Urine metabonomic profiling of a female adolescent with PIT-1 mutation before and during growth hormone therapy: insights into the metabolic effects of growth hormone.

OBJECTIVE: Growth hormone (GH) is a protein hormone with important roles in growth and metabolism. The objective of this study was to investigate the metabolism of a human subject with severe GH deficiency (GHD) due to a PIT-1 gene mutation and the metabolic effects of GH therapy using Nuclear Magnetic Resonance (NMR)-based metabonomics. NMR-based metabonomics is a platform that allows the metabolic profile of biological fluids such as urine to be recorded, and any alterations in the profile modulated by GH can potentially be detected. DESIGN: Urine samples were collected from a female subject with severe GHD before, during and after GH therapy, and from healthy age- and sex-matched controls and analysed with NMR-based metabonomics. SETTING: The samples were collected at a hospital and the study was performed at a research facility. PARTICIPANTS: We studied a 17 year old female adolescent with severe GHD secondary to PIT-1 gene mutation who had reached final adult height and who had ceased GH therapy for over 3 years. The subject was subsequently followed for 5 years with and without GH therapy. Twelve healthy age-matched female subjects acted as control subjects. INTERVENTION: The GH-deficient subject re-commenced GH therapy at a dose of 1 mg/day to normalise serum IGF-1 levels. MAIN OUTCOME MEASURES: Urine metabolic profiles were recorded using NMR spectroscopy and analysed with multivariate statistics to distinguish the profiles at different time points and identify significant metabolites affected by GH therapy. RESULTS: NMR-based metabonomics revealed that the metabolic profile of the GH-deficient subject altered with GH therapy and that her profile was different from healthy controls before, and during withdrawal of GH therapy. CONCLUSION: This study illustrates the potential use of NMR-based metabonomics for monitoring the effects of GH therapy on metabolism by profiling the urine of GH-deficient subjects. Further controlled studies in larger numbers of GH-deficient subjects are required to determine the clinical benefits of NMR-based metabonomics in subjects receiving GH therapy.

Effects of growth hormone deficiency and rhGH replacement therapy on the 6beta-hydroxycortisol/free cortisol ratio, a marker of CYP3A activity, in growth hormone-deficient children.

OBJECTIVE: To determine the effect of both growth hormone deficiency (GHD) and rhGH replacement therapy on CYP3A activity as well as the potential influence of gender. METHODS: The sample consisted of 35 GHD children (16 males and 19 females), aged 2.9-13.1 years, and a control group of 35 healthy children matched for age and sex. The urinary ratio 6beta-hydroxycortisol/free cortisol was used as a marker of CYP3A activity. In patients, urine samples were collected at two times, prior to starting rhGH replacement treatment and 30 days after beginning therapy. RESULTS: A significantly higher metabolic activity in GHD children was observed in relation to controls ( P=0.0001) without sex differences. Paired comparisons demonstrated a sexually dimorphic effect of rhGH therapy on the CYP3A activity. While boys displayed a significant decrease ( P=0.003), girls showed no significantly different values of CYP3A marker ( P>0.05). Unpaired comparison between controls and GHD children after therapy demonstrated absence of significant differences in boys ( P>0.05) and a strikingly higher activity in girls ( P=0.0001). CONCLUSIONS: The data suggests that: (a) GHD in children increases CYP3A activity in a non-sex-dependent manner, (b) rhGH treatment for 30 days to GHD children results in a sexually dimorphic effect on CYP3A activity, with a significant decrease in males toward normalization in relation to controls and non-significant changes in females. The results of this study may have important clinical implications for GHD children, since changes in CYP3A activity importantly affect the metabolism of both steroid hormones and CYP3A substrate drugs.

Correlation of urinary albumin and beta-2-microglobulin and growth hormone excretion in patients with diabetes mellitus and short stature.

