Propylthiouracil treatment reduces long-term potentiation in area CA1 of neonatal rat hippocampus.

Rat pups were made hypothyroid by exposure to propylthiouracil in drinking water beginning at 1 week of age, and the degree of long-term potentiation (LTP) in hippocampal area CA1 determined from brain slices of animals ranging in age from 2 to 6 weeks. Serum T3 levels were less than 20% of that of age matched controls after 3 weeks of treatment, and remained at that level. Relative to the age-matched controls, LTP was reduced significantly after 2 weeks of treatment. These observations are consistent with the conclusion that LTP magnitude is a reflection of cognitive function, which is known to be depressed in hypothyroid conditions in both animals and man.

Human ventricular myosin light chain isotype 1 as a marker of myocardial injury.

A monoclonal enzyme immunoassay for measuring human ventricular myosin light chain isotype 1 (HVMLC1) in serum has been developed. To evaluate the method in patients with suspected myocardial injury, we studied 51 patients (16 acute myocardial infarction (AMI), 19 unstable angina pectoris (UAP), 9 stable angina pectoris, 3 nonischemic heart disease, 4 hip surgery patients), and 190 controls (blood donors). Serial blood-samples were drawn from patients; a single blood-sample from controls. The diagnostic value of the HVMLC1 assay was compared with total creatine kinase (CK), CKMB activity, CKMB mass concentration, lactate dehydrogenase isoenzyme 1 (LD1), troponin T (TnT) and mitochondrial-aspartate aminotransferase (m-ASAT). The detection limit of HVMLC1 was 0.4 microgram/l (linear range 0-20 micrograms/l). Sera from 190 reference persons did not contain detectable levels of HVMLC1 (< 0.4 microgram/l; 99% percentile). The coefficients of variation were 13% (1.0 microgram/l) and 3.1% (17.7 micrograms/l). Cross-reactivity with myosin from skeletal muscle was seen. Times to peak value were: CK 19.3 +/- 2.0, LD1 43.4 +/- 3.2, HVMLC1 72.9 +/- 7.0, and m-ASAT 67.3 +/- 5.6 h. Time-curves of HVMLC1 and m-ASAT were similar, whereas time-curves for HVMLC1 and TnT were quite different in most cases. Peak value of HVMLC1 was five times higher than CK peak value and eight times that of LD1. HVMLC1 appeared in the blood within hours after the onset of chest pain and in the majority remained for more than a week after AMI. Among patients with UAP 16% (3/19) had elevated HVMLC1 in serum, whereas elevated TnT was seen in 26% (5/19) and elevated CKMB mass in 26% (5/19). We conclude that the new HVMLC1 assay offers a sensitive diagnosis of myocardial injury. It is characterized by a wide diagnostic time window. The similarity of the HVMLC1 and m-ASAT curves indicates that it may be used to estimate the extent of myocardial necrosis.

Accurate enzyme activity measurements. Two decades of development in the commutability of enzyme quality control materials.

Commercial serum preparations are integral components of both internal and external quality control programs for enzyme activity measurements. However, properties of these materials may differ significantly from those of clinical specimens. Differences from clinical specimens may include the following: species origin of the enzyme; isoenzyme form(s); integrity of the molecular species; matrix of the solution; processes such as lyophilization; and addition of preservatives. There are also significant differences among methods measuring the activity of a single enzyme including a diversity of compounds that may serve as substrate(s); variable cofactor or metal supplementation; and differences in the substrate concentration(s), buffer substances, pH, and temperature. The measured response to each of these variations in assay technique may differ among these types of specimens. To be acceptable, quality control materials must have properties similar to those of clinical specimens. Thus, the concept of commutability that we originated and first applied to enzyme activity measurements remains useful, and its further application to the problem of "matrix effects" is reviewed here. Multivariate display techniques are applied to the specific examples of aspartate aminotransferase, alpha-amylase, and alkaline phosphatase to judge the commutability of quality control materials for these enzymes.

How good are clinical laboratories? An assessment of current performance.

