Changes in Erythrocyte Transketolase Activity and the Thiamine Pyrophosphate Effect during Storage of Blood

Abstract

SUMMARY. Whole blood and washed erythrocytes from nutritionally normal and abnormal elderly patients were stored at room temperature, 4 c e and -20 c e for up to 14 days. In both groups, and under all storage conditions, the erythrocyte transketolase (ETK) activity and the thiamine pyrophosphate (TPP) effect decreased with time of storage. The ETK activity for specimens stored at -20 c e decreased significantly by day 14 (P<0·05). The TPP effect in specimens stored at -2OCe decreased significantly in the nutritionally abnormal group at 4 days (P<0·05) and in the nutritionally normal group by day 14 (P<0·05). These significant changes in ETK activity and TPP effect with storage can lead to misleading interpretation of thiamine nutritional status.Erythrocyte transketolase (ETK) assays are widely used to assay thiamine status indirectly. There is, however, no clear consensus on the stability of the enzyme under different storage conditions. Brin' reported that haemolysed specimens are stable for only a matter of hours at room temperature, 2-3 days at 4 believed, this could perhaps contribute to these variations.We investigated, therefore, possible changes in the saturated and unsaturated ETK activities and the TPP effect after storage at different temperatures. Method SUB1ECTSThe nutritional status of 22 elderly in-patients was assessed on the basis of their dietary history, weight and anthropometric measurements. Eleven were considered to be nutritionally normal, with a mean age of 74 (67-90) years whereas 11 were regarded to be nutritionally abnormal, with a mean age of 76 (6&-84) years. None of the subjects had any evidence of infection. SAMPLE PREPARATION AND STORAGETwenty millilitres of heparinised blood was centrifuged at 3000g for 5 min, following which the plasma and buffy coat were discarded. An equal volume of cold sodium chloride solution (140 mmoUL) was added to the erythrocytes and then centrifuged at 3000 g for 5 min; the supernatant was discarded and the washing procedure repeated twice.

Clinically Unapparent Infantile Thiamin Deficiency in Vientiane, Laos

“…Two laboratories had to be used for ETK determinations and haemoglobin concentrations were not assayed in Oxford and therefore basal EKTA could not be expressed for these samples. The long sample storage period is unlikely to have affected α [35], [36] and if it did, it would have tended to underestimate thiamin deficiency [42]. It is likely that infants with thiamin deficiency have other clinically important nutritional deficiencies, such as vitamin A, riboflavin and folate.…”

Vitamin Analysis

“…Several authors 62,63 have identified potential problems with the performance and interpretation of this assay, including a gradual deterioration of samples during several months of storage at -20°C and a loss of reactivatable apoenzyme during chronic deficiency in vivo. 62 This necessitates the calculation and recording of basal enzyme activity per unit of haemoglobin or blood volume, as well as alpha or ETKAC,63 since a low basal activity can provide additional evidence of deficiency, in circumstances where the activation coefficient may prove either unreliable, or insufficiently discriminatory.…”

A Fluorimetric Method for Measurement of Erythrocyte Transketolase Activity

“…Stability data were gathered in response to information kindly supplied by Puxty et al, 11 principally concerning serious deterioration of ETKA and 'TPP Effects' during two weeks' storage of haemolysate at -20°C. Leunis et al have recently reported similar losses of activity.r" With -70°C refrigeration available, it seemed sensible to see if this temperature gave better results.…”