Determination of Total Cholesterol in Hypertriglyceridemic Serums

MICROCHEMICAL JOURNAL ARTICLE NO.

59, 315–322 (1998)

MJ981579

Determination of Total Cholesterol in Hypertriglyceridemic Serums1 B. R. Morgan,* J. D. Artiss,† and B. Zak†,‡,2 *Department of Pathology, Carle Clinic Associates, Champaign, Illinois; †Department of Pathology, Wayne State University School of Medicine, Detroit, Michigan; and ‡Department of Pathology, Detroit Medical Center, Detroit, Michigan Received December 15, 1997; accepted January 4, 1998 The determination of serum total cholesterol by a direct reaction involving a Trinder-type enzymic pathway can be severely hampered by the turbidity caused by excessive concentrations of triglyceride-rich lipoproteins such as very-low-density lipoproteins and chylomicrons. This paper describes a clarification procedure that effectively removes such turbidity by a process involving lipolysis of triglycerides and entrapment of the liberated nonesterified fatty acids by alpha cyclodextrin (a-CD) as a transparent host– guest complex. Many elevated triglyceride conditions can be treated by this enzymic clarification procedure as exemplified by spectral investigations. It may still be necessary to determine triglycerides in order to estimate and correct for the dilution of the serum by large lipoproteins containing the triglycerides. The cloudy or milky appearance of a fasting serum suggests that triglyceride determination probably should be performed in any case simply because the appearance of the specimen provides this clue to such an abnormality. © 1998 Academic Press

INTRODUCTION Current technology involving enzymic reagents for the determination of serum cholesterol without any pretreatment of the sample can be handicapped by the light-scattering caused by the turbidity of severe hypertriglyceridemia. Our results indicate that such assays are subject to interference because the light-scattering problem cannot be handled well by current procedures for removing the turbidity caused by excessive concentrations of triglycerides. In fact, any analyte that is determined spectrophotometrically by a direct reaction of serum with reagent could be problematic for accurate assay when the sample is severely turbid. Ultracentrifugation cannot be used to clear samples for the determination of cholesterols because this process of clarification results in the loss of part of the total cholesterol. Extraction is obviously not tenable unless the determination is carried out on the extract itself as in the Abell et al. procedure (1). However, this procedure is no longer suitable, if it ever was, as an approach for the routine hospital determination of cholesterol, and furthermore, it is not amenable to automation. Clarification by detergent treatment is probably limited to relatively low concentrations of triglyceride. Enzymic clearing can overcome much of the turbidity problem, even though it may still be necessary to determine triglyceride present in order to correct for the dilution of the serum by voluminous lipoproteins containing the triglycerides. The purpose of the present report is to demonstrate the effect on cholesterol measurements of interference by elevated levels of triglyceride. We will also elucidate the 1 2

We thank the Carle Clinic Foundation for support of this project. To whom correspondence should be addressed. 315 0026-265X/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.

