Development and Application of a Modified Method to Determine the Encapsulation Efficiency of Proteins in Polymer Matrices.

A modified method to determine protein encapsulation efficiency in polymer matrices has been developed and applied to two proteins and two polymers to demonstrate its wide range of applicability. This study was pursued due to the wide variation in reported protein encapsulation efficiency of polymer-based microcapsules, even when the protein, the polymer, and the microcapsule manufacturing method were consistent. Hemoglobin (Hb) and bovine serum albumin (BSA) were chosen as model proteins and ethylcellulose and poly(lactic-co-glycolic acid) (PLGA) as model polymers. The polymer of the microcapsule was dissolved in dichloromethane/ethanol or dichloromethane/ethyl acetate for ethylcellulose or PLGA microcapsules, respectively. Liberated proteins were simultaneously precipitated, pelleted by centrifugation, isolated by decanting the polymer solution, redissolved in 10% w/v sodium dodecyl sulfate in 0.8 N sodium hydroxide, and quantified using a modified Lowry assay. Blank microcapsules and exogenously added proteins demonstrated ≥ 93.8% recovery of proteins. The mean encapsulation efficiency of ethylcellulose or PLGA microcapsules was 52.4 or 76.9% for Hb and 86.4 or 74.7% for BSA, respectively. This demonstrates the effective use of centrifugation and the importance of an appropriate cosolvent system in the measure of encapsulation efficiency where one solvent dissolves the polymer while the other solvent quantitatively precipitates the liberated protein. It is evident that an alkaline solution of sodium dodecyl sulfate is efficient at quantitatively dissolving precipitated proteins. Remediation of problems observed with current methods and high reproducibility suggest that this modified method is generally applicable to the measure of protein encapsulation efficiency of polymer microcapsules.

Decreased liver triglyceride content in adult rats exposed to protein restriction during gestation and lactation: roles of hepatic lipogenesis and lipid utilization in muscle and adipose tissue.

Protein restriction throughout pregnancy and lactation reduces liver triglyceride (TG) content in adult male rat offspring. The study determined the contribution of hepatic lipogenesis to the reduction in liver TG content. Rats received either control or protein-restricted diets throughout pregnancy and lactation. Offspring were sacrificed on day 65. Hepatic fatty acid uptake and de novo fatty acid and TG biosynthesis were similar between control and low-protein (LP) offspring. These results indicate that hepatic lipogenesis cannot mediate the decrease in liver TG content in LP offspring. We then determined whether increased lipid utilization in adipose tissue and muscle was responsible for the decrease in liver TG content. There was suggestive evidence of increased sympathetic nervous system tone in epididymal adipose tissue of LP offspring that increased fatty acid uptake, TG lipolysis, and utilization of fatty acids in mitochondrial thermogenesis. Measurement of similar parameters demonstrated that such alterations do not occur in gastrocnemius muscle, another major lipid-utilizing tissue. Our results suggest that the decrease in liver TG content in LP offspring is likely due to increased diversion of fatty acids to white and brown adipose tissue depots and their enhanced utilization to fuel mitochondrial thermogenesis.

Administration of Fenofibrate Markedly Elevates Fabp3 in Rat Liver and Plasma and Confounds Its Use as a Preclinical Biomarker of Cardiac and Muscle Toxicity.

Proteins involved in lipid homeostasis are often regulated through the nuclear peroxisome proliferator-activated receptors (PPAR). PPARα is the target for the fibrate-class of drugs. Fenofibrate has been approved for its lipid-lowering effects in patients with hypercholesterolemia and hypertriglyceridemia. We were interested in understanding the expression of the energy transporters in energy-utilizing tissues like liver, heart, muscle, and adipose tissues in rat with the hypothesis that the change in transporter expression would align with the known lipid-lowering effects of PPARα agonists like fenofibrate. We found that several fatty-acid transporter proteins had significantly altered levels following 8 days of fenofibrate dosing. The mRNA levels of the highly abundant Fatp2 and Fatp5 in rat liver increased approximately twofold and decreased fourfold, respectively. Several fatty-acid-binding proteins and acyl-CoA-binding proteins had a significant increase in mRNA abundance but not the major liver fatty-acid-binding protein, Fabp1. Of particular interest was the increased liver expression of Fabp3 also known as heart-fatty acid binding protein (H-FABP or FABP3). FABP3 has been proposed as a circulating clinical biomarker for cardiomyopathy and muscle toxicity, as well as a preclinical marker for PPARα-induced muscle toxicity. Here, we show that fenofibrate induces liver mRNA levels of Fabp3 ~5000-fold resulting in an approximately 50-fold increase in FABP3 protein levels in the whole liver. This increased liver expression complicates the interpretation and potential use of FABP3 as a specific biomarker for PPARα-induced muscle toxicities.

