PRODUCT OPTIMIZATION AND THE ACCEPTOR SET SIZE

The acceptability of a product, measured by the acceptor set size (percentage of consumers rating the product acceptable), is a function of the perception of its attributes. The attributes are themselves a function of the inputs to the product (such as ingredients, processing or storage variables). These assumptions lead to the following model:
Acceptor set size = F (attribute₁, attribute₂, …, attribute_n)
Attribute j = f (input₁, input₂, …, input_m)
If we assume that these functions are differentiable, we can estimate the partial derivatives of the acceptor set size, with respect to the input variables. The gradient vector obtained indicates the fastest way to maximize the acceptor set size. The gradient search method, using the acceptor set size as an objective measure, can be applied in a variety of situations: to improve existing products, to maximize the acceptability of new products, and to study the relationship between shelf-life and acceptability.     

SURVIVAL ANALYSIS TO ESTIMATE SENSORY SHELF LIFE USING ACCEPTABILITY SCORES

ABSTRACT

In the present study, survival analysis was applied on consumers' acceptability scores, focusing the shelf‐life risk on the consumers disliking the product. Shelf life was estimated as the time necessary to reach a fixed percentage of consumers disliking the product, that is, the percentage of consumers scoring the product's overall acceptability below 6 in a 9‐point hedonic scale. In the present work, this methodology was applied to estimate the sensory shelf life of whole pan bread and alfajores. Shelf lives estimated considering 50% of the consumers disliking the product were shorter than those estimated considering consumers' rejection to consume. These results suggest that a proportion of consumers might dislike the sample but still answer “yes” when asked if they would consume the product at their homes. This could be attributed to the fact that when consumers are asked whether they would consume the product, they might think about consuming it after being stored at their homes. In this situation, consumers might be more tolerant toward sensory defects because they do not want to discard the product.

PRACTICAL APPLICATIONS

Sensory shelf life can be estimated using survival analysis considering the percentage of consumers disliking the product. This methodology seems to be a conservative criterion in order to assure the product's quality throughout its storage.     

A Generic Model to Solve Tactical Planning Problems in Flexible Manufacturing Systems

This paper is an attempt to develop a generic modeling framework that addresses tactical planning problems of flexible manufacturing systems in a coherent manner. We propose a generic 0-1 mixed integer programming formulation, that integrates batching, loading, and routing problems with their critical aspects related to a system's performance. For this purpose, a thorough analysis is made to determine and relate system components, their attributes, and alternatives together with performance measures specific to tactical planning. This provided the justification to support our argument about generality of the model. A linear programming formulation is provided to approximate the mixed integer formulation proposed so as to overcome the problem's combinatorial complexity. The potential capability of the linear approximation proposed also is demonstrated via a small set of test problems.     

Use of Polyvinyl Alcohol as a Solubility-Enhancing Polymer for Poorly Water Soluble Drug Delivery (Part 1)

Polyvinyl alcohol (PVAL) has not been investigated in a binary formulation as a concentration-enhancing polymer owing to its high melting point/high viscosity and poor organic solubility. Due to the unique attributes of the KinetiSol® dispersing (KSD) technology, PVAL has been enabled for this application and it is the aim of this paper to investigate various grades for improvement of the solubility and bioavailability of poorly water soluble active pharmaceutical ingredients. Solid amorphous dispersions were created with the model drug, itraconazole (ITZ), at a selected drug loading of 20%. Polymer grades were chosen with variation in molecular weight and degree of hydroxylation to determine the effects on performance. Differential scanning calorimetry, powder X-ray diffraction, polarized light microscopy, size exclusion chromatography, and dissolution testing were used to characterize the amorphous dispersions. An in vivo pharmacokinetic study in rats was also conducted to compare the selected formulation to current market formulations of ITZ. The 4-88 grade of PVAL was determined to be effective at enhancing solubility and bioavailability of itraconazole.     

