Non-esterified fatty acids


Non-esterified ("free" or unsaturated) fatty acids (NEFAs) are the major component of triglycerides (the fat stores in the body), which consist of three fatty acids linked to a glycerol backbone. Hydrolysis of stored triglycerides (fat) in adipose tissue by hormone sensitive lipase liberates NEFAs and glycerol. Hormone sensitive lipase (which is found within the cytosol of adipocytes) is stimulated by various hormones, including glucagon (which is released from α-cells in pancreatic islets in response to low glucose). NEFAs can be used as an energy source by many tissues, including skeletal muscle and hepatocytes. In hepatocytes, their fate differs depending on energy needs, hormone balance and substrate availability (see figure below), i.e. they can be used for energy production, re-packaged into triglycerides and exported as very low density lipoproteins (VLDL) or stored within the liver, or converted to ketones.

Lipolysis of fat stored as triglycerides in adipose tissue occurs in response to increasing energy demands that cannot be adequately supplied by glucose. Hormones, such as glucagon, catecholamines, ACTH, corticosteroids and growth hormone, stimulate hormone sensitive lipase, whereas insulin inhibits this enzyme. Lipolysis of triglycerides releases NEFAs (which are usually long-chain fatty acids) and glycerol. Glycerol is taken up by cells and used for glucose production or can be used to re-form triglycerides. NEFAs are water-insoluble and are transported bound to albumin. Once taken up by hepatocytes, NEFAs are esterified. The esterified fatty acids then have several fates:
1) They can recombine with glycerol to form triglycerides, which are packaged into VLDL. The VLDL are exported from the liver or (if produced in excess) are stored as fat within the hepatocyte (eventually causing lipidosis).
2) They can enter the mitochondria (in a reaction that requires carnitine) and be used for energy production (through the Kreb's cycle) or ketone formation. Within the mitochondria, esterified fatty acids undergo β-oxidation to acetyl CoA. Acetyl CoA combines with oxaloacetate in the Kreb's cycle (tricarboxylic acid cycle) to form citrate. Continued oxidation in this cycle leads to energy (ATP) production. If oxaloacetate supplies are low (oxaloacetate is used as a substrate for gluconeogenesis in states of negative energy balance), acetyl CoA is then used to form ketones.

Low concentrations of NEFAs are found in the blood of healthy animals. Increased concentrations indicate breakdown of fat (lipolysis), which occurs in response to increased energy demand. Thus, NEFAs are considered a biomarker of negative energy balance, where the supply of glucose is insufficient to meet energy needs. Negative energy balance can be detrimental because it predisposes animals to hepatic lipidosis (excess NEFAs are stored as triglyceride within hepatocytes) and ketosis. In veterinary medicine, NEFAs are mostly used for energy metabolite assessment of periparturient (transition) dairy cows and for detecting negative energy balance in camelids (llamas and alpacas), both of which are predisposed to hepatic lipidosis. NEFAs can be measured in small animals and are increased in states of negative energy balance (anorexia, inappetance) or where there is increased lipolysis (diabetes mellitus), however testing is rarely performed in these species.

Sample collection guidelines for transition cow energy metabolite assessment

  • Collection tube: A red top (non-anticoagulant) or purple top (EDTA anticoagulant) tube is recommended for sample collection. Serum/plasma should be separated from cells ASAP after collection and the serum.plasma placed in a separate tube. Collection tubes containing heparin (green top) should be avoided because NEFA values increase rapidly in these samples with storage. Similarly, corvac (serum-separator) tubes are not recommended, because values are slightly (but significantly) higher in these tubes compared to non-anticoagulant (red top) tubes.
  • Time of collection: Sample cows as they are coming into the feeding stalls (see below).
    • Prepartum NEFAs: Collect samples from cows 2-14 days before calving.
    • Postpartum NEFAs: Collect samples from cows 3-14 days in milk. Measurement of both postpartum NEFAs and BHB is performed in a transition cow energy profile for assessment of energy status in lactating cows at the Clinical Pathology Laboratory in Cornell University.
  • Sample storage: Samples MUST be kept cool to minimize false changes in results (NEFAs are particularly unstable). Submit ASAP to the laboratory. For more information, see below.
  • Number of cows: A minimum of 12 animals per herd should be sampled for herd level testing. This can be a mixture of heifers and >2 parity cows.
  • Pooled samples: Pooling of individual samples from cows to assess energy status of a herd is NOT recommended. Studies at Cornell University have shown that results from pooled samples are insensitive when using prepartum or postpartum NEFA for the detection of excessive negative energy balance in transition dairy cows.


