1.1 ± 0.2), and pig (1.5 ± 0.1), the two omnivores, was intermediate. Rodents and lagomorphs (mouse, rat, rabbit) had P _urea intermediate between carnivores and omnivores (3.3 ± 0.4, 2.5 ± 0.3 and 2.4 ± 0.3, respectively). Birds that do not excrete urea and do not express UT-B in their erythrocytes had very low values (<0.1 × 10⁻⁵ cm/s). In contrast to P _urea, water permeability, measured simultaneously, was relatively similar in all mammals. The species differences in erythrocytes P _urea most probably reflect adaptation to the different types of diet and resulting different needs for concentrating urea in the urine.     

安徽某铁矿不同矿山废水库中微生物群落结构特征

【目的】研究安徽某铁矿不同矿山废水库中微生物群落结构特征及其影响因素。【方法】对比分析了该铁矿3个大型废水库的地球化学特征,并用高通量测序技术研究了水体中微生物群落组成,进而用统计学方法解析了环境因子对微生物群落结构的影响。【结果】3个废水库中有2个为酸性,1个为中性,理化性质有明显的差异。近年形成的塌方采场废水库(TF) pH仅为2.55±0.01,Fe浓度高达154.95±0.78mg/L,SO_4~(2–)浓度为3374.86±3.81mg/L;形成于20世纪70年代的排土场废水库(PT)酸性略弱(pH 2.9±0.02),Fe浓度(34.57±4.00 mg/L)与TF相比明显降低,SO_4~(2–)浓度则高达10398.98±626.70 mg/L;东沙采场废水库(DS)则为中性(pH7.55),但SO_4~(2–)仍高达4162.99mg/L,主要的金属离子为Mg(594.90 mg/L)、Ca (650.10 mg/L)。3个废水库的原核生物多样性随pH的升高而升高。两个酸性废水库的原核生物组成较为接近,但TF的化能自养菌含量较高(69.54%±2.89%),PT的化能异养菌含量较高(64.45%±13.81%)。自养铁氧化菌Ferrovum在TF中的比例高达(64.17±1.84)%,在PT中则下降为(35.39±13.74)%。但PT中含有丰富的化能异养嗜酸菌如Acidicapsa(15.75%±3.99%)、Acidiphilium(10.65%±2.05%)、Acidisphaera (6.34%±1.02%)等。DS中虽然也含有较高的金属离子和SO_4~(2–),但其中的原核生物组成与TF和PT截然不同,主要为Limnohabitans (18.47%)、Rhodobacter (8.42%)等。3个废水库的真核生物群落主要由藻类组成,酸水库TF和PT中主要为棕鞭藻属(Ochromonas)和胶球藻属(Coccomyxa),棕鞭藻属在TF中(53.65%±2.02%)占优势,胶球藻属在PT中(68.84±10.4%)占优势,中性废水库DS中则主要是小环藻属(Cyclotella)(49.85%)。经统计学分析,pH是影响矿山废水微生物多样性和群落组成的主要环境因素。     

Self-selected fluid volume and flavor strength does not alter fluid intake,body mass loss,or physiological strain during moderate-intensity exercise in the heat

IntroductionThe purpose of this study was to determine the effects of ad libitum flavor and fluid intake on changes in body mass (BM) and physiological strain during moderate intensity exercise in the heat.MethodsTen subjects (24±3yrs, 7M/3F) performed 60 min of treadmill walking at 1.3 m/s and 7% grade in an environmental chamber set to 33 °C and 10% relative humidity while carrying a 22.7 kg pack on two different occasions. Subjects consumed either plain water or water plus flavor (Infuze), ad libitum, at each visit. Pre and post exercise, fluid consumption (change in fluid reservoir weight) and BM (nude) were measured. During exercise, heart rate (HR), systolic blood pressure (SBP), rate of perceived exertion (RPE), oxygen consumption (VO₂), respiratory exchange ratio (RER), core temperature (T_C), and physiological strain index (PSI) were recorded every 15 min during exercise.ResultsNo significant differences were observed for fluid consumption between fluid conditions (512 ± 97.2 mL water vs. 414.3 ± 62.5 mL Infuze). Despite a significant decrease from baseline, there were no significant differences in overall change of BM (Δ −1.18 vs. −0.64 Kg) or percent body weight loss for water and Infuze conditions, respectively (1.58 ± 0.6 and 0.79 ± 0.2%). Furthermore, there were no significant differences in HR (144 ± 6 vs. 143 ± 8 bpm), SBP (157 ± 5 vs. 155 ± 5 mmHg), RPE, VO₂ (27.4 ± 0.9 vs. 28.1 ± 1.2 ml/Kg/min), RER, T_C (38.1 ± 0.1 vs. 37.0 ± 0.1 °C), and peak PSI (5.4 ± 0.4 vs. 5.7 ± 0.8) between conditions.ConclusionsOffering individuals the choice to actively manipulate flavor strength did not significantly influence ad libitum fluid consumption, fluid loss, or physiological strain during 60 min of moderate intensity exercise in the heat.     

