Biomonitoring of ochratoxin A in grain workers

Handling agricultural commodities such as grain can result in an inhalation of mycotoxin-containing dusts. Ochratoxin A (OTA) is particularly well suited for biomonitoring studies due to its long half-life in blood, and served as a marker toxin to investigate whether or not exposure to dusts in occupational contexts may result in elevated OTA blood serum levels. OTA analysis was performed for blood samples (n=61) obtained from a cohort of male workers employed at granaries of several grain handling companies in Germany. OTA was analyzed in plasma extracts by HPLC with fluorimetric detection; calibration curves were run for each batch of samples collected between July 2005 and March 2006, and the level of detection was 0.05 ng/ml plasma. The OTA plasma levels of the 61 grain workers ranged between 0.07 ng/ml and 0.75 ng/ml. The mean (0.28±0.13 ng/ml) and median (0.26 ng/ml) OTA value for this cohort was similar to average values previously reported for the German population. Our results gave no indication that OTA in excess of those originating from typical dietary sources was ingested by these workers. Although measurable OTA concentrations have been found in dust samples collected at the corresponding workplaces (Mayeret al, this issue), the biomonitoring data do not provide evidence for a significant inhalatory burden of OTA in grain workers. Since deoxynivalenol and zearalenone were also detected in the dust samples in concentrations much higher than that of OTA, additional research should try to assess the potential relevance of an inhalation exposure to these mycotoxins. Presented at the 28^th Mykotoxin-Workshop, Bydgoszcz, Poland, May 29–31, 2006     

Serum progesterone and corpus luteum function in pregnant pigtailed monkeys (Macaca nemestrina)

Corpus luteum (CL) function and control during pregnancy and early lactation in the pigtailed macaque was investigated. Peripheral concentrations of progesterone (P) on day 10 of pregnancy were 12.98 ± 2.21 ng/ml and decreased progressively to 7.96 ± 1.27 ng/ml by day 21 of pregnancy. The concentration of P increased around day 27 of gestation and reached peak levels of 18.48 ± 2.45 ng/ml on day 37, there-after gradually decreasing to a nadir at about midgestation. Ten days before parturition P concentrations increased again (P < 0.05). Concentrations of P decreased from 6.62 ± 1.48 ng/ml on the day of delivery to 2.16 ± 0.43 ng/ml on day 2 of lactation and remained low thereafter. Ovariectomy on day 35 did not affect the normal course of gestation or the patterns of P secretion during pregnancy. However, in these ovariectomized animals, in spite of suckling, P was not detectable after parturition. In intact monkeys, serum concentrations of P in the uteroovarian vein at days 80 and 159 of pregnancy were higher relative to the uterine vein. Incubation studies utilizing ³H-cholesterol as a substrate revealed that the CL were capable of synthesizing P on days 35 and 159 of gestation. Histologically, the CL contained active luteal cells at late pregnancy.Low serum concentrations of chorionic gonadotropin were detected on day 10 of gestation; concentrations of this hormone reached high levels between days 18 and 24 and the titers were nondetectable after day 40 of pregnancy. Luteinizing hormone was present in constant amounts in the circulation during pregnancy and lactation.These data suggest that the CL of pregnancy in the pigtailed monkey is functional or capable of functioning during various stages of pregnancy. However, the fetoplacental unit is the primary source of P during the latter 4.5 months of gestation. As in other primates, a functional CL is not required for maintenance of pregnancy after implantation nor for lactation. Thus, the physiological significance of CL function during pregnancy is unclear.     

Circulating isoflavonoid levels in CD-1 mice: effect of oral versus subcutaneous delivery and frequency of administration

