Ni Putu Yunita Sri Lestari, Widya Kusuma Wardani, Linda Ratnawati, Tatit Nurseta, and

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CORRELATION OF LEUCINE INTAKE WITH SERUM

PROLACTIN AND BREAST MILK PROTEIN LEVELS IN

BREASTFEEDING WOMEN

Ni Putu Yunita Sri Lestari

, Widya Kusuma Wardani

, Linda Ratnawati

, Tatit Nurseta

, and Bambang

Rahardjo

12345

Brawijaya University, Malang, East Java, Indonesia

niputuyunitaa@gmail.com

, widyawardani02@gmail.com

, bar_obg.fk@ub.ac.id

, tns_obg.fk@ub.ac.id

linda.ratna@ub.ac.id

KEYWORDS

Amino Acid, Leucine,

Prolactin, Milk Protein,

Breastmilk

ABSTRACT

Breast milk is the best choice food for a baby’s growth and development of infants

at some point of the primary 6 months of life. Fulfillment of nutrition during

breastfeeding is an important requirement, one of which is the intake of the amino

acid leucine which is said to affect prolactin as a regulator of breast milk

production and protein synthesis in breast milk. This can support the quality and

quantity of breast milk to meet the nutritional needs of infants support infant

growth and development and reduce the incidence of stunting at the age of

toddlers. This research aims to determine the relationship between leucine intake

and levels of prolactin and protein in breast milk in breastfeeding mothers. This

research is an analytic observational study with a cross-sectional method. This

study carried out normality using the Kolmogorov-Smirnov test and using the

Pearson correlation test to determine the relationship between variables. Based on

the test results of the relationship between leucine intake and serum prolactin

levels, a significance value of 0.005 (p<0.05) was obtained with a correlation

coefficient value of 0.183 and the relationship between leucine intake and breast

milk protein levels obtained a significance value of 0.033 (p<0.05) with a

correlation coefficient of 0.147. The results of this test showed a significant

relationship between these variables. There is a weak correlation between leucine

intake and levels of prolactin and protein in breast milk

INTRODUCTION

Lactation is defined as the secretion of milk from the mammary glands and is

influenced by a complex hormonal network. Breastfeeding or lactation is one of the effective

ways to ensure the health and survival of a child (Lyons, Ryan, Dempsey, Ross, & Stanton,

2020). Breast milk contains all the energy, macronutrients, and micronutrients needed for a

baby's growth and development during the first 6 months of life. Breast milk also provides

various immunological factors and bioactive components (Thai & Gregory, 2020).

Milk production is regulated by the mother's hypothalamus and hormones produced

by the anterior pituitary gland. Prolactin is a polypeptide hormone that is responsible for

lactation. Prolactin promotes the growth of the alveoli, which are components of the

mammary glands, where milk production occurs. Prolactin stimulates breast alveolar

epithelial cells to synthesize components of breast milk, including lactose, casein, and lipids

(Rudolph, Russell, Webb, Neville, & Anderson, 2011).

Breast milk protein plays a role in providing nutrition and contributing to infant

development. If the consumption of energy and protein is less, it will cause the body to lack

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Milk Protein Levels in Breastfeeding Women]

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Linda Ratnawati, Tatit Nurseta, and Bambang Rahardjo

nutrients, so the body will use the available energy and protein stores. If this condition lasts

for a long time, energy and protein stores will be depleted, resulting in tissue damage that

causes children to experience stunting (Wells et al., 2020).

During lactation, a diet high in red meat, cereals, and eggs were associated with

higher protein and energy content in breast milk (Zhang et al., 2021). These findings indicate

that the diet of breastfeeding mothers can affect the macronutrient composition of breast milk

including breast milk protein and provide a basis for improving child health .

Proteins are composed of amino acids linked to one another. These amino acids play

an important role in the development and growth of cells in the body. Certain amino acids,

such as leucine, serve as substrates for protein synthesis and are important factors in

regulating protein metabolism (Li, Yin, Tan, Kong, & Wu, 2011).

