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Human livers with histologically proven cirrhosis were assessed using in vitro 31P NMR spectroscopy. Spectra were compared with those from histologically normal livers and showed significant elevations in phosphoethanolamine (PE) and phosphocholine (PC) and significant reductions in glycerophosphorylethanolamine (GPE) and glycerophosphorylcholine (GPC). There were no significant differences in spectra from livers with compensated and decompensated cirrhosis. These results help to characterise the alterations in membrane metabolism in cirrhosis of the liver.

Results of magnetic resonance spectroscopic (MRS) studies of the chemical patterns in brain tumors have been inconsistent. Actual biochemical correlations are needed. In 2 patients with heterogeneous intracranial tumors, in vivo 1H MRS and in vitro biochemical analyses were correlated. Histology confirmed the tumor heterogeneity. Choline was elevated in the cellular portion of both tumors but decreased in the necrotic or cystic portions. Creatine was diffusely decreased while lactate was elevated in all regions of both tumors. Furthermore, the increase in the choline peak on 1H MRS appeared to be due to increases in water-soluble choline compounds. This study illustrates the value of small localized voxels for differentiating regional chemical differences in tumors.

Studies of the muscle phospholipid metabolism of Ascaris suum suggest an effect of cholinergic drugs on the turnover of phosphatidylcholine and the generation of glycerophosphorylcholine (GPC). 31P-nuclear magnetic resonance (NMR) studies of helminths revealed the presence of a major peak that was assigned to GPC. The primary effect of the cholinergic drugs on the parasites' phosphate profile appeared to be on the level of GPC. In in vivo studies, decreases in internal GPC concentrations occurred prior to any decrease in the concentration of ATP. The importance of these studies relies on the correct identity of this major 31P-NMR resonance. More recently, the identity of this resonance as GPC was questioned by experimental data obtained from C. elegans dauer larvae using the NMR technique. Because studies from our laboratory suggested that phospholipid metabolism may be intimately connected with the parasite's responses to drugs, the identity of the assigned resonance in the 31P-NMR spectrum as GPC in Ascaris suum was reexamined and found to be correct. Similar studies with C. elegans indicate the presence of both GPC and GPE.

Urea is known to be disruptive to proteins, yet high levels occur in a variety of tissues. It has been suggested that trimethylamines counteract the effects of urea on protein. We have, therefore, directly investigated whether elevating tissue urea levels produces an increase in trimethylamine concentrations by using 31P and 14N nuclear magnetic resonance (NMR) spectroscopy to detect two trimethylamines (glycine betaine and glycerylphosphorylcholine) in intact, living gastrocnemius muscle from Xenopus laevis. Xenopus laevis naturally elevates its tissue urea levels under dry conditions. This ability was used to obtain plasma urea levels ranging from 1 to 110 mmol l-1. This procedure did not alter pH or levels of ATP or phosphocreatine in the muscles, but did raise trimethylamine levels. We find that there is a significant relationship between plasma urea concentrations and the trimethylamine levels studied. This relationship was, however, limited to the lower range of urea levels. We propose that other trimethylamines or mechanisms come into play at urea concentrations above 20 mmol l-1.

Quantitative, single-voxel proton NMR spectroscopy of normal brain was performed in five adult beagle dogs using the cerebral water signal as an internal intensity reference. The same brain regions were then rapidly isolated and frozen using a pneumatic biopsy drill, perchloric acid extracted, and analyzed by biochemical assay and high-resolution NMR spectroscopy. The concentrations of the major resonances in the in vivo and in vitro spectra were compared, and good agreement was found between the different measurements. The in vivo spectra contained three peaks at 3.21, 3.04, and 2.02 ppm, which are usually assigned to trimethylamines (TMA), creatines, and N-acetyl derivatives (NAc), which corresponded to be the following metabolite concentration values: 1.7 +/- 0.6, 7.7 +/- 2.1, and 10.9 +/- 2.7 mumol/g wet weight respectively. In vitro, the following metabolite concentrations were measured: glycerophosphocholine (GPC) 1.3 +/- 0.2, phosphocholine (PC) 0.5 +/- 0.1, phosphocreatine (PCr) 2.6 +/- 0.4, creatine (Cr) 5.9 +/- 1.4, and N-Acetyl aspartate (NAA) 8.9 +/- 1.8 mumol/g wet weight. Therefore, the 3.21 ppm resonance observed in the in vivo spectrum is predominantly GPC and PC in a ratio of 2.6:1, the 3.04 ppm resonance is Cr and PCr in a ratio of 2.3:1, and the 2.02 ppm resonance is predominantly (approximately 80%) NAA with small contributions from N-acetylaspartylglutamate (NAAG) and glutamate. The data presented here validate the technique of water referencing as a simple and convenient means of quantitating single-voxel in vivo proton NMR spectra of the brain.

