Mutations in MCT1 cDNA in patients with symptomatic deficiency in lactate transport.

We identified 5 patients with subnormal erythrocyte lactate transport plus symptoms and signs of muscle injury on exercise and heat exposure. All had transport rates below the 95% envelope for normals. Three cases had rates 40-50% of mean normal. One was found to have a missense mutation in monocarboxylate transporter 1 (MCT1), the gene for the red cell lactate transporter (also expressed in skeletal muscle), at a conserved site, which was not mutated in a cohort of 90 normal humans. The other 2 cases had a different missense mutation (at a nonconserved site), which was also not mutated in the normal cohort. All 3 patients were heterozygotes. We presume that these mutations are responsible for their subnormal lactate transport, and hence their muscle injury under environmental stress; homozygous patients should be more seriously compromised. The other 2 cases had lactate transport rates 60-65% of mean normal, and their MCT1 revealed a third mutation, which proved to be a common polymorphism in the normal cohort. These 2 patients may be physiologic outliers in lactate transport, with their muscle damage arising from some other genetic defect.

Detection of very-rare-copy DNA in 0.2-ml dried human blood blots.

A nested PCR assay for chromosome 7 inversions (as identified by the presence of T-cell receptor trans-rearrangements) can detect as rare a frequency as 1 copy in 300,000 leukocytes. To identify such rare occurrences from dried blood blots, the most conveniently obtained and stored samples for field population studies, demands a DNA extraction method that will provide both high quality and high yield. We have satisfied this requirement by extracting proteins and other components directly from the minced filter with phenol, before extracting the DNA with Chelex-water. This provides a near maximal yield of denatured DNA of sufficient quality to detect these translocations with a sensitivity equivalent to that of DNA purified from whole blood samples. Blots stored 6 months worked as well as fresh blots. In addition, we present a method for obtaining native DNA from the dried blots, although at a much lower yield. The successful use of blood blots to detect such rare events signals the feasibility of large-scale field studies involving diagnostic molecular epidemiology.

Comparative enzymology of AMP deaminase, adenylate kinase, and creatine kinase in vertebrate heart and skeletal muscle: the characteristic AMP deaminase levels of skeletal versus cardiac muscle are reversed in the North American toad.

The specific activity of three characteristic enzymes, adenylate deaminase, adenylate kinase, and creatine kinase, in the skeletal muscles and heart of a variety of vertebrate land animals, including the human, are surveyed. Data from this study and available studies in the literature suggest that adenosine monophosphate deaminase in land vertebrates is quite high in white skeletal muscle, usually somewhat lower in red muscle, and 15- to 500-fold lower in cardiac muscle. Adenosine monophosphate deaminase is active primarily under ischemic or hypoxic conditions which occur frequently in white muscle, only occasionally in red muscle, and ought never occur in heart muscle, and this may therefore account for observed enzyme levels. The common North American toad, Bufo americanus, provides a striking exception to the rule with cardiac adenosine monophosphate deaminase as high as in mammalian skeletal muscle, whereas its skeletal muscle level of adenosine monophosphate deaminase is several times lower. The exceptional levels in the toad are not due to a change in substrate binding and are not accompanied by comparable change in the level of adenylate or creatine kinase. Nor do they signal any major change in isozyme composition, since a human muscle adenosine monophosphate deaminase-specific antiserum reacts with toad muscle adenosine monophosphate deaminase, but not with toad heart adenosine monophosphate deaminase. They do not represent any general anuran evolutionary strategy, since the bullfrog (Rana catesbeiana) and the giant tropic toad (Bufo marinus) have the usual vertebrate pattern of adenosine monophosphate deaminase distribution.(ABSTRACT TRUNCATED AT 250 WORDS)

Medical implications of the lactate and ammonia relationship in anaerobic exercise.

The genesis of the modern ischemic forearm exercise test (IFET) employing the measurement of lactate and ammonia as countervailing metabolites is briefly reviewed, along with the application of the lactate ammonia exercise ratio in the diagnosis of myoadenylate deaminase deficiency and disorders of glycolysis and glycogenolysis. Two cases are presented to illustrate the response patterns elicited, their reproducibility, the types of parameters that can be quantified, and the role the IFET may play in the differential diagnosis of fitness failures. The role of the ammonia measurement is emphasized here because the lactate response is more familiar. The roles of hypoxanthine responses and of muscle ammonia measurements in the evaluation of cases are examined, and some pertinent and recently introduced analytic methods are cited. The applications of newer approaches, such as aspiration biopsy and N-14 NMR spectroscopy, are discussed, along with an example of the new clinical defects in enzymes and membrane carriers that should be anticipated during the utilization of the IFET.

Clinical assay of the human erythrocyte lactate transporter. II. Analysis and display of normal human data.

