THE PATTERN OF URINARY EXCRETION OF INDIVIDUAL 17-KETOSTEROIDS IN RHESUS MONKEYS

1971 ◽  
Vol 67 (4) ◽  
pp. 711-720 ◽  
Author(s):  
S. B. Pal

ABSTRACT A systematic procedure for the extraction, separation, purification and colorimetric determination of urinary individual 17-ketosteroids in normal rhesus monkeys is described. Dehydroepiandrosterone was extracted after hot hydrolysis of the urine at neutral pH and the remaining steroid conjugates were extracted at pH 2 by the addition of ammonium sulphate. The liberated steroids were extracted after enzyme hydrolysis and the ketonic steroids were separated from the non-ketones by the Girard T reaction. The 11-deoxy steroids were separated from the 11-oxygenated steroids on a Celite column. They were further separated by paper chromatography and after elution, the individual 17-ketosteroids were estimated by the Zimmermann reaction. Normal mean excretion values in 30 female monkeys for total neutral 17-ketosteroids were 1.8 mg/24 h (range 1.5–2.6); total 17-hydroxycorticosteroids, 3.1 mg/24 h (range 2.2–4.8); androsterone, 38.6 μg/kg/24 h (range 9.3–60.2); aetiocholanolone, 19.2 μg/kg/24 h (range 6.8–30.7); dehydroepiandrosterone, 30.8 μg/kg/24 h (range 13.4–50.6); 11-ketoandrosterone, 7.5 μg/kg/24 h (range 5.1–14.1); 11-ketoaetiocholanolone, 10.2 μg/kg/24 h (range 6.8–18.3); 11β-hydroxyandrosterone, 15.4 μg/kg/24 h (range 8.8–26.7); 11β-hydroxyaetiocholanolone, 19.6 μg/kg/24 h (range 12.2–33.1). For total neutral 17-ketosteroids in 20 males, they were 2.5 mg/day (range 2.3–4.1); total 17-hydroxycorticosteroids, 3.7 mg/day (range 2.8–5.3); androsterone, 51.5 μg/kg/24 h (range 12.3–76.7); aetiocholanolone, 22.6 μg/kg/24 h (range 8.7–42.3); dehydroepiandrosterone, 41.3 μg/kg/24 h (range 16.9–59.7); 11-ketoandrosterone, 9.6 μg/kg/24 h (range 6.4–16.3); 11-ketoaetiocholanolone, 12.3 μg/kg/24 h (range 8.2–20.5); 11β-hydroxyandrosterone, 17.5 μg/kg/24 h (range 11.7–29.2); 11β-hydroxyaetiocholanolone, 21.7 μg/kg/24 h (range 14.5–36.4).

1984 ◽  
Vol 30 (4) ◽  
pp. 534-537 ◽  
Author(s):  
J D Artiss ◽  
M W McGowan ◽  
D R Strandbergh ◽  
E Epstein ◽  
B Zak

Abstract We describe a procedure for the enzymic, colorimetric determination of phosphatidylglycerol in amniotic fluid. After extraction into chloroform:methanol (2:1 by vol) and evaporation, the phospholipid-containing residue is redissolved in a non-ionic detergent, which thus provides an aqueous sample. The subsequent enzymic reaction sequence involves phospholipase-catalyzed hydrolysis of glycerol from its phospholipid. Subsequent enzyme-catalyzed reactions phosphorylate this glycerol and oxidize the resulting glycerol phosphate to produce hydrogen peroxide, which is reacted to produce an intense red chromogen in the peroxidase-catalyzed coupling of 4-aminoantipyrine and 2-hydroxy-3,5-dichlorobenzenesulfonate. When used in conjunction with previously reported enzymic techniques for determination of lecithin and sphingomyelin, this procedure may provide an accurate and precise "lung profile" for assessment of fetal lung maturity.


