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.

Immunolocalization of AMP-deaminase isozymes in human skeletal muscle and cultured muscle cells: concentration of isoform M at the neuromuscular junction.

The three major isoforms of AMP-deaminase (AMPda) were localized in human skeletal muscle and cultured muscle cells by immunocytochemistry. The M isoform was mainly located in Type II muscle fibers and showed a clear cross-striation. Particularly strong staining was present at the neuromuscular junction. Capillaries were also immunoreactive. The L isoform was predominantly observed in nerve bundles and to a minor extent in smooth muscle cells and endothelial cells. The E isoform was predominantly present in smooth muscle cells, and to a lesser extent in Type I muscle fibers and nerve bundles. In quadriceps muscle of patients with myoadenylate deaminase deficiency, no immunostaining for the M isozyme was observed, whereas reactivity for the L and E isoforms was unaltered. In human muscle cell cultures, mononuclear cells, including myoblasts, were immunoreactive for the L isoform and to a lesser extent the E isoform, whereas the M isoform was absent. In myotubes, diffuse or fibrillar staining was present for all three isoforms, but only the M isoform showed a clear cross-striation pattern in highly differentiated myotubes.

Natural gastric infection with Helicobacter pylori in monkeys: a model for spiral bacteria infection in humans.

BACKGROUND/AIMS: There is no generally accepted model for Helicobacter pylori infection in humans. The aim of this study was to examine the natural history and effect of treatment in rhesus monkeys and sequentially define the immune response to H. pylori in relation to treatment. METHODS: Infection and gastritis were graded blindly by histological analysis and culture of biopsy specimens harvested during gastroduodenoscopies in 26 anesthetized colony-bred monkeys. Plasma H. pylori-specific immunoglobulin (Ig) G levels were determined by enzyme-linked immunosorbent assay. RESULTS: H. pylori and Gastrospirilum hominis-like organisms were present in 13 and 9 monkeys, respectively; 3 animals harbored both organisms, whereas 4 monkeys were not infected. Gastritis score was < or = 1.5 in animals uninfected or infected only with G. hominis-like organisms and > or = 2.0 in all H. pylori-infected animals. IgG ratios were > or = 0.5 in 12 of 13 H. pylori-infected animals and in 2 of 13 H. pylori-negative animals (P < 0.001). One monkey became infected with H. pylori during the observation period, with concurrent increase of gastritis and plasma IgG levels. In untreated animals, infection, gastritis, and plasma IgG levels remained unchanged over 7-15 months. Triple therapy eradicated H. pylori at 6 months in 4 of 6 animals while suppressing gastritis and plasma IgG levels. CONCLUSIONS: Rhesus monkeys harboring H. pylori are persistently infected and have gastritis and elevated specific IgG levels, all of which may respond to appropriate therapy, whereas G. hominis infection is associated with little inflammation.

Immunologic evidence for three isoforms of AMP deaminase (AMPD) in mature skeletal muscle.

Four rabbit polyclonal antisera to purified AMP deaminase (AMPD) isozymes were used to precipitate homogenate AMPD activity from dissected gracilis, soleus and gastrocnemius muscles of the cat, rabbit, rat, mouse, Rhesus monkey, human and toad. The antisera were also tested against other unusual muscles: autonomically innervated striated muscle of the upper esophagus (UEM), skeletal muscle of patients with myo-AMPD deficiency and extraocular muscles (EOM) of humans and Rhesus monkeys. The reference antiserum, M, prepared against human psoas muscle AMPD, precipitated > 90% AMPD from all primate skeletal muscles tested, and from type-2 muscles of all mammals tested, but < 75% from cat and rodent soleus, toad gastrocnemius and primate UEM, EOM and myo-AMPD deficient muscles. Thus, a second isozyme was clearly indicated. Antibody B, against rat liver and kidney AMPD, had no effect with any muscle specimen. Antibody C, against rat heart AMPD, produced additive precipitation of AMPD from soleus of rat and mouse, while antibody E1, against human red cell (and heart) AMPD, produced additive AMPD precipitation from toad gastrocnemius, cat soleus and muscles of several AMPD-deficient humans. A second AMPD isozyme thus accounted for as much as 25% of total activity in some animal red muscles, but no more than 5% in human mixed muscles. At least one more isozyme is needed to account for muscle AMPD unreactive with all antibodies tested in rabbit soleus, toad gastrocnemius and primate UEM and EOM. A list is appended of the approximate AMPD activity in various human cells and tissues.