Detection of NAD:arginine ADPribosyltransferases in animal tissues using 125I-labeled 1-(p-hydroxyphenyl) 2-guanidinoethane as ADPribose acceptor

125I-labeled 1-(p-hydroxyphenyl) 2-guanidinoethane (N-guanyltyramine), previously used to assay for the bacterial toxin choleragen (Mekalanos, J.J., Collier, R.J. and Romig, W.R. (1979) J. Biol. Chem. 254, 5849-5854) was utilized to identify NAD:arginine ADPribosyltransferases in animal tissues. The use of this radiolabelled ADPribose acceptor, rather than radiolabelled NAD, would bypass the problem posed by the almost ubiquitous presence of enzymes that degrade NAD. With a homogeneous ADPribosyltransferase from turkey erythrocytes, NAD and 125I-labeled guanyltyramine as ADPribose acceptor, formation of ADPribosyl 125I-guanyltyramine was linear with time and enzyme concentration. The product was indistinguishable on both thin-layer and high-performance liquid chromatography from that formed by choleragen. Using 125I-guanyltyramine, ADPribosyltransferase activity was also demonstrated in crude turkey erythrocyte cytosolic and membrane fractions. When rat liver was fractionated, apparent activity was detected primarily in the microsomes. The NAD-dependent product of the microsomal reaction was, however, distinguished from the turkey erythrocyte transferase product by thin-layer and DEAE-Sephadex chromatography; this product had a retention time identical to that of free 125I on high-performance liquid chromatography. In addition to NAD, the microsomal deiodinase activity was supported by NADH, NADP and NADPH. Phenyl boronate selectively bound ADPribosyl 125I-guanyltyramine and other metabolites of 125I-guanyltyramine which were formed by microsomes in a NAD-dependent process. These metabolites were distinguished from ADPribosyl 125I-guanyltyramine by high-performance liquid chromatography. These results indicate that in some cases, for example, turkey erythrocyte cytosolic and membrane fractions, 125I-guanyltyramine can be used to quantify ADPribosyltransferases in crude mixtures, whereas in others, for example, rat liver microsomes, high-performance liquid chromatographic analysis must be used to identify products.     

Family-size distribution and Ewens'' equivalence theorem.

Segregation analysis of a data set containing nuclear families of more than one sibship size is considered, and two different formulations of the likelihood are examined. One is the "separate-multinomials" formulation, which treats each family size as representing a separate multinomial distribution; the other is the "grand-multinomial" formulation, which treats the entire data set as representing one distribution. It is shown that these two formulations are equivalent, if and only if the population distribution of family sizes is completely unknown. However, if anything is known about the family-size distribution, the grand-multinomial formulation, although more cumbersome, makes more complete use of the data; moreover, it enables the use of one-child families in a segregation analysis. The relationship of this work to Ewens' equivalence theorem concerning "unconditional" and "conditional" likelihoods is discussed. The findings are illustrated with a simple example, and their practical relevance to real-life segregation analysis is discussed.     

Flexibility and Pricing Decisions for High-Volume Products with Short Life Cycles

We examine the competitive implications of a firm's ability to change over its facility for the manufacture of successive generations of high-volume products with short life cycles. This ability is known as changeover flexibility. The model introduced extends the existing literature in several directions. First, the model offers explicit treatment of the critical relationships between market entry time, changeover flexibility, product life cycles, and profit. Second, the model explicitly considers the effect of early market entry on the accumulation of manufacturing experience (learning), which reduces the unit production cost. Third, the product's optimal selling price is determined and its relation to the firm's changeover flexibility is examined. Last, facility flexibility is permitted to vary over a continuum. Therefore, we are able to capture decision making concerning the optimal degree of changeover flexibility. Both analytic and numerical results are reported, demonstrating the link between the operations and marketing domains in the context of a firm's optimal entrance and exit strategies. Among the key findings are (1) a firm more capable of reducing operating costs through learning over short life cycles optimally invests in more changeover flexibility, charges higher prices, and obtains greater profit; and (2) as the cost of flexible technologies decrease, a firm optimally increases its investment in changeover flexibility, enters markets earlier, and charges higher average prices over the product's life cycle.     

Exact and approximation algorithms for DNA tag set design.

