Blood Biochemistry - Science topic

Dear Luay A. Al-Helaly,

In the country where I live, blood is drawn from the arm in a sitting position. Vaccinations against the SARS-CoV-2 (Covid-19) coronavirus are also performed in a similar way, also in a sitting position. The point is for the patient to be comfortable, blood collection or vaccination to go smoothly. According to current standards and recommendations, vaccination against the coronavirus SARS-CoV-2 (Covid-19) is made in the left shoulder. So I do not know why in the information campaign encouraging to vaccinate, in the spot of the film information and advertising campaign, which involved, among others, of a famous actor, people known from other commercials appear, and there is a situation that suggests that vaccinations are supposedly being made in the right shoulder. Apparently, the authors of this government-commissioned information campaign did not take the above-mentioned knowledge into account when directing it.

Regards,

Dariusz Prokopowicz

The environment that's supposed to be made more alkaline is only the endosomal compartment, not the entire cell or body. Naturally, endosomes slowly become more acidic, on their way to becoming lysosomes. Many viruses sense this acidification, and use it as a trigger to fire their endosome escape functions and enter the cytoplasm. If this fails, the virus will likely be destroyed once the lysosomes become mature and loaded with protease & RNAse.

To prevent endosome acidification, a traditional chemical buffering system is likely not the correct mechanism. This would more likely be achieved by a specific phamacological mechanism, like interrupting membrane integrity or inhibiting a proton transporter. I have seen remarkably little work on this, and all of it only in cell culture.

Thanks dear

Thank you Keagile,

following

iAUC (incremental ...) is the most recommended and approved method. Follow iAUC

Merry TL, Ristow M. Mitohormesis in exercise training. Free Radic Biol Med 2016;98:123-130. https://www.researchgate.net/publication/285392332_Mitohormesis_in_exercise_training

I think that 10-15 units (IU)/ml of blood is enough for anti-coagulation !

The term "Artificial blood" is a misnomers and can't be prepared in laboratory. There is a limitation of stem cells culture in vitro and component in whole blood is much more complex.

Dear Dr. Ali F Almehemdi

Blood types help us to fight diseases and tell us what major disease epidemics a given population suffered in its recent history, (e.g. in the last thousand years). For example, most native Americans belong to group O. It is believed that this is due to a syphilis epidemic and that the O type were better at fighting off the disease. Group A and B make people more resistant to cholera, while AB confers the most resistance. O offers virtually no immunity against cholera. B confers weaker protection against plague. This is probably why B is more common in North-East Europe, which was virtually unaffected by the Black Death during the Middle Ages.

A-type carriers are the most likely to survive plague, but suffer from a higher rate of heart disease, because their blood is more likely to clot. They are also at increased risk of contracting smallpox and developing cancer of the esophagus, pancreas, and stomach. Type O, contrarily to A, is slightly protective against cardiovascular problems. It also boosts resistance against tuberculosis, but increases the risk of venous thromboembolism and developing duodenal and peptic ulcers. It also attracts more mosquitoes (through which malaria is transmitted). A recent study revealed that people with type O blood are less likely to get pancreatic cancer, but also stomach, breast, ovarian and cervical cancer.

The ABO blood group system isn't the only antigen system found in humans. There are about 30 human blood type systems: Rhesus, Kell, Diego, Duffy, Kidd, and so on. Each have a role in immunity. Some are found only in some specific populations and completely absent elsewhere. This is the case of Diego antigens, found only (at low frequency) among Mongolic people and Amerindians.

Twice the concentration used for human blood, since the hemostasis system of mice is pretty procoagulant when compared with the human one.

Dear Olivia,

Try HNP-1, CD66b and MPO to confirm presence of PMNs.

Short answer, no markers that are not also found on other cell types in the periphery.

In the absence of a condition which perturbs the BBB there are no microglia in the blood. Your question is important and one with active investigation because it would be useful for neuroinflammation biomarker research.

At our Institution we have a protocol (WHO/LAB 97.2) for whole blood preservation (glutaraldehide 0,37%, formaldehide 2,7%, trisodic citrate 26%, and antibiotics) which is intended, precisely, for CBC blood counters. You can seek it in the WHO page. Success!

