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Enzyme activity as an indicator of red cell age
CLINIC.4 CHIMICS
ENZYME
ACTIVITY
.4CT.4
21
AS AN INDICATOR
OF RED CELL AGE MARTIN
D.
SASS,
ERIC
VORSANGER
TTeteransAdministration (Received
AND
Hospital, Brooklyn, August
Igth,
PAUL
N.Y.
W.
SPEAR*
(U.S.A .)
1963)
SUMMARY
Young red cells obtained from the upper layers of a centrifuged column of blood have been shown to exhibit a higher activity of certain enzymes than that found in the heavier and presumably older red cells at the bottom of the column. Of the enzymes studied, glutamic-oxaloacetic transaminase activity appeared to provide the most sensitive reflection of a change in red cell age, Its determination is proposed as the most appropriate for use as an aid in distinguishing the presence of a young red cell population.
Recent studies have provided extensive evidence that young red cells are characterized by variably increased activity of certain enzymesl-S. Thus, a decrease in the average age of the peripheral erythrocytes is reflected in a measurable increment in the activity of certain enzymes over that found in a population of cells with a normal age distribution. Ever since our demonstration that glutamic-oxaloacetic transaminase (GOT) was included in the group of enzymes whose activity is increased in young cells l we have been using this enzyme as an indicator of red cell proliferation 4$5 in the evaluation of a variety of anemias of questionable etiology. The adequacy with which increased enzyme levels reflect decreased red cell age, however, is simply a question of the magnitude of the difference between the enzyme activity found in young and old cells. Although there seems to be no doubt that such differences are encountered with numerous enzymes 6, little attention has been directed toward the selection of the most appropriate enzyme for this purpose. It was this consideration that stimulated the comparison of a number of enzymes which forms the basis for the present report.
METHODS
Samples of blood were obtained from patients with and without hematological disease. In the latter group, an effort was made to include a number of patients with chronic and acute blood loss to provide red cell populations with large numbers of young erythrocytes. Heparin was used as an anticoagulant throughout the study. * Present address:
Director of Medicine,
Morrisania Hospital,
Bronx,
New York.
M. I). SASS et al.
22
For the first phase of the study whole blood was subjected to centrifugation at 3,000 rev./min (2,000 g) for 2 h to affect a distribution in accordance with the varying density of young and old cells?. The column of red cells was subdivided into various samples by aspiration of successive portions of cells using a new technique to be reported elsewhere *. This separation provided fractions containing cells of progressively increasing density in accordance with the order in which they were removed from the red cell column. Including the heaviest cells at the bottom of the tube, eight fractions were obtained containing, respectively, 5%, IO%, IO%, 25O/o, 25%, IO%, IO”/ and5% of thecells in the total sample centrifuged. The cells were washed three times with normal saline, an effort being made to remove the buffy coat after each washing. The cells were then hemolyzed by dilution with water and particulate matter was removed by centrifugation at 2,000 rev./min for IO min. The enzyme activity and hemoglobin content of the clear supernatant solutions were determined as indicated below. In the second phase, enzyme analyses were carried out on washed red cell samples from a variety of patients with and without hematologic disease. In order to provide sufficient data, subject selection continued until elevated enzyme activity was encountered in a sufficient number of patients to permit statistical comparison. Blood samples were treated and hemolysates prepared as indicated above. In order to avoid variable losses of activity due to storage of hemolysates prior to analysis, determinations of enzyme activity were carried out on fresh samples with only rare exception. Since GOT has been shown to be stable on storage at -20’ as well as at 4’, analyses for this enzyme were occasionally carried out on material stored a maximum of 3 days at -20’. GOT activity was always estimated’ together with another single enzyme for each group. Catalase activity was determined by a titrimetric procedure 8, with millimoles of hydrogen peroxide destroyed as the unit of measurement. Spectrophotometric techniques were used for the estimation of lactic acid dehydrogenase (LDH) lo, glucosed-phosphate dehydrogenase (G-6-PD) 11, 6-phosphogluconic acid dehydrogenase (6-PGD) la and purine nucleoside phosphorylase (PNP) Is. For these, a unit represents a change of 1.0 O.D. units per minute *. All activities were corrected to the same temperature (25’) * * and calculations were based on the hemoglobin content of the hemolysate analyzed. Hemoglobin content was estimated in the Beckman DU spectrophotometer at 540 m,u. RESULTS
The distribution of activities of GOT and G-6-PD in the various red cell fractions separated after centrifugation is presented in Table I. In accordance with previous reportsB, the young cells are concentrated in the upper layers and contain increased activity of both of these enzymes 1l 2. As progressively heavier fractions are removed this activity decreases to the lowest level in the highest density fraction at the bottom of the tube. The differences in enzyme activity are significant at the 17; level for fractions I and 2 and 6,7 and 8 when compared to the activity of the whole population of cells prior to centrifugation. The percentage of the total hemoglobin in the various fractions (Table I) is in general agreement with the planned distribution. In a number * Total volume: 3.0 ml * * except for catalase. C[in. Chitrt. Ada, IO (1963) ~1-26
ENZYME ACTIVITY AND RED CELL AGE TABLE ACTIVITY
OF
FRACTIONS
VARIOUS
I
ENZYMES
SEPARATED
BY
NO.
