Kinetics Of Acid Uptake Research Articles

A controlled human intervention trial to study protein quality by amino acid uptake kinetics with the novel Lemna protein concentrate as case study

A human intervention trial was conducted to study amino acid uptake of the novel Lemna protein concentrate (LPC) in comparison to whey (WPC). The study was a cross-over, double-blind, controlled trial in which 12 healthy participants received 20 grams of LPC and WPC in randomised order. The LPC consumption resulted in a significant lower postprandial increase in almost all individual amino acids, total amino acid (TAA) and total essential amino acids (TEAA) compared to WPC based on area under the curve (AUC) calculations. When the AUC after WPC consumption was set at 100%, LPC showed a relative AUC of 60.4% for TAA and 66.3% for the TEAA. Interindividual variation for LPC was high with an uptake of TEAA of LPC compared to WPC ranging from 18.2 to 94.2%. Human intervention trials can partly replace animal trials as they fully reflect the human situation and provide estimates on individual variations.

Real-time microscopic assessment of fatty acid uptake kinetics in the human term placenta

IntroductionThe placenta employs an efficient and selective fatty acid transport system to supply lipids for fetal development. Disruptions in placental fatty acid transport lead to restricted fetal growth along with cardiovascular and neurologic deficits. Nevertheless, little is known about the molecular mechanisms involved in human placental fatty acid trafficking during the initial steps of uptake, or the importance of fatty acid chain length in determining uptake rates. MethodsWe employed BODIPY fluorophore conjugated fatty acid analogues of three chain lengths, medium (BODIPY-C5), long (BODIPY-C12), and very-long (BODIPY-C16), to study fatty acid uptake in isolated human trophoblast and explants using confocal microscopy. The three BODIPY-labeled fatty acids were added to freshly isolated explants and tracked for up to 30 min. Fatty acid uptake kinetics were quantified in trophoblast (cytotrophoblast and syncytiotrophoblast together) and the fetal capillary lumen. ResultsLong- (BODIPY-C12) and Very long-chain (BODIPY-C16) fatty acids accumulated more rapidly in the trophoblast layer than did medium-chain (BODIPY-C5) whereas BODIPY-C5 accumulated more rapidly in the fetal capillary than did the longer chain length fatty acids. The long-chain fatty acids, BODIPY-C12 and BODIPY-C16, are esterified and stored in lipid droplets in the cytotrophoblast layer, but medium-chain fatty acid, BODIPY-C5, is not. DiscussionFatty acids accumulate in trophoblast and fetal capillaries inversely according to their chain length. BODIPY-C5 accumulates in the fetal capillary in concentrations far greater than in the trophoblast, suggesting that medium-chain length BODIPY-labeled fatty acids are capable of being transported against a concentration gradient.

Hepatic uptake of conjugated bile acids is mediated by both sodium taurocholate cotransporting polypeptide and organic anion transporting polypeptides and modulated by intestinal sensing of plasma bile acid levels in mice.

The Na+‐taurocholate cotransporting polypeptide (NTCP/SLC10A1) is believed to be pivotal for hepatic uptake of conjugated bile acids. However, plasma bile acid levels are normal in a subset of NTCP knockout mice and in mice treated with myrcludex B, a specific NTCP inhibitor. Here, we elucidated which transport proteins mediate the hepatic uptake of conjugated bile acids and demonstrated intestinal sensing of elevated bile acid levels in plasma in mice. Mice or healthy volunteers were treated with myrcludex B. Hepatic bile acid uptake kinetics were determined in wild‐type (WT), organic anion transporting polypeptide (OATP) knockout mice (lacking Slco1a/1b isoforms), and human OATP1B1‐transgenic mice. Effects of fibroblast growth factor 19 (FGF19) on hepatic transporter mRNA levels were assessed in rat hepatoma cells and in mice by peptide injection or adeno‐associated virus–mediated overexpression. NTCP inhibition using myrcludex B had only moderate effects on bile acid kinetics in WT mice, but completely inhibited active transport of conjugated bile acid species in OATP knockout mice. Cholesterol 7α‐hydroxylase Cyp7a1 expression was strongly down‐regulated upon prolonged inhibition of hepatic uptake of conjugated bile acids. Fgf15 (mouse counterpart of FGF19) expression was induced in hypercholanemic OATP and NTCP knockout mice, as well as in myrcludex B–treated cholestatic mice, whereas plasma FGF19 was not induced in humans treated with myrcludex B. Fgf15/FGF19 expression was induced in polarized human enterocyte‐models and mouse organoids by basolateral incubation with a high concentration (1 mM) of conjugated bile acids. Conclusion: NTCP and OATPs contribute to hepatic uptake of conjugated bile acids in mice, whereas the predominant uptake in humans is NTCP mediated. Enterocytes sense highly elevated levels of (conjugated) bile acids in the systemic circulation to induce FGF15/19, which modulates hepatic bile acid synthesis and uptake. (Hepatology 2017;66:1631–1643).

