- Email: [email protected]
The relationship between erythrocyte transketolase activity and the ‘TPP effect’ in Wernicke's encephalopathy and other thiamine deficiency states
Clinica Chimica Acta, 192 (1990) Elsevier
89
89-98
CCA 04834
The relationship between erythrocyte transketolase activity and the ‘TPP Effect’ in Wernicke’s encephalopathy and other thiamine deficiency states Peter F. Nixon, John Price, Maureen Norman-Hicks, and Ray A. Kerr Departments
of Biochemistry
and Psychiatry
University of Queensland
Gail M. Williams Brisbane (Australia)
(Received 13 November 1989; revision received 24 July 1990; accepted 27 July 1990) Key words: Erythrocyte; Transketolase; Thiamine diphosphate; Thiamine deficiency; TPP effect; Wemicke’s encephalopathy
Patients (n = 104) were judged to be thiamine deficient by the criteria of erythrocyte transketolase activity (ETK) less than 0.6 U/g of hemoglobin, or greater than 17% increase in this activity on addition of thiamine pyrophosphate in vitro (TPP effect). ETK activated by TPP in vitro (AETK) was related to ETK by a linear regression of slope 2 1, implying that transketolose apoenzyme (apoTK) was constant or decreased as ETK decreased. For most patient groups the value of apoTK was 0.1 U/g and the slope 1.033 to 1.050. In the subgroup of non-vomiting drinkers with Wernicke’s encephalopathy (WE), the slope of the linear regression of AETK on ETK was 1.21, so that apoTK decreased as ETK decreased. Comparison of these data is consistent with a difference in the TK of WE drinkers from that of others. Generally, any variation of TPP effect was due only to variation of ETK. We recommend measurement of ETK, without TPP effect, for the assessment of thiamine nutrition.
Introduction
The two laboratory criteria used most frequently for the evaluation of thiamine (vitamin B,) nutrition are measurements of (i) the erythrocyte transketolase activity Correspondence and requests for reprints to: Dr P.F. Nixon, Department of Biochemistry, University of Queensland, St. Lucia, Queensland 4067, Australia. 0009-8981/90/$03.50
0 1990 Elsevier Science Publishers B.V. (Biomedical Division)
90
(ETK; EC 2.2.1.1) and (ii) its activation by the addition of thiamine pyrophosphat~ in vitro (the ‘TPP effect’), introduced by Brin [l-3] and Dreyfus [4] and endorsed by Sauberlich [5]. Since thiamine diphosphate (TPP) is a required cofactor for transketolase, decreased ETK can be due to deficiency of either the cofactor (and hence of its precursor vitamin B,) or the transketolase protein (apoTK) or both but is usually interpreted as being due to thiamine deficiency [l-18]. Addition of TPP to a hemolysate in vitro allows measurement of an ‘activated’ ETK (AETK) which is presumed to measure the sum of holoTK and apoTK in the hemolysate; the difference AETK-ETK is presumed to measure apoTK alone. The term ‘TPP effect’ is calculated [3] by the formula (AETK-ETK/ETK) percent. Many authors regard the TPP effect as the most reliable and sensitive single criterion for the diagnosis of thiamine deficiency [3-$7-141 whilst others report that the TPP effect is less reliable than ETK for the ev~uation of thiamine nutrition in those most frequently deficient, namely chronic alcoholics, particularly those suffering mifd or severe liver disease [15-181. The first purpose of this paper is to explore the relationship between the AETK value and the ETK value in patients presumed to be thiamine deficient by either of the above stated criteria, and to comment upon the significance of these relationships and of the derived TPP effect as measures of thiamine nutrition. The second purpose is to document the relationship of AETK with ETK in a subset of patients, namely alcohol drinkers suffering Wemicke’s encephalopathy (WE), in whom this relationship appears to be different from that in other patients studied. Materials and methods ETK and AETK were measured f19] in the Chemical Pathology laboratories of two Brisbane hospitals. Blood samples were collected into tubes containing lithium heparin then stored at 5 “C for up to 2 h, or up to 10 h in the case of samples collected late at night. The samples were then centrifuged at 1200 x g for 10 mm and the plasma and buffy coat removed by aspiration. The erythrocytes were then lyzed by addition of an equal volume of deionized, R.O. purified water containing Triton X-100, 2 ml/l, then stored at 5°C until assay the same day. Samples were deemed thiamine deficient and entered into this anaIysis whenever either of the following criteria were met: ETK < 0.6 U/g of hemoglobin, or TPP effect greater than 17%, or both. To limit the sample size, data from male alcoholics which met these criteria were arbitrarily restricted to those from patients in whom the measurements were repeated during the same hospital admission, although the repeat measurements are not analyzed here. The 104 patients were divided into a group of 56 drinkers (28 of each sex) and of 48 non-drinkers (24 of each sex). The non-drinkers included ex-drinkers whose alcohol consumption had been zero for at least 2 yr prior to admission. Alcohol consumption was assessed and recorded by the admitting medical officer, and corroborated by relatives and local medical officers. Laboratory measurements were used as a check on alcohol consumption: the mean erythrocyte corpuscular volume (MCV) was 101.3 fI (SE 1.01) in drinkers and 89.5 fl (SE 1.23) in non-drinkers, and
91
the median plasma gamma-ghnamyl transferase activity (yGT) in these respective groups was 114 U/l and 26.5 U/l. The differences between drinkers and nondrinkers were significant at the 0.001 level for the normally distributed, untransformed MCV values, and for the yGT values after log transformation to normalize the distribution. Two patients were excluded from further study because these data were not fully consistent. Of the non-drinkers, the primary diagnosis was psychiatric illness in 13 (depression, psychosis, bulimia) and drug dependence in 5. The remaining non-drinking patients were elderly and on a poor diet, or suffered a wide variety of medical illnesses, frequently multiple, or a combination of these possible causes of thiamine deficiency. The presence or absence of vo~ting at the time of admission was recorded for all but 4 patients, because vomiting might have led to the cessation of drinking. Of the 104 patients studied, 35 suffered Wemicke’s encephalopathy (WE) by meeting two or more of the following criteria: ataxia, ophthalmoplegia (including nystagmus) and global confusion [20]. Results and discussion
Mean ETK and AETK values Mean ETK and AETK values did not differ between males and females, between drinkers and non-drinkers or between vomiters and non-vomiters (data not shown). However, the mean ETK and AETK values for the WE patients, respectively 0.39 (SE 0.17) and 0.55 (SE 0.19), were both lower (P < 0.05) than the respective values for the group of other patients, 0.52 (SE 0.16) and 0.65 (SE 0.17); and WE was much more common (P -c 0.001) among drinkers than among non-drinkers. Of the group of drinkers, 50% had WE whereas only 15% of non-drinkers had WE.
