Thyrotropin-releasing hormone immunoreactivity in human blood, urine, spinal fluid, amniotic fluid, saliva, and gastric juice

1987 ◽  
Vol 114 (4) ◽  
pp. 552-558 ◽  
Author(s):  
Anna-Stina Suhonen-Malm
Keyword(s):  
Amniotic Fluid ◽  
Gastric Juice ◽  
Human Blood ◽  
Biological Fluid ◽  
Biological Fluids ◽  
Exchange Chromatography ◽  
Spinal Fluid ◽  
Reverse Phase ◽  
Blood And Urine Samples ◽  
Radio Immunoassay

Abstract. TRH immunoreactivity levels were measured in human blood, urine, saliva, spinal fluid, amniotic fluid and gastric juice. Urinary TRH excretion during a 48-h period was measured in 11 healthy persons. Blood and urinary TRH immunoreactivity were measured at 2 and 5 h, respectively, after administration of 40 mg of TRH. All the samples were prepurified by SP-Sephadex-C-25 cation-exchange chromatography and subjected to reverse-phase high-pressure liquid chromatography (HPLC). TRH immunoreactivity levels were then measured by our TRH radio-immunoassay. The TRH immunoreactivity (TRH-ir) levels found in urine were 14.6 ± 2.5 pmol/l; in blood 7.5 ± 2.0 pmol/l; in spinal fluid 2.8 ± 1.4 pmol/l, and in gastric juice 23.2 ± 7.1 pmol/l. In all of the amniotic fluid and saliva samples, in almost one half of the blood and spinal fluid samples, and in almost one third of the gastric juice samples, TRH-ir was below the detectable limit. In blood and urine samples taken after oral administration of TRH, TRH-ir was eluted at the same time as synthetic TRH. The recovery of synthetic TRH added to the samples ranged from 36 to 99%. In all of the biological fluid samples, endogenous TRH-ir was eluted at the same time in HPLC, at 15–18 min, as was synthetic TRH which had been added to the samples. Urine was found to contain two TRH immunoreactive peaks, the second of which was eluted at the same time as synthetic TRH. No diurnal variation in urinary TRH excretion or TRH-ir levels was found. The levels of TRH-ir measured in blood, urine, spinal fluid and amniotic fluid are lower than most of those reported earlier. This study suggests that our method can be used to measure TRH immunoreactivity more accurately in a variety of biological fluids.

Preparation and Characterization of NH2-Terminal Fibrinogen Bβ Fragments from N-DSK of Human Fibrinogen

1984 ◽  
Vol 51 (01) ◽  
pp. 016-021 ◽  
Author(s):  
S Birken ◽  
G Agosto ◽  
B Lahiri ◽  
R Canfield
Keyword(s):  
High Performance ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Reverse Phase ◽  
Human Fibrinogen ◽  
Human Volunteers ◽  
Cnbr Cleavage ◽  
Two Peaks

SummaryIn order to investigate the early release of NH2-terminal plasmic fragments from the Bβ chain of fibrinogen, substantial quantities of Bβ 1-42 and Bβ 1-21 are required as immunogens, as radioimmunoassay standards and for infusion into human volunteers to determine the half-lives of these peptides. Towards this end methods that employ selective proteolytic cleavage of these fragments from fibrinogen have been developed. Both the N-DSK fragment, produced by CNBr cleavage of fibrinogen, and Bβ 1-118 were employed as substrates for plasmin with the finding of higher yields from N-DSK. Bβ 1-42 and Bβ 1-21 were purified by gel filtration and ion-exchange chromatography on SP-Sephadex using volatile buffers. When the purified preparation of Bβ 1-42 was chromatographed on reverse-phase high performance liquid chromatography, two peaks of identical amino acid composition were separated, presumably due either to pyroglutamate or to amide differences.

Review on the Application of Mixed-mode Chromatography for Separation of Structure Isoforms

2018 ◽  
Vol 20 (1) ◽  
pp. 56-60 ◽  
Author(s):  
Tsutomu Arakawa
Keyword(s):  
Ion Exchange ◽  
Mixed Mode ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Charged State ◽  
Partial Structure ◽  
Reverse Phase ◽  
Oligomer Formation ◽  
Structure Rearrangement ◽  
Disulfide Scrambling

Proteins often generate structure isoforms naturally or artificially due to, for example, different glycosylation, disulfide scrambling, partial structure rearrangement, oligomer formation or chemical modification. The isoform formations are normally accompanied by alterations in charged state or hydrophobicity. Thus, isoforms can be fractionated by reverse-phase, hydrophobic interaction or ion exchange chromatography. We have applied mixed-mode chromatography for fractionation of isoforms for several model proteins and observed that cation exchange Capto MMC and anion exchange Capto adhere columns are effective in separating conformational isoforms and self-associated oligomers.

