Immunol. Cell Bid. (1990) 68, 75-79
A semi-automated method for 5'-ectonucleotidase m^suremj in lymphocytes A. H. Chalmers and Cavelyn Hare Department of Pathology. Mater Misericordiae Public Hospitals, South Brisbane, Qld4I0/. Australia (Submitted
22 June 1989. Accepted for publication
14 November
1989.)
Summary We describe a simple, non-radioactive, semi-automated method for measuring lymphocyte ccto-5'-nucleotidase activity in isolated cell homogenates. The method which uses inosine 5'-monophosphate(IMP)asa subslrate was optimized and requires a total of 4x 10*' lympbocytes. The reference range obtained (38 7-180 0 nmol/h per mg protein; .v= 106-6) agrees with other isotopic methods which use ''*C-IMP. The simplicity, sensitivity (lower limit 10, upper limit 400 nmol/h per mL) and precision (at 114 nmol/h per mL, interday4-2%s.d.,M = 20; intraday 5-8%, n * 10) makes this method suitable for routine eliniea! laboratory use.
INTRODUCTION Ecto-5'-nucleotidase (E.C.3.1.3,5: NT) located on the external surface of cells catalyses the conversion of purine and pyrimidlne ribo- and deoxyribonucleotide tnonophosphates to their corresponding nucleosides (I). This enzyme which occurs in both T and B lymphocytes is a marker for the maturation of these cells (2-4). Because NT is reduced in a number of congenital and acquired immunodeficiency disorders its measurement is now clinically desirable (5-8). Most methods for the estimation of NT in lympbocytes are time consuming, involve cbromatographic procedures and use expensive radioisotopes (2.8-16). Also most methods use intact cells and are therefore difficult to assay when there are geographical constraints. We describe one assay which measures NT in lymphocyte ceil homogenates by a semi-automated method. The biochemical conversions in this assay are as follows:
Manual: IMP
NT
• Inosme
PNP Phosphate
Hypoxanthine Automated: Hypoxanthine XO
Uric acid
Xanthine 293 nm)
In the manual part of the assay, NT from lymphocytes converts the substrate inosine 5'monophospbate (IMP) to inosine which, in the presence of phosphate buffer and high levels of endogenous purine nucieoside phospborylase in lymphocytes (17), is converted to hypoxanthine. In the automated part of the assay the conversion of hypoxanthine to uric acid with xanthine oxidase (XO) is measured at 293 nm in a centrifugal analyser.
MATERIALS AND METHODS Correspondence: Dr A. H. Chalmers. Depanment of Haematology. Flinders Medieal Centre, Bedford Park. SA 5042, Australia. .Ahhrevialion.'i used in this paper: AMP, Adenosine 5'-monophosphate; AOPCP. Q./3-mcthylcne adenosine 5'-dipbosphate: Con-A, Concanavalin-A; dTTP, deoxylbymidine 5'-triphosphate; E293, extinction or absorbance change at 293 nm; EDTA. cthylencdiaminetetraacetate; IMP, inosine 5'-monophosphate; NT, 5'-ectonueleotidase; PNP, purine nucieoside phosphorylase; XO, xanthine oxidase.
Inosine 5'-monophosphatc (Na +: IMP), deoxythymidine triphosphate (dTTP). ^glycerophosphate, a.^methylene adenosine diphosphate (AOPCP), Concanavaiin-A (Con-A). 5'-ND Control E were all purchased from Sigma Chemical Co. (St Louis, MO, USA). Xanthine oxidase (XO: Cow's milk; 20 units/mL. 10 units/mg protein), purine nucieoside phosphorylase (PNP; bovine calf spleen; 20 units/mg protein) were purchased from Boehringcr Mannheim (Sydney. Australia). All other reagents were of analytical grade quality.
