Effect of Honey on 50% Complement Hemolytic Activity in Infants with Protein Energy Malnutrition: A Randomized Controlled Pilot Study

Abstract

Protein energy malnutrition (PEM) is associated with a significant impairment of cell-mediated immunity and complement system, which may be responsible for the high incidence of infections among these patients. This study was designed to examine the effect of honey, as a natural substance, on the 50% complement hemolytic activity (CH50) in patients with PEM. Thirty patients with PEM and 20 healthy infants serving as controls participated in this study. The patients were randomized to receive either honey (group 1) or placebo (group 2), in addition to conventional nutritional rehabilitation therapy. Measurements of weight, midarm circumference, skin fold thickness, serum albumin, and CH50 were done for all patients before and after 2 weeks of rehabilitation. Before nutritional rehabilitation, the CH50 was significantly lower in the PEM groups compared with the control. However, after rehabilitation, the CH50 increased significantly in both PEM groups, compared with the pre-interventional state and with the controls. Moreover, the rise of CH50 was significantly more in the honey group compared with the placebo. On the other hand, the improvement in the anthropometric measures and serum albumin did not differ significantly between the honey and placebo groups after rehabilitation. Thus honey supplementation in patients with PEM increased the level of CH50. Whether this would have an effect on the frequency and severity of infections in patients with PEM needs further studies.

Introduction

P rotein energy malnutrition(PEM) remains as one of the most important health problems in developing countries.¹ In Egypt, the syndrome is relatively common. It stands as a major threat to health, growth, and development of infants.² PEM is associated with a significant impairment of cell-mediated immunity, phagocyte function, complement system, secretory immunoglobulin A antibody concentration, and cytokine production.³ The frequent dissemination of Candida and herpes simplex infection among malnourished infants and the high incidence of Gram-negative infections were suggested to be due to impaired cell-mediated immunity or the complement deficiency.⁴ The complement system plays an important role in host defense against infection and in most inflammatory processes. The standard 50% hemolytic complement activity (CH50) assay is the most commonly used method of screening patient sera for functional activity of the classical complement pathway.⁵ Availability of complement components is restricted by malnutrition, thereby affecting the capacity of professional phagocytes to engulf and eliminate pathogens.⁶ Haller et al.⁷ found that the plasma levels of CH50, C3, C9, and factor B were decreased—without a concomitant decrease of C4 and C5—in patients with PEM.

Honey is a natural substance with many benefits for nutrition and health.⁸ Among honey's benefits are its anti-inflammatory,⁹ antioxidant,^10
–12 and antimicrobial^13

–17 properties. Honey can stimulate the release of tumor necrosis factor-α and cytokines in monocytic cells.^18,19 It also increases the proliferation of B and T lymphocytes and neutrophils in vitro.²⁰ In addition, honey has been demonstrated to stimulate antibody production during primary and secondary immune responses against thymus-dependent and thymus-independent antigens.²¹ The aim of the present study was to verify whether the intake of honey, as a natural substance, during nutritional rehabilitation of patients with PEM could affect the plasma levels of CH50.

Subjects and Methods

All infants suffering from PEM and admitted to the Children's Hospital of Ain Shams University, Cairo, Egypt to receive conventional nutritional rehabilitation therapy were candidates for inclusion in the study. They were not enrolled in the study if either of the following criteria was present: (1) clinical evidence of infection or (2) use of any drug affecting the immune system within the past 3 months. Thirty eligible patients were recruited from the Inpatient Department of the Hospital in the period from February 2008 to March 2009. No one was lost to follow-up. Patients were randomized to receive either honey (group 1) or placebo (group 2), in addition to conventional nutritional rehabilitation therapy primarily based on the World Health Organization classification.²² Each group consisted of 15 patients. The patients were compared with 20 age- and sex-matched clinically healthy infants. The mean (±SD) ages in the honey and placebo groups were 12.27 (±5.28) and 13.4 (±4.2) months, respectively. The age and sex did not differ significantly between groups. The socioeconomic score (according to Park and Park²³), however, was significantly lower in the two PEM groups compared with the control group (5.80, 5.13, and11.02, respectively; P < .001).

