Nitrite and nitrate analyses: a clinical biochemistry perspective.

OBJECTIVE: To review the assays available for measurement of nitrite and nitrate ions in body fluids and their clinical applications. DESIGN AND METHODS: Literature searches were done of Medline and Current Contents to November 1997. RESULTS: The influence of dietary nitrite and nitrate on the concentrations of these ions in various body fluids is reviewed. An overview is presented of the metabolism of nitric oxide (which is converted to nitrite and nitrate). Methods for measurement of the ions are reviewed. Reference values are summarized and the changes reported in various clinical conditions. These include: infection, gastroenterological conditions, hypertension, renal and cardiac disease, inflammatory diseases, transplant rejection, diseases of the central nervous system, and others. Possible effects of environmental nitrite and nitrate on disease incidence are reviewed. CONCLUSIONS: Most studies of changes in human disease have been descriptive. Diagnostic utility is limited because the concentrations in a significant proportion of affected individuals overlap with those in controls. Changes in concentration may also be caused by diet, outside the clinical investigational setting. The role of nitrite and nitrate assays (alongside direct measurements of nitric oxide in breath) may be restricted to the monitoring of disease progression, or response to therapy in individual patients or subgroups. Associations between disease incidence and drinking water nitrate content are controversial (except for methemoglobinemia in infants).

Interference in triiodothyronine (T3) analysis on the Immuno 1 Analyzer.

OBJECTIVE: To evaluate the interference in Triiodothyronine (T3) analysis on the Immuno 1 Analyzer. METHODS: We analyzed 686 samples for T3 using the Miles Technicon Immuno 1 Analyzer. We compared the results of 318 samples with those given by radioimmunoassay (RIA) and the remaining 368 results with those given by the Ciba-Corning ACS 180 analyzer. RESULTS: On the Immuno 1 correlated with those by RIA or chemiluminescence immunoassay. However, results on eight patients by the Immuno 1 method were anomalously elevated. We attempted to find and eliminate the cause of the interference on the Immuno 1. Although the method uses an alkaline phosphatase labelled T3 analog and fluoresceinated monoclonal antibody, serum binding of fluorescein or alkaline phosphatase did not appear to be the major causes of the interference. Ethanol extraction of samples followed by reconstitution in zero calibrator was the only reliable way to eliminate the interference. CONCLUSION: The Immuno 1 assay was more prone to interference than other methods. Until it is reformulated, we recommend that users assay ethanol extracts of samples with unexpectedly high T3.

Sensitive time-resolved fluorescence immunoassay of somatotropin in serum.

We describe a new "sandwich"-type non-isotopic immunoassay for human somatotropin (GH, growth hormone) in serum. In the assay, GH is captured by a monoclonal antibody immobilized in a white microtiter well and simultaneously reacted with a second biotinylated monoclonal antibody. The degree of binding of biotinylated antibody, which increases with increasing amount of GH in the sample, is quantified by adding streptavidin labeled with the europium chelate of 4.7 - bis(chlorosulfophenyl) - 1.10 - phenanthroline - 2.9 - dicarboxylic acid. The fluorescent complex on the solid phase is then measured by excitation at 337.1 nm (nitrogen laser) and monitoring the emission at 615 nm in a gated fluorometer/analyzer. The proposed procedure has short incubation times (less than 4 h protocol), uses only 25 microL of serum per microtiter well, and gives precise and accurate results. The method was clinically evaluated with samples obtained from pediatric patients undergoing investigation for growth abnormalities and from a patient with acromegaly.

Nonspecificity of a direct 17 alpha-hydroxyprogesterone radioimmunoassay kit when used with samples from neonates.

