蛋氨酸.Methionine

Describe:

The importance of adequate stores of the amino acid methionine cannot be underestimated. Methionine is particularly important because it supplies sulfur--a mineral--that helps to maintain healthy skin tone, well-conditioned hair, and strong nails. Because your body can't produce this essential amino acid on its own, you must get it from methionine-rich foods, such as cheddar cheese, eggs, chicken, and beef. Supplements are also a source.

Methionine is thought to keep fat from building up in the liver, and it's often included in liver-detoxifying products called lipotropic combinations. These formulations are believed to accelerate the flow of bile and cell-damaging toxins away from the liver. They commonly contain the B vitamins choline and inositol in addition to methionine.

Methionine in lipotropic combinations has been proposed for treating endometriosis, a condition in which patches of endometrial tissue from the uterine lining grow outside the uterus. The nutrient is believed to help by expediting the removal of excess estrogen from the liver. The bleeding, cramps, pain, and other complications of this common ailment may subside as a result. More research is needed, however.

Reproducibility of the Methionine Breath Test: 

The objective of this study was to assess variability of the methionine breath test by administering the test twice within 7 days to healthy volunteers and patients with previously diagnosed liver disease. The study included 20 healthy volunteers, 11 females and 9 males, with a mean age of 33 years (range 18-59). The 22 liver patients included (10 females and 12 males) and the mean age was 51 years (range 26-71). The liver patients included 11 patients with stable cirrhosis with no alcohol intake for > 1 year, 5 actively drinking alcoholic cirrhotic patients, and 6 patients with cirrhosis and alcoholic hepatitis. The methionine breath test was performed while participants were at rest after an 8-hour fast. Reproducibility of the tests was evaluated using a paired Student’s t-test. A linear correlation coefficient was also calculated for the two tests. The null hypothesis was that the difference between the two tests was equal to zero. The null hypothesis was tested at p < 0.05.  

Reproducibility of the replicate tests was excellent as the paired t-test accepted the hypothesis that no difference was detectable between the tests (p=0.735). There was a significant correlation (r = 0.966, p<0.001) between methionine breath test 1 and test 2. The correlation plot is given in figure 1. These results show the reproducibility of the methionine breath test over a broad clinical range of liver function.  

Figure: Methionine Breath Test 1 vs Test 2 data for healthy controls and liver patients.

 

 

We found that the reproducibility of the methionine breath test was excellent in 20 volunteers as well as in the 20 patients with liver disease. Short-term reproducibility proved satisfactory, as differences between test 1 and test 2 were not significantly different from zero at 95% confidence with a paired t-test. Correlation analysis yielded (r = 0.966, p<0.001) across the entire clinical range.

 

Figure 2: Effect of alcohol and Aspirin on methionine metabolis

 

When alcohol or aspirin change the NAD/NADH ratio this either decreases or increases methionine oxidation, respectively. When alcohol is metabolized to acetaldehyde, NAD is reduced to NADH and thus NAD is not available for the metabolism of 13C-methionine. Therefore, 13CO2 production is decreased. When ASA is metabolized, NADH is oxidized to NAD making more NAD available for methionine metabolism. Therefore, 13CO2 production is increased.

Initially 20 healthy volunteers were evaluated with the methionine breath test. Then, the next day, participants were randomized to ingest either alcohol (n=9) or aspirin (n=11) 15 minutes prior to the methionine breath test. Subjects ingested 60 cc (2 oz) vodka (Smirnoff, 86 proof) dissolved in 200 ml orange juice. The dose of aspirin ingested was 30 mg/kg body weight (5-8 tablets) and was swallowed with 100 ml water. A paired Student t-test compared the mean of methionine breath test tests 1 and 2 to the methionine breath test performed during the ingestion of alcohol or aspirin.

Data for the two treatment groups is shown in table 1. The mean maximum % oxidation per hour for all subjects was 6.71 ± 2.55 (mean ± 1 SD). In subjects who ingested alcohol, the methionine breath test decreased to 3.01 ± 0.81 (mean change from baseline -59 ± 17%, p < 0.003) and in those who ingested aspirin, the methionine breath test increased to 8.43 ± 1.16 (mean change from baseline 36 ± 36%, p<0.001).
Table 1: Effect of alcohol and ASA ingestion on the Methionine Breath Test in healthy controls. Results are expressed as maximum percent dose per hour.

