Steps Of High-performance Liquid Chromatography Research Articles

Culex quinquefasciatus Late Trypsin Biosynthesis Is Translationally Regulated by Trypsin Modulating Oostatic Factor

Trypsin is a serine protease that is synthesized by the gut epithelial cells of female mosquitoes; it is the enzyme that digests the blood meal. To study its molecular regulation, Culex quinquefasciatus late trypsin was purified by diethylaminoethyl (DEAE), affinity, and C18 reverse-phase high performance liquid chromatography (HPLC) steps, and the N-terminal amino acid sequence was determined for molecular cloning. Five overlapping segments of the late trypsin cDNA were amplified by PCR, cloned, and the full sequence (855 bp) was characterized. Three-dimensional models of the pro-trypsin and activated trypsin were built and compared with other trypsin models. Trypsin modulating oostatic factor (TMOF) concentrations in the hemolymph were determined by ELISA and compared with trypsin activity in the gut after the blood meal. The results showed that there was an increase in TMOF concentrations circulating in the hemolymph which has correlated to the reduction of trypsin activity in the mosquito gut. Northern blot analysis of the trypsin transcripts after the blood meal indicated that trypsin activity also followed the increase and decrease of the trypsin transcript. Injections of different amounts of TMOF (0.025 to 50 μg) decreased the amounts of trypsin in the gut. However, Northern blot analysis showed that TMOF injections did not cause a decrease in trypsin transcript abundance, indicating that TMOF probably affected trypsin translation.

Novel Angiotensin-I Converting Enzyme Inhibitory Peptides Isolated From Rice Wine Lees: Purification, Characterization, and Structure-Activity Relationship.

The bioactive peptides that can inhibit angiotensin-I converting enzyme (ACE, EC. 3. 4.15.1) are considered as possible cures of hypertension. Food-derived angiotensin-I converting enzyme inhibitory (ACEi) peptides have gained more attention because of their reduced side effects. In this study, we reported the method for purifying ACEi peptides from the lees of traditional Chinese rice wine and evaluated the product's biochemical properties. After three steps of reversed-phase high-performance liquid chromatography (RP-HPLC), for the first time, we isolated, purified, and identified two novel peptides: LIIPQH and LIIPEH, both of which showed strong ACEi activity (IC50-values of 120.10 ± 9.31 and 60.49±5.78 μg/ml, respectively). They were further categorized as mixed-type ACE inhibitors and were stable against both ACE and gastrointestinal enzymes during in vitro digestion. Together, these results suggest that the rice wine lees that produced as a by-product during rice wine production can be utilized in various fields related to functional foods and antihypertensive medicine.

Large scale purification and characterization of A21 deamidated variant-most prominent post translational modification (PTM) for insulins which is also widely observed in insulins pharmaceutical manufacturing and storage

Biopharmaceutical development demands appropriate understanding of product related variants, which are formed due to post-translational modification and during downstream processing. These variants can lead to low yield, reduced biological activity, and suboptimal product quality. In addition, these variants may undergo immune reactions, henceforth need to be appropriately controlled to ensure consistent product quality and patient safety. Deamidation of insulin is the most common post‐translational modification occurring in insulin and insulin analogues. AsnA21 desamido variant is also the most prominent product variant formed during human insulin manufacturing process and/or during the storage. Often, this deamidated variant is used as an impurity standard during in-process and final product analysis in the QC system. However, purification of large quantity of purified deamidated material is always being challenging due to highly similar mass, ionic, hydrophobic properties, and high structural similarity of the variant compared to the parent product. Present work demonstrates the simplified and efficient scalable process for generation of AsnA21 deamidated variant in powder form with ~96% purity. The mixed-mode property of anion exchange resin PolyQuat was utilized to purify the deamidated impurity with high recovery. Subsequent reversed-phase high performance liquid chromatography (RP-HPLC) step was introduced for concentration of product in bind elute mode. Elution pool undergone isoelectric precipitation and lyophilisation. The lyophilized product allows users for convenient use of the deamidated impurity for intended purposes. Detailed characterization by Mass spectrometry revealed deamidation is at AsnA21 and further confirmed that, structural and functional characterization as well as the biological activity of isolated variant is equivalent to insulin.

