Comparative studies of the in vitro metabolism and covalent binding of 14C-benzene by liver slices and microsomal fraction of mouse, rat, and human.

Metabolism of benzene by the liver has been suggested to play an important role in the hepatotoxicity of benzene. The role of the different benzene metabolites and the causes of species differences in benzene hepatotoxicity are, however, not known. The metabolism and covalent binding of 14C-benzene by liver microsomal fractions and liver slices from rat, mouse, and human subjects have been studied. Rat microsomal fraction formed phenol at a rate of 0.32 nmol/min/mg of protein; mouse microsomal fraction formed phenol at 0.64 nmol/min/mg and hydroquinone at 0.03 nmol/min/mg; and human microsomal fraction formed phenol at 0.46 nmol/min/mg and hydroquinone at 0.07 nmol/min/mg. Covalent binding of 14C-benzene metabolites to rat, mouse, and human liver microsomal protein was 29, 113, and 169 pmol/min/mg of protein, respectively. The rates of metabolite formation from benzene by liver slices in nmol/min/g of tissue were: rat, phenol 0.15, hydroquinone 0.26, and phenylsulfate 1.22; mouse: phenol 0.13, hydroquinone 0.29, phenylsulfate 1.37, and phenylglucuronide 1.34; and human: phenol 0.16, hydroquinone 0.27, phenylsulfate 0.83, and phenylglucuronide 0.52. trans,trans-Muconic acid formation was not detected with liver slices of any species. Covalent binding of 14C-benzene metabolites to rat, mouse, and human liver slices was 8.2, 79.7, and 27.3 pmol/min/g liver, respectively. There was no correlation between ascorbic acid levels in the human liver slices and covalent binding of 14C-benzene metabolites. The results show that phenol and hydroquinone found in extrahepatic tissues, including bone marrow, of animals exposed to benzene could originate from the liver. There was no evidence for the release of highly reactive benzene metabolites such as trans,trans-muconaldehyde or p-benzoquinone from liver cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Human and dog, but not rat, isolated hepatocytes have decreased foreign compound-metabolizing activity compared to liver slices.

A comparison has been made of the metabolism of biphenyl by isolated hepatocytes and liver slices from rat, dog, and human. Hepatocytes were prepared by low Ca2+ and enzyme digestion of the perfused liver of rat or liver slices from the rat, dog, and human. The ratio of free to total hydroxybiphenyl formation (R) was a sensitive measure of hepatocyte functional viability in perfusion-isolated rat hepatocytes, showing a significant negative correlation (r = -0.920, p less than 0.01) with trypan blue exclusion (TBE). Rs for rat hepatocytes prepared by the perfusion and slice-digestion techniques were not significantly different. Biphenyl metabolism and TBE in rat, dog, and human hepatocytes isolated by the slice-digestion technique were compared. Total hydroxybiphenyl formation by dog and human hepatocytes was 21% and 4% of that seen with rat hepatocytes. Rs for rat, dog, and human hepatocytes were 0.19, 0.46, and 0.63, respectively. TBE for all the hepatocyte preparations was approximately 90%. In contrast to the hepatocytes, total hydroxybiphenyl formation by slices of dog and human liver was 106% and 108%, respectively, of that seen with slices of rat liver. Rs for rat, dog, and human liver slices were 0.11, 0.21, and 0.26, respectively. These results suggest that hepatocytes prepared by the slice-digestion technique from dog and human but not rat liver have lost some of their ability to oxidize biphenyl and form biphenyl conjugates. This may be due to damage to the hepatocytes during isolation. TBE does not appear to be an accurate measure of hepatocyte functional viability between species. It is concluded that liver slices may provide a better model than isolated hepatocytes for foreign compound metabolism studies with dog and human liver.

Oxidative metabolism of 14C-pyridine by human and rat tissue subcellular fractions.

