Human milk stimulates prostacyclin production by cultured human vascular endothelial cells.

Prostacyclin (PGI2) is an antithrombotic and vasodilatory factor, which is produced mainly by the vascular endothelium. Little is known about how this process is regulated. We investigated the effect of human milk on PGI2 synthesis by human vascular endothelial cells by measuring its stable metabolite, 6-keto-prostaglandin F1 alpha, by RIA. Human milk induced dose- and time-dependent stimulation of PGI2 production, whereas cow's milk was ineffective. The lowest concentration of human milk that stimulated the production of PGI2 was 0.1%, and 10% induced a 2.4- to 3.4-fold increase. The effect of human milk was detectable after 2 h and was blocked by inhibitors of transcription, translation, and cyclooxygenase. Boiling abolished the activity, but acetone extraction enhanced it. A 10% concentration of acetone-extracted human milk stimulated the release of endothelial cell PGI2 by 6.6-fold. In human milk samples we found no correlation between the amount of immunoreactive epidermal growth factor (EGF) and the activity stimulating PGI2 synthesis. Furthermore, EGF antibodies did not inhibit the activity. This is the first demonstration that human milk stimulates PGI2 production by endothelial cells. We conclude that human milk is a potent inducer of PGI2 production by human vascular endothelial cells and that the stimulatory activity is not due to EGF.

Epidermal growth factor in human urine from birth to puberty.

The highest concentrations of epidermal growth factor (EGF) are found in urine, but the physiological role of urinary EGF is unknown. We studied human urinary EGF excretion, by measuring its concentration with a specific homologous RIA, in 265 healthy children from birth until age 16 yr. The absolute concentrations varied widely between individuals. Mean values were approximately 10 ng/ml in 1- to 30-day-old infants; 2.5-fold higher values were found in infants aged 2 to 12 months. During the second year there was a further rise to about 70 ng/ml, and urinary EGF excretion was in the same range in older subjects. The EGF/creatinine concentration ratio was less variable. The mean ratio increased 6-fold from birth to the second year of life. Thereafter, the EGF/creatinine ratio decreased gradually to one-third of the peak level at puberty. No sex difference was found.

Characterization of material with epidermal growth factor immunoreactivity in human serum and platelets.

We investigated the molecular nature of the epidermal growth factor immunoreactivity (ir-EGF) in serum and platelets of normal subjects. In serum, ir-EGF appeared and increased during spontaneous blood coagulation, reaching a plateau in 2 h. The mean plateau measured by time-resolved immunofluorometric assay was 778 pg/mL [130 pmol/L; range, 465-1352 pg/mL (78-225 pmol/L)] for men (n = 66), and 774 pg/mL (129 pmoL/L; range, 521-1114 pg/mL (87-186 pmol/L)] for women (n = 33). Serum samples (n = 9), when examined under nonreducing conditions by high performance liquid chromatography, contained three mol wt components. Their approximate proportions averaged 56 +/- 6% (+/- SD) for a 140K component, 22 +/- 9% for a 67K component, and 22 +/- 6% for a component coeluting with the 6K EGF standard. The molecular size distribution of ir-EGF released from isolated platelets varied with the treatment of the platelets. After stimulation with a Ca2+ ionophore, the proportions of the 140K, 67K, and 6K components were 61 +/- 11%, 20 +/- 8%, and 17 +/- 4% (n = 5), respectively, as in serum. In addition, we found a small amount, (approximately 1%) of a 17K component. When platelets (n = 6) were ruptured by sonication and repeated freeze-thawing, the 67K component formed 53 +/- 7% of the total; the proportions of the 140K, 17K, and 6K components were 14 +/- 7%, 2.1 +/- 0.7%, and 31 +/- 4%, respectively. Under reducing conditions the 17K and 6K components remained intact, but part of the 140K component and all of the 67K component were cleaved to a 35-37K form. After protease digestion of the higher mol wt components, the ir-EGF was exclusively of the 6K form. The 67K and 6K components bound to the EGF receptor, whereas the 140K component bound inconsistently. We conclude that treatment of platelets with Ca2+ ionophore produces ir-EGF components similar to those found in incubated serum but different from those obtained by freeze-thawing. The 67K mol wt component appears to be the main storage form of EGF in platelets; at least two independent mechanisms appear to exist for EGF release.

