Polyamines

We have developed a quantitative LC-MS/MS assay, the Polyamine and 1-Carbon Panel, which quantifies several metabolites in the arginine and proline metabolism (KEGG HSA00330).

These metabolites include

Alzheimer’s disease (AD) has been associated with alterations in arginine metabolism as well changes in the SAM/SAH ratio and polyamines. Increased arginase and homocysteine with decreased ornithine and NOS activities were found to be AD specific in the brain and blood. The acetylated metabolites of spermidine and spermine have also been implicated in several types of cancers and have been proposed as tumor markers in urine. Polyamines, including spermidine and spermine are growth factors necessary for cell division and cell proliferation and have been linked to cancerous tumor growth, Alzheimer’s disease and stress resistance. 

Sample chromatograms of the analysis of a standard mixture (A), urine (B) and a tissue sample (C) are below; the total analysis is seven minutes per sample.

Sample chromatograms of the analysis of a standard mixture (A), urine (B) and a tissue sample (C)

The assay has been validated for quantifying compounds from several matrices including tissue and urine and can be performed with <50 mg of tissue. Analysis is performed using LC-MS/MS operating in multiple reaction monitoring mode, and Skyline quantifies the metabolites based on external calibration curves with the use of deuterated internal standards (standards are available from commercial vendors). Example calibration curves are shown below for MTA and Diacetylspermine, followed by a table of the average concentrations of polyamines in tissues (lung, liver, brain) as well as plasma.

calibration curve for MTA

calibration curve for Diacetylspermine

 

Lung (µM)

Liver (µM)

Plasma (µM)

Brain (uM)

Agmatine

0.023 ± 0.013

0.013 ± 0.003

0.053 ± 0.013

N/A

GABA

0.67 ± 0.12

1.96 ± 0.42

0.78 ± 0.90

217.0 ± 40.2

N1 Acetylspermidine

0.093 ± 0.023

0.103 ± 0.033

0.053 ± 0.043

0.77 ± 0.27

Spermidine

2.4 ± 0.6

3.3 ± 0.8

2.5 ± 1.6

0.05 ± 0.01

Acetylspermine

N/A

N/A

N/A

0.11 ± 0.34

Acetylputrescine

0.043 ± 0.013

0.033 ± 0.013

N/A

N/A

N8 Acetylspermidine

0.083 ± 0.023

0.093 ± 0.023

0.053 ± 0.043

0.02 ± 0.01

Spermine

0.34 ± 0.05

0.34 ± 0.07

1.3 ± 1.5

N/A

Diacetylspermine

N/A

N/A

N/A

0.00 ± 0.00

Homocysteine

N/A

N/A

N/A

N/A

MTA

0.11 ± 0.03

0.27 ± 0.09

0.023 ± 0.013

0.27 ± 0.05

SAH

0.46 ± 0.12

7.0 ± 1.2

0.073 ± 0.063

0.47 ± 0.13

SAM

8.2 ± 1.5

18.3 ± 3.1

0.79 ± 0.31

4.9 ± 1.3

Methionine

2.4 ± 0.8

2.2 ± 0.9

2.1 ± 0.7

1.9 ± 0.4

Arginine

24.1 ± 4.8

8.1 ± 2.4

58.8 ± 9.3

20.8 ± 6.6

Ornithine

9.0 ± 1.8

59.1 ± 11.1

29.6 ± 5.8

0.43 ± 0.11

Sources:

  1. Minois N, Carmona-Gutierrez D, Madeo F. Polyamines in aging and disease. Aging. 2011;3(8):716.
  2. Liu P, Fleete MS, Jing Y, Collie ND, Curtis MA, Waldvogel HJ, Faull RL, Abraham WC, Zhang H. Altered arginine metabolism in Alzheimer's disease brains. Neurobiology of aging. 2014;35(9):1992-2003.
  3. Morrison LD, Kish SJ. Brain polyamine levels are altered in Alzheimer's disease. Neuroscience letters. 1995;197(1):5-8.
  4. Linnebank M, Popp J, Smulders Y, Smith D, Semmler A, Farkas M, Kulic L, Cvetanovska G, Blom H, Stoffel-Wagner B. S-adenosylmethionine is decreased in the cerebrospinal fluid of patients with Alzheimer’s disease. Neurodegenerative Diseases. 2010;7(6):373-8.
  5. Graham SF, Chevallier OP, Elliott CT, Hölscher C, Johnston J, McGuinness B, Kehoe PG, Passmore AP, Green BD. Untargeted metabolomic analysis of human plasma indicates differentially affected polyamine and L-arginine metabolism in mild cognitive impairment subjects converting to Alzheimer’s disease. PLoS One. 2015;10(3):e0119452.
  6. Seshadri S, Beiser A, Selhub J, Jacques PF, Rosenberg IH, D'agostino RB, Wilson PW, Wolf PA. Plasma homocysteine as a risk factor for dementia and Alzheimer's disease. New England Journal of Medicine. 2002;346(7):476-83.
  7. Häkkinen MR, Roine A, Auriola S, Tuokko A, Veskimäe E, Keinänen TA, Lehtimäki T, Oksala N, Vepsäläinen J. Analysis of free, mono-and diacetylated polyamines from human urine by LC–MS/MS. Journal of Chromatography B. 2013;941:81-9.
  8. Samejima K, Hiramatsu K, Takahashi K, Kawakita M, Kobayashi M, Tsumoto H, Kohda K. Identification and determination of urinary acetylpolyamines in cancer patients by electrospray ionization and time-of-flight mass spectrometry. Analytical biochemistry. 2010;401(1):22-9.
  9. Hiramatsu K, Takahashi K, Yamaguchi T, Matsumoto H, Miyamoto H, Tanaka S, Tanaka C, Tamamori Y, Imajo M, Kawaguchi M. N1, N12-Diacetylspermine as a sensitive and specific novel marker for early-and late-stage colorectal and breast cancers. Clinical cancer research. 2005;11(8):2986-90.