Decreased serum 19-nor-deoxycorticosterone (21-hydroxy-19-nor-4-pregnene-3, 20-dione) level in adrenal regeneration hypertensive rats

An enzyme immunoassay of 19-nor-deoxycorticosterone in rat serum was established. The normal value of 19-nor-DOC in rat serum obtained from 9:00 am to 10:00 am was 148 +/- 30 ng/dl (mean +/- SE,n = 10). Serum levels of this steroid decreased in rats with adrenal regeneration hypertension during the course of the experiment up to 8 weeks, while systolic blood pressure rose progressively. We concluded that this mineralocorticoid is not involved at least as a circulating hormone in the pathogenesis of adrenal regeneration hypertension in rats.     

Comparative studies on the detection of mycotoxins in barley using enzyme immunoassays

Barley samples (n = 128) from Central Canada (Manitoba) of the 1993 and 1994 crop years were analysed using enzyme immunoassays for the detection of the mycotoxins deoxy-nivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, and zearalenone. For this study enzyme immunoassays, which were developed at the Lehrstuhl für Hygiene und Technologie der Milch as well as a commercial testkit (Ridascreen® DON, specific for deoxynivalenol and its acetylated derivatives) were used. In addition, some samples were analysed by using a combination of HPLC and enzyme immunoassay (immunochromatography). Results obtained by the enzyme immunoassays for these samples were compared with other analytical data on mycotoxin contamination (analysed by GC/MS) and with the mycological conditions, respectively.     

Selective 19-hydroxylase inhibition by an aromatase inhibitor, 4-hydroxyandrostenedione

19-Nor-deoxycorticosterone is a newly recognized mineralocorticoid which has been associated with some forms of genetic, experimental, and human hypertension. To further examine this relationship, specific inhibitors of 19-nor-deoxycorticosterone biosynthesis must be developed. Since 19-hydroxylation is the pivotal step in both 19-nor-deoxycorticosterone biosynthesis and aromatization of androgens to estrogens, we evaluated an aromatase inhibitor, 4-hydroxyandrost-4-ene-3,17-dione on the inhibition of 19-hydroxylation in both rat and human adrenal mitochondria in vitro and 19-nor-deoxycorticosterone production and blood pressure in spontaneously hypertensive rats in vivo. Adrenal mitochondria from 48 male Sprague-Dawley rats and 1 patient with an aldosterone-producing adenoma were incubated in the presence of deoxycorticosterone substrate both with and without 4-hydroxyandrost-4-ene-3,17-dione. 4-Hydroxyandrost-4-ene-3,17-dione produced significant inhibition of 19-hydroxy-deoxycorticosterone production in both rat and human adrenal mitochondria, with a smaller and not significant inhibition of corticosterone and 18-hydroxy-corticosterone. 4-Hydroxyandrost-4-ene-3,17-dione given subcutaneously to spontaneously hypertensive rats lowered 19-nor-deoxycorticosterone by 69% and completely abolished hypertension compared to Wistar-Kyoto controls. These data demonstrate that 4-hydroxyandrost-4-ene-3,17-dione is a specific inhibitor of 19-hydroxylase, that it lowers 19-nor-deoxycorticosterone production and prevents hypertension in the spontaneously hypertensive rat. These studies reinforce the possible pathogenic significance of 19-nor-deoxycorticosterone in hypertension in spontaneously hypertensive rats.     

