Transketolase activities in human lymphocytes and granulocytes

NUTRITION RESEARCH, Vol. 10, pp. 1367-1373,1990 0271-5317/90 $3.00 + .00 Printed in the USA. Copyright (c) 1990 Pergamon Press plc. All rights reserved.

TRANSKETOLASE ACTIVITIES IN HUMAN LYMPHOCYTES AND GRANULOCYTES Richard C. Chu, Ph.D. and Charles A. Hall, M.D. Nutrition Laboratory for clinical Assessment and Research, Department of Veterans Affairs Medical Center and Department of Medicine, Albany Medical College, Albany, New York 12208

ABSTRACT Transketolase activities (TKA) were measured in human lymphooytes, granulocytes and erythrocytes by a NADH-dependent method. Lymphocyte and granulocyte TKA were 125 and 152 times greater, respectively, than erythrocyte. Xylulose-5-phosphate, at a final concentration of 3.6 mmol/L, was needed for optimal lymphocyte and granulocyte TKA, but had no apparent effect on erythrocyte TKA. Key Words:

Transketolase; Lymphocyte;

Granulocyte

INTRODUCTION Thiamine ( B I ) deficiency in humans results in Beriberi (i) and Wernicke's encephalopathy (2). A variety of biochemical methods have been used to assess B 1 nutriture of humans (3). This includes measurements of blood B 1 level (4), urinary excretion of B 1 with or without B 1 loading (5), blood pyruvate level (6), erythrocyte B 1 pyrophosphate level (7) and erythrocyte or whole blood transketolase activity (TKA) with or without the addition of B 1 pyrophosphate (8). Measurement of blood pyruvate level is not sufficiently sensitive and may be affected by other factors (9, I0). Erythrocyte TKA determination is now the method of choice because it reflects the function of B~ rather than the concentration of B 1 in tissues. However, aaequacy of using erythrocyte TKA as an indicator of B1 nutriture was questioned by Cheng et al. (ii). Brin (12) reported that it took 14 days for erythrocyte TKA to normalize after correcting B 1 deficiency. Cheng et al. (ii) in their study of rat leukocyte TKA concluded

1367

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R.C. CHU and C.A. HALL

that it is a s e n s i t i v e and s p e c i f i c i n d i c a t o r of B 1 nutriture. Measurement of l e u k o c y t e T K A as an a s s e s s m e n t of B 1 s t a t u s in humans is r a r e l y u s e d but has b e e n r e p o r t e d by Markkanen and Peltola (13, 14). L e u k o c y t e T K A w a s found to be 100-150 times higher t h a n t h a t of red cells. I n f o r m a t i o n on h u m a n lymphocyte and g r a n u l o c y t e TKA, however, is lacking. The purpose of this study w a s to m e a s u r e T K A in h u m a n l y m p h o c y t e s and granulocytes and c o m p a r e t h e i r a c t i v i t i e s w i t h r e d b l o o d cells. The n e e d of xylulose-5-phosphate for a s s a y i n g l y m p h o c y t e a n d g r a n u l o c y t e TKA in v i t r o w a s a l s o evaluated. Information gained will provide clinical a p p l i c a b i l i t y of t h e s e cells to the m e a s u r e m e n t s of B1 status.

