Continued improvement in pressure-flow parameters in men receiving finasteride for 2 years. Finasteride Urodynamics Study Group.

OBJECTIVES: To assess the long-term effects of finasteride on pressure-flow parameters in men with urodynamically documented bladder outflow obstruction (BOO). METHODS: One hundred twenty-one men with benign prostatic enlargement (BPE) and lower urinary tract symptoms (LUTS) underwent a pressure-flow study (PFS) at 1 of 11 clinical centers. The PFS technique was standardized, and all tracings were read by a single reader unaware of the treatment group. Patients who were obstructed according to a modified Abrams-Griffiths nomogram were randomized to 5 mg finasteride (n = 81) or placebo (n = 40) for 12 months; all patients continuing into an open extension received finasteride during the second 12 months of therapy. Results of the initial 12-month study demonstrated the benefit of finasteride treatment on PFS parameters. To examine the continuing effects over time, an analysis of the data from 54 patients who completed 24 months of treatment with finasteride is provided. RESULTS: Detrusor pressure at maximum flow (PdetQmax) continued to decrease during the second 12 months of therapy (decreases of 5.3 and 11.7 cm H2O at months 12 and 24, respectively). The percentage of patients obstructed by Abrams-Griffiths classification decreased from 76.2% at baseline to 66.7% at month 12 and 59.6% at month 24. An intention-to-treat analysis yielded similar results. CONCLUSIONS: Finasteride improves urodynamic measures of obstruction in men with BPE and LUTS, with continued improvement during the second 12 months of therapy.

Oral administration of the growth hormone secretagogue MK-677 increases markers of bone turnover in healthy and functionally impaired elderly adults. The MK-677 Study Group.

Growth hormone (GH) stimulates osteoblasts in vitro and increases bone turnover and stimulates osteoblast activity when given to elderly subjects. Probably a major effect of GH on bone is mediated through stimulation of either circulating or locally produced insulin-like growth factor I (IGF-I). We determined the effect of chronic administration of the GH secretagogue, MK-677, on serum IGF-I and markers of bone turnover in 187 elderly adults (65 years or older) enrolled in three randomized, double-blind, placebo-controlled clinical studies lasting 2-9 weeks. Urine was collected for determination of N-telopeptide cross-links (NTXs), a marker of bone resorption, and blood was collected for determination of serum osteocalcin and bone-specific alkaline phosphatase (BSAP), as bone formation markers, and serum IGF-I levels pre- and post-treatment. Dose response data were initially obtained in healthy elderly subjects who received oral doses of 10 mg or 25 mg of MK-677 or placebo for 2 weeks (n = 10-12/group). Treatment with 10 mg and 25 mg of MK-677 for 2 weeks increased mean urine NTXs 10% and 17%, respectively (p < 0.05 vs. placebo). Additionally, 50 healthy elderly subjects received either placebo (n = 20) for 4 weeks or 25 mg of MK-677 (n = 30) daily for 2 weeks followed by 50 mg daily for 2 weeks. MK-677 increased mean serum osteocalcin by 8% (p < 0.05 vs. placebo). In both studies, MK-677 increased serum IGF-I levels significantly (55-94%). Subsequently, the biological effects of MK-677 were studied in 105 elderly subjects who met objective criteria for functional impairment. Subjects were randomized to receive oral doses of placebo for 9 weeks or either 5, 10, or 25 mg of MK-677 daily for an initial 2 weeks followed by 25 mg of MK-677 daily for the next 7 weeks(n = 63 on MK-677 and n = 28 on placebo completed 9 weeks of therapy). Treatment with MK-677 (all MK-677 groups combined) for 9 weeks increased mean serum osteocalcin by 29.4% and BSAP by 10.4% (p < 0.001 vs. placebo) and mean urinary NTX excretion by 22.6% (p < 0.05 vs. placebo). The change from baseline serum osteocalcin correlated with the change from baseline serum IGF-I in the MK-677 group (r = 0.37; p < 0.01). In conclusion, once daily dosing with MK-677, an orally active GH secretagogue, stimulates bone turnover in elderly subjects based on elevations in biochemical markers of bone resorption and formation.

Improvement of pressure flow parameters with finasteride is greater in men with large prostates. Finasteride Urodynamics Study Group.

