A novel progranulin mutation associated with variable clinical presentation and tau, TDP43 and alpha-synuclein pathology.

Mutations in the progranulin (GRN) gene have recently been reported as a cause of the frontotemporal dementia (FTD) syndrome. We performed a clinical, neuropathological and molecular genetic study of two families with FTD and the same novel mutation in GRN. Age of onset ranged from 35 to 75 years and all individuals progressed to a severe dementia syndrome with a mean disease duration of approximately 6-10 years. Variable clinical presentations included language impairment, behaviour change or parkinsonism. Seven total autopsies in the families (five in Family 1, two in Family 2) showed gross and microscopic evidence of neuronal loss in the neocortex, striatum, hippocampus and substantia nigra. All cases with material available for immunohistochemistry had cytoplasmic and intranuclear ubiquitin positive, tau negative inclusions that stained best with an antibody to the TDP43 protein. In addition, all but one had evidence of distinctive tau pathology. Two cases in Family 1 also had alpha-synuclein (SNCA) pathology, one with diffuse neocortical inclusions and neurites and unusual striatal cytoplasmic inclusions. Affected persons in both families had the same mutation in GRN (c.709-2A>G). A minigene construct showed that this mutation alters splicing of exon 7 and results in reduced mRNA message in brain. A single GRN mutation in these two families was associated with variable clinical presentations consistent with the FTD syndrome. All cases had ubiquitin/TDP43 immuno-positive inclusions and most had additional tau pathology. Two cases had SNCA pathology. These findings suggest a link between mutations in GRN and aggregation of tau, TDP43 and SNCA.

Mutations in exon 3 of the lipoprotein lipase gene segregating in a family with hypertriglyceridemia, pancreatitis, and non-insulin-dependent diabetes.

A proband with chylomicronemia, pancreatitis, and non-insulin-dependent diabetes (NIDDM) bears two different mutations in exon 3 of the lipoprotein lipase (LPL) gene: a missense mutation, 75Arg-->Ser, inherited through the paternal line and a truncation, 73Tyr-->Ter, through the maternal line. NIDDM appeared to be independently segregating. The R75S mutant was studied in extracts and media from transfected COS-1 cells. Detectable amounts of catalytically competent R75S LPL suggested destabilization of the active homodimer as with exon 5 mutants (Hata et al. 1992. J. Biol. Chem. 267:20132-20139). Hydrolysis of a short-chain fatty acid ester indicated that R75S does not directly affect activation of LPL by apoC-II. Subjects with NIDDM and wild-type LPL, and nondiabetic middle-aged carriers of the 73Tyr-->Ter truncation had moderate hypertriglyceridemia (260-521 mg/dl) and reduced high density lipoprotein cholesterol. A maternal aunt with NIDDM carried the truncation. Her phenotype (triglycerides of 5,300 mg/dl, eruptive xanthomatosis, and recurrent pancreatitis) was as severe as that in homozygotes or compound heterozygotes. We conclude: (a) diabetic carriers of dysfunctional LPL alleles are at risk for severe lipemia; and (b) the physiologic defects in NIDDM may be additive or synergistic with heterozygous LPL deficiency.

Antiproliferative agents and differential survival between normal and cancer cells.

A basic difference in response between normal cells (primate fibroblasts) and colonic cancer cells (human and rodent) to the antiproliferative action of both N-(phosphonacetyl)-L-aspartate and thymidine is described in this report. Both normal and colonic cancer cells, when cultured in the presence of these agents, cease to increase their cell numbers. Evidence is presented to show that the normal cells respond to deprivation of pyrimidine nucleotide induced by these agents by simple growth arrest, in which a quiescent state may be maintained for prolonged periods without cell death. Cancer cells are shown to respond in a characteristically different manner in which cell division continues accompanied by limited cell survival, with the surviving population representing a balance of these opposing processes. The extent to which these in vitro findings, based on a limited number of comparisons under restrictive artificial conditions, relate to the in vivo state remains to be established.

Selective inhibition of pyrimidine biosynthesis and effect on proliferative growth of colonic cancer cells.

