A Novel Injection-Molded Capsular Device for Oral Pulsatile Delivery Based on Swellable/Erodible Polymers

The feasibility of injection molding was explored in the preparation of a novel capsular device for oral pulsatile/delayed delivery based on swellable/erodible polymers. For this purpose, a mold intended to be coupled with a bench-top injection-molding press was designed. This was expected to enable the preparation of matching capsule cap and body items within a single manufacturing cycle and the selection of differing shell thicknesses (300, 600, and 900 μm). Hydroxypropylcellulose (Klucel^® EF, LF, and GF) was employed as the release-controlling polymer in admixture with polyethylene glycol 1500 (10%, w/w) as the plasticizer. After preliminary trials aimed at the setup of operating conditions, Klucel^® EF and LF capsule shells with satisfactory technological properties were manufactured. The performance of capsular devices filled with a tracer drug powder was studied by means of a modified USP31 disintegration apparatus. Typical in vitro delayed release patterns were thereby obtained, with lag time increasing as a function of the wall thickness. A good correlation was found between the latter parameter and t _10%, i.e., the time to 10% release, for both polymer grades employed. On the basis of the overall results, the investigated technique was proven suitable for the manufacturing of an innovative pulsatile release platform.     

QbD-Oriented Development and Characterization of Effervescent Floating-Bioadhesive Tablets of Cefuroxime Axetil

The objective of the present studies was systematic development of floating-bioadhesive gastroretentive tablets of cefuroxime axetil employing rational blend of hydrophilic polymers for attaining controlled release drug delivery. As per the QbD-based approach, the patient-centric target product profile and quality attributes of tablet were earmarked, and preliminary studies were conducted for screening the suitability of type of polymers, polymer ratio, granulation technique, and granulation time for formulation of tablets. A face-centered cubic design (FCCD) was employed for optimization of the critical material attributes, i.e., concentration of release controlling polymers, PEO 303 and HPMC K100 LV CR, and evaluating in vitro buoyancy, drug release, and ex vivo mucoadhesion strength. The optimized formulation was embarked upon through numerical optimization, which yield excellent floatation characteristic with drug release control (i.e., T _60%?>?6 h) and bioadhesion strength. Drug-excipient compatibility studies through FTIR and P-XRD revealed the absence of any interaction between the drug and polymers. In vivo evaluation of the gastroretentive characteristics through X-ray imaging and in vivo pharmacokinetic studies in rabbits revealed significant extension in the rate of drug absorption (i.e., T _max, K _a, and MRT) from the optimized tablet formulation as compared to the marketed formulation. Successful establishment of various levels of in vitro/in vivo correlations (IVIVC) substantiated high degree of prognostic ability of in vitro dissolution conditions in predicting the in vivo performance. In a nutshell, the studies demonstrate successful development of the once-a-day gastroretentive formulations of cefuroxime axetil with controlled drug release profile and improved compliance.     

Particle Size Distribution Equivalency as Novel Predictors for Bioequivalence

The use of particle size distribution (PSD) similarity metrics and the development and incorporation of drug release predictions based on PSD properties into PBPK models for various drug administration routes may provide a holistic approach for evaluating the effect of PSD differences on in vitro drug release and bioavailability of disperse systems. The objectives of this study were to provide a rational approach for evaluating the utility of in vitro PSD comparators for predicting bioequivalence for subcutaneously administered test and reference drug emulsions. Two types of in vitro comparators for test and reference emulsion products were evaluated: PSD characterization comparators (overlap metrics, median, and span ratios) and release profile comparators (f₂ and various fractional time ratios). A subcutaneous-input PBPK disposition model was developed to simulate blood concentration-time profiles of reference and test emulsion products and pharmacokinetic responses (e.g., AUC, C_max, and T_max) were used to determine bioequivalence. A pool of 10,440 pairs of test and reference products was simulated using Monte Carlo experiments. The PSD and release profile comparators were correlated to pass/fail bioequivalence metrics using logistical regression. Based on the use of single in vitro comparators, the f₂ method was the best predictor of bioequivalence prediction. The use of combinations of f₂ and PSD overlap comparators (e.g., OVL or PROB) improved bioequivalence prediction to about 90%. Simulation procedures used in this study demonstrated a process for developing reliable in vitro BE predictors.     

