The growth factor from plerocercoid larvae of the tapeworm, Spirometra mansonoides, stimulates growth but is not diabetogenic

A factor produced by plerocercoids of the tapeworm Spirometra mansonoides is similar to human growth hormone (hGH) in that it stimulates body growth, binds to hGH receptors, cross-reacts with anti-hGH antibodies, and has lactogenic and insulin-like activities. The purpose of this study was to determine whether plerocercoid growth factor (PGF) is similar to hGH in expressing diabetogenic activity in the genetically obese (ob/ob) mouse. To determine an effective dose for use in the obese mice, the ability of daily injections of PGF to stimulate growth of phenotypically normal mice of the same strain was assessed in a 10-day weight gain assay. Injections of PGF stimulated a dose-dependent weight gain (r = 0.83) and 25 ng eq/day of PGF stimulated a response not significantly different from that produced by 100 micrograms of bovine growth hormone/day. Diabetogenicity was assessed using fasting blood glucose and glucose tolerance tests in obese mice that had been injected for 3 days with saline, hGH, or PGF. Human growth hormone caused a significant increase (P less than 0.005) in fasting blood glucose and glucose tolerance of the obese mice was impaired (P less than 0.01). All of the doses of PGF used to test diabetogenicity in the obese mice were at least twice that required to stimulate a maximal growth response in normal mice, yet none of the doses of PGF increased fasting blood glucose or decreased glucose tolerance. These results show that PGF was a potent growth stimulant but was not diabetogenic.     

Study of human growth hormone structure with a radioimmunoassay specific for the fifteen amino acid fragment comprising hGH (32–46)

A radioimmunoassay for the 15-amino acid fragment comprising residues 32–46 of the 22,000-dalton human growth hormone has been devised. The radioimmunoassay detects from 1 to 150 fmol of the synthetic peptide. The 12-amino acid peptide hGH (35–46) shows parallel displacement, but the sensitivity decreased 50% due to the deletion of the three amino acids. Displacement activity is lost from the synthetic peptides hGH (38–46) and hGH (43–46), and intact hGH itself shows no displacement. Disrupting the large loop of hGH by subtilisin cleavage or by reduction and alkylation of the S-S bridges imparts displacement activity parallel to that of hGH (32–46), and hGH (1–139) has similar activity. A plausible interpretation for these results is that native hGH has a tertiary structure that masks or obscures the sequence 32–46, the sequence being unmasked by release of constraints imposed by an intact large loop.     

Down-regulation of prolactin receptors in the liver, mammary gland and kidney of female virgin rat, infused with ovine prolactin or human growth hormone

Down regulation of prolactin (PRL) receptors resulting from i.v. infusion of oPRL or human growth hormone (hGH) into female virgin rats was demonstrated. A decrease of over 85% in the number of free receptors was achieved within 15 - 30 min using infusion of oPRL or hGH at 25 micrograms/h and remained at this level until the end of infusion. Ovine growth hormone or recombinant bovine growth hormone at ten-fold higher concentration had no effect at all. The decrease in the specific binding resulted from a lower number of binding sites and not from change in the dissociation constants. The decrease in the total receptors in the liver was more gradual and leveled off at 40 - 50% of the initial value. Our results suggest that a change in blood PRL or hGH level may lead to a new steady state in the number, occupancy and distribution of prolactin receptors.     

Human growth hormone enhances pertussis toxin-stimulated ADP-ribosylation of Gi in Nb2 cell membrane.

