Evaluation of total Plasma Homocysteine in Indian newborns using Heel-prick samples
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《美国医学杂志》
Diagnostic Division, Center for DNA Fingerprinting and Diagnostics, Hyderabad, India
Abstract
Objective. To estimate total plasma homocysteine levels in Indian newborns by modifying the existing SBD-F based High performance liquid chromotography (HPLC) method in order to enable analysis in newborn heel-prick samples and assess the prevalence of hyperhomocysteinemia in Indian newborns who are exclusively breast-fed. Methods. Reverse-phase HPLC with fluorescence detection for plasma homocysteine estimation and statistical analysis using student t-test. Results. SBD-F based HPLC method was modified and Bland and Altman analysis was carried out to assess agreement between original and modified methods. The correlation co-efficient was 0.994. The limits of agreement (-5.9, 6.3) were small enough to apply new method in place of the old for heel-prick sample analysis. Total plasma homocysteine analysis was carried out on heel-prick samples of 607 randomly selected newborns (331 males and 276 females). The mean plasma homocysteine estimated by this method in Indian newborns was 6.99 (95% CI: 6.48-7.49) with no appreciable gender effect (P=0.74). Elevated homocysteine levels were observed in 31 males and 21 females. Conclusions. Modified HPLC method is validated and can be used for homocysteine analysis on newborn heel-prick samples. Using this method, the prevalence of hyperhomocysteinemia in Indian newborns is 8.6%.
Keywords: Homocysteine; Indian newborns; HPLC; Prevalence
Homocysteine, a sulfur-containing aminoacid gained lot of focus and attention in recent years due to its association in various pathological conditions. This non-dietary aminoacid is a by-product of methionine metabolism and its elevation is due to defective remethylation or defective trans-sulfuration pathway in methionine metabolism. The underlying cause for this metabolic disorder is either genetic (mutations in crucial genes) or non-genetic (cofactor i.e. B6, B12 and folate deficiency).
Hyperhomocysteinemia is known to cause multi-disease manifestations such as: premature occlusive vascular disease[1], smooth muscle proliferation[2], progressive arterial stenosis[3], and haemostatic changes[4], spontaneous early abortion[5], placental vasculopathy[6], birth defects[7], impaired cognitive function[8] and dementia.[9] The age of onset may range from early fetal life to adulthood. The pathophysiology could be due to direct toxicity of homocysteine on tissues, low S-adenosyl methionine or high S-adenosyl homocysteine or due to thrombotic events triggered by stimulation of procoagulant factors and suppression of anticoagulant factors by homocysteine.
Supplementation of cofactors viz. B6, B12 and folic acid orally is effective in reducing total plasma homocysteine levels,[10] which in turn reduce the risk for adverse clinical conditions caused by homocysteine.
The incidence of anemia, B6 deficiency and folate deficiency in Indian women was reported to be 15%, 84% and 24.5% in a case-control study from India.[11]
A recent study also showed a prevalence of 47% cobalamin deficiency and 5% folate deficiency among Asian Indians.[12]
These deficiencies in the pregnant women are responsible for the acquired impairment of remethylation leading to elevated neonatal total homocysteine and methyl malonic acid at birth. The condition worsens during infancy if the baby is exclusively breast-fed. Thus an elevated total homocysteine and low B12 status or elevated homocysteine and methyl malonic acid in babies indicate biochemical evidence of impaired B12 function. Such babies are at risk of developing neurological, cognitive or vascular complications later in life.
Neonatal screening for total homocysteine is recommended in countries following vegetarian diet. Through early detection of hyperhomocysteinemia and early intervention disability and death can be prevented. However, data on homocysteine levels in Indian newborns and children is sparse and identification of both the genetic and nutritional causes of hyper homocysteinemia is lacking.
The present study, the first of its kind in Indian newborns, aims to assess the prevalence of hyperhomocysteinemia and provide cut-off values for homocysteine in the newborns. Reverse-phase HPLC method using fluorescent derivatisation technique is modified to enable analysis of homocysteine with minimum amount of plasma, to enable its use for large-scale newborn screening.
Materials and Methods
Out of the 19, 000 newborns screened in Expanded Newborn Screening Program[13] during the period of October 1999 to June 2004, 607 (331 males and 276 females) newborns were randomly selected without any discretion for region, age, sex or religion. The age groups studied were between 2 to 21 days.
Modified SBD-F method (Ammonium 7 - fluorobenzo-2-oxa-1, 3 - diazole - 4 - sulphonate)
Chemicals
L-homocysteine, tri-n-butyl phosphine, dimethyl formamide, boric acid, EDTA (Sigma); ammonium 7-fluorobenzo-2-oxa-1, 3-diazole -4-sulphonate (SBD-F) (Fluka chemie); tri-chloro acetic acid, sodium hydroxide, KH2PO4 and acetonitrile (Qualigens).
Instrumentation
The Waters- HPLC system containing solvent delivery system 515 pump, Autosampler717, fluorescent detector 474 was used for analysis with excitation and emission wavelengths 385 nm and 515 nm respectively. Millennium software was used for acquiring the data.
Sample Collection
Blood Samples were collected from heel prick in micro-EDTA Tube. Plasma was separated immediately after centrifugation of the blood at 3000 RPM for 15 min at 4 °C.
