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Clinical Biochemistry - Science topic

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Questions related to Clinical Biochemistry
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methods
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The ELISA method can realize the quantification of AP without distinguishing the cell state. The ELISA kit manufactured by Cusabio does not require fixation or cell activity. It only analyzes the target protein.
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Most of the dissolution tests use 900 mL of medium. How was this volume defined? Is there any correlation with the volume of gastric fluid in humans?
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The volume of dissolution medium to be used is defined considering "sink condition." The solubility of the drug substance is quantitatively determined in several dissolution media within the physiological pH range at 37 °C. Using this value, the volume of dissolution medium necessary to obtain a saturated solution of the highest dose of the product to be marketed is calculated. Sink condition is considered as at least 3 times this volume. Some companies work with 5 times or 10 times this value. There are some instances where the dissolution test is more discriminative if sink condition is not followed. The volume of dissolution medium has no relationship with the volume of gastric fluids in humans. According to information available in the literature, a realistic volume to simulate the total fluid available in the stomach in the fasted state is in the range of 250-300 mL.
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Imagine, you have no problem choosing one of the following disciplines.
-Analytical Chemistry
-Organic Chemistry
-Physical chemistry
-Inorganic chemistry
-Applied Chemistry
-Medicinal Chemistry
-toxicology
-Clinical Biochemistry
-Nano chemistry
Which of these disciplines do you choose? Be sure to state the reason for your choice.
Thanks a lot
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Inorganic chemistry. But..in PhD i will change to biochemistry ☺
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I have approached various companies but they aren't dealing with an Elisa kit which can measure corticosterone level in brain tissue homogenate. Can anyone suggest me such kit? Is it possible( considering sensitivity) to use a kit for brain tissue homogenate which is marketed for plasma, serum, urine, milk etc?
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Hi guys, anyone has good results with corticosterone ELISA Kit DetectX by Arbor Assays on brain tissue homogenate?
¡Cheers!
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Biological techniques are methods or procedures that are used to study living things. They include experimental and computational methods, approaches, protocols and tools for biological research.
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looking for some best ref. books on Viva in Clinical Biochemistry / Biochemical Analysis / Biochemistry PDF ??
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What do you mean 'on Viva' : Viva forum?
I never heard of good books clin biochem books free available as pdf, please be more specific
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Dear colleagues;
I look for young scientists for authoring a book deals with subjects belongs to medical biochemistry. Are you interested?
A priority is to the English native speakers
Regards;
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Am very much interested. please how do i contact you . And again what are the subjects of interest
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Dear colleagues;
What are the novel and accurate methods for testing the following:
  1. Total cholesterol conc.
  2. Triglycerides conc.
  3. HDL-C conc.
  4. LDL-C & VLDL-C
  5. Lipid peroxidation marker (MDA)
Best regards;
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Hello, see the info below you can find some news there.
Good luck!
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We are trying to collect a sample before and after exercise. Plasma Dihydroxyphenylalanine (DOPA) is a precursor of urine free dopamine. If one collects the urine sample then what is the time frame to match your urine dopamine concentration to plasma?
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You can collect urine and serum samples and make the Comparison between them results.
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Good day to everyone.
Can somebody provide me please, to get the answer to this question?
What is the reason for the number of wells in Microtiter plate that uses in Elisa technique is 96 wells or its multiples in some cases? Nor more neither less of that.
Thank you! I appreciate the help.
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There are 48 wells in Elisa technique for small samples like in animal researches.
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I'm trying to measure the heme content of plasma with and without the hemoglobin depleted to better understand heme trafficking. I've looked into Hemoglobind but the price is prohibitive given the number of samples I would like to run. Does anyone have a chemical method for removing Hemoglobin? 
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This is a message for Matthew Kuruc,
Is the product HemogloBind is useful to remove hemoglobin from the antibody elution from the sensitized RBCs without affecting antibody strength? Also it availability in Mumbai, India.
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Soluble fms-like tyrosine kinase 1 (sFlt-1) and placental growth factor (PlGF) are not only observed in serum and plasma in preeclampsia. Both are biomarkers of angiogenesis and cardiovascular risk in the metabolic syndrome.
"Placental growth factor is a vascular endothelial growth factor involved in angiogenesis, vascular inflammation and plaque formation. Soluble Fms-like tyrosine kinase 1 is a decoy receptor for placental growth factor, reducing its activity. " (Theilade S, and col. in Diabet Med. 2012 Mar;29(3):337-44)
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Dear Marcos, I think this paper will be useful.
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In my lectin haemaglutination assay by doing 2-fold serial dilution I have to add 50ul of trypsinized rabbit erythrocytes instead of normal rabbit erythrocytes and also I have heard that the wells need to be coated with EDTA. Can we do the EDTA coating ourselves? Please explain. Thank you. Also can we use direct human blood for the assay. If yes then how to store it till we do the assay?
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Hi did you find any answer for your question?
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I will be testing serum and plant extracts. Thank you.
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Any of following:
  1. Lonza
  2. ACC CapeCode
  3. Charlesriver
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I need a methodology to apply as I want to the project in clinical biochemistry
I also need suggestions about softwares to use
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Dear all,
thanks for your replies. I will go on the project trying to use different infos you provided and I will let you know how far I progress.
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I have an in vitro dissolution profile, and I would like to predict the plasmatic concentration or the absorbed percentaje, for a bioequivalence study.
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Dear Dr. Gouveia,
Thank yoiu very much for your response and useful comments.
