Quantification Of Lactoferrin Research Articles

A Magnetic Beads-Based Sandwich Chemiluminescence Enzyme Immunoassay for the Rapid and Automatic Detection of Lactoferrin in Milk.

Lactoferrin (LF), an iron-binding glycoprotein with immunological properties and a high nutritional value, has emerged as a prominent research focus in the field of food nutrition. Lactoferrin is widely distributed in raw milk and milk that has undergone low-temperature heat treatment during pasteurization, making its rapid and accurate detection crucial for ensuring the quality control of dairy products. An enzyme-linked immunosorbent assay-based analytical protocol has often been referred to for the detection of LF in real samples. Signal amplification was accomplished using the streptavidin-biotin system. Here, an automated magnetic beads-based sandwich chemiluminescence enzyme immunoassay (MBs-sCLEIA) system was developed for the quantification of lactoferrin in pasteurized milk. The MBs-sCLEIA system consists of an automated chemiluminescence-based analyzer and a lactoferrin MBs-sCLEIA assay kit. Notably, our proposed method eliminates the need for pretreatment procedures and enables the direct addition of milk samples, allowing for the automatic quantitative detection of lactoferrin within a rapid 17 min timeframe for up to eight samples simultaneously. The MBs-sCLEIA was linear over the range of 7.24-800 ng/mL and displayed a limit of detection (LOD) of 2.85 ng/mL. As its good recovery and CV values indicate, the method exhibited high precision and accuracy. Furthermore, it was verified that it was selective towards five additional common milk proteins. A good correlation was observed between the results from the MBs-sCLEIA and heparin affinity column-HPLC (r2 = 0.99042), which proves to be a useful and practicable way of conducting an accurate analysis of lactoferrin in dairy products.

Quantification of lactoferrin in dietary supplements using a Heparin affinity clean-up column by HPLC PDA detector

The use of dietary supplements containing lactoferrin is a growing trend. As a result, there is a need for a simple and reliable analytical method to determine the content of lactoferrin in these supplements to ensure food quality. In this study, an HPLC method was developed and validated for determining lactoferrin in dietary supplements using a HiTrap Heparin clean-up column in combination with HPLC. The study involved selecting optimal conditions for extracting lactoferrin from the sample matrix, followed by chromatography conditions using a C4 column (150 mm × 4.6 mm, 3.5 µm). The limit of detection (LOD) and limit of quantification (LOQ) were determined, with the achieved values being 1.2 mg/100g and 4.0 mg/100g, respectively. The method has shown good recovery (99.5–103.4%) and precision (RSD 1.76–3.44%), meeting the method performance requirements outlined in AOAC SMPR 2020.005. The validated method was applied to determine the contents of lactoferrin in dietary supplement samples.

Contractile Hairpin DNA-Mediated Dual-Mode Strategy for Simultaneous Quantification of Lactoferrin and Iron Ion by Surface-Enhanced Raman Scattering and Fluorescence Analysis.

DNA-mediated self-assembly technology with good sensitivity and affinity ability has been rapidly developed in the field of probe sensing. The efficient and accurate quantification of lactoferrin (Lac) and iron ions (Fe3+) in human serum and milk samples by the probe sensing method can provide useful clues for human health and early diagnosis of anemia. In this paper, contractile hairpin DNA-mediated dual-mode probes of Fe3O4/Ag-ZIF8/graphitic quantum dot (Fe3O4/Ag-ZIF8/GQD) NPs were prepared to realize the simultaneous quantification of Lac by surface-enhanced Raman scattering (SERS) and Fe3+ by fluorescence (FL). In the presence of targets, these dual-mode probes would be triggered by the recognition of aptamer and release GQDs to produce FL response. Meanwhile, the complementary DNA began to shrink and form a new hairpin structure on the surface of Fe3O4/Ag, which produced hot spots and generated a good SERS response. Thus, the proposed dual-mode analytical strategy possessed excellent selectivity, sensitivity, and accuracy due to the dual-mode switchable signals from "off" to "on" in SERS mode and from "on" to "off" in FL mode. Under the optimized conditions, a good linear range was obtained in the range of 0.5-100.0 μg/L for Lac and 0.01-5.0 μmol/L for Fe3+ and with detection limits of 0.14 μg/L and 3.8 nmol/L, respectively. Finally, the contractile hairpin DNA-mediated SERS-FL dual-mode probes were successfully applied in the simultaneous quantification of iron ion and Lac in human serum and milk samples.

