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3
The
*
Nutrition and Genomics Laboratory, the
Vitamin Metabolism and Aging Laboratory, Jean Mayer U.S. Department of Agriculture Human Nutrition Research Center on Aging at Tufts University, Boston, MA 02111; and the
**
Framingham Heart Study, Boston University School of Medicine, Framingham, MA
3To whom correspondence should be addressed. E-mail: cvargas{at}hnrc.tufts.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • absorption • folate • genetics • glutamate carboxypeptidase II • homocysteine
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). The bioavailability of these folates depends in part on the ability of the animal to hydrolyze the polyglutamate chain because only the monoglutamate form of folate can be absorbed in the small intestine. In the human small intestine, this hydrolysis is catalyzed by glutamate carboxypeptidase II (GCPII),4
previously known as folypolyglutamate carboxypeptidase (1
), an enzyme that is located in the brush border membrane of the jejunum (2
).
The GCPII gene has been localized to chromosome 11p11.2 (3
). It has a genomic sequence containing 19 exons, encoding a 750 amino acids polypeptide (4
). The GCPII mRNA is highly expressed in the human small intestine (5
,6
). Its expression is greater in the proximal than in the distal small intestine (7
). It is also expressed in other tissues (prostate, brain, kidney, liver, spleen, lung, heart and thyroid gland) (5
,6
).
Recently, Devlin et al. (5
) analyzed DNA samples obtained from 75 healthy elderly white men and women (group of controls selected from a British case-control study of dementia) and identified a polymorphism in GCPII. This polymorphism (1561C>T) is located in exon 13 within the putative catalytic region of GCPII with an allelic frequency of 5%. The T allele was associated with a lower enzyme activity in vitro, and the data from these 75 healthy subjects suggested that this polymorphism is associated with lower serum folate and higher serum homocysteine levels.
These findings, suggesting that genetic variability in the GCPII locus compromises folate absorption, are important but need confirmation because of the limited number of participants. In this study, we analyzed the association of this polymorphism with plasma folate and homocysteine concentrations in a larger cohort (n = 1913), consisting of subjects attending the fifth examination of the Framingham Offspring Study. This is a well-characterized cohort representative of the general population in whom dietary habits and other lifestyle and biochemical variables have been extensively documented.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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This study was carried out using data from 1913 subjects (958 men and 955 women) who attended the fifth examination of the Framingham Offspring Study conducted between January 1991 and December 1994. From the 1913 subjects studied in this population, only 680 men and 644 women had complete information for all the variables analyzed and were included in the analysis. We have chosen this period of examination because it took place before the implementation of the folic acid fortification program (8
). Folic acid is a monoglutamyl derivative whose intestinal absorption is not dependent on GCPII. The details of the design and methods of the Framingham Offspring Study have been reported elsewhere (9
).
Dietary information.
Individuals who participated in the fifth examination of the Framingham Offspring Study received and completed a semiquantitative food frequency questionnaire (10
), and their dietary intake was estimated using this questionnaire. We restricted our analysis to those subjects whose source of folate intake is primarily from food and not from any multivitamin or vitamin supplements (958 men and 955 women).
DNA isolation and genotyping.
Leukocyte DNA was extracted from 5 to 10 mL whole blood as previously described (11
). GCPII genotypes were determined by allelic discrimination, as previously described (12
). For this technique we used TaqMan minor groove binder (MGB®) probes (Applied Biosystems, Foster City, CA) [FAM-CTTGGTACACAACCTAA (wild type); VIC-AGCTTGGTATACAACCT (mutant allele)] and primers (5'-GAGTTGATTGTACACCGCTGATG and 3'-CCACCTATGTTTAACATAATACCTCAAGA). PCR was carried out in the ABI Prism 7700 sequence detection system (Applied Biosystems). The thermal cycler program was set up at 50°C for 2 min, 95°C for 10 min, and 45 cycles, each of 95°C for 20 s and 60°C for 1 min.
