Cardiovascular Journal of Africa: Vol 28 No 3 (May/June 2017)

CARDIOVASCULAR JOURNAL OF AFRICA • Volume 28, No 3, May/June 2017

160

AFRICA

dysfunction in the porcine carotid and coronary arteries.

15,16

Individuals with elevated Hcy levels are therefore at increased risk

of atherosclerosis and cardiovascular disease.

17-19

It is not clear whether ghrelin protects vascular tissue from

injury that is secondary to risk factors such as high levels of

Hcy. Levels of vitamin B

and folic acid, which form part of

Hcy metabolism, are in inverse correlation with the total Hcy

level. Nutritional deficiency or insufficiency of these vitamins

increases the risk of hyperhomocysteinaemia.

20,21

Saliva, which is among the biological fluids used in the

diagnosis of diseases, is also used to monitor treatment.

Companies that manufacture devices for diagnostic purposes

allocate a significant share to research and development efforts

geared towards the measurement of multiple parameters in the

saliva. This is because saliva is the most important biological

fluid that could be an alternative to using blood for analysis.

This study aimed to compare ghrelin, obestatin, Hcy, vitamin

and folate levels in the serum and saliva samples of ischaemic

heart disease patients and those of healthy individuals.

Methods

Patients who had acute coronary syndrome or types 1 and

2 diabetes mellitus, were extremely obese [body mass index

(BMI)

35 kg/m

], or had undergone invasive revascularisation

in the previous three months were not included in the study.

Additionally, individuals were excluded if they were found to

have a normal coronary anatomy on angiography but had signs

of heart failure and branch blocks on electrocardiography.

The study was carried out on 33 patients (female/male:

48.5/51.5%) with IHD and 28 BMI- and age-matched healthy

subjects (female/male: 45.5/54.5%). All subjects, including the

controls, underwent coronary angiography. IHD was defined

as a

≥

50% diameter stenosis, as diagnosed by angiography. The

control group was defined as those having normal coronary

arteries, as diagnosed by angiography. Written consent was

obtained before the study, together with the institutional ethics

committee’s approval of the study protocol (dated 29 August

2007, issue no. 13).

Hypertension, family history of IHD and smoking were

present in 48.5, 36.4 and 33.4% of patients, respectively, in the

IHD group. In the control group, 25, 21.4 and 21.4% of patients

demonstrated these risk factors. None of the healthy controls

had a family history of obesity or a history of abdominal surgery

or gastrointestinal disease. They had taken no medication for at

least seven days before sample collection.

All subjects were advised not to eat, smoke or drink (except

water) during the night before the saliva and blood sample

collection. Approximately 2 ml of saliva and 5 ml of blood

were taken from the enrolled subjects by the standard procedure

described by Hosoda

et al

Total and acylated ghrelin levels in the serum and saliva

samples were measured using an enzyme linked immunosorbent

assay (ELISA) kit (Linco Research). As desacylated ghrelin level

was calculated by subtracting the acylated ghrelin value from the

total ghrelin value, care was taken to use the same commercial

kits throughout the study.

Serum and saliva obestatin levels were determined using a

human obestatin enzyme immunoassay (EIA) kit made by the

Bachem brand (Peninsula Laboratories, LLC: a member of the

Bachem group, California, USA). Serum and saliva Hcy levels

were measured using an axis homocysteine EIA kit. Levels of

vitamin B

and folate were determined by a Roche Elecsys 2010

hormone analyser.

Statistical analysis

The data were statistically analysed using the SPSS for Windows

15.0 software package. First, continuous variables were

checked for normality. Upon finding deviations from a normal

distribution, the Mann–Whitney

-test was used to compare

the groups. Correlations between blood and saliva values were

established by calculating the Spearman correlation coefficient.

Mean

SD and median (min, max) values are presented

as descriptive statistics. Frequencies and percentage values

are presented for the categorical data. Pearson’s chi-squared

continuity correlation analysis was used in the comparison. A

value

0.05 was accepted as significant.

Results

While the age, BMI, diastolic blood pressure, and triglyceride,

total cholesterol and low-density lipoprotein (LDL) cholesterol

levels were not significantly different between the control and

IHD groups (

0.05), systolic blood pressure (

0.019) and

high-density lipoprotein (HDL) cholesterol (

0.042) were

significantly lower in the IHD group (Table 1).

An intra-group comparison of serum and saliva levels of

total acylated and desacylated ghrelin, obestatin, vitamin B

and folate was conducted. These parameters were also compared

between the groups. Some differences were seen between serum

and saliva levels of the biochemical parameters when we carried

out intra- and inter-group comparisons (Table 1).

In the control group, saliva levels of total acylated and

desacylated ghrelin were higher than in the serum (

0.001).

Conversely, in the IHD group, serum levels were found to be

higher than in the saliva (

0.001) (Table 1, Fig. 1).

When serum and saliva levels of these parameters were

compared between the two groups, total ghrelin, desacylated

ghrelin and serum levels of Hcy and acylated ghrelin were found to

be higher in the IHD group (

0.001) (Table 1, Figs 1, 2). While

Control

IHD

Acyl ghrelin (pg/ml)

AG serum AG saliva

Fig. 1.

Serum and saliva acylated ghrelin levels of controls

and patients with ischaemic heart disease.