Detection of a single nucleotide polymorphism in the human alpha-lactalbumin gene: implications for human milk proteins

Variability in the protein composition of breast milk has been observed in many women and is believed to be due to natural variation of the human population. Single nucleotide polymorphisms (SNPs) are present throughout the entire human genome, but the impact of this variation on human milk composition and biological activity and infant nutrition and health is unclear. The goals of this study were to characterize a variant of human alpha-lactalbumin observed in milk from a Filipino population by determining the location of the polymorphism in the amino acid and genomic sequences of alpha-lactalbumin. Milk and blood samples were collected from 20 Filipino women, and milk samples were collected from an additional 450 women from nine different countries. alpha-Lactalbumin concentration was measured by high-performance liquid chromatography (HPLC), and milk samples containing the variant form of the protein were identified with both HPLC and mass spectrometry (MS). The molecular weight of the variant form was measured by MS, and the location of the polymorphism was narrowed down by protein reduction, alkylation and trypsin digestion. Genomic DNA was isolated from whole blood, and the polymorphism location and subject genotype were determined by amplifying the entire coding sequence of human alpha-lactalbumin by PCR, followed by DNA sequencing. A variant form of alpha-lactalbumin was observed in HPLC chromatograms, and the difference in molecular weight was determined by MS (wild type=14,070 Da, variant=14,056 Da). Protein reduction and digestion narrowed the polymorphism between the 33rd and 77th amino acid of the protein. The genetic polymorphism was identified as adenine to guanine, which translates to a substitution from isoleucine to valine at amino acid 46. The frequency of variation was higher in milk from China, Japan and Philippines, which suggests that this polymorphism is most prevalent in Asia. There are SNPs in the genome for human milk proteins and their implications for protein bioactivity and infant nutrition need to be considered.     

The reaction of ethanolamine O-sulphate with 4-aminobutyrate aminotransferase.

Homogeneous rat alpha-lactalbumin was prepared from whey by chromatography on DEAE-Sephadex A-50 and Ultrogel AcA 44. Two biologically active forms of alpha-lactalbumin were apparent after ion-exchange chromatography, but on gel filtration the combined forms were eluted as a single peak with a molecular weight of approx. 33000. The molecular weight when determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was 15100. Antiserum to alpha-lactalbumin was prepared from rabbits, and single radial immunodiffusion was used to measure the concentration of alpha-lactalbumin in milk expressed from rats during lactation and for 2 days after the cessation of lactation. A significant positive correlation (r = + 0.89) between the concentrations of alpha-lactalbumin and lactose was obtained for the first 20 days of lactation. This is consistent with the suggestion that alpha-lactalbumin may control the concentration of lactose in milk. However, a significant negative correlation (r = -0.91) between the concentration of alpha-lactalbumin and lactose was obtained for 2 days after the cessation of lactation on day 20.     

Macronutrient, mineral and trace element composition of breast milk from Japanese women

