Erythrocyte thiamin deficiency

In humans, thiamine is both actively and passively absorbed to a limited level in the intestines, is transported as the free vitamin, is then taken up in actively metabolizing tissues, and is converted to the phosphate esters via ubiquitous thiamine kinases. During thiamine deficiency all tissues stores are readily mobilhed. Because depletion of thiamine levels in erythrocytes parallels that of other tissues, erythrocyte thiamine levels ate used to quantitate severity of the deficiency. As deficiency progresses, thiamine becomes indetectable in the urine, the primary excretory route for this vitamin and its metaboHtes. Six major metaboHtes, of more than 20 total, have been characterized from human urine, including thiamine fragments (7,8), and the corresponding carboxyHc acids (1,37,38). 

Brin, M. (1963). Thiamine deficiency and erythrocyte meiabohsm. Am. /. Clin. Nutr. 12, 107-116. 

As a thiamine deficiency develops, there is a rather rapid loss of the vitamin from aU tissue except the brain. The decrease of TPP in the erythrocyte roughly parallels the decrease of this coenzyme in other tissue. During this time, thiamine levels in urine fall to near zero the urinary metabolites remain high for some time before decreasing. 

Brin M, Tai M, Ostashever AS, Kalinslcy H. The effect of thiamine deficiency on the activity of erythrocyte hemolysate transketolase. J Nutr 1960 71 273-81. 

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. 

Thiamine deficiency is most frequently assessed by assaying erythrocyte transketolase activity in the presence and absence of added TPP. If the red blood cells have sufficient thiamine, the transketolase will be fully saturated with TPP, and no increase in activity will be observed when TPP is added to the assay system. An increase in transketolase activity indicates that the patient is thiamine deficient. 

Elderly people living on their own frequently have an inadequate diet. This is particularly true of men if they are unused to cooking for themselves. This patient may have a number of micronutrient deficiencies but, acutely, the most important would be possible thiamine deficiency. This can be detected by demonstrating an increase in the percentage activation of erythrocyte transketolase in vitro by the addition of thiamine or the measurement of thiamine pyrophosphate in erythrocytes. 

Chronic renal failure patients on hemodialysis and peritoneal dialysis are at risk for thiamine deficiency due to inadequate nutrition in part and possible thiamine loss during the dialysis process. Renal failure patients are often on a diet restricted in protein and potassium, which increases the risk of thiamine deficiency (Masud, 2002 Piccoli et al, 2006). Studies with detailed dietary surveys have shown poor oral intake of thiamine in chronic renal failure patients (Hung et al., 2001). There is no convincing evidence that thiamine levels are significantly altered by either hemodialysis or peritoneal dialysis (Reuler et al, 1985). DeBari et al (1984) measured thiamine levels of granulocytes, erythrocytes and plasma. They found no significant differences in thiamine levels in dialysis patients compared to controls. Further research in this area would benefit chronic renal failure patients and help determine possible need for supplementation of water-soluble vitamins. 

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 . 

The changes in a-ketoglutarate decarboxylase and pyruvic acid decarboxylase activities are not the only biochemical alterations observed in thiamine deficiency. The activities of some other enzymes were found to be altered, for example, erythrocyte transke-tolase and adenosine-5 -phosphatase activities are decreased in chicken brain. 

ETK AC = Erythrocyte Transketolase Activity Coefficient TDP = thiamine diphosphate N.S. = not specified MTD = moderate thiamine deficiency TD = thiamine... 

Brin, M., 1962. Erythrocyte transketolase in early thiamine deficiency. Annals of the New York Academy of Sciences. 98 528-541. 

Thiamine pyrophosphate is required as a cofactor for transketolase activity. Decreased erythrocyte transketolase activity is therefore found in thiamine deficiency and activity is increased by the addition of thiamine pyrophosphate to the assay system. 

Beriberi is caused by a deficiency of thiamin (also called thiamine, aneurin(e), and vitamin Bj). Classic overt thiamin deficiency causes cardiovascular, cerebral, and peripheral neurological impairment and lactic acidosis. The disease emerged in epidemic proportions at the end of the nineteenth century in Asian and Southeast Asian countries. Its appearance coincided with the introduction of the roller mills that enabled white rice to be produced at a price that poor people could afford. Unfortunately, milled rice is particularly poor in thiamin thus, for people for whom food was almost entirely rice, there was a high risk of deficiency and mortality from beriberi. Outbreaks of acute cardiac beriberi still occur, but usually among people who live under restricted conditions. The major concern today is subclinical deficiencies in patients with trauma or among the elderly. There is also a particular form of clinical beriberi that occurs in patients who abuse alcohol, known as the Wer-nicke-Korsakoff syndrome. Subclinical deficiency may be revealed by reduced blood and urinary thiamin levels, elevated blood pyruvate/lactate concentrations and a-ketoglutarate activity, and decreased erythrocyte transketolase (ETKL) activity. Currently, the in vitro stimulation of ETKL activity by thiamin diphosphate (TDP) is the most useful functional test of thiamin status where an acute deficiency state may have occurred. The stimulation is measured as the TDP effect. 

