Canine Hyperadrenocorticism Due to Adrenocortical Neoplasia: Pretreatment Evaluation of 41 Dogs

This retrospective study identifies parameters that might separate dogs with hyperadrenocorticism caused by adrenocortical tumors from dogs with pituitary-dependent hyperadrenocorticism. Further, an attempt was made to identify factors that could separate dogs with adrenocortical adenomas from dogs with carcinomas. The records of 41 dogs with hyperadrenocorticism caused by adrenocortical neoplasia were reviewed. The history, physical examination, urinalysis, hemogram (CBC), chemistry profile adrenocorticotrophic hormone (ACTH) stimulation and low dose dexamethasone test results were typical of the nonspecific diagnosis of hyperadrenocorticism. The preceding information on the 41 dogs with adrenocortical tumors was compared with that from 44 previously diagnosed pituitary-dependent hyperadrenocorticoid dogs. There was no parameter which aided in separating these two groups of dogs. Thirty dogs with adrenocortical tumors were tested with a high-dose dexamethasone test and none had suppressed plasma cortisol concentrations 8 hours after IV administration of 0.1 mg/kg of dexamethasone. In 29 of the 41 adrenal tumor dogs, plasma endogenous ACTH was not detectable on at least one measurement (less than 20 pg/ml). The remaining 12 dogs from this group had nondiagnostic concentrations (20-45 pg/ml). Thirteen of 22 dogs (59%) with adrenocortical carcinomas had adrenal masses identified on abdominal radiographs and seven of 13 dogs (54%) with adrenocortical adenomas had radiographically visible adrenal masses. Thirteen of 17 adrenocortical carcinomas (76%) and five of eight adenomas (62%) were identified with ultrasonography. Radiographs of the thorax and ultrasonography of the abdomen identified most of the dogs (8 of 11) with metastatic lesions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hyperadrenocorticism in cats: seven cases (1978-1987)

Hyperadrenocorticism was diagnosed in 7 cats with concurrent diabetes mellitus. Four cats had pituitary adenoma with bilateral adrenocortical hyperplasia, 1 cat had pituitary carcinoma with bilateral adrenocortical hyperplasia, 1 cat had adrenocortical carcinoma, and 1 cat had adrenocortical adenoma of the left adrenal gland. One year later, adrenocortical adenoma involving the right adrenal gland also was diagnosed in this cat. Clinical signs included polyuria and polydipsia (n = 7), development of pot-bellied appearance (n = 5), dermatologic alterations (n = 5), lethargy (n = 3), weight loss (n = 3), dyspnea/panting (n = 2), and recurrent bacterial infections (n = 2). In 6 cats, the diagnosis of hyperadrenocorticism was established before death on the basis of results of the ACTH stimulation test (n = 3) and the dexamethasone screening test (n = 5). Pituitary-dependent hyperadrenocorticism was differentiated from adrenocortical neoplasia on the basis of results of the dexamethasone suppression test (n = 4), endogenous ACTH concentration (n = 3), results of abdominal radiography and ultrasonography (n = 3), and exploratory celiotomy (n = 1). Four cats died or were euthanatized without treatment attempts. Treatment with mitotane followed by 60Co teletherapy was ineffective in one cat with pituitary adenoma. One cat with pituitary carcinoma died one week after bilateral adrenalectomy. Bilateral adrenocortical adenomas were removed surgically in the affected cat.     

Use of a low dose synthetic ACTH challenge test in normal and prednisone-treated dogs

The utility of a low dose (1 microgram/kg) synthetic ACTH challenge test in detecting moderate reductions in adrenocortical sensitivity in dogs was examined. First, the adrenocortical responses to an intravenous bolus of either 1 microgram/kg or 0.25 mg per dog of synthetic ACTH were compared in two groups of normal dogs. While plasma cortisol concentrations were similar in both groups 60 minutes after ACTH injection, dogs given 0.25 mg ACTH showed continued elevations in plasma cortisol concentrations at 90 and 120 minutes after ACTH injection. Later, the dogs previously tested with the 1 microgram/kg ACTH challenge were given a single intramuscular dose of prednisone (2.2 mg/kg) and retested with 1 microgram/kg of ACTH one week later. Plasma cortisol levels were significantly reduced after ACTH injection in dogs previously given prednisone demonstrating that a single intramuscular prednisone dose causes detectable adrenocortical suppression one week after administration. The 1 microgram/kg synthetic ACTH challenge test provides a sensitive means for evaluating adrenocortical suppression in dogs.     

