Clinical assay of the human erythrocyte lactate transporter. I. Principles, procedure, and validation.
A clinically applicable method for the assay of lactate efflux from human red cells has been developed and described in detail. It requires only small volumes of blood and routine chemicals, and evaluates the process under physiological conditions and direction of lactate loading and transport. The decline of red cell lactate level fit a first order decay curve reasonably well, and better than the fit to zero order or second order plots. Bias is controlled by the use of least-squares curve fitting for all assays, and constraints on the elimination of outlier points. The assay shows a variety of inhibitor effects that may be considered typical for this transporter: potent inhibition by p-hydroxymercuribenzoate, but not by other types of sulfhydryl reagents; marked inhibition by phloretin, quercitin, and 1-fluoro-2,4-dinitrobenzene; lack of inhibition by the amine-reactive agents that block the chloride/carbonate exchanger, DIDS and SITS; and reversible competitive inhibition by alpha-cyano-4-OH-cinnamic acid. Harmaline and N-I-succinimide also produced effective inhibition. The assay also demonstrated transacceleration of L-lactate efflux in the presence of external additions of D-lactate, glycollate, iodoacetate, fluoropyruvate, and bromopyruvate, which are substituted monocarboxylates like lactate, but not by iodoacetamide or L-alanine. Such activation is a manifestation of a macromolecular carrier in operation, and cannot be explained by a pore or channel. These findings satisfy all reasonable criteria for a satisfactory and sensitive lactate transporter assay, which should be adequate to evaluate volunteers and patients for the normal range of this carrier, and to seek possible deficient states.
Fishbein
W N
WN
Division of Biochemistry, Armed Forces Institute of Pathology, Washington, D.C. 20306.
Foellmer
J W
JW
Davis
J I
JI
Fishbein
T M
TM
Armbrustmacher
P
P
D002352:Carrier Proteins / Q000097:blood*; D004910:Erythrocyte Membrane / Q000378:metabolism; D004912:Erythrocytes / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007202:Indicators and Reagents; D007700:Kinetics; D007770:L-Lactate Dehydrogenase; D008565:Membrane Proteins / Q000097:blood*; D027501:Monocarboxylic Acid Transporters; D013056:Spectrophotometry, Ultraviolet / Q000379:methods
D002352:Carrier Proteins; D007202:Indicators and Reagents; D008565:Membrane Proteins; D027501:Monocarboxylic Acid Transporters; D007770:L-Lactate Dehydrogenase