Immune Function Is Impaired With a Mini Nutritional Assessment Score Indicative of Malnutrition in Nursing Home Elders With Pressure Ulcers
From the * Food Science and Human Nutrition and Correspondence: Bobbi Langkamp-Henken, PhD, RD, Food Science and Human Nutrition, University of Florida, Newell Drive, PO Box 110370, Gainesville, FL 32611-0370. Electronic mail may be sent to Henken{at}ufl.edu.
Background: Malnutrition is prevalent in elders with pressure ulcers and is associated with increased morbidity and mortality. This study compared nutritional status, assessed by the Mini Nutrition Assessment (MNA), to immune function in nursing home elders with pressure ulcers. Methods: Nutritional status was assessed in nursing home residents (>65 years) with a stage II or more severe pressure ulcer. Subjects were classified as well nourished, at risk of malnutrition, or malnourished according to MNA score. Blood was drawn to assess whole blood mitogen-induced lymphocyte proliferation and neutrophil respiratory burst. Delayed-type hypersensitivity to 3 antigens was measured. MNA status was compared with immune parameters using the Kruskall-Wallis test. Results: Of the 24 subjects (23 men, 1 woman) who completed the study protocol, only 4 (17%) were classified as well nourished, whereas 7 (29%) were at risk and 13 (54%) were malnourished according to MNA score. Whole blood lymphocyte proliferation was significantly lower in the malnourished vs at risk subjects with both pokeweed (median [25th,75th percentile], 0.6 [0.3, 0.9] vs 1.8 [1.2, 2.1] disintegrations per minute [dpm]/cell, p < .05); and concanavalin A (1.7 [0.9, 2.0] vs 2.8 [2.6, 3.9] dpm/cell, p < .05) mitogens. Neutrophil respiratory burst normalized to a young control was significantly lower in malnourished subjects vs well-nourished subjects (0.8 [0.5, 0.9] vs 1.4 [1.0, 1.7], p < .05). Total induration to 3 skin-test antigens was 13.4 ± 4.6, 3.5 ± 2.6, and 3.8 ± 1.8 (mean ± SEM) for well-nourished, at risk, and malnourished, respectively (p = .059). Conclusions: Immune function is impaired with an MNA score indicative of malnutrition in nursing home elders with pressure ulcers. The prevalence of malnutrition increases with age and among those with pressure ulcers.1,2 Malnutrition, as defined by the Mini Nutritional Assessment (MNA), a highly sensitive (96%), specific (98%), predictive (97%) and reproducible assessment tool designed and validated for use with elders,3–6 is associated with morbidity and mortality. Van Nes et al7 assessed nutritional status of 1319 older patients (mean age 84 years) upon admit to a tertiary-care geriatric hospital. Patients classified by the MNA as being malnourished had a longer hospital stay and a 3-fold increase in mortality and in rate of discharge to a nursing home compared with patients classified as well nourished.7 In a prospective study, elders assessed by the MNA as being at risk of malnutrition during a general practitioner office visit were more likely to be hospitalized over the next 6 months compared with well-nourished elders.8 The increased morbidity and mortality associated with malnutrition may be caused by the impact of protein, energy, or micronutrient deficiencies on immune function. In "optimally aged" healthy elders as defined by the SENIEUR protocol, protein undernutrition (serum albumin between 30 to 35 g/L) is associated with impaired cell-mediated immunity.9 Protein-undernourished elders have fewer positive responses and lower total induration to delayed type hypersensitivity (DTH) antigens and lower mitogen-induced lymphocyte proliferation compared with healthy elders with normal serum albumin.9 Both anergy (lack of response to DTH testing) and the inability of lymphocytes to proliferate to mitogens are associated with mortality.10–12 Neutrophils, one of the first lines of defense against invading microbes, show impaired chemotactic and Candida-killing activity with advancing age, which may contribute to a higher incidence of infection in elders.13,14 Generation of superoxide, one of the mechanisms by which neutrophils kill microbes, appears to be less predictable in elders. Di Lorenzo et al14 showed an age-related decrease in superoxide generation, whereas Corberand et al13 showed an increase in superoxide generation in subjects over 80 years of age. Differences in neutrophil function among elders may be caused by the presence of chronic disease or the associated nutritional consequences of chronic disease because carefully selected healthy elders do not seem to have age-related declines in neutrophil function.15 Regardless, impaired neutrophil function is associated with increased susceptibility to infection. Numerous tools are available to assess nutritional status in a nursing home population; however, no noninvasive tools are available to assess immune function. Because malnutrition and impaired immune function are interdependent, it may be possible that an assessment tool, such as the MNA, could be used to identify nursing home residents with impaired immune function. The purpose of this study was to compare MNA score to immune function in nursing home elders with pressure ulcers.
