Prostaglandins in human seminal fluid: two novel compounds.

Human seminal fluid frozen immediately after ejaculation contains two novel prostaglandins. These are present in larger quantities than the previously reported prostaglandins. They are characterized by gas chromatography and mass spectrometry as 19-hydroxyprostaglandins E1 and E2. Most of the previously identified prostaglandins may be artifacts.

Cardiovascular effects of antihypertensive polar and neutral renomedullary lipids.

Two antihypertensive lipids can be extracted from fresh renal medulla. One is polar (the antihypertensive polar renomedullary lipid, or APRL) and the other is nonpolar (the antihypertensive neutral renomedullary lipid, or ANRL). APRL and ANRL differ in their biologic activities: APRL in bolus intravenous injections causes a very rapid decline in the arterial pressure (AP) while ANRL, after a lag of 2 minutes, causes a slower decline in AP. APRL increases heart rate and sympathetic activity. ANRL decreases heart rate and sympathetic activity. ANRL appears to convert to APRL, under certain in vitro circumstances, suggesting that the structure of the two molecules is related. ANRL and APRL appear in the renal venous effluent after unclipping; biologically, ANRL seems dominant. The renal venous effluent of the unclipped isolated kidney lowers the HR and sympathetic activity of the normal rat. Unclipping degranulates the renomedullary interstitial cells (RIC). The antihypertensive effect of unclipping appears due to the secretion of ANRL and APRL by the kidney. It is concluded that ANRL seems to be the antihypertensive hormone of the RIC.

Fast atom bombardment mass spectrometry of synthetic peptides.

Fast atom bombardment mass spectrometry plays a continuing and effective role in the rapid and efficient analysis of synthetic peptides. In this article, the basic principles of FAB-MS and FAB-MS/MS are reviewed, and the limitations and pitfalls of the method are discussed. The potential of the technique is illustrated by several selected applications. The molecular weight of a synthetic peptide can be readily and accurately determined by FAB-MS. The sensitivity of the FAB-MS method also makes it extremely useful for the evaluation of the purity of a peptide. FAB-MS/MS allows the elucidation of the primary structure of the target peptide, even in a mixture, and also permits the rapid identification of synthetic side products. Hence, FAB-MS and FAB-MS/MS aid in the unequivocal characterization of synthetic peptides. Furthermore, the information that is gained from the FAB analysis can help to unravel potential problems that may be associated with the synthesis.

Structure-function studies of amphiphilic antibacterial peptides.

The synthesis of 11 peptides, ranging in composition from 9 to 17 amino acid residues, by solid-phase methodology was accomplished with the purpose of studying how the amphiphilic and hydrophobic character, the size of the molecule, and the charge distribution modulate the antibacterial activity. It was found that peptides composed of 16 and 17 amino acid residues, with high hydrophobic (mainly due to Trp or Phe) and hydrophilic (due to Lys) character distributed along opposite amphiphilic faces, showed considerable antibacterial activity against clinically isolated bacteria together with Gram positive and Gram negative ATCC bacterial strains. However, the hemolytic capacity of the peptides was also significant. Decreasing the hydrophobic character of the molecule by replacing Trp or Phe with Leu residues while maintaining the basic contribution of Lys drastically reduced the hemolytic activity and only slightly decreased the bioactivity. Peptides composed of 9-10 amino acid residues with high hydrophobic and basic nature possess antibacterial activity but, in general, are less active than the larger counterpart peptides. By replacing all Trp residues of a short peptide by Leu residues, the activity was considerably reduced. Circular dichroism studies and antibacterial assays showed that shorter peptides with very low helical content, and thus deprived of amphiphilic character, still have appreciable bioactivity. This observation, coupled with the fact that due to their small size they cannot span the bacterial outer lipid bilayer, may suggest different mechanisms of action for long-chain vis-a-vis short-chain peptides.

Characterization of an opioid peptide-containing protein and of bovine α-lactalbumin by electrospray ionization and liquid secondary ion mass spectrometry.