Prenatal diagnosis of Duchenne muscular dystrophy by radio-immunoassay of myoglobin in amniotic fluid

2008 ◽  
Vol 21 (5) ◽  
pp. 354-355 ◽  
Author(s):  
O. Török ◽  
K. Norregaard-Hansen ◽  
M. Szokol ◽  
K. Csécsei ◽  
A. Harsányi ◽  
...  
Keyword(s):  
Prenatal Diagnosis ◽  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Amniotic Fluid ◽  
Radio Immunoassay

scholarly journals Human Glandular Kallikrein in Breast Milk, Amniotic Fluid, and Breast Cyst Fluid

1999 ◽  
Vol 45 (10) ◽  
pp. 1774-1780 ◽  
Author(s):  
Angeliki Magklara ◽  
Andreas Scorilas ◽  
Carlos López-Otín ◽  
Francisco Vizoso ◽  
Alvaro Ruibal ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Amniotic Fluid ◽  
Biological Fluids ◽  
Cyst Fluid ◽  
Cystic Disease ◽  
Breast Cyst Fluid ◽  
Breast Cyst ◽  
Glandular Kallikrein ◽  
Female Breast

Abstract Background: Human glandular kallikrein (hK2) belongs to the serine protease family of enzymes and has high sequence homology with prostate-specific antigen (PSA). The physiological role of hK2 has not as yet been determined, but there is evidence that it can regulate the proteolytic activity of PSA through processing and activating pro-PSA, an inactive precursor. Thus, it is conceivable that these two secreted proteins may coexist in biological fluids. Currently, hK2 is considered an androgen-regulated and prostate-specific protein. Recently, it has been demonstrated that hK2 is expressed in the breast cancer cell line T-47D after stimulation by steroid hormones, and we reported that hK2 can be detected in a subset of breast tumor extracts. These data suggest that hK2 may be expressed in tissues other than the prostate, such as those in which PSA has already been detected. Because hK2 is a secreted protein, it may be present in various biological fluids. Methods: We analyzed milk samples from lactating women, amniotic fluid from pregnant women, and breast cyst fluid from patients with gross breast cystic disease, using a highly sensitive and specific immunoassay for hK2. Results: hK2 was present in all three biological fluids. We suggest that the female breast may produce hK2 and provide evidence that hK2 may have value as an additional marker for the discrimination between type I and type II breast cysts. Conclusions: The female breast produces hK2 in addition to PSA. More studies are necessary to establish the role of this kallikrein in nondiseased breast, gross breast cystic disease, and breast cancer.

Clinical Determination of Methemalbumin

1975 ◽  
Vol 21 (10) ◽  
pp. 1506-1510 ◽  
Author(s):  
Scott F Andres ◽  
Thomas J Kregoski ◽  
Charles F Frey ◽  
Ramon R Joseph ◽  
Daisy S McCann
Keyword(s):  
Colorimetric Assay ◽  
Biological Fluids ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Original Sample ◽  
Initial Screening ◽  
Albumin Fraction ◽  
Hemorrhagic Pancreatitis ◽  
Clinical Determination

Abstract We developed an assay for methemalbumin in biological fluids by using diethylaminoethyl-Sephadex ion-exchange chromatography to separate this protein from interfering components, including hemopexin, transferrin, hemoglobin, and haptoglobin/hemoglobin complex. Initial screening of the samples requires measurement of A280/A405 ratios of the peak tubes of the isolated albumin fraction. Values exceeding 30 indicate that methemalbumin is absent, and no further work is required. Values of less than 30 suggest that methemalbumin is present in the original sample, whereupon the presence and amount of methemalbumin can be ascertained by colorimetric assay for iron with use of ferrozine. Results may be expressed either in terms of micrograms of methemalbumin iron per gram of albumin or in milligrams of methemalbumin per liter. The reproducibility of the method is of the order of ±7% (SD). Normal persons have essentially no methemalbumin iron in their serum. Three individuals with hemorrhagic pancreatitis showed values of 65, 98, and 198 µg of methemalbumin Fe per gram of albumin.

Automated analysis for taurine in biological fluids and tissues.

