Increased serum mitochondrial creatine kinase activity as a risk for hepatocarcinogenesis in chronic hepatitis C patients.

Serum mitochondrial creatine kinase (MtCK) activity was reportedly increased in cirrhotic patients although less prominent than that in hepatocellular carcinoma (HCC) patients. To elucidate the clinical significance of serum MtCK activity in chronic liver disease, 171 chronic hepatitis C patients were enrolled. Serum MtCK activity in study subjects was correlated with serum albumin, platelet counts, liver stiffness values and serum aspartate and alanine aminotransferase. In mouse fibrotic liver induced by bile duct ligation, ubiquitous MtCK mRNA and protein expressions were significantly enhanced and its immunoreactivity was increased, predominantly in hepatocytes. During the mean follow-up period of 2.7 years, HCC developed in 21 patients, in whom serum MtCK activity was significantly higher than that in patients without HCC development. Multivariate Cox regression analysis revealed that higher serum MtCK activity was a risk for HCC development. A cutoff value of MtCK for the prediction of HCC development was determined as 9.0 U/L on receiver operating characteristics analysis, where area under receiver operating characteristics curve was 0.754, with a sensitivity of 61.9%, a specificity of 92.8% and a high negative predictive value of 94.2%. Cumulative incidence of HCC was significantly higher in patients with serum MtCK activity of >9.0 U/L compared to those with serum MtCK activity of ≤ 9.0 U/L even in patients with elevated liver stiffness value, >15 kPa. In conclusion, serum MtCK activity may be increased correlatively with the stage of liver fibrosis and hepatocellular damage. Increased serum MtCK activity is an independent risk for hepatocarcinogenesis in chronic hepatitis C patients.

Macro CK2 accumulation in tenofovir-treated HIV patients is facilitated by CK oligomer stabilization but is not predictive for pathology.

BACKGROUND: Ubiquitous mitochondrial creatine kinase (uMtCK) accumulates as macroenzyme creatine kinase type 2 (macro CK2) in the serum of HIV-infected patients under a tenofovir disoproxil fumarate (TDF)-containing antiretroviral regimen. The genesis and clinical significance of this finding is unclear. METHODS: A prospective observational 5-year follow-up study was performed on those patients in which macro CK2 appearance was initially described ('TDF switch study' cohort). In addition, tenofovir (TFV), its prodrug TDF and its active, intracellular derivative TFV diphosphate (TDP) were tested in vitro for their effects on different key properties of uMtCK to clarify possible interactions of uMtCK with TFV compounds. RESULTS: In just under 5 years of continuous TDF treatment, only 4/12 (33%) patients remained macro CK2-positive, whereas 8/12 (66%) originally positive patients were macro CK2-negative at the end of follow-up. Prospective clinical follow-up data indicate that macro CK2 appearance under TDF is not associated with significant cell damage or occurrence of malignancies. A trend towards grade 1 hypophosphataemia suggests subclinical proximal tubular dysfunction in macro-CK2-positive patients, although it was not associated with a significant decrease in estimated glomerular filtration rate. In vitro, TFV, TDF and TDP did not interfere with uMtCK enzyme activity as competitive inhibitors or pseudo-substrates, but TFV and TDF stabilized the native uMtCK octameric structure in dilute solutions. CONCLUSIONS: Appearance of octameric uMtCK as macro CK2 in the serum of TDF-treated patients is suggested to result from a combination of low-level mitochondrial damage caused by subclinical renal tubular dysfunction together with possible compensatory uMtCK overexpression and a putative concomitant stabilization of uMtCK octamers by higher levels of TFV in proximal tubules.

[Discovery of novel blood markers for liver diseases].

We have recently discovered three novel blood markers for liver diseases; one for predicting hepatocarcinogenesis, the second for predicting liver cirrhosis and the third for predicting hepatocellular carcinoma. In this review, the usefulness of these markers is summarized.

Increased activity of serum mitochondrial isoenzyme of creatine kinase in hepatocellular carcinoma patients predominantly with recurrence.

