Antimalarial drug susceptibility testing of Plasmodium falciparum in Brazil using a radioisotope method

From March 1996 to August 1997, a study was carried out in a malaria endemic area of the Brazilian Amazon region. In vivo sensitivity evaluation to antimalarial drugs was performed in 129 patients. Blood samples (0.5 ml) were drawn from each patient and cryopreserved to proceed to in vitro studies. In vitro sensitivity evaluation performed using a radioisotope method was carried out with the cryopreserved samples from September to December 1997. Thirty-one samples were tested for chloroquine, mefloquine, halofantrine, quinine, arteether and atovaquone. Resistance was evidenced in 96.6% (29/30) of the samples tested for chloroquine, 3. 3% (1/30) for quinine, none (0/30) for mefloquine and none for halofantrine (0/30). Overall low sensitivity was evidenced in 10% of the samples tested for quinine, 22.5% tested for halofantrine and in 20% tested for mefloquine. Means of IC 50 values were 132.2 (SD: 46. 5) ng/ml for chloroquine, 130.6 (SD: 49.6) ng/ml for quinine, 3.4 (SD: 1.3) ng/ml for mefloquine, 0.7 (SD: 0.3) ng/ml for halofantrine, 1 (SD: 0.6) ng/ml for arteether and 0.4 (SD: 0.2) ng/ml for atovaquone. Means of chloroquine IC 50 of the tested samples were comparable to that of the chloroquine-resistant strain W2 (137.57 ng/ml) and nearly nine times higher than that of the chloroquine-sensitive strain D6 (15.09 ng/ml). Means of quinine IC 50 of the tested samples were 1.7 times higher than that of the low sensitivity strain W2 (74.84 ng/ml) and nearly five times higher than that of the quinine-sensitive strain D6 (27.53 ng/ml). These results disclose in vitro high resistance levels to chloroquine, low sensitivity to quinine and evidence of decreasing sensitivity to mefloquine and halofantrine in the area under evaluation.     

Simultaneous quantitative analysis of methyl salicylate,ethyl salicylate and salicylic acid from biological fluids using gas chromatography–mass spectrometry

A gas chromatographic–mass spectrometric (GC–MS) assay was developed for the quantitative analysis of methyl salicylate (MeS), ethyl salicylate (ES) and salicylic acid (SA) from biological fluids. The method was validated from 100-μl rat liver homogenate preparations (5 mg/ml protein) in 70 mM KH₂PO₄ (pH 7.4) buffer and from 100 μl rat plasma. The samples were extracted with chloroform, derivatized with BSTFA and quantitated by GC–MS in the SIM mode. The standard curves ranged from 31 ng/ml to 800 or 1250 ng/ml. Relative standard deviations and bias were less than 11% in plasma and homogenate for all compounds except SA which evidenced greater variability. The assay was used in preliminary experiments to characterize the pharmacokinetics of MeS in rats.     

Quantification of steroid glycosides from Hoodia gordonii in porcine plasma using high performance liquid chromatography-mass spectrometry

An HPLC-ESI-MS/MS method using collision induced dissociation - multiple reaction monitoring was developed for the quantification of eight Hoodia gordonii steroid glycosides and their metabolites in porcine plasma samples. The method was validated for the three most important glycosides and was successfully applied also for the related glycosides and metabolites. The limits of quantification were 0.04 ng ml(-1) for the two main steroid glycosides and 0.1 ng ml(-1) for the detiglated metabolites. These limits are sufficiently low to allow monitoring the concentration-time profiles in plasma after feeding H. gordonii. The standard deviations of the intra-day measurements were better than 20% for concentrations below 5 ng ml(-1) and better than 10% for concentrations above 5 ng ml(-1). The method was successfully applied to plasma samples collected from a porcine pharmacokinetics study.     

Headspace solid-phase microextraction in combination with gas chromatography and tandem mass spectrometry for the determination of organochlorine and organophosphorus pesticides in whole numan blood

A method for the determination of several organochlorine and organophosphorus pesticides in human whole blood samples was developed. The combination of solid-phase microextraction in headspace mode with gas chromatography with tandem mass spectrometry allowed the determination of 11 selected pesticides at ppb levels, minimizing the sample treatment. Quantitation was carried out by means of calibration curves prepared in blood using labelled surrogate/internal standards. The method showed good linearity between 1 and 50 ng ml(-1) (0.5-25 ng ml(-1) for HCB) using second-order calibration curves. Precision was found to be better than 20% at the three concentration levels assayed in the range of ng ml(-1). The detection limits obtained were in the range 0.02-0.7 ng ml(-1), except for p,p'-DDT (3 ng ml(-1)). The developed procedure was applied to blood and serum samples obtained from agricultural workers. HCB. beta-HCH and p,p'-DDE were most frequently detected in the samples analyzed.     

