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(Circulation. 1995;92:2848-2854.)
© 1995 American Heart Association, Inc.

Articles

Discrimination Between Myocardial and Skeletal Muscle Injury by Assessment of the Plasma Ratio of Myoglobin Over Fatty Acid–Binding Protein

Frans A. Van Nieuwenhoven, MSc; Appie H. Kleine, PhD; K. Will H. Wodzig, PhD; Wim T. Hermens, PhD; Hans A. Kragten, MD; Jos G. Maessen, MD, PhD; Cees D. Punt, MD; Marja P. Van Dieijen, PhD; Ger J. Van Der Vusse, PhD; Jan F.C. Glatz, PhD

From the Departments of Physiology (F.A.V.N., K.W.H.W., J.F.C.G.) and Motion Sciences (G.J.V.D.V.), University of Limburg; the Cardiovascular Research Institute Maastricht (CARIM) (A.H.K., W.T.H.); the Departments of Cardiothoracic Surgery (J.G.M.), Anesthesiology (C.D.P.), and Clinical Chemistry (K.W.H.W., M.P.V.D.), Academic Hospital Maastricht; and the Department of Cardiology (H.A.K.), De Wever Hospital, Heerlen, The Netherlands.

Correspondence to J.F.C. Glatz, PhD, Department of Physiology, Cardiovascular Research Institute Maastricht (CARIM), University of Limburg, PO Box 616, 6200 MD Maastricht, The Netherlands.

	Abstract

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Background Myoglobin and fatty acid–binding protein (FABP) each are useful as early biochemical markers of muscle injury. We studied whether the ratio of myoglobin over FABP in plasma can be used to distinguish myocardial from skeletal muscle injury.

Methods and Results Myoglobin and FABP were assayed immunochemically in tissue samples of human heart and skeletal muscle and in serial plasma samples from 22 patients with acute myocardial infarction (AMI), from 9 patients undergoing aortic surgery (causing injury of skeletal muscles), and from 10 patients undergoing cardiac surgery. In human heart tissue, the myoglobin/FABP ratio was 4.5 and in skeletal muscles varied from 21 to 73. After AMI, the plasma concentrations of both proteins were elevated between 1 and 15 to 20 hours after the onset of symptoms. In this period, the myoglobin/FABP ratio was constant both in subgroups of patients receiving and those not receiving thrombolytics and amounted to 5.3±1.2 (SD). In serum from aortic surgery patients, both proteins were elevated between 6 and 24 hours after surgery; the myoglobin/FABP ratio was 45±22 (SD), which is significantly different from plasma values in AMI patients (P<.001). In patients with cardiac surgery, the ratio increased from 11.3±4.7 to 32.1±13.6 (SD) during 24 hours after surgery, indicating more rapid release of protein from injured myocardium than from skeletal muscles.

Conclusions The ratio of the concentrations of myoglobin over FABP in plasma from patients with muscle injury reflects the ratio found in the affected tissue. Since this ratio is different between heart (4.5) and skeletal muscle (20 to 70), its assessment in plasma allows the discrimination between myocardial and skeletal muscle injury in humans.

Key Words: myoglobin • fatty acids • myocardial infarction • aorta • surgery

	Introduction

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Biochemical markers of myocardial injury are helpful tools in differentiating patients with and those without AMI, thereby defining the small percentage (10% to 20%) of patients with symptoms consistent with ischemia who indeed have had an AMI.¹ Important characteristics determining the utility of a biochemical marker are its cellular localization, aqueous solubility, clearance from the circulation, specificity for myocardium, and detectability in plasma.¹

Recently, heart-type FABP has been introduced as a plasma marker for the early assessment of myocardial tissue injury^{2 3 4 5} and estimation of infarct size⁶ in humans. This small (15 kD) cytoplasmic protein is abundant in cardiomyocytes and is assumed to be involved in myocardial lipid homeostasis.⁷ Heart-type FABP is distinct from other types of FABP such as those found in liver and intestine.^{7 8} The plasma concentration of heart-type FABP is significantly increased within 3 hours after AMI,⁴ similar to that of myoglobin (18 kD), which has been described previously as an early biochemical marker for myocardial injury.^{9 10 11}

