Birth weight related essential, non-essential and conditionally essential amino acid blood concentrations in 12,000 breastfed full-term infants perinatally

Essential, non-essential and conditionally essential amino acid blood concentrations play a critical role in newborns. We aimed to quantitate most of these amino acids in the blood of full-term breastfed infants, perinatally and correlate the obtained values with their birth weight. Breastfed full-term infants (n = 12,000; 6000 males, 6000 females) with birth weight 2000–4000 g were divided into 4 equal groups: Group A, 2000–2500 g; B, 2500–3000 g; C, 3000–3500 g and D, 3500–4000 g. Blood samples as Dried Blood Spots (DBS) were collected on the 3rd day of life and analyzed via liquid chromatography-tandem mass spectrometry (LC-MS/MS) protocol. Blood concentrations of the amino acids, Phenylalanine, Leucine, Glutamine, Ornithine, Alanine, Tyrosine and Glycine in full-term breastfed newborns, were found to be related to their birth weight, perinatally. On the contrary, no relationship between birth weight and blood concentrations of the amino acids Valine, Methionine, Citrulline and Arginine was found. Due to the number of the samples, data from this study could be applied as neonatal screening reference values for full-term breastfed newborns in relation to their birth weight.

Keywords: Newborn screening; essential amino acids; non-essential amino acids; conditionally essential amino acids; reference values

Introduction

Newborn screening (NBS) is a useful public health tool for the early diagnosis and treatment of most inborn errors of metabolism, including aminoacidopathies. Aminoacidopathies are caused by genetic mutations in specific genes and most of them are monogenic disorders, leading to impaired protein synthesis and enzyme activities which affect physiologically important metabolic pathways [[1]]. Beyond this application, however, amino acid concentrations have the potential to convey information about energy utilization, nutrition, liver function, nitrogen balance, and connective tissue turnover [[2]].

Based on dietary needs for growth or nitrogen balance, amino acids (AAs) have traditionally been classified as nutritionally essential or nonessential [[2]]. Essential AAs (EAAs; Table 1) are those whose carbon skeletons cannot be synthesized or those that are inadequately synthesized de novo by the body, relative to needs and which must be provided from the diet to meet requirements. Except for lysine and threonine, metabolic needs for EAAs can be also covered through the intake of the corresponding keto acids, as they can be transaminated in the body to yield the respective amino acid [[3]]. Non-essential AAs (NEAAs; Table 1) are those AAs that can be synthesized de novo in adequate amounts by the body from carbon skeletons, derived from lipid and carbohydrate sources or from transformations that involve EAAs. NΕΑΑs and their precursors are: glutamic acid (α-ketoglutaric acid), aspartic acid (oxaloacetic acid), serine (3-phosphoglyceric acid), glycine (serine), tyrosine (phenylalanine), proline (glutamic acid), alanine (pyruvic acid), cysteine (methionine and serine), arginine (glutamate-γ-semialdehyde), glutamine (glutamic acid), and asparagine (aspartic acid). From a nutritional, as well as metabolic view, all the 20 amino acids must be present in sufficient quantities to support growth and individuals' health. The absence of any essential amino acid leads to the cessation of protein synthesis, catabolism of unused amino acids, increased loss of nitrogen in urine, and reduced growth [[3]].

Table 1. Essential, non-essential and conditionally essential amino acids.

Essential	Non-essential
Histidine* Isoleucine * Leucine Valine Lysine* Methionine Phenylalanine Threonine* Tryptophan*	Arginine** Ornithine Citrulline Alanine Aspartic acid* Asparagine* Cysteine* Glutamic acid** Glutamine** Glycine** Proline* Serine* Tyrosine**

1 *Not quantified in this study; **conditionally essential amino acids.

Under certain conditions, some ΝΕΑΑs may become essential or conditionally essential (CEAA) (Table 1), meaning that the body is not capable of producing them in sufficient quantities during a specific physiologic or pathologic condition. For example, when liver function is compromised by disease or premature birth, Cysteine (Cys) and Tyrosine (Tyr) become essential, since they cannot be formed from their usual precursors Methionine (Met) and Phenylalanine (Phe), respectively. Dietary Arginine (Arg) is also required for maximum neonatal growth and embryonic survival and thus is essential in infancy, while dietary Glutamine (Gln) is indispensable for intestinal mucosal integrity [[4]]. Therefore, recent research has revealed that some of the traditionally classified as NEAAs, such as Gln, Glutamate (Glu), and Arg are considered CEAAs, as they play important roles in multiple signaling pathways, regulating gene expression, intracellular protein turnover, nutrient metabolism, and oxidative defence [[2], [5]].

Adequate fetal growth is primarily determined by nutrient availability, which in turn depends on nutrient transport from the maternal to the fetal circulation across the placenta. The significant roles of AAs during pregnancy were recently reviewed by Manta-Vogli et al. [[6]].

