Partial enrichment of phospholipids by enzymatic hydrolysis and membrane filtration of whey protein phospholipid concentrate.

Whey protein phospholipid concentrate (WPPC) contains high amounts of phospholipids (PL; 4.5 ± 1%) but there is interest in further enriching the PL content for nutritional and functional applications. Chemical methods were unsuccessful in separating PL from proteins due to the presence of protein-fat aggregates. Instead, we explored hydrolysis of the proteins to peptides with the objective of removing peptides, thereby concentrating the PL fraction. We used microfiltration (MF) with a pore size of 0.1 µm to help reduce protein/peptide retention. Hydrolyzing proteins should facilitate passage of low molecular weight peptides through the MF membrane, while concentrating fat and PL in the MF retentate. Bench-top experiments were performed to select the proteolytic enzyme that resulted in the most extensive hydrolysis of proteins in WPPC from among 5 different commercial proteases. Sodium dodecyl sulfate-PAGE analysis was performed to measure the extent of protein hydrolysis over a period of 4 h. Alcalase enzyme was found to exhibit the highest proteolytic activity at conditions of pH 8 and temperature 55°C. The intensity of major protein bands (milkfat globule membrane proteins, caseins, ß-lactoglobulin) in WPPC decreased in sodium dodecyl sulfate-PAGE profiles as hydrolysis progressed, along with the appearance of low molecular weight bands. Pilot-scale MF production, coupled with diafiltration (DF), of the hydrolyzed sample aided in the removal of peptides that caused an ~18% reduction in protein content with the final retentate having a total PL content of 9.3% dry basis (db) with protein and fat contents at approximately 43.8 ± 0.4% (db) and 48.9 ± 1.2% (db), respectively. The MF permeate had minimal fat content, indicating that there was no transmission of lipids or PL through the membrane during the MF/DF process. Confocal laser scanning microscopy and particle size analysis of enzyme hydrolyzed solution revealed that protein aggregates were still present after 1 h of hydrolysis. Complete removal of proteins and peptides was not achieved by this process, suggesting that a combination of enzymes would be needed for further hydrolysis of protein aggregates in WPPC solution to further enrich the PL content.

A study of various chemical pretreatments to fractionate lipids from whey protein phospholipid concentrate.

Dairy-derived lipids such as phospholipids (PL) have been gaining interest due to their functional and nutritional properties. Our research goal was to develop a separation process (nonsolvent based) to produce an enriched dairy lipid fraction from whey protein phospholipid concentrate (WPPC). Various chemical pretreatments (i.e., adjustment of pH, calcium, or temperature) were applied to rehydrated commercial WPPC solutions. These treatments were done on a bench-top scale to aid in the precipitation of proteins or PL. The chemically treated solutions were centrifuged and fractionated into the following 3 layers: (1) top fat layer, (2) supernatant in the middle zone, and (3) sediment at the bottom of the centrifuge tubes. The thickness and size of the layers varied with the treatment parameters. Compositional analysis of each layer showed that the proteins, fat, and PL always appeared to fractionate in similar proportions. The proteins in each layer were characterized using sodium dodecyl sulfate-PAGE under reducing and nonreducing conditions. Different proteins including whey proteins, caseins, and milk fat globule membrane proteins and lipoproteins were identified, and no specific type of protein had an affinity for either the top or bottom layer. All types of proteins were present in each of the layers after centrifugation, and there were no major differences in fractionation of the proteins between layers with respect to the chemical treatment applied. The microstructure of protein and fat in WPPC was investigated using confocal laser scanning microscopy. Dual staining of the rehydrated WPPC solution with Fast Green FCF (proteins) and Nile Red (lipids) showed the presence of very large protein aggregates that varied in size from 20 to 150 µm, with fat trapped within these aggregates. The confocal laser scanning microscopy images of liquid WPPC revealed fine strands of a weak protein network surrounding the fat globules. This indicated that there were specific interactions between the proteins, as well as between the fat and proteins in WPPC. Sodium dodecyl sulfate treatment was performed to understand the nature of the interactions between protein and fat. We found that about 35% of the fat present in WPPC was in the form of free fat, which was only physically entrapped within the protein aggregates. The remaining fat had some form of association with the proteins in WPPC. Other fractionation techniques would be needed to obtain an enriched dairy lipid fraction.

