Measurement of bile acid in serum and bile with arylamine-glass-bound 3alpha-hydroxysteroid dehydrogenase and diaphorase

3Alpha-hydroxysteroid dehydrogenase (3-HSD) from Pseudomonas testosteroni and diaphorase (lipoyl dehydrogenase) from Clostridium spp. have been immobilized individually onto arylamine glass beads through diazotization. A cost-effective enzymic colorimetric method for determination of bile acid in serum and bile employing a mixture of these immobilized enzymes was developed. The method is based on measurement of reduced nicotinamide adenine dinucleotide generated from bile acid in serum/bile by immobilized 3alpha-HSD with a color reagent consisting of nitro blue tetrazolium chloride salt, oxidized nicotinamide adenine dinucleotide, and immobilized lipoyl dehydrogenase in 0.065 M sodium phosphate buffer, pH 7.0. Analytical recovery of added bile acid (50 and 200 micromol/L) was 95.57 and 85.46% in serum and 97.6 and 91.6% in bile, respectively. Within- and between-batch coefficients of variation (CV) for bile acid determination were <1.2 and <0.2% in serum and >0.1 and <0.1% in bile, respectively. Good correlations for bile acid in serum (r1=0.92) and in bile (r2=0.97) were obtained by use of a standard chemical method and the present method. The mixture of immobilized 3alpha-HSD dehydrogenase and lipoyl dehydrogenase lost 50% of its initial activity after 6 months of regular use. The cost of bile acid determination in 100 serum and bile samples by the present method has been compared with that of the Sigma kit method.     

A bioluminescent assay for 12-alpha-hydroxy bile acids using immobilized enzymes

A bioluminescent assay for 12-alpha-hydroxy bile acids was developed using enzymes coimmobilized onto Sepharose 4B. The immobilized enzymes used were a bacterial 12-alpha-hydroxysteroid dehydrogenase, bacterial luciferase, and NADPH:FMN oxidoreductase or bacterial diaphorase. The assay was specific for 12-alpha-hydroxy bile acids and the lower limit of detection was 4 pmol/0.5 ml assay volume with a linear range of 4 to 2000 pmol. Intraassay precision was from 7.8 to 8.2%. Values obtained with this assay showed good agreement with those obtained by gas-liquid chromatography. The system using diaphorase was not stable at 4 degrees C in the absence of added thiol compounds, but could be stabilized by the addition of glutathione (0.5 mM). The assay is a convenient, a rapid, and an extremely sensitive method for the measurement of 12-alpha-hydroxy bile acid concentrations in the serum of patients or experimental animals.     

Serum bile acid determination for assessment of hepatic injury in the woodchuck

A direct spectrophotometric assay for determination of the serum bile acid concentration in the woodchuck (Marmota monax) has been validated. The assay relies on the conversion of 3-hydroxy bile acids to 3-oxo bile acids by 3 alpha-hydroxysteroid dehydrogenase with concomitant reduction of NAD+ to NADH. Reduction of NAD+ is coupled via a diaphorase catalyst to the formation of a diformazan dye from nitrotetrazolium blue and the diformazan product is measured spectrophotometrically at 540 nm. Interfering endogenous dehydrogenase activity present in woodchuck sera was inactivated with sodium pyruvate. Mean recovery of seven exogenous bile acids added to woodchuck sera was 102.0 +/- 2.2%. Intra-assay precision was determined with ten replicate samples giving a mean +/- standard error of the mean of 1.94 +/- 0.12 micron/L with a coefficient of variation of 3.9%. The mean serum bile acid concentration determined in 33 clinically healthy animals was 5.52 +/- 0.81 micron/L. The serum bile acid concentration increased following surgical ligation of the bile duct from 3.78 +/- 0.58 micron/L to a maximum value of 148.0 +/- 30.7 micron/L and remained increased for the 42 day study period. In woodchucks treated with carbon tetrachloride, the serum bile acid concentration peaked at 16 hours following treatment at 72.7 +/- 29.3 micron/L, and returned to pretreatment concentration within 6 days. The serum bile acid concentration therefore appears to be a sensitive biochemical test of cholestasis and hepatocellular forms of hepatic injury and of potential value in the clinical assessment of hepatic disease associated with woodchuck hepatitis virus infection.     

