The lack of purine salvage enzyme, adenine phosphoribosyltransferase (APRT), leads to 2,8-dihydroxyadenine stone formation and/or crystalluria because it is insoluble in urine. Urolithiasis composed of 2,8-dihydroxyadenine is not only formed in a complete defect of APRT, but also in a partial deficiency of this enzyme. The defect is inherited as an autosomal recessive trait, the homozygous state is associated with high urinary levels of 2,8-dihydroxyadenine and with crystalluria, calculus formation, and potential nephrotoxicity. Determination of the APRT activity will facilitate quantification of the enzyme deficiency and elucidation of the hereditary history. 2,8-dihydroxyadenine excretion in the 24-hour urine and its circadian rhythm were determined using a new method of high performance liquid chromatography determination. By means of a standard case presentation, we illustrate the analysis of urinary sediments and calculi as well as the scanning electron microscopic images of this kind of stone

Hesse, A.

Klocke, K.

Schaefer, R. M.

Winter, P.

English

DigitalCommons@USU

Scanning Microscopy Volume 7 Number 3 Article 31 6-5-1993 Scanning Electron Microscopy of 2,8-Dihydroxyadenine Crystals and Stones P. Winter Urologische Universitätsklinik Bonn A. Hesse Experimentelle Urologie der Universitätsklinik Bonn K. Klocke Urologische Universitätsklinik Bonn R. M. Schaefer Urologische Universitätsklinik Bonn Follow this and additional works at: https://digitalcommons.usu.edu/microscopy  Part of the Biology Commons Recommended Citation Winter, P.; Hesse, A.; Klocke, K.; and Schaefer, R. M. (1993) "Scanning Electron Microscopy of 2,8-Dihydroxyadenine Crystals and Stones," Scanning Microscopy: Vol. 7 : No. 3 , Article 31. Available at: https://digitalcommons.usu.edu/microscopy/vol7/iss3/31 This Article is brought to you for free and open access by the Western Dairy Center at DigitalCommons@USU. It has been accepted for inclusion in Scanning Microscopy by an authorized administrator of DigitalCommons@USU. For more information, please contact digitalcommons@usu.edu. Scanning Microscopy, Vol. 7, No. 3, 1993 (Pages 1075-1080) 0891-7035/93$5.00+ .00 Scanning Microscopy International, Chicago (AMF O'Hare), IL 60666 USA SCANNING ELECTRON MICROSCOPY OF 2,8-DIHYDROXYADENINE CRYSTALS AND STONES P. Winter*, A. Hesse 1, K. Klocke, and R.M. Schaefer U rologische U niversi tatsklinik Bonn, 1 Experimentelle U rologie der U niversitatsklinik Bonn, 53105 Bonn, Federal Republic of Germany (Received for publication August 7, 1992, and in revised form June 5, 1993) Abstract The lack of purine salvage enzyme, adenine phos-phoribosyltransferase (APRT), leads to 2,8-dihydroxy-adenine stone formation and/or crystalluria because it is insoluble in urine. Urolithiasis composed of 2,8-dihy-droxyadenine is not only formed in a complete defect of APRT, but also in a partial deficiency of this enzyme. The defect is inherited as an autosomal recessive trait, the homozygous state is associated with high urinary levels of 2,8-dihydroxyadenine and with crystalluria, calculus formation, and potential nephrotoxicity. Deter-mination of the APRT activity will facilitate quantifica-tion of the enzyme deficiency and elucidation of the he-reditary history. 2, 8-dihydroxyadenine excretion in the 24-hour urine and its circadian rhythm were determined using a new method of high performance liquid chro-matography determination. By means of a standard case presentation, we illustrate the analysis of urinary sedi-ments and calculi as well as the scanning electron mi-croscopic images of this kind of stone. Key Words: 2,8-Dihydroxyadenine, stone analysis, urinary sediment, scanning electron microscopy. * Address for correspondence: Peter Winter Urolog. Universitatsklinik Bonn Sigmund-Freud Str. 25 53105 Bonn, FRG Telephone Number: (0228) 280-2109 FAX Number: (0228) 280-3616 1075 Introduction In 1968, Kelly et