Paul Finglas

Determination of folate vitamers in food and in Italian reference diet by high-performance liquid chromatography☆

A trienzyme treatment (conjugase, alpha-amylase, protease) followed by affinity chromatography and reversed-phase HPLC with UV and fluorescence detection was performed for the quantification of folate vitamers in legumes (chickpea and beans), processed meats (salami Milano and Parma ham) and in an Italian reference diet. This method allowed a good separation of six folate vitamers: 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, folic acid, 10-formylfolic acid, 10-formyldihydrofolate and tetrahydrofolate within 30 min. Recovery, reproducibility and limits of detection of the method are reported. HPLC results were 24-52% lower than the microbiological assay findings.

The effect of β-carotene supplementation on the immune function of blood monocytes from healthy male nonsmokers

Although there is strong epidemiologic evidence that diets rich in carotenoids such as beta-carotene are associated with a reduced incidence of cancer, the cellular mechanisms underlying this phenomenon remain unknown. This article describes the effect of dietary beta-carotene supplementation on both the expression of functionally associated surface molecules on human monocytes and on the secretion of the cytokine tumor necrosis factor-alpha (TNF-alpha) by monocytes, all of which are involved in the initiation and regulation of immune responses involved in tumor surveillance. A double-blind, placebo-controlled, crossover study was undertaken in which 25 healthy, adult male nonsmokers were randomly assigned to receive beta-carotene (15 mg daily) or placebo for 26 days, followed by the alternative treatment for a further 26 days. The expression of functionally related monocyte surface molecules was quantified by flow cytometry, and ex vivo secretion of TNF-alpha was quantified by an enzyme-linked immunosorbent assay, before and after each treatment period. After dietary supplementation there were significant increases in plasma levels of beta-carotene and in the percentages of monocytes expressing the major histocompatibility complex class II molecule HLA-DR and the adhesion molecules intercellular adhesion molecule-1 and leukocyte function-associated antigen-3. In addition, the ex vivo TNF-alpha secretion by blood monocytes was significantly increased after supplementation. These findings suggest that moderate increases in the dietary intake of beta-carotene can enhance cell-mediated immune responses within a relatively short period of time, providing a potential mechanism for the anticarcinogenic properties attributed to beta-carotene.

Interlaboratory studies of HPLC procedures for the analysis of carotenoids in foods

This paper describes the use of a potential reference material in interlaboratory studies for the analysis of carotenoids in a mixed vegetable material. Seventeen European laboratories have carried out collaborative studies to assess the accuracy of HPLC procedures for the measurement of lutein, zeaxanthin, lycopene, α-carotene and β-carotene in a vegetable mix. The studies investigated possible problem areas including chromatographic systems, standardization of carotenoid stock solutions, extraction procedures and data handling. The results suggested that the effect of the chromatographic system is probably not a major variable, although some systems achieved a more discrete separation of carotenoid isomers than others. In the more experienced laboratories, variation in the standardization of the carotenoid solution was not thought to be a significant problem. However, there were greater variations for lycopene calibration and measurement. Preliminary conclusions from these studies suggested that the preparation of the carotenoid extract may account for about 13% of the overall variance of around 23%.

Standardisation of HPLC techniques for the determination of naturally-occurring folates in food

The aim of this work was to evaluate current in-house HPLC procedures for the determination of naturally-occurring folates in food, and to identify problem areas for further improvement. Five intercomparison studies were completed over the period 1990–1997 in which nine participants from six countries took part. Through careful validations and detailed discussions held at evaluation meetings, possible biases and sources of systematic error have been identified and reduced. The use of ascorbic acid and nitrogen flushing during extraction, sample clean-up using strong anion exchange columns, spectrophometrically calibrated standards and fluorescence detection are all recommended. Both in-house hog kidney and human plasma deconjugase enzymes gave similar results to the circulated common hog kidney enzyme which was prepared from fresh pig’s kidneys. The most consistently reported values were for 5-CH3H4-PteGlu, and to a lesser extent, for H4PteGlu. Four candidate reference materials (CRM 121, wholemeal flour; CRM 421, milk powder; CRM 485, lyophilised mixed vegetables, and CRM 487, lyophilised pig’s liver) have been proposed with both indicative values (mean ± uncertainty) for 5-CH3H4-PteGlu in CRM 421 (0.25; ± 0.02 mg/kg) and CRM 485 (2.14; ±0.42 mg/kg), and information values (mean; range) for 5-CH3H4-PteGlu in CRM 121 (0.04; 0.03–0.08 mg/kg) and CRM 487 (2.6; 1.9–3.8 mg/kg). Certified values are also given for total folate by microbiological assay: CRM 121 (0.50; ±0.07 mg/kg), CRM 421 (1.42; ±0.14 mg/kg), CRM 485 (3.15; 0.28 mg/kg), and CRM 487 (13.4; 1.3 mg/kg). Average recovery of 5-CH3H4-PteGlu, added prior to extraction and deconjugation, was 91% (84–95%) for the four CRMs. The average within- and between-laboratory variations were 6 and 15% for the determination of 5-CH3H4-PteGlu by HPLC, and 9 and 18% for the determination of total folate by microbiological assay. These CRMs will be used for quality control of folate measurements for nutritional labelling, and validation of new techniques. Further methodology work is required for the HPLC analyses of folate forms other than 5-CH3H4-PteGlu.1

