GLYCATED HEMOGLOBIN IN CAMEL : MINIMAL CORRELATION WITH BLOOD GLUCOSE LEVEL

Glucose and glycated hemoglobin (Hb) in the blood of camel (Camelus dromedarius) and cow (Bos taurus) were analyzed and compared with human values. Camel displayed high blood glucose concentration (9.7±2.8 mM) but a low level of glycated-Hb (3.4± 0.23%).Cow blood samples did not show sufficient variations in glucose concentrations (5.7±0.73 mM) or glycated Hb levels (3.2± 0.11%) compared to human values. The low glycation of camel Hb at higher glucose concentrations suggest that certain factors protect the Hb from glycation at high glucose concentrations. Camel Hb also exhibited a higher electrophoretic mobility than normal hemoglobin of human or cow. Camel Hb migrated at a rate corresponding to that of human Hb-C. Bioinformatics tools were used to explore the biochemical basis for the difference in camel Hb migratory position and its apparent resistance to glycation.


INTRODUCTION
The Arabian camel is important in the Arabian and African deserts for economical and cultural reasons.The camel has many interesting physiological adaptations that enable it to survive the harsh desert environment (Bogin, 2000;Gaughan, 2011;Ouajd and Kamel, 2009).This fact has promoted several investigations to determine the "normal" values of several biochemical parameters in the camel, including blood enzymes, hematological parameters, blood metabolites, and others.There are considerable discrepancies in the reported values, possibly due to differences in the genetic, nutritional, and environmental conditions of the animal (Al-Rehaimi et al., 1989;Amin et al., 2007;Barakat and Abdel-Fattah, 1971;Bogin, 2000;Kataria et al., 2002;Mohamed and Hussein, 1999;Ouajd and Kamel, 2009).This paper focuses on two aspects of camel hemoglobin, the universal oxygen carrier in the blood of all living organisms, its glycation status and electrophoric mobility.Glycation proceeds via the non-enzymatic reaction of glucose with proteins, including Hb, resulting in the formation of glycation adducts (Bunn, 1981;Peacock, 1984).Several reports suggest that camel blood has normal to high concentrations of glucose compared to other animals and human (Al-Ali et al., 1988;Ali et al., 2006;Mohamed and Hussein, 1999;Nazifi et al., 1998;Yagil and Berlyne, 1977).However, no attempts have been made to correlate the extent of Hb glycation with blood glucose concentration in the camel.
The present study reveals that the normal blood glucose concentration in the camel is significantly higher than in human blood: despite this, camel Hb showed a lower degree of glycation.The mobility of camel Hb observed in capillary electrophoresis was consistent with human HbC, an inherited variant of HbA.

MATERIALS AND METHODS
Blood samples were collected from 11 camels and 5 cows in tubes containing Na 2 EDTA (10mg/ml of blood) as an anticoagulant, from a slaughterhouse in the Riyadh area, Saudi Arabia.Blood samples were placed on ice while transported to the laboratory for analysis.Hb typing was determined in all blood samples by capillary electrophoresis.These measurements were carried out on CAPILLARYS™ 2, Sebia.All measurements were made in accordance with the manufacturer's instructions.Blood glucose and glycated hemoglobin were analyzed by the Dimension® RxL Max® clinical chemistry system (Siemens Healthcare Diagnostics Inc.).Multiple sequence alignment was carried out using MAFFT Multiple Sequence Alignment.The sequence of the camel Hb chain A (NCBI accession no P63106.1)was aligned with human Hb Chain A (NP_000508.1) and cow Hb chain A (NP_001070890.2).Similarly, the B chain of camel Hb (P68231.2) was aligned with human HbB chain (000509.1)and cow HbB chain (NP_001103977.1).The alignment output was color-coded according to ther identities.
Sequence homologies between the chains of Hb (camel versus human or cow) were calculated by using Blast-2-seq (http://blast.ncbi.nlm.nih.gov).The query and subject sequences were added in FASTA format and BLASTp was conducted.
The isoelectric points of A and B chains of camel, human and cow were computed using the ProtParam tool (http://web.expasy.org/protparam/).pKa values and surface exposure of lysines were calculated for the A and B chains of camel and hu-man using the PROPKA Web Interface (http://propka.ki.ku.dk/)