___ G-6-PD
u/g Hb 6-PGD
(6):
(6)
(6)
(21
3 4 5 6
2.2
;
7.4 10.3
4.8 3.7 3.0 2.6 1.7
WEP Ratio
3.5
Fraction
I
Fraction
8
* Represents
4.5
‘7.3 16.5 13.6 II.4 IO.5 9.7 9.2 8.7 12.0
CELLS
activity
GOT
7.7 6.2
2
RED
Total Hb
5.6 9.5 10.3 23.1 22.3 II.5
I
IN
CENTRIFUGATION
Enzyme
%
Fraction
23
ro.4 8.1
LDH (2)
PNP 12) .--
190 ‘47 ‘49
165 165
6.4 6.3
I33 ‘44 ‘23 ‘37 I.32
149 154 143 132
7.3
‘43
149
8.1
7.0 6.6 6.8
I.7
162
I21
I.3
I.4
number of experiments.
GOT-",qn “b
Fig. I. Relationship between the activity of GOT and that of either G-6-PD, 6-PGD, PNP. LDH or catalase in the red cells from patients with and without hematologic disease.
of experiments, however, the last fraction to be removed contained a proportion of the total sample in excess of the 5% originally planned. Thus, the mean enzyme activity of these fractions (No. 8, Table I) represents an overestimation due to the presence of lighter, younger cells. Clin. Chim. Acta, 10 (1964) 21-26
24
M.
I).
SAss et al.
Similar experiments were carried out to compare the changes in GOT activity with those of PNP, 6-PGD and LDH (Table I). Of this latter group, only 6-PGD showed a definite progressive drop in activity with increasing red cell density and age. Changes in LDH and PNP activity were sporadic and not so striking. In the second phase of this study, GOT activity was compared with the activity of a different enzyme in the red cells from each of five groups of patients. Scatter diagrams of the results and their regression lines (Fig. I, A-E) indicate the degree to which increases in GOT activity are associated with increases in activity of G-6-PD, 6-PGD, PNP, LDH and catalase. The correlation coefficients (Table II) indicate a statistically significant positive correlation between the activity of GOT and each of the enzymes except catalase. GOT and G-6-PD showed the highest degree of correlation. An examination of the respective regressions of G-6-PD, 6-PGD, PNP, TABLE CORRELATION
_~_.._
_~
AND
G-6-PD,
REGRESSION
6-PGD,
COEFFICIENTS
PNI’,
G-6-PD b-PGD PNP LDH Catalase .____~~~
LDH
Correlntior~ cocfficie,lt
Ellcyme
_ ~_~___
II
0.77 0.45 0.68 0.63 -0.2, _~
COMPARING AND
GOT
WITH
CATALASE
Hegrcssion coefficirwt 0.79 0.19
0..54 7.3 -0.19.5
_~
LDH and catalase on GOT activity indicate that the change in GOT activity is associated with a numerically smaller change in the activity of G-6-PD, 6-PGD, PNP and catalase. This is illustrated by regression coefficients less than unity for these comparisons (Table II) despite the fact that the units of G-6-PD and PNP activity are substantially larger than the units of GOT activity. The relatively large regression coefficient for LDH becomes insignificant when full consideration is given to the fact that its unit is approximately ~5 times greater than that of GOT. Extensive experience with red cell GOT activity in this laboratory has established a normal mean of 3.4 U/g Hb with a standard deviation of _t_ 0.96. This has permitted the selection of 5.3 U/g Hb as the upper limit of normal with a 9~7~ level of confidence that values in excess of this figure represent samples from an other than normal red cell population. Applying this criterion we have subdivided the data in each group on the basis of normal or increased GOT activity, reflecting the presence of two different red cell populations. In one, represented by GOT activity within the normal range (A, Table III), the distribution of cells of different ages is normal; in the other (B, Table III), elevated GOT activity indicates a skewed age distribution with increased numbers of younger cells. The five major groups differ only in that, in each, GOT activity was compared with the activity of another enzyme. A comparison of the ratios of the enzyme activities of the two sub-groups (ratio B/A, Table III) emphasizes the fact that a decrease in the age of the red cell population is associated with at least a two to three-fold increase in GOT activity. Although statistically significant to a varying degree, changes in the activities of the other enzymes appear to be less striking.