Formulation and <Emphasis Type="Italic">In Vitro</Emphasis> and <Emphasis Type="Italic">In Vivo</Emphasis> Evaluation of Lipid-Based Terbutaline Sulphate Bi-layer Tablets for Once-Daily Administration

The objective of this study was to prepare and evaluate terbutaline sulphate (TBS) bi-layer tablets for once-daily administration. The bi-layer tablets consisted of an immediate-release layer and a sustained-release layer containing 5 and 10 mg TBS, respectively. The sustained-release layer was developed by using Compritol®888 ATO, Precirol® ATO 5, stearic acid, and tristearin, separately, as slowly eroding lipid matrices. A full 4?×?2² factorial design was employed for optimization of the sustained-release layer and to explore the effect of lipid type (X ₁), drug–lipid ratio (X ₂), and filler type (X ₃) on the percentage drug released at 8, 12, and 24 h (Y ₁, Y ₂, and Y ₃) as dependent variables. Sixteen TBS sustained-release matrices (F1–F16) were prepared by melt solid dispersion method. None of the prepared matrices achieved the targeted release profile. However, F2 that showed a relatively promising drug release was subjected to trial and error optimization for the filler composition to develop two optimized matrices (F17 and F18). F18 which consisted of drug–Compritol®888 ATO at ratio (1:6 w/w) and Avicel PH 101/dibasic calcium phosphate mixture of 2:1 (w/w) was selected as sustained-release layer. TBS bi-layer tablets were evaluated for their physical properties, in vitro drug release, effect of storage on drug content, and in vivo performance in rabbits. The bi-layer tablets showed acceptable physical properties and release characteristics. In vivo absorption in rabbits revealed initial high TBS plasma levels followed by sustained levels over 24 h compared to immediate-release tablets.     

<Emphasis Type="Italic">In Vivo</Emphasis> Evaluation of a PEO-Gellan Gum Semi-Interpenetrating Polymer Network for the Oral Delivery of Sulpiride

In this study, an optimized epichlorohydrin-crosslinked semi-interpenetrating polymer network xerogel matrix system (XePoMas) for the controlled delivery of sulpiride was prepared. The ability of XePoMas to sustain drug release was determined by in vitro and in vivo drug release experiments. Swelling of the xerogel over the 24-h experimental period ranged from 346 to 648%; swelling was observed to increase exponentially over the initial 8 h. In vitro drug release depicted a linear zero order drug release profile with an R ² value of 0.9956. The ability of the fabricated XePoMas to sustain drug release and enhance bioavailability of sulpiride in vivo was investigated by evaluating the plasma drug concentration over 24 h in the large pig model. The optimized XePoMas formulation was shown to increase intestinal absorption of sulpiride to a greater extent than the marketed product in vivo, with a C _max of 830.58 ng/mL after 15 h.     

Oral fast-dissolving films containing lutein nanocrystals for improved bioavailability: formulation development, <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis> evaluation

Lutein is widely used as diet supplement for prevention of age-related macular degeneration. However, the application and efficacy of lutein in food and nutritional products has been hampered due to its poor solubility and low oral bioavailability. This study aimed to develop and evaluate the formulation of oral fast-dissolving film (OFDF) containing lutein nanocrystals for enhanced bioavailability and compliance. Lutein nanocrystals were prepared by anti-solvent precipitation method and then encapsulated into the films by solvent casting method. The formulation of OFDF was optimized by Box-Behnken Design (BBD) as follows: HPMC 2.05% (w/v), PEG 400 1.03% (w/v), Cremophor EL 0.43% (w/v). The obtained films exhibited uniform thickness of 35.64 ± 1.64 μm and drug content of 0.230 ± 0.003 mg/cm² and disintegrated rapidly in 29 ± 8 s. The nanocrystal-loaded films with reconstituted particle size of 377.9 nm showed better folding endurance and faster release rate in vitro than the conventional OFDFs with raw lutein. The microscope images, thermograms, and diffractograms indicated that lutein nanocrystals were highly dispersed into the films. After administrated to SD rats, t _max was decreased from 3 h for oral solution formulation to less than 0.8 h for OFDF formulations, and C _max increased from 150 ng/mL for solution to 350 ng/mL for conventional OFDF or 830 ng/mL for nanocrystal OFDF. The AUC _0-24h of conventional or nanocrystal OFDF was 1.37 or 2.08-fold higher than that of the oral solution, respectively. These results suggested that drug nanocrystal-loaded OFDF can be applied as a promising approach for enhanced bioavailability of poor soluble drugs like lutein.     

