Cow milk fat interferes with prolactin radioimmunoassay

The concentration of prolactin (PRL) in fat-free cows milk was one-third that of whole milk while the values were similar for milk from the goat. A number of experiments were performed to determine if PRL was present in cow milk fat. The results indicate that cow milk fat introduces an artifact in the double antibody PRL RIA which overestimates the amount of prolactin in whole cow milk.     

Rat lymphoma cell bioassay for prolactin: Observations on its use and comparison with radioimmunoassay

The rat Nb₂ node lymphoma cell bioassay (BA) for prolactin (PRL) was validated for use in our laboratories. During the course of this validation we observed that rat prolactin (NIAMDD-RP-1) stimulated cell division by as much as 16.5 fold over the range of 0.04 to 40.0 ng/ml at the end of 72 hours of incubation. We also observed a dose related increase in the size of the lymphoma cells. Prolactin concentrations in rat plasma, serum, anterior pituitary (AP) homogenates and milk were measured by both radioimmunoassay (RIA) and BA. In individual BA's there was parallelism between samples and standard; but when several dilutions of the same plasma and pituitary homogenates were assayed repeatedly, higher PRL levels were consistently observed for the more concentrated samples. At low or moderate levels of plasma PRL there was excellent agreement between RIA and BA; however, at high levels plasma PRL bioactivity exceeded radioimmunoactivity by a small, but significant, amount. A comparison of pituitary PRL concentrations measured by RIA and BA were in good agreement when homogenization was done at pH 10.6. However, when homogenization was done at pH 7.6, slightly but significantly more PRL was extracted when assayed by BA than when assayed by RIA.     

Studies on prolactin in human serum, urine and milk.

Prolactin activity was measured in serum, urine and milk using a specific human prolactin radioimmunoassay (RIA). Serum, urine and milk were parallel with the human prolactin standard in the RIA. There was no correlation between serum prolactin levels and urinary prolactin activity. Dialysis of urine samples resulted in complete loss of human prolactin activity while the addition of human prolactin to the urine resulted in the recovery of over 50% of the hormone after dialysis. Thus it was concluded that prolactin is not present in urine. In additional experiments it was observed that the RIA prolactin activity in urine was significantly correlated with the osmolality of the urine and that Na+ and K+ were contributory elements. On the other hand, prolactin was found in human milk and correlated well with the expected serum levels of this hormone. This latter finding is interesting because prolactin receptors have been shown to exist on the serosal side of the mammary epithelial cells. The presence of prolactin in milk suggests the possibility of other sites of action for this hormone in addition to the cell membrane.     

Negligible release of cardiolipin during milk secretion by the ruminant

The presence of cardiolipin (diphosphatidyl glycerol) in lactating mammary tissue (cow and goat) was investigated. The tissue was separated into subcellular fractions by sedimentation; the identities of the fractions were confirmed by electron microscopy. Polar lipids recovered from the fractions, the whole tissues, and milks were analyzed by two-dimensional thin-layer chromatography and the percentages of cardiolipin were determined. The phospholipids of whole mammary tissue from the cow and goat contain 3-5% cardiolipin which is concentrated largely, if not exclusively, in the mitochondria. Although milk may on occasion have up to 1% cardiolipin in its phospholipids, some normal milks contain less than 0.15%. Since tissue contains 20-30 times the amount (mg/g) of phospholipids in milk, the quantitative ratio of tissue to milk cardiolipin is several hundred to one. We interpret this to mean that the mechanism of milk secretion is highly selective and insures retention of mitochondria within the cell even though they are decidedly smaller than milk fat globules which are continuously secreted. Our findings substantiate the conception that there is very little disintegration of the cell or disruption of the plasma membrane during milk secretion. The fatty acids of cardiolipin from lactating mammary tissue of cow, goat, and pig are highly unsaturated; they contain 50% or more octadecadienoic acid.     