Measurement of NEFAs

Critical care should be paid to sample collection and handling when measuring NEFA concentrations. The following factors impact NEFA values, independently of negative energy balance:

  • Hemolysis: Depending on the method used by the laboratory, hemolysis will either decrease or increase NEFA values. In the method used at Cornell University, severe hemolysis (hemolytic index > 800 units) will mildly decrease NEFA concentrations in bovine blood.
  • Storage: NEFA values in bovine blood are stable for 24 hours as whole blood and for 72 hours as separated serum or EDTA plasma, as long as the samples are kept cool. NEFA values increase steadily at room temperature in all sample types. NEFA concentrations are also stable in serum stored frozen for one month in a dedicated freezer at or below -40C.
  • Excitement/exercise/stress: These conditions, which induce catecholamine or ACTH release, increase NEFA concentrations by stimulating lipolysis through hormone sensitive lipase. These conditions should be minimized when collecting blood samples for NEFA measurement.
  • Timing of sample collection: NEFA values are likely to be higher in cows that are coming in for daily feeding (likely to be in some degree of negative energy balance) versus those cows which have just been fed (lipolysis will be inhibited in these latter cows). Collection just before feeding is recommended.


Negative energy balance in transition dairy cows

Dairy cows in the periparturient (transition) period are always in a state of negative energy balance due to high energy demands from the developing fetus and milk production (particularly with the emphasis on selection for high milk-producers). However, this state of negative energy balance can be excessive and affected cows are at risk of gastrointestinal (displaced abomasum), metabolic (clinical ketosis), and infectious (e.g. metritis) diseases in the early postpartum period. Thus, dairy practitioners frequently monitor dairy herds for excess negative energy balance by testing for NEFAs, either alone in pre- or postpartum cows or as a component of a metabolic profile in postpartum cows. Results of these tests can be interpreted at the individual cow level (i.e. a NEFA value above a certain cut-off indicates excess negative energy balance) or at the herd level (i.e. a proportion of tested cows have NEFA values over a certain cut-off value). Identification of excess negative energy balance in individual cows (and more importantly) in the herd indicates the need for changes in nutrition (e.g. increase bunk feed space, increase energy density of ration) and transition cow management to decrease energy demands and stresses on transition cows.

The following intepretation guidelines are based on studies done at Cornell University and are valid for samples collected from 'at risk' TMR-fed cows between 2-14 days precalving (prepartum NEFAs) or 3-14 days post-calving (postpartum NEFAs). We recommend sampling at least 12 'at risk' cows when evaluating total mixed ration (TMR)-fed herds for subclinical ketosis.

Cow level testing

  • Prepartum NEFAs: There is an increased incidence of postcalving diseases (displaced abomasum, metritis/retained placenta and clinical ketosis), decreased milk yield and
    decreased reproductive performance in the first 30 days in milk in Holstein dairy cows (fed TMR) with NEFA values > 0.30 mEq/L when tested 2-14 days before calving.
  • Postpartum NEFAs: There is an increased incidence of postcalving diseases (displaced abomasum, metritis/retained placenta and clinical ketosis), decreased milk yield and
    decreased reproductive performance in the first 30 days in milk in Holstein dairy cows (fed TMR) with NEFA values > 0.60-0.70 mEq/L when tested 3-14 days after calving. In the Cornell studies, postcalving NEFAs were actually a better predictor of than postcalving β-hydroxybutyrate concentrations or precalving NEFAs.

Herd level testing

  • Prepartum NEFAs: At the herd-level, there is a significantly increased risk of post-calving metabolic and infectious diseases, decreased milk production or decreased reproductive performance if >15% of tested precalving cows have NEFA values > 0.30 mEq/L. Note, that as indicated above, pooling samples from individual cows is not recommended for herd-level testing.
  • Postpartum NEFAs: At the herd-level, there is a significantly increased risk of post-calving metabolic and infectious diseases, decreased milk production or decreased reproductive performance if >15-20% of tested postcalving cows have NEFA values > 0.70 mEq/L. Note, that as indicated above, pooling samples from individual cows is not recommended for herd-level testing.

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