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IOSR Journal of Pharmacy and Biological Sciences (IOSR-JPBS)
e-ISSN: 2278-3008, p-ISSN:2319-7676. Volume 6, Issue 1 (Mar. – Apr. 2013), PP 51-55
www.iosrjournals.org
www.iosrjournals.org 51 | Page
Estimation of G6pd Status in the Rajbangshi Population of
Sushrutanagar
Anindya Dasgupta1
, Tanushree Mondal2
, Asok Kumar Datta3
1. Professor, Department of Biochemistry, National Medical College, Kolkata, West Bengal
2. Assistant Professor, Department of Community Medine, N.R.S. Medical College, Kolkata, West Bengal
3. Associate Professor, Department of Pediatrics, Bankura Sammilani Medical College, Bankura, West
Bengal
Abstract: The Glucose 6 Phosphate Dehydrogenase (G6PD) deficiency renders the cells susceptible to severe
hemolysis under oxidative stress producing conditions. Due to considerable genetic heterogeneity it is found to
be distributed in an unequal manner in different parts of the world including India. The present study was
undertaken to find the distribution of G6PD deficiency in the Rajbangshi population group of Sushrutanagar,
Darjeeling district. The Rajbangshis are one of the oldest tribes residing in North Bengal. A quantitative assay
of the G6PD activity was performed in the Rajbangshi population and the non Rajbangshi control population of
the same area. The G6PD deficiency was found in 12 percent of the Rajbangshi population studied which is
significantly higher than the value of 3.3 percent found in the controls with the lower and upper confidence limit
for the population Odds ratio being 3.58 and 25.93 respectively at 95% confidence interval. This high rate of
G6PD deficiency in the population group studied poses them to a greater risk for several oxidative stress
producing conditions including some commonly used antimalarial drug like Primaquin.
Key Words : Glucose 6 Phosphate Dehydrogenase (G6PD) deficiency, Rajbangshis, Ethnicity.
I. Introduction
Glucose 6 Phosphate Dehydrogenase (G6PD) is an important enzyme in the hexose monophosphate
(HMP) shunt pathway of glucose utilization1
. The HMP shunt provides NADPH which along with other cellular
antioxidants is utilized to neutralize the hydrogen peroxide (H2O2) and hence protect the Red Blood Corpuscle
(R.B.C) membrane from hemolysis, particularly under the conditions which produce oxidative stress1
in the
cells. The enzyme is present abundantly in R.B.Cs, liver, adipose tissues, adrenal cortex and gonads 1
. In the
persons having low G6PD activity the R.B.Cs are much prone to hemolysis, particularly under conditions
which produce oxidative stress in the cells, e.g. bacterial infection and exposure to certain drugs like aspirin,
mepacrine, primaquine, sulphadoxine etc2,3
. On the other hand the G6PD deficiency is found to protect the
population against the Falciparum Malaria1
. So, this enzyme has been an important sector of research all
throughout the ages and throughout the world. The deficiency of this enzyme is the most common congenital
defect in the HMP shunt pathway4
and is transmitted in an X linked recessive manner by heterozygous females,
while the sufferers are mainly the males5
. This deficiency is found to be distributed unequally among different
races in the world. Highest incidence is in Kurdish Jews which equals to about 60 percent, followed by 15-30
percent in Negroes, 13 percent in Parsees of Mumbai2
. The deficiency is also prevalent in the West and East
Africa, Greece, Mediterranean people, China(specially the Li population)6
, Japan, North West India, Srilanka2
etc. Among these the black people of Africa and the Japanese (10.8 percent Shan Group of Indonesia and
Malaysia) are more susceptible7
and about 100 million people are deficient in this enzyme8
. India is a country
belonging to a heterogeneous population group for ages. In India this deficiency has been found to be present in
2-5 percent of the general population9
. The Nagas and Hmars of the North Eastern India have reported G6PD
deficiency to a substantial amount.10
. The other groups of the Indian population where G6PD deficiency is
prevalent are the Punjabis, the Sindhis, the Lohanas, and the Kutchis apart from the Parsees11
.
Keeping these facts in mind, we hypothesized that the indigenous population group living in North
Bengal might exhibit a different pattern of G6PD deficiency also. Accordingly the study on the distribution of
G6PD deficiency was carried out in the „Rajbangshi‟ population of Sushrutanagar of District Darjeeling of the
West Bengal state. The total area of the Sushrutanagar has a population of 1,299,91 covering an area of 30.75
sq.km (as per census, 1991, data obtained from Matigara No.1 village Panchayat) 15-20 percent of which
comprise of the “Rajbhansis”, a particular tribe residing here for decades. They are, in fact, one of the oldest
tribes of the North Bengal adding significantly to the cultural and economical growth of the area. G6PD activity
in them was measured to find out the prevalence of the deficiency of this enzyme in them.
Estimation Of G6pd Status In The Rajbangshi Population Of Sushrutanagar
www.iosrjournals.org 52 | Page
II. Materials And Methods
Selection of patients
The present study was carried out in 100 persons selected randomly from the Rajbangshi population.