Effect of temperature and pH on the properties of skim milk gels made from a tamarillo (Cyphomandra betacea) coagulant and rennet.

Reconstituted skim milk was gelled with a crude protease extract from tamarillo [Cyphomandra betacea or Solanum betacea (syn.)] fruit and compared with gels prepared with calf rennet. The effects of temperature and pH on the gelation of skim milk were investigated by small deformation oscillatory rheology. The tamarillo extract-induced gels had a faster rate of increase in the elastic modulus (G') at the early stage of gelation than rennet-induced milk gels. This was probably due to the broader proteolytic activity of tamarillo protease extracts as shown by sodium dodecyl sulfate-PAGE analysis. Confocal microscopy also showed that the milk gels resulting from the addition of tamarillo extracts had larger voids than rennet-induced milk gels. The proteolytic activity of tamarillo extracts was found to be optimal at pH 11. For both rennet and tamarillo extracts, the aggregation time was similar between pH 6.7 and 6.5, but the aggregation time of rennet-induced milk gels was lower than that of milk gels obtained by the addition of tamarillo extracts at pH lower than 6.5. An increase in temperature was found to have a significant effect on aggregation time, particularly at 20°C, where rennet did not coagulate milk in 3 h but the tamarillo extracts coagulated milk within 2 h. The results of this study suggest that extracts from tamarillo fruit could be used for milk gelation, particularly under lower temperature or high pH conditions.

Milk production and composition, nitrogen utilization, and grazing behavior of late-lactation dairy cows as affected by time of allocation of a fresh strip of pasture.

Eighty late-lactation dairy cows were used to examine the effects of allocating a new pasture strip of a sward based on ryegrass (Lolium perenne L.) in the morning (a.m.; ∼0730 h) or in the afternoon (p.m.; ∼1530 h) on milk production and composition, nitrogen (N) utilization, and grazing behavior. Cows grazed the same pasture strips for 24 h and were offered the same daily herbage allowance. Herbage composition differed among treatments; p.m. herbage had greater dry matter (DM; 22.7 vs. 19.9%), organic matter (OM; 89.5 vs. 88.9%), and water-soluble carbohydrate (10.9 vs. 7.6%) concentrations and lesser crude protein (20.5 vs. 22.2%) and neutral detergent fiber (48.8 vs. 50.4%) concentrations compared with a.m. herbage. Total fatty acids (FA), α-linolenic acid, and polyunsaturated FA (PUFA) were greater in a.m. herbage, whereas monounsaturated FA were greater in p.m. herbage. Estimates of herbage DM intake did not differ among treatments. Daily milk yields and milk fat and milk protein concentrations were similar among treatments, whereas milk fat (684 vs. 627 g/cow), milk protein (545 vs. 505 g/cow), and milk solids (milk fat + milk protein) yields (1,228 vs. 1,132 g/cow) tended to be greater for cows on p.m. herbage. Rumenic acid and total PUFA in milk were greater for cows on a.m. herbage, whereas oleic acid was greater for cows on p.m. herbage. Estimates of urinary N excretion (g/d) did not differ among treatments, but urinary N concentrations were greater for cows on a.m. herbage (5.85 vs. 5.36 g/L). Initial herbage mass (HM) available (kg of DM/ha) and instantaneous HM disappearance rates (kg of DM/ha and kg of DM/h) did not differ, but fractional disappearance rates (0.56 vs. 0.74 per hour for a.m. vs. p.m., respectively) differed. Under the current conditions, timing of pasture strip allocation altered the herbage nutrient supply to cows; allocating a fresh strip of pasture later in the day resulted in moderate increases in milk and milk solids yields in late-lactation dairy cows. Conversely, a greater concentration of precursor FA in a.m. herbage resulted in a greater concentration of beneficial FA in milk, compared with cows on p.m. herbage.