A highly selective inhibition of de novo pyrimidine synthesis in the intact cell has been demonstrated by the action of N-(phosphonacetyl)-L-aspartate (PALA), a transition-state analog inhibitor of the reaction catalyzed by asparate transcarbamylase. The effect of pyrimidine deprivation induced by this agent on the viability and survival of human normal (WI-38) and colonic cancer cells (HT-29) was examined. The PALA-treated, pyrimidine-deprived cells failed to grow but demonstrated a normal rate of glucose utilization with impaired glycogen synthesis. Pyrimidine deprivation and lack of cell growth were maintained long after PALA removal. Growth inhibition of HT-29 cells by PALA was found to reflect an apparent steady state between newly formed and dying cells induced by limited pyrimidine availability. The highly selective nature of PALA action was confirmed by the ability of an exogenous source of pyrimidine to restore the normal growth pattern of the cell. Significant antitumor activity of PALA was found against a transplantable colonic tumor (line 26) carried in mice.

Sugar hydrolases of the infant rat intestine and their arrangement of the brush border membrane.

Lactase and maltase, the predominant sugar hydrolases associated with the intestinal brush bordermembrane of the suckling rat, were purified essentially free of the other to near homogeneity (lactase at specific activity 23, maltase at specific activity 58), and their specific physiocochemical properties determined. Antisera prepared to each showed by immunodiffusion a single common precipitin line with pure enzyme and solubilized proteins of the brush border membrane. Brush border membranes were purified 26--35-fold from infant rat intestine. Membranes prepared from 10-day-old rats contained 32% protein, 43% lipid and 25% carbohydrate with lactase and maltase estimated to comprise in excess of 10% and 2%, respectively, of the membrane protein. Immunotitration curves of lactase and maltase showed equivalent antibody binding by the membrane-bound and free enzyme forms. Furthermore, antibody binding to one enzyme did not affect the immunotitration curve or the extractability (by papain or Triton X-100) of the other membrane-bound enzyme. It was concluded that the lactase and maltase molecules are attached singly on the external membrane surface in a spatially independent manner with their antigenic sites as freely available to antibody binding as exhibited by their papain-solubilized counterparts.

The nature of maturational decline of intestinal lactase activity.

We have examined the nature of the decline of lactase (EC 3.2.1.23) activity in the maturing rat intestine. It was established in an initial study that the activity decline reflected a proportional reduction in the concentration of the enzyme protein. Accumulation patterns of label into lactase, total intestinal proteins and sucrase (EC 3.2.1.48)-isomaltase (EC 3.2.1.10) were compared, 4 h following administration of a tracer dose of [3H]leucine to weanling rats exhibiting a wide range of lactase decline. Accumulation of increasing amounts of label in total intestinal proteins and sucrase-isomaltase pools was found to accompany the lactase decline, in contrast to accumulation of a constant amount of label in the declining lactase pools. The pattern of increased label accumulation in total intestinal proteins was shown in a corollary study to reflect a corresponding acceleration of total protein synthesis. On this basis, the finding of a constant amount of label in the declining lactase pools suggested a constant synthesis of lactase. We proposed earlier that associated reductions in enterocyte life-span (leading to correspondingly less lactase accumulation) rather than suppressed synthesis may provide the primary causal basis of lactase decline in the postweaned mammal.

Intestinal and hepatic cholesterogenesis in hypercholesterolemic dyslipidemia of experimental diabetes in dogs.

We previously reported that dog diabetes results in hypercholesterolemia and the accumulation of a high-density lipoprotein (HDL) subclass, HDL1. Hypercholesterolemic diabetic rodents exhibit hyperphagia, intestinal hypertrophy, and increased intestinal cholesterol synthesis and absorption; intestinal 3-hydroxy-3-methylglutaryl (HMG) CoA reductase activity is increased, whereas hepatic activity is unchanged or reduced. To determine whether similar mechanisms operate in the hypercholesterolemic diabetic dog, we measured hepatic and intestinal cholesterologenesis. Streptozocin-alloxan-induced diabetic dogs allowed access to food ad libitum were hyperphagic and hypercholesterolemic (10.1 vs. 4.47 mM) but normotriglyceridemic. Plasma HDL1 concentrations were markedly increased. Differences in renal and hepatic function were not statistically significant, except serum alkaline phosphatase, which was elevated 4-fold (P = 0.0003). Urinary mevalonate, an index of whole-body cholesterol synthesis, was increased 6-fold. Intestinal and hepatic weights were both increased, and direct measurements showed crypt and villus thickening. The activity of HMG CoA reductase per gram organ weight was increased 1.7-fold in liver and 2.1-fold in intestine. Calculated whole-organ activity in intestine was nearly twice that in liver. These observations provide strong evidence that intestinal cholesterogenesis is involved in the pathogenesis of hypercholesterolemia in dog diabetes and support the conclusion that increased cholesterol synthesis plays a role in the hypercholesterolemia of diabetes.