Both heart-type FABP and myoglobin are low-molecular-mass, cytoplasmic proteins present not only in the heart but also in skeletal muscle.^{7 12} This feature makes it difficult to discriminate between heart and skeletal muscle injury when plasma levels of these proteins are used as markers for loss of muscle cell viability. However, the myoglobin content of human heart is lower than that of skeletal muscle,¹² while studies in humans¹³ and rats¹⁴ have shown that the FABP content is at least twice as high in heart as in skeletal muscle. Therefore, we hypothesized that when the ratio of the contents of myoglobin and FABP in heart and skeletal muscle would differ significantly, and upon muscle injury both proteins would be released into and cleared from the blood to a similar extent, then the ratio of the increased plasma concentrations of myoglobin and FABP would be a useful index to identify the type of injured muscle.

The aims of this study were (1) to investigate whether the ratio of myoglobin over FABP in human myocardial tissue is substantially different from the ratio in skeletal muscle tissue and, if this is the case, (2) whether the assessment of this ratio in plasma can be used to discriminate between myocardial and skeletal muscle injury. To this end, the myoglobin and FABP contents were assessed in samples from human heart and various types of human skeletal muscle. Subsequently, the myoglobin and FABP concentrations were assessed in blood samples from patients after AMI and from patients after either aortic or cardiac surgery. These latter patients were suspected to have skeletal muscle damage alone (aortic surgery) or in combination with myocardial muscle damage (cardiac surgery). During aortic surgery, the aorta is clamped just beneath the renal arteries, rendering the lower part of the body ischemic and thus leading to appreciable skeletal muscle injury. Previous studies have shown that surgery alone can lead to detectable skeletal muscle injury.¹⁵ The ratios of the blood concentrations of the two biochemical markers then were compared for myocardial injury, skeletal muscle injury, and the occurrence of a combination of myocardial and skeletal muscle injury. Portions of these studies have been published previously in abstract form.^{16 17}

	Methods

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Patients With Acute Myocardial Infarction
Twenty-two patients with an initial clinical diagnosis of AMI were included in this study and were divided into three subgroups: (1) a subgroup of 10 patients not receiving thrombolytic therapy (8 women, 2 men; age, 71±10 years, mean±SD; ischemia located anteriorly in 8 cases and inferioposteriorly in 2 cases), (2) a subgroup of 9 patients receiving streptokinase (1.5 million U) (1 woman, 8 men; age, 58±10 years, mean±SD; ischemia located anteriorly in 2 cases and inferioposteriorly in 7 cases), and (3) 3 special cases (men with inferior myocardial infarction): a patient (45 years) who underwent cardioversion, a patient (59 years) who developed a recurrent AMI, and a patient (68 years) with AMI in combination with severe renal insufficiency. AMI was positively diagnosed when patients showed elevation of more than 1 mm of the ST segment in the recorded cardiogram and typical presentation of chest pain (often combined with radiation of pain to the left arm) in combination with transpiration, nausea, and/or shortness of breath. Reasons for omitting treatment with thrombolytic agents were increased risk of bleeding or previous coronary bypass surgery. Starting from admission of the patients to the coronary care unit of the hospital (2.1±1.1 hours and 2.6±1.0 hours after onset of symptoms for patients not receiving and receiving thrombolytic treatment, respectively; mean±SD), blood samples were taken every hour during the first 10 hours (except for the patient with renal insufficiency, every 3 hours). Thereafter, blood samples were taken every 6 or 12 hours, in accordance with the hospital routine. Blood samples were collected in glass tubes coated with ethylenediamine tetra-acetic acid (EDTA) (Sherwood Medical) and centrifuged at 1500g for 10 minutes. Plasma was collected and stored at -70°C until use.