al. described a formerly un-known disorder in purine metabolism caused by a defi-ciency of the enzyme adenine phosphoribosyltransferase (APRT) [8]. This defect is inherited as an autosomal recessive trait [l, 7). The complete or partial lack of the salvage en-zyme obstructs the conversion of adenine to adenosine monophosphate. The alternative metabolic route availa-ble for urinary excretion is the oxydation of the in-creased concentration of adenine to 8-hydroxyadenine (Fig. 1). The inadequate solubility of 2,8-dihydroxy-adenine (2,8-DHA) in the normal pH-range of human urine (namely 4.8-8.0) leads to crystalluria and stone formation [ 12]. Homozygotes tend to develop urinary calculi in early childhood. As stone formation in chil-dren does not occur frequently, the possibility of 2,8-DHA stones should come to mind when X-ray negative stones are found in children [I, 6, 7, 13). 2,8-DHA urolithiasis in homozygotes was first described in 1974 [2]. Since that time, 20 cases have been described in medical literature [9]. By means of a standard case presentation, we illustrate here the analysis of 2,8-DHA urinary sediments and calculi as well as the scanning electron microscopic (SEM) appearance of this kind of stone [11]. Materials and Methods A 30-year-old male patient, who had produced X-ray negative calculi in his left kidney since infancy, lost the kidney at 8 years of age (no stone analysis). At the age of 22, first spontaneous excretion of stones from the remaining right kidney was observed. The first stone analysis by infrared (IR) spectroscopy was made after a further 3 years and showed 2,8-DHA. In 1987, 8 con-crements in the right kidney were sonographically de-tected. The majority of the stones were removed percu-taneously. The remaining concrements were passed spontaneously. The stone analysis by IR spectroscopy revealed also 2,8-DHA. There was no known family history of lithiasis. Laboratory Investigations Urinary sediment: Microscopic examination of P. Winter, A. Hesse, K. Klocke, R.M. Schaefer nucleotide nucleoside IMlP~ ---- . /r HGPRT /one - adenosone T APRT purine hypoxanlhine adenine l XO XO I • xanthine 8-hydrox yadenine l XO XO I ' uric acid 2,8-dihydrox yadenine Figure 1. Diagram of purine metabolism. XO = xan-thine oxidase, APRT = adenine phosphoribosyltrans-ferase, HGPRT = Hypoxanthineguaninephosphoribosyl-transferase, IMP = Inosinemonophosphat, AMP = Ade-ninemonophospat. 0 • Figure 2. Microscopic examination of the crystalline urinary sediment containing 2,8-dihydroxyadeninuria spherical crystals in a radial corona. Bar = ??? µm. Figure 3. Scanning electron micrograph of 2,8-dihy-droxyadenine stone crystals in urinary sediment. Bar = 10 µm. 1076 b Figure 4. Infrared spectra of a 2,8-DHA (a) and uric acid (b). At wavelengths 3045, 3238 and 3338 cm-1, there are characteristic absorptions which do not occur in uric acid. 100 f----4 without therapy 1----1 3 x 1 00 mg Allopurinol 0 E :::i. 50 <f-I 0 CX) ,...; ----, .._ __ - -- __ .--- -, ___ -- ----- -- ---0 8-11 11-2 2-5 5-8 8-11 11-2 2-5 5-8 a.m. + p.m. ------a.m. Time of day, h Figure 5. 2,8-DHA concentration in urine with (solid line) and without 3 x 100 mg allopurinol treatment (dotted line). the crystalline urinary sediment was made after centrifu-gation. A large quantity of crystalline material was ob-tained for quantitative IR analysis. IR spectroscopic concrement analysis of the urinary sediment and the stone was performed using the KBr press process [14]. APRT activity: The activity of the enzyme was assayed in the erythrocyte hemolysat by a radiochemical technique [3]. Quantitative 2,8-DHA determination in the urine was performed using high performance liquid chromatography (HPLC) in conj unction with a separat-ing system specially developed for purine [4]. SEM investigations: In order to study the mor-phology of the concrements, their surfaces, stone inter-spaces, fracture surfaces, and a commercially available synthetic test substance of 2,8-DHA were examined by scanning electron microscopy (SEM). 