In Vitro Antifungal Susceptibility of Oral <Emphasis Type="Italic">Candida</Emphasis> Isolates from Patients Suffering from Caries and Chronic Periodontitis

Caries and chronic periodontitis are common oral diseases where a higher Candida colonization is reported. Antifungal agents could be adjuvant drugs for the therapy of both clinical conditions. The aim of the current study has been to evaluate the in vitro activities of conventional and new antifungal drugs against oral Candida isolates from patients suffering from caries and/or chronic periodontitis. In vitro activities of amphotericin B, fluconazole, itraconazole, miconazole, nystatin, posaconazole and voriconazole against 126 oral Candida isolates (75 Candida albicans, 18 Candida parapsilosis, 11 Candida dubliniensis, six Candida guilliermondii, five Candida lipolytica, five Candida glabrata, four Candida tropicalis and two Candida krusei) from 61 patients were tested by the CLSI M27-A3 method. Most antifungal drugs were highly active, and resistance was observed in less than 5% of tested isolates. Miconazole was the most active antifungal drug, being more than 98% of isolates susceptible. Fluconazole, itraconazole, and the new triazoles, posaconazole and voriconazole, were also very active. Miconazole, fluconazole and voriconazole have excellent in vitro activities against all Candida isolates and could represent suitable treatment for a hypothetically adjunctive therapy of caries and chronic periodontitis.     

Colon-Targeted Delivery of IgY Against <Emphasis Type="Italic">Clostridium difficile</Emphasis> Toxin A and B by Encapsulation in Chitosan-Ca Pectinate Microbeads

This study investigated the use of a newly developed chitosan-Ca pectinate microbead formulation for the colon-targeted delivery of anti-A/B toxin immunoglobulin of egg yolk (IgY) to inhibit toxin binding to colon mucosa cells. The effect of the three components (pectinate, calcium chloride, and chitosan) used for the microbead production was examined with the aim of identifying the optimal levels to improve drug encapsulation efficiency, swelling ratio, and cumulative IgY release rate. The optimized IgY-loaded bead component was pectin 5% (w/v), CaCl₂ 3% (w/v), and chitosan 0.5% (w/v). Formulated beads were spherical with 1.2-mm diameter, and the drug loading was 45%. An in vitro release study revealed that chitosan-Ca pectinate microbeads inhibited IgY release in the upper gastrointestinal tract and significantly improved the site-specific release of IgY in the colon. An in vivo rat study demonstrated that 72.6% of biologically active IgY was released specifically in the colon. These results demonstrated that anti-A/B toxin IgY-loaded chitosan-Ca pectinate oral microbeads improved IgY release behavior in vivo, which could be used as an effective oral delivery platform for the biological treatment of Clostridium difficile infection (CDI).     

