Blood thiamin measurement

Table 16.2 Thiamine measures in blood (ETKAC and TDP effect). 

Techniques using HPLC and fluorescent detection are now the standard for direct quantification of thiamine in blood (Lynch and Young 2000). Plasma contains less than 10% of whole blood thiamine (in the form of T" and TMP) and levels are strongly influenced by recent food consumption (Tallaksen et al. 1993). Following thiamine intake, plasma thiamine levels peak at 50 minutes (range 20-120 minutes) and return to baseline within 12 hours (Tallaksen et al. 1993). Thus, a fasting state is often recommended to increase accuracy of plasma thiamine measures. However, measurements in whole blood or erythrocytes are preferred as plasma measures suffer from poor sensitivity and specificity. 

Table 16.3 Thiamine measures in blood (total thiamine in ng/mL and nmol/L).

Table 16.4 Thiamine measures in blood (free thiamine and its phosphate derivative).

Numerous reports of prodrugs in the literature show improved drug effects. Prodrugs that have shown some measure of success for site-specific delivery include L-3,4-dihydroxyphenylalanine (L-dopa) to the brain [56], dipivaloyl derivative of epinephrine to the eye [57], /-glutamyl-L-dopa to the kidney [58], fi-n-glucoside dexamethasone and prednisolone derivatives to the colon [59], thiamine-tetrahydrofuryldisulfide to red blood cells, and various amino acid derivatives of antitumor agents such as daunorubicin [61,62], acivicin [63], doxorubicin [63], and phenylenediamine [63] to tumor cells. 

In experimental animals and in depletion studies, measurement of the concentration of thiamin in plasma or whole blood provides an indication of the progression of deficiency. The normal method is by the formation of thiochrome, which is fluorescent ordy free thiamin, and not the phosphates, undergoes... 

The most reliable method for assessing thiamin status involves the measurement of red blood cell transketolase. This enzyme is measured with and without the addition of TPP to the enzyme assay mixtures. In dietary thiamin deficiency, synthesis of transketolasc continues, but conversion of the apoet zyme to the holoenzyme in the cell is inhibited, resulting in the accumulation of the enzyme in the apoenzyme form. Addition of TPP to cell homogenates results in the conversion of apoenzyme to holoenzyme. This conversion can easily be detected by enzyme assays. The amount of shmulation of enzyme activity by the added TPP is used to assess thiamin status. A deficiency is indicated by a shmulation of over 20%, The TPP-dependent stimulation, using red blood cells from normal subjects, ranges from 0 to 15%. 

The existence of apoenzyme and holoenzyme forms of various enzymes is of use to the clinician. The proportion of a specific enzyme occurring in apoenzyme and holoenzyme forms is used to assess vitamin status in the cases of vitamin Bg, thiamin, and riboflavin. Vitamin status is determined by measuring the percentage stimulation of enzyme activity that occurs after adding the appropriate cofactor to a biological sample (blood) containing the enz3me of interest. 

Thiamin status has been assessed by direct tests involving the measurement of thiamin levels in the blood or urine. The vitamin can be assayed by the thiochrome method or by microbiological assays. The disadvantage of these methods is that thiamin levels in normal individuals can vary greatly. The test organism used for microbiological assays may be Lactobacillus viridescens or Lactobacillus fermenti. 

Direct measurement of circulating thiamine concentration may be made in plasma, erythrocytes, or whole blood. The plasma (or serum) concentration is thought to reflect recent intake and is mainly unphosphorylated thiamine at low concentration (around 10 to 20nmol/L). Because the erythrocyte contains approximately 80% of the total thiamine content of whole blood,mainly as the pyrophosphate, and erythrocyte thiamine stores deplete at a similar rate to other major organs, HPLC measurement of TPP in erythrocytes is a good indicator of body stores. Typical HPLC methods include a protein precipitation step, precolumn or postcolumn formation of the fluorophore thiochrome, usually with alkaline ferricyanide and isocratic separation. The method is easily standardized with pure... 

Talwar D, Davidson H, Cooney J, St JO Reilly D. Vitamin B(l) status assessed by direct measurement of thiamin pyrophosphate in erythrocytes or whole blood by HPLC comparison with erythrocyte transketolase activation assay. Clin Chem 2000 46 704-10. 

Thiamine deficiency can be assessed by measuring blood levels. Increased blood levels of pyruvate and lactate suggest thiamine deficiency. Measurement of erythrocyte transketolase activity, which requires TPP as a coenzyme, confirms the deficiency. 