1. The oxidative metabolism of 14C-pyridine by human and rat microsomal fractions has been studied. Metabolites were separated by h.p.l.c. employing continuous radioactivity monitoring of the column effluent. 2. Human liver microsomal fractions incubated with an NADPH-generating system and oxygen formed pyridine N-oxide (PNO) at an average rate of 275 pmol/min per mg protein, 2-pyridone (2PO) at 207 pmol/min per mg and 4-pyridone (4PO) at 154 pmol/min per mg. One human subject formed 3-hydroxypyridine N-oxide in addition to the other metabolites. 3. Human kidney microsomal fractions formed PNO, 2PO and 4PO at rates similar to those with human liver microsomal fractions, whereas human lung microsomal fractions formed the metabolites at less than half the rate. 4. Metabolism of pyridine by human liver microsomal fractions was inhibited 54% by nitrogen, 34% by 80% carbon monoxide-20% oxygen, and 20% by metyrapone. 2-Diethylaminoethyl-2,2-diphenylvalerate HCl (SKF-525A) did not inhibit pyridine metabolism. 5. Liver microsomal fractions from non-induced rats metabolized pyridine to PNO at a rate of 19 pmol/min per mg protein, 2PO at 17 pmol/min per mg and 4PO at 61 pmol/min per mg. 6. There was no pyridine metabolism by human or rat tissue cytosolic fractions incubated under the same conditions.

Preclinical pharmacologic studies of the new antitumor agent carmethizole (NSC-602668) in the mouse and beagle dog.

The chemical breakdown of carmethizole [1-methyl-2-methylthio-4,5-bis-(hydroxymethyl)imidazole-4',5'- bis(N-methylcarbamate)hydrochloride] and its pharmacokinetics in the mouse and beagle dog were studied. Carmethizole was relatively unstable in aqueous media, having a half-life of less than or equal to 1 h in 0.9% sodium chloride, human whole blood, human plasma, and dog urine at 37 degrees C. Its major breakdown product in 0.9% sodium chloride and pH 5.0 sodium phosphate buffer was carmethizole diol. When carmethizole was added to pH 7.0 or pH 9.0 sodium phosphate buffer, the major breakdown product was carmethizole diol-4'-monophosphate. Carmethizole reacted directly with glutathione at pH 8.0, forming a glutathione adduct of carmethizole monocarbamate. Elimination of the drug from the plasma of the beagle dog following i.v. bolus doses of 22.4 and 4.3 mg/kg was biphasic. At these doses the terminal half-life was 39 and 46 min, respectively, and the respective total body clearance was 4.6 and 7.7 ml/min per kg. The 22.4 mg/kg dose was lethal to the beagle dog by day 4. Elimination of carmethizole from the plasma of mice following an i.v. bolus dose of 115 mg/kg was monoexponential, with a half-life of 11.6 min and a total body plasma clearance of 43.6 ml/min per kg. When the drug was infused at 230 mg/kg over 8 h into mice, the total body clearance was 40.8 ml/min per kg. Following the i.v. bolus administration of carmethizole to mice, 30% of the total dose was excreted in urine over 3 h as carmethizole diol, 10%, as carmethizole diol-sulfate, 3.4%, as carmethizole 4'-monocarbamate, and 2.4%, as unchanged drug.

The role of precision in determining the performance of a thyrotropin assay in diagnosing hyperthyroidism.

We examined the relationship between analytical sensitivity, precision at the lower limit of the reference interval, and diagnostic performance in hyperthyroidism for one radioimmunoassay and five immunometric assay kits for thyrotropin. The analytical sensitivity of these kits extended from 0.05 to 1.56 milli-int. units/L. Diagnostic efficiencies of the immunometric assays, in discriminating between euthyroidism and hyperthyroidism, ranged between 93% and 98%. There was a highly significant correlation (r = 0.99, P less than 0.001) between analytical sensitivity and diagnostic efficiency. The between-assay coefficients of variations, at the lower limit of the reference interval, ranged from 26% to 87%. There was no correlation (r = 0.36) between precision, at this concentration, and diagnostic efficiency. We conclude that analytical sensitivity and not precision is the major determinant in controlling the diagnostic performance of a thyrotropin assay in hyperthyroidism.

In vitro cytotoxicity of pyrazine-2-diazohydroxide: specificity for hypoxic cells and effects of microsomal coincubation.