76
A. H. CHALMERS AND C. HARE
Substrate reagent 1MP 5 mniol/L, ^glycerophosphate2 mmol/L, magnesium chloride 4 mmol/L, manganese chloride 0-24 mmol/L were stored as I mL aliquots at —20°C in phosphate buffer (Na ^; 100 mmol/L; pH 8 0). Conirol 5'-ND Control E (75 U/L) is a commercial nucleotidase (reptile) in bovine serum albumin. To each vial was added 200 mLTris(10 mmol/L, pH 7-4)/isotonic saline buffer at 4°C and this solution was stored in 0-5 mL aliquots at -2O''C, Before each assay, the control was thawed and 10 juL PNP (0 2 units) was added. Lymphocyte separation Lymphocytes from 10 mL EDTA-collected blood were isolated in >80% purity using lymphocyte Separation Medium (Flow Laboratories, Sydney) and washed twice with tris/saline before suspending at 1 •5-2-5x10' cells/mL. Cells were stored frozen at — 20°C for batch analysis and. after thawing, sonicated at 4°C with 4x5 s pulses (Branson B30 Sonifier Cell Disrupter) at energy levels 5-7 on the day of analysis (!7). Protein was estimated by the Lowry method (18). Enzyme analysis The manual part of the assay was done in Cobas Bio specimen cups (Roche, Sydney) and the automated analysis in a Cobas Bio centrifugal analyser. Both assays were carried out at 37°C. Manual assay To 20 ^L substrate reagent add 50 pL of lymphocyte extract. The reaction was done at 37''C and stopped with 20 /iL HC! (I -0 mol/L) at 0, 30 and 60 min after addition of lymphocyte extract. The acidified reaction mixture was then spun 1-5 min in a microfuge centrifuge and then transferred to the circular Cobas Bio analysis tray. Automated assay The reagent buffer used in the assay was potassium phosphate (0-25 mol/L; pH 8-0). The start reagent. XO diluted I/lOO with reagent buffer to give 0-2 units/mL, was prepared prior to use and stored at 4''C. The Cobas Bio was programmed as follows: Units, absorbance change; Calculation factor 1790; Standards I, 2, 3 were 0 concentration; Limit 400; Temperature 37°C; Type of analysis 6 (end-point); Wavelength 293 nm;Sampievolume 20^L; Diluent volume 20/JL; Reagent buffer volume 200 ^L; Incubation time 20 s; Start reagent volume 20/jL; Time of first reading 60 s; Time interval 20 s; Number of readings 5; Blanking mode 1; Printout mode I. Calculation The calculation factor (1790) was derived as follows: MT, wv. ,. E293 117 1000 NT (nmol/h per mL) = 1-04 _ '" x 5 '• 12-6
= £293x1790
where I 04 is the pathlength of the curvettes in centimetres; 12-6 the millimolar extinction coefficient of uric acid at 293 nm; 117/5 is the dilutioti factor; and E293 is the change in extinction at 293 nm after XO addition. The time zero result is subtracted from both the 30 and 60 min reactions. The net time 30 min reading is then multiplied by 2 and this figure is averaged with the net time 60 min readmg as follows: Average NT (nmol/h/mL) = NT(t30 - tO)x2 H- NT(t60 - tO) 2 This figure divided by the mg protein/mL of lymphocyte fiomogenate gives NT activity in nmol/h per mg protein. RESULTS AND DISCUSSION Because of the low NT activity in lymphocytes the assay was optimized by using a large protein enzyme mass (005-01 mg/50 /iL) to low substrate volume (20 ^L). The reaction was linear to 60 min and hence was monitored at time 0, 30 and 60 min. The purity of lymphocytes obtained was consistently above 80%. however, the possibility that other contaminating cells may contribute to the activity was considered. Red blood cell represented the largest group of contaminating cells. The activity of NT in these cells was very low (1-5 nmo!/h per mg protein) and represented only I-5% of the lymphocyte activity in normals. Treatment of cell preparations with 0-84% NH4CI to lyse red blood cells did not affect NT activities and so this step was omitted from the method. NT is virtually absent in peripheral blood monocytes and null cells (2,19). The effect of IMP concentrations on the reaction rate indicated that IMP levels > 3 mmol/L
S
80
E
5 60
tMP ( mmol/L )
Fig. i. The eifect of increasing substrate reagent concentrations of IMP on NT activity. The IMP assay concentration is 3 5-fo!d less. The mean and spread of values for assays done in triplicate are indicated.
