Healthy Human Plasma Research Articles

Establishment and evaluation of direct assay for plasma microRNAs without RNA extraction by quantitative PCR

Objective To establish a new method for the detection of plasma miRNAs by real-time fluorescence quantitative PCR without RNA extraction. Methods 7.5 μl plasma separated from normal human blood was treated with equal amount of buffer containing Tween 20, which was used directly as sample for the following reverse transcription and PCR reaction to establish a direct assay for plasma microRNAs. U6 snRNA, miR-24-1-5p and miR-4634 were selected as target microRNAs for the determination of RT-qPCR. Both specificity and amplification efficiency were assessed. The difference of Cq values between traditional quantitative PCR detection method and our new method was also observed. Furthermore, the results of mixed primer or individual primer by RT-qPCR were compared in new established method. Results The amplification products of U6, miR-24-1-5p and miR-4634 show a single melting peak at 81 ℃, 83 ℃ and 84.2 ℃, respectively, without primer dimer peak or non-specific peak in all 8 cases of healthy human plasma samples. Amplification efficiency for miR-24-1-5p reached 1.1 detected in plasma direct assay. The amplification curve of U6, miR-24-1-5p and miR-4634 is parallel in each sample, which demonstrated that the amplification of slope is consistent. The Cq value of miR-4634 determined by direct assay is higher than the result of the method by RNA extraction（plasma direct assay 30.98±0.17，RNA extraction 29.22±0.21；t=8.475，P 0.05）and miR-24-1-5p(plasma direct assay 20.73±0.14，RNA extraction 21.06±0.21；t=1.749, P>0.05) although they had significant correlation（r=0.860 5,0.728 9,0.748 5,P<0.05）. No significant difference of Cq value between mixed primer reverse transcription and individual primer reverse transcription. Conclusion A novel RT-qPCR direct assay of plasma miRNAs is established, which can improve the efficiency of plasma miRNAs determination in potential clinical applications. （Chin J Lab Med，2014,37：119-122） Key words: MicroRNAs; Real-time polymerase chain reaction; Plasma

Hyperhomocysteinemia in patients with cardiovascular disease

Homocysteine (Hcy) is an endogenous, non-structural protein, a sulfur-containing amino acid emerging on the pathway of methionine and cysteine, actively involved in numerous biochemical reactions. Total concentration of homocysteine in plasma of healthy humans is low and its level is between 5.0 and 15.0 mmol/l, assessed with the use of HPLC, or 5.0-12.0 mmol/l, using immunoassay methods. Higher concentration of this amino acid in blood is called hyperhomocysteinemia. Hyperhomocysteinemia is significantly correlated with cardiovascular disease and its complications: heart attacks and strokes. It is believed that hyperhomocysteinemia damages endothelial cells, reduces the flexibility of vessels, and adversely affects the process of hemostasis. In addition, hyperhomocysteinemia enhances the adverse effects of risk factors such as hypertension, smoking, and impaired glucose, lipid and lipoprotein metabolism, as well as promoting the development of inflammation. The concentration of homocysteine can be effectively lowered by supplementation with folic acid and vitamins B12 and B6. However, intervention studies conducted in the past decade did not confirm the clinical benefit of vitamin therapy lowering the level of homocysteine in blood of patients with cardiovascular disease. Moreover, there is not clear evidence from genetic studies that the presence of the gene for MTFHR polymorphism 677C>T, which is one of the most common causes of hyperhomocysteinemia, is also associated with the development of cardiovascular disease. These results led the researchers to discuss the role of homocysteine in the development and treatment of cardiovascular disease as well as the need for further research on this issue.

A multiple-reaction-monitoring mass spectrometric method for simultaneous quantitative analysis of five plasma apolipoproteins

A multiplexed targeted proteomic assay using a mTRAQ-MRM/MS-based approach was developed and assessed to systematically quantify the relative expressions of five candidate plasma apolipoproteins that have been previously shown to be dysregulated in neuropsychiatric disorders and cognitive dysfunction: apolipoprotein H (APOH), apolipoprotein J (APOJ), apolipoprotein A4 (APOA4), apolipoprotein E (APOE), and apolipoprotein D (APOD). The peptides and transitions of each APO were carefully selected according to the tandem MS signals acquired on a TripleTOF™ 5600, followed by optimization of the declustering potential and collision energy voltages for transitions on a QTRAP 5500. Our results showed that the collision energies of mTRAQ-labeled peptides were approximately 15%–20% higher than corresponding non-labeled peptides. Through optimized transitions and parameters, we analyzed the relative abundances of the five APOs in human plasma with and without depletion of high abundant proteins. The results indicated that the MRM signals of four target APOs were significantly increased after depletion, while the MRM signal of one APO, APOD, was decreased. Furthermore, the relative abundances of the five target APOs in healthy human plasma were stable, and the ranking of these proteins according to their MS responses changed slightly. Therefore, we deduced that the rank order of the MS signals for these target proteins can be developed as a diagnostic signature for diseased plasma.

