Synthesis and Application of Calcium beta-hydroxy-beta-methylbutyrate

Physicochemical properties

Calcium beta-hydroxy-beta-methylbutyrate is a white powdery solid, melting point >215°C, slightly soluble in water.

Fig. 1 The structure of Calcium beta-hydroxy-beta-methylbutyrate.

Synthetic routes

Fig. 2 The synthetic step 1 of Calcium beta-hydroxy-beta-methylbutyrate.

In the there-necked flask of 2L, add 60.98 g (1.05 mol) acetone, 122.55 g (1 mol) ethyl chloroacetate and 610 g ethanol, be cooled to 3 DEG C, slowly drip 30% sodium ethoxide ethanol solution 249.52 g (1.1mol), the reaction was incubated for 2 hours after dropping, and the detection reaction was completed [1].

Fig. 3 The synthetic step 2 of Calcium beta-hydroxy-beta-methylbutyrate.

Prepare lye, 48 g (1.2 mol) sodium hydroxide is added in 432 g water, dissolve clear, the prepared alkali is dropped into the reaction solution after the condensation finishes, and it is warming up to 50 DEG C after dripping, insulation reaction is 3 hours , the reaction is completed, be cooled to 10 DEG C, adjust pH to acidity with concentrated hydrochloric acid, pH=3, add 200 g of ethyl acetate and extract once, layering, the water phase is extracted once with 200 g of ethyl acetate again, and the organic phases are combined, Washed twice with water, once with saturated brine, and concentrated to dryness to obtain 89.5 g (77%) of an off-white solid, the yield determined by HPLC was 99.3% [1].

Fig. 4 The synthetic step 3 of Calcium beta-hydroxy-beta-methylbutyrate.

Add 700 g methanol, 5 g Pt/C, 89.5 g hydrolysis product 3,3-dimethyloxirane-2-carboxylic acid to the hydrogenation kettle, close the lid, replace with nitrogen three times, pressurize the hydrogen to 2 Mpa, Heat up to 60 ° C, replenish the hydrogen pressure at intervals, keep at 2 Mpa, until no longer absorb hydrogen, the reaction is terminated after 8 h, filter, the filtrate is concentrated to dryness, and the methanol solution state of β-hydroxy-βmethylbutyric acid was maintained [1].

Fig. 5 The synthetic step 4 of Calcium beta-hydroxy-beta-methylbutyrate.

In the methanol solution of β-hydroxy-β-methyl butyric acid, add water 700 g, calcium carbonate 38.57 g, calcium chloride 0.90 g, be warming up to 50 ℃, insulation reaction 6 hours, after the reaction finishes, be heated to 80 ℃ ℃ of dissolved clear, filtered while hot, the filtrate was cooled to 5 ℃, separated out a large amount of white crystals, filtered, and oven-dried to obtain 100.52 g (95%) white crystalline HMB-Ca solid. The total yield was 73.2% [1].

Application

As a phosphate binder in uremia

The binding capacity of calcium beta-hydroxy-beta-methylbutyrate (calcium HMB), compared to other binders, was investigated in an in vitro study. Fifty milli-equivalents of either calcium HMB, calcium acetate, calcium carbonate, aluminum hydroxide gel or non-gel aluminum hydroxide was added to a phosphate solution, titrated (HCl or NaOH), shaken and centrifuged to four different pH levels at 37 degrees C (simulating the gastrointestinal milieu). The difference in phosphate concentration between that of the initial and that of the supernatant represented from the bound phosphate in the precipitate. After 4 h at a pH of 6 (representing the intestinal condition after a meal), the binding percentage was: calcium acetate = 95.6%, calcium HMB = 92.6%, calcium carbonate = 46.4%, aluminum hydroxide gel = 33.4% and non-gel aluminum hydroxide = 17.8%. There was no significant difference (p > 0.05) between calcium HMB and calcium acetate. These results suggest that calcium HMB is an efficient phosphate binder in vitro, which may predict its effective role in vivo [2].

As dietary supplementation

Intra-uterine growth restriction (IUGR) impairs postnatal growth and skeletal muscle development in neonatal infants. This study evaluated whether dietary beta-hydroxy-beta-methylbutyrate Ca (HMB-Ca) supplementation during the early postnatal period could improve muscle growth in IUGR neonates using piglets as a model. A total of twelve pairs of IUGR and normal-birth-weight (NBW) male piglets with average initial weights (1.85 (SEM 0.36) and 2.51 (SEM 0.39) kg, respectively) were randomly allotted to groups that received milk-based diets (CON) or milk-based diets supplemented with 800 mg/kg HMB-Ca (HMB) during days 7-28 after birth. Blood and longissimus dorsi (LD) samples were collected and analysed for plasma amino acid content, fibre morphology and the expression of genes related to muscle development. The results indicate that, regardless of diet, IUGR piglets had a significantly decreased average daily weight gain (ADG) compared with that of NBW piglets (P < 0.05). However, IUGR piglets fed HMB-Ca had a net weight and ADG similar to that of NBW piglets fed the CON diet. Irrespective of body weight (BW), HMB-Ca supplementation markedly increased the type II fibre cross-sectional area and the mRNA expression of mammalian target of rapamycin (mTOR), insulin-like growth factor-1 and myosin heavy-chain isoform IIb in the LD of piglets (P < 0.05). Moreover, there was a significant interaction between the effects of BW and HMB on mTOR expression in the LD (P< 0.05). In conclusion, HMB-Ca supplementation during the early postnatal period could improve skeletal muscle growth and maturity by accelerating fast-twitch glycolytic fibre development in piglets [3].

Subchronic toxicity study

Calcium beta-hydroxy-beta-methylbutyrate-monohydrate (CaHMB) is a dietary supplement used as an ergogenic aid and in functional and medical foods. A new delivery form has been developed, beta-hydroxy-beta methylbutyric free acid (HMBFA), which has improved bioavailability. While the safety of CaHMB is well documented, there are few published studies demonstrating the safety of HMBFA. Because HMBFA results in greater serum levels of beta-hydroxy-beta-methylbutyrate (HMB) and greater clearance rates, a 91-day subchronic toxicity study was conducted in male and female Sprague-Dawley Crl:CD rats assigned to HMBFA treatments at either 0%, 0.8%, 1.6%, or 4% of the diet by weight. No deaths or untoward clinical observations, and no negative clinical chemistry or hematology were attributed to the administration of HMBFA. Gross pathology and histopathology results showed no tissue abnormalities due to HMBFA and all measures were within a normal physiological range for the animals or were expected in the population studied. In conclusion, the no-observed-adverse-event-level (NOAEL) for HMBFA was the highest level administered, 4% of the diet, which corresponded to an intake of 2.48 and 2.83 g/kg BW d^-1 in the males and females, respectively. The equivalent human dosage using body surface area conversion would be 402 and 459 mg/kg BW d^-1 for men and women, respectively [4].

References

[1] Liu Y. Preparation of calcium beta-hydroxy-beta