The amino acid content of human milk (HM) is comprised of total amino acids (TAAs), as well as free amino acids (FAAs). TAAs decline in the first two months of lactation and then remain relatively unchanged, whereas some FAAs such as, Alanine (Ala), Gly, Serine (Ser), Gln and Glu, increase with progressing lactation. Leu and Met are the most and the least abundant essential amino acids across all the lactation stages, whereas Glu plus Gln comprise nearly 50% of total FAAs [[7]]. The concentration of free Gln increases to the greatest extent among all FAAs as lactation progresses, whereas Glu has been reported to be 20 times higher in mature milk than its lowest value in colostrum [[7]]. The secretion of these FAAs in milk is an actively regulated process, where branched-chain amino acids (BCAAs) are catabolized in lactating mammary tissue to provide Glu and Gln.

Breastfeeding during the first week of life is related to colostrum which is produced by the mammary gland, starting immediately after delivery. Systemic changes in metabolic profiles in neonates related to breastfeeding with colostrum depend on composition, amounts, time and duration of feeding [[10]]. One research indicates that maternal diet quality may influence TAA and FAA concentrations in mothers' milk [[11]], yet in other studies, protein content is well preserved in mothers consuming protein-deficient diets [[12]].

In a very recent pilot study on Arginine's family amino acid blood concentrations in 2000 breastfed full-term newborns, statistically, significant differences were demonstrated in Glu + Gln and Ornithine (Orn) concentrations in relation to infants birth weight (BW), whereas Citrulline (Cit) and Arg blood levels remained unaltered [[14]].

The aim of the current study was to demonstrate most EAA (Phe, Met, Leu, Valine (Val)), NEAA (Orn, Cit, Ala) and CEAA (Arg, Glu + Gln, Gly, Tyr) blood concentrations in a great number (12,000) of full-term breastfed infants in relation to their BW, perinatally and reach to conclusions that can be taken into account during the evaluation of the results of newborn screening.

Materials and methods

Subjects

All newborns of Greek origin (n = 12,000), males (n = 6000) and females (n = 6000), were randomly selected. They were born after an uncomplicated pregnancy (37+ weeks) and vaginal delivery, with BW range 2000–4000 g. Males (M) and Females (F) were divided into four equal-sized groups (n = 1500), A with BW = 2000–2500 g, B with BW = 2500–3000 g, C with BW = 3000–3500 g and D with BW = 3500–4000 g. All newborns were on breastfeeding and their mothers were healthy omnivores, aged 24 to 36-years-old. Placental and fetus echograms, as well as laboratory investigations, were normal throughout pregnancies. Τhis analysis was part of the expanded newborn screening program, which has been approved by the Institutional Review Board of IASO maternity hospital for the last twelve years. Informed written consent was obtained from the parents of the aforementioned newborns and in general, the whole study was in accordance with the current revision of the Helsinki Declaration.

Blood samples

Blood samples were collected on Guthrie cards (Whatman 903, HVD, Athens, Greece) from newborns on 3rd day of life at 9.00–10.00am after heel prick forming dried blood spots (DBS) and were analyzed within 48 h. It should be underlined that these 12,000 Guthrie cards selected from an equal number of newborns correspond to more than 10% of the live births per year in Greece [[15]].

Quantitation of amino acids in DBS

DBS samples were analysed by LC-MS/MS, using a protocol previously described [[16]]. Amino acids were determined as butyl esters derivatives. MS/MS analysis was conducted on a Sciex API 4000Qtrap mass spectrometer (Antisel, Athens, Greece) by multiple reaction monitoring (MRM) transitions. A group of isotopically labelled amino acids (Sigma-Aldrich, Athens, Greece) were utilized as internal standards. A gradient elution LC program controlling the flow rate of the mobile phase (acetonitrile: formic 0.1%, 80:20) was employed via an Agilent LC system (Hellamco, Athens, Greece). Analyst 1.6 software was utilized to operate LC-MS/MS, while data processing and quantitation of amino acids were performed by Chemoview software (Antisel, Athens, Greece).

Statistics

Data were analyzed using IBM SPSS v. 25.0 statistical packages. The normality of residuals was tested and ANOVA with or without Tukey's test, when needed, was utilized for the statistical analysis of the results. p-Values <.05 were considered statistically significant.

Results

As presented in Table 1, the essential amino acids, Phe, Leu, Val and Met were quantitated in the studied groups of newborns, whereas histidine, lysine, threonine and tryptophan were not measured. Regarding NEAAs, Orn, Cit and Ala were quantified, while aspartic acid, asparagine, cysteine, proline and serine were not. The CEAAs Glu and Gln, Arg, Tyr and Gly were also investigated in all studied groups.

The first step of the statistical analysis included a normality test of residuals using SPSS. Such analyses were conducted for all amino acids and the results were interpreted by Kolmogorov–Smirnov tests and the relevant normal Q–Q plots. In all cases p-values were >.05, while in the respective normal Q–Q plots all dots are very close to the line, depicting that residuals for all amino acids are normally distributed. Therefore, ANOVA could be utilized, providing the results that follow.

As shown in Table 2 mean values of Phe between males and females were found non statistically significantly different in groups A–D. Additionally, the lowest mean value of Phe in males was observed in group A and the highest in group D. Statistically significant differences in males were detected in groups A vs D, B vs D and C vs D. In contrast, non-statistically significant differences were observed in the mean values of groups A vs B, A vs C and B vs C. In females, the lowest mean values were measured in groups A and C and the highest in group D. Statistically significantly different values were demonstrated between groups A vs D, as well as C vs D groups.

