Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author. The micronutrient status of long-term Home Enteral Nutrition (HEN) patients of Te Whatu Ora Counties Manukau A thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Nutrition and Dietetics Massey University, Albany, New Zealand. Marcos Mantovani 2023 2 Abstract Background: Iron, folate, vitamin B12, copper, and zinc are essential nutrients that play a role in metabolic processes associated with disease prevention and improving health complications and well-being. Enteral feeds contain adequate nutrients that patients may require. However, these nutrients may be digested and absorbed differently than in whole foods. Nutrient deficiencies can only be treated once recognised and confirmed, so it is in our best interest to know if deficiency is present in patients receiving long-term home enteral nutrition (HEN). Aim: To investigate the nutritional status of long-term enterally fed patients over 18 years of age in Te Whatu Ora Counties Manukau. Methods: Data from 42 patients receiving long-term enteral nutrition for ≥ 4 weeks were collected. The blood concentrations of iron, copper, zinc, folate and vitamin B12 of only 21 participants were obtained and compared to recognised cut-offs for adults. For all participants, a detailed 5 x 24-hour recall of dietary intake, including enteral nutrition (EN) and oral food sources, was determined and compared to the Recommended Daily Intake (RDI) for age appropriate. A physical assessment of nutritional signs and symptoms determined the presence of deficiency for the selected micronutrients. The Charlson comorbidity index score (CCIS) was evaluated and categorised by summing the weight of 17 comorbidities, severe (>5), moderate (3-4), mild (1-2), or no comorbidities (0), indicating the degree of mortality within the next 10-years. Descriptive statistical analyses were completed for participants' characteristics, demographics, and health characteristics by gender. Generalised linear models and binary regression estimated the association between dietary intake, biomarker status and physical signs and symptoms of deficiency. A stepwise regression method was performed on the model residuals to confirm normality (histogram and Shapiro-Wilk), independence (Durbin-Watson), equality of variance (scatter plot) and multi-collinearity (VIF and tolerance). Results: The rates of total participants with blood results lower than the reference ranges for iron and zinc were 19.04% (n=4) and 66.7% (n=14), respectively. Folate, vitamin B12 and copper were all within their respective reference ranges. Dietary intake for women only was below the RDIs for iron (n=9, 47%), dietary folate equivalent (DFE) (n=3, 15.9%), vitamin B12 (n=3, 26%), zinc (n=1, 5.3%), and copper (n=1, 6.3%). Most participants had adequate dietary intake via EN feeds, excluding iron, with a mean±standard deviation of 17.1±8.3 mg/d (RDI <18mg/d for women). Alopecia was correlated with decreased dietary intake of iron and zinc and reduced serum zinc concentrations. The presence of eczema, dermatitis and perioral 3 stomatitis and the absence of wound healing and alopecia combined were associated with lower blood zinc concentration. Increased age, cerebrovascular accident (CVA) & transient ischemic attack (TIA), liver disease, solid tumour and myocardial infarction were contributor comorbidities found to decrease the risk of 10-year survival rates among the participants. Conclusion: Blood results below the reference range values for iron and zinc were significant. Given that the total dietary intake for iron and zinc was insufficient to meet the RDIs for women, it may suggest that dietary iron and zinc are lacking in the diets of HEN women patients. Strategies to improve this, including fortification and supplementations, may positively impact the nutritional status of HEN patients and should be investigated further. The physical signs and symptoms of deficiency were largely unrelated to their biomarker status and dietary intake; however, it is essential to regularly monitor the nutrition physical examination of HEN patients to detect any clinical symptoms of nutrient-related deficiencies. 4 Acknowledgements I would like to acknowledge all the incredible people that have supported me on this journey to fulfil my Master of Science in Nutrition and Dietetics, without whom this research project would not have been possible. Firstly, I would like to thank my supervisors, Professor Rozanne Kruger and Andrew Xia, for their invaluable patience and feedback. Without their critique, excellence and constant motivation, this project would not be what it is today, and I am deeply grateful to have had the opportunity to complete this project with both of you as my supervisors. I am also thankful to my cohort members for their moral support throughout the challenging times during this journey. Thanks should also go to the research assistants and participants from the university, especially Sophie Turner and Sally Pattison who contributed significantly with data collection and made this thesis possible. To my wonderful partner, Thomas Russell, for being incredibly patient and understanding on this journey. I would not be where I am now without your love, emotional and financial support. To all, thank you for your words of encouragement at times when I needed them the most. Thank you for always encouraging and pushing me towards excellence. 5 Table of Contents Abstract ...................................................................................................................................... 2 List of Tables ............................................................................................................................. 8 List of Figures ........................................................................................................................... 8 1. Chapter 1: Introduction .................................................................................................... 9 Background ................................................................................................................ 9 Purpose of Study ....................................................................................................... 13 Aims and Objectives ................................................................................................. 14 Structure of thesis ..................................................................................................... 15 Research's contributions ........................................................................................... 15 2. Chapter 2: Review of Literature ..................................................................................... 17 Enteral nutrition and home care ............................................................................... 17 Malnutrition ............................................................................................................. 18 Various enteral feeding methods ............................................................................... 21 Non-Pathophysiological Factors Affecting Micronutrient Deficiency ........................ 22 Age ................................................................................................................................................ 23 Gender ........................................................................................................................................... 24 Mouth Health ................................................................................................................................ 25 Enteral feeding formulations ......................................................................................................... 26 Micronutrients Assessment ....................................................................................... 27 Iron ................................................................................................................................................ 27 Vitamin B12 ................................................................................................................................... 30 Folate ............................................................................................................................................. 31 Zinc ............................................................................................................................................... 32 Copper ........................................................................................................................................... 33 24-hour Diet-Recall Assessment ................................................................................ 34 Nutrition-Focused Physical Assessment .................................................................... 35 Skin ............................................................................................................................................... 36 Nails .............................................................................................................................................. 37 Hair/Head ...................................................................................................................................... 37 Mouth ............................................................................................................................................ 37 Eyes ............................................................................................................................................... 38 Te Whatu Ora Counties Manukau............................................................................ 38 6 3. Chapter 3: Research Study Manuscript ......................................................................... 41 Abstract .................................................................................................................... 41 Background .............................................................................................................. 42 Materials and Methods ............................................................................................. 43 Study Design and Participant’s Recruitment ................................................................................ 43 Ethical considerations ................................................................................................................... 44 Dietary assessment – 24-hour diet recall ...................................................................................... 44 Biomarker analysis ........................................................................................................................ 45 Nutrition Focused Physical Examination ...................................................................................... 45 Charlson Comorbidity Index Score ............................................................................................... 46 Statistical Analysis ........................................................................................................................ 46 Results ...................................................................................................................... 47 Demographics and participants' characteristics ............................................................................ 48 Charlson Comorbidity Index Score by Gender ............................................................................. 49 Total dietary intake of Energy, iron, folate, vitamin B12, copper and zinc. .................................. 51 Biomarker status............................................................................................................................ 51 Enteral nutrition and oral intake food sources by gender ............................................................. 55 Nutrition-Focused Physical Assessment ....................................................................................... 