Analysis of 6-keto PGF1 alpha, 5-HETE, and LTC4 in rat lung: comparison of GM/MS, RIA, and EIA

The recent availability of fast and sensitive radioimmunoassay (RIA) and enzyme immunoassay (EIA) procedures to measure icosanoids has led to utilization of these techniques by many investigators. A major concern has been that techniques based on immunoreactivity may lack specificity, in particular if complex biologic fluids or tissue extracts are evaluated. The purpose of this investigation was the comparison of icosanoid measurements obtained either with EIA or RIA with those obtained by gas chromatography/mass spectrometry (GC/MS). Rats were injected with Salmonella enteritidis endotoxin, killed at various times after the injection and the lung extract assayed for 6-keto-PGF1 alpha, 5-HETE and LTC4. By EIA lung tissue was found to contain large quantities of 6-keto-PGF1 alpha after endotoxin stimulation. Comparisons made between EIA and GC/MS analysis showed good correlation between 6-keto-PGF1 alpha amounts in lung as determined by each technique. It was also determined that little purification of lung extract was needed to obtain reliable quantitation of 6-keto-PGF1 alpha, probably due to the specificity of the antibody and the large quantity of this prostaglandin produced. Crudely purified (Sep-Pak) lung extracts gave 5-HETE levels by RIA which were highly correlated with GC/MS values, but RIA values were 70% higher than those obtained by GC/MS. The presence of other components in lung extract which cross react with this 5-HETE antibody was probably responsible for the higher values obtained by RIA. LTC4 was measured by immunoassay in crude lung extracts, as well as after Sep-Pak purification and HPLC purification. LTC4 levels were identical in unpurified lung extract and after Sep-Pak purification, but decreased substantially after HPLC purification. Thus, by validating the icosanoid immunoassays, we have found that they can give accurate and reproducible results in lung tissue, although LTC4 and 5-HETE must be purified prior to analysis.     

Renal 21-hydroxylation of 19-hydroxy-progesterone to 19-hydroxy-deoxycorticosterone

19-Nor-deoxycorticosterone (19-nor-DOC) is a mineralocorticoid present in both rat and human urine, and it is elevated in some forms of experimental and human hypertension. Although the exact steps in the biosynthesis of 19-nor-DOC are uncertain, it is probably produced from a 19-oxygenated derivative of DOC, which undergoes 19-desmolation in the kidney. Since DOC biosynthesis is partly due to renal 21-hydroxylation of progesterone (Prog), we sought to determine whether a parallel pathway could exist for the biosynthesis of 19-hydroxy-DOC, a precursor to 19-nor-DOC. In order to test this hypothesis, authentic 19-hydroxy-progesterone was incubated with homogenized renal tissues from either rat or human sources. Formation of 19-hydroxy-DOC was found to be the major metabolite in both rat and human incubations, as demonstrated by an HPLC retention time identical to authentic 19-hydroxy-DOC. 19-Hydroxy-DOC formation was further verified by GC/MS analysis with highly sensitive selected ion recording. Since it has been demonstrated that the placenta can convert progesterone to 19-hydroxy-progesterone, the renal 21-hydroxylation of 19-hydroxy-progesterone to 19-hydroxy-DOC could be an alternate pathway of 19-nor-DOC production especially during pregnancy.     

Enzyme immunoassay of serum testosterone.

An enzyme immunoassay of serum testosterone using the testosterone-glucoamylase complex is described. Testosterone was estimated by the enzyme immunoassay after extraction with hexan: ether (4:1) for serum from men and additional thin layer chromatographic step for serum from women. The within and between assay errors, measured as the coefficient of variation were 11.1 percent (n=8) and 12.0 percent (n=12). The sensitivity of this assay was 0.25 ng. The mean testosterone concentration (+/- SD) in 19 normal men and 4 normal cycling women were 5.3 +/- 1.8 and 0.52 +/- 0.12 ng/ml, respectively. The level of testosterone found by the present assay compared favorably with those obtained by other methods.     