Cytoskeleton-dependent release of human platelet epidermal growth factor.

To study the source of immunoreactive epidermal growth factor (ir-EGF) released by thrombin formation we removed 99.9% of the leukocytes normally present in platelet-rich plasma and induced coagulation with 30 mM of Ca2+. The absence of leukocytes did not reduce the amount of ir-EGF released; thus platelets are most likely the only source of the ir-EGF released during aggregation. To identify the site of ir-EGF in platelets we exposed washed platelets to collagen or thrombin and compared the kinetics of releases of ir-EGF, beta-thromboglobulin (bTG, an alfa-granule marker), ATP (dense granule marker), N-acetyl-beta-D-glucosaminidase (NAGA, a lysosome marker) and lactate dehydrogenase (LDH, a cytoplasmic marker). Release of ir-EGF started immediately and continued linearly. The process differed clearly from the releases of the granule markers, which occurred readily, and were completed in a few minutes. The release of ir-EGF also differed from the leakage of LDH, the start of which was delayed greater than 5 min, but then proceeded linearly. Cytochalasin B inhibited the release of hEGF, but demecolcine had no effect. We conclude that the ir-EGF released from platelets during aggregation derives neither from the granules nor the cytoplasma. The assembly of cytoskeleton is needed for its release.

Size heterogeneity of epidermal growth factor in human body fluids.

We measured the concentration of immunoreactive (IR) hEGF in various body fluids by radioimmunoassay (RIA) and evaluated its size heterogeneity by size exclusion high performance liquid chromatography combined with RIA or with time-resolved immunofluorometric assay (TR-IFMA). Mean concentration was 80 ng/ml in urine, 65 ng/ml in milk, 50 ng/ml in seminal plasma, 25 ng/ml in armpit sweat, 1 ng/ml in breast sweat, 0.3 ng/ml in third-trimester amniotic fluid, 3 ng/ml in saliva, 1.5 ng/ml in tears and 0.3 ng/ml in gastric juice. All the fluids except armpit sweat and gastric juice contained two to five molecular sizes of IR-hEGF. As well as the 6200-dalton (6.2 kDa) hEGF we found at least four other different molecular sizes with approximate weights of greater than or equal to 300, 150, 70 and 20 kDa. The authentic 6.2 kDa form made up greater than 90% of the total IR-hEGF in all except the amniotic fluid where its proportion was 71%, and the seminal plasma where the proportion could not be determined.

Ocular disease leads to decreased concentrations of epidermal growth factor in the tear fluid.

The concentration of epidermal growth factor (EGF) in tear fluid (TF) was recently shown to decrease with increasing tear fluid flow (TFF). The purpose of the present study was to clarify the effects of ocular surface disease on the TF EGF concentrations. Tear fluid samples (n = 243) were collected from diseased eyes by means of blunted glass capillaries. The time of collection was measured for each sample, and the tear fluid flow in the capillaries (TFFc) was calculated. The concentration of human EGF (hEGF) was determined using a time-resolved immunofluorometric assay (TR-IFMA). For statistical analysis diagnosis-dependent multigrouping was performed and the data of the patient groups were compared to the data for a control group. The control material consisted of 271 TF samples collected from healthy eyes before (n = 59) and after stimulation of reflex tearing (n = 212). It was shown that TF specimens of patients (n = 243) contained significantly (p less than 0.001) less EGF (mean 952 pg/ml) than the TF of healthy control individuals before (n = 59 samples; mean 6589 pg/ml) or after stimulation of reflex tearing (n = 212 samples; mean 2762 pg/ml). The EGF concentration of every patient group was significantly lower than that found in the TF of control individuals both before and during reflex tearing (p less than 0.001). The rate of EGF released with TF during collection did not differ significantly between the various groups of patients or from that released with the TF of normal individuals before induction of reflex tearing.(ABSTRACT TRUNCATED AT 250 WORDS)

Variation of hydrophobicity of human urinary epidermal growth factor.