Maternal protein restriction during pregnancy and lactation alters central leptin signalling, increases food intake, and decreases bone mass in 1 year old rat offspring.

The effects of perinatal nutrition on offspring physiology have mostly been examined in young adult animals. Aging constitutes a risk factor for the progressive loss of metabolic flexibility and development of disease. Few studies have examined whether the phenotype programmed by perinatal nutrition persists in aging offspring. Persistence of detrimental phenotypes and their accumulative metabolic effects are important for disease causality. This study determined the effects of maternal protein restriction during pregnancy and lactation on food consumption, central leptin sensitivity, bone health, and susceptibility to high fat diet-induced adiposity in 1-year-old male offspring. Sprague-Dawley rats received either a control or a protein restricted diet throughout pregnancy and lactation and pups were weaned onto laboratory chow. One-year-old low protein (LP) offspring exhibited hyperphagia. The inability of an intraperitoneal (i.p.) leptin injection to reduce food intake indicated that the hyperphagia was mediated by decreased central leptin sensitivity. Hyperphagia was accompanied by lower body weight suggesting increased energy expenditure in LP offspring. Bone density and bone mineral content that are negatively regulated by leptin acting via the sympathetic nervous system (SNS), were decreased in LP offspring. LP offspring did not exhibit increased susceptibility to high fat diet induced metabolic effects or adiposity. The results presented here indicate that the programming effects of perinatal protein restriction are mediated by specific decreases in central leptin signalling to pathways involved in the regulation of food intake along with possible enhancement of different CNS leptin signalling pathways acting via the SNS to regulate bone mass and energy expenditure.

Decreased liver triglyceride content in adult rats exposed to protein restriction during gestation and lactation: role of hepatic triglyceride utilization.

We have previously demonstrated that protein restriction throughout gestation and lactation reduces liver triglyceride content in adult rat offspring. However, the mechanisms mediating the decrease in liver triglyceride content are not understood. The aim of the current study was to use a new group of pregnant animals and their offspring and determine the contribution of increased triglyceride utilization via the hepatic fatty-acid oxidation and triglyceride secretory pathways to the reduction in liver triglyceride content. Pregnant Sprague-Dawley rats received either a control or a low protein diet throughout pregnancy and lactation. Pups were weaned onto laboratory chow on day 28 and killed on day 65. Liver triglyceride content was reduced in male, but not female, low-protein offspring, both in the fed and fasted states. The reduction was accompanied by a trend towards higher liver carnitine palmitoyltransferase-1a activity, suggesting increased fatty-acid transport into the mitochondrial matrix. However, medium-chain acyl coenzyme A dehydrogenase activity within the mitochondrial matrix, expression of nuclear peroxisome proliferator activated receptor-α, and plasma levels of ß-hydroxybutyrate were similar between low protein and control offspring, indicating a lack of change in fatty-acid oxidation. Hepatic triglyceride secretion, assessed by blocking peripheral triglyceride utilization and measuring serum triglyceride accumulation rate, and the activity of microsomal transfer protein, were similar between low protein and control offspring. Because enhanced triglyceride utilization is not a significant contributor, the decrease in liver triglyceride content in male low-protein offspring is likely due to alterations in liver fatty-acid transport or triglyceride biosynthesis.

Elucidation of thrifty features in adult rats exposed to protein restriction during gestation and lactation.

Since the introduction of the thrifty phenotype hypothesis, the potential traits of thrift have been described in increasingly broad terms but biochemical and behavioral evidence of thrift has not been well demonstrated. The objective of our studies was to use a rodent model to identify features of thrift programmed by early life protein restriction. Robust programming of thrifty features requires a thrifty nutritional environment during the entire window of developmental plasticity. Therefore, pregnant rats were exposed to a low protein diet throughout the window of developmental plasticity spanning the period of gestation and lactation and its effects on energy acquisition, storage and expenditure in the adult offspring were examined. Maternal protein restriction reduced birth weight and produced long term reductions in body and organ weights in the offspring. Low protein offspring demonstrated an increased drive to seek food as evidenced by hyperphagia that was mediated by changes in plasma leptin and ghrelin levels. Hyperphagia was accompanied by increased efficiency in converting caloric intake into body mass. The higher feed efficiency was mediated by greater insulin sensitivity. Energy expenditure of low protein offspring in locomotion was not affected either in the light or dark phase. However, low protein offspring exhibited higher resting and basal metabolic rates as evidenced by higher core body temperature in the fed and fasted states. The increased thermogenesis was not mediated by thyroid hormones but by an increased sympathetic nervous system drive as reflected by a lower areal bone mineral density and bone mineral content and lower plasma adiponectin and triglyceride levels. Elevated thermogenesis in the low protein offspring possibly offsets the effects of hyperphagia, minimizes their chances of weight gain, and improves survivability. This constellation of metabolic features in the low protein offspring will maximize survival potential in a post natal environment of nutritional scarcity and constitute a thrifty phenotype.

