Protein-free filtrate

Over thirty different elements have been determined in medical and biological materials by atomic absorption spectroscopy. The popularity of the technique is due to a number of factors, including sensitivity, selectivity, and ease of sample preparation. With biological fluids, often no preparation at all is required. The techniques employed usually involve simple dilution of the sample with water or with an appropriate reagent to eliminate interference. Alternatively, the element to be determined is separated by solvent extraction. Either an untreated sample, a protein free filtrate, or an ashed sample is extracted. 

Savory et al. 3S) measured calcium and magnesium directly in protein-free filtrates of serum or urine. Baker et al. 36) found that both trichloroacetic acid and hydrochloric acid suppress calcium absorption and that uniform acid content is therefore required for the determination of calcium. Okuda and Sasamoto37) determined calcium by adjusting solution conditions to 20 — 50 % methanol and 650 mg % lanthanum serum is diluted 21-fold and urine is diluted 10—21 fold to bring the calcium concentration into the optimum range of 1 to 0.5 mg %. 

Willis 93) extracted lead directly from 200 ml of urine with APDC into 1.5 ml of methyl-n-amyl ketone. He was able to determine as little as 0.02 ppm of lead. Kopito and Shwachman 141>, on the other hand, co-precipitate the lead from urine with bismuth nitrate by adding ammonia. The precipitated bismuth hydroxide is dissolved in acid and this solution is aspirated. Coprecipitation of the lead is not quantitative, and so standards should be prepared in the same manner. It should be possible to employ this procedure with protein free filtrates of blood without the necessity of close pH control. 

Several investigators have described the indirect determination of orthophosphate by extraction of the phosphomolybdic acid complex and the measuring the molybdenum extracted. Zaugg and Knox 2921 first applied this technique to the determination of phosphate in urine. A protein-free filtrate was formed and the complex was extracted into 2-octanol. More recently, Devoto 293) determined 0 to 25 pg of phosphate in 50 ml of urine by extracting the complex from acidified urine into isobutyl acetate. 

The uses of constant-current coulometry for the determination of drugs in biological fluids are few, basically due to sensitivity restriction. Monforte and Purdy [46] have reported an assay for two allylic barbituric acid derivatives, sodium seconal and sodium sandoptal, with electrogenerated bromine as the titrant and biamperometry for endpoint detection. Quantitative bromination required an excess of bromine hence back titration with standard arsenite was performed. The assay required the formation of a protein-free filtrate of serum with tungstic acid, extraction into chloroform, and sample cleanup by back extraction, followed by coulometric titration with electrogenerated bromine. The protein precipitation step resulted in losses of compound due to coprecipitation. The recoveries of sodium seconal and sodium sandoptal carried through the serum assay were approximately 81 and 88%, respectively. Samples in the concentration range 7.5-50 pg/mL serum were analyzed by this procedure. 

Chromatography of Bile Pigments In 1953 Cole and Lathe (C5) subjected protein-free filtrates of icteric serum to reverse phase partition chromatography. Using silicone-treated... 

In a protein-free filtrate of blood bromide was oxidized to bromine by potassium permanganate in acid solution and extracted into cyclohexane-containing cyclohexene to give l,2-dibromocyclohexane ... 

One of the earliest methods for determining CP in biological fluids is mentioned here for historical purposes. A protein-free filtrate of specimen is titrated with mercuric nitrate solution in the presence of diphenylcarbazone as an indicator. Free Hg combines with CP to form soluble but essentially nonionized mercuric chloride ... 

Movements of water are due mainly to osmosis and filtration. In osmosis, water moves to the area of highest solute concentration. Thus, active movement of salts into an area creates a concentration gradient down which water flows passively. In filtration, hydrostatie pressure in arterial blood moves water and nonprotein solutes through specialized membranes to produce an almost protein-free filtrate This process occurs in formation of the renal glomerular filtrate. Filtration also accounts for movement of water from the vascular space into the interstitial compartment, which is opposed by the osmotic (oncotic) pressure of plasma proteins. 

Procedure Starting with 0.2 ml of plasma, prepare a protein-free filtrate by the method of Somogyi (S8). Copenhaver (CIO) has pointed out that the choice of a method for deproteinization which yields an alkaline filtrate is important because of the finding of Wells et al. (W5) that acidic filtrates inhibit trimethylsilylation. 

Lyophilize 1 ml of protein-free filtrate (or 1 ml of galactose and glucose standard). Add 0.5 ml of anhydrous pyridine, 100 /il of hexamethyl-... 

Glucose is stable in a protein-free filtrate because the glycolytic enzymes are removed. 

The inorganic phosphorus in a protein-free filtrate is reacted with ammonium molybdate [Mo(VI)] to form ammonium phosphomolybdate. This is reduced with a mild reducing agent to produce molybdenum blue, a heteropoly molybdenum(V) species. Molybdates are not reduced under these conditions. The blue color of the solution is measured spectrophotometrically. 

Plot the net absorbance of the standards against concentration using a spreadsheet (Chapters 3 and 16). From this plot and the net absorbance of the sample, determine the concentration of phosphorus in the protein-free filtrate. Multiply by 20 to obtain the concentration in the original serum sample. The normal range of phosphorus in serum is about 3.0 to 4.5 mg/dL for adults and 4.5 to 6.5 mg/dL for children. 

A protein-free filtrate of a 0.5-mL sample of whole blood, serum, or plasma is prepared by precipitating proteins with zinc hydroxide. The glucose in an aliquot of this is reacted with a mixture of the enzymes glucose oxidase and horseradish peroxidase, and the hydrogen peroxide produced is coupled with the chromogenic hydrogen donor o-dianisidine to form a color with absorption maximum at 540 nm. 

Depending on the procedure employed for the determination of lead and the particular sample, the organic matter in biological samples may need to be removed prior to analysis. In some cases, simple preparation of a protein-free filtrate for fluid samples is satisfactory. In other cases, the organic matter must be destructively removed. 

Lead has been determined in whole blood by precipitating proteins with TCA and then extracting the lead in the protein-free filtrate with APCD and MIBK (P3, Bl). The pH is adjusted to 2.8 with sodium hydroxide. Usually about 5 ml of heparinized blood is taken with 5 ml of MIBK. The limit of the determination is about O.I ppm. 

It should be possible to apply the coprecipitation methods of Kopito and Shwachman (K7) or Zurlo et al. (Z2) to protein free filtrates of blood samples. This would eliminate problems of pH control, and interferences from complexing agents in the latter procedure. The former method has, in fact, been applied to digested blood samples (K8). 

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