Superiority of gas chromatography/tandem mass spectrometry assay (GC/MS/MS) for estradiol for monitoring of aromatase inhibitor therapy

Currently available radioimmunoassay methods for estradiol in serum lack sufficient sensitivity and precision to monitor estradiol levels in patients placed on third generation aromatase inhibitors. We recently validated a gas chromatography/tandem mass spectrometry assay (GC/MS/MS) for estradiol and determined estrogen levels in normal post-menopausal women and in women with breast cancer before and during administration of aromatase inhibitors. Validation of the GC/MS/MS assay in human plasma and human serum included determination of assay sensitivity (<0.63 pg/ml), precision (all CVs less than 17.8%), recovery (98-103%), and linearity of recovery (R=0.998). Levels of estradiol were lower when assayed by GC/MS/MS compared to RIA under all conditions (7.26+/-4.82 pg/ml versus 11.9+12.0 pg/ml in normal post-menopausal women; 5.88+/-3.43 pg/ml versus 13.8+/-7.5 pg/ml in breast cancer patients prior to treatment; and<0.63 pg/ml versus 5.8+/-4.1 pg/ml during aromatase inhibitor therapy). Fifty-five women treated either with atamestane/toremiphene or letrozole/placebo were monitored for estradiol levels at 4, 8 and 12 weeks of therapy. The mean levels of estradiol during aromatase inhibitor therapy was 5.8+/-4.1 pg/ml as measured by RIA and <0.63 pg/ml by GC/MS/MS. The degree of suppression with the aromatase inhibitors as detected by RIA was 58% versus >89% by GC/MS. These results suggest that most RIA methods detect cross-reacting estrogen metabolites and yield higher measured levels than GC/MS/MS. Several pharmacological and clinical considerations suggest that GC/MS/MS should become the preferred method for monitoring aromatase inhibitor therapy.     

Basal level of prostaglandin D2 in rat brain by a solid-phase enzyme immunoassay

A solid-phase enzyme immunoassay for prostaglandin D2 (PGD2) was developed in which PGD2 was labeled with horseradish peroxidase. After competitive binding to the immobilized antibody between enzyme-labeled and free PGD2, the activity of the enzyme bound to the antibody was assayed fluorometrically using 3-(p-hydroxyphenyl)-propionic acid and hydrogen peroxide as substrates. The procedure allowed determinations of 3-100 pg for PGD2. The IC50 value for PGD2 in the solid-phase enzyme immunoassay was about 25 pg and the sensitivity was improved about 10 times compared to those in radioimmunoassay and in solution-phase enzyme immunoassay. The solid-phase enzyme immunoassay was applied to the measurement of PGD2 content in rat brain and thereby an octadecylsilyl silica cartridge and a reversed-phase HPLC were sequentially used for sample preparations. Heads were immediately frozen in liquid nitrogen after decapitation to avoid a postmortem formation of PGD2. PGD2 contents measured by solid-phase enzyme immunoassay correlated well with the values obtained by radioimmunoassay (r = 0.966) after raising its contents by intravenous administration of PGD2. The in vivo level of PGD2 in rat brain was extremely low but determined to be 0.11 +/- 0.03 ng/g tissue (mean +/- S.E.M.) with this enzyme immunoassay. The result was equal to the value extrapolated to zero time from the postmortem change.     

A radioimmunoassay for the measurement of aminopeptidase (microsomal) in human serum

A radioimmunoassay for the measurement of aminopeptidase (microsomal) (AP) in human serum was developed by using antiserum to human kidney AP. AP purified from kidney and AP present in normal serum and in serum from a patient with obstructive jaundice gave parallel logit-log transformation lines, suggesting immunological identity. The mean concentration of AP in normal serum (n = 104) was 1.33 +/- 0.30 (mean +/- SD) micrograms/ml. Men had significantly higher serum AP levels (1.41 +/- 0.30 micrograms/ml) (p less than 0.005) than women (1.24 +/- 0.28 micrograms/ml). Serum AP levels of patients with hepatoma (2.26 +/- 0.87 micrograms/ml) and cancer of the pancreas or the biliary tract (2.90 +/- 0.67 micrograms/ml) were significantly higher (p less than 0.005) than those of normal subjects. Patients with acute and chronic hepatitis (2.06 +/- 0.66 micrograms/ml) also had significantly higher serum AP levels (p less than 0.005) than normal subjects. In pregnant women, however, the increase in AP activity without the increase in AP concentration showed that the increased AP activity was due to an enzyme other than AP. The enzyme levels and activities in normal serum as well as in patients' sera were significantly correlated (normal, r = 0.77; patients, r = 0.95). Based on the specific activity of AP purified from human plasma, the enzyme activity splitting L-alanyl-beta-naphthylamide is due almost completely to AP in normal subjects and in patients with hepatobiliary diseases.     

