Enzyme reagents from unusual sources.

Although microbes are frequent sources of clinically useful enzymes, there are certain biocatalysts which appear to be enriched in, or more naturally available from nonmicrobes. This paper highlights several of these enzyme reagents, and illustrates their potential or actual clinical diagnostic uses. These reagents include: an enzyme extract containing both glucose oxidase and mutarotase activities from peppers; a bilirubin-degrading enzyme from orange peels; a gentisic acid metabolizing activity from onions; and enzymes from snails which catalyze the detoxification of cyanide by divergent mechanisms.

Delta and conjugated bilirubin as complementary markers of early rejection in liver-transplant recipients.

For 51 liver allograft recipients, we evaluated whether serum profiles of delta (Bd) and conjugated bilirubins (Bc) could be used to diagnose rejection during the first 30-50 postoperative days, in comparison with histology as the "gold standard." Daily measurements of aspartate aminotransferase, alkaline phosphatase, total bilirubin, Bd, and Bc were made, the last two by liquid chromatography. In 34 patients without any biochemical or histological evidence of rejection, within seven to 10 postoperative days Bd increased to greater than 40-50% of total bilirubin, while Bc decreased to less than 10%.

New prostaglandin synthetase inhibitors--di-and triphenylacrylonitriles.

16 triphenylacrylonitriles (TPE) or diphenylacrylonitriles (DPE) were synthesized by condensation of various benzophenones or benzaldehydes with various phenylacetonitriles. The pharmacological potency of these compounds was studied by incubation of bovine seminal vesicle microsomes and PG-RIA. The results showed that the potency of inhibition of PG biosynthetase of DPE was stronger than that of TPE.

Effect of oxygen tension on the anti-oxidant enzyme activities of tetralogy of Fallot ventricular myocytes.

Since the chronically cyanotic tetralogy of Fallot (TOF) myocardium is more sensitive to reperfusion injury after cardiac surgery than the adult myocardium, we decided to study the regulation of myocardial superoxide dismutase (SOD), catalase and glutathione peroxidase by oxygen tension. TOF myocytes were cultured at a Po2 of 150 mmHg for 30 days to establish the culture. The cells were then cultured at Po2 of 150 and 40 mmHg and the myocyte antioxidant enzymes measured at days 3, 7, 14 and 21.

The quantitation of human ventricular myosin light chain 1 in serum after myocardial necrosis and infarction.

To determine the clinical utility of human ventricular myosin light chain 1 (HVLC1) in the diagnosis of myocardial necrosis, we established a radioimmunoassay for serum HVLC1 using polyclonal antibodies raised against the purified human protein. HVLC1 levels were measured in sera from 110 control patients and 38 patients immediately after cardiovascular surgery and in serial specimens from 10 patients with uncomplicated myocardial infarctions. The HVLC1 assay was found to be equal in sensitivity for the first 48 hours to the CK-MB for myocardial necrosis after cardiovascular surgery and for myocardial infarction.

Myocardial salvage with trolox and ascorbic acid for an acute evolving infarction.

Both Trolox (a water-soluble analogue of alpha-tocopherol) and ascorbic acid were more effective than superoxide dismutase or catalase in protecting myocyte cell cultures from free radical attack (induced by hypoxanthine and xanthine oxidase). In a canine model of two hours of left anterior descending coronary artery occlusion followed by four hours of reperfusion, Trolox and ascorbic acid reduced the area of infarction within the area at risk. The Trolox group received 500 mL of deoxygenated saline solution containing 2.

A new bilirubin-degrading enzyme from orange peels.

A novel enzyme activity that catalyzes the degradation of unconjugated bilirubin (Bu) has been demonstrated in extracts of the peels of edible oranges. Unlike the few known bilirubin-oxidizing enzymes, the orange enzyme does not produce biliverdin as a product, does not seem to require oxygen, and has a unique absorption spectrum of its products. Even at the crude stage, the enzyme has a specific activity that is 10 and 20 times higher, respectively, than those reported for the crude or partially purified Bu-degrading enzymes from mushrooms and rat liver.

Delta bilirubin: absorption spectra, molar absorptivity, and reactivity in the diazo reaction.

