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"Tiamin (Vitamin B1) adalah vitamin B yang pertama kali diidentifikasi. Tiamin berperan sebagai koenzim untuk beberapa enzim yang terlibat dalam metabolisme energi.Uji laboratorium terhadap kekurangan tiamin dapat dilakukan dengan mengukur kadar tiamin dalam darah. Penelitian ini bertujuan untuk mengetahui kadar tiamin serum pada alkoholic dan penderita DM dengan teknik ELISA, HPLC, dan menggunakan protein ikat tiamin kacang hijau (PITKH) dengan teknik enzyme-labeled protein ligand assay (ELPLA). Untuk melihat faktor-faktor yang mempengaruhi ikatan antara PITKH dengan tiamin digunakan teknik dialisis kesetimbangan. Validitas teknik ELPLSBA dilakukan dengan uji presisi dan akurasi. Teknik ELISA dan HPLC digunakan sebagai pembanding pada pengukuran tiamin serum. Konsentrasi PITKH pasca kromatografi afinitas hasil pengenceran liofilisat stabil selama 30 hari pada suhu -20°C dan 3 hari pada suhu 4°C. Aktifitas pengikatan PITKH dengan tiamin optimum pada pH 7,5. Aktifitas pengikatan ini juga dipengaruhi oleh senyawa alkilasi, oksidator, dan reduktor, tetapi kurang dipengaruhi oleh ion kalsium dan logam-logam berat. Kemampuan PITKH dalam mengukur kadar tiamin serum dengan teknik ELPLA memiliki presisi dengan CV 4,1% dan akurasi dengan nilai R 96-98%. Pengukuran dengan ELISA memberikan hasil yang lebih rendah dari teknik ELPLA, sedangkan uji banding dengan HPLC diperoleh p = 0,102 (p > 0,05) ; artinya tidak terdapat perbedaan yang signifikan antara metode ELPLA dan HPLC. Pengukuran tiamin serum dengan teknik ELISA, HPLC dan ELPLA pada alkoholic dan penderita DM, lebih rendah dari serum normal.

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Thiamine (vitamin B1) was the first B vitamin to have been identified. It serves as a coenzyme for several enzymes involved in energy metabolism. The laboratory test against thiamine deficiency can be done by measuring thiamine levels in the blood. The aim of this study was to determine the serum thiamine levels in alcoholics and DM by ELISA, HPLC, and using mung bean thiamine binding protein (MBTBP) with the development of enzyme-labeled protein ligand assay (ELPLA) method. The equilibrium dialysis technique was used to see the factors affecting the bond between TBP and thiamine. The ELPLA method validity was performed with precision and accuracy tests. ELISA and HPLC methods were used as comparators for measurements of serum thiamine. The MBTBP concentration of post-chromatographic affinity resulted from dilution of lyophilisate was stable for 30 days at -20°C and 3 days at 4°C. The optimal pH for binding MBTBP to thiamine was 7,5. This binding activity was also affected by alkylation, oxidizing, and reducing agents, but it was less affected by calcium ions and heavy metals. MBTBP ability to measure serum thiamine levels with the ELPLA technique has precision with CV 4,2% and accuracy with R 96-98%. Measurements by ELISA has lower result than ELPLA. The comparison test with HPLC method obtained p = 0,102 (p > 0,05); meaning no significant difference between ELPSLBA and HPLC methods. Serum thiamine level by ELISA, HPLC and ELPLSBA techniques in alcoholic and DM patients were lower than normal serum."

"Tiamin berfungsi sebagai koenzim untuk beberapa enzim yang terlibat dalam metabolisme karbohidrat. Mengingat pentingnya peran tiamin, maka dilakukan pengembangan teknik pengukuran tiamin yang analog dengan enzyme linked immunosorbent assay ( ELISA), dimana antibodi diganti dengan protein pengikat spesifik yaitu protein ikat tiamin kacang hijau (PITKH). Teknik pengukuran ini dilakukan secara kompetitif, kompetitor akan dikompetisikan dengan tiamin bebas yang akan diukur. Kompetitor tersebut berupa konjugat antara tiamin-biotin. Tiamin murni diikatkan dengan biotin menggunakan senyawa perangkai yaitu glutaraldehid. Pada analisis LC-MS ditemukan 3 puncak, . Puncak ke 3 merupakan konjugat tiamin-biotin. Dibuat kurva standar dan diperoleh persamaan garis lurus dengan nilai R2= 0,9986. Uji validasi menggunakan konjugat tiamin-biotin menunjukan nilai coefficient of variation (CV) = 3,81%, nilai ini lebih kecil dari CV Horwitz = 8,12%, akurasi dengan nilai Recovery (R) =94 %-98%. Hasil ini menunjukan syarat pengukuran dengan teknik ELPLA sudah terpenuhi, dengan presisi dan akurasi yang baik. Aplikasi pengukuran kadar tiamin pada serum normal sebanyak 23 sampel didapatkan kadar tiamin berkisar 2,62-9,76 μg/ml. Dengan demikian, teknik ELPLA dengan konjugat tiamin-biotin sebagai kompetitor dapat digunakan pada pengukuran kadar tiamin dalam serum

