Marshall L. Stoller, Maxwell V. Meng-Urinary Stone Disease

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Table 1

The Chemical Name, Composition, Mineral Name, and Abbreviation

of the Most Common Components of Human Urinary Tract Calculi

Chemical name Mineral name Chemical formula Abbreviation

Oxalates

• Calcium oxalate monohydrate Whewellite CaC

·H

OWH

• Calcium oxalate dihydrate Weddellite CaC

·(2+x)H

OWE

Phosphates

• Basic calcium phosphate Apatite Ca

(PO

)

(OH) AP

• Calcium hydrogen phosphate Brushite CaHPO

·2H

OBR

• Magnesium ammonium phosphate hexahydrate Struvite MgNH

·6H

OST

Purines

• Uric acid C

• Monosodium urate monohydrate NaC

·H

O MSU

Other

•

-Cystine (-SCH

CHNH

COOH)

Chapter 5 / Kidney Stones 71

stones suggests multiple physiologic conditions that must be unraveled in the process of

defining the optimal medical management and the avoidance of stone recurrence.

The major crystalline components of human urinary tract stones are listed in Table 1.

Prevalence of crystalline admixtures from multiple studies of multicomponent stones is

presented in Table 2. Currently, there are two analysis methods that are routinely used

for stone analysis, namely X-ray powder diffraction crystallography (XRD) and Fourier

transform infrared spectroscopy (FTIR). These two analysis methods differentiate stone

crystalline composition based on either the uniqueness of crystalline structures or sub-

tle energy differences associated with the motion of atoms in chemical bonds found in

the various stone components. It is worthwhile to first address the chemistry of stone

components and aspects of various crystalline structures seen in stones before discussing

the unique aspects and capabilities of the two primary methods of stone analysis.

CHEMISTRY AND STRUCTURES FOUND IN STONES

The primary component of human stones, calcium oxalate, crystallizes in two differ-

ent chemical and crystallographic forms: a monohydrate and a dihydrate structural com-

plex. Calcium oxalate is a calcium salt of a dicarboxylic acid, oxalic acid. The differences

in the degree of water of crystallization included in the crystalline lattice results in two

different crystalline structures, leading to different growth morphologies, different

atomic structures on the crystal surfaces of the two hydrated structures, and different

effective biologic activities.

Table 2

Relative Frequency of Occurrence of Admixed Stones

Country USA (13) Glasgow (15) Berlin (11) USA (5)

No. Stones 1,000 149 10,000 3,833

WH 13.7 4 24.9 23.4

WE 0.4 1 0.6 4.7

WH/WE 18.6 21 33.2 29.4

WH/WE/AP 22.4 45

13.5 5.2

WH/AP 7.2 45

2.9 2.7

WE/AP 4.7 45

0.3 1.3

AP 3.4 5 1.1 6.6

ST/AP 15.5 14 7.0 6.4

ST 0.4 — 0.3 4.3

WH/ST/AP — 3 1.4 0.0

UA 4.7 — 2.4 5.2

UA/UD — 1 3.9 1.6

WH/UA — — 2.9 1.5

WH/UA/UD — — 1.8 0.2

BR — 2 0.4 1.2

CY 2.2 1 0.2 0.4

No. stones represents number of samples included in study.

Frequency of occurrence of both whewellite and weddelite mixed with

apatite reported as a single group.

72 Mandel and Mandel

The second major class of components of stones includes phosphate salts. Four

different calcium phosphate salts and two magnesium phosphate salts are found in

stones. The calcium phosphate salts vary in calcium-to-phosphate ratio, hydroxyl-vs-

hydrogen ion content, and the degree of water of crystallization included in the crystal-

line lattice structure. The magnesium phosphate salts differ by inclusion of either

ammonium or hydrogen ions and the degree of water of crystallization in the crystalline

lattice structure. Similarities in chemistry, but differences in crystalline structures, often

create challenges in composition analysis using infrared spectroscopic analysis that are

focused at differentiating chemistry.

