infrared and raman spectroscopy of urinary calculi: a review

Infrared and Raman Spectroscopy of Urinary Calculi:A Review

PEDRO CARMONA,1 JUANA BELLANATO,1 ELENA ESCOLAR2

1 Instituto de Estructura de la Materia (CSIC), Serrano 121, 28006-Madrid, Spain

2 Facultad de Veterinaria, Universidad Complutense, 28040-Madrid, Spain

Received 2 January 1997; revised 11 April 1997; accepted 29 May 1997

ABSTRACT: The application of infrared and Raman spectroscopic techniques to theanalysis of urinary calculi is reviewed and their relative efficiency and adaptability toroutine analysis are discussed. Using the classification of urinary calculi based on theirmain constituents, infrared and Raman spectra of calcium oxalates, phosphates, uricacid, urates, and cystine are reported. Some characteristic bands are suggested asuseful for analytical purposes. References to other constituents such as drugs are in-cluded. Although this review is aimed principally at human stones, it also extends toliterature references dealing with urinary calculi from canine, feline, and equine animalspecies. q 1997 John Wiley & Sons, Inc. Biospectroscopy 3: 331–346, 1997

Keywords: urinary calculi; FTIR spectroscopy; Raman spectroscopy; infrared; calculianalysis

INTRODUCTION able. On the other hand, light microscopy andelectron microscopy without diffraction are lim-

Among the outstanding achievements of the last ited in that they do not generally provide informa-three decades has been the establishment of tion on the nature of the phases present.methods for the determination of the structure Information about the identity of the mineraland composition of urinary calculi, which is of in- components of calculi can be obtained without dif-terest both for the correct identification of the type ficulty by infrared spectroscopy. The current gen-of stone disease and to provide clues to etiopatho- eration of Fourier transform infrared (FTIR)geny. spectrophotometers provide data of extremely

Techniques aiming at these objectives include high signal-to-noise ratio that can be manipulatedlight and electron microscopies, X-ray diffraction, easily by computer, enabling, for example, sub-and infrared and Raman spectroscopies. Tradi- traction of spectra, scaling to account for concen-tionally, X-ray diffraction is the technique of tration differences, calculation of integratedchoice for the determination of particle size and areas, and analyses of the components of complexcomposition of biological crystals. However, overlapping peaks.1,2 Second derivative spectraground samples are required, thereby prohibiting can be computed, permitting better defined bandsthe analysis of the morphological distribution of and thus facilitating the measurement of peak po-components. Microbeam X-ray diffraction can be sitions. Moreover, Fourier deconvolution tech-performed on microscopic sections of urinary cal- niques, through which the width of the spectralculi. This technique, however, is not readily avail- peaks may be reduced, permit the separation of

overlapping bands into their constituent sub-bands. Through the coupling of an optical micro-Correspondence to: P. Carmona.

q 1997 John Wiley & Sons, Inc. CCC 1075-4261/97/050331-16 scope to a Fourier transform spectrometer, these

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8W0C 96-062/ 8W0C$$6062 08-12-97 09:26:22 biosa W: Bio Spec

332 CARMONA, BELLANATO, AND ESCOLAR

spectral methods can be used to evaluate each different layers separately and a quantitative, orat least a semiquantitative, analysis, if necessary.component at discrete sites within the calculi at

a spatial resolution of 10–20 mm (the diffraction The use of pellets can be replaced by diffusereflectance methods, in which the detector collectslimit of infrared radiation).1

Raman microprobe spectroscopy also permits the light reflected diffusely by a powder instead ofthat transmitted by a pellet.5 Diffuse reflectanceevaluation of the structure of the components at

the microscopic level.3 The main disadvantage of overcomes some disadvantages associated withthe use of KBr pellets. There is no exchange ofRaman spectroscopy is the inherent interference

from the fluorescence arising in this case from the ions and no phase changes because KBr and calcu-lus are mixed at ordinary pressure. Organic com-organic matrix of urinary calculi when subjected

to visible or near-UV laser excitation. However, pounds adsorbed by KBr are automatically sub-tracted from the calculus spectrum through userecent innovations in Raman technology have led

to the development of an approach whereby the of the ratio R Å I /I0 in Kulbelka-Munk’s function,and atmospheric water is quickly desorbed fromspectra are excited with near infrared radiation

at 1.06064 mm. The loss in scattering intensity the powdered KBr by the stream of dry air flowingthrough the sample compartment. Another obvi-(which is approximately to the fourth power of

the excitation frequency) is overcome by the mul- ous advantage of diffuse reflectance is the rapiditywith which the sample can be prepared. In routinetiplex advantage of sending the radiation through

an interferometer for generation of the frequency analysis, only two short steps (grinding the calcu-lus and mixing the power with KBr) are requiredspectrum. In addition, the fluorescence is not ex-

cited because of the long wavelength. The tech- for diffuse reflectance, whereas preparing a pelletinvolves a further, more complicated, procedure.nique, termed FT–Raman spectroscopy, has not

yet been widely applied to urinary calculi but may For microscopic measurements, FTIR micros-copy can be used to identify components of calculieventually prove to be a useful complement to

