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Divisions of Endocrinology (X.-D.Q., I.T.G.G., M.Y.A.S., P.C., P.D.C., R.S.S., C.W.) and Neurosurgery (D.F.K.), Departments of Medicine and Surgery, Harbor-University of California Los Angeles (UCLA) Medical Center and Los Angeles Biomedical Research Institute, Torrance, California 90509; and David Geffen School of Medicine at UCLA (X.-D.Q., I.T.G.G., M.Y.A.S., P.C., P.D.C., R.S.S., D.F.K., C.W.), Los Angeles, California 90024
Address all correspondence and requests for reprints to: Christina Wang, M.D., General Clinical Research Center, Harbor-University of California Los Angeles (UCLA) Medical Center, 1000 West Carson Street, Torrance, California 90509. E-mail: wang{at}labiomed.org.
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Abstract |
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Introduction |
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The biochemical diagnosis of adult GHD is established by the provocative tests of GH secretion. IGF-I, although very useful in the diagnosis of GH excess, is not used for the diagnosis of GHD (3). The insulin tolerance test (ITT) is presently regarded as the gold standard, with a peak GH value of less than 3–5 µg/liter representing the diagnostic cut-off point for adult GHD (4). However, this test is less commonly used because it has to be carefully supervised due to the potential risk of induction of hypoglycemia in patients, especially those who are elderly and those who have coronary artery disease or seizure disorders. Several other diagnostic tests are currently available that are easier to administer. However, the precise diagnostic cut-off points for adult GHD remain controversial. Among these provocative tests, the combined administration of GHRH and arginine (ARG) is considered the most promising alternative and has been commonly used in the United States (4).
Many factors affect GH secretion, such as sleep, nutritional status, exercise, and stress. Obesity is known to influence the GH response to the provocative tests (5, 6), but body mass index (BMI) is not considered in the current peak GH cut-off points used in the diagnosis of GHD. Gender is another factor that is often overlooked in interpreting results of GH-provocative tests. This is unfortunate because the regulatory mechanisms of GH secretion in humans are sexually dimorphic (7), and estrogen has well-known priming effects on GH-provocative tests in children and postmenopausal women (8, 9, 10, 11, 12).
Given these unresolved controversies regarding diagnostic criteria for adult GHD, the aim of this study was to evaluate and compare the effects of BMI and gender on GH responses to the GHRH + ARG and the gold standard ITT-provocative tests in normal healthy subjects.
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Subjects and Methods |
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Healthy subjects were recruited by advertisements posted at each medical center. Separate groups of subjects received either GHRH + ARG or ITT stimulation. The GHRH + ARG group comprised 39 healthy volunteers (19 males and 20 females), ages 21–50 yr, with BMIs from 16.0 to 32.5. The ITT group comprised another 27 healthy subjects (19 males and eight females), ages 20–49 yr, with BMIs from 17.6 to 37.1. Subjects with previous diagnoses of endocrine diseases who were currently on hormone replacement therapy were excluded, as well as females who were postmenopausal or pregnant or used birth control pills for the past 3 months. Although female subjects were not studied at the same phase of the menstrual cycle, they all reported a history of regular menses. Their serum FSH, LH, and estradiol values ranged from 1–9 IU/liter, 1–17 IU/liter, and 20–150 pg/ml (SI units, 73–551 pmol/liter), respectively, and the values were not different between the two stimulation groups (P > 0.23). All male subjects had normal serum total testosterone concentrations.
Study procedures
All subjects were tested in the General Clinical Research Centers (GCRC) at either Harbor-University of California Los Angeles (UCLA) Medical Center or UCLA Center of Health Sciences between 8 and 12 h after an overnight fast.
The GHRH + ARG stimulation test was performed as follows: 1 µg/kg of GHRH was administered by an iv bolus at time 0, followed by a 30-min infusion of 30 g of ARG. Blood was drawn 30 min before (–30 min) and at the time of iv bolus of GHRH (0 min) to obtain the baseline GH values, and at 15, 30, 60, 90, and 120 min thereafter.
