Urinary hGH Research Articles

Analysis of urinary human growth hormone (hGH) using hydrogel nanoparticles and isoform differential immunoassays after short recombinant hGH treatment: Preliminary results

Successful application clinical-grade human growth hormone (hGH) immunoassays to the discovery of illegal doping cases has been rare. Indeed, the preferred biological matrix in doping control is urine, where the estimated baseline concentration of hGH falls well below the linear range and sensitivity threshold of all commercially available immunoassays, including hGH isoform differential immunoassays which can discriminate pituitary endogenous hGH from recombinant hGH. We employed hydrogel nanoparticles as a pre-processing step that concentrate urinary hGH into the linear range of isoform differential immunoassays.We explored the characteristics of immunoassays in urine spiked with both phGH or rhGH, after pre-treatment with the nanoparticles. Subsequently, pre-treatment was applied to urine obtained from 3 healthy volunteers administered during three days with daily subcutaneous injections of 0.026mg/kg/day rhGH, Genotonorm®.Linearity between both rhGH and phGH concentrations in urine measured by a chemoluminescent assay (Immulite) and in the particle eluate was evident for differential immunoassays (R square higher than 0.999). In case of treated individuals the recombinant/pituitary concentration ratios remained above the established World Anti-Doping Agency (WADA) criterion for hGH misuse up to 24h after the last administration dose, using both assays for volunteer 1 and 2 while in case of volunteer 3 results were inconclusive.The use of nanoparticles appears to open the possibility of assessing rhGH misuse in urine.

Beyond Punishment: Doping, Deterrence, and Moral Disengagement

Urine represents a valuable biofluid for noninvasive measurement of Human Growth Hormone (HGH) secretion. Unfortunately, currently available commercial HGH immunoassays do not achieve the sensitivity needed for urinary HGH measurement in the low picogram per milliliter range, the expected normal concentration range of HGH in urine. A nanotechnology based sample preprocessing step was used to extract and concentrate HGH in urine so that urinary HGH could be measured with a clinical grade standard immunoassay designed for serum (Immulite 1000, Siemens). We applied the nanoparticle enhanced immunoassay to evaluate the baseline value of urinary HGH in a population of healthy young adults (age 18-30, N=33, median 21, M: F=39%:61%, with no reported medical therapies). Nanoparticle sample preprocessing effectively improved the lower limit of detection of the Immulite HGH assay by more than 50 fold, shifting the linear range of the assay to encompass the expected value of urinary HGH. The full process between run and within run CV% was 7.9 and 9.0%, respectively. On 33 healthy volunteers, the 95% reference values for hGH in spot urine normalized to specific gravity were 0.64 - 16.85 pg/mL (0.05-5.82 ng/g creatinine). Nanoparticle preprocessing constitutes a reliable means of measuring urinary HGH with a clinical grade immunoassay, now establishing a normal baseline value for HGH in urine. Nanoparticles can be used to study the kinetics of HGH excretion in urine, and the factors that influence urinary HGH secretion and HGH isoform proportions.

Application of Analyte Harvesting Nanoparticle Technology to the Measurement of Urinary HGH in Healthy Individuals

Application of Analyte Harvesting Nanoparticle Technology to the Measurement of Urinary HGH in Healthy Individuals

Nanoparticle technology: amplifying the effective sensitivity of biomarker detection to create a urine test for hGH

Several clinical-grade immunoassays exist for the specific measurement of hGH or its isoforms in blood but there is an urgent need to apply these same reliable assays to the measurement of hGH in urine as a preferred 'non-invasive' biofluid. Unfortunately, conventional hGH immunoassays cannot attain the sensitivity required to detect the low concentrations of hGH in urine. The lowest limit of sensitivity for existing hGH immunoassays is >50 pg/mL, while the estimated concentration of urinary hGH is about 1 pg/m-50 times lower than the sensitivity threshold. We have created novel N-isopropylacrylamide (NIPAm)-based hydrogel nanoparticles functionalized with an affinity bait. When introduced into an analyte-containing solution, the nanoparticles can perform, in one step, (1) complete harvesting of all solution phase target analytes, (2) full protection of the captured analyte from degradation and (3) sequestration of the analyte, effectively increasing the analyte concentration up to a hundredfold. N-isopropylacrylamide nanoparticles functionalized with Cibacron Blue F3GA bait have been applied to raise the concentration of urinary hGH into the linear range of clinical grade immunoassays. This technology now provides an opportunity to evaluate the concentration of hGH in urine with high precision and accuracy.

