Clin Chem Lab Med 2015; aop
Jörg Jahnel, Evelyn Zöhrer, Hubert Scharnagl, Wolfgang Erwa, Günter Faulera and Tatjana Stojakovica,*
Reference ranges of serum bile acids in children and adolescents DOI 10.1515/cclm-2014-1273 Received December 23, 2014; accepted February 10, 2015
mechanisms underlying these variations remain to be determined.
Abstract
Keywords: children; mass spectrometry; neonates; reference ranges; serum bile acids.
Background: Bile acids (BA) are found predominantly in bile but also in serum, where they can be used as markers for inborn and acquired hepatobiliary disorders. We measured serum BA levels by mass spectrometry to determine reference ranges for healthy children and adolescents in different age groups. Methods: In 194 healthy children and adolescents (0–19 years) concentrations of serum BA and BA composition were determined using high-performance liquid chromatography high-resolution mass spectrometry. Individuals were classified by ages into five groups: 0–5 months, 6–24 months, 3–5 years, 6–11 years, and > 11 years. Results: The 95% confidence interval of serum total BA values in newborns was 3.85–6.32 μmol/L. In the cohort aged 6–24 months total BA values were significantly higher (6.61–9.43 μmol/L; p 11 years (n = 91)
BMI
GGT
ALT
AST
13±4
97±49
41±59
65±47
16±2
13±4
19±9
42±8
16±2
27±68
30±47
45±33
18±4
22±21
51±85
45±36
23±6
25±23
22±17
24±8
Laboratory analysis In a volume of 10 μL of serum BA levels were determined as unconjugated acids and as taurine- (T) and glycine-conjugated (G) acids using HPLC-HRMS (Table 2). HPLC was performed on a reversedphase (C18) column, using a methanol/water gradient for chromatographic solution of isobaric BA and 2,2,4,4-d4-internal standards for quantification (all Sigma-Aldrich, Taufkirchen, Germany; Figure 2). Mass-spectrometer Q Exactive™ MS/MS (Thermo Fisher Scientific, Waltham, MA, USA) follows HPLC by the use of high-resolution,
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Jahnel et al.: Bile acids in pediatrics 3
Table 2: Serum concentrations of individual BA (μmol/L) are given as means. Ranges represent 95% confidence intervals.
0–5 months (n = 23) Mean Range 6–24 months (n = 15) Mean Range 3–5 years (n = 23) Mean Range 6–11 years (n = 42) Mean Range > 11 years (n = 91) Mean Range
Total BA
G-conjugates
T-conjugates
5.09 3.85–6.32
2.96 0.75–6.88
1.97 0.00–5.43
8.02 6.61–9.43
5.57 2.53–15.34
2.08 0.09–9.99
5.35 4.27–6.43
3.59 0.33–7.73
1.01 0.00–3.80
4.37 3.61–5.14
3.33 0.39–7.01
0.50 0.00–1.92
3.61 3.09–4.12
2.50 0.44–5.91
0.35 0.00–1.52
accurate-mass Orbitrap™ detection providing qualitative and quantitative data (Amplatz et al., submitted).
Statistical analysis Serum BA concentrations are represented as means±standard deviation (SD) within a 95% confidence interval. All statistical tests were two-tailed and p-values of 11 years) had BA profiles similar to that in adults Figure 3D and E). Glycoconjugates predominated over tauroconjugates (G:T = 6.5:1) and unconjugated BA attained a maximum in the fifth age group. Interestingly, no significant gender-related differences were observed in children’s BA profiles (data not shown).
Primary BA (CA, CDCA) In neonates conjugated primary BA predominated in serum, with taurine and glycine conjugates in balanced proportions; GCA was most abundant (Table 3). GCDCA predominated in the age group 6–24 months (up to 50% of tBA) and still remained high in older age groups. In addition, the percentage of unconjugated BA slightly increased with age. Interestingly, TCDCA was the most abundant taurine-conjugated primary BA within the first 5 months.
Secondary BA (DCA, LCA, UDCA) Serum levels of most secondary BA in newborns did not substantially vary from those in other age groups. However, DCA, GDCA, and GUDCA values continually rose with increasing age.