The aim of the study was to determine the concentrations of macronutrients and the mineral and trace element composition in maternal milk of Japanese women. We collected human milk samples from mothers living throughout Japan from December 1998 to September 1999, and defined as group A the 1197 samples among them that met the following conditions: breast milk of mothers who were under 40 years old, not in the habit of smoking and/or using vitamin supplements, and whose babies showed no symptoms of atopy and whose birth weights were 2.5 kg or more. We then analyzed their contents individually. We also analyzed the amino acid and free amino acid composition of the breast milk of pooled samples from various lactation stages. Large differences were found to exist among the contents of individual human milk samples. The mean contents of each component were as follows: energy, 66.3+/-13.3 kcal/100 mL; solid matter, 12.46+/-1.56 g/100 mL; ash, 0.19+/-0.06 g/100 mL; total nitrogen, 0.19+/-0.04 g/100 mL; lipids, 3.46+/-1.49 g/100 mL; carbohydrates, 7.58+/-0.77 g/100 mL; lactose, 6.44+/-0.49 g/100 mL; pH, 6.5+/-0.3; osmotic pressure, 299+/-14 mOsm/kg.H2O; chloride, 35.9+/-16.2 mg/100 mL; sodium, 13.5+/-8.7 mg/100 mL; magnesium, 2.7+/-0.9 mg/100 mL; phosphorus, 15.0+/-3.8 mg/100 mL; potassium, 47.0+/-12.1 mg/100 mL; calcium, 25.0+/-7.1 mg/100 mL; chromium, 5.9+/-4.7 microg/100 mL; manganese, 1.1+/-2.3 microg/100mL; iron, 119+/-251 microg/100 mL; copper, 35+/-21 microg/100 mL; zinc, 145+/-135 microg/100 mL; and selenium, 1.7+/-0.6 microg/100 mL. The content of each component varied greatly as the duration of lactation increased. In conclusion, it appears to be necessary to evaluate individual differences of human milk in order to perform valid research regarding infant formula.     

Isolation, partial sequence and asynchronous appearance during lactation of lysozyme and alpha-lactalbumin in the milk of a marsupial, the common ringtail possum (Pseudocheirus peregrinus)

1. Lysozyme and alpha-lactalbumin from the milk of the common ringtail possum have been purified and partially sequenced. 2. The lysozyme had similar enzymic activity to the c-type lysozyme of the domestic hen and 43% homology over the N-terminal 49 residues. 3. alpha-Lactalbumin was present in the milk in two biologically active forms; the more acidic form had 66% sequence homology with the N-terminal 35 residues of red-necked wallaby, 54% with human and 43% with bovine alpha-lactalbumin. 4. SDS polyacrylamide-gel electrophoresis of milk samples showed that alpha-lactalbumin was present in the milk throughout lactation but that lysozyme first appeared only in mid-lactation. The implications of this functional adaptation are discussed.     

Heat-treatment method for producing fatty acid-bound alpha-lactalbumin that induces tumor cell death

HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells), which was identified in human breast milk as an alpha-lactalbumin (LA)-oleic acid complex, kills tumor cells, selectively. Although it may have potential as a therapeutic agent against various tumor cells, only low-volume methods for its production exist. In this study, heat treatment was used to produce complexes from LAs and oleic acid using a simple method. In the case of human LA and oleic acid, heat-treated samples apparently showed much stronger activities than those treated at room temperature, with cytotoxicities equal to that of HAMLET. Furthermore, circular dichroism spectroscopy revealed that heat-treated samples lost their tertiary structure, suggesting a molten globule as oleic acid-bound LA. BLA samples also showed strong activities by heat treatment. Batch production with heat treatment can efficiently convert LAs into tumoricidal complexes.     

An expression profile of human α-lactalbumin in the milk of transgenic mouse

Five female transgenic mice were produced by microinjection using a construct made up of a 7.3-kb-5′ flanking region and a 2.0-kb coding region of human α-lactalbumin, as well as a 227-bp 3′-flanking region from bovine growth hormone gene. A founder female expressed human α-lactalbumin as much as 0.3 g per liter of its milk, approximately a 3-fold increase in the total α-lactalbumin concentration of the transgenic mouse milk. Compared with the normal mice, the expression profile of the hα-Lac transgene in the transgenics is different during the lactation, showing low level in the first 3 days and becoming increased from day 4, then gradually reaching and stabilizing at the highest level from day 13. In addition, the milk yielding volume in the transgenics tended to be higher than in normal mice, suggesting higher concentrations of α-lactalbumin might boost more milk output.     

