Globin Digest Research Articles

Effect of Globin Digests on Physical Fatigue in Mice.

Globin digest (GD) inhibits dietary hypertriglyceridemia; however, its effects on physical fatigue remain unknown. Therefore, this study aimed to investigate the potential anti-fatigue effects of GD. Repeated administration of GD and valine (Val)-Val-tyrosine (Tyr)-proline (Pro), a component of GD, for five days prevented the forced walking-induced decrease in locomotion. Furthermore, GD treatment reversed the forced walking-induced increase in blood lactate levels in mice and increased phosphorylated AMP-activated protein kinase (p-AMPK) in the soleus muscle, suggesting that the anti-fatigue effect of GD involves AMPK activation in the soleus muscle through reduced blood lactate.

Globin Digest Improves Visceral Adiposity Through UCP1 Upregulation in Diet-Induced Obese Zebrafish and Mice.

Globin digest (GD), a bioactive oligopeptide derived from porcine hemoglobin proteins, has been demonstrated to have beneficial effects on improving postprandial hyperlipidemia, hyperglycemia, and liver injury. We previously reported the lipid-lowering effects of GD using a zebrafish obesogenic test. Here, we sought to evaluate the effect of GD on visceral adiposity and the underlying molecular mechanisms using zebrafish and mouse obesity models. GD ameliorated dyslipidemia and suppressed the accumulation of visceral adipose tissue (VAT) in adult obese zebrafish. Transcriptomic analysis by RNA sequencing of GD-treated adult zebrafish revealed that GD upregulated UCP1-related pathways. Further, we performed mouse experiments and found that GD intake (2 mg/g body weight/day) was associated with lowered plasma triglyceride and total cholesterol levels, decreased VAT accumulation, and improved adipocyte hypertrophy with the upregulation of Ucp1 expression in white adipose tissue at both the mRNA and protein levels. Taken together, these results indicate that GD improves visceral adiposity by upregulating UCP1 expression, providing a novel perspective on combating obesity.

Val-Val-Tyr-Pro protects against non-alcoholic steatohepatitis in mice by modulating the gut microbiota and gut-liver axis activation.

Val‐Val‐Tyr‐Pro (VVYP) peptide is one of the main active components of Globin digest (GD). Our previous studies indicated that VVYP could protect against acetaminophen and carbon tetrachloride‐induced acute liver failure in mice and decrease blood lipid level. However, the effects and underlying mechanisms of VVYP in the treatment of non‐alcoholic steatohepatitis (NASH) have not been discovered. Our present study was designed to investigate the preventive effect of VVYP on NASH and its underlying specific mechanisms. We found that VVYP inhibited the cytotoxicity and lipid accumulation in L‐02 cells that were exposed to a mixture of free fatty acid (FFA). VVYP effectively alleviated the liver injury induced by methionine‐choline‐deficient (MCD) diet, demonstrated by reducing the levels of serum alanine aminotransferase (ALT)/aspartate aminotransferase (AST)/triglycerides (TG)/non‐esterified fatty acids (NEFA) and improving liver histology. VVYP decreased expression levels of lipid synthesis‐related genes and reduced levels of the proinflammation cytokines in the liver of mice fed by MCD diet. Moreover, VVYP inhibited the increased level of LPS and reversed the liver mitochondria dysfunction induced by MCD diet. Meanwhile, VVYP significantly increased the abundance of beneficial bacteria such as Eubacteriaceae, coriobacteriacease, Desulfovibrionaceae, S24‐7 and Bacteroidia in high‐fat diet (HFD)‐fed mice, however, VVYP reduced the abundance of Lactobacillus. Moreover, VVYP conferred the protective effect of intestinal barrier via promoting the expression of the mucins and tight junction (TJ)‐associated genes and inhibited subsequent liver inflammatory responses. These results indicated that the protective role of VVYP on NASH is mediated by modulating gut microbiota imbalance and related gut‐liver axis activation. VVYP might be a promising drug candidate for NASH.

Globin Digest Exerts Anti-Obesity Effects through UCP1 Upregulation in Zebrafish and Mouse Obesity Models

Globin Digest Exerts Anti-Obesity Effects through UCP1 Upregulation in Zebrafish and Mouse Obesity Models

Structure-function properties of hypolipidemic peptides.