Analysis of 3-sulfated and nonsulfated bile acids by one-step solvolysis and high performance liquid chromatography

A facile solvolysis procedure of 3-sulfated bile acid was devised using trifluoroacetic acid, tetrahydrofuran, and methanol. The sulfate esters were completely solvolyzed within only 2 hr by the present method. The clinical utility of the solvolysis procedure and high performance liquid chromatography using immobilized 3 alpha-hydroxysteroid dehydrogenase was demonstrated in the analysis of bile acids in serum of patients with obstructive jaundice. The quantities of 3-sulfated bile acids were calculated from the difference in the amount of bile acids before and after solvolysis. A significantly large proportion of 3-sulfated glycochenodeoxycholic acid, i.e., 21.9 to 31.3% of total glycochenodeoxycholic acid, was found in the serum of patients with obstructive jaundice. Thus, the present method permits simultaneous quantitation of 3-sulfated as well as nonsulfated bile acids in biological samples.     

Assay of free and glycine- and taurine-conjugated bile acids in serum by high-pressure liquid chromatography by using post-column reaction after group separation.

An accurate and sensitive method that involves the group separations of serum bile acids (i.e. free and glycine- and taurine-conjugated bile acid fractions) by ion-exchange chromatography on piperidinohydroxypropyl-Sephadex LH-20 is described. Each group was then analysed by high-pressure liquid chromatography by using the post-column reaction technique with immobilized 3 alpha-hydroxy steroid dehydrogenase. The bile acid patterns in the umbilical venous serum samples were analysed by this method. Taurochenodeoxycholate predominated in the umbilical blood.     

Flow injection determination of glucose,bile acid and ATP using immobilized enzyme reactor and chemiluminescent assay of NAD(P)H

We have developed a chemiluminescent flow injection method for analysis of bile acid, glucose and ATP using the chemiluminescent assay of NADH using 1-methoxy-5-methylphenazinium methyl sulphate (1-MPMS)/isoluminol(IL)/microperoxidase (m-POD) system and immobilized enzyme reactors such as 3α-hydroxysteroid dehydrogenase, glucosedehydrogenase, hexokinase and glucose-6-phosphate dehydrogenase. The standard curves were obtained in the range of 5 ～ 100 pmol for bile acid, 0.5 ～ 5.0 nmol for glucose and 10^?7 ～ 10^?5 mol/L for ATP. The coefficient of variation for each assay was not more than 4.1% for bile acid, 2.3% for glucose and 5.3% for ATP, respectively.     

Foetomaternal relationships of serum bile acid pattern estimated by high-pressure liquid chromatography.

The bile acid patterns in the maternal and umbilical vein and artery serum samples were analysed by a two-step chromatographic method involving group separation by piperidinohydroxypropyl-Sephadex LH-20 and high-pressure liquid chromatography using immobilized 3 alpha-hydroxy steroid dehydrogenase. Glycochenodeoxycholate predominates in the maternal blood and taurochenodeoxycholate in the umbilical blood. In cases where a free bile acid was detected in the maternal blood, the same bile acid was also demonstrated in the corresponding cord blood. The concentrations of taurocholate and taurochenodeoxycholate were found to be significantly higher in the umbilical artery than in the corresponding umbilical vein. Our data suggest that there is a bidirectional placental transfer of free bile acids and that there is a transfer of taurine-conjugated primary bile acids from the foetus to the mother.     

Development of a micro-planar amperometric bile acid biosensor for urinalysis

The determination of bile acid concentration in urine is useful for the screening and diagnosis of various hepatobiliary diseases. Currently, there is no concise method to determine bile acid concentration in urine. This study describes a bile acid biosensor fabricated by electrochemical technique for urinalysis. The micro-planar electrodes employed for the study consisted of a working electrode (platinum), a counter electrode (platinum) and a reference electrode (silver/silver chloride (Ag/AgCl)). The sensor chip was coated with Nafion using a spin-coater in order to both eliminate many interference species in urine and achieve long-term stability of the reference electrode. Nafion coating allowed the sensor chip to prevent the electrode reaction from interference species in urine, because it is charged negative strongly (Nafion contains sulfonic acid group). Three enzymes (bile acid sulfate sulfatase: BSS, beta-hydroxysteroid dehydrogenase: beta-HSD, and NADH oxidase: NHO) were immobilized by glutaraldehyde (GA: cross-linker) onto the sensor chip, because the immobilization of enzymes by GA is simple and commonly carried out. The sensor chip was able to detect bile acid in buffer solution. The optimum enzyme ratio immobilized onto the sensor chip was BSS:beta-HSD:NHO=4:4:20 U/1 chip. There was a relationship between the concentration of bile acid and the response current value. The dynamic range of the sensor chip was 2-100 microM for bile acid. Additionally, bile acid in the urine specimen could be detected using this bile acid biosensor. We present a simple and rapid bile acid biosensor with high sensitivity and high reproducibility.     