Discussion
Mean levels of tBA in the first 5 months of life were 5.09±2.20 μmol/L (Table 2). In the age group from 6 to 24 months tBA values were higher and reached a peak of 8.02±5.58 μmol/L. In age groups of 3–5 years, 6–11 years, and > 11 years mean tBA levels gradually decreased to 5.35±2.68, 4.37±2.67 μmol/L, and 3.61±1.89 μmol/L, respectively, reaching concentrations like those in healthy adults (0.28–6.50 μmol/L) [8].
BA composition During the first 5 months conjugated BA greatly predominated over unconjugated unconjugated BA and similar
In this study we established a comprehensive serum BA profile measured by tandem mass spectrometry. To our knowledge this is the first study using HPLC-HRMS to determine reference ranges of tBA for different age groups in children and adolescents. So far, tBA have been assessed in adults and children with different methods [9]. High tBA concentrations in neonates (19.6±5.2 μmol/L) were reported by Polkowska et al., with peak values of 22.2±5.1 μmol/L at the age of 1 month, determined using an enzymatic-colorimetric test [10]. However, after 1 year tBA levels declined to nearly adult concentrations (5.1±2.9 μmol/L) [10]. Using highperformance liquid chromatography Niijima et al. also
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4 Jahnel et al.: Bile acids in pediatrics 11.23
d4-DCA
m/z=395.3105
11.24
CDCA
8.21
UDCA
DCA
m/z=391.2854
10.82
9.02
m/z=407.2803
CA 12.70
m/z=379.3156
d4-LCA 12.70
m/z=375.2905
LCA
8.38
m/z=452.3320
d4-GCDCA 8.39 5.58
GUDCA
Relative abundances
8.74
m/z=448.3068
GDCA
GCDCA
6.81
m/z=468.3269
d4-GCA
6.81
m/z=464.3018
GCA 9.99
m/z=432.3119
GLCA
8.45
m/z=502.3140
d4-TDCA 8.13 5.34
TCDCA
TUDCA
8.45
m/z=498.2895
TDCA
6.55
m/z=514.2844
TCA 9.71
m/z=482.2946
TLCA
5
6
7
8
9
10
11
12
13
14
15
Time, min Figure 2: Representative HPLC-HRMS chromatograms of unconjugated as well as taurine- (T) and glycine-conjugated (G) bile acids and corresponding m/z values.
found high levels of tBA in neonates (11.0±8.7 μmol/L; 0–4 weeks) which continuously decreased with growth [11]. Primary BA predominated during the first year of age, whereas secondary BA were low in general. Moreover, tauroconjugated BA were high in neonates [11]. In contrast to these studies, we measured BA profiles using HPLC-HRMS. In our study population we observed high serum tBA concentrations from birth until
age 24 months. However, in older children and adolescents tBA levels continually decreased and children aged 11 years and older had tBA values like those in adults. We also found age-dependence in the composition of BA. Interestingly, in neonates the BA pool differed substantially from that in adolescents. Varying serum BA levels in newborns and in infants might result from physiological immaturity in enterohepatic circulation,
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Jahnel et al.: Bile acids in pediatrics 5
A
B
CA
GLCA
GUDCA
6–24 months (group 2)
0–5 months (group 1)
UDCA
CDCA
CA
DCA LCA
GDCA
GLCA
UDCA
CDCA LCA DCA TUDCA
GUDCA
TUDCA GDCA
TCA
TCA
TCDCA GCA
GCA
TDCA
TCDCA
TLCA GCDCA
GCDCA TDCA TLCA
3–5 years (group 3)
C UDCA
GUDCA
DCA CDCA
CDCA
TUDCA TDCA
LCA TCA
GLCA
6–11 years (group 4)
D
CA
TCDCA
TLCA
GDCA
UDCA GLCA GUDCA
CA
DCA
LCA
TUDCA
TCDCA TDCA
TCA
TLCA GDCA
GCA
GCDCA
GCDCA
GCA
>11 years (group 5)
E CA
CDCA
TUDCA TCA DCA LCA
TCDCA TDCA TLCA
UDCA GLCA GCDCA GUDCA
GDCA GCA
Figure 3: BA pool composition in (A) age group 1, (B) age group 2, (C) age group 3, (D) age group 4, and (E) age group 5. In neonates, BA were primarily conjugated with taurine; however, after age 6 months glycoconjugates clearly predominated. The presence of secondary BA is notable after age 2 years. After age 11 years BA pool composition resembles that in healthy adults. White: unconjugated BA; light-gray: glycine conjugates; dark-gray: taurine conjugates.
