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Oviduct fluid, collected from 65 mature, female rabbits in estrus and in which a modified copper intrauterine device (IUD) was introduced by laparotomy in one of the uterine horns, was examined for chemical composition. No differences were noted for sodium, potassium, calcium, chloride and bicarbonate between the two sides. The copper content was found to be significantly higher in the IUD side (12.80 +/- 3.01 microgram/100 ml), as compared to the control side (9.0 +/- 1.0 microgram/100 ml), the toxicity of which, to blastocyst development, has been reported in some cases. Total protein in the experimental side was significantly higher (258.52 +/- 36.22 mg/100 ml) than in the control side (156.67 +/- 26.45 mg/100 ml). The same was found for sialic acid, 5.30 +/- 0.62 microgram/ml, compared to 3.66 +/- 0.27 microgram/ml, suggesting that a part of the increased protein can be ascribed to glycoprotein. Oviduct fluid glucose was found to be lower in the copper IUD side than in the control side (10.5 +/- 0.2 vs. 17.7 +/- 0.3 mg/100 ml). These findings may be explained on the basis of an inflammatory process, taking place in the side of the copper IUD, and should be used with cautiousness in adolescent, and nulligravidae women.

A high-performance liquid chromatographic analysis of temelastine in biological fluids with UV detection at 229 nm is presented. The method involves ether extraction followed by re-extraction into phosphoric acid and chromatography on a reversed-phase column. The method has a lower limit of detection of 0.05 mg L-1 and is adequate for use in pharmacokinetic studies following oral administration.

The distribution in the dog prostate and other tissues of ciprofloxacin, a quinoline carboxylic acid derivative, was investigated in an experimental model. The concentrations in prostatic tissue, prostatic interstitial fluid (PIF), and prostatic secretion (PS) were lower than the corresponding plasma (P) concentrations, as would be expected for an acidic compound. The experiments were carried out under steady state conditions during intravenous infusion in one group of dogs and following gastric administration in another group. During steady state the ciprofloxacin concentrations were significantly higher in PS than in PIF, and the median PS/P ratios were significantly higher than the PIF/P ratios. These concentrations and ratios were compared with those of two other quinoline carboxylic acid derivatives, rosoxacin and norfloxacin. The concentrations of ciprofloxacin in prostatic tissue, PIF, PS, and urine were several times higher than the minimum inhibitory concentrations for most gram-negative pathogens that cause bacterial prostatitis and urinary tract infections. Clinical trials of ciprofloxacin in these diseases are therefore indicated.

Pseudocholinesterase activity of sweat, lacrimal fluid, urine, parotid saliva, whole saliva, whole blood and plasma was estimated using butyrylthiocholine iodide and propionylthiocholine iodide as substrates. There was similarity in activity between erythrocytes and plasma, but considerable differences between whole blood and other body fluids. The level of enzyme activity was approximately the same in sweat, lacrimal fluid, urine, parotid fluid and whole saliva, but it was only about 1/1500 of that of plasma. The enzyme activity in either whole blood or plasma is a good measure of organophosphate exposure but in other fluids activity can hardly give more than an approximate indication of acute poisoning.

For determination of a drug in biological fluids, accuracy and sensitivity of detection of the adopted method are decisive parameters, but practical aspects such as time and necessary instrumentation are equally important. In the case of N-acetyl-L-cysteine, sample pretreatment can be kept at a minimum if sufficiently selective modes of derivatization, chromatography and detection are employed. Its concentration in serum is between 1 and 20 mumol/l; for quantitative analysis capillary gas chromatography and mass fragmentographic detection are employed. For urine with its ten-fold higher concentration the preferred method is reversed-phase high-performance liquid chromatography after thiol-selective derivatization.

