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ARAB REPUBLIC OF EGYPT ATOMIC ENERGY ESTABLISHMENT NUCLEAR CHEMISTRY DEPARTMENT
NEUTRON ACTIVATION ANALYSIS CF URINARY CALCULI
By
\\ SOUKA, S. SOUKA, M\ SANAU 1974 SCIENTIFIC INFORMATION DIVISION ATOMIC ENERGY POST OFFICE CAIRO, A.R.E. CONTENTS A B S T R A C T i INTRODUCTION , . 1 EXPERIMENTAL 2 RESULTS AND DISCUSSION 5 ACKNOM'IEDGEMTWT?? 10 APPENDIT 11 REFERENCES 14 NEUTKON ACTIVATION ANALYSIS Of U!UNAT>V CALCULI By N. SOUKA, S. SOUKA+, W. SANAD and A.A. ABT)EL-RASSOUL ABSTRACT Neutron activation analysis of u collection of urinary calculi has been undertaken,, Search for silver, selenium, zinc and cobalt -was carried out. Cobalt presented a measurable concentration in all the investigated calculi. The cobult content in oxalate stones (around 1200+93 ppn>)> was found to be approximately five times its value in phosphate stones (around 200+21 ppm). The cobalt content in the examined calcium oxalate- triple phosphate mixed calculi lies betireen both concentration levels. Clinical pathology Laboratory Sidnawy Health Ensurance Hospital, Cairo. IflEHODUCTlOfi Recently, several investigatore have shown that a defi nite relationship exists between the concentration level of several trace elements in human body and certain diseases. The determination of trace eiemenos is further considered as a helpful tool to clarify many physioiogical processes. In this connection, activation analysis offers considerable pote ntialities relevant to both reliability and high detection sensitivity of various elements. Trace analysis of urine, was one of the subjects treated by neutron activation analysis. Determination of Ha, G.l,' Al, Br, I, Au, Mn, Sr, as well as radioactive deposits and several toxic elements was made with, great precision^ K xuie^ * , has determined the detection limits for 72 elements in'urine, these limits were in some cases as low as 0,000^ ppm.• Urinary calculi resulting from disorders in the urinary system and formed in any part of the urinary tract, have been classified by biochemists into simple calculi and mixed cal culi^'* '. In general, these stones are mostly composed of substances normally excreted in the urine, together with a certain amount of protein material. On rare occasions, for eign bodies may be found and may form the nucleus of a stone. Substances found in calculi include : uric acid, urates, cal cium oxalate, alkaline earth phosphates (Ua and-MgJ, triple phosphate (.magnesium ammonium phosphate.), calcium carbonate, cystine, and xanthine^"''. It is unusual to find a stone com posed of only one urinary constituent-, though it is often that one of the substances present is- in marked preponderance. .Procedures for the determination of macro constituents in uri nary calculi have been well established^'"*"'. No attempts, apart from the study reported by herring et al. , have been undertaken'to determine trace elements in urinary stones. _ o „ In a radiological surveillance of inorganic urinary calculi Herring et al. , indicated "that urolithiasis patients con sume approximately three times the concentration of strontium jii the national diet data (.^r concentration was measured with reierence to Ga concentration;. The present v.ox'ii. is a uon-'Ue?aoruc't»ive neutron activation analysis of urinary calculi, ana it aims maimy at studying the variation in concentration of certain trace elements with the chemical form or the calculus«, EJCWSHIMia'AL Physical and_OhemicaI Analysis of Oaicuii: A collection of urinary calculi, surgically removed from urolithiasis patients, and amounting to about ii? stones, has been analyzed physically and chemically^°»"'(see Appendix I;. These calculi were characterised by certain differences in physical and chemical properties* Pending on these differen ces, the collection was divided into three groups, see Table (i;. uroup I s oxalate stones consisting mainly of calcium oxa latef Group II s phosphate stones containing either triple phos phate or calcium phosphate© Group .III i mixed stones of the two f orementioned types. Neutron Activation Analysis of Calculit