Na-LACTATE & LACTIC ACID-BUFFERED PHYSIOLOGICAL SOLUTION (Lac-SBF)

SBF (synthetic or simulated body fluid) is made popular by Prof. Tadashi Kokubo (J. Non-crystalline Solids, 120, 138-151, 1990). Prof. Kokubo has since published hundreds of profile-raising articles on SBF solutions.

The original Kokubo solution is named as SBF, and this 50 mM Tris-buffered solution has a HCO₃^- ion concentration of only 4.2 mM (in stark contrast to the human blood plasma value of 27 mM), and in this sense, c-SBF solutions are carbonate-deficient solutions.

(Just to mention here, Hanks’ Balanced Salt Solution (HBSS) also has the same 4.2 mM bicarbonate ion concentration.)

Moreover, Kokubo-SBF solutions do also possess an excess of Cl^- ions (148 mM) in them, in comparison to that of the human blood plasma (103 mM). Therefore, Kokubo-SBF solutions are quite far from mimicking the human blood plasma.

SBF solutions of Kokubo were prepared by using K₂HPO₄∙3H₂O. The selection of dipotassium hydrogen phosphate trihydrate, as the phosphate source, was causing the above-mentioned deviations in c-SBF from the electrolyte concentration of blood plasma.

The use of Na₂HPO₄∙2H₂O, as shown below, in preparing the 50 mM Tris-buffered SBF solutions would easily help to increase the HCO₃^- concentration to 27 mM and to reduce the Cl^- concentration to 125 mM. (Reference: Biomaterials, 21, 1429-1438, 2000)

Tris (or Hepes) is not found in human metabolism. Using 50 mM Tris (or 50 mM Hepes) in preparing SBF solutions is not the best way to follow if such solutions are required to mimic the human blood plasma. 50 mM is a large quantity of a non-metabolic organic substance to be present in a solution which claims itself to “simulate the body fluid.”

We have published, for the first time in 2010,

how to prepare a Na-L-lactate and lactic acid-buffered SBF (or call it as a physiological solution) solution, with the abbreviation of Lac-SBF, being able to “perfectly match” the concentrations of all the ions present in human blood plasma.

Is lactate present in blood plasma as well as in extracellular and intracellular fluids? YES! → download the reference to this answer

These solutions (Lac-SBF) are much easier to prepare than the Tris-buffered or Hepes-buffered SBF solutions. (Lac-SBF solutions were inspired by the Lactated Ringer solution (LRS) or Hartmann solutions which contain 28 mM Na-L-lactate.) The Lac-SBF solution we developed contains 22 mM Na-L-lactate. Lactated Ringer injections are already used in hospitals around the world. On the other hand, a solution which contains 50 mM Tris or 50 mM Hepes (such as SBF) cannot be injected intravenously.

Lac-SBF solutions display an enhanced ability of inducing biomimetic calcium phosphate layers on ceramics, metals and polymers in comparison to the conventional solutions. This is because Lac-SBF solutions do not have any cation-chelating (i.e., complexation or binding of Ca²⁺ present in a solution by or to the organic molecules), and non-metabolic, organics such as Tris or Hepes.

Please see the below articles via their web-link for Lac-SBF solutions,

which surely mimic the human blood better than c-SBF solutions:

1.) Acta Biomaterialia, 6, 2282-2288 (2010)

2.) Journal of The American Ceramic Society, 95(7), 2178-2188 (2012)

3.) Journal of Non-Crystalline Solids, 400, 27-32 (2014).

Ion Human Blood Plasma (mM) Lac-SBF (mM)

Na⁺ 142 142

K⁺ 5 5

Mg²⁺ 1.5 1.5

Ca²⁺ 2.5 2.5

HPO₄^2- 1 1

HCO₃^- 27 27

Cl^- 103 103

SO₄^2- 0.5 0.5

Buffering agent: Na-lactate/lactic acid pair

Protected by a European patent:

“Calcium Phosphate Coating of Ti6Al4V by a Na-lactate and Lactic Acid-buffered Body Fluid Solution” ® Patent Document

European Patent No: 2,296,718 B1 Patent Granted on: March 27, 2013

PCT Patent; Appl. No: WO 2009/145741 A2 (December 3, 2009)

