Using of HPLC coupled with coulometric detector for the determination of biotin in pharmaceuticals.

The method for the determination of biotin by high performance liquid chromatography (HPLC) coupled with coulometric detector is presented here. Chromatographic and detection conditions were tested. A LiChrospher 60RP-select B column (250 mm x 4 mm; 5 microm) and the mobile phase containing 0.24 mol/L aqueous solution of acetic acid and acetonitrile in the ratio 85:15 (v/v) were found as the most suitable. The flow rate was 1 mL/min and the injected volume of the sample was 20 microL. The hydrodynamic voltammogram of biotin was measured and according to obtained data the detection parameters were set--channel I 600 mV, channel II 900 mV, sensitivity 1 microA. The developed method has been validated. The calibration curve is linear in the range 15-3600 ng/mL, correlation coefficient is 0.9998, limits of detection and quantification are 5 and 15 ng/mL, respectively. Recovery of the spiked samples was 98.67% with R.S.D. 0.255% on average. The developed method has been successfully applied for determination of biotin in pharmaceutical preparations.

Somatotropin has no effect on the quantity of guanine nucleotide binding proteins Gqalpha/G11alpha in goat adipose tissue in vivo.

The decapeptide QLNLKEYNLV corresponding to the C-terminus of Gq/G11alpha guanine nucleotide-binding proteins (G-proteins) was synthesized by the solid-phase method and conjugated to keyhole limpet hemocyanin. The rabbits were immunized with these conjugates and an antiserum that reacted specifically with the alpha subunit of Gq/G11 proteins was used in this study. The antiserum exhibited no cross-reactivity with the alpha subunits of stimulatory (Gs) or inhibitory (Gi) G-proteins associated with adenylate cyclase. Immunoblots with the antiserum showed that it specifically recognized the Gq/G11 alpha-proteins in cholate extracts of adipose tissue membranes of goats. Treatment of young castrated male goats with bST had no effect on the quantity of Gq/G11 alpha subunits in adipose tissue and the results thus obtained did not support the idea that the bST signal in adipose tissue is transmitted via Gq/G11 alpha-proteins.

Electron microscopic demonstration of the penetration of liposomes through skin.

To verify the penetration of liposomes through skin, we have used liposomes with encapsulated protein G-gold conjugate in a gel vehicle. Skin samples were examined 2, 4, 24, 48, and 72 h after liposome application. Our findings show that the penetration of liposomes through skin depends mainly on their size. Liposomes up to 600 nm in diameter penetrate through skin rather easily, whereas liposomes 1000 nm and more in diameter remain interiorized in the stratum corneum. The main penetration of liposomes proceeds along the hair sheaths as indicated by larger amounts of free liposomes in the corium of guinea pigs.

Plasma beta-endorphin, bombesin and pancreatic polypeptide immunoreactivity in healthy subjects and ulcer patients and changes during treatment with pirenzepine.

A lower level of BEI was demonstrated in duodenal ulcer patients in both acute and chronic cases (P less than 0.01). Treatment of patients with PI increases BEI up to values found in healthy subjects, in accordance with our previous findings (17). The basal level of BOI in ulcer patients did not differ from that in healthy subjects. A transient increase of BOI was recorded during the first phase after PI application, however, BOI decreased to basal levels at the end of the therapy (P less than 0.05). The basal PPI level was higher in both groups, primarily in acute ulcer patients (P less than 0.01). During the treatment with PI the PPI level slightly increased. Mathematical correlation between BEI and BOI, and BEI and PPI revealed a significant negative correlation (P less than 0.001). However, this correlation was found in healthy subjects only, indicating that healthy subjects with a high BEI level have lower BOI and PPI values and this relation depends on BEI. This dependence is absent in ulcer patients.

