**3. Subjects and methods**

#### **3.1 Subjects**

Twenty patients with type 1 diabetes mellitus, aged 3–18 (mean 10.95 years) and ten healthy non-diabetic children and adolescents, aged 1–17 (mean 8.5 years) were studied. All subjects were within 68–118% and 77–125% of their ideal body weight and height, respectively. The mean BMI of patients and controls were 22.60 and 23.15, respectively. All patients with diabetes had a mean glycosylated hemoglobin of 9.9% (range = 7–15%). The sex ratio in patients and controls was 1:1. The patients were recruited from the regular attendants of the children clinic of the National Institute of Diabetes in Cairo, Egypt. The study was approved by the local ethical committee, and an informed written consent was obtained from at least one parent of each subject before the study. All patients were receiving three insulin injections per day, each consisting of a mixture of a medium-acting insulin (isophane NPH) and a short-acting soluble insulin (human Actrapid).

#### **3.2 Methods**

All patients were primarily diagnosed with type 1 insulin-dependent diabetes mellitus by measuring the serum level of C-peptide on presentation [the patient was considered suffering from insulin-dependent diabetes mellitus type 1 if the C-peptide level was below 0.4 ng/dl (Connors, 2000)]. All subjects were subjected to the following:


inflammatory (Al Waili & Boni, 2003), anti- oxidant (Frankel et al., 1998; Gheldof & Engeseth, 2002; Gross et al., 2004) and anti-microbial effects (Molan, 1992; Steinberg et al., 1996; Molan, 1997; Theunissen et al., 2001). Further-more, several studies have shown that honey produced an attenuated postprandial glycemic response when compared with sucrose in both patients with diabetes and normal subjects (Ionescu-Tirgoviste et al., 1983;

C-peptide is considered to be a good marker of insulin secretion and has no biologic activity of its own (Ido et al., 1997). Measurement of C-peptide, however, provides a fully validated means of quantifying endogenous insulin secretion. C-peptide is co-secreted with insulin by the pancreatic cells as a by-product of the enzymatic cleavage of proinsulin to insulin. Consequently, serum C-peptide level can be used as a true indicator of any change in the

Several studies were performed in healthy and in type 2 diabetic patients to evaluate the effects of honey on the insulin and C-peptide levels, and the results were controversial

The aim of this work was to compare the effects of honey, sucrose and glucose on plasma glucose and C-peptide levels in children and adolescents with type 1 diabetes mellitus.

Twenty patients with type 1 diabetes mellitus, aged 3–18 (mean 10.95 years) and ten healthy non-diabetic children and adolescents, aged 1–17 (mean 8.5 years) were studied. All subjects were within 68–118% and 77–125% of their ideal body weight and height, respectively. The mean BMI of patients and controls were 22.60 and 23.15, respectively. All patients with diabetes had a mean glycosylated hemoglobin of 9.9% (range = 7–15%). The sex ratio in patients and controls was 1:1. The patients were recruited from the regular attendants of the children clinic of the National Institute of Diabetes in Cairo, Egypt. The study was approved by the local ethical committee, and an informed written consent was obtained from at least one parent of each subject before the study. All patients were receiving three insulin injections per day, each consisting of a mixture of a medium-acting insulin (isophane NPH)

All patients were primarily diagnosed with type 1 insulin-dependent diabetes mellitus by measuring the serum level of C-peptide on presentation [the patient was considered suffering from insulin-dependent diabetes mellitus type 1 if the C-peptide level was below

1. Anthropometric measures including weight in kg and height in cm which were plotted

2. Oral sugar tolerance tests using glucose, sucrose and honey in three separate sittings: After an overnight fast (8 h) and omission of the morning insulin dose, a calculated amount of each sugar (amount = weight of subject in kg X 1.75 with a maximum of 75 g) (William & Ruchi, 2005) was diluted in 200 ml water and then ingested over 5 min in a

0.4 ng/dl (Connors, 2000)]. All subjects were subjected to the following:

Shambaugh et al., 1990; Samanta et al., 1985; Al Waili, 2004; Agrawal et al., 2007).

insulin level, which is the main determinant of plasma glucose level.

(Bornet et al., 1985; Elliott et al., 2002; Watford, 2002; Al-Waili, 2003).

**2. Aim of the study** 

**3.1 Subjects** 

**3.2 Methods** 

**3. Subjects and methods** 

and a short-acting soluble insulin (human Actrapid).

against percentiles for age and sex.

random order, on separate mornings 1 week apart. The honey dose for each patient was calculated based on the fact that each 100 gm of the honey used in this study contained 77.3 gm sugars. So if a patient weighs for example 20 kg, he/she should receive 20 x 1.75 = 35 gm sugar which will be present in (35 x 100) ÷ 77.3 = 45.3 gm honey. Venous blood was sampled just before ingestion and then every 30 min postprandial for 2 h thereafter. Samples were left to clot, centrifuged and glucose assay was performed chemically on the Synchron CX5 autoanalyzer (Beckman instruments Inc.)1.


Area under glycemic curve of test food Glycemic index◌ً of the food Jenkins, 1987<sup>=</sup> Area under glycemic curve of glucose


Maximal increment produced by the sugar tested Peak incremental index Maximal increment produced by glucose

Maximal increment is the difference between the peak point and the fasting point.

#### **3.3 Statistical analysis**

Standard computer program SPSS for Windows, release 13.0 (SPSS Inc., USA) was used for data entry and analysis. All numeric variables were expressed as mean ± standard deviation (SD). Comparison of different variables in various groups was done using student t-test and Mann–Whitney test for normal and non-parametric variables, respectively. Wilcoxon signed

<sup>1</sup> Beckman: 2005, kraemerBLW, Brew, CA 92621, USA.