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rationale behind the enzymic clearing process. The only difference in reagent and procedure between the treated and the uncleared samples will be the incorporation of the clearing ingredients into the reagent. Since no sample treatment is necessary and no additional procedural steps are required, this system is ideally suited to automation. MATERIALS AND METHODS Apparatus A Beckman DU-640 scanning spectrophotometer equipped with thermostated cuvettes (Beckman Instruments, Inc., Fullerton, CA) was used throughout the study. Chemicals Total cholesterol reagent was obtained from Beckman Instruments, Inc. (Brea, CA). Cholesterol, alpha-cyclodextrin, and 2-methoxyethanol were obtained from Sigma Chemical Co. (St. Louis, MO). Liposyn III intravenous lipid emulsion was obtained from Abbott Laboratories (North Chicago, IL) to be used as the triglyceride contaminent. Lipase (glycerol ester hydrolase) was obtained from Genzyme Diagnostics (Cambridge, MA). Reagents Cholesterol reagent. A Beckman one-piece total cholesterol reagent was prepared by mixing reagent A and reagent B in a 29:1 ratio (working reagent) as recommended by Beckman reagent instructions. Modified cholesterol reagent. Beckman one-piece total reagent was modified for clearing triglycerides by dissolving the clearing components to give the following concentrations per liter of working reagent: lipase 443 kU, alpha-cyclodextrin 4 g. Triton X-100, a required clearing component, is already present in the commercial reagent at a concentration of 3.3 g/L. Cholesterol standards. A 6000 mg/L cholesterol standard was prepared by dissolving 300 mg of cholesterol in 2-methoxyethanol in a 50-mL volumetric flask. Serial dilution with 2-methoxyethanol give standards of 3000, 1500, and 750 mg/L. Triglyceride standards. Triglyceride standards were prepared with deionized water by diluting Liposyn III to give triglyceride concentrations ranging from 10 to 40 g/L. Procedure Twenty microliters of sample were mixed with 2 mL of working reagent and incubated at 37°C for 10 min. The absorbance was measured at 498 nm against a reagent blank. Procedure with Sample Blanking Twenty microliters of sample were treated as under Procedures above. A sample was prepared by mixing 20 microliters of sample with 2 ml of Reagent A, modified to contain lipase and a-CD. The mixture was incubated at 37°C for 10 min and its absorbance measured at 498 mm against a reagent blank. RESULTS AND DISCUSSION It has recently been pointed out that interference caused by lipemia, exemplified by hypertriglyceridemia, is an ‘‘underrated problem’’ of the clinical laboratory (2). This

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source of perplexity is undoubtedly exacerbated by the increased use in assay methodology of direct approaches in which untreated serum is added directly to a reagent or reagents. That problem was obviated earlier in clinical chemistry history by dialysis, obviously autoanalyzer technology (3) limited to diffusibles, or by the manual off-line preparation of protein-free filtrates (4). At present, a sample blank (5), a kinetic approach (6), or bichromatics (7) could aid in eliminating or minimizing the interference. The use of detergent clarification is also a limited adversary to interference although a time factor could create a side effect that might cause an even more significant error if the method were improperly designed. An example involves centrifugal assays with the blank read early in the process (8) followed by some clearing by detergent action during the time it takes to bring the desired reaction to equilibrium. This results in a final absorbance reading that is too low as the turbidity has been reduced by the detergents. All turbidity problems cannot easily be solved by reagent makeup. However, a number of procedures involving hypertriglyceridemia are amenable to enzymic treatment by changing the character of the reagent, allowing the turbidity caused by the sample to be eliminated or at least greatly minimized, thereby providing essential clarity for spectrophotometric measurement (9 –12). However, enzymic clarification only solves one of the two important problems caused by severe hypertriglyceridemia. These two problematic circumstances involve light-scattering owing to the turbidity of the serum analytes and their dilution by the voluminous chylomicrons or very-low density lipoproteins that are the source of the turbidity. Thus a diluted sample in which the desired analyte concentration is low owing to hypertriglyceridemia can still create a higher-than-true answer because of the light-scattering caused by the turbidity of the specimen even though the true answer in the diluted specimen is actually lower than it would be if the dilution phenomenon were accounted for and corrected mathematically (13). Therefore, an anomolous situation exists with a hypertriglyceridemic specimen, which causes all analyte concentrations to fall into a pseudohypo classification because of a dilution effect. Ironically, the concentrations can be and usually are distorted pseudohyper concentrations by light-scattering. Nevertheless, even if the answer obtained for the serum could be absolutely accurate in terms of assay measurement mechanics, a mathematical correction to overcome the dilution effect would still be required in order to change that answer to a clinically useful one (14). Of the several processes proposed to minimize or eliminate turbidity from lipemia, including ultracentrifugation, chemical precipitation, detergent treatment, extraction, chromatography, and enzymic clearing (9 –12), with the possible exception of detergent treatment, only enzymic clearing is currently amenable to incorporation into automated instrumentation and furthermore does not otherwise alter the methodological treatment of a sample. Like many photometric determinations, Trinder type endpoint methods for total cholesterol are susceptible to interference from turbidity. Beckman total cholesterol reagent is typical of this type of reagent, and because it is our routine method, it was chosen for this study. This procedure is based on the use of 4-aminoantipyrene and phenol (11) to react with the hydrogen peroxide generated under catalysis by peroxidase to produce a quinoneimine product whose absorbance is measured. The enzymic clearing technique, previously applied to a number of analytes, has been discussed, but will be briefly summarized (11). Lipase and a-CD, a scavenging agent for fatty acids in this

