Amino Acids in Urine

In some diseases, e. g., cystinuria, an unequivocal diagnosis is possible only on the basis of the pathological pattern of amino acid distribution in the urine. The reference amount is important for evaluating the results of thin-layer chromatography of urine amino acids. Use of standard volumes in TLC is unsatisfactory since the daily urine volumes passed can vary widely, e. g., from 600—2000 ml for adults. In order to circumvent this difficulty, some investigators refer the results to the amount of creatinine contained in the same sample the elimination of creatinine is largely independent of the volume of urine. Others use volumes per time unit thus Jepson [81] has proposed 2-second portions for TLC in cases of severe aminoaciduria. For a period of collection of, say, 2 hours and a 

Dpienska-Blauth and co-workers [114, 115] and Rokkones [133, 134] have investigated the thin-layer chromatographic separation of free amino acids in urine. Using 10 xl of urine which had not been previously treated, the former [ll4] were able to detect 3 to 6 ninhydrin-positive spots in two-dimensional TLC on silica gel G with the solvents biitanol-acetic acid-water (60 + 15 + 15) and phenol-water (75 + 25) 100 [xl of the same but demineralised urine yielded 14—16 visible spots. Rokkones [134] has obtained 9—12 ninhydrin-positive spots from an 

Dittmann [30, 31] has given procedures for TLC of amino acids without prior demineralisation. He used cellulose layers and triple development. The solvents must be buffered if compounds are present like phosphoethanolamine which are unstable in acid solution [31]. 

Tancredi and Curtius [165] have investigated the ether-soluble DNP-amino acids in the urine of healthy and sick children. In a case of hyperglycinaemia, one of hypoaminoaciduria and one of renal insufficiency, they were able to discover several amino acids which could not be found in the urine of a healthy girl, using the same technique. 

---

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. 

V2. Van Slyke, D. D., MacFadyen, D. A., and Hamilton, P. B., The gasometric determination of amino acids in urine by the ninhydrin-carbon dioxide method. J. Biol. Chem. 150, 251-258 (1943). 

Amino acids in urine may be preserved by a bacteriostatic such as chloroform or toluene during the 24-h collection. Aliquots of urine should be stored subsequently at -20°C. Heat inactivation at 55°C for 20 min has proven to be effective in suppressing bacterial activity consequently, the sample can be shipped at room temperature. 

Table 2,1,6 Reference values for amino acids in urine in different age groups (mmol/ mol creatinine)... 

Pentz et al. (P5) estimate taurine with fluorodinitrobenzene in urine passed through Dowex 50 H+ columns, but there are doubts as to whether this procedure is really specific for taurine (B38). Dent et al. have compared results obtained for the estimation of sulfur-containing amino acids in urine of cystinuric patients, by polarographic and microbiological methods (D18, D19). Hier (H12) and Schreier and Pliickthun (S10, Sll) have published data on amino acid excretion as determined microbiologically. Enzymatic methods have been used with success in the case of histidine in urine with specific decarboxylase preparations (S23). 

Determination of oxidized amino acids in urine is usually performed by isotope dilution gas chromatography-mass spectrometry (L9). DOPA is estimated by HPLC separation of acid protein hydrolysates with fluorescence detection (excitation 280 nm, emission at 320 nm) (A15). Other methods are based on borate-hydrochloric acid difference spectroscopy (this method suffers interference from tyrosine and tryptophan) (W2), derivatization of DOPA with nitrite and subsequent coulometric determination (W3), and fluorometric detection after derivatization with ethylenediamine (A15). 3-Hydroxylysine is quantitated by HPLC with 9-fluorenylmethyl chloroformate precolumn derivatization (M25) of amino acids obtained by gas-phase hydrolysis of proteins (F21). Other general methods to detect amino acid damage are mass spectometry methods applied to protein hydrolysates, such as tandem mass spectrometry (F6). 

Enrichment of amino acids in urine can be performed by extracting the lyophilized sample (10 mL) with ImL methanol-lM HCl mixture (4 1 v/v) and applying an ahquot of supernatant liquid to the plate after centrifugation. 

Screening Test for Increased a-Amino Acids in Urine. 160... 

D6. Dent, C. E., Detection of amino acids in urine and other fluids. Lancet if, 637-639 (1946). 

Jll. Juul, P., Quantitative measurement of individual free amino acids in urine by means of high voltage paper electrophoresis. Investigations of a group of mentally retarded patients. Scand. J. Clin. Lab. Invest. 18, 629-637 (1966). 

P20. Philpott, D., Tlun-layer chromatography of trinitrophenyl derivatives of amino acids in urine and plasma. J. Clin. Pathol. 23, 315-318 (1970). 

Spinella, C. J., Two dimensional paper chromatographic determination of amino acids in urine. Clin. Chem. 16, 1011-1016 (1969). 

Conservation of amino acids filtered at the glomerulus is made possible by the existence of four main transport systems for specific amino acids that facilitate active reabsorption of these amino acids from the proximal tubule. A lack or deficiency of the transport system responsible for the absorption of valine, alanine, cystine, and tryptophan, and of the transport system for arginine, lysine, cystine, and ornithine, leads to excretion of these specific amino acids in urine, which is characterized as renal aminoaciduria to distinguish it from overflow aminoaciduria. In the latter situation, the production of amino acids far exceeds the proximal tubular reabsorption capacity, thus leading to overflow of amino acids into urine. This can occur due to defective metabolism of amino acids, as is the case when phenylalanine cannot be metabolized due to the deficiency of the enzyme phenylalanine hydroxylase, or to the inability to deaminate amino acids in liver disease. 

Nl. Nardi, G. L., Essential and non-essential amino acids in urine of severely burned patients. J. Clin. Invest. 33, 847-854 (1954). 

Amino acids in urine Nucleosil C-18 A 0.1 M phosphate (pH 7.5) B Methanol 40-75%B Pre-column derivation with o-phthalaldehyde Fluorescence at 330/418 nm 99... 

Separation of peptides from amino acids in urine... 

C. Carducci, M. Birarelli, V. Leuzzi, G. Santaga, P. Serafini and I. Antonozzi, Automated method for the measurement of amino acids in urine by high-performance liquid chromatography, /. Chromatogr., A, 1996, 729, 173-180. 

An Analytical Ion Exchange-Gas Liquid Chromatographic Methodology for Studying the Glycosides of Amino Acids in Urine in Health and Disease Univ. Microfilms (Ann Arbor, Mich.), Order No. 76-9422, 232 pp. Diss. Abstr. Int. 36B(10) 5001 (1976) CA 85 1980m... 

As an example, the determination of amino acids in urine or neutral lipids in serum can be approached in a number of different ways, depending on the loading capacity of the layer and the sensitivity of the detection method. Urine, saliva, or serum can sometimes be directly spotted on the layer and compounds of interest separated with no additional cleanup steps. More often, compounds of interest must be extracted from the sample the extract may then require further cleanup by liquid-liquid partition or column chromatography the final extract is then concentrated prior to TLC. 

Table B.2. Amino acids in urine by age group (mmol per mol of creatinine) ...

---