JSrperxments were conducted along the same lines set forth for activation analysis^ \ The different calculi were weighed and encapsulated in thin aluminium foils. Known weights (. yug concentrations; of the following standards : silvert selenium, zine and cohalt, were separately encapsulated in the same man ner. Both the test samples and monitoring standards were.irr- - 3 - Table (l): physical and Chemical Analysis of Urinary Calculi. Group I Group II Group ITT Physical Analysis : Colour dark brown •white yellow LayerB numerous dense numerous white concenteric yellow brown concen- yellow concen layers frith white teric layers ter ic layers. nucleus und surface with vhite shows fine man*illa nucleus. tions . JJaulitative Chemical Analysis: Carbonate — trace truce Calcium moderate nil truce Magnesium nil triple phosphate triple phosphate Oxalate +++ nil + Phosphorus trace in +++++ +++ nucleus Uric acid ++ ++ Urates ++ ++ Ammonia trace + trace Fibrine + nil nil Cystine trace nil nil Xanthine nil nil nil .„ 4 - 13 2 adiated in the UA-KR-1 at a mean neutron flux of 10 yn/cm sec. for 25 hours. After a suitable cooling period (about 6 weeks.), each of the activated calculi was transferred to a fx^esh plastic vial and set for gamma pulse height analysis. The first step in the analysis, is to identify the com ponents in the gamma ray spectra o± the different calculi. This has been undertaken by tracing the gamma spectra of each calculus using a true coaxial Ge(Li) detector, 30 cnr intre- nsic volume^ "•', connected to a multichannel (512 channels) counting assembly, model E.D* 120o Standard gamma sources of Am241 (59.568 KeV), Co^7 (122.05 KeV and 136.406 KeV) and Gs *' (661o59 KeV) were used to calibrate the energy scale. The counting period was 10 minutes in all cases. The second step in the analysis, is to determine the amounts of the elements of interest in the different calculi. This is carried out by a comparison of the photopeak areas in the compound spectra of the calculi with those of the res pective irradiated monitoring standards, accounting for any differences in experimental conditions (cooling time and coun ting geometries). - 5 - HBSUI/TS AND DISCUSSION Tracing the gamma spectra of the irradiated calculi, in dicated that the neutron induced nuclides Ag (£•, = 260 d9 main X-energies 0.45, 0.68, 0.88 and 1.39 MeV), Se75 (T-^ 120 d, main X -energies 0.12, 0.14, 0.28 and 0.4C MeV;, and Zn65 (Ti/ = 24-5 dp /6 -enrgy 0.31 annih., 1.11 MeV) could not be qualitatively detected in the complex measurements carried, oirtr • On the other hand, Go proved to be pre sent in all calculi, as characterised by peak energies 1.17 fin Me? and 1.32 MeV. '-d© identity of Co was further evidenced. by giving an almost constant GE/ 1.17 J CE^ 1.33 ratio in. the spectral measurements of the different samples analyzed and that of the monitoring Co-standard (where: G,represents the net integral counts under respective photoelectric pealc)* An example of the produced spectra is given in. Fig. (1J>. From the fore-lined considerations, it is clear that no accurate estimates for Ag, Se and Zn, could be given. This is probably due to their absence or presence in concentrations beyond the detection limits under used experimental conditions. For the quantitative estimation of cobalt, the half area under the higher side of the photoelctric peak of the 1.^3 MeV line has been accurately measured, and compared with that of the irradiated cobalt standard. This eliminates the pos sible interference of contaminants that might exist at lower energies. Fig,(2), represents an enlarged scale of this peak, for three different calculi,, each belongs to one of the investi gated, groups, i'he integrated, counts, have been normalized to one gram of the investigated, samples, and the analytical results are represented in. , Table (.2). Prom the tabulated data, it is obvious that the cobalt content changes with the chemical form, of the s-cone. Gobalt is more concentrated in calcium ... 6 _. 