Erythrocyte transketolase activity coefficient (ETKAC) assay protocol for the assessment of thiamine status.

Vitamin B1 (thiamine) is an essential nutrient that acts as a cofactor for a number of metabolic processes, particularly in energy metabolism. Symptoms of classic thiamine deficiency are recognized as beriberi, although clinical symptoms are nonspecific and recognition of subclinical deficiency is difficult. Therefore, reliable biomarkers of thiamine status are required. Thiamine diphosphate is a cofactor for transketolase, including erythrocyte transketolase (ETK). The ETK activity assay as an indirect, functional marker of thiamine status has been used for over 50 years. The ETK activity assay provides a sensitive and specific biomarker of thiamine status; however, there is a lack of consensus over the cutoffs for deficiency, partly due to a lack of assay harmonization. Here, we provide a step-by-step protocol for the measurement of ETK activity and the calculation of the ETK activity coefficient, including detailed explanations of equipment and chemicals required and guidance for quality control procedures. Harmonization of the protocol will provide the basis for the development of internationally recognized cutoffs for thiamine insufficiency. The establishment of quality control materials and a quality assurance scheme are recommended to provide reliability. This will ensure that the ETK activity assay remains an important method for the assessment of thiamine status.

Untargeted metabolomics as an unbiased approach to the diagnosis of inborn errors of metabolism of the non-oxidative branch of the pentose phosphate pathway.

Inborn errors of metabolism (IEM) involving the non-oxidative pentose phosphate pathway (PPP) include the two relatively rare conditions, transketolase deficiency and transaldolase deficiency, both of which can be difficult to diagnosis given their non-specific clinical presentations. Current biochemical testing approaches require an index of suspicion to consider targeted urine polyol testing. To determine whether a broad-spectrum biochemical test could accurately identify a specific metabolic pattern defining IEMs of the non-oxidative PPP, we employed the use of clinical metabolomic profiling as an unbiased novel approach to diagnosis. Subjects with molecularly confirmed IEMs of the PPP were included in this study. Targeted quantitative analysis of polyols in urine and plasma samples was accomplished with chromatography and mass spectrometry. Semi-quantitative unbiased metabolomic analysis of urine and plasma samples was achieved by assessing small molecules via liquid chromatography and high-resolution mass spectrometry. Results from untargeted and targeted analyses were then compared and analyzed for diagnostic acuity. Two siblings with transketolase (TKT) deficiency and three unrelated individuals with transaldolase (TALDO) deficiency were identified for inclusion in the study. For both IEMs, targeted polyol testing and untargeted metabolomic testing on urine and/or plasma samples identified typical perturbations of the respective disorder. Additionally, untargeted metabolomic testing revealed elevations in other PPP metabolites not typically measured with targeted polyol testing, including ribonate, ribose, and erythronate for TKT deficiency and ribonate, erythronate, and sedoheptulose 7-phosphate in TALDO deficiency. Non-PPP alternations were also noted involving tryptophan, purine, and pyrimidine metabolism for both TKT and TALDO deficient patients. Targeted polyol testing and untargeted metabolomic testing methods were both able to identify specific biochemical patterns indicative of TKT and TALDO deficiency in both plasma and urine samples. In addition, untargeted metabolomics was able to identify novel biomarkers, thereby expanding the current knowledge of both conditions and providing further insight into potential underlying pathophysiological mechanisms. Furthermore, untargeted metabolomic testing offers the advantage of having a single effective biochemical screening test for identification of rare IEMs, like TKT and TALDO deficiencies, that may otherwise go undiagnosed due to their generally non-specific clinical presentations.

Biomarkers Apo10 and TKTL1: Epitope-detection in monocytes (EDIM) as a new diagnostic approach for cholangiocellular, pancreatic and colorectal carcinoma.