On-line two-dimensional capillary strong anion exchange/reversed phase liquid chromatography–tandem mass spectrometry for comprehensive lipid analysis

An on-line two-dimensional liquid chromatography method was developed for comprehensive lipid profiling by coupling strong anion exchange (SAX) and nanoflow reversed-phase liquid chromatography (nRPLC) prior to electrospray ionization-tandem mass spectrometry (2D-SAX/nRPLC–ESI-MS/MS). Lipids can be classified into four different types according to the electrical propensities of the lipids: anionic, weak anionic, neutral polar, and special lipids. In 2D-SAX/nRPLC, various lipids can be fractionated in the first dimension (SAX: 5μm to 100Å, 5.0cm×75μm i.d.) by step elution (methanol and salt solution), followed by the molecular separation of lipids in the second dimension (RP: 3μm to 100Å, 7.0cm×75μm i.d.) with binary gradient LC. Since the elution of lipids from SAX can be achieved with a very small volume of eluent delivered from an autosampler, it can be simply implemented with an LC–ESI-MS instrument for full automation, and the salt step elution, including the two-step injection procedure, can be used for the selective analysis of the desired lipid fraction. For nRPLC–ESI-MS/MS run in either positive or negative ion mode, a common ionization modifier (0.05% ammonium hydroxide with 5mM ammonium formate) was introduced into the binary mobile phase solutions so that 2D-LC–MS could be operated in both ion modes without changing the mobile phase solutions. The developed on-line 2D-SAX/nRPLC–ESI-MS/MS was evaluated with 22 different standard lipids for the optimization of the salt step elution and was applied to a healthy human plasma lipid extract, resulting in the identification of a total of 303 plasma lipids, including 14 different classes.

Differential cytokine expression profile of healthy human bone marrow plasma samples from the endosteal and vascular region

Differential cytokine expression profile of healthy human bone marrow plasma samples from the endosteal and vascular region

Acute appearance of fatty acids in human plasma – a comparative study between polar-lipid rich oil from the microalgae Nannochloropsis oculata and krill oil in healthy young males

BackgroundThe long-chain n-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have human health benefits. Alternatives to fish as sources of EPA and DHA are needed. Oil from the micro-algae Nannochloropsis oculata contains a significant amount of EPA conjugated to phospholipids and glycolipids and no DHA. Krill oil contains EPA and DHA conjugated to phospholipids. We compare the appearance of fatty acids in blood plasma of healthy humans after consuming a high fat meal followed by either algal oil or krill oil.MethodsTen healthy males aged 18-45 years consumed a standard high fat (55 g) breakfast followed by either algal oil (providing 1.5 g EPA and no DHA) or krill oil (providing 1.02 g EPA and 0.54 g DHA). All participants consumed both oils in random order and separated by 7 days. Blood samples were collected before the breakfast and at several time points up to 10 hours after taking the oils. Fatty acid concentrations (μg/ml) in plasma were determined by gas chromatography.ResultsFatty acids derived mainly from the breakfast appeared rapidly in plasma, peaking about 3 hours after consuming the breakfast, and in a pattern that reflected their content in the breakfast. There were time-dependent increases in the concentrations of both EPA and DHA with both algal oil (P < 0.001 for EPA; P = 0.027 for DHA) and krill oil (P < 0.001 for both EPA and DHA). The concentration of EPA was higher with algal oil than with krill oil at several time points. DHA concentration did not differ between oils at any time point. The maximum concentration of EPA was higher with algal oil (P = 0.010) and both the area under the concentration curve (AUC) and the incremental AUC for EPA were greater with algal oil (P = 0.020 and 0.006). There was no difference between oils in the AUC or the incremental AUC for DHA.ConclusionThis study in healthy young men given a single dose of oil indicates that the polar-lipid rich oil from the algae Nannochloropis oculata is a good source of EPA in humans.

