Purification of five creatine kinase-MM variants from human heart and skeletal muscle.

Variants of creatine kinase-MM (variant of ATP:creatine N-phosphotransferase, EC 2.7.3.2), present in human heart and skeletal muscle, have been purified to homogeneity using DEAE-Sepharose column chromatography and column chromatofocusing techniques. Creatine kinase-MM I-IV were present in both heart and skeletal muscle, while MM-V was found only in heart. The number, ratio and elution profile of the variants during chromatofocusing remained identical even when they were purified in the presence of proteinase inhibitors. MM-I-V, on chromatofocusing, were eluted at pH 8.3, 7.9, 7.6, 7.2 and 6.8, respectively. Isoelectric focusing revealed the pI of MM-I-V to be 7.2, 6.9, 6.7, 6.4 and 6.2. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis showed a doublet pattern for creatine kinase-MM variants III-V. However, polyacrylamide gel electrophoresis without SDS indicated homogeneity because each variant showed a single band. The doublet pattern observed in the presence of SDS may reflect the presence of two subunits of slightly different mass.

Transient right but not left ventricular dysfunction after strenuous exercise at high altitude.

OBJECTIVES: We sought to evaluate whether prolonged exercise in ultramarathon runners results in left ventricular (LV) damage. BACKGROUND: Strenuous exercise has been reported to cause LV damage. METHODS: Fourteen runners who completed an ultramarathon at high altitude underwent echocardiography, finger-tip oximetry and blood measurements of cardiac troponin I (cTnI) and creatine kinase, MB fraction (CK-MB) levels before, immediately after and 1 day after the race. RESULTS: At baseline, the echocardiograms showed normal LV and right ventricular (RV) size and function in all subjects, as well as mild tricuspid regurgitation in nine subjects, with normal estimated pulmonary artery systolic pressure (mean 28 mm Hg). At baseline, all oxymetric readings and CK-MB measurements were normal, and cTnI was undetectable. Immediately after the race, the echocardiograms showed the expected augmentation of global and segmental LV function in all subjects. Although the RV was normal in nine subjects, five developed marked RV dilation and hypokinesia, paradoxic septal motion, pulmonary hypertension and wheezing. CK-MB values were elevated in all subjects. In all but one subject cTnI was undetectable. In that subject there was a small elevation in cTnI accompanied by severe RV dysfunction and pulmonary hypertension. At the 1-day follow-up study, the echocardiographic measurements had normalized in all subjects. CONCLUSIONS: In trained athletes, strenuous exercise at high altitude did not result in LV damage. However, wheezing, reversible pulmonary hypertension and RV dysfunction occurred in a third of those completing the race. The incidence and pathogenesis of these findings remain to be determined.

Cardiac troponin I levels are normal or minimally elevated after transthoracic cardioversion.

OBJECTIVES: The present study was designed to assess the impact of direct current shocks on cardiac troponin I (cTnI), which has greater sensitivity and specificity than creatine kinase (CK) for the diagnosis of myocardial injury. BACKGROUND: Transthoracic direct current shocks can cause myocardial injury. They also cause elevations of total CK and CK-MB fraction (CK-MB). METHODS: We obtained measurements of cTnI total CK and CK-MB before and after elective cardioversions in 38 patients. Blood samples were drawn before and 8, 16, 24 and 48 h after cardioversion. Shock energy, current, impedance and number of shocks delivered were tabulated. RESULTS: Patients received a mean (+/-SD) of 2.1 +/- 1.2 shocks with a median cumulative energy of 300 J (range 50 to 1,580). Three patients had minimal elevations (1.5, 1.2 and 0.8 ng/ml, normal < or = 0.6 ng/ml) of cTnI. Two of these patients had impaired left ventricular contractility by echocardiography. Thirty-five of the 38 patients had no elevations of cTnI. Sixty-two percent of patients had an increase in CK after cardioversion, but CK-MB was elevated to an abnormal level of 12.7 ng/ml (normal < 6.7) in only one patient after cardioversion. CONCLUSIONS: Cardiac troponin I levels are either normal or minimally elevated after elective direct current cardioversion, suggesting that subtle myocardial injury can be caused by direct current transthoracic shocks. However, substantial elevations of cTnI after cardioversion suggest the presence of myocardial injury from causes unrelated to the direct current shocks administered for cardioversion.