Elimination of macro elements from inoculation and co-cultivation media enhances the efficiency of <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation in <Emphasis Type="Italic">Petunia</Emphasis>

In order to evaluate the effect of inoculation and co-cultivation media elements on transformation frequency in Petunia hybrida, modified MS media with different elements were tested on Alvan and Large Flower Alvan (LF Alvan), two local cultivars. Leaf explants of both cultivars were inoculated with Agrobacterium tumefaciens strain LBA4404 (pBI121) containing neomycin phosphotransferase (nptII) and an intron-containing β-glucuronidase (gus) genes. When medium lacking KH₂PO₄, NH₄NO₃, KNO₃, and CaCl₂ was used as inoculation and co-cultivation medium, a higher frequency of transformation for Alvan (22%) and LF Alvan (16%) was obtained. Kanamycin resistant plantlets were stained blue by GUS assay. Furthermore, polymerase chain reaction (PCR) analysis revealed the presence of both gus and nptII genes in all putative transformants. Finally, southern blot hybridization confirmed insertion of 1–4 copies of gus gene in transgenic plants.     

The characterization of anti-<Emphasis Type="Italic">T. cruzi</Emphasis> activity relationships between ferrocenyl,cyrhetrenyl complexes and ROS release

Trypanosoma cruzi (T. cruzi) is the parasite that causes Chagas disease. Nifurtimox is the most used drug against the T. cruzi, this drug increases intermediaries nitro group, being mainly responsible for the high toxicity component, for this reason it is important to study new organic compounds and thus improve therapeutic strategies against Chagas disease. The electronic effects of ferrocenyl and cyrhetrenyl fragments were investigated by DFT calculation. A close correlation was found between HOMO–LUMO gap of nitro radical NO ₂ ^? with the experimental reduction potential found for nitro group and IC₅₀ of two forms the T. cruzi (epimastigote and trypomastigote). The IC₅₀ on human hepatoma cells is higher for both compounds compared to IC₅₀ demonstrated in the two forms the T. cruzi, and additionally show reactive oxygen species release. The information obtained in this paper could generate two new drugs with anti-T. cruzi activity, but additional studies are needed.     

Modeling of Progesterone Release from Poly(3-Hydroxybutyrate) (PHB) Membranes

Poly(3-hydroxybutyrate) (PHB) biodegradable polymeric membranes were evaluated as platform for progesterone (Prg)-controlled release. In the design of new drug delivery systems, it is important to understand the mass transport mechanism involved, as well as predict the process kinetics. Drug release experiments were conducted and the experimental results were evaluated using engineering approaches that were extrapolated to the pharmaceutical field by our research group. Membranes were loaded with different Prg concentrations and characterized by scanning electron microscopy (SEM), differential scanning calorimetry (DSC), and Fourier transform infrared spectroscopy (FTIR). SEM images showed that membranes have a dense structure before and after the progesterone addition. DSC and FTIR allowed determining the influence of the therapeutic agent in the membrane properties. The in vitro experiments were performed using two different techniques: (A) returning the sample to the receptor solution (constant volume of the delivery medium) and (B) extracting total volume of the receptor solution. In this work, we present a simple and accurate “lumped” second-order kinetic model. This lumped model considers the different mass transport steps involved in drug release systems. The model fits very well the experimental data using any of the two experimental procedures, in the range 0?≤?t?≤?∞ or 0?≤?M _t ?≤?M _∞. The drug release analysis using our proposed approaches is relevant for establishing in vitro–in vivo correlations in future tests in animals.     

Impacts on Micro- and Macro-Structure of Thermally Stabilised Whey Protein-Pectin Complexes: A Fluorescence Approach

Stability of whey protein-pectin complexes is an essential criterion for their application in different food matrices. The impact of process parameters on micro- and macro-structural characteristics of thermally stabilised whey protein-pectin complexes was investigated using fluorescence spectroscopy, ζ-potential measurements, dynamic light scattering and phase separation. Complexes prepared from whey protein isolate (WPI) and pectins with different degrees of esterification (HMP, LMP) were generated at different biopolymer concentrations (WPI + pectin: 5.0 % + 1.0 %, c _{h i g h} ; 2.75 % + 0.55 %, c _{m e d} ; 0.5 % + 0.1 %, c _{l o w} ), heating temperatures (80-90^°C) and pH levels (6.1-4.0). Micro- and macro-structural characteristics of the complexes depended on concentration level and degree of esterification, with complexes being more sensitive towards environmental changes at c _{l o w} than at c _{m e d} and c _{h i g h} . WPI-LMP complexes exhibited sizes <1 μm suitable for micro-encapsulation, whereas WPI-HMP complexes at c _{m e d} achieved sizes from 1-10 μm and at c _{h i g h} from 10-200 μm underlining their potential as fat-replacers and structuring agents, respectively. Slopes and intercepts derived from intensity ratios of fluorescence spectra gave insights into the state of unfolding of β-lactoglobulin within the complexes and thus about the protective effect of pectin addition.     

