Lippia origanoides essential oil: an efficient and safe alternative to preserve food, cosmetic and pharmaceutical products.

AIMS: The aim of this work was to evaluate the efficacy and safety of Lippia origanoides essential oil as a preservative in industrial products. METHODS AND RESULTS: The composition, antimicrobial activity, mutagenic and toxic potential of L. origanoides were determined. Then, the effect of essential oil as a preservative in food, cosmetics and pharmaceutical products was evaluated. The essential oil of L. origanoides consisted mainly of oxygenated monoterpenes (38·13%); 26·28% corresponded to the compound carvacrol. At concentrations ranging from 0·312 to 1·25 µl ml-1 and in association with polysorbate 80, the essential oil of L. origanoides inhibited the growth of all the tested micro-organisms. The medium lethal dose in mice was 3·5 g kg-1 , which categorizes it as nontoxic according to the European Union criteria, and negative results in the Ames test indicated that this oil was not mutagenic. In combination with polysorbate 80, the essential oil exerted preservative action on orange juice, cosmetic and pharmaceutical compositions, especially in the case of aqueous-based products. CONCLUSIONS: Lippia origanoides essential oil is an effective and safe preservative for orange juice, pharmaceutical and cosmetic products. SIGNIFICANCE AND IMPACT OF THE STUDY: This study allowed for the complete understanding of the antimicrobial action and toxicological potential of L. origanoides essential oil. These results facilitate the development of a preservative system based on L. origanoides essential oil.

Electrosprayed Janus Particles for Combined Photo-Chemotherapy.

This work is a proof of concept study establishing the potential of electrosprayed Janus particles for combined photodynamic therapy-chemotherapy. Sub-micron-sized particles of polyvinylpyrrolidone containing either an anti-cancer drug (carmofur) or a photosensitiser (rose bengal; RB), and Janus particles containing both in separate compartments were prepared. The functional components were present in the amorphous form in all the particles, and infrared spectroscopy indicated that intermolecular interactions formed between the different species. In vitro drug release studies showed that both carmofur and RB were released at approximately the same rate, with dissolution complete after around 250 min. Cytotoxicity studies were undertaken on model human dermal fibroblasts (HDF) and lung cancer (A549) cells, and the influence of light on cell death explored. Formulations containing carmofur as the sole active ingredient were highly toxic to both cell lines, with or without a light treatment. The RB formulations were non-toxic to HDF when no light was applied, and with photo-treatment caused large amounts of cell death for both A549 and HDF cells. The Janus formulation containing both RB and carmofur was non-toxic to HDF without light, and only slightly toxic with the photo-treatment. In contrast, it was hugely toxic to A549 cells when light was applied. The Janus particles are thus highly selective for cancer cells, and it is hence proposed that such electrosprayed particles containing both a chemotherapeutic agent and photosensitiser have great potential in combined chemotherapy/photodynamic therapy.

A novel solid dispersion system for natural product-loaded medicine: silymarin-loaded solid dispersion with enhanced oral bioavailability and hepatoprotective activity.

A surface-attached silymarin-loaded solid dispersion with improved dissolution profile and enhanced oral bioavailability was formulated using silymarin, polyvinylpyrrolidone (PVP) and Tween 80 in water. In this solid dispersion, hydrophilic PVP was adhered onto the surface of crystalline drug rendering silymarin hydrophilic without changing its crystallinity. The drug solubility from the optimised solid dispersion prepared with silymarin/PVP/Tween 80 at the weight ratio of 5/2.5/2.5 increased by almost 650-fold compared to drug powder. The drug was physically and chemically stable in the solid dispersion for at least 6 months. Moreover, the solid dispersion enhanced the oral bioavailability of the drug in rats by almost 3-fold compared to the commercial product. The silymarin-loaded solid dispersion also exhibited advanced hepatoprotective bioactivity against CCl4-induced liver damage compared to silymarin or the commercial product. Thus, this silymarin-loaded solid dispersion would be useful for the enhancement of oral bioavailability and hepatoprotective activity of poorly water-soluble silymarin.

Application of armodafinil-loaded microneedle patches against the negative influence induced by sleep deprivation.