The antitumor drug pyrazine-2-diazohydroxide exhibits cytotoxicity to A204 tumor cells in vitro under acid conditions. The IC50 with a 1 hr drug exposure at pH of 7.4 was 61 micrograms/ml and at pH of 6.0 it was 31 micrograms/ml. It is suggested that the increased cytotoxicity is due to the acid catalyzed formation of a reactive pyrizinyldiazonium ion from pyrazine-2-diazohydroxide. Pyrazine-2-diazohydroxide is also more cytotoxic to A204 cells under hypoxic conditions in the presence of glucose with an IC50 at pH 7.4 of 22 micrograms/ml. The increased cytotoxicity of pyrazine-2-diazohydroxide under acid and hypoxic conditions may favor selective toxicity to solid tumors in vivo. Coincubation with rat hepatic microsomes increased the cytotoxicity of pyrazine-2-diazohydroxide to A204 cells. The effect did not require NADPH and was not due to formation of metabolites. There was an increased rate of degradation of pyrazine-2-diazohydroxide in the presence of microsomes, presumably with formation of the pyrizinyldiazonium ion. The final degradation product 2-hydroxypyrazine was not cytotoxic to A204 cells. The effect of microsomes on pyrazine-2-diazohydroxide cytotoxicity is probably of little in vivo significance.

Disposition and metabolism of the antitumor agent pyrazine-2-diazohydroxide in mouse and beagle dog.

The pharmacokinetics and metabolism of pyrazine-2-diazohydroxide have been studied in the beagle dog and mouse. When pyrazine-2-diazohydroxide was administered to beagle dogs at a dose of 18.6 mg/kg (428 mg/m2) by i.v. bolus, the plasma half-life (t1/2) was 7.3 min, the apparent volume of distribution (Vd) 577 ml/kg, and the total body clearance (Cl) 55 ml/min per kg. In mice given pyrazine-2-diazohydroxide by i.v. bolus at 100 mg/kg (428 mg/m2), the t1/2 was 5.8 min, the Vd 250 ml/kg, and the Cl 30 ml/min per kg. When [2-14C]pyrazine-2-diazohydroxide was infused i.v. to mice at 100 mg/kg over 8 h, the Cl for parent drug was 122 ml/min per kg. The major product formed from pyrazine-2-diazohydroxide was 2-hydroxypyrazine, which accounted for 80% of the total radioactivity in the plasma after a 6-h drug infusion. There were three other metabolites in plasma, two more polar than pyrazine-2-diazohydroxide, which accounted for 7% of the radioactivity, and one less polar, which accounted for 5% of the radioactivity. Following an i.v. bolus dose of [2-14C]pyrazine-2-diazohydroxide, 79% of the radioactivity was excreted in the urine in 24 h, 3% in the feces, and 0.4% in the expired air; 18% remained in the carcass. The liver and kidney showed the highest tissue levels of radioactivity. 2-Hydroxypyrazine accounted for 45% of the urinary radioactivity, pyrazine-2-diazohydroxide for 14%, and a glucuronide or sulfate conjugate of 2-hydroxypyrazine for 17%. Twenty-four percent of the radioactivity eluted near the void volume on high-performance liquid chromatography and was not identified.

Pharmacokinetics and metabolism of the antitumor agent sulfamic acid 1,7-heptanediyl ester (sulfamic acid diester) in the mouse and beagle dog.

The pharmacokinetics and metabolism of sulfamic acid diester were studied in the beagle dog and mouse. Elimination of sulfamic acid diester from the plasma and whole blood following i.v. administration at a dose of 193 mg/m2 was best approximated by a three-compartment model in both species. The compound was relatively rapidly cleared from the plasma, with a plasma beta half-life of 2.3 h and 0.9 h and a gamma half-life of 16 h and 3 h in the dog and the mouse, respectively. Sulfamic acid diester was taken up by blood cells and only slowly eliminated with a whole blood gamma half-life of 42 h in the dog and 32 h in the mouse. When sulfamic acid diester was infused i.v. to mice at 15 mg/kg over 8 h, the clearance for the parent drug was 13.2 ml/min kg from the plasma and 3.3 ml/min kg from the whole blood. Urine collected from mouse and dog contained the parent drug and three metabolic/breakdown products, namely, sulfamic acid 1,7-heptanemonoyl ester, sulfamic acid 3-hydroxyl-1,7-heptanediyl ester, and an unidentified product. Excretion of unchanged drug and products in mouse urine over 8 h accounted for less than 16% of the dose of sulfamic acid diester. Sulfamic acid diester did not react with glutathione in buffer, whole blood, or 100,000 g rat liver cytosol.

