Use of a simplified spectrophotometric method
89
ORIGINAL ARTICLE
Use of a simplified spectrophotometric method for
quantitative determination of glucose-6-phosphate
dehydrogenase activity in normal children from two day-care
centers of the city of São Paulo
Uso de um método espectrofotométrico simplificado na determinação quantitativa da
atividade da glicose-6-fosfato desidrogenase em crianças normais de duas creches da cidade
de São Paulo*
Roberto Muller1
ABSTRACT
Objective: To evaluate the applicability of a simplified method for
quantitative determination of glucose-6-phosphate dehydrogenase
activity in normal children; to determine the mean, standard
deviation and threshold value under which the enzyme activity is
considered deficient. Methods: Blood samples were collected from
201 children from two day-care centers in the city of São Paulo.
The subjects were considered normal based on physical
examination and laboratory tests. The enzyme activity was
determined in red blood cells of normal children using the “Test
Combination G-6-PDH®” kit. The following statistical analyses were
carried out: the results were submitted to Student’s t test,
Kolmogorov-Smirnov test, lower confidence interval (one-tailed
test) and Spearman’s correlation coefficient. Results: The mean
hemoglobin value for girls was slightly higher than the mean value
for boys, but this difference was not statistically significant. There
was no statistical difference in mean enzyme activities for Caucasian
and non-Caucasian children. There was no significant correlation among
enzyme activity levels, red blood cells, hemoglobin levels,
hematocrit, reticulocytes, white blood cells and age of patients.
The mean enzyme activity for boys was 4.448 U/g Hb, standard
deviation = 1.380 U/g Hb. For girls, the mean enzyme activity was
4.531 U/g Hb, standard deviation = 1.386 U/g Hb, and the difference
was not statistically significant. Therefore, the two population
groups were considered as one single population, presenting a
mean enzyme activity of 4.490 U/g Hb, standard deviation = 1.380 U/g Hb.
Since the distribution curve of enzyme activity values was normal,
a lower confidence interval was determined (one-tailed test), with
a cutoff point of 2.227 U/g Hb. Conclusion: The method used by
Solem proved to be simple, fast, very accurate and useful to detect
glucose-6-phosphate dehydrogenase activity and to identify
children with enzyme deficiency.
Keywords: Spectrophotometry; Glucose-6-phosphate dehydrogenase;
Quantitative analysis; Favism; Jaundice, neonatal; Anemia,
hemolytic; Child day care centers; Child; Child, preschool;
Adolescent
RESUMO
Objetivo: Avaliar a aplicabilidade de um método simplificado para
determinação quantitativa da atividade da glicose-6-fosfato
desidrogenase em crianças consideradas normais, determinar a
média e o desvio padrão e o valor abaixo do qual a atividade possa
ser considerada deficiente. Métodos: Foram analisadas amostras
de sangue colhidas de 201 crianças de duas creches da cidade de
São Paulo, consideradas normais, clínica e laboratorialmente. A
atividade enzimática foi determinada nas hemácias das crianças
normais, utilizando-se os reagentes do “kit Test Combination G-6-PDH®”.
Análises estatísticas: os resultados foram submetidos ao teste
“t” de Student, teste de Kolmogorov-Smirnov, teste monocaudal do
limite inferior de confiança e teste do coeficiente de correlação de
Spearman. Resultados: A média da hemoglobina das meninas foi
ligeiramente superior à média dos meninos, mas a diferença não
foi significativa. Não houve diferença significativa entre as
atividades médias da enzima para brancos e não-brancos. Não
houve correlação significativa entre os níveis de atividade enzimática, número de glóbulos vermelhos, concentração de hemoglobina,
hematócrito, reticulócitos, glóbulos brancos e idade dos pacientes.
A média da atividade da enzima para crianças do sexo masculino
foi de 4,448 U/g Hb, com desvio padrão de 1,380 U/g Hb. Para as
meninas foi de 4,531 U/g Hb, com desvio padrão de 1,386 U/g Hb,
diferença não significativa. Assim, considerou-se essas duas populações como população única, cuja média da atividade enzimática
foi de 4,490 U/g Hb, com desvio padrão de 1,380 U/g Hb. Como a
* Department of Pediatrics – Universidade Federal de São Paulo (SP).