Changes in alpha-lactalbumin, total lactose, UDP-galactose hydrolase and other factors in tammar wallaby (Macropus eugenii) milk during lactation

alpha-Lactalbumin was isolated from milk of M. eugenii and its concentration in milk samples taken at various times during lactation (0-40 weeks post partum) was determined by single radial immunodiffusion using rabbit antiserum to the purified protein. The alpha-lactalbumin concentration remained almost constant throughout lactation even though the concentration of total lactose (free lactose plus lactose contained in oligosaccharides) fell to zero after 34 weeks post partum. This fall in lactose was accompanied by a rise in the free galactose and glucose concentrations and marked increases in UDP-galactose hydrolase, nucleotide pyrophosphatase, alkaline phosphatase and acid beta-galactosidase activities. It is suggested that the in vitro hydrolysis of UDP-galactose was due to nucleotide pyrophosphatase and that this enzyme may also play a role in vivo late in lactation by making UDP-galactose unavailable for the synthesis of lactose. Alternatively, lactose and lactose-containing oligosaccharides might be degraded by the acid beta-galactosidase during or after secretion.     

Immunocytochemical localization of alpha-lactalbumin in the male reproductive tract

The immunocytochemical localization of the milk protein alpha-lactalbumin in the male reproductive tract is described. Using a primary antiserum raised against highly purified rat milk alpha-lactalbumin, specific staining was consistently shown in the supranuclear Golgi region of the principal cells of the proximal caput epididymidis but only occasionally in epithelial cells from other regions of the duct. Staining was also found in the epididymal lumen and associated with spermatozoa. This luminal staining persisted throughout the distal caput, corpus and cauda epididymidis. Staining was rarely associated with spermatozoa in the efferent ducts and initial segment. Alpha-lactalbumin immunoreactivity was also detected in the seminiferous epithelium. Staining was confined to the Golgi-acrosome region of spermatids. These results indicate that an alpha-lactalbumin-like molecule, or molecules, is present in the male reproductive tract and that it is localized specifically in principal cells from the proximal caput epididymidis and germ cells from the seminiferous epithelium.     

Variation of the cholesterol content in breast milk during 10 days collection at early stages of lactation

More and more research is done concerning nutritional programming. Human milk nutrients which are consumed by infants can influence their health in later life. High level of cholesterol in human milk paradoxically lowers the cholesterol concentration in blood in adults. During the course of human lactation the cholesterol concentration decreases from 31 mg/100cm(3) (colostrum) to 16 mg/100 cm(3) (mature milk). According to Scopesi et al., 2002, Clin Nutr 21: 379-384, cholesterol concentration in mature milk ranged from 6.5 to 18.4 mg/100 cm(3). The aim of the study was to assess the variations in breast milk cholesterol content during 10 day collection at early lactation. 48 samples of human milk were analyzed. Mean age of women was 31 years. Women were collecting samples during 10 days of an early lactation stage (1-3 months after delivery). An Attenuated Total Reflectance Fourier Transformed Infrared (FTIR-ATR) method for easy and rapid determination of cholesterol in human milk was elaborated. Cholesterol content assessed by the FTIR method ranged from 3.36 to 12.98 mg/100 cm(3). Results indicate that milk cholesterol concentration during 10 consecutive days of early lactation is highly variable. Cholesterol content depends on an individual. Therefore it is suggested that not only the period of lactation but also mother's diet, age, season and place of residence are important factors determining cholesterol content.     

Milk composition of three free-ranging African elephant (Loxodonta africana africana) cows during mid lactation

Data are presented that indicate the dynamic changes of nutrients in milk from three free ranging African elephant (Loxodonta africana africana) cows during lactation. At the respective collection times of 12, 14 and 18 months of lactation the nutrient content was 47.3, 52.0 and 68.6 g protein; 60.7, 87.4 and 170.8 g fat; 1.6, 2.1 0.5 g lactose and 20.9, 21.5 and 8.6 g oligosaccharides per kg milk. The protein fraction respectively consisted of 18.0, 31.7 and 45.9 g caseins/kg milk and of 29.3, 20.3 and 22.7 g whey proteins/kg milk. Electrophoresis and identification of protein bands showed that polymorphs of one whey protein may be present in elephant's milk similar to polymorphs of alpha-lactalbumin found in cow's milk. From the middle of the lactation time lactose was replaced by oligosaccharides as major carbohydrate, and the major compound of these was identified as isoglobotriose by 1H NMR spectroscopy. The lipid fraction contains a high content, of capric and lauric acids, approximately 70% of the total fatty acids, and low content of myristic, palmitic and oleic acids. During these lactation times the content of short chain fatty acids, capric and caprylic acids increased, while fatty acids lauric acid and longer decreased.     

