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Adenylate deaminase (AMP aminohydrolase, EC 3.5.4.6) was purified 500-fold from the human erythrocytes by DEAE-cellulose adsorption and chromatography on hydroxylapatite. Its isoelectric point is 5.0 and the pH of optimal activity is near 7.0. Although the enzyme is markedly activated by ATP and by monovalent cations, it is able to deaminate AMP in the absence of activators. At pH 7 the order of effectiveness for activation by monovalent cations is K+ > Rb+ > Li+ > Na+ > Cs+. The presence of ATP or changes in pH somewhat alter their relative activities but not the order. dATP activates the enzyme as effectively as ATP. ADP activates to a lesser degree and GTP is without effect. The enzyme displays a cooperative effect with substrate and also with its activators, monovalent cations and ATP. The activators thus increase the apparent affinity of the enzyme for AMP without affecting the maximum reaction velocity. When both cation and ATP are added, the substrate-velocity curve is changed from a pronounced sigmoidal curve to a rectangular hyperbola. The apparent affinity for AMP increases as the concentration of cation is raised. Conversely, the affinity for cation increases when the concentration of AMP is elevated or ATP is added. Halide anions are non-competitive inhibitors and decrease in their effectiveness as follows: F − > I − > Br > Cl − . Pi and 2,3-diphosphglyceric acid inhibit the enzyme competitively and ATP counteracts that inhibition. In the presence of 1 mM ATP and 100 mM KCl the K i values for Pi and 2,3-diphosphoglyceric acid are between 4 and 11 mM.

From corn leaves, two phosphoenolpyruvate (PEP) carboxylase isoenzymes were separated and purified on DEAE-cellulose and hydroxylapatite columns. Both isoenzymes exhibited non-Michaelis—Menten kinetics with respect to Mg2+ ion concentrations. The Lineweaver—Burk double reciprocal plots were concave downward and consisted of at least three linear portions with different slopes. The Rs values were significantly higher (above 225) than a Michaelis—Menten type enzyme. The Hill coefficients were less than 1.0 at Mg2+ ion concentrations below 0.5 mM. The kinetics thus show a negative cooperativity with respect to the binding of Mg2+ to phosphoenolpyruvate carboxylases but a positive cooperativity with respect to catalysis by Mg2+.

An α-glucosidase was purified from flint corn by precipitation with ammonium sulfate, chromatographies on CM-cellulose and Hydroxylapatite and gel-filtrations on Sephadex G-100. The purified enzyme was homogeneous in ultracentrifugal and disc electrophoretic analysis. The sedimentation coefficient was calculated to be 6.5 S. The molecular weight was estimated to be approximately 6.5×104 by gel-filtration technique.The optimal pH was found to be 3.6 for both maltose and soluble starch. The enzyme lost about 80% of the activity by incubation at 60°C for 10 min.The ratio of velocity of hydrolysis for maltose, phenyl-α-glucoside and soluble starch was estimated to be 100:14.3:6.1 in this order. The αglucosidase hydrolyzed soluble starch exo-wisely.

C1-Esterase was purified from the euglobulin fraction of human plasma by successive column chromatography on DEAE-cellulose, hydroxylapatite and TEAE-cellulose. The final product, purified 3500-fold with respect to serum, hydrolyzed 1,155 μmoles of Nα-acetyl-l-tyrosine ethyl ester per milligram of protein at pH 7.4 and 37°C in 15 min. The homogeneity of the purified C1-esterase was confirmed by ultracentrifugation and disc-electrophoresis. Its s20,w value was 4.3 and its molecular weight was determined as 113,000 by gel filtration on Sephadex G–200.Cl-Esterase possesses esterolytic activity for both Nα-acetyl-l-tyrosine ethyl ester and Nα-tosyl-l-arginine methyl ester, and acts on human kininogen I and II releasing kinin very slowly.

Synthesis of chromatin proteins is studied in T and B lymphocytes during activation with leucoagglutinin. The lymphocyte populations are prepared from human peripheral blood by rosette formation with sheep erythrocytes. Stimulation is accompanied by a relative increase in the amount of nonhistones (hydroxylapatite fraction 2) as compared with histones in T cells, while no change is observed in B cells. Incorporation of 3 H-leucine in T cell histones and nonhistones is stimulated 1.8-fold; no change is observed in the labelling of B cell chromatin proteins. Gel electrophoretic analysis reveals preferential labelling of two groups of T cell nonhistones at 40 000 and 95 000 D.