OBJECTIVE: The EDIM (Epitope detection in monocytes) blood test is based on two biomarkers Apo10 and TKTL1. Apo10 is responsible for cell proliferation and resistance to apoptosis. TKTL1 plays a major role in anaerobic glycolysis of tumor cells, leading to destruction of the basal membrane and metastasis as well as in controlling cell cycle. For the first time we analyzed Apo10 and TKLT1 in patients with cholangiocellular (CCC), pancreatic (PC), and colorectal carcinoma (CRC). METHODS: Blood samples of 62 patients with CCC, PC, and CRC were measured and compared to 29 control patients. We also investigated 13 patients with inflammatory conditions, because elevated TKTL1 and Apo10 have been previously described in affected individuals. Flow cytometry was used to detect surface antigens CD14+/CD16+ (activated monocytes/macrophages). Percentages of macrophages harboring TKTL1 and Apo10 were determined. A combined EDIM score (EDIM-CS: TKTL1 plus Apo10) was calculated. Results were correlated with serum tumor markers CEA and CA19-9. RESULTS: Patients with CCC had 100% positive EDIM-CS but CEA and CA19-9 were positive in only 22.2% and 70%, respectively. Patients with PC had 100% positive EDIM-CS but positive tumor markers in only 37.5% (CEA) and 72.7% (CA19-9). Patients with CRC had 100% positive EDIM-CS but only 50% positive CEA. EDIM-CS was positive in 100% (62/62) of all cancer patients and in 0% of healthy individuals. Of the individuals with inflammation, 7.7% had a positive EDIM-CS. CONCLUSION: The sensitivity of the EDIM blood test and the comparison with traditional tumor markers indicate that this new test might improve the detection of carcinomas (CCC, PC and, CRC) and might be relevant for the diagnosis of all tumor entities.

B Vitamins Can Reduce Body Weight Gain by Increasing Metabolism-related Enzyme Activities in Rats Fed on a High-Fat Diet.

B vitamins are enzyme cofactors that play an important role in energy metabolism. The aim of this study was to elucidate whether B vitamin administration can reduce body weight (BW) gain by improving energy metabolism-related enzyme activities in rats fed on a highfat diet. Fifty rats were randomly assigned to one of the following five groups: control group (C), including rats fed on standard rat chow; four treatment groups (HO, HI, H2, and H3), in which rats were fed on a high-fat diet. Rats in the HI group were treated daily with 100 mg/kg BW thiamine (VB1), 100 mg/kg BW riboflavin (VB2), and 250 mg/kg BW niacin (VPP); rats in the H2 group were treated daily with 100 mg/kg BW pyridoxine (VB6), 100 mg/kg BW cobalamin (VB12), and 5 mg/kg BW folate (FA); and rats in the H3 group were treated daily with all of the B vitamins administered to the HI and H2 groups. After 12 weeks, the BW gains from the initial value were 154.5±58.4 g and 159.1±53.0 g in the HI and C groups, respectively, which were significantly less than the changes in the HO group (285.2±14.8 g, P<0.05). In the HO group, the plasma total cholesterol (CHO) and triglyceride (TG) levels were 1.59±0.30 mmol/L and 1,55±0.40 mmol/L, respectively, which were significantly greater than those in the HI group (1.19±0.18 mmol/L and 0.76±0.34 mmol/L, respectively, P<0.05). The activities of transketolase (TK), glutathione reductase, and Na+/K+ adenosine triphosphatase were significantly increased in the B vitamin-treated groups and were significantly greater than those in the HO group (P<0.05). Furthermore, the glucose-6-phosphate dehydrogenase, pyruvic acid kinase, and succinate dehydrogenase activities also were increased after treatment with B vitamins. Supplementation with B vitamins could effectively reduce BW gain and plasma levels of lipids by improving energy metabolism-related enzyme activities in rats, thus possibly providing potential benefits to humans.

Dysfunctional protection against advanced glycation due to thiamine metabolism abnormalities in gestational diabetes.

While the pathogenic role of dicarbonyl stress and accelerated formation of advanced glycation end products (AGEs) to glucose intolerance and to the development of diabetic complications is well established, little is known about these processes in gestational diabetes mellitus (GDM), a condition pathogenically quite similar to type 2 diabetes. The aims of the present study were (i) to determine plasma thiamine and erythrocyte thiamine diphosphate (TDP) and transketolase (TKT) activity in pregnant women with and without GDM, (ii) to assess relationships between thiamine metabolism parameters and selected clinical, biochemical and anthropometric characteristics and, finally, (iii) to analyse relationship between variability in the genes involved in the regulation of transmembrane thiamine transport (i.e. SLC19A2 and SLC19A3) and relevant parameters of thiamine metabolism. We found significantly lower plasma BMI adjusted thiamine in women with GDM (P = 0.002, Mann-Whitney) while levels of erythrocyte TDP (an active TKT cofactor) in mid-trimester were significantly higher in GDM compared to controls (P = 0.04, Mann-Whitney). However, mid-gestational TKT activity - reflecting pentose phosphate pathway activity - did not differ between the two groups (P > 0.05, Mann-Whitney). Furthermore, we ascertained significant associations of postpartum TKT activity with SNPs SLC19A2 rs6656822 and SLC19A3 rs7567984 (P = 0.03 and P = 0.007, resp., Kruskal-Wallis). Our findings of increased thiamine delivery to the cells without concomitant increase of TKT activity in women with GDM therefore indicate possible pathogenic role of thiamine mishandling in GDM. Further studies are needed to determine its contribution to maternal and/or neonatal morbidity.