Effect of Kampo Extract Formulation (Japanese Traditional Medicine) on Pro- and Anti-angiogenic Factors Secreted from Primary Endothelial Cells and Placental Cells

The placenta employs an efficient and selective fatty acid transport system to supply lipids for fetal development. Disruptions in placental fatty acid transport lead to restricted fetal growth along with cardiovascular and neurologic deficits. Nevertheless, little is known about the molecular mechanisms involved in human placental fatty acid trafficking during the initial steps of uptake, or the importance of fatty acid chain length in determining uptake rates.We employed BODIPY fluorophore conjugated fatty acid analogues of three chain lengths, medium (BODIPY-C5), long (BODIPY-C12), and very-long (BODIPY-C16), to study fatty acid uptake in isolated human trophoblast and explants using confocal microscopy. The three BODIPY-labeled fatty acids were added to freshly isolated explants and tracked for up to 30 min. Fatty acid uptake kinetics were quantified in trophoblast (cytotrophoblast and syncytiotrophoblast together) and the fetal capillary lumen.Long- (BODIPY-C12) and Very long-chain (BODIPY-C16) fatty acids accumulated more rapidly in the trophoblast layer than did medium-chain (BODIPY-C5) whereas BODIPY-C5 accumulated more rapidly in the fetal capillary than did the longer chain length fatty acids. The long-chain fatty acids, BODIPY-C12 and BODIPY-C16, are esterified and stored in lipid droplets in the cytotrophoblast layer, but medium-chain fatty acid, BODIPY-C5, is not.Fatty acids accumulate in trophoblast and fetal capillaries inversely according to their chain length. BODIPY-C5 accumulates in the fetal capillary in concentrations far greater than in the trophoblast, suggesting that medium-chain length BODIPY-labeled fatty acids are capable of being transported against a concentration gradient.

Growth and Nitrogen Uptake Kinetics in Cultured Prorocentrum donghaiense

We compared growth kinetics of Prorocentrum donghaiense cultures on different nitrogen (N) compounds including nitrate (NO3 −), ammonium (NH4 +), urea, glutamic acid (glu), dialanine (diala) and cyanate. P. donghaiense exhibited standard Monod-type growth kinetics over a range of N concentraions (0.5–500 μmol N L−1 for NO3 − and NH4 +, 0.5–50 μmol N L−1 for urea, 0.5–100 μmol N L−1 for glu and cyanate, and 0.5–200 μmol N L−1 for diala) for all of the N compounds tested. Cultures grown on glu and urea had the highest maximum growth rates (μm, 1.51±0.06 d−1 and 1.50±0.05 d−1, respectively). However, cultures grown on cyanate, NO3 −, and NH4 + had lower half saturation constants (Kμ, 0.28–0.51 μmol N L−1). N uptake kinetics were measured in NO3 −-deplete and -replete batch cultures of P. donghaiense. In NO3 −-deplete batch cultures, P. donghaiense exhibited Michaelis-Menten type uptake kinetics for NO3 −, NH4 +, urea and algal amino acids; uptake was saturated at or below 50 μmol N L−1. In NO3 −-replete batch cultures, NH4 +, urea, and algal amino acid uptake kinetics were similar to those measured in NO3 −-deplete batch cultures. Together, our results demonstrate that P. donghaiense can grow well on a variety of N sources, and exhibits similar uptake kinetics under both nutrient replete and deplete conditions. This may be an important factor facilitating their growth during bloom initiation and development in N-enriched estuaries where many algae compete for bioavailable N and the nutrient environment changes as a result of algal growth.