Lactoferrin concentration in bovine milk: validation of radial immunodiffusion technique, sources of variation, and association to udder health status

The present study aimed to evaluate analytical performances of radial immunodiffusion (RID) technique for the quantification of milk lactoferrin (LF), and to investigate the main sources of variation of LF concentration in individual milk samples of Holstein Friesian (HF, n = 1516) and Simmental (SI, n = 230) cows. Repeatability (RSDr) and reproducibility (RSDR) of RID method were assessed as relative standard deviation of 15 measurements within the same day and 45 measurements across 3 d, respectively. Sources of variation of milk LF were investigated through a mixed linear model which included the fixed effects of classes of stage of lactation and parity (and their interaction), cow breed, udder health status (UHS, based on somatic cell count and differential somatic cell count), and the random effects of the herd-test day nested within breed and the residual. Method performances were satisfactory in terms of repeatability (RSDr < 9%) and reproducibility (RSDR < 8%), suggesting that the RID can be considered as a consistent cow-side test for the quantification of milk LF. Milk LF was positively associated with somatic cell score (r = 0.40) and negatively with lactose content (r = −0.33). Lower concentration of LF was observed in milk of ‘healthy’ and ‘susceptible’ cows (13.03 and 12.87 mg/dL, respectively) compared to ‘mastitic’ and ‘chronic’ cows (17.91 and 17.33 mg/dL, respectively). In this light, milk LF has the potential to be a useful biomarker to detect early mastitis. Further research is advisable to deepen the association between milk LF content and observed clinical mastitis. Highlights Radial immunodiffusion method for milk lactoferrin quantification is repeatable and reproducible. Somatic cell count is positively associated with milk lactoferrin content. Milk lactoferrin is less concentrated in milk of healthy and susceptible than potentially mastitic and chronic cows.

Quantification of lactoferrin in human milk using monolithic cation exchange HPLC

Human lactoferrin (hLF) is one of the most important whey proteins in human milk, known for its ability to modulate innate host immunity and multifunctional activities for neonatal growth. The objective of this study was to validate an efficient method for the detection and quantification of hLF using a unique technology of cation-exchange high-performance liquid chromatography (HPLC) on CIM® monolithic columns. Human milk samples were collected using manual expression or a breast pump, at different weeks of lactation. After sample preparation, hLF was detected and measured by HPLC method and further confirmed by SDS-PAGE. Selected fractions were analysed also by LC-MS/MS. Presumably, due to the high density of positive charge on the surface of the N-terminal domain, hLF binds strongly to the column and elutes last, enabling the high specificity of this method. The LC-MS/MS analysis indicated that hLF eluted in two clearly separated peaks, presumably representing two different molecular species of hLF. hLF concentration in the human milk samples ranged from 2.03 mg/mL to 5.79 mg/mL and was not significantly affected by the sample collection method whereas it was negatively correlated with the stage of lactation. These results suggest that cation exchange chromatography is an accurate, efficient, and robust method for the detection and quantification of hLF.

S1980 Cocaine-Induced Pseudomembranous Colitis With Negative Clostridium difficile Infection

