Assessment of the Physicochemical, Antioxidant, in-vitro Anti-diabetic and Nutritional Characteristics of Pigeon Pea Protein Hydrolysates

Background and Objective: Cajanus cajan (pigeon pea) seeds include special characteristics that can serve as alternative vegan protein sources. The aim of this study was to investigate bioactive peptides in the pigeon pea using economically feasible method of acid and enzymatic hydrolysis. Material and Methods: In this study, pigeon pea was subjected to hydrolysis by two methods of acid and enzymatic hydrolysis. The generated hydrolysates were characterized by result analysis of the protein content and yield, degree of hydrolysis, anti-nutritional profile, Fourier transform infrared spectroscopy, antioxidant assay of 2,2-diphenyl-1-picryl hydrazyl, hydroxyl radical scavenging assay, metal chelating ion assay and reducing power. Moreover, antidiabetic effects were assessed using α-amylase inhibition assay. Results and Conclusion: Pigeon pea was digested by acid (pH 4) and enzyme hydrolysis, further subjected to membrane filtration to achieve peptide fractions with bioactive characteristics. The hydrolyzed pigeon pea showed good increased protein contents and degree of hydrolysis, compared with the control. Degree of hydrolysis were 62.7% for acid, 68.42% for enzyme and 34.32% for unhydrolyzed proportion. Hydrolyzed samples included Fourier transform infrared  peaks at 3500–4000 cm-1, showing amides I and II. The resulting peptides after the hydrolysis showed a higher range in acid hydrolysis (250–20 kDa), whereas the EH fractions showed a very low molecular weight of less than 15 kDa. Peptides produced by AH demonstrated considerable bioactive characteristics, compared to EH antioxidant and anti-diabetic characteristics against the standards. This study highlights production of pigeon pea protein hydrolysates using two methods of traditional (acid) and modern (enzymatic), showing that acid hydrolysate can be a cheap economical method for generating protein hydrolysate with good bioactive characteristics.  1. Introduction Recently, high demand for plant proteins have been reported due to production of plant-based meat, shifting consumer preferences and increased awareness of their role in health and fitness. Legume seeds include a critical place in the human diet worldwide as a rich source of proteins. Legumes are classically called the poor man’s meat when animal proteins are limited or when poverty, spiritual or holy preferences prevent consumption of meat. One of the most essential dietary legumes is Cajanus cajan, commonly known as pigeon pea and red gram in English [1, 2]. Production statistics of pigeon pea reveal that India contributes to nearly 90% of the global production. Despite having a high nutritional profile, pigeon pea is underused and received little attention from research and development to unlock its potential uses in food industries. Cajanus cajan is a significant source of proteins, vitamins and minerals rich in essential amino acids (EAA), with large quantities of lysine, which is often a limiting factor in plant-based proteins within the dietary legumes playing critical roles in human nutrition. Numerous studies on pigeon pea have revealed significant findings such as antioxidant, anti-hypertensive, anti-diabetic, anti-carcinogenic, anti-coagulant, anti-inflammatory and certain satiety effects. It is the most appropriate alternative for individuals with allergies or sensitivities to other popular sources such as soy, dairy, or wheat. It offers a hypoallergenic option for incorporating proteins into various food systems. Pulse proteins are a rich source of potential bioactive sites with the help of hydrolysis, autolysis, gastrointestinal digestion and fermentation. Complex protein in pigeon pea when subjected to artificial hydrolysis, natural gastrointestinal digestion or fermentation hydrolysis forms peptides with good bioactive characteristics that can be used as functional foods, benefiting human health [1]. Multiple processes have been used for deriving protein hydrolysates, chemical and modified, of which, enzymatic protein hydrolysis is the most common process. Currently, most proteins are hydrolysed using proteolytic enzymes at the ideal temperature and pH. These often target particular peptide bonds, resulting in the release of AAs and peptides of various sizes [3]. In chemical methods, acid/alkaline hydrolysis are a