In this paper, we propose new solution methods for designing tag sets for use in universal DNA arrays. First, we give integer linear programming formulations for two previous formalizations of the tag set design problem. We show that these formulations can be solved to optimality for problem instances of moderate size by using general purpose optimization packages and also give more scalable algorithms based on an approximation scheme for packing linear programs. Second, we note the benefits of periodic tags and establish an interesting connection between the tag design problem and the problem of packing the maximum number of vertex-disjoint directed cycles in a given graph. We show that combining a simple greedy cycle packing algorithm with a previously proposed alphabetic tree search strategy yields an increase of over 40% in the number of tags compared to previous methods.     

The Role of Formulation Additives in Increasing the Potency of Cydia pomonella Granulovirus for Codling Moth Larvae,in Laboratory and Field Experiments

Studies were undertaken to improve the biological efficacy of the granulovirus (CpGV) of the codling moth, Cydia pomonella , by evaluating the performance of some formulation additives that might improve virus persistence and/or virus uptake by first instar larvae. Laboratory studies, using a leaf disc bioassay, demonstrated that 15% cane molasses incorporated within a formulation of purified CpGV dramatically reduced the median lethal exposure time (LET 50 ) to CpGV for neonate larvae at a CpGV dosage rate of 10 7 occlusion bodies (OBs) ml -1 . Screening of a range of other compounds showed that sucrose, fructose and sorbitol (at 10% concentrations) and extracts of apple flesh and skin also gave significant reductions in the LET 50 of CpGV formulations containing these ingredients. Pectin, malic acid and &#102 -farnesene did not significantly reduce the LET 50 . In a field trial, molasses included at 15% (v/v) in a CpGV formulation, containing a dosage rate of 10 12 OBs ha -1 , gave as good control of codling moth damage as virus formulations containing the 'sticker' 0.2% skimmed milk at higher dosage rates of 10 13 and 10 14 OBs ha -1 . Studies of CpGV persistence on foliage revealed no significant improvement of virus persistence on apple foliage using 10% or 15% molasses formulations. A second field trial demonstrated that 10% molasses, 10% sorbitol or 0.08% &#102 -farnesene significantly reduced codling moth deep damage to fruit when these ingredients were added to formulations of pure CpGV. Substantial sooty-mould growth ( Cladosporium spp.) was observed on apple foliage treated with formulations containing molasses, indicating that this formulation additive has secondary consequences that would need to be taken into account if molasses was to be used in commercial CpGV formulations. Nonetheless, these studies clearly demonstrate that major biological improvements in CpGV performance can be achieved by the incorporation of formulation additives, including molasses and several other compounds, that probably function as attractants and/or feeding stimulants for codling moth larvae.     

Stereospecific determination of the in vitro dissolution of modified release formulations of (±)-verapamil

The determination of the in vitro dissolution profiles of three different modified-release formulations of (±)-verapamil were determined and compared to a reference (Calan SR). The determination of (±)-verapamil utilized a microwave-facilitated derivatization reaction with an enantiomerically pure reagent followed by measurement of drug by reversed-phase high-performance liquid chromatography (HPLC). The dissolution profile of each of the four modified-release formulations was followed for the first hour in simulated gastric fluid (pH 1.2) and then in simulated intestinal fluid (pH 7.5) for up to a total of 12 h. The resulting dissolution profiles of each modified-release formulation suggested that only one of the three test formulations had a similar dissolution to the Calan SR reference. Interestingly, the (S:R)-enantiomer ratios of two of the test products were significantly different from unity, and the third test product was also significantly different from unity if 4 out of 5 outliers were omitted. It is suggested that dissolution testing of modified-release formulations containing chiral active ingredients must be stereospecific in order to discern whether a drug-excipient interaction occurs. Additionally, it may be suggested that dissolution techniques relying more upon diffusion of medium through the tablet matrix, rather than erosion of the tablet, may accentuate enantiomeric differences in release rates. © 1993 Wiley-Liss, Inc.     