Introduction: Pseudomembranous colitis (PMC) is most commonly caused by Clostridium difficle (C.Diff). The incidence of C.Diff related PMC is 3-8% and increasing. Other than Clostridium difficile, ischemia, infections, medications, and inflammatory conditions can cause PMC. We report a case of pseudomembranous colitis-tested negative PCR for C. difficile. Case Description/Methods: A 52-year-old female with a past medical history of hypothyroidism, asthma, and polysubstance abuse (history of chronic heroin, cocaine, benzodiazepine, alcohol, and cigarette use) with Hx of chronic intermittent diarrhea due to opioid withdrawal presented to the emergency department with bloody watery diarrhea of 3-4 times in a day for 5 days. On initial evaluation, the patient's vital signs are stable. The abdomen is diffusely tender on physical examination with increased tenderness in the left lower quadrant. Labs showed WBC count of 15,900/μL, 90.2% neutrophils, AST of 80 U/L, ALT of 41 U/L, ALP of 97 U/L, total bilirubin of 0.5 mg/dL, lactic acid of 0.9 mmol/L and c-reactive protein of 11.9 mg/dL. The urine drug screen was positive for benzodiazepine, cocaine, opiate, and methadone. CT scan of the abdomen showed mild diffuse concentric colonic wall thickening representing colitis. The patient was empirically treated with IV ceftriaxone, IV metronidazole, and PO vancomycin. Stool for C. difficile PCR was negative 3 times on 3 different days. Stool culture was negative for Salmonella, Shigella, Campylobacter, or E. coli O157:H7. Fecal lactoferrin quantification was 320.72 μg/mL. The patient underwent sigmoidoscopy, which showed discontinuous areas of ulcerated mucosa covered with yellowish punctate membrane with stigmata of recent bleeding in the entire examined colon. Biopsy of the colon showed focal active colitis with superficial crypt erosion and focal adherent mucopurulent material negative for crypt abscesses and granulomas, consistent with pseudomembranous colitis. The patient was continually treated with supportive care including IV fluids and bowel rest. Her diet was advanced, and her diarrhea improved (Figure). Discussion: Other than C. difficile, we propose keeping other differentials of pseudomembranous colitis is essential. Other infections from S. aureus, E. coli, Shigella and Strongyloides may also cause PMC. Non-infectious causes of PMC include chemical endoscope cleaning agents, intestinal ischemia, drug abuse from cocaine, inflammatory bowel disease, and microscopic colitis to avoid over-usage of antibiotics and focus on targeted therapy.Figure 1.: Discontinuous areas of ulcerated mucosa covered with yellowish punctate membrane with stigmata of recent bleeding in the entire examined colon.

Lactoferrin quantification in cattle faeces by ELISA.

BackgroundPromoting and maintaining health is critical to ruminant welfare and productivity. Within human medicine, faecal lactoferrin is quantified for routine assessment of various gastrointestinal illnesses avoiding the need for blood sampling. This approach might also be adapted and applied for non-invasive health assessments in animals.MethodsIn this proof-of-concept study, a bovine lactoferrin enzyme-linked immunosorbent assays (ELISA), designed for serum and milk, was applied to a faecal supernatant to assess its potential for quantifying lactoferrin in the faeces of cattle. Faecal lactoferrin concentrations were compared to background levels to assess the viability of the technique. A comparison was then made against serum lactoferrin levels to determine if they were or were not reflective of one another.ResultsThe optical densities of faecal samples were significantly greater than background readings, supporting the hypothesis that the assay was effective in quantifying faecal lactoferrin (T13, 115 = 11.99, p < 0.0005). The mean faecal lactoferrin concentration was 0.269 µg mL−1 (S.E. 0.031) and the mean serum concentration 0.074 µg mL−1 (S.E. 0.005). Lactoferrin concentrations of faecal and serum samples, taken from the same animals on the same day, were significantly different (T21 = 2.20, p = 0.039) and did not correlate (r = 0.2699, p = 0.238).ConclusionResults support the hypothesis that lactoferrin can be quantified in cattle faeces by ELISA. Whilst further research is required to determine the physiological source of the lactoferrin, this highlights the potential of the method for non-invasive assessment of cattle immunology and pathology.

Determination of native lactoferrin in milk by HPLC on HiTrapTM Heparin HP column

Foods supplemented with lactoferrin are an increasing trend owing to its multiple functions. Therefore, a reliable analytical method for the nutritional assessment and quality control of lactoferrin containing foods must be established. A method for the determination of native lactoferrin in milk using a HiTrapTM Heparin HP column coupled with HPLC was developed and validated. The conditions for lactoferrin enrichment in milk using the HiTrapTM Heparin HP column were optimized. A linear range of 2 to 100 mg L-1 was obtained for lactoferrin standards, with a R2 value of 0.9989. The detection limit (LOD) and quantification limit (LOQ) of the lactoferrin standards were 2.00 and 6.67 mg L-1, respectively. Considering the enrichment factor involved in the pre-treatment procedures, LOD and LOQ for detection of milk sample were 0.57 and 1.90 mg L-1, respectively. In addition, milk samples spiked with lactoferrin at three concentration levels (2, 5, and 10 mg/L) showed overall mean recovery of 88.3%, 90.2% and 95.1%, respectively. The relative standard deviations of intra-day and inter-day precision were 1.3% ~ 4.8%, and 2.1% ~ 5.7%, respectively, indicating that the present method showed good performances. Furthermore, the successful quantification of lactoferrin in raw milk and processed milk demonstrated the usefulness and effectiveness of the proposed method.