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FIG. 1. Panel A shows calibration spectra of cholesterol standards (a– d) with unmodified reagent concentrations of 750, 1500, 3000, and 6000 mg/L. Panel B shows near superimposable calibration spectra (a1– d1) cholesterol standards of left panel reacted with modified reagent containing a-CD and lipase.

circumstance, are incorporated into the reagent. In the presence of a detergent such as Triton X-100, which serves as an activator of lipase and an interface between enzyme and triglyceride, lipase cleaves the triglyceride into glycerol and free fatty acids. The liberated fatty acids, which themselves may form soaps that might precipitate, giving rise to a second turbidity, instead form transparent inclusion complexes with a-CD during this process of clarification. Figure 1A shows the spectra of cholesterol standards graphed after the use of unmodified reagent as provided by the manufacturer when a 20-mL sample was incubated with 2 mL of reagent for 10 min at 37°C and then measured against a reagent blank to achieve an absorbance maximum at 498 nm. Figure 1B shows the spectra of the same cholesterol standards, now obtained using modified reagents containing the clearing components. The curves are identical, resulting in a linear and reproducible calibration curve, as is found in both panels of the figure. It can be seen from these superimposed graphs that the cholesterol reaction is not affected by the presence of the clearing components in the reagent. The potential interference of lipemia was then studied by spiking cholesterol standards with triglyceride using Abbott Liposyn III intravenous lipid emulsion as the source. This substance is considered to provide a reasonable simulation of lipemia in such a perturbed sample with respect to spectrophotometry. Figure 2 demonstrates the degree of distortion caused by varying degrees of lipemia. Panel 3A was generated by spiking a group of 1500 mg/L cholesterol standards with Liposyn III ranging from 10 g/L for spectrum b to 40 g/L of triglyceride for spectrum e in increasing additions of 10 g/L. Samples were incubated with unmodified reagent which did not contain clearing components. Spectrum a was graphed using an uncontaminated standard. With increasing lipemia, there is increasingly severe distortion of spectrum a, more exaggerated at the ultraviolet end of the spectrum.

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FIG. 2. Panel A shows spectrum a of a standard cholesterol containing 1500 mg/L versus distorted spectra (b– e) of the same standard to which 10.0, 20.0, 30.0, and 40.0 g/L of triglyceride was preadded. Panel B shows distorted spectra of cholesterol standards of 750, 1500, 3000, and 6000 mg/L (f–i) to each of which 40 g/L of triglyceride was preadded.

Indeed, at 40 g/L, spectrum e, the 1500 mg/L standard would result in an apparent cholesterol concentration of 6600 mg/dL. Panel 2B shows the spectra graphed from several cholesterol standards each contaminated with triglyceride set at 40 g/L. By comparison to the spectra of the uncontaminated standards shown in Fig. 1, the extreme distortions of all the spectra are clearly demonstrated. The spectral distortion is greater at the ultraviolet end of the spectrum, as would be expected from the relationship of light-scattering and wavelength. In addition, it can also be seen here that as the absorbance from increasing concentrations of analyte rises there is less distortion because the scatter effect is measured by the inverse relationship of absorbance to scatter. Figure 3 shows spectra of triglyceride standards alone incubated with unmodified reagent, which, in the most simplistic view, approximates the lipemic component of the spectrum of the triglyceride-contaminated cholesterol standards of panels A and B of Fig. 2. As the optimum concentrations of lipase, a-CD, and Triton X-100 have been previously discussed in detail, this cholesterol reagent was arbitrarily modified to contain lipase concentration set at 443 kU/L, plus a-CD at 4 g/L, whereas Triton X-100, already present in the reagent at 3.3 g/L, was not changed. From previous reports, increasing the lipase concentration can achieve faster clearing, albeit at greater cost. Little improvement in clearing is achieved with higher alpha-cyclodextrin concentrations, as high concentration may lead to increased absorbance (15). Triton X-100 concentration, at 3.3 g/L, is considerably higher than the 0.5 g/L recommended by previous reports, but did not appear to hinder the rapidity of clearing by reagent. As the instrument protocol necessitates incubation of reagent and sample for 10 min for equilibrium of the color reaction, complete clearing of lipemic samples within this time frame was required. Figure 4 shows the clearing of triglyceride standards using the modified reagent. In