Derivatisation
To 5 ml of plasma 20 ml of borate buffer (125 mM boric acid, 4 mM EDTA) was added to make the final volume to 25 ml. 10% (V/V) of tri-n-butyl phosphine in dimethyl formamide was added to the sample and vortexed. The resulting mixture was incubated at 4 °C for 30 min. Deproteinisation was done with 25 ml 10% (W/V) TCA, followed by vortexing and centrifugation at 3000 RPM for 15 min at 4°C. To 20 ml of clear supernatant taken in a fresh tube, 4 ml of (1.55N) NAOH was added and vortexed. This tube was kept at 4 °C for 10 min and 50 ml of borate buffer was added and vortexed. Finally 20 ml of SBDF (ammonium 7-flurobenzo-2-oxa-1, 3-diazole-4-sulphonate 1mg/ml in 125mM boric acid) was added and incubated at 60°C for one hour. After incubation sample was kept at ambient temperature for immediate processing. Standards and controls were prepared simultaneously in similar manner.
Chromatography and Conditions of HPLC
The derivatised sample was injected into a column equilibrated with mobile phase at a flow rate of 1.5 ml per min. The column equilibration time was 15 minutes at ambient temperature. The retention time of each sample was calculated by using standards and calibrators and a calibration curve was plotted for different concentrations.
Stationary phase: Octa decyl silane (ODS) 250X4.6 mm column, Mobile phase: 4% acetonitrile in potassium di-hydrogen orthophosphate (0.2 M, pH: 2.1), Detector: fluorescent with Ex/Em being 385 nm/515 nm
Recovery and precision
In plasma samples, known concentrations of homocysteine were added. The concentrations in samples with added calibrators were determined in replicates and analytical recoveries were calculated. The intra-assay precision was obtained by analysing replicates of sample in a day. Mean recoveries of homocysteine were 95 - 107%. Bland and Altman statistical analysis was carried out with the original method and the modified method and found to be in good correlation.
Optimisation of derivatization procedure
We have slightly modified the SBD-F method[14] to adapt it for small quantity of samples. The major alterations for the existing procedure are addition of borate buffer to less volume of sample and still maintaining the sensitivity by increasing injection volume. However neither the sample dilution nor the increased injection volume altered the sensitivity, specificity, accuracy and precision of chromatographic analysis. The incubation temperature if deviated from 4°C may adversely affect the analysis.
Statistical Analysis
Plasma variables were analyzed using raw data and also logarithmically transformed data as there is skewed distribution. The variables are presented as geometric mean and 95% confidence intervals (CIs). Online t-test was performed for one sample using Graph Pad Quick Calcs software. The mean, SEM and P values were obtained. To assess any possible gender-bias we performed a t-test for two-sets of samples (males and females). A P value of <0.05 was considered statistically significant.
Results
Two modifications were made in the SBD-F based HPLC method i.e. diluting the plasma with borate buffer and increasing the injection volume. Before adopting this method in place of original method, Bland and Altman analysis[15] was carried out to assess agreement between two methods of homocysteine measurement.
Bland and Altman analysis
1. Plotting Data
Plasma homocysteine values estimated by original method and modified method were plotted on X and Y axes respectively Figure1. The correlation coefficient (r) between the two methods was 0.994 concluding that homocysteine measurements by original and modified methods are related.
2. Measuring Agreement
Average homocysteine by two methods (X) was plotted against the difference in homocysteine (Y) in Figure2. The mean difference was found to be 0.183. The limits of agreement between these methods were -5.943 and 6.309.
3. Precision of estimated limits of agreement
The precisions of estimated limits of agreement were calculated. 95% CI for the lower limit of was found to be - 9.30 to - 2.58, whereas for the upper limit of agreement 2.94 to 9.68
4. Relation between difference and mean
Logarithmic transformation of the average homocysteine by both methods and difference in homocysteine between the original and modified was carried out and Log-Log graph was plotted Figure3. Limits of agreement for logarithmic data were -0.032 and 0.13 and antilog for it being 0.92 to 1.34.
5. Repeatability
Applying modified method, two consecutive analyses on a sample set were carried out and average homocysteine was plotted against the difference in homocysteine Figure4. The limits of agreement between two consecutive analyses were -4.81 and 2.55.
Linearity
The modified method was found to be linear Figure5 for concentrations ranging from 5 to 200 mmol/L (r=0.999).
Analysis of newborns table1
The mean plasma homocysteine concentration in newborns was 6.99±0.26. When data was segregated according to gender, the plasma homocysteine levels in male and female newborns were found to be 6.91±0.3 and 7.08±0.43 respectively (P=0.74). Increased homocysteine (>15 mmol/L) was observed in 31 males and 21 females out of the 331 males and 276 females tested respectively. The prevalence in males and females when compared with each other, there was no statistically significant gender-specific risk was observed (P=0.56). The overall incidence of hyperhomocysteinemia is 52 per 607 (8.5%) newborns. When hyperhomocysteinemia was segregated into mild (15-30 mmol/L), moderate (30-100 mmol/L) and severe(>100 mmol/L) table2, the most prevalent category was the milder one followed by moderate hyperhomocysteinemia. There were no newborns with severe hyperhomocysteinemia.
Discussion
Original method of homocysteine estimation by HPLC was modified to enable analysis on heel prick samples of newborns. Bland and Altman analysis showed good agreement between original and modified methods. The limits of agreement (-5.943 and 6.309) were small enough to use this technique for lesser volume of samples. This method has good repeatability and linearity (r=0.999).
