Daily maintenance dose of a long-acting theophylline from a single theophylline serum level.

A single point method has recently been described whereby the daily maintenance dose of theophylline required to achieve a desired steady state serum concentration can be established from a nomogram with the aid of a single blood sample drawn after a test dose. This approach has been validated for intravenous administration and for a rapidly absorbed elixir (coefficient of absorption ka 2.3 h-1) given to children. In order to study whether this method could be applied to a slowly absorbed preparation (ka 0.44 h-1), we gave theophylline (Theo-Dur) to nine adults as a single dose and for one week. In applying a nomogram, we found that a sample taken nine hours after a test dose would more accurately predict the daily dose required to achieve a desired steady state concentration than a sample taken at six hours as has previously been recommended (error nine hours -16.8 percent to +29 percent, six hours -59.7 percent to +29.4 percent).

Accelerated gallstone dissolution in methyl tert-butyl ether by sonication. An in vitro study.

OBJECTIVES: The authors tested the effect of 195 KHz therapeutic ultrasound energy on gallstone dissolution in concert with methyl tert-butyl ether (MTBE) in vitro. METHODS: Sixteen sets of three gallstones matched for weight and appearance were selected from 16 surgically resected human gallbladders. One stone from each set was analyzed for its density pattern by computed tomography (CT) and biochemically for cholesterol content. Based on CT appearance, the stones were classified into eight noncalcified, four partially calcified, and four heavily calcified sets. The three stones were subjected to dissolution with MTBE: one with simultaneous sonication via an experimental ultrasound unit, one with manual stirring, and one acted as control without added treatment. RESULTS: Sonication reduced the dissolution time of noncalcified stones by 96% (range, 94%-98%; standard deviation [SD], 2%) relative to controls, and it was three to four times more effective than manual stirring. It was similarly effective in helping to dissolve partially calcified stones, but not heavily calcified stones. CONCLUSIONS: This study demonstrates the positive effect of sonication in accelerating gallstone dissolution with MTBE in vitro for stones without heavy calcification.

The catalytic function of active site amino acid side chains in well-characterized enzymes.

Although the kinetics and types of reactions carried out by enzymes have been established for some time, the detailed chemistry performed by these catalysis is largely unknown. Their geometries and compositions are very different from those found in conventional chemistry and understanding their mechanisms will open new areas of chemistry and make important contributions to the rational design of pharmaceuticals. We have formulated a computational method for determining the electronic role of amino acid residues in the active sites of enzymes that have been well characterized by high resolution spectroscopy and other physical chemistry techniques. Ab initio electronic structure calculations with a good basis set were employed, and solvent and dielectric effects were taken into account. Applications were made to ribonuclease A, the serine proteases, the labile hydrogen bonds in acid proteases (pepsin), and carbonic anhydrase.

Ultrasound and amniotic fluid alpha-fetoprotein in the prenatal diagnosis of spina bifida.

Amniotic fluid alpha-fetoprotein (AFP) assays and detailed ultrasound examinations were performed in 376 prenatal patients at risk for a neural tube defect (high-risk group). In addition, 2436 patients who underwent amniocentesis for other indications underwent preamniocentesis ultrasound screening and amniotic fluid AFP assays (low-risk group). There were 10 neural tube defects in the high-risk group (7 open and 3 closed) and 3 in the low-risk group (all open). Two of the 3 closed defects were detected prenatally. The predictive value of an elevated AFP level for an abnormal fetus was much higher in the high-risk (6 of 6, 100%) than in the low-risk group (1 of 6, 17%). When both ultrasound and AFP assay results were normal, the chance of a normal outcome was very high in both the high- and low-risk groups (99.7 and 100%, respectively). It was of particular interest that in the low-risk group, the likelihood of an abnormal outcome in women with elevated AFP and a normal ultrasonogram was low (0 of 5).

Effects of hemoglobin variants on Hb A1c determinations using Bio-Rad columns.

In the column chromatographic determination of Hb A1c, hemoglobin variants affect Hb A1c results and column patterns. Samples from several patients with hemoglobin variants were run on Bio-Rad Hb A1c columns to demonstrate these patterns. Columns should be examined during the run to detect the presence of Hb F which migrates very rapidly, and after the run to detect abnormal column patterns. Hb A1c levels in patients heterozygous for Hb S or C are low but may be interpreted in relation to the patient's level of Hb A. On the other hand, Hb A1c results on patients with homozygous Hb S, Hb C, or high levels of Hb F cannot be interpreted.

