Cytosolic modulators of activities of microsomal enzymes of cholesterol biosynthesis. Effects of Acyl-CoA inhibition and cytosolic Z-protein.

Physiological concentrations of long-chain fatty acyl-CoAs have now been shown to inhibit microsomal methyl sterol oxidase. Acyl-CoA inhibition of hydroxymethylglutaryl-CoA reductase as well as methyl sterol oxidase can be either prevented or reversed by the addition of purified Z-protein (fatty acid-binding protein). Concomitantly, Z-protein addition decreases the extent of binding of radioactively labeled oleoyl-CoA to microsomal membranes. Free heme also inhibits hydroxymethylglutaryl-CoA reductase, and Z-protein reverses the extent of observed inhibition by binding heme analogous to the effect observed with acyl-CoAs. Similarly, Z-protein reverses substrate inhibition of acyl-CoA:cholesterol acyltransferase at high concentrations of acyl-CoA substrate. All these observations are consistent with the suggestion that, by binding acyl-CoAs and other enzyme effectors such as free heme, Z-protein modulates the effects of fluctuations of concentrations of major cellular metabolites. Furthermore, because the concentration of Z-protein is very low in rapidly growing hepatomas, such tumors may be very poorly buffered against the effects of acyl-CoAs, free fatty acids, heme and other effectors that may vary markedly by either altered metabolism or release of metabolites from necrotic tumor tissue.

Duration of positive urine for cocaine metabolite after ophthalmic administration: implications for testing patients with suspected Horner syndrome using ophthalmic cocaine.

PURPOSE: To determine the duration of positive urine for benzoylecgonine, the major metabolite excreted in the urine, after topical ophthalmic administration of cocaine as one would perform for testing the presence of Horner syndrome. METHODS: Two drops of cocaine 10% were applied to each eye of 50 normal subjects. Urine samples were collected 4 to 6, 24, 48, 72, and 96 hours later. Each sample was assayed for benzoylecgonine using a screening competitive enzyme donor immunoassay followed by a highly specific and sensitive confirmatory gas chromatography-mass spectrometry assay. We employed assays and cutoff levels that fulfilled guidelines required by the Substance Abuse and Mental Health Services of the United States Department of Health and Human Services to mimic measures that exist for federal workplace drug testing. RESULTS: Of the 50 subjects, there were 25 women and 25 men, ranging in age from 19 to 59 years (median age, 40 years). Positive screening samples were obtained in 47 of 50 subjects (94%) 4 to 6 hours, 35 of 50 (70%) 24 hours, and 1 of 50 (2%) 48 hours after topical application of cocaine. None of the samples tested positive at 72 hours or beyond. Using the confirmatory assay's cutoff as the gold standard for a positive test, the sensitivity of the screening assay was 96% 4 to 6 hours, 90% 24 hours, and 14% 48 hours after topical application. Urine benzoylecgonine concentration was negatively correlated with body mass index and positively correlated with urine creatinine concentration. CONCLUSIONS: Patients should be informed that their urine may test positive for cocaine, if assayed according to US federal guidelines and using the protocol employed in this study, up to 2 days after undergoing testing for Horner syndrome.

A closer look at acetyl and pentafluoropropionyl derivatives for quantitative analysis of morphine and codeine by gas chromatography/mass spectrometry.

PFPA and acetic anhydride derivatives of morphine and codeine were evaluated with respect to stability, chromatography, potential for analytical interferences by other opiates, and suitability of major fragment ions for analysis by GC/MS with deuterated internal standards and selected ion monitoring (SIM). The PFPA derivatives showed acceptable stability and could be analyzed without interference from other opiates, but the codeine derivative had relatively poor chromatography and its mass spectrum had only two ions suitable for SIM. The acetic anhydride derivatives were stable and chromatographed well, but diacetyl hydromorphone enol, a minor product of derivatization of hydromorphone, interfered with analysis of morphine. 3-Monoacetylmorphine, a minor product of derivatization of morphine, prevented use of the abundant m/z 285 ion of derivatized D3-codeine as a qualifying ion in quantitative assays. The acetic anhydride derivative of morphine cannot be distinguished from the corresponding derivative of the heroin metabolite 6-monoacetylmorphine.

Heterogeneity of lipoprotein Lp(a) and apolipoprotein(a).

