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T Williamson. the Desalting of Urines Prior to Amino Acid Chromatography

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T Williamson. the Desalting of Urines Prior to Amino Acid Chromatography " ' J R Army Med Corps: first published as 10.1136/jramc-112-03-12 on 1 January 1966. Downloaded from , " THEDESAL TING OF UiRINES P,RIOR TO AMINO ACID CHROMATOGRAPHY Sergeant T. WILLIAMSON, A.I,M.L.T;, RAM.C. Royal ArmyMedical Colleg~, Millbank Introduction THE need for a simple and inexpensive method for identifying urinar.yamino acids has been fulfilled to date by the use of paper chromatography; Before the urine can be chroniatographed for amino acids, inorganic ions a.nd, large ,neutral molecules such as , proteins must be renlOved. Ifleft in solution they will seriously impair the ~potsepa.~~i.ti,On. Several methods of effecting deionisation have been described and fll.ll into three main -categories: Organic solvent extraction ' , , guest. Protected by copyright. Verghese and Ram~krishnan (1957) described a method in which -urine isev'apor~ted: . to dryness and the amino aci.ds extracted from the residue with a mixture of phenoL ',.' '. and h~tanol. This become~ a lengthy procedure since dessication over~night is involve~( . Electrolytic diionisationand dialysis , . These two methods,described respectively by Consden etal (1947) 'and Wood (1956) . are very efficient, but fequife equipment not readily available. They are expensive,., taking .into account the factthat since 'the Army is a relatively healthy comnmnity, identification of urinary amino acids is a comparatively uncommon request. , Ion exchange resins ~mith (1958) indicated how a cation exchange resin could be used to adsorbinorganic-' iOrisandamino acids from urine ,followed by selective -elution, cbncentnltionand http://militaryhealth.bmj.com/ -chromatography of the amino acids. ' . " _. Thus a method fordeionising urines based onthis thirdcategqry has been found . suitable-and is subsequently descfibed.· ' " MateriaIsand Methods This' method depends on the fact, that amip:~ a~ids in soluti~n are capable of acting' as weak bases or weak adds: , . I ' . They contain both basIc (-NH2-:'-) and acidic (-COOH~) radicles. , , Thus is acid solution they a.ct 'as weak 'bases and are adsOrbed onto the resin, in alike on September 27, 2021 by . manner to the inorganic cations. , . R. CHNH2 COOH+ H + CI:- R. CHNH 3' CO OH +Cl'- Slightly acid urine is passed thro~gh thecatio'n exchange re~in " ZEOKARs'225 ". Proteins and aniOns, are washed out of the column and, the amino acids eluted with ammonia, leaving the inorganic ca.tions absorbed on the resin. '~ZEOKARB 225 " is a " ' J R Army Med Corps: first published as 10.1136/jramc-112-03-12 on 1 January 1966. Downloaded from 172 - The desalting of Urines Prior to Amino Acid Chromatography stable sulphonated polystyrene resin, operational over a wide pH rang~ and is used for this purpose at a mesh size of 52-lOp. It is supplied by British Qrug Houses inthe sodium, form so conversionto-t}:le hydrogen or acid form is n~ctls~ary. ' . This is done by soaking a quantity ofresin in twice its volume of warm 10 per cent v /v hydrochloric acid. The resin settles out and the hydmchloric acid plus any remaining floating partiCles is decanted to waste. This is repeated and followed by three similar washings in double distilled water. The resin may be stored at this stage in an airtight contaIner, preferably polythene, as an aqueous slurry. '. A column of 25 m!. of resin is set up in a: burette after first plugging the bottom with a small wad of glass wool. Any bubbles trapped in. thecoltimn maybe disturbed by back ftusWng ,with water. frQm a high reservoir connected to the uoz?:le. The wl;J,ter standing qn the ~ohlffin is allowed to run away and the. ~;urfac;e of the resin plugged with anotheJi glass wool plug, Cotton wool is unsa:tis(actery. At no timtlduring or afterus~is the. re~in allowed, to dry. The'deionisation Qf the urine is carried out as follows:~ , 5 m!. of slightly acid filtered urine (pH about 6.0) are pipetteclonto the resin and . allowed to percolate into the column. This is followed by 30-35 ml. of double distilled water which is allowed t~ run to waste. 30-ml. of 3N ammonia are poured into the guest. Protected by copyright. column. As the ammonia travels dow:nwards the resin will be seen-to change colour from '. a'light to a-dark amber. When,the'ammonia approaches the nozzle the drops are allowed to[a11 onto red litmus paper in a Wml. measuring cylinder. On the, litmus paper changing . cololfr I' ml .. of the eluate is measured and discarcled. The remaining eluate is collected . .