Best regards
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The dissolution test results for a particular product have a high variability and are very high. What is the upper limit for the amount of drug released in dissolution testing?
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@Vipul, Would you be so kind to clarify what do you actually want to know (please take a look to my previous post)?
@Md R Khayer. Would you be so kind to let us know to which "dissolution guideline" are you  referring to? And where is the 85-115% drug dissolved (wrt label claim) requirement stated?
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Parameters to be analyze : Glucose, Triglyceride,ALT,AST,ALP,Calcium,T.Protein,Urea,T.Bil , Phosphorous,GGT,LDH ,Creatinine ,Cholesterol .
Analysis to be processed immediately after sample separation.
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 Dear Jayalakshmi.
We apologize for the analysis of the plasma seems to be suitable is the use of ELISA instruments, but to provide input I have not dared, because next week I will follow the training on the use of ELISA in the laboratory of Biochemistry Hasanuddin University, Makassar, Indonesia
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PCR-RT, ELISA or both
in blood, urine or other like cephaloraquid liquid, seminal or saliva
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Thanks a lot  i´m going to  review it
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I need the protocol of Vitamin B9 quantification to determine the Vit B9 level in liver of fishes treated with increasing concentrations of this vitamin for a long period.
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Thanks a lot my friend for the interesting document
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the capsule if very small and not dissolved in water
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Dose administration  of drug or any chemical test item in chicken is very simple comparing with other Lab animals. Test item can be suspended or dissolved in suitable vehicle and put directly in to the esophagus or in the mouth cavity with the help of catheter. Chicken has tendency to gulp whatever you put in the mouth. Only thing you have be cautious  about regurgitation  of dosed material .     
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I am looking for the information on the normal concentration of butyrylcholinesterase in the blood serum of a healthy horse in µg/mL.
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Dear Maja Sopotnik,
I am attaching the file. Hope it might be useful to you
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I see serum leptin concentrations reported, but am curious about the accuracy of non-invasive methods when urine isn't available.
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The present study was shown the leptin levels measurements and all its details. 
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Dear Pr Soldin,
1- What are the advantages of quantifying free thyroids in urine matrix?
2- which technique will be used for that purpose?
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Thank you.
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Metabolites to be extracted from blood plasma and urine samples
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Or if your compounds are volatile, which in your case they probably are not. You can readily evaporate away MTBE and hexane at low (<50 C) temperatures; this should allow you to effect a solvent exchange without losing your analytes.
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Hi.
I want to know what different methods exsist to determine the gene polymorphisms of the CYP2C9 and VKORC1, which are inportant in warfarin therapy.
What are the straightness and weaknesses of each method? Which one is the "gold standard"? Which was the first one?
Does the final price of the test relates to the method that test is based on? How?
it would be very helpfull if you can back up your answers with references.
Thank you.
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Evaluating the effect of gold nanoparticles on kidney tissue
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Thak you Robert and Amanda.
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What is the best internal control to qt PCR. to study obesity mice model in Brain?
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thank you
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INR, labile, simpler parameter, vitamin K antagonist, new research 
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There is a new (patented US 9234902 B2) method called Fiix, that measures only FII and FX. I don't know if it is commercially available.
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I have some incomplete data regarding creatinine in mouse urine that I would like to use to evaluate proteinuria. I would like to know what is the usual quantity of creatinine found in 24h urine to compare it to my results, does anyone know it?
Thank you!
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Thank you for your answers! Apparently, in human there is a fixed quantity (not concentration) of creatinine that can be found in 24h-urine samples. I think it is around 1 to 1.5 g so I was expecting something similar for mice. Thank you again
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I have to estimate eNOS in blood & skin
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Thanks Manoj
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I am looking for human proven alternative cholesterol ( LDL)  lowering drugs ( no drugs like statins/plantsterols/ezetimbe/pcsk9).
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Add more fibery foods to your diets, as soy bean, chickpea,  vegetable, nuts etc.
Besides Oat meal, you may also try Psullium husk, Konjac powder,
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I am planning to do a drug discrimination task in rats with Isoproterenol hydrochloride. As the risk of heart faillur is very high with that drug, I wanted to be aware of any precautions I should take to prepare the drug in 0.9% Saline.
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Inject slowly and monitor heart rate.  Do 1-2 test rats before doing the total population to get an idea of how the rats will respond.  You might be able to save some animals with high HRs in the "test doses" with a beta blocker and use them again.
Remember the old saying in pharmacology, "Start low, go slow."
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I just want to determine the TNF-α levels in human blood plasma and what is the most reliable, recent method for this? 
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Hi Izquierdo,
Thank you so much for the help. It means a lot.
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Hi,
I want to measure metabolic hormones in mouse samples using Multiplex (Magpix).
Which is better to use: plasma or serum? And why.
Thank you
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There are little differences between plasma ore serum in most cases , but i think that plasma is superior, because the coagulation process can modify to some degree some molecules to be trapped by the clot.
