Accepted Manuscript

Cholesterol concentrations in lipoprotein fractions separated by

anion-exchange–high-performance liquid chromatography in
healthy dogs and dogs with hypercholesterolemia

Hitomi Oda, Akihiro Mori, Yuji Hirowatari, Toshie Takoura,

Daisuke Manita, Tomoya Takahashi, Saori Shono, Eri Onozawa,
Hisashi Mizutani, Yohei Miki, Yukiko Itabashi, Toshinori Sako

PII: S0034-5288(17)30402-2
DOI: doi: 10.1016/j.rvsc.2017.04.004
Reference: YRVSC 3299
To appear in: Research in Veterinary Science
Received date: 26 April 2016
Revised date: 28 March 2017
Accepted date: 7 April 2017

Please cite this article as: Hitomi Oda, Akihiro Mori, Yuji Hirowatari, Toshie Takoura,
Daisuke Manita, Tomoya Takahashi, Saori Shono, Eri Onozawa, Hisashi Mizutani, Yohei
Miki, Yukiko Itabashi, Toshinori Sako , Cholesterol concentrations in lipoprotein fractions
separated by anion-exchange–high-performance liquid chromatography in healthy dogs
and dogs with hypercholesterolemia. The address for the corresponding author was
captured as affiliation for all authors. Please check if appropriate. Yrvsc(2017), doi:
10.1016/j.rvsc.2017.04.004

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 ACCEPTED MANUSCRIPT

Research Paper

Cholesterol concentrations in lipoprotein fractions separated by

anion-exchange–high-performance liquid chromatography in healthy dogs and

dogs with hypercholesterolemia

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Hitomi Oda1 , Akihiro Mori 1* , Yuji Hirowatari 2 , Toshie Takoura 3 , Daisuke Manita 3 ,

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Tomoya Takahashi 1 , Saori Shono 1 , Eri Onozawa 1 , Hisashi Mizutani 4 , Yohei Miki 5 ,

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Yukiko Itabashi 5 , Toshinori Sako 1 NU
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School of Veterinary Nursing & Technology, Nippon Veterinary and Life Science

University, Tokyo, Japan

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Department of Health Sciences, Saitama Prefectural University, Saitama, Japan
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Tosoh Corporation, Tokyo, Japan
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School of Veterinary Science, Nippon Veterinary and Life Science University,
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Tokyo, Japan
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Monolis Incorporated, Tokyo, Japan
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*Corresponding author: Tel.: +81 422314151; fax: +81 422317841

E-mail: [email protected]

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ABSTRACT

Anion-exchange (AEX)–high-performance liquid chromatography (HPLC) for

measurement of cholesterol can be used to separate serum lipoproteins (high-density

lipoprotein (HDL); low-density lipoprotein (LDL); intermediate-density lipoprotein

(IDL); very-low-density lipoprotein (VLDL)) in humans. However, AEX–HPLC has

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not been applied in veterinary practice. We had three objectives: (i) the validation of

AEX-HPLC methods including the correlation of serum cholesterol concentration in

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lipoprotein fraction measured by AEX–HPLC and gel permeation–HPLC (GP–

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HPLC) in healthy dogs and those with hypercholesterolemia was investigated; (ii)
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the reference intervals of lipoprotein fractions measured by AEX –HPLC from

healthy dogs (n=40) was established; (iii) lipoprotein fractions from the serum of
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healthy dogs (n=12) and dogs with hypercholesterolemia (n=23) were compared .

Analytic reproducibility and precision of AEX–HPLC were acceptable. Positive

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correlation between serum concentrations of total cholesterol (Total-Chol), HDL

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cholesterol (HDL-Chol), LDL cholesterol (LDL-Chol) +IDL cholesterol (IDL-Chol),

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and VLDL cholesterol (VLDL-Chol) was noted for AEX–HPLC and GP–HPLC in
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healthy dogs and dogs with hypercholesterolemia. Reference intervals measured by

AEX–HPLC for serum concentrations of Total-Chol, HDL-Chol, and LDL-Chol

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were determined to be 2.97–9.32, 2.79–6.57, 0.16–3.28 mmol/L (2.5–97.5% interval),

respectively. Furthermore, there was significant difference in lipoprotein profiles

between healthy and dogs with hypercholesterolemia. These results suggest that

AEX–HPLC can be used to evaluate lipoprotein profiles in dogs and could be a new

useful indicator of hyperlipidemia in dogs.

