**6. Patterns of LDH isoenzymes and their interpretation**

thoroughly three times in twice their volume, and the supernatant discarded. The cells were then lysed with the addition of three volumes of cold distilled water and centrifuged at

Chicken embryonic organs/tissues were taken from 9-day-old chicken embryo (n = 6), washed in buffer saline, and homogenated in 50 mM Tris-HCl, pH 7.5, with the content of 0.01% EDTA using Precellys 24—Dual homogenizer (Bertin Technologies, France). The homogenates were centrifuged at 25,000 × *g* for 30 min at 4°C, and the supernatants served as the source of the

Catalytic activity of LDH was assayed colorimetrically at 37°C using lactate as the substrate

(A505). Protein concentration for the calculation of specific enzyme activity (U/g) was determined using Bradford method [26]. The activity of LDH in erythrocytes was expressed in

Lactate dehydrogenase isoenzymes can be separated using various supporting media such as starch, agarose, cellulose acetate, and polyacrylamide gel. The separations should be carried out with cooling to 4°C in order to prevent the possibility of enzyme inactivation, particularly

Two types of polyacrylamide gel electrophoresis for the separation of lactate dehydrogenase isoenzymes of mammalian and bird origin were used in our laboratory (PhastSystem,

**1.** Gradient polyacrylamide gel electrophoresis with continuous 10–15% gradient gel zone and 2% cross-linking (PAGE 10–15) as well as with continuous 8–25% gradient gel zone (PAGE 8–25). Separation conditions: 400 V, 10.0 mA, 45 min separation time at 4°C and

**2.** Isoelectric focusing technique with a pH range of 3–9 in homogeneous 5% polyacrylamide gel containing Pharmalyte carrier ampholytes (IEF 3–9). Separation conditions: 2000 V,

As many proteins were separated by electrophoresis, lactate dehydrogenase isoenzymes were detected (stained) specifically using a colorimetric method with an assay system in 0.1 M Gly-

nitro blue tetrazolium (NBT) (6.0 ml), and 2.0 mg/ml phenasine methosulfate (PSM) (0.6 ml)

**5. Detection and quantification of lactate dehydrogenase isoenzymes**

and LDH5

) [3].

(1.5 ml), 1.0 mg/ml

enzyme units per gram of hemoglobin (U/g of hemoglobin).

heat-sensitive slow-moving isoenzymes (LDH4

2.5 mA, 15°C, 20 min separation time.

**4. Electrophoretic techniques used for separation of LDH** 

with a buffer system of 0.88 mol/L l-alanine/0.25 mol/L Tris pH 8.8.

NaCl-NaOH, pH 8.3: 1.0 mol/l sodium lactate (1.0 ml), 10 mg/ml NAD<sup>+</sup>

as the coenzyme and brownish-red pyruvate hydrazone as the measured product

19,000 × *g* for 60 min at 4°C.

84 Electrophoresis - Life Sciences Practical Applications

enzyme.

with NAD+

**isoenzymes**

Pharmacia LKB, Sweden):

Four to five isoenzymes of LDH are usually present in normal sera of animal and human beings as a result of natural degradation of cells of various tissues/organs. Their quantitative distribution in the serum is different and relatively characteristic for a particular biological species (**Figure 1**) [8]. After their separation in a concentration gradient of polyacrylamide (10–15%) and at pH 8.8, mammalian lactate dehydrogenases were separated with a good resolution, whereas bird serum (chicken) produced only one, somewhat diffuse enzymatic zone [8].

A good and clear resolution of bird LDH (chicken and pheasant) isoenzymes with all five isoenzymes zones was achieved using isoelectric focusing technique in a pH range of 3–9 [8]. A similar pattern was also produced by turkey isoenzymes [27]. A comparison of LDH catalytic activity and relative distribution (%) of its isoenzymes in the sera of the investigated animals (**Figure 1**) revealed that the predominant portion of chicken serum LDH activity was concentrated in the muscle form of the enzyme (LDH5 ) (66%), followed by LDH<sup>4</sup> (23%). LDH<sup>1</sup> to

**Figure 1.** Lactate dehydrogenase isoenzyme patterns in the serum of some birds and mammals.

LDH3 occurred in low amounts with fairly similar proportions (3–6%). The muscle isoenzyme was also the main fraction in pheasant and turkey serum [8, 27] (44 and 50%, respectively). Heart homotetramer as well as hybrid forms were present in smaller amounts. Most mammals showed a reverse pattern: the main portion of serum lactate dehydrogenase activity migrated in the first three anodic fractions, while LDH<sup>4</sup> and LDH5 made only minor contributions (**Figure 1**). The serum pattern of LDH isoenzymes probably reflects the pattern of the main LDH organ donors (heart, skeletal muscle, and liver) and differs from animal to animal species. As an example, the quantitative analysis can serve organ/tissue pattern and relative distribution of LDH isoenzymes in chicken (**Figure 2**) [28]. The patterns of lactate dehydrogenase isoenzymes in various chicken tissues, erythrocytes, and serum can be divided into three groups: (1) those with cathodic domination, (2) those with anodic domination, and (3) those distributed in the entire electrophoreogram. The anodic domination (LDH1

breast muscle. Other tissues and serum exhibit a more spread pattern with some anodic tendency (kidney, liver), cathodic tendency (skeletal muscle, serum), and more even distribution (spleen, pancreas, lung, and brain). Similar distribution and pattern of LDH isoenzymes were

