Administration of recombinant bovine somatotropin to dairy cows for four consecutive lactations

Effects of long-term administration of recombinant bovine somatotropin (bST) to dairy cows on complete lactational performance [60 (+/- 3) to 284 (+/- 3) d in milk (DIM)] were studied for four consecutive lactations. Beginning on d 60 (+/- 3) postpartum, Holstein cows received biweekly injections (500 mg) of bST (n = 39) or a placebo (control; n = 39) during the first lactation of the study. Cows either continued on the same treatment (n = 26) or were switched to the opposite treatment (n = 29) during the second lactation. Cows that changed treatments were injected for only 16 wk during the second lactation. Six cows per treatment completed four consecutive lactations. Treatment with bST during the first lactation did not have a residual effect on milk yields during the second lactation. Injections of bST during the second lactation increased milk yield 6.5 kg/d from 60 (+/- 3) to 172 DIM. For the four lactations, cows receiving bST yielded 3.7 kg/d (14%) more milk and gained 52 kg (37%) more body weight than did controls. Pretreatment (from 0 to 56 DIM) milk yields in yr 2, 3, and 4 were not affected by previous bST treatment. Milk yield, efficiency of feed utilization, and body weights were enhanced in cows injected with bST for four consecutive lactations. Previous bST treatment did not diminish milk yields in subsequent lactations.     

Rapid assay of dinitrophenyl derivative of taurine by high-performance liquid chromatography

A rapid and simple method for the determination of taurine (2-aminoethanesulphonic acid) in complex samples is described. It is based on the HPLC separation of the dinitrophenyl (DNP) derivative of taurine. The reaction conditions are selected to allow complete derivatization of taurine within 15 min. DNP-taurine samples are stable for at least 3 days. DNP-taurine was separated by reversed-phase liquid chromatography within 12 s. The recovery of taurine was 102 +/- 3% (S.D. = 2.5%, n = 6) and the detection limit was 10 pmol for taurine (signal-to-noise ratio of 10). The method was applied to the determination of taurine levels in different samples including marine products, infant formulas and fermentation media of different bacterial species.     

Structural characterization of the two refold dimers of recombinant bovine somatotropin (bST)

Two major dimers are generated during the folding/oxidation of inclusion bodies of recombinant bovine somatotropin (bST). These dimers represent the major part of the inactive high molecular weight species that are formed in this process. The structures of the two dimers are unambiguously determined by peptide mapping using trypsin, thrombin cleavage, and selective DTT reduction experiments. Results indicate that the formation of both dimers involves the large disulfide loop cysteines. The latter-eluting dimer from RP-HPLC, previously reported as a large loop concatenated dimer, was revised to be an antiparallel disulfide-linked dimer. On the other hand, the first eluting dimer is a concatenane in which two monomers are held together by the interlocking of the two large disulfide loops.     

Separation from related compounds and assay of calcipotriol by high-performance liquid chromatography

In this paper, two methods are presented. One involves the separation of calcipotriol, a new synthetic analogue, from two related compounds, specifically cholecalciferol (Vitamin D3) and calcitriol (1,25-dihydroxyvitamin D3). The other involves the isolation and assay of calcipotriol from a topical ointment. The study was performed with reversed-phase high-performance liquid chromatography using an RP18 column and ultraviolet detection. Applying the method of Snyder, a mobile phase mixture containing methanol-acetonitrile-water (67:23:10, v/v) was found which achieved a total separation within 18 min. A mobile phase of methanol-water (80:20, v/v) attained a slower elution of calcipotriol. For isolation and assay of calcipotriol from an ointment (Daivonex), dissolution in chloroform gave the highest recovery (> 98%). The isolation and assay process can be performed within 2 h.     

Simultaneous determination of azathioprine and 6-mercaptopurine by high-performance liquid chromatography

We investigated whether linear whole-body acceleration along the interaural y-axis influenced the concurrent perception of visual motion direction as has been shown for angular accelerations. A sled running on air bearings along a 7.5-m track was used to accelerate 18 subjects at two different linear accelerations. These young, healthy volunteers, aged 25.50 +/- 7.38 years, used a joystick to indicate whether or not they perceived visual motion to the left within a random-dot kinematogram continuously presented on a monitor moving with them. The percentage of coherently leftward moving pixels presented for a 640-ms period during acceleration was adjusted according to a Modified Binary Search (MOBS) procedure. Six conditions were tested, two acceleration levels of 1 and 2 m/s2 to both left and right with, at the higher acceleration, two different times of visual motion presentation. Conditions were sequenced by means of a 6 x 6 Latin square balanced for order and carry over. A MANOVA did not show any statistically significant effects either for the independent variables acceleration, velocity, and direction of motion of the sled or for their interactions. The results obtained are in clear contrast to those obtained under rotatory stimulation. We conclude that the otolithic contribution to vestibular-visual motion processing is negligible.     