Cognition maintenance is essential for healthy and safe life if sleep deprivation happens. Armodafinil is a wake-promoting agent against sleep deprivation related disorders. However, only the tablet formulation is available, which may limit its potential in some circumstances. Here, we report the synthesis of a new formulation of armodafinil, microneedle patches, which can be conveniently used by any individual and removed in time if not wanted. To produce the needles of higher mechanical strength and higher drug loading, polyvinylpyrrolidone (PVP) K90 was used to fabricate armodafinil-loaded microneedles by applying the mold casting method after dissolving in methanol and drying. The higher mechanical strength was validated by COMSOL Multiphysics® software stimulation and universal mechanical testing machines. The obtained armodafinil microneedles can withstand a force of 70 N and penetrate the skin to a depth of 230 µm, and quickly released the drug within 1.5 h in vitro. The pharmacokinetic analysis showed that microneedle administration can maintain a more lasting and stable blood concentration as compared to oral administration. After the treatment of sleep deprived mice with microneedles, the in vivo pharmacodynamics study clearly demonstrated that armodafinil microneedles could eliminate the effects of sleep deprivation and improve the cognitive functions of sleep-deprived mice. A self-administered, high drug-loaded microneedle patch were prepared successfully, which appeared to be highly promising in preserving cognition by transdermal administration.

Esters of terpene alcohols as highly potent, reversible, and low toxic skin penetration enhancers.

Skin penetration/permeation enhancers are compounds that improve (trans)dermal drug delivery. We designed hybrid terpene-amino acid enhancers by conjugating natural terpenes (citronellol, geraniol, nerol, farnesol, linalool, perillyl alcohol, menthol, borneol, carveol) or cinnamyl alcohol with 6-(dimethylamino)hexanoic acid through a biodegradable ester linker. The compounds were screened for their ability to increase the delivery of theophylline and hydrocortisone through and into human skin ex vivo. The citronellyl, bornyl and cinnamyl esters showed exceptional permeation-enhancing properties (enhancement ratios up to 82) while having low cellular toxicities. The barrier function of enhancer-treated skin (assessed by transepidermal water loss and electrical impedance) recovered within 24 h. Infrared spectroscopy suggested that these esters fluidized the stratum corneum lipids. Furthermore, the citronellyl ester increased the epidermal concentration of topically applied cidofovir, which is a potent antiviral and anticancer drug, by 15-fold. In conclusion, citronellyl 6-(dimethylamino)hexanoate is an outstanding enhancer with an advantageous combination of properties, which may improve the delivery of drugs that have a limited ability to cross biological barriers.

Effect of sonophoresis and chemical enhancers on testosterone transdermal delivery from solid lipid microparticles: an in vitro study.

The main objective of the study was to investigate the effect of permeation enhancers and application of low frequency (LUS) and high frequency ultrasound (HUS) on testosterone (TS) transdermal permeation after application of testosterone solid lipid microparticles (SLM). SLM formulations contained 10% compritol and 5 mg TS /g of SLM. The permeation experiments were performed using Franz diffusion cells and abdomen rat skin. The examined permeation enhancers were 1% oleic acid (OA) or 1 % dodecylamine (DA). HUS (1 MHz) was applied in a continuous mode for 1h at intensity 0.5 W/cm(2). Different intensities and application time of pulsed LUS (20 kHz) were also examined. Additionally, the effect of combination of US and OA or DA was investigated. Skin irritation and histological changes were also evaluated. The results revealed that SLMs have an occlusive effect on the skin. Statistical analysis revealed the following order for the permeation of TS: 1% DA for 30 min>HUS +1% DA for 30 min= HUS=HUS + SLM containing 1% OA> SLM containing 1% OA=control. At total application time of LUS 6, 12, and 15 min the flux increased by 1.86, 4.63, and 4.77 fold, respectively. The enhancement effect of different intensities of LUS was not directly proportional to the magnitude of intensity. Skin exposure to HUS or LUS before application of 1% DA for 30 min had no superior enhancement effect over application of either LUS or HUS alone. Application of drug loaded SLM offered skin protection against the irritation effect produced by TS and 1% DA. Histological characteristics of the skin were affected to various extents by application of enhancers or ultrasound. In general, application of LUS gave higher TS permeation than HUS. However, safe application of LUS should be practiced by careful selection of exposure parameters.

Therapeutic Intervention of Aloe Gel Containing Nano-Sized and Micron-Sized Silver Sulfadiazine Gel on Second-Degree Burn: A Comparative Study.