The mean plasma homocysteine estimated by this method was 6.99mmol/L (95% CI: 6.48-7.49) [males and females being 6.907 (95% CI: 6.31-7.51) and 7.08 (95% CI: 6.23-7.93) respectively]. Gender differences were not observed in the mean plasma homocysteine concentration among newborns (P=0.74). Gender differences becoming prominent after puberty probably indicate the role of reproductive hormones[16] and body mass index (BMI).[17]
Mild to moderate hyperhomocysteinemia in newborns (52 per 607) studied indicate a high prevalence of abnormal homocysteine metabolism among the Indian newborns. Earlier studies on the mutation analysis of dried blood spots of the newborns collected in the newborn screening program showed 9% 677T allele frequency of Methylene tetrahydrofolate reductase (MTHFR) gene. In 80 male newborns, 8 were heterozygous and out of 108 female newborns, 22 were heterozygous and 2 were homozygous for C677T mutation.[18] With these frequency and a background of B12 and folate deficiency among the Indian mothers, a high total homocysteine in the newborns could be possible as has been observed in the present study. However, correlation with the cobalamin and folate status at the same time is required to confirm the finding.
The median homocysteine in UK newborns was reported to be 6.8 (4.2-12.8 mmol/L) with 2.54% newborns having homocysteine >15mmol/L.[19] The mean homocysteine in Japanese[20] and Italian newborn[21] were found to be 2.5 mmol/L and 2.92 mmol/L (Dried blood spot method) respectively. Another study on Italian newborns using capillary blood obtained by heel puncture showed a mean plasma homocysteine concentration of 4.9mmol/L.[22] The mean homocysteine in Indian newborns is higher than Japanese and Italian newborns. There is higher prevalence of hyperhomocysteinemia in Indian newborns in comparison to UK newborns (OR: 3.92, P=0.0001).
The significance of elevated homocysteine has been well studied in causing multi-disease manifestation at all ages. More so in the newborn period where studies indicate a direct correlation between cobalamin status and homocysteine levels and such newborns continue to have further increase in the concentration when they are exclusively breast-fed by mothers who themselves are deficient in B12. The clinical significance of elevated homocysteine becomes significant in later infancy and childhood in terms of mild to moderate mental retardation, cognitive failure, seizures, cerebro-vascular accidents etc.
Elevation in homocysteine could be due to mutations in genes regulating the crucial biochemical reactions in methionine metabolism or due to deficiencies of cofactors such as B6, B12 and folic acid. The consequences of this elevation are: low methionine levels, less production of S-adenosyl methionine (SAM), and high S-adenosyl homocysteine (SAH). SAM and SAH have antagonistic functions. The former acts as a universal methyl donor for transmethylases and the later acts as competitive inhibitor for transmethylases. High SAH/SAM ratio results in decreased cellular methylation. The functional consequences of decreased cellular methylation are significant and include central nervous system demyelination,[23] reduced neurotransmitter synthesis,[24] decreased chemotaxis and macrophage phagocytosis,[25] altered membrane phospholipid composition and membrane fluidity,[26] altered gene expression[27] and cell differentiation.[28], [29]
High prevalence of hyperhomocysteinemia in the newborn period, availability of reliable testing method, simple and less expensive treatment, justify the newborn screening for homocysteine in developing countries.
Follow-up of newborns with hyperhomocysteinemia showed a good biochemical and clinical improvement with supplementation of cofactors B12 and folate. One of the newborn who did not respond to recall for re-testing, developed acute infantile hemiplegia at the age of 1 yr. Although we did not find any case that is suggestive of Cystathionine Beta Synthase (CBS) deficiency, most of the cases studied were probably due to acquired defects in the remethylation as was evident by the response of the screen positive babies to cofactor supplementation.
In conclusion, our data along with other published data suggest homocysteine measurement in newborn period in order to reduce the mortality and morbidity associated with it. Although routine homocysteine measurement for the newborns is not recommended worldwide but total homocysteine is a better indicator rather than methionine for different types of hyperhomocysteinemia. Further work is required to prove the importance of screening in developing countries following vegetarian diet and high incidence of malnutrition. The need for routine cofactor supplementation in the first one year of life need to be assessed through large scale screening of newborns.
Acknowledgements
We thank all the newborns and their parents/guardians, who participated and cooperated in this study. We thank Dr. Reddy's foundation for Human and Social Development for funding Newborn screening program. We thank Department of Biotechnology, Government of India for the core grant.
References
1. Boers GH, Smals AG, Trijbels FJ, Fowler B, Bakkeren JA, Schoonderwaldt HC, Kleijer WJ, Kloppenborg PW. Heterozygosity for homocystinuria in premature peripheral and cerebral occlusive arterial disease. N Engl J Med 1985 Sep 19; 313(12) : 709-715.
2. Majors A, Ehrhart LA, Pezacka EH. Homocysteine as a risk factor for vascular disease. Enhanced collagen production and accumulation by smooth muscle cells. Arterioscler Thromb Vasc Biol. 1997 Oct; 17(10):2074-81.
3. Mueller T, Furtmueller B, Aigelsdorfer J, Luft C, Poelz W, Haltmayer M.Total serum homocysteine-a predictor of extracranial carotid artery stenosis in male patients with symptomatic peripheral arterial disease. Vasc Med. 2001; 6(3):163-7.
4. McCully KS. Vascular pathology of homocysteinemia: implications for the pathogenesis of arteriosclerosis. Am J Pathol. 1969 Jul; 56(1):111-28.
5. Wouters MG, Boers GH, Blom HJ, Trijbels FJ, Thomas CM, Borm GF, Steegers-Theunissen RP, Eskes TK. Hyperhomocysteinemia: a risk factor in women with unexplained recurrent early pregnancy loss. Fertil Steril. 1993 Nov; 60(5):820-5.
6. Van der Molen EF, Verbruggen B, Novakova I, Eskes TK, Monnens LA, Blom HJ. Hyperhomocysteinemia and other thrombotic risk factors in women with placental vasculopathy. BJOG. 2000 Jun; 107(6):785-91.
