Analysis of caffeine: comparison of the manual enzyme multiplied immunoassay (EMIT), automated EMIT, and high-performance liquid chromatography procedures.

Caffeine has been recommended for the treatment of apnea in newborn children. Most of the current methods for measurement of caffeine in newborns have used high-performance liquid chromatography (HPLC) because analysis can be performed on as little as 50 microliter of serum, an important factor when obtaining multiple blood samples for routine drug monitoring in infants and children. We evaluated the manual Syva enzyme multiplied immunoassay technique (EMIT) caffeine procedure and compared the assay results with our HPLC caffeine method. The EMIT procedure was also automated, and these results were compared with those of the manual and HPLC procedures. The 7-micrograms/ml calibrator was analyzed 20 times to establish within-run precision for the manual procedure. The mean +/- SD was 6.97 +/- 0.33, and the coefficient of variation (CV) was 4.80%. Reproducibility was tested by multiple analysis of the 11-micrograms/ml control. The mean +/- SD (CV) for the manual system was 11.3 +/- 0.45 (4%), n = 17; for the HPLC system, 10.58 +/- 0.89 (8.4%), n = 10; and for the automated system, 10.4 +/- 0.78 (7.5%), n = 12. Samples from 32 patients were analyzed by each of the above procedures. There was a good correlation among all three procedures. The correlation coefficient (r) for HPLC vs. manual EMIT was 0.98; for HPLC vs. automated EMIT, 0.97; and for automated EMIT vs. manual EMIT, 0.96. We conclude that the new Syva EMIT caffeine method is reliable and reproducible and can be applied to automated analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

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

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.

Metabolism of palmitaldehyde in human cardiac muscle-II. Fatty chain elongation and shortening.

1. The metabolism of [1-14C[ palmitaldehyde has been studied in human heart homogenates. 2. Incorporation of the radioactivity into the individual acyl chains of nonpolar (NPL) and polar (PL) lipid fractions was assessed as well as changes in the weight percentage distribution of these chains. 3. Incorporation of the radioactivity into specific lipid fractions of free fatty acids (FFAcd), free fatty aldehydes (FFAld) and cholesterol esters were also studied. 4. A significant amount of incorporation into fatty chains of both fewer and more than 16 carbons was observed. 5. Chain shortening was found to be favored by NAD while the chain elongation required NADPH.

Benign symmetrical lipomatosis (Madelung's disease).

Two patients with benign symmetrical lipomatosis (madelung's disease) have been followed for 7 and 9 years. Despite extensive surgical procedures, both patients have developed multiple recurrences. Lipomas and normal adipose tissue from the same patients were compared using chromatographic techniques. The lipomas contained significantly increased triglyceride fractions. Conservative surgical debulking is the recommended treatment.

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

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 ADP levels: direct determination with luciferase luminescence using a biometer.

A method is described for the determination of low plasma levels of adenosine-5'-diphosphate (ADP) using a Dupont Biometer to measure luminescence produced by the luciferin-luciferase reaction. Endogenous ATP is removed by incubation with luciferase. The remaining ADP is then quantitated, following its conversion to ATP, after incubation with creatine phosphate and creatine kinase. The mean coefficient of variation for 0.02 and 2.2 micromol/liter ADP standards were 2.1 and 1.8% respectively. The method has been applied to human and rabbit plasma. Human plasma ADP concentrations were found to be 0.13 +/- 0.025 (10) micromol/liter and rabbit plasma concentration were 0.07 +/- 0.05 (5) micromol/liter. Several other possible applications of the method are discussed.

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

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.

Variables in the measurement of serum cholesterol by gas-liquid chromatography.

Gas-liquid chromatography (GLC) is an effective method for the measurement of total serum cholesterol. Its use in the clinical laboratory, however, requires careful attention to many variables (flow rate, sample size, sensitivity of solvent and choice of the quantitation method, i.e. peak-height ratio vs. peak height). This report evaluates the effect of these variables on the quantitation of serum cholesterol by GLC.

Synthesis and properties of 35S, 14C and 3H labeled S-alkyl glycerol ethers and derivatives.

Radioactive S-alkyl glycerol ethers have been synthesized with 35S, 14C and 3H labels as well as 3H/35S double labels. The synthesized compounds were converted to various derivatives which can serve to characterize the S-alkyl glycerol ethers. These included the isopropylidene derivative, oxidation with periodate to the aldehyde followed by reduction with LiAlH4 to the alcohol, and reaction of the alcohol with acetic anhydride to form the acetate derivative. Chemical analysis, IR, NMR, zonal TLC profile scans and GLC showed all the products to be greater than 99% pure. The GLC behavior of the aldehyde and acetate derivatives of both S-alkyl glycerol ethers and O-alkyl glycerol ethers on EGSS-X was compared.

Reductive and oxidative synthesis of saturated and unsaturated fatty aldehydes.

Saturated and unsaturated fatty aldehydes were synthesized 99+% pure with yields of up to 80% by the reduction of 1-acylaziridines with lithium aluminium hydride, and in yields of up to 87% by oxidation of the corresponding alcohol with 1-chlorobenzotriazole. It was found for the reduction that optimum aldehyde yield was obtained with a mole ratio of reactants, consisting of acid chloride-ethylenimine-triethylamine-LiAlH(4), equal to 1:2:2:2. Optimum conditions for alcohol oxidation were found to be a mole ratio of oxidant to alcohol of 1:1.3 with refluxing for 45 min in methylene chloride containing 25% pyridine. Methods for the purification of the final product are also described. Purity criteria were thin-layer and gas-liquid chromatography and infrared and nuclear magnetic resonance spectroscopy.