The current work focuses on the formulation development, optimization, and in vivo assessment of nano-sized silver sulfadiazine ( nSSD) and micron-sized silver sulfadiazine ( mSSD) topical gel composed of Aloe vera gel ( Aloe gel) and Carbopol 940 for the management of second-degree burn wound. The optimized concentration of gel-forming agent (Carbopol 940) was chosen based on best possible consistency and spreadability of the gel. The second-degree burn infliction was developed in the posterior region of rats followed by anesthesia. Afterward, the created wounds were further treated individually by both the gel formulation (1 application daily) for 14 days and observations were recorded. The nSSD gel showed better wound healing and a higher degree of tissue hyperplasia as compared with mSSD gel in rats. In vitro drug release study showed better drug release from nSSD gel (74.25 ± 3.331%) as compared with mSSD gel formulation (61.32 ± 2.112%) after 24 hours. The nSSD and mSSD topical gel-treated rats showed 95.63% and 78.75% wound healing after 14 days, while in the case of control group rats, 48.65% wound contraction was seen after 14 days. Furthermore, the histopathological study revealed that the nSSD gel was more efficient in controlling the wound infection and showed better wound healing as compared with mSSD gel formulation.

Chronic exposure to free fatty acid reduces pancreatic beta cell insulin content by increasing basal insulin secretion that is not compensated for by a corresponding increase in proinsulin biosynthesis translation.

The pancreatic beta cell normally maintains a stable balance among insulin secretion, insulin production, and insulin degradation to keep optimal intracellular stores of the hormone. Elevated levels of FFA markedly enhance insulin secretion; however, the effects of FFA on insulin production and intracellular stores remain unclear. In this study, twofold elevation in total circulating FFA effected by infusion of lard oil and heparin into rats for 6 h under normoglycemic conditions resulted in a marked elevation of circulating insulin levels evident after 4 h, and a 30% decrease in pancreatic insulin content after a 6-h infusion in vivo. Adding 125 muM oleate to isolated rat pancreatic islets cultured with 5.6 mM glucose caused a 50% fall in their insulin content over 24 h, coupled with a marked enhancement of basal insulin secretion. Both effects of fatty acid were blocked by somatostatin. In contrast to the stimulatory effects of oleate on insulin secretion, glucose-induced proinsulin biosynthesis was inhibited by oleate up to 24 h, but was unaffected thereafter. This result was in spite of a two- to threefold oleate-induced increase in preproinsulin mRNA levels, underscoring the importance of translational regulation of proinsulin biosynthesis in maintaining beta cell insulin stores. Collectively, these results suggest that chronically elevated FFA contribute to beta cell dysfunction in the pathogenesis of NIDDM by significantly increasing the basal rate of insulin secretion. This increase in turn results in a decrease in the beta cell's intracellular stores that cannot be offset by commensurate FFA induction of proinsulin biosynthesis.

Activity of sodium metabisulfite against planktonic and biofilm Staphylococcus species.

Biofilm-forming staphylococci cause a majority of intravascular catheter-related infections. We evaluated the effect of sodium metabisulfite, a preservative commonly added to intravenously administered pharmaceuticals as an antioxidant and previously used as a catheter lock solution, on planktonic and biofilm staphylococci at clinically encountered concentrations. Sodium metabisulfite exhibited bactericidal activity against planktonic Staphylococcus aureus, Staphylococcus lugdunensis, and Staphylococcus epidermidis at concentrations of 512, 512, and 1024 microg/mL, respectively. A concentration of 720 microg/mL inhibited cell growth by all 3 species in a biofilm formation assay. However, established S. aureus and S. lugdunensis biofilms showed less than 1.5 log10 decreases in viable cell counts when treated with 720 microg/mL of sodium metabisulfite for 24 h. These in vitro results suggest that the use of sodium metabisulfite as a catheter lock may inhibit staphylococcal colonization of catheters, thereby preventing catheter-related infection.

Evaluation of chemical enhancers in the transdermal delivery of lidocaine.

The effect of various classes of chemical enhancers was investigated for the transdermal delivery of the anesthetic lidocaine across pig and human skin in vitro. The lipid disrupting agents (LDA) oleic acid, oleyl alcohol, butenediol, and decanoic acid by themselves or in combination with isopropyl myristate (IPM) showed no significant flux enhancement. However, the binary system of IPM/n-methyl pyrrolidone (IPM/NMP) improved drug transport. At 2% lidocaine dose, this synergistic enhancement peaked at 25:75 (v/v) IPM:NMP with a steady state flux of 57.6 +/- 8.4 microg cm(-2) h(-1) through human skin. This observed flux corresponds to a four-fold enhancement over a 100% NMP solution and over 25-fold increase over 100% IPM at the same drug concentration (p < 0.001). NMP was also found to co-transport through human skin with lidocaine free base and improve enhancement due to LDA. These findings allow a more rational approach for designing oil-based formulations for the transdermal delivery of lidocaine free base and similar drugs.