Simultaneous determination of catechins in human saliva by high-performance liquid chromatography

Green tea extracts have been suggested to possess a preventive effect against dental caries. A quantitative method for their anticariogenic substances, catechins, was developed to evaluate their concentrations in human saliva after mouthrinsing with green tea extract. Salivary catechins were extracted to the organic phase after forming a complex with diphenylborate and an ion-pair with tetra-n-butylammonium, and then back-extracted to the acidic aqueous phase. The extract was analyzed by high-performance liquid chromatography using diode array detection at absorption wavelengths ranging from 269 to 278 nm. In reversed-phase chromatography by a gradient elution, eight catechins originating from green tea and an internal standard were separated in 15 min without interfering peaks. All the catechins were simultaneously and selectively determined in the concentration range 0.05-25.0 microg/ml. In replicate spiking experiments with standards, the mean recovery ranged between 86 and 99%, and both intra- and inter-assay C.V.s were within 2.3%. When mouthrinsing with an aqueous solution of green tea extract (5.0 mg/ml) containing eight catechins, the quantitative results revealed that each catechin was retained at microg/ml levels in saliva for up to 60 min.     

Simultaneous determination of the stereoisomers of guggulsterone in serum by high-performance liquid chromatography

Simultaneous separation of E- and Z-guggulsterone, which is the main ingredient of 'Guggulip', an ayurvedic drug, was accomplished by HPLC on a C18 column using methanol, acetonitrile, buffer and tetrahydrofuran as a mobile phase. The compounds were monitored at 248 nm on a photodiode array detector. The assay method was used for the simultaneous determination of stereoisomers (E and Z) of guggulsterone in spiked serum and dosed (50 mg/kg, p.o.) rats. The recoveries of E- and Z-isomers from serum samples were always greater than 90%. The calibration graph was linear over the range of 25-2500 ng/ml for Z- and E-isomers. Lowest quantitation limit of Z- and E-guggulsterones was 25 ng/ml.     

Sensitive determination of nitrotyrosine in human plasma by isocratic high-performance liquid chromatography

A subfamily of small GTP-binding proteins, rab, has been shown to be involved in regulation of vesicular traffic in eukaryotic cells. The goal of this study was to identify the rab proteins associated with atrial secretory granules. A [32P]GTP-overlay assay showed the presence of multiple small GTP-binding proteins on the atrial granules. By biochemical analysis, we have demonstrated that one of the small GTP-binding proteins associated with the atrial granules is a rab12 protein (rab12p), one of the rab proteins that are most closely related to a Sec4 protein of yeast. Association of rab12p with the atrial granules was confirmed by immunogold electron microscopy. Immunoprecipitation followed by immunoblot analysis with anti-rab12 antibody showed that in addition to atria, rab12p was expressed in multiple other organs and cell lines. These results suggest that rab12p may function in vesicular traffic in multiple diverse types of cells.     

Stability and determination of aflatoxins by high-performance liquid chromatography with amperometric detection

A method based on reversed-phase high-performance liquid chromatography (RP-HPLC) with amperometric detection with a glassy carbon electrode at a constant potential of 1.4 V is reported for the separation and identification of aflatoxins B1, B2, G1 and G2 in a model mixture. The chromatography was performed on a PAH-Baker column with a ternary mobile phase containing methanol, acetonitrile and aqueous LiClO4 electrolyte. Aflatoxin G1 showed the highest electroactivity in the compound series studied. Calibration curves of aflatoxins G1 and B2 were linear up to 0.2 and 0.3 mmol/l, respectively. Sensitivity varied between 7 and 10 ng for the different aflatoxins. The combination of different HPLC detectors in the analysis of these compounds was applied to investigate the stability of aflatoxins G1 and B2.     