Assay of erythrocyte glucose-6-phosphate dehydrogenase on the GEMSAEC analyser.

A procedure for the assay of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) in erythrocytes on the GEMSAEC centrifugal analyzer using a modification of Beutler's procedure is described. Glucose-6-phosphate dehydrogenase activity is corrected for the contributing activity of 6-phosphogluconate dehydrogenase (EC 1.1.1.44) by using a two-cuvet system in which the activity of the sample incubated with 6-phosphogluconic acid is subtracted from the activity produced in the presence of the combined substrates, glucose-6-phosphate and 6-phosphogluconic acid. An overlay program on the GEMSAEC computer for calculation of results, and the use of a yeast glucose-6-phosphate dehydrogenase control which is stable frozen in dilute solution are discussed.

Comparison of the Ames, Randox and Roche methods with the Synermed method for the determination of serum iron concentrations on nondialysis and dialysis specimens.

OBJECTIVES: To evaluate the Ames, Randox, Roche, and Synermed methods for the measurement of serum iron and to investigate patterns of possibly discrepant results in dialysis patients. METHODS: Assays were performed on the Cobas Fara II analyzer. Precision and accuracy studies were conducted; recovery studies were done by adding pooled serum from dialysis patients to an assayed human serum-based control. Patient comparisons included over 150 nondialysis patients and 30 dialysis patients. RESULTS: For the Ames, Randox, Roche and Synermed methods, the between-run precision was less than 2.80% with the normal aqueous iron standard; 2.00, 2.70, 0.80, and 2.00% for the four methods with the high serum iron control, respectively, and less than 2.30% with the serum pool. Using a pooled serum from dialysis patients, between-run precision was higher with all four methods. With an abnormal assayed human serum-based control, accuracy was over 98% for the four methods. Recoveries were 121% for the Ames and Randox methods and 104-105% for the Roche and Synermed methods. Accuracy as assessed with Murex EQAS specimens ranged from 71 to 80%, 71 to 96%, 98 to 99.5%, and 42 to 50% for the four methods, respectively. For comparisons of the Ames, Randox, and Roche methods with the Synermed method, difference analyses revealed biases (SD) for nondialysis patients of 1.9 (2.7), 1.5 (3.3), and 1.8 (2.2) mumol/L, respectively; and for dialysis patients of 8.2 (13.3), 5.1 (5.4), and 1.4 (1.7) mumol/L. Standard linear regression analyses and correlation coefficients are also provided. CONCLUSIONS: The Roche method was slightly more precise than the other methods. Using an abnormal assayed serum-based control, all methods showed good accuracy. Recovery studies with pooled serum from dialysis patients showed interferences with the Ames and Randox methods and good recovery with the Roche and Synermed methods. With the bovine serum-based Murex samples, all but the Roche method yielded some low results; the Synermed method has been reported to suffer from matrix problems with bovine serum albumin. Based on recovery studies and difference analyses, the Ames and Randox methods revealed discrepancies in iron results for samples from dialysis patients. The Roche and Synermed methods appeared to be suitable for measurement of serum iron in dialysis patients.

Postdoctoral training in clinical chemistry: laboratory training aspects.

OBJECTIVE: To provide a general outline for a 2-year postdoctoral training program in clinical chemistry, and a detailed outline of the first year laboratory training program. METHODS & RESULTS: Essential elements of the 2-year Postdoctoral Training Program in Clinical Chemistry at the University of Toronto are its didactic courses and a comprehensive, structured laboratory rotation in the first year. Residents rotate in hospital laboratories in both years of the Program. The hospital laboratory rotation in first year includes a 36-week laboratory rotation based on the Laboratory Training Program Manual. In the second year, they consolidate the basic knowledge acquired in first year and gain experience in pediatric testing and other specialty areas. In both years, residents attend teaching and ward rounds on a regular basis, investigate unusual test requests and patient results, and make regular presentations at case presentation and journal club sessions. They undertake research and development projects which lead to presentations at scientific meetings and to publication. Residents attend departmental management meetings, arrange discussions on management topics, and attend a short course on key management topics. Approaches for strengthening the knowledge and skills of residents in the areas of hematology, microbiology and pathology are being developed. CONCLUSION: The program outline described should provide a useful framework for other such programs both nationally and internationally.

