Fast internal dynamics in alcohol dehydrogenase.

Large-scale domain motions in alcohol dehydrogenase (ADH) have been observed previously by neutron spin-echo spectroscopy (NSE). We have extended the investigation on the dynamics of ADH in solution by using high-resolution neutron time-of-flight (TOF) and neutron backscattering (BS) spectroscopy in the incoherent scattering range. The observed hydrogen dynamics were interpreted in terms of three mobility classes, which allowed a simultaneous description of the measured TOF and BS spectra. In addition to the slow global protein diffusion and domain motions observed by NSE, a fast internal process could be identified. Around one third of the protons in ADH participate in the fast localized diffusive motion. The diffusion coefficient of the fast internal motions is around two third of the value of the surrounding D2O solvent. It is tempting to associate the fast internal process with solvent exposed amino acid residues with dangling side chains.

Bending elastic properties of a block copolymer-rich lamellar phase doped by a surfactant: a neutron spin-echo study.

The influence of the short alkyl-chain ionic surfactant OTAB on the dynamic behavior of an inverse block copolymer-rich lamellar phase was investigated by neutron spin-echo spectroscopy (NSE). The observed intermediate scattering function can be described by a sum of two contributions. For high scattering vectors the model of Zilman-Granek plus a slow diffusional mode can be used to describe the experimental data and the bending elastic modulus κ for a polymer-rich membrane is calculated. At low scattering vectors the relaxation curves are strongly influenced by de Gennes narrowing arising from the structure factor of the Lα phase. Hence, the computed relaxation rates in this q-range are inversely proportional to the static structure factor. The present study demonstrates the necessity to directly investigate the dynamic behavior of lamellar phases and that an analysis of the width of the Bragg peaks can be insufficient to derive information about the single membrane elasticity, especially when both κ and B[combining macron] depend on the composition of the membrane.

Functional domain motions in proteins on the ~1-100 ns timescale: comparison of neutron spin-echo spectroscopy of phosphoglycerate kinase with molecular-dynamics simulation.

Protein function often requires large-scale domain motion. An exciting new development in the experimental characterization of domain motions in proteins is the application of neutron spin-echo spectroscopy (NSE). NSE directly probes coherent (i.e., pair correlated) scattering on the ~1-100 ns timescale. Here, we report on all-atom molecular-dynamics (MD) simulation of a protein, phosphoglycerate kinase, from which we calculate small-angle neutron scattering (SANS) and NSE scattering properties. The simulation-derived and experimental-solution SANS results are in excellent agreement. The contributions of translational and rotational whole-molecule diffusion to the simulation-derived NSE and potential problems in their estimation are examined. Principal component analysis identifies types of domain motion that dominate the internal motion's contribution to the NSE signal, with the largest being classic hinge bending. The associated free-energy profiles are quasiharmonic and the frictional properties correspond to highly overdamped motion. The amplitudes of the motions derived by MD are smaller than those derived from the experimental analysis, and possible reasons for this difference are discussed. The MD results confirm that a significant component of the NSE arises from internal dynamics. They also demonstrate that the combination of NSE with MD is potentially useful for determining the forms, potentials of mean force, and time dependence of functional domain motions in proteins.

Material properties and bioactivity of a resin infiltrant functionalized with polyhedral oligomeric silsesquioxanes.

OBJECTIVES: To investigate the effect of methacrylate polyhedral oligomeric silsesquioxanes (POSS-8) on various material properties and mineral precipitation potential of a resin infiltrant. METHODS: A TEGDMA-based resin infiltrant was mixed with 0.5, 1, 3, 5 or 10 wt% POSS-8 or left unchanged (control). Degree of conversion (DC), water sorption (WS), viscosity, elastic modulus (E-modulus), flexural strength (FS), Knoop microhardness (KHN) and softening ratio (SR) were assessed. Growth of calcium phosphate (Ca/P) precipitates infiltrant-treated bovine enamel and dentin specimens immersed in artificial saliva or artificial dentinal fluid, respectively, for 28 days was analyzed by scanning electron microscopy and energy-dispersive X-ray spectroscopy. For viscosity assessment, pure TEGDMA filled with 0-10 wt% POSS-8 was used. Statistical analyses were performed using ANOVA and Tukey's post-hoc tests (p < 0.05). RESULTS: POSS-8 did not change the flexural strength, water sorption and softening ratio. The apparent degree of conversion was increased at lower concentrations only while E-modulus remained constant in almost all groups. The particles led to a slight decrease of KHN at concentrations below 3%. The effect on viscosity is comparable to the reinforcement effect. Ca/P precipitates formed on dentin specimens treated with POSS-8-filled infiltrant after 4 weeks of immersion, but were not detected on the control infiltrant. The mineral precipitation on enamel was not improved by POSS-8. SIGNIFICANCE: POSS-8 particles did not worsen the material properties of the resin infiltrant, while the Ca/P precipitation on dentin was stimulated.