Simultaneous determination of five macrolide antibiotics in meat by high-performance liquid chromatography

A simple and rapid method using high-performance liquid chromatography (HPLC) for the simultaneous determination of five macrolides (josamycin, kitasamycin, mirosamicin, spiramycin and tylosin) in meat has been developed. The drugs were extracted with 0.3% metaphosphoric acid-methanol (7:3, v/v), and the extracts were cleaned up on a Bond Elut SCX (500 mg) cartridge. The HPLC separation was performed on a Puresil 5C18 column (150 x 4.6 mm I.D.) with a gradient system of 0.025 M phosphate buffer (pH 2.5)-acetonitrile as the mobile phase at a flow-rate of 1.0 ml/min. The drugs were detected at 232 mn for josamycin, kitasamycin, mirosamicin and spiramycin, and 287 mn for tylosin. The calibration graphs were rectilinear from 2.5 to 100 ng for each drug. The recoveries at the level of 1.0 microgram/g were 70.8-90.4%, and detection limits were 0.05 microgram/g for each drug.     

Continuous purification of porcine lipase by rotating annular size-exclusion chromatography

Non-accidental head injury, be it shaking, impact(s) or a combination of the two, is characterised by subdural and/or subarachnoid haemorrhages with retinal haemorrhages, but minimal or absent external cranio-facial trauma. The classical assault scenario depicts the infant being gripped around the head, face, chest and abdomen and shaken or being gripped by a limb and swung. This gripping might be expected to leave physical evidence in the form of bruising. A study was undertaken to establish the prevalence, distribution and pathological association of external bruising in 24 cases of fatal non-accidental head injury in children. At autopsy, 17 cases had new external bruises, 15 old external bruises and 13, a combination of both. However, seven (29%) cases showed no fresh external bruising and five (21%) showed no external bruising at all. Thus, external bruising may be absent in children with fatal intracranial injury. The face was shown to be the commonest site of bruising followed by the forehead and buttocks. Limb, chest and abdominal bruising were found to be uncommon. Retinal haemorrhages were confirmed in 23 (96%) cases. It is hypothesised that bruising, when present, may be a result of abuse in the form of punches and slaps rather than due to gripping during the assault. We discuss why gripping does not necessarily result in external bruising.     

A routine high-performance size-exclusion chromatography to determine molecular size distribution of Haemophilus influenzae type b conjugate vaccines

High-performance size exclusion chromatography has been used to determine the molecular size distribution of Haemophilus influenzae type b (Hib) conjugate vaccines. Both high molecular weight preparations of native Hib capsular polysaccharide coupled to tetanus toxoid and low molecular weight vaccines with Hib oligosaccharides linked to the CRM197 nontoxic mutant diphtheria protein were analysed. Columns with different fractionation ranges were used for the two kinds of vaccines. This method showed to be rapid, accurate and reproducible for different lots of Hib vaccine of different composition produced by various manufacturers. It could replace more time-consuming chromatographic methods enabling control authorities to employ a single methodological approach for different Hib vaccines.     

Enantiomeric determination of amines by high-performance liquid chromatography using chiral fluorescent derivatization reagents

BACKGROUND: Despite increased awareness of risk factors, wound complications continue to be a problem following coronary artery bypass graft (CABG) surgery. A minimally invasive alternative was therefore developed to reduce the risk of complications while providing the same benefits as the standard open vein harvest procedure. METHODS: Video-assisted endoscopic technique for vein harvest was introduced in our medical center in October 1996. The procedure was evaluated and compared with the standard open vein harvest procedure. With the endoscopic technique, small incisions were made, each about 2-3 cm at the selected access sites (groin and above and below the knee). An endopath subcutaneous dissector was subsequently inserted along the anterior surface of the saphenous vein with the assistance of an endoscope and video monitor. The venous side branches were detected and positioned using a vessel dissector. A ligaclip was applied and the branches were divided using endopath-scissors. In some cases, the venous branches were divided directly using the endopath-scissors. Therefore, the distal and proximal ends of the saphenous vein were isolated, ligated and divided. The harvested veins were used for CABG. Each patient was evaluated for length of surgery, hospital stay and morbidity. RESULTS: From October 1996 through May 1997, we performed 50 procedures using video-assisted endoscopic vein harvest. The results were compared with those from 106 patients who underwent standard open vein harvest during the same period. The rate of complications was 2% in the endoscopic group compared with 13.2% in the open group (p < 0.05). The average hospital stay was 7.2 days in the endoscopic group and 11.5 days in the open group (p < 0.05). Twelve weeks after the operation, all of the incisions healed with good cosmetic results in the endoscopic group. However, long visible scars were found in the patients in the open group. CONCLUSIONS: Endoscopic saphenous vein harvest provides a minimally invasive alternative to open vein harvest. It provides good cosmetic results without a hypertrophic scar and enables the patient to regain early ambulation.     

