Journal of Life Sciences
Volume 7, Number 5, May 2013 (Serial Number 61)
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Journal of Life Sciences
Volume 7, Number 5, May 2013 (Serial Number 61)
Contents
Molecular Biology and Medical Sciences
443 Evaluation of Gray Leaf Spot Tolerant Genotypes from CIMMYT in the Highland Maize
Production Eco-systems of Bhutan
Tirtha Bdr. Katwal, Dorji Wangchuk, Lhap Dorji, Namgay Wangdi and Rinzin Choney
453 Old Drug for New Use: Searching for Mitogen-Activated Protein Kinase Kinase 1 (MEK1)
Inhibitor by the Computer Aided Drug Design
Po-Yuan Chen, Hong-Jye Hong, Mien-De Jhuo, Tzu-Ching Shih, Yu-Chi Wu, Chia-Hsing Cheng, Yen-Yu Huang and Tzu-Hurng Cheng
459 Relationship of Circulating High-Density Lipoprotein Cholesterol and Anemia
Md. Aminul Haque Khan, Mst. Rokshana Rabeya, Muhammad Saiedullah, Rukhsana Parvin, Sohel Ahmed and Md. Rezwanur Rahman
464 Development of in-vitro Susceptibility Testing for Pathogenic Bacteria
Fouad Houssein Kamel, Chiman Hameed Saeed, Ashti M. Amin and Saleem Saaed Qader
468 Inhibitory Effect of Alcoholic Extract of Sage Leaves on the Growth of Pathogenic Fungi Causing External Ear and Skin Infections
Maha Akram Al-Rejaboo and Omar Mu’ayad Al-Obaidy
475 External Gastric Balloon in Obesity Treatment
Mesut Basak, H. Erdem Gozden, Gulay Turan, Hayrettin Mutlu and Emine Pakir
Biotechnology and Biological Engineering
483 Chromatographic Analysis of Thiophenes in Calli and Suspension Cultures of Tagets spp.
Hussein S. Taha, Hamida A. Osman, Mahmoud M.M.A. Youssef, Abdel Monem Y. El-Gindi, Hoda H. Ameen and Asmahan M.S. Lashein
491 Bioluminescence and Chitinase Production during Chitin Fermentation by Vibrio harveyi
495 Promising Additives to Protect the Activity of Baculovirus Biocontrol Agent under Field -Sunlight Conditions, in Egypt
Alexandra El-Helaly, Magda Khattab, Said El-Salamouny, Mohammed El-Sheikh and Salah Elnagar
Botany and Zoology
501 Study of Behavior Germination and Essays the Removing Tegumentary Inhibition of Seeds of Chamaerops humilis L. var. argentea André (Arecaceae)
Nadjat Médjati, Okkacha Hasnaoui, Nouria Hachemi, Brahim Babali and Mohammed Bouazza
507 Evaluation of Regeneration Stock Alternatives for Optimization of Growth and Survival of Field-Grown Forest Trees
Titus Fondo Ambebe, Lum Ayeoffe Fontem, Balgah Roland Azibo and Njoya Moses Tita Mogho
517 Microalgal Epibiontic Communities on Some Brachyuran Crabs in Suez Canal, Egypt
Nesreen K. Ibrahim and Abeer S. Amin
527 Comparative Study of Two Methods of Induction of Estrus and Fertility Following Artificial Insemination in Azawak Zebu in Niger
Issa Moumouni, Marichatou Hamani, Semita Carlo, Nervo Tiziana, Yénikoye Alhassane, Cristofori Francesco and Trucchi Gabriella
532 The Channel Catfish in Georgian Aquaculture
Rezo K. Goradze, Akaki Komakhidze and Irakli Goradze
539 Advocacy for Camel Research and Development in Kenya
Kisa Juma Ngeiywa and James Chomba Njanja
Nutritional Sciences
547 Lipids Data Composition of Edible Ant Eggs Liometopum apiculatum M. Escamoles
Melo Ruiz Virginia, Sánchez Herrera Karina, Sandoval Trujillo Horacio, Quirino Barreda Tomás and
Calvo Carrillo Concepción
553 Development of Blast Chilling Method for Cooked Meat Dishes
May 2013, Vol. 7, No. 5, pp. 443-452
Journal of Life Sciences, ISSN 1934-7391, USA
Evaluation of Gray Leaf Spot Tolerant Genotypes from
CIMMYT in the Highland Maize Production Eco-systems
of Bhutan
Tirtha Bdr. Katwal1, Dorji Wangchuk1, Lhap Dorji1, Namgay Wangdi1 and Rinzin Choney2
1. Renewable Natural Resources Research and Development Centre, Wengkhar, Mongar, Bhutan
2. Department of Agriculture, Ministry of Agriculture and Forests, Thimphu, Bhutan
Received: March 26, 2013 / Accepted: May 07, 2013 / Published: May 30, 2013.
Abstract: Bhutan is a small landlocked country located in the eastern Himalayas. Over 69% of the population is engaged in
agriculture. Rice, maize, wheat, barley, buckwheat and millets are the major cereal crops cultivated. Rice is the most preferred food crop of the Bhutanese. Maize is a primary food crop after rice and it ranks first among food crops in production. The cultivation ranges from less than 300 m asl (metres above sea level) nearly up to 2,800 m asl. In 2007, a new, extremely serious problem of GLS (gray leaf spot) in maize that was previously never reported in Bhutan was confirmed. This disease spread rapidly in the highland maize growing areas causing production losses of over 50% to 70%. All the maize varieties cultivated in the country were found to be highly susceptible to the disease. In order to contain this devastating disease, the national maize program drew short and long term strategies with the help of a CIMMYT Expert. As an immediate short term action to contain GLS, systemic fungicide Tilt 25 EC (active ingredient propiconazole) was supplied free of cost to the farmers. A longer term strategy pursued was the introduction, evaluation and selection of GLS tolerant genotypes for the highland ecosystem. Over 100 GLS tolerant genotypes were introduced from CIMMYT Colombia, Mexico, Zimbabwe and Nepal. These materials were initially evaluated in a disease hotspot sites and then further tested in multi-location trials in GLS affected areas across the country. Farmers were engaged for Participatory Variety Selection by organizing farmer’s field days at the trial sites. Finally, in 2011 considering the need of GLS tolerant varieties for farmers, two GLS tolerant genotypes ICAV305 and S03TLYQAB05 were provisionally released. In the 2011 season, these two provisionally released genotypes were put under large scale demonstration in the GLS affected areas in nine districts across the country. In 2012, the two genotypes were formally released by the Technology Release Committee of the Ministry of Agriculture and Forest. Rapid seed increase of the new varieties was initiated through farmers from Community Based Seed Production groups and so far 75% seed replacement of GLS affected farmers has been accomplished.
Key words: Gray leaf spot, hotspot, yield loss, participatory variety selection, community based seed production and seed
replacement.
1.
Introduction
Bhutan is a small landlocked mountainous country located in the southern slopes of eastern Himalayas. It is sandwiched between the two great Asian civilizations, China to the north and India in the east, west, and south. The country lies between latitudes
26°45′N and 28°10′N, and longitudes 88°45′E and
Corresponding author: Tirtha Bdr. Katwal, M.Sc., research
fields: tropical agriculture development, crop production. E-mail: tirthakatwal@gmail.com.
92°10′E. The country has a total geographical area of
38,394 km2 with a population of 745,600 people [1].