7. Steegers-Theunissen RP, Boers GH, Trijbels FJ, Eskes TK. Neural-tube defects and derangement of homocysteine metabolism. N Engl J Med. 1991 Jan 17; 324(3):199-200.
8. Gussekloo J, Heijmans BT, Slagboom PE, Lagaay AM, Knook DL, Westendorp RG. Thermolabile methylenetetrahydrofolate reductase gene and the risk of cognitive impairment in those over 85. J Neurol Neurosurg Psychiatry. 1999 Oct; 67(4): 535-8.
9. McCaddon A, Davies G, Hudson P, Tandy S, Cattell H. Total serum homocysteine in senile dementia of Alzheimer type. Int J Geriatr Psychiatry. 1998 Apr; 13(4): 235-9.
10. Maike Wolters, Silke Hermann, and Andreas Hahn. B vitamin status and concentrations of homocysteine and methylmalonic acid in elderly German women. Am J Clin Nutr. 2003; 78:765-72.
11. Neela J, Raman L. The relationship between maternal nutritional status and spontaneous abortion. Natl Med J India 1997 Jan-Feb; 10(1) : 15-16.
12. Helga Refsum, Chittaranjan S Yajnik, Milind Gadkari, J φrn Schneede, Stein E Vollset, Lars rning, Anne B Guttormsen, Anjali Joglekar, Mehmood G Sayyad, Arve Ulvik and Per M Ueland. Hyperhomocysteinemia and elevated methylmalonic acid indicate a high prevalence of cobalamin deficiency in Asian Indians. American Journal of Clinical Nutrition, August 2001; 74(2) : 233-241.
13. Radha Rama Devi A, Naushad S.M. Newborn Screening in India. Indian Journal of Pediatrics Vol 71-Feb 2004; (2) : 157-160.
14. Ubbink JB, Hayward Vermaak WJ, Bissbort S. Rapid high-performance liquid chromatographic assay for total homocysteine levels in human serum. J Chromatogr 1991 Apr 19; 565(1-2) : 441-6.
15. Bland J M and Altman D G. Statistical methods for assessing agreement between two methods of clinical measurements. Lancet 1986; I : 307-310.
16. Selhub J, Jacques PF, Wilson PWF et al. Vitamin status and intake as primary determinants of homocysteinemia in an elderly population. JAMA 1993; 270: 2693-2698.
17. Mudd SH and Poole JR. Labile methyl balances for normal humans on various dietary regimens. Metabolism 1975; 24: 721-735.
18. RadhaRamaDevi A, Govindaiah V, Ramakrishna G and Naushad SM. Prevalence of Methylene tetra hydro Folate reductase polymorphism in South Indian population. CurrSci 2003; 86 (3): 101-104.
19. Refsum H, Grindflek AW, Ueland PM, Fredriksen A, Meyer K, Ulvik A, Guttormsen AB, Iversen OE, Schneede J, Kase BF. Screening for serum total homocysteine in newborn children. Clin Chem 2004 Oct; 50(10) : 1769-1784.
20. Febriani AD, Sakamoto A, Ono H, Sakura N, Ueda K, Yoshii C, Kubota M, Yanagawa J. Determination of total homocysteine in dried blood spots using high performance liquid chromatography for homocystinuria newborn screening. Pediatr Int 2004 Feb; 46(1) : 5-9.
21. Accinni R, Campolo J, Parolini M, De Maria R, Caruso R, Maiorana A, Galluzzo C, Bartesaghi S, Melotti D, Parodi O. Newborn screening of homocystinuria: quantitative analysis of total homocyst(e)ine on dried blood spot by liquid chromatography with fluorimetric detection. J Chromatogr B Analyt Technol Biomed Life Sci 2003 Mar 5; 785(2): 219-26.
22. Bartesaghi S, Accinni R, De Leo G, Cursano CF, Campolo J, Galluzzo C, Vegezzi PG, Parodi O. A new HPLC micromethod to measure total plasma homocysteine in newborn. Pharm Biomed Anal 2001 Mar; 24(5-6): 1137-1141.
23. Scott JM, Molloy AM, Kennedy DG, Kennedy S, Weir DG. Effects of the disruption of transmethylation in the central nervous system: an animal model. Acta Neurol Scand Suppl 1994; 154 : 27-31.
24. Schatz RA, Wilens TE, Sellinger OZ Decreased transmethylation of biogenic amines after in vivo elevation of brain S-adenosyl-l-homocysteine. J Neurochem 1981; 36(5): 1739-1748.
25. Garcia-Castro I, Mato JM, Vasanthakumar G, Wiesmann WP, Schiffmann E. and Chiang PK. Paradoxical effects of adenosine on neutrophil chemotaxis. J Biol Chem 1983; 258: 4345-4349.
26. Chiang PK. Biological effects of inhibitors of S-adenosylhomocysteine hydrolase. Pharmacol Ther 1998; 77(2): 115-134.
27. Dizik M, Christman JK and Wainfan E. Alterations in expression and methylation of specific genes in livers of rats fed a cancer promoting methyl-deficient diet. Carcinogenesis 1991; 12(7): 1307-1312.
28. Chiang PK. Conversion of 3T3-L1 fibroblasts to fat cells by an inhibitor of methylation: effect of 3-deazaadenosine. Science 1981; 211(4487): 1164-1166.
