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Background: The anterior wall index (AWI) and posterior wall index (PWI) have been proposed to quantify anterior and posterior acetabular coverage using AP pelvic radiographs. However, these indices have only been reported in symptomatic patients with apparent pathomorphologies (dysplasia, overcoverage, and retroversion) undergoing osteochondroplasty or reorientation osteotomy., Questions: (1) What are the ranges for AWI and PWI from measurements obtained on AP pelvic radiographs of asymptomatic senior athletes with well-functioning hips? (2) Is there a difference between the AWI and PWI in asymptomatic athletes with acetabular morphology consistent with acetabular dysplasia, overcoverage, and retroversion when compared with asymptomatic hips that do not meet the radiographic definitions for those morphologies (controls)?, Methods: Five hundred five athletes (998 asymptomatic native hips) were independently evaluated by two readers on AP pelvic radiographs for AWI and the PWI after excluding hips with prior surgery, inadequate radiographs, or poor function (modified Harris hip score < 80). Hips with a lateral center-edge angle (LCEA) ≥ 20° and ≤ 38° and without acetabular retroversion, based on a positive crossover sign, were used as controls. Hips were categorized as developmental dysplasia of the hip (DDH; undercoverage) if the LCEA was < 20°. Finally, overcoverage was defined as an LCEA > 38°. The mean age of the athletes was 67 years (range, 50-91 years) and 55% were men. Linear generalized estimating equation regression was used to compare each individual diagnosis (DDH, retroversion, overcoverage) with the controls for both AWI and PWI adjusting for age and sex., Results: The mean AWI in the study population was 0.36 (range, -0.02 to 0.91). The mean PWI was 1.13 (range, 0.12-1.74). The mean AWI and PWI in controls (n = 740) was 0.35 (range, -0.02 to 0.91) and 1.13 (range, 0.64-1.70), respectively. There were 25 (3%) with DDH in whom the mean AWI was 0.26 (range, 0.05-0.5) and the mean PWI was 1.03 (range, 0.71-1.3). There were 112 (11%) retroverted hips in whom the mean AWI was 0.42 (range, 0.1-0.89) and PWI was 1.02 (range, 0.61-1.5). There were 121 (12%) overcovered hips in whom the mean AWI was 0.43 (range, -0.18 to 0.85) and PWI was 1.22 (0.12-1.74). The AWI in the control hips was no different than that of DDH hips (β -0.06; 95% confidence interval [CI], -0.12 to 0.002; p = 0.059) but was found to be lower than retroverted hips (β 0.08; 95% CI, 0.04-0.11; p < 0.001) and overcovered hips (β 0.05; 95% CI, 0.03-0.08; p < 0.001). The PWI in control hips was greater than that of DDH hips (β -0.08; 95% CI, -0.14 to -0.02; p = 0.013) and retroverted hips (β -0.07; 95% CI, -0.11 to -0.04; p < 0.001) but was less than overcovered hips (β 0.07; 95% CI, 0.04-0.10; p < 0.001)., Conclusions: The measurements of AWI and PWI in well-functioning, asymptomatic hips may be useful in assessing anterior and posterior acetabular coverage because it was able to distinguish between different types of known pathologic morphology. Despite evidence of these morphologic variances, these senior athletes continued to function at a high level. Thus, the identification of morphologic variance was not consistent with signs of pathology, which further supports that early screening of morphology may not predict the development of symptomatic pathology. Future work should focus on comparing these indices for morphologic variance in both symptomatic and asymptomatic hips to determine whether these measurements can be used in identifying problematic hips and as reference ranges for surgical correction., Level of Evidence: Level III, diagnostic study.

