Rat Weight Gain Research Articles

Variability in rat weight gain during development.

The rat is one of the most employed animal models in biomedicine. Traditionally, weight gain has been utilized to gauge development and compare across species. Numerous studies have conducted longitudinal analyses of rat development, with emphasis on weight gain analysis. Given the high variability in these patterns, experimental data from a single laboratory may not be reliable for generalized estimation. This study aimed to analyze the effect of different factors on the pattern of weight gain during rat development. A literature survey was conducted to compile a database comprising nearly 300 data points of age and weight from 15 longitudinal studies. The database comprised both pre- and postnatal data. Utilizing the Gompertz equation, the data was analyzed to formulate a comprehensive model describing rat development. Differences in growth patterns became increasingly evident at later developmental stages, when significant differences in the maximum asymptote between sexes and strains were reached.

Supplementation with Bovine Milk or Soy Beverages Recovers Bone Mineralization in Young Growing Rats Fed an Insufficient Diet, in Contrast to an Almond Beverage

ABSTRACTBackgroundNondairy beverages, produced from soy, rice, oat, almond, or coconut, are increasingly being used as alternatives to dairy milk, with the perception that they are healthier and/or more sustainable products than dairy products.ObjectiveThe aim of this study was to compare the effects of supplementing either bovine milk, soy, or almond-based beverages to young, growing rats fed an intact-protein diet or a diet that had protein substituted with amino acids (AA-diet).MethodsThree-week-old male Sprague-Dawley rats were randomly assigned to 5 groups (n = 10/group) and fed ad libitum for 4 wk. Two control groups were fed either standard AIN-93G food [20% casein (CN) protein] or AIN-93G with amino acids (AAs) equivalent to CN protein, and water to drink. Three treatment groups were fed AIN-93G AA and supplemented with either bovine ultra-heat treatment (UHT) milk or soy or almond UHT beverages. Rat weight gain and food intakes were recorded. During week 4, body composition was assessed using DEXA to determine lean soft tissue, fat, and bone mass. At trial end, bone biomechanical properties and blood plasma mineral concentrations were measured.ResultsAt the end of the trial, animals supplemented with almond beverage were lightest (P > 0.05), with higher plasma calcium concentrations (P > 0.05) and lower bone mineral content (BMC) and bone density (P > 0.05) than animals supplemented with milk or soy beverage. Soy-supplemented animals had similar BMC and bone density compared with milk-supplemented animals, although the soy group gained most weight (P > 0.05) and had the highest fat:lean ratio (P > 0.05) compared with other groups.ConclusionsIn the model tested, supplementing rats with bovine UHT milk and soy UHT beverage provided favorable bone health outcomes. Conversely, almond UHT beverage was not an effective supplement and could be detrimental to bone mineralization and strength outcomes.

A Cell-Based Strategy for Bioactivity Determination of Long-Acting Fc-Fusion Recombinant Human Growth Hormone.

The long-acting growth hormone (LAGH) is a promising alternative biopharmaceutical to treat growth hormone (GH) deficiency in children, and it was developed using a variety of technologies by several pharmaceutical companies. Most LAGH preparations, such as Fc fusion protein, are currently undergoing preclinical study and clinical trials. Accurate determination of bioactivity is critical for the efficacy of quality control systems of LAGH. The current in vivo rat weight gain assays used to determine the bioactivity of recombinant human GH (rhGH) in pharmacopoeias are time-consuming, expensive, and imprecise, and there are no recommended bioassays for LAGH bioactivity in pharmacopoeias. Therefore, we developed a cell-based bioassay for bioactivity determination of therapeutic long-acting Fc-fusion recombinant human growth hormone (rhGH-Fc) based on the luciferase reporter gene system, which is involved in the full-length human GH receptor (hGHR) and the SG (SIE and GAS) response element. The established bioassay was comprehensively validated according to the International Council for Harmonization (ICH) Q2 (R1) guidelines and the Chinese Pharmacopoeia, and is highly precise, time-saving, simple, and robust. The validated bioassay could be qualified for bioactivity determination during the research, development, and manufacture of rhGH-Fc, and other LAGH formulations.