29. Aarbakke J, Miura GA, Prytz PS, Bessesen A, Slordal L, Gordon RK and Chiang PK. Induction of HL-60 cell differentiation by 3-deaza- (+/-)-aristeromycin, an inhibitor of S-adenosylhomocysteine hydrolase. Cancer Research 1996; 46(11): 5469-5472.(Rama Devi Radha A, Nausha)
Abstract
Objective. To estimate total plasma homocysteine levels in Indian newborns by modifying the existing SBD-F based High performance liquid chromotography (HPLC) method in order to enable analysis in newborn heel-prick samples and assess the prevalence of hyperhomocysteinemia in Indian newborns who are exclusively breast-fed. Methods. Reverse-phase HPLC with fluorescence detection for plasma homocysteine estimation and statistical analysis using student t-test. Results. SBD-F based HPLC method was modified and Bland and Altman analysis was carried out to assess agreement between original and modified methods. The correlation co-efficient was 0.994. The limits of agreement (-5.9, 6.3) were small enough to apply new method in place of the old for heel-prick sample analysis. Total plasma homocysteine analysis was carried out on heel-prick samples of 607 randomly selected newborns (331 males and 276 females). The mean plasma homocysteine estimated by this method in Indian newborns was 6.99 (95% CI: 6.48-7.49) with no appreciable gender effect (P=0.74). Elevated homocysteine levels were observed in 31 males and 21 females. Conclusions. Modified HPLC method is validated and can be used for homocysteine analysis on newborn heel-prick samples. Using this method, the prevalence of hyperhomocysteinemia in Indian newborns is 8.6%.
Keywords: Homocysteine; Indian newborns; HPLC; Prevalence
Homocysteine, a sulfur-containing aminoacid gained lot of focus and attention in recent years due to its association in various pathological conditions. This non-dietary aminoacid is a by-product of methionine metabolism and its elevation is due to defective remethylation or defective trans-sulfuration pathway in methionine metabolism. The underlying cause for this metabolic disorder is either genetic (mutations in crucial genes) or non-genetic (cofactor i.e. B6, B12 and folate deficiency).
Hyperhomocysteinemia is known to cause multi-disease manifestations such as: premature occlusive vascular disease[1], smooth muscle proliferation[2], progressive arterial stenosis[3], and haemostatic changes[4], spontaneous early abortion[5], placental vasculopathy[6], birth defects[7], impaired cognitive function[8] and dementia.[9] The age of onset may range from early fetal life to adulthood. The pathophysiology could be due to direct toxicity of homocysteine on tissues, low S-adenosyl methionine or high S-adenosyl homocysteine or due to thrombotic events triggered by stimulation of procoagulant factors and suppression of anticoagulant factors by homocysteine.
Supplementation of cofactors viz. B6, B12 and folic acid orally is effective in reducing total plasma homocysteine levels,[10] which in turn reduce the risk for adverse clinical conditions caused by homocysteine.
The incidence of anemia, B6 deficiency and folate deficiency in Indian women was reported to be 15%, 84% and 24.5% in a case-control study from India.[11]
A recent study also showed a prevalence of 47% cobalamin deficiency and 5% folate deficiency among Asian Indians.[12]
These deficiencies in the pregnant women are responsible for the acquired impairment of remethylation leading to elevated neonatal total homocysteine and methyl malonic acid at birth. The condition worsens during infancy if the baby is exclusively breast-fed. Thus an elevated total homocysteine and low B12 status or elevated homocysteine and methyl malonic acid in babies indicate biochemical evidence of impaired B12 function. Such babies are at risk of developing neurological, cognitive or vascular complications later in life.
Neonatal screening for total homocysteine is recommended in countries following vegetarian diet. Through early detection of hyperhomocysteinemia and early intervention disability and death can be prevented. However, data on homocysteine levels in Indian newborns and children is sparse and identification of both the genetic and nutritional causes of hyper homocysteinemia is lacking.
The present study, the first of its kind in Indian newborns, aims to assess the prevalence of hyperhomocysteinemia and provide cut-off values for homocysteine in the newborns. Reverse-phase HPLC method using fluorescent derivatisation technique is modified to enable analysis of homocysteine with minimum amount of plasma, to enable its use for large-scale newborn screening.
Materials and Methods
Out of the 19, 000 newborns screened in Expanded Newborn Screening Program[13] during the period of October 1999 to June 2004, 607 (331 males and 276 females) newborns were randomly selected without any discretion for region, age, sex or religion. The age groups studied were between 2 to 21 days.
Modified SBD-F method (Ammonium 7 - fluorobenzo-2-oxa-1, 3 - diazole - 4 - sulphonate)
Chemicals
L-homocysteine, tri-n-butyl phosphine, dimethyl formamide, boric acid, EDTA (Sigma); ammonium 7-fluorobenzo-2-oxa-1, 3-diazole -4-sulphonate (SBD-F) (Fluka chemie); tri-chloro acetic acid, sodium hydroxide, KH2PO4 and acetonitrile (Qualigens).
Instrumentation
The Waters- HPLC system containing solvent delivery system 515 pump, Autosampler717, fluorescent detector 474 was used for analysis with excitation and emission wavelengths 385 nm and 515 nm respectively. Millennium software was used for acquiring the data.
Sample Collection
Blood Samples were collected from heel prick in micro-EDTA Tube. Plasma was separated immediately after centrifugation of the blood at 3000 RPM for 15 min at 4 °C.
