ORIGINAL ARTICLE

Chemical analysis of stone in patients

with nephrolithiasis

Authors

ABSTRACT

Luis Alberto Batista

Peres1

Introduction: Nephrolithiasis is a multifactorial disease and it has relation with genetic

disorders and environmental factors. Stones
are most common in adults and are associated with several metabolic disorders. Calcium
oxalate is the most common type of stone.
The objective of this study is to evaluate chemical analysis of calculi in our region. Methods:
We made a retrospective study on 1,342 patients with evidence of recent formation of renal stones. Laboratory investigation included
chemical analysis when the stones were available. Results: 1,342 patients with nephrolithiasis were consulted, among whom only 109
(8.1%) were submitted to chemical analysis
of stones. Mean age of those patients were
38.9+13.4 years, and 55 (50.5%) were male.
Familial history occurred in 65% of the cases.
Calcium oxalate stones were found in 87% of
the cases. Hypercalciuria and hyperuricosuria
were the most associated metabolic disturb in
patients with calcium oxalate and uric acid
in the stones (60%). Conclusions: Chemical
analysis has demonstrated that calcium oxalate
is the most common component found in our
region, according to the literature.

Mnica Tereza
Suldofski2
Paulino Yassuda Filho2
Ana Paula Kazue Beppu3
Everaldo Roberto de
Arajo Junior3
Gustavo Vicenzi3
Ricardo Yukiharu Tsuge
Yamamoto3
Nephrology Division,
School of Medicine
(UNIOESTE)

School of Pharmacy
(UNIOESTE)
3
School of Medicine
(UNIOESTE)
2

Keywords: nephrolithiasis, stone composition.

[ J Bras Nefrol 2009;31(2):94-97]
Submitted: 01/21/2009
Approved: 05/15/2009

Correspondence to:
Luis Alberto Batista Peres
Rua So Paulo, 769/Ap. 901
Centro
Cascavel Paran Brazil
CEP: 85801-020
Fax: 55 (45) 3327-3413
E-mail: peres@certto.com.br
We declare no conflict of
interest.

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INTRODUCTION
Nephrolithiasis is a high prevalence disease
affecting mainly adults aged between 20 and
60 years.1,2 It is estimated that up to 12%
of the population will suffer an episode of
nephrolithiasis during lifetime and, depending on the type of stone, more than 50%
of them will have a recurrence within 10
years.2,3,4,5,6,7 Caucasians are more affected.2,8
Stone formation is related to hereditary fac-

tors, as well as to environmental factors such

as geographical area, climate, dietary habits,
and socioeconomical situation.2 Regarding
the gender, it is believed that males are more
affected, as there was a proportional decrease
in the prevalence of nephrolithiasis compared
to female gender.2,9,10
The composition of urine is a major factor
in crystal formation. An imbalance between
promoters and inhibitory factors is responsible for nucleation and further development of
the stone.4,11,12 States of known supersaturation are: hypercalciuria, hyperphosphatemia,
hyperoxaluria, hyperuricosuria, and cystinuria. Hypocitraturia is the main condition of
crystallization inhibitor deficiency. Other
factors that may influence in the stone formation are pH changes and/or a reduction in
urinary volume.2,13
The main chemical component found in
renal calculi was calcium oxalate, as shown
by several authors.1,2,3,14 Calcium oxalate and
uric acid calculi are more frequent in males,
while calcium phosphate and struvite calculi
are more frequent in females.1 The chemical
analysis of the renal calculus is diagnosed in
cystinuria events and calculi secondary to urinary infection.1,8 The aim of this study was
to demonstrate the prevalence of the main
chemical components found in urinary calculi in patients with nephrolithiasis in the west
region of Paran.