Background: It is not known whether morphological abnormalities of the hip are compatible with lifelong hip function and avoidance of osteoarthritis (OA). Our purpose was to investigate the prevalence of radiographic findings consistent with femoroacetabular impingement (FAI) and dysplasia (DDH) in senior athletes with well-functioning hips., Questions/purposes: (1) What is the prevalence of FAI and DDH in senior athletes with well-functioning hips? (2) Are radiographic findings of FAI and DDH associated with OA? (3) Is a history of longer duration or more intense activity associated with hip pathomorphology? (4) Were the modified Harris hip scores and the Hip Outcome Scores lower (legacy scales) in patients with evidence of hip pathomorphology than those without?, Methods: Five hundred forty-seven individuals (55% men, 45% women; 1081 hips, 534 bilateral and 13 unilateral), mean age 67 years (SD 8 years), gave consent and qualified for this institutional review board-approved cross-sectional study of senior athletes. Hips were independently evaluated for radiographic signs of FAI, DDH, and OA. Additionally, a lifetime activities questionnaire and outcome instruments were used to assess pain and function. Hips that had previously undergone arthroplasty or fracture surgery were excluded., Results: Eighty-three percent (898 of 1081) of hips had radiographic abnormalities consistent with FAI, of which 67% (599 of 898) were cam-type FAI. Ten percent (103 of 1081) of hips had radiographic evidence for dysplasia. Radiographic findings of FAI were not predictive of OA after controlling for age and sex (odds ratio [OR], 1.79; 95% confidence interval [CI], 0.48-6.62; p = 0.390). Similarly, radiographic findings of DDH were not predictive of OA (OR, 1.48; 95% CI, 0.31-7.21; p = 0.62). Our data suggest an increased risk of FAI-type morphologies in athletes who participated in competitive sporting events during early adult years (OR, 1.49; 95% CI, 1.04-2.11; p = 0.020). Additionally, participants who reported lifetime participation in competitive sports were at an increased risk of OA compared with those who did not (OR, 1.75; 95% CI, 1.14-2.69; p = 0.007). There were no differences in outcome scores between athletes with and without morphologic abnormalities., Conclusions: Radiographic findings consistent with FAI in these senior athletes were common and were not associated with the presence of OA. These data suggest that the need to screen for asymptomatic young athletes for radiographic evidence of FAI and DDH may not be necessary., Level of Evidence: Level III, prognostic study.

Background: Abnormal anatomy frequently results in the use of a modular stem in patients undergoing primary total hip arthroplasty (THA) for developmental dysplasia of the hip (DDH). However, because these stems are not always available in the operating room, it would be helpful if standard radiographic views could be analyzed in such a way that patients whose femoral anatomy might call for stem modularity could be anticipated before surgery. To our knowledge, no such parameters have been defined., Questions/purposes: In the senior author's practice, we used femoral neck anteversion of more than 25° as a determinant for use of a modular stem. Given this criterion, we asked if we could reliably identify plain film radiographic parameters of the femur that predict the use of modular stems. We looked at the following: (1) the neck-shaft angle based on the anteroposterior (AP) radiograph (alpha); (2) the neck-shaft angle from the crosstable lateral radiograph (beta); and (3) the calculated femoral anteversion angle., Methods: We reviewed preoperative radiographs from 50 of 67 patients (79 hips) who had a primary diagnosis of DDH and underwent primary THA from January 1999 to February 2007 inclusive. Hips with prior femoral-sided surgery (n = 2) or without preoperative films (n = 19) were excluded. Furthermore, patients with bilateral hips had the second hip excluded (n = 8). Twenty-one of 50 received a modular femoral stem based on the criterion of intraoperative neck-shaft anteversion of greater than 25° as measured by the senior surgeon (CLP), whereas the remainder received tapered nonmodular stems. There were no differences in age, sex, height, or weight between the modular stem group and tapered stem group. Radiographs were evaluated to record the parameters listed., Results: Patients in whom modular femoral stems were used had a greater mean AP (alpha) neck-shaft angle compared with patients who received tapered nonmodular stem (152°; 95% confidence interval [CI], 146°-157° versus 137°; 95% CI, 134°-141°; p < 0.001) with an optimal cutoff point for determining the use of modular stems of ≥ 142° (receiver operating characteristic [ROC] area = 73%). Hips in which modular femoral stems were chosen had a smaller mean lateral (beta) neck-shaft angle (152°; 95% CI, 148°-157° versus 161°; 95% CI, 158°-164°; p = 0.003) with an optimal cutoff point of ≤ 153° (ROC area = 65%). Hips in which modular femoral stems were used had a higher femoral anteversion angle (mean 45°; 95% CI, 37°-54° versus 21°; interquartile range, 17°-25°; p < 0.001) with an optimal cutoff of ≥ 32° (ROC area = 80%)., Conclusions: Preoperative radiographs anticipated the use of modular stems during THA for DDH in a practice where modular stems were chosen on the basis of a neck-shaft angle of greater than 25° measured at surgery. We found that this could be predicted on preoperative radiographs based on smaller lateral neck-shaft angles, steeper AP radiographic neck-shaft angles, and increased femoral anteversion calculated using these angles. Prospective studies are needed to better determine if these cutoff values adequately predict the use of modular stems.