Chronic ethanol consumption increases cardiomyocyte fatty acid uptake and decreases ventricular contractile function in C57BL/6J mice

Alcohol, a major cause of human cardiomyopathy, decreases cardiac contractility in both animals and man. However, key features of alcohol-related human heart disease are not consistently reproduced in animal models. Accordingly, we studied cardiac histology, contractile function, cardiomyocyte long chain fatty acid (LCFA) uptake, and gene expression in male C57BL/6J mice consuming 0, 10, 14, or 18% ethanol in drinking water for 3months. At sacrifice, all EtOH groups had mildly decreased body and increased heart weights, dose-dependent increases in cardiac triglycerides and a marked increase in cardiac fatty acid ethyl esters. [3H]-oleic acid uptake kinetics demonstrated increased facilitated cardiomyocyte LCFA uptake, associated with increased expression of genes encoding the LCFA transporters CD36 and Slc27a1 (FATP1) in EtOH-fed animals. Although SCD-1 expression was increased, lipidomic analysis did not indicate significantly increased de novo LCFA synthesis. By echocardiography, ejection fraction (EF) and the related fractional shortening (FS) of left ventricular diameter during systole were reduced and negatively correlated with cardiac triglycerides. Expression of myocardial PGC-1α and multiple downstream target genes in the oxidative phosphorylation pathway, including several in the electron transport and ATP synthase complexes of the inner mitochondrial membrane, were down-regulated. Cardiac ATP was correspondingly reduced. The data suggest that decreased expression of PGC-1α and its target genes result in decreased cardiac ATP levels, which may explain the decrease in myocardial contractile function caused by chronic EtOH intake. This model recapitulates important features of human alcoholic cardiomyopathy and illustrates a potentially important pathophysiologic link between cardiac lipid metabolism and function.

Molecular Modeling and Functional Confirmation of a Predicted Fatty Acid Binding Site of Mitochondrial Aspartate Aminotransferase

Molecular interactions are necessary for proteins to perform their functions. The identification of a putative plasma membrane fatty acid transporter as mitochondrial aspartate aminotransferase (mAsp-AT) indicated that the protein must have a fatty acid binding site. Molecular modeling suggests that such a site exists in the form of a 500-Å3 hydrophobic cleft on the surface of the molecule and identifies specific amino acid residues that are likely to be important for binding. The modeling and comparison with the cytosolic isoform indicated that two residues (Arg201 and Ala219) were likely to be important to the structure and function of the binding site. These residues were mutated to determine if they were essential to that function. Expression constructs with wild-type or mutated cDNAs were produced for bacteria and eukaryotic cells. Proteins expressed in Escherichia coli were tested for oleate binding affinity, which was decreased in the mutant proteins. 3T3 fibroblasts were transfected with expression constructs for both normal and mutated forms. Plasma membrane expression was documented by indirect immunofluorescence before [3H]oleic acid uptake kinetics were assayed. The Vmax for uptake was significantly increased by overexpression of the wild-type protein but changed little after transfection with mutated proteins, despite their presence on the plasma membrane. The hydrophobic cleft in mAsp-AT can serve as a fatty acid binding site. Specific residues are essential for normal fatty acid binding, without which fatty acid uptake is compromised. These results confirm the function of this protein as a fatty acid binding protein.