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)

Molecular analysis of the myoadenylate deaminase deficiencies.

Myoadenylate deaminase (mAMPD) deficiency in a clinically heterogeous metabolic myopathy consisting of primary (inherited) and secondary (acquired) forms based on a variety of clinical and laboratory findings. To provide a basis for delineating the underlying molecular defects in mAMPD deficiency, and as a means to test the proposal for multiple forms of the resulting disease, Northern blot analyses were performed with RNA isolated from individual patients with classified primary and secondary deficiency utilizing human mAMPD cDNA probes isolated from adult skeletal muscle libraries. Analysis of nine patients with primary mAMPD deficiency indicates normal abundance of mAMPD transcript. No immunoreactive mAMPD polypeptide is detected in Western blot analyses of skeletal muscle extracts prepared from these patients. Specificity to mAMPD is demonstrated by normal creatine kinase (CK) activities and M-creatine kinase (M-CK) transcript abundance. Similar analyses of four individuals with secondary mAMPD deficiency reveal heterogeneity in this subgroup of patients. Whereas two of these patients exhibit normal mAMPD transcript abundance, two others associated with inflammatory myopathy display reductions in mAMPD and M-CK transcript abundance. Examination of tissue sections derived from the same biopsies utilized in the isolation of RNA demonstrates the integrity of the skeletal muscle in those patients with associated inflammatory myopathy. Combined, these data support the proposal for multiple forms of mAMPD deficiency, and indicate that the primary condition is most commonly characterized by specific point mutations or small deletions/rearrangements in the ampd1 gene, whereas some patients with secondary mAMPD deficiency display more generalized aberrations in gene expression.

A glucose-6-phosphate dehydrogenase stain for frozen human skeletal muscle biopsy specimens. A sensitive indicator of fiber degeneration.

The glucose-6-phosphate dehydrogenase (G6PD) stain was adapted to skeletal muscle by using homogenate assays and quantitative cytochemical stains to determine the "correct" localization. For both feline and human skeletal muscles, the appropriate level of phenazine methosulfate eliminated fiber typing, which was falsely localizing the rate-limiting, bound reduced form of NADPH rather than the soluble G6PD. Use of a viscous solution of polyvinyl alcohol was necessary to prevent enzyme diffusion. Under these conditions, G6PD produced a mild myoplasmic stain with even sarcoplasmic reticulum granularity in human biopsy specimens. Fibers that were degenerating or regenerating by hematoxylin-eosin or alkaline phosphatase stains yielded an intense myoplasmic G6PD stain. Additional degenerating fibers were also often detectable with G6PD staining. No increased staining was found in denervated or atrophic fibers. Absence of staining (after 2 hours) was not a reliable indicator of G6PD deficiency, although it could be used for preliminary screening of muscle biopsy specimens.

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.

Calcium uptake in frozen muscle biopsy sections compared with other predictors of malignant hyperthermia susceptibility.