Vitellogenin in the eggs of Blattella germanica was solubilized with solutions at high salt concentrations and high pH. This protein was purified by ammonium sulfate precipitation, acetic acid precipitation, DEAE-cellulose chromatography, and hydroxylapatite chromatography, into a chromatographically homogeneous state. By sucrose density gradient centrifugation, the purified vitellogenin was resolved into two components. The relative amounts of the two components varied according to the pH of the solution. An equilibrium seemed to exist in the interconversion between them when the conditions of the solution were fixed. It is suggested that aggregation and disaggregation of the vitellogenin molecules may account for the apparent heterogeneity.

— –A preparation of the photochemical reaction center of Chromatiwn has been obtained by chromatography of lauryldimethylamineoxide-solubilized chromatophores on hydroxylapatite and Sepharose columns. The procedure has yielded a reaction center preparation from both carotenoid-containing and carotenoid-deficient Chromatium cells. Preliminary analysis of the isolated component indicates that the photochemical reaction center of the Thiorhodaceae is homologous to that of the Athiorhodaceae. In particular, the near infrared absorption spectrum of the Chromatium reaction center preparation shows the same triple-peaked spectrum observed for reaction center preparations from the Athiorhodaceae. The Chromatium preparation undergoes a rapid light-induced oxidation and dark reduction of the reaction center. The ratio of the reaction center to the two membrane-bound cytochromes (cytochrome c552 and c555) is greatly increased over the ratio observed in chromatophores or in other previously isolated, reaction center-enriched subchromatophore fractions of this organism.

Prostaglandin Δ13-reductase was purified extensively by ammonium sulfate fractionation, and DEAE-Sephadex-, hydroxylapatite-, and phosphocellulose chromatography. Enzyme activity was followed by radioimmunoassay with the use of an antiserum directed toward 15-keto-prostaglandin F2α and antibodies directed toward 13, 14 dihydro-15-keto-prostaglandin F2α. The purified enzyme used NADPH as a cofactor much more effectively than NADH. It specifically reduced 15-keto-prostaglandins but not 15-hydroxy-prostaglandins. The enzyme was inhibited by p-chloromercuribenzoate. It had a relatively broad pH optimum (pH 7.4 to pH 8.5), and has a molecular weight estimated to be 70,000 to 80,000.

Fragmented and mitomycin C cross-linked E. coli DNA was fractionated according to base composition by means of hydroxylapatite chromatography and density-gradient centrifugation in order to determine the effect of compositional heterogeneity on the breadth of the helix–coil transition. The transitions of some of the fractions are broader than that of the unfractionated DNA, due, presumably, to nonrandom sequences in molecules of 5 × 105 daltons. Analysis of the transition breadths in terms of the known heterogeneity leads to reconsideration of current DNA helix–coil transition theory. We propose that partially denatured states include those for which the chains do not remain in strict register. Denaturation profiles are comprehensible only if this multitude of entropically favorable, degenerate states is included in the theory.

Internal proteins from T2 coliphages have been purified by a new method, involving osmotic shock and chromatography on hydroxylapatite columns. Purified T2 internal proteins interacted specifically with T2 DNA causing folding of the DNA as reflected by significant changes in its viscosity. Internal proteins from T4 phages, similarly affected T4 DNA.

An improved purification method for the glial fibrillary acidic protein from normal human brain is reported. Preparations of high purity were obtained by substituting DEAE and phosphocellulose chromatography with one step of hydroxylapatite chromatography. The glial fibrillary acidic protein from normal and gliosed brain was separated into 4 bands (components 1-4) ranging in molecular weight from 54 000 plus or minus 1000 to 40500 plus or minus 1000 by sodium dodecylsulfate gel electrophoresis at 7.5% and 12.5% acrylamide concentration. A better separation of the components was obtained on 12.5% acrylamide gels by increasing the time of electrophoresis to 15-17 h. In these conditions each component was split into a doublet. Preparations identical to those previously reported, i.e. 2-band preparations with an average molecular weight of 43 000, were obtained by incubating multiple sclerosis plaques at 24C for 48 h. These 2-band preparations co-migrated with the 2 lower molecular weight components (component 3, 45 000 plus or minus 1000; component 4, 40 500 plus or minus 1000) in 4-band preparations. The components cross-reacted with antisera against different preparations with an immunodiffusion pattern of complete identity and appeared to be chemically related. Most cyanogen bromide peptides were common to 2-band and 4-band preparations. A unique amino-terminal sequence of alanine-glycine-phenyl-alanine was found in all preparations, regardless of the source and of the number of components. The amino acid composition of 2-band and 4-band preparations was similar.

