Adrenalectomized Patients Research Articles

Hormone-sensitive lipase (HSL) expression and regulation in skeletal muscle.

Because the enzymatic regulation of muscle triglyceride metabolism is poorly understood we explored the character and activation of neutral lipase in muscle. Western blotting of isolated rat muscle fibers demonstrated expression of hormone-sensitive lipase (HSL). In incubated soleus muscle epinephrine increased neutral lipase activity by beta-adrenergic mechanisms involving cyclic AMP-dependent protein kinase (PKA). The increase was paralleled by an increase in glycogen phosphorylase activity and could be abolished by antiserum against HSL. Electrical stimulation caused a transient increase in activity of both neutral lipase and glycogen phosphorylase. The increase in lipase activity during contractions was not influenced by sympathectomy or propranolol. Training diminished the epinephrine induced lipase activation in muscle but enhanced the activation as well as the overall concentration of lipase in adipose tissue. In agreement with the in vitro findings, in adrenalectomized patients an increase in muscle neutral lipase activity was found at the end of prolonged exercise only if epinephrine was infused. In accordance with feedforward regulation of substrate mobilization in exercise, our studies have shown that HSL is present in skeletal muscle cells and is stimulated in parallel with glycogen phosphorylase by both epinephrine and contractions. HSL adapts differently to training in muscle compared with adipose tissue.

Adrenaline and psychometric mood factors: A controlled case study of two patients with bilateral adrenalectomy

Previous studies have suggested that adrenaline subserves many emotions and that adrenaline is the physiological basis for fear, but these observations have been difficult to replicate. Acute insulininduced hypoglycaemia can be used to induce tense arousal in normal humans, and is one of the most potent stimuli for the secretion of adrenaline. Adrenalectomized Ss do not secrete adrenaline in response to hypoglycaemia, so it was hypothesized that a study of these patients might clarify the role of circulating plasma adrenaline in the generation of changes in psychometric mood states. Measurements of mood state, and plasma catecholamine concentrations were made during hypoglycaemia induced by an intravenous infusion of insulin, in 25 healthy subjects (12 male and 13 female) and in two women who had previously undergone surgical removal of both adrenal glands. Hedonic tone fell in the control subjects, but in both adrenalectomized patients hedonic tone was unaffected. Energetic arousal decreased significantly both in the control subjects and in the two adrenalectomized patients. Tense arousal rose in the control subjects to a peak which coincided with the onset of the acute autonomic reaction, identified by the onset of symptoms and the rapid rise in heart rate and sweating. Conversely, in the adrenalectomized patients no substantial change was observed in tense arousal from basal levels and it appeared to be lower before the induction of hypoglycaemia than in the control subjects. This experimental model has demonstrated that adrenaline release from the adrenal glands is a necessary requisite for the increase in tense arousal induced by hypoglycaemia and may contribute to the change in hedonic tone. The alteration in energetic arousal was unaffected and this is probably mediated by the direct effect of glucose deprivation on cerebral functioning.

B5 - Effects of acute saline loading on plasma ouabain before and after high sodium intake in adrenalectomized patients and in controls

B5 - Effects of acute saline loading on plasma ouabain before and after high sodium intake in adrenalectomized patients and in controls

Electron microscopical morphometry of well-differentiated and undifferentiated ACTH secreting adenomas in Cushing's disease and Nelson's syndrome.