Determination of midazolam and its major metabolite 1'-hydroxymidazolam by high-performance liquid chromatography-electrospray mass spectrometry in plasma from children

We have developed a sensitive, selective and reproducible reversed-phase high-performance liquid chromatography method coupled with electrospray ionization mass spectrometry (HPLC-ESI-MS) for the simultaneous quantification of midazolam (MDZ) and its major metabolite, 1'-hydroxymidazolam (1'-OHM) in a small volume (200 microl) of human plasma. Midazolam, 1'-OHM and 1'-chlordiazepoxide (internal standard) were extracted from alkalinised (pH 9.5) spiked and clinical plasma samples using a single step liquid-liquid extraction with 1-chlorobutane. The chromatographic separation was performed on a reversed-phase HyPURITY Elite C18 (5 microm particle size; 100 mm x 2.1mm i.d.) analytical column using an acidic (pH 2.8) mobile phase (water-acetonitrile; 75:25% (v/v) containing formic acid (0.1%, v/v)) delivered at a flow-rate of 200 microl/min. The mass spectrometer was operated in the positive ion mode at the protonated-molecular ions [M+l]+ of parent drug and metabolite. Calibration curves in spiked plasma were linear (r2 > or = 0.99) from 15 to 600 ng/ml (MDZ) and 5-200 ng/ml (1'-OHM). The limits of detection and quantification were 2 and 5 ng/ml, respectively, for both MDZ and 1'-OHM. The mean relative recoveries at 40 and 600 ng/ml (MDZ) were 79.4+/-3.1% (n = 6) and 84.2+/-4.7% (n = 8), respectively; for 1'-OHM at 30 and 200 ng/ml the values were 89.9+/-7.2% (n = 6) and 86.9+/-5.6% (n = 8), respectively. The intra-assay and inter-assay coefficients of variation (CVs) for MDZ were less than 8%, and for 1'-OHM were less than 13%. There was no interference from other commonly used antimalarials, antipyretic drugs and antibiotics. The method was successfully applied to a pharmacokinetic study of MDZ and 1'-OHM in children with severe malaria and convulsions following administration of MDZ either intravenously (i.v.) or intramuscularly (i.m.).     

Quantitation of tamsulosin in human plasma by liquid chromatography-electrospray ionization mass spectrometry

A highly sensitive method for quantitation of tamsulosin in human plasma using 1-(2,6-dimethyl-3-hydroxylphenoxy)-2-(3,4-methoxyphenylethylamino)-propane hydrochloride as the internal standard (I.S.) was established using liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). After alkalization with saturated sodium bicarbonate, plasma were extracted by ethyl acetate and separated by HPLC on a C18 reversed-phase column using a mobile phase of methanol-water-acetic acid-triethylamine (620:380:1.5:1.5, v/v). Analytes were quantitated using positive electrospray ionization in a quadrupole spectrometer. LC-ESI-MS was performed in the selected ion monitoring (SIM) mode using target ions at m/z 228 for tamsulosin and m/z 222 for the I.S. Calibration curves, which were linear over the range 0.2-30 ng/ml, were analyzed contemporaneously with each batch of samples, along with low (0.5 ng/ml), medium (3 ng/ml) and high (30 ng/ml) quality control samples. The intra- and inter-assay variability ranged from 2.14 to 8.87% for the low, medium and high quality control samples. The extraction recovery of tamsulosin from plasma was in the range of 84.2-94.5%. The method has been used successfully to study tamsulosin pharmacokinetics in adult humans.     

Analgesic responses to intrathecal morphine in relation to CSF concentrations of morphine-3,beta-glucuronide and morphine-6,beta-glucuronide.

