A novel panel of yeast assays for the assessment of thiamin and its biosynthetic intermediates in plant tissues.

Thiamin (or thiamine), known as vitamin B1, represents an indispensable component of human diets, being pivotal in energy metabolism. Thiamin research depends on adequate vitamin quantification in plant tissues. A recently developed quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is able to assess the level of thiamin, its phosphorylated entities and its biosynthetic intermediates in the model plant Arabidopsis thaliana, as well as in rice. However, their implementation requires expensive equipment and substantial technical expertise. Microbiological assays can be useful in deter-mining metabolite levels in plant material and provide an affordable alternative to MS-based analysis. Here, we evaluate, by comparison to the LC-MS/MS reference method, the potential of a carefully chosen panel of yeast assays to estimate levels of total vitamin B1, as well as its biosynthetic intermediates pyrimidine and thiazole in Arabidopsis samples. The examined panel of Saccharomyces cerevisiae mutants was, when implemented in microbiological assays, capable of correctly assigning a series of wild-type and thiamin biofortified Arabidopsis plant samples. The assays provide a readily applicable method allowing rapid screening of vitamin B1 (and its biosynthetic intermediates) content in plant material, which is particularly useful in metabolic engineering approaches and in germplasm screening across or within species.

Patient-Centric Assessment of Thiamine Status in Dried Blood Volumetric Absorptive Microsamples Using LC-MS/MS Analysis.

Many populations in low- and middle income countries are at a higher risk of thiamine deficiency, mainly due to the lack of dietary diversification and their reliance on staple crops low in thiamine content, such as polished rice. Unfortunately, symptoms of thiamine deficiency are variable and clinical determination of thiamine status is essential for early diagnosis. Currently, the diagnosis of thiamine deficiency in remote regions is hampered due to several drawbacks related to venous blood collection, for example, cold chain transport. Therefore, we here describe the first liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of thiamine diphosphate (TDP) in dried blood, using volumetric absorptive microsampling (VAMS). Moreover, by setting up an additional method in liquid blood, the results in VAMS samples could be compared to liquid blood samples. Both methods, employing a simple one-step extraction and fast (2 min) chromatography, were fully validated based on international guidelines. Accuracy (% bias) was below 6.5% for all QC levels. The total imprecision (% CV) was below 13% for both QCs and native blood samples. The recovery of the VAMS samples was not impacted by the hematocrit, within the hematocrit range of 0.20-0.60. Additionally, we showed improved TDP stability in dried blood compared to liquid blood. VAMS samples were stable for 1 week at 60 °C or at high humidity (80%) and for at least 1 month at room temperature. Finally, we demonstrated the commutability of commercial calibrators with authentic blood samples. The validity and applicability of both methods were demonstrated via their successful application on blood samples from healthy volunteers.