Peripheral arterial disease and methylenetetrahydrofolate reductase (MTHFR) C677T mutations: A case-control study and meta-analysis

OBJECTIVE Hyperhomocysteinaemia is associated with peripheral arterial disease (PAD). There are inter-individual variations in the metabolism of homocysteine because of genetic polymorphisms. This study analyzed the role of one polymorphism that is associated with raised homocysteine, as a risk factor for PAD. METHODS This study considered the association of methylenetetrahydrofolate reductase (MTHFR) C677T polymorphisms with the incidence of PAD by performing a case-control study and a cross sectional study of homocysteine levels. We recruited 133 patients with PAD in Norfolk and compared the MTHFR allele distribution with 457 healthy individuals. We also carried out a meta-analysis to place our data within the context of other published studies. We searched Medline, Embase, and Cochrane databases up to March 2008 for any studies on the association between MTHFR C677T polymorphism and PAD. RESULTS The MTHFR C677T allele frequencies in the cases and controls were 0.37 and 0.33, and the odds ratios for the association of the 677 T allele or TT genotype with PAD were 1.18 (95% Confidence Interval [CI] 0.89, 1.58) and 1.99 (95% CI 1.09, 3.63). Homozygotes for the MTHFR C677T mutation had higher concentrations of plasma total homocysteine, odds ratio 2.82 (95% CI 1.03, 7.77) compared to homozygotes for the MTHFR 677 CC genotype. Twelve of 72 articles retrieved from the database search reported the prevalence of mutations in PAD patients. A meta-analysis of 9 appropriate studies, including our own, showed that being homozygous for the C677T allele was associated with an increased risk of PAD, pooled odds ratio 1.36 (95% CI 1.09, 1.68). CONCLUSION We have found a strong association between raised homocysteine, the TT genotype, and PAD.

Dietary intake and micronutrient status of adolescents: effect of vitamin and trace element supplementation on indices of status and performance in tests of verbal and non-verbal intelligence

Relationships between micronutrient intake and status, and micronutrient status and performance in tests of intelligence were investigated in a group of adolescents (13-14 years old). Dietary intakes were assessed using a 7 d weighed dietary record method, coupled with the collection of duplicate diets. Vitamin and trace mineral intakes calculated using food composition tables were compared with those obtained by direct analysis of duplicate diets. Micronutrient status was judged via a range of biochemical indices measured in blood samples taken after a 12-15 h fast. Blood samples were taken both before and after a 16-week period of vitamin and trace mineral supplementation. Individual tests of verbal and nonverbal intelligence were also performed pre- and post-supplementation. The results of this study indicate that the use of food table data may lead to substantial over- or underestimation of the intake of several micronutrients. In general, the total calculated or analysed amount of a specific micronutrient consumed did not adequately predict status, as judged by a range of biochemical indices. There were significant changes in status measurements over the 16-week study period, irrespective of supplementation, and these changes were markedly influenced by the initial status of the subject. There was no effect of supplementation on performance in tests of intelligence. However, there was a significant association between plasma ascorbic acid and initial non-verbal intelligence quotient (IQ) in the boys, and between whole blood glutathione peroxidase (EC 1.11.1.9) activity and non-verbal and verbal IQ in both sexes. These findings are discussed in relation to other recent studies of the influence of micronutrient supplementation on the psychological performance of children.