ENZYME
ACTIVITY
AND RED CELL BGE
TABLE ACTIVITY OF VARIOUS ENZYMES IN
III
NORMAL
No.
Group
AND
of
Normal Young Ratio B/A
19 IO
P
II A B
Normal Young Ratio B/A
I2
*4
P
III A B
YOUNG
Normal Young Ratio B/A
*4 IO
P
A B
Normal Young Ratio B/A
34 23
G-6-PD
3.3 9.0 2.72 <.OI
13.1 18.1 1.38 .06
GOT
6.PGD
3.7 10.4 2.81 <.or
5.0 8.5 1.70
GOT
LDH
3.6 II.2 3.11
16.6 21.9
3.55 7.8 <.OI
GOT
V A B
Normal Young Ratio B/A
13 16
3.9 8.0 2.O.j
P
POPULATIONS
u/g Hb
2.20
P
CELL
GOT
GOT
IV
RED
Enzyme activity
subjects
I A B
2.5
<.OI
1.32 c.057
PNP 12.0
13.9 1.16 <.or
Catalase 11.82 1o.*g
.86 .06
DISCUSSION
The present data obtained by centrifugation of normal red cells confirm previous observations on the relative changes in the activities of G-6-PD, 6-PGD, LDH and PNP associated with erythrocyte aging 2. Of these, only the former two exhibit a definite and consistent increase in activity with decreasing age of the erythrocyte fraction. Under comparable conditions, decreased red cell age is associated with at least a two-fold greater increase in GOT activity, providing a far more sensitive reflection of the age differences found in the various red cell fractions of different densities. The findings on red cells from patients with a variety of disorders corroborate the view that, of the enzymes studied, GOT activity represents the most sensitive indication of a decrease in the average age of an erythrocyte population. The high coefficient of correlation between GOT and G-6-PD activity (Table II) suggests that both parameters may be reflecting the same phenomenon. On the basis of the results with centrifuged cells as well as previous studies on increased enzyme activity in young erythrocytes21 14116, it is probable that this phenomenon is the aging process. It is conceivable that some other factor apart from age is responsible for part of the more marked increases in GOT activity encountered in the various groups studied. Extensive experience in approximately 2,000 patients has confirmed previous indications* that red cell GOT activity is within the normal range in a variety of Clitz. Chit% Artn, IO (1964) ?I--26