METHIONINE BREATH TEST Group 1

METHIONINE BREATH TEST Group 2

Participant

Baseline

Alcohol

Participant

Baseline

ASA

1

6.10

3.36

10

8.28

9.47

2

13.05

4.54

11

5.92

10.08

3

5.72

2.76

12

4.42

8.88

4

7.04

2.22

13

6.32

8.73

5

4.89

2.97

14

7.30

7.77

6

4.32

3.39

15

6.02

7.34

7

5.37

1.86

16

8.72

9.24

8

13.66

3.52

17

5.92

6.47

9

6.17

2.45

18

4.55

8.61

 

19

5.27

6.88

20

5.23

9.27

Mean

7.37

3.01

 

6.17

8.43

SD

3.48

0.81

1.41

1.16

The results indicate that the methionine breath test significantly decreases with ingestion of alcohol and increases with aspirin ingestion. Changes in the methionine breath test, therefore, reflect changes in hepatic mitochondrial function. This study suggests that the methionine breath test is a sensitive and specific measure of changes in hepatic mitochondrial function induced by alcohol and aspirin and can be used as a quantitative measure of mitochondrial function.

Ability of the Methionine Breath Test to detect Liver Disease:

The goal for this experiment was to determine if the methionine breath test is capable of distinguishing healthy controls from patients with liver disease. We studied 27 healthy controls and 46 patients with well-characterized cirrhosis. All patients were diagnosed with cirrhosis based on clinical symptoms, standard serum liver tests, radiological testing, and/or liver histology. All patients were clinically stable. The current practice guideline is to determine liver disease severity using the Child-Pugh (CP) classification that uses standard liver blood test results and presence of other symptoms. The CP score was determined on all liver patients at the time of the first test. and were calculated as shown in table 2.  The CP score designations are CP A, CP B, and CP C representing mild, moderate and severe liver dysfunction, respectively.

Differences between healthy controls, CP A, CP B, and CP C were compared using a One Way Analysis of Variance with Bonferroni Group Mean Comparisons. Comparisons were made between groups as follows; healthy controls and CP A, CP A and CP B, and CP B and CP C.

Analysis of the data showed that the methionine breath test distinguished healthy controls and patients with different degrees of liver disease severity. Healthy controls (9.16 ± 2.62, mean ± 1 SD) had methionine breath test results that were significantly different (p<0.001) from liver patients characterized as CP A (4.10 ± 4.53). Liver patients classified as CP A (4.10 ± 4.53) were significantly different (p=0.003) from CP B (2.57 ± 1.37) patients. CP B patients had methionine breath test values that were less dramatic but still significantly different (p=0.02) from CP C (1.33 ± 0.76) patients.  

Using the threshold of 6% (maximum % dose oxidized /h) patients were classified as either having normal or abnormal mitochondrial function. Abnormal function was classified as an methionine breath test < 6% and normal function if > 6%. Utilizing the threshold of 6%, sensitivity was 96%, specificity 100%, and accuracy 97% (see figure 3 and table 2).  

 

Figure 3: Methionine Breath Test in controls and patients showing the threshold level of 6% differentiates controls from liver patients classified as CP A, CP B, or CPC.

Table 2: Sensitivity and Specificity of the METHIONINE BREATH TEST for all participants.

 

METHIONINE BREATH TEST (%)
Child Pugh A, B, C
Controls

<6.0

44 Total Positive

0 False  Positive

>6.0

2 False Negative

27 True Negative

The sensitivity, specificity, and accuracy of the test were calculated as follows:

Sensitivity = No. of True Positives/Total with Disorder

               = 44/46 x 100 = 96%

Specificity = No of True Negatives/Total free of Disorder

                  = 27/27 x 100 = 100%

Accuracy = True Positives + True Negatives/ Total Investigated

              = [(44+27)/73] x 100 = 97%

 

The methionine breath test was able to distinguish healthy controls, CP A, CP B, and CP C patients with high specificity and sensitivity. Our data suggests that mitochondrial function is compromised as liver disease progresses either through the CP scoring system or from stable cirrhosis to alcoholic hepatitis. In addition, serial testing of patients over time may be useful as a prognostic indicator of liver disease progression and provide information for optimal timing of therapeutic measures including liver transplantation. Serial testing may indicate a response to treatment and therefore allow the clinician to employ a cost effective treatment strategy by either continuing useful therapy or discontinuing ineffective therapy.

It was not surprising that some overlap of the methionine breath test results occurred for each of the Child’s classifications. This is predicted by existing literature that reported CP A patients have a wide range of hepatic functional impairment ranging from nearly normal to severely abnormal. The Child’s score is only for classification purposes and is not a true measure of liver function. The Child’s classification should not be considered as a reference test of liver function. In fact, the methionine breath test shows some patients with CP A and CP B have normal function. The clinical occurrence of normal liver function in a CP A or CP B occurs frequently. This is the reason that the Child’s scoring does not offer the clinician insight into quantitative liver function reserves.

Determining Normal Range of the Methionine Breath Test:

One hundred and fifty (150) individuals without liver disease were administered the methionine breath test to understand the inter-subject variation and normal range of the test. Subjects were determined to be free of liver disease if they had normal liver chemistries, no blood alcohol levels prior to testing, no Hepatitis C antigens, and no substance abuse in the urine.

