Formulation of L-asparaginase-loaded poly(lactide-co-glycolide) nanoparticles: influence of polymer properties on enzyme loading, activity and in vitro release.

This paper describes the preparation and characterisation of poly(lactide-co-glycolide) (PLG) nanoparticles containing the enzyme L-asparaginase. L-Asparaginase was encapsulated in PLG nanospheres using a water-in-oil-in-water solvent evaporation technique. The effect of the copolymer molecular weight and the presence of carboxyl-end groups in the copolymer chain on the physicochemical and in vitro release properties of the nanoparticles was investigated. Results indicated that size, encapsulation efficiency and in vitro release properties (enzymatic activity retention and protein quantification) of the nanoparticles were affected by the PLG molecular weight. As expected, nanoparticles made of high-molecular-weight PLG had a larger size, a higher loading and la slower release rate than those made od a low-molecular-weight PLG. Nevertheless, the most relevant factor affecting the entrapment and release of L-asparaginase from PLG nanoparticles was the presence of free carboxyl-end groups in the PLG chain. The nanoparticles made of PLG with free carboxyl-end groups had a high protein loading (4.86%, w/w) and provided a continuous delivery of the active enzyme for 20 days. However, the enzyme loading was lower (2.65%, w/v) and no active enzyme was detected in the release medium after a 14-day incubation period when nanoparticles were made of PLG with carboxyl-end groups esterified. These results give evidence of the potential of PLG nanospheres for the continuous delivery of L-asparaginase for extended periods of time and show the effect of the PLG chain end-groups in the amount and activity of the enzyme loaded into the nanospheres.

Comparison and modeling study of vancomycin, ristocetin A, and teicoplanin for CE enantioseparations.

The structurally related glycopeptide antibiotics vancomycin, ristocetin A, and teicoplanin can all be used as chiral selectors in capillary electrophoresis (CE). Both experimental and modeling studies were done to elucidate their similarities and differences. There are identifiable morphological differences in the aglycon macrocyclic portions of these three compounds. In addition, there are other structural distinctions that can affect their CE enantioselectivity, migration times, and efficiency. Teicoplanin is the most distinct of the three and is the only one that is surface active. Its aggregational properties appear to affect its enantioselectivity among other things. The similar but not identical structures of the three glycopeptides produce similar but not identical enantioselectivities. This leads to the empirically useful "principle of complementary separations", in which a partial resolution with one chiral selector can be brought to baseline with one of the others. Overall, ristocetin A appears to have the greatest applicability for CE enantioseparations.

Highly enantioselective capillary electrophoretic separations with dilute solutions of the macrocyclic antibiotic ristocetin A.

Ristocetin A is one of a series of structurally related amphoteric, glycopeptide, macrocyclic antibiotics. These compounds have several features that make them attractive as chiral selectors. These include spatially oriented functional groups that are known to provide the types of interactions that are conducive to enantio-recognition, a somewhat rigid "pocket" that can provide a site for hydrophobic interactions and polar, flexible arms (i.e., pendent sugar moieties) that can rotate to hydrogen bond and otherwise interact with a variety of chiral analytes. In addition, these compounds are sufficiently soluble in water, aqueous buffers and aqueous-organic solvents that are commonly used in capillary electrophoresis (CE). The use and optimization of ristocetin A as a chiral selector in CE is discussed. Over 120 racemates are resolved including a variety of N-blocked amino acids, non-steroidal anti-inflammatory compounds and a large number of biologically important compounds containing carboxylic acid groups (e.g., mandelic acid derivatives, lactic acid derivatives, folinic acid, tropic acid).

Stereochemistry of pipecolic acid found in the urine and plasma of subjects with peroxisomal deficiencies.

Recently it was found that normal adults excrete pipecolic acid primarily as the D-enantiomer even though it is present in the blood stream mainly as the L-enantiomer (i.e. > 98% L). This study of pipecolic acid stereochemistry was extended to subjects with peroxisomal deficiencies since they are known to have high levels of pipecolic acid in their physiological fluids. Also, pipecolic acid stereochemistry was examined in young normal subjects since this group was not considered previously. It was found that the stereochemical composition of pipecolic acid in plasma was very similar for all subjects tested (i.e. > 98% of the L-enantiomer). However, the stereochemical composition of excreted pipecolic varied considerably. Urine samples from subjects with the most severe peroxisomal deficiency, i.e. cerebralhepatorenyl (Zellweger) syndrome (CHRS) contained little D-pipecolic acid. In fact the enantiomeric ratios for pipecolic acid in the urine and plasma of these subjects were very similar. This was not the case for normal subjects. Levels of D-pipecolic acid in the urine of subjects with 'less severe' peroxisomal deficiencies tended to be somewhat higher but they did not approach the levels found in normal adults. Several possible reasons for these results are discussed.