Casein polymorphism heterogeneity influences casein micelle size in milk of individual cows.

Milk samples from individual cows producing small (148-155 nm) or large (177-222 nm) casein micelles were selected to investigate the relationship between the individual casein proteins, specifically κ- and ß-casein phenotypes, and casein micelle size. Only κ-casein AA and ß-casein A1A1, A1A2 and A2A2 phenotypes were found in the large casein micelle group. Among the small micelle group, both κ-casein and ß-casein phenotypes were more diverse. κ-Casein AB was the dominant phenotype, and 3 combinations (AA, AB, and BB) were present in the small casein micelle group. A considerable mix of ß-casein phenotypes was found, including B and I variants, which were only found in the small casein micelle group. The relative amount of κ-casein to total casein was significantly higher in the small micelle group, and the nonglycosylated and glycosylated κ-casein contents were higher in the milks with small casein micelles (primarily with κ-casein AB and BB variants) compared with the large micelle group. The ratio of glycosylated to nonglycosylated κ-casein was higher in the milks with small casein micelles compared with the milks with large casein micelles. This suggests that although the amount of κ-casein (both glycosylated and nonglycosylated) is associated with micelle size, an increased proportion of glycosylated κ-casein could be a more important and favorable factor for small micelle size. This suggests that the increased spatial requirement due to addition of the glycosyl group with increasing extent of glycosylation of κ-casein is one mechanism that controls casein micelle assembly and growth. In addition, increased electrostatic repulsion due to the sialyl residues on the glycosyl group could be a contributory factor.

Fatty acid profile differs between organic and conventionally produced cow milk independent of season or milking time.

Differing amounts of fresh forage and concentrates fed, and level of input contributes to the differences reported in fatty acid (FA) composition of organic and conventionally produced cow milk. In many previous studies designed to investigate this phenomenon, comparisons were made between grazed organic cows and housed conventional cows. In the present study, we have investigated differences between organic and conventional milk produced using year-round pasture grazing, as practiced in New Zealand. The FA composition was determined in milk sampled at morning and evening milking in both spring and autumn. Samples were taken from 45 cows from the Massey University organic herd and compared with 50 cows from the corresponding conventional herd grazed and managed similarly at the same location. Forty-three out of 51 analyzed FA were influenced by season, whereas 28 were different between production systems. In addition, one-half were also different due to time of milking. Levels of linoleic acid and α-linolenic acid were higher in organic milk, whereas conjugated linoleic acid (CLA) and vaccenic acid were higher in conventional milk. The first 3 FA (linoleic acid, α-linolenic acid, and CLA) were more abundant in milk harvested during autumn, and the CLA concentration was also significantly influenced by time of milking. Our results confirm reports that the FA profile is affected by season and time of milking, and we also showed an effect due to the production system, when both sets of cows were kept continuously on pasture, even after taking milking time and seasonal effect into account.

Invited review: organic and conventionally produced milk-an evaluation of factors influencing milk composition.

