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Japanese Journal of Clinical Oncology Pages 13-17

Measurement of Lactate Levels in Serum and Bile Using Proton Nuclear Magnetic Resonance in Patients with Hepatobiliary Diseases: its Utility in Detection of Malignancies
Introduction
Subjects and Methods
   Patients
   Sample Collection
   Measurement of Serum and Bile Lactate Levels by 1H-NMR
   Enzymatic Measurement of Serum Lactate Levels
   Biochemical Laboratory Tests and Serum Tumor Marker Assays
   Statistical Analysis
Results
   Serum Lactate Levels Measured by 1H-NMR
   Serum Lactate Levels Measured by the Enzymatic Method
   Bile Lactate Levels Measured by 1H-NMR
   Time Required for 1H-NMR measurements
Discussion
References

Measurement of Lactate Levels in Serum and Bile Using Proton Nuclear Magnetic Resonance in Patients with Hepatobiliary Diseases: its Utility in Detection of Malignancies

Measurement of Lactate Levels in Serum and Bile Using Proton Nuclear Magnetic Resonance in Patients with Hepatobiliary Diseases: its Utility in Detection of Malignancies Tatsushige Nishijima1, Masami Nishina2 and Kenji Fujiwara1

1Third Department of Internal Medicine, 2Department of Medical Zoology, Saitama Medical School, Irumagun, Saitama, Japan

Proton nuclear magnetic resonance (1H-NMR) has been utilized for qualitative and quantitative measurement of the components of nonhomogeneous biological specimens, as it can analyze sensitively the chemical structure of organic compounds without pretreatment of the materials. Levels of lactate in serum and bile were measured by 1H-NMR in healthy volunteers and patients with non-malignant or malignant diseases of the liver and biliary tract, and the usefulness of such measurements for the diagnosis of hepatobiliary malignancies was determined. The mean (+/-SD) serum lactate levels were 0.52 +/- 0.33 mmol/l in five healthy volunteers, 1.38 +/- 1.59 mmol/l in 30 patients with non- malignant diseases and 2.95 +/- 2.00 mmol/l in 21 patients with malignant diseases, the differences among the three groups being significant. Biochemical enzymatic measurement of serum lactate levels revealed no such difference. In bile, the spectrum of lactate was observed in all of 16 patients with malignant diseases, but in none of two healthy volunteers and 12 patients with non-malignant diseases. The mean time required for the measurement was 36.77 min for serum and 6.40 min for bile. The measurement of lactate levels in serum and bile using 1H-NMR may be useful for the detection of hepatobiliary malignancies.

Key words: 1H-NMR - lactate - hepatocellular carcinoma - carcinoma of the biliary tract

Introduction

Proton nuclear magnetic resonance (1H-NMR) is a sensitive technique for analysis of chemical structures, paticularly those of organic compounds (1). Recently, 1H-NMR has drawn attention as a tool for medical and laboratory studies, since it can measure the components of nonhomogeneous biological specimens with high specificity and requires no specimen preparation (2-4).

Lactate accumlates in cells under anaerobic conditions (5). In tumor tissues where cell proliferation is active, a large amount of glucose is converted to lactate, leading to an increase of lactate in the extracellular space of tumors and in serum (6).

In the present study using lH-NMR, we measured the levels of lactate in serum and bile of patients with hepatobiliary diseases to test its possible application for the diagnosis of malignancies.

Subjects and Methods

Patients

All the studied patients had hepatobiliary diseases with no major complications. There were 30 patients with nonmalignant diseases (16 men and 14 women; mean age 53.0 years) comprising six with bile duct stones, three with intrahepatic gallstones, four with fatty liver, one with acute hepatitis A, five with chronic hepatitis C, eight with liver cirrhosis and three with primary biliary cirrhosis. Twenty-one patients had malignant diseases (nine men and 12 women; mean age 58.6 years) comprising five with cholangiocarcinoma, two with gallbladder carcinoma and 14 with hepatocellular carcinoma complicating liver cirrhosis. The diagnoses were made by blood biochemical tests, histological studies and imaging diagnostic aids. Five healthy volunteers (three men and two women; mean age 37.0 years) were also studied. All the studied patients and healthy volunteers gave their informed consent. Serum was collected from all subjects, and bile from 12 patients with non malignant diseases, 16 patients with malignant diseases and two volunteers.

Sample Collection

Blood was collected from all subjects at rest before breakfast with a plastic syringe, and centrifuged immediately at 4°C for 15 min to separate the serum. Bile samples were collected from healthy volunteers (A bile) by the Meltzer-Lyon method and from patients by puncture of the gallbladder or bile duct at surgery or immediately after percutaneous transhepatic cholangiographic drainage. Serum and bile samples for measurements of lactate levels were stored at -80°C immediately after collection until use, which was within 1 month.