Anticancer Efficacy of Self-Nanoemulsifying Drug Delivery System of Sunitinib Malate

Sunitinib malate (SM) is reported as a weakly soluble drug in water due to its poor dissolution rate and oral bioavailability. Hence, in the current study, various “self-nanoemulsifying drug delivery systems (SNEDDS)” of SM were prepared, characterized and evaluated for the enhancement of its in vitro dissolution rate and anticancer efficacy. On the basis of solubilization potential of SM in various excipients, “Lauroglycol-90 (oil), Triton-X100 (surfactant) and Transcutol-P (cosurfactant)” were selected for the preparation of SM SNEDDS. SM-loaded SNEDDS were developed by spontaneous emulsification method, characterized and evaluated for “thermodynamic stability, self-nanoemulsification efficiency, droplet size, polydispersity index (PDI), zeta potential (ZP), surface morphology, refractive index (RI), the percent of transmittance (% T) and drug release profile.” In vitro dissolution rate of SM was significantly enhanced from an optimized SNEDDS in comparison with SM suspension. The optimized SNEDDS of SM with droplet size of 42.3 nm, PDI value of 0.174, ZP value of ?36.4 mV, RI value of 1.339, % T value of 97.3%, and drug release profile of 95.4% (after 24 h via dialysis membrane) was selected for in vitro anticancer efficacy in human colon cancer cells (HT-29) by MTT assay. MTT assay indicated significant anticancer efficacy of optimized SM SNEDDS against HT-29 cells in comparison with free SM. The results of this study showed the great potential of SNEDDS in the enhancement of in vitro dissolution rate and anticancer efficacy of poorly soluble drug such as SM.     

Preparation and Optimization of Immediate Release/Sustained Release Bilayered Tablets of Loxoprofen Using Box–Behnken Design

The aim of our current study was to characterize and optimize loxoprofen immediate release (IR)/sustained release (SR) tablet utilizing a three-factor, three-level Box–Behnken design (BBD) combined with a desirability function. The independent factors included ratio of drug in the IR layer to total drug (X ₁), ratio of HPMC to drug in the SR layer (X ₂), and ratio of Eudragit RL PO to drug in the SR layer (X ₃). The dependent variables assessed were % drug released in distilled water at 30 min (Y ₁), % drug released in pH 1.2 at 2 h (Y ₂), and % drug released in pH 6.8 at 12 h (Y ₃). The responses were fitted to suitable models and statistical validation was performed using analysis of variance. In addition, response surface graphs and contour plots were constructed to determine the effects of different factor level combinations on the responses. The optimized loxoprofen IR/SR tablets were successfully prepared with the determined amounts of ingredients that showed close agreement in the predicted and experimental values of tablet characterization and drug dissolution profile. Therefore, BBD can be utilized for successful optimization of loxoprofen IR/SR tablet, which can be regarded as a suitable substitute for the current marketed formulations.     