57 Association analysis between C-reactive protein, albumin, and haemoglobin with blood levels for iron, folate, vitamin B12, copper and zinc. ................................................................................................... 58 Association between blood test results of zinc, iron, vitamin B12, folate and copper by their nutrition-focused physical signs and symptoms of deficiency. ................................................................... 59 Association between dietary intake of zinc, iron, vitamin B12, folate and copper by their nutrition- focused physical signs and symptoms of deficiency. .................................................................................. 60 Linear regression analysis to predict zinc, iron and copper blood levels using combined physical signs and symptoms of deficiency and dietary intake. ................................................................................ 61 Discussion ................................................................................................................. 62 Iron ................................................................................................................................................ 64 Zinc ............................................................................................................................................... 65 Copper ........................................................................................................................................... 66 Vitamin B12 ................................................................................................................................... 68 Folate ............................................................................................................................................. 68 Inflammatory markers ................................................................................................................... 69 Charlson comorbidity index .......................................................................................................... 71 7 4. Chapter 4: Conclusions and Recommendations ............................................................ 73 Strengths .................................................................................................................. 75 Limitations ............................................................................................................... 75 Final Recommendations............................................................................................ 76 Conclusion ................................................................................................................ 76 Abbreviations ........................................................................................................................... 78 5. References ....................................................................................................................... 79 6. Appendices ....................................................................................................................... 90 Appendix A – Supplementary Results ................................................................................... 91 Multiple linear regression analysis to predict blood zinc level using zinc’s physical signs of deficiency and dietary intake. .............................................................................................................................................. 91 Multiple linear regression analysis to predict blood iron level using iron’s physical signs of deficiency and dietary intake. .............................................................................................................................................. 92 Simple regression analysis to predict blood copper levels using copper’s physical signs of deficiency and dietary intake. .............................................................................................................................................. 93 Length of enteral feeding received for all participants, including participants with lower biomarker for zinc and iron. 94 Appendix B: Participant Information Sheet.......................................................................... 95 Appendix C: Charlson Comorbidity Index Form ................................................................. 98 Appendix D: Nutrition Focused Physical Examination Form (NFPE)................................. 100 Appendix E: 24-hours Diet Recall Assessment Form .......................................................... 106 Appendix F: FoodWorks Analysis (SOP) ............................................................................ 108 Appendix G: Journal requirements: Nutrition Journal ....................................................... 109 8 List of Tables Table 1: Researcher's contributions .......................................................................................................15 Table 2: Iron status measure and cut-offs. .............................................................................................30 Table 3: Vitamin B12 status measure and cut-off. ..................................................................................31 Table 4: Folate status measure and cut-off. ...........................................................................................32 Table 5: Zinc status measure and cut-off. ..............................................................................................33 Table 6: Copper status measure and cut-off. .........................................................................................34 Table 7: Table of variables in each model .............................................................................................47 Table 8: Participant characteristics, demographics, and health characteristics by gender. ...................48 Table 9: Prevalence of comorbid conditions in each category of the Charlson Comorbidity Index. ....50 Table 10: Iron, folate, vitamin B12, copper and zinc: total intake and nutritional biomarkers in relation to cut-off values. ....................................................................................................................................53 Table 11: Comparison of micronutrient intake distribution, oral intake food sources vs enteral feeding sources by gender in relation to RDIs. ...................................................................................................56 Table 12: Frequency of micronutrient deficiency: Nutrition focused physical findings by gender. .....57 Table 13: Correlation coefficients between blood levels of micronutrients, c-reactive protein, albumin, and haemoglobin levels. ........................................................................................................................58 Table 14: Regression analysis of blood results: zinc, iron, vitamin B12, folate and cooper with its expected physical signs and symptoms of deficiency. ..........................................................................60 Table 15: Regression analysis of total dietary intake of zinc, iron, vitamin B12, and folate and cooper with its expected physical signs and symptoms of deficiency. .............................................................61 Table 16: Multiple linear regression analysis for association between blood zinc, copper and iron level and physical signs of deficiency and dietary intake. .............................................................................62 List of Figures Figure 1. Distribution of Charlson Comorbidity Index Score for all participants. ................................49 Figure 2. Comparison of Estimated 10-year Survival Rate by CCIS for all participants. .....................51 Figure 3: Positive linear regression by albumin with iron and zinc. ....................................................58 Figure 4: Negative linear regression by albumin with copper..............................................................59 9 1. Chapter 1: Introduction Background Eating is essential for humans, both from a biological and a social perspective (Rolandelli, 2005). When being affected by a disease, the ability and pleasure of eating can be reduced or impaired. The risk of being malnourished is apparent for patients who cannot eat and take in their daily need for nourishment (Boullata et al., 2017). Individuals who rely on nourishment through feeding tubes encounter difficulties with oral eating due to issues related to chewing and swallowing. These challenges often stem from neurological and gastrointestinal diseases, cancer, and brain injuries (Rolandelli, 2005; Santos, Fonseca, Carolino, & Guerreiro, 2016). Malnutrition implies deviations from normal nutrition (Morrow & Raymond, 2021), but there is no common consent about a uniform definition (Soeters et al., 2017). In addressing malnutrition, the treatment can range from oral support, including food fortification and oral supplements (Boullata et al., 2017), to more intensive measure. For severe cases, the treatment may be initiated with a feeding tube. If enteral feeding fails, parenteral nutrition may be required (Baxter, Speight, & Weir, 2021). For patients unable to meet their nutritional needs orally (i.e., patients with eating problems having their gut preserved), enteral feeding tube is the preferred mode for delivery of nourishment (Boullata et al., 2017; Liley & Manthorpe, 2003). Historically, feeding through a tube has been around as early as 1800 (Minard, 2006). During the 1950s and 1960s, attention was paid to developing feeding formulas containing complete nutrition, which were shown to positively impact the patient's wellbeing (Phillips, 2006). Enteral nutrition (EN) is one of the most efficient nutritional methods in medical nutrition therapy and refers to the intake of food directly into the gastrointestinal tract bypassing the oral cavity (Boullata et al., 2017; Nishiwaki et al., 2011; Oliver, Allen, & Taylor, 2005). Worldwide, enteral nutrition is considered to be the act of providing nutrition intake through the gut, orally or through an enteral access device (Boullata et al., 2017; Ferrie et al., 2018). There are several routes for the delivery of feeding tubes. The most frequent tube methods used in long-term HEN include; percutaneous endoscopic gastrostomy (PEG), PEG with jejunal extension (PEG-J), nasojejunal tube (NJT), percutaneous endoscopic jejunostomy (PEJ), radiologically inserted gastrostomy (RIG), and jejunostomy tube (JJ) (Boullata et al., 2017; Pearce, 2002). These feeding tubes appear safe, well-tolerated, and effective in treating various nutrition-related deficiencies (Boullata et al., 2017; Oliver et al., 2005; Pearce, 2002). 