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 6). The presumption that the cat was fasted should be verified, to ensure that all access to food has been withheld. Once fasting hyperlipidemia has been confirmed, the causes of hyperlipidemia secondary to other disorders should be ruled out. If no secondary disorder resulting in hyperlipidemia is evident, then a primary hyperlipidemia 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. Algorithm to determine the cause of hyperlipidemia.

Serum Turbidity

Visual evaluation of the degree of serum turbidity can provide an estimation of serum triglyceride concentration:

• Normal, clear serum: typical triglyceride concentration < 200 mg/dL (2.3 mmol/L)
• Hazy serum: triglyceride concentration around 300 mg/dL (3.4 mmol/L)
• Opacity of the serum: triglyceride concentration approaches 600 mg/dL (6.8 mmol/L)
• Serum with the appearance of skim milk: triglyceride concentration is usually around 1000 mg/dL (11.3 mmol/L)
• Serum with the appearance of whole milk: triglyceride concentration as high as 2500 (28.2 mmol/L) to 4000 mg/dL (45.2 mmol/L)

Refrigeration Test

To ascertain the lipoprotein classes that may be present in excess, a simple refrigeration test can be performed (Figure 7). 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 the chylomicron layer is clear, then only chylomicrons are present in excess, and either a nonfasted 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.

Figure 7. Refrigeration test of hyperlipidemic serum. On the left, a fasting serum sample 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).

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 semiquantify classes of lipoproteins (Figure 8). Lipoprotein electrophoresis should be performed on fresh, not-previously-frozen serum, and the scan interpreted by someone knowledgeable of feline lipoprotein characteristics (i.e., not a human laboratory), since differences exist between humans and cats in electrophoretic pattern. Lipoprotein electrophoresis is not quantitative, but is useful to identify an excess in a particular lipoprotein class.

Figure 8. Densitometric tracing of a lipoprotein electrophoretogram from a normal cat.

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

Depending on the 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 a1-antitrypsin concentration may also be falsely increased (Bossuyt & Blanckaert, 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 & Solter, 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 & Paulson, 1987).