Development and Characterization of Sodium Hyaluronate Microparticle-Based Sustained Release Formulation of Recombinant Human Growth Hormone Prepared by Spray-Drying

The purpose of this study was to develop and characterize a sodium hyaluronate microparticle-based sustained release formulation of recombinant human growth hormone (SR-rhGH) prepared by spray-drying. Compared to freeze-drying, spray-dried SR-rhGH showed not only prolonged release profiles but also better particle property and injectability. The results of size-exclusion high-performance liquid chromatography showed that no aggregate was detected, and dimer was just about 2% and also did not increase with increase of inlet temperature up to 150°C. Meanwhile, the results of reversed-phase high-performance liquid chromatography revealed that related proteins increased slightly from 4.6% at 100°C to 6.3% at 150°C. Thermal mapping test proved that product temperature did not become high to cause protein degradation during spray-drying because thermal energy was used for the evaporation of surface moisture of droplets. The structural characterization by peptide mapping, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and circular dichroism revealed that the primary, secondary, and tertiary structures of rhGH in SR-rhGH were highly comparable to those of reference somatropin materials. The biological characterization by rat weight gain and cell proliferation assays provided that bioactivity of SR-rhGH was equivalent to that of native hGH. These data establish that spray-dried SR-rhGH is highly stable by preserving intact rhGH and hyaluronate microparticle-based formulation by spray-drying can be an alternative delivery system for proteins.

The Histone Deacetylase 1 and 2 (HDAC1/2) Inhibitor ACY-957 Increases Epsilon (HbE) and Gamma (HbG) Globin mRNA in the Peripheral Blood of Non-Anemic Rats and Monkeys

The Histone Deacetylase 1 and 2 (HDAC1/2) Inhibitor ACY-957 Increases Epsilon (HbE) and Gamma (HbG) Globin mRNA in the Peripheral Blood of Non-Anemic Rats and Monkeys

Dietary Docosahexaenoic Acid Improves Cognitive Function, Tissue Sparing, and Magnetic Resonance Imaging Indices of Edema and White Matter Injury in the Immature Rat after Traumatic Brain Injury.

Traumatic brain injury (TBI) is the leading cause of acquired neurologic disability in children. Specific therapies to treat acute TBI are lacking. Cognitive impairment from TBI may be blunted by decreasing inflammation and oxidative damage after injury. Docosahexaenoic acid (DHA) decreases cognitive impairment, oxidative stress, and white matter injury in adult rats after TBI. Effects of DHA on cognitive outcome, oxidative stress, and white matter injury in the developing rat after experimental TBI are unknown. We hypothesized that DHA would decrease early inflammatory markers and oxidative stress, and improve cognitive, imaging and histologic outcomes in rat pups after controlled cortical impact (CCI). CCI or sham surgery was delivered to 17 d old male rat pups exposed to DHA or standard diet for the duration of the experiments. DHA was introduced into the dam diet the day before CCI to allow timely DHA delivery to the pre-weanling pups. Inflammatory cytokines and nitrates/nitrites were measured in the injured brains at post-injury Day (PID) 1 and PID2. Morris water maze (MWM) testing was performed at PID41-PID47. T2-weighted and diffusion tensor imaging studies were obtained at PID12 and PID28. Tissue sparing was calculated histologically at PID3 and PID50. DHA did not adversely affect rat survival or weight gain. DHA acutely decreased oxidative stress and increased anti-inflammatory interleukin 10 in CCI brains. DHA improved MWM performance and lesion volume late after injury. At PID12, DHA decreased T2-imaging measures of cerebral edema and decreased radial diffusivity, an index of white matter injury. DHA improved short- and long-term neurologic outcomes after CCI in the rat pup. Given its favorable safety profile, DHA is a promising candidate therapy for pediatric TBI. Further studies are needed to explore neuroprotective mechanisms of DHA after developmental TBI.