Long-chain fatty acid uptake is upregulated in omental adipocytes from patients undergoing bariatric surgery for obesity

To determine the impact of obesity on adipocyte cell size and long-chain fatty acid (LCFA) uptake kinetics in human subjects undergoing laparoscopic abdominal surgery. A total of 10 obese patients (BMI 49.8+/-11.9 (s.d.) kg/m(2)) undergoing laparoscopic bariatric surgery, and 10 nonobese subjects (BMI 24.2+/-2.3 kg/m(2)) undergoing other clinically indicated laparoscopic abdominal surgical procedures. Cell size distribution and [(3)H]oleic acid uptake kinetics were studied in adipocytes isolated from omental fat biopsies obtained during surgery. Adipocyte surface area (SA) was calculated from the measured cell diameters. Plasma leptin and insulin concentrations were measured by RIA in fasting blood samples obtained on the morning of surgery. The mean SA of obese adipocytes (41 508+/-5381 mu(2)/cell) was increased 2.4-fold compared to that of nonobese adipocytes (16 928+/-6529 mu(2)/cell; P<0.01). LCFA uptake in each group was the sum of saturable and nonsaturable components. Both the V(max) of the saturable component (21.3+/-6.3 vs 5.1+/-1.9 pmol/s/50,000 cells) and the rate constant k of the nonsaturable component (0.015+/-0.002 vs 0.0066+/-0.0023 ml/s/50 000 cells) were increased (P<0.001) in obese adipocytes compared with nonobese controls. When expressed relative to cell size, V(max)/mu(2) SA was greater in obese than nonobese adipocytes (P<0.05), whereas k/mu(2) SA did not differ between the groups. The data support the concepts that (1) adipocyte LCFA uptake consists of distinct facilitated (saturable) and diffusive processes; (2) increased saturable LCFA uptake in obese adipocytes is not simply a consequence of increased cell size, but rather reflects upregulation of a facilitated transport process; and (3) the permeability of adipocyte plasma membranes to LCFA is not appreciably altered by obesity, and increased nonsaturable uptake in obese adipocytes principally reflects an increase in cell SA. Regulation of saturable LCFA uptake by adipocytes may be an important control point for body adiposity.

Effect of iron deficiency on diatom cell size and silicic acid uptake kinetics

We studied the silicic acid uptake kinetics of the pennate diatom Cylindrotheca fusiformis grown under a wide range of iron concentrations (from Fe‐limiting to Fe‐sufficient conditions) to assess the effect of iron availability on diatom cell size, silicon content, and silicic acid uptake kinetic parameters. As the iron stress increased, the growth rate slowed, cell size decreased, and silicification increased. A series of Si kinetic uptake experiments (from Si‐limiting to Si‐sufficient conditions) performed at different iron concentrations demonstrated the extent of the colimitation domain, where the specific Si uptake rate (VSi) varied as a function of both silicic acid and Fe availability. A decrease in maximal specific uptake rate of silicic acid (VSi‐max) under iron limitation was observed along with a decrease in half‐saturation constant for silicic acid uptake (KSi). Because VSi‐max and KSi vary in the same direction, the specific affinity for silicic acid does not change under iron stress. The variation in cell size is an acclimation to low nutrient concentrations. Our study shows that Si uptake kinetics parameters, especially VSi‐max, are strongly related to cell size, which is itself constrained by the degree of iron limitation. Thus, the surface to volume ratio should be related to silicic acid flows in size‐based biogeochemical models of planktonic ecosystems. Another way to describe the observed variations of Si uptake over the full range of Si and Fe concentrations tested in the present study would be to implicitly take into account the variations in cell size through a scaling of the maximal specific uptake rate (Vmax) by the iron‐limitation term in a multiplicative manner.

Spectroscopic Study of Nitric Acid and Water Adsorption on Oxide Particles: Enhanced Nitric Acid Uptake Kinetics in the Presence of Adsorbed Water