Sandwich Enzyme Immunoassay for Rat Retinol-Binding Protein Using Antibody against Recombinant Antigen and Its Application

The development of a sandwich enzyme immunoassay for rat retinol-binding protein using molecular biological techniques was described. Rat retinol-binding protein gene cloned by the PCR method was expressed by a fusion vector pEZZI8 in Escherichia coli strain HB101. A recombinant retinol-binding protein fused with IgG-binding domain ZZ of protein A was purified with IgG-Sepharose. Antibody against the recombinant protein was found to be specific to rat retinol-binding protein in plasma by immunoblot analysis. Affinity-purified anti-recombinant protein IgG was biotinylated and used for the sandwich enzyme immunoassay. In this assay, the measurable range is 1.9-60 ng/ml and the coefficients of variation within and between the assay series (assay range: 4-30 ng/ml) are 4.30 ± 4.33 and 5.32 ± 1.45%, respectively. Cross-reactivity of the immunoassay was examined using bovine, human, and mouse serum. There was a cross-reaction only with mouse serum. In an in vitro experiment, retinol-binding protein produced by rat hepatocytes could be measured by the sandwich enzyme immunoassay.     

Development of enzyme immunoassay for serum 13,14-dihydro-15-ketoprostaglandin F2 alpha

An enzyme immunoassay was developed for a convenient and sensitive assay of 13,14-dihydro-15-ketoprostaglandin F2 alpha, a metabolite of prostaglandin F2 alpha appearing in human blood. The compound was chemically conjugated to beta-galactosidase from Escherichia coli. The enzyme-labeled antigen was mixed with a sample containing 13,14-dihydro-15-ketoprostaglandin F2 alpha, and the mixture was allowed to react competitively with the antibody immobilized in a polystyrene tube. The activity of beta-galactosidase bound to the antibody was assayed by fluorometry. The enzyme activity was plotted against the amount of authentic 13,14-dihydro-15-ketoprostaglandin F2 alpha to obtain a calibration curve, and the compound was detectable over a range of 10 fmol to 10 pmol. Prostaglandins were extracted from human serum by the use of an octadecylsilyl silica column, and the extract gave an abnormally high level of 13,14-dihydro-15-ketoprostaglandin F2 alpha by enzyme immunoassay due to the presence of unidentified interfering substance(s), which was removed by high-performance liquid chromatography (HPLC). The purified material gave a value in the order of 0.1 pmol per ml of human serum. Validity of the enzyme immunoassay was confirmed by radioimmunoassay and gas chromatography/mass spectrometry (GC-MS) of a methyl ester n-butoximedimethylisopropylsilyl ether derivative.     

Measurement of 25-hydroxyvitamin D in the clinical laboratory: Current procedures, performance characteristics and limitations