 It depends of the cytokine to be analyzed in multiplex assay
Reference
James Drummond in another question (Serum or Plasma is the best way to detect cytokines via ELISA?) recommends plasma vs serum
References
Comparison of serum, EDTA plasma and P100 plasma for luminex-based biomarker multiplex assays in patients with chronic
obstructive pulmonary disease in the SPIROMICS study (2014) O Neal WK, Anderson W, Basta PV, Carretta EE, Doerschuk CM,
Barr RG, Bleecker ER, Christenson SA, Curtis JL, Han MK, Hansel NN, Kanner RE, Kleerup EC, Martinez FJ, Miller BE, Peters SP,
Rennard SI, Scholand MB, Tal-Singer R, Woodruff PG, Couper DJ, Davis SM J Transl Med. 2014 Jan 8;12(1):9
Analysis of serum and plasma identifies differences in molecular coverage, measurement variability and candidate biomarker
selection (2012) Alsaif M, Guest PC, Schwarz E, Reif A, Kittel-Schneider S, Spain M, Rahmoune H, Bahn S Proteomics Clin Appl. 2012
May 29
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Hi there, I want to measure the 15d-PGJ2 titer in endothelial cell culture medium supernatant and therefore I extracted it using solid phase extraction. I eluted the samples in ethyl acetate and stored them temporarily until I evaporated the solvent using a speed vac. I tried to reconstitute the dried residues in assay buffer for subsequent ELISA. In contrast to my anticipation, only part of it dissolved. Neither increase of the buffer volume nor repeated vortexing could make a difference. Now my question is: does this precipitate hint to an incorrectly performed SPE/evaporation or is it a common co-observation, that should be ignored.
Thanks for your help
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I eluted in 100% ethyl acetate and the assay buffer contains Tris buffered saline and proteins/sodium azide as preservative...
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aromatase inhibition
HPLC-UV
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I've done estrogen it in female urine/blood in 1995, with a buffer nearly equal to the one described by http://www.phenomenex.com/Application/Detail/19680 on Hypersil ODS (150x2mm), however detection was performed with an electrochemical detector (glassy carbon electrode).  The electrochemical detector offers a lot of selectivity.
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We want to confirm the presence of acarbose in our sample.. How do we do that? 
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Actinoplanes spp. produce acarbose. The biosynthetic genes for its production have been identified.
Check the genome of Actinoplane missouriensis to see if those genes are present.
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I need charcoal stripped urine and saliva for spiking steroids  for LC-MS analysis
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Charcoal stripping may add extra components and black debris to your samples. If you want to strip your sample from lipophilic substances, then use SPE columns with e.g. C18 coating. Consider adding some buffer, to standardize the process. In acid urine the amount of weak acids being adsorbed by C18 column is pH dependent. I used a similar approach as clean up step for urine samples in HPLC analysis.
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LDL cholesterol (LDL-C) is the main culprit for the CHD, sometimes is called "bad" cholesterol. This is because it carries cholesterol to tissues, including heart arteries. A high LDL cholesterol level raises the risk of CHD potentially.
Since 1972 the LDL-C has been estimated using this  Formula called Friedewald. LDL-c = TC-HDL-c- TG/5 (in mg/dl)
But the limitation of Friedewald Formula is as follows:
1)Inability to measure LDL-C in samples containing chylomicrons.
2)Erroneously high results in patient with Type III hyperlipoproteinemia.
3)Only applicable with plasma TG concentration <400mg/dL.
Moreover, studies showed that FW formula significantly overestimated LDL-C values at TG <100mg/dL, while increasingly underestimated at >200mg/dL.
Global attempts are taken to overcome the problem:
1. Several LDL-C assays; expensive, labor-intensive and time consuming
2. New formulas and modification of FW formula
None of them could satisfy the degree of accuracy compared to expensive direct measurement.
I have developed a new formula that overcome all limitations associated with LDL-C estimation. Implication of this formula will save the time and money and reduce burden of CHD management
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How can we help you? We can cooperate if you want.
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I want to check CRP levels of stored serum samples. I want to know whether it is possible or whether we need fresh samples
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It depends on the period of storage. The CRP half life is 19 days but under the temperature of 4oC. My suggestion is this; if can lay your hands a fresh serum, please use that for accurate result.
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Interleukins can be very unstable and their degradation is related to temperature as well. I'm looking for a protocol who can give a reliable measurement of interleukins in patient with chylothorax or pleural effusion. In detail I would like to understand:
1. How collect the sample? Usually we can not define for how long the fluid remain in the chest cavity or in the chest drain before collection.
2. How prepare the sample, I know have been seen to be collected in heparin container and put in ice and centrifuged and frozen -80. Is there any preparation protocol for how centrifugate and store the sample?
 
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I'm going out of the box of your queries to suggest an analogous solution. The problem of brain analysis for another labile molecule was solved by developing an  in situ electrochemical probe calibrated to the concentration of analyte in vivo. In their case acsorbate was very close to the electrochemical signal. The 40 y/o technique is not on PubMed but I read in OMNI magazine back then. Terry Christian's group at Tulane developed it. Palo Alto library has the OMNI issue but I'm sure the technique would be well recognized by now. Calibration would need to encompass LT a,b,c,d,e 4 at physiological milieu.
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I need to perform the measurement of ATP in cardiac extracellular milieu without disaggregating the organ to prevent not to release intracellular ATP. How can I perfuse it?
(ATP colorimetric/fluorometric assay kit- Sigma Aldrich)
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I think you can use ATP Assay Kit (Colorimetric/Fluorometric) (ab83355). Best wishes.
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Most of the literature refers to increased troponin in serum as a biomarker but I am interested in knowing if decreased levels in the heart indicates disease.