The forest (tree) cover of the country is about 70.46%, arable land 2.93%, meadow land 4.10%, shrub land 10.43%, snow cover land 7.44% and bare areas 3.20% of the total geographic area [2]. Agriculture is the mainstay of the people with an estimated 69% of the population engaged in farming. Rice, maize, wheat, barley, buckwheat and millets are the major cereal crops cultivated in Bhutan and rice is by far the most
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Evaluation of Gray Leaf Spot Tolerant Genotypes from CIMMYT in the Highland Maize Production Eco-systems of Bhutan
444
important and preferred food crop of the Bhutanese. Maize (Zea mays L.) is a major food crop cultivated by 69% of the rural households for subsistence. The total area under maize in 2010 was 61,476 acres; the total production was 57,666 Mt with a national average yield of 2.38 t·ha-1 [3]. Maize ranks first in the extent of area cultivated amongst the food crops. Maize cultivation in the country ranges from less than 300 m to nearly up to 2,800 m asl owing to its versatile capacity to adapt to different environments. It plays a critical role in ensuring the household food security. It is estimated that 80% of the total production is consumed at the household level by the farmers which is valued at Nu. 353 Million (1 USD = 54 Ngultrum (Nu.)) annually [4]. About 6% of the total production is sold which is an important source of household income. The rest of the production is used as seed, processed into different products and fed to the livestock. The maize production environment in the country is broadly categorized into three zones mainly based on the altitude. The three production zones are: sub-tropical maize production zone I (< 1,200 m asl) or low altitudes; sub-tropical maize production zone II (1,200-1,800 m asl) or mid altitudes; and the highland maize production zone (> 1,800 m asl) [5]. These different production zones vary widely in their production potentials and constraints.
Bhutanese maize farmers in the Highland and Sub-tropical Zone II are facing a new, extremely serious problem of GLS (Gray Leaf Spot), fungal disease which was previously never reported in the country [6]. According to [7], the incidences of GLS has increased during past two decades and today it is one of the greatest threats to global maize production [8]. In Bhutan, GLS and TLB (Turcicum leaf blight) have become economically important diseases especially in areas above 1,200 m asl. Serious outbreaks of these diseases were reported from 12 major maize growing districts in 2006. GLS spread rapidly in the highland maize growing areas and
attained epidemic scale in 2007. It affected 4,193 maize growing households. The total area affected was 4,821.89 acres and the total production loss due to the disease was 6,504.12 Mt [3]. The estimated production loss of the affected farmers ranged from 50% to 70% [3]. In Zambia, when similar GLS epidemic occurred in the mid 1990s, the average yielded losses ranged from 28% to 54% with an average of 33.5% [9]. The impact of this disease, farmers crop husbandry practices, disease management strategies and the disease reaction on the GLS resistant genotypes introduced from CIMMYT Colombia are discussed in this paper.
2. Materials and Methods
and observed under the microscope and finally GLS and TLB were confirmed in 2007. GLS was observed to be more serious and damaging as compared to TLB. Immediately after the confirmation of the disease, the expert recommended an immediate and a long term strategy for the management of the disease considering the serious impact of the disease on the household food security of the maize dependent farmers. All the traditional maize varieties including the most popular improved variety Yangtsipa (Suwan 1) were found to be highly susceptible to GLS.
The immediate and short term strategy adopted was the spray of systemic fungicide Tilt 25 EC (active ingredient propiconazole). The longer term and a more sustainable strategy was the introduction, evaluation and selection of GLS tolerant maize genotypes as the majority of Bhutanese farmers cannot effort expensive fungicides. Moreover, repeated use of fungicides are not feasible both economically and environmentally [10] besides physical constraints due to steep terrain slopes more than 50%. The use of genetic resistance has been noted as the most sustainable means to prevent maize production losses from GLS in Africa, especially for subsistence farmers who cannot afford to purchase expensive fungicides [11].
To initiate the development and selection of GLS tolerant genotypes for the country, GLS tolerant germplasm were requested from CIMMYT International. Accordingly in 2007, 45 GLS tolerant genotypes from CIMMYT Colombia, 23 from Mexico, eight from Zimbabwe and six from the National Maize Program of Nepal were introduced to the country. As the quantity of seeds was small, the first step was the off season increase of the seed at Lingmethang research farm (640 m asl) through controlled pollination. The first batch of materials to arrive in the country was 45 entries from CIMMYT Colombia. From the 45 entries, 39 entries were planted at Lingmethang Research Sub Centre (640 m asl) in October 2007 as winter nurseries for seed increase through controlled pollination. From the 45 entries,
four entries had enough seed and one which was a hybrid was dropped from seed increase. To screen the introduced materials under disease pressure, a disease hotspot site at Chaskar (1,960 m asl), in Mongar district where the disease occurs naturally and in abundance due to continuous cropping of maize was identified. Verma [9] reported that new materials were screened for tolerance to GLS in the Zambian Seed Company’s farm at Lusaka that had turned into a good “hotspot” for GLS due to continuous cropping of GLS susceptible maize varieties. The screening and evaluation for tolerance to GLS in Bhutan started from March 2008. To facilitate the screening of the materials, two acres of land was leased at Chaskar from a farmer by the maize research program. After initial evaluation, promising materials were promoted to the nationally coordinated trials for evaluation in multi-location trials across the country through the Regional Research Centers. At the trial sites, farmer’s field days were organized to engage farmers for PVS (Participatory Variety Selection). As GLS was new to the country, on the job training were provided to the researchers and extension staff in monitoring the disease. In the trial sites, GLS was constantly monitored by the researchers. At the disease hotspot trial site in Chaskar, the introduced materials were planted in the third week of March and harvested in late September. GLS was scored based on the scale of 1-5 where 1 = no lesions visible, 2 = few lesion seen on two lower leaves, 3 = lesions visible on most leaves below the ear, 4 = many lesions visible on leaves above the ear and 5 = all leaves dead. All other agronomic parameters were recorded at the time of harvest. Existing improved and released variety Yangtsipa and a local variety from Chaskar were used as the check varieties in the trial site.
3. Results and Discussions
3.1 Disease Confirmation and Conditions That Favoured Disease Development
Evaluation of Gray Leaf Spot Tolerant Genotypes from CIMMYT in the Highland Maize Production Eco-systems of Bhutan
446
subsequent observation of spores under a simple microscope confirmed the disease to be GLS (Fig. 1)
caused by the fungus Cercospora zeae-maydis (Tehon
and Daniels 1925). Another fungal disease TLB
caused by Exserohilium turcicum (Synonymous;
Helminthosporium turcicum) locally known as
Songsongma was also confirmed. TLB was present in the country, but serious infection was noticed only after 2006. GLS was previously never reported in the country and was confirmed for the first time. Severe incidences of GLS were observed at elevations nearly and above 1,500 m asl. At these elevations, GLS was found to be causing the most damaging effects with some farmers losing almost 100% of their crop [6]. At the elevations 1,500-1,800 m asl, both GLS and TLB were prevalent. Below 1,700 m asl, TLB was more prevalent but with incidences of lesser economic significance. GLS symptoms were also seen at 600 m asl, however, the impact was much lesser as compared to areas above 1,500 m asl.
The cause for outbreak of these two diseases in Bhutan is attributable to the farming practices which are congenial for the development of the diseases. The predominant farming practices adopted by the maize growers in the country are: (1) mono-cropping of maize with occasional rotation with potato; (2) use of maize stalks and crop residues as livestock bedding, and subsequently as FYM (farm yard manure)—a major source of nutrients for the crop; (3) reduced or minimum tillage due to steep terrain.