Mass spectrometry methods have been used to characterize two proteins: an opioid peptide-containing protein extracted from bovine pituitary, and bovine α-lactalbumin (BAL). A protein that contains ß-endorphin was found in bovine pituitary, and that protein was characterized with electrospray ionization mass spectrometry (ESIMS), gel permeation chromatography, reversed-phase high performance liquid chromatography (RP-HPLC), radioimmunoassay, trypsinolysis, and liquid secondary ion mass spectrometry (LSIMS).BAL is a protein that was used as a model to develop analytical methods to study opioid peptide-containing proteins. Commercial BAL was purified by RP-HPLC, and its molecular weight (M.W.) was determined by ESIMS. The shift in mass observed following dithiothreitol (DTT) reduction estimated the number of disulfide bonds.For all of the data obtained for BAL with or without RP-HPLC separation, ESIMS determined the M.W. of the peptides produced by trypsin treatment of BAL, and LSIMS selected a precursor ion, the protonated molecule ion [M + H](+), of a tryptic peptide, which was analyzed by tandem mass spectrometry. Following DTT reduction, ESIMS and LSIMS detected each peptide that contained disulfide bonds in that mixture of tryptic peptides.

Electrospray mass spectrometry for the analysis of opioid peptides and for the quantification of endogenous methionine enkephalin and ß-endorphin.

Electrospray ionization mass spectrometry was used to characterize several different neuropeptides, whose molecular weights ranged from 555 to 3463 Da, and to quantify endogenous methionine enkephalin (ME) and ß -endorphin (ß E) extracted from a human pituitary gland. Methionine enkephalin and leucine enkephalin both yield only an [M + H] + ion with electrospray mass spectrometry; the other peptides produce a series of multiply charged even-electron molecular ions of the general nature [M + nH](n)+ in proportion to the number of basic amino acid units present, with no evidence of fragmentation. The electrospray mass spectra are characterized by low background noise. The quantiftcation of ME is based on a comliarison of the ion current due to the [M + H] + ion of native and of a deuterated ME ([(2)H5 s-(4)Phe]-ME) internal standard. The calibration curve is linear in the range of ca. 1-35 pmol synthetic ME. The amounts of ME determined in three separate human pituitary extracts were 9.1, 8.2, and 4.7 pmol/mg protein. The corresponding amount of ME in a canine pituitary was 39.8 pmol/mg protein. To quantify ß E, the ion current due to the [M + 5H](5) + ion was monitored and compared to an external calibration curve obtained by analyzing solutions of synthetic ß E in the range 5 µmol-50 pmol. The analysis of a human pituitary yielded 660 fmol ß E/mg protein.

Comparative lipoprotein metabolism of myristate, palmitate, and stearate in normolipidemic men.

This project was designed to test the hypothesis that long-chain saturated fatty acids (myristate, palmitate, and stearate) are metabolized differently in human subjects, and that these differences may therefore account for the changes in plasma lipoprotein composition when these fatty acids are altered in the diet. Ethyl esters of each of the stable-isotope-labeled fatty acids (2H3- or 2H4-myristate, 13C16-palmitate, and 13C18-stearate) were fed to five nonhyperlipidemic men. The concentration of each labeled fatty acid was monitored for up to 72 hours as the fatty acids were assimilated into the lipid components (phospholipid [PL], triglyceride [TG], and cholesteryl ester [CE]) of the plasma lipoproteins (TG-rich lipoproteins [TRL], intermediate-density [IDL], low-density [LDL], and high-density lipoprotein [HDL]). Over 95% of the myristate was incorporated into TG, whereas 33% and 9% of the stearate and 18% and 7% of the palmitate were incorporated into PL and CE, respectively. The mean residence times (MRTs) for myristate in TG (8.6 to 9.9 hours) and PL (6.7 to 10.9 hours) in the individual lipoprotein subfractions were significantly shorter than for either palmitate (TG, 12.7 to 15.3 hours; PL, 19.6 to 21.3 hours) or stearate (TG, 10.7 to 15.5 hours; PL, 17.8 to 19.9 hours). The MRTs for stearate were shorter than for palmitate in PL. These data indicate that TG fatty acid in general, and myristate TG in particular, is the most rapidly cleared of the saturated fatty acids. There was a rapid transfer of labeled TG and PL between the lipoproteins. We were unable to detect any significant amount of stearate desaturation or elongation. In conclusion, these data demonstrate that myristate, palmitate, and stearate are metabolized in unique ways, and that it may therefore be inappropriate to continue to regard all "saturated fatty acids" as metabolically similar in clinical studies. Rather, it is important that we elucidate more clearly the specific metabolic pathway of each fatty acid to understand the mechanisms by which it alters plasma lipoprotein concentrations and composition and influences atherogenesis.