This study was performed to determine whether variations in analgesic responses to intrathecal morphine could be explained by cerebrospinal fluid (CSF) concentrations of morphine metabolites. Twenty-four CSF samples were collected at the beginning, middle and end of treatment periods in seven cancer patients with pain of malignant origin. CSF concentrations of morphine-3,beta-glucuronide (M3G) and morphine-6,beta-glucuronide (M6G) metabolites were measured by gas chromatography/mass spectrometry. Analgesic responses to morphine were estimated concurrent with CSF collection using a visual analog scale representing percentages of pain relief. Effective analgesia was defined as > or = 75% pain relief. CSF concentration of M3G and M6G in the 24 samples were 722 +/- 116 ng/ml and 699 +/- 158 ng/ml, respectively. CSF samples were categorized into two groups: (1) those collected during effective analgesia (N=14), and (2) those collected during ineffective analgesia (N=10). M6G levels detected in group 1 samples (effective analgesia) were significantly greater than those found in group 2 samples (ineffective analgesia) (978 +/- 243 ng/ml vs 309 +/- 68 ng/ml, P<0.05). Intergroup differences in CSF M3G concentrations and M3G/M6G ratios were not significant. It is concluded that CSF M6G may be indicative of effectiveness of analgesia in cancer patients subjected to intrathecal morphine.     

Determination of testosterone in saliva and blow of bottlenose dolphins (Tursiops truncatus) using liquid chromatography-mass spectrometry

A rapid, accurate and reproducible assay utilising high performance liquid chromatography-mass spectrometry (LC-MS) has been developed and validated for determining testosterone concentrations in saliva and blow of bottlenose dolphins. Sample preparation used solid phase extraction with specific preconditioning of cartridges. Analytes were eluted with 100% acetonitrile, dried under nitrogen and stored at -80 degrees C. Samples were reconstituted in 60% acetonitrile for LC-MS analysis. Chromatographic separation was achieved with an Alltech Macrosphere C8 stainless steel analytical column (2.1 mm x 150 mm i.d., 5 microm particle size, 300 angstroms pore size) using a 55% mobile phase B isocratic method (mobile phase A = 0.5% acetic acid; mobile phase B = 0.5% acetic acid, 90% acetonitrile). Samples were analysed in SIM at m/z 289.20 (testosterone mw 288.40) and a positive ion ESI. The limit of quantification was 0.5 ng/ml with a limit of detection of 0.2 ng/ml. The concentration curve was linear from 0.5 to 50 ng/ml (y = 0.01x + 0.0045, r(2) = 0.959, r = 0.979, p < 0.001). The R.S.D.s of intra- and inter-batch precision were less than 15% for saliva and 11% blow. Recovery of the assay for saliva was 93.0 +/- 7.9% (50 ng/ml) and 91.5 +/- 3.72% (1 ng/ml), and for blow was 83.3 +/- 6.8% (50 ng/ml) and 85.8 +/- 4.6% (1 ng/ml). Recovery of the internal standard in saliva was 73.0 +/- 14.2% and in blow was 78.63 +/- 4.29. The described assay was used to determine the presence of endogenous testosterone in saliva (9.73-23 ng/ml, n = 10) and blow (14.71-86.20 ng/ml, n = 11) samples of captive bottlenose dolphins.     

Social suppression of ovarian cyclicity in captive and wild colonies of naked mole-rats, Heterocephalus glaber