1
Master’s degree in Pediatrics by Escola Paulista de Medicina/Unifesp. Physician-geneticist at Hospital do Servidor Público Estadual de São Paulo (SP).
Corresponding author: Roberto Muller - R. Araporé, 758 - Jardim Guedala - CEP 05608-001 - São Paulo (SP), Brazil - Tel./Fax: 288-4139 - e-mail: [email protected]
Received on September 30, 2003 – Accepted on November 24, 2003
einstein 2003; 1:89-94
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Muller R
distribuição dos valores dessa atividade aderiu à curva normal, foi
realizado o teste monocaudal de limite de confiança inferior, que
mostrou valor de corte de 2,227 U/g Hb. Conclusão: O método de
Solem mostrou ser simples, rápido, bastante preciso e útil para
detecção de atividade da glicose-6-fosfato desidrogenase e
identificação de crianças deficientes na enzima.
Descritores: Espectrofotometria; Glucose-6-fosfato desidrogenase;
Análise quantitativa; Favismo; Icterícia, neonatal; Anemia
hemolítica; Creches; Criança; Pré-escolar; Adolescente
INTRODUCTION
Glucose-6-phosphate dehydrogenase (G-6-PD) is an
enzyme composed of a polypeptidic chain of 515 amino
acids found in all tissues. Its role in the metabolism is
to catalyze the first step of the hexose-monophosphate
pathway that produces pentose, which is a precursor
of nucleic acids and some coenzymes; moreover, it
supplies reduced nicotinamide adenine dinucleotide
phosphate (NADPH), which is necessary for numerous
biological and detoxification(1) reactions. It was first
identified in 1931, as described by Van Noorden, in
1984(2), and its activity is regulated by a balance between
the active dimeric and the inactive monomer forms.
The gene that codifies G-6-PD is located in the
telomeric region of the long arm of chromosome X, in
band Xq28, at 400kb of factor VIII gene, being
composed of 13 exons and 12 introns(3).
As from 1956, many essays on G-6-PD(4) were carried
out and soon demonstrated the existence of several
variations of this enzyme, most of which cause no clinical
repercussion; some have markedly decreased activity, others,
diminished activity and some have increased activity.
Glucose-6-phosphate dehydrogenase deficiency is
the most common disease in humans, affecting
approximately 400,000,000 people worldwide(3) and has
been found in practically every race. The most frequent
types occurring in human populations are A+ (quick
electrophoretic migration) and B+ (slow electrophoretic
migration) phenotypes(5). The A+ and A- phenotypes are
found, mainly, in Africa and in areas where African
migration took place. Therefore, in the Afro-American
population, the genetic frequency of the A- allele is
10%-15% in males and approximately 2% in females(6).
The B - type is more frequent in Mediterranean
populations, such as Italian, Greek, Sephardi Jews,
whereas in populations from Southeastern Asia and
South Pacific the Canton variation prevails(7-8).
In Brazil, the A-African variation is the most
frequent (9-11) type, whereas the B -Mediterranean
variation stands out only among the Italian descendants
living in the southern states and in the State of São
Paulo. Population studies, conducted in several regions
einstein 2003; 1:89-94
of the country, showed this disease in 10% of negroids
and in 2% of Caucasians. The latter, specifically, reside
in the Southern and Southeastern states(5-6).
Numerous laboratory methods were described to
diagnose G-6-PD deficiency; some are only useful to
detect activity deficiency (qualitative), whereas others
quantify enzyme activity, or quantify their electrophoretic
migration pattern (quantitative). In 1972, WHO(12), in
Technical Report No. 509, classified the different
laboratory methods to investigate G-6-PD deficiency
activity as detection tests and confirmation tests.
The detection tests are as follows:
1. brilliant cresyl blue discoloration;
2. methemoglobin reduction;
3. tetrazoleum;
4. glutation reduction;
5. fluorescence.
The confirmation tests are as follows:
1. starch gel electrophoresis;
2. spectrophotometric quantification of enzyme activity
3. cytochemical methods
According to some authors, detection of a suspected
case must always be complemented by a confirmation
test(12), preferably spectrophotometric quantification(3),
in order to make an accurate diagnosis of G-6-PD
deficiency.