Effects of Chronic Bryophyllum pinnatum Administration on Wistar Rat Pregnancy

Background: Drugs used in preterm labor (PTL) have side effects. Research into new tocolytic agents is recommended. The plant Bryophyllum pinnatum (Lam.) Oken (Kalanchoe pinnata (Lam.) Pers) is used in Anthroposophic Medicine for PTL, insomnia, and emotional disturbances. The aim of this study was to evaluate the effects of B. pinnatum mother tincture (MT) on Wistar rats and their fetuses throughout pregnancy. Method: Sixty animals were divided into 6 equal groups: controls C1 and C2 received 1 and 25 × the maximum daily dose of a 30% ethanol / water solution, serving as vehicle of B. pinnatum MT; B1 and B2 received 1 and 25 × the maximum daily dose of B. pinnatum MT; and B3 and B4 received 50 and 100 × B. pinnatum concentrate. The following parameters were assessed: weight gain; maternal and fetal mortality; implantations and resorptions; number and weight of fetuses and placentas; major external fetal malformations. Results: Rat weight gain (excluding fetal and placental weight) was higher in group B4 and lower in group B2. There were no maternal or fetal deaths and no group differences in implantations and resorptions or number and weight of fetuses and placentas. No macroscopic fetal abnormalities were observed at the 4 dosage levels investigated. Conclusion: Daily administration of B. pinnatum MT at high doses to pregnant Wistar rats interfered with maternal weight gain and did not interfere with fetal compartment.

High Fatty Diet Effects on Rat Liver

Aim of this study was to investigate effects of high fat diet on rat liver and weight gain. By this purpose 30 Wistar Albino rats were divided into 4 groups. 1. High carbonhydrate diet group for 16 weeks (K16), 2. High fatty diet group for 16 weeks (D16), 3. High carbonhydrate diet group 20 weeks (K20), 4. High fatty diet group 20 weeks (D20). High fatty diet; %60 fat (1/3 canola, 1/3margarine, 1/3 sunflower oil), %20 protein, %20 carbohydrate and high carbohydrate control diet; %69 carbohydrate, %20 protein and %11 margarine was composed. There weren’t any significant differences between groups as compared to body weight, liver weight and epididymal fat weight. At the result of biochemical analysis, LDH in 16 weeks high carbohydrate diet and ALT in 20 weeks high carbonhydrate diet was significant higher than high fatty diet groups. In histological research, even though fibrosis, inflamation, steatosis findings observed at portal area of all groups, there wasn’t significant statistical differences. Similar to this, α-SMA and TGF-β accumulation in all groups were similar interms of immunohistological investigation. In conclusion; this study showed that comparing to relatively high fatty diet rich with omega-9, high carbonhydrate feeding caused liver injury and anointment and there wasn’t significant differences in terms of weight gain. By this way, we think that decreasing of carbonhydrate and increasing amount of olive oil, canola oil and hazelnut oil at which containing omega-9 in diet, liver can be protected.

Green juice as a protector against reactive species in rats.

Green juice is popularly known for introducing antioxidants, improving intestinal function and reducing weight gain. In the present study we determine the antioxidant effect of green juice comparing it with orange juice. Rats were divided into three experimental groups and submitted to supplementation for 15 days: the (GJ) group received green juice, the (OJ) group received orange juice and the control group received water. We evaluated the antioxidant activity and total phenolic content of green and orange juices, as well as rat weight gain. We also investigated some oxidative stress parameters, namely thiobarbituric acid-reactive substances (TBARS), superoxide dismutase and catalase in rat cerebral cortex. Results showed that GJ had significantly less weight gain than the control group. With respect to antioxidant activity screening, the remaining percentage of DPPH at dilutions 1:10, 1:100 and 1:1000 of green juice was 22.8%, 58% and 78%, and orange juice, at the same dilutions, was 5.6%, 5.6% and 77.2%, respectively. The ability of juices to reduce the ABTS radical was 3.5 mmol trolox/L for green juice and 5.2 mmol trolox/L for orange juice. Additionally, the green juice did not present any difference in total phenolic acid content when compared to orange juice. TBARS were reduced in GJ and OJ. Besides, GJ supplementation decreased catalase activity. In conclusion, our data showed that green juice reduced weight gain, lipoperoxidation and catalase activity, suggesting that this supplementation may have a protective effect against reactive species.

