In Vitro Stability of Human Fibrinopeptide B

1979 ◽  
Vol 42 (04) ◽  
pp. 1316-1323
Author(s):  
G D Qureshi ◽  
William C Vennart
Keyword(s):  
Molar Ratio ◽  
Temperature Dependent ◽  
Carboxypeptidase B ◽  
Enzyme Substrate ◽  
Substrate Molar Ratio ◽  
Normal Urine ◽  
The Stability ◽  
In Vitro Stability ◽  
Plasma Ultrafiltrate

SummaryIn anticipation of a future clinical application of plasma fibrinopeptide B (FPB) measurement, we studied the stability of FPB in an ultrafiltrate of normal plasma, normal urine and alkaline buffer by measuring the immunoreactivity of the peptide by FPB radioimmunoassay using anti FPB serum (R-29). FPB was unstable in an ultrafiltrate of plasma and urine and demonstrated a temperature dependent loss of activity. In plasma ultrafiltrate the loss of immunoreactivity was not significant during the first 24 hours, however, 92% of the peptide activity was lost at the end of seven days at 25° C and 37° C. The rate of FPB degradation in urine was comparable. The peptide was stable in an alkaline buffer (pH 8.5) at temperatures ranging from �10° C to 37° C or in plasma ultrafiltrate or urine when incubated at �10° C. Treatment with carboxypeptidase B or leucine aminopep- tidase for two hours at 37° C (enzyme/substrate molar ratio of up to 1:100) did not cause a loss of FPB immunoreactivity. EDTA (1.0 mM) and Trasylol (500 units/ml) completely stabilized the peptide in a plasma ultrafiltrate.

Kinetics of Various 99mTc-Sn-Pyrophosphate Compounds in the Rat

1977 ◽  
Vol 16 (04) ◽  
pp. 157-162 ◽  
Author(s):  
C. Schümichen ◽  
B. Mackenbrock ◽  
G. Hoffmann
Keyword(s):  
Normal Saline ◽  
Half Life ◽  
Compound A ◽  
Short Half Life ◽  
Low Concentrations ◽  
The Stability ◽  
Kinetics Of ◽  
In Vitro Stability

SummaryThe bone-seeking 99mTc-Sn-pyrophosphate compound (compound A) was diluted both in vitro and in vivo and proved to be unstable both in vitro and in vivo. However, stability was much better in vivo than in vitro and thus the in vitro stability of compound A after dilution in various mediums could be followed up by a consecutive evaluation of the in vivo distribution in the rat. After dilution in neutral normal saline compound A is metastable and after a short half-life it is transformed into the other 99mTc-Sn-pyrophosphate compound A is metastable and after a short half-life in bone but in the kidneys. After dilution in normal saline of low pH and in buffering solutions the stability of compound A is increased. In human plasma compound A is relatively stable but not in plasma water. When compound B is formed in a buffering solution, uptake in the kidneys and excretion in urine is lowered and blood concentration increased.It is assumed that the association of protons to compound A will increase its stability at low concentrations while that to compound B will lead to a strong protein bond in plasma. It is concluded that compound A will not be stable in vivo because of a lack of stability in the extravascular space, and that the protein bond in plasma will be a measure of its in vivo stability.

scholarly journals A Pretargeted Imaging Strategy for EGFR Positive Colorectal Carcinoma via the Modulation of Tz-radioligand Pharmacokinetics

2020 ◽  
Author(s):  
Lin Qiu ◽  
Hui Tan ◽  
Qingyu Lin ◽  
Zhan Si ◽  
Jun Zhou ◽  
...  
Keyword(s):  
Tumor Imaging ◽  
Molecular Probes ◽  
Ideal Situation ◽  
Imaging Results ◽  
Imaging Strategy ◽  
Xenograft Mice ◽  
The Stability ◽  
In Vitro Stability

