Severe Adult Growth Hormone Deficiency Research Articles

Absorption, metabolism and excretion of once-weekly somapacitan, a long-acting growth hormone derivative, after single subcutaneous dosing in human subjects

Somapacitan is a reversible albumin-binding growth hormone (GH) derivative in clinical development for once-weekly administration in patients with adult GH deficiency (AGHD) and children with GH deficiency (GHD). To date, the use of somapacitan in AGHD or severe AGHD has been approved in the USA and Japan, respectively. This study (ClinicalTrials.gov, NCT02962440) investigated the absorption, metabolism and excretion, as well as the pharmacokinetics (PK), of tritium-labelled somapacitan ([3H]-somapacitan). Seven healthy males received a single subcutaneous dose of 6 mg somapacitan containing [3H]-somapacitan 20 MBq. Blood, serum, plasma, urine, faeces, and expired air were collected for radioactivity assessment. Metabolites were identified and quantified in plasma and urine collected. The PK of plasma components were determined, and the radioactive peaks of the most abundant plasma metabolites and urine metabolites were selected for analysis. Twenty-eight days after dosing, 94.0% of the administered dose was recovered as [3H]-somapacitan-related material, most of which was excreted in urine (80.9%); 12.9% was excreted in faeces, and an insignificant amount (0.2%) was exhaled in expired air. PK properties of [3H]-somapacitan-related material appeared to be consistent across plasma, serum and blood. Three abundant plasma metabolites (P1, M1 and M1B) and two abundant urine metabolites (M4 and M5) were identified. The total exposure of intact somapacitan accounted for 59% of the total exposure of all somapacitan-related material, P1 accounted for 21% and M1 plus M1B accounted for 12%. M4 and M5 were the most abundant urine metabolites and accounted for 37% and 8% of the dosed [3H]-somapacitan radioactivity, respectively. No intact somapacitan was found in excreta. Two subjects had six adverse events (AEs); all were mild in severity and unlikely to be related to trial product. The majority of dosed [3H]-somapacitan (94%) was recovered as excreted metabolites. Urine was the major route for excretion of somapacitan metabolites, followed by faeces, and exhalation in expired air was negligible. The low molecular weights of identified urine metabolites demonstrate that somapacitan was extensively degraded to small residual fragments that were excreted (fully biodegradable). The extensive metabolic degradation and full elimination of metabolites in excreta were the major clearance pathways of somapacitan and the key elements in its biological fate. A single dose of 6 mg somapacitan (containing [3H]-somapacitan) in healthy male subjects was well tolerated with no unexpected safety issues identified.

SUN-310 Growth Hormone Deficiency and Replacement Therapy: Association with Health-Related Physical Fitness

Objective: To compare health-related physical fitness (HRPF) in patients with severe adult growth hormone deficiency (AGHD) according to the deficiency onset phase, and to evaluate the effects of a six-months human growth hormone (rhGH) replacement therapy on HRPF, in a subgroup of patients. Methods: First arm: cross-sectional observational study at baseline of naive rhGH multiple pituitary hormonal deficiency (MPHD) hypopituitarism patients - adult-onset growth hormone deficiency (AO-GHD) versus child onset growth hormone deficiency (CO-GHD). Second arm: a 6-month intervention clinical trial in a selected group of a non-randomized, non-controlled cohort. HRPF was evaluated by measuring isokinetic and isometric torque stensor strength at the knee using an isokinetic dynamometer, handgrip strength and six-minute walk test. Body composition was assessed by DXA. Results: Patients who presented AO-GHD had higher BMI than CO-GHD (28.1±3.5 x 22.4±4.8; p=0.017), but body composition (lean body mass%:57.9±7.9 x 58.9±8.6;p=0.816/fatty body mass%:39.3±6.8 x 36.0±9.1;p=0.434), stensor peak torque/body weight at 60, 90 and 180deg/s (2.18±0.6 x 2.18±0.6; p=0.580/1.99±0.5 x 2.14±0.5;p=0.546/1.52±0.4 x 1.64±0.4;p=0.547), isometric torque/body weight at the knee (2.62±0.7 x 2.91±0.6;p=0.357) and six-minute walk test (570.2±76.0cm x 554.1±91.0cm;p= 0.703) did not differ between groups. Handgrip strength test also showed significant reduction in scores for age and gender in both groups of GHD patients. After six months of rhGH, no improvement in muscular strength tests was found. There was a significant worsening in the six-minute walk test (575.1±84cm x 545.4±90.6cm; p=0.033) despite the improvement in body composition (lean body mass%:59.7±8.6 x 63.6±11.1;p=0.005/fatty body mass%:35.7±9.2 x 32.9±10.0;p=0.003). Conclusion: Despite differences in BMI, there were no other differences in HRPF between AO-GHD and CO-GHD patients. The decrease of the six-minute walking test performance after rhGH replacement therapy supports the clinical evidence that the GH regulates bioenergetics in human skeletal muscle fibers. Although the treatment had a short period, GH might have stimulated the anaerobic and suppressed the aerobic energy system.

