Introduction

Diabetes mellitus (DM) is characterized by impaired glucose metabolism which is attributed either to inadequate synthesis of insulin as in Type I DM or by severe insulin resistance associated with defects in insulin receptor as in Type II DM [1, 2]. Increased glycosylated hemoglobin (HbA1c) is a characteristic feature of diabetes [3, 4] and pathies associated with diabetes have been well documented [5, 6]. However, more recently it has also been noted that the erythrocytes from Type II diabetic patients show significantly altered characteristics. These include: decreased/reduced life-span, change in deformability, increased red cell aggregation, and reduced membrane cholesterol, sphingomylein and phosphotidylcholine, and decreased sialic acid content [7–9]. It has also been reported that the intracellular concentrations of intermediates of glycolysis change significantly in the RBCs of diabetic patients. In particular the concentration of glucose, G6P, F6P and lactate increased significantly, whereas those of FBP, DHAP + GAP, and 3PGA decreased [10]. More recent studies from our laboratory have shown that the activities of enzymes of glycolytic pathway changed significantly in RBCs from Type II diabetic patients. In particular the levels of lactate dehydrogenase (LDH) increased significantly [11].

Lactic acidosis is an established feature of diabetes and LDH plays a crucial role in conversion of pyruvate to lactate; as cited above the levels of lactate are significantly high which is consistent with our observation on increased levels of LDH [11, 12]. In normal healthy individuals erythrocytes contribute to about 20% of the plasma levels of lactate [13]. Obviously, as cited above [12] increased levels of lactate in the diabetic RBCs will lead to lactic acidosis.

The increased levels of LDH which we reported earlier could be either due to intrinsic changes and/or altered kinetics properties of the enzyme. We tried to illustrate these points by studying the substrate saturation, substrate binding and substrate inhibition characteristic of the enzyme from diabetic erythrocytes in comparison with those from normal healthy individuals. The results of these investigations are described in the present communication which indicates that indeed significant changes were noted in the kinetics properties of LDH from diabetic RBCs.

Materials and Methods

Chemicals

Sodium salt of pyruvic acid and NADH (disodium salt) were purchased from SRL, Mumbai, India. All other chemicals were of analytical reagent grade and were purchased locally.

Sample Collection

3.0 mL blood samples were collected in vacutainer tubes containing EDTA from six normal healthy individuals and six Type II diabetic patients after written informed consent, as per guidelines approved by the Institutional Ethics Committee. For the normal male volunteers the mean age was 40.3 ± 2.01 years (ranging from 33 to 45 years) and the mean blood sugar level was 95.30 ± 3.75 mg/dL (ranging from 91 to 107 mg/dL). The mean age of the diabetic patients was 45.8 ± 3.89 years (ranging from 33 to 53 years) and the mean blood sugar level was 326.9 ± 22.32 mg/dL (ranging from 223 to 422 mg/dL). The samples were centrifuged immediately at 0–4 °C at 4000 rpm to sediment RBCs. The RBCs were washed 3 times with physiological saline (0.9% NaCl) and then were reconstituted to original volume with physiological saline. Known aliquots were diluted 1:10 in 10 mM sodium phosphate buffer, pH 7.4 as described previously and the lysate was used within 2–3 h for kinetics studies [11, 14]. The RBC count was determined in hemocytometer using separate aliquots.

Enzyme Kinetics

The basic assay mixture contained: 50 mM sodium phosphate buffer, pH 6.5, 0.8 mM sodium pyruvate and 0.18 mM NADH in a total volume of 1.0 mL. 10 μL RBC lysate was used as the source of the enzyme [11, 14].

With pyruvate as the substrate, concentration of NADH was kept constant at 0.18 mM and the concentration of pyruvate was varied from 0.08 to 1.44 mM in 12 steps. To study the kinetics parameters for NADH as substrate, concentration of sodium pyruvate was kept constant at 0.8 mM and the concentration of NADH was varied from 0.015 to 0.27 mM in 12 steps. The reaction was monitored at 25 °C by recording decrease in optical density at 340 nm at 5 s intervals for up to 1 min in a LABINDIA analytical UV/VIS spectrophotometer model UV 3000+ and the linear rates of reaction were taken into consideration for calculations of the activity. The activity is expressed as nmoles of substrate transformed/min/106 RBCs.