Among them sixty were males and forty were females. All of them were free from any disease at the time of test
with no history of any chronic disease. 60 non Rajbangshi people were selected from the non- Rajbangshi
populations residing in the same area in a randomized manner. Forty of them were males and twenty of them
were females. All of them were healthy and were free from any diseases at the time of test with no history of
any chronic disease. Informed consent was obtained from the participants and the study was carried out
according to the Helsinki declaration 1975. The study was approved by the institutional ethical committee.
Measurement of G6PD activity
The quantitative assay for the G6PD activity in the present study was done by the reagents supplied in
the Lyozyme kinetic kit provided by the Vital Diagnostics (P) Ltd, Mumbai12,
.
Principle of the test
Glucose 6 phosphate is oxidized in presence of NADP+
and the enzyme Glucose 6 Phosphate
dehydrogenase into 6 phosphogluconate. In this reaction the NADP+
is reduced into NADPH and H+
. The
absorbance of the NADPH is measured in a spectrophotometer at 340 nm at one minute interval for four times.
The rate of change of the absorbance is calculated and calibrated against a factor provided in the kit manual and
the hemoglobin concentration to calculate the activity of the G6PD enzyme per gram of hemoglobin.
Reagents provided
1) G6PD reagent 1 : Coenzyme and substrate.
2) G6PD reagent 2 : Buffer.
3) G6PD reagent 3 : Lysing reagent.
Preparation of working reagent
1.0 ml. of G6PD reagent 2 (buffer) is added to the bottle of G6PD reagent 1 (Coenzyme and substrate),
mixed well and used after five minutes.
Method of the test
Preparation of the hemolysate
For both cases and controls, 2 ml. of blood sample were collected in an EDTA vial. From this 0.1 ml.
of whole blood was added to 2 ml. aliquots of physiological saline (0.9percentNacl) and washed three times.
Now 0.5 ml. of G6PD reagent 3 (Lysing reagent) precooled at 2-8 degrees Celsius is added to the packed cells.
After mixing well by vigorous shaking the solution was kept at 2-40
C for half an hour. It was centrifuged then
at 3000 rpm for 5 minutes prior to use.
Determination of the hemoglobin concentration
This was done by the Cynomethemoglobin method (CMG method). For this 20µl of blood was added
to 5 ml. of Drabkin‟s solution and mixed well. After keeping at room temperature for five minutes the
absorbance of the solution was determined in a spectrophotometer 530 nm, and the haemoglobin content was
determined by calibrating it against the standard curve of hemoglobin.
Determination of the G6PD activity
In a test tube marked (T) the reagents are added in following sequence
Addition Sequence Test
Working reagent 1000µl.
Hemolysate 25µl.
After mixing well and after an initial delay of 180 seconds, the absorbance of the test is measured at a
wavelength of 340 nm. This measurement is continued at an interval of one minute for next three minutes and
the mean absorbance change per minute (∆A/min) is calculated.
Calculations
G6PD activity in U/g Hb = ∆A/Min X
224 X 100
6.22 X Hb(g/dl).
Hb(g/dl).
Estimation Of G6pd Status In The Rajbangshi Population Of Sushrutanagar
www.iosrjournals.org 53 | Page
= ∆A/Min X
Where; 100 = Factor to convert to 100ml.
224 = Total assay volume to sample volume,
6.22 = Millimolar absorptivity of the NADPH at 340 nm,
Hb(g/dl) = Hemoglobin concentration.
Normal reference range : G6PD activity (at 300
C) in U/g Hb = 4.6-13.5 U/g Hb.
Statistical analysis
The deficient numbers of G6PD deficiency were segregated for both case and
non case population. Odds ratio and chi square values were calculated and the lower and upper limit of the Odds
ratio (OR) was found at 95% confidence interval. Significance of the lower and upper range of OR was found
out at 95% confidence interval.
III. Results
Among the 100 cases, 12 persons were found to have a G6PD activity below the normal reference
range (Table-1). Among the 60 non case people this deficiency was found in 2 persons. So, in the present study
the G6PD deficiency among the Rajbanshis is found to be 12 percent, and about 3.33 percent in the non case
population. Chi square (χ2
) and Odds ratio (OR) was found to be 2.05 and 3.95 respectively. The lower and
upper confidence limit for the population Odds ratio were 3.951-1.96/√2.05
= 3.58 and 3.951+1.96/√2.05
= 25.93
respectively. We concluded with 95% confidence that the population OR was somewhere between 3.58 and
25.93. Since the interval did not include 1, we concluded that in the Rajbangshi population (cases) G6PD
deficiency is more likely to be present than in the non Rajbangshi (noncase) group.
The overall distribution of the G6PD activity in both Cases and Controls are shown in the figure 1 and
table 1 respectively.
Figure 1
Distribution of G6PD activity in cases (n = 100) and control (n = 60) groups
Table 1
The distribution of the G6PD activity in the Cases and Controls (Normal reference range of G6PD activity in
U/g Hb=4.6-13.5 U/g hemoglobin).