conventional method. In chemical method, it is seen that mostly non-essential amino acids (asparagine, glutamine, cysteine, and tryptophan) are destroyed that are difficult to recover by acidic hydrolysis; hence, neutralization with a base (hydroxide) is recommended after heating. The hydrolysed protein is further subjected to membrane filtration or purification method [4]. The major problem of enzymatic hydrolysis is its expensive costs as well as presence of enzyme inhibitors in raw materials. The need of careful optimization and handling is essential, which can lead to enzyme denaturation or inactivation, resulting in incomplete hydrolysis with lower yields. An alternate economical method that can be used for protein hydrolysis includes acid hydrolysis. Studies have shown that essential amino acids (EAAs) such as aspartic acid, glutamic acid, proline, glycine, alanine, leucine, phenylalanine, histidine, and arginine can be achieved by acid hydrolysis (AH) [4]. Therefore, the aim of this study was to investigate the best cheap method for the production of pigeon pea protein hydrolysates via AH or enzymatic hydrolysis (EH) with good bioactive characteristics. Bioactive peptides are addressed as nutraceuticals with health advantages associated with illness prevention or therapy. Studies on the antioxidant characteristics of crude protein hydrolysates have been carried out by several researchers [5–8]. Nutritional characteristics of pigeon pea have been associated with decrease in occurrence of various cancers, HDL cholesterol, type-2 diabetes, and heart diseases. These include compounds such as protease inhibitors, non-antinutritional components and angiotensin I-converting enzyme (ACE) inhibitor with possible beneficial characteristics [5,9,10]. Studies have shown that foods packed with antioxidants provide functional health benefits by acting as exogenous sources of antioxidants to neutralize oxidants. Recently, foods rich in protein-derived peptides, typically achieved through the hydrolysis of food proteins, have been assessed for potential therapeutic functions in preventing cellular damages from oxidative stress that promotes human health. These pulse proteins are the most investigated naturally occurring alternatives to synthetic antioxidants and antidiabetic characteristics. Pulse proteins have become popular due to their availability, accessibility, affordability and simpler derivative [11]. Furthermore, the novelty of this study is to highlight advantages, disadvantages and cost-effectiveness of various methods for producing superior bioactive peptides from pigeon pea. Research on the specific effects of AH on pulse hydrolysates is in its early stages, however presenting an exciting opportunity to tailor their functional characteristics. 2. Materials and Methods Unpolished pigeon pea seeds were purchased from a local market in Karnataka, India. Olive oil was purchased from a local supermarket in Vishakhapatnam, India. All the chemicals and reagents were in analytical grade  from Sigma, Germany, Merck, Germany, and Himedia, India, and provided by a local vendor. 2.1. Preparation of the protein hydrolysate Sample preparation was carried out based on a method described previously [12]. Seeds purchased were washed, sun-dried and powdered into fine meal using 40-mesh sieve. Roasted seeds were subjected to fat extraction process for nearly 6 h using an ethanol extraction-based system. Mixture was further subjected to distillation to achieve the final fat-free product for hydrolysis. For acid hydrolysis, hydrolysates were prepared using 6 M of HCl and were then added to defatted pigeon pea flour. Extracted samples were  with 6 M hydrochloric acid per mL for various time constraints under high pressure at 121 PSI for 3–4 h. Precipitated protein at isoelectric pH was removed from the suspension by centrifugation at 12298 g for nearly 20–30 min, adjusted to pH 7.0 with 0.1 M NaOH, lyophilized and stored at 4 °C [13]. For EH, pigeon pea sample was hydrolysed enzymatically using protease pigeon pea and fat-free suspensions were prepared using 100 g of the samples that was brought to a volume of 1 L with 0.10 M phosphate buffer (pH 7–7.4 under optimum conditions with the enzyme). The enzyme complex was added for each of the experiments at room temperature (RT), based on the enzyme/substrate (E/S) ratios (m/m) (0.1, 0.3, 0.5, 0.7, and 1). The reaction mixture was transferred into a bioreactor with a capacity of 20 L. Ranges used to assess variables of pH, temperature, and time included 1–9, 50–70 °C, and 100–180 min, respectively. Separation of the solid from the supernatant was carried out for each sample when the enzymatic activity ended via heating at 90 °C for 20 min. After decanting the samples, supernatant was centrifuged at 2490 g. Further selective filtration was carried out using 0.4-µm membrane filtration and the supernatant was freeze-dried and stored at 4 °C until further use [10,11,14]. 