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Clinical Biochemistry 41 (2008) 914 – 916
Post-methionine load test: A more sensitive tool to reveal hyperhomocysteinemia in Alzheimer patients? Gloria Galimberti a,b,⁎, Elisa Conti a,b , Michela Zini a,c , Fabrizio Piazza a , Francesco Fenaroli d , Valeria Isella a,c , Maurizio Facheris a,c , Vittoria Perlangeli e , Laura Antolini f , Francesco DeFilippi d , Carlo Ferrarese a,b,c a
Department of Neuroscience and Biomedical Technology, University of Milano-Bicocca, via Cadore 48, 20052 Monza (MI), Italy b Scientific Institute “E.Medea”, via don Luigi Monza 20, 23842 Bosisio Parini (LC), Italy c Department of Neurology, San Gerardo Hospital, via Donizetti 106, 20052 Monza (MI), Italy d Geriatric Division, San Gerardo Hospital, via Donizetti 106, 20052 Monza (MI), Italy e Laboratory of Analyses, San Gerardo Hospital, via Donizetti 106, 20052 Monza (MI), Italy f Department of Clinical Medicine and Prevention, University of Milano-Bicocca, via Cadore 48, 20052 Monza (MI), Italy Received 21 November 2007; received in revised form 14 March 2008; accepted 29 March 2008 Available online 11 April 2008
Abstract Objective: To identify the real number of hyperhomocysteinemic Alzheimer's patients who may benefit from homocysteine-lowering therapy. Methods: Basal and post-methionine load homocysteine levels were assessed by rp-HPLC system. Results: PML test revealed twice as many hyperhomocysteinemic AD subjects with respect to the fasting analysis. Conclusion: PML test resulted useful in detecting higher number of hyperhomocysteinemic AD patients who may have the chance of an early folate treatment. © 2008 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. Keywords: Homocysteine; Post-methionine load test; Folate lowering therapy; Alzheimer's disease
Introduction Homocysteine (hcy) is a sulphur-containing aminoacid involved in methionine cycle and in trans-sulphuration pathway. To keep low hcy levels, the re-methylation pathway requires vitamin B12 as a co-factor of methionine synthase, and folate as co-enzymes. Vitamin B6 as a co-factor for cystathionine β-syntase (CβS) and cystathionine γ-lyase is necessary for trans-sulphuration. Hyperhomocysteinemia is a condition characterized by an abnormally high level of plasma hcy commonly associated with genetic disorders, such as mutations in CβS or methylenetetrahydrofolatereductase (MTHFR) genes, and vitamin deficiencies leading to
⁎ Corresponding author. Department of Neuroscience and Biomedical Technologies, University of Milano-Bicocca, via Cadore 48, 20052 Monza (MI), Italy. Fax: +39 02 64488108. E-mail address:
[email protected] (G. Galimberti).
metabolic dysfunction in methionine cycle [1]. Elevated hcy plasma levels and low vitamin B serum content have been recognized as risk factors for vascular disorders [2] and, more recently, for dementia [3]. In particular, various studies focused on the association between hyperhomocysteinemia and Alzheimer's disease (AD), where vascular damage might contribute to disease onset and progression [4,5]. Reductions in number of cerebral microvessels, aberrant capillary features and morphometric dysfunction of blood brain barrier have already been demonstrated in AD patients [6]. Hcy plasma levels are usually measured following an overnight fasting, but studies demonstrated that post-methionine load test (PML) is more efficacious in evidencing hyperhomocysteinemia in both vascular disease patients and controls [7]. Homocysteine measurement after overnight fasting may lead to false negative results. For these reasons, we aimed to identify the true number of hyperhomocysteinemic AD subjects comparing PML measurement with traditional techniques. In fact, correct hyperhomocysteinemia detection may lead to an early
0009-9120/$ - see front matter © 2008 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2008.03.015