Molecular screening of the lipoprotein lipase gene in hypertriglyceridemic members of familial noninsulin-dependent diabetes mellitus families.

Hypertriglyceridemia is common among individuals with noninsulin-dependent diabetes mellitus (NIDDM), and heterozygous lipoprotein lipase (LPL) mutations may result in the syndrome of familial hypertriglyceridemia and low levels of high density lipoprotein (HDL) cholesterol. To test the hypothesis that heterozygous LPL mutations predispose to the hypertriglyceridemia and low HDL cholesterol levels observed among members of familial NIDDM families, we examined 36 members and 3 unrelated spouses selected from members of 20 pedigrees for triglyceride levels exceeding the age- and sex-specific 95th percentile. Eighteen pedigree members and 2 spouses were diabetic. LPL exons 1-9 were screened by single strand conformation polymorphism analysis. Six different variants were detected in exons 2, 3, 4, 8, and 9, including 4 (exons 3, 4, and 8) silent nucleotide substitutions. A common nonsense mutation (exon 9; Ser-->Ter) was present in 2 pedigrees, and a missense mutation (exon 2; Asp-->Asn) was also present in members of 2 pedigrees. Analysis of members of these families suggested an association of the exon 2 variant with hypertriglyceridemia, although this trend was no longer significant when individuals with diabetes were excluded from the analysis. The variant enzyme was not present among 83 random control individuals, and when expressed in COS-1 cells, it was similar to the wild type with respect to specific activity, heparin binding, and heat stability. Our data suggest that coding region mutations of the LPL gene cannot account for the elevated triglyceride and low HDL levels noted in diabetic individuals and their relatives in most NIDDM pedigrees, but the exon 2 Asn variant may contribute to the hypertriglyceridemia in some families.

Acute dyslipoproteinemia induced by interleukin-2: lecithin:cholesteryl acyltransferase, lipoprotein lipase, and hepatic lipase deficiencies.

Recombinant human interleukin-2 (rIL-2) is used to treat refractory cancers. During such treatment, patients develop severe hypocholesterolemia along with striking alterations in the concentration and composition of the circulating lipoproteins. The present study was undertaken to gather information about the pathogenesis of these abnormalities. Patients were studied before-, during- and after a 5-day course of high dose i.v. rIL-2. Whole plasma cholesterol was markedly reduced by rIL-2 administration (52%; P < 0.001), whereas the triglyceride concentration did not change. Thus, the lipoproteins became triglyceride enriched (P = 0.004). Low density lipoprotein cholesterol, apolipoprotein B (apoB), high density lipoprotein cholesterol, and apoA-I concentrations all decreased. Esterified cholesterol levels were markedly reduced. Total plasma apoE increased markedly, and two kinds of abnormal particles appeared: 1) beta-migrating, very low density lipoproteins; and 2) discoidal, apoE- and phospholipid-containing particles with abnormal density and electrophoretic mobility. The activities of two lipoprotein triglyceride hydrolases, lipoprotein lipase and hepatic lipase, fell significantly during treatment and returned promptly to pretreatment levels after rIL-2 was discontinued. Lecithin:cholesteryl acyltransferase (LCAT) activity also decreased significantly (64%) during treatment, but in contrast to the lipases, remained low for at least 5 days after the last dose of rIL-2 (P < 0.001). High dose i.v. rIL-2 induces severe dyslipidemia with deficiencies of both postheparin lipases and acute LCAT deficiency. Most, if not all, of the lipoprotein changes observed are explained by the LCAT deficiency that follows IL-2-induced hepatocellular injury and cholestasis.

Effect of age and caloric restriction on bleomycin-chelatable and nonheme iron in different tissues of C57BL/6 mice.