Measurement of Serum and Bile Lactate Levels by 1H-NMR

Frozen serum and bile samples were thawed at room temperature. Measurements were made at 400 MHz using 32 to 64 scans with a Model JEOL-EX 400 (Tokyo, Nihon Denshi) according to the homogated decoupling method (HMG) (7) and the Carr-Purcell-Meiboom-Gill method (CPMG) (4). The time required for measurements was calculated from the number of scans and min read as ACQTM (acquisition time) and PD (pulse delay time).

450 µ1 of the serum sample or bile sample was placed in the NMR column (5 mm diameter). After the site of the NMR signals had been confirmed, the proton of the methyl group of lactic acid was quantified by adding 50 µl of a solution of methionine in heavy water for serum samples or a solution of sodium tetrasilyl propionate (TSP) in heavy water for bile samples as a reference for the intensity of the signals. The final level of methionine and TSP in serum and bile samples for analysis was 1 mmol/l.

Enzymatic Measurement of Serum Lactate Levels

Frozen serum samples were thawed at room temperature. The enzymatic lactate measurement was made using lactate oxidase (8).

Biochemical Laboratory Tests and Serum Tumor Marker Assays

Biochemical laboratory tests were performed by an autoanalyser. Alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9) were measured by RIABEAD (solid-phase assay method using polystyrene beads and protein- induced vitamin K absence or antagonist-II (PIVKA-II) by enzymatic immunoassay.

Statistical Analysis

Results were expressed as mean +/- SD and analyzed statistically by Mann-Whitney U test.

Results

Serum Lactate Levels Measured by 1H-NMR

In all healthy volunteers, no spectrum of lactate was evident by the HMG method, but a minimal lactate peak was recorded by the CPMG method. Although a lactate peak was detected by the HMG method in patients with non-malignant as well as malignant diseases, it was much smaller than the peak detected by the CPMG method. Fig. 1 shows representative spectra obtained by the CPMG method.


Figure 1. Spectra of serum samples determined by 1H-NMR using the CPMG method. Upper: patient with liver cirrhosis. Lower: patient with hepatocellular carcinoma (Table 1, Case 3). M, methionine; L, lactate; W, water.

Serum lactate levels in healthy volunteers and patients detected by the CPMG method are shown in Fig. 2. The mean (+/- SD) levels were 0.52 +/- 0.33 mmol/l in five healthy volunteers, 1.38 +/- 0.59 mmol/l in 30 non-malignant patients, and 2.95 +/- 2.00 mmol/l in 21 malignant patients, with significant differences among three groups (P < 0.01). There were no differences in the levels according to sex, age, or presence of jaundice.

Table 1 shows the demographic and clinical features and serum lactate levels of 14 patients with hepatocellular carcinoma complicating liver cirrhosis. The lactate levels had no correlation with tumor size. There were eight patients (57.1%) whose serum lactate levels were <2.80 mmol/l, the upper limit in nonmalignant diseases. In four of these patients, the serum AFP levels were below 1000 ng/ ml, and two of them had serum PIVKA-II levels below 0.1 arbitrary units (AU)/ml.

Table 2 shows the demographic and clinical features and serum lactate levels of seven patients with biliary tract carcinoma. Two patients (28.6%) showed serum lactate levels above the upper limit for non-malignant diseases, with high serum CA19-9 levels.

Table 1 Demographic and clinical features and serum lactate levels measured by 1H-NMR in patients with hepatocellular carcinoma
Case Sex Age AFP PIVKA II Lactate (mmol/l) Tumor size*
    (yr) (ng/ml) (AU/ml) Serum Bile (cm)
1 F 67 1600 2.56 4.86 + Multiple
2 F 57 21 <0.06 1.44 + <3
3 F 52 2700 2.80 3.80 + <3, 5
4 F 64 180 0.08 3.20 + <3
5 M 52 460 0.08 3.01 + 3
6 M 64 880 1.60 2.96 + 4.5
7 F 59 15000 2.88 5.72 + 5, 7
8 F 69 20 <0.06 2.12 + <3
9 F 49 220 1.02 2.04 + 3
10 F 59 680 1.90 2.88 + 3
11 M 52 810 2.01 1.98 + 2, 3
12 M 56 2200 2.80 6.40 ND 4
13 M 60 160 <0.06 1.76 ND 3
14 F 57 80 <0.06 1.40 ND 3
AFP, alpha fetoprotein; PIVKA II, protein induced by vitamin K absence or antagonist-II; AU, arbitrary units; M, male; F, female; +, presence of a lactate peak; ND, not determined; * measured by ultrasonography.