The CD-1 mouse is a commonly used animal model to understand the biological effects of early-life exposure to soy isoflavones in infants. Most studies using CD-1 mice have administered isoflavones by daily subcutaneous injection, while infants receive oral feeds every few hours. The study objectives were to compare the total serum levels of genistein (GEN), daidzein (DAI) and the DAI metabolites equol and O-desmethyl-angolensin (O-DMA), after subcutaneous injection and oral dosing and to determine if frequency of oral administration results in different circulating levels of isoflavones using the CD-1 mouse model. From postnatal days 1 to 5, pups randomly received corn oil or soy isoflavones (total daily dose, 0.010 mg DAI+0.025 mg GEN) by subcutaneous injection once a day, orally once a day or orally every 4 hours. On postnatal day 5, 1 h posttreatment, mice were killed and serum was collected. Mice treated with soy isoflavones had higher (P<.05) serum GEN (female: 1895–3391 ng/ml and male: 483–578 ng/ml) and DAI (female: 850–1580 ng/ml and male: 248–322 ng/ml) concentrations versus control (5–20 ng/ml) mice, regardless of route or frequency of administration, and were similar among dosing strategies. Total serum concentrations of GEN and DAI were higher (P<.05) among females (GEN: 2714 ± 393 ng/ml and DAI: 1205 ± 164 ng/ml) than males (GEN: 521 ± 439 ng/ml and DAI: 288 ± 184 ng/ml) across treatment groups. Serum equol and O-DMA concentrations were negligible (<3 ng/ml) across groups. In conclusion, different routes of delivery and frequency of administration resulted in similar total serum levels of GEN, DAI¸ equol or O-DMA.     

Effect of calf removal on serum luteinizing hormone and cortisol concentrations in postpartum beef cows

Serum luteinizing hormone (LH) and cortisol concentrations were measured in ten fall calving, Angus cows averaging 38 +/- 8 days postpartum. Calves from five cows were weaned at the beginning of the study. Blood samples were collected at 20 min. intervals for 48 h after weaning and for 8 h on day 4 and day 6 postweaning. Mean serum LH concentrations increased (P<0.01) in weaned cows (W) from 0.55 +/- 0.01 ng/ml at time of calf removal to 1.3 +/- 0.04 ng/ml 48 h afterwards. Comparable LH concentrations for suckled cows (S) were 0.65 +/- 0.08 ng/ml and 0.62 +/- 0.03 ng/ml respectively. Average serum LH concentrations at 48 h after weaning were greater (P<0.01) for W cows than S cows and a treatment by time interaction occurred (P<0.01) with serum LH concentrations increasing (P<0.01) from time of calf removal to 48 h after calf removal in W cows. Frequency of LH peaks increased (P<0.01) in W cows and by 48 h after weaning was greater (P<0.01) in W cows than in S cows. Magnitude of LH peaks did not differ between the two groups. Serum cortisol concentrations were not different between W and S cows except for a transient elevation (P<0.01) in W cows from 7.6 +/- 0.9 ng/ml to 11.9 +/- 1.0 ng/ml 9 to 12 h after calf removal. Since serum LH concentrations were increased in W cows but not in S cows at 48 h and serum cortisol concentrations increased transiently in W cows we suggest that circulating cortisol levels may not be a physiological inhibitor of LH secretion in the suckled postpartum beef cow.     

Pharmacokinetic and pharmacodynamic interactions between simvastatin and diltiazem in patients with hypercholesterolemia and hypertension

Pharmacokinetic and pharmacodynamic interactions between simvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, and diltiazem, a calcium antagonist, were investigated in 7 male and 4 female patients with hypercholesterolemia and hypertension. The patients were given, for one in a three consecutive 4-week periods, oral simvastatin (5 mg/day), oral simvastatin (5 mg/day) combined with diltiazem (90 mg/day), and then oral diltiazem (90 mg/day), respectively. The area under the plasma concentration versus time curve up to 6 hours post-dose (AUC0-6h) and maximum plasma concentrations (Cmax) of the drugs, serum lipid profiles, blood pressures and liver functions were assessed on the last day of each of the three 4-week periods. After the combined treatment period, Cmax of HMG-CoA reductase inhibitor was elevated from 7.8 +/- 2.6 ng/ml to 15.4 +/- 7.9 ng/ml (P < 0.01) and AUC0-6h from 21.7 +/- 4.9 ng x hr/ml to 43.3 +/- 23.4 ng x hr/ml (P < 0.01), while Cmax of diltiazem was decreased from 74.2 +/- 36.4 ng/ml to 58.6 +/- 18.9 ng/ml (P < 0.05) and its AUC0-6h from 365 +/- 153 ng x hr/ml to 287 +/- 113 ng x hr/ml (P < 0.01). Compared to simvastatin monotherapy, combined treatment further reduced LDL-cholesterol levels by 9%, from 129 +/- 16 mg/dl to 119 +/- 17 mg/dl (P < 0.05). No adverse events were observed throughout the study. These apparent pharmacokinetic interactions, namely the increase of HMG-CoA reductase inhibitor concentration by diltiazem and the decrease of diltiazem concentration by simvastatin, enhance the cholesterol-lowering effects of simvastatin during combined treatment.     