The Clinical Laboratory Improvement Act of 1967 and Amendments of 1988 (CLIA '67 and CLIA '88) were enacted to ensure that clinical laboratories within the U.S. provide a quality of service that meets clinical needs for good patient care. Approved proficiency-testing programs are to judge the quality of laboratory testing by promulgated performance criteria. We examine the quality of analytical results reported in 1991 to the New York State Department of Health Proficiency Testing program in light of these criteria and analytical goals, based on medical usefulness. Analytical performance is examined for cholesterol, potassium, sodium, calcium, glucose, aspartate aminotransferase, digoxin, and theophylline. In general, proposed CLIA '88 performance standards are compatible with the current state of practice for the population of laboratories examined. Exceptions appear to be digoxin and sodium (failure rate exceeding average) and most therapeutic substances (low failure rate). Sources of analytical bias relative to an accuracy-based target value must be characterized as method-, laboratory-, or matrix-dependent if regulatory programs are to achieve the objective of improving analytical accuracy across all testing sites.

Application of clinical laboratory measurements to issues of environmental health.

Monitoring of biochemical constituents in serum is an important component in revealing potential toxicity in humans and experimental animals due to exposure to a variety of xenobiotic agents. The relative toxicity of pure compounds, usually at large doses, has helped elucidate the mode of action of these compounds and their relative risk. However, most actual cases of environmental exposure present an extensive range of components and the potential for synergistic or inhibitory interactions. In this paper we review two such environmental cases: The Love Canal chemical dump site in Niagara Falls, NY, and the transformer fire at the State Office Building in Binghamton, NY. We focus on the clinical laboratory measurements obtained in these studies (including serum glucose, triglycerides, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma-glutamyltransferase, lactate dehydrogenase, sodium and potassium), their usefulness, limitations, and application to such cases. Significant alterations in serum triglyceride and alanine aminotransferase levels were found in guinea pigs due to exposure to dioxins. These two tests were useful in estimating the 'equivalent' concentration of 2,3,7,8-tetrachlorodibenzo-p-dioxin in complex chemical mixtures.

A novel method for differentiating dextran sulfate from related sulfated polysaccharides.

A method is described for unequivocal identification of dextran sulfate, based on combined chemical desulfation and dextranase enzymolysis of dextran sulfate moieties to isomaltose, a specific indicator of dextran-type precursors. The method was developed using high-resolution (300 MHz) 1H NMR spectroscopy for assurance of the molecular transformations, identification, and estimation of the hydrolysis products. Overall conversion of approximately 80% of highly sulfated and moderately sulfated dextran sulfates was realized. Both 2-D 1H and 13C NMR spectra of a dextran sulfate (MW 500,000) clarified the extent of sulfation (75%) at C-4 and confirmed that sulfation at positions C-2 and C-3 was virtually complete. Estimation of the hydrolysis products (isomaltose, major; alpha-D-glucose, minor) is not restricted to 1H NMR now that the desulfation-enzymolysis methodology has been established; rather, it can be performed using HPLC or GLC (with derivatization).

Sulfation of some chemically-modified heparins. Formation of a 3-sulfate analog of heparin.

A modified form of heparin containing residues of nonsulfated alpha-L-idopyranosyluronic acid (7) in place of the normal 2-sulfate (1) was sulfated with sulfur trioxide-trimethylamine in dimethylformamide at 0 and 25 degrees. Examination of the reaction products by n.m.r. spectroscopy showed that sulfation occurred selectively at C-3 of residue 7, to give a new polymer that may be described as a 3-sulfate analog of heparin. A slower substitution reaction led subsequently to sulfation at C-3 of 2-deoxy-2-sulfamino-alpha-D-glucopyranosyl 6-sulfate residues (2), although this was accompanied by partial N-desulfation of 2. An analogous pattern of O-sulfation-N-desulfation was observed for the residues of 2 in two other modified heparins, one containing residues of 2,3-anhydro-alpha-L-gulopyranosyluronic acid and the other residues of alpha-L-galactopyranosyluronic acid, in place of residues of 1. The galacto diastereomer exhibited relatively low regioselectivity, as it was found to be sulfated at C-2 or C-2.3, or both. Selective resulfation of free amino groups gave the products that were examined for anticoagulant activity and susceptibility to enzymolysis by heparinase. Antithrombin-binding affinity measurements were also carried out. Although none of the materials had significant anti-Xa activity, nor were they affected by heparinase, their patterns of binding to antithrombinagarose were not dissimilar to that of heparin.