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Dear Rachael i am afraid i havent come across any evidence that is suggestive of Trop reduction in context of DCM. Trop could indeed be significantly raised in a a wide variety of DCM such as Ischemic cardiomyopathy, Hypertrophic CM, Cardiac Sarcoid/Amyloid & Vasculitis with Cardiac Involvement etc as i am sure you must be aware.
very best wishes
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How clinical biochemistry analyzers remove effects of some interference by measuring absorbence at two different wave lengths. E.g serum calcium 650 nm & 700 nm.
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Thanks a lot Ken 
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We use GFP (Green fluorescent protein) to ensure implantation of mesenchymal stem cells injected into the vein.
I want to know in the intratumoral injection of mesenchymal stem cells,  use of the GFP is necessary ?
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Some say 24 hours (UK Minimum retesting interval), whereas others say 48 hours (Waldron JL, et al. An automated minimum retest interval rejection rule reduces repeat CRP workload and expenditure, and influences clinician-requesting behaviour. J Clin Pathol. 2014 Aug;67(8):731-3.).
Who is right?
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In acute inflammation such as postoperatively, CRP rise may be due to trauma or infection. When trauma is mild the CRP is a useful indicator of infection (J Otolaryngol Head Neck Surg. 2015 Apr 2;44:13). In more traumatic surgery procalcitonin is a more sensitive and specific marker of infection (Biomed Res Int. 2014;2014:565080, Pediatr Res. 2013 Oct;74(4):413-9), but since the motivation for restricting CRP is economical, it is hard to argue for sequential procalcitonin in its place. Although generally speaking, when there is a risk of infection, it may take CRP 3 or 4 days to clearly rise (Crit Care. 2011 Jul 15;15(4):R169), CRP can change significantly in one day. I have added a 'raw data' graph of ninety thousand CRP's repeated the next day (from our laboratory database over 5 years) to my publication list. While the interquartile ranges for CRP the next day generally show stability, the 95% confidence intervals are very wide e.g., an initial CRP of 20 mg/L has a 95% confidence interval of 8-130 mg/L the next day. I personally think that CRP is the most underutilised test in medicine and particularly at an analytical cost around $1USD, it is hard not to argue it has major value in detecting and monitoring acute inflammation.
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From the FRAP assay, why use mM of FeSO4/g of sample as unit, can I use g of FeSO4 /g of sample ?
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Dear Wasin,
In biochemical reactions, we wish to know how many molecules are reacting with your reagent. If you express in terms of moles/mmoles/micromoles/nanomoles it is easy for us to know how many molecules or ions in the reagent are interacting with your sample (for instance in your case you want to determine antioxidant status of some samples/homogenates and accordingly you can express antioxidant status in mole/mmole/micromole/nanomole equivalence). [Of course, you can determine also in terms of g or mg or micrograms or ng, but for discussing your findings/results in apt manner you might have to convert g values into mole values.]
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What are the arguments for non-fasting lipid profiles? Are there any cut-offs? Which group of patients stand to benefit most from it?
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Meals have little effect on total cholesterol, HDLC and LDLC levels, and in fact, the levels are usually lower after a meal, probably because fluid shifts are more significant than cholesterol changes (Miida T, et al, .LDL-cholesterol and HDL-cholesterol concentrations decrease during the day.Ann Clin Biochem. 2002 May;39(Pt 3):241-9.).
The main reason for fasting is therefore to get a consistent triglyceride value, which does increase variably after meals. Although elevated triglycerides are very important (!), the strong reciprocal relationship between high triglycerides and low HDLC, and the low variability of HDLC, paradoxically means that low HDLC is a more reliable indicator of high triglycerides than high triglycerides! (See Tenenbaum A, Klempfner R, Fisman EZ. Hypertriglyceridemia: a too long unfairly neglected major cardiovascular risk factor. Cardiovasc Diabetol. 2014 Dec 4;13:159.)
The end result is that the reciprocal of HDLC is a reliable indicator of high triglycerides whether fasting or not, and the use of the Total Cholesterol / HDLC ratio can be applied on fasting or non fasting samples as the most powerful simple cardiovascular predictor (and the difference between TC-HDLC is a good marker for monitoring pharmacological treatment whether fasting or not). One word of warning though; HDLC is gender dependent and affected by steroid hormones with the differences appearing at puberty and altering also at menopause. Therefore the use of a single TC/HDLC cutoff (or single TC-HDLC cutoff), will introduce various gender dependent biases which are seldom appreciated in the literature even though metabolic syndrome definitions clearly have different HDLC cutoffs for men and women.
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I want to perform some biochemical experiments(cancer effect of ibuprofen on the liver) on ibuprofen, but these experiments require dissolve ibuprofen in a solvent for injection it to the rats. The ibuprofen not dissolved in water, 0.9%NaCl, DMSO, but dissolve in ethanol. Is ethanol a suitable solvent for injection?
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Hi Hossam,
As ibuprofen structurally contains both hydrophilic and hydrophobic functional groups it is not straightforward to dissolve in water. Ethanol as a solvent is no problem externally but it may have different affect on the liver function (as ethanol is absorbed by the liver) if used as absolute ethanol (high concentration) - so I would recommend dilution to the point where ethanol is just enough to dissolve the ibuprofen but water content is higher for injection to ascertain ibuprofen effect.  
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Dragendorff’s reagent is used for visualization of alkaloids. Its modifications (e.g. Munier-Macheboeuf) with alkaloids and other compounds are unclear.
What is that modification, can someone elaborate the details please?
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Dear Dr. Sergio Leite,
Thanking you for additional details with link.