PURPOSE: We assess the effect of finasteride, a 5alpha-reductase inhibitor, on objective voiding parameters in men with lower urinary tract symptoms and benign prostatic enlargement on digital rectal examination (known as clinical benign prostatic enlargement) in a double-blind placebo controlled multicenter study using strict standard pressure flow study techniques. MATERIALS AND METHODS: A modification of the Abrams-Griffiths nomogram was used by 1 reader to ensure that all patients met objective criteria for bladder outlet obstruction at baseline. After performing a pressure flow study patients with obstruction were randomized 2:1 to receive 5 mg. finasteride (81) or placebo (40) daily. A second pressure flow study was performed at month 12. At baseline and month 12 free urinary flow studies and transrectal ultrasound were performed, and International Prostate Symptom Score questionnaires were completed. Patients were treated between May 1994 and July 1996. RESULTS: Finasteride caused a significant decrease (-8.1 cm. water) in detrusor pressure at maximum flow (p <0.05 versus placebo p = 0.02), increase (+1.1 ml. per second) in maximum flow rate (p <0.05 versus placebo p = 0.02) and decrease (-22.8%) in prostate volume (p <0.05 versus placebo p <0.001). Men with prostates larger than 40 cc had greater improvement in detrusor pressure at maximum flow (between group difference -14.5 cm. water, 95% confidence interval -26.2 to -2.6, p = 0.02) and maximum flow rate (mean treatment effect +1.6 ml. per second, 95% confidence interval -0.2 to 3.0, p = 0.02) compared to those with prostates 40 cc or less (between group differences not significant). CONCLUSIONS: Finasteride treatment resulted in improvements in urodynamic parameters, which were greater in men with large prostates.

Repeated pressure-flow studies in the evaluation of bladder outlet obstruction due to benign prostatic enlargement. Finasteride Urodynamics Study Group.

Test-retest reliability of repeated voids in pressure-flow studies and the influence on maximum flow rate (Q(max)pQ), detrusor pressure at maximum flow rate (p(det)Qmax), voided volume, and residual urine were studied. Also the agreement in interpretation of pressure-flow tracings between investigators and a single blinded central reader acting as a quality control center (QCC) were assessed. In addition, correlations between p(det)Qmax and patient age, International Prostate Symptom Score (IPSS), free maximum flow rate (Qmax), and prostate volume were calculated. Using suprapubic pressure recording, 216 men with lower urinary tract symptoms (LUTS) due to benign prostatic enlargement (BPE) were investigated in 11 centers. In each pressure-flow study, three sequential voids were performed, and quality controlled recordings were analyzed for Q(max)pQ and p(det)Qmax by the QCC. Trans rectal ultrasound was used to measure the prostate volume. Mean Q(max)pQ did not change, but p(det)Qmax decreased significantly in the second and third sequential voids. Using the Abrams-Griffiths nomogram definition of obstruction, 125 patients (67%) were classified as obstructed from the first void, but only 111 patients (59%) from the third void. The agreement between the investigator assessment and that of a single blinded reader was good. There was no significant correlation between p(det)Qmax and patient age, IPSS, and Qmax, whereas a modest correlation was found between p(det)Qmax and prostate volume. In summary, there was no significant change in Q(max)pQ, but p(det)Qmax decreased for the three consecutive voids, which can be explained by a decrease in outlet resistance. The agreement between the investigator and QCC interpretations shows the value of a standardized technique, supporting the feasibility of multicenter urodynamic studies. There is a modest, but statistically significant, correlation between detrusor pressure and prostate size, supporting the hypothesis that prostate size is a contributing factor in symptomatic BPH.

Are performance-based measures sufficiently reliable for use in multicenter trials? Musculoskeletal Impairment (MSI) Study Group.