Consumer perception of organic cow milk is associated with the assumption that organic milk differs from conventionally produced milk. The value associated with this difference justifies the premium retail price for organic milk. It includes the perceptions that organic dairy farming is kinder to the environment, animals, and people; that organic milk products are produced without the use of antibiotics, added hormones, synthetic chemicals, and genetic modification; and that they may have potential benefits for human health. Controlled studies investigating whether differences exist between organic and conventionally produced milk have so far been largely equivocal due principally to the complexity of the research question and the number of factors that can influence milk composition. A main complication is that farming practices and their effects differ depending on country, region, year, and season between and within organic and conventional systems. Factors influencing milk composition (e.g., diet, breed, and stage of lactation) have been studied individually, whereas interactions between multiple factors have been largely ignored. Studies that fail to consider that factors other than the farming system (organic vs. conventional) could have caused or contributed to the reported differences in milk composition make it impossible to determine whether a system-related difference exists between organic and conventional milk. Milk fatty acid composition has been a central research area when comparing organic and conventional milk largely because the milk fatty acid profile responds rapidly and is very sensitive to changes in diet. Consequently, the effect of farming practices (high input vs. low input) rather than farming system (organic vs. conventional) determines milk fatty acid profile, and similar results are seen between low-input organic and low-input conventional milks. This confounds our ability to develop an analytical method to distinguish organic from conventionally produced milk and provide product verification. Lack of research on interactions between several influential factors and differences in trial complexity and consistency between studies (e.g., sampling period, sample size, reporting of experimental conditions) complicate data interpretation and prevent us from making unequivocal conclusions. The first part of this review provides a detailed summary of individual factors known to influence milk composition. The second part presents an overview of studies that have compared organic and conventional milk and discusses their findings within the framework of the various factors presented in part one.

Determination of immunoglobulin G in bovine colostrum and milk powders, and in dietary supplements of bovine origin by protein G affinity liquid chromatography: collaborative study.

An AOAC collaborative study was conducted to evaluate an affinity LC procedure for measuring immunoglobulin G (IgG) in selected dairy powders. The powders were extracted with 0.15 M sodium chloride solution and the pH was adjusted to 4.6 to precipitate caseins, which would otherwise lead to an overestimation of IgG. The analyte was then bound to a commercially available Protein G affinity cartridge and selectively eluted with a glycine buffer at pH 2.5. Detection was at 280 nm and quantification was made against a calibration curve prepared from bovine serum IgG. The samples analyzed included the likely matrixes for which this assay will find commercial use, namely, high- and low-protein-content colostrum powders, tablets containing colostrum powder, and some IgG-containing dairy powders; milk protein isolate, whey protein concentrate, and skim milk powder. Eleven laboratories provided data for the study and assayed blind duplicates of six materials. The repeatability RSD values ranged from 2.1 to 4.2% and the reproducibility RSD values ranged from 6.4 to 18.5%. The Protein G method with casein removal has adequate reproducibility for measuring IgG in colostrum-derived powders that are traded on the basis of IgG content as a colostral marker.

Direct measurements of interfacial interactions between pectin and kappa-casein and implications for the stabilisation of calcium-free casein micelle mimics.

Using Surface Plasmon Resonance (SPR) it has been shown that the fine structure of the anionic polysaccharide pectin strongly influences its interfacial interaction with a kappa-casein layer coated onto a gold surface (via a dextran linker) in the pH range 3.5-6.8, with the highest SPR signal being observed for pectin with the lowest charge density tested (a degree of methylesterification (DM) around 90%). Furthermore, the Brownian motions of kappa-casein coated polystyrene beads (used to provide calcium-free 'model casein micelles') were studied in pectin solutions using Diffusing Wave Spectroscopy (DWS) and microscopy, and were compared with measurements made on naked beads. At every pH value studied (with the exception of 3.5), bridging of the protein-covered probe particles was observed for pectins of both DM 28 and DM 78. However, no aggregated complexes were found in these model casein micelle systems when pectin of an unusually high DM was used (90%). It was hypothesised that having a limited number of binding regions of spatially limited extent maximises the number of chains binding to the protein layer (as found with the SPR measurement), encourages the formation of loops and trains, and additionally limits the potential for destabilisation via bridging.

Simultaneous separation and quantitation of the major bovine whey proteins including proteose peptone and caseinomacropeptide by reversed-phase high-performance liquid chromatography on polystyrene-divinylbenzene.