causing ineffective digestion of lipids, prolonged hepatic clearance of BA, and cholestasis [1, 12]. In 1980 Barbara et al. reported a predominance of primary BA in neonates [13]. In our study, 97% of the BA pool in neonates consisted of conjugates of primary BA (GCA, GCDCA, TCA, and TCDCA). Poley et al. suggest an immature intestinal microflora in the colon as reason for low BA concentrations in the intestinal lumen despite high serum BA levels [14]. However, in neonates remarkably high concentrations of tauroconjugates were measured. This may be due to nutrition, since taurine is the most abundant free amino acid in breast milk. In the age groups 6–24 months and
older, levels of tauroconjugates dropped and accordingly glycine-conjugated BA predominated. In our study the presence of secondary BA proceeded pari passu with bacterial colonization of the large bowel within the first postnatal year. In the two youngest age groups (age 11 years (n = 91)
5.09±2.20 0.03±0.03 0.06±0.07 0.02±0.02 0.02±0.04 0.01±0.03 0.59±0.66 1.53±1.21 0.08±0.16 0.01±0.03 0.02±0.06 1.67±1.61 0.90±0.69 0.10±0.13 0.02±0.06 0.04±0.09
8.02±5.58 0.05±0.03 0.20±0.19 0.08±0.11 0.01±0.02 0.02±0.04 0.16±0.19 1.74±2.43 0.11±0.13 0.04±0.05 0.03±0.07 1.44±0.58 3.60±3.11 0.25±0.20 0.08±0.09 0.21±0.23
5.35±2.68 0.16±0.35 0.31±0.29 0.15±0.19 0.03±0.02 0.10±0.30 0.19±0.27 0.63±0.87 0.08±0.09 0.05±0.08 0.06±0.09 1.00±0.75 2.04±1.51 0.23±0.22 0.09±0.12 0.24±0.24
4.37±2.67 0.08±0.14 0.23±0.23 0.19±0.14 0.03±0.03 0.05±0.07 0.06±0.11 0.23±0.35 0.10±0.14 0.07±0.13 0.05±0.08 0.84±0.66 1.71±1.00 0.41±0.44 0.07±0.11 0.28±0.39
3.61±1.89 0.15±0.36 0.30±0.39 0.26±0.24 0.03±0.03 0.08±0.12 0.06±0.10 0.18±0.30 0.05±0.10 0.04±0.07 0.04±0.06 0.55±0.44 1.23±0.76 0.32±0.29 0.09±0.12 0.25±0.29
in serum, are excreted into bile but, unlike primary BA and DCA, are not efficiently absorbed from the small intestine. As a result, they are eliminated in feces and never constitute more than 5% of tBA [1]. This may be beneficial, as LCA is highly hepatotoxic in animal studies. In addition, concentrations of UDCA are also low; however, its glycoconjugate constitutes up to 4%–6% of tBA. Of importance is that recent studies have just begun to elucidate the complex interactions among the liver, BA, and the gut microbiome. Buffie et al. and Weingarden et al. showed that fecal microbiota transplantation had a great impact on secondary BA concentrations [15, 16]. This might be a connecting factor for further studies focusing on the relationship between the development of the intestinal microflora in neonates and shifts in BA profiles. In summary, this is the first study to determine standard value ranges of serum tBA in children and adolescents using HPLC-HRMS. Our data show that tBA concentrations vary substantially in the first years of life, warranting further investigation in future studies. These age-dependent reference ranges may provide useful information to assess the diagnostic value of BA profiles in neonatal cholestatic disorders and in prematurely born infants. Acknowledgments: The excellent technical assistance of Maria Schäffer is gratefully acknowledged. We thank A.S. Knisely, Institute of Liver Studies, King’s College Hospital, London, UK, for helpful comments on the manuscript. Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Financial support: None declared. Employment or leadership: None declared. Honorarium: J. Jahnel has received a grant. Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.