All of the calcium and magnesium salts have different crystalline structures and are

easily differentiated by XRD methods of analysis, but sensitivity of detection can com-

plicate and challenge a complete analysis. Similarly, the purine structural moieties

include subtle chemical and structural variations of the uric acid-like six/five-member

aromatic ring structure. Addition of carbonyl groups to the purine ring moiety separates

related structures such as hypoxanthine, xanthine, and uric acid. The salts of the purine

organic acids and variations in the degree of water of crystallization induce additional

chemical and structural variation in the identification criteria for definitive composi-

tional analysis. The preceding description regarding the method of analysis for the

oxalates and phosphates in stones is also applicable to the purines and their salts. Similar

chemistry leads to very subtle differences in infrared spectra, but they lead to significant

differences in XRD data. The miscellaneous components are sufficiently unique that

analyses by either the XRD or the FTIR method is straightforward, assuming accurate

standards are available.

Need for instrumentation sensitivity and more importantly accurate standards for

comparison are critical for successful stone analysis using either XRD or FTIR analysis

methods. The two methods of analysis used separately have unique capabilities depend-

ing on the type of stone being analyzed. It is clear that the two analysis methods used

collectively yield the highest degree of certainty in compositional analysis of urinary

tract stones.

CRYSTALLURIA AND STONE MORPHOLOGY

Determination of crystalluria and probable crystalline composition must be accom-

plished using freshly voided urine and a polarizing microscope. The different crystalline

components found in crystalluria reveal different growth morphologies when viewed

with a polarizing microscope (1,2). Many of the crystals demonstrate birefringence so

observation of growth morphology is often more definitive. Calcium oxalate monohy-

drate can be observed as ovals or dumbbells and calcium oxalate dihydrate as bipyramids.

Apatite crystals usually appear as an amorphous precipitate, and frequently grow as

clumps of very small crystallites. Struvite crystals grow in a characteristic coffin lid

shape. Uric acid crystals appear as flat parallelepiped plates, and cystine crystals appear

as hexagonal plates. The most common growth morphologies of mature stones com-

posed of components listed in Table 1 are described in the following sections.

Calcium Oxalate Monohydrate

These stones are frequently hard, dark brown, and often have a dull gray exterior.

When sectioned, the stones often appear to grow in radial fashion from a nidus with

wedges rounding off at their extremities creating a generally smooth exterior.

Chapter 5 / Kidney Stones 73

Calcium Oxalate Dihydrate

Pure dihydrate stones are usually small and spherical consisting of a tan or yellow

cluster of platelets. The platelets are sharp and are arranged in various orientations.

Admixed monohydrate/dihydrate stones frequently have many of the characteristics

of a dihydrate stone because dihydrate most frequently appears on the exterior of the

admixed stone. These stones are normally larger than pure dihydrate stones, are often

spherical, and have a cluster of yellow platelets surrounding a hard dark brown interior.

The platelets are sharp and in various orientations. Occasionally, the interior is light

brown in color and has a granular consistency.

Apatite

Pure apatite stones are usually small, white in color with a very fine granular surface,

and they have a soft white chalklike interior. Occasionally, these stones are also light

brown with a smooth shiny surface.

The most frequently occurring stone admixture of apatite or calcium oxalate mono-

hydrate and calcium oxalate dihydrate is generally smooth, spherical, and has light

brown platelets on the surface. The interior is normally layered with both white and

light brown sections. Frequently, papillary casts are observed on one side of the stone

with apatite located in the cast because it is often the first crystalline substance depos-

ited. The dihydrate plates are normally observed on the opposing side from the papillary

casts.

Struvite

Pure struvite stones are usually off-white to light brown in color with a rough textured

surface. When sectioned, the interior of the stone contains white concentric rings, and

occasionally the interior contains white porous granulated material. Struvite stones fre-

quently grow in a staghorn shape.

Admixed struvite/apatite stones are usually light brown in color with a coarse, granu-

lar surface. The interior is normally intermixed with white and light brown layers.

Brushite

Pure brushite stones are normally clusters of beige, nodular material surrounding a

crystalline interior with a cauliflower-like growth pattern. Occasionally, the surface has

a yellow or white tinge.

Uric Acid

Uric acid stones are spherical with a smooth yellow-orange surface. When sectioned,

the interior of the stone appears as orange concentric rings with little to no macroscopic

substructure.

Uric Acid Dihydrate

The surface of uric acid dihydrate stones is often dark orange and the stone is com-

posed of small spherical regions. When sectioned, the stones have a well-defined nidus

with thick concentric rings composed of small needles that radiate from the center of the

stone.

74 Mandel and Mandel

L-Cystine

Pure L-cystine stones are homogeneously composed of very small yellow spheroids.

Matrix

Matrix stones are noncrystalline and take on a variety of shapes and colors. The stones

are composed of a variety of organic molecules including urinary macromolecules and

membrane fragments (3).