Sensitive assay for midazolam and its metabolite 1'-hydroxymidazolam in human plasma by capillary high-performance liquid chromatography

A sensitive high-performance liquid chromatographic method is described for the quantification of midazolam and 1'-hydroxymidazolam in human plasma. Sample (1 ml plasma) preparation involved a simple solvent extraction step with a recovery of approximately 90% for both compounds. An aliquot of the dissolved residue was injected onto a 3 microm capillary C18 column (150 mm x 0.8 mm I.D.). A gradient elution was used. The initial mobile phase composition (phosphate buffer-acetonitrile, 65:35) was maintained during 16 min and was then changed linearly during a 1-min period to phosphate buffer-acetonitrile, 40:60. The flow-rate of the mobile phase was 16 microl/min and the eluate was monitored by UV detection. The limits of quantification for midazolam and 1'-hydroxymidazolam were 1 ng/ml and 0.5 ng/ml, respectively. The applicability of the method was demonstrated by studying the pharmacokinetics of midazolam, and its major metabolite 1'-hydroxymidazolam, in human volunteers following i.v. bolus administration of a subtherapeutic midazolam dose (40 microg/kg).     

Sensitive and selective determination of methylenedioxylated amphetamines by high-performance liquid chromatography with fluorimetric detection

A rapid, sensitive and selective liquid chromatographic method with fluorimetric detection was developed for the separation and quantification of four methylenedioxylated amphetamines without interference of other drugs of abuse and common substances found in illicit tablets. The method was validated by examining linearity, precision and accuracy as well as detection and quantification limits. Methylenedioxylated amphetamines were quantified in eight tablets from illicit drug seizures and results were quantitatively compared to HPLC-UV analyses. To demonstrate the better sensitivity of the fluorimetric detection, methylenedioxylated amphetamines were analyzed in serum after a liquid-liquid extraction procedure and results were also compared to HPLC-UV analyses.     

Quantitative determination of domperidone in rat plasma by high-performance liquid chromatography with fluorescence detection

A sensitive and selective analytical method for the determination of domperidone in rat plasma is described. The procedure involves liquid-liquid extraction followed by reversed-phase high-performance chromatographic analysis with fluorometric detection at 282 nm for excitation and 328 nm for emission. The detection limit was 1 ng ml(-1) using 1 ml of plasma. This assay procedure should be useful for the pharmacokinetic study of domperidone in small animals such as rats.     

Automated and sensitive method for the determination of formoterol in human plasma by high-performance liquid chromatography and electrochemical detection

An automated high-performance liquid chromatography (HPLC) method for the determination of formoterol in human plasma with improved sensitivity has been developed and validated. Formoterol and CGP 47086, the internal standard, were extracted from plasma (1 ml) using a cation-exchange solid-phase extraction (SPE) cartridge. The compounds were eluted with pH 6 buffer solution-methanol (70:30, v/v) and the eluate was further diluted with water. An aliquot of the extract solution was injected and analyzed by HPLC. The extraction, dilution, injection and chromatographic analysis were combined and automated using the automate (ASPEC) system. The chromatographic separations were achieved on a 5 microm, Hypersil ODS analytical column (200 mm x 3 mm I.D.), using (pH 6 phosphate buffer, 0.035 M + 20 mg/l EDTA)-MeOH-CH3CN (70:25:5, v/v/v) as the mobile phase at a flow-rate of 0.4 ml/min. The analytes were detected with electrochemical detection at an operating potential of +0.63 V. Intra-day accuracy and precision were assessed from the relative recoveries of calibration/quality control plasma samples in the concentration range of 7.14 to 238 pmol/l of formoterol base. The accuracy over the entire concentration range varied from 81 to 105%, and the precision (C.V.) ranged from 3 to 14%. Inter-day accuracy and precision were assessed in the concentration range of 11.9 to 238 pmol/l of formoterol base in plasma. The accuracy over the entire concentration range varied from 98 to 109%, and precision ranged from 8 to 19%. At the limit of quantitation (LOQ) of 11.9 pmol/l for inter-day measurements, the recovery value was 109% and C.V. was 19%. As shown from intra-day accuracy and precision results, favorable conditions (a newly used column, a newly washed detector cell and moderate residual cell current level) allowed us to reach a LOQ of 7.14 pmol/l of formoterol base (3 pg/ml of formoterol fumarate dihydrate). Improvement of the limit of detection by a factor of about 10 was reached as compared to the previously described methods. The method has been applied for quantifying formoterol in plasma after 120 microg drug inhalation to volunteers. Formoterol was still measurable at 24 h post-dosing in most subjects and a slow elimination of formoterol from plasma beyond 6-8 h after inhalation was demonstrated for the first time thanks to the sensitivity of the method.