Study Design and Subjects Data for this cross-sectional study were collected prospectively and represent baseline data from an interventional trial. Thirty nursing home residents from the Veteran's Administration Medical Center (VAMC), Lake City and Gainesville Nursing Homes, Lake City and Gainesville, Florida, were recruited for the study from January 2001 to March of 2003. Eligible subjects were 65 years of age or older and had a stage II or more severe pressure ulcer. Residents were excluded if they were receiving immunosuppressive medications or had egg or thimersal allergies, diabetes treated by insulin, or an immune deficiency disease. Additionally, renal failure (serum creatinine >220 umol/L [>2.5 mg/dL]) or hepatic failure (abnormally elevated liver enzymes) were means for exclusion because this population was chosen for a larger study that supplemented residents with protein. Informed consent was obtained from all subjects or their power of attorney after the nature of the study and procedures had been explained. The study protocol and informed consent were approved by the institutional review board at the University of Florida and the Malcolm Randall VAMC Research and Development Committee, Gainesville, Florida. Once the subjects were enrolled in the study, demographical data and medical history were obtained from the medical record, and the nutritional status of the subject was assessed using the MNA. Within a week of assessing nutritional status, venous blood was drawn on one occasion at 9:00 AM (nonfasting) for immune function tests, serum albumin, and erythrocyte sedimentation rate, and skin-test antigens were applied to measure DTH.
Nutrition Assessment To maintain consistency between interviewers, interpretations and clarifications of several questions of the MNA were agreed upon by all researchers, and the MNA scoring was discussed by all researchers before assigning a final score. The question clarifications are listed below. For the question, "Has food intake declined over the past 3 months due to loss of appetite, digestive problems, chewing or swallowing difficulties?,"16 the scoring was interpreted as severe loss, moderate loss, or no loss of food intake instead of loss of appetite. The intent of the question regarding mobility is poorly defined for a nursing home population where leaving the home is not required to obtain groceries or meals. We interpreted this question to be that subjects who are able to get out of their room or the nursing home (whether on their own account or via a wheelchair) and socialize will have a better nutritional intake. Clarification of this question was as follows: subjects received no points if they were in contracture or could not hold themselves up if placed in a chair (bed or chair bound). Subjects received a score of 1 if they were able to go out of their room either with assistance or independently but did not or chose not to go out of the nursing home. Subjects received 2 points if they did go out of the nursing home either with assistance or independently. For questions requiring weight and height information, current and past weights were obtained from the medical record. For amputees, the weight used in BMI calculations was derived from the percent total body weight contributed by the amputated limb.17 All heights were derived from knee height measurements using a knee height caliper (Ross Products, Columbus, OH) and equations based on gender, race, and age >60 years.18 When knee height could not be measured because of double below-the-knee amputation or severe contracture, height was obtained from the medical record. All medications are prescribed at nursing homes. Therefore, to answer the question "Takes >3 prescription drugs per day,"16 prescription drugs were interpreted as medications not available over the counter to the general public. For "How many full meals does the patient eat daily?... 1 meal = 0 points, 2 meals = 1 point, 3 meals = 2 points,"16 a full meal was considered to be 75% or more of a meal as recorded by nursing personnel in the medical record. Subjects received 2 points if they were receiving 100% of their energy needs though a feeding tube alone, through a tube feeding and oral diet, or through meals and betweenmeal supplements. Subjects also received full points for the questions regarding protein, fluid, and fruit or vegetable intake if they were meeting nutrient needs via tube feeding. For the question designed to assess the subject's mode of feeding, the subject receives no points if unable to self-feed without assistance.16 In an institutionalized setting, subjects receive additional help with all meals and often receive supplements between meals. For these reasons, the researchers agreed to assign no points only if the subject completely refuses to eat and full points if the resident was meeting nutrient needs via tube feeding. If subjects could not answer the subjective questions independently, because of either cognitive impairment or physical impediment, the primary caregiver for that resident was consulted. Serum albumin was measured by the photometric bromocresol green complex method using a Hitachi 917 (Roche Diagnostics Corp, Indianapolis, IN) by the VAMC Laboratory (Lake City, FL) from blood collected into a lithium Vacutainer (Becton Dickinson, Franklin Lakes, NJ).