Complete amino acid sequencing and immunoaffinity clean-up can facilitate screening of various chemical modifications on human serum albumin

This manuscript describes a simple and practical strategy for screening of various chemical modifications of human serum albumin (HSA). Serum albumin is the most abundant blood plasma protein in humans (HSA, 66.5kDa, t1/2 = 19d), constituting about 60% of total proteins. Therefore, it is believed to be the main target of chemical stresses during physiological events such as increased oxidative stress from the degenerative diseases of aging, and higher glucose stress in diabetes mellitus. Consequently, chemical modifications can provide significant information about these biological events. In this study, a complete and robust sequencing method was attained by the peptide mass fingerprinting (PMF) technique using two different complementary proteases (trypsin and Glu-C) and matrix-assisted laser desorption ionization-time of flight/mass spectrometry (MALDI-TOF/MS) in both positive and negative ionization modes. Using this strategy, several modified peptides, 12 oxidations, 25 glycations, 6 lipoxidations, and 5 nitrations have been identified on HSA treated with chemical reactions in vitro. Combined with immunoaffinity clean-up, this method was able to detect in vivo chemical modifications of HSA and found oxidized Trp(214) and glycated Lys(525) in healthy human plasma.

The Study of the Proteome of Healthy Human Blood Plasma under Conditions of Long-Term Confinement in an Isolation Chamber

We identified changes in the proteome of healthy human blood plasma caused by exposure to 105-day confinement in an isolation chamber. After removal of major proteins and concentration of minor proteins, plasma fractions were analyzed by two-dimensional electrophoresis followed by identification of significantly different protein spots by mass spectrometric analysis of the peptide fragments. The levels of α- and β-chains of fibrinogen, a fragment of complement factor C4, apolipoproteins AI and E, plasminogen factor C1 complement, and immunoglobulin M changed in participants during the isolation period. These changes probably reflect the adaptive response to altered conditions of life.

Even and carbon dioxide independent distribution of nitrite between plasma and erythrocytes of healthy humans at rest

In the literature, the distribution of nitrite and nitrate, the major metabolites of nitric oxide (NO), between plasma and erythrocytes and its dependency on partial CO2 pressure (pCO2) in mammalian blood are uncertain. By means of a previously reported fully validated stable-isotope dilution gas chromatography-mass spectrometry (GC–MS) method, we measured nitrite and nitrate concentrations in heparinized plasma from venous, arterial and arterialized blood donated by five healthy non-exercising volunteers at three different time points (0, 15, 30min). pCO2, pH and oxygen saturation were measured by standard techniques. The nitrite and nitrate concentrations and the nitrite-to-nitrate ratio in plasma did not correlate with pCO2 (r=−0.272, P=0.07). Nitrite was found to be almost evenly distributed between plasma and erythrocytes of another eleven healthy non-exercising subjects. In a rabbit model of ARDS, no differences were found in the plasma nitrite and nitrate concentrations comparing normoventilation with hypercapnia. Our studies suggest that the distribution of nitrite between plasma and erythrocytes at rest is largely even and independent of pCO2 in blood of healthy humans and rabbits with ARDS.

Determination of plasma protein binding rate of arctiin and arctigenin with ultrafiltration

To determine the plasma protein binding rate of arctiin and arctigenin. The ultrafiltration combined with HPLC was employed to determine the plasma protein binding rate of arctiin and arctigenin as well as rat plasma and healthy human plasma proteins. The plasma protein binding rate of arctiin with rat plasma at the concentrations of 64. 29, 32.14, 16.07 mg x L(-1) were (71.2 +/- 2.0)%, (73.4 +/- 0.61)%, (78.2 +/- 1.9)%, respectively; while the plasma protein binding rate of arctiin with healthy human plasma at the above concentrations were (64.8 +/- 3.1)%, (64.5 +/- 2.5)%, (77.5 +/- 1.7)%, respectively. The plasma protein binding rate of arctigenin with rat plasma at the concentrations of 77.42, 38.71, 19.36 mg x L(-1) were (96.7 +/- 0.41)%, (96.8 +/- 1.6)%, (97.3 +/- 0.46)%, respectively; while the plasma protein binding rate of arctigenin with normal human plasma at the above concentrations were (94.7 +/- 3.1)%, (96.8 +/- 1.6)%, (97.9 +/- 1.3)%, respectively. The binding rate of arctiin with rat plasma protein was moderate, which is slightly higher than the binding rate of arctiin with healthy human plasma protein. The plasma protein binding rates of arctigenin with both rat plasma and healthy human plasma are very high.