In 1955, a quantitative analysis (13) method was
described based on increased absorbance that takes
place when NADPH is formed from nicotinamide
adenine dinucleotide phosphate (NADP), by the action
of G-6-PD on glycose-6-phosphate. This absorbance
was measured by a spectrophotometer using ultra-violet
light of 340 nanometers. This method was widely
accepted and is still used today in commercial “kits”.
In 1984, however, this method(14) was submitted to
changes in order to improve, such as eliminating several
erythrocyte washings, making the method simpler,
quicker and more accurate, by avoiding hemolysis
caused by these washings.
The search for a simpler, easier, and quicker, yet
more accurate method stimulated testing the method
proposed by Solem in children considered normal, in
São Paulo.
OBJECTIVES
This essay had the following objectives:
• to evaluate the applicability of Solem method in
determining G-6-PD enzyme quantitative activity;
• to measure G-6-PD activity in children enrolled in two
day-care centers located in the city of São Paulo and
considered normal regarding physical examination and
hematimetric parameters;
Use of a simplified spectrophotometric method
• to determine the mean and standard deviation for
G-6-PD activity in this population;
• to determine the lower confidence interval for
G-6-PD activity in this population; the values below
this interval may be considered deficient.
METHODS
Patients and setting
Blood samples were collected from 201 children,
selected among 245, age range 18 months-13 years and
10 months (four girls in this oldest range), from several
origins, descending, mainly, from Afro-Brazilians,
Indigenous and Europeans and enrolled in two daycare centers located downtown, in the of the city of
São Paulo, were analyzed. There were one hundred
male children, 101 were female, 117 Caucasian and 84
considered non-Caucasian, that is, having any physical
characteristic that would lead to suspecting the child
was in fact Mestizo, Black or Oriental.
Inclusion and exclusion criteria
The criteria were based on clinical examination and
laboratory tests.
a. Clinical examination
The history of each child in the initial group of 245
children was thoroughly taken, in order to rule out
possible infectious processes, anemia or jaundice of
any origin. Next, these children went through a detailed
medical examination, in search of morbid processes that
may interfere with normal G-6-PD activity. Those
presenting signs consistent with infectious processes
or suspect of suffering from anemia were excluded from
the study.
b. Laboratory tests
Full blood count
The children included in the study through physical
examination were submitted to a full blood test with
peripheral vein puncture. Using a Coulter T890 model
automatic counter, the number of red blood cells per
cubic millimeter, amount of hemoglobin in grams per
deciliter of blood, number of leukocytes/mm 3 were
determined; reticulocyte percentage was determined
by brilliant cresyl blue (15) method. Children whose
parameters were within the normal limits found in the
literature were maintained in the study, totalling 201
subjects.
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G-6-PD quantitative dosage
G-6-PD activity was determined in normal child red
blood cells using the “Test Combination G-6-PDH®”
kit, manufactured by Boehringer Mannheim GmbH
Diagnostics and sold, in Brazil, by Merck do Brasil(16).
A 1-ml sample of peripheral venous blood was
collected from each child in vials containing 1.5 mg of
the anticoagulant EDTA. These samples were
withdrawn by the same person, always in the morning,
and while children were selected.
Later the samples were submitted to a quantitative
enzyme assay, according to the method described in
1984(14), which is based on the following reaction:
G-6PD
Glucose-6-phosphate + NADP Ý 6-phosphogluconate + NADPH
As NADPH is formed from NADP, there is an increase
in absorbance of ultraviolet light at 340 nm. Quantifying
this increase, since it translates raise in NADPH
concentration, indirectly quantifies the activity of
glucose-6-phosphate dehydrogenase, the enzyme that
catalyzes this reaction.
The blood samples were submitted to ultracentrifugation
at 15.000 g (approximately 11.000 rpm), for five minutes,
by means of a Beckman L3-50 Preparative Ultracentrifuge,
manufactured by Beckman Instruments Inc. (Palo Alto,
Ca., USA). The aim was to separate old red blood cells
from new ones based on higher specific weight of the
older cells, thus eliminating the influence exerted by
greater enzyme activity of G-6-PD in the neocytes and
improving accuracy of the method. Due to their higher
specific weight, the old red blood cells sediment stayed
in the bottom of the flask, from where it was carefully
aspirated using a 5-mL Oxford automatic pipette.
Afterwards, 10 mL of red blood cells were placed in a
test tube, and 100 mL of digitonine (solution No. 4,
according to manufacturer’s specifications(16)) were added.