FTIR spectroscopy. at discrete sites with good spatial (10–20 mm)and spectral resolutions, excellent signal-to-noiseIn this review, we survey the infrared and Ra-

man spectroscopies used as analytical techniques ratios, and minimal sample preparation.1,6

All relevant components can be identified fromfor urinary calculi. After discussing the maincharacteristics of stone analysis methods, we at- the spectra, using the techniques described above,

and even components with different amounts oftempt to show that these methods are a usefulmeans of classifying urinary calculi on the basis water crystallization can be determined. Ac-

cording to the literature, about 30 distinct compo-of their chemical composition. Such informationcan point to a specific process of stone nucleation nents have been found in urinary calculi. More-

over, the number of drugs or drug metabolitesor stone etiopathogeny. Although this review con-cerns human urinary calculi, a section referring appearing in calculi appears to be increasing. The

components most commonly found in urinaryto the applications of these techniques to animalcalculi is also included. stones are calcium oxalate (monohydrate and di-

hydrate), calcium phosphate (particularly car-bonate apatite), magnesium ammonium phos-phate hexahydrate (struvite), uric acid and

METHODS OF SPECTROSCOPIC ANALYSIS urates, cystine, and others. Besides, it is wellknown that although a particular component usu-ally predominates, a mixed mineral compositionInfrared Spectroscopyis found in many calculi. With reference to theinfrared spectra of the most common calculi com-With infrared spectroscopy, a transparent pellet

is prepared out of a small amount of fine powder of ponents, the interested analyst may consult theappropriate literature, where the spectra of thesethe calculi material and potassium bromide under

pressure using a specially evacuated die.4 Ab- components have been compiled.4,7–13

sorbance is then recorded against energy (cm01) .Infrared spectroscopy also has advantages over

Raman Spectroscopyinstrumental methods such as X-ray and electrondiffraction that are not valid in the case of amor- This spectroscopic technique is finding increasing

application to the study of urinary calculi.14–16 Asphous or poorly crystalline materials. Further-more, this technique allows the ready study of a general rule, the Raman examination of calculi


IR AND RAMAN SPECTROSCOPY OF URINARY CALCULI 333

is experimentally simple. Raman spectra can be aging of their signals using computer-as-sisted Raman spectrometers.measured either from powdered specimens con-

tained in capillary tubes or through direct focus- 3. Sometimes there is an advantage to usingdifferent laser excitation lines. Fluorescenceing of the laser beam at discrete sites on the calcu-

lus surface. Another alternative is the laser Ra- depends on the frequency of excitation. Atsome frequencies, fluorescence is produced,man microprobe,3,11,17 which is a combination of a

microscope and a Raman spectrometer. The total whereas at other frequencies, there is nofluorescence. Thus, there exists an optimumimage of the sample can be obtained in the micro-

scope when the laser light (n0) is used. This image choice of incident frequency to achieve amaximum signal-to-noise ratio for any givenis the same as that seen with a conventional mi-

croscope. The light can be filtered with the micro- sample. In this connection, exciting radia-tion from the near-infrared range is usuallyprobe, and the sample is observed with Raman-

scattered light. The only part of the sample that used. The energy of the quanta at 1064 nmof the Nd : YAG laser is only 46% of that ofappears in the second case is the one that exhibits

the Raman effect at the particular frequency un- the 488 nm Ar/ laser line.20 This is notenough to excite fluorescence in most sam-der study. Also, the Raman spectrum of a sample

from a narrowly focused area, down to 1 1 1 mm ples. Raman spectroscopy in the near-infra-red region is therefore much less prone toin size, can be obtained under perfect optical con-

ditions. From the spectrum we can determine the fluorescence due to organic impurities.4. Instead of using a continuous-wave source,type of compound present in that particular area.

Another more recent technique is Raman laser a pulsed laser may improve the quality ofthe spectra. The purpose is to record the Ra-fiber optics spectroscopy, which has been used to

determine the composition of different calculi.18 man scattered light before fluorescencetakes place. The lifetime of Raman scatter-Good results are obtained in comparison with

classical Raman spectroscopy, the calculus compo- ing is of the order 10013 to 10011 s, whereasthat of fluorescence is around 1009 to 1007sition being correctly identified with relatively

good test quality. This is an in situ analytical s. To achieve temporal resolution of the Ra-man scattering and fluorescence signals, itmethod that may be directly applied inside the

urinary tract by endoscopy. is possible to create a short-lived Raman sig-nal by using a pulsed laser.19,20There are several ways to eliminate or suppress

the fluorescence19 that masks the classical Ramanspectra of stones: Raman spectroscopic studies of the composition

of urinary calculi use the spectra of the differ-ent chemical components reported in the litera-1. Prolonged exposure to laser illuminationture.9,11,21,22suppresses the fluorescence, probably due to

the fact that the laser beam bleaches thefluorescent moiety.