We sought to determine if HbA1c and perioperative hyperglycemia were positively associated with the incidence of PJI. We retrospectively reviewed the VA VINCI database on patients who underwent primary joint arthroplasty between 2001 and 2011 and had HbA1c and perioperative blood glucose levels. Of 13,272 patients, 38% (n=5035) had an elevated perioperative HbA1c ≥7%. While there was no increased risk of infection associated with elevated HbA1c (HR 0.86, P=0.23), mortality was increased (HR 1.3, P=0.01). Preoperative hyperglycemia was associated with an increased incidence of PJI (HR 1.44, P=0.008). While HbA1c did not perfectly correlate with the risk of PJI, perioperative hyperglycemia did, and may be a target for optimization to decrease the burden of PJI., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Opioid pain medications are the basis for analgesia after orthopaedic injuries and procedures. However, opioids have many adverse effects, including opioid-induced androgen deficiency., Questions/purposes: We evaluated the occurrence and affect of opioid-induced androgen deficiency on osseous union and its ability to be reversed. Additional focus was placed on its perioperative onset and its duration in an orthopaedic animal model., Methods: A femoral osteotomy was created in 75 Sprague-Dawley rats. Postoperatively, animals were randomized into three treatment groups: control, morphine, and morphine plus testosterone. Testosterone levels were recorded preoperatively and at 48 hours, 4 weeks, and 8 weeks postoperatively. Some animals were euthanized at 4 weeks and others at 8 weeks postoperatively. Histology and micro-CT scans were used to evaluate callus. Three-point bend testing was performed to evaluate callus strength., Results: Serum testosterone levels in the morphine group showed decreased baseline levels of 2.2 ng/mL, 1.4 ng/mL, and 1.4 ng/mL (p < 0.001), whereas the morphine plus testosterone supplementation group showed increased serum levels at 41.7 ng/mL, 11.8 ng/mL, and 19.8 ng/mL (p < 0.001) compared with control animals (3.3 ng/mL, 5.8 ng/mL, 5.2 ng/mL) at 48 hours, 4 weeks, and 8 weeks, respectively. Compared with control animals, histology and micro-CT showed an impedance of callus maturation in the two experimental groups. Morphine-treated animals showed reduction in callus strength at 8 weeks (30% of the contralateral unfractured femur strength compared with 49% seen in the control animals at 8 weeks; p = 0.048); this finding was not fully reversed by testosterone supplementation (33% of the contralateral femur strength; p = 0.171)., Conclusions: Opioid-induced androgen deficiency occurred in this orthopaedic animal model. Although we previously showed that morphine inhibits callus maturation, the current study did not show a rescue of the morphine-treated animals with testosterone supplementation in either morphologic or mechanical testing. Testosterone suppression associated with opioid administration occurred almost immediately (within 48 hours) and was suppressed continually throughout the 8-week duration of study., Clinical Relevance: Opioid-induced androgen deficiency occurs during the perioperative orthopaedic period. Although its clinical relevance remains unknown, further evaluation is needed to determine if supplementation is warranted during the perioperative period.