Large domain fluctuations on 50-ns timescale enable catalytic activity in phosphoglycerate kinase.

Large-scale domain motions of enzymes are often essential for their biological function. Phosphoglycerate kinase has a wide open domain structure with a hinge near the active center between the two domains. Applying neutron spin echo spectroscopy and small-angle neutron scattering we have investigated the internal domain dynamics. Structural analysis reveals that the holoprotein in solution seems to be more compact compared to the crystal structure but would not allow the functionally important phosphoryl transfer between the substrates if the protein were static. Brownian large-scale domain fluctuation dynamics on a timescale of 50 ns was revealed by neutron spin echo spectroscopy. The dynamics observed was compared to the displacement patterns of low-frequency normal modes. The displacements along the normal-mode coordinates describe our experimental results reasonably well. In particular, the domain movements facilitate a close encounter of the key residues in the active center to build the active configuration. The observed dynamics shows that the protein has the flexibility to allow fluctuations and displacements that seem to enable the function of the protein. Moreover, the presence of the substrates increases the rigidity, which is deduced from a faster dynamics with smaller amplitude.

Efficacy of supplemental 1 alpha-hydroxycholecalciferol and microbial phytase for young pigs fed phosphorus- or amino acid-deficient corn-soybean meal diets.

Young pigs (5 wk of age and 8 kg) were used to test the efficacy of 1 alpha-hydroxycholecalciferol (1 alpha-OH D3) and microbial phytase for improving the utilization of phytate phosphorus (P) and amino acids present in corn-soybean meal (SBM) diets. Phytase supplementation (1,200 units/kg) to a vitamin D3-adequate, P-deficient corn-SBM diet elicited a marked response (P < .05) in weight gain and ash content of fibula, scapula, and metatarsal bones, but dietary addition of 1 alpha-OH D3 (20 micrograms/kg) was without effect. A P- and vitamin D3-adequate, amino acid-deficient corn-SBM diet (15.5% CP) also was supplemented with 1,200 units/kg of phytase to evaluate the efficacy of phytase in improving amino acid utilization. Pigs gained faster (P < .05) and more efficiently (P < .05) when this diet was supplemented with limiting amino acids, and phytase addition also increased (P < .05) weight gain, regardless of whether the diet was deficient or adequate in amino acids. Feed efficiency was improved (P < .05) by phytase addition to the amino acid-deficient diet but not to the amino acid-adequate diet. Pigs fed the low-CP, amino acid-fortified diet gained as fast and as efficiently as those fed a 19.5% CP (1.19% lysine) positive-control diet.

Utilization of phytate and nonphytate phosphorus in chicks as affected by source and amount of vitamin D3.

Commercial and laboratory-strain crossbred chicks responded (P < .01) markedly to 1alpha-hydroxycholecalciferol (1alpha-OH D3) during the 2nd and 3rd wk of life. Bone-ash responses exceeded 50% when this compound was added at 20 microg/kg to phosphorus (P)-deficient corn-soybean meal diets containing surfeit levels (25 microg/kg) of cholecalciferol (D3). Phosphorus excretion was decreased (P < .01) and, thus, retention was increased (P < .01) when 1alpha-OH D3 was supplemented. A P-deficient (.10% P) casein-amino acid purified diet, devoid of D3, was used to determine whether 15 microg/kg of D3 was sufficient to facilitate optimal absorption of the nonphytate P contained in this diet. Bone ash responded to .075% P addition (KH2PO4), and chicks fed diets with .175% nonphytate P exhibited further bone-ash responses to 15 microg/kg of D3 or 10 microg/kg 1alpha-OH D3. Higher levels of either of these D3 compounds did not produce additional responses. This suggested that 15 to 25 microg/kg of D3 in a P-deficient corn-soybean meal diet (.28% phytate P and .14% nonphytate P) is more than adequate to facilitate optimal absorption of the nonphytate P present in the diet. A P-deficient casein-dextrose diet (.13% nonphytate P and 15 microg/kg D3) was fed in the final chick assay, and chicks fed this diet did not show bone ash responses to 1alpha-OH D3 or to microbial-derived phytase (1,470 units/kg). Thus, with P-deficient corn-soybean meal diets containing at least 15 microg D3/kg, 1alpha-OH D3 supplementation markedly increased weight gain and bone ash because it increased the utilization of phytate P.