This paper describes the use of positive and negative ion electrospray mass spectrometry (MS) and MS/MS (tandem mass spectrometry) to identify glycerophospholipid and ceramide headgroups and their alkyl, alkenyl and acyl constituents. Molecular ion adducts were the primary products formed by positive ionization, occurring as [M+H]+, [M+Na]+, [M+K]+, [M+formate]+, or [M+acetate]+, depending upon the class of glycerophospholipid and the presence or absence of these ionization-promoting species. Similar (negatively charged) ions corresponding to the loss of the groups listed above were formed in negative ion MS. Positive ion electrospray MS/MS provides information on the nature of the headgroup, with the formation of an ion corresponding to the headgroup itself, or the loss of the headgroup from the molecular ion H+ or Na+ adduct. Acyl constituents are identified during negative ion MS/MS from the formation of their RCOO- ions. The nature of alkyl or alkenyl substituents in glycerophosphoethanolamine (PE) molecular species can be identified from residual ions following the loss of ethanolamine plus loss of the acyl moiety in the sn-2 position, and cyclization of a phosphate oxygen with C-2 of glycerol. In glycerophosphoinositol (PI) species, it appears that an RCO- ion is formed during negative ion MS/MS, possibly to steric interference from the bulky phosphoinositol headgroup that prevents cyclization (and subsequent stabilization) of the ion described for PE species. Positive and negative ion electrospray MS spectra for molecular species of commercial preparations of PE, PI, phosphatidylserine (PS), glycerophosphocholine (PC) and sphingomyelin (SM) produced similar profiles. For phospholipids occurring as Na+ adducts, concentrations above ca. 1 ng/microliter produced significant quantities of both [M+H]+ and [M+Na]+ ions for those molecular species present in the largest quantities, complicating interpretation of the spectra. Complete profiles of molecular species were obtained from as little as 10 picograms of material. Major components of PE were identified from 0.1 picogram total lipid. Using single ion monitoring of the Na+ adduct of beta-acetyl-gamma-O-hexadecyl L-alpha-phosphatidylcholine, 10 femtograms of material was detected. A mixture of 1 nanogram each of PE, PI, PS, and PC was readily resolved into individual molecular species, with little apparent loss of resolution or preferential ionization. Electrospray MS did not provide information on the position (sn-1 or sn-2) of fatty acids, and was not capable of differentiating in all instances between alkyl-acyl and alkenyl-acyl substituents without prior separation of these lipid subclasses.(ABSTRACT TRUNCATED AT 400 WORDS)

A method for the determination of the relative amounts of the diacyl, alkylacyl, and alk-1-enylacyl subclasses of choline glycerophospholipids by benzoolysis is described. The procedure consists of simply heating the phospholipid with benzoic anhydride in the presence of boric acid for 5 h at 100 degrees C, followed by reaction with 4-dimethylaminopyridine for 2 h at room temperature and HPLC analysis of the lipidic products formed. With model compounds of the three subclasses it is shown that diacyl- and alkylacylglycerophosphocholines are completely dephosphorylated by this procedure, yielding quantitatively the corresponding diacyl- and alkylacylglycerobenzoates. The same procedure applied to a model alk-1-enylacylglycerophosphocholine gives only 53.5% of dephosphorylation, while the dephosphorylated products in turn are quantitatively converted into the corresponding acylglycerodibenzoates. The latter figure is shown to be fairly reproducible. The reduced dephosphorylation rate of plasmalogens appears to be due to complete disruption of the vinyl ether bond. The liberated fatty aldehyde gives rise to an addition product with benzoic anhydride, which was identified by gas chromatography-mass spectrometry. It is demonstrated that acyl migration occurring during the benzoolysis does not interfere with the HPLC separation of the glycerobenzoates and dibenzoates derived from the three distinct subclasses. Results of subclass determinations by benzoolysis of several natural diradylglycerophosphocholines are in good accordance with literature values. The agreement between the plasmalogen contents, determined by benzoolysis and by phosphorus determination following exposure to HCl and separation by thin-layer chromatography, is satisfactory. The reliability of the benzoolysis method is generally discussed.

The presence and localization of glycerylphosphorylcholine (GPC) on the surface of human sperm, as well as the metabolism of its breakdown product L-glycerol 3 phosphate (G3P), were investigated. GPC was found to be associated with sperm after penetrating cervical mucus and was present after repeated washing of the sperm. GPC was partially released by treatment with 0.4 M NaCl in 0.01 M sodium phosphate buffer (pH 7.4) and localized to the head region after sperm fractionation. G3P did not increase O2 uptake of uncapacitated human sperm. However, under aerobic conditions, lactate accumulated when exogenous G3P or uterine GPC diesterase was added to sperm in suspension. The uptake of O2 by washed capacitated sperm pre-incubated with 1 unit of rat uterine GPC diesterase for 30 min was significant. This effect was inhibited by 2 microM oligomycin indicating that oxidative phosphorylation had occurred. The present study indicates that GPC may play a role in the metabolism of human sperm after capacitation.

The content of the phospholipid metabolites, phosphocholine, phosphoethanolamine, glycerophosphorylcholine and glycerophosphorylethanolamine was measured in chemical extracts from 46 human breast carcinoma using 31P NMR spectroscopy. Some patients had received therapy prior to tumour resection. The data were therefore stratified into two groups: (i) all tumours; and (ii) untreated tumours. Three indices of tumour proliferation i.e., mitotic index, Ki67 and S-phase fraction were determined on tissue from the same tumours and were found not to correlate with the content of any of these metabolites. In addition oestrogen-receptor status and density, tumour grade and DNA ploidy were obtained on some tumours. The phosphocholine content was higher in high grade tumours when compared with low grade tumours. There was no apparent relationship between DNA ploidy and the content of any of these metabolites. Glycerophosphorylcholine content of oestrogen-receptor positive tumours correlated with receptor density. However, there was no significant difference between receptor positive and negative tumours in the content of any of the phospholipid metabolites measured.