The samples were homogenized and left to rest for 15
minutes, at 4°C. Later the samples were centrifuged at
3.000 rpm, for 10 minutes and at the same temperature,
using a Sorwall refrigerated centrifuge, model RC5-B,
manufactured by Ivan Sorwall, Inc. (Norwalk, Co., USA).
Hemoglobin concentration was measured in the resulting
hemolysate using the cyanomethemoglobin method(17).
In a test tube containing 3 mL of solution No. 1 and
0.1mL of solution N. 2, we added 0.02 mL of the
hemolysate. Sixty seconds later, 0.05mL of solution N. 3
was added, according to manufacturer’s specifications(16).
After a thirty-second pre-incubation period, the reading
was made using a Spectronic 2000 spectrophotometer,
manufactured by Bausch & Lomb (Rochester, NY,
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Muller R
USA), with ultraviolet light at 340 nm. The reading
accuracy control was made using the standard serum
Precinorm E, manufactured by Boehringer Mannheim
GmbH Diagnostics(16).
Four readings at 25°C were made for each sample,
always by the same person, with a 60-second interval
between readings. The enzyme activity was calculated
using the following formula:
children, 100 were boys and 101 were girls, and 117
were Caucasian and 84 non-Caucasian (Mestizo, Black
and Oriental) (table 1).
Table 1. Composition of the sample of children studied by sex and race
Race
Key: U/g Hb = G-6-PD enzyme activity
E/min = mean of the differences between each
absorbance reading
Hb = hemoglobin concentration in hemolysate, in g/%
4531 = ratio between enzyme activity and hemoglobin
volume in the hemolysate
Statistical methods
The mean glucose-6-phosphate dehydrogenase activity
for Caucasians and non-Caucasians(5,18), and for boys and
girls were submitted to Student’s t test for two independent
means according to Sokal, 1969(19), in order to verify if
there was any statistically significant difference between
these populations or not. The red blood cell count,
hemoglobin, hematocrit, reticulocyte percentage and
white blood cell count mean values, for boys and girls,
were also submitted to the same test.
Next, the individual values of G-6-PD activity were
submitted to Kolmogorov-Smirnov test(20) to verify if
there was any significant deviation from the normal
distribution. The lower confidence interval was then
applied to obtain the reference value from which G-6-PD
activity should be considered deficient.
The Spearman’s correlation coefficient (20) was
calculated to study the relation between age and numerical
values of the variables studied.
For all tests, a value of 0.05 or 5% (significance
level) was established to reject null hypothesis.
The parents were requested to sign a written consent
authorizing their children to participate in the study,
pursuant to regulations of the Research Ethics
Committee, Postgraduate Courses, Escola Paulista de
Medicina, Universidade Federal de São Paulo.
RESULTS
The 201 children who presented blood parameters
within the normal range (21-22) were submitted to
quantitative analysis of the G-6-PD activity. Of these
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Sex
Total
Boys
%
Girls
%
56
47,9
61
52,1
NON CAUCASIAN
44
52,4
40
47,6
TOTAL
100
CAUCASIAN
E/min
U/g Hb = ————————————— x 4531
Hb
Sex
101
117
84
201
The mean hemoglobin value of the girls was slightly
lower than that of boys, but this difference was not
considered significant. There was also no difference
between the mean G-6-PD activities for Caucasian and
non-Caucasian children (tables 2 and 3).
Table 2. G6PD Activity in U/g Hb - ordenated data, means and standarddev for boys and girls, student´s t test (Sokal, 1969)
Race
Sex
Sex
Total
Boys
%
Girls
%
56
47,9
61
52,1
NON CAUCASIAN
44
52,4
40
47,6
TOTAL
100
CAUCASIAN
101
117
84
201
Table 3. G6PD Activity in U/g Hb - ordenated data, means and standarddeviations caucasian and non-caucasian, student´s t test (Sokal, 1969)
SAMPLE
MEAN
STANDARD DEVIATION
STUDENT´S t TEST
Caucasian
Non-caucasian
117
4.622
1.404
84
4.305
1.333
calculated t = 0.346
critical t (5%) = 1.66
There was no significant correlation between G-6-PD
activity levels, red blood cell count, hemoglobin
concentration, hematocrit, reticulocyte count, white
blood cell count and age, as shown by the linear
regression tests.