1964 ◽  
Vol 47 (2) ◽  
pp. 283-286
Author(s):  
D P Johnson

Abstract Collaborative study of a method for Sevin insecticide residues was repeated with apples and lettuce as test crops. The method is based on alkaline hydrolysis of Sevin and colorimetric determination of the resulting 1-naphthol with p-nitrobenzenediazonium fluoborate as chromogenic agent. Data from 5 collaborators were in good agreement with an average recovery of 87.8% at 2 concentration levels. It is recommended that the method be adopted as official, first action.


1967 ◽  
Vol 13 (4) ◽  
pp. 281-289 ◽  
Author(s):  
Kirsten Hviid

Abstract The manual procedure of Babson et al. (1) for the determination of serum alkaline phosphatase has been automated. The assay is based on the colorimetric determination of phenolphthalein formed on hydrolysis of phenolphthalein monophosphate. The procedure utilizes 0.16 ml. of serum without dialysis. Blanks are required only for turbid sera. Results are compared with those obtained by the manual procedure, and data relating to sample interaction, precision, blank values, and normal values are presented.


1958 ◽  
Vol 41 (3) ◽  
pp. 485-500 ◽  
Author(s):  
M. Castañeda-Agulló ◽  
Luz M. Del Castillo

A direct titration method for the determination of proteolytic activity is discussed. This involves the potentiometric measurement of the volume of 0.08 N NaOH required to maintain a constant pH (8.0) during the time of the hydrolysis. It is a sensitive method which presents several advantages; viz., it measures simultaneously protease and esterase activity, it follows the hydrolysis very closely and from the first stages; the titration is continuous and on the same sample. This method determines a constant fraction of the groups titratable by formol titration. The ratio formol: direct titration is represented by a factor "f" which is presumed to be distinct for each protein-enzyme system. Kinetic studies, using this method, revealed that the rates of hydrolysis of mixtures casein-gelatin on one hand, casein-BAEE or gelatin-BAEE on the other, are always larger than those of the corresponding isolated substrates. In many cases the resulting rates are equal or nearly equal to the sum of the individual rates, even though the mentioned rates have been determined within the saturation zones for every substrate. The former observations are inconsistent with the theory of the formation of an intermediary enzyme-substrate compound, unless it is assumed that the enzyme has a specific active group for each substrate.


1993 ◽  
Vol 289 (2) ◽  
pp. 475-480 ◽  
Author(s):  
R de Cristofaro ◽  
B Rocca ◽  
B Bizzi ◽  
R Landolfi

A method derived from the analysis of viscosity effects on the hydrolysis of the amide substrates D-phenylalanylpipecolyl-arginine-p-nitroaniline, tosylglycylprolylarginine-p-nitroanaline and cyclohexylglycylalanylarginine-p-nitroalanine by human alpha-thrombin was developed to dissect the Michaelis-Menten parameters Km and kcat into the individual rate constants of the binding, acylation and deacylation reactions. This method was used to analyse the effect of the C-terminal hirudin (residues 54-65) [hir-(54-65)] domain on the binding and hydrolysis of the three substrates. The results showed that the C-terminal hir-(54-65) fragment affects only the acylation rate, which is increased approx. 1.2-fold for all the substrates. Analysis of the dependence of acylation rate constants on hirudin-fragment concentration, allowed the determination of the equilibrium binding constant of C-terminal hir-(54-65) (Kd approximately 0.7 microM). In addition this peptide was found to competitively inhibit thrombin-fibrinogen interaction with a Ki which is in excellent agreement with the equilibrium constant derived from viscosity experiments. These results demonstrate that binding of hir-(54-65) to the fibrinogen recognition site of thrombin does not affect the equilibrium binding of amide substrates, but induces only a small increase in the acylation rate of the hydrolysis reaction.