Adrenocorticotrophic hormone (ACTH)-secreting adenomas of patients with Cushing's disease (undifferentiated and well-differentiated ACTH-cell adenomas) were studied ultrastructurally and analysed morphometrically by a computer-supported quantitative image-analysing system. They were compared with identically prepared ACTH tumours (undifferentiated and well-differentiated ACTH-cell adenomas) of pituitaries from bilateral adrenalectomised patients with Nelson's syndrome. The aim of our study was to look for significant differences in ultrastructure and to evaluate these findings statistically regarding adenoma types and clinical syndromes. Clinical syndromes aside, more secretory granules and larger-sized prosecretory granules were measured in the well-differentiated ACTH-cell adenomas. The undifferentiated adenomas showed a greater content of nucleoli and prosecretory granules. Within the adenoma types, comparison of well-differentiated ACTH-cell adenomas showed that the clinical group of Cushing's disease contained larger areas of cytofilaments, whereas the clinical group of Nelson's syndrome had a larger tumour size and more lysosomes. Comparing the undifferentiated adenomas of both clinical groups the adenomas in Cushing's disease contained larger nuclei and more lysosomes, whereas the adenomas in Nelson's syndrome were larger in tumour size and contained larger prosecretory granules. Comparison of well-differentiated and undifferentiated adenomas in Cushing's disease showed more secretory granules and bigger prosecretory granules in well-differentiated adenomas whereas in undifferentiated adenomas the total area of the nuclei is larger, the nucleoli increase in number and size and the lysosomes are more frequent. Comparison of well-differentiated and undifferentiated adenomas in Nelson's syndrome demonstrated more lysosomes in well-differentiated adenomas and a larger total area of the nuclei in undifferentiated adenomas. The differences between the well-differentiated adenomas (mainly more secretory granules and larger prosecretory granules) and undifferentiated adenomas (mainly more and larger nuclei and nucleoli and more prosecretory granules) prove the clear separability between the adenoma types, not demonstrated in the literature up to now. The significant differences between adenomas in Cushing's disease (mainly more cytofilaments) and Nelson's syndrome (mainly more ribosomes and larger prosecretory granules) may be interpreted as different cell reactions due to the hypercortisolism present in Cushing's disease and lacking in Nelson's syndrome following adrenalectomy. Despite the fact that both clinical syndromes are based on the same adenoma types, indistinguishable by light microscopy, significant morphometrical findings in ultrastructure allow a clear discrimination of both clinical types.

LONG‐TERM FOLLOW‐UP OF LOW‐DOSE EXTERNAL PITUITARY IRRADIATION FOR CUSHING'S DISEASE

Twenty-four patients (three male) with Cushing's disease, aged between 11 and 67 years, were treated with low-dose external pituitary irradiation (20 Gy in eight fractions over 10-12 days) and followed for between 13 and 171 months (median 93 months). Eleven patients (46%) went into remission 4-36 months after irradiation, but five subsequently relapsed. Two of these received no further active treatment, one underwent successful pituitary surgery, one underwent a second course of low-dose external irradiation (as yet unsuccessful) and one has been treated with metyrapone for a total of 75 months. One of the 13 patients who did not respond received a further course of low-dose pituitary irradiation with prompt remission and two have received metyrapone for 41 months and 15 years without ill effect. One patient died from cerebrovascular disease. The remaining nine patients underwent bilateral adrenalectomy (one after unsuccessful pituitary surgery) with rapid resolution of hypercortisolism. Five of these patients have developed hyperpigmentation and elevated ACTH levels (range 505-1150 ng/l). A pituitary microadenoma has been demonstrated on CT scan in three and successfully removed by microadenomectomy. In the present series, the low incidence of radiation-induced hypopituitarism and absence of other complications attributable to radiotherapy suggest that low-dose pituitary irradiation may be a useful treatment option in selected patients. However, long-term follow-up has demonstrated a high relapse rate and failure to prevent Nelson's syndrome in adrenalectomized patients, indicating that it should not be used as primary treatment in preference to selective adenomectomy.

Cortisol production rates measured by liquid chromatography/mass spectrometry

Cortisol production rates (FPRs) in physiologic and pathologic states in humans have been investigated over the past 30 years. However, there has been conflicting evidence concerning the validity of the currently accepted value of FPRs in humans (12 to 15 mg/m 2/d) as determined by radiotracer methodology. The present study reviews previous methods proposed for the measurement of FPRs in humans and discusses the applications of the first method for the direct determination of 24-hour plasma FPRs during continuous administration of a stable isotope, using a thermospray high-pressure liquid chromatography-mass spectrometry technique. The technique is fast, sensitive, and, unlike gas chromatography-mass spectrometry methods, does not require derivatization, allowing on-line detection and quantification of plasma cortisol after a simple extraction procedure. The results of determination of plasma FPRs by stable tracer/mass spectrometry are directly in units of mass/time and, unlike radiotracer methods, are independent of any determination of volume of distribution or cortisol concentration. Our methodology offers distinct advantages over radiotracer techniques in simplicity and reliability since only single measurements of isotope ratios are required. The technique was validated in adrenalectomized patients. Circadian variations in daily FPRs were observed in normal volunteers and, to date, results suggest a lower FPR in normal children and adults than previously believed.