The mean and median rate of methionine oxidation was 4.00% per hour in this larger group of subjects. We felt it was necessary to revise the scoring of the test because the differences in the numbers were very small. We have converted the methionine oxidation rate to a “Methionine Breath Test Score” by multiplying all results by 25. This made the results easy to interpret and easier to inspect true differences between healthy subjects and those with liver disease. Therefore, the mean methionine breath test Score is now 100. A histogram of the results revealed a normal distribution as shown below.

 

 

Establishment for a cutoff of Cirrhosis by the Methionine Breath Test: 

A retrospective analysis of our database was used to define a methionine breath test cutoff value that would predict the presence of cirrhosis. In the methionine breath test analysis, 165 subjects without any known liver disease and 22 biopsy-proven cirrhotics were used in the analysis. Using ROC analysis, a methionine breath test cutoff score of less than 38 was indicative of cirrhosis. The sensitivity of the methionine breath test was 91% (20/22 correct) and the specificity was 98% (161/165). These results showed an overall accuracy of the methionine breath test to predict cirrhosis at 97%. This level of accuracy is compelling to evaluate further whether the methionine breath test can replace or reduce the use of liver biopsies.

A reduction in liver biopsies will not only reduce healthcare costs but also improve quality of life of patients. Liver biopsies are associated with pain in 30% of patients, severe complications in 0.3%, and death in 0.03%. It has also been reported that the duration of the pain after biopsy extends beyond the day of the biopsy in 40% of patients and extends for over 1 week in a small number.7 In fact, when questioned after the biopsy, 15% of the patients said that they would not have agreed to the procedure if they knew ahead how they would feel during and after the procedure.  

Reduction of the Number of Breath Samples:

 We recently developed a way to reduce the number of breath samples taken with the methionine breath test, which is currently every 10 minutes for one hour. Using a database of over 400 subjects, we have found an excellent correlation between the 40 minute breath collection and the area-under-the-curve for the entire hour. The correlation was found to be r2 = 0.986, p < 0.0001. There was no statistical difference in the final methionine breath test score calculated with the one point (40 minute time point) method or the six point (every 10 minutes for 1 hour) method. Since the database is so large, we are convinced that the methionine breath test can be performed with just one breath collection after administering the methionine dose.  

Summary of experiments:

 1)Reproducibility of the methionine breath test was excellent in all participants with a correlation of (r = 0.97).  

2)The statistically significant changes in the methionine breath test after alcohol and aspirin ingestion, agents that are known to inhibit or induce mitochondrial metabolism respectively, indicate that the methionine breath test monitors mitochondrial function.  

3)Methionine breath test results were significantly different for healthy controls and patients classified as Child-Pugh A, B, and C. The methionine breath test was also able to identify patients with normal and abnormal function with a high degree of sensitivity, specificity, and accuracy.  

4)An average methionine breath test Score is 100 and we have established a cutoff for cirrhosis of 38.  

5)The methionine breath test had an accuracy of 97% of predicting cirrhosis suggesting it was useful to reduce the number of liver biopsies performed.  

6) We have established that the methionine breath test can be performed with just two breath collections, a pre-breath sample and a 40-minute post-methionine dose sample. 

Methionine

Systematic name

(S)-2-Amino-4-(methyl-
sulfanyl)butanoic acid

Abbreviations

Met
M

Chemical formula

C5H11NO2S

Molecular mass

149.21 g mol-1

Melting point

281 °C

Density

1.340 g cm-3

Isoelectric point

5.74

pKa

2.16
9.08

CAS number

[63-68-3]

EINECS number

200-562-9

SMILES

C(N)(C(O)O)CCSC

 

 

 

 

 

 

 

 

 

 

 

Biosynthesis:

Since methionine is an essential amino acid, it cannot be synthesized in humans. However, in plants and microorganisms, methionine is synthesized from aspartic acid and cysteine. First, aspartic acid is converted to β-aspartyl-semialdehyde, an important intermediate in the biosynthesis of methionine, lysine, and, threonine. Of homoserine by homoserine acyltransferase, puts a good leaving group on homoserine allowing it to react with cysteine to produce cystathionine. Enzymatic cleavage of cystathionine yilds homocysteine, which can then be methylated by folates to give methionine. Both cystathionine-γ-synthase and cystathionine-β-lyase require Pyridoxyl-5'-phosphate as a cofactor, while homocysteine methyltransferase requires Vitamin B12 as a cofactor.

 

  

Enzymes invovled in m

1.          aspartokinase

2.          β-aspartate semialdehyde dehydrogenase

3.          homoserine dehydrogenase

4.          homoserine acyltransferase

5.          cystathionine-γ-synthase

6.          cystathionine-β-lyase

7.          methionine synthase (in mammals, this step is performed by homocysteine methyltransferase)