Apparently, the main source of inoculum for GLS was the infected crop residues especially the leaves and the leaf sheath left on the soil surface. Bhutanese farmers in the highlands practice the continuous cultivation of maize, use maize stover for feeding the cattle as animal bedding and also collect and heap the stover in the field to be spread in the field at the time of planting. The leftover residues from the feeding stalls go into the compost yard which is later spread in the maize field. These practices seemed to greatly favor the development of the disease.
Majority of Bhutanese farmers use locally made ploughs drawn by the bullocks which do not penetrate very deep in the soil. The plough does not adequately incorporate the crop residues deep in the soil. Farmers in the highlands (> 1,800 m asl) normally practice continuous cultivation of maize without crop rotation due to the limited land holdings, short growing season and lack of other suitable optional crops. As a result of the disease, the maize production has substantially declined from 2005 (Fig. 2).
All these cropping practices like continuous mono-cropping of maize [12-14] and use of minimum or conservation tillage practices [12, 15, 16] have been found to increase the incidence of the disease. In
Fig. 1 GLS conidia as observed under simple microscope,
2007.
Fig. 2 Maize production trend in Bhutan [3]. 93.97
71.06
61.79 66.78 61.16
57.67
0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00
2005 2006 2007 2008 2009 2010 Year
Production (Mt,
metric
Uganda, Africa where maize is one of the main crops, leaving the disease infected stover in the soil surface and planting of susceptible variety were identified as the key factors responsible for the perpetuating of GLS [17]. Further, in most maize growing areas of the country, cloudy weather with high humidity and extended period of wetness prevails with the onset of monsoon from late May till September, which favours the growth and development of the pathogens [18].
3.2 Immediate Management of the Disease
Since the diseases had a serious threat on the household food security of the farmers, spraying of systemic fungicide Tilt (active ingredient propiconazole) was recommended as an immediate measure. Tilt 25 EC (active ingredient propiconazole) was imported from India and was recommended to be sprayed at 2 mL per litre of water with minimum of one spray, two weeks before flowering. The DoA (Department of Agriculture) with fund support from the EU-ASSP (EU-supported Agriculture Sector Support Project) supplied 1,650 L of fungicide Tilt (propaconizole) worth Nu. 1.65 Million the affected farmers free of cost. However, spraying was cumbersome and ineffective due to steep terrain where water had to be fetched from downhill, lack of appropriate spraying machines and accessibility of far flung maize fields.
3.3 Introduction, Evaluation and Selection of Disease
Tolerant Varieties
The use of genetic resistance has been noted as the most sustainable means to prevent maize production losses from GLS, especially for subsistence farmers who cannot afford to purchase expensive fungicides [11]. Currently, there are no GLS tolerant varieties released for the highland maize growing areas in the country and therefore, the introduction, evaluation and selection of GLS tolerant maize genotypes were given the highest priority. The 45 GLS tolerant genotypes from CIMMYT Colombia, 23 from Mexico, eight
from Zimbabwe and six from the National Maize Program of Nepal were the primary source for adapting and releasing GLS tolerant genotypes. In 2008 season, most of the genotypes appeared to be promising with reasonably good level of field tolerance to GLS and TLB. In the first season, the main selection parameter was the tolerance to GLS and TLB and not too much emphasis was given to other agronomic traits. Among all the genotypes, the genotypes from CIMMYT Colombia were most stable and exhibited better tolerance to GLS while genotypes from CIMMYT Mexico appeared to be inbred lines with poor plant type. All the materials from CIMMYT Zimbabwe were white maize which is less preferred by farmers and the ones from Nepal were all susceptible to GLS. From the 45 genotypes from CIMMYT Colombia, 15 genotypes which received an average GLS and TLB score of less than 2.5 [19] were selected for further evaluation (Table 1).
In 2009 maize season, the 15 selected entries were evaluated in five different locations above 1,500 m asl where GLS occurs in abundance. The results indicated significant difference on GLS and TBL incidences and yield among the different genotypes evaluated (Table 2). The results also showed that there was no large difference on disease tolerance and yield among the different genotypes as compared to the check varieties. This is mainly because the introduced genotypes are new to the highland maize ecosystem and are still undergoing adaptation whereas the check varieties are well adapted to the highland ecosystem.
Evaluation of Gray Leaf Spot Tolerant Genotypes from CIMMYT in the Highland Maize Production Eco-systems of Bhutan
448
Table 1 The 15 promising GLS tolerant genotypes selected from CIMMYT Colombia materials, 2008.
Si No. Entries No. Pedigree GLS score* TLB score* Yield t/ha
1 1 Cap. Miranda 99Bact1F-1 1.8 2.2 7.91
2 13 Menegua 01 Phaeo 2.2 1.5 5.13
3 2 Granada 01Phaeo1AS2 2.3 2.3 4.85
4 23 Villavicencio 03Phaeo1A(SA4) 2.3 1.7 4.58
5 35 S03TLYQ AB05 2.3 2.0 8.58
6 6 GLSIY01/SPMAT 2.3 1.4 5.07
7 38 ICA V305 2.4 2.3 7.58
8 21 Villavicencio 03Asp1C(LET-EARLY) 2.5 1.3 7.58
9 33 Cimcali 03HCG1A 2.5 1.8 6.10
10 3 Turipana 01DMR 1D(1) 2.5 2.5 6.01
11 5 GLSIY01HG"A" 2.5 1.6 4.53
12 15 Granada 03Poly1A(SA4) 2.5 1.7 4.43
13 17 Villavicencio 03Asp1C(QPM) 2.5 1.8 3.88
14 25 Villavicencio 03Phaeo1A(Elites) 2.5 1.6 4.16
15 9 ACROSS S9624-1 2.0 2.0 4.05
16 Yangtsipa (Improved Check) 3.1 4.0 4.74
*: disease score based on scale of 1-5, where: 1 = No lesions are visible; 2 = Few lesion seen on two lower leaves; 3 = Lesions visible on most leaves below the ear; 4 = Many Lesions visible on leaves above the ear; 5 = All Leaves dead.
Table 2 GLS, TLB and yield of 15 selected genotypes from CIMMYT Colombia at five different locations, 2009.
Entry Pedigree Mean of 5 locations
GLS* TLB* Yield (t·ha-1)
1 Cap. Miranda 99Bact1F-1 1.9 1.9 5.94
2 Granada 01Phaeo1AS2 1.7 1.7 5.31
3 Turipana 01DMR 1D(1) 1.9 1.7 5.4
5 GLSIY01HG"A" 2.0 1.6 4.16
6 GLSIY01/SPMAT 1.9 1.7 4.76
9 ACROSS S9624-1 1.8 1.7 4.35
13 Menegua 01 Phaeo 1.7 1.6 4.03
15 Granada 03Poly1A(SA4) 1.8 1.8 3.57
17 Villavicencio 03Asp1C(QPM) 2.2 1.7 4.05
21 Villavicencio 03Asp1C(LET-EARLY) 1.9 1.8 4.51
23 Villavicencio 03Phaeo1A(SA4) 1.9 1.7 5.28
25 Villavicencio 03Phaeo1A(Elites) 2.2 1.7 3.90
33 Cimcali 03HCG1A 1.9 1.7 5.26
35 S03TLYQ AB05 1.9 1.8 4.15
38 ICA V305 1.9 1.9 5.35
Yangtsipa 2.0 1.7 5.57
Local 2.2 1.8 5.45
Location (L) ** ** **
Entry (E) ** ns **
L E ** ns ns
S.E.D 0.2435 0.3122 1.339
CV 13 18 28
** P > 0.01.
as fourth choice. Farmer’s selection criteria included plant height, husk cover, tolerance to GLS, yellow
Entry No. 1, 35 and 21 were selected for further evaluation. The genotypes Entry No. 6 which was preferred by the farmers was dropped due to less quantity of seed. One additional genotypes, ICA V305 (Entry No. 38) that was not selected by the farmers during the PVS, was included for further evaluation based on the advice of the CIMMYT experts. This genotype ICA V305 is a stable variety released in Colombia and one of the parents is Suwan 1 (Yangtsipa) which is a popular variety in Bhutan. All the selected genotypes showed higher tolerance to GLS than the check varieties (Table 2).