Lactate Dehydrogenase Isoenzyme Electrophoretic Pattern in Serum and Tissues of Mammalian…

Comparing the isoenzyme patterns of birds (chicken, turkey) [27, 28] to human and other mammals reveals some differences. While the heart and skeletal muscle patterns are similar in birds and mammalians, it differs in other tissues, especially in the liver. While in most mammals, the liver pattern is quantitatively dominated by the slower moving cathodic isoenzymes

turkey, the patterns are opposite, with the prevalence of anodic isoenzymes similar to the aerobic heart muscle isoenzyme [27, 28]. The reason for these differences is not clear but it may suggest that the bird internal organs are geared to function better under aerobic metabolism. Besides the serum and tissue LDH isoenzymes of adult birds, we also succeeded in separating lactate dehydrogenases originated from selected chicken embryo tissues using IEF technique

An interesting pattern was found in the muscle of chicken embryo where all five isoforms

(**Figure 2**). This difference can be related to the developmental changes of LDH isoenzymes, similar to heart chicken embryonic lactate dehydrogenases. They were also relatively equally distributed on the electrophoreogram producing a pattern different from adult chicken heart

A suitable method for electrophoretic separation of bird lactate dehydrogenase isoenzymes is isoelectric focusing technique in a pH range of 3–9. It enabled to determine patterns and relative distribution (%) of LDH isoforms in bird sera and tissues (chicken adult and embryonic,

**Table 2.** Relative distribution (%) of lactate dehydrogenase isoenzymes in skeletal muscle and heart of chicken embryo

both species probably originated from breast muscle that distinguish them from mammalian

as well as turkey). They were characterized by a prevalence of slow moving LDH5

Skeletal muscle 18 21 21 20 20 Heart 12 21 23 21 18

isoform prevails over cathodic ones that are either absent or present only

**1 2 3 4 5**

; rabbit, horse, lamb, dog, humans) [29], in chicken and

the cardiac muscle and erythrocytes, while the cathodic domination (LDH5

, LDH5

were present (**Table 2**), while in adult animal tissue, only one, LDH5

**Tissue Lactate dehydrogenase (%)**

described in turkey [27].

similar to skeletal muscle (LDH4

in the pH range of 3–9 (**Table 2**).

where anodic LDH1

in a trace amount.

**7. Conclusion**

(n = 6).

) is seen in

87

) is seen in the

http://dx.doi.org/10.5772/intechopen.76322

isoenzyme, was detected

in sera in

**Figure 2.** Lactate dehydrogenase isoenzymes pattern in chicken tissues and blood.

those distributed in the entire electrophoreogram. The anodic domination (LDH1 ) is seen in the cardiac muscle and erythrocytes, while the cathodic domination (LDH5 ) is seen in the breast muscle. Other tissues and serum exhibit a more spread pattern with some anodic tendency (kidney, liver), cathodic tendency (skeletal muscle, serum), and more even distribution (spleen, pancreas, lung, and brain). Similar distribution and pattern of LDH isoenzymes were described in turkey [27].

Comparing the isoenzyme patterns of birds (chicken, turkey) [27, 28] to human and other mammals reveals some differences. While the heart and skeletal muscle patterns are similar in birds and mammalians, it differs in other tissues, especially in the liver. While in most mammals, the liver pattern is quantitatively dominated by the slower moving cathodic isoenzymes similar to skeletal muscle (LDH4 , LDH5 ; rabbit, horse, lamb, dog, humans) [29], in chicken and turkey, the patterns are opposite, with the prevalence of anodic isoenzymes similar to the aerobic heart muscle isoenzyme [27, 28]. The reason for these differences is not clear but it may suggest that the bird internal organs are geared to function better under aerobic metabolism.

Besides the serum and tissue LDH isoenzymes of adult birds, we also succeeded in separating lactate dehydrogenases originated from selected chicken embryo tissues using IEF technique in the pH range of 3–9 (**Table 2**).

An interesting pattern was found in the muscle of chicken embryo where all five isoforms were present (**Table 2**), while in adult animal tissue, only one, LDH5 isoenzyme, was detected (**Figure 2**). This difference can be related to the developmental changes of LDH isoenzymes, similar to heart chicken embryonic lactate dehydrogenases. They were also relatively equally distributed on the electrophoreogram producing a pattern different from adult chicken heart where anodic LDH1 isoform prevails over cathodic ones that are either absent or present only in a trace amount.


**Table 2.** Relative distribution (%) of lactate dehydrogenase isoenzymes in skeletal muscle and heart of chicken embryo (n = 6).
