Comparison of availability and plasma clearance rates of-hydroxy--methylbutyrate delivery in the free acid and calciumsalt forms

John C. Fuller Jr1, Rick L. Sharp2, Hector F. Angus2, Paul Y. Khoo2 and John A. Rathmacher1,3*1Metabolic Technologies Inc., Iowa State University Research Park, Ames, IA 50010, USA2Department of Kinesiology, Iowa State University, Ames, IA 50010, USA3Department of Animal Science, Iowa State University, Ames, IA 50010, USA

(Submitted 22 February 2015 Final revision received 14 July 2015 Accepted 17 July 2015 First published online 16 September 2015)

Abstract-Hydroxy--methylbutyrate (HMB), a leucine metabolite, has long been supplemented as a Ca salt (Ca-HMB) to increase strength and performancegains with exercise and to reduce recovery time. Recently, the free acid form of HMB (HMB-FA) has become commercially available in capsule form(gelcap). The current study was conducted to compare the bioavailability of HMB using the two commercially available capsule forms of HMB-FA andCa-HMB. We also compared the pharmacokinetics of each form when administered mixed in water. Ten human subjects (five male and five female)were studied in a randomised crossover design. There was no significant sex by treatment interaction for any of the pharmacokinetic parametersmeasured. HMB-FA administered in capsules was more efficient than Ca-HMB capsule at HMB delivery with a 37% increase in plasma clearance rate(748 (SEM 40) v. 545 (SEM 32)ml/min, P

in offspring of sows fed HMB for the final 2 weeks of preg-nancy; increased bone density and plasma amino acid con-centrations in pigs with fundectomy-induced osteopaenia(23);and improved somatotrophic axis and accelerated bone meta-bolism in lambs fed HMB for the first 21 d after birth(24). Theimprovement in somatotrophic axis function was furtherdemonstrated in rats when chronic HMB administration wasshown to stimulate the GH/IGF-1 axis(25). Recently, HMB sup-plementation was shown to enhance the exercise-stimulatedincrease in GH and IGF-1 response in humans, similar to whatthe previous studies in animals had demonstrated(26).The pharmacokinetics of orally ingested Ca-HMB in

capsules were originally described by Vukovich et al.(14)

and showed a time to peak plasma level of HMB of 2 h aftera 1 g dosage. Recently, Fuller et al.(15) demonstrated thatHMB-FA gel form increased plasma HMB concentrationsin a shorter period of time (3040 min) with a greaterblood clearance rate of HMB compared with the Ca-HMBcapsule. A recent 12-week exercise study by Wilson et al.(27)

used these favourable advantages to test the efficacy ofHMB-FA administered 30min before exercise in a periodisedtraining programme and showed increased strength, lean bodymass gain and muscle power in a highly trained athletepopulation(27), demonstrating the importance of dosage timingand peak levels of HMB for maximisation of exercisebenefits. The current human study was thus designed tocompare the pharmacokinetics of the current commerciallyavailable delivery form of Ca-HMB in a capsule and comparedthis with the newly designed delivery form of HMB-FA in agelcap. Our results confirmed the previous findings of Fulleret al.(15) and showed that HMB-FA had superior bioavailabilityin humans as assessed by blood levels, and improvedclearance.

Methods

Human subjects

We performed a randomised crossover study consisting offive men and five women (aged 2132 years). The study wasconducted according to the guidelines laid down in theDeclaration of Helsinki, and all procedures involving humansubjects were approved by the Iowa State UniversityInstitutional Review Board. Written informed consent wasobtained from all subjects before participation. The study waslisted on ClinicalTrials.gov (NCT01914952, https://clinicaltrials.gov). The subjects were not currently taking amino acid, proteinor HMB supplements.