DNA synthesis was studied in human peripheral blood lymphocytes that had been stimulated with phytohemagglutinin. DNA pulse-labeled with [3H]thymidine was fractionated by sucrose gradient centrifugation or by chromatography on hydroxylapatite columns. Nascent DNA was identified as a single-stranded species that sedimented at 4-5S in neutral sucrose gradients and appeared to be precursor to chromosomal DNA in pulse-chase experiments. At least two-thirds of the nascent DNA was released as single strands from high molecular weight DNA without employing a denaturation step. It is concluded that synthesis of DNA by phytohemagglutinin-stimulated lymphocytes involves a low molecular weight, single-stranded, short-lived intermediate similar to that described for other eukaryotic cells.

Comparisons were made among the fluorescence spectra of DNA-bound benzo[a]pyrenes which were produced in vivo and in vitro. DNA from mouse skin treated with benzo[a]pyrene had a maximum emission beyond 400 nm, which was clearly distinguished from that of DNA-bound benzo[a]pyrene 4,5-oxide. The emission spectra from mouse skin were classified into two groups, type I and type II. The former was similar to the spectrum of benzo[a]pyrene, although the two maxima were shifted to longer wavelengths (410 and 435 nm). Type II was characterized by a broad peak around 430 nm. Type I and type II were obtained from different fractions of hydroxylapatite chromatography, but type I was changed into type II during storage. This suggests that type II is a modified product of type I. The emission spectra of both groups also were detected in in vitro activating systems, including photoirradiation, iodine treatment, and hydrogen peroxide treatment. Treatment of Escherichia coli with benzo[a]pyrene during culture produced only fluorescence of type I. Although the relationship between types I and II remains to be established, both types of fluorescence evidently indicate that the conjugated ring structure of the parent compound, benzo[a]pyrene, is preserved intact in DNA-bound benzo[a]pyrene. Several lines of evidence suggest that the proximate (active) form is an unidentified hydroxylated product including an oxy radical, but a cation radical cannot be completely excluded.

Mineral phosphate (apatite) is activated for the synthesis of nucleotides when dilute solutions containing nucleoside and ammonium oxalate are evaporated in its presence. A natural, igneous flourapatite was found to be even more effective in nucleotide synthesis than the more soluble hydroxylapatite. The phosphorylation is considerably more efficient if urea or cyanamide is also present. Hydrolysis of solutions of cyanogen to form oxalate and urea among other products is a spontaneous process that provides a geologically plausible model for nucleotide synthesis on the primitive earth.

Successive extracts of a precipitated hydroxylapatite were made with water and each extract was analyzed for calcium, phosphate, sulfate, and pH. The sulfate impurity rapidly decreased to a non-detectable quantity. Calcium and phosphate were released in concentrations decreasing with successive extracts and in a mole ratio that depended on the proportion of solid and water used (slurry density) and the number of water extracts. The total ions in the water extracts was expressed as the fraction of the hydroxylapatite contained in the boundary layer and this fraction never exceeded a monolayer of 1 unit cell depth. The results are compatible with the formation of a surface complex having the composition Ca2(HPO4) (OH)2, this being a product of hydrolysis of the boundary planes of hydroxylapatite. The complex has a small solubility that yields Ca, HPO4, and OH ions in the water extracts and a value for pKc has been calculated.