Biochemical measurements.
Individuals had their blood drawn after 12 h of fasting. Plasma folate was determined by microbial assay using Lactobacillus casei (13
,14
). Plasma total homocysteine was determined using HPLC with fluorometric detection (15
). Plasma pyridoxal 5'-phosphate (PLP) was determined by tyrosine decarboxylase apoenzyme method (16
). Plasma vitamin B-12 was measured by radioimmunoassay (Quantaphase II; Bio-Rad, Hercules, CA). Creatinine was determined by the Jaffe method (17
).
Statistical analyses.
The Statistical Package for Social Sciences, version 10.1 (SPSS Inc., Chicago, IL) was used for the analysis. Dietary and plasma levels of PLP, vitamin B-12 and folate were log10-transformed, and alcohol consumption and smoking status were square-root-transformed to improve normality for statistical testing. We used Student’s t test for independent groups to assess mean differences for continuous variables between men and women. We used one-way analysis of covariance to evaluate in men and women the relationships between plasma total homocysteine and folate levels and GCPII genotypes, using as covariates age, plasma levels of vitamin B-12 and pyridoxal-5-phosphate (PLP), dietary intake of folate, body mass index (BMI), alcohol (g/d) and caffeine (mg/d) consumption, smoking status (smokers were defined as those who smoked cigarettes regularly in the past 1 y), and serum creatinine levels. All statistical tests were two-tailed, and differences with a P value < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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shows the demographic, anthropometric, dietary and plasma vitamin levels of this population, which was divided on the basis of the 1561 C>T polymorphism in the GCPII.
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shows the relationship between the 1561 C>T polymorphism in the GCPII and plasma folate and homocysteine concentrations. Male carriers of the T allele had higher plasma folate concentrations (14.79 ± 0.88 nmol/L) than homozygotes of the C allele (12.55 ± 0.38 nmol/L; P < 0.05). In women, plasma folate concentrations did not differ between genotypes (P = 0.8). Neither male (P = 0.54) nor female (P = 0.33) carriers of the T allele differed from respective homozygotes of the C allele in plasma total homocysteine levels.
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). In women, we found a borderline interaction between the T allele and age (P = 0.054, Table 3
) after controlling for folate, alcohol and caffeine intake, plasma PLP and B-12 levels, creatinine levels, smoking status and BMI. Carriers of the T allele had lower plasma folate levels than homozygotes for the C allele in both older and younger women. However, the difference tended to be greater among older women (P = 0.1). We did not observe any significant interaction between age and homocysteine levels in men (P = 0.98) or women (P = 0.6; Table 3
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) found in a British population that carriers of the GCPII T allele had significantly lower serum folate concentrations (7.0 ± 1.2 µg/L) and higher plasma homocysteine levels (15.0 ± 1.10 µmol/L) than homozygotes for the wild-type allele (10.9 ± 0.5 µg/L for folate and 12.6 ± 0.93 µmol/L for homocysteine). The same study also found that the in vitro activity of the enzyme from mammalian COS-7 cells transfected with the mutant GCPII cDNA was 53% lower than the enzyme from cells transfected with the wild-type GCPII cDNA. Based on these data, Devlin et al. proposed that this mutation compromises the bioavailability of food folate and, hence, it is a determinant of both folate and homocysteine plasma concentrations. An important limitation of the study by Devlin et al. (5
) is the small number of subjects who participated in the study. Only 6 of the 75 subjects (8%) were heterozygotes for the T allele. Therefore, it was important to replicate these findings in a larger representative population. Our study examined this association in a population of 1913 subjects of which 200 had the T allele. This population was selected carefully to exclude those who took vitamin supplements to ensure that folate status in these individuals derives primarily from dietary folate sources. The GCPII T allele is not associated with lower plasma folate concentrations, and it is also not related to higher plasma total homocysteine concentrations. If anything, male carriers of the mutation (T allele) had significantly higher folate concentrations (14.79 ± 0.88 nmol/L) than men homozygous for the wild-type allele (12.55 ± 0.38 nmol/L). However, because plasma total homocysteine concentrations did not differ between these groups, we regard this difference in plasma folate concentrations as biologically inconsequential.