Corticotropin-Releasing Hormone (CRH) Stimulation in Nelson''s Syndrome: Response of Adrenocorticotropin Secretion to Pulse Injection and Continuous Infusion of CRH*

Nelson's syndrome develops in 10-15% of patients with Cushing's disease who undergo bilateral adrenalectomy. Whether the pituitary tumors of Nelson's syndrome are autonomous or are regulated by hypothalamic signals or glucocorticoids is controversial. We, therefore, compared the plasma ACTH responses to synthetic ovine corticotropin-releasing hormone (CRH) in 11 patients with Nelson's syndrome, 1 patient with Cushing's disease who had had bilateral adrenalectomy and did not have Nelson's syndrome, 14 patients with Cushing's disease, and 27 normal subjects. The plasma ACTH response to CRH in 10 patients with Nelson's syndrome was markedly increased and prolonged compared to the responses of normal subjects or patients with Cushing's disease. In 4 patients with Nelson's syndrome, plasma ACTH and cortisol concentrations also were determined at frequent intervals for 10-24 h during continuous infusions of 0.15 M saline or CRH (1 microgram/kg X h). There was no desensitization of ACTH secretion during short term continuous infusion of CRH. Exogenous cortisol inhibited CRH-stimulated ACTH secretion. These findings suggest that the ACTH response to CRH of patients with ACTH-secreting tumors of Nelson's syndrome differs from the response of those who have the microadenomas of Cushing's disease in two ways: the magnitude is greater, and the response is prolonged. These differences can be explained by the greater size of the tumor and the reduced glucocorticoid feedback in adrenalectomized patients with Nelson's syndrome.

445 ISOLATION OF AN IMHJNOREACTIVE SUBSTANCE IN PATIENTS WITH DEXAHETHASONE-SUPPRESSIBLE HYPERALDOSTERONISM (DSH)

We have isolated a substance immunoreactive (IR) with the aldosterone (aldo) antibody in patients with DSH. Substantial data exists that an unidentified steroid with mineralocorticoid properties plays an important role in the etiology of the hypertension in patients with DSH. This IR substance is more polar than 18-oxocortisol by paper chromatography. It is also more polar than aldo, facilitating separation from aldo utilizing high-pressure liquid chromatography. This substance was detected in both the free (930 pg IR/ml urine) extracts of urine in patients with DSH and was not detected in non-hypertensive controls. In addition, this IR substance was not detected in an adrenalectomized patient and was not detected in two patients with 11β-hydroxylase deficiency with and without hypertension. In patients with DSH, the IR substance increased substantially after a 6 hr infusion of ACTH (3400 pg IR/ml urine). In normal controls, no appreciable amount of immunoreactivity was detected in the baseline state. After ACTH stimulation a small amount was detected. Dexamethasone administration results in suppression of aldosterone and suppression of the IR substance in patients with DSH. With the techniques we have developed, we are now able to isolate a sufficient amount of purified material to submit for mass spectrometry analysis.

Effect of CRF on the release of anterior pituitary hormones in normal subjects and patients with Cushing's disease

Patterning of plasma ACTH, beta-EP/beta-LPH and cortisol in response to ovine CRF (1 microgram/kg b.w.t. injected i.v.), was studied in three normal subjects and in five patients with Cushing's disease, two of whom had undergone total bilateral adrenalectomy. CRF caused in all subjects a prompt and concurrent rise of plasma hormone levels. The hormonal response was of the same magnitude in normal controls and in the three untreated Cushing's patients, but was much greater in the two adrenalectomized subjects. No changes were noted, after CRF, in plasma levels of GH, PRL, TSH, LH and FSH. These findings indicate that CRF specifically promotes the pituitary release of ACTH and ACTH-related peptides both in normal subjects and patients with Cushing's disease. In view of the likely CRF hypersecretion of the adrenalectomized patients, their ACTH and beta-EP/beta-LPH hyperresponsiveness to exogenous CRF would denote that the peptide does not down regulate its own pituitary receptors.