To investigate the endocrine cause of reproductive suppression in nonbreeding female naked mole-rats, animals from 35 colonies were studied in captivity. Urinary and plasma progesterone concentrations were elevated in pregnant females (urine: 10.0-148.4 ng/mg Cr, 27 samples from 8 females; plasma: 3.6-30.0 ng/ml, 5 samples from 5 females; Days 21-40 of pregnancy) and cyclic breeding females (urine: 0.5-97.8 ng/mg Cr, 146 samples from 7 females; plasma: less than 1.0-35.4 ng/ml, 25 samples from 7 females). The latter group showed cyclic patterns of urinary progesterone, indicating a mean ovarian cycle length of 34.4 +/- 1.6 days (mean +/- s.e.m.) with a follicular phase of 6.0 +/- 0.6 days and a luteal phase of 27.5 +/- 1.3 days (19 cycles from 9 breeding females). In non-breeding females urinary and plasma progesterone values were undetectable (urine: less than 0.5 ng/mg Cr, 232 samples from 64 females; plasma: less than 1.0 ng/ml, 7 samples from 6 females). Breeding females had higher (P less than 0.001) plasma LH concentrations (3.0 +/- 0.2 mi.u./ml, 73 samples from 24 females) than did non-breeding females (1.6 +/- 0.1 mi.u./ml, 57 samples from 44 females). Urinary and plasma progesterone concentrations in non-breeding females from wild colonies situated near Mtito Andei, Kenya, were either below the assay sensitivity limit (urine: less than 0.5 ng/mg Cr, 11 females from 2 colonies; plasma: less than 1.0 ng/ml, 25 females from 4 colonies), or very low (plasma: 1.6 +/- 0.6 ng/ml, 15 females from 4 colonies). In captivity, non-breeding females removed from their colonies (i.e. the dominant breeding female) and either paired directly with a non-breeding male (N = 2), or removed and housed singly for 6 weeks before pairing with a non-breeding male (N = 5) may develop a perforate vagina for the first time in as little as 7 days. Urinary progesterone concentrations rose above 2.0 ng/mg Cr (indicative of a luteal phase) for the first time 8.0 +/- 1.9 days after being separated. These results suggest that ovulation is suppressed in subordinate non-breeding female naked mole-rats in captive and wild colonies, and show that plasma LH concentrations are significantly lower in these non-breeding females. This reproductive block in non-breeding females is readily reversible if the social factors suppressing reproduction are removed.     

Circulating tumour necrosis factor-alpha bioactivity in rheumatoid arthritis patients treated with infliximab: link to clinical response

Our objective was to clarify the heterogeneity in response to infliximab treatment in rheumatoid arthritis (RA); to this end, a bioassay was designed to explore the contribution of circulating tumour necrosis factor (TNF)-alpha bioactivity and its possible link to response. The bioassay is based on the induction of IL-6 and osteoprotegerin (OPG) production by synoviocytes in response to TNF-alpha. RA synoviocytes were cultured with TNF-alpha (5 ng/ml) and 42 RA plasma samples collected just before starting therapy. Levels of IL-6 and OPG were measured in supernatants. In 20 of the patients, plasma samples collected before and 4 hours after the first and the ninth infusions were tested in the same way. Plasma concentrations of TNF-alpha and p55 and p75 soluble receptors were measured using ELISA. TNF-alpha induced IL-6 and OPG production by synoviocytes, which was further increased with patient plasma dilutions and inhibited by infliximab. With plasma samples obtained before the first infusion, the IL-6-induced production was greater in patients with a good clinical response than in the poor responders (44.4 +/- 23.3 ng/ml versus 27.4 +/- 20.9 ng/ml; P = 0.05). This high circulating TNF-alpha bioactivity was strongly inhibited with the first infliximab infusion. The difference between IL-6 levels induced with plasma samples obtained before and 4 hours after the first infusion was greater in patients with a good clinical response (40.0 +/- 23.7 ng/ml versus 3.4 +/- 10.0 ng/ml; P = 0.001). Similar findings were obtained for OPG production (7.0 +/- 6.2 ng/ml versus 0.0 +/- 3.0 ng/ml; P < 0.05). Levels of circulating TNF-alpha bioactivity were predictive of clinical response to TNF-alpha inhibition, confirming a key role for TNF-alpha in these RA patients.     

Prostacyclin metabolism in adults and neonates. Urinary profiles of 6-ketoprostaglandin F1 alpha and 2,3-dinor-6-ketoprostaglandin F1 alpha studied by gas chromatography-mass spectrometry

Metabolism of endogenous prostacyclin was studied in adults and neonates by measuring urinary levels of 6-ketoprostaglandin F1 alpha (spontaneous hydrolysis product) and 2,3-dinor-6-ketoprostaglandin F1 alpha (enzymatically formed by beta-oxidation). Quantification of prostanoids was achieved by capillary gas chromatography-mass spectrometry using the stable isotope dilution technique. Purification of the urinary lipid extract included silicic acid column chromatography and reverse- and straight-phase high-pressure liquid chromatographies. Accuracy of the method was proven by recovery experiments for both metabolites. Partial mass spectra of endogenous 6-ketoprostaglandin F1 alpha and 2,3-dinor-6-ketoprostaglandin F1 alpha were obtained from urine samples. In neonates (third day of life, n - 5 pooled urines) levels of 2,3-dinor-6-ketoprostaglandin F1 alpha (0.28 +/- 0.18 ng/ml) were much lower than those of 6-ketoprostaglandin F1 alpha (2.13 +/- 1.10 ng/ml), indicating low beta-oxidation activity at high prostacyclin formation. In adults (n = 7), levels of 2,3-dinor-6-ketoprostaglandin F1 alpha (0.27 +/- 0.21 ng/ml) and levels of 6-ketoprostaglandin F1 alpha (0.20 +/- 0.11 ng/ml) were about the same, indicating relatively high beta-oxidation at low prostacyclin formation. Values are expressed as mean +/- S.D.     

