Urinary Aldosterone to Creatinine Ratio in Cats before and after Suppression with Salt or Fludrocortisone Acetate
S.C. Djajadiningrat-Laanen, S. Galac, S.E. Cammelbeeck, K.J.C. van Laar, P. Boer, and H.S. Kooistra
Background: The endocrine diagnosis of primary hyperaldosteronism in cats currently is based on an increased plasma aldosterone to renin ratio, which has several disadvantages for use in veterinary practice.
Objectives: To establish a reference range for the urinary aldosterone to creatinine ratio (UACR) and to determine whether oral administration of either sodium chloride or fludrocortisone acetate is effective for use in a suppression test.
Animals: Forty-two healthy cats from an animal shelter and 1 cat with primary hyperaldosteronism from a veterinary teaching hospital.
Methods: Morning urine samples for determination of the basal UACR were collected from 42 healthy cats. For the sup- pression tests, urine samples for the UACR were collected after twice daily oral administration for 4 consecutive days of either sodium chloride, 0.25 g/kg body weight (n = 22) or fludrocortisone acetate, 0.05 mg/kg body weight (n = 15).
Results: The median basal UACR was 7.2 x 10-9 (range, 1.8-52.3 x 10-9), with a calculated reference range of <46.5 x 10-9. Administration of sodium chloride resulted in adequate salt loading in 10 of 22 cats, but without significant reduction in the UACR. Administration of fludrocortisone resulted in a significant decrease in the UACR (median, 78%; range, 44-97%; P < . 001) in healthy cats. In the cat with an aldosterone-producing adrenocortical carcinoma, the basal UACR and the UACR after fludrocortisone administration were 32 × 10-9 and 36 x 10-9, respectively.
Conclusions and Clinical Importance: Using the UACR for an oral fludrocortisone suppression test may be useful for the diagnosis of primary hyperaldosteronism in cats.
Key words: Adrenal cortex; Conn’s syndrome; Feline; Primary hyperaldosteronism.
P rimary hyperaldosteronism has been diagnosed with increasing frequency in cats since first being reported in 1983. It may be caused by adrenocortical neoplasia or bilateral adrenocortical hyperplasia.1 6 Excessive secre- tion of aldosterone causes increased renal reabsorption of sodium and water and increased renal excretion of po- tassium. These aldosterone-induced changes may result in systemic arterial hypertension and potassium deple- tion, signs of which can include hypokalemic paroxysmal flaccid paresis, acute blindness due to retinal detachment or intraocular hemorrhage, and other changes attribut- able to hypertensive damage in target organs such as the kidney, heart, or brain.
The diagnosis of primary hyperaldosteronism in cats is at present mainly based upon the relation between the plasma aldosterone concentration (PAC) and plasma renin activity (PRA), ie, an increased plasma aldosterone to renin ratio (ARR).6 8 Practical disadvantages associ- ated with determining the ARR include the large volume
of blood (4mL) that is required and the necessity to instantly freeze the plasma sample after collection. PRA measurements are time consuming and there is large vari- ation in reference values among laboratories. Also, due to fluctuations in the PAC and the PRA, a single ARR result within the reference range does not exclude hyper- aldosteronism and repeated measurements may be re- quired to demonstrate this condition in cats.6 The solution could lie in the determination of aldosterone excretion in urine, as has proved useful in humans.9 The urinary aldosterone to creatinine ratio (UACR) repre- sents an integrated measure of aldosterone secretion over time. Moreover, urine for measurement of aldosterone does not require immediate freezing and can be collected quite easily.
The diagnosis of an endocrine hyperfunction fre- quently is based on the results of a suppression test. The suppressive agent is administered in a dose that reduces secretion of the hormone in healthy individuals, while causing little or no reduction in those affected with the disorder. In human medicine, oral or IV salt loading and oral administration of fludrocortisone are used in suppression tests for the diagnosis of primary hyper- aldosteronism.10-1 10-13
The aims of this study were to establish a reference range for the UACR in cats and to determine whether PO administered sodium chloride or fludrocortisone ac- etate is effective in suppressing urinary aldosterone excretion in healthy cats.