G6PD inadequate G6PD adequate
Cases 12 88
Non cases 2 58
Chi square (χ2) = 2.05, OR = 3.95
0
2
4
6
8
10
12
14
16
0 50 100 150
G6PD Activity in CASES ,
G6PD Activity in CONTROLS
3601_
Hb(g/dl).
Estimation Of G6pd Status In The Rajbangshi Population Of Sushrutanagar
www.iosrjournals.org 54 | Page
The lower and upper confidence limit for the population Odds ratio are 3.951-1.96/√2.05
= 3.58 and 3.951+1.96/√2.05
=
25.93 respectively. We conclude with 95% confidence that the population OR is somewhere between 3.58 and
25.93.
IV. Discussion
More than 200 million people all over the world suffer from G6PD deficiency 13
and this deficiency is
found to be distributed in a heterogeneous manner among different population groups of the world. In India also
the G6PD deficiency is found to be distributed in a heterogeneous pattern as already discussed above. In the
present study the G6PD deficiency in the Rajbangshis of Sushrutanagar area of the Darjeeling district of West
Bengal is found to be about 15 percent which is definitely higher than the overall rate of 2-5 percent of G6PD
deficiency in the general population of India9
. Although this study comprised of about one hundred cases
selected randomly from about 15000 populations of Rajbangshis of the Sushrutanagar area, yet the high
percentage of G6PD deficiency in the studied cases indicates an overall high rate of G6PD deficient people
among the Rajbangshi population of Sushrutanagar. Simultaneously, the distribution rate is found to be only 3
percent in the non Rajbangshi population of the same area. The genetic heterogeneity which is found in some
enzymopathies may be one of the important causes for this noted heterogeneous distribution of the G6PD
deficiency among the individuals, and indeed over 400 variants of G6PD have been described already13
. A
strong evidence of significant difference between frequency of G6PD deficiency in Tribal and non-tribal group
of newborns with more frequency of G6PD deficiency in tribal newborns with neonatal hyperbilirubinemia.
Prevalence ratios among Non-tribal and Tribal neonates were 12.00% and 23.07% respectively. This difference
is statistically significant (χ2 = 2.31; df=1 p <0.05 )14
.
Other reports of G6PD deficiency are shown variability in different ethnic populations like Parsee16
,
Brahmin17
, Jat18
and Vataliya Prajapati19
etc. The frequency is higher among the tribals than the caste
populations20
. According to Bhasin and Walter 21
, the frequency of G6PD among Indian Population as a whole
ranges from complete absence to 27%. It is higher among scheduled tribes as compared to other ethnic groups.
G6PD deficient allele frequency is comparatively higher in North and West Indian Zones, whereas in South
India it is uniformly low except in Andhra Pradesh and Tamil Nadu. Prevalence of G6PD deficiency is generally
0-10%, although some community may have higher prevalence: 27.5% for the Angami Nagas, a tribal group in
Northern India22
Abnormalities in primary DNA or protein sequence have been established in a number of the G6PD
variants and in almost all cases the alteration there is one or more base substitutions, leading to an amino acid
replacement and not a deletion in the protein 13
. Evidence in the abnormality in structure is seen in the
differences in electrophoresis mobility, enzyme kinetics, pH optimum, and heat stability; and these differences
result in great variation of clinical severity, ranging from nonspherocytic hemolytic anemia without
demonstrable oxidant stress (particularly shortly after birth), through hemolytic anemia only when stimulated by
marked to mild oxidant stress, to no clinically detectable abnormality23
. The normal G6PD is designated as type
B, but about 20% of individuals of African descent have a G6PD , designated as type A+ that differs by a single
amino acid and is electrophoretically distinguishable but functionally normal. Among the clinically significant
G6PD variants, the most common, the A- type, is due to two base substitutions and is encountered primarily in
individuals with ancestral origins in Central Africa24
. This variety of the G6PD enzyme has abnormal kinetic
property and is found in about 11 percent of African descended males in the United States. A second variety of
G6PD is encountered in the peoples of the Mediterranean circumlittoral, particularly the Sardinians and
Sephardic Jews. This variant is more severe than the A- variety and may result in non spherocytic hemolytic
anemia 24
. A third relatively common and slightly less severe variant occurs in southern Chinese population. Till
now no such genetic data is available for the population of the Rajbangshis of the Sushrutanagar and so, a
detailed study is required in this area to properly characterize the G6PD variety of this region where the G6PD
deficiency has been found to be relatively higher than the general population of India.
References
[1]. Vasudevan, D.M. and Srikumari, S. (1998), Text Book of Biochemistry for Medical Students, 2nd edn. Jaypee Brothers Medical
Publishers Ltd., New Delhi, p 59.
[2]. Ghai, O.P. (2001), Essentials of Paediatrics, 5th
edn. Mehta Publishers, New Delhi, p 102-103.