Prolactin as a regulator of breast milk production consists of 199 amino acids that

form a single polypeptide chain with three intramolecular disulfide bonds (Bai et al., 2012).

Previous studies have shown that leucine regulates prolactin secretion through a mechanism

by which tyrosine levels decrease, which regulates dopamine synthesis. The low dopamine

hormone will trigger the production of prolactin. It was also mentioned that leucine directly

regulates the release of prolactin from pituitary mammotrophs. Increased intake of leucine is

also said to increase plasma prolactin concentrations.

A maternal diet with good nutritional content is very important in determining the

quality and quantity of breast milk, one of which is leucine. Leucine is one of the essential

amino acids that make up protein and plays an important role in protein synthesis in breast

milk by activating the rapamycin pathway and regulating prolactin secretion (Rezaei, Wu,

Hou, Bazer, & Wu, 2016). Leucine is one of the nutrients needed by mothers during

breastfeeding which will support the protein content of breast milk and milk production so

that breastfeeding mothers can provide exclusive breastfeeding with good breast milk protein

content. Meeting the protein needs of children during breastfeeding is expected to reduce the

incidence of stunting in children (Dewey, 2016).

METHOD RESEARCH

This research is quantitative research with an analytical observational research design.

This research was carried out from December 2021 to February 2022 in the city of Surabaya,

namely in the Benowo Health Center and Kenjeran District working area The research

method used purposive sampling. The number of samples in this study was 110 people who

were determined based on the inclusion criteria (mothers who have babies aged 1 to 6

months, babies are given exclusive breastfeeding, mothers and babies are in good health,

mothers have low-stress levels, and are willing to be respondents) and criteria exclusion (not

present at the time of data collection, refusal during follow-up, mothers taking drugs that

affect prolactin production such as domperidone). The researcher studied the intake of

leucine with direct interviews multiple 24h food recall methods for 3 days, then the

researchers converted the food ingredients data according to the 2014 Individual Food

Consumption Survey Book. Intake of the leucine using the USDA National Nutrient

Database for Standard Reference Legacy (2018). The researchers collaborated with the

Surabaya Prodia Clinical Laboratory to take blood samples and check serum prolactin levels

(ECLIA test), and the Faculty of Mathematics and Natural Sciences, University of Brawijaya

[ Correlation of Leucine Intake with Serum Prolactin and Breast

Milk Protein Levels in Breastfeeding Women]

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laboratory to examine breast milk protein levels (Lowry test). The data is processed using

SPSS Type 22.0 Software. This study uses the Kolmogorov-Smirnov test to test for

normality, and numerical data obtained were then tested using the Pearson correlation

statistical test to determine the relationship between variables.

RESULT AND DISCUSSION

A. RESULT

During the study, 110 breastfeeding mothers who had babies aged 1 to 6 months with

several characteristics of the respondents were assessed

Table 1.1. Respondents Characteristics

Sample Characteristics

Amount (people)

Percentage (%)

Age

<20

21-30

31-40

41-50

1.8%

68.2%

28.2%

1.8%

Educational level

Elementary School

Middle School

High School

Diploma

Bachelor

8.2%

11.8%

50.9%

8.2%

20.9%

Profession

Working mom

Housewife

28.2%

71.8%

Parity

Primipara

Multipara

32.7%

67.3%

Total

110

100%

Table 1 shows that the majority of respondents are aged 21-30 years (68.2%),

followed by ages 31-40 years (28.2%) and the last age <20 years and ages 41-50 years having

the same number (1.8%) . based on education level, the majority of respondents have high

school education (50.9%) and bachelor’s (20.9%), there are 8.2% of respondents with

elementary school and diploma educational background. Based on occupation, most of the

respondents studied were housewives as many as 79 respondents (71.8%). Respondents who

work are dominated by private employees and health workers (doctors, nurses, midwives,

dentists). Meanwhile, based on parity, the majority of respondents were multiparous (67.3%).

