Measurement of cholesterol concentrations of major serum lipoprotein classes in haemodialysis patients by anion-exchange chromatography

This version was published on 1 November 2008

Ann Clin Biochem 2008;45:571-574
doi:10.1258/acb.2008.008018
© 2008 Association for Clinical Biochemistry

 

This Article
Right arrow
Abstract

Freely available
Right arrow
Figures Only
Right arrow

Full Text (PDF)

Right arrow
All Versions of this Article:

acb.2008.008018v1

45/6/571

most recent

Right arrow
Alert me when this article is cited
Right arrow
Alert me if a correction is posted
Services
Right arrow
Email this article to a friend
Right arrow

Similar articles in this journal

Right arrow

Similar articles in Web of Science
Right arrow
Similar articles in PubMed
Right arrow
Alert me to new issues of the journal
Right arrow
Download to citation manager
Right arrow
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow
Articles by Hirowatari, Y.
Right arrow
Articles by Miida, T.
Right arrow Search for Related Content
PubMed
Right arrow
PubMed Citation
Social Bookmarking

What’s this?

Original Articles


Yuji Hirowatari1,
Hiroshi Yoshida2,
Yuriko Fueki3,
Masayuki Ito3,
Yutaka Ogura1,
Nobuyuki Sakurai4 and
Takashi Miida5


1 Bioscience Division, Tosoh Corporation, Kanagawa, Japan;
2 Department of Laboratory Medicine, Jikei University Kashiwa Hospital, Chiba, Japan;
3 Graduate School of Medical and Dental Sciences Medicine, Niigata University, Niigata, Japan;
4 Murakami Memorial Hospital, Niigata, Japan;
5 Department of Clinical Laboratory Medicine, Juntendo University Hospital, Tokyo, Japan


Corresponding author: Dr Yuji Hirowatari. Email: hirowata{at}tosoh.co.jp




Abstract

Go to previous sectionTop

 Abstract
Go to next sectionIntroduction

Go to next sectionMethods

Go to next sectionResults

Go to next sectionDiscussion

Go to next sectionGRANT/FUNDING SUPPORT

Go to next sectionACKNOWLEDGEMENTS

Go to next sectionREFERENCES

Background: Increased triglyceride (TG)-rich lipoproteins and decreasedHDL that are implicated in the progression of atheroscleroticvascular diseases, are present in serum samples of patientsundergoing haemodialysis (HD) therapy. Therefore, it is importantto measure serum TG-rich lipoprotein concentrations to preventthe diseases.

Methods: The cholesterol concentrations of lipoprotein classes in serum samples from the HD patients (n = 18) and healthy subjects (n= 18) were analysed by our recently developed method of high-performanceliquid chromatography (HPLC), in which the lipoprotein classeswere separated using an anion-exchange column, and the cholesterolconcentrations of each of those were measured enzymaticallyusing a post-column reaction. The ability of fractionated lipoproteincholesterol determination by this HPLC method is mostly equivalentto the determination ability of an ultracentrifugation (UC).

Results: HDL, LDL, and TG-rich lipoproteins, i.e. IDL, VLDL and chylomicrons, were well separated in the chromatograms. HDL cholesterol concentrations in the HD patients were significantly lower than in the healthy subjects (P < 0.0001), and IDL cholesterol concentrations and VLDL cholesterol concentrations in the HD patients were significantly higher than in the healthy subjects (P < 0.05).Profiles of these measured lipoprotein values were consistentwith the previously reported lipoprotein values, measured ultracentrifugallycharacteristic of HD patients.

Conclusion: These results suggest that the HPLC method may be sufficientlyapplied to the assessment of serum lipoprotein profile in HDpatients in place of the other method including an UC.





Introduction

Go to previous sectionTop

Go to previous sectionAbstract

 Introduction
Go to next sectionMethods

Go to next sectionResults

Go to next sectionDiscussion

Go to next sectionGRANT/FUNDING SUPPORT

Go to next sectionACKNOWLEDGEMENTS

Go to next sectionREFERENCES

 