Preoperative growth hormone (GH) peak values during a GH releasing peptide-2 test reflect the severity of hypopituitarism and the postoperative recovery of GH secretion in patients with non-functioning pituitary adenomas.

Non-functioning pituitary adenoma (NFPA) is one common cause of adult growth hormone deficiency (AGHD). In Japan, a GH-releasing peptide (GHRP)-2 test is used to evaluate GH secretion. Although the cut-off for peak GH during a GHRP-2 test for severe AGHD is ≤9 ng/mL, severe AGHD may further diminish responses (range, nearly no-response to ≤9 ng/mL). We studied whether the peak GH responses during a GHRP-2 test could be predicted based on clinical characteristics of patients with NFPA. We compared patients with almost no-response during a GHRP-2 test with other patients considered as severe AGHD. Among the 76 patients with NFPA who were admitted to our institution, 36 patients (mean age, 61 years; male/female, n = 23/n = 13) were diagnosed with severe AGHD based on a preoperative GHRP-2 test. Based on the preoperative median peak GH concentration (2.83 ng/mL), patients were divided into two groups (<median = low or group L, n = 18; ≥median = moderate or group M, n = 18). Clinical manifestations, body mass index, severity of hypopituitarism and tumor size, volume, and extension were analyzed retrospectively. Compared with group M, group L patients were significantly older and more gonadotropin and ACTH deficient. A lower peak GH release during a GHRP-2 test was associated with a higher number of anterior pituitary hormone deficiencies across all 76 patients. Postoperatively, seven in group M and no patient in group L were assessed as having no longer severe AGHD, respectively. Preoperative peak GH concentrations assessed during a GHRP-2 test reflected the severity of hypopituitarism and the recovery of postoperative GH secretion.

Transient Elastography and Controlled Attenuation Parameter (CAP) in the Assessment of Liver Steatosis in Severe Adult Growth Hormone Deficiency.

Non-alcoholic fatty liver disease (NAFLD) is common in patients with growth hormone deficiency (GHD). Some noninvasive techniques have been used to quantify liver fat, such as the controlled attenuation parameter (CAP).Objective: To evaluate CAP as a tool to identify liver steatosis and its relationship with different clinical and biochemical metabolic parameters in a group of patients with severe adult growth hormone deficiency (AGHD), and to compare the evolution of metabolic profiles after 6 months of human growth hormone (rhGH) replacement therapy in a subgroup of patients.Methods: Cross-sectional observational study at baseline of naive rhGH multiple pituitary hormonal deficiency (MPHD) hypopituitarism patients. A 6-month intervention clinical trial in a selected group of a non-randomized, non-controlled cohort was also applied.Results: Liver stiffness measurement (LSM) was normal in severe AGHD patients. CAP evaluation showed steatosis in 36.3% of baseline patients (8/22), associated with higher BMI, waist circumference, insulin, and alanine aminotransferase (ALT) levels. According to steatosis degree by CAP, child-onset growth hormone deficiency (CO-GHD) was graded as 68.75% (11/16) S0, 12.5% (2/16) S1, and 18.75% (3/16) S3, whereas AO-GHD was graded as 50% (3/6) S0, 16.66% (1/6) S2, and 33.33% S3. After 6 months of hrGH replacement, CAP measurements did not change significantly, neither on group without hepatic steatosis at baseline (194.4 ± 24.3 vs. 215.4 ± 51.3; p = 0.267) nor on the group with hepatic steatosis (297.2 ± 32.3 vs. 276.4 ± 27.8; p = 0.082). A significant improvement of body composition was observed only in the first group.Conclusions: We have demonstrated the importance of CAP as a non-invasive tool in the liver steatosis identification on hypopituitary patients. This method may be an important indicator of the severity of metabolic disorders in MPHD patients. In our study, no liver health modification in LSM at baseline or after 6 months of rhGH replacement was found. Longer studies can help to establish the potential repercussions of growth hormone replacement therapy on liver steatosis.

