Association between Haemoglobin and Growth Hormone peak in Chinese Children and Adolescents with Short Stature: A Cross-Sectional Study

Background: This research aimed to investigate the relationship between haemoglobin (Hb) and growth hormone (GH) peak in children and adolescents with short stature. Methods: This cross-sectional study included a total of 787 children and adolescents with short stature. Anthropometric and biochemical indicators were measured at baseline. All patients underwent GH provocation tests with L-dopa and insulin to assess GH peak levels. Results: The univariate analysis results showed that Hb was positively associated with GH peak (β 0.07, P=0.001). Furthermore, a non-linear relationship was detected between Hb and GH peak in multivariate piecewise linear regression analysis, and the breakpoint was 123 g/L. GH peak increased with Hb elevation when the Hb level was greater than 123 g/L (β 0.08, 95% CI 0.01, 0.14; P=0.0207). However, when the Hb level was lower than 123 g/L, there was no significant relationship between Hb and GH peak (β -0.12, 95% CI -0.30, -0.06; P = 0.1867). Conclusion: In children and adolescents with short stature, we found a non-linear association between Hb and GH peak. These findings suggest that clinicians should pay more attention to Hb levels in patients with short stature.


Introduction
Short stature refers to individuals in a similar living environment and of the same race, same sex and age who are 2 standard deviations lower than the average height of the normal population [1]. Short stature can be caused by many factors, such as endocrine, genetic, environmental, nutritional, psychological, metabolic, and clinical factors. The GH/insulin-like growth factor-1 (IGF-1) axis plays an important role during critical periods of child growth and development [2,3].
GH is secreted in pulses, with obvious fluctuations occurring during day and night.
Thus, GH levels at a single time point cannot accurately reflect GH secretion status in the body, and peak-stimulated GH after provocation testing is therefore used to determine GH status. Many studies have shown that GH level in children is closely related to the nutritional status of the body [4,5]. Nutrition plays a critical role in programming and shaping linear growth through influencing the neuroendocrine somatotropic (GH/IGF-1) axis [6].
Hb is an indicator of the nutritional status of the human body, and Hb levels are largely affected by diet [7,8]. Partial eating, picky diet, and single nutrient intake, which are detrimental eating habits common among patients with short stature, may lead to low Hb levels. In recent years, Hb has been found to be associated with linear growth [9][10][11]. Previous studies have shown that low Hb levels are associated with growth hormone deficiency (GHD) [12], but the association between Hb and GH peak after a growth hormone stimulation test is not clear. This study retrospectively explored the relationship between Hb and GH peak.

Study population
Participant data were collected from the Department of Endocrinology, Affiliated Hospital of Jining Medical University. To protect patient privacy, identifiable data were not included. This study was approved by the Human Ethics Committee of the Affiliated Hospital of Jining Medical University (Shandong, China). All procedures were performed in accordance with the ethical standards laid out in the Declaration of Helsinki. Written informed consent was obtained from each participant before data collection.
This cross-sectional study analysed data collected from a total of 787 participants between March 1, 2013 and February 28, 2019. Inclusion criteria were as follows: each participant was completely in accordance with short stature; body weight and length at birth were in the normal range; and all patients agreed to undergo two GH provocation tests with L-dopa and insulin. Exclusion criteria included patients with any of the following: thyroid dysfunction, brain tumour, Turner syndrome, congenital heart disease, chronic liver or kidney disease, malnutrition, congenital metabolic disease, chromosome abnormalities, and blood system diseases except anaemia. All tests were performed in a short-stay ward by experienced nurses according to established criteria [13].

Anthropomorphic measurements
All participants were measured with the same height-measuring instrument (Nantong Best Industrial Co. Ltd. Jiangsu, China), with an allowable error range of 0.1 cm. Participant height was measured after participants removed their hat and shoes. Height SDS was calculated based on the normal range for Chinese children [14]. Weight of all participants was measured with the same electronic scale (Xiangshan Weighing Apparatus Co. Ltd. Guangdong, China) and was accurate within ± 0.1 kg. Participant weight was measured in a fasting state. Body mass index (BMI) was calculated as weight (kilograms)/height (metres squared). The stage of puberty was assessed by physical examination according to the Tanner stages [15]. The following criteria were used to define prepuberty: for boys, a testicular volume of less than 4 mL and no pubic hair; for girls, no breast development and no pubic hair [16][17][18].