In 2009 season, a very close monitoring of GLS was done for the four selected genotypes and the two check varieties. The GLS incidence starts by the last week of July and reaches the peak in the last week of August. All the four new genotypes including the check varieties were infected by GLS, however, the incidences of GLS was much higher in the two check varieties right from the initial stage and continued to increase until all the leaves were dead by end August (Fig. 3).
This strongly indicates that in the event of an early incidence of GLS, the new genotypes can tolerate
much belter as compared to the check varieties. The results from multi-location trial of four selected genotypes in four locations in 2010 revealed that new genotypes showed much higher tolerance to GLS as compared to the local check variety (Table 3). There was significant difference in yield and the highest
yield of 5.33 t·ha-1 was recorded for Yangtsipa, the
improved check variety. Among the new genotypes, the highest yield of 5.27 t·ha-1 was recorded for Cap. Miranda 99Bact1F-1 (Entry No. 1) (Table 3). This genotype has semi dent grains and higher percentage of open husk which is less preferred by the farmers. The mean data of three years indicated that this genotypes had high incidence of Turcicum Leaf blight and yield was comparable to that of genotypes S03TLYQ AB05 (Entry No. 35) and ICAV305 (Entry No. 38) and therefore was not considered for immediate release (Tables 4 and 5). Although the yield of ICA V305 (Entry No. 38) is lower than that of the check varieties, it had better tolerance to GLS and good husk cover. Due to the urgency for GLS tolerant varieties for maize growing areas above 1,500 m asl, the two genotypes S03TLYQ AB05 (Entry No. 35) and ICAV305 (Entry No. 38) were selected for large
Fig. 3 GLS progression at Chaskhar, 2009. 1.0
1.5 2.0 2.5 3.0 3.5
115 123 130 135 148
GLS Score (1-5)
Assessment Time (Days after planting)
Entry # 21
Entry # 33
Entry # 35
Entry # 38
Yangtsipa
Evaluation of Gray Leaf Spot Tolerant Genotypes from CIMMYT in the Highland Maize Production Eco-systems of Bhutan
450
Table 3 Mean GLS, TLB score and yield t/ha for 2010 season (mean of four locations).
Entry No. Pedigree GLS, score* TLB, score Yield (t·ha-1)
1 Cap. Miranda 99Bact1F-1 2.1 2.2 5.27
21 Villavicencio 03Asp1C(LET-EARLY) 1.9 2.1 2.48
35 S03TLYQ AB05 2.1 2.1 4.78
38 ICA V305 2.1 2.2 3.83
Yangtsipa 2.0 2.2 5.33
Local 2.5 2.4 3.96
Fprob ***
LSD 1.606
CV (%) 25
*** P > 0.001.
Table 4 Other agronomic traits of selected varieties, 2010 season.
Entry No. Pedigree Average plant height
(cm)
Average ear height (cm)
Husk cover** (1-5)
Ear aspect *** (1-5)
1 Cap. Miranda 99Bact1F-1 217.4 93.8 2.5 2.6
21 Villavicencio 03Asp1C(LET-EARLY) 222.8 108.6 2.8 2.9
35 S03TLYQ AB05 226.6 94.5 2.3 2.2
38 ICA V305 242.6 116.7 2.4 2.7
Yangtsipa 257.7 124.9 2.4 2.4
Local 281.7 145.7 1.6 1.6
** Husk cover based on scale of 1-5 where 1 = best (fully covered) and 5 = open husk; *** Ear aspect based on scale of 1-5 where 1 = best quality with uniform ear size and good grain filling 5 = poor quality.
Table 5 Mean GLS, TLB and yield of four selected genotypes.
Entry No. Pedigree Mean of three years
GLS TLB Yield (t·ha-1)
1 Cap. Miranda 99Bact1F-1 2.1 2.4 4.96
21 Villavicencio 03Asp1C(LET-EARLY) 2.2 2.1 3.80
35 S03TLYQ AB05 2.3 2.3 4.75
38 ICA V305 2.3 2.3 4.81
Yangtsipa 2.4 2.3 5.57
Local 2.6 2.5 4.41
scale demonstration and provisional release in 2011 season considering the mean performances of three years (Table 4). Although the yield of ICA V305 (Entry No. 38) is lower than that of the check varieties, it had better tolerance to GLS and good husk cover (Tables 4 and 5).
In 2011, the TRC (Technology Release Committee) of the MOAF (Ministry of Agriculture and Forest) in its 15th meeting endorsed the provisional release of the two GLS tolerant genotypes S03TLYQ AB05 (Entry No. 35) and ICAV305 (Entry No. 38). The urgency of GLS tolerant variety was one important consideration for the release.
Further in the 2011 season, the two provisionally
released genotypes were put under large scale demonstration in the GLS affected areas in nine districts across the country. The mean yield reported from the large scale demonstrations for the two genotypes S03TLYQ AB05 (Entry No. 35) and
ICAV305 (Entry No. 38) were 3.73 t·ha-1 and 4.43
of the farmers, the two provisionally released genotypes were proposed for final release to the TRC of the MoAF. The TRC finally endorsed the release of the two genotypes in April 2012. After the release the two genotypes were given local names as
Chaskarpa for genotype ICAV305 (Entry No. 38) and
Shafangma Ashom for genotype S03TLYQ AB05 (Entry No. 35).
The main justification for the release were that GLS epidemics has gained the status of a national emergency as all the available maize varieties were found to be highly susceptible to the disease. The two new genotypes have shown much higher field tolerance to GLS as compared to the existing varieties. Further, in the event of early outbreak of GLS, the two new genotypes will perform much better in terms of disease tolerance and production. Both are open pollinated varieties for which seed production is easy and the two genotypes have a higher yield potential and have shown comparable yields with the check varieties despite the fact that they are relatively new to the highland ecosystem and are undergoing adaptation.
Both have yellow flint grains, and Shafangma Ashom
S03TLYQ AB05 (Entry No. 35) is a QPM (Quality Protein Maize) which is more nutritious than the traditional maize varieties. It is the first QPM maize variety released in the country and will immensely contribute to the nutritional requirement of the maize farmers particularly that of the children [21].
The two new varieties are recommended for the maize production zone II (1,200-1,800 m asl) and Highland maize production Zone (> 1,800 m asl) where the two disease GLS and TLB have severely affected maize production. Both the varieties also perform well in Sub-tropical maize production zone I (< 1,200 m asl) or low altitudes. By 2013 season, the maize program has accomplished around 75% seed replacement of the GLS affected farmers with the two new varieties mostly with the supports of DRDP-WB, DoA MoAF (World Bank through the Decentralized Rural Development Project).