Table 2 Demographic and clinical features and serum lactate levels measured by 1H-NMR in patients with cholangiocellular carcinoma
Case Sex Age CEA CA19-9 Lactate (mmol/l) Tumor type,
    (yr) (mg/dl) (U/ml) Serum Bile size* (cm)
1 F 32 45 1400 10.10 + H, ~4
2 F 64 ND ND 1.23 ND H, ~4
3 M 64 8 880 2.26 + H, 3-4
4 M 59 4 ND 2.13 + P, ~3
5 M 62 6 ND 2.57 + P, ~3
6 F 54 ND ND 2.14 ND G, ~3
7 M 78 6 340 2.88 + G, ~4
CEA, carcinoembryonic antigen; CA19-9, carbohydrate antigen 19-9; ND, not determined; M, male; F, female; H, hilar cholangiocarcinoma; P, peripheral cholangiocarcinoma; G, gallbladder carcinoma; * gross tumor size was measured by ultrasonography and CT scan.

Serum levels of sodium, potassium, chloride, creatinine and blood urea nitrogen were all within the normal ranges in all patients with hepatocellular carcinoma and biliary tract carcinoma.


Figure 2. Serum lactate levels measured by 1H-NMR using the CPMG method in healthy volunteers and patients with hepatobiliary diseases. Clear circles indicate icteric patients with serum total bilirubin levels >2.0 mg/dl and solid circles non-icteric patients. There were significant differences among the three groups (P < 0.01).

Serum Lactate Levels Measured by the Enzymatic Method

The mean (+/- SD) serum lactate levels measured by the enzymatic method were 36.22 +/- 6.42 mg/dl in five healthy volunteers, 33.46 +/- 11.48 mg/dl in 30 patients with nonmalignant diseases, and 32.50 +/- 6.35 mg/dl in 21 patients with malignant diseases. There were no significant differences among the three groups.

Bile Lactate Levels Measured by 1H-NMR

Lactate was not detected in any of the bile samples obtained from two healthy volunteers and 12 patients with non-malignant diseases by the HMG method or CPMG method. However, it was detected in all of the samples from 11 patients with hepatocellular carcinoma and five patients with carcinoma of the biliary tract even by the HMG method. Fig. 3 shows representative spectra obtained by the HMG method and the CPMG method in a patient with biliary tract carcinoma.


Figure 3. Spectra of bile samples determined by 1H-NMR in a patient with biliary tract carcinoma (Table 2, Case 1). Upper, HMG method; lower, CPMG method; T, TSP (sodium tetrasilyl propionate); L, lactate; W, water.

Time Required for 1H-NMR measurements

The mean times required for measurements of serum and bile samples by 1H-NMR were 6.40 and 36.77 min by the HMG and CPMG methods respectively.

Discussion

The present study revealed that serum lactate levels measured by 1H-NMR were significantly increased in patients with malignant hepatobiliary diseases in comparison with healthy adults and patients with nonmalignant hepatobiliary diseases. Conventional enzymatic measurement showed no such difference. Moreover, bile lactate was detectable only in hepatobiliary malignancy.

NMR spectroscopy, particulary 1H-NMR and l3C-NMR, is an excellent method for investigating the chemical structure of organic compounds, since it can measure qualitatively and quantitatively various organic acids, amino acids, and glucose in nonhomogeneous biological materials such as blood and urine non-destructively and with high specificity (9-10).

In the present study, lactate in serum and bile samples was measured by 1H-NMR using both the HMG method and the CPMG method, since when used to measure the constituents of biological samples containing abundant water, the HMG method sometimes fails to detect the constituents due to the prominent water peak. In such cases, therefore, the CPMG method is preferable (4, 7) despite being rather time-consuming (36.77 min versus 6.40 min by the HMG method in the present study). In fact, serum lactate was detectable in healthy adults only by the CPMG method.

In the case of lactate in bile, which contains various materials other than water and protein, even the HMG method was able to differentiate clearly malignant from non-malignant diseases, as shown in Fig. 3.

Of 14 patients with hepatocellular carcinoma complicating liver cirrhosis, eight showed serum lactate levels above the upper limit for non-malignant diseases including liver cirrhosis (Table 1). In four of them, the serum AFP levels were below 1000 ng/ml, as sometimes observed in patients with only liver cirrhosis (11).