Evaluation of a six-hour combined dexamethasone suppression/ACTH stimulation test in dogs with hyperadrenocorticism

Seventeen dogs with hyperadrenocorticism were studied. Three dogs had functioning adrenocortical tumors and 14 had pituitary-dependent hyperadrenocorticism. Each dog was evaluated by determining the endogenous plasma ACTH concentration and by performing 4 tests: ACTH stimulation, dexamethasone screening, dexamethasone suppression, and a 6-hour combined dexamethasone suppression/ACTH stimulation test. The combined test was less reliable as a screening test in diagnosing hyperadrenocorticism than was the dexamethasone screening test or the ACTH stimulation test. Compared with the endogenous plasma ACTH concentration, results of the dexamethasone suppression portion of the combined test were less reliable in distinguishing dogs with adrenocortical tumors from those with pituitary-dependent hyperadrenocorticism. It was concluded that the combined test cannot be recommended for use.     

Evaluation of a combined dexamethasone suppression/ACTH stimulation test in dogs with hyperadrenocorticism

Twenty-one dogs with hyperadrenocorticism were studied. Six dogs had functioning adrenocortical tumors and 15 had pituitary-dependent hyperadrenocorticism. Each dog was evaluated, using endogenous plasma ACTH, ACTH stimulation, dexamethasone screening, dexamethasone suppression, and combined dexamethasone suppression/ACTH stimulation tests. The ACTH stimulation portion of the combined test was less reliable as a screening test in diagnosing hyperadrenocorticism than was the isolated ACTH stimulation test or the dexamethasone screening test. The dexamethasone suppression portion of the combined test was less reliable in distinguishing dogs with adrenocortical tumors from those with pituitary-dependent hyperadrenocorticism than was the endogenous ACTH or isolated dexamethasone suppression test. The combined test is not recommended for use. The ACTH stimulation test is the recommended screening test because of its diagnostic reliability and its subsequent importance as a base line in determining success of mitotane therapy.     

Intermittent Stress in Pigs: Behavioural and Pituitary-Adrenocortical Reactivity

Abstract

Ninetysix pigs, half females and half castrated males from 12 litters, were housed in 24 groups of 4 litter mates (2 females, 2 castrates). The pigs were weaned at 4 weeks of age and from the age of 115 days half of the groups were subjected to a schedule of unpredictable, inescapable electroshocks for 33 days, and half served as control. The adrenocortical reactivity to ACTH stimulation and to an emotional stressor (the procedure associated with initiation of blood collection) was measured in females, whereas the behavioural and pituitary-adrenocortical reactivity to an open field test was measured in castrates, as was the behavioural reactivity in a social test. Intermittent stress increased adrenocortical reactivity to ACTH stimulation significantly. This effect was present within the first week of intermittent stress (p < 0.06), but no difference was found after 4–5 weeks of stress. In contrast, the adrenocortical reactivity to additional emotional stressors was unaffected by stress after one week of intermittent stress, but increased after 4–5 weeks. In the open field test, centre location increased after one week of intermittent stress, whereas exploration decreased and walking and ambulation increased after 4–5 weeks of stress. In addition, 4–5 weeks of intermittent stress decreased aggressiveness in a social test. In conclusion, the change in reactivity to an additional acute stressor during intermittent stress appeared in different variables and contexts at different times during the course of intermittent stress. Thus, assessment of stress based on changed reactivity has to include several distinct measures of behavioural and hormonal reactivity.     

Hyperadrenocorticism in four cats.