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Thiamine has a coenzyme function in several enzymes involved in carbohydrate metabolism. Considering the important role of thiamine, a thiamine measurement technique analogous to the enzyme linked immunosorbent assay (ELISA) was developed, that the antibody was replaced by a specific binding protein named mung bean thiamine binding protein (MBTBP). The measurement technique was carried out competitively in which competitors would be competed with free thiamine to be measured. The competitor is a thiamine-biotin bond. Pure thiamine was bound to biotin using a coupling compound called glutaraldehyde. In the LC-MS analysis we found 3 peaks. The third peak was the thiamine-biotin conjugate. A standard curve was made and the value of its straight line equation was obtained R2= 0,9986. The validation test using thiamine-biotin conjugate showed coefficient of variation (CV) value = 3,81% which was smaller than Horwitz CV = 8,12%, with the accuracy of the Recovery (R) value = 94% – 98%. These results indicated that the measurement requirements for the ELPLA technique had been met with good precision and accuracy. The application of the serum measurements to 23 samples showed thiamine levels ranging from 2,62- 9,76 μg/ml. Thus, the ELPLA technique with thiamine-biotin conjugate as a competitor could be used in the measurement of serum thiamine levels"

"Latar BelakangTiamin merupakan mikronutrien yang dibutuhkan dalam proses metabolisme energi. Defisiensi tiamin dapat menyebabkan berbagai kelainan jangka pendek dan jangka panjang, terutama pada populasi khusus, seperti pasien dengan diabetes atau stunting. Meskipun pentingnya kecukupan tiamin, belum terdapat standar tiamin serum bagi populasi Indonesia. Hal ini disebabkan oleh metode pengujian yang belum optimal untuk dilakukan pada sampel besar. Karena itu dikembangkan metode pengukuran yang lebih ekonomis dan praktis menggunakan prinsip ELISA namun menggunakan protein ikat tiamin. Protein ini telah ditemukan dalam berbagai spesies kacang-kacangan. Penelitian ini mengeksplorasi keberadaannya pada spesies kacang yang masih kurang diteliti, yaitu kacang lima (Phaseolus lunatus)MetodeKacang lima dihaluskan dan dibuat tiga sampel kacang, masing-masing sebanyak 5 gram. Tiap sampel dilarutkan menggunakan dapar fosfat. Larutan tersebut disentrifugasi dan supernatan diambil. Dilakukan proses salting out dengan ammonium sulfat, lalu larutan kembali disentrifugasi. Presipitat diisolasi dan dilarutkan dengan dapar fosfat lalu menjalani proses dialisis. Kadar protein diukur menggunakan spektrofotometer. Ditambahkan larutan tiamin pada sampel hasil salting out, kemudian dilakukan dialisis kesetimbangan untuk mengukur aktivitas pengikatan tiamin. Absorbansi dialisat dibaca untuk menghitung jumlah tiamin terikat.HasilPenelitian ini menunjukkan bahwa kacang lima mengandung rata-rata 7,61 gram protein/100 gram kacang. Ditemukan aktivitas pengikatan tiamin yang bervariasi antara 573,16 dan 878,09 μg/10 gram kacang dengan rasio tiamin terikat per jumlah protein 0,95μg/mg protein.KesimpulanDitemukan protein ikat tiamin pada kacang lima dengan aktivitas yang tinggi dibandingkan dengan sumber lainnya.

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Introduction

Thiamine is a micronutrient essential for energy metabolism, and its deficiency may lead to various disorders, especially on certain populations, such as diabetic and stunting patients. In Indonesia, there is no established serum thiamine standard, partly due to the unoptimal testing methods available. Therefore, a more economical and practical measurement method was developed using the ELISA principle but using thiamine binding protein found in various species of beans. This research explores its presence in a lesser-studied lima bean (Phaseolus lunatus)

Method

Lima beans were ground and three samples are prepared, each containing 5 grams of bean. Each sample is dissolved using phosphate buffer. The solution was centrifuged and the supernatant was collected. Ammonium sulfate was added to precipitate protein, then the solution was centrifuged again. The precipitate was isolated and dissolved in phosphate buffer, then dialysed. Protein levels were measured using a spectrophotometer. Thiamine solution was added to the salted out sample, then equilibrium dialysis was carried out to measure the thiamine binding activity. Absorbance reading of the dialysate were used to quantify bound thiamine.

Results

Lima beans contain an average of 7.61 grams of protein/100 grams. Thiamine binding activity ranged from 573.16 to 878.09 μg per 10 grams of beans with a ratio of bound thiamine per amount of protein of 0.95 μg/mg protein.

Conclusion

Thiamine binding protein was found in Lima beans with high activity compared to other sources."