[3]. Vasudevan, D.M. and Srikumari, S. (1998), Text Book of Biochemistry for Medical Students, 2nd
edn. Jaypee Brothers Medical
Publishers Ltd. New Delhi, p103-104.
[4]. Wendell, Ross. and H, Franklin Bunn. (2001), Harrison‟s Principle of Internal Medicine, vol 1,15th
edn. (International edition).
MCgraw Hills, Singapore, p 685.
[5]. Vasudevan, D.M.and Srikumari, S. (1998), Text Book of Biochemistry for Medical Students, 2nd
Edition. Jaypee Brothers Medical
Publishers Ltd. New Delhi, p 305.
[6]. Cai, W., Filosa, S., Martini, G. (1994), DNA haplotypes in the G6PD Gene Cluster studied in the Chinese Li Population and their
relationship to G6PD. HumHered. 44 (5), 279-86.
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[7]. Iwak, K., Hiron , A., Matsuoka, H., Kawamoto, F., Herie, T Link., Tantular, I S., Dachlan, I P., Notopuro, H., Hidayah, N I .,
Salim, A M., Fujii, H., Miwa, S., Ishii, A. (2001), Distribution of Glucose 6 Phosphate Dehydrogenase Mutations in South East
Asia. HumGenet. 108(6), 445-9.
[8]. Robert, K Murray. (2000), Harper‟s Biochemistry, 25th
edn. Appleton & Lange, Stanford, Connecticut, p 767.
[9]. Talwar, G.P., Srivastava, L M., Moudgil, U D. (1989), Text Book of Biochemistry and Human Biology, 2nd
edn. Elements of
Human Genetics, Chapter-53,Section –VII, Page-636.
[10]. Saha, N., Tay, J S. (1990), Genetic studies among the Nagas and Hmars of Eastern India. Am J Phys Antropol. 82(1), 101-12.
[11]. Chatterjee, M.N. and Shinde, Rana. (1997), 2nd
edition, Test Book of Medical Biochemistry, Jaypee Brothers Medical Publishers Ltd.
New Delhi, page: 401.
[12]. Kachmar , J. F. and Moss, D. W. (1976), Enzymes in fundamentals of Clinical Chemistry. N.W.Teitz , Saunders, Philadelphia, p 666-
670.
[13]. Gross, R T., Hurwitz, R E., Marks, P A. (1978), Red cell Glucose 6 Phosphate Dehydrogenase Deficiency. J.Clin. Invest. 37, 176.
[14]. Manik Mondal, Asok Kumar Datta, Syamali Mandal, Pradeep Kumar Das. Study of Glucose 6 phosphate dehydrogenase deficiency
in neonatal jaundice. IOSR Journal of Pharmacy and Biological Sciences. Volume 1, Issue 5 (July-August 2012), PP 30-36.
[15]. Baxi, V. Balakrishnan, J. V. Undevia, and L. D. Sanghvi, “Glucose-6-phosphate dehydrogenase deficiency in the Parsee community,
Bombay.,” Indian journal of medical sciences, vol. 17, pp. 493–500, 1963.
[16]. A. Santhi and M. P. Sachdeva, “Glucose-6-phosphate dehydrogenase deficiency among the jats and brahmins of sampla
(Haryana),” The Anthropologist, vol. 6, no. 4, pp. 291–292, 2004.
[17]. K. N. Saraswathy and S. Aggarwal, “Study of glucose-6- phosphate dehydrogenase deficiency and sickle cell anemia among the
Tamil Brahmins of Delhi,” The Anthropologist, vol. 7, no. 1, pp. 45–47, 2005.
[18]. A. Santhi and M. P. Sachdeva, “Glucose-6-phosphate dehydrogenase deficiency among the jats and brahmins of sampla
(Haryana),” The Anthropologist, vol. 6, no. 4, pp. 291–292, 2004.
[19]. S. C. Gupte, A. N. Shaw, and K. C. Shah, “Hematological findings and severity of G6PD deficiency in Vataliya Prajapati
subjects,” Journal of Association of Physicians of India, vol. 53, pp. 1027–1030, 2005.
[20]. V. Tripathy and B. Reddy, “Present status of understanding on the G6PD deficiency and natural selection,” Journal of Postgraduate
Medicine, vol. 53, no. 3, pp. 193–202, 2007.
[21]. M. K. Bhasin and H. Walter, Genetic of Caste and Tribes of India, Kamla-Raj Enterprises, Delhi, India, 2001.
[22]. P. K. Seth and S. Seth, “Biogenetical studies of Nagas: glucose-6-phosphate dehydrogenase deficiency in Angami Nagas.,” Human
Biology, vol. 43, no. 4, pp. 557–561, 1971.
[23]. Wendell, Ross., H, Franklin Bunn. (1998), Harrison‟s Principle of Internal Medicine, vol 1, 14th
edn. (International edition).
MCgraw Hills, Singapore, p 663.