Patients undergoing haemodialysis (HD) therapy over a long term are susceptible to premature cardiovascular diseases.1 It is also known that cardiovascular disease is a major cause of mortality in HD patients.2,3 HD patients have a characteristic dyslipidaemia typified by hypertriglyceridaemia.46 Serum lipoprotein fractions of HD patients with dyslipidaemia is characterized by increased concentrations of VLDL and intermediate-density lipoproteins (IDL), mostly normally ranged concentrations of LDL and decreased HDL.4,5 The increased triglyceride (TG)-rich lipoproteins and decreased HDL are implicated in the progression of atherosclerotic cardiovascular diseases.46 Therefore, it is important to monitor the concentrations of TG-rich lipoproteins in HD patients to prevent cardiovascular diseases. However, TG-rich lipoproteins in HD patients are basically analysed using an ultracentrifugation (UC) method, which is time-consuming and laborious. It is known that the other methods, including gel-permeation chromatography and electrophoresis, have a poor ability to separate heterogeneous TG-rich lipoproteins.7,8 We have recently developed a convenient method for analysing serum fractionated lipoprotein classes by high-performance liquid chromatography (HPLC) with an anion-exchange column, which has a high ability to separate TG-rich lipoproteins, IDL, VLDL and chylomicron.9 In our developed HPLC method, cholesterol concentrations of HDL, LDL, IDL, VLDL and chylomicron were highly correlated with those estimated by the UC method.9 Therefore, we attemptedto measure cholesterol concentrations of TG-rich lipoproteinclasses in HD patients using the HPLC method.





Methods

Go to previous sectionTop

Go to previous sectionAbstract

Go to previous sectionIntroduction

 Methods
Go to next sectionResults

Go to next sectionDiscussion

Go to next sectionGRANT/FUNDING SUPPORT

Go to next sectionACKNOWLEDGEMENTS

Go to next sectionREFERENCES

 

The study samples were obtained from HD patients (n = 18) and the same number of age-matched healthy subjects after a 12 hour-overnight fast. Their clinical characteristics were shown in Table 1. The HD patients included subjects with diabetes mellitus (n = 11) and chronic glomerulonephritis (n = 7), and were undergoingthrice-weekly three- to four-hour HD therapy using high-fluxpolysulphone dialysis membranes (Toray Medical Co., Tokyo, Japan)and standard heparin doses for anti-coagulation.



View this table:
[in this window]
[in a new window]
Table 1 Characteristics and lipoprotein profiles of haemodyalysis patients and healthy subjects

 

The column size and the flow rates in the HPLC method reported previously9 was partially modified, and used for measuring cholesterol concentrations of serum lipoprotein classes (HDL, LDL, IDL, VLDL and chylomicron) The anion-exchange column, which contained 2.5 µm of non-porous polymer-based gel with diethylaminoethyl ligands, and 3.0 mm ID x 25 mm in size, and a post-column reactor, which contained an enzymatic cholesterol reagent (TCHO-CL, Serotec Co., Hokkaido, Japan), were used for the modified HPLC method. The flow rates of eluent and the enzymatic cholesterol reagent were 0.5 mL/minute and 0.2 mL/minute, respectively. It took 23 minutes to complete the assay of one sample. Serum sample (4.5 µL) was injected to the column, and lipoprotein classes in the sample were eluted by a step-gradient of perchlorate ion concentrations in order. Serum sample (500 µL) was mixed with 250 µL of the stock solution (sucrose 760 g/L, EDTA2K 1.5 g/L), and stored at –40°C until measured. The stored samples were analysed within one month. The coefficient data of variation in between-day assay (n = 10) and within-day assay (n = 10) using hyperlipidaemic serum samples by this HPLCmethod were as follows: HDL cholesterol 3.2 and 1.8%, LDL cholesterol3.2 and 2.0%, IDL cholesterol 7.3 and 3.4%, VLDL cholesterol5.0 and 3.3% and chylomicron cholesterol 7.5 and 6.1%, respectively.

Total cholesterol, TG, and creatinine concentrations were measuredenzymatically with commercially available kits (Daiichi PureChemical Industries, Tokyo, Japan). Apolipoprotein concentrationswere measured by immuno-nephelometry with commercial kits (DaiichiPure Chemical Industries).

Results were expressed as mean ± SD. The comparative significance of variables between the different groups was assessed with an unpaired Welch’s t-test. A value of P < 0.05 wasconsidered statistically significant.





Results

Go to previous sectionTop

Go to previous sectionAbstract

Go to previous sectionIntroduction

Go to previous sectionMethods

 Results
Go to next sectionDiscussion

Go to next sectionGRANT/FUNDING SUPPORT

Go to next sectionACKNOWLEDGEMENTS

Go to next sectionREFERENCES

 

Figure 1 shows the typical chromatograms of serum samplesfrom the two HD patients and the healthy subject. In the chromatograms,HDL, LDL, IDL, VLDL and chylomicron were sufficiently separated.



