Metabolism of selenocyanate in the rat

Rats injected subcutaneously with 2 mg Se/kg body weight of [75Se]selenocyanate or [14C, 75Se]selenocyanate excreted dimethylselenide (DMSe) in the breath and trimethyl-selenonium ion (TMSe) in the urine. The 24-h respiratory DMSe and urinary TMSe excretions were 26.8 +/- 8.1 and 14.5 +/- 5.1% of the dose, respectively. Tissue concentrations of 75Se were highest in the kidneys (1.89 +/- 0.2% dose/g), liver (1.46 +/- 0.2% dose/g), and blood (0.50 +/- 0.05% dose/ml), and lower (greater than 0.3% dose/g) in the other tissues. Trimethyl-selenonium was the major form (61%) of selenium in urine. Approximately 2% of the dose of doubly labeled SeCN- was excreted unchanged in urine (about 12% of urinary Se). 14C from doubly labeled SeCN- was not present in the methylated selenium metabolites, but a major 14C urinary metabolite was identified as thiocyanate. These results indicate that a substantial part of selenocyanate in the body undergoes metabolism and Se is excreted in methylated forms following scission of the C-Se bond.     

Determination of cadmium in blood, plasma, and urine by electrothermal atomic absorption spectrophotometry after isolation by anion-exchange chromatography

The determination of cadmium in whole blood, urine, or plasma by atomic absorption using electrothermal atomization is described. In preparation for atomic absorption analysis, cadmium was concentrated on an anion-exchange column, significantly lowering the limit of detection and allowing for the first time the accurate and precise determination of plasma cadmium concentrations in persons/animals with low-level cadmium exposures. Recovery of 109Cd from spiked whole blood, plasma, and urine into supernatants of nitric acid-deproteinated samples averaged 99, 100, and 95%, respectively. Anion-exchange isolation of the anionic chlorocadmium complex removed 99.8% of the major elements associated with a deproteinated whole blood sample. The recovery of 109Cd from the anion-exchange column was 92.2 +/- 0.9% (mean +/- SE, N = 35). The separation of cadmium from constituents in blood, urine, or plasma in this manner allowed comparison of unknown samples to aqueous standards with a defined acid matrix using commercially available acids. The mean intra-assay coefficient of variation (CV) was 12 +/- 3% (mean +/- SE, N = 6) for blood, plasma, and urine samples having cadmium concentrations of 0.1-0.8 microgram/liter. The interassay CV was 13% (N = 7) for a blood sample containing 0.6 microgram Cd/liter. The recovery of known amounts of cadmium added to blood, plasma, and urine in the range of 0.2 to 5.0 micrograms Cd/liter was 97 +/- 6% (mean +/- SE, N = 4).     

Specific and rapid assay of urinary oxalic acid using high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method was developed to determine the levels of oxalic acid in the urine. This acid was extracted from urine with tri-n-butyl phosphate and converted into the fluorescent derivative by esterification with 9-anthryldiazomethane (ADAM). The reaction mixture containing the oxalic acid derivative can be directly chromatographed on HPLC using octadecylsilane reverse-phase column monitoring with a fluorophotometric detector. A linear relationship was observed in the range from 1 to 100 micrograms/ml of standard oxalic acid dissolved in saline. Disease-free Japanese adults excrete 23.8 +/- 9.0 mg (mean +/- SD) of oxalic acid per day. This method should prove valuable for routine measurements of urinary oxalic acid as it is accurate, simple, and specific.     

Selenium serum and urine is associated to mild asthma and atopy. The SUS study.