Calcium determination in primary hyperparathyroidism.

Calcium is the most closely controlled substance in the blood. The biologic variation of total calcium is approximately 2% and of the biologically active free (ionized, ionic) calcium only 1%. Thus, the monitoring of calcium in blood requires analytic procedures of high precision and accuracy. For patients with asymptomatic primary hyperparathyroidism, calcium monitoring involves the measurement of total calcium and free calcium. This review first considers the measurement of total calcium and then free calcium.

Creatine kinase brain isoenzyme (BB-CK) presence in serum distinguishes osteopetroses among the sclerosing bone disorders.

Creatine kinase (CK) isoenzyme BB-CK is predominantly found in brain and is not normally detected in the blood. A few recent reports, however, have described BB-CK in serum from several patients with osteopetrosis (OP). To evaluate the presence and specificity of BB-CK in serum in the osteopetroses among disorders that increase skeletal mass, we quantitated total CK activity and CK isoenzymes in 15 patients representing the five major clinical forms of OP (2 infantile, 3 intermediate, 7 adult [2 type I, 5 type II], and 3 carbonic anhydrase II [CA II] deficiency cases) and in 22 patients representing 14 other types of sclerosing bone disease. All OP patients (except the two adult type I subjects) had BB-CK readily detected in their serum. Conversely, only 1 of the 22 patients with other sclerosing bone disorders had detectable BB-CK in serum (1 of 3 patients with fibrodysplasia [myositis] ossificans progressiva who had barely measurable activity). In three OP patients (one of two with the infantile form and two of five with adult, type II disease), BB-CK values were sufficiently high that serum total CK activity was elevated. In a newborn with malignant OP, both cord blood plasma and peripheral blood serum had substantial amounts of BB-CK. In three subjects (with adult type II OP), who were restudied 2-6 years later, BB-CK was still elevated in their blood. BB-CK in serum appears to distinguish the osteopetroses among the sclerosing bone disorders. Absence of serum BB-CK in adult type I disease suggests that this condition may not be a genuine form of OP. Assay of BB-CK in fetal blood could be studied as a means for prenatal diagnosis of malignant OP. Why the osteoclast failure that characterizes all true forms of OP is associated with BB-CK in the circulation is a new question for skeletal biologists.

The effects of extracellular calcium and epinephrine on cytosolic-free calcium in single rat adipocytes.

Changes in cytosolic calcium concentration ([Ca2+]i) in response to extracellular calcium and epinephrine were monitored in individual rat adipocytes by both photon counting and digital imaging techniques utilizing the intracellular fluorescent calcium probes Fura-2 and Indo-1. Adipocytes containing Fura-2 were attached to coverslips and shown to be as hormonally responsive to insulin as adipocytes in suspension [3.5 +/- 0.8 (n = 5) vs. 4.2 +/- 0.6 (n = 8)-fold increase in glucose oxidation over basal in response to 0.7 nM insulin]. Basal [Ca2+]i in single rat adipocytes was found to be 128 +/- 6 nM (n = 100). The addition of either extracellular calcium or epinephrine elicited transient, concentration-dependent increases in [Ca2+]i. Although the characteristics of calcium- and epinephrine-induced calcium transients are generally similar, the peak [Ca2+]i increase over basal is higher in response to calcium vs. epinephrine [37 and 64% (1 and 27 microM epinephrine), vs. 132 and 236% (2 and 4 mM calcium)]. All the cells tested responded to calcium but only 67% responded to epinephrine. Both alpha- and beta-adrenergic agonists were able to increase [Ca2+]i. The epinephrine-induced [Ca2+]i transients appear to be dependent upon extra-cellular calcium. Neither cholera nor pertussis toxin treatments altered basal [Ca2+]i. However, after treatment of adipocytes with either pertussis or cholera toxin, epinephrine stimulated oscillations in [Ca2+]i. Digital imaging revealed that adipocytes demonstrate a high degree of intracellular spatial heterogeneity and intercellular variability in the magnitude of response to both calcium and epinephrine. These studies demonstrate the feasibility of using single rat adipocytes to monitor intracellular free calcium, using both photon counting and digital imaging.

Failure of total calcium corrected for protein, albumin, and pH to correctly assess free calcium status.