The Nb2 node lymphoma cell line has been widely used as a model for investigating lactogen cellular actions. Both pertussis (PTX) and cholera (CTX) toxins modulate lactogen-stimulated Nb2 cell mitogenesis, suggesting G protein involvement in lactogen signal transduction. The following studies were performed to further investigate this possibility. Both PTX-sensitive (41 kDa) and CTX-sensitive substrates (42 and 45 kDa) were identified in Nb2 cell membrane and recognized by specific anti-Gi and anti-Gs antibodies, respectively. Equal numbers of Nb2 cells were then incubated with the lactogen human growth hormone (hGH, 10 ng/ml) for 0-72 h. Membrane protein prepared from each time point (50 micrograms) was compared in toxin-stimulated ADP-ribosylation studies. CTX-stimulated ADP-ribosylation was unaffected by prior hGH incubation. PTX-stimulated ADP-ribosylation increased 237 +/- 69% (X +/- S.E.) compared with 0-h controls (n = 11; p less than 0.01) after 4-7 h of hGH incubation then decreased toward 0-h samples by 24 and 72 h. No change in Gi alpha concentration was observed, but beta subunit concentration increased (145 +/- 14% at 7 h; p less than 0.01; n = 3) in a time course that paralleled the changes in PTX-stimulated ADP-ribosylation. In summary, 1) both Gi and Gs were present in Nb2 cell membrane, 2) incubation of cells with a lactogen, hGH, for 4-7 h markedly enhanced PTX-stimulated ADP-ribosylation of Gi alpha in vitro, whereas CTX-stimulated ADP-ribosylation of Gs alpha was unchanged, and 3) although no change in Gi alpha concentration was observed, beta subunit concentration increased in parallel with the increase in PTX-stimulated ADP-ribosylation of Gi alpha. These results suggest that hGH may modify PTX-stimulated ADP-ribosylation of Gi not by changing Gi alpha concentration, perhaps by increasing beta subunit concentration, enhancing association of Gi alpha by beta gamma subunits, which, in turn, is preferentially ADP-ribosylated. This may represent a late signal transduction event and may also have implications for other effectors dependent on Gi-mediated events.     

Targeted proteomics of low-level proteins in human plasma by LC/MSn: using human growth hormone as a model system

This paper describes the profiling of human growth hormone (hGH) in human plasma in order to assess the dynamic range of the ion-trap mass spectrometer for proteomic studies of complex biological samples. Human growth hormone is an example of a low-level plasma protein in vivo, present at subfemtomole levels. This study was performed on a plasma sample in which hGH has been spiked at 10-fold above the natural level, that is approximately 16 pg/microL of plasma. Initially, the measurement was carried out without any sample enrichment and consisted of the following steps: the full set of plasma proteins were reduced, alkylated, and digested with trypsin, and the resulting peptides were separated on a capillary C-18 column and then detected by ion-trap mass spectrometry (1D LC/MS). In addition, this study provided a global view of the serum proteome with over 200 plasma proteins being preliminarily identified. In the MS/MS analysis, hGH was detected by characterization of the first tryptic peptide (T1). The initial identification was confirmed by alternative approaches, which also allowed the evaluation of different sample purification protocols. First, the plasma sample containing hGH was fractionated on a reversed-phase HPLC column and digested, and hGH could now be identified by MS/MS measurements of two tryptic peptides (T1 and T4) by the same 1D LC/MS protocol. In addition, the assignment of peptide identity was made with higher certainty (as measured by an algorithm score). The plasma sample was also fractionated by 1D and 2D gel electrophoresis, the selected bands were digested and analyzed again by the 1D LC/MS protocol. In both cases using the gel prepurifications, hGH was identified with additional peptides. Finally, the plasma sample was analyzed by 2D chromatography (ion exchange and reversed phase) on a new instrumental platform (ProteomeX), and hGH was identified by the observation of five tryptic peptides. In conclusion, these experiments were able to detect growth hormone in the low femtomole level with a dynamic range of 1 in 40 000 by several independent approaches. The amount of growth hormone, while 10-fold above normal in vivo levels, represents concentrations that may be present in disease states (such as acromegaly) and also in doping control measurements. These studies have demonstrated that shotgun sequencing approaches (LC/MS/MS) not only can profile high-abundance proteins in complex biological fluids but also have the potential to identify and quantitate low-level proteins present in such complex mixtures without extensive prepurification protocols. A key to such studies, however, is to use targeted approaches that reduce the complexity of the solute mixture that is presented to the mass spectrometer at a given time point. The various sample preparation protocols described here all improved the quality of the hGH measurement, although in this study the 2D chromatographic approach gave the greatest sequence coverage.     

