Biochemical Assessment

• Depletion stages

  • Dietary inadequacy
  • Decreased level in reserve tissue store
  • Decreased level in body fluids
  • Decreased functional level in tissues
  • Decreased activity of nutrient-dependent enzyme or mRNA for some proteins
  • Functional changes
  • Clinical signs/symptoms
  • Anatomical signs
• Biochemical Assessment


  Measurement of a nutrient and its metabolite in blood, feces, urine, or blood which has a relationship to nutritional status
• Biochemical Assessment

  • More objective and precise approach than the other methods
  • Can identify borderline nutritional condition prior to appearance of symptoms
  • Reveal marginal or acute deficiencies when clinical signs are normal since clinical signs usually occur only after prolonged inadequate intakes
• Limitations of Biochemical Assessment

  • May not necessarily reflect presence of pathological lesion nor show health significance in relation to nutritional status
  • Affected by biological or technical factors (confounders) rather than nutrient status
  • May not always reflect total body nutrient content or tissue stores which is most sensitive to nutritional change
  • Interpretation of results are not always simple
• Laboratory Methods are Used to Determine Deficiencies in

  • Serum protein particularly albumin level
  • Blood-forming nutrients – iron, folacin, vitamin B6, and vitamin B12
  • Water-soluble vitamins – thiamine, riboflavin, niacin, and vitamin C
  • Fat-soluble vitamins – A, D, E, K
  • Minerals – iron, iodine and other trace minerals
  • Lipids and glucose – cholesterol, triglycerides, various enzymes which are implicated in heart disease, diabetes, and other chronic diseases
• Types of Biochemical Tests

  • Static Biochemical Tests
  • Functional Test
• Static biochemical tests


  • Measure the level rather than the function of a nutrient
  • Provide information on the degree of deficiency of the particularly body pool sampled rather than the whole-body status
  • Measure either a nutrient in biological fluids or tissues or the urinary excretion rate of the nutrient or its metabolite
• Static biochemical tests

  • Useful for identifying the second and third stages in the development of a nutritional deficiency when the tissue stores followed by the body fluids levels become gradually depleted of the nutrient(s)
  • Some static biochemical tests measure levels of the nutrient in biological fluids and tissues on the assumption that such tests reflect the total body nutrient content or the nutrient tissue store most sensitive to depletion
  • Show the change of nutrient concentration in any given specimen which is most sensitive to nutritional change
• Functional tests

  • Functional physiological tests
  • Functional biochemical tests
• Functional physiological tests


  • Assess the physiological performance of an individual in vivo such as immune competence, taste acuity, night blindness, muscle function, and work capacity
  • None of the tests are specific and must be interpreted along with biochemical measurements
  • Determine the changes in the activities of enzymes dependent on a specific nutrient or in the concentrations of specific blood components dependent on a given nutrient
• Functional biochemical tests


  Assess the consequences of the nutrient deficiency by measuring changes in the activities of a specific enzyme or in the concentrations of specific blood components dependent on a given nutrient
• Potential Confounders

  • Subject characteristics
  • Health condition
  • Biological
  • Sampling errors
  • Method
  • Specimen
  • Measurement errors
• Factors to Consider in Specimen Collection

  • Ease of data/sample collection
  • Sample storage and transport
  • Transportability and ruggedness of field equipment
  • Expertise available for collection and analysis
  • Equipment available and its maintenance
  • Culture and religious context
  • Costs (capital and recurring costs, training, personnel, maintenance of equipment
• Commonly Used Specimen (1)

  • Venous blood
  • Capillary blood
  • Erythrocyte
  • Leucocyte
• Commonly Used Specimen (2)


  • Breast milk
  • Sweat
  • Semen
  • Hair
  • Nails
  • Mucosal swab
  • Stool
  • Biopsy materials
  • Liver
  • Amniotic fluid
• Urine


  • Readily available, and non-invasive
  • A normal renal function is a prerequisite when used as specimen to determine nutritional status
  • Analyses on fasting morning, casual, or 24-hr collections
  • 24-hour urine is the most preferred as concentration of nutrient metabolite is not equally distributed throughout the day
• Factors to Consider in Selection of Laboratory Methods for Nutritional Impact Evaluation

  • Nature of the intervention/program
  • Kind
  • Severity
  • Prevalence of nutritional problems in the population
• Nutritional biomarkers


  • Biological measurements (e.g., in blood or urine) that are used to indicate normal biological processes, pathogenic processes, or pharmacologic responses to therapeutic intervention
  • Defined as biological characteristics that can be objectively measured and evaluated as indicators of normal biological or patho genic processes, or as responses to nutrition inter ventions
  • Can be measure ments based on biological tissues and fluids, on physio logical or behavioral functions, and more recently, on metabolic and genetic data that in turn influence health, well-being and risk of disease
• Uses of nutritional biomarkers

