Our research group was appointed Research LOCUS at the University of Bergen in May 1998. The «LOCUS for Homocysteine and Related Vitamins» was authorized as a result of evaluation of 15 research groups under the Medical Faculty, University of Bergen (UoB).
The criteria are 1) a well defined research area, 2) the research activity exploits identifiable local unique resources or expertise, 3) the research area must have a long-term potential, 4) the core staff must comprise at least 5 senior scientists affiliated at UoB, and mainly dedicated to the actual research activity, 5) the research group should have the potential to function for at least 10 years, 6) the group should be localized in a defined area, and 7) documented scientific publications within a restricted and defined area are paramount.
The senior scientists of the Homocysteine LOCUS are listed in the Table below

The group comprises persons affiliated at different departments/institutes, i.e. Institute of Medicine (IoM) and Department of Public Health and Primary Health Care (DoP), University of Bergen (UoB), and Laboratory of Clinical Biochemistry (LoC),  Haukeland University Hospital (HUS).

The actual research field is homocysteine and functional markers for co-factors (vitamins) involved in homocysteine metabolism. Accordingly, the vitamins include folates, cobalamin (B12), pyridoxal phosphate (B6), riboflavin (B2), and recently choline. Deficiency states involving these vitamins are more commonly occurring than previously anticipated, and are associated with four conditions, i.e. cardiovascular disease, (colon)cancer, neuropsychiatric symptoms and impaired cognitive function, and pregnancy complications and adverse pregnancy outcome.

The research activities of the Homocysteine LOCUS can be categorized into two areas:

Area A: Development of biochemical analyses to measure levels of vitamins and functional markers of vitamin status (i.e. methylmalonic acid (MMA), homocysteine, cystathionine etc.) in blood and tissues, and genetic analyses to detect polymorphisms and genetic defects related to vitamin function. .

Area B: Clinical and epidemiological studies of homocysteine and vitamin status, and the associations with life style, diseases and use of drugs. Clinical homocysteine lowering trials.

The group covers expertise from analytical technology to large-scale population studies, as detailed in the table below.

Four post-docs and ten fellows are currently working at LOCUS Homocysteine. Their research activities are listed in the table below.

A close collaboration within this multi-disciplinary group has already existed for years. Ueland and Lien has worked together for 15 years. Dr. Schneede joined the group as a fellow in 1991, and got a tenure position in 1996. Dr. Svardal, a recognized expert in mass spectrometry, has been involved in instrumentation and method development. Vollset and Nygård have been collaborating since 1991, within the frame of the Hordaland Homocysteine study. Prof. Tell has been responsible for the planning and conduction of the large Homocysteine-HUSK study since she came to UoB in 1995.

• 1984- Construction of a radioenzymic method for determination of Hcy in tissues (Ueland et al. 1984).

• 1985- Reporting that 75 % of plasma Hcy is bound to albumin, and that by determining both the bound and free form of Hcy, denoted total homocysteine (tHcy), stored samples can be used for epidemiological studies (Refsum et al. 1985). The method is the basis for the assay marketed by Axis Biochemicals, i.e., the first commercial immunoassay for tHcy determination.

• 1989- Introduction of the first fully automated tHcy assay with high sample throughput (Fiskerstrand et al. 1993; Refsum et al. 1989). This assay has been used for performing the largest epidemiological study on tHcy to date (the Hordaland study), for the European concerted action project (COMAC) on homocysteine and vascular disease, in the clinical routine here in Norway, and has been established in several other laboratories.

• 1991- Introduction of tHcy into clinical routine. In 2000, 46660 tHcy analyses were performed at Haukeland University Hospital.

• 1992- Development of a method for the various Hcy species in plasma, and demonstrating that changes in Hcy is closely related to other thiols, such a cysteine and glutathione (Mansoor et al. 1992). Some of these Hcy species, in particular the thiol form, are particularly responsive to changes in the overall Hcy status (Mansoor et al. 1993; Mansoor et al. 1992; Mansoor et al. 1993; Mansoor et al. 1994; Ueland 1995).

• 1994- Introduction of MMA into clinical routine. Capillary electrophoresis was used in routine clinical diagnostics for the first time. In 2000, 46424 MMA analyses were performed by GC-MS at the Haukeland University Hospital.