Evaluation of a biomarker based blood test for monitoring surgical resection of oral squamous cell carcinomas.

INTRODUCTION: The potential use of determination of biomarkers in blood for the monitoring of surgical removal of oral squamous cell carcinomas (OSCC) was evaluated using the epitope detection in monocytes (EDIM) technology. MATERIALS AND METHODS: In tumor specimen, elevated Apo10 and transketolase-like 1 (TKTL1) expression was analyzed by immunohistochemistry. Apo10 and TKTL1 biomarkers have been used prospectively for EDIM blood test in patients with primary and/or recurrent OSCC (n = 92) before surgery and after curative tumor resection (n = 45). RESULTS: There were highly significant (p < 0.0001) correlations found between EDIM blood scores and the tissue expression of both biomarkers measured by immunohistochemistry (Apo10: n = 89/92, 97%; TKTL1: n = 90/92, 98%). EDIMApo10 and EDIM-TKTL1 scores were positive in 92% (EDIM-Apo10: n = 85/92) and 93% (EDIM-TKTL1: n = 86/92), respectively, in patients with OSCC before surgery. The combined score EDIM-Apo10/EDIM-TKTL1 increased significantly the detection rate of tumors to 97% (n = 89/92). After surgery, the EDIM-TKTL1 and EDIMApo10 scores significantly decreased in 75.6 and 86.7% of the patients (p < 0.0001), respectively, in the aftercare. CONCLUSIONS: The correlation of TKTL1 and Apo10 immunohistochemistry with the blood test results indicates that the EDIM blood test could serve as a non-invasive diagnostic tool (liquid biopsy) to assess surgical removal of OSCC by determination of two biomarkers. CLINICAL RELEVANCE: This is the first study that has been demonstrated a reliable and successful monitoring of OSCC cancer patients by a blood test. The specific and significant decrease of EDIM-TKTL1 and EDIM-Apo10 scores after surgery could serve as a new tool for monitoring surgical removal of OSCC.

A biomarker based detection and characterization of carcinomas exploiting two fundamental biophysical mechanisms in mammalian cells.

BACKGROUND: Biomarkers allowing the characterization of malignancy and therapy response of oral squamous cell carcinomas (OSCC) or other types of carcinomas are still outstanding. The biochemical suicide molecule endonuclease DNaseX (DNaseI-like 1) has been used to identify the Apo10 protein epitope that marks tumor cells with abnormal apoptosis and proliferation. The transketolase-like protein 1 (TKTL1) represents the enzymatic basis for an anaerobic glucose metabolism even in the presence of oxygen (aerobic glycolysis/Warburg effect), which is concomitant with a more malignant phenotype due to invasive growth/metastasis and resistance to radical and apoptosis inducing therapies. METHODS: Expression of Apo10 and TKTL1 was analysed retrospectively in OSCC specimen (n = 161) by immunohistochemistry. Both markers represent independent markers for poor survival. Furthermore Apo10 and TKTL1 have been used prospectively for epitope detection in monocytes (EDIM)-blood test in patients with OSCC (n = 50), breast cancer (n = 48), prostate cancer (n = 115), and blood donors/controls (n = 74). RESULTS: Positive Apo10 and TKTL1 expression were associated with recurrence of the tumor. Multivariate analysis demonstrated Apo10 and TKTL1 expression as an independent prognostic factor for reduced tumor-specific survival. Apo10+/TKTL1+ subgroup showed the worst disease-free survival rate in OSCC.EDIM-Apo10 and EDIM-TKTL1 blood tests allowed a sensitive and specific detection of patients with OSCC, breast cancer and prostate cancer before surgery and in after care. A combined score of Apo10+/TKTL1+ led to a sensitivity of 95.8% and a specificity of 97.3% for the detection of carcinomas independent of the tumor entity. CONCLUSIONS: The combined detection of two independent fundamental biophysical processes by the two biomarkers Apo10 and TKTL1 allows a sensitive and specific detection of neoplasia in a noninvasive and cost-effective way. Further prospective trials are warranted to validate this new concept for the diagnosis of neoplasia and tumor recurrence.