Bioactivity of human growth hormone in urine from acromegalic patients

Bioactivity of hGH in urine from five acromegalic patients was determined by Nb2 bioassay and IM-9 receptor modulation assay (RMA). Urine samples were concentrated by immunoadsorbent chromatography, dialysis and lyophilization. The ratio of the bioactivity by Im-9 RMA and the immunoactivity by RIA was between 0.81 and 1.24 (1.01 +/- 0.19, mean +/- SD). The ratio of the bioactivity by Nb2 bioassay and the immunoactivity ranged from 0.45 to 0.60 (1.37 +/- 0.85). Gel chromatography of hGH in urine samples measured by sensitive sandwich enzyme immunoassay showed that more than 97% of hGH activity was the 22K form. Urinary hGH from acromegalic patients was bioactive in Nb2 bioassay and IM-9 RMA and the bioactivity showed a close correlation with the immunoactivity. The major immunoactive form of hGH (22K) seems to correspond to the bioactivity.     

Homology modeling of rabbit prolactin hormone complexed with its receptor

The pituitary hormone prolactin (prl) is implicated in a number of biological functions, especially lactation, which is mediated through specific lactogenic receptors (PrlR). Human growth hormone (hGH) is also a pituitary hormone responsible for linear growth. While the growth hormone receptor (hGHR) binds only hGH, hPrlR can interact with both hGH and hPrl. Using structural information from the human growth hormone (hGH)/receptor (hGHR) complex, we modeled by homology a complex between rabbit prolactin hormone (rbPrl) and its receptor (rbPrlR). While the somatogenic hormone/somatogenic receptor (hGH/hGHR) and somatogenic hormone/lactogenic receptor (hGH/hPrlR) interactions are now known and well studied, here we propose a model for the interaction of the lactogenic hormone with its receptor (rbPrl/rbPrlR), and compare these three kinds of ligand/receptor interaction. We identified residues contributing to the active site and tested the potential dimerization of the receptor. Biochemical studies and information deduced from the modeled complex do not exclude a homodimeric form but point to a functional heterodimeric complex. Proteins 27: 459–468, 1997. © 1997 Wiley-Liss, Inc.     

Effects of hypophysectomy and the insulin-like and anti-insulin pituitary peptides on carbohydrate metabolism in yellow Avy/A (BALB/c x VY)F1 hybrid mice

The amino-terminal portion of human growth hormone, residues 1-43 (hGH1-43), has insulin-potentiating action, while a hyperglycemic pituitary peptide (HP), which co-purifies with human growth hormone (hGH), is antagonistic to the action of insulin. The effects of hGH, hGH1-43, and HP on glucose metabolism were assessed in young (4-5 weeks) and adult (6-8 months) hypophysectomized yellow Avy/A mice which lacked any interfering endogenous pituitary hormones, and compared with age-matched intact obese yellow Avy/A and lean agouti A/a mice. Treatment with hGH1-43 or HP did not promote body growth in hypophysectomized yellow mice; but after 2 weeks of treatment with hGH, there was a significant increase in body weight (P less than 0.05). Treatment with HP raised blood glucose and lowered insulin concentrations in obese yellow mice, but not in agouti or hypophysectomized yellow mice. The severely impaired glucose tolerance of the hypophysectomized yellow mice was improved by acute (60 min) and chronic (3 days) treatment with hGH1-43 as well as by 2 weeks of treatment with hGH; in contrast, HP had no effect. Glucose oxidation in adipose tissue from obese yellow mice was low and showed essentially no response to stimulation by insulin at doses lower than 1000 microunits/ml. Basal glucose oxidation rates in adipose tissue taken from agouti and hypophysectomized yellow mice were significantly higher (P less than 0.001) than those in tissue from obese yellow mice, and the rates responded significantly (P less than 0.05) to 100 microunits/ml insulin. The insulin binding affinities in liver membranes from agouti mice were higher than those from either obese or hypophysectomized yellow mice. The insulin receptor densities were similar in both agouti and obese yellow mice, but higher in hypophysectomized yellow mice (P less than 0.05). Treatment with hGH1-43 slightly increased, although not significantly, the insulin receptor density in yellow obese mice while hGH showed essentially no change. Therefore, hypophysectomy appeared to increase tissue response and decrease insulin resistance by increasing receptor numbers and lowering the circulating insulin levels. Furthermore, the insulin-like action of hGH was elicited directly in vivo by hGH1-43 in hypophysectomized yellow mice.     

