Zinc stoichiometry in Escherichia coli alkaline phosphatase. Studies by 31P NMR and ion-exchange chromatography.

31P nuclear magnetic resonance spectra and enzymatic activities are compared for alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) species with different zinc contents. The enzyme containing two Zn2+ per protein dimer exists in two forms; one, prepared by dialysis of native enzyme, has full enzymatic activity and a 31P magnetic resonance spectrum similar to but distinguishable from that of the native enzyme containing four or more Zn2+. The other form, prepared by restoring two Zn2+ to apoenzyme, has low enzymatic activity and a 31P magnetic resonance spectrum that indicates stoichiometric binding of phosphate, but otherwise altered properties. Reconstituted enzyme with four Zn2+ is similar to but distinguishable from native enzyme with four Zn2+. Chromatography on DEAE-cellulose can separate apoenzyme and enzyme containing two Zn2+ and suggests that the binding of a pair of Zn2+ is cooperative.

Further studies on alterations in magnesium binding during cold storage of erythrocytes.

Free intracellular Mg2+ concentration ([Mg2+]i) was measured in cold-stored human erythrocytes by the method of null-point titration with ionophore A23187. [Mg2+]i was 311 +/- 41 microM (mean +/- S.D.) for cells stored 0-10 days, increasing to 458 +/- 64 microM for cells stored 22-48 days. The values for stored cells were higher than those previously determined by a 31P-NMR method (Bock et al. (1985) Blood 65, 1526-1530); however, the null-point method requires extensive washing of the cells, which we have found to increase NMR-measured [Mg2+]i. The null-point values still represent a small fraction of total cell Mg2+, and confirm that binding of Mg2+ to ligands other than ATP and 2,3-bisphosphoglycerate must increase during storage. As an initial test of whether this may imply suboptimal availability of Mg2+ for cell preservation, we used A23187 to prepare erythrocytes with altered Mg2+ content, then removed ionophore and stored the cells in plasma-free medium for up to 2 weeks. Higher Mg2+ content had a very significant positive correlation (P less than 0.0001) with higher cell ATP concentrations. Storage did not significantly affect basal or Na+-stimulated efflux of Mg2+ from Mg2+-loaded red cells.

Studies on the mechanism of decreased NMR-measured free magnesium in stored erythrocytes.

31P-NMR spectra have been recorded on erythrocytes stored at 4 degrees C in various preservation media. Storage was always associated with an upfield shift of the inorganic phosphate (Pi) resonance and a pronounced upfield shift of the ATP beta resonance, indicating decreased intracellular pH (pHi) and decreased intracellular free magnesium ([Mg2+]i). The decreased [Mg2+]i occurred in preservation media not containing citrate and even in media supplemented with Mg2+. It could not be attributed to the changes in pHi, Na+, K+, lactate, Pi or 2,3-diphosphoglycerate, that occur with storage. The decrease in [Mg2+]i was largely reversed when stored cells were incubated for 1 h at 37 degrees C in fresh plasma. Stored cells were found to contain significant amounts of inorganic pyrophosphate, up to about 200 mumol per liter cell water. Being a tight binder of Mg2+, pyrophosphate could account for some of the observed decrease in [Mg2+]i. Additional mechanisms may involve precipitation of some other Mg2+ complex during cold storage or enhancement of Mg2+ binding to membrane components.

Clinical implications of 2-hydroxydesipramine plasma concentrations.

The clinical utility of 2-hydroxydesipramine (2-OH-DMI) measurements was evaluated by examining the relationship of 2-OH-DMI concentrations in plasma to clinical outcome and side effects in depressed inpatients treated with desipramine (DMI). Studies were performed in responders and nonresponders to treatment and in patients experiencing subjective side effects or major adverse reactions necessitating interruption of treatment. Unlike DMI concentrations, 2-OH-DMI concentrations did not correlate with response. Summing the concentrations of parent drug and metabolite (DMI + 2-OH-DMI) did not improve the correlation over that achieved with DMI alone. Neither DMI, 2-OH-DMI, nor their sum correlated with subjective side effect totals or major adverse reactions. While our data do not permit any conclusions regarding the clinical activity of 2-OH-DMI, they suggest that its routine measurement in plasma is not likely to be useful in the management of depression.

Steady-state plasma concentrations of cis- and trans-10-OH amitriptyline metabolites.