Ideal digestible lysine level for early- and late-finishing swine.

Two hundred sixteen crossbred (PIC line 26 x Camborough 15) pigs were used in three trials to determine optimal digestible lysine levels during early (EF = 50 to 95 kg) and late (LF = 90 to 110 kg) finishing periods. Pigs were self-fed in sex groups of two in all trials. The assay diets for EF and LF periods were 11 and 10% CP corn-soybean meal diets, respectively, supplemented with threonine, methionine, tryptophan, valine, and isoleucine. Corn-soybean meal positive-control diets were included in each trial (14.5% CP for EF and 13.5% CP for LF). This dietary CP regimen was shown to give the same performance and carcass quality as a 17% CP corn-soybean meal diet fed during both EF and LF. Plateau portions of the lysine response curves resulted in performance levels that were equal to or greater than those achieved with pigs fed the 14.5/13.5% CP positive-control diets. Early-finishing pigs responded (P < .05) to graded doses of digestible lysine (.41 to .71%) for daily weight gain, gain:feed, longissimus muscle area, 10th-rib fat depth, lean gain, and plasma urea N. Digestible lysine requirement estimates based on average plateau points were .58% for EF barrows and .64% for EF gilts. Late-finishing pigs responded (P < .05) to digestible lysine doses (.35 to .65%) for daily weight gain, gain:feed, lean gain, and plasma urea N. Digestible lysine requirement estimates based on average plateau points were .49% for LF barrows and .52% for LF gilts.

Hepatic and renal betaine-homocysteine methyltransferase activity in pigs as affected by dietary intakes of sulfur amino acids, choline, and betaine.

In Exp. 1, young pigs were fed a basal diet containing .17% methionine (Met) (.14% digestible Met), and .48% cystine (.38% digestible cystine) for 14 d (34 to 48 d of age). Treatment additions were .25% DL-Met, .34% betaine, .30% choline, or .25% DL-Met and .34% betaine. Methionine, but not betaine or choline supplementation, increased (P < .05) weight gain and feed efficiency. Hepatic betaine-homocysteine methyltransferase (BHMT) activity was increased (P < .05) by betaine and choline supplementation but was not affected by Met deficiency. Renal BHMT activity was increased (P < .05) by Met deficiency and was further increased (P < .05) by betaine supplementation. In Exp. 2, 10-kg pigs were fed the basal diet from Exp. 1 supplemented with enough DL-Met to bring the total basal Met to .24% (.20% digestible Met). Treatment additions consisted of .20% DL-Met or .34% betaine, and diets were fed for 16 d (34 to 50 d of age). Feed efficiency increased (P < .05) in response to Met, but not to betaine, supplementation. Hepatic BHMT activity increased (P < .05) in response to betaine and Met, but no changes in renal BHMT activity occurred. Although statistically significant changes in hepatic and renal BHMT activity occurred in both experiments, the magnitude of the responses was probably not physiologically important. Therefore, in contrast to previous findings with rats and chicks, it does not seem that hepatic and renal BHMT activity in pigs is influenced substantially by Met deficiency, or by surfeit levels of choline or betaine.

Microbial phytase improves amino acid utilization in young chicks fed diets based on soybean meal but not diets based on peanut meal.