Table 2. Essential amino acid blood concentrations (mean ± SD, μmol/L) in full term breastfed newborns in relation to their birth weight and sex, perinatally.

Groups	Phenylalanine	Leucine	Valine	Methionine
Mean ± SD	Range	Mean ± SD	Range	Mean ± SD	Range	Mean ± SD	Range
Group A
(M)	51.78 ± 18.88a	31.90–70.20	114.55 ± 44.42a	69.75–160.30	115.87 ± 44.01a	69.86–161.29	28.02 ± 11.94a	15.83–40.38
(F)	51.24 ± 16.83a	34.30–67.50	113.66 ± 45.59a	67.50–160.20	116.53 ± 45.52a	70.04–162.68	28.20 ± 16.92a	10.50-46.12
Group B
(M)	52.92 ± 18.11b	33.80–70.48	106.84 ± 36.83b	69.84–143.14	115.56 ± 44.68b	70.42–161.29	27.59 ± 12.48b	14.62–40.32
(F)	52.92 ± 18.23b	34.00–70.65	105.25 ± 39.06b	65.64–148.50	115.55 ± 43.20b	71.05–158.15	29.36 ± 13.47b	14.50–43.97
Group C
(M)	53.01 ± 17.10c	35.80–69.49	104.60 ± 38.09c	65.50–142.58	115.21 ± 44.38c	70.42–161.42	30.35 ± 16.99c	13.16–48.35
(F)	51.21 ± 18.85c	32.00–69.73	106.83 ± 38.61c	67.85–146.32	114.18 ± 42.11c	72.07–160.20	30.93 ± 12.07c	18.56–43.99
Group D
(M)	57.64 ± 13.77d	43.00–71.20	104.93 ± 38.56d	65.50–145.59	116.57 ± 39.45d	76.59 –155.88	30.74 ± 11.85d	17.52–43.45
(F)	54.81 ± 17.76d	36.93–72.03	109.41 ± 35.10d	73.80–144.02	118.33 ± 44.12d	74.28–162.68	30.34 ± 14.86d	15.20–46.99
Statistics	(M): a/b ΝS, a/c NS, a/d p <.001, b/c NS, b/d p <.01, c/d p<.01	(M): a/b p <.001 , a/c p <.0001, a/d p <.0001, b/c NS, b/d NS, c/d NS	(M): a/b NS, a/c NS, a/d NS, b/c NS, b/d NS, c/d NS	(M): a/b NS, a/c NS, a/d NS, b/c NS, b/d p <.05, c/d NS
(F): a/b NS, a/c NS, a/d p <.05, b/c NS, b/d NS, c/d p <.05	(F): a/b p <.0001, a/c p <.001, a/d p <.01, b/c NS , b/d p <.01 c/d NS	(F): a/b NS, a/c NS, a/d NS, b/c NS, b/d NS, c/d p <.01	(F): a/b NS, a/c NS, a/d NS, b/c NS, b/d NS , c/d NS
(M) vs (F): a/a NS, b/b NS, c/c NS, d/d NS	(M) vs (F): a/a NS, b/b NS, c/c NS, d/d p <.01	(M) vs (F): a/a NS, b/b NS, c/c NS, d/d NS	(M) vs (F): a/a NS, b/b NS, c/c NS, d/d NS

• 2 Abbreviations. M: males; F: females; NS: non statistically significant.
• 3 a/b means that there is comparison between group A and B etc.

The mean values of Leu between males and females were found statistically significantly different in group D only. Additionally, the lowest mean Leu values in males were observed in groups C and D, whereas the highest in group A. Statistically significant differences in Leu concentrations were demonstrated in groups A vs B, A vs C and A vs D. In females, the lowest mean value was measured in group B and the highest in group A. Statistically significantly different values of the amino acid were found in groups A vs B to D.

Mean values of Val between males and females were found non statistically significantly different in all the studied groups. The lowest mean Val value in males was observed in group C and the highest in group D. Statistically significant differences in Val levels were not found between all the studied groups in males. In females, the lowest, as well as the highest Val mean values were demonstrated in the same groups as above. On the contrary, statistically significant different values of the amino acid were found in groups C vs D.

Mean values of Met between males and females were found non statistically significantly different in all the studied groups. Additionally, the lowest mean value of Met in males was measured in group B and the highest in group D. Statistically significantly different Met values were detected in groups B vs D only. Oppositely, in females non-statistically significant differences were observed between all the studied groups.

As illustrated in Table 3 mean values of Orn were found statistically significantly different between males vs females of the same BW in groups B and C. With regards to the mean values measured in both males and females with different BW, the lower mean value was determined in group A, whereas the highest in group D. Statistically significant differences of the amino acid were determined between the groups of both sexes, with the exception of females in group C vs D.

Table 3. Non-essential amino acid blood concentrations (mean ± SD, μmol/L) in full term breastfed newborns in relation to their birth weight and sex, perinatally. Abbreviations: M: males, F: females, NS: non statistically significant.