Patients Undergoing Aortic or Cardiac Surgery
We studied a group of 9 patients undergoing aortic surgery (1 woman, 8 men; age, 72±11 years) and a group of 10 patients undergoing cardiac surgery (1 woman, 9 men; age, 63±8 years). The aortic surgery patients had either an aneurysm of the abdominal aorta (5 patients) or occlusive arterial disease (4 patients) and were given protheses of part of the abdominal aorta, the aorta bifurcation, or the common iliac artery. For this, the aorta was clamped distal of the renal arteries, rendering the lower part of the body ischemic (period of ischemia, 73±55 minutes). This intervention was expected to lead to significant skeletal muscle damage. None of the patients had a recent history of myocardial injury.

The other group consisted of patients with left ventricular dysfunction undergoing coronary bypass surgery alone (8 patients) or in combination with valve replacement surgery (2 patients). Patients were recruited from those operated on in the period July 1994 to February 1995 and were selected on the basis of a postsurgery increase in plasma activity of creatine kinase isoenzyme MB of more than 20 U/L, indicating the occurrence of significant myocardial injury. The period of ischemia was 74±37 minutes.

Serial blood samples obtained immediately before and after the surgery were collected in CORVAC separator tubes (aortic surgery) or glass tubes coated with EDTA (cardiac surgery) (Sherwood Medical). After centrifugation at 1500g for 10 minutes, serum or plasma, respectively, was collected and stored at -70°C until use.

Study Approval
For each substudy, the experimental protocol was thoroughly explained to the patients, and informed consent was obtained. The study protocols were approved by the Medical-Ethical Committees of the Academic Hospital Maastricht and the De Wever Hospital Heerlen.

Tissue Samples
Tissue samples of intact human heart and various skeletal muscles were obtained after autopsy (performed within 12 hours after death) from the Academic Hospital Maastricht. The samples were stored at -20°C until use. All steps of the tissue homogenization procedure were performed at 4°C or on ice. The tissue samples were homogenized (5% wt/vol) in PBS (pH 7.4) containing 3% (wt/vol) BSA (Sigma) with the use of an Ultra-Turrax homogenizer (IKA Werke). Thereafter, the samples were centrifuged at 2000g for 15 minutes, and the supernatants were stored at -70°C until use.

Sandwich ELISA for FABP
BSA, A7888, horseradish peroxidase (HRP, P8375), N-hydroxysuccinimidobiotin (NHS-d-biotin, H1759), and o-phenylenediamine dihydrochloride (OPD, P1526) were obtained from Sigma.

FABP was determined in plasma, serum, and supernatants of tissue sample homogenates using an enzyme linked immunosorbent assay of the antigen capture type (sandwich ELISA). This assay was developed essentially according to that described by Börchers et al¹⁸ for bovine heart FABP and that described by Vork et al¹⁴ for rat heart FABP.

In short, rabbit antibodies directed to human heart-type FABP were coated on 96-well microtiterplates (Falcon type 3912, Becton Dickinson) in 0.1 mol/L carbonate buffer pH 9.6 at 37°C for 2 hours. All further steps were performed at room temperature in PBT (phosphate-buffered saline, pH 7.2, supplemented with 0.1% (wt/vol) BSA and 0.05% (vol/vol) Tween-20). Between each step, the plate was washed 5 times with PBT. After coating and washing, 50 µL of sample or standard was incubated for 90 minutes, allowing the FABP to bind to the antibodies attached to the plates. A second antibody, either directly conjugated with HRP or biotinylated, then was incubated for 90 minutes. When conjugated antibody was used, 100 µL of substrate mixture containing 20 mmol/L OPD and 6 mmol/L H₂O₂ in 0.1 mol/L citrate buffer (pH 5) was added to each well. The biotinylated antibody required an additional incubation for 60 minutes with streptavidine-HRP (Pierce). In both cases, the enzyme reaction was stopped after 5 to 10 minutes with 50 µL 2 mol/L H₂SO₄, and the absorbance at 492 nm was measured with the use of a microplate reader (Titertek Multiskan MKII). Detection limit of the assay was 0.5 µg/L (25 pg per well). Recovery experiments (n=11) using normal human plasma spiked with purified human heart FABP yielded an average recovery of 93%. The interassay coefficient of variation was on the order of 7%.