TABLE I Linear regressions of AETK on ETK by patient groups Patient groups (and subgroups)
n
Slope
Intercept
Mean
SE
Mean
SE
Correlation coefficient (r)
1.045 1.050 1.047 1.062 1.084 1.217 1.116 1.033
0.033 0.070 0.038 0.071 0.082 0.059 0.160 0.081
0.101 0.103 0.097 0.121 0.121 0.039 0.151 0.104
0.018 0.036 0.021 0.030 0.034 0.027 0.058 0.037
0.9690 0.9465 0.9767 0.9338 0.9321 0.9860 0.9186 0.9846
U/g NonWE Drinkers Non-drinkers WE Drinkers Non-vomiters Vomiters * Non-drinkers
69 28 41 35 28 14 11 7
* Vomiting status was uncertain for 3 WE drinkers, who are not included in the subgroups. Note that the slopes for the two subgroups of WE drinkers are both greater than the slope of the whole group. Tbis results from the clustering of data and consequent unequal weighting of the contributions of each separate subgroup to each end of the regression for the whole group.
92
ETK, AETK
and TPP effect
For each group of patients classified by the presence of WE and by alcohol consumption (Table I), the AETK was closely related to ETK by a linear regression of slope in the range 1.033 to 1.084 and intercept on the AETK axis in the range 0.097 to 0.121. Division of the groups by vomiting status did not affect the regressions except in the case of WE drinkers, who are discussed later. Since in all groups the value for the slope was not different from 1 (Table I) for all values of ETK, the potential activity of apoTK able to be reactivated by the addition of TDP in vitro was constant and equal to the intercept on the AETK axis (Table I). The regressions for the total nonWE patients (Fig. l), nonWE drinkers, nonWE nondrinkers and WE non-drinkers could be superimposed (Table I). In the case of the WE drinkers (Fig. 2), the intercept and slope values were not significantly greater than those for all other groups (Table I). The good fit of this regression in all groups (0.9321 I I I 0.9846) is consistent with the proposition that this model accurately represents the relationship of AETK and ETK in any sample of blood. The very small residual differences from a perfect fit of the regression to the data (Figs. 1 and 2) show the maximum limits to variation of apoTK, since they represent the sum of both this variation and the
1.2I ,
,’
I’ I’
021 02
I 0.4
06 ETK
1 0.6
,fl
,’
I 1
( U/g)
Fig. 1. Linear regression of AETK on ETK for 69 nonWE patients, together with 95% confidence limits. Mean values for the slope and intercept on the AETK axis are listed in Table I, together with the correlation coefficient r for the fit of the regression to the data.
93
0.8-
06
0 0
0.1
0.2
03 ETK
0.4
05
0.6
0.7
(
8
(o/g)
Fig. 2. Linear regression of AETK on ETK for 28 WE drinkers. Other details are as for Fig. 1.
measure of lack of fit of the model. Thus the SEM values for the intercept are m~mum vahtes for the standard errors which measure the variation of apoTK When the attempt was made to relate TPP effect to ETK (Figs. 3 and 4), by fitting to the same data the hyperbolic expression for an inverse relationship TPP effect = c/ETK, which assumes that AETK-ETK is constant, the scatter of data was much greater and the goodness of fit much less than was the case for Figs. 1 and 2, as has been found by others [10,11,13,17~ who have d~umented the same relationship. The correlation coefficients for the fit of this model to the data were 0.7314 for Fig. 3 and 0.4735 for Fig, 4. There are good reasons to expect that the relations~p of TPP effect to ETK should be less close than the relations~p of AETK to ETK. Since the TK activity of erythrocytes is low, it is difficult to achieve very high precision for the measurement of AETK and ETK. Moreover, since the calculation of TPP effect requires first a calculation of the difference between these two values, each of indifferent precision, and then calculation of the ratio of that difference to one of these measurements, the precision of the final result must be much lower than the precision of either one measurement. This imprecision is reflected in the relatively poor correlation coefficients for the relationship of TPP effect and ETK [10,11,13,17, Figs 3 and 41. Calculation of TPP effect appears to add only imprecision to the assessment of thiamine nutrition, since our results show that variation of TPP effect is due only to
94
. I
I
02
I
1
03
04 ETK
05
I
06
I
07
08
09
(u/g)
Fig. 3. Hyperbolic regression of TPP effect on ETK for 69 nonWE patients, together with 95% confidence limits. The regression equation was TPP effect = c/ETK where c is a constant, thus assuming that apoTK is constant. The correlation coefficient r was 0.7314.
variation of ETK, the denominator in the calculation of TPP effect. The difference AETK-ETK, the measure of reactivatable apoTK and the numerator in the calculation of TPP effect, remained constant (Fig. 1) for all groups of patients (Table I).