Erythrocyte transketolase activity in patients with diabetic and alcoholic neuropathies.

INTRODUCTION: The activity of erythrocyte transketolase induced by thiamine pyrophosphate and normalized to age of the patient is a marker of thiamine metabolism disturbances with pathological consequences in the central and peripheral nervous system. The measurement of erythrocyte transketolase activity enables evaluation of the thiamine status and therapeutic decisions in the disorders of the nervous system related to its deficiency. The aim of the study was to compare different modes of expression of transketolase activity in the most frequent acquired neuropathies. MATERIAL AND METHODS: The study included 29 patients with type 2 diabetes mellitus (21 males, 8 females) aged 48.3 years (range: 20-70 years) and 31 subjects with a history of alcohol dependence (23 males, 8 females) aged 54.5 years (range: 28-60 years). All participants of the study showed signs and symptoms of neuropathy. The cases in both groups presented the involvement of either axon, myelin, or both, what was evidenced by electrophysiological tests (electromyography and estimation of nerve conduction velocity). The control group consisted of 20 healthy persons aged 49.1 years (range: 23-70 years). Transketolase activity in erythrocytes (TK) was assessed by means of the spectrophotometric method. Basic TK activity was expressed as units per gram of haemoglobin (g Hb), moreover the normalized transketolase activity ratio (NTKZ) and percentage of activation of thiamine pyrophosphate (TPP) were calculated. RESULTS: The basal TK activity was decreased in cases with diabetic neuropathy (0.64 ± 0.342 U/g Hb, p = 0.0131), whereas in patients with alcoholic neuropathy only a trend (p = 0.058) of the decrease was noticed (0.85 ± 0.484 U/g Hb), in relation to the control group (1.005 ± 0.390 U/g Hb). Normalized transketolase activity ratio in erythrocytes has not shown any statistically significant differences. The median TPP did not indicate any thiamine deficiency, both in the group of diabetic and alcoholic neuropathy. CONCLUSIONS: The decrease in transketolase activity in diabetic neuropathy may be independent of thiamine deficiency. Abnormalities in transketolase activity, when expressed in three modalities: basal activity, normalized transketolase activity ratio and the activity after the stimulation with thiamine pyrophosphate may be differentiated as thiamine-dependent or resulting from posttranslational modification.

EDIM-TKTL1 blood test: a noninvasive method to detect upregulated glucose metabolism in patients with malignancies.

AIM: To determine whether the TKTL1 protein epitope detection in monocytes (EDIM) test allows detection of upregulated glucose metabolism in malignancies. MATERIALS & METHODS: The EDIM-TKTL1 blood test was conducted in 240 patients with 17 different confirmed or suspected malignancies. Test scores were compared with (18)F-fluoro-2-deoxy-D-glucose (FDG)-PET/computed tomography (CT) results. RESULTS: EDIM-TKTL1 score and FDG-PET results showed a concordance of 90% with a sensitivity of 94% and specificity of 81%. Including CT data, all values were enhanced. A subgroup analysis of non-small-cell lung cancer patients showed a significant correlation between the EDIM-TKTL1 score and the primary tumor size determined by FDG-PET/CT. CONCLUSION: EDIM-TKTL1 blood test revealed good concordance with FDG-PET/CT results in patients with malignancies demonstrating its efficacy to detect upregulation of glucose metabolism in primary tumors or metastases.

[Anti-heat shock protein 70 (anti - Hsp 70) antibodies in alcohol use disorder patients]. / Przeciwciala przeciw bialku wstrzasu termicznego 70 kDa (anty- Hsp 70) u osób z zespolem zaleznosci alkoholowej.