Data Analysis

Computer analysis of substrate kinetics data was carried out using Sigma Plot version 6.1 to obtain Lineweaver–Burk, Eadie–Hofstee and Eisenthal and Cornish-Bowden plots for calculation of Km and Vmax values [15]. The Km and Vmax values obtained by the three methods were in close agreement and were averaged for final presentation of the data. Component with low Km is designated as component I and the component with high Km designated as component II (e.g. see Tables 1, 2; Figs. 2, 3).

Table 1 Km and Vmax Values of Erythrocyte Lactate Dehydrogenase from Type II Diabetic Patients
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Table 2 Apparent Kcat and Kcat/Km values of erythrocyte lactate dehydrogenase from Type II diabetic patients
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Hill plot analysis was carried out to find out the substrate binding properties while Murray plot analysis was performed to determine the values of Ki; the concentration of substrate inhibition constant [15].

The apparent Kcat values were calculated from the corresponding Vmax values essentially according to methods described previously [16–18]. Thus the apparent Kcat represents

$$Apparent\,Kcat = \frac{{\text{Vmax}}\,{\text{in}}\,{\upmu {\text {mole}}}\,{\text{of}}\,{\text{substrate}}\,{\text{transformed}}\,{\text{per}}\,{\text{second}}}{1.42 \times 10^{4}}$$

where 1.42 × 104 is the molecular weight of lactate dehydrogenase [19], and the values are expressed as Kcat × 10−8. Similarly, values of Kcat/Km are given as Kcat/Km × 10−8. Kcat represents the number of substrate molecules turned over per enzyme molecule per second and hence Kcat may also be considered as the turnover number. Kcat/Km is thought to be a measure of enzyme efficiency. Either a high value of Kcat (rapid turnover) or a low value of Km (high affinity for substrate) will increase the catalytic efficiency [16].

Statistical analysis of the data was carried out using GraphPad Prism version 5 and p values less than 0.05 were considered statistically significant.

Results

The substrate saturation curves for pyruvate and NADH are shown in Fig. 1. The corresponding Lineweaver–Burk, Eadie–Hofstee and Eisenthal-Cornish-Bowden plots were obtained from substrate saturation kinetics data. These plots for pyruvate as the substrate for control and diabetic RBCs are shown in Fig. 2a–c respectively. Similar plots for NADH as the substrate are shown in Fig. 3a–c respectively. As is evident, (Fig. 2) for pyruvate as the substrate the activity resolved in a single component in both the groups. By contrast, the picture was very different for NADH as the substrate. Thus in the control group the activity resolved in the single component whereas for the diabetic group two components, one with low Km low Vmax and second one with high Km and high Vmax was apparent (Fig. 3). The Km and Vmax values were calculated by the above three methods and averaged for final presentation of data.

Fig. 1
figure 1

Substrate saturation curves for Na Pyruvate and NADH for lactate dehydrogenase from Control and Diabetic erythrocytes. The enzyme activity v (nmoles substrate transformed/min/106 RBCs) is plotted on abscissa versus substrate concentration (mM) on the ordinate. The results are typical of 6 independent observations

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Fig. 2
figure 2

Corresponding Lineweaver–Burk (a), Eadie-Hoffstee (b) and Eisenthal-Cornish-Bowden (c) plots for lactate dehydrogenase from Control and Diabetic erythrocytes with Na pyruvate as the substrate. For Lineweaver–Burk plots (a) the reciprocal of enzyme activity (1/v) is plotted on abscissa versus reciprocal of substrate concentration (1/[S]) where substrate concentration is in mM. For Eadie-Hoffstee plot (b) the reaction velocity, v is plotted on abscissa versus v/[S] on the ordinate. In the Eisenthal-Cornish-Bowden plots (c) the reaction velocity, v is plotted on abscissa versus substrate concentration is in mM on negative scale. The results are typical of 6 independent observations