COMPOSITION OF URINARY CALCULI2. If the degree of fluorescence is small, signalaveraging will improve the signal-to-noiseratio. Thus, the quality of spectra markedly Epidemiological data on stone composition con-

tribute to the knowledge of the main causes ofimproves with repetitive scanning and aver-

Figure 1. Infrared spectrum of calcium oxalate monohydrate, CaC2O4.H2O.



Figure 2. Infrared spectrum of calcium oxalate dihydrate, CaC2O4.2H2O.

urolithiasis disease. Although we describe here the presence of weddellite is detected by a high-frequency shoulder of the sharp band at 1315the stone composition from the point of view of

the main components, it may be very important cm01 and a decrease in the 780 cm01 /520 cm01

absorbance ratio relative to that found for pureto identify small quantities of minor componentslocated in the nucleus of the stone or in a given whewellite under the same experimental condi-

tions.4,23 On the other hand, whewellite may bearea, because such information can point to a spe-cific process of stone nucleation or growth. Sepa- detected in a nearly pure weddellite calculus by

a sharpening and increase in relative intensity ofrate analyses of nucleus and peripheral layersshould be performed on all calculi. the 780 cm01 band and by the presence of charac-

teristic bands of whewellite at 3492, 3430, 3340,3058, 952, 885, and 665 cm01 .

Calcium Oxalate Calculi These calcium oxalates are easier to distin-guish in Raman spectra. The most intense spec-The two calcium oxalates frequently appearing in

urinary calculi are calcium oxalate monohydrate tral feature of the monohydrate is a 1493/1468doublet due to the symmetrical COO0 stretch(whewellite) and dihydrate (weddellite) . In many

cases, both oxalates appear together in varying (Fig. 3). The less intense 1632 cm01 band is proba-bly due to the asymmetric stretch and the 898proportions and identification may be difficult

when one component is in excess. Careful exami- cm01 band to the C{C stretch. This contrastswith calcium oxalate dihydrate (Fig. 4), whichnation of the 3600–3000, 1400–1300, and 800–

400 cm01 infrared regions allows the detection of only shows a sharp singlet near 1477 cm01 closeto the mean of the monohydrate doublet and athe minor component (Figs. 1 and 2).

In the case of a nearly pure whewellite calculus, shifted C{C stretch at 915 cm01 . The proportions

Figure 4. Raman spectrum of calcium oxalate dihy-Figure 3. Raman spectrum of calcium oxalate mono-hydrate. drate.



Figure 5. Infrared spectrum of nonstoichiometric hydroxyapatite, Ca100x(PO4)60x(H-PO4)x(OH)20x .xH2O, 0 õ x õ 2.

of mono- and dihydrate can be found if synthetic 1100 cm01 . Bellanato et al.26,27 studied the originof this band aided by in vitro experiments andmixtures are used to calibrate the peak heights

of the nsCOO0 and n C{C bands. concluded that it was due to the presence ofH2PO0

4 ions adsorbed onto the calcium oxalateThe difference in the Raman spectra of whewel-lite and weddellite reflects the difference in the crystals.

This type of pure or oxalate-rich calculi sug-structures. Whewellite contains two types of oxa-late unit, one planar and the other barely twisted gests high urine oxalate concentration as the

main pathophysiological factor.17with a shorter C{C distance, whereas weddellitehas equivalent planar units with C{C distancesthe mean of those in whewellite.24 Planarity of

Calcium Phosphate Calculithe oxalate ensures that the Raman and infraredfrequencies are mutually exclusive. The crystal Other important constituents of calculi are cal-

cium phosphates, the spectra of which show char-structures also have a bearing on the formationof mixed oxalate stones found to be richer in acteristic PO30

4 and HPO204 vibrations. Different

types of calcium apatites have been found in uri-monohydrate toward the center.25 These authorsbelieve that the monohydrate is deposited ini- nary calculi. These include nonstoichiometric hy-

droxyapatites of the general formula Ca100xtially and then partly converted to the dihy-drate.25 An alternative theory is that the dihy- (PO4)60x (HPO4)x (OH)20x .xH2O and carbonate-

containing apatites where CO203 ions may substi-drate is the precursor.16 If oxalates are formed by

substitution of the Ca2/ hydration sphere, then tute for PO304 and/or OH0 ions.