The expression of the most important chaperone protein - Hsp70 and autoimmunity directed against it is a risk factor of cardiovascular diseases, increased in subjects with alcohol use disorder (AUD). The aim of the study was to evaluate the level of anti-Hsp 70 protein antibodies (anti-Hsp 70) in sera of AUD patients during abstinence period. Material and methods. The study included 54 subjects with AUD diagnosed basing on DSM IV criteria. In the studied group clinimetric evaluation was performed, plasma lipids, basic transketolase activity in erythrocytes (TK), thiamine pyrophosphate (TPP) activation of transketolase and the level of anti-Hsp 70 antibodies were evaluated as well. Results. In AUD subjects anti-Hsp 70 level was decreased during abstinence period. During first month of abstinency it correlated negatively with total cholesterol concentration (rS=-0.8857, p=0.0188) and the percentage of TPP stimulation (rS=-0.5960, p<0.05), and during 6 months of abstinence with HDL cholesterol (rS=-0.6848, p=0.0289). After 1 year of abstinence anti-Hsp 70 correlated positively with basic TK activity (rS=0.9550, p=0.0008). Sex is an independent factor influencing anti-Hsp 70 level in AUD subjects (B=60.9469, p=0.0435). In multiple regression model including results of clinimetric evaluation and its effect on the level of anti-Hsp 70 antibodies in AUD patients during 1 month of abstinency anti-Hsp 70 correlated with TWEAK scale score (BETA=-1.4543, p=0.0144) and AUDIT score (BETA-=1.2255, p=0.0224). In 2-6 months of abstinency anti-Hsp 70 correlated with TWEAK score (BETA=1.1110, p=0.0418). After 1 year of abstinency anti-Hsp 70 correlated with AUDIT score (BETA=-1.2161, p=0.0210). Conclusion. The autoimmune reaction against Hsp 70 is decreased during abstinency in AUD patients. Its relation with plasma lipids and thiamine deficiency may lead to increased risk of cardiovascular disorders. TWEAK and AUDIT scoring seem to be most useful for clinimetric evaluation in the context of the role of anti-Hsp 70 antibodies.

Altered heavy metals and transketolase found in autistic spectrum disorder.

Autism and autism spectrum disorder (ASD) are developmental brain disorders with complex, obscure, and multifactorial etiology. Our recent clinical survey of patient records from ASD children under the age of 6 years and their age-matched controls revealed evidence of abnormal markers of thiol metabolism, as well as a significant alteration in deposition of several heavy metal species, particularly arsenic, mercury, copper, and iron in hair samples between the groups. Altered thiol metabolism from heavy metal toxicity may be responsible for the biochemical alterations in transketolase, and are mechanisms for oxidative stress production, dysautonomia, and abnormal thiamine homeostasis. It is unknown why the particular metals accumulate, but we suspect that children with ASD may have particular trouble excreting thiol-toxic heavy metal species, many of which exist as divalent cations. Accumulation or altered mercury clearance, as well as concomitant oxidative stress, arising from redox-active metal and arsenic toxicity, offers an intriguing component or possible mechanism for oxidative stress-mediated neurodegeneration in ASD patients. Taken together, these factors may be more important to the etiology of this symptomatically diverse disease spectrum and may offer insights into new treatment approaches and avenues of exploration for this devastating and growing disease.

Benfotiamine reduces genomic damage in peripheral lymphocytes of hemodialysis patients.

Hemodialysis patients have an elevated genomic damage in peripheral blood lymphocytes (PBLs) and an increased cancer incidence, possibly due to accumulation of uremic toxins like advanced glycation end products (AGEs). Because the vitamin B1 prodrug benfotiamine reduces AGE levels in experimental diabetes, and dialysis patients often suffer from vitamin B1 deficiency, we conducted two consecutive studies supplementing hemodialysis patients with benfotiamine. In both studies, genomic damage was measured as micronucleus frequency of PBLs before and at three time-points after initiation of benfotiamine supplementation. AGE-associated fluorescence in plasma, and in the second study additionally, the antioxidative capacity of plasma was analyzed. Benfotiamine significantly lowered the genomic damage of PBLs in hemodialysis patients of both studies independent of changes in plasma AGE levels. The second study gave a hint to the mechanism, as the antioxidative capacity of the plasma of the treated patients clearly increased, which might ameliorate the DNA damage.

B-vitamin status and concentrations of homocysteine in Austrian omnivores, vegetarians and vegans.

