Pre-analytical challenges from adsorptive losses associated with thiamine analysis.

Thiamine (vitamin B1) is an essential vitamin serving in its diphosphate form as a cofactor for enzymes in the citric acid cycle and pentose-phosphate pathways. Its concentration reported in the pM and nM range in environmental and clinical analyses prompted our consideration of the components used in pre-analytical processing, including the selection of filters, filter apparatuses, and sample vials. The seemingly innocuous use of glass fiber filters, glass filter flasks, and glass vials, ubiquitous in laboratory analysis of clinical and environmental samples, led to marked thiamine losses. 19.3 nM thiamine was recovered from a 100 nM standard following storage in glass autosampler vials and only 1 nM of thiamine was obtained in the filtrate of a 100 nM thiamine stock passed through a borosilicate glass fiber filter. We further observed a significant shift towards phosphorylated derivatives of thiamine when an equimolar mixture of thiamine, thiamine monophosphate, and thiamine diphosphate was stored in glass (most notably non-silanized glass, where a reduction of 54% of the thiamine peak area was observed) versus polypropylene autosampler vials. The selective losses of thiamine could lead to errors in interpreting the distribution of phosphorylated species in samples. Further, some loss of phosphorylated thiamine derivatives selectively to amber glass vials was observed relative to other glass vials. Our results suggest the use of polymeric filters (including nylon and cellulose acetate) and storage container materials (including polycarbonate and polypropylene) for thiamine handling. Losses to cellulose nitrate and polyethersulfone filters were far less substantial than to glass fiber filters, but were still notable given the low concentrations expected in samples. Thiamine losses were negated when thiamine was stored diluted in trichloroacetic acid or as thiochrome formed in situ, both of which are common practices, but not ubiquitous, in thiamine sample preparation.

Understanding and mitigating thiaminase activity in silver carp.

A deficiency of thiamine (vitamin B1), an essential cofactor for enzymes involved in metabolic processes, can be caused by the enzyme thiaminase. Thiaminase in food stocks has been linked to morbidity and mortality due to thiamine depletion in many ecologically and economically important species. Thiaminase activity has been detected in certain bacteria, plants, and fish species, including carp. The invasive silver carp (Hypophthalmichthys molitrix) presents an enormous burden to ecosystems throughout the Mississippi River watershed. Its large biomass and nutritional content offer an attractive possibility as a food source for humans, wild animals, or pets. Additionally, harvesting this fish could alleviate some of the effects of this species on waterways. However, the presence of thiaminase would detract from its value for dietary consumption. Here we confirm the presence of thiaminase in several tissues from silver carp, most notably the viscera, and systematically examine the effects of microwaving, baking, dehydrating, and freeze-drying on thiaminase activity. Certain temperatures and durations of baking and microwaving reduced thiaminase activity to undetectable levels. However, caution should be taken when carp tissue is concentrated by processes without sufficient heat treatment, such as freeze-drying or dehydration, which results in concentration, but not inactivation of the enzyme. The effects of such treatments on the ease of extracting proteins, including thiaminase, and the impact on data interpretation using the 4-nitrothiophenol (4-NTP) thiaminase assay were considered.

Dietary factors potentially impacting thiaminase I-mediated thiamine deficiency.

Fish population declines from thiamine (vitamin B1) deficiency have been widespread in ecologically and economically valuable organisms, ranging from the Great Lakes to the Baltic Sea and, most recently, the California coast. Thiamine deficiencies in predatory fishes are often attributed to a diet of prey fishes with high levels of thiamine-degrading (e.g., thiaminase) enzymes, such as alewives, rainbow smelt, and anchovies. Since their discovery, thiaminase I enzymes have been recognized for breaking down thiamine into its pyrimidine and thiazole moieties using various nucleophilic co-substrates to afford cleavage, but these studies have not thoroughly considered other factors that could modify enzyme activity. We found the thiaminase I enzyme from Clostridium botulinum efficiently degrades thiamine in the presence of pyridoxine (vitamin B6) as a co-substrate but has relatively limited activity in the presence of nicotinic acid (vitamin B3). Using fluorescence measurements, thiamine degradation in an over-the-counter complete multivitamin formulation was inhibited, and a B-complex formulation required co-substrate supplementation for maximal thiamine depletion. These studies prompted the evaluation of specific constituents contributing to thiaminase I inhibition by both chromatography and fluorescence assays: Cu2+ potently and irreversibly inhibited thiamine degradation; ascorbic acid was a strong but reversible inhibitor; Fe2+, Mn2+ and Fe3+ modulated thiamine degradation to a lesser degree. The enhancement by pyridoxine and inhibition by Cu2+ extended to thiaminase-mediated degradation from Burkholderia thailandensis, Paenibacillus thiaminolyticus, and Paenibacillus apiarius in tryptic soy broth supernatants. These co-substrate limitations and the common presence of inhibitory dietary factors complement recent studies reporting that the intended function of thiaminase enzymes is to recycle thiamine breakdown products for thiamine synthesis, not thiamine degradation.