This review addresses the structure-function properties of hypolipidemic peptides. The cholesterol-lowering peptide (lactostatin: IIAEK) operates via a new regulatory pathway in the calcium-channel-related mitogen-activated protein kinase (MAPK) signaling pathway of cholesterol degradation. The bile acid binding peptide (soystatin, VAWWMY) inhibits the micellar solubility of cholesterol in vitro and cholesterol absorption in vivo. VVYP is the most effective peptide having hypotriglyceridemic action in globin digests. The suppressive effect of globin digest on postprandial hyperlipidemia has been reported in humans. The ability of peptides (KRES, Apolipoprotein A-I mimetic peptides) to interact with lipids, remove LOOH and activate antioxidant enzymes associated with high-density lipoprotein determines their anti-inflammatory and anti-atherogenic properties. The β-conglycinin derived peptides KNPQLR, EITPEKNPQLR, and RKQEEDEDEEQQRE inhibit fatty acid synthase in vitro. These promising findings indicate the need for more conclusive molecular, cellular, and animal and human studies to design innovative new peptides that ameliorate cholesterol and lipid metabolism. PRACTICAL APPLICATIONS: Prevention and amelioration of hypercholesterolemia by dietary regulation are important. Dietary protein and peptides are very useful as regulators of serum cholesterol concentration. Diets low in saturated fat and cholesterol that include soy protein may reduce the risk of heart disease. In Japan, the concept of "food for specified health use" has been introduced for the prevention and treatment of life-style related disease. Thus, peptides derived from food proteins and sources other than food proteins such as peptide-rich functional foods and nutraceutical products, have considerable potential to prevent lifestyle-related diseases, especially hyperlipidemia, as discussed in this review. Furthermore, various strategies have been used for the efficient screening, development, and application of new hypolipidemic peptides. These include the use of phage display (for anti-obesity peptide), peptide mimetics (for anti-atherogenic peptide), and molecular targets such as CYP7A1 (for hypocholesterolemic peptide) and prohibitin (for anti-obesity peptide).

Efficacy and Safety of Globin Digest as a Functional Beverage for the Treatment of Childhood Obesity

<b>Background and aims</b>: Childhood obesity is a serious health concern in many countries in the world. In addition to lifestyle education, functional foods or beverages may be useful for preventing and treating obesity. Here we evaluated the effectiveness and safety of a globin digest (GD) functional beverage for the treatment of childhood obesity. <b>Methods</b>: Twenty-eight obese Japanese children (median age, 12.4 years; range, 6.0–17.3 years; 18 males) were randomly assigned to receive orally either placebo beverage (n = 14) or GD beverage containing 6 mg of the amino acid residue Val-Val-Tyr-Pro (n = 14) within 1 h of eating dinner daily for 3 months. Changes in body weight, body mass index percentile, and percentage overweight were based on Japanese age-, sex-, and height-specific body-weight standards. The prevalence of adverse events was also evaluated. <b>Results</b>: The changes in body weight, body mass index percentile, or percentage overweight were not significantly different between the two groups at 3 months or at 6 months after the start of GD intake. No adverse events were reported in any of the subjects. <b>Conclusions</b>: Although the effectiveness of GD beverages for the treatment of childhood obesity needs further evaluation, the GD beverage used in the present study was safe and had good compliance in obese Japanese children.

Effects of globin digest and its active ingredient Trp-Thr-Gln-Arg on galactosamine/lipopolysaccharide-induced liver injury in ICR mice

AimsWe investigated the effects of globin digest (GD) and its active ingredient Trp-Thr-Gln-Arg (WTQR) on galactosamine/lipopolysaccharide (GalN/LPS)-induced liver injury in imprinting control region (ICR) mice. Main methodsThe effects of WTQR and GD on the liver injury were examined by measuring the survival rate, serum aminotransferase activities, hepatic components, antioxidant enzyme activities, histopathological analysis, serum levels and hepatic gene expression of tumor necrosis factor-alpha (TNF-α), macrophage inflammatory protein-2 (MIP-2), and nitric oxide (NO) or inducible nitric oxide synthase (iNOS), and nuclear factor-kappa B (NF-κB) p65 content in GalN/LPS-treated ICR mice. RAW264 mouse macrophages were used to confirm the anti-inflammatory effects of WTQR and GD on the macrophages. Key findingsWTQR and GD increased the survival rate, suppressed the serum aminotransferase activities, serum levels and hepatic gene expression of TNF-α, MIP-2, and NO or iNOS, and nuclear NF-κB p65 content in GalN/LPS-treated mice; decreased the oxidized glutathione content, increased the superoxide dismutase activity, and decreased the histopathological grade values of the hepatocyte necrosis and lobular inflammation in GalN/LPS-injured liver; and suppressed the release levels and gene expression of TNF-α, MIP-2, and NO or iNOS, and nuclear NF-κB p65 content in LPS-stimulated RAW264 macrophages. WTQR and GD may improve the antioxidant defense system and inflammatory status in GalN/LPS-injured liver. SignificanceThese findings indicate that WTQR and GD have hepatoprotective effects on GalN/LPS-induced liver injury in ICR mice.