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Fig. 3
figure 3

Corresponding typical Lineweaver–Burk (a), Eadie-Hoffstee (b) and Eisenthal-Cornish-Bowden (c) plots for lactate dehydrogenase from Control and Diabetic erythrocytes with NADH as the substrate. For Lineweaver–Burk plots (a) the reciprocal of enzyme activity (1/v) is plotted on abscissa versus reciprocal of substrate concentration (1/[S]) where substrate concentration is in mM. For Eadie-Hoffstee plot (b) the reaction velocity, v is plotted on abscissa versus v/[S] on the ordinate. In the Eisenthal-Cornish-Bowden plots (c) the reaction velocity, v is plotted on abscissa versus substrate concentration is in mM on negative scale. The results are typical of 6 independent observations

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As can be noted from data in Table 1, in the diabetic group, the Km for pyruvate was unchanged. However, consistent with our earlier report [11] the value of Vmax was significantly high (53% increase). With NADH as the substrate, in the control group the Km was 69 μM and the Vmax was comparable to that noted with pyruvate as the substrate. In the diabetic group the component with low Km (Component I) showed a very low Km i.e. 9 μM. However, the Vmax was almost comparable to that noted for pyruvate in the control group as well as that noted for control with NADH as the substrate. Even for component II, the Km was significantly lower than that noted for the control group (47 µM against 69 µM), however the Vmax was significantly high (Table 1).

In view of the observed changes in the Km and Vmax, it was of interest to find out how these factors influenced the catalytic efficiency of the enzyme. This was determined in terms of apparent Kcat and apparent Kcat/Km values. The results are given in Table 2. As can be noted, with pyruvate as the substrate the apparent Kcat as well as apparent Kcat/Km values increased significantly in the diabetic patients (53 and 51% increase respectively). With NADH as the substrate the apparent Kcat value for component I was close to that noted for pyruvate in the control group but the value of apparent Kcat/Km was about seven folds higher. For component II the value for apparent Kcat was comparable to that of pyruvate in the control group but the apparent Kcat/Km values almost doubled (Table 2).

The substrate binding characteristics as derived from Hill Plots (Fig. 4) are given in Table 3. Thus for sodium pyruvate as the substrate, the enzyme from both the groups bound one molecule of substrate in low concentration range whereas in the high substrate concentration range three molecules of the substrate were bound. The transition substrate concentration for the two groups was comparable i.e. 250 and 240 μM. With NADH as the substrate the enzyme from both the groups bound one molecule of the substrate in the low concentration range. In the high concentration range the enzyme bound two molecules of substrate in the control group; by contrast in the diabetic group three molecules of substrate were bound. The transition concentrations also differed significantly. For the control group the transition concentration was 54 μM whereas in the diabetic group this value shifted to higher concentration of 91 μM (e.g. see Fig. 4).

Fig. 4
figure 4

Corresponding Hill plots for lactate dehydrogenase from Control and Diabetic erythrocytes with Na pyruvate and NADH as the substrate. The values of log[v/Vmax − v] on abscissa are plotted against log substrate concentration (mM). Perpendicular line indicates point of Transition Concentration. The results are typical of 6 independent observations

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Table 3 Hill plot analysis of erythrocyte lactate dehydrogenase activity from Type II diabetic patients
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In the next set of experiments we tried to evaluate substrate inhibition characteristics based on Murray plots. Typical Murray plots are shown in Fig. 5 and the values of Ki are given in Table 4. As can be noted, there was marginal but reproducible decrease in the Ki value for Na Pyruvate (23% decrease). The picture was opposite for NADH as the substrate. Thus the value of Ki in the control group was 1.15 mM; this value became almost three fold higher in the diabetic group.

