Thrombosis Research 123 (2009) 800–802

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Thrombosis Research

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Letter to the Editors-in-Chief

Table 1Characteristics, biochemical and mutational analytic data of the study population

Features Patients Controls

Total number of study subjects 50 67Age range 26–40 26–40Male:Female ratio 4.5:1 4.5:1Subjects with normal fHcy 15 (30.0%) 22 (32.8%)Subjects with HHC 35 (70.0%) 45 (67.2%)Number of individuals given methionine load 50 27HHC after methionine load (PML unmasking) 39/50 (78.0%) 22/27 (81.5%)MTHFR heterozygote 17 (34.0%) 9 (33.3%)Individuals given vitamin supplements 49 20Hyperhomocysteinemic individuals benefitedby vitamin supplements

20/35 (57.1%) 11/20 (55.0%)

Hyperhomocysteinemic individuals withnormalization of Hcy levels

05/35 (14.3%) –

High prevalence of hyperhomocysteinemia in young population of NorthIndia - A potential risk factor for coronary artery disease?

To the Editor,

Incidence of coronary artery disease (CAD) amongst younger agegroup is constantly rising in Indian population. Recent trends show thatIndia is on course to being the largest contributor to global CADmortality [1,2]. The conventional risk factors like hypertension, diabetesmellitus, or dyslipidemia do not play a significant role in CAD affectingthe young. Among the other factors, hyperhomocysteinemia (plasmahomocysteineN13 µ mol/L) has been found to be associated withincreased risk of CAD [2]. It may be caused by acquired or genetic factorsor both. The hyperhomocysteinemia (HHC) due to acquired causes likevitamin B12 or folic acid deficiency is reversible and vitamin supple-mentation in these patients may reduce the CAD mortality. The aim ofthe study was to compare the plasma homocysteine (Hcy) levels inyoung patients of CAD to age and sex matched healthy population ofNorth India and to investigate the role of acquired and genetic factors inthe causation of young CAD.

The study was conducted in the department of Hematology incollaboration with the department of Cardiology of a tertiary careinstitute of North India. Fifty young patients (b40 years of age) of CADadmitted in the cardiology unitwere chosen for the study. A diagnosis ofCAD was strictly based on WHO criteria i.e. ischemic type chestdiscomfort, evolutionary changes on serially obtained EKG tracings, riseor fall of serum cardiac markers, echocardiograms or angiography.Patients with no definite acute myocardial infarction, age more than40years, hypertension, impaired renal function, diabetesmellitus, hypo/hyperthyroidism, hyperlipidemia, smoking or with family history ofpremature CAD were excluded from the study. For comparison 67normal healthy subjectsmatched for age, sex andwithout any history ofCAD were selected from the general population. Of these 27 subjectswere volunteers from the hospital staff. Both the patients and controlswere advised to avoid substances whichmight interfere with Hcy levelsi.e. tea, coffee, alcohol at least 24 hours before sampling. It was madesure that patients or controls were not on drugs (e.g. theophylline, oralcontraceptives,methotrexate, anticonvulsants, penicillamine, acetylcys-teine and vitamins) known to affect the Hcy levels. To minimize theimmediate effects of high protein diet all the study subjects wererequested to remain fasting overnight. An informed consent was takenfrom both the patients and controls and ethical clearance was obtainedfrom the institute.

An analysis of fasting homocysteine (fHcy) was done in both thestudy and control groups. Post methionine load homocysteine (PMLHcy) levels were also estimated as administration of methionineincreases the specificity and sensitivity of detecting HHC (up to 100%)[3]. TheMTHFR gene (C677T)mutationwas tested by PCR-RFLPmethod.

