Concentration of Free Amino Acids in Human Milk of Women with Gestational Diabetes Mellitus and Healthy Women

Abstract

Objective:

It is generally agreed that breastfeeding has a positive effect on the metabolic situation in diabetic mothers. However, negative long-term effects are described for breastfed offspring of diabetic women. It is unknown if the composition of free amino acids (FAAs) in breastmilk of women with gestational diabetes mellitus (GDM) differs from that in milk of healthy women. We studied the amount of FAAs in breastmilk of women with GDM and women with normal glucose tolerance (NGT).

Subjects and Methods:

Human milk samples of 68 women (21 GDM and 47 NGT) were analyzed. Contents of FAAs in milk samples, obtained within the first 4 days after delivery (colostrum) and 6 weeks later (mature milk), were analyzed using high-performance liquid chromatography. Total amounts of FAAs in colostrum and in mature milk were compared between the groups. The impact of maternal age, body mass index (BMI), gestational age at birth, birth weight, and diagnosis of GDM on the total amount of FAAs was evaluated.

Results:

Overall, the total amount of FAAs increased significantly from colostrum to mature milk in both groups (p<0.001). The total amount of FAAs did not significantly differ between GDM and NGT in colostrum and in mature milk (1,560 μmol/L vs. 1,730 μmol/L and 2,440 μmol/L vs. 2,723 μmol/L, respectively). No significant influence on the total amount of FAAs at both measurements of maternal age, BMI, gestational age at birth, birth weight, and diagnosis of GDM could be observed by regression analyses.

Conclusion:

The content of FAAs of human milk does not significantly differ between women with GDM and women with NGT.

Introduction

Gestational diabetes mellitus (GDM) is a common pregnancy complication, with a prevalence of up to 15%, and its prevalence is increasing.¹ It is defined as carbohydrate intolerance of variable severity, with onset or first recognition during pregnancy.² GDM is associated with several short- and long-term complications for the mother and child, such as development of infant adiposity and type 2 diabetes.^3–5

It is generally agreed that breastfeeding has a positive effect on the metabolic situation in diabetic mothers.⁶ A possible decrease in the incidence of maternal and infant diabetes due to breastfeeding in women with GDM has been indicated.⁷ However, negative long-term effects are described for breastfed offspring of diabetic women.⁸ Early neonatal ingestion of breastmilk from diabetic mothers may increase the risk of adiposity and impaired glucose tolerance during childhood.

It has been shown that glucose and insulin levels in human milk of diabetic women correlate with plasma levels.⁹ Strict metabolic control in women with GDM leads to comparable concentrations of lactose, proteins, triglycerides, cholesterol, glucose, myo-inositol, and fatty acids in human milk as in healthy women.¹⁰

It is unknown if the composition of free amino acids (FAAs) in human milk of women with GDM is different from that of healthy women. Therefore, we evaluated the total concentrations of FAAs in colostrum (human milk obtained within the first 4 days after delivery) and in mature milk (obtained 6 weeks later) of women with GDM and of healthy women.

Subjects and Methods

This study was conducted at the Medical University of Vienna and at the Hanusch Hospital of Vienna and was approved by the local ethics committee.

Women who booked for delivery at these hospitals were prospectively screened for GDM by using a 75-g oral glucose tolerance test (OGTT) at 24–28 weeks of gestation. The OGTT was evaluated using the guidelines of the Austrian Society for Diabetes.¹¹ Upper normal limits for fasting, 1-hour, and 2-hour serum glucose levels were 92 mg/dL, 180 mg/dL, and 153 mg/dL, respectively. If one of these limits was exceeded, patients were diagnosed as having GDM and were referred to the Department of Internal Medicine for dietary counseling and if necessary for insulin treatment. If blood glucose self-assessments exceeded at fasting more than 90 mg/dL glucose and 1 hour after a meal more than 140 mg/dL glucose more than five times per week, insulin treatment was started or adapted, respectively. GDM patients receiving insulin therapy, under good metabolic control, and with normal fetal biometry were seen every 2 weeks. Patients with poor metabolic control were examined weekly, and insulin therapy was adapted. Labor was induced at term in patients with GDM and 10 days after the estimated date of birth in healthy pregnant women, unless labor occurred spontaneously.

We calculated a total sample size of 42 to be able to detect a difference of 15% in the total amino acid concentration between both groups with 80% power at the 5% significance level. Assuming a dropout rate of 30%, 30 consecutive women with GDM and 60 women with normal glucose tolerance (NGT) with uncomplicated singleton pregnancies without preexisting medical conditions and no fetal anomalies who underwent uncomplicated vaginal birth at term and who intended to breastfeed their babies were asked to participate in the study on the first day after delivery. Twelve women refused participating. After signing an informed consent, 25 women with GDM and 53 women with NGT were included in the study.

