591338
research-article2015
JHLXXX10.1177/0890334415591338Journal of Human LactationEscuder-Vieco et al
Original Research: Brief Report
Determination of Acidity in Donor Milk: Comparison of Results Obtained by 2 Different Analytical Methods
Journal of Human Lactation 1–3 © The Author(s) 2015 Reprints and permissions: sagepub.com/journalsPermissions.nav DOI: 10.1177/0890334415591338 jhl.sagepub.com
Diana Escuder-Vieco, BSc1,2, Sara Vázquez-Román, MD1, Juan Sánchez-Pallás, BSc3, Noelia Ureta-Velasco, MD1, Rocío Mosqueda-Peña, MD1, and Carmen Rosa Pallás-Alonso, PhD1,2
Abstract Background: There is no uniformity among milk banks on milk acceptance criteria. The acidity obtained by the Dornic titration technique is a widely used quality control in donor milk. However, there are no comparative data with other aciditymeasuring techniques, such as the pH meter. Objective: The objective of this study was to assess the correlation between the Dornic technique and the pH measure to determine the pH cutoff corresponding to the Dornic degree limit value used as a reference for donor milk quality control. Methods: Fifty-two human milk samples were obtained from 48 donors. Acidity was measured using the Dornic method and pH meter in triplicate. Statistical data analysis to estimate significant correlations between variables was carried out. The Dornic acidity value that led to rejecting donor milk was ≥ 8 Dornic degrees (°D). Results: In the evaluated sample size, Dornic acidity measure and pH values showed a statistically significant negative correlation (τ = −0.780; P = .000). A pH value of 6.57 corresponds to 8°D and of 7.12 to 4°D. Conclusion: Donor milk with a pH over 6.57 may be accepted for subsequent processing in the milk bank. Moreover, the pH measurement seems to be more useful due to certain advantages over the Dornic method, such as objectivity, accuracy, standardization, the lack of chemical reagents required, and the fact that it does not destroy the milk sample. Keywords breastfeeding, donor milk, human milk banking, quality control, acidity
Well Established It is essential to achieve quality control of donor human milk.The determination of acidity by the Dornic method is the traditional quality control used in milk banks to assess the nutritional and technological conditions.
Newly Expressed This study determined the pH cutoff corresponding to the Dornic degree limit used as a reference for donor milk quality control.The calculation was performed using a mathematical expression that linearly correlates the results obtained from both testing methods.
Background Donor human milk is recommended as the first alternative when maternal milk is not available, especially for sick or premature infants, due to the vast potential benefits that it provides compared with breast milk substitute.1 To date, there are no worldwide uniform guidelines for the screening, processing, storage, and handling of donor
milk among milk banks. Because of this, the current techniques to check donor milk are under constant review. The determination of acidity is one of the common controls performed on milk donated to milk banks.2 The overall acidity of breast milk comprises natural acidity (provided by casein, minerals, and organic acids) and developed acidity (due to lactic acid and other organic acids from the microbial degradation of lactose and, occasionally, from the lipids from milks in decline). The acidification of breast milk has been shown to cause a decrease in white blood cells, lipase 1
Department of Neonatology, Hospital 12 de Octubre, Madrid, Spain SAMID Network (Spanish Collaborative Maternal and Child Health Research Network), RD12/0026/0007, Spain 3 Environmental Science Degree, Universidad Autónoma de Madrid, Madrid, Spain 2
Date submitted: September 22, 2014; Date accepted: May 21, 2015. Corresponding Author: Diana Escuder-Vieco, BSc, Department of Neonatology, Hospital 12 de Octubre, Edificio Materno-Infantil, Avda de Cordoba s/n, 28041 Madrid, Spain. Email: [email protected]
Downloaded from jhl.sagepub.com by guest on November 15, 2015
2
Journal of Human Lactation
activity, and total protein.3 Lipoprotein lipase is not stable at pH below 5, and bile salt–stimulated lipase has a neutral to alkaline pH optimum value (pH 7.3-8.6).4,5 On the other hand, it is likely that in an acidic environment, the casein fraction of the milk will denature and precipitate, resulting in an unstable milk to heat treatment.6 Holder pasteurization (62.5°C, 30 minutes) is the most widely used method to achieve the microbiological safety of donor milk.1 Thus, measuring the acidity of donor milk is an indicator not only of nutritional quality but also of technological quality. Currently, milk banks determine acidity by the Dornic method, accepting that the donor milk is suitable for heat treatment when acidity is 8° Dornic degrees (°D) or less.7 However, direct measurement of pH using a pH meter also provides information on total acidity, but there are no data concerning the correlation between pH measurement and Dornic acidity. The aim of this study is to assess the correlation between the Dornic titration method and the pH measurement with calibrated electrodes to determine the pH cutoff corresponding to the Dornic degree limit value used as a reference for donor milk quality control.