LYMPHOCYTE ECTONUCLEOTIDASE ANALYSIS 190
S 170
^
-
•
/
150
t30
/ /
110 0 20
0 40
0 60
0 80
100
MnCI, ( mmol/L )
Fig. 2. The effect of increasing substrate reagent concentrations of MnCI: on NT activity. The MnCh assay concentrations is 3-5-fold less. The mean and spread of values for assays done in triplicate are indicated.
in the substrate reaction (assay concentration >O-85 mmol/L) were required for maximal velocity {Fig. I). Similar studies using manganese indicated maximal activation with 024 mmol/L in the substrate reagent (assay concentration 007 mmol/L) confirming similar findings made for serum NT measurement (Fig. 2; ref. 20). Exogenous PNP added to the assay failed to increase NT activity confirming the presence of sufficient endogenous PNP levels in lymphocytes, even in immunodeficient patients 0.05
0.04
•
003
-
002
-
0.01
80
120
160
200
Time ( s)
Fig. 3. Time course for the conversion of hypoxanthine lo uric acid with XO (4 mU/assay) measured by the extinction change at 293 nm (E293 nm) in a Cobas Bio centrifugal analyser. The manual part of the reaction (conversion of IMP to hypoxanthine) was stopped at time 0 (•), 15 (A). 30 (D) and 60 min. (•). The reaction in phosphate bulfer is also shown (o).
77
(17). PNP must however be added to the control which lacks PNP activity. The time course with XO is shown in Fig. 3 and indicate 100 s is sufficient for monitoring the automated phase of the reaction. To test the specificity of the reaction, Con-A, dTTP and AOPCP, all inhibitors of NT (1), when added to the reaction resulted in >90% inhibition and also confirmed that ^glycerophosphate present in the reaction mixture was sufficient to inhibit, by substrate competition, any alkaline phosphatase activity present in the assay. To determine NT distribution in the cytosol and membrane fractions of the cell, we homogenized lymphocytes from three donors and centrifuged them at 120 000.^ for 60 min (Beckman L-8 ultracentrifuge). The clear supernatant (cytosol) fraction was carefully removed and the cell pellet (membrane) re homogenized in tris/saline bulfer with sonication and PNP (0-2 units) added to the sonicate. Assays indicated 15-3% of the total NT activity (101 3 nmol/h per mg protein) was present in the cytosol with a major portion (84-7%) present in the membrane fraction. The within-day precision in the control sample gave a mean activity of 114-4 ±6-6 nmol/h per mL (5 8% s.d.. « = 10). This same sample analysed 20 times over a 4 month period gaveameanof 118-0 ± 5-0(4 2%s.d.)indicating good precision and stability of the QC sample over this period. The lower limit of detect ion for NT was 10 nmo!/h per mL which was about 3fold above baseline values when no enzyme was added. The maximal activity which could be determined before substrates and ancillary enzymes became limiting was 400 nmol/h per mL. The reference range obtained on 20 immunocompetent persons (11 females, 9 males) with ages ranging from 19 to 73 years was 38 7-180-0 nmol/h per mg protein with a mean of 106-6 (±41-4 s.d.). There was no significant difference in NT in the age groups 19-39 years (n= 11), 40-60 years (« = 7) and >60 years (3). This differs from findings made by others who found a gradual loss of NT with increasing age {13) and may be explained by the small numbers in our age groups. The female values were on average higher than males, though there was no significant difference detected {mean ± s.d.: 120-0±45 7 vs90-2±30-l). A comparison of NT reference ranges obtained for different lymphocyte preparations is shown in Table 1 {some of the values sbown in this table have been recalculated from graphed data for purposes of comparison). The ranges
A. H. CHALMERS AND C. HARE
78
Table 1. A comparison of reference ranges obtamed for lymphoid NT activities Cell line
n
Assay
T-enriched Lymphocytes T cells B cells Lymphocytes Lymphocytes Lymphocytes
27 10
[i4C]-AMP/whole cells p-^n-AMP/wholc cells