The role of adrenal medullary catecholamine release in the response to a cold pressor test

We studied the effects of a cold pressor test on the plasma catecholamine levels of ten patients undergoing coronary angiography, to determine whether the pressor changes were related to adrenergic activity. To investigate the relative contribution of adrenal medullary catecholamine release, we subjected two adrenalectomised volunteers to the same test. Arterial blood was assayed for dopamine (DA), adrenaline (A) and noradrenaline (NA). We found significant rises in the levels of all three catecholamines in the angiography patients, accompanied by a significant elevation in arterial blood pressure. In both the adrenalectomised patients a rise in blood pressure was seen but no significant rise in plasma catecholamines could be demonstrated. We postulate that although adrenal medullary catecholamine release occurs in response to the cold pressor test, the blood pressure elevation is independent of such adrenal activity. Sampling radial arterial blood may not reflect changes in plasma levels of peripherally released NA.

Comparison of four-combination chemotherapy programs in metastatic breast cancer. Comparison of multiple drug therapy with cytoxan, 5-fu and prednisone versus cytoxan and adriamycin, versus cytoxan, 5-Fu and adriamycin, versus cytoxan, 5-Fu and prednisone alternating with cytoxan and adriamycin

In 126 postmenopausal women with metastatic breast cancer, four chemotherapeutic combination programs were tested. Patients were stratified and randomized to one of the four programs: (1) fluorouracil, cytoxan and prednisone (CFP): (2) fluorouracil, cytoxan and Adriamycin (CAF); (3) cytoxan and Adriamycin (CA); or (4) alternating combinations of CFP-CA. Objective response rates were 17% for 18 patients on CFP, 25% for 40 patients on CAF, 42% for 41 patients on CA and 63% for 19 patients on CFP-CA. CFP-CA provided a significantly higher response rate than either CFP or CAF. The response rate of CA was intermediate. The duration of remission and survival were similar for all four groups. When progression or relapse following remission was demonstrated, patients were rerandomized to either adrenalectomy or tamoxifen. There were no responders either in the 15 patients undergoing adrenalectomy or the 11 patients receiving tamoxifen. In a crossover study, nine adrenalectomized patients received tamoxifen and four tamoxifen-treated patients underwent adrenalectomy. None responded. It appears that response rates to hormonal modalities are reduced in patients previously treated by combination chemotherapy.

RESIDUAL ADRENOCORTICAL FUNCTION AFTER BILATERAL "TOTAL" ADRENALECTOMY FOR CUSHING'S DISEASE

6 patients who had been treated for pituitary-dependent Cushing's syndrome by intended complete removal of the adrenal glands are described. There were clinical grounds for attempting to reduce maintenance steroid therapy in each case, and it was found possible to withdraw steroids completely without adverse effects. These findings illustrate the importance of clinically assessing the minimum steroid requirements of adrenalectomised patients and the need for continued surveillance for persistent or recurrent Cushing's syndrome

Studies on circulating met-enkephalin and beta-endorphin: normal subjects and patients with renal and adrenal disease.

SUMMARYStudies were performed to define the responses of plasma met‐enkephalin to various endocrine and pathological stimuli and to determine the relationship between plasma β‐endorphin and met‐enkephalin. During insulin‐induced hypoglycaemia ACTH, β‐LPH and β‐endorphin immunoreactivity rose in parallel, but plasma met‐enkephalin did not change significantly. Sephadex G75 chromatography of samples taken at the time of the peak response (45 min) confirmed the rise in both β‐LPH and β‐endorphin. During administration of dexamethasone, 0·5mg 6 hourly for 48 h, plasma cortisol and ACTH became undetectable at 24 h, and β‐LPH and β‐endorphin fell significantly by 24 h and were undetectable by 48 h; plasma met‐enkephalin, however, showed no significant change. Nine adrenalectomized patients with Cushing's disease and four patients with Addison's disease had elevated plasma ACTH, β‐LPH and β‐endorphin but normal plasma met‐enkephalin levels. Each of ten patients with renal failure had markedly elevated plasma met‐enkephalin immunoreactivity which co‐eluted with synthetic met‐enkephalin on BioGel P4 chromatography. Trypsin and carboxypeptidase‐B digestion of the P4 chromatography fractions generated met‐enkephalin immunoreactivity in earlier fractions, indicating the presence of a potential high molecular weight met‐enkephalin precursor.The results imply that different control mechanisms govern β‐endorphin and met‐enkephalin plasma levels and that the adrenal is not the only source of plasma met‐enkephalin. The elevated levels of met‐enkephalin in renal failure may be due to impaired clearance of met‐enkephalin or of its precursor or to increased met‐enkephalin production.