1.17 MeV 1.33 MeV - • ^ , n 13 -2 j3f ~ 10 n,,CB) „sec t. irr ^± 25 hours, 'cooling 6 weeks. In minutes, counting Ge(Li) 30 car I ± 40 80 120 160 200 240 280 320 Channel Number Fig. (l)s An example of Produced Gamma Spectra. - 7 - 270 275 280 Channel Nuiuber Fig. (2): The 1.33 MeV $ -peak for Representative Samples of the Different Calculi. -- 8 - oxalate calculi (giving an average cobalt content of 1200 +, y> ppiii), ana is less concentrated in the triple phosphate calculi (having a mean value of 200 ± 'dl ppnO. Calculi of xiiixed calcium oxalate and triple phosphate (group III), con tain cobalt in concentrations that lie between the two fore- mentioned concentration levels, Tabic (2)., Hue ooserved llu- uvuai-iona in me cobalt contents for this type of calculi, could be attributed to differences in the relative proportions of the oxalate and phosphate components. Generally speaking,, oxalate stones are formed in acidic urine, while phosphate stones are formed in alkaline urine„ The acidity of urine, however, could not explain the observed inarmed variation in cobalt concentration* On the other hand, the etiology of urinary calculi is by no means clear9 possible factors may bei the high concentration of urinary salts, var ious infections, deficiency in vitamin A, para thyroid tumor ana nephrocalcinosis* Unfortunately, no clinical interpreta tion could be set in the light of the available data, and further work is necessary to clarify this point. The analysis of urine, serum, diet and the follow up of patients may be of help in this concern,, - 9 Table (a)s Neutron Activation Analysis of Urinary Calculi. Sample No, Weight of Irradiated4"' Cobalt Content**' Stone (g) (ug/g) Group I : Calcium oxalate atones; 1-1 0.4838 1179 1-2 1.3204 1302 1-3 0.6245 1052 1-4 0.8653 1170 1-5 1.0263 1304 mean 1201+93 Group II s Triple phosphate stone as II-l 3.2556 201 II-2 2.1429 176 II-3 1.2658 234 II-4 0.3437 184 II-5 0.7627 212 mean 20l+2l Group III s Mixed calcium oxalate & triple phosphate stones; III-l 0.9342 736 111-2 0.8667 465 ITI-3 0.9428 592 III-4 0.8732 879 III-5 0.6548 940 +) Pile activation -was carried out in the 2 MW Research Reactor, UA-RR-1, for 25 hours, with a mean flux of I0*^n/cm2sec; followed by a cooling period of about 6 weeks. ++) Nuclear measurements were done through 10 minutes counting under the same geometry, using a 30 cm^ Ge(Li) detector, connected to a multi channel analyzer. The analytical data are based on the half area under the higher energetic side of the photoelectric peak of 1.33 MeV line. ACKNOWLEDGEMENTS The authors would lilce to acknowledge Dr. £• Foda for furnishing the calculi, and Dr. E.A.Eissa for providing facilities for pulse-height analysis. - 11 - APPENDIX Chemical Examination of Urinary Calculi^ ^: The stone is washed with water and then dried in an in cubator,fcJince most stones are mixtures and may consist of several layers, including a nucleus, it is desirable when ever possible to separate these out and examine them sepa rately. So the stone should be cut into two halves and exa mined for the presence of such layers and nucleus. Reagentsi 1 - Dilute HG1, 2 - Concentrated HNC%, 3 - Ammonium molybdate solution, freshly prepared by dissolving a few crystals in water, 4- - KOH, 10% solution, 5 - Nessler's reageent, 6 - Dilute ammonia solution, 7 - Concentrated ammonia solution9 8 - Acetic acid, y — Ammonium oxalate 4 per cent solution, 10- Potassium dihydrogen phosphate 10 per cent solution, 11- Alcohol, 12- Ether Tehnlque: The calculus is powdered and examined as follows: Heat a small amount of the powder in a small crucible. Since most calculi contain at least a small amount of organic mat ter there is always a little charring, but if the stone is almost entirely inorganic this will be small in amount and a greyish powder will be left. If the stone is purely organic as in uric acid and crystine stones it will burn away entirely or leave only a trace of ash. - 12 - Watch carefully for the presence of a flame. Uric acid amm onium urates and xanthine burn away without producing a flames but cystine gives a pale blue flame with a rather sharp smell, while fibrin and urostealith give yellow flame, the former with a smell of burnt