A more specific type of chemical assay is based on enzymatic measurement of vitamin co-enzyme activity. This approach is designed to detect a vitamin deficiency in tissues, and is only feasible for those vitamins that serve as co-enzymes. For instance, thiamin depletion in a subject can be diagnosed by measuring the transketolase activity in red blood cells with and without the addition of thiamin pyrophosphate (TPP) in vitro. If TPP increases the activity by more than a given amount, thiamin deficiency is indicated. Similarly, a subnormal level of riboflavin is indicated in tissues if the activity of erythrocyte glutathione reductase is increased after the addition of flavin adenine dinucleotide (FAD). Erythrocyte transaminase activation by pyridoxal-5 -phosphate (PLP) can be measured to establish a deficiency of vitamin B . 

Measurement of Thiamine Levels in Human Tissue Table 16.1 Thiamine reference standards in blood. 

The erythrocyte transketolase (ETK) activation assay (also known as the saturation test) measures the functional capacity of the enzyme transketolase in red blood cells i.e. erythrocytes). Transketolase is a thiamine-dependent enzyme in the non-oxidative branch of the pentose phosphate pathway (PPP), a process of glucose turnover that produces nicotinamide adenine dinucleotide phosphates (NADPH) as reducing equivalents and pentose sugars as essential components of nucleotides. In the absence of adequate thiamine, the PPP output is compromised. 

The measurement of thiamine levels in human tissue other than blood is limited to biopsy or autopsy samples. This limitation translates into high sample variability due to factors such as age and nutritional, disease and medication status. Another limitation associated with measurement of thiamine in human tissue is the biological instability of thiamine derivatives. Because the turnover time of TTP is approximately one hour, significant hydrolysis can occur during extended delays between death and sample retrieval, preparation, and storage (Bettendorff et al. 1996a). 

The most accurate way to measure thiamine status is to quantify the biologically active form of thiamine, thiamine diphosphate, in whole blood or isolated red blood cells. 

Lu, J., and Frank, E.L., 2008. Rapid HPLC measurement of thiamine and its phosphate esters in whole blood. Clinical Chemistry. 54 901-906. 

Nutritional status assessment for thiamine is generally carried out by assaying the total thiamine in whole blood or erythrocytes, or by measuring the activity of erythrocyte transketolase before and after incubation with exogenous thiamine pyrophosphate. The latter serves as the sensitive index of thiamine nutritional status (Brin 1980). In addition to the enzymatic test, a measure of urinary thiamine in relation to dietary intake has been the basis for balance studies to assess the adequacy of intake. When thiamine excretion is low, a larger portion of the test dose is retained, indicating a tissue s need for thiamine. A high excretion indicates tissue saturation. In the deficient state, excretion drops to zero. Plasma pyruvate and lactate concentrations have also been used to assess thiamine status. 

There is a claim that low TDP in blood may be a laboratory marker for alcoholism and a predictor for overt thiamine deficiency (Ceccanti et al. 2005). However, this marker is apparently secondary to earlier, direct and diverse elfects of ethanol on all systems of the human body which pave the road to alcoholic-TD encephalopathies of the Wernicke-Korsakolf type (Tables 33.1 and 33.2) (Pitel et al. 2011). Therefore, preventive measures are undertaken in several countries, including mandatory supplementation of bread flour with thiamine. However, there is no evidence as to whether such treatment delays onset of dementia in the population of alcoholics. 

In thiamine deficiency, pyruvate accumulates in the blood and tissues and there is a change in the lactate-pyruvate ratio. These findings have been used in detecting thiamine deficiency in man. MIorwitt has proposed as a test of thiamine nutrition the simultaneous measurement of lactic acid, pyruvic acid, and glucose in the blood 5 min. after mild exercise and 60 min. after ingestion of glucose. The carbohydate index (C.I.) is determined from the formula... 

Gerrits et al. (20) determined thiamine and its phosphates in whole blood by reversed-phase HPLC with precolumn derivatization. Good resolution of the elution profile for thiamine and its phosphate esters was obtained for a standard sample (Fig. 8) and for a human blood sample (Fig. 11). Reference values obtained from the whole blood samples of 65 healthy volunteers were in nM (mean SD) thiamine, 4.3 1.9 TMP, 4.1 1.6 TPP, 120 17.5. For TTP, all measurements were <4.0 nM. However, a few patients with low TPP and relatively high TTP were found, and in most cases these patients were alcoholics. The precise role of TTP in a clinical setting remains to be elucidated. 

A test, used mainly for the diagnosis of thiamine deficiency, which consists of giving an oral load of glucose and measuring the subsequent blood pyruvate levels (see pyruvate). In normal individuals only a small rise in the level occurs. A larger increase, however, occurs in thiamine deficiency and in diseases where there is disturbed carbohydrate metabolism such as liver diseases. An absent response may be found in untreated diabetics. 

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