Effects of Growth Hormone Replacement on Peripheral Muscle and Exercise Capacity in Severe Growth Hormone Deficiency.

The aim of this study is to evaluate the effect of growth hormone therapy (rGH) on mitochondrial function on peripheral muscle and to correlate with exercise capacity in subjects with severe adult growth hormone deficiency (GHD). Six months, double-blind, randomized, crossover, placebo-controlled trial of subcutaneous rGH in 17 patients with GHD. Quadriceps muscle biopsies were obtained at baseline, 3 months, and 6 months to measure succinate dehydrogenase (SDH) to assess mitochondrial activity. Exercise capacity was measured with cardiopulmonary exercise testing. Lipids, glycemic parameters, and body fat levels were also measured. Serum insulin-like growth factor 1 (IGF1) levels reduced fat mass by 3.2% (p < 0.05) and normalized with rGH in the active phase (p < 0.005). Patients showed an increase in SDH (p < 0.01) from base line that differed between placebo and rGH therapy treatment groups (p < 0.05): those treated by rGH followed by placebo showed a significant increase in SDH (p < 0.001) followed by a decrease, with a significant between group difference at the end of 6 months (p < 0.05). No significant improvements or correlation with exercise capacity was found. Short-term rGH for 3 months normalized IGF1 levels, reduced fat mass, and had a significant effect on mitochondrial function, but exercise capacity was unchanged. Number ISRCTN94165486.

Effects of growth hormone on thyroid function are mediated by type 2 iodothyronine deiodinase in humans.

Growth hormone (GH) therapy in adults alters thyroid function, and acromegaly often involves thyroid disease. The present study aimed to elucidate roles and mechanisms of GH in regulating thyroid function. We performed two retrospective observational studies, which focused on consecutive patients with severe adult GH deficiency who received recombinant human GH (rhGH) therapy (n = 20) and consecutive patients with acromegaly who underwent transsphenoidal surgery (TSS) (n = 25). In both studies, serum free triiodothyronine (fT3), free thyroxine (fT4), and fT3/fT4 ratio were examined before and after the interventions. We subsequently administered GH to four human cell lines (HepG2, TSA201, MCF7, and HTC/C3) in vitro, and examined changes in mRNA levels of iodothyronine deiodinases (D1, D2, and D3). Median serum fT3 level significantly increased after rhGH therapy from 2.38 to 2.78 pg/mL (p < 0.001), and fT4 decreased from 1.115 to 1.065 ng/dL (p = 0.081). TSS significantly decreased median serum fT3 from 3.03 to 2.53 pg/mL (p < 0.001), and increased fT4 from 1.230 to 1.370 ng/dL (p < 0.001). In vitro, GH significantly increased D2 expression at the mRNA level in HTC/C3 cells (p < 0.01), as well as D2 protein and its activity. GH increased serum fT3 level and decreased serum fT4 level in humans. Our results suggest that its mechanism involves D2 upregulation. Considering this GH effect on thyroid hormone metabolism, data on thyroid function could be useful in the management of GH deficiency and acromegaly.

Assessment of quality of life on 4-year growth hormone therapy in Japanese patients with adult growth hormone deficiency: A post-marketing, multicenter, observational study