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specific vitamin treatment, which may eventually limit Hcycorrelated damages. Methods After informed consent, approved by local ethical committee, 29 AD patients and 23 healthy controls were recruited at San Gerardo Hospital, Monza (Italy). The mean age and standard deviation in years were 78.45 ± 4.63 in AD and 70.13 ± 3.01 in controls. Diagnosis of AD was made according to NINCDS-ADRDA criteria [8]. All patients underwent brain CT or NMR imaging and a complete neuropsychological test battery. Blood samples were collected after overnight fasting for basal evaluations and after 4 h methionine administration for post-load total hcy analysis [9,10]. Methionine amount was calculated as follows: methionine (gr) = 3.8 × m2 body surface area (m2 = 10Log10S and Log10S = [0.45 × Log10 weight (kg)]+ [0.75 × Log10 height (cm)] − 2.144). Total hcy levels were measured in blood samples (K3EDTA) immediately placed on ice and centrifuged (1100 ×g) within 1 h from collection. Plasma hcy was analyzed by isocratic rp-HPLC system (BioRad, Richmond, CA, USA), using a commercial kit (Bio-Rad, Richmond, CA, USA). Folate was assessed in serum of fasting subjects after blood centrifugation (2800 ×g) by chemiluminescent competitive test (Roche Diagnostics, Indianapolis, IN, USA) using the Immuno-analyzer Elecsys 2010. Fasting hcy b 15 µM, PML hcy b 30 µM, and folate 2–9.1 ng/mL were considered as cut off values for normality, established by the Laboratory of Analyses (S. Gerardo Hospital, Monza) based on the company's instructions. All results are expressed as median and interquartile range. Mann Whitney test was used for means comparison, whereas McNemar test was used to verify if the PML assay could discriminate hyperhomocysteinemic subjects not detected by the fasting test. The strength of cor-
Fig. 2. Folate plasma content evaluated in CTRL and AD patients. Minimum, lower quartile (25%), median, upper quartile (75%) and maximum are shown. Horizontal lines represent upper (9.1 ng/mL) and lower (2 ng/mL) limits of normality range values.
relation between variables was expressed as Pearson's correlation coefficient (r). Results The test after overnight fasting identified eight hyperhomocysteinemic subjects among AD cases (28%) and three among controls (13%). Conversely, PML test revealed sixteen hyperhomocysteinemic individuals among AD cases (56%) and four within healthy controls (17%). The usefulness of PML test in our study was further corroborated by the McNemar test (p = 0.013). The probability to find a false negative by the fasting test (i.e. undetected hyperhomocysteinemic subject) was 0.44 (95% confidence interval: 0.21, 0.67). Most of the AD subjects showed PML hcy levels above the normality range (Fig. 1). Hcy plasma levels, both fasting and after methionine load, were higher in AD patients (14.60 µM ± 5.90, 34.91 µM ± 10.84) compared to controls (11.53 µM ± 3.90, 26.84 µM ± 5.80). However values appeared statistically different between patients and controls for the PML analysis only (p = 0.0052). No influence of age and sex on hcy plasma levels was found in patients and controls. Folate plasma levels appeared lower (p b 0.0001) in AD subjects (3.81 ng/mL ± 1.24) than in controls (8.75 ng/mL ± 2.72), yet both groups showed values within normality (Fig. 2). An inverse correlation (r = 0.40, p = 0.0469 ) between folate and Hcy levels was found in AD patients only (data not shown). Discussion
Fig. 1. Hcy plasma content evaluated after overnight fasting and postmethionine load (PML) in controls (CTRL) and Alzheimer's disease (AD) patients. Minimum, lower quartile (25%), median, upper quartile (75%) and maximum are shown. Horizontal lines represent upper limits of normality range values for fasting (15 µM) and PML test (30 µM).
Hyperhomocysteinemia is now considered as a key risk factor for several pathologies, but most of the studies in patients population seem to perform their hcy plasma level measurements using the fasting test only [11]. However, the PML test seems to better identify subjects with hyperhomocysteinemia otherwise assessed as normal by the fasting test [7]. Our results confirm this last evidence, since we found twice as many hyperhomocysteinemic subjects among the AD group using this test with respect to the fasting one.
