A biomarker refers to a measurable indicator or ‘marker’ of some biological state or condition. Lab values are often referred to as analytes. In the development of laboratory tests two main issues are generally considered, the sensitivity and specificity of identifying a condition or situation, and the accuracy and reproducibility of the laboratory test in getting the ‘right’ answer. It’s important to note that nutritional laboratory tests do not generally diagnose a condition, they give nutritional levels and metabolic status. Laboratory tests in functional nutrition rely heavily on understanding and knowing biochemical pathways. Nutrients are often the substrate that start a pathway, and act as cofactors in supporting the function and flow of the pathways.
A full assessment, including a diet evaluation, anthropometrics, a nutrition physical and review of associated systems, should give a good picture of what is going on in an individual. The next step is to assess biomarkers in areas that may be of concern. Is there a specific nutrient need that could impact biochemical or physiologic function? Many functional clinicians do a full laboratory nutrition panel on all new patients as a starting point. Combining the full assessment with laboratory results can help to better identify what the main nutrition issues are, and if there are impairments in biochemical pathways. There are several good resources to review biochemical pathways:
- ExPASy: http://biochemical-pathways.com/#/map/1 (they do a nice job of using standard language and identify nutrient cofactors needed by the enzymes. The example below shows tryptophan breakdown to serotonin, and melatonin, and the required cofactors.)
- Wikipedia: https://en.wikipedia.org/wiki/Metabolic_pathway
- Sigma Aldrich: http://www.sigmaaldrich.com/technical-documents/articles/biology/interactive-metabolic-pathways-map.html
Types of Laboratory Testing
There are two primary types of laboratory tests, functional (or indirect) and direct. A direct measure is the analyte itself, and a functional test is the evaluation of markers that are dependent on the nutrient. Vitamin D is a direct measure, it looks at the actual level of vitamin D (25-hydroxyvitamin D). An example of a functional marker is methylmalonic acid (MMA), it is a functional measure of vitamin B12. The enzyme that takes MMA to succinyl Coenzyme A is dependent on vitamin B12 as a cofactor. If vitamin B12 is not available the step cannot move forward and MMA builds up in blood and urine. There is a good correlation between MMA and serum vitamin B12. It can be difficult to identify when a nutrient is insufficient or deficient for each individual, thus ideally getting both direct and indirect tests may provide the most comprehensive data.
One of the best places to find information on biochemical/metabolic analytes is:
- Human Metabolome Data Base (HMDB): http://www.hmdb.ca/ In an example of Tryptophan it gives extensive information on tryptophan, and if you scroll down you will see it list the biofluids used to assess tryptophan status, it gives conditions associated with the laboratory assessment and the pub med reference it was pulled from. http://www.hmdb.ca/metabolites/HMDB00929
Functional Laboratory Testing
Laboratory testing helps to identify discrepancies in physiologic function or biochemical pathways. Additionally, comparing intake and lab values lets you identify possible digestive issues or increased needs. Let’s use protein as an example. While someone may consume enough protein based on standard diet calculations, they may have inadequate stomach acid production, leading to a decreased ability to digest or absorb all the protein they are taking in, or they may have increased needs of a specific amino acid or nutrient cofactors. For example, someone with depression and sleep issues may have low tryptophan (which goes to make serotonin and melatonin) or a lack of vitamin B6 a co-factor in the breakdown pathway of protein. [ Arch Psychiatr Nurs. 2015 Apr;29(2):102-7. The effects of dietary tryptophan on affective disorders. Lindseth, G, Helland B, Caspers J.] Vitamin B6 has the highest level of deficiency in the US population, according to NHANES data. They could also be interfering factors such as an increased level of large neutral amino acids (LNAA) that compete with tryptophan to cross the BBB. Experimentally this is achieved by giving collagen supplements since they contain LNAA and no tryptophan. Imbalanced levels of individual nutrients, especially those related to key biological pathways can be linked to clinical findings.
When a lab test is done it is compared against a reference range and identifies if your value is within or out of range. Laboratories can develop their own tests or purchase an established kit, both are acceptable to regulatory agencies. If the lab develops its own test, they will need to establish a reference range. Statistically it is acceptable and often done to test ~120 healthy individuals and calculate 1 and 2 standard deviations from the mean. Two standard deviations accounts for variability so a result outside is out of range. Two standard deviations include 95% of the population tested. A patient is considered abnormal if they are beyond 2 standard deviations from the mean. The more ‘healthy’ subjects that were included in determining the range the more accurate it should be. Because different labs set their own reference ranges on different populations it is important that you only compare a lab value with its own reference range. Additionally, you can’t always assume that 95% of the population has adequate nutrient levels. For example, some 40% of the population doesn’t meet the recommended intake of magnesium, so simply identifying the cut point of 95% may assume some people have an adequate level when they may not.