Materials and Methods
Animals
Forty-two cats from an animal shelter were enrolled in this study. The inclusion criteria were age ≥5 months, no remarkable findings on physical examination, systemic arterial blood pressure ≤160 mm Hg, plasma ARR below the upper reference limit (< 3.8
From the Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands ( Djajadiningrat-Laanen, Galac, Kooistra); Dierenartsen Lochem en omstreken, Lochem, The Netherlands (Cammelbeeck); Haags Dierencentrum, Den Haag, The Netherlands (van Laar); and the Department of Nephrology, University Medical Center Utrecht, Utrecht, The Netherlands (Boer). The study was performed at the Haags Dierencentrum, Den Haag, The Netherlands. Findings were presented in part at the 17th ECVIM-CA Congress, Budapest, Hungary, September 13-15, 2007.
Corresponding author: Sylvia C. Djajadiningrat-Laanen, Depart- ment of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 108, 3584 CM Utrecht, The Netherlands; e-mail: s.c.djajadiningrat@uu.nl.
Submitted January 1, 2008; Revised May 1, 2008; Accepted June 23, 2008.
Copyright @ 2008 by the American College of Veterinary Internal Medicine 10.1111|j.1939-1676.2008.0166.x
× 10-9),8 and laboratory results within the following reference ranges: plasma urea concentration 6.1-12.8 mmol/L, plasma creati- nine concentration 76-164 umol/L, plasma sodium concentration (Na) 146-158 mmol/L, plasma potassium concentration (K) 3.4- 5.2 mmol/L, urine specific gravity (SG) >1.020, urinary total protein to creatinine ratio <10 × 10-5, and no remarkable abnor- malities in the urine sediment.
The cats were housed and fed individually in a separate, quiet room and had access to natural light and ventilation, toys in the cages, and daily free exercise. They were groomed by and had daily social interaction with the nurses of the animal shelter. One cat was an oriental shorthair and the other 41 were domestic shorthair. Sex and age were recorded as stated by the former owner or, in the case of stray animals, age was estimated by physical appearance, the condition of the teeth, and the presence or absence of nuclear scle- rosis of the lens. The sexual integrity in female cats was judged by examining the abdominal midline for a scar indicating a previous ovariectomy or ovariohysterectomy. There were 16 intact females, 6 spayed females, 11 intact males, and 9 castrated males. Their ages (known or estimated) ranged from 5 months to 9 years, with a mean of 2.6 years and a median of 2 years. Their mean body weight was 3.3 kg (median, 3.2 kg; range, 2.0-5.0 kg). All cats were fed a single variety of commercial canned cat foodª starting at least 1 week be- fore the onset of the experiments.
A privately owned, 15-year-old, male castrated Burmese cat with primary hyperaldosteronism due to a metastasized adrenocor- tical adenocarcinoma also was studied. The diagnosis of pri- mary hyperaldosteronism was confirmed by the finding of a PAC of 2,780 pmol/L (reference range, 110-540 pmol/L) and a PRA of 270 fmol/L/s (reference range, 60-630 fmol/L/s), resulting in an ARR of 10.3 × 10-9 (reference range, 0.3-3.8 x 10-9). The cat’s only medication at the time of diagnosis was amlodipine besylateb at an oral dosage of 0.2 mg/kg body weight q24h to decrease systemic arterial blood pressure.
Blood Pressure Measurements
Systemic arterial blood pressure was measured with an ultrasonic Doppler flow detector in combination with a 5cm cuffd and a hand-held sphygmomanometer.e The cats were allowed to sit or stand and were restrained as little as possible. The sphygmomano- meter cuff was placed just above the right elbow and the Doppler flow detector was applied with ultrasound transmission gelf to the skin just above the carpus on the medial side, from which the hair had been clipped. The probe was moved around until a clear signal was obtained from the median artery and then the cuff was gently inflated to 10-20 mmHg above the pressure at which blood flow could no longer be detected. The cuff then was slowly deflated and the pressure at which a clearly audible signal first reappeared was recorded as the systolic blood pressure. The mean value of 3 con- secutive measurements was recorded.
UACR and Suppression Tests
Morning urine samples were collected at least 24 hours after completion of the initial physical and laboratory examinations for measurements of urinary aldosterone and creatinine concentrations (day 1). The UACR was the quotient of the urinary aldosterone concentration (pmol/L) divided by the urinary creatinine concen- tration (umol/L).