Effect of globin digest on the liver injury and hepatic gene expression profile in galactosamine-induced liver injury in SD rats

Aims We investigated the effect of globin digest (GD) on the liver injury and hepatic gene expression profile in galactosamine (GalN)-induced liver injury. Main methods The effect of GD on the liver injury was examined by measuring the activities of serum transferases and hepatic antioxidant enzymes, histopathological analysis, gene expression profile, and proteins of the peroxisome proliferator-activated receptor alpha (PPARα) and met proto-oncogene (c-Met) in SD rats at 24 h after GalN administration. The effect of GD on the expression of PPARα and its target gene in AML-12 mouse hepatocytes was also examined. Key findings GD suppressed the elevated activities of serum transferases in GalN-induced liver injury in SD rats. The thiobarbituric acid reactive substance content in GalN-injured liver was a decreasing tendency by GD. GD suppressed the increased oxidized glutathione content, and increased the decreased protein, reduced glutathione contents, and catalase activity in GalN-injured liver. GD may improve the antioxidant defense system and protein synthesis in GalN-injured liver. GD suppressed the elevated expression of the genes related to the inflammation, and decreased the histopathological grade value of inflammatory cell infiltration in GalN-injured liver. GD increased the expression of PPARα protein in GalN-injured liver, and also increased the expression of PPARα and its target gene in AML-12 hepatocytes. The total and phosphorylated c-Met proteins in GalN-injured liver were the increasing tendencies by GD. Significance These findings indicate that GD has the hepatoprotective effect on GalN-induced liver injury in SD rats.

Anti-diabetic effects of globin digest and its active ingredient Leu-Ser-Glu-Leu in ICR mice, streptozotocin-induced diabetic mice and KK-Ay mice

Aims Leu-Ser-Glu-Leu (LSEL) is the main active ingredient of globin digest (GD) that has an anti-diabetic effect. Here, we investigated the anti-diabetic effect of LSEL for the first time. Main methods The anti-diabetic effects of GD and LSEL in ICR mice, streptozotocin (STZ)-induced diabetic mice and KK-Ay mice were examined. Key findings GD and LSEL suppressed the elevation of blood glucose in an oral glucose tolerance test (OGTT) in ICR mice, STZ-induced diabetic mice and KK-Ay mice as well as in an oral sucrose tolerance test in ICR mice and in an insulin tolerance test (ITT) in KK-Ay mice. GD and LSEL decreased the blood glucose levels in the basal state in STZ-induced diabetic mice and KK-Ay mice. Furthermore, GD and LSEL elevated the serum insulin levels in an OGTT in ICR mice and KK-Ay mice and promoted the use of insulin in an ITT in KK-Ay mice. GD and LSEL increased the translocation or expression of the glucose transporter 4 in the muscle of ICR mice, STZ-induced diabetic mice and KK-Ay mice and increased the expression of the uncoupling protein 2 (UCP2) in the muscle of ICR mice. Significance These results indicate that GD and LSEL control blood glucose through the promotion of glucose uptake in the muscle of the mice. The acceleration of glucose uptake by GD and LSEL may be controlled by the promotion of insulin secretion and the up-regulation of UCP2 expression. GD and LSEL seem to be useful for lowering the incidence of hyperglycemia.