Methods Samples Donor samples (n = 52) were obtained from 48 donors who fulfilled the requirements of the Human Milk Bank (negative serology for breast milk transmission of viral disease and a healthy lifestyle). Some donors provided more than one sample for this study, taken at different times. Before taking the sample, donors had to wash their hands and sterilize their breast pump or the breast pump borrowed from the milk bank. The samples were collected in sterile glass containers provided by the bank, which were kept chilled at −18°C to −20°C until brought into the milk bank by the donors themselves, without breaking the cold chain. Subsequently, the samples were thawed at 37°C in a shaking water bath (LabCompanion, Seoul, Korea) and divided into aliquots to analyze acidity. This study was reviewed and approved by the institutional review board of the Hospital 12 de Octubre de Madrid, and informed consent was obtained from each donor.
pH measurement. The pH human milk samples (20-25°C) were measured with a calibrated pH meter (pH-Meter BASIC 20; Crison, Barcelona, Spain) equipped with a glass electrode (5011T; Crisol, Barcelona, Spain). The sensitivity value of this technique is 0.01. Each sample was analyzed in triplicate using both methods.
Statistical Analysis Prior to analysis, the sample size was estimated with PASS 13 software (NCSS, Kaysville, Utah, USA). Thus, assuming nonnormal distribution of data, a sample size of 27 was expected to be sufficient when a 2-sided 95% confidence interval with a width equal to 0.2 and an estimated Kendall τ correlation of 0.800 were considered. The Shapiro-Wilk test was used to assess normality of data distribution. The Kendall τ correlation coefficient was used to test the correlation for the measured variables, using the statistical package R 2.15.3 (R Core Team, Vienna, Austria).
Results The relevant donor data and the samples analyzed in this study were as follows: median gestational age of the infants at birth was 39 weeks (interquartile range [IQR], 38-40.5), the median time period from delivery to the expression date of the donor samples was 129 days (IQR, 69-258), and the median freezing period was 15 days (IQR, 10-24). Of the 52 samples analyzed, 94% (49/52) were under the limits recommended for consumption (acidity ≤ 8°D); of these, 83% (41/49) of the samples were of top quality (acidity ≤ 4°D). Furthermore, median Dornic acidity was 3 (IQR, 2-4), and median pH was 7.23 (IQR, 7-7.34). The correlation between both acidity-measuring methods was obtained by the Kendall τ correlation coefficient calculation; thus, a high significant inverse correlation was observed (τ = −0.780, P < .000). Figure 1 shows the graphical representation of the linear model obtained. In addition, the expression of the model was acidity = 56.170 – 7.321 * pH. A pH of 6.57 corresponds to an acidity of 8°D, and a pH of 7.12 corresponds to top quality milk (acidity of 4°D).
Discussion
Analytical Methods Titratable acidity expressed in Dornic degrees (°D). One drop of 1% (w/v) phenolphthalein in 95% (v/v) ethanol (turning point at pH 8.2-8.4) was added to each 1-mL sample of human milk. The mixture was titrated with N/9 NaOH (Dornic solution) until the first color change (to pink) persisted for 30 seconds. Each 0.01 mL of Dornic solution expended to neutralize 1 mL of human milk was equivalent to 1°D. Milk with ≥ 8°D was rejected and milk with ≤ 4°D was considered of excellent quality.
The aim of this study was to determine the pH cutoff corresponding to the Dornic degree limit value used as a reference for donor milk quality control. Donor milk with 8°D acidity had a pH of 6.57, a limit below which it is not ideal to pasteurize donor milk. Moreover, a pH of 7.12 indicates an acidity value of 4°D. As a result, donor milk with a pH of 7.12 or above could be considered top quality. To establish these cutoffs, the correlation between the traditionally used Dornic titration method vs the measuring of
Downloaded from jhl.sagepub.com by guest on November 15, 2015
3
Escuder-Vieco et al Figure 1. Linear Correlation Analysis of Dornic Acidity and pH.