Effect of hydrocortisone on ACTH, growth hormone, insulin and glucose in the blood of bilaterally adrenalectomized patients.

This study is aimed at elucidating the mechanism of paradoxical rise in plasma ACTH levels in response to glucocorticoids, observed by several authors in bilaterally adrenalectomized patients with Cushing's disease. Six control subjects and fourteen patients bilaterally adrenalectomized for Cushing's disease were given a dose of 200 mg hydrocortisone sodium succinate by 3-5 mm i.v. injection. Plasma ACTH (in 6 patients), serum cortisol, growth hormone (GH) and insulin and blood glucose levels were estimated at 0, 30, 60, 90, and 120 minutes. The administration of hydrocortisone significantly suppressed plasma ACTH levels only at 60 min. In one case a slight rise in ACTH level during the test was observed. A significant fall in blood glucose levels was found only in the adrenalectomized patients. No significant changes in serum insulin and GH levels were noted. The possible mechanisms are discussed, especially the potential role of transient glucose deficiency in the pathophysiology of plasma ACTH increase in response to hydrocortisone in the bilaterally adrenalectomized patients.

Effect of mineralocorticoid replacement therapy on renal acid-base homeostasis in adrenalectomized patients

Chronic balance studies were performed in six adrenalectomized patients to investigate the renal and systemic acid-base consequences of mineralocorticoid deficiency in the absence of either glucocorticoid deficiency or parenchymal renal disease. Constant glucocorticoid replacement was provided with dexamethasone, 750 to 875 micrograms/day, administered orally. Creatinine clearance averaged 98 +/- 8 ml/min/1.73 m2. Following a control period, mineralocorticoid replacement with fludrocortisone (100 to 200 micrograms/day) was either discontinued (N = 3) or initiated (N = 2). In an additional patient, mineralocorticoid replacement was initiated and sustained (5 days) by continuous i.v. infusion of aldosterone, at a dose approximating the normal secretion rate (120 micrograms/day). Net acid excretion (NAE) and plasma total carbon dioxide decreased in each patient in whom mineralocorticoid was discontinued and increased in each patient in whom mineralocorticoid was initiated. The cumulative change in NAE (sigma delta NAE) independent of direction averaged 66 +/- 20 mEq (P less than 0.05) by the fifth experimental day in the six patients, and the corresponding change in plasma total CO2 averaged 1.2 +/- 0.3 mmoles/liter (P less than 0.02). The magnitude of sigma delta NAE correlated with the basal rate of NAE (r = 0.87, P less than 0.05), which averaged 0.9 +/- 0.1 mEq/kg body wt per day. The change in plasma total CO2 correlated with sigma delta NAE (r = 0.83, P less than 0.05). The changes in NAE correlated positively with the corresponding changes in sodium balance and negatively with the corresponding changes in potassium balance. These findings provide the first evidence that renal acidification is under tonic stimulation by mineralocorticoid at levels not exceeding those in normal subjects ingesting acid-producing diets of normal sodium and potassium content. The extent to which the tonic stimulation of renal acidification is mediated by a direct effect of mineralocorticoid on renal hydrogen ion transport or by an indirect effect dependent on altered renal sodium and/or potassium transport requires further investigation. The findings implicate mineralocorticoid deficiency as a significant renal acidosis-producing condition not dependent on the presence of renal disease or glucocorticoid deficiency, and potentially amplified when endogenous acid production is increased by diet or disease.

Synthetic 1-24 ACTH-stimulated insulin release in bilaterally adrenalectomized patients.

The plasma insulin and blood glucose responses to synthetic 1-24 ACTH were studied in 21 patients bilaterally adrenalectomized for pituitary-dependent Cushing's syndrome and in 8 healthy adults. In the adrenalectomized patients, intravenous 1-24 ACTH administration was followed by an increase in plasma insulin concentrations after 15 and 30 min and a fall in blood glucose after 30 min. In healthy subjects no significant changes in plasma insulin and blood glucose levels were found. The presence of intact adrenals seems to be the cause of the different responses of insulin to 1-24 ACTH injection in these two groups.

