K0 : sebagai kelompok kontrol normal, terdiri dari 6 tikus putih yang diberikan diet

standar dan akuades 3mL selama 28 hari.

K(-): sebagai kelompok kontrol negatif, terdiri dari 6 tikus putih yang diberikan diet
standar dan formaldehid sebanyak 0,03 mL per oral selama 14 hari.
K1 : sebagai kelompok perlakuan dosis I, terdiri dari 6 ekor tikus putih yang diberi
diet standar. Tikus diberi formaldehid sebanyak 0,03 mL per oral selama 2
minggu dilanjutkan dengan pemberian astaxanthin dosis 0,216 mg/200gBB tikus
putih / hari selama 2 minggu.
K4 : sebagai kelompok perlakuan dosis II, terdiri dari 6 ekor tikus putih yang diberi
diet standar. Tikus diberi formaldehid sebanyak 0,03 mL per oral selama 2
minggu dilanjutkan dengan pemberian astaxanthin dosis 0,432 mg/200gBB tikus
putih / hari selama 2 minggu.
K5 : sebagai kelompok perlakuan dosis III, terdiri dari 6 ekor tikus putih yang diberi
diet standar. Tikus diberi formaldehid sebanyak 0,03 mL per oral selama 2
minggu dilanjutkan dengan pemberian astaxanthin dosis 0,864 mg/200gBB tikus
putih / hari selama 2 minggu.

Lipid peroxidation refers to the oxidative degradation of lipids. In this

process free radicals take electrons from the lipids (generally in cell
membranes), resulting in cell damage. Quantification of lipid
peroxidation is essential to assess oxidative stress in pathophysiological
processes. The end products of lipid peroxidation are reactive aldehydes
such as malondialdehyde (MDA) and 4-hydroxynonenal (4- HNE), as
natural bi-products. Measuring the end products of lipid peroxidation is
one of the most widely accepted assays for oxidative damage.

ab118970
Lipid Peroxidation
(MDA) Assay kit
(Colorimetric/
Fluorometric). 2016. Abcam

Metode Penelitian Pengukuran MDA

Konsentrasi MDA dalam material biologi telah digunakan secara luas sebagai indikator dan
kerusakan oksidatif pada lemak tak jenuh sekaligus merupakan indikator keberadaan radikal bebas
( Zakaria, 1996 ). Analisis MDA merupakan analisis radikal bebas secara tidak langsung dan mudah
dalam menentukan jumlah radikal bebas yang terbentuk. Analisis radikal bebas secara langsung
sangat sulit dilakukan karena senyawa radikal sangat tidak stabil dan bersifat elektrofil dan reaksinya
pun berlangsung cepat (Halliwel and Gutteridge, 1989).

Pengukuran MDA dapat dilakukan dengan pereaksi thiobarbituric acid ( TBA) dengan melalui
reaksi dengan penambahan nukleofilik membentuk senyawa MDA – TBA ( Conti et al., 1991).
Senyawa ini berwarna merah jambu yang dapat diukur intensitasnya dengan menggunakan
spektrofotometer pada panjang gelombang 532 nm. Senyawa 1,1,3,3-tetraetoksipropana atau
malondialdehyde tetrebutylammonium salt digunakan dalam pembuatan kurva standard karena
1,1,3,3-tetraetoksipropana dapat dioksidasi dalam suasana asam menjadi senyawa aldehid yang
dapat bereaksi dengan TBA ( Conti et al., 1991). Walaupun metoda ini tidak spesifik namun metoda
ini diterima sebagai petanda ( marker) peroksidasi lemak dari banyak peneliti ( Finand, 2006)

Prosedur pemeriksaan MDA:

1. Timbang 0.25 gram sampel.

2. Selanjutnya ditambah 4.5 ml larutan PBS dingin, lalu digerus.
3. Disentrifuse dengan kecepatan 3000 rpm selama 15 menit.
4. Setelah itu diambil 4 ml supernatan
5. Kemudian supernatan tersebut ditambah 1 ml larutan TCA 15%
6. Selanjutnya diberikan 1 ml larutan TBA 0,37% dalam HCl 0,25 N
7. Setelah itu dipanaskan dalam waterbath 80 o C selama 15 menit.
8. Kemudian didinginkan pada suhu ruang selama 60 menit.
9. Setelah didinginkan disentrifuse dengan kecepatan 3000 rpm selama 15 menit.
10.Kemudian mengukur absorbansi supernatan MDA sampel pada spektrofotometer
dengan λ = 532 nm.
11.Dihitung kadar MDA dengan menggunakan persamaan garis regresi dari kurva
standar (baku) larutan MDA
- The analysis of MDA by the thiobarbituric acid (TBA) assay has been widely employed over the many years
in biological systems for the assessment of lipid peroxidation.

- There was the formation of non-lipidrelated, malondialdehyde like, TBA-reactive substance that leads to
overestimation of the extent of lipid peroxidation. On the contrary, by direct HPLC method, there was a
decrease of MDA from plasma and tissue serving as control.

- Although simple and reproducible, the frequently used TBA method is fairly sensitive but not specific

- This reaction, although simple and reproducible, is unfortunately rather non-specific as TBA reacts with
many other carbonyl-containing compounds. Plasma fatty acids can also be oxidized during the 95ºC
heating step with TBA, generating artificially high results

- When they were measured as triplicate in the TBA method, an important difference between MDA levels
were observed (Figs. 4,5,6,7). As indicated in Table 1, the mean±SD values of plasma MDA levels with
HPLC and TBA method were 0.066±0.021 nmol/ml and 14.46±0.712 nmol/ml respectively, at a significance
of p<0.001. Brain MDA level with HPLC was 0.0323±0.011 nmol/mg protein whereas with TBA method it
was 5.30±1.578 nmol/mg protein (at a significance of p<0.05). The MDA level of kidney cortex with HPLC
was 0.028±0.006 nmol/mg protein, while with TBA it was 0.321±0.029 nmol/mg protein (at a significance of
p<0.05). In the same way, liver MDA level when measured with HPLC was 0.028±0.004 nmol/mg protein,
whereas with TBA this value was 0.196±0.051.nmol/mg protein (at a significance of p<0.05).

- We found 0.066 nmol/ml of total MDA in plasma with our method. This value is approximately 40% below

then those reported by Pilz et al 5 despite the fact that the sample preparation is quite similar. In the other

hand Yeo et al 15 found only 0.03 nmol/ml. THIS DIFFERENCE MAY BE RELATED TO THE FACTORS
SUCH AS AGE, TOBACCO SMOKING, AND NUTRITION OF HEALTHY VOLUNTEERS.
- In this reaction TBA may react with many other carbonyl-containing compounds and results in high MDA
levels

- As our data demonstrated, the concentration of TBA reactive material is higher than the real concentration
of MDA.

- Pilz et al 5 (Pilz J, Meineke I, Gleiter CH. Measurement of free and bound malondialdehyde in plasma by
high-performance liquid chromatography as the 2,4-dinitrophenylhydrazine derivative. J Chromatogr B
Biomed Sci Appl 2000; 742: 315-25 )also found 100 fold difference of MDA level between the two methods.
This can be due to the lack of the specificity of the reaction of TBA with MDA or the artificial formation of
MDA-like material during the heating stage. As seen in Table 1, the brain MDA levels with TBA method were
higher than the other tissues MDA. As it is well known that the brain tissue contains more phospholipids and
fatty acids than the other tissues.