BACKGROUND: The literature contains few reports of the test-retest reliability of performance-based measures. The purpose of this study was to determine the test-retest reliability of a battery of seven timed, performance-based measures used to assess the functional limitations of frail, older adults. METHODS: One hundred and five frail, elderly subjects were twice administered a battery of timed tests approximately 2 weeks apart: 8-foot walk, get-up-and-go test, stair climb, single and repetitive standing from a chair, and single and repetitive 10-pound lifts with the upper limbs. Agreement between the mean times recorded for accomplishing each task at the two administrations was assessed. RESULTS: Intraclass correlation coefficients ranged from .25 for the single chair stand to .79 for the 8-foot walk. Only the time taken for the single 10-pound lift was significantly greater at the first administration as compared with the second. CONCLUSIONS: Timed performance-based measures have a wide range of test-retest reliability. Performance-based protocols that reflect familiar tasks with discrete starting and ending points may achieve higher reliability than tasks that are unfamiliar to subjects or may have ambiguous elements in them.

Growth hormone secretagogues: mechanism of action and use in aging.

GH secretagogues present a tool for furthering our understanding of the control of GH secretion, as well as a unique therapeutic opportunity. These compounds activate the receptors of a putative endogenous ligand in the hypothalamus and pituitary. Acting as functional somatostatin antagonists, GH secretagogues potentiate the actions of GHRH on GH secretion, enhancing pulsatile GH secretion. The clinical target of the elderly population presents significant challenges to drug development. Age-related musculoskeletal impairment as a result of muscle wasting (sarcopenia) is not well recognized as a clinical syndrome. In addition, given the inherent day to day variability in function in the "frail" target population as well as the presence of a host of concomitant conditions, the appropriate patient population to be studied remains to be defined, and demonstration of clinically meaningful efficacy may be difficult. It is not clear whether it will be useful to restore to young levels the activity of the GHIGF-I axis in aging. Nevertheless, if beneficial effects on strength, similar to those demonstrated with GH79 can be shown, GH secretagogues could provide a well-tolerated clinical approach for treating or preventing sarcopenia, and perhaps, even forestall the inevitability of age-associated decline in function and independence. Such efficacy would have a great social impact.

Building the French Muscular Dystrophy Association: the role of doctor/patient interactions.

The process of creating the French Muscular Dystrophy Association (AFM) is analysed through the interactions between the medico-scientific community on the one hand, and patients and their families on the other, from the 1950s to 1986. Each stage of its development was characterized by a particular mode of co-operation between lay people and doctors. Starting in 1958, the Association built a close relationship with a single partner, Jean Demos, a paediatrician and biochemist who developed a new vasodilation therapy based on his controversial vascular theory of muscular dystrophy. Around 1966, some AFM members, disappointed by Demos' treatment, decided to collaborate with other specialists, primarily neurologists, but channelled most of their resources in social action. Two other organizations were then created around Dr. Demos: the first (Union de Myopathes de France (UMF) acted as a "grass-roots organization" for maintaining "therapeutic orthodoxy" among patients and supporting his research through political lobbying; the other, composed of a handful of wealthy individuals, raised private funds for his laboratory. In the late 1970s, some UMF members questioned Demos' approach. They united with AFM to form a single association and created a Scientific Council representing all French groups interested in neuromuscular diseases. The co-operation established between these two collective partners proved to be most fruitful for both parties.

Stimulation of the growth hormone (GH)-insulin-like growth factor I axis by daily oral administration of a GH secretogogue (MK-677) in healthy elderly subjects.

Aging is associated with declining activity of the GH axis, possibly contributing to adverse body composition changes and increased incidence of cardiovascular disease. The stimulatory effects on the GH-insulin-like growth factor I (IGF-I) axis of orally administered MK-677, a GH-releasing peptide mimetic, were investigated. Thirty-two healthy subjects (15 women and 17 men, aged 64-81 yr) were enrolled in a randomized, double blind, placebo-controlled trial. They received placebo or 2, 10, or 25 mg MK-677, orally, once daily for 2 separate study periods of 14 and 28 days. At baseline and on day 14 of each study period, blood was collected every 20 min for 24 h to measure GH, PRL, and cortisol. Attributes of pulsatile GH release were assessed by 3 independent algorithms. MK-677 administration for 2 weeks increased GH concentrations in a dose-dependent manner, with 25 mg/day increasing mean 24-h GH concentration 97 +/- 23% (mean +/- SE; P < 0.05 vs. baseline). This increase was due to an enhancement of preexisting pulsatile GH secretion. GH pulse height and interpulse nadir concentrations increased significantly without significant changes in the number of pulses. With 25 mg/day MK-677 treatment, mean serum IGF-I concentrations increased into the normal range for young adults (141 +/- 21 microgram/L at baseline, 219 +/- 21 micrograms/L at 2 weeks, and 265 +/- 29 micrograms/L at 4 weeks; P < 0.05). MK-677 produced significant increases in fasting glucose (5.4 +/- 0.3 to 6.8 +/- 0.4 mmol/L at 4 weeks; P < 0.01 vs. baseline) and IGF-binding protein-3. Circulating cortisol concentrations did not change, and PRL concentrations increased 23%, but remained within the normal range. Once daily treatment of older people with oral MK-677 for up to 4 weeks enhanced pulsatile GH release, significantly increased serum GH and IGF-I concentrations, and, at a dose of 25 mg/day, restored serum IGF-I concentrations to those of young adults.

