Characterization of cardiac troponin subunit release into serum after acute myocardial infarction and comparison of assays for troponin T and I. American Association for Clinical Chemistry Subcommittee on cTnI Standardization.

We examined the release of cardiac troponin T (cTnT) and I (cTnI) into the blood of patients after acute myocardial infarction (AMI). Three postAMI serum samples were applied in separate analytical runs onto a calibrated gel filtration column (Sephacryl S-200), and the proteins were separated by molecular weight. Using commercial cTnT and cTnI assays measured on collected fractions, we found that troponin was released into blood as a ternary complex of cTnT-I-C, a binary complex of cTnI-C, and free cTnT, with no free cTnI within the limits of the analytical methodologies. The serum samples were also examined after incubation with EDTA and heparin. EDTA broke up troponin complexes into individual subunits, whereas heparin had no effect on the assays tested. We added free cTnC subunits to 24 AMI serum samples and found no marked increase in the total cTnI concentrations, using an immunoassay that gave higher values for the cTnI-C complex than free cTnI. To characterize the cross-reactivity of cTnT and cTnI assays, purified troponin standards in nine different forms were prepared, added to serum and plasma pools, and tested in nine quantitative commercial and pre-market assays for cTnI and one approved assay for cTnT. All nine cTnI assays recognized each of the troponin I forms (complexed and free). In five of these assays, the relative responses for cTnI were nearly equimolar. For the remainder, the response was substantially greater for complexed cTnI than for free cTnI. Moreover, there was a substantial difference in the absolute concentration of results between cTnI assays. The commercial cTnT assay recognized binary and ternary complexes of troponin on a near equimolar basis. We conclude that all assays are useful for detection of cardiac injury. However, there are differences in absolute cTnI results due to a lack of mass standardization and heterogeneity in the cross-reactivities of antibodies to various troponin I forms.

Simultaneous detection of seven drugs of abuse by the Triage panel for drugs of abuse.

This novel, competitive immunoassay simultaneously detects seven drugs of abuse in urine. A urine sample is placed in contact with lyophilized reagents, the reaction mixture is allowed to come to equilibrium (10 min), and then the whole mixture is applied to a solid phase that contains various immobilized antibodies in discrete drug-class-specific zones. After a washing step, the operator visually examines each zone for the presence of a red bar. The method incorporates present threshold concentrations that are independent for each drug. In the absence of drug or in the presence of drug in quantities less than the threshold concentration, no colored bar is visible. Samples containing drug(s) at or above the threshold concentration cause a red bar to appear for the appropriate drug(s). Positive and negative procedural control zones are incorporated into each determination. The performance of the assay methodology matches that of instrumented immunoassay systems.

The involvement of carnitine intermediates in peroxisomal fatty acid oxidation: a study with 2-bromofatty acids.

Metabolism-dependent inactivators of 3-ketothiolase I and carnitine acyltransferase I (CAT I) have been used to study the oxidation of fatty acids in intact hepatocytes. 2-Bromooctanoate inactivates mitochondrial and peroxisomal 3-ketothiolases I in a time-dependent manner. During the first 5 min of incubation, inactivation of 3-ketothiolase in mitochondria is five times faster than its inactivation in peroxisomes. Almost complete inactivation of 3-ketothiolase I in both types of organelle is achieved after incubation with 1 mM 2-bromooctanoate for 40 min. The inactivation is not affected by preincubating hepatocytes with 20 microM tetradecylglycidate (TDGA), an inactivator of CAT I, under conditions which cause greater than 95% inactivation of CAT I. 2-Bromododecanoate (1 mM) causes 60% inactivation of mitochondrial and peroxisomal 3-ketothiolases I in 40 min. These inactivations are greatly reduced by preincubating hepatocytes with 20 microM TDGA, demonstrating that 2-bromododecanoate enters both mitochondria and peroxisomes via its carnitine ester. 2-Bromopalmitate (1 mM) causes less than 5% inactivation of mitochondrial and peroxisomal 3-ketothiolases I in 40 min, but causes 95% inactivation of CAT I during this time. Incubation of hepatocytes with 10-200 microM 2-bromopalmitoyl-L-carnitine causes inactivation of mitochondrial and peroxisomal 3-ketothiolases I at similar rates. This inactivation is decreased by palmitoyl-D-carnitine during the first 5 min of incubation. Pretreating hepatocytes with 20 microM TDGA does not affect the inactivation of mitochondrial or peroxisomal 3-ketothiolase I by 2-bromopalmitoyl-L-carnitine. These results demonstrate that in intact hepatocytes, peroxisomes oxidize fatty acids of medium-chain length by a carnitine-independent mechanism, whereas they oxidize long-chain fatty acids by a carnitine-dependent mechanism.