Maternal protein restriction during pregnancy and lactation in rats imprints long-term reduction in hepatic lipid content selectively in the male offspring.

Maternal protein restriction during pregnancy and lactation reduces whole body lipid stores and alters lipid homeostasis in the adult offspring. Lipid homeostasis in the body is regulated, in part, by the liver via the metabolic processes of synthesis and utilization of lipids. The present study tested the hypothesis that maternal protein restriction will imprint changes in hepatic lipid metabolism and thereby alter the hepatic lipid content of the adult offspring. Pregnant rats were fed purified diets containing 19% protein (control group) or 8% protein (low-protein group) throughout pregnancy and lactation. On day 28, pups from both groups were weaned onto regular laboratory chow. On days 65 and 150, male and female pups from each litter in both groups were killed and blood and liver collected. Maternal protein restriction was found to reduce birth weight and produce long-term reduction in the body weight of the offspring. On day 65, liver triglyceride content was decreased by 40% in the male offspring that were fed a low-protein diet. The reduction in liver triglyceride content persisted until day 150, at which time it was accompanied by decreases in hepatic cholesterol content. No such changes were observed in the female offspring. To determine if the alterations in liver lipid content resulted in compensatory changes in liver carbohydrate stores, hepatic glycogen content was measured in male offspring. Hepatic glycogen content was similar between the 2 groups on days 65 and 150. In conclusion, the present study in rats showed that maternal protein restriction during pregnancy and lactation imprints long-term changes in hepatic lipid content selectively in the male offspring.

Strategies to maximize the encapsulation efficiency of phenylalanine ammonia lyase in microcapsules.

The activity of phenylalanine ammonia lyase (PAL) encapsulated in cellulose nitrate microcapsules is only 23% of the activity of PAL in Tris buffer. This lower activity is partially due to its incomplete encapsulation. The objective of the study was to maximize the encapsulation efficiency (EE) of PAL by optimizing different formulation parameters. PAL was purified using size exclusion chromatography and then radioiodinated using the Iodo-gen reaction. Use of (125)I-PAL showed that PAL had an EE of 45% in cellulose nitrate microcapsules. Formulation parameters including concentration of polymer in solution, stirring speed and ratio of aqueous phase volume to organic phase volume were individually optimized to maximize the EE of PAL. The reformulated microcapsules showed an EE of PAL of 80%. The dramatic increase in EE was reflected in a marked (119%) increase in the activity of encapsulated PAL compared to its activity in the original microcapsules. Eighty two percent of the encapsulated PAL was physically present in the aqueous core while 18% was entrapped in the microcapsule membrane. Distribution of PAL activity in the microcapsule was in concordance with its physical distribution.

Effect of perinatal low protein diets on the ontogeny of select hepatic cytochrome p450 enzymes and cytochrome p450 reductase in the rat.

In the present study, we administered two low protein diets (LPDs) to rats during pregnancy and lactation and determined their effect on the ontogeny of select hepatic cytochrome P450 (P450) isoforms in their offspring. The L93 and LM76 LPDs were derived from the American Society of Nutrition recommended AIN93G and a modified version of the AIN76A purified control diets, respectively. The LPDs contained 8% crude protein in the form of casein, whereas the purified control diets contained 19% casein. A regular cereal-based diet (NP) was also included, and, therefore, a total of five groups were tested. Pups in all five groups were weaned onto a regular NP diet on postnatal day 28. Perinatal LPD altered the activities of a number of P450 isoforms in 28-day-old male and female offspring. However, nutritional rehabilitation abolished most of these changes as evidenced by lack of differences between the five groups in the activities of P450 isoforms in either 65- or 150-day-old offspring. Interestingly, 58-day-old female offspring in the LM76 group but not those in the L93 group exhibited shorter hexobarbital sleep time than the purified control group. However, hexobarbital hydroxylase activity and the amount of CYP2C12 protein, an important P450 isoform involved in hexobarbital metabolism in females, were unchanged. This suggests that the decrease in hexobarbital sleep time in this group is not due to an increase in the activity of hexobarbital-metabolizing enzymes. In summary, perinatal LPDs produced transient alterations in activities of select hepatic P450s and resulted in a gender- and diet-dependent long-term alteration in hexobarbital pharmacodynamics.