Study participants were asked to express at least 4 mL of their breastmilk into sterile polypropylene vials at the end of a feeding within the first 4 days after delivery (colostrum) and 6 weeks later (mature milk). Overall, 10 women (four women with GDM and six women with NGT) were excluded from the study; seven of these 10 women (three women with GDM and four with NGT) had not attended the second study visit, and three patients (one woman with GDM and two with NGT) stopped breastfeeding before the second study visit.

Finally, milk samples of 21 women with GDM and 47 women with NGT were analyzed. The contents of FAAs in human milk were determined by using high-performance liquid chromatography (HPLC).

Immediately after extraction the expressed human milk was put on dry ice and deproteinized with 20 μL of 5-sulfosalicylic acid (30%) containing 1 mM β-(2-thienyl)-dl-alanine as the internal standard. The samples were kept on –20°C for 30 minutes and then centrifuged at 17,970 ×g for 5 minutes after equilibration to room temperature.

For HPLC analysis of α-amino acids, 7 μL of the plasma extracts was diluted with 0.75 mL of Milli-Q^® water (Waters Millipore, Austria), and a precolumn derivatization was done by o-phthaldehyde reagent. After 1 minute of derivatization, 7 μL of the derivate was injected onto the column with a Spark (The Netherlands) Triathlon autosampler.

Separation of the amino acids was obtained with a Beckman (USA) HPLC apparatus equipped with an ODS Hypersil 3-μm (particle size) 125-×3.0-mm column (Thermo) and a Chromguard SS 10-×3-mm HPLC column (Chrompack, The Netherlands). In brief, separation was obtained at 22°C using a step gradient formed by the following buffers: Buffer A, 0.96 g of sodium acetate, 0.58 mL of acetic acid, and 2.8 mL of tetrahydrofuran dissolved in 1 L of Milli-Q water; Buffer B, 450 g of Milli-Q water, 450 g of acetonitrile (HPLC grade), and 9 mL of tetrahydrofuran. The flow rate was 0.5 mL/minute.

Detection was performed fluorometrically by a Jasco (Tokyo, Japan) fluorescence detector with an excitation wavelength of 330 nm and an emission wavelength of 440 nm.

First we compared the total amount of FAAs in human milk of patients with GDM and women with NGT in colostrum and in mature milk.

Additionally, we separately analyzed concentrations of the 11 most abundant FAAs in human milk and compared patients with GDM and healthy women.

Statistical analyses were performed with SPSS version 17.0 software (SPSS, Chicago, IL). We performed a Kolmogorov–Smirnov test to verify the use of tests for normally distributed variables. Normally distributed variables were summarized as mean (±SD) values, not normally distributed variables as medians (minimum and maximum), and categorical data as percentages. Differences between patients with or without GDM were analyzed by Student's unpaired t test. Differences between the two longitudinal measurements of amino acids in human milk were analyzed by Student's paired t test. Amino acid values were found to be normally distributed using the Kolmogorov–Smirnov test. Data were tested by multiple linear regression analyses to demonstrate the compound effect of maternal age, maternal body mass index (BMI), gestational age at birth, birth weight, and diagnosis of GDM on the total amount of amino acids in colostrum and mature milk. The total amount of amino acids was used as the dependent variable. Additionally, we performed a univariate variance analysis for repeated measurements using the total amount of amino acids in colostrum and mature milk as within-subjects factors and diagnosis of GDM as between-subject factor. All tests were two-tailed, and an α level of p<0.05 was considered statistically significant.

Results

In 68 white women (21 patients with GDM and 47 healthy women), FAAs in breastmilk were analyzed.

Patient characteristics are shown in Table 1. Women with GDM were significantly older than healthy women (32.3±4.2 years vs. 28.9±6.0 years, p=0.013 by t test). They did not significantly differ from healthy women concerning BMI at the time of the OGTT (p=0.13 by t test) and at birth (p=0.91 by t test). Weight gain between the time of the OGTT and birth was significantly higher in women with NGT compared with women with GDM (15.6±6.1 kg vs. 10.6±3.8 kg, p=0.04 by t test).

Table 1.