- (Tukozkan, N., Erdamar, H., & Seven, I. (2006). Measurement of total malondialdehyde in plasma

and tissues by high-performance liquid chromatography and thiobarbituric acid assay. Firat Tip

Dergisi, 11(2), 88-92. )

- UNTUK SARAN PAKAI MEODE HPLC YANG LEBIH SPESIFIK

For critical comparison of the MDA values it pmol/ml [7] or failed to detect free MDA in human
is necessary to realize that plasma MDA may plasma [9,10]. Therefore, we conclude that values
originate from different sources that is (I) free MDA; for free MDA in human plasma must be very low
(II) protein bound MDA and (III) MDA that may (,0.2 nmol/ml) and all values reported to be higher
have been liberated from endogenous lipoperoxide are most likely the sum of free and some fraction of
molecules during the assay, and even minor modi- bound MDA or stem from additional MDA liberated
fications of a given assay procedure may change the from lipoperoxide precursors even though results
contribution from the sources I, II or III to the MDA were declared to represent free MDA.
signal measured. Furthermore, we must recognise Bound MDA in plasma samples can only be
that the measured values of MDA reflect the sum of measured after acid or alkaline hydrolysis of the
the enzymatically catalyzed PUFA oxygenation (par- protein binding. We used alkaline hydrolysis with 1
ticularly the arachidonic acid pathway) and the M NaOH for 30 min under heating to 608C which
metabolically uncoupled ‘‘autooxidation’’ of PUFAs was found to be optimal. This is in good agreement
and PUFA-esters if we inferred from the MDA with the findings of Chighetti et al. [8] who demonvalues about
oxidative stress
- (Pilz, J., Meineke, I., & Gleiter, C. H. (2000). Measurement of free and bound malondialdehyde in
plasma by high-performance liquid chromatography as the 2, 4-dinitrophenylhydrazine
derivative.Journal of Chromatography B: Biomedical Sciences and Applications, 742(2), 315-

325.)
-
- MDA/TBARS Lipid peroxides are unstable and decompose to form a series of compounds,
including reactive carbonyl compounds. MDA is the by-product of the arachidonate cycle and a
principle aldehyde product of lipid peroxidation in vivo, that is being widely used as an indicator
of oxidative stress in biological systems [54]. The TBA assay is the simplest and most popular
method for quantifying lipid peroxidation in biological samples. The assay works on the reaction
of TBA with MDA to produce a pink coloured MDA-(TBA2) Schiff base adduct. The basic principle
involves heating the sample to high temperature (95-100°C) with TBA under acidic conditions to
allow the formation of MDA-(TBA2) adduct. Molecules of MDA and TBA bind in 1:2 ratio in this
thermo energy driven reaction and the amount of pink coloured MDA-(TBA2) complex produced
can be measured colorimetrically by a spectrophotometer with absorbance at 530-540 nm or
fluorometerically using an excitation wavelength of 525nm and emission wavelength of 547nm.
For increased sensitivity, the complex can be extracted into an organic solvent such as butanol
and measured fluorimetrically. Compared to colorimetric methods, fluorimetric measurements
have been shown to be more sensitive and specific than colorimetric measurement [55]. A
standard curve can be constructed using MDA prepared by the acid hydrolysis of TEP (1,1,3,3,-
tetraethoxypropane) as one molecule of TEP yields one molecule of MDA plus four molecules of
ethanol when acidified [56]. TBA may react with other aldehydes in the biological sample and in
uncharacterised systems it is usual to refer to the assay of TBA reactive substances (TBAR) as the
test may not be not specific to MDA [44]. Apart from directly heating the sample, variations of
the TBARS assays techniques have been described. For example, distillation of the samples
followed by reaction of the distillate with TBA or by extraction of MDA using aqueous
trichloroacetic or perchloric acid and reaction with TBA [44]. Criticisms for the application of
TBARS assay to biological samples relate to the issue of specificity. Firstly, TBARS assays measure
the MDA generated by decomposition of lipid peroxides in the biological samples rather than
the free MDA content of the biological system. Secondly, many different aldehydes are formed
in the lipid peroxidation process and aldehydes other than MDA can form chromogens with TBA
and form complex with similar absorbance or emission wavelengths [57]. In addition, a variety
of TBA reactive “materials”, including sugars, amino acids, and bilirubin is generated and can
interfere with the sensitivity of the assay [55]. The iron content of the reagents used for analysis
may also interfere with the measurement and the use of EDTA as a chelating agent has been
shown to reduce variability of the assay [58]. The sensitivity of the assay can be increased by
combining it with HPLC to separate such compounds after the heating stage. However, very
delicate sample handling is required hence greatly reduces the throughput of this technique
[59]. Finally, MDA does not just reflect lipid peroxidation but is also a by-product of
cyclooxygenase activity in platelets [60]. Therefore the measurement of MDA level in serum may
lead to overestimation of the MDA formation ex vivo.
2.