Fig. 5
figure 5

Corresponding Murray plots for lactate dehydrogenase from Control and Diabetic erythrocytes with Na pyruvate and NADH as the substrate. The values of reciprocal of reaction velocity (1/v) is plotted on abscissa versus substrate concentration (mM) on the ordinate. The results are typical of 6 independent observations

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Table 4 Murray plot analysis of erythrocyte lactate dehydrogenase activity from Type II diabetic patients
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Discussion

From the data presented it is clear that with pyruvate as the substrate the Km of LDH from diabetic erythrocytes was unchanged. Our observations on the Km for pyruvate agree with the values reported in the literature [20]. Interestingly, however, the Vmax value increased by 52% in the diabetic group which agrees well with our previously reported observation [11]. Kinetics studies with NADH as the substrate displayed an entirely different picture. Thus the control group showed presence of a single component whereas in the diabetic group the system resolved in two components: one with low Km and the second one with high Km.

From survey of literature we could not get information regarding the Km of LDH from RBCs; apparently the major emphasis has been to delineate the kinetics properties with pyruvate as the substrate. Thus it would appear that we are reporting for the first time the substrate saturation kinetics properties with NADH as the substrate. As is evident (Table 1; Fig. 3) in the control group the Km for NADH was about half that for pyruvate. This would imply that NADH may not be rate limiting and reaction can proceed in the forward direction towards formation of lactate. In the case of diabetic RBCs component I showed about 7 times lower Km compared to that in the control groups. Even for component II the Km was lower by 32%, however, the Vmax was high. The results thus suggest that the catalytic efficiency of the enzyme from diabetic RBCs is significantly high. This is exemplified by data in Table 2. Thus for pyruvate the Kcat and Kcat/Km values were significantly high. However, most importantly with NADH even component I showed Kcat values comparable to that of control and the Kcat/Km value increased seven folds. For component II also Kcat value was about 30% higher and that of Kcat/Km increased by 86%. Even substrate binding characteristics for NADH changed significantly. Thus the transition concentration increased from 54 to 91 μM but in higher concentration range the enzyme from diabetic erythrocytes bound 3 molecules of NADH as against 2 molecules bound by enzyme from control group; in lower concentration range enzyme from both the group bound only one molecule of NADH. These changes are once again consistent with enhanced catalytic activity of the enzyme from diabetic RBCs (Table 3). One additional feature was increased resistance to inhibition by pyruvate in the diabetic group where the Ki almost doubled; there was only marginal decrease in Ki for NADH (Table 4).

Taken together the results suggest that in diabetic erythrocytes there are significant alterations in the kinetic properties of lactate dehydrogenase which are favorable for efficient conversion of pyruvate to lactate. As is well recognized, the tissues which have predominately anaerobic glycolysis convert pyruvate to lactate which is released in the plasma. In normal individual RBCs contribute about 20% of the plasma lactate level [13]. Obviously these levels would be significantly higher in diabetes. As can be inferred from the data presented increased catalytic efficiency together with increased resistance to inhibition by higher concentrations of pyruvate are the major influencing factors.

It has been reported that LDH 1 and LDH 2 are the major isoforms in the erythrocytes from normal individuals and make up about 80% of the total activity. It has been reported that the Km for heart type of LDH (LDH 1 and LDH 2) is in the range of 0.1 mM whereas that of muscle type of LDH (LDH 4 and LDH 5) is in much higher ranging from 0.35 to 3 mM. It may therefore be inferred that the enzyme in the diabetic erythrocyte shows entirely different and unique characteristics especially with NADH as the substrate. Non-enzymatic glycosylation is a distinguishing feature of diabetes. It is possible but not clear if such structural changes could influence the catalytic activity of LDH. Alternately, it is possible that the LDH isozyme pattern might have changed in the diabetic RBCs. These possibilities need to be ascertained by more direct experiment. Nonetheless data of our present studies emphasize the significant role of altered kinetic properties of LDH in diabetic lactic acidosis.