[24]. Wendell, Ross., H, Franklin Bunn. (1998), Harrison‟s Principle of Internal Medicine, vol 1, 14th
edn. (International edition). MCgraw
Hills, Singapore, p 663-64.

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Estimation of G6pd Status in the Rajbangshi Population of Sushrutanagar

  • 1. IOSR Journal of Pharmacy and Biological Sciences (IOSR-JPBS) e-ISSN: 2278-3008, p-ISSN:2319-7676. Volume 6, Issue 1 (Mar. – Apr. 2013), PP 51-55 www.iosrjournals.org www.iosrjournals.org 51 | Page Estimation of G6pd Status in the Rajbangshi Population of Sushrutanagar Anindya Dasgupta1 , Tanushree Mondal2 , Asok Kumar Datta3 1. Professor, Department of Biochemistry, National Medical College, Kolkata, West Bengal 2. Assistant Professor, Department of Community Medine, N.R.S. Medical College, Kolkata, West Bengal 3. Associate Professor, Department of Pediatrics, Bankura Sammilani Medical College, Bankura, West Bengal Abstract: The Glucose 6 Phosphate Dehydrogenase (G6PD) deficiency renders the cells susceptible to severe hemolysis under oxidative stress producing conditions. Due to considerable genetic heterogeneity it is found to be distributed in an unequal manner in different parts of the world including India. The present study was undertaken to find the distribution of G6PD deficiency in the Rajbangshi population group of Sushrutanagar, Darjeeling district. The Rajbangshis are one of the oldest tribes residing in North Bengal. A quantitative assay of the G6PD activity was performed in the Rajbangshi population and the non Rajbangshi control population of the same area. The G6PD deficiency was found in 12 percent of the Rajbangshi population studied which is significantly higher than the value of 3.3 percent found in the controls with the lower and upper confidence limit for the population Odds ratio being 3.58 and 25.93 respectively at 95% confidence interval. This high rate of G6PD deficiency in the population group studied poses them to a greater risk for several oxidative stress producing conditions including some commonly used antimalarial drug like Primaquin. Key Words : Glucose 6 Phosphate Dehydrogenase (G6PD) deficiency, Rajbangshis, Ethnicity. I. Introduction Glucose 6 Phosphate Dehydrogenase (G6PD) is an important enzyme in the hexose monophosphate (HMP) shunt pathway of glucose utilization1 . The HMP shunt provides NADPH which along with other cellular antioxidants is utilized to neutralize the hydrogen peroxide (H2O2) and hence protect the Red Blood Corpuscle (R.B.C) membrane from hemolysis, particularly under the conditions which produce oxidative stress1 in the cells. The enzyme is present abundantly in R.B.Cs, liver, adipose tissues, adrenal cortex and gonads 1 . In the persons having low G6PD activity the R.B.Cs are much prone to hemolysis, particularly under conditions which produce oxidative stress in the cells, e.g. bacterial infection and exposure to certain drugs like aspirin, mepacrine, primaquine, sulphadoxine etc2,3 . On the other hand the G6PD deficiency is found to protect the population against the Falciparum Malaria1 . So, this enzyme has been an important sector of research all throughout the ages and throughout the world. The deficiency of this enzyme is the most common congenital defect in the HMP shunt pathway4 and is transmitted in an X linked recessive manner by heterozygous females, while the sufferers are mainly the males5 . This deficiency is found to be distributed unequally among different races in the world. Highest incidence is in Kurdish Jews which equals to about 60 percent, followed by 15-30 percent in Negroes, 13 percent in Parsees of Mumbai2 . The deficiency is also prevalent in the West and East Africa, Greece, Mediterranean people, China(specially the Li population)6 , Japan, North West India, Srilanka2 etc. Among these the black people of Africa and the Japanese (10.8 percent Shan Group of Indonesia and Malaysia) are more susceptible7 and about 100 million people are deficient in this enzyme8 . India is a country belonging to a heterogeneous population group for ages. In India this deficiency has been found to be present in 2-5 percent of the general population9 . The Nagas and Hmars of the North Eastern India have reported G6PD deficiency to a substantial amount.10 . The other groups of the Indian population where G6PD deficiency is prevalent are the Punjabis, the Sindhis, the Lohanas, and the Kutchis apart from the Parsees11 . Keeping these facts in mind, we hypothesized that the indigenous population group living in North Bengal might exhibit a different pattern of G6PD deficiency also. Accordingly the study on the distribution of G6PD deficiency was carried out in the „Rajbangshi‟ population of Sushrutanagar of District Darjeeling of the West Bengal state. The total area of the Sushrutanagar has a population of 1,299,91 covering an area of 30.75 sq.km (as per census, 1991, data obtained from Matigara No.1 village Panchayat) 15-20 percent of which comprise of the “Rajbhansis”, a particular tribe residing here for decades. They are, in fact, one of the oldest tribes of the North Bengal adding significantly to the cultural and economical growth of the area. G6PD activity in them was measured to find out the prevalence of the deficiency of this enzyme in them.