Analytical Method for Lactoferrin in Milk-Based Infant Formulas by Signature Peptide Quantification with Ultra-High Performance LC-Tandem Mass Spectrometry.

Background: There is a need for a standardized method for quantification of lactoferrin in infant formulas, and manufacturers have started fortifying lactoferrin to mimic the higher levels found in human milk. A variety of current methods exist, but specificity and accuracy are challenging with the infant formula matrix. The use of signature peptides and MS is becoming more prevalent in the realm of analytical chemistry for quantification of proteins. Objective: The objective of this work was to develop and validate a method through a single-laboratory validation for quantification of lactoferrin in milk-based infant formula and begin to lay the foundation for a standardized method. Methods: The method presented uses signature peptides to quantify lactoferrin in milk-based infant formulas by ultra-high performance LC-tandem mass spectrometry (MS/MS). These peptides are produced through tryptic digestion, and fragments produced from these peptides through MS/MS allow the specific quantification using correlating isotopically labeled peptides. Results: The validation parameters were all met with precision RSDr ranging from 2.1 to 7.1 and intermediate RSDR ranging from 7.0 to 10.4 across different fortified milk-based infant formulas. Accuracy with certified reference material resulted in mean recoveries of 91.7-96.4%. Conclusions: The results from this study demonstrate the method is fit for purpose to support manufacturing specifications and nutritional labeling requirements.

A rapid HPLC method for the determination of lactoferrin in milk of various species.

This Research Communication describes the adaptation and testing of an RP-HPLC method, previously tested for the determination of lactoferrin (LF) in whey, for its applicability to determine milk lactoferrin content. Milk samples of various species, namely, ovine, caprine, bovine, donkey and human milk, were tested. The advantage of this RP-HPLC method includes speed and convenience, as it does not include extensive pretreatment or separation steps. A simple pre-treatment step was added in order to remove fat and proteins of the casein family and the samples were tested. The results varied in terms of elution of the LF peak both between the milk of the different species as well as from the initial application on whey. The peak resolution was satisfactory in the cases of ovine, bovine and donkey milk samples while for caprine and human milk an interference with other peaks was observed. Nevertheless, quantification of LF was found possible for all samples. This new application of the modified method will allow the determination of LF in milk samples of the tested species either for everyday analysis or as a useful qualitative screening for presence or absence of LF.

A Simple, Sensitive, and Label-Free Platform for the Quantification of Lactoferrin in Camel and Goat Milk Based on Thin-Layer Chromatography

Lactoferrin is a multifunctional glycoprotein secreted in most body fluids, especially in milk. It has been identified in many mammalian species, such as human, goat, cow, and camel. Lactoferrin exerts a significant protective biological role by acting as an antimicrobial, antiviral, immunomodulatory, and anti-inflammatory agent. This work describes a simple, sensitive, direct, inexpensive, and label-free method for quantifying lactoferrin in camel and goat milk based on thin-layer chromatography (TLC). Analysis and quantification of lactoferrin were done through simple protein extraction procedure followed by subjecting the obtained extract to TLC using RP-18 TLC silica gel plates and a ternary mobile phase composed of 2-propanol: TFA: water (40:5:55, v:v:v). Densitometric detection was carried out at 250 nm. The proposed method showed linear calibration curves over the range of 10.0–205.0 µg/mL. The limit of detection and limit of quantitation were found to be 3.50 and 10.0 µg/mL, respectively. The method was fully validated according to the ICH guidelines. The average lactoferrin concentrations in camel and goat milk were determined as 29.70 ± 0.69 and 38.12 ± 0.53 μg/mL, respectively. The reliability of the assay was demonstrated through direct comparison with a previously reported high-performance liquid chromatography (HPLC) method for lactoferrin analysis. The presented study represents the first report for the application of TLC in quantifying lactoferrin in natural extracts.

Quantification of lactoferrin in breast milk by ultra-high performance liquid chromatography-tandem mass spectrometry with isotopic dilution

We developed a LC-MS/MS method for quantification of human lactoferrin in breast milk based on tryptic peptides and a synthetic isotopic peptide standard.