10 According to guidelines from the European Society for Clinical Nutrition and Metabolism (ESPEN), PEG is a safe and comfortable long-term enteral feeding method for patients considered appropriate for PEG (Löser et al. 2005). However, there are still different methods for hospitals for the choice of route for enteral feeding. Home enteral nutrition (HEN) significantly improves patients' quality of life in community healthcare settings (Ojo & Brooke, 2016). Home enteral nutrition is considered short-term when administrated for up to 4-6 weeks, beyond which time it is regarded as a long-term feeding strategy (Bischoff et al., 2020). In previous studies, micronutrient deficiency is said to have become evident during HEN treatment. The writers (Kang, Baik, & Chung, 2014; Nishiwaki et al., 2011; Oliver et al., 2005; Vural, Avery, Kalogiros, Coneyworth, & Welham, 2020) have identified trace elements such as zinc (Zn), iron (Fe), copper (Cu), vitamin B12, and folate to be in deficit in a patient receiving HEN over different timeframes. According to Khan & Jialal (2022), iron and zinc biomarker status is decreased within 1-2 months. This author discovered an increase in types of micronutrients becoming deficient after 2-6 months of HEN, including iron, zinc, copper, and folate, decreased subsequent to vitamin B12 deficiency and became evident within 8-16 weeks. It is well known that a deficiency of micronutrients can lead to physical signs and symptoms. However, few studies have explored the relationship between HEN, dietary intake, and the nutrition physical signs and symptoms of micronutrient deficiency within a single study design. This area remains largely unexplored. To date, the existing literature indicates that significant deficiencies in micronutrients, such as Zn, Fe, Cu, vitamin B12, and folate, can lead to physical manifestations of nutrient deficiency. For instance, particularly when the deficiency is prolonged and severe. Such deficits can thereby worsen health outcomes (Boland et al., 2017; Milne, 1994; Oliver et al., 2005). Physical signs and symptoms of these nutrients can become apparent when the nutritional status is deprived (MacQuillan, Ford, & Baird, 2021). The functional improvement of the body as micronutrient status improves, counteract the physical signs of deficiency. It also enhances metabolism and tissue function, which supports the reversing process of health-related disorders such as anaemia, wound healing, bone health, oxidative damage, skin and eye disease, inflammation and overall improved immune system function (Johnson, 2002; Kang et al., 2014; Okada et al., 2001; Shenkin, 2006). It is, then, vital to consider the impact of adequate nutrient intakes on health status in patients undergoing long- term HEN. 11 Zinc deficiency leads to skin rashes and delayed wound healing (Maxfield, Shukla, & Crane, 2022). Mild iron, vitamin B12, and folate deficiency may result in hair loss, weight loss, tiredness and irritability. Severe deficiency of these nutrients is associated with anaemia and neurological defect disorders such as progressive wasting of the muscles controlling movement, depression, Alzheimer's disease, Parkinson's disease and most commonly, stroke (Ferrie et al., 2018; Khan & Jialal, 2022; Stavroulakis & McDermott, 2016; Vural et al., 2020). Patients with a copper deficiency may have sparse, brittle, and kinky hair (Altarelli, Ben- Hamouda, Schneider, & Berger, 2019). Other factors, such as increasing age and altered gastrointestinal absorption, may attenuate the bioavailability of the selected nutrients studied in this thesis (Bailey et al., 2011). Inadequate nutrient intakes also have been associated with lower biomarkers of these nutrients (Fraser et al., 1989; Khan & Jialal, 2022; Nishiwaki et al., 2011; Oliver et al., 2005). Studies suggest that patients who cannot eat orally and rely on HEN for their daily requirements might be at a higher risk of developing micronutrient deficiency than patients who solely get their nutrients orally (Kajiyama et al., 2001; Kang et al., 2014; Okada et al., 2001; Oliver et al., 2005). Dietary intake in the EN population is also shown to be affected by compliance and adherence to EN treatment (Alicia Gea, María, Javier, & Elsa, 2019). According to Kraft et al. (2012), patients undergoing long-term EN feeding regimes show noncompliance to EN intake. The authors did not present figures; however, their system made it possible to receive alerts such as that the patients were not taking EN supplements since they disliked the flavour and texture of EN formulas. Alicia et al. (2019), in agreement, state that the EN population commonly experience a decrease in appetite caused by nausea sensations, barriers of beliefs, ignorance and forgetfulness or carelessness, which can lead to neglecting compliance with EN and reduction of dietary intake (Boland et al., 2017; Boullata et al., 2017). This ultimately has implications affecting dietary patterns, thereby lowering serum biomarker concentrations of Zn, Cu, Fe, vitamin B12 and folate (Gille & Schmid, 2015; Kang et al., 2014; Osland, Polichronis, Madkour, Watt, & Blake, 2022). Trace elements such as Fe, folate and vitamin B12 are intertwined in the production of red blood cells in the haematopoietic system; in particular, they are the most common nutrient deficiencies associated with anaemia (Snow, 1999). Copper plays many essential roles in the body by acting as a cofactor for the enzyme function of ceruloplasmin and assisting in haemoglobin formation, cell signalling, and cellular respiration (Myint, Oo, Thein, Tun, & Saeed, 2018; Osland et al., 2022). Zinc's role within the 12 human body is extensive in reproduction, immune function, and wound repair (Maxfield et al., 2022). The Nutrient Values Reference Values (NRVs) for Australia and New Zealand refer to the levels of the recommended intake of essential nutrients, such as vitamins and minerals (NHMRC, 2017). The NRVs include a level known as the recommended dietary intake (RDI), which is the average daily intake level of a particular nutrient sufficient to meet the requirements of nearly all healthy individuals in a specific life stage and gender group. These include zinc, iron, vitamin B12 and Folate. For Cu, an adequate intake (AI) is used when an RDI cannot be determined, which estimates of nutrient intake of apparently healthy people that are assumed to be adequate (NHMRC, 2017). Inflammatory markers, C-reactive protein (CRP) and albumin are pentameric proteins synthesised by the liver (Morrow & Raymond, 2021). For decades, these markers have been used as indicators of inflammation or malnutrition, especially in patients in clinically unstable conditions. During the acute phase response, serum levels of negative proteins (e.g., albumin and pre-albumin) decrease, whilst positive acute-phase proteins (e.g., CRP) increase. This shift in protein concentrations can be attributed to the body prioritising the production of immune mediators during stress, and downregulating the production of non-essential proteins for immune function. Since albumin levels can decline in inflammation regardless of nutrition status, both CRP and albumin are used as an independent marker to assess the presence of inflammation. The Charlson Comorbidity Index Score (CCIS) is a valuable tool that quantifies the mortality risk for patients with various comorbidities. It provides an estimate of their life expectancy, which is particularly important for HEN patients (Kobayashi et al., 2002). Patients who require feeding tubes often suffer from multiple concurrent conditions, such as cancer and organ failure (Blumenstein, 2014). Furthermore, micronutrient deficiencies can develop despite receiving HEN due to their existing medical conditions (e.g., gastrointestinal cancer). Such deficiencies can further exacerbate the comorbidities identified by the CCIS (Jorgensen et al.,2012; Voelkle et al., 2022). These comorbidities can independently lead to malnutrition, potentially reducing life expectancy (Jorgensen et al., 2012). Therefore, it is in our interest to understand the interrelation between micronutrient deficiencies and the presence of comorbidities in predicting life expectancy for this group. A deeper understanding of these factors can offer 13 evidence to improve quality of life, enhance clinical outcomes, and potentially extend life expectancy for these patients (Jorgensen et al., 2012; Kobayashi et al., 2002). In this study, the population investigated are patients from Te Whatu Ora – Health New Zealand Counties Manukau. For short, Counties Manukau District (CMD). The CMD play an essential role in promoting health and wellbeing among a variety of ethnic groups in New Zealand. The most recent CMD population projection for 2020/21 anticipated a resident population of 578,658 persons serviced by CMD (Lees J, 2021). The population comes from various ethnic backgrounds: 16.3% of CMD patients identified as Māori, 22% as Pacific, and 61.8% as other ethnicities (Lees J, 2021). A study, Nutrition and Indigenous Health in New Zealand, identified that 12% of Māori women aged 15-49 showed iron deficiency compared to European/other 7% (Grant, Wall, Yates, & Crengle, 2010), which demonstrates that Māori women, a population group that is a large part of CMD, are at risk of nutrient depletion. In summary, adequate nutrition intake is critical for people undergoing long-term HEN and assists with decreasing the risk of prolonged health outcomes that may lead to worsening clinical status (Mundi, Patel, McClave, & Hurt, 2018; Vural et al., 2020). The lack of literature comparing HEN and NFPF is evident and requires attention. Although evidence shows that clinical signs and symptoms are apparent in patients with trace elements deficiency, there is not enough evidence linking it to HEN. The population group studied in this thesis are prone to develop nutrient deficiency. The RDI and AI are used to compare adequate nutrient intake for each element and age-gender appropriately. The selected biomarker will indicate a deficiency, and the CCIS will predict life expectancy for the population. Purpose of Study The nutrition of patients undergoing long-term enteral nutrition hasn’t been thoroughly studied and is an area that requires attention. Enteral feeding is known to help people who have trouble eating and cannot meet their nutrient requirements. Trace elements such as zinc and copper appear not to be regularly tested in clinical settings in New Zealand (Song, 2010; University of Otago, 2011a), and iron, vitamin B12 and folate are the three essential nutrients associated with the development of anaemia and have also been associated with the dietary patterns linked to higher malnutrition risk in older adults in New Zealand (Green, Venn, Skeaff, & Williams, 2005; University of Otago, 2011a). 14 Therefore, whether or not patients on tube feeding receive adequate nutrition through their feeding regimes is still uncertain. Despite the degree of health implications requiring tube feeding, patients should be aware if they are prone to micronutrient deficiency. When patients require long-term home enteral nutrition (HEN) and have received preparatory guidance at the hospital, they can be discharged to their homes. When researching the scientific literature, it was evident that some studies described how HEN care should be performed, but few studies explored the nutrient intake and status of those patients. Therefore, this thesis focuses on understanding more about HEN patients and their micronutrient status. This thesis, in particular, aims to investigate the effect of selected micronutrient intakes (zinc, iron, copper, vitamin B12, and folate) on biomarker status and physical signs and symptoms of deficiency in patients receiving long-term HEN from Te Whatu Ora Counties Manukau. The CCIS is used to evaluate the level of comorbidities that may affect the life expectancy of HEN patients. It also helps compare if there is an association between CCIS and nutritional status, particularly regarding micronutrient deficiencies. To our knowledge, this is the first study conducted in New Zealand, focusing primarily on the association of micronutrient intake of zinc, iron, copper, vitamin B12, and folate on biomarker status and physical signs and symptoms of long-term HEN patients. Aims and Objectives Aim: To investigate the nutritional status of long-term home enterally fed patients older than 18 years in patients of Te Whatu Ora Counties Manukau. Objectives: 1. To assess the biomarker status of the selected minerals (iron, zinc and copper) and vitamins (folate and vitamin B12) in relation to recognized reference ranges. 2. To investigate the dietary adequacy of iron, vitamin B12, folate, zinc and copper in relation to the RDIs. 3. To investigate the presence of the nutrition-related physical signs and symptoms of deficiency of the selected micronutrients. 15 4. To investigate the estimated 10-year survival rate using the Charlson Comorbidity Index Score. 