The position of renal denervation in treatment of hypertension: an expert consensus statement.

Hypertension is an important risk factor for cardiovascular disease. In the Netherlands, there are approximately 2.8 million people with hypertension. Despite treatment recommendations including lifestyle changes and antihypertensive drugs, most patients do not meet guideline-recommended blood pressure (BP) targets. In order to improve BP control and lower the risk of subsequent cardiovascular events, renal sympathetic denervation (RDN) has been introduced and studied as a non-pharmacological approach. While early data on the efficacy of RDN showed conflicting results, improvements in treatment protocols and study design resulted in robust new evidence supporting the potential of the technology to improve patient care in hypertensive subjects. Recently, 5 randomised sham-controlled trials demonstrated the safety and efficacy of the technology. Modelling studies have further shown that RDN is cost-effective in the Dutch healthcare setting. Given the undisputable disease burden along with the shortcomings of current therapeutic options, we postulate a new, clearly framed indication for RDN as an adjunct in the treatment of hypertension. The present consensus statement summarises current guideline-recommended BP targets, proposed workup and treatment for hypertension, and position of RDN for those patients with primary hypertension who do not meet guideline-recommended BP targets (see central illustration).

Volumetric evaluation of CT images of adrenal glands in primary aldosteronism.

OBJECTIVES: To investigate whether adrenal volumetry provides better agreement with adrenal vein sampling (AVS) than conventional CT for subtyping PA. Furthermore, we evaluated whether the size of this contralateral adrenal was a prognostic factor for clinical outcome after unilateral adrenalectomy. METHODS: We retrospectively analyzed volumes of both adrenal glands of the 180 CT-scans (88/180 with unilateral and 92/180 with bilateral disease) of the patients with PA included in the SPARTACUS trial of which 85 also had undergone an AVS. In addition, we examined CT-scans of 20 healthy individuals to compare adrenal volumes with published normal values. RESULTS: Adrenal volume was higher for the left than the right adrenal (mean and SD: 6.49 ± 2.77 ml versus 5.25 ± 1.87 ml for the right adrenal; p < 0.001). Concordance between volumetry and AVS in subtyping was 58.8%, versus 51.8% between conventional CT results and AVS (p = NS). The volumes of the contralateral adrenals in the patients with unilateral disease (right 4.78 ± 1.37 ml; left 6.00 ± 2.73 ml) were higher than those of healthy controls reported in the literature (right 3.62 ± 1.23 ml p < 0.001; left 4.84 ± 1.67 ml p = 0.02). In a multivariable analysis the contralateral volume was not associated with biochemical or clinical success, nor with the defined daily doses of antihypertensive agents at 1 year follow-up. CONCLUSIONS: Volumetry of the adrenal glands is not superior to current assessment of adrenal size by CT for subtyping patients with PA. Furthermore, in patients with unilateral disease the size of the contralateral adrenal is enlarged but its size is not associated with outcome.

Pregnancy and phaeochromocytoma/paraganglioma: clinical clues affecting diagnosis and outcome - a systematic review.

BACKGROUND: Phaeochromocytoma and paraganglioma (PPGL) in pregnancy, if not diagnosed antepartum, pose a high risk for mother and child. OBJECTIVE: To examine the clinical clues of antepartum and postpartum/postmortem diagnosis of PPGL. SEARCH STRATEGY: Case reports on PPGL in pregnancy published between 1 January 1988 and 30 June 2019 in English, German, Dutch or French. SELECTION CRITERIA: Case reports containing a predefined minimum of clinical data on PPGL and pregnancy. DATA COLLECTION AND ANALYSIS: Two authors independently performed data extraction and assessed data quality. We calculated odds ratios (OR) (with 95% confidence intervals) and used uni- and multivariable logistic regression analysis. MAIN RESULTS: Maternal and fetal/neonatal mortalities were 9.0% (18/200) and 14.2% (29/204), respectively. Maternal mortality was 42-fold higher with PPGL diagnosed postpartum/postmortem (17/58; 29.3%) than antepartum (1/142; 0.7%) (adjusted OR 45.9, 95% CI 5.67-370, P = 0.0003). Offspring mortality was 2.6-fold higher with PPGL diagnosed postpartum/postmortem than antepartum (OR 3.1, 95% CI 1.38-6.91, P = 0.0044). Hypertension at admission (OR 2.29, 95% CI 1.12-4.68, P = 0.022), sweating (OR 3.14, 95% CI 1.29-7.63, P = 0.014) and a history of PPGL, a known PPGL-associated gene mutation or adrenal mass (OR 8.87, 95% CI 1.89-41.64, P = 0.0056) were independent factors of antepartum diagnosis. Acute onset of symptoms (OR 8.49, 95% CI 3.52-20.5, P < 0.0001), initial diagnosis of pre-eclampsia (OR 6.34, 95% CI 2.60-15.5, P < 0.0001), admission for obstetric care (OR 10.71, 95% CI 2.70-42.45, P = 0.0007) and maternal tachycardia (OR 2.72, 95% CI 1.26-5.85, P = 0.011) were independent factors of postpartum diagnosis. CONCLUSION: Several clinical clues can assist clinicians in considering an antenatal diagnosis of PPGL in pregnancy, thus potentially improving outcome. TWEETABLE ABSTRACT: Systematic review of 204 pregnant patients with phaeochromocytoma identified clinical clues for a timely antepartum diagnosis.