Oxygen oxidizes ferrous to ferric. Does have affinity towards ferrous. This property is used for oxygen transport by hemoglobin. But iron is buried in a pocket guarded by histidine residues. This prevents oxidation of ferrous by oxygen but the affinity helps carrying oxygen. In ferric form, affinity is lost and cannot carry oxygen.

does anyone have this protocol?

Cholecystokinin-8 regulates hepatic glucose production through a CNS-dependent CCKAR mechanism which may be defective in the setting of obesity.

The difficulty with freezing enzymes is what the defrost cycle enzymes are degraded. It is then necessary to use a cryoprotectant such as glycerol before freezing. Than we can storage 6 months or plus

Is this paper helpful at any extent ? 

Small amount like 5-30 ml don't have significant role in changing any parameter within human body . 

Hi Mansai, the choice of anticogulant may have more to do with being able to compare the microparticle results with other lab tests that have used citrated-blood.

Regards, Rosemary  

A good place to start looking is the Mouse Phenome Database at phenome.jax.org. 

From the MPD main page, click on "Phenotype" and browse the blood measures, or click on  "Strains"  and follow the top links on the next three pages to get to the available phenotypes for C57BL/6 mice.

Blood pH does indeed affect the RI. The percentage of HbA1C in the erythrocyte is directly related to the RI. pH affects the charge on the Hb molecule. This is well established and you can find more information in most text books. *Try a keyword search (Google or Bing) for texts or articles on hemoglobin and/or glucose levels in blood as related to pH and RI. You will find plenty of sources.

Partha - coagulation is slow in plastic containers. Can you collect the blood in sterilised glass tubes, or transfer the blood to such tubes and store forr at least one hour before centrifugation?

If you collect in plastic tubes you can add glass beads or thrombin and CaCl2 to your blood after collection. This will speed up the clotting process.

Whar is the serum used for?

Best regards

Johannes

Dear Martin: Though proteins in the aqueous humor resembles those in the plasma, they are actively secreted by ciliary epithelium cells.  The secretion rate of a specific protein in the humor may be proportional to the liver production, the change(s) of which should be more subtle than the plasma.  As immunoglobulins are the main proteins, I'd suggest to monitor sIgA (secreted) in plasma to establish any correlation.

I am in agreement with the doses mentioned in the diferent answers. However, you should consider that EDTA is not adequate anticoagulant for PRP preparation. Because this substance could be harmful for live cells and technically is very hard to concentrate platelets when this substance is used. I strongly recomend you to use anticoagulants as sodium citrate or ACD-A.

You must also consider taht many calcium chloride preparations could produce calcium precipitates in gels from PRP. We prefer to use calcium gluconate (9.3 mg/mL) in a ratio 1:10 for PRP activation.

Thrombin in blood froms stable complexes with antithrombin (AT3), fibrin (AT1), heparin cofactor 2, or alpha-2-macroglobulin. In the latter complex thrombin still has amidolytic activity. In the other complexes thrombin has lost its enzymatic activity.

The standard reference for Hematology is by Williams. It covers both Blood Chemistry and Blood Histology.

Hope this helps.

I am thankful for your response. I think that if new devices are used to detect the capillary refill time to provide more accurate measurements, it is more valuable physical sign and it may be considered as a vital sign. I am sending two articles about this topic as attachment.

The pathlength in a microplate well is the height of the liquid in the well, which is directly proportional to the volume (if the wells have vertical sides, which is usually the case). You can measure the height of the liquid, approximately, in wells at the edge of a transparent plate. However, it isn't really necessary if you can make a standard curve.

The greater the volume in the well, the greater the absorbance for a given concentration of substance. So you might want to use the largest practical volume. However, large absorbances aren't as important as you might think. An absorbance of 0.1 is usually sufficient. There will be a slight well-to-well variation in the baseline absorbance of up to +/- 0.01. You can do a pre-read of the empty plate to record this baseline and subtract it from the final measurement, if necessary.

Another consideration is mixing. It's difficult to mix the contents of the 384-well plate when the wells are nearly full. Trituration (pipetting the solution up and down a few times) is not a good way because it is usually causes bubbles to form, and you can't make a good measurement with bubbles in the well. You can mix the plate by holding it with one hand while tapping it several times with the other. Mixers designed for 96-well plates don't work well for 384-well plates. The Eppendorf MixMate is a good instrument for mixing 384-well plates. You have to be careful with it not to spill the contents from too-vigorous mixing, especially if you have solutions with a lot of protein or detergent, because of the low surface tension. It's a good idea to practice a little before committing your precious samples.