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Is supersaturation relative to Ca oxalate, for example, defined relative to the saturation value in pure water at a given T and pH, or relative to empirically measured or calculated saturations in actual urine with its host of Ca binding ligands?
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you can observe the ca oxalate crystals in urine sediment with light microscopy.  This phenomenon may be linked increased parathormone.
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analysis, biologists and microbiologists How can I quantify the quinolic acid in the serum/plasma sample?
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there are various references
Heyes MP, Markey SP. Quantification of quinolinic acid in rat brain, whole blood, and plasma by gas chromatography and negative chemical ionization mass spectrometry: effects of systemic L-tryptophan administration on brain and blood quinolinic acid concentrations. Anal Biochem. 1988 Oct;174(1):349-59.
Xia C, Dang Y, Brown OR. HPLC analysis of quinolinic acid, a NAD biosynthesis intermediate, after fluorescence derivatization in an aqueous matrix. Microbios. 1998;94(379):167-81.
Junichi Odo · Masahiko Inoguchi · Akihito Hirai. Fluorometric Determination of Quinolinic Acid Using the Catalytic Activity of Horseradish Peroxidase. Journal of health science 04/2009; 55(2):242-248. DOI:10.1248/jhs.55.242
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I need your help to get some good protocol for extracting metabolites (volatile and non volatile both) from urine and serum samples. Generally I have found through literature surveys that people are using methanol to extract metabolites from serum. Can anybody suggest some good protocols for metabolite extraction from urine and serum. I would be using LC-MS & GC-MS for my studies.
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Dear Khushman, 
The following may be of help!
Szultka M, Pomastowski P, Railean-Plugaru V, Buszewski B. Microextraction
sample preparation techniques in biomedical analysis. J Sep Sci. 2014
Nov;37(21):3094-105. doi: 10.1002/jssc.201400621. Epub 2014 Sep 25. PubMed PMID: 25132413.
Salting-out assisted liquid-liquid extraction for bioanalysis.
Tang YQ, Weng N. Salting-out assisted liquid-liquid extraction for
bioanalysis. Bioanalysis. 2013 Jun;5(12):1583-98. doi: 10.4155/bio.13.117.
Review. PubMed PMID: 23795935.
http://europepmc.org/abstract/MED/2585385  Metabolomic method: UPLC-q-ToF polar and non-polar metabolites in the healthy rat cerebellum using an in-vial dual extraction. (PMID:25853858 PMCID:PMC4390242)
Review: Targeted Metabolite Profiling: Sample Preparation Techniques for GC-MS-Based Steroid Analysis.  Krishna Chaitanya Sadanala.  Mass Spectrometry Letters / V.3, No.1, 2012 year, pp.4
Best...
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We have a patient with raised (6x the upper limit) urine normetanephrine. However, this patient had noradrenaline 5 days previous. The half-life of noradrenaline is very short (minutes)- but is metabolised to normetanephrine which is what we measure in our assay. Could this just be the drug?
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Hi, 
I found this
The half-lives of plasma free metanephrines
Kirsten A. Campbell, Shantha P. Joseph, Malcolm J. Whiting, Matthew P. Doogue, Clinical Endocrynology, Volume 76, Issue 5, May 2012 
Free Normetanephrine should have half-life between 1 and 2 hours.
In general, only drugs which undergo specific organ partition (e.g. pesticides in the adipe) have long half lives (being "kidnapped and stored away", they do not undergo metabolism).
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Zn and Cu levels have to be estimated from blood samples
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You can use nitric acid and high temperature about 450oC in muffle oven, or the best way the microwave digestion with the use of teflon test-tube and nitric acid in microwave oven until a clear liquid.
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Hi All
I am conducting an RCT for my PhD measuring Oxytocin Levels in mothers and their babies. After my previous post I have decided to do this with Urine samples as it feels more ethical than Saliva samples as then baby and mother can still eat. I need to cost the analysis of these samples and I was wondering if anyone else has done this analysis, particularly in England, GB? Does anyone know the cost of having the samples analysed? I am surmising that the kits are not DIY as all the research talks about centrifugal force in the lab? Any advice, recommendations would be gratefully received.
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Hi Jacquie, I have done OT assay in Urine for trial a long time ago and my assay is working well for that, but we needed to extract OT, as for blood sample, to avoid any cross reaction with hormone. But...I am not sure that it could be scientifically relevant. OT is not secreted as another gland hormon with a tonical release. It is a neuro hormone secreted by episodes during short period (By example not during all a nursing duration in human that is your model) and very rapidly destroyed by Oxytocinase and other protease in blood. In consequence, OT urine level are very very limited in concentration and sometimes under the detection threshold. Additionaly, Corpus luteum is another OT source that produce very important concentrations of OT in a tonical manner. If and ovulation occurred in the very fisrt time after parturition, you can have very high OT level during the 28 days after LH surge that should totally overlap OT relased during nursing. Finnaly, If you want to follow OT release due to Mother/ypoung interaction, you can forgot because OT releases are lower in concentration that during nursing episodes. Only blood and sometimes LCR or microdialysis will be able to follow this activity..and it is not ethically possible to perform that in Human mother. Sorry to give you negative advice but it is to avoid a long work. If you need any precision please, do not hesitate to contact me via RG or my professional mail adress (Marnet(at)agrocampus-ouest.fr). Best regards
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i want to estimate total antioxidant(with FRAP test) in the liver tissue homogenate but the frap test usually done in serum or plasma.whats difference between method of meassuring FRAP in serum and tissue homogenate?