We analyzed 240 samples for 17 alpha-hydroxyprogesterone (17-OHP) with the direct-assay kit ("Coat-A-Count" method for serum samples) from Diagnostic Products Corp. (DPC). The specimens were from 50 patients with known or suspected congenital adrenal hyperplasia (CAH); 74 mostly hospitalized neonates and infants, ages three days to three months; and 116 other patients, ages six months to 23 years. Samples from the CAH group were also analyzed with our in-house assay. For 39 of the neonatal samples, the analysis with the DPC assay was repeated with re-solubilized material that had been extracted from the serum with organic solvents. Values for "17-OHP" measured with the DPC direct assay were high, not only in CAH patients, but also in many of the unaffected neonates and infants. The extraction properties of the cross-reacting immunoreactive material into various organic solvent systems were different from those of 17-OHP, and were more like those of steroid sulfates. Because of this significant cross-reactivity, we recommend that the DPC kit not be used for sera from children younger than six months of age, unless the method is modified to include an extraction step.

Myocardial ischaemia in asphyxia neonatorum. Electrocardiographic, enzymatic and histological correlations.

Serial electrocardiograms and creatine kinase (CK) isoenzyme activities were studied prospectively in 20 asphyxiated term newborn infants and 43 normal neonates. By adapting a previously described grading system for ischaemic changes, a degree of electrocardiographic ischaemia was defined which occurred almost solely in asphyxiated infants. Infants with this degree of abnormality had significantly higher mean CK-MB and MM activities than other asphyxiated infants at 0, 8 and 28 hours. Histological changes of peripartum myocardial necrosis were seen in 4 of the 5 infants on whom an autopsy was performed, and either electrocardiogram or CK-MB was abnormal in all four. It is concluded that myocardial injury in the newborn period is often associated with CK-MB release, but in view of the lack of cardiac-specificity of CK-MB in newborn infants, caution is urged in the interpretation of elevated isoenzyme activity in the neonate.

Assessment of neurologic outcome in asphyxiated term infants by use of serial CK-BB isoenzyme measurement.

Brain-type isoenzyme of creatine kinase was measured serially in 45 healthy and 22 severely asphyxiated term infants. The enzyme was measured in cord blood and in venous, capillary, or arterial blood at six to eight hours, 24 to 30 hours, and 72 to 80 hours after birth. In the healthy infants a brief rise of CK-BB occurred at six to eight hours; CK-BB activities were greater than 2.5 log-transformed standard deviations above the mean of the control values in ten of the asphyxiated infants and in none of the control infants. When normal CK-BB activity was used as a predictor of good neurologic outcome and elevated CK-BB as a predictor of subsequent neurologic abnormality, the outcome was predictable from the CK-BB activity in 17 of 22 cases (77%) and in 11 of the 12 survivors (92%). Eight of the 12 surviving infants had neonatal seizures and outcome was predictable from CK-BB activity in all cases. We conclude that serum CK-BB activity, especially when measured in cord blood and at six to 12 hours of life, correlates with neurologic outcome after severe asphyxia, and that measurement of CK-BB compares favorably with radionuclide and computerized tomographic scanning as a method of predicting neurologic outcome after asphyxia.

Creatine kinase isoenzymes in serum from cord blood and the blood of healthy full-term infants during the first three postnatal days.

Isoenzymes of creatine kinase (ATP:creatine phosphotransferase; EC 2.7.3.2; CK) were measured by electrophoresis in serum from cord blood and skin-puncture blood taken from 45 healthy full-term infants during the first three postnatal days. Mean total CK activities (in U/L at 30 degrees C) were 185 in cord samples, 536 in samples taken between 5--8 h postnatally, 494 between 24--33 h, and 288 in the 72-100 h samples. Values for all three isoenzymes increased to a peak over this period, with the highest values generally being found in the samples taken 5--33 h after birth; the subsequent decline was most rapid for CK-BB. Serum CK isoenzymes in cord samples and those taken at 72--100 h in the 11 babies delivered by cesarian section did not differ significantly from those of babies delivered vaginally. However the postnatal increases in total CK, CK-MM, and CK-MB (but not in CK-BB) were significantly greater in those patients born by vaginal delivery. The reasons for the increases in CK isoenzymes after birth are not clear, but our results and reported studies on the ontogeny of CK suggest that CK-MB cannot be regarded as a "cardiac-specific" isoenzyme in the neonatal period.