Endocrine and ultrasonographic characterization of a successful pregnancy in a Sumatran rhinoceros (Dicerorhinus sumatrensis) supplemented with a synthetic progestin

A Sumatran rhinoceros with a history of early pregnancy loss was supplemented with a synthetic progestin, altrenogest (Regu‐Mate^®), and delivered a healthy, full‐term calf 475 days after mating. Serum hormone concentrations were measured throughout gestation, and ultrasonography was used to monitor embryo/fetal growth and viability. The embryonic vesicle growth curve was characterized by three phases: rapid expansion, plateau, and a final rapid expansion, and was similar to that in the domestic horse. Fetal sex was determined by ultrasound on day 73 of gestation. After day 80 of gestation, transabdominal examinations were more useful than rectal examinations for imaging the fetus. Serum progesterone concentrations remained at luteal levels (1.5±0.5 ng/ml) for the first 2 months of pregnancy, and then they gradually increased. However, progesterone decreased almost to luteal levels during the fifth month before it increased again, and eventually reached peak concentrations (13.3±1.9 ng/ml) shortly before parturition. Relaxin concentrations remained basal (≤0.5 ng/ml) for the first half of the pregnancy, increased to 2.7±1.2 ng/ml and stabilized until 2 weeks before parturition, when relaxin spiked to unusually high concentrations (800–1300 ng/ml). Prolactin concentrations were at baseline (7.2±1.7 ng/ml) throughout most of the gestation, but rose markedly 2 weeks before parturition, reaching concentrations as high as 75 ng/ml. Attempts to measure serum estrogen concentrations were unsuccessful. These data represent the first attempt to characterize pregnancy in the critically endangered Sumatran rhinoceros, a species that heretofore had not successfully reproduced in captivity for 112 years. Zoo Biol 23:219–238, 2004. © 2004 Wiley‐Liss, Inc.     

Effects of kepone on growth and respiration of several estuarine bacteria

The toxicity of Kepone to mixed populations of estuarine microorganisms was determined by standard plate assays on Zobell marine medium containing 0.02, 0.20, and 2.0 mg of Kepone per liter. Under aerobic conditions, Kepone reduced the number of colony-forming units at all concentrations tested, but had no effect on the number of anaerobic microorganisms. Gram-positive organisms were more sensitive to Kepone than were gram-negative organisms. Growth of gram-negative isolates was not inhibited in nutrient broth, but was significantly inhibited in a minimal salts broth. Oxygen uptake by most isolates was reduced 25 to 100% by 20 ppm (20 mg/ml) of Kepone. Oxygen evolution was observed when several gram-positive isolates were exposed to Kepone concentrations of 20 ppm. Pentachlorophenol at concentrations above 28 ppm produced effects similar to those produced by Kepone. Inhibition of electron transport by Kepone was demonstrated by a significant reduction in the specific activities of NADH oxidases and succinooxidase.     

Effects of kepone on growth and respiration of several estuarine bacteria.

The toxicity of Kepone to mixed populations of estuarine microorganisms was determined by standard plate assays on Zobell marine medium containing 0.02, 0.20, and 2.0 mg of Kepone per liter. Under aerobic conditions, Kepone reduced the number of colony-forming units at all concentrations tested, but had no effect on the number of anaerobic microorganisms. Gram-positive organisms were more sensitive to Kepone than were gram-negative organisms. Growth of gram-negative isolates was not inhibited in nutrient broth, but was significantly inhibited in a minimal salts broth. Oxygen uptake by most isolates was reduced 25 to 100% by 20 ppm (20 mg/ml) of Kepone. Oxygen evolution was observed when several gram-positive isolates were exposed to Kepone concentrations of 20 ppm. Pentachlorophenol at concentrations above 28 ppm produced effects similar to those produced by Kepone. Inhibition of electron transport by Kepone was demonstrated by a significant reduction in the specific activities of NADH oxidases and succinooxidase.     