  • 2. Estimation Of G6pd Status In The Rajbangshi Population Of Sushrutanagar www.iosrjournals.org 52 | Page II. Materials And Methods Selection of patients The present study was carried out in 100 persons selected randomly from the Rajbangshi population. Among them sixty were males and forty were females. All of them were free from any disease at the time of test with no history of any chronic disease. 60 non Rajbangshi people were selected from the non- Rajbangshi populations residing in the same area in a randomized manner. Forty of them were males and twenty of them were females. All of them were healthy and were free from any diseases at the time of test with no history of any chronic disease. Informed consent was obtained from the participants and the study was carried out according to the Helsinki declaration 1975. The study was approved by the institutional ethical committee. Measurement of G6PD activity The quantitative assay for the G6PD activity in the present study was done by the reagents supplied in the Lyozyme kinetic kit provided by the Vital Diagnostics (P) Ltd, Mumbai12, . Principle of the test Glucose 6 phosphate is oxidized in presence of NADP+ and the enzyme Glucose 6 Phosphate dehydrogenase into 6 phosphogluconate. In this reaction the NADP+ is reduced into NADPH and H+ . The absorbance of the NADPH is measured in a spectrophotometer at 340 nm at one minute interval for four times. The rate of change of the absorbance is calculated and calibrated against a factor provided in the kit manual and the hemoglobin concentration to calculate the activity of the G6PD enzyme per gram of hemoglobin. Reagents provided 1) G6PD reagent 1 : Coenzyme and substrate. 2) G6PD reagent 2 : Buffer. 3) G6PD reagent 3 : Lysing reagent. Preparation of working reagent 1.0 ml. of G6PD reagent 2 (buffer) is added to the bottle of G6PD reagent 1 (Coenzyme and substrate), mixed well and used after five minutes. Method of the test Preparation of the hemolysate For both cases and controls, 2 ml. of blood sample were collected in an EDTA vial. From this 0.1 ml. of whole blood was added to 2 ml. aliquots of physiological saline (0.9percentNacl) and washed three times. Now 0.5 ml. of G6PD reagent 3 (Lysing reagent) precooled at 2-8 degrees Celsius is added to the packed cells. After mixing well by vigorous shaking the solution was kept at 2-40 C for half an hour. It was centrifuged then at 3000 rpm for 5 minutes prior to use. Determination of the hemoglobin concentration This was done by the Cynomethemoglobin method (CMG method). For this 20µl of blood was added to 5 ml. of Drabkin‟s solution and mixed well. After keeping at room temperature for five minutes the absorbance of the solution was determined in a spectrophotometer 530 nm, and the haemoglobin content was determined by calibrating it against the standard curve of hemoglobin. Determination of the G6PD activity In a test tube marked (T) the reagents are added in following sequence Addition Sequence Test Working reagent 1000µl. Hemolysate 25µl. After mixing well and after an initial delay of 180 seconds, the absorbance of the test is measured at a wavelength of 340 nm. This measurement is continued at an interval of one minute for next three minutes and the mean absorbance change per minute (∆A/min) is calculated. Calculations G6PD activity in U/g Hb = ∆A/Min X 224 X 100 6.22 X Hb(g/dl). Hb(g/dl).
  • 3. Estimation Of G6pd Status In The Rajbangshi Population Of Sushrutanagar www.iosrjournals.org 53 | Page = ∆A/Min X Where; 100 = Factor to convert to 100ml. 224 = Total assay volume to sample volume, 6.22 = Millimolar absorptivity of the NADPH at 340 nm, Hb(g/dl) = Hemoglobin concentration. Normal reference range : G6PD activity (at 300 C) in U/g Hb = 4.6-13.5 U/g Hb. Statistical analysis The deficient numbers of G6PD deficiency were segregated for both case and non case population. Odds ratio and chi square values were calculated and the lower and upper limit of the Odds ratio (OR) was found at 95% confidence interval. Significance of the lower and upper range of OR was found out at 95% confidence interval. III. Results Among the 100 cases, 12 persons were found to have a G6PD activity below the normal reference range (Table-1). Among the 60 non case people this deficiency was found in 2 persons. So, in the present study the G6PD deficiency among the Rajbanshis is found to be 12 percent, and about 3.33 percent in the non case population. Chi square (χ2 ) and Odds ratio (OR) was found to be 2.05 and 3.95 respectively. The lower and upper confidence limit for the population Odds ratio were 3.951-1.96/√2.05 = 3.58 and 3.951+1.96/√2.05 = 25.93 respectively. We concluded with 95% confidence that the population OR was somewhere between 3.58 and 25.93. Since the interval did not include 1, we concluded that in the Rajbangshi population (cases) G6PD deficiency is more likely to be present than in the non Rajbangshi (noncase) group. The overall distribution of the G6PD activity in both Cases and Controls are shown in the figure 1 and table 1 respectively. Figure 1 Distribution of G6PD activity in cases (n = 100) and control (n = 60) groups Table 1 The distribution of the G6PD activity in the Cases and Controls (Normal reference range of G6PD activity in U/g Hb=4.6-13.5 U/g hemoglobin). G6PD inadequate G6PD adequate Cases 12 88 Non cases 2 58 Chi square (χ2) = 2.05, OR = 3.95 0 2 4 6 8 10 12 14 16 0 50 100 150 G6PD Activity in CASES , G6PD Activity in CONTROLS 3601_ Hb(g/dl).