Derivatisation
To 5 ml of plasma 20 ml of borate buffer (125 mM boric acid, 4 mM EDTA) was added to make the final volume to 25 ml. 10% (V/V) of tri-n-butyl phosphine in dimethyl formamide was added to the sample and vortexed. The resulting mixture was incubated at 4 °C for 30 min. Deproteinisation was done with 25 ml 10% (W/V) TCA, followed by vortexing and centrifugation at 3000 RPM for 15 min at 4°C. To 20 ml of clear supernatant taken in a fresh tube, 4 ml of (1.55N) NAOH was added and vortexed. This tube was kept at 4 °C for 10 min and 50 ml of borate buffer was added and vortexed. Finally 20 ml of SBDF (ammonium 7-flurobenzo-2-oxa-1, 3-diazole-4-sulphonate 1mg/ml in 125mM boric acid) was added and incubated at 60°C for one hour. After incubation sample was kept at ambient temperature for immediate processing. Standards and controls were prepared simultaneously in similar manner.
Chromatography and Conditions of HPLC
The derivatised sample was injected into a column equilibrated with mobile phase at a flow rate of 1.5 ml per min. The column equilibration time was 15 minutes at ambient temperature. The retention time of each sample was calculated by using standards and calibrators and a calibration curve was plotted for different concentrations.
Stationary phase: Octa decyl silane (ODS) 250X4.6 mm column, Mobile phase: 4% acetonitrile in potassium di-hydrogen orthophosphate (0.2 M, pH: 2.1), Detector: fluorescent with Ex/Em being 385 nm/515 nm
Recovery and precision
In plasma samples, known concentrations of homocysteine were added. The concentrations in samples with added calibrators were determined in replicates and analytical recoveries were calculated. The intra-assay precision was obtained by analysing replicates of sample in a day. Mean recoveries of homocysteine were 95 - 107%. Bland and Altman statistical analysis was carried out with the original method and the modified method and found to be in good correlation.
Optimisation of derivatization procedure
We have slightly modified the SBD-F method[14] to adapt it for small quantity of samples. The major alterations for the existing procedure are addition of borate buffer to less volume of sample and still maintaining the sensitivity by increasing injection volume. However neither the sample dilution nor the increased injection volume altered the sensitivity, specificity, accuracy and precision of chromatographic analysis. The incubation temperature if deviated from 4°C may adversely affect the analysis.
Statistical Analysis
Plasma variables were analyzed using raw data and also logarithmically transformed data as there is skewed distribution. The variables are presented as geometric mean and 95% confidence intervals (CIs). Online t-test was performed for one sample using Graph Pad Quick Calcs software. The mean, SEM and P values were obtained. To assess any possible gender-bias we performed a t-test for two-sets of samples (males and females). A P value of <0.05 was considered statistically significant.
Results
Two modifications were made in the SBD-F based HPLC method i.e. diluting the plasma with borate buffer and increasing the injection volume. Before adopting this method in place of original method, Bland and Altman analysis[15] was carried out to assess agreement between two methods of homocysteine measurement.
Bland and Altman analysis
1. Plotting Data
Plasma homocysteine values estimated by original method and modified method were plotted on X and Y axes respectively Figure1. The correlation coefficient (r) between the two methods was 0.994 concluding that homocysteine measurements by original and modified methods are related.
2. Measuring Agreement
Average homocysteine by two methods (X) was plotted against the difference in homocysteine (Y) in Figure2. The mean difference was found to be 0.183. The limits of agreement between these methods were -5.943 and 6.309.
3. Precision of estimated limits of agreement
The precisions of estimated limits of agreement were calculated. 95% CI for the lower limit of was found to be - 9.30 to - 2.58, whereas for the upper limit of agreement 2.94 to 9.68
4. Relation between difference and mean
Logarithmic transformation of the average homocysteine by both methods and difference in homocysteine between the original and modified was carried out and Log-Log graph was plotted Figure3. Limits of agreement for logarithmic data were -0.032 and 0.13 and antilog for it being 0.92 to 1.34.
5. Repeatability
Applying modified method, two consecutive analyses on a sample set were carried out and average homocysteine was plotted against the difference in homocysteine Figure4. The limits of agreement between two consecutive analyses were -4.81 and 2.55.
Linearity
The modified method was found to be linear Figure5 for concentrations ranging from 5 to 200 mmol/L (r=0.999).
Analysis of newborns table1
The mean plasma homocysteine concentration in newborns was 6.99±0.26. When data was segregated according to gender, the plasma homocysteine levels in male and female newborns were found to be 6.91±0.3 and 7.08±0.43 respectively (P=0.74). Increased homocysteine (>15 mmol/L) was observed in 31 males and 21 females out of the 331 males and 276 females tested respectively. The prevalence in males and females when compared with each other, there was no statistically significant gender-specific risk was observed (P=0.56). The overall incidence of hyperhomocysteinemia is 52 per 607 (8.5%) newborns. When hyperhomocysteinemia was segregated into mild (15-30 mmol/L), moderate (30-100 mmol/L) and severe(>100 mmol/L) table2, the most prevalent category was the milder one followed by moderate hyperhomocysteinemia. There were no newborns with severe hyperhomocysteinemia.
Discussion
Original method of homocysteine estimation by HPLC was modified to enable analysis on heel prick samples of newborns. Bland and Altman analysis showed good agreement between original and modified methods. The limits of agreement (-5.943 and 6.309) were small enough to use this technique for lesser volume of samples. This method has good repeatability and linearity (r=0.999).