Delta bilirubin (B delta), isolated from serum, has an absorption maximum near 440 nm and a molar absorptivity of 72,000 L mol-1cm-1 in either Tris HCl (0.1 mol/L, pH 8.5) or phosphate (0.

A multilayer element for determining hemoglobin in whole blood: principles and analytical performance.

We described the structure and analytical performance of a thin-film element for determining total hemoglobin in whole blood. The element can be used with the Kodak Ektachem DT60 analyzer. The method, analogous to the standard Drabkin's method, provides accurate and precise results on 10-microL samples of undiluted whole blood.

Chemical nature of a synthetic bilirubin conjugate and its reactivities in the total and direct reactions by the Jendrassik-Gróf method.

Using liquid chromatography, nuclear magnetic resonance spectroscopy, and elemental analyses, we verified that a commercially available synthetic bilirubin conjugate is predominantly a ditaurobilirubin (DTB) disodium salt. The Jendrassik-Gróf total bilirubin (TBIL) method quantitatively measures unconjugated bilirubin (Bu) and the Bu-equivalent content in DTB, from which we infer that the azopigments of Bu and DTB have identical molar absorptivities, which do not change in the presence of either serum or serum albumin of human or bovine origin. However, based on the ratio of direct bilirubin (DBIL) to TBIL, the DBIL reaction in dilute HCI is incomplete (even up to 20 min), with lower yields in a matrix of bovine serum albumin than in human serum.

Inaccurate values for direct bilirubin with some commonly used direct bilirubin procedures.

We compared five methods for the determination of total and direct bilirubins in serum samples from normal controls, subjects with Gilbert's syndrome, and serum pools containing about 50 and 150 mg of total bilirubin per liter. The Kodak Ektachem method and a diazotized sulfanilic acid method with 0.15 mmol/L sodium nitrite concentrations are the only methods that gave accurate direct bilirubin values, as judged by liquid-chromatographic results.

An anion-exchange chromatographic method for measuring bilirubin covalently bound to albumin.

After alkalinizing the diazo products of serum bilirubins, we apply them to a column of anion-exchange resin. The azodipyrroles derived from unconjugated and sugar-conjugated bilirubins, as well as from one half of the tetrapyrrole covalently linked to albumin ("Bil-Alb"), are anionic and protein-free and adsorb to the resin. The other half of the azo product of Bil-Alb, with absorptivity similar to that of the protein-free azodipyrroles, remains attached to albumin (Clin Chem 28:629-637, 1982) and passes through the resin unadsorbed.

Origin of mammalian biliprotein and rearrangement of bilirubin glucuronides in vivo in the rat.

In hepatobiliary disease bilirubin becomes bound covalently to serum albumin, producing a nondissociable bile pigment-protein complex (biliprotein). To elucidate the mechanism of biliprotein formation we studied the bile pigment composition of blood from animals with experimental cholestasis and carried out comparative studies on the rate of biliprotein formation in vivo and in vitro during incubation of bilirubin glucuronides with albumin. Bile duct ligation in the rat and guinea pig led to rapid accumulation in the circulation of bilirubin, heterogeneous bilirubin esters of glucuronic acid, and a biliprotein that migrated along with albumin on high performance liquid chromatography.

Estimation of unconjugated, conjugated, and "delta" bilirubin fractions in serum by use of two coated thin films.

We used two coated thin films to measure the concentrations of unconjugated, conjugated, and total bilirubin as well as bilirubin covalently bound to albumin ("delta" bilirubin) in more than 400 serum samples. We measured the unconjugated and conjugated species by determining their reflection densities at two wavelengths (400 and 460 nm) on a coating designed for the enhanced spectral measurement of bilirubin but which does not register the delta form. Total bilirubin was measured by use of a diazo-based thin film (Clin Chem 29: 37-41, 1983).

Diazo-based assay for total bilirubin in a coated thin film evaluated.

We compared results for total bilirubin as measured on a coated thin film and by the Evelyn-Malloy and Jendrassik-Gróf methods. We examined serum samples from patients and studied the effects of protein, hemoglobin, and lipids on bilirubin measurement. Results from the thin-film assay compared favorably with those of the other methods.

