Bennie Zak

Glucose-6-phosphatase

Publisher Summary This chapter focuses on glucose-6-phosphatase (G6Pase), which was first described by Fantl et al. Numerous attempts have been made to determine G6Pase activity in blood and so relate its activity to liver damage. G6Pase is applied in biochemistry and clinical chemistry. The inorganic phosphate liberated is determined with ammonium molybdate; ascorbic acid is used as the reducing agent. The amount of phosphate liberated per unit time, determined as the blue phosphomolybdous complex at 700 or 840 nm, is a measure of the G6Pase activity. The optimum pH for the enzyme reaction lies between 6.2 and 6.51. Only doubly distilled water should be used. The sucrose, G-6-P solution and the cacodylate buffer should be stored at 0–4°C. Freshly obtained tissue should be homogenized first in a precooled blender and then in glass-Teflon homogenizer with ice-cold sucrose solution. The enzyme is stable for several months at −35°C in the absence of EDTA. The instability of the enzyme causes serious errors, it is therefore necessary to assay the suspensions immediately after thawing. The enzyme is very specific for G-6-P, although other phosphate esters can be hydrolyzed.

Micro-determination of serum phosphate and phospholipids.

Abstract A simple, sensitive and accurate micro procedure for the determination of serum phosphorus, phospholipid or phosphatase activity has been described. The several facets of the investigation included order of reagents, stability of the chromogen formed, linearity of spectrophotometric response, recoveries and precision. A novel feature of the method includes the use of citrate and arsenite as stabilizers for the phosphomolybdate reduced by ascorbic acid. The color formed is stable for 24 hours. A procedure for the destruction of organic matter has been included which is simpler to carry out than sulfuric acid-peroxide destructions and which leaves a residue whose acidity presents no problem in the final formation of color.

Simple procedure for the single sample determination of serum copper and iron.

Abstract A simple procedure is proposed for sensitive, reproducible determination of both copper and iron on a single aliquot of scrum filtrate. The additive nature of the ferrous-bathophenanthroline sulfonate and cuprous-bathocuproine sulfonate complexes enables one to analyze both metals individually on the same sample by virtue of the fact that one constituent is determined directly and the second constituent is determined after a small subtractive correction is made to the subscqucnt total absorbance found. The additive nature of the color complexes formed, cuprousbathocuproine sulfonate and ferrous-bathophenanthroline sulfonate. makes the technique possible. The color forms instantaneously for both metals, is stable, sensitive and reproducible. With the use of micro attachments for the spectrophotometer and micro techniques it is possible to reduce the sample size employed to 500μl.

Determination of rat liver microsomal glucose-6-phosphatase activity: Study of citrate and G-6-P inhibition

Abstract Experiments were carried out to study the effect of citrate, G-6-P concentration, and pH on the G-6-Pase activity of liver microsomes. It was shown that, under the described experimental conditions, the following hold: 1. 1. Citrate ion neither inhibited nor enhanced G-6-Pase activity, and did not interfere with the determination of inorganic phosphate. 2. 2. Cacodylate buffer was more suitable for these enzymes studies than citrate buffer, but both buffers could be omitted from the incubation mixture if the pH of G-6-P substrate is preadjusted to 6.2. 3. 3. Maximum enzyme activity was obtained between G-6-P concentration of 20 and 50 μmoles present in 0.4 ml of reaction mixture at a pH between 6.2 and 6.5. 4. 4. The degree of nonenzymic acid hydrolysis of G-6-P was insignificant.

PROBLEMS WITH MEASUREMENTS CAUSED BY HIGH CONCENTRATIONS OF SERUM SOLIDS

There have been numerous reports of spectrophotometric and volume problems caused by elevated levels of lipids in blood. The offending lipids, primarily triglycerides, not only cause turbidity leading to optical aberrations when added to analytical reagents, but also result in short-sampling errors leading to the measurement of inaccurate volumes of sample. Numerous methods have been developed to clear the lipemia, including ultracentrifugation organic solvent extraction, chemical precipitation and, most recently, enzymic hydrolysis. Although the latter procedures eliminate the optical problems, they do not deal with the volume dilution error created by the triglycerides. In turn, corrective mathematics have been developed to compensate for the inaccurate pipetting caused by the elevated lipids in a sample; however, these empirical calculations are not truly accurate at high concentrations of total lipids. This monograph will describe the problems caused by the presence of elevated lipids and the means available for treating them.

Determination of ethanol in fingertip quantities of blood

Abstract A simple procedure has been described which involves the use of the Conway cell for the “isothermal distillation” of ethanol into an oxidizing scrubber of a strong sulfuric acid solution of dichromate. The residual dichromate is then reacted with brucine which yields a stable, sensitive chromophore. The increased sensitivity enables the technique to be performed on amounts of blood easily obtainable from the fingertip.

Determination of human erythrocyte zinc: hemoglobin ratios.

Abstract A procedure for the determination of erythrocyte zinc and its ratio to the hemoglobin present has been described. The several phases of study included replication, recoveries, interference, contamination and the determination of a range of Zn:Hb ratios in normal persons. This technic appears to be suitable to multiple analyses in everyday operation

A STUDY ON SERUM HEMOGLOBIN AND HEMOGLOBIN-BINDING CAPACITY.

Abstract Methods have been proposed to measure hemoglobin in serum in the range of 2–20 mg/100 ml. The effect of haptoglobin-binding on the peroxidase activity of hemoglobin and the need for proper calibration standards have been discussed. Serum hemoglobin and haptoglobin concentrations should be performed simultaneously.

Automation for copper, iron, and zinc in mixtures

Summary A procedure has been described for the flow spectrophotometric determinations of copper, iron, and zinc when these metals were present in mixtures. A high resolution monochromator attached to a sensitive multi-range recorder through a simple log converter served as the detection device for the three colored complexing reactions. Graphic exemplication of the accuracy of the determination of copper, iron, and zinc is shown for each metal in the presence of the other two metals.

The analysis of blood iron

Abstract A procedure has been presented which makes possible the analysis of blood iron by three short and simple steps which include: ( a ) partial digestion of the material with a reagent which contains both oxidizing and protein-precipitating reagents, ( b ) filtering and washing into a buffer containing reducing agent and a color-complexing agent for the reduced form of iron, ( c ) and finally measuring spectrophotometrically the resulting chromophore which follows Beers law over a wide range.