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yes u can do FRAP assay on both animal tissue and plant extract.
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Is there any published papers that support the above question?
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See attached link: Clin Biochem. 2013 Aug;46(12):979-82. doi: 10.1016/j.clinbiochem.2013.04.016. Epub 2013 Apr 26.
A few additional nuances: you specified ACS. Since, ACS includes unstable angina which is by definition, troponin negative it is technically incorrect to think about the sensitivity and specificity for ACS but rather, for acute MI. In addition, specificity for AMI will depend on whether one is interested in capturing all MI's or more importantly type I MI's. I would argue, it is important to quickly and accurately diagnose type I MI but the same is not true for type II MI. The management of these two conditions is very different. Type I MI is characterized by acute intracoronary thrombosis and the backbone of treatment is prompt recognition followed by anti-coagulation and anti-platelet therapy +/- mechanical intervention or thrombolysis. Type II MI has many causes but the backbone of treatment requires, most importantly, treatment of the underlying condition. Furthermore, ordering troponin without reference to the clinical context can lead to misdiagnosis of type I MI and treatment that not only fails to address the underlying condition but could be dangerous and make the condition worse. See attached manuscripts which provide more detail.
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I see most people running urinary steroid hormone and oxytocin assays are publishing that SPE is required as sample prep, but I have been unable to replicate results reliably. I can use a commercially available EIA kit that does not require SPE prior to running, but I'm looking for good resources on how to know what sample prep is appropriate for your sample matrix and testing method as well as how to choose the correct SPE column. There are multiple types in the literature that are used. For example, oxytocin extractions interchangeably report using Phenomenex StrataX, Waters Oasis HLB, and Sep Pak C18,
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You're correct that various investigators use different SPE cleanup methods for steroids and oxytocin, and indeed it doesn't matter which method you use as long as you verify the results yourself. There are two main things you need to do to check out for a particular method: (1) determine recovery from the SPE column (using known amounts of pure standards, spiking samples with known amounts of a standard, or using radiolabeled materials), and (2) determine whether the clean-up procedure works to give you reliable results. If you have access to a mass spectrometry facility, you can directly check the purity of the column eluate and how well the EIA values compare to values from LC-MS/MS measurement. More commonly, accuracy of an antibody-based assay (e.g., EIA) is assessed by preparing serial dilutions of your unknowns (purified and non-purified) and determining whether the readout from the set of serially diluted samples parallels the readout from your standard curve. One final note is that in my lab we have been happy with using the Phenomenex StrataX cleanup method for salivary oxytocin measurement. Good luck!
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Hi, I want to find out change in concentration of six different proteins due aging in muscle tissue. I am performing western blot right now.  Is there another technique available to get accurate protein concentration?  I don't have purified proteins to use as standers so can't perform ELISA. 
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You should read an excellent new paper about important factors for quantitative immunoblot analysis. It recommends use of multiple internal loading controls, for maximum precision and reproducibility. Also suggests diagnostic experiments you can run to look for sources of variability.
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In general lipase protocol-
Lipase activity assay
Determination of liberated free fatty acid was measured by colorimetry method (Kwon and Rhee, 1986) using olive oil as substrate. The reaction mixture, consisting of 1 ml crude enzyme (culture filtrate), 2.5 ml olive oil emulsion (properly mixed of an equal volume olive oil with 50 mM sodium phosphate buffer, pH 7.0) and 0.02 ml of 20 mM CaCl2, was incubated in a water bath shaker for 30 min at 50°C under 200 rpm agitation. The enzyme reaction in the emulsion system was stopped by adding 6 M HCl (1 ml) and isooctane (5 ml), followed by mixing for 1 min. The upper isooctane layer (4 ml) containing the free fatty acid was transferred to a test tube and properly mixed with 1 ml copper reagent.
The reagent was prepared by adjusting the solution of 5% (w/v) copper (II) acetate-1-hydrate to pH 6.1 with pyridine. The free fatty acid dissolved in isooctane was determined by measuring the absorbance of the upper layer at 715 nm after mixture settlement. Lipase activity was determined by measuring the amount of free fatty acid released based on the standard curve of oleic acid (0-50.0 µmole) in isooctane. One unit of lipase activity was defined as 1.0 μmol of free fatty acid liberated min−1 and reported as Uml−1.
I would like to know if same can be done using palm oil as substrate.
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kindly use esters rather than acids for lipse assay, Since reaction equilibrium will be well balanced 
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Is it HPLC, ELISA or Chromatography?
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There are three new methods with superior performance that need to be considered. HbA1c by (i) Capillary electrophoresis (Sebia Capillarys) (ii) by enzymatic digestion (Abbott Chemistry) and (iii) by ion exchange (BioRad D100). All three methods have excellent imprecision CV(SD) < 1.5%. The reference method is enzymatic digestion and mass spectrometry (only for purists!), so the Abbott method is closest by incorporating the enzymatic digestion but using colorimetric reaction rather than mass spec to estimate the N terminal glycation.
However, quality shouldn't be judged by imprecision alone as accuracy and specificity are also issues. In terms of accuracy, all methods are fairly accurate around the diagnostic decision point of 6.5% (48 mmol/mol). However, they do tend to drift in accuracy when you get to much lower values (eg biases of up to 0.5% @ 5.0% ie in the healthy reference interval) or higher values (biases if 1.0% @8.0% or higher ie in the poorly controlled diabetic ranges).