Isolation of a starfish myorelaxant peptide (SMP) isotype from the pyloric caeca of Patiria pectinifera

Peptides are naturally occurring biological molecules that are found in all living organisms. These biologically active peptides play a key role in various biological processes. The aim of this study is the extraction and the purification of bioactive materials that induce relaxation of an apical muscle from the pyloric caeca of Patiria pectinifera. The acidified pyloric caeca extract was partially separated by the solid phase extraction using a stepwise gradient on Sep-Pak C18 cartridge. Among the fractions, materials eluted with 60% methanol/0.1% trifluoroacetic acid was put a thorough of a series of high performance liquid chromatography (HPLC) steps to isolate a neuropeptide with relaxation activity. The purified compound was eluted at 28% acetonitrile in 0.1% trifluoroacetic acid with retention time of 25.8 min on the CAPCELL-PAK C18 reversed-phase column. To determine the molecular weight and the amino acid sequence of the purified peptide, LC-MS and Edman degradation method were used, respectively. The primary structure of the peptide was determined to be FGMGGAYDPLSAGFTD which corresponded to the amino acid sequence of a starfish myorelaxant peptide (SMP) isotype (SMPb) found in the cDNA sequence encoding SMPa and its isotypes. In this study, a muscle relaxant neuropeptide (SMPb) has been isolated from pyloric caeca of starfish P. pectinifera. This is the first report of SMPb isolation on the protein level from P. pectinifera.

Separation and Purification of Glucose in Sake for Carbon Stable Isotope Analysis

An existing procedure for the separation and purification of glucose in sake for compound-specific carbon stable isotope analysis was examined with a view to reducing the sample amount required and process complexity, with a focus on the freeze-drying, solid-phase extraction (SPE), and high-performance liquid chromatography (HPLC) steps. In the revised procedure, the carbon stable isotopic composition, as indicated by δ13C values, of glucose was not significantly affected by glucose concentration in the range 0.25–1.00 mg mL−1, solvent (water or 80% aqueous acetonitrile), or HPLC procedure. Glucose freeze-dried after separation and purification by SPE and HPLC exhibited procedure-induced carbon isotopic discrimination of ≤ 0.1‰. The isolation procedure achieved the same accuracy in glucose δ13C values as that achieved with traditional ion-exchange procedures while requiring a sample volume (1 mL) only 4% that of the usual volume. The improved method enables cost-effective, labor-efficient, small-scale, compound-specific carbon stable isotope analyses of glucose in sake.

Identification and characterization of antioxidant peptides from Chinese dry-cured mutton ham.

Chinese mutton ham is a dry-cured meat product with a long ripening time. The aim of this study was to identify and characterize antioxidant peptides from Chinese mutton ham. Mutton ham peptides (MHPs) were purified by gel filtration, anion exchange and reversed-phase high-performance liquid chromatography steps. The 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) free radical scavenging capacity was used to guide the purification of MHPs. Three antioxidant peptides were identified by liquid chromatography with tandem mass spectrometry (LC-MS/MS) as Met-Trp-Thr-Asp (MWTD), Ala-Pro-Tyr-Met-Met (APYMM) and Phe-Trp-Ile-Ile-Glu (FWIIE), with molecular weights 551.61, 611.76, and 706.84 Da, respectively. Among them, APYMM exhibited the highest ABTS radical scavenging activity. The three peptides had the ability to inhibit lipid oxidation and Fenton's reagent-induced protein oxidation and DNA damage. After simulated gastrointestinal digestion, FWIIE and APYMM showed increased antioxidant activity, while MWTD showed decreased activity. Three novel peptides isolated from Chinese mutton ham had strong biological activity. Chinese mutton ham is potentially a functional food and an excellent source of natural antioxidants. © 2019 Society of Chemical Industry.