ObjectiveImprovement of quality of life (QOL) by growth hormone (GH) therapy was not demonstrated in Japanese adult growth hormone deficiency (AGHD) patients by either the QOL Assessment of Growth Hormone Deficiency in Adults or the Questions on Life Satisfaction-Hypopituitarism, which are widely used to evaluate QOL in Western AGHD patients. We therefore evaluated QOL in Japanese AGHD patients receiving recombinant GH, Norditropin® (Novo Nordisk A/S, Denmark), using the newly developed Adult Hypopituitarism Questionnaire (AHQ). DesignThis multicenter, non-interventional, observational study in Japanese patients with severe AGHD was conducted from 1 October 2009 to 30 September 2014. Patients with severe AGHD already receiving somatropin and somatropin-naïve patients were included. GH therapy (Norditropin®) was initiated as injections of 0.021mg/kg/week divided into 6–7 doses/week, and was adjusted according to clinical responses. Demographic/clinical data were obtained from medical records or by patient recall. QOL was assessed using the AHQ at baseline; 3, 6, and 12months; and annually up to 4years. ResultsOf 387 registered patients, 161 were eligible for QOL analysis. AHQ scores significantly improved after 3months of treatment. Improvements in the psycho-social and physical domains were statistically significant throughout the 4-year study period. Although the GH dose was increased in females such that insulin-like growth factor-1 levels reached those of males, QOL improvements in females did not reach those of males. Despite the greater GH dose in child-onset patients, limited QOL improvements were observed in child-onset vs adult-onset cases. ConclusionsFour-year GH treatment in Japanese AGHD patients elicits sustained improvement in QOL as assessed by AHQ scores.

Adult Growth Hormone Deficiency: Current Concepts

The clinical syndrome of adult growth hormone deficiency (AGHD) was widely recognized in the 1980s. In this review, we first describe the clinical features and diagnosis of AGHD and then state the effects of growth hormone (GH) therapy for these patients. The main characteristics of AGHD are abnormal body composition, dyslipidemia, insulin resistance, and an impaired quality of life (QoL) due to decreased psychological well-being. For diagnosing AGHD, the international consensus guidelines have suggested that an insulin tolerance test (ITT) is the gold standard, but in Japan, the growth hormone releasing peptide-2 (GHRP-2) test is available and is recommended as a convenient and safe GH stimulating test. The cut-off for diagnosing severe AGHD is a peak GH concentration of 9 g/L during the GHRP-2 test. Since 2006, GH therapy has been approved for Japanese patients with severe AGHD. For adults, GH replacement therapy should be initiated at a low dose (3 g/kg body weight/day), followed by individualized dose titration while monitoring patients' clinical status and serum insulin-like growth factor-I (IGF-I) concentrations. A variety of favorable effects of GH replacement have been indicated; however, it has not yet been established fully whether there is a direct effect of GH treatment on reducing mortality.

Growth hormone replacement in adults with severe growth hormone deficiency is effective even if baseline IGF-1 levels are in the normal range

Replacement of human growth hormone (GH) in adults with severe adult growth hormone deficiency (GHD) is associated with improvement in quality of life, body composition, bone mineral density and lipid profile. IGF-1 levels below the normal range are used as a screening tool to identify patients with GHD and it is recommended to titrate the GH dose to achieve normal IGF-1 levels. However, IGF-1 levels in the normal range do not exclude severe GHD. Up to now it is not clear whether patients with normal IGF-1 levels prior to replacement therapy will benefit from GH therapy. Therefore we analysed the KIMS international database and identified 575 patients with IGF-1 levels in the normal range (-2 SDS to +2 SDS) before treatment (Group NIB) and compared those with 271 patients with low IGF-1 levels (< -2 SDS) at baseline (Group LIB). There was no significant difference regarding gender distribution in the two groups. The percentage of childhood onset of GHD was significantly higher in the LIB group (35% vs. 4%). The medium age of entering into KIMS was 38,8 years in the LIB group versus 49,0 years in the NIB group. Baseline medium height was significantly lower in the LIB group (167.7cm vs. 170.7cm) while there were no significant differences regarding BMI and waist to hip ratio. The GH starting dose did not differ between the groups, however after one year the medium dose in the NIB group was significantly lower (0.3mg/d vs. 0.42mg/d). Disease specific quality of life measured by Quality of Life-Assessment of Growth Hormone Deficiency in Adults (QoL-AGHDA) was worse in the NIB group (13 vs. 10.25). After one year of treatment QoL-AGHDA Score improved in both groups, however the improvement in the NIB group was even higher (-4.41 vs. -3.52). The results of this analysis show that GH replacement therapy is effective even if IGF-1 levels prior to treatment are in the normal range.