Cardiovascular and hematologic effects of diphenylhydantoin in maternal and fetal sheep.

Anticonvulsant therapy often includes diphenylhydantoin (Dilantin) and is usually advocated during the pregnancy of an epileptic woman. The cardiovascular and hematologic effects of diphenylhydantoin (5 mg/kg body weight/5 min) were studied in 12 experiments on 5 ewes and their fetuses in which catheters were chronically implanted and an electromagnetic flow probe was continually around the uterine artery. Slight but significant transient decreases in fetal blood pressure (P less than .001), oxygen percent saturation (O2%, P less than .02) and O2 content (P less than .01) were observed. These parameters recovered to baseline values within 75 minutes after infusion was begun, and no other fetal changes were noted. Maternal metabolic alkalosis was evidenced by significant increases in pH (P less than .001) bicarbonate (P less than .05), and base excess (P less than .02). A slight decrease in the percent hemoglobin was observed at 15, 60, and 90 minutes (P less than .02, P less than .05, and P less than .01, respectively). No changes were observed in maternal heart rate or uterine blood flow, though an increase in blood pressure was observed at 30 minutes (P less than .05). From these observations, it is concluded that the administration of diphenylhydantoin intravenously causes transient fetal hypotension, reduction of O2%, maternal metabolic alkalosis, and transient hypertension without any changes in fetal pH, PCO2, or uterine blood flow. Therefore, it can be considered a relatively safe drug to be used during pregnancy.

Substance P inactivating enzymes in human cerebrospinal fluid.

The Km and Vmax values were determined for enzymes in human lumbar cerebrospinal fluid (CSF) that inactivate synthetic substance P (SP = RPKPQQFFGLM-NH2) and produce metabolic products. For the human lumbar CSF samples analyzed in this study, Km = 2.24 +/- 0.93 mM and Vmax = 0.113 +/- 0.035 nmol/ml/min (n = 10; mean +/- SEM) for the rate of decrease of SP. HPLC analysis of the incubated synthetic peptide fragments demonstrated that the primary enzymatically produced fragment is SP(3-11), with minor amounts in decreasing order of SP(1-4), SP(1-7), and SP(1-9). Electrospray ionization mass spectrometry (ESI-MS) confirmed the appropriate molecular weights for the four peptides, SP(3-11), SP(1-4), SP(1-7), and SP(1-9). These data demonstrate that the primary enzyme in human lumbar CSF that acts on synthetic SP is a post-proline cleaving enzyme (PPCE).

Beta-endorphin-containing proteins in the human pituitary.

Two new proopiomelanocortin (POMC)-derived beta-endorphin (BE)-containing proteins were detected in the human pituitary, using HPLC, trypsin digestion, and a high sensitivity search with liquid secondary ion mass spectrometry (LSIMS) for the protonated molecule ion, (M + H)+, of tryptic peptides that are unique to BE. Proteins were extracted from pituitary tissues and were purified by solid phase extraction (SPE) chromatography and RP-HPLC. Each HPLC fraction was treated with trypsin, and each unseparated peptide mixture was analyzed by LSIMS to detect the two selected marker peptides (BE 20-24 and BE 10-19) that have excellent LSIMS desorption-ionization properties. The detection of both of those peptides indicated the presence of BE-containing proteins in two HPLC fractions (number 47 and 51). Tandem MS determined the amino acid sequence of the marker peptide BE 20-24 (NAIIK), and those sequence data optimized the specificity of the method. The two new BE-containing proteins derive from the C-terminal region of POMC, and were minor components in the two HPLC fractions. The major component in fraction 51 derived from the vasopressin-neurophysin 2-copeptin precursor.

Purification, characterization, and localization of neuropeptides in the cornea.

The immunologically detected neuropeptides methionine enkephalin (ME), substance P (SP), beta-endorphin (beta-End), and alpha-melanocyte stimulating hormone (alpha-MSH) were purified from bovine corneal extracts by gradient, followed by isocratic, reversed phase-high performance liquid chromatography (RP-HPLC) and characterized, after both chromatographic steps, by radioimmunoassay (RIA). Immunologically detected ME and SP were purified from canine corneal extracts by gradient RP-HPLC and characterized by RIA. An anatomical study of the bovine cornea separated the cornea into an epithelium-enriched and a stroma-enriched portion. After gradient RP-HPLC, RIA demonstrated that all the ME-like immunoreactivity was located in the corneal epithelium, whereas the SP-like immunoreactivity was distributed between the stroma and epithelium in an approximate two-to-one ratio.