To examine the associations between selenium (Se) status, asthma, bronchial hyperresponsiveness (BHR), and atopy in 154 male subjects (72 with mild asthma, 41 with BHR and 41 with no respiratory symptoms) aged 18 (range 17-22) years. Each subject underwent a medical interview and FEV1 and FVC were recorded. Histamine bronchial reactivity (Yan method) was measured, skin prick test (inhalant allergens) was performed and Se in urine and serum was analysed (AOAC modified fluometric method). Se in serum 74.04 (10.58) micrograms/L (mean (SD)) was lower in subjects with asthma and the logarithm of the ratio of Se in serum (microgram/L) and urine standardised to creatinine excretion (ng/mg creatinine) 0.748 (0.096) (mean (SD)) was lower in subjects with asthma and atopy compared to subjects with no allergic symptoms 77.79 (10.16) micrograms/L and 0.808 (0.111) respectively (p < 0.05). In subjects with asthma atopy was significantly associated to urine Se 0.24 (0.73) (beta (SE)) (p < 0.05). Subjects with BHR had the same Se status as subjects with no respiratory symptoms and heavy smokers had a lower concentration of Se in serum 73.80 (9.56) micrograms/L than non-smokers 78.16 (10.74) micrograms/L (p < 0.05), Se status was associated to asthma and smoking. Measuring Se in urine might add further information to possible relations between Se status, atopy and asthma.     

Renal function and fractional clearances of American river otters (Lutra canadensis)

The finely lobulated kidneys of American river otters (Lutra canadensis) are not visualized on plain abdominal radiographs. Similar values for blood urea nitrogen (BUN), creatinine, and uric acid were obtained on different analytical systems used in 1984 and 1985. The mean +/- SD for measured plasma osmolalities (309.80 +/- 8.86 mOsmol/kg) of otters in 1985 was significantly (P less than 0.01) less than that of calculated serum osmolalities in the same 1985 specimens (321.61 +/- 5.64 mOsmol/kg) and in 1984 specimens (322.20 +/- 7.16 mOsmol/kg). Urine specific gravities and osmolalities were highly correlated (r = 0.92). On routine urinalysis, protein and bilirubin were frequent chemical findings, and urobilinogen was present in all urine samples. White and red blood cells and epithelial cells were frequent findings on urine microscopic examinations. Proteus mirabilis was cultured from four of four female otters with genitourinary infections. The mean +/- SD creatinine values for paired serum and urine samples (n = 13) were serum creatinine (Scr) 0.66 +/- 0.09 mg/dl and urine creatinine (Ucr) 186.9 +/- 55.6 mg/dl. Corresponding values for serum electrolytes (Se) and urine electrolytes (Ue) yielded mean +/- SD calculated renal fractional clearances (FC = Ue/Se x Scr/Ucr) of sodium 9.65 +/- 5.81 x 10(-4), potassium 4.15 +/- 2.01 x 10(-2), chloride 10.81 +/- 5.33 x 10(-4), calcium 4.52 +/- 4.46 x 10(-3), and phosphate 6.58 +/- 3.44 x 10(-3).     

Measurement of endogenous lithium levels in serum and urine by electrothermal atomic absorption spectrometry: a method with potential clinical applications

A highly sensitive flameless atomic absorption method has been adapted for the determination of endogenous trace lithium levels in serum and urine. With ammonium nitrate as the only matrix modifier, serum levels of Li as low as 0.03 mumol/liter are measured accurately and there is no requirement for standard additions. The need for background correction during analysis was clearly established, and tungsten and Zeeman-effect background corrections were compared. The tungsten correction offered superior sensitivity and linearity of standards. Recoveries in urine and serum average 94.8 +/- 7.7 and 95.3 +/- 6.1% (+/- SD), respectively. The endogenous serum Li levels were 0.16 +/- 0.08 mumol/liter for normal subjects dwelling in the Denver metropolitan area. The mean 24-h excretion rate was 5.24 +/- 1.4 mumol/day. The mean fractional excretion of endogenous Li (clearance Li/clearance creatinine) was 23.2 +/- 3.0%, a value similar to values published for exogenously administered Li and measured by conventional methods.     

Quantification of apolipoprotein D in human urine by zone immunoelectrophoresis assay: a methodological and clinical study.

A zone immunoelectrophoresis assay (ZIA) has been developed for the quantification of apolipoprotein D (apo D) in unconcentrated native human urine. A standard curve, linear between 1 and 8 mg apo D/l was obtained with ZIA. The relative coefficients of variation for this method were 5-9% (n = 15 x 6) with a mean +/- SD of 7 +/- 1.4% and below 11% (n = 6 x 15) for within-run and between-run reproducibility, respectively. Equal amounts of apo D in unconcentrated and diluted urines, in serum and of the purified protein produced the same zone migration distances indicating parallelism between the immunologic reactions of apo D in different sample matrixes. Storage experiments with normal urines demonstrated good stability of apo D in both acidic and alkalinized urine over at least 2 days at +5 degrees C and during several days at -20 degrees C to -40 degrees C. Using ZIA, urine samples from 50 normal healthy men aged 23-65 years were analyzed for apo D. Mean and SD were: 2.8 +/- 2.1 mg/l, 2.6 +/- 1.8 micrograms/min and 0.24 +/- 0.13 mg/mmol for concentration, rate of excretion and mass/creatinine concentration, respectively.     