Electron probe analysis by x-ray spectrometry is used in biology for simultaneous determination of the concentrations of any elements with a higher atomic number than that of carbon in single samples with volumes of 0.01 to 0.5 nl. In this technique, deposits prepared from identical volumes of biological fluids and standard solutions are totally covered by the electron beam, and the measured x-ray intensities for each element directly compared. The possibility of intensity quantification depends on the thinness of the dried deposits obtainable by various preparatory techniques. Factors affecting the accuracy of the results include droplet stability under the electron beam, the identity of the degree of oxidation of the elements in biological fluids and standards, sample mass thickness, beam voltage, and matrix effects. Minimum detectable concentrations in the 0.05 mmol.l-1 range are now achievable. This technique is the only one applicable in cases where available volumes are too small to determine the concentrations of several elements on the same sample (for instance of Na, Mg, S, P, Cl, K, Ca, Fe and Co), or even to determine the concentration of a single element (e.g. Mg). Although the droplet technique has so far mainly been used in renal physiology, it has also been applied in reproductive and digestive physiology. Isolated cells are analyzed according to the same principle of totally covering the cell by the electron beam. During the last decade, the vast increase in the relevant literature has testified to the contribution of the microdroplet technique to various fields of biology.

Nuclear magnetic resonance (NMR) which now has widespread clinical use as an imaging technique, may also have significant application to diagnostic pathology. High-field proton NMR spectroscopy can perform rapid multicomponent analysis of serum, urine, cerebrospinal fluid, and even intact cells. This technique allows unusually detailed studies of certain metabolic derangements and toxic ingestions. Phosphorus NMR spectroscopy in vivo can measure ATP and other phosphometabolites, intracellular pH, and free Mg2+. It has been applied to diagnosing inborn errors of metabolism such as glycogen storage disease, monitoring growth and regression of tumors, and assessing tissue viability. The basics of NMR spectroscopy are reviewed in this article, along with a survey of applications in human pathology.

A gas-liquid chromatography (tlc) method was developed to assay individual ginsenosides and sapogenins in rabbit plasma and urine samples. A flavonoid, panasenoside, and a sterol, stigmasterol, were used as internal standards for ginsenosides and their sapogenins, respectively. Linear relationships of peak height ratio to weight ratio were obtained for ginsenosides (A1, 20-350 microgram; A2, 20-400 microgram; B2, 20-300 microgram; C, 20-500 microgram), and sapogenins (panaxadiol or panaxatriol, 10-200 microgram) in 0.1 ml of the silylation mixture. The glc assay method developed was sensitive to 0.2 microgram of ginsenosides and 0.1 microgram of sapogenins.

Prolactin concentrations were assayed in vaginal fluid, urine and plasma in 20 women between 38 and 41 weeks gestation. Plasma concentrations of 12.3 (7.3-20.8) mU/ml (n = 40) were found whereas urine concentrations were low or undetectable. Vaginal fluid concentrations were generally lower than 0.25 mU/ml but occasionally elevated up to 3 mU/ml in women with unruptured membranes. Half an hour after artificial rupture of the membranes, vaginal fluid prolactin concentrations were always higher than paired maternal plasma concentrations (p less than 0.001). In women suspected of premature rupture of the membranes, prolactin concentrations in vaginal fluid were always higher than the plasma concentrations except in those in whom the clinical outcome showed that the membranes had not ruptured. A rapid prolactin radioimmunoassay is therefore recommended as a new clinical tool for the diagnosis or exclusion of prematurely ruptured membranes.

Intracellular adenosine triphosphate (ATP) determination appears to be a rapid, and reliable technique for the detection of complement-dependent cytolysis mediated by cytotoxic anti-HLA sera. Only a few minutes after the addition of complement to anti-HLA-coated target lymphocytes, a striking loss of intracellular ATP is observed. The technique can be easily performed with minute amounts of cells, serum, and complement, and exactly following the technical conditions of the standard HLA typing microcytotoxicity test (MCT). One of its main advantages over MCT is that the reading of results can be performed automatically. Since in most tissue typing laboratories all steps preceding the reading of MCT results have already been automated, this method could be of great interest, with a view to permitting the complete automation of HLA typing.