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FIG. 3. Light-scattering spectra (a– d) of triglyceride concentrations of 10.0, 20.0, 30.0, and 40.0 g/L in a milieu of unmodified cholesterol reagent.

panel A, each standard was incubated in the spectrophotometer and monitored for 12 min at 37°C and 498 nm, the wavelength of measurement of the quinoneimine product. Triglyceride standards as high as 40 g/L were cleared in the 10-min time period, with no subsequent rise in absorbance seen up to 12 min. However, when the triglyceride

FIG. 4. Panel A shows absorbance versus time curves for 10, 20, 30, and 40 g/L of triglyceride (a– d) cleared by modified cholesterol reagent during a total time period of 12 min. Absorbance-time period for 60 g/L triglyceride is shown by curve e. Panel B shows effectively cleared spectra (f–i) of identical standards of distorted spectra of Fig. 3 (panel B) subjected to treatment by lipase and a-CD in the modified reagent.

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FIG. 5. Comparison of clear, triglyceride-contaminated, and enzymatically cleared serums. Samples 1– 6 were not serum-blanked but samples 7–11 were.

concentration was increased to 60 g/L, demonstrated by time study e, there was initial clearing followed by a modest rise in absorbance at 12 min. This can be seen when fatty acid inclusions with a-CDs precipitate and may explain this finding. Thus, it appears that 40 g/L of triglyceride was the limit for complete clearing of samples for this reagent. Panel B of Fig. 4 shows the contaminated calibration standards of Fig. 2B when treated with the modified reagent containing lipase and a-CD. These compare favorably to those of panels A and B of Fig. 1. The degree of interference with cholesterol concentrations that could be caused in serum samples was studied by contaminating clear serum samples with Liposyn III to give triglyceride concentrations ranging from 20 to 40 g/L. These samples are only an approximation of true lipemia from chylomicrons; however, they do show significant turbidity. The triglyceride concentrations of all serum samples prior to contamination by lipid emulsion were less than 2 g/L, that is, normal for humans. The cholesterol concentration was first determined for the clear serum sample using reagents without clearing components. The cholesterol concentrations were then determined on the same but now contaminated samples using modified reagents containing the clearing components. The results are summarized graphically in Fig. 5. There is a dramatic elevation of the apparent cholesterol concentration when samples are made lipemic. Some samples (7–11) were serum-blanked, whereas the others were not. In both circumstances, the original cholesterol concentrations of the serums as determined with modified reagent are very close to

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the uncontaminated concentrations. Samples 5 and 6 are the same sample, but differ in the concentration of triglyceride added. It seems reasonable to assume that serum clarification of hypertriglyceridemic serums can be achieved by reaction with modified reagents reinforced with a-CD and lipase as described here. These additions result in clearing by allowing lipolysis of the turbiditycausing triglycerides with subsequent entrapment of liberated nonesterified fatty acids into a transparent, soluble guest– host complex. It may also be reasonable to conclude that hypertriglyceridemia, which is known to dilute electrolytes (14), also dilutes the other analytes of serum including cholesterol. Therefore, corrective mathematical treatment can be easily applied to overcome this phenomenon of short sampling. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

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