Dynorphin A(1-8) in human placenta: amino acid sequence determined by tandem mass spectrometry.

Presence of the kappa receptor-preferring neuropeptide dynorphin A(1-8) in human placenta has been demonstrated by mass spectrometry to establish rigorously the appropriate molecular weight and amino acid sequence. Liquid secondary ionization mass spectrometry produced the protonated molecule ion, (M + H)+, at m/z 981 of the endogenous peptide, and tandem mass spectrometry collected the product ion spectrum that contained the appropriate amino acid sequence-determining fragment ions produced from the precursor ion (M + H)+. The amino acid sequence of the peptide is YGGFLRRI.

Opioid and tachykinin neuropeptides in prolactin-secreting human pituitary adenomas.

Two opioid neuropeptides, methionine enkephalin (ME) and beta-endorphin (BE), and one tachykinin neuropeptide, substance P (SP), were quantified in 10 prolactin (PRL)-secreting human pituitary adenomas and in 10 control human pituitaries. Immunohistochemical techniques provided appropriate staining for PRL. Reversed-phase high performance liquid chromatography (RP-HPLC) was used to purify these three neuropeptides before their analysis, radioimmunoassay (RIA) was used for the quantification of SP-like immunoreactivity (SP-LI), and liquid secondary-ion mass spectrometry (LSIMS) was used for the qualitative and quantitative analysis of ME and a tryptic peptide of BE. This study shows that, for 90% of the cases studied here (excluding one hypothyroidism case), the tachykinin A neuropeptide SP-LI level is decreased, the POMC peptide BE level is not altered, and the proenkephalin A neuropeptide ME level is increased in these PRL-secreting tumors.

Structures of biologically active muramyl peptides from peptidoglycan of Streptococcus sanguis.

The structures of major muramyl peptides derived from peptidoglycan of the oral pathogen Streptococcus sanguis were determined and the biological activity of the peptides was tested in vitro on human monocytes. The muramyl peptides, produced by muramidase digestion of the purified peptidoglycan, were separated by reversed-phase high-performance liquid chromatography, either in their native form or after reduction with sodium borohydride. Chemical structures of the peptides were elucidated by a combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, amino acid analysis, post-source decay analysis and Edman sequencing. The study revealed two distinct monomers: N-acetylglucosaminyl-N-acetylmuramyl-Ala-iGln-Lys(Ala-Ala) (1), where the Ala-Ala is connected to the epsilon-amino group of lysine, and N-acetylglucosaminyl-N-acetylmuramyl-Ala-iGln-Lys(Ala-Ala)-Ala-Ala (2), where an additional dialanyl residue is attached to the lysine alpha-carboxyl group. Two sets of higher oligomers (di-, tri- and tetramers), related structurally to monomers 1 or 2 were also detected. In these oligomers, the monomeric subunits are linked together by Ala-Ala-Ala bridges. The native muramyl peptides primed human monocytes in vitro for the increased production of the microbicidal superoxide radical.

Matrix-assisted laser desorption/ionization mass spectrometric quantification of the mu opioid receptor agonist DAMGO in ovine plasma.

The synthetic opioid peptide analog Tyr-D-Ala-Gly-N-methyl-Phe-Gly-ol (DAMGO), which is a mu opioid receptor-selective agonist, was quantified in ovine plasma samples with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), using delayed extraction and a reflectron. The internal standard was pentadeuterated DAMGO. Timed-ion selection was used to select the precursor ion. The analysis of the post-source decay fragments improved the detection sensitivity, and the use of the precursor-product ion relationship optimized the specificity. For plasma samples, the inter-assay variability of this method was 6.4% (n = 79) and the intra-assay variability was 6.0% (n = 10). The variability for controls was 3.4% (n = 43). The profile of DAMGO amount versus time was determined in sheep plasma, and the corresponding pharmacokinetic data were calculated.

Measurement of dynorphins with 3H-etorphine and canine limbic system receptors.