Sodium and osmolar concentrations of porcine folliclar fluid in the cyclic pig did not vary significantly during the oestrous cycle, and were similar to those in plasma. The K+ concentration was greater in small (Days 12-13) and medium-sized (Day 16) follicles than in plasma or large (Day 18-oestrus) follicles of cyclic sows. In contrast, follicular fluid obtained from slaughterhouse material had higher potassium and osmolality, and lower sodium values, which are assumed to be due to post-mortem changes.

Immature female rats were injected sc with a single dose of PMSG to induce growth and maturation of ovarian follicles. In the morning of prooestrus the rats were given a single ip injection of LH (10 micrograms/rat) or 0.154 M NaCl, 2 h prior to sacrifice. Granulosa cells were isolated fro the pre-ovulatory follicles and incubated in Krebs bicarbonate buffer, for 1 h with or without in vitro addition of various test substances. Following incubation the amounts of cAMP in tissue plus medium were determined. It was found that the isolated granulose cells exposed to LH in vivo responded to the addition of LH in vitro with a production of high amounts of cAMP, i.e. these cells were not refractory to LH stimulation and in fact responded better than granulosa cells isolated from ovaries not exposed to LH in vivo. The addition to the incubation medium of follicular fluid (FFl) obtained from pre-ovulatory follicles decreased the effect of LH in vitro when added at a final concentration of 1% and completely abolished it at a concentration of 3%. Removal of steroids from the FFl did not influence the inhibitory effect and the addition of a phosphodiesterase inhibitor (IBMX) in vitro did not alter the results in principle. These results point to the existence of a factor in the FFl which interacts with the sensitivity of the isolated pre-ovulatory granulosa cells to repeated exposures to LH. Characterization of this factor is subject to further investigations.

Relaxin was measured by radioimmunoassay in follicular fluid from sows which were of different reproductive states. In non-pregnant animals, follicular fluid relaxin values ranged from 0.23 to 666 ng/ml. For pregnant animals the range was 172.6-32 707 ng/ml and for sows with polycystic ovaries this range was 5.0-2800 ng/ml. For polycystic ovaries only there was a correlation (r = 0.74) between the diameter of the follicle and relaxin concentration in the respective fluid. Gel filtration species of relaxin, of molecular weights 27500, 19000 and 6000. Relaxin was extracted from 1 kg of ovaries collected from prepubertal pigs and gel filtration studies indicated the presence of large molecular weight species in addition to a molecule of approximately 6000. These experiments suggest that the corpus luteum is not the sole source of relaxin in the pig.

Patients' nonadherence or low adherence to low adherence to prescribed medication regimes threatens the satisfactory conduct of controlled clinical trials for the efficacy of drug treatment. Suboptimal adherence is costly since an increased sample size must be used to achieve and maintain study power. Accurate measurements of patients' adherence to test medications are not generally available. Current, popular, indirect methods provide uncertain estimates of adherence. As a result, behavioral strategies for management of adherence during trials have been handicapped, and analyses of trial results utilizing adherence levels are poorly developed. These problems could be solved by the ingestion, concomitant with the test medication, of a metabolically and pharmacologically inert compound (adherence marker) whose measurement in body fluids or secretions would indicate the extent of ingestion of the prescribed dose of the test medication. An adherence marker with proven wide applicability has not yet been developed or identified. The detailed requirements for an adherence marker include that it be pharmacologically and chemically inert, nontoxic, unaffected by the physical and chemical properties of body tissues and fluids, not accumulated in the body, and undetectable by the patient. The pharmacodynamics and tissue distribution of the marker should be appropriate for the medication being evaluated. The interindividual and intraindividual variances of marker metabolism should be small enough to permit precise and accurate estimates of adherence. For each trial, the choice of using an adherence marker in lieu of direct analysis of the test medication must be evaluated considering the circumstances of the trial and the pharmacokinetics of the test medication.