Furthermore, two of these four patients had hepatocellular carcinoma <= 3 cm in diameter, with low serum PIVKA-II levels. None of the 14 patients had conditions causing lactic acidosis. The measurement of serum lactate levels by 1H-NMR would thus be a useful screening test for early detection of hepatocellular carcinoma. Similarly, a high serum lactate level would be a helpful indicator of the presence of malignant leisions in the biliary tract, as shown in Table 2.

Bile lactate was detected only in patients with malignant hepatobiliary diseases. This was done using the HMG method, which requires only 6.4 min for the measurement. Furthermore, collection of bile by the Meltzer-Lyon method is simple, and so the measurement of bile levels by 1H-NMR may be useful for the diagnosis of hepatobiliary malignancies.

Ohsaka et al. (12) reported that serum lactate levels measured by 1H-NMR were significantly increased in patients with various malignant tumors in comparison with patients with non-malignant diseases. However, only two patients with hepatobiliary malignant diseases were included in their series, and thus the utility of the measurement, which was done according to the HMG method using serum after deproteinzation, was unclear. The present measurement by the CPMG method required no pretreatment of the serum samples, and represents the first attempt to determine bile lactate levels by lH-NMR. Lactate levels might be increased in other fluids caused by malignancies, such as pleural effusion and ascites.

In conclusion, the measurement of serum and bile lactate levels by 1H-NMR may be useful as a screening test or a diagnostic aid in the detection of hepatobiliary malignancies.

References

1. Farrar, TC, Becker ED. Pulse and Fourier Transform NMR. New York: Academic Press, 1971

2. Nicholson JK, JK, Sadler PJ, Bales JR, Juul SM, MacLeod AF, Sonken PH. Monitoring metabolic diseases by proton NMR of urine. Lancet 1984; 2:751-52. MEDLINE Abstract

3. Bell JD, Brown JCC, Nicholson IK, Sadler PJ. Assignment of resonance for acute phase glycoproteins in high resolution proton NMR spectra of human blood. FEBS Lett 1987;215:311-5. MEDLINE Abstract

4. Rabenstein DL, Millis KK, Strauss EJ. Proton NMR spectroscopy of human blood plasma and red cells. Anal Chem 1988;60:1380A-91A. MEDLINE Abstract

5. Cori CF, Cori GT. The carbohydrate metabolism tumors. 1. The free sugar, lactic acid, and glycogen content of malignant tumors. J Biol Chem 1925; 64:11-22.

6. Haug H, Schramm C. Metabolic pattern of free amino acids, glucose, lactic acid, pyruvic acid, fatty acids, BUN, uric acid, etc. in serum of 21 healthy persons and 24 patients with malignant tumors. Clin Chem 1975;21:1025.

7. Bales JR, Higham M, Howe I, Nicholson JK, Sadler PJ. Use of high resolution nuclear magnetic resonance spectroscopy for rapid multi- component analysis of urin. Clin Chem 1984;30:426-32. MEDLINE Abstract

8. Hadjivassiliou AG, Pieder SV. The enzymatic assay of pyruvic and lactic acids. A diffinitive procedure. Clin Chim Acta 1968;19:357-61. MEDLINE Abstract

9. Nicholson JK, Wilson ID. High resolution proton magnetic resonance spectroscopy of biological fluid. Prog NMR Spectr 1989;21:449-501.

10. Halvorsen RA Jr, Riberio A, Blinder R, Waters C, Thompson WM. Magnetic resonance spectroscopy of bile. In vitro stability over time and component identity. Invest Radiol 1989;24:903-08. MEDLINE Abstract

11. Lee H-S, Chung YH, Kim CY. Specificities of serum alphafetoprotein in HBsAg+ and HBsAg- patients in the diagnosis of hepatocellular carcinoma. Hepatology 1991;14:68. MEDLINE Abstract

12. 0hsaka A, Yoshikawa K, Matsuhashi T. Detection by proton nuclear magnetic resonance of elevated lactate concentration in serums from patients with malignant tumors. Jpn J Med Sci Biol 1979;32:305-9.

Received May 29, 1996; accepted July 19, 1996
For reprints and all correspondence: Kenji Fujiwara, Third Department of Internal Medicine, Saitama Medical School, 38, Morohongo, Moroyama, Iruma-gun, Saitama 350-04, Japan
Abbreviations: 1H-NMR, proton nuclear magnetic resonance; HMG, homogated decoupling method; CPMG, Carr-Purcell-Meiboom-Gill method; AFP, alpha-feto protein; CEA, carcinoembryonic antigen; CA19-9, carbohydrate antigen 19-9; PIVKA-II, protein-induced vitamine K absence or antagonist-II; Au, arbitrary units; TSP, sodium tetrasilyl propionate.

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