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Key words; Cholesterol, dogs, HPLC, hypercholesterolemia, serum

1. Introduction

Lipoproteins are classified according to their size, composition, and physical

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characteristics. Major classes of lipoproteins are: chylomicrons, very-low-density

lipoproteins (VLDL), low-density lipoproteins (LDL), intermediate-density

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lipoproteins (IDL) and high-density lipoproteins (HDL). LDL are the major

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lipoproteins in human, meanwhile predominant HDL and very few VLDL are
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characteristic of canine lipoproteins. Several methods have been applied to separate

lipoproteins depending on their particle size, form, electrophoretic mobility, and

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density (Mizutani et al., 2010; Usui et al., 2002).

Ultracentrifugation is the “gold standard” method for separating lipoproteins;

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however, it is cumbersome and time-consuming (Hirowatari et al., 2012). Mizutani

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et al. reported that lipoprotein fractions obtained using gel permeation (GP)–
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high-performance liquid chromatography (HPLC) are closely related to

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ultracentrifugation methods, and that GP–HPLC could be useful for the study of

lipid metabolism in dogs (Mizutani et al., 2010). As such, GP–HPLC is used widely
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for the diagnosis of hyperlipidemia in dogs in Japan.

Anion-exchange–high-performance liquid chromatography (AEX–HPLC) is a

relatively new method applicable to lipoprotein analyses in humans. AEX–HPLC can

be used to determine IDL in specimens of human blood (Hirowatari et al., 2003;

Hirowatari et al., 2008; Hirowatari et al.,2012; Kon et al., 2010). However, analyses

of lipoprotein have not been investigated by AEX–HPLC in dogs. In particular, IDL

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concentrations are considered to be a risk factor for arteriosclerotic disease in

humans (Krauss et al., 1987; Tatami et al., 1981). However, serum concentrations of

IDL have not been evaluated in veterinary practice. Arteriosclerotic disease is

studied less often in veterinary medicine than in human medicine, but disease of the

endocrine system (e.g., hypothyroidism, hyperadrenocorticism, diabetes mellitus

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(DM)) are related to hyperlipidemia and coronary artery disease (Hess et al., 2003;

Xenoulis and Steiner, 2015). Furthermore, primary hypercholesterolemia has been

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reported in some canine breeds such as Beagle, Miniature Schnauzer and Shetland

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Sheepdogs (Sato et al., 2000). NU
The first objective of the current study was to investigate the validation of AEX–

HPLC methods including correlation of serum cholesterol concentration in lipoprotein

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fractions measured by AEX–HPLC and GP–HPLC in healthy dogs and those with

hypercholesterolemia. Secondarily, we wished to establish the reference intervals of

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lipoprotein fractions measured by AEX–HPLC from healthy dogs. Finally, lipoprotein

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fractions from the serum of healthy dogs and dogs with hypercholesterolemia were
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compared.
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2. Materials and Methods

2.1.Animals

A total of 75 dogs were used for the present study. Twelve healthy control dogs

maintained in our laboratory for research as well as 23 client -owned dogs with

hypercholesterolemia (cholesterol >7.76 mmol/L) who visited our veterinary medicine

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teaching hospital were used for studies on reproducibility, linearity, correlation and

comparison study. Profiles and concurrent diseases in the 12 healthy dogs and 23

canine patients with hypercholesterolemia are shown in Table 1.

For the determination of reference intervals, 40 serum specimens were obtained

from healthy dogs maintained in our laboratory and client-owned dogs which visited

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local veterinary hospital. Data for individual dogs (breed, age, sex, neuter status and

body weight) from these 40 dogs was shown in Table 2. Complete blood counts and

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serum biochemistry (glucose, total protein, albumin, aspartate aminotransferase,

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alanine aminotransferase, alkaline phosphatase,
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triglyceride, blood urea nitrogen, creatinine and phosphate) were within the reference

intervals classified as “healthy”, and used for the determination of reference intervals.
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Furthermore, there was no abnormality in the physical examinations diagnosed by

veterinarian and no signs of illness in the 2weeks preceding specimen collection. The
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study protocol was approved by the Animal Research Committee of the Nippon
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Veterinary and Life Science University (approval number; 27S-72) (Tokyo, Japan).
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Written informed consent was obtained from all dogs-owners after detailing the
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purpose, nature, and potential risks/benefits of the study.

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2.2.Blood sampling and lipoprotein assay

Random blood specimens (2-5 mL) were collected from the peripheral veins of

individual animals. Specimens were collected into polypropylene tubes and allowed to

clot at room temperature for 15 min before being centrifuged (1,700 × g for 10 min at

4°C) to separate the serum. All serum specimens were sent to Monolis Incorporated

(Tokyo, Japan) for AEX–HPLC. Serum specimens for the correlation study were sent
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to Skylight Biotech (Akita, Japan) for GP–HPLC. Serum specimens were stored at

4°C. AEX–HPLC (Manita et al., 2015) and GP–HPLC (Mizutani et al., 2010) was

carried out as described previously. Lower limit cholesterol concentrations of the

measuring interval for cholesterol measurement by AEX-HPLC was 0.026 mmol/L.