Associations of HSP90AA2 gene polymorphisms with disease susceptibility,glucocorticoids efficacy and health-related quality of life in Chinese systemic lupus erythematosus patients

Although the current glucocorticoids (GCs) treatment for systemic lupus erythematosus (SLE) is effective to a certain extent, the difference in therapeutic effect between patients is still a widespread problem. Some patients can have repeated attacks that greatly diminish their quality of life. This study was conducted to investigate the relationship between HSP90AA2 polymorphisms and disease susceptibility, GCs efficacy and health-related quality of life (HRQoL) in Chinese SLE patients. A case–control study was performed in 470 SLE patients and 470 normal controls. Then, 444 patients in the case group were followed up for 12 weeks to observe efficacy of GCs and improvement of HRQoL. Two single nucleotide polymorphisms (SNPs) of HSP90AA2 were selected for genotyping: rs1826330 and rs6484340. HRQoL was assessed using the SF-36 questionnaire. The minor T allele of rs1826330 and the TT haplotype formed by rs1826330 and rs6484340 showed associations with decreased SLE risk (T allele: P_BH?=?0.022; TT haplotype: P_BH?=?0.033). A significant association between rs6484340 and improvement of HRQoL was revealed in the follow-up study. Five subscales of SF-36 were appeared to be influenced by rs6484340: total score of SF-36 (additive model: P_BH?=?0.026), physical function (additive model: P_BH?=?0.026), role-physical (recessive model: P_BH?=?0.041), mental health (dominant model: P_BH?=?0.047), and physical component summary (additive model: P_BH?=?0.026). No statistical significance was found between HSP90AA2 gene polymorphisms and GCs efficacy. These results revealed a genetic association between HSP90AA2 and SLE. Remarkably, HSP90AA2 has an impact on the improvement of HRQoL in Chinese population with SLE.     

3D-Printed Network Structures as Controlled-Release Drug Delivery Systems: Dose Adjustment,API Release Analysis and Prediction

3D printing evolved as a promising technique to improve individualization of drug therapy. In particular, when printing sustained release solid dosage forms, as for instance implants, inserts, and also tablets, estimation of the drug release profile in vivo is necessary. In most cases, corresponding analyses cannot be performed at hospital or community pharmacies. Therefore, the present study aimed to develop a sustained release drug delivery system produced via 3D printing, which allows dose adaption and estimation of drug release at the same time. Filaments as feedstock for the printer were produced via hot-melt extrusion and consisted of Eudragit® RL as sustained release polymer, 30% theophylline as model active pharmaceutical ingredient, and stearic acid as solid plasticizer. Assuming that the surface/mass ratio was constant, network structures of different densities were printed as novel solid dosage form. Their weight (263 to 668 mg), thereby their dose, and surface area, determined using X-ray microcomputed tomography, showed a linear correlation with the fill density. The specific surface area of the network hardly varied with changing fill density. Dissolution studies showed a slower drug release for dosage forms with a denser network. Higuchi’s model was used for prediction of drug release and showed limited applicability due to different release kinetics for different fill densities. However, using linear interpolation for the prediction resulted in good RMSEP values between 1.4 and 3.7%. These findings might be useful to enable customized production of sustained release solid dosage forms via 3D printing in hospital and community pharmacies in the future.     

Pharmacokinetic Evaluation of Improved Oral Bioavailability of Valsartan: Proliposomes <Emphasis Type="Italic">Versus</Emphasis> Self-Nanoemulsifying Drug Delivery System

The objective of this study was to develop proliposomes and self-nanoemulsifying drug delivery system (SNEDDS) for a poorly bioavailable drug, valsartan, and to compare their in vivo pharmacokinetics. Proliposomes were prepared by thin-film hydration method using different lipids such as soy phosphatidylcholine (SPC), hydrogenated soy phosphatidylcholine (HSPC), distearyl phosphatidylcholine (DSPC), dimyristoylphosphatidylcholine (DMPC), and dimyristoyl phosphatidylglycerol sodium (DMPG) and cholesterol in various ratios. SNEDDS formulations were prepared using varying concentrations of capmul MCM, labrafil M 2125, and Tween 80. Both proliposomes and SNEDDS were evaluated for particle size, zeta potential, in vitro drug release, in vitro permeability, and in vivo pharmacokinetics. In vitro drug release was carried out in purified water and 0.1 N HCl using USP type II dissolution apparatus. In vitro drug permeation was studied using parallel artificial membrane permeation assay (PAMPA) and everted rat intestinal permeation techniques. Among the formulations, the proliposomes with drug/DMPG/cholesterol in the ratio of 1:1:0.5 and SNEDDS with capmul MCM (16.0% w/w), labrafil M 2125 (64.0% w/w), and Tween 80 (18.0% w/w) showed the desired particle size and zeta potential. Enhanced drug release was observed with proliposomes and SNEDDS as compared to pure valsartan. Valsartan permeability across PAMPA and everted rat intestinal permeation models was significantly higher with proliposomes and SNEDDS. Following single oral administration of proliposomes and SNEDDS, a relative bioavailability of 202.36 and 196.87%, respectively, was achieved compared to pure valsartan suspension. The study results indicated that both proliposomes and SNEDDS formulations are comparable in improving the oral bioavailability of valsartan.     