Studies were conducted to evaluate the ability of supplemental microbial phytase to improve performance of young chicks fed phytate-containing, amino acid-deficient diets. Diets based on corn and peanut meal or soybean meal (SBM) and dextrose were fed to young chicks housed in battery cages for 10- and 13-d experimental periods. Assays were designed to evaluate phytase supplementation of both amino acid-deficient and amino acid-adequate diets. Weight gain, feed intake, and gain:feed values of chicks increased (P < 0.05) when deficient amino acids were serially supplemented to either the corn-peanut meal or SBM-dextrose diets. Phytase supplementation (600 and 1,200 U/kg) to the corn-peanut meal diet resulted in no significant differences in weight gain, feed intake, or gain:feed values of chicks when the diet was either deficient or adequate in amino acids. However, phytase supplementation (1,200 U/kg) produced significant increases (P < 0.05) in gain:feed values, but not weight gain, of chicks when they were fed the amino acid-deficient, but not the amino acid-adequate, SBM-dextrose diet. This gain:feed response to phytase supplementation of the amino acid-deficient SBM-dextrose diet occurred in both crossbred (New Hampshire x Columbian) and commercial (Ross x Hubbard) chicks. A digestibility assay was also performed using cecectomized roosters fed dehulled SBM containing three levels of phytase (0, 600, and 1,200 U/kg). When averaged across nine essential amino acids and cystine, true amino acid digestibility (TAAD) values were increased by approximately 2% when 1,200 U/kg phytase was included with SBM and administered to cecectomized roosters. However, neither TAAD values nor TMEn were significantly improved by the phytase addition.

Iron bioavailability in soybean meal as affected by supplemental phytase and 1 alpha-hydroxycholecalciferol.

An Fe depletion-repletion chick bioassay was conducted to determine whether supplemental microbial phytase or 1 alpha-hydroxycholecalciferol (1 alpha-OH D3) would improve the bioavailability of Fe in soybean meal (SBM). Weight gain, hemoglobin, and hematocrit were markedly improved when increasing levels (0, 10, 20, and 80 mg/kg) of Fe from analytical grade ferrous sulfate (FeSO4.7H2O) were added to the Fe-deficient casein-dextrose basal diet containing 20 mg Fe/kg. Addition of 19 mg Fe/kg from SBM to the basal diet improved (P < 0.05) hemoglobin and hematocrit, but the response was less than that obtained from 10 mg Fe/kg from FeSO4.7H2O. Phytase (1,430 units/kg), 1 alpha-OHD3 (10 micrograms/kg), or the combination, added to the SBM-fortified basal diet did not further improve hematocrit or hemoglobin, indicating that Fe bioavailability of SBM was not increased by either of these feed additives. Based on standard-curve methodology, and using hemoglobin as a criterion, the relative bioavailability of Fe was 38.5% for SBM, 21.0% for SBM+phytase, 23.2% for SBM+1 alpha-OHD3, and 29.2% for SBM+phytase+ 1 alpha-OHD3.

Bioavailability of iron in cottonseed meal, ferric sulfate, and two ferrous sulfate by-products of the galvanizing industry.

Iron depletion-repletion assays were carried out with young chicks to establish Fe bioavailability values for Fe2(SO4)3.7H2O (22.7% Fe), Fe-ZnSO4.H2O (20.2% Fe, 13.0% Zn), Zn-FeSO4.H2O (20.2% Zn, 14.2% Fe), and cottonseed meal (200 mg Fe/kg). Standard hemoglobin response curves were established using feed-grade FeSO4.H2O (28.8% Fe) or reagent-grade FeSO4.7H2O (20.1% Fe) as standards such that relative bioavailability (RBV) could be assessed for the experimental sources of Fe. Weight gain, hemoglobin, and hematocrit responded linearly (P < 0.05) to Fe supplementation in all assays. Using hemoglobin as the response criterion, slope-ratio calculations established Fe RBV values of 126% for Fe-ZnSO4.H2O and 93% for Zn-FeSO4.H2O. The 126% value for Fe-ZnSO4.H2O was greater (P < 0.05) than the FeSO4.H2O standard (100%), but the 93% value for Zn-FeSO4.H2O was not different (P > 0.10) from the standard. However, evaluation of all criteria of response (hemoglobin, hematocrit, weight gain) suggested that neither Fe-ZnSO4.H2O nor Zn-FeSO4.H2O had different Fe RBV values than FeSO4.H2O. Standard-curve calculations were used for assessment of Fe RBV in Fe2(SO4)3.7H2O and cottonseed meal, as only a single level of Fe addition was studied for each of these products. Iron RBV in Fe2(SO4)3.7H2O was estimated to be 37%, whereas Fe RBV in cottonseed meal was found to be 56%. Both of these values were lower (P < 0.05) than the FeSO4 standard. The data suggest that the two new products, representing combinations of FeSO4.H2O and ZnSO4.H2O by-products of the galvanizing industry, are excellent sources of bioavailable Fe, whereas ferric sulfate and cottonseed meal are relatively poor sources of usable Fe.