References 1. Hofmann AF. Bile acids: the good, the bad, and the ugly. News Physiol Sci 1999;14:24–9. 2. Colombo C, Okolicsanyi L, Strazzabosco M. Advances in familial and congenital cholestatic diseases. Clinical and diagnostic implications. Dig Liver Dis 2000;32:152–9. 3. Suzuki M, Muraji T, Obatake M, Nio M, Ito K, Suzuki K, et al. Urinary sulfated bile acid analysis for the early detection of biliary atresia in infants. Pediatr Int 2011;53:497–500. 4. Chiang JY. Bile acids: regulation of synthesis. J Lipid Res 2009;50:1955–66. 5. Brunner H, Northfield TC, Hofmann AF, Go VL, Summerskill WH. Gastric emptying and secretion of bile acids, cholesterol, and pancreatic enzymes during digestion. Duodenal perfusion studies in healthy subjects. Mayo Clin Proc 1974;49:851–60. 6. Galatola G, Jazrawi RP, Bridges C, Joseph AE, Northfield TC. Direct measurement of first-pass ileal clearance of a bile acid in humans. Gastroenterology 1991;100:1100–5. 7. Makishima M, Okamoto AY, Repa JJ, Tu H, Learned RM, Luk A, et al. Identification of a nuclear receptor for bile acids. Science 1999;284:1362–5. 8. Tagliacozzi D, Mozzi AF, Casetta B, Bertucci P, Bernardini S, Di Ilio C, et al. Quantitative analysis of bile acids in human plasma by liquid chromatography-electrospray tandem mass
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Jahnel et al.: Bile acids in pediatrics 7
9. 10.
11. 12.
spectrometry: a simple and rapid one-step method. Clin Chem Lab Med 2003;41:1633–41. Griffiths WJ, Sjovall J. Bile acids: analysis in biological fluids and tissues. J Lipid Res 2010;51:23–41. Polkowska G, Polkowski W, Kudlicka A, Wallner G, ChrzastekSpruch H. Range of serum bile acid concentrations in neonates, infants, older children, and in adults. Med Sci Monit 2001;7:268–70. Niijima S. Studies on the conjugating activity of bile acids in children. Pediatr Res 1985;19:302–7. Balistreri WF. Fetal and neonatal bile acid synthesis and metabolism – clinical implications. J Inherit Metab Dis 1991;14:459–77.
13. Barbara L, Lazzari R, Roda A, Aldini R, Festi D, Sama C, et al. Serum bile acids in newborns and children. Pediatr Res 1980;14:1222–5. 14. Poley JR, Dower JC, Owen CA Jr, Stickler GB. Bile acids in infants and children. J Lab Clin Med 1964;63:838–46. 15. Buffie CG, Bucci V, Stein RR, McKenney PT, Ling L, Gobourne A, et al. Precision microbiome reconstitution restores bile acid mediated resistance to Clostridium difficile. Nature 2015;517:205–8. 16. Weingarden AR, Chen C, Bobr A, Yao D, Lu Y, Nelson VM, et al. Microbiota transplantation restores normal fecal bile acid composition in recurrent Clostridium difficile infection. Am J Physiol Gastrointest Liver Physiol 2014;306:G310–9.
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Jörg Jahnel, Evelyn Zöhrer, Hubert Scharnagl, Wolfgang Erwa, Günter Faulera and Tatjana Stojakovica,*
Reference ranges of serum bile acids in children and adolescents DOI 10.1515/cclm-2014-1273 Received December 23, 2014; accepted February 10, 2015
mechanisms underlying these variations remain to be determined.