Globin Digest: No Evidence for a Weight Loss Mechanism

This study was designed to document the mechanism through which globin digest, a dietary herbal supplement, might cause weight loss by exploring possible fat malabsorption, calorie malabsorption, energy expenditure, and fat oxidation. Six healthy subjects were placed on an outpatient diet for 14 days and given a meal containing 40.9 g of fat on days 5 and 11, and stools were collected for 72 hours after each meal for analysis of fecal fat content. Four grams of globin digest was given with one meal and placebo with the other. In another separate study, six subjects were placed on a 100-g fat, weight-maintaining diet for 14 days. All food was prepared by the Pennington Center (Baton Rouge, LA) metabolic kitchen. Globin digest (2 g) or placebo was given with each of three meals per day, and stool was collected for calorie determinations during the last 72 hours of each week. Subjects received globin digest during one of the 2 weeks and placebo during the other. Resting metabolic rate and respiratory quotient were measured on the last day of each 1-week period. There was no increase in 72-hour fecal fat or fecal calories by bomb calorimetry during either of the studies. There was no difference in the respiratory quotient. Globin digest did result in an increase in resting metabolic rate. However, this increase was not statistically significant. Globin digest, if effective, does not cause weight loss or fat loss through fat malabsorption or a relative increase in fat oxidation. Future studies are needed to document the efficacy of globin digest for weight loss in humans before further mechanistic investigation is attempted.

Hb MOLFETTA [β126(H4)Val→Leu, GTG→CTG]: A NEW, SILENT, NEUTRAL β CHAIN VARIANT FOUND IN AN ITALIAN WOMAN

We have identified a new neutral hemoglobin variant in a pregnant Italian woman, that resulted from a GTG → CTG replacement at codon 126 of the β chain, corresponding to a Val → Leu amino acid change at position β126(H4). Thermal and isopropanol stability tests were normal and there were no abnormal clinical features. Routine electrophoretic and ion exchange chromatographic methods for hemoglobin separation failed to show this variant, but reversed phase high performance liquid chromatography revealed an abnormal peak eluting near the normal β chain. No abnormal tryptic peptide was revealed on the high performance liquid chromatographic elution pattern of the total globin digest. The mutation was determined at the DNA level by amplification of the three β exons by polymerase chain reaction and direct sequencing of one exon that showed an abnormal migration on single strand conformational polymorphism analysis.

A liquid chromatography tandem mass spectrometric method for in vivo dose monitoring of diepoxybutane, a metabolite of butadiene.

1,3-Butadiene, a common air pollutant formed in the combustion of organic matter, has been assessed by the U.S. EPA to be a strongly carcinogenic compound. This risk assessment is very uncertain because of the lack of information on the dose of the powerful carcinogenic metabolite diepoxybutane (DEB). This report presents an analytical method for in vivo dose monitoring of a unique marker for DEB. For a large number of alkylating agents in vivo doses are monitored by measurement by gas chromatography/mass spectrometry (GC/MS) of adducts to N-terminal valine in hemoglobin (Hb), using a modified Edman degradation method. This method is applicable to monofunctional epoxides from butadiene. However, in reaction with N-terminal valine, DEB forms an adduct which is ring-closed to a pyrrolidine, N,N-(2,3-dihydroxy-1,4-butadiyl)valine, with a tertiary amino group that prevents detachment of the alkylated valine by the Edman reagent. Therefore a method has been developed based on the analysis by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) of the N-modified N-terminal peptides enriched after trypsin digestion of globin. In this study Hb samples from mice injected intraperitoneally with (+/-)-DEB were examined qualitatively and quantitatively with regard to the ring-closed adduct. The N-terminal pyrrolidine-heptapeptide was identified in treated mice. The highest adduct levels were obtained in samples from animals given the highest dose of DEB and the adduct levels were below the detection level in control mice.

In vitro adduct formation of phosgene with albumin and hemoglobin in human blood.

The development of procedures for retrospective detection and quantitation of exposure to phosgene, based on adducts to hemoglobin and albumin, is described. Upon incubation of human blood with [(14)C]phosgene (0-750 microM), a significant part of radioactivity (0-13%) became associated with globin and albumin. Upon Pronase digestion of globin, one of the adducts was identified as the pentapeptide O=C-(V-L)-S-P-A, representing amino acid residues 1-5 of alpha-globin, with a hydantoin function between N-terminal valine and leucine. Micro-LC/tandem MS analyses of tryptic as well as V8 protease digests identified one of the adducts to albumin as a urea resulting from intramolecular bridging of lysine residues 195 and 199. The adducted tryptic fragment could be sensitively analyzed by means of micro-LC/tandem MS with multiple-reaction monitoring (MRM), enabling the detection in human blood of an in vitro exposure level of >/=1 microM phosgene.