2.2 Proximate composition of pigeon pea Pigeon pea seeds (unhydrolysed, defatted, and hydrolysate) were analyzed for crude fiber, ash, and relative humidity using standardized methods by the Association of Official Analytical Chemists (AOAC) [15]. Crude fat was assessed using Soxhlet solvent extraction system [13]. Crude fat was calculated using the Eq. 1:                                   Eq. 1 Where, W1 was the empty thimble, W2 was weight of the sample and thimble and P was weight of the sample. 2.3. Protein content assessment Kjeldahl protocol was used as described previously [16]. Briefly, 200 mg of the samples were used in the analysis. Kjeldahl method was used to assess the protein content based on the protocol of AOAC IS 7219 [15]. The assessed nitrogen content was multiplied by 6.25 (the nitrogen-to-protein conversion factor for legumes) to achieve the final protein concentration of the samples [13] using Eq. 2:                                                                                        Eq. 2 Where, a was the volume in 0.5 N acid assessed for distillation, b was the volume in 0.1 N base used for back titrating a, c was the volume in 0.5 N acid used for blank distillation a, and d was the volume in 0.1 N alkali used for back titrating c. 2.4. Degree of hydrolysis The O-phthalaldehyde assay (OPA) procedure was used for assessing degree of hydrolysis (DH) as described by Nielsen [13]. The OPA was a sensitive technique widely used for pulse proteins [14] and DH was calculated using Eq. 3:                                        Eq. 3 Where, V was the sample volume (0.1 lL), X was the sample weight (0.125 g), P was the soluble protein content (90%) of the sample and serine-NH2 was in meqv serine-NH2·g-1 protein (Eq. 4).                                                              Eq. 4 Where, α and β were respectively the constants of 1.00 and 0.40 for the raw materials that were not assessed. The DH was calculated using Eq. 5                                                       Eq. 5 Where, htot was the total number of peptide bonds per protein equivalent. 2.5. Protein yield assessment The yield of protein pigeon pea samples (defatted, AH, and EH) was assessed as per modifications and was calculated using Eq. 6:                                       Eq. 6 2.6. Assessment of anti-nutritional factors Anti-nutritional factors of tannic acid, total phenol content, phytic acid, and trypsin inhibition were carried out as previously described [15]. 2.6.1. Total phenolic content assessment The total phenol content of various samples was assessed quantitatively using Folin-Ciocalteu method; in which, gallic acid was taken as standard and the absorbance was measured at 760 nm [15]. 2.6.2. Tannins assessment Presence of tannins was assessed using Folin-Denis reagent spectrophotometric method at 700 nm [15]. 2.6.3. Phytic acid content assessment Phytic acid content of the samples was assessed spectrophotometrically at 480 nm, where Fe (NO3)3 was used as standard [15]. 2.6.4. Trypsin inhibition assessment Trypsin inhibition activity was assessed indirectly by inhibiting activity of the synthetic substrate [Nα-benzoyl-D,L-arginine p-nitroanilife hydrochloride (BAPNA)], which was subjected to hydrolysis by trypsin to produce yellow colored p-nitroanilide at 400 nm [17]. 2.6.5. Saponin assessment Saponins were assessed spectrophotometrically at 430 nm using saponin (0–40 µg) as standard [17]. 2.7. Fourier transform infrared spectroscopy The Fourier transform infrared (FTIR) spectroscopy spectra of pigeon pea AH were recorded using ALPHA-II FTIR spectrometer (Bruker Optics, Germany) fitted with an ATR (attenuated total reflectance) sampling device containing diamond crystals. The absorbance spectra were 4000–400 cm-1 (with a standard KBr beam splitter) at a spectral resolution of 4 cm-1 with 16 scans co-added and averaged. Additionally, 1–2 g of the sample were finely powdered for the scan. Data were translated into transmittance units [18]. 