The objective of this study was to test the hypothesis that the widely observed age-associated increase in the amounts of macromolecular oxidative damage is due to an elevation in the availability of redox-active iron, that is believed to catalyze the scission of H2O2 to generate the highly reactive hydroxyl radical. Concentrations of bleomycin-chelatable iron and nonheme iron were measured in various tissues and different regions of the brain of mice fed on ad libitum (AL) or a calorically restricted (to 60% of AL) diet at different ages. The concentrations of these two pools of iron varied markedly as a function of tissue, age, and caloric intake. There was no consistent ratio between the amounts of nonheme and the bleomycin-chelatable iron pools across these conditions. Nonheme iron concentration increased with age in the liver, kidney, heart, striatum, hippocampus, midbrain and cerebellum of AL animals, whereas bleomycin-chelatable iron increased significantly with age only in the liver. Amounts of both nonheme and bleomycin-chelatable iron remained unaltered during aging in the cerebral cortex and hindbrain of AL mice. Caloric restriction had no effect on iron concentration in the brain or heart, but caused a marked increase in the concentration of both bleomycin-chelatable and nonheme iron in the liver and the kidney. The results do not support the hypothesis that accumulation of oxidative damage with age, or its attenuation by CR, are associated with corresponding variations in redox-active iron.

Altered maturation of small intestinal function in the absence of intraluminal nutrients: rapid normalization with refeeding.

The absence of intraluminal nutrients during weaning in rats was shown to result in altered intestinal growth and maturation. In this study intestinal length, mucosal weight, DNA, protein, and total disaccharidase activities were significantly lower in animals sustained by intravenous nutrients over the normal weaning age than were normally weaned controls but were greater than preweaning values. Absorptive capacity for sucrose (assessed by hydrogen-gas production) was diminished, directly linking incomplete maturation of sucrase to diminished intestinal function. To determine whether these alterations were permanent, rats previously deprived of intraluminal nutrients over the weaning period were refed. Eight days after refeeding, all variables except total lactase had attained values found in normally weaned age-matched controls, including absorptive capacity for sucrose. Although intestinal growth and maturation is abnormal in the absence of intraluminal nutrients during weaning, the abnormalities are not permanent and are rapidly corrected upon refeeding.

Maturational patterns of carbohydrases in the ileal remnant of rats after jejunectomy at infancy.

The enteric epithelium of suckling rat undergoes dramatic functional and cytokinetic changes (redifferentiation) with maturation. Ileal epithelial maturation was studied in infant rats subjected to 60% proximal enterectomy at age 10 d in an effort to examine redifferentiation mechanisms. Two months after resection the residual ileal remnant was increased in diameter, weight, total protein, and DNA per unit length compared with ileal segments from control littermates that had laparotomy without resection. The residual ileum demonstrated increased sucrase activity per unit length but was indistinguishable from control ileal segments in activity per unit DNA or villus distribution. Lactase activity was negligible in all segments of the residual intestine. Villus height and crypt depth were increased in the residual ileum with slight increases in cell turnover and cell-migration rates. These results show the presence of an intrinsic program for regulation of ileal epithelial maturation and its resistance to alteration by a major stimulus applied before its expression.

Hydrogen gas excretion after sucrose gavage in the fasted rat.

The effect of a 3-day fast on the functional ability of the adult rat to hydrolyze and absorb sucrose was determined. The evaluation was based on previous studies which have shown the total amount of hydrogen gas (H2) excreted by the animal to reflect the extent of undigested carbohydrate entering the colon from the small intestine. H2 excretion was measured using a gas chromatographic technique in experimental (72 h fasted) and control (12 h fasted) animals after administration of sucrose by gastric gavage. Total H2 excretion was 3-fold higher in the experimental animals (n = 5) than in the controls (n = 5) (p less than 0.005) indicating a significant increase of sucrose malabsorption in the experimental animals. Administration of a second dose of sucrose 8 to 9 h after the first dose (refeeding) resulted in markedly decreased malabsorption relative to the first administration in both experimental (n = 2) and control (n = 2) animals. These results suggest that a 3-day fast markedly impairs the ability of the intestine to hydrolyze and absorb sucrose and that refeeding rapidly restores the ability to utilize this substrate. H2 excretion was similar between experimental and control animals after the administration of lactulose, a nonabsorbed and nondigested carbohydrate, suggesting that the observed results of the sucrose studies were independent of any possible changes in the intestinal microflora.

Diabetes increases hepatic hydroxymethyl glutaryl coenzyme A reductase protein and mRNA levels in the small intestine.