Patients' Characteristics (n=68)

	Controls	GDM	p value^a
Patients (n)	47	21
Age (years)	28.9±6.0	32.3±4.2	0.013
Parity	1.6±1.0	2.0±1.1	0.16
At OGTT
Body mass index (kg/m²)	25.1±5.1	27.3±5.9	0.13
Gestational age (days)	174±20	163±54	0.62
Glucose (mg/dL)
Fasting	82.1±6.7	94.6±16.9	< 0.01
After 1 hour	129.9±24.2	199.5±37.3	< 0.001
After 2 hours	108.3±19.6	171.2±39.2	< 0.001
Body mass index at birth	29.3±3.1	29.5±5.1	0.91
Weight gain between OGTT and birth (kg)	15.6±6.1	10.6±3.8	0.04
Breastmilk (days after birth)
Sample 1	3.0±1.1	4.1±1.4	0.11
Sample 2	45.6±6.6	44.4±4.9	0.62
Time between sample 1 and sample 2 (days)	42.5±6.5	40.3±4.9	0.36

Data are mean±SD values.

By t test.

GDM, gestational diabetes mellitus; OGTT, oral glucose tolerance test.

There were no significant differences in absolute birth weights, birth weight percentiles, Apgar scores after 5 minutes, and pH values of the umbilical artery of newborns of patients with GDM and newborns of healthy women. Birth outcome is shown in Table 2.

Table 2.

Birth Outcome

	Controls	GDM	p value^a
Birth weight (g)	3,155±522	3,213±406	0.66
Birth weight percentile (%)	22.2±21.4	34.4±33.9	0.08
Gestational age at birth (days)	279.6±9.9	274.0±12.4	0.08
pH value	7.20±0.10	7.26±0.09	0.12
Apgar score at 5 minutes	9.8±0.3	9.8±0.4	0.65

Data are mean±SD values.

By t test.

Overall, the total amount of amino acids increased significantly from colostrum to mature milk (p<0.001 by paired t test). When women with GDM and without GDM were analyzed separately, a significant increase was also observed in both groups (p<0.001 by paired t test).

Total amount of amino acids did not significantly differ between women with GDM and healthy controls in colostrum and in mature milk (1,560 μmol/L vs. 1,730 μmol/L and 2,440 μmol/L vs. 2,723 μmol/L, p not significant by t test). Furthermore, the increase of the total amount of amino acids between both measurements did not significantly differ between women with GDM and without GDM (p=0.71 by univariate variance analysis for repeated measurements).

The two most abundant amino acids in colostrum of diabetic mothers and healthy mothers were glutamic acid and taurine. In diabetic women the combined concentration of glutamic acid and taurine was 1,159 μmol/L out of a total of 1,560 μmol/L, and in women with NGT it was 1,009 μmol/L out of 2,440 μmol/L.

Overall, from the 11 analyzed amino acids, glutamic acid, serine, glutamine, threonine, and alanine increased significantly between both measurements, and asparagine, arginine, and taurine decreased significantly between both measurements (by paired t test) (Table 3).

Table 3.

Separate Analyses of 11 Amino Acids in Patients with Gestational Diabetes Mellitus and Patients with Normal Glucose Tolerance

	Level (μmol/L)
	All patients (n=68)			Controls (n=47)			GDM (n=21)
Amino acid	I	II	p	I	II	p	I	II	p
Aspartic acid	44±34	102±22	0.06	45±8	125±27	0.08	43±20	55±34	0.13
Glutamic acid	603±357	1,112±363	<0.001	591±39	1,159±323	<0.001	624±28	1,025±42	0.01
Asparagine	18±14	13±7	0.04	20±2	14±8	0.13	15±6	12±5	0.05
Serine	53±27	101±57	<0.001	53±13	111±64	<0.001	54±17	82±35	0.02
Glutamine	106±10	432±198	<0.001	95±10	452±207	<0.001	127±90	396±180	<0.001
Glycine	75±17	110±44	0.21	48±31	121±44	<0.001	126±29	90±38	0.64
Threonine	67±33	82±32	0.04	68±30	88±34	0.06	65±21	72±28	0.41
Alanine	119±69	243±82	<0.001	118±74	252±69	<0.001	122±62	228±10	<0.01
Arginine	35±31	17±15	0.001	37±5	17±3	0.01	32±11	17±13	0.01
Taurine	459±202	281±123	<0.001	418±187	290±107	0.01	535±215	265±51	0.001
Valine	53±11	65±9	0.61	61±14	50±16	0.69	38±20	95±16	0.23

Data are mean±SD values.

I, measurement of the first milk sample, 4 days after birth; II, measurement of the second milk sample, 6 weeks after birth.

In women with NGT the difference between both measurements from the 11 analyzed amino acids showed a significant increase of glutamic acid, serine, glutamine, glycine, and alanine and a significant decrease of arginine and taurine (by paired t test) (Table 3).

