Quantitation of folates and their catabolites in blood plasma, erythrocytes, and urine by stable isotope dilution assays

Mönch, Sabine; Netzel, M.; Netzel, Gabriele; Rychlik, Michael

doi:10.1016/j.ab.2009.11.007

Cited by 36 publications

(67 citation statements)

References 26 publications

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“…isotopologues, the compliance with the former two prerequisites has been confirmed 14 in previous method validation studies [6,13]. However, the latter prerequisite of 15 absent spectral overlaps had to be verified for the [ …”

Section: Data Analysis 1mentioning

confidence: 60%

“…When comparing fractionated elution and elution with 1.5 mL, the 17 latter was the reasonable compromise in terms of sensitivity and work load. The 18 ratios of unlabelled or [ 13 C 5 ]-labelled folates to deuterated standards, however, did 19 not differ between all elution patterns indicating that no discrimination between the 20 isotopologues occurs as had been reported during affinity chromatography earlier [6]. (Table 4).…”

Section: Plasma Analysis 11mentioning

confidence: 70%

“…Stable isotope 13 dilution assays (SIDA) using stable isotope-labelled folates and LC-MS/MS detection 14 have proven their superiority over conventional methods in analysis of clinical and 15 food samples [6]. For dietary recommendations, knowledge on food folate content is 16 as essential as on folate bioavailability.…”

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confidence: 99%

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Quantification of isotope-labelled and unlabelled folates in plasma, ileostomy and food samples

et al. 2010

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New stable isotope dilution assays were developed for the simultaneous quantitation 2 of [ 13 C 5 ]-labelled and unlabelled 5-methyltetrahydrofolic acid, 5-formyltetrahydrofolic 3 acid, folic acid along with unlabelled tetrahydrofolic acid and 10-formylfolic acid in 4 clinical samples deriving from human bioavailability studies, plasma, ileostomy 5 samples and food. The methods were based on clean up by strong anion exchange 6 followed by LC-MS/MS detection. Deuterated analogues of the folates were applied 7 as the internal standards in the stable isotope dilution assays. Assay sensitivity was 8 sufficient to detect all relevant folates in the respective samples as their limits of 9 detection were below 0.62 nmol/L in plasma and below 0.73 µg/100g in food or 10 ileostomy samples. 11

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Section: Data Analysis 1mentioning

confidence: 60%

Section: Plasma Analysis 11mentioning

confidence: 70%

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Quantification of isotope-labelled and unlabelled folates in plasma, ileostomy and food samples

et al. 2010

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“…This modification makes it possible to reanalyze a sample from a specimen volume ≥ 500-μL in case of a quality control failure or the need to confirm a low or high concentration. The 150-μL test volume is lower compared to most published multi-analyte folate LC-MS/MS methods, which require anywhere from 200 μL to 2 mL of serum or plasma [15][16][17][18][19]. The method by Hannisdal et al requires only 60 μL of serum [20], however using such a low specimen volume may come at the cost of not being able to detect minor folate forms because of inadequate analytical sensitivity.…”

Section: Resultsmentioning

confidence: 86%

A high-throughput LC-MS/MS method suitable for population biomonitoring measures five serum folate vitamers and one oxidation product

et al. 2013

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Small specimen volume and high sample throughput are key features needed for routine methods used for population biomonitoring. We modified our routine 8-probe solid phase extraction (SPE) LC-MS/MS method for the measurement of five folate vitamers (5-methyltetrahydrofolate , folic acid [FA], plus three minor forms: THF, 5-formylTHF, 5,10-methenylTHF) and one oxidation product of 5-methylTHF (MeFox) to require less serum volume (150 μL instead of 275 μL) by using 96-well SPE plates with 50-mg instead of 100-mg phenyl sorbent and to provide faster throughput by using a 96-probe SPE system. Total imprecision (10 days, 2 replicates/day) for three serum quality control (QC) pools was 2.8-3.6% for 5-methylTHF (19.5-51.1 nmol/L), 6.6-8.7% for FA (0.72-11.4 nmol/L), and ≤11.4% for the minor folate forms (<1-5 nmol/L). Mean (±SE) recovery of folates spiked into serum (3 days, 4 levels, 2 replicates/level) was: 5-methylTHF, 99.4±3.6%; FA, 100±1.8%; minor folates, 91.7-108%); SPE extraction efficiencies were ≥85% except for THF (78%). Limits of detection were ≤0.3 nmol/L. The new method correlated well with our routine method (n=150; r=0.99 for 5-methylTHF, FA, and total folate [tFOL, sum of folate forms]) and produced slightly higher tFOL (5.6%) and 5-methylTHF (7.3%) concentrations, likely due to the faster 96-probe SPE process (1 vs. 5 h) resulting in improved SPE efficiency and recovery compared to the 8-probe SPE method. With this improved LC-MS/MS method, 96 samples can be processed in ~2 h and all relevant folate forms can be accurately measured using a small serum volume.