We examined the correlation between urinary GH, urinary albumin, and beta-2-microglobulin excretion to determine how the excretion of GH relates to markers of renal glomerular and tubular function. Urinary albumin and GH excretion was determined in timed daytime and nighttime urine collections obtained from both subjects with diabetes mellitus and subjects with short stature. For subjects with diabetes, urinary GH excretion rate correlated highly with urinary albumin concentration and excretion rate in both the range of 0 to 1.6 g/L (r = 0.75), P less than 0.001) and in the microalbuminuria range, 0 to 0.4 g/L (r = 0.53, P less than 0.001). Changes in GH and albumin excretion occurred in parallel in 71% of the subjects with diabetes and elevated albumin excretion. The mean GH excretion rate was higher in the group with elevated albumin excretion rate (AER) during both day and night compared to the group with microalbuminuria during the day and normal AER at night. For subjects with short stature, the mean albumin excretion rate was 0.7 +/- 1.3 micrograms/min (range 0.05-8.3 micrograms/min) using a sensitive enzyme-linked immunosorbent assay to measure albumin concentration. The correlation of GH and albumin excretion rates for the subjects with short stature was not statistically significant (r = 0.14, P less than 0.5). About half of the subjects with diabetes and elevated AER (greater than 10 micrograms/min) had a GH excretion rate within the range observed in subjects with short stature. The GH and albumin excretion rate were not correlated in this group. There was a positive correlation of both albumin and GH excretion rate with age in the subjects with diabetes. Urinary GH and beta-2-microglobulin excretion rates were determined in a larger group of subjects with diabetes and a separate group with short stature. Urinary GH and beta-2-microglobulin excretion were correlated both in subjects with diabetes (r = 0.46, P less than 0.001) and with short stature (r = 0.64, P less than 0.001). The association was present in urine collected either during the day or night. The mean GH excretion rate of the group with diabetes was greater than the group with short stature. In conclusion, there was an association of urinary GH and albumin excretion rate in subjects with abnormal glomerular function as indicated by elevated albumin excretion rate. An association of urinary GH and beta-2-microglobulin excretion was observed in subjects with normal tubular function.(ABSTRACT TRUNCATED AT 400 WORDS)

A highly sensitive sandwich enzyme immunoassay of urinary growth hormone in children with short stature, Turner's syndrome, and simple obesity.

GH values were determined by a highly sensitive sandwich enzyme immunoassay in the 1st morning and/or 24-h accumulated urine samples in 94 children (short stature 70, including 14 with complete GH deficiency, 9 with partial GH deficiency, and 47 with GH-normal short stature; Turner's syndrome, 10, and simple obesity, 14). GH values were also determined in the 2nd to 4th urine samples taken on the same day together with the 1st morning urine in 5 of them. GH values in the 1st morning urine correlated significantly with those of the 24-h urine and with serum peak and mean GH values during nocturnal sleep as a physiological GH secretion test. The 2nd to 4th urines had lower GH concentrations than the 1st morning urine. The GH value of the 1st morning urine in complete GH deficiency was significantly lower than those in GH-normal short stature, partial GH deficiency and Turner's syndrome. However, no significant difference was detected in urinary GH values between complete GH deficiency and simple obesity. We conclude that 1st morning urinary GH estimation may be useful for differentiation of complete GH deficiency from other causes of short stature, but may be difficult for the distinction between complete GH-deficiency and obesity with normal GH secretory ability.

Thyroid hormone-induced reduction of urinary 5-hydroxyindole acetic acid (5 HIAA) in obese children. Comparison with hypothyroid patients of similar age having either pituitary dwarfism or congenital myxedema.

