Background: Measurement of plasma total homocysteine has become common as new methods have been introduced. A wide range of disorders are associated with increased concentrations of total homocysteine. The purpose of this review is to provide an international expert opinion on the practical aspects of total homocysteine determinations in clinical practice and in the research setting and on the relevance of total homocysteine measurements as diagnostic or screening tests in several target populations. Methods: Published data available on Medline were used as the basis for the recommendations. Drafts of the recommendations were critically discussed at meetings over a period of 3 years. Outcome: This review is divided into two sections: (a) determination of homocysteine (methods and their performance, sample collection and handling, biological determinants, reference intervals, within-person variability, and methionine loading test); and (b) risk assessment and disease diagnosis (homocystinuria, folate and cobalamin deficiencies, cardiovascular disease, renal failure, psychiatric disorders and cognitive impairment, pregnancy complications and birth defects, and screening of elderly and newborns). Each of these subsections concludes with a separate series of recommendations to assist the clinician and the research scientist in making informed decisions. The review concludes with a list of unresolved questions.

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Facts and Recommendations about Total Homocysteine Determinations: An Expert Opinion

The Biomarkers of Nutrition for Development (BOND) project is designed to provide evidence-based advice to anyone with an interest in the role of nutrition in health. Specifically, the BOND program provides state-of-the-art information and service with regard to selection, use, and interpretation of biomarkers of nutrient exposure, status, function, and effect. To accomplish this objective, expert panels are recruited to evaluate the literature and to draft comprehensive reports on the current state of the art with regard to specific nutrient biology and available biomarkers for assessing nutrients in body tissues at the individual and population level. Phase I of the BOND project includes the evaluation of biomarkers for 6 nutrients: iodine, iron, zinc, folate, vitamin A, and vitamin B-12. This review represents the second in the series of reviews and covers all relevant aspects of folate biology and biomarkers. The article is organized to provide the reader with a full appreciation of folate's history as a public health issue, its biology, and an overview of available biomarkers (serum folate, RBC folate, and plasma homocysteine concentrations) and their interpretation across a range of clinical and population-based uses. The article also includes a list of priority research needs for advancing the area of folate biomarkers related to nutritional health status and development.

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Biomarkers of Nutrition for Development (BOND)-Iron Review.

Background/Aim: Immunosuppressants are now used much earlier in the course of Crohn’s disease; however their effect on the natural history of the disease, especially on the need for surgery, is not known. The aim of this study was to assess the evolution of the need for surgery in Crohn’s disease during the last 25 years. Patients and Methods: The medical charts of 2573 patients were reviewed retrospectively. The use of immunosuppressants (azathioprine or methotrexate), the need for intestinal resection, and the occurrence of intestinal complications were assessed using Kaplan-Meier analysis in five consecutive cohorts of patients defined by the date of diagnosis of Crohn’s disease (1978–82; 1983–87; 1988–92; 1993–97; 1998–2002). Results: In 565 patients seen in the authors’ unit within the first three months after diagnosis, characteristics of Crohn’s disease at diagnosis did not differ from one cohort to another. The five year cumulative probability to receive immunosuppressants increased from 0 in the 1978–82 cohort to 0.13, 0.25, 0.25, and 0.56 in the 1983–87, 1988–92, 1993–97, and 1998–2002 cohorts, respectively (p Conclusion: Although immunosuppressants have been used more frequently over the last 25 years, there was no significant decrease of the need for surgery, or of intestinal complications of Crohn’s disease.

https://gut.bmj.com/content/gutjnl/54/2/237.full.pdf

Impact of the increasing use of immunosuppressants in Crohn’s disease on the need for intestinal surgery

Logistical constraints associated with the collection and analysis of biological samples in community-based settings have been a significant impediment to integrative, multilevel biodemographic and biobehavioral research. However, recent methodological developments have overcome many of these constraints and have also expanded the options for incorporating biomarkers into population-based health research in international as well as domestic contexts. In particular, using dried blood spot (DBS) samples—drops of whole blood collected on filter paper from a simple finger prick— provides a minimally invasive method for collecting blood samples in nonclinical settings. After a brief discussion of biomarkers more generally, we review procedures for collecting, handling, and analyzing DBS samples. Advantages of using DBS samples—compared with venipuncture—include the relative ease and low cost of sample collection, transport, and storage. Disadvantages include requirements for assay development and validation as well as the relatively small volumes of sample. We present the results of a comprehensive literature review of published protocols for analysis of DBS samples, and we provide more detailed analysis of protocols for 45 analytes likely to be of particular relevance to population-level health research. Our objective is to provide investigators with the information they need to make informed decisions regarding the appropriateness of blood spot methods for their research interests.

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What a Drop Can Do: Dried Blood Spots as a Minimally Invasive Method for Integrating Biomarkers Into Population-Based Research

T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene. This variant, with mildenzymatic deficiency, is associated with an increased risk for neural tube defects and pregnancy complicationsand with a decreased risk for colon cancer and leukemia. Although many studies have reported that this variant isalso a risk factor for vascular disease, this area of investigation is still controversial. Severe MTHFR deficiencyresults in homocystinuria, an inborn error of metabolism with neurological and vascular complications. Toinvestigate the in vivopathogenetic mechanisms of MTHFR deficiency, we generated mice with a knockout ofMthfr. Plasma total homocysteine levels in heterozygous and homozygous knockout mice are 1.6- and 10-foldhigher than those in wild-type littermates, respectively. Both heterozygous and homozygous knockouts haveeither significantly decreased S-adenosylmethionine levels or significantly increased S-adenosylhomo-cysteine levels, or both, with global DNA hypomethylation. The heterozygous knockout mice appear normal,whereas the homozygotes are smaller and show developmental retardation with cerebellar pathology. Abnormallipid deposition in the proximal portion of the aorta was observed in older heterozygotes and homozygotes,alluding to an atherogenic effect of hyperhomocysteinemia in these mice.INTRODUCTION