Optimization of an online heart-cutting multidimensional gas chromatography clean-up step for isotopic ratio mass spectrometry and simultaneous quadrupole mass spectrometry measurements of endogenous anabolic steroid in urine.

Measuring carbon isotope ratios (CIRs) of urinary analytes represents a cornerstone of doping control analysis and has been particularly optimized for the detection of the misuse of endogenous steroids. Isotope ratio mass spectrometry (IRMS) of appropriate quality, however, necessitates adequate purities of the investigated steroids, which requires extensive pre-analytical sample clean-up steps due to both the natural presence of the target analytes and the high complexity of the matrix. In order to accelerate the sample preparation and increase the automation of the process, the use of multidimensional gas chromatography (MDGC) prior to IRMS experiments, was investigated. A well-established instrumental configuration based on two independent GC ovens and one heart-cutting device was optimized. The first dimension (1D) separation was obtained by a non-polar column which assured high efficiency and good loading capacity, while the second dimension (2D), based on a mid-polar stationary phase, provided good selectivity. A flame ionization detector monitored the 1D, and the 2D was simultaneously recorded by isotope ratio and quadrupole mass spectrometry. The assembled MDGC set-up was applied for measuring testosterone, 5α- and 5β-androstanediol, androsterone, and etiocholanolone as target compounds and pregnanediol as endogenous reference compound. The urine sample were pretreated by conventional sample preparation steps comprising solid-phase extraction, hydrolysis, and liquid-liquid extraction. The extract obtained was acetylated and different aliquots were injected into the MDGC system. Two high performance liquid chromatography steps, conventionally adopted prior to CIR measurements, were replaced by the MDGC approach. The obtained values were consistent with the conventional ones. Copyright © 2016 John Wiley & Sons, Ltd.

Analysis of the sesquiterpene fraction of citrus essential oils by using the off‐line combination of high performance liquid chromatography and gas chromatography‐based methods: a comparative study

AbstractThe present investigation is based on the off‐line combination of high‐performance liquid chromatography (HPLC), with different high‐resolution gas chromatography (GC) methods (LC//GC), for the analysis of the sesquiterpene hydrocarbon (SH) fraction of various Citrus essential oils. Attention was directed to such constituents because the SH profile is often highly specific and can vary considerably between different Citrus oil‐types. The (normal phase) HPLC step was exploited for the separation of the hydrocarbons, from the oxygenated compounds, hence generating a simplified sub‐sample. After, the hydrocarbon fraction was concentrated and subjected to analysis, using conventional GC‐quadrupole mass spectrometry (qMS) and GC‐flame ionization detection (FID), comprehensive two‐dimensional GC‐qMS and comprehensive two‐dimensional GC‐FID, and cold‐injection GC‐qMS. The latter approach was exploited to avoid and evaluate solute rearrangements occurring under hot‐injection conditions. The data derived from each approach were compared, to evaluate each analytical approach. Apart from the comparative scopes, an overall highly‐detailed view (both qualitative and quantitative) was attained on the SH fraction of each essential oil, with many constituents reported here for the first time. Copyright © 2015 John Wiley & Sons, Ltd.

Sequence analysis and feeding responses evoked by the large molecular form of gastrin releasing peptide (GRP) in the rat GRP-29