5. To determine the association between the serum zinc, copper plasma and serum iron concentrations, physical signs and symptoms of deficiency and dietary intake. Structure of thesis This thesis includes four key chapters with additional sections for references and appendices. Chapter one presents the introduction and purpose of the study and outlines the scope and justification of this research regarding iron, vitamin B12, folate, zinc and copper of long-term home enteral nutrition patients of Te Whatu Ora – Health New Zealand Counties Manukau. This chapter also outlines the study aims, objectives, and researcher contributions. Chapter two provides a review of the current literature regarding the health and nutritional status of enteral nutrition and home care, malnutrition and various enteral feeding methods of tube-fed patients, non-pathophysiological factors affecting micronutrient deficiency and investigation of micronutrient deficiency for iron, vitamin B12, folate, zinc and copper, with particular focus on literature relating to adults age >18 years old. Chapter three provides the research design and participants recruitment, ethical considerations, dietary assessment (24-hour diet recall, biomarkers analysis, nutrition-focused physical examination, Charlson Comorbidity Index and statistical analysis, results and discussion. Chapter four outlines the conclusions and recommendations based on the presented research findings, including the study's strengths and limitations. Appendices include the supplementary methods relating to the Participant Information Sheet, the Charlson Comorbidity Index Score Assessment form, the standard operating procedures (SOPs) for the Nutrition-Focused Physical Examination Form, the 24- hour Diet Recall Assessment Form, FoodWorks Analysis for iron, vitamin B12, folate, zinc and copper, and Nutrition Journal requirements. Research's contributions Table 1: Researcher's contributions Marcos Mantovani Master's student; - Primary researcher/writer - Data collection - Literature review - Statistics analysis - Writing of the thesis Andrew Xia Primary supervisor and co-investigator; 16 - Research design and funding acquisition - Thesis guidance and review - Ethics application Professor Rozanne Kruger Co-supervisor and principal investigator; - Research design and funding acquisition Thesis guidance and review - Ethics application Sally Pattison Research Fellow - Data collection Sophie Turner Research Assistant - Data collection - FoodWorks Software data analysis Dr Owen Mugride (Nutrition and Dietetics research trial manager, Massey University Laboratory supervisor - Biomarker processing advisor (teaching technician) Dr Thomas Russell George King (NZRN) Liam Perrell (NZRN) Yongsijia Wei (Research assistant, Massey University) Phlebotomists; - Biomarker collection 17 2. Chapter 2: Review of Literature Enteral nutrition and home care Individuals who are unable to safely consume enough food by mouth to meet their nutritional requirements are at high risk of malnutrition (Sánchez-Rodríguez et al., 2019). Since it is essential to prevent and combat malnutrition, enteral nutrition (EN) should be considered when a patient is not safe for oral intake and/or oral intake cannot meet complete nutritional requirements (Bischoff et al., 2020; Boullata et al., 2017; Gramlich, Hurt, Jin, & Mundi, 2018). Enteral nutrition provides nourishment through the stomach or small intestine via an enteral access device for these patients (Ferrie et al., 2018). Enteral nutrition has been shown to be safe, cost-effective, and compatible with the body's normal processes (A. Wong, Goh, Banks, & Bauer, 2018). Home enteral nutrition (HEN) refers to the provision of complete or partial feeding for patients whose acute medical condition has stabilised after being discharged from a healthcare facility (i.e., hospital) and continuing their enterally nutritional care at home (Gramlich et al., 2018). Often patients with long-term HEN have chronic medical conditions that reduce their ability to swallow, digest or absorb nutrients (Silver, Wellman, Arnold, Livingstone, & Byers, 2004). For example, neurological diseases, head and neck cancer, gastrointestinal cancer, cerebral palsy, non-neoplastic gastrointestinal disease (e.g., fistulae, oesophageal stenosis, inflammatory bowel disease), head injury, malabsorptive syndromes (e.g., short bowel syndrome), severe intestinal motility disorders, inherited metabolic diseases, and cystic fibrosis (Bischoff et al., 2020; Gramlich et al., 2018; Liley & Manthorpe, 2003). According to Flood et al. (2021), the absence of a centralised registration or reporting system for HEN services in Australia and New Zealand has resulted in a lack of data to understand the HEN care process and the number of patients receiving EN in the community is currently unknown. Interestingly, a study has suggested that patients, when discharged home, may lack sufficient understanding of the feeding regime (Liley & Manthorpe, 2003). The lack of knowledge on adherence to EN treatment could lead to underestimating treatment effects, such as lower dietary intake of prescribed oral nutrition supplements. Moreover, the effectiveness of nutritional supplementation depends on patient acceptance and compliance. (Alicia Gea et al., 2019). It is still a relatively scarcely explored area regarding patients receiving preparatory guidance about the feeding regime at home following discharge from the hospital (DeBruyne & Pinna, 2020; Gramlich et al., 2018; Mcwhirter & Pennington, 1996). 18 Overall, HEN can offer comprehensive nourishment to long-term EN patients. However, the number of individuals receiving HEN in Australia and New Zealand is unclear (Flood et al., 2021). There is also a need for more research on patients' compliance with HEN feeding and their nutrition status. Therefore, further investigation is necessary to enhance our understanding of HEN treatment and care. Malnutrition Malnutrition remains to be a major public health problem worldwide. It occurs when a population's diet lacks sufficient macronutrients like protein, carbohydrates, and fat (Millward & Jackson, 2004), or is deficient in specific micronutrients such as minerals and vitamins (Mette, Olivier, Antoine, & Nawfel, 2019). Globally, about 2 billion people are affected by micronutrient deficiency. The highest prevalence is monitored in South-East Asia and Sub- Saharan Africa, but developed countries are not excluded (WHO, 2022). In New Zealand, a study found that 56.5% of community-living older adults were at some degree of nutritional risk, and Māori were 5.2 times more likely to be at nutritional risk compared to non-Māori (McElnay et al., 2012). Also, in 2010, the Australasian Nutrition Care Survey (ANCDS) reported that 40% of the patients in acute care across New Zealand hospitals were at risk of malnutrition (Agarwal et al., 2013). Approximately 32% of them were identified as malnourished (Agarwal et al., 2013). Malnutrition, if untreated, may result in a prolonged and complicated recovery from illness or surgery (Goldstein, Katona, & Katona-Apte, 2008), an increased risk of infection, compromised wound healing and persisting functional deficits (Agarwal et al., 2013; Mette et al., 2019). This, in turn, leads to a more extended hospital stay with a possible increase in cost and a negative impact on quality of life (Volkert, Pauly, Stehle, & Sieber, 2011). Iron deficiency anaemia is a common clinical diagnosis related to iron deficiency (WHO, 2020). To show the aggrievance of micronutrient deficiency in New Zealand, the Activity & Nutrition Aotearoa (2020) reported the prevalence of inadequate iron intake in the overall population of 5.6%. It was shown to be higher in women (9.7%) and even higher for Māori and Pacific women (18.4% and 19.9%, respectively) compared to New Zealand Europeans and Others (9.3%) (Young et al., 2020). Micronutrient deficiency, if untreated, can cause visible and dangerous health conditions, but it can also lead to less clinically notable reductions in 19 energy level, mental clarity and overall capacity (Shenkin, 2006). This can further lead to an increased risk of developing other diseases and health conditions and cutaneous manifestations of micronutrient deficiency (i.e., hair, nails and skin) (Dibaise & Tarleton, 2019). In New Zealand, one in fourteen adults (6.9%) (5.0% in men and 8.7% in women) in 2014/15 was shown to have iron deficiency anaemia (Young et al., 2020). Zinc deficiency targets more than 2 billion people worldwide (Prasad, 2013). The leading causes of zinc deficiency include insufficient intake, increased requirements, malabsorption, increased losses and impaired utilization. Inadequate intake of zinc is considered to be one of the most significant determinants of the development of zinc deficiency (Maxfield et al., 2022). Zinc deficiency is commonly seen in developing regions and is attributable to malnutrition; however, it is associated with aging and many chronic illnesses and can be acquired or inherited (Maxfield et al., 2022). Zinc deficiency can occur from decreased intake, inability of absorption, increased metabolic demand, or excessive loss (Maxfield et al., 2022). Patients with an acquired form of zinc deficiency usually have a combination of various factors: inadequate nutritional intake of zinc-rich foods (e.g., meat, legumes, seeds, soy products and whole grains) and chronic illnesses (e.g., gastrointestinal diseases, liver disease, excess of alcohol or chronic infections) (Maxfield et al., 2022). A review of zinc status in Australia and New Zealand found that certain groups were at risk, including toddlers, adolescents (particularly those of Pacific Islander descent), and older people living in institutions (Anna & Samir, 2012; Gibson & Heath, 2011). While these findings may also apply to New Zealand, as the two countries share similar dietary habits, further research is needed to understand zinc intake levels among individuals in New Zealand. Copper is involved in various physiological functions, and deficiency impairs growth and reproduction and can cause biochemical alterations, leading to health problems (Altarelli et al., 2019; Leslie, 2022). Biochemical or clinical copper deficiency is common in infants recovering from malnutrition, children with chronic diarrhea, malabsorption cases, and areas with low copper content in soil or food availability (Altarelli et al., 2019; Leslie, 2022). Medical publications revealed poor copper nutrition in over 2,500 individuals with cardiovascular, musculoskeletal, and nervous diseases (Leslie, 2022). Sixty-thousand more individuals with common disorders have abnormally low copper concentrations or compromised metabolic pathways dependent on copper, which are diagnostic of deficiency (Leslie, 2022). 