Southern elephant seal,Mirounga leonina: composition of milk during lactation

An analysis of milk constituents during various stages of lactation in the southern elephant seal Mirounga leonina was carried out. Forty-six milk samples were taken from 30 females throughout lactation during 1985, 1987, 1990 and 1991 on Stranger Point, King George Island, South Shetland Islands. Total nitrogen (TN), non-protein nitrogen (NPN), sugar, fat, ash and water were measured, and from some of these data true protein and energy content were calculated. The results showed a high degree of variation in water and fat concentrations among samples at different stages of lactation. During the first 20 days the fat content of milk increased from about 12 to approximately 52%, while water content fell from 70 to 33%. The composition of milk changes rapidly during the first days post-partum. Protein, minerals and sugar appear to remain stable after the fourth day of lactation. Milk samples contain significant levels of sugars; thin layer chromatography indicates the presence of lactose and glucose together with other unidentified components. There is evidence of a striking change in composition of the milk in the later part of lactation; the progressive increase in the fat:water ratio is abruptly reversed just prior to weaning.     

Synthesis and evolution of concentration of beta-lactoglobulin and alpha-lactalbumin from cow and sheep colostrum and milk throughout early lactation

Synthesis of beta-lactoglobulin and alpha-lactalbumin by explants of ovine mammary gland and evolution of concentration of these proteins in cow and sheep colostrum and milk throughout early lactation have been studied. The evolution of both proteins was similar in cow and sheep species. The highest concentration was found in the first milking (19 and 2 mg/ml for beta-lactoglobulin and alpha-lactalbumin, respectively). Then, levels of beta-lactoglobulin decreased sharply and those of alpha-lactalbumin slowly during the first days of lactation, reaching stable values during the second week postpartum (4 and 1.5 mg/ml). The concentration ratio beta-lactoglobulin/alpha-lactalbumin was four times greater in colostrum than in mature milk. On the other hand, synthesis of these proteins represented about 25-30% of the synthesized total soluble proteins. The synthesis ratio beta-lactoglobulin/alpha-lactalbumin in explants obtained at 12 and 30 hours postpartum was found to be 3.5 and 1.7. These results indicate that the synthesis and secretion of beta-lactoglobulin are comparatively greater than those of alpha-lactalbumin during colostral period, suggesting that beta-lactoglobulin could have some specific function during this period.     

Selenium, zinc, and copper concentrations in the blood and milk of lactating women

The aim of the study was to determine Se, Zn, and Cu concentrations in blood plasma and milk of lactating women from central Poland who were in different stages of lactation and to investigate the relationship between the content of trace elements in mothers’ blood and concentrations of microelements in their milk. Se and Zn concentrations in blood plasma of mothers were the lowest and Cu was the highest on the first 4 d of lactation (colostrum, n=43) and were found to be 34.9±11.8 μg/L, 0.51±0.13 mg/L, and 1.70±0.55 mg/L, respectively. The highest plasma level of Se and Zn and the lowest content of Cu could be observed between d 10 and 30 of lactation (mature milk, n=41), and were found to be 54.3±14.6 μg/L for Se (p<0.001), 0.76±0.20 mg/L for Zn (p<0.001), and 1.03±0.30 mg/L (p<0.001) for Cu. The results of Se, Zn, and Cu determination in breast milk samples demonstrate a pattern of decline in their concentration with advancing stages of lactation. We found out that Se, Zn, and Cu concentrations were the highest in colostrum (n=43) and amounted to 24.8±10.1 μg/L, 8.2±2.8 mg/L, and 0.45±0.11 mg/L, respectively. The content of all determined microelements declined significantly during the time of lactation. Statistically significant linear correlation was found between concentrations of Zn in blood plasma and milk in the first stage of lactation. Weak but statistically significant linear correlations were also found between plasma Se content in plasma and in transitional and mature milk of breast-feeding women.     