Tissue Factor De-encryption, Thrombus Formation, and Thiol-disulfide Exchange

Tissue factor (TF) by forming a complex with factor VIIa (FVIIa) initiates blood coagulation. It was traditionally believed that the separation of FVIIa in circulation from subendothelial TF was the main control that was preventing spontaneous initiation of thrombosis and that circulating cells and endothelium did not express TF protein at rest in healthy individuals. However, TF has been detected in healthy human plasma and animal models of thrombosis, which indicate that TF in circulation can contribute to thrombin generation and fibrin formation after an activation event. Circulating TF-and indeed, most of the TF on the cell surface-is "encrypted" or coagulation inactive. The de-encryption step involves exposure of phosphatidylserine (PS), but PS exposure alone is insufficient for full TF activity. Allosteric disulfide bonds control protein function by mediating conformal change through the formation and breaking of disulfide bonds. TF contains a typical surface exposed allosteric bond in the membrane proximal fibronectin type III domain. Thiol-disulfide exchange involving this disulfide is implicated in TF activation with the formation of the disulfide bond corresponding with the active conformation of TF and free thiol or thiol-modified forms corresponding with encryption. Although the exact mechanism by which TF de-encryption occurs remains a subject of debate, thiol blockade and inhibition of oxidoreductases show an important role for thiol-disulfide reactions in platelet-independent pathways of coagulation in vitro and in vivo. In particular, redox active extracellular protein disulfide isomerase is involved in the earliest stages of thrombus initiation and has proven to be a potential target for antithrombotic drug development.

Haemolysis Effect Estimation of Doxycycline Hyclate 150 mg Delay Release Tablet in Bio-analysis (Human Plasma) by Liquid Chromatography-Tandem Mass Spectrometry

A fast, simple and particular method for estimation of Doxycycline in healthy human plasma was validated using Minocycline as IS. The analyte and IS were extracted from plasma using SPE. The compound was estranged on a RP column with an isocratic mobile phase consisting of 0.1% formic acid in water and acetonitrile (12:88, v/v), and detected by tandem mass spectrometry in positive ion mode. The ion transition recorded in several reaction monitoring mode were m/z 294.1→225.1 for Doxycycline and m/z 286.1→217.1 for IS. Linearity in plasma was observed over the concentration range 0.3-30 ng/mL for Doxycycline. The mean recovery for Doxycycline was 83.7%, with a lower limit of quantification of 0.3 ng/mL. The coefficient of variation of the assay was less than 6.8%, and accuracy of 96.1% to 102.2%. The validated method was applied to bioequivalence study of 150 mg Doxycycline Hyclate tablet in healthy human volunteers. The validated method was used to expose study samples of bioequivalence study of 150 mg Doxycycline Hyclate delay release tablet in 36 healthy human volunteers. Total 50 samples from individual volunteers identified as Haemolyzed, which were analyze initial and repeat again to cross check the method reproducibity for Haeamolysis effect and compared which found acceptable range. Bioequivalence was prove for test and reference using validated method to experimental samples (Figure 1).

Conjugated Linoleic Acid Is a Preferential Substrate for Fatty Acid Nitration

The oxidation and nitration of unsaturated fatty acids by oxides of nitrogen yield electrophilic derivatives that can modulate protein function via post-translational protein modifications. The biological mechanisms accounting for fatty acid nitration and the specific structural characteristics of products remain to be defined. Herein, conjugated linoleic acid (CLA) is identified as the primary endogenous substrate for fatty acid nitration in vitro and in vivo, yielding up to 10(5) greater extent of nitration products as compared with bis-allylic linoleic acid. Multiple enzymatic and cellular mechanisms account for CLA nitration, including reactions catalyzed by mitochondria, activated macrophages, and gastric acidification. Nitroalkene derivatives of CLA and their metabolites are detected in the plasma of healthy humans and are increased in tissues undergoing episodes of ischemia reperfusion. Dietary CLA and nitrite supplementation in rodents elevates NO(2)-CLA levels in plasma, urine, and tissues, which in turn induces heme oxygenase-1 (HO-1) expression in the colonic epithelium. These results affirm that metabolic and inflammatory reactions yield electrophilic products that can modulate adaptive cell signaling mechanisms.