20 A review of the magnitude of folate and vitamin B12 deficiency worldwide states that folate and vitamin B12 deficiencies have long been known to have adverse effects on health, including anemia and neuropathy (McLean, de Benoist, & Allen, 2008). This study identified folate and vitamin B12 status most frequently assessed in women of reproductive age in 34 countries and all adults in 27 countries. Their findings show that in most countries worldwide for which national surveys were available, folate and vitamin B12 deficiencies appeared to be a public health problem; six out of eight countries were deficient in folate, and five out of seven were deficient in vitamin B12. Deficiency was identified in preschool children in Venezuela (33.8%), pregnant women in Costa Rica (48.8%) and Venezuela (25.5%), and older adults in the United Kingdom (15.0%) are the main groups affected by folate deficiency. Vitamin B12 deficiency is prevalent in school-age children in Venezuela (11.8%), pregnant women in Venezuela (10.9%) and Costa Rica (5.3%), and older adults in the United Kingdom (31.8%) and New Zealand (12.0%) (McLean et al., 2008). Deficiencies in folate and vitamin B12 may be of public health consequences, but it is unclear how prevalent these deficiencies are (Allen, 2008; McLean et al., 2008). The authors Mette et al. (2019) report that the leading causes of micronutrient malnutrition include reduction of food intake, lower bioavailability and malabsorption. Many of these deficiencies are preventable through nutrition education, a healthy diet containing diverse foods, and food fortification and supplementation as needed (DeBruyne & Pinna, 2020). Boullata et al. (2017) reports that the importance of adequate nutrition among critically ill and HEN patients is amplified by an increase in the metabolic stress response, impaired immune function and the severity of illness. This author states that it is often challenging to deliver nutrition in this population with increased requirements in the context of an altered metabolic and immune response, which lead to a cumulative energy and protein deficit, resulting in muscle wastage (Millward & Jackson, 2004), as well micronutrient deficiency (Osland et al., 2022). The influence of co-existing illnesses on prognosis, therapy and patient outcomes has been recognized since the 1970s (Lu, Barratt, Vitry, & Roughead, 2011). In epidemiological studies, clinical trials and health services research, controlling for additional co-existing diseases or comorbidity is essential (Feinstein, 1970). Comorbidity refers to the presence of one or more other health conditions co-occurring with a primary condition (Lu et al., 2011). The Charlson index (CCI) was developed in 1984 by Charlson et al. and categorizes patients' comorbidity 21 conditions based on medical diagnoses and has successfully predicted mortality in various patient populations (Lu et al., 2011). This instrument identifies comorbid conditions, which may, singly or in combination, affect the short-term mortality risk for patients (Feinstein, 1970; Lu et al., 2011). Mortality rates by the CCI can be evaluated by categorising the sum of 17 comorbidities (Lu et al., 2011) into four categories of the Charlson Comorbidity Index Score (CCIS): severe (>5), moderate (3-4), mild (1-2), or no comorbidities (0) (Huang et al., 2014). The CCIS indicates the degree of mortality within the next ten years. The CCIS has been used to evaluate the long-term prognosis of EN and the parenteral nutrition population (Kobayashi et al., 2002). A study discovered using the CCIS instrument that of 3.548 participants, 2,384 (67%) died within 730 days after the initiation of gastrostomy (GS) and nasogastric tube feeding (NGT) and parenteral nutrition (PN) in those with malignancies and enteral feeding including secondary GS, primary GS, and NGT had a better 2-year prognosis than PN in older patients with and without malignant disease (Kobayashi et al., 2002). Thus, trauma and critically ill patients receiving HEN might be at risk of micronutrient malnutrition. Vitamin and mineral deficiencies are widespread, affecting the vulnerable population and worsening clinical status. Various enteral feeding methods Enteral Nutrition is a valuable intervention for patients of all ages in various care settings and enters the body through various routes (Boullata et al., 2017). Different tubes differ based on how the tube is inserted, through the nose or abdomen and where the tube ends in the digestive system, stomach or intestine (Bischoff et al., 2020). The most commonly used enteral feeding delivery methods include nasogastric tube (NG), percutaneous endoscopic gastrostomy (PEG), and percutaneous endoscopic gastrojejunostomy (PEGJ) (Gossum, 2005). Several studies have looked at the benefits and challenges of various delivery methods, including NG and PEG. Though NG has been mostly used for short-term treatment, whilst PEG is reported as a long-term pathway (Boullata et al., 2017; Crosby & Duerksen, 2005; Ferrie et al., 2018). According to ESPEN (2020), PEG is a safe and comfortable method for long-term HEN patients (Bischoff et al., 2020). Enterally feeding methods can be described as intermittent (allocated in shorter or longer periods throughout the day) (Ferrie et al., 2018). A continuous feeding regime is defined as 22 feeding for 24 hours continuously either by gravity drip or feeding pump method (Bischoff et al., 2020; Ferrie et al., 2018). Bolus feeding delivers a liquid meal for a short feeding time (100-400ml over 15-60 minutes) using a syringe and may be repeated at intervals to achieve the required intake (Ferrie et al., 2018). Bolus feeding mainly delivers food to the stomach due to the higher capacity to tolerate a larger feed volume. The literature shows no consensus found on which method is preferred (Minard, 2006; Pearce, 2002; Phillips, 2006). Studies show that enterally fed patients are prone to under-feeding due to improper use of the tubes and gastric intolerance, leading to negative implications for nutritional status (Boullata et al., 2017; Crosby & Duerksen, 2005). The results of a prospective cohort study show that patients often do not take their enteral nourishment, causing a lack of nutrition intake (De Jonghe et al., 2001). This study concluded that, out of the 100 kcal that were required, 78.3% were prescribed; however, only 71.2% were taken. This means there was a difference between the theoretical requirement and the actual delivery of calories. The difference resulted from under-prescription, which accounted for about two-thirds of the difference, and under-delivery, which accounted for about one-third, attributed to wasted volume through interruptions caused by digestive intolerance, airway management and diagnostic procedures (i.e., tracheal tube repositioning) and mechanical issues (i.e., electric feeding pump dysfunction, gastric tube occlusion or malposition). Interestingly, other researchers (Boullata et al., 2017; Crosby & Duerksen, 2005; Pearce, 2002) agree that enteral nutrition may have shortcomings, including underfeeding, perceived intolerance, aspiration, access-related complications, vomiting and diarrhoea, which leads to under intake of nutrition. To exemplify, a previous study by Layec et al. (2011) found that patients who receive exclusive feeding through a tube in the jejunum are at a high risk of copper deficiency. This is because the duodenal and gastric mucosa are critical in copper absorption. When the duodenal site is bypassed during jejunal feeding, the absorption area of copper is reduced, leading to impaired absorption and potential copper deficiency. It has been observed that jejunal supplementation is ineffective in correcting the deficiency, but using the gastric route has been a successful and definitive solution. Non-Pathophysiological Factors Affecting Micronutrient Deficiency Several factors may influence the dietary intake of micronutrients such as iron, zinc, copper, vitamin B12 and folate. Studies in older adults have shown a positive correlation between dietary intake of iron, zinc, copper, vitamin B12 and folate and their respective serum biomarkers (Kang et al., 2014; Shi et al., 2020; Suchdev et al., 2016). However, numerous 23 factors, including age-related changes to the gastrointestinal tract, gender, oral health, enteral feeding formulations, bioavailability, and cooking methods, may hinder this association (S. Lee, Choi, Jeong, Lee, & Sung, 2017; Milne & Johnson, 1993; Russell, 2001; WHO, 2020). Non-pathophysiological factors such as poverty, lack of access to a variety of foods, and lack of knowledge of optimal dietary practices are some factors that lead to deficiency. Inadequate intake of these micronutrients can be seen with strict vegetarian diets, anorexia nervosa and when receiving exclusively enteral nutrition. Research confirms that inadequate enterally intake leads to micronutrient deficiency due to inconsistency between the prescribed and delivered nutrition methods (De Jonghe et al., 2001; Flood et al., 2021; Gregg, Reddy, & Prchal, 2002; Kang et al., 2014; Myint et al., 2018). Age Micronutrient intake in older persons can be affected by physiological changes associated with ageing and changes in health status and lifestyle (Organization, 2020; Shenkin, 2006; WHO, 2020). The KORA-Age Study conducted in Germany suggests that micronutrient deficiency tends to increase beyond age 65 and even more so after 80. Their population study identified that older adults aged 85 and over were two times more likely to have low folate and low vitamin B12 levels than those younger than that age due to problems with the acids and stomach enzymes needed to process the vitamins (Snow, 1999). As a result, reduced gastric acid secretion prevents the release of vitamin B12 from foods, affecting absorption (Conzade et al., 2017). Epidemiological evidence suggests that subclinical micronutrient deficiencies in older adults are associated with chronic age-related diseases and adverse functional outcomes (Morrow & Raymond, 2021). Absorption and the bioavailability of nutrients decrease with ageing due to changes in the gastrointestinal tract and polypharmacy, leading to malabsorption of macronutrients and micronutrients (Morrow & Raymond, 2021; Saboor, Zehra, Qamar, & Moinuddin, 2015). Plasma copper has been identified as affected by age; men and women older than 50 showed higher concentrations than men and women under 40 (Milne & Johnson, 1993). The ZENITH study assessed zinc intake and status in European males and females of age 55- 70 years (middle-aged group) and older-aged subjects (>70 years). They documented that individuals presenting a serum zinc concentration below the cut-off level were 4.8% in middle- aged and 5.6% in older subjects (Andriollo-Sanchez et al., 2005). 24 Research indicates that older adults aged ≥70 years are more likely to require EN care as they experience higher levels of disability and chronic diseases explained by the demographic transition associated with aging (Menezes & Fortes, 2019; Okada et al., 2001). Okada et. (2001) found that 44 bed-ridden patients who received tube feeding had a higher incidence of protein malnutrition than 41 free-eating elderlies. Their findings indicate a decrease in arm muscle circumference (<80% of normal) and hypoalbuminemia (<35 g/L) in EN patients higher than in healthy elders. Interestingly, the study also revealed that orally fed bed-ridden patients were malnourished, indicating that bed-ridden patients are susceptible to malnutrition despite receiving energy and protein similar to calculated predicted values. Therefore, ageing becomes a risk factor for developing diseases, and older adults have the highest risk of being at nutritional risk or becoming malnourished. Gender Recognising nutritional differences between men and women is critical to prevent gender- related micronutrient deficiency. There has been a lack of research on how gender differences impact nutrient status in tube-feeding patients. This is likely because men and women have different nutrient needs. However, we attempted to connect existing research on the relationship between gender differences and nutrient status in patients receiving EN. For example, the KORA-Age Study found that subclinical deficiencies were more common in women for folate in gender-specific analysis, 9.4% vs. 8.0 % for men. They also identified a high prevalence of deficiency in vitamin B12 (28.5% vs. 26.0%) and iron (13.5% vs. 8.4%) for women and men, respectively. Folate deficiency was also identified to be lower in the male participants, 8.2%, compared to 9.2% in females (Conzade et al., 2017). A study stated that the overall range of plasma copper concentrations was 8.8-17.5 μmol/L for men, 10.7-26.6 μmol/L for women who were not taking oral contraceptives, and 15.7-31.5 umol/L for women who were taking oral contraceptives or who were on oestrogen therapy (Milne & Johnson, 1993). In agreement, an investigation study on gender effects on plasma and brain copper discovered that plasma copper was higher in women, 1008 ± 51 ng/mL, than in male (836 ± 41) control subjects (Quinn et al., 2011). Lower serum zinc was identified in the ZENITH study showing in the middle-age group 55-70 years (6.4% men, 3.1% women) and the older group aged 70 + years (9% men and 6.2%), showing zinc deficiency higher in men compared to women despite the age difference (Andriollo-Sanchez et al., 2005). 