First BCR-intercomparison on the determination of folates in food

Abstract The first BCR intercomparison on the determination of folate in food was designed to study the state-of-the-art in folate analysis in a group of experienced vitamin laboratories in Europe. In all 15 participants from 8 countries took part using microbiological and HPLC procedures, enzyme protein-binding assays (EPBA) and commercial radioprotein-binding assay kits (RPBA). The participants were asked to quantify folate levels in a lyophilised Brussels sprout material, which had been specifically prepared as a candidate reference material for vitamin work, using their preferred method of analysis. Three types of deconjugation were also investigated, human plasma (HP), chicken pancreas (CP) and hog kidney (HK). Generally good agreement was obtained by participants using the microbiological procedures. Folate levels determined after CP deconjugate treatment (mean = 984 μ g per 100 g dry matter, SD = 237, n = 6) were 19% higher than those levels found after HP deconjugation (mean = 824 μ g per 100 g dry matter, SD = 147, n = 6). The use of autoclaving followed by deconjugation with either HP or CP enzymes gave lower (10–20%) folate levels (determined by microbiological assay) when compared to refluxing and deconjugation with the same enzymes. Hog kidney deconjugase enzyme and autoclaving/refluxing was not as effective. Although the HPLC results from the 2 participants who were able to complete the study agree reasonably well with the microbiological data, there were differences in the proportion of the individual folate forms measured. One participant found 5-CH 3 THF (55%), THF (25%) and 5-CHOTHF (20%), whereas the other only initially reported 5-CH 3 THF but later confirmed small amounts (10–15%) of THF and 5-CHOTHF forms. Despite the use of HPLC with fluorometric detection, there were some problems in peak identification and calibration. The use of HPLC with UV detection gave unsatisfactory results due to difficulties in resolution of folate compounds and these results were excluded. RPBA results were generally higher (50–60%) than both the microbiological and HPLC results but more variable. EPBA results also varied between the three laboratories using this format but the mean folate content (HP only) agreed favourably with both the HPLC and microbiological results. The major problem identified with these methods was the response of the individual folate forms to the binding-protein used. Careful control of assay pH and calibrant are required if these methods are to be applied to the determination of food folates. Future work will focus on improvements in methodology of each type of assay prior to further intercomparisons.

The HPLC analysis of thiamin and riboflavin in potatoes

Abstract A simple HPLC method for the analysis of thiamin (as thiochrome) and riboflavin in both raw and cooked potato is described. The potato extract, after a minimum clean up, is injected onto a C 18 reverse phase column and the vitamins are separated isocratically with water:methanol. The use of fluorescence detection, which is highly specific and sensitive, minimises the number of interfering peaks. Recoveries of both vitamins, when taken through the method or added to potato samples before extraction, are consistently better than 90%. The results for thiamin in the potato are slightly higher than those obtained by the AOAC (1980) chemical method, whereas the reverse is true for riboflavin. The method may have application to other food matrices and is more rapid than the AOAC (1980) chemical method.

Proposed mandatory fortification of the UK diet with folic acid: have potential risks been underestimated?

Abstract This article discusses the recent consultation by the UK Department of Health and Food Standards Agency on the risk-benefit of a whole population strategy for increased intake of folic acid via the fortification of flour. The potential benefit of folic acid in relation to decreased incidence of Neural Tube Defects in the newly born is contrasted to the potential risk of increasing permanent neuropathy (nerve damage) in those suffering from vitamin B12 deficiency. Questions are also raised with regard to the potential risk of folic acid addition in the context of pre-existing malignant neoplasms, and the incidence of dichorionic twin births.

Effects of Lycopene and Lutein Supplementation on the Expression of Functionally Associated Surface Molecules on Blood Monocytes from Healthy Male Nonsmokers

It has been suggested that dietary carotenoids can enhance immune function. Supplementation with beta-carotene (15 mg daily) was previously shown to enhance human monocyte function. To examine the effect of other dietary carotenoids, two similar independent studies were done. Healthy adult male nonsmokers were randomly assigned to receive lycopene (study 1), lutein (study 2), or placebo for 26 days, followed by the alternative treatment for another 26 days. The expression of functionally related monocyte surface molecules was quantified by laser flow cytometry before and after each treatment period. There was a significant increase in plasma levels of each carotenoid following dietary supplementation, but the effects on monocyte surface molecule expression were not as striking as those observed after beta-carotene supplementation. These findings emphasize that it cannot be assumed that the effect of one carotenoid will be the same as another, even at the same level of intake.