The honey was supplied in an empirical dose of 2 mL/kg/day in two divided doses; each dose was diluted in an equal amount of water just before ingestion. Placebo was identically supplied and formulated except that it contained no bee honey. For placebo we used molasses, which is a honey-like substance made when cane sugar is processed. Molasses contains no fat and is composed mainly of sucrose, besides some fructose and glucose. Clover honey is composed mainly of fructose and glucose in about equal proportions.²⁴ One gram of molasses gives 4 kcal, compared with 3 kcal for 1 g of bee honey. So 1.8 g/kg (7.2 kcal/kg) molasses was used as a placebo, which is equivalent to the same caloric value of the bee honey given (2 mL of bee honey = 2.4 g, which also gives 7.2 kcal). Although this caloric contribution from either honey or molasses is very low in comparison with the total calories given, yet it was taken into consideration during the nutritional rehabilitation program.

The honey used was a pure unprocessed clover honey, collected from Al Mahala-Gharbia Governorate, Egypt. It was supplied directly from the beekeeper without heating or gamma irradiation and stored in dark containers at room temperature for use in the study. The honey was tested for the presence of Clostridium botulinum spores before use (no spores were detected). Examination of honey for C. botulinum spores was done by centrifugation and filtration of the supernatant, followed by culture on cooked meat.²⁵

Because the usual policy in our hospital is not to keep PEM patients who show improvement for more than 2 weeks, the primary end point of the study was 2 weeks after the start of treatment in order not to lose patients to follow-up and to ensure compliance with honey intake. The study was approved by the local ethical committee, and an informed consent was obtained from at least one parent or guardian of each subject before enrollment in the study. The patients studied were subjected to the following procedures:

Accurate measurement of weight, midarm circumference (MAC), and skin fold thickness (SFT). We used the weighted Z score as an indicator for wasting.²⁶ The Z score (or SD score) is the difference between the actual measurement and the median age- and sex-matched reference population divided by the SD of the reference population.²⁷ Wasting is defined as a Z score of less than −2 in a normally distributed reference population. The MAC (%) and the SFT (%) were also calculated as follows: the actual measure/expected measure for age (median or 50th percentile) × 100 according to the World Health Organization classification.²⁸

Laboratory investigations: complete blood count, total serum proteins and serum albumin, and complement hemolytic activity assay (CH50) using a CH50 enzyme-linked immunosorbent assay kit (purchased from DRG^® International, Inc., Mountainside, NJ, USA). The laboratory procedure was according to the instructions for use given in the DRG kit. The DRG CIC immune complex CH50 enzyme-linked immunosorbent assay is an immunoenzymatic colorimetric method for the quantitative determination of complement functionality. The normal CH50 value is 101–300.

After 2 weeks of nutritional rehabilitation the two groups of PEM patients studied were reevaluated using the same study procedures mentioned before.

Statistical analysis

The standard computer program SPSS for Windows (release 13.0; SPSS Inc., Chicago, IL, USA) was used for data entry and analysis. All numerical variables were expressed as mean ± SD values. Comparison of different variables in various groups was done using Student's t test and the Mann–Whitney test for normal and nonparametric variables, respectively. Wilcoxon signed rank tests were used to compare multiple readings of the same variables. The χ² test was used to compare frequency of qualitative variables among the different groups.²⁹

Results

Before nutritional rehabilitation the weighted Z score, MAC (%), SFT (%), percentages of hemoglobin (Hb) and lymphocytes, and serum levels of total protein and albumin were significantly lower in the PEM groups compared with the control, and there was no significant difference between the two PEM groups regarding these parameters (Table 1). After 2 weeks of nutritional rehabilitation, the weight, MAC (%), SFT (%), percentages of Hb and lymphocytes, and serum levels of proteins and albumin showed significant increase in both PEM groups compared with the pre-interventional state. The weight, MAC (%), SFT (%), and percentage of Hb in both PEM groups and the percentage of lymphocytes in the placebo group remained significantly lower than the control. On the other hand, no significant difference remained between the control and the two PEM groups regarding serum protein and albumin levels and between the control and the honey group regarding the percentage of lymphocytes (Tables 2 and 3).

Table 1.