Other

Other substances that have been reported in stones include the drugs sulfamethoxaz-

ole, crixivan, guaifenesin, triamterene and 5-fluorocytosine, xanthine, 2,8-dihydroxy-

adenine, gypsum, and silicates following antacid therapy. Growth morphology for these

components is often variable.

STONE ANALYSIS

XRD (1–5) and FTIR (6–8) are the most frequently used methods of stone analysis.

Both XRD and FTIR methods require a very comprehensive and accurate library of

standard diffraction or spectroscopic patterns for comparative analysis. The focus of

these methods is the identification of the crystalline mineral composition of the stone

with little emphasis on the macromolecular or other noncrystalline entities that may be

present. XRD should be viewed as a semiquantitative method of analysis. XRD analysis

should be presented in rank order of relative amounts of the stone components in mul-

ticomponent stones. FTIR spectral libraries allow for the reporting of relative percentage

compositional analyses in multicomponent stones using computer algorithms that math-

ematically admix spectra of pure components at specified percent admixtures. Computer

addition of spectra from components with significantly different infrared absorption

characteristics can artificially and inappropriately skew spectral standard comparison

methodology.

Virtually all crystal structures are unique is some structural aspect and their diffrac-

tion patterns can be differentiated from other structures and diffraction patterns. Highly

sensitive and accurate XRD instruments are often necessary to differentiate some of the

structures seen in stones as their chemistry and crystal structures can be similar.

The XRD data for the most common components of human kidney stones are pre-

sented in Table 3. The diffraction data are presented in crystallographic language as

interplanar d-spacings in Ångstroms (d[Å]) associated with the distances between atoms

in the structure and as diffraction intensities either relative to a weak vs strong scale or

on a maximum of 100 scale (4,9,10).

In practice, the experimental XRD patterns are compared with those for the standard

patterns presented in Table 3. All diffraction lines of a given standard pattern, especially

the strongest lines, must be matched with diffraction lines in the sample pattern. If some

lines of a given intensity are thought to match a standard, then all lines with equal or

greater intensity must also match the standard lines. Remaining unmatched lines are used

to determine any other crystalline components in the sample. With the advent of high

resolution XRD cameras utilizing focusing monochromators and high flux X-ray gen-

erators, the ability to detect minor stone components has greatly increased as the diffrac-

tion patterns appear sharper and diffraction lines are easily differentiated from

Chapter 5 / Kidney Stones

Table 3

XRD Interplanar Spacings and Relative Intensities of the Components of Human Urinary Calculi

COM COD AP BR ST UA MSU CY

d (Å) I d (Å) I d (Å) I d (Å) I d (Å) I d (Å) I d (Å) I d (Å) I

5.93 100 8.70 12 8.15 29 7.59 100 5.90 38 6.56 38 10.60 8 4.70

5.79 25 6.31 5.26 6 4.24 100 5.60 45 5.63 20 9.30 30 4.68 100

4.64 7 100 4.08 8 3.80 8 5.38 22 4.91 48 7.50 25 4.63

4.52 6 6.15 3.89 10 3.04 75 4.60 6 4.76 6 5.28 10 4.45 5

3.78 4.40 45 3.44 45 2.93 70 4.25 100 3.86 4.88 22 4.20 7

13 3.89 14 3.17 10 2.85 15 4.14 34 + 55 4.63 4 3.32 8

3.65 100 3.09 2.81 100 2.60 30 3.29 24 3.70 5 3.45 15 3.18 19

3.00 10 18 2.78 62 2.56 6 3.02 10 3.28 14 3.38 20 3.13 30

2.97 46 3.07 2.72 58 2.96 3.18 70 3.26 5 3.06 16

2.91 12 2.81 20 2.63 31 + 16 3.09 100 3.12 100+ 2.71

2.89 10 2.77 2.53 5 2.95 2.87 3.01 1 2.70 32

2.84 14 85 2.40 7 2.92 46 + 25 2.87 12 2.68

2.48 30 2.75 2.26 20 2.80 26 2.86 2.84 8 2.60 17

2.41 5 2.41 14 2.15 9 2.72 8 2.80 2.78 5

2.34 90 2.39 14 2.06 7 2.69 37 + 10 2.63 40

2.33 10 2.00 5 2.66 37 2.79 2.59 5

1.94 30 2.57 18

1.89 15

1.87 5

1.81 20

76 Mandel and Mandel

neighboring diffraction lines separated by as little as 0.01–0.03 Å interplanar spacings.