Impact of growth hormone (GH) treatment on circulating Nt-proBNP concentrations and on cardiac function in adult GH-deficient patients

Severe adult growth hormone deficiency (AGHD) is associated with increased cardiovascular risk. We have therefore investigated levels of amino terminal pro-brain natriuretic peptide (Nt-proBNP), a well established biomarker for cardiac failure, in adult GHD patients before and after GH replacement therapy, and potential parallel variations in cardiac function. Nt-proBNP concentrations were determined at baseline and after GH treatment in two studies including 28 and 12 patients with severe AGHD, respectively. In the second study, a maximal exercise test and a doppler echocardiography were performed to assess cardiac functional parameters. At baseline, Nt-proBNP levels were higher in AGHD patients than in controls (median: 7.8 vs. 3.7 pmol/L; p < 0.01 in study 1; 8.4 vs. 4.1 pmol/L; p < 0.01 in study 2). Following GH treatment, Nt-proBNP levels decreased significantly in both studies. None of the AGHD patients had signs of cardiac dysfunction at baseline and no significant effect of GH replacement therapy was observed on cardiac functional parameters, independent of changes in Nt-proBNP. In conclusion, GH treatment markedly reduces Nt-proBNP concentrations in adult GHD patients without obvious parallel changes in cardiac functional parameters. These results suggest that Nt-proBNP may appear as a biomarker of GH status and GH treatment efficiency.

Similar Clinical Features Among Patients with Severe Adult Growth Hormone Deficiency Diagnosed with Insulin Tolerance Test or Arginine or Glucagon Stimulation Tests

To determine whether insulin tolerance tests (ITTs), arginine stimulation tests (ASTs), and glucagon stimulation tests (GST) identify patients who have similar clinical features of growth hormone (GH) deficiency when a diagnostic GH threshold of 3 μg/L is used. Data were obtained from the KIMS database (Pfizer International Metabolic Database). Comparisons were made between patients who underwent ITT, AST, or GST for GH peak, body mass index, lipids, waist circumference, waist-to-hip ratio, and quality of life. A total of 5453 tests were available from 4867 patients registered in the database (ITT = 3111, AST = 1390, GST = 952). Significant (P<.001) intraindividual correlations were observed between the GH peaks for ITT vs AST (r = 0.655), ITT vs GST (r = 0.445), and AST vs GST (r = 0.632). GH peaks in response to all tests were negatively correlated to the number of additional pituitary hormone deficiencies and positively correlated to the insulinlike growth factor 1 standard deviation score. Body mass index had a negative influence on all 3 tests. Most clinical variables did not differ between the groups when comparing GH-deficient patients according to the diagnostic test used. The only exceptions that showed any difference were body mass index (slightly higher in the AST and GST groups), triglyceride levels (increased in the GST group), and insulinlike growth factor 1 (standard deviation score) (lower in the ITT and AST groups than in the GST group). Waist circumference was greater and quality of life was worse in the GST group than in the other groups. The ITT, AST, and GST produce similar GH peaks, are influenced by similar clinical factors, and identify patients with similar features of GH deficiency at a diagnostic threshold of 3 μg/L.

Effects of GH replacement on 24-h ambulatory blood pressure and its circadian rhythm in adult GH deficiency.