• 1995- Design and construction of an in-house build laser-induced fluorescence (LIF) detector for capillary electrophoresis, based on sheath-flow technology (see Fig. 1). This instrument was built as part of the EU contract BMH4-CT95-0505 (completed July 1999) for high throughput genotyping of mutations predisposing to elevated tHcy. The performance in terms of sensitivity out ranges several commercial detectors available on the market.

• 1996- Development of various methods for mutation analyses based on multiinjection capillary electrophoresis with LIF detection (Ren 1997; Ren and Ueland 1999; Ulvik 1997; Ulvik et al. 1998). The methods allow rapid genotyping of samples (average analysis time is about 2.5 min per sample), and the sensitivity is sufficiently high to allow genotyping based on DNA remnants in serum/plasma. This novel technology opens up for genetic studies based on plasma/serum biobanks (Ulvik 1997)

• 1999- Completion of the Biomed project LIF detection in mutation analyses (contract BMH4-CT95-0505, co-ordinator Per Magne Ueland). The project's primary aim was to develop fast assays for mutation analysis based on capillary electrophoresis.

• 1999- Development of technology for mutation detection based on MALDI-TOF mass spectrometry (Harksen et al. 1999).

• 1999- Development of a CE-LIF method for the quantitation of the low concentration (in the nanomolar range) of vitamin B2 (riboflavin, FMD and FAD) in human plasma (Hustad et al. 1999)

• 2001- Completion of the Biomed project Demonstration of the diagnostic utility of a novel and fast method for plasma total homocysteine in representative target populations (contract BMH4983549, co-ordinator Helga Refsum). The primary objective was to evaluate the performance and compare of assays for tHcy.

• 2001- Development of a sensitive, high throughput LC-MS/MS method for the simultaneous determination of six vitamin B6 and three riboflavin species in human blood (Midttun et al, 2001, 2005).

• 2003- Determination of choline, betaine and related compounds by LC-MS/MS (Holm et, al 2003).

• 2004- A multiplex assay for the determination of 13 SNPs related to one carbon metabolism by MALDI-TOF mass spectrometry (Meyer et al, 2004).

• 2005- An automated assay for the determination of methylmalonic acid, tHcy, and related amino acids by GC-MS (Windelberg et al., 2005).

• 2006- Established a panel of 40 markers and metabolites related to one-carbon metabolism, based on mass-spectrometry. This metabolite profile is measured in less than 1 mL of serum/plasma.

• 1985- Numerous reports based on cell culture experiments, animal models and clinical studies on factors influencing Hcy formation, cellular export and elimination were published. The effect of vitamins, methionine and various drugs, inborn errors of Hcy metabolism and various diseases, such as cancer, psoriasis, vascular disease and renal failure, were documented. These data provided basic knowledge on Hcy metabolism and regulation, i.e. the fundamentals for the clinical utility of plasma tHcy as a indicator or risk factor for common diseases.

• 1986- One of the first report that tHcy is a sensitive indicator of folate status, by demonstrating that the antifolate effect of MTX leads to increased tHcy in humans (Refsum et al. 1986). tHcy is now world wide routinely used to evaluate functional folate deficiency.

• 1991- Demonstrating that even short term nitrous oxide anaesthesia may lead to a significant increase in tHcy, reflecting a functional vitamin B12 deficiency (Christensen et al. 1994; Ermens et al. 1991).

• 1993- Design of the Hcy loading test, documenting that this test is an adequate tool for investigating turnover of Hcy in plasma, and demonstrating the pivotal role of intact renal function for the elimination of Hcy from plasma (Guttormsen et al. 1993; Guttormsen et al. 1996; Guttormsen et al. 1997; Mansoor et al. 1993).

• 1998- Demonstrating stability and utility of tHcy under experimental field conditions. These results suggest that determination of plasma tHcy is as reliable as for instance that of total serum cholesterol (Clarke et al. 1998).

• 2000- Demonstrating that vitamin B2 status is an independent determinant of plasma homocysteine in subjects with the CT or TT genotype of the MTHFR 677C->T polymorphism (Hustad et al. 2000).

• 2001- Report on low serum cobalamin and metabolic evidence of impaired cobalamin status (elevated MMA and tHcy) in neonates (Bjorke-Monsen et al. 2001).

• 2004, 2005- The first publications reporting that circulating betaine is an important determinant of plasma homocysteine (Holm et al. 2004, 2005).

• 1993- Prospective studies demonstrating that tHcy within the normal range is related to myocardial infarction (Arnesen et al. 1995) and to stroke (Perry et al. 1995).