Conclusion
8
10
The results of this study revealed that donor milk with a pH over 6.57 may be accepted for subsequent processing in a milk bank. In addition, measuring acidity using a pH meter offers certain advantages over the traditional Dornic degree method.
6
The authors declare no potential conflicts of interest with respect to this research, authorship, and/or publication of this article.
Funding 4
Dornic Acidity (°D)
Declaration of Conflicting Interests
2
The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was funded by Spanish Health Research Funding (grant FIS 09/00040).
6.6
6.8
7.0
7.2
7.4
7.6
pH
Dornic acidiy degrees in donor milk were negative correlated with pH values (r = – 0.780, P = .000)
pH with calibrated electrode devices was assessed. A high coefficient of correlation value was obtained between Dornic degrees and pH units in the total donor milk samples analyzed. This finding was expected because both techniques measure the overall acidity in the milk. Moreover, the pH values obtained in this study are similar to those found by other authors who analyzed breast milk samples that had been collected and stored under controlled conditions.8-11 Although the Dornic method is the traditional test to check the quality of donor milk, this technique has some disadvantages because it is an indirect and subjective method (the detection of the turning point varies depending on the training of the clinical staff), has low sensitivity, requires the use of chemical reagents, and destroys the samples. On the other hand, the determination of pH with calibrated electrode devices is standardized and objective, with high sensitivity and accuracy. Furthermore, it does not require the use of chemical reagents and does not destroy the milk sample during the analysis, allowing the sample to be reused if necessary. All of these characteristics highlight that the use of pH meters might be a more convenient alternative than Dornic titration to determine the overall acidity of milk. One of the most significant limitations of this study is the fact that the correlation has been established with a limited number of milk samples. These samples have low acidity values as a result of the careful sampling and transportation process performed by donors to hinder sample deterioration.
References 1. ESPGHAN Committee on Nutrition, Arslanoglu S, Corpeleijn W, Moro G, et al. Donor human milk for preterm infants: current evidence and research directions. J Pediatr Gastroenterol Nutr. 2013;57(4):535-542. 2. Vázquez-Román S, García-Lara NR, Escuder-Vieco D, et al. Determination of Dornic acidity as a method to select donor milk in a milk bank. Breastfeed Med. 2013;8(1):99-104. 3. Erickson T, Gill G, Chan GM. The effects of acidification on human milk’s cellular and nutritional content. J Perinatol. 2013;33(5):371-373. 4. Olivecrona T, Hernell O. Human milk lipases and their possible role in fat digestion. Padiatr Padol. 1976;11(4): 600-604. 5. Freed LM, York CM, Hamosh P, et al. Bile salt–stimulated lipase of human milk: characteristics of the enzyme in the milk of mothers of premature and full-term infants. J Pediatr Gastroenterol Nutr. 1987;6(4):598-604. 6. McMahon DJ, Du H, McManus WR, et al. Microstructural changes in casein supramolecules during acidification of skim milk. J Dairy Sci. 2009;92(12):5854-5867. 7. Agência Nacional de Vigilância Sanitária. Banco de leite humano: funcionament, prevenção e controle de riscos. Brasília, Brazil: Anvisa; 2008. 8. Hamosh M, Ellis LA, Pollock DR, et al. Breastfeeding and the working mother: effect of time and temperature of short-term storage on proteolysis, lipolysis, and bacterial growth in milk. Pediatrics. 1996;97(4):492-498. 9. Morriss FH Jr, Brewer ED, Spedale SB, et al. Relationship of human milk pH during course of lactation to concentrations of citrate and fatty acids. Pediatrics. 1986;78(3):458-464. 10. Slutzah M, Codipilly CN, Potak D, et al. Refrigerator storage of expressed human milk in the neonatal intensive care unit. J Pediatr. 2010;156(1):26-28. 11. Miranda M, Gormaz M, Romero FJ, et al. Stability of the antioxidant capacity and pH of human milk refrigerated for 72 hours: longitudinal study. Nutr Hosp. 2011;26(4):722-728.
