The analytical repertoire of BEVITAL is composed of methods based on cutting edge analytical technology. Most assays are developed by scientists and technicians at BEVITAL with more than 20 years experience in gass-liquid chromatography and/or mass spectrometry. One dedicated scientist is responsible for each platform, its upgrade, performance and ruggedness. Such long-term experience enables the prevention of common mistakes, including unrecognized assay interference and matrix effects (ion-suppression).
Loss of analytical precision with non-matching internal standards
BEVITAL employs a strategy of using a dedicated (matching) internal standard for most analytes (only a few exceptions apply). Recently, we quantitated the consequences of substituting matching with non-matching internal standards on analytical precision. The use of non-matching internal standards, as used by some laboratories, may result in lower precision leading to wider confidence intervals in statistical analyses, so that biological important associations may escape detection.
Extensive upgrade of platforms
Metabolites have been added to platforms A (TCA metabolites and intermediates, short-chain fatty acids (SCFAs)), platform B (beta-alanine), platform C (short-chain carnitines), platform D (imidazole propionate and microbiota-derived indoles) and platform H (long-chain carnitines).
A novel functional marker of vitamin B6 status
By combining five metabolites in the kynurenine pathway into a simple index, HKr, a sensitive and specific indicator of intracellular vitamin B6 status is obtained. The index also underscores the merit of evaluating alterations in kynurenine metabolism when investigating vitamin B6 and health.
Inflammation markers, cystatin C and its proteoforms in 20 µL of sample
A new method (platform G) for the combined quantification of C-reactive protein (CRP), serum amyloid A (SAA), and calprotectin (S100A8/9) and the kidney function marker, cystatin C (CysC), and a number of proteoforms (11 for SAA, 4 for S100A8/9 and 4 for CysC) has been developed at BEVITAL. The method is characterized by low sample consumption, high capacity, and high precision, and has been designed for the assessment of biomarker status in precious samples from large biobanks.
Saving biobank material by combining platforms
A procedure involving combined sample workup for platforms B and H enabling quantification of 6 lipid-soluble and 26 water soluble biomarkers in 50 µL serum/plasma has been established. The method utilizes liquid-liquid extraction of the lipid soluble vitamins and further processing of the aqueous phase through the steps established for platform B.
Two review articles on vitamin B6 biomarkers
A review on vitamin B6 biomarkers has been published in Annual Review of Nutrition. It deals with both direct vitamin B6 biomarkers that measure B6 vitamers in plasma, serum and urine and functional biomarkers reflecting enzymatic or metabolic functions of vitamin B6, including novel markers like the PAr index and the HK:XA ratio. Another review on inflammation, vitamin B6 and related pathways has recently been published in Molecular Aspects of Medicine. Both articles are co-authored by scientists at BEVITAL.
Recovery of degraded biomarkers
The combined measurements of the degradation product and parent compound may correct for moderate biomarker decomposition. Such recovery assays have been validated and published for the conversion pyridoxal 5′-phosphate (PLP) to pyridoxal (PL), flavin mononucleotide (FMN) to riboflavin, for the oxidation of methionine to methionine sulfoxide and for the oxidation of 5-methyltetrahydrofolate to 4-alfa-hydroxy-5-methyltetrahydrofolate (hmTHF). In samples where folate has been extensively degraded beyond hmTHF, folate can be measured as p-aminobenzoylglutamate (pABG) equivalents by an assay involving oxidation and mild acid hydrolysis.
Vitamin and biomarker status across countries and continents
Biomarkers that are related to vitamin status, nutrition, inflammation and lifestyle have been measured in subjects from 20 cohorts recruited from the United States, Nordic countries, Asia, and Australia, participating in the Lung Cancer Cohort Consortium. The biochemical analyses have been carried out in a centralized laboratory, i.e. BEVITAL.
Analysis of metabolites and biomarkers in extract from cells or tissues
The methods used by BEVITAL have been developed and validated for analyses of metabolites in plasma, serum and/or urine. Occasionally, customers request analysis in cell or tissue extracts. In such cases, the method has to be validated and optimized for the actual extract and extraction procedure, to secure adequate performance of the method. Such validation and optimization include the following steps and considerations:
Initially, the customer must provide representative extracts. This will be used to determine the approximate concentration of metabolite. Such information is required for method validation (steps 2 to 3).
Preferably, the extraction should be carried out using the same procedure and precipitating agent as used for the method optimized for serum/plasma. Extraction could be carried out by the customer according to agreed procedure. Alternatively, extraction of frozen cell pellet or frozen tissue specimen could be carried out by BEVITAL. In both cases, the extraction solution (trichloric acid or perchloric acid) should be supplemented with deuterated internals standard for each metabolite, the concentration of which must be similar to the concentration of each metabolite, as obtained in step 1.
The stability of the metabolite prior to extraction is the responsibility of customer, but if rapid changes in concentration are expected, BEVITAL recommends freeze clamping of tissue or treatment cell pellet with liquid nitrogen. Metabolite stability after protein precipitation in extraction solution is expected to be similar for serum/plasma, cells and tissues.
Interference from endogenous compounds may differ between different sample matrices. Interference will be considered by inspection of elution profile, mass fragmentation pattern and comparisons of different ion pairs.
Assessment of linearity is obtained by serial dilution of the extract up to about 30-fold dilution or until Lower Limit of Detection (LOD). In addition, a standard addition procedure should be carried out by adding unlabelled metabolite to obtain a final metabolite concentration of 2 to about 30 times the endogenous levels. An indication of linearity will be obtained by plotting the ratio of ion intensity of metabolite and deuterated internal standard versus dilution for both experiments.
A measure of analytical recovery will be obtained by comparing metabolite concentrations from the standard addition experiment with concentrations detected by adding the same amounts to the extraction solution, assuming 100 % recovery in the absence of cell/tissue components. Comparison of the absolute ion intensities of metabolites with and without cell/tissue extraction, will give a measure of ion suppression.
A 3 points calibration curve covering expected metabolite concentrations will be set up either by adding metabolite to the extraction solution, or, in case of substantial ion suppression (> 50 %) or low analytical recovery (< 70 %) to a cell/tissue extract (standard addition method).
If requested by the customer, assessment of within-day and between-day precision of the method will be done.
The cost for validation and optimization may be difficult to predict, must be covered by customer, and is calculated from man-months required to accomplish tasks 1 to 8. The current rate is 12000 EURO per man-month.
BEVITAL is partner in numerous projects
These include two EU projects, “Metabolomic profiles throughout the continuum of colorectal carcinogenesis” (MetaboCCC) and ”Biomarkers related to folate-dependent one-carbon metabolism in CRC recurrence and survival” (FOCUS), and one large multicenter study on lung cancer, organized within the frame of the lung cancer consortium (LC3), funded by US NCI. The latter study includes 11000 cases and controls recruited from 20 centers, in Europe, US, Asia and Australia.