With aseptic precautions 10 ml fasting venous sample wascollected from both the patients and control subjects. Plasma wasseparated for fHcy and the samples were stored at −20 °C for analysis.After this, methionine (0.1 g/kg body weight, SRL, Sisco Research

0049-3848/$ – see front matter © 2008 Elsevier Ltd. All rights reserved.doi:10.1016/j.thromres.2008.08.021

Laboratories Pvt. Ltd., Mumbai, India) was administered to all thepatients and 27 hospital staff volunteers in 200 ml of orange juice.After 4 hours of methionine load a repeat 2 ml sample was collectedfor PML Hcy estimation. After initial tests all the patients and controlsubjects who took methionine were followed up for 3 months. Duringthis period they were given a similar dose of folic acid (1.5 mg) andvitamin B12 (0.5 mg) as vitamin supplement, twice a day. All wereadvised to take regular diet or as advised by their dietician. Some ofthe patients who were discharged were instructed to visit weekly toout patient department. The control subjects were also regularlyinteracted for the feedback. After completion of 3 months, a repeat2 ml fasting blood sample for fHcy was obtained in 49 patients and 20control subjects. Thereafter the therapy was discontinued and nofurther samples were taken related to the study. Post methionine load(PML) though initially planned but could not be done due to poorcompliance to methionine (methionine has a bitter taste).

Plasma fHcy estimation was done by using commercial ELISA kits(Bio-Rad Laboratories Inc. Hercules, CA. 94547, USA) as per instruc-tions of the manufacturer. For detection of MTHFR gene (C677T)mutation, genomic DNA was isolated from peripheral blood leuko-cytes by standard phenol chloroform extraction method and PCR-based RFLP (restriction fragment length polymorphism) was carriedout. The study subjects were labeled as hyperhomocysteinemic withfHcy above 13 µ mol/L. After PML, if the Hcy levels were N38.0 µ mol/Lthe subject was considered under high risk group and hyperhomo-cysteinemic [4,5].

Statistical analysis was carried out using SPSS statistical package(Version 10, SPSS Inc, and Chicago, IL). Student's t-test was used toanalyze the homocysteine lowering effects of vitamin B12 and folicacid. The Chi-square test was applied to assess the association offasting and PML Hcy with MTHFR gene mutation in the study groups.The Pb0.05 was considered significant.

On data analysis, majority of the patients were males (M: F=4.5: 1)with age range 26–40 years (Table 1). Plasma Hcy levels obtainedbefore and after methionine load were compared between patientsand control subjects. With fHcy measurement, 70.0% (35/50) of the

Table 2Comparison of some of the relevant studies on Indian population

Slno

Authors No. of patientsof CAD

No. of healthycontrols

Age group(years)

HHC PrevalenceFasting Hcy (%)

HHC PrevalencePML Hcy (%)

Vitaminsupplements

MTHFR genemutation (%)[patients/controls]CAD Controls CAD Controls

1. Chacko K A16 1998, Cochin 56 53 N40 41.1 35.9 – – – –

2. Deep et al17 2001, Chennai 41 36 N40 19 5.6 – – – –

4. Nair et al18 2002, Mumbai 115 115 N40 19 18 – – – 54.5/-5. Mishra et al19 2002, Delhi – 72 N40 – 84.0 – – – –

6. Puri et al20 2003, Lucknow 51 15 b45 72.5 26.7 – – – –

7. Radha et al21 2004, Hyderabad – 608 – – – – – – -/24.98. Khare et al22 2004, Mumbai 120 100 b40 25.1 2.0 – – – 3.3/b19. Current study Chandigarh 50 67 b40 58.0 67.2 74.0 88.9 + 34.0/33.3

801Letter to the Editors-in-Chief

CAD patients and 67.2% (45/67) controls subjects were found to behyperhomocysteinemic. The HHC was much more prevalent amongstthe 27 medical staff personnel as 74.0% (20/27) of them had elevatedfHcy. With PML, the prevalence of HHC increased further as 4 patientsand 2 controls with normal fHcy were found to have PML Hcy abovethe high risk cut off value (N38 µ mol/L). The actual prevalence afterPML was, therefore 78% (39/50) in patients and 81.5% (22/27) in themedical staff personnel. Moreover the PML Hcy levels were more thanthe fasting Hcy in majority of the patients (range 16.5–94.0 µ mol/L)and control subjects (range 22.0–74.0 µ mol/L).