The effects of subcutaneous insulin-like growth factor-I infusion in insulin-dependent diabetes mellitus.

Insulin-dependent diabetes can be associated with low insulin-like growth factor-I (IGF-I) levels despite normal or even high GH secretion. The basis of the diabetic abnormalities in GH-IGF dynamics that contribute to insulin resistance and impaired fuel metabolism are not well understood. To further investigate these matters, this study evaluated baseline IGF system parameters and responses to recombinant human IGF-I in four diabetic adolescents and six pubertal stage-matched controls. Spontaneous overnight and arginine-stimulated GH secretion, insulin, IGF-I, IGF-II, IGF-binding protein-1 (IGFBP-1), and IGFBP-3 levels were measured before, during, and after daily 10-h sc infusions of saline or IGF-I (20 micrograms/kg.h). Baseline overnight GH secretion and IGFBP-1 and -3 levels were not significantly different in the two groups, but IGF-I levels were significantly lower and IGF-II levels were higher in diabetic subjects. IGF-I infusion produced a 3-fold increase in serum IGF-I levels and a reciprocal profound reduction in IGF-II levels in both groups. IGFBP-1 levels increased dramatically in diabetics and modestly in normal subjects in response to IGF-I infusion, but IGFBP-3 levels were not significantly altered. Spontaneous overnight and arginine-stimulated GH secretion were suppressed by about 50% in both groups after IGF-I infusion. Insulin requirements were substantially reduced in diabetics receiving IGF-I, and insulin secretion was suppressed in normal subjects, with no evidence of a change in insulin half-life. Blood glucose remained stable in both groups throughout saline and IGF-I infusions, and no hypoglycemia or other adverse effect occurred during IGF-I infusions. Further studies are necessary to determine whether the addition of IGF-I to insulin replacement therapy may stably reduce the insulin requirement, maintain normal GH levels, and perhaps achieve better metabolic and anabolic balance in the treatment of insulin-dependent diabetes.

Healthy monozygous twins do not recognize identical T cell epitopes on the myelin basic protein autoantigen.

The T cell response against myelin basic protein (MBP) has been extensively studied in humans because of its putative role in the pathophysiology of multiple sclerosis (MS). Higher concordance rates in monozygous twins as well as an increased risk in relatives suggest the role of genetic factors in MS susceptibility. Very little is known about the shaping of T cell repertoire towards self antigens in humans and their contribution to disease susceptibility in autoimmune disorders. Here we report the comparative T cell epitope recognition patterns towards the MBP auto-antigen in healthy identical twins. We have established MBP-specific T cell lines from eight sets of twins and characterized their fine epitope specificity. Intra-pair comparison showed the co-existence of shared as well as distinct epitopes in six of eight pairs and a complete absence of concordant epitope recognition within two other pairs. These findings indicate that important differences in T cell repertoires against a self antigen may be observed between genetically identical healthy individuals, rendering difficult the interpretation of the differences which may be observed between identical twins discordant for an autoimmune disease.

Clinical uses of insulin-like growth factor I.