We have purified Lp(a) lipoproteins from sera of four subjects by ultracentrifugation, selective precipitation, and chromatofocusing. Each subject had two forms of serum Lp(a) that were separable by chromatofocusing. We purified apolipoprotein (a) [apo(a)] from the eight isolated Lp(a)s and obtained only one form of apo(a) from each subject. The four apo(a)s seen on sodium dodecyl sulfate-polyacrylamide gel electrophoresis had different apparent molecular masses, ranging from 275 to 440 kDa. Chemical deglycosylation of the smallest apo(a) yielded a 235 kDa protein, which may be a core protein structure common to all apo(a)s. We conclude that there are many forms of serum Lp(a) and apo(a). The heterogeneity of serum Lp(a) particles can be ascribed in part to differences in size of apo(a), but other factors must account for the existence within a single patient of different Lp(a)s that contain apparently identical apo(a). One must consider the heterogeneity of Lp(a) when designing assays for this lipoprotein.

Total enzymic synthesis of cholesterol from lanosterol. Cytochrome b5-dependence of 4-methyl sterol oxidase.

Methyl sterol oxidase of microsomal synthesis of cholesterol from lanosterol is a mixed-function oxidase that is dependent upon reduced pyridine nucleotide. The methyl sterol oxidase, as well as NADH-cytochrome c reductase, in intact rat liver microsomes are inhibited by anti-cytochrome b5 immunoglobulin, but NADPH-cytochrome c reductase is not affected. There is a decreased time lag prior to onset of reoxidation of steady state levels of reduced cytochrome b5 when 4-methyl sterol oxidase substrates are present. Trypsin treatment of microsomes destroys cytochrome b5 with loss of methyl sterol oxidase activity. Activity is restored by addition of purified cytochrome b5 to trypsin-treated microsomes. Initial attempts to solubilize and purify 4-methyl sterol oxidase have been only partially successful due to the extreme lability of the oxidase. However, DEAE-cellulose column chromatography of a detergent extract of microsomes yields a fraction that contains the oxidase, lipids, and NADH-cytochrome b5 reductase but is free of cytochrome b5. Oxidation of 4 alpha [30-3H] methyl-5 alpha-cholest-7-en-3 beta-ol by methyl sterol oxidase in this isolated fraction can be fully restored by the addition of purified liver microsomal cytochrome b5. These results strongly support the suggestion that membrane-bound cytochrome b5 of rat liver microsomes is an obligatory electron carrier from NADH to 4-methyl sterol oxidase.

The Ames Clinitek 200/Multistix 9 urinalysis method compared with manual and microscopic methods.

We assayed 315 urine specimens by the Ames Clinitek 200/Multistix 9 semi-automated method and by corresponding standard methods (listed in parentheses) for the following analytes: protein (sulfosalicylic acid precipitation), glucose (Lilly Tes-Tape), ketones (Boehringer Mannheim Chemstrip K), leukocyte esterase (microscopic examination for leukocytes), blood (microscopic examination for erythrocytes), nitrite (microscopic examination for bacteria, Gram stain, or culture), and pH (pH meter). The Clinitek and standard methods agreed well at all concentrations for protein, ketones, and glucose. The Clinitek leukocyte esterase, nitrite, and blood methods were less sensitive than microscopic methods for detection of pyuria, bacteriuria, and hematuria, respectively. The Clinitek pH method produced falsely low pH values for urines with true pH less than 6.5, and falsely high values for urines with true pH greater than 6.5.

Melanogenuria: laboratory evaluation of the qualitative Thormählen and ferric chloride tests and their clinical utility.

Malignant melanoma, a disease that is increasing in occurrence and medical concern, is characterized by the excretion of melanogens. Two qualitative tests are recommended for melanogen detection, the Thormählen test and the ferric chloride test. We evaluated the laboratory and clinical performance of these tests by subsequently re-evaluating 201 urine samples that had been submitted for routine melanogen analysis. We used (a) Thormählen, (b) ferric chloride, (c) small-scale thin-layer chromatography, and (d) spectrophotometry. Nearly 30% of Thormählen test results were equivocal. The ferric chloride test was of no value in itself or in categorizing equivocal Thormählen results as positive or negative. The small-scale chromatography was irreproducible. Prompt scanning of the Thormählen reaction product was helpful in classifying equivocal results. History review of 121 histopathologically diagnosed melanoma patients indicated that these qualitative assays were of no clinical value in the diagnosis or monitoring of melanoma patients.

Measurement of low-density-lipoprotein cholesterol in serum: a status report.

Current recommendations of the Adult Treatment Panel and the Children and Adolescents Treatment Panel of the National Cholesterol Education Program make the concentration of low-density lipoproteins cholesterol (LDL-C) in serum the basis for the classification and treatment of hypercholesterolemia. Numerous methodologies for the determination of serum LDL-C concentrations, in research and clinical laboratories, have been described. Here, we review the principles, performance, and limitations of major current methodologies for determining LDL-C concentrations. These methods include sequential and density-gradient ultracentrifugation, chromatographic and electrophoretic techniques, and precipitation methods. In addition, the advantages and disadvantages of estimating LDL-C concentration by the Friedewald equation, the most commonly used approach in clinical laboratories, are addressed.