Downloaded from jhl.sagepub.com by guest on November 15, 2015
research-article2015
JHLXXX10.1177/0890334415591338Journal of Human LactationEscuder-Vieco et al
Original Research: Brief Report
Determination of Acidity in Donor Milk: Comparison of Results Obtained by 2 Different Analytical Methods
Journal of Human Lactation 1–3 © The Author(s) 2015 Reprints and permissions: sagepub.com/journalsPermissions.nav DOI: 10.1177/0890334415591338 jhl.sagepub.com
Diana Escuder-Vieco, BSc1,2, Sara Vázquez-Román, MD1, Juan Sánchez-Pallás, BSc3, Noelia Ureta-Velasco, MD1, Rocío Mosqueda-Peña, MD1, and Carmen Rosa Pallás-Alonso, PhD1,2
Abstract Background: There is no uniformity among milk banks on milk acceptance criteria. The acidity obtained by the Dornic titration technique is a widely used quality control in donor milk. However, there are no comparative data with other aciditymeasuring techniques, such as the pH meter. Objective: The objective of this study was to assess the correlation between the Dornic technique and the pH measure to determine the pH cutoff corresponding to the Dornic degree limit value used as a reference for donor milk quality control. Methods: Fifty-two human milk samples were obtained from 48 donors. Acidity was measured using the Dornic method and pH meter in triplicate. Statistical data analysis to estimate significant correlations between variables was carried out. The Dornic acidity value that led to rejecting donor milk was ≥ 8 Dornic degrees (°D). Results: In the evaluated sample size, Dornic acidity measure and pH values showed a statistically significant negative correlation (τ = −0.780; P = .000). A pH value of 6.57 corresponds to 8°D and of 7.12 to 4°D. Conclusion: Donor milk with a pH over 6.57 may be accepted for subsequent processing in the milk bank. Moreover, the pH measurement seems to be more useful due to certain advantages over the Dornic method, such as objectivity, accuracy, standardization, the lack of chemical reagents required, and the fact that it does not destroy the milk sample. Keywords breastfeeding, donor milk, human milk banking, quality control, acidity
Well Established It is essential to achieve quality control of donor human milk.The determination of acidity by the Dornic method is the traditional quality control used in milk banks to assess the nutritional and technological conditions.
Newly Expressed This study determined the pH cutoff corresponding to the Dornic degree limit used as a reference for donor milk quality control.The calculation was performed using a mathematical expression that linearly correlates the results obtained from both testing methods.
Background Donor human milk is recommended as the first alternative when maternal milk is not available, especially for sick or premature infants, due to the vast potential benefits that it provides compared with breast milk substitute.1 To date, there are no worldwide uniform guidelines for the screening, processing, storage, and handling of donor
milk among milk banks. Because of this, the current techniques to check donor milk are under constant review. The determination of acidity is one of the common controls performed on milk donated to milk banks.2 The overall acidity of breast milk comprises natural acidity (provided by casein, minerals, and organic acids) and developed acidity (due to lactic acid and other organic acids from the microbial degradation of lactose and, occasionally, from the lipids from milks in decline). The acidification of breast milk has been shown to cause a decrease in white blood cells, lipase 1
Department of Neonatology, Hospital 12 de Octubre, Madrid, Spain SAMID Network (Spanish Collaborative Maternal and Child Health Research Network), RD12/0026/0007, Spain 3 Environmental Science Degree, Universidad Autónoma de Madrid, Madrid, Spain 2
Date submitted: September 22, 2014; Date accepted: May 21, 2015. Corresponding Author: Diana Escuder-Vieco, BSc, Department of Neonatology, Hospital 12 de Octubre, Edificio Materno-Infantil, Avda de Cordoba s/n, 28041 Madrid, Spain. Email: [email protected]
Downloaded from jhl.sagepub.com by guest on November 15, 2015
2
Journal of Human Lactation
activity, and total protein.3 Lipoprotein lipase is not stable at pH below 5, and bile salt–stimulated lipase has a neutral to alkaline pH optimum value (pH 7.3-8.6).4,5 On the other hand, it is likely that in an acidic environment, the casein fraction of the milk will denature and precipitate, resulting in an unstable milk to heat treatment.6 Holder pasteurization (62.5°C, 30 minutes) is the most widely used method to achieve the microbiological safety of donor milk.1 Thus, measuring the acidity of donor milk is an indicator not only of nutritional quality but also of technological quality. Currently, milk banks determine acidity by the Dornic method, accepting that the donor milk is suitable for heat treatment when acidity is 8° Dornic degrees (°D) or less.7 However, direct measurement of pH using a pH meter also provides information on total acidity, but there are no data concerning the correlation between pH measurement and Dornic acidity. The aim of this study is to assess the correlation between the Dornic titration method and the pH measurement with calibrated electrodes to determine the pH cutoff corresponding to the Dornic degree limit value used as a reference for donor milk quality control.