Cholinergic Stimulation of Norepinephrine Release in Man

Amplification of endogenous cholinergic activity-produced by the intravenous injection of edrophonium, an acetylcholinesterase inhibitor which does not enter the central nervous system, into normal subjects-resulted in significant and briefly sustained increments in the plasma concentrations of norepinephrine (153+/-15-234+/-29 pg/ml, P < 0.01) and epinephrine (16+/-3-34+/-5 pg/ml, P < 0.01) measured with a single-isotope derivative method. These increments were not attributable to reflex responses to hemodynamic changes and similar increments in plasma norepinephrine occurred in adrenalectomized (epinephrine deficient) patients. Thus, cholinergic activation results in direct stimulation of sympathetic postganglionic neurons, with augmented norepinephrine release, and of the adrenal medullae, with augmented epinephrine release, in man. Four diabetic patients with hypoadrenergic postural hypotension exhibited blunted sympathetic postganglionic neural responses, and normal adrenomedullary responses, to cholinergic stimulation (and to standing) indicative of the presence of a sympathetic postganglionic axonal lesion in diabetic adrenergic neuropathy. Nondiabetic patients with hypoadrenergic postural hypotension due to documented or probable central nervous system lesions exhibited normal responses to cholinergic stimulation produced in this fashion demonstrating the presence of intact sympathetic postganglionic neurons and adrenal medullae in these patients and providing further support for the conceptual soundness of this approach to the study of human adrenergic physiology and pathophysiology.

Secretory B-Cell Activity in Insulin Dependent Maturity-Onset Diabetic

In 10 insulin dependent maturity onset diabetics we found elevated basal C-Peptide levels (4.78 +/- 0.5 ng/ml. Normal range 1.1--3.6 ng/ml), which could be suppressed by insulin injection to the same extent as in sulfonylurea treated diabetics could be demonstrated. C-Peptide immunoreactivity in these patients therefore seems to be newly secreted rather than accumulated material. Since adrenalectomized patients could be suppressed in the same way, it is likely, that catecholamines are not the major factor in the mechanism of suppression. Therefore only decrease of bloodsugar levels seems to be accountable for the decrease of C-Petide levels. High C-Petide levels in insulin dependent maturity onset diabetics which cannot be stimulated but suppressed may be explained by a loss of glucoreceptor molecules.

The role of the adrenergic innervation to the pancreatic islets in the control of insulin release during exercise in man

The normal depression of plasma insulin concentration during exercise has been ascribed to adrenergic inhibition of insulin release and the role of humoral catecholamines in this hormonal adjustment has repeatedly been stressed. In the present study this contention has been investigated in 6 bilaterally adrenalectomized patients and in 6 sex- and age-matched controls who undertook exercise on an ergometer until they were exhausted. No differences were observed in any cardiovascular or metabolic adjustments between the two groups during strenous exercise. Mean plasma insulin concentration fell by about 50% in both groups. Phentolamine effectively abolished the fall in plasma insulin concentration during exercise in 2 adrenalectomized patients. The results suggest that the adrenergic nerves that supply the B-cells have a functional role in man during exercise.

A study on the pathophysiology and diagnosis of adrenocortical insufficiency using angiotensin II blocker (author's transl)

Angiotensin II analogue (AIIA), 1-sarcosine, 8-isoleucine angiotensin II (Sar1, Ile8-AII), was given in a graded dose to patients with adrenal hypofunction of various etiologies, and the blood pressure response to AIIA was observed to investigate the role of the renin-angiotensin system for the maintenance of blood pressure in the state of adrenal insufficiency. An agonistic pressor response to AIIA was observed in the control subjects without adrenal hypofunction. In contrast with this, patients with Addison's disease showed a blood pressure fall to AIIA. When the test was repeated on the same patients after hydrocortisone replacement, these patients showed a neutral or pressor response. Three patients with adrenalectomy and hydrocortisone replacement showed a pressor response to AIIA in a supine position. When these patients were tilted, fall in blood pressure to AIIA was observed in two of them who had been on a regular diet, whereas one patient who had been on a high sodium diet showed no fall in blood pressure with this procedure. We concluded that the renin-angiotensin system plays an important role in the maintenance of normal or subnormal blood pressure in Addison's disease and in the postural change of adrenalectomized patients on hydrocortisone and regular salt intake. The response of blood pressure to AIIA may be a clue for the diagnosis of Addison's disease and the evaluation of the adequacy of replacement therapy with regard to these patients.