1987 ◽  
Vol 33 (6) ◽  
pp. 835-837 ◽  
Author(s):  
H O Goodman ◽  
Z K Shihabi
Keyword(s):  
Ion Exchange ◽  
Continuous Flow ◽  
Biological Fluids ◽  
Automated Analysis ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Fluorescent Compound ◽  
Automated Method ◽  
Tissue Homogenates ◽  
Proteins And Peptides

Abstract We have developed an automated method of analysis for taurine, based on incorporating an ion-exchange chromatography column into the continuous-flow AutoAnalyzer (Technicon). After removal of proteins and peptides by dialysis, taurine is selectively eluted from an ion-exchange column and reacted with o-phthaldialdehyde to yield a fluorescent compound. The advantages of this method are: full automation with no need for sample deproteinization or cleanup; sensitivity, detecting as little as 5 mumol/L; speed (20 samples per hour); and flexibility. It can be used for assaying taurine in urine, plasma, cerebrospinal fluid, and tissue homogenates. This method can be adapted for assays of other metabolites.

Effect of Deproteinating Agents (Picric Acid and Sulfosalicylic Acid) on Analysis for Free Amino Acids in Swine Blood and Tissue

1969 ◽  
Vol 52 (5) ◽  
pp. 981-984 ◽  
Author(s):  
J E Knipfel ◽  
D A Christensen ◽  
B D Owen
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Ion Exchange ◽  
Free Amino ◽  
Biological Fluids ◽  
Picric Acid ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Sulfosalicylic Acid ◽  
Coefficients Of Variation

Abstract Amino acid analyses were performed on samples of blood, liver tissue, loin muscle, and ham muscle by ion exchange chromatography after deproteination of the samples with picric acid or sulfosalicylic acid (SSA). Resolution of threonine and serine from the ion exchange column was poor when SSA was used as the deproteinating agent. Twelve of sixteen amino acids were higher (P < 0.05) in serum deproteinated with picric acid as compared to concentrations determined after SSA deproteination. Amino acid values for ham muscle tended to be higher after deproteination with picric acid; however, with liver and loin muscle samples, the values were somewhat higher after SSA deproteination. In both serum and tissue analyses, coefficients of variation were lower for niGSt amino acids when picric acid was utilized as the deproteinating agent. The latter observation, in particular, suggests that picric acid is preferable to SSA as a deproteinating agent before amino acid analyses of biological fluids. Standardization of methods of deproteination is needed to allow meaningful comparisons of data.

scholarly journals Human Kallikrein 13: Production and Purification of Recombinant Protein and Monoclonal and Polyclonal Antibodies, and Development of a Sensitive and Specific Immunofluorometric Assay

2003 ◽  
Vol 49 (1) ◽  
pp. 77-86 ◽  
Author(s):  
Carl Kapadia ◽  
Albert Chang ◽  
Georgia Sotiropoulou ◽  
George M Yousef ◽  
Linda Grass ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Amniotic Fluid ◽  
Polyclonal Antibodies ◽  
Biological Fluids ◽  
Expression System ◽  
Immunohistochemical Analysis ◽  
Cross Reactivity ◽  
Yeast Expression System ◽  
Tissue Extracts ◽  
Cancer Tissues

Abstract Background: The aims of this study were to develop immunologic reagents and a sensitive and specific immunoassay for human kallikrein 13 (hK13) and to examine the presence of hK13 in human tissues and biological fluids. Methods: Recombinant hK13 protein was produced and purified with use of a Pichia pastoris yeast expression system. The protein was used as an immunogen to generate mouse monoclonal and rabbit polyclonal anti-hK13 antibodies. A sandwich-type immunoassay was developed with these antibodies. The assay was used to measure hK13 in various biological fluids and tissue extracts. Immunohistochemical analysis was also performed on nondiseased and cancerous prostatic sections. Results: The hK13 immunoassay had a detection limit of 0.05 μg/L and showed no cross-reactivity with homologous kallikreins. The assay was linear at 0–20 μg/L, and within-and between-run CVs were <10% (n = 12). hK13 was detected in tissues, including esophagus, tonsil, trachea, lung, cervix, and prostate. hK13 was also found in seminal plasma, amniotic fluid, follicular fluid, ascites of ovarian cancer patients, breast milk, and cytosolic extracts of ovarian cancer tissues. hK13 was immunohistochemically localized in epithelial cells of both nondiseased and cancerous prostate. hK13 appears to be overexpressed in 50% of ovarian cancer tissues compared with healthy ovarian tissues. Recovery of active enzyme added to milk or amniotic fluid was 70–98%, but was <20% when added to serum, suggesting rapid sequestration by protease inhibitors. In fluids and tissue extracts, hK13 was found in its free (∼30 kDa) form. Conclusions: This immunofluorometric assay for hK13 may be used to examine the value of hK13 as a disease biomarker and to further explore the physiologic and pathobiologic role of this enzyme in human disease.

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