Methods Samples Donor samples (n = 52) were obtained from 48 donors who fulfilled the requirements of the Human Milk Bank (negative serology for breast milk transmission of viral disease and a healthy lifestyle). Some donors provided more than one sample for this study, taken at different times. Before taking the sample, donors had to wash their hands and sterilize their breast pump or the breast pump borrowed from the milk bank. The samples were collected in sterile glass containers provided by the bank, which were kept chilled at −18°C to −20°C until brought into the milk bank by the donors themselves, without breaking the cold chain. Subsequently, the samples were thawed at 37°C in a shaking water bath (LabCompanion, Seoul, Korea) and divided into aliquots to analyze acidity. This study was reviewed and approved by the institutional review board of the Hospital 12 de Octubre de Madrid, and informed consent was obtained from each donor.
pH measurement. The pH human milk samples (20-25°C) were measured with a calibrated pH meter (pH-Meter BASIC 20; Crison, Barcelona, Spain) equipped with a glass electrode (5011T; Crisol, Barcelona, Spain). The sensitivity value of this technique is 0.01. Each sample was analyzed in triplicate using both methods.
Statistical Analysis Prior to analysis, the sample size was estimated with PASS 13 software (NCSS, Kaysville, Utah, USA). Thus, assuming nonnormal distribution of data, a sample size of 27 was expected to be sufficient when a 2-sided 95% confidence interval with a width equal to 0.2 and an estimated Kendall τ correlation of 0.800 were considered. The Shapiro-Wilk test was used to assess normality of data distribution. The Kendall τ correlation coefficient was used to test the correlation for the measured variables, using the statistical package R 2.15.3 (R Core Team, Vienna, Austria).
Results The relevant donor data and the samples analyzed in this study were as follows: median gestational age of the infants at birth was 39 weeks (interquartile range [IQR], 38-40.5), the median time period from delivery to the expression date of the donor samples was 129 days (IQR, 69-258), and the median freezing period was 15 days (IQR, 10-24). Of the 52 samples analyzed, 94% (49/52) were under the limits recommended for consumption (acidity ≤ 8°D); of these, 83% (41/49) of the samples were of top quality (acidity ≤ 4°D). Furthermore, median Dornic acidity was 3 (IQR, 2-4), and median pH was 7.23 (IQR, 7-7.34). The correlation between both acidity-measuring methods was obtained by the Kendall τ correlation coefficient calculation; thus, a high significant inverse correlation was observed (τ = −0.780, P < .000). Figure 1 shows the graphical representation of the linear model obtained. In addition, the expression of the model was acidity = 56.170 – 7.321 * pH. A pH of 6.57 corresponds to an acidity of 8°D, and a pH of 7.12 corresponds to top quality milk (acidity of 4°D).
Discussion
Analytical Methods Titratable acidity expressed in Dornic degrees (°D). One drop of 1% (w/v) phenolphthalein in 95% (v/v) ethanol (turning point at pH 8.2-8.4) was added to each 1-mL sample of human milk. The mixture was titrated with N/9 NaOH (Dornic solution) until the first color change (to pink) persisted for 30 seconds. Each 0.01 mL of Dornic solution expended to neutralize 1 mL of human milk was equivalent to 1°D. Milk with ≥ 8°D was rejected and milk with ≤ 4°D was considered of excellent quality.
The aim of this study was to determine the pH cutoff corresponding to the Dornic degree limit value used as a reference for donor milk quality control. Donor milk with 8°D acidity had a pH of 6.57, a limit below which it is not ideal to pasteurize donor milk. Moreover, a pH of 7.12 indicates an acidity value of 4°D. As a result, donor milk with a pH of 7.12 or above could be considered top quality. To establish these cutoffs, the correlation between the traditionally used Dornic titration method vs the measuring of
Downloaded from jhl.sagepub.com by guest on November 15, 2015
3
Escuder-Vieco et al Figure 1. Linear Correlation Analysis of Dornic Acidity and pH.