In this review we describe procedures, performance characteristics and limitations of methods available for the measurement of 25-hydroxyvitamin (25OHD) since the year 2000. The two main types of methods are competitive immunoassay and those based on chromatographic separation followed by non-immunological direct detection (HPLC, LC-MS/MS). Lack of a reference standard for 25OHD has, until recently, been a major issue resulting in poor between-method comparability. Fortunately this should soon improve due to the recent introduction of a standard reference material in human serum (SRM 972) from the National Institute of Standards and Technology (NIST). For immunoassay, specificity can be an issue especially in relation to the proportion of 25OHD2 that is quantified whereas HPLC and LC-MS/MS methods are able to measure the two major vitamin D metabolites 25OHD2 and 25OHD3 independently. HPLC and LC-MS/MS require more expensive equipment and expert staff but this can be offset against lower reagent costs. Increasingly procedures are being developed to semi-automate or automate HPLC and LC-MS/MS but run times remain considerably longer than for immunoassays especially if performed on automated platforms. For most HPLC and LC-MS/MS methods extraction and procedural losses are corrected for by the inclusion of an internal standard which, in part, may account for higher results compared to immunoassay. In general precision of immunoassay, HPLC and LC-MS/MS are comparable and all have the required sensitivity to identify severe vitamin D deficiency. Looking to the future it is hoped that the imminent introduction of a standard reference method (or methods) for 25OHD will further accelerate improvements in between method comparability.     

1‐Acetyl‐3‐[(3R)‐hydroxyfatty acyl]glycerols: Lipid Compounds from Euphrasia rostkoviana Hayne and E. tetraquetra (Bréb.) Arrond.

Five homologous acetylated acylglycerols of 3‐hydroxyfatty acids (chain lengths C(14) – C(18)), named euphrasianins A – E, were characterized for the first time in Euphrasia rostkoviana Hayne (Orobanchaceae) by gas chromatography/mass spectrometry (GC/MS) and high‐performance liquid chromatography/atmospheric pressure chemical ionization‐mass spectrometry (HPLC/APCI‐MSⁿ). In addition to mass spectrometric data, structures of euphrasianins were verified via a three‐step total synthesis of one representative homologue (euphrasianin A). The structure of the latter was confirmed by 1D‐ and 2D‐NMR experiments as well as high‐resolution electrospray ionization‐mass spectrometry (HR‐ESI‐MS). The absolute configuration of the 3‐hydroxyfatty acid moiety at C(3) was found to be R in the natural euphrasianins, which was determined by alkaline hydrolysis and methylation of a purified fraction, followed by chiral GC analysis. Furthermore, in extracts of Euphrasia tetraquetra (Bréb .) Arrond . euphrasianins C and E were detected exclusively, indicating that this subclass of lipid constituents is possibly valuable for fingerprinting methods.     

A simple procedure for the preparation and purification of the oligosaccharide components of the glycosylphosphatidylinositol anchor of membrane proteins

The oligosaccharide components of the glycosylphosphatidylinositol anchors of Trypanosoma brucei variant surface glycoproteins have been prepared and purified by treatment with hydrolytic enzymes and solvent extraction procedures followed by HPLC purification using a specific oligosaccharide binding matrix (Glyco-Pak N, by Waters). Three oligosaccharide peaks (peaks I, II and III) were resolved by a single isocratic HPLC step (70% acetonitrile in water). The material from these peaks was hydrolyzed in acid and analyzed by GC/MS. GC/MS analysis of the material obtained from each peak demonstrated the presence of inositol, glucosamine, and mannose in a 1:1:3 ratio. A variable number of galactose residues were detected in each peak. The galactose:inositol ratios of the purified components were 1:1, 2:1, and 3:1 for peaks I, II and III, respectively, suggesting that the separation obtained depends primarily on the number of sugar residues present in each fraction.     

Identification of plasma protein biomarkers associated with idiopathic pulmonary arterial hypertension

We have employed SELDI-TOF MS to screen for differentially expressed proteins in plasma samples from 27 patients with idiopathic pulmonary arterial hypertension (IPAH) and 26 healthy controls. One ion (m/z approximately 8600) that was found to be elevated in IPAH was validated by SELDI-TOF MS analysis of a second and separate set of plasma samples comprising 30 IPAH patients and 19 controls. The m/z 8600 was purified from plasma by sequential ion exchange and reverse-phase chromatographies and SDS-PAGE. It was identified, following trypsin digestion, by MS peptide analysis as the complement component, complement 4a (C4a) des Arg. Plasma levels of C4a des Arg measured by ELISA confirmed that the levels were significantly higher (p < 0.0001) in IPAH patients (2.12 +/- 0.27 microg/mL) compared with normal controls (0.53 +/- 0.05 microg/mL). A cut-off level of 0.6 microg/mL correctly classified 92% of IPAH patients and 80% of controls. Further studies will be needed to determine its performance as a diagnostic biomarker, whether used alone or in combination with other biomarkers. Nevertheless, this study demonstrates that putative biomarkers characteristic of IPAH can be identified using a conjoint SELDI-TOF MS - proteomics approach.     