Previous studies have shown that both cholesterol synthesis and the activity of hepatic hydroxymethyl glutaryl coenzyme A (HMG CoA) reductase, the rate-limiting enzyme in cholesterol synthesis, are increased in the small intestine of a wide variety of different animal models of diabetes. In the present study, we demonstrate that the mass of HMG CoA reductase protein is increased in the small intestine of both streptozocin-induced diabetic rats (2.5-fold) and streptozocin/alloxan-induced diabetic dogs (2.4-fold). These increases in HMG CoA reductase protein mass are of a magnitude similar to the previously observed increases in either HMG CoA reductase activity and/or cholesterol synthesis in the small intestine of diabetic animals. Furthermore, mRNA levels for HMG CoA reductase in the small intestine of diabetic rats and diabetic dogs are increased 2.1- and 1.7-fold, respectively. These results suggest that the increase in HMG CoA reductase protein levels in the small intestine of diabetic animals is due to an increase in mRNA levels. In contrast, mRNA levels for HMG CoA reductase in the liver of diabetic rats are not increased. Additionally, mRNA levels for the low-density lipoprotein (LDL) receptor are also increased in the small intestine of diabetic animals (rats, 43%; dogs, 59%). The increase in small-intestinal cholesterol synthesis has the potential for adversely affecting lipoprotein metabolism and increasing the risk of atherosclerosis in diabetes.

The role of bilirubin production in breast-fed infants with elevated serum bilirubin concentrations at 2 weeks of life.

The carboxyhemoglobin level (COHb), an accepted qualitative index of bilirubin production, was measured in normal, full-term, breast-fed (n = 9) or formula-fed (n = 11) infants at 2 days and 2 weeks of life. The mean COHb did not differ significantly at 2 days and 2 weeks in either of the groups, nor did the mean COHb differ between the groups at 2 weeks. The mean serum bilirubin concentration was lower in the formula-fed infants compared to the breast-fed infants at 2 weeks (p less than 0.05). The mean serum bilirubin concentration decreased by only 14 percent among the breast-fed infants, and actually increased in three infants by 2 weeks. In comparison, the mean serum bilirubin concentration of the formula-fed infants decreased by 61 percent (p less than 0.05), with the serum bilirubin concentration decreasing in each infant by 2 weeks. These findings are consistent with the generally held belief that bilirubin production is not the primary etiology of elevated serum bilirubin concentrations associated with breast-feeding in the second week of life. However, continued high bilirubin production at 2 weeks may contribute to the potential for significant jaundice in some infants with impaired hepatic function or increased enterohepatic circulation of bilirubin.

Substrate and site specificity of hydrogen peroxide generation in mouse mitochondria.

The objective of this study was to elucidate the mechanisms of mitochondrial H2O2 generation in mouse organs by determining the nature of their differences in substrate utilization, inhibitor sensitivity, and the site specificity affecting H2O2 production. Mitochondria were isolated from heart, brain, and kidney and the rate of H2O2 generation was measured using the FADH-linked substrates succinate and alpha-glycerophosphate as well as the NADH-linked substrates pyruvate/malate, beta-hydroxybutyrate, and glutamate. Respiratory inhibitors, antimycin and rotenone, were added singly and sequentially to each substrate-supported H2O2 generation reaction mixture to determine the mitochondrial site(s) of generation and the optimal condition(s) for maximal rates of generation. Succinate supported the highest rate of mitochondrial H2O2 generation. Moreover, it was the preferred substrate for the heart mitochondria. alpha-Glycerophosphate is a poor substrate for H2O2 generation in heart mitochondria. Inhibitor studies showed that heart mitochondria were the most sensitive and responsive to antimycin, while brain was the most sensitive to rotenone. A surprising finding was that NADH-linked substrate-supported H2O2 generation in kidney mitochondria was not responsive to rotenone. The contribution from each of the three sites (ubiquinone, NADH dehydrogenase, and alpha-glycerophosphate dehydrogenase) of mitochondrial H2O2 generation to the total was both substrate and organ dependent. Results indicate that assay conditions must be considered before comparisons of sites and rates of mitochondrial H2O2 generation among different organs can be made.

Age-related changes in activities of mitochondrial electron transport complexes in various tissues of the mouse.