Specificity is also an issue as mentioned by others. Genetic haemoglobin variants including the persistence of fetal haemoglobin can affect the HbA1c value given, so I agree that having a chromatogram, by ion exchange or capillary electrophoresis is preferrable so that abnormal haemoglobins that affect the reliability of HbA1c measurement, can be registered. In general terms, all the new methods have been designed to be robust so that haemoglobin variants generally affect only 1% of all HbA1c analyses - which is very good (unless you happen to belong to the 1% who are mislead).
I would be happy to use any of the three new methods mentioned above, however I would prefer the new BioRad D100 ion exchange method for it's overall imprecision, accuracy and specificity.
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On our high throughput hospital clinical chemistry analysers we measure haemolysis, icterus and lipaemia using simple spectrophotometric indices. This is to avoid analysing tests where these interference are know to lead to factitious results. 
We have recently observed that some patients with persistent raised triglycerides have high haemolysis indices and there is a relatively strong correlation!
Has anyone else seen this? I am not sure if this represents increased invitro haemolysis in lipaemic samples or a direct analytical interference. The strong correlation suggests the later to me?
All help gratefully received.
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Although we find no correlation between the lipaemic index and haemolysis index on our Roche analysers (c701), this may be due to the Roche spectral correction mentioned by Wesley above.
Goce Dimeski from Brisbane did publish on a possible effect of lipid on RBC membrane fragility a few years ago;
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 I am going to measure this activity but I don't know the exact protocol (quantity and concentration of PA or buffer...)
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Hello 
The best method for Arylesterase activity was determined spectrophotometrically at 270 nm with phenyl acetate used as the substrate. The assay mixture included 1.0 mmol/L of phenyl acetate and 0.9 mmol/L CaCl2 in 20 mmol/L Tris HCl, pH 8.0, at 25°C. Nonenzymatic hydrolysis of phenyl acetate was subtracted from the total rate of hydrolysis. The E270 for the reaction is 1310 mol/L−1 · cm−1 and 1 unit of arylesterase activity is equal to 1 micromole of phenyl acetate hydrolyzed per milliliter per minute.
Good Luck 
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It is possible that the absorption of fat soluble vitamins have not affected? What happens with their plasmatic transport if the truncated form causes that plasma concentration of LDL is very low?
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Dear Juan,
Adsorption of fat soluble vitamins should not be affected, as apoB48 is derived from apoB transcripts that are RNA-edited in exon 26. Any transcript longer than the RNA-editing point will therefore result in functional apoB48.
For the answer to your other question, please have a look at these papers:
You may also find this publication interesting:
Best wishes,
Petra
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Free radicals and mitochondrial dysfunction are proportionally correlated.
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Yes there is a correlation. Use ETC inhibitors and check the modulation in ROS using mitosox red. Also simultaneously monitor the changes in antioxidant levels. You will definitely get your answer.
Regards
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Newly discovered hormones or markers, that have relation with fertility and some glands cancers.
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perhaps you can think about inhibin B and AMH which can be elevated in ovarian and testicular cancer and are used as a marker of gonads function
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I need to know the method how industrial enzymes are produced, because industrial enzymes are involved in everyday life aspect.
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This can be done in two ways by using solid state fermentation (SSF) technique or by Submerged fermentation (SmF). These two have their own advantages and limitations. If u r searching for an enzyme from an economical point of view then SSF is the one by which u can standardize a production medium for enzyme production using agro wastes. But in those case where need of enzyme in more purified form then SmF is preferred as there is a smooth and easy extraction process but which cost more also. What ever it may be by any method of two discussed above, industrial enzymes are produced and applied in various purposes.
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What is exact dose of STZ to induce diabetic type 2 in rats?
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Doses given on the based of Type I or Type II induction
35 mg STZ with diet for Type II
50 mg STZ  (minimum 2 times 1st day and 3rd day) This is my personal experience as getting rats diabetic first stretch is bit tough so have to give 2nd time STZ.
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Often we find markedly lipemic serum in which biochemical analyzes suffer from strong interference, including the concentration of the analytes in "free water". What could be the pretreatment in order to analyze them properly avoiding the positive interference that produce the turbidity and the effect of the volume due to chylomicrons usually present?
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A few points here.
1. Not analysing such specimens can be clinically inappropriat. What do we tell the patient with potential pancreatitis, came back in a week when they might be dead?
2. Some solvents are not allowed/available on health and safety grounds. How many have been compared to the gold standard of high speed centrifugation?
3. Lipoclear is available but again there is limited data available compared to high speed centrifugation.
Hujjjjjjj4. Polyethylene glycol produces similar results to Lipoclear apart from bicarbonate if my memory is correct when we checked this many years ago.
5. Availability of any of the above when you have lipaemia is important and a protocol to follow!
6. Investigation and treatment are important! The highest triglyceride that I saw in a patient was 180 mmol/L. As a Chemical Pathologist, I was lucky enough to also have treated him (diagnosing his diabetes and treating this and his dyslipidaemia at our clinic next day).
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Some recent studies have demonstrated a strong reduction of the cLDL with diverse molecules that have this activity. But does someone know about studies directed to the cardiovascular effect of the inhibiting PCSK9? What do you think about possible collateral effects?
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To my knowledge at least two antibodies against PCSK9 are now in phase III studies (AMG145 or evolocumab and REGN727or alirocumab) and we have to wait until 2016/7 for the answer whether the reduction of LDL-C corresponds to a similar reduction in cardiovascular endpoints.