A selected ion monitoring method for the determination of 6-oxo-PGF1 alpha, the stable end-product of prostacyclin, in biological fluids has been developed. In this method, biosynthetically prepared [2H6]-6-oxo-PGF1 alpha is used as internal standard. The method involves extraction, thin-layer chromatography purification and derivatization into the methyl ester, methoxime, trimethylsilyl ether derivatives by carrying out the methoximation first. Quantitative gas chromatographic mass spectrometric analysis is performed in the electron impact mode by monitoring the [M - (TMSOH + CH3O)]+ fragment ions. The use of this method in the measurement of 6-oxo-PGF1 alpha in serous fluids and in incubation media of serous tissues is described.

Blood and breast cyst fluid were drawn simultaneously for hormonal determination. There was no difference between serum and cyst fluid values of PRL and TSH. A significant difference was noted for LH (p less than 0.01) and FSH (p less than 0.05), serum concentrations being higher than cyst fluid concentrations.

Reconstructed mass chromatography using methane as a carrier and reagent gas for chemical ionization gas chromatography-mass spectrometry of the derived methyl esters allows rapid, quantitative microdeterminations of complete cholesteryl ester fatty acid profiles. The sensitivity of this method is consistent with completely specific, multicomponent assay at the picomole level. Introduction of two homologues as internal standards, one into the intact biological specimen and the other after derivatization, provides a measure of the net efficiency of the processes of extraction and derivatization. This procedure may be extended readily to the determination of fatty acid profiles in most biological fluids.

The use of a column switching system for direct injection of samples and of a sample clean-up on reversed phase pre-columns is described. The pre-columns were filled with spherical C-18 silica gel of particle size 30 microns. Two applications are reported on: (1) the direct injection of serum samples for the simultaneous analysis of nine antiepileptic drugs and metabolites and (2) the determination of phenytoin and of carbamazepine in serum ultra-filtrates. The purge liquid for the sample clean-up was diluted phosphoric acid, and the eluent mixture for the chromatographic separation was water/acetonitrile. The analytical column (length 12.5 cm) was filled with C-18 silica gel of particle size 5 micron. A gradient elution was chosen for the first application, while the second application was carried out using isocratic chromatographic conditions.

Identification of semen in vaginal fluid may provide documentation of sexual contact in alleged victims of rape. We describe an enzyme-linked immunosorbent assay for a semen glycoprotein of prostatic origin, designated p30. This test detects as little as 3 ng of the p30 antigen per milliliter in various body fluids. Semen from normal and vasectomized men contains high levels of p30 (mean, 1.55 mg per milliliter of seminal plasma), and urine from men contains low levels (mean, 260 ng per milliliter). However, the antigen cannot be detected in body fluids from women, including vaginal fluid and urine, suggesting that p30 may be a male-specific antigen. The p30 antigen was detectable in vaginal fluid for a mean period of 27 hours after coitus, as compared with 14 hours for prostatic acid phosphatase. Of 27 vaginal fluid samples from women who were allegedly raped in which the acid phosphatase test was negative, 7 (26 per cent) were unequivocally positive for p30 by our assay. We conclude that the assay for p30 offers a more sensitive and specific method of semen detection in rape investigation than the enzyme assay for prostatic acid phosphatase.

The studies reviewed here indicate that follicle regulatory protein (FRP) alters aromatase and 3B-hydroxysteroid dehydrogenase activity in porcine, human, and rat granulosa cells. The inhibitory effect of FRP on granulosal aromatase activity depend upon the response of the cell to FSH: large amounts of FSH can partially overcome FRP inhibition while relatively small amounts of FSH sensitize the granulosal aromatase system to FRP. Although androgens potentiate FSH-mediated granulosal functions, they also sensitize granulosa cell steroidogenic enzymes to inhibition by FRP. The demonstration that FRP acts primarily on granulosa cells of less mature antral follicles to inhibit aromatase supports the hypothesis that FRP may facilitate follicle selection and suggests a role for FRP in atresia. Most of the effects of FRP on granulosal activities reflect an interplay between the systemic endocrine and local paracrine systems. That FRP functions, at least in part, by modulating follicular response to FSH is consistent with the hypothesis that paracrine effectors are important mediators of folliculogenesis in the presence of gonadotropins.