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In our study, we also showed higher hcy levels in AD patients compared to controls. These data are in good keeping with other studies that recently proposed hyperhomocysteinemia among the risk factors for dementia in AD [4]. Higher hcy levels, both at fasting and PML, may reflect a compromised genetic and biological picture, which might be present only in subjects suffering from AD. A correct detection of hyperhomocysteinemia could lead to a rapid therapeutic intervention aimed to lower hcy plasma content. If it is true for the elderly, even more so for subjects with AD, where a vascular damage may in turn worsen an already compromised clinical picture. Among commonly used agents, folate and vitamin B12 seem to be the most efficacious [12]. The finding of decreased folate levels in AD patients and its correlation with hyperhomocysteinemia further supports the possible positive outcome for vitamin supplementation in subjects showing elevated hcy levels. Even with the practical burden and the costs that limit its worldwide application [7], we recommend the use of PML test to identify and eventually treat hyperhomocysteinemic AD subjects. Despite the mild side effects of PML test, to reveal a hyperhomocysteinemic status in AD subjects would allow an early folate administration as a treatment for at least one pathogenetic aspect of a multifactorial disease, which still lacks of an efficacious therapy. Acknowledgments This work was supported by grant 2002067518 (MIURCOFIN 2002–2004) from the Italian Ministry of Education, University and Research and by grants PS/03/4, PS/03/9 (Ricerche finalizzate 2003–2005) from the Italian Ministry of Health. We thank the associations Alzheimer Monza and Brianza and AVIS Monza for the AD and healthy subject recruitment.
References [1] Pietrzik K, Bronstrup A. Causes and consequences of hyperhomocysteinemia. Int J Vitam Nutr Res 1997;67:389–95. [2] Castro R, Rivera I, Blom HJ, Jakobs C, Taveres de Almeida I. Homocysteine metabolism, hyperhomocysteinemia and vascular disease: an overview. J Inherit Metab Dis 2006;29(1):3–20. [3] Haan MN, Miller JW, Aiello AE, et al. Homocysteine, B vitamins, and the incidence of dementia and cognitive impairment: results from the Sacramento Area Latino Study On Aging. Am J Clin Nutr 2007;85:511–7. [4] Ravaglia G, Forti P, Maioli M, et al. Homocysteine and folate as risk factors for dementia and Alzheimer disease. Am J Clin Nutr 2005;82: 636–43. [5] Seshadri S. Elevated plasma homocysteine levels: risk factor or risk marker for the development of dementia and Alzheimer's disease? J Alzh Dis 2006;9:393–98. [6] Zhu X, Smith MA, Honda K, et al. Vascular oxidative stress in Alzheimer disease. J Neurol Sci 2007;257:240–6. [7] van der Griend R, Biesma DH, Banga JD. Post-methionine load homocysteine determination for the diagnosis hyperhomocysteinemia and efficacy of homocysteine lowering treatment regimens. Vasc Med 2002;7: 29–33. [8] McKhann G, Drachman D, Folstein M, Katzman R, Price D, Stadlan EM. Clinical diagnosis of Alzheimer disease: report of the NINCDS-ADRDA Work Group under the auspices of Department of Health and Human Services Task Force on Alzheimer's Disease. Neurology 1984;34(7): 939–44. [9] Rasmussen K, Moller J. Total homocysteine measurement in clinical practice. Ann Clin Biochem 2000;37:627–48. [10] de Jonge R, Griffioen PH, van Zelst B, Brouns RM, Visser W, Lindemans J. Evaluation of a shorter methionine loading test. Clin Chem Lab Med 2004;42(9):1027–31. [11] Molero AE, Altimari CC, Duran DA, Garcia E, Pino-Ramirez G, Maestre GE. Total plasma homocysteine values among elderly subjects: findings from the Maracaibo Aging Study. Clin Biochem 2006;39(10): 1007–15. [12] Homocysteine Lowering Trialists' Collaboration. Dose-dependent effects of folic acid on blood concentrations of homocysteine: a meta-analysis of the randomized trials. Am J Clin Nutr 2005;82:806–12.