Infrared spectra allow the distinction betweenthe dihydrate would be the obvious first stage.Moreover, synthetic specimens of dihydrate are different apatites. The broad band at ca. 870 cm01

indicates the presence of HPO204 groups [P{O-found to slowly convert to the monohydrate.18

It must be mentioned that the infrared spectra (H)] and the bands at ca.1460, 1420, and 875 cm01

indicate CO320 groups (Figs. 5 and 6). Differencesof most whewellite calculi, which are considered

pure when studied by other methods, show a in frequency are caused by differences in thestructure of apatites. In this context, it may bebroad band with weak to medium intensity near

Figure 6. Infrared spectrum of carbonate apatite, Ca100x(PO4)603x/2(CO3)B3x/2(CO3)A

x/4-(OH)20x .(3x /4)H2O.



Figure 7. Infrared spectrum of whitlockite-type calculus, (Ca, Mg)3(PO4)2.

noted that the infrared spectra of the calcination depend on the anions, adjacent cations, and waterof crystallization molecules. Consequently, theproducts of apatites give valuable information

about the original structure.28 different types of calcium phosphates can be dis-tinguished using Raman spectroscopy.Tricalcium phosphate, or whitlockite, which is

rarely found in renal calculi, is stabilized by the Crystallization of calcium phosphates stronglydepends on the urine pH. Thus, stones predomi-incorporation of some magnesium into the struc-

ture. This compound gives rise to characteristic nantly composed of calcium phosphate develop inalkaline or weakly acidic urine. Such pH depen-bands at 1138, 1100, 1078, 1028, and 993 cm01

(Fig. 7). The presence of the band at 993 cm01 dency is not found in common idiopathic calciumoxalate lithiasis.29 Several workers emphasizedmay be used to distinguish this component from

octacalcium phosphate, Ca8H2(PO4)6.5H2O, which the relationship between calcium phosphate cal-culi and disorders in renal acidification.30–32is rarely found in urinary calculi.23

Amorphous or poorly crystalline calcium phos-phates give characteristic infrared spectra, show-

Magnesium Phosphate Calculiing broad absorption bands (Fig. 8). Band split-ting in the 650 to 550 cm01 region indicates the Crystals of struvite (magnesium ammoniumdegree of crystallization.23 phosphate hexahydrate, MgNH4PO4.6H2O) in

Brushite, CaHPO4.2H2O, is another rare com- urinary calculi and in crystalline urine sedimentsponent of urinary calculi (Table I) sometimes ap- are familiar as concomitant manifestations of uri-pearing in the nidus of oxalate stones. Several nary tract infection. A high ammonia concentra-infrared bands (e.g., bands at 3545, 3495, 1218, tion in urine is produced by urea-cleaving bacte-1140, 1065, 990, and 875 cm01) allow rapid identi- ria, which mostly results in a pH rise to abovefication. 7.5. At this pH value, the normal concentrations

Raman spectra of calcium phosphates are dom- of magnesium and phosphate in the urine are suf-inated by strong nsHPO20

4 and nsPO304 bands ap- ficient to produce supersaturation of struvite.33

pearing in the 1000–900 cm01 spectral range and This is the most common magnesium orthophos-phate found in human lithiasis and gives rise tothe phosphate bending and nasPO20

4 bands in the600–400 and 1100–1000 cm01 ranges, respec- a characteristic infrared spectrum (Fig. 10). This

is easily recognized, even in mixed stones, bothtively (Fig. 9). The frequencies of these bands

Figure 8. Infrared spectrum of a carbonate apatite calculus.



Table I. Characteristic Infrared Bands of Some Rare Human Urinary Calculi

Compound Bands (cm01)

Octacalcium phosphate; 1123 s 1105 s 1076 s 1038 vs 1025 vs 963 mCa8H2(PO4)6r5H2O 917 w 864 m

Brushite; CaHPO4r2H2O 3545 s 3495 s 1650 m 1218 m 1140 vs 1065 vs990 s 875 m 793 m 578 m

Bobierrite; Mg3(PO4)2r8H2O 3498 s 3095 s 1085 vs 1046 vs 1002 vs 957 m862 m

Magnesium phosphate 2260 b, vw 2020 b, w 1072 vs 990 vs 854 w 795 wpentahydrate, Mg3(PO4)2r5H2O 598 m

Newberyite; MgHPO4r3H2O 3522 s 3484 s 1708 m 1648 m 1237 m 1165 s1060 vs 1020 vs 900 m

Magnesium ammonium phosphate 3420 s 1467 m 1429 m 1101 s 1056 vs 977 vsmonohydrate, MgNH4PO4rH2O 948 vs 770 m 628 m 570 m

Potassium hydrogen urate, 1395 s 1350 m 1280 w 1010 w 882 w 795 mKC5H3N4O3 770 m 743 w 720 w 598 m 525 m 483 w

2,8-Dihydroxyadenine, C5H4N4O2 3356 s 3254 s 3066 vs 1532 m 1466 m 1445 m1391 m 1348 m 980 m 882 m 797 m 765 m