Plasma concentrations of the geometric isomers of 10-OH amitriptyline (10-OH AT) and 10-OH nortriptyline (10-OH-NT) were determined by reversed-phase high-pressure liquid chromatography. Steady-state concentrations of At, NT, and the four 10-OH metabolites were measured in 27 patients taking AT for depression. All of the unconjugated hydroxylated metabolites were usually detectable and trans-10-OH NT always predominated. Mean concentrations, expressed as percentage of the sum of all six compounds, were: AT 30%, NT 27%, cis-10-OH AT 1.1%, trans-10-OH AT 4.0%, cis-10-OH NT 4.0%, and trans-10-OH NT 33%. Repeated measurements on 10 patients over several weeks indicated that interindividual variations in absolute and relative 10-OH metabolite concentrations are much greater than day-to-day variations. Five patients who also received a phenothiazine had a lower proportion of 10-OH metabolites.

Desipramine hydroxylation: variability and effect of antipsychotic drugs.

Steady-state plasma concentrations of desipramine (DMI), unconjugated 2-OH DMI, and total 2-OH DMI were measured in 82 depressed inpatients, 35 of whom were concurrently receiving a phenothiazine or butyrophenone antipsychotic drug. In the patients not on an antipsychotic, the ratio of unconjugated metabolite to parent varied from 0.01 to 1.5, with a median of 0.48, and was inversely related to the parent drug level. Antipsychotic drug was associated with higher DMI levels and a lower proportion of OH-metabolite (median, 0.23). In both groups the unconjugated form accounted for only about 10% of the total metabolite. No relationship of age, sex, drinking history, or smoking to DMI or 2-OH DMI levels was found.

Effects of commonly prescribed nonsteroidal anti-inflammatory drugs on thyroid hormone measurements.

PURPOSE: To assess the effects of nonsteroidal anti-inflammatory drugs (NSAIDs) on thyroid function tests. PATIENTS AND METHODS: Eighty-nine patients receiving NSAIDs and 22 control subjects not taking NSAIDs were studied in a cross-sectional survey at Veterans Affairs and University hospitals. Measurements of serum thyroxine (T4), free T4 index, triiodothyronine (T3), and thyrotropin (thyroid-stimulating hormone [TSH]) were obtained for all subjects. RESULTS: Serum T4 measurements were lowered only in salsalate-treated patients, while serum T3 was depressed in patients receiving salsalate, diclofenac sodium, and naproxen. Serum T4 and T3 were unchanged in patients treated with diflunisal, ibuprofen, indomethacin, piroxicam, or sulindac. Serum TSH was normal in all subjects. CONCLUSIONS: Several NSAIDs can lower serum thyroid hormone concentrations, principally by interfering with the binding of T4 and T3 to serum carrier proteins; patients taking these drugs remain euthyroid. Awareness of these interactions may prevent unnecessary diagnostic or therapeutic interventions.

The new era of automated immunoassay.

Use of immunoassays and other ligand-binding assays in clinical diagnosis has increased dramatically during the last several years. Despite impressive technical advances, "mass production" of these assays in a routine laboratory still presents many difficulties. This review of ligand-binding assay technology highlights some recent developments, emphasizing challenges and possible solutions for cost-effective patient care.

Current issues in maternal serum alpha-fetoprotein screening.

Measurement of maternal serum alpha-fetoprotein originated in the early 1970s as a means to screen for fetal neural tube defects, a relatively common and devastating class of malformations. Since that time, assay methods have improved, interpretation has been refined, follow-up testing for neural tube defects has advanced, and many other disease associations have been uncovered. It is a unique test, both in its clinical application and its laboratory implementation. The present review outlines current procedures for maternal serum alpha-fetoprotein screening and summarizes recent developments.

Evaluation of a sensitive immunoenzymetric assay for maternal-serum alpha-fetoprotein screening. Comparison with radioimmunoassay.

An automated immunoenzymetric assay (IEMA) using monoclonal antibodies (Tandem-E, Hybritech) was evaluated for maternal serum alpha-fetoprotein (MSAFP) testing, in comparison with a classical radioimmunoassay (RIA) (Clinical Assays). The IEMA was superior in terms of sensitivity, precision, and linearity. Systematic bias between the two methods resulted in a narrower spread of clinical results, expressed as multiple-of-median, with the IEMA. Consequently, the IEMA produced fewer results that by usual criteria would be considered high (suggesting increased risk of neural tube defect) or low (suggesting increased risk of Down's syndrome). Although both methods may be comparably suitable for neural tube defect screening, the IEMA is advantageous for Down's syndrome risk assessment.