Concentration and preservation of very low abundance biomarkers in urine, such as human growth hormone (hGH), by Cibacron Blue F3G-A loaded hydrogel particles

Urine is a potential source of diagnostic biomarkers for detection of diseases, and is a very attractive means of non-invasive biospecimen collection. Nonetheless, proteomic measurement in urine is very challenging because diagnostic biomarkers exist in very low concentration (usually below the sensitivity of common immunoassays) and may be subject to rapid degradation. Hydrogel nanoparticles functionalized with Cibacron Blue F3G-A (CB) have been applied to address these challenges for urine biomarker measurement. We chose one of the most difficult low abundance, but medically relevant, hormones in the urine: human growth hormone (hGH). The normal range of hGH in serum is 1 to 10 ng/mL but the urine concentration is suspected to be a thousand times less, well below the detection limit (50 pg/mL) of sensitive clinical hGH immunoassays. We demonstrate that CB particles can capture, preserve and concentrate hGH in urine at physiological salt and urea concentrations, so that hGH can be measured in the linear range of a clinical immunometric assay. Recombinant and cadaveric hGH were captured from synthetic and human urine, concentrated and measured with an Immulite chemiluminescent immunoassay. Values of hGH less than 0.05 ng/mL (the Immulite detection limit) were concentrated to 2 ng/mL, with a urine volume of 1 mL. Dose response studies using 10 mL of urine demonstrated that the concentration of hGH in the particle eluate was linearly dependent on the concentration of hGH in the starting solution, and that all hGH was removed from solution. Thus if the starting urine volume is 100 mL, the detection limit will be 0.1 pg/mL. Urine from a healthy donor whose serum hGH concentration was 1.34 ng/mL was studied in order detect endogenous hGH. Starting from a volume of 33 mL, the particle eluate had an hGH concentration of 58 pg/mL, giving an estimated initial concentration of hGH in urine of 0.175 pg/mL. The nanotechnology described here appears to have the desired precision, accuracy and sensitivity to support large scale clinical studies of urine hGH levels.

The bladder as a bioreactor: urothelium production and secretion of growth hormone into urine.

Uroplakin genes are expressed in a bladder-specific and differentiation-dependent fashion. Using a 3.6-kb promoter of mouse uroplakin II gene, we have generated transgenic mice that express human growth hormone (hGH) in their bladder epithelium, resulting in its secretion into the urine at 100-500 ng/ml. The levels of urine hGH concentration remain constant for longer than 8 months. hGH is present as aggregates mostly in the uroplakin-delivering cytoplasmic vesicles that are targeted to fuse with the apical surface. Using the bladder as a bioreactor offers unique advantages, including the utility of all animals throughout their lives. Using urine, which contains little protein and lipid, as a starting material facilitates recombinant protein purification.

Detection of human growth hormone doping in urine: out of competition tests are necessary.

The misuse of human growth hormone (hGH) in sport is deemed to be unethical and dangerous because of various adverse effects. Thus, it has been added to the International Olympic Committee list of banned substances. Until now, the very low concentration of hGH in the urine made its measurement difficult using classical methodology. Indeed, for routine diagnosis, only plasma measurements were available. However, unlike blood samples, urine is generally provided in abundant quantities and is, at present, the only body fluid allowed to be analysed in sport doping controls. A recently developed enzyme-linked immunosorbent assay (Norditest) makes it now possible, without any extraction, to measure urinary hGH (u-hGH) in a dynamic range of 2-50 ng hGH/l. In our protocol, untreated and treated non-athlete volunteers were followed. Some of them received therapeutical doses of recombinant hGH (Norditropin) for one week either intramuscularly (three increasing doses) or subcutaneously (12 i.u. every day). The u-hGH excretion after treatment showed dramatic increases of 50-100 times the basal values and returned to almost the mean normal level after 24 h. u-hGH was also measured in samples provided by the anti-doping controls at major and minor competitions. Depending on the type of efforts made during the competition, the hGH concentration in urine was dramatically increased. Insulin-like growth factor binding proteins and beta 2-microglobulins in urine and/or in blood could be necessary for the correct investigation of any hGH doping test procedure.