Cryopreservation of rat and dog hepatocytes for studies of xenobiotic metabolism and activation.

Isolated human hepatocytes offer a unique way of studying the metabolism and mechanisms of action of drugs and toxic chemicals. Because of the irregular availability of human liver, a way of storing the hepatocytes until they can be conveniently used is required. Using rat and dog isolated hepatocytes, we have developed a procedure for cryopreserving hepatocytes in large numbers such as are needed for metabolism and toxicity studies. Hepatocytes were frozen in medium containing 10% dimethyl sulfoxide using a microcomputer-controlled freezing gradient and stored at -196 degrees C. Upon thawing, the total cell recovery for rat hepatocytes was 67%. Cell viability measured by trypan blue (TB) exclusion was 67%, 7-ethoxycoumarin (7-EOC) dealkylation 33%, and cytochrome P-450 75%, compared to fresh hepatocytes. With cryopreserved dog hepatocytes, the total cell recovery was 75%. TB exclusion was 62%, 7-EOC dealkylation 37%, and cytochrome P-450 68%, compared to fresh hepatocytes. The viability of cryopreserved hepatocyte preparations could be improved by density separation on Percoll giving a TB exclusion for rat hepatocytes of 85%, and 7-EOC dealkylation of 69% compared to fresh hepatocytes, with 67% of the viable cells recovered. Biphenyl was used as a substrate to measure integrated xenobiotic metabolizing activity by the hepatocytes. Total hydroxybiphenyl (OHB) formation, a mixed function oxygenase activity, was maintained in cryopreserved Percoll-treated rat hepatocytes at 86%, OHB glucuronide formation at 85%, and OHB sulfate formation at 20% of the values in fresh hepatocytes. In cryopreserved dog hepatocyte, total OHB formation was 39%, and OHB glucuronide and sulfate formation less than 10% of the values in fresh hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

A high-performance liquid chromatography assay for measuring integrated biphenyl metabolism by intact cells: its use with rat liver and human liver and kidney.

A rapid, sensitive high-performance liquid chromatography assay with fluorescence detection for measuring biphenyl metabolism by intact cells has been developed. The assay does not require organic solvent extraction or enzymatic digestion for the measurement of hydroxybiphenyl conjugates. The lower limit of detectability for 4-hydroxybiphenyl is 5 pmol injected. Rat hepatocytes incubated with biphenyl form predominantly 4-hydroxybiphenyl sulfate with lesser amounts of 4-hydroxybiphenyl glucuronide and free hydroxybiphenyls, and small amounts of 3-hydroxybiphenyl sulfate and 3-hydroxybiphenyl glucuronide. Slices of fresh human liver incubated with biphenyl form predominantly 4-hydroxybiphenyl glucuronide with some free hydroxybiphenyl and small amounts of 4-hydroxybiphenyl sulfate. 4-Hydroxybiphenyl glucuronide formation by human liver shows a lag time that is not abolished by preincubating the liver without substrate. Human kidney slices incubated with biphenyl form 4-hydroxybiphenyl glucuronide and 4-hydroxybiphenyl sulfate at rates less than one-tenth those seen with human liver. Human kidney slices do not form detectable free hydroxybiphenyl. There is wide intersubject variability in the rates of hydroxybiphenyl metabolite formation by human liver and kidney.

Total testosterone, free-androgen index, calculated free testosterone, and free testosterone by analog RIA compared in hirsute women and in otherwise-normal women with altered binding of sex-hormone-binding globulin.