• The ammonia re~aining in' the column is displaced with distille'd water and' collected: . The total: ehiateshcnild measurk abo}1t 25 mh This is evaporated: down toJ ml. on,a 1:ioiling ·waterbath or oyera low bunsen. flame, then cooled. This drives off the ammonia ancl . concentrates the original sample content five times. The eluate 'can then be spotted onto, the chromatography paper. Results . ; Rec6:v.ery: e)(,pe~iments 'Y;el,'e ~ar-ried out usipg solution,s o( aminQ acids in: electrolyte http://militaryhealth.bmj.com/ " s'oiutions, in normal and known abnormal samples, of llrin~; In b~tween:sl;lillp1.e.s the resin colUllln: was rech;lrged: with 5 per Geritv.!v }:lydrochIQric, acid, and, washed WIth double distilled water. The s,ubsequent chromato'gntphy was, capried, out "t\Y.Q way."~' using ,Smith's n butanol/acetic/water and phenol/ammonia solvents with ninhydrin or isatin in pyricline and acetone as locating reagent~. It w;as found that an ar.ninoacids, likely to: OGClfr in ur:ine"normlil andabnorma:l, wererecoveredf~om the column with the on~ exception of taJlTwe, This was, either corn,,: pletely absent or g~yea, very. faint spot, depe~dip.gonits ol!igina1iconeentration. , " -", , . The concentration of ammonia for eltltion, was' found to'. be criticaf-2N, ammonia on September 27, 2021 by . } gave excellent, discrete spOts but failed to elute arginine. 4N 'ammonia gave· the best . 'recovery of arginin~. but the diffuse spots suggested that inorganic Gations· were also being eluted., 3N anmionia gave a reasonable recovery of arginine with good spots and is used routinely. -.. l'his'meth~d h,as been 'used routinely for amino acid' chromatography of urines at' . the Royal Army Medical College fo,r the past four years andchasproved' satisfactory .. Ij 1""0 • \," J R Army Med Corps: first published as 10.1136/jramc-112-03-12 on 1 January 1966. Downloaded from , T. Williamsori 173 Summary Deionising methods for urine amino acid chromatography are briefly reviewed with . the purpose of elucidating a satisfactory method using simple equipment suitable for routine use. A method suggest~d by Smith (1958) using an ion exchange resin has been investigated, modified and found satisfactory. REFERENCES CONSDEN, R., GORDON:A. H. and MARTIN, A. J. P. (1947). Biochem. J. 41, 590. SMITH, I. (1958) Chromatographic and Electrophoretic-Techniques. Vol. 1, ch 3. Heinemann. WOOD, T. (1956). Biochem. J. 62, 611. VERGHESE, N. and RAMAKRISHNAN, P. N. (1957). J. Clin. Path. 10,104. Honorary Conspltants To the Army ,Endocrinology Dr. J. D. N. Nabarro, M.D.~F.R.C.P., in succession to Dr. A. W. Spence who has retired. Oto-Rhino-Laryngology Mr. G. H. Bateman, M.A., RM., B.Ch., F.R.C.S., in guest. Protected by copyright. succession to Mr. Myles LFormby who has retired. To the Army in .Scotland ' Thoracic Surgery 'Mr. Andrew Logan, M.A., M.B., F.R.C.S.; F.R.C.S. (Edin.), in succession to Mr. B. W. Dick who has retired. To the Queen Alexandra Military Hospital (jto-Rhino~Laryngology Mr. R. A. Wiiliams; M.A.,M:B.;' B.Chir., F.R.CS;, as , " from 25th March. 1966. " President of the Royal College of Physicians oCLondon , " - " '. .:- "- Professor Max Leonard Rosenheim has been elected to succeed Sir Charles Dodds. http://militaryhealth.bmj.com/ " Commencing a military career at an ea~ly age in Shrewsbury School O.T:C., Professor,. Rosenheim was not ,deterred from a brilliant career at Cambridge and at University CoIIegeHospital. The War of 1939~45 saw him involved as ,Officer in Charge Medical Divisions of General Hospitals at home and in the Middle Easr. Before th~ close of hqstilities he applied' his great knowledge and experience as.a consultant in India in the' , \ " . rank of Brigadier. A much valued ~md reaaily 'given opinion at Millbank and, a much acclaimed guestlecturer at R.A.M.College, the Journal congratulates him on attaining high office.' ' on September 27, 2021 by Chair of Medicine-Post .Graduat~ Medical School, Hammersmith·· Dr.Christopher Charles Booth i~tosucceed Professor Sir John McMichaeI. As arating in the RoyalNavy in the 1939-45 War he was commissioned and later ob­ tained a watch keeping certificate before turning to medicine at st. Andrews., As lecturer in medicine. at the Post· Graduate Medical School he has. given gr~at support to Army:Medical researchesciri sprue.. ,\. .
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