Radiometric
Assays of Lymphocyte Carbohydrate Metabolism in Response to Mitogens
By Ngo Manh Tran, M. Chen,
P. McIntyre, S. Larson, and Henry N. Wagner.
The Johns Hopkins Medical Institutions, Baltimore, Maryland
After the initial report of stimulation of lymphocyte
(lymphs) carbohydrate metabolism by phytohemagglutinin (PHA) (J. Nucl.
Med. 15: 510, 1974), it was discovered that oxidation of glucose-1-14C
(G-1-14C) to 14CO2 by contaminating for up to 50% of the 14CO2.
The present study was done to evaluate radiometric
detection of changes in carbohydrate metabolism in purified lymphs in
response to mitogens, i.e. PHA and concavanalin A (Con. A).
A Ficoll-Hypaque technique was used for preparation
of fractions containing 90-95% lymphs and less than 1-2% PMN.
14CO2 output was quantified by an ionization chamber.
(Another radiometric system was used at the Johns
Hopkins Medical Institutions including the following components: cap,
needles, culture mixture in inner closed vial, scintillation vial with
fluor liquid plus hyamine hydroxide).
2x10millions lymphs or 0.1x10millions PMN were incubated
with or without mitogens with 1.0µCi G-1-14C, G-6-14C or G-U-14C in modified
Hanks plus 5.0mM Mg at 37ºC for 360 minutes.
Results showed:
1) Lymphs 14CO2 production from G-1-14C plus PHA (Gibco
3.12-6.25mg) was increased at 0-2 hours (n=9, p < 0.001);
2) Lymphs 14CO2 production from glucose was increased
by incubation with PHA mediate from G-U-14C and least from G-1-14C, intermediate
from G-U-14C and least from G-6-14C;
3) Similar changes in lymphs carbohydrate metabolism
were noted after incubation with Con. A;
4) No significant change in glucose oxidation was detected
in PMN with PHA; a slight increase in 14CO2 (p<0.025) occurred only
from G-1-14C when PMN were counted for less than 2% of the 14CO2 produced
by lymphs under identical conditions.
These results show that both PHA and Con. A stimulate
lymphs carbohydrate metabolism and that glucose oxidation by contaminating
PMN did not contribute significantly to 14CO2 detected.
Thus study may be of potential importance for development
of automated in vitro tests of lymph immune responsiveness.
(We note furthermore that this enclosed vial system
is of importance for radiometric detection of bacterial growth in both
aerobic and anaerobic conditions, as well as in other gaseous atmospheres).
References:
1) Ngo Manh Tran, M Chen, P. McIntyre, S. Larson,
and Henry Wagner: Radiometric Assays (ionization chamber) of Lymphocyte
Carbohydrate Metabolism in Response to Mitogens, Journal of Nuclear Medicine,
16: 576, 1975
2) Ngo Manh Tran and Henry N. Wagner, Jr.:
Liquid Scintillation Vial For Radiometric Assay
of Lymphocyte Carbohydrate Metabolism in Response to Mitogens, Journal
of Nuclear Medicine, 19: 61-63, 1977
Tran Manh Ngo, M.D., Ph.D.