Rapid analysis of human fecal bile acids

A rapid, accurate, precise method for determining human fecal bile acids is reported. Feces are homogenized and then briefly extracted with boiling absolute ethanol. A portion of the extract is evaporated to dryness and the residue heated with mild alkali to hydrolyze bile acid 3α-hydroxyl esters. Aliquots of hydrolyzed crude extract are treated with resazurin reagent which effects a series of enzyme catalyzed reactions in which bile acid free 3α-hydroxyls are first oxidized to 3-oxo-groups in a reaction catalyzed by 3α-hydroxysteroid dehydrogenase. Resulting protons are transferred to β-nicotinamide adenine dinucleotide, yielding reduced β-nicotinamide adenine dinucleotide (β-NADH). β-NADH then reduces nonfluorescent resazurin to fluorescent resorufin in a reaction catalyzed by diaphorase. Developed fluorescence, which is proportional to the extract aliquots bile acid content, is excited at 565 nm and read at 580 nm, wavelengths which lie in a spectral region in which there is minimal fecal pigment absorption. 3-Oxo-bile acids and bile acid 3α-sulfates are extracted in the procedure but reduction and/or solvolysis is necessary before quantification.     

Determination of bile acids in biological fluids by liquid chromatography-electrospray tandem mass spectrometry

A simple, sensitive, and specific liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) method for the determination of bile acids in human bile has been developed. The bile acids were extracted with a C(18) (octadecyl) reversed-phase column and identified and quantified by simultaneous monitoring of their parent and daughter ions, using the multiple reaction monitoring mode. Identification and quantification of conjugated bile acids in bile was achieved in 5 min. The detection limit was 1 ng, and the determination was linear for concentrations up to 100 ng. The percent recovery of standards made of single conjugated (glycine and taurine) bile acid or of mixture of glycine- or taurine-conjugated cholic acid, chenodeoxycholic acid, deoxycholic acid, ursodeoxycholic acid, and lithocholic acid averaged 71.73% to 95.92%. The percent recovery of the same standard bile acids was also determined by gas chromatography-mass spectrometry (GC-MS), using the selected ion monitoring mode, and averaged 66% to 96%. A biliary bile acid profile of human gallbladder bile was obtained by LC-MS/MS and GC-MS.The results showed a good correlation between the two techniques and no significant differences between the two methods were observed. The LC-MS/MS method was also used for the analysis of serum, urine, and fecal bile acids. In conclusion, LC-MS/MS is a simple, sensitive, and rapid technique for the analysis of conjugated bile acids in bile and other biological samples. - Perwaiz, S., B. Tuchweber, D. Mignault, T. Gilat, and I. M. Yousef. Determination of bile acids in biological fluids by liquid chromatography-electrospray tandem mass spectrometry. J. Lipid Res. 2001. 42: 114;-119.     

Continuous-flow automated assay of steroids with nylon-tube-immobilized hydroxysteroid dehydrogenases

Several NAD(P)⁺-dependent hydroxysteroid dehydrogenases, namely 3α-hydroxysteroid dehydrogenase, β-hydroxysteroid dehydrogenase, 7α-hydroxysteroid dehydrogenase, and 12α-hydroxysteroid dehydrogenase were separately immobilized on nylon tubes for the continuous-flow automated assay of hydroxysteroids. 3α-Hydroxysteroid dehydrogenase was also immobilized on pore glass. Spectrophotometric monitoring in the visible region, where blank values were markedly reduced, was achieved through the Meldola blue catalyzed transfer of hydrogen from NAD(P)H to a tetrazolium salt. Nylon-tube-immobilized enzymes maintained 45–55% of the original activity after 1 month of intermittent use. The operational range, using the “end point” approach, was 1–25 nmol of steroid and the assay speed 10–15 samples/h. Reliable results were obtained in the determination of 3α-hydroxysteroids and 3β,17β-hydroxysteroids in urine and total bile acids in serum.     