A canine limbic system preparation was used as the source of opioid peptide receptors to screen biologic extracts for the presence of opioid receptoractive peptides following their gradient RP-HPLC separation. Eight synthetic dynorphin peptides were studied for their ability to displace the commonly-used ligand 3H-etorphine from the canine limbic system P2 preparation. The peptides studied included the dynorphins 1-7, 1-8, 1-9, 1-10, 1-12, 1-13, 1-17, and dynorphin B. Two different types of opioid peptide molecules were utilized for the determination of the level of non-specific binding. In one study, methionine enkephalin, and in the second study each one of the eight corresponding dynorphins, was used for determination of non-specific binding. The experimental data indicated that 3H-etorphine bound to the canine limbic system P2 receptors, and that those dynorphins displaced effectively the 3H-etorphine from those receptors.

Metabolic profiling of opioid peptides in tooth pulp by HPLC and radioreceptor assay.

A analytical system using a combination of gradient RP-HPLC and radioreceptor assay as the HPLC-detector is used to analyze the peptide-rich fraction extracted from a canine tooth pulp homogenate and to provide a metabolic profile of endogenous receptoractive peptides. The gradient RP-HPLC effectively separates the endogenous peptide mixture into a range of hydrophobicities that corresponds to a spectrum of peptide sizes. The receptor preparation is derived from a canine limbic system synaptosome fraction. 3H-DADL serves as the ligand in the RRA. The RP-HPLC/RRA data indicate canine tooth pulp contains a wide range of peptides that interact with the opioid peptide receptor preparation and displace the delta-receptor preferring ligand D2-ala, D5-Leu leucine enkephalin.

Enkephalinase 'A' inhibition by thiorphan: central and peripheral cardiovascular effects.

On the basis of the distribution of enkephalins within the central and peripheral nervous systems as well as on responses to their administration, it has been suggested that these peptides participate in the regulation of the circulation. The present series of experiments examined the effects of thiorphan, an inhibitor of enkephalinase A, on cardiovascular responses to intracerebroventricular (i.c.v.) administration of [D-Ala2,Met5]enkephalin (DAME) and its amide and on peripheral interactions with the sympathetic nervous system and vasoactive peptides. Thiorphan (30 micrograms i.c.v.) potentiated the pressor response to i.c.v. DAME and DAMEamide in conscious spontaneously hypertensive rats. Responses to i.c.v. angiotensin I (AI) were unaffected suggesting lack of inhibition of central angiotensin converting enzyme (ACE). Peripheral administration of relatively large doses of thiorphan (30 and 100 mg/kg s.c.) attenuated the pressor response to i.v. AI by 30-40% and enhanced the depressor effect of i.v. bradykinin in anesthetized normotensive rats indicating inhibition of peripheral ACE. Pressor and tachycardic responses to activation of spinal sympathetic outflow were not altered by thiorphan in pithed normotensive rats. Thiorphan itself did not affect baseline blood pressure or heart rate in any of these experiments. In conclusion, inhibition of central enkephalinase A by i.c.v. administration of thiorphan potentiates the pressor response to i.c.v. DAME. The compound inhibits peripheral ACE but has little direct cardiovascular activity in its own right.

Mass spectrometric quantification of neuropeptides.

The molecular specificity of MS/MS methods for the accurate quantification of endogenous neuropeptides, such as ME and BE, exceeds that of all other methods because: 1. MS/MS qualitative analysis first establishes the amino acid sequence of the endogenous peptide; 2. MS/MS establishes and maintains the structural link between the (M + H)+ ion and its corresponding amino acid sequence-determining ion(s) during quantification; and 3. A stable isotope-incorporated synthetic peptide internal standard is used. Endogenous ME and BE have been measured in human pituitary tissue by this quantitative analytical MS method.

Quantitative analytical mass spectrometry of endogenous neuropeptides in human pituitaries.

This manuscript reviews state-of-the-art mass spectrometric (MS) methodology for the qualitative (amino acid sequence determination) and quantitative analysis of opioid neuropeptides in human pituitary tissue. Those analytical data are required for the elucidation of the basic molecular mechanisms involved in tumor formation and to test the hypothesis that metabolic defects in neuropeptidergic system processing is a contributing factor to human anterior pituitary tumor formation. Several different neuropeptide products that derive metabolically from the proenkephalin A and proopiomelanocortin (POMC) precursors have been analyzed separately and together in human pituitaries, including post-mortem controls and post-surgical tumors. The quantification, with optimal molecular specificity, of a peptide in a tissue is an important measurement because the amount of an endogenous peptide reflects the ratio of its synthesis to its degradation and thus, any defects in those processes may be reflected in the amount of a peptide.