Cytokine profile in cases with premature elevation of progesterone serum concentrations during ovarian stimulation

The aim of this study was to investigate the concentrations of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), leptin, tumor necrosis factor-alpha, interleukin (IL)-1beta and IL-6, in cycles with a premature rise of serum progesterone. 25 intracytoplasmic sperm injection (ICSI) cycles with (Group 1) and 25 ICSI cycles without a premature progesterone elevation (Group 2) were included. The cut-off value of serum progesterone on the day of human chorionic gonadotropin (hCG) administration was 0.9 ng/ml. The indication for ICSI was male factor infertility exclusively. On the day of hCG injection, serum IL-6, VEGF and bFGF were significantly higher in Group 1 (7.7+/-24.5 pg/ml, 290.2+/-161.4 pg/ml and 15.7+/-8.2 ng/ml respectively) than in Group 2 (1.7+/-0.7 pg/ml, 175.2+/-92.1 pg/ml, and 9+/-1.6 ng/ml respectively). On the day of follicular puncture, serum cytokine concentrations were similar in the two groups. IL-6 intrafollicular concentrations were higher in Group 1 (14.7+/-20.7 pg/ml) than in Group 2 (9+/-9.3 pg/ml, p=0.031). There were no differences regarding the ICSI outcome. Patients with serum progesterone above 0.9 ng/ml, have elevated serum concentrations of IL-6, VEGF, and bFGF, as well as elevated intrafollicular concentrations of IL-6. The outcome of ICSI cycles is not associated with premature elevation of progesterone when the cut-off value is set at 0.9 ng/ml.     

Radioimmunoassay of [D-Trp6]-luteinizing hormone-releasing hormone: its application to animal pharmacokinetic studies after single injection and long-acting formulation administration

A sensitive radioimmunoassay (RIA) for [D-Trp6]-luteinizing hormone-releasing hormone (LHRH) has been developed. This assay allowed measurement of the LHRH analog in unextracted plasma with a minimum detectable concentration of 10 pg/ml. Validation of plasma assays was performed through Sep-Pak and HPLC purification. The in vivo fate of the peptide was investigated in dogs after subcutaneous or intravenous injections. In both cases, the LHRH analog showed longer plasma half-life than native LHRH with an elimination half-life superior to 80 min. Long-acting formulations were tested in dogs and rats: the day following administration, [D-Trp6]-LHRH plasma level rose to 2.9-4.6 ng/ml in dogs and 0.8-3.8 ng/ml in rats. From day 4 to day 30, [D-Trp6]-LHRH plasma level followed a plateau with concentrations of 0.3-0.8 ng/ml in dogs and 0.2-0.4 ng/ml in rats. In parallel, testosterone plasma concentration was reduced to castrate level between day 4 and day 7 in dogs and was significantly lowered in rats. This sensitive [D-Trp6]-LHRH RIA will be particularly useful for the evaluation of long-acting formulations in patients with advanced prostate cancer.     

Artificial insemination with frozen,thawed semen and pregnancy diagnosis in addax (Addax nasomaculatus)

Nonsurgical artificial insemination (AI) procedures were performed using frozen, thawed semen in two adult female addax. Pregnant mares' serum gonadotropin (PMSG) (300 IU) and prostaglandin (PG) (125 μg) was administered intramuscularly to female 1 on days ?3 and ?1, respectively, where day 1 was the first day of AI. Only one injection of PG was used, as a low serum progesterone (PROG) concentration (0.5 ng/ml) and a low pregnanediol glucuronide (PdG) concentration (0.36 ng PdG/mg creatinine) had been recorded on day ?13. A total of 219 × 10⁶ motile sperm was deposited in the os cervix or anterior vagina on days 1–4. High serum PROG levels (9.4–15.8 ng/ml) and high urinary PdG levels (1.35–1.63 ng PdG/mg creatinine) were observed on days 23, 31, and 39, and the fetal heart was seen beating during an ultrasound exam on day 45. A male calf was born on day 266, 8.7 mo after insemination. This is the first live birth resulting from AI of an antelope with frozen, thawed semen. The Synchro-mate-B system of estrous control was used in female 2. The implant was inserted, and the injection was given on day ?11 per the manufacturer's instructions. The implant was removed on day ?1, and a total of 220 × 10⁶ motile sperm was deposited in the os cervix or anterior vagina on days 1 and 2. High serum PROG levels (17.5–24.5 ng/ml) were observed on days 13, 21, and 29. Although the fetal heartbeat was observed during an ultrasound examinataion on day 42, the fetus was not carried to term. Details of the radioimmunoassay used to determine the luteinizing hormone immunoreactivity of urine samples are presented.     