Insulin-like growth factor I (IGF-I) has acute insulin-like metabolic effects and long-term anabolic actions. The therapeutic potential of recombinant human IGF-I treatment is being investigated in various growth hormone-resistant and insulin-resistant disorders. Recent studies have shown that IGF-I may substitute for growth hormone in promoting linear growth in children with growth hormone insensitivity. The anabolic, protein-sparing action of IGF-I is being evaluated as a potential therapy for adults with catabolic diseases. Patients with insulin-dependent diabetes mellitus have reduced endogenous IGF-I production, and studies are in progress to determine whether treatment with IGF-I in addition to insulin may improve their metabolic/anabolic status. Insulin-like growth factor I treatment may reduce glucose and triglyceride levels in adults with non-insulin-dependent diabetes mellitus and in some patients with extreme insulin resistance. Further studies are needed to evaluate the efficacy and safety of IGF-I treatment in these and other conditions and to provide a better understanding of this hormone's normal physiologic role(s) and complex relations with growth hormone and insulin.

[Familial hemiplegic migraine. Localization of a responsible gene on chromosome 19]. / Migraine hémiplégique familiale. Localisation d'un gène responsable sur le chromosome 19.

Familial hemiplegic migraine is an autosomal dominant disorder of unknown pathogenesis in which the migrainous attacks are marked by the occurrence of a transient hemiplegia during the aura. The aim of our study was the identification of the affected gene. The first step was the chromosomal mapping of the affected gene, for which we used a "candidate gene" strategy. The first candidate gene was the gene responsible for CADASIL. While investigating CADASIL, mapped previously to chromosome 19, we observed that some patients had recurrent attacks of migraine with aura. Although the clinical and neuroimaging features of familial hemiplegic migraine differ markedly from CADASIL, we hypothesized that the same gene could be involved in the pathogenesis of both conditions. We chose two large pedigrees for linkage analysis of familial hemiplegic migraine. A maximum lodsore > 8 was found with two markers that are strongly linked to CADASIL. Multilocus linkage analysis located the affected gene within an interval of about 30 cM on chromosome 19, containing the gene responsible for CADASIL. At this step it's not possible to conclude that CADASIL and familial hemiplegic migraine are due to the same mutated gene. It will be necessary to analyse other familial hemiplegic migraine and CADASIL families in order to reduce the size of their respective interval and ultimately identify the mutated gene(s).

A gene for familial hemiplegic migraine maps to chromosome 19.

Familial hemiplegic migraine is an autosomal dominant disorder of unknown pathogenesis in which the migrainous attacks are marked by the occurrence of a transient hemiplegia during the aura. While investigating CADASIL, mapped previously to chromosome 19, we observed that some patients had recurrent attacks of migraine with aura. Although the clinical and neuroimaging features of familial hemiplegic migraine differ markedly from CADASIL, we hypothesized that the same gene could be involved in the pathogenesis of both conditions. We chose two large pedigrees for linkage analysis of familial hemiplegic migraine. A maximum lod score > 8 was found with two markers that are also strongly linked to CADASIL. Multilocus linkage analysis suggested that the loci responsible for the two diseases reside within an interval of about 30 cM on chromosome 19.

Anatomy of the pituitary insulin-like growth factor system.

In situ hybridization was used to map patterns of gene expression for components of the insulin-like growth factor (IGF) system, including IGF-I and -II, IGF-binding proteins 1-5 (IGFBP1-5), the IGF-I receptor, and GH in the rat pituitary. IGF-I mRNA was concentrated in isolated cells scattered throughout the gland with features typical of nonendocrine folliculo-stellate cells. IGF-II mRNA was abundant in neural (NL) and intermediate lobe (IL) capillaries, and low levels were present in endocrine cells of anterior lobe (AL) and IL. IGFBP1 mRNA was not detected in the pituitary. IGFBP2 mRNA was concentrated in epithelial cells lining AL follicles and in astroglial-like cells (pituicytes) of the NL. IGFBP3 mRNA was localized in isolated cells scattered throughout the AL and NL. IGFBP4 mRNA was relatively abundant in NL pituicytes and was diffusely expressed in the AL. IGFBP5 mRNA was equally abundant in NL and AL, and was localized in folliculo-stellate and epithelial cells of the AL and pituicytes and capillaries of the NL. Neither IGF-I nor IGFBP1-5 were detected in the IL. IGF-I receptor mRNA was abundant and homogeneously distributed throughout the AL and IL, compatible with expression by endocrine cells. There was overlap, but no particular correlation, between IGF system gene expression and GH-producing cells, which were clustered in the dorsal-lateral wings of the AL. In summary, IGF system gene expression is bountiful in the rat pituitary, but does not correlate with sites of GH synthesis. IGF-I receptor mRNA, which might have been expected to localize to somatotrophs, appears to be equally abundant in all of the endocrine cells of both AL and IL; the other constituents of the IGF system are localized in connective tissue and support elements that demonstrate no special anatomical relationship to somatotrophs. Finally, there is remarkably abundant gene expression for IGFBP2, -4, and -5 in the NL.

