Separation Of Isoenzymes Research Articles

LACTATE DEHYDROGENASE ISOENZYMES IN TISSUES OF CARNIVOROUS MAMMALS DURING POSTNATAL ONTOGENY

The electrophoretic separation of isoenzymes of lactate dehydrogenase (LDH, EC 1.1.1.27) was performed in the kidneys, skeletal muscle, lungs and spleen in mink Neovison vison Schreber and blue fox Alopex lagopus L. pups at 15, 25, 60, 90, 120, 150, 180, and 210 days of age. Significant changes in the isoenzyme spectra of LDH in the tissues of mink and blue fox pups were observed in the initial period of the animals’ intensive growth and development –at the age of 15–0 days. In the skeletal muscles, lungs and spleen, the content of M-subunits of LDH during postnatal ontogeny was higher in mink than in foxes. The isozyme profile of renal LDH in 4‑onth-old pups of mink and blue foxes corresponded to that of 7‑onth-old animals. The tissue specificity of the LDH isoenzyme pattern reflects the metabolic profile of the tissues and is detectable already in early developmental stages.

Kinetic Studies of Na+/K+-ATPase in Tissue Aerobic Thyroid Patients

Na+/K+-ATPase is a prevalent enzyme that maintains the Na+ and K+ gradients across the cell membrane by transporting three Na+ out and two K+ into the cell, the aim of this study is to provide detailed mechanistic insights, potentially with important effects on physiological regulation of active Na and K transport in tissues of Aerobic Thyroid Patient. Thyroid tissues were obtained from a 35 year old patients, the operation was carried out at the Al-Hadi Specialist Hospital in Samarra city, the sample was stored at -20ºC until used. The purification protocol included Salt Precipitation, Ion Exchange Chromatography, Gel Filtration and Electrophoresis, a spectrophotometric method was used to determine the enzyme activity. kinetic parameters was also obtained for the enzyme. Partial purification of Na+/K+-ATPase revealed two isoenzymes (I ,II). The purity of separated isoenzymes were proved by SDS-PAGE electrophoresis. The kinetic characteristics of Na+/K+-ATPase showed that optimum substrate concentration about 1.5mM, Km 1.052mM, and Vmax 6.062, optimum temperature was 37 ºC, optimum pH 7.4 and optimum time in 25 min. Na+/K+-ATPase purified from Thyroid tissue has distinct kinetic characteristic that reflects the importance of intracellular regulation of specific Na+/K+-ATPase pump which gives cells the ability to precisely coordinate to their physiological requirements .

Fructose-associated hepatotoxicity indexed by the lactate dehydrogenase isoenzyme LDH-5

Modern diets have become increasingly rich in fructose, for example through the addition of high-fructose corn syrup to many foods and drinks. It has been suggested that this might lead to hepatotoxicity, including the development of non-alcoholic fatty liver disease. After entering hepatocytes via insulin-independent glucose transporter 2 transmembrane carrier proteins, fructose is phosphorylated to fructose-1-phosphate in a reaction catalysed by fructokinase (ketohexokinase). In turn, fructose-1-phosphate is hydrolysed by aldolase B to glyceraldehydes. Glyceraldehydes may enter gluconeogenesis via fructose-1,6-bisphosphate and fructose-6-phosphate; glyceraldehydes may also enter glycogenolysis via pyruvate. The last pathway involves conversion of pyruvate to acetyl-CoA. Alternatively, pyruvate may be converted, via the action of the hepatic lactate dehydrogenase isoenzyme LDH-5, into lactate. In liver damage, the LDH-5 isoenzyme becomes elevated, predominantly in serum/plasma. We therefore hypothesised that if dietary fructose is associated with hepatotoxicity, there should be a positive correlation between erythrocyte fructose-6-phosphate and plasma LDH-5. This hypothesis was tested by assaying venous blood samples taken from 39 patients at rest, three hours after eating. Quantitative Fourier transform infrared spectrometry following gel electrophoresis was used to assay erythrocyte fructose-6-phosphate levels. Similarly, plasma LDH-5 concentrations were spectrophotometrically analysed, using the pyruvate-lactate reaction, following electrophoretic separation of the LDH isoenzymes. A significant positive correlation was found between the two variables (r = 0.44, p = 0.0047). This result, which supports our hypothesis, is evidence in favour of the possibility that dietary fructose is associated with hepatotoxicity. In addition to being a marker of hepatic damage, LDH-5 may play a more direct epigenetic role in causing liver damage; acute hepatic injury is associated with nuclear translocation of LDH, causing the production of lactate from pyruvate in the nucleus; in turn, the lactate inhibits histone deacetylase and is associated with upregulation of genes associated with the damage response, leading to cell death.

