The stability of L-valine in biological samples

L-valine is an essential branched-chain amino acid widely present in biological samples such as blood, urine, and tissue homogenates. The determination of its content is often used in nutritional assessment, diagnosis of metabolic diseases, and analysis of physiological states. However, biological samples have complex matrices (containing enzymes, reactive oxygen species, metal ions, etc.), and L-valine is prone to degradation or transformation, leading to deviations in detection results. Therefore, studying its stability in biological samples and optimizing preservation conditions are crucial to ensuring the accuracy of analysis.

I. Mechanisms of Instability of L-valine in Biological Samples

The degradation of L-valine is mainly related to endogenous factors in biological samples and environmental conditions, with the core mechanisms including:

Enzymatic degradation

Biological samples (such as blood and tissues) contain proteases (e.g., peptidases, aminopeptidases) that can catalyze the hydrolysis or transamination of L-valine. For example, alanine transaminase (ALT) in serum can promote the transamination of L-valine with α-ketoglutarate to generate the corresponding keto acid (α-ketoisovalerate), resulting in a decrease in its content over time. This enzymatic reaction is most active at 37°C (physiological temperature) and can still proceed slowly at room temperature (25°C).

Oxidation reaction

Reactive oxygen species (e.g., hydrogen peroxide, superoxide anions) or transition metal ions (e.g., Fe²⁺, Cu²⁺) present in samples can trigger oxidative degradation of L-valine. The isopropyl structure of its side chain is easily oxidized to form peroxides or hydroxyl derivatives, and the oxidation rate accelerates significantly especially under neutral or alkaline conditions. In addition, light (especially ultraviolet light) promotes the generation of reactive oxygen species, further accelerating the oxidation reaction.

Adsorption and precipitation

L-valine is a polar amino acid (isoelectric point pI≈5.96) and tends to exist in ionic form under acidic or alkaline conditions. It may adsorb on the inner wall of containers (e.g., the hydrophobic surface of plastic centrifuge tubes) or combine with proteins and lipids in samples to form precipitates, leading to a decrease in the content of free L-valine. This adsorption is more obvious in low-concentration samples (such as urine).

II. Impact of Preservation Conditions on L-valine Stability and Optimization Strategies

To address the above instability mechanisms, preservation conditions need to be optimized in terms of temperature, pH value, additives, and container materials to minimize degradation.

Temperature control

Low temperature can significantly inhibit enzyme activity and oxidation reaction rate, which is a core means to extend the stability of L-valine:

Short-term storage (≤24 hours): Refrigeration at 4°C can delay degradation. At this temperature, enzyme activity decreases by approximately 80%, and the oxidation reaction rate drops by more than 50%, which is suitable for temporary storage from sample collection to detection. However, it should be noted that even at 4°C, L-valine in serum will still lose about 5%-10% daily, so analysis should be performed as soon as possible.

Medium-term storage (≤1 month): Freezing at -20°C can further inhibit degradation, and enzymatic reactions are basically stagnant. However, repeated freeze-thaw cycles (more than 3 times) will destroy cell structures due to ice crystals, releasing more endogenous enzymes and metal ions, resulting in a 15%-20% decrease in L-valine content. Therefore, samples should be aliquoted before freezing to avoid repeated freeze-thawing.

Long-term storage (≥3 months): Ultra-low temperature freezing at -80°C can maximize stability. At this temperature, oxidation reactions and enzyme activity are almost completely inhibited, and the annual loss rate of L-valine can be controlled within 5%, which is suitable for sample storage in large-scale cohort studies.

pH adjustment

Adjusting the sample pH to acidic conditions can inhibit enzyme activity and reduce oxidation:

Add a small amount of formic acid (final concentration 0.1%-0.5%) or trichloroacetic acid (TCA, final concentration 5%) to blood or tissue homogenates to lower the pH to 2-3, which can inactivate proteases and inhibit metal ion-catalyzed oxidation reactions. However, it should be noted that strong acids may cause protein denaturation and precipitation, so the supernatant should be saved after centrifugation to remove precipitates before storage.

Urine samples are inherently weakly acidic (pH≈5.5-6.5) and can be directly frozen for storage; if the pH is too high (>7.0), hydrochloric acid can be added dropwise to adjust it to pH 5.0-6.0 to reduce oxidative degradation under alkaline conditions.

Use of additives

Adding stabilizers can specifically inhibit degradation pathways:

Enzyme inhibitors: For example, adding ethylenediaminetetraacetic acid (EDTA, final concentration 1-5 mM) to chelate metal ions and inhibit metalloprotease activity; or adding phenylmethylsulfonyl fluoride (PMSF, final concentration 0.1 mM) to irreversibly inhibit serine proteases, which is suitable for tissue samples.

Antioxidants: Adding vitamin C (final concentration 10-50 μM) or butylated hydroxyanisole (BHA, final concentration 10 μM) can scavenge reactive oxygen species and reduce oxidative degradation, especially suitable for sample processing under light conditions.

Preservatives: For urine and other samples prone to microbial growth, sodium azide (final concentration 0.05%) can be added to inhibit bacterial growth and avoid microbial metabolism consuming L-valine.

Container material and treatment

Choosing containers with low adsorption properties can reduce the loss of L-valine:

Priority is given to polypropylene (PP) or glass containers, avoiding polyethylene (PE) plastics (which have strong adsorption on hydrophobic surfaces). Glass containers need to be silanized in advance to reduce the adsorption of amino acids by polar inner walls.

Samples should be fully mixed before storage to avoid precipitation caused by excessive local concentration; the aliquot volume should be suitable for a single detection (e.g., 0.5-1 mL) to reduce the number of freeze-thaw cycles.

III. Stability Verification Methods

The stability of L-valine under optimized conditions is evaluated through the following methods:

Accelerated test: Place samples at 37°C, 25°C, and 4°C for 0, 2, 6, 24, and 48 hours respectively, determine the L-valine content at different time points, calculate the degradation rate (based on 0 hours), and determine the critical time for short-term storage.

Freeze-thaw cycle test: Repeatedly freeze and thaw samples between -20°C and room temperature 1-5 times, determine the content change after each cycle, and evaluate freeze-thaw stability.

Long-term stability test: Store samples at -80°C for 1, 3, 6, and 12 months, and regularly detect the content to verify the long-term preservation effect.

Generally, it is considered stable if the degradation rate of L-valine under the set preservation conditions is ≤10%.

The stability of L-valine in biological samples is affected by factors such as enzymatic reactions, oxidation, and adsorption. Effective measures such as low temperature (-80°C for long-term storage, 4°C for short-term storage), acidic pH adjustment, addition of enzyme inhibitors and antioxidants, and use of low-adsorption containers can effectively extend its stable period. In practical applications, appropriate conditions should be selected according to the sample type (blood, urine, tissue) and storage duration, and stability verification should be performed to ensure the reliability of detection results, providing accurate data support for clinical diagnosis and metabolic research.