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Clinical Case Study |
1 Clinical Laboratory Science Department, Northern Michigan University, Marquette, MI; 2 Department of Laboratory Medicine and Pathology and 3 Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN.
aAddress correspondence to this author at: Mayo Clinic, 200 First Street, Rochester, MN, 55905. Fax 507-284-1399; e-mail mcconnell.joseph{at}mayo.edu.
CASE DESCRIPTION
A 47-year-old woman was referred to us for follow-up of primary biliary cirrhosis (PBC).1 She reported nausea, malaise, weight loss, and new-onset jaundice. The patient had recently had an episode of melena with anemia requiring blood transfusions, but upper endoscopy and colonoscopy did not reveal a bleeding source. She reported a smoking history of 30 years and was currently smoking. The patient’s family history included rheumatoid arthritis, hyperlipidemia, and cardiovascular disease. Her medical history included fibromyalgia and treated Hashimoto thyroiditis.
A diagnosis of PBC had been made elsewhere 5 years previously on the basis of positive antimitochondrial antibody and liver biopsy findings showing stage 1 PBC. The patient was started on ursodeoxycholic acid, but she did not tolerate this medication. Four years later, she remained unable to tolerate ursodeoxycholic acid and was referred to our clinic.
When the case patient presented to our clinic 1 year ago her laboratory studies showed alkaline phosphatase 1416 U/L, aspartate aminotransferase 120 U/L, alanine aminotransferase 81 U/L, total bilirubin 18.9 µmol/L (1.1 mg/dL), direct bilirubin 10.3 µmol/L (0.6 mg/dL), albumin 35 g/L (3.5 g/dL), total cholesterol 17.2 mmol/L (665 mg/dL), triglycerides 2.01 mmol/L (178 mg/dL), HDL cholesterol (HDL-C) 0.67 mmol/L (26 mg/dL), and calculated LDL-C 15.6 mmol/L (603 mg/dL). Ursodeoxycholic acid treatment was recommended, and the patient’s lipids were to be rechecked in 3 months. The patient returned 1 year later, off medication, after the episode of gastrointestinal bleeding. Physical examination showed scleral icterus, jaundice, and sclerodactyly, as well as a palpable liver 2 cm below the costal margin and multiple firm, whitish dermal papules on her left forearm. The most recent laboratory results are shown in Table 1
. Computed tomographic enterography of her abdomen was negative for a bleeding source in the small bowel. In addition, extensive atherosclerotic disease, especially in the aortoiliac vasculature, and focal ectasia in the distal right common iliac artery were noted. Because of the patient’s high LDL-C concentration and family history of atherosclerosis, she was referred for a cardiology evaluation.
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DISCUSSION
laboratory evaluation of increased ldl-c
This patient’s marked elevations in total cholesterol and LDL-C, far above optimal ranges as defined by the National Cholesterol Education Program Adult Treatment Panel III guidelines, were of particular interest. The total cholesterol of 27.4 mmol/L (1060 mg/dL) and LDL-C of 25.9 mmol/L (1002 mg/dL), measured on a Roche-Hitachi modular system, prompted further evaluation. The automated Roche direct method uses a multidetergent enzymatic system for HDL quantification, and LDL-C was determined by use of the Friedewald equation: total cholesterol – (HDL-C + triglyceride/5). We also performed the triglyceride assay with an automated, enzymatic, colorimetric method on the Roche-Hitachi modular system. A cardiovascular risk-marker panel, which included lipoprotein (Lp) electrophoresis, was performed. Agarose Lp electrophoresis (SPIFE® Lp electrophoresis, Helena Laboratories) and polyacrylamide gel electrophoresis (Quantimetrix Lipoprint® LDL Subfractions Kit) were performed (Fig. 1
). Abnormal electrophoresis results prompted additional testing using an alternate method. Ultracentrifugation with β-quantification of LDL-C and HDL-C determined by precipitation of the lower fraction with dextran sulfate and Ca++ was performed (Table 1
). We did not detect Lp(a) or chylomicrons.
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We observed discrepancies between the initial automated lipid concentration measurement results and the concentrations obtained by using the ultracentrifugation method. Ultracentrifugation gave substantially lower, although still markedly increased, LDL-C measurement results compared to the calculated concentration from the Roche-Hitachi instrument. In contrast, ultracentrifugation with precipitation of the lower fraction gave a substantially higher HDL-C concentration. Agarose electrophoresis showed an atypical pattern that did not resolve with dilution (Fig. 1A
).
LDL-C appeared to be present on the agarose gel, but the dark, smearing nature of the gel with slight reverse migration was consistent with the presence of LpX. Neither Lp(a) nor HDL-C was visible on the gel. The electrophoresis findings suggest that the ultracentrifugation result of 5.21 mmol/L (201 mg/dL) for HDL-C was erroneous, but we cannot rule out the possibilities that the HDL-C was in some way modified, resulting in a change in migration, or that HDL-C is not bound to LpX particles. We also performed LDL-C electrophoresis on polyacrylamide gel, followed by densitometry (Fig. 1B
). Although apolipoprotein (apo)B was increased, this was unlikely due to LDL-C, because apoB was not detected on this gel. ApoB is found on chylomicrons (apoB-48), VLDL, IDL, LDL, and Lp(a) but is not reported to be contained on LpX.