After 3 months of vitamin B12 and folic acid supplementation, thefHcy levels were reduced in 20/35 CAD patients with previous HHC. Asimilar response was seen in 11/20 hospital staff personnel with HHC.The beneficial effect of vitamin supplementation was found to bestatistically significant (P=0.018) with reduction of Hcy in 57.1% ofpatients and 55.0% controls. In addition 05/35 (14.3%) of the HHCpatients showed normalization of their fHcy value.

The prevalence of heterozygote MTHFR gene (C677T) mutationwas 34.0% and 33.3% in patient and control group respectively. Nohomozygote was noted. On statistical analysis there was no associa-tion of MTHFR gene (C677T) mutation with high Hcy levels either incases or controls (PN0.05).

The world wide prevalence of HHC in CAD ranges from 5% to 40%[1,6]. The data available from India is meager and shows regionalvariation [7–13]. Most of the studies in our country have beenconducted on older patients (Table 2). Their results are based on fHcyonly and show a variable prevalence of HHC ranging from 19%–72.5%[9–11,13]. Puri et al from eastern part of India showed HHC prevalenceof 72.5% in CAD patients below 45 years [11]. A single Indian study onpatients b40 years showed HHC in 25.1% of CAD patients. We in ourstudy found 70.0% of the CAD patients and 67.2% controls to behyperhomocysteinemic. The prevalence of HHC increased further withPML. The results however could not be compared as no Indian studieson PML Hcy are available. A surprising finding of the study was a highprevalence of HHC in healthy population. Other authors have alsoshown HHC to be present in 2.0% to 84% of general population [13].The higher prevalence amongst the medical staff personnel (81.5%) inour study however remains unexplained.

The prevalence of MTHFR (C677T) gene mutation in westerncountries varies from 9%–78% [14]. The Indian studies show a lowprevalence of the mutation [9,12,13]. Our study also supports the dataas only heterozygote form of MTHFR gene (C667T) mutation wasfound with equal distribution in both the study groups. The otherpolymorphisms i.e. MTHFR gene (A1298C), cystathione β synthase,methionine synthase, methionine synthase reductase etc. are alsoknown but have only a minor effect on Hcy levels [15].

The 3 months vitamin supplementation (B12 and folic acid) led tostatistically significant (P=0.037) lowering of the Hcy levels in boththe study groups. The lowering effect was more in CAD patients thanthe controls. Although normal levels of Hcy could not be achieved incontrol subjects, majority reached near normal Hcy levels. It is

possible that if higher dosage or the same dose was given for littlelonger duration most of the subjects might have reached to normal ornear normal levels.

Beneficial effect of vitamin supplements suggests that acquiredfactors like nutritional deficiency of vitamin B12 and folic acid maycontribute to HHC in this part of country. The results are alsostrengthened by the fact that a large group of Indian population isdeficient in both vitamin B12 and folic acid [16,17].

The extent of HHC and its contribution to the causation of CAD inIndian population is not clear. From the present study also due to smallstudy groups, little follow up and a surprising HHC in both patient andcontrol groups, the influence of homocysteine reduction on the risk ofCAD could not be calculated. Because of high prevalence of HHC inhealthy young people of north India as shown by the present study,similar but larger and population based studies with longer follow upare needed to prove the association of HHC and the CAD risk.

In summary it is suggested that HHC is highly prevalent in Indianpopulation which might be one of the reasons for high incidence ofCAD in younger population. The vitamin deficiency rather thanMTHFR gene (C677T) mutation is the likely etiology for HHC. Vitaminsupplement helps in reducing Hcy and may play an important role inprevention of young CAD exclusively due to HHC. However its role inestablished CAD cases or individuals who have CAD due to othercauses needs to be proven by further studies.

References

[1] Muray CJ, Lopez AD. Mortality by cause for eight regions of the world. Globalburden of disease study. Lancet 1997;349:1269–76.

[2] Chambers JC, Wander G, Kooner JS. Homocysteine and heart disease among IndianAsians. Indian Heart J 2000;52:5–8.