2.8. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of unhydrolyzed and hydrolyzed samples was carried out using 5% stacking gel and 15% separating gel using Biobase gel system (Shandong, China) at 100 V. Gels were fixed in Coomassie brilliant blue (CBB) under RT and destained to visualize bands [18]. 2.9. Amino acid composition  The pigeon pea fat-free unhydrolysed control and protein hydrolysates (AH and EH) were digested with 6 M of HCl for 24 h. Then, AA composition was assessed using HPLC 1260 Infinity Agilent system, USA, based on a method previously described [19]. Cysteine and methionine contents were assessed post performic acid oxidation as described previously [20] and the tryptophan content was assessed as described by Li et al. [8]. 2.10. Assessment of functional characteristics 2.10.1. Water and oil absorption capacities Water absorption capacities (WAC) of the protein was assessed [17, 18] and then calculated using Eq. 7:                                     Eq. 7 Where, W was the protein weight, V1 was quantity of the distilled water (DW) and V2 was volume of the water. For oil absorption capacities (OAC), 0.5 g of the samples were mixed with 5 mL of the appropriate vegetable oil and vortexed for 5 min. Slurry was centrifuged at 1968 g for 30 min and then weight of the adsorbed oil was assessed. The OAC was calculated using Eq. 8:                                                  Eq. 8 Where, W was weight of the protein sample, W1 was weight of the oil and W2 was quantity of the free oil. 2.10.2.    Emulsifying activity index (EAI) Emulsifying ability of the samples (unhydrolyzed, defatted and hydrolyzed) was carried out based on a method with modifications [19]. The EAI was calculated using Eq. 9:                                       Eq. 9 Where, A0 was the absorption at 500 nm, φ was volume of the oil fraction, and C was protein concentartion of the sample. 2.10.3.    Emulsifying stability index For emulsifying stability index (ESI), samples at a protein concentration of 1% were mixed with 9 g of vegetable oil (e.g. olive oil) and then homogenized for 5 min using ultrasonic cell crusher noise isolating chamber [19]. Two aliquots were pipetted at 0 and 10 min and further diluted with 5 ml of 0.1% SDS solution. Absorbance of the solution was recorded at 500 nm (in min) and was calculated using Eq. 10:                                                    Eq. 10 Where, A0 was the absorbance at 0 min., A10 was the absorbance at 10 min, ∆t was the time difference of 10 min. 2.10.4 Foaming characteristics Foaming characteristics were assessed, including foaming capacity (FC) and foaming stability (FS). Briefly, 200 mL of the sample solutions (unhydrolysed, defatted, and hydrolysates) with 0.5% protein concentration at various ranges of pH (2–10) were homogenized for 10 min using ultrasonic cell crusher noise isolating chamber and φ 6 probes to induce air at RT [20]. Generally, FC and FS were calculated using Eqs. 11 and 12:                         Eq. 11                          Eq. 12 Where, A was the volume before whipping (mL), B was the volume of foam at 0 min after whipping (mL) and C was the volume of foam at 20 min after whipping (mL). 2.11. Antioxidant characteristics 2.11.1 DPPH radical scavenging activity For DPPH radical scavenging activity (DRSA), antioxidant activity was assessed using samples with a concentration of 1 mg·mL-1 that were mixed in water; 0.1 mg of DPPH-1:1 (v:v) was dissolved in 99.5% methanol [14]. Potential of the radical scavenging activity was assessed using absorbance at 517 nm and Shimadzu UV-1800, Nakagyo-Ku, Japan. In general, DPPH radical scavenging activity was calculated based on Eq. 13:                    Eq. 13 Where, Asample was absorbance of the sample and Acontrol was absorbance of the control. 2.11.2. Hydroxyl radical scavenging activity For hydroxyl radical scavenging activity (HRSA), nearly 100 μl of each sample with concentrations of 20–200 g were collected and mixed with 100 μl of ferrous sulphate (3 mM) and 100 μl of 1,10-phenanthroline (3 mM; dissolved in 0.1 M phosphate buffer; pH 7.4). Then, 100 μl of 0.01% hydrogen peroxide were added to the mixture to initiate the reaction. Mixture was incubated at 37 oC for 1 h and absorbance was measured at 536 nm using Shimadzu UV-1800 spectrophotometer, Nakagyo-Ku, Kyoto, Japan [14]. Equation 14 was used to assess hydroxyl radical scavenging capacity.                            