Membrane Adaptations and Cellular Responses of Sulfolobus acidocaldarius to the Allylamine Terbinafine.

Cellular membranes are essential for compartmentalization, maintenance of permeability, and fluidity in all three domains of life. Archaea belong to the third domain of life and have a distinct phospholipid composition. Membrane lipids of archaea are ether-linked molecules, specifically bilayer-forming dialkyl glycerol diethers (DGDs) and monolayer-forming glycerol dialkyl glycerol tetraethers (GDGTs). The antifungal allylamine terbinafine has been proposed as an inhibitor of GDGT biosynthesis in archaea based on radiolabel incorporation studies. The exact target(s) and mechanism of action of terbinafine in archaea remain elusive. Sulfolobus acidocaldarius is a strictly aerobic crenarchaeon thriving in a thermoacidophilic environment, and its membrane is dominated by GDGTs. Here, we comprehensively analyzed the lipidome and transcriptome of S. acidocaldarius in the presence of terbinafine. Depletion of GDGTs and the accompanying accumulation of DGDs upon treatment with terbinafine were growth phase-dependent. Additionally, a major shift in the saturation of caldariellaquinones was observed, which resulted in the accumulation of unsaturated molecules. Transcriptomic data indicated that terbinafine has a multitude of effects, including significant differential expression of genes in the respiratory complex, motility, cell envelope, fatty acid metabolism, and GDGT cyclization. Combined, these findings suggest that the response of S. acidocaldarius to terbinafine inhibition involves respiratory stress and the differential expression of genes involved in isoprenoid biosynthesis and saturation.

Naftifine: A Topical Allylamine for Superficial Dermatophytosis.

Dermatophytosis is a very common public health problem with high prevalence. Dermatophytes are a highly specialized set of filamentous fungi, which are adapted to keratinized tissues of humans and animals. Dermatophytosis is the most common fungal infection worldwide, affecting approximately 20-25% of the world's population. The etiological agents of dermatophytosis, called dermatophytes, change with geography and socioeconomic status. Trichophyton rubrum (T. rubrum) is the prime species for skin and nail infections followed by T. mentagrophytes/ T. interdigital complex. There is a shift from T. rubrum to T. mentagrophytes in India for superficial fungal infections. In order to deal with fungal infections, treatment strategies involve the use of systemic antifungals and/or topical antifungal agents. Naftifine is a synthetic allylamine antifungal first reported in 1974 and in 1985 became the first commercially available allylamine. The highly lipophilic nature of allylamine allows efficient penetration and reasonably high concentrations in the stratum corneum (SC) and hair follicles. Naftifine is fungicidal as well as fungistatic. The higher efficacy rates of allylamines over imidazoles for the treatment of fungal infections, even for months after cessation of treatment, is thought to be due to their fungicidal effect, as well as to potentially greater keratin binding and slower release from the SC. The effectiveness of naftifine is also demonstrated against various bacteria belonging to both gram-negative and gram-positive classes. The antiinflammatory property of naftifine has been reported in various preclinical studies where it has been shown to target the prostaglandin pathway. Naftifine 1 and 2% gel and cream is approved by The United States Food and Drug Administration (USFDA), recently naftifine has been approved in India by the Indian regulatory authority Drug Controller General of India (DCGI) for the treatment of dermatophytosis. Naftifine 2% also appears to be a promising treatment, requiring fewer applications than the 1% formulation. Naftifine appears to be effective in a single dose and has a shorter treatment duration than azoles. Naftifine demonstrated its efficacy and safety in various clinical studies of tinea infections. Naftifine offers a very useful and promising option for treating dermatophytosis.

Effects of colesevelam on glucose absorption and hepatic/peripheral insulin sensitivity in patients with type 2 diabetes mellitus.