Xanthine, C5H4N4O2 1567 m 1419 m 1335 m 1207 m 1150 m 1120 w850 m 765 m 723 m 613 m 537 s 497 m

Hipoxanthine, C5H4N4O 1420 s 1365 m 1348 m 1214 s 1150 m 1136 m966 m 893 s 791 m 645 m 633 m 565 m

Silicon dioxide (amorphous) 1095 vs 940 w 805 w 470 mDrugsa See Refs. 9, 10, 23, 40, 63–73

s, strong; m, medium; w, weak; b, broad; v, very.a As drugs generate multiple bands, which are near those of other organic compounds, the identification of these components

requires the examination of the whole spectrum.

by the position of the strong band at 1010 cm01 (Table I) . It generally appears as a result of struv-ite decomposition on the surface of struvite stonesand by the presence of characteristic weaker

bands at 2370, 760, and 572 cm01 . removed months or years earlier, after long expo-sure to atmospheric conditions. The infrared spec-Magnesium hydrogen phosphate trihydratetrum shows characteristic bands at 1237, 1165,(newberyite) is a rare component of renal stones1060, 1020, and 900 cm01 .

Magnesium ammonium phosphate monohy-drate, MgNH4PO4.H2O, is rarely found in urinarycalculi8–10 (Table I) . It may also be a dehydrationproduct of struvite. The bands between 1150 and550 cm01 allow distinction from other magnesiumphosphates.

Another magnesium phosphate found in renalcalculi is trimagnesium orthophosphate pentahy-drate.34 Chemical and spectroscopical data indi-cate that this phosphate could also be a decompo-sition product of struvite. Its infrared spectrumshows characteristic identification bands at ca.2020 (broad absorption), 1072, 990, 795, and 598cm01 (Fig. 11). It must be stated that both new-beryite and magnesium phosphate pentahydratecan be formed by the transformation of struviteby formol; therefore, care must be taken when astone is washed with formol before spectroscopicFigure 9. Raman spectrum of nonstoichiometric hy-

droxyapatite. analysis.



Figure 10. Infrared spectrum of a struvite calculus.

Other magnesium phosphates have been re- Anhydrous I and dihydrate uric acids are oftenfound associated with other stone componentsported as very rare components of urinary calculi:

hannayite Mg3(NH4)2H4(PO4)4.8H2O, bobierrite (calcium oxalates, calcium phosphates, ammo-nium urate, etc.) . The occurrence of uric acid dihy-Mg3(PO4)2.8H2O, and so on.34 Infrared spectros-

copy allows the identification of all these calculi drate and uric acid II on a stone surface has sug-gested35 the probability of uric acid II beingcomponents.

Concerning the Raman spectra of magnesium formed during storage by dehydration of the dihy-drate. Anhydrous uric acid I is the thermodynami-phosphate components in renal calculi, the strong-

est bands correspond to the deformation and cally more stable form. The dihydrate crystal isrelatively unstable and undergoes spontaneousstretching motions of the PO30

4 ion (Fig. 12) as incalcium phosphates. However, magnesium phos- dehydration to form the anhydrous form I. De-

spite this, both forms were identified in uric acidphate bands are well separated from those of cal-cium phosphates, thus making it possible to dis- calculi. Hesse et al.38 reported the presence of uric

acid dihydrate in up to 20% of uric acid stones,tinguish between both types of salt.ancient and recent,39 occasionally as the sole com-ponent of the stone.38 According to Sperling,40 this

Uric Acid Calculi may reflect the presence of some substances inurine that favor the formation of this particularPhysical methods of analysis allow differentiation

into uric acid dihydrate and two anhydrous forms: crystal form and protect it from transformationinto the more stable anhydrous crystal. Thisa monoclinic pseudorhombic (I) and a monoclinic

(II) unstable form.35,36 Uric acid stones are char- agrees with previous studies of Dosch41 that con-cluded that the cation-stabilized uric acid dihy-acterized by a high degree of mineralization and

by a conchoidal structure consisting of character- drate is not a pure compound but contains vari-able quantities of different cations.istic radii, comparable with that of whewellite.

Within this structure, growth fronts may be prom- Infrared spectra are very convenient for theanalysis of uric acid calculi. Infrared bands thatinent and can be made even more pronounced by

the incorporation of pigments and organic sub- may be used for the characterization of anhydrousuric acid23 are at 2020, 1592, 1440, 1403, 1350,stances.37

Figure 11. Infrared spectrum of magnesium orthophosphate pentahydrate,Mg3(PO4)2.5H2O.



The causes of uric acid calculus formation arewell documented33 and consist of stable acidity(pH 5.5–6.0), hyperuricaemia, and hyperurico-suria.