The influence of blinding, olfactory bulbectomy and pinealectomy on plasma and anterior pituitary prolactin levels and on uterine and anterior pituitary weights in normal and neonatally androgenized rats

Lactating Sprague-Dawley rats had their litters adjusted to 8-10 pups on day 3 of lactation favoring females and some litters were injected with 1.25 mg of testosterone propionate to neonatally androgenize (NA) them. At 22-25 days of age both normal and NA animals were ovariectomized (OVX) and subjected to either sham olfactory bulbectomy (ANOS) + sham pinealectomy (PX), blinding (BLD) + ANOS or BLD + ANOS + pinealectomy (PX). At 13 weeks of age all animals were injected with 0.5 mg of polyestradiol phosphate. At 15 weeks of age the animals were fitted with atrial catheters and at 16 weeks of age blood samples (0.3 ml) were obtained every 3 hours over a 24 hour period. Uterine and anterior pituitary (AP) weights were recorded at sacrifice. Plasma and AP were assayed for prolactin (PRL) by RIA and AP were also assayed for PRL using the Nb2 lymphoma cell PRL bioassay. In both normal and NA animals, BLD + ANOS suppressed plasma PRL levels and PX partially prevented this response. Uterine weights were similar among groups while AP weights were significantly lower for sensory deprived animals. Anterior pituitaries extracted at pH 7.6 had a PRL concentration that was higher for the BLD + ANOS groups when estimated by either RIA or BA, a result that was not observed when the AP were extracted at pH 10.6. The amount of PRL extracted at pH 10.6 was twice that obtained at pH 7.6. Sensory deprived animals that were OVX prepubertally and administered estrogen as adults had a small but significant increase in mean plasma prolactin at 1700 hr. Both normal and NA animals responded in a similar manner to experimental manipulation.     

Serum prolactin heterogeneity in the cow and goat

Jugular blood samples were taken from Friesian cows and British Saanen goats and the serum prolactin (PRL) heterogeneity profile obtained using a 2.5 × 92 cm Sephadex G-100 column. Two PRL components were identified by radioimmunoassay in cow and goat serum from virgin, pregnant and lactating animals. Using the nomenclature developed for human serum, one component, which eluted off the column just after the void volume (Vf/Vt = 0.09), was called big-big PRL and the second component eluting at a Vf/Vt value of 0.50 was called little PRL. In two out of 5 lactating cows before milking a PRL component with a Vf/Vt value of 0.27 was observed and this component was called big PRL; no big-big PRL was observed for these two cows. After milking the latter two cows the big component disappeared and the big-big component appeared. When PRL was released in response to milking in cows there was a significant decrease (P < 0.01) in the proportion of larger molecular weight PRL forms and an increase in the smaller form as compared to the values observed before milking. No such shift in either the number or proportions of heterogeneous PRL was observed in lactating goats as a result of the milking stimulus.     

Effects of some thermal, chemical and mechanical treatments on lipase activity in Shammi goat milk

The effects of heating (20, 37 or 50 °C), cooling (5 °C), pasteurisation (71 °C for 15 s), boiling (100 °C), agitation (5 or 10 min), pH (acid or alkaline), and addition of chemicals such as silver and lead nitrates, copper sulphate and sodium chloride on lipase activity in Shammi goat milk were studied. There were non-significant differences (P < 0.01) in chemical composition between Shammi goat milk and Arabi cow milk. Lipase activity in Shammi goat milk was non-significantly (P < 0.01) lower than in Arabi cow milk. Lipase activity in milk of Shammi goats and Arabi cows was reduced when the milk was subjected to heating, cooling, pasteurisation, boiling, or when chemicals or acid was added, whereas in agitated and alkaline milk, the lipase activity was increased. The increase following agitation was greater after 10 min than 5 min. It can be concluded that heating, pasteurising, boiling, cooling, addition of certain chemicals and acidity are means by which lipase activity in milk can be reduced.     