Abstract Objective: Previously, we successfully developed a pretargeted imaging strategy (Atezolizumab-TCO/99mTc-HYNIC-PEG11-Tz), which is a powerful tool for evaluating Programmed Cell Death Ligand-1 (PD-L1) expression in xenograft mice tumor models. However, the surplus unclicked 99mTc-HYNIC-PEG11-Tz is cleared somewhat sluggishly through the intestines. This is certainly not an ideal situation for imaging for colorectal cancer (CRC). In order to shift the excretion of the Tz-radioligand to the renal system, we have sought to develop a novel Tz-radioligand by adding a polypeptide linker between HYNIC and PEG11. Methods: Pretargeted molecular probes 99mTc-HYNIC-Polypeptide-PEG11-Tz and Cetuximab-TCO were synthesized. The stability of 99mTc-HYNIC-Polypeptide-PEG11-Tz was evaluated in vitro, and its blood pharmacokinetic test was performed in vivo. In vitro ligation reactivity of 99mTc-HYNIC-Polypeptide-PEG11-Tz towards Cetuximab-TCO was tested. The biodistribution and imaging of 99mTc-HYNIC-Polypeptide-PEG11-Tz was performed to observe the clear pathway of this novel Tz-radioligand. Pretargeted biodistribution of three different accumulation intervals was performed to determine the optimal pretargeted interval time. Comparison of pretargeted (Cetuximab-TCO 48 h/99mTc-HYNIC-PEG11-Tz 6 h) and (Cetuximab-TCO 48 h/99mTc-HYNIC-Polypeptide-PEG11-Tz 6 h) imagings was performed to show the effect of the two Tz-radioligands with different excretion pathway on tumor imaging. Results: 99mTc-HYNIC-Polypeptide-PEG11-Tz showed favorable in vitro stability and rapid blood clearance in mice. SEC-HPLC revealed almost complete reaction between Cetuximab-TCO and 99mTc-HYNIC-Polypeptide-PEG11-Tz in vitro, with the 8:1 Tz-to-mAb reaction providing a conversion yield of 87.83 ± 3.27%. The biodistribution and imaging of 99mTc-HYNIC-Polypeptide-PEG11-Tz demonstrated that the Tz-radioligand was cleared through kidneys. After allowing 24 h, 48 h and 72 h for accumulation of Cetuximab-TCO in HCT116 tumor, pretargeted biodistribution revealed the tumor-to-blood ratio was 0.83 ± 0.13, 1.40 ± 0.31, and 1.15 ± 0.21, respectively. Both pretargeted (Cetuximab-TCO 48 h/99mTc-HYNIC-PEG11-Tz 6 h) and (Cetuximab-TCO 48 h/99mTc-HYNIC-Polypeptide-PEG11-Tz 6 h) imaging delineated the HCT116 tumor clearly. However, pretargeted imaging strategy using Cetuximab-TCO/99mTc-HYNIC-Polypeptide-PEG11-Tz could be used for diagnosing CRC since the surplus unclicked 99mTc-HYNIC-Polypeptide-PEG11-Tz is cleared through urinary system and produces low abdominal uptake background. Conclusion: We developed a novel pretargeted imaging strategy (Cetuximab-TCO/99mTc-HYNIC-Polypeptide-PEG11-Tz) for imaging CRC since the surplus unclicked 99mTc-HYNIC-Polypeptide-PEG11-Tz produces low abdominal uptake background, which broadens the application scope of pretargeted imaging strategy.

The Effects of the Spacer on Radiochemical and Biological Properties of New Radiolabeled Bombesin(7-14) Derivative

2020 ◽  
Vol 13 (2) ◽  
pp. 149-158
Author(s):  
Farzaneh Rezazadeh ◽  
Sara Karoubian ◽  
Saied Abediankenari ◽  
Nourollah Sadeghzadeh ◽  
Manouchehr Jandaghi ◽  
...  
Keyword(s):  
Radiochemical Purity ◽  
Renal Excretion ◽  
Specific Binding ◽  
Biological Properties ◽  
Radiolabeled Peptides ◽  
The Stability ◽  
Grp Receptors ◽  
In Vitro Stability ◽  
Grp Receptor

Objective: The aim of this study was to develop 99mTc-[HYNIC-X-D-Phe13]-BBN(7-14)NH2 derivatives using two different tripeptidic spacer groups (X=GGG and X=SSS) in order to improve its pharmacokinetics, in vitro stability, specific binding, and affinity. Background: Bombesin (BBN), a 14-aminoacid amphibian peptide homolog of mammalian gastrinreleasing peptide (GRP), has demonstrated the ability to bind with high affinity and specificity to GRP receptor, which is overexpressed on a variety of human cancers. Methods: Peptide conjugates labeled with 99mTc using tricine-EDDA and radiochemical purity was assessed by TLC and HPLC. The stability of radio conjugates was evaluated in the presence of saline and human serum. Affinity, internalization, and also dissociation Constant was evaluated using MDAMB- 231 and PC-3 cell line. Biodistribution study was performed in BALB/C mice. Results: Labeling yield of ˃95% was obtained. The change introduced in the BBN sequence increased plasma stability. In vitro blocking studies showed that binding and internalization of both radiolabeled peptides are mediated by their receptors on the surface of MDA-MB-231 and PC-3 cells. Biodistribution results demonstrated a rapid blood clearance, with predominantly renal excretion. Specific binding in GRP receptor-positive tissues, such as pancreas was confirmed with a blocking study. Conclusions: The introduction of the spacer sequence between chelator and BBN(7-14) led to improved bidistribution. Analog with tri-Gly spacer is the more promising radiopeptide for targeting GRP receptors than Ser conjugates. : Therefore, these analogs can be considered as a candidate for the identification of bombesin-positive tumors.