  • Program managers and policymakers use population-level biomarkers for screening, surveillance, and monitoring and evaluation of interventions
  • Clinicians use biomarkers mainly for diagnosis, prognosis, and treatment; and researchers use biomarkers for any or all of these purposes based on their needs
• Classification of nutritional biomarkers

  • Biomarkers of exposure
  • Biomarkers of status
  • Biomarkers of function
• Biomarkers of exposure


  • Biomarkers of food or nutrient intakes; dietary patterns; sup ple ment usage
  • Can be based on direct measurements of nutrient intake using traditional dietary assessment methods, or indirect measurements using surrogate indicators termed "dietary bio markers"
• Classification and properties of dietary biomarkers

  • Recovery biomarkers
  • Concentration biomarkers
  • Predictive biomarkers
• Biomarkers of status


  • Measure ments of concentrations of a nutrient in biological fluids or tissues, or the urinary excretion rate of a nutrient or its metabolite
  • Biopsy material most frequently used is whole blood or some fraction of blood
  • Other body fluids and tissues, less widely used, include urine, saliva, adipose tissue, breast milk, semen, amniotic fluid, hair, toenails, skin, and buccal mucosa
• Functional biomarkers

  • Functional biochemical
  • Functional physiological/behavioral
• Functional biochemical biomarkers


  Measure the extent of the functional con sequences of a nutrient deficiency, such as enzyme stimulation assays, abnormal metabolites, DNA damage
• Functional physiological/behavioral biomarkers


  More directly related to health status or disease such as vision, growth, immune function, taste acuity, cognition, depression. These biomarkers impact on clinical and health outcomes.
• Biomarkers of function


  • Measure the extent of the functional consequences of a nutrient deficiency
  • Have greater biological significance than static biomarkers
  • Some are used as substitutes for chronic disease outcomes (surrogate biomarkers)
• Functional biochemical biomarkers

  • Serve as early biomarkers of subclinical deficiencies
  • Measure abnormal metabolic products in blood/urine from nutrient-dependent enzyme deficiency
  • Measure reduced activity of nutrient-dependent enzymes
• Functional biochemical biomarkers

  • Urinary excretion of xanthurenic acid, formiminoglutamic acid (FIGLU), and methylmalonic acid for vitamin B6 and B12 deficiency
  • Erythrocyte glutathione reductase activity for riboflavin
  • Erythrocyte transketolase activity for thiamin
  • Erythrocyte glutamic oxaloacetic transaminase for vitamin B6
  • Whole blood hemoglobin for iron
  • Thyroglobulin for iodine
  • Retinol-binding protein for vitamin A
  • Holo transcobalamin for vitamin B12
• Functional physiological and behavioral biomarkers

  • In vitro tests of in vivo functions (e.g. lymphocyte proliferation for protein-energy, zinc, iron)
  • Load and tolerance tests and induced responses in vivo (e.g. Relative Dose Response for vitamin A, CobaSorb test for vitamin B12 absorption)
  • Spontaneous in vivo responses (e.g. dark adaptation/vision at low intensity for vitamin A, taste acuity for zinc, handgrip strength for lower-body strength)
  • Growth or developmental responses (e.g. growth velocity for protein-energy, zinc, cognitive performance for iron, iodine, vitamin D, folate, vitamin B12, motor development for micronutrients, depression for folate and zinc)
• Biomarkers should be selected with care, and their limitations under conditions of health, inflammation, genetic and disease states understood
• Biomarkers are affected by non-biological sources of variation arising from specimen collection and storage, seasonality, time of day, contamination, stability, and laboratory quality assurance
• Both biological and non-biological sources of variation will impact the validity, precision, accuracy, specificity, sensitivity, and predictive value of the biomarker
• Personnel should use calibrated equipment and standardized, validated techniques which are continuously monitored by appropriate quality-control procedures
• Factors affecting choice of nutritional biomarkers

  • Study objectives
  • Population and setting
  • Validity
  • Precision
  • Sensitivity and specificity
  • Analytical sensitivity and analytical specificity
  • Predictive value
• Validity


  • How well the biomarker correctly describes the nutritional parameter of interest
  • Valid biomarkers are free from random and systematic errors and are both sensitive and specific
• Action of inflammation, stress, or certain medications on enzyme activity and nutrient metabolism may alter nutrient status and affect the validity of a nutritional biomarker
• To adjust for the presence of systemic inflammation, WHO recommends concurrent measurement of serum C-reactive protein and α-1-acid glycoprotein
• Impact of inflammation on micronutrient biomarkers

  • Ferritin: 44.9% at risk without adjustment, 64.9% at risk with Brinda adjustment
  • Retinol binding protein: 24.3% at risk without adjustment, 12.4% at risk with Brinda adjustment
  • Zinc: 13.0% at risk without adjustment, 10.4% at risk with Brinda adjustment
• Precision


  • The degree to which repeated measurements of the same biomarker give the same value
  • Assessed by coefficient of variation (CV) of replicates