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Biomarkers of Nutritional Status

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Some of the changes in gene expression are specific to arginine. Changes in these genes may be potentially used as 'biomarkers' of arginine deficiency in vivo. ... – PowerPoint PPT presentation

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Title: Biomarkers of Nutritional Status


1
Biomarkers of Nutritional Status
  • Juan B. Ochoa, MD, FACS
  • Associate Professor of Surgery and Critical Care
  • University of Pittsburgh

2
WWW.CCM.UPMC.EDU/OCHOA
3
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Infections after Trauma or Surgery
  • Occurs in up to 30 of critically ill traumatized
    patients
  • Is responsible for late mortality in up to 25 of
    the patients.
  • Nutrition support is the only therapy that has
    shown effects

5
Objectives
  • Determine
  • What is a biomarker
  • Why are they important
  • When and where to use a biomarker
  • Evaluate some examples of Classic Biomarkers
  • Do we have biomarkers of amino acid availability?
  • Can molecular biology help us?
  • Genomics, proteomics
  • Can enzymes that metabolize nutrients serve as
    biomarkers?
  • Future directions and conclusions

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Monitoring Nutrition
Physiologic Effect
Immunity
Intake
Dietary History
Metabolism
Nitric Oxide
Distribution
Absorption
Vitamin B 12
Amino acid (HPLC)
Effect on Outcome
Decreased Infections
8
Nutrition Assessment How do we do it?(Clinical
approach)
  • Dietary History
  • Anthropometric Measures
  • Laboratory data
  • Nitrogen
  • Serum Proteins
  • Immune Function
  • Total Lymphocyte Count
  • Delayed type hypersensitivity

9
Nutrition Assessment How do we do it?(Research
approach)
  • Combinations of previous measurements
  • Statistical Tools
  • Prognostic Nutritional Index
  • Direct measurements of specific nutrients in
    tissue samples
  • Amino acid levels in plasma

10
Limitations of Tools of Nutrition Assessment
  • Lack sensitivity
  • They do not detect all cases of nutrition
    deficiency
  • Lack specificity
  • They are affected by processes other than
    nutrition deficiency
  • Albumin - Affected by surgery, sepsis, critical
    illness
  • Cumbersome and difficult to use
  • Standardize
  • Inter-observer variability
  • Institutional variations
  • Technical difficulties
  • Measuring amino acids by HPLC
  • Expensive

11
As a result
  • No nutritional assessment tool is ideal.
  • No clinically useful (in the ICU) new nutritional
    assessment tool has been developed recently.
  • Increased demands on accountability for
    Nutritional Support.
  • Outcomes
  • Decreased revenue increases demands on Nutrition
    Support team

12
ExampleA Critical Approach to Nutritional
Assessment in Critically Ill Patients
  • Rvasco et. Al. Clinical Nutrition 2002
    21(1)73-77
  • Goals - Evaluate anthropometric and biochemical
    tests in critical illness
  • 44 patients Medical ICU.
  • NO trauma or surgical patients evaluated.
  • Length of stay gt 48 hours.

13
Ravasco et. Al.- Abnormal Values
Percent Patients
14
Mid arm circumference (MAC) as a valid tool to
assess malnutrition in the ICU
Ravasco, et. Al. Clinical Nutrition 2002.
21(1)73-77.
15
Adverse Consequences of Malnutrition Mortality
  • Mullen, J. Surg. Clin. N.A. 61 (3)465- . 1981
  • 100 Consecutive patients
  • All had non-emergency gastrointestinal surgery
  • Assessment of Malnutrition was done using
    Prognostic nutritional Index
  • Prospective study
  • Careful follow-up of patients
  • Complex statistical analysis
  • - Multivariate

16
Is there a solution?
  • Development of New Methods of nutritional
    evaluation and monitoring

How do cells sense nutrient availability?
17
What is a "biomarker"
  • Is an integrated measure of metabolism of a
    specific nutrient.
  • Potischman - J. Nutr. 133875S-880S, March 2003.
  • Is an indicator of amount of nutrient available.
  • Should reflect the physiologic effects mediated
    by a specific nutrient.
  • Should ultimately allow correlation with outcome.
  • Should be sensitive.
  • Should be specific to a given nutrient.

18
Why use a biomarker ?
  • Identify patients nutritional status
  • Develop quantitative tools to evaluate
    nutritional risk
  • (e.g. Folate deficiency)
  • Monitor effectiveness of therapy
  • Allow research for better and more effective
    nutrition intervention strategies

19
How do we develop a biomarker
  • Accessible samples or tissues
  • Easily measured by laboratory techniques used in
    the clinical laboratory
  • Can be standardized for different laboratories
  • Validated at different levels
  • In vitro
  • In vivo - animal models
  • Humans
  • Health
  • Disease
  • Should respond to a deficiency of a specific
    nutrient
  • Should be better than available methods of
    evaluation of that nutrient.
  • Should have clinical impact.