Treatments

The treatments were given to each subject in random order withat least a 48 h washout period between treatments. The treat-ments were as follows: (1) Ca-HMB in a commercial gelatincapsule (Optimum Nutrition); (2) HMB-FA capsule (gelcap)(Capsugel); (3) HMB-FA as a gel as previously described(15);(4) Ca-HMB mixed in water or (5) HMB-FA mixed in water.All treatments delivered 08 g of HMB orally, confirmed by

HPLC analysis, and were given with 355 ml of water. Althoughthe pH of the free acid itself is

Statistical analysis and calculations

The AUC was calculated for each subject using the trapezoidalmethod that sums the area above the baseline(29). The followingequations were used in the calculation of the half-life ofplasma HMB:

Kel ln Cpeak ln Ctrough

Tinterval

; (1)

t1=2

0693Kel

: (2)

The peak plasma concentration was the highest HMBconcentration at a measured time point for each subject andwas used for Cpeak. Trough concentrations, Ctrough, were theconcentrations measured at 12 h, which were not significantlydifferent from baseline concentrations. The Tpeak was the timeat which Cpeak was measured for each subject and Tinterval was12 h (time at Ctrough) minus Tpeak. The extracellular fluidcompartment was assumed to be 20 % of body weight. Vd wascalculated using equation (3) below(30). The plasma clearanceof HMB was calculated by multiplying the extracellular fluidcompartment, Vd, by the elimination constant, Kel, as shown inequation (4)(31).

Vd Bodyweight 020 ; (3)Clearance Vd Kel : (4)

The data for comparable treatments were analysed using acrossover design with the general linear models (GLM) proce-dure in SAS(32). A priori power analysis (G*Power, version 3.1.7)was completed based on our previously published pharmaco-kinetic study(15). Assuming a similar degree of variability, apower analysis indicated that ten subjects were required todetect a P value of 005 at a power of 090 in a crossoverdesigned study. A repeated-measures polynomial model wasused for the timed sampling of plasma HMB, and the modelincluded subject, treatment order, treatment main effects andtime by treatment interaction where appropriate. For otherparameters, Proc GLM was used with subject, treatment orderand treatment main effects included in the model, and theP values are reported for the treatment main effect. Althoughthe study was neither designed nor powered to detect sexdifferences, sex by treatment interaction was determined byGLM. In addition, the main effects of Ca-HMB and HMB-FAwhen given in water and by capsule were determined usingGLM and least square means, and these data are shown inonline Supplementary Table S3. Statistical significance wasdetermined for P< 005.

Results

Subject characteristics

Subject characteristics of the five male and five female subjectsare shown Table 1. The average age of the subjects was251 (SEM 10) years and the average weight and BMI were691 (SEM 31) kg and 230 (SEM 08) kg, respectively. The malesubjects had greater body weights (P< 005) and BMI (P< 005)than the female subjects; however, there were no significant sex

by treatment interactions for any of the pharmacokinetic para-meters measured, even though all subjects were administeredthe same HMB dose.

Free acid form of -hydroxy--methylbutyrate gel kinetics

The HMB-FA gel treatment in the present study showedsimilar pharmacokinetics as we had previously shown(15).HMB-FA in a gel had no comparator in the current study. TheCpeak was 2617 (SEM 150) mol/l at 355 (SEM 40)min post-administration. The half-life of HMB delivered in the gel form was2.24 (SEM 010) h and the AUC was 541 (SEM 31) mol/l1440min. The clearance from plasma was 717 (SEM 36)ml/min, and246 (SEM 44) % of the dosage was excreted in urine over the24 h study.

Acute -hydroxy--methylbutyrate kinetics for capsule andwater administration

The 3 h plasma HMB pharmacokinetics for subjects adminis-tered the HMB-FA and Ca-HMB capsule treatments are shownin Fig. 1(a). Compared with Ca-HMB capsules, HMB-FAadministration in capsules resulted in significantly greaterplasma levels at all time points from 15 to 120min afteradministration (P< 0001 at 15, 25, 35, 45, 60 and 90 min andP< 005 at 120min). The 180 min AUC was 75 % greater(P< 0001) for the HMB-FA in capsule form. In addition,administration of HMB-FA in capsule form resulted in 76 %greater peak levels (P< 0006) in one-third the time (P< 0009).The dissolution time of the capsules could not account for theseresults; it was 13 min for the HMB-FA capsules and 5min for theCa-HMB capsules, thus favouring the release of Ca-HMB fromthe capsules.