Role of high-affinity folate-binding protein in the plasma distribution of tetrahydrofolate in pigs

1996 ◽  
Vol 270 (1) ◽  
pp. R105-R110 ◽  
Author(s):  
K. Sasaki ◽  
M. Natsuhori ◽  
M. Shimoda ◽  
Y. Saima ◽  
E. Kokue
Keyword(s):  
Protein Binding ◽  
Binding Capacity ◽  
Binding Protein ◽  
Folate Binding Protein ◽  
High Affinity ◽  
Plasma Distribution ◽  
The Stability ◽  
Plasma Ultrafiltrate

Stability and protein-binding properties of tetrahydrofolate (THF) in pig plasma were studied in vitro. THF in plasma was stable for more than 120 min when it existed in a bound form, whereas THF both in plasma ultrafiltrate and in plasma ultrafiltrate plus porcine albumin was degraded rapidly and disappeared soon after its addition. These results suggest that high-affinity folate-binding protein (HFBP) is related to the stability of THF. THF-protein binding kinetic analysis showed that porcine plasma had HFBP and low-affinity binding protein (albumin) for THF. Dissociation constant and maximal binding capacity of HFBP were calculated to be 0.4 and 70 nM, respectively, indicating that > 98% of endogenous plasma THF existed in bound form with HFBP. Porcine albumin was not essentially a protein that binds and protects endogenous THF from degradation. We conclude that most endogenous THF binds to HFBP and only the unbound form of THF is rapidly degraded in pig plasma. HFBP protects THF from degradation and allows THF to exist stably in pig plasma. In addition, HFBP may govern the species specificity of plasma folate distribution in pigs.

Effect of Acid Pretreatment on the Stability of EDTA, Cysteine, Lactic and Succinic Acid Complexes of Various Iron Sources in a Wheat Flake Cereal

1987 ◽  
Vol 50 (7) ◽  
pp. 587-597
Author(s):  
D. B. NADEAU ◽  
F. M. CLYDESDALE
Keyword(s):  
Succinic Acid ◽  
Ferrous Sulfate ◽  
Iron Complex ◽  
Molar Ratio ◽  
Acid Pretreatment ◽  
Gastrointestinal Ph ◽  
Iron Solubilization ◽  
The Stability ◽  
Reducing Potential

An in vitro incubation at pH 2 of EDTA, cysteine, lactic or succinic acids with each of five iron sources, [hydrogen (HRI) and electrolytically reduced elemental iron (ERI), ferric chloride (FeCl3), ferrous sulfate (FeSO4) and ferric orthophosphate (FOP)] at a 10:1 molar ratio (ligand:iron) was evaluated for its effect on iron solubilization in a wheat flake cereal subjected to a sequential gastrointestinal pH treatment from endogenous pH (E) to 2 to 6. Incubation significantly enhanced the iron solubilizing potential of EDTA at each pH with HRI and ERI, while lactic and succinic acids were similarly effective with FeSO4 and FeCl3 at pH 2. The reducing potential of cysteine, along with its role as a ligand, generated substantial amounts of Fe+ 2 (pH 2) at the apparent expense of complexed iron. However, with the exception of ERI (pH E), incubation did not increase cysteine's effectiveness in producing more soluble iron (ionic + complexed). This indicates that pH was the major solubilizing factor. Due to FOP's relative insolubility, incubation proved ineffective in all instances. These in vitro results indicate that acid incubation to form a ligand-iron complex has the potential to improve bioavailability of iron.