Based on the study of leucine intake for 3 days, it was shown that of 110 respondents

in the Benowo and Kenjeran Health Centers, the average daily intake of leucine was 5.57

grams, the lowest daily intake was 1.35 grams and the largest intake was 10.12 grams.

Assessment of serum prolactin levels was carried out by examining blood samples in

breastfeeding mothers. Data on serum prolactin levels showed an average value of 129.2

ng/mL with the lowest level of 13.10 ng/mL and the highest level of 345.9 ng/mL. This

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Linda Ratnawati, Tatit Nurseta, and Bambang Rahardjo

shows that on average breastfeeding mothers in the Benowo and Kenjeran health centers have

normal prolactin levels. While the assessment of breast milk protein levels was carried out by

examining breast milk samples in the laboratory, the results showed that the average value of

breast milk protein levels in respondents was 1.12 g/dL with the lowest level being 0.93 g/dL

and the highest level being 1.47 g/dL. This data shows that breastfeeding mothers in the

Benowo and Kenjeran health centers have normal milk protein levels on average.

Table 1.2. Results of Kolmogorov-Smirnov test

Leucine intake

Serum prolactin

Breastmilk protein

110

Asymp. Sig

.079

.200

Note: The data is normally distributed if the results of the one-sample Kolmogorov-Smirnov test show

a significance value of > 0.05.

The results of the data normality test showed that the value of leucine intake, serum

prolactin levels, and breast milk protein levels were normally distributed with a significance

value >0.05.

Table 1.3. Relationship of leucine intake with serum prolactin and breastmilk protein

levels

Independent variable

Dependent variable

p-value

Leucine intake

Serum prolactin

0.005

0.183

Breastmilk protein

0.033

0.147

A bivariate test was performed using the Pearson correlation test. The correlation

value or relationship between the intake of leucine and serum prolactin levels was obtained at

a significance value of 0.005 which indicates that the p-value <0.05 means that the intake of

leucine has a relationship with serum prolactin levels. Based on the results of the regression

test between the intake of leucine and serum prolactin levels, it is known that there is a

correlation of 0.183 (18.3%). Assessment of the correlation between intake of leucine and

protein content of breast milk showed a significant value of 0.033 which indicated a

relationship between intake of leucine and protein content of breast milk. The results of the

regression test showed that there was a correlation of 0.147 (14.7%) between the intake of

leucine and the protein content of breast milk.

B. DISCUSSION

Research conducted on 110 respondents showed a significant relationship between

leucine intake and serum prolactin levels. Based on the study by de Ridder et al. (2014) stated

that a mild increase in dietary leucine can also affect the synthesis and release of prolactin,

and the normal development of the estrous cycle. Leucine is also said to regulate the release

of prolactin from the pituitary either directly or by lowering dopamine levels in the brain.

Leucine is one of the amino acids that make up protein, consumption of foods with a high

[ Correlation of Leucine Intake with Serum Prolactin and Breast

Milk Protein Levels in Breastfeeding Women]

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protein diet can increase prolactin concentrations through the mechanism of increasing the

expression of SNAT2 simultaneously not only in the mammary glands during lactation but

also in the liver and adipose tissue.

The relationship between amino acid supply and milk production is complex.

Previous studies in experimental animals have shown that amino acid intake has been shown

to affects circulating hormone concentrations. One possible mechanism of action of amino

acids is through stimulation of endocrine responses, in particular through increased

endogenous production of insulin and prolactin, two hormones are known to be important in

the control and maintenance of lactation (De Ridder, Farmer, De Lange, Shoveller, &

Luimes, 2014). The nutritional needs of breastfeeding mothers increase during breastfeeding

to support the growth and development of the baby. The amount of breast milk consumed by

infants and the nutritional content of breast milk is often used to assess nutritional adequacy

during the lactation process. However, the volume of breast milk produced can be

significantly reduced if the mother is malnourished.