Fibrinolysis inhibitors in plaque stability: a morphological association of PAI-1 and TAFI in advanced carotid plaque.

Essentials Fibrinolysis inhibitors are localized in advanced atheroma by immunohistology of endarterectomies. Neovascular endothelium/neocapillaries show thrombin-activatable fibrinolysis inhibitor (TAFI). Macrophage areas show free plasminogen activator inhibitor (PAI-1), notably in the vulnerable part. Free PAI-1 and TAFI stabilize active plaque area by inhibition of fibrinolysis and inflammation. SUMMARY: Background Fibrinolysis plays an important role in destabilization of atherosclerotic plaques and is tightly regulated by specific inhibitors. Objective The fibrinolysis inhibitors plasminogen activator inhibitor type-1 (PAI-1) and thrombin-activatable fibrinolysis inhibitor (TAFI) were quantified and described in the morphological context of advanced carotid plaques American Heart Association VI-VIII to elucidate their role in plaque stability. Methods Immunohistochemistry in serial sections along the longitudinal axis of endarterectomies from patients with symptomatic carotid stenosis (n = 19) were studied using an antibody specific for free PAI-1 (I205), an antibody with high affinity for TAFI/TAFIa (CP17) and established antibodies for smooth muscle cells (α-actin), endothelial cells (von Willebrand factor [VWF]), macrophages (CD68) and platelets (CD42). Results PAI-1 and TAFI show a specific distribution in these advanced plaques with a maximum corresponding to the internal carotid artery (ICA). Free PAI-1 was mainly detected in macrophages and in intravascular thrombi, and TAFI in endothelial cells (ECs) but also macrophages. The one-way ANOVA analysis with Bonferroni's correction showed a significant increase of macrophages and ECs, TAFI and PAI-1 in areas with high neovascularization in endarterectomy sections corresponding to ICA. High Spearman factors for TAFI, PAI-1 and VWF indicate neovascularization as the main source of plasma proteins, transported by platelets into the atheroma (PAI-1) or expressed by ECs (TAFI). CD68 was highly associated with VWF, PAI-1 and especially TAFI, underlining the role of macrophages in fibrinolytic activity and inflammation. Conclusion The abundance of free PAI-1 and TAFI in the plaque may inhibit plasmin generation and thereby counteract plaque destabilization by fibrinolysis, cell migration and inflammation.

Evaluating the learning curve for retroperitoneoscopic adrenalectomy in a high-volume center for laparoscopic adrenal surgery.