Characterization and differentiation of some complex dextran sulfate preparations of medicinal interest.

Dextran sulfate samples from different sources were examined by 1H and 13C NMR spectroscopy to differentiate the samples on the basis of extent and sites of sulfation. The anomeric (H-1) signal proved to be a good indicator ranging from no sulfation (as in dextran) to virtually complete sulfation at positions 2 and 3, whereas the relative intensity of the H-4 signal afforded a measure, conversely, of the degree of sulfation at position 4. Three different dextran sulfate tablet formulations were found by NMR to contain similar dextran sulfate material that was characterized by a high degree of sulfation at positions 2 and 3. Position 4 of dextran appears to be less readily amenable to substitution. Quasi-elastic light scattering (QELS) analysis of aqueous dispersions of the bulk dextran sulfate samples and formulated tablets permitted additional particle size and homogeneity differentiation. None of the dextran sulfate materials showed either anti-factor Xa activity or marked anticoagulant activity.

The nature of calibrators in immunoassays: are they commutable with test samples? Must they be?

Immunoassay systems require calibration protocols that are normally more sophisticated than many analytical techniques in routine clinical use. Calibrators used in such assays may differ significantly from the analyte in clinical specimens. Differences in the properties of calibrators, or reference materials, from those of clinical specimens may include: species origin of the calibrator for an analyte; integrity of the molecular species; matrix of the calibration solution; addition of preservative agents. Owing to the large number of potential differences in the properties of calibrators and those of serum specimens that may affect immunoassay results, the concept of commutability that we originated and first applied to enzyme activity measurements can readily be applied to immunoassay determinations. We specifically examined the properties of calibration materials in nine commercial immunoassay tests for human thyrotropin. Significant non-commutability of materials was demonstrated. The measured results obtained with authentic patient sera differed by a factor of approximately two fold between the techniques exhibiting the lowest and greatest numeric results. Considerably larger intermethod biases were found for calibration materials. Multivariate analysis revealed that the patient sera formed a highly focussed pattern. The calibration materials for one instrument system also focussed in this group. Other calibrators formed three foci indicating similar patterns of commutability within each of the three groups. Clustering was independent of the amount of thyrotropin in the patient specimens, but appeared to be concentration-dependent for at least some of the calibrators. Thus the availability of a common calibration material appears feasible, but not presently available in many commercial products. A processed human serum, a candidate material for use in our proficiency testing program, was projected in the same cluster as authentic patient sera indicating that this material has intermethod properties identical to patient sera (i.e. fully commutable).

Marked stereoselectivity in the binding of copper ions by heparin. Contrasts with the binding of gadolinium and calcium ions.

Heparin forms a complex with cupric ion (Cu2+) at a level of less than or equal to 10(-3) mol of the metal ion per dimeric unit of the polymer, as evidenced by paramagnetic relaxation effects on its 1H- and 13C-n.m.r. spectra. No interaction occurred with heparin derivatives modified either by desulfation of the residues of alpha-L-iduronic acid 2-sulfate, or by hydrolysis of the sulfamino group of the residues of 2-deoxy-2-sulfamino-alpha-D-glucose 6-sulfate, although binding was induced by N-acetylation of the latter derivative. Under the same experimental conditions, no alternative type of glycosyluronic acid structure tested, including the other glycosaminoglycans, showed significant relaxation enhancement by Cu2+. These results are in contrast to those obtained with gadolinium ion (Gd3+), another paramagnetic probe, or with calcium ion (Ca2+), which promotes chemical-shift displacements. The binding selectivities of those two cations are much broader than that of Cu2%, although they also differ notably in their relationship to the structure of heparin.

Effects of exercise on serum amino-transferase activity and pyridoxal phosphate saturation in Thoroughbred racehorses.