AIM: Colesevelam lowers glucose and low-density lipoprotein cholesterol levels in patients with type 2 diabetes mellitus. This study examined the mechanisms by which colesevelam might affect glucose control. METHODS: In this 12-week, randomized, double-blind, placebo-controlled study, subjects with type 2 diabetes and haemoglobin A(1c) (HbA(1c)) ≥7.5% on either stable diet and exercise or sulphonylurea therapy were randomized to colesevelam 3.75 g/day (n = 16) or placebo (n = 14). Hepatic/peripheral insulin sensitivity was evaluated at baseline and at week 12 by infusion of (3) H-labelled glucose followed by a 2-step hyperinsulinemic-euglycemic clamp. Two 75-g oral glucose tolerance tests (OGTTs) were conducted at baseline, one with and one without co-administration of colesevelam. A final OGTT was conducted at week 12. HbA(1c) and fasting plasma glucose (FPG) levels were evaluated pre- and post-treatment. RESULTS: Treatment with colesevelam, compared to placebo, had no significant effects on basal endogenous glucose output, response to insulin or on maximal steady-state glucose disposal rate. At baseline, co-administration of colesevelam with oral glucose reduced total area under the glucose curve (AUC(g)) but not incremental AUC(g). At week 12, neither total AUC(g) nor incremental AUC(g) were changed from pre-treatment values in either group. Post-load insulin levels increased with colesevelam at 30 and 120 min, but these changes in total area under the insulin curve (AUC(i)) and incremental AUC(i) did not differ between groups. Both HbA(1c) and FPG improved with colesevelam, but treatment differences were not significant. CONCLUSIONS: Colesevelam does not affect hepatic or peripheral insulin sensitivity and does not directly affect glucose absorption.

Bile acid salt binding with colesevelam HCl is not affected by suspension in common beverages.

It has been previously reported that anions in common beverages may bind to bile acid sequestrants (BAS), reducing their capacity for binding bile acid salts. This study examined the ability of the novel BAS colesevelam hydrochloride (HCl), in vitro, to bind bile acid sodium salts following suspension in common beverages. Equilibrium binding was evaluated under conditions of constant time and varying concentrations of bile acid salts in simulated intestinal fluid (SIF). A stock solution of sodium salts of glycochenodeoxycholic acid (GCDC), taurodeoxycholic acid (TDC), and glycocholic acid (GC), was added to each prepared sample of colesevelam HCl. Bile acid salt binding was calculated by high-performance liquid chromatography (HPLC) analysis. Kinetics experiments were conducted using constant initial bile acid salt concentrations and varying binding times. The affinity, capacity, and kinetics of colesevelam HCl binding for GCDC, TDC, and GC were not significantly altered after suspension in water, carbonated water, Coca-Cola, Sprite, grape juice, orange juice, tomato juice, or Gatorade. The amount of bile acid sodium salt bound as a function of time was unchanged by pretreatment with any beverage tested. The in vitro binding characteristics of colesevelam HCl are unchanged by suspension in common beverages.

Colloidal nanocarriers for the enhanced cutaneous delivery of naftifine: characterization studies and in vitro and in vivo evaluations.