Prandial Ghrelin Attenuation Provides Evidence That Des-Acyl Ghrelin May Be An Artifact of Sample Handling in Human Plasma

Plasma acyl and des-acyl ghrelin are thought of as components of total ghrelin, but this has never been validated using ex vivo spiking experiments, human sample collection comparisons and fit-for-purpose translatable assays. Acyl ghrelin plasma stability was analyzed by LC-MS/MS and it revealed that acyl ghrelin is enzymatically and chemically converted to des-acyl ghrelin in the presence of active serine proteases and HCl. ELISAs with less than 30% total error were used to assess acyl ghrelin behavior in matched authentic human samples. Acyl and total ghrelin were not statistically different in 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride samples and acyl ghrelin losses in K(2)EDTA plasma were accounted for in des-acyl ghrelin formation. Acyl ghrelin is total ghrelin and des-acyl ghrelin should not be detectible in healthy human plasma under optimal sample handling and assaying conditions.

An Efficient Sample Preparation Method for High-Throughput Analysis of 15(S)-8-iso-PGF2α in Plasma and Urine by Enzyme Immunoassay

Although several methods have been reported on the analysis of the oxidative stress marker 15(S)-8-iso-prostaglandin-F2alpha (8-iso-PGF2α) in biological fluids, they either involve extensive sample preparation and costly technology or require high sample volume. This study presents a sample preparation method that utilizes low sample volume for 8-iso-PGF2α analysis in plasma and urine by an enzyme immunoassay (EIA). In brief, 8-iso-PGF2α in deproteinized plasma or native urine sample is complexed with an antibody and then captured by molecular weight cut-off filtration. This method was compared with two other sample preparation methods that are typically used in the analysis of 8-iso-PGF2α by EIA: Cayman's affinity column purification method and solid-phase extraction on C-18. The immunoaffinity purification method described here was superior to the other two sample preparation methods and yielded recovery values of 99.8 and 54.1% for 8-iso-PGF2α in plasma and urine, respectively. Analytical precision (relative standard deviation) was ±5% for plasma and ±15% for urine. The analysis of healthy human plasma and urine resulted in basal 8-iso-PGF2α levels of 31.8 ± 5.5 pg/mL and 2.9 ± 2.0 ng/mg creatinine, respectively. The robustness and analytical performance of this method makes it a promising tool for high-throughput screening of biological samples for 8-iso-PGF2α.

180. Circadian rhythmicity, variability and correlation of Interleukin-6 in plasma and cerebrospinal fluid of healthy humans

Interleukin-6 (IL-6) is a pleiotropic proinflammatory cytokine circulating in the periphery and central nervous system, however, little is known about its circadian characteristics. Main objective of this study was the assessment of IL-6 levels in plasma and CSF of healthy humans across the 24-h period, in order to characterize physiological circadian variation, within- and between-individual variability and correlation of IL-6 levels in plasma and CSF. Simultaneous sequential measurements of plasma and CSF IL-6 concentrations were performed hourly in 10 healthy male volunteers over 24 h. Circadian rhythmicity of IL-6 levels was assessed by a harmonic analysis. Our results showed a considerably high within- and between-individual variability of both CSF and plasma IL-6 levels and deviating circadian profiles between the two compartments. Plasma IL-6 levels demonstrated a biphasic 12-h circadian pattern, while CSF IL-6 levels showed only a main 24-h circadian periodicity. No correlation between IL-6 levels in plasma and CSF was found. Our novel findings expand considerably the relevant literature on the physiology of IL-6 humans. We provide first evidence for a circadian IL-6 rhythm in CSF in humans and confirm prior results on the biphasic plasma IL-6 pattern. The high circadian and between-subject variability in IL-6 levels suggests that single time-point measurements of IL-6 levels can lead to significant bias and should be considered in further research.

Quantification of l-arginine, asymmetric dimethylarginine and symmetric dimethylarginine in human plasma: A step improvement in precision by stable isotope dilution mass spectrometry

The amino acid l-arginine and its metabolites ADMA and SDMA are important markers for a range of diseases in humans. Increased levels of ADMA and SDMA in plasma point to endothelial dysfunction, hypertension, renal impairment and other pathological states. We present here a method to quantify l-arginine, ADMA and SDMA in human plasma, which is suitable to support clinical research in this field. Sample preparation consisted only of protein precipitation and the analytes were separated using a silica based HILIC column. The analytes were detected by ESI MS/MS, providing high selectivity and sensitivity. The calibration functions were linear in the ranges of 7.5–150μmol/l for l-arginine, 0.15–3μmol/l for ADMA and 0.2–4μmol/l for SDMA. These ranges cover the concentrations encountered in healthy and pathological human plasma. The method employs 13C6-arginine, D7-ADMA and, for the first time in LC–MS/MS, D6-SDMA as internal standards for l-arginine, ADMA and SDMA. Therefore, matrix independency and a high intra-day precision of 0.82% for l-arginine, 2.12% for ADMA and 2.83% for SDMA, were achieved at basal plasma concentrations. The respective inter-day precision values were 4.01% for l-arginine, 3.77% for ADMA and 3.86% for SDMA.