The mean G-6-PD activity for boys was 4.448 U/g Hb,
standard deviation = 1.380 U/g Hb. For girls, the mean
activity was 4.531 U/g Hb, standard deviation = 1.386 U/Hb
(table 2). Such difference was not considered significant
using Student’s t test (p = 0.05). Therefore, these two
groups were considered as one single population, with
a mean G-6-PD activity of 4.490 U/g Hb, standard
deviation = 1.380 U/g Hb.
Use of a simplified spectrophotometric method
The distribution of the G-6-PD activity values was
assessed by Kolmogorov-Smirnov test and did not show a
significant deviation from the normal distribution (table 4
and chart 1). Therefore, lower confidence interval (onetailed) was performed and resulted in a cutoff point of
2.227 U/g Hb.
Table 4. G6PD Activity in U/g Hb - ordenated data, means and standarddeviations for boys+girls, caucasian+non-caucasian kolmogorov-smirnov
test (Siegel, 1975)
Boys+Girls, Caucasian+Non-caucasian
SAMPLE
MEAN
STANDARD DEVIATION
KOLMOGOROV-SMIRNOV
TEST
dmax. = 0.051
201
4.49
1.38
critical SD (1%) = 0.115
critical SD (5%) = 0.959
DISCUSSION
The children of the study were considered normal based
on clinical and hematological examinations and fulfilled
the criteria for normality(21-22). A detailed history, complete
physical examination and laboratory tests were the bases
used to exclude the subjects with infectious processes of
any etiology and any type of jaundice.
The population of this study is different from that
studied by Solem(14) since only pediatric subjects (aged
18 months to 13 years and 10 months) were taken into
account, whereas Solem evaluated individuals aged one
month to 48 years of age. The size of both samples is
also very different - the present study (101 girls and
100 boys) sample is much larger than that used by Solem
(55 women and 76 men). Another difference to be
considered is the genetic composition of the two
populations, since Solem(14) selected individuals who
came from the Mediterranean area, whereas the
children in this study had several origins, descending,
mainly, from Afro-Brazilians, Indigenous and
Europeans(5).
In the literature, there are many studies on several
laboratory methods to evaluate G-6-PD activity(23-34); in
that, some are simpler and less expensive, but require
fresh samples; other methods are either too sophisticated
and require longer performance time, or are not
sufficiently accurate, giving false-positive and falsenegative results, specially regarding heterozygote
detection.
Unlike the traditional methods, such as Körnberg,
Solem offers a quantitative analysis method(14) that is
recommended by the kit manufacturer, requires not
many erythrocyte washings, rendering the test simpler,
93
quicker and more accurate, since it avoids hemolysis
caused by these procedures(19). Moreover, considering
the specific hemoglobin concentration of each patient,
it deals with the influence of reticulocytosis observed
in newborns and in pathologic processes of other
etiology.
In both studies - Solem(14) and the present one, great
care was taken to select normal individuals based on
hematological parameters; nevertheless, in our study,
there was some difference in mean hemoglobin
concentration between males and females, and the girls
had lightly higher concentration. This difference,
however, was not significant. This study included girls
who could have menses; however, there was no
influence of menses on enzyme activity result, which
was always given as “units per gram of hemoglobin”,
especially because the number of girls at this age range
was minimum.
The values found in this study had a normal
distribution and fit Gauss curve, as shown in the
statistical analysis. Therefore, it is possible to calculate
mean, standard deviation, and consequently, the lower
confidence interval to be the reference value under
which G-6-PD activity could be considered deficient.
Moreover, this method could easily be performed to
test G-6-PD activity in children with hemolytic anemia,
which may be caused by deficiency of this enzyme.
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Muller R
CONCLUSIONS
The method proposed by Solem, which is used to
quantitatively determine glucose-6-phosphate
dehydrogenase activity is a simple, fast, and very
accurate method that may be useful to detect children
with deficiency of this enzyme.
In this study, G-6-PD activity values did not
significantly deviate from the normal distribution and
the mean value found for G-6-PD activity was
4.490 U/g Hb ± 1.380 U/g Hb. The lower confidence
interval for G-6-PD was 2.227 U/g Hb (95% confidence
interval). The values below this interval should be
considered as deficient by this method.
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