• 1994- Demonstrating that children of vegan mothers had elevated MMA and tHcy levels consistent with a functional vitamin B12 deficiency, predisposing to neurological damage in infancy and even in adolescence (Schneede et al. 1994; van Dusseldorp et al. 1999; Louwman et al. 2000).

• 1995- Demonstrating that several life-style factors such as smoking, coffee drinking, intake of fruits and vegetables and physical activity are among the important determinants of tHcy (the Hordaland Homocysteine Study) (Nygård et al. 1995).

• 1996- Showing that markedly elevated tHcy (>40 µM) in the general population is usually due to a combination of a genetic disposition (MTHFR 677C->T mutation) and life-style factors predisposing to high tHcy levels. The majority of these individuals obtained a normal tHcy level by increasing their folate intake (the Hordaland Homocysteine Study) (Guttormsen 1996).

• 1996- tHcy in children was found to be associated with cardiovascular mortality of their male relatives, suggesting a strong genetic predisposition of high tHcy levels (Tonstad et al. 1996; Tonstad et al. 1997).

• 1997- Results from the European concerted action (COMAC) project "Homocysteine and Vascular Disease", demonstrated for the first time that tHcy strongly interacts with established risk factors for vascular disease, especially hypertension and smoking (Graham et al. 1997).

• 1997- tHcy was reported to be a strong predictor of mortality in patients with confirmed coronary artery disease. The mortality risk within 5 years was 25 % in patients with tHcy >20 µM, vs 4 % in patients with tHcy <9 µM. The finding was published in the NEJM (Nygård et al. 1997).

• 1997- Finding that the inborn error homocystinuria is probably much more common (>1/20,000) than previously anticipated (1/300,000). Moreover, in Norway, homocystinuria is most often related to one particular 'Norwegian mutation'. Patients with this mutation respond to treatment with pyridoxine (Kim et al. 1997). This finding motivates new-born screening for homocystinuria in Norway (pilot study starting in 1998/99).

• 1998- The first report on a substantial increase in tHcy in subjects consuming large amount of filtered coffee (Nygård et al. 1997).

• 2000- Reporting that plasma tHcy was associated with pregnancy complications and adverse outcomes in the large Hordaland homocysteine cohort (Vollset et al. 2000).

• 2001-Reporting that Plasma tHcy is a strong predictor of both cardiovascular and noncardiovascular mortality in a general population of 65–72-y-olds (Vollset et al. 2001).

• 2003- Reporting that hyperhomocysteinemia and the MTHFR 677TT genotype are related to depression in 6000 subjects from the Hordaland homocysteine cohort (Bjelland et al. 2003).

• 2004- Demonstration that change in lifestyle has a modest (compared to folate supplementation) but significant effect on tHcy in 7000 subjects over 6 years period (Nurk et al. 2004).

• 2004- The largest study to date (2168 case-control pairs) on the MTHFR 677C->T polymorphism and (colorectal ) cancer, demonstrating reduced risk of 27% and 35%, respectively, of colorectal cancer in subjects with the MTHFR 677 TT and MTR 2756 GG genotypes (Ulvik et al. 2004).

• 2006- Report of the results from the NORVIT (Norwegian Vitamin Trial), a secondary trial in patients with a history of recent myocardial infarction (Bonaa et al. 2006).

• 2007- Report of the results from a randomized clinical trial on folic acid for the prevention of colorectal adenomas (Cole et al. 2007).

• 2007- Metabolite profiling in large-scale epidemiological studies. All B-vitamins (folate, vitamin B12, riboflavin, vitamin B6), a panel of related metabolites / markers and single polymorphisms (SNPs, including MTHFR 677C->T) related to one-carbon metabolism were determined in 10700 healthy subjects enrolled in the NORCCAP study (Ulvik et al. 2007; Holm et al. 2007; Hustad et al. 2007; Midttun et al. 2007; Fredriksen et al. 2007).

In 1999 and 2000 the Research Council of Norway carried out a comprehensive review of Norwegian basic biological research. The report on Homocysteine LOCUS is as follows:

Description: All the research of the department is focused in a Homocysteine LOCUS. A LOCUS is a group identified as having unique resources and clear long-term potential. It is headed by Per Magne Ueland and in addition has four professors - Helga Refsum, Ernst Lien, Grethe Tell and Stein Emil Vollset (the latter two are not at the department) - two associate professors including Einar Solheim, who acts as departmental chairman.