Background: The current mainstay of orthopaedic pain control is opioid analgesics but there are few studies in the literature evaluating the effects of opioids on bone healing., Questions/purposes: The purpose of this study was to use a rat fracture model to evaluate the effects of opioid administration on osseous union in the acute (4 weeks) and subacute (8 weeks) setting in an operatively stabilized fracture. We asked the following question: does morphine administration alter (1) fracture callus strength; (2) callus volume and formation; and (3) morphology and early remodeling to final osseous union?, Methods: A 0.4-mm femoral osteotomy gap was created in 50 Sprague-Dawley rats using an established model. Postoperatively, rats were randomized to control versus morphine-treated study groups. Equal numbers of rats from each group were euthanized at 4 weeks and 8 weeks postoperatively. Three-point bend biomechanical testing was performed to evaluate postoperative callus strength. Micro-CT scans and histological analyses were used to evaluate postoperative callus volume and formation, morphology, and features of early remodeling., Results: Biomechanical testing identified a statistically significant (p = 0.048) reduction in callus strength in morphine-treated animals 8 weeks postoperatively compared with controls. Radiographic and histological analysis showed delayed callus maturation and lack of remodeling in the morphine group compared with control animals at 8 weeks. Micro-CT analysis expressed remodeling and resorption as a decrease in callus volume over the two time points. The control group had significant levels of resorption decreasing 29% (p = 0.023) over the 4-week to 8-week time interval. Morphine administration inhibited callus resorption and remodeling with only a 13% decrease (p = 0.393) in callus volume comparing these time points. The callus inhibition associated with morphine administration was not as evident in the acute, 4-week time setting., Conclusions: Morphine administration inhibited callus strength in this animal model. This finding is likely consistent with the observation that the callus and healing bone appear to have a decreased rate of maturation and remodeling seen at 8 weeks., Clinical Relevance: This study identifies that administration of an opioid pain medication leads to weaker callus and impedes callus maturation compared with controls. These findings may provide the impetus to alter our current orthopaedic analgesic gold standard toward more multimodal and opioid-limiting pain control regimens.

Study Design: Systematic review., Objective: The objectives of this review are to examine the spectrum of complications that have been reported in the literature after posterior interbody fusions of the lumbar spine augmented with bone morphogenetic proteins (BMPs) and discuss potential methods for their prevention., Summary of Background Data: The use of BMPs for spinal arthrodesis procedures has increased dramatically during the past decade. These products are commonly used in "off-label" fashion in posterior lumbar interbody fusion (PLIF) and transforaminal lumbar interbody fusion (TLIF) constructs. Recently, a number of adverse events have been attributed to the use of BMP for PLIF and TLIF surgical procedure, including heterotopic ossification within the epidural space or neuroforamina, postoperative radiculitis, and endplate osteolysis with interbody device subsidence., Methods: A computer aided literature search was performed on the electronic databases PubMed, MEDLINE, EMBASE, CINAHL, and Cochrane Database of Systematic Reviews. Key words of interest included BMPs, PLIF, TLIF, complications, heterotopic ossification, radiculitis, and osteolysis. All articles, in the English language, between 1990 and 2011 were considered relating to the use of BMPs in PLIF and TLIF constructs., Results: Seventeen articles discussing the use and potential complications of BMPs in PLIF and TLIF constructs were identified and reviewed. The studies were in the range of a level I prospective randomized trial to case reports of complications. There is a wide variation of published complications associated with BMP-augmented PLIF and TLIF constructs., Conclusion: Despite high fusion rates there is a growing body of evidence that the use of BMP in PLIF and TLIF constructs does not come without potential complication. There are appreciable rates of BMP-specific complications, which include heterotopic ossification within the epidural space or neuroforamina, postoperative radiculitis, and endplate osteolysis with interbody device subsidence.

Posterior lumbar interbody fusion and transforaminal lumbar interbody fusion are commonly performed to obtain a 360° arthrodesis through a posterior-only approach. These techniques are currently used in the management of spondylolisthesis, degenerative scoliosis, pseudarthrosis, recurrent disk herniation, and chronic low back pain with associated degenerative disk disease. Several adverse events have been described, including intraoperative neurologic injury, implant migration or subsidence, dural tears, infection, heterotopic ossification, BMP-related radiculitis, and osteolysis. Although the use of newer materials (eg, bone morphogenetic proteins) and procedures (eg, minimally invasive surgery) is on the rise, they are associated with unique concerns. Understanding the potential adverse events and steps that can be taken to prevent, detect, and manage complications is critical in patient counseling and perioperative decision making.