MATERIALS

AND METHODS

Blood from ten h e a l t h y a d u l t s (6 w o m e n and 4 men) was c o l l e c t e d in h e p a r i n i z e d tubes. All s u b j e c t s w e r e v o l u n t e e r s and gave informed consent. This study was conducted under the a p p r o v a l of t h e i n s t i t u t i o n a l c o m m i t t e e o v e r s e e i n g h u m a n studies. Isolation of lymphocytes, granulocytes and erythrocytes were p e r f o r m e d a c c o r d i n g to the m e t h o d of B o y u m (15). A l i q u o t s of e a c h fraction were taken for cell counts. Cell viability for lymphocytes and granulocytes w e r e t e s t e d by the Trypan Blue method (16). L y m p h o c y t e s , g r a n u l o c y t e s and erythrocytes were f r o z e n a n d t h a w e d t h r e e t i m e s and c e n t r i f u g e d at 2,500 x g at 4~ for i0 m i n u t e s . Supernates obtained were diluted appropriately for T K A m e a s u r e m e n t s by the N A D H - d e p e n d e n t m e t h o d of Smeats et al. (17). Hemoglobin concentration was determined by the c y a n m e t h e m o g l o b i n m e t h o d u s i n g the h e m o g l o b i n a s s a y kit of Sigma C h e m i c a l Company. For xylulose-5-phosphate requirement studies, increasing amounts of xylulose-5-phosphate (Sigma Chemical Company) at 0, 1.8, 3.6 and 6.9 mmol/L, were added to the reaction tubes. All r e s u l t s w e r e s t a t i s t i c a l l y a n a l y z e d by the Student "t" t e s t (18). Differences were considered significant at P < 0 . 0 1 level.

RESULTS The activity of lymphocyte, granulocyte and erythrocyte transketolase of i0 h e a l t h y a d u l t h u m a n s is s h o w n in TABLE i. Lymphocyte TKA r a n g e d f r o m 31.8 to 199.3 ~mol/h/1 x 109 cells w i t h a m e a n of 107.4 • 14.1. G r a n u l o c y t e T K A r a n g e d f r o m 86.6 to 252 ~mol/h/l x 109 cells w i t h a m e a n of 130 • 10.4. Erythrocyte T K A r a n g e d f r o m 0.3 to 1.5 ~mol/h/l x 109 c e l l s w i t h a m e a n of 0.9 • 0.4. W h e n e r y t h r o c y t e t r a n s k e t o l a s e a c t i v i t y was expressed as U / g Hb, it r a n g e d f r o m 0.31 to 1.09 w i t h a m e a n of 0.68 • 0.07 w h i c h is c o m p a t i b l e w i t h t h a t found by S m e a t s et al. (17) who reported a m e a n v a l u e of 0.77 U / g Hb from 47 b l o o d donors. The differences in T K A b e t w e e n l y m p h o c y t e s and g r a n u l o c y t e s w a s not statistically significant. Lymphocyte and granulocyte TKA were 125 and 152 times greater (P<0.005), respectively, than e r y t h r o c y t e s TKA.

TRANSKETOLASE ACTIVITIES

1369

The e f f e c t s of a d d i n g x y l u l o s e - 5 - p h o s p h a t e to t h e reaction t u b e s on e r y t h r o c y t e , l y m p h o c y t e a n d g r a n u l o c y t e T K A a r e s h o w n in T A B L E 2. X y l u l o s e - 5 - p h o s p h a t e is not n e e d e d for t h e erythrocyte TKA. H o w e v e r , a l o w e r T K A w e r e f o u n d in h u m a n lymphocytes and granulocytes w h e n x y l u l o s e - 5 - p h o s p h a t e was n o t i n c l u d e d in the assay system. T h e final c o n c e n t r a t i o n of xylulose-5-phosphate n e e d e d for t h e o p t i m a l T K A w a s f o u n d to b e 3.6 mmol/L.

TABLE

1

T r a n s k e t o l a s e A c t i v i t i e s in H u m a n L y m p h o c y t e s , Granulocytes and Erythrocytes

Subject

Lymphocyte

Granulocyte

~mol/h/l

Erythrocyte

x 109 c e l l s

Erythrocyte

U/g Hb

i.

199.3

106.3

0.6

0.42

2.

134.2

134.1

1.4

0.98

3.

~13.7

86.8

1.2

0.61

4.

108.1

98.7

1.5

0.74

5.

99.5

132.8

1.2

0.57

6.

123.4

113.7

0.6

0.55

7.

118.9

99.1

0.3

0.31

8.

31.8

103.3

0.6

1.09

9.

62.0

252.0

0.3

0.83

i0.