RESULTS AND DISCUSSION
Table 1 shows blood glucose and glycated Hb (HbA1c) values for camel and cow.Camel showed higher blood glucose levels than cow (9.7 mM versus 5.7 mM), but a similar concentration of HbA1c (3.7% versus 3.2%).Even though the blood parameters in cattle are subjected to seasonal variations, the results were consistent with other studies, suggesting that camel had high blood glucose and a low level of Hb glycation (Abdalla et al., 1988;Al-Ali et al., 1988;al-Ali et al., 1990;Amin et al., 2007).
The glucose concentration in camel blood is high by human standard, and is observed only in unmanaged diabetic cases.However, the HbA1c in camel was lower than expected for a human with similar high blood glucose level.In humans, the HbA1c fraction is universally accepted as a long-term indicator The square shows the actual data and the dashed line shows the calculated values for human, based on equation as described by (Nathan et al., 2008).
Glycation of hemoglobin has been reported to proceed via a non-enzymatic reaction; a reaction between amino groups of Hb and sugars leading to the formation of stable derivatives via the Amadori reaction (Bunn, 1981;Peacock, 1984).The linkage of sugar to the N-terminal valine residue of the β-chain of Hb gives rise to HbA1c, a type of Hb commonly associated with diabetes (Nathan et al., 1984;Rohlfing et al., 2002;Soranzo, 2011).The extent of HbA1c formation is directly related to the blood glucose levels in humans (Nathan et al., 2008), and possibly in other animals (Shahbazkia and Nazifi, 2008;Shahbazkia et al., 2010).
In this study, the observed range of glucose in camel blood was wide enough to permit the examination of a possible correlation between HbA1c and A number of possible scenarios, physiological and/or chemical, could account for such results.For example, camel erythrocytes may restrict glucose transport and therefore camel hemoglobin might be exposed to lower glucose concentrations than suggested by the blood glucose.It is also possible that the redox state of camel blood or the internal environment of erythrocytes as well as variations of blood pH may contribute to the reduction of HbA1c levels (Kurata et al., 1993).Other scenarios are also possible, but these factors, together with other posttranslational modification, might be responsible for protecting camel Hb against glycation.
The mobility of camel Hb was examined by capillary electrophoresis (Fig. 2).Camel Hb consistently migrated at a position very close to that of human Hb C (Fig. 2A): camel Hb migrated 250±5 seconds in all samples examined (n=11).By contrast, cow Hb migrated to a position comparable to that of normal human HbA: all samples examined (n=5) migrated 155±5 seconds (Fig. 2B).
The biochemical basis for the migratory position of camel Hb, as well as the apparent resistance of camel Hb to glycation was investigated further via comparative analyses using bioinformatics tools: for sequence alignments (Fig. 3), charged amino acids compositions (Table 2), and 3D structural similari-ties (Fig. 4) between camel and human Hb.The difference in electrophoretic mobility between camel versus human Hb could be due to a difference in net electrical charges, shape, or exposure of amino acids.Protein glycation could be modulated by a number of glycation sites, their pKa values, and their surrounding environment and surface exposure.
The alignment of camel, cow and human hemoglobin chain A (Fig. 3A) and chain B (Fig. 3b) suggests that these chains are fairly conserved in these species, with more than 84% identity.A particularly interesting observation was that the amino acids around the positively charged amino groups are fairly conserved; it has been reported that such residues play an important role in the susceptibility of glycation (Shapiro et al., 1980;Baynes et al., 1989) Camel Hb appeared to be more basic than either that of humans or cow (Table 2).It also contains more positively and negatively charged amino acids than either humans or cow.However, minor differences in the charges alone may not account for the difference in electrophoretic mobility between camel and human Hb: the cow and human hemoglobin migrated nearly to the same position, despite the net differences in the number of charged amino acids as well as their calculated isoelectric points.
Fig. 4 superimposed 3D structures of camel and human Hb, with special emphasis on the lysine residues, the most probable sites of glycation.The two structures were virtually identical.All lysines of chain A and B of human versus the corresponding chains of camel Hb were very similar in pKa values and burial percentage; only one lysine of human chain A and two lysines of chain B had significant variations in pKa values (1 unit lower) than corresponding residues in camel Hb.These data indicated that the lysines of camel Hb might be more nucleophilic than those of human Hb, and thus more prone to glycation.
In summary, camel Hb appeared to be less susceptible to glycation than human Hb, and it also migrated to a position close to that of human HbC.An analysis of the number of charged residues, their pKa values, and surface exposure failed to provide an explanation for either of these observations.

Fig. 1 .
Fig. 1.Extent of glycation versus blood glucoseThe figure shows actual data of camel blood samples (n=11).Camel blood glucose vs extent of glycation was determined.The square shows the actual data and the dashed line shows the calculated values for human, based on equation as described by(Nathan et al., 2008).

Figs. 2
Figs. 2 A & B: Panel A-migratory position of camel Hb.Panel B shows migration of cow Hb under the same conditions.In both panels, the migratory positions of human Hb are shown for comparison.

Table 1 .
Blood glucose and glycated Hb values for camel and cow.

Table 2 :
Comparison of positively and negatively charged residues in the camel, human and cow Hb.