The purpose of this study was to investigate whether the measurement by NMR of some biochemical markers such as glycerylphosphorylcholine (GPC), glycerylphosphorylethanolamine (GPE), citrate, and lactate in human seminal plasma may be able to differentiate spermatogenic failure from obstructive azoospermia. The peak area ratios citrate/lactate, between normozoospermic and spermatogenic failure or obstructive azoospermia groups were significantly different. We have also found significant differences in GPE/GPC peak intensity ratio between spermatogenic failure men and obstructive azoospermia patients (P < 0.001). This ratio appears to be a very important parameter to differentiate spermatogenic failure from obstructive azoospermia. These results demonstrate the potential use of 1H NMR on human seminal plasma in male infertility evaluation and such analysis may be used to elucidate the molecular basis of the human pathologic seminal plasma disturbances.

Proton-decoupled, 31P three-dimensional (3-D) chemical shift imaging (CSI) spectra have been acquired from the entire human brain using a new dual tuned resonator. The resonator operates in quadrature mode to provide improved sensitivity, excellent B1 homogeneity and reduced power deposition at both frequencies. Proton-decoupled and fully NOE enhanced, 31P spectra were acquired from normal volunteers using Waltz-4 proton decoupling with continuous wave bi-level excitation applied through a second radio frequency channel. Well resolved peaks in the phosphomonoester (PME) and phosphodiester regions were obtained from nonlocalized FIDs and spectra localized with 3-D CSI without processing for resolution enhancement. pH measurements made over large regions of the brain using the P(i) resonance show no significant variations (6.9 +/- 0.02) for a single individual. The improved spectral resolution and sensitivity of the PME resonances results in more well defined metabolite images of the PME peak region.

Glycerylphosphorylcholine (GPC), glycerylphosphorylethanolamine (GPE), citrate, and lactate content of human seminal plasma was analysed by measuring the peak area of 1H nuclear magnetic resonance (NMR) spectra in samples from four groups of patients: 21 spermatogenic failure subjects; 14 obstructive azoospermic subjects (vasectomized); seven patients presenting very severe oligoasthenozoospermia (OAT) and 18 normozoospermic subjects (control). The peak areas for GPC, citrate and lactate in seminal plasma were significantly smaller for patients with azoospermia than for the control group: 16.79, 8.18 and 2.28 versus 23.38, 10.58 and 4.30, respectively (P < 0.01). The peak area ratios for citrate:lactate and GPC:lactate were significantly different (P < 0.01) between the control group and the spermatogenic failure or obstructive azoospermia groups. A significant difference was also found in GPE:GPC peak intensity ratio between spermatogenic failure and obstructive azoospermia subjects (P < 0.001). These results provide some quantitative markers which may be used for examining infertility by using 1H NMR of seminal plasma samples.

Sodium absorption in distal tubule segments was stimulated by increasing the distal delivery via infusion of hypertonic saline. In these animals, and in control rats, electrolyte concentrations in thick ascending limb cells, light and dark cells of the collecting duct in the outer and inner stripe of the outer medulla and in cells of the proximal straight tubule (outer stripe only) were studied. The measurements were performed by electron microprobe analysis of freeze-dried cryosections of the outer medulla. In addition, organic osmolytes (glycerophosphorylcholine, betaine and myo-inositol) were measured by high performance liquid chromatography in cortex and outer medulla. Augmented delivery of sodium chloride to the distal tubule was associated with increased sodium concentrations of thick ascending limb cells both in the outer and inner stripe and of medullary collecting duct light and dark cells in the outer stripe. While the sum of organic osmolyte concentrations was 28% higher in the outer medulla of the salt-loaded animals compared with controls, this value was unchanged in the renal cortex. These findings indicate that the primary event underlying stimulation of sodium absorption along the thick ascending limb during increased distal sodium delivery is enhanced entry of sodium across the apical cell membrane. This would be expected to lead to higher cell sodium concentrations and stimulation of basolateral active Na-K-exchange. The enhanced transport activity of outer medullary tubules may be associated with increased interstitial tonicities and intracellular retention of organic osmolytes.

The abundance of the "phosphodiester" peak in differentiating or proliferating tissues, including reproductive organs and tumors, warrants further investigations of its metabolic role(s), which would require a rigorous confirmation of its identity. The assignment of this peak to glycerophosphorylcholine in 31P NMR spectra of biological samples has been largely based on chemical shift, which can result in ambiguities. We employed a combination of two-dimensional 31P-1H heteronuclear shift correlation and 1H total correlation spectroscopies to trace the spin connectivities of glycerophosphorylcholine and thus to identify its structure directly from crude ovarian extracts of mussels without ambiguities and the need for extensive purification. This approach can be applied generally to the identification of molecules containing heteroatoms in crude tissue extracts.