Besides nutrition, many other factors such as milk production are controlled through a

neuroendocrine feedback system. The baby's sucking stimulates mechanoreceptors in the

nipple and areola, which send nerve signals to the brain. The anterior pituitary then releases

prolactin, which stimulates milk production. Stimulus during breastfeeding can inhibit the

release of dopamine by the hypothalamus thus allowing for higher prolactin production

(Romano et al., 2013).

Previous research has consistently shown a decrease in prolactin levels in response to

physical and psychosocial stress. Prolonged exposure to stress is said to affect milk ejection,

leading to suppression of lactation secondary to inhibition of milk synthesis as a result of

incomplete emptying of the breast. The breast emptying rate is a factor that regulates milk

synthesis.

The low correlation value of 18.3% can be caused by several factors that can affect

prolactin production. In this study, only food intake (leucine) was studied, and controlled

stress factors were eliminated as confounding factors by selecting respondents who only had

a high level of stress. low. Other factors such as breastfeeding time before blood sampling,

menstrual phase, especially the luteal and follicular phases, sleep quality, sexual activity,

duration of breastfeeding, and breastfeeding frequency could not be controlled by

researchers.

The relationship between leucine intake and breast milk protein also showed

significant results. The results of this study are supported by the research of (Reitelseder et

al., 2011) who showed that leucine significantly affects the regulation of casein (milk

protein). Milk protein is an important component of milk and a critical index that affects

milk quality. It is reported that amino acids can increase the protein content of milk. Previous

research found that leucine itself regulates the synthesis of milk proteins in the epithelial cells

of the cow's mammary glands. It was noted that leucine enhances milk protein synthesis via

the mammalian target of rapamycin (mTOR) and Janus kinase 2/signal transducer and

activator of the transcriptional signaling pathway 5 (JACK2/STAT5). In the mammary gland,

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Linda Ratnawati, Tatit Nurseta, and Bambang Rahardjo

leucine regulates various biological processes, such as cell proliferation and milk synthesis

(αs-casein, β-casein, and к-casein).

Individually, each amino acid has a different effect on protein signaling and the rate

of synthesis of the casein fraction. A strategy to increase post-absorption efficiency is to

decrease the present percentage of food and supplement it with specific amino acids that have

the greatest effect on the regulation of translation and protein synthesis. This will increase or

maintain mammary gland requirements and amino acid extraction and reduce the recycling of

splanchnic tissue (Apelo, Knapp, & Hanigan, 2014).

The amount of essential amino acids in the diet is important for milk protein

synthesis, but the ratio of essential amino acids in the diet is more important (Appuhamy et

al., 2014). In a previous study, a significant interaction was observed between methionine and

leucine indicating a higher response to β-casein. The use of optimal concentration ratios of 4

essential amino acids or optimal concentrations of single essential amino acids substantially

increased β-casein expression. The four amino acids are histidine, lysine, methionine, and

leucine with a ratio of 5:6:1:7 which efficiently increase the expression of β-casein.

The balance of the proportion of amino acid intake in the diet is very important in

protein nutrition (Hoffer, 2016). Imbalances between chemically or structurally similar amino

acids lead to amino acid antagonism, which is manifested as reduced food intake, abnormal

behavior, and impaired growth. This was also stated by (Hoffer, 2016) that when increasing

milk protein, the level of supply of a single amino acid is important, but it is also necessary to

evaluate the supply of balanced amino acids. Concerns with amino acid balance have

increased. Several authors have shown that increased milk production may be related to an

increased or balanced supply of amino acids that results from protein supplementation.

Protein synthesis in milk is a very complex and high-cost process because the conversion

efficiency of food protein into milk protein is very low (Carr & Golding, 2016).

CONCLUSION

There is a weak relationship between leucine intake and serum prolactin and breast

milk protein levels in breastfeeding mothers in the Benowo and Kenjeran health centers,

Surabaya. Suggestions for future researchers, further research is needed on other amino

acids/micronutrients that can affect prolactin and protein synthesis in breast milk.

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Nurseta, and Bambang Rahardjo (2022)

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