Biomedical subjects
Publications and source records attributed to C P Price.
Enzymes are now used in a wide range of analytical methods, primarily for the measurement of substrates and as labels in immunoassays. Enzymes from microbial sources are becoming the preferred choice because of ease of extraction, catabolic activities and opportunities for enhancing yields. Protein engineering of bacterial enzymes will also play a role in future enzyme design with respect to improvements in specificity, reaction kinetics and stability.
Serum cystatin C has been suggested as a new marker of GFR. For the introduction of this marker into clinical use a rapid and automated method is required. We have developed and validated an assay for serum cystatin C using latex particle-enhanced immunoturbidimetry. Intra- and inter-assay precision were < 3% and < 5% across the assay range. Analytical recovery was 93 +/- 3.8% and no lack of parallelism was demonstrated. Regression analysis of a method comparison with an enzyme-enhanced radial-immunodiffusion method, gave PETIA = 0.074 + 0.93 x SRID, r = 0.98, N = 100. Inter-assay precision profiles showed cystatin C was measured with two-fold better precision than creatinine on the same analyzer. Cystatin C measurement was neither interfered with by icterus nor by hemolysis. 1/cystatin C versus 1/creatinine concentrations gave r = 0.67, N = 469. Comparison of Cr EDTA GFR with 1/cystatin C and 1/creatinine gave r = 0.81 and 0.50, respectively, N = 206. Calculating diagnostic sensitivity for abnormal GFR showed cystatin C to be significantly (P < 0.05) more sensitive than creatinine (71.4 vs. 52.4%). Cystatin C measurement using PETIA technology can be automated on the same instruments used routinely for the measurement of creatinine and offers better analytical performance and probably improved clinical sensitivity as a screening test for early renal damage.
Inhaled steroids are increasingly advocated as first line treatment for mild asthma. Some studies suggest that inhaled steroids suppress bone formation as reflected by a fall in plasma osteocalcin. Spacers have been shown to increase the proportion of inhaled aerosol that is deposited in the lungs and to reduce the amount swallowed. We measured plasma osteocalcin levels to determine the effect on bone formation of inhaled beclomethasone dipropionate (BDP) with and without a 750 ml spacer in a double-blind, randomised, placebo-controlled, cross-over study. Twenty-six healthy male volunteers took BDP 500 micrograms (two puffs of Becloforte) together with two puffs of placebo, inhaled twice daily for seven days. One inhaler was taken directly while the other was inhaled through a 750 ml spacer. After a two week washout period, the inhalers were exchanged so that BDP was taken by the alternate route for a further seven days. Fasting plasma osteocalcin levels were measured at 09.00 h before and at the end of each week. After a week of BDP taken directly (without a spacer), osteocalcin levels fell from 11.8 (SEM 0.6) ng/ml to 9.5 (SEM 0.5) ng/ml (p < 0.001). After a week of BDP taken through a spacer, osteocalcin levels fell from 12.1 (SEM 0.5) ng/ml to 11.1 (SEM 0.5) ng/ml (p < 0.001). The fall in osteocalcin when a spacer was used was significantly less than when BDP was taken directly (p < 0.005). This is likely to be because the systemic effects on bone are caused by swallowed rather than inhaled BDP, and this is limited by the use of a spacer. Spacers should be more widely prescribed with inhaled steroids. Further prospective studies are indicated to evaluate whether spacers protect bone mass.
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A detailed investigation of the performance of an immunometric assay for the quantitation of bone alkaline phosphatase (ALP) in serum showed that precision of this assay was similar to that of lectin precipitation and electrophoretic methods. Relationships for mass and activity measurements of the bone isoform using samples from children and patients with Paget's disease were similar for lectin and electrophoretic methods. Purified liver enzyme showed 100% cross reactivity in the immunometric assay. However, comparison of the slopes between bone ALP mass and total ALP activity using only samples with predominantly liver or bone isoforms showed that the cross reactivity of the liver isoform in serum was 18.3%. Experiments in which increasing amounts of a sample containing 90% of liver isoform adding to a serum sample from a patient with Paget's disease showed a cross reactivity of 16.5%. The reference range for bone ALP mass was 7-28 micrograms/L for men (n = 77) and 5-20 micrograms/L for women (n = 110) in the age group 20-50 years. In women over 50 years bone ALP was 28% higher. Increased bone ALP mass was also demonstrated in patients with Paget's disease (n = 59), liver disease (n = 95), chronic renal failure (n = 41) and hyperthyroidism (n = 17).
We have developed computer-based clinical case histories incorporating multimedia elements to aid the learning of medicine in a problem-based manner. Topics have been developed in the specialty of Clinical Biochemistry but the approach used is suitable for any branch of clinical medicine. Each topic has material aimed at medical students and also postgraduate candidates for professional examinations. A browser program is also incorporated. Emphasis is made on interaction through the case and modeling of real-life decisions in diagnosis and treatment. Advantages of the program are self-paced learning, assessment of understanding, feedback, and emphasis on deep understanding of the basic physiological and biochemical processes underlying clinical problems.