After collection of the first urine sample, either sodium chloride or fludrocortisone acetate was administered to 40 of the 42 cats on 4 consecutive days (days 1-4), and the second urine sample was col- lected on the morning after the last dose (day 5). Tablets of sodium chloride® were divided in half and mixed in the meals of 22 cats to provide a dosage of 0.25 g/kg body weight q12h on 4 consecutive days. The cats were carefully observed to insure that each dose was
ingested completely. Morning urine samples for determination of urinary aldosterone, creatinine, and Na concentrations were collected on days 1 and 5. Oral salt loading was considered successful if the urinary Na to creatinine ratio (USCR) increased by at least 100%.
Eighteen cats received fludrocortisone acetate (0.0625 mg tab- letsh) at a dosage of 0.025 mg/kg body weight q12h (n = 3) or 0.05 mg/kg body weight q12h (n = 15) on 4 consecutive days. The cat with primary hyperaldosteronism also received fludrocorti- sone at an oral dosage of 0.05 mg/kg body weight q12h for 4 days. Morning samples for measurement of UACR were collected on days 1 and 5.
Blood and Urine Collection, Sample Handling, and Clinical Biochemistry
For measurements of PAC and PRA, 4-mL blood was collected by jugular venipuncture into ice-chilled EDTA-coated tubes. Sam- ples were centrifuged at 4 ℃ for 12 minutes at 919xg and plasma was stored at -20℃ until assayed. One milliliter blood was col- lected in a heparin-coated tube for measurement of plasma urea, creatinine, Na, and K concentrations at the University Veterinary Diagnostic Laboratory, Utrecht, the Netherlands. Na and K were measured with a blood gas and electrolyte analyzer and urea and creatinine were measured on a Beckman Synchron CX7ª with Beck- man-Coulter reagents.k
Morning urine samples of 10mL were collected from the litter boxes, which had been cleaned, dried, and bedded with shredded plastic the night before. The samples were divided into 2 tubes. One sample was stored at -20 ℃ for measurement of urine aldosterone and the other was used for urine SG, pH, hemoglobin, glucose, sed- iment, Na, creatinine, and total protein. Urine SG was estimated with a refractometer1 and pH with indicator paper.” Urine Na, creatinine, and total protein concentrations were measured on a Beckman Synchron CX7.ª Urine glucose and hemoglobin were mea- sured with test strips.” The USCR was the quotient of the sodium concentration in mmol/L divided by the creatinine concentration in umol/L. The total protein to creatinine ratio was the quotient of the total protein concentration in g/L divided by the creatinine concen- tration in umol/L.
Hormone Measurements
PAC and PRA were measured at the Department of Nephrology of the University Medical Center, Utrecht, the Netherlands, as described previously14 and validated for the cat.8 Briefly, for mea- surement of PRA, 0.5 mL of plasma was incubated at 37 ℃ and pH 6.0 for 1 hour, in the presence of inhibitors of angiotensinases and angiotensin I converting enzyme. After incubation, the samples were deproteinized with 4mol/L acetone and ammonia (9: 1 vol/ vol) and centrifuged. The supernatants were evaporated and re- dissolved in assay buffer and angiotensin I was measured by radio- immunoassay.º Aldosterone was extracted from 1 mL of plasma using dichloromethane. The extracts were evaporated and redis- solved in assay buffer, and aldosterone was quantitated by radioimmunoassay.P A similar procedure, with additional acid hy- drolysis of the aldosterone-18-glucuronide binding, was applied to a 2 mL urine sample for measurement of the urine aldosterone con- centration. For acid hydrolysis, 1 mL of a 0.2 N HCl solution was added to 0.5 mL supernatant of a urine and dichloromethane mix- ture (1:5 vol/vol). The sensitivity of the aldosterone assay was 10 pmol/L and urine aldosterone concentrations below the sensitiv- ity (n = 3) were set at 10 pmol/L. Pooled control samples were included in each aldosterone and renin assay. The within-assay and between-assay coefficients of variation were 8 and 15%, respec- tively, for the renin assay and 6 and 14%, respectively, for the aldosterone assay. The plasma ARR was the quotient of the PAC in pmol/L divided by the PRA in fmol/L/s.