Slow internal protein dynamics in solution.

Large-scale domain dynamics in proteins are found when flexible linkers or hinges connect domains. The related conformational changes are often related to the function of the protein,for example by arranging the active center after substrate binding or allowing transport and release of products. The adaptation of a specific active structure is referred to as 'induced fit' and is challenged by models such as 'conformational sampling'. Newer models about protein unction include some flexibility within the protein structure or even internal dynamics of the protein. As larger domains contribute to the configurational changes, the timescale of the involved motions is slowed down. The role of slow domain dynamics is being increasingly recognized as essential to understanding the function of proteins. Neutron spin echospectroscopy (NSE) is a technique that is able to access the related timescales from 0.1 up to several hundred nanoseconds and simultaneously covers the length scale relevant for protein domain movements of several nanometers distance between domains. Here we focus on these large-scale domain fluctuations and show how the structure and dynamics of proteins can be assessed by small-angle neutron scattering and NSE.

Fast microscopic method for large scale determination of structure, morphology, and quality of thin colloidal crystals.

We present a novel fast microscopic method to analyze the crystal structures of air-dried or suspended colloidal multilayer systems. Once typical lattice spacings of such films are in the range of visible light, characteristic Bragg scattering patterns are observed. If in microscopic observations these are excluded from image construction, a unique color coding for regions of different structures, morphologies, and layer numbers results. Incoherently scattering defect structures, however, may not be excluded from image construction and thus remain visible with high resolution.

1alpha-hydroxycholecalciferol does not increase the specific activity of intestinal phytase but does improve phosphorus utilization in both cecectomized and sham-operated chicks fed cholecalciferol-adequate diets.

Two chick assays were conducted in an attempt to understand how 1alpha-hydroxylated cholecalciferol compounds [1,25-(OH)2 D3 and 1alpha-OH D3] function in chicks to improve utilization of phytate-bound phosphorus (P) and trace minerals. Mucosal tissue from chicks fed a P-deficient corn-soybean meal diet, with or without supplemental 1alpha-OH D3, was incubated with sodium phytate. Inorganic P (Pi) release from sodium phytate, a measure of mucosal phytase activity, was not influenced by 1alpha-OH D3 presence in the diet. Increasing doses of mucosal protein in tubes containing sodium phytate resulted in marked increases (P < 0.01) in Pi release, but 1alpha-OH D3 in the diet from which the duodenal mucosal tissue was obtained had no effect on Pi release. Similarly, addition of either 1alpha-OH D3 or 1,25-(OH)2 D3 directly to the incubation tubes had no effect on Pi production. Efficacy of supplemental 1alpha-OH D3 and phytase was also tested in cecectomized vs. sham-operated chicks that were fed P-deficient and cholecalciferol-adequate corn-soybean meal diets. Removal of the twin ceca was done in an attempt to remove much of the intestinal microbial activity, and in turn, much of the gut microbial phytase activity. Marked increases (P < 0.01) in bone ash occurred in response to phytase or 1alpha-OH D3 supplementation, and cecectomized birds responded to either addition in the same manner as sham-operated controls. The data suggest that the marked phytate-P releasing capacity of dietary 1alpha-OH D3 or 1, 25-(OH)2 D3 is not caused by an increased specific activity of intestinal phytase.

1 alpha-Hydroxylated cholecalciferol compounds act additively with microbial phytase to improve phosphorus, zinc and manganese utilization in chicks fed soy-based diets.