Immobilized enzymatic fluorescence capillary biosensor for determination of sulfated bile acid in urine

The authors have proposed an immobilized enzymatic fluorescence capillary biosensor (SBAs-IE-FCBS) for the determination of sulfated bile acids (SBAs). The reaction principle of the biosensor is that under the catalysis of the bile acid sulfate sulfatase (BSS) and beta-hydroxysteroid dehydrogenase (beta-HSD) immobilized on inner surface of a medical capillary, SBAs desulfates to 3beta-hydroxyl bile acids, then the latter reacts with nicotinamide adenine dinucleotide (NAD(+)), and is converted into 3-ketosteroid; meanwhile, NAD(+) is converted to reduced nicotinamide adenine dinucleotide (NADH). NADH continuously reacts with 1-methoxy-5-methylphenazinium methyl sulfate (1-MPMS) and is converted into NAD(+) circularly and 1-MPMSH(2). Finally resazurin is reduced into resorufin by 1-MPMSH(2), the formed resorufin (lambda(ex)/lambda(em): 540 nm/580 nm) is used for quantifying the concentration of SBAs. Optimized conditions being suitable with the biosensor are as follows: the concentrations of BSS and beta-HSD used for the immobilization all are 5 kUL(-1); the concentrations of 1-MPMS and resazurin all are 25 micromolL(-1); the concentrations of Tris-HCl buffer and NAD(+) are 100 and 400 micromolL(-1), respectively; total volume of the enzyme, reagent and sample is only 18 microL per time for determining; the reaction temperature is 37 degrees C; the reaction time is 15min. The concentration of SBAs is directly proportional to the fluorescence intensity of the biosensor measured from 0.5 to 5.0 micromolL(-1). The relative standard deviation is less than 3.4%, and the detection limit was 0.16 micromolL(-1). The recoveries are in the range 95.5-106%. This SBA-IE-FCBS can be used for quantifying SBAs in urine to diagnose and judge hepatobiliary diseases, etc.     

Crystal structure of human type III 3alpha-hydroxysteroid dehydrogenase/bile acid binding protein complexed with NADP(+) and ursodeoxycholate

The crystal structure of human type III 3alpha-hydroxysteroid dehydrogenase (HSD)/bile acid binding protein (AKR1C2) complexed with NADP(+) and 3alpha,7beta-dihydroxy-5beta-cholanic acid (ursodeoxycholate) at 3.0 A resolution is presented. Thus, the three-dimensional structure has now been solved for a human HSD member of the aldo-keto reductase superfamily. AKR1C2 is implicated in the prostatic production of the potent androgen 5alpha-dihydrotestosterone and the hepatic transport of bile acids. It also catalyzes the formation of the neurosteroid 3alpha-hydroxy-5alpha-pregnan-20-one in the central nervous system, and its allosteric modulation by fluoxetine has been linked to the use of this drug for premenstrual dsyphoria. Like other members of the superfamily, AKR1C2 folds into an alpha/beta-barrel and binds NADP(+) in an extended conformation. The carboxylate of ursodeoxycholate binds to AKR1C2 in the oxyanion hole at the active site. More interestingly, the orientation of ursodeoxycholate is essentially "backwards" and "upside-down" from that observed for testosterone in the related rat 3alpha-HSD.NADP(+).testosterone ternary complex, where testosterone assumes the position of a 3-ketosteroid substrate. The orientation of ursodeoxycholate is thus similar to that expected of a 17beta-HSD substrate. The ternary structure explains the ability of AKR1C2 to catalyze 3alpha-, 17beta-, and 20alpha-HSD reactions. Comparison of the steroid binding pocket of AKR1C2 with that of rat 3alpha-HSD reveals significant differences in the positions of conserved and nonconserved loop residues, providing insights into the structural basis for the functional flexibility that is observed in all the human 3alpha-HSD isoforms but not in the rat isoform.     