The urine corticoid concentration was measured in the day 1 urine of 11 of the 15 cats that were to receive fludrocortisone acetate at a dosage of 0.05 mg/kg body weight q12h. The measurements were performed at the University Veterinary Diagnostic Labora- tory, Utrecht, the Netherlands, by radioimmunoassay as described previously.15 The intra- and interassay coefficients of variation were 6 and 8%, respectively, and the sensitivity was 1 nmol/L. The uri- nary corticoid to creatinine ratio (UCCR) was the quotient of the corticoid concentration in nmol/L divided by the creatinine concen- tration in umol/L.
Data Analysis
Results are expressed numerically as median and range and graphically as box-and-whisker-plots. In the latter, the box repre- sents the interquartile range from the 25th to 75th percentile, the horizontal bar through the box indicates the median, and the whis- kers represent the main body of the data. Outliers are indicated by an O and extreme outliers by an asterisk.
Statistical analyses were performed using SPSS for Mac OS X.ª The Kolmogorov-Smirnov test was used to test the data for normal distribution. The UACRs and USCRs before and after suppression were compared using Wilcoxon’s signed ranks test. Reference ranges were determined by the nonparametric method of percentile estimates with nonparametric confidence intervals for the true per- centile. P < . 05 was considered significant.
Results
The basal UACR in healthy cats ranged from 1.8 x 10-9 to 52.3 x 10-9, with a median of 7.2 × 10-9 (Fig 1) and a calculated reference range of <46.5 × 10-9. Oral sodium chloride administration resulted in a significant increase (P < . 001) in the median USCR (Fig 2). The USCR increased by 4-582% (median, 103%) in 20 cats, but decreased by 19 and 29%, respectively, in 2 others. Sodium chloride administration resulted in a 2-fold or higher increase in the USCR (and thereby successful salt loading) in only 10 of the 22 cats (Fig 3A). In these 10 cats, the basal UACR (median, 8.4 x 10-9; range, 3.3- 52.3 × 10-9) did not differ significantly (P = . 78) from the UACR after oral salt loading (median, 9.25 x 10-9; range, 2.8-86.7 x 10-9) (Fig 3).
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Fludrocortisone acetate at an oral dosage of 0.025 mg/ kg body weight q12h in 3 healthy cats caused a reduction in the UACR by 23, 56, and 67%, respectively. Given PO at a dosage of 0.05 mg/kg body weight q12h in 15 healthy cats it resulted in a significant decrease (P < . 001) in the UACR from a median basal UACR of 6.9 x 10-9 (range, 2.7-17.8 × 10-9) to a median suppressed UACR of 2.2 × 10-9 (range, 0.9-5.4 × 10-9). The UACR after fludrocortisone administration was below 6.0 × 10-9 in all 15 cats. The median suppression was 78% (range, 44-97%; n = 15) (Fig 4). In 11 cats receiving fludrocor- tisone acetate PO at a dosage of 0.05 mg/kg body weight q12h the basal UCCR was within the reference range16 (< 42 ×10-6).
In the patient with confirmed primary hyper- aldosteronism, the administration of 0.05 mg fludrocor- tisone per kilogram body weight q12h on 4 consecutive days was associated with an increase in the UACR from 32.3 to 36.0 x 10-9.
Discussion
The main aim of this study was to determine whether a new urine-based diagnostic test, less sensitive to fluc- tuations in aldosterone secretion than the ARR, may be advantageous in the diagnosis of primary hyper- aldosteronism in cats. Urinary aldosterone excretion rel- ative to urine creatinine concentration was evaluated in 42 healthy cats, before and after administration of so- dium chloride or fludrocortisone acetate. The calculated reference range for the basal UACR in healthy cats was <46.5 × 10-9. Sodium chloride administration resulted in successful oral salt loading in only 10 of 22 cats and did not significantly suppress the UACR in any. In contrast, oral administration of fludrocortisone acetate at a dosage of 0.05 mg/kg body weight q12h in 15 healthy cats resulted in a significant decrease (P < . 001) in the UACR from a median basal value of 6.9 x 10-9 to a median of 2.2 x 10-9. The median suppression was 78% (range, 44-97%). However, administration of 0.05 mg fludrocortisone per
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kilogram body weight q12h for 4 consecutive days to a cat with confirmed primary hyperaldosteronism did not suppress urinary aldosterone excretion.