In topical administration of antifungals, the drugs should pass the stratum corneum to reach lower layers of the skin in effective concentrations. Thus, the formulation of antifungal agents into a suitable delivery system is important for the topical treatment of fungal infections. Nanosized colloidal carriers have gained great interest during the recent years to serve as efficient promoters of drug penetration into the skin. Microemulsions are soft colloidal nanosized drug carriers, which are thermodynamically stable and isotropic systems. They have been extensively explored for the enhancement of skin delivery of drugs. This study was carried out to exploit the feasibility of colloidal carriers as to improve skin transport of naftifine, which is an allylamine antifungal drug. The microemulsions were formulated by construction of pseudoternary phase diagrams and composed of oleic acid (oil phase), Kolliphor(®) EL or Kolliphor(®) RH40 (surfactant), Transcutol(®) (cosurfactant), and water (aqueous phase). The plain and drug-loaded microemulsions were characterized in terms of isotropy, particle size and size distribution, pH value, refractive index, viscosity, and conductivity. The in vitro skin uptake of naftifine from microemulsions was studied using tape stripping technique in pig skin. The drug penetrated significantly into stratum corneum from microemulsions compared to its marketed cream (P<0.05). Moreover, the microemulsion formulations led to highly significant amount of naftifine deposition in deeper layers of skin than that of commercial formulation (P<0.001). Microemulsion-skin interaction was confirmed by attenuated total reflectance - Fourier transformed infrared spectroscopy data, in vitro. The results of the in vivo tape stripping experiment showed similar trends as the in vitro skin penetration study. Topical application of the microemulsion on human forearms in vivo enhanced significantly the distribution and the amount of naftifine penetrated into the stratum corneum as compared to the marketed formulation (P<0.05). The relative safety of the microemulsion formulations was demonstrated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide viability test. This study indicated that the nanosized colloidal carriers developed could be considered as an effective and safe topical delivery system for naftifine.

In vivo metabolism of the cardiovascular toxin, allylamine.

Previous evidence from this laboratory demonstrated that allylamine, a known cardiovascular toxin, is metabolized in vitro to acrolein, which has been hypothesized to act as a distal toxin. In this study, 3-hydroxypropylmercapturic acid was isolated and identified by MS, NMR, and 2D-NMR spectroscopy as the sole urinary metabolite of allylamine metabolism in vivo. Parallel experiments showed reduced glutathione (GSH) depletion in several organs (most marked in aorta, blood, and lung), which is consistent with GSH conjugation of the proposed acrolein intermediate. These findings indicate that allylamine was metabolized in vivo to a highly reactive aldehyde which was converted to a mercapturic acid through a GSH conjugation pathway; the exact mechanisms of cellular damage remain unclear.

Allylamine cardiotoxicity--IV. Metabolism to acrolein by cardiovascular tissues.

Acrolein was detected in homogenates of rat aorta, lung, skeletal muscle, and heart incubated with allylamine. Semicarbazide and hydralazine, which protect against allylamine-induced myocardial injury in vivo in the rat, inhibited acrolein formation. Hydrogen peroxide, a product of oxidative deamination, was generated during allylamine oxidation. Acrolein was also produced from allylamine by bovine plasma amine oxidase and porcine kidney diamine oxidase but not by rat liver or brain homogenates. Allylamine competitively inhibited benzylamine oxidation in rat aorta, but pargyline-sensitive monoamine oxidase was not involved in acrolein production. The high activity in aorta, the competition with benzylamine, and the sensitivity to benzylamine oxidase inhibitors indicate that benzylamine oxidase is the active enzyme in oxidizing allylamine. The formation of acrolein may be the basis of the cardiotoxic action of allylamine.

Effects of cloud-point grafting, chain length, and density of PEG layers on competitive adsorption of ocular proteins.

The effects of pinning density, chain length, and 'cloud point' (CP) versus non-CP grafting conditions have been studied on the ability of polyethylene glycol (PEG) layers to minimize adsorption from a multicomponent (lysozyme, human serum albumin (HSA), IgG and lactoferrin) protein solution. Methoxy-terminated aldehyde-PEG (M-PEG, MW 5000) and dialdehyde-PEG (PEG(ald)2, MW 3400) were grafted by reductive amination onto two surfaces of different amine group density, generated by radiofrequency glow discharge (r.f.g.d.) deposition of n-heptylamine (HA) (low density) or allylamine (AlA) (high density) r.f.g.d. polymer layers. The PEG graft density was varied also by increasing the temperature and salt (K2SO4) content of the grafting solution; it reached a maximum at the CP of the PEGs. The CP reaction conditions were critical for producing PEG layers capable of minimizing protein adsorption. X-ray photoelectron spectroscopy (XPS) showed that under these conditions, PEG(ald)2 produced a thick linear PEG layer, most likely by aldol condensation. Protein adsorption was assessed using XPS and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF-MS) in the surface mode (Surface-MALDI-MS). Coatings grafted at non-CP conditions showed some protein adsorption, as did the HA/M-PEG layer grafted at the CP. On the other hand, no protein adsorption was detected on the HA/PEG(ald)2, AlA/M-PEG, and AlA/PEG(ald)2 surfaces when grafted at the CP. Thus, the effects of pinning density and chain length are interrelated, but the key factor is optimization of PEG chain density by use of the CP conditions, provided that a sufficient density of pinning sites exists.