BACKGROUND: Laparoscopic adrenalectomy is an effective method for benign adrenal tumor removal. In the literature, both lateral transperitoneal (TLA) and posterior retroperitoneoscopic (RPA) approaches are described. Since 2007, the number of patients increased significantly in our center. Therefore, RPA was introduced in 2011 because of its potential advantages in operating and recovery times. The learning curve of RPA is now evaluated. METHODS: All data of patients undergoing laparoscopic adrenalectomy from 2007 until 2014 were prospectively collected. Patients were eligible for RPA with a tumor <7 cm, with BMI < 35 kg/m2, and with low suspicion of malignancy. The learning curve of RPA was measured by operating time. Furthermore, blood loss, preoperative complications and hospital stay were analyzed. Descriptive statistics were performed using SPSS 20.0. RESULTS: In the study period, 290 patients underwent surgery, of whom 113 underwent RPA. After starting with RPA, operating times decreased significantly (median 100 min in the first 20 patients to 60 min after 40 patients, p < 0.05). There was a significant difference in operating times (median 108 vs. 62 min, p < 0.05) and hospital stay (median 4 vs. 3 days, p < 0.05) in unilateral surgery in favor of RPA, compared to TLA. Also in bilateral surgery, operating times were significantly shorter (median 236 vs. 117 min, p < 0.05). In both groups, few major complications occurred. CONCLUSION: After the introduction of RPA, a short learning curve was seen for a single surgeon with extensive experience in laparoscopic adrenal surgery. Compared to TLA, RPA has significant advantages in operating times and hospital stay. Therefore, RPA may be the preferred approach for patients with BMI < 35 kg/m2 and small benign adrenal tumors (<7 cm).

Safety and long-term effects of renal denervation: Rationale and design of the Dutch registry.

BACKGROUND: Percutaneous renal denervation (RDN) has recently been introduced as a treatment for therapy-resistant hypertension. Also, it has been suggested that RDN may be beneficial for other conditions characterised by increased sympathetic nerve activity. There are still many uncertainties with regard to efficacy, safety, predictors for success and long-term effects. To answer these important questions, we initiated a Dutch RDN registry aiming to collect data from all RDN procedures performed in the Netherlands. METHODS: The Dutch RDN registry is an ongoing investigator-initiated, prospective, multicentre cohort study. Twenty-six Dutch hospitals agreed to participate in this registry. All patients who undergo RDN, regardless of the clinical indication or device that is used, will be included. Data are currently being collected on eligibility and screening, treatment and follow-up. RESULTS: Procedures have been performed since August 2010. At present, data from 306 patients have been entered into the database. The main indication for RDN was hypertension (n = 302, 99%). Patients had a mean office blood pressure of 177/100 (±29/16) mmHg with a median use of three (range 0-8) blood pressure lowering drugs. Mean 24-hour blood pressure before RDN was 157/93 (±18/13) mmHg. RDN was performed with different devices, with the Simplicity™ catheter currently used most frequently. CONCLUSION: Here we report on the rationale and design of the Dutch RDN registry. Enrolment in this investigator-initiated study is ongoing. We present baseline characteristics of the first 306 participants.

Steroid Hormone Production in Patients with Aldosterone Producing Adenomas.

Primary aldosteronism encompasses 2 major underlying causes: (1) aldosterone producing adenoma and (2) bilateral adrenal hyperplasia. In addition to the aldosterone excess, increased production of other compounds of the steroidogenic pathways may be involved. Until recently, most studies examined the production of steroids other than aldosterone in tumor tissue, urine, or peripheral plasma samples, but several new studies have also addressed steroid levels in adrenal venous blood samples using liquid chromatography tandem mass spectrometry. Plasma and tissue levels of several precursors of aldosterone with mineralocorticoid activity are higher in patients with aldosterone producing adenomas than in those with bilateral hyperplasia. These include corticosterone, deoxycorticosterone, and their 18-hydroxylated metabolites. Similarly, urinary, peripheral, and adrenal venous concentrations of the hybrid steroids 18-oxocortisol and 18-hydroxycortisol are higher in patients with aldosterone producing adenomas than in bilateral hyperplasia. Differences in the pathophysiology and in clinical and biochemical phenotypes caused by aldosterone producing adenomas and bilateral adrenal hyperplasia may be related to the differential expression of steroidogenic enzymes, and associated to specific underlying somatic mutations. Correct appreciation of differences in steroid profiling between aldosterone producing adenomas and bilateral adrenal hyperplasia may not only contribute to a better understanding of the pathogenesis of primary aldosteronism but may also be helpful for future subtyping of primary aldosteronism.

Ten years experience with oral immunosuppressive treatment in adult patients with atopic dermatitis in two academic centres.