The polyols, sorbitol and myo-inositol, seem to be involved in the development of diabetic complications of different organs. High concentrations of both polyols were found in kidney medulla in addition to trimethylamines. To investigate the influence of diabetes mellitus on the regulation of both polyols and glycerophosphorylcholine in kidney, these osmolytes were quantitated enzymatically along the corticopapillary axis in untreated, streptozotocin-diabetic and insulin-treated streptozotocin-diabetic rats. In control animals three individual osmolyte patterns were found: a steep gradient of sorbitol in the papilla, increasing amounts of glycerophosphorylcholine from the outer medulla to the papilla, and nearly equal amounts of myo-inositol in the renal medulla, decreasing towards the cortex. Diabetic rats exhibit an up to fourfold increase of inner medullary sorbitol, whereas myo-inositol was only elevated in the outer medulla. Glycerophosphorylcholine was lowered in the papillary tip and elevated in the outer medulla and cortex. Insulin treatment reduced sorbitol to a concentration between those of diabetic and control rats, caused a restoration of glycerophosphorylcholine in the papillary tip and outer medulla to control values, and increased cortical myo-inositol. These data confirm previous in vitro data, which show that papillary sorbitol specifically increases in hyperglycaemic states, thereby counteracting the increased extracellular tonicity due to elevated tissue glucose concentrations. Imbalance of extra- vs intracellular osmolality during insulin treatment may be involved in the pathomechanism of renal papillary necrosis.

This study investigates the potential of in vivo 31P magnetic resonance spectroscopy (MRS) to characterize musculoskeletal tumors and to determine preoperative levels of histological necrosis, which is an important clinical indicator of patient response. Pretherapy MRS was performed on 28 patients with large musculoskeletal tumors: 13 with osteosarcoma, 3 with chondrosarcoma, 5 with malignant fibrous histiocytoma, 1 with desmoid tumor, 1 with Ewing's, 2 with hemangioendothelioma, 1 with myxoid liposarcoma, 1 with synovial cell sarcoma, and 1 with rhabdomyosarcoma. Fifteen patients had follow-up MRS examinations after commencement of chemotherapy (mean of five/patient), eight of whom have now had surgery. Elevated levels of PMEs (P < 0.01), P(i) (P < 0.01), and PDEs (P < 0.02) as well as elevated tumor pH (P < 0.05) were observed in all patients. The synovial cell sarcoma was characterized by high levels of PMEs (> 20%) and low pH (pH 6.76). This contrasted with the spectra obtained from the malignant fibrous histiocytomas which had high levels of PDEs (17 +/- 5%). Reductions in PDE levels postchemotherapy were associated with a high degree of necrosis (> 90%) at surgery, while an increase in PDE levels was associated with a low level of histological necrosis. Likewise, reductions in the ratios PDE/NTP and PDE/PCr and an increase in P(i)/PDE were also associated with a high level of necrosis.

Histopathological and biochemical effects of gossypol acetate (GA) on pituitary-gonadal axis were investigated. 10 and 25 mg GA/kg were administered orally to sexually mature adult male Wistar rats for 4 and 5 weeks, respectively. STH and LTH/PRL cells showed no significant changes as compared to those of controls while TSH cells showed hypertrophy, hyperplasia and degranulation in both experimental groups. Pituitary FSH, LH/ICSH cells showed progressive regression. Gonosomatic indices of sex accessory glands at 25 mg showed significant reduction in the experimental animals as compared to those of controls. The diameter of seminiferous tubules reduced and azoospermia developed. Sertoli and Leydig cells also regressed. At 10 and 25 mg GA treatment, spermatogenesis ceased at secondary spermatocytes and spermatogonia stages, respectively. Epididymis and prostate regressed. Seminal vesicle showed no significant histological variations as compared to that of control except reduction in secretion. Biochemical observations revealed increased levels of acid phosphatase, fructose and citric acid and significant reduction in glycerylphosphoryl choline in reproductive glands of both experimental groups as compared to those of controls. Possible mechanism of antifertility action of GA is discussed.

In vivo 31P magnetic resonance spectroscopy (MRS) was undertaken in 28 healthy adult individuals and 32 patients with hepatic malignancies of varying histology, using chemical shift imaging techniques. The mean peak area ratio (total range) of phosphomonoester (PME) to phosphodiester (PDE) in the health adult group was 0.23 (0.15-0.41). The mean (total range) PME/PDE ratio of the total patient group was 0.68 (0.15-2.38), which was significantly elevated (P less than 0.001) compared to the mean of the healthy adult group. Liver biopsies, obtained at operation, were analysed using high-field in vitro MRS techniques in order to identify the contributions of aqueous-soluble metabolites to the multicomponent PME and PDE in vivo signals. Concentrations of phosphorylethanolamine (PE), phosphorylcholine (PC), glycerophosphorylethanolamine (GPE) and glycerophosphorylcholine (GPC) were measured. The in vitro spectrum of six samples of liver of normal histological appearance all showed a similar pattern of PE, PC, GPE and GPC. The in vitro spectrum of seven liver tumours of differing histology all showed an increase in PE and PC signals and a decrease in GPC and GPE signals. The in vitro results were compared with in vivo findings in five patients. The increase in PME/PDE observed in vivo represented, in part, an increase in PE and PC in the PME region and a decrease in GPE and GPC in the PDE region.