Zimelidine: a therapeutic and pharmacokinetic study in depression.

Zimelidine, a bicyclic compound with a strong effect on the neuronal reuptake of 5-hydroxy-tryptamine and with weak anticholinergic actions, was evaluated for its antidepressant efficacy in a double-blind comparative trial with amitriptyline. In doses of 200 mg a day it was found to be as effective as 150 mg amitriptyline, but with significantly less subjective side-effects. The plasma concentration of zimelidine and its metabolite, norzimelidine, showed no significant correlation with therapeutic outcome.

Action of polyamine aminotransferase on norspermidine.

The norspermidine-pyruvate reaction catalyzed by polyamine aminotransferase from Arthrobacter sp. TMP-1 formed N-3-aminopropyl-3-aminopropionaldehyde (APAPAL), L-alanine, 1,3-diaminopropane (DAP), allylamine, and acrolein, and the relative rates of formation of the latter four products were 24, 3.3, 2.3, and 1.2%, respectively, of the rate of the DAP-pyruvate transamination. The identification of APAPAL was done by 13C-NMR after it had been enzymatically oxidized to N-3-aminopropyl-beta-alanine followed by isolation of the oxidized product. The DAP was also isolated and identified by 13C-NMR. The allylamine and acrolein were identified by HPLC and a specific color reaction with m-aminophenol, respectively. In the absence of pyruvate, the enzyme catalyzed the elimination of DAP from norspermidine to yield allylamine, and the addition of DAP to allylamine to yield norspermidine with relative rates of 0.007 and 0.095%, respectively. When allylamine was incubated with the enzyme as the sole substrate, it was converted to N-allyl-1,3-diaminopropane and an unidentified product.

Allylamine cardiovascular toxicity.

The chemistry, industrial usage, general toxicity, and experimental use of allylamine are briefly reviewed. This highly reactive unsaturated alkylamine has had industrial applications in a variety of organic processes, and continues to be utilized, although accurate data concerning production is not readily available. The general toxic effects of the freebase form are primarily related to irritation of the mucous membranes, whereas the relatively long history of experimental use of this chemical has emphasized its' extraordinarily deleterious effects on heart and vascular tissue. Allylamine has been given by a variety of routes to many species in attempts to cause lesions which mimic human acute vasculitis, acute myocardial necrosis, and atherosclerosis; examples of typical lesions are illustrated. More recent in vivo and in vitro experimental work concerning the cellular toxicity of allylamine are summarized, and possible mechanisms of this chemicals' toxic action are discussed.

Allylamine cardiovascular toxicity: V. Tissue distribution and toxicokinetics after oral administration.