We did not find any differences in plasma folate (13.27 ± 0.42 nmol/L for the C allele and 12.93 ± 0.95 nmol/L for the T allele) or total homocysteine levels (10.29 ± 0.15 umol/L for the C allele and 9.93 ± 0.34 umol/L for the T allele) between women carrying the mutation compared with those homozygous for the wild-type allele.
Our data did show that in its relationship to plasma folate levels, the 1561 C>T polymorphism in GCPII exhibits borderline interactions with age and gender. These interactions, however, are weak and inconsistent: plasma folates are higher in older men with the T allele than in men with the wild-type C allele. In older women, this trend was opposite. Those with the T allele had lower plasma folates than those with the wild-type C allele. Neither difference, however, was statistically significant, and more relevant, neither difference was accompanied by parallel changes in plasma total homocysteine levels.
The conflicting findings of this and the previous study might be explained by differences in population and study design. Devlin et al (5
). studied a relatively small group of controls, mainly women, selected from a British case-control study of dementia (n = 75; age > 50 y). Their analysis of the population did not take into account dietary intake of folate or other vitamins known to be determinants of plasma total homocysteine concentration.
In this study, we also took into account several factors known to be determinants of folate and homocysteine status (18
,19
). These include dietary intake of folate and other B vitamins (B-6 and B-12), smoking status, alcohol and caffeine ingestion and serum creatinine. Adjusting for these factors did not alter our findings. Therefore, we conclude that in the Framingham Offspring Cohort the 1561 C>T polymorphism in GCPII is inconsequential as far as folate and total homocysteine plasma levels are concerned.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Any opinions, findings, conclusion, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the U.S. Department of Agriculture.
4 Abbreviations used: BMI, body mass index; GCPII, glutamate carboxypeptidase II; PLP, pyridoxal 5'-phosphate.
Manuscript received 23 January 2002. Initial review completed 10 March 2002. Revision accepted 22 March 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Rimm, E. B., Giovannucci, E. L., Stampfer, M. J., Colditz, G. A., Litin, L. B. & Willett, W. C. (1992) Reproducibility and validity of an expanded self-administered semiquantitative food frequency questionnaire among male health professionals. Am. J. Epidemiol. 135:1114-1136.
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Miller, S. A., Dykes, D. D. & Polesky, H. F. (1988) A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 16:1215.
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Osgood-McWeeney, D., Galluzzi, J. R. & Ordovas, J. M. (2000) Allelic discrimination for single nucleotide polymorphisms in the human scavenger receptor class B type 1 gene locus using fluorescent probes. Clin. Chem. 46:118-119.
13.
Horne, D. W. & Patterson, D. (1988) Lactobacillus casei microbiological assay of folic acid derivatives in 96-well microtiter plates. Clin. Chem. 34:2357-2359.
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16. Shin, Y. S., Rasshofer, R., Friedrich, B. & Endres, W. (1983) Pyridoxal-5'-phosphate determination by a sensitive micromethod in human blood, urine and tissues; its relation to cystathioninuria in neuroblastoma and biliary atresia. Clin. Chim. Acta 127:77-85.[Medline]
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18.
Selhub, J., Jacques, P. F., Rosenberg, I. H., Rogers, G., Bowman, B. A., Gunter, E. W., Wright, J. D. & Johnson, C. L. (1999) Serum total homocysteine concentrations in the third National Health and Nutrition Examination Survey (1991–1994): population reference ranges and contribution of vitamin status to high serum concentrations. Ann. Intern. Med. 131:331-339.
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