A precise, sensitive and reliable RP-HPLC method was developed to enable not only unequivocal determination of alpha-lactalbumin and beta-lactoglobulin in bovine whey samples, but also simultaneous measurement of proteose peptone, caseinomacropeptide, bovine serum albumin and immunoglobulin G. The optimised method on the Resource RPC column allowed separation of the proteins in 30 min and could be applied to the analysis of soluble proteins in a variety of commercial and laboratory whey products. Furthermore, some qualitative information on protein heterogeneity and quality could be derived from the RP-HPLC analyses with additional data available from on-line electrospray mass spectrometry. Within- and between-day repeatability over a wide range of concentrations was excellent (RSD< or =5%) for all proteins except immunoglobulin G and bovine serum albumin where RSD was 7-10%. Analysis of grouped data from whey protein concentrate and whey protein isolate samples gave a limit of detection of < or =0.3% powder mass and a limit of quantitation of < or =1.0% powder mass for all proteins except immunoglobulin G. Limits of detection and quantitation were 0.6% and 2.0%, respectively, for this protein. Quantitative data obtained by the RP-HPLC method compared very favourably with data obtained by alternative methods of whey protein analysis.

Simultaneous monitoring of vascular contractility, intracellular pH and intracellular calcium in isolated rat mesenteric arteries; effects of weak bases.

We report the first simultaneous measurements of pHi, [Ca2+]i and tension, upon alteration of pHi, in isolated rat mesenteric arteries loaded with both carboxy SNARF and indo-1. In these vessels (pre-contracted by 30 mM KCl) alterations of pHi, by addition and subsequent washout of weak bases, produced complicated effects on tone. Although the changes in contractility did not mirror the changes in pHi they were, at all times, accompanied by parallel changes in [Ca2+]i.

Hypoxia, metabolic inhibition, and isolated rat mesenteric tone: influence of arterial diameter.

The present study examined and compared the effects of severe hypoxia (induced by either substitution of O(2) in the gassing medium with N(2) or by addition of the O(2) scavenger sodium dithionite) and metabolic inhibition (induced by addition of sodium cyanide) on the tone of isolated rat mesenteric resistance vessels. The influence of vessel diameter and the endothelium on responses to these maneuvers was investigated. Hypoxia (due to both substitution of O(2) with N(2) and by addition of 2 mM sodium dithionite) caused near maximal relaxation of all tissues studied. Addition of 10 mM dithionite, however, produced a significantly smaller response. Two mM cyanide also relaxed mesenteric arteries. In small vessels a near maximal relaxation to cyanide was observed, however, in larger vessels the relaxation to metabolic inhibition was significantly less than that observed to hypoxia. Increasing the concentration of cyanide had no further effect on responses. All responses were found to be endothelium-independent. Thus, as the effects of hypoxia and cyanide are not always similar, care must be taken when extrapolating the effects of metabolic inhibition to those of hypoxia. The results of this study suggest that, in large mesenteric vessels at least, an "O(2) sensing step," in addition to effects of metabolism, may be involved in the hypoxic response.

Mechanisms of hypoxic vasodilatation of isolated rat mesenteric arteries: a comparison with metabolic inhibition.

1. Hypoxia (PO2 < 5 mmHg) decreased vessel tone in isolated rat mesenteric arteries precontracted with either high [K+] or the thromboxane analogue U46619. This response was not altered by N-nitro-L-arginine (L-NA) and indomethacin. 2. Simultaneous measurement of pHi and tension showed that the decrease in vessel tone was accompanied by an intracellular acidification. Similar reductions in tone and pHi were observed with the metabolic inhibitors 2,4-dinitrophenol (DNP) and sodium azide. 3. The presence of the lactate transport inhibitor alpha-cyano-4-hydroxy-cinnamic acid (CHC) increased the magnitude of the acidification and resulted in a significantly faster reduction in tone in response to hypoxia. Addition of CHC to normoxic tissues caused both a vasodilatation and a reduction of pHi. 4. A decrease in pHi induced on washout of ammonium chloride (NH4Cl) resulted in an increase in tone. 5. Relaxation to hypoxia or metabolic inhibition was unaffected when the change in pHi was neutralized by addition of the weak base trimethylamine (TMA). 6. It is concluded that severe hypoxia decreases tone in isolated rat mesenteric arteries by a mechanism which is independent of nitric oxide and prostaglandins. Both severe hypoxia and metabolic inhibition reduced pHi, although this does not appear to be contributing to the changes in tone observed.