MATERIALS AND METHODS

The present study was approved by the Ethics Committee for Research in Humans at
the UNIOESTE. Hospital charts from patients with evidence of urinary lithiasis within the last six months were evaluated from
December 2001 to December 2008, and the
following laboratory tests were performed:
urinalysis, qualitative cystinuria, determina-

Chemical analysis of stone with nephrolithiasis

tion of oxalate, citrate, calcium, sodium and uric acid in

a 24-hour urine sample, in addition to serum creatinine,
calcium, uric acid, and parathormone. Laboratory methods used and adopted reference range adopted for 24hour urine samples were: Calcium: Atomic absorption
spectrophotometry method (<4.0 mg/kg); Uric acid: uricase enzymatic method (>15 mg/kg); citrate: citrate enzymatic method (>320 mg). For plasma determinations
the methods used were as follow: Calcium: colorimetric
method (8.5-10.5 mg/dL), uric acid: uricase colorimetric
method (2.0 to 7.0 mg/dL); creatinine: alkaline picrate
method (0.7 to 1.4 mg/dL); and parathormone: intact
molecule assay. For qualitative cystinuria test: Sodium
nitroprusside test. Chemical analysis of renal calculi
was performed when it was available.
The method used for calculi chemical analysis, determined by the manufacturer of reagent Bioclin, is described as follows:
1. Preparing the sample being analyzed: Spraying the
calculus. Transfer a small amount (40-50 mg) of the homogeneous powder into a test-tube of 13100 mm and
add 10 drops of reagent # 5+10 drops of distilled or
deionized water. Heat under immersion in water at 56C
for 5 minutes, and shake the tube 2 or 3 times during this
period. Centrifuge to 3,000 rpm for 3 minutes. Transfer all supernatant to another test-tube of 13100 mm;
Mark with letter S (to be utilized on phase 3) and mark
the letter P for the other tube with the precipitated (to be
utilized on phase 2).
Precipitated analysis: Carbonate: In the tube marked
with letter P add 10 drops of reagent # 1, simultaneously
observing if there was any gas separation. In affirmative
case, the test is positive for carbonate. Add then 10 drops
of distilled water and homogenize. Heat the tube in direct flame up to the first sign of boiling. Wait to be cold.
This solution will serve as sample (sample P) for oxalate,
calcium and magnesium tests described below. Oxalate:
0.1 ml of sample P+3 drop of reagent # 2. The formation
of an intensive turvation or a white precipitate indicates
the presence of oxalate. Calcium: 0.1 mL of sample P+5
drops of reagent # 6. The formation of white precipitation
indicates the presence of calcium. Magnesium: Transfer
0.02 mL of sample P to an Erlenmeyer and add 20 mL of
distilled or deionized water. Add to this solution 1 drop of
reagent # 5. Homogenize. This will be the diluted sample.
Use a test-tube 1275 mm and add 7 drops of reagent #
7+10 drops of reagent #8. Homogenize. Add 0.05 mL of
the diluted sample and homogenize. The appearance of
violet color indicates the presence of magnesium.
Supernatant analysis: Urate: Transfer 0.1 mL of the
supernatant from stage B.1 to a test-tube of 1275 mm.
Add 5 drops of reagent # 10 and 5 drops of reagent #

11. The appearance of an intense blue color indicates the

presence of urate. Cystine: Transfer 0,1 mL of supernatant from stage B.1 to a test-tube of 1275 mm. Add 1
drop of reagent # 1 and 1 drop of reagent # 13. Wait for 5
minutes. Add 2 drops of reagent # 14. The presence of an
intense red color indicates the presence of cystine. Note:
The color formed quickly disappears. Remember to take
care while manipulating reagent # 13. It contains cyanide. Ammonium: Transfer 0.1 ml of supernatant, from
B1 stage to a test-tube of 1275 mm. Add 10 drops of
distiled or deionizaded water. Homogenize. Add 5 drops
of reagent # 9. The appearance of orange-yellow color
indicates the presence de ammonium. Note: The color
formed may acquire a dark tone if there is a high level of
ammonium in the sample. Phosphate: Transfer 0.1 mL of
supernatant, from stage B.1 to a test-tube of 1275 mm.
Add 1 mL of distiled or deionizaded water + 1 drop of
reagent # 1. Homogenize. Add two drops of reagent # 3.
Homogenize. Add 2 drops of reagent # 4. Let it rest for 2
minutes and add 2 drops of reagent # 5. The appearance
of a blue color indicates the presence of phosphate.
Data was stored in a Microsoft Excel database and
analyzed using descriptive statistics: Arithmetic means,
standard deviation, minimum and maximum values, raw
and percentage frequencies.