2009 ◽  
Vol 60 (2) ◽  
pp. 147-156 ◽  
Author(s):  
Lida Bagdonienė ◽  
Danutė Labeikytė ◽  
Ivars Kalviņš ◽  
Veronika Borutinskaitė ◽  
Aleksandrs Prokofjevs ◽  
...  

Rat Serum Carboxylesterase Partly Hydrolyses Gamma-Butyrobetaine EstersAlthough described some time ago, gamma-butyrobetaine esters and related compounds have not gained much attention from researchers, and their physiological function remains obscure. Formerly we detected GBB-esterase enzymatic activity in rat blood serum using phenylated gamma-butyrobetaine as an artificial substrate of the enzyme and HPLC. The aim of the present work was to develop an assay that would enable spectrophotometric or colorimetric determination of the reaction products of GBB-esterase activity and to reveal individual proteins performing GBB-esterase activity in rat blood serum. For this purpose gamma-butyrobetaine 1-naphthyl ester was synthesised. Hydrolysis of this ester releases 1-naphthol, which increases the optical absorbance at 322 nm. We have shown that the enzymatic hydrolysis of GBB 1-naphthyl ester to 1-naphthol in rat blood serum is due to GBB-esterase activity. An attempt was done to purify the enzyme from rat blood serum. By combining DEAE Sepharose at pH 4.2 and affinity chromatography with procainamide we achieved a 68-fold enrichment of GBB-esterase activity in our preparations. Separation of fraction proteins in 2D protein electrophoresis with following mass-spectrometry indicated that GBB esterase activity in rat blood serum is performed in part by carboxylesterase.


1983 ◽  
Vol 66 (1) ◽  
pp. 105-107 ◽  
Author(s):  
Kalapanda M Appaiah ◽  
Omprakash Kapur ◽  
Krishnarajpet V Nagaraja

Abstract A method has been developed for the determination of propoxur (o-isopropoxyphenyl N-methylcarbamate) based on the hydrolysis of propoxur with methanolic potassium hydroxide to its phenol and coupling with diazotized 4,4-diaminodiphenyl sulfone. The orange complex formed has an absorption maximum at 500 nm and obeys Beer’s law in the range 0.25-5.0 μg/mL. The method can be applied to levels as low as 0.5 ppm propoxur from vegetables.


Author(s):  
Е. М. Serba ◽  
М. B. Overchenko ◽  
N. I. Ignatova ◽  
М. Е. Medrish ◽  
L. V. Rimareva

Enzyme preparations - sources of β-galactosidase, are widely used in the dairy industry. However, there is no standardized methods to determine their activity. Existing methods differ by the type of substrate used, conditions of analysis and how to define the products of the hydrolysis. The aim of this work is to study the process of conversion biocatalytic milk sugar and experimental substantiation of methods of analysis of hydrolysis products, providing reliable results determine activity of β-galactosidase. Found that of chemical methods glucose oxidase showed a greater sensitivity towards low concentrations of the product of hydrolysis is glucose formed in the conversion process lactose. The results of testing showed that the mass concentration of the glucose formed in hydrolysis of lactose, HPLC tested, consistent with data obtained using the glucose oxidase method. Set optimum range, which was observed directly proportional to the level of glucose concentration rise in reaction mixture from the lengthy process and the concentration of enzyme:  hydrolysis time - from 10 to 20 min, produced   glucose - from 20 µcg to 40 mcg, while the mass of enzyme - 25.0·10-6, and from 40 mcg to 80 mcg, when mass -5,0·10-5 g.  When the values of absorbance of the reaction mixture ranged 0,045-0,300 to ensure reliable performance. Studies on determination of the activity of the enzyme preparations confirmed received experimental data and allowed to choose optimum parameters and conditions analysis for introducing them to the method for determining the activity β-galactosidase using glucose oxidase reagent: time of inactivation enzyme (control) -10 min.  in a boiling water bath, substrate - 2% solution of lactose, pH of the substrate-6,0, hydrolysis time 15 min. at 30° c.


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