Fig. 1(b) shows the 3 h plasma HMB pharmacokineticsfor the administration of HMB-FA and Ca-HMB when mixed inwater. Administration of HMB-FA in water resulted in greaterplasma HMB levels at 15, 25, 35 and 45min (P< 005, 0001,0004, and P< 005, respectively). The inset shows that HMB-FAadministered in water had a significantly greater AUC during thefirst hour of administration (P< 003). Compared with Ca-HMBadministered in water, the Cpeak tended to be greater for

Table 1. Subject descriptors(Mean values with their standard errors)

Mean SEM

Sex (n)Male/female 5/5

Age (years) 251 10Body weight (kg)

All 691 31Female 643 30Male 738 49

BMI (kg/m2)All 230 08Female 219 08Male 242 13

-Hydroxy--methylbutyric acid clearance 1405

HMB-FA in water, whereas the Tpeak level was not differentbetween the HMB-FA and Ca-HMB treatments.

24 h -hydroxy--methylbutyrate kinetics for capsule andwater administration

Fig. 2(a) shows the 24 h plasma HMB levels in subjects giveneither Ca-HMB or HMB-FA in capsule form. As shown in theinset, the HMB-FA capsule administration resulted in a 37 %increased clearance of HMB from plasma (P< 00001), resultingin a 29 % decrease in half-life (P< 00003). The 24 h AUC forHMB and for urinary excretion rates were the same for bothforms.Similarly, Fig. 2(b) shows the 24 h plasma HMB levels in

subjects given either Ca-HMB or HMB-FA mixed in water. Wenoted significant differences in plasma HMB levels during thefirst hour, with no further observed differences during the 24 hmeasurement period. As seen in the inset, there were also nodifferences observed for plasma HMB half-life, clearance ratesor AUC over the 24 h measurement period. In addition, 24 hurinary excretion as a percentage of the initial dose was thesame for both treatments.

Blood chemistry and haematology

There were no untoward effects of the treatments on bloodchemistries or haematology and the data are presented inonline Supplementary Tables S1 and S2.

Discussion

In this study, we tested the human pharmacokinetics of the twocommercially available forms of HMB, used for the promotionof muscular strength and mass. Our results demonstrate that,irrespective of the capsule delivery form used, HMB-FA is morereadily available and has a higher clearance rate than Ca-HMB(Fig. 2(a)), indicative of higher efficiency and faster utilisation.All human studies to date have consistently demonstrated thesuperiority of the HMB-FA form to reach a higher HMB plasmapeak in a much shorter time than the Ca-HMB form, with similarurinary excretion of HMB(14,15). When Ca-HMB was adminis-tered, peak plasma HMB levels were 115(14), 131(15) and131 mol/l(15), which were comparable to the 154 mol/l deter-mined in the current study. Previous studies with HMB-FA foundthe peak plasma HMB levels to be 259(15) and 239 mol/l(15)

300

250

200

150

100

50

0

300

250

200

150

100

50

00 1 2 3

Time (h)

0 1 2 3

Time (h)

Pla

sma

HM

B (

mol

/l)P

lasm

a H

MB

(m

ol/l)

Cpeak (mol/l)

Tpeak (min)

AUC (mol/l 180 min)

Cpeak (mol/l)

Tpeak (min)

AUC (mol/l 60 min)

HMB-FA water Ca-HMB water

HMB-FA capsule Ca-HMB capsule

Mean SEM Mean SEM P

Mean SEM Mean SEM P

270.2

44.5

28.3

17.8

5.5

1.4

153.9

133.5

16.2

17.9

29.2

1.8

0.006

0.009

0.001

274.4

36.0

10.9

24.6

2.8

0.9

247.4

42.5

9.1

15.2

4.1

0.9

0.11

0.23

0.03

***

***

***

****** *** ***

*

**

*

*

(b)

(a)

Fig. 1. (a) The 3 h plasma levels of -hydroxy--methylbutyrate (HMB) after administration of either free acid form of HMB (HMB-FA) gelcaps (--) or Ca salt of HMB(Ca-HMB) capsule (- --). (b) The 3 h plasma levels of HMB after administration of either HMB-FA mixed in water (--) or Ca-HMB mixed in water (- --). All treatmentscontained 08 g of HMB. Values are means with their standard error of means for five men and five women. Significance between the treatments is indicated as follows:* P< 005; ** P< 0004; and *** P< 0001. The inset table shows the peak concentration reached (Cpeak), time to reach peak concentration (Tpeak) and AUC.The P value indicated is for differences between the treatment means.