Epidermal growth factor is present in human urine in large amounts, but its biological significance is not known. The results of this study indicate that the predominant 6000-dalton form of epidermal growth factor in human urine is divided by hydrophobic interaction chromatography into four fractions; only 3% of the total 6000-dalton epidermal growth factor coeluted with the biosynthetic epidermal growth factor and the rest was separated into three different peaks. These different forms may lack one or two amino or carboxy terminal amino acids from the 53 amino acids present in epidermal growth factor, or they may be products of deamidation or oxidation of amino acid(s). Further knowledge of these micromodifications of epidermal growth factor secreted in urine may reveal the origin and function of epidermal growth factor in humans.

Effect of sulprostone on concentrations of progesterone, 20 alpha-dihydroprogesterone, estrone, and estradiol in human placental tissue in the first trimester of pregnancy.

Placentas were obtained from 35 healthy women whose pregnancy was terminated in the first trimester. Seventeen of them had no prostaglandin medication whereas in 18 women 500 micrograms of sulprostone was administered for cervical softening 12-14 h prior to the termination of pregnancy. Concentrations of progesterone and 20 alpha-dihydroprogesterone were determined from the extract of the placenta by liquid chromatography and those of estrone and estradiol by a radioimmunoassay. Placental progesterone concentration (6,829 +/- 469 ng/g wet weight, mean +/- SEM) was higher in the sulprostone group than in the control group (5,070 +/- 539 ng/g, p less than 0.02), but the mean concentrations of 20 alpha-dihydroprogesterone, estrone and estradiol were similar in both groups. Thus the small dose of sulprostone used in this study did not result in any great changes in placental steroidogenesis which could bring about the initiation of abortion. The increase of placental progesterone may, however, indicate that uterine contractions and impaired uteroplacental blood flow secondarily decreased the direct transfer of steroids from the placenta to the myometrium.

Serum transferrin receptor for assessment of iron status in healthy prepubertal and early pubertal boys.

A recently introduced test measures the concentration of transferrin receptor (TfR) in serum, which increases shortly after the onset of iron deficiency. In adults this increase reflects the degree to which tissue iron availability is impaired. We developed a fluoroimmunoassay to quantify TfR. The purpose of this study was to evaluate the role of TfR as an index of iron sufficiency in 62 healthy prepubertal or early pubertal boys. The mean concentration of serum TfR was 3.8 (-1 SEM = 3.6, +1 SEM = 3.9) mg/L. No associations were observed between the serum TfR and the concentration of Hb, the values of packed cell volume, reticulocyte production index, mean corpuscular Hb, mean corpuscular volume, or the concentrations of serum iron, transferrin, or ferritin. Because none of the subjects had signs of iron deficiency, we determined the 95% reference intervals for Hb, red blood cell indices, and the above-mentioned serum concentrations. The reticulocyte count and reticulocyte production index were higher than expected. Our results indicated that the individual concentration of TfR in serum does not depend on any of the several other parameters of iron status in a group of healthy individuals.

Increased rate of salivary epidermal growth factor secretion in patients with juvenile periodontitis.

We compared salivary epidermal growth factor (EGF) concentrations in patients with juvenile periodontitis (JP) and periodontally healthy controls. In initial screening of 45 JP patients and a group of healthy controls, significantly higher salivary EGF concentrations were measured in the JP patients. Subsequently, 17 JP patients who had high EGF concentrations in some of their salivary samples were chosen, and a group of age- and sex-matched controls was selected. We then examined their EGF concentrations and EGF secretion rates under standardized conditions in stimulated and unstimulated saliva and studied the expression of EGF receptor (EGF-R) in their gingival tissues. The results showed that the mean EGF concentration (pmol/ml) was slightly higher in JP patients than in controls. However, the difference was statistically significant only in stimulated saliva and when calculated per milligram salivary protein. When EGF release was measured as the rate of EGF secretion (pg/min), significantly higher values were observed in JP patients than in controls both in unstimulated and stimulated saliva. Immunofluorescence microscopy (IF) of gingival samples from JP patients and their controls revealed no quantitative or qualitative differences in the expression of EGF-R. Our results demonstrate the complex nature of salivary EGF release. The elevated rate of salivary EGF secretion in JP patients may be associated with the pathogenetic mechanisms of juvenile periodontitis.