Microisolation techniques utilizing several reverse phase high performance liquid chromatography (HPLC) steps have resulted in the purification of two rat gastrin releasing peptide (GRP) forms suitable for microsequence and mass spectral analysis. The sequence of the larger form is APVSTGAGGGTVLAKMYPRGSHWAVGHLM-amide and the smaller form is GSHWAVGHLM-amide which is the carboxyl terminal decapeptide of the larger peptide. The peptides were synthesized and their feeding patterns e.g. first meal size (MS), intermeal interval (IMI) and satiety ratio (SR, IMI/MS) were determined in overnight food-, but not water deprived, male Sprague Dawley rats. The peptides were administered in the femoral vein (0, 0.21, 0.41 and 1.03nmol/kg) immediately before presenting the rats with a 10% sucrose solution. We found that (1) GRP-10 (all doses) and GRP-29 (0.41nmol/kg) reduced first MS, (2) both peptides prolonged IMI length and (3) both peptides increased the SR to similar extents. In conclusion, GRP-10 and GRP-29 are the two endogenous forms of GRP in the rat intestine and they reduce short term feeding to similar extents when administered intravenously.

Abstract 631: Big Angiotensin-25 (Bang-25): A Novel Glycosylated Angiotensin-related Peptide Isolated From Human Urine

The renin-angiotensin system (RAS) plays key roles in the regulation of blood pressure and body fluid dynamics. It is now recognized that a wide variety of tissues and organs, including the heart, vasculature, kidney and nervous system, among others, contain a local tissue RAS, which exerts autocrine/paracrine effects and acts independently of the systemic RAS. In 2006, we isolated and characterized proangiotensin-12, a component of the tissue RAS in rat small intestine. Similar angiotensin-related peptides had never been reported in humans. When we screened human urine gel filtration fractions for bioactive peptides using an antibody raised against the N-terminal portion of Angiotensin II (Ang II), we found a highly immunoreactive peptide with an apparent molecular weight of about 5,000. Therefore, we purified this peptide from 5.5 L of human urine using ion-exchange, gel-filtration, and affinity chromatography, and finally reverse-phase high-performance liquid chromatography (RP-HPLC) steps. We isolated and identified a novel Aogen-derived peptide, Big angiotensin-25 (Bang-25), which is composed of 25 amino acids and is N-glycosylated on its 14th amino acid (Asn) and has a cysteine linked to its 18th amino acid (Cys). We then assessed the distribution of Bang-25 in tissues by immunostaining with an antiserum raised against the C-terminal portion of the peptide. We found that Bang-25 was abundantly expressed in a number of human tissues, including kidney, heart, adrenal gland, pancreas and placenta. In the kidney, Bang-25 is localized predominantly to podocytes. Then, compared with angiotensinogen, Bang-25 is rapidly cleaved by chymase to Ang II, but is resistant to cleavage by renin. In the present study, we have isolated and identified a novel Angiotensin-derived peptide, Bang-25, which is composed of 25 amino acids and is N-glycosylated on its 14th amino acid (Asn) and has a cysteine linked to its 18th amino acid (Cys). The identification of Bang-25 suggests the existence of a RAS processing cascade different from the renin-catalyzed cleavage of Angiotensin to Ang I, and provides a potential target for assessing tissue RAS status and for the development of new therapeutic approaches to related diseases.

Purification and identification of lipolysis-stimulating peptides derived from enzymatic hydrolysis of soy protein

The aim of this study was to purify and identify lipolysis-stimulating peptides derived from Flavourzyme®-soy protein isolate (SPI) hydrolysate (F-SPIH). Glycerol release was employed as a marker for lipolysis in 3T3-L1 adipocytes. A higher glycerol release represents a better lipolysis-stimulating activity. The peptide fraction with highest glycerol release obtained from F-SPIH fractionated by sequential ultrafiltration membranes was further purified using gel filtration chromatography and two steps of reverse-phase high-performance liquid chromatography. The peptides were identified using liquid chromatography–tandem mass spectrometry (LC/MS/MS). Three lipolysis-stimulating peptides were obtained, and the amino acid sequences were ILL, LLL and VHVV, respectively. The in vitro effect of gastrointestinal proteases on lipolysis-stimulating activity of synthetic ILL, LLL and VHVV, respectively, was also investigated. The result suggested that the gastrointestinal protease did not affect lipolysis-stimulating activity of the three novel peptides, which reveals their potential to act as anti-obesity ingredients.

