Thiamine status in humans and content of phosphorylated thiamine derivatives in biopsies and cultured cells

Abstract

Background: Thiamine (vitamin B1) is an essential molecule for all life forms because thiamine diphosphate (ThDP) is an indispensable cofactor for oxidative energy metabolism. The less abundant thiamine monophosphate (ThMP), thiamine triphosphate (ThTP) and adenosine thiamine triphosphate (AThTP), present in many organisms, may have still unidentified physiological functions. Diseases linked to thiamine deficiency (polyneuritis, Wernicke-Korsakoff syndrome) remain frequent among alcohol abusers and other risk populations. This is the first comprehensive study on the distribution of thiamine derivatives in human biopsies, body fluids and cell lines.

Methodology and principal findings: Thiamine derivatives were determined by HPLC. In human tissues, the total thiamine content is lower than in other animal species. ThDP is the major thiamine compound and tissue levels decrease at high age. In semen, ThDP content correlates with the concentration of spermatozoa but not with their motility. The proportion of ThTP is higher in humans than in rodents, probably because of a lower 25-kDa ThTPase activity. The expression and activity of this enzyme seems to correlate with the degree of cell differentiation. ThTP was present in nearly all brain and muscle samples and in ∼60% of other tissue samples, in particular fetal tissue and cultured cells. A low ([ThTP]+[ThMP])/([Thiamine]+[ThMP]) ratio was found in cardiovascular tissues of patients with cardiac insufficiency. AThTP was detected only sporadically in adult tissues but was found more consistently in fetal tissues and cell lines.

Conclusions and significance: The high sensitivity of humans to thiamine deficiency is probably linked to low circulating thiamine concentrations and low ThDP tissue contents. ThTP levels are relatively high in many human tissues, as a result of low expression of the 25-kDa ThTPase. Another novel finding is the presence of ThTP and AThTP in poorly differentiated fast-growing cells, suggesting a hitherto unsuspected link between these compounds and cell division or differentiation.

Figure 1. Interconversion of thiamine derivatives in a model human cell.

1, cytosolic thiamine diphosphokinase (TPK); 2, cytosolic thiamine diphosphatase (also hydrolyzes nucleoside diphosphates); 3, thiamine monophosphatase (hypothetical); 4, mitochondrial membrane-associated ThTP synthase; 5, cytosolic adenylate kinase; 6, cytosolic 25-kDa ThTPase; 7, cytosolic ThDP adenylyl transferase; 8, AThTP hydrolase (hypothetical). The mechanisms of AThDP synthesis and degradation are unknown. Δp, transmembrane H⁺ gradient. Updated from .

Figure 2. 25-kDa ThTPase activities in the brain and cultured cell lines from humans and mice.

(A) ThTPase specific activities measured at pH 8.5, at 37°C and at a Mg²⁺ concentration of 10 mM (means ± SD, n = 3) in mouse and human brain and cell lines. (B) pH profile of ThTPase activity from various sources (means ± SD, n = 2). The buffers (50 mM final concentration) used were: Pipes (pH 6.5), Mops (pH 7.1), Hepes (pH 7.5), Taps (pH 8.0), Bis-Tris-Propane (pH 8.5) and Ches (pH 9.1 and 9.5).

Figure 3. Amplification of 25-kDa ThTPase mRNA in different cell lines by RT-PCR.

(SL, standard ladder; S, SN-K-BE; C, C2C12; L, LN-18).

Figure 4. Western blots of human tissues and cultured cell lines using a commercial mouse polyclonal antibody.

The antibody was raised against human recombinant 25-kDa hThTPase. The samples were homogenized and centrifuged (20 000× g, 30 min). In the case of the biological samples, 40 µg of cytosolic protein were loaded on the gel; for hThTPase, the amount was 0.5 µg. The arrow indicates the location of 25-kDa hThTPase. (Ex, ectocervix; En, endometrium; hT, human recombinant ThTPase; L, human LN-18 glioblastoma cells; S, human SK-N-BE neuroblastoma cells; U, uterus; Tr, trophoblast).