Nuclear magnetic resonance in the clinical laboratory.

Nuclear magnetic resonance (NMR) which now has widespread clinical use as an imaging technique, may also have significant application to diagnostic pathology. High-field proton NMR spectroscopy can perform rapid multicomponent analysis of serum, urine, cerebrospinal fluid, and even intact cells. This technique allows unusually detailed studies of certain metabolic derangements and toxic ingestions. Phosphorus NMR spectroscopy in vivo can measure ATP and other phosphometabolites, intracellular pH, and free Mg2+. It has been applied to diagnosing inborn errors of metabolism such as glycogen storage disease, monitoring growth and regression of tumors, and assessing tissue viability. The basics of NMR spectroscopy are reviewed in this article, along with a survey of applications in human pathology.

Evaluation of the Technicon Immuno 1 free thyroxine assay.

The Technicon Immuno 1 free thyroxine (fT4) assay, a modified two-step procedure that is fully automated on a random access analyzer, was evaluated-at two clinical sites. The method had excellent precision and correlated well overall with three other estimates of free thyroxine: free thyroxine index (FTI) measured on the Immuno 1; the Abbott IMx fT4 assay (Abbott Park, IL); and the Clinical Assays two-step manual fT4 assay. Using a combination of thyrotropin and FTI assay results as a "gold standard" for defining thyroid status, the Immuno 1 fT4 method had a sensitivity of 100% and specificity of 98.3% for hyperthyroidism, versus 93.8% and 99.3%, respectively, for hypothyroidism. In conclusion, the Immuno 1 fT4 assay is useful in screening for thyroid disease.

False positive immunometric assays caused by anti-immunoglobulin antibodies: a case report.

A serological phenomenon causing aberrant results with monoclonal immunoenzymetric assays (IEMA's) is reported. Two different commercial IEMA kits detected low levels of choriogonadotropin (hCG) in the serum of a non-pregnant woman. These assays detected between 32 and 55 IU/l of serum hCG over a 3-wk period; however, an RIA for beta-subunit and two monoclonal immunoradiometric assays (IRMA's) detected no hCG (less than 5 IU/l). An IEMA measurement of creatine kinase MB isozyme was also elevated. Antisera to either human immunoglobulin or specifically to human IgM, added to the serum prior to assay, substantially decreased these IEMA reactions. Addition of either mouse serum or purified mouse IgG totally abolished them. It is concluded that these spurious reactions were most likely caused by an IgM antibody which binds to native and enzyme-labelled mouse IgG, but not to iodinated IgG.

The binding of metal ions to ATP: a proton and phosphorus nmr investigation of diamagnetic metal--ATP complexes.

Phosphorus and proton nmr spectra were recorded for complexes of ATp with Mg(II), Ca(II), Sr(II), Zn(II), Cd(II), Pb(II), Hg(II), Ag(I), and Tl(I) ions. Each of these ions except Hg(II) affected the 31P nmr of ATP, usually by shifting all three resonances downfield and decreasing the 31P-31P coupling constants. Pb(II) exerted the greatest shifts, while Mg(II) caused the greatest change in coupling constants. Effects on the adenine proton resonances were generally small and attributable to base stacking, but a direct metal-adenine binding is likely for Zn(II), Cd(II), and Ag(I). Effects on the ribose proton resonance were small in all of the ATP complexes, but were much larger in Zn(II)ADP and Cd(II)ADP. Formation of metal-bis(nucleotide) complexes occurred with Sn(II), Zn(II), and Cd(II).

25Mg NMR studies of magnesium binding to erythrocyte constituents.