Quantification of metabolites is a goal of many biomedical NMR studies. To obtain absolute measurements of metabolite concentrations is often both difficult and time-consuming. In this paper a method for determining metabolite concentrations directly is described and validated. It is applicable to studies of amphibian muscles, and with suitable precautions, to other isolated organs and tissues. The method is based upon using 14N NMR and nitrate-containing solutions to determine what fraction (F) of the sensitive volume of the RF coil is occupied by tissue. As the concentration of nitrate is known it can be used to calibrate other 14N metabolites in the tissue. Moreover, once F is determined, it can be used to calibrate metabolites in spectra from other nuclei e.g., 31P or 31C. All that is required is that a spectrum from a standard for that nucleus is obtained. Thus this method does not require any 'internal' (intrinsic to the tissue) standard, and is extremely quick and simple to use.

A method has been developed for the analysis of derivatized diradylglycerols obtained from glycerophosphocholine (GPC) of transformed murine bone marrow-derived mast cells that provided high performance liquid chromatography (HPLC) separation of GPC subclasses and molecular species separation with on-line quantitation using UV detection. In addition, the derivatized diradylglycerol species were unequivocably identified by continuous flow fast-atom bombardment mass spectrometry. GPC was initially isolated by thin-layer chromatography (TLC), the phosphocholine group was hydrolyzed, and the resultant diradylglycerol was derivatized with 7-[(chlorocarbonyl)-methoxy]-4-methylcoumarin (CMMC). After separation of the derivatized subclasses by normal phase HPLC, the individual molecular species of the alkylacyl and diacyl subclasses were quantitated and collected during a subsequent reverse phase HPLC step. With an extinction coefficient of 14,700 l mol-1 cm-1 at a wavelength detection of 320 nm, the CMMC derivatives afforded sensitive UV detection (100 pmol) and quantitation of the molecular species. Continuous flow fast-atom bombardment mass spectrometry of the alkylacyl CMMC derivatives yielded abundant [MH]+ ions and a single fragment ion formed by loss of alkylketene from the sn-2 acyl group, [MH-(R = C = O)]+. No fragmentation of the sn-1 alkyl chain was observed. Diacyl derivatives also produced abundant [MH]+ ions plus two fragment ions arising from loss of RCOOH from each of the acyl substituents and two fragment ions from the loss of alkyketene from each acyl group. Individual molecular species substituents were assigned from these ions.

The aims of this study were two-fold: to characterize the in vivo 31P NMR spectrum of human breast tumours by identifying the metabolites contributing to each peak; and to demonstrate changes in the detectability of the phosphodiester (PDE) peak at varying field strengths. This was achieved by obtaining 31P spectra from 23 patients in vivo at 1.5 T and also of 11 perchloric acid (PCA) extracts and 3 chloroform-methanol (CM) extracts of tumour biopsy samples at 9.4 T. In spectra acquired in vivo the percentage areas for each peak were: phosphomenoester (PME), 15%; Pi, 11%; PDE 35%; phosphocreatine, 5%; gamma-NTP, 11%; alpha-NTP, 11% and beta-NTP, 12% of the total spectral area. PCA extracts showed the PME peak to be greater than 70% phosphorylethanolamine whilst the PDE peak included almost equal proportions of glycerophosphorylethanolamine and glycerophosphorylcholine. CM extracts of tumours revealed additional metabolites in the PDE region suggesting that the large PDE peak observed in vivo could arise mainly from phospholipids. Spectra of human breast tumour xenografts examined in vivo at both 1.9 T and 9.4 T confirmed that the presence of a large PDE peak in vivo was a function of field strength. Further experiments with microsomal membranes from rat mammary tumours at 1.5 and 9.4 T demonstrated that phospholipids are more clearly visible at the lower field strength due to a substantial decrease in linewidth.

Glycerylphosphorylcholine (GPC), one of the major phosphorus-containing-choline compounds of seminal plasma, is secreted mainly by the epididymal epithelium under androgenic control. This study reports a new method that uses chemiluminescence to determine seminal GPC content, comparing it with a spectrophotometric technique. The results, obtained with both techniques studying 20 fertile patients (as control), 35 infertile patients with normospermia, 23 infertile patients with oligozoospermia and impaired motility and 9 patients with excretory azoospermia, demonstrate that the GPC chemiluminescent assay is more sensitive, simple and rapid than the spectrophotometric assay. Our data confirm that GPC may be used as a marker of vas deferens and ejaculatory duct perviousness, suggesting a possible role of this glycerophosphodiester in sperm motility.

An oestrogen sensitive rat mammary tumour was grown in two groups of female and one group of male hooded rats. The male group and one of the female groups were supplemented with oestrogen. The tumours grew most rapidly in the female supplemented group. When the tumours reached 1.5 cm in diameter they were harvested and the cell cycle distribution and number of cells actively synthesising DNA (bromodeoxyuridine (BrdU) labelling index) determined in each case. Chemical extracts were prepared from each tumour and the concentration of phosphorus-containing metabolites determined using high resolution NMR spectroscopy. The concentration of phosphocholine was found to correlate strongly with the number of cells in S-phase and the number of cells labelled with BrdU, whilst a highly significant negative correlation was observed between these two parameters and glycerophosphocholine. The concentration of phosphoethanolamine did not correlate with either of these measures of proliferation rate. The concentration of glycerophosphorylethanolamine showed a weak negative correlation with the number of cells in S-phase.