BACKGROUND: A vegetarian diet is considered to promote health and longevity and reduce the risk of cardiovascular diseases and cancer. However, a vegetarian diet may be deficient in some nutrients. Exclusion of animal products in vegetarian diets may affect the status of certain B-vitamins, and further cause the rise of plasma homocysteine concentration. OBJECTIVE: The nutritional status of various B-vitamins (B(1), B(2), B(6), B(12), folic acid) and the concentration of homocysteine in blood plasma of omnivores (n = 40), vegetarians (n = 36) and vegans (n = 42) in Austria was evaluated. METHODS: The evaluation was done using the functional parameters erythrocyte transketolase (ETK), glutathione reductase (EGR) and glutamic oxaloacetic transaminase (EGOT) activation coefficients. Enzyme activity was measured photometrically. The quantity of vitamins B(1), B(2) and B(6) in urine and the concentrations of vitamin B(6) and homocysteine in plasma were determined by HPLC methods with fluorescence detection. Plasma concentration of vitamin B(12) and folic acid were measured with radioimmunoassay. RESULTS: Most of the subjects showed a satisfying vitamin B(1) status. Vegans presented a significantly lower mean plasma vitamin B(12) concentration than omnivores and vegetarians and deficiency in 2.4% of the volunteers but the highest mean value of plasma folate among the investigated groups. A deficient status of folate was found in 18% of omnivores and in approximately 10% of vegans and vegetarians. The status of riboflavin is considered to be deficient in about 10% of omnivores and vegetarians and in over 30% of vegans. According to the activation coefficient of GOT, approximately one third of all subjects showed vitamin B(6) deficiency. Elevated homocysteine concentration in plasma was observed in 66% of the vegans and about 45-50% of the omnivores and vegetarians. Vegan subjects had significantly higher mean plasma homocysteine levels than omnivores. CONCLUSION: Thiamin and folate need not be a problem in a well-planned vegan diet. Vitamins B(12) and B(2) may need attention in the strict vegan diet, especially regarding elevated homocysteine levels in plasma. Pyridoxine status appeared to be independent of the diet.

Faecal thiaminase, plasma lactate and pyruvate concentrations and erythrocyte transketolase activity changes in apparently normal replacement ewes after the initiation to the pasture.

A study was made to investigate faecal thiaminase and the thiamine-related biochemical changes in apparently normal replacement ewes with a feed change, after the initiation without adaptation to the new pasture. Twenty-four female ewes were divided into two groups. Group A was managed in a system based on pasture and was compared with group B system based on a diet of concentrate and straw until moving to pasture 9 weeks after. Blood samples for lactate, pyruvate and erythrocyte transketolase activity determinations and faeces for thiaminase estimation were evaluated chronologically. At the end of a 126 days experimental period, live weights of groups were similar. We confirmed that clinically normal sheep may have thiaminase activity in the faeces and concluded that the thiaminase release increased during the diet changes, from concentrate to pasture, and that their continued excretion could develop some degree of thiamine deficiency.

Thiamin status during the third trimester of pregnancy and its influence on thiamin concentrations in transition and mature breast milk.

Thiamin deficiency remains an important public health problem in some populations. The aim of the present investigation was to study thiamin status during the third trimester of pregnancy and its influence on the concentration of this vitamin in transition (days 13-14 of lactation) and mature breast milk (day 40 of lactation) in a group of Spanish women. The pregnancies and lactation periods of fifty-one healthy women 18-35 (mean 26.7 (SD 3.7)) years old were monitored. Vitamin intake during the third trimester was determined by recording the consumption of foods over 5 d and of the quantities provided by dietary supplements. Thiamin status during this stage of pregnancy was determined by measuring the activation coefficient of erythrocyte transketolase (alpha-ETK). Milk thiamin content was estimated (in 41% of the subjects) by oxidizing thiamin to thiocrome and measuring fluorescence. Subjects with thiamin intakes above that recommended (group H) had more satisfactory serum alpha-ETK coefficients (1.01 (SD 0.19)) than did those with lower intakes (group L) (1.21 (SD 0.30); P<0.05). Mature milk thiamin concentrations were significantly higher in group H subjects (0.59 (SD 0.44) micromol/l) than group L subjects (0.25 (SD 0.07) micromol/l). Subjects with alpha-ETK coefficients >1.25 in the third trimester had significantly lower mature milk thiamin concentration (0.31 (SD 0.10) micromol/l) than did subjects with more satisfactory alpha-ETK levels at this time (0.55 (SD 0.42) micromol/l; P<0.05). The thiamin status of women can be improved since 25.5% of subjects took less than that recommended and 13.7% showed signs of severe deficiency (alpha-ETK >1.25). The influence of maternal thiamin intake on alpha-ETK coefficients and on mature breast milk thiamin concentration is confirmed.