Shared biochemical properties of glucotoxicity and lipotoxicity in islets decrease citrate synthase activity and increase phosphofructokinase activity.

Diabetic states are characterized by a raised serum/islet level of triglycerides and a lowered EC50 (concentration at half-maximal stimulation) for glucose-induced insulin secretion. Culturing islets with long-chain fatty acids (FAs) replicates the basal insulin hypersecretion. In a previous study, we showed that the mechanism involved deinhibition of hexokinase by a 60% decrease in glucose-6-phosphate (G-6-P). The key event was proposed to be an increased phosphofructokinase (PFK) Vmax secondary to an upregulatory effect of the FA metabolite, long-chain acyl-coenzyme A (LC-CoA). We now show another contributory factor, a lowered content of the PFK inhibitor citrate. Citrate synthase Vmax and citrate levels were lowered 45% in rat islets cultured with 250 micromol/l oleate for 24 h. Both effects were reversed by triacsin C, an inhibitor of fatty acyl-CoA synthetase, the enzyme that generates LC-CoA. Culturing islets with high doses of glucose (16.7 mmol/l) for 48 h should also raise cytosolic LC-CoA. As predicted, citrate synthase Vmax was lowered and PFK Vmax was increased, both in a triacsin C-reversible fashion. These results show shared selected functional and biochemical properties in beta-cells of so-called glucotoxicity and lipotoxicity.

Chlorbutol, a new inhibitor of aldosterone biosynthesis identified during examination of heparin effect on aldosterone production.

This study was designed to examine the mechanism whereby routine heparin therapy inhibits adrenal aldosterone production. In bovine adrenal glomerulosa cell suspensions, pure heparin, in concentrations up to 500 U/ml, had no significant effect on basal or angiotensin II-stimulated aldosterone production. A therapeutic preparation of heparin for parenteral use containing the preservative chlorbutol (2.8 X 10(-2) M) inhibited aldosterone production [67 +/- 8.7% (+/- SE); P less than 0.005]. Chlorbutol alone, in a dose-dependent manner, inhibited basal aldosterone production from 1548 +/- 355 to 316 +/- 152 pg/ml (P less than 0.001) and inhibited angiotensin II-stimulated production from 4950 +/- 724 to 589 +/- 257 pg/ml (P less than 0.001). To elucidate the inhibitory mechanism of chlorbutol, we used trilostane, an inhibitor of the conversion of pregnenolone to progesterone, and aminoglutethimide, an inhibitor of the conversion of cholesterol to pregnenolone. Aldosterone production was completely suppressed by each inhibitor. Pregnenolone accumulation in trilostane-treated cells fell from 9.70 +/- 1.66 to 1.40 +/- 0.28 ng/ml (P less than 0.005) with the addition of chlorbutol. Aldosterone accumulation from corticosterone added to aminoglutethimide-treated cells fell from 715 +/- 96 to 348 +/- 59 pg/ml (P less than 0.02) in cells incubated with chlorbutol. Thus, chlorbutol is a potent inhibitor of aldosterone production, inhibiting both the early biosynthetic phase and, to a lesser extent, the late phase. Since chlorbutol is a widely used pharmaceutical preservative and has a slow metabolic clearance, these findings may be of toxicological significance and may account for the inhibition of aldosterone production previously attributed to heparin.

Oleic acid and angiotensin II induce a synergistic mitogenic response in vascular smooth muscle cells.

Oleic acid and angiotensin II (Ang II) are elevated and may interact to accelerate vascular disease in obese hypertensive patients. We studied the effects of oleic acid and Ang II on growth responses of rat aortic smooth muscle cells (VSMCs). Oleic acid (50 micromol/L) raised thymidine incorporation by 50% at 24 hours and cell number by 55% at 6 days (P<.05). Ang II (10(-11) to 10(-6) mol/L) did not significantly increase thymidine incorporation or VSMC number. Combining Ang II and 50 micromol/L oleic acid doubled thymidine incorporation and VSMC number. Losartan, an angiotensin type 1 (AT1) receptor antagonist, blocked the synergistic interaction between Ang II and oleic acid, whereas the AT2 receptor antagonist PD 123319 did not. Protein kinase C inhibition and downregulation, as well as inhibition of extracellular signal-regulated kinase (ERK) activation by PD 98059, eliminated the rise of thymidine incorporation in response to oleic acid and the synergistic interaction with Ang II. However, the response to 10% fetal bovine serum was unaffected. An antisense oligodeoxynucleotide to ERK-1 and ERK-2 reduced ERK protein expression and activation by 83% and 75%, respectively. Antisense prevented the rise of thymidine incorporation in response to oleic acid and the synergy with Ang II. Antisense reduced but did not prevent increased thymidine incorporation in response to serum. The data indicate that oleic acid and Ang II exert a synergistic mitogenic effect in VSMCs and suggest an important role for the AT1 receptor, PKC, and ERK in this synergy. The observations raise the possibility that a synergistic mitogenic interaction between oleic acid and Ang II accelerates vascular remodeling in obese hypertensive patients.

