Analysis of Intermediary Metabolites - MetBio.Net – sample collection & handling ... mainline lab analysers? ... • Special Chemistry - UHW. Bonham 1993. Subject 1 Subject 2

Analysis of Intermediary Metabolites

Stuart J MoatMedical Biochemistry, University

Hospital of WalesMetBioNet April 2009

Intermediary Metabolites

• Glucose• Lactate• NEFA’s• 3-OH Butyrate

• Pyruvate• Acetoacetate

Overview

• Specimen collection and handling

• Sample stability

• Methodology – interferences & performance

Glucose

Glucose – sample collection & handling

• Continued glycolysis in vitro – decrease glucose ~7%/hour in un-separated blood

• Glycolytic inhibitors (Flox/FLHep)• PCA, TCA – denatures & precipitates

proteins• Manufacturers recommend serum

samples!

Effect of storage and specimen type on plasma glucose concentrations

Moat & Bonham 1995 - Unpublished Observations

-100-90-80-70-60-50-40-30-20-10

0

0 10mins

30mins

1 hour 2hours

6hours

24hours

48hours

72hours

1week

Storage time at toom temperature

Con

cent

ratio

n %

Cha

nge

FloxLiHepEDTAPCA

Effect of storage and specimen type on plasma glucose concentrations

2

2.5

3

3.5

4

4.5

5

0 10 mins 30 mins 1 hour 2 hours 6 hours

Flox

LiHep

PCAGluc

ose

mmol/L

Storage time at room temperature


Subject 1

Subject 2

Time (mins)

Gluc

ose

mmol/L Glucose m

g/L

Changes in plasma glucose concentrations with time

Flox plasma

LiHep plasma

Mikesh & Bruns. Clin Chem 2008

Effect of increasing haematocrit and time on blood glucose

0

1

2

3

4

5

6

0 1 2 3 4 5 6

Hct 0.51Hct 0.60Hct 0.71Hct 0.81

Gluc

ose

mmol/L

Time (Hours)Sidebottom et al. Clin Chem 1982

Glucose - Methodology

• Hexokinase• Glucose oxidase• Glucose dehydrogenase

• Pyrroloquinoline quinone glucose dehydrogenase

• PQQ glucose POCT systems – NOT glucose specific

WEQAS Glucose – Method Bias

-1.00

-0.80

-0.60

-0.40

-0.20

0.00

0.20

0.40

0.00 5.00 10.00 15.00 20.00 25.00

ID-GCMS Reference Targets

Bia

s

OverallGOD-PAPHexokinaseO2/peroxide electVitros

Bias

(mmol/l)

Glucose – Interpretation

• Pre-analytical factors have greater influence on blood glucose result than assay bias and imprecision

• Threshold glucose for investigation?

Lactate

Lactate – specimen collection & sample handling

• RBC’s metabolise glucose to lactate• Glycolytic inhibitors• Patient should be rested and avoid hand

clenching • Venous stasis – no effect• Indwelling catheters

Effect of storage and specimen type on plasma lactate concentrations


-100

100

300

500

700

900

0

10 m

ins

30 m

ins

1 ho

ur

2 hou

rs

6 hou

rs

24 ho

urs

48 ho

urs

72 ho

urs

1 wee

k


Con

cent

ratio

n %

Cha

nge

FloxLiHepEDTAPCA

0

1

2

3

4

5

6

0 10mins

30mins

1hour

2hours

6hours

24hours

48hours

72hours

1week

FloxLiHepPCA

Effect of specimen type on plasma lactate concentrations

Lact

ate

mmol/L


Effect of storage temperature on blood lactate concentrations

4.64*(2.06)

4.6*(2.10)

4.54*(2.10)

4.5*(2.10)

4.42*(2.10)

4.37*(2.08)

4.29(2.07)

RT (n=26)

3.89(1.07)

3.9(1.07)

3.89(1.10)

3.88(1.10)

3.88(1.12)

3.86(1.12)

3.83(1.1)

Ice (n=26)

302520151050Time

Sinn J et al. J Paed Child Health 2001

Lactate - Methodology

• Mainline chemistry analysers

Lactate + NAD+ Pyruvate + NADH + H+pH 9.0, LDH

Lactate + O2 Pyruvate + H2O2LO

H2O2 + 4-aminoantipyrine Red chromogenPeroxidase

Lactate - Methodology

• Blood gas analysers

Lactate + O2 Pyruvate + H2O2LO

H2O2 2H+ +O2 + 2e-Plt electrode

– Whole blood heparinised tubes– Analysis within 20 minutes of collection

WEQAS Lactate EQA scheme (Feb 2009)

11.110.911.8CV%

0.781.460.17SD

7.0513.341.44MeanmMol/L

All methods n=680 returns

5.15.95.2CV%0.330.730.07SD

6.4412.251.35MeanmMol/L

POCT ABL systems n=216

13.110.911.8CV%0.921.00.11SD

7.0513.221.47MeanmMol/L

Enzymatic methods n=35

Karon B et al. Am J Clin Pathol 2007

Lactate – Interpretation• Interpretation often difficult • Plasma vs whole blood & Blood vs CSF• Comparison of lactate between POCT and

mainline lab analysers?

• Age related reference intervals• Within subject variation ~30%• Threshold value for investigation?