4. Conclusion
The high altitude farmers in Bhutan are facing the serious problem of GLS and TLB. Although, use of fungicide Tilt 25 EC seems to be quite effective, yet the use of chemicals will not be sustainable. The small and subsistence Bhutanese farmers cannot afford expensive fungicides, besides the difficulty to spray manually in steep slopes where most of the maize is grown and the detrimental impact of continuous use of fungicides on environment does not make the use of fungicide a suitable disease management option. The release of two GLS and TLB tolerant maize varieties has come as a big respite for the Bhutanese maize famers particularly those above 1,500 m asl. With rapid seed increase through the CBSP groups, 75% seed replacement of the affected farmers with the two GLS tolerant varieties has been accomplished by 2013 planting season. The seed increase and replacement of the affected farmers is rigorously being pursued in collaboration with the National Seed Center and the district extension services.
Acknowledgments
Evaluation of Gray Leaf Spot Tolerant Genotypes from CIMMYT in the Highland Maize Production Eco-systems of Bhutan
452
contributed in improving the seed production system and technical capacity building of researchers, extension and farmers. We also acknowledge the financial support of CIMMYT International and the SLMP (Sustainable Land Management Project)-NSSC (National Soil Services Center) for supporting the participation of maize researchers in the 11th Asian Maize Conference at Nanning, China, where an abstract of this paper was presented as a poster on GLS management.
References
[1] MOIC, Bhutan Portal [Online], available: http://www.bhutan.gov.bt/government/abt_geography.ph p (accessed Jan. 15, 2013).
[2] LCAR, Land cover assessment report, in: Agriculture, The National Soil Service Center and the Policy and Planning Division, Thimphu, Bhutan, 2010.
[3] DOA, Agriculture Statistics, in: Agriculture, Thimphu, Bhutan, 2010.
[4] T.B. Katwal, P. Dem, G.B. Chhetri, L. Bockel, M. Punjabi, Maize commodity chain analysis, A working document, Department of Agriculture, Ministry of Agriculture, Thimphu, Bhutan, 2006.
[5] S. Shrestha, T.B. Katwal, B.B. Ghalley, Adoption and Impact Assessment of Improved Maize Technologies in Bhutan, Council of RNR Research of Bhutan, RNR RDC Wengkhar, 2006.
[6] C. de Leon, Report of activities and recommendations on a consultancy to Bhutan, August 12-26th, 2007, RNR Research Centre, Bajo, Wangdiphodrang, Council of RNR Research of Bhutan, Bhutan, 2007.
[7] J. Wang, M. Levy, L.D. Dunkle, Sibling species of Cercospora associated with gray leaf spot of maize, Phytopathology 88 (1998) 1269-1275.
[8] J.M.J. Ward, E.L. Stromberg, D.C. Nowell, F.W. Nutter, Gray leaf spot: A disease of global importance in maize production, Plant Disease 83 (1999) 884-895.
[9] B.N. Verma, Gray leaf spot disease of maize-loss assessment, genetic studies and breeding for resistance in Zambia, in: 7th Eastern and Southern Africa Regional Maize Conference, 2001.
[10] F.M. Latterell, A.E. Rossi, Gray leaf spot of corn: A disease on the move, Plant Disease 67 (1983) 842-847. [11] A. Menkir, M. Ayodele, Genetic analysis of resistance to
gray leaf spot of midaltitude maize inbred lines, Crop Sci. 45 (2005) 163-170.
[12] P.M. Beckman, G.A. Payne, External growth, penetration, and development of Cercospora zeae-maydis in corn leaves, Phytopathology 72 (1982) 810-815.
[13] J.C. Rupe, M.R. Siegel, J.R. Hartman, Influence of environment and plant maturity on gray leaf spot of corn caused by Cerscopsora zeae-maydis, Phytopathology 72 (1982) 1587-1591.
[14] S.I. Harlapur, Epidemiology and management of Turcicum leaf blight of maize caused by Exserohilum turcicium (Pass.) Leonard and Suggs, Ph.D., University of Agricultural Sciences, Dharward, India, 2005.
[15] C.W. Roane, Observations on gray leaf spot of maize in Virginia, Plant Disease 58 (1974) 456-459.
[16] N.R.X. de Nazareno, P.E. Lipps, L.V. Madden, Survival of Cercospora zeae-maydis in corn residues in Ohio, Plant Disease 76 (1992) 560-563.
[17] G. Bigirwa, R.C. Pratt, P.E. Lipps, E. Adipala, Farming components for gary leaf spot disease severity in districts of contrasting incidence, in: Seventh Eastern and South Africa Regional Maize Conference, 2001.
[18] P.A. Paul, G.P. Munkvold, Influence of temperature and relative humidity on sporulation of Cercospora zeae-maydis and expansion of gray leaf spot lesions on maize leaves, Plant Disease 89 (2005) 624-630.
[19] T.B. Katwal, D. Wangchuk, N.B. Adhikari, N. Wangdi, S. Wangdi, P.B. Biswa, First year report on maize breeding and selection for tolerance to gray leaf spot and turcicum leaf blight diseases: A working document, RNR RDC Wengkhar, Field Crops Sector, Mongar, 2009.
[20] T.P. Tiwari, G. Ortiz-Ferrara, C. Urrea, R.B. Katuwal, K.B. Koirala, R.C. Prasad, et al., Rapid gains in yield and adoption of new maize varieties for complex hillside environments through farmer participation. II. Scaling-up the adoption through community-based seed production (CBSP), Field Crops Research 111 (2009) 144-151. [21] K. Mbuya, K.K. Nkongolo, A. Kalonji-Mbuyi,
May 2013, Vol. 7, No. 5, pp. 453-458
Journal of Life Sciences, ISSN 1934-7391, USA
Old Drug for New Use: Searching for MEK1 (Mitogen-
Activated Protein Kinase Kinase 1) Inhibitor by the
Computer Aided Drug Design
Po-Yuan Chen1, Hong-Jye Hong2, Mien-De Jhuo1, Tzu-Ching Shih3, Yu-Chi Wu1, Chia-Hsing Cheng1, Yen-Yu Huang1 and Tzu-Hurng Cheng1
1. Department of Biological Science and Technology, China Medical University, Taichung 404, Taiwan
2. School of Chinese Medicine, College of Chinese Medicine, China Medical University, Taichung 404, Taiwan
3. Department of Biomedical Imaging and Radiological Science, China Medical University, Taichung 404, Taiwan
Received: November 27, 2012 / Accepted: January 24, 2013 / Published: May 30, 2013.
Abstract: An old drug with a new use can significantly reduce the cost and time for new drug research and development. MAPK
(Mitogen-activated protein kinase) plays a very important key role in signal transduction pathways of cell proliferation and differentiation. According to the statistics, there are about 30% persons who suffered from cancers related to the MAPK signal transduction pathways. Therefore, many researchers are focused on blocking these pathways in cancers therapies. Ras/Raf/MEK/ERK, however, is one of very important pathways among MAPK message transduction pathways. More and more information about MEK protein inhibitors are unveiled in several recent years. In the present study, the authors utilized MEK inhibitors which were already published and their activities were available to construct 2D-QSAR model by using CADD (multiple linear regression). Then, the authors searched certified FDA drugs (Drugs@FDA 6184 drugs) making preliminary screening. The secondary screening on 3D structures were followed by using Docking, Scoring and Pharmacophore analysis to find out most suitable MEK inhibitors to become a fundamental database in drug discovery. The results are shown the ALogP, number of aromatic rings, number of hydrogen bond acceptors and number of hydrogen bond donors are all in positive correlation. According to the equation from 2D-QSAR model, the results conform to the previous description.
Key words: MEK (Mitogen-activated protein kinase kinase), MAPK (mitogen-activated protein kinase), pharmacophore, QSAR
(quantitative structure-activity relationship), PHP (hypertext preprocessor).