Effect of medroxyprogesterone acetate on serum levels of LH, FSH, cortisol, and estrone in patients with endometrial carcinoma.

Medroxyprogesterone acetate (MPA) was administered orally for at least 6 months to 25 patients with endometrial adenocarcinoma starting 1 week after hysterectomy and bilateral ovariectomy. We measured serum concentrations of MPA during treatment and serum levels of LH, FSH, cortisol, and estrone before and during treatment with MPA. The serum levels of MPA 3--5 h after ingestion of a daily 100-mg dose of MPA at 08.00 h, varied considerably, the mean value (+/- SE) being 43 +/- 3.6 nmol/l range 15--119 nmol/l. Serum FSH levels rose after operation in the five premenopausal patients and remained elevated despite MPA treatment. In the 20 postmenopausal patients, however, initially high serum FSH and LH levels declined to half the starting values as a result of MPA treatment. After the administration of MPA for 6 months the mean serum cortisol and estrone concentrations at 08.00 a.m. had fallen to 78% and 82% of the mean values before treatment.

An ultrasensitive time-resolved immunofluorometric assay of human epidermal growth factor.

We have developed a sandwich-type time-resolved immunofluorometric assay (TR-IFMA) for human epidermal growth factor (hEGF) in body fluids. A two-step solid-phase technique was used. The assay utilizes a polyclonal anti-hEGF attached to the solid phase, and a monoclonal anti-hEGF labeled with Europium (III) as a tracer. The sensitivity of the assay (2.5 pg/ml) is at least 20 times better than what has been achieved by radioimmunoassay (RIA), and the measuring range is much wider: 2.5-5000 pg/ml. The feasibility of TR-IFMA was tested by assaying urine containing large amounts and amniotic fluid containing small amounts (mostly undetectable by RIA) of immunoreactive hEGF. The correlation between urine hEGF concentrations (1-100 ng/ml) measured by RIA and TR-IFMA was good: r = 0.96.

Metabolic control and serum hormone levels in relation to retinopathy in diabetic pregnancy.

The occurrence and progression of retinopathy were related to the mean blood glucose levels and the serum concentrations of prolactin, human placental lactogen, oestradiol and progesterone in 57 pregnant insulin-dependent diabetic patients. Fifteen patients had frank retinopathy, of whom eight showed a marked increase in retinopathy. The initial blood glucose levels were significantly higher in patients whose retinopathy progressed, whereas during the second and third trimester similar blood glucose levels were achieved in all groups. Serum concentrations of progesterone and human placental lactogen were significantly increased in diabetic patients during the last trimester when compared with those in normal pregnancies, and during the second trimester, patients with retinopathy showed significantly higher concentrations than those without, but no significant difference was found in oestradiol values. The eight patients with progressive retinopathy showed progesterone, human placental lactogen and oestradiol levels at or above the upper limit of the normal range. Throughout gestation, serum prolactin concentrations were significantly lower in diabetic patients than in healthy subjects. No correlation was found between serum prolactin values and the occurrence of retinopathy.

Purification and characterization of a tumor-associated trypsin inhibitor from the urine of a patient with ovarian cancer.