用聚丙烯酰胺凝胶电泳和微流体芯片电泳鉴别6种液态奶

目的:研究6种液态奶制品蛋白电泳图谱的区别,建立奶制品的蛋白质学鉴别方法。方法:以5种纯牛奶、羊奶、水牛奶、骆驼奶、牦牛奶、黄豆浆为研究对象,通过SDS-PAGE和Agilent 2100微流体芯片电泳法进行分析比较。结果:骆驼奶、黄豆浆与其他研究对象的图谱有明显区别,而牛奶、羊奶、水牛奶、牦牛奶的差异却不是很大;采用微流体芯片电泳可有效地对豆奶、骆驼奶进行区分,还可在一定程度上鉴别牛奶、羊奶、水牛奶和牦牛奶。结论:Agilent2100系统作为一种新型半自动微流体芯片技术,可以快速、高效、准确地应用于液态奶制品的蛋白成分分析及鉴别。     

The effect of steroids on the size heterogeneity of pituitary prolactin in castrate male rats

Male Sprague-Dawley rats were castrated and given daily sc. injections of estradiol (E2, 10 micrograms/day), testosterone propionate (TP, 1.0 mg/day), dihydrotestosterone (DHT, 1.0 mg/day) or sesame oil (SO, 0.2 ml/day). A group of sham castrate males received daily sc. injections of SO (0.2 ml/day). On day 8 of steroid treatment animals were decapitated and anterior pituitaries were removed and hemisected. Each half was homogenized in PBS buffer (0.01 M Na2HPO4-NaH2PO4; 0.14 M NaCl; 0.1% bovine serum albumin) at either pH 7.6 or 10.6. Homogenates were chromatographed on Sephadex G-100 columns and eluted fractions were assayed for prolactin (PRL) by RIA. Four immunoreactive forms of PRL, designated as "void volume," "big big," "big" and "little," were eluted from the pituitary homogenates of each experimental group. Homogenates obtained at pH 7.6 contained a greater percentage of PRL in the "void volume" and less activity in the "big" and "little" forms than pH 10.6 homogenates in all experimental groups. Pituitaries from SO- and TP-treated castrate animals contained significantly greater percentages of activity in the "void volume" at pH 7.6 compared to the other groups. At pH 10.6, the pituitary homogenates from the E2-treated group eluted a significantly greater percentage of "big" PRL and a smaller percentage of "little" PRL compared to all other groups. These findings suggest that androgenic and estrogenic steroids may play a role in the pituitary PRL molecular size profile of the male rat.     

Beneficial effect of goat milk on bioavailability of copper, zinc and selenium in rats

We studied the effects of dietary inclusion of freeze-dried goat and cow milk on the utilization of copper, zinc and selenium, and on the metabolic fate of copper and zinc, in rats using a standard (non-milk) control diet recommended by the American Institute of Nutrition and diets based on goat or cow milk. For animals given the goat milk diet, the apparent digestibility coefficient (ADC) of copper is similar to that obtained with the standard diet and higher than that in animals given the cow milk diet. The copper balance was higher among the rats given the goat milk and the standard diets than among those given cow milk. The ADC and retention of zinc and selenium were higher for the goat milk diet than for the other two diets. The copper content in the kidneys and in the femur was greater when the animals consumed a goat milk diet than a cow milk diet. Zn deposits in femur, testes, liver, kidney, heart and longissimus dorsi muscle were greatest with the goat-milk diet, followed by the standard diet and were lowest for the rats given cow-milk diet. This study shows that the goat-milk has an important and beneficial effect on the bioavailability of copper, zinc and selenium.     

Simultaneous radioimmunoassay for luteinizing hormone and prolactin

A combined radioimmunoassay (RIA) for the measurement of the anterior pituitary proteins luteinizing hormone (LH) and prolactin (PRL) is described and compared with individual RIAs for these hormones. The standard curves and the sample values for LH and PRL were identical when determined in a combined or in an individual RIA. This technique may prove useful to a number of laboratories where it is desirable to determine levels of more than one hormone in limited sample volumes.     