References ranges can also be established ranges, like cholesterol, which are generally comparable from one lab to another. Ranges can also be matched to ranges found in the literature or in population studies such as NHANES. Lab values are often identifying what is in a normal range, and do not identify the ‘best’ or ‘ideal’ value. There is also often disagreement among experts as to what an ideal level is, for example, 25-hyroxyvitamin D. The central 95% reference range for serum 25-hydroxyvitamin D in NHANES was 23-124 nmol (9.2-49.6 ng). Though everyone would agree that 9.2, while inside the range of ‘normal’ is certainly deficient. In a review article by Bischoff-Ferrari in 2014 they found serum 25-hydroxy vitamin D at a level of 75 nmol/l (30ng/dl) was the starting level associated with the best health outcomes, in relation to bone mineral density (BMD), lower extremity function, dental health, risk of falls, fractures, cancer prevention, incident hypertension and mortality. [Adv Exp Med Biol. 2014;810:500-25. Optimal serum 25-hydroxyvitamin D levels for multiple health outcomes. Bischoff-Ferrari HA.] Researchers have not settled on an ideal vitamin D level, and the reference range can vary from lab to lab.
What’s the best specimen for what you are looking at?
Sometimes there is an ideal specimen, though often different specimens are identifying different things. It is best to use a specimen type that is based in the literature and correlates to population studies, such as NHANES. Being able to compare nutrient values to population ranges gives you a better idea of where your values falls. Additionally, different specimen types do not always correlate. Urine amino acids can tell you what a person has eaten the last few days, while serum amino acids can tell you more about amino acid status. Whole blood may not be the best way to assess lead levels but it is the way all public health organization check for lead, and if you are checking lead you would be best to check whole blood so you can make comparisons. There are usually a variety of analytes used to assess a nutrient, it is up to you to find the one that is most valid. You should find out how much research has been done correlating a specific specimen type with the specific condition you are evaluating. There are many specimen types and they include:
– RBC -- Urine -- Stool – Whole blood -- Plasma -- Saliva – Finger stick -- Serum - WBC
Laboratory Tests and Dietary Intake
The recommended dietary allowance (RDA) is the amount of a nutrient to meet the needs of 97-98% of the population. It is set based on the Estimated Average Requirement (EAR), which is the amount of a nutrient that will meet the requirement of half the healthy individuals, the median. The RDA is set at the EAR plus twice the standard deviation. In setting the RDAs the Institute of Medicine (IOM) reviewed an array of laboratory assessments. They identify what they choose in determining the population standard as well as review the pros and cons of all the laboratory assessments identified. It does not mean the other markers are not valid they are simply noting what worked best in setting a population standard. I find the easiest way to see what laboratory test the IOM used in setting the RDA for an individual nutrient is to google ‘the nutrient I’m interested in’ plus ‘IOM’.
Here is vitamin B12 as an example: http://www.nap.edu/read/6015/chapter/10
- Summary: The Recommended Dietary Allowance (RDA) for vitamin B12is based on the amount needed for the maintenance of hematological status and normal serum vitamin B12 values.
- Selection of Indicators for Estimating the Requirement for Vitamin B12: Search of the literature revealed numerous indicators that could be considered as the basis for deriving an Estimated Average Requirement (EAR) for vitamin B12 for adults. These include but are not limited to hematological values such as erythrocyte count, hemoglobin concentration or hematocrit, and mean cell volume (MCV), blood values such as plasma B12, and the metabolite methylmalonic acid (MMA).
Discrepancies with Laboratory Assessment and Nutrient Intake
Nutrient intake and laboratory values found to be out of the recommended range, should be noted. If there is a significant discrepancy between laboratory value and intake it can identify an issue with digestion and absorption, or increased need. It’s also important to review what laboratory test was used and how well it correlates to intake. Some correlate better than others. Generally fat-soluble nutrients correlate better with laboratory values. Once I collect a full nutrition assessment and all the laboratory data I run through each of the following areas: Metabolomics/Metabolic, Mind-Body, Immune, Inflammation, Nutrition, Nutrigenomics, Dietoxification and Digestions (MIND2).