The ITT test was performed as follows: 0.1 U/kg of human regular insulin was administered as an iv bolus at time 0 to induce a fall in the blood glucose level to 40 mg/dl or less. An additional 0.025 U/kg of insulin was given if the glucose level did not reach the goal of equal or less than 40 mg/dl within 30 min, or if the subject did not demonstrate hypoglycemic symptoms. If the subjects did not meet these criteria of attaining clinically significant hypoglycemia, the ITT results were not used for analyses. Blood was drawn before testing (–30 min and 0 min) to obtain the baseline GH and cortisol values, and at 30, 60, 90, and 120 min after administration of the test. Experienced physicians or nurses were present throughout the procedure. The results of serum cortisol will not be discussed here.
The GHRH + ARG and ITT tests were performed along with other anterior pituitary function stimulation tests, including TRH and GnRH stimulation tests. Serum samples for GH were stored at –20 C before assay by the GCRC Core Laboratory at Harbor-UCLA Medical Center. The study had been approved by the institutional review boards of both the Harbor-UCLA Medical Center and Los Angeles Biomedical Research Institute and by the UCLA School of Medicine. All subjects gave written informed consent to participate in this study.
Hormonal assays
GH was measured by an enzymatically amplified, two-step sandwich-type immunoassay with reagents that were obtained from Diagnostic Systems Laboratory (DSL-10–1900 hGH ELISA, Webster, TX) and validated at the Core Laboratory of the Harbor-UCLA Medical Center GCRC. The lower limit of quantitation is 0.1 µg/liter. The intraassay and interassay precision is less than 8 and 14%, respectively. The recovery of GH from serum spiked with 0.1–15 µg/liter of GH was between 92 and 112%. All samples from a subject were measured in the same assay.
Statistics
Peak and area under the curve (AUC) GH responses to stimulation by GHRH + ARG and to ITT were logarithmically transformed to achieve approximate normal distributions. Geometric means of GH concentrations are reported in the text. The AUC of the GH response was calculated by the trapezoid method. Two-sample t tests were used to compare separate subject groups on GH responses. Adjustment of GH responses for subject differences in BMI was performed using analysis of covariance.
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Results |
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GH responses were significantly higher in subjects stimulated with GHRH + ARG than in subjects stimulated with ITT (Fig. 1
). The geometric mean peak GH levels were 29.0 (range, 2.8–120.8) µg/liter for the GHRH + ARG group and 15.9 (range, 2.3–52.1) µg/liter for the ITT group (P = 0.005). GH AUC responses were also higher in the GHRH + ARG group compared with the ITT group [1983 µg/liter·120 min (range, 145-8034) vs. 979 µg/liter·120 min (range, 100-3464), respectively; P = 0.002]. These differences in the provocative tests remained after adjusting for gender, age, and BMI (P = 0.005 for peak GH, and P = 0.002 for GH AUC). Because these data may be used in future studies to define GHD, the fifth (possible cut-off point for GHD) and 10th percentiles (possible cut-off point of GH insufficiency) of peak GH responses are reported in Table 1 as 5.9 and 12.0 µg/liter to GHRH + ARG and 6.0 and 7.0 µg/liter to ITT, respectively.
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GH responses to the combined GHRH + ARG and to the ITT stimulation were negatively correlated with the BMI of the subjects (Fig. 2). Subjects in the GHRH + ARG group had negative correlations between BMI and both peak GH (r = –0.76; P < 0.0001) and GH AUC (r = –0.75; P < 0.0001) that were similar to (P > 0.10) subjects in the ITT group (r = –0.65, P = 0.0002 for peak GH; and r = –0.52, P = 0.005 for GH AUC).
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, subjects with normal weight (BMI < 25 kg/m2) had higher fifth and 10th percentiles of peak GH responses to both GHRH + ARG and ITT stimulation than the subjects who were overweight (BMI 
25 kg/m2). No significant differences of BMI were observed between genders (P = 0.81) in the ITT group. It is noteworthy that the males in the GHRH + ARG group had marginally higher BMI than the females (25.8 vs. 23.6 kg/m2, respectively; P = 0.09).
Gender effects
When subjects were divided into two subgroups by gender, a difference of GH responses was noticed only in subjects stimulated with the combined GHRH + ARG (Fig. 3). As shown in Table 2, the mean peak GH level for females is 44.2 µg/liter, which was more than twice the level of 18.6 µg/liter in males (P = 0.004). Similar results are observed for the GH AUC (3023 µg/liter·120 min for females, vs. 1272 µg/liter·120 min for males; P = 0.004). No significant (P > 0.15) gender difference in GH responses in either peak GH or GH AUC occurred in subjects stimulated with ITT (Fig. 3 and Table 2).
