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Diagnostic Test J Clin Pathol 1984;37:1305-1307 J Clin Pathol: first published as 10.1136/jcp.37.11.1305 on 1 November 1984. Downloaded from Immune deficiency due to adenosine deaminase and purine nucleoside phosphorylase deficiency: a simple diagnostic test JL MADDOCKS, SA AL-SAFI, G WILSON From the University Department of Therapeutics, Royal Hallamshire Hospital, Sheffield SJ0 2JF SUMMARY A simple method is described for diagnosing adenosine deaminase and purine nu- cleoside phosphorylase deficiency using urine. Cellulose thin layer chromatography of 1 of urine from affected children was performed and deoxyadenosine and deoxyguanosine were easily detected by phosphorescence at the temperature of liquid nitrogen. This test is not expensive and can be done in any laboratory. It should be suitable for diagnostic screening in patients with immune deficiency. Severe recurrent infections in childhood may occur detecting deoxyadenosine and deoxyguanosine. It is as a result of defects in the immune system. A major an extremely sensitive detection method. advance by Giblett et al was the discovery that these may be due to a deficiency in enzymes involved in Material and methods (EC recycling purines: adenosine deaminase' copyright. 3.5.4.4) and purine nucleoside phosphorylase2 (EC Urine samples from a child (SY) aged 2 months with 2.4.2.1). Without treatment both these conditions adenosine deaminase deficiency and another (SB) are usually fatal. aged 3 months with purine nucleoside phosphoryl- Adenosine deaminase deficiency results in lack of ase deficiency were supplied by Dr Anne Simmonds, both humoral and cellular immunity. It is associated Guy's Hospital, London. Both were obtained before with recurrent bacterial, viral, fungal and protozoan drug treatment. They were transported in solid car- infections starting within weeks or months of birth. bon dioxide and stored at -20°C until used, as Patients pass urine containing abnormally high con- deoxyadenosine and deoxyguanosine are unstable in centrations of deoxyadenosine.35 In purine nu- acid urine at room temperature.'3 Control urine http://jcp.bmj.com/ cleoside phosphorylase deficiency children have a samples (50) were obtained from children with vari- selective defect in cellular immunity and suffer from ous diseases treated at Sheffield Children's Hospital. repeated viral and fungal infections. Deoxy- Deoxyadenosine, deoxyguanosine and other guanosine in high concentrations is found in their chemicals used were obtained from Sigma Chemical urine.6 ' Company, Dorset. Stock solutions of 1 mmol/I were Two methods of diagnostic screening are available prepared in water and in urine and stored at 4°C. for these conditions: enzyme analysis8e" and detec- They were replaced every two weeks. Cellulose thin tion and measurement of abnormal purines by layers (Art 5552) supplied by Merck, Darmstadt, on October 1, 2021 by guest. Protected isotachophoresis and high performance liquid were 0-1 mm thick. They were non-activated, with- chromatography.3 They require technical expertise out fluorescent indicator, and mounted on and special equipment. aluminium sheets. Those mounted on plastic were We report a simple and cheap method for diag- less satisfactory owing to high background phos- nostic screening of urine for these disorders by thin phorescence. layer chromatography. Adenosine phosphoresces One microlitre samples of urine and standard sol- strongly at the temperature of liquid nitrogen utions of nucleosides were applied with a microlitre (-1960C),'2 and we have used this property for syringe over a 1 cm line, which was 2 cm from the edge of the sheet. The most effective solvent system for separation Accepted for publication 24 July 1984 of deoxyadenosine and deoxyguanosine from other 1305 J Clin Pathol: first published as 10.1136/jcp.37.11.1305 on 1 November 1984. Downloaded from 1306 Maddocks, Al-Safi, Wilson urinary constituents was sodium acetate (3%) con- Results taining lithium chloride (3 g/100 ml): methanol (30:70 vol/vol). Development of the chromatogram Deoxyadenosine was easily detected in the urine of to 12 cm took 100 minutes. Detection of phos- the child (SY) with adenosine deaminase deficiency phorescent compounds was carried out in a poly- and deoxyguanosine in the urine of the child (SB) styrene box which had been painted with black.'4 A with purine nucleoside phosphorylase deficiency central well had been cut out of the floor of the box (Figure). These compounds were absent in 30 to hold liquid nitrogen and this was covered by a samples of normal urine and in the urine of 50 chil- black painted steel plate. The chromatogram was dren with various disorders used as controls. They then placed on the steel plate in the box and liquid were undetectable by ultraviolet light at room temp- nitrogen was poured over it until it was just covered. erature. Their Rf values, colour of emitted light, The chromatogram was viewed in a dark room after duration of phosphorescence, and detection sensitiv- exposing it to ultraviolet light (254 nm) for a few ity are shown