Identification, characterisation and cDNA cloning of two caseins from the common brushtail possum (Trichosurus vulpecula)1.

Two major caseins have been isolated from the milk of the common brushtailed possum (Trichosurus vulpecula). These have been identified as alpha- and beta-casein on the basis of the similarity of their N-terminal sequences to those of the caseins of another marsupial (Macropus eugenii). Both proteins appear to exist in multiple forms. Possum alpha-casein is glycosylated mainly in the form of sialic acid residues and was shown by electrospray mass spectrometry to have multiply phosphorylated forms of three families with molecular masses 22700 and 23200 Da that may represent genetic variants. Two-dimensional electrophoresis showed that beta-casein exists as a complex of five or six proteins of identical N-terminal sequence but differing pI. Electrospray mass spectrometry indicated that the beta-caseins also are multiply phosphorylated with masses between 32300 and 32600 Da. A subfamily with mass values 1530 greater was also detected. The patterns were not affected by stage of lactation and quantitative analysis of two-dimensional gels of whole milk shows that alpha- and beta-caseins are present at a constant ratio throughout lactation. cDNA clones for the possum alpha- and beta-caseins have been isolated from an early lactation mammary cDNA library and sequenced.

Quantification of glutamine in proteins and peptides using enzymatic hydrolysis and reverse-phase high-performance liquid chromatography.

An enzymatic method for hydrolyzing bovine milk proteins was developed. Purified milk proteins (alpha-lactalbumin, beta-lactoglobulin, and beta-casein) were hydrolyzed in 0.1 M Hepes buffer (pH 7.5) containing pronase E, aminopeptidase M, and prolidase at 37 degrees C for 20 h. Free glutamine and other amino acids were derivatized with phenylisothiocyanate and separated using a C18 Pico-Tag column. Amino acids were eluted from the column with an aqueous sodium acetate-acetonitrile gradient with detection at 254 nm. Glutamine recoveries from hydrolyzed alpha-lactalbumin, beta-lactoglobulin, and beta-casein were 78 +/- 4, 98 +/- 3, and 101 +/- 3% of the theoretical values, respectively. The recoveries of most amino acids were comparable with those obtained using acid hydrolysis, except for the recoveries of proline and acidic amino acids. These peptide bonds appeared to be resistant to enzymatic hydrolysis and also to inhibit the hydrolysis of adjacent amino acids. Free glutamine was found to be very stable (97% recovery) under the enzymatic hydrolysis conditions.

Effects of 17beta-oestradiol on rat isolated coronary and mesenteric artery tone: involvement of nitric oxide.

Pre- and post-menopausal women receiving oestrogen replacement therapy have a significantly reduced risk of cardiovascular disorders. It has been suggested that this protection might be partly a result of a direct relaxant effect of oestrogens on coronary arteries. This study examines and directly compares the effects of 17beta-oestradiol on rat isolated coronary and mesenteric vessels. The influence of nitric oxide on these responses was also investigated. 17Beta-oestradiol caused similar concentration-dependent relaxation of isolated coronary and mesenteric resistance arteries pre-contracted with either KCl (60 mM) or 9,11-dideoxy-11alpha,9alpha-epoxymethanoprostaglandin (U46619; 1 microM). The relaxation responses to 17beta-oestradiol were significantly reduced, but not totally inhibited, in the presence of N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase; they were not altered by indomethacin, an inhibitor of prostaglandin synthesis. The responses to 17beta-oestradiol in the presence of L-NAME were not dependent on the vessel studied or the pre-contracting agent used. These results suggest that nitric oxide might contribute to the vasodilatory effects of 17beta-oestradiol in rat isolated coronary and mesenteric resistance arteries.

Application of capillary electrophoresis in the identification of phenotypes containing the beta-lactoglobulin C variant.