Determination of Myoglobin
Myoglobin was determined in plasma and tissue samples with the use of a turbidimetric immunoassay (Turbiquant immunoassay, code No. OWNL, Behring, Hoechst Holland) on a Turbitimer analyzer (Behring, Hoechst Holland). Assay of myoglobin was performed according to the method of Delanghe et al.¹⁹ Turbiquant myoglobin is a freeze-dried reagent consisting of polystyrene latex particles (size, 100 nm) coated with rabbit anti-human myoglobin. The lyophilized reagent is resuspended with 10 mL of citrate buffer (pH 7.8). In the assay, the cuvette is filled with 50 µL of plasma, serum, or tissue homogenate and 500 µL of suspended latex particles. Dilutions of plasma, serum, and tissue homogenate were made in saline (0.9% NaCl). The myoglobin concentration is determined by turbidimetric measurement of the maximum reaction velocity (peak-rate method). The bar code on the package insert contains the calibration information needed for the assay. These data are stored by the instrument and can be used as long as the reagent lot number remains unchanged. Detection limit of the method is 50 µg/L. The preprogrammed measuring range covers myoglobin concentrations from 50 to 650 µg/L.

Internal quality control was performed with the use of the human Apolipoprotein Control Serum CHD (Behring, OUPH 06/07; lot No. 063617; assigned value, 95 µg/L; confidence limit, 81 to 109 µg/L). Day-to-day variation was obtained by measuring the control serum on 22 subsequent days, resulting in a mean concentration of 97.4 µg/L and a day-to-day variation of 4.8%.

Calculation of Myoglobin/FABP Ratio
The ratio (g/g) of myoglobin over FABP in cardiac and skeletal muscle tissue was calculated directly from the tissue contents of these proteins. The ratio of myoglobin over FABP in plasma or serum upon muscle injury was calculated from the increased levels of myoglobin and FABP. The basic levels of both proteins were subtracted from the plasma or serum levels measured. For FABP, individually measured basic values were used. For the AMI patients, the basic value was the FABP concentration measured in the first sample taken after arrival in the hospital or, in case this sample already showed a significantly raised FABP level (>19 µg/L, Reference 4), the plasma level more than 36 hours after AMI or the average FABP basic level of 9 µg/L was used.⁴ The basic FABP levels of patients undergoing aortic or cardiac surgery were determined by measuring blood samples before surgery.

Because the practical lower detection limit of the currently used myoglobin assay is 50 µg/L, the basic plasma level of myoglobin was assumed to be 30 µg/L.^{10 11 20 21}

The ratios of myoglobin over FABP were calculated only for those samples in which both proteins were raised at least twice above their basic value. For this reason, calculations were not performed for some time points.

Statistical Analysis
Data are expressed as mean±SD as indicated. Release curves of proteins into plasma or serum and curves of ratios of myoglobin over FABP are presented as mean±SEM for sake of clarity. Pearson's correlation coefficient was calculated to show relations between the myoglobin and FABP contents of human heart and skeletal muscle. A t test for independent samples was used to assess statistically significant differences. The level of significance was set at P<.05.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Tissue Contents of FABP and Myoglobin
The FABP and myoglobin contents were measured in five different areas of the human heart and in various types of skeletal muscle (Table). The FABP content of left ventricular tissue is similar to that found in an earlier study (0.56±0.07 mg/g wet wt for 17 individuals).⁶ For the calculation of the average contents of both proteins in the total heart, the differences in total mass of the left and right ventricle were taken into account. The weight ratio of right/left ventricles was taken as 1 to 3.²² It is apparent from the Table that the FABP values (normalized on gram of wet weight of muscle) are higher in heart than in any of the skeletal muscles examined, while in most cases the myoglobin levels show the opposite. This type of FABP (heart or muscle type) is also expressed in some other organs and tissues such as smooth muscle, kidney, and mammary gland but always in markedly lower quantities than that found in heart and skeletal muscle.^{23 24}