Immune Studies The leukocyte-rich plasma layer was isolated from whole blood by mixing blood 9:1 with a 6% dextran (Accurate Chemical and Scientific Corp, Westbury, NY) and 9% NaCl solution and allowed to stand at room temperature for 40 minutes. The leukocyte-rich plasma layer (top fraction) was then layered on a prepared density gradient. The density gradient consisted of 4 mL of the 1.077 g/mL Optiprep (Accurate Chemical and Scientific Corp) in 0.85% NaCl, 1 mmol/L ethylenediaminetetraacetic acid (EDTA) and 20 mmol/L HEPES (Mediatech, Inc), pH 7.4, layered on top of 4 mL of the 1.095 g/mL Optiprep solution in the same buffer. The layered sample was centrifuged at 800 x g for 30 minutes. Centrifugation results in the formation of 2 distinct bands: the upper band contains lymphocytes and the lower band contains neutrophils. The neutrophil layer was removed and washed with 0.85% NaCl, 1 mmol/L EDTA and 20 mmol/L HEPES, pH 7.4, and centrifuged for 10 minutes at 350 x g. If red blood cell contamination was noted in the pellet, 3 mL lysing buffer (0.83% NH4Cl with 10 mmol/L HEPES, pH 7.0) was added and the sample was incubated at 37°C for 7 minutes. After lysing, the sample was centrifuged at 260 x g for 10 minutes and the resulting pellet was washed twice in 10 mL Hanks' Balanced Salt Solution without Mg2+ or Ca2+ (Mediatech, Inc, Herndon, VA) and centrifuged at 350 x g for 10 minutes. The supernatant was removed and 1 mL of 1 g/L glucose in Dulbecco's Phosphate-Buffered Saline (DPBS, Mediatech, Inc) was added to the pellet and the neutrophils were counted in a Beckman Coulter Z Series Instrument (Beckman Instruments, Inc). Neutrophil concentration was adjusted to 5.0 x 106 cells/mL. Fifty microliters of neutrophils were plated per well for a final concentration of 2.5 x 105 cells/well in a 96-well flat bottom plate. To each well, 100 µL DPBS with glucose (as prepared above), 10 µL of 1 µg/mL phorbol 12-myristate 13-acetate (Sigma Chemical Company) in DPBS with glucose, and 10 µL of 18.6 mg/mL ferricytochrome c, horse heart muscle (Sigma Chemical Company), were added for a final volume of 170 µL/well. Neutrophil oxidative burst was measured using a kinetic dual wavelength 540 nm to 490 nm from 3 to 10 minutes using UV Max (Molecular Devices, Sunnyvale, CA). The Vmax results were normalized with a young control. Erythrocyte sedimentation rate was obtained as an indirect measure of inflammation. Erythrocyte sedimentation rate was measured by the VAMC laboratory using whole blood collected from a K3EDTA Vacutainer. Normal values for this laboratory for males and females >50 years of age are 0 to 20 mm/h and 0 to 30 mm/h, respectively. DTH testing was completed with the use of 3 antigens and a saline control purchased from ALK-Abello (Round Rock, TX); these antigens were Candin (Candida albicans), MSTA (mumps antigen), and tetanus toxoid. These antigens were chosen because of their common exposure in this population. Candin and MSTA were purchased ready to use, but the tetanus toxoid was diluted 1:10 with sterile 0.9% normal saline and 0.4% phenol mixture (ALK-Abello). A test dose of 0.1 mL of all 3 antigens and the control were injected intradermally. Induration was measured at 48 and 72 hours postadministration. Measurements were taken by averaging the 2 widest points of the induration. Erythema without induration was considered not significant. Total induration was calculated by adding the greatest average diameters of all positive responses from either time point. Subjects were classified as responding if they had a minimum induration of 5 mm to any of the 3 antigens at either time point, or anergic if they did not respond to any of the antigens.