Are the vascular effects of naloxone attributable to the preservatives methyl- and propylparaben?

In vitro, the Nalonee preparation of naloxone caused a concentration-dependent relaxation of human pial cortical arteries contracted by potassium, noradrenaline, serotonin, prostaglandin F2 alpha (PGF2 alpha), and haemorrhagic cerebrospinal fluid, or inhibited contractions elicited by these agents. However, the preservatives in the Nalonee preparation, methyl- and propylparaben, had similar effects. Pure naloxone alone had no effect on potassium or PGF2 alpha-induced contractions. It is suggested that the relaxant effects on vascular smooth muscle of Nalonee can be attributed to the alkylparabens rather than to naloxone. The pronounced relaxations induced by the alkylparabens had a rapid onset, and they were stable and could easily be cleared after rinsing.

Polyethylene glycol-mediated infection of non-permissive mammalian cells with semliki forest virus: application to signal transduction studies.

Semliki Forest Virus (SFV) vectors allow the subcloning of a gene of interest directly in the expression vector, thus avoiding the need to select and purify viral recombinants, making this viral expression system attractive over many others for mammalian protein expression. We now describe a novel and generally applicable method for infection of non-permissive mammalian cells with SFV, that greatly enhances the utility of this expression system. We demonstrate that the hygroscopic polymer poly (ethylene glycol), PEG, promotes the infectivity of cells by SFV under conditions that did not promote cell-cell fusion. We also found that the PEG-induced infection and expression of an exogenous protein (green fluorescent protein, GFP) did not elevate the basal tyrosine kinase activity, induce a stress-activated responses, or result in aberrant cell responses. Expression of GFP tagged-Vav, an activator of stress-activated protein kinase (SAPK/JNK), resulted in the expected induction of JNK activity and in the normal redistribution of Vav in response to engagement of the high affinity receptor for IgE (FcepsilonRI). Thus, our findings that PEG allows the infection of non-permissive cells by SFV makes this system extremely attractive for expression of proteins in mammalian cells, and studies on signal transduction and cellular localization in immune and non-immune cells.

Assessment of sperm cryodamage and strategies to improve outcome.

Sperm cryopreservation still represents a valuable clinical aid in the management of infertility. Its current principal indications include (1) donor sperm insemination; (2) freezing before cancer therapy to maintain reproductive capacity; (3) patient's convenience; and (4) because of the outstanding success with ICSI, even patients with different degrees of oligo-asthenoteratozoospermia can now be offered the use of frozen/thawed sperm for oocyte micromanipulation. Although sperm cryopreservation/thawing and results of insemination and IVF have been consistently good using donor semen, results of infertile men (with or without various degrees of oligoasthenoteratozoospermia) have yielded remarkably lower rates of survival and pregnancy. Freezing/thawing techniques have not been subjected to major changes in the last years, Furthermore, the exact nature of sperm cryodamage still remains to be elucidated. Various aspects of sperm freezing are revisited here (1) development of new technical approaches for cryopreservation; (2) analysis of the stimulatory effect of putative cryoprotectant additives; (3) the use of intrauterine insemination-ready processed samples; and (4) selection and optimization of end-points for analysis of cryodamage. It is expected that advances in such areas will improve significantly the cryopreservation/thawing outcome particularly as related to semen samples of subfertile men.

Preservation of canine hearts after warm ischemia (zero to thirty minutes) and one to two days of hypothermic storage. A comparative analysis of crystalloid and colloid solutions with different osmolarity and ion composition.