View this table: [in this window] [in a new window]   Table 1. Myoglobin and FABP Contents of Human Heart and Skeletal Muscle

The ratio (g/g) of myoglobin over FABP appeared to be rather constant among the various parts of the human heart and was found to be about 10 times lower in heart than in skeletal muscle (Table). In addition, a significant correlation was found for the contents of myoglobin and FABP in the individual muscle samples both from heart (r=.82; n=23; P<.001) and from skeletal muscle (r=.66; n=13; P=.014). The relation between the myoglobin and FABP contents in the individual heart and skeletal muscle samples is shown in Fig 1.

View larger version (13K): [in this window] [in a new window]   Figure 1. Plot shows relation between myoglobin and FABP content in individual samples from different areas of human heart (, n=23) and various types of skeletal muscle (, n=13). A significant correlation is found for the contents of myoglobin and FABP among the muscle samples from heart (r=.82; P<.001) and those from skeletal muscle (r=.66; P=.014).

Protein Release After AMI
In 19 patients with AMI, 9 receiving and 10 not receiving thrombolytic therapy, the plasma concentrations of myoglobin and FABP were measured in serial samples obtained during the first 25 hours after the onset of symptoms. All patients showed a marked and simultaneous release of myoglobin and FABP into plasma within a few hours after onset of pain, with peak values being reached at about 8 hours (patients not receiving thrombolytics) (Fig 2A1) and about 4 hours after the onset of symptoms (patients receiving thrombolytics) (Fig 2B1). In both groups of patients, the ratio of myoglobin over FABP appeared constant during the time of elevated plasma concentrations, amounting to 6.2±1.0 (126 samples; range, 2.2 to 10.5) for 10 patients not receiving thrombolytics and 4.4±1.4 (93 samples; range, 2.1 to 6.5) for 9 patients receiving thrombolytics (Figs 2A2 and B2). For individual patients, the relative maximal scatter of the ratio in time after AMI amounted to 19% to 41% around the mean value (all patients).

View larger version (24K): [in this window] [in a new window]   Figure 2. Plots show mean plasma concentrations of myoglobin (MYO, ) and FABP () (left panels) and the myoglobin/FABP ratio () (right panels) in 10 patients after AMI and not receiving thrombolytic therapy (A) and in 9 patients after AMI and receiving thrombolytic therapy (B). Data refer to mean±SEM. Myoglobin/FABP ratios on each time point were calculated as means of the ratios found for each patient; the small standard error, however, is not larger than the size of the symbols used.

Protein Release After Aortic or Cardiac Surgery
Myoglobin and FABP concentrations were measured in blood samples obtained immediately before surgery and during the first 24 hours after surgery. For these patients, the mean release curves are shown in Fig 3. The ratio of myoglobin over FABP was calculated for those samples in which both myoglobin and FABP concentrations were raised to at least twice their basic values. In case of aortic surgery, 7 of the 9 patients showed release of both proteins to a level twice above their individual basic level in samples between 6 and 24 hours after surgery. The mean serum ratio of myoglobin over FABP varied from 35 to 50 (Fig 3A2) and for all samples examined amounted to 45±22 (26 samples, 7 patients). In the cardiac surgery cases, all 10 patients showed a marked increase in plasma concentrations of both proteins (Fig 3B1). In these patients, the ratio of myoglobin over FABP was 11.3±4.7 at 0.5 hours after surgery; this decreased to 6.7±3.7 at 8 hours after surgery and then increased again to 32.1±13.6 at 24 hours after surgery (values at 8 and 24 hours each significantly different from value at 0.5 hours; P<.05) (Fig 3B2).