Statistics
Thirty nursing home residents were recruited for this study. Of the 30 residents recruited, 2 did not meet the inclusion criteria and 4 dropped out before initiating the study protocol. Reasons for drop out included unexpected discharge to home (n = 1), MD request (n = 1), and blood draw refusal (n = 2). The final number of residents included in the study was 24. Subjects were categorized by MNA score as malnourished (<17), at risk for malnutrition (17–23.5), and well nourished (>23.5). Of the 24 subjects who met the inclusion criteria, only 4 were classified as well nourished, more than half the subjects (n = 13, 54%) were malnourished, and 7 (29%) were classified as at risk of malnutrition (Table I). There were no differences in age, pressure ulcer stage, number of pressure ulcers, albumin, or erythrocyte sedimentation rate among subjects within each of the 3 MNA groups. No subjects were receiving anabolic steroids or enteral supplements or tube feedings promoted as "immune-enhancing." Six subjects were receiving all or part of their enteral intake via tube feedings. Of those receiving tube feedings, 0, 1, and 5 were categorized as well nourished, at risk for malnutrition, and malnourished, respectively. BMI, which is a component of the MNA score, was significantly lower (p < .05; Table I) in the subjects identified as being malnourished compared with those classified as well nourished and at risk for malnutrition.
Whole blood lymphocyte proliferation to PHA was not different among well-nourished, at-risk, and malnourished subjects (Fig. 1A). Whole blood lymphocyte proliferation to PW and ConA was significantly lower in the malnourished subjects compared with the subjects at risk for malnutrition (p < .05; Fig. 1B, C). Normalized neutrophil burst was significantly lower (p < .05) in subjects categorized as malnourished compared with those categorized as well nourished (Fig. 2). There were no differences in the mean number of blood lymphocytes or neutrophils among subject groups (data not shown). The number of subjects who responded to DTH was not different among MNA groups (Table II); however, there was a trend toward lower total induration with lower MNA score (p = .059).
The purpose of this study was to compare MNA score to immune function in nursing home elders with pressure ulcers. Subjects who were identified as being malnourished had lower whole blood proliferative responses to ConA and PW mitogens and reduced neutrophil respiratory burst. Whole blood proliferative responses to PHA were not different in elders among the 3 MNA groups. These data are in agreement with Schiffrin et al,19 who were unable to demonstrate any differences between MNA score in elders and PHA/phorbol myristate acetate–induced lymphocyte proliferation but did show a trend toward lower interleukin-2 mRNA in at-risk vs well-nourished elders. Interleukin-2, a cytokine that is secreted by activated T cells, stimulates T- and B-cell proliferation and the development of the DTH responses. Delayed-type hypersensitivity responses in this study followed the same trend (ie, lower responses with poorer nutritional status). The immune system is important for wound healing. Neutrophils are key in the initial stages of wound healing. Neutrophils migrate into wounds and provide host defense against microorganisms by bacterial killing via oxidative burst. Cell-mediated immunity is important in the healing process itself. T cells activate fibroblasts, which play a major role in wound healing. Identifying impaired immune function and malnutrition and improving immune and nutritional status in patients with pressure ulcers may enhance wound healing. Wissing et al20 showed that MNA scores decreased over time in subjects with open ulcers but not in those who were healed. This suggests that a worsening MNA score is not compatible with wound healing; however, the role of immune or nutritional status (other than MNA score) in wound healing was not examined.20 An assumption of the present study was that the MNA distinguished between well-nourished and malnourished nursing home elders with pressure ulcers. Only 1 other study, to our knowledge, has used the MNA to assess nutritional status of elders with ulcers. Wissing and Unosson1 showed that in free-living elders with leg ulcers of any cause, 32 (46%) and 2 (3%) were at risk of malnutrition and malnourished, respectively. In the present study, using a more frail nursing home population with pressure ulcers, only 4 of 24 subjects (17%) were classified as well nourished and 7 (29%) and 13 (54%) were classified by MNA as at risk of malnutrition and malnourished, respectively. Because malnutrition is one of a number of risk factors for development of pressure ulcers, it is not unrealistic that 83% of the subjects were at risk of malnutrition or malnourished. Serum albumin and BMI are 2 parameters commonly used to assess nutritional status. Serum albumin was low in all 3 MNA groups and was not different among the groups (Table I). This lack of effect of nutritional status on serum albumin and MNA score could be attributed to chronic inflammation that is associated with pressure ulcers21–23 and that is suggested by the elevated erythrocyte sedimentation rates, a crude indicator of inflammation (Table I).24 BMI, which is a component of the MNA, was significantly lower with a lower MNA score (ie, malnutrition). A low