Determination of eleven small selenium species in human urine by chromatographic-coupled ICP-MS methods

BackgroundThe determination of various selenium species in urine enables a specific biomonitoring of the exposure to different selenium compounds.MethodsFor this task a coupling of three chromatographic techniques with ICP-MS was developed for the separate quantification of eleven species in urine. The first procedure was based on reverse phase chromatography and was designed for the separate determination of methyl-2-acetamido-2-deoxy-1-seleno-b-d-galactopyranoside (SeSug1), methyl-2-acetamido-2-deoxy-1-seleno-b-d-glucopyranoside (SeSug2), selenomethionine (SeMet), methylselenocysteine (MeSeC), seleno-D,L-ethionine (SeEt), methylselenic acid (MeSeA) and methylselenoglutathione (MeSeG); the second procedure was based on anion exchange chromatography and measured selenate (Se (VI)) and selenite (Se (IV)); the third procedure was based on cationic exchange chromatography and determined methyl-2-amino-2-deoxy-1-seleno-b-d-galactopyranoside (SeSug3) and the trimethylselenium ion (TMSe). A fourth method for the more sensitive determination of TMSe was upgraded by an on-line after-column reaction process.ResultsThe validation of the methods yielded sensitive detection limits of the species between 0.03 and 0.10 μg Se/L. For TMSe a detection limit of 0.02 μg Se/L resulted by the fourth method. An intra-day precision of 2.7–10.6% and a relative recovery between 87 % and 108 % confirm the robustness of the methods.ConclusionThe developed procedures enable a separate and sensitive determination of eleven selenium species in urine and thus permit the exploring of metabolic factors in the general population and particularly exposed individuals.     

Isolation of a yeast heptaglucoside that inhibits monocyte phagocytosis of zymosan particles

To isolate a unit ligand recognized by human monocyte beta-glucan receptors, acid-solubilized oligoglucosides were prepared by partial acid hydrolysis of purified yeast cell walls, gel filtered sequentially on Bio-Gel P-4 and P-2, derivatized with 2-aminopyridine, and separated by normal-phase HPLC. Ligand recognition was assessed by quantitating the effect of pretreatment with isolated materials on the capacities of adherent monocytes to phagocytose zymosan particles. Partial acid hydrolysis solubilized 23 +/- 4% (mean +/- SD; n = 7) of the cell wall glucans; at an input of 50 micrograms/ml, the solubilized products reduced the numbers of monocytes ingesting zymosan by an average of 44%. Gel filtration of acid-solubilized glucans on Bio-Gel P-4 revealed several peaks with phagocytosis-inhibiting activity, and fractions from the peak containing the smallest oligoglucosides, which accounted for 10 +/- 2% (mean +/- SD; n = 7) of the carbohydrate applied, were pooled. Further purification on Bio-Gel P-2 resolved this phagocytosis-inhibiting activity to a single peak that contained apparent heptaoses and accounted for 8 +/- 2% (mean +/- SD; n = 6) of the carbohydrate applied. At a concentration of 0.5 microgram/ml, the oligoglucosides pooled from the Bio-Gel P-4 and P-2 columns reduced the numbers of ingesting monocytes by 45 +/- 1% and 42 +/- 7% (mean +/- SD; n = 3), respectively. When derivatized with 2-aminopyridine, the oligoglucosides were resolved by HPLC to a number of peaks; a peak that eluted as an apparent heptaglucoside contained virtually all the inhibitory activity and accounted for only 6.6 +/- 0.7% (mean +/- SD, n = 7) of the carbohydrate applied. Gas chromatography analysis revealed only glucose and FAB-mass spectrometric analysis showed only heptaglucoside and no noncarbohydrate molecules. At a concentration of 1.6 ng/ml, the derivatized yeast heptaglucoside reduced the numbers of monocytes ingesting zymosan and glucan particles by 44 +/- 9% (mean +/- SD; n = 5) and 45 +/- 6% (n = 3), respectively. Thus, a heptaglucoside present in yeast cell walls is a unit ligand for human monocyte beta-glucan receptors.     