Luteinizing hormone,total estrogens and progesterone secretion during lactation and after weaning in sows

Two experiments were conducted to determine changes in serum concentrations of LH, total free estrogens and progesterone before and after weaning in sows. Blood was collected either via indwelling anterior vena cava cannula or by venipuncture and serum hormones were measured by radioimmunoassay. In Exp. I, blood was collected at 15-min intervals for 4 hr on day 7 and day 21 postpartum from three sows on each day. In addition, individual samples were collected from 10 sows on days 4 and 14 postpartum and from 11 sows on days 1, 3 and 5 after weaning (day 23 postpartum). Serum LH ranged from .2 to .8 ng/ml during lactation and averaged 1.1 ± .7, 1.1 ± .7 and 2.7 ± .7 on days 1, 3 and 5 after weaning, respectively. Progesterone was low (< 1 ng/ml) during lactation and averaged 1.9 ± .3, .6 ± .3 and 1.2 ± .3 on days 1, 3 and 5 after weaning. Estrogens were variable during lactation, averaged 121 ± 36 pg/ml on day 1 after weaning and decreased thereafter. Estrus began on day 3 after weaning in 1 sow and on day 5 in the remaining 10 sows.In Exp. II, blood was collected from seven sows at 12 to 24 hr intervals from 2 days before until 5 days after weaning (day 26 postpartum). Mean serum LH was .7 ± .1 ng/ml during 48 hr before weaning and remained unchanged after weaning until day 3 when LH increased to 6.1 ± .8 ng/ml. Serum LH concentrations then declined to 1.3 ± .8 and .9 ± .8 ng/ml on days 4 and 5 after weaning. Total estrogens averaged 31 ± 4 pg/ml during 48 hr prior to weaning and 32 ± 4, 43 ± 17, 28 ± 1, 30 ± 2, 16 ± 2 and 18 ± 2 on days 0 to 5 after weaning. Progesterone increased from 1.0 ± .3 ng/ml 24 hr before weaning to 3.0 ± .3 at weaning and then remained low (< 1 ng/ml) until after ovulation when progesterone increased. Estrus began on day 4 after weaning in all seven sows.Results from these two experiments indicate that in sows: (1) LH is suppressed during early lactation (day 7), gradually increases during late lactation (day 21) and then reaches peak concentrations after weaning near the onset of estrus, (2) estrogens increase between weaning and estrus and decline thereafter, and (3) progesterone rises transiently at weaning and then increases after estrus and ovulation.     

Plasma progesterone concentrations derived from the administration of exogenous progesterone to ovariectomized mares.

Six ovariectomized mares were divided into 3 groups to determine the effects of exogenous progesterone in oil and repositol progesterone on plasma progesterone concentrations. Progesterone in oil was administered in 7 daily injections in Exp. I. Progesterone concentrations were not maintained greater than 1.0 ng/ml for 24 h with 50 mg/day. However, they remained greater than 1.0 ng/ml during the last 4 days of 100 mg/day and greater than 1.5 ng/ml throughout the injection sequence of 200 mg/day. Repositol progesterone was administered on Days 1 and 7 in Exp. II. At 500 mg, progesterone concentrations peaked in 6 h but returned to near 1.0 ng/ml in 2 days. At 1000 mg and 2000 mg, plasma progesterone was maintained at approximately 2.0 and 4.0 ng/ml respectively for 7 days after injection on Day 1 and was 1.5 and 3.5 ng/ml respectively, 11 days after injection on Day 7. An indication of a cumulative effect on plasma progesterone was observed following repeated dosages of both progesterone in oil and repositol progesterone.     