I 180
160
, 140-
: \
a
\ ‘\
120-
20m 0 0
. I 0.2
I 04 ETK
.‘--------__________ , 06
(
(U/g)
Fig. 4. Hyperbolic regression of TPP effect on ETK for 35 WE patients. The correlation coefficient 0.4735. Other details are as for Fig. 3.
r was
95
The constant value for apoTK implies that changes in the concentration of total TK protein, i.e. synthesis or catabolism of apoTK, or both, are dependent upon cofactor concentration; otherwise apoTK would increase as thiamine nutrition and ETK decreased, and the slope of the regression of AETK on ETK would be closer to zero rather than 1. We conclude that calculation of the TPP effect adds nothing to the single measurement of ETK as a criterion of thiamine nutrition, and that the reputed sensitivity and reliability of the TPP effect [3-5,7-141 is due to its inverse relationship to ETK in the face of a constant value for apoTK. Therefore calculation of the TPP effect is without value, and argument [cf. 7-14 and 15-181 as to the relative sensitivity and reliability of the TPP effect versus the measurement of ETK for the evaluation of thiamine nutrition in alcoholics is irrelevant. Nevertheless, we show below that there might be small differences between the WE drinkers and all other patient groups in respect to the relationship of AETK and ETK, and suggest that those differences might provide insight into the pathogenesis of WE in these patients, without affecting the evaluation of thiamine nutrition. AETK and ETK in drinking WE patients Classification of WE drinkers into those who were vomiting on admission and those who were not resulted in different regressions for these subgroups. In the case of the WE drinkers who were not vomiting on admission, the linear regression (Fig. 5) of AETK on ETK was particularly tight (regression coefficient 0.986) and yielded a slope value of 1.22 (Table I), significantly greater (0.01 < P < 0.02) than the value for the nonWE group. Moreover, in this subgroup, the 95% confidence limits for the value of the slope (1.088 and 1.346) excluded unity. Thus in this subgroup, alone, the apoTK value was not constant, but ranged from a lowest experimental value of 0.05 U/g to a highest experimental value of 0.18 U/g, and theoretical values read from the linear regression ranged from 0.04 to 0.26 U/g as the value for ETK ranged from 0 to 1.0 U/g. To test for divergence between the non-vomiting WE drinker subgroup and other groups, we have plotted in Fig. 5 the 95% confidence limits for the linear regression of AETK on ETK for that group together with the regression, from Fig. 1, for all nonWE patients (which is identical to that for WE non-drinkers also). In the range ETK > 0.5 U/g, the regression for nonWE patients lies outside and below the 95% confidence limits of the regression for non-vomiting WE drinkers (Fig. 5). The latter regression line also lies above the 95% confidence limits for the former (not shown in Fig. 5 to avoid confusion). Thus the regression for the non-vomiting WE drinkers emerges as different from those for both the nonWE groups and the non-drinking WE group. In the case of the subgroup of vomiting WE drinkers, the linear regression of AETK on ETK (Table I) yielded a slope value of 1.116 and intercept of 0.15 U/g. The correlation coefficient of 0.9186 reflects the number and scatter of data points yielding this regression; although the difference AETK-ETK is greater at all points on this regression than on that for any other group, a significant difference for the slope value or intercept value has not been established. Thus in both subgroups of drinkers who develop WE, the regression of AETK on ETK could be different from that of other groups. Unlike the case of the subgroups of
96
06-
? 3
=
04-
:
0
0
I
I
I
02
04
06
ETK
(I. 6
(U/g)
Fig. 5. Linear regression of AETK on ETK for 14 non-vomiting WE drinkers (m) together with 95% confidence limits (. . . . .); and the regression, (- - - - - -), without data points, for 69 nonWE patients shown previously in Fig. 1. Other details are as for Fig. 1.