A liquid chromatography tandem mass spectrometric method forin vivodose monitoring of diepoxybutane, a metabolite of butadiene

1,3-Butadiene, a common air pollutant formed in the combustion of organic matter, has been assessed by the U.S. EPA to be a strongly carcinogenic compound. This risk assessment is very uncertain because of the lack of information on the dose of the powerful carcinogenic metabolite diepoxybutane (DEB). This report presents an analytical method for in vivo dose monitoring of a unique marker for DEB. For a large number of alkylating agents in vivo doses are monitored by measurement by gas chromatography/mass spectrometry (GC/MS) of adducts to N-terminal valine in hemoglobin (Hb), using a modified Edman degradation method. This method is applicable to monofunctional epoxides from butadiene. However, in reaction with N-terminal valine, DEB forms an adduct which is ring-closed to a pyrrolidine, N,N-(2,3-dihydroxy-1,4-butadiyl)valine, with a tertiary amino group that prevents detachment of the alkylated valine by the Edman reagent. Therefore a method has been developed based on the analysis by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) of the N-modified N-terminal peptides enriched after trypsin digestion of globin. In this study Hb samples from mice injected intraperitoneally with (+/-)-DEB were examined qualitatively and quantitatively with regard to the ring-closed adduct. The N-terminal pyrrolidine-heptapeptide was identified in treated mice. The highest adduct levels were obtained in samples from animals given the highest dose of DEB and the adduct levels were below the detection level in control mice.

Suppressive Effect of Globin Digest on Postprandial Hyperlipidemia in Male Volunteers1,2

We have reported previously that various edible protein digests inhibit dietary hyperlipidemia in mice, rats, pigs and dogs. Of the various digests tested, globin digest had the most potent inhibitory activity, and a tetrapeptide extracted from globin digest, Val-Val-Tyr-Pro, had activity 7000-fold greater than that of the parent digest. In this clinical study, we investigated the influence of globin digest on serum chylomicron triglyceride concentrations as an indicator of the effect of globin digest on fat absorption and catabolism in humans. Parallel and crossover trials were conducted in which men consumed a control high fat diet (25 g fat, 7.6 g carbohydrate, 1.9 g protein and 0.7 g sodium chloride) or the same diet supplemented with globin digest. The supplemented dosages were 1 and 4 g globin digest. In the parallel trial, 22 men were divided into three groups: control, globin digest 1 g and globin digest 4 g. The increases in chylomicron triglyceride concentrations at 1 h after ingestion of 1 or 4 g globin digest were significantly lower (P< 0.05) compared with the control group. The crossover trial involved six subjects who consumed the control high fat diet and the same diet supplemented with 4 g globin digest. Serum chylomicron triglyceride levels increased in both groups at 1 and 2 h after ingestion, but when subjects consumed 4 g globin digest the increases were suppressed to 75 (P< 0.05) and 42% (P< 0.05) of the increases in controls at the corresponding times, respectively. The areas under the curves of chylomicron and serum total triglyceride concentrations during the 4 h after ingestion of 4 g globin digest were 46 (P< 0.05) and 34% (P< 0.05) lower, respectively, than when the men consumed the high fat control diet. In these trials, globin digest reduced the increase in serum chylomicron triglyceride concentrations as a result of the ingestion of a high fat diet. This hypotriglyceridemic effect of globin digest may be valuable for preventing obesity and in lowering the incidence of cardiovascular diseases.

Aggregate Formation of Peptic Globin Digest.

Aggregate Formation of Peptic Globin Digest.

Synthesis and mass spectrometric identification of the major amino acid adducts formed between sulphur mustard and haemoglobin in human blood.

As part of a program to develop methods for the verification of alleged exposure to sulphur mustard, we synthesized and characterized three amino acid adducts presumably formed by alkylation of haemoglobin: 4-(2-hydroxyethylthioethyl)-L-aspartate, 5-(2-hydroxyethylthioethyl)L-glutamate and N1- and N3-(2-hydroxyethylthioethyl)-L-histidine. Suitable derivatization methods for GC/MS analysis were developed for these adducts as well as for the cysteine and the N-terminal valine adduct. Incubation of human blood with [35S]sulfur mustard in vitro followed by acidic hydrolysis of isolated globin and derivatization with Fmoc-Cl afforded three radioactive peaks upon HPLC analysis, one of which coeluted with the synthetic Fmoc derivative of N1/N3-(2-hydroxyethylthioethyl)-L-histidine. After pronase digestion of globin the adducts of histidine, glutamic acid, aspartic acid, cysteine and N-terminal valine could be tentatively identified and quantitated. Final identification was obtained from GC/MS analysis. The most abundant adduct, N1/N3-(2-hydroxyethylthioethyl)-L-histidine, could not be sensitively analysed by GC/MS. A convenient LC-tandem MS procedure was developed for this compound, enabling the detection of exposure of human blood to 10 microM sulphur mustard in vitro.