This paper describes four cats with hyperadrenocorticism. Cat 1 showed polydipsia and polyphagia. Diabetes mellitus was initially diagnosed. As the animal appeared to be insulin resistant, pituitary and adrenocortical function tests were performed and the diagnosis of hyperadrenocorticism was made. Resistance to the high-dose dexamethasone suppression test was noticed in this cat. Pathological examination revealed a pituitary chromophobe adenoma. Cat 2 presented with diabetes mellitus, which was treated with insulin. The animal had a pendulous abdomen and its coat was in a poor condition. The low-dose dexamethasone suppression test demonstrated hyperadrenocorticism. Necropsy findings of pituitary tumour and hyperplasia of the adrenal cortex confirmed the diagnosis. Cat 3 showed clinical abnormalities indicative of hyperadrenocorticism, for instance, muscle weakness, alopecia, multiple abscesses. The diagnosis of hyperadrenocorticism was confirmed by the results of the lowe-dose dexamethasone suppression test. Pathological examination revealed an adrenocortical carcinoma. Cat 4 presented with polydipsia. The cause of this symptom was not found initially. One and a half years later additional symptoms, such as nephritis and polyphagia developed. Hyperadrenocorticism was diagnosed because of a palpable mass cranial to the left kidney. The diagnosis was confirmed by the results of the lowe-dose dexamethasone suppression test and the necropsy findings.     

Radiographic features of canine hyperadrenocorticism caused by autonomously functioning adrenocortical tumors: 23 cases (1978-1986)

Radiographic features of 23 dogs with functioning adrenocortical tumors are described. In 56% of the dogs, radiographic detection of adrenomegaly, with or without calcification, was an aid in the diagnosis of adrenocortical neoplasia. Adrenal gland calcification did not distinguish adrenocortical adenoma from carcinoma.     

Hyperadrenocorticism in four cats

Summary

This paper describes four cats with hyperadrenocorticism. Cat 1 showed polydipsia and polyphagia. Diabetes mellitus was initially diagnosed As the animal appeared to be insulin resistant, pituitary and adrenocortical function tests were performed and the diagnosis of hyperadrenocoricism was made. Resistance to the high‐dose dexamethasone suppression test was noticed in this cat. Pathological examination revealed a pituitary chromophobe adenoma.

Cat 2 presented with diabetes mellitus, which was treated with insulin. The animal had a pendulous abdomen and its coat was in a poor condition. The low‐dose dexamenthasone suppression test demonstrated hyperadrenocorticism. Necropsy findings of pituitary tumour and hyperplasia of the adrenal cortex confirmed the diagnosis.

Cat 3 showed clinical abnormalities indicative of hyperadrenocorticism, for instance, muscle weakness, alopecia, multiple abcesses. The diagnosis of hyperadrenocorticism was confirmed by the results of the lowe‐dose dexamethasone suppression test. Pathological examination revealed an adrenocortical carcinoma.

Cat 4 presented with polydipsia. The cause of this symptom was not found initially. One and a half years later additional symptoms, such as nephritis and polyphagia developed Hyperadrenocorticism was diagnosed because of a palpable mass cranial to the left kidney. The diagnosis was confirmed by the results of the lowe‐dose dexamethasone suppression test and the necropsy findings     

Plasma cortisol response to exogenous ACTH in 22 dogs with hyperadrenocorticism caused by adrenocortical neoplasia

The plasma cortisol response to exogenous ACTH (ACTH stimulation test) was evaluated in 22 dogs with hyperadrenocorticism caused by adrenocortical neoplasia. The mean basal cortisol concentration (6.3 microgram/dl) was high, but 7 dogs had basal cortisol concentrations that were within normal range. Administration of exogenous ACTH increased the plasma cortisol concentrations in each dog. Normal post-ACTH cortisol concentrations were found in 9 (41%) of the 22 dogs; 13 (59%) had an exaggerated increase in cortisol concentrations after ACTH administration. In 9 of 13 dogs with carcinoma and in 4 of 9 with adenoma, the cortisol response was exaggerated. The mean post-ACTH cortisol concentration in the dogs with carcinoma was approximately 4 times that of the dogs with adenoma; the 7 dogs with the highest concentrations had carcinoma. Repeat studies were performed in 6 dogs 2 to 8 weeks after initial testing. In 5 of the 6 dogs, repeat testing yielded data of similar diagnostic significance. One dog, however, had an abnormally high post-ACTH cortisol concentration at initial evaluation, but had only a minimal response to ACTH administration, with a normal post-ACTH cortisol concentration, at time of resting. Although ACTH stimulation testing is useful in diagnosing hyperadrenocorticism, it can not reliably separate dogs with hyperfunction adrenocortical tumors from clinically normal dogs or from dogs with pituitary-dependent hyperadrenocorticism (bilateral adrenocortical hyperplasia).     