Effect of periodontal therapy on lactoferrin levels in gingival crevicular fluid.

The aim of this study was to evaluate lactoferrin quantification as a sensitive and objective method of detecting the degree of periodontal inflammation, oxidative stress and to monitor the effects of periodontal therapy. Fifty subjects were divided into two groups based on gingival index, probing pocket depth, clinical attachment loss and alveolar bone loss: healthy group and periodontitis group with generalized chronic periodontitis. Non-surgical periodontal therapy was rendered and crevicular fluid samples collected at baseline and four weeks after therapy for lactoferrin quantification using enzyme linked immunosorbent assay. The correlation between clinical parameters and lactoferrin levels was drawn and analysed for both groups. The mean level of crevicular lactoferrin in the periodontitis group was 1857.21 ng/ml. The mean level decreased to 1415.03 ng/ml after treatment. The lowest lactoferrin concentration was seen in the healthy group (75.34 ng/ml). All clinical parameters correlated positively with lactoferrin levels. The lactoferrin level was higher in the periodontitis group compared to the healthy group, and reduced with periodontal therapy. Higher levels were associated with higher values of clinical parameters, both before and after therapy. The data indicates that Lactoferrin plays an important role in periodontal disease and crevicular lactoferrin quantification can be a marker for detecting periodontal inflammation, oxidative stress and monitoring periodontal therapy.

An antibody-free microfluidic paper-based analytical device for the determination of tear fluid lactoferrin by fluorescence sensitization of Tb3+

An inkjet-printed microfluidic paper-based analytical device (μPAD) for the detection of lactoferrin has been developed. The analyte concentration dependent fluorescence emission, caused by the sensitization of pre-deposited terbium (Tb(3+)) upon complexation with lactoferrin on the paper device, is captured using a digital camera. The dynamic response range (0.5-3 mg mL(-1)) and the limit of detection (0.30 mg mL(-1)) of the μPAD are suitable for the analysis of normal human tears and the detection of eye disorders. Finally, lactoferrin concentrations in human tear samples were analyzed by the μPADs and the assay results corresponded within 6% error to those obtained by an immunoassay (ELISA). The μPADs provide a simple, rapid and accurate method for lactoferrin detection in tear fluid. Results are obtained within 15 min of a single application of 2.5 μL of sample. To the best of our knowledge, this is the first report of a device for lactoferrin quantification relying neither on an immunoassay nor on high cost analytical instrumentation.

An evaluation of the expression profiles of salivary proteins lactoferrin and lysozyme and their association with caries experience and activity

PURPOSE: Saliva contains both specific and non-specific protective factors of the immune system, such as antimicrobial proteins, which can inhibit the adhesion and viability of cariogenic microorganisms. The association between caries experience/activity and the electrophoretic profiles of salivary proteins lactoferrin and lysozyme was evaluated. METHODS: Eighty 12-year-old students from public schools in Londrina, PR, Brazil, were selected and divided into two groups: Group A - with decayed teeth and Group B - with caries-free teeth. The parent/guardian of each child signed a consent form and filled out a questionnaire regarding the oral and systemic health of his/her child. A clinical examination to diagnose the presence or absence of dental caries, by means of the DMFT index, was conducted. A total of 1 mL of saliva was collected for protein analysis using a polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: A total of 58.8% of the children were caries-inactive; in contrast, 63.3% showed caries experience. There was a slight association between lysozyme concentrations and DMFT. Lactoferrin was positively correlated with both DMFT and restored teeth. CONCLUSION: The quantification of lactoferrin and lysozyme enabled an assessment of possible associations with caries status, thus improving the understanding of the biological and etiological aspects of caries.

Purification and quantification of lactoferrin in equine seminal plasma.

Lactoferrin with a molecular mass of 80 kDa was purified from equine seminal plasma by heparin-Agarose affinity chromatography and Sephacryl S-200 gel filtration. Purified lactoferrin was found to be highly homogeneous on the bases of its migration as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and of the monospecificity of rabbit antibodies to the purified protein in immunoblotting of seminal plasma proteins. A sandwich enzyme-linked immunosorbent assay was developed for quantifying lactoferrin in equine seminal plasma. Seminal plasma lactoferrin concentrations in 23 normal stallions ranged from 42 to 453 microg/ml, with a mean value of 157 +/- 118 microg/ml (S.D.).