25 Based on the results, the difference in nutrient status between gender is evident and may be translated to EN patients; however, it still needs further study to comprehend gender-specific and EN. Mouth Health Dental health is a key indicator of overall health, well-being and quality of life. Dental health has been shown to reduce the nutritional intake of foods essential for optimal health and plays a critical role in a person’s ability to consume adequate nutrition (Sheiham et al., 2001). The absence of oral health is known to cause avoidance of some nutritional foods such as whole grains, fruits, vegetables and meat (Beaudette, Fritz, Sullivan, & Ward, 2017). Other issues associated with oral health include loose and painful teeth, decreased saliva production, and changes in sensory perceptions of taste and smell (Su, Yuki, Hirayama, Sato, & Han, 2020). In elderly subjects, tooth deterioration is not necessarily due to aging but the oral disease (dental caries, periodontal diseases), usage of drugs, and cumulated physical and psychic disorders (such as loss of autonomy, mobility, or dexterity). As reported in The British National Diet and Nutrition Survey and the Dentistry Journal, foods containing iron, zinc, vitamin B12, folate and copper are less consumed by elderly with fewer teeth or adults with dentures due to these nutrients being found primarily in meat, seafood, nuts and seeds, vegetables and green leaves (Beaudette et al., 2017; Sheiham et al., 2001). Individuals with poor chewing abilities or ill-fitting dentures have been proven in the literature to consume smaller amounts of veggies and have overall reduced nutrient intake (Sheiham et al., 2001; Su et al., 2020). Enterally fed patients, such as those with head and neck cancers and dysphagic patients, usually lose swallowing function, subsequently leading to the inability to meet nutritional requirements (Santos et al., 2016). Dysphagia may occur due to an obstructive disease or in the setting of a neurological disorder and can affect oral intake (Lopes et al., 2022). It reduces oral intake by decreasing swallowing efficacy and safety, leading to depletion of nutrient intake and, consequently, to malnutrition and decreased quality of life (Lopes et al., 2022). When oral intake is insufficient, an individual cannot eat or drink safely, and there is no other digestive tract disorder, tube feeding is the obvious feeding option (Lopes et al., 2022; Nerina, Marco, & Elvio, 2013). It is worth noting that many patients undergoing percutaneous endoscopic gastrostomy (PEG) have limited ability to care for themselves (Lopes et al., 2022). As a result, 26 those responsible for caring for long-term enteral feeding patients, including caregivers and professional teams, often overlook the significance of dental health as a critical aspect of overall health (Lopes et al., 2022) . Maintaining good dental health can help prevent low-level oral inflammation linked to adverse outcomes in various systemic disorders (Lopes et al., 2022; Touger-Decker & Mobley, 2013). Therefore, all of these features may contribute to poor oral care and impaired oral health in patients unable to adequately use their mouth for food consumption, leading to an incapacity to meet nutritional requirements. Enteral feeding formulations The content of standard enteral mixtures should contain a micronutrient content that aligns with the nutrient reference range values (NRVs) for a healthy population (Iacone et al., 2015). These mixtures are designed for people on standard enteral formulas; as outlined in the guidelines on foods for special medical purposes, these formulas are intended for individuals who cannot meet their nutritional needs through regular food consumption due to their medical condition and who do not have any specific nutritional requirements (Gazette, 2015) . A study compared Sixty-two nutritionally complete enteral formulas (Iacone et al., 2015). This study evaluated the micronutrient content of enteral formulas grouped as standard- and disease- specific at 1500 and 2000 Kcal/day. Their findings were that micronutrients supplied in EN mixtures were often above the NRVs for a healthy population, below the upper limit (UL), and within the range of the relevant European standards; it was reported as suitable for patients on long-term total EN. As an interest of our research, the micronutrient content for some enteral formulas showed a greater zinc content than the tolerable UL levels. They conclude that it would be more appropriate to keep zinc content at the limit set by the European Commission (Iacone et al., 2015). The literature shows that most patients with long-term EN are in stable clinical conditions, and metabolic diseases are reduced, making micronutrient requirements similar to the general population under a standard diet (Iacone et al., 2015). Enteral formulas supply sufficient micronutrient intake for patients with long-term HEN. 27 Micronutrients Assessment Iron Iron is an essential micronutrient that plays a crucial role in erythropoiesis, oxidative metabolism and cellular immune function (Munoz, Villar, & Antonio Garcia-Erce, 2009). In addition, it is essential for growth, neurodevelopment, myelination of neurons, and neuronal energy metabolism (López & Martos, 2004; Thomas, Raghavendra, Phu, & Michael, 2020). Iron exists in states of reduced ferrous form (Fe 2+ ) and oxidised ferric form (Fe 3+ ); however, ferrous iron is more bioavailable than ferric iron (Palacios, 2012). According to Anderson et al. (2005), iron is also present in the diet in two forms, haem iron and inorganic, non-haem iron. The authors complement that non-haem iron is the most abundant in the diet; however, it is poorly absorbed compared to haem iron, derived primarily from haemoglobin and myoglobin and thus primarily associated with meat intake. Iron deficiency (ID) is the most common micronutrient deficiency, affecting approximately two billion people worldwide (WHO & CDC, 2007). Iron deficiency is characterised by a state in which there is a reduction in iron stores and a disparity between serum iron levels and cellular requirements but adequate iron for erythropoiesis (WHO & CDC, 2007). According to a critical review, iron deficiency anaemia is associated with underlying gastrointestinal pathogenic conditions. The study states that mucosal malabsorption, such as chronic pancreatitis, cystic fibrosis, Zollinger-Ellison syndrome and obstructive jaundice, can lead to iron deficiency (Saboor et al., 2015). Iron status in an average population exhibits varying body iron levels ranging from replete stores to overt iron deficiency anaemia. From a clinical standpoint, three stages of iron deficiency can identify a decrease in iron storage. The first stage is reduced iron stores, measured by ferritin concentration (storage depletion). The second stage is early iron deficiency, identified by reduced transferrin saturation or excess free protoporphyrin or transferrin receptors (mild deficiency). The most severe phase is when erythropoiesis is impaired, leading to iron deficiency anaemia (Brito et al., 2020; Kang et al., 2014). A study of 44 patients who received tube feeding longer than four weeks showed that 13 patients presented lower blood iron (38.5%) (Kang et al., 2014). This study identified that significant iron deficiency in long-term tube-fed patients could increase after two or >6 months (Kang et al., 2014). (Santos et al., 2016) showed similar results and identified that n=69 (47%) of (n=146) participants receiving HEN longer than 3-4 weeks showed low serum iron concentration of 7-44 mg/dl (normal range: 45-160 mg/dl). 28 With such results, the most common reference range for iron ranges between 50 to 120μg/dl (equivalent to approximately 10 to 30μmol/L) (WHO & CDC, 2007). The Canterbury Health Laboratories used the cut-off value <10-30μmol/L as sufficient serum iron (Canterbury Health Laboratories, 2023). From a clinical standpoint, a decrease in storage iron can be identified by a reduction in serum ferritin concentration and a decrease in stainable iron in the bone marrow. When iron stores are exhausted by low ferritin levels (lower than 157μg/L), and demand continues to exceed supply, it is considered diagnostic of iron deficiency (R. D. Lee & Nieman, 2007). Hemoglobin in the blood primarily depends on the number of red blood cells and, to a lesser extent, on the amount of hemoglobin in each red blood cell. The results of impaired hemoglobin synthesis lead to a concentration below the widely accepted threshold level for iron deficiency of 135g per litre for men and 120g per litre for women (R. D. Lee & Nieman, 2007). In the case of iron-deficiency anaemia, blood iron is severely depleted, resulting in decreased red blood cell production and low hemoglobin concentrations. Serum transferrin distinguishes between iron deficiency anaemia and iron deficiency due to an acute phase response. Total iron-binding capacity (TIBC) represents the total number of iron atoms binding on transferrin, and it is a more stable indicator of iron status; however, it only appears once iron stores are depleted (WHO & CDC, 2007). The normal range for TIBC levels is 45-71μmol/L, increasing when iron stores are depleted. Transferrin saturation is calculated using serum iron and TIBC, a measure of the iron supply to increase red blood cells. The WHO defines the normal range of serum transferrin as 1.9–2.58 g/L. The transferrin concentration may increase to 9mg/L in non-anaemic iron deficiency and reach higher levels of 25mg/L in iron deficiency anaemia. Therefore, studies suggest that transferrin saturation of <15% is insufficient to meet standard red blood cell production requirements and, coupled with TIBC, indicates iron deficiency (WHO & CDC, 2007). The Biomarkers Reflecting Inflammation and Nutrition Determinants of Anaemia (BRINDA) established guidelines on assessing anaemia and micronutrient status, including serum iron. This project identified a correlation between iron status for determining iron deficiency which involves the acute phase response triggered by infection and trauma. They stated that during the inflammation response, there is an increase in the concentration of some positive acute phase protein (APPs) in plasma, and the increased APPs lead to an over- or under-estimation of iron deficiency. Examples of such positive APPs include CRP, AGP, and ferritin levels. 29 Negative APPs such as albumin is suppressed in the presence of inflammation (Suchdev et al., 2016; WHO, 2020). During infection and inflammation, iron is necessary for the synthesis of new red cells and for replenishing iron enzymes and transferrin transports myoglobin to the bone marrow or body tissues in the plasma (Hurrell, 2012). Iron primarily enters the plasma through macrophages when senescent red cells are destroyed, and the remaining amount is absorbed through mucosal cells or taken from the stored ferritin in the hepatocytes during times of necessity. Iron enters the plasma through the transport protein ferroportin on the cell membrane (Donovan et al., 2005). The hormone hepcidin regulates its entry and maintains adequate iron levels in the body. When iron levels are sufficient, the liver secretes hepcidin, inhibiting iron transport from macrophages and intestinal cells into the plasma (Ganz, 2005). Hepcidin binds ferroportin on the cell membrane, leading to its internalization and degradation (Ganz, 2005). In contrast, hepcidin release from the liver decreases when iron levels are low and iron absorption is maximize (Nemeth et al., 2004). When the body is infected with microbes, the innate immune response increases hepcidin through an inflammatory response, which restricts microbial growth by limiting iron entry into the plasma (Wander, Shell-Duncan, & McDade, 2009). The anaemia of infection occurs when red cell iron recycling is disrupted. Macrophage iron is not released, leading to a shortage of iron for erythropoiesis. The outcome of many infectious diseases depends on preventing the invading pathogen from accessing its iron supply. Providing high doses of iron to an infected patient can worsen the infection since the inflammatory response prevents iron release from macrophages and mucosal cells, thus restricting iron absorption (Hurrell, 2012). Recent findings suggest that the inflammatory response to malarial parasitemia reduces iron absorption from iron-fortified sorghum gruel (Cercamondi et al., 2010), and inflammation associated with obesity and overweight reduces iron absorption and diminishes the effectiveness of iron-fortified foods (Zimmermann et al., 2008), which shows how inflammation in the individuals’ body can decrease iron absorption efficacy. Anaemia was defined as HB <120 g/L (>18years old female–non-pregnant) and 120 g/L (18 years old male) (R. D. Lee & Nieman, 2007). Depleted iron stores were defined using common cut-offs, including serum iron <10-30 μmol/L, serum ferritin 20-400 μg/L, transferrin 2.0-3.5 g/L and transferrin saturation 16-45% (Canterbury Health Laboratories, 2023). In conclusion, inflammation is known to affect iron biomarkers and can lead to incorrect diagnosis and overestimation or underestimation of the prevalence of deficiency, and serum ferritin is poor marker of iron stores in chronic inflammation. 