SUMMARY: A 5′-nucleotidase, widely distributed in teleost fish muscles, was purified about 20-fold from Pacific cod (Gadus macrocephalus) by chromatography of a dialyzed aqueous extract of the muscle on DEAE-cellulose. The enzyme was unstable and lost 85% of its activity in 1 hr at 37°C 53% in 10 min at 42°C and 40% in 1 hr at 30°C. It was stable for 6 days at 0°C, could be dialyzed for up to 3 days at 0°C against 1 mM tris buffer pH 7.5 and quickly frozen and thawed without loss of activity. However, it was inactivated rapidly when held at −30°C. Brief exposure to pH 4.0 or 5.0 effected marked destruction. Attempts at further purification by means of chromatography on hydroxylapatite, adsorption using alumina Cγ and starch gel electrophoresis failed due to instability. The enzyme was strongly inhibited by EDTA, pyrophosphate, KF and ZnCl2 (1-10 mM); less markedly inhibited by GSH, 2-mercaptoethanol, carbonate and CaCl2 (10 to 100 mM). It was strongly activated by Mn++ and weakly activated by Mg++. The optimum pH was 7.6, and the Km was 5 × 10−4M with UMP and 8 −4M with IMP. It hydrolyzed, in order of effectiveness, LJMP, IMP, CMP, d-AMP, GMP, d-IMP, d-GMP, d-UMP and AMP, but not p-nitro phenylphosphate, sugar phosphates or a number of other compounds including 2′,3′-nucleotides.

Soluble proteins were extracted from Triton WR 1339-filled rat liver lysosomes and were chromatographed on hydroxylapatite by stepwise elution with increasing concentrations of phosphate buffer (pH 6.8). The distribution of the activities of the cathepsins A, B, C, and D; β-galactosidase; α-mannosidase; β-glucuronidase; β-N-acetylglucosaminidase; acid phosphatase and arylsulfatase in the resulting fractions was determined. Ammonium sulfate fractionation of lysosomal proteins was combined with separation on hydroxylapatite in an attempt to improve the resolution of the cathepsins. Lowering the pH of the phosphate buffers to 5.8 greatly increased the affinity of lysosomal proteins for hydroxylapatite. By a combination of these manipulations fractions were obtained which contained cathepsin D without contamination by other cathepsins.

A purification technique developed for the glutamine dependent carbamoyl phosphate synthetase from mouse spleen resulted in the copurification of aspartate transcarbamoylase. The two activities remained associated through (NH 4 ) 2 SO 4 precipitation, hydroxylapatite adsorption and sucrose density gradient centrifugation. The pH curve of the aspartate transcarbamoylase exhibited two optima, one at pH 9.4 and a much higher one at pH 10.2. At pH 7.4 and 37°, aspartate transcarbamoylase had a K m for aspartate of 8.4 mM and V max of 0.35 μmoles product formed per min. per mg. protein. The K m for carbamoyl phosphate was 1.8 × 10 −3 mM. Activity of aspartate transcarbamoylase was not affected by any one of a number of pyrimidine nucleotides.

Hydroxylapatite (calcium phosphate) chromatography was investigated as a method for separating protein subunits dissociated with sodium dodecyl sulfate (SDS). The proteins used included: a series of 11 well characterized proteins composed of single or identical subunits varying in molecular weight from 11,700 to 165,000; hemoglobin, composed of α and β chains of 146 and 141 amino acids; and vaccinia virus structural proteins, composed of more than 15 different polypeptide subunits. Reduced proteins were complexed with SDS and adsorbed to columns of hydroxylapatite. Using linear gradients of sodium phosphate, pH 6.4, containing 0.1% SDS and 1mm dithiothreitol, all tested proteins eluted between 0.2 and 0.5 m phosphate. A useful feature of the method is that proteins do not all elute in order of molecular weight. Thus polypeptide separations on hydroxylapatite are different from those obtained by polyacrylamide gel electrophoresis or gel filtration in SDS. The high resolution of the method was demonstrated by separating the α and β chains of hemoglobin and complex mixtures of viral polypeptides. By using polyacrylamide gel electrophoresis and hydroxylapatite chromatography in succession, it has been possible to separate vaccinia virus structural polypeptides that had not previously been resolved.

Bovine liver homogentisicase can exist in two forms which are interpreted as being apo-and holoenzymes. It is obtained in an unenriched form (apoenzyme) from bovine liver by acetone and ammonium sulfate fractionations and chromatography on hydroxylapatite column. This form of the enzyme can be enriched to an active form (holoenzyme) by incubation with ferrous ion under anaerobic conditions. The extent of activation is completely time dependent and the rate is extremely sensitive to the pH. Reducing substances do not affect the restoration of activity of the apoenzyme under anaerobic conditions. Properties of the apo- and holoenzymes are compared with respect to absorption spectrum, effects of reducing agents and pH on activity (or on reconstitution), stability, and molecular weight. The activity of homogentisicase is strongly inhibited by various diphenolic compounds as well as chelating agents specific for ferrous ion. However, the reconstitution of homogentisicase from the apoenzyme with ferrous ion is strongly prevented only by the substrate, homogentisate or 2,5-dihydroxybenzoate, a compound structurally similar to the substrate.