in the Table. Spraying the chromato- seconds. The colour of the phosphorescent bands gram with 1N hydrochloric acid abolished phosphor- was noted and the duration of light emission was escence due to deoxyadenosine and deoxy- measured by eye using a stop watch. As the guanosine. phosphorescence of deoxyadenosine and deoxy- There were several phosphorescent compounds in guanosine is affected by pH, the effect of spraying control urine which we have not yet identified. It is the chromatogram with 1N hydrochloric acid was of interest that the urine of child (SB) lacked all of also noted. these compounds, which were regularly present in A permanent record of the phosphorescence was control urine. Standards have been chromato- obtained by laying a medical x ray film (Singul-X graphed of the following compounds: adenine, RP, Ceaverken AB, Sweden) on to the chromato- guanine, hypoxanthine, xanthine, adenosine, gram as soon as the ultraviolet light had been guanosine, inosine, deoxyinosine, thymine, switched off and exposing the film for 20 s. thymidine, uracil, uridine, cytosine, uric acid, copyright. http://jcp.bmj.com/ on October 1, 2021 by guest. Protected 2 !..: 3 78 ....N:. Photograph ofan x ray fim which was placed on a cellulose thin layer chromatogram, immersed in liquid nitrogen, after exposure to ultraviolet light at 254 nm. One microlitre was applied ofeach ofthe following: (1) Deoxyadenosine (1 n mole) in water; (2) deoxyadenosine (I n mole in normal urine; (3) adenosine deaminase deficiency urine (SY); (4) deoxyguanosine (I n mole) in water; (5) deoxyguanosine (I n mole) in normal urine; (6) purine nucleoside phosphorylase deficiency urine; (7-9) control urines. J Clin Pathol: first published as 10.1136/jcp.37.11.1305 on 1 November 1984. Downloaded from Immune deficiency due to adenosine deaminase and purine nucleoside phosphorylase deficiency 1307 Properties ofsubstances detected in urine in adenosine References deaminase deficiency (SY) and purine nucleoside phosphorylase deficiency (SB) 'Giblett ER, Anderson JE, Cohen F, Pollara B, Meuwissen HJ. Adenosine deaminase deficiency in the patients with severely Rf Band Decay Detection impaired cellular immunity. Lancet 1972;ii: 1067-9. value colour time (s) sensitivity 2Giblett ER, Amman AJ, Sandman R, Wara DW, Diamond IK. (p mole) Nucleoside phosphorylase deficiency in a child with severely defective T-celi immunity and normal B-cell immunity. Lancet Deoxyadenosine in water 0-44 Blue 16 32 1975;ii: 1010-3. Deoxyadenosine in urine 0-44 Blue 16 32 3Simmonds HA, Sahota A, Potter CF, Cameron JS. Purine Urine (SY) 0-44 Blue 14 Deoxyguanosine in water 0-50 Blue 7 60 metabolism immunodeficiency: urinary purine excretion as a Deoxyguanosine in urine 0-50 Blue 7 60 diagnostic screening test in adenosine deaminase and purine Urine (SB) 0-49 Blue 7 nucleoside phosphorylase deficiency. Clin Sci Mol Med 1978;54:579-84. RSvalue = distance run by substance divided by distance run by 4Donofrio J, Coleman MS, Hutton JJ, Daoud DA. Overproduc- so vent. tion of adenosine deoxynucleosides and deoxynucleotides in adenosine deaminase deficiency with severe combined immunodeficiency disease. J Clin Invest 1978;62:884-7. 2,8-dihydroxyadenine, 1-methylhypoxanthine, 5 Mills GC, Goldbnum RM, Newkirk KE, Schmalstieg FC. Uri- 7-methylhypoxanthine, and caffeine. None of these nary excretion of purines, purine nucleosides and corresponded to the phosphorescent compounds in pseudouridine in adenosine deaminase deficiency. Biochem control urine. Med 1978;20 180-99. were in 6 Cohen A, Doyle D, Martin DWJ, Amman AJ. Abnormal purine No phosphorescent compounds detected metabolism and purine overproduction in a patient deficient in one sample of normal amniotic fluid by this method, purine nucleoside phosphorylase. N Engl J Med 1976; although added standards of deoxyadenosine and 295:1449-54. deoxyguanosine were easily detected. Stoop JW, Zegers BJM, Hendrickx GFM. Purine nucleoside phosphorylase deficiency and cellular immunodeficiency. N Engl J Med 1977;296:651-5. Discussion Kalckar HM. Differential spectrophotometry of purine com- pounds by . means of specific enzymes. J Biol Chem The absence of deoxyadenosine and deoxy- 1947; 167:461-75. in 9 Spencer N, Hopkinson DA, Harris H. Adenosine deaminase guanosine unaffected individuals allows the polymorphism in man. Ann Hum Genet 1968;32:9-14. simple qualitative detection of these compounds to '°Hopkinson DA, Cook PJL, Harris H. Further data on the copyright. be used for diagnosis. Four of their physicochemical adenosine deaminase (ADA) polymorphism and a report of a properties can be easily recorded so that high new phenotype. Ann Hum Genet 1969;32:361-7. Edwards JH, Hopkinson DA, Harris H. Inherited variants of specificity is obtained with little effort. This test can human nucleoside phosphorylase. Ann Hum Genet be done using less than 1 jul of urine and the abnor- 1971;34:395-408. mal urinary purines are easy to detect. 12 Steele RH, Szent-Gyorgy A. On excitation of biological sub- The absence of all the phosphorescent compounds stances.
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