[3] Bostom AG, Roubenoff R, Dellaripa P, Nadeau MR, Sutherland P, Wilson PW, et al.Validation of abbreviated oral methionine-loading test. Clin Chem 1995;41:948–9.

[4] Jacobsen DW. Homocysteine and vitamins in cardiovascular disease. Clin Chem1998;44:1833–43.

[5] Fokkema MR, Weijer JM, Dijck-Brouwer DAJ, van Doormaal JJ, Muskiet FAJ.Influence of Vitamin-optimized Plasma Homocysteine Cutoff Values on thePrevalence of Hyperhomocysteinemia in Healthy Adults. Clin Chem 2001;47:1001–7.

[6] Corinne DL, Jean-Claude W, Daniel B, Michèle D, Jean-Marie B, Jean D, et al. Plasmahomocysteine concentrations in a Belgian school-age population. Am J Clin Nutr1999;69:968–72.

[7] Chacko KA. Plasma homocysteine levels in patients with coronary artery disease.Indian Heart J 1998;50:295–9.

[8] Deepa R, Velmurugan K, Sarvanan G, Karkuzhah, Diwakanath V, Mohan V. Absenceof association between serum homocysteine levels and coronary artery disease inSouth Indian males. Indian Heart J 2001;53:44–7.

[9] Nair K, Nair S, Ashavaid T, Dalal J, Eghlim F. Methylenetetrahydrofolate reductasegene mutation and hyperhomocysteinemia as a risk factor for coronary heartdisease in the Indian population. J Assoc Phys India 2002;50:9–15.

[10] Mishra A, Vikram NK, Pandey RM, Dwivedi M, Ahmad FU, Luthra K, et al.Hyperhomocysteinemia, and low intakes of folic acid and vitamin B12 in urbanNorth India. Eur J Nutr 2002;41:68–77.

[11] Puri A, Gupta OK, Dwivedi RN, Bharadwaj RPS, Narain VS, Singh S. Homocysteineand Lipid Levels in Young Patients with Coronary Artery Disease. JAPI 2003;51:681–5.

[12] Radha A, Govindaiah V, Ramakrishna G, Naushad SM. Prevalence of Methylene-tetrahydrofolate reductase polymorphism in South Indian population. Curr Sci2004;86:440–3.

802 Letter to the Editors-in-Chief

[13] Khare A, Ghosh K, Shetty S, Kulkarni B, Mohanty D. Combination of thrombophiliamarkers in acute myocardial infarction of the young. Indian J Med Sci2004;58:381–8.

[14] Abu-Amero KK, Carol A, Wyngaard, Dzimiri N. Prevalence and role of Methylene-tetrahydrofolate Reductase 677 CTand 1298 AC Polymorphisms in Coronary ArteryDisease in Arabs. Arch Pathol Lab Med 2003;127:1349–52.

[15] Kluijtmans LAJ, Young IS, Boreham CA, Murray L, McMaster D, McNulty H, et al.Genetic and nutritional factors contributing to hyperhomocysteinemia in youngadults. Blood 2003;101:2483–8.

[16] Nath I, Reddy KS, Dinshaw KA. Country profile: India. Lancet 1998;351:1265–75.[17] Antony AC. Prevalence of cobalamine (Vitamin B-12) and folate deficiency in India.

Am J Clin Nutr 2001;74:157–9.

Yashwant Kumar⁎Reena Das

Gurjeevan GarewalDepartment of Hematology, Postgraduate Institute of Medical Education

and Research, Chandigarh, India 160012⁎Corresponding author. The Pine, Near Ashiana Regency, Chhota

Shimla, Shimla (HP), India – 171002. Fax: +91 172 2744401.E-mail addresses: yashwantk74@yahoo.com (Y. Kumar),

reenadaspgi@hotmail.com (R. Das),ykabh@hotmail.com (G. Garewal) .

Harinder Kumar BaliDepartment of Cardiology, Postgraduate Institute of Medical Education

and Research, Chandigarh, India 160012E-mail address: hkbali_pgi@rediffmail.com.

15 May 2008