(Black Africans) Lymphocytes 'CD8 CD4
Lymphocytes ' T cells B cells Lymphocytes (female) Lymphob'lasioid Lymphocvtes Age years 10-19 26-29 30-39 40-49 50-59 60-75 75-85 Lymphoeytes Lymphoeytes
10
10 14 6 5 18 24 12 9 7 4 8 8 24 18 6 7 13 4 6 7 6 20 20
Activity (nmol/li per 10'^ cells ±s.d. or range)
[•'H]-AMP/homogenate t-'Hi-AMP/whole cells [3H)-AMP/whole cells [^H]-AMP/whole cells ['H]-AMP/wholeeeIls [-^-P]-AMF/whole cells [-'-P]-AMP/whole cells ["THMP/whole cells
[i^q-IMP/wholeeelis IM P/h om ogena I c
446 ±212 208(150-280) 187(140-290) 234(160-280) 270-6 (±119)* 180(120-240) 69-6(41-2-1111 108(60-174)* 42 (24-66)• 2I7(±7O) 240 (±100) 80(±41) 272 (230-320) 229(189-286) 379(320-518) 48'8±(18-H) 70 (±65)* 83 (±30) 93 (± 40) 105 (±35) 53 (±27) 50 (± 30) 34 (± 38) 17 (±5) 141 ± 6 3 106(38-7-180-0)*
Reference
8
6 12 16 11 11 11 15 10 9 14 13
2 This study
•Results expressed as nmol/h per mg protein.
differ widely possibly reflecting the different subfractions of^cells used as well as the different methodologies and isotopic substrates. For example, values obtained with [3H]-AMP are in general higher than IMP and may be explained by the greater activity of membrane NT toward AMP than to IMP (21). Also NT activities obtained with [-^-P]-AMP appear lower than those obtained with [-'H]-AMP and may reflect recycling of liberated ^^P into other substrates. The values obtained by our method is about the same order of magnitude obtained by others using [ '4c]-IMP as a substrate (2,13). In conclusion we describe an assay for lymphocyte NT which has been optimized and is simple and cheap (reagent cost/assay
A semi-automated method for 5'-ectonucleotidase m^suremj in lymphocytes A. H. Chalmers and Cavelyn Hare Department of Pathology. Mater Misericordiae Public Hospitals, South Brisbane, Qld4I0/. Australia (Submitted
22 June 1989. Accepted for publication
14 November
1989.)
Summary We describe a simple, non-radioactive, semi-automated method for measuring lymphocyte ccto-5'-nucleotidase activity in isolated cell homogenates. The method which uses inosine 5'-monophosphate(IMP)asa subslrate was optimized and requires a total of 4x 10*' lympbocytes. The reference range obtained (38 7-180 0 nmol/h per mg protein; .v= 106-6) agrees with other isotopic methods which use ''*C-IMP. The simplicity, sensitivity (lower limit 10, upper limit 400 nmol/h per mL) and precision (at 114 nmol/h per mL, interday4-2%s.d.,M = 20; intraday 5-8%, n * 10) makes this method suitable for routine eliniea! laboratory use.
INTRODUCTION Ecto-5'-nucleotidase (E.C.3.1.3,5: NT) located on the external surface of cells catalyses the conversion of purine and pyrimidlne ribo- and deoxyribonucleotide tnonophosphates to their corresponding nucleosides (I). This enzyme which occurs in both T and B lymphocytes is a marker for the maturation of these cells (2-4). Because NT is reduced in a number of congenital and acquired immunodeficiency disorders its measurement is now clinically desirable (5-8). Most methods for the estimation of NT in lympbocytes are time consuming, involve cbromatographic procedures and use expensive radioisotopes (2.8-16). Also most methods use intact cells and are therefore difficult to assay when there are geographical constraints. We describe one assay which measures NT in lymphocyte ceil homogenates by a semi-automated method. The biochemical conversions in this assay are as follows:
Manual: IMP
NT
• Inosme
PNP Phosphate
Hypoxanthine Automated: Hypoxanthine XO
Uric acid
Xanthine 293 nm)
In the manual part of the assay, NT from lymphocytes converts the substrate inosine 5'monophospbate (IMP) to inosine which, in the presence of phosphate buffer and high levels of endogenous purine nucieoside phospborylase in lymphocytes (17), is converted to hypoxanthine. In the automated part of the assay the conversion of hypoxanthine to uric acid with xanthine oxidase (XO) is measured at 293 nm in a centrifugal analyser.