The clinical effectiveness of published algorithms in correcting serum total calcium (CaT) for the effects of protein, albumin, and pH was tested. Corrected calcium (CaC) values obtained by 13 of these methods were compared with values of measured free calcium (CaF) in 55 samples from normal controls and 404 samples from patients with various disorders of calcium metabolism. Three criteria were used to compare either CaC or CaT with measured CaF: 1) the correlation coefficient, 2) the average absolute deviation from measured CaF of the values of CaF predicted by the linear regression of CaF on each CaC, and 3) the number of samples in which CaC or CaT gave a different impression of normality than measured CaF. Application of the 13 published algorithms produced varied results, but none produced substantially better agreement between CaC and CaF than was found between CaT and CaF. The application of additional algorithms derived by multiple linear regression using our data base gave slightly better results than any of the published algorithms, but many values of CaC remained which were disparate from the measured value of CaF. Correction of measured total calcium by using other concurrently obtained chemistry values does not seem to adequately predict calcium status as measured by free calcium.

Relationship of free and total calcium in hypercalcemic conditions.

An assessment of free and total calcium measurements was made in 691 patients with suspected hypercalcemia or disorders often associated with hypercalcemia. In 18.9% of the 1049 specimens analyzed from nine different patient groups, a different impression of hypercalcemia was obtained depending on whether the free or total calcium was considered. Analysis of the ratio of free to total calcium indicated that there are two main factors which influence the distribution of calcium in the serum of hypercalcemic patients: the concentrations of albumin and parathyroid hormone. A lowered albumin concentration accounted for the altered distribution of calcium in patients with malignancies and partially accounted for the altered distribution in patients postrenal transplantation. In patients with confirmed primary hyperparathyroidism a higher ratio of free to total calcium was found, which could not be explained by alterations in protein, albumin, pH, or CO2 content but was related to parathyroid hormone concentration. Free calcium appears to be a slightly better indicator of elevated calcium states than total calcium. Measurements of free calcium should be particularly useful in patients with altered albumin concentration, with multiple myeloma in whom a calcium-binding protein could be present, after renal transplantation, and with suspected hyperparathyroidism and normal or slightly elevated total calcium values.

Value of laboratory tests in the differential diagnosis of hypercalcemia.

Eighteen laboratory tests were compared in the differentiation of primary hyperparathyroidism from hypercalcemia associated with malignancy. Statistical comparisons of the test results were carried out in four patient groups and two control groups. The patient groups evaluated were those with confirmed primary hyperparathyroidism, those with malignancy with hypercalcemia, those with malignancy without hypercalcemia, and those with surgically cured primary hyperparathyroidism. These groups allowed determination of the relative diagnostic values of the tests and a rationale for their value. After exclusion of patients with renal failure from the patient and control groups, these data indicated that the laboratory tests with the greatest differential diagnostic value, in order of efficacy, were: albumin, carboxy-terminal parathyroid hormone, venous pH, cholesterol, chloride, alkaline phosphatase, phosphorus, and the chloride/phosphate ratio. Hemoglobin, hematocrit, and red blood cell count also had some value, particularly in male patients. However, none of these tests individually achieved better than an 81 percent classification accuracy. With application of logistic discriminant analysis, only three tests--albumin, parathyroid hormone, and chloride--were identified as statistically significant in jointly improving the diagnostic separation between these two patient groups. Although the 94.4 percent classification accuracy achieved by use of these three variables in a logistic discriminant function was better than that obtained with any individual variable, incorrect classification was still a significant problem, particularly in the case of patients with malignancy and high concentrations of parathyroid hormone. With the exception of albumin and chloride measurements, the commonly available ancillary laboratory tests proposed to aid this differential diagnosis do not give any more information than the analysis of parathyroid hormone alone and merely add to the increased cost of medical care.

Clinically recognized cardiac dysfunction: an independent determinant of mortality among critically ill patients. Is there a role for serial measurement of cardiac troponin I?