2,8-Dihydroxyadenine crystals and stones Results In the presence of APRT deficiency, and depend-ent upon the scale of the enzyme deficiency, crystals were detected in the urine. Masses of crystals were al-ways present; they were spherical in shape and brown-red in color. Under a light microscope, the crystalline arrangement was spherolitic (Figure 2). Under the SEM, nests of individual crystals were detected, some 10 µm in diameter, which also aggregated to form microliths (Figure 3). 2,8-DHA can only be detected using modern chemophysical methods of analysis [5, I 0]. The chemi-cal method will always produce a false result indicating uric acid. Infra-red spectroscopic analysis indicated that both urinary sediments and stones consisted of pure 2, 8-dihydroxyadenine. The characteristic absorptions observed at wavelengths 3045, 3238 and 3338 cm- 1 do not occur in any other type of urolith, e.g., uric acid (Figures 4a, 4b). In order to obtain a clear picture of the extent of the enzyme deficiency, the lack of APRT activity in haemolysed erythrocytes must be determined. Caution is advisable if the patient has just received blood trans-fusions, which can lead to a temporary increase in APRT activity. The determination of 2,8-DHA in urine was car-ried out by the HPLC method. In the 24-hour urines of healthy test persons, no "-,8-DHA could be traced. If 2,8-DHA is found, it can always be regarded as an indi-cator of APRT deficiency. 2,8-DHA excretion rates in our patient ranged be-tween 125 and 160 µmol/1 (i.e., 245-414 µmol per 24 hours). Treatment with 300 mg allopurinol per day re-duced the daily excretion to 40 µmol/day. The excre-tion of 2,8-DHA shows a characteristic day/night rhythm. Increased rates were registered at night and in the morning hours. The administration of the xanthino-xydase inhibitor allopurinol reduced excretion rates to a constantly low level, while adenine concentration in urine increased (Figure 5). SEM examination of the synthetic test material after fast recrystallization revealed plaque-shaped structures (Figure 6), and after slow recrystallization spherical, matted structures (Figure 7). With increasing magnification, the 2,8-DHA stone shows a spherical surface, a felt-like surface and spherolite surface with matted needle-shaped crystals (Figures 8, 9, 10 and 11). The SEM examination of structures in stone interspaces revealed mostly needle-shaped crystals (Figure 12), although, sometimes lance-shaped crystals were also observed (Figure 13). After fragmentation of a 2,8-DHA stone and SEM examination of individual fragments, microliths with an irregular surface, and sometimes a fracture surface with peripher-al regular crystals, were observed (Figures 14, 15, 16 and 17). 1077 Discussion Only the introduction of new methods of urinary stone analysis such as X-ray diffraction and IR spectros-copy have made it possible to identify 2,8-DHA calculi. There is no structural difference between 2,8-DHA and uric acid. Since there is still no way to heal this "in-born error of metabolism", recurrence of stone forma-tion can only be prevented by strict life-long treatment. The SEM has been instrumental in yielding valua-ble new data in the field of stone research. The variety of microscopic forms and the different structures of urine stones can be observed in details with the SEM. Whereas examinations have been carried out with the SEM on most types of urinary stones, an analysis of the 2,8-DHA stone, due to its extreme rarity, was lacking. This paper presents SEM analysis of the test substance after recrystallization, urinary sediment, stone surface, stone interfaces, and fracture surface of the 2,8-DHA stone. Once a 2,8-DHA stone has been diagnosed, the enzyme deficiency can be determined by measuring APRT activity in erythrocyte