26
M. D. SASS et al.
disease states unassociated with increased red cell proliferation. To our knowledge, pyridoxine administration is the only other factor beside decreased age that may be responsible for increased red cell GOT activity 18. The few patients encountered receiving this vitamin were eliminated from the study. The desirability of an evaluation of mean red cell age as an indication of the proliferative response of the bone marrow needs no justification. Reticulocytosis is notoriously erratic and, at best, too short-lived. Several investigators have utilized cholinesterase activity for this purpose 3. An adequate selection of the most appropriate enzyme requires a comparative evaluation of the technical simplicity of an analytical procedure, sensitivity of the enzyme activity to change in mean cell age and stability of the enzyme on storage of the erythrocyte hemolysate. GOT apparently fits all requirements. The instability of G-6-PD and the reduced sensitivity of both G-6-PD and 6-PGD make these enzymes undesirable for routine use. No comparison with cholinesterase activity has been attempted. Its methcdology would appear to bc far more laborious than any of the other enzymes we have studied. In addition, an examination of the reported data on the increased cholinesterase activity in diseases characterized by young red cell populations3 supports the view that GOT activity provides a much more adequate response to the changes in rtd cell age in similar diseases 4?5. REFERENCES 1 M. D. SASS AND P. W. SPEAR, J. Lab. Clin. Med., JI (1958) 926. * P. A. MARKS AND A. B. JOHNSON, J. Clin. Ir~vesl., 37 (1958) 1542. 9 J. C. SABINE, Am. J. Med., 27 (rgsg) 81. 4 M. D. SASS AND P. W. SPEAR, J. Lab. Clin. Med., 58 (1961) 580. 5 M. D. SASS AND P. W. SPEAR, J. Lab. Clin. Med., $3 ($961) 586. * T. A. 1. PRANKERD, The Red Cell, Chas. C. Thomas, Sprin&eld. III., r9G1. 7 E. R. “BORWN, W. G. FIGUEROA AND S. M. PERRY, J. ~lin~lnwst., 36 (1957) 676. * D. J. O’CONNELL .+ND M. D. SASS, to be published. @ E. BEUTLER AND R. K. BLAISDELL, J. Clin. Invest., 37 (1958) 833. 10 F. WROBLEWSK~ AND J. S. LADUE, Proc. Sot. Exptl. Biol. Med., go (1955) 210. 11 A. KORNBERG AND B. L. HORECKER, in S. P. COLOWICK AND M. 0. KAPLAN (Eds.), Methods in E~zy~ology~ Vol. I, 1955, p. 323. I8 B. L. HORECKER AND P. Z. SMYRNIOTIS, J. Viol. Chem., 193 (1951) 37r. 13 Y. E. PRICE, M. C. OTEY AND P. PLESNES, in S. P. COLOWICK AND M. 0. KAPLAN (Eds,), Methods in Enzymology, Vol. 2, 1955, p. 448. I4 L. M. LEVY, W. WALTER AND M. D. SASS, Nature, 184 (1959) 643. 15 M. D. SASS, L. M. LEVY AND H. WALTER, Can. J. Biochem. Physiol., 41 (1963) 2287. I8 M. D. SASS AND G. T. MURPHY, .4m. J. CLin. Nutr., 6 (1958) 424 C&.
China. A&,
IO (x964) ~I-26
ENZYME
ACTIVITY
.4CT.4
21
AS AN INDICATOR
OF RED CELL AGE MARTIN
D.
SASS,
ERIC
VORSANGER
TTeteransAdministration (Received
AND
Hospital, Brooklyn, August
Igth,
PAUL
N.Y.
W.
SPEAR*
(U.S.A .)
1963)
SUMMARY
Young red cells obtained from the upper layers of a centrifuged column of blood have been shown to exhibit a higher activity of certain enzymes than that found in the heavier and presumably older red cells at the bottom of the column. Of the enzymes studied, glutamic-oxaloacetic transaminase activity appeared to provide the most sensitive reflection of a change in red cell age, Its determination is proposed as the most appropriate for use as an aid in distinguishing the presence of a young red cell population.
Recent studies have provided extensive evidence that young red cells are characterized by variably increased activity of certain enzymesl-S. Thus, a decrease in the average age of the peripheral erythrocytes is reflected in a measurable increment in the activity of certain enzymes over that found in a population of cells with a normal age distribution. Ever since our demonstration that glutamic-oxaloacetic transaminase (GOT) was included in the group of enzymes whose activity is increased in young cells l we have been using this enzyme as an indicator of red cell proliferation 4$5 in the evaluation of a variety of anemias of questionable etiology. The adequacy with which increased enzyme levels reflect decreased red cell age, however, is simply a question of the magnitude of the difference between the enzyme activity found in young and old cells. Although there seems to be no doubt that such differences are encountered with numerous enzymes 6, little attention has been directed toward the selection of the most appropriate enzyme for this purpose. It was this consideration that stimulated the comparison of a number of enzymes which forms the basis for the present report.
METHODS
Samples of blood were obtained from patients with and without hematological disease. In the latter group, an effort was made to include a number of patients with chronic and acute blood loss to provide red cell populations with large numbers of young erythrocytes. Heparin was used as an anticoagulant throughout the study. * Present address:
Director of Medicine,
Morrisania Hospital,
Bronx,
New York.