We compared the clinical value of information on free testosterone as measured with the Coat-A-Count (Diagnostic Products Corp.) radioimmunoassay kit involving a ligand analog with that of total testosterone, the free-androgen index, and free testosterone calculated from concentrations of testosterone, sex-hormone-binding globulin, and albumin, in hirsute women, pregnant women, oral-contraceptive users, women with thyroid disease, and epileptic women taking phenytoin. Total testosterone, the free-androgen index, calculated free testosterone, and free testosterone by RIA were increased in 41-68% of hirsute women. Values for free testosterone increased in the first and third trimesters of pregnancy but remained within normal limits in all non-hirsute groups. Total testosterone was increased in patients having increased sex-hormone-binding globulin, whereas the free-androgen index and, to a lesser extent, calculated free testosterone were significantly decreased. Free testosterone measured by analog RIA not only has greater diagnostic efficiency than total testosterone, it also is technically simpler to determine than the free-androgen index and calculated free testosterone.

High-K diets reduce brain haemorrhage and infarcts, death rate and mesenteric arteriolar hypertrophy in stroke-prone spontaneously hypertensive rats.

Male stroke-prone spontaneously hypertensive rats (SHRSP) were fed 4% NaCl diets containing either 0.75% normal K or 2.11% high K, starting at 6 weeks of age. After 8 months on these diets 69% of 58 SHRSP rats on 0.75% K had died, whereas 2% of 95 rats of 2.11% K died, a 98% reduction in mortality, P less than 0.000 001. After 20 weeks the daytime and night-time blood pressure (BP) of each rat were measured intra-arterially. We selected two groups precisely matched for BP. One matched SHRSP group (BP 182 mmHg) ate the 0.75% K diet and 30 of 47 rats died (64% mortality). The other matched SHRSP group (BP 182 mmHg) ate the 2.11% K diet, and two of 35 died (6% mortality, a 91% reduction of mortality, P less than 0.0001). Seemingly, the striking reduction in mortality rate with the 2.11% hig-K diet does not depend on a lowering of BP. High-K diets do not change body Na or K. The dry weight of mesenteric arterioles was reduced by 22% on 2.11% K diet versus 75% K (7.5 versus 9.7 mg) (P less than 0.001), indicating a greatly reduced hypertensive hypertrophy. In nine surviving SHRSP on 0.75% K, 13 of 36 brain hemisphere slides (four slides per rat) showed infarcts (36%). In 11 surviving SHRSP on 2.11% K, one of 44 brain slides showed infarcts (2%, a 94.5% reduction, P less than 0.0001). Brain haemorrhage was reduced by 92% on the 2.11% K diet. High-K diets allow cerebral arteries to carry very high BPs without sustaining damage to the artery wall, thereby drastically reducing brain infarcts and lowering the death rate.

Gas chromatographic assay for the new antitumor agent pyrazine-2-diazohydroxide (diazohydroxide) and its stability in buffer, blood and plasma.

Diazohydroxide is a new antitumor agent being considered for clinical trial. A sensitive and specific assay for diazohydroxide in physiological media, plasma and blood has been developed based on conversion of diazohydroxide to 2-chloropyrazine in the presence of strong hydrochloric acid. The 2-chloropyrazine is extracted into the ethyl acetate and separated by capillary gas chromatography with nitrogen-phosphorus detection. Using 0.2 ml plasma the assay was linear up to 100 micrograms/ml diazohydroxide and had a lower limit of detectability for diazohydroxide of 50 ng/ml. The coefficient of variation of the assay at 1 micrograms/ml was 6.7%. Breakdown of diazohydroxide was rapid under mild acid conditions but slower under alkaline conditions,. The half-life of diazohydroxide in 0.1 M sodium phosphate buffer, pH 6.0, at room temperature was 5 min and at pH 8.0, 480 min. Breakdown of diazohydroxide in plasma was biphasic. In fresh mouse plasma diazohydroxide had a terminal half-life at 37 degrees C of 72 min while in fresh human plasma the terminal half-life was 23 min and in fresh blood 21 min. Diazohydroxide accumulated in red blood cells at 37 degrees C to a concentration 68% above the concentration in plasma. Diazohydroxide was 49% bound to human plasma proteins at room temperature.

Discriminative ability of tests for free and total thyroid hormones in diagnosing thyroid disease.