Are oestrogen receptors and protein tyrosine kinases involved in phytoestrogen-modulated steroid secretion by porcine adrenocortical cells?

The phytoestrogens genistein and daidzein had been found to affect the function of some tissues via oestrogen receptors (ER). In addition, genistein, but not daidzein, is considered to be a protein tyrosine kinase (PTK) inhibitor. Thus, the involvement of oestrogen receptors and PTK in phytoestrogen action on adrenocortical porcine steroidogenesis was examined in this study. The aims of the experiment were to test the effects of (i) ICI 182, 780 (ICI), an ER antagonist, on genistein- and daidzein-modulated cortisol and androstenedione (A4) secretion by adrenocortical cells isolated during the luteal and follicular phases of the porcine oestrous cycle; (ii) tyrphostin AG 957 (TAG), a nonsteroidal PTK inhibitor, on cortisol and A4 secretion by the cells and (iii) the phase of the porcine oestrous cycle on the mechanism of phytoestrogen action. Adrenals were harvested during the luteal (n = 5 animals) and follicular (n = 5 animals) phases of the oestrous cycle from locally slaughtered crossbred gilts. The isolated adrenocortical cells were incubated for 8 h (37 °C, 95% air, 5% CO2) with genistein (5 or 10 μM) or daidzein (5 or 10 μM) in the presence or absence of ICI (0.5 μM) or TAG (5 or 10 μM). Genistein and daidzein inhibited cortisol secretion and stimulated A4 secretion by porcine adrenocortical cells harvested during both the luteal and follicular phases of the oestrous cycle. The ER antagonist ICI did not eliminate phytoestrogen-induced changes in steroidogenesis. In contrast to genistein, TAG reduced the secretion of A4 and did not affect cortisol secretion. There was no observable effect due to the phase of the cycle. It is suggested that the mechanism of genistein and daidzein action in the adrenocortical cells of pigs is independent of ER and PTK. It is possible that PTK are involved in A4 secretion by porcine adrenocortical cells.     

Effects of social isolation and restraint on adrenocortical responses and hypoalgesia in loose-housed dairy cows

Effects of social isolation or restraint, applied outside the home pen, on adrenocortical and nociceptive responses were examined in 28 loose-housed dairy cows. Treatments lasted 15 min and consisted of social isolation in novel surroundings or restraint by the head in a test pen. A control treatment was applied in the test pen as well. Each cow was exposed to all treatments in a balanced order, with 3 to 4 d between treatments. Compared with the control treatment, social isolation in novel surroundings led to increased plasma concentration of cortisol (P < 0.001) as well as to indications of hypoalgesia [posttreatment lack of decrease in latency to respond toward nociceptive laser stimulation, a tendency for decreased frequency of kicking in the pauses between laser stimulations (P = 0.06), and an increased proportion of leg moving (least possible active response) after treatment (P = 0.04)]. Indications of hypoalgesia were also observed after restraint (reduced kicking in response to laser stimulation, P = 0.04); however, the indications were to a lesser extent than after social isolation, and restraint treatment did not lead to increased plasma concentration of cortisol. For control and restraint treatment, an initial increase (P < 0.02) in plasma concentration of cortisol was found, suggesting effects of pretreatment factors such as handling. No correlations between adrenocortical and nociceptive responses toward social isolation were found. The results confirm earlier reports stating that nociceptive changes induced by environmental challenges can be shown in dairy cows, even when they are kept in groups and removed from the home pen during the study of stress responses. However, testing outside the home pen seemed to affect the nociceptive and adrenocortical responses, thereby suggesting that care should be taken to avoid effects of pretreatment situational factors.     

Effects of dexamethasone infusion on the plasma cortisol response to cosyntropin (synthetic ACTH) injection in normal dogs

Graded dosages of cosyntropin (synthetic corticotropin) were injected into groups of normal dogs on consecutive days. On the first day, cosyntropin was administered alone and, on the second, dogs were infused with dexamethasone three hours before cosyntropin injection. Adrenocortical function was assessed by sequential measurement of plasma cortisol (hydrocortisone) concentration. While no response differences were noted to the various amounts of cosyntropin injected with or without dexamethasone pretreatment, the magnitude of adrenocortical response was significantly greater in dogs infused with dexamethasone. It is concluded that dexamethasone pretreatment renders the canine adrenal cortex more responsive to a subsequent injection of cosyntropin. The combined dexamethasone infusion-cosyntropin injection test produces consistent adrenocortical responses in normal dogs, and has potential value in evaluation of adrenopathic dogs.     