Serum creatine kinase isoenzymes and macroenzymes in dogs with different neurologic diseases.

Increased serum activity of CK isoenzymes and macroenzymes, and in particular of the brain isoenzyme (CK-BB) has been reported in dogs with central nervous system (CNS) disorders. However, no studies on the possible differences in serum activities of CK iso- or macroenzymes (Macro-CK1 and Macro-CK2) in different neurologic diseases are available. The aim of this study was to describe the electrophoretic distribution of CK iso- and macroenzymes in dogs with CNS disorders in order to assess whether this distribution depends on a specific neurologic disease. This study was done on sera from 45 dogs with neurologic diseases (degenerative, n = 7; idiopathic epilepsy [IE], n = 14; inflammatory, n = 16; space occupying lesions [SOL], n = 8) and from 10 clinically healthy dogs. The separation of serum CK isoenzymes and macroenzymes was performed using an automated electrophoretic method already validated in dogs. Compared with healthy dogs, dogs with CNS disorders had significantly higher total CK and CK-BB activities, and a significantly lower Macro-CK2 activity (P < .001). Comparison of pathologic subgroups and healthy dogs revealed significant differences (P < .01) in dogs with IE and inflammatory disorders for total CK activity, in all the subgroups for CK-BB (P < .01), and in dogs with IE and SOL for Macro-CK2 (P < .01). The results of this study suggest that CK-BB is released by neurons damaged by inflammatory or degenerative conditions or due to compressive effects of SOL. However, the neurologic diseases cannot be differentiated based on CK-BB or Macro-CK2 activities, unless further studies allow the definition of diagnostic thresholds.

Separation of turkey lactate dehydrogenase isoenzymes using isoelectric focusing technique.

Native polyacrylamide gel electrophoresis at pH 8.8 did not allow to separate lactate dehydrogenase (LDH) isoenzymes of turkey origin. Five electrophoretically distinguishable forms of the enzyme were detected in serum and tissues of turkey using IEF technique in a pH range of 3-9. Generally, three different groups were seen: (i) those having an anodic domination (heart, kidney, pancreas, and erythrocytes) with mainly LDH-1 fraction, (ii) those having a cathodic domination (breast muscle and serum) with prevalence of LDH-5, and (iii) those with a more uniform distribution (liver, spleen, lung, and brain). The specific enzyme activity was the highest in the breast muscle, followed by heart muscle, and brain. Low activities were detected in serum, kidney, and liver.

Separation of the Isoenzymes of Rat Liver Glutathione Transferase by Chromatofocusing

Separation of the Isoenzymes of Rat Liver Glutathione Transferase by Chromatofocusing

Some observations on the attempted separation of isoenzymes of monoamine oxidase.

Some observations on the attempted separation of isoenzymes of monoamine oxidase.

L-lactate metabolism can occur in normal and cancer prostate cells via the novel mitochondrial L-lactate dehydrogenase

Both normal (PTN1A) and cancer (PC3) prostate cells produce high levels of L-lactate because of a low energy supply via the citric cycle and oxidative phosphorylation. Since some mammalian mitochondria possess a mitochondrial L-lactate dehydrogenase (mLDH), we investigated whether prostate cells can take up L-lactate and metabolize it in the mitochondria. We report here that externally added L-lactate can enter both normal and cancer cells and mitochondria, as shown by both the oxygen consumption and by the increase in fluorescence of NAD(P)H which occur as a result of L-lactate addition. In both cell types L-lactate enters mitochondria in a carrier-mediated manner, as shown by the inhibition of swelling measurements due to the non-penetrant thiol reagent mersalyl. An L-lactate dehydrogenase exists in mitochondria of both cell types located in the inner compartment, as shown by kinetic investigation and by immunological analysis. The mLDHs proved to differ from the cytosolic enzymes (which themselves differ from one another) as functionally investigated with respect to kinetic features and pH profile. Normal and cancer cells were found to differ from one another with respect to mLDH protein level and activity, being the enzyme more highly expressed and of higher activity in PC3 cells. Moreover, the kinetic features and pH profiles of the PC3 mLDH also differ from those of the PNT1A enzyme, this suggesting the occurrence of separate isoenzymes.

