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Increased Plasma Homocyst(e)ine after Withdrawal of Ready-to-Eat Breakfast Cereal from the Diet: Prevention by Breakfast Cereal Providing 200 µg Folic Acid

Department of Pathobiology, Oregon Regional Primate Research Center, Beaverton (M.R.M., B.M.U., E.E.G.), Portland, Oregon
Department of Medicine, Oregon Health Sciences University (M.R.M., P.B.D.), Portland, Oregon
Providence St. Vincent Hospital (A.I.-J.), Portland, Oregon

Address reprint requests to: M.R. Malinow, MD, Oregon Regional Primate Research Ctr, 505 NW 185^th Ave, Beaverton, OR 97006-3448. E-mail: malinowr{at}ohsu.edu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIAL AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
Objective: We tested the hypothesis that cessation of habitual ingestion of breakfast cereals would be associated with elevated plasma homocyst(e)ine concentrations. We anticipated that those subjects who reported consuming breakfast cereals containing 100 to 400 µg of folic acid per serving before entering the study would achieve higher plasma homocyst(e)ine concentrations if, in addition to their regular diet, they began ingesting a daily serving of breakfast cereal that contained less than 10 µg of folic acid per serving.

Design: Seventy-nine subjects consumed a daily serving of breakfast cereal containing either <10 µg or folic acid per serving (placebo) or breakfast cereal containing 200 µg of folic acid per serving (folic acid fortified).

Results: Cessation of intake of commercially available breakfast cereal was associated with homocyst(e)ine elevation. Breakfast cereal containing 200 µg folic acid per day was sufficient to maintain the homocyst(e)ine lowering effects of commercial cereals.

Conclusions: Habitual consumption of commercially available fortified breakfast cereals, usually containing 100 to 400 µg folic acid per serving, had significant homocyst(e)ine-lowering effects as shown by the homocyst(e)ine increase after cessation of habitual intake of commercial breakfast cereal. Substitution of breakfast cereal containing only 200 µg folic acid per day was sufficient to maintain the homocyst(e)ine-lowering effects of commercial cereals.

Key words: homocysteine, folic acid, breakfast cereal, atherosclerosis

	INTRODUCTION

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Elevated plasma homocyst(e)ine* is an independent risk factor for coronary artery disease (CAD) [1], peripheral vascular disease [1], carotid artery intimal medial thickening [2], carotid artery stenosis [3], coronary artery stenosis [4] and stroke [1,5]. Rather than showing a threshold effect, numerous studies have shown a graded risk relationship across the range of homocyst(e)ine concentrations [6]. However, the results of on-going clinical trials are needed to determine whether lowering homocyst(e)ine concentrations will result in a decreased occurrence of complications of atherosclerotic diseases.

The results of previous studies have shown that plasma homocyst(e)ine concentrations are decreased by the intake of supplemental folic acid in mean amounts between 0.5 and 5 mg/d [7], by multivitamin tablets containing inter alia 0.4 mg of folic acid [8] or by intake of breakfast cereals containing as little as 200 µg [9] or 400 µg [10] of folic acid.

Habitual intake of fortified breakfast cereals is an important source of folic acid and other vitamins and, thus, is an important determinant of plasma homocyst(e)ine [11]. We hypothesized that withdrawal of habitual intake of breakfast cereals, which are currently fortified with folic acid, would increase concentrations of homocyst(e)ine. We also hypothesized that a daily serving of breakfast cereal containing 200 µg folic acid/serving would be sufficient to block the elevation of plasma homocyst(e)ine after cereal withdrawal.

	MATERIAL AND METHODS

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Healthy subjects and patients with stable coronary heart disease (CHD) were recruited from internists or family-practice physicians associated with Providence St. Vincent Hospital in Portland Oregon, by word of mouth advertising or from a cohort of patients discharged with the diagnosis of ischemic heart disease (ICD 9 codes 410–414).

Letters of invitation and informational flyers included information about the study that required participants to self-select themselves as study participants by meeting the following criteria: not currently taking multivitamins or vitamin supplements containing folic acid or vitamins B₆ or B₁₂, the ability to ingest wheat products, no history of stroke or recent hospital admission (within the previous three months) for treatment of a coronary condition, no diagnosis of significant (as determined by the primary physician) liver, thyroid or kidney diseases, no cancer (except non-melanoma cancer), gastric resection, alcohol or substance abuse or psychiatric illness, not currently taking medication(s), such as methotrexate, tamoxifen, anticonvulsants, nicotinic acid, theophylline, bile acid sequestrants (or nitrous oxide anesthesia within seven days prior to entering the study). Two groups of subjects were included: habitual consumers and non-consumers of breakfast cereals.