[Efficient diagnosis of hypothalamo-hypophyseal nanism: 5 years' experience with the clonidine stimulation test in the diagnosis of growth hormone secretion]. / Na pomoc úcelné diagnostice hypotalamo-hypofyzárního nanismu: petileté zkusenosti s clonidinovým stimulacnim testem v diagnostice sekrece rustového hormonu.

The clonidine stimulation test, using doses of 37.5--75 micrograms clonidine and assessment of growth hormone levels at times 0, 60 minutes, is a suitable, safe and relatively reliable screening test for ambulatory work. As ensues from examinations of 156 children, it is possible to select by this test, as compared with the test of insulin induced hypoglycaemia, more than 70% children with a normal post-stimulation response of growth hormone.

Mutual correlation between levels of plasma beta-endorphin, bombesin and pancreatic polypeptide in healthy subjects and ulcer patients.

A lower level of beta-endorphin (BE) was demonstrated in patients with both acute and chronic duodenal ulcer (P less than 0.01), while the basal level of bombesin (BO) in such patients did not differ from that in healthy subjects. The basal pancreatic polypeptide (PP) level was higher in both groups of patients, primarily in those with acute ulcer (P less than 0.01). A significantly negative correlation (P less than 0.001) between the levels of BE and BO, and between those of BE and PP, was found in healthy subjects. Similar interrelation was absent in ulcer patients.

[Analogs of gonadoliberin in the treatment of endometriosis and uterine myoma. Comparison of daily subcutaneous treatment and intramuscular depot therapy]. / Analoga gonadoliberinu v lécbe endometriózy a delozních myomu.Srovnání s.c. lécby denní a i.m. lécby depotní.

Seven women with endometriosis and three women with uterine myomas were treated for six months with tryptoreline (Decapeptyl, Decapeptyl Depot, Ferring). In six patients with endometriosis improvement was achieved according to the AFS classification and the finding on palpation and ultrasonic examination. In three patients with uterine myoma a reduction by 25-100% was recorded. Dysmenorrhoea and pain in the hypogastrium disappeared completely in all patients except one. The LH and FSH levels declined and E2 levels were similar as in the menopause. The menstrual cycle disappeared in the course of treatment and all women experienced flushes. Daily s. c. and monthly depot doses gave similar results. Four months after treatment the hormonal levels and the menstrual cycle were normal. Endometriosis disappeared twice, four times minor endometriotic nodes persisted, once a uterine myoma of half the size reappeared. Treatment with LH-RH agonists in endometriosis matches danazole treatment in uterine myomas it is suitable for special cases.

Monoclonal antibodies against human alpha-fetoprotein. Exploitation of an unusual calcium-dependent interaction with the antigen for analytical and preparative purposes.

Seven murine hybridomas were established producing IgG1 monoclonal antibodies (mAbs) against three different epitopes of human alpha-fetoprotein (alpha FP). The interaction of two of these mAbs (AFP-01 and AFP-02) with the antigen was strongly inhibited by calcium-chelating agents. The mAb AFP-11 could be used for estimation of alpha FP by RIA in the concentration range of 5-100 ng/ml. Several combinations of the mAbs could be used in a two-site sandwich-ELISA for similar purposes. Immunoaffinity chromatography on a column of immobilized mAb AFP-01 permitted the purification of alpha FP.

A simple and rapid method for the isolation of human alpha-fetoprotein from human cord serum.

A description is given of a simple and time-saving method for the isolation of human alpha-fetoprotein (AFP) by means of liquid chromatography. The isolation was made in three steps: Step 1--affinity chromatography on column CNBr-Sepharose 4 B coupled with sheep antibody against human alpha-fetoprotein. AFP was eluted from the column by a change pH. Step 2--application of AFP to column Blue-Sepharose CL-6B for the removal of albumin remnants. Step 3--additional purification of the AFP preparation on column Con-A-Sepharose. AFP was eluted from the column by using methyl-alpha-D-glucepyranoside gradient. The isolation yield was 20%. The purity of the AFP preparation was satisfactory for RIA.