Screening for growth hormone deficiency using urinary growth hormone measurement

The diagnostic approach in growth hormone deficiency (GHD) is complicated. Two or more provocative tests are essential for definitive diagnosis of GHD. However, such testing cannot be carried out routinely on all subjects with short stature because of the need for hospitalization and blood sampling. A simple screening method for GHD would be of great value. Human growth hormone (hGH) levels were measured in the early morning urine of 192 children aged 7-15 years with height 2.0 s.d. below the mean for their ages. Sixty-eight subjects were selected because they showed a urinary hGH level < 10 ng/g creatinine. They were further examined in terms of bone age and plasma insulin-like growth factor (IGF-I) levels. In 30 subjects, the ratio of bone age: chronological age was < 0.8 and/or plasma IGF-I level was < 0.7 U/mL. Finally 24 of these subjects were examined with provocative tests and other endocrinological tests. Eleven subjects proved to have poor growth hormone secretion and one subject was diagnosed as having Turner syndrome. In conclusion, 11 patients with GHD were diagnosed from 192 children with short stature using urinary hGH measurement as the first screening method. These findings suggests that urinary hGH measurement could be a useful and simple method for detecting GHD.

RENAL FRACTIONAL EXCRETION (FE) OF HUMAN GROWTH HORMONE (HGH) IS CORRELATED TO INULIN CLEARANCE (GFR) AND PROXIMAL TUBULAR FUNCTION

Urinary HGH has been proposed to evaluate HGH secretion. Renal excretion of microproteins, including HGH, depend on GFR and proximal tubular reabsorption. In 15 children with various renal diseases HGH excretion was compared to inulin clearance (GFR) with a mean±SEM of 108115 ml/min*1.73m2 (range 22-238) and renal tubular function determined by albumin (ALB) and α1-microglobulin (α1-MG) excretion under standardized conditions (fasting, volume load 2% of body weight per h). HGH concentrations (n=44) were measured with two different antibodies by RIA, Nordisk and own (Girard) method. With both assays HGH was significantly correlated to ALB (mean±SEM: 628±342mg/l) with R of 0,50 (p<0.001, Nordisk) and R=0,41 (p<0.01, Girard), and α1-MG (7.9±3.0 mg/l) with R=0,42 and R=0,52 (p<0.01 and p<0.001). FE of HGH was inversely correlated to GFR (R=0.61, p<0.001) but independent of urinary flow rate. Conclusion: To exclude disturbed renal handling urinary HGH concentrations should be evaluated in conjunction with albumin and α1-MG to establish glomerular and proximal tubular integrity.

Low urinary growth hormone values in patients with Turner's syndrome.

Short stature is one of the major symptoms in Turner's syndrome (TS). The cause of short stature is not clearly known at present. In this study we initially assessed GH secretory status in TS by determinations of urinary human (h) GH excretion for 2 consecutive days. Secondly, the therapeutic dose of hGH used for treatment of short stature in TS was evaluated by measurements of urinary hGH after recombinant hGH (r-hGH) injections. Twenty-four-hour urinary hGH excretion for the 2 days combined was significantly lower in patients with TS than in normal children [2.3 +/- 1.8 ng/day (n = 7) vs. 13.4 +/- 3.2 (n = 16); P less than 0.001], although four of seven patients with TS had normal GH responses to the provocative tests. The mean level of urinary hGH in TS after 2 days was comparable to that in complete GH deficiency (1.9 +/- 0.9 ng/day; n = 14) that we previously reported. Treatment with daily sc injections of 1.0 IU (0.37 mg)/kg.week r-hGH, given in seven divided doses, normalized urinary hGH excretion and induced remarkable catch-up growth in all patients with TS. These results indicate that the 24-h endogenous GH secretion in seven patients with TS is impaired. The measurement of 24-h urinary hGH excretion may prove to be useful as a marker to assess the abnormal GH secretion and the adequacy of treatment with hGH in patients with TS. The therapeutic dose of hGH in TS is approximately 0.37 mg/kg.week, given in seven divided doses. To convert international units of r-hGH to milligrams, divide by 2.7.