Seven experiments were conducted to determine the efficacy of 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] and microbial phytase in improving P, Zn and Mn utilization of chicks fed P, Zn- and Mn-deficient soy protein diets containing surfeit levels of cholecalciferol. Efficacy of 1 alpha-hydroxycholecalciferol (1 alpha-OH D3) was also studied. A dose titration study indicated that supplemental phytase at 1200 units/kg diet would increase bone ash by at least 65% when added to a corn-soybean meal diet containing 0.43 g P/100 g (0.1 g nonphytate P/100 g). These responses were similar to those obtained from supplemental P (0.1 g/100 g) as KH2PO4 or from added 1,25-(OH)2D3 (10 micrograms/kg). Dietary addition of both 1200 units phytase and 10 micrograms/kg 1,25-(OH)2D3 elicited bone ash responses that were near 100%. When chicks were fed a Zn-deficient soy-concentrate diet (13 mg Zn/kg), diet supplementation with 1,25-(OH)2D3 or phytase increased growth rate by 40% and tibia Zn content by > 100%; adding 1,25-(OH)2D3 together with phytase increased tibia Zn content by 160%. Utilization of both Zn and Mn contained in the corn-soybean meal diet also was markedly enhanced by supplemental phytase, 1,25-(OH)2D3, or the combination. The cholecalciferol analog 1 alpha-OH D3 was found to improve dietary P utilization maximally (70% bone ash response) at a dose of 20 micrograms/kg diet, and effects were additive when 1 alpha-OH D3 was fed in the presence of phytase.(ABSTRACT TRUNCATED AT 250 WORDS)

Activity of various hydroxylated vitamin D3 analogs for improving phosphorus utilisation in chicks receiving diets adequate in vitamin D3.

1. Young chicks were used to evaluate the efficacy of 2 new vitamin D3 analogs relative to 1 alpha-hydroxycholecalciferol (1 alpha-OH D3) for their ability to improve the bioavailability of phytate-bound phosphorus (P) when added to P-deficient maize-soyabean meal diets that were superadequate in cholecalciferol. 2. Both 20-epi-19-nor-1,25-(OH)2 D3 and 20-epi-19-nor-1 alpha-OH D3 were observed to have phytate-P releasing activity, as measured by bone ash responses. 3. However, the bioactivity of the 2 new analogs differed when compared with 1 alpha-OH D3. The 20-epi-19-nor-1 alpha-OH D3 analog had a lower (P < 0.05) phytate-P releasing activity (45%) than either 1 alpha-OH D3 of 20-epi-19-nor-1,25-(OH)2 D3, which did not differ. 4. A dietary concentration of 10 to 15 micrograms/kg 1 alpha-OH D3 was found to optimize phytate-P utilisation in 2-week-old chicks.

Vitamin D3 requirement of young chicks receiving diets varying in calcium and available phosphorus.

1. Three battery experiments were conducted with broiler chicks during the 2nd and 3rd week of life. Graded amounts of cholecalciferol (D3) were added to maize-soyabean meal diets that were designed to be (a) severely deficient in available phosphorus (P), (b) marginally deficient in calcium (Ca) or (c) adequate in both available P and Ca. 2. With diets containing 1.0 g available P and 6.3 g Ca/kg (assay 1), graded doses of D3 between 0 and 37.5 mu/kg produced linear (P < 0.05) positive responses in both weight gain and tibia ash. With a D3 concentration of 1250 micrograms/kg, 250 times the requirement recommended by the NRC, bone ash was increased (P < 0.05) over that of birds fed 37.5 micrograms/kg, and neither weight gain nor food intake were reduced. 3. With a P-adequate diet (4.5 g available P/kg) containing 8.5 g Ca/kg (assay 2), weight gain and bone ash increased linearly (P < 0.05) upon supplementing the basal diet with 0, 2.5 and 5.0 micrograms D3/kg. Higher doses of D3 did not elicit further responses, and chicks fed on a diet containing 1250 micrograms D3/kg gained as fast and had bone ash values that did not differ from those of chicks receiving 5, 10, 20 or 40 micrograms D3/kg. 4. When the maize-soyabean meal basal diet was fortified with Ca and P to achieve adequate amounts of Ca (10.1 g/kg) and P (4.5 g available P/kg) in assay 3, dietary additions produced results similar to those obtained in assay 2 where P was adequate and Ca was slightly deficient. Again, chicks receiving a surfeit of D3 (1250 micrograms/kg) exhibited weight gains and bone ash values that were as great as those of chicks receiving 5, 10, 15 or 30 micrograms D3/kg. 5. It is apparent that young chicks have a high tolerance for excess D3, and chicks fed on diets that are severely deficient in available P continue to respond to D3 in excess of 37.5 micrograms/kg.