If you can lay your hands on multichannel pipettors pitched for 384-well plates, your work will go a lot faster than if you use single-channel pipettes. But you can also use multichannel pipettors intended for 96-well plates. They dispense into alternate wells of a 384-well plate.

Be careful not to dispense air bubbles into the wells. You can avoid this with proteinaceous or detergent solutions by pre-coating the pipet tips. Draw up the solution and dispense it back into the reservoir once or twice before doing the actual dispensing.

If you do get some air bubbles at the top of the wells, you can usually burst them by blowing a stream of methanol or ethanol vapor gently over them. Get a laboratory squeeze bottle, the kind with a straw inside. Remove the straw and put in a small amount of solvent. That way, only the vapor comes out, not the liquid.

Once you have experience with regular 384-well plates, and if your absorbance is more than sufficient, you may be able to switch to low-volume 384-well plates and use even less reagent. Pipetting is trickier, however, because of the small size of the wells.

Alex,

Table 1 of the following paper might be of help http://www.nature.com/ni/journal/v15/n2/full/ni.2787.html 

Fructosamine can replace the glycated hemoglobin for example in pregnant women because it represents the glycation of proteins, especially albumin, and reflects the glycemia of 15-20 days before the determination. 

Your question involves three different enzymes, so the discussions and answers might not be easy to follow. I would suggest you to ask a different question for each enzyme, next time.

Below are some references for catalase and superoxide dismutase, with detailed protocols. I could not find a PDF for the oldest references, but the papers with more recent modifications of the protocols might be more useful anyway.

As for para-oxydase, were you referring to the PON1 enzyme? I attached a few papers on that as well.

I also link to other threads in researchgate with similar questions.

All the best with your work!

Dear Shamsul, 

Check this link out: 

Dear Gaurav Kumar I wish to help you to realize your research about the methemoglobin argument, which is also  my field of research interest.

I cite the article which explain the procedure appliable to human or animal blood:

CLIN.CHEM.25/8,1388-1393(1979) F.Lee Rodkey, Thomas A. Hill, L.Loring Pitts and Robert F. Robertson.Spectrophotometric Measurement of Carboxyhemoglobin and Methemoglobin in blood.

With regards

Lucijan Mohorovic,MD,PhD

Hello Bradi

Concerning the DNA extraction, using cow's blood, i suggest you use the Buffy coat, avoiding excessive red cells. Just like 300 microlitres is ok for the extraction.

Cytogenetic analysis after 10-16 hr of sampling usually still work pretty well. The heparinized samples should be mixed well by inverting the tubes several times and best be shipped at room temperature.

As a reminder, if you want to really quantify (meaning absolute quantification in terms of numbers of molecules of specifically bound MAb to this Ag, aka sABC) an internal Ag, you have to correctly choose your fluorochrome and optimize permeabilization/washing steps*.

Also remember that (act.) monocytes do harbour high autofluorescence (higher than any other blood cells) from flavins/NADP ... Maybe you want to prefer red-emitting fluors. One trick I have seen (long ago) in the litterature to reduce autofluorescence of monocytes is the addition of Trypan blue as a quencher (though I presently do not find the - old -ref.).  Also avoid PE since PE is the fluor most prone to interferences from counter-staining reagents (tandems, FITC ...).

If you do want to go to sABC, then you may consider using our CellQuant Calibrator kit, or QIFIkit from DAKO. This may work even for intra-cellular staining. see e.g. *) "Involvement of Peripheral Benzodiazepine Receptors in the Protection of Hematopoietic Cells Against Oxygen Radical Damage. Carayon et al Blood 1996:3170-78 or "Expression of Bcl-2 and Bax ..." Sermadiras et al Br J Dermatol 1997; 137:883-9.

Hope that helps. PP

Hi Jim! pH is a bit acid, and progressively more so with time. In LR blood slight increase in PCO2 with thime, big increases with non-LR blood over time. O2 vewry different between LR and non-LR blood, more than 250 mmHg in LR blood by end of week one. Thank you for the suggestion re: nitrogen in bag. :-)

Thank you very much, people!