OBJECTIVE: To determine the relative importance of clinically recognized cardiac dysfunction and unrecognized cardiac injury to hospital mortality. DESIGN: Prospective, blinded, single-center study. SETTING: Medical ICU of Barnes-Jewish Hospital, St. Louis, a university-affiliated teaching hospital. PATIENTS: Two hundred sixty adult patients requiring admission to the medical ICU. INTERVENTIONS: Daily blood collection. MAIN OUTCOME MEASURES: The presence of cardiac dysfunction (myocardial infarction, unstable angina, cardiac arrest, or congestive heart failure) as determined by the physicians responsible for the care of the patient. Daily measurement of levels of cardiac troponin I, a sensitive, highly specific, and long-lived marker of myocardial injury. RESULTS: Fifty-five (21.2%) patients had clinical evidence of cardiac dysfunction, among whom 22 (40%) had an elevated level of cardiac troponin I. A total of 41 (15.8%) patients had evidence of acute myocardial injury based on elevated levels of cardiac troponin I. Patients with clinically recognized cardiac dysfunction had a significantly greater hospital mortality rate compared to patients without clinically recognized cardiac dysfunction (45.5% vs 10.2%; p < 0.001). Similarly, patients with elevated blood levels of cardiac troponin I had a greater hospital mortality rate compared to patients without elevated blood levels of cardiac troponin I (26.8% vs 16.0%; p = 0.095). Multiple logistic-regression analysis controlling for potential confounding variables demonstrated that the presence of clinically recognized cardiac dysfunction was independently associated with hospital mortality (adjusted odds ratio = 3.0; 95% confidence interval = 1.9 to 4.8; p = 0.016). However, having an elevated blood level of cardiac troponin I was not found to be an independent determinant of hospital mortality. CONCLUSION: Among critically ill medical patients, clinically recognized cardiac dysfunction is an independent determinant of hospital mortality. The identification of unrecognized cardiac injury, using serial measurements of cardiac troponin I, did not independently contribute to the prediction of hospital mortality.

A clinically applicable high-pressure liquid chromatographic method for measurement of serum theophylline, with detailed evaluation of interferences.

A high-pressure liquid chromatographic method for the routine measurement of serum theophylline that is accurate, specific, and sensitive is presented. It is technically simple, requires only 50 microliter serum, and takes relatively little technician time. Values for theophylline in sera obtained by this method correlate well with those obtained by use of a gas-liquid chromatographic method (r = 0.980) and with values obtained by the commonly used spectrophotometric method (r = 0.939) when the latter is free of interference. The assay has been employed for two years in a routine clinical chemistry laboratory, with interassay coefficients of variation of 2.4-3.2%. Interferences have been identified at an incidence rate of 3.8% of all specimens, and interfering substances include cephazolin, chloramphenicol, sulfamethoxazole, and sulfisoxazole. A calculation technic that allows a reliable and clinically useful estimation of a range of concentrations of theophylline in the serum when these interferences are present is described. In contrast, the incidence of interferences with the spectrophotometric method is 4.9%, and in this method the interferences prevent the analysis of theophylline. Of 33 samples having interferences by use of the spectrophotometric method, the high-pressure liquid chromatographic technic yielded precise values for theophylline in 21 and reliable estimates of ranges of concentrations of theophylline in the remaining 12.

Prolonged exercise decreases serum leptin concentrations.

Serum leptin and free fatty acid concentrations were determined in two groups of subjects undergoing strenuous exercise: 12 men who fasted overnight and then pedaled a stationary ergometer for 2 hours, and 14 nonfasting ultramarathon runners. Blood samples were collected before exercise, immediately after cessation of exercise, and 6 to 24 hours after the end of the exercise period. Two hours of strenuous pedaling following an overnight fast significantly reduced mean leptin levels by 8.3%; free fatty acids were highly increased and correlated well with the decrease in serum leptin (r = .737, P = .01). After 6 hours of rest and refeeding, leptin concentrations recovered to preexercise levels and free fatty acid concentrations were decreased to less than preexercise levels. A similar decrease in serum leptin levels (12.3%) occurred in subjects who fasted overnight and then for a period corresponding to the cycle exercise period. The prolonged exercise of an ultramarathon significantly reduced leptin concentrations by 32% in comparison to prerace levels; free fatty acid concentrations were highly increased, but did not correlate with the change in serum leptin concentrations (r = .366, P = .20). Leptin and free fatty acid concentrations all trended toward prerace levels in blood samples collected 18 to 24 hours after cessation of racing. The results suggest that the negative energy balance of exercise can reduce serum leptin concentrations, but that the significant decrease occurs only at extremes of severity/duration of the exercise-induced negative balance. The possible physiological role of reduced leptin concentrations in response to energy balance and the role of free fatty acids in mediating the response are discussed.