Neuroinflammatory disorders (including human immunodeficiency virus-1 encephalitis, HIVE) are associated with oxidative stress and inflammatory brain injury, and excessive alcohol use can exacerbate tissue damage. Using a murine model of HIVE, we investigated the effects of alcohol abuse on the clearance of virus-infected macrophages and neuroinflammation. Severe combined immunodeficient mice were reconstituted with human lymphocytes, and encephalitis was induced by intracranial injection of HIV-1-infected monocyte-derived macrophages (HIV-1(+) MDM). Animals were fed an ethanol-containing diet beginning 2 weeks before lymphocyte engraftment and for the entire duration of the experiment. Lymphocyte engraftment was not altered by ethanol exposure. Alcohol-mediated immunosuppression in ethanol-fed mice was manifested by a significant decrease in CD8(+)/interferon-gamma(+) T lymphocytes, a fivefold increase in viremia, and diminished expression of immunoproteasomes in the spleen. Although both groups showed similar amounts of CD8(+) T-lymphocyte infiltration in brain areas containing HIV-1(+) MDMs, ethanol-fed mice featured double the amounts of HIV-1(+) MDMs in the brain compared to controls. Ethanol-exposed mice demonstrated higher microglial reaction and enhanced oxidative stress. Alcohol exposure impaired immune responses (increased viremia, decreased immunoproteasome levels, and prevented efficient elimination of HIV-1(+) MDMs) and enhanced neuroinflammation in HIVE mice. Thus, alcohol abuse could be a co-factor in progression of HIV-1 infection of the brain.

Indoleamine 2,3-dioxygenase (IDO) is the rate-limiting enzyme in the kynurenine pathway of tryptophan metabolism. IDO activity is linked with immunosuppression by its ability to inhibit lymphocyte proliferation, and with neurotoxicity through the generation of quinolinic acid and other toxins. IDO is induced in macrophages by HIV-1 infection, and it is up regulated in macrophages in human brain tissue with HIV-1 encephalitis (HIVE). Using a model of HIVE, we investigated whether IDO inhibitor 1-methyl-d-tryptophan (1-MT) could affect the generation of cytotoxic T lymphocytes (CTLs) and clearance of virus-infected macrophages from the brain. Severe combined immunodeficient mice were reconstituted with human peripheral blood lymphocytes, and encephalitis was induced by intracranial injection of autologous HIV-1-infected monocyte-derived macrophages (MDMs). Animals treated with 1-MT demonstrated increased numbers of human CD3+, CD8+, CD8+/interferon-gamma+ T cells, and HIV-1(gag/pol)-specific CTLs in peripheral blood compared with controls. At week 2 after MDM injection in the basal ganglia, mice treated with 1-MT showed a 2-fold increase in CD8+ T lymphocytes in the areas of the brain containing HIV-1-infected MDMs compared with untreated controls. By week 3, 1-MT-treated mice showed 89% reduction in HIV-infected MDMs in brain as compared with controls. Thus, manipulation of immunosuppressive IDO activity in HIVE may enhance the generation of HIV-1-specific CTLs, leading to elimination of HIV-1-infected macrophages in brain.

