| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Technical Briefs |
ebeková1,a
oviová-Kudláková1
íek2
1 Institute of Preventive and Clinical Medicine,
2
Hospital of Ministry of Defense of Slovak Republic, and
3
St. Cyril and Method Hospital, 833 01 Bratislava, Slovakia
4 University of Wuerzburg, 97070 Wuerzburg, Germany
aaddress correspondence to this author at: Institute of Preventive and Clinical Medicine, Limbová 14, 833 01 Bratislava, Slovakia; fax 421-2-59369-170, e-mail sebekova{at}upkm.sk
Vegetarians are at risk of developing hyperhomocysteinemia (HHcy). The predominant or selective consumption of proteins of plant origin shifts homocysteine (Hcy) metabolism to the remethylation pathway (1), which requires vitamin B12 as a cofactor and methyltetrahydrofolate as a substrate. In the vegetarian diet, the intake of folic acid exceeds the recommended dietary allowance (RDA), whereas intake of vitamin B12 is inadequate or even absent (2).
HHcy represents an independent risk factor for cardiovascular disease (3). Autooxidation of Hcy produces reactive oxygen species (ROS) (4), which may stimulate lipid peroxidation and formation of advanced oxidation protein products (AOPPs) and advanced glycation end products (AGEs). Interaction of AGEs with their specific receptor, RAGE, induces formation of ROS (5). In mice, HHcy was shown to enhance the expression of RAGE (6). AGEs, AOPPs, and lipid peroxidation products are implicated in the pathogenesis of degenerative and inflammatory diseases, including atherosclerosis (7).
In vegetarians, plasma concentrations of AGEs are mildly but significantly increased compared with populations on a Western mixed diet (8). We therefore investigated (a) whether there is an association between Hcy and plasma AGE concentrations or markers of protein oxidation and lipid peroxidation, and (b) whether supplementation of vitamin B12 affects the mentioned analytes in vegetarians with HHcy produced by a potential vitamin B12 deficit.
The study was approved by the Institutional Ethics Board and was conducted according to the Declaration of Helsinki. All participants gave written consent to participate.
We investigated 63 healthy vegetarians in whom HHcy had been revealed previously. The normohomocysteinemic (NHcy; Hcy <12.0 µmol/L) subgroup (with plasma folate and vitamin B12 concentrations within the appropriate reference intervals) was compared with the subgroup with functional HHcy (Hcy >12.0 µmol/L) attributable to vitamin B12 deficiency (plasma B12 <220 pmol/L) who had plasma folate and iron concentrations and blood values within the appropriate reference intervals. This subgroup was then administered intramuscular doses of vitamin B12 (Léiva), with an initial dose of 1000 µg and four 300-µg doses over the next 14 days.
Hcy (9), vitamin B12, and folate concentrations (Elecsys 2010 System; Boehringer); AGE-associated fluorescence (
350/450 nm) (10); carboxymethyllysine (CML; competitive ELISA; Roche Diagnostics) (11)(12); AOPPs (13); and lipid conjugated dienes (CDs) (14) were measured in all participants before and in the treated group 4 weeks after the last intervention. Plasma concentrations of ß-carotene (15); vitamins A (15), C(16), and E(15); thyrotropin (IRMA); triiodothyronine (RIA); and thyroxine (RIA; all assays from Immunotech) were determined. Routine chemistries were performed on a COBAS Integra 700 analyzer (Roche), and blood profiles were determined on a Sysmex KX-21 analyzer.
The participants’ nutritional regimens were evaluated by use of dietary interviews and food frequency questionnaires. The intake of vitamin B12 was determined with use of the Alimenta database (Food Research Institute).
The results of the above analyses are presented as the mean (SE) in Table 1
. For statistical evaluation, we used unpaired and paired Wilcoxon tests. Regression analysis was performed. P <0.05 was considered significant.
|
For the whole group, plasma Hcy (by 15%) was slightly increased, whereas plasma vitamin B12, folate, and iron concentrations and the blood profiles (data not given) were within the appropriate reference intervals. Mean (SE) daily intake of vitamin B12 was 2.41 (0.11) µg (RDA = 2.0 µg/day). All participants had normal kidney and thyroid gland function (data not given). Plasma albumin and vitamin concentrations indicated a balanced diet. Stepwise multiple regression analysis revealed that plasma vitamin C, vitamin B12, and iron concentrations correlated significantly with Hcy (ANOVA, F = 12.12; P <0.0001).
NHcy vegetarians had significantly higher vitamin B12 concentrations than the HHcy group because the latter were vitamin B12-deficient. Other investigated variables did not differ significantly. Stepwise multiple regression analysis suggested that CML, vitamin B12, and folate concentrations (inverse relationship) correlated significantly with Hcy (ANOVA, F = 7.399; P <0.007).
Hcy concentrations were >12.0 µmol/L (HHcy) in 27 of the vegetarians. HHcy was not associated with a vitamin B12 deficiency in 6 of these individuals, whereas in the remaining 21, the daily intake of vitamin B12 was 1.48 (0.09) µg. Seven individuals were not evaluated in the clinical study (refusal of participation or exclusion because of concomitant anemia and iron deficiency). Plasma albumin and vitamin concentrations and body mass index were within the appropriate reference intervals, thus allowing the exclusion of protein energy malnutrition as a source of HHcy.