Comparative Analysis of Human Growth Hormone in Serum Using SPRi,Nano-SPRi and ELISA Assays

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml⁻¹ and minimum levels of 0.03 ng ml⁻¹) in circulating blood are essential as variable levels may indicate altered physiology. For example, growth disorders occurring in childhood can be diagnosed by measuring levels of hGH in blood. Also, the misuse of recombinant hGH in sports not only poses an ethical issue it also presents serious health threats to the abuser. One popular strategy for measuring hGH misuse, relies on the detection of the ratio of 22 kDa hGH to total hGH, as non-22 kDa endogenous levels drop after exogenous recombinant hGH (rhGH) administration.Surface plasmon resonance imaging (SPRi) is an analytical tool that allows direct (label-free) monitoring and visualization of biomolecular interactions by recording changes of the refractive index adjacent to the sensor surface in real time. In contrast, the most frequently used colorimetric method, enzyme-linked immunosorbent assay (ELISA) uses enzyme labeled detection antibodies to indirectly measure analyte concentration after the addition of a substrate that induces a color change. To increase detection sensitivity, amplified SPRi uses a sandwich assay format and near infrared quantum dots (QDs) to increase signal strength. After direct SPRi detection of recombinant rhGH in spiked human serum, the SPRi signal is amplified by the sequential injection of detection antibody coated with near-infrared QDs (Nano-SPRi). In this study, the diagnostic potential of direct and amplified SPRi was assessed for measuring rhGH spiked in human serum and compared directly with the capabilities of a commercially available ELISA kit.     

Sensitive sandwich enzyme immunoassay of human growth hormone (hGH) in serum and urine using monoclonal antibody-coated polystyrene balls

A sensitive sandwich enzyme immunoassay for human growth hormone (hGH) using monoclonal antibody is described. A monoclonal anti-hGH IgG-coated polystyrene ball was incubated with hGH and subsequently with affinity-purified rabbit anti-hGH Fab'-horseradish peroxidase conjugate. Peroxidase activity bound to the polystyrene ball was assayed by fluorimetry using 3-(4-hydroxyphenyl) propionic acid as a substrate. The detection limits of hGH in serum and urine were 1.5 ng/l using 20 microliters of serum and 0.2 ng/l using 0.15 ml of urine, respectively. The specificity and assay precision were satisfactory. hGH levels in serum and urine determined by the present sandwich enzyme immunoassay using monoclonal anti-hGH IgG-coated polystyrene balls were well correlated to those determined by the previous sandwich enzyme immunoassay using rabbit anti-hGH IgG-coated polystyrene balls. Levels of hGH in urine collected as first morning voids from healthy subjects aged 19-28 yr were 6.4 +/- 3.2 (SD) ng/g creatinine. However, the present assay gave lower hGH levels than the previous assay. This was at least partly explained by the fact that hGH in urine was less efficiently bound to monoclonal anti-hGH IgG-polystyrene balls than standard hGH, while the binding of hGH in urine and standard hGH to rabbit anti-hGH IgG-coated polystyrene balls was equally efficient. In addition, gel filtration showed that 22K hGH, a major component, in urine was less efficiently bound to monoclonal anti-hGH IgG-coated polystyrene balls than standard 22K hGH. The nature of hGH in serum and urine remains to be investigated.     