View larger version (23K): [in this window] [in a new window]   Figure 3. Plots show mean serum or plasma concentrations of myoglobin () and FABP () (left panels) and the myoglobin/FABP ratio () (right panels) in 9 patients after aortic surgery (A) and in 10 patients after cardiac surgery (B). Data on myoglobin/FABP ratios on each time point were calculated as means of the ratios found for each patient. Data refer to mean±SEM.

Special Cases
To assign the clinical significance of the plasma ratio of myoglobin over FABP for patients with myocardial infarction, we also studied three special cases. One patient underwent cardioversion (defibrillation) approximately 4 hours after the first onset of symptoms of AMI, a treatment that could very well have resulted in skeletal muscle damage (most likely of the intercostal and pectoral muscles). The release curves of myoglobin and FABP of this defibrillated patient (Fig 4A1) are different from the mean release curves of the nondefibrillated patients (Fig 2B1). Interestingly, the plasma ratio of myoglobin over FABP increased from 8 at 4 hours after AMI to >50 at 24 hours after AMI (Fig 4A2).

View larger version (24K): [in this window] [in a new window]   Figure 4. Plots show mean plasma concentration of myoglobin () and FABP () (left panels) and the myoglobin/FABP ratio () (right panels) in one patient after AMI and subsequent defibrillation (A), in one patient who developed a recurrent AMI (B), and in one patient after AMI in combination with severe renal insufficiency (C). All patients received routine treatment with thrombolytic agents.

Another patient developed a recurrent myocardial infarction soon (<10 hours) after the initial AMI. The appearance of this recurrent infarction is reflected clearly in the plasma curves for myoglobin and FABP (Fig 4B1). However, the plasma ratio of myoglobin over FABP is constant in time, amounting to 4.6±0.8 (8 samples) (Fig 4B2). A third patient suffered from AMI in combination with severe renal insufficiency, which caused the plasma concentrations of both myoglobin and FABP to remain elevated during the entire period of blood sampling (Fig 4C1) but did not affect the myoglobin over FABP ratio in this time interval; the ratio was 2.9±0.4 (12 samples) (Fig 4C2).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
One of the primary characteristics of a biochemical marker of myocardial damage is a specificity for myocardium.¹ Myoglobin and FABP each are early biochemical markers present in substantial amounts not only in the aqueous cytoplasm of cardiomyocytes but also in that of skeletal muscle,^{12 13 14} thus limiting their use as plasma markers of myocardial injury. However, the present study shows that the ratio of the tissue contents of myoglobin and FABP is markedly different between heart and skeletal muscles and that in patients with muscle injury, the ratio of the plasma concentrations of myoglobin and FABP reflects that of the injured tissue, indicating the use of this ratio to discriminate myocardial from skeletal muscle tissue injury.

The immunochemically assessed FABP content is found to be higher in human heart than in various skeletal muscles, whereas the myoglobin content in the majority of cases shows the opposite. These differences are best reflected in the ratios of the tissue contents of myoglobin over FABP, being 4.5 for heart and 21 to 73 for the skeletal muscles studied, which covers the entire range of types of skeletal muscle.