Groups	Ornithine	Citrulline	Alanine
Mean ± SD	Range	Mean ± SD	Range	Mean ± SD	Range
Group A
(M)	66.88 ± 42.89a	24.10–111.35	14.37 ± 6.44a	7.95–21.32	253.35 ± 81.73a	170.80–332.74
(F)	69.75 ± 42.65a	26.90–113.82	14.03 ± 5.28a	8.50–20.43	256.01 ± 74.52a	180.59–329.61
Group B
(M)	73.98 ± 44.81b	28.83–120.24	13.15 ± 6.11b	6.75–19.72	251.90 ± 97.91b	153.85–349.46
(F)	80.41 ± 42.73b	36.89–124.06	14.12 ± 5.22b	8.56–20.02	271.32 ± 86.75b	184.26–357.87
Group C
(M)	80.66 ± 44.21c	36.57–125.94	14.52 ± 5.92c	8.70–20.99	266.55 ± 83.27c	183.34–349.76
(F)	84.11 ± 44.55c	39.29–129.60	14.16 ± 4.81c	9.50–20.67	274.62 ± 85.10c	190.10–359.10
Group D
(M)	85.82 ± 44.51d	40.95–131.20	15.59 ± 6.45d	9.12–22.68	273.53 ± 99.38d	174.20–370.93
(F)	85.76 ± 43.13d	42.10–129.60	15.18 ± 5.30d	9.57–20.65	277.97 ± 86.19d	191.83–364.50
Statistics	(M): a/b p <.001, a/c p <.00001, a/d p <.000001, b/c p <.001, b/d p <.00001, c/d p <.001	(M): a/b NS, a/c NS, a/d NS b/c NS, b/d NS, c/d NS	(M): a/b NS, a/c p <.00001, a/d p <.0000001, b/c p <.00001, b/d p <.0000001, c/d p <.001
(F): a/b p <.00001, a/c p <.000001, a/d p <.000001, b/c p <.01, b/d p <.001, c/d NS	(F): a/b NS, a/c NS, a/d NS, b/c NS, b/d NS, c/d NS	(F): a/b p <.00001, a/c p <.0000001, a/d p <.0000001, b/c p <.05 , b/d p <.001, c/d p <.05
(M) vs (F): a/a NS, b/b p <.001, c/c p <.01, d/d NS	(M) vs (F): a/a NS, b/b NS, c/c NS, d/d NS	(M) vs (F): a/a p <.05, b/b p <.0000001, c/c p <.001, d/d p <.01

Non-statistically significant p-values of Cit were determined in males and females with different BW, as well as in males vs females of the same BW.

The mean values of Ala were found statistically significantly different between males vs females in all the studied groups. The lowest mean value of the amino acid in males was found in group B, whereas the highest in group D. Statistically significantly different Ala values were obtained between the groups A vs C and D, B vs C and D and C vs D in males. The lowest mean value of Ala in females was detected in group A and the highest in group D. Statistically significantly different values of the amino acid were determined between in almost all the female groups.

As presented in Table 4, the mean values of Glu plus Gln were found statistically significantly different between males and females in all the studied groups. With regards to the mean values measured in both males and females, the lowest mean value was determined in group A, whereas the highest in group D. Both in males and females statistically significant differences of Glu plus Gln were measured between groups of different BW.

Table 4. Conditionally essential amino acid blood concentrations (mean ± SD, μmol/L) in full term breastfed newborns in relation to their birth weight and sex, perinatally. Abbreviations: M: males, F: females, NS: non statistically significant.