3. Lee, R., Margaritis, M., M Channon, K., & Antoniades, C. (2012). Evaluating oxidative
stress in human cardiovascular disease: methodological aspects and
considerations. Current medicinal chemistry, 19(16), 2504-2520.
- MDA content between unstressed control and BDNF
heterozygous groups M/mgM/mg tissue and 13.86 ±
0.98 (12.75 ± 0.61 tissue, respectively) (Figure 2). On
the other hand, MDA values were significantly
increased in the M/mggroups exposed to acute stress
(16.82 ± 1.01 M/mgtissue for WT-Stress group and
20.88 ± 1.2 tissue for BDNF (+/-)-Stress group)
compared with the control group (Hacioglu, G.,
Senturk, A., Ince, I., & Alver, A. (2016). Assessment of
oxidative stress parameters of brain-derived
neurotrophic factor heterozygous mice in acute stress
model. Iranian journal of basic medical sciences, 19(4),
388.)>>>>>> masih sumber yang sama

- The increased serum corticosterone level in stressed

group is in accordance with previous studies (30-33)
showing that serum corticosterone is an important
indicator of stress
- It is reported that exposure to glucocorticoids or stress
may cause oxidative injury in various tissues (Fontella
FU, Siqueira IR, Vasconcellos AP, Tabajara AS, Netto CA,
Dalmaz C. Repeated restraint stress induces oxidative
damage in rat hippocampus. Neurochem Res. 2005;
30:105-111.)
 - [INI KATA KATA NYA BAGUS NAK] In the brain cortex of
this animal model, the expression of BDNF levels was
shown to be reduced by nearly 50% (29). The results of
our study indicated that there is no significant
difference in oxidative stress biomarkers between BDNF
heterozygous and wild-type cerebral cortexes of mice
that did not undergo stress. This finding suggests the
possibility that the levels of BDNF expression in
heterozygotes are sufficient to maintain oxidative
homeostasis under non-stress conditions. Moreover, it
is also conceivable that lack of change in oxidative
markers may be due to signaling by other growth
factors that compensate the effects of reduced BDNF
under normal physiological conditions.
- The possible impairing effect of corticosterone on brain
antioxidant capacities may be causal factor in the
unchanged SOD activity (Sahin E, Gümüşlü S.
Immobilization stress in rat tissues: alterations in
protein oxidation, lipid peroxidation and antioxidant
defense system. Comp Biochem Physiol C Toxicol
Pharmacol 2007; 144:342- 347.)
- Fontella, F. U., Siqueira, I. R., Vasconcellos, A. P. S., Tabajara, A. S., Netto, C. A., & Dalmaz, C.
(2005). Repeated restraint stress induces oxidative damage in rat hippocampus. Neurochemical
research, 30(1), 105-111. >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> ke bawah