We studied the uptake, tissue distribution, toxicokinetics, and excretion of allylamine by giving rats [14C]allylamine (1.5 microCi/kg; 150 mg/kg) by gavage. Rats were killed at intervals up to 2 h, and multiple tissues were sampled. Aorta showed the highest counts of 14C-label at most times (5-10-fold higher than most other organs, 100-fold higher than blood), although a minority of aortas had very low counts. Coronary arteries dissected from the hearts showed consistently higher 14C-label than myocardium. Liver counts, which were high at 5 min, decreased rapidly; kidney counts slowly increased until 45 min, then decreased rapidly, consistent with an excretory function for this organ. Counts of 14C-label were lower in all other organs, including lung, skeletal muscle, brain, testes, pancreas, adrenal, spleen, fat, and blood. Toxicokinetic study showed a very rapid absorption rate and short half-lives (less than 1 h) for those organs which reasonably fit a toxicokinetic one-compartment model. 14C-label was rapidly excreted in the urine; approximately 60% of the dose given was recovered by 24 h. No counts were found in feces. These studies indicate that allylamine--or its metabolite(s)--has a unique predilection for elastic and muscular arteries, such as aorta and coronary arteries. This relatively specific cardiovascular toxin acts as a highly polar, highly water soluble substance, which is rapidly absorbed from the gastrointestinal tract, has a short half-life in most tissues, and is rapidly excreted in the urine. The actual mechanisms by which allylamine injures tissue, especially in view of its rapid sequestration in vascular tissue, remain to be uncovered.

Binding of [14C] allylamine to isolated mitochondria from rat heart and aorta.

This study demonstrates specific and saturable binding of [14C] allylamine to mitochondria derived from rat aorta and heart. Specific binding is linear with respect to mitochondrial concentration and has a pH optimum of 7.0. Saturation isotherms reveal anomalous kinetics of specific binding on heart mitochondria with a high affinity site (KD 16 nM) and a lower affinity site (KD 80 nM); Scatchard plots have a common intercept. Exhaustive flow dialysis in the presence of SDS demonstrates that as much as 23.5% of bound radioactive moieties in aorta mitochondria are covalently bound, and as much as 42.6% are covalently bound in heart mitochondria. Hydrolysis of heart mitochondria with phospholipase C markedly enhances saturation of [14C] allylamine, and greatly increases the quantity of covalently bound radioactive ligand. Phospholipase C hydrolysis of heart mitochondria increased monoamine oxidase B activities and unmasked a small amount of benzylamine oxidase activity, whereas hydrolysis of mitochondria with phospholipases A2 and D diminish MAO-B activity. The monoamine oxidase B inhibitor, deprenyl, significantly reduced both specific and covalent binding of the 14C-activity from [14C] allylamine to phospholipase hydrolyzed mitochondria. The benzylamine oxidase inhibitor, phenelzine, significantly decreased specific binding but had no effect on the degree of covalent binding of [14C] allylamine to phospholipase C hydrolyzed mitochondria. The benzylamine oxidase inhibitor, semicarbazide, had no effect in inhibiting [14C] allylamine binding. Covalent binding of 14C-moiety from [14C] allylamine to mitochondria--which express specific binding sites for the [14C] allylamine--and inhibition of binding by monoamine oxidase inhibitors, suggest the formation of highly reactive intermediates.

Allylamine cardiovascular toxicity: VI. Subcellular distribution in rat aortas.

The cardiovascular toxin allylamine (3-aminopropene) has been shown to concentrate in elastic and muscular tissues. In this study the 14C-moiety of [14C]allylamine was traced in aortas of adult Sprague-Dawley rats after intravenously injecting 30 microCi of [14C]allylamine (spec. act. = 0.4 mCi/mM). At 5, 10, 15 and 20 min after injection 33.3-29.8% of the 14C-moiety was sequestered in aortas; at 30 min 16.8% was still present. Subcellular fractionation of the postnuclear supernatant by isopycinic centrifugation in sucrose demonstrated that 5 min after administration of [14C]allylamine, the 14C-moiety displayed a modal density peak of 1.20 g/ml. Similar activities were observed up to 30 min exposure. This modal density was similar to the distribution pattern of mitochondria based on analysis of malate dehydrogenase activities. As early as 20 min post-exposure, mitochondrial malate dehydrogenase activities of aortic mitochondria decreased, while cytosolic malate dehydrogenase activities increased, suggesting mitochondrial membrane perturbation. We suggest that the subcellular site for allylamine injury to the aorta is the mitochondrion.