Tolnidamine (50 mg/kg body weight; twice a week; oral) was administered for 90 days to adult male langur monkeys (Presbytis entellus entellus Dufresne) to assess its contraceptive potential. Semen weight, volume, seminal fluid volume, colour, pH and libido remained unchanged. Sperm motility, vitality and morphology were impaired with the advancement of treatment. Sperm density reduced to severe oligospermia following 75-90 days of treatment. Increased number of immature germ cells were also noticed. Resumption of changes to pretreatment range was observed following 90 days of cessation of treatment. However, sperm density remained low all through the recovery period of 150 days. Seminal fructose, ACP, LDH and citric acid concentrations did not change markedly. A significant depletion in GPC and magnesium levels was recorded during treatment and early recovery periods. Alterations in germ cells and Sertoli cells were also observed. A progressive but reversible rise in serum creatinine was evident. Other clinical parameters and body weight response revealed no drug-related alterations. In conclusion, tolnidamine medication induced irreversible inhibition of spermatogenesis.

1. Phospholipase B-like activity was found in rabbit brain membranes. 2. The activity was greatly enhanced by 0.025% (w/v) Triton X-100 and was inhibited by both Ca2+ and Mg2+. 3. With increasing pH of the reaction mixture, the activity was augmented. 4. The characteristics of the enzyme activity possibly suggest that phospholipase B in rabbit brain may be distinct from those previously reported.

The phosphodiester peak in 31P nuclear magnetic resonance spectra of human brain in vivo is often the most prominent feature of the spectrum. We have demonstrated that this resonance exhibits bi-exponential spin-spin relaxation, giving relaxation times of 2 and 10 ms. We interpret this in terms of the two components which make up the peak, bilayer lipids and the small cytosolic phosphates glycerophosphoethanolamine and glycerophosphocholine. Using the relaxation times and the relative peak heights of the two components we have also been able to quantitate the concentration of the bilayer lipids as 20-40 times that of ATP.

The individual molecular species composition of diacyl, alkylacyl and alkenylacyl glycerophospholipids was determined in mouse peritoneal macrophages. A marked heterogeneity in the relative composition (mol%) of macrophage ether and ester phospholipid individual species was noted. High concentrations of 16:0-20:4 were found in ether phospholipids such as alkenylacyl glycerophosphoethanolamine (GPE; 27.5 mol%) and alkylacyl glycerophosphocholine (GPC; 16.6%) as compared to mol% levels of 16:0-20:4 in diacyl GPE (5.7%) and diacyl GPC (8.1%), respectively. Interestingly, alkenylacyl GPE was highly enriched in 1-ether (16:0) relative to alkylacyl GPC. The predominant diacyl molecular species in glycerophosphoinositol (GPI) and glycerophosphoserine (GPS) were 18:0-20:4 (59.1%) and 16:0-18:1 (41.1%), respectively. It is noteworthy that the level of 18:0-20:4 was several times higher in diacyl GPI (59.1%) than in diacyl GPS (11.1%), diacyl GPE (25.7%), and diacyl GPC (3.7%). The most abundant molecular species in diacyl GPC and diacyl GPE were 16:0-18:1 (29.9%) and 18:0-20:4 (25.7%), respectively. The abundance of 20:4 in ether phospholipids, specifically 16:0-20:4 and 18:0-20:4, in alkylacyl GPC is significant in view of the role these antecedents play in the biosynthesis of platelet-activating factor (PAF) and 20:4-derived eicosanoids in stimulated macrophages. The unique molecular species composition of the peritoneal macrophage distinguishes this cell type from others.

The effects of vitamin B-12 deficiency on lipids and fatty acids of spinal cord myelin were studied in control and vitamin B-12-deficient fruit bats. Very low plasma and brain vitamin B-12 concentrations were present in animals fed the vitamin B-12-free, all fruit diet. Myelin was isolated from the spinal cord of control and vitamin B-12-deficient animals (n = 3 pools) by means of a flotation method in a discontinuous sucrose gradient. The molar concentration of cholineglycerophosphatide was significantly lower in the deficient bats than in the controls. The molar cholesterol: phospholipid ratio in the deficient bats was also significantly lower. The odd-chain fatty acid 15:1 was not detected in cholineglycerophosphatide from the controls, but comprised 1.4% of the total fatty acids in the deficient group. The odd-chain fatty acids 15:0, 17:1 and 19:0 were present in slightly higher amounts in cholineglycerophosphatide of deficient bats. Lipid and fatty acid differences were related to the vitamin B-12-requiring methionine synthetase and methylmalonyl CoA mutase reactions.

Skeletal muscle contains appreciable lysophospholipase activity which is differentiated by muscle type with red muscle subcellular fractions having greater activity than the corresponding white ones.

Stallion semen has several chemical characteristics not commonly encountered in other animals. It contains very little fructose and the spermatozoa possess only limited fructolytic ability under anaerobic conditions. By determining some of the characteristic constituents such as ergothioneine, citric acid, glycerylphosphorylcholine and certain glycosidases, it is possible to obtain a quantitative measure of the secretory contribution of the ampulla, seminal vesicle and epididymis, respectively, towards the final composition of the whole ejaculate. By analysing separately the various fractions of a split ejaculate, including the so-called tail-end sample, it is possible to detect and pinpoint abnormalities in the functional state of the various male accessory organs and in the entire process of ejaculation.

