Urine fractionation procedure

Introduction of microbiological methods for the determination of amino acids made possible the estimation of the amount of both free and combined amino acids in urine. Dunn et al. (D4), Thompson and Kirby (Tl), Eckhard and Davidson (El), and Woodson et al. (W3) estimated the amount of amino acids liberated in the course of acid or, as in the case of tryptophan determination, alkaline hydrolysis. Microbiological and colorimetric methods used for the determination of certain amino acids present very little opportunity for evaluating the proper quantitative relations between free and combined amino acids, since under the applied condition both combined and free amino acids are equally involved in the reaction. In 1949 Albanese et al. (A3) applied such methods to the quantitative determination of free and combined amino acids in the nondiffusible fraction of urine, and subjected the procedures to broad criticism from just this point of view. 

A similar ion-exchange resin method was used by Ling in 1955 (LI) for the examination of combined amino acids in urine. According to this procedure urine was desalted and simultaneously freed from amino acids by using Amberlite IR-112, H+-form resin. The effluent collected from the column was then fractionated on Amberlite IRA, OH--form resin, by successive elution with 0.16 N acetic acid, 0.08 N formic acid, 0.25 N formic acid, 0.08 N hydrochloric acid, and finally with 0.16 N formic acid. The solutions of all acids contained 10% of acetone. The collected fractions were hydrolyzed with hydrochloric acid and the liberated amino acids identified by means of paper chromatography. 

In 1952 Carsten (Cl) developed a method, which allowed him to isolate and characterize several lower peptides contained in normal and pathological urine. According to this procedure, urine was desalted on the Amberlite IR-100 column and the adsorbed substances washed out with 2 M ammonia solution. The eluate was then passed through the column of Amberlite IRA-400. This column retained the ampholytes and rejected the weak bases. The former were recovered by elution with 1 M hydrochloric acid and the eluate was subsequently fractionated on Dowex 50 resin with 2M and later 4M hydrochloric acid as the eluents. By applying two-dimensional paper chromatography to further analysis of... 

The simplest methods are usually restricted to the estimation of the amount of combined amino acids as a whole or of some definite fraction thereof separated from urine in certain fixed conditions. More efficient separation procedures permit identification of some simple peptides, which represent in many cases the nonphysiological constituents of abnormal urine. 

Recently introduced procedures for the examination of peptides in urine, though restricted only to a certain limited peptide fraction, make the separation of this fraction and analysis of its individual components possible. 

The first fractionation of urinary ampholytes in this way was carried out by Boulanger et al. (BIO) in 1952 with the use of ion-exchange resins. They had designed this procedure previously for the fractionation of ampholytes in blood serum (B8). According to this method, deproteinized urine was subjected to a double initial procedure aiming at the separation of low-molecular weight substances from macro-molecular ones. One of the methods consisted of the fractionation of urinary constituents by means of dialysis, the second was based on the selective precipitation of urinary ampholytes with cadmium hydroxide, which, as had previously been demonstrated, permits separation of the bulk of amino acids from polypeptides precipitated under these circumstances. Three fractions, i.e., the undialyzable part of urine, the dialyzed fraction, and the so-called cadmium precipitate were analyzed subsequently. 

By means of a procedure described above, Hanson and Fittkau (HI) isolated seventeen different peptides from normal urine. One of them, not belonging to the main peptide fraction, consisted of glutamic acid, and phenylalanine with alanine as the third not definitely established component. The remaining peptides contained five to ten different amino acid residues and some unidentified ninhydrin-positive constituents. Four amino acids, i.e., glutamic acid, aspartic acid, glycine, and alanine, were found in the majority of the peptides analyzed. Twelve peptides contained lysine and eight valine. Less frequently encountered were serine, threonine, tyrosine, leucine, phenylalanine, proline, hydroxyproline, and a-aminobutyric acid (found only in two cases). The amino acid composi-... 