Determination of 7-methylguanine, N2,N2-dimethylguanosine, and pseudouridine in ultrafiltrated serum of healthy adults by high-performance liquid chromatography

7-Methylguanine (m7Gua), N2,N2-dimethylguanosine (m2(2)Guo), and pseudouridine (psi) are degradation products from RNA turnover and can be used as markers for the whole-body turnover of mRNA-cap, tRNA, and rRNA (in healthy individuals, urinary excretion of these catabolites follows a regular pattern; the relative molar ratio of psi:m7Gua:m2(2)Guo is approximately 100:19:6). HPLC methods were developed to measure serum concentrations of these RNA catabolites after deproteinization of the samples by ultrafiltration through microcollodion bags with a nominal exclusion Mr of 12,400. For healthy adults the following values (mean +/- SD) were found: psi, 2760 +/- 460 nmol/liter (n = 10); m7Gua, 129.7 +/- 24.0 nmol/liter (n = 13); m2(2)Guo, 31.0 +/- 3.7 nmol/liter (n = 9). The relative molar ratio of these substances in serum derived from our data is approximately 100:4.7:1.1. 7-Methylguanosine (m7Guo) added to serum is to a large extent converted to the corresponding free base, m7Gua, the form which is excreted in urine.     

Selenium concentration in human urine

In this work we have determined selenium concentration in urine in two groups of healthy subjects. Selenium content in the younger group, aged 11-15 years (n = 41), was 13.7 +/- 6.5 micrograms/g-1 creatinine. In the older group, aged 17-97 years (n = 62), slightly but statistically significant lower selenium concentration in urine (11.4 +/- 4.9 micrograms/g Ct, p less than 0.05) was found. We have also shown a significant difference in the excretion of the element between the group of boys and men (p less than 0.05). Concentration of selenium excreted in urine in the population of healthy people (11-97 years, n = 103) is 12.3 +/- 5.4 micrograms Se/g Ct.     

Changes in ovarian blood flow and secretion of progesterone and 20 alpha-hydroxypregn-4-en-3-one on day 16 and the morning and afternoon of day 22 of pregnancy in the rat

During late pregnancy in rats, ovarian secretion of progesterone decreases and that of its reduced metabolite, 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OHP), increases. The present study was undertaken to determine whether changes in ovarian blood flow are consistent with changes in progestin secretion. Rats (n = 5 per group) were examined on Day 16, the time of maximal progesterone secretion, and in the morning (AM) and afternoon (PM) of Day 22, the day prior to parturition. Ovarian blood flow was monitored continuously for 60 to 80 min, and serial samples of arterial and ovarian venous blood were obtained at 20-min intervals for determination of ovarian secretion rates of progesterone and 20 alpha-OHP. Ovarian blood flow increased from 0.38 +/- 0.04 ml/min (mean +/- SEM) on Day 16, to 0.77 +/- 0.05 and 0.78 +/- 0.04 ml/min on Day 22 AM and PM, respectively, whereas the secretion of progesterone decreased from 26.9 +/- 4.0 to 4.5 +/- 1.0 and 3.2 +/- 0.3 micrograms/h per ovary. The secretion of 20 alpha-OHP was similar on Day 16 and Day 22 AM (5.6 +/- 1.7 and 5.4 +/- 1.3 micrograms/h per ovary) but then increased to 18.9 +/- 1.2 micrograms/h per ovary by Day 22 PM. Thus the amount of total progestins secreted per unit rate of blood flow relative to that on Day 16 (100%) fell to 15% and 34% on the morning and afternoon of Day 22, respectively. Clearly, the relative changes in ovarian progestin secretion and blood flow in the rat near term to not conform to patterns observed at luteal regression in some other species.(ABSTRACT TRUNCATED AT 250 WORDS)     

Formation of dimethyl selenide and trimethylselenonium from selenobetaine in the rat