View larger version (12K):
[in this window]
[in a new window]
Figure 1 Chromatograms in serum samples from haemodialysis (HD) patient and healthy subject. Part figures a, b, and c are chromatograms of a healthy serum (a) and serum samples of two HD patients’ (b and c), respectively. The basic data of sex, age and cholesterol concentrations of HDL, LDL, IDL, VLDL and chylomicrons were as follows: (a) male, 65, 1.58 mmol/L, 3.06 mmol/L, 0.305 mmol/L, 0.148 mmol/L and 0.031 mmol/L; (b) male, 62, 0.94 mmol/L, 2.83 mmol/L, 0.506 mmol/L, 0.344 mmol/L and 0.039 mmol/L; (c) female, 61, 1.12 mmol/L, 2.23 mmol/L, 0.393 mmol/L, 0.155 mmol/L and 0.144 mmol/L, respectively

 

Body mass index, total cholesterol and apolipoprotein A1 of the HD patients were significantly lower than those of age- and sex-matched healthy subjects (P < 0.05, P < 0.05 and P < 0.0001, respectively), while systolic BP, creatinine and TG of the HD patients were significantly higher than those of the healthy subjects (P < 0.001, P < 0.0001 and P < 0.001, respectively) as shown in Table 1. In the HPLC analysis, HDL cholesterol concentrations of the HD patients were significantly lower than those of the healthy subjects (P < 0.0001), and IDL and VLDL cholesterol concentrations of the HD patients were significantly higher than those of the healthy subjects (P < 0.05) (Table 1).





Discussion

Go to previous sectionTop

Go to previous sectionAbstract

Go to previous sectionIntroduction

Go to previous sectionMethods

Go to previous sectionResults

 Discussion
Go to next sectionGRANT/FUNDING SUPPORT

Go to next sectionACKNOWLEDGEMENTS

Go to next sectionREFERENCES

 

HDL, LDL, VLDL and chylomicron in human serum samples can be well separated using gel-permeation chromatography or agarose-gel electrophoresis, but IDL cannot be sufficiently separated.7,8 Therefore, we conducted the separation of lipoprotein classes in human serum samples by the HPLC method with the anion-exchange column, and two HDL peaks, one broad LDL peak, one small IDL peak and one broad VLDL peak can be insufficiently separated in a hyperlipidaemic serum using the anion-exchange chromatography with 0–155 mmol/L linear gradient of sodium perchlorate (data not shown). Hence, we established the novel anion-exchange chromatographic method with a step-gradient of sodium perchlorate concentration for the sufficient separation of HDL, LDL, IDL, VLDL and chylomicron in human serum.9 The present study attemptedto analyse lipoprotein profiles of HD patients using the HPLCmethod.

In the present study, HDL cholesterol concentrations were significantly lower, and IDL and VLDL cholesterol concentrations were significantly higher in the HD patients than age- and sex-matched control subjects. In addition, LDL cholesterol concentrations were modestly but insignificantly lower in the HD patients compared with the control subjects. Using the UC method, Shoji et al.4,5 reported that HDL and LDL cholesterol concentrations were significantly lower, and IDL and VLDL cholesterol values were significantly higher in HD patients than in control subjects. Quaschning et al.6 reported that when compared four groups of HD patients(HD-NTG group, normotriglyceridaemic HD patients without diabetesmellitus; HD-HTG group, hypertriglyceridaemic HD patients withoutdiabetes mellitus; HD-NIDDM-NTG group, normotriglyceridaemicHD patients with non-insulin-dependent diabetes mellitus; HD-NIDDM-HTG,hypertriglyceridaemic HD patients with non-insulin-dependentdiabetes mellitus) with control subjects, HDL cholesterol concentrationswere significantly lower in HD-HTG and HD-NIDDM-HTG groups thanin control subjects, LDL cholesterol concentrations were similarlycalculated between the four groups of HD patients and controlsubjects, and VLDL cholesterol concentrations were significantlyhigher in HD-HTG, HD-NIDDM-NTG and HD-NIDDM-HTG groups thanin control subjects, revealed by the UC method.

In the UC method, 200 µL of plasma was mixed with the appropriate density solution with potassium bromide, and the sample was spun in three hours.4,5 The HPLC method required only a small volume of sample (4.5 µL) to determine thecholesterol concentrations of major lipoprotein classes. Inaddition, the assay time of one sample was merely 23 minutesin the HPLC method, so that subsequently any alteration of samplelipoproteins may be little found during the course of lipoproteinseparation in the HPLC assay in contrast to the UC method whichmay have the possibility of altering lipoproteins because ofrequiring long assay times.