83.6

181.7

1.0

0.68

Mean •

107.4 •

130.8 •

0.9 •

0.68 •

l

I N.So

I

I P<0.005

9

! P<0.005

R.C. CHU and C.A. HALL

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TABLE Effect

2

of X y l u l o s e - 5 - P h o s p h a t e on E r y t h r o c y t e r L y m p h o c y t e and Granulocyte Transketolase Activities

Final C o n c e n t r a t i o n of X y l u l o s e - 5 - P h o s p h a t e in Reaction Mixture

mmol/L Erythrocyte

Lymphocyte

Granulocyte

Activity

~mol/h/l

x 109 c e l l s

0

1.1

1.8

i. 1

3.6

l.l

6.9

1.1

0

90.0

1.8

157.0

3.6

165.3

6.9

158.7

0

49.4

i. 8

205.6

3.6

267.3

6.9

246.8

DISCUSSION Measurement of erythrocyte TKA with or without thiamine pyrophosphate saturation is a p o p u l a r m e t h o d for assessing B1 s t a t u s of h u m a n (8). It m e a s u r e s t h e f u n c t i o n of B 1 r a t h e r than the c o n c e n t r a t i o n of B 1 in tissues, s i n c e B 1 a c t s as a cofactor of transketolase. The use of leukocytes, particularly l y m p h o c y t e s a n d g r a n u l o c y t e s , for t h e e v a l u a t i o n of! B 1 n u t r i t u r e

TRANSKETOLASE ACTIVITIES

1371

in humans has not been well investigated. This may be due to the technically more difficult procedures involved in cell separation. Our findings of higher TKA in lymphocytes and granulocytes than erythrocytes is compatible with the earlier studies on leukocyte TKA from healthy human subjects reported by AMarkkanen and Peltola (13, 14). TKA averaged 7.7 ~mole/30 min/108 cells in man and 8.0 for woman and were about 100-150 times higher than that of the red cells. The higher TKA in lymphocytes and granulocytes in our study suggests that they may be more sensitive samples than erythrocytes for assessing B 1 nutriture. They probably will respond more rapidly to small changes of B 1 status than other cells of the body since they have a rapid turnover rate and are metabolically active (19, 20). The usefulness of m e a s u r i n g lymphocyte and granulocyte TKA in comparison with erythrocyte TKA in B 1 deficient human has not been reported. However, Cheng et al. (ii) recently evaluated rat leukocyte TKA as an indicator of B 1 adequacy and demonstrated that it is a practical, sensitive and specific test. Both hepatic and leukocyte TKA varied with dietary B 1 intake. A d m i n i s t r a t i o n of oxythiamin, resulted in decreases in ~KA in both the liver and leukocytes. Erythrocyte TKA measured by Smeat et al.'s method (17) is based on the formation of glyceraldehyde-3-phosphate from substrate ribose-5-phosphate as indicated in the following reaction: Ribose-5-P + xylulose-5-P = sedoheptulose-7-P + glyceraldehyde The need for xylulose-5-phosphate in the assay of erythrocyte TKA was investigated by Wiliams (21) who found that xylulose-5phosphate had little effect on erythrocyte TKA. Erythrocytes evidently are able to convert some of the ribose-5-phosphate to xylulose-5-phosphate by the enzymes ribose phosphate isomerase and ribulose phosphate-3-epimerase via ribulose-5-phosphate. Our findings of a lack of any apparent effect of xylulose-5-phosphate on erythrocyte TKA support the study of Williams (21). TKA, however, was found to be lower in human lymphocytes and granulocytes when xylulose-5-phosphate was not added in the assay system. This suggests that conversion of ribose-5-phosphate to xylulose-5-phosphate by lymphocytes and granulocytes was limited. Our study Clearly demonstrates the need for xylulose-5-phosphate, at a final concentration of 3.6 mmol/L, for optimal lymphocyte and granulocyte TKA in the NADH-dependent method. ACKNOWLEDGMENT This study was supported by the Medical Research Service of the Department of Veterans Affairs. Thanks to Ms Jean Polansky for her technical help.

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Accepted for publication August 27, 1990.

W a s h i n g t o n DC: Hematology.