In this study, the heterogeneous reactivity of nitric acid on oxide particles of some of the most abundant crustal elements is investigated at 296 K. The oxide particles are used as models for mineral dust aerosol found in the atmosphere. Transmission FT-IR spectroscopy is used to probe changes in the spectrum of the oxide particle surface following adsorption of HNO3 on SiO2, α-Al2O3, TiO2, γ-Fe2O3, CaO, and MgO. It is found that HNO3 molecularly and reversibly adsorbs on SiO2. For the other oxides investigated, HNO3 dissociatively and irreversibly adsorbs to form surface nitrate. There is also a small amount of molecularly adsorbed nitric acid (<10% of the adsorbed nitrate) on the oxide particle surface in the presence of gas-phase nitric acid. Because adsorbed water may play a role in the heterogeneous uptake of nitric acid in the atmosphere, transmission FT-IR spectroscopy is used to investigate H2O adsorption on SiO2, α-Al2O3, TiO2, γ-Fe2O3, CaO, and MgO particles as well. Uptake of water on the oxide particles can be described by a multilayer adsorption isotherm. Water uptake on nitrate-coated oxides remained similar for α-Al2O3 and TiO2 compared to the uncoated surface; however, for γ-Fe2O3, CaO, and MgO, the shape of the adsorption isotherm changed for the nitrate-coated particles with an increased amount of water adsorption at a given relative humidity. The infrared spectrum of the surface nitrate shows that water adsorbed on the particle surface can solvate the nitrate ion. The rate of nitric acid uptake on α-Al2O3 and CaO is found to increase by nearly 50-fold when going from conditions near 0 to 20% relative humidity, indicating that the nitric acid dissociation kinetics on the wetted particle surface is significantly enhanced. In the case of MgO and CaO, the amount of nitric acid uptake is increased in the presence of water and is not limited to the surface of the particles, producing saturated solutions of Mg(NO3)2 and Ca(NO3)2. From the studies presented here, atmospheric implications of heterogeneous reactions of HNO3 with mineral dust aerosol are discussed.

Systemic application of photosensitizers in the chick chorioallantoic membrane (CAM) model: photodynamic response of CAM vessels and 5-aminolevulinic acid uptake kinetics by transplantable tumors

The aim of this study is to modify the chick chorioallantoic membrane (CAM) model into a whole-animal tumor model for photodynamic therapy (PDT). By using intraperitoneal (IP) photosensitizer injection of the chick embryo, use of the CAM for PDT has been extended to include systemic delivery as well as topical application of photosensitizers. The model has been tested for its capability to mimic an animal tumor model and to serve for PDT studies by measuring drug fluorescence and PDT-induced effects. Three second-generation photosensitizers have been tested for their ability to produce photodynamic response in the chick embryo/CAM system when delivered by IP injection: 5-aminolevulinic acid (ALA), benzoporphyrin derivative monoacid ring A (BPD-MA), and Lutetium-texaphyrin (Lu-Tex). Exposure of the CAM vasculature to the appropriate laser light results in light-dose-dependent vascular damage with all three compounds. Localization of ALA following IP injections in embryos, whose CAMs have been implanted with rat ovarian cancer cells to produce nodules, is determined in real time by fluorescence of the photoactive metabolite protoporphyrin IX (PpIX). Dose-dependent fluorescence in the normal CAM vasculature and the tumor implants confirms the uptake of ALA from the peritoneum, systemic circulation of the drug, and its conversion to PpIX.

The interactive effects of temperature and silicon limitation on the psychrophilic ice diatomPseudonitszchia seriata

Pseudonitzschia seriata, isolated from sea ice in the Canadian Arctic, was grown in silicon-limited batch and semi-continuous culture to determine the effects of temperature on its nutrient utilization. Resource-saturated growth rate (μmax) increased significantly with temperature from -1.5 to 6°C with a Q10 of 1.63. The efficiency of the algae in using exogenous silicic acid for growth had no significant monotonic relationship with temperature but was significantly (P 0°C than in those at lower temperatures. Silicic acid uptake kinetics did not differ between high and low temperatures. Silicon cell quotas were significantly higher at temperatures <0°C, indicating greater silicon demand at low temperatures.P. seriata should not suffer silicon limitation in its natural ice habitat based on the observed kinetics, but its behaviour provides some support for the suggestion that temperatures <0°C are associated with diminished efficiency of nutrient utilization by cold ocean microalgae.