Myocardial Infarction
The myoglobin over FABP ratio found in plasma after myocardial injury (AMI) was constant during the entire sampling period and for patients not receiving thrombolytics amounted to 6.2±1.0 (n=126, 10 patients) and for those receiving thrombolytics to 4.4±1.4 (n=93, 9 patients). These mean ratios are not significantly different despite the differences in shape of the release curves found for the two groups of patients. It is important to note that the ratio of myoglobin over FABP found in plasma agrees with the ratio found in heart tissue. In serial blood samples obtained from a larger but less frequently sampled group of patients with AMI and who had been treated with thrombolytic agents,⁶ we also found the plasma ratio of myoglobin over FABP to be constant with time after AMI and of similar magnitude as the tissue ratio (from 4.1±0.6 at 3 hours to 4.4±0.7 at 24 hours after AMI; 49 patients).²⁵ Further confirmation of these observations was obtained in a recent study with 23 patients with AMI, for which the plasma myoglobin over FABP ratio amounted to 6.2±0.4 (n=23).²⁶

Because the release curves of myoglobin and FABP show a similar pattern for each group of patients, our findings indicate that both myoglobin and FABP are released from the heart and cleared from the bloodstream essentially in the same manner. In view of their low molecular masses, it is most likely that myoglobin (18 kD) as well as FABP (15 kD) are eliminated from the circulation mainly by renal clearance.^{27 28} Indeed, both proteins have been found in urine from patients with AMI^{3 4 5} and show elevated plasma levels during a longer period of time in case of renal insufficiency (Fig 4C).

Aortic and Cardiac Surgery
Patients who underwent aortic surgery also showed a simultaneous release and clearance of myoglobin and FABP even though peak values were recorded at a longer time period after muscle injury than in patients with AMI. We expected the former patients to have skeletal muscle injury resulting from the ischemic period during aortic surgery. Indeed, in these patients the serum peak values of myoglobin and FABP generally were higher in patients with a longer period of occlusion (data not shown). In addition, the serum ratio of myoglobin over FABP was 45±22 (n=26, 7 patients), which is within the range of ratios monitored in skeletal muscle tissue and significantly different from the ratio found in blood plasma after AMI (t test for independent samples; AMI, n=93; aortic surgery, n=26; P<.001). The slow release of proteins from skeletal muscle will relate to a lower blood flow during rest, a smaller lymph flow, and a lower permeability of the endothelial barrier in skeletal muscle than in heart.²⁹ In a study with volunteers after skeletal muscle overload caused by strenuous exercise, we also observed the ratio of myoglobin over FABP in plasma to be comparable with the ratios found in skeletal muscle tissue.¹⁶

Patients undergoing cardiac surgery were studied because they can be expected to have both myocardial and skeletal muscle injury.^{15 30} In these patients, the postoperative plasma curves of myoglobin and FABP were markedly different, with highest FABP concentrations found between 5 and 15 hours and highest myoglobin concentrations between 10 and 25 hours after surgery (Fig 3B1). As a result, during this entire time period the ratio of myoglobin over FABP first decreased from 11 (0.5 hours after surgery) to 7 (8 hours after surgery) and then increased to over 30 (24 hours after surgery) (Fig 3B2). These data agree with earlier observations that after cardiac surgery, release of enzymes from myocardium is more rapid and completed earlier in time (within 24 hours) than is release from injured skeletal muscle (>40 hours).¹⁵ The initial decrease of the ratio may reflect a higher relative contribution of proteins released from injured skeletal muscles (a result of the operation) than from myocardial necrosis at this early point in time. Thus, assessment of postoperative changes in the ratio of myoglobin over FABP in dependence of time will give insight into the relative contribution of myocardial and skeletal muscle injury to total muscle loss.