feathers. (.A; If the substance burns away completely it contains only organic matter. Oxalate, phosphate and carbonate are absent. Proceed as follows: (.IJ Test for uric acid and ammonium urate by the murexide test: Add 2 or J drops of concentrated nitric acid to a small amount of the substance in a small evaporating dish and eva porate to dryness. The test is positive if a red or yellow residue is obtained which after being allowed to cool changes to a puplish-red on addition of a drop of dilute ammonium hydroxide„ Xanthine does not give a murexide test. It dissolves in nitric acid leaving a yellow residue which on addition of alkali changes to orange and to red on warming. {2) Test for cystine : if the murexide test is negative. Dissolve some of the calculus in concentrated ammonia and al low this to evaporate off on a watch glass. Hexagonal crys tals of cystine will be deposited if it is present and should be examined microscopically. Cystine crystals are insoluble in acetic acid and in water, alcohol, ether and acetone but dissolve in dilute mineral acids, unlike uric acid crystals, which do not* ($) Urostealith : in addition to burning with a yellowish, flame with a resinous odour, is distinguished by its solubi lity in alcohol and ether, CO Fibrin : is insoluble in alcohol and ether but dis solves in hot potassium hydroxide solution from, which it is ~ 13 - precipitated by acetic acid, hydrogen sulphide being liberated. {H) If a deposit remains after heating on platinum, inorganic material is present* Test as follows: (.1J Add a little dilute hydrochloric acid to a small por tion of the stone. An effervescence shows the presence of carbonate, {'d) After allowing to coox, add a little dilute hydro chloric acid to the ash remaining after heating on platinum. An effervescence now, in the absence of one before heating shows the presence of oxalate. To confirm, treat a small amount of the powder with warm dixute hydrochloric acid and filter. Only phosphates, cystine and oxalates dissolve. Maice alkaline with ammonia,, Oxalates and phosphates will be precipitated* The former are insoluble, the latter soluble, in acetic acidB (3) Dissolve a little of the powdered stone in a few ml. of concentrated nitric acid and then add an equal volume of ammonium molybdate solution., Heat to boiling. If phosphates are present, a yellow precipitate of ammonium phosphomoiybdate is obtained. (4) Heat a little of the powder with 10 per cent. Potas sium hydroxide solution, involution of ammonia shows the pre sence of either triple phosphate or ammonium urate. Messier's reagent may be used in testing for ammonia. (5; Phosphates, if present when test for ammonia are ne gative are those of calcium ana magnesium. The presence of these elements can he confirmed by ordinary methods of analysis. Jtor calcium adjust the pH to > with ammonia and acetic acid, and add ammonium oxalate solution., A precipitate shows the presence of calcium. Eilter this off, make alkaline with amm onia and add a solution of potassium dihydrogen phosphate. A precipitate on standing indicates magnesium. The red colour with titan yellow and sodium hydroxide can he used to test for mag- - 14 - REFERENCES ( 1) D.E.Bryan, V.P.Ciuinn and M.Dorothy, .Nuclear Activation Techniques in Life Sciences, Vienna, Internation al Atomic Energy Agency, (1967)-. ( 2) B.Moav, Intern. J. Appl. Radiation Isotopes, 16, 365(1965). V. j>) EoP.Elkas and A.G.Soulistis, Analyst, ^1, 199 (196o)„ ( 4) Hoi-oYule, Anal. Ghem. , £8,818 (1966). C 5) SoSolvsten, Scan. J". Clin. Lab. Invest., 16, 39 (1964). ( 6) AoAoSmales and B.D.Pate (AKRB-C/R-859/3) (1952). ( 7) WoBoyd, "Text Book of Pathology", Henergy Kimpton, London (1961)«, ( 8) HoVariey, "Practical Clinical Biochemistry", Interscience Books Inc., New York (1969). ( 9) E0JpKing, "Micro Analysis in Medical Biochemistry", J.A. Ghurchil Ltd., London (1970). (10) Herring, Louis, Keefer and Souglas, Arch. Envirom., Health, 22, 251 (1971). (11) BoA.Eissa and H„M. A"bou-Zeid, ARE ABB/Rep.-I61 (1961). (12) D.Strominger, J.M.Hollander and G.T.Seaborg, Review of Modern Physics, *>o , 2 (195^).