Inhibitory effects of alkaline extract of Citrus reticulata on pulmonary fibrosis

Ethnopharmacological relevanceThe pericarp of Citrus reticulata possesses medical functions of regulating Qi and expelling phlegm, and has been clinically used for the treatment of lung related diseases in traditional Chinese medicine for a long time. Our previous research revealed that Citrus reticulata exhibited inhibitory effects on pulmonary fibrosis; however, its active principles are still unclear. Aim of the studyTo investigate the inhibitory effects on pulmonary fibrosis of alkaline extract from ethanol extract of Citrus reticulata and clarify its possible mechanism. Materials and methodsThe citrus alkaline extract (CAE) was prepared from ethanol extract of Citrus reticulata and MRC-5 cells were used for the evaluation of inhibitory activity in vitro. CAE was further orally administrated to bleomycin (BLM)-induced pulmonary fibrosis rats. The rat body weight, hydroxyproline levels in serum and lung, pathological changes of lung, as well as mRNA and protein expressions of matrix metalloproteinase-9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1) and tumor necrosis factor-α (TNF-α) in rat lung tissues were analyzed. ResultsCAE dose-dependently inhibited the proliferation of MRC-5 cells, and the LDH assay clearly revealed that the inhibitory activity of CAE was not due to its cytotoxicity. CAE treatment significantly increased rat weight gain, ameliorated alveolitis and pulmonary fibrosis degree, and lowered hydroxyproline contents in both serum and lung tissues. RT-PCR and western blot revealed that mRNA and protein expressions of MMP-9 were significantly elevated, while mRNA and protein levels of TIMP-1 and TNF-α were markedly decreased in lung tissues of CAE treated rats. ConclusionsThe results collectively demonstrated that CAE possessed an inhibitory activity on the proliferation of MRC-5 and a preventive effect on BLM-induced pulmonary fibrosis in rats. The preliminary mechanisms of the effects may be through upregulation of MMP-9 expression and inhibition of the expressions of TNF-α and TIMP-1.

Bioavailability of Beta Carotene in Traditional Fermented, Roasted Granules,<i> Gari</i> from Bio-Fortified Cassava Roots

Vitamin A Deficiency (VAD) is a major public health issue and of global concern, as it affects millions of preschool children and pregnant women worldwide. Bio-fortification has emerged as a technology with potential to sustainably alleviate VAD especially in the sub-Saharan Africa, using staples like cassava. This work studied bioavailability of beta carotene (BC) in two processed gari samples from bio-fortified cassava varieties: 01/1412 and 01/1371, using 40 weanling albino rats, grouped into four, acclimatized for 1 week and fed experimentally for 4 weeks. Plasma beta carotene (PBC) was determined with HPLC while bioavailable BC calculated using conventional linear dose response plot. The mean rat weight gain was 5.3 g with significant difference (p< 0.001) among them while mean PBC was 60.5 and 61.2 μg/dL for 01/1412 and 01/1371 respectively. From this study, a large variation of PBC among animals was found with a weak linear relationship between feed and PBC, showing that BC bioavailability is not limited to intake. The BC bioavailability of the samples was between 11% and 18% with sample from variety 01/1371 recording higher percentage (18%). Gari from bio-fortified cassava roots processed traditionally, had appreciable bioavailable BC, which can contribute to the fight against VAD and improve nutritional status in developing countries although the magnitude of the problem requires a combination of strategies, of which bio-fortification is just one. However, further work is necessary on public awareness and adoption of the product.