Eq. 14 Where, Asample was absorbance of the sample, Acontrol was absorbance of the control and Ablank was absorbance of the blank. 2.11.3. Metal ion-chelating assay For metal ion-chelating assay (MCA), samples (100 μg) were mixed with 250 μl of 100 mM Na acetate buffer (pH 4.9) and 30 μl of FeCl2 (0.01%, w v-1). Ferrozine (12.5 μl, 40 mM) was added to the mixture after incubation at RT for 30 min. Generally, EDTA was used as positive control. Binding of Fe (II) ions to ferrozine generated a colored complex that was measured at 562 nm using Shimadzu UV-1800, Nakagyo-Ku, Kyoto, Japan [14,21,22]. The ferrous ion chelating ability was calculated using Eq. 15:                                                                                 Eq. 15 Where, Acontrol was absorbance of the control and Asample was absorbance of the sample. Reduced glutathione (GSH) was assessed (1 mg·ml-1) concurrently with the samples as positive control for all the antioxidant activity assays (DRSA, HRSA, and MCA). 2.11.4.    Assessment of reducing power Reducing power of the protein was assessed based on a modified method [8]. Protein hydrolysate solutions with various concentrations (1, 5, 10 and 15 mg·ml-1) were prepared. Then, 1 ml of the vortexed sample was added to 2.5 ml of 0.2 M phosphate buffer (pH 6.6) and 2.5 ml of 1% potassium ferricyanide. Reaction mixture was rapidly vortexed and incubated at 50 °C for 20 min. Then, 2.5 ml of 10% TCA were added to the mixture and centrifuged at 12298 g for 10 min. Supernatant of 2.5 ml was mixed with 200 µl of deionized water and 40 µl of 0.1% FeCl3. This was set to react for 10 min and absorbance was measured at 700 nm using Shimadzu UV-1800, Nakagyo-Ku, Japan. 2.12. Assessment of anti-diabetic characteristic using α-amylase inhibition activity Inhibition of α-amylase activity was assessed using a protocol with slight modifications [14]. Briefly, 6 mM of NaCl and 100 μl of 20 ml l-1 sodium phosphate buffer (pH 6.9) were mixed with 100 μl aliquot of the sample containing 1 mg ml -1 of α-amylase solution. Further, 100 μl of 1% starch solution in 20 mM sodium phosphate buffer (pH 6.9, 6 mM) were mixed with the sample and incubated. Reaction was terminated by the addition of 200 μl of dinitrosalicylic acid and incubated at 100 °C for 5 min using boiling water bath. The reaction mixture was cooled down to RT, followed by the addition of 3 ml of double DW. Absorbance of the samples, Control 1 and Control 2 was measured at 540 nm. A standard synthetic drug (Glyciphage) was used to compare the values. Inhibition proportion of α-amylase activity was calculated using Eq. 16:        Eq. 16 Where, Control 1 represented mixture of starch solution, protein sample excluding α-amylase enzyme and Control 2 represented mixture of starch solution, α-amylase enzyme excluding protein sample. 2.13. Statistical analysis Analyses were carried out in three independent replications, with the outcomes subjected to one-way variance analysis. Statistically significant differences (p ≤ 0.05) between the mean values were assessed using Tukey test and Origin Pro software v.8.1. 3.Results and Discussion 3.1. Proximate composition of pigeon pea Crude fiber contents of unhydrolysed, defatted, acid, and enzyme hydrolysed pigeon pea seed samples were 7.56% ±0.96, 7.45% ±0.90, 7.10% ±0.80 and 6.95% ±0.80, respectively (Table 1). There was a little difference within the samples. The total ash contents were 3.76% ±0.46, 3.45% ±0.60, 3.97% ±0.70, and 4.05% ±0.60 for the highlighted samples, respectively. For crude fat content, decreases in fat concentration were reported with a decreasing order from unhydrolysed to hydrolysate samples due to the extraction of defatted seeds using Soxhlet ethanol-based extraction, leading to the removal of fat in outer and inner pods of the seeds, which resulted in 4.45% ±0.50, 2.78% ±0.40, 2.18% ±0.50, and 1.95% ±0.50, respectively. Moisture contents of the samples were respective

ASSESSMENT OF ENDOTRACHEAL TUBE CUFF PRESSURE: FINGER PRESSURE TECHNIQUE VERSUS MINIMUM LEAK TECHNIQUE