BACKGROUND & AIMS: Mitochondrial isoenzyme of creatine kinase (MtCK) is reportedly highly expressed in hepatocellular carcinoma (HCC). Clinical relevance of serum MtCK activity in patients with HCC was assessed using a novel immuno-inhibition method. METHODS: Among patients with cirrhosis caused by hepatitis B or C virus, 147 patients with HCC (12 with the first occurrence and 135 with recurrence) and 92 patients without HCC were enrolled. RESULTS: Serum MtCK activity was higher in cirrhotic patients with HCC than in those without HCC or healthy subjects. Elevated serum MtCK activity in HCC patients decreased after radiofrequency ablation. In case of prediction of HCC, MtCK had a sensitivity of 62.6% and a specificity of 70.7% at a cut-off point of 8.0 U/L, with an area under the receiver operating curve of 0.722 vs. 0.713 for alpha-fetoprotein (AFP) and 0.764 for des-gamma-carboxy prothrombin (DCP). Among the HCC patients, serum MtCK activity was elevated in 52.9% individuals with serum AFP level < 20 ng/ml and 63.2% individuals with serum DCP level < 40 mAu/ml. Even in patients with a single HCC ≤ 2 cm, the sensitivity of serum MtCK activity for the prediction of HCC was 64.4%, which was comparable to the overall sensitivity. This increased activity was due to an increase in ubiquitous MtCK, not sarcomeric MtCK, and the enhanced mRNA expression of ubiquitous MtCK was observed in cell lines originating from HCCs in contrast to healthy liver tissues. CONCLUSIONS: Serum MtCK activity merits consideration as a novel marker for HCC to be further tested as for its diagnostic and prognostic power.

Increase in serum mitochondrial creatine kinase levels induced by tenofovir administration.

Recently, 2 monoclonal antibodies that specifically inhibit mitochondrial creatine kinase (MtCK) activity have been developed. In this study, we measured the serum MtCK activity in HIV-1-infected individuals (n = 100) by employing a novel method using these antibodies. The mean serum MtCK activity in 44 patients treated with highly active antiretroviral therapy (HAART) including tenofovir disoproxil fumarate (TDF) was 16.0 IU/L. The MtCK activity was significantly higher in patients receiving TDF than in those receiving HAART without TDF (3.4 IU/L) or in naïve patients (6.9 IU/L) (Tukey-Kramer test, p < 0.0001 and p = 0.0029, respectively). The serum MtCK activity reached a plateau at 1 month after the initiation of TDF administration and decreased upon discontinuation. It showed no significant correlation with the trough plasma TDF concentration, serum creatinine level, or red blood cell count. The activity was elevated in 75% of the patients receiving TDF, and this elevation was specific to TDF; it was not observed with other anti-HIV drugs. In addition, our report emphasizes the careful interpretation of creatine kinase-MB (CK-MB) test results in patients receiving TDF because MtCK in serum could cause false-positive results on a conventional CK-MB test, which does not include MtCK-specific inhibitory antibodies.

[Clinical evaluation of the new creatine kinase MB reagent kit "L-System CK-MB MtO"].

The creatine kinase MB (CK-MB) activity assay, which is based on the immunoinhibition method, has long been used in the diagnosis of acute myocardial infarction (AMI) because of its good cost-performance ratio and simplicity. However, the immunoinhibition method can not differentiate between CK-MB and MtCK, and therefore, CK-MB activity determined using this method is higher than the actual value in the sample which MtCK appears; this may lead to the misdiagnosis of AMI. We, therefore, evaluated the analytical and clinical performance of a new CK-MB reagent kit "L-System CK-MB MtO," which can inhibit MtCK. The kit yielded good precision and linearity and no interference from hemolysis, bilirubin or chyle. A good correlation was observed between the values determined using this kit and those determined using the conventional kit for samples of patients with acute coronary syndromes. However, differences were observed in the CK-MB values determined for samples from patients with malignancy. CK isoenzyme analysis indicated that MtCK was present in all these samples. The new method permits the accurate estimation of CK-MB activity in samples of patients with high serum levels of MtCK activity and indicates that the conventional method has a high false-positive rate for CK-MB activity. CK-MB activity in the serum of healthy individuals measured using the new and the conventional kits was 1.9-9.5 U/l and 4.5-15.3 U/l, respectively. The new kit, enables accurate estimation of CK-MB activity and is, therefore, more useful than the conventional kit in the diagnosis of acute coronary syndromes.

Development and performance of an enzyme immunoassay to detect creatine kinase isoenzyme MB activity using anti-mitochondrial creatine kinase monoclonal antibodies.