Abbreviations
MAPK—Mitogen-activated protein kinase MEK—Mitogen-activated protein kinase kinase ERK—Extracellular signal-regulated kinase QSAR—Quantitative structure-activity relationship CADD—Computer aided drug design
FDA—Food and drug administration PHP—Hypertext preprocessor
1. Introduction
The message transduction routes often influence the downstream gene expression inside cells thus affect
Corresponding author: Po-Yuan Chen, Ph.D., assistant
professor, research field: bioinformatics. E-mail: pychen@mail.cmu.edu.tw.
the protein activation, suppression all together with cell behavior and surviving. Ras/Raf/MEK/ERK MAPK (Mitogen-activated protein kinase) is the most crucial pathway that controls cell proliferation and differentiation among all message transduction pathways [1, 2]. It is inevitable to think of tumor while mentioning the function of MAPK that can control cell proliferation. Thus, many related researches even point out that there are about 30% persons who suffered from cancers that are related to the MAPK signal transduction pathways [3] which includes pancreatic cancer, colon cancer and lung cancer [4].
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Old Drug New Use 454
In recent news, an old drug with new use can dramatically reduce the cost and time for new drug development. A new drug released on market in America needs 15 years and 0.8 billion USD in average and the average number of drugs that can pass the FDA certification to release on market is only around 30. But the use of old drug for new use only need two years and 17,000,000 USD, compared with new drug developing, the cost is extremely low. Nobel Prize winner James Black said: “start from old drug to develop new is the most effective way”.
The main purpose of this study is to search for possible MAPK message transduction pathway inhibitor with fast and effective way for cancer therapy. MEK phosphorylation can enhance ERK phosphorylation in MAPK message transduction pathway. While MEK stop expressing, ERK is also stopped to express, and the downstream gene expressions are also suppressed (Fig. 1) [2, 3]. Additionally, MEK inhibitor PD184352 already had clinical trial in 1999 [5] and great amounts of research data have been accumulated during recent year. Thus, the use of known drug activity statistical data to test new unknown drug (old drug new use) is applicable.
The main series of transduction pathway among all MAPK message transduction pathways compose of three: (1) the c-jun kinase pathway which regulates many transcription factors; (2) the p-38 pathway which manly activates inflammatory response; (3) the most important pathway is the Ras/Raf/MEK/ERK pathway which influences cell proliferation, differentiation, living and apoptosis. The MEK protein plays a critical role in the pathway: firstly, the MEK will be affected by the upstream Raf and phosphorylate at serine, and then induce the downstream ERK to phosphorylate at specific tyrosine to complete message transduction. MEK contains two kinds of protein MEK1 and MEK2 which have 79% similarities. Both of them can activate ERK [2, 3, 6].
MEK inhibitor, PD184352 (a.k.a. CI-1040) (Fig. 2a), already has very high selectivity over EMK1
Fig. 1 MAPK message transduction pathway chart. MEK will not activate downstream protein ERK and ERK could not further express the downstream gene if MEK inhibitor suppresses MEK expression thus achieve the inhibit effect.
Fig. 2 Famous MEK inhibitor recently and PD184352 analogues: (a) PD184352; (b) PD318088; (c) PD98059; (d) U0126.
Van der Waal force between the hydrophobic area of Met143, Ile141, Leu118 and Phe209, electrostatic force on the hydrogen atom on Vall27 and finally the H-bonding on Ser212 then block the MEK phosphorylation by ATP (Fig. 3) [3].
More analogous medicines (PD318088 (Fig. 2b)) based on the same backbone structures were developed for activity tests in order to study more about the related mechanism between PD184352 and MEK [3]. Recently, there are more and more medicine that have been synthesized for MEK like PD98059 (Fig. 2c) (MEK1 Inhibitor, Cell Signaling Technology), U0126 (Fig. 2d), and etc.. This kind of medicine used the same mechanism as PD184352 to block MEK.
2. Materials and Methods
This research used 2D-QSAR (2D-quantitative structure-activity relationships) as reference for drug screening. 2D-QSAR is a drug design method to build up the model of relationship between chemical structure and activity (pIC50). The theory is to take the overall structure of known active molecular as parameters and input the test value for regression analysis (this research topic: active pIC50) to estimate the drug activity. Once the real activity is in linear relation with estimate activity and with acceptable correlation, the result can be applied on unknown active molecule to predict the real activity. Computer calculations with rapid in speed and great amounts of molecules are the merits for this method. Reversely, the predicted results are only data after calculation that needed further verification.
Refer to Fig. 4 for experiment procedure:
The 2D-QSAR model is constructing under the Windows OS with multiple linear regression in drug design package software, Discovery Studio 2.0, to build up the regression equation. The MEK inhibitor with known activity is required for building up the QSAR model specifically for MEK protein. Collecting those activity test results of known MEK inhibitor on
Fig. 3 Illustration of 3D structure of amino acids neighboring PD318088 and MEK protein (PDBid:1S9J). Green: MgATP; Blue arrow: entrance of MEK protein for PD318088; Purple: fluorine; Red: oxygen.
PubMed from other researcher is applicable for next step. There are some limitations for using these materials and keeping the literature from sample experiment, same author, same method etc., and to reduce error.
The drugs are divided into two groups:
The purpose of first group is to build up 2D-QSAR model and take it as Training Set. The drug parameters such as molecular weight, H-bond donor, H-bond acceptor, AlogP and molecular solubility will be calculated with multiple linear regressions for linear regression curves before the model is built up.
The purpose of second group is to build up 2D-QSAR efficacy and take it as Test Set. The 2D-QSAR needs to be reconstructed if the predicted activity and real activity have enormous difference (or with non-linear relation). Those parameters involve in calculation which needed to be modified till linear relation established in order to go through as many MEK inhibitor test as possible. The ongoing test also includes famous clinical MEK inhibitor such as PD184352, PD98059, U0126, CI-1040, and etc..
Old Drug New Use 456
Fig. 4 Research flow chart and estimate schedule.
chemACX chemical molecular database after the calibrated 2D-QSAR built. This step is accomplished by using PHP (hypertext preprocessor) program design to search for FDA drug database and download the 2D structure within chemACX. Calculate the character value and input into 2D-QSAR for preliminary screening.
This primary screening is just the result from 2D plane which could not be applied on stereoscopic MEK protein and inhibitor. The Ligand Fit program in Discovery Studio 2.0 drug design software is used for molecular docking. This program can calculate the stereo structure change of drugs, simulate the binding site while drug enters proteins (as mentioned previously, the MEK inhibitor binding site is nearby Met143, Ile141, Leu118 and Phe209) and also
the similar properties to perform secondary screening that can be able to exclude those unqualified drugs.
3. Results
The primary 2D-QSAR model (Fig. 5), since the model has not been tested, therefore the result may still have some changes which includes Training Set reselection or parameters change, and etc..
As previously mentioned, PD184352 analogue have hydrophobic property on MEK binding site, the formation of electrics and hydrogen bonds. Addition of more effective MEK inhibitor can not only go deep into Phe209 hydrophobic region but produce pi-pi interaction between the aromatic ring on Phe209 (Fig. 6). This kind of interaction is a frequently discussed force between drugs and proteins in recent years. The interaction takes place at the inner part of MEK protein, so the pi-pi interaction can have attractive force over drug internally. Additionally, the electrostatic force and hydrogen bond formation can further stabilize the structure between drugs and protein.
4. Discussion
The ALogP, number of aromatic rings, number of hydrogen bond acceptors and number of hydrogen bond donors are all in positive correlation according to the equation from primary 2D-QSAR model which confirms to the previous description. However, the model is built up by several simple parameters without it being examined with test set. Thus, the 2D-QSAR model will basically be reconstructed and perform the examination to verify the precision to further reduce some ineffective parameters to simplify the model.