AZT-Induced Oxidative Cardiovascular Toxicity: Attenuation by Mg-Supplementation

Cardiovascular effects of chronic AZT treatment on SD male rats (185 g) fed either a normal Mg diet (0.1% MgO) or a high Mg diet (0.6% MgO) were examined. AZT treatment (1 mg/ml drinking water) for 3 weeks led to a 5.5-fold (0.88 +/- 0.11 nmol/min/10(6) cells, P < 0.05) elevation in neutrophil basal activity of O2(-) production versus controls (0.16 +/- 0.03 nmol/min, assayed ex vivo as SOD-inhibitable cytochrome c reduction). Concomitantly, plasma 8-isoprostane and PGE(2) levels rose 2.1-fold and 3-fold (both P < 0.05), respectively, compared to control; however, RBC GSH decreased 28% (P < 0.02) with GSSG content increased 3-fold, indicative of systemic oxidative stress. High Mg diet substantially attenuated the AZT-induced neutrophil activation by 70% (0.26 +/- 0.05 nmol/min, P < 0.05); reduced plasma 8-isoprostane and PGE(2) to levels comparable to normal; and RBC GSH was restored back to 92% of control. AZT alone caused moderate, but significant vascular inflammatory lesions in the heart (assessed by H&E staining). Immunohistochemical staining revealed significantly higher (about 4-fold) infiltration of CD11b positive cells (WBC surface marker) in the atria and ventricles of AZT-treated rats. However, these inflammatory pathological markers were minimal in samples of rats treated with AZT plus high Mg diet. Moreover, AZT alone significantly (P < 0.02) decreased rat weight gain by 21% at 3 weeks; Mg-supplementation completely prevented (P < 0.05) the weight gain loss due to AZT intake. It is concluded that high dietary Mg may provide beneficial effects against AZT toxicity due to its systemic antioxidative/anti-inflammatory properties.

The effects of polybrominated diphenyl ethers (PBDEs) on adipocyte functionality in vitro and in vivo

PBDEs are flame retardants found in furniture, carpets and electronics. These organic pollutants are highly lipophilic and accumulate in the adipose tissue of animals and humans. However, the effects of PBDEs on adipocyte metabolism are unknown. The purpose of this study was to determine the effects of penta-PBDE on lipolysis and glucose oxidation in rat adipocytes in vitro and in vivo. In vitro, adipocytes from 12 male Sprague-Dawley rats were isolated and incubated for 90 minutes with either 0, 15 or 30ug/ml PBDEs (4 rats/group.) In vivo, male Sprague-Dawley rats weighing 120g were gavaged daily for 4 weeks with either 18mg/kg PBDEs or corn oil (12 rats/group). Adipocytes from in vivo and in vitro treatments were tested for lipolytic response to epinephrine and [U-14C] glucose oxidation response to insulin. Rat weight gain was similar in all groups throughout the study. In vitro data show that incubation with PBDEs for 90 minutes had no effect on lipolysis or glucose oxidation in rat adipocytes. In vivo data show that after 4 weeks, basal lipolysis was increased by 58% and maximal lipolysis was increased by 22%, compared to control. There was a 41% decrease in maximal glucose oxidation in rats treated with PBDEs for 4 weeks, compared to controls. In summary, although incubation of adipocytes with PBDEs does not change adipocyte functionality, a low daily dose of PBDEs to rats increased basal and maximal lipolysis and decreased maximal glucose oxidation in freshly isolated rat adipocytes. Whether these changes persist and contribute to permanently altered adipocyte function remains to be determined. Supported by Agr. Exp. Stn. Grant NH00432.

Characterization of Nα-acetyl methionyl human growth hormone formed during expression in Saccharomyces cerevisiae with liquid chromatography and mass spectrometry

We found a new variant of human growth hormone (hGH) from the recombinant hGH expression process in Saccharomyces cerevisiae. The variant was identified as N α-acetyl methionyl hGH which may be formed by N α-acetylation of met-hGH during the intracellular expression of hGH in S. cerevisiae. The variant was isolated from manufacturing process of LG Life Sciences’ hGH product. The variant was subjected to trypsin digestion and RP-HPLC analysis, resulting in a delayed retention time and an increased mass (173 Da) of T1 tryptic peptide. The amino acid composition and amino acid sequence of the peptide showed the same result with T1 peptide of met-hGH except the N-terminal modification on methionine in the variant peptide. With collision induced dissociation (CID) experiments of the variant T1 tryptic peptide, we found the sequence and the a 1 fragment of N-terminal residue matched with those of acetyl-methionyl hGH. Within our production process, we produce the methionyl hGH first and then use the aminopeptidase to cut the N-terminal methionine. So the acetylation may inhibit the aminopeptidase to remove methionine and produces N α-acetyl methionyl hGH. And the biological activity of the variant was comparable to one of the unmodified hGH when tested by rat weight gain bioassay.

