Differential diagnosis in patients with suspected bile acid synthesis defects

Table 1 Reference ranges of bile acid metabolites which accumulate in different bile acid biosynthesis defects

Defect	Metabolite	m/z	Median	Range
3β-hydroxy-Δ5-C27-steroid dehydrogenase deficiency	Glyco-dihydroxy-5-cholenoic acid	526	0.05	0.0-0.9
HSD3B7	Glyco-trihydroxy-5-cholenoic acid	542	0.07	0.0-0.4
16p11.2-12	Dihydroxy-5-cholenoic acid sulphate	469	0.71	0.15-8.1
OMIM: 23110	Trihydroxy-5-cholenoic acid sulphate	485	0.08	0.0-2.0
Δ4-3-oxosteroid 5-β-reductase deficiency	Glyco-hydroxy-oxo-cholenoic acid	444	0.03	0.0-0.4
AKR1D1	Glyco-dihydroxy-oxo-cholenoic acid	460	0.04	0.0-2.4
7q31	Tauro-hydroxy-oxo-cholenoic acid	494	0.02	0.0-0.5
OMIM: 235555	Tauro-dihydroxy-oxo-cholenoic acid	510	0.04	0.0-5.0
Defects of peroxisomal biogenesis	Tauro-tri-hydroxycholestanoic acid	556	0.02	0.0-0.3
Multiple	Tauro-tetra-hydroxycholestanoic acid	572	0.02	0.0-0.2
OMIM: 214100
Sterol 27-hydroxylase deficiency	Glucuronide-5β-cholestane-tetrol	611	0.24	0.0-1.6
(cerebrotendinous xanthomatosis)	Glucuronide-5β-cholestane-pentol	627	0.37	0.1-4.3
CYP27A1
2q33-qter
OMIM: 213700
Cholestatic liver disease	Glyco-chenodeoxy-cholic acid	448	0.49	0.0-5.2
Multiple	Glyco-cholic acid	464	0.64	0.1-14.6
	Tauro-chenodeoxy-cholic acid	498	0.05	0.0-0.8
	Tauro-cholic acid	514	0.08	0.0-8.4

The disorders are listed with their Online Mendelian Inheritance in Man^TM (OMIM) and their genetic defect, respectively. Reference ranges for bile acid metabolites which are increased in different bile acid synthesis defects were established in 100 healthy children, adolescents and young adults of age 10 d to 20 years. The metabolites are arranged to their appearance in different pathological conditions. Data are peak area ratios obtained for conjugates versus internal standard normalized to mol creatinine. The characteristic fragment ions used for detection of the metabolites are given as the mass-to-charge ratio (m/z).

Table 2 Electrospray ionization-tandem mass spectrometry operating parameters for bile acid intermediates

	Glycine conjugates (IS included)	Taurine conjugates	Sulfatides	Glucuronides
Nebulizer gas (au)	11	11	11	11
Curtain gas (au)	9	9	9	9
Collision gas (au)	5	5	5	5
Ion spray voltage (V)	-4100	-4100	-4100	-4100
Temperature (°C)	200	200	200	200
Declustering potential (V)	-35	-35	-35	-35
Focusing potential (V)	-31	-31	-31	-31
Entrance potential (V)	-10	-10	-10	-10
Prefilter (V)	23	23	23	23
Collision energy (V)	-55	-129	-60	-75
Collision cell exit potential (V)	-9	-9	-9	-9
Daughter ion (m/z)	74	80	97	85
Scan range of precursor ions (m/z)	430 to 550	490 to 590	460 to 490	600 to 630

The mass spectrometer was operated in a negative precursor ion scan mode with a step-size of 0.1 amu. Nitrogen was used as nebulizer, curtain and collision gases. au: Arbitrary units; IS: D₄-glycocholic acid.

Table 3 Peak area ratios of specific glycine and taurine conjugates in controls, patients with cholestatic liver disease (cholestasis) and patients AS and MK with suspected 5β-reductase deficiency

Metabolite	m/z	Controls(n = 100)	Cholestasis(n = 10)	AS	MK	MK1
Glyco-hydroxy-oxo-cholenoic acid	444	0-0.4	1-210	34	104	14
Glyco-dihydroxy-oxo-cholenoic acid	460	0-2.4	8-251	15	1331	1
Tauro-hydroxy-oxo-cholenoic acid	494	0-0.5	1-26	39	6	17
Tauro-dihydroxy-oxo-cholenoic acid	510	0-5	8-133	19	779	0
Glyco-chenodeoxy-cholic acid	448	0-5.2	2-10  754	4	9	25
Glyco-cholic acid	464	0.1-14.6	36-2814	17	49	47
Tauro-chenodeoxy-cholic acid	498	0-0.8	1-718	9	45	10
Tauro-cholic acid	514	0-8.4	6-749	9	10	1

Elevation of glycine and taurine conjugates of dihydroxy-oxo-cholenoic acid (m/z 460 and 510) and hydroxy-oxo-cholenoic acid (m/z 444 and 494) point to 3β-HSD. Elevation of glycine and taurine conjugates of cholic acid (m/z 448 and 464) and chenodeoxycholic acid (m/z 498 and 514) point to cholestatic liver disease. In patient AS, 5β-reductase and 3β-hydroxy-Δ⁵-C₂₇-steroid dehydrogenase deficiency were excluded by mutation analysis. The cholanoid profiles of patient MK with cystic fibrosis are in a decompensated state with severe cholestatic liver disease and

¹after treatment with ursodeoxycholic acid, pancreatic enzymes and fat-soluble vitamins.