haemolysate. Making use of this relatively simple test, a possible hereditary factor may be determined in other family members. The HPLC method permits a quantitative determi-nation of 2,8-DHA excretion in urine. With this meth-od, the monitoring of treatment is easy and reliable. In addition to allopurinol therapy, the dilution of urine throughout the day, sufficient fluid intake, and a low-purine diet are advised to eliminate 2,8-DHA excretion in urine. References 1. Bruh I P, Hesse A, Gu R ( 1987). Harnsteiner-krankungen im Kindesalter. Atiologie, Diagnostik, Therapie und Metaphylaxe. (Urinary stone disease in Childhood. Etiology, Diagnosis, Therapy and Metaphylaxis). Wiss. Verl.-Ges, 63-68. 2. Cartier P, Hamel M (1974). Une nouvelle maladie metabolique: Le deficit complet en adenine-phosphoribosyltransferase avec lithiase de 2,8-dihy-droxyadenine (A new metabolic disease: The complete deficiency of adenine-phosphoribosyltransferase with lithiasis of 2,8-dihydroxyadenine). C. R. Acad. Sc. Paris. 279, 883-886. 3. Doppler W, Hirsch-Kauffmann M, Schweiger M ( 1981). Characterization of the biochemical basis of a complete deficiency of the adenine phosphoribosyl-transferase (APRT). Hum. Genet. 57, 404-410. 4. Hesse A, Thon A, Classen A, Birwe H (1987). Diagnostic and therapy-control of inborn metabolic dis-orders by high-performance liquid chromatography: 2, 8 dihydroxy-adeninuria, xanthinuria. Chromatographia25, 205-209. 5. Hesse A, Sanders G (1988). Atlas of Infrared Spectra for the Analysis of Urinary Concrements. Thieme Stuttgart. pps. 22. P. Winter, A. Hesse, K. Klocke, R.M. Schaefer .. ,. 7'. f.:. •... f. "' Figure 6. Synthetic test material. Plaque-shaped structure. Fast crystallisation. Bar = 10 µm. Figure 7. Synthetic test material. Spherical, matted structure. Slow crystallisation. Bar = 10 µm. Figure 8. Spherical surface. Bar = 100 µm. Figure 9. Spherical, now felt-like surface. Bar = 100 µm. Figure 10. Spherolite surface with matted needle-shaped crystals. Bar Figure 11. Similar structures with regular crystals. Bar = 10 µm. 1078 10 µm. 2,8-Dihydroxyadenine crystals and stones Figure 12. Needle-shaped crystals. Bar = 10 µm. Figure 13. Lance-shaped crystals. Bar = 10 µm. Figure 14. Microlith with irregular surface. Bar = 10 µm. Figure 15. Doubled magnification shows a similar surface structure. Bar = 10 µm. Figure 16. Fracture surface of a 2,8-DHA stone. Bar = 10 µm. Figure 17. Fracture surface with peripheral regular crystals. Bar = 10 µm. 1079 P. Winter, A. Hesse, K. Klocke, R.M. Schaefer 6. Joost J, Doppler W (1982) 2,8-Dihydroxy-adenine stone in Children. Urology 20, 67-70. 7. Joost J, Hesse A (1987). Der 2,8-Dihydroxy-adeninstein. In: Das Harnsteinleiden. Ursachen, Diag-nose, Therapie (The 2,8-dihydroxyadenine stone. In: Urolithiasis. Causes, Diagnosis, Therapy). Vahlensieck W (ed.). Springer-Verlag. 542-549. 8. Kelley WN, Levy RI, Rosenbloom FM, Henderson JF, Seegmiller JE (1968). Adenine phospho-ribosyltransferase deficiency: A previously undescribed genetic defect in man. J. Clin. Invest. 47, 2281-2285. 9. Leusmann DB, Schmidt G (1990). Urolithiasis bei 2,8-Dihydroxyadeninurie: Vorstellung von drei weiteren Fallen (Urolithiasis of 2,8-dihydroxyadenin-uria: Presentation of further cases) . Z. Urol. Nephrol. 83, 383-389. 10. Leusmann DB (1982). Erste zusammenfas-sende Ergebnisse der kombinierten Phasen-und Gefilge-analyse von Harnsteinen mittels Rontgenbeugung und Rasterelektronenmikroskopie (First results of combined phase- and structure analysis of urinary calculi with X-ray diffraction and scanning electron microscopy). Fortschr. Urol. Nephrol. 17, 275-305. 11. Leusmann DB (1983). Routine analysis of urinary calculi by scanning electron microscopy. Scanning Electron Microsc. 1983, 387-396. 12. Peck CC, Bailey FJ, Moore GL (1977). En-hanced solubility of 2,8-dihydroxyadenine in human urine. Transfusion 17, 383-390. 