The frozen-section Ca45 uptake procedure of Mabuchi and Sreter has been applied to 51 frozen human muscle biopsies previously tested for halothane and for caffeine contracture responses. In preliminary tests with cat muscle, uptake varied with skeletal muscle fiber type, was absent from smooth muscle, and was blocked by agents which damage or extract sarcoplasmic reticulum. In the human biopsies, however, the calcium uptake values showed no significant correlation with any of the following parameters: time of frozen-biopsy storage up to 5 yr; fiber type distribution; positive versus negative response in the halothane contracture test; positive versus negative response in the caffeine contracture test; positive versus negative response in the combined halothane/caffeine contracture test; and presence of a documented clinical episode of malignant hyperthermia. In contrast, a positive halothane or caffeine contracture test each correlated highly with the presence of a prior clinical episode. We conclude that the evidence fails to support the use of the Ca45 uptake procedure in frozen sections of skeletal muscle biopsies as a valid indicator of malignant hyperthermia susceptibility.

Lactate transporter defect: a new disease of muscle.

New methods were used to identify the abnormality in a patient who showed evidence of neuromuscular dysfunction on extensive clinical examination. The methods revealed that the lactate content of the patient's skeletal muscle does not decline normally after exercise and that his red cells are defective in lactate transport. These results suggest that skeletal muscle and erythrocyte membranes share the same genetic lactate transporter (or a common subunit), which is deficient in this patient. This defect may be a common cause of elevated serum creatine kinase levels, as seen in the patient described here and of unexplained episodes of rhabdomyolysis and myoglobinuria.

Myoadenylate deaminase deficiency: primary and secondary types.

Myoadenylate deaminase deficiency, the most common of the known enzyme deficits of muscle, appears to occur in two forms. The primary type seems to be inherited as a complete gene block in an autosomal recessive pattern. Although occasionally diagnosed in infancy, when muscle biopsy is performed on a hypotonic but normoreflexic child, the deficiency is usually not symptomatic until adult or middle age, when muscle cramping and exercise intolerance develop. The skeletal muscle isozyme is immunologically, and presumably genetically, unique, and these patients have normal levels of adenylate deaminase in their other cells and tissues. A presumptive diagnosis can usually be made by an ischemic forearm exercise test, which shows a negligible increase in blood ammonia, despite a normal rise in lactate. Despite the absence of more than 99% of normal adenylate deaminase activity, the muscle biopsy shows no anatomic pathology, and other enzymes are at normal levels. These patients do not suffer progressive disease, and should be reassured and encouraged to maintain physical activity. The heterozygous state is probably asymptomatic, except, perhaps, on extreme exercise, but may be associated with an increased incidence of malignant hyperthermia susceptibility. Since the gene defect is not rare, it is not surprising that some cases of the deficiency will be coincidentally associated with other neuromuscular disease. However, there is also a secondary form of myoadenylate deaminase deficiency, consequent to muscle damage from other disease. In this form, the residual activity is higher (1-10% of normal), may present rare foci of positive stain in the section, and reacts normally with antibody to the muscle isozyme. Other muscle enzymes are also depleted, although not as severely, and the prognosis in such cases is dictated by the primary disease. Since the heterozygous state is common, these patients might have been carriers whose adenylate deaminase levels have been lowered to the deficient category by the advent of other neuromuscular disease.

Myoadenylate deaminase deficiency and malignant hyperthermia susceptibility: is there a relationship?

Muscle biopsies from 35 patients referred for possible malignant hyperthermia were subjected to contracture testing with halothane, caffeine, and the combined agents, histopathological and fiber-type-distribution analysis, and quantitative assay of three major muscle enzymes: adenylate deaminase, adenylate kinase, and creatine kinase. Adenylate kinase and creatine kinase were in the normal range in all biopsies and each averaged 92% of expected normal value when corrected for their fiber-type distribution. Of the 14 cases with a positive halothane test, 2 had primary myoadenylate deaminase deficiency, and 5 others had low levels of this enzyme (less than one-third normal). In contrast, only 3 of 21 cases negative to halothane testing had low adenylate deaminase levels, and none were deficient. This association was significant by several statistical tests, although it would not be highly predictive for an individual case. A positive halothane test also correlated with a high type 2 fiber contribution, but this was probably secondary, since cases with low enzyme levels had significantly higher type 2 fiber areas. Caffeine contractures did not correlate with either low enzyme levels or with fiber-type distribution. Sixty percent of the biopsies were entirely normal histologically, and showed a significant correlation with a negative combined contracture test. Data on the one family included in this study suggest separate inheritance of the trait for myoadenylate deaminase deficiency and the trait for positive contracture tests. The present findings suggest that patients with myoadenylate deaminase deficiency (and the carrier state as well) may be at increased risk of malignant hyperthermia when subjected to anesthesia.