Time-Resolved fluorometric sandwich immunoassay for human growth hormone in serum and urine

A specific and sensitive time-resolved fluorometric sandwich immunoassay for human growth hormone (hGH) in serum and urine is described. A monoclonal anti-hGH IgG1 (5802)-coated polystyrene ball was incubated with serum or dialyzed urine and subsequently with europium ion-labeled monoclonal anti-hGH IgG1 (5801). Europium ion bound to the polystyrene ball was measured by time-resolved fluorometry. The detection limit of hGH was 0.3 pg/tube, which was 15-fold higher than that by sandwich enzyme immunoassay using horseradish peroxidase as label. The assay range of serum hGH was 15-15,000 ng/liter using 20 microliters of serum. The assay range of urinary hGH was 2-2,000 ng/liter using 150 microliters of dialyzed urine. The detection limits of hGH in serum and urine by this immunoassay were satisfactory for measuring hGH levels in serum and urine of healthy children and in serum of healthy adults and higher levels but not in urine of healthy adults and in serum and urine of patients with hGH deficiency.

Urinary Human Growth Hormone Measurement Using a Highly Sensitive Sandwich Enzyme Immunoassay: Diagnostic and Therapeutic Uses in Patients with Growth Hormone Deficiency*

Several reports indicate that urinary hGH excretion is significantly lower in patients with either partial (PGHD) or complete GH deficiency (CGHD) than in normal but short children (NSC) or normal children (NC). However, there is an overlap between the NSC and NC groups and the PGHD group. Using a highly sensitive sandwich enzyme immunoassay, we investigated whether the measurement of urinary hGH can clearly separate the PGHD and CGHD groups from the NSC and NC groups. In addition, we measured the urinary excretion of synthetic methionyl-hGH (met-hGH) in PGHD and CGHD after sc injections of 2 and 4 IU and im injections of 4 IU in an attempt to determine the optimal replacement dose. Total 24-h urinary hGH excretion in each patient examined for 2 consecutive days varied from 1 day to the next. There were no differences in urinary hGH excretions between the NSC group and the NC group. The lower values for daily urinary hGH excretion in the NSC group overlapped some of the higher values in the PGHD group. However, when the mean urinary hGH level of both days was used, the 24-h urinary hGH excretion clearly separated the PGHD (5.5 +/- 2.3 ng/day; range, 1.3-9.2; n = 21) and CGHD (1.9 +/- 0.9 ng/day; range, 0.6-3.6; n = 14) groups from the NSC (12.8 +/- 3.1 ng/day; range, 9.3-17.5; n = 10) and NC (14.6 +/- 3.1 ng/day; range, 10.6-19.0; n = 6) groups without any overlap. A mean urinary hGH value less than 9.0 ng/day during a 2-day collection strongly suggested GH deficiency. Ten of 16 patients with PGHD and CGHD who received 2 IU met-hGH, sc, had urinary hGH levels within the range of the mean +/- SD in NSC. These patients received daily sc 0.097 +/- 0.024 IU/kg hGH injections. These results suggest that the measurement of 24-h urinary hGH excretion is noninvasive, accurate, and useful for the screening of GH deficiency. The mean value on 2 days of 24-h urinary hGH excretion for the screening of GH deficiency is estimated to be less than 9.0 ng/day. The optimal dose of GH as therapy for GH deficiency is demonstrated as daily sc injection of 0.1 IU/kg hGH, 0.7 IU/kg/week. To convert international units of met-hGH to milligrams, divide by 2.4.

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.