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2.3.Intra and inter- assay precision and linearity test measured by AEX–HPLC

Intra-assay precision (repeatability) for serum concentrations of lipoprotein was

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estimated using pooled serum specimens with normal concentrations of lipoprotein

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with 10 replicates. Other pooled specimens stored at - 80°C with normal
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concentrations of lipoprotein were used for evaluation of inter-assay precision

(reproducibility) between assays, with 6 replicates run independently over 1 month.

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Linearity was assessed by serial dilution of a normal- concentration serum pool (up

to fivefold) to obtain various lipoprotein values.

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2.4.Statistical analyses
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Values are the mean ± standard deviation (SD) or median and

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minimum-maximum range. For the determinnation of reference intervals, the

Reference Value Advisor macroinstructions for Excel were used (Friedrichs et al.,
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2012; Geffré et al., 2011). Outliers were evaluated by the Turkey’s test using the

Reference Value Advisor. Normality of the distributions of native or transformed

values was tested by the Anderson-Darling test using the Reference Value Advisor.

Reference intervals and 90% confidence intervals of the limits were determined

using the Reference Value Advisor and the nonparametric method. Spearman's

correlation coefficient by rank was used to identify significant correlations between

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measurements, and Spearman's correlation coefficient (r) is reported using GraphPad

Prism (GraphPad Prism 6, GraphPad Software, La Jolla, CA). The Mann–Whitney

U-Test was employed to assess the significance of the difference of lipoprotein

fractions between healthy dogs and those suffering from hypercholesterolemia using

GraphPad Prism. P<0.05 was considered significant.

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3. Results

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AEX–HPLC was not previously used to evaluate lipoproteins in canine
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specimens, so assessment of its reproducibility and precision was important.

Analytic evaluation of the assay of HDL-cholesterol (HDL-Chol) and

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LDL-cholesterol (LDL-Chol) using AEX–HPLC suggested acceptable analytic

precision, with intra- and inter-assay coefficient of variation (CV) values less than
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the commonly accepted limit of 5% (Table 3). Analytic evaluation of

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IDL-cholesterol (IDL-Chol) and VLDL-cholesterol (VLDL-Chol) showed intra- and

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inter-assay CV values of below 6.7% and 14.2%, respectively (Table 3). Excellent
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linearity was demonstrated with all of the dilutions tested, up to fivefold dilution

(Fig 1). A positive correlation between AEX–HPLC and GP–HPLC was observed for
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Total-Chol, HDL-Chol, LDL-Chol + IDL-Chol and VLDL-Chol (r=0.974, 0.928,

0.973, 0.939, respectively, P<0.001, Fig. 2). GP–HPLC cannot be used to evaluate

IDL-Chol concentrations, so LDL-Chol + IDL-Chol measured by AEX–HPLC was

employed to estimate LDL-Chol concentrations.

The raw data of cholesterol concentration in each lipoprotein fraction s in 40

healthy dogs for reference intervals are shown in Table 2. The reference interval for
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Total-Chol, HDL-Chol and LDL-Chol in the serum of dogs was shown in Table 4.

Outliers were not detected by the Turkey’s test. The dog population representing the

reference intervals of total-cholesterol (Total-Chol) and HDL-Chol passed the

Anderson-Darling test, revealing a Gaussian distribution of values ( P=0.116, 0.194,

respectively). Distribution of native LDL-Chol values did not fit a Gaussian

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distribution but was not significantly different from Gaussian after Box -Cox

transformation (Anderson- Darling test, P <0.001 and 0.921, respectively). With

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regard to concentrations of IDL-Chol and VLDL-Chol, we could not establish the

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reference intervals because some measurement results (17 of 40 specimens for
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IDL-Chol; 23 of 40 specimens for VLDL-Chol) were below the lower limit of the

measuring interval (0.026 mmol/L).

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Lipoprotein fractions from the serum of 12 healthy and 23 dogs suffering from

hypercholesterolemia were compared (Fig. 3). There were significantly higher

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concentration (P<0.001) of Total-Chol, HDL-Chol, LDL-Chol, IDL-Chol and

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VLDL-Chol with dogs suffering from hypercholesterolemia than those with healthy
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dogs. Dogs suffering from hypercholesterolemia had a significantly lower (P<0.001)

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HDL-Chol:LDL-Chol ratio compared with healthy dogs. Median and

minimum-maximum range of Total-Chol, HDL-Chol, LDL-Chol, IDL-Chol,

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VLDL-Chol and HDL:LDL ratio are shown in Table 5.