Groups	Glutamine + glutamic acid	Arginine	Tyrosine	Glycine
Mean ± SD	Range	Mean ± SD	Range	Mean ± SD	Range	Mean ± SD	Range
Group A
(M)	191.15 ± 64.80a	125.90–254.80	7.74 ± 5.51a	2.26–13.89	65.53 ± 31.01a	35.10–96.86	325.11 ± 108.92a	217.12–435.64
(F)	196.68 ± 85.75a	110.62–282.60	7.58 ± 5.37a	2.23–13.19	69.49 ± 26.26a	44.10–95.37	328.13 ± 111.73a	217.15–440.65
Group B
(M)	203.72 ± 90.72b	113.50–294.82	6.80 ± 4.54b	2.28–11.52	73.21 ± 30.39b	43.12–103.50	368.53 ± 109.61b	259.61–480.27
(F)	207.54 ± 89.95b	117.30–297.56	7.74 ± 4.95b	2.76–12.88	72.76 ± 32.92b	40.10–105.87	431.79 ± 108.27b	322.82–542.52
Group C
(M)	207.09 ± 92.62c	114.52–300.15	7.47 ± 5.01c	2.47–12.60	72.76 ± 29.97c	43.15–102.60	422.61 ± 90.86c	332.54–514.54
(F)	212.90 ± 91.21c	122.12–304.11	7.69 ± 5.37c	2.34–13.30	75.33 ± 32.05c	42.80–107.37	437.95 ± 100.82c	337.50–539.82
Group D
(M)	212.62 ± 81.81d	130.15–293.40	7.22 ± 5.15d	2.02–12.60	71.82 ± 29.97d	42.12–101.70	430.71 ± 100.95d	330.15–532.96
(F)	227.37 ± 106.08d	122.20–334.80	7.74 ± 5.43d	2.40–13.41	73.50 ± 30.06d	43.50–103.39	444.11 ± 108.12d	336.22–556.10
Statistics7	(M): a/b p <.0001, a/c p <.0001, a/d p <.00001, b/c p <.05, b/d p <.0001, c/d p <.001	(M): a/b NS, a/c NS, a/d NS, b/c NS, b/d NS, c/d NS	(M): a/b p <.0001, a/c p <.001, a/d p <.0001, b/c NS, b/d NS, c/d NS	(M): a/b p <.0000001, a/c p <.0000001, a/d p <.0000001, b/c p <.0000001, b/d p <.0000001, c/d p<.0001
(F): a/b p <.0001, a/c p <.00001, a/d p <.00001, b/c p <.001, b/d p<.00001, c/d p<.00001	(F): a/b NS, a/c NS, a/d NS, b/c NS, b/d NS, c/d NS,	(F): a/b p <.05, a/c p <.001, a/d p <.01, b/c NS, b/d NS, c/d NS	(F): a/b p <.0000001, a/c p <.0000001, a/d p <.0000001, b/c p <.001, b/d p <.00001, c/d p <.001
(M) vs (F): a/a p <.001, b/b p <.01, c/c p <.001, d/d p <.00001	(M) vs (F): a/a NS, b/b NS , c/c NS, d/d NS	(M) vs (F): a/a p <.05, b/b NS, c/c NS, d/d NS	(M) vs (F): a/a p <.05, b/b p <.0000001, c/c p <.000001, d/d p <.00001

Non-statistically significantly p-values of Arg were determined in males and females with different BW, as well as in males vs females of the same BW.

Mean values of the CEAA Tyr, were found statistically significantly different between males and females in group A only. In males, the lowest mean value was measured in group A and the highest in group B. Non statistically significant differences were found between groups B vs C and D and C vs D. Statistically significant differences were detected among groups A vs groups B, C and D. In females the lowest Tyr values were determined in group A whereas the highest in group C. Statistically significantly different values were found between groups A vs B to D.

Μean values of the CEAA Gly, between males and females, were found just statistically significantly different in group A only. On the contrary, statistically significant differences were found between the rest of the studied groups (groups B, C and D). In males, the lowest mean value was measured in group A and the highest in group D. In both males and females statistically significant differences were detected between the studied groups.

Discussion

According to WHO, infants with BW <2500 g, irrespective of gestation, are defined as LBW [[17]]. So, group A infants, both males and females could be characterized as LBW. Their Phe mean values were lower, but not significantly lower than those demonstrated in groups B and C. This could be due to the kind of feeding, for example, HM composition and the immaturity of enterocytes which take part in the absorption and further Phe metabolism via enterohepatic utilization. Additionally, the observed statistically significantly different values of Phe between groups A vs D and C vs D, could be due to the maturity of the gastrointestinal tract cells and the activity of the hepatic phenylalanine hydroxylase (PAH) system in infants with BW 3000–4000 g, who might consume a larger amount of HM. Other studies regarding LBW infants, referred to a wide range of BW: 1000–2500 g [[7]]. In these studies, the kind and the amount of feeding were not reported, not even the characteristics of protein intake/kg/day and in none of them, the reported values of Phe were related to breastfeeding only. Consequently, the little lower but not significantly different mean values between the studied groups A–C, could be due to the quantity and quality of HM composition and to normal hepatic Phe metabolism. These findings are further supported by the significant differences detected between Phe values measured in groups A vs D. Infants of group D, males and females, were not only benefited intrauterine with their mothers' amino acid concentrations but also from breastfeeding perinatally, as this group of infants is accustomed to consuming larger quantities of HM. Obviously, their Phe levels were normal, compared to current reference values, but higher than those measured in A to C groups. BW is closely related to the metabolic status of infants perinatally. The maturity of gastrointestinal enzymes in combination with PAH status could be related to better absorption and enterohepatic Phe metabolism.

Recent studies have disclosed that free BCAAs in the tissue amino acid pool function not only as substrates for protein synthesis but also as regulators of protein and energy metabolism [[18]]. Furthermore, BCAAs are actively used as an amino group donor to synthesize Glu in the brain and may also exert an important role in intestinal integrity and maturity [[19]].

BCAAs are metabolized via reversible transamination to produce their respective branched-chain α-keto acids (BCKAs), BCKAs in turn undergo irreversible decarboxylation to produce their respective branched-chain acyl-CoA derivatives by branched-chain keto dehydrogenase complex (BCKDC). The regulation of the activity state of BCKDC may be critically important for growth, tissue repair, and maintenance of body protein. In addition, branched-chain aminotransferase (BCAT) catalyzing the transamination of BCAAs with α-ketoglutarate contributes to the formation of a number of NEAAs such as Gln, Glu, Ala and thus participates in the interorgan shuttle of nitrogen [[20]]. As BCAAs are essential AAs, any dietary limitation can affect protein synthesis and other metabolic processes. Oppositely, excess BCAAs, and their metabolites are associated with neurological dysfunction and acidemias. The most studied inborn error of BCAA metabolism is Maple Syrup Urine Disease (MSUD) [[20]].