Patterns and potential value of cardiac troponin I elevations after pediatric cardiac operations.

BACKGROUND: Perioperative myocardial injury is a major determinant of postoperative cardiac dysfunction for congenital heart disease, but its assessment during this period is difficult. The objective of this study was to determine the suitability of using postoperative serum concentrations of cardiac troponin I (cTnI) for this purpose. METHODS: Cardiac troponin I levels were measured serially in the serum of patients undergoing uncomplicated repairs of atrial septal defect (n = 23), ventricular septal defect (n = 16) or tetralogy of Fallot (n = 16). The concentrations were correlated with intraoperative parameters (cardiopulmonary bypass time, aortic cross-clamp time, and cardiac bypass temperature), and postoperative parameters (magnitude of inotropic support, duration of intubation, and postoperative intensive care and hospital stay). RESULTS: Postoperative absolute cTnI levels were lesion specific, with a pattern of increase and decrease similar for each lesion. For the total cohort, significant correlations between postoperative cTnI levels at all times (r = 0.43 to 0.83, p < 0.05) until 72 hours were noted for all parameters, except for cardiac bypass temperature. When evaluated as individual procedure groups, no significant relationships were noted in the atrial septal defect group, whereas postoperative cTnI levels were more strongly correlated with all intraoperative and postoperative parameters in the ventricular septal defect group than in the tetralogy of Fallot group. CONCLUSIONS: This study suggests that cTnI values immediately after operation reflect the extent of myocardial damage from both incisional injury and intraoperative factors. Cardiac tropinin I levels in the first hours after operation for congenital heart disease are a potentially useful prognostic indicator for difficulty of recovery.

Comparison of hemoglobin A1c and hemoglobin A1 in diabetic patients.

Hemoglobin A1 (HbA1) and hemoglobin A1c (HbA1c) values were compared in 934 samples from 686 subjects with diabetes mellitus. Linear regression analysis revealed the two parameters to be highly related with HbA1 = 1.18 HbA1c + 1.67, and a correlation coefficient of 0.97. Samples with HbA1 greater than predicted from the measured HbA1c had been stored significantly longer (approximately 1 day) than other samples, suggesting that sample storage may slightly alter the relationship between these parameters. Serial HbA1 and HbA1c values in all 9 patients for whom data was available from at least 5 time points changed in parallel regardless of the time interval between sampling or the magnitude of the interval change of HbA1c and HbA1. These data indicate that the potentially simpler measurement of HbA1 gives results equivalent to HbA1c for clinical applications.

Influence of fatty acids on the binding of calcium to human serum albumin.

Fatty acids have been shown to influence the binding of calcium to human serum albumin. The calcium binding to albumin was enhanced when long-chain fatty acids were added to albumin prepared by two different methods and decreased when fatty acids were removed from albumin. Palmitic, stearic and oleic acids all exhibited this phenomenon. The effects of long-chain fatty acids on the binding of calcium to albumin in vitro appears to be of sufficient magnitude to have in vivo implications in calcium homeostasis and in determining the ratio of free to total calcium. Preliminary in vivo experiments have confirmed the calcium binding of fatty acids in serum and suggest a physiological role for this phenomena.

The effects of stasis with and without exercise on free calcium, various cations, and related parameters.

The changes in free calcium, total calcium, albumin, protein, pH, lactate, sodium, magnesium, and potassium values during and following venous stasis (3 min at an external pressure of 100 mmHg) with and without forearm exercise have been measured. The pattern of changes observed with time were dependent on the presence or absence of forearm exercise. Without exercise significant changes are only observed at 1 min following stasis and then the increases are only moderate (free calcium 2.0%, total calcium 2.4%, albumin 6.9%, and protein 5.9%). However, when the forearm is exercised, larger increases (free calcium 8.6%, total calcium 8.4%, albumin and protein 12.4%) were observed for all parameters following stasis. These increases took 1 to 3 min to return to baseline for most parameters. Only potassium and magnesium went below baseline during the recovery period. It is recommended that when a tourniquet is used to aid in obtaining venous blood samples for these analytes, exercise of the forearm be avoided. If forearm exercise is unavoidable, then the sample should be obtained 1 to 3 min after release of the tourniquet.