It is estimated that the primary screening of released drug database may result in too many drugs match the condition because 2D-QSAR only input several molecular parameters for calculation. Further docking calculation can exclude those molecules with great volume that could not enter the protein. Finally, applying with Pharmacophore to perform the analysis
Fig. 5 Primary 2D-QSAR model (N = 102, r2 = 0.69,
least-squared error = 0.178) using 3-hydroxy-4carboxyalkylamidino-5-arylamino-isothiazoles and isothiazole-4-carboxamidines derivatives as training set
built model. Y-axis as real activity (pIC50) and X-axis as
predicted activity and related equation (pIC50).
MLRTempModel (predicted activity) = -2.346 + 0.138 *
ALogP + 1.0952 * Energy 2.735 *
Molecular_PolarSurfaceArea 1.306 *
Molecular_Solubility 1.322 * Molecular_Volume + 1.026 *
Molecular_Weight + 0.171 * Num_AromaticRings + 0.109 * Num_H_Acceptors + 0.198 * Num_H_Donors (published on 5th International Symposium of Biocatalysis and Biotechnology, 2009, poster 1-4).
Old Drug New Use 458
is more objective, but the screening procedure still base on the former conditions. Further screening of clinical drug database may be required if the result from approved drug database screening is not as expected.
5. Conclusions
In dealing with old drug for new use and computational pharmacology, most researchers will take care about system biology and drug database design. However, most of them are not necessarily proven to be effective. It is because the extensive information will disrupt the concentration about the true issue on this disease therapy. By choosing suitable dataset and focusing on important drug, candidates will be helped to MAPK drug design and computational chemical analysis as well. Rapidly screen the FDA drug database for drugs that receive FDA certification (Drugs@FDA 6184 of drugs) and transform the name into 2D structure with chemACX chemical molecular database after the calibrated 2D-QSAR built will be the best choice to accomplish this mission. On the other hand, 2D-QSAR is a well know method to evaluate these drugs activities.
For further study, the authors will introduce PC clusters to calculate these drug candidates. Traditionally, supercomputers have only been built by a selected number of vendors. A company or organization that required the performance of such a machine had to have a huge budget required for its supercomputer. The concept of cluster computing was introduced when people first tried to spread different jobs over more computers and then gather back the data from these systems. In general, clustering refers to technologies that allow multiple computers to work together to solve common computing programs. It will be greatly helped to optimize the drug database screening.
Acknowledgments
The corresponding authors would like to thank their parents, all colleagues and friends who contributed to this study. The authors thank the support of China Medical University (CMU100-TC-05) in this research.
References
[1] N.G. Ahn, R. Seger, R.L. Bratlien, C.D. Diltz, N.K. Tonks, E.G. Krebs, Multiple components in an epidermal growth factor-stimulated protein kinase cascade. In vitro activation of a myelin basic protein/microtubule-associated protein 2 kinase, J Biol Chem 266 (1991) 4220-4227.
[2] H.J. Schaeffer, M.J. Weber, Mitogen-activated protein kinases: Specific messages from ubiquitous messengers, Mol Cell Bio l19 (1999) 2435-2444.
[3] J.F. Ohren, Structures of human MAP kinase kinase 1 (MEK1) and MEK2 describe novel noncompetitive kinase inhibition, Nat Struct Mol Biol 11 (2004) 1192-1197.
[4] H. El Abdellaoui, C.V. Varaprasad, D. Barawkar, S. Chakravarty, A. Maderna, R. Tam, et al., Identification of isothiazole-4-carboxamidines derivatives as a novel class of allosteric MEK1 inhibitors, Bioorg Med Chem Lett 16 (2006) 5561-5566.
[5] C.R. Chong, D.J. Jr Sullivan, New uses for old drugs, Nature 448 (2007) 645-646.
[6] J.S. Sebolt-Leopold, D.T. Dudley, R. Herrera, K. Van Becelaere, A. Wiland, R.C. Gowan, et al., Blockade of the MAP kinase pathway suppresses growth of colon tumors in vivo, Nat Med 5 (1999) 810-816.
[7] N. Dhanasekaran, R.E. Premkumar, Signaling by dual specificity kinases, Oncogene 17 (1998) 1447-1455.
[8] C.V. Varaprasad, D. Barawkar, H. El Abdellaoui, S. Chakravarty, M. Allan, H. Chen, et al., Discovery of 3-hydroxy-4-carboxyalkylamidino-5-arylamino-isothiazo les as potent MEK1 inhibitors, Bioorg Med Chem Lett 16 (2006) 3975-3980.
May 2013, Vol. 7, No. 5, pp. 459-463
Journal of Life Sciences, ISSN 1934-7391, USA
Relationship of Circulating High-Density Lipoprotein
Cholesterol and Anemia
Md. Aminul Haque Khan1, Rokshana Rabeya2, Muhammad Saiedullah3, Rukhsana Parvin4, Sohel Ahmed5 and Md. Rezwanur Rahman6
1. Department of Biochemistry, Enam Medical College, Dhaka 1340, Bangladesh
2. Biochemistry Laboratory, Enam Medical College Hospital, Dhaka 1340, Bangladesh
3. Department of Applied Laboratory Sciences, Bangladesh University of Health Sciences, Dhaka 1216, Bangladesh
4. Department of Medicine, Enam Medical College & Hospital, Dhaka 1340, Bangladesh
5. Department of Biochemistry & Molecular Biology, Jahangirnagar University, Dhaka 1342, Bangladesh
6. Department of Biochemistry, Delta Medical College, Dhaka 1216, Bangladesh
Received: February 16, 2013 / Accepted: April 19, 2013 / Published: May 30, 2013.
Abstract: Circulating level of low HDLC (high-density lipoprotein cholesterol) represents a common critical risk factor for IHD (ischemic heart disease) and may further aggravate the condition in anemic subjects, as the presence of anemia itself is a threat to cardiovascular consequences. To investigate the relationship of circulating HDLC with anemia, first we determined the levels of total hemoglobin (Hb) in a total of 301 subjects (male, n = 158; female, n = 143) randomly, and then examined the circulating levels of HDLC in fasting condition. Age of the study subjects was 47.9 ± 16.6 (mean ± SD) years. Both the male and female subjects were divided into three groups according to their levels of Hb. The relationship of circulating levels of HDLC with the levels of total Hb was statistically analyzed. In case of the male subjects, we found that the levels of HDLC differed significantly among the three groups with different levels of Hb (P = 0.0233) and decrease in the levels of HDLC correlated significantly with the gradual decrease of total Hb level (r = 0.2504; P = 0.0015). In female subjects, we observed a similar trend of difference among the three groups (P = 0.0685). However, decrease in the levels of HDLC correlated significantly with the gradual decrease of Hb level (r = 0.2199; P = 0.0083). Altogether, this study demonstrates that decrease in the circulating HDLC is related to the gradual decrease of Hb level. This study also indicates that circulating level of HDLC may be influenced by the level of total Hb and reveals the cardiovascular risks in anemia as well.
Key words: High density lipoprotein cholesterol, hemoglobin, anemia.