Aminotransferase activities were measured in the serum of two- to three-year-old Thoroughbred fillies and colts during a four week period of peak training for flat racing. Aspartate aminotransferase (AspAT, EC 2.6.1.1), mitochondrial aspartate aminotransferase (m-AspAT) and alanine aminotransferase (AlaAT, EC 2.6.1.2) activities in serum were measured and the relative proportions of apoenzyme and holoenzyme were determined. The aminotransferase activities were increased only slightly immediately following exercise. This small and immediate post exercise increase in activity did not vary greatly over the period of peak training. Measured in the presence of exogenous pyridoxal 5'-phosphate, mean enzyme activities (iu/litre at 30 degrees C) before exercise were: AspAT, 291; m-AspAT, 13; AlaAT, 18. After exercise they were: AspAT, 317; m-AspAT, 16; AlaAT, 23. Nearly all of the AspAT activity was present in the holoenzyme form (94 per cent holoenzyme) indicating excellent vitamin B6 status in these animals. Paradoxically, the AlaAT in serum from the same highly trained Thoroughbred horses was poorly saturated with pyridoxal phosphate, with nearly half of the AlaAT in most horses present in the inactive apoenzyme form (61 per cent that of holoenzyme). It is critical therefore, that exogenous pyridoxal phosphate be included in aminotransferase assays to determine the amounts of enzyme release into the peripheral circulation.

Physicochemical characterization of the First World Health Organization International Standard for low molecular weight heparin derivatives.

High-field (300 MHz) 1H NMR spectral analysis and particle size distribution analysis employing the quasielastic light scattering (QELS) technique were performed on samples of the 1st International Standard for low molecular weight (LMW) heparin derivatives recently selected by the World Health Organization (WHO). We propose that the results of these analyses, which showed that the material is highly homogeneous in particle size and retains spectral features characteristic of its porcine mucosal origin, form an appropriate basis for physicochemical comparison between the "Standard" and other LMW heparin preparations.

Mitochondrial aspartate aminotransferase determined by "Fast Protein Liquid Chromatography".

We describe an improved separation of the isoenzymes of aspartate aminotransferase (EC 2.6.1.1), based on ion-exchange chromatography. Involving the "Fast Protein Liquid Chromatography" system (Pharmacia) with a MonoQ column, this rapid, reproducible method for quantifying the mitochondrial enzyme shows good resolution and sensitivity, and results correlate well with those by an established immunochemical method.

Aminotransferases in disease.

Aspartate and alanine aminotransferases are two of the enzymes most frequently measured by the clinical laboratory. They are most commonly used in the differential diagnosis of various liver diseases where the ratio of the two enzymes provides additional clinical insight. AST is also useful in many cases for diagnosis, or estimating severity, of myocardial infarction. The mitochondrial isoenzyme of AST has a growing significance in the diagnosis of alcoholism and other conditions.

Treatment of infantile spasms with high-dose ACTH: efficacy and plasma levels of ACTH and cortisol.

Fifteen children with infantile spasms and a hypsarrhythmic EEG defined by EEG-videotelemetry monitoring received a regimen of high-dose (150 IU/m2/d) ACTH for their seizures. We carried out an endocrinologic evaluation before and after initiation of the ACTH and conducted a time course study of plasma ACTH and cortisol levels after ACTH dosing. Spasms were controlled and the EEG normalized in 14 of the 15 children. Prior to starting ACTH therapy all the patients had normal prolactin, insulin, cortisol, and ACTH levels in plasma and normal thyroid function. Although the pattern of rise of ACTH levels in plasma after ACTH dosing was similar in all the children, there was great individual variation in the absolute concentrations. However, both the pattern of rise and absolute level of cortisol in plasma after ACTH was highly predictable in all patients. Plasma cortisol rose rapidly within 1 hour of ACTH administration and continued a slower rise for 12 to 24 hours after the ACTH dose. High-dose ACTH therapy seems quite effective in infantile spasms, perhaps because of a sustained high level of plasma cortisol. This sustained plateau of cortisol may be more effective in controlling infantile spasms than the pulse effect expected with oral steroids or lower doses of ACTH.