This research has developed from basic research and the development of unique research tools into patient based and epidemiological clinical research. The research is centered around: a) development of precise methods to measure key variables in patient materials; such as laser-induced fluorescence (LIF) with a customs built super-sensitive detector developed. This has developed into a multi-injection capillary electrophoresis coupled to LIF useful for genotypic screening of large patient materials. b) Experimental studies on folates, vitamins B12, B6 and B2. The group has shown that Hcy predicts folate deficiency and that the levels are also controlled by anesthesia and renal function. c) Clinical and epidemiological studies: The highest impact (five papers since 1995 cited more than 100 times) has been of the epidemiological studies. In particular, they have shown that homocysteine is the best predictor of mortality of patients with coronary heart disease, and that it strongly interacts with other established risk factors.

Evaluation: The members of the research group represent complementary competencies that are required to achieve the research goals. One very interesting ongoing study is an examination of folate administration with potential major health implications. In addition, there are ongoing studies on the implications of polymorphisms and markers of vitamin status on colonic cancer. The group has very strong links with several outstanding international groups, partly via different EU projects. The most striking and impressive aspect is the continuous development of cutting edge analytical methodology and the very clear focus on areas where the group can have a decisive competitive edge to maintain the high international standing. Altogether this group is ranked as excellent. It should continue to receive good funding at a high level. In particular, it is suggested that this group, which provides excellent training, is given one or several open graduate student positions.

Objective 1. To validate the technical performance of the holoTC assay (throughput, robustness, precision and variability) outside the manufacturer’s laboratory in different laboratory settings in four different countries reflecting future end-users of the assay and to provide advice to the company on improvements needed for the commercialisation of the product..
Objective 2. To show the diagnostic utility of the holoTC assay compared with the conventional approach (determination of plasma B12 and haematological markers) for the early identification of individuals with impaired vitamin B12 status. The current gold standard for the assessment of B12 status (a combination of plasma B12, MMA and tHcy) will be used as a reference.
Objective 3. To demonstrate the ability of holoTC to identify, at an early stage, the development of vitamin B12 deficiency in relevant target populations, including elderly subjects with depression, or cognitive impairment , as well as other conditions known to be associated with vitamin B12 deficiency.
Objective 4. To demonstrate the socio-economic benefits of the holoTC assay as an early marker of vitamin B12 deficiency in selected populations.
Objective 5. To disseminate the results in order to promote the transfer of knowledge into daily clinical practice and to advance its exploitation by European industry.

Hordaland Homocysteine Study III.
We will also consider to invite the second half of the 1992-93 homocysteine cohort to a second cardiovascular screening in co-operation with the National Health Screening Service.

10.3.4. The Hordaland Homocysteine studies: Novel markers and determinants
Some novel biomarkers related to homocysteine metabolism are probably stable under the storage conditions (- 20 oC) used for the Hordaland Homocysteine biobank. Among these, betaine and related factors (choline, DMG) have recently been identified as important determinants of plasma tHcy. These and other tHcy determinants (neopterine) will be determined in samples from The Hordaland Homocysteine Study II. The associations between these parameters, tHcy, lifestyle factors and the MTHFR 677C->T polymorphism will be investigated.

10.3.5. WENBIT - Western Norway B-vitamin Intervention Trial
This is a randomized 2x2 factorial trial of homocysteine-lowering B-vitamins in 3000 patients undergoing coronary angiography at the Haukeland University Hospital, Bergen, and at Stavanger University Hospital. The four treatment arms are placebo / folic acid (0.8 mg) plus vitamin B12 (0.4 mg) / vitamin B6 (40 mg) / folic acid plus vitamin B12 plus vitamin B6. The major endpoint is long-term outcome (mortality or major cardiovascular events). The study was initiated in year 2000 and randomization was completed early 2004. WENBIT was stopped prematurely in November 2005 due to reduced compliance from patients after the results of the NORVIT trial had been presented in media.
Extensive clinical and lifestyle information as well as biological samples have been collected from all recruited patients, whereas information on dietary habits was obtained from 2400 patients at baseline. These data will constitute a valuable research resource for studies of lifestyle and cardiovascular health long after the trial has been terminated. At Haukeland University Hospital‚s Department of Heart Disease, the biobank also includes data from 2000 cardiac patients not included in WENBIT.
At baseline, about 1300 patients in WENBIT were treated with angioplasty (PCI) and about 650 with bypass surgery. Major substudies include evaluation of restenosis after PCI with repeat angiography and intravascular ultrasound of the treated segment 9 months after the baseline procedure. The angiographic data in these 350 patients will also be used to study the progression of coronary artery disease. Among 40 of these patients, the treatment effects on coronary endothelial function and on atherosclerosis progression (evaluated by intravascular ultrasound) are investigated.