1. Introduction
Anemia is an important potential nontraditional risk factor for CVD (cardiovascular diseases) [1]. There are pathophysiologic reasons why the presence of anemia may lead to adverse cardiovascular consequences. In theory, the presence of anemia may also exacerbate cardiac ischemia as a result of decreased supply or increased demand for oxygen, such as in patients with underlying coronary disease
Corresponding author: Md. Aminul Haque Khan, MBBS, MCPS, FCGP, FCPS, MD, professor, research field: clinical biochemistry. E-mail: aminhkhan@yahoo.com.
or those with LVH (left ventricular hypertrophy), respectively [1]. Chronic anemia may increase preload, reduce afterload and lead to increased cardiac output [2]. In the long term, this may result in maladaptive LVH which in turn is a well recognized risk factor for CVD outcomes and all-cause mortality [3-5]. Several studies in various populations have evaluated the importance of anemia as a risk factor for adverse outcomes. In patients with heart failure, anemia has for the most part been found to be associated with an increased risk for hospitalization and all-cause mortality [4, 6-9]. In patients with coronary artery
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Relationship of Circulating High-Density Lipoprotein Cholesterol and Anemia 460
disease, the relationship between anemia and adverse outcomes is not consistent. Some studies have demonstrated an adverse effect of anemia on CVD outcomes while others have demonstrated no adverse effect of anemia on CVD outcomes [10, 11]. In a study of atherosclerosis risk in communities, anemia was found associated with CVD outcomes [12]. In United States, in the NHANES II (Second National Health and Nutrition Examination Survey) mortality study, after adjustment for traditional CVD risk factors and other covariates, there was no significant association between anemia and CHD (coronary heart disease) mortality [13].
HDL (high-density lipoprotein) enhances cholesterol transport from peripheral tissues to liver and thus maintains blood cholesterol level at normal level and reduces the risk of IHD (ischemic heart disease) and other atherosclerotic disorders [14]. Nascent HDL particles released from the liver pick up cholesterol from peripheral tissues and then come back to liver to release cholesterol. In doing so, some of the HDL particles in liver are assaulted by hepatic lipase and turn into lipid-depleted nascent HDL particles which along with newly produced nascent particles repeat the process of cholesterol extraction from peripheral tissues [14]. HDL contains paraoxonase, an antioxidant enzyme which shows native antioxidant activity to prevent initiation of atherogenic events [14]. Apo A-1 of HDL stabilizes PGI2 (prostacyclin I2) and thus prevents coronary artery constriction [14]. As noted by the National NCEP-ATP III (Cholesterol Education Program-Adult Treatment Panel III) guidelines, low HDLC (high-density lipoprotein cholesterol) represents a key determinant of the Framingham risk score and a common critical risk factor for IHD [15]. Low HDLC levels, defined as below 40 mg/dL for men and 50 mg/dL for women, remain prevalent [15-17]. Numerous prospective cohort studies support a powerful inverse correlation between circulating HDLC levels and coronary risk [18-25].
Earlier studies supported an inverse correlation between circulating HDLC and coronary risk in patients with normal or elevated LDLC. In theory, the presence of anemia may also exacerbate cardiac ischemia as a result of decreased supply or increased demand for oxygen, such as in patients with underlying coronary disease or those with LVH, respectively [1]. The combination of anemia and decrease in HDL may, therefore, be more detrimental. In this study, we have assessed the relationship of circulating HDLC with anemia.
2. Materials and Methods
2.1 Study Design
The present research was a cross sectional study.
2.2 Study Subjects
Between July 2009 and December 2010, 301 adult subjects, both males and females, were randomly selected from patients attending the out-patient department (OPD) of Enam Medical College Hospital, Savar, Dhaka advised for hematological investigations. Out of the total subjects, 158 were males and 143 were females. Age of the subjects was 47.9 ± 16.6 years. Both classes of subjects were further divided into 3 (three) groups based on their total hemoglobin (Hb). Subjects with Hb level < 10 g/dL were categorized as Group A, with Hb level from 10-14 g/dL as Group B and Hb level > 14 g/dL as Group C. Following the determination of total Hb, fasting levels of circulating HDLC were measured.
2.3 Biochemical Analysis
2.4 Statistical Analysis
Statistical analysis was done using the GraphPad Prism Version 5.04 for Windows. One way analysis of variance (ANOVA) test was applied to compare the levels of HDLC among different hemoglobin groups in males and females. The relationship between the levels of total Hb and fasting levels of circulating HDLC was determined by Pearson’s r-test. A P value less than 0.05 was considered as significant.
3. Results and Discussion
Tables 1 and 2 show the results of ANOVA analyses to compare the levels of circulating HDLC and the levels of total Hb among the three different groups of male and female subjects, respectively. We found that the levels of circulating HDLC differed significantly with the levels of total Hb among the three groups of male subjects as shown in Table 1 (P = 0.0233). In case of female subjects, the difference among the three groups was close to be significant statistically (P = 0.0685) and we observed a similar trend.
Figs. 1A and 1B show the correlation between the circulating HDLC and the levels of total Hb in male and female subjects, respectively. We found that decrease in the levels of circulating HDLC positively associated with the gradual decrease in the levels of total Hb in both the male (r = 0.2504) and female (r = 0.2199) subjects. The correlation was found highly significant in both male (P = 0.0015) and female (P = 0.0083) subjects.
Circulating low HDLC represents a common critical risk factor for IHD. The presence of anemia itself is a threat to cardiovascular consequences. Low HDLC in anemic subjects may be more detrimental. In the present study, the relationship of circulating HDLC and anemia has been investigated. Results of this study clearly demonstrate that circulating levels of HDLC differed significantly with different levels of total Hb (P = 0.0233) and decrease in circulating HDLC was positively associated with the gradual decrease of total Hb levels (r = 0.2504; P = 0.0015) in
Table 1 Circulating levels of HDLC in different groups of male subjects (n = 158).
Table 2 Circulating levels of HDLC in different groups of female subjects (n = 143).
Fig. 1 Relationship between HDL cholesterol (HDLC) and Hb levels in male (A) and female (B).
Relationship of Circulating High-Density Lipoprotein Cholesterol and Anemia 462
0.2199; P = 0.0083). These findings reveal that decrease in the circulating HDLC was strongly related to the gradual decrease of total Hb levels. This study indicates that circulating levels of HDLC may be influenced by the levels of total Hb. The possible aggravation of cardiovascular risks with low circulating HDLC in anemia may be assumed.
In a study involving 67 patients with homozygous beta-thalassemia, circulating HDLC was found low compared to that of healthy or heterozygous subjects of the same families [26]. In another study, levels of HDLC were found low with a mean of only 70% of the controls, where plasma and erythrocyte lipids were estimated in children with different types of anemia and healthy control and a positive correlation of Hct (hematocrit) with HDLC was reported [27]. These findings are consistent with the findings of our study. However, a study on scavenger receptor class B, type I (SR-BI) knockout mice showed that HDLC increased dramatically with simultaneous increase in osmotic fragility of red blood cells and also erythropoiesis. The investigator concluded that increased HDLC levels due to SR-BI deficiency induced erythrocyte cholesterol to phospholipid ratio, resulting in increased osmotic fragility of red blood cells leading to anemia [28]. Report of this study [28] contradicts the findings of our study. However, from this study, it is clear that circulating levels of HDLC may be regulated by the levels of total Hb. Here in this study, the authors did not investigate the causes of low circulating HDLC in subjects with low levels of total Hb. It can only be suggested that the diluting effect of increased plasma volume could partly explain the lowering of HDLC [27]. To understand the mechanisms of how circulating levels of HDLC may be influenced by the levels of total Hb, the authors recommend more investigations in different settings.
4. Conclusion
From the findings of this study, it can be concluded that hemoglobin level may be an important factor for
HDL cholesterol levels. But more studies with larger number of subjects are required to come to a decision whether there is significant association of hemoglobin levels with HDL cholesterol levels.
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