Partial purification and characterization of rhinoceros gonadotropins, growth hormone, and prolactin: comparison with the horse and sheep

The rhinoceros is an endangered species related to the horse family. Little is known of its reproductive endocrinology. The objectives of this study were to partially purify rhinoceros pituitary hormones, determine which assays could be used for their assessment, and to ascertain whether rhinoceros LH possesses the intrinsic FSH activity of equine LH. A single pituitary each from a White (1.3 g) and a Black (1.2 g) Rhinoceros was homogenized and extracted (pH 9.5), then subjected to pH and salt fractionation, and ion-exchange chromatography (DEAE and Sephadex SP-C50) to yield partially purified fractions of LH, FSH, growth hormone (GH), and prolactin (PRL). LH was readily measured by a rat Leydig cell assay (0.1-1% x equine LH) and an RIA using a monoclonal antibody to bovine LH (6-11% x equine LH). FSH activity detected in the LH by either an FSH RIA or a calf testis radioreceptor assay (RRA) was extremely low. No FSH activity could be detected in the White Rhinoceros pituitary "FSH" fraction, but was readily detected in the Black Rhinoceros fraction (RIA: 0.2% x equine FSH: RRA: 0.8% x equine FSH). The presence of GH and PRL was determined by SDS-PAGE and Western blots. Results showed a single immunoreactive GH band and multiple immunoreactive PRL bands. Adsorption with Concanavalin A-Sepharose indicated that some of the PRL bands are glycosylated.     

Hormones, IgG and lactose changes around parturition in plasma, and colostrum or saliva of multiparous sows

Blood, colostrum and saliva samples were serially taken from 6 multiparous sows from day 109 of gestation until day 3 postpartum. Plasma was assayed for oestradiol-17beta (E2), progesterone (P4), prolactin (PRL), cortisol, immunoglobulin G (IgG) and lactose. Colostrum was assayed for E2, P4, IgG and lactose. Lactoserum, obtained after ultra centrifugation of colostrum, was assayed for PRL. Saliva was assayed for cortisol. Time-related variations in hormone, IgG and lactose concentrations measured in plasma were parallel to those measured in colostrum, lactoserum or saliva. However, the concentrations were higher in colostrum or lactoserum and lower in saliva than in plasma. Ratios of concentrations of cortisol in saliva and PRL in lactoserum over those in plasma did not vary with time and averaged 0.2 and 1.6, respectively. Conversely, the ratios of concentrations of E2 and P4 in colostrum over those in plasma varied with time (P < 0.05) but were quite constant before the end of parturition, averaging 2.7 and 3.6, respectively. The ratios of concentrations of IgG and lactose in colostrum over those in plasma also varied with time (P < 0.05). The concentrations of hormones in plasma on the one hand and in colostrum, lactoserum or saliva on the other hand were significantly correlated but correlations varied with time (PRL across periods: r = 0.31; cortisol across periods: r = 0.60; E2 during parturition: r = 0.83; P4 before parturition: r = 0.82; P4 during parturition: r = 0.67). The present results indicate that around parturition, assays of hormones in colostrum or saliva can be used to study the hormonal status of sows. Furthermore, variations in colostrum and plasma concentrations of IgG and lactose are good indicators of the transition from colostrum to milk synthesis.     

Mammary glucose uptake in the lactating ewe and the use of methionine arterio-venous difference for the calculation of mammary blood flow

(1) The validity of using the arterio-venous concentration difference of methionine to calculate mammary blood flow in the ewe, on the basis of the Fick principle, is discussed. (2) Calculation of mammary blood flow in the lactating Merino ewe indicated that blood flow per unit weight of tissue and the ratio of blood flow : milk yield were approximately twice that found in the lactating cow and goat. (3) Calculated mammary blood flow in Merino ewes was used in conjunction with glucose arterio-venous difference to determine mammary glucose uptake. Glucose uptake per unit weight of tissue in the ewe was almost double that found in the cow and goat. The ratio of mammary glucose uptake to lactose output was also higher in the ewe than that found in the cow and goat. The utilization of glucose by the mammary gland of the ewe is discussed in relation to the possible greater requirement for NADPH and glycerol for milk fat synthesis in this species.     