Gingival crevicular fluid lactoferrin levels in adult periodontitis patients.

The present study was designed to determine in a cross-sectional study whether there was any relationship between the levels of lactoferrin in gingival crevicular fluid and clinical periodontal parameters. Crevicular fluid was collected from individual sites using standardized filter paper strips (clinically healthy sites, N = 23; periodontitis sites, n = 66) and evaluated for lactoferrin by enzyme-linked immunosorbent assay. The data showed that: (1) the total amounts of lactoferrin were 0.003-0.021 ng (30 second sample) (average 0.009 +/- 0.005 ng) in a clinically healthy periodontium group and 0.016-3.847 ng (30 second sample) (average 0.575 +/- 0.069 ng) in adult periodontitis patients (statistically significantly higher in adult periodontitis patients); and (2) the total amounts of lactoferrin were significantly correlated with clinical parameters, especially a strong positive correlation with gingival crevicular fluid volume (r = 0.85, p < 0.01) and with probing depth (r = 0.71, p < 0.01). These results indicated that quantification of lactoferrin in gingival crevicular fluid may be a more sensitive indicator of periodontal pathology than traditional clinical indices.

Microparticle-enhanced nephelometric immunoassay of lactoferrin in human milk

A microparticle-enhanced nephelometric immunoassay was developed for lactoferrin quantitation in human milk. It is based on the nephelometric measurement of the light scattered during the competitive immuno-agglutination of a microparticle-lactoferrin conjugate with an antilactoferrin antiserum. This immunoassay is sensitive (detection limit in reaction mixture, 0.2 mg/L) and can be performed in diluted milk (1/3,000 in reaction mixture), excluding any interference or sample pretreatment. It allowed the quantification of lactoferrin on a large range of concentrations (0.675-21.6 g/L) with accuracy (linear recovery in dilution-overloading assay) and precision (within- and between-run coefficients of variation from 3% to 6%). Changes in the lactoferrin concentration of human milk during lactation were determined in 190 samples. The concentration and ratio of lactoferrin vs. total protein were found to be significantly higher in colostrum (5.9 g/l, 29%) than in transitional milk (2.9 g/L, 22%) or mature milk (2.5 g/L, 24%).

Microparticle‐enhanced nephelometric immunoassay of lactoferrin in human milk

A microparticle-enhanced nephelometric immunoassay was developed for lactoferrin quantitation in human milk. It is based on the nephelometric measurement of the light scattered during the competitive immunoagglutination of a microparticle-lactoferrin conjugate with an antilactoferrin antiserum. This immunoassay is sensitive (detection limit in reaction mixture, 0.2 mg/L) and can be performed in diluted milk (1/3,000 in reaction mixture), excluding any interference or sample pretreatment. It allowed the quantification of lactoferrin on a large range of concentrations (0.675–21.6 g/L) with accuracy (linear recovery in dilution-overloading assay) and precision (within- and between-run coefficients of variation from 3% to 6%). Changes in the lactoferrin concentration of human milk during lactation were determined in 190 samples. The concentration and ratio of lactoferrin vs. total protein were found to be significantly higher in colostrum (5.9 g/l, 29%) than in transitional milk (2.9 g/L, 22%) or mature milk (2.5 g/L, 24%). J. Clin. Lab. Anal. 11:239–243, 1997. © 1997 Wiley-Liss, Inc.

Neutrophil Activation Subsequent to Cardiopulmonary Bypass: A Sensitive Method for Determining Neutrophil Activation by Quantification of Lactoferrin Concentrations

Neutrophils have been implicated as contributors to the pathogenesis of cardiopulmonary bypass (CPB). We report a method to quantify the extent of neutrophil activation during CPB through measuring the serum concentrations of lactoferrin. We developed a modified enzyme-linked immunosorbent assay (ELISA) for the quantification oflactoferrin that is sensitive to 1 ng/ml. The preliminary data acquired to support this method confirm that there is a time-dependent increase in neutrophil activity during CPB. This was determined by measuring lactoferrin serum concentrations, and the lactoferrin concentrations appear to be higher in the pediatric patient compared to the adult patient.