The Ektachem clinical chemistry slide for simultaneous determination of unconjugated and sugar-conjugated bilirubin.

Using dual-wavelength spectrometry, we have refined the mordant-based bilirubin slide method (Clin Chem 28:2366-2372, 1982) to co-detect unconjugated bilirubin (Bu) and its sugar conjugates (Bc) in 10 microL of serum. The assay is based on three principles: (a) mono- and diconjugated bilirubins behave spectrally like one fraction (Bc) when bound to the mordant, (b) Bu and Bc are spectrally distinct, and (c) B delta (the bilirubin-albumin complex) is not measured in the film. With known bilirubin mixtures, results by the assay agree with those by nuclear magnetic resonance, by a Jendrassik-Gróf method for total bilirubin, and by a liquid-chromatographic procedure.

Bilirubin analysis--the state of the art and future prospects.

Progress in separating and identifying different bile pigments in serum has led to the recognition of a bilirubin fraction (delta) distinct from unconjugated bilirubin and its (mono- and di-) sugar conjugates. Delta bilirubin reacts directly diazo-positive and is strongly linked to an albumin-like protein, presumably via an amide bond between a propionic acid side-chain of the tetrapyrrole and a functional group (e.g.

A diazo-based dry film for determination of total bilirubin in serum.

We have prepared a diazo-based dry film for use in determining total serum bilirubin. On a transparent support are a buffered gelatin layer containing a polymeric quaternary amine (the mordant) and a white, reflective spreading layer that contains all of the components necessary for detection of bilirubin. The method is based on the use of dyphylline and Triton X-100 surfactant to dissociate bilirubin from albumin and subsequent reaction of bilirubin with a diazonium salt [4-(N-carboxymethylsulfamyl)benzenediazonium hexafluorophosphate].

Assessment of the fundamental accuracy of the Jendrassik-Gróf total and direct bilirubin assays.

Using unconjugated bilirubin (Bu) and authentic human disconjugated bilirubin (dBc) supplemented in low-bilirubin serum pools, we have directly verified for the first time that the Jendrassik-Gróf total bilirubin assay modified after Doumas et al. (Clin. Chem.

Biliprotein in adult icteric serum--demonstrated by extension of the alkaline methanolysis procedure.

We confirmed that the alkaline methanolysis procedure of Blanckaert (Biochem. J. 185: 115-128, 1980) converts the sugar conjugates of bilirubin (Bc) into their corresponding methyl and dimethyl esters, which can be extracted into chloroform along with underivatized unconjugated bilirubin (Bu).

The Kodak Ektachem clinical chemistry slide for measurement of bilirubin in newborns: principles and performance.

In this slide, unconjugated bilirubin and its sugar conjugates interact with a cationic polymeric mordant to form spectrally enhanced complexes having similar absorptivities at approximately 400 nm. With reflection densitometry and appropriate mathematical transformation, readings at this wavelength are linearly related to bilirubin concentrations up to 260 mg/L. The slide requires 10 microL of serum, is precise (total CV less than 2% determined over 20 days for the analyte range 39-184 mg/L), gives results that correlate well with the Doumas et al.

Dry film for the enzymic determination of total cholesterol in serum.

We have prepared a dry film for the enzymic determination of total serum cholesterol. It consists of a transparent support bearing a buffered gelatin layer, and a white reflective spreading layer that contains all of the necessary components for the detection of cholesterol. The method is based on (a) hydrolysis of cholesterol esters to cholesterol by cholesterol ester hydrolase (EC 3.

Isolation and preliminary characterization of a fraction of bilirubin in serum that is firmly bound to protein.

We have isolated from pathological sera a bilirubin fraction (delta) that is very tightly, if not covalently, bound to protein, most likely albumin. This delta fraction absorbed at a lambda max of 433 nm in the visible spectrum, between the lambda max of unconjugated (alpha) and that of conjugated (Bc) bilirubin when measured in solutions containing albumin. However, unlike the other bilirubin species, this fraction could not be separated from the proteins in serum by exhaustive ultrafiltration in the presence of caffeine/benzoate solution.