Elevated growth hormone levels in sera from breast cancer patients

The concentrations of growth hormone (GH) and prolactin (PRL) in the serum of 42 breast cancer patients were determined by radioimmunoassay (RIA). Forty percent of the patients had elevated GH levels while only 17% had elevated PRL levels. These findings suggest a relationship between GH and breast cancer; a weaker correlation exists between PRL and this malignancy. In addition, total lactogens in the serum were measured by a bioassay (BA). The BA/RIA (GH + PRL) ratio was greater in the breast cancer patients than the controls, indicating that variant forms of the hormones with higher than normal biological activity might be present.     

Evidence for a dose-dependent effect in the sex-specific plasma prolactin response to naloxone in humans

In attempt to ascertain if the sex specific plasma PRL response to naloxone, that we suggested in previous studies, was a dose dependent effect, 26 healthy volunteers were studied. They received naloxone 2 mg and 4.8 mg or a volume matched of saline i.v. as a bolus. Blood samples were collected and plasma PRL was measured by double antibody RIA. Naloxone, at dose of 2 mg, was able to decrease significantly plasma PRL levels in normally menstruating women (p less than 0.05 at 60 min.; p less than 0.01 at 120 min.), but not in post-menopausal ones and in men. In addition, the dose of 4.8 mg of the drug did not change plasma PRL in any group. These results suggest a dose-dependent effect in the sex specific PRL response to naloxone in humans.     

Ovariectomized Sprague-Dawley and Long-Evans rats release prolactin differently in response to estrogen

Mature female Sprague-Dawley (SD) and Long-Evans (LE) rats were ovariectomized (OVX), fitted with indwelling atrial catheters and given a single sc injection of either 25 or 100 μg polyestradiol phosphate (PEP); seven days later blood samples were withdrawn at two hour intervals from 1100 to 2100 hours to detect the presence of an afternoon surge of prolactin (PRL). Other groups of OVX rats of both strains also treated with PEP and catheterized as above were sampled before and at 2, 5, 10 and 30 min after iv administration of 1 μg synthetic thyrotropin releasing hormone (TRH). Pituitary (AP) and uterine weights were determined following sacrifice one day after TRH treatment. Separate groups of OVX rats of both strains treated with 100 μg PEP were decapitated 7 days later and each AP was removed and homogenized. The AP homogenates and plasma samples were assayed for PRL by radioimmunoassay. Rats of both strains had afternoon PRL surges and in both strains the magnitude and/or duration of the surges were enhanced by the higher dose of PEP. However, within each PEP dose LE rats released significantly more PRL during the surge than did SD rats. Rats of both strains also released PRL in response to TRH and this response was enhanced in both strains by the higher of the two doses of PEP. However, there were no differences between the strains at 25 μg PEP and at 100 μg PEP SD rats released significantly more PRL to TRH than did LE rats. Pituitary weight and PRL concentration were not different between the strains at either dose of PEP but LE rats had significantly heavier uteri at both doses of PEP compared to SD rats. These data not only show that strain differences exist in estrogen-induced or mediated PRL release in the rat but also indicate that the differences are not uniform. This latter observation suggests that the estrogen-induced mechanisms examined in this study are for the most part independent of each other.     