Conclusion
8
10
The results of this study revealed that donor milk with a pH over 6.57 may be accepted for subsequent processing in a milk bank. In addition, measuring acidity using a pH meter offers certain advantages over the traditional Dornic degree method.
6
The authors declare no potential conflicts of interest with respect to this research, authorship, and/or publication of this article.
Funding 4
Dornic Acidity (°D)
Declaration of Conflicting Interests
2
The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was funded by Spanish Health Research Funding (grant FIS 09/00040).
6.6
6.8
7.0
7.2
7.4
7.6
pH
Dornic acidiy degrees in donor milk were negative correlated with pH values (r = – 0.780, P = .000)
pH with calibrated electrode devices was assessed. A high coefficient of correlation value was obtained between Dornic degrees and pH units in the total donor milk samples analyzed. This finding was expected because both techniques measure the overall acidity in the milk. Moreover, the pH values obtained in this study are similar to those found by other authors who analyzed breast milk samples that had been collected and stored under controlled conditions.8-11 Although the Dornic method is the traditional test to check the quality of donor milk, this technique has some disadvantages because it is an indirect and subjective method (the detection of the turning point varies depending on the training of the clinical staff), has low sensitivity, requires the use of chemical reagents, and destroys the samples. On the other hand, the determination of pH with calibrated electrode devices is standardized and objective, with high sensitivity and accuracy. Furthermore, it does not require the use of chemical reagents and does not destroy the milk sample during the analysis, allowing the sample to be reused if necessary. All of these characteristics highlight that the use of pH meters might be a more convenient alternative than Dornic titration to determine the overall acidity of milk. One of the most significant limitations of this study is the fact that the correlation has been established with a limited number of milk samples. These samples have low acidity values as a result of the careful sampling and transportation process performed by donors to hinder sample deterioration.
References 1. ESPGHAN Committee on Nutrition, Arslanoglu S, Corpeleijn W, Moro G, et al. Donor human milk for preterm infants: current evidence and research directions. J Pediatr Gastroenterol Nutr. 2013;57(4):535-542. 2. Vázquez-Román S, García-Lara NR, Escuder-Vieco D, et al. Determination of Dornic acidity as a method to select donor milk in a milk bank. Breastfeed Med. 2013;8(1):99-104. 3. Erickson T, Gill G, Chan GM. The effects of acidification on human milk’s cellular and nutritional content. J Perinatol. 2013;33(5):371-373. 4. Olivecrona T, Hernell O. Human milk lipases and their possible role in fat digestion. Padiatr Padol. 1976;11(4): 600-604. 5. Freed LM, York CM, Hamosh P, et al. Bile salt–stimulated lipase of human milk: characteristics of the enzyme in the milk of mothers of premature and full-term infants. J Pediatr Gastroenterol Nutr. 1987;6(4):598-604. 6. McMahon DJ, Du H, McManus WR, et al. Microstructural changes in casein supramolecules during acidification of skim milk. J Dairy Sci. 2009;92(12):5854-5867. 7. Agência Nacional de Vigilância Sanitária. Banco de leite humano: funcionament, prevenção e controle de riscos. Brasília, Brazil: Anvisa; 2008. 8. Hamosh M, Ellis LA, Pollock DR, et al. Breastfeeding and the working mother: effect of time and temperature of short-term storage on proteolysis, lipolysis, and bacterial growth in milk. Pediatrics. 1996;97(4):492-498. 9. Morriss FH Jr, Brewer ED, Spedale SB, et al. Relationship of human milk pH during course of lactation to concentrations of citrate and fatty acids. Pediatrics. 1986;78(3):458-464. 10. Slutzah M, Codipilly CN, Potak D, et al. Refrigerator storage of expressed human milk in the neonatal intensive care unit. J Pediatr. 2010;156(1):26-28. 11. Miranda M, Gormaz M, Romero FJ, et al. Stability of the antioxidant capacity and pH of human milk refrigerated for 72 hours: longitudinal study. Nutr Hosp. 2011;26(4):722-728.
Downloaded from jhl.sagepub.com by guest on November 15, 2015