The 24-h respiratory excretion of dimethyl selenide (DMSe) and urinary excretion of trimethylselenonium (TMSe) were studied in adult male rats injected with 2 mg Se/kg as selenobetaine [(CH3)2Se+CH2COOH] or its methyl ester, labeled with 75Se and 14C. The DMSe was trapped by means of 20% benzyl chloride in xylene. TMSe was measured by cation exchange high performance liquid chromatography. There was extensive respiratory excretion of DMSe from selenobetaine methyl ester (about 50% of the dose) and from selenobetaine (about 25%). About 12% of the dose was converted to TMSe for both compounds. When the Se-methyl carbons were labeled with 14C and the selenium with 75Se, doubly labeled DMSe and TMSe were formed; the 14C/75Se ratio in DMSe formed from selenobetaine methyl ester was almost unchanged from that administered, and the ratio in TMSe was only slightly lower than in DMSe. In contrast to its ester, doubly labeled selenobetaine yielded DMSe having a lower 14C/75Se ratio (approximately one-half of that administered) and a further decrease was observed between DMSe and TMSe. These data indicate that the (CH3)2Se moiety in selenobetaine methyl ester undergoes facile release to form DMSe, which is directly methylated to form TMSe. Selenobetaine, however, appears to lose a methyl group prior to scission of the Se-CH2COOH bond. The results with selenobetaine also suggest that TMSe generated metabolically is not inert, and can undergo demethylation followed by remethylation; confirmatory evidence for this metabolic instability is provided by the exhalation of [75Se]DMSe after the direct administration of [75Se]TMSe. When [75Se]selenobetaine or its ester was given with the methylene carbon in the acetic acid moiety labeled with 14C, only 75Se was present in the DMSe and TMSe, indicating that TMSe did not arise by decarboxylation of selenobetaine. It is concluded that both selenobetaine and its methyl ester are readily converted to DMSe and TMSe by pathways that do not involve decarboxylation or the formation of hydrogen selenide as an intermediate, and DMSe is a direct precursor of TMSe.     

Ultrasonographic evaluation of the conceptus from days 10 to 60 of pregnancy in jennies

Daily ultrasound examinations were conducted from Days 10 to 60 (ovulation = Day 0) of pregnancy to monitor the conceptus in jennies (n = 12). The embryonic vesicle was first detected on Day 11.5 +/- 0.9 (mean +/- SD; range 10 to 13 d) and was mobile until movement ceased (fixation) on Day 15.5 +/- 1.4 (range, 13 to 18 d). The vesicle was spherical from Days 10 to 18 (mean growth rate, 3.2 mm/d), non spherical (irregular) with a reduced growth rate (0.5 mm/d) from Days 19 to 29, and then grew at a moderate rate (1.6 mm/d) up to Day 46. On average, detection of the embryo proper (consistently located on the ventral aspect of the yolk sac) and embryonic heartbeat were Days 20.7 +/- 1.2 and 23.5 +/- 1.3, respectively. Formation of the allantoic sac was first detected on Day 24.4 +/- 1.7 and was complete on Day 36.8 +/- 1.6. Descent of the fetus (and formation of the umbilical cord) began on Day 37.9 +/- 1.7 and was complete on Day 44.1 +/- 2.1. Crown-rump length averaged 3.7, 15.4, 22.7, 37.5 and 59.6 mm on Days 20, 30, 40, 50 and 60, respectively. In general, morphologic features and dates of occurrence were similar to those reported previously in the mare.     

Periodate-oxidized adenosine inhibits the formation of dimethylselenide and trimethylselenonium ion in mice treated with selenite