1406 J. C. Fuller et al.

for the gel form, which was equivalent to the levels, 262 and270 mol/l for the gel and gelcap, respectively, in the currentstudy. Similarly, the times to peak HMB level previously deter-mined for Ca-HMB administration were 120(14), 122(15) and 135min(15), which are comparable to the 134min in the current study.When HMB-FA was administered in a gel, the times to peak levelswere 33(15) and 42min(15), and in the current study the times topeak level for HMB-FA as a gel and gelcap were 36 and 45min,respectively. Urinary losses were similar across all studies anddosage forms, and including our present study were between 14and 25% of the dosage(14,15). The findings have several practicalimplications for the average athlete. Although both forms of HMBare equally effective on muscle function and are equally retainedby the body over a 24 h period, the use of HMB-FA allows formore predictive timing of intake of HMB in relation to the plannedexercise, thus achieving better outcomes, as was recently con-firmed by Wilson et al.(27).Our human data differ considerably from the recently pub-

lished data in rodents(33), which reported that Ca-HMB hadbetter availability as measured by peak blood levels whencompared with HMB-FA. In that study, the investigators com-pared Ca-HMB with HMB-FA administration both orally in aliquid suspension and by intravenous injection,(33) and showed

that similar amounts of HMB given as either Ca-HMB orHMB-FA resulted in the Ca-HMB having greater peak bloodHMB concentrations. There are several concerns with com-parison with the rodent study; the most notable is the concernrelated to species differences. For example, the half-life of HMBin blood after Ca-HMB dosing is reported as approximately 1, 2and 3 h for the rat, pig and sheep, respectively(3335), comparedwith humans, with a half-life of 24 to 32 h for a 1 g dosage ofCa-HMB(14,15). Other concerns relate to the experimentaldesign, and the analytical methods used, as well as methodo-logical differences related to the accuracy of HMB intake. Forexample, in the rodent study, there is no mention of themethodology for the administration of the oral forms in themethods section of the paper; hence, the method and accuracyof the amount given is in question. In addition, both forms ofHMB were dissolved in solutions containing carboxy-methylcellulose and Tween 80 with the pH adjusted to 45. Thistype of solution is likely to affect the bioavailability of theCa-HMB by increasing its ionisation, dissociation and absorp-tion, thus leading to overestimates of the true pharmacokineticparameters measured. Last, but not least, there is concernrelated to the accuracy of the measured plasma HMB levels.The authors used warfarin, which is structurally different fromthe natural HMB form, as an internal standard for the LC-MS/MSmeasurements, in contrast to our use of a similar HMBcompound, namely d6-HMB, as an internal standard

(36).To reconcile any potential differences attributed to the

delivery forms, we carried out a head-to-head comparison of anoral dose of HMB-FA and Ca-HMB given mixed in water insteadof in capsule form. The data from the current study confirm ourprevious findings(15) and demonstrate that even when deliveredmixed in water HMB-FA is still superior to Ca-HMB in deliveringHMB, albeit to lesser degree than when both forms are deliv-ered in capsules. This was quite evident within the first hour ofthe absorptive period (Fig. 1(b)). In addition, we noted nodifference in 24 h urinary excretion of HMB (Fig. 2(b)),suggesting an earlier and greater tissue availability of HMBwhen given in the free acid form.

Our study has few limitations, albeit minor in nature. The firstlimitation relates to the few number of subjects studied (n 10);this limitation, however, is countered by the use of the subjectsas their own control in a crossover design. Another theoreticallimitation may relate to our calculation of the amount of HMBavailable for tissue utilisation, as the difference between theamount consumed minus the amount excreted in the urine. Thisassumption would be accurate with additional measurement ofHMB oxidation rates, which were not examined in this study.Wilkinson et al.(11) recently demonstrated that after an oraldosage of HMB-FA intramuscular HMB concentrationsincreased almost 14-fold from baseline at 150 min after thedose, suggestive of enhanced utilisation of HMB by humanmuscle following intake of HMB-FA.