Measurements were made on micropuncture samples of luminal fluids. The concentration of GPC was highest in the caput and corpus regions of the epididymis, that of phosphocholine changed little along the length of the rat epididymis and there was approximately a 3-fold increase of concentration of inorganic phosphate in the luminal fluid from caput epididymidis to ductus deferens. The estimated total phosphate appeared to equal that of the sum of the phosphate derived from the above compounds. The concentrations of these compounds in the luminal fluid of the testis and in blood plasma were very low.

Small unilamellar vesicles containing alkenylacyl-, alkylacyl-, dialkyl- or diacyl-glycerophosphocholine were prepared by sonication. Their size was determined from the average internal volume after chromatography on Sepharose 2B and from 31P-NMR linewidths. Alkenylacyl glycerophosphocholine (choline plasmalogen) was found to form the largest vesicles. By addition of 30 mol% cholesterol, the size of plasmalogen vesicles, but not of those containing the alkyl and acyl analogue lipids, was significantly increased. The presence of 50 mol% sterol led to highly increased vesicle sizes of alkylacyl, dialkyl and diacyl-glycerophosphocholine. Mixtures of plasmalogens with 50 mol% cholesterol did not form unilamellar vesicles upon sonication. Bilayer thickness and surface area per phospholipid molecule were determined by small angle X-ray scattering and measurement of partial specific volumes. There is little difference between alkenylacyl glycerophosphocholine and the corresponding diacyl-analog, whereas bilayers consisting of dioleoyl glycerophosphocholine are significantly thinner. Correspondingly their molecular surface area is by about 8% larger than that of the mixed-chain diradyl glycerophosphocholine, since the partial molar volumes are similar for all vesicles tested.

Seminal plasma from 20 azoospermic and 4 vasectomized men and 75 samples from normal ejaculates were quantitated colorimetrically for zinc, fructose, and glycerylphosphorylcholine (GPC) contents. The results were similar to those obtained in the normal ejaculates, in 17 of 21 azoospermic ejaculates, whereas in the vasectomized ejaculates, GPC values were reduced. In the remaining 4 azoospermic ejaculates, less than 15% of fructose and GPC and a zinc content three times higher than normal were detected. Vasogram performed in these 4 azoospermic men revealed occlusion or aplasia of the ampula. The data suggest that biochemical evaluation may aid in differential diagnosis of the etiology of azoospermia.

Pituitary, testicular and accessory gland functions were assessed at intervals of 10-20 days in adult male rhesus monkeys given 10, 100 or 1000 micrograms dihydrotestosterone (DHT). Circulating levels of LH and testosterone were suppressed. Ejaculated spermatozoa showed morphological abnormalities and decrease in motility but sperm counts were unaffected. Seminal fructose was decreased in animals receiving DHT. Sexual behaviour was maintained in these animals.

Glycerol-3-phosphorylcholine (GLY-3-PrC) has a role in determining the epididymal environment, but its function is not yet completely clear. This lack of knowledge might be partially due to the limitations of techniques currently employed in the GLY-3-PrC determination. In this paper we have studied and adjusted to human seminal plasma a spectrophotometric assay initially devised for bull and rabbit ejaculates. By means of column-chromatography and solvent extraction the specific interferences (up to 30%) present in human ejaculate, probably due to phosphatidylcholine, were avoided. The technique is precise, simple, cheap and shows GLY-3-PrC recovery higher than 90%.

Glycerophosphorylcholine (GPC)--an organic solute which is considered to be involved in cellular osmoregulation in the renal medulla--was determined by means of an enzymatic assay in various zones of the rat kidney and in papillary tubule suspensions. In antidiuresis, GPC content in cortex, outer medulla and papillary tip was 0.64, 14.6, and 108.9 mmol/kg fresh weight, respectively. Significant concentrations of GPC could not be detected in the urine or in the peripheral plasma. The sharp increase in GPC concentration from cortex to papillary tip was partially abolished by the induction of diuresis by either waterloading or furosemide. These manoeuvres, however, did not change cortical GPC content. Papillary tubule suspensions prepared from hydropenic rats contained only slightly less GPC per g protein than whole papillae from antidiuretic animals. Incubation of tubules over 120 min did not lead to a significant loss of GPC which is in accordance with the low activity of GPC degrading enzymes in papillary tissue. The results confirm the intracellular localization of GPC and provide further evidence that this substance plays a substantial role in the osmoregulation of renal papillary cells.

The concentrations of some inorganic ions (sodium, potassium, chloride and phosphate) and organic compounds (carnitine, myo-inositol, glycerophosphocholine, phosphocholine and total phosphate) were estimated in the luminal fluid collected from sections of human ductus deferens removed at vasectomy. From the data presented it would appear that in contrast to non-primate species, in the human the inorganic ions contribute more to the total osmolarity of the luminal fluid than do the organic compounds.