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). After adjusting for BMI differences, the adjusted mean peak GH difference between males and females of 25.6 µg/liter was reduced to a nonsignificant (P = 0.13) difference of 15.2 µg/liter. The gender difference of GH AUC was also reduced from 1751 (P = 0.004) to 1057 (P = 0.11) µg/liter·120 min.
We used another approach to compare gender differences after GHRH + ARG stimulation while accounting for BMI differences by matching men and women based on BMI, rather than statistically adjusting for BMI differences. Thus, we compared 16 women with the highest BMIs (mean BMI = 25.0 kg/m2) with the 16 men with the lowest BMIs (mean BMI = 24.9 kg/m2). The mean BMI of these groups differed by only 0.1 kg/m2. Geometric mean peak GH responses to GHRH + ARG, unadjusted for BMI, were 37.5 for women and 24.0 µg/liter for men (female to male ratio = 1.6; P = 0.11). Geometric mean AUC GH responses to GHRH + ARG, unadjusted for BMI, were 2624 µg/liter·120 min for females and 1644 µg/liter·120 min for males, respectively (female to male ratio = 1.6; P = 0.08). These ratios are almost identical with those obtained when adjusting for BMI using analysis of covariance, with similar statistical significance (Table 2).
The fifth and 10th percentiles of peak GH responses to the combined GHRH + ARG were 13.2 and 15.9 µg/liter for females and 2.8 and 3.0 µg/liter for males, respectively. In the ITT group, fifth and 10th percentiles were 2.3 and 2.3 µg/liter for females and 6.0 and 7.0 µg/liter for males, respectively (Table 1).
There was no association between age and GH responses to either GHRH + ARG or ITT stimulation among all subjects, or for males and females separately (P > 0.20).
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Discussion |
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In the present study, we assessed the effects of BMI and gender on GH responses to two provocative tests of GH secretory reserve, ITT and GHRH + ARG, in healthy individuals. The negative correlation between BMI and GH responses was confirmed in both study cohorts, with no significant difference between the two stimuli. The female to male ratio of mean GH responses to GHRH + ARG was reduced from 2.4 to 1.6 and became not significantly different after the application of analysis of covariance to eliminate the BMI influence. No gender influence on GH responses to ITT was demonstrated. This lack of gender differences could result from insufficient sample size of female subjects in the subjects tested with ITT. Similar to other studies (14), we found that GHRH + ARG is a more potent stimulator of GH release than insulin-induced hypoglycemia. This more potent stimulation may have been because of differences in the characteristics of subjects receiving GHRH + ARG stimulation compared with subjects receiving ITT stimulation. However, differences between the two provocative tests in GH responses remained unchanged after adjusting for age, gender, and BMI, suggesting that GHRH + ARG are indeed more potent stimuli of GH secretion.
Obesity is known not only to affect basal GH secretion but also to be associated with an impairment of GH response to all stimuli. Not all of the GH-provocative tests are able to distinguish between obesity-associated perturbations (blunting of the GH response) of the GH axis and true GHD resulting from hypothalamic-pituitary disease. Cordido et al. (5) recently evaluated the diagnostic capability of four different stimuli of GH secretion: ITT, GHRH, GHRH + acipimox, and GHRH + GH-releasing peptide-6 (GHRP-6). They reported that GHRH alone was not able to distinguish organic GHD from obesity. Free fatty acid reduction by acipimox, an inhibitor of lipolysis, was able to enhance spontaneous GH secretion and GH responses to various stimuli, including the most potent GHRH + GHRP-6 (15, 16, 17). Both acipimox and GHRP-6 only partially enhance the GH responses to GHRH in obesity. Other studies have demonstrated that ARG is capable of enhancing, but not completely restoring, the blunted GHRH-induced GH response in obesity (18, 19). Markedly decreased 24-h GH secretion, including both basal rate and burst frequency, was observed in nonhypopituitary obese subjects when compared with the nonobese healthy subjects (6). Increased GH clearance may also contribute to the low GH response in obesity (6, 20). High-circulating free fatty acid concentration may be involved in the pathogenesis of obesity-associated impaired GH secretion (21, 22, 23, 24). Although obesity is associated with high plasma level of leptin, a hormone produced by the adipocytes, a recent study did not show any association of high plasma level of leptin in obesity to reduced GH secretion (25). The current study showed strong negative correlation between BMI and GH responses in both GHRH + ARG and ITT groups. Normal-weight subjects had significantly higher fifth and 10th percentiles of peak GH responses to both stimuli than overweight subjects.