MATERIALS AND METHODS Correspondence: Dr A. H. Chalmers. Depanment of Haematology. Flinders Medieal Centre, Bedford Park. SA 5042, Australia. .Ahhrevialion.'i used in this paper: AMP, Adenosine 5'-monophosphate; AOPCP. Q./3-mcthylcne adenosine 5'-dipbosphate: Con-A, Concanavalin-A; dTTP, deoxylbymidine 5'-triphosphate; E293, extinction or absorbance change at 293 nm; EDTA. cthylencdiaminetetraacetate; IMP, inosine 5'-monophosphate; NT, 5'-ectonueleotidase; PNP, purine nucieoside phosphorylase; XO, xanthine oxidase.
Inosine 5'-monophosphatc (Na +: IMP), deoxythymidine triphosphate (dTTP). ^glycerophosphate, a.^methylene adenosine diphosphate (AOPCP), Concanavaiin-A (Con-A). 5'-ND Control E were all purchased from Sigma Chemical Co. (St Louis, MO, USA). Xanthine oxidase (XO: Cow's milk; 20 units/mL. 10 units/mg protein), purine nucieoside phosphorylase (PNP; bovine calf spleen; 20 units/mg protein) were purchased from Boehringcr Mannheim (Sydney. Australia). All other reagents were of analytical grade quality.
76
A. H. CHALMERS AND C. HARE
Substrate reagent 1MP 5 mniol/L, ^glycerophosphate2 mmol/L, magnesium chloride 4 mmol/L, manganese chloride 0-24 mmol/L were stored as I mL aliquots at —20°C in phosphate buffer (Na ^; 100 mmol/L; pH 8 0). Conirol 5'-ND Control E (75 U/L) is a commercial nucleotidase (reptile) in bovine serum albumin. To each vial was added 200 mLTris(10 mmol/L, pH 7-4)/isotonic saline buffer at 4°C and this solution was stored in 0-5 mL aliquots at -2O''C, Before each assay, the control was thawed and 10 juL PNP (0 2 units) was added. Lymphocyte separation Lymphocytes from 10 mL EDTA-collected blood were isolated in >80% purity using lymphocyte Separation Medium (Flow Laboratories, Sydney) and washed twice with tris/saline before suspending at 1 •5-2-5x10' cells/mL. Cells were stored frozen at — 20°C for batch analysis and. after thawing, sonicated at 4°C with 4x5 s pulses (Branson B30 Sonifier Cell Disrupter) at energy levels 5-7 on the day of analysis (!7). Protein was estimated by the Lowry method (18). Enzyme analysis The manual part of the assay was done in Cobas Bio specimen cups (Roche, Sydney) and the automated analysis in a Cobas Bio centrifugal analyser. Both assays were carried out at 37°C. Manual assay To 20 ^L substrate reagent add 50 pL of lymphocyte extract. The reaction was done at 37''C and stopped with 20 /iL HC! (I -0 mol/L) at 0, 30 and 60 min after addition of lymphocyte extract. The acidified reaction mixture was then spun 1-5 min in a microfuge centrifuge and then transferred to the circular Cobas Bio analysis tray. Automated assay The reagent buffer used in the assay was potassium phosphate (0-25 mol/L; pH 8-0). The start reagent. XO diluted I/lOO with reagent buffer to give 0-2 units/mL, was prepared prior to use and stored at 4''C. The Cobas Bio was programmed as follows: Units, absorbance change; Calculation factor 1790; Standards I, 2, 3 were 0 concentration; Limit 400; Temperature 37°C; Type of analysis 6 (end-point); Wavelength 293 nm;Sampievolume 20^L; Diluent volume 20/JL; Reagent buffer volume 200 ^L; Incubation time 20 s; Start reagent volume 20/jL; Time of first reading 60 s; Time interval 20 s; Number of readings 5; Blanking mode 1; Printout mode I. Calculation The calculation factor (1790) was derived as follows: MT, wv. ,. E293 117 1000 NT (nmol/h per mL) = 1-04 _ '" x 5 '• 12-6