Long-lasting rat growth enhancement by an immunoregulating synthetic peptide

We have studied the long-lasting effects on rat weight gain of an active immunization with a peptide construction consisting of the covalent linkage of the 104–113 GH sequence to the 323–339 sequence of ovalbumin and to adjuvant muramyl dipeptide (MDP). The 104–113 GH sequence has already been identified as a potential epitope capable of enhancing growth hormome (GH) activity when complexed with the specific antibodies. Our results show that: a) 104–113 peptide antibodies after reacting with the endogenous GH produced a weight gain up to 7% higher than that observed in unimmunized rats; and b) the position of the adjuvant sequence in the molecule was critical for the immune response. Antibodies elicited in hypophysectomised rats had no effect on weight gain, thus confirming that the increase in hormonal activity is actually linked to the binding of specific peptide antibodies to endogenous GH.

A novel bioassay based on human growth hormone (hGH) receptor mediated cell proliferation: measurement of 20K-hGH and its modified forms.

Previously we introduced the full-length hGH receptor (hGHR) into the mouse pro-B cell line, Ba/F3, and obtained stable transfectant (Ba/F3-hGHR), which could grow in response to 20K- and 22K-hGH in a dose-dependent manner(1). In the present study, we established a new bioassay system based on the proliferation of the Ba/F3-hGHR in combination with the eluted stain assay (ESTA). The Ba/F3-hGHR assay is completed in 18 h and requires only 10(-6)-fold amount of GH sample (1.8 ng) as compared with the rat weight gain assay. The validation study shows that the Ba/F3-hGHR assay is specific for hGH, precise (RSD = 1.1-19.7%) and ultrasensitive (lower limit of working range = 18.7 pg/mL). Four modified forms of recombinant 20K-hGH (oxidized, deamidated, des-Phe(1)and cleaved form) all of which are newly identified were measured by the Ba/F3-hGHR assay and the rat weight gain assay with our in-house recombinant 20K-hGH as standard. The oxidized and deamidated 20K-hGH were fully active, however the des-Phe(1)and cleaved 20K-hGH had significantly reduced activities in both assays. These findings suggest that the Ba/F3-hGHR assay is useful as an alternative to the rat weight gain assay.

Cellular Activities of 20K- and 22K-hGH Do Not Necessarily Correlate with Their Binding Affinities for Rat GH Receptor

Even though 20K human growth hormone (20K-hGH) has 3–10% binding affinity for the rat liver and adipose tissue microsomes as compared to 22K-hGH, it was also reported that 20K-hGH has the same potency as 22K-hGH in the hypophysectomized rat weight gain assay. In order to investigate the reason why such controversial data exist, we have studied 20K- and 22K-hGH using the rat GH receptor extracellular domain (rGHR-ECD) and full-length rGHR. When we examined the complex formation of rGHR-ECD with 20K- and 22K-hGH in gel filtration assay, 20K-hGH formed no complex while 22K-hGH formed a 1:1 complex. Next, rGHR cDNA was introduced into Ba/F3 cells and CHO-K1 cells, and stable transfectants (Ba/F3-rGHR and CHO-rGHR) were established. In the proliferation of Ba/F3-rGHR cells, 20K-hGH had 10-fold lower activity than 22K-hGH, which is consistent with their affinities for rGHR. But surprisingly, in the Spi2.1 gene promoter activation in CHO-rGHR cells, 20K- and 22K-hGH had the same activity, which was found not only in stable CHO-rGHR clones but also in CHO-K1 cells transiently expressing rGHR. In conclusion, these results indicate that cellular activities of 20K- and 22K-hGH do not necessarily correlate with their binding affinities for rGHR.