The binding of Mg2+ ion to ATP, ADP, AMP, 2,3-bisphosphyoglycerate (DPG), and hemoglobin has been studied by 25Mg NMR spectroscopy at 9.4 T. Addition of any of these ligands to a solution of 2 mM 25MgCl2 at pH 7.2 caused a progressive increase in linewidth, with no discernible chemical shift. ATP and ADP, which form tight 1:1 complexes with Mg2+, did not cause maximal broadening until present in several-fold excess, implying that bis(nucleotide) complexes also form. The studies showed progressively weaker Mg2+ binding to ATP, ADP, DPG, and AMP, consistent with published binding constants. Hemoglobin cause fairly little broadening, consistent with its known weak affinity for Mg2+. Competition studies determined ATP affinities for Ca2+ and H+ that were also in good agreement with published values. 25Mg NMR spectra of 2 mM bound 25Mg2+ were obtained with good signal to noise in less than 1 hr. The technique may now be a practical means for studying the binding of Mg2+ within erythrocytes and other cells.

Evaluation of cardiac STATus CK-MB/myoglobin device for rapidly ruling out acute myocardial infarction.

The Cardiac STATus CK-MB/Myoglobin device is highly sensitive and has a high negative predictive value within 3 hours of patient presentation. The device may play a role in the re-triage of patients from the CCU to less intensive settings, resulting in a net cost savings.

Evaluation of a new assay for cardiac troponin I vs creatine kinase-MB for the diagnosis of acute myocardial infarction. Biochemical Markers for Acute Myocardial Ischemia (BAMI) Study Group.

OBJECTIVE: To compare a new assay for cardiac troponin 1 (cTn-1) with an assay for creatine kinase-MB (CK-MB) for the diagnosis of acute myocardial infarction (AMI). METHODS: A prospective cross-sectional study of patients presenting with symptoms consistent with cardiac ischemia was performed at a university teaching hospital. Serum sampling for cTn-1 and CK-MB was performed at 0, 1, 3, 8, and 16 hours after presentation. Normal values were defined as CK-MB < or = 7 ng/mL and a relative index < or = 2%, cTn-I < or = 1.4 ng/mL. Final diagnosis was made using World Health Organization criteria, including standard enzyme sampling. Consecutive patients with AMI were compared with a randomly selected subset of patients without AMI to determine the sensitivity and specificity of cTn-I and CK-MB assays for AMI, stratified by time from symptom onset. The ability of the biochemical cardiac markers obtained within 6 hours of symptom onset to predict later complications or need for interventions was assessed using odds ratios (ORs). RESULTS: Thirty-five patients who had AMI were compared with 136 patients who did not have AMI. The sensitivities and specificities of the cTn-I and CK-MB assays, stratified by time from symptom onset, were: [table: see text]. Patients who had elevations in either CK-MB or cTn-I within 6 hours of symptom onset were at increased risk for cardiovascular complications and/or interventions (CK-MB, OR 5.8; cTn-I, OR 6.3). CONCLUSION: cTn-I was as sensitive and specific for AMI as was CK-MB in ED patients who presented within 24 hours of symptom onset. However, cTn-I was more sensitive in patients who presented > or = 24 hours after symptom onset. Elevations of either marker within 6 hours of symptom onset predict an increased risk of complications and/or need for interventions.

Evaluation of CK-MB isoform analysis for early diagnosis of myocardial infarction.

Measurement of CK-MB and its isoforms by high-voltage electrophoresis has been proposed as a sensitive test for early detection of myocardial infarction (MI). We performed a prospective study of this test in 231 patients presenting to the Emergency Department with symptoms consistent with ischemic chest pain. Blood specimens were obtained at 0, 1, and 3 h following presentation, and plasma was immediately frozen and analyzed within 1 week by high-voltage electrophoresis for total CK-MB and isoforms. The test was considered positive whenever total CK-MB was elevated (>6 U/L) or the cardiac isoform MB2 was relatively increased (MB2 > 2 U/L and MB2/MB1 > 1.7). This test had a sensitivity of 68% overall and 55% for specimens collected within 3 h of symptom onset. It was positive within 3 h of presentation in 36/39 (92%) of patients with confirmed MI. Specificity was 92% overall and did not vary with time after symptoms. The CK-MB alone, at the cutoff of 6 U/L, had lower sensitivity overall (56%; p = 0.01) and within 3 h of onset (39%; p = 0.03), and higher specificity overall (98%; p < 0.001). Lowering the cutoff for CK-MB alone to match the sensitivity of the isoform test caused a greater loss of specificity. It is concluded that analysis of CK-MB by high-voltage electrophoresis is an effective method for rapid diagnosis of MI, with the isoform analysis enhancing early sensitivity.