We assessed the sensitivity, specificity, predictive value of a positive result, and efficiency of tests for total thyroxin, free thyroxin index, free thyroxin, total triiodothyronine, free triiodothyronine index, and free triiodothyronine in serum from 1619 consecutive new patients with suspected thyroid dysfunction. Multivariate discriminant analysis was also used. Free thyroxin index and free thyroxin were clearly the most sensitive indicators of hypothyroidism. In contrast, all of these tests identified hyperthyroidism with similar efficiencies. By stepwise discriminant analysis, the free thyroxin index was the most efficient test for distinguishing between euthyroidism and hyperthyroidism and between euthyroidism and hypothyroidism. The combination of tests for total thyroxin, free thyroxin index, triiodothyronine, and free triiodothyronine was optimal for separating euthyroidism, hyperthyroidism, and hypothyroidism. We conclude that the free thyroxin index, despite the introduction of newer technologies, is still the best thyroid hormone test for screening for thyroid disease.

Estimation of free thyroid hormone concentrations in the clinical laboratory.

A multiplicity of tests have come into routine use for the estimation of the concentrations of free thyroid hormones in serum and plasma since the introduction, almost three decades ago, of the hypothesis that only the free thyroid hormone is metabolically active. Most of these tests give estimates of considerable diagnostic value, although results may be unreliable in some clinical situations. This review outlines the methods currently available for estimating free thyroid hormones and highlights the extrathyroidal and physiological factors influencing the results of such tests, particularly for the analog-based techniques.

Laboratory and clinical experience with a monoclonal antibody-based radioimmunoassay for serum total thyroxine.

The performance characteristics and diagnostic value of a monoclonal antibody-based radioimmunoassay for serum total thyroxine (Mallinckrodt) are described. Between-batch precision (coefficient of variation) was 10.4% at 87 nmol/l and 3.3% at 185 nmol/l. Scatchard analysis revealed a linear plot with a Ka of 5.4 X 10(8) l/mol. Sensitivity was 4.5 nmol/l of thyroxine. An association study showed that the assay reached equilibrium well within the specified incubation time. Cross-reaction of triiodothyronine and reverse triiodothyronine in the assay was 0.6% and 25.0% respectively. Analytical recovery was 91-110%. Linearity was well demonstrated but dilutions of a high concentration of thyroxine in serum did not parallel the standard curve. The correlation coefficient for comparison with a polyclonal antibody assay was 0.95 for 83 patients. The diagnostic accuracy of the monoclonal antibody assay was adequate for most patients with thyroid disease, pregnant women, oral contraceptive users and subjects on thyroxine-replacement therapy. Measurement of total thyroxine by a monoclonal antibody-based method shows no definite advantage over the conventional polyclonal antibody assay.

PIP: The performance characteristics and diagnostic value of a monoclonal antibody-based radioimmunoassay for serum total thyroxine (Mallinckrodt) are described. Between-batch precision (coefficient of variation) was 10.4% at 87 nmol-l and 3.3% at 185 nmol/l. Scatchard analysis revealed a linear plot with a Ka of 5.4 x 108 1/mol. Sensitivity was 4.5 nmol/l of thyroxine. An association study showed that the assay reached equilibrium well within the specified incubation time. Cross-reaction of triiodothyronine and reverse triiodothyronine in the assay was 0.6% and 25.0% respectively. Analytical recovery was 91 -- 110%. Linearity was well demonstrated but dilutions of a high concentration of thyroxine in serum did not parallel the standard curve. The correlation coefficient for comparison with a polyclonal antibody assay was 0.95 for 83 patients. The diagnostic accuracy of the monoclonal antibody assay was adequate for most patients with thyroid disease, pregnant women, oral contraceptive users and subjects on thyroxine-replacement therapy. Measurement of total thyroxine by a monoclonal antibody-based method shows no definite advantage over the conventional polyclonal antibody assay. (author's)

Study of a mechanism responsible for anomalous results for free thyroxin by some analog assays for subjects with albumin deficiency.