Production and secretion of C-19 steroids by rat and guinea pig adrenals

The concentrations of C-19 steroids were measured in guinea pig and rat adrenals before and after castration as well as after stimulation with adrenocorticotropin hormone (ACTH). Characterization of adrenal C-19 steroids was also carried out by isolation with high-performance liquid chromatography and gas chromatography/mass spectrometry (GC/MS). From radioimmunoassay (RIA) data, androstenedione (4-DIONE) and 11 beta hydroxyandrostenedione (11 beta-DIONE) were the major C-19 steroids found in guinea pig adrenals, and castration induced a decrease of 4-DIONE levels only while all other C-19 steroids remained unchanged. In rat adrenals, the major C-19 steroids were 4-DIONE and testosterone, and they were also markedly inhibited after castration. With the exception of 11 beta-DIONE, all other C-19 steroids in circulation were eliminated after castration in both animals species. After ACTH administration in the guinea pig, adrenal 4-DIONE and 11 beta-DIONE levels were markedly stimulated, while an increase of only 11 beta-DIONE was observed in plasma. In the rat, ACTH had a small stimulatory effect on adrenal 52-androstane-3 alpha, 17 beta-diol (3 alpha-DIOL) and plasma 11 beta-DIONE levels. Analysis of guinea pig adrenal steroids by GC/MS confirmed the presence of C-19 steroids in adrenals (namely, 4-DIONE and 11 beta-DIONE) while, in the rat, this could not be confirmed. Our data indicate that production of C-19 steroids occurs in guinea pig adrenals, and 11 beta-DIONE is the major C-19 steroid as well as the only C-19 steroid secreted into the circulation. In the rat, the production of C-19 steroids detected by RIA is not supported by GC/MS data.     

The determination of 11 beta-hydroxyandrostenedione in human follicular fluid and plasma

The development of a chromatographic/immunoassay method is presented for the measurement of 11 beta-hydroxyandrostenedione (11 beta-OH-A4) in ovarian follicular fluid (FFL) and plasma from women undergoing embryo transfer for in vitro fertilization. This method incorporates high-performance liquid chromatography (HPLC) and permits the simultaneous measurement of other steroids from a single sample in order to assess the intraovarian environment. Authenticity of 11 beta-OH-A4 in follicular fluid was confirmed using selected ion monitoring (SIM) gas chromatography/mass spectrometry (GC/MS). Our results demonstrate a mean concentration of 18.6 nmol/l in follicular fluid compared with 3.2 nmol/l in plasma. The origin of 11 beta-OH-A4 in follicular fluid requires further investigation but these findings supports the hypothesis of ovarian 11 beta-hydroxylase activity on C19 steroids.     

Quantitative assessment of tyrosine nitration of manganese superoxide dismutase in angiotensin II-infused rat kidney