Among BCAAs, Leu is a signaling molecule that stimulates protein synthesis in the periphery by activation of the mammalian target of rapamycin (mTORC1) pathway, the master regulator of cell growth and proliferation in selected tissues, particularly skeletal muscle [[20]] that represents 30% of neonatal body mass and is the most rapidly growing body compartment [[21]].

In this study, we may underline the high Leu levels measured in group A of both sexes, which is probably explained by the immaturity of the enzyme(s) for further Leu metabolism. Additionally, the observed statistically significantly higher Leu concentrations in females in group D as compared to those of males in the same group, maybe due to the increased volume of HM intake which is rich in Leu [[7]] and its metabolites [[22]], as well as to sex-specific differences in HM composition [[23]]. Furthermore, other reasons should be considered such as placental function and/or mothers' dietary habits during pregnancy that has an influence on BW [[24]]. Additionally, the statistically significantly higher concentrations of Val in group D in females only could be due to the quantity and quality of human milk consumption [[23]].

Met is an indispensable sulfur-containing amino acid required for synthesizing protein as well as a number of other critical nutrients via the methionine cycle. The methionine cycle transfers the terminal methyl group from Met to various methylated products to form homocysteine, which can irreversibly transfer its sulfur atom for cysteine synthesis [[26]].

In this report non statistically significantly different mean values of Met were measured in almost all the studied groups. These findings are in agreement with previous studies by Thomas et al. and by Kalhan and Bier [[27]]. Consequently, Met responsible enzyme activities for its further metabolism are present at birth [[27]], except in the case of inborn errors of Met metabolism. In addition, Met concentrations in HM colostrum are considered high and constant during early life [[7], [9]] (Table 2).

The biosynthesis of NEAAs (Table 3), is heavily dependent on Gln. Apart from its primary use for energy production in the gut, enteral Glu + Gln are also metabolized by enterocytes via conversion to other AAs such as Proline (Pro), Orn, Cit and Arg [[14]]. Glu + Gln as well as Arg, would be discussed in detail below, with the other CEAAs (Table 4).

In this study, the high Glu + Gln concentrations measured in groups C and especially in group D were further metabolized via L-Glutamyl-γ-phosphate into Glutamate-5-semialdehyde (GSA) which subsequently was transformed into Orn via pyrroline-5-carboxylate (P-5-C). Ornithine aminotransferase (OAT) catalyzes the conversion of Orn into GSA and vice versa using α-ketoglutarate (aKG) and Glu as co-substrates. The intermediary product P-5-C in this metabolic pathway is the main precursor of Pro [[29]]. Considering the equilibrium constant of the reaction taking this direction requires either a large amount of GSA and Glu, or fast consumption of Orn (or aKG). This is in line with its conversion into Cit [[14]]. Additionally, Pro is found in great amounts in HM and is also converted into P-5-C through proline oxidase and further metabolized into Orn via OAT. Consequently, Orn blood concentrations that are measured high in groups C and D, come from two sources, GSA and P-5-C [[14]]. It can be assumed that Orn blood levels come from two different pathways and the high concentrations measured should be catabolized into Cit via carbamoyl phosphate synthetase I (CPS I) and ornithine carbamoyltransferase (OCT). So, it can be considered that the activities of these enzymes are not able enough to metabolize all the amount of Orn offered to produce Cit. This suggestion is further supported by previous experimental studies in humans and animals in which Orn supplementation resulted in constant and not a further elevation of Cit blood concentrations [[30]]. This report could explain the constant Cit blood levels measured in all the studied groups, independently of Orn concentrations. The main source of circulating Cit is coming from the intestine because Cit produced in the liver is catabolized in situ [[31]]. For this reason, plasma Cit has been proposed as a marker for gut mass and function, not only in neonates [[31]] but also in adult subjects [[32]]. Thus in humans, Cit production has been shown to be rather constant and independent from feeding or Arg content of the diet [[33]]. The present results in 12,000 newborn measurements are in agreement with these reported in a pilot study [[14]] (Table 3).

According to the literature, high concentrations of Orn in the blood are highly implicated with gyrate atrophy which is a serious neuro-degenerative inherited disease [[29]]. Additionally, elevated Cit levels appear in citrullinemia type I, argininosuccinic aciduria, and citrine deficiency, whereas, low serum citrulline concentration is associated with ornithine transcarbamylase (OTC) deficiency [[34]] and CPS I deficiency concomitant with hyperammonemia.