4. DISCUSSION

Hyperlipidemia is associated with the development of arteriosclerotic disease in

humans (Jacobson et al., 2007; Kagawa et al., 1998; Stein, 2002). In dogs,
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hyperlipidemia is often related to diseases of the endocrine system, such as DM,

Cushing syndrome or hypothyroidism (Hess et al., 2003; Xenoulis and Steiner, 2015).

Hyperlipidemia in dogs has been reported to induce pancreatitis, liver disease,

atherosclerosis, ocular disease, and seizures (Xenoulis and Steiner, 2015). Therefore,

correct diagnosis of hyperlipidemia is also necessary for dogs.

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Mizutani et al. (2010) investigated the usefulness of GP–HPLC. Lipoprotein

concentrations measured by GP–HPLC have been used widely for the diagnosis of

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hyperlipidemia in dogs in Japan. GP-HPLC separates lipoproteins on the basis of

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size. Meanwhile, AEX-HPLC separates lipoproteins on the basis of charges using
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ion-exchange resin. AEX-HPLC is considered an accurate method for separating

lipoproteins because each lipoprotein is composed of several apolipoproteins with

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different ion-charges (Hirowatari et al., 2010). Lipoprotein-A consists of LDL

particles apolipoprotein B-100 and the specific apolipoprotein-A. Lipoprotein-A

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overlaps with VLDL in GP-HPLC; however, AEX-HPLC can detect the specific
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apolipoproteins of VLDL (Hirowatari et al., 2010). In the present study, a high level

of correlation for Total-Chol, HDL-Chol, LDL-Chol + IDL-Chol and VLDL-Chol

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between AEX–HPLC and GP–HPLC was observed, which supports the notion that
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AEX–HPLC is useful for the determination of lipoprotein fractions in dogs.

In the present study, intra- and inter-assay CV values for canine cholesterol

concentration in each lipoprotein fractions were <10% except for inter-assay CV

values for IDL-Chol and VLDL-Chol (14.2% and 10.7 %, respectively). The

specimens used for the assay were from healthy dogs, so the serum concentrations of

IDL-Chol and VLDL-Chol might have been too low for evaluation of inter-assay

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differences. Furthermore, serum specimens for separating lipoproteins by

AEX-HPLC should be measured within 5 day and without frozen storage

(manufacturer’s recommendations). As such, high inter-assay CV values for

IDL-Chol and VLDL-Chol might not be a clinical issue. Overall, in the present study,

excellent linearity was demonstrated with all dilutions tested . Furthermore,

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repeatability and reproducibility with canine serum using AEX–HPLC were

satisfactory.

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Because validation of AEX–HPLC was established, whether differences in

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lipoprotein fractions were observed between healthy dogs and dogs with
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hypercholesterolemia needed investigation. We found that dogs suffering from

hypercholesterolemia had significantly higher concentrations of Total-Chol,

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HDL-Chol, LDL-Chol, IDL-Chol, VLDL-Chol and a lower HDL:LDL ratio than

those of healthy dogs. High concentrations of HDL-Chol and low concentrations of

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LDL-Chol and VLDL-Chol are characteristics of healthy lipoprotein profiles in dogs

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(Bailhache et al., 2003; Mizutani et al., 2010). In the present study, 23 dogs with

hypercholesterolemia had concurrent diseases: Cushing syndrome (including

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complications of DM, n=8), DM (n=4), under treatment of prednisolone (n = 3), liver

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disorder (n=3), hypothyroidism (n=2), neoplasia (n=2), orthopedic disease (n=1). As

such, 11 out of 23 dogs with hypercholesterolemia had endogenous or exogenous

glucocorticoids. Administration of glucocorticoids is related to hepatic secretion of

VLDL (Jericó et al., 2009), and induces reductions in the activity of LDL receptors

(Al Rayyes et al., 1999). Therefore, AEX–HPLC could be used to detect changes in

cholesterol metabolism induced by endogenous or exogenous glucocorticoids.

Primary hypercholesterolemia has been reported in breeds of Shetland sheepdogs

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(Sato et al., 2010). In the present study, 2 dogs with neoplasia were Shetland

sheepdogs, so their hypercholesterolemia may not have been related to concurrent

disease. Recently, IDL was shown to be associated with atherosclerosis progression

in humans (Ito et al., 2013; Hodis et al., 1997). In the present study, dogs suffering

from hypercholesterolemia had higher concentrations of IDL-Chol than healthy

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dogs.

One limitation of our study was using random (non-fasted) specimens for

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evaluating lipoprotein profiles, since almost of our canine specimens were

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client-owned dogs (including disease dogs). However, no significant differences
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were observed in Total-Chol and HDL-Chol concentrations between fasted and

non-fasted human specimens (Nigam, 2011). Another study also demonstrated that
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HDL-Chol concentration in humans were 95% agreement between fasted and

non-fasted status (Craig et al., 2000). As such, cholesterol concentration might be

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less affected by fasting state.