M. I). SASS et al.
22
For the first phase of the study whole blood was subjected to centrifugation at 3,000 rev./min (2,000 g) for 2 h to affect a distribution in accordance with the varying density of young and old cells?. The column of red cells was subdivided into various samples by aspiration of successive portions of cells using a new technique to be reported elsewhere *. This separation provided fractions containing cells of progressively increasing density in accordance with the order in which they were removed from the red cell column. Including the heaviest cells at the bottom of the tube, eight fractions were obtained containing, respectively, 5%, IO%, IO%, 25O/o, 25%, IO%, IO”/ and5% of thecells in the total sample centrifuged. The cells were washed three times with normal saline, an effort being made to remove the buffy coat after each washing. The cells were then hemolyzed by dilution with water and particulate matter was removed by centrifugation at 2,000 rev./min for IO min. The enzyme activity and hemoglobin content of the clear supernatant solutions were determined as indicated below. In the second phase, enzyme analyses were carried out on washed red cell samples from a variety of patients with and without hematologic disease. In order to provide sufficient data, subject selection continued until elevated enzyme activity was encountered in a sufficient number of patients to permit statistical comparison. Blood samples were treated and hemolysates prepared as indicated above. In order to avoid variable losses of activity due to storage of hemolysates prior to analysis, determinations of enzyme activity were carried out on fresh samples with only rare exception. Since GOT has been shown to be stable on storage at -20’ as well as at 4’, analyses for this enzyme were occasionally carried out on material stored a maximum of 3 days at -20’. GOT activity was always estimated’ together with another single enzyme for each group. Catalase activity was determined by a titrimetric procedure 8, with millimoles of hydrogen peroxide destroyed as the unit of measurement. Spectrophotometric techniques were used for the estimation of lactic acid dehydrogenase (LDH) lo, glucosed-phosphate dehydrogenase (G-6-PD) 11, 6-phosphogluconic acid dehydrogenase (6-PGD) la and purine nucleoside phosphorylase (PNP) Is. For these, a unit represents a change of 1.0 O.D. units per minute *. All activities were corrected to the same temperature (25’) * * and calculations were based on the hemoglobin content of the hemolysate analyzed. Hemoglobin content was estimated in the Beckman DU spectrophotometer at 540 m,u. RESULTS
The distribution of activities of GOT and G-6-PD in the various red cell fractions separated after centrifugation is presented in Table I. In accordance with previous reportsB, the young cells are concentrated in the upper layers and contain increased activity of both of these enzymes 1l 2. As progressively heavier fractions are removed this activity decreases to the lowest level in the highest density fraction at the bottom of the tube. The differences in enzyme activity are significant at the 17; level for fractions I and 2 and 6,7 and 8 when compared to the activity of the whole population of cells prior to centrifugation. The percentage of the total hemoglobin in the various fractions (Table I) is in general agreement with the planned distribution. In a number * Total volume: 3.0 ml * * except for catalase. C[in. Chitrt. Ada, IO (1963) ~1-26
ENZYME ACTIVITY AND RED CELL AGE TABLE ACTIVITY
OF
FRACTIONS
VARIOUS
I
ENZYMES
SEPARATED
BY
NO.
___ G-6-PD
u/g Hb 6-PGD
(6):
(6)
(6)
(21
3 4 5 6
2.2
;
7.4 10.3
4.8 3.7 3.0 2.6 1.7
WEP Ratio
3.5
Fraction
I
Fraction
8
* Represents
4.5
‘7.3 16.5 13.6 II.4 IO.5 9.7 9.2 8.7 12.0
CELLS
activity
GOT
7.7 6.2
2
RED
Total Hb
5.6 9.5 10.3 23.1 22.3 II.5
I
IN
CENTRIFUGATION
Enzyme
%
Fraction
23
ro.4 8.1
LDH (2)
PNP 12) .--
190 ‘47 ‘49
165 165
6.4 6.3
I33 ‘44 ‘23 ‘37 I.32
149 154 143 132
7.3
‘43
149
8.1
7.0 6.6 6.8
I.7
162
I21
I.3
I.4
number of experiments.
GOT-",qn “b
Fig. I. Relationship between the activity of GOT and that of either G-6-PD, 6-PGD, PNP. LDH or catalase in the red cells from patients with and without hematologic disease.
of experiments, however, the last fraction to be removed contained a proportion of the total sample in excess of the 5% originally planned. Thus, the mean enzyme activity of these fractions (No. 8, Table I) represents an overestimation due to the presence of lighter, younger cells. Clin. Chim. Acta, 10 (1964) 21-26
24
M.