OBJECTIVE: The MB fraction of creatine kinase (CK-MB) has long been used as a cardiac marker. It is known that the CK-MB immunoinhibition method lacks selectivity and accuracy, because the appearance of macro CK type 2, corresponding to mitochondrial creatine kinase (MtCK) in some patient serum may render CK-MB activity measured by conventional method abnormally high. Thus, to improve the specificity and accuracy of the CK-MB assay, we developed two types of monoclonal anti-MtCK antibodies against sarcomeric MtCK and ubiquitous MtCK, and present herein the performance of a new method using these antibodies. MATERIAL AND METHODS: The performance of our test for detecting CK-MB activity was compared with other methods, and the range of CK-MB activities in normal human serum was investigated. RESULTS: The two types of monoclonal antibodies developed by us were isoenzyme-specific to sMtCK or uMtCK. The correlation coefficients of our method and conventional method to electrophoresis were 0.973 and 0.873, respectively. The mean CK-MB activity in normal human serum by our method and the conventional method was 2.4 and 11.7 U/L, respectively. Thus, our data indicated that about 80% of CK-MB activity, determined using the conventional method, seems to correspond to the MtCK activity. CONCLUSION: Our method is novel in offering higher accuracy of measuring true CK-MB contents in human serum as compared to the conventional method. The possibility of accurately estimating CK-MB activity by our method which can inhibit MtCKs in healthy person and patient serum is likely to bring a break-through in clinical diagnostics.

A multi-marker approach for the prediction of adverse events in patients with acute coronary syndromes.

BACKGROUND: Cardiac troponin T (cTnT), high sensitivity C-reactive protein (hs-CRP) and N-terminal pro-B-type natriuretic peptide (NT-proBNP) have emerged as strong predictors of adverse events among patients presenting with acute coronary syndromes (ACS). We evaluated the prognostic performance of each of these markers, individually, and in combination in patients presenting to the emergency department (ED) with ACS symptoms. METHODS: Serum samples were obtained from 422 consenting patients presenting to the ED with symptoms of acute coronary syndrome (ACS) and subsequently tested for cTnT, NT-proBNP, myoglobin, CK-MB, and hs-CRP. Adverse events (AEs) occurring within 30 days (death, myocardial infarction, unstable angina and the need for revascularization procedures) were recorded and ROC curves were constructed. RESULTS: AEs occurred in 42 patients (10%). Relative risk, cut-off, and predictive values for each biomarker were determined statistically, with the exception of cTnT, where the concentration meeting the 99th percentile of a healthy population with a 10% coefficient of variation (0.03 ng/ml) was used. These cut-off values, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and relative risk (RR) were calculated. Sensitivity and RR for a panel of cTnT and NT-proBNP were 78.6% (66.2-91.0) and 4.7 (2.3-9.5), respectively. CONCLUSIONS: If used alone, cTnT appeared to have greater prognostic value when compared to hs-CRP, NT-proBNP, myoglobin or CK-MB. The combination of cTnT and NT-proBNP performed better than the combination of cTnT and hs-CRP. When cTnT, NT-proBNP and hs-CRP were used as a panel, there was no significant improvement in prognostic performance over using cTnT and NT-proBNP together. Thus, in patients with suspected ACS, the measurement of both cTnT and NT-proBNP may have enhanced prognostic performance over using either marker in isolation.

Biochemical evidence of cardiac damage following transvenous implantation of a permanent antibradycardia pacemaker lead.

OBJECTIVES: We tested the hypothesis that transvenous permanent pacemaker lead implantation causes clinically detectable myocardial damage. BACKGROUND: Histological evidence of myocardial damage has been reported after antibradycardia pacemaker lead implantation. METHODS: We studied 30 patients undergoing implantation of a full antibradycardia pacemaker system (pulse generator plus leads) and 10 patients in whom only a generator was implanted. Blood samples for cardiac troponin-I (CTNI), CK-MB mass, and myoglobin measurement were drawn at baseline, at the end of the procedure, and at 2, 6, 12, 24, 48, and 72 hours thereafter. RESULTS: Abnormal CTNI levels were noted only in 24 of the 30 patients undergoing a full system implantation. CTNI levels were already abnormal at the end of the procedure in 16 and became so in all 24 during the next 6 hours. Peak levels were reached within 6 hours in 21 patients and were compatible with "minimal" necrosis (CTNI < 1.5 pg/mL) in 20. Maximum ventricular lead diameter and number of implanted leads were independent predictors of peak CTNI levels. CK-MB mass also increased after the procedure, but exceeded the normal range in only 10 patients. Myoglobin levels increased significantly both in patients undergoing a complete system implantation and in those where only a pulse generator was implanted. CONCLUSIONS: Transvenous insertion of endocardial leads for permanent pacing is accompanied in most patients by "minimal" myocardial damage. In this setting CTNI level kinetics are fast, characterized by early elevation and peak.