Sarnecka-Keller (SI), by use of Bondzynski s method modified by Gawinski, isolated from normal urine a well-defined peptide fraction easily reproducible without change of qualitative composition. Throughout the isolation procedure all factors which may have caused any changes in peptide structure were taken into account. The isolated preparation was substantially similar in general properties to that obtained originally by Bondzynski et al. (B7). 

Prior to the chromatographic separation of amino acids on Dowex 50 columns, Carsten (C5) first desalts the urine sample on Amberlite IR 100 or Duolite C 3 and removes most of the nitrogenous bases on Amberlite IRA 400. This preliminary treatment allows for amino acid separations at ordinary temperatures using 2M and 4M HC1 on H+ columns for elution, instead of buffer mixtures a single column of 25 g of Dowex 50 is sufficient for all amino acids and 350-375 one-milliliter fractions are collected. The resolving power of this method does not seem to be as satisfactory as Moore and Stein s procedures, and it is not less time nor labor consuming. 

We shall not endeavor to describe in this chapter the various types of fraction collectors that have been used in column chromatography. For urine analysis 1-ml fractions are necessary ( °/o error). In Moore and Stein s 1958 procedure (see above (c) Amberlite IR 120-X8 columns), 2-ml fractions can also be collected. 

Up to the present time, Moore and Stein s 1954 procedure has given the most satisfactory results, both qualitative and quantitative, for the study of amino aciduria. Paper chromatography is useful as a screening process for routine urine analysis prior to the use of column chromatography for more accurate and complete analysis. It is also most useful for the analyses of fractions containing unknown constituents obtained by column chromatography or to check whether such fractions are single substances or mixtures. 

Two independent research groups at our hospital have pursued the development of analytical methodology for THC and its metabolites in urine. One group, under L. E. Hollister, has directed its efforts primarily toward fractioning the in vivo THC metabolites by solvent partitioning and by TLC. The other group, under I. S. Forrest and D. E. Green, has been concerned with the development of an automated GC/MS quantitative procedure. ... 

Certain of the tests described here may be carried out by the micro technique (E. G. C. Clarke and M. Williams, J. Pharm. Pharmac., 1955, 7, 255-262). This applies particularly to Mandelin s Test, Marquis Test, and the Sulphuric Acid test. Information on the direct application of colour tests to urine, blood, and stomach contents can be found under Hospital Toxicology and Drug Abuse Screening (p. 4). Where an extraction procedure has been carried out with the production of acidic and basic fractions (see p. 11), the tests below may be applied to the evaporated extracts. 

Volumes of urine of 10-15 ml at pH 2.10 are added directly to the column and elution is carried out with the formic acid-pyridine buflFers referred to above. Fractions of 25 ml are collected, rapidly evaporated to dryness in vacuo, and after dilution to known volume spotted on paper sheets. At this point the procedure of Coppini et al. (CIO) is followed the results obtained by analyzing urine of normal individuals are tabulated below (Section 2.1.1). 

Analysis of urine samples for excreted radionuclides is the most prevalent in-vitro bioassay procedure in use today. It is particularly useful for radionuclides that enter the body in a relatively transferable (i.e. soluble) form. When a radionuclide in such a form reaches the bloodstream, fractions will be deposited in various body organs and the remainder excreted, predominantly in the urine. 

The column was washed with 5 mL of water and dried with a gentle stream of forced air for 1 min. The column was eluted with 9 mL of methanol. The eluate was evaporated to dryness and the residue was redissolved in 10.00 mL of Pi buffer. Human urine was fortified with an acetone solution of picloram. The urine was analyzed without further processing by the RIA procedure. For the enzyme immunoassays, 10.00 mL aliquots were acidified to pH 2 with 3 N H2SO4. The picloram was extracted three times with 3 mL portions of diethyl ether. The ether fractions were pooled and evaporated to dryness. The residue was redissolved in 10.00 mL Pi buffer, and cetrifuged at 12 000 x g for 10 min. 

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