ORIGINAL RESEARCH: Electrophoretic fractionation of creatine kinase isoenzymes and macroenzymes in clinically healthy dogs and cats and preliminary evaluation in central neurologic disease

Background: Information about the electrophoretic distribution of CK‐MM, CK‐MB, and CK‐BB, serum creatine kinase (CK) isoenzymes that are indicators of skeletal muscle, cardiac muscle, and brain lesions, respectively, and CK macroenzymes (macro‐CK1 and macro‐CK2) in dogs and cats with and without central neurologic disease is scant and equivocal. Objectives: The objectives of this study were to describe the electrophoretic distribution of CK isoenzymes and macroenzymes in healthy dogs and cats and to provide a preliminary assessment of the utility of CK enzymatic electrophoresis in dogs and cats with central neurologic disease. Methods: Electrophoretic separation of serum CK isoenzymes and macroenzymes was performed on freeze‐thawed serum samples from 20 healthy dogs and 3 dogs with central neurologic disease and from 14 healthy cats and 6 cats with neurologic feline infectious peritonitis (FIP). Electrophoretic separation was also performed on supernatants of homogenized brain, skeletal muscle, and cardiac muscle from both species, to assess the tissue distribution of isoenyzmes in dogs and cats. Results: CK‐MM was the predominant isoenzyme in the serum of healthy dogs and cats, followed by macro‐CK2 and CK‐BB in dogs and by both macroenzymes in cats. In dogs, CK‐MB was essentially absent from both serum and homogenized hearts. CK‐BB increased in dogs with neurologic disease. In cats, CK‐BB was essentially absent from serum, but was present in brain homogenates. Two of 6 cats with FIP had increased macro‐CK1 and increased CK‐BB activity. Conclusions: This study identified the electophoretic distribution of CK isoenzymes and macroenzymes of dogs and cats and provided encouraging data about the possible use of CK‐BB as a biomarker for canine neurologic disorders, but not for FIP.

Effect of vitamin E alone and in combination with lycopene on biochemical and histopathological alterations in isoproterenol-induced myocardial infarction in rats

Background:The present study has been designed to evaluate the combined cardioprotective effect of vitamin E and lycopene on biochemical and histopathological alteration in isoproterenol-induced myocardial infarction in rats.Materials and Methods:Adult male albino rats of Wistar strain were treated with isoproterenol (200 mg/kg, s.c.) for 2 days at an interval of 24 h to develop myocardial infarction. Vitamin E (100 mg/kg/day, p.o.) and lycopene (10 mg/kg/day, p.o.) were administered alone and in combination for 30 days. Change in body weight and organ weight were monitored. Levels of serum marker enzymes (AST, ALT, LDH and CK-MB), lipid peroxidation, endogenous antioxidants (GSH, GPX, GST, SOD and CAT), membrane bound enzymes (Na+/K+ ATPases, Mg2+ ATPases and Ca2+ ATPases) were evaluated. LDH isoenzyme separation was carried out using gel electrophoresis. Histopathology of heart tissue was performed.Results:Induction of rats with isoproterenol resulted in a significant elevation in organ weight, lipid peroxidation, serum marker enzymes (AST, ALT, CK-MB and LDH), and Ca 2+ ATPases, whereas it caused a significant (P < 0.001) decrease in body weight, activities of endogenous antioxidants (GSH, GPx, GST, SOD and CAT), Na+/K+ and Mg2+ ATPases. ISO treated rats showed high intensity band of LDH1-LDH2 isoenzymes. Treatment with the combination of Vitamin E and lycopene for 30 days significantly attenuated these changes as compared to the individual treatment and ISO treated groups. Histopathological observations were also in correlation with the biochemical parameters.Conclusion:These findings indicate the synergistic cardioprotective effects of vitamin E and lycopene during ISO-induced myocardial infarction in rats.