Longitudinal study of the dietary selenium intake of exclusively breast-fed infants during early lactation in Korea and Japan.

213 samples of human breast milk were collected from 51 healthy Korean women. Selenium content of the samples was determined by atomic absorption spectrometry with hydride generation. The selenium content of Korean milk decreased with increase of days after birth: The arithmetic mean of selenium content was higher in colostrum (< 4 days) 34 micrograms/kg (SD +/- 11, n = 44) than in transitional milk 21 micrograms/kg (SD +/- 8, n = 78) or in mature milk (> 10 days) 13 micrograms/kg (SD +/- 6, n = 91). The daily dietary selenium intake of 0-1 month aged Korean infants fed on breast milk is estimated to be around 10 micrograms per day (3 micrograms/kg body weight) regardless of days postpartum, resulting from the calculation of our selenium data and daily milk intake during early lactation. The same result on selenium intake for Japanese newborns, as well as Korean infants, is also estimated to be around 10 micrograms per day (3 micrograms/kg body weight) regardless of days postpartum.     

Southern elephant seal (Mirounga leonina) II. Studies of milk protein fractions by gel electrophoresis

 Milk protein fractions during various stages of lactation in the southern elephant seal Mirounga leonina were analysed. Twelve milk samples were taken from ten females throughout the lactation period during 1990 and 1991 at Stranger Point, King George Island, South Shetland Islands. Milk samples were subjected to polyacrylamide gel electrophoresis (PAGE). Samples from different days of lactation gave similar qualitative electrophoretic patterns. True protein content was significantly higher (P<0.05) at the beginning of lactation, and then remained constant until weaning. Caseins and whey proteins each consisted of several protein entities (four and five distinct bands respectively). Casein constituted only about 30% of the protein nitrogen, the remaining 70% being derived from whey proteins. There was some variation in concentration of casein and whey proteins as a function of time (P<0.0.5). Received: 28 July 1993/Accepted: 25 July 1995     

Partial purification of rat alpha-lactalbumin mRNA.

Alpha-lactalbumin messenger RNA was partially purified from RNA extracted from 3-5 day lactating rat mammary glands on a poly(U)-sepharose column followed by sucrose gradient centrifugation. Apha-Lactalbumin mRNA activity was assayed in wheat germ cell-free translational system by immunoprecipitation of the in vitro synthesized protein using specific antiserum prepared against purified rat alpha-lactalbumin. In the purified mRNA preparation alpha-lactalbumin mRNA activity comprised approximately 85% of the total mRNA activity.     

"Whey" proteins of milk of the red (Macropus rufus) and eastern grey (Macropus giganteus) kangaroo

1. A comparison is made of gel electrophoretic patterns of the "whey" proteins of the milk of red (Macropus rufus) and eastern grey (Macropus giganteus) kangaroos at various stages of lactation. Qualitative and quantitative changes occur with time during the mature phase of lactation of both types. Their onset is related solely to the stage of lactation. "Whey" proteins are isolated and characterised and the nature of protein changes determined for the first time. 2. The anodic electrophoretic pattern is divided into 6 main zones (designated A F in order of decreasing mobility) and 2 cathodic zones (G and H) that are only detected in the milk of M. giganteus. 3. Zones A, B and C are milk specific. Zone B is present throughout lactation in both species and is an alpha-lactalbumin. Zones A and C are present only in late lactation, zone C, usually, but not always, appearing first. Zone A is an alpha-lactalbumin in M. giganteus, but is not an alpha-lactalbumin in M. rufus. Zone C appears to be the same protein in both species and is possibly a beta-lactoglobulin. 4. Zone D is kangaroo serum albumin and zone E is possibly a beta 2-microglobulin. Zone F contains three main iron (III) binding bands whose relative intensity varies with stage of lactation. Their intensity differs from the corresponding blood serum transferrin bands. 5. Zone H of Macropus giganteus is a lysozyme. 6. Lactose is present in the milk, but is not the principal sugar. 7. The significance of the results is discussed.     