Novel Assays Based on Human Growth Hormone Receptor as Alternatives to the Rat Weight Gain Bioassay for Recombinant Human Growth Hormone

Abstract.Two methods, High-Performance Receptor Binding Chromatography (HPRBC) and Cell Proliferation (CP), have been developed as alternatives to the classical hypophysectomized rat weight gain bioassay for the determination of potency for recombinant human growth hormone (rhGH). In the HPRBC assay, rhGH is combined with an excess of the soluble extracellular domain of the recombinant human growth hormone receptor (referred to as ‘receptor’ in the discussion of the HPRBC assay). Nondenaturing size-exclusion chromatography is used to analyze the resulting complex, which forms in a 2:1 receptor to rhGH ratio. The 2:1 complex is assayed at a concentration near the K d(0.4 nM), providing high specificity for rhGH and detection of rhGH variants with reduced activity. In the CP assay, a mouse myeloid leukaemia cell line (FDC—P1) transfected with the full-length receptor is exposed to varying levels of rhGH for 16–20 h. The incorporation of 3H-thymidine into DNA is used as an index of cell proliferation. The results show that the HPRBC assay provides significantly improved precision with a relative standard deviation (RSD) of ≤5% vs.an RSD of 23% for the rat bioassay. The CP assay has RSDs of 4–16% . Analysis of rhGH variants and mutants shows that the potencies measured by both the HPRBC and CP assays are in general agreement with the rat weight gain bioassay. Both of the HPRBC and CP assays are sufficiently rugged for operating in a Good Manufacturing Practices (GMP) routine batch release testing environment. In vitroalternatives such as the HPRBC and CP assays build a foundation for replacing the hypophysectomized rat weight gain bioassay by correlating receptor dimerization, binding specificity and signal transduction with the biological activity of rhGH.

Properties of a cleaved two‐chain form of recombinant human growth hormone

Escherichia coli cells transformed with plasmids engineered for the expression of recombinant human growth hormone as a secreted product also produced a proteolytically cleaved form of rhGH. This variant is isolated at a high resolution anion exchange chromatography stage during the manufacturing process. The higher isoelectric point of this form is demonstrated by isoelectric focusing and chromatofocusing and the two-chain nature by tryptic mapping, N- and C-terminal sequence analyses, and sodium dodecyl sulfate polyacrylamide gel electrophoresis. These data indicate that the single site of cleavage is between Thr-142 and Tyr-143, in contrast to the two-chain variant isolated from human pituitary glands, which has a clip after residue Phe-139. The recombinant two-chain form was further characterized by reversed-phase high performance liquid chromatography at both acidic and basic pHs. The assay utilizing bicarbonate-containing mobile phases was determined to be the most efficient and sensitive method. The bioactivity of this two-chain form was measured by the in vivo rat weight gain assay and by the in vitro Nb2 cell bioassay. Its immunological similarity to intact one-chain rhGH was demonstrated with an enzyme-linked immunosorbent assay.

Effects of Germination and Dietary Calcium on Zinc Bioavailability from Peas

ABSTRACTBioavailability of zinc was determined by rat weight gain and tibia Zn response from whole pea sprouts (WPS), heat treated peas (HTP) and purified diets (PD1 and 2) supplemented with Zn and phytate for similar phytate/Zn molar ratios (phy/Zn). With diets adequate Ca (0.75%), weight gains from WPS (phy/Zn = 24) and HTP (phy/Zn = 39) were equivalent or better to a nonphytate control (NPC). Tibia Zn decreased in order from: NPC, WPS, PD1, HTP and PD2. At the 1.5% Ca level reduced growth responses from WPS and HTP indicated interaction between Ca and phytate. Zn availability from peas may be superior to that from grains and other legumes previously reported. The decreased phytate and increased phytase resulting from germination may explain the enhanced responses from pea sprouts.