I analyzed the samples and the results were similar from those that I found on the literature..

Hi, I am not sure, but maybe you will find the answer at macs miltenyi Homepage, they offer mitochondria Isolation kit, I used it for cell culture. But I don't know if it is useful with blood, ask the technical Service. cheers.

I agree with Suzanne – Room temperature storage increases viable cell yield and reduces activation and cell death induced by 4°C storage overnight. I believe  4°C makes membrane RAFT areas more rigid - locking signaling molecules together that may have been mean to be a transient interactions. People who work with granulocytes do NOT use blood stored overnight at 4°C because that process  activates the cells.

Thank you very much!

Dear Christmann, 

Thanks for your help. Can you please tell me your protocol for fixing primary cells? Thank you. 

I ' m glad I could help you.

Calcium is necessary for the activity of complexes which participate in thrombin generation but not for its activity.

In fact we buy prepared QC from Arvecon. In our centrifuge evaporator, the max temperature that we can use is 80°C and this is not the real temp. in the sample because this the value of the evaporation chamber. THC is boiling at 157°C in its pure state so I think this will be ok. I'll say you that in a few days :)

I think both might be important, depending on your biological or scientific question. The % abundance may be more signifficant for some metbolic disease (like phenylalanine ratio). However,  the absolute concentration is very importantant in relation to the concentration of other metabolites or the value of the hematocrit.

of course you can. i frequently use regularcentrifuge to measure hematocrit. just ad 2 - 3 cm long styrofoam into the centrifuge tube to help the capillary tube stand up. turn the sentrifuge on, measure it, done :D i wish this is helpful

Plasma is derived from samples of blood collected using an anti-coagulant and spinning the cells down. Serum is collected after clotting of the blood.

So in my opinion you need either to collect two tubes of blood one with and one without anti-coagulant or divide the sample after collection over the two different tubes.

It should be efficient. Just follow step by step the protocol, paxgene tubes shouldn't be influential.

The Srere procedure you used for assaying citrate synthase in muscle should work in serum though I would expect very, very low levels in serum unless you had some sort of muscle damage and/or mitochondrial damage.

Dear Devan,

I frequently isolate DNA from both frozen and fresh blood samples of 2 ml using simple salt extraction method. Add 5 volumes of RBC lysis buffer to the thawed sample and vortex for 2 min and incubate at RT for 10 min. Spin at 2000 g for 20 min, discard the supernatant but leave the pellet with tiny amount of buffer. Then add 1 ml of RBC lysis buffer and repeat the above step. Then add 2 ml of WBC lysis buffer with Proteinase k and vortex for 2 min, incubate at 50 deg C for 3-4 hrs or until the pellet get solubilized. Add 200 ul of 3M sodium acetate, and incubate at -20 deg C for 1 hour, spin at 12000g for 10 min, transfer the supernatant and precipitate the DNA with double volume of cold ethanol. Wash the DNA pellet with 70% ethanol, and dissolve the semi-dried pellet in required vol of Tris, pH 8.5 or sterile ultrapure water.

Now your DNA is ready to be estimated and other down stream process. This method is applicable for both frozen and fresh blood.

all the best wishes,

regards,

Rajkumar

What kind of solutions you want to know .

On both soluble and membrane protéine extracts from RBCs, I successfully depleted Hb through IMAC chromatography with nickel-loaded phase, the same way as for the purification of His-tag proteins. Have a look at this paper : 

Journal of Proteomics

5 December 2012, Vol.76:181–193, doi:10.1016/j.jprot.2012.05.004

hope that can help you ;-)

From my experience the biggest problem will be if the brake acts due to a power failure as this will ruin your layer on the Ficoll (or whatever you use). But how your centrifuge reacts to the power cut (break on or off) could depend on the type of centrifuge you use.

Hi Ivana

Yes, the way you are doing is also valid, where in you first normalize all samples to the same protein concentration and then dilute either 1:10 or 1:2.

The way I did was, I started with the same weight of tissue across different tissue samples. Then, I did different dilutions of the samples to check and see what dilution falls within 25%-75% of the standard curve. I also did a BCA assay to determine the protein concentration of the samples. Once I got the protein concentration of the specific antigen from the ELISA data, I divided that with the total protein concentration to appreciate the actual differences of the specific antigen between samples.