The purpose of the present study was to examine the role of mitochondria in the aging process by determining whether the activities of various electron transport chain oxidoreductases are deleteriously affected during aging and whether the hypothesized age-related alterations in different tissues follow a common pattern. Activities of respiratory complexes I, II, III, and IV were measured in mitochondria isolated from brain, heart, skeletal muscle, liver, and kidney of young (3.5 months), adult (12-14 months), and old (28-30 months) C57BL/6 mice. Activities of some individual complexes were decreased in old animals, but no common pattern can be discerned among various tissues. In general, activities of the complexes were more adversely affected in tissues such as brain, heart, and skeletal muscle, whose parenchyma is composed of postmitotic cells, than those in the liver and kidney, which are composed of slowly dividing cells. The main feature of age-related potentially dysfunctional alterations in tissues was the development of a shift in activity ratios among different complexes, such that it would tend to hinder the ability of mitochondria to effectively transfer electrons down the respiratory chain and thus adversely affect oxidative phosphorylation and/or autooxidizability of the respiratory components.

Synthesis and accumulation of protein and carbohydrases along the rat villus column.

Enterocytes of the intestinal mucosa of infant and adult rats continuously proliferate in the crypt, mature as they migrate along the villus column, and are discharged from the villus tip. We examined the synthesis patterns of total protein, lactase-phlorizin hydrolase, sucrase-isomaltase, and maltase-glucoamylase as well as the accumulation of these enzymes in cells during migration along the villus. Labeled leucine was administered intraperitoneally to suckling and young adult rats, and radioactivity was determined in protein and digestive carbohydrase pools of developing villus cells separated sequentially from tip to base of the villus column. The developing cells were found to continuously accumulate protein and carbohydrates as they ascended the villus column. In addition, incorporation of radioactivity into total protein and carbohydrase pools occurred at generally constant rates along the length of the villus. These studies showed that the differentiated enterocyte of both infant and young adult rat intestine exhibits a pattern of continuous growth while migrating the length of the villus column and maintains synthesis of protein and digestive carbohydrates at generally constant rates during this time.

Pseudouridine-deficient transfer RNAs from Escherichia coli B and their use as substrates for pseudouridine synthetase.

Transfer RNAs isolated from Escherichia coli B grown in the presence of 2-thiouracil are deficient in pseudouridine. Much of this deficiency is from the T psi C region, which has only about 50% of its normal pseudouridine content. The other modified nucleoside from this region, ribothymidine, is reduced by only about 10%. Studies showed that 2-thiouracil is incoproated into the RNA of E. coli during growth in the presence of the analog. This incorporation appears to result from the replacement of uracil, occur in a random manner, and involve all RNA species. The extent of incorporation varies from 1 to 3 mol %, depending upon the preparation and RNA species examined. Electrophoresis on polyacrylamide gels and chromatography on Sephadex G-75 and reverse phase (Systen 5) columns of normal and 2-thiouracil-containing tRNAs revealed no profile differences. No accumulation of any precursor tRNA in the thiopyrimidine-treated cells is found. A partial recovery of the pseudouridine content of 2-thiouracil-containing tRNAs can be achieved in vivo by removal of the 2-thiouracil from the culture media. These transfer RNAs have also been used as substrates to study the properties of a partially purified preparation of pseudouridine synthetase II invitro and should be useful as substrates in the further purification of this enzyme.

Carbon monoxide in blood: an improved microliter blood-sample collection system, with rapid analysis by gas chromatography.

We examined the sensitive assay for carboxyhemoglobin based on reaction with K3Fe(CN)6 and gas chromatography of the liberated CO. Our improvements included increased baseline stability, shorter analysis time, and simpler standardization. EDTA-containing Vacutainer Tubes (lavender-stoppered) increase the carboxyhemoglobin content of blood stored in them. The carboxyhemoglobin content of blood stored in capillary tubes containing solid heparin and saponin remained stable for two weeks. Using our improved procedures, we measured the carboxyhemoglobin content of blood from adults and neonates collected via venipuncture or heel or fingersticks. We observed no significant difference in carboxyhemoglobin content of blood obtained by venipuncture or heel stick for premature infants, 0.19 +/- 0.04 vs 0.18 +/- 0.03 mL of CO per 100 mL of blood, respectively (mean +/- SD). Nonsmoking adults (n = 19) had CO values (mean +/- SD) of 0.19 +/- 0.03 and 0.17 +/- 0.04 mL per 100 mL of blood, and smoking adults (n = 7) gave CO values of 0.96 +/- 0.49 and 0.91 +/- 0.49 mL/dL, for venipuncture and fingerstick, respectively.