The epididymal and seminal vesicular contributions to split-ejaculate fractions from boars were analysed for sperm concentration, glycerylphosphorylcholine (GPC), total-N, ethanol-soluble and insoluble N, citrate, zinc and haemagglutinin. The same components were also determined in epididymal plasma (EP), vesicular secretion (VS) and whole seminal plasma (SP). Isoelectric focusing of protein patterns was studied in the fractions. With the exception of haemagglutinin, the components were present to a major extent in either VS or EP and in lower concentrations in the other secretion. The parameters in VS or EP were positively correlated among themselves and negatively correlated with most of the parameters of the other fluid. The correlation coefficients were not significant in all cases for individual animals, but the degree of significance was greater for the over-all correlations. The EP components were mainly secreted in the first three or four fractions, but occasionally from fraction four onwards. Those of VS were emitted during the entire ejaculation, the maximum occurring in the sperm-rich fraction or the immediately succeeding fraction. The first fractions were devoid of VS components in only one case. The majority of the EP proteins could be identified electrophoretically in the sperm-rich fractions, but the protein patterns in the other fractions were similar to those of VS. The results are discussed and compared with previous findings.

One- and two-dimensional proton (1H) nuclear magnetic resonance (NMR) spectroscopic techniques have yielded detailed in vitro profiles of the metabolites present in the parasitic nematode Toxocara canis. The major intracellular metabolites were found to include trehalose, alanine, succinate, acetate, propionate and alpha-glycerophosphorylcholine.

Alpha-glucosidase, glycerophosphocholine, and L-carnitine were measured in sperm-free seminal plasma to determine whether these markers reflected the epididymal function of men attending an infertility clinic. The putative markers correlated well with each other (r = 0.66 to 0.70) and in 92% of 283 cases were accurate in categorizing semen as containing normal or subnormal amounts of markers. Glucosidase was considered the best index of epididymal function and was used for a further 306 samples. The ejaculate content of epididymal markers was correlated with testicular volume and serum testosterone below values of 30 ml and 30 nmol/l, respectively. Markers were also correlated with the concentration and motility of spermatozoa in semen. Seventy-one of 425 patients (17%) displayed subnormal epididymal secretions, mainly in association with hypogonadism (Klinefelter syndrome, Kallman syndrome, idiopathic hypogonadotropic hypogonadism) but also in cases of obstructed ducts, maldescended testicles, and local irradiation following hemicastration. Because azoospermic patients had reduced epididymal markers with both high and low FSH levels and a large proportion of men with reduced glucosidase and normal FSH suffered from testicular failure, it is suggested that other indices of testicular function are required for correct interpretation of reduced epididymal markers. Thirteen patients (3%) had low markers for which no cause was apparent; these may be cases of infertility due to isolated epididymal dysfunction.

The phospholipids from subcellular fractions of human intracranial tumors were examined. For comparison, microsomes were isolated from a fetal human brain and from the gray matter of adult human brains. The subcellular membranes of tumors had a higher protein-to-phospholipid ratio than the normal brain membranes. The microsomes from tumors had a lower proportion of diacylglycerophosphorylethanolamine and higher proportions of alkenylacylglycerophosphorylcholine and sphingomyelin (plus diacylglycerophosphorylinositol) than microsomes from the gray matter. Also, the ratios of alkenylacylglycerophosphorylethanolamine to diacylglycerophosphorylethanolamine were higher in the tumors than in the normal controls. The acyl groups of ethanolamine phosphoglycerides in tumor microsomes had relatively more 18:1, 18:2, and 20:4(n - 6) and less 18:0, 22:4(n - 6), and 22:6(n - 3) than the adult brain gray matter. Except for the increase in 18:2, acyl group changes in choline phosphoglycerides between tumors and controls were not as extensive as in the ethanolamine phosphoglycerides. The characteristic features of phospholipids and their constituent acyl groups of tumors were often present in all the subcellular fractions. Although the acyl group profiles of the tumor phosphoglycerides were in closer resemblance to the fetal brain than to the adult brain, other differences were observed. Results indicate that neoplastic brain cells are unique in their cellular composition, and consequently they deviate from the normal neurons and glials in metabolism and functions.

The relationship between the antifertility effect of alpha-chlorohydrin and changes in composition of luminal plasma from the cauda epididymidis of rats and rabbits has been investigated. At each dose regimen studied, the fertilizing capacity of rats treated with alpha-chlorohydrin was reduced to zero. The levels of sodium, potassium, glycerylphosphorylcholine (GPC), acid phosphatase and alkaline phosphatase in epididymal plasma were not markedly affected by drug treatment. The most noticeable change was a considerable increase in the concentration of lactic dehydrogenase (LDH) at all dose levels and of glutamic-oxaloacetic transaminase (GOT) after 7 days of treatment with 8 and 16 mg/kg. The effect of cold shock on the composition of epididymal plasma showed that LDH and GOT are, at least in part, derived from spermatozoa. In contrast, alpha-chlorohydrin did not have an antifertility action in the rabbit, and the only notable change in the compositon of epididymal plasma was an increase in the level of GPC. These results provide evidence that, in the rat, alpha-chlorohydrin or a metabolite primarily exerts its antifertility effect by a direct action on the spermatozoa, whilst in the rabbit a barrier may exist to the entrance of the drug into the lumen of the epididymal duct.