Immunochemical studies on urine from a patient with ovarian cancer revealed the presence of a tumor-associated peptide. This peptide occurred in elevated concentrations in the urine of some patients with gynecologic cancer, in early amniotic fluid, and in some cancer tumor extracts from these patients as described previously (Stenman, U.-H., Huhtala, M.-L., Koistinen, R., and Seppälä, M. (1982) Int. J. Cancer 30, 53-47). The peptide has now been purified from the urine of a patient with ovarian cancer by gel chromatography, ion exchange chromatography, and reverse phase liquid chromatography. The amino acid composition of the peptide is: Lys (4), Arg (3), Asx (8), Thr (4), Ser (3), Glx (6), Pro (3), Gly (5), Ala (1), Val (2), Ile (3), Leu (4), Tyr (3), Phe (1), and Cys (6). These 56 amino acids correspond to Mr = 62000 for the peptide, a value that is in agreement with the molecular weight established by gel chromatography. The molecule contains no carbohydrate. It is microheterogeneous in charge, the isoelectric point of the main component being 5.8. The purity of the peptide was confirmed by determination of the NH2-terminal amino acid sequence. The 21 residues determined were found to be identical with the corresponding ones of human pancreatic trypsin inhibitor. The purified peptide also inhibited bovine trypsin effectively.

Epidermal growth factor in mice: effects of estradiol, testosterone and dexamethasone.

To clarify the influence of steroids on the metabolism of epidermal growth factor, we studied the effects on its concentrations in adult male and female mice of 1. gonadectomy, 2. postgonadectomy treatments with estradiol and testosterone, and 3. treatment with dexamethasone. We also measured its mRNA levels in submandibular salivary glands and kidneys after ovariectomy. After gonadectomy, the male mice had 1.4-fold higher mean epidermal growth factor concentration in the urine than the female, in contrast to a 1.5-fold reverse difference in intact mice; the female mice had 2.5-fold higher concentration in the submandibular glands than the male animals, in contrast to a 4.5-fold reverse difference in intact mice. The kidney sex difference of intact mice (male greater than female) was abolished. In both gonadectomized sexes, treatment with testosterone increased the concentration of epidermal growth factor in plasma and the submandibular gland; treatment with estradiol increased the concentration in urine and decreased it in the submandibular gland. Treatment with dexamethasone decreased the concentration of epidermal growth factor in plasma of the male mice, and in urine of the female mice, thus decreasing the sex differences. In the submandibular gland and the kidneys, dexamethasone increased the concentration. The mRNA levels were higher in the submandibular gland and lower in the kidneys in the ovariectomized than in the intact female mice. The effects of sex steroids on epidermal growth factor concentrations are mediated through modulation of its gene activity. Testosterone has an increasing and estradiol a decreasing effect in the submandibular gland. Estradiol has also an increasing effect in the kidneys.

Epidermal growth factor is a constant component of normal human tear fluid.

Epidermal growth factor (EGF) is a mitogenic polypeptide, which was first isolated from mouse submaxillary gland. Since EGF also stimulates the growth of corneal epithelial cells and only preliminary data exist on its presence in tear fluid, we studied the occurrence of human EGF (hEGF) in the tear fluid of 36 healthy persons (31 women and 5 men from 20 to 59 years of age; 60 eyes). hEGF, as measured by an immunofluorometric assay, was present in all tear fluid samples investigated. Its concentration varied from 200 to 2860 pg/ml (median, 705 pg/ml). The tear fluid hEGF concentrations differed less between the eyes of one individual than between individuals. The total amount of hEGF released to the tear fluid increased with fluid flow, but the higher the flow was, the lower the concentration of hEGF. We could not find any evidence of sex dependency in the hEGF concentrations. In demonstrating that hEGF is a normal component of human tear fluid, the results of this study suggest that hEGF may be important for conjunctival and corneal epithelial integrity.

Rapid chromatographic quantitation of glycosylated haemoglobins.

We have developed a rapid chromatographic method for determination of glycosylated haemoglobins by high-performance liquid chromatography with a new cation-exchange column. The haemoglobins are eluted with a three-step gradient in 7 min, and total assay time including re-equilibration of the column is 15 min. The method permits separation and quantitation of HbAlc, even in the presence of elevated levels of HbF. HbA1a, A1b and A2 can also be determined. The results correlate well (r = 0.94) with those obtained by the macro-column method of Trivelli et al. [New Engl. J. Med., 284 (1971) 353] for determination of HbA1c. The method has been fully automated by the use of an automatic injector. The within-assay and between-assay coefficient of variation of the method is 2-3%.