Quality by Design Approach: Application of Artificial Intelligence Techniques of Tablets Manufactured by Direct Compression

The publication of the International Conference of Harmonization (ICH) Q8, Q9, and Q10 guidelines paved the way for the standardization of quality after the Food and Drug Administration issued current Good Manufacturing Practices guidelines in 2003. “Quality by Design”, mentioned in the ICH Q8 guideline, offers a better scientific understanding of critical process and product qualities using knowledge obtained during the life cycle of a product. In this scope, the “knowledge space” is a summary of all process knowledge obtained during product development, and the “design space” is the area in which a product can be manufactured within acceptable limits. To create the spaces, artificial neural networks (ANNs) can be used to emphasize the multidimensional interactions of input variables and to closely bind these variables to a design space. This helps guide the experimental design process to include interactions among the input variables, along with modeling and optimization of pharmaceutical formulations. The objective of this study was to develop an integrated multivariate approach to obtain a quality product based on an understanding of the cause–effect relationships between formulation ingredients and product properties with ANNs and genetic programming on the ramipril tablets prepared by the direct compression method. In this study, the data are generated through the systematic application of the design of experiments (DoE) principles and optimization studies using artificial neural networks and neurofuzzy logic programs.KEY WORDS: artificial neural networks (ANNs), gene expression programming (GEP), optimization, quality by design (QbD)     

Theoretical and Experimental Considerations Related to Reaction-Based Modeling: A Case Study Using Iron(III) Oxide Bioreduction

The kinetics of reductive dissolution of hematite ( f -Fe 2 O 3 ) by the dissimilatory iron-reducing bacterium Shewanella putrefaciens strain CN32 under nongrowth conditions with H 2 as the electron donor was measured and then modeled using a reaction-based biogeochemical model. Minimum data needs and a reaction matrix decomposition procedure are presented from a reaction-based modeling perspective and used to design subsequent experiments. Detailed step-by-step modeling methodology is presented. Independent experiments were performed to determine if Fe 2+ sorption to S. putrefaciens CN32 or hematite could be described as either kinetic or equilibrium reactions (i.e., slow or fast, respectively, relative to the time-scale of the bioreduction experiments). Fe 2+ sorption to S. putrefaciens CN32 was an equilibrium reaction and a linear adsorption isotherm was used to determine the associated equilibrium constant. Fe 2+ sorption to hematite was a kinetic reaction and an elementary rate formulation was independently determined from abiotic experiments. The ratio of the forward rate divided by the backward rate [log(k f /k b )] for the sorption of Fe 2+ to hematite was 6.33 - 0.14 (n = 2) and the corresponding log(k f ) was 6.66 - 0.28 (n = 2, M -1 h -1 ). Three different kinetic reaction rate formulations were used to model hematite bioreduction, an elementary rate law for the overall reaction, an empirical rate law physically based on hematite "free" surface sites, and an empirical rate law physically based on hematite free surface sites and bacterial inhibition caused by Fe(II) biosorption. All rate formulations modeled the measured results reasonably well (R 2 values ranged from 0.83 to 0.99). For the elementary rate formulation, log(k f /k b ) was 24.37 - 0.15 (n = 4) and the corresponding forward rate [log(k f )] was 26.46 - 0.27 (n = 4, M -4 h -1 ). These results demonstrate that independently determined reaction-based rate formulations were applicable in another experimental system, as theoretically expected. Therefore, the simulation and prediction of complex biogeochemical systems may eventually be able to be performed using reaction-based models.     

Application of thermal effusivity as a process analytical technology tool for monitoring and control of the roller compaction process

The aim of this study was to examine the relationship between physical characteristics of compacted ribbons and their thermal effusivity in an attempt to evaluate the feasibility of using effusivity for in-process monitoring of roller compaction. In this study, thermal effusivity, solid fraction, tensile strength, and Young's modulus of ribbons of microcrystal-line cellulose (MCC), anhydrous lactose, and placebo (PBO) formulations containing various ratios of MCC to anhydrous lactose (75∶20, 55∶40, 40∶55, and 20∶75) were determined at various compaction pressures (25–150 bars). The effusivity-square root of solid fraction relationship was linear for MCC and all the PBO formulations but was a second-order polynomial function for lactose. This could be due to the predominant deformation of lactose by brittle fracture, which might have significantly increased the number and size of contact points between particles, causing a change in thermal conductivity along with a density change. The effusivitytensile strength and effusivity-Young's modulus relationships were best described by logarithmic functions for MCC but were linear for lactose up to a compaction pressure of 65 bars. There were similar relationships for effusivity with tensile strength and Young's modulus for all PBO formulations except PBO IV, which might have been due to the deformation of lactose, the largest component in this formulation. Strong correlations between effusivity and physical properties of ribbons were established. Although these correlations were formulation-dependent, they demonstrate the possibility of using effusivity as a tool in monitoring roller compaction. Published: March 23, 2007     