  • 4. Estimation Of G6pd Status In The Rajbangshi Population Of Sushrutanagar www.iosrjournals.org 54 | Page The lower and upper confidence limit for the population Odds ratio are 3.951-1.96/√2.05 = 3.58 and 3.951+1.96/√2.05 = 25.93 respectively. We conclude with 95% confidence that the population OR is somewhere between 3.58 and 25.93. IV. Discussion More than 200 million people all over the world suffer from G6PD deficiency 13 and this deficiency is found to be distributed in a heterogeneous manner among different population groups of the world. In India also the G6PD deficiency is found to be distributed in a heterogeneous pattern as already discussed above. In the present study the G6PD deficiency in the Rajbangshis of Sushrutanagar area of the Darjeeling district of West Bengal is found to be about 15 percent which is definitely higher than the overall rate of 2-5 percent of G6PD deficiency in the general population of India9 . Although this study comprised of about one hundred cases selected randomly from about 15000 populations of Rajbangshis of the Sushrutanagar area, yet the high percentage of G6PD deficiency in the studied cases indicates an overall high rate of G6PD deficient people among the Rajbangshi population of Sushrutanagar. Simultaneously, the distribution rate is found to be only 3 percent in the non Rajbangshi population of the same area. The genetic heterogeneity which is found in some enzymopathies may be one of the important causes for this noted heterogeneous distribution of the G6PD deficiency among the individuals, and indeed over 400 variants of G6PD have been described already13 . A strong evidence of significant difference between frequency of G6PD deficiency in Tribal and non-tribal group of newborns with more frequency of G6PD deficiency in tribal newborns with neonatal hyperbilirubinemia. Prevalence ratios among Non-tribal and Tribal neonates were 12.00% and 23.07% respectively. This difference is statistically significant (χ2 = 2.31; df=1 p <0.05 )14 . Other reports of G6PD deficiency are shown variability in different ethnic populations like Parsee16 , Brahmin17 , Jat18 and Vataliya Prajapati19 etc. The frequency is higher among the tribals than the caste populations20 . According to Bhasin and Walter 21 , the frequency of G6PD among Indian Population as a whole ranges from complete absence to 27%. It is higher among scheduled tribes as compared to other ethnic groups. G6PD deficient allele frequency is comparatively higher in North and West Indian Zones, whereas in South India it is uniformly low except in Andhra Pradesh and Tamil Nadu. Prevalence of G6PD deficiency is generally 0-10%, although some community may have higher prevalence: 27.5% for the Angami Nagas, a tribal group in Northern India22 Abnormalities in primary DNA or protein sequence have been established in a number of the G6PD variants and in almost all cases the alteration there is one or more base substitutions, leading to an amino acid replacement and not a deletion in the protein 13 . Evidence in the abnormality in structure is seen in the differences in electrophoresis mobility, enzyme kinetics, pH optimum, and heat stability; and these differences result in great variation of clinical severity, ranging from nonspherocytic hemolytic anemia without demonstrable oxidant stress (particularly shortly after birth), through hemolytic anemia only when stimulated by marked to mild oxidant stress, to no clinically detectable abnormality23 . The normal G6PD is designated as type B, but about 20% of individuals of African descent have a G6PD , designated as type A+ that differs by a single amino acid and is electrophoretically distinguishable but functionally normal. Among the clinically significant G6PD variants, the most common, the A- type, is due to two base substitutions and is encountered primarily in individuals with ancestral origins in Central Africa24 . This variety of the G6PD enzyme has abnormal kinetic property and is found in about 11 percent of African descended males in the United States. A second variety of G6PD is encountered in the peoples of the Mediterranean circumlittoral, particularly the Sardinians and Sephardic Jews. This variant is more severe than the A- variety and may result in non spherocytic hemolytic anemia 24 . A third relatively common and slightly less severe variant occurs in southern Chinese population. Till now no such genetic data is available for the population of the Rajbangshis of the Sushrutanagar and so, a detailed study is required in this area to properly characterize the G6PD variety of this region where the G6PD deficiency has been found to be relatively higher than the general population of India. References [1]. Vasudevan, D.M. and Srikumari, S. (1998), Text Book of Biochemistry for Medical Students, 2nd edn. Jaypee Brothers Medical Publishers Ltd., New Delhi, p 59. [2]. Ghai, O.P. (2001), Essentials of Paediatrics, 5th edn. Mehta Publishers, New Delhi, p 102-103. [3]. Vasudevan, D.M. and Srikumari, S. (1998), Text Book of Biochemistry for Medical Students, 2nd edn. Jaypee Brothers Medical Publishers Ltd. New Delhi, p103-104. [4]. Wendell, Ross. and H, Franklin Bunn. (2001), Harrison‟s Principle of Internal Medicine, vol 1,15th edn. (International edition). MCgraw Hills, Singapore, p 685. [5]. Vasudevan, D.M.and Srikumari, S. (1998), Text Book of Biochemistry for Medical Students, 2nd Edition. Jaypee Brothers Medical Publishers Ltd. New Delhi, p 305. [6]. Cai, W., Filosa, S., Martini, G. (1994), DNA haplotypes in the G6PD Gene Cluster studied in the Chinese Li Population and their relationship to G6PD. HumHered. 44 (5), 279-86.