Potential variant of multiple endocrine neoplasia in a dog

This report describes multiple endocrine neoplasia in a dog, which is a rare hereditary disorder characterized by the presence of two or more neoplasms of different endocrine tissues within a patient. A 14 yr old dog was evaluated for polyuria/polydipsia, polyphagia, and abdominal enlargement. Adrenal-dependent hyperadrenocorticism with concomitant left thyroid enlargement and a presumed abdominal metastatic lesion were diagnosed by an adrenocorticotropic hormone stimulation test, ultrasonography, and computed tomography. Trilostane therapy was initiated and resolved the clinical signs for 2 yr at which time the dog presented with left testicular enlargement. The dog was euthanized and was diagnosed with adrenocortical carcinoma, thyroid carcinoma, an abdominal mass compatible with a metastatic lymph node, and bilateral interstitial cell testicular adenomas. To the authors' knowledge, this is the first report to describe the concomitant association of these types of endocrine neoplasms in a dog. The concomitant presence of these neoplasms could represent a potential variant of multiple endocrine neoplasia; however, the presence of the interstitial cell testicular adenomas may have only been an incidental finding. If any of these tumors are diagnosed, veterinarians should perform a thorough clinical assessment to evaluate for the presence of additional endocrine neoplasms or hyperplasia.     

Comparison of two low-dose dexamethasone suppression protocols as screening and discrimination tests in dogs with hyperadrenocorticism

Two low-dose dexamethasone suppression test protocols were evaluated in 18 dogs with hyperadrenocorticism (14 dogs with pituitary-dependent hyperadrenocorticism [PDH] and 4 dogs with adrenocortical tumor) and in 5 healthy control dogs. Blood was obtained immediately before and 2, 4, 6, and 8 hours after IV administration of either 0.01 mg of dexamethasone sodium phosphate/kg of body weight or 0.015 mg of dexamethasone polyethylene glycol/kg. At 8 hours after dexamethasone administration, 18 of 18 (100%) dogs with hyperadrenocorticism given the sodium phosphate preparation and 16 of 18 (89%) affected dogs given the polyethylene glycol preparation failed to have suppression of plasma cortisol concentration (less than 1.4 micrograms/dl). Plasma cortisol concentration was suppressed to less than 1.4 micrograms/dl at 2, 4, and/or 6 hours after administration of either dexamethasone preparation in 5 of 14 dogs with PDH and to less than 50% of baseline cortisol concentration in 10 of 14 dogs with PDH. Suppression, as identified by these 2 criteria, was not observed at 2, 4, 6, or 8 hours after administration of either dexamethasone preparation in dogs with adrenocortical tumor. For both protocols, the 8-hour plasma cortisol concentration was suppressed to less than 1.4 micrograms/dl and to less than 50% of baseline in the 5 control dogs. Both protocols were comparable for use as screening tests in establishing a diagnosis of hyperadrenocorticism. Suppression of plasma cortisol concentration to less than 50% of baseline (or less than 1.4 micrograms/dl) during the test was consistent with diagnosis of PDH. Failure to have such suppression, however, was observed in dogs with PDH as well as in those with adrenocortical tumor.     

Pituitary and adrenocortical responses to corticotropin-releasing factor in pigs

A study was conducted to determine whether differences in adrenocortical response to exogenous adrenocorticotrophin (ACTH) were an accurate reflection of an animal's perception of and response to stressful stimuli, or whether the pituitary gland might modulate adrenocortical responsiveness. Sixteen Large White x Landrace female pigs, of which 8 had high adrenocortical response to ACTH and the other 8 had low response, were administered IV a bolus of synthetic human corticotropin-releasing factor (hCRF) at dose rates ranging from 0.002 to 2 micrograms/kg of body weight. Blood samples were collected at known times for up to 2 hours after administration of hCRF. Plasma ACTH and cortisol concentrations were measured by radioimmunoassay. Results indicate that hCRF stimulated the pituitary gland of high- and low-responding pigs to secrete ACTH, which in turn stimulated the adrenal cortex to secrete cortisol. Plasma ACTH concentration, before or after hCRF administration, was not significantly different between the high and low responders. However, high-responding pigs had higher cortisol concentration after hCRF administration than did low-responding pigs. Thus, the differences in adrenocortical response to ACTH between the 2 groups of pigs were not attenuated by variation in pituitary response. It is concluded that adrenocortical responsiveness to ACTH is an accurate indicator of the perception of and the response to stress.     