Additional criteria for entry and completion of the study included a screening plasma homocyst(e)ine concentration less than 30 µmol/L, serum creatinine concentration less than 1.7 mg/dL, no intake of multivitamins or vitamin supplements, no pregnancy during the study, good health throughout the study and no hospitalization for CAD. All patients signed an Informed Consent Form and completed a Medical History Form. Participants were instructed to ingest daily one serving of the provided study breakfast cereal throughout the 15-week study and were excluded if they did not consume at least five servings of cereal per week throughout the study.

Study participants who reported non-compliance or who were observed to be non-compliant while enrolled in the study (n=19) were withdrawn from the study and excluded from analysis. Final results of the study were determined from the analyses of data from 79 study participants who completed the study.

Participants were block randomized by age (65 years vs. >65 years) and gender into one of two study groups to eat a daily serving of one of two breakfast cereals containing either 10 µg folic acid per serving (group A) or 237±21 µg folic acid per serving (group B). Both blends of cereal contained 25% of the recommended daily allowance (RDA) per serving of vitamins B₂, B₆, B₁₂ and <10% of the RDA of vitamin C. Both blends were similar in taste, texture, color, shape and size. Participants were provided with a five-week supply, plus five extra servings of breakfast cereal at the beginning at each of three five-week study phases (weeks 0, 5, and 10) for a total of 40 serving packets dispensed to each subject. Participants were instructed to return all unused servings and not to eat any other breakfast cereal or additional B-vitamins, folic acid or multivitamins containing B vitamins throughout their 15-week participation in the study. Participants were allowed to eat their daily serving at any time during the day, mixed with milk, fruit, fruit juice, yogurt or plain. Participants were advised also to continue medical treatment prescribed by their physician(s) throughout the 15-week study.

Venous blood samples were obtained at screening and at the end of weeks 5, 10 and 15 of the study. All samples were drawn at the Outpatient Clinical Laboratory (Providence St. Vincent Hospital). Samples were drawn in EDTA-containing vacutainers, placed immediately on wet ice, centrifuged at 4°C within one hour and the plasma stored at -20°C for duplicate homocyst(e)ine analysis by high-pressure liquid chromatography and electrochemical detection [12,13]. Additional plasma aliquots were protected from light and frozen at -20°C for single radioassay of folate and Vitamin B₁₂ (Bio-Rad Quantaphase II, Bio-Rad Diagnostics). The inter-run coefficient of variation for replicate plasma samples for homocyst(e)ine was 6.0%, for single samples of plasma folate, 13.0% and for plasma vitamin B₁₂, 14.9%.

Study participants who missed a laboratory appointment by more than ten days or who reported missing daily cereal intake by more than a total of five consecutive days during a five-week period were deleted from the study.

Individual compliance with cereal ingestion was estimated at the end of the participant’s 15-week study by comparing the number of cereal servings left over with the total number of servings dispensed during the entire study.

The study protocol was approved by the Cardiology Section at Providence St. Vincent Hospital and the Providence Health System Institutional Review Board.

The distribution of study variables was examined using computer data analysis programs for independent subjects. Study variables were compared using chi-square tests for categorical variables and t and repeated measures ANOVA tests for continuous variables. p-values were 2-sided, and p<0.05 was considered statistically significant. Statistical analyses were conducted using SAS, Version 6.1 (SAS Institute, Cary, NC), and Graph Pad Prism (Version 2.0; San Diego, CA).

	RESULTS

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The characteristics of subjects at entry are shown in Table 1. 53% were male, 28% had CAD, and 63% reported habitual consumption of breakfast cereal (>5 days/week prior to entering the study). As demonstrated, the characteristics of subjects in groups A and B were comparable. Moreover, compliance with breakfast cereal consumption was similar in both groups (87.3%, group A (placebo); 87.7%, group B (folic acid fortified), respectively (not shown in the tables).