d-Aspartate binding sites in rat Harderian gland

Radioligand binding of d-[³H]aspartic and l-[³H]glutamic acids to plasma membranes from rat Harderian gland was evaluated. Binding was optimal under physiological conditions of pH and temperature, and equilibrium was reached within 50 min. Specific binding for d-Asp and l-Glu was saturable, and Eadie–Hofstee analysis revealed interaction with a single population of binding sites (for d-Asp K _d = 860 ± 28 nM, B _max = 27.2 ± 0.5 pmol/mg protein; for l-Glu, K _d = 580 ± 15 nM and B _max = 51.3 ± 0.8 pmol/mg protein). l-[³H]glutamate had higher affinity and a greater percentage of specific binding than did d-[³H]aspartate. The pharmacological binding specificity of l-[³H]glutamate indicated an interaction with NMDA-type receptors. Specifically, the order of potency of the displacing compound tested was l-Glu > d-Asp > NMDA > MK801 > d-AP5 > glycine. For d-[³H]aspartate, the data revealed an interaction of d-Asp with either NMDA-type receptors or putative specific binding sites.     

Application of liquid chromatography-tandem mass spectrometry (LC–MS/MS) for the analysis of stable isotope enrichments of phenylalanine and tyrosine

Whole-body protein synthesis and breakdown are measured by a combined tracer infusion protocol with the stable isotope amino acids l-[ring-²H₅]-phenylalanine, l-[ring-²H₂]-tyrosine and l-[ring-²H₄]-tyrosine that enable the measurement of the phenylalanine to tyrosine conversion rate. We describe a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the measurement of very low tracer–tracee ratios (TTR) of the amino acids l-phenylalanine and l-tyrosine in human plasma. TTR calibration curves of the tracers l-[ring-²H₅]-phenylalanine, l-[ring-²H₂]-tyrosine and l-[ring-²H₄]-tyrosine were linear (r² > 0.99) in the range between 0.01% and 5.0% TTR and lowest measurable TTR for the tracers was 0.01% at a physiological concentration of 60 μM. The method was applied successfully to plasma samples from a clinical study reaching a steady state enrichment plateau (mean ± SD) of 3.33 ± 0.19% for l-[ring-²H₅]-phenylalanine, 2.40 ± 0.43% for l-[ring-²H₂]-tyrosine and 0.29 ± 0.07% for l-[ring-²H₄]-tyrosine, respectively. The LC–MS/MS method can be applied for measurement of very low plasma enrichments of phenylalanine and tyrosine for the determination of whole-body protein synthesis and breakdown rates in humans.     

Electron paramagnetic studies of the copper and iron containing soluble ammonia monooxygenase from <Emphasis Type="Italic">Nitrosomonas europaea</Emphasis>

Soluble ammonia monooxygenase (AMO) from Nitrosomonas europaea was purified to homogeneity and metals in the active sites of the enzyme (Cu, Fe) were analyzed by electron paramagnetic resonance (EPR) spectroscopy. EPR spectra were obtained for a type 2 Cu(II) site with g_|| = 2.24, A_|| = 18.4 mT and g_⊥ = 2.057 as well as for heme and non heme iron present in purified soluble AMO from N. europaea. A second type 2 Cu(II) EPR signal with g_|| = 2.29, A_|| = 16.1 mT and g_⊥ = 2.03 appeared in the spectrum of the ferricyanide oxidized enzyme and was attributed to oxidation of cuprous sites. Comparison of EPR-detectable Cu²⁺ with total copper determined by inductively coupled plasma-mass spectrometry (ICP-MS) suggests that there are six paramagnetic Cu²⁺ and three diamagnetic Cu¹⁺ per heterotrimeric soluble AMO (two paramagnetic and one diamagnetic Cu per αβγ-protomer). A trigonal EPR signal at g = 6.01, caused by a high-spin iron, indicative for cytochrome bound iron, and a rhombic signal at g = 4.31, characteristic of specifically bound Fe³⁺ was detectable. The binding of nitric oxide in the presence of reductant resulted in a ferrous S = 3/2 signal, characteristic of a ferrous nitrosyl complex. Inactivation of soluble AMO with acetylene did neither diminish the ferrous signal nor the intensity of the Cu²⁺-EPR signal.