scholarly journals In Vitro Study of MefenamateStarch as Drug Delivery System

2013 ◽  
Vol 10 (3) ◽  
pp. 954-964
Author(s):  
Baghdad Science Journal
Keyword(s):  
In Vitro Study ◽  
Controlled Drug Release ◽  
Gastric Fluid ◽  
Molar Ratio ◽  
Ph Values ◽  
Ft Ir ◽  
Uv Visible ◽  
The Stability ◽  
Hydrolysis Of

Mefenamic acid was esterified with starchwith[1:1] Molar ratio, as drug substituted with natural polymer, to prolongthe period of hydrolysis of drug polymer with other advantages. The new prodrug starch was characterized by FT-IR and UV-Visible and 1H-NMR spectroscopies. The physical properties were studied and controlled drug release was studied in different pH values at 37oC. The stability of drug was carried out by measuring the absorbance of mefenamic starch which hydrolyzed in HCl solution of pH 1.1 (artificial gastric fluid) and phosphate buffer of pH 7.4 (simulating intestinal fluid SIF) at 37oC for several days. The thermal analysis such as DSC was studied.

scholarly journals Distribution of manganese in rat pancreas and identification of its primary binding protein as pro-carboxypeptidase B

1991 ◽  
Vol 278 (3) ◽  
pp. 857-862 ◽  
Author(s):  
H Kodama ◽  
N Shimojo ◽  
K T Suzuki
Keyword(s):  
Binding Protein ◽  
Gel Filtration ◽  
Chemical Characterization ◽  
Molar Ratio ◽  
Emission Spectrometry ◽  
Repeated Injection ◽  
Plasma Atomic Emission Spectrometry ◽  
Carboxypeptidase B ◽  
Rat Pancreas

Distribution of manganese (Mn) and its binding to specific proteins were examined in rat pancreas. A MnCl2 solution was injected subcutaneously into Wistar rats daily at a single dose of 15 mg of Mn/kg body weight for 10 days and the animals were killed 1 day after the last injection. The concentration of Mn in the pancreas increased considerably from 1.4 +/- 0.2 (control) to 13.3 +/- 3.7 micrograms/g wet tissue by the repeated injection of Mn. The distribution of Mn in the soluble fraction of the pancreas (170,000 g supernatant) was determined on a gel-filtration column (Asahipak GST-520) using an h.p.l.c.-inductively coupled argon plasma atomic-emission spectrometry (i.c.p.) technique. The metal was eluted as a single peak in the high-molecular-mass protein fraction, where Mn had been observed as a small peak in the control profile, suggesting that the administered Mn was bound to the same Mn-binding component as that in the control. On the basis of enzymic and chemical characterization of the protein, it was identified as a zymogen of carboxypeptidase B (pro-carboxypeptidase B, pro-CPB). The elution profiles of the protein by h.p.l.c.-i.c.p. indicated that Mn and zinc (Zn) were bound to the zymogen with a molar ratio of 1:4 in normal rat pancreas. Mn bound to the zymogen was easily replaced by Zn in vitro, suggesting that Mn was bound to the Zn-binding site and that the binding affinity to Zn was higher than that to Mn. The present results indicate that pro-CPB is the primary Mn-binding protein in the pancreas of control and also Mn-administered rats.

scholarly journals Co-Ingestion of Black Carrot and Strawberry. Effects on Anthocyanin Stability, Bioaccessibility and Uptake

Foods ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 1595
Author(s):  
Celia Carrillo ◽  
Senem Kamiloglu ◽  
Charlotte Grootaert ◽  
John Van Camp ◽  
Marc Hendrickx
Keyword(s):  
Cell Model ◽  
In Vitro Digestion ◽  
Food Sources ◽  
Food Matrix ◽  
Reciprocal Effect ◽  
Black Carrot ◽  
The Stability ◽  
Real Scenario ◽  
In Vitro Stability

Although the fate of anthocyanins along digestion has been a matter of research over the last decade, their bioaccessibility so far has been mainly assessed for single administered fruits or vegetables, which is far from the real scenario where they are co-ingested in a meal. Accordingly, the aim of this study was to evaluate the effect of simultaneous intake of fruit and vegetable on in vitro stability, bioaccessibility and uptake of anthocyanins. Black carrot and strawberry were used as food sources of anthocyanins. Anthocyanin identification and quantification were performed using HPLC-Qtof/HPLC-UV. Single matrices and mixtures thereof, were submitted to a standardized in vitro digestion procedure. Anthocyanin uptake was evaluated through an intestinal Caco-2 cell model. Our results showed an increased intestinal stability for specific anthocyanins as a consequence of co-digestion. The presence of the strawberry food matrix positively affected the bioaccessibility of the carrot associated cyanidin-based anthocyanins, whereas no reciprocal effect was observed for pelargonidin-based derivatives in the presence of the black carrot food matrix. Anthocyanin transport was maintained after co-administration. Overall, co-ingestion of black carrot and strawberry did not negatively affect the stability, bioaccessibility or uptake of cyanidin-based anthocyanins, although the effect on pelargonidin-based anthocyanins depended on the type of pelargonidin derivative.