WE drinkers, the small group of non-drinkers who presented with WE yielded a regression of AETK on ETK which was not different from that of the nonWE group (Table I). Thus our data are consistent with the hypothesis that the TK of drinkers (but not that of non-drinkers) who develop WE is different from that of other patients. Evidence of a variant TK associated with WE has been previously reported [21-241 without specification of drinking status. Whether the putative variant is characterized by a lower affinity for cofactor [21,24], an altered turnover number, or by decreased stability of apoTK [23] must await measurements of TK protein in WE and nonWE patients, perhaps by immunoassay [25,26], and more sophisticated analysis of the activation of apoTK by cofactor [27]. Since the regressions for the non-vomiting and vomiting WE drinkers diverged at very low values of ETK (Table I, compare intercepts of 0.039 and 0.151, respectively), it might be that alcohol consumption in the period immediately before presentation has the effect, increasing in severity as ETK values decrease, of inactivating residual apoTK. Certainly others have observed [15-181 an unusually low TPP effect, despite decreased ETK, in the erythrocytes of thiamine-deficient chronic alcoholics; and acetaldehyde, the proximal product of ethanol metabolism,
91
has been shown [28] to inactivate apoTK much more readily than holoTK. Accurate records of drinking, by period before presentation with thiamine deficiency, are important to studies such as this despite the difficulty of their collection. Conclusion
We recommend measurement of ETK, without AETK or TPP effect, for evaluation of thiamine nutrition. However, measurement of AETK and ETK together has provided further evidence that some WE patients may have a variant of TK, and has suggested that the putative variant apoTK has increased susceptibility to inactivation by ethanol or its metabolites. Acknowledgements
We are grateful for the support of the National Health and Medical Research Council of Australia, to Dr. H. Kalant for constructive comments on the manuscript, and to Ms. N. Lee for typing the manuscript. References 1 Brin M, Tai M, Ostashever AS, Kalinsky H. The effect of thiamine deficiency on the activity of erythrocyte hemolysate transketolase. J Nutr 1960;71:273-281. 2 Brin M. Erythrocyte transketolase in early thiamine deficiency. Ann NY Acad Sci 1962;98:528-541. 3 Brin M. Erythrocyte as a biopsy tissue for functional evaluation of thiamine adequacy. J Am Med Assoc 1964;187:762-766. 4 Dreyfus PM. Clinical application of blood transketolase determinations. N Engl J Med 1962;267:596-598. 5 Sauberlich HE. Biochemical alterations in thiamine deficiency: their interpretation. Am J Clin Nutr 1967;20:528-542. 6 Wolfe SJ, Brin M, Davidson CS. Effect of thiamine deficiency on human erythrocyte metabolism. J Clin Invest 1958;37:1476-1484. 7 Tanphaichitr V, Vimokesant SL, Dhanamitta S, Valyasevi A. Clinical and biochemical studies of adult beriberi. Am J Clin Nutr 1970;23:1017-1026. 8 Sauberlich HE, Dowdy RP, Skala JH. Laboratory tests for the assessment of nutritional status. Thiamine (vitamin B,) CRC Crit Rev Clin Lab Sci 1973;4:236-244. 9 Kuriyama M, Yokomine R, Arima H, Hamada R, Igata A. Blood vitamin B,, transketolase and thiamine pyrophosphate (TPP) effect in beriberi patients, with studies employing discriminant analysis. Clin Chim Acta 1980;108:159-168. 10 McLaren DS, Docherty MA, Boyd DHA. Plasma thiamine pyrophosphate and erythrocyte transketolase in chronic alcoholism. Am J Clin Nutr 1981;34:1031-1033. 11 Waldenlind L, Borg S, Vikander B. Effect of peroral thiamine treatment on thiamine content and transketolase activity of red blood cells in alcoholic patients. Acta Med Stand 1981;209:209-212. 12 Graudal N, Torp-Pedersen K, Hanel H, Kristensen M, Thomsen AC, Nerrg&rd G. Assessment of thiamine nutritional status. An evaluation of erythrocyte transketolase activity, the stimulated erythrocyte transketolase activity and the thiamine pyrophosphate effect. Int J Vit Nutr Res 1985;55:399-403. 13 Anderson SH, Vickery CA, Nicol AD. Adult thiamine requirements and the continuing need to fortify processed cereals. Lancet 1986;2:85-89. 14 Fidanza F, Simonetti MS, Floridi A, Codini M, Fidanza R. Comparison of methods for thiamine and riboflavin nutriture in man. Int J Vit Nutr Res 1989;59:40-47.
98 15 Fermelly J, Frank 0, Baker H, Leevy CM. Red blood cell transketolase activity in malnourished alcoholics with cirrhosis. Am J Clin Nutr 1967;20:946-949. 16 Konttinen A, Louhija A, HSLrtel G. Blood transketolase in assessment of thiamine deficiency in alcoholics. Ann Med Exp Fenn 1970;40:172-175. 17 Camilo ME, Morgan MY, Sherlock S. Erythrocyte transketolase activity in alcoholic liver disease. Stand J Gastroent 1981;16:273-279. 18 Majumdar SK, Shaw GK, O’Gorman P, Aps EJ, Offerman EL, Thomson AD. Blood vitamin status (B,,B&, folic acid and B,,) in patients with alcoholic liver disease. Int J Vit Nutr Res 1982;52:266271. 19 Bayoumi RA, Rosa&i SB. Evaluation of methods of coenzyme activation of erythrocyte enzymes for detection of deficiency of vitamins B,, & and 4. Clin Chem 1976;22:327-335. 20 Reuler JB, Girard DE, Cooney TG. Wemicke’s encephalopathy. N Engl J Med 1985;312:1035-1039. 21 Blass JP, Gibson GE. Abnormality in a thiamine-requiring enzyme in patients with Wernicke-Korsakoff syndrome. N Engl J Med 1977;297:1367-1370. 22 Nixon PF, Kaczrnarek MJ, Tate J, Kerr R, Price J. An erythrocyte transketolase pattern associated with the Wemicke-Korsakoff syndrome. Eur J Clin Invest 1984;14:278-281. 23 Kerr RA, Clague AE, Morris DJ, Price J. The rapid decline in erythrocyte transketolase on cessation of high-dose thiamine administration in Korsakoff patients. Ale Ale 1986;21:257-262. 24 Mukheriee AB, Svoronos S, Ghazanfari A, et al. Transketolase abnormality in cultured fibroblasts from familial chronic alcoholic men and their male offspring. J Clin Invest 1987;79:1039-1043. 25 Takeuchi T, Jung EH, Nishino K, Itokawa Y. Western blotting assay of transketolase concentration in human hemolysates. Analyt Biochem 1988;168:470-475. 26 Paoletti F, Mocali A, Marchi M, Truschi F. Analysis of transketolase and identification of an enzyme variant in human leucocytes. Biochem Biophys Res Commun 1989;161:150-155. 27 Tate JR, Nixon PF. Measurement of Michaelis constant for human erythrocyte transketolase and thiamine diphosphate. Analyt Biochem 1987;160:78-87. 28 Atukorala TMS, Duggleby RG, Nixon PF. Effect of acetaldehyde upon catalysis by human erythrocyte transketolase. Biochem Pharmacol 1988;37:2100-2101.