Vascular amine oxidase activities during synergistic vasculotoxicity.

Allylamine (AA) and beta-aminopropionitrile (beta APN) are well known vascular toxins with a demonstrated synergistic toxic effect, i.e. given together they cause extensive smooth muscle cell necrosis of the aortic media. In this study, we investigated the possibility that the enzymes involved in the separate toxicity of AA (semicarbazide-sensitive amine oxidase, or SSAO) and beta APN (lysyl oxidase, or LyO), could be the target(s) of their synergistic toxicity. Adult male Sprague-Dawley rats were given AA alone (AA), 100 mg/kg/day, beta APN alone (beta APN), 1 g/kg/day, or both chemicals (AA + beta APN) by gavage for 1, 2, 5 or 10 days. SSAO ahd LYO were assayed in aorta, lung, and bone. SSAO activity in aortas of rats treated with AA + beta APN showed a maximal decrease (40%) at 10 days; more moderate depression of SSAO was seen in lung and bone. LyO changes were most marked in aorta, where activities were consistently and markedly depressed in all rats receiving beta APN (either alone or in combined treatment). Similarly, the lung and bone LyO activity was depressed at all time points in rats receiving beta APN, but to an apparently lesser degree than in aorta. The most striking changes in in vivo enzyme activities were seen in the aorta, the major target organ in this model. No synergistic effect of the two toxins was seen in the depression of LyO enzyme activity, since there was no difference in the degree of enzyme inhibition present between rats given beta APN alone or AA + beta APN, indicating that inhibition of this enzyme is mainly due to the effect of beta APN. We suggest that AA is the primary toxin in this synergistic vasculotoxic effect. It is likely that some effect of beta APN on AA metabolism or detoxification mechanisms results in synergism.

Role of benzylamine oxidase in the cytotoxicity of allylamine toward aortic smooth muscle cells.

In this study we demonstrate that by inhibiting benzylamine oxidase (BzAO) with either semicarbazide or phenelzine, aortic smooth muscle cells (ASMCs) are protected from cytolethal injury by the cardiovascular toxin allylamine. We find that although both semicarbazide and phenelzine inhibit BzAO or ASMCs grown in vitro, phenelzine is the more effective inhibitor. We further demonstrate that although semicarbazide--at concentrations inhibiting BzAO--protects ASMCs from cytolethal concentrations of allylamine, it does not fully protect ASMCs from sublethal injury as assessed by [3H]uridine uptake. In contrast, phenelzine appears to afford complete protection of ASMCs from allylamine injury. Although semicarbazide and phenelzine pretreatment does not interfere with [14C]allylamine uptake by ASMCs, retention time of the 14C-moiety from radiolabeled allylamine is less in pretreated ASMCs. Subcellular distribution studies of ASMCs exposed to [14C]allylamine demonstrate that inhibiting BzAO activity in ASMCs results in marked derangement of the distribution pattern of 14C-moiety in subcellular fractions of ASMCs, with 14C-moiety not localized to mitochondrial/endoplasmic reticulum enriched fractions.

Colesevelam HCl: a non-systemic lipid-altering drug.

Colesevelam HCl (WelChol, Sankyo Pharmaceuticals Inc.) is a bile acid sequestrant polymer, which has been shown to significantly lower low density lipoprotein cholesterol and favourably affect high-density lipoprotein cholesterol blood levels in monotherapy and in combination with statins (HMG-CoA reductase inhibitors). Although it is similar to other bile acid sequestrants in that it binds bile acids and is non-systemic, colesevelam HCl differs in that it has a unique polymer structure that allows for greater tolerability with less potential drug interactions than with resins. Currently, statins are the most commonly prescribed lipid-altering drugs. However, it is not uncommon that patients demonstrate true or perceived intolerances to statin therapy, that are often dose-related and may include elevations in liver or muscle enzyme blood levels, or myalgias or muscle weakness without muscle enzyme elevation. In rare circumstances, myopathy and even rhabdomyolysis can occur with statins. In addition, many statins also have important potential drug interactions. Finally, statin monotherapy is often not sufficient in achieving lipid treatment goals in many severely dyslipidaemic patients and the availability of colesevelam HCl provides a lipid-altering treatment addition to other lipid-altering drugs. From a clinical perspective, such combination therapy is often required to achieve treatment goals [1] in patients with more complicated or severe dyslipidaemia. Colesevelam HCl may also be an alternative in monotherapy for many patients with mild-to-moderate hypercholesterolaemia, as well as in some patients at potential risk from systemic exposure to alternative lipid-altering drugs (such as young children and fertile women).