DIAGNOSIS: PBC WITH LpX
overview of lPx
Although not uncommonly present in patients with cholestatic liver diseases such as PBC, LpX is seldom discussed or considered by the clinical laboratorian or physician. The term LpX was coined in 1969 by Seidel and colleagues after they demonstrated the presence in patients with PBC of an abnormal Lp that was immunochemically distinct from Lp(a) and Lp(B) (1). Previously, this Lp had been referred to as Lp "d <1.063," based on its density as determined via centrifugation, and OLP (obstruction Lp). LpX is a unique Lp characterized by a vesicular structure that consists of a lipid bilayer of 30–70 nm that encloses an aqueous compartment. LpX has a uniquely high content of unesterified cholesterol and phospholipids and carries apoC, apoE, apoA-I, and albumin but is devoid of the structural protein apoB (2)(3). Although the density of LpX is in the LDL range, the physical size is in the range of VLDL or larger. Like VLDL, LpX is large enough to scatter light and cause visual turbidity. This atypical Lp migrates with β-globulins. The mechanism of formation of LpX remains poorly understood. LpX is also seen in lecithin-cholesterol acyltransferase deficiency and may contribute to nephropathy related to lecithin-cholesterol acyltransferase deficiency (4). Interestingly, LpX is not thought to be very atherogenic. The risk of coronary artery disease does not seem to be increased in patients with PBC (5). Some researchers believe LpX has antiatherogenic properties and may in fact decrease atherosclerotic risk (6).
interaction of lPx with ldl-c and hdl-c measurements
Homogeneous assays for LDL-C are known to be markedly affected by LpX (7)(8). In the case we report, lipid analysis with ultracentrifugation gave substantially lower LDL-C and higher HDL-C concentration results than those obtained from the automated Roche-Hitachi instrument. The ultracentrifugation (modified β-quantification) procedure requires precipitation of LDL in the bottom fraction with dextran sulfate and Ca2+, followed by enzymatic measurement of the remaining HDL-C. We reason that LpX present in the bottom fraction was not fully precipitated along with LDL and thus was measured as HDL-C. The HDL-C concentration determined this way was 4.58 mmol/L (177 mg/dL) higher than that found with the automated Roche direct HDL method. Because LDL-C is determined by subtracting HDL-C from the total cholesterol in the bottom fraction, the LDL-C concentration obtained by ultracentrifugation (740 mg/dL) was lower than the calculated LDL-C from the Roche-Hitachi instrument (1002 mg/dL). By either method, the LDL-C determinations produced inaccurate results when LpX was present at high concentrations, because most of the LDL-C measured is derived from the LpX particles. Because we do not fully understand the composition of the LpX particle and the nature of its interactions with other Lps, we cannot accurately assess LDL status in a patient with increased LpX. Although apoB measurement is one potential option, we cannot rule out the possibility that samples with high concentrations of LpX interfere with the methods for apoB determination.
importance of lPx
It is important for clinical chemists to note that high concentrations of LpX may affect several laboratory tests. In addition to the effects on Lp analyses described here, high LpX may result in pseudohyponatremia (9) and falsely increased laboratory measurement of Lp(a) (10). These effects can have important clinical implications for treatment recommendations.
diagnosis
On the basis of the electrophoresis patterns observed and the diagnosis of PBC, we concluded that the discrepant results in this patient were due to the presence of LpX. Results from the Roche-Hitachi analyzer were released with the comment that LpX was present in the sample and that it accounted for the majority of cholesterol present. The true LDL-C concentration in the sample could not be calculated, owing to the presence of LpX, but both LDL-C and HDL-C appeared to be markedly decreased when the gels were examined.
case resolution
Although LpX is not thought to be atherogenic, this patient had marked atherosclerosis revealed on computed tomographic examination and was referred for a cardiology consultation. The patient was counseled on smoking cessation, and further testing was ordered to assess coronary calcification. Specific antilipemic medications were not thought to be necessary until reassessment of the patient’s lipid status after appropriate treatment and improvement of her cholestasis. At the time of this report the patient was scheduled to return for further laboratory testing after 3 months of therapy with ursodeoxycholic acid.
CONCLUSION
LpX is often present in association with PBC and cholestatic liver disease. In the presence of LpX, lipid and Lp analyses cannot be expected to yield reliable results. HDL-C may be falsely increased or decreased, depending on the method used, and LDL-C may be falsely and markedly increased. It is not common knowledge to either the clinician or the clinical laboratorian that these abnormalities and inaccuracies in measurement are usually the result of LpX. If the presence of LpX is suspected, confirmation by electrophoresis is recommended. In addition to the laboratory abnormalities observed for lipid testing, other laboratory tests may also be affected. Further characterization of LpX and its effects on laboratory testing is warranted.
POINTS TO REMEMBER
Acknowledgments
Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.
Authors’ Disclosures of Potential Conflicts of Interest: Upon manuscript submission, all authors completed the Disclosures of Potential Conflict of Interest form. Potential conflicts of interest:
Employment or Leadership: None declared.
Consultant or Advisory Role: K.D. Lindor, Intercept Pharmaceuticals.
Stock Ownership: None declared.
Honoraria: None declared.
Research Funding: K.D. Lindor, Axcan Pharma.
Expert Testimony: None declared.
Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, preparation or approval of manuscript.
Footnotes
1 Nonstandard abbreviations: PBC, primary biliary cirrhosis; C, cholesterol; Lp, lipoprotein; apo, apolipoprotein. 
References
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