In conclusion, the use of the commercially available forms ofHMB as either the HMB-FA or Ca-HMB resulted in earlier andincreased plasma concentrations, with increased clearancerates, when the HMB was given as HMB-FA. This finding can beused favourably by athletes to gauge their intake of HMB beforean exercise bout, as recently observed by Wilson et al.(27).

300

250

200

150

100

50

0

300

250

200

150

100

50

0

0 6 12 18 24

0 6 12 18 24

Time (h)

Time (h)

Pla

sma

HM

B (

mol

/l)P

lasm

a H

MB

(m

ol/l)

HMB-FA water Ca-HMB water

Mean SEM Mean SEM P

HMB-FA capsule Ca-HMB capsule

Mean SEM Mean SEM P

2.23

72.0

52.3

20.0

0.10

3.2

3.4

3.3

2.30

70.7

54.6

21.6

0.10

4.4

3.3

3.0

0.47

0.69

0.65

0.77

Half-life (h)

Clearance(ml/min)

AUC(mol/l 1440 min)

24h Urine HMB(% initial dose)

Half-life (h)

Clearance(ml/min)

AUC(mol/l 1440 min)

24h urine HMB(% initial dose)

2.15

74.8

53.8

21.0

0.07

4.0

2.7

3.0

3.02

54.5

48.8

20.3

0.24

3.2

3.7

3.2

0.0003

0.0001

0.16

0.65

(b)

(a)

Fig. 2. (a) The 24 h plasma levels of -hydroxy--methylbutyrate (HMB) afteradministration of either free acid form of HMB (HMB-FA) gelcaps (--) or Casalt of HMB (Ca-HMB) capsule (- --). (b) The 24 h plasma levels of HMB afteradministration of either HMB-FA mixed in water (--) or Ca-HMB mixed in water(- --). All treatments contained 08 g of HMB. See Fig. 1 for significantdifferences in plasma HMB concentrations in the initial 3 h. Values are meanswith their standard error of means for five men and five women. The inset tableshows the half-life, clearance, 24 h AUC and 24 h urinary excretion of HMB.The P value indicated is for differences between the treatment means.

-Hydroxy--methylbutyric acid clearance 1407

Acknowledgements

The present study was funded by Metabolic Technologies, Inc.,Ames, IA.The authors contributions were as follows: J. A. R. designed

the research. J. A. R., H. F. A., P. Y. K. and R. L. S. conductedthe research including sample analysis. J. C. F. Jr analysed thedata and wrote the paper. J. A. R. had primary responsibility forfinal content. All authors read and approved the final manuscript.J. C. F. Jr and J. A. R. are employed by Metabolic Technolo-

gies Inc., Ames, IA. R. L. S. received funds from MetabolicTechnologies to conduct the study. H. F. A. and P. Y. K. had noconflicts of interest to declare.

Supplementary material

For supplementary material/s referred to in this article, pleasevisit http://dx.doi.org/doi:10.1017/S0007114515003050

References

1. Nissen S, Sharp R, Ray M, et al. (1996) Effect of the leucinemetabolite -hydroxy -methylbutyrate on muscle metabo-lism during resistance-exercise training. J Appl Physiol 81,20952104.

2. Panton LB, Rathmacher JA, Baier S, et al. (2000) Nutritionalsupplementation of the leucine metabolite -hydroxy-methylbutyrate (HMB) during resistance training. Nutr 16,734739.

3. Knitter AE, Panton L, Rathmacher JA, et al. (2000) Effects of-hydroxy--methylbutyrate on muscle damage following aprolonged run. J Appl Physiol 89, 13401344.

4. Wilson GJ, Wilson JM & Manninen AH (2008) Effectsof beta-hydroxy-beta-methylbutyrate (HMB) on exerciseperformance and body composition across varying levels ofage, sex, and training experience: a review. Nutr Metab(Lond) 5, 1.

5. Wilson JM, Fitschen PJ, Campbell B, et al. (2013) InternationalSociety of Sports Nutrition Position Stand: beta-hydroxy-beta-methylbutyrate (HMB). J Int Soc Sports Nutr 10, 6.

6. Eley HL, Russell ST & Tisdale MJ (2008) Mechanism ofattenuation of muscle protein degradation induced by tumornecrosis factor alpha and angiotensin II by beta-hydroxy-beta-methylbutyrate. Am J Physiol Endocrinol Metab 295,E1417E1426.