The mean plasma homocysteine estimated by this method was 6.99mmol/L (95% CI: 6.48-7.49) [males and females being 6.907 (95% CI: 6.31-7.51) and 7.08 (95% CI: 6.23-7.93) respectively]. Gender differences were not observed in the mean plasma homocysteine concentration among newborns (P=0.74). Gender differences becoming prominent after puberty probably indicate the role of reproductive hormones[16] and body mass index (BMI).[17]
Mild to moderate hyperhomocysteinemia in newborns (52 per 607) studied indicate a high prevalence of abnormal homocysteine metabolism among the Indian newborns. Earlier studies on the mutation analysis of dried blood spots of the newborns collected in the newborn screening program showed 9% 677T allele frequency of Methylene tetrahydrofolate reductase (MTHFR) gene. In 80 male newborns, 8 were heterozygous and out of 108 female newborns, 22 were heterozygous and 2 were homozygous for C677T mutation.[18] With these frequency and a background of B12 and folate deficiency among the Indian mothers, a high total homocysteine in the newborns could be possible as has been observed in the present study. However, correlation with the cobalamin and folate status at the same time is required to confirm the finding.
The median homocysteine in UK newborns was reported to be 6.8 (4.2-12.8 mmol/L) with 2.54% newborns having homocysteine >15mmol/L.[19] The mean homocysteine in Japanese[20] and Italian newborn[21] were found to be 2.5 mmol/L and 2.92 mmol/L (Dried blood spot method) respectively. Another study on Italian newborns using capillary blood obtained by heel puncture showed a mean plasma homocysteine concentration of 4.9mmol/L.[22] The mean homocysteine in Indian newborns is higher than Japanese and Italian newborns. There is higher prevalence of hyperhomocysteinemia in Indian newborns in comparison to UK newborns (OR: 3.92, P=0.0001).
The significance of elevated homocysteine has been well studied in causing multi-disease manifestation at all ages. More so in the newborn period where studies indicate a direct correlation between cobalamin status and homocysteine levels and such newborns continue to have further increase in the concentration when they are exclusively breast-fed by mothers who themselves are deficient in B12. The clinical significance of elevated homocysteine becomes significant in later infancy and childhood in terms of mild to moderate mental retardation, cognitive failure, seizures, cerebro-vascular accidents etc.
Elevation in homocysteine could be due to mutations in genes regulating the crucial biochemical reactions in methionine metabolism or due to deficiencies of cofactors such as B6, B12 and folic acid. The consequences of this elevation are: low methionine levels, less production of S-adenosyl methionine (SAM), and high S-adenosyl homocysteine (SAH). SAM and SAH have antagonistic functions. The former acts as a universal methyl donor for transmethylases and the later acts as competitive inhibitor for transmethylases. High SAH/SAM ratio results in decreased cellular methylation. The functional consequences of decreased cellular methylation are significant and include central nervous system demyelination,[23] reduced neurotransmitter synthesis,[24] decreased chemotaxis and macrophage phagocytosis,[25] altered membrane phospholipid composition and membrane fluidity,[26] altered gene expression[27] and cell differentiation.[28], [29]
High prevalence of hyperhomocysteinemia in the newborn period, availability of reliable testing method, simple and less expensive treatment, justify the newborn screening for homocysteine in developing countries.
Follow-up of newborns with hyperhomocysteinemia showed a good biochemical and clinical improvement with supplementation of cofactors B12 and folate. One of the newborn who did not respond to recall for re-testing, developed acute infantile hemiplegia at the age of 1 yr. Although we did not find any case that is suggestive of Cystathionine Beta Synthase (CBS) deficiency, most of the cases studied were probably due to acquired defects in the remethylation as was evident by the response of the screen positive babies to cofactor supplementation.
In conclusion, our data along with other published data suggest homocysteine measurement in newborn period in order to reduce the mortality and morbidity associated with it. Although routine homocysteine measurement for the newborns is not recommended worldwide but total homocysteine is a better indicator rather than methionine for different types of hyperhomocysteinemia. Further work is required to prove the importance of screening in developing countries following vegetarian diet and high incidence of malnutrition. The need for routine cofactor supplementation in the first one year of life need to be assessed through large scale screening of newborns.
Acknowledgements
We thank all the newborns and their parents/guardians, who participated and cooperated in this study. We thank Dr. Reddy's foundation for Human and Social Development for funding Newborn screening program. We thank Department of Biotechnology, Government of India for the core grant.
References
1. Boers GH, Smals AG, Trijbels FJ, Fowler B, Bakkeren JA, Schoonderwaldt HC, Kleijer WJ, Kloppenborg PW. Heterozygosity for homocystinuria in premature peripheral and cerebral occlusive arterial disease. N Engl J Med 1985 Sep 19; 313(12) : 709-715.
2. Majors A, Ehrhart LA, Pezacka EH. Homocysteine as a risk factor for vascular disease. Enhanced collagen production and accumulation by smooth muscle cells. Arterioscler Thromb Vasc Biol. 1997 Oct; 17(10):2074-81.
3. Mueller T, Furtmueller B, Aigelsdorfer J, Luft C, Poelz W, Haltmayer M.Total serum homocysteine-a predictor of extracranial carotid artery stenosis in male patients with symptomatic peripheral arterial disease. Vasc Med. 2001; 6(3):163-7.
4. McCully KS. Vascular pathology of homocysteinemia: implications for the pathogenesis of arteriosclerosis. Am J Pathol. 1969 Jul; 56(1):111-28.
5. Wouters MG, Boers GH, Blom HJ, Trijbels FJ, Thomas CM, Borm GF, Steegers-Theunissen RP, Eskes TK. Hyperhomocysteinemia: a risk factor in women with unexplained recurrent early pregnancy loss. Fertil Steril. 1993 Nov; 60(5):820-5.
6. Van der Molen EF, Verbruggen B, Novakova I, Eskes TK, Monnens LA, Blom HJ. Hyperhomocysteinemia and other thrombotic risk factors in women with placental vasculopathy. BJOG. 2000 Jun; 107(6):785-91.