Background: Brain endothelial cells form the blood-brain barrier (BBB) that regulates solute and macromolecule flux in and out of the brain, leukocyte migration, and maintains the homeostasis of the central nervous system. BBB dysfunction is associated with disruption of tight junctions (TJ) in the brain endothelium. We propose that alcohol abuse may impair BBB permeability through TJ modification., Methods: Primary cultured bovine brain microvascular endothelial cells (BBMEC) were treated with 50 mM ethanol (EtOH), and monolayer tightness was assessed by measurement of transendothelial electrical resistance (TEER). Changes in TEER were correlated with alterations in TJ protein distribution [occludin, zonula occludens-1 (ZO-1), claudin-5] using immunofluorescence (IF). Expression of myosin light chain (MLC) kinase (MLCK), ZO-1, claudin-5, and phosphorylated MLC, occludin and claudin-5 were determined by immunoprecipitation and Western blot. EtOH-induced changes in monocyte migration across in vitro BBB constructs were also examined., Results: EtOH induced a decrease in TEER of BBMEC monolayers that was reversed by EtOH withdrawal. Treatment of BBMEC with EtOH or its metabolite, acetaldehyde, prior to monocyte application resulted in a 2-fold increase in monocyte migration across the BBB. IF demonstrated decrease in claudin-5 staining, occludin translocation from cell borders to cytoplasm and gap formation in EtOH-treated BBMEC monolayer. These changes paralleled significant increase in phosphorylation of MLC, occludin and claudin-5. EtOH-treated BBMEC showed reduction of total occludin and claudin-5 without changes in ZO-1 or MLC. TEER decrease, changes in occludin/claudin staining, increase in MLC, occludin and claudin-5 phosphorylation and enhanced monocyte migration across the BBB were all reversed by inhibition of MLCK. Inhibition of EtOH metabolism in BBMEC also reversed these events., Conclusion: These results suggest that EtOH activates MLCK leading to phosphorylation of MLC, occludin and claudin-5. Cytoskeletal alterations (MLC) and TJ changes (occludin and claudin-5 phosphorylation) result in BBB impairment (decrease in TEER). TJ compromise is associated with increased monocyte migration across the BBB.

A new method for the analysis of organic structural groups from absorption spectra is described. The method is based on the integrated intensity of absorption. It requires only micrograms of analyzed compound, which may be fully recovered after analysis from its solution. The method can be used for different kinds of structural and/or kinetic studies. For example, the reaction kinetics and tautomeric equilibria can be easily studied by this method. The method can also be used for the determination of molecular weight and quantitative composition of polymers, nucleotides, and/or peptides. Hydrolysis or derivatization of the studied compounds is not necessary. On the basis of this method, automatic molecular weight, nucleotide, and peptide analyzers can be constructed.

A spectrophotometric method for the determination of cysteine and cystine in urine is described. This method is a modification of that reported for the determination of cysteine and cystine in proteins. The determination in urine is specific and simple. The colour develops at room temperature and no pre-treatment of urine is needed. Other amino acids, urea, uric acid, ascorbic acid, bilirubin, biliverdin, carbohydrates, hormones, acetone, salicylic acid, small amounts of protein and other common components of urine do not interfere. The method is particularly suitable for the diagnosis of hepatic cystinuria and other diseases characterised by high sulphur-containing amino acids in urine.

Gentisuric acid was synthesized from gentisic acid and glycine ethyl ester. NMR, mass spectrometric and elemental analysis confirmed the product as GU, and physicochemical characteristics were determined. A TLC-densitometric technique was developed to estimate GU and other metabolites of aspirin. Conjugation of gentisic acid with glycine to form GU was catalyzed by a mitochondrial fraction of rat and beef liver. GU was also formed by the rat liver microsomal hydroxylation of salicyluric acid, and phenobarbital pretreatment increased this formation. A random survey showed GU in 76% of SA-positive urines from aspirin-treated patients. Identity of GU in urine from two aspirin-treated patients was confirmed by TLC and mass spectrometric analysis, and hydrolysis of the compound from one patient yielded glycine and gentisic acid. Urine from controls or post-aspirin treatment patients did not show GU by TLC analysis. These results demonstrate for the first time the metabolic formation of GU in animals and its occurrence as a metabolite of aspirin in man.

Time course studies of rho-nitroanisole O-demethylation revealed formaldehyde production in excess of rho-nitrophenol (PNP) and 4-nitrocatechol (NTC) formation by rat liver microsomes. This indicated that these products (PNP, NTC) were metabolised further. The hydroxylation reaction PNP yields NTC showed substrate and product inhibition and a requirement for reduced nicotinamide adenine dinucleotide phosphate and O2 and was localized in liver microsomes. It was strongly activated by ascorbic acid, cysteine, adenosine triphosphate or hydroxylamine in vitro and enhanced by phenobarbital treatment in vivo. Mercapturic derivatives were metabolized to the corresponding hydroxy compounds with the same speed as their parent compounds. Both PNP and NTC were metabolized to the corresponding glucuronide and sulfate conjugates. On the other hand, the PNP or NTC glucuronides and sulfates were metabolized with liver microsomes to PNP and NTC.