Urate Calculi

The uric acid salt most commonly found in urinarycalculi is anhydrous monoammonium urate (Fig.14), which is frequently accompanied by struviteand calcium oxalate monohydrate. The conditionsof formation for monoammonium urate differ interms of urinary pH in contrast with those of uricacid. Monoammonium urate requires a high am-monia excretion for precipitation to occur. Thisarises as a result of urea cleavage due to infection

Figure 12. Raman spectrum of struvite, MgNH4- and a pH value ¢ 7.0. It is mostly accompaniedPO4.6H2O. by hyperuricosuria.

Infrared spectroscopy has been used for theanalysis of the different constituents of this typeof heterogeneous stone.4 Moreover, infrared has1312, 1123, 993, 878, 783, 745, 705, 620, 578, 525,

and 475 cm01 . The characterization bands depend shown that ammonium urate may be a minor con-stituent in many cases, for example, in uric acidon the accompanying constituents.8,9 The exis-

tence of uric acid dihydrate with the anhydrous stones. The medium band at 600 cm01 and thebands appearing between 1600 and 1250 cm01 canacid in mixtures can be deduced by the presence

of water bands (Fig. 13) at 3515 and 3445 cm01 , be used for the distinction of ammonium urateand uric acid.23the disappearance of the band at 878 cm01 , and

changes in the relative intensities of the bands in Although monosodium urate monohydrate hasalways been considered to be a rare component,42the 1500–1300 and 650–450 cm01 range.23

In connection with the infrared detection of uric Cifuentes-Delatte et al.43 found this urate inabout 2% of cases in the examination of aboutacid dihydrate in calculi, it should be mentioned

that its presence may escape the analyst when 3000 urinary stones and the sole component ina few stones. They also found Randall’s plaquesthe KBr pellet technique is used because halide

absorbs the water of the unstable uric acid dihy- consisted of monosodium urate or uric acid.44 Theinfrared bands at 3600 and 1740 cm01 (waterdrate. Therefore, to detect this compound, a pellet

must be made very rapidly, preferentially without bands) and those at 1613, 1532, 1432, 1385, 1352,and 1260 cm01 among others may be used for dis-the use of vacuum and pressure.23

The Raman spectrum of uric acid shows many tinction; the bands chosen depend on the otherconstituents present.23bands due to the in-plane bending motions of the

purine rings,9,16 and, as in infrared, the anhy- Anhydrous monopotassium urate is considereda very rare constituent of human stones42 (Tabledrous and dihydrate forms can be distinguished.

Figure 13. Infrared spectrum of a uric acid dihydrate calculus, C5H4N4O3.2H2O.



Figure 14. Infrared spectrum of ammonium urate, C5H7N5O3.

I) . Cifuentes-Delatte and co-workers45,46 reported complex urates containing two or more cations inmany areas of near pure uric acid and of ammo-the existence of nearly ‘‘pure’’ potassium urate in

selected areas of a few urinary stones, although nium and sodium urate calculi. Frequently, potas-sium ammonium urate and ammonium urate con-most of the urates studied could not be considered

as stoichiometric salts (see below). Later, the taining both potassium and calcium in varyingamounts were found in so-called ‘‘ammoniumsame group47,48 concluded from a study of more

urate samples that although the existence of pure urate calculi.’’ For these ‘‘complex urates,’’ the au-thors proposed the general formula (NH4)xKyCaz-potassium urate could not be excluded, it could

only be considered an exception. However, they H20x0y02zU.nH2O, where U is the urate ion, x /2y/ 2z ! 1 and n ! 2. In the case of canine stones,later reported the possible presence of a mixture

of potassium and ammonium urates in the urate Escolar et al.52 found ammonium potassium and/or potassium enriched ammonium urate andzone of calcium oxalate oolitic granules (milk of

calcium).49 In the case of dogs, Hesse et al.50 ana- other complex urates in 8% of the cases studied.Sodium was also present in many urate stones,lyzed 741 canine urinary calculi using infrared

spectroscopy and scanning electron microscopy and Cifuentes-Delatte et al.43 first thought thatthis cation only formed monosodium urate mono-and in some cases found besides monoammonium

urate, monosodium and monopotassium urates, hydrate. However, in a later study of a series ofsupposedly pure sodium urate calculi, the resultsand also calcium urate. Later studies using infra-

red spectroscopy confirmed by scanning electron revealed the presence of potassium in many areasof the stones.48 For this type of urate, the authorsmicroscopy and energy dispersive X-ray analysis

(EDX) of 171 specimens of canine and a few sam- proposed the general formula NaxKyH20x0y-U.nH2O, where U is urate ion, x / y Ç 1 and nples of cat urinary calculi51,52 showed that the

presence of nearly pure potassium urate in somezones of urate stones are more common in dogsand cats than in humans.

