Evaluation of Very Long Chain Fatty Acids by UPLC-MS/MS in A Normal Pediatric Population

Liu A 1, Bunker A 1, Robert W 1,2, Longo N 1,2,3, Pasquali M 1,2,3

1 ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT 84108, USA2 Department of Pathology, University of Utah School of Medicine, Salt Lake City, Utah 84132, USA3 Department of Pediatrics, University of Utah School of Medicine, Salt Lake City, Utah, 84132, USA

ABSTRACTBackground: Peroxisomal disorders are caused by defective activity of one (single enzyme deficiencies) or multiple peroxisomal enzymes (peroxisome biogenesis disorders). These diseases are biochemically characterized by accumulation in tissues and body fluids of very long chain fatty acids (VLCFAs), hexacosanoic (C26:0), tetracosanoic (C24:0), and docosanoic (C22:0) acids, and branched chain fatty acids, pristanic (C19:0-br) and phytanic (C20:0-br) acids. Analysis of VLCFAs in plasma is used in the evaluation of patients with peroxisomal disorders.Methods: In this study we have evaluated VLCFAs in 419 normal controls, ranging in age from 3 weeks to 18 years, using Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS). Plasma samples were collected as part of the CHILDx pediatric reference range study. VLCFAs were converted to trimethylaminoethyl iodide esters prior to chromatographic separation and MS/MS analysis. Results: The concentration of straight chain VLCFAs (C26:0, C24:0, and C22:0) did not change significantly with age. By contrast, the concentration of pristanic and phytanic acids (BCFA) peaked around 1-2 years of age, then decreased and remained constant after 7 years of age. This may reflect dietary changes with age (phytanic acid is present in products derived from ruminant animals). There was, however, a clear separation in the concentration of VLCFAs and/or their ratios between normal controls and patients with peroxisomal disorders.

INTRODUCTIONPeroxisomal disorders are a heterogeneous group of progressive multisystem diseases caused by defective peroxisomal function. Peroxisomal disorders are divided in two main groups: peroxisome biogenesis disorders (PBDs) and single enzyme deficiencies. Among these disorders, X-linked adrenoleukodystrophy (X-ALD) is the most common, and Zellweger syndrome (ZWS) is the most severe. These diseases are biochemically characterized by accumulation in tissues and body fluids of very long-chain (C22 – C26) fatty acids (VLCFA), and branched-chain fatty acids (BCFA), pristanic (Pri) and phytanic (Phy) acids. Measurement of total VLCFA and BCFA in plasma screens for peroxisomal disorders. We have developed a new UPLC-MS/MS method for the analysis of VLCFA and BCFA and have established reference ranges in a normal pediatric population.

METHOD:Sample preparation: 20 µL plasma, 1 hr acid hydrolysis, Hexane extraction, TMAE derivatizationUPLC chromatography: Acquity BEH C18, 50 mm; H2O/Acetonitrile/TFADetection/Quantitation: Waters Quattro Premier, ESI/MS/MS in SRM mode (deuterated fatty acids as Internal Standards)Samples: Normal controls, age range: 3 weeks – 18 years (n=419);

Normal controls, age range: > 20 years (n=30) This study was approved by the IRB of the University of Utah.

CONCLUSIONS

We have developed a UPLC-MS/MS method for the measurement of VLCFAs in plasma and established reference ranges for the pediatric and adult population.

The concentration of phytanic acid and its degradation product, pristanic acid, changes with age with a peak at 1-3 years of age likely reflecting dietary changes.

The concentration of straight-chain fatty acids C26:0 and C22:0 does not change significantly with age. Patients with peroxisomal disorders can be easily identified by this method.

Time3.0 3.5 4.0 4.5 5.0 5.5

%

0

100

C26d3-Pri d3-Phyd3-C20Pri Phy

C20

d4-C22 d4-C24d4-C26

C22

C24

Normal

Time3.0 3.5 4.0 4.5 5.0 5.5 6.0

%

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C26d4-C24

C24

d4-C22

C22

d4-C20

C20

d3-Phy

Prid3-Pri Phy C22:1

d4-C26

RESULTSFigure 1 shows the chromatographic profile of VLCFAs obtained from plasma of a normal control (1A) and a patient with Zellweger syndrome (1B). The run time (6.5 min, injection to injection) is greatly reduced compared to the more traditional gas-chromatographic methods used for this analysis.

Pri

0.0

0.3

0.6

0.9

-2 8 18 28 38 48 58

Age (Yrs)

um

ol/L

Pri

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0.6

0.9

0 1 10 100Age (Yrs)

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ol/

L

Phy

0.0

2.0

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8.0

-2 8 18 28 38 48 58

Age (Yrs)

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ol/L

Phy

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0 1 10 100Age (Yrs)

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ol/

L

C22:0

0.0

30.0

60.0

90.0

120.0

-2 8 18 28 38 48 58

Age (Yrs)

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ol/

L

C26:0

0.00

0.50

1.00

1.50

-2 8 18 28 38 48 58

Age (Yrs)

umol

/L

C26:0

0.0

5.0

10.0

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-2 8 18 28 38 48 58

Age (Yrs)

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ol/

L C26:0/C22:0

0.00

0.01

0.02

0.03

-2 8 18 28 38 48 58

Age (Yrs)

Rat

io

C26:0/C22:0

0.000.100.200.300.400.500.600.70

-2 8 18 28 38 48 58

Age (Yrs)

Rat

io

BA

Figure 1. UPLC-MS/MS analysis of VLCFAs in plasma from a normal control (A) and a patient with Zellweger syndrome (B).

Figures 2 and 3 show the changes in the concentration of VLCFAs and BCFAs with age in the normal pediatric and adult population (blue dots). The concentration of phytanic (Phy) and pristanic (Pri) acid shows more variability between 1 and 3 years of age, with a significantly (p< 0.05) higher mean value; likely reflecting the relatively high intake of cow milk during these years, then remained stable after 7 years of age. The concentration of C26:0 and C22:0 or their ratio did not show any significant change with age (Fig. 3). The concentration of C22:0 was decreased while that of C26:0 and the C26:0/C22:0 ratio were increased in patients with peroxisomal disorders (pink squares).

Figure 2. Variation in the concentration of phytanic (B) and pristanic (A) acids with age. Blue dots represent normal controls. Pink squares represent patients with peroxisomal disorders. The small insert represents the same graph with the age on a logarithmic scale.

Figure 3. Variation in the concentration of C22:0 (A), C26:0 (B) and their ratio (C) with age . Blue dots represent normal controls. Pink squares in the insert represent values from patients with peroxisomal disorders compared to normal controls.

A

A B

B C

Zellweger