In women with GDM the difference between both measurements from the 11 analyzed amino acids showed a significant increase of glutamic acid, serine, glutamine, and alanine and a significant decrease of asparagine, arginine, and taurine (by paired t test) (Table 3).

When comparing patients with GDM and women with NGT for each amino acid separately, there were no significant differences at both measurements, except for glycine at the second measurement (p=0.03 by paired t test).

After correction of these results of differences between both measurements for multiple testing, in the group of women with NGT as well as in the group of women with GDM no significant result was seen.

Patients with GDM did not significantly differ from healthy women regarding the change of each amino acid between both measurements (tested by univariate variance analysis for repeated measurements).

No significant influence on the total amount of amino acids at both measurements of BMI, maternal age, gestational age at delivery, birth weight, and diagnosis of GDM could be observed by using multivariate stepwise back linear regression analysis.

Discussion

We have analyzed the amount of FAAs in human milk of patients with GDM and women with NGT in colostrum and in mature milk. Our results show that the total amount of amino acids increases from colostrum to mature milk in all patients. The total amount of amino acids, as well as the concentrations of each amino acid separately, did not significantly differ between women with GDM and women with NGT.

The milk of each mammalian species has a distinctive FAA profile, which may reflect the relative importance of FAAs during early postnatal development.¹² An inadequate mixture of amino acids in nutrition may affect protein synthesis, and ultimately body composition and growth could be adversely affected.¹³

Human milk contains a pool of FAAs, twofold to fivefold higher than that in infant formulas.¹⁴ Ferreira¹⁵ observed that the two amino acids with the highest concentration, glutamic acid and taurine, are significantly lower in levels in infant formulas compared with human milk. This is in line with our results for the concentrations of glutamic acid and taurine in human milk, which were found in the highest levels. This was also described by Chuang et al.,¹⁶ who measured FAA concentrations in human milk of preterm infants, term infants, and infant formulas. In contrast to our results, they showed a decrease in total amino acids within the first 3 weeks of lactation, whereas we observed a significant increase in the whole study population, as well as in each group (GDM and controls) separately. This observational difference might be due to different times of measurement. We collected “mature” milk 6 weeks after birth, whereas Chuang et al.¹⁶ collected at 3 weeks after birth. Because we analyzed breastmilk at the end of the feeding, we cannot rule out that the amino acid content of human milk of an entire breast emptying could differ between patients with GDM and patients with NGT.

It has been suggested that the differences in FAAs between human milk and infant formula may contribute to differences in enteral mucosa protection, neurotransmitters, and nitrogen supply in formula-fed infants.¹⁴

Previously, Plagemann et al.⁸ reported that early ingestion of human milk may increase the risk of adiposity and impaired glucose tolerance in offspring of diabetic mothers (mainly patients with type 1 diabetes). It is unclear what component of human milk could cause the increased body weight. It has been shown that breastmilk from diabetic mothers has higher energy content.¹⁷ Glucose concentrations in breastmilk have been demonstrated to follow maternal blood glucose concentrations.

Nutrition, social, and economic factors also influence the risk for adiposity and impaired glucose tolerance.¹⁸ Furthermore, it is well established that intrauterine exposure to increased glucose and insulin levels leads to an increased risk of obesity and impaired glucose tolerance later in life.^4,19,20

In animal studies it has been suggested that body weight and glucose metabolism are “programmed” by early neonatal nutrition.²¹ Furthermore, overnutrition early after birth has been described to enhance a genetic disposition of adiposity during childhood.²²

Although we did not measure glucose levels of the breastmilk of our study population, it seems unlikely that glucose levels in the milk of diabetic women were higher than in the milk of healthy women, given that our GDM patients were well controlled and showed NGT 8–12 weeks after birth.

Because we did not observe any significant difference in the amino acid amount of colostrum and human milk 6 weeks after birth in women with well-controlled GDM and healthy women, it seems unlikely that early breastfeeding influences the risk of adiposity and impaired glucose tolerance in the offspring of mothers with well-controlled GDM.

Conclusions

Our results did not show any significant difference in the content of FAAs of human milk between women with GDM with good metabolic control and women with NGT. Although it has been described that early ingestion of milk from patients with overt diabetes could lead to a higher risk of adiposity and impaired glucose tolerance, it remains unclear if a different milk composition or other factors increase these risks.⁸ The current recommendations, that diabetic women should breastfeed their offspring as often as healthy women, should be maintained.²³

Footnotes

Disclosure Statement

No competing financial interests exist.

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