Determination of O-benzylguanine in human plasma by reversed-phase high-performance liquid chromatography

A high-performance liquid chromatographic assay for O⁶-benzylguanine utilizing liquid-liquid extraction and reversed-phase chromatography has been developed. Plasma samples were alkalinized, extracted into ethyl acetate, evaporated, and the residues were constituted and chromatographed. Separation was accomplished by gradient elution with a mobile phase of methanol, acetonitrile, and phosphate buffer, pH 3.2. Eluted compounds were detected spectrophotometrically at 280 nm. Sample quantitation was obtained from the regression line of six-point standard curves ranging from 25 to 400 ng/ml. O⁶-Benzylguanine peak heights were compared to peak heights of O⁶-(p-chlorobenzyl)guanine (internal standard). The average regression coefficient was 0.999 (n = 4). High concentration (305 ng/ml) and low concentration (38 ng/ml) quality control samples were determined with a day-to-day relative standard deviation of 7 and 8%, respectively (n = 18). The within-day relative standard deviations were 2.7 and 3.0% (n = 18) for the high and low concentration quality control specimens, respectively. Sample quantitation was reliable to 25 ng/ml with a signal-to-noise ratio of 8:1. This method was applied to plasma samples obtained from patients in a clinical trial of O⁶-benzylguanine.     

Development and validation of a reverse-phase HPLC with fluorescence detector method for simultaneous determination of CZ48 and its active metabolite camptothecin in mouse plasma

A simple and sensitive high-performance liquid chromatography (HPLC) assay for the analysis of CZ48, a potent anticancer candidate, and its active metabolite camptothecin (CPT) in mouse plasma was developed and validated. CZ44 was used as an internal standard (IS). The samples were injected onto a C18 Synergi Polar-RP column (4 microm, 150 mm x 4.60 mm) maintained at 30 degrees C. The identification of peaks showed high specificity. Shimadzu RF-10AXL fluorescence detector was used at the excitation and emission of 380 and 418 nm, respectively. The mean recoveries were 81.41+/-0.035%, 86.00+/-0.053% and 82.21+/-0.020% for CZ48 and 76.01+/-0.028%, 77.04+/-0.042% and 85.93+/-0.023% for CPT at three concentrations of 10, 100 and 900 ng/ml, respectively. The calibration curve was linear (r(2)=0.9999) over CZ48 and CPT concentrations ranging from 5 to 1000 ng/ml and 10-1000 ng/ml (n=6), respectively. The method had an accuracy of >95% and intra- and inter-day precision (RE%) of <1.2% and <2.2% for CZ48 and CPT, respectively, at three different concentrations (10, 100 and 900 ng/ml). The lower limit of quantification (LLOQ) using 0.1 ml mouse plasma was 10 ng/ml for CZ48 and 5 ng/ml for CPT. Stability studies showed that CZ48 and CPT were stable in mouse plasma after 4h incubation at room temperature or after 1 month storage at -80 degrees C with three freeze/thaw cycles. The method reported is simple, reliable, precise and accurate and confirmed by the determination of plasma samples in the mice after oral administration of CZ48.     

Liquid chromatography-tandem mass spectroscopy assay for quercetin and conjugated quercetin metabolites in human plasma and urine

A sensitive and specific method was developed and validated for the quantitation of quercetin in human plasma and urine. The application of liquid chromatography-tandem mass spectrometry (LC/MS/MS) with a TurboIonspray (TIS) interface in negative mode under multiple reactions monitoring was investigated. Chromatographic separation was achieved on a C12 column using a mobile phase of acetonitrile/water with 0.2% formic acid (pH 2.4) (40/60, v/v). The detection limit was 100 pg/ml and the lower limit of quantification was 500 pg/ml for plasma samples; the detection limit was 500 pg/ml and the lower limit of quantification was 1 ng/ml for urine samples. The calibration curve was linear from 1 to 800 ng/ml for plasma samples and was linear from 1 to 200 and 50 to 2000 ng/ml for urine samples. All the intra- and inter-day coefficients of variation were less than 11% and intra- and inter-day accuracies were within +/-15% of the known concentrations. This represents a LC/MS/MS assay with the sensitivity and specificity necessary to determine quercetin in human plasma and urine. This assay was used to determine both parent quercetin and the quercetin after enzymatic hydrolysis with beta-glucuronidase/sulfatase in human plasma and urine samples following the ingestion of quercetin 500 mg capsules.     