The reference range for the basal UACR is relatively wide, indicating marked interindividual variation in uri- nary aldosterone excretion. The UACR of 32.3 x 10-9 in the patient with confirmed primary hyperaldosteronism was within the reference range. Also, Syme et al17 re- cently reported that the differences in the basal UACR among healthy cats, cats with normotensive chronic renal failure, and cats with chronic renal failure and ar- terial hypertension were not significant. These findings illustrate that the basal UACR does not always reveal hyperaldosteronism. A suppression test may have better discriminating power.
Although an increased basal UACR may be regarded as a positive screening test, the diagnosis of primary hyperaldosteronism may require confirmation by a sup- pression test.18 Oral or IV administration of sodium chloride (salt loading) and the oral fludrocortisone
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suppression test are widely used for the diagnostic sup- pression of aldosterone secretion in humans suspected of primary hyperaldosteronism. 10-13 Because the aim of this study was to explore the potential of function tests that are practical, noninvasive, and easy to perform in veter- inary practice, attention was given to the suppressive effects of PO-administered sodium chloride and fludro- cortisone acetate.
Sodium chloride was mixed into a commercial canned cat food. Although the full dose of sodium chloride was ingested in all cases, oral salt loading was successful (ie, causing a 2-fold or higher increase in USCR) in only 10 of 22 cats. In these cats, aldosterone excretion was not significantly suppressed. Cats likely excrete an excessive sodium chloride load rapidly, and consequently the effect on aldosterone secretion could no longer be detected in urine collected the morning after the last evening dose of sodium chloride. Although 24-hour urine collection would identify the sodium and aldosterone excretion af- ter sodium chloride loading, full 24-hour urine collection is not a realistic, practical option in most cats. Therefore, based on the results of this study, it does not appear that oral salt loading is a useful test for cats suspected of pri- mary hyperaldosteronism.
Fludrocortisone administered PO in a dosage of 0.025 mg/kg body weight q12h to 3 cats caused urinary aldosterone excretion to decrease by 23, 56, and 67%,
respectively. In view of the small decrease in 1 cat, the dose was doubled in the remaining 15 cats in this study. In all of them, the oral dosage of 0.05 mg/kg body weight q12h had a marked effect on urinary aldosterone con- centration. The UACR was decreased significantly, by a minimum of 44% and a median of 78%. In contrast, oral administration of fludrocortisone in the same dose to the cat with confirmed primary hyperaldosteronism was followed by an increase rather than a decrease in the UACR. Although limited to a single case, the mag- nitude of the difference suggests that this suppression test may prove useful in the diagnosis of primary hyper- aldosteronism in cats.
The main application of the fludrocortisone suppression test is in cats suspected of primary hyperaldosteronism. Because fludrocortisone acetate can potentially have ad- verse effects on systemic arterial blood pressure and plasma potassium concentration in patients already prone to systemic arterial hypertension or hypokalemia, it is essential to monitor patients for these adverse effects.
The reference group differed from cats with primary hyperaldosteronism in at least 2 respects: age distribu- tion and environmental circumstances. Primary hyper- aldosteronism has been reported in cats ≥6 years of age.16 Although the median age of the cats in this study was 2 years, the median PAC in cats does not differ sig- nificantly among age groups,8 and thus the age mismatch may not be relevant.
Basal levels of stress are presumably different between cats in an animal shelter and those in private homes (the source of patient populations). ACTH release in re- sponse to stress may enhance the secretion of both aldosterone and cortisol. The effect of stress on cortisol secretion was illustrated in a study showing that the UCCR was increased in 12 of 97 cats in an animal shel- ter.19 Aldosterone secretion is mainly regulated by angiotensin II and plasma K concentration. An acute in- crease of plasma ACTH concentration also leads to temporary stimulation of aldosterone secretion.20,21 However, in cats that were likely to have been acutely stressed by transport, handling, and sampling by veni- puncture, Javadi et al8 found that plasma ACTH concentration was correlated positively with plasma cor- tisol concentration, but not with PAC. Thus, in the study reported here the influence of ACTH-induced aldoste- rone release on the UACR can be expected to have been small. Moreover, McCobb et al19 noted that the UCCR was lower in cats housed in modern, enriched, animal shelters than in cats housed in traditional animal shelters. The animal shelter in this study was comparable to the modern, enriched, animal shelters described by McCobb et al, implying that stress responses were probably not as extreme as reported for some of the cats in traditional animal shelters. This was confirmed by the fact that the UCCR was within the reference range in all 11 cats in which this variable was measured.