Cytochrome b5 expression in gonadectomy-induced adrenocortical neoplasms of the domestic ferret (Mustela putorius furo)

Whereas the adrenal glands of healthy ferrets produce only limited amounts of androgenic steroids, adrenocortical neoplasms that arise in neutered ferrets typically secrete androgens or their derivative, estrogen. The 17,20-lyase activity of cytochrome P450 17alpha-hydroxylase/17,20-lyase (P450c17) must increase to permit androgen biosynthesis in neoplastic adrenal tissue. We screened ferret adrenocortical tumor specimens for expression of cytochrome b(5) (cyt b(5)), an allosteric regulator that selectively enhances the 17,20-lyase activity of P450c17. Cyt b(5) immunoreactivity was evident in 24 of 25 (96%) adrenocortical adenomas/carcinomas from ferrets with signs of ectopic sex steroid production. Normal adrenocortical cells lacked cyt b(5), which may account for the low production of adrenal androgens in healthy ferrets. Other markers characteristic of gonadal somatic cells, such as luteinizing hormone receptor, aromatase, and GATA4, were coexpressed with cyt b(5) in some of the tumors. We concluded that cyt b(5) is upregulated during gonadectomy-induced adrenocortical neoplasia and is a marker of androgen synthetic potential in these tumors.     

Nephrotomography and ultrasonography for the localization of hyperfunctioning adrenocortical tumors in dogs

Nephrotomography and ultrasonography were used in 11 dogs with hyperadrenocroticism to assess the value of these techniques for the localization of biochemically diagnosed hyperfunctioning adrenocortical tumors. Both techniques enabled accurate localization of a unilateral adrenal mass in each of the dogs. Cross-sectional diameters of the masses ranged from 1 to 4 cm. In 1 dog, expansion of tumor into the caudal vena cava was revealed by caudal venacavography and ultrasonography. Mineralization in the tumor mass in 2 dogs was easily recognized by nephrotomography, but not by ultrasonography. Paracostal laparotomy confirmed the presence of an adrenocortical tumor in each dog, and expansion of tumor into the caudal vena cava in 1 dog. Cross-sectional diameters of the tumors ranged from 1.2 to 4.5 cm and corresponded well with cross-sectional measurements by nephrotomography and ultrasonography. It was concluded that nephrotomography and ultrasonography have similar diagnostic accuracies for the detection and localization of hyperfunctioning adrenocortical tumors.     

Altered adrenocortical response to acute stressors or ACTH(1–24) in intensively housed pigs

In two experiments, the effect of intensive housing on behavior and adrenocortical reactivity was studied in 68 castrated male German Landrace pigs. In both experiments, half of the pigs were tethered on a partially slatted floor without straw and half were kept, as controls, in groups on straw. Prior to the study, 32 of the pigs used in the first experiment were selected according to their reaction to halothane anesthesia; for the second experiment, 36 pigs were selected according to their adrenocortical response to azaperone-metomidate anesthesia. During the 4- to 6-wk experimental period, behavioral activity was observed and adrenocortical reactivity was analyzed after stimulation with synthetic ACTH(1–24), azaperone-metomidate anesthesia, insulin-induced hypoglycemia and neostigmine-atropine. Although no difference was found in general activity between groups, tethered pigs revealed a significantly higher cortisol increase after stimulation with ACTH(1–24) in both experiments and after insulin stimulation in the second experiment. Similarly, the area below the cortisol response curve after ACTH stimulation was significantly larger in tethered pigs in the second experiment. The individual reaction pattern was positively correlated between tests, indicating this pattern is a relatively constant, individual characteristic. In contrast, no correlation was found between behavior or adrenocortical reaction pattern and sensitivity to halothane. The results suggest that tethering on a partially slatted floor constitutes a chronic stressor which changes the adrenocortical response to an additional acute stressor. This increased reactivity is revealed most clearly after stimulation with ACTH(1–24) when individual responsiveness is taken into consideration.