Yes, you are right. If your samples after background subtraction are negative, then you should extend the lower end of your standard curve to determine the lower limit of quantification (LLOQ) of that assay. To do this, I ran 3 sets of standard curves in triplicate on two different plates, calculated the % CV (a sister of SD) and % recovery for each set of the standard curve. If % CV (SD/Mean*100) is 10% or 15% and % recovery (observed conc/true conc *100) is between 80%-120%, then that lowest point on the standard curve (across all standard curve sets) that obeyed the rules you set for %CV and % recovery cut-off was considered the LLOQ. Again, you can determine if you want the % recovery to be 80-120% or 90-110% and for the % CV to be below 10% or below 15% or below 20% (which is the max).

It is also possible that your antigen is not completely soluble in the sample diluent. Adding 0.1% SDS or 0.1% Triton-X-100 to the sample and standard diluent might help solubilize your antigen better, thereby help interact with the capture/detection Abs. When doing this, I would first test just the standard curve, with or without SDS/Triton-X-100, and see how the signal improves and then extend it to the samples, at which point you should test the standard too.

Another possible explanation is that your antigen may not be completely extracted while processing the plasma. This happened with my antigen. I used a number of different methods (RIPA/SDS/Triton) to extract the antigen. SDS worked very well for my antigen. Mine was a transporter protein, which is a transmembrane protein. Because it is not a soluble protein, but a membrane protein, it needed 10% SDS during the sample processing step to completely solubilize the protein.

Also, unless the kit that you purchased from the company has passed good quality control, and is a reliable company (like Innotest or Covance), ELISA may not work, just because the company sells. I had a not-so-good experience with one company out in the market, and ended up developing my own. Sometimes, companies sell products with a part of the recombinant protein (fragment), rather than using the full length protein, whose folding will be different from that of the full-length antigen in the sample.

I hope these tips help improve the ELISAs. Always, please  feel free to contact if you have any further questions.

Best wishes,

Chandana

You are likely to get less inter-animal variation if your rats were fasted.

The reason we use fasting is to eliminate some of the variability between different animals. When you leave rodents with unlimited access to food, not all of them eat at the same time or even the same amounts. If you have multiple animals in a single cage, you will often notice some differences in their weights. All of this plays into what you may see in serum levels of various measurements, especially parameters that are known to be effected by feeding status, such as the ones you are measuring.

If your experiment requires levels from animals that fed ad libitum, that's a different matter.

Thanks Jonathan. It's a valid point to double check when reading publications.

I personally think that the practice of adding RNA carrier to RNA extraction is a bad one, and I have been using either linear acrylamide or glycogen successfully for many years - not with serum, but with low numbers of cells.

A

Hi Chunying Li,

I have so far never seen an anti-U - but maybe the article in the link might help.

I am not aware of an PCR kit determining U variants - however, in Germany this is probably seldomly needed. You should ask African colleagues, maybe they are more experienced with this problem due to the "higher" proportion of U negative people there...

As we pointed out in our paper in AJPCELL in 2008, clotrimazole interacts with P450 proteins, hence with heme group containing molecules, like hemoglobin. We do not know the role of P450 in the regulation of IK channels. It would be interesting to use hemoglobin as a simpler accessible molecule  and study its interaction with the heme group as function of its oxidation state. 

From Page C830 of AJPCell 294, 2008:

Neither typical inhibitors of Big conductance (BK), other

voltage-gated K channels, Small conductance (SK) channels,

nor known blockers of the connexin family were effective

(Table 2) (15). Only CTZ with an approximate IC50 of 25 M

and completely at 50 M prevented Kc loss (Fig. 10). This

concentration range is comparable to published CTZ doses in

other epithelial cell systems, with intermediate conductance

KCa3.1 (IK) channels involved in RVD (29, 60). TRAM-34, an

inhibitor of IK channels not involving the P450 protein like

CTZ (56, 66), also reduced Kc loss by 50% and less Rb influx

suggesting that the two inhibitors act through different mechanism

either at the channel or regulatory level.

Thanks for all of you to answer me

RiboPure™ RNA Purification Kit for blood (Life Technologies) works very well. Trizol LS Reagent (also Life Technologies) also works well. Here it is the protocol: https://tools.lifetechnologies.com/content/sfs/manuals/trizol_ls_reagent.pdf

I was co-chair of the conference, and am editing the proceedings. let me know if there is a more specific area of interest for you.