Stabilization of phenylalanine ammonia lyase against organic solvent mediated deactivation.

A potential novel therapy for phenylketonuria involves oral administration of microencapsulated phenylalanine ammonia lyase (PAL), an enzyme that converts phenylalanine to transcinnamic acid. A drawback of this potential therapy is that encapsulated PAL retains only 23% of its activity. Microcapsules are prepared by emulsifying PAL in 10% hemoglobin solution with water-saturated ether (WSE) and subsequent addition of cellulose nitrate dissolved in ether:ethanol (E:E) mixture. The objective of this paper was to determine the contribution of emulsification to the overall loss in activity of encapsulated PAL, and to devise strategies to protect PAL against such loss in activity. Emulsification was simulated by stirring the aqueous phase containing PAL with the organic phase. The mixture was then centrifuged, and the protein content and catalytic activity of PAL in the aqueous phase was measured. Emulsification of PAL solution with WSE caused no loss in activity but resulted in a loss in protein content of aqueous phase. Size exclusion chromatography and gel electrophoresis studies showed that the loss was primarily due to the specific loss of impurities in the PAL sample. Emulsification of PAL solution with E:E resulted in a 50% decrease in its activity. Among the additives, hydroxypropyl-gamma-cyclodextrin and hydroxy propyl-beta-cyclodextrin protected PAL against emulsion mediated loss in activity.

Two low protein diets differentially affect food consumption and reproductive performance in pregnant and lactating rats and long-term growth in their offspring.

We fed 2 low protein diets (LPD) to rats during pregnancy and lactation, and compared food intake and reproductive performance in the dams, and long-term growth in their offspring. The L93 and LM76 LPDs were derived from the American Society of Nutrition's recommended AIN93G and a modified version of the AIN76A purified control diets, respectively. The LPDs contained 8% crude protein in the form of casein and differed in their fat and carbohydrate sources. The purified control diets contained 19% crude protein. A regular cereal-based diet was also included, therefore, a total of 5 groups were tested. Blood urea nitrogen concentrations in dams of both LPD groups were lower than their respective controls, confirming decreased protein intake. The LM76 diet lowered food consumption of dams and produced energy malnourishment during pregnancy that persisted throughout lactation. In contrast, the L93 diet produced energy malnourishment only during lactation. Offspring of both LPD groups exhibited lower birth weights than their respective controls. Despite initiating nutritional rehabilitation at weaning (d 28), perinatal administration of both low protein diets produced long-term reductions in the body weight of male offspring. Interestingly, in the female offspring, the LM76 diet reduced birth weight for the entire duration of the study (180 d), whereas the L93 diet produced a relatively short-term (up to 58 d) reduction in body weight. This suggests that the imprinting effect of the perinatal nutritional environment on body weight is diet and gender dependent. The performance of the purified control diet groups were similar to the nonpurified diet group in most measured biochemical indices, with the notable exception of a decrease in the body-weight normalized kidney weight of the dams.

Effects of maternal immobilization stress on birth weight and glucose homeostasis in the offspring.

Recent epidemiological studies have shown strong associations between low birth weight and the incidence of diabetes in the adult offspring. It has been hypothesized that exposure to maternal glucocorticoids programs cellular changes in the fetus which increases the susceptibility of the offspring to diabetes. Stressors produces large increases in maternal glucocorticoids. The present study determined the effects of immobilization stress during weeks one, two or three of pregnancy on offspring birth weight, glucose homeostasis, and the ability of the offspring to cope with metabolic stress. Immobilization stress produced large increases in maternal levels of ACTH and corticosterone, but did not affect birth weight of the pups. Chronic administration of high fructose diet, a metabolic stressor, to 60 days old control and prenatally stressed offspring produced large increases in plasma levels of triglyceride and insulin. However, there were no differences between the groups either in peak levels, or in the rates of increase and decrease (upon discontinuation of the diet) of plasma triglyceride and insulin concentrations. Basal levels of glucose and insulin, and areas under the glucose and insulin plasma concentration-time curves after an i.p. glucose dose were similar between 120 days old control and prenatally stressed offspring. These results suggest that in young adult rats prenatal immobilization stress did not affect glucose homeostasis or the ability of these rats to cope with chronic metabolic stress.