Finally, the existence of several ‘‘urates’’ withlow or very low cation/urate ratios has been re-ported by some authors.41,53

As is the case for uric acid, the Raman spectraof urates (Fig. 15) show many bands, most ofthem stemming from the in-plane vibrations ofthe purine rings. The intensities and frequenciesof these bands are cation dependent, thus makingit possible for urates containing different cationsto be distinguished.

Complex Urates

Bellanato and co-workers47,48,53,54 reported in a se-ries of articles their results concerning the fre-

Figure 15. Raman spectrum of ammonium urate.quent appearance of a series of nonstoichiometric



Figure 16. Infrared spectrum of a cystine calculus, C6H12N2O4S2.

! 1. Taking into account the results obtained with showed that the ignited rest of apparently purecystine calculi contained calcium phosphate in al-synthetic urates containing sodium and potas-

sium, it was suggested that in some cases it is most all the samples analyzed.A congenital renal defect in the reabsorption ofpossible that potassium substitutes for sodium

and water within the crystalline structure of the dibasic amino acids is responsible for the forma-tion of cystine calculi. As a result, cystine excre-monosodium urate monohydrate salt. These re-

sults have been recently confirmed by Rgz-Minon tion in humans can rise from 200 to more than1000 mg/day.Cifuentes and Bellanato in a careful study of uric

acid and urate stones using scanning electron mi-croscopy, EDX, and infrared spectroscopy. Cal- Miscellaneous Calculicium is also present in potassium sodium urates

2,8-Dihydroxyadenine Calculi(unpublished results) . Calcium sodium uratewith random substitution of magnesium for cal- The lack or partial deficiency in the purine sal-cium and potassium for sodium has been identi- vage enzyme, adenine phosphoribosyltransferase,fied in chicken kidney stones.55

leads to 2,8-dihydroxyadenine stone formationDaudon56 also reported the presence of alumi- and/or crystalluria.60 The stones usually present

num magnesium complex urate found in six cases in childhood and may be easily confused with uricafter treatment with aluminum hydroxide in the acid stones when analyzed chemically.61 However,study of renal stones from 30 chronic hemodialy- infrared spectroscopic analysis allows the easysis patients. identification of this compound8 (Table I) and its

Finally, in many cases, an almost white super- distinction from any other type of urolith (e.g.,ficial layer, in stones containing anhydrous uric uric acid).23,60

acid and less frequently uric acid dihydrate, wasfound. The infrared and atomic absorption spec- Xanthine Calculitrophotometic analysis showed that it was mainly

An increased urinary excretion of the purine de-composed of complex urates.47

rivative xanthine may lead to the formation ofxanthine calculi. Conditions for the formation of

Cystine Calculi these very rare calculi were reported by Robertsonand Peacock.62 The infrared spectrum of xanthineMost human cystine calculi are nearly pure inallows its rapid identification. Characteristiccomposition. For example, the infrared spectrumbands appear at 1695, 1207, 1150, 1120, 850, 613,(Fig. 16) of a cystine calculus shows characteris-537, and 497 cm01 . Xanthine is frequently accom-tic bands at 1623, 1585, 1487, 1410, 845, 542, 455,panied by hypoxanthine, which can be detectedand 395 cm01 that may be used for identificationby key bands at 645 and 565 cm01 . Quantitativeof this component.23

infrared results for binary samples of both compo-Martin et al.57 used infrared spectroscopy tonents are quite good.4study the impact of a new treatment on 15 cysti-

nuric patients. Although it is generally acceptedDrug Calculithat cystine calculi are homogeneous in nature,

other components have been found in the nuclei of Depending on clearance rate and solubility, drugsare more or less prone to crystallization in urine.cystine stones.58 Bellanato et al.59 using infrared



Whereas urinary microcrystals of drug compo- male dogs, in percentages between 5 and 16%.Both whewellite and weddellite coexist in thenents are frequently observed, stone formation it-

self is an extremely rare event.63 However, ac- same calculus, whewellite being located inside thecalculus and weddellite in the more superficialcording to Daudon and Reveillaud,64 70% of the

drugs involved in crystalluria can in fact induce layers. Pure calcium oxalate calculi are not com-monly found,52 and as a general rule it can be saidkidney stones or promote their growth. These au-

thors emphasize the increasing importance of that this component is accompanied by apatiticcalcium phosphate. As in human calculi, a non-the detection and diagnosis of drug-induced cal-

culi.64–66 The identification of relevant constit- apatitic calcium phosphate is also observed in‘‘pure’’ whewellite calculi. Other minor compo-uents requires reference substances and/or refer-

ence spectra. Otherwise, the identification of new nents detected in these uroliths are brushite andstruvite.drug-induced calculi, which most probably are

medication metabolites, is very difficult. Calculi composed mainly of urates account forabout 7% of all canine calculi studied. The mostAmong the products reported in the literature

and readily identified by infrared spectroscopy are common urates are ammonium urate and sodiumurate. Escolar et al.52 found that potassium wasglafenine and its metabolites,65–67 flumequine,65,68