Increased prevalence of hypertension and cardiovascular mortality have been reported in hypopituitary patients who had been appropriately replaced with conventional pituitary hormones except GH. Growth hormone replacement (GHR) results in improvement of surrogate markers of cardiovascular function. Data on effects of GHR on blood pressure (BP) in adult growth hormone deficiency (AGHD), however, remain contradictory. There are as yet no reports on BP circadian rhythms in untreated or treated AGHD. Therefore, in a 12-month follow-up study, we evaluated the effects of GHR on ambulatory blood pressure (ABP) in AGHD patients. A prospective, open treatment design study to determine the effects of GHR on ABP and heart rate in AGHD patients. GH was commenced at a daily dose of 0.5 IU, and titrated up by increments of 0.25 IU at 4-weekly intervals to achieve and maintain IGF-I standard deviation score (IGF-I SD) between the median and upper end of the age-related reference range. Twenty-two, post-pituitary surgery, severe AGHD patients (11 men), defined as peak GH response < 9 mU/l to provocative testing were recruited. The mean age +/- SEM was 48.8 +/- 2.5 years. Twenty-one patients required additional pituitary replacement hormones following pituitary surgery and were on optimal doses at recruitment. Twenty-four-hour ABP and heart rate (HR), body mass index (BMI), waist hip ratio (WHR) and total body water (TBW) were measured before and after 12 months on GHR. Cosinor analysis was used to analyse BP and HR circadian rhythm parameter estimates. Target IGF-I SD was achieved within 3 months of commencement of GHR in all patients (-3.5 +/- 0.4 at baseline vs. 0.8 +/- 0.2 at 3 months, P < 0.001) and remained within range at 12 months (1.1 +/- 0.2, P < 0.001 compared to baseline). A significant increase in TBW (45.8 +/- 1.2 vs. 47.8 +/- 1.5 kg, P < 0.05) but no significant change in BMI (30.7 +/- 2.2 vs. 31.8 +/- 2.7, P = NS) or WHR (0.95 +/- 0.02 vs. 0.93 +/- 0.02, P = NS) was observed after 12 months on GHR. The 24-h mean systolic ABP (SBP; 126.2 +/- 2.8 vs. 120.1 +/- 2.7 mmHg, P < 0.001) and diastolic ABP (DBP; 78.2 +/- 1.6 vs. 71.4 +/- 1.8 mmHg, P < 0.001) significantly decreased following GHR with a parallel increase in 24-h mean HR (69.6 +/- 2.5 vs. 73.8 +/- 2.5 beats/min; P < 0.001). A significant nocturnal decrease in SBP and DBP was observed both before (SBP; daytime, 129.1 +/- 2.8 vs. night time, 115.9 +/- 3.0 mmHg, P < 0.001 and DBP; daytime, 80.7 +/- 1.6 vs. night time, 69.2 +/- 1.8 mmHg, P < 0.001) and following GHR (SBP; daytime, 122.8 +/- 2.6 vs. night time, 110.0 +/- 3.6 mmHg, P < 0.001 and DBP; daytime, 73.9 +/- 1.8 vs. night time, 62.0 +/- 2.3 mmHg, P < 0.001). Individual and population-mean cosinor analysis demonstrated significant circadian rhythms for SBP, DBP and HR before and after 12 months on GHR (P < 0.001), suggesting that SBP, DBP and HR circadian rhythms were not altered by GHR. There was, however, a significant reduction in SBP (124.2 +/- 2.8 vs. 118.4 +/- 2.8 mmHg, P < 0.001) and DBP (77.0 +/- 1.6 vs. 70.2 +/- 1.8 mmHg, P < 0.001) MESOR with an increase in HR MESOR (68.9 +/- 2.5 vs. 72.2 +/- 2.4 beats/min, P < 0.01) following GHR. Systolic and diastolic BP and HR circadian rhythms are preserved in AGHD following 12 months of GHR. However, there is a significant decrease in 24-h mean SBP and DBP and increase in 24-h mean HR after 12 months on GHR. We postulate that this decrease in 24-h mean SBP and DBP may result in a reduction of cardiovascular morbidity and mortality and may explain the beneficial effects of GHR on cardiovascular system previously reported in AGHD patients.

Is the somatotropic axis related to sympathetic nerve activity in healthy ageing men?

The mechanisms underlying the age-related increase in blood pressure and sympathetic nerve activity remain largely unknown. The decline in growth hormone (GH) secretion and insulin-like growth factor-I (IGF-I) with age has been related to several cardiovascular risk factors. Low serum IGF-I levels in severe adult GH deficiency is associated with markedly increased sympathetic nerve activity. This study evaluates whether a relationship between serum IGF-I and sympathetic nerve traffic exists in healthy aging men. Sympathetic nerve activity to the muscle vascular bed (MSA) was recorded in 56 healthy normotensive males, and related to age (range 21-71 years), body mass index (BMI, range 18.4-32.2), serum IGF-I and plasma nitrate levels. Blood pressure, BMI and MSA increased with age, whereas IGF-I and plasma nitrate decreased. In a forward stepwise multiple regression analysis, age explained 40% of the variability in MSA and excluded other variables. Omitting age, IGF-I became the strongest independent predictor, explaining 23% of the variability in MSA. MSA was an independent predictor of diastolic blood pressure, but its influence (10%) was less than that of BMI (28%). BMI was not related to MSA or IGF-I. Decreased serum IGF-I levels are coupled to increased MSA during ageing, an effect independent from the impact of increased body weight. Although MSA is a weak predictor of rising blood pressure with age, it constitutes one possible pathway for the somatotropic axis to affect cardiovascular function in ageing.