Table 3 Effects of thiamin deficiency on urinary thiamin, the erythrocyte transketolase TDP effect, and eariy clinical symptoms of thiamin deficiency in human volunteers...

Whole blood total thiamin below 150 nmol per L is considered to indicate deficiency. However, the changes observed in depletion studies are small. Even in patients with frank beriberi, the total thiamin concentration in erythrocytes is only 20% lower than normal whole blood thiamin is not a sensitive index of status. 

The most commonly used enzyme for the functional assay is transketolase. Transketolase catalyzes two reactions in the pentose phosphate pathway (Figure 30-10). As an enzyme within the erythrocyte, transketolase is independent of nonspecific changes in the extracellular plasma. As vitamin Bi deficiency becomes more severe, (1) thiamine becomes limiting in the body cells, (2) the amount of the coenzyme is depleted, and (3) the transketolase activity sub-... 

The transketolase activation test is in reality two tests one a measurement of basal activity and the other the degree to which the basal activity can be increased by exogenous thiamine pyrophosphate, and each may be influenced by different factors. There is evidence that chronic deficiency states of thiamine may down regulate synthesis of the apoen-zyme. In comparison studies against erythrocyte TPP concentrations, better correlations were obtained with basal activity rather than the activation coefficient. ... 

Reference intervals for thiamine and its esters depend upon whether (1) erythrocytes, whole blood, or plasma are used as a sample (2) cellular concentrations are expressed per liter of packed red cells or grams of Hb and (3) mass or SI units are used. Some guidance intervals are, for erythrocyte trans-ketolase activity 0.75 to 1.30U/g Hb (48.4 to 83.9kU/mol Hb) and for percent TPP effect (activation), 0 to 15% is normal, 16% to 25% marginally deficient and >25% severely... 

Thiamin (vitamin Bi) Thiamin in the body is chiefly found in the phosphorylated form thiamin pyrophosphate (TPP) which is a coenzyme. The majority (80%) of thiamin in the blood is found in the erythrocytes and assay of blood thiamin is a more reliable indicator of deficiency than assay of erythrocyte transketolase. The phosphorylated vitamers are enzymically converted to thiamin in samples using diastase following deproteinization. To reach the low picomolar concentrations the thiamin compounds are oxidized by ferricyanide to form thiochromes, which are highly fluorescent. The thiochromes are then separated by reversed-phase HPLC and detected by their emission at 425-450 nm. 

Standard methods for assessment of thiamine status used to be determination of erythrocyte transketolase (a-ETK) activity (EC 2.2.1.1) with and without stimulation of this enzyme by addition of TDP cofactor (TOP TK effect). A TDP TK effect >15% is considered to show some degree of deficiency, whereas values >22% are considered to indicate severe deficiency. Technical difficulties, including standardization of the assay, instability of the enzyme during storage, and various conditions possibly influencing apoenzyme concentrations led to an increasing use of direct determination of TDP in whole blood, e.g., by HPLC in order to assess thiamine status. The HPLC assay is more robust and easier to perform. Thiamine... 

Erythrocyte transketolase activity was the classic method to assess thiamine status. Two samples of blood are incubated with excess substrate for the pentose phosphate pathway to one is also added excess thiamine diphosphate while the other serves as the control. The amount of substrate remaining and product formed are quantified, and any enhancement in activity resulting from the added thiamine diphosphate indicates that the sample was originally deficient in thiamine to some extent. 

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. 

The activation of apo-transketolase in erythrocyte lysate by thiamin diphosphate added in vitro has become the most widely used and accepted index of thiamin nutritional status. An activation coefficient > 1.25 is indicative of deficiency, and < 1.15 is considered to reflect adequate thiamin nutrition. 

In general, the recommended allowances are based (1) on assessments of the effects of varying levels of dietary thiamin on the occurrence of clinical signs of deficiency, (2) on the excretion of thiamin or its metabolites, and (3) on erythrocyte transketolase activity. Most studies have been conducted on subjects fed diets with ratios of carbohydrate and fatsimilar to those commonly consumed in the United States. There is evidence that dietary fat spares thiamin to some extent... 

---