Urinary hGH, Albumin, α&lt;SUB&gt;1&lt;/SUB&gt; MG and β&lt;SUB&gt;2&lt;/SUB&gt; MG in Normal and Diabetic Children

I measured urinary hGH level in children with insulin dependent diabetes mellitus (DM) and studied the relation with urinary albumin alpha 1-microglobulin (alpha 1 MG), beta 2-microglobulin (beta 2MG) and plasma HbA1. 24-hour urine or night-time urine was collected in 54 normal children (NC) and 61 DM. Urinary hGH was estimated by using a sensitive sandwich enzyme immunoassay. Urinary albumin, alpha 1 MG and beta 2MG were measured by RIA. HGH concentration in 24-hour urine in NC significantly correlated with urinary albumin and beta 2MG concentrations (p less than 0.01). In DM, hGH concentration in 24-hour urine also correlated with urinary albumin concentration (p less than 0.05). To avoid the effects of posture and exercise, night-time urine was used in the following study. Urinary hGH concentration was significantly higher in DM for all age (p less than 0.05-0.01) than that in NC. Urinary alpha 1MG and beta 2MG concentrations were also significantly higher in DM than those in NC. HGH concentration in night-time urine in NC has not correlated with urinary albumin, alpha 1MG nor beta 2MG. In DM, however hGH significantly correlated with urinary albumin and beta 2MG. Even in DM with normal concentrations of urinary albumin and beta 2MG, urinary hGH was significantly higher than in NC. Urinary hGH in poorly controlled DM was higher than in well controlled DM. Urinary hGH concentration showed positive correlation with plasma HbA1 in DM. We conclude that urinary hGH relates to not only serum hGH concentration but also renal function. In DM with normal renal function, hGH concentration in night-time urine was higher than in NC, which suggested high serum hGH concentration in DM.

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.

Bioactivity and heterogeneity of human growth hormone in urine from acromegalic patients

Bioactivity of hGH in urine from five acromegalic patients was determined by Nb2 rat lymphoma bioassay (Nb2BA) and IM-9 receptor modulation assay (RMA). One urine sample (case 1) was concentrated by dialysis and lyophilization. Bioactivity by IM-9 RMA showed close correlations with immunoactivity by RIA in serial dilutions (RMA/RIA; 0.66-1.77). The estimates of bioactivity by Nb2BA were higher in all diluted samples except undiluted one than those of immunoactivity. Four samples were concentrated by affinity chromatography, dialysis and lyophilization. The ratio of RMA and RIA estimates was between 0.81-1.24, and that of Nb2BA and RIA estimates was between 0.46-2.62. Heterogeneity of urinary hGH was analyzed by gelfiltration using TSK-G-3000SW column by high performance liquid chromatography (HPLC) and by sensitive sandwich enzyme immunoassay. In addition to the main peak of 22K molecular weight (more than 97%), very small peaks of 40-60K (big hGH) and 150K (big-big hGH) were detected in chromatographic profile. Urinary hGH from acromegalic patients was bioactive in IM-9 RMA and Nb2BA. The predominant form of urinary hGH from acromegalic patients was little hGH, but big and big-big hGH were detected by sensitive sandwich enzyme immunoassay.

Measurement of urine human growth hormone levels by ultra-highly sensitive enzyme immunoassay