Quantitation of serum lactate dehydrogenase-5 with monoclonal antibodies.

Spleen cells from BALB/cJ mice which had been immunized with human lactate dehydrogenase-1 (LDH-1) were fused with SP2/0-Ag14. Two hybridomas were produced which recognized the antigen. Competitive RIA revealed that one antibody ('Smit-LDH') recognized the H subunit of LDH while the other ('Hem-LDH') recognized both H and M subunits of LDH. With the use of these antibodies we developed an assay for LDH-5 activity in which serum is incubated for 30 min at room temperature with the two antibodies ('Smit-LDH' and 'Hem-LDH' in the ratio 64:1.3, micrograms/ml) immobilized on latex beads to extract LDH-1 through LDH-4. After centrifugation, the LDH activity of the supernatant is measured and represents LDH-5 activity. Latex beads coated with bovine serum albumin were used as control. The LDH-5 activity as determined by our assay correlated well (r = 0.98) with the values obtained by an electrophoresis method. There was no interference due to LDH-1 through LDH-3 up to 3,000 U/l and LDH-4 up to 350 U/l. Serum samples with total LDH activity above 1,000 U/l were appropriately diluted in order to avoid interference by LDH-4. Use of these monoclonal antibodies allows precise, rapid and direct measurement of LDH-5 activity in serum.

Effects of beta-mercaptoethanol and chelating agents on the stability and activation of creatine kinase in serum.

We investigated the effects of sulfhydryl compounds on the stability of creatine kinase (CK) in unfrozen human serum and found that both beta-mercaptoethanol and N-acetylcysteine led to accelerated loss of the endogenous serum enzyme activity. This is in contrast to the results of others who have either studied the stability of exogenous enzyme added to human serum or studied endogenous enzyme in frozen serum. The addition of cation chelators to serum markedly improved the stability of the endogenous CK activity. The enhanced stability was independent of chelation of calcium, iron, manganese, copper, or zinc. In addition, cation chelators caused a 16% increase in the CK activity of fresh samples. This latter effect was independent of the activation of CK by BME and could be accounted for by chelation of calcium ions during the assay. The data suggest that addition of cation chelators prior to storage may be useful in enhancing the stability of CK in serum whereas sulfhydryl compounds should be added prior to assay rather than prior to storage.

A high-pressure liquid chromatography method for serum quinidine and (3S)-3-hydroxyquinidine.

A bonded reversed phase, high pressure liquid chromatography (HPLC) method for serum quinidine and its major metabolite in serum, (3S)-3-hydroxyquinidine, is presented. The method employs toluene, rather than benzene for extraction of 100 microliter of serum, can utilize either absorbance or fluorescence detectors, and utilizes a column and equipment which is also suitable for theophylline analyses. Quinidine values in sera by the present method correlated well with fluorometric non-chromatographic methods using ethylene chloride (r = 0.932) or benzene-sulfuric acid (r = 0.950) for extraction of the quinidine. The comparison data suggest a therapeutic range for quinidine of 1.3--5.0 mg/liter when measured by HPLC. In over one year of routine use in a clinical chemistry laboratory, the method has proven to be rapid and precise with interassay coefficients of variation of 2.5--5.5%. No interferences with the HPLC method have yet been identified.

Assay of theophylline: comparison of EMIT on the ABA-100 to HPLC, GLC and UV procedures, with detailed evaluation of interferences.

The EMIT technique for theophylline measurement as adapted to the Abbott Bichromatic Analyzer (ABA-100), was compared to two other established procedures. EMIT results correlated well with those obtained with high pressure liquid chromatographic (HPLC) (r = 0.995, N = 54) and spectrophotometric (UV) (r = 0.95, N = 37) methods when the patient samples used were free of apparent interferences in the comparison methods. On samples exhibiting interferences with HPLC and/or UV methods, the EMIT assay gave the same results as a gas-liquid chromatographic procedure which showed no interferences. In addition, theophylline metabolites, xanthine analogs and many commonly administered drugs did not interfere with the EMIT theophylline assay. An interassay coefficient of variation of 3.5--6.5% was demonstrated. Our data indicate that the EMIT technique exhibits adequate precision and specificity for routine theophylline determinations. Unlike the HPLC or UV procedures, we have not encountered any interferences with the EMIT technique.