7. Smith HJ, Wyke SM & Tisdale MJ (2004) Mechanism of theattenuation of proteolysis-inducing factor stimulated proteindegradation in muscle by beta-hydroxy-beta-methylbutyrate.Cancer Res 64, 87318735.

8. Smith HJ, Mukerji P & Tisdale MJ (2005) Attenuationof proteasome-induced proteolysis in skeletal muscle by-hydroxy--methylbutyrate in cancer-induced muscle loss.Cancer Res 65, 277283.

9. Eley HL, Russell ST, Baxter JH, et al. (2007) Signaling path-ways initiated by -hydroxy--methylbutyrate to attenuate thedepression of protein synthesis in skeletal muscle in responseto cachectic stimuli. Am J Physiol Endocrinol Metab 293,E923E931.

10. Eley HL, Russell ST & Tisdale MJ (2008) Attenuation ofdepression of muscle protein synthesis induced by lipopoly-saccharide, tumor necrosis factor and angiotensin II by-hydroxy--methylbutyrate. Am J Physiol Endocrinol Metab295, E1409E1416.

11. Wilkinson DJ, Hossain T, Hill DS, et al. (2013) Effects ofleucine and its metabolite beta-hydroxy-beta-methylbutyrateon human skeletal muscle protein metabolism. J Physiol 591,29112923.

12. Wilson JM, Kim JS, Lee SR, et al. (2009) Acute and timingeffects of beta-hydroxy-beta-methylbutyrate (HMB) on indir-ect markers of skeletal muscle damage. Nutr Metab (Lond)6, 6.

13. Wilson JM, Lowery RP, Joy JM, et al. (2013) ) -Hydroxy--methylbutyrate free acid reduces markers of exercise-induced muscle damage and improves recovery inresistance-trained men. Br J Nutr 110, 538544.

14. Vukovich MD, Slater G, Macchi MB, et al. (2001) ) -Hydroxy--methylbutyrate (HMB) kinetics and the influence of glucoseingestion in humans. J Nutr Biochem 12, 631639.

15. Fuller JC Jr, Sharp RL, Angus HF, et al. (2011) Free acid gelform of -hydroxy--methylbutyrate (HMB) improves HMBclearance from plasma in humans compared to the calciumHMB salt. Br J Nutr 105, 367372.

16. Dreyer HC, Fujita S, Cadenas JG, et al. (2006) Resistanceexercise increases AMPK activity and reduces 4E-BP1 phos-phorylation and protein synthesis in human skeletal muscle.J Physiol 576, 613624.

17. Phillips SM, Tipton KD, Aarsland A, et al. (1997) Mixed muscleprotein synthesis and breakdown after resistance exercisein humans. Am J Physiol 273, E99E107.

18. Kerksick C, Harvey T, Stout J, et al. (2008) InternationalSociety of Sports Nutrition Position Stand: nutrient timing. J IntSoc Sports Nutr 5, 17.

19. American College of Sports Medicine, American DieteticAssociation & Dieticians of Canada (2000) Joint positionstatement: nutrition and athletic performance. Med Sci SportsExerc 32, 21302145.

20. Coyle EF, Coggan AR, Hemmert MK, et al. (1985) Substrateusage during prolonged exercise following a preexercisemeal. J Appl Physiol (1985) 59, 429433.

21. Tatara MR (2009) Effect of beta-hydroxy-beta-methylbutyrate(HMB) administration on volumetric bone mineral density,and morphometric and mechanical properties of tibia in maleturkeys. J Anim Physiol Anim Nutr (Berl) 93, 669677.

22. Tatara MR, Krupski W, Tymczyna B, et al. (2012) Effects ofcombined maternal administration with alpha-ketoglutarate(AKG) and beta-hydroxy-beta-methylbutyrate (HMB) on pre-natal programming of skeletal properties in the offspring. NutrMetab (Lond) 9, 39.

23. Tatara MR, Sliwa E, Krupski W, et al. (2008) 3-Hydroxy-3-methylbutyrate administration diminishes fundectomy-induced osteopenia of the lumbar spine in pigs. Nutr 24,753760.