Prediction of Drug Solubility in Lipid Mixtures from the Individual Ingredients

The purpose of this research was to investigate the relationship of drug solubility in a complex lipid mixture to that of the individual ingredients with the goal of substantiating a quantitative equation that can be applied in formulation development of lipid dosage forms. To this end, the solubility of four drugs, which span a large range of physicochemical properties, was evaluated in 18 lipid ingredients that cover the major lipid classes. To assess the solubility relation in complex lipid mixtures in an unbiased manner, the experiments were created as an experimental design with the ability to detect cubic curvature in the solubility-lipid composition space. The results demonstrated that for all drugs, irrespective of their significantly distinct physicochemical properties, solubility in lipid mixtures can be readily estimated as a simple weighted average of the drug solubility in the individual ingredients. This result is of great value to formulators who can minimize a large number of solubility experiments once a basis set of solubility is determined in individual lipids.     

Storage stability of Anagrapha falcifera nucleopolyhedrovirus in spray-dried formulations

A multiply embedded nucleopolyhedrovirus isolated from Anagrapha falcifera (Kirby) (AfMNPV) can lose insecticidal activity during months of dry storage in ambient room conditions. We tested the spray-dried AfMNPV formulations after storage for up to 1 year at room temperatures for insecticidal activity against neonate Trichoplusia ni (Hübner). Experimental formulations were made using combinations of corn flours, lignin, and sucrose, and were selected based on previous work which demonstrated that these formulations resisted solar degradation in field experiments. Twelve experimental formulations (organized in three groups of four formulations) compared the effect of (1) the ratio of formulation ingredients (lignin and corn flour) to virus concentration, (2) different sources of lignin, or (3) different corn flours and sugar. Based on a single-dose plant assay with these 12 formulations, none of the formulations lost significant activity due to the drying process, when compared with the unformulated wet AfMNPV. Samples of the 12 dried formulations were stored at room (22+/-3 degrees C) and refrigerated (4 degrees C) temperatures. Insecticidal activity (LC(50)) was determined with a dosage-response assay for neonates fed on treated cotton-leaf disks. After 6 (or 9) and 12 months storage, refrigerated samples maintained insecticidal activity better than corresponding samples stored at room temperatures with LC(50)s that averaged 2.0 x 10(6) polyhedral inclusion bodies per milliliter (pibs/ml) for refrigerated samples and 5.4 x 10(6) pibs/ml for samples stored at room temperatures. Compared with unformulated stock virus stored frozen, six formulations stored at room temperature and 10 formulations stored in the refrigerator did not lose significant insecticidal activity after 1 year based on overlapping 90% confidence intervals. Changing the ratio of virus to formulation ingredients did not provide a clear trend over the range of concentrations tested, and may be less important for shelf-life of virus activity compared with formulations made with different ingredients. Two of the four formulations made with different lignins were about 15 times less active after 1 year at room temperature compared with refrigerated samples, indicating that specific formulation ingredients can affect storage stability. Formulations that contained sugar generally maintained activity during storage better than formulations without sugar. Unformulated virus stock maintained insecticidal activity (ranged from 0.20 to 2.5 x 10(6) pibs/ml) better during storage than dried formulations with LC(50)s that ranged from 0.39 to 27 x 10(6) pibs/ml. Unformulated virus stock, which is essentially a suspension of virus occlusion bodies in homogenized insect cadavers, did not lose activity when stored at refrigerated or room temperature. We believe that stability of AfMNPV insecticidal activity during storage as dry formulations is related to the general composition of the formulation and that sugar may play a critical role in maintaining insecticidal activity.     