Guanosine triphosphate and colchicine affect the activity and the polymeric state of acetyl-CoA carboxylase.

Acetyl-CoA carboxylase was purified 300-fold from rat liver, in the absence of added citrate, by precipitation from an 18,000g supernatant in the presence of Triton X-100 at 105,000g and 20 degrees C, followed by chromatography on phosphocellulose. Acetyl-CoA carboxylase activity in this preparation was activated by preincubation with GTP (0.1-2.0 mM) and with citrate (20 mM). Colchicine (10(-6)-10(-3) M) inhibited enzyme activity and counteracted the effects of GTP and citrate. Sucrose density gradient centrifugation demonstrated that GTP and citrate preincubation promoted the formation of the polymeric, active enzyme, while colchicine engendered disassembly. Preincubation of the purified acetyl-CoA carboxylase at 4 degrees C caused inactivation and disassembly, which was countered by preincubation at 37 degrees C in the presence of GTP or citrate. These results suggest that GTP, like citrate, activates acetyl-CoA carboxylase by enhancing the conversion of the protomeric form of the enzyme to its more active, polymeric state.

Studies on the assay, activity and sedimentation behaviour of acetyl-CoA carboxylase from isolated hepatocytes incubated with insulin or glucagon.

The activity of acetyl-CoA carboxylase, measured in various ways, was studied in 15000g extracts of rat liver hepatocytes and compared with the rate of fatty acid synthesis in intact hepatocytes incubated with insulin or glucagon. Hepatocyte extracts were prepared by disruption of cells with a Dounce homogenizer or by solubilization with 1.5% (v/v) Triton X-100. Sucrose-density-gradient centrifugation demonstrated that the sedimentation coefficient of acetyl-CoA carboxylase from cell extracts was 30-35S, regardless of the conditions of incubation or disruption of hepatocytes. Solubilization of cells with 1.5% Triton X-100 yielded twice as much enzyme activity (measured by [14C]bicarbonate fixation) in the sucrose-gradient fractions as did cell disruption by the Dounce homogenizer. Analysis by high-performance liquid chromatography of acetyl-CoA carboxylase reaction mixtures showed that [14C]malonyl-CoA accounted for 10-60% of the total acid-stable radioactivity, depending on the method for disrupting hepatocytes and on the preincubation of the 15000g extract, with or without citrate, before assay. Under conditions in which incubation of cells with insulin or glucagon caused an activation or inhibition, respectively, of acetyl-CoA carboxylase, only 25% of the acid-stable radioactivity was [14C]malonyl-CoA and enzyme activity was only 13% (control), 16% (insulin), and 57% (glucagon) of the rate of fatty acid synthesis. Under conditions when up to 60% of the acid-stable radioactivity was [14C]malonyl-CoA and acetyl-CoA carboxylase activity was comparable with the rate of fatty acid synthesis, there was no effect of insulin or glucagon on enzyme activity.

Inhibition of hepatic lipogenesis by salicylate.