I thing saranya bharathi method is most common method to extract proteins but most of the case it won't work.,

Some protein extraction methods we compare means we can get enough idea.,

if i found any i will get back here...

Hi Stephanie, for albumin depletion, Blue Sepharose is the easiest and best.

Hüseyin Bey merhaba

Ekte sorunuz ile ilgili bir literatür gönderiyorum, -80 ^oC de analiz zamanına kadar örnek saklanmış, iyi çalışmalar dilerim.

Türkan Yurdun

Does low pentraxin-3 levels associate with polycystic ovary syndrome and obesity?

Figen Kir Sahin,1 Serap Baydur Sahin,2 Gulsah Balik,1 Ulku Mete Ural,1 Yesim Bayoglu Tekin,1 Medine Cumhur Cure,3 Senol Senturk,1 Suleyman Yuce,4 and Erkan Cure5

Tubes containing EDTA with 2 ml were used collecting blood. The blood then was immediately transferred to centrifuge tubes which contain aprotinin (0. 6 TIU/ml of blood) inside. The tubes were gently rocked several times to inhibit the activity of proteinases before being centrifuged. The centrifuge process was carried out at 1600 g rate for 15 min at 4°C condition to obtain the plasma that was stored in -80°C freezer until the time of assay.

Score software. Check Olerup homepage!!

May recommendation to froze the serum.

 see ; http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2679668/

In Ron de Kloets lab we routinely used the CORT RIA from MP Biomedicals. We adjusted the standard curve by adding two lower concentrations (6,25 and 12,5 ng/ml) to make sure we would detect low basal circulating levels of corticosterone. Also you just need 5-10 microliters of plasma to run you assay.

Hi Steingrimur, thanks for your additional comment. I agree with you on the fact that plasma contains a balance of proteases and anti-proteases. As a matter of precaution, aprotinin could be added before freezing (ideal) and, if not, right after thawing to limit the risks of protease activation that results from thawing. The choice of the anti-proteases can include aprotinin, that inhibits fibrinolysis and the intrinsic coagulation pathway, or others. Benzamidine has been used at lab scale for its anti-protease activity. Best, Thierry

Hi Augusto, I found this paper looking at gpIIbB3 antagonists:

However, I cant find the mechanism by which Tirofiban (Aggrastat) inhibits gpIIbB3. It is not an RGD containing small molecule like other gpIIbB3 inhibitors.

Do you know the mechanism of how Tirofiban inhibits gpIIbB3?

Plus, I found this paper saying that Eptifibatide and 7E3, but Not Tirofiban, Inhibit αvβ3 Integrins on smooth muscle cells

Eptifibatide is an RGD mimetic peptide and 7E3 is an anti-B3 monoclonal.

I'm bringing this up because I found some papers showing that platelets express avB3

Albeit that avB3 expression on platelets is much lower than gpIIaB3, it could have an effect in your assay.

actually i have isolated few phytochemicals which boost-up platelet count very rapidly and maintain optimum level. so i want to study the mechanism that how they are doing so. there may be 2 possibility. one is regarding growth factors and another is regarding morphological modification. I am not very sure. but i really want to do this study to know the mechanism.

It will not interfere because in ELISA, we are always detecting immune substances.

@ Aaron, thanks for the answe

Dear Mohammad,

Blood can be collected from the retero-orbital plexus (ROP) into heparinised or non-heparinized eppedorf’s tubes as per your requirement, You may see some presentations on this on google too. It is a very easy technique of blood collection. I am sure you will be easily learning the same.

We have used this technique during pharmacokinetic studies in rats as well as efficacy studies.

Nitric oxide was identified as EDRF (endothelium derived relaxation factor) in 1980, using aortic rings. This establishes a link between NO and endothelial damage (impaired vasodilation). A gradient of the NO synthesizing enzyme, endothelial nitric oxide synthase (eNOS), has been detected in the aorta, which correlates with the gradient of lesions (endothelial damage). Endothelial damage and atherosclerosis are, therefore, linked with NO.

I doubt that you can measure by any method the release of zinc from nanoparticles into the blood of mice. The normal zinc concentration in blood is so high that a contribution from nanoparticles could only me measured for unrealistic high NP concentrations