The metabolic detoxification of selenite and many other selenium compounds involves a series of S-adenosylmethionine-dependent methylations yielding dimethylselenide (DMSe), which is exhaled, and trimethylselenonium ion (TMSe), which is excreted in the urine. This paper shows that periodate-oxidized adenosine (Adox) inhibits these methylation reactions in vivo and increases the toxicity of selenite. When Adox was injected in mice at 100 mumol/kg 30 min before injection of [75Se]selenite at 0.4 mg Se/kg the appearances of [75Se]DMSe in the breath and [75Se]TMSe in the liver were completely inhibited for 90 min. This was mediated by accumulation of S-adenosylhomocysteine, the methyltransferase inhibitor, in the livers of Adox-treated mice due to inhibition of its hydrolase enzyme. During 24 h, Adox-treated mice excreted no detectable urinary [75Se]TMSe and exhaled only 20% as much [75Se]DMSe as controls. The urine of Adox-treated mice also contained S-adenosylhomocysteine at a level (ca. 4 mM), 200 times that of untreated mice, which provided a convenient index of methylation potential in the intact animal. When three groups of three mice each were injected with 100 mumol Adox/kg, selenite at 4 mg Se/kg, or a combination of the two, the mice receiving the combination were dead within 2 days, while the mice in the other two groups all survived at least 4 days. These results verify the enzymatic nature of selenium methylation in vivo, support its importance in detoxification, and indicate the value of Adox in further studies of selenium metabolism.     

The renal excretion of selenium.

The excretion of selenium in urine was determined in West German healthy volunteers. Women excrete 17.7 +/- 4.2 micrograms Se/d and men 19.0 +/- 9.0 micrograms Se/d. The daily selenium excretion per gram creatinine is 13.5 +/- 3.8 micrograms Se/g crea for women and 9.8 +/- 3.3 micrograms Se/g crea for men. The clearance of selenium from the plasma is calculated with 0.18 mL/min. The selenium excretion per day is positively correlated with the 24 h excretion of urea and creatinine. The correlation of the selenium excretion with the urea excretion is most probably owing to the fact that the selenium intake of West Germans is linked primarily to foods with high protein contents. That the selenium excretion is directly correlated with the creatinine excretion is an indicator that the muscle, which accounts for nearly 50% of the whole body selenium in West German adults, influences the selenium excretion in urine. The positive correlation of the selenium excretion with the potassium excretion also indicates that the muscle mass contributes significantly to the selenium excretion in urine. Another indicator that the selenium excretion is influenced by the muscle is that after intensive muscular activity (running), selenium excretion is enhanced. The 24 h selenium excretion is dependent on the glomerular filtration rate of the kidney characterized by the creatinine clearance. This result is important, because if the selenium excretion is used as parameter for the selenium status of humans, the kidney function should be known. This is a limitation for the use of the urinary selenium excretion as parameter for the selenium status. This is especially important for patients whose glomerular filtration rate is low. The 24 h selenium excretion is further influenced by the 24 h urine volume. Selenium losses via urine may be concomitant with protein losses in urine.     

Chemical immobilization of free-ranging North American bison (Bison bison) in Badlands National Park, South Dakota

Twenty-six free-ranging North American bison (Bison bison) (22 adult bulls, one yearling male and three adult females) were immobilized using a combination of carfentanil and xylazine. For carfentanil the dose range (mean +/- SD) was 1.8-5.0 micrograms/kg (2.4 +/- 0.7 micrograms/kg) and for xylazine 0.004-0.125 mg/kg (0.07 +/- 0.03 mg/kg). Induction time (mean +/- SE) was 14.2 +/- 2.9 min (median 8 min), while the total mean reversal time after administration of a narcotic antagonist was 9.0 +/- 1.4 min (median 8 min). Only one animal that received the highest initial dose of carfentanil (2.5 mg) showed evidence of becoming "re-narcotized." Five animals required two or more doses of carfentanil before becoming immobilized. Overall, small volumes of drug used (mean = 0.62 ml for carfentanil, 0.53 ml for xylazine) enabled the use of 1 to 2 ml darts, increasing both accuracy and impact safety. Darting success approached 100%.     

Perturbation of beta-glucan receptors on human neutrophils initiates phagocytosis and leukotriene B4 production