BACKGROUND: There is a lack of information on the use oral immunosuppressive drugs in atopic dermatitis (AD) daily practice. OBJECTIVE: A 10-years overview of the use of oral immunosuppressive drugs in patients with severe AD. METHODS: Medical charts of patients with AD, who received oral immunosuppressive drugs at the Academic Medical Center Amsterdam and in the University Medical Center Utrecht between January 2001 and January 2011, were analysed. Particular attention was paid to patient characteristics, prior treatment, prescribed oral immunosuppressive drugs, the order of use, doses and treatment durations and reasons for discontinuation of treatment. RESULTS: Of 334 patients [53% male, mean age at start of an oral immunosuppressive drug 36.9 years (SD 13.6)] with AD received oral immunosuppressive treatment of which 102 (31%) participated in clinical trials. Cyclosporine A (CyA) was given in 80% of the patients, mycophenolate mofetil or enteric-coated mycophenolate (MMF/EC-MPS) in 31%, azathioprine (AZA) in 14%, methotrexate (MTX) in 11%, systemic glucocorticosteroids in 7% and systemic tacrolimus in 5%. In these academic centra, CyA was the first choice oral immunosuppressive in 252 patients. Reasons for discontinuation of oral immunosuppressive drugs were controlled AD disease, ineffectiveness and adverse events. CONCLUSION: Various types of oral immunosuppressive drugs have been used over the past 10 years for the treatment of severe AD with a prominent first choice for CyA. Adverse events and ineffectiveness were frequent reasons for discontinuation. A prospective database of patients using oral immunosuppressive treatments in daily practice will give more insight in the effectiveness and safety and may help to formulate future recommendations.

The effect of recombinant and plasma-derived prothrombin on prothrombin time in human plasma.

INTRODUCTION: When investigating coagulation assays to measure the effect of infused prothrombin (FII) in in vivo coagulopathy models, we found that addition of FII, plasma-derived human FII (pd-hFII) or recombinant human FII (r-hFII), to normal plasma resulted in a concentration-dependent increase in prothrombin time (PT) initiated with Innovin(®) . METHODS: The effect on PT by addition to plasma of either pd-hFII or r-hFII, using different commercial PT reagents, was studied both by turbidimetry and viscometry. RESULT: Addition of FII to plasma resulted in increased PT when initiated with Innovin(®) : PT increased with 20% by doubling the concentration. The prolongation of PT became more pronounced with 2-6000 times diluted Innovin(®) . However, by adjustment of the final free Ca(2+) concentration in the assay with diluted Innovin(®) from 8.3 to 1.3 mmol/L, no FII-dependent increase in PT was found. In contrast, no prolongation of PT was found with other commercial PT reagents. A KM = 3 nmol/L was obtained with pd-hFII, respectively, r-hFII with FII-depleted plasma using Thromborel(®) to initiate PT. CONCLUSION: At normal plasma concentration of FII, addition of FII should not have an effect on PT. The prolonged PT with Innovin(®) , but not with other PT reagents, at supranormal FII concentration is an artefact.

The effect of corn trypsin inhibitor and inhibiting antibodies for FXIa and FXIIa on coagulation of plasma and whole blood.

BACKGROUND: Corn trypsin inhibitor (CTI), an inhibitor of FXIIa, is used to prevent plasma coagulation by contact activation, to specifically investigate tissue factor (TF)-initiated coagulation. OBJECTIVE: In the present work the specificity of CTI for factor (F) XIIa is questioned. METHODS AND RESULTS: In the commercial available plasma coagulation assays CTI was found to double activated partial thromboplastin time (APTT) at a plasma concentration of 7.3 ± 1.5 µm CTI (assay concentration 2.4 µm). No effect was found on the prothrombin time (PT) when high TF concentrations were used. Also, with specific antibodies for FXIIa and for FXIa only APTT was found to be extended but not PT. With specific enzyme assays using chromogenic substrates CTI was shown to be a strong inhibitor of FXIIa and a competitive inhibitor of FXIa with Ki  = 8.1 ± 0.3 µm, without effect on the coagulation factors FVIIa, FIXa, FXa and thrombin. In thrombin generation and coagulation (free oscillation rheometry, FOR) assays, initiated with low TF concentrations, no effect of CTI (plasma concentrations of 4.4 and 13.6 µm CTI, 25 resp. 100 mg L(-1) in blood) was found with ≥ 1 pm TF. At ≤ 0.1 pm TF in the FOR whole blood assay the coagulation time (CT) concentration dependently increased while the plasma CT became longer than the observation time. CONCLUSION: To avoid inhibition of FXIa and the thrombin feedback loop we recommend that for coagulation assays the concentration of CTI in blood should be below 20 mg L(-1) (1.6 µm) and in plasma below 3 µm.