Abstract
Keywords: children; mass spectrometry; neonates; reference ranges; serum bile acids.
Background: Bile acids (BA) are found predominantly in bile but also in serum, where they can be used as markers for inborn and acquired hepatobiliary disorders. We measured serum BA levels by mass spectrometry to determine reference ranges for healthy children and adolescents in different age groups. Methods: In 194 healthy children and adolescents (0–19 years) concentrations of serum BA and BA composition were determined using high-performance liquid chromatography high-resolution mass spectrometry. Individuals were classified by ages into five groups: 0–5 months, 6–24 months, 3–5 years, 6–11 years, and > 11 years. Results: The 95% confidence interval of serum total BA values in newborns was 3.85–6.32 μmol/L. In the cohort aged 6–24 months total BA values were significantly higher (6.61–9.43 μmol/L; p 11 years (n = 91)
BMI
GGT
ALT
AST
13±4
97±49
41±59
65±47
16±2
13±4
19±9
42±8
16±2
27±68
30±47
45±33
18±4
22±21
51±85
45±36
23±6
25±23
22±17
24±8
Laboratory analysis In a volume of 10 μL of serum BA levels were determined as unconjugated acids and as taurine- (T) and glycine-conjugated (G) acids using HPLC-HRMS (Table 2). HPLC was performed on a reversedphase (C18) column, using a methanol/water gradient for chromatographic solution of isobaric BA and 2,2,4,4-d4-internal standards for quantification (all Sigma-Aldrich, Taufkirchen, Germany; Figure 2). Mass-spectrometer Q Exactive™ MS/MS (Thermo Fisher Scientific, Waltham, MA, USA) follows HPLC by the use of high-resolution,
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Jahnel et al.: Bile acids in pediatrics 3
Table 2: Serum concentrations of individual BA (μmol/L) are given as means. Ranges represent 95% confidence intervals.
0–5 months (n = 23) Mean Range 6–24 months (n = 15) Mean Range 3–5 years (n = 23) Mean Range 6–11 years (n = 42) Mean Range > 11 years (n = 91) Mean Range
Total BA
G-conjugates
T-conjugates
5.09 3.85–6.32
2.96 0.75–6.88
1.97 0.00–5.43
8.02 6.61–9.43
5.57 2.53–15.34
2.08 0.09–9.99
5.35 4.27–6.43
3.59 0.33–7.73
1.01 0.00–3.80
4.37 3.61–5.14
3.33 0.39–7.01
0.50 0.00–1.92
3.61 3.09–4.12
2.50 0.44–5.91
0.35 0.00–1.52
accurate-mass Orbitrap™ detection providing qualitative and quantitative data (Amplatz et al., submitted).
Statistical analysis Serum BA concentrations are represented as means±standard deviation (SD) within a 95% confidence interval. All statistical tests were two-tailed and p-values of 11 years) had BA profiles similar to that in adults Figure 3D and E). Glycoconjugates predominated over tauroconjugates (G:T = 6.5:1) and unconjugated BA attained a maximum in the fifth age group. Interestingly, no significant gender-related differences were observed in children’s BA profiles (data not shown).
Primary BA (CA, CDCA) In neonates conjugated primary BA predominated in serum, with taurine and glycine conjugates in balanced proportions; GCA was most abundant (Table 3). GCDCA predominated in the age group 6–24 months (up to 50% of tBA) and still remained high in older age groups. In addition, the percentage of unconjugated BA slightly increased with age. Interestingly, TCDCA was the most abundant taurine-conjugated primary BA within the first 5 months.
Secondary BA (DCA, LCA, UDCA) Serum levels of most secondary BA in newborns did not substantially vary from those in other age groups. However, DCA, GDCA, and GUDCA values continually rose with increasing age.
Discussion
Mean levels of tBA in the first 5 months of life were 5.09±2.20 μmol/L (Table 2). In the age group from 6 to 24 months tBA values were higher and reached a peak of 8.02±5.58 μmol/L. In age groups of 3–5 years, 6–11 years, and > 11 years mean tBA levels gradually decreased to 5.35±2.68, 4.37±2.67 μmol/L, and 3.61±1.89 μmol/L, respectively, reaching concentrations like those in healthy adults (0.28–6.50 μmol/L) [8].