Normal human neutrophils were stimulated with the yeast cell wall product, zymosan, and examined for two biologic responses, ingestion of particles and production of leukotriene B4 (LTB4), under conditions that were comparable and optimal for the quantitation of each response. Monolayers of adherent neutrophils ingested unopsonized zymosan particles, at particle-to-cell ratios of 12.5:1 to 125:1, in a dose- and time-related manner. At a ratio of 125:1, the percentages of neutrophils ingesting greater than or equal to 1 and greater than or equal to 3 zymosan particles reached plateau levels of 55 +/- 6 and 32 +/- 9% (mean +/- SD, n = 8), respectively, within 30 min. At this same ratio, neutrophils during gravity sedimentation with zymosan particles synthesized LTB4 in a time-dependent manner for at least 45 min. The maximum amount of immunoreactive LTB4 released into supernatants was 3.8 +/- 1.2 ng per 10(6) neutrophils (mean +/- SD, n = 5) and the corresponding total immunoreactive LTB4 was 6.2 +/- 1.9 ng per 10(6) neutrophils. Treatment of 2 x 10(7) suspended neutrophils with 250 micrograms of trypsin for 20 min before concurrent assessment of neutrophil phagocytosis and LTB4 production reduced both of these responses by about 50%. Pretreatment of neutrophils with 800 micrograms/ml of soluble yeast beta-glucan inhibited their ingestion of zymosan by 84% (mean +/- SD, n = 3), with 50% inhibition occurring with 100 micrograms/ml of soluble beta-glucan; 800 micrograms/ml of soluble yeast alpha-mannan had no inhibitory effect. Pretreatment of neutrophils with 400 micrograms/ml of soluble yeast beta-glucan inhibited neutrophil synthesis of LTB4 by 90%, with 50% occurring with 200 micrograms/ml; 400 micrograms/ml of soluble yeast alpha-mannan had no inhibitory effect. The presence of 1.25 micrograms/ml of cytochalasin B during incubation with zymosan particles reduced neutrophil phagocytosis from 65 to 6%, and neutrophil synthesis of LTB4 from total levels of 6.0 +/- 0.3 ng/10(6) cells to zero (mean +/- SD, n = 3). Pretreatment with either cytochalasin B or vinblastine did not alter neutrophil generation of LTB4 induced by calcium ionophore. Neutrophils pretreated with vinblastine, at 4 x 10(-6) to 4 x 10(-4) M, and then maintained at one-half these concentrations during incubation with unopsonized zymosan particles exhibited no diminution in particle ingestion, but were markedly reduced in zymosan-induced synthesis of LTB4.(ABSTRACT TRUNCATED AT 400 WORDS)     

High-performance liquid chromatographic determination of alpha-acetyldigoxin in Digitalis lanata leaves.

An analytical method for the determination of alpha-acetyldigoxin in Digitalis lanata leaves by HPLC has been developed. The procedure consists of extraction of dry leaf powder with 50% methanol and cleanup by a Sep-Pak C18 cartridge prior to HPLC analysis. The quantitation is carried out by the incorporation of beta-methyldigoxin as an internal standard. HPLC is performed on an octylsilyl bonded silica column with acetonitrile/methanol/water (100/11/188, v/v). The effluent is monitored by uv absorption at 220 nm. The amount of alpha-acetyldigoxin per 100 mg of dry leaf powder is estimated at 5.55 +/- 0.21 micrograms (mean +/- SD). The average recovery of alpha-acetyldigoxin from added samples is 97.2%. The present method is sensitive, reliable, and relatively simple. Application of this HPLC method to the analysis of samples obtained by fermentation of the leaf powder is also demonstrated.     

Determination of CGS 16617 and stable isotope-labeled CGS 16617, an angiotensin-converting enzyme inhibitor, in human plasma by gas chromatography/mass spectrometry

An analytical method has been developed for the simultaneous determination of a novel orally active angiotensin-converting enzyme inhibitor (CGS 16617) and a stable isotope-labeled analog. Both compounds are isolated from human plasma using an ion-exchange column, derivatized with pentafluoropropionic anhydride and pentafluoropropanol, and analyzed by gas chromatography/mass spectrometry. After splitless injection on a methyl-silicon column, the compound is detected using negative ion chemical ionization with nitrous oxide as a reagent gas. CGS 16617 labeled with four deuteriums and two 13C is used as an internal standard. The accuracy and precision of the method, expressed as the overall mean +/- SD recovery obtained from two sets of 36 quality-control samples used during a clinical study (concentration range 0.2-100 ng ml-1 plasma), was 96.1 +/- 16.2% for unlabeled drug and 97.6 +/- 14.4% for the D4-labeled drug (concentration range 0.2-100 ng ml-1 plasma). The limit of quantification using 1 ml plasma is 0.2 ng ml-1 for both labeled and unlabeled drug.