89
89-98
CCA 04834
The relationship between erythrocyte transketolase activity and the ‘TPP Effect’ in Wernicke’s encephalopathy and other thiamine deficiency states Peter F. Nixon, John Price, Maureen Norman-Hicks, and Ray A. Kerr Departments
of Biochemistry
and Psychiatry
University of Queensland
Gail M. Williams Brisbane (Australia)
(Received 13 November 1989; revision received 24 July 1990; accepted 27 July 1990) Key words: Erythrocyte; Transketolase; Thiamine diphosphate; Thiamine deficiency; TPP effect; Wemicke’s encephalopathy
Patients (n = 104) were judged to be thiamine deficient by the criteria of erythrocyte transketolase activity (ETK) less than 0.6 U/g of hemoglobin, or greater than 17% increase in this activity on addition of thiamine pyrophosphate in vitro (TPP effect). ETK activated by TPP in vitro (AETK) was related to ETK by a linear regression of slope 2 1, implying that transketolose apoenzyme (apoTK) was constant or decreased as ETK decreased. For most patient groups the value of apoTK was 0.1 U/g and the slope 1.033 to 1.050. In the subgroup of non-vomiting drinkers with Wernicke’s encephalopathy (WE), the slope of the linear regression of AETK on ETK was 1.21, so that apoTK decreased as ETK decreased. Comparison of these data is consistent with a difference in the TK of WE drinkers from that of others. Generally, any variation of TPP effect was due only to variation of ETK. We recommend measurement of ETK, without TPP effect, for the assessment of thiamine nutrition.
Introduction
The two laboratory criteria used most frequently for the evaluation of thiamine (vitamin B,) nutrition are measurements of (i) the erythrocyte transketolase activity Correspondence and requests for reprints to: Dr P.F. Nixon, Department of Biochemistry, University of Queensland, St. Lucia, Queensland 4067, Australia. 0009-8981/90/$03.50
0 1990 Elsevier Science Publishers B.V. (Biomedical Division)
90
(ETK; EC 2.2.1.1) and (ii) its activation by the addition of thiamine pyrophosphat~ in vitro (the ‘TPP effect’), introduced by Brin [l-3] and Dreyfus [4] and endorsed by Sauberlich [5]. Since thiamine diphosphate (TPP) is a required cofactor for transketolase, decreased ETK can be due to deficiency of either the cofactor (and hence of its precursor vitamin B,) or the transketolase protein (apoTK) or both but is usually interpreted as being due to thiamine deficiency [l-18]. Addition of TPP to a hemolysate in vitro allows measurement of an ‘activated’ ETK (AETK) which is presumed to measure the sum of holoTK and apoTK in the hemolysate; the difference AETK-ETK is presumed to measure apoTK alone. The term ‘TPP effect’ is calculated [3] by the formula (AETK-ETK/ETK) percent. Many authors regard the TPP effect as the most reliable and sensitive single criterion for the diagnosis of thiamine deficiency [3-$7-141 whilst others report that the TPP effect is less reliable than ETK for the ev~uation of thiamine nutrition in those most frequently deficient, namely chronic alcoholics, particularly those suffering mifd or severe liver disease [15-181. The first purpose of this paper is to explore the relationship between the AETK value and the ETK value in patients presumed to be thiamine deficient by either of the above stated criteria, and to comment upon the significance of these relationships and of the derived TPP effect as measures of thiamine nutrition. The second purpose is to document the relationship of AETK with ETK in a subset of patients, namely alcohol drinkers suffering Wemicke’s encephalopathy (WE), in whom this relationship appears to be different from that in other patients studied. Materials and methods ETK and AETK were measured f19] in the Chemical Pathology laboratories of two Brisbane hospitals. Blood samples were collected into tubes containing lithium heparin then stored at 5 “C for up to 2 h, or up to 10 h in the case of samples collected late at night. The samples were then centrifuged at 1200 x g for 10 mm and the plasma and buffy coat removed by aspiration. The erythrocytes were then lyzed by addition of an equal volume of deionized, R.O. purified water containing Triton X-100, 2 ml/l, then stored at 5°C until assay the same day. Samples were deemed thiamine deficient and entered into this anaIysis whenever either of the following criteria were met: ETK < 0.6 U/g of hemoglobin, or TPP effect greater than 17%, or both. To limit the sample size, data from male alcoholics which met these criteria were arbitrarily restricted to those from patients in whom the measurements were repeated during the same hospital admission, although the repeat measurements are not analyzed here. The 104 patients were divided into a group of 56 drinkers (28 of each sex) and of 48 non-drinkers (24 of each sex). The non-drinkers included ex-drinkers whose alcohol consumption had been zero for at least 2 yr prior to admission. Alcohol consumption was assessed and recorded by the admitting medical officer, and corroborated by relatives and local medical officers. Laboratory measurements were used as a check on alcohol consumption: the mean erythrocyte corpuscular volume (MCV) was 101.3 fI (SE 1.01) in drinkers and 89.5 fl (SE 1.23) in non-drinkers, and
91
the median plasma gamma-ghnamyl transferase activity (yGT) in these respective groups was 114 U/l and 26.5 U/l. The differences between drinkers and nondrinkers were significant at the 0.001 level for the normally distributed, untransformed MCV values, and for the yGT values after log transformation to normalize the distribution. Two patients were excluded from further study because these data were not fully consistent. Of the non-drinkers, the primary diagnosis was psychiatric illness in 13 (depression, psychosis, bulimia) and drug dependence in 5. The remaining non-drinking patients were elderly and on a poor diet, or suffered a wide variety of medical illnesses, frequently multiple, or a combination of these possible causes of thiamine deficiency. The presence or absence of vo~ting at the time of admission was recorded for all but 4 patients, because vomiting might have led to the cessation of drinking. Of the 104 patients studied, 35 suffered Wemicke’s encephalopathy (WE) by meeting two or more of the following criteria: ataxia, ophthalmoplegia (including nystagmus) and global confusion [20]. Results and discussion
Mean ETK and AETK values Mean ETK and AETK values did not differ between males and females, between drinkers and non-drinkers or between vomiters and non-vomiters (data not shown). However, the mean ETK and AETK values for the WE patients, respectively 0.39 (SE 0.17) and 0.55 (SE 0.19), were both lower (P < 0.05) than the respective values for the group of other patients, 0.52 (SE 0.16) and 0.65 (SE 0.17); and WE was much more common (P -c 0.001) among drinkers than among non-drinkers. Of the group of drinkers, 50% had WE whereas only 15% of non-drinkers had WE.