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“…Tissue Levels of 5-mTHF, Hcy and Methionine in C57BL/6J Mice 5-mTHF is the predominant folate species in both humans and rodents [131][132][133]. It is utilized in the conversion of Hcy to methionine, which is a precursor for the synthesis of SAM.…”

Section: Nd-not Detected Pabae2-e5 = Polyglutamated Pabgmentioning

confidence: 99%

Human arylamine n-acetyltransferase 1: pursuit of an endogenous role

Witham¹

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It has been hypothesized that the ubiquitously expressed Phase II drug metabolizing enzyme human arylamine N-acetyltransferase 1 (NAT1) has a role in cell biology other than the metabolism of xenobiotics. The identification of p-aminobenzoylglutamate (pABG) as an endogenous substrate for NAT1 led to speculation that the enzyme might be involved in folate regulation. NAT1 is overexpressed in human luminal breast cancers and is a proposed biomarker for estrogen receptor positive breast cancers in women as well as luminal M2 breast cancers in men. Moreover, NAT1 has been shown to support human cancer cell growth, survival and invasion both in vitro and in vivo. Overall, the aim of this work was to identify possible endogenous roles for NAT1 that might explain its wide tissue expression.Initially, mouse Nat2 (homologue to human NAT1) was considered, and the levels of pABG and 5-methyltetrahydrofolate, the predominant circulating folate, were measured in wild-type and Nat2-deleted (Nat2-/-) mice. Both metabolites were unaltered in Nat2-/-mice suggesting the enzyme did not regulate either of these folate products in vivo under normal conditions. Therefore, the effect of human NAT1 on pABG and 5-methyltetrahydrofolate in the human colon adenocarcinoma cell line, HT-29, was investigated to determine if NAT1 regulated folate in cancer cells. As with the mouse study, pABG and 5-methyltetrahydrofolate were unaltered when NAT1 was knocked down in these cells. Overall, these results suggests that NAT1 is not essential for the regulation of 5-methyltetrahydrofolate nor is the acetylation of pABG physiologically relevant in these models.Metabolomic analysis following human NAT1 inhibition was also carried out. Firstly, changes in the S-adenosylmethionine (SAM) cycle was also investigated because 5-methyltetrahydrofolate provides the methyl group to the SAM cycle necessary for methylation reactions of DNA, proteins and lipids. Secondly, differences in amino acids present in cell culture medium were investigated following human NAT1 knocked down.SAM cycle homeostasis was maintained in HT-29 cells independent of human NAT1 activity. Furthermore, there were very few changes in SAM cycle components in Nat2-/-tissues compared to wild-type. This suggests that NAT1 was not involved in regulating methylation reactions. However, changes in alanine and homocysteine concentrations in cell culture medium between control and human NAT1 knock-down cells were identified. 3Homocysteine levels in the medium initially increased with time for both control and knockdown cells. However, by day 3 in culture, levels began to decrease in the knocked-down cells whereas they continued to increase in the controls. This correlated with the depletion of methionine from the medium suggesting the knocked down cells required homocysteine whereas the control cells did not. The methionine salvage pathway is a multi-enzyme multi-reaction process that recycles methylthioadenosine (MTA), a by-product of polyamine synthesis, back to methionine. This pathway...

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Quantitation of folates and their catabolites in blood plasma, erythrocytes, and urine by stable isotope dilution assays

Cited by 36 publications

References 26 publications

Quantification of isotope-labelled and unlabelled folates in plasma, ileostomy and food samples

Quantification of isotope-labelled and unlabelled folates in plasma, ileostomy and food samples

A high-throughput LC-MS/MS method suitable for population biomonitoring measures five serum folate vitamers and one oxidation product

Human arylamine n-acetyltransferase 1: pursuit of an endogenous role

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