  • 5. Estimation Of G6pd Status In The Rajbangshi Population Of Sushrutanagar www.iosrjournals.org 55 | Page [7]. Iwak, K., Hiron , A., Matsuoka, H., Kawamoto, F., Herie, T Link., Tantular, I S., Dachlan, I P., Notopuro, H., Hidayah, N I ., Salim, A M., Fujii, H., Miwa, S., Ishii, A. (2001), Distribution of Glucose 6 Phosphate Dehydrogenase Mutations in South East Asia. HumGenet. 108(6), 445-9. [8]. Robert, K Murray. (2000), Harper‟s Biochemistry, 25th edn. Appleton & Lange, Stanford, Connecticut, p 767. [9]. Talwar, G.P., Srivastava, L M., Moudgil, U D. (1989), Text Book of Biochemistry and Human Biology, 2nd edn. Elements of Human Genetics, Chapter-53,Section –VII, Page-636. [10]. Saha, N., Tay, J S. (1990), Genetic studies among the Nagas and Hmars of Eastern India. Am J Phys Antropol. 82(1), 101-12. [11]. Chatterjee, M.N. and Shinde, Rana. (1997), 2nd edition, Test Book of Medical Biochemistry, Jaypee Brothers Medical Publishers Ltd. New Delhi, page: 401. [12]. Kachmar , J. F. and Moss, D. W. (1976), Enzymes in fundamentals of Clinical Chemistry. N.W.Teitz , Saunders, Philadelphia, p 666- 670. [13]. Gross, R T., Hurwitz, R E., Marks, P A. (1978), Red cell Glucose 6 Phosphate Dehydrogenase Deficiency. J.Clin. Invest. 37, 176. [14]. Manik Mondal, Asok Kumar Datta, Syamali Mandal, Pradeep Kumar Das. Study of Glucose 6 phosphate dehydrogenase deficiency in neonatal jaundice. IOSR Journal of Pharmacy and Biological Sciences. Volume 1, Issue 5 (July-August 2012), PP 30-36. [15]. Baxi, V. Balakrishnan, J. V. Undevia, and L. D. Sanghvi, “Glucose-6-phosphate dehydrogenase deficiency in the Parsee community, Bombay.,” Indian journal of medical sciences, vol. 17, pp. 493–500, 1963. [16]. A. Santhi and M. P. Sachdeva, “Glucose-6-phosphate dehydrogenase deficiency among the jats and brahmins of sampla (Haryana),” The Anthropologist, vol. 6, no. 4, pp. 291–292, 2004. [17]. K. N. Saraswathy and S. Aggarwal, “Study of glucose-6- phosphate dehydrogenase deficiency and sickle cell anemia among the Tamil Brahmins of Delhi,” The Anthropologist, vol. 7, no. 1, pp. 45–47, 2005. [18]. A. Santhi and M. P. Sachdeva, “Glucose-6-phosphate dehydrogenase deficiency among the jats and brahmins of sampla (Haryana),” The Anthropologist, vol. 6, no. 4, pp. 291–292, 2004. [19]. S. C. Gupte, A. N. Shaw, and K. C. Shah, “Hematological findings and severity of G6PD deficiency in Vataliya Prajapati subjects,” Journal of Association of Physicians of India, vol. 53, pp. 1027–1030, 2005. [20]. V. Tripathy and B. Reddy, “Present status of understanding on the G6PD deficiency and natural selection,” Journal of Postgraduate Medicine, vol. 53, no. 3, pp. 193–202, 2007. [21]. M. K. Bhasin and H. Walter, Genetic of Caste and Tribes of India, Kamla-Raj Enterprises, Delhi, India, 2001. [22]. P. K. Seth and S. Seth, “Biogenetical studies of Nagas: glucose-6-phosphate dehydrogenase deficiency in Angami Nagas.,” Human Biology, vol. 43, no. 4, pp. 557–561, 1971. [23]. Wendell, Ross., H, Franklin Bunn. (1998), Harrison‟s Principle of Internal Medicine, vol 1, 14th edn. (International edition). MCgraw Hills, Singapore, p 663. [24]. Wendell, Ross., H, Franklin Bunn. (1998), Harrison‟s Principle of Internal Medicine, vol 1, 14th edn. (International edition). MCgraw Hills, Singapore, p 663-64.