An analytical procedure, which allows the determination and quantitation of the R(-)- and S(+)-enantiomers of gamma-vinyl-gamma-aminobutyric acid (gamma-vinyl-GABA; MDL 71.754) in body fluids was developed. The method is based on a combined gas chromatographic--mass spectrometric technique. A glass capillary column coated with a chiral phase enabled the separation of the enantiomers of gamma-vinyl-GABA as their N-trifluoroacetyl-O-methyl ester derivatives. This was followed by quantitation using a selected ion monitoring technique in the electron-impact mode of ionization. The internal standard, gamma-acetylenic GABA, was included throughout the work-up of the samples. The assay was shown to be reproducible, specific and sensitive. No interferences were encountered from plasma, urine or cerebrospinal fluid constituents. The method was applied to the analysis of plasma samples obtained from a human volunteer who had received racemic gamma-vinyl-GABA. Significant differences in the plasma concentrations and plasma half-lives of the two enantiomers were seen, clearly illustrating the need for a specific assay technique capable of distinguishing between the enantiomers of this drug.

Cefsulodin is a third-generation cephalosporin with a unique specificity for Pseudomonas aeruginosa. To study the pharmacokinetics of this agent in children, a rapid and sensitive high-performance liquid chromatographic micromethod was developed for plasma and urine. Protein was precipitated from plasma with one volume of cold methanol, and 20 microliter of the resulting supernatant solution was injected into a Zorbax C-8 reversed-phase column. The mobile phase was composed of 4.5 parts acetonitrile and 95.5 parts of 0.035 M ammonium acetate buffer (pH 5.2). Flow rate was 1.0 ml/min and peaks were detected at 265 nm. A flow gradient from 0.3 to 2.0 ml/min over 34 min was employed for urine. The analysis had a limit of detectability of 1 microgram/ml and a between-day coefficient of variation of 4.4 and 5.0 for 100 and 10 micrograms/ml samples, respectively.

The concentration of 5 different proteins in suction blister fluid and serum was determined by immunotechniques. These proteins, varying in size and molecular weight (6,600-2,300,000) were insulin, albumin, high density lipoprotein determined as apoprotein A-I, alpha 2-macroglobulin and low density lipoprotein measured as apoprotein B. The difference in the blister fluid/serum concentration ratio of the proteins was dependent on the molecular weight and followed mainly the law of diffusion. Moreover, the amounts of insulin, albumin and apoproteins A-I and B in suction blister fluid were the same as those reported in peripheral lymph. The results indicate that the sieve function of the capillary basement membrane remains intact during the formation of the suction blisters. Suction blister fluid might therefore be regarded as representative of interstitial fluid. The concentrations of 4 different lipids (cholesterol, cholesterolesters, triglycerides and phospholipids) were also determined and their blister fluid/serum concentration ratio proved to have a fairly constant value of 0.25.

A selected ion monitoring gas chromatography/mass spectrometric method for the quantitative determination of 3,4,4'-trichlorocarbanilide (TCC) and its major metabolites (the 2'-hydroxy sulfate and the N- and N'-glucuronides) in human plasma and urine was developed using the deuterium-labelled compounds as internal standards. Limits of detection of 3 ng/mL in urine for the N-glucuronides and of 1.5 ng/mL in plasma for the 2'-hydroxy sulfate were attained. Use of the method was illustrated in a study in human subjects employing TCC-containing bar soaps.