antrafenine,69 N,4-acetylsulphamethoxazole,70 N- generally present almost constantly in ammo-nium urate and sodium urate calculi, suggestingacetylsulphamethoxazole hydrochloride,23,64 N,4-

acetylsulfadiazine,70 mefenamic acid,70 triamterene the presence of potassium urate and/or ammo-nium-potassium or sodium-potassium complexand its derivatives,9,65,67,71 piridoxylate,67 oxolinic

acid,72 5-paracetamol,63 silicon dioxide,9,70,73 and urates in these stones. As described above, theseurates have also been observed in human andothers.10,65

avian urolithiasis.Other less frequent types of calculi found in

Animal Calculi dogs consist of calcium phosphate (2–3%) and sil-ica (around 1%). Apatitic calcium phosphate andThe composition of animal calculi is similar tobrushite are the principal forms of calcium phos-that of humans except that the prevalence of uro-phate that have been detected. Struvite and oxa-lith types is significantly different. The use of in-lates are common compounds associated with cal-frared spectroscopy for the analysis of animalcium phosphate in this type of urolith. Silica cal-stones has been increasing over the last two de-culi appear normally as pure silica. However,cades52,55,74–82 and has been applied to calculi fromEscolar et al.52 also found an external layer ofvarious animal species. Here we refer particularlycalcium phosphate covering a silica calculus.to canine, feline, and equine species.

Feline SpeciesCanine Species

Several studies of canine uroliths have revealed Feline calculi cannot be considered as an isolatedprocess, because their formation is closely relatedthat struvite is the major constituent (46–

70%).52,78,81–83 Of the minor components accompa- to a feline urological syndrome. This syndromerepresents a constellation of clinical signs associ-nying struvite, apatitic calcium phosphate was

found to be the most frequent. In addition, cal- ated with lower urinary tract disease in male andfemale cats, the consequence of which, in manycium oxalates, calcium hydrogen phosphate dihy-

drate (brushite), magnesium hydrogen phos- cases, is the formation of urethral plugs. Theseare composed of relatively large quantities of anphate trihydrate (newberyite), and ammonium

urate and/or ammonium potassium complex organic matrix in addition to minerals. The chem-ical composition of feline calculi and urethralurates were also found in canine struvite calculi.

Stones having cystine as the major constituent plugs have been studied by many investiga-tors.77,80,81,85–88 In most reports, struvite is theare of great significance in dogs, although the inci-

dence varies widely for different countries (2– mineral component most frequently found bothin feline uroliths and urethral plugs, up to 76%26%).52,78,81,83,84 In a study of cystine calculi,52,84

about 45% were composed of nearly pure cystine. for calculi77,80,85–88 and up to 94% for urethralplugs.81,85–89 The most common secondary mineralOther minor components were calcium oxalate,

struvite, calcium phosphates, and urates. component in struvite uroliths is calcium apatite,although ammonium urate may be also present.Calcium oxalate calculi occur mainly in old



Other primary minerals much less frequently analytical results with the appropriate diagnosisand therapeutic regimen to prevent recurrence ofidentified in feline uroliths are uric acid, calcium

phosphate, and calcium oxalate.77,80,81,87–89 In ure- calculi. Several techniques (infrared and Ramanspectroscopies, X-ray diffraction, etc.) have beenthral plugs, minor components detected are cal-

cium phosphate, calcium oxalate, ammonium used for the analysis of calculi. Furthermore, ev-ery urinary stone that is surgically removed orurate, newberyite, and in exceptional cases xan-

thine and sulphadiazine.81,85,86,89 spontaneously expulsed must also be analyzed toget information about conditions of crystalline nu-cleation and growth and about ions and moleculesEquine Speciesthat can take part in the formation and evolution

In equine calculi, the main component mentioned of the diverse crystalline phases. On the clinicalin the literature is calcium carbonate in the form level, these data can contribute to a better com-of calcite, although minor quantities of two other prehension of the lithogenic factors such as meta-polimorphs of calcium carbonate (vaterite and, bolic disorders and drug-induced lithiasis.less frequently, aragonite) have also been Infrared and Raman microspectroscopies andfound.74–76,86,90–92 In the calculi recently studied the computerized analysis of the spectra are mod-by Dıaz-Espineira et al.,74–76 calcite, the major ern techniques particularly well suited to theconstituent, was found in 93.3% of the samples above aims. Moreover, Raman laser fiber-opticsand vaterite in 13%. Whewellite, weddellite, and spectroscopy is being applied in urine and urinaryapatitic calcium phosphate were minor compo- tract diagnosis as an additional promising tech-nents associated with calcium carbonate. Other nique to obtain analytical information under incompounds less frequently found in the uroliths vivo conditions.studied were calcium sulphate and amorphous sil-ica.

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