Salicylate has been found to be an inhibitor of fatty acid synthesis in isolated rat hepatocytes. Half-maximal inhibition of fatty acid synthesis occurs at approximately 2 mM. The inhibitory effect of salicylate on fatty acid synthesis is not relieve by the addition of acetate, suggesting that salicylate inhibits the conversion of acetate into fatty acids. Acetyl-CoA carboxylase activity in homogenates of hepatocytes is not influenced by previous exposure of the intact cells to salicylate. Partially purified acetyl-CoA carboxylase, isolated and assayed in the absence of citrate, is markedly inhibited by salicylate. However, in the presence of 0.5 mM citrate, which is the concentration of this metabolite in the cytosol of the liver cell, salicylate activates the enzyme. Upon treatment of acetyl-CoA carboxylase with salicylate (in the absence or presence of citrate), followed by separation of enzyme and effector on a Sephadex G-25 column, the enzyme activity is enhanced as compared to the salicylate-free control, demonstrating that the inhibitory effect of salicylate (in the absence of citrate) is reversible, but not the stimulatory effect (in the presence of citrate). Salicylate inhibition of fatty acid synthesis by hepatocytes is not rapidly reversible; hepatocytes preincubated with salicylate followed by a wash procedure (centrifugation and resuspension) still show depressed rates of fatty acid synthesis from acetate upon further incubation. Salicylate was found to prevent pyruvate accumulation in hepatocyte suspensions observed in the absence of this compound; salicylate even induces the disappearance of pyruvate and lactate initially present in the cell suspension. This suggests that salicylate activates pyruvate and lactate consumption, which is most likely related to the well-known fact that salicylate uncouples oxidative phosphorylation. The latter action of the drug will stimulate citric acid-cycle activity. This causes an inhibition of fatty acid and cholesterol synthesis since acetyl units will be specifically channelled into the citric acid cycle and not into the lipogenic pathway. It is concluded that part of the inhibitory effect of salicylate on fatty acid biosynthesis is exerted at (a) step(s) in the conversion of acetate into fatty acids, acetyl-CoA carboxylase not being a target of this compound. In addition, salicylate prevents that pyruvate, generated by glycolysis, enters the lipogenic pathway. The latter effect of salicylate would also explain the observed inhibition of cholesterol synthesis by this compound.

The effects of lactate and acetate on fatty acid and cholesterol biosynthesis by isolated rat hepatocytes.

1. The present study demonstrates that lactate and acetate stimulate fatty acid synthesis and inhibit cholesterogenesis by isolated rat hepatocytes. 2. Exposure of the intact cells to lactate increases the activity of acetyl-CoA carboxylase, as can be measured in homogenates of these cells. A similar effect by acetate was not observed. 3. Both acetate and lactate drastically increase the cellular level of citrate. 4. Possible mechanisms underlying the difference in response of fatty acid and cholesterol synthesis to an increase in substrate availability are discussed. Futhermore, a mechanism is proposed for the lactate effect on acetyl-CoA carboxylase.

De novo fatty acid synthesis in the perfused rat lung. Incorporation of palmitate into phospholipids.

1. The incorporation of exogenously derived [14C]palmitate and endogenously synthesized [3H]palmitate (from 3H2O) was measured in the isolated perfused lung. 2. Over 40% of the fatty acid esterified into lung disaturated phosphatidylcholine was derived from de novo synthesis. 3. A major portion of the palmitate synthesized de novo was incorporated in the 2 position of disaturated phosphatidylcholine. 4. Streptozotocin-induced diabetes and the compound 5-(tetradecyloxy)-2-furoic acid markedly inhibited de novo fatty acid synthesis while the incorporation of exogenously supplied palmitate increased into disaturated phosphatidylcholine, primarily in the 2 position. 5. Treatment with insulin resulted in an increase in [14C]glucose incorporation into lung phospholipid, with the largest increase appearing in the glyceride-glycerol fraction of the phosphatidylcholine species. 6. Insulin neither stimulated de novo fatty acid synthesis nor increased exogenous palmitate incorporation. 7. These data show: (1) that de novo fatty acid synthesis in the perfused rat lung is involved in the remodeling reactions in the synthesis of phosphatidylcholine, and (2) that diabetes affects the relative contribution of de novo synthesized and exogenously supplied palmitate available for the esterification of lung phospholipid.

Inhibition of lipogenesis in isolated hepatocytes by 3-amino-1,2,4-triazole.

3-Amino-1,2,4-triazole has been found to be an inhibitor of fatty acid synthesis by isolated rat hepatocytes. Half-maximal inhibition of fatty acid synthesis occurs at approximately 20mM. Acetyl-CoA carboxylase activity in homogenates of hepatocytes is not affected by previous exposure of the intact cells to 3-amino-1,2,4-triazole. The drug opposes the activation of partially purified acetyl-CoA carboxylase by citrate, but does not influence enzyme activity in the absence of citrate. As compared to fatty acid synthesis, cholesterol synthesis by the hepatocytes is more drastically depressed by incubation of the cells with 3-amino-1,2,4-triazole. Half-maximal inhibition of cholesterogenesis occurs at approximately 5 mM 8-amino-1,2,4-triazole.