24. Tatara MR (2008) Neonatal programming of skeletal develop-ment in sheep is mediated by somatotrophic axis function.Exp Physiol 93, 763772.

25. Gerlinger-Romero F, Guimaraes-Ferreira L, Giannocco G,et al. (2011) Chronic supplementation of beta-hydroxy-betamethylbutyrate (HM) increases the activity of the GH/IGF-Iaxis and induces hyperinsulinemia in rats. Growth Horm IGFRes 21, 5762.

26. Townsend JR, Hoffman JR, Gonzalez AM, et al. (2015) Effectsof beta-hydroxy-beta-methylbutyrate free acid ingestion andresistance exercise on the acute endocrine response. Int JEndocrinol 2015, article ID 856708.

27. Wilson JM, Lowery RP, Joy JM, et al. (2014) The effects of12 weeks of beta-hydroxy-beta-methylbutyrate free acidsupplementation on muscle mass, strength, and power inresistance trained individuals: a randomized, double-blind,placebo-controlled study. Eur J Appl Physiol 114, 12171227.

1408 J. C. Fuller et al.

http://dx.doi.org/doi:10.1017/S0007114515003050

28. Nissen S, Van Koevering M & Webb D (1990) Analysisof -hydroxy--methyl butyrate in plasma by gaschromatography and mass spectrometry. Anal Biochem 188,1719.

29. Urso R, Blardi P & Giorgi G (2002) A short introductionto pharmacokinetics. Eur Rev Med Pharmacol Sci 6,3344.

30. Guyton AC & Hall JE (2006) The body fluid compartments:extracellular and intracellular fluids: interstitial fluid andedema. In Textbook of Medical Physiology, pp. 291306.Philadelphia, PA: W B Saunders Company.

31. Thalhammer F, Schenk P, Burgmann H, et al. (1998) Single-dose pharmacokinetics of meropenem during continuousvenovenous hemofiltration. Antimicrob Agents Chemother42, 24172420.

32. SAS Institute Inc. (1985) SAS Users Guide: Statistics, 5th ed.Cary, NC: SAS Institute Inc.

33. Shreeram S, Johns PW, Subramaniam S, et al. (2014) Therelative bioavailability of the calcium salt of beta-hydroxy-beta-methylbutyrate is greater than that of the free fatty acidform in rats. J Nutr 144, 15491555.

34. Nissen SL & Abumrad NN (1997) Nutritional role of the leu-cine metabolite -hydroxy--methylbutyrate (HMB). J NutrBiochem 8, 300311.

35. Van Koevering M & Nissen S (1992) Oxidation of leucine and-ketoisocaproate to -hydroxy--methylbutyrate in vivo. AmJ Physiol (Endocrinol Metab) 262, E27E31.

36. Nissen S, Van Koevering M & Webb D (1990) Analysis of-hydroxy--methyl butyrate in plasma by gas chromato-graphy and mass spectrometry. Anal Biochem 188, 1719.

-Hydroxy--methylbutyric acid clearance 1409

Comparison of availability and plasma clearance rates of -hydroxy--methylbutyrate delivery in the free acid and calcium saltformsMethodsHuman subjectsTreatmentsStudy designPlasma and urine -hydroxy--methylbutyrate analysisStatistical analysis and calculations

ResultsSubject characteristicsFree acid form of -hydroxy--methylbutyrate gel kineticsAcute -hydroxy--methylbutyrate kinetics for capsule and water administration

Table 1Subject descriptors(Mean values with their standard errors)24h -hydroxy--methylbutyrate kinetics for capsule and water administrationBlood chemistry and haematology

DiscussionFig. 1(a) The 3h plasma levels of -hydroxy--methylbutyrate (HMB) after administration of either free acid form of HMB (HMB-FA) gelcaps (--) or Ca salt of HMB (Ca-HMB) capsule (- --). (b) The 3h plasma leFig. 2(a) The 24h plasma levels of -hydroxy--methylbutyrate (HMB) after administration of either free acid form of HMB (HMB-FA) gelcaps (--) or Ca salt of HMB (Ca-HMB) capsule (- --). (b) The 24h plasma AcknowledgementsACKNOWLEDGEMENTSReferencesReferences