Mice deficient in methylenetetrahydrofolate reductase exhibit hyperhomocysteinemia and decreased methylation capacity, with neuropathology and aortic lipid deposition

BACKGROUND Inflammatory bowel disease (IBD) is associated with an increased incidence of thromboembolic disease. Hyperhomocysteinaemia (hyper-tHcy), a condition associated with the C677T variant of 5,10-methylenetetrahydrofolate reductase (MTHFR), is linked with an increased incidence of thromboembolic disease. Hyper-tHcy has been reported in patients with IBD. AIMS To assess the prevalence of the C677T MTHFR genotype and the contribution of this genotype to hyper-tHcy in patients with IBD. METHODS Patients with established IBD (n=174) and healthy controls (n=273) were studied. DNA samples were genotyped for the MTHFR (C677T) mutation. Subjects were categorised as homozygous for the thermolabile variant (TT), heterozygous for wild type and variant (CT), or homozygous for the wild type (CC). RESULTS Plasma homocysteine concentrations were significantly higher in patients with IBD than in healthy controls. A total of 17.5% of ulcerative colitis and 16.8% of Crohn’s disease patients were homozygous for the C677T variant compared with 7.3% of controls. Homozygosity (TT) for the variant was associated with higher plasma tHcy levels in patients with IBD and in healthy controls. When all subjects who were TT for the variant were excluded, median plasma tHcy was still significantly higher in IBD than controls. Plasma vitamin B 12 levels were lower in patients with IBD irrespective of MTHFR genotype. CONCLUSIONS There is an association between the thermolabile MTHFR C677T variant and IBD. This accounts in part for the raised plasma tHcy found in patients with IBD and may contribute to the increased incidence of thromboembolic complications. All patients with IBD should receive low dose folic acid and vitamin B 12 therapy to protect against the thromboembolic complications of raised tHcy.

https://gut.bmj.com/content/gutjnl/45/3/389.full.pdf

Increased prevalence of methylenetetrahydrofolate reductase C677T variant in patients with inflammatory bowel disease, and its clinical implications

Background: There is a need for systematic evaluation of methods before their release to the market. We addressed this problem in novel homocysteine assays as part of an European Demonstration Project involving six centers in four countries.

Methods: Two immunological methods for measurement of plasma total homocysteine (P-tHcy), the fluorescence polarization immunoassay (FPIA) and the enzyme immunoassay (EIA), were compared with two comparison methods, HPLC and gas chromatography–mass spectrometry (GC-MS). All laboratories performed the following procedures: ( a ) familiarization; ( b ) determination of linearity and precision by analyzing five plasma samples with interrelated concentrations for 20 days; ( c ) correlation using patients’ samples; and ( d ) assessment of long-term performance.

Results: Both immunological methods were linear for P-tHcy between 5 and 45 μmol/L. The intralaboratory imprecision (CV) was <5% for FPIA and <9% for EIA used with a sample processor. The bias was −2% to 3% for FPIA and 2–4% for EIA used with a sample processor.

Conclusions: The immunological methods provide results with little bias compared with HPLC and GC-MS. The imprecision of the assays must be considered in the context of their intended use(s).

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Evaluation of Novel Assays in Clinical Chemistry: Quantification of Plasma Total Homocysteine

The relationship between increased folate catabolism and the increased requirement for folate in pregnancy *John R. Higgins Research Registrar, fEoin P. Quidivan Graduate Research Student,

The benefit of the introduction of mandatory folic acid fortification of all flour products in the USA in 1998 has been amply demonstrated in a reduction of neural tube defect births. Doubt has been cast on the actual level of fortification and recent calculations have shown that the level of folic acid fortification is likely to have been over twice the amount mandated. The implication of this is that a greater proportion of the population are likely to have consumed folic acid at >1 mg/d, the Food and Drug Administration safe upper level of intake. Using the criteria of appearance of synthetic folic acid in serum, the objective of this pilot study was to investigate the consequences of consumption of baked bread preparations containing 1 mg folic acid. Four healthy adult volunteers undertook each dosing schedule 2 weeks apart. This consisted of a single dose of 1000 microg, two doses of 500 microg, three doses of 333 microg, five doses of 200 microg and, finally, ten doses of 100 microg. Serum was collected pre- and postprandially and analysed for synthetic folic acid by a combined HPLC-microbiological assay for folic acid. Folic acid appeared in all subjects at all test doses, with the effect more pronounced as the standard dose was administered in smaller amounts over the test period. Approaches to optimise folic acid intake in target populations as part of a universal fortification strategy should take into consideration the potential hazard of over-exposure in groups consuming high amounts of flour-based products.

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Joseph McPartlin

Papers

Increased prevalence of methylenetetrahydrofolate reductase C677T variant in patients with inflammatory bowel disease, and its clinical implications

Evaluation of Novel Assays in Clinical Chemistry: Quantification of Plasma Total Homocysteine

The relationship between increased folate catabolism and the increased requirement for folate in pregnancy *John R. Higgins Research Registrar, fEoin P. Quidivan Graduate Research Student,

Postprandial serum folic acid response to multiple doses of folic acid in fortified bread.

Measurements of sub-nanomolar concentrations of unmetabolised folic acid in serum.