The increased sensitivity has allowed for the identification of smaller amounts of poorly

crystalline materials such as apatite. However, the most severe limitation of XRD is

sensitivity when a very limited amount of sample is available. Also, if the stone material

is a drug, or drug metabolite whose XRD pattern or single crystal structure has not been

published, XRD methods fail to definitively characterize the sample. In those cases, the

XRD method can only tell you what the stone is not composed of.

In FTIR spectral analysis, spectral data is related to the vibrational motions of atoms

in bonds (e.g., bond stretching, bond contracting, or bond wagging, etc.). Classically,

the powdered sample is admixed with powdered potassium bromide, compressed into

a nearly transparent wafer, and the IR beam is passed through the wafer. Recently,

advances in other sample preparation methods have allowed powdered samples to

simply be ground to ensure optimal sampling of a multicomponent stone and then the

IR beam is directed at the sample surface (attenuated total reflectance). The reflected

IR beam containing spectral data specific to the sample is then recorded. Standard IR

patterns for common stone components are presented in Figures 1–8. The IR pattern

contains absorption bands representing specific energies (presented as wavelengths in

units of cm-1, or more commonly known as wavenumbers) corresponding to molecular

motions in molecules. It is therefore possible to differentiate molecular motions in

similar organic groups. The IR pattern of a mixed component stone is frequently very

complex, but the advent of computer controlled IR spectrometers, especially modern

FTIR spectrometers has allowed for computer assisted pattern stripping and compara-

tive standards library matching. Frequently, the assignment of weak absorbance bands

in the spectra to specific structures is very difficult and often requires careful back-

ground and noise level adjustments in the computer data smoothing functions. The

specific choice of mathematical calculations used in the standards library search/match

routines can provide markedly different results, so operator training and experience is

critical.

The real benefit of FTIR is the high sensitivity of the new computer controlled spec-

trometers that can take many repetitive spectra of the same sample and mathematically

enhance the sample signal to experimental noise ratio. It is often possible to obtain a

definitive FTIR spectra with less material than is needed for XRD. FTIR is the method

of choice for the characterization of noncrystalline samples or drug related samples

because the IR absorbance is related to independent chemical bonds and IR data related

to a majority of the molecule is still definitive. Metabolism of a drug resulting in an

altered chemical structure in a stone compared with the ingested molecule has far less

impact in FTIR analysis compared with XRD. The drawback to FTIR is that the differ-

entiation of spectral signals from more than one component with similar molecular

bonds can be difficult. All too often the operator of modern FTIR instruments may rely

too heavily on the computer-assisted analysis of the spectra as the computer algorithms

scan and interpolate data from computer based spectral libraries. In the case of weak

spectra or multicomponent stones, the computer often indicates a positive identification,

but in fact the component identification is false.

The analysis of stone composition with microscopic inspection (including polarizing

microscopy) is very inaccurate and unfortunately too frequently used for the routine

analysis of stones (3,11,12). The assumption is that all components of stones always

appear the same regardless of unique urine chemistry and different admixtures with other

Chapter 5 / Kidney Stones 77

Fig. 3. The FTIR spectra of hydroxyapatite.

Fig. 2. The FTIR spectra of calcium oxalate dihydrate.

Fig. 1. The FTIR spectra of calcium oxalate monohydrate.

78 Mandel and Mandel

Fig. 6. The FTIR spectra of uric acid.

Fig. 5. The FTIR spectra of struvite.

Fig. 4. The FTIR spectra of brushite.

Chapter 5 / Kidney Stones 79

stone components. This is not a sound assumption. After fracturing the calculi to reveal

the internal structure, samples are taken from various portions of the stone and are

visually inspected and identified using a polarizing microscope. This technique is not

capable of identifying small amounts of crystalline materials in admixed samples. A

significant contribution to the potentially low level of accuracy using this method is that

the accuracy is entirely dependent on the level of sophistication and experiences of the

technicians conducting the analyses (12).

CONCLUSIONS AND RECOMMENDATIONS

Numerous treatises have been written on the crystalline composition of renal calculi

and the frequency of occurrence of the varied crystalline components in stones observed

in industrialized countries (3,5,13–17). The majority of urinary stones are admixtures of

Fig. 8. The FTIR spectra of cystine.

Fig. 7. The FTIR spectra of monosodium urate monohydrate.