Detection and quantitation of forty eight cytokines, chemokines, growth factors and nine acute phase proteins in healthy human plasma, saliva and urine

Cytokines, chemokines, growth factors (CCGFs) and other low abundance proteins/peptides in human body fluids or in tissues are potential biomarkers. Human body fluids such as plasma, saliva, urine, etc. are being analyzed more frequently than tissues primarily because of ease of sample collection. However, available information on concentrations of a large number of CCGFs in various body fluids of the same healthy individuals and gender-specific CCGFs is limited. In this work concentrations of 48 CCGFs were measured using multiplex bead assays and compared between plasma, saliva and urine collected from 20 male and female healthy volunteers. Forty three CCGFs were detected at least in one sample type of which 37 were in plasma, 41 were in saliva, and 34 were in urine; five CCGFs were not detected in any sample. Concentrations of detected CCGFs differed significantly between sample types but similar between gender groups. Gender-specific CCGFs were also observed. Concentrations of nine acute phase proteins were also measured from plasma, saliva and urine to determine general health conditions of the volunteers. This work will provide an idea of which CCGFs are detectable and their relative concentrations in healthy human plasma, saliva and urine and which CCGFs are gender-specific.

Measurement of nitrite in plasma and serum: still a challenging analytical task

Measurement of nitrite in plasma and serum: still a challenging analytical task

Allium ursinum L.: Bioassay-Guided Isolation and Identification of a Galactolipid and a Phytosterol Exerting Antiaggregatory Effects

Aims: We wanted to investigate the possible antithrombotic effects and elucidate the chemical identity of the active principles involved in inhibitory effects against adenosine diphosphate (ADP)-induced aggregation of human platelets by wild garlic, Allium ursinum L. Methods: For this purpose, a bioassay-guided isolation procedure was used followed by spectrometric identification of pure active compounds. For the bioassay, blood was taken from healthy human volunteers and platelet-rich plasma was prepared for turbidimetric platelet aggregation tests. Platelet-rich plasma, stimulated with 20 µmol/l of ADP, was treated with extracts of different polarities, fractions and isolated single compounds from A. ursinum. The extracts were investigated by thin-layer chromatography (TLC), HPLC, mass spectroscopy, electrospray ionization mass spectrometry (ESI-MS) and 1/2-dimensional ¹H/¹³C-nuclear magnetic resonance (NMR) spectroscopic techniques. Results: Fresh A. ursinum leaves were extracted with ethanol, which was the potent form that effectively inhibited ADP-induced aggregation of human platelets. This ethanolic extract was subjected to liquid-liquid partition. Whilst the aqueous phase, containing the moiety of cysteine sulphoxide and thiosulphinate derivatives, showed only weak activity on platelet aggregation, the ethyl-acetate and particularly the chloroform partitions showed the highest aggregation-inhibiting potency. Thus, in our bioassay, the effects of alliins/allicins could be neglected. The chloroform phase, possessing the strongest activity, was separated into 28 fractions by gradient-elution open column chromatography on silica gel. The most active fractions 11–17 were separated again, yielding 10 subfractions. This afforded 1,2-di-O-α-linolenoyl-3-O-β-D-galactopyranosyl-sn-glycerol and β-sitosterol-3-O-β-D-glucopyranoside, the structures of which were determined by ESI-MS and 1/2-dimensional ¹H/¹³C NMR spectroscopic techniques. Furthermore, the minute amounts of volatile oil of A. ursinum leaves obtained by steam distillation according to Ph. Eur. could be evaluated as a third aggregation-inhibiting principle. Conclusion: In our study, for the first time, 2 active, non-sulphur-containing constituents of wild garlic, namely a galactolipid and a phytosterol, could be identified exhibiting inhibitory action on ADP-induced aggregation in human blood platelets. As a major constituent, the galactolipid, 1,2-di-O-α-linolenoyl-3-O-β-D-galactopyranosyl-sn-glycerol, not yet found in Allium sp., appears as a new, highly useful marker substance for A. ursinum drugs, or their pharmaceutical or food preparations, as shown by our orientating TLC analyses.