Effect of a recombinant human growth hormone preparation on the urinary 15nitrogen balance in growth-hormone-deficient children

In 10 patients with idiopathic growth hormone (GH) deficiency (9 boys and 1 girl, aged 7.5-14.5 years, mean 12.1 +/- 2.2 years), urinary 15N-balance studies were performed before and on recombinant hGH (2 x 3 IU/m2 of body surface area subcutaneously on consecutive days). Before and on the 2nd day of recombinant hGH, 99% 15N-labeled ammonium chloride (0.05 g/kg, divided in 3 doses per day, corresponding to 389 +/- 30 mg/m2 of 15N) was administered and 24 h urine was collected. In urine, total nitrogen and the percentage of 15N were measured. From the ingested and excreted quantity, a urinary 15N balance was calculated. Mean 15N percentage from total N was 3.3 +/- 0.5. In 9 patients, basal 15N balance was +79 +/- 15 mg/m2 or +2.9 +/- 0.4 mg/kg. On recombinant hGH, it was +166 +/- 16 mg/m2 or +6.1 +/- 0.6 mg/kg (p less than 0.001). The recombinant hGH-induced positive 15N balance change was +87 +/- 17 mg/m2 or +3.2 +/- 0.6 mg/kg. 1 patient with a higher basal 15N balance (+196 mg/m2, +7.1 mg/kg) had no positive 15N balance change due to latent hypothalamic hypothyroidism. In previous similar studies with pituitary hGH the change of 15N balance was +80 +/- 27 mg/m2 or +2.8 +/- 1.1 mg/kg. It is concluded that the acute nitrogen-retaining effect of recombinant hGH is at least equal to that of pituitary hGH.     

Non-conventional use of growth hormone: European experience

Results from several centres in Europe using biosynthetic human growth hormone (hGH) for the promotion of growth in a variety of conditions other than classical hGH deficiency were evaluated. Significant increments in growth rates were achieved by daily administration of hGH in doses appropriate for body size without disproportionate skeletal advances in short normals, Turner syndrome, low birth weight, skeletal dysplasia, central precocious puberty (reared with gonadotrophin-releasing hormone analogue) and renal failure.     

Human growth hormone peptide 1–43: Isolation from pituitary glands

A procedure is described for isolation from human pituitary glands of a peptide with the amino acid sequence of the first 43 residues of human growth hormone, hGH (1–43). The peptide was recovered in a yield of 12 mg per 1000 pituitary glands. In a radioimmunoassay for hGH the peptide was unreactive when tested at 1 µg/ml and therefore if it does circulate, it probably has gone underected in blood. Growth-promoting activity as measured by the tibial line procedure was low but detectable; however, the log dose response was not parallel to that of hGH. Separate studies indicated the hGH (1–43) was a potent potentiator of insulin action suggesting physiologic significance. Further, because the peptide can be a cleavage product of the major form of hGH but not of its 20,000-dalton variant, importance is indicated for the multiple forms of the hormone.     

Variation in protein levels obtained from human blood cells and biofluids for platelet, peripheral blood mononuclear cell, plasma, urine and saliva proteomics

Blood cells and biofluid proteomics are emerging as a valuable tool to assess effects of interventions on health and disease. This study is aimed to assess the amount and variability of proteins from platelets, peripheral blood mononuclear cells (PBMC), plasma, urine and saliva from ten healthy volunteers for proteomics analysis, and whether protein yield is affected by prolonged fasting. Volunteers provided blood, saliva and morning urine samples once a week for 4 weeks after an overnight fast. Volunteers were fasted for a further 24 h after the fourth sampling before providing their final samples. Each 10 mL whole blood provided 400–1,500 μg protein from platelets, and 100–600 μg from PBMC. 30 μL plasma depleted of albumin and IgG provided 350–650 μg protein. A sample of morning urine provided 0.9–8.6 mg protein/dL, and a sample of saliva provided 70–950 μg protein/mL. None of these yields were influenced by the degree of fasting (overnight or 36 h). In conclusion, in contrast to the yields from plasma, platelets and PBMC, the protein yields of urine and saliva samples were highly variable within and between subjects. Certain disease conditions may cause higher or lower PBMC counts and thus protein yields, or increased urinary protein levels.     