Ein einfaches Verfahren zur Gc-Globulindarstellung aus nativem menschlichen Serum wird beschrieben. Die Auftrennung des Serums erfolgt an Hydroxylapatit-Saulen unter Verwendung vonSorensen-Puffern. Die Gc-Fraktion enthalt noch einen Teil des Albumins, der durch Rechromatographie bis auf sehr geringe Reste eliminiert wird. Die Gc-Proteinausbeute ist nahezu quantitativ.

A proenzyme (hereafter propeptidase), with a high activity on N-acetyl-l-tyrosine ethyl ester, was found associated with proelastase through several steps of purification. Propeptidase is distinct from chymotrypsinogen A as shown by its electrophoretic mobility and its high resistance, in the activated form, to inhibition by l-1-tosylamido-2-phenyl-ethyl chloromethyl ketone. Evidence indicates that propeptidase is probably the proenzyme form of elastomucase. Activation of proelastase and propeptidase with trypsin changed their electrophoretic mobilities. The activated proelastase migrated at the same rate as authentic, pure elastase. The proenzymes that were originally highly insoluble could be solubilized by treatment with alumina Cγ gel. The solubilizing effect of alumina Cγ gel was reversible. The solubilized proenzymes could be reprecipitated by recombination with the eluate of alumina Cγ gel. Activation of the proenzymes had no effect on their precipitation with Cγ eluate. To a lesser degree, trypsin and chymotrypsinogen A also precipitated upon mixing them with Cγ eluate. Proelastase and propeptidase each were obtained in an electrophoretically pure state by column chromatography on hydroxylapatite.

Apatitische Calciumphate mit vom Hydroxylapatit abweichenden Ca/P-Verhaltnissen wurden chemisch und physikalisch untersucht und ihre Zusammensetzung bestimmt. Fehlstellen konnen nicht nur in Calcium-, sondern auch in OH-Positionen vorhanden sein. Die allgemeine Formel fur Apatite dieser Art ist Ca10-x-y(HPO4, CO3)x (PO4)6-x(OH)2-x-2y. in Carbonatapatiten konnen Defekte 1. Ordnung teilweise wieder aufgefullt sein: Ca10-x+y (CO3)x(PO4)6-x(OH)2-x+2y. Apatitic calcium phosphates with Ca/P ratios different from that of hydroxylapatite were investigated by chemical and physical methods and their compositions established. Defects occur not only in calcium, but also in OH-positions. the general formula for apatites of this kind is Ca10-x-y(HPO4)6-x(OH)2-x-2y. In carbonateapatites first order defects can partially be refilled: Ca10-x+y(CO3)x (PO4)6-x(OH)2-x+2y.

Studies have been made on the antigen-antibody reaction with PE and its antibody when the immune γ-globulin is strongly adsorbed on chromatographic adsorbents such as hydroxylapatite and sulphomethyl-cellulose. The reverse case has also been studied with the anionic cellulose exchanger diethylaminoethyl-cellulose where PE remains rather firmly bound to the adsorbent. Frontal analysis and the stepwise elution technique show that both antigen and antibody react with their complementary reactants while adsorbed on column material. Increasing the salt concentration causes some of the PE-ASPE complex to dissociate, which shows that PE-ASPE combination varies in magnitude. Some possible implications have been discussed.