Levels of free and total cholesterol were quantified in control sera and human urine using [2H7]cholesterol or [13C2]cholesterol as internal standard and field desorption mass spectrometry for the quantification. These analyses are performed using a crude chloroform extract without chromatographic separation of the constituents. Due to the extremely soft ionization conditions in field desorption, the constituents of the crude lipid extract predominantly form molecular ion species and only a very low amount of fragment ions of low relative abundance thus minimizing the possibility of ion overlap in the mass spectrum. Mass spectrometric quantification of cholesterol in two control sera resulted in data within the range of assigned concentrations. The standard deviations of the mass spectrometric data were around +/- 2%. Generally, 50 microliters of serum or plasma or 500 microliters of urine are extracted and the mass spectrometric quantification performed using an aliquot of 2-10%. A characteristic pattern of intact cholesteryl esters is also obtained.

A major 68-kDa protein in ram rete testis fluid (RTF) is shown to be chemically and immunologically indistinguishable from albumin in ovine serum. Data obtained with two-dimensional gel electrophoresis of RTF demonstrate the presence of additional proteins with a molecular mass of 68 kDa that do not react with antisera against sheep serum albumin. Biochemical characteristics of albumin preparations isolated by immunoaffinity chromatography from ovine serum and from RTF were compared. Albumin from both sources had the same apparent molecular mass of 68 kDa, the same isoelectric point of approximately 4.2, and neither bound specifically to Concanavalin A. Analysis of tryptic peptide maps, obtained with reverse-phase high-pressure liquid chromatography, indicated no significant differences between digests of the two purified albumin preparations. Results indicate that RTF albumin and serum albumin are the same protein, which implies that RTF albumin may originate from serum. Albumin levels in RTF, collected from different rams and measured by radioimmunoassay, varied between 46 and 164 micrograms/ml, constituting between 11 and 17% of total RTF protein, while albumin levels in sheep plasma were 40,000 micrograms/ml. The protein composition of RTF is discussed in relation to the relative amounts of various components contributed by testis cells and the amounts derived from serum.

As a primary feasibility study for the selection of biomarkers for more extended clinical evaluation, 17 potential biomarker candidates were measured in the body fluids of patients with ovarian carcinoma. For comparative purposes, patients were staged and separated into three groups: those considered completely free of disease, those with residual but minimal tumor, and those with advanced disease. Individual markers included plasma carcinoembryonic antigen, serum human chorionic gonadotrophin, urinary beta-aminoisobutyric acid, serum UDP-galactosyltransferase, and urinary hydroxyproline. Structurally related groups of biomarkers included six modified nucleosides and three polyamines analyzed in urine, and three serum protein-bound neutral carbohydrates. The respective chromatographic methods developed for these latter biochemical materials enabled all the individual compounds in each group to be quantitated simultaneously in one analytical run. The general frequency and degree of elevation for the total number of biomarkers was directly proportional to increasing tumor burden with specific exceptions, human chorionic gonadotropin and beta-aminoisobutyric acid. Galactosyltransferase was the most frequently elevated in the limited disease categories. Several of the biomarkers were elevated in the majority of patients with advanced disease and appeared potentially superior to carcinoembryonic antigen or human chorionic gonadotrophin.

Flow cytometry can be performed on testicular aspirates of vasovasostomy candidates preoperatively. On the basis of ploidy ratios and debris components, DNA histograms can be classified as normal or abnormal. Using this method, the likelihood of the presence of sperm may be predicted.

Two specific antigens of human spermatic plasma have been identified: thermostable alpha 1-globulin and alpha 2-microglobulin of fertility. The thermostable alpha 1-globulin presents a glycoprotein containing sialic acids with a molecular weight of 60000 +/- 7000. In addition to the sperm, it has been also identified in the saliva of men and women. Alpha 2-microglobulin of fertility is identical to placental alpha 2-microglobulin. This protein is specific for the reproductive system of men and women. The physicochemical properties of the test antigens are described.