Simple generalisation of a mesophyll resistance model for various intracellular arrangements of chloroplasts and mitochondria in C<Subscript>3</Subscript> leaves

The classical definition of mesophyll conductance (g _m) represents an apparent parameter (g _m,app) as it places (photo)respired CO₂ at the same compartment where the carboxylation by Rubisco takes place. Recently, Tholen and co-workers developed a framework, in which g _m better describes a physical diffusional parameter (g _m,dif). They partitioned mesophyll resistance (r _m,dif = 1/g _m,dif) into two components, cell wall and plasmalemma resistance (r _wp) and chloroplast resistance (r _ch), and showed that g _m,app is sensitive to the ratio of photorespiratory (F) and respiratory (R _d) CO₂ release to net CO₂ uptake (A): g _m,app = g _m,dif/[1?+?ω(F?+?R _d)/A], where ω is the fraction of r _ch in r _m,dif. We herein extend the framework further by considering various scenarios for the intracellular arrangement of chloroplasts and mitochondria. We show that the formula of Tholen et al. implies either that mitochondria, where (photo)respired CO₂ is released, locate between the plasmalemma and the chloroplast continuum or that CO₂ in the cytosol is completely mixed. However, the model of Tholen et al. is still valid if ω is replaced by ω(1?σ), where σ is the fraction of (photo)respired CO₂ that experiences r _ch (in addition to r _wp and stomatal resistance) if this CO₂ is to escape from being refixed. Therefore, responses of g _m,app to (F?+?R _d)/A lie somewhere between no sensitivity in the classical method (σ =1) and high sensitivity in the model of Tholen et al. (σ =0).     

Nitrogen-induced variations in leaf gas exchange of spring triticale under field conditions

Nitrogen (N) is the key factor limiting photosynthetic processes and crop yield. Little is known about the response of leaf gas exchange of spring triticale (Triticosecale Wittm.) to N supply. The effect of N fertilizers on different gas exchange variables, i.e., photosynthetic rate (A), transpiration rate (E), stomatal conductance (g _s), instantaneous water use efficiency (WUE) and maximum quantum yield of photosystem II (PSII) (F _v/F _m), chlorophyll index (SPAD, soil–plant analysis development), and the relationship of these variables with yield were studied in spring triticale grown under field conditions. Six treatments of N—0, 90, 180, 90 + 30, 90 + 30 + 30 kg ha^?1 (applied as ammonium nitrate, AN) and one treatment of N 90 + 30 + 30 kg ha^?1 (applied as urea ammonium nitrate solution, UAN) were compared. The analysis of variance showed that throughout the triticale growing season, N fertilization had significant effects on A, WUE, g _s and SPAD. On average, N fertilizer application increased A values by 14–70%. E and F _v/F _m values were not influenced by N fertilization levels. The effect of growth stage and year on gas exchange variables and F _v/F _m and SPAD was found to be significant. At different growth stages, A values varied and maximum ones were reached at BBCH 31–33 (decimal code system of growth stages) and BBCH 59. With aging, values of A decreased independently of N fertilization level. The gas exchange variables were equally affected by both fertilizer forms. The interplay among grain yield, leaf gas exchange variables, F _v/F _m and SPAD of spring triticale was estimated. The statistical analysis showed that grain yield positively and significantly correlated with A and SPAD values throughout the growing season.