Simple enzymatic detection method for urinary sulfated 7α-hydroxy bile acids in normal subjects and in patients with acute hepatitis

Urinary sulfated primary bile acids, 7α-hydroxy bile acids, are detected by an enzymatic method using 7α-hydroxysteroid dehydrogenase (EC 1.1.1.-, 7α-HSD) after chromatographic fractionation on Sephadex G-25. Urinary sulfated or glucuronated bile acids are hydrolyzed by β-glucuronidase/sulfatase (EC 3.2.1.31/EC 3.1.6.1) from Helix pomatia and then released 7α-hydroxy bile acids are detected with 7α-HSD in the presence of β-NAD⁺, diaphorase (EC 1.6.99.2, from Clostridium kluyveri) and 2-p-iodophenyl-3-p-nitrophenyl-5-phenyltetrazolium chloride. The absorbance of formazan formed during the enzymic reaction is measured at 500 nm. Excretion values of 7α-hydroxy bile acids in normal subjects and in patients with acute hepatitis were compared. This enzymatic detection method for the excretion pattern of urinary 7α-hydroxy bile acids may be useful for clinical diagnosis.     

Molecular structure of rat hepatic 3 alpha-hydroxysteroid dehydrogenase. A member of the oxidoreductase gene family.

3-alpha-Hydroxysteroid dehydrogenase (3 alpha-HSD) (EC 1.1.1.50) is an important multifunctional oxidoreductase capable of metabolizing steroid hormones, polycyclic aromatic hydrocarbons, and prostaglandins. 3 alpha-HSD is also required for bile acid synthesis and has been suggested to play an important role in net bile acid transport across the hepatocyte (Stolz, A., Takikawa, H., Ookhtens, M., and Kaplowitz, N. (1989) Annu. Rev. Physiol. 51, 166-177). In order to characterize molecular forms and begin to determine its regulation, we now report the nucleotide sequence, tissue distribution, and homology to other members of the oxidoreductase superfamily. Rat hepatic 3 alpha-HSD cDNA encodes for a 322-amino acid protein with a predicted molecular weight of 37,022 expressed in a 2.4-kilobase (kb) message size. Northern blot analysis of total RNA revealed equivalent steady-state levels in liver and intestine in male rats with lower levels of expression in the colon and minimal expression in stomach, lung, and testis. Female liver contained approximately 2-3-fold greater steady-state levels of mRNA as compared to the male liver with equivalent intestinal expression. Two hybridizing bands, 2.4 and 1.4 kb, were identified in total RNA from the ovary. 3 alpha-HSD exhibits 75% amino acid sequence homology with bovine lung prostaglandin F synthetase and 50% homology with human aldose reductases. Amino acid sequence analysis with short chain alcohol dehydrogenases identified a possible NADP(H) cofactor-binding site at the amino terminus. The significant homology of 3 alpha-HSD with both prostaglandin F synthetase and aldose reductases suggest a subdivision of monomeric, NADPH reductases within the larger oxidoreductases superfamily.     

Determination of conjugated bile acids in human urine by high-performance liquid chromatography with chemiluminescence detection

A qualitative and quantitative analysis of the conjugated 1β- and 6α-hydroxy bile acids, including common bile acids, in human urine using high-performance liquid chromatography with chemiluminescence detection is described. After extraction of urine with C₁₈ silica cartridges, the bile acids were separated into non-conjugated, glycine, taurine and sulphate fractions by ion-exchange chromatography on a lipophilic gel. Solvolysis of the sulphate was carried out by treatment with trifluoroethanol in acetone containing hydrochloric acid, and the liberated amino acid conjugates were fractionated again. The individual bile acids were separated on a reversed-phase C₁₈ column (Bile Pak II), with detection by an immobilized 3α-hydroxysteroid dehydrogenase enzyme reactor and chemiluminescence reaction of the generated NADH using 1-methoxy-5-methylphenazinium methylsulphate—isoluminol—microperoxidase system. The assay method showed the detection limits ranging from 8 to 250 pmol for the bile acids tested. Analysis of urine samples obtained from newborns, non-pregnant women and women in late pregnancy showed a large difference in bile acid composition and conjugation mode, suggesting that bile acid metabolism is different during fetal and neonatal periods.     