NEFA’s

NEFA’s – sample handling

• Stability – contradictory data

• Manufacturers recommend:– EDTA or serum samples NOT heparin– Immediate analysis

Effect of storage and specimen type on plasma NEFA concentrations


-80-60-40

-200

2040

6080

100

0 10mins

30mins

1 hour 2hours

6hours

24hours

48hours

72hours

1week


Con

cent

ratio

n %

Cha

nge

Flox

LiHep

EDTA

Effect of specimen type on plasma NEFA concentrations

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

0 10 mins 30 mins 1 hour 2 hours 6 hours

Flox

LiHep

EDTA

PCA

Time

NEF

A’s

mmol/L


Storage time

NEF

A’s

mmol/L

Effect of storage temperature on NEFA’sin serum and EDTA plasma

RT 20-25°C

-20°C

4°C

Menendez L et al. Ann Clin Biochem 2001

0

0.5

1

1.5

2

2.5

0 10 20 30 40 50 60

Heparin n=8

Dextrose n=8

Effect of therapeutic heparin administration on plasma NEFA’s

Time (Mins)

NEF

A’s

(mM

ol/L

)

NEFA’s – Methodology (1)• Wako, Randox & Biovision Kits

NEFA + ATP + CoA Acyl CoA + AMP + PPi

2H2O2 4-AAP + MEHA Purple Adduct (550nm)

Acyl CoA synthase

Acyl CoA + O2 H2O2 + 2,3-trans-enoyl-CoAAcyl CoA Oxidase

Peroxidase

NEFA’s – Methodology (2)•Wako – linearity 4mmol/L•Freeze thaw analysis?•Wako vs Randox

Randox – 15% positive bias and problems with reagent stability

•QC Material – Randox & Wako

•No EQA scheme

NEFA’s - Interpretation• Separated within 6hrs and stored -20°C• Heparin• Catecholamines• Haemolysis • Bilirubin• Assay precision ~6%

• Use of reference intervals?

3-Hydroxybutyrate

3-OH Butyrate – sample handling

• Stable in whole blood at room temp ~48 hours

• Plasma & PCA extracts stable long term at -20°C

• Freeze-thaw cycles no effect

Effect of storage and specimen type on plasma 3-OH Butyrate concentrations

Stokol & Nydam 2005

n=10n=10 n=10

1.882.122.152.2524 Hours1.902.142.202.286 Hours1.932.082.132.02BLPCAEDTALiHepFloxMoat & Bonham 1995

- Unpublished Observations

3-OH Butyrate - Methodology

• Randox, Wako, Thermo Kits & in house assays

D-3-OH Butyrate + NAD Acetoacetate + NADH + H+pH8.5, HBD

NADH + INT (oxi) NAD + INT (reduced) Diaphorese

• POCT devices

3-OH Butyrate - Methodology• D-3-OH-Butyrate standards• Assay linearity up to 10mmol/L• Good reagent stability (Randox)• Unaffected by haemolysis, increased lactate &

LDH (Randox)• QC material - Randox

• Randox vs Wako kits?• EQA schemes – Randox, ERNDIM & NEQAS

RANDOX 3-OH Butyrate EQA scheme

6.60.0330.5025126.90.0220.322911

4.60.1132.452710

3.70.0300.81299

3.40.0411.202785.00.0160.322875.70.0180.32296

5.40.1332.46285

3.80.0360.95264

8.10.0240.303034.60.0551.20292

5.70.0290.51281

CV%SDMean 3-OHBut concentration

mmol/LNumber of

labsSample Number

3-OH Butyrate - Interpretation

• Stable analyte• Robust assay (analytical CV~5%)• 3-OH-But should NOT be interpreted

in isolation

• Use of reference intervals?

Summary

• IM’s useful analytical tools

• Simple commercially available assays

• Lack of pre-analytical/sample collection guidelines

Discussion• Further studies:

– effect of anticoagulants and storage on IM’s and to assess any concentration dependent effects

– Freeze thaw stability studies

• Validation of kits/assays in use• EQA scheme for NEFA’s• MetBioNet Guidelines for the analysis of

IM’s

Acknowledgements

• Metabolic Section - Sheffield Children’s Hospital• Special Chemistry - UHW

Bonham 1993

Subject 1

Subject 2

Time (mins)

Lact

ate

mmol/L

Changes in plasma Lactate concentrations with time

Flox plasma

LiHep plasma

Mikesh & Bruns. Clin Chem 2008

Glucose – Method Bias (WEQAS)Glucose Bias plot

-1.2

-1

-0.8

-0.6

-0.4

-0.2

0

0.2

0.4

0 5 10 15 20 25 30

Method mean

Bia

s

O2/peroxide electHexokinaseGOD-PAPVitros

Glucose – Method BiasGlucose Bias plot

-1.2

-1

-0.8

-0.6

-0.4

-0.2

0

0.2

0.4

0 5 10 15 20 25 30

Method mean

Bia

s


-1.00

-0.80

-0.60

-0.40

-0.20

0.00

0.20

0.40

0.00 5.00 10.00 15.00 20.00 25.00

ID-GCMS Reference Targets

Bia

s

OverallGOD-PAPHexokinaseO2/peroxide electVitros

Glucose Methods

46

239

83

58


Effect of storage and specimen type on plasma 3-OH Butyrate concentrations

0.0

20.0

40.0

60.0

80.0

100.0

120.0

140.0

160.0

0 10mins

30mins

1hour

2hours

6hours

24hours

48hours

72hours

1week

Time

Con

cent

ratio

n %

Cha

nge

FloxLiHepEDTAPCA

Moat & Bonham - Unpublished Observations

Analysis of Intermediary Metabolites - MetBio.Net – sample collection & handling ... mainline lab analysers? ... • Special Chemistry - UHW. Bonham 1993. Subject 1 Subject 2

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