Several, but not all, studies reported that baseline GH secretion in humans was different between genders, as manifested by higher GH secretory rates in women than in men (26, 27, 28). There are strong clinical data implicating the central role of estrogen in mediating the gender difference in GH secretion. Faria et al. (29) reported a positive correlation between maximal GH-pulse amplitude and serum estradiol level throughout the menstrual cycle. Short-term therapy with low-dose estrogen was shown to double the GH production rate in prepubertal girls with Turner’s syndrome (11). Estrogen supplementation in postmenopausal women has been demonstrated to increase GH secretion in numerous studies (8, 9, 10, 12). Gender differences also exist in GH responses to certain discrete physiological and pharmacological stimuli (30, 31, 32, 33, 34, 35). As early as 1969, Merimee et al. (36, 37) reported the gender difference in GH responses to ARG. They noticed that young, nonobese women had greater GH secretion after iv infusion of ARG than men of the same age. The greater GH responses occurred during the menstrual cycle, during the time of increased estrogen production. Men, after short-term exposure to estrogen, also showed elevated ARG-induced GH secretion. Recently, Wideman et al. (38) studied the gender effect of L-arginine and GHRP-2 on GH release in 18 young, nonobese subjects (nine men and nine early follicular-phase women). Women had greater peak GH (2.8-fold), GH AUC (3.3-fold), GH production rate (3.0-fold), and GH-secretory burst amplitude (2.9-fold) than men when stimulated with L-arginine. No gender differences were noticed on GH responses to GHRP-2 or GHRP-2 + L-arginine. Aimaretti et al. (14) compared 10 GH-provocative tests in 178 normal subjects (ages 20–50 yr, ±15% ideal body weight). Gender differences were only observed in subjects stimulated with ARG (15 men, 22 women) or GHRH + ARG (20 men, 28 women). Similar results of gender-dependent GH responses to ARG or GHRH + ARG were also noted by Biller et al. (13). Estrogen is likely to modulate the inhibitory effect of L-arginine on hypothalamic somatostatin and potentiates the GHRH-induced GH secretion (39). In our study, females in the GHRH + ARG group had a consistent 1.6- to 1.7-fold greater response than males, after adjusting for BMI, regardless of measure (GH peak or AUC) or of method of statistical adjustment, with marginal significance (0.08 
P 0.13). On the other hand, in the ITT group, females and males had similar GH responses. The number of female subjects in the ITT group was smaller, and thus only sensitive for detecting greater gender differences (80% power for 2.6-fold differences), but females had a lower response than males in this group, so it is doubtful that females would show a significantly greater response than males, even in a much larger study of ITT stimulation. Although all female subjects in the present study reported normal menses, the timing of testing was not standardized to a particular phase of the menstrual cycle. The baseline serum estradiol, FSH, and LH concentrations were similar in the two groups. Thus, from the current study limited by the sample size of the ITT group, we cannot determine whether gender should be considered as a contributing factor to determine GH normal cut-off points in response to stimulation by GHRH + ARG or ITT.
In conclusion, the current study demonstrates a strong negative correlation between BMI and GH responses to both ITT and GHRH + ARG stimulation. Probable gender difference of GH responses to GHRH + ARG above that explained by BMI is suggested. Our data indicate that BMI should be considered when test-specific cut-off points are used in diagnosis of adult GHD, but the data are inconclusive as to whether gender should also be considered. Although not large enough to definitively specify those cut-off points, this study suggests that the peak GH cut-off after either GHRH + ARG or ITT should be at least severalfold higher in leaner individuals compared with those who are overweight.
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Acknowledgments |
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Footnotes |
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Results from this work were presented in part at the Annual Meeting of The Endocrine Society, New Orleans, LA, June 2004.
First Published Online December 21, 2004
Abbreviations: ARG, Arginine; AUC, area under the curve; BMI, body mass index; GHD, GH deficiency; GHRP-6, GH-releasing peptide-6; ITT, insulin tolerance test.
Received July 23, 2004.
Accepted December 14, 2004.
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) in normal subjects.