The multidrug resistance (mdr1) gene product, P-glycoprotein, is responsible for the ATP-dependent extrusion of a variety of compounds, including chemotherapeutic drugs, from cells. The data presented here show that cells with increased levels of the P-glycoprotein release ATP to the medium in proportion to the concentration of the protein in their plasma membrane. Furthermore, measurements of whole-cell and single-channel currents with patch-clamp electrodes indicate that the P-glycoprotein serves as an ATP-conducting channel in the plasma membrane. These findings suggest an unusual role for the P-glycoprotein.

https://www.pnas.org/content/pnas/90/1/312.full.pdf

The multidrug resistance (mdr1) gene product functions as an ATP channel.

This study aims to improve the health of patients suffering from chronic fatigue syndrome (CFS) by interventions based on the biochemistry of the illness, specifically the function of mitochondria in producing ATP (adenosine triphosphate), the energy currency for all body functions, and recycling ADP (adenosine diphosphate) to replenish the ATP supply as needed. Patients attending a private medical practice specializing in CFS were diagnosed using the Centers for Disease Control criteria. In consultation with each patient, an integer on the Bell Ability Scale was assigned, and a blood sample was taken for the "ATP profile" test, designed for CFS and other fatigue conditions. Each test produced 5 numerical factors which describe the availability of ATP in neutrophils, the fraction complexed with magnesium, the efficiency of oxidative phosphorylation, and the transfer efficiencies of ADP into the mitochondria and ATP into the cytosol where the energy is used. With the consent of each of 71 patients and 53 normal, healthy controls the 5 factors have been collated and compared with the Bell Ability Scale. The individual numerical factors show that patients have different combinations of biochemical lesions. When the factors are combined, a remarkable correlation is observed between the degree of mitochondrial dysfunction and the severity of illness (P<0.001). Only 1 of the 71 patients overlaps the normal region. The "ATP profile" test is a powerful diagnostic tool and can differentiate patients who have fatigue and other symptoms as a result of energy wastage by stress and psychological factors from those who have insufficient energy due to cellular respiration dysfunction. The individual factors indicate which remedial actions, in the form of dietary supplements, drugs and detoxification, are most likely to be of benefit, and what further tests should be carried out.

https://www.ijcem.com/files/IJCEM812001.pdf

Chronic fatigue syndrome and mitochondrial dysfunction

Background: Human plasma ATP concentration is reported in many studies as roughly 1000 nmol/L. The present study tested the hypothesis that the measured plasma ATP concentration is lower if ATP release from formed blood elements is inhibited during blood sample processing. A second hypothesis was that pretreatment with aspirin to inhibit platelets would reduce the measured plasma concentration of ATP.

Methods: Blood was sampled from the antecubital vein in 20 healthy individuals 30 and 60 min after ingestion of aspirin (325 mg) or placebo. Aliquots of each blood sample were added to the usual EDTA/saline solution to inhibit ATP catabolism, or to a new stabilizing solution designed to both stop ATP catabolism and inhibit ATP release from blood elements. The stabilizing solution contained NaCl, EDTA, tricine buffer, KCl, nitrobenzylthioinosine, forskolin, and isobutylmethylxanthine. Plasma ATP was measured with the luciferin–luciferase assay with standard additions in each sample to determine ATP content. Hemoglobin concentration was used as an index of sample hemolysis, and the plasma ATP concentration was corrected for the hemolysis component.

Results: Aspirin pretreatment had no effect on plasma ATP concentrations. However, use of the stabilizing solution resulted in mean (SD) ATP concentrations 8-fold lower than the use of EDTA alone [28 (16) vs 236 (201) nmol/L; P <0.001].

Conclusion: When precautions are taken to inhibit ATP release from blood elements during sample preparation, human venous plasma ATP concentration is much lower than previously reported.

Human plasma ATP concentration.