= £293x1790
where I 04 is the pathlength of the curvettes in centimetres; 12-6 the millimolar extinction coefficient of uric acid at 293 nm; 117/5 is the dilutioti factor; and E293 is the change in extinction at 293 nm after XO addition. The time zero result is subtracted from both the 30 and 60 min reactions. The net time 30 min reading is then multiplied by 2 and this figure is averaged with the net time 60 min readmg as follows: Average NT (nmol/h/mL) = NT(t30 - tO)x2 H- NT(t60 - tO) 2 This figure divided by the mg protein/mL of lymphocyte fiomogenate gives NT activity in nmol/h per mg protein. RESULTS AND DISCUSSION Because of the low NT activity in lymphocytes the assay was optimized by using a large protein enzyme mass (005-01 mg/50 /iL) to low substrate volume (20 ^L). The reaction was linear to 60 min and hence was monitored at time 0, 30 and 60 min. The purity of lymphocytes obtained was consistently above 80%. however, the possibility that other contaminating cells may contribute to the activity was considered. Red blood cell represented the largest group of contaminating cells. The activity of NT in these cells was very low (1-5 nmo!/h per mg protein) and represented only I-5% of the lymphocyte activity in normals. Treatment of cell preparations with 0-84% NH4CI to lyse red blood cells did not affect NT activities and so this step was omitted from the method. NT is virtually absent in peripheral blood monocytes and null cells (2,19). The effect of IMP concentrations on the reaction rate indicated that IMP levels > 3 mmol/L
S
80
E
5 60
tMP ( mmol/L )
Fig. i. The eifect of increasing substrate reagent concentrations of IMP on NT activity. The IMP assay concentration is 3 5-fo!d less. The mean and spread of values for assays done in triplicate are indicated.
LYMPHOCYTE ECTONUCLEOTIDASE ANALYSIS 190
S 170
^
-
•
/
150
t30
/ /
110 0 20
0 40
0 60
0 80
100
MnCI, ( mmol/L )
Fig. 2. The effect of increasing substrate reagent concentrations of MnCI: on NT activity. The MnCh assay concentrations is 3-5-fold less. The mean and spread of values for assays done in triplicate are indicated.
in the substrate reaction (assay concentration >O-85 mmol/L) were required for maximal velocity {Fig. I). Similar studies using manganese indicated maximal activation with 024 mmol/L in the substrate reagent (assay concentration 007 mmol/L) confirming similar findings made for serum NT measurement (Fig. 2; ref. 20). Exogenous PNP added to the assay failed to increase NT activity confirming the presence of sufficient endogenous PNP levels in lymphocytes, even in immunodeficient patients 0.05
0.04
•
003
-
002
-
0.01
80
120
160
200
Time ( s)
Fig. 3. Time course for the conversion of hypoxanthine lo uric acid with XO (4 mU/assay) measured by the extinction change at 293 nm (E293 nm) in a Cobas Bio centrifugal analyser. The manual part of the reaction (conversion of IMP to hypoxanthine) was stopped at time 0 (•), 15 (A). 30 (D) and 60 min. (•). The reaction in phosphate bulfer is also shown (o).