Laboratory measurements
To measure GH peak, two types of GH provocation tests were performed in sequence. On the first day, a provocation test with L-dopa was administered; participants with a body weight < 30 kg received 0.25 g orally and those with a body weight ≥ 30 kg received 0.5 g orally. Blood was collected at 0, 30, 60, 90, 120 min, and GH level was measured by radioimmunoassay. On the second day, a second GH provocation test was performed with an intravenous bolus of ordinary insulin. The insulin dose was 0.1-0.15 U/kg, and both blood glucose and GH levels were monitored at 0, 15, 30, 60, 90, and 120 min. The success rate of the test was determined by the minimum value of blood glucose lowered by 50% or ≤ 2.8 mmol/L. Before the trial, all participants and their guardians received health education, in which they were informed that the participant would undergo the tests while sedated. All participants fasted for 8 hours before the test. Both tests were performed while the participant was in a quiet state.

Statistical analysis.
All analyses were performed with the statistical software packages R (http://www.Rproject.org, The R Foundation) and EmpowerStats (http://www.empowerstats.com, X&Y Solutions, Inc, Boston, MA). Continuous variables were presented as mean ± SD and categorical variables were presented as a percentage (%). A univariate analysis model (Table 3) was used to determine the association between GH peak and Hb, as well as any association with other independent variables. We then investigated the relationship between Hb and GH peak using smooth curve fitting after adjustment for potential confounders (Fig. 1). P values less than 0.05 (two-sided) were considered statistically significant.

Baseline characteristics
Data of 787 participants were selected for the final analysis. Table 1 shows baseline characteristics of participant anthropometric measurements and biomarkers. The mean age of participants was 10.3 ± 3.8 years old. A total of 558 (70.90%) males and 229 (29.10%) females were included. The mean height SDS of participants was − 2.66 ± 0.58. GH peak value was defined as the highest level of GH, regardless of time point or provocation test. We divided GH peak levels into three groups: GH < 5 ng/mL; GH 5 ng/mL-10 ng/mL; and GH ≥ 10 ng/mL ( Table 2). BMI decreased across the tertiles (P < 0.001), but E2, T, TC, IGF-1 and Hb increased in the three groups (all P < 0.05). However, there were no obvious significant differences in age, CR, UA, or TG (all P > 0.05). Abbreviations: Height SDS, height standard deviation scores; BMI, body mass index; IGF-1, insulin-like growth factor-1; IGF-1 SDS, insulin-like growth factor-1 standard deviation scores; IGFBP-3, insulin-like growth factor-binding protein-3; Hb, haemoglobin; CR, creatinine; BUN, blood urea nitrogen; UA, uric acid; TG, triglyceride; TC, total cholesterol. Correlations between GH peak and anthropometrical and biochemical 8 variables The univariate analysis results of GH peak and all tested variables are shown in were positively correlated with GH peak. There was no significant correlation between GH peak and sex, age, height SDS, CR, or UA. Table 3 Correlation between GH peak level and different variables

Non-linear relationship between Hb and GH peak
In the present study, smooth curve fitting showed a non-linear relationship between Hb and GH peak after adjusting for age, sex, height SDS, BMI SDS, UA, TC, TG, CR, E2, T, and Tanner stage (Fig. 1). There was a two-stage change and one inflection point in this curve. When the Hb level was greater than the breakpoint, there was a positive relationship between Hb and GH peak; however, if the value was less than the breakpoint, there was a negative relationship between Hb and GH peak. Next, threshold saturation based on the curve fitting was analysed (Table 4) however, there was no statistical significance. On the right side of the inflection point, GH peak increased with Hb elevation (β 0.08; 95% CI 0.01, 0.14; P = 0.0207). Table 4 The independent correlation between Hb and GH peak by multivariate piecewise linear regression.  Declarations standards laid out in the Declaration of Helsinki. All of the families of the patients were informed of the aims of the study, and written informed consent was obtained from the parents of the patients.

Consent for publication
All authors have read and approved the content, and they agree to submit it for consideration for publication in the journal.

Figure 1
Smooth curve fitting showed a non-linear relationship between Hb and GH peak after adjustin Figure 1 Smooth curve fitting showed a non-linear relationship between Hb and GH peak after adjustin Declarations authors upon reasonable request.

Figure 1
Smooth curve fitting showed a non-linear relationship between Hb and GH peak after adjusting for Figure 1 Smooth curve fitting showed a non-linear relationship between Hb and GH peak after adjusting for