RESULTS
One hundred and nine (109) patients underwent an
investigation of the chemical analysis of calculi, corresponding to 8.1% of the total cases considered for metabolic investigation of nephrolithiasis. The mean age of
these patients was 38.9+13.4 years (ranging from 5 to
77 years). In relation to the gender, 55 patients (50.5%)
were male. Regarding family history we obtained that
information from 100 patients, and it was positive in 65
(65%) of the cases.
Regarding the chemical analysis of the stone, 95 patients
(87.2%) had calcium oxalate, and this was the most frequent composition found. Table 1 shows the results of the
chemical analysis performed in these stones. In relation to
the results of metabolic investigation in patients who performed the chemical analysis, among those with calcium
oxalate calculi or uric acid calculi, hypercalciuria and hyperuricosuria were the most common metabolic disorders.

DISCUSSION
In this study, 1,342 patients with nephrolithiasis were assisted; of these 109 performed the chemical analysis of their
calculi. Calcium oxalate was the most prevalent chemical
component, which is consistent with findings from other
regions and corroborated by the literature.2,3

J Bras Nefrol 2009;31(2):94-97

95

Chemical analysis of stone with nephrolithiasis

Table 1

CHEMICAL ANALYSIS OF URINARY STONES IN 109 PATIENTS

Chemical composition

Calcium oxalate

95

87%

Calcium carbonate

44

40%

Uric acid

23

21%

Ammonia

18

17%

Calcium phosphate

7%

Cystine

1%

Struvite

1%

Total of chemical constituents

Urinary calculi formation may affect 5% to 12% of

population. Several metabolic changes have been identified in the urine of patients with idiopathic recurrent
nephrolithiasis, particularly hypercalciuria, hyperoxaluria, hypocitraturia, and hyperuricosuria, among others.
These changes depend on a complex relationship between
genetic and environmental factors.15 Data from the literature has demonstrated that, among patients with urinary
lithiasis, 40% of them presented hypercalciuria as the
ethiopathogenic factor.16
Calcium oxalate calculi are more frequent than calcium phosphate calculi. The most important physiopathologic factor in formation of these calculi is hypercalciuria,
which is a defect in at least one of the following organs:
kidney, bones, or gout. Hypercalciuria increases the
saturation and crystallization of calcium salts, reducing
citraturia.17,18,19 In previous studies from our group, hypercalciuria was the most prevalent metabolic disorder in our
region,20,21 but, as far as we know, no study evaluating the
prevalence of chemical components of urinary calculi have
been performed in the west region of Paran.
The main chemical component in renal calculi is calcium oxalate.1 Baker et al.2 have found a 68% prevalence of calcium oxalate in urinary calculi studied in their
series. Calcium oxalate calculi and uric acid calculi are
more frequent in males, while calcium phosphate and
struvite are more common in females.1,3,23
Urinary composition is an important factor in crystal
formation. An imbalance between promoter and inhibitory crystallization factors is responsible for nucleation
and posterior development of the calculus.3,24 States of
known supersaturation are: hypercalciuria, hyperphosphatemia, hyperoxaluria, hyperuricosuria, and cystinuria. Hypocitraturia is the main condition of crystallization inhibitor deficiency. Other factors that may influence
the calculus formation are pH changes and/or reduction
in urinary volume.3,4

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J Bras Nefrol 2009;31(2):94-97

190

The interaction among renal tubular cells, calcium

oxalate crystal and oxalate ions are an early event in
lithogenesis. Urine contains ions, glucoproteins and
glucasominoglicans, which inhibit the crystallization
process, protecting the kidney against lithogenesis. Renal
epithelial tubular cells are the main target to the effects
induced by hyperoxaluria- and by calcium oxalate crystal incorporation. Since tubular cells damage predisposes
to nucleation, maybe an increase in glucosaminoglicans
synthesis may protect the tubular epithelium against
crystal adhesion and toxic effect of hyperoxaluria, what
could limit the formation of renal calculi.25
This study was the base to the knowledge of chemical
components of urinary calculi in patients with nephrolithiasis in the west region of Paran.

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