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We showed that serum concentrations of lipoprotein in dogs can be separated by

AEX–HPLC. In particular, IDL fractions can be measured, which has never been
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reported. Validation of AEX–HPLC was established by examining its linearity and

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precision. Correlation of lipoprotein fractions between AEX–HPLC and GP–HPLC

was confirmed. Reference intervals were also established, which results could be

considered useful for the diagnosis and treatment of dogs suffering from

hypercholesterolemia. Furthermore, dogs suffering from hypercholesterolemia had

higher values of each lipoprotein fractions as compared with healthy dogs. Therefore,

AEX–HPLC may be useful method for evaluating canine hyperlipidemia.

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Acknowledgements

The authors would like to thank Dr Shinogu Hasegawa, Kazuo Yamauchi, Akihiro

Suda and Keita Matsumoto for taking blood specimens from healthy dogs for the

determination of reference intervals.

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FIGURE LEGENDS

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Figure 1.

Linearity of the lipoprotein fractions (HDL cholesterol, LDL cholesterol, IDL

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cholesterol, VLDL cholesterol) in pooled serum specimens of healthy dogs measured

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by AEX–HPLC. Serum specimens underwent serial dilution up to fivefold.
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Figure 2.
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Correlation between lipoprotein fractions (A. Total cholesterol; B. HDL cholesterol ;

C. LDL + IDL cholesterol ; D. VLDL cholesterol) measured by AEX–HPLC and GP–

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HPLC in random blood specimens in healthy dogs (n=12) and dogs with
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hypercholesterolemia (n=23). Slope and intercept of each lipoprotein fraction s were:

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Total cholesterol, y = 1.038x – 0.829; HDL cholesterol, y = 1.043x – 0.246;

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LDL+IDL cholesterol, y = 0.770x + 0.040; VLDL cholesterol, y = 1.337x – 0.065.

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Figure 3.

Comparison of lipoprotein fractions (A. Total cholesterol; B. HDL cholesterol ; C.

LDL cholesterol; D. IDL cholesterol; E. VLDL cholesterol; F. HDL:LDL ratio)

between healthy dogs (n=12) and dogs with hypercholesterolemia (n=23). Individual

“raw” values are expressed as dots. The horizontal line denotes the median value.

Asterisk denotes significance (P<0.001, Mann–Whitney U-test) when compared with

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healthy dogs.

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DE
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CE
AC

20
 ACCEPTED MANUSCRIPT

Table 1. Profiles of 35 dogs used in intra - and inter-assays, dilution test,

correlation and comparison study

Total-C
Class Age Body B
hol
ificat Breed (yea Sex weight C Concurrent disease
(mmol/
ion rs) (kg) S

PT
L)