Interaction of selected cosolvents with bovine alpha-lactalbumin

Alpha-lactalbumin constitutes about 3% of bovine milk proteins. The preferential solvent interactions between selected cosolvents (sorbitol, sucrose and glycerol) and alpha-lactalbumin at pH 7.5 was determined using precision densitimetry. The preferential interaction parameter (xi(3)) and other thermodynamic parameters were calculated at different solvent concentrations. The xi(3) parameter was maximum at 30%, 45% and 40% (w/v) concentrations with the values of -0.282g/g, -0.171g/g and -0.299g/g for sorbitol, sucrose and glycerol, respectively. Thus the principal driving energy in the system being preferential hydration and mutual exclusion of bulk solvent. There was only a marginal change in the CD spectra of the protein with these cosolvents indicating the integrity of secondary structures. The results of thermal denaturation measurements indicated an increase in thermal stability of alpha-lactalbumin with these cosolvents. In the presence of 30% sorbitol there was an increase in the apparent thermal transition temperature (apparent T(m)) from 65 to 71 degrees C. These results indicate that the selected cosolvents in this study stabilizes alpha-lactalbumin without altering the structure of the protein.     

Copper,selenium, and zinc concentrations in human milk during the first three weeks of lactation

Human milk samples were analyzed by neutron activation analysis (NAA) for three essential trace elements (Cu, Se, and Zn). Mothers' milk samples were collected from 25 lactating mothers from the Central Hospital, Tripoli, Libya. The average concentration level of Cu, Se, and Zn declined from 0.84 +/- 0.06 mg/L, 104 +/- 9.46 microg/L, and 16.1 +/- 2.67 mg/L at d 0 to 0.39 +/- 0.045 mg/L, 41.8 +/- 6.66 microg/L, and 4.95 +/- 1.3 mg/L, respectively, at d 20 of lactation. Copper and Zn levels in the Libyan mothers' milk were in agreement with reported levels from other countries, whereas Se was at a higher level. The Cu daily intake level is slightly higher than the RDA (Recommended Daily Allowance) value. Selenium and Zn have higher intake levels than the RDA values but within the tolerable upper intake levels.     

Gelsolin expression in sheep milk somatic cells during lactation

The identification of genes involved in phenotypes related to milk quality is important for both economic and health aspects in livestock production. The aim of this study was to assess the level of gelsolin gene expression in two breeds of dairy sheep – Sarda and Gentile – with pronounced differences in quantitative and qualitative milk traits. Gelsolin, a type of actin-modulating proteins is involved in the processes of actin remodeling during cell growth and apoptosis; therefore a role of this protein in mammary changes during lactation was here hypothesized. Individual milk samples were collected three times during lactation from 26 ewes of the two breeds. The differential gene expression of gelsolin in the two breeds and the three lactation times was estimated by quantitative PCR on RNA extracted from milk somatic cells. Correlations of gelsolin gene expression with milk yield and quality and days of lactation were also estimated. The results showed that gelsolin gene expression was significantly higher in the Sarda compared to the Gentile at each lactation stage, in agreement with the longer lactation duration and the higher daily milk yield of the first breed. Significant correlations of gelsolin gene expression were found with milk fat content in Sarda breed (−0.46, P<0.05). Gelsolin expression analysis confirmed the link between gelsolin gene function and milk fat content of sheep.