At the moment a strong reduction in LDL-C on top of statins has been published for all inhibitors, in some patients even below 50 mg/dl. Whether this strong reduction in LDL-C might lead to other problems is not clear. Some years ago it was discussed whether strong LDL-C lowering by statins might increase the risk for certain cancers but in the meantime this is no longer a topic.
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In some recent studies there seems to be a greater occurrence of diabetes in patients treated with high doses of statins. Do you think that it is an artifact or actually exists, and if so, why?
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According to Kohl (Circulation, see enclosed) statins are “ inhibiting the synthesis of isoprenoid and suppressing ubiquinone (CoQ10) biosynthesis and thus delaying formation of ATP by pancreatic β-cells leading to impaired insulin secretion, inhibiting
glucose-induced insulin secretion from pancreatic islets, reducing sensitivity to insulin, altering glycemic control by decreasing various isoprenoids that enhance glucose uptake via glucose transporter (GLUT) 4 in adipocytes, or other mechanisms, including potential
central nervous system actions to impair glucose homeostasis.”
 Another very good explanation of possible mechanism is described below:
 
Metabolism. 2014 Jun;63(6):735-45. doi: 10.1016/j.metabol.2014.02.014. Epub 2014 Feb 25.
Statin treatment and new-onset diabetes: a review of proposed mechanisms.
Brault M1, Ray J2, Gomez YH3, Mantzoros CS4, Daskalopoulou SS5.
Author information
Abstract
New-onset diabetes has been observed in clinical trials and meta-analyses involving statin therapy. To explain this association, three major mechanisms have been proposed and discussed in the literature. First, certain statins affect insulin secretion through direct, indirect or combined effects on calcium channels in pancreatic β-cells. Second, reduced translocation of glucose transporter 4 in response to treatment results in hyperglycemia and hyperinsulinemia. Third, statin therapy decreases other important downstream products, such as coenzyme Q10, farnesyl pyrophosphate, geranylgeranyl pyrophosphate, and dolichol; their depletion leads to reduced intracellular signaling. Other possible mechanisms implicated in the effect of statins on new-onset diabetes are: statin interference with intracellular insulin signal transduction pathways via inhibition of necessary phosphorylation events and reduction of small GTPase action; inhibition of adipocyte differentiation leading to decreased peroxisome proliferator activated receptor gamma and CCAAT/enhancer-binding protein which are important pathways for glucose homeostasis; decreased leptin causing inhibition of β-cells proliferation and insulin secretion; and diminished adiponectin levels. Given that the magnitude of the risk of new-onset diabetes following statin use remains to be fully clarified and the well-established beneficial effect of statins in reducing cardiovascular risk, statins remain the first-choice treatment for prevention of CVD. Elucidation of the mechanisms underlying the development of diabetes in association with statin use may help identify novel preventative or therapeutic approaches to this problem and/or help design a new generation statin without such side-effects.
Also, please find enclosed a very good review from Chung regarding the mechanism of statin induced DM.
Best regards, Michaela
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Searching to discover lysosomal storage diease
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Until the enzyme is specified, the problem cannot be solved.
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In general sense, all the drugs have an expiry date (shelf life). After the expiry date the drug is recommended not to use as it may produce toxic effects in biological system and/or lose its potency in lesser or greater extent i.e. deviation from the optimum specification is seen. My question is what will be happened in case of a toxicant (toxin)? Is there any recommendation for toxiants' using? What will be there toxicological efficacy after expiry?
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Depends entirely on the class of compound, the process by which it degrades and what are the intermediaries produced. Without knowing something about the type of toxin you are asking about, there is no way to say.
Some biological venom, for example, will eventually break down into completely harmless degradation products. Some industrial or agricultural chemical toxins tend to break down into sometimes even more potent or harmful secondary compounds.
Remember, pretty much anything is toxic, at some dose or some concentration. Or if not the native compound itself, its metabolites are.
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In our laboratory, we tried to measure iodine levels by ion selective electrodes. However, our direct assays were subject to interferences from other anions like fluorine, bromine. We would like to use a resin and solvent extraction to discard the interference molecules.
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Hallo!
Actually, you don't need to extract the iodine. You have to digest proteins in the urine by hydrochloric acid, for example, and the use a colorimetric method. See Clinical Chemistry 46 2000 529-535. As color reaction. Sandell-Kolkoff is usually used.
greetings
Sighart Walter Golf
April 2000 vol. 46 no. 4 529-536
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I need information about the right amount of the reagents and sample
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I have sent you a link where you can find materials and method to measure IMA.. we have published paper on it.. Hope it will help you to measure with less cost..
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As you know chylomicrons or vldls take apo E from hdl in the blood during circulation. Does anyone know its mechanism? Is there any molecule or whatever else that provides this transfer between lipoproteins?
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It is affinity mediated mechanism. ApoE can bind more preferably to chylomicrones when come closer to HDL. Most probably due to receptor on chylomicrones for apoE. because ApoE itselp is a protein and chylomicrones have a receptor for it.
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I want this information to my PhD project.
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there are two publications on betatrophin in humans. I do not believe either found a connection to diabetes or dyslipidemia
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I am interested in the microparticles' functions in blood. I want to determine the levels of some microRNAs included microparticles by real-time qPCR. As the level of microparticles in blood is limited, I want to know if the level of the included microRNA is sufficient to be detected?
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Thank you, all. I will try it.