Pharmacokinetics of free catecholestrogens and catecholestrogen benzoates

To provide a definite basis for studies on the biological effects of exogenously administered catecholestrogens, the time courses of the concentrations of these estrogens in serum, pituitary and CNS-tissues were studied in male rats after s.c. injection of either 150 μg of 4-hydroxyestradiol or 2-hydroxyestradiol (dissolved in 200 μl sesame oil/ethanol/ascorbic acid; 97.5/2.5/0.1; vol/vol/wt) or equimolar amounts of 4-hydroxyestradiol 3,4-dibenzoate or 2-hydroxyestradiol 2,3-dibenzoate (dissolved in 200 μl sesame oil). The injection of free catecholestrogens resulted in bolus-like elevations of the serum and tissue concentrations of the respective compound (max. values up to 9 ng/ml, half-life below 1 h) whereas the injection of catecholestrogen benzoates gave lower (max. values about 1 ng/ml) but prolonged elevations (half-life approx. 24 h and 32 h for 4-OHE₂ and 2-OHE₂) of the respective free catecholestrogen.     

Distribution and characterization of kepone-resistant bacteria in the aquatic environment

Effects of the chlorinated insecticide Kepone on the ecology of Chesapeake Bay and James River bacteria were studied. Kepone-resistant bacteria present in a given environment were found to reflect the degree of fecal and/or high organic pollution of the sampling sites, based on total numbers and generic composition of the populations of Kepone-resistant bacteria. The presence of Kepone-resistant bacteria was found to be correlated (alpha = 0.01) with total coliforms, fecal coliforms, and total aerobic viable heterotrophic bacteria, but not with Kepone concentration, since Kepone-resistant bacteria were present in locations where Kepone could not be detected by the analytical methods used in this study. Only gram-negative bacteria, predominantly Pseudomonas, Vibrio, and Aeromonas spp., were found to be resistant to >/=10 mug of Kepone per ml. Gram-positive bacteria, i.e., Bacillus and Corynebacterium spp., were generally sensitive to >/=0.1 mug of Kepone per ml. From results of cluster analysis of taxonomic data, we determined that characteristics of Kepone-resistant bacteria included: resistance to pesticides and heavy metals; degradation of oil; positive oxidase and catalase reactions; and nitrate reduction. From results of the ecological and taxonomic analyses, we conclude that Kepone resistance in estuarine bacteria is due to the physicochemical composition of the gram-negative cell wall and not prior exposure to Kepone. Therefore, the presence of Kepone-resistant bacteria cannot serve as an indicator of Kepone contamination in the aquatic environment where gram-negative bacteria are predominant.     

Growth hormone secretion in the rat as a function of age.

The first appearance of rat growth hormone (RGH) in serum was in the 19 day-old foetus. The level was high on the 21st day of gestation (129 +/- 7 ng/ml serum), it decreased after birth and descended to 17 +/- 2 ng/ml in the 15 day-old rat. After weanling it again rose to reach a plateau at 80 cays. The half-life and metabolic clearance rate (MCR) of RGH were compared in the 4 and 15 day-old rat and in normal and hypophysectomized adult rats after a single intravenous injection of hormone. As the MCR was essentially the same in all groups, serum RGH levels reflect the rate of hormonal secretion of the pituitary gland.     

Effect of Kepone on estuarine microbial activity

In situ heterotrophic uptake of mixed¹⁴C-amino acids and direct viable cell (DVC) count of Chesapeake Bay water samples were not significantly affected by the insecticide Kepone at concentrations 0.01 mg/1. Maximum inhibition of heterotrophic uptake,ca. 85–90%, and DVC count, 45–97%, was evident at concentrations of Kepone exceeding 0.2 mg/1. A specific activity index (Metabolic Activity/DVC or Kepone-resistant DVC), heterotrophic uptake, and DVC count were found to be statistically correlated (a=0.05) to one another, but negatively correlated with concentration of Kepone. The direct viable cell count proved to be a rapid, simple method for estimating the effect of Kepone on in situ estuarine microbial activity.     