I).
SAss et al.
Similar experiments were carried out to compare the changes in GOT activity with those of PNP, 6-PGD and LDH (Table I). Of this latter group, only 6-PGD showed a definite progressive drop in activity with increasing red cell density and age. Changes in LDH and PNP activity were sporadic and not so striking. In the second phase of this study, GOT activity was compared with the activity of a different enzyme in the red cells from each of five groups of patients. Scatter diagrams of the results and their regression lines (Fig. I, A-E) indicate the degree to which increases in GOT activity are associated with increases in activity of G-6-PD, 6-PGD, PNP, LDH and catalase. The correlation coefficients (Table II) indicate a statistically significant positive correlation between the activity of GOT and each of the enzymes except catalase. GOT and G-6-PD showed the highest degree of correlation. An examination of the respective regressions of G-6-PD, 6-PGD, PNP, TABLE CORRELATION
_~_.._
_~
AND
G-6-PD,
REGRESSION
6-PGD,
COEFFICIENTS
PNI’,
G-6-PD b-PGD PNP LDH Catalase .____~~~
LDH
Correlntior~ cocfficie,lt
Ellcyme
_ ~_~___
II
0.77 0.45 0.68 0.63 -0.2, _~
COMPARING AND
GOT
WITH
CATALASE
Hegrcssion coefficirwt 0.79 0.19
0..54 7.3 -0.19.5
_~
LDH and catalase on GOT activity indicate that the change in GOT activity is associated with a numerically smaller change in the activity of G-6-PD, 6-PGD, PNP and catalase. This is illustrated by regression coefficients less than unity for these comparisons (Table II) despite the fact that the units of G-6-PD and PNP activity are substantially larger than the units of GOT activity. The relatively large regression coefficient for LDH becomes insignificant when full consideration is given to the fact that its unit is approximately ~5 times greater than that of GOT. Extensive experience with red cell GOT activity in this laboratory has established a normal mean of 3.4 U/g Hb with a standard deviation of _t_ 0.96. This has permitted the selection of 5.3 U/g Hb as the upper limit of normal with a 9~7~ level of confidence that values in excess of this figure represent samples from an other than normal red cell population. Applying this criterion we have subdivided the data in each group on the basis of normal or increased GOT activity, reflecting the presence of two different red cell populations. In one, represented by GOT activity within the normal range (A, Table III), the distribution of cells of different ages is normal; in the other (B, Table III), elevated GOT activity indicates a skewed age distribution with increased numbers of younger cells. The five major groups differ only in that, in each, GOT activity was compared with the activity of another enzyme. A comparison of the ratios of the enzyme activities of the two sub-groups (ratio B/A, Table III) emphasizes the fact that a decrease in the age of the red cell population is associated with at least a two to three-fold increase in GOT activity. Although statistically significant to a varying degree, changes in the activities of the other enzymes appear to be less striking.
ENZYME
ACTIVITY
AND RED CELL BGE
TABLE ACTIVITY OF VARIOUS ENZYMES IN
III
NORMAL
No.
Group
AND
of
Normal Young Ratio B/A
19 IO
P
II A B
Normal Young Ratio B/A
I2
*4
P
III A B
YOUNG
Normal Young Ratio B/A
*4 IO
P
A B
Normal Young Ratio B/A
34 23
G-6-PD
3.3 9.0 2.72 <.OI
13.1 18.1 1.38 .06
GOT
6.PGD
3.7 10.4 2.81 <.or
5.0 8.5 1.70
GOT
LDH
3.6 II.2 3.11
16.6 21.9
3.55 7.8 <.OI
GOT
V A B
Normal Young Ratio B/A
13 16
3.9 8.0 2.O.j
P
POPULATIONS
u/g Hb
2.20
P
CELL
GOT
GOT
IV
RED
Enzyme activity
subjects
I A B
2.5
<.OI
1.32 c.057
PNP 12.0
13.9 1.16 <.or
Catalase 11.82 1o.*g
.86 .06
DISCUSSION
The present data obtained by centrifugation of normal red cells confirm previous observations on the relative changes in the activities of G-6-PD, 6-PGD, LDH and PNP associated with erythrocyte aging 2. Of these, only the former two exhibit a definite and consistent increase in activity with decreasing age of the erythrocyte fraction. Under comparable conditions, decreased red cell age is associated with at least a two-fold greater increase in GOT activity, providing a far more sensitive reflection of the age differences found in the various red cell fractions of different densities. The findings on red cells from patients with a variety of disorders corroborate the view that, of the enzymes studied, GOT activity represents the most sensitive indication of a decrease in the average age of an erythrocyte population. The high coefficient of correlation between GOT and G-6-PD activity (Table II) suggests that both parameters may be reflecting the same phenomenon. On the basis of the results with centrifuged cells as well as previous studies on increased enzyme activity in young erythrocytes21 14116, it is probable that this phenomenon is the aging process. It is conceivable that some other factor apart from age is responsible for part of the more marked increases in GOT activity encountered in the various groups studied. Extensive experience in approximately 2,000 patients has confirmed previous indications* that red cell GOT activity is within the normal range in a variety of Clitz. Chit% Artn, IO (1964) ?I--26