10.4.2. Various form of vitamins B2 and B6 in healthy and hyperhomocysteinemia subjects. An assay has been developed which includes 7 vitamin B6 species (pyridoxal phosphate, pyridoxal, pyridoxic acid, pyridoxine, pyridoxine phosphate, pyridoxamine, and pyridoxamine phosphate) and 3 vitamin B2 species (riboflavin, FMN and FAD). This multiplexing assay is based on LC-MS/MS and will be used for the assessment of B6 and B2 status and and health indicators in the NORCCAP, WENBIT and NORVIT biobanks.

10.4.3. Markers of folate status in archival material. Folate is degraded in stored serum/plasma and erythrocyte extract, even when stored in at – 20 ^oC. Furthermore, there are large inter assay variations between folate methods. We therefore are developing an assay measuring folate equivalents as paraaminobenzoyl-glutamate (PABA-Glu) in serum/plasma after controlled acid hydrolysis. The method is based LC-MS/MS and will also include other folate degradation products.

10.4.4. Alkylchloroformates as general derivatizing reagents in gas chromatography. Alkylchloroformate derivatization allows the simultaneous acylation of amine and thiol groups creating alkoxycarbonyl forms and esterification of carboxylic group. Such a strategy was proposed by Husek in 1995. Simultaneous determination of MMA, tHcy and related metabolites in the same serum or plasma specimen is practical because of the complementarity of these two tests in the diagnostics of cobalamin and folate deficiencies. We are developing methods for such markers, based on derivatization with novel chloroformates (CFs), included halogenated CFs. These analyses are carried out in microtiter plates, using a robotic work station. This project is a collaboration with Drs. Petr Husek (Prague) and Petr Simek (Ceské Budejovice).

10.4.5. Thyroid status. Thyroid status as a strong determinant of plasma homocysteine was first reported by Nedrebø and colleagues in 1998. Several mechanisms have been suggested, including altered renal function or folate status. However, thyroid function affects the metabolism and function of several other B-vitamins involved in homocysteine metabolism, in particular vitamin B2. One projects is investigating change in choline according thyroid function, and the effect modification by genetics and life-style factors.

10.5.2. Cobalamin intervention in newborns. There is high prevalence of elevated MMA and tHcy in newborns. Studies are underway which investigate the effect of cobalamin intervention on cobalamin status in newborns, including premature/SGA infants. Other study outcomes are growth and development.

10.5.3. The Norwegian Mother and Child Cohort. This is a joint project of the Bergen and Oslo Departments of the Norwegian Institute of Public Health and is the largest epidemiologic study ever conducted in Norway. The goal of the study is to gain new knowledge on lifestyle and genetic causes of pregnancy complications and adverse outcomes. By mid-2005, the study has recruited and collected data from more than 50,000 pregnant women and their children and 80 % of fathers. The plan is to reach a size of 100,000 women, 70,000 partners and their children in 2008. A project has started August 2005 to study B-vitamin status in a sample of about 3000 pregnant women in the Mother and Child cohort. This is a PhD project funded by the Norwegian Research Council.

10.5.4. Norwegian Orofacial Cleft Study. This data set consists of 574 Norwegian case-parent triads (child, mother, father) and 763 randomly selected controls recruited from the Medical Birth Registry in the period 1996-2001. With 574 cases and complete triads, this study will address intake of folate and other B-vitamins and the risk of orofacial cleft.

10.6.3. Norwegian Colorectal Cancer Prevention (NORCCAP). This is a screening study using flexible sigmoidoscopy. It includes extensive clinical data, B-vitamin/homocysteine status and genetic data on > 10000 participants. All key metabolites involved in the B-vitamin network (homocysteine, vitamins B2, B6, B12, betaine, choline, methionine and others) have been measured together with the 13 related SNPs. These data are a rich and unique source for studying the interplay between normal genetic variation and levels of B-vitamin and related metabolites.

10.5.4. The Hordaland Homocysteine Studies. See 10.2.4.