Characteristics of the Studied Groups Before Nutritional Rehabilitation

Parameter	Group 1 (honey group)	Group 2 (placebo)	Control
Socioeconomic status (%)	5.80 ± 1.69	5.13 ± 1.40	11.02 ± 2.89^a
Weighted Z score	−3.07 ± 1.28	−3.84 ± 0.88	0.94 ± 0.67^a
MAC (%)	69.47 ± 13.07	65.00 ± 9.56	87.08 ± 7.50^a
SFT (%)	54.30 ± 12.99	55.54 ± 9.44	98.87 ± 13.65^a
Hb (g %)	8.41 ± 1.43	8.82 ± 1.08	12.81 ± 0.79^a
TLC (× 10³)	9.98 ± 2.85	9.46 ± 2.69	8.47 ± 1.49
Lymphocytes (%)	52.93 ± 9.12	50.8 ± 9.71	59.95 ± 3.72^a
PLT (× 10³)	296.8 ± 103.80	321.93 ± 69.90	333.95 ± 45.06
Protein (g/dL)	5.11 ± 0.77	5.56 ± 0.59	6.24 ± 0.39^a
Albumin (g/dL)	2.50 ± 0.68	2.92 ± 0.61	3.83 ± 0.25^a

Data are mean ± SD values.

P < .05 compared with either group 1 or 2.

Hb, hemoglobin; MAC, midarm circumference; PLT, platelets; SFT, skin fold thickness; TLC, total leukocytic count.

Table 2.

Anthropometric Measures and Laboratory Results of Groups After 2 Weeks of Nutritional Rehabilitation

Parameter	Group 1 (honey group)	Group 2 (placebo)	Control
Weighted Z score	−2.56 ± 1.21	−3.29 ± 0.77	0.94 ± 0.67^a
MAC (%)	73.09 ± 12.75	68.68 ± 9.24	87.08 ± 7.50^a
SFT (%)	63.37 ± 12.59	66.30 ± 8.57	98.87 ± 13.65^a
Hb (g %)	10.39 ± 0.79	10.4 ± 0.96	12.81 ± 0.79^a
TLC (× 10³)	10.59 ± 1.27	10.40 ± 1.69	8.47 ± 1.49^a
Lymphocytes (%)	57.53 ± 5.27^b	52.53 ± 6.51	59.95 ± 3.72^c
PLT (× 10³)	290.13 ± 76.34	330.4 ± 59.73	333.95 ± 45.06
Protein (g/dL)	6.30 ± 0.36	6.44 ± 0.28	6.24 ± 0.39
Albumin (g/dL)	3.70 ± 0.30	3.84 ± 0.41	3.83 ± 0.25

Data are mean ± SD values.

P < .05 compared with either group 1 or 2.

P < .05 compared with group 2.

Table 3.

Anthropometric Measures and Laboratory Results of Group 1 (Honey Group) Before and After 2 Weeks of Nutritional Rehabilitation

Parameter	Before	After	P^*
Weighted Z score	−3.07 ± 1.28	−2.56 ± 1.21	<.001
MAC (%)	69.47 ± 13.07	73.09 ± 12.75	<.001
SFT (%)	54.30 ± 12.99	63.37 ± 12.59	<.001
Hb (g %)	8.41 ± 1.43	10.39 ± 0.79	<.001
TLC (× 10³)	9.98 ± 2.85	10.59 ± 1.27	>.05
Lymphocytes (%)	52.93 ± 9.12	57.53 ± 5.27	<.001
PLT (× 10³)	296.8 ± 103.80	290.13 ± 76.34	>.05
Protein (g/dL)	5.11 ± 0.77	6.30 ± 0.36	<.001
Albumin (g/dL)	2.50 ± 0.68	3.70 ± 0.30	<.001

Data are mean ± SD values.

P > .05 is not significant; P < .001 is highly significant.

Before nutritional rehabilitation the initial CH50 was significantly lower in the PEM groups compared with the control group. However, the initial CH50 did not differ significantly between the honey and placebo groups (P > .05). Two weeks after rehabilitation, a significant rise of CH50, compared with the pre-interventional state and as well with the controls, occurred in both PEM groups. On the other hand, the rise of CH50 was significantly more in the honey group compared with the placebo (Table 4).

Table 4.

50% Complement Hemolytic Activity in the Protein Energy Malnutrition Groups Before and After 2 Weeks of Nutritional Rehabilitation

	CH50 (mean ± SD)
Group	Before	After	P
Group 1 (honey group)	77.0 ± 10.61	639.06 ± 121.43	<.01
Group 2 (placebo)	70.80 ± 8.72	287.93 ± 54.26	<.01
Control	155.55 ± 21.16^a

P < .001 compared with either group 1 or 2 before and after rehabilitation.

CH50, 50% complement hemolytic activity.