Adrenal nodularity and somatic mutations in primary aldosteronism: one node is the culprit?

CONTEXT: Somatic mutations in genes that influence cell entry of calcium have been identified in aldosterone-producing adenomas (APAs) of adrenal cortex in primary aldosteronism (PA). Many adrenal glands removed for suspicion of APA do not contain a single adenoma but nodular hyperplasia. OBJECTIVE: The objective of the study was to assess multinodularity and phenotypic and genotypic characteristics of adrenals removed because of the suspicion of APAs. DESIGN AND METHODS: We assessed the adrenals of 53 PA patients for histopathological characteristics and immunohistochemistry for aldosterone (P450C18) and cortisol (P450C11) synthesis and for KCNJ5, ATP1A1, ATP2B3, and CACNA1D mutations in microdissected nodi. RESULTS: Glands contained a solitary adenoma in 43% and nodular hyperplasia in 53% of cases. Most adrenal glands contained only one nodule positive for P450C18 expression, with all other nodules negative. KCNJ5 mutations were present in 22 of 53 adrenals (13 adenoma and nine multinodular adrenals). An ATP1A1 and a CACNA1D mutation were found in one multinodular gland each and an ATP2B3 mutation in five APA-containing glands. Mutations were always located in the P450C18-positive nodule. In one gland two nodules containing two different KCNJ5 mutations were present. Zona fasciculata-like cells were more typical for KCNJ5 mutation-containing nodules and zona glomerulosa-like cells for the other three genes. CONCLUSIONS: Somatic mutations in KCNJ5, ATP1A1, or CACNA1D genes are not limited to APAs but are also found in the more frequent multinodular adrenals. In multinodular glands, only one nodule harbors a mutation. This suggests that the occurrence of a mutation and nodule formation are independent processes. The implications for clinical management remain to be determined.

[Antihypertensives: better evenings than mornings]. / Antihypertensiva: beter's avonds dan's ochtends?

In a reasonably large proportion of patients who take antihypertensive drugs (10-30%), hypertension appears to be therapy resistant; even the use of three antihypertensives does not lower the blood pressure sufficiently. The average nocturnal blood pressure is a better predictor of cardiovascular events than blood pressure measured during the day. Antihypertensives have a stronger effect on nocturnal blood pressure when taken in the evening rather than in the morning. The exact mechanism of this has not yet been unravelled, but randomised, non-blinded studies suggest that this so-called chronotherapy does indeed lower cardiovascular risk in certain groups. The authors regard this as a promising strategy for patients with therapy-resistant hypertension in whom a nocturnal blood pressure dip does not occur.

The paradoxical stimulation by a reversible thrombin inhibitor of thrombin generation in plasma measured with thrombinography is caused by alpha-macroglobulin-thrombin.

BACKGROUND: Thrombin generation (TG) in plasma can be monitored continuously with a fluorogenic thrombin substrate using calibrated automated thrombinography (CAT). In the presence of low concentrations of a reversible direct thrombin inhibitor (DTI), CAT shows an unexpected effect: the endogenous thrombin potential (ETP) increases at low concentrations of the inhibitor to subsequently decrease concentration dependently at higher concentrations (> approximately 100 nm). OBJECTIVES: To find an explanation for this phenomenon, we measured the concentrations of free thrombin and alpha(2)-macroglobulin-thrombin complex (alpha(2)MT) with a sub-sampling technique in the presence of AR-H067637, a selective DTI. RESULTS: At all concentrations of the DTI there was a gradual dose-dependent decrease in the concentration of free, not-inhibited thrombin but a transient increase in free alpha(2)MT due to competition of thrombin and alpha(2)MT for the inhibitor. Because the CAT technique uses an algorithm to subtract alpha(2)MT activity from the total amidolytic activity, this transient increase in alpha(2)MT activity is not subtracted and erroneously attributed to thrombin itself. CONCLUSIONS: This study explains the spurious increase in ETP observed at low DTI concentrations. The results obtained in plasma were corroborated by observations in a thrombin generating system reconstituted with purified factors. In practise, the effect of DTIs on TG can be reliably evaluated from the area under the curve till time-to-peak.