Inventors: A. Pasinli, M. Yuksel, H. Havitcioglu, A. C. Tas, R. S. Aksoy, E. Celik, H. Yildiz, M. Toparli, A. Canatan, S. Sener

Proprietors: A. Pasinli, M. Yuksel, H. Havitcioglu, A. C. Tas, R. S. Aksoy, E. Celik, H. Yildiz, M. Toparli, A. Canatan, S. Sener

Preparation of Lac-SBF solution

Notes: (1) only use high purity deionized water (free of dissolved carbon dioxide, it is advised to boil the water just before using it, then cool it down to RT and store it in a sealed environment free of atmospheric carbon dioxide) and use chemicals of the highest possible purity your research budget can afford.

1000 mL-capacity glass beaker (use a hot-plate/magnetic stirrer); use a Teflon-coated magnetic stirrer

+ 997 mL deionized water (18.2 MΩ.cm)

+ 5.2599 g NaCl (Sigma, S9888)

stir vigorously at RT for 3 min

+ 2.2682 g NaHCO₃ (Fisher, S233)

stir for 2 min

+ 0.3728 g KCl (Sigma, P3911)

stir for 2 min

+ 0.3049 g MgCl₂×6H₂O (Acros, 19753)

stir for 10 min

+ 0.071 g Na₂SO₄ (Acros, 21875)

stir for 3 min

+ 0.3675 g CaCl₂×2H₂O (EMD, CX0130)

stir 3 min

+ 0.1419 g Na₂HPO₄ (Fisher, S374)

stir for 3 min

+ 2.4653 g NaCH₃CH(OH)COO (Sodium L-lactate, Sigma L7022) (= 22 mM)

+ stir for 3 min

+ add approximately 1.5 to 2 mL of 1 M lactic acid solution, Fluka, 35202 (add intermittently, in 0.25 mL portions / aliquots)

to adjust the solution pH at 7.4 @ 37°C

Note: Increase the temperature to 36-37°C only after you started adding lactic acid solution (otherwise you would observe irreversible turbidity in solution)

+ stir for 15 min while monitoring the pH stability

+ measure the total volume of the transparent solution, if it is not exactly equal to 1000 mL, you shall add deionized water to complete the volume to 1000 mL

Always keep your Lac-SBF solution in a clean glass media bottle (of 1 L-capacity), tightly capped, in a refrigerator at +4° to 5°C (i.e., a regular refrigerator);

write the date of preparation on your glass bottle; do not use any SBF solution older than 30 to 35 days.

When you repeat this preparation procedure at least two times, you will see that it is actually very easy!

+++ Perform the SBF experiments in clean, glass media bottles;

do NOT use plastic bottles, if you do so you may grow bacteria during the period of your experiment, because plastic surfaces are rough, they may have numerous microscopic crevices or protrusions and these serve as nice hosts to bacteria; glass surfaces are smooth on the other hand; SBF solutions kept at 37°C typically forms a pleasant habitat for numerous bacteria to grow!

NaN₃ additions (approx 10 mg per liter) to the solutions may be a good alternative to completely prevent bacteria growth.

For possible different uses of Lac-SBF solutions (in biomimetic coating or testing of metals, ceramics and polymers), you may visit the following weblinks:

http://www.cuneyttas.com/hasbf37-citat.htm

http://www.cuneyttas.com/haurea-citat.htm

http://www.cuneyttas.com/SBF-compare-citat.htm

http://www.cuneyttas.com/ha-enzyme-citat.htm

SBF solutions CANNOT test the bioactivity of synthetic biomaterials, bioglasses, biopolymers, biometals, bioceramics, etc.,

in strong opposition to what Dr. Kokubo promoted or advocated in his articles.

It should be quite easy to comprehend that a solution which cannot keep cells (in it) alive shall not be used for the testing of bioactivity of synthetic biomaterials.

The word “bioactivity” is the abbreviation for the phrase “biological activity.” Bioactivity of synthetic materials could only be evaluated by using in vitro cell culture tests or, better, by using in vivo tests. The medium for bioactivity testing must contain biological entities, SBF solutions don’t.

For learning more about what SBF solutions really are or are not;

Contact: Prof. A. Cuneyt Tas, PhD c_tas@hotmail.com www.cuneyttas.com