Urinary excretion of monoamine metabolites (noradrenaline-NA, adrenaline-A, 3-methoxy-4-hydroxyphenyl glycol-MHPG, homovanillic acid-HVA, 5-hydroxyindole acetic acid-5 HIAA) was studied in four groups of children as follows: Group I consisting of obese children subjected to caloric restriction and to a short term course of thyroid extract in "low" dosage (1-2 mg/kg bwt), Group II consisting of obese children subjected to diet alone, Group III consisting of children myxedema and subjected to a short term course of thyroid extract given in the "high" dosage (3-5 mg/kg bwt) and Group IV consisting of GH deficient short children having (many of them) thyrotropin deficiency and subjected to a short term course of thyroid extract in "very high" dosage (5-10 mg/bwt). In obese, calorie-restricted children, the previously low mean level of 5 HIAA excretion was further lowered by thyroid extract. In obese children subjected to calorie restriction alone no urinary abnormality was noted. The congenitally hypothyroid patients had low levels of basal 5 HIAA when compared to controls. The degrees of 5-hydroxy tryptamine (5 HT) deficiency in Group III was similar to the obese groups. The thyroid extract course did not influence, at least in short term administration, the low 5 HIAA levels in group III. In GH deficient, short children (group IV) thyroid extract had no significant effect on urinary pattern of monoamine metabolites. A central 5 HT deficiency may tentatively explain the mood disturbances and possibly the other psychic disorders in both the obese and myxedematous patients. The different effects of thyroid extract on 5 HIAA may also witness the differences in the food intake behaviour in these two conditions.

The assessment of the dopaminergic tonus by urinary determinations of homovanillic acid (HVA) and dihydroxyphenylacetic acid (DOPAC) in normal, obese and GH-deficient short children.

In order to assess the dopaminergic tonus, urinary determinations of HVA and DOPAC and also of noradrenaline (NA), adrenaline (A), and methoxyhydroxyphenylglycol (MHPG) were performed in 86 obese children, 11 growth hormone (GH)-deficient short children and also in 40 control children. Part of the obese patients were subjected to a low carbohydrate, low calorie diet and also to short-term (9-14 days) courses of diethylpropion (DEP) 50 mg/day, meclofenoxate (MEC) 100 mg/day and thyroid extract (THE) 1-2 mg/kg/day. The GH-deficient patients received only THE in substitutive (5-10 mg/kg/day) doses. Significative correlations between DOPAC and age, weight and height were found in controls. In the obese group a significantly increased mean level of HVA was found (1.45 +/- 0.09 mg/24 h vs 1.15 +/- 0.10 in controls). The excretion of DOPAC was slightly greater but far from significance. There was also a significant decrease of HVA but not DOPAC in the DEP-treated obese. The rest of the drugs and the diet alone were not effective in any way. Normal levels in all metabolites except NA and A were found in GH-deficient short children. The therapy with thyroid extract did not alter the excretion levels. These findings indicate that in infantile obesity the dopaminergic tonus is somewhat increased but its pathophysiological significance is doubtful. In GH-deficient short children of standard appearance the dopaminergic tonus seems to be undistinguishable from normal.

Immunoreactive somatomedin C/insulin-like growth factor I in urine from normal subjects, pituitary dwarfs, and acromegalics.

Using antibodies to somatomedin C/insulin-like growth factor I (SmC) produced in rabbits using the recombinant hormone, we have developed a radioimmunoassay for SmC. Gel-chromatography of urine revealed that the vast majority of immunoreactive SmC was eluted coincident with 125I-SmC and a small portion eluted with fractions having a mol. wt. range of 30,000-40,000. The SmC concentration in urine was determined by radioimmunoassay after ammonium sulfate extraction. Values did not ordinarily exceed 1 ng/ml. When the values from normal subjects were expressed as ng/mg creatinine, high levels were observed in the neonatal period. These values fell rapidly in infancy, declined more gradually in childhood, were slightly elevated at early puberty, and were lowest in adulthood. Urine SmC concentrations in 15 pituitary dwarfs were lower than the averages obtained from agematched control subjects, and six of them showed abnormally low values. Three patients with active acromegaly had high SmC values in urine. In conclusion, 1) SmC, mainly of monomeric form, was immunologically detected in urine. 2) Radioimmunoassay for urine SmC revealed that values varied considerably with age in normal subjects and were partially dependent on the human growth hormone status. However, the full meaning of the findings remains to be elucidated.