Chromium and Manganese Levels in Biological Samples of Normal and Night Blindness Children of Age Groups (3–7) and (8–12) Years

This study was designed to compare the levels of chromium (Cr) and manganese (Mn) in scalp hair, blood, and urine of night blindness in children age ranged (3–7) and (8–12) years of both genders, comparing them to sex- and age-matched controls. A microwave-assisted wet acid digestion procedure, was developed as a sample pretreatment, for the determination of Cr and Mn in biological samples of night blindness children. The proposed method was validated by using conventional wet digestion and certified reference samples of hair, blood and urine. The digests of all biological samples were analyzed for Cr and Mn by electrothermal atomic absorption spectrometry. The results indicated significantly higher levels of Cr, whilst low level of Mn in the biological samples (blood and scalp hair) of male and female night blindness children, compared with control subjects of both genders. These data present guidance to clinicians and other professional investigating deficiency of Mn and excessive level of Cr in biological samples (scalp hair and blood) of night blindness children.     

Analysis of methanol or formic acid in body fluids by headspace solid-phase microextraction and capillary gas chromatography

Methanol and its metabolite formic acid have been found extractable from human whole blood and urine by headspace solid-phase microextraction (SPME) with a Carboxen/polydimethylsiloxane fiber. The headspace SPME for formic acid was carried out after derivatization to methyl formate under acidic conditions. The determinations of both compounds were made by using acetonitrile as internal standard (IS) and capillary gas chromatography (GC) with flame ionization detection. The headspace SPME–GC gave sharp peaks for methanol, methyl formate and I.S.; and low background noises for whole blood and urine samples. Extraction efficiencies were 0.25–1.05% of methanol and 0.38–0.84% formic acid for whole blood and urine. The calibration curves for methanol and formic acid showed excellent linearity in the range of 1.56 to 800 and 1.56 to 500 μg/0.5 ml of whole blood or urine, respectively. The detection limits were 0.1–0.5 μg/0.5 ml for methanol and 0.6 μg/0.5 ml for formic acid for both body fluids. The within-day relative standard deviations in terms of extraction efficiency for both compounds in whole blood and urine samples were not greater than 9.8%. By using the established SPME method, methanol and formic acid were successfully separated and determined in rat blood after oral administration of methanol.     

Renal Tubule-Specific Expression and Urinary Secretion of Human Growth Hormone: A Kidney-Based Transgenic Bioreactor

Tissue-specific expression of human genes and secretion of human proteins into the body fluids in transgenic animals provides an important means of manufacturing large-quantity and high-quality pharmaceuticals. The present study demonstrates using transgenic mice that a 3.0 kb promoter of the mouse Tamm-Horsfall protein (THP, or uromodulin) gene directs the specific expression of human growth hormone (hGH) gene in the kidney followed by the secretion of hGH protein into the urine. hGH expression was detected in renal tubules that actively produce the THP, that is, the ascending limb of Henle's loop and distal convoluted tubules. Up to 500 ng/ml of hGH was detected in the urine, and this level remained constant throughout the 10-month observation period. hGH was also detectable in the stomach epithelium and serum in two of the transgenic lines, suggesting position-dependent effects of the transgene and leakage of hGH from the site of synthesis into the bloodstream, respectively. These results indicate that the 3.0 kb mouse THP promoter is primarily kidney-specific and can be used to convert kidney into a bioreactor in transgenic animals to produce recombinant proteins. Given the capacity of urine production independent of age, sex and lactation, the ease of urinary protein purification, and the potentially distinct machinery for post-translational modifications in the kidney epithelial cells, the kidney-based transgenic bioreactor may offer unique opportunities for producing certain complex pharmaceuticals.     