[Treatment of hypertension in people over 80 years should not be discontinued]. / Behandeling van hypertensie bij mensen boven de 80 jaar niet stopzetten.

The 'Hypertension in the very elderly trial' (HYVET) was designed to answer the question whether antihypertensive treatment reduces strokes (both fatal and non-fatal strokes) without increasing total mortality. A total of 3845 patients were assigned to active treatment or placebo. About 90% had a history of hypertension, 65% of which were being treated. At the start of the study all antihypertensive treatment was stopped and the subjects were randomized to either indapamide 1.5 mg with or without perindopril 2-4 mg or to identical looking placebo. After about 2 years the trial was discontinued for ethical reasons as there was less death from any cause in the intervention group. Blood pressure decreased with 30/13 mmHg in the treatment versus 15/7 mmHg in the placebo group. There was a 30% decrease of all strokes (p = 0.06) but a significant reduction in fatal strokes (p < 0.05). Unexpected was the 21% reduction in all-cause mortality (p < 0.02). The 23% reduction in the rate of cardiovascular death was not significant (p < 0.06). Heart failure and total cardiovascular events decreased (p < 0.001). There were fewer adverse effects in the treatment group (p = 0.001). In our opinion HYVET proves that antihypertensive medication should not be stopped when patients pass the age of 80 years. However, it remains to be established whether treatment should be started in new patients who present themselves with hypertension above the age of 80 years.

[Determining aldosterone in the adrenal veins in order to establish uni- or bilateral aldosterone production in patients with primary hyperaldosteronism]. / Aldosteronbepaling in bijniervenen voor het vaststellen van enkel- of dubbelzijdige aldosteronoverproductie bij patiënten met primair hyperaldosteronisme.

In 3 patients, men aged 60, 55 and 60, respectively, with hypertension due to primary hyperaldosteronism, the aldosterone level in the adrenal veins was determined for the purpose of further diagnosis. In two patients, unilateral adrenal enlargement on the CT-scan was accompanied by overproduction ofaldosterone, in one case in a non-enlarged adrenal gland and in the other case in both adrenals. The first patient underwent adrenalectomy of the non-enlarged adrenal gland, while in the second patient surgery was decided against. The third patient had bilateral adrenal gland enlargement on the CT-scan with a surgically treatable, unilateral overproduction ofaldosterone. Now that determination ofthe aldosterone:renin ratio in plasma as a screening method in selected patients with hypertension is being used more often, primary hyperaldosteronism turns out to be more common than was previously thought. For differentiation between unilateral and bilateral overproduction of aldosterone, imaging of the adrenals, for example with CT, is insufficiently accurate. Aldosterone determination in the adrenal veins can distinguish between unilateral and bilateral overproduction of aldosterone with great accuracy, which has important therapeutic consequences.

[From gene to disease; 'apparent mineralocorticoid excess' syndrome, a syndrome with an apparent excess of mineral corticoids]. / Van gen naar ziekte; 'apparent mineralocorticoid excess'-syndroom, een syndroom met ogenschijnlijke overmaat aan mineralocorticoïden.

Apparent mineralocorticoid excess (AME) is an autosomal recessive disease caused by deficiency of the enzyme 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2). 11beta-HSD2 converts cortisol into inactive cortisone and prevents the stimulation of the mineralocorticoid receptor by cortisol. In patients with AME, an enhanced stimulation of mineralocorticoid receptors by cortisol in the distal nephron causes an elevated sodium reabsorption and increased potassium excretion. Sodium retention leads to severe low renin hypertension. The diagnosis of AME is based on the detection of an increased concentration of cortisol metabolites and a low or undetectable concentration of cortisone metabolites in urine. Molecular analysis of the HSD11B2 gene confirms the diagnosis. AME is successfully treated by potassium-sparing diuretics, sometimes in combination with loop diuretics (furosemide). Mild forms of AME might occur more frequently than is currently known and should be suspected in patients with hypertension, hypokalemia and decreased plasma renin concentration. Since liquorice can induce the clinical symptoms of AME due to reversible inhibition of the 11beta-HSD2 enzyme by glycyrrhetinic acid, the active ingredient of liquorice, patients suspected of having AME should not consume liquorice.