Hypertension caused by angiotensin II is characterized by an increase in tissue oxidant stress as evidenced by increased quantities of reactive oxygen and nitrogen species. Manganese superoxide dismutase (MnSOD) is a key mitochondrial antioxidant enzyme that is inactivated in conditions of oxidant stress by reacting with peroxynitrite to form 3-nitrotyrosine in its active site. The increase in 3-nitrotyrosine content in MnSOD in the kidney of angiotensin II-infused rats was assessed in this study by immunohistochemistry, Western blotting, immunoprecipitation, and HPLC with UV detection (HPLC-UV). MnSOD activity decreased approximately 50% in angiotensin II-infused rat kidneys (24 +/- 4.6 vs. 11 +/- 5.2 U/mg) without a change in protein expression. Immunohistochemical staining showed 3-nitrotyrosine predominantly in distal tubules and collecting duct cells in the angiotensin II-infused rat kidneys. By two-photon microscopy, 3-nitrotyrosine colocalized with MnSOD. Total 3-nitrotyrosine content in kidney homogenates was increased in angiotensin II-infused rat kidney [3.2 +/- 1.9 (sham treated) vs. 9.5 +/- 2.3 ng/mg protein by HPLC-UV detection]. With tracer amounts of tyrosine-nitrated recombinant MnSOD, the most sensitive technique to detect tyrosine nitration of MnSOD was immunoprecipitation from tissue with anti-MnSOD antibody, followed by detection of 3-nitrotyrosine by Western blotting or HPLC. By HPLC, 3-nitrotyrosine content of kidney MnSOD increased 13-fold after angiotensin II infusion, representing an increase from approximately one-twentieth to one-fifth of the total 3-nitrotyrosine content in sham-treated and angiotensin II-infused rat kidney, respectively. Angiotensin II-induced hypertension is accompanied by increased tyrosine nitration of MnSOD, which, because it inactivates the enzyme, may contribute to increased oxidant stress in the kidney.     

Current methodologies for the analysis of aminoglycosides

The aminoglycosides are a large and diverse class of antibiotics that characteristically contain two or more aminosugars linked by glycosidic bonds to an aminocyclitol component. Structures are presented for over 30 of the most important members of this family of compounds. The use of aminoglycosides in clinical and veterinary medicine and in agriculture is described. Qualitative methods for aminoglycoside analysis include X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS). The major part of this article comprises a comprehensive review of quantitative methods for the determination of aminoglycosides. These are microbiological assay, radiochemical assay, radioimmunoassay, enzyme immunoassay, fluoroimmunoassay and other immunoassays, spectrophotometric and other non-separative methods, gas chromatography (GC), thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and capillary electrophoresis (CE). Simple spectrophotometric methods may be adequate for the assay of bulk pharmaceuticals and their formulations. Microbiological assays make useful semi-quantitative screening tests for the analysis of veterinary drug residues in food, but rapid enzyme immunoassays are more suitable for accurate measurements of aminoglycosides in complex matrices. Automated immunoassays are the most appropriate methods for serum aminoglycoside determinations during therapeutic drug monitoring. HPLC techniques provide the specificity and sensitivity required for pharmacokinetic and other research studies, while HPLC–MS is employed for the confirmation of veterinary drug residues. The potential for further development of chromatographic and CE methods for the analysis of biological samples is outlined.     

Immunochemical detection of 3-deoxyglucosone in serum

3-Deoxyglucosone (3-DG) is a metabolite of glucose that is thought to lead to the production of advanced glycation end products in diabetes. The previous assay for 3-DG in serum was based on a multi-step protocol, including derivatization, extraction, HPLC separation, and detection. In the current studies, we established a monoclonal antibody that recognizes the 3-DG-derivative, which is generated by the reaction of 3-DG and a 2,3-diamino-benzene derivative. Attachment of a biotin moiety to the 2,3-diamino-benzene ring via a linker allowed development of a highly sensitive chemiluminescent enzyme immunoassay for 3-DG equivalents. Unlike the previous assay, this method does not require extraction of 3-DG derivatives from serum. Treatment of 3-DG in serum with the DAB-link-biotin produced a quinoxaline derivative, which was specifically recognized by the monoclonal antibody. Using this assay, we found that serum 3-DG was higher in streptozotocin-induced diabetic rats than in normal control rats (25+/-5.6 vs. 9.8+/-1.1 microg/L). This simple assay may allow the monitoring of conditions leading to the accumulation of advanced glycation end products and evaluation of the risk of complications in diabetic patients.