MNA score is associated with morbidity and mortality.7,8 This relationship may be in part because a low score is also associated with impaired immune function, as shown by this study. Impaired immune function and specifically the inability of lymphocytes to proliferate to mitogens is associated with death from any cause.12 The inability to produce a DTH reaction and impaired neutrophil function are associated with increased mortality and sepsis, respectively.10,11 In elders admitted to long-term care, Donini et al4 showed that variations in MNA score over time were minimally correlated with variations in nutritional parameters but that a low MNA score was a better predictor of adverse clinical events and of higher mortality. When scores from MNA assessment sections (ie, anthropometric, global, diet, subjective) were compared between elders who survived or died, there was a significant difference in scores from questions in the global and diet assessment parameters with global and diet scores being lower in those who died compared with those who survived.4 Questions within the global assessment section address independence, medications taken, psychological stress, acute disease, mobility, neuropsychological problems, and skin lesions or ulcers. These global parameters may be indicators of chronic stress. Chronic stress is associated with elevated cortisol levels and depressed lymphocyte proliferation and interleukin-2 production.25 Pressure ulcers, which are scored as part of the MNA global assessment, are also associated with increased cortisol levels in elders. Bonnefoy and colleagues22 showed that blood cortisol, C-reactive protein, and interleukin-6 levels were elevated and serum albumin levels were decreased in elders with stage IV pressure ulcers compared with elders with stage III ulcers. Although there was no statistical difference in the median stage of the most severe pressure ulcer among subjects classified in the present study as well nourished, at risk for malnutrition, and malnourished, there may have been a biologic difference. Subjects classified as well nourished had a median stage II pressure ulcer, whereas those classified as at risk and malnourished had a median stage III and stage IV, respectively. It might be tempting to say that MNA is more predictive of frailty than nutritional status and that is why MNA score is predictive of morbidity and mortality; however, other studies demonstrate that nutritional repletion improves MNA score and reduces morbidity and mortality.26,27 There are a number of limitations associated with this study. This study population consisted of all men and 1 woman. This is in contrast to the original study population used to develop the MNA, which consisted of 66% women.28 Additionally, the original validation studies were done in relatively healthy elders, which is reflected by the lack of guidance for scoring nutritional status of elders receiving tube feedings to meet nutrient needs. In this study, ambiguous MNA questions were interpreted by the researchers to promote consistency among assessments. These interpretations, which were discussed within the methods, may not have been in line with how the MNA questions were interpreted in developmental and validation studies. Future studies are required to address the potential limitations of this study and to determine whether nutritional repletion of elders with pressure ulcers improves nutritional status, MNA score, immune function, and ultimately morbidity and mortality. This material is the result of work supported with resources, the use of facilities, and the help of Cynthia Dooley, MT(ASCP), and Ruby Steele, RN, MSN, at the Lake City and Gainesville, Florida, VAMC nursing homes. This research was supported by the Florida Agricultural Experiment Station and a grant from the National Institute of Nursing Research R15 NR05056 and approved for publication as Journal Series No. R-10057. The study was presented at Nutrition Week 2004, Las Vegas, NV, and was published in an abstract format in JPEN 2004;28:S20.
1 Current affiliation: Food and Nutrition Services, Lake City Medical Center, Lake City, Florida.  Received for publication March 8, 2004. Accepted for publication June 18, 2004.
Journal of Parenteral and Enteral Nutrition, Vol. 28, No. 6,
416-422 (2004) This article has been cited by other articles:
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MATERIALS AND METHODS
RESULTS
2 test for independence. Results were
considered statistically different at the p < .05 level.
Statistical analyses were performed using GraphPad Instat (version 3.05,
GraphPad Software, San Diego, CA). Data in figures are represented with box
plots. Each box is set up so that the bottom of the box represents the 25th
percentile and the top of the box represents the 75th percentile. The
horizontal line inside the box is the median. The mean can be estimated by
finding the point equidistant from the bottom (25th percentile) and the top
(75th percentile) of the box and drawing a horizontal line through it so that
it bisects the box. The whisker extending from the top of the box represents
the top quartile (75% to 100%), and the highest horizontal line on the top
whisker represents the highest data point. The whisker below the box
represents the lowest quartile (0% to 25%) and the lowest horizontal line on
the whisker represents the smallest data point. 