Recently, non-HDL cholesterol (total cholesterol – HDL cholesterol) has been proposed as a risk marker for cardiovascular diseases.10,11 Several epidemiologic studies have shown that non-HDL cholesterol is a better predictor of cardiovascular events than LDL cholesterol, and that non-HDL cholesterol is a potential predictor of risk for carotid atherosclerosis in the elderly.1214 Nishizawa et al.15 have reported that non-HDL cholesterol was a significant and independent predictor of cardiovascular mortality in HD patients. Precise measurement of non-HDL cholesterol may be more useful in HD patients with elevated TG, because HD patients have normal concentrations of LDL cholesterol in contrast to increased TG-rich lipoproteins. In HD patients, IDL and VLDL cholesterol presumably account for major part of non-HDL cholesterol. Furthermore, several studies have reported that increased concentrations of VLDL and IDL were associated with the increased cardiovascular risk.1621 Koba et al.16 reported that an increased number of VLDL particles was strongly associated with coronary heart disease in men. Tatami et al.17 also reported that moderately increased concentrationsof IDL cholesterol were closely associated with a high frequencyof coronary heart disease in consecutive patients evaluatedby selective coronary cineangiography. Therefore, because theprecise measurement of IDL and VLDL cholesterol is likely tobe useful for cardiovascular risk assessment in HD patients,our convenient HPLC assay for IDL and VLDL cholesterol may behelpful in clinical practice for HD patients.

In conclusion, HD patients have the impaired lipoprotein metabolismcharacteristic of the elevated TG-rich lipoproteins (IDL andVLDL) in lipoprotein profiles analysed by our developed HPLCmethod, of which data were consistent with those shown by theother study groups with the UC method. HPLC chromatograms showthe excellent separation of TG-rich lipoproteins. Taken together,our developed HPLC method may be sufficiently applied to theroutine assay for TG-rich lipoproteins in clinical practicefor the diagnosis and treatment of dyslipidaemia to preventatherosclerotic cardiovascular diseases in HD patients.





GRANT/FUNDING SUPPORT

Go to previous sectionTop

Go to previous sectionAbstract

Go to previous sectionIntroduction

Go to previous sectionMethods

Go to previous sectionResults

Go to previous sectionDiscussion

 GRANT/FUNDING SUPPORT
Go to next sectionACKNOWLEDGEMENTS

Go to next sectionREFERENCES

 

The present study was supported by a grant from Tosoh Corporationand the Jikei University Research Fund.

 

 




ACKNOWLEDGEMENTS

Go to previous sectionTop

Go to previous sectionAbstract

Go to previous sectionIntroduction

Go to previous sectionMethods

Go to previous sectionResults

Go to previous sectionDiscussion

Go to previous sectionGRANT/FUNDING SUPPORT

 ACKNOWLEDGEMENTS
Go to next sectionREFERENCES

 

We wish to thank Mika Komori and Yukako Uchihashi for theirexcellent technical assistance.

(Accepted May 20, 2008)