30 Table 2: Iron status measure and cut-offs. Status Measures and cut-offs Iron stores Serum Iron 10-30μmol/L Transferrin 2.0-3.5 g/L Transferrin saturation 16-45% Serum Ferritin 20-350 ug/L (>15yr-30yr men) 20-400 ug/L (>30yr men) 20-150 ug/L (>15yr-30yr women) 20-300 ug/L (>30yr women) Haemoglobin Haemoglobin (men) < 130 g/L Haemoglobin (women) < 120 g/L References for cut-offs: iron stores and haemoglobin (Canterbury Health Laboratories, 2023). Vitamin B12 Vitamin B12 is the term used to describe the group of compounds that exhibit the biological activity of cyanocobalamin. Many cobalamin compounds are required to synthesise fatty acids in myelin and, together with folate, for DNA synthesis (NHMRC, 2017). Vitamin B12 is synthesised only in certain bacteria and is subsequently concentrated in the tissue of ascending organisms in the food chain. As such, animal-based foods, including milk, fish, and egg, are the main contributors of vitamin B12 to the human diet (Watanabe & Bito, 2018). Certain strains of bacteria in the human colon also produce vitamin B12; however, the contribution of bacterial synthesised vitamin B12 to human physiology is unknown (Ankar & Kumar, 2021; Koury & Ponka, 2004; Watanabe & Bito, 2018). The median intake of vitamin B12 as part of a Western diet contains 5-30μg of vitamin B12 per day, which covers the average requirement of 1-4μg lost daily through normal physiological processes (Finglas, 2000). Vitamin B12 can also be stored in the liver and be sufficient for up to 4 years without further supply (NHMRC, 2017). Vitamin B12 is bound to food proteins, serving as a coenzyme. Many events need to occur for successful vitamin B12 absorption, and any interruption results in malabsorption (Nielsen, Rasmussen, Andersen, Nexø, & Moestrup, 2012). Inside the stomach, vitamin B12 is liberated from the protein by the action of stomach acid and pepsin, allowing vitamin B12 to bind to small proteins secreted by the stomach called R-binders (Russell, 2001). Vitamin B12, bound to the R-binders, is then transported to the proximal small intestine, where the R-binders are removed from the vitamin through the action of pancreatic proteases (Russell, 2001). Vitamin B12 is then bound to a small glycoprotein secreted by the stomach called intrinsic factor, which transports vitamin B12 to the terminal ileum, where it can be actively absorbed (Nielsen et al., 2012; Russell, 2001). 31 The average bioavailability of vitamin B12 is commonly said to be around 50%; therefore, it may be better for older adults to have multiple low-vitamin B12 dietary sources, such as milk and dairy, rather than one large source of dietary vitamin B12 from liver or meat (Gille & Schmid, 2015). The absorption of vitamin B12 can be reduced due to impaired gastrointestinal absorption due to decreased gastric acid and intrinsic factors (Ankar & Kumar, 2021). The authors of Clinical Nutrition Enteral and Tube Feeding (Rolandelli, 2005) state that generally, humans maintain a significant vitamin B12 reserve that can last 3 to 5 years in individuals with optimal status. A review article states that, except for strict vegetarians, vitamin B12 deficiency implies the presence of an absorptive problem (Snow, 1999). This study indicates that the body stores a large amount of vitamin B12 (2-5 mg) relative to daily requirements, and it takes 2 to 5 years to develop vitamin B12 deficiency in the presence of severe malabsorption (Snow, 1999). There is currently no universally accepted serum vitamin B12 cut-off to define deficiency; however, several have been proposed in the literature ranging from 148-250pmol/L (Ankar & Kumar, 2021; De Benoist, Darnton-Hill, Davidsson, Fontaine, & Hotz, 2007; Devi, Rush, Harper, & Venn, 2018; Wong, 2015). The literature suggests that the cut-off values used to define vitamin B12 deficiency is <148 pmol/L and depletion was defined as 148–221 pmol/L (Devi et al., 2018), since value is widely used in the epidemiologic setting to indicate insufficiency (Carmel, 2011). This thesis applied cut-off value based on the Canterbury Health Laboratories (Canterbury Health Laboratories, 2023). Table 3: Vitamin B12 status measure and cut-off. Status Measures and cut-offs Vitamin B12 80-675 pmol/L References for cut-off: vitamin B12 (Canterbury Health Laboratories, 2023). Folate Folate, a water-soluble B vitamin, has many roles within the body, with one of the most important roles as a coenzyme within single-carbon metabolism (Koury & Ponka, 2004). Folate comprises a base structure of a pteridine ring linked to para-aminobenzoic acid, with a different number of glutamate residues attached. Folic acid is one of several forms of folate. It is a synthetic folate isomer most commonly used for supplementation and food fortification. As such, it is required for the synthesis of DNA and is a key nutrient for growth, making it a nutrient of great concern for reproductive-aged females (Koury & Ponka, 2004; Snow, 1999). 32 Folate is abundant in dark leafy greens, lentils, and the liver. Other sources include fortified foods, supplementation and the folate produced by the gut microbiome (Medicine et al., 2002). Folate present in dietary supplements and fortified foods is in the form of folic acid. The folate fortification of foods continues to be a voluntary process in New Zealand, with approval to fortify breakfast cereals, bread, and fruit juices (MOH, 2003). Generally, the bioavailability of natural food folate and synthetic folic acid is estimated to be 50 and 85%, respectively. Folate turnover results from losses due to catabolism, excretion and skin (Suh, Herbig, & Stover, 2001). Due to low storage capacity, serum folate decrease after only a few weeks of inadequate dietary intake and deficiency can develop in weeks to months (Jialal., 2021; Sriram & Lonchyna, 2009). Jialal (2021) state that folate deficiency can become evident in 8-16 weeks. A negative folate balance reduces pyrimidines and purines and results in an accumulation in homocysteine levels, leading to adverse effects on health. Megaloblastic anaemia is a common feature of folate deficiency (Koury & Ponka, 2004). The reduced cell division causes significant, nucleated erythrocyte precursors called macrocytes and hypersegmented neutrophils, resulting in decreased DNA synthesis and delayed maturation of bone marrow. Common characteristics of anaemia are weakness, fatigue, irritability, breathlessness and lack of concentration. Folate deficiency will also affect other areas with higher cell turnover rates, like the intestinal epithelium. This is shown as megaloblastic of the enterocytes, which can cause increased problems such as malabsorption and diarrhoea (Datta Mitra, Gupta, & Jialal, 2016; Jialal., 2021). Folate status can be assessed by directly measuring serum folate and red blood cells. Red blood folate concentration indicates long-term status, while serum folate indicates folate status when the blood sample is collected. Serum folate of <7nmol/L mmol/L indicates negative folate balance (R. D. Lee & Nieman, 2007), which value was used in this research to indicate deficiency (Canterbury Health Laboratories, 2023). Table 4: Folate status measure and cut-off. Status Measures and cut-offs Folate >7nmol/L References for cut-off: serum folate (Canterbury Health Laboratories, 2023). Zinc Zinc plays a role in T-cell activation and natural killer (NK) cell and beta cell production (Prasad, 2009). Zinc deficiency may reduce immunity response by decreasing NK cell and B- 33 cell function. These actions are seen to be associated with infections such as tuberculosis, pneumonia, pulmonary infections, rough skin and delayed wound healing (Lowe, Fekete, & Decsi, 2009; Prasad, 2009). However, trials in older people have observed zinc supplementation assists with wound healing time and infection resistance (Barney & Perkinson, 2015). This study showed that in older populations, normal zinc levels (>70ug/dL) were associated with a decrease in the incidence and duration of pneumonia (Barney & Perkinson, 2015). A study on serum trace elements in dysphagic gastrostomy candidates before endoscopic gastrostomy for long-term enteral feeding identified that 122 (84%) patients showed low zinc, while 24 (16%) presented normal values, zinc in the range (normal range: 70-120 mg/dl) (Santos et al., 2016). According to the International Zinc Nutrition Consultative Group (ZiNCG), <10.7 μmol/L of serum zinc is recommended to assess zinc status at the population level (De Benoist et al., 2007). For the matter of assessing zinc status, the cut-off value used in this research is based on the Canterbury Health Laboratories reference range of total zinc 10.0-17.0 μmol/L (10 μmol/L = 650 ug/L (1 ppb), being with <10.0 μmol/L considered deficiency (Canterbury Health Laboratories, 2023). Table 5: Zinc status measure and cut-off. Status Measures and cut-offs Serum zinc 10.0-17.0 μmol/L References for cut-off: serum zinc (Canterbury Health Laboratories, 2023). Copper As one of the essential minerals, copper (CU) plays many important roles in the body by acting as a cofactor for the enzyme function of ceruloplasmin (Cp) and superoxide dismutase 1 (SOD1). It also assists in haemoglobin formation, cell signalling, and cellular respiration (Myint et al., 2018; Osland et al., 2022). Copper is absorbed as Cu+ forms at the apical membrane of the enterocyte. A metalloreductase localised in the brush border membrane and involved in this process is expressed in the duodenum. Intestinal absorption is influenced by the chemical form that Cu is in and by interactions that Cu has with other components of the diet (Harvey & McArdle, 2008). Most healthy adults meet dietary Cu requirements because it is found in commonly consumed foods such as mushrooms, leafy green vegetables, and even cocoa (Medicine et al., 2002). However, copper deficiency in humans is rare but has been found in particular conditions, such as a result of long-term tube feeding (Ferrie et al., 2018; Kang et 34 al., 2014). To exemplify, individuals who rely solely on jejunal tube feeding are at significant risk of developing copper deficiency due to the duodenal and gastric mucosa's crucial role in copper absorption. Serum copper levels are used to diagnose a deficiency. During the inflammatory response, ceruloplasmin, an acute-phase protein that increases during inflammation and transports 80- 95% of copper, can lead to elevated blood copper levels (Altarelli et al., 2019). This author suggests using low serum ceruloplasmin (<20 mg/dL) in addition to low serum copper levels with an elevated C-reactive protein to diagnose deficiency (Altarelli et al., 2019). Per the present value, this study uses a serum copper concentration range of 11.0-20μmol/L and a lower of <11.0μmol/L to diagnose copper deficiency. Table 6: Copper status measure and cut-off. Status Measures and cut-offs Copper (plasma) 11.0-20.0 μmol/L References for cut-off: copper plasma (Canterbury Health Laboratories, 2023). 