New therapies on the horizon.

Statins have proved to be potent drugs for reducing low-density lipoprotein cholesterol (LDL-C) levels. However, because the response to current statin therapy regimens is not always sufficient to reach defined goal levels, additional drugs to lower LDL-C are needed. New drugs may soon be available to lower LDL-C levels by mechanisms that differ from those of the statins. Among these new agents are a bile acid binding resin, inhibitors of bile acid transport, inhibitors of cholesterol transport, inhibitors of cholesterol esterification, and triglyceride-lowering agents.

[Safety of animal husbandry products produced by using allyl isothiocyanate-preserved feeds]. / Izuchenie bezvrednosti producktov zhivotnovodstva, poluchennykh pri ispol'zovanii kormov, konservirovannykh allilizotiotsianatom.

To examine the safety of foods (meat and milk) obtained from animals whose feeds were preserved with allyl isothiocyanate (AITC), the authors investigated the status and development of animals, some aspects of protein, lipid and carbohydrate metabolism, some enzymes, hemopoiesis and reproduction function of Wistar rats fed diets containing the above products (55 g dry milk or 50 g boiled meat per 100 g diet). To measure the residual quantity of allylamine (AA), the main metabolite of AITC, a chromato-fluorometric method with the use of dansyl chloride was devised, permitting the detection of 2 ng AA in the spot. The relative standard deviation was 0.14-17. The degree of AA extraction amounted to 96%. The method made it possible to detect the residual quantity of AA in the kidneys (up to 12.2 +/- 2.0 micrograms/kg) and in the liver (up to 83.1 +/- 12.4 micrograms/kg). The data obtained evidence that AA cannot be used as conservant for manufacturing feeds for agricultural animals.

Time-dependent metabolic activity and adhesion of human osteoblast-like cells on sensor chips with a plasma polymer nanolayer.

PURPOSE: To improve orthopedic implant ingrowth, knowledge of the effect of chemical surface modifications on vital cell function in vitro is of importance. Early in our investigations we recognized that amino groups, positively charged via plasma polymerized allylamine, increased cell growth and the actin-filament formation in the initial cell-material contact phase. To gain insight into continuous vital cell behavior on this plasma polymer layer, here we present the metabolic activity of osteoblasts and their time-dependent adhesion using the sensor chip technology. METHODS: We demonstrate a new method for continuous 24 hour-measurements with vital human osteoblast-like cells (MG-63, ATCC) on sensor chips (Bionas® SC 1000) modified with plasma polymerized allylamine (PPAAm). The PPAAm film deposited on the chip is a cross-linked, strongly fixed plasma polymer with relatively high amino functionality and well defined chemical surface composition. We assessed continuous cell adhesion and the metabolic activity, i.e., oxygen consumption and acidification. RESULTS: We determined that adhesion of vital cells on PPAAm is not only enhanced shortly (1 h) after cell seeding but remained continuously higher for 24 h, which is significant. This nanometer-thin PPAAm layer did not change the overall metabolic activity of MG-63 cells during 24 h. CONCLUSION: This tool--using adhesion and metabolic sensor chips--appears to be a suitable method for the recognition of vital cell physiology in biocompatibility measurements of plasma chemical treated surfaces.