1) Water-soluble substances of the triturated equine and bovine dentins and bovine bone, when suspended in distilled water and N/100 Veronal buffer, are studied by the determinations of calcium, magnesium, phosphorus and nitrogen.2) The dissolution of magnesium ions of dentin considerably differs from that of bone i.e. the ratio of magnesium and calcium dissolved from dentin (Mg/Ca) is much greater than that of bone, and in the case of bone it keeps nearly constant throughout the experiment period, but in the case of dentin the ratio shows an increasing inclination.From these phenomenon it is suggested that dentin and bone hold magnesium ions in different manners respectively.3) The ratio of water-soluble magnesium and calcium is much greater than that of the composition of magnesium and calcium in the both tissues.4) The ratio of calcium and phosphorus dissolved from dentin (Ca/P) is fairly small (below 1.0) . It seems that other phosphate salts are more soluble than calcium phosphate, since the ratio of calcium and phosphorus in Ca3 (PO4) 2 gives 1.94 and in hydroxylapatite gives 2.15. But (Mg+Ca) /P gives nearly 2.0 when magnesium ions are also multiplied by the ratio of both molecular weights (40/24.3) .So it can be considered that the soluble magnesium ions are driven from some phosphate compounds such as tricalcium phosphate or hydroxylapatite in which calcium ions are replaced by magnesium ions.On the contrary, the ratio of calcium and phosphorus dissolved from bone is greater than 2.3, especially in N/100 Veronal buffer, and it seems that other calcium salts are dissolved more than calcium phosphate is.5) According to the nitrogen determination, little difference is observed with the amount of dissolved organic compounds between dentin and bone.

Jelly coat substances of Anthocidaris crassispina and Pseudocentrotus depressus were fractionated on a hydroxylapatite column into several components. However, the immunological test of Outhterlony suggests that in the case of Anthocidaris jelly, such fractions may not be homogeneous. These fractions, rich in sialic acid, poor in protein, and with very little sulfate, belong to the sialopolysaccharides. Since Anthocidaris sialopolysaccharides show similar amino acid histograms, the peptide portion is considered to form the backbone structure of these components. Jelly sialopolysaccharides tightly agglutinate the homologous sperm, and, generally speaking, there is some relationship between the sialic acid content and the sperm-agglutinating activity of the sialopolysaccharides.

The adsorption of CO 2 on carbon adsorbents (FT carbon, coconut charcoal, acid-washed bone char) and adsorbents containing basic calcium phosphate (hydroxylapatite, bone char, ash of bone char) was studied. Special consideration was given to the pretreatment of the materials. The carbons equilibrated as rapidly as the temperature; the basic calcium phosphates showed a rapid initial adsorption followed by a very slow rate which continued for days. Linear adsorption isotherms were found on FT carbon and the isosteric heats varied slightly with coverage. The isotherms for the remaining materials had varying curvature and were for the most part in the same sequence as the estimated surface areas. The isosteric heats of CO 2 correlated very well with the magnitude of surface hydroxyl groups, an estimate of which was made from the chemical composition. There appeared to be three increasing levels of interaction: 1. (1) pure physical adsorption; 2. (2) an adsorption complex having “bicarbonate structure” and 3. (3) an adsorption complex having “carbonate structure”.

The properties of purified liver hyaluronidase have been compared with those of testis hyaluronidase. The lysosomal enzyme showed a similar action pattern to the testis enzyme (1), but had a lower pH optimum at pH 3.5 and reduced affinity for hexasaccharide. The Km for hyaluronate was 8 x 10-2 mg per ml. Sodium chloride was not required for enzyme activity but served to prevent inhibition by sulfated polysaccharides, notably chondroitin sulfate B, which had a Ki of 3.8 x 10-4 mg per ml. The polycation, protamine sulfate, also prevented polyanion inhibition. Liver hyaluronidase, like the testis enzyme (12), exhibited transglycosylase activity.

The action of B. subtilis saccharifying amylase on starch has been examined. It has been found that high yields of reducing sugar and glucose are produced from both starch solutions and starch gels upon prolonged reaction. The enzyme has also been found to have maltase and cyclodextrinase activities and to produce an almost quantitative yield of glucose from β-cyclodextrin. Attempts to separate or differentiate the amylase, maltase and cyclodextrinase activities by electrophoresis, inhibitors, heat inactivation and hydroxylapatite chromatography have been unsuccessful, and it is concluded that the enzyme activities are all due to one molecular species.

Wax bean agglutinin prepared according to Liener was separated by chromatography on hydroxylapatite into three fractions, two of which were biologically active. Both fractions are glycoproteins with essentially identical amino acid composition and molecular weight, each consisting of four subunits, molecular weight 30 000. The isolectins agglutinated transformed cells at a concentration about 100 times lower than that required to agglutinate normal cells. Agglutination was inhibited by fetuin but not by any of the simple sugars tested.