TABLE I Linear regressions of AETK on ETK by patient groups Patient groups (and subgroups)
n
Slope
Intercept
Mean
SE
Mean
SE
Correlation coefficient (r)
1.045 1.050 1.047 1.062 1.084 1.217 1.116 1.033
0.033 0.070 0.038 0.071 0.082 0.059 0.160 0.081
0.101 0.103 0.097 0.121 0.121 0.039 0.151 0.104
0.018 0.036 0.021 0.030 0.034 0.027 0.058 0.037
0.9690 0.9465 0.9767 0.9338 0.9321 0.9860 0.9186 0.9846
U/g NonWE Drinkers Non-drinkers WE Drinkers Non-vomiters Vomiters * Non-drinkers
69 28 41 35 28 14 11 7
* Vomiting status was uncertain for 3 WE drinkers, who are not included in the subgroups. Note that the slopes for the two subgroups of WE drinkers are both greater than the slope of the whole group. Tbis results from the clustering of data and consequent unequal weighting of the contributions of each separate subgroup to each end of the regression for the whole group.
92
ETK, AETK
and TPP effect
For each group of patients classified by the presence of WE and by alcohol consumption (Table I), the AETK was closely related to ETK by a linear regression of slope in the range 1.033 to 1.084 and intercept on the AETK axis in the range 0.097 to 0.121. Division of the groups by vomiting status did not affect the regressions except in the case of WE drinkers, who are discussed later. Since in all groups the value for the slope was not different from 1 (Table I) for all values of ETK, the potential activity of apoTK able to be reactivated by the addition of TDP in vitro was constant and equal to the intercept on the AETK axis (Table I). The regressions for the total nonWE patients (Fig. l), nonWE drinkers, nonWE nondrinkers and WE non-drinkers could be superimposed (Table I). In the case of the WE drinkers (Fig. 2), the intercept and slope values were not significantly greater than those for all other groups (Table I). The good fit of this regression in all groups (0.9321 I I I 0.9846) is consistent with the proposition that this model accurately represents the relationship of AETK and ETK in any sample of blood. The very small residual differences from a perfect fit of the regression to the data (Figs. 1 and 2) show the maximum limits to variation of apoTK, since they represent the sum of both this variation and the
1.2I ,
,’
I’ I’
021 02
I 0.4
06 ETK
1 0.6
,fl
,’
I 1
( U/g)
Fig. 1. Linear regression of AETK on ETK for 69 nonWE patients, together with 95% confidence limits. Mean values for the slope and intercept on the AETK axis are listed in Table I, together with the correlation coefficient r for the fit of the regression to the data.
93
0.8-
06
0 0
0.1
0.2
03 ETK
0.4
05
0.6
0.7
(
8
(o/g)
Fig. 2. Linear regression of AETK on ETK for 28 WE drinkers. Other details are as for Fig. 1.
measure of lack of fit of the model. Thus the SEM values for the intercept are m~mum vahtes for the standard errors which measure the variation of apoTK When the attempt was made to relate TPP effect to ETK (Figs. 3 and 4), by fitting to the same data the hyperbolic expression for an inverse relationship TPP effect = c/ETK, which assumes that AETK-ETK is constant, the scatter of data was much greater and the goodness of fit much less than was the case for Figs. 1 and 2, as has been found by others [10,11,13,17~ who have d~umented the same relationship. The correlation coefficients for the fit of this model to the data were 0.7314 for Fig. 3 and 0.4735 for Fig, 4. There are good reasons to expect that the relations~p of TPP effect to ETK should be less close than the relations~p of AETK to ETK. Since the TK activity of erythrocytes is low, it is difficult to achieve very high precision for the measurement of AETK and ETK. Moreover, since the calculation of TPP effect requires first a calculation of the difference between these two values, each of indifferent precision, and then calculation of the ratio of that difference to one of these measurements, the precision of the final result must be much lower than the precision of either one measurement. This imprecision is reflected in the relatively poor correlation coefficients for the relationship of TPP effect and ETK [10,11,13,17, Figs 3 and 41. Calculation of TPP effect appears to add only imprecision to the assessment of thiamine nutrition, since our results show that variation of TPP effect is due only to
94
. I
I
02
I
1
03
04 ETK
05
I
06
I
07
08
09
(u/g)
Fig. 3. Hyperbolic regression of TPP effect on ETK for 69 nonWE patients, together with 95% confidence limits. The regression equation was TPP effect = c/ETK where c is a constant, thus assuming that apoTK is constant. The correlation coefficient r was 0.7314.
variation of ETK, the denominator in the calculation of TPP effect. The difference AETK-ETK, the measure of reactivatable apoTK and the numerator in the calculation of TPP effect, remained constant (Fig. 1) for all groups of patients (Table I).