Uptake of organic acids byClostridium acetobutylicum B18 under controlled pH and reduced butanol inhibition

Uptake of organic acids byClostridium acetobutylicum B18 was studied at controlled pH and under reduced butanol inhibition conditions. A pervaporative membrane module was placed in the fermentor to remove butanol from the fermentation broth. Uptake of added butyric acid followed zero order kinetics at pH 4.75 and first order kinetics at pH 5.75. At pH 5.25 the kinetic order shifted from zero to first order as the butyric acid was taken up. At the point of order shift undissociated butyric acid (UBA) concentration was approximately 0.5 g/L. Unlike butyric acid, uptake of acetic acid followed first order kinetics regardless of pH. The difference in acid uptake kinetics could be explained by the combined effect of acid diffusion across the cell membrane and intracellular enzymatic reaction. The acid concentration for kinetic order shift seemed to be dependent upon pH and the kind of the acid used. Glucose was consumed simultaneously with added acids. Both butyric and acetic acids were taken up simultaneously but the rate was faster for butyric acid. Added butyric acid was completely assimilated whereas acetic acid uptake was incomplete.

Kinetics of silicic acid uptake by natural diatom assemblages in two Gulf Stream warm-core rings

We performed Si tracer experiments to measure the uptake rates of silicic acid by natural diatom assemblaged in 2 Gulf Stream warm-core rings as a function of the extracellular silicic acid concentration. The experiments were performed a t and near the centers of rings 81-D and 82-B, and lncluded 5 sampling dates in late spring, summer and autumn, when silicic acid was undetectable to conventional analyses (< 0.2 yM) throughout the upper 20 to 30 m Uptake was measurably substratelimited only at those stations where silicic acid was undetectable. Concentrations of 2.5 to 3 1rM Si(OH)4, which typlfied ring 82-B before the development of the seasonal thermocline, were high enough to support maximum uptake rates. When substrate 1i.mitation was present the dependence of the uptake rate on sllicic acid concentration exhibited hyperbolic saturation kinetics, with half-saturation constants (K,) ranging from 0.53 to 0.90 \&M. This range is considerably lower than that reported previously for natural diatom asselnblages (1.5 to 3.7 pM). A numerical sllnulation indicates that a diatom species with a K, value typical of the ring assemblages would outcompete most diatom species whose silicon kinetics have been examined in culture, not only under conditions of continuously low sll~cic a c ~ d concentration but with the sporadic, pulsed nutrient injections that appear to characterne warm-core ocean eddies. Prevlous studies of silicic acid uptake kinetics in natural waters have all been conducted In relatively nutnent-rlch systems and the lower K, values we report here for 2 Gulf Stream warm-core rings Indicate that diatoms growing in severely nutrient-depleted surface waters have a higher afflnlty for slliclc acid than those growing in more eutrophic habitats. If this is true for other nutrient-depleted environments such as the tropics and mid-ocean gyres, the likelihood of significant slhcon limitation in such systems is diminished.

Kinetics of acid uptake by weak-base anion exchangers

The sorption of acids by unifunctional weak-base anion exchangers of conventional gel type from solutions below about 0·1N in acid follows neither classical ion-exchange kinetics nor those appropriate to ion-exchange accompanied by reaction. In many such resins, the particle diameters of which ranged from 400 to 900 microns, the sorption of a number of acids has been found to occur by a chemically controlled process in which the rate of attainment of equilibrium is independent of particle size, but depends upon, amongst other things, the basic strength of the active group.This behaviour contrasts with that found by earlier workers for multifunctional resins, and with that found in the present work for unifunctional macroporous resins, both of which show a conventional dependence of rate on particle size corresponding to particle-diffusion controlled kinetics at least down to about 0·001N.Similar particle-diffusion controlled kinetics, characterised by apparent activation energies of ca. 3 kcal./mole, are shown by the unifunctional gel resins at higher acid concentrations at and above 0·1N. The apparent activation energies for the chemically controlled processes are 2 ½ to 3 times higher.Change in the valence of the counter-ion affects rates of uptake on conventional gel resins markedly, while on macroporous resins the change is small. The importance of electrolyte invasion as a controlling factor in such processes is emphasised, and an approximate analysis of both particle-diffusion and chemically controlled kinetics has been attempted taking electrolyte invasion parameters into account.