We have applied a lactate efflux assay to human red cells at two temperatures and with initial lactic acid loads up to 8 mM, metabolically generated. Efflux was about 1.5 times faster at external pH of 8.5 than at 7.5; the latter was the standard pH used thereafter. Multiple lactate loads in a single blood specimen demonstrated clear evidence of saturation kinetics at both pH levels, since the efflux rate did not increase proportionally with the lactate load. Best-fitting rectangular hyperboles were determined for 129-131 assays from 43 volunteers at 20 degrees and 30 degrees. In most cases high and low lactate loads permitted a two-point evaluation of saturation kinetics, and a positive indication was obtained in 88 of 89 tests. The apparent efflux Km and Vm values may be influenced by pH as well as by lactate levels and cannot be taken as rigorous, although they agree reasonably well with literature data on influx and exchange velocities. The data displayed a Hill constant of 1, a 30 degrees/20 degrees velocity ratio of 2.7, and no significant clustering by sex or age. A single assay with initial lactate level above 5 mM at 30 degrees should be sufficient to identify cases with a defective transporter, using the 95% tolerance limits developed in this report.

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.

Kinetic and immunologic evidence for a complete gene block in myoadenylate deaminase deficiency.

Muscle biopsies from patients with myoadenylate deaminase deficiency (mADD) have been evaluated kinetically and immunologically to ascertain the origin of residual enzyme activity. Kinetic evaluation employed 5 mM AMPS/1 mM AMP ratios, which were 0.7-0.8 for the human muscle isozyme, but 0-0.25 for the isozyme(s) of all other human blood cells and tissues examined. Of 14 control biopsies, 13 showed a ratio greater than 0.60 (one gave 0.47) regardless of the enzyme specific activity, while all 14 mADD biopsies showed a ratio less than 0.24, suggesting that a fetal muscle isozyme and/or blood cell isozyme were responsible for the residual activity. Confirmation was provided by rabbit antisera to purified human muscle AMP deaminase. These antisera fully precipitate the isozyme from crude human muscle biopsy homogenates, regardless of fiber-type composition, and cross-react effectively with the muscle isozyme of Rhesus monkeys and thoroughbred horses, but are inactive toward the isozymes of all other human blood cells and tissues examined. Of 18 mADD homogenates tested, 14 showed less than 20% reactivity with the antisera, at levels that precipitated 10 x more enzyme in control specimens. The residual activity in most cases of mADD must therefore arise from some source other than normal AMP deaminase. To evaluate the possibility of a single common determinant, 9 mADD homogenates were tested for soluble immune complexes. Seven of the 9 then showed 20-42% reactivity, suggesting that part of their residual activity may be due to an isozyme sharing one antigenic determinant with the normal muscle isozyme. Competitive antigen binding was used to assess whether catalytically inactive AMP deaminase was present in mADD. The method was demonstrated effective in identifying spontaneously inactivated purified enzyme and alkaline-inactivated crude enzyme. Nevertheless, homogenates from 17 mADD cases failed to produce more than 14% activation, under conditions in which 63-99% activation was expected. Triton X-100 extracts of homogenate residues of 11 mADD cases were also tested, in a search for insoluble antigen; none produced significant competition. The evidence thus indicates that most cases of mADD are due to a complete gene block, with total absence of all normal muscle AMP deaminase protein.

Cell-mediated immune responses in sarcoidosis.

In an attempt to understand divergent observations regarding the in vitro lymphocyte responsiveness to antigens and mitogens, the frequency of cutaneous reactivity, and the relationship of these variables to clinical features of sarcoidosis, we examined cell-mediated immune responses in 75 untreated patients. In vitro lymphocyte responses to phytohemagglutinin and concanavalin A were significantly decreased among the group of patients with chronic active disease. In vitro lymphocyte responses to streptococcal antigen paralleled the patient's cutaneous reactivity; however, when compared to healthy persons, patients with sarcoidosis responded significantly less often to streptococcal skin tests. Twelve of 13 persons expected to be tuberculin positive by clinical history were found to be dermally reactive, and 11 these 12 exhibited positive in vitro lymphocyte responses to purified protein derivative. Among the remaining 62 patients, failure to respond to all stimuli in vivo and in vitro occurred in only 10. No evidence was found to support the general view that sarcoidosis is characterized by anergy to specific antigens. The patterns of responses to mitogens were neither characteristic nor unique for sarcoidosis. In patients in whom diminished specific cellular immune reactivity was observed, no correlation was evident with specific clinical features of the disease.

Rifampin and cell-mediated immune responses in tuberculosis.

To estimate the potential adverse consequences of rifampin therapy on cell-mediated immunity in tuberculosis, we measured in vitro lymphocyte responses to phytohemagglutinin, concanavalin A, pokeweed mitogen, and in vitro and in vivo responses to purified protein derivative tuberculin. Thirty-seven patients treated with therapeutic combinations containing rifampin were compared with 13 persons who had never received the drug. After initial improvement, responses to phytohemagglutinin became depressed in patients receiving rifampin for periods of 4 to 24 months. No significant changes were noted in lymphocyte responses to concanavalin A or pokeweed mitogen. In vitro and in vivo responses to purified protein derivative tuberculin were not altered. Because a favorable therapeutic outcome was achieved in all subjects, we concluded that rifampin does not have clinically significant immunosuppressive activity.