13. Sakamoto K, Fujisawa Y, Ohmori A, Minoda K, Yamanaka H, Nishioka K (1981). Dihydroxyadenine urolithiasis in children with partial deficiency of adenine phosphoribosyltransferase. Urol. Int. 36, 274-280. 14. Simmonds HA, Acker KJ van, Cameron JS, Snedden W ( 1976). The identification of 2 ,8-dihydroxy-adenine, a new component of urinary stones. Biochem. J. 157, 485-491 Discussion with Reviewers H. Iwata: Was dissolution carried out by heating or changing pH? Was the substance dissolved in pure water, buffer solution, and urine? Authors: 2,8-DHA was obtained from Sigma (Article no. D7382). The substance was heated in pure water at neutral pH at 80°C. After filtration, one part was cooled down to room temperature (slow crystallization), and the other part in ice water (fast crystallization). D.B. Leusmann: Is there a possibility to resolve 2,8-DHA stones either by oral medication (similar to uric acid stones) or by direct dissolution after pyelostomy and/or indwelling ureteral stents? Authors: The dissolution of 2,8-DHA stones by oral medication is not possible. Solubility of 2,8-DHA stones in the pH-range of 4.8-8.0 varies between 3 and 5 mg/I. A considerable increase in solubility can be achieved at a pH-range less than 2 or more than 9. 5; however, such extensive changes in human pH-values 1080 cannot be achieved through oral medication. In any case, attempts to change the pH-value by means of per-cutaneous chemolitholysis will fail because of incompat-ible tissue tolerance; even if this problem was irrele-vant, the many potential side effects of this kind of therapy would forbid its application. S.R. Khan: What are the some of the other features associated with this disease? Authors: Other features associated with APRT-defi-ciency on its own are not known. APRT-deficiency, when not treated, may lead to nephrocalcinosis with resultant renal failure. A. Rogers: In the Materials and Methods section, you mention that the first stone analysis showed 2,8-DHA. What technique was used for this analysis? Authors: We used infrared spectroscopy. A. Rogers: Do you think that the brown red colour of the 2,8-DHA crystals is due to blood stains? Uric acid microliths are often red orange. Were the 2,8-DHA crystals similar in colour to uric acid crystals? Authors: 2,8-DHA crystals of brown-red colour ap-peared in several patients and cannot be attributed to blood stains in our opinion. It is most likely that urine colouring matter may be adsorbed by 2,8-DHA, which causes this kind of stone to appear very similar to a uric acid stone. A. Rogers: Figures 11, 12 and 13 show crystals which are very similar to the morphological features of sodium urate. Were no other stone components besides 2,8-DHA detected (e.g., uric acid and salts thereof)? Authors: Analyses by IR proved 2,8-DHA stones to be extremely pure. Components of uric acid or salts thereof are rather unlikely. A. Rogers: How sensitive are your IR measurements? Authors: The sensitivity of IR spectroscopic measure-ments is dependent on the composition of substances in-vestigated. All components exceeding 5 % can be un-mistakably determined. A. Rogers: Did you consider verifying your results by subjecting the crystals to another analytical technique such as as X-ray powder diffraction or mass spec-trometry? Authors: Results achieved through IR spectroscopy were confirmed by X-ray diffraction. Mass spectroscopy was not applied. Editor: Am I correct in understanding that Figures 8 to 17 are from different stones collected from your 30 year old patient? Authors: Yes; Figures 6 and 7 are scanning electron micrographs from synthetic crystals; while Figures 8 to 17 are from stones collected from the patient. 

Scanning Electron Microscopy of 2,8-Dihydroxyadenine Crystals and Stones

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Scanning Electron Microscopy of 2,8-Dihydroxyadenine Crystals and Stones

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