Purification of a Novel Angiotensin I-Converting Enzyme (ACE) Inhibitory Peptide with an Antihypertensive Effect from Loach (Misgurnus anguillicaudatus)

To isolate and characterize novel angiotensin I-converting enzyme (ACE) inhibitory peptide from loach (Misgurnus anguillicaudatus), six proteases, pepsin, α-chymotrypsin, bromelain, papain, alcalase, and Neutrase, were used to hydrolyze loach protein. The hydrolysate (LPH) generated by bromelain [ratio of enzyme to substrate, 3:1000 (w/w)] was found to have the highest ACE inhibitory activity (IC(50), 613.2 ± 8.3 μg/mL). Therefore, it was treated by ultrafiltration to afford fraction of LPH-IV (MW < 2.5 kDa) with an IC(50) of 231.2 ± 3.8 μg/mL, having higher activity than the other fractions. Then, LPH-IV was isolated and purified by consecutive purification steps of gel filtration chromatography and reverse-phase high-performance liquid chromatography to afford a purified peptide with an IC(50) of 18.2 ± 0.9 μg/mL, an increase of 33.7-fold in ACE inhibitory activity as compared with that of LPH. The purified peptide was identified as Ala-His-Leu-Leu (452 Da) by Q-TOF mass spectrometry and amino acid analyzer. An antihypertensive effect in spontaneously hypertensive rats revealed that oral administration of LPH-IV could decrease systolic blood pressure significantly.

Quantification of chitinase and thaumatin-like proteins in grape juices and wines

Chitinases and thaumatin-like proteins are important grape proteins as they have a great influence on wine quality. The quantification of these proteins in grape juices and wines, along with their purification, is therefore crucial to study their intrinsic characteristics and the exact role they play in wines. The main isoforms of these two proteins from Chardonnay grape juice were thus purified by liquid chromatography. Two fast protein liquid chromatography (FLPC) steps allowed the fractionation and purification of the juice proteins, using cation exchange and hydrophobic interaction media. A further high-performance liquid chromatography (HPLC) step was used to achieve higher purity levels. Fraction assessment was achieved by mass spectrometry. Fraction purity was determined by HPLC to detect the presence of protein contaminants, and by nuclear magnetic resonance (NMR) spectroscopy to detect the presence of organic contaminants. Once pure fractions of lyophilized chitinase and thaumatin-like protein were obtained, ultra-HPLC (UHPLC) and enzyme-linked immunosorbent assay (ELISA) calibration curves were constructed. The quantification of these proteins in different grape juice and wine samples was thus achieved for the first time with both techniques through comparison with the purified protein calibration curve. UHPLC and ELISA showed very consistent results (less than 16% deviation for both proteins) and either could be considered to provide an accurate and reliable quantification of proteins in the oenology field.

High-performance liquid chromatography ultraviolet assay for human erythrocytic catalase activity by measuring glutathione as o-phthalaldehyde derivative

The most frequently used catalase (CAT) activity assay is based on the spectrophotometric measurement of hydrogen peroxide (H2O2) absorbance decrease at 240nm. Here we report an alternative high-performance liquid chromatography (HPLC) assay for human erythrocytic CAT (heCAT) activity measurement based on glutathione (GSH) analysis as a highly stable, H2O2-insensitive o-phthalaldehyde (OPA) derivative. The method was developed and validated using an isolated heCAT in phosphate-buffered saline at pH 7.4 and was applied to measure CAT activity in lysed human erythrocytes. heCAT activity was measured at initial concentrations of 5nM for heCAT, 5mM for H2O2, and 10mM for GSH, and the incubation time was 10min. Nitrite (NO2−) was found to be an uncompetitive inhibitor of heCAT activity (IC50=9μM) and of CAT activity in hemolysate (IC50∼750μM). Nitrate (NO3−) at concentrations up to 100μM did not inhibit heCAT activity. Azide (N3−) was found to be a very strong inhibitor of the heCAT (IC50=0.2nM) but a relatively weak CAT inhibitor (IC50∼10μM) in human hemolysates. The novel CAT activity assay works under redox conditions that more closely resemble those prevailing in cells and allows high-throughput analysis despite the required HPLC step.