Design and Characterization of Topical Formulations: Correlations Between Instrumental and Sensorial Measurements

The interaction between cosmetic emulsions and the skin’s surface is an important factor to consider in the development of topical formulations. Two important ingredients in cosmetic formulations are waxes and polymers. The physical and mechanical properties of formulations directly impact the interface skin-formulation. To evaluate this interaction, it is important to study the rheology, texture, and sensory properties. In this context, the aim of the study was to evaluate the influence of waxes and polymers on the rheological behavior, texture profile, and sensorial properties of topical formulations and the correlation between these parameters. The best combination of a wax and a polymer was determined by full factorial design of experiments and applied to develop eight formulations that were tested in relation to rheological, mechanical, and sensorial properties. The polymer helps with the spreadability of the formulation, and the wax had a strong influence on the parameters related to the structure of emulsions. A correlation between these parameters was observed. This way, it was possible to compare theoretical and practical data, except between the flow index and the work of shear. Finally, it was possible to predict sensorial aspects from rheological and texture parameters, making the formulation process easier and more integrated with all stages of the development of new topical formulations. Thus, the present study introduces a new proposal in the development of cosmetics.     

Multi‐criteria manufacturability indices for ranking high‐concentration monoclonal antibody formulations

The need for high‐concentration formulations for subcutaneous delivery of therapeutic monoclonal antibodies (mAbs) can present manufacturability challenges for the final ultrafiltration/diafiltration (UF/DF) step. Viscosity levels and the propensity to aggregate are key considerations for high‐concentration formulations. This work presents novel frameworks for deriving a set of manufacturability indices related to viscosity and thermostability to rank high‐concentration mAb formulation conditions in terms of their ease of manufacture. This is illustrated by analyzing published high‐throughput biophysical screening data that explores the influence of different formulation conditions (pH, ions, and excipients) on the solution viscosity and product thermostability. A decision tree classification method, CART (Classification and Regression Tree) is used to identify the critical formulation conditions that influence the viscosity and thermostability. In this work, three different multi‐criteria data analysis frameworks were investigated to derive manufacturability indices from analysis of the stress maps and the process conditions experienced in the final UF/DF step. Polynomial regression techniques were used to transform the experimental data into a set of stress maps that show viscosity and thermostability as functions of the formulation conditions. A mathematical filtrate flux model was used to capture the time profiles of protein concentration and flux decay behavior during UF/DF. Multi‐criteria decision‐making analysis was used to identify the optimal formulation conditions that minimize the potential for both viscosity and aggregation issues during UF/DF. Biotechnol. Bioeng. 2017;114: 2043–2056. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Perodicals, Inc.     

Influence of fruit traits on the infestation of Dacus persicus in two fruit morphs of Calotropis procera

The larvae of Dacus (Leptoxyda) persicus (aak fruit fly) are key predispersal seed predators in Calotropis procera (Asclepiadaceae). Based on fruit characteristics, two morphs are distinguishable in C. procera viz., the soft-fruited morph (SF morph) and the hard-fruited morph (HF morph). The work reported here examined whether the fruit characteristics influenced the infestation by the aak fruit fly and, if so, what mechanism(s) were operative. Fruits in the SF morph were significantly more acceptable to the aak fruit fly than those of the HF morph irrespective of their size class and availability or fly population density. A general ranking of fruit acceptability for oviposition by the aak fruit fly within the fruit size class was: size class III ≥ size class II > size class I and IV. The negative relationship between fruit infestation and pericarp toughness, which is suggestive of trade-offs between the fly’s oviposition obligation and energy/time (predation risk) constraint, was found to correlate with the requirement of greater force to puncture the pericarp in the hard fruits. Lower penetrability of the pericarp in the hard fruits appeared to be primarily due to the thickness of pericarp and secondarily on account of the thickened walls of endocarpic–mesocarpic cells in the inner pericarpic layer. The present data point to the existence of two fruit morphs in C. procera differing in the acceptability of fruits for oviposition by the aak fruit fly primarily on account of toughness and internal structure of the pericarp.     