Objective: The purpose of this study is to compare routinely used cuff insufflation techniques to finger-pressure and minimal leak procedures for achieving safe endotracheal tube (ETT) intracuff pressures in patients undergoing endotracheal intubation. Methods: It is a prospective observational study conducted in patients undergoing elective surgical procedures under general anaesthesia at GITAM Institute of Medical Sciences and Research, Visakhapatnam from January 2019 to June 2020. In Group FP, which includes 50 patients, the ETT cuff (ETTc) was inflated by palpating the pilot balloon between the index finger and thumb until it became taut. When this point was reached, the syringe was detached from the pilot balloon, and a cuff manometer was attached. The pressure reading on the cuff manometer is noted. In Group ML, which includes 50 patients, the ETTc was inflated fully, and then the air was withdrawn slowly from the cuff with auscultation over the trachea until a small leak was heard. When the point was reached, the syringe was detached, and a cuff manometer was attached; pressure readings were noted. Results: Mean inflation cuff pressure in the FP group was 45.40±21.74 cm H2O and in the ML group was 28.68±8.35 cm H2O. In Group FP, out of 50 patients, cuff pressure in 14 (28%) patients was in the normal range; in 32 (64%) patients, the cuff was over inflated, and in 4 patients (8%) cuff was under inflated. In the group ML, 24 (48%) patients have cuff pressure within the normal range; in 18 (36%) patients, the cuff has been over inflated, and 8 (16%) patients have low cuff pressures. Cuff pressure adjustment was required in 36 patients (72%) in the FP group, whereas 26 patients (52%) in the ML group. ML group has a low incidence of postoperative complications, i.e., 10%, compared to the FP group, i.e., 18%. A positive correlation was seen between the measured cuff pressure and body mass index, Volume of air insufflated. Conclusion: The main conclusion is to realize the need to use manometers or better-automated controllers during routine anaesthetic procedures

ASSESSMENT OF TOTAL INTRAVENOUS ANESTHESIA BY PROPOFOL AND INHALATIONAL ANESTHESIA WITH ISOFLURANE FOR CONTROLLED HYPOTENSION IN FUNCTIONAL ENDOSCOPIC SINUS SURGERY

Objective: The study’s key objective is to compare the propofol-based total intravenous anesthesia (TIVA) with isoflurane-based inhalational anesthesia for controlled hypotension during functional endoscopic sinus surgery (FESS). Methods: This study was a prospective randomized and controlled single-blinded clinical study. The study involved 40 patients posted for elective FESS surgery, selected randomly from the ENT department. Anesthesia was induced with Inj. Midazolam 2 mg, Inj. Fentanyl 2 μg/kg, Inj. Propofol 2 mg/kg, Inj. Vecuronium 0.1 mg/kg was ventilated using oxygen, air, and Isoflurane (FiO2 of 0.5) in patients with isofurane. Injections of 2 mg of midazolam, 2 μg/kg of fentanyl,2 mg/kg propofol, and 0.1 mg/kg vecuronium, as well as oxygen and air for ventilation, were used to induce anesthesia (FIO2 of 0.5) in TIVA group patients. Fromme boezaart scale was used as an evaluation scale for surgical site bleeding. Results: The average blood loss in the isoflurane group was 134.25±4.65 ml and in the propofol group was 66.95±4.28 ml. The quality of the surgical field in the propofol group is (3.13±0.9), and in the isoflurane group is (3.13±0.8). The results are significant. Conclusion: Total intravenous anesthesia using propofol provides notable advantages over the traditionally used inhalational anesthetic technique using isoflurane in surgical field conditions and intraoperative blood loss

A Rare Case of Cavernous Haemangioma Mimicking Arteriovenous Malformation of the Arm

Cavernous haemangiomas are venous malformations that are commonly localised defects in the vasculature. These haemangiomas are typically asymptomatic but can adversely affect Quality of Life (QoL). Arteriovenous Malformations (AVMs) are also vascular defects with a congenital origin. AVMs are characterised by direct abnormal connections with high blood flow. Cavernous haemangiomas and AVMs can be differentiated using diagnostic modalities such as Magnetic Resonance Imaging (MRI) and computed tomography angiography. This case report aims to document a rare clinical presentation of cavernous venous malformations in a 30-year-old female presenting with swelling of the arm and intermittent pain for the past two years. This case illustrates an atypical presentation of cavernous haemangioma, which initially appeared as an AVM but was gradually confirmed to be a ‘cavernous haemangioma’. A 30-year-old female presented with complaints of swelling in her upper arm, which was managed by embolisation followed by surgical resection. The diagnosis was confirmed through histopathology. Histopathological examination played a crucial role in the definitive diagnosis. This case highlights a rare diagnostic challenge where imaging findings initially suggested an AVM, but histopathology confirmed a cavernous haemangioma, emphasising the importance of thorough diagnostic workup in vascular anomalies