小麦中脱氧雪腐镰刀菌烯醇酶联免疫吸附测定方法的研究

用B淋巴细胞杂交瘤技术制备了能稳定分泌抗脱氧雪腐镰刀菌烯醇(DON)单克隆抗体的杂交瘤细胞株,命名为3D7.3D7的亚类为lgG_1.运用该抗体,建立了检测小麦中DON的间接竞争性酶联免疫吸附测定法.该法最低检出量为5ng/ml,敏感范围为5—1000ng/ml;平均回收率为96.6—107.3%;精密度为6.2—13.3%.运用该方法,检测了10份小麦样品.均为阳性,其含量为56.4—1002.0ppb.     

Selective and sensitive liquid chromatographic assay of amodiaquine and desethylamodiaquine in whole blood spotted on filter paper

We have developed a sensitive, selective and reproducible reversed-phase HPLC method with ultraviolet detection (340 nm) for the simultaneous quantification of amodiaquine (AQ) and its major metabolite, desethylamodiaquine (AQm) in a small volume (200 microl) of whole blood spotted on filter paper. The method involves liquid-liquid extraction with diethyl ether followed by elution from a reversed-phase phenyl column with an acidic (pH 2.8) mobile phase (25 mM KH2PO4-methanol; 80:20% (v/v) +1% (v/v) triethylamine). Calibration curves in spiked whole blood were linear from 100-2500 ng/ml (r2 > or = 0.99) for AQ and 200-2500 ng/ml (r2 > or = 0.99) for AQm. The limit of detection was 5 ng for AQ and 10 ng for AQm. The relative recovery at 150 ng/ml of AQ (n = 6) was 84.0% and at 300 ng/ml of AQm the relative recovery was 74.3%. The intra-assay coefficients of variation at 150, 600 and 2250 ng/ml of AQ and 300, 600 and 2250 ng/ml of AQm were 7.7, 8.9 and 6.2% (AQ) and 10.1, 5.4 and 3.9% (AQm), respectively. The inter-assay coefficient of variation at 150, 600 and 2250 ng/ml of AQ and 300, 600 and 2250 ng/ml of AQm were 5.2, 8.1 and 6.9% (AQ) and 3.3, 2.3 and 4.6% (AQm). There was no interference from other commonly used antimalarial and antipyretic drugs (chloroquine, quinine, sulfadoxine, pyrimethamine, artesunate, acetaminophen and salicylate). The method is particularly suitable for pharmacokinetic studies in settings where facilities for storing blood/plasma samples are not available.     

Serum progesterone concentration in pyometritic and normal postpartum dairy cows

Two studies were conducted to evaluate the concentration of serum progesterone in pyometritic cows and relate it to palpation of ovarian structures per rectum . In Trial 1, serum samples from 34 pyometritic cows were assayed for progesterone. Mean serum progesterone concentrations were 6.8 +/- 0.7 ng/ml. In Trial 2, each of 54 pyometritic cows was paired with a control cow on the basis of days post partum (18-50 days). Mean concentration of progesterone was 9.7 +/- 1.0 ng/ml for the pyometritic cows and 5.7 +/- 0.8 ng/ml in control cows (P<0.005). Progesterone concentration was greater (P<0.005) in both groups of cows with palpable corpora lutea (CLs). Ninety-six percent of the pyometritic cows had palpable CLs compared to 57% of the control group. Comparing serum progesterone only in cows with a palpable CL, the mean concentration was still greater (P<0.005) in the pyometritic group (10.6 +/- 1.0 ng/ml) than the control group (6.6 +/- 1.0 ng/ml). Compatability of rectal palpation findings and concentrations of serum progesterone were 92.6% for the pyometritic group and 72.2% for the control group. Progesterone concentration increased (P<0.05) by increased days post partum in Trial 2 (n=54) but not in Trial 1 (n=23). In both Trials 1 and 2, uterine size due to pyometra increased (P<0.05) with increased days post partum. No other associations were found.     

