Potassium bicarbonate, assay

The crude ketal from the Birch reduction is dissolved in a mixture of 700 ml ethyl acetate, 1260 ml absolute ethanol and 31.5 ml water. To this solution is added 198 ml of 0.01 Mp-toluenesulfonic acid in absolute ethanol. (Methanol cannot be substituted for the ethanol nor can denatured ethanol containing methanol be used. In the presence of methanol, the diethyl ketal forms the mixed methyl ethyl ketal at C-17 and this mixed ketal hydrolyzes at a much slower rate than does the diethyl ketal.) The mixture is stirred at room temperature under nitrogen for 10 min and 56 ml of 10% potassium bicarbonate solution is added to neutralize the toluenesulfonic acid. The organic solvents are removed in a rotary vacuum evaporator and water is added as the organic solvents distill. When all of the organic solvents have been distilled, the granular precipitate of 1,4-dihydroestrone 3- methyl ether is collected on a filter and washed well with cold water. The solid is sucked dry and is dissolved in 800 ml of methyl ethyl ketone. To this solution is added 1600 ml of 1 1 methanol-water mixture and the resulting mixture is cooled in an ice bath for 1 hr. The solid is collected, rinsed with cold methanol-water (1 1), air-dried, and finally dried in a vacuum oven at 60° yield, 71.5 g (81 % based on estrone methyl ether actually carried into the Birch reduction as the ketal) mp 139-141°, reported mp 141-141.5°. The material has an enol ether assay of 99%, a residual aromatics content of 0.6% and a 19-norandrost-5(10)-ene-3,17-dione content of 0.5% (from hydrolysis of the 3-enol ether). It contains less than 0.1 % of 17-ol and only a trace of ketal formed by addition of ethanol to the 3-enol ether. 

Sodium, potassium and chloride sensors were evaluated using undiluted serum specimens with no sample pretreatment of any kind. The performance data set, summarized by Table II, comprises a minimum of 8 sensors and 60 samples. The bicarbonate assays were performed separately on serum specimens buffered at pH 9.0. Clinical efficacy is normally judged by the response linearity, precision and... 

The incubation mixture contained in a Anal volume of 100 /iL 400 fiM DL-homocysteine, 500 (iM ( )-L-A/5-methyltetrahydrofolate, 50 fiM cyanocobalamin, 300 fiM 5-adenosylmethionine, 125 mM 2-mercaptoethanol, 20 fiM L-norvaline, 50 mM potassium phosphate buffer (pH 7.4), and 50 /xL of liver or cell extract. The incubation mixture was immediately flushed with nitrogen and overlayered with 50 /tL of bis(3,5,5-trimethylcyclohexyl)-phthalate. The incubation, carried out at 37°C in the dark, was stopped by the addition of 10 / L of 4 TV perchloric acid. The acid was then neutralized by addition of 10 / L of 4 TV KOH containing 3.3 M potassium bicarbonate. After centrifugation, 90 /iL of supernate was mixed with 175 fiL of o-phthaldialdehyde reagent (prepared by mixing 1 mL of 56 mM o-phthaldialdehyde in methanol with 9 mL of 0.1 M sodium borate buffer, pH 9.5, then adding 40 fiL of 2-mercaptoethanol). After 2 minutes at 23°C, 220 /xL of this mixture was used for HPLC analysis. The assay is linear for at least 2 hours. 

Ribulosebisphosphate carboxylase (isolated by a published procedure ) (250 mg, 3.57 /imoles of protomeric unit > ) in 50 ml of metal-free 0.1 Af Bicine/60 inM potassium bicarbonate/0.1 mAf EDTA (pH 8.0) was treated at 25° with four successive 0.25-ml additions, at 20-min intervals, of 20 mAf Br-butanone- P2- Twenty minutes after the fourth addition, less than 10% of the initial activity (as determined by the method of Racker ) remained, and the reaction was terminated by the addition of 2-mercaptoethanol (10 mAf). A duplicate enzyme solution containing ribulosebisphosphate (1 mAf) was treated with reagent in an identical manner 95% of the initial enzymic activity was retained. A third enzyme solution under the same conditions but lacking both the reagent and substrate served as control. The three protein solutions were dialyzed against 0.1 M sodium chloride at 4° after dialysis the protein samples were made 0.1 M in sodium bicarbonate and 0.01 Af in sodium [ H]borohydride in order to reduce the carbonyl of the protein-bound reagent. The mixtures were maintianed in an ice bath for 30 min and then dialyzed exhaustively at 4° against 50 mAf sodium chloride. The samples then were assayed for protein concentration, radioactivity ( H and P), and sulfhydryl content (see the table). 

Fig. 4 Superfusion neurotransmitter release assay in synaptosomes. (a) Schematic drawing of a superfusion setup. Synaptosomes are preloaded with radioactive neurotransmitter and captured on fiberglass filters in superfusion chambers under continuous superfusion with gassed physiological salt solution (e.g., Krebs bicarbonate buffer) using a peristaltic pump. After a 10- to 15-minute wash, neurotransmitter release is triggered by rapid switching of superfusion lines to a stimulating buffer (e.g., high-potassium solution). Superfusate is collected on a fraction collector, and radioactivity is measured by liquid scintillation, (b) Typical trace recording of tritium-labeled norepinephrine fractional release in rat cortical synaptosomes stimulated by high potassium and a-latrotoxin in the presence or absence of external calcium.

Assay Transfer about 2 g of sample, accurately weighed, into a 250-mL volumetric flask, dissolve in 15 mL of hydrochloric acid, dilute to volume with water, and mix. Transfer 50.0 mL of this solution into a 500-mL flask, add 5 g of sodium potassium tartrate, and mix. Make the solution alkaline to litmus with a cold saturated solution of sodium bicarbonate, and titrate at once with 0.1 A iodine, using starch TS as the indicator. Each milliliter of 0.1 A iodine is equivalent to 9.48 mg of SnCl2 or 11.28 mg of SnCl2-2H20. 

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