Diagnostic Approach to the Hyperlipidemic Patient

2. Diagnostic Approach to the Hyperlipidemic Patient

When a patient exhibits serum hyperlipidemia after a 10 to 12 hour fast (Figure 5), investigation into the cause is warranted (Figure 7). The presumption that the dog was fasted should be verified to ensure that all food and treats have been withheld. Once fasting hyperlipidemia has been confirmed, hyperlipidemia secondary to other disorders should be investigated. If no secondary disorder resulting in hyperlipidemia can be identified, a primary lipid abnormality should be considered.

Figure 5. The appearance of normal and hyperlipidemic serum. Normal serum should be clear, with no evidence of turbidity (left tube). Fasting serum that is turbid indicates the presence of excess lipid in the serum (right tube).  

Figure 6. Refrigeration test of canine hyperlipidemic serum. On the left, a fasting serum sample from a dog shows hyperlipidemia. After the refrigeration test, there is the appearance of a lactescent layer ("cream layer") floating on top of the serum. This layer is due to increased chylomicron particles present in the serum sample. Note that the serum below the top lactescent layer is also turbid, indicating the presence of other lipoproteins in excess (in addition to the excess chylomicron particles).

Figure 7. Algorithm to aid in the determination of cause of serum hyperlipidemia.

Serum Turbidity

Visual evaluation of the degree of serum turbidity can provide an estimation of serum triglyceride concentration. Normal, clear serum typically has a triglyceride concentration of less than 200 mg/dL, while hazy serum may have a triglyceride concentration of approximately 300 mg/dL. Opacity is seen when triglyceride concentration approaches 600 mg/dL, and if the serum has the appearance of skim milk, the triglyceride concentration is usually around 1000 mg/dL. Serum with the appearance of whole milk can have triglyceride concentration as high as 2500 to 4000 mg/dL.

Refrigeration Test

To ascertain the lipoprotein classes that may be present in excess, a simple refrigeration test can be performed (Figure 6). The serum sample is refrigerated and left undisturbed overnight. Chylomicrons, being the least dense lipoprotein, will "float" forming a "cream layer" on the top of the serum sample (Rogers, 1977). If the serum below is clear, only chylomicrons are present in excess, and either a non-fasted sample, or primary hyperchylomicronemia should be suspected. If the serum below the chylomicron layer is turbid, then other lipoproteins are present in excess in addition to the hyperchylomicronemia. If a "cream layer" does not form after refrigeration, then chylomicrons are not present, and the visible hyperlipidemia is due to an excess of other lipoproteins.

Lipoprotein Electrophoresis

Lipoprotein electrophoresis can be used to characterize lipoproteins in serum. With electrophoresis, lipoproteins separate based on their charge and mobility on agarose gel. The agarose gel is then stained and scanned using a densitometer to semi-quantify classes of lipoproteins (Figure 8). Lipoprotein electrophoresis should be performed on fresh, not-previously-frozen serum, and the scan interpreted by someone knowledgeable of canine lipoprotein characteristics (i.e., not a human laboratory), since major differences exist between humans and dogs in electrophoretic pattern. Lipoprotein electrophoresis is not specific since there is some overlap in electrophoretic migration, but is useful especially for monitoring effectiveness of treatment of lipid abnormalities.

Figure 8. Densitometric tracing of lipoprotein electrophoretogram of a normal dog.

Ultracentrifugation

Ultracentrifugation can be utilized to separate lipoproteins based on density. Ultracentrifugation is time-consuming, requires expensive equipment, and considerable skill to produce reliable results, and is rarely available except in the research setting.

Serum Interferences

Excess of other analytes present in serum may interfere with the measurement of lipids.

- Hyperbilirubinemia may cause a false lowering of cholesterol measurement.
- Hypertriglyceridemia may also result in a falsely lower cholesterol concentration (Cobbaert et al., 1993).
- If cholesterol is present at a concentration of greater than 700 mg/dL, the measured triglyceride concentration may be falsely lowered (Shephard et al., 1990).
- Pentobarbital may also falsely increase triglyceride measurement (Hata et al., 1978), but phenobarbitone has no effect on cholesterol concentration (Foster et al., 2000).

Depending on methodology utilized for analysis, hyperlipidemia may interfere with a number of assays. Hyperlipidemia may result in an approximately 2% increase in sodium, urea, glucose, chloride, and total protein measurement (Miyada et al., 1982). Total calcium measurement may be slightly elevated (Darras et al., 1992), and cortisol may be slightly elevated, but not clinically significant (Lucena et al., 1998). Bilirubin concentration may be falsely increased (Ng et al., 2001), and immunoglobulin A, immunoglobulin M, haptoglobin and α1-antitrypsin concentration may also be falsely increased (Bossuyt et al., 1999). Concentration of LDH is decreased and AST and ALT concentrations are increased (Miyada et al., 1982). Hypertriglyceridemia may interfere with WBC, RBC, hemoglobin and platelet measurements (Peng et al., 2001), and causes a false increase in haptoglobin concentration (Weidmeyer et al., 1996). Glycated hemoglobin measurement may be falsely decreased (Garrib et al., 2003), and free thyroxine measured by ELISA may be increased (Lucena et al., 1998). However, triglyceride concentration up to 1000 mg/dL will not interfere with phenobarbital measurement (Baer et al., 1987).