Biomarkers of Nutritional Status

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Biomarkers of Nutritional Status

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

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WWW.CCM.UPMC.EDU/OCHOA
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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

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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
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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

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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

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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

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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

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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.

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Ravasco et. Al.- Abnormal Values
Percent Patients
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Mid arm circumference (MAC) as a valid tool to
assess malnutrition in the ICU
Ravasco, et. Al. Clinical Nutrition 2002.
21(1)73-77.
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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

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Is there a solution?

• Development of New Methods of nutritional
  evaluation and monitoring

How do cells sense nutrient availability?
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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.

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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

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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
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HypothesisChanges in the concentrations of
specific GENES reflect availability of certain
nutrients
Gene expression as a biomarker of nutrient
availability
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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
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ARGININE

• Conditionally essential amino acid
• Part of most immune enhancing diets
• Precursor of Nitric oxide
• Vasodilator
• Signaling molecule (memory)
• Immune competence

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Amino acid - Arginine
Ochoa et al Ann. Surg.
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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
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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.
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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.
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? Chain Expression is Dependent on Arginine
Arginine
No Arginine
No Glutamine
Repletion of Arginine
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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
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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
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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
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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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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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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.

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Open new avenues of therapeutic
intervention.New types of nutrition
interventionNew disease processes to
treatPharmacologic or even genetic intervention
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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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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.

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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.

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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)

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THANK YOUCanadian Society for Clinical Nutrition

• Dr. Daren Heyland