Isolation and characterization of novel pI 4.8 MnP isoenzyme from white-rot fungus Irpex lacteus

Growth of Irpex lacteus on polyurethane solid support led to secretion of various manganese-dependent peroxidase (MnP) isoenzymes with pI in the range of 3.8–6.7. The highest MnP activity was obtained at 8.9 mmol l −1 Mn 2+ when predominantly a pI 4.8 isoenzyme was produced. Optimization of anion exchange chromatography (Mono Q) allowed the separation of MnP isoenzymes and collection of extensive sequence information by tandem LC–MS. The pI 4.8 isoenzyme was purified 109-fold (6% activity yield) by ion exchange chromatography, its specific activity of Mn 2+ oxidation was 2800 nkat mg −1, molecular mass 37 kDa and pH optimum 5. The other properties were: respective half-lives (pH 4.5) of 575, 325 and 7 min at 40, 50 and 60 °C, K m values 46.7; 9.5; 21.4 μmol l −1 and k cat values 69.0; 64.2; 15.7 s −1 for Mn 2+, H 2O 2 and DMP with Mn 2+, respectively. The pI 4.8 isoenzyme was shown to be a true MnP affecting secondary substrates via the oxidation of Mn 2+. Its biochemical properties and production by I. lacteus make the enzyme a candidate for biotechnological applications.

Profile of peroxidase isoforms influenced by spongy tissue disorder in Alphonso mango (Mangifera indica L.) fruits

This paper describes the profile of peroxidase (POX) isoenzymes induced due to the natural infection of Staphylococcus xylosus in spongy Alphonso mango fruits. Very low levels of protein and POX activity was observed in non-spongy unripe fruits, and when these fruits turned table-ripe, the levels of both the protein content and POX activity increased several fold. The spongy fruits, however, showed further 2-fold increase in POX activity; although drastic decrease in protein content was observed. Anionic and cationic PAGE, and isoelectric focusing (IEF), resulted in separation of various isoenzymes of POX. Both, anionic and cationic PAGE indicated that, at unripe stage, only basic isoforms were present in trace amounts. In non-spongy ripe fruits, increased levels of both anionic and cationic isoforms were observed after staining the gel with o-dianisidine, the POX substrate. In spongy fruits, however, an anionic PAGE showed appearance of four acidic isoforms with relative electrophoretic mobility (REM) of 0.52, 0.73, 0.78, and 0.84 and an isoenzyme (REM 0.52), showed further activation, as indicated by the intense dark color formation. Cationic PAGE also indicated higher levels of two basic isoforms (REM 0.56 and 0.62), in the spongy fruits. Isoelectric focusing resolved these isoenzymes in acidic, neutral, and basic isoforms. Two acidic isoforms in the pI range of 2–3.5 were detected toward the anode region and two cationic isoforms of pI 7.8 and 8.7, toward the cathode, giving visible indication of increased levels of these isoforms. The increased intensities of the POX bands observed in anionic and cationic PAGE, and IEF, gave confirmatory evidence for the up regulation of anionic and cationic isoforms in spongy fruits. These isoenzymes could have been overexpressed as a defense response of the spongy fruits against the Staphylococcus infection.

Evaluation of Acid Phosphatase Isoenzymes in Seminal Fluid from Normozoospermic, Oligozoospermic, Azoospermic and Asthenoteratozoospermic Men

Acid phosphatase was measured in the semen of normozoospermic, oligozoospermic, azoospermic and asthenoteratozoospermic men: there was no significant difference between oligozoospermic and control groups whereas we observed an increase in enzyme levels in the azoospermic group and a reduction in the asthenoteratozoospermic subjects. Furthermore, an isoelectric focusing method for the separation of isoenzymes was developed. Only prostatic isoenzymes 2 and 4 were found in semen. Isoenzyme 2 sialylation profile was determined in the four groups. The sperm count did not influence this profile. These findings are discussed.

Microchip‐based capillary electrophoresis for determination of lactate dehydrogenase isoenzymes

This article describes a novel microchip-based capillary electrophoresis and oncolumn enzymatic reaction analysis protocol for lactate dehydrogenase (LDH) isoenzymes with a home-made xenon lamp-induced fluorescence detection system. A microchip integrated with a temperature-control unit is designed and fabricated for low-temperature electrophoretic separation of LDH isoenzymes, optimal enzyme reaction temperature control, and product detection. A four-step operation and temperature control are employed for the determination of LDH activity by on-chip monitoring of the amount of incubation product of NADH during the fixed incubation period and at a fixed temperature. Experiments on the determination of LDH standard sample and serum LDH isoenzymes from a healthy adult donor are carried out. The results are comparable with those obtained by conventional CE. Shorter analysis times and a more stable and lower background baseline can be achieved. The efficient separation of different LDH forms indicates the potential of microfluidic devices for isoenzyme assay.