Castr

RI
Healt
1 Beagle 7 ated 11.5 3 -

SC
hy
male NU 6.2

Castr
Healt
2 Beagle 3 ated 12.1 3 -
MA

hy
male 4.5

Castr
D

Healt
3 Beagle 7 ated 11.5 3 -
E

hy
PT

male 4.6

Spay
CE

Healt ed
4 Beagle 10 10.2 3 -
AC

hy femal

e 4.1

Spay

Healt ed
5 Beagle 7 10.5 3 -
hy femal

e 4.9

21
 ACCEPTED MANUSCRIPT

Spay

Healt ed
6 Beagle 7 11.2 3 -
hy femal

e 3.8

Castr
Healt

PT
7 Beagle 7 ated 11.1 3 -
hy
male 5.3

RI
Castr

SC
Healt
8 Beagle 5 ated 8.7 3 -
hy
NU
male 6.3

Castr
MA

Healt
9 Beagle 5 ated 10.3 3 -
hy
male 4.8
D

Castr
E

1 Healt
Beagle 5 ated 10.4 3 -
PT

0 hy
male 4.9
CE

Castr
1 Healt
Beagle 7 ated 13.1 3 -
AC

1 hy
male 4.0

Spay

1 Healt ed
Beagle 8 8.1 3 -
2 hy femal

e 3.4

22
 ACCEPTED MANUSCRIPT

Castr
1 Patie Toy
8 ated 6.0 3 Hypothyroidism 22.2
3 nt poodle
male

Jack
1 Patie Fema
Russell 11 7.3 4 Hepatic fibrosis 11.3
4 nt le

PT
Terrier

Spay

RI
Jack
1 Patie ed

SC
Russell 10 6.2 3 Cushing syndrome 13.9
5 nt femal
Terrier
NU
e

Spay
MA

1 Patie Siberia ed
11 27.6 4 Diabetes meliltus 8.8
6 nt n husky femal
D

e
E

Yorkshi Castr
PT

1 Patie
re 12 ated 4.0 3 Cushing syndrome 8.2
CE

7 nt
terrier male

Castr
AC

1 Patie Shih Diabetes mellitus,

13 ated 7.3 4 12.1
8 nt Tzu Cushing syndrome
male

1 Patie Toy
8 Male 3.6 4 Hypothyroidism 9.9
9 nt poodle

2 Patie Jack 7 Castr 8.2 3 Cushing syndrome 8.9

23
 ACCEPTED MANUSCRIPT

0 nt Russell ated

Terrier male

Castr
2 Patie
Mix 3 ated 21.9 3 Patellar dislocation 10.2
1 nt
male

PT
Castr
2 Patie Toy
9 ated 7.1 3 Cushing syndrome 8.6

RI
2 nt poodle
male

SC
Castr
2 Patie Shih
NU
8 ated 3.7 3 Hepatic fibrosis 10.8
3 nt Tzu
male
MA

Spay

2 Patie Toy ed Undergoing treatment

D

9 4.7 3 24.4
4 nt poodle femal with prednisolone
E

e
PT

2 Patie Toy
CE

9 Male 7.6 3 Diabetes mellitus 23.9

5 nt poodle

Miniatu Spay
AC

2 Patie re ed
10 4.9 3 Diabetes mellitus 8.7
6 nt Schnau femal

zer e

2 Patie
Mix 13 Male 14.5 4 Diabetes mellitus 11.3
7 nt

24
 ACCEPTED MANUSCRIPT

Spay
Jack
2 Patie ed Elevation of hepatic
Russell 11 7.8 3 9.0
8 nt femal enzymes
Terrier
e

Spay

PT
2 Patie Shih ed
14 5.9 3 Cushing syndrome 9.9
9 nt Tzu femal

RI
e

SC
Spay NU
3 Patie Shih ed
12 4.1 3 Cushing syndrome 8.6
0 nt Tzu femal
MA

e
D

Shetlan Castr
E

3 Patie Mucosal cyst in

d 11 ated 14.0 3 11.5
PT

1 nt gallbladder
Sheepd male
CE

og
AC

Spay
Shetlan
3 Patie ed Vaginal
d 9 12.1 3 27.0
2 nt femal leiomyosarcoma
Sheepd
e
og

3 Patie Miniatu 8 Castr 12.2 4 Diabetes mellitus, 31.5

25
 ACCEPTED MANUSCRIPT

3 nt re ated Cushing syndrome

Schnau male

zer

Spay
Addison's disease,
3 Patie Toy ed
7 3.5 3 undergoing treatment 10.3

PT
4 nt poodle femal
with prednisolone
e

RI
Miniatu Spay

SC
Arthritis, undergoing
3 Patie re ed
10 6.3 3 treatment with 14.1
NU
5 nt Schnau femal
prednisolone
zer e
MA

BCS, body condition score. The BCS was determined on a five -point
D

scale: 1. thin; 2. lean; 3. optimal; 4. obese; 5. gross.

E

Total-Chol, total cholesterol. Total -Chol was measured by

PT

anion-exchange–high-performance liquid chromatography.

CE
AC

26
 ACCEPTED MANUSCRIPT

Table 2. Profiles and raw data of 40 healthy dogs used for the determine of refernce

intervals.

Clas Total- HDL-c LDL-C IDL-C VLDL-

Age Body
sific Chol hol hol hol Chol
Breed (ye Sex weigh

PT
atio (mmol/ (mmol (mmol (mmol (mmol/
ars) t (kg)
n L) /L) /L) /L) L)