I 180
160
, 140-
: \
a
\ ‘\
120-
20m 0 0
. I 0.2
I 04 ETK
.‘--------__________ , 06
(
(U/g)
Fig. 4. Hyperbolic regression of TPP effect on ETK for 35 WE patients. The correlation coefficient 0.4735. Other details are as for Fig. 3.
r was
95
The constant value for apoTK implies that changes in the concentration of total TK protein, i.e. synthesis or catabolism of apoTK, or both, are dependent upon cofactor concentration; otherwise apoTK would increase as thiamine nutrition and ETK decreased, and the slope of the regression of AETK on ETK would be closer to zero rather than 1. We conclude that calculation of the TPP effect adds nothing to the single measurement of ETK as a criterion of thiamine nutrition, and that the reputed sensitivity and reliability of the TPP effect [3-5,7-141 is due to its inverse relationship to ETK in the face of a constant value for apoTK. Therefore calculation of the TPP effect is without value, and argument [cf. 7-14 and 15-181 as to the relative sensitivity and reliability of the TPP effect versus the measurement of ETK for the evaluation of thiamine nutrition in alcoholics is irrelevant. Nevertheless, we show below that there might be small differences between the WE drinkers and all other patient groups in respect to the relationship of AETK and ETK, and suggest that those differences might provide insight into the pathogenesis of WE in these patients, without affecting the evaluation of thiamine nutrition. AETK and ETK in drinking WE patients Classification of WE drinkers into those who were vomiting on admission and those who were not resulted in different regressions for these subgroups. In the case of the WE drinkers who were not vomiting on admission, the linear regression (Fig. 5) of AETK on ETK was particularly tight (regression coefficient 0.986) and yielded a slope value of 1.22 (Table I), significantly greater (0.01 < P < 0.02) than the value for the nonWE group. Moreover, in this subgroup, the 95% confidence limits for the value of the slope (1.088 and 1.346) excluded unity. Thus in this subgroup, alone, the apoTK value was not constant, but ranged from a lowest experimental value of 0.05 U/g to a highest experimental value of 0.18 U/g, and theoretical values read from the linear regression ranged from 0.04 to 0.26 U/g as the value for ETK ranged from 0 to 1.0 U/g. To test for divergence between the non-vomiting WE drinker subgroup and other groups, we have plotted in Fig. 5 the 95% confidence limits for the linear regression of AETK on ETK for that group together with the regression, from Fig. 1, for all nonWE patients (which is identical to that for WE non-drinkers also). In the range ETK > 0.5 U/g, the regression for nonWE patients lies outside and below the 95% confidence limits of the regression for non-vomiting WE drinkers (Fig. 5). The latter regression line also lies above the 95% confidence limits for the former (not shown in Fig. 5 to avoid confusion). Thus the regression for the non-vomiting WE drinkers emerges as different from those for both the nonWE groups and the non-drinking WE group. In the case of the subgroup of vomiting WE drinkers, the linear regression of AETK on ETK (Table I) yielded a slope value of 1.116 and intercept of 0.15 U/g. The correlation coefficient of 0.9186 reflects the number and scatter of data points yielding this regression; although the difference AETK-ETK is greater at all points on this regression than on that for any other group, a significant difference for the slope value or intercept value has not been established. Thus in both subgroups of drinkers who develop WE, the regression of AETK on ETK could be different from that of other groups. Unlike the case of the subgroups of
96
06-
? 3
=
04-
:
0
0
I
I
I
02
04
06
ETK
(I. 6
(U/g)
Fig. 5. Linear regression of AETK on ETK for 14 non-vomiting WE drinkers (m) together with 95% confidence limits (. . . . .); and the regression, (- - - - - -), without data points, for 69 nonWE patients shown previously in Fig. 1. Other details are as for Fig. 1.