Enzyme immunoassays are attaining increased usage for the direct detection of microbial antigens in body fluids. Advantages of enzyme immunoassays include a high degree of sensitivity resulting from the inherent magnification of the enzyme-substrate reaction and the use of objective end points without the need for radioactivity. Enzyme immunoassays have been developed for the reliable detection of several important microbial antigens in body fluids, including antigens of rotavirus, hepatitis B virus, and Haemophilus influenzae type b. However, standard enzyme immunoassay techniques are not sufficiently sensitive for the measurement of some antigens from other viruses, bacteria, and parasites in concentrations that commonly occur in body fluids during the course of infectious diseases. This review examines some of the limitations of currently available enzyme immunoassay technology and discusses approaches to increasing the sensitivity and specificity of enzyme immunoassay systems. Methods for improving these assay systems include the use of monoclonal antibodies, improved methods of enzyme-immunoreactant conjugation, more sensitive substrate systems, improved methods of antigen-antibody access, and the direct measurement of microbial enzymes. The use of such techniques should lead to the development of efficient enzyme immunoassay systems for the direct detection of a wide range of bacterial, viral, and parasitic infections.

Enzyme Immunoassays for the Detection of Infectious Antigens in Body Fluids: Current Limitations and Future Prospects

The various types of chemiluminescent and bioluminescent reactions are described. Applications of luminescence in the analysis of substancs of clinical interest are surveyed. The advantages, disadvantages, and prospects for luminescent assays are discussed.

Analytical luminescence: its potential in the clinical laboratory.

We optimized conditions for determination of adenosine-5'-triphosphate (ATP) and creatine phosphate from plasma extracted with ethanol/water (96/4 by vol). The procedures utilize the firefly luciferin/luciferase reaction, the bioluminescence being measured with a Du Pont Biometer. ATP is quantitated directly and creatine phosphate is quantitated by reaction with creatine kinase and ADP, after plasma ATP is removed by incubation with the enzyme apyrase. The method is applied to plasma from humans, rabbits, and rats, and possible clinical applications are discussed.

Microdetermination of plasma ATP and creatine phosphate concentrations with a luminescence biometer.

Decisive patterns have been demonstrated in plasma adenosine 5' triphosphate (ATP) levels in both endotoxin and anaphylactic shock which correlate with periods of low platelet counts, low arterial pressures and abnormal electrocardiograms. When these irregularities were occurring, the plasma ATP level was low; when improvement occurred, the plasma ATP level rose. Plasma ATP levels appear to be an index to the metabolic state of the animal. The plasma creatine phosphate (CP) level showed a tendency to decrease when the ATP level dropped in anaphylactic shock, although the CP level did not recover to the same extent as the ATP level. In endotoxin shock, the plasma CP level increased on an average of six-fold. It is proposed that this rise resulted from either CP mobilization from tissues, for the purpose of replenishing the energy deficient myocardial muscle, or possible leakage from damaged cells. Adenosine diphosphate (ADP) plasma values were measured in both anaphylactic and endotoxin shock. High initial ADP values were prone towards a more severe anaphylactic reaction and a shorter survival time in the endotoxin shock experiments.

Plasma changes in endotoxin and anaphylactic shock (ATP, ADP and Creatine phosphorus).

Cinephotomicrography shows emboli are formed in the lung during stages of anaphylactic and endotoxin shock and platelet counts are decreased. Rabbits in anaphylactic shock had a 50 percent mortality while 100 percent mortality was associated with endotoxin shock. During the terminal phase, the platelet count recovers in anaphylactic shock but not in endotoxin shock. This suggests a difference in the emboli formed during these two forms of shock. This study was directed at determining what might be the difference. Periodic measurement of electrocardiographic tracings, fibrinogen, factor VIII and fibrin degradation products (FDP) were made using standard equipment and test kits. The results of the study showed no changes in any of the measured clotting factors during anaphylactic shock. However, in endotoxin shock FDP appeared after seven to 10 hours, fibrinogen levels decreased from 221 mg per dl to 85 mg per dl and factor VIII time increased significantly. From these results, it is concluded that platelet aggregation in anaphylactic shock does not involve fibrin deposition, whereas in endotoxin shock platelet aggregation and fibrin deposition are both a factor in the formation of the emboli, possibly explaining the irreversible aggregation observed in terminal endotoxin shock.

Comparative study of blood clotting factors in anaphylactic and primary and secondary endotoxin shock.

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Microdetermination of plasma ATP and creatine phosphate concentrations with a luminescence biometer.

Plasma changes in endotoxin and anaphylactic shock (ATP, ADP and Creatine phosphorus).

Comparative study of blood clotting factors in anaphylactic and primary and secondary endotoxin shock.