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DNA
S
Sensor
RNA
S
S
S
Ribosome
Signal Transduction
Protein
22
HypothesisChanges in the concentrations of
specific GENES reflect availability of certain
nutrients
Gene expression as a biomarker of nutrient
availability
23
Genes regulated by amino acid regulation
  • Genes Induced by amino acid supplementation
  • Argininosuccinate synthase
  • Ornithine decarboxylase
  • Collagenase
  • Tissue inhibitor of metalloproteinases
  • Genes induced by amino acid deprivation
  • IGFB-P, CHOP, Asparagine synthase, ApoB

Biochem. J. 2000. 3511-12
24
ARGININE
  • Conditionally essential amino acid
  • Part of most immune enhancing diets
  • Precursor of Nitric oxide
  • Vasodilator
  • Signaling molecule (memory)
  • Immune competence

25
Amino acid - Arginine
Ochoa et al Ann. Surg.
26
T cell receptor expression increases in response
to Arginine concentration
  • Mouse T cells cultured in vitro
  • Proliferation induced by stimulation with
    anti-CD3
  • MGchF measured by Flow cytometry
  • Arginine controlled in media

Plt0.05
Ochoa et. Al JPEN
27
Effect of L-Arginine on T Lymphocyte Surface
Receptor Expression




Blue - CD 3 Red - IL-2R plt0.05
From Ochoa, et al. JPEN. Vol 25 No 1 pp 23-29
2001.
28
The TCR
APC
MHC
Antigen
??
T Lymphocyte
From Hudrisier D and Luescher IF. Antigen
Recognition by CD 8 CTLs. In Sitkovsky MV and
Henkart PA. Cytotoxic Cells. Lippincott Williams
Wilkins. Philadelphia, 2000. Page 28.
29
? Chain Expression is Dependent on Arginine
Arginine
No Arginine
No Glutamine
Repletion of Arginine
30
Arginine
- Arginine
Mn Y 67.2
Mn Y 19.0
TCR? PE
CD3? FITC
Mean Gated Channel Fluorescence (MGchF- MnY) is a
reflection of the amount of protein expressed on
a T lymphocyte. In this case we are looking at
two peptides of the T cell receptor - CD3? and ?
chain
31
Flow cytometry measures individual "cells" by
binding a fluorescent antibody to the protein of
interest - Number of cells that are "" or "-" -
Amount of protein expressed in a specific cell -
Mean Gated Channel fluorescence (MGchF)
A- No Arginine
32
Effect of other amino acids on T cell
Proliferation
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Can T lymphocytes upregulate gene expression in
the presence of arginine deficiency ?
AL
AS
35
Arginino-succinate Synthase Expression Effects of
Arginine depletion - sqRT-PCR
1- Complete Media 2- No Arginine 3- No Arg,
Citrulline 4- Arg and Citrulline
Ochoa et. Al. Submitted
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37
IL-2
- - - -
Anti-CD3
0 0 0 0 100 100 100 100 L-arginine
18S
1 2 3 4 5 6 7 8
9 10
1. Control 2. - Control
Fig 3
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39
Conclusions
  • Some of the changes in gene expression are
    specific to arginine.
  • Changes in these genes may be potentially used as
    biomarkers of arginine deficiency in vivo.
  • Some of these genes may be useful as biomarkers
    to therapeutic repletion of arginine with the use
    of IEDs.

40
Open new avenues of therapeutic
intervention.New types of nutrition
interventionNew disease processes to
treatPharmacologic or even genetic intervention
41
IL-2
- - - -
Anti-CD3
0 0 0 0 100 100 100 100 L-arginine
18S
1 2 3 4 5 6 7 8
9 10
1. Control 2. - Control
Fig 3
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43
Conclusions
  • Adequate Nutrition assessment is paramount for
    the decision making process of nutritional
    intervention.
  • The tools of molecular biology may allow for the
    development of tools that can better assess and
    monitor nutritional status and Intervention.

44
Conclusions(Arginine as an example)
  • There appears to be a direct effect of arginine
    on the expression of specific genes in the T
    lymphocyte.
  • Roughly arginine-dependent gene expression can be
    classified into
  • Genes that are not affected by the absence of
    arginine
  • Genes whose expression is de-repressed by the
    absence of arginine.
  • Genes that are repressed by the absence of
    arginine.

45
Thank You
  • Sources of funding and support
  • National Institutes of Health United States
  • Frederick Coller Society
  • Firstnotebook/Patientrak
  • University of Pittsburgh
  • University of Kentucky
  • Individuals
  • Dr. Sidney Morris
  • Dr. Augusto Ochoa (LSU Med. Center)

46
THANK YOUCanadian Society for Clinical Nutrition
  • Dr. Daren Heyland
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