77
(17). PNP must however be added to the control which lacks PNP activity. The time course with XO is shown in Fig. 3 and indicate 100 s is sufficient for monitoring the automated phase of the reaction. To test the specificity of the reaction, Con-A, dTTP and AOPCP, all inhibitors of NT (1), when added to the reaction resulted in >90% inhibition and also confirmed that ^glycerophosphate present in the reaction mixture was sufficient to inhibit, by substrate competition, any alkaline phosphatase activity present in the assay. To determine NT distribution in the cytosol and membrane fractions of the cell, we homogenized lymphocytes from three donors and centrifuged them at 120 000.^ for 60 min (Beckman L-8 ultracentrifuge). The clear supernatant (cytosol) fraction was carefully removed and the cell pellet (membrane) re homogenized in tris/saline bulfer with sonication and PNP (0-2 units) added to the sonicate. Assays indicated 15-3% of the total NT activity (101 3 nmol/h per mg protein) was present in the cytosol with a major portion (84-7%) present in the membrane fraction. The within-day precision in the control sample gave a mean activity of 114-4 ±6-6 nmol/h per mL (5 8% s.d.. « = 10). This same sample analysed 20 times over a 4 month period gaveameanof 118-0 ± 5-0(4 2%s.d.)indicating good precision and stability of the QC sample over this period. The lower limit of detect ion for NT was 10 nmo!/h per mL which was about 3fold above baseline values when no enzyme was added. The maximal activity which could be determined before substrates and ancillary enzymes became limiting was 400 nmol/h per mL. The reference range obtained on 20 immunocompetent persons (11 females, 9 males) with ages ranging from 19 to 73 years was 38 7-180-0 nmol/h per mg protein with a mean of 106-6 (±41-4 s.d.). There was no significant difference in NT in the age groups 19-39 years (n= 11), 40-60 years (« = 7) and >60 years (3). This differs from findings made by others who found a gradual loss of NT with increasing age {13) and may be explained by the small numbers in our age groups. The female values were on average higher than males, though there was no significant difference detected {mean ± s.d.: 120-0±45 7 vs90-2±30-l). A comparison of NT reference ranges obtained for different lymphocyte preparations is shown in Table 1 {some of the values sbown in this table have been recalculated from graphed data for purposes of comparison). The ranges
A. H. CHALMERS AND C. HARE
78
Table 1. A comparison of reference ranges obtamed for lymphoid NT activities Cell line
n
Assay
T-enriched Lymphocytes T cells B cells Lymphocytes Lymphocytes Lymphocytes
27 10
[i4C]-AMP/whole cells p-^n-AMP/wholc cells
(Black Africans) Lymphocytes 'CD8 CD4
Lymphocytes ' T cells B cells Lymphocytes (female) Lymphob'lasioid Lymphocvtes Age years 10-19 26-29 30-39 40-49 50-59 60-75 75-85 Lymphoeytes Lymphoeytes
10
10 14 6 5 18 24 12 9 7 4 8 8 24 18 6 7 13 4 6 7 6 20 20
Activity (nmol/li per 10'^ cells ±s.d. or range)
[•'H]-AMP/homogenate t-'Hi-AMP/whole cells [3H)-AMP/whole cells [^H]-AMP/whole cells ['H]-AMP/wholeeeIls [-^-P]-AMF/whole cells [-'-P]-AMP/whole cells ["THMP/whole cells
[i^q-IMP/wholeeelis IM P/h om ogena I c
446 ±212 208(150-280) 187(140-290) 234(160-280) 270-6 (±119)* 180(120-240) 69-6(41-2-1111 108(60-174)* 42 (24-66)• 2I7(±7O) 240 (±100) 80(±41) 272 (230-320) 229(189-286) 379(320-518) 48'8±(18-H) 70 (±65)* 83 (±30) 93 (± 40) 105 (±35) 53 (±27) 50 (± 30) 34 (± 38) 17 (±5) 141 ± 6 3 106(38-7-180-0)*
Reference
8
6 12 16 11 11 11 15 10 9 14 13
2 This study
•Results expressed as nmol/h per mg protein.
differ widely possibly reflecting the different subfractions of^cells used as well as the different methodologies and isotopic substrates. For example, values obtained with [3H]-AMP are in general higher than IMP and may be explained by the greater activity of membrane NT toward AMP than to IMP (21). Also NT activities obtained with [-^-P]-AMP appear lower than those obtained with [-'H]-AMP and may reflect recycling of liberated ^^P into other substrates. The values obtained by our method is about the same order of magnitude obtained by others using [ '4c]-IMP as a substrate (2,13). In conclusion we describe an assay for lymphocyte NT which has been optimized and is simple and cheap (reagent cost/assay