A new microbioassay for the measurement of lactogenic hormones in human serum

The standard Nb2 assay for biologically active prolactin has been modified to allow a rapid convenient microbioassay without loss of specificity or accuracy. Lactogenic hormones specifically stimulate the replication of Nb2 node rat lymphoma cells in suspension culture and form the basis of a currently available bioassay to measure prolactin and growth hormone in human serum. A new microbioassay was developed using microtest plates enabling a large number of samples to be assayed simultaneously whilst maintaining the overall sensitivity of the bioassay for lactogenic hormones. Growth of the Nb2 node lymphoma cells, measured by a light scattering technique using optical density on a spectrophotometer, was shown to be closely correlated with the cell number determined on a Coulter counter. Addition of excess anti-human prolactin and anti-human growth hormone completely inhibited the growth stimulatory effects of both human prolactin and human growth hormone. This new microbioassay (BA) and conventional radioimmunoassay (RIA) were used to measure lactogenic hormones in 48 normal subjects. There was a close correlation between the results of both assays for each hormone studied in the control sera. The mean basal BA/RIA ratio was 1.5 (range 0.8-2.0) for prolactin, 0.7 (range 0-4.5) for growth hormone and 1.3 (range 0.5-1.9) for total lactogenic activity.     

Bioactivity of plasma prolactin in ovariectomized, diethylstilbestrol-treated Long-Evans and Holtzman rats after thyrotropin-releasing hormone or bromocriptine administration

The objective of this study was to determine the effects of thyrotropin-releasing hormone (TRH) and bromocriptine on plasma levels of biologically active prolactin in ovariectomized, diethylstilbestrol (DES)-treated rats. Female Long-Evans and Holtzman rats were ovariectomized and each was given a subcutaneous implant of diethylstilbestrol (DES). One week later, groups of DES-treated rats were fitted with indwelling intra-atrial catheters, and 2 days later blood samples were withdrawn before and at 1, 2, 5, 10, and 20 min after intravenous administration of TRH (250, 500, or 1000 ng/rat). Blood samples were obtained from other groups at 4 weeks of DES treatment by orbital sinus puncture under ether anesthesia before and at 30, 60, and 120 min after bromocriptine administration (2.5 mg/rat sc). Plasma was assayed for prolactin by conventional radioimmunoassay (RIA) and by Nb2 lymphoma bioassay (BA). Holtzman rats released significantly more prolactin following TRH than did Long-Evans rats when the RIA was used to measure prolactin. However, when the BA was used to assay prolactin in the same samples, the Long-Evans rats released more prolactin than did the Holtzman rats. In addition, the ratio of the BA to RIA values was significantly increased in both strains following TRH, but the greatest increase was observed in the Long-Evans rats, in which the ratio was 4.5 at the peak of the TRH-induced rise in plasma prolactin. Gel filtration chromatography of plasma obtained at 5 min after TRH treatment in Long-Evans rats revealed large molecular forms of prolactin with BA to RIA ratios of 4-5. In addition, monomeric prolactin had a BA to RIA ratio of 2. Bromocriptine treatment reduced prolactin levels in both strains, but the effect was more rapid in Holtzman than in Long-Evans rats. In addition, bromocriptine treatment of Holtzman, but not Long-Evans, rats significantly reduced the BA to RIA ratio of plasma prolactin. The results indicate that TRH and bromocriptine affect the release of biologically active prolactin to a greater extent than prolactin detected by antibody in the RIA, and that Long-Evans and Holtzman rats respond to these secretagogues differently with regard to BA to RIA comparisons.     

大熊猫乳汁蛋白组成

测定了2只大熊猫（ailuropoda melanoleuca)乳汁中蛋白含量,乳糖含量,乳蛋白组成,并与成都麻羊（Capra hircus)初乳和中国荷斯坦牛（Bos taurus)乳进行了比较,大熊猫乳蛋白含量平均为41．52g/L,与中国荷斯坦牛乳接近;乳糖平均含量为15．41g/L,极显著低于牛乳和成都麻羊初乳,乳蛋白SDS－PAGE分析表明,大熊猫乳中酪蛋白（CN）含量明显低于羊乳和牛乳;乳中存在3条蛋白含量的区带,而在成都麻羊和中国荷斯坦牛乳电泳图谱的相应位置未见明显的区带,乳中上皮粘蛋白MUC1仅存在1条分子量约为196kDa的区带,未发现多态现象。