Plasma progesterone levels during the estrous cycle of Holstein and Brahman cows, Carora type and cross-bred heifers

Daily plasma progesterone (P(4)) was determined during one estrous cycle of 19 cows and 18 heifers of four different breeds: Holstein (H), Brahman (B), Carora-type (C) and crossbred (CB) females. Estrus detection was made by visual observation and using a teaser bull with a chin-ball marker. The P(4) profiles showed no differences among groups. In Group 1 (H), P(4) levels ranged from 0.5 ng/ml plasma on the day of estrus (Day 0) to 5.1 ng/ml at the luteal phase peak (Day 13). In Group 2 (B), P(4) levels ranged from 0.5 ng/ml on Day 0 to 9.2 ng/ml on Day 13. In Groups 3 (C) and 4 (CB), P(4) levels ranged from 0.5 ng/ml, on Day 0, to 13.7 ng/ml on Day 12 and 8.8 ng/ml on Day 13. These last two groups were moved to the same location and then compared. It was found that P(4) concentrations were significantly higher (P < 0.025) in Group 3 between Days 7 and 14 of the estrous cycle. In all groups, P(4) levels were lower than 1 ng/ml one day before the next estrus, and levels of 0.4, 0.5, 0.4 and 0.4 ng/ml were obtained the day of estrus in Groups 1 to 4, respectively. Results indicated that the pattern of P(4) for each one of the groups was similar to those reported by other investigators.     

Quantitative analysis of two opioid peptides in plasma by liquid chromatography–electrospray ionization tandem mass spectrometry

Quantitative analysis of two opioid peptides, DSLET [(d-Ser²)Leu-enkephalin-Thr⁶] and Met-enkephalin-Arg-Gly-Leu, was performed using microbore liquid chromatography interfaced to electrospray ionization tandem mass spectrometry. Validation of the methodology was demonstrated for each peptide in plasma. Quantitative analyses were performed through the use of a deuterium labelled peptide analog as an internal standard. Linearity was observed for the analysis of DSLET (5–1000 ng/ml) and Met-enkephalin-Arg-Gly-Leu (1–1000 ng/ml) in plasma with a limit of detection of 0.25 ng/ml for Met-enkephalin-Arg-Gly-Leu and 1.0 ng/ml for DSLET. In general, the observed concentrations showed good reproducibility with coefficients of variation of within 15%. In the concentration range studied, only 0.5 ml of plasma was required for optimal detection of Met-enkephalin-Arg-Gly-Leu and 0.25 ml for DSLET. Application of this method was demonstrated by studying the disposition of DSLET in a rat. DSLET administered to a rat exhibited a short half-life and a high clearance value.     

Interaction of circulating cell-derived and plasma growth factors in stimulating cultured smooth muscle cell replication

Multiple growth factors that circulate in plasma have been shown to stimulate cellular growth in vitro. The plasma growth factors appear to stimulate DNA synthesis in cultured fibroblasts only after prior exposure of cell growth factors derived from circulating cell types, such as platelets and macrophages. The purpose of these studies was to investigate the role of the plasma growth factors in stimulating smooth muscle cell replication following exposure to platelet-derived growth factor (PDGF). Following transient exposure to PDGF, insulin stimulated smooth muscle cell replication but only when supraphysiologic concentrations were used (i.e., greater than 1.0 μg/ml). Somatomedin-C (Sm-C), in contrast, was found to stimulate a 320% increase in [³H]thymidine incorporation when concentrations that are present in extracellular fluids were used (i.e., 0.5–10 ng/ml). Epidermal growth factor (EGF), an important mitogen for multiple cell types, caused a 70% increase in [³H]thymidine incorporation when added to quiescent cells following PDGF exposure, and EGF caused a substantial increase in the absolute level of [³H]thymidine incorporation when coincubated with Sm-C. When EGF (1 ng/ml) was incubated simultaneously with concentrations of Sm-C between 1 and 10 ng/ml plus Sm-C-deficient plasma, maximal [³H]thymidine incorporation was 2.1-fold greater in the presence of EGF. In contrast, insulin (20 ng/ml), when coincubated with Sm-C under similar conditions, had no enhancing effect on the cellular response to Sm-C. None of the plasma factors tested was an effective stimultant of replication when incubated either in serum-free medium or in the presence of Sm-C-deficient plasma without prior PDGF exposure. Hydrocortisone was shown to inhibit smooth muscle cell replication in concentrations between 10^?7 and 10^?5M. In summary, multiple plasma growth factors can stimulate the smooth muscle cell replication, and Sm-C appears to be most effective of those tested. Insulin and EGF appear to work by different mechanisms; that is, EGF can facilitate the cellular response to Sm-C, whereas insulin is effective only at supraphysiologic concentrations at which it will directly bind to Sm-C receptors.