Detection of NAD: arginine ADPribosyltransferases in animal tissues using I-labeled 1-(p-hydroxyphenyl) 2-guanidinoethane as ADPribose acceptor

¹²⁵I-labeled 1-(p-hydroxyphenyl) 2-guanidinoethane (N-guanyltyramine), previously used to assay for the bacterial toxin choleragen (Mekalanos, J.J., Collier, R.J. And Romig, W.R. (1979) J. Biol. Chem. 254, 5894-5854) was utilized to identify NAD: arginine ADPribosyltransferases in animal tissues. The use of this radiolabelled ADPribose acceptor, rather than radiolabelled NAD, would bypass the problem posed by the almost ubiquitous presence of enzymes that degrade NAD. With a homogeneous ADPribosyltransferase from turkey erythrocytes, NAD and ¹²⁵I-labelled guanyltyramine as ADPribose acceptor, formation of ADPribosyl ¹²⁵-I-guanyltyramine was linear with time and enzyme concentration. The product was distinguishable on both thin-layer and high-performance liquid chromatography from that formed by cholerangen. Using ¹²⁵I-guanyltyramine, ADPribosyltransferase acitivity was also demonstrated in crude turkey erythrocyte cytosolic and membrane fractions. When rat liver was fractioned, apparent activity was detected primarily in the microsomes. The NAD-dependent product of the microsomal reaction was, however, distinguished from the turkey erythrocyte transferase by thin-layer and DEAE-Sephadex chromatography; this product had a retention time identical to that of free ¹²⁵I on high-performance liquid chromatography. In addition to NAD, the microsomal deiodinase activity was supported by NADH, NADP and NADPH. Phenyl boronate selectively bound ADPribosyl ¹²⁵I-guanyltyramine and other metabolites of ¹²⁵I-guanyltyramine which were formed by microsomes in a NAD-dependent process. These metabolites were distinguished from ADPribosyl ¹²⁵I-guanyltyramine by high-performance liquid chromatography. These results indicate that in some cases, for example, turkey erythrocyte cytosolic and membrane fractions, ¹²⁵I-guanyltyramine can be used to quantify ADPribosyltransferases in crude mixtures, whereas in others, for example, rat liver microsomes, high-performance liquid chromatographic analysis must be used to identify products.     

Detection of NAD: arginine ADPribosyltransferases in animal tissues using 125I-labeled 1-(p-hydroxyphenyl) 2-guanidinoethane as ADPribose acceptor

¹²⁵I-labeled 1-(p-hydroxyphenyl) 2-guanidinoethane (N-guanyltyramine), previously used to assay for the bacterial toxin choleragen (Mekalanos, J.J., Collier, R.J. And Romig, W.R. (1979) J. Biol. Chem. 254, 5894-5854) was utilized to identify NAD: arginine ADPribosyltransferases in animal tissues. The use of this radiolabelled ADPribose acceptor, rather than radiolabelled NAD, would bypass the problem posed by the almost ubiquitous presence of enzymes that degrade NAD. With a homogeneous ADPribosyltransferase from turkey erythrocytes, NAD and ¹²⁵I-labelled guanyltyramine as ADPribose acceptor, formation of ADPribosyl ¹²⁵-I-guanyltyramine was linear with time and enzyme concentration. The product was distinguishable on both thin-layer and high-performance liquid chromatography from that formed by cholerangen. Using ¹²⁵I-guanyltyramine, ADPribosyltransferase acitivity was also demonstrated in crude turkey erythrocyte cytosolic and membrane fractions. When rat liver was fractioned, apparent activity was detected primarily in the microsomes. The NAD-dependent product of the microsomal reaction was, however, distinguished from the turkey erythrocyte transferase by thin-layer and DEAE-Sephadex chromatography; this product had a retention time identical to that of free ¹²⁵I on high-performance liquid chromatography. In addition to NAD, the microsomal deiodinase activity was supported by NADH, NADP and NADPH. Phenyl boronate selectively bound ADPribosyl ¹²⁵I-guanyltyramine and other metabolites of ¹²⁵I-guanyltyramine which were formed by microsomes in a NAD-dependent process. These metabolites were distinguished from ADPribosyl ¹²⁵I-guanyltyramine by high-performance liquid chromatography. These results indicate that in some cases, for example, turkey erythrocyte cytosolic and membrane fractions, ¹²⁵I-guanyltyramine can be used to quantify ADPribosyltransferases in crude mixtures, whereas in others, for example, rat liver microsomes, high-performance liquid chromatographic analysis must be used to identify products.