Shredded plastic was used as litter box bedding for urine collection. In an earlier study, nonabsorbant litter box material did not affect the UCCR (Kooistra, unpub- lished observation). Because aldosterone is structurally related to cortisol, it was assumed that this bedding
material would not significantly influence the UACR either. Finding a major, fludrocortisone-induced decline in the UACR from baseline levels in all healthy cats, but not in the cat with confirmed primary hyper- aldosteronism, further supported the assumption that shredded plastic is a suitable cat box filler for urine col- lection for the UACR.
At the time the aldosterone assays for this study were performed, acid hydrolysis was included routinely in order to free aldosterone from its 18-glucuronide bind- ing. In a recent study by Syme et al,17 however, acid hydrolysis did not lead to significant increases in aldoste- rone recovery from feline urine. From the latter study, it may be concluded that acid hydrolysis probably is not an essential step in aldosterone measurement in feline urine.
In conclusion, measurement of the UACR is a prac- tical, noninvasive method which, combined with fludro- cortisone-induced suppression, may be a useful tool in the diagnosis of primary hyperaldosteronism in cats. Administration of fludrocortisone acetate caused a sig- nificant reduction in the UACR in healthy cats but not in a cat with confirmed primary hyperaldosteronism.
Footnotes
ª Whiskas beef, Mars Inc, Veghel, the Netherlands
b Norvasc, Pfizer BV, Capelle aan den IJssel, the Netherlands
” Parks Model 811-B ultrasonic Doppler flow detector, Parks Medical Electronics Inc, Aloha, OR
d Babyphon infant, Rudolf Riester GmbH & Co KG, Jungingen, Germany
e Precisa N, Rudolf Riester GmbH & Co KG
f Aquasonic 100 ultrasound gel, Parker Laboratories Inc, Fairfield, NJ
Natrii Chloridum 1000 mg, Genfarma BV, Zaandam, the Nether- lands
h Fludrocortison, Aesculaap BV, Boxtel, the Netherlands
ABL, Radiometer Nederland BV, Zoetermeer, the Netherlands jBeckman Synchron CX7, Beckman-Coulter, Mijdrecht, the Netherlands
k Beckman-Coulter reagents, Beckman-Coulter
l Atago SPR-T2, Atago Co Ltd, Tokyo, Japan
m Dual-Tint, Mallinckrodt Baker Inc, Phillipsburg, NJ
” Combur5Test D, Roche, Basel, Switzerland
° Antibody from Peninsula Laboratories Inc, Belmont, CA; tracer from NEN Life Sciences Products, Boston, MA
PICN Pharmaceuticals Inc, Costa Mesa, CA
9 SPSS 11.0.4 for Mac OS X, SPSS Benelux BV, Gorinchem, the Netherlands
Acknowledgments
The authors are grateful for the assistance of the tech- nicians of the Haags Dierencentrum, The Hague. The authors thank Mrs N. Willekes-Koolschijn, Mr T. Altena, Mrs FHC Altena-van Drunen, Mrs L. Bijlsma- Heijting, Mrs HC Boswijk-Appelhof, Mr F. Frederiksz, Ms S. van Ginkel, Ms BE den Hartog, Ms M. van Houten, Mr H. Koeslag, Mr MW van Leeuwen, Ms AE Meijer, Mrs CSJM Meijnen-Klijn, Ms L. Overbeek, Ms GFCS Purimahuwa, Ms M. van der Schaar, Ms PCE Spithoven,
and Mr JPHM Vossen for their valuable technical assis- tance, and Mars Inc, Veghel, the Netherlands, for provid- ing the Whiskas beef. The authors thank Ms YWEA Pollak for creating the figures and Dr BE Belshaw and Prof Dr A. Rijnberk for their critical review of this paper. The study was not supported by a grant or otherwise.
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