Isolation of an Antihypertensive Peptide from Alcalase Digest of Spirulina platensis

An angiotensin I-converting enzyme (ACE) inhibitory peptide Ile-Gln-Pro with an IC(50) value of 5.77 +/- 0.09 microM was purified from the alcalase digests of Spirulina platensis by gel filtration chromatography and two steps of reverse-phase high-performance liquid chromatography (RP-HPLC). The peptide was synthesized and showed resistance to in vitro digestion by gastrointestinal proteases. Kinetics studies indicated that the peptide was a noncompetitive inhibitor and that the K(i) value was 7.61 +/- 0.16 microM. Oral administration of Ile-Gln-Pro at a dosage of 10 mg/kg showed significant decreases of the weighted systolic blood pressure (SBP) and diastolic blood pressure (DBP) in spontaneously hypertensive rats (SHR) at 4, 6, and 8 h after treatment. The results showed that the ACE inhibitory peptide from Spirulina platensis may have potential for use in the prevention and treatment of hypertension.

Nitro‐fatty Acids Occur in Human Plasma in the Picomolar Range: a Targeted Nitro‐lipidomics GC–MS/MS Study

First studies on the occurrence of nitrated fatty acids in plasma of healthy subjects revealed basal concentrations of 600 nM for free/nonesterified nitro-oleic acid (NO(2)-OA) as measured by liquid chromatography tandem mass spectrometry (LC-MS/MS). We recently showed by a gas chromatography tandem mass spectrometry (GC-MS/MS) method the physiological occurrence of two isomers, i.e., 9-NO(2)-OA and 10-NO(2)-OA, at mean basal plasma concentrations of 880 and 940 pM, respectively. In consideration of this large discrepancy we modified our originally reported method by replacing solid-phase extraction (SPE) by solvent extraction with ethyl acetate and by omitting the high-performance liquid chromatography (HPLC) step for a more direct detection and with the potential for lipidomics studies. Intra-assay imprecision and accuracy of the modified method in human plasma were 1-34% and 91-221%, respectively, for added NO(2)-OA concentrations in the range 0-3,000 pM. This method provided basal plasma concentrations of 306 +/- 44 pM for 9-NO(2)-OA and 316 +/- 33 pM for 10-NO(2)-OA in 15 healthy subjects. Nitro-arachidonic acid and nitro-linolenic acid were not detectable in the plasma samples. In summary, our studies show 9-NO(2)-OA and 10-NO(2)-OA as endogenous nitrated fatty acids in human plasma in the pM range; HPLC is recommendable as a sample clean-up step for reliable quantification of nitro-oleic acids by GC-MS/MS.

Purification of homoharringtonine and removal of residual solvents by spray drying

Homoharringtonine was purified from Cephalotaxus koreana by a combination of extraction, synthetic adsorbent treatment, low-pressure chromatography, and high performance liquid chromatography (HPLC). A crude extract was obtained by methanol extraction of biomass, followed by liquid-liquid extraction using chloroform. The waxy compounds were efficiently removed by adsorbent (active clay P-1) treatment. The extract was purified to greater than 52% with an 86.4% step yield by silica gel low-pressure chromatography. High performance liquid chromatography steps, which were composed of an HPLC step with silica column and an HPLC step with ODS column, were applied to give 98% purity with high yield. Amorphous homoharringtonine, with a fine particle size, was simply made by dissolving homoharringtonine in methylene chloride/methanol (98/2, v/v), followed by spray drying. Residual solvents, methylene chloride and methanol, could be reduced to 250 ppm and 1,160 ppm by spray drying and successive drying in a vacuum oven.