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We report a fully automated method for determining dibucaine number (DN) in a single-run procedure involving Dimension cholinesterase (CHE) Flex pseudo-(P)CHE reagents. The method was developed and optimized with the "open channels" and "kinetic" software facilities of the Dimension-ES instrument, where the DN is calculated automatically by an algorithm from the ratio of the uninhibited and inhibited rates, measured bichromatically, from a single analysis. The protocol was satisfactorily assessed for substrate depletion, linearity, reagent stability. and the effects of different dibucaine concentrations. Validation was performed across a range of CHE activities (1.5-22 kU/L) representing the three main genotypes, UU, UA, and AA. The respective DNs (mean +/- SD), determined on the Dimension-ES, were 82.0 +/- 1.6 (n = 32), 71.0 +/- 3.1 (n = 10), and 23.0 +/- 2.7 (n = 14), with corresponding imprecisions (CV) of 0.3%, 0.6%, and 5.2% (intraassay) and 0.7%, 0.7%, and 8.6% (interassay). Comparisons with reference (x) laboratory values and the DuPont aca (x') procedure (n = 53) gave regression equations of: y = 0.88x + 11.2, r = 0.99, and y = 0.85x' + 11.9, r = 0.99. A separate trial conducted with a Dimension-AR instrument gave similar performances. We conclude that the new DN method is fast, efficient, and appropriate for clinical use.
We have investigated the replacement of urine total protein estimations for the assessment of glomerular permeability, by the measurement of urine albumin excretion using a latex particle enhanced immunoturbidimetric assay. An initial screen was performed using Albustix to assess the sample pre-dilution necessary for immunoanalysis. A total of 167 24-hour urine samples were analysed and urine albumin concentration correlated well with that of urine total protein (r = 0.93) over the range 0-16,800 mg/l. This protocol provides a more cost effective and analytically valid assessment of glomerular permeability.
We have evaluated a dry reagent stick test for the quantitation of creatinine in plasma employing a whole blood sample. The assay was found to be specific for creatinine and correlated well (r = 0.993) with an established laboratory procedure. The precision found indicated that the method was suitable for use in the diagnosis and monitoring of renal disease.
In the United Kingdom, clinical biochemistry is practised by medical and non-medical graduates. Their training is postgraduate, led by the profession and has a strong vocational orientation. Although there is considerable overlap between the training of medical and non-medical graduates, each group has a different career structure and different training requirements. The training of non-medical biochemists has recently been restructured. Their new training programme is described in detail; for comparison, the training of medical graduates is outlined.
We describe the optimization of a rapid procedure for the elution of phenylalanine from blood spots and the estimation of the amino acid eluted using an enzyme-mediated assay linked to a colorimetric detection system. The method is rapid, fully quantitative, interference-free, accurate and precise unlike many of the methods currently employed for phenylalanine determination. The performance of the proposed method may necessitate the review of reference ranges and cut-off assignment strategies.
The DCA 2000 clinical analyser for the measurement of haemoglobin A1c was evaluated for analytical quality. The analyser, which utilises inhibition of latex agglutination immunoassay, demonstrated good within-batch (1.9-3.1% CV) and between-batch (2.2% CV) imprecision, and was not affected by haemoglobin concentration. The analyser was linear throughout the analytical range, and was found to correlate well with agar electroendosmosis (r = 0.93), affinity chromatography (r = 0.97), HPLC (r = 0.90) and EIA (r = 0.98). The analyser was found to give reliable analytical results, and with its ease of use, will provide the diabetologist with HbA1c results in the clinic; although an analysis time of 9 min will limit sample throughput.
There is a limited amount of data on the binding of paracetamol to plasma proteins. It has been suggested that binding might influence the ability of some analytical methods to quantify the total amount of drug present in the plasma fraction--the basis of clinical experience in risk assessment and antidote usage. We have investigated the binding of paracetamol to plasma proteins using an ultrafiltration technique. In overdose and spiked uraemic plasma samples the mean percentage of paracetamol bound was 24.1 (1SD = 7.0) with no significant correlation with drug levels or degree of uraemia. There is a small but significant increase in binding with increasing serum albumin concentration both in plasma (rs = 0.549, P = 0.014) and in pure serum albumin solutions (rs = 0.848, P < 0.001).
The peroxidase activity of haemoglobin A was characterized for non-glycated and glycated haemoglobin (HbA1) within the pH range 4.5 to 6.0, by measuring the rate of oxidation of 5-aminosalicylic acid following the degradation of H2O2. Glycation was found to significantly lower the pH activity of haemoglobin peroxidase throughout the pH range. However, in the presence of 100 mmol/l sorbitol the pH activity profile of glycated haemoglobin was significantly elevated whilst that of non-glycated haemoglobin remained unchanged.
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The specificity of a phenylalanine dehydrogenase, particularly with respect to cross reactivity toward tyrosine, has been shown to be pH dependent, being minimal at high pH. The dehydrogenase step has been coupled to colorimetric detection of NADH using a tetrazolium salt. The assay shows no significant cross reactivity towards a range of amino acids or drugs and correlates well with an established HPLC technique.
An enzyme-mediated assay has been developed for the measurement of salicylate using salicylate monooxygenase purified from Pseudomonas cepacia ATCC 29351. Two assay formulations were produced, based on either a multiple-reagent or a single-reagent formulation, to allow sufficient flexibility for automated use. The multiple-reagent formulation was especially suited to diagnostic laboratories performing infrequent manual salicylate estimation where stability of the reconstituted reagent is of paramount importance. This was achieved by preparing the enzyme and color reagents in separate vials, so keeping the enzyme at a stable pH. For more frequent assay use where a reconstituted reagent shelf life was less important, the single-reagent system offers advantages of convenience. However, the working reagent required a pH of 10.0 upon reconstitution. Although the enzyme was sufficiently active at this pH to give a reliable assay, its storage stability was poor at pH 10.0, preventing lyophilization of the reagent at a pH suitable for immediate use on reconstitution. This incompatibility was overcome by use of a layering technique. The enzyme was separated from the buffering solution in the same vial by freezing the buffering solution and then overlayering with the enzyme reagent prior to a second freezing cycle and subsequent freeze drying.