24-hour Diet-Recall Assessment Traditionally, food intake and behaviours have been assessed using subjective methods such as 24-hour diet recalls, food frequency questionnaires, and food records. All of these require participants to remember their past food intake or record it as it occurs (Gemming, Utter, & Ni Mhurchu, 2015). In the 24-hour diet recall, the respondent is asked to list and quantify all foods eaten during the previous day. The 24-hour diet recall has many advantages: it is inexpensive, easy to implement and has a low respondent burden (Bailey, 2021). However, the 24-hour recall is more feasible among some people than others. For example, older individuals must have sharp memories, honesty, and willingness to record and remember what they consumed. It can be burdensome and has the potential for social desirability bias, leading to intentional or unintentional misreporting of food intake and dietary habits. Therefore, there is potential for both researcher and technical errors in the data collected from using these methods (Bailey, 2021; Boushey, Spoden, Zhu, Delp, & Kerr, 2017). Technological devices, such as mobile phones, have been used to increase the accuracy of dietary data collection. Mobile phones are reported to eliminate recall bias and reduce participant burden by prompting them to record and send their food intake at typical eating times. However, all these methods are prone to error and bias and require considerable time 35 and effort from the participants and researchers (Boushey et al., 2017). According to Martin et al. (2014), the 24-hour diet recall over the phone is a valuable method for when participants forget to record their food intake by reminding them when no recent information had been received at a specific time to confirm types and amounts of foods in poor quality images received and to prompt uncertain details. This researcher concluded that using mobile phone calls to ask questions and prompt participants regarding their 24-hour diet recall resulted in a compliance rate of over 95% (Martin et al., 2014). In agreement, a systematic review by Schembre et al. (2018) found that using a mobile phone to report intake and images of their food reduced participant burden and recall bias due to less reliance on participants remembering to record their food intake. This highlights an opportunity for using images of food to estimate portion sizes and food intake without burdening participants to remember everything they consume. In summary, the 24-hour diet recall is an efficient interview method that captures detailed information regarding food intake. Using images to estimate portion sizes and food intake is an advantage for more precise dietary recall data collection. Using a mobile phone for prompting information, with images sent to the researchers, decreases bias and burden on participants and improves high-quality information from participants. However, it is still challenging for researchers to estimate portion sizes and the food's nutritional composition. Nutrition-Focused Physical Assessment The nutrition-focused physical exam (NFPE) is a system-based examination of each body region to evaluate nutrition status conducted by a registered dietitian, which involves inspection, palpation, percussion, and auscultation of physical parameters (Esper, 2015). The examination focuses on anthropometric measurements, visualization, subcutaneous fat and muscle stores, and assessment of the hair, eyes, oral cavity, skin, and nails for micronutrient deficiencies (Esper, 2015; MacQuillan et al., 2021). In contrast, the nutrition-focused physical assessment (NFPA) interprets the NFPE findings (Desjardins, Brody, & Touger-Decker, 2018). The NFPE involves a head-to-toe evaluation for micronutrient deficiencies, malnutrition, digestive health, and functional status (MacQuillan et al., 2021). According to Esper (2015), it is the clinician’s job to gather all the patient’s clinical characteristics, relate them to nutrition status, and use clinical judgment to determine the severity of malnutrition. Compared with 36 other techniques or the use of biomarker values, clinical histories such as 24-hour diet recall and physical examination provide more accurate information about the nutrition status of patients (MacQuillan et al., 2021). Malnutrition and micronutrient abnormalities can widely affect organs and tissues, resulting in physical sequela that can quickly be detected by a trained practitioner using NFPE (Esper, 2015). Fischer et al. (2015) state that unlike physical examinations conducted by nurses or physicians, the NFPE primarily focuses on changes to muscle and fat stores and physical signs that can result from micronutrient deficiencies or excesses. Currently, micronutrient assessment is not part of the American Society of Parental and Enteral Nutrition and Academy of Nutrition and Dietetics (ASPEN/AND) clinical characteristics to diagnose malnutrition (White, Guenter, Jensen, Malone, & Schofield, 2012). Although, NFPE should also consider the various parts of the body where high cell turnover occurs (e.g., hair, skin, mouth, tongue) because they are among the most likely to rapidly show signs of possible nutrient deficiencies (Jensen, Hsiao, & Wheeler, 2012). Symptoms of nutrition abnormalities may be non-specific, so other possible causes need to be considered (Fischer et al., 2015). For example, anaemia is associated with reduced serum albumin in haemodialysis patients in an inflammatory response setting, but this is not related to nutritional adequacy (Behzad, Hasan, Karimollah, Mehdi, & Roghayeh, 2015). In the NFPE, system components-based examination of each body area includes the following: general inspection, vitals, skin, nails, head/hair, eyes/nose, mouth, neck/chest, abdomen, and musculoskeletal (Esper, 2015). Skin When observing the skin, the clinician should inspect for changes in colour, texture, temperature, moisture, lesions, mobility and turgor. The physical finding of pallor can be noted in overall appearance, conjunctiva (lower eyelid), nail beds, and tongue. These findings correlate with iron or B-complex vitamin deficiencies (they are involved in hematologic processes) (Esper, 2015). Researchers also complements that depigmentation of skin, hair and anaemia are common manifestations of copper deficiency (Dibaise & Tarleton, 2019; Esper, 2015; Vanek et al., 2012). Seborrheic dermatitis (red, inflamed spots on the skin) is seen in zinc deficiency (Aktas Karabay & Aksu Cerman, 2019) and correlates with B-complex vitamins (Esper, 2015). A vitamin B deficiency is rarely seen alone and is often accompanied 37 by other B-complex deficiencies (R. D. Lee & Nieman, 2007). Eczema, a reddish, scaly rash found on the face, neck, and hands, is a physical finding correlated with zinc deficiency (R. D. Lee & Nieman, 2007; Osland et al., 2022). Nails Inspection and palpation of the nail should be performed to assess for colour and texture. Nail textures of thinness, brittleness, or rigidity can indicate iron deficiency anaemia or inadequate dietary protein intake. In the koilonychia condition, the nail will appear concave and flat, like a spoon-shaped fingernail, and the central ridge (appearance of vertical ridge lines) indicates iron deficiency (Montgomery, Streit, Beebe, & Maxwell, 2014). Beau’s line, for instance, is related to acute and severe disease and can occur in cases of zinc deficiency (Debra & Shari, 2022). Hair/Head Physical findings of hair loss or patchy areas on the scale can relate to protein deficiencies, malnutrition, and iron and zinc deficiency (Dibaise & Tarleton, 2019; Esper, 2015). Sparse hair (alopecia) and hair loss can be caused by the depletion of zinc and iron (Samer, Abdulla, & Ausama Ayob, 2018). These authors explain that zinc is a structural or regulatory factor in the hair follicle by accelerating recovery and inhibiting hair follicle regression. Iron, for instance, is essential in forming part of the enzymes implicated in DNA synthesis and cell respiration. A study found that the mean ferritin level in patients with androgenetic alopecia was significantly lower than that in normal individuals without hair loss (Park et al., 2013). Mouth According to Touger-Decker & Mobley (2013), the mouth has a three-to-seven-day turnover rate of most oral mucosal cells. Vitamin and mineral deficiencies can manifest within the oral cavity relatively quickly. These researchers complement that cellular changes can also occur from periodontal disease, infections, viruses, injury, or trauma, which should be considered when noting physical changes in the oral cavity (Touger-Decker & Mobley, 2013). Angular stomatitis or cheilitis (bilateral cracks and redness at the corners of the lips and mouth) are common deficiencies of B-complex vitamins and iron (Radler & Lister, 2013). Glossitis (a beefy, red tongue) and atrophied papillae can also be significant findings for these micronutrient deficiencies (R. D. Lee & Nieman, 2007). Hypogeusia (decreased sensitivity to 38 taste), dysgeusia (unpleasant perception of taste) and ageusia (complete loss of taste) are conditions that are characterized by changes in taste or total loss of tastiness (Rathee & Jain, 2022). If changes in taste and dryness are reported, the possibility of zinc deficiency can be investigated since zinc deficiency can affect cell structure and function (Dibaise & Tarleton, 2019; Gooding, Packer, & Pensiero, 2019). Eyes Pallor conjunctiva is a traditional sign used in the physical diagnosis of anaemia (Sheth, Choudhry, Bowes, & Detsky, 1997). A study suggests that pallor conjunctiva may be a more accurate indicator of the presence or absence of anaemia and has been reported to appear more frequently in patients with severe anaemia. It may be more sensitive than other signs, such as pallor of the palms or nail beds (Sheth et al., 1997), a physical sign related to iron and vitamin B12 deficiency due to anaemia (Sheth et al., 1997). Vitamin B12 deficiency anaemia and pallor conjunctiva are probably due to the common pathology of capillaries disruptions (Sheth et al., 1997), whether the iron deficiency is commonly associated with microcytic anaemia results from processes that impair haemoglobin synthesis in the RBC (Chai, Huang, Rakočević, & Chung, 2021). Thus, the present evidence suggests that severe anaemia causes the conjunctiva to appear abnormally pale due to reduced amounts of red-coloured oxyhaemoglobin that circulate in the dermal and subconjunctival capillaries. Te Whatu Ora Counties Manukau Counties Manukau Health is one of twenty District Health Boards (DHBs) in New Zealand and was established in 2001 and is primarily funded by the government (Lees J, 2021). On 1 July 2022, the public health Counties Manukau District Health Board (CMDHB) were merged into Te Whatu Ora – Health New Zealand (all health services, including hospitals, specialist services, and primary and community care). Counties Manukau DHB is now called Te Whatu Ora – Health New Zealand Counties Manukau. For short, Counties Manukau District (CMD) (Commissioner, 2023). Counties Manukau District is responsible for providing strategic direction for health and disability services in the northern region of NZ in collaboration with other Te Whatu Ora, services providers, the community, and other stakeholders. The CMD provides and funds most health and stability services through contracts with health and disability providers and non- governmental organizations (CMH, 2021). Counties Manukau District offers hospital-based 39 services for its population, and some patients referred from other Te What Ora have access to specialist or highly complex services. It is committed to promoting, protecting and improving the health of its population thr