Ala is a type of glucogenic AA that can facilitate glucose metabolism, help alleviate hypoglycemia, and improve the body's energy. Alanine aminotransferase (ALT), is an enzyme catalyzing the reversible transamination between pyruvate and Glu to form Ala and 2-oxoglutarate, contributing to gluconeogenesis. [[35]]. In the skeletal muscle, ALT transfers the alpha-amino group from Glu to pyruvate to form Ala. Ala, released from the skeletal muscle, is used as a substrate for gluconeogenesis in the liver. ALT catalyzes the reaction to form pyruvate from Ala in the liver, which is known as the 'glucose-alanine cycle' [[35]]. Ala deficiency tends to cause hypoglycemia, and high Ala leads to the risk of gout or diabetes [[36]].

In this study, Ala blood concentrations were statistically significantly higher in females vs males. The same results were also reported by Yamamoto et al. [[37]]. In males, Ala blood concentrations were demonstrated statistically significantly lower in groups A and B than those measured in groups C and D. These findings could be due to the immaturity of the gastrointestinal tract in infants of group A and/or to the amount of HM intake, as well as to the low activity of ALT1 in the colon in relation to the low activity of ALT2 in the liver and skeletal muscles [[35]]. Oppositely, neonates of groups C and D may have better absorption of HM due to the maturity of the intestinal enzymes, ALT1 and/or to their increased skeletal muscle mass in relation to the maturity of ALT2 enzyme [[35]]. Additionally, in females, statistically significant differences were detected between A vs B–D groups, as well as between B vs C and D and C vs D. It may be assumed that in newborns with BW 2000–2500 g the maturity of intestinal enzymes and the quantity of HM intake may play a significant role in the measured lower Ala blood concentrations, as compared to those of newborns with BW > 2500 g in whom the higher muscle mass may play an additional role in Ala blood levels (Table 3).

CEAAs (Table 1) can be synthesized in adequate amounts by the organism, but in cases of preterm birth, LBW or perinatal nutritional problems that impose parenteral nutrition their quantities that can be synthesized could be quite limited [[3]]. Perinatally, Gln is one of the most important amino acids for energy production and gut maturity [[38]]. Additionally, Glu + Gln are the main precursors for intestinal Arg synthesis in neonates [[30]]. In this metabolic pathway, the enzymes phosphate-dependent glutaminase, OAT, arginosuccinate synthetase (ASS), arginosuccinate lyase (ASL) and aspartate aminotransferase, are widely distributed in animal tissues, whereas CPS I, OCT and N-acetyl glutamate synthase are restricted in the liver and intestinal mucosa. Proline oxidase is present mainly in the small intestine, liver and kidneys but P-5-C is located almost exclusively in the intestinal mucosa, with only trace amounts in other tissues [[30]].

As shown in Table 4, statistically significant differences of the AAs Glu + Gln were measured in the blood of the studied infants, both males, and females, with BW 2000–4000 g. The highest values of Glu + Gln determined in females with BW 3500–4000 g and the lowest in group A of both sexes were also reported by Ivorra et al. [[39]]. These important findings could be due to placental function and/or mothers' dietary habits during pregnancy that affects BW [[24]].

During pregnancy, glucose and AAs, especially Arg and Gln, are crucial for proper energy metabolism and growth and are key regulators of mTOR, which is linked to cell growth and proliferation, resulting in increased fetal growth [[40]]. Additionally, it has been reported that vaginal delivery is associated with high levels of BCAAs, both in mothers and neonates as compared to those measured during caesarean section [[41]]. These findings may be related to uterine and skeletal muscle contractions during the vaginal delivery process. Consequently, the high levels of BCAAs in infants mirror those of their mothers' and the high BCAA levels in mothers reflect those found in their milk, for further Gln plus Glu production [[42]]. Consequently, maternal plasma AA profile is a major factor in determining the delivery not only to the fetus but also to the newborn via breastfeeding [[43]]. At this point, the significant role of maternal nutrition support during pregnancy [[6]] and lactation should be mentioned.

In this study, the observed differences of Glu + Gln concentrations among groups of different BW, breastfed full-term newborns, both males, and females, could be due to the quantity and quality of maternal milk composition [[10]]. As HM is rich in these AAs, it can be assumed that their concentrations reflect those of mothers'. The observed high concentrations of these AAs in groups D in both sexes could be due not only to the content of Glu plus Gln in HM but also to the muscle mass and mature anatomically longer intestine which takes part in the production and utilization of these AAs [[38]]. Furthermore, the significant role of human colostrum, which is rich in these AAs, should be emphasized, contributing to enteral utilization of Glu plus Gln [[7]]. Recent studies confirm that human colostrum supports and promotes the regular development of both the systemic nervous system (SNC) and ENS (enteric nervous system), which take part in Glu + Gln production and their neurotransmission action [[44]]. Therefore, it can be suggested that newborns with BW 3500–4000 g in whom high levels of Glu + Gln were measured could have greater opportunities for neurotransmission in their early life [[45]].

Τhe constant Cit blood concentrations measured in all the studied groups were described above in detail. Conversion of Cit into Arg via ASS and ASL in enterocytes is the only pathway for Cit utilization and net synthesis of Arg in neonates with no functional intestinal-renal axis [[30]]. It has been estimated based on the kinetics of whole body flux that one-half of the neonate's Arg needs are met by gut first-pass de novo synthesis and this does not change when Arg is deficient or in excess [[46]]. This is the reason because of which Arg levels in group D are not different compared to those of the other studied groups, since it is mainly produced in the intestine from Cit (Table 4).