Some practical considerations in quantitative absorbance microspectrophotometry. Preparation techniques in DNA cytophotometry.

An experimental review of the Feulgen and gallocyanine-chrome-alum stains for quantitative cytophotometry of DNA in tissue sections yielded information on the preparation and staining of tissue for quantitative absorbance microspectrophotometry. (1) Tissues routinely fixed in formalin are suitable for either stain. Specimens fixed with glutaraldehyde-containing fixatives are not satisfactory for Feulgen staining, nor are ethanol-fixed specimens, unless they are post-fixed in formalin. (2) The pararosaniline dyes, used in the Feulgen stain, are sufficiently pure to use if a solution of the dye in ethanol shows an absorbance peak at 543 to 546 nm. (3) The Feulgen stain provides good reproducibility when the staining solution is adjusted to pH 1.5. (4) Gallocyanine is the best stain to use on Bouin-fixed or glutaraldehyde-fixed tissues. (5) Where fixation is an option, Carnoy and methanol-formalin-glacial acetic acid are excellent fixatives that can be followed by either stain. (6) Selection of the thickness of a tissue section involves a compromise. Requirements of minimum nuclear overlap and sharp focusing favor a section thickness of 4 micron to 6 micron. On the other hand, the requirement for full nuclear thickness, as judged by absorbance equivalent to that of a touch preparation, demands sections as thick as 8 micron in the case of mouse liver. Within this range, the optimum thickness, therefore, is determined by the particular tissue, the range of its nuclear sizes and its packing density. (7) The refractive index of the mounting medium should be closely matched to that of the background structure of the tissue sections. For animal tissues, we found that media with refractive indices of 1.54 to 1.56 are suitable.

Myoadenylate deaminase deficiency: inherited and acquired forms.

Myoadenylate deaminase deficiency, the most common of the known enzyme deficits of muscle, appears to occur in two forms. The primary type seems to be inherited as a complete gene block in an autosomal recessive pattern. Although occasionally diagnosed in infancy, when muscle biopsy is performed on a hypotonic but normoreflexic child, the deficiency is usually not symptomatic until adult or middle age, when muscle cramping and exercise intolerance develop. The skeletal muscle isozyme is immunologically, and presumably genetically, unique, and these patients have normal levels of adenylate deaminase in their other cells and tissues. A presumptive diagnosis can usually be made by an ischemic forearm exercise test, which shows a negligible increase in blood ammonia, despite a normal rise in lactate. Despite the absence of more than 99% of normal adenylate deaminase activity, the muscle biopsy shows no anatomic pathology, and other enzymes are at normal levels. These patients do not suffer progressive disease, and should be reassured, and encouraged to maintain physical activity. The heterozygous state is probably asymptomatic, except, perhaps, on extreme exercise, but may be associated with an increased incidence of malignant hyperthermia susceptibility. Since the gene defect is not rare, it is not surprising that some cases of the deficiency will be coincidentally associated with other neuromuscular disease. However, there is also a secondary form of myoadenylate deaminase deficiency, consequent to muscle damage from other disease. In this form, the residual activity is higher (1-10% of normal), may present rare foci of positive stain in the section, and reacts normally with antibody to the muscle isozyme. Other muscle enzymes are also depleted, although not as severely, and the prognosis in such cases is dictated by the primary disease. Since the heterozygous state is common, these patients might have been carriers, whose adenylate deaminase levels have been lowered for the deficient category by the advent of other neuromuscular disease.