WE drinkers, the small group of non-drinkers who presented with WE yielded a regression of AETK on ETK which was not different from that of the nonWE group (Table I). Thus our data are consistent with the hypothesis that the TK of drinkers (but not that of non-drinkers) who develop WE is different from that of other patients. Evidence of a variant TK associated with WE has been previously reported [21-241 without specification of drinking status. Whether the putative variant is characterized by a lower affinity for cofactor [21,24], an altered turnover number, or by decreased stability of apoTK [23] must await measurements of TK protein in WE and nonWE patients, perhaps by immunoassay [25,26], and more sophisticated analysis of the activation of apoTK by cofactor [27]. Since the regressions for the non-vomiting and vomiting WE drinkers diverged at very low values of ETK (Table I, compare intercepts of 0.039 and 0.151, respectively), it might be that alcohol consumption in the period immediately before presentation has the effect, increasing in severity as ETK values decrease, of inactivating residual apoTK. Certainly others have observed [15-181 an unusually low TPP effect, despite decreased ETK, in the erythrocytes of thiamine-deficient chronic alcoholics; and acetaldehyde, the proximal product of ethanol metabolism,
91
has been shown [28] to inactivate apoTK much more readily than holoTK. Accurate records of drinking, by period before presentation with thiamine deficiency, are important to studies such as this despite the difficulty of their collection. Conclusion
We recommend measurement of ETK, without AETK or TPP effect, for evaluation of thiamine nutrition. However, measurement of AETK and ETK together has provided further evidence that some WE patients may have a variant of TK, and has suggested that the putative variant apoTK has increased susceptibility to inactivation by ethanol or its metabolites. Acknowledgements
We are grateful for the support of the National Health and Medical Research Council of Australia, to Dr. H. Kalant for constructive comments on the manuscript, and to Ms. N. Lee for typing the manuscript. References 1 Brin M, Tai M, Ostashever AS, Kalinsky H. The effect of thiamine deficiency on the activity of erythrocyte hemolysate transketolase. J Nutr 1960;71:273-281. 2 Brin M. Erythrocyte transketolase in early thiamine deficiency. Ann NY Acad Sci 1962;98:528-541. 3 Brin M. Erythrocyte as a biopsy tissue for functional evaluation of thiamine adequacy. J Am Med Assoc 1964;187:762-766. 4 Dreyfus PM. Clinical application of blood transketolase determinations. N Engl J Med 1962;267:596-598. 5 Sauberlich HE. Biochemical alterations in thiamine deficiency: their interpretation. Am J Clin Nutr 1967;20:528-542. 6 Wolfe SJ, Brin M, Davidson CS. Effect of thiamine deficiency on human erythrocyte metabolism. J Clin Invest 1958;37:1476-1484. 7 Tanphaichitr V, Vimokesant SL, Dhanamitta S, Valyasevi A. Clinical and biochemical studies of adult beriberi. Am J Clin Nutr 1970;23:1017-1026. 8 Sauberlich HE, Dowdy RP, Skala JH. Laboratory tests for the assessment of nutritional status. Thiamine (vitamin B,) CRC Crit Rev Clin Lab Sci 1973;4:236-244. 9 Kuriyama M, Yokomine R, Arima H, Hamada R, Igata A. Blood vitamin B,, transketolase and thiamine pyrophosphate (TPP) effect in beriberi patients, with studies employing discriminant analysis. Clin Chim Acta 1980;108:159-168. 10 McLaren DS, Docherty MA, Boyd DHA. Plasma thiamine pyrophosphate and erythrocyte transketolase in chronic alcoholism. Am J Clin Nutr 1981;34:1031-1033. 11 Waldenlind L, Borg S, Vikander B. Effect of peroral thiamine treatment on thiamine content and transketolase activity of red blood cells in alcoholic patients. Acta Med Stand 1981;209:209-212. 12 Graudal N, Torp-Pedersen K, Hanel H, Kristensen M, Thomsen AC, Nerrg&rd G. Assessment of thiamine nutritional status. An evaluation of erythrocyte transketolase activity, the stimulated erythrocyte transketolase activity and the thiamine pyrophosphate effect. Int J Vit Nutr Res 1985;55:399-403. 13 Anderson SH, Vickery CA, Nicol AD. Adult thiamine requirements and the continuing need to fortify processed cereals. Lancet 1986;2:85-89. 14 Fidanza F, Simonetti MS, Floridi A, Codini M, Fidanza R. Comparison of methods for thiamine and riboflavin nutriture in man. Int J Vit Nutr Res 1989;59:40-47.
98 15 Fermelly J, Frank 0, Baker H, Leevy CM. Red blood cell transketolase activity in malnourished alcoholics with cirrhosis. Am J Clin Nutr 1967;20:946-949. 16 Konttinen A, Louhija A, HSLrtel G. Blood transketolase in assessment of thiamine deficiency in alcoholics. Ann Med Exp Fenn 1970;40:172-175. 17 Camilo ME, Morgan MY, Sherlock S. Erythrocyte transketolase activity in alcoholic liver disease. Stand J Gastroent 1981;16:273-279. 18 Majumdar SK, Shaw GK, O’Gorman P, Aps EJ, Offerman EL, Thomson AD. Blood vitamin status (B,,B&, folic acid and B,,) in patients with alcoholic liver disease. Int J Vit Nutr Res 1982;52:266271. 19 Bayoumi RA, Rosa&i SB. Evaluation of methods of coenzyme activation of erythrocyte enzymes for detection of deficiency of vitamins B,, & and 4. Clin Chem 1976;22:327-335. 20 Reuler JB, Girard DE, Cooney TG. Wemicke’s encephalopathy. N Engl J Med 1985;312:1035-1039. 21 Blass JP, Gibson GE. Abnormality in a thiamine-requiring enzyme in patients with Wernicke-Korsakoff syndrome. N Engl J Med 1977;297:1367-1370. 22 Nixon PF, Kaczrnarek MJ, Tate J, Kerr R, Price J. An erythrocyte transketolase pattern associated with the Wemicke-Korsakoff syndrome. Eur J Clin Invest 1984;14:278-281. 23 Kerr RA, Clague AE, Morris DJ, Price J. The rapid decline in erythrocyte transketolase on cessation of high-dose thiamine administration in Korsakoff patients. Ale Ale 1986;21:257-262. 24 Mukheriee AB, Svoronos S, Ghazanfari A, et al. Transketolase abnormality in cultured fibroblasts from familial chronic alcoholic men and their male offspring. J Clin Invest 1987;79:1039-1043. 25 Takeuchi T, Jung EH, Nishino K, Itokawa Y. Western blotting assay of transketolase concentration in human hemolysates. Analyt Biochem 1988;168:470-475. 26 Paoletti F, Mocali A, Marchi M, Truschi F. Analysis of transketolase and identification of an enzyme variant in human leucocytes. Biochem Biophys Res Commun 1989;161:150-155. 27 Tate JR, Nixon PF. Measurement of Michaelis constant for human erythrocyte transketolase and thiamine diphosphate. Analyt Biochem 1987;160:78-87. 28 Atukorala TMS, Duggleby RG, Nixon PF. Effect of acetaldehyde upon catalysis by human erythrocyte transketolase. Biochem Pharmacol 1988;37:2100-2101.