Preservation of Quality of Life in HER2+ Metastatic Breast Cancer Patients Treated with Tucatinib or Placebo when Added to Trastuzumab and Capecitabine (The HER2CLIMB Trial)

Aims: In HER2CLIMB, tucatinib significantly improved progression-free and overall survival in patients with human epidermal growth factor receptor 2epositive (HER2þ) metastatic breast cancer. We evaluated the impact of tucatinib on health-related quality of life (HR-QoL) in HER2CLIMB. Methods: Patients were randomised 2:1 to tucatinib or placebo combined with trastuzumab and capecitabine. Starting with protocol version 7, the EuroQol 5 Dimensions 5 Levels (EQ-5D-5L) questionnaire and EQ visual analogue scale (VAS) were administered at day 1 of cycle 1, every two cycles during cycles 3e9, every three cycles during cycle 12 and thereafter and at each patient’s 30-day follow-up visit. Results: Among 364 patients eligible for HR-QoL assessment, 331 (91%) completed 1 assessment. EQ-VAS scores were similar for both arms at baseline and maintained throughout treatment. EQ-5D-5L scores were similar between the treatment arms, stable throughout therapy and worsened after discontinuing treatment. Risk of meaningful deterioration (7 points) on EQ-VAS was reduced 19% in the tucatinib vs. placebo arm (hazard ratio [HR]: 0.81; 95% confidence interval [CI]: 0.55, 1.18); the median (95% CI) time to deterioration was not reached in the tucatinib arm and was 5.8 months (4.3, -) in the placebo arm. Among patients with brain metastases (n Z 164), risk of meaningful deterioration on EQ-VAS was reduced 49% in the tucatinib arm (HR: 0.51; 95% CI: 0.28, 0.93); the median (95% CI) time to deterioration was not reached in the tucatinib arm and was 5.5 months (4.2, -) in the placebo arm. Conclusions: HR-QoL was preserved for patients with HER2þ metastatic breast cancer who were treated with tucatinib added to trastuzumab and capecitabine and maintained longer with tucatinib therapy than without it among those with brain metastases

Preservation of quality of life in patients with human epidermal growth factor receptor 2–positive metastatic breast cancer treated with tucatinib or placebo when added to trastuzumab and capecitabine (HER2CLIMB trial)

AIMS: In HER2CLIMB, tucatinib significantly improved progression-free and overall survival in patients with human epidermal growth factor receptor 2–positive (HER2+) metastatic breast cancer. We evaluated the impact of tucatinib on health-related quality of life (HR-QoL) in HER2CLIMB. METHODS: Patients were randomised 2:1 to tucatinib or placebo combined with trastuzumab and capecitabine. Starting with protocol version 7, the EuroQol 5 Dimensions 5 Levels (EQ-5D-5L) questionnaire and EQ visual analogue scale (VAS) were administered at day 1 of cycle 1, every two cycles during cycles 3–9, every three cycles during cycle 12 and thereafter and at each patient's 30-day follow-up visit. RESULTS: Among 364 patients eligible for HR-QoL assessment, 331 (91%) completed ≥1 assessment. EQ-VAS scores were similar for both arms at baseline and maintained throughout treatment. EQ-5D-5L scores were similar between the treatment arms, stable throughout therapy and worsened after discontinuing treatment. Risk of meaningful deterioration (≥7 points) on EQ-VAS was reduced 19% in the tucatinib vs. placebo arm (hazard ratio [HR]: 0.81; 95% confidence interval [CI]: 0.55, 1.18); the median (95% CI) time to deterioration was not reached in the tucatinib arm and was 5.8 months (4.3, -) in the placebo arm. Among patients with brain metastases (n = 164), risk of meaningful deterioration on EQ-VAS was reduced 49% in the tucatinib arm (HR: 0.51; 95% CI: 0.28, 0.93); the median (95% CI) time to deterioration was not reached in the tucatinib arm and was 5.5 months (4.2, -) in the placebo arm. CONCLUSIONS: HR-QoL was preserved for patients with HER2+ metastatic breast cancer who were treated with tucatinib added to trastuzumab and capecitabine and maintained longer with tucatinib therapy than without it among those with brain metastases