Discussion

Socioeconomic scoring showed significantly lower values in the PEM patients compared with controls. A low socioeconomic score increases the incidence of poor hygiene, poor housing, and inappropriate feeding; all these factors contribute to the development of malnutrition. Ferrari et al.³⁰ have previously discussed the role of the previous factors in the development of malnutrition.

In agreement with the Wellcome Trust's classification of PEM,³¹ the present study showed that the weight, before starting nutritional rehabilitation, was significantly lower in the PEM groups compared with the controls. Before nutritional rehabilitation, the SFT and the MAC, which reflect the state of muscle and subcutaneous fat,³² were also significantly lower in both PEM groups compared with the control group. However, after 2 weeks of nutritional rehabilitation, the weight, MAC, and SFT increased significantly in both PEM groups but still remained significantly lower than in the controls, probably because of the short period of follow-up. The significant increase of weight, MAC, SFT, and levels of serum proteins and albumin in both PEM groups after 2 weeks of rehabilitation indicates not only successful nutritional rehabilitation, but also that honey intake did not adversely affect the growth and serum proteins of PEM infants.

Before rehabilitation, the CH50 was significantly lower in both PEM groups compared with the healthy infants (controls). The same finding was previously observed by Chandra,³³ who found that serum hemolytic complement activity and C3 were significantly decreased in 35 malnourished children. Also, McMurray et al.³⁴ reported that both moderate and severe nutritional deprivations have been associated with impairment of complement functions. Similarly, Haller et al. ³⁵ noted a significant decrease of CH50, C3, C9, and factor B in PEM, and they attributed the increased susceptibility to infections in malnourished children to relative complement deficiency. It is hypothesized that reduced complement function in malnutrition may result from impaired synthesis and/or impaired complement activation.

In the present study there was a significant rise of CH50 in both PEM groups compared with the pre-interventional state. This agrees with Chandra,³³ who found that nutritional rehabilitation of malnourished children reversed the abnormalities of complement functions and that there was a consistent rise in CH50 and C3. He added that the low level of complement in undernourished children and its increase to normal range after dietary treatment suggest that the nutritional status of the individual can influence the complement system. Suskind et al.³⁶ found that the mean CH50 titer of children with kwashiorkor was significantly less than the control values on both day 1 and day 4 and that the titers rose, however, after day 4 and were significantly greater than the controls by day 50; on day 71 they fell to near control levels. These researchers suggested that the elevated CH50 activity levels on days 29 and 50 may express functionally the “rebound” elevation of serum complement protein concentrations previously observed by Sirisinha et al.³⁷ during convalescence of malnourished Thai children. In the present study, the levels of CH50 became significantly higher than the controls by day 14, and because the follow-up period was short, we were not be able to determine for how long the CH50 levels remained higher than the control values.

The results of the present study showed a significant increase of the CH50 and the lymphocyte percentage in the PEM group supplemented with honey compared with the placebo group. Moreover, the two PEM groups did not show a significant difference regarding the improvement in weight, MAC, SFT, and levels of serum proteins and albumin. This indicates that honey intake had a beneficial effect on the immune system of PEM patients. To our knowledge, no previous studies evaluated the effects of honey on the complement or the immune system in patients with PEM. However, in vitro immunomodulatory effects of honey were demonstrated,³⁸ and in the study of Al Waili and Haq²¹ oral honey was found to stimulate antibody production during primary and secondary immune responses against thymus-dependent (Escherichia coli) and thymus-independent (sheep red blood cells) antigens in mice. Honey was also found to stimulate inflammatory cytokine production from monocytes.¹⁹ In conclusion, honey supplementation in patients with PEM increased the level of CH50. Further studies are recommended to study the effects of honey on other immunologic markers in patients with PEM and to see whether this would have an effect on the frequency and severity of infections in such patients.

Footnotes

Author Disclosure Statement

M.A.A. developed the main idea, planned the work, supervised the clinical work, and edited the manuscript. M.F.N. helped establishing the main idea, supervised patient enrollment and follow-up, interpreted the data, and helped editing the manuscript. H.W.M. helped in planning the work. Z.A.E.-K. did the laboratory assessment and wrote the relevant section in the article. M.W.A.E.N. was responsible for follow-up of patients and helped interpreting the patient data. The authors have no financial or personal interests in any company or organization sponsoring the research currently or at the time the research was done.

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