Globin digest, acidic protease hydrolysate, inhibits dietary hypertriglyceridemia and Val-Val-Tyr-Pro, one of its constituents, possesses most superior effect

Globin digest (GD), prepared from globin by acidic protease treatment, suppressed the elevation of serum triglyceride level in not only total but also chylomicron fraction after oral administration of olive oil By screening with this lowering activity, we concluded that Val-Val-Tyr-Pro (VVYP) would be most effective constituent having hypotriglyceridemic action in GD The mode of their action was dose dependent and did not show species specificity Neither the repression of peristaltic movement of intestine nor the delaying of gastric emptying was caused by intake of GD or VVYP, however, the excretion of administered lipid was much more than that of control. Furthermore, administration of GD caused more prominent activation of hepatic triglyceride lipase (HTGL) and the increase of hepatic free fatty acid (FFA) concentration in early phase after administration of fat. From these results, it could be elucidated that GD, and also VVYP, inhibited fat absorption from digestive tract and enhanced activity of HTGL, so that more rapid clearance of dietary hypertriglyceridemia was caused.

ＢＭＳ 質量分析による変異蛋白質の迅速検出と臨床検査への応用

The soft ionization mass spectrometry such as MALDI-TOF and ESI are powerful tools for the detection of proteins, peptides and other biological molecules in body fluids. Since biologically active compounds occur at very low concentrations in mixtures containing salts, there has arisen the need for some special sample handling techniques. In this study, we described some simple preparations for the detection of abnormal proteins in blood and urine prior to the analysis by MALDI-TOF and ESI mass spectrometry.First, by only dilution with distilled water to 10-150 times, albumin and the incomplete immunoglobulin heavy chain in sera and albumin, Bence Jones protein, β2-microglobulin, hemoglobin and lysozyme in urine from patients with nephritis, monoclonal gammopathy and monocytic leukemia were clearly detected. Second, adsorption of albumin with Blue gel or IgG with Protein G Sepharose 4FF could be improved the sensitivities and signal-to-noise ratios for the detections of these proteins. Finally, by immunoprecipitations with specific antisera, proteins at lower concentrations, such as carbohydrate deficient transferrin, could be easily detected using MALDI-TOFMS as well as on-line HPLC/ESIMS analysis.As to the analysis of variant hemoglobinopathy, the peptides prepared by digestion of intact globin were analyzed and nearly 90 significant peaks were detected by a single spectrum from ESI analysis. Ion peaks which were assigned with some degree of certainly covered whole sequence from a- and b-subunits. Especially, at the analysis of a variant hemoglobin “core”, the free cystein residue would be converted into cysteic acid by performic acid oxidation before enzymatic cleavage, which would increase the recoveries of the peptides in the “core” regions. The tandem mass spectrometry with oxidized globin mixture digested with trypsin and lysyl endopeptidase will offer a standard method to detect substitution in the “core” region of hemoglobin.

Separation and tryptic digest mapping of normal and variant haemoglobins by capillary electrophoresis

Characterization of haemoglobin (Hb) through whole protein separations and tryptic digest mapping allows the identification of structural Hb variants which result in haemoglobinopathies. Tryptic digest mapping by conventional two-dimensional paper chromatograph-electrophoresis provides high resolution but requires 48 h, while gradient elution reversed-phase high-performance liquid chromatography (HPLC) is faster (1.5 h), there is decreased resolution. CE analysis provides a fast separation with high resolution. We have used CE to optimise the tryptic digestion of globin purified from normal human haemoglobin A and to analyze tryptic digests from normal Hb. The separations were optimised and peak identification performed using UV scanning detection. In the optimised tryptic digest separation up to 28 peaks could be resolved in < 20 min. These peaks were identified as far as possible and a high-resolution map of the digest was constructed. The optimised analytical conditions were used to observe the separation pattern obtained from normal adult haemoglobin (HbA), common variant haemoglobins and some rarer haemoglobin variants.