Alkaline phosphatase isoenzymes in children with respiratory disease

Aim: To present the electrophoretic pattern of alkaline phosphatase (ALP) isoenzymes in serum of infants and children exhibiting increased total ALP catalytic activity in the course of acute respiratory disease. Subjects and methods: Results obtained in 21 children (17 of them infants), including 13 male and eight female children aged 2 months to 8 years, hospitalized for respiratory diseases are presented. Total ALP catalytic activity was determined and electrophoretic separation of ALP isoenzymes was performed in children's sera. Results: Increased total ALP catalytic activity (range, 528-5622 U/L) during hospital stay was recorded in eight (38.1%) children. A typical picture of be-nign transient hyperphosphatasemia (TH), which implies the occurrence of fast anodal fraction (faster than hepatic fraction) and near-cathode fraction (faster than bone fraction), was recorded in five children. The placental-like isoenzyme was detected in two children. Expression of bone fraction and placental-like fraction was recorded in a rachitic child. Prehepatic ALP was expressed in two children, and hepatic ALP isoenzyme in one child. Conclusion: Acute respiratory disease in infants and children may entail transient increase in the ALP catalytic activity with the occurrence of various isoenzyme bands such as fast anodal and near-cathode fraction (in TH), prehepatic fraction and placental-like fraction. TH is verified when total ALP activity has decreased and returned to reference intervals. In this case, no additional testing is required.

Separation of serum lactate dehydrogenase isoenzymes by capillary electrophoresis

目的 利用还原型辅酶I(NADH)在340 nm波长处特异性吸收峰,建立用毛细管区带电泳在线检测人血清中乳酸脱氢酶 (LDH) 同工酶的方法.方法实验中采用未涂层毛细管,长60 cm,内径75 μm,pH 9.4(23 ℃) 二氨基二甲基1,3丙二醇(AM2P)缓冲液含20 mmol/L氧化型辅酶I(NAD+ )、51.6 mmol/L乳酸锂.结果在25 min内完成电泳分离,乳酸脱氢酶同工酶1(LDH1)、乳酸脱氢酶同工酶5(LDH5)纯品各自均可得到较好峰形,而且LDH1、 LDH5纯品的混合物亦可得到完全分离,在分析正常人血清与肝癌患者血清时,其结果与美国Helena公司REP全自动琼脂糖电泳结果一致,取得满意效果.结论该法快速、低耗,具有较好的推广应用价值。

Regio- and Stereoselectivity of Malt Lipoxygenases LOX1 and LOX2

J. Inst. Brew. 111(3), 265–274, 2005 The characterisation of lipoxygenases LOX1 and LOX2 and hydroperoxyoctadecadienoic acid (HPODE) degrading enzymes from barley green malt is reported. Hydroxylapatite chromatography (HAC) and isoelectric focussing (IEF) were performed to separate and purify LOX isoenzymes. The regio- and stereoselectivity of LOX1 and LOX2 towards linoleic acid as substrate was characterised. HAC purified isoenzyme LOX1 showed a 9-HPODE:13-HPODE ratio of 75:25 and LOX2 a ratio of 39:61. IEF separated LOX1 and LOX2 transformed linoleic acid to 9-:13-HPODE ratios of 90:10, and 13:87, respectively. 9HPODE stereoisomers from LOX1 exhibited a S:R ratio of 93:7 and 13-HPODE from LOX2 a S:R ratio of 89:11. However, the minor regioisomers were analysed with S:R = 48:52 (LOX1, 13-HPODE) and 40:60 (LOX2, 9-HPODE). These results indicate a complete LOX isoenzyme separation by IEF. Hydroperoxide-metabolising enzymes, which were investigated in the IEF fractions, did not interfere with the dual position specificities of LOX isoenzymes.

Targeted disruption of the inosine 5'-monophosphate dehydrogenase type I gene in mice.