26
M. D. SASS et al.
disease states unassociated with increased red cell proliferation. To our knowledge, pyridoxine administration is the only other factor beside decreased age that may be responsible for increased red cell GOT activity 18. The few patients encountered receiving this vitamin were eliminated from the study. The desirability of an evaluation of mean red cell age as an indication of the proliferative response of the bone marrow needs no justification. Reticulocytosis is notoriously erratic and, at best, too short-lived. Several investigators have utilized cholinesterase activity for this purpose 3. An adequate selection of the most appropriate enzyme requires a comparative evaluation of the technical simplicity of an analytical procedure, sensitivity of the enzyme activity to change in mean cell age and stability of the enzyme on storage of the erythrocyte hemolysate. GOT apparently fits all requirements. The instability of G-6-PD and the reduced sensitivity of both G-6-PD and 6-PGD make these enzymes undesirable for routine use. No comparison with cholinesterase activity has been attempted. Its methcdology would appear to bc far more laborious than any of the other enzymes we have studied. In addition, an examination of the reported data on the increased cholinesterase activity in diseases characterized by young red cell populations3 supports the view that GOT activity provides a much more adequate response to the changes in rtd cell age in similar diseases 4?5. REFERENCES 1 M. D. SASS AND P. W. SPEAR, J. Lab. Clin. Med., JI (1958) 926. * P. A. MARKS AND A. B. JOHNSON, J. Clin. Ir~vesl., 37 (1958) 1542. 9 J. C. SABINE, Am. J. Med., 27 (rgsg) 81. 4 M. D. SASS AND P. W. SPEAR, J. Lab. Clin. Med., 58 (1961) 580. 5 M. D. SASS AND P. W. SPEAR, J. Lab. Clin. Med., $3 ($961) 586. * T. A. 1. PRANKERD, The Red Cell, Chas. C. Thomas, Sprin&eld. III., r9G1. 7 E. R. “BORWN, W. G. FIGUEROA AND S. M. PERRY, J. ~lin~lnwst., 36 (1957) 676. * D. J. O’CONNELL .+ND M. D. SASS, to be published. @ E. BEUTLER AND R. K. BLAISDELL, J. Clin. Invest., 37 (1958) 833. 10 F. WROBLEWSK~ AND J. S. LADUE, Proc. Sot. Exptl. Biol. Med., go (1955) 210. 11 A. KORNBERG AND B. L. HORECKER, in S. P. COLOWICK AND M. 0. KAPLAN (Eds.), Methods in E~zy~ology~ Vol. I, 1955, p. 323. I8 B. L. HORECKER AND P. Z. SMYRNIOTIS, J. Viol. Chem., 193 (1951) 37r. 13 Y. E. PRICE, M. C. OTEY AND P. PLESNES, in S. P. COLOWICK AND M. 0. KAPLAN (Eds,), Methods in Enzymology, Vol. 2, 1955, p. 448. I4 L. M. LEVY, W. WALTER AND M. D. SASS, Nature, 184 (1959) 643. 15 M. D. SASS, L. M. LEVY AND H. WALTER, Can. J. Biochem. Physiol., 41 (1963) 2287. I8 M. D. SASS AND G. T. MURPHY, .4m. J. CLin. Nutr., 6 (1958) 424 C&.
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