Quantitation of Muscle Injury
Since in patients with AMI as well as with surgery nearly full protein release curves were recorded, it is possible to globally estimate the total amount of muscle injury, expressed as gram equivalents of healthy muscle, for each group of patients. As described elsewhere,⁶ the cumulative release of FABP (and of myoglobin) from muscle can be calculated with the use of a one-compartment model, thus neglecting extravascularization of protein. Using a value of 2.6 h^-1 for the fractional clearance rate of FABP⁶ and a plasma volume of 3 L, in the group of patients with AMI and not receiving thrombolytics the mean cumulative release of FABP is 18 mg and in the patients receiving thrombolytics 7.5 mg, which is equivalent to 34 g and 14 g of myocardial tissue, respectively. Similarly, in the patients who underwent aortic surgery the mean cumulative release of FABP (up to 24 hours after surgery) amounts to 2.3 mg, which is equivalent to 30 g skeletal muscle tissue (estimated average FABP content, 0.07 mg/g). Assuming the lower body to contain about 10 kg of skeletal muscle, aortic surgery is found to cause an estimated mean injury of <0.5% of muscle mass. In patients undergoing uncomplicated coronary bypass surgery, cardiac injury has been estimated to amount to 1.5 g of myocardium compared with a loss of 13 g of skeletal muscle.¹⁵ Since in our study, patients were selected on the basis of a postsurgery increase in plasma activity of creatine kinase isoenzyme MB of more than 20 U/L, the contribution of myocardial necrosis to total muscle loss will be higher.

Clinical Application and Significance
The use of the ratio of myoglobin over FABP to determine the origin of protein release is illustrated by the patient who was defibrillated at arrival in the coronary care unit, an intervention resulting in a steady increase in the plasma ratio of myoglobin over FABP caused by additional skeletal muscle injury (Fig 4A). The latter is reflected more in the plasma curve of myoglobin than that of FABP (Fig 4A), indicating that in this case myocardial infarct size can be estimated better from the cumulative release of FABP than from that of myoglobin.

The data from the patient who developed a recurrent infarction and the patient with AMI and renal failure show that the plasma ratio of myoglobin over FABP may help in discriminating myocardial injury alone from the situation that additional skeletal muscle injury had occurred simultaneously or shortly after AMI. After all, in the latter case similar plasma curves for myoglobin and FABP could have been observed, but the myoglobin over FABP ratio would have been significantly different.

Since the plasma clearance rate of both myoglobin and FABP is rapid,⁶ application of the myoglobin/FABP ratio as discriminator of myocardial versus skeletal muscle injury requires a frequent blood sampling scheme and rapid assay procedures for both proteins, which for FABP is not yet available. However, now that a rapid and sensitive monoclonal antibody–based enzyme immunosensor assay system for FABP in plasma is being developed,^{31 32} the application of the myoglobin over FABP ratio can soon enter clinical practice.

Concluding Remarks
The present study indicates that both myoglobin and FABP in plasma can be used as markers of loss of cardiac and/or skeletal muscle cell integrity. Both proteins show a similar pattern of release into and clearance from plasma. However, the ratio of the plasma or serum concentrations of myoglobin over FABP after myocardial injury is significantly different from that found when skeletal muscles are most likely injured, as the plasma ratio reflects the ratio in which the proteins occur in the injured tissue. Hence, measurement of myoglobin and FABP in the same blood sample and expression of their ratio is useful to determine the origin of the proteins that are released into the vascular compartment. In this way, one can discriminate between damage inflicted upon cardiac and skeletal muscle tissue.

	Selected Abbreviations and Acronyms

 

AMI = acute myocardial infarction BSA = bovine serum albumin FABP = fatty acid–binding protein MYO = myoglobin PBS = phosphate-buffered saline

	Acknowledgments

 
This work was supported by StiPT, Executive Agency for Technology Policy, grant MTR88002, and the Netherlands Heart Foundation, grant D90.003. Jan F.C. Glatz is an Established Investigator of the Netherlands Heart Foundation. The authors wish to thank the employees of the Coronary Care Units of the Academic Hospital Maastricht and the De Wever Hospital Heerlen and the Departments of Pathology, Cardiology, and Anesthesiology of the Academic Hospital Maastricht for collecting tissue and blood samples. We would also like to thank Drs Harm Kuipers, Frits Prinzen, Eric van Breda, and Eric Fransen for their help and stimulating discussions during this study and Maurice Pelsers for expert technical assistance.

Received June 24, 1994; revision received June 15, 1995; accepted July 5, 1995.

	References

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