Inosine 5'-monophosphate dehydrogenase (IMPDH) is the critical, rate-limiting enzyme in the de novo biosynthesis pathway for guanine nucleotides. Two separate isoenzymes, designated IMPDH types I and II, contribute to IMPDH activity. An additional pathway salvages guanine through the activity of hypoxanthine-guanine phosphoribosyltransferase (HPRT) to supply the cell with guanine nucleotides. In order to better understand the relative contributions of IMPDH types I and II and HPRT to normal biological function, a mouse deficient in IMPDH type I was generated by standard gene-targeting techniques and bred to mice deficient in HPRT or heterozygous for IMPDH type II. T-cell activation in response to anti-CD3 plus anti-CD28 antibodies was significantly impaired in both single- and double-knockout mice, whereas a more general inhibition of proliferation in response to other T- and B-cell mitogens was observed only in mice deficient in both enzymes. In addition, IMPDH type I(-/-) HPRT(-/0) splenocytes showed reduced interleukin-4 production and impaired cytolytic activity after antibody activation, indicating an important role for guanine salvage in supplementing the de novo synthesis of guanine nucleotides. We conclude that both IMPDH and HPRT activities contribute to normal T-lymphocyte activation and function.

Significance of Carbohydrate Epitopes in a Latex Allergen with β-1,3-Glucanase Activity

Background: One of the latex allergens, Hev b 2, has β-1,3-glucanase activity. The entire sequence of this allergen is already known. There is one potential N-glycosylation site in this molecule (²⁷Asn). Heterogeneous glycosylation of this Asn residue could be a source of the multiplicity of natural Hev b 2. Possible participation of the carbohydrate epitopes of latex β-1,3-glucanase isoenzymes in their IgE-binding capacity and cross-reactivity was investigated in this study. Methods: β-1,3-Glucanase isoenzymes were separated based on their affinities for concanavalin A. IgE-binding capacity and cross-reactivity were examined by immunoblotting and enzyme-linked immunosorbent assay (ELISA). Sequence heterogeneity among the isoenzymes was probed by peptide mass mapping after lysyl endopeptidase digestion. To clarify the relation to Hev b 2, N-terminal sequencing was performed on a fragmented peptide common to the separated isoenzymes. Results: Basic β-1,3-glucanase was subdivided into two glycosylated isoenzymes (GI and GII) and one non-glycosylated isoenzyme (GIII). IgE antibodies in latex-positive sera chiefly recognized the glycosylated isoenzymes. Inhibition ELISA supported the significance of the carbohydrate epitopes for the IgE recognition and cross-reactivity. However, non-glycosylated GIII, as well as GI and GII, produced positive results in a skin prick test. The three β-1,3-glucanase isoenzymes shared a partial sequence in common with Hev b 2. Conclusions: Our results suggest that the carbohydrate epitopes in Hev b 2 homologues are relevant to an in vitro diagnosis of latex allergy and the accompanying cross-reactivity. Carbohydrate epitopes do not necessarily provoke allergic symptoms. Therefore, the actual allergenicity of Hev b 2 and its homologues should be carefully evaluated not only by in vitro IgE tests but also by in vivo tests.

Separation of serum LD isoenzymes by capillary electrophoresis

Separation and quantitative estimation of the isoenzymes of lactate dehydrogenase(LD) in serum were accomplished with capillary electrophoresis system. An uncoated fused silica capillary column 50 cm long, 75μm I.D. and substrate containing running buffer including L-lactic acid and NAD(+) were used for the separation of serum LD isoenzymes. The resulting product of "NADH" was detected at 340 nm. Injection of 10 nL of five fold diluted serum sample were performed by pressure injection within 2 seconds. The isoenzymes were separated at 10 kV of voltage for 5 min, by turning off the voltage applied for 30 min incubation at 24°C for reaction between substrate and isoenzymes, and applying voltage of 30 min. Under these conditions, the isoenzymes of LD were detected by a NADH generated as isoenzyme of LD-5 emerged at 20 min, LD-1 peak at 23.5 min with close to baseline separation of the other isoenzymes which emerge between LD-5 to LD-1, after the emergence to LD-1 peak, followed another peak, termed "sample shock": The results obtained by the proposed method correlated well with those by gel electrophoresis systemes (r=0.92∼0.98) for each five LD isoenzymes, respectively. Within-run precision CVs for 5 replicate analysis were 3.01 (LD-3, mean 14.6%)%∼7.82% (LD-4, mean 4.22%.), respectively.