In-vitro stability of human alpha-fetoprotein.

1985 ◽  
Vol 31 (10) ◽  
pp. 1692-1697 ◽  
Author(s):  
J T Wu ◽  
J A Knight
Keyword(s):  
Incubation Temperature ◽  
Enzyme Inhibitor ◽  
Alpha Fetoprotein ◽  
Proteolytic Enzyme Inhibitor ◽  
Different Temperatures ◽  
Normal Human ◽  
The Stability ◽  
Phosphate Buffered Saline ◽  
In Vitro Stability

Abstract We assessed the stability of alpha-fetoprotein (AFP) in clinical specimens in the presence and absence of serum and albumin, at different temperatures and concentrations. We find it depends on both AFP concentration and incubation temperature. Dilution of most specimens with either phosphate buffer or phosphate-buffered saline or by immunoelectrodiffusion resulted in some loss of AFP. Attempts to stabilize AFP during either sample dilution or incubation by use of albumin in concentrations up to 1 g/L did not protect it from inactivation unless normal human serum was also included. Frozen AFP solutions were less stable than solutions stored at 4 degrees C. AFP was most stable when lyophilized and stored desiccated. The AFP-inactivation curves were usually nonlinear. Apparently both polymerization and degradation occur simultaneously as AFP loses its activity. Proteolytic enzyme inhibitor and sulfhydryl reagent not only failed to protect it from inactivation, they appeared to speed it.

Purification and characterization of ensconsin, a novel microtubule stabilizing protein

1994 ◽  
Vol 107 (10) ◽  
pp. 2839-2849 ◽  
Author(s):  
J.C. Bulinski ◽  
A. Bossler
Keyword(s):  
Biochemical Characterization ◽  
Cell Types ◽  
Molar Ratio ◽  
Purification And Characterization ◽  
Protein Map ◽  
Tight Association ◽  
The Stability

In previous studies (Bulinski and Borisy (1979). Proc. Nat. Acad. Sci. 76, 293–297; Weatherbee et al. (1980). Biochemistry 19, 4116–4123) a microtubule-associated protein (MAP) of M(r) approximately 125,000 was identified as a prominent MAP in HeLa cells. We set out to perform a biochemical characterization of this protein, and to determine its in vitro functions and in vivo distribution. We determined that, like the assembly-promoting MAPs, tau, MAP2 and MAP4, the 125 kDa MAP was both proteolytically sensitive and thermostable. An additional property of this MAP; namely, its unusually tight association with a calcium-insensitive population of MTs in the presence of taxol, was exploited in devising an efficient purification strategy. Because of the MAP's tenacious association with a stable population of MTs, and because it appeared to contribute to the stability of this population of MTs in vitro, we have named this protein ensconsin. We examined the binding of purified ensconsin to MTs; ensconsin exhibited binding that saturated its MT binding sites at an approximate molar ratio of 1:6 (ensconsin:tubulin). Unlike other MAPs characterized to date, ensconsin's binding to MTs was insensitive to moderate salt concentrations (< or = 0.6 M). We further characterized ensconsin in immunoblotting experiments using mouse polyclonal anti-ensconsin antibodies and antibodies reactive with previously described MAPs, such as high molecular mass tau isoforms, dynamin, STOP, CLIP-170 and kinesin. These experiments demonstrated that ensconsin is distinct from other proteins of similar M(r) that may be present in association with MTs. Immunofluorescence with anti-ensconsin antibodies demonstrated that ensconsin was detectable in association with most or all of the MTs of several lines of human epithelial, fibroblastic and muscle cells; its in vivo properties and distribution, especially in response to drug or other treatments of cells, were found to be different from those of MAP4, the predominant MAP found in these cell types. We conclude that ensconsin, a MAP found in a variety of human cells, is biochemically - and perhaps functionally - distinct from other MAPs present in non-neuronal cells.

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