The electrophilic properties of the quinone-hydroquinone configuration of anthracycline antibiotics suggests a possible influence on cytochrome P-450-mediated mono-oxygenase reactions. Both doxorubicin and triferric-doxorubicin (a derivative in which the quinone groups are blocked with iron) showed a similar dose-dependent inhibition of liver microsomal drug metabolism. A doxorubicin concentration-related stimulation of NADPH oxidase activity was found to be linear but that for triferric-doxorubicin was asymptotic. Neither inhibitor affected the activity of cytochrome c reductase, cytochrome b5 reductase or cytochrome P-450 reductase. However, doxorubicin did potentiate the inhibitory effect of aniline on cytochrome P-450 reductase and on ethylmorphine metabolism. It is concluded that these anthracyclines inhibit drug metabolism in vitro not by their electron-withdrawing potential but in a manner more similar to that described for type II compounds.

1. The enzymic product formation curves for several enzymes have been studied. 2. The product formation kinetics was related to the initial velocity kinetics and to the diffusion rate limited kinetics. 3. The time curves revealed new constants characterizing structural and binding properties of the enzymic systems which are not revealed from initial velocities. 4. The influence of selected inhibitors on the time curves has been studied. 5. The time curves revealed the specific substrate-receptor binding which was not revealed from initial velocities. 6. The product formation kinetics of acid phosphatase, beta-amylase and NADPH2 cytochrome-c reductase in the absence and in the presence of inhibitors, mercuric acetate and o-iodosobenzoate is described. 7. The time curves revealed the binding of cytochrome-c to the specific natural protein receptors. 8. The activation energies of acid phosphatase and beta-amylase were determined from the time curves.

A rapid thin-layer densitometric method for the quantitative determination of salicylic acid and its metabolites in urine or plasma is described. The method is specific and very sensitive. Nanogram quantities of salicylic acid and its metabolites, both free and conjugated, may be estimated. Known metabolites, as well as the newly described gentisuric acid, were estimated quantitatively in urine from a patient treated with aspirin.

Hydroxylation systems containing cytochromes, proteins and ascorbic acid were studied at physiological pH (7.4) under O2 or N2 with added H2O2. Proteins inhibited aromatic hydroxylation of p-nitrophenol or oxidative demethylation of ethylmorphine in ascorbic acid-containing systems incubated under O2, but strongly activated the systems containing H2O2. Cytochrome c and partially purified cytochrome P-450 from rat liver microsomal preparations activated the system in either O2 or H2O2. The systems needed ascorbic acid (or other enol structures) for activation. Cytochrome iron participated probably in the activation of O2, whereas cytochrome protein participated in a free radical activation of H2O2 (or of O2).

A new spectrophotometric method for quantitative determination of tryptophan and tyrosine in peptides and proteins is described. It is based on two specific color reactions, the reaction of tryptophan with formaldehyde and the reaction of tyrosine and tryptophan with hydroxylamine and ceric cations. By combination of these two reactions both tyrosine and tryptophan can be determined simultaneously. Tyrosine and/or tryptophan bound in peptides and/or proteins react independently of the rest of the peptide or protein molecule. The method is simple, accurate, and sensitive. Hydrolysis is not necessary.

1. The dependency of product formation on reaction time and on enzyme concentration was studied with certain purified enzymes and liver microsomal mixed-function oxidase system. 2. The product-formation-time relation was estimated with different enzyme and/or substrate concentrations for the reactions limited by diffusion. 3. The kinetic constants of the product-time relations in these diffusion systems have been verified experimentally to give a more specific and detailed characterization of an enzyme system under a variety of physiologic and reaction conditions. 4. The influence of inhibitors and activators in vitro, the effect of enzyme preparation technic, aging, starvation, pretreatment with somatotropic hormone, phenobarbital or 3-methylcholanthrene, and the effect of a tumor were studied. 5. The influence of vitamin C deficiency in guinea pig was also studied.