BA composition During the first 5 months conjugated BA greatly predominated over unconjugated unconjugated BA and similar
In this study we established a comprehensive serum BA profile measured by tandem mass spectrometry. To our knowledge this is the first study using HPLC-HRMS to determine reference ranges of tBA for different age groups in children and adolescents. So far, tBA have been assessed in adults and children with different methods [9]. High tBA concentrations in neonates (19.6±5.2 μmol/L) were reported by Polkowska et al., with peak values of 22.2±5.1 μmol/L at the age of 1 month, determined using an enzymatic-colorimetric test [10]. However, after 1 year tBA levels declined to nearly adult concentrations (5.1±2.9 μmol/L) [10]. Using highperformance liquid chromatography Niijima et al. also
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4 Jahnel et al.: Bile acids in pediatrics 11.23
d4-DCA
m/z=395.3105
11.24
CDCA
8.21
UDCA
DCA
m/z=391.2854
10.82
9.02
m/z=407.2803
CA 12.70
m/z=379.3156
d4-LCA 12.70
m/z=375.2905
LCA
8.38
m/z=452.3320
d4-GCDCA 8.39 5.58
GUDCA
Relative abundances
8.74
m/z=448.3068
GDCA
GCDCA
6.81
m/z=468.3269
d4-GCA
6.81
m/z=464.3018
GCA 9.99
m/z=432.3119
GLCA
8.45
m/z=502.3140
d4-TDCA 8.13 5.34
TCDCA
TUDCA
8.45
m/z=498.2895
TDCA
6.55
m/z=514.2844
TCA 9.71
m/z=482.2946
TLCA
5
6
7
8
9
10
11
12
13
14
15
Time, min Figure 2: Representative HPLC-HRMS chromatograms of unconjugated as well as taurine- (T) and glycine-conjugated (G) bile acids and corresponding m/z values.
found high levels of tBA in neonates (11.0±8.7 μmol/L; 0–4 weeks) which continuously decreased with growth [11]. Primary BA predominated during the first year of age, whereas secondary BA were low in general. Moreover, tauroconjugated BA were high in neonates [11]. In contrast to these studies, we measured BA profiles using HPLC-HRMS. In our study population we observed high serum tBA concentrations from birth until
age 24 months. However, in older children and adolescents tBA levels continually decreased and children aged 11 years and older had tBA values like those in adults. We also found age-dependence in the composition of BA. Interestingly, in neonates the BA pool differed substantially from that in adolescents. Varying serum BA levels in newborns and in infants might result from physiological immaturity in enterohepatic circulation,
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Jahnel et al.: Bile acids in pediatrics 5
A
B
CA
GLCA
GUDCA
6–24 months (group 2)
0–5 months (group 1)
UDCA
CDCA
CA
DCA LCA
GDCA
GLCA
UDCA
CDCA LCA DCA TUDCA
GUDCA
TUDCA GDCA
TCA
TCA
TCDCA GCA
GCA
TDCA
TCDCA
TLCA GCDCA
GCDCA TDCA TLCA
3–5 years (group 3)
C UDCA
GUDCA
DCA CDCA
CDCA
TUDCA TDCA
LCA TCA
GLCA
6–11 years (group 4)
D
CA
TCDCA
TLCA
GDCA
UDCA GLCA GUDCA
CA
DCA
LCA
TUDCA
TCDCA TDCA
TCA
TLCA GDCA
GCA
GCDCA
GCDCA
GCA
>11 years (group 5)
E CA
CDCA
TUDCA TCA DCA LCA
TCDCA TDCA TLCA
UDCA GLCA GCDCA GUDCA
GDCA GCA
Figure 3: BA pool composition in (A) age group 1, (B) age group 2, (C) age group 3, (D) age group 4, and (E) age group 5. In neonates, BA were primarily conjugated with taurine; however, after age 6 months glycoconjugates clearly predominated. The presence of secondary BA is notable after age 2 years. After age 11 years BA pool composition resembles that in healthy adults. White: unconjugated BA; light-gray: glycine conjugates; dark-gray: taurine conjugates.