- Inti argumennya tu bahwa, rendahnya K- adalah

karena, sebanarnya bukan rendah tapi tidak lebih
tinggi dari k2, makanya pas lihat k3 lebih rebdah dari
k-, jadi k2 itu dosisnya belum mampu turunkan
stresor 28 hari itu, sedangkan k3 sudah mampu, dan
MDA yang terukurtinggi di k0 k1 dan k2 itu akibat
 respons stress yng 28 hari itu, sedangkan k- hanya
14 hari, dan terbukti lagi bahwa stresor yang tinggi
mnyebabkan reles dari glukokortikoid ke otak yang
tinggi, jadi formaldehid juga nda banyak interfer ke
k- karena habis di babat di hepar, sedagkan yang
berperan disini sbenarnya faktor stress dan di tambah
lagi korteks ini hanya beberapa jenis glial yang bisa
hambat stres oksidatif, jadi terlihat penurunan di k3
sangat tergantiung dengan astaxanthin dosisi efektif,
beda dengan k- yang nda di kasih astaxanthin, karna
juga dosis satu dan 2 kemungkinan habis bantuin di
hepar jadi kadar MDAnya tetap tinggi, nah masalah
k2 kenapa lebih tinggi dari k1 karena memang ga
efektif k2 nya walupun terlohat lebih banyak dari k1
tetepaja ga “berefek”
- se of glucocorticoids (GCs), steroid hormones released by the adrenals; these substances play an
adaptive role, mobilizing energy to critical tissues during an emergency and suppressing
unessential anabolism. Nevertheless, there is strong evidence suggesting that, in the brain, high
levels of GCs may produce deleterious effects including damage to neurons
-
INI LAIN LAGI
Formaldehyde is readily combined with cellular constituents in all exposed tissues. It is rapidly
oxidized to formic acid largely in the liver by the catalytic action of alcohol dehydrogenase, and to a
lesser extent in erythrocytes, brain, kidney, and muscle. Formic acid is further oxidized to carbon
dioxide and water via a folate-dependent enzymatic pathway. The conversion of formaldehyde to
formic acid is very rapid: the estimated half-life is 1.5 min [3]. NIH GUNAKAN KALIMAT INI<<<<
LARGELY IN THE LIVER, jadi yang di otak sisa sedikit lebih banyak efek stres psikologi
 >>>>>

PENTING NIH JURNAL INI

Physiologically, the HPA axis regulates diverse body functions (such as digestion, sexual behavior, etc.)
and controls the reactions to stress. Anatomically, the key element of this axis is the paraventricular
nucleus of the hypothalamus, which contains neuroendocrine neurons synthesizing and secreting
vasopressin and corticotropin-releasing hormone (CRH). These two peptides mainly act on the
anterior lobe of the pituitary gland, stimulating the secretion of the adrenocorticotropic hormone
(ACTH) (69). ACTH, in turn, acts on the adrenal cortices, which respond to ACTH stimulation
producing glucocorticoid hormones (mainly cortisol in humans) (Fig. 2). Cortisol is considered the
main stress hormone that targets a variety of both peripheral and central systems, including
metabolic, cardiovascular, and immune responses (104).
Schiavone, S., Jaquet, V., Trabace, L., & Krause, K. H. (2013). Severe life stress and oxidative stress
in the brain: from animal models to human pathology. Antioxidants & redox signaling, 18(12), 1475-

1490.
Although the exact mechanisms linking oxidative stress
to the HPA axis is relatively unknown, ROS may affect
the HPA axis via several molecular mechanisms (Fig. 4):

LIHAT LIHAT< PEMBENTUKKAN ROS AIBAT

STRESS BEGITU BANYAK< BANDINGAKAN
DNEGAN FORMALDEHYDE YANG HNY
MELALUI DEPLESI GSH< DI TAMBAH LAGI
YANG KE OTAK SISA SEDIKIT
Lihat tuh stres kaya gitu jak tuh, apa lagi di sonde tiap
hari... nah tambahan lagi, yang negatif tu ada masa
istirahatnya selama 14 hari jadi kemungkinan sistem
nya seimbang lagi

Ketidakmungkinan mengukur kadar MDA normal

- faktor-faktor yang mempengaruhi kadar MDA, sehingga penelitian ini hanya meneliti satu
sisi saja, dan sednagkan hasilnya merupakan akumulasi
- formaldehid melewati BBB?
- mekanisme stres menigkatkan MDA?
- bahwa MDA yang dikukuradalah sebagian besar produk endogen stress, bukan dari formal
dehid
- sudah banyak yang di proses di hati dan hanya “sedikit” yang ke korteks, sedikit ini
berapa,dan bagaimana prosesnya di hati dan apakah sama prosesnya di korteks?
- bahwa faktor tress lebih itnggi meningkatkan MDA dari pada formaldehid
- mekanisme penetralan MDA oleh antioksidan endogen, baik cukup baik sangat baik?