Neonates have a particularly high requirement for Arg because of the abundance of Arg in tissue proteins and its utilization by multiple pathways [[47]]. Besides serving as a building block for tissue proteins, Arg is a physiological precursor for the synthesis of important molecules such as nitric oxide (NO), creatine, and polyamines [[30]]. Moreover, Arg stimulates the secretion of growth hormone and insulin in mammals including preterm infants, thereby playing an important role in regulating protein, lipid, and carbohydrate metabolism. Arg deficiency results in hyperammonemia as well as cardiovascular, pulmonary, intestinal, immunological, and neurological dysfunction, particularly in preterm infants [[47]]. Therefore, this AA is considered to be a nutritionally essential amino acid for neonates, particularly under stress conditions [[31]].

The primary route of the EAA Phe metabolism is its conversion to Tyr by the phenylalanine hydroxylase system, as mentioned. During the neonatal period, the enzymatic activity of PAH might be suboptimal in newborns and even older infants, which makes Tyr a CEAA [[48]]. Full-term infants with BW 2500–4000 g were characterized with almost similar Tyr concentrations. The same results were found either in males or in females with the same BW (Table 4). The observed statistically significantly lower values of Tyr in group A vs groups B to D in both males and females, could be due to the immaturity of the enteric cells, resulting in poor absorption of Phe and Tyr, as well as to the immature hydroxylation system of Phe. It may also be suggested that LBW infants consume a smaller amount of breast milk, and their protein intake was lower than that of the infants with birth weight >2500 g. Additionally, Tyr levels in females in group A were higher than those measured in males of the same group. These findings are in accordance with a recent study by Yang et al. [[49]].

An elevated level of Tyr detected in newborn screening by tandem mass spectrometry is usually due to transient neonatal hypertyrosinemia. To differentiate, Tyrosinemia type I can be diagnosed by the presence of succinylacetone in serum or urine [[50]].

Gly is synthesized from choline, serine, hydroxyproline, and threonine through interorgan metabolism [[51]]. However, in common feeding conditions, Gly is not sufficiently synthesized in humans and animals [[51]]. This AA is a precursor for a variety of important metabolites such as glutathione, porphyrins, purines, haem, creatine and plays a significant role in gluconeogenesis, as well as in absorption and digestion of lipid-soluble vitamins and lipids. In the central nervous system, Gly acts as a neurotransmitter controlling the intake of food, behavior, and complete body homeostasis. Gly also regulates the immune function, production of superoxide, and synthesis of cytokines exerting its antioxidant, anti-inflammatory and immunomodulatory role in peripheral and nervous tissues [[53]].

Human colostrum is characterized by poor concentrations of Gly but it is rich in its precursor's serine, choline and threonine [[7], [54]]. Consequently, the lower Gly concentrations measured in group A both in males and females could be due to the small amount of HM intake and/or the immaturity of the responsible enzymes for Gly production from its precursors and degradation. On the contrary, the higher Gly concentrations demonstrated in the other studied groups of newborns could be due to the higher amount of HM intake and the maturity of the responsible enzymes for further Gly production from threonine, choline and serine. Overall, it may be underlined that maternal glycine nutrition is of great importance not only during pregnancy but also during lactation. From another point of view toxic values in blood and cerebrospinal fluid (CSF) due to a defect in the Gly cleavage enzyme complex, leads to Gly encephalopathy [[55]].

In the present study, detailed information regarding maternal diet during pregnancy and lactation, other than that participating mothers consumed a free-living, omnivorous diet is lacking. A weak point of this study could be considered the lack of total amount of human milk consumed by the newborns.

The current results may refer to newborns of Greek origin, however, it is not irrational to assume that these findings could apply to other populations as well, at least in the Mediterranean region. It needs to be confirmed, though.

Regarding reference cut off values used for newborn screening [[16]], the current data suggest that specific changes could be considered based on neonate's birth weight. These changes are mainly focused on neonates belonging to Group A (LBW) and for some cases to Group B. Special attention should be paid to Phe, Orn, Ala, Glu + Gln, Tyr, and Gly where new (lower) cut off values could be set for LBW, while this could be an option for Orn, Ala, Glu + Gln and Gly for neonates belonging to group B. On the contrary, since existing cut-off values [[16]] have been estimated for all newborns, regardless BW, some changes (increase) could be considered for full-term neonates belonging to group D (or even C) for the aforementioned amino acids.

Conclusions

The amino acid blood concentrations of Phe, Leu, Gln, Orn, Ala, Tyr and Gly in full-term breastfed newborns, were found to be related to their birth weight perinatally and the observed significant differences were attempted to be interpreted. The studied concentrations of the amino acids in DBS from 12,000 full-term newborns can be applied as neonatal screening reference values not only for preterm and LBW infants but also for full-term newborns with BW > 3000 g.

Disclosure statement

no potential conflict of interest was reported by the author(s).

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By Penelope D. Manta-Vogli; Kleopatra H. Schulpis; Yannis L. Loukas and Yannis Dotsikas

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