Characterization of Fusarium graminearum inhibitory lipopeptide from Bacillus subtilis IB

Bacillus subtilis strain IB exhibiting inhibitory activity against the Fusarium head blight disease fungus Fusarium graminearum was isolated and identified. The major inhibitory compound was purified from the culture broth through anion exchange, hydrophobic interaction, and reverse phase high-performance liquid chromatography (RP-HPLC) steps. It was a 1,463-Da lipopeptide and had an amino acid composition consisting of Ala, Glx, Ile, Orn, Pro, Thr, and Tyr at a molar ratio of 1:3:1:1:1:1:2. Electrospray ionization mass spectrometry/mass spectrometry (ESI MS/MS) analyses of the natural and the ring-opened peptides showed the antagonist was fengycin, a kind of macrolactone molecule with antifungal activity produced by several Bacillus strains. Fluorescence microscopic analysis indicated this peptide permeabilized and disrupted F. graminearum hyphae.

A simple purification protocol for the detection of peptide hormones in the hemolymph of individual insects by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

The endocrine system of insects is largely based on peptide hormones. Nevertheless, an unequivocal chemical demonstration of the occurence in the hemolymph (the 'insect blood') is still lacking for most if not all insect peptide hormones, although this is the only way to prove their hormonal status. Focusing on peptides released during ecdysis behavior of the tobacco hornworm Manduca sexta, we developed a purification protocol based on ultrafiltration and a single reversed-phase high-performance liquid chromatography (RP-HPLC) step that for the first time allowed the mass spectrometric and chemical identification of a peptide hormone in the hemolymph of single specimens. Since this method is simple, relatively cheap and fast, it should be useful for routine endocrinological analyses and for monitoring peptide release during different physiological conditions and behaviors in insects.

Flight fuel and neuropeptidergic control of fuel mobilisation in the twig wilter, Holopterna alata (Hemiptera, Coreidae)

The corpus cardiacum of the twig wilter Holopterna alata contains a factor that elicits increases in the concentration of lipids in the haemolymph of twig wilters and migratory locusts and causes hypertrehalosaemia in American cockroaches. A hyperlipaemic neuropeptide was isolated from corpora cardiaca of H. alata in a single high-performance liquid chromatography step. The primary sequence of this peptide was assigned by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry, biological assay and co-elution with the synthetic peptide. The adipokinetic peptide of H. alata is an octapeptide with the sequence pGlu–Leu–Asn–Phe–Ser–Thr–Gly–Trp amide denoted Schgr–AKH-II which was sequenced previously from the corpora cardiaca of a number of Caelifera, Ensifera and some Hymenoptera. A dose of 1 pmol of synthetic Schgr–AKH-II causes a pronounced hyperlipaemic effect in the twig wilter. Physiological experiments with the twig wilter reveal that during flight periods of 3 min, the normally low carbohydrate concentration in the haemolymph is significantly diminished, whereas the lipid concentration stays constant in most cases. During a subsequent rest period of 60 min after a 3 min flight episode, however, the concentration of lipids in the haemolymph increases substantially and significantly, indicating that lipids, too, are a major fuel during flight of twig wilters. This is corroborated by the activation of the enzyme triacylglycerol (TAG) lipase in the fat body, but not in the flight muscles, by injection of 5 pmol of synthetic Schgr–AKH-II, the endogenous adipokinetic hormone that is thought to be released during flight. Moreover, in the thorax there is a significant decrease in the concentration of glycogen and lipids measured after flight plus 60 min of rest compared to non-flown twig wilters, whereas no significant changes were monitored for these substrates stored in the abdomen. When the change in lipid class composition was analysed during flight plus 60 min of rest, TAG which comprised the major class in all compartments analysed (thorax, abdomen, haemolymph) was significantly reduced in abdomen and thorax, and diacylglycerol was significantly increased in all three compartments. From all the data collected, it is concluded that lipids are the major fuel class for flight in H. alata and that the contribution of carbohydrates is minimal.