RI
Cast

SC
Heal
1 Beagle 1 rated 8.2 3.6 3.4 0.2 ND ND
thy
NU
male

Spay
MA

Heal ed
2 Beagle 1 7.6 5.2 4.4 0.8 0.07 ND
thy fema
D

le
E
PT

Spay

Heal ed
CE

3 Beagle 2 11.1 5.1 4.2 0.8 0.09 ND

thy fema
AC

le

Cast
Heal
4 Beagle 5 rated 14.0 5.2 4.2 0.9 0.10 ND
thy
male

Heal Cast
5 Beagle 5 11.6 5.9 4.8 1.0 0.09 0.04
thy rated

27
 ACCEPTED MANUSCRIPT

male

Cast
Heal
6 Beagle 5 rated 13.0 4.7 4.1 0.6 0.05 ND
thy
male

Cast
Heal

PT
7 Beagle 5 rated 12.2 3.6 3.3 0.3 ND 0.04
thy
male

RI
Spay

SC
Heal ed
8 Beagle 7 13.8 3.5 3.2 0.3 0.03 0.04
NU
thy fema

le
MA

Cast
Heal
9 Beagle 7 rated 10.8 4.9 4.2 0.6 0.05 ND
D

thy
male
E

Cast
PT

1 Heal
Beagle 7 rated 11.5 3.0 2.8 0.2 ND ND
CE

0 thy
male

Cast
AC

1 Heal
Beagle 7 rated 10.2 3.2 0.3 ND ND
1 thy
male 3.5

Flat-Co
1 Heal fema
ated 3 29.0 7.7 4.5 3.0 0.13 0.03
2 thy le
Retriev

28
 ACCEPTED MANUSCRIPT

er

Flat-Co

1 Heal ated
3 male 38.0 4.7 4.0 0.7 ND ND
3 thy Retriev

er

PT
Flat-Co

1 Heal ated

RI
3 male 35.0 5.9 3.5 2.2 0.08 ND
4 thy Retriev

SC
er NU
Flat-Co
Cast
1 Heal ated
MA

6 rated 27.2 8.3 4.6 3.3 0.38 0.05

5 thy Retriev
male
er
D

Flat-Co
E

Cast
1 Heal ated
PT

7 rated 35.3 6.7 5.5 1.2 0.05 ND

6 thy Retriev
CE

male
er

Flat-Co
AC

Cast
1 Heal ated
7 rated 33.0 7.3 5.2 1.9 0.16 ND
7 thy Retriev
male
er

1 Heal Labrad fema

1 15.7 6.0 4.7 1.3 0.05 ND
8 thy or le

29
 ACCEPTED MANUSCRIPT

Retriev

er

Labrad
Cast
1 Heal or
1 rated 21.3 3.5 3.1 0.4 ND ND
9 thy Retriev
male

PT
er

Labrad Spay

RI
2 Heal or ed

SC
1 21.4 3.7 3.1 0.6 ND ND
0 thy Retriev fema NU
er le

Labrad
MA

2 Heal or
2 male 29.9 6.0 3.8 1.9 0.26 0.04
1 thy Retriev
D

er
E

Labrad
PT

Cast
2 Heal or
CE

2 rated 24.0 4.0 3.6 0.3 ND 0.03

2 thy Retriev
male
er
AC

Miniatu Spay

2 Heal re ed
2 8.5 6.6 4.5 1.8 0.22 0.11
3 thy Schnau fema

zer le

2 Heal Miniatu 8 Spay 8.1 5.6 4.0 0.6 0.40 0.51

30
 ACCEPTED MANUSCRIPT

4 thy re ed

Schnau fema

zer le

Spay

2 Heal Toy ed
6 4.3 4.1 3.7 0.4 ND ND

PT
5 thy Poodle fema

le

RI
Spay

SC
2 Heal Toy ed
8 3.1 4.1 3.9 0.2 ND ND
NU
6 thy Poodle fema

le
MA

German
2 Heal fema
Shephe 1 24.8 4.4 3.9 0.5 ND ND
D

7 thy le
rd Dog
E

Gorlde
PT

2 Heal n
CE

1 male 33.1 5.2 1.1 0.04 ND

8 thy Retriev

er 6.4
AC

2 Heal Siberia
1 male 19.8 3.6 3.1 0.4 0.05 ND
9 thy n husky

Spay
3 Heal Border
2 ed 13.0 4.0 3.7 0.3 ND 0.03
0 thy Collie
fema

31
 ACCEPTED MANUSCRIPT

le

3 Heal Bull fema

3 11.6 7.1 5.4 1.6 0.05 ND
1 thy Terrier le

3 Heal Great
3 male 70.0 9.3 6.0 3.1 0.18 0.05
2 thy Dane

PT
3 Heal Shih
3 male 5.9 4.4 3.9 0.5 ND ND
3 thy Tzu

RI
Miniatu

SC
3 Heal re
4 male 5.5 5.0 4.5 0.4 ND 0.05
NU
4 thy Pinsche

r
MA

Cast
3 Heal Chihua
6 rated 3.8 4.5 4.0 0.4 ND 0.12
D

5 thy hua
male
E

Cast
PT

3 Heal
Pug 7 rated 5.0 8.0 6.6 1.3 0.08 0.04
CE

6 thy
male

Spay
AC

3 Heal ed
MIX 2 10.5 7.2 6.0 1.2 ND 0.09
7 thy fema

le

3 Heal
MIX 2 male 8.1 5.5 4.9 0.6 ND ND
8 thy

32
 ACCEPTED MANUSCRIPT

Cast
3 Heal
MIX 4 rated 4.5 6.7 5.3 1.0 0.05 0.26
9 thy
male

Cast
4 Heal
MIX 7 rated 16.0 4.7 4.1 0.5 0.04 0.05
0 thy

PT
male

RI
Total-Chol, total cholesterol; HDL -Chol, high-density lipoprotein cholesterol;

SC
LDL-Chol, low-density lipoprotein cholesterol; IDL -Chol, intermediate -density
NU
lipoprotein cholesterol; VLDL -Chol, very-low-density lipoprotein cholesterol

Total-Chol, HDL-Chol, LDL-Chol, IDL-Chol and VLDL-Chol were measured by

MA

anion-exchange–high-performance liquid chromatography.