D-amino acid levels in human physiological fluids.

Plasma, urine, cerebrospinal fluid (CSF), and amniotic fluid were examined to determine whether free D-amino acids were present and if so at what levels. It was found that D-amino acids exist in all physiological fluids tested, but that their level varied considerably. The lowest levels of D-amino acids were usually found in amniotic fluid or CSF (almost always < 1% of the corresponding L-amino acid). The highest levels were found in urine (usually tenth percent to low percent levels). Pipecolic acid seemed to be different from the other amino acids tested in that it was excreted primarily as the D-enantiomer (often > 90%). Correspondingly high levels of D-pipecolic acid were not found in plasma. Some of the trends found in this work seemed to be analogous to those found in a recent rodent study.

Factors controlling the level and determination of D-amino acids in the urine and plasma of laboratory rodents.

Unambiguous methodologies were developed for the accurate and reproducible determination of specific D-amino acids in the physiological fluids of common laboratory rodents. Depending on the strain of rodent and the type of amino acid examined, excreted D-amino acids ranged from the low percent levels to over 40 percent of the total specific amino acid level. Relative plasma levels tended to be considerably lower, typically an order of magnitude less. A number of factors were found to alter the relative amounts of excreted D-amino acids. This included: diet, age, pregnancy, advanced cancer, and antibiotics. The two factors that seemed to result in substantially lower levels of excreted D-amino acids were fasting and young age. Pregnancy was the only factor that consistently resulted in higher relative D-amino acid excretion. Much of the observed data are believed to be related to the efficiency with which the kidney reabsorbs L-amino acids. No claims are made as to the meaning and/or importance of free D-amino acids in regards to pathology, age, clinical usefulness and so forth. However, a knowledge of normal D-amino acid levels and dynamics is necessary before it is possible to identify perturbations caused by either natural or pathological conditions. The techniques are now available that should allow these topics to be addressed properly.

The involvement of oxygen radicals in acute pancreatitis.

The purpose of this study was to assess the involvement of oxygen radicals in acute edematous and hemorrhagic pancreatitis. Acute pancreatitis was induced in rats by the CCK-analogue cerulein (5 micrograms/kg/h) and by retrograde injection of 5% sodium taurocholate for 30 min, 3.5 h, and 12 h. At the end of the infusion and observation time, serum enzymes, conjugated dienes, and malondialdehyde in the tissue were measured. Moreover, the tissue samples underwent light microscopical examination. In cerulein pancreatitis, an interstitial edema and intravascular margination of granulocytes in the pancreatic gland were observed after 3.5 h. After 12 h, the histological evaluation revealed a pronounced zymogen degranulation, extensive tissue necrosis and migration of granulocytes into the tissue. Parallelly, amylase and lipase increased by 15 and 35 times, respectively. In contrast, conjugated dienes and malondialdehyde increased in cerulein pancreatitis and reached their highest level after 3.5 h and decreased to normal levels after 12 h. The development of the histological damages and serum enzyme levels with sodium taurocholate pancreatitis was similar as compared to the cerulein pancreatitis, however, the development was faster and more traumatic. Already after 3.5 h an extensive zymogen degranulation and cell necrosis was observed. Concomitantly, the amylase and lipase levels increased by 90 and 30 times, respectively. Treatment with superoxide dismutase (100,000 U/kg/h) and catalase (400,000 U/kg/h) prevented lipid peroxidation and reduced zymogen degranulation and tissue necrosis. Tissue edema and inflammatory response were not affected in both models of acute pancreatitis.(ABSTRACT TRUNCATED AT 250 WORDS)

The response of one primigravida to the situation of persistent anemia during pregnancy.

This descriptive longitudinal case study examines the nature and purposes of the responses of one primigravida to the situation of persistent anemia during pregnancy. Selected literature in the following topic areas relevant to pregnancy complicated by serious illness is reviewed: stress, adaptation, defense and coping; pregnancy, illness and uncertainty; and body image implications of pregnancy and blood loss. Following unstructured interviews and observations in natural settings, process recordings were prepared and analyzed by the nurse-investigator to identify major behavioral themes. After repeated analysis and refining of categories, six mutually exclusive groups of responses were defined, and their frequency of use calculated: conditional compliance (34%), expressing affective states (24%), monitoring (23%), using support (11%), replenishing (5%), and using knowledge (3%). The subject used these responses, singly or in combination, to deal with various elements of perceived threat inherent in her anemic condition, the treatment of that condition, and her relationship with caregivers. The findings reveal one person's creative use of resources (inner and environmental) to develop a flexible response pattern effective in limiting or preventing stress associated with a potentially life-threatening condition.