causing ineffective digestion of lipids, prolonged hepatic clearance of BA, and cholestasis [1, 12]. In 1980 Barbara et al. reported a predominance of primary BA in neonates [13]. In our study, 97% of the BA pool in neonates consisted of conjugates of primary BA (GCA, GCDCA, TCA, and TCDCA). Poley et al. suggest an immature intestinal microflora in the colon as reason for low BA concentrations in the intestinal lumen despite high serum BA levels [14]. However, in neonates remarkably high concentrations of tauroconjugates were measured. This may be due to nutrition, since taurine is the most abundant free amino acid in breast milk. In the age groups 6–24 months and
older, levels of tauroconjugates dropped and accordingly glycine-conjugated BA predominated. In our study the presence of secondary BA proceeded pari passu with bacterial colonization of the large bowel within the first postnatal year. In the two youngest age groups (age 11 years (n = 91)
5.09±2.20 0.03±0.03 0.06±0.07 0.02±0.02 0.02±0.04 0.01±0.03 0.59±0.66 1.53±1.21 0.08±0.16 0.01±0.03 0.02±0.06 1.67±1.61 0.90±0.69 0.10±0.13 0.02±0.06 0.04±0.09
8.02±5.58 0.05±0.03 0.20±0.19 0.08±0.11 0.01±0.02 0.02±0.04 0.16±0.19 1.74±2.43 0.11±0.13 0.04±0.05 0.03±0.07 1.44±0.58 3.60±3.11 0.25±0.20 0.08±0.09 0.21±0.23
5.35±2.68 0.16±0.35 0.31±0.29 0.15±0.19 0.03±0.02 0.10±0.30 0.19±0.27 0.63±0.87 0.08±0.09 0.05±0.08 0.06±0.09 1.00±0.75 2.04±1.51 0.23±0.22 0.09±0.12 0.24±0.24
4.37±2.67 0.08±0.14 0.23±0.23 0.19±0.14 0.03±0.03 0.05±0.07 0.06±0.11 0.23±0.35 0.10±0.14 0.07±0.13 0.05±0.08 0.84±0.66 1.71±1.00 0.41±0.44 0.07±0.11 0.28±0.39
3.61±1.89 0.15±0.36 0.30±0.39 0.26±0.24 0.03±0.03 0.08±0.12 0.06±0.10 0.18±0.30 0.05±0.10 0.04±0.07 0.04±0.06 0.55±0.44 1.23±0.76 0.32±0.29 0.09±0.12 0.25±0.29
in serum, are excreted into bile but, unlike primary BA and DCA, are not efficiently absorbed from the small intestine. As a result, they are eliminated in feces and never constitute more than 5% of tBA [1]. This may be beneficial, as LCA is highly hepatotoxic in animal studies. In addition, concentrations of UDCA are also low; however, its glycoconjugate constitutes up to 4%–6% of tBA. Of importance is that recent studies have just begun to elucidate the complex interactions among the liver, BA, and the gut microbiome. Buffie et al. and Weingarden et al. showed that fecal microbiota transplantation had a great impact on secondary BA concentrations [15, 16]. This might be a connecting factor for further studies focusing on the relationship between the development of the intestinal microflora in neonates and shifts in BA profiles. In summary, this is the first study to determine standard value ranges of serum tBA in children and adolescents using HPLC-HRMS. Our data show that tBA concentrations vary substantially in the first years of life, warranting further investigation in future studies. These age-dependent reference ranges may provide useful information to assess the diagnostic value of BA profiles in neonatal cholestatic disorders and in prematurely born infants. Acknowledgments: The excellent technical assistance of Maria Schäffer is gratefully acknowledged. We thank A.S. Knisely, Institute of Liver Studies, King’s College Hospital, London, UK, for helpful comments on the manuscript. Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Financial support: None declared. Employment or leadership: None declared. Honorarium: J. Jahnel has received a grant. Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.
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