<sup>2</sup> Biosource Europe S.A—Rue de lindustrie, 8-B-1400-Nivelles-Belgium.

Honey and Type 1 Diabetes Mellitus 427

No significant difference was found between patients (diabetics) and controls (nondiabetics) as regards the age and anthropometric measures (table 4.1). The mean age of subjects in the diabetic and non- diabetic groups was 11.3 and 8.5 years, respectively, with no statistically significant difference between groups (P > 0.05). The mean weight %, height % and body mass index did not also differ significantly between diabetics and nondiabetics (93.6%, 99.2%, 22.6 versus 94%, 98.2%, 23.1, respectively; P > 0.05). The mean plasma glucose level at 0 (fasting) and 30 min postprandial (i.e. 30 min after intake of glucose, sucrose or honey) did not differ significantly between subjects in both groups (diabetics and non-diabetics) (Tables 4.2 - 4.5) (P > 0.05). In non-diabetics (control), as shown in tables 4.2 and 4.3, the mean plasma glucose level 60, 90 and 120 min after intake of honey became significantly lower than after either glucose or sucrose (P< 0.05). Similarly, as shown in tables 4.4 and 4.5, there was a statistically significant decrease of plasma glucose in diabetics at 60, 90 and 120 min after honey intake, when compared with either glucose or sucrose (P< 0.05). The glycemic index (GI) and the peak incremental index (PII) of either sucrose or honey did not differ significantly between patients and controls (P > 0.05). On the other hand, both the GI and PII of honey were significantly lower when compared with sucrose in patients and controls. In non-diabetics, the glycemic index (GI) of honey was 0.69 compared to 1.32 for sucrose, with statistically significant difference (P< 0.05). In diabetics, the GI of honey was also significantly lower than that of sucrose (o.61 versus 1.19, respectively; P< 0.001) (table 4.6; figure 4.1). The PII of honey in non-diabetics was 0.61, compared to 1.25 for sucrose (P< 0.05). In diabetics, the PII of honey was also significantly lower than that of sucrose (0.60 versus 1.10,

The mean (±SD) fasting C-peptide of patients and controls were 0.15 (±0.13) and 1.91 (±0.77) ng/ml, respectively (P< 0.001). All diabetic patients had a basal C-peptide level less than 0.7 ng/ml. In diabetics, although honey intake resulted in increase in the mean level of Cpeptide, yet this increase was not statistically significant when compared with either glucose or sucrose (P> 0.05) (Table 4.8; figure 4.3). On the other hand, in non-diabetics, honey produced a statistically significant higher C-peptide level, when compared with either

Variable Diabetics Non-diabetics P Age (yr) 11.30 ± 4.80 8.50 ± 5.38 >0.05 Weight % 93.60 ± 13.82 94.00 ± 14.28 >0.05 Height % 99.20 ± 13.01 98.20 ± 11.14 >0.05 BMI 22.59 ± 5.50 23.14 ± 2.90 >0.05

Table 4.1 Age and anthropometric measures in diabetics and non-diabetic controls

**4. Results** 

respectively; P< 0.001) (table 4.7; figure 4.2).

glucose or sucrose (P< 0.05) (Table 4.8; figure 4.4).

P > 0.05 is non significant BMI: Body Mass Index

(mean ± SD)

rank tests were used to compare multiple readings of the same variables. Chi-square (χ2) test was used to compare frequency of qualitative variables among the different groups (Daniel, 1995).

Fig. 3.1 Oral glucose tolerance curve of one of our patients

For calculation of the area under honey curve (AUC) =A1+A2+A3+A4

A1 is a triangle = 1/2 base x height = 1/2(X2 - X1) x (Y1 - X2) = ½(30) x (144 – 89) = 15 x 55 = 825

A2 is a trapezoid = 1/2 sum of the parallel sides (heights) x base

$$= 1/2[(\mathbf{Y}\_1 \cdot \mathbf{X}\_2) + (\mathbf{Y}\_2 \cdot \mathbf{X}\_3)] \times (\mathbf{X}\_4 \cdot \mathbf{X}\_5) = 1/2[(144 - 89) + (225 - 89)] \times 30 = 1/2(55 + 136) \times 30 = 1/2(191) \times 30 = 2865$$

A3 is a trapezoid = 1/2 sum of the parallel sides (heights) x base

$$= 1/2[(\mathbf{Y}\_2 - \mathbf{X}\_3) + (\mathbf{Y}\_3 - \mathbf{X}\_4)] \times (\mathbf{X}\_4 \cdot \mathbf{X}\_3) = 1/2[(225 - 89) + (245 - 89)] \times 30 = 1/2(136 + 156) \times 30 = 1/2(292) \times 30 = 146 \times 30 = 4380$$

A4 is a trapezoid = 1/2 sum of the parallel sides (heights) x base

$$\mathbf{x} = 1/2 [(\mathbf{Y}\_3 - \mathbf{X}\_4) + (\mathbf{Y}\_4 - \mathbf{X}\_5)] \times (\mathbf{X}\_3 \cdot \mathbf{X}\_2) = 1/2 [(245 - 89) + (128 - 89)] \times 30 = 1/2 (156 + 39) \times 30 = 21/2 (195) \times 30 = 2925$$

$$\text{AUC} = \text{A}\_1 + \text{A}\_2 + \text{A}\_3 + \text{A}\_4 = 825 + 2865 + 4380 + 2925 = 10995$$