Interference of IgM paraproteins in the Olympus AU800 uric acid assay.

INTRODUCTION: In the Olympus uric acid procedure, uric acid is converted by uricase to allantoin and hydrogen peroxide, which is reacted in a Trinder reaction to produce a chromophore read bichromatically at 520 and 660 nm. Repeated difficulty was encountered in obtaining uric acid results on samples from myeloma patients with known IgM paraproteins. Large absorbances in sample blanks were due to a visible precipitation observed in the reaction cuvettes. OBJECTIVE: To alter the Olympus method (OM) to eliminate the interference by IgM, and to verify the modified method (MM). METHODS: Dilution of the sample blank by saline was substituted for water in the MM, with small alterations in the reaction timing sequence necessary to accommodate the instrument requirements. RESULTS: A comparison of uric acid results obtained from nonmyeloma patient samples using the OM and the MM showed a good correlation (r = 0.970), and no statistical difference between the two means using a paired t-test. A similar comparison performed using the samples containing IgA and IgG paraproteins also revealed a good correlation (r = 0.981), and no statistical difference between the two means. Results on IgM containing specimens were assessed indirectly because the samples could not be assayed with the OM. First, removal of detectable levels of proteins using a 20% TCA solution did not affect the measurement of uric acid. Second, protein-free supernatants from IgM containing samples were measured by the OM and compared with the corresponding serum samples measured by the MM. There was good correlation between the two methods (r = 0.945), and no statistical difference between the means using a paired t-test. CONCLUSION: The modified method is satisfactory for routine analysis of samples, including those with IgM paraproteins.

Prostate-specific antigen in amniotic fluid of normal and abnormal pregnancies.

OBJECTIVE: To examine if prostate-specific antigen (PSA) is present in amniotic fluid or maternal serum during pregnancy and if its presence is associated with fetal abnormalities. METHODS: Samples tested included amniotic fluids from 853 pregnant women for whom amniocentesis was performed; 312 nonpregnant women who donated blood; 259 pregnant women who donated blood at various gestational ages. Amniotic fluid or serum PSA was measured with an ultrasensitive time-resolved immunofluorometric procedure. 372 pregnancies were studied for the presence of genotypic or phenotypic fetal abnormalities. RESULTS: PSA was present in most amniotic fluids; the median PSA concentration increased from gestational week 11 to 22 and stabilized thereafter until delivery. The most prominent PSA concentration change occurred during gestational weeks 13-14. Pregnant women had significantly higher serum PSA concentrations than nonpregnant women; the pattern of serum PSA concentration change during pregnancy was similar to that of amniotic fluid; however, serum PSA concentrations were lower by a factor of 20-40. No association existed between amniotic fluid PSA and maternal age, gender of fetus, or length of abstinence of mother from sexual intercourse. After gestational week 15, fetuses with trisomy 21 or 18, anencephaly, or renal disorders were associated with low amniotic fluid PSA levels. CONCLUSION: Our data suggest that PSA may play a role in fetal development, especially at gestational ages between 13-20 weeks. The diagnostic usefulness of PSA in identifying fetal abnormalities remains to be determined.

High performance liquid chromatographic analysis of urinary catecholamines employing amperometric detection: references values and use in laboratory diagnosis of neural crest tumors.

We have developed an high performance liquid chromatographic procedure employing amperometric detection for the measurement of urinary free norepinephrine, epinephrine and dopamine. The between-day precision (C.V.) at various concentrations of the above analytes varied from 4.8-11.6%. There was negligible between-urine difference in the percent recovery of norepinephrine and epinephrine but considerable between-urine difference for dopamine. The procedure has been used to determine reference ranges in infants, children and adults. Its preliminary application to the laboratory detection of neural crest tumors is also described.

Comparison of two immunoassay methods for the determination of alpha-fetoprotein in amniotic fluid.

We compared two immunoassay methods for determination of alpha-fetoprotein in amniotic fluid. The two methods showed a very good correlation and this is further confirmed by the clinical study. The electroimmunoassay (rocket immunoelectrophoresis) seems suitable for a small laboratory, whereas radioimmunoassay may be a better choice for a large laboratory.