A comparison of subcutaneous and intramuscular administration of human growth hormone (hGH) and increased growth rate by daily injection of hGH in GH deficient children

Plasma human growth hormone (hGH) profiles and biological activities of recombinant hGH were compared after im and sc injection in 8 normal volunteers. The time to reach maximal plasma GH and plasma hGH concentrations and the areas under the curve of hGH profiles did not differ significantly after im and sc injections. The biological effect of hGH in increasing nonesterified fatty acid and insulin-like growth factor-I (IGF-I) was the same after both im and sc injections. During 6 months of daily sc administration of recombinant hGH in 20 naive patients, their height increased between 5 and 16.5 cm with a mean of 11.0 +/- 3.0 cm/year. In 27 patients who switched from hGH injections of 2-4 times/week to daily injections, the height increased between 5.3 and 16.5 cm with a mean of 8.3 +/- 2.2 cm/year. These values were greater than those observed in a previous study in which the same amount of hGH was injected in 2-4 doses per week. Plasma IGF-I increased more with daily sc administration than with 2-4 doses per week. The rate of appearance of an antibody to hGH was low (0.5%) and there were no notable changes in blood cell count, urinalysis and/or routine chemistries during the 6 months of daily recombinant hGH treatment. These results show that sc daily administration of hGH is safe, has a greater growth promoting effect, and can be recommended for the treatment of patients with GH deficiency.     

Urinary zinc excretion and zinc status of patients with Β-thalassemia major

In this study, zinc status and urinary zinc excretion with and without desferrioxamine (DFO) infusion and the relationship between urinary zinc excretion and renal tubular dysfunction in thalassemia major (TM) patients were investigated. Forty TM patients were given four DFO infusions on alternate days over a 1-wk period prior to the transfusion. On each day that DFO was given, a 24-h urine collection initiated. DFO was omitted for 1-wk before the following transfusion and during the period four 24-h urine collections were performed. Twenty healthy children provided 24-h urine collection as controls. Blood samples were taken on each of two consecutive transfusion days of the patients and from the controls. Urinary zinc excretion was measured and plasma and red blood cell (RBC) zinc analysis were performed by inductively coupled plasma-atomic emission spectrophotometry. UrinaryN-acetyl-Β-D-glucosaminidase (NAG) activity and creatinine were determined in morning urine specimens. The mean plasma zinc concentration was significantly lower in the patients not given DFO compared to the values of the patients given DFO and the control group. The mean RBC zinc concentration (Μmol/g Hb) in the patients (with and without DFO) and the control group were similar. Urinary zinc excretion was significantly higher in the patients receiving DFO compared to the control group, whereas urinary zinc excretion in the patients not given DFO was not different from the controls. Urinary NAG indices (U/g Cr) were significantly higher in the patients compared to controls. Urinary zinc excretion was correlated with the urinary NAG indices.     

Characterization of human epidermal growth factor in human serum and urine under native conditions

The objective of this study was to investigate the molecular nature of the human epidermal growth factor (EGF) in serum and urine samples of normal subjects. Recombinant EGF emerged as a single peak and did not interact with human IgG1 and albumin up to the concentration of 12 microg/ml. Freshly separated human serum contained only trace amounts of EGF. However, EGF appeared and increased in serum separated from blood after spontaneous overnight clotting. The authentic 6 kDa form of EGF made up nearly 40% of the total EGF in serum and revealed relatively homogeneous feature. The remaining immunoreactive fractions corresponded to 160 kDa proEGF. Immunoreactive EGF in blood seemed to be associated with the EGF release from platelets. TSKgel G3000SW chromatography of freshly-voided morning and day urines revealed that urine samples mainly contained two major form of EGF; a high-molecular-weight (HMW) and low-molecular-weight (LMW) forms. In the sense of molecular nature of EGF contents, morning urine was more heterogeneous than day urine of the same individuals. The LMW form of EGF in morning urine, in which its proportion was more than 90% of the total EGF, revealed further heterogeneous feature generally containing three to four different components.