I examined the effect of albumin deficiency, as might occur in pregnancy and nonthyroidal illness, on four analog-based assays for free thyroxin (Amersham's Amerlex, Diagnostic Products Corporation's Coat-A-Count "one-step," Clinical Assays' Gammacoat "one-step," and Corning's Immophase "single-step"). In subjects with albumin deficiency per se, Amerlex and Coat-A-Count results for free thyroxin were not significantly different from normal, whereas free thyroxin values by Gammacoat and Immophase assays were lower than that of the control. Furthermore, in the albumin-deficient group, the mean amount of thyroxin associated with antisera in each assay was significantly higher than that of the control and correlated well with the degree of decrease in free thyroxin. I conclude that subnormal results for thyroxin by analog assays, in euthyroid patients with albumin deficiency, is a function of the specific assays and is not a characteristic of analog assays per se. In addition, the study suggests that albumin deficiency alters the distribution between thyroxin and its binders in all four analog assays, but only in the Gammacoat and Immophase methods is this alteration sufficiently large as to distort assay results.

The evaluation of five commercial serum cortisol kits regarding precision and accuracy.

We evaluated Amerlex (Amersham International), Corti-Shure (Nuclear Medical Laboratories), Gammacoat (Clinical Assays), Coat-A-Count and DPC-direct (Diagnostic Products Corporation) radioimmunoassay kits for determination of cortisol in sera. The between- and within-batch precision (coefficient of variation) ranged between 4.4-12.5% and 3.0-10.9% respectively. No one kit exhibited a clearly better precision throughout the assay range. All kits displayed good sensitivity and parallelism. Cortisol concentrations determined by kit methods, in two assayed control sera, were 3-14% higher than by gas chromatography/mass spectrometry. Coat-A-Count and Gammacoat were technically the simplest assays while Corti-Shure and DPC-direct kits had the best designed standard curves over the diagnostic range. The Amerlex kit had the greatest sensitivity, showed least shift in the dose-response curve between assay and was the only assay that came to equilibrium within the specified incubation time.

Performance characteristics and diagnostic value (vs other tests) of two radioimmunoassay kits for estimating free triiodothyronine in serum.

We compared the clinical value of "Amerlex" (Amersham International) and "Pre-release Coat-A-Count" (Diagnostic Products Corp.) radioimmunoassay kits for free triiodothyronine with that of triiodothyronine, triiodothyronine/thyroxin-binding globulin ratio, and free triiodothyronine index in patients with thyroid disease, pregnant women, oral contraceptive users, thyroxin-binding globulin-deficient subjects, and patients receiving phenytoin. Assay of free triiodothyronine in hyperthyroidism provided diagnostic information similar to that from the indirect indices, whereas the free triiodothyronine index was the most sensitive index of hypothyroidism. With respect to diagnostic value, free triiodothyronine assay was comparable with the free triiodothyronine index and superior to triiodothyronine assay and the triiodothyronine/thyroxin-binding globulin ratio in correcting for alterations in thyroid-hormone binding capacity. In neither kit for free triiodothyronine was the equilibrium between bound and free hormone significantly disturbed during assay. Overall, as a diagnostic index of thyroid status, free triiodothyronine is as good as the free triiodothyronine index and better than either triiodothyronine or the triiodothyronine/thyroxin-binding globulin ratio.

Studies on the accuracy of a quantitative kit radioimmunoassay for free thyroxine.

Experiments designed to determine the accuracy of the Corning Immophase Free T4 assay revealed that there was a marked perturbation of thyroxine binding to the serum proteins during the assay; binding of 125I-T4 to the immobilised T4 antibody in the absence of merthiolate increased considerably with buffer dilution of serum; and the free thyroxine concentration declined significantly during pregnancy. These changes in measured fT4 were not observed with an equilibrium dialysis-radioimmunoassay procedure. The assay was precise, easily performed, and as effective as the free thyroxine index (FTI) in diagnosing thyroid disease. We conclude that the assay does not provide an accurate quantitative estimation of serum fT4 concentration in samples with elevated TBG concentration and that the kinetic principles on which the assay is based are altered as serum binding-protein concentration is reduced (serum dilution). Both FTI and fT4 (Corning) data in pregnant patients should be interpreted with caution and with reference to clinical symptomatology and other thyroid function tests.