A method for the separation of the beta-LG variants A, B, and C was developed using free zone capillary electrophoresis. A separation buffer consisting of 50 mM 2-(N-morpholino)ethane sulfonic acid (pH 8.0) and polyoxyethylene (20)-sorbitan monolaurate was used for the separation. Milk samples from 424 Jersey cows were phenotyped using this method, and the gene frequencies for beta-LG A, B, and C were .41, .53, and .06, respectively. These frequencies were different from those found in studies of Jersey populations in other countries, where the beta-LG B allele had a significantly higher frequency, and the beta-LG A allele a significantly lower frequency, than those of this present study. The frequency of the beta-LG C allele was intermediate to those of other studies. The concentration of beta-LG in the milk was different among the beta-LG phenotypes and was, from greatest to least: AA, AB, AC, BB, and BC.

Use of capillary zone electrophoresis in an investigation of peptide uptake by dairy starter bacteria.

A capillary zone electrophoresis (CZE) method to separate the peptide series val-glyn, where n is 1 to 4, has been evaluated and compared to separation by reversed-phase high-performance liquid chromatography (RP-HPLC). The method was able to quantitate peptides present a very low concentrations (down to 0.05 mM) with high reproducibility and accuracy and was capable of separating peptides differing in size by only a single glycine residue. It could also separate the peptides val-gly and leu-gly which differed in only a single side-chain methylene group. The method was fast, required small sample volumes, and proved to be superior to RP-HPLC. The suitability of the CZE method to analyze peptide uptake by dairy starter bacteria is discussed.

Influence of adrenoceptor stimulation on aggregation of platelets from diabetic and control rats.

1. Studies of cardiac and vascular responses have previously demonstrated that diabetes influences the sensitivity of these tissues to adrenoceptor stimulation. Adrenoceptors are also present on platelets where they modulate aggregatory responses. The present study investigates the influence of diabetes on these platelet adrenoceptor-mediated responses. 2. Rats were made diabetic with streptozotocin and platelet aggregatory responses to ADP were examined 2 or 12 weeks later. Aggregation to ADP of platelets from 2-week-diabetic rats was similar to that of platelets from age-matched controls, but platelets from 12-week-diabetic animals exhibited an enhanced aggregation to ADP. 3. In all groups studied, beta-adrenoceptor stimulation with isoprenaline caused a concentration-dependent inhibition of aggregation to ADP, whilst alpha-adrenoceptor stimulation with adrenaline was found to potentiate aggregation to ADP. The degree of inhibition or potentiation was found to remain unchanged by diabetes of either 2 or 12 weeks duration. 4. Previously reported increases in cardiac beta-adrenoceptor sensitivity and aortic alpha-adrenoceptor sensitivity were confirmed 2-week-diabetic animals, but these sensitivity changes were not observed in 12-week-diabetic rats. 5. The results indicate that, unlike the heart and vasculature, the influences of adrenoceptor stimulation on platelet aggregation are not altered by diabetes, even when aggregation to ADP is enhanced.

Separation of beta-lactoglobulin A, B and C variants of bovine whey using capillary zone electrophoresis.

beta-Lactoglobulin is a whey protein that affects milk composition and product functionality and which can be present in up to eight genetic variant forms. A free zone capillary electrophoresis method has been developed to separate and identify the beta-lactoglobulin A, B and C variants. Three buffer systems [borate, 2-(N-morpholino)-ethanesulphonic acid (MES) and bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane (Bistris)] were examined over a range of pH values and with the addition of the separation buffer modifiers Tween 20 and/or ethanolamine. The most successful combination of these was 50 mM MES at pH 8.0 with the addition of 0.1% Tween 20 which clearly resolved the three variants from both each other and from the other whey proteins even though the MES buffer was acting well outside its pKa range (pH 5.3-7.3). The retention times and identification of the individual variants were verified by spiking with commercially purified beta-lactoglobulin A and B proteins and a beta-lactoglobulin AC whey. The method was then used to phenotype beta-lactoglobulin in a sample population of New Zealand Jersey cows.