An enzymatic cycling method for the determination of serum total bile acids with recombinant 3alpha-hydroxysteroid dehydrogenase

A highly sensitive enzymatic cycling method was developed for the serum total bile acids assay. We constructed a prokaryotic expression system to prepare the recombinant 3alpha-hydroxysteroid dehydrogenase in place of the natural enzyme and for the first time used it in the total bile acids assay. The production rate of thio-NADH correlated with the bile acids concentration and was measured by the change of absorbance at 405/660 nm. The enzymatic cycling method could detect 0.22 micromol/L total bile acids in serum. Within-run and between-run imprecisions were 1.2-3.7% and 2.3-4.8%, respectively. The calibration curve for total bile acids in serum was linear between 0.5 and 180 micromol/L. This method was free from interference by bilirubin, hemoglobin, ascorbate, and lactate dehydrogenase. In conclusion, serum total bile acids could be measured by the enzymatic cycling method with recombinant 3alpha-hydroxysteroid dehydrogenase as the tool enzyme.     

Determination of individual conjugated bile acids in human bile

A method has been developed and validated for the determination of the six major conjugated bile acids, cholesterol, and total phospholipids in bile of human subjects previously injected with 4-(14)C-cholesterol. The procedure is designed for use with 5-10 ml of duodenal or T-tube bile and eliminates difficulties associated with existing methods for bile acid determination, in particular the requirement for preliminary saponification under pressure or the use of paper chromatography. Saponification under pressure is employed only in steps where partial destruction of the steroid moiety of conjugated bile acids is not a crucial matter. A preliminary Folch extraction and washing step separated free cholesterol and phospholipids (bottom layer) from the six major conjugated bile acids (top layer). The conjugated bile acids were then fractionated cleanly by thin-layer chromatography to give four groups, the (14)C content of each of which was determined. A second aliquot of the top layer was used to determine (after deconjugation) the radioactivity ratio of deoxycholic acid to chenodeoxycholic acid for the two unresolved groups (dihydroxycholanoic acid conjugates with glycine and taurine, respectively). A third aliquot was used for determination of specific activities of the methyl esters of cholic, chenodeoxycholic, and deoxycholic acids derived from the total bile salts. Appropriate calculations yielded the concentration in bile of all six major bile acid conjugates.     

Transient-phase kinetic studies on the nucleotide binding to 3alpha-hydroxysteroid dehydrogenase from Pseudomonas sp. B-0831 using fluorescence stopped-flow procedures.

The dual nucleotide cofactor-specific enzyme, 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) from Pseudomonas sp. B-0831, is a member of the short-chain dehydrogenase/reductase (SDR) superfamily. Transient-phase kinetic studies using the fluorescence stopped-flow method were conducted with 3alpha-HSD to characterize the nucleotide binding mechanism. The binding of oxidized nucleotides, NAD(+), NADP(+) and nicotinic acid adenine dinucleotide (NAAD(+)), agreed well with a one-step mechanism, while that of reduced nucleotide, NADH, showed a two-step mechanism. This difference draws attention to previous characteristic findings on rat liver 3alpha-HSD, which is a member of the aldo-keto reductase (AKR) superfamily. Although functionally similar, AKRs are structurally different from SDRs. The dissociation rate constants associated with the enzyme-nucleotide complex formation were larger than the k(cat) values for either oxidation or reduction of substrates, indicating that the release of cofactors is not rate-limiting overall. It should also be noted that k(cat) for a substrate, cholic acid, with NADP(+) was only 6% of that with NAD(+), and no catalytic activity was detectable with NAAD(+), despite the similar binding affinities of nucleotides. These results suggest that a certain type of nucleotide can modulate nucleotide-binding mode and further the catalytic function of the enzyme.     

Serum bile acid analysis: a rapid, direct enzymatic method using dual-beam spectrophotofluorimetry.

The direct quantitative measurement of total bile acids in serum has been achieved using an enzymatic fluorescent method with a dual-beam spectrophotofluorimeter. By use of a 3alpha-hydroxysteroid dehydrogenase, oxidation of bile acids with NAD is completed in 200 seconds with the observed NADH fluorescence being proportional to the concentration of serum bile acids. This method is rapid (8 minutes per individual sample), has an intrinsic sensitivity of +/- micronM of total bile acids, requires no sample preparation and less than 0.8 ml of serum. Paired data analysis using enzymatic fluorescence and gas-liquid chromatographic methods gives a correlation coefficient (r) of 0.99 for 34 samples ranging from 2 to 530 micronM.