Determination of riboflavin by high-performance liquid chromatography with riboflavin-depleted urine as calibration and control matrix

A simple method, exposure to natural-light, was developed to remove riboflavin from urine to enhance its use as the biological matrix for the preparation of calibration and control samples. Riboflavin-depleted urine containing less than 1 ng/ml of riboflavin was used to validate a high-performance liquid chromatography with fluorescence detection method for the determination of urinary riboflavin. The linearity of the assay (r2=0.999) was acceptable over the range of 10-5000 ng/ml. The intra-assay and inter-assay CVs were 3.3% and 9%, respectively. Subsequent stability studies found that urine riboflavin was stable for up to 6 months at 4 or -20 degrees C.     

Incidence and characteristics of microprolactinomas (3-5 mm) in 4199 women assayed for prolactin.

Difficulties and controversies still exist in the diagnosis of small (3-5 mm) prolactinomas (micro-PRL-omas). In the present study serum prolactin (PRL) was assayed in 4199 women aged 14-43 years belonging to 4 groups: A: 753 women with normal cycles (NC) and infertility (control group), B: 2523 with menstrual disorders, C: 519 with NC and hirsutism, D: 404 with galactorrhoea. The distribution of PRL values from 1 to 30 ng/ml was almost similar in the subjects of group A, B and C. Within this range the vast majority of subjects (91%, 92.2% and 88% respectively in these 3 groups and 83% in group D) had PRL levels from 1 to 15 ng/ml and together with the proportion of subjects with PRL values 16 to 20 ng/ml they included 96.7% of the entire mixed population. A proportion of scattered outlying PRL values above 30 ng/ml was found in each group (A = 2%, B = 3%, C = 1% and D = 28.7%) and in this subset 117 prolactinomas (PRL-omas) were found, 19 (23%) in the 83 subjects with PRL levels 31-49 ng/ml and 98 (75.3%) in the 130 subjects with PRL values greater than or equal to 50 ng/ml. Of the 117 PRL-omas 9 were bigger than 10 mm and 4 had a size from 6 to 9 mm. In the remaining 104 the size was presumed from direct or indirect radiological evidence to be 3-5 mm.(ABSTRACT TRUNCATED AT 250 WORDS)     

A key functional role for the insulin-like growth factor 1 N-terminal pentapeptide.

In order to elucidate the role of the N-terminus of insulin-like growth factor 1 (IGF-1) with respect to its biological properties, we chemically synthesized analogues of IGF-1 truncated by one to five amino acid residues from the N-terminus. In a bioassay that measured the stimulation of protein synthesis in rat L6 myoblasts, the concentrations required to produce a half-maximal response were: IGF-1, 13 ng/ml; des-(1)-IGF-1, 10 ng/ml; des-(1-2)-IGF-1, 13 ng/ml; des-(1-3)-IGF-1, 1.5 ng/ml; des-(1-4)-IGF-1, 5.1 ng/ml; des-(1-5)-IGF-1, 1200 ng/ml. When tested for their abilities to compete with 125I-IGF-1 binding to L6 myoblasts at 3 degrees C, the concentrations required for 50% competition were: IGF-1, des-(1)-IGF-1 and des-(1-2)-IGF-1, 20 ng/ml; des-(1-3)-IGF-1, 14 ng/ml; des-(1-4)-IGF-1, 40 ng/ml; des-(1-5)-IGF-1, greater than 1000 ng/ml. Receptor-binding experiments at 25 degrees C, however, gave results suggesting that the myoblasts were secreting a binding protein selective for the three longest peptides. This interpretation was confirmed by binding studies with medium conditioned by the L6 myoblasts as well as binding protein purified from MDBK-cell-conditioned medium. In both cases IGF-1, des-(1)-IGF-1 and des-(1-2)-IGF-1 competed for tracer IGF-1 binding at least 60-fold better than did the three shorter peptides. The results obtained account for the increased potency of des-(1-3)-IGF-1 and des-(1-4)-IGF-1, since their activities are not attenuated by the binding protein, and the relatively lower potency of des-(1-4)-IGF-1 is a consequence of this peptide binding less well to the L6-myoblast receptor.