The effect of extracellular crystalloid (Ringer's) and colloid (silica gel fraction [SGF]) solutions, and intracellular crystalloid (Sacks) and colloid (modified silica gel fraction [MSGF]) solutions for canine heart preservation in a 24 to 48 hour model of hypothermic storage and zero to 30 minutes of warm ischemia was compared. Canine hearts flushed with an intracellular colloid solution (MSGF) had better survival rates after transplantation than did the hearts flushed with intracellular crystalloid solutions (Sacks). Better survival results also were observed in the group of hearts flushed with extracellular colloid (SGF) solutions than extracellular crystalloid (Ringer's) solutions. The most important theoretical factor in heart preservation appears to be hyperosmolarity and elevated concentration of potassium, proteins, and glucose.

Characterization of amiodarone pneumonitis as related to inflammatory cells and surfactant apoprotein.

STUDY OBJECTIVE: To characterize the inflammation observed in amiodarone-induced pneumonitis. DESIGN: The density of inflammatory cells in BAL fluid (BALF) and lung interstitium was quantified in a rat model of amiodarone pneumonitis. Immunoperoxidase staining for surfactant apoprotein was evaluated in lung tissue. ANIMALS AND INTERVENTIONS: Male Fischer 344 rats weighing 170 to 180 g received amiodarone, 150 mg/kg/d, suspended in 0.5% methylcellulose by gavage 5 d/wk. Control animals were given only methylcellulose. Rats were killed after 3, 5, 7, 9, and 12 weeks. Histologic sections were prepared for hematoxylin-eosin staining and the immunoperoxidase method. MEASUREMENTS AND RESULTS: Significant positive correlations between the density of neutrophils in BALF and the interstitium were seen at 5 weeks (r=0.90, p<0.05) and 7 weeks (r=0.90, p<0.05). Significant positive correlations were observed between the density of lymphocytes in BALF and the interstitium at 9 weeks (r=0.90, p<0.05) and 12 weeks (r=0.90, p<0.05). The density of type II pneumocytes was significantly increased in the amiodarone-fed rats. Extracellular surfactant apoprotein was found in the alveolar space and the cytoplasm of type II pneumocytes, Clara cells, and large, foamy macrophages throughout drug treatment. Extracellular surfactant apoprotein filled some alveoli at 9 weeks. CONCLUSIONS: The density of lymphocytes and neutrophils increased significantly in the BALF and the lung interstitium throughout amiodarone administration. The relationship between the density of lymphocytes in BALF and in the interstitium differed from that of neutrophils. In addition, amiodarone caused hyperplasia of type II pneumocytes and deposition of conglomerated, extracellular surfactant apoprotein in the alveolar space.

Rabbit endothelial response to ophthalmic preservatives.

The endothelial surface of isolated rabbit corneas were perfused for three hours with varying concentrations of benzalkonium chloride and cetylpyridinium chloride. The threshold for physiological and ultrastructural alteration of corneal endothelium is approximately 0.0001% for benzalkonium chloride and 0.01 mM for cetylpyridinium chloride. The effects of the surfactants can be induced with as little as a 15-minute exposure without subsequent recovery. Use of ophthalmic medications or irrigating solutions containing these agents inside the eye is potentially hazardous to the corneal endothelium. Topical administration of 0.133% benzalkonium chloride to the anterior surface of deepithelialized in vivo corneas (five doses, seven minutes apart) caused no alterations of corneal endothelial cell function or ultrastructure.

Storage of red blood cells: new approaches.

Longer red blood cell (RBC) storage can improve blood logistics, increase the usefulness of autologous blood storage, and reduce donor exposure in neonatal intensive care. Better RBC storage can prevent membrane loss and preserve the secretion of adenosine 5'-triphosphate (ATP) in response to deformation. Better RBC storage may also reduce the formation of proinflammatory membrane breakdown products that lead to transfusion-related acute lung injury and the systemic inflammatory response syndrome. To improve RBC storage, the authors have attempted to maximize the production of ATP by the manipulation of pH and to minimize membrane loss through the use of membrane protectants and the manipulation of tonicity. Increasing the initial storage pH led to successively higher RBC ATP concentrations until pH 7.2 was reached, when the synthesis of 2,3-diphophoglycerate was initiated at the expense of ATP. Synthesis of ATP could be maintained by buffering the fall of pH with increased storage solution volume or the addition of bicarbonate. Maintaining RBC ATP turns out to be an important way of preventing membrane microvesiculation or blebbing, as does the manipulation of tonicity and the addition of mannitol. These experiments show that it is possible to store RBC in experimental additive solutions containing only saline, adenine, glucose, mannitol, sodium bicarbonate, and disodium phosphate for 11 weeks or longer with little loss of membrane and high in vivo recovery.