REFERENCES

Go to previous sectionTop

Go to previous sectionAbstract

Go to previous sectionIntroduction

Go to previous sectionMethods

Go to previous sectionResults

Go to previous sectionDiscussion

Go to previous sectionGRANT/FUNDING SUPPORT

Go to previous sectionACKNOWLEDGEMENTS

 REFERENCES


  1. Igel-Korcagova A, Raab P, Brensing KA, et al. Cholesterol metabolism in patients with chronic renal failure on hemodialysis. J Nephrol 2003;16:850–4[Medline]
  2. Cheung AK, Sarnak MJ, Yan G, et al. Atherosclerotic cardiovascular disease risks in chronic hemodialysis patients. Kidney Int 2000;58:353–62[Medline]
  3. Sarnak MJ, Levey AS, Schoolwerth AC, et al. Kidney disease as a risk factor for development of cardiovascular disease. Circulation 2003;108:2154–69[Free Full Text]
  4. Shoji T, Nishizawa Y, Kawagishi T, et al. Atherogenic lipoprotein changes in the absence of hyperlipidemia in patients with chronic renal failure treated by hemodialysis. Atherosclerosis 1997;131:229–36[Medline]
  5. Shoji T, Nishizawa Y, Kawagishi T, et al. Intermediate-density lipoprotein as an independent risk factor for aortic atherosclerosis in hemodialysis patients. J Am Soc Nephrol 1998;9:1277–84[Abstract]
  6. Quaschning T, Schomig M, Keller M, et al. Non-insulin-dependent diabetes mellitus and hypertriglyceridemia impair lipoprotein metabolism in chronic hemodialysis patients. J Am Soc Nephrol 1999;10:332–41[Abstract/Free Full Text]
  7. Usui S, Hara Y, Hosaki S, Okazaki M. A new on-line dual enzymatic method for simultaneous quantification of cholesterol and triglycerides in lipoproteins by HPLC. J Lipid Res 2002;43:805–14[Abstract/Free Full Text]
  8. Sato I, Taniguchi T, Ishikawa Y, et al. The lipoprotein fraction between VLDL and LDL detected by biphasic agarose gel electrophoresis reflects serum remnant lipoprotein and Lp(a) concentrations. J Atheroscler Thromb 2006;13:55–61[Medline]
  9. Hirowatari Y, Yoshida H, Kurosawa H, et al. Measurement of cholesterol of major serum lipoprotein classes by anion-exchange HPLC with perchlorate ion-containing eluent. J Lipid Res 2003;44:1404–12[Abstract/Free Full Text]
  10. Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults. Executive summary of the third report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). JAMA 2001;285:2486–97[Free Full Text]
  11. American Diabetes Association. Management of dyslipidemia in adults with diabetes. Diabetes Care 2003;26:S83–6[Medline]
  12. Cui Y, Blumenthal RS, Flaws JA, et al. Non-high-density lipoprotein cholesterol level as a predictor of cardiovascular disease mortality. Arch Intern Med 2001;161:1413–9[Abstract/Free Full Text]
  13. Lu W, Jain AK, Resnick HE, et al. Non-HDL cholesterol as a predictor of cardiovascular disease in type 2 diabetes. Diabetes Care 2003;26:16–23[Abstract/Free Full Text]
  14. Kawamoto R, Oka Y, Tomita H, Kodama A. Non-HDL cholesterol as a predictor of carotid atherosclerosis in the elderly. J Atheroscler Thromb 2005;12:143–8[Medline]
  15. Nishizawa Y, Shoji T, Kakiya R, et al. Non-high-density lipoprotein cholesterol (non-HDL-C) as a predictor of cardiovascular mortality in patients with end-stage renal disease. Kidney Int 2003;63(Suppl. 84):S117–S120
  16. Koba S, Hirano T, Sakaue T, Takeuchi H, Adachi M, Katagiri T. An incresaed number of very-low-density lipoprotein particles is strongly associated with coronary heart disease in Japanese men, independently of intermediate-density lipoprotein or low-density lipoprotein. Coron Artery Dis 2002;13:255–62[Medline]
  17. Tatami R, Mabuchi H, Ueda K, et al. Intermediate-density lipoprotein and cholesterol-rich very low density lipoprotein in angiographically determined coronary artery disease. Circulation 1981;64:1174–84[Abstract/Free Full Text]
  18. Steiner G, Schwartz L, Shumak S, Poapst M. The association of increased levels of intermediate-density lipoproteins with smoking and with coronary artery disease. Circulation 1987;75:124–30[Abstract/Free Full Text]
  19. Sacks FM, Alaupovic P, Moye LA, et al. VLDL, apolipoproteins B, CIII, and E, and risk of recurrent coronary events in the cholesterol and recurrent events (CARE) Trial. Circulation 2000;102:1886–92[Abstract/Free Full Text]
  20. Whayne TF, Alaupovic P, Curry MD, Lee ET, Anderson PS, Schechter E. Plasma apolipoprotein B and VLDL-, LDL-, and HDL-cholesterol as risk factors in the development of coronary artery disease in male patients examined by angiography. Atherosclerosis 1981;39:411–24[Medline]
  21. Krauss RM, Lindgren FT, Williams PT, et al. Intermediate-density lipoproteins and progression of coronary artery disease in hyperchole sterolaemic men. Lancet 1987;2:62–6[Medline]


CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What’s this?






This Article
Right arrow
Abstract

Freely available
Right arrow
Figures Only
Right arrow

Full Text (PDF)

Right arrow
All Versions of this Article:

acb.2008.008018v1

45/6/571

most recent

Right arrow
Alert me when this article is cited
Right arrow
Alert me if a correction is posted
Services
Right arrow
Email this article to a friend
Right arrow

Similar articles in this journal

Right arrow

Similar articles in Web of Science
Right arrow
Similar articles in PubMed
Right arrow
Alert me to new issues of the journal
Right arrow
Download to citation manager
Right arrow
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow
Articles by Hirowatari, Y.
Right arrow
Articles by Miida, T.
Right arrow Search for Related Content
PubMed
Right arrow
PubMed Citation
Social Bookmarking

What’s this?