7. Steegers-Theunissen RP, Boers GH, Trijbels FJ, Eskes TK. Neural-tube defects and derangement of homocysteine metabolism. N Engl J Med. 1991 Jan 17; 324(3):199-200.
8. Gussekloo J, Heijmans BT, Slagboom PE, Lagaay AM, Knook DL, Westendorp RG. Thermolabile methylenetetrahydrofolate reductase gene and the risk of cognitive impairment in those over 85. J Neurol Neurosurg Psychiatry. 1999 Oct; 67(4): 535-8.
9. McCaddon A, Davies G, Hudson P, Tandy S, Cattell H. Total serum homocysteine in senile dementia of Alzheimer type. Int J Geriatr Psychiatry. 1998 Apr; 13(4): 235-9.
10. Maike Wolters, Silke Hermann, and Andreas Hahn. B vitamin status and concentrations of homocysteine and methylmalonic acid in elderly German women. Am J Clin Nutr. 2003; 78:765-72.
11. Neela J, Raman L. The relationship between maternal nutritional status and spontaneous abortion. Natl Med J India 1997 Jan-Feb; 10(1) : 15-16.
12. Helga Refsum, Chittaranjan S Yajnik, Milind Gadkari, J φrn Schneede, Stein E Vollset, Lars rning, Anne B Guttormsen, Anjali Joglekar, Mehmood G Sayyad, Arve Ulvik and Per M Ueland. Hyperhomocysteinemia and elevated methylmalonic acid indicate a high prevalence of cobalamin deficiency in Asian Indians. American Journal of Clinical Nutrition, August 2001; 74(2) : 233-241.
13. Radha Rama Devi A, Naushad S.M. Newborn Screening in India. Indian Journal of Pediatrics Vol 71-Feb 2004; (2) : 157-160.
14. Ubbink JB, Hayward Vermaak WJ, Bissbort S. Rapid high-performance liquid chromatographic assay for total homocysteine levels in human serum. J Chromatogr 1991 Apr 19; 565(1-2) : 441-6.
15. Bland J M and Altman D G. Statistical methods for assessing agreement between two methods of clinical measurements. Lancet 1986; I : 307-310.
16. Selhub J, Jacques PF, Wilson PWF et al. Vitamin status and intake as primary determinants of homocysteinemia in an elderly population. JAMA 1993; 270: 2693-2698.
17. Mudd SH and Poole JR. Labile methyl balances for normal humans on various dietary regimens. Metabolism 1975; 24: 721-735.
18. RadhaRamaDevi A, Govindaiah V, Ramakrishna G and Naushad SM. Prevalence of Methylene tetra hydro Folate reductase polymorphism in South Indian population. CurrSci 2003; 86 (3): 101-104.
19. Refsum H, Grindflek AW, Ueland PM, Fredriksen A, Meyer K, Ulvik A, Guttormsen AB, Iversen OE, Schneede J, Kase BF. Screening for serum total homocysteine in newborn children. Clin Chem 2004 Oct; 50(10) : 1769-1784.
20. Febriani AD, Sakamoto A, Ono H, Sakura N, Ueda K, Yoshii C, Kubota M, Yanagawa J. Determination of total homocysteine in dried blood spots using high performance liquid chromatography for homocystinuria newborn screening. Pediatr Int 2004 Feb; 46(1) : 5-9.
21. Accinni R, Campolo J, Parolini M, De Maria R, Caruso R, Maiorana A, Galluzzo C, Bartesaghi S, Melotti D, Parodi O. Newborn screening of homocystinuria: quantitative analysis of total homocyst(e)ine on dried blood spot by liquid chromatography with fluorimetric detection. J Chromatogr B Analyt Technol Biomed Life Sci 2003 Mar 5; 785(2): 219-26.
22. Bartesaghi S, Accinni R, De Leo G, Cursano CF, Campolo J, Galluzzo C, Vegezzi PG, Parodi O. A new HPLC micromethod to measure total plasma homocysteine in newborn. Pharm Biomed Anal 2001 Mar; 24(5-6): 1137-1141.
23. Scott JM, Molloy AM, Kennedy DG, Kennedy S, Weir DG. Effects of the disruption of transmethylation in the central nervous system: an animal model. Acta Neurol Scand Suppl 1994; 154 : 27-31.
24. Schatz RA, Wilens TE, Sellinger OZ Decreased transmethylation of biogenic amines after in vivo elevation of brain S-adenosyl-l-homocysteine. J Neurochem 1981; 36(5): 1739-1748.
25. Garcia-Castro I, Mato JM, Vasanthakumar G, Wiesmann WP, Schiffmann E. and Chiang PK. Paradoxical effects of adenosine on neutrophil chemotaxis. J Biol Chem 1983; 258: 4345-4349.
26. Chiang PK. Biological effects of inhibitors of S-adenosylhomocysteine hydrolase. Pharmacol Ther 1998; 77(2): 115-134.
27. Dizik M, Christman JK and Wainfan E. Alterations in expression and methylation of specific genes in livers of rats fed a cancer promoting methyl-deficient diet. Carcinogenesis 1991; 12(7): 1307-1312.
28. Chiang PK. Conversion of 3T3-L1 fibroblasts to fat cells by an inhibitor of methylation: effect of 3-deazaadenosine. Science 1981; 211(4487): 1164-1166.
29. Aarbakke J, Miura GA, Prytz PS, Bessesen A, Slordal L, Gordon RK and Chiang PK. Induction of HL-60 cell differentiation by 3-deaza- (+/-)-aristeromycin, an inhibitor of S-adenosylhomocysteine hydrolase. Cancer Research 1996; 46(11): 5469-5472.(Rama Devi Radha A, Nausha)