Since folic-acid-fortified breakfast cereal consumption has been shown to be a prominent source of dietary folate and of contribution to homocyst(e)ine lowering [11], the data were further analyzed by subgroups on the basis of reported cereal consumption prior to entering the study. Table 3 shows a statistically significant increase (23.6%) in plasma homocyst(e)ine in the subgroup of regular users of breakfast cereal among the placebo group (subgroup A1) (p, ANOVA test <0.0001), but a non-significant (7%) increase was observed in those eating the folic-acid-fortified breakfast cereal (subgroup B1) (p, ANOVA test=0.17). Plasma folate concentrations did not change significantly compared to baseline in subgroups A1 or B1 (p, ANOVA=0.38 and 0.48, respectively), but the folate concentrations in subgroup B1 subjects were significantly higher than in subgroup A1 subjects (placebo) at weeks 10 and 15.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIAL AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

In this study, we endeavored to test the hypothesis that cessation of habitual ingestion of fortified breakfast cereals would be associated with elevated plasma homocyst(e)ine concentrations. In addition, we anticipated that subjects in this (placebo) group A consuming breakfast cereal containing <10 µg folic acid per serving would achieve higher plasma homocyst(e)ine concentrations compared to those in group B (folic acid fortified).

Among the subgroup of subjects who habitually consumed breakfast cereal prior to entering the study, plasma homocyst(e)ine concentrations elevated 23.6% after their consuming placebo cereal, whereas the homocyst(e)ine concentrations elevated non-significantly by only 7% in the group who received experimental cereal providing 200 µg folic acid/serving (ANOVA tests p<0.0001 and 0.17, respectively).

In contrast, no significant changes in plasma homocyst(e)ine after placebo cereal were seen in the subgroup who reported only sporadic breakfast cereal consumption prior to entering the study. Despite randomization of patients to receive placebo or cereal fortified with folic acid, non-consumers of breakfast cereal in the fortified cereal group had significantly lower homocyst(e)ine at baseline. This difference may have been related to a trend for higher folate concentrations in the fortified cereal group. During treatment, the homocyst(e)ine concentrations in this subgroup rose non-significantly from 7.8 to 9.0 µmol/L despite ingestion of an additional 200 µg folic acid daily. This unexpected finding is in contrast to the results of previous studies that showed significant lowering of homocyst(e)ine after supplementation with 200 µg per day of folic acid [9]. The aberrant results in this study could be related to regression to the mean, since the final homocyst(e)ine concentrations among non-regular consumers of breakfast cereals were comparable in the placebo and fortified cereal groups. In addition, the lack of difference in final homocyst(e)ine concentrations between placebo and fortified cereal treatment in this study could be related to greater intake of folic acid as a consequence of folic-acid-fortification of foods since January 1, 1998 [14,15].

	CONCLUSIONS

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In summary, these results support the following conclusions. First, despite wide-spread fortification of grains and cereal products in the U.S., daily consumption of commercially available fortified breakfast cereals, usually containing 100 to 400 µg folic acid per serving, had significant homocyst(e)ine-lowering effects among the subjects in this study as shown by the homocyst(e)ine increase after cessation of habitual intake of commercial breakfast cereal. Second, substitution of breakfast cereal containing only 200 µg folic acid per day was sufficient to maintain the homocyst(e)ine-lowering effects of commercial cereals. Our findings suggest that the current concentrations of folic acid fortification mandated by the FDA, which would provide additional 70 to 120 µg folic acid per day [14,15], may be sufficient to achieve near marginal folate-mediated homocyst(e)ine lowering in many individuals. Such a conclusion is supported by the findings in Framingham subjects reported by Jacques et al. [16]. Additional studies are required to assess the possible beneficial effects of supplemental intake of vitamins B₆ and B₁₂ in combination with folic-acid-fortified foods.

	ACKNOWLEDGMENTS

 
Supported in part by grants from the National Institutes of Health P51-RR00163, by an Established Investigator Grant from the American Heart Association, grants from General Mills, Inc., and Bio-Rad Laboratories, Inc., Diagnostics Group. The authors are indebted to Dr. Len Marquart, from General Mills, Inc., for providing the breakfast cereals used in this study and for the analyses of the vitamins in the cereals.

	FOOTNOTES

 
* Plasma total homocysteine (tHcy) or homocyst(e)ine is the sum of the concentrations of the reduced amino acid homocysteine and the homocysteinyl moieties of the disulfides homocystine and homocysteine-homocystine, whether free or bound to plasma proteins.

Received March 6, 2000. Accepted April 26, 2000.

	REFERENCES

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