A highly sensitive enzyme immunoassay (EIA) for measurement of urine hGH was set up by a modification of the method of Hashida and Ishikawa, which was a sandwich enzyme immunoassay using anti-hGH antibody coated polystyrene balls and anti-hGH antibody-peroxidase conjugate. Anti-hGH serum was obtained in rabbits by subcutaneous injections of hGH emulsified in complete Freund's adjuvant. In order to reduce non-specific binding to the solid phase, anti-hGH IgG was precipitated from rabbit serum followed by digestion to F(ab')2 and affinity purification. Fab'-peroxidase conjugate was produced by maleimide method. The assay procedure was as follows. 1. 100 microliters of urine samples or hGH standard were incubated with anti-hGH IgG coated polystyrene balls. 2. Polystyrene balls were then incubated with Fab'-peroxidase conjugate. Polystyrene balls were carefully washed three times in saline after incubation with Fab'-peroxidase conjugate, which reduced contamination and non-specific binding. 3. Peroxidase activity bound to the balls was assayed by enzyme reaction using 3(p-hydroxyphenyl) propionic acid as a substrate, and fluorescence intensity was measured by a spectrofluorophotometer (Shimadzu RF-540). Reducing the energy of excitation by setting the slit width of the spectrofluorophotometer at 2nm made it possible to gain stable fluorescence. The minimum detectable quantity of hGH was 30fg/tube in the assay, so that the detection limit was 0.3 pg/ml when 100 microliters of urine samples were used. Coefficient of intra- and inter-assay variation was 6.0% and 8.6%, respectively. The recovery was 98.8 +/- 2.8 (+/- SE) on average. Multiple dilution of acromegalic urine and urine after insulin injection produced dose-response curves parallel to those of the standards. Urine hGH levels in acromegalic patients were significantly greater than those in normal subjects. These findings indicate that sensitive EIA of urine hGH is potentially useful for evaluating the pituitary function.

Level of Human Growth Hormone (hGH) in Urine Determined by A Highly Specific and Sensitive Sandwich Enzyme Immunoassay

Abstract Levels of human growth hormone (hGH) in urine samples were determined by a highly specific and sensitive sandwich enzyme immunoassay, which had little cross-reaction with human luteinizing hormone, human follicle-stimulating hormone, human thyroid-stimulating hormone and human prolactin. The volume of urine samples used was 0.05 ml. Urine hGH levels in normal subjects, aged less than 2 yr, 2-5 yr, 6-15 yr and 28-35 yr (male) were 78-113, 19-51, 7.8-25 and 1.1-5.2 ng/g of creatinine, respectively. In dwarfism patients aged 6-15 yr, urine hGH levels were < 0.2-6.0 ng/g of creatinine.

Demonstration of Human Growth Hormone in Normal Urine by a Highly Specific and Sensitive Sandwich Enzyme Immunoassay

Abstract Human growth hormone (hGH) was demonstrated in normal urine by a highly specific and sensitive sandwich enzyme immunoassay. The molecular weight of hGH in normal urine was shown to be 22,000 by gel filtration, and levels of urine hGH were 15–64 ng/1 or 50–83 ng/g of creatinine in normal subjects aged 1.2–5.9 yr.

Urinary growth hormone in man: evidence for multiple molecular forms.

Human GH (hGH) consists of several molecular forms. Monomeric forms present in pituitary extracts include the single chain, 22,000 mol wt form (22K; hGH-B); a 20,000 mol wt, single chain variant (20K); three proteolytically cleaved, two-chain forms (hGH-C, -D, and -E), acetylated, deaminated, and slow-migrating forms. It is not known which of these forms are secreted in vivo or whether peripheral organs contribute to the interconversion between some of these hGH forms. To answer these questions, we studied the molecular forms of hGH excreted in urine from normal volunteers and from an acromegalic patient, as urinary hGH presumably reflects integrated plasma hGH. hGH was extracted from urine by hollow fiber diafiltration and concentration, followed by immunoadsorbent chromatography. The extracted hGH was examined by polyacrylamide gel electrophoresis under native as well as denaturing and reducing conditions and by isoelectric focusing. The predominant form of hGH in both normal urine and urine from the acromegalic patient was 22K, with small quantities (approximately 10%) of 20K and an unidentified acidic form also present. Cleaved forms with enhanced bioactivity (hGH-D and -E) and big hGH forms were not detectable. We conclude that 1) the pattern of urinary hGH suggests that spontaneously secreted and circulating hGH is composed of at least three hGH forms, with 22K predominating; 2) the pattern of urinary hGH is similar to that of plasma hGH after L-dopa stimulation; 3) hGH excreted in acromegaly is indistinguishable from hGH excreted by normal subjects; 4) only a minute fraction (less than 0.01%) of the hGH secreted reaches the final urine; and 5) renal interconversion among hGH forms does not appear quantitatively important.