The electrostatic potential of the alpha helix (electrostatic potential/alpha-helix/secondary structure/helix dipole).

The active sites of many enzymes are very close to the N-terminus of an alpha-helix. The helix dipole has been postulated to enhance the binding of anions and speed charge relays in catalysis. We present electrostatic potential maps of alpha-helices of various lengths using a point charge model. We show that the potential field of the helix can be mimicked by two equal and opposite charges, one at each terminus. The magnitude of these equivalent charges reaches its limiting value of +/- 0.2 to 0.3 electron at a helix length of approximately 7-10 residues. We also comment on the relative importance of the helix dipole to that of ionized residues in determining the electrostatics of a protein and discuss what consequences this has for enzymology.

Effects of vestibular stimulation on nystagmus response and motor performance in the developmentally delayed infant.

The effects were studied of 10 days' exposure to daily repetitive, specific vestibular stimulation on motor performance of children with Down's syndrome and normal children. A quantitative assessment of vestibular function was made in these children including the habituation response of postrotatory nystagmus. Control groups were included. Both the children with Down's syndrome and the normal children who received vestibular therapy demonstrated positive effects when evaluated using a quantitative motor skills assessment test. Control and treatment children showed vestibular habituation, with treatment children evidencing the greater change. This change may reflect the acquisition of an increased level of central nervous system inhibitory control.

Acetaldehyde hydrate and carbonic anhydrase: possible roles in the inhibition of brain aldehyde dehydrogenase.

One biochemical explanation for the chronic and addictive effects of ethanol involves a relationship between biogenic aldehydes, brain aldehyde dehydrogenase and acetaldehyde, the principal metabolic product of ethanol. We suggest here the possibility that acetaldehyde hydrate may act as an especially strong inhibitor of aldehyde dehydrogenase. Aldehyde hydrates are known to strongly inhibit aldehyde dehydrogenase as well as a number of other aldehyde oxidizing enzymes and it may be that acetaldehyde hydrate acts as a transition state or activated intermediate inhibitor. It is also suggested that carbonic anhydrase, which catalyzes the very rapid equilibrium between acetaldehyde and its hydrate, may play a role in this process.

A comparison of two techniques for the determination of serum bone-specific alkaline phosphatase activity in dogs.

Bone-specific alkaline phosphatase (BALP) shows potential as a marker of bone formation in the dog. Recent studies have indicated that serum BALP may provide a useful, non-invasive indicator of skeletal health in dogs, and as a diagnostic and prognostic marker in the management of dogs with musculoskeletal or metabolic disorders. Two assay techniques (one based on wheatgerm lectin precipitation followed by a simple enzymatic reaction, the second on a specific enzyme-linked immunoassay) were used to measure serum levels of BALP in 35 dogs of different ages. As expected, BALP concentrations decreased with age. For the enzymatic assay, mean (+/-SD) serum concentrations of BALP activities were 100.3 (+/-11.6) U/liter in dogs under 1 year of age, 25.3 (+/-6.8) U/L in dogs 1 to 2 years of age, 16.5 (+/-7.3) U/L in dogs 2 to 3 years of age, 14.3 (+/-5.6) U/L in dogs 3 to 7 years of age, and 12.3 (+/-4.8) U/L in dogs aged 8 years and older. Corresponding results from the immunoassay were 56.3 (+/-9.8) U/L, 10.7 (+/-4.5) U/L, 7.0 (+/-2.5) U/L, 6.7 (+/-3.6) U/L and 7.0 (+/-2.9) U/L. There was excellent correlation between the results from the two assay techniques (r = 0. 96; P < 0.0001). The correlation between BALP and total ALP activities was poor (r = 0.20 for enzymatic BALP, r = 0.31 for immunoreactive BALP), indicating that total ALP should be considered unreliable as an indicator of BALP activity in canine serum. The immunoassay demonstrated acceptable (13 per cent) cross-reactivity with the liver isoform of ALP. The commercial immunoassay kit is simple and provides fast results. Although the wheatgerm lectin/enzymatic technique is preferred in situations where the activities of all three isoforms of ALP are required, the immunoassay should be considered whenever the activity of BALP is the focus of interest.