ND means Not Detected.

E D
PT
CE
AC

33
 ACCEPTED MANUSCRIPT

Table 3. Assesment of intra - and inter-assay coefficient of variation (CV) values for

lipoprotein analyses in pooled serum samples of healthy dogs measured by AEX –

HPLC

PT
Intra-assay (n=10) Inter-assay (n=6)

VLD VLD

RI
Units HDL LDL IDL HDL LDL IDL
L L

SC
Mean mmol/L 3.72 0.36 0.05
NU 0.04 4.32 0.59 0.03 0.06

0.11 0.01 0.00

SD mmol/L 0.031 0.005 0.003 0.002 0.007
0 9 5
MA

CV % 0.8 1.4 6.7 5.3 2.5 3.2 14.2 10.7

D

HPLC, high-performance liquid

E

chromatography
PT

HDL, high-density lipoprotein cholesterol

CE

LDL, low-density lipoprotein cholesterol

IDL, intermediate -density lipoprotein cholesterol

AC

VLDL, very-low-density lipoprotein cholesterol

34
 ACCEPTED MANUSCRIPT

Table 4. Means and reference intervals for serum total, HDL and LDL cholesterol in

40 healthy dogs.

2.5th 97.5th

units mean* Percentile Percentile Normality (P)

PT
(90% CI) (90% CI)

RI
Total 2.97 9.32 N: 0.116
mmol/L 5.33

SC
cholesterol (2.96-3.46) (7.98-9.35) Box-Cox: 0.628

HDL 2.79 6.57 N: 0.194

NU
mmol/L 4.25
cholesterol (2.79-3.12) (5.96-6.58) Box-Cox: 0.897
MA

LDL 0.16 3.28 N: <0.001

mmol/L 0.97
cholesterol (0.16-0.26) (2.98-3.29) Box-Cox: 0.921
D

*Calculated using untransformed

E
PT

data.

n = 40 dogs. Outliers were not detected by the Turkey’s test.

CE

Normality testing was assessed using the Anderson -Darling test on untransformed (N)
AC

and Box-Cox transformed values.

CI means confidence intervals.

The CI of the limits of the nonparametric reference interval was determined using a

bootstrap method.

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 ACCEPTED MANUSCRIPT

Table 5. Serum concentrations of total, HDL, LDL, IDL and VLDL cholesterol and

HDL:LDL ratio in healthy dogs and dogs with hypercholesterolemia

Hypercholesterolemia

Healthy (n=12) (n=23)

Media minimum-maximu Media minimum-maximu

PT
Unit n m range n m range

Total mmol/

RI
cholesterol L 4.69 3.39 - 6.25 10.80 8.22 - 27.00

SC
mmol/ NU
HDL L 3.98 3.00 - 5.29 5.99 2.83 - 7.15

mmol/
MA

LDL L 0.47 0.26 - 0.80 3.09 0.65 - 15.49

mmol/
D

IDL L 0.08 0.03 - 0.16 0.48 0.14 - 10.01

E
PT

mmol/

VLDL L 0.06 0.04 - 0.09 0.70 0.13 - 11.78

CE

HDL：LDL
AC

ratio Ratio 7.9 6.5 - 11.4 2.2 0.4 - 6.2

HDL, high-density lipoprotein cholesterol

LDL, low-density lipoprotein cholesterol

IDL, intermediate-density lipoprotein cholesterol

VLDL, very-low-density lipoprotein cholesterol

Since distributions were significantly different from Gaussian (Anderson -Darling

36
 ACCEPTED MANUSCRIPT

test, P<0.05) for many analytes, values were expressed as median and

minimum-maximum range.

PT
RI
SC
NU
MA
E D
PT
CE
AC

37
 ACCEPTED MANUSCRIPT

Highlights

 Anion-exchange high-performance liquid chromatography (AEX–HPLC) for

lipoprotein analysis was evaluated in dogs.

PT
 Analytic reproducibility and precision of AEX–HPLC were acceptable in dogs.

RI
 Reference intervals of lipoprotein measured by AEX–HPLC were established in

SC
dogs.

 There is significant difference in lipoprotein profiles between healthy and

NU
hypercholesterolemia dogs.


MA

AEX–HPLC can be used to evaluate lipoprotein profiles in dogs .

E D
PT
CE
AC

38