In vegetarians with HHcy attributable to vitamin B12 deficiency, HHcy was not associated with increases in plasma AGE, AOPP, or CD concentrations. However, the pretreatment Hcy concentrations did correlate with AOPP and CD concentrations (r = 0.598; P <0.03 and r = 0.575; P <0.03, respectively).
In individuals with a vitamin B12 deficiency, plasma vitamin B12 concentrations normalized and Hcy concentrations decreased to within reference values by 4 weeks after initiation of vitamin B12 treatment. The higher the pretreatment Hcy value, the more profound the observed decrease (r = 0.872; P <0.001). None of the other investigated variables was affected significantly. Posttreatment Hcy concentrations and changes in Hcy during treatment did not correlate with any of the investigated data.
In a general population on a Western mixed diet, folate deficiency represents the main risk factor for development of HHcy. Its supplementation produces only a mild decrease in Hcy (17). In long-term vegetarians, vitamin B12 deficiency plays a key role in the pathogenesis of HHcy because it is lacking in the vegan diet (2). In vegans, the only sources of vitamin B12 are bacteria in the lower intestinal tract. Intake of food of animal origin (milk, dairy products, and eggs) contributes to vitamin B12 concentration in lacto-ovo-vegetarians. In HHcy vegetarians, the estimated intake of vitamin B12 (74% of RDA) was insufficient to maintain a balance of this vitamin.
The high plasma folate observed in vegetarians might be attributable to higher folate intake. Moreover, the "methyl folate trap" might be a contributing factor. In vitamin B12 deficiency, 5-methyltetrahydrofolate and folic acid may accumulate (18). Cobalamin deficiency renders folate largely biologically ineffective, although its plasma concentrations and distribution appear sufficient. In the present study, plasma folate concentrations did not differ between the NHcy and pre- and posttreatment HHcy individuals. Treatment with vitamin B12 decreased Hcy by 41.8%, much more than might be expected in mild HHcy. Thus, the methyl folate trap explains the rapid and substantial correction of HHcy after vitamin B12 supplementation in vegetarians with vitamin B12 deficiency.
In NHcy vegetarians with sufficient plasma vitamin B12 and folate, CML appeared to correlate with Hcy concentrations. This relationship is of particular interest because this chemically defined AGE results not only from the classic pathway of AGE formation via Amadori products but also from autooxidation of glucose and from lipid peroxidation (5). The direct relationship between CML and Hcy could be of interest with regard to the mild increase in circulating AGE in healthy vegetarians (8). However, the other markers associated with oxidative stress (fluorescent AGEs, AOPPs, and CDs) showed no correlation with Hcy concentrations. There are limited data available on the association of Hcy and oxidative stress in the NHcy population. Powers et al. (19) found a correlation between plasma Hcy and malondialdehyde in young men, but because a methionine-load test produced a significant increase in Hcy but not malondialdehyde, they considered it unlikely that the oxidative stress could be a direct effect of Hcy.
In contrast to NHcy vegetarians, in the subgroup with functional HHcy, Hcy correlated directly with AOPPs and CDs. These data agree with the findings of Voutilainen et al. (20), who reported an association between Hcy and F2-isoprostanes in men with HHcy. In spite of its long duration, HHcy in our study group was not associated with increased AOPP or CD concentrations. It is conceivable that the high plasma concentrations of antioxidants in vegetarians may partially abrogate the potentially enhanced formation of ROS induced by HHcy and AGEs.
Even the short-term intervention with vitamin B12 effectively normalized Hcy concentrations in vegetarians with functional HHcy. This action was not associated with a change in plasma AGE, AOPP, or CD concentrations, indicating that high Hcy concentrations in vegetarians do not support the concept of a causal link to markers of oxidative stress. It should be emphasized that long-term maintenance of normohomocysteinemia in long-term vegetarians may be achieved only by their adherence to a recommended consumption of food of animal origin or by supplementation with vitamin B12 preparations sufficient to saturate the body. Otherwise, vitamin B12 concentrations decrease and hyperhomocysteinemia is reestablished within the next 6 months (21).
It must be considered that the HHcy state is a reflection of metabolic events within a cell. Thus, our in vivo investigation carries limitations in that measurements in plasma may not accurately reflect intracellular oxidative modifications.
In summary, this study provides the first data indicating that, in vegetarians with functional HHcy attributable to vitamin B12 deficiency, increases in Hcy may be paralleled by increases in markers of lipid peroxidation and oxidation of proteins, without their overt increase. Additional studies are needed to elucidate the relationship between Hcy and AGEs, AOPPs, and lipid oxidation products in a population on a standard Western mixed diet and with various disease states.
Acknowledgments
We wish to acknowledge support from Léiva SK (Bratislava, Slovakia) and the Verein zur Bekämpfung der Hochdruck-und Nierenkrankheiten (Wuerzburg, Germany) as well as the excellent assistance of André Klassen in preparing the manuscript. A portion of this study was presented as a poster at the 2002 AACC Annual Meeting (Orlando, FL).
References
es a Slov Farm 1994;43:166-168.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