T-cell receptor identification of an oligodendrocyte-specific autoreactive cytotoxic T-cell clone without self restriction.

In addition to myelin basic protein (MBP) and proteolipid protein (PLP), oligodendrocyte (Od) membrane autoantigens, such as the glycoprotein M2/MOG, could participate in the pathogenesis of autoimmune demyelinating diseases of the central nervous system (CNS), such as experimental allergic encephalomyelitis (EAE) or multiple sclerosis (MS). We have described an Od-specific autoreactive and cytotoxic T-cell clone, named C2, which recognized M2/MOG without conventional MHC restriction. In order to analyse the Od/C2 interaction, we determined the alpha/beta T-cell receptor (TCR) variable region usages and structures of C2. Monoclonal antibody stainings of C2 and nucleotide sequences show that the alpha chain is composed of a V alpha 5 and a J alpha identical to J alpha 18BBM142 gene segments, and that the TCR beta chain is composed of V beta 17a, D beta 2.1 and J beta 2.2 gene segments indicating that C2 used a conventional alpha/beta TCR for M2/MOG recognition.

Anti-peripheral nerve antibodies in leprosy patients recognize an epitope shared by the M. leprae 65 kDa heat shock protein.

Binding of leprosy sera to peripheral nerve from different species (mouse, guinea pig and rabbit) was evaluated by ELISA. A majority of sera, whatever the clinical form of leprosy, bind to these antigens. Absorption with Mycobacterium bovis BCG demonstrated that these antibodies recognize cross-reactive epitopes between peripheral nerve and mycobacteria. In immunoblot analysis, both leprosy patient sera and a monoclonal antibody directed at the 65 kDa heat shock protein of M. leprae were shown to react with a heat-shock 67-68 kDa sciatic nerve protein. Binding of the monoclonal antibody to this sciatic nerve antigen was prevented by incubation with lepromatous patient sera, showing that some peripheral nerve epitopes recognized by patient antibodies are shared by the 65 kDa heat shock protein of M. leprae.

Structural and functional analysis of the insulin-like growth factor I receptor gene promoter.

The insulin-like growth factor I receptor (IGF-I-R) gene is expressed in most body tissues. The levels of IGF-I-R mRNA, however, are regulated by a number of physiological conditions (development, differentiation, and hormonal milieu) as well as in certain pathological states (diabetes and tumors). To understand the molecular mechanisms which control the transcription of the IGF-I-R gene, we have cloned the promoter of the rat receptor gene and have characterized its activity by transient expression assays. Different fragments of the 5'-flanking region (subcloned upstream of a luciferase reporter gene) were transfected into buffalo rat liver 3A cells (a cell line with a low number of IGF-I binding sites) and Chinese hamster ovary cells (a cell line with a higher number of cell-surface receptors). In both cell lines, most of the promoter activity was located in the proximal 416 base pairs of 5'-flanking region. However, further dissection of this proximal fragment revealed a cell type-specific pattern of promoter activity. Thus, in buffalo rat liver 3A cells, subfragments of this region each contributed to total activity, suggesting that contiguous cis-elements can act together to activate transcription. In Chinese hamster ovary cells, on the other hand, subfragments of the proximal promoter region partially substituted for the proximal 416 base pairs of 5'-flanking region. Coexpression studies using an IGF-I-R promoter reporter construct together with an Sp1 expression vector (under the control of an ADH promoter) were performed in SL2 cells, a Drosophila cell line which lacks endogenous Sp1. The results obtained showed that Sp1 can trans-activate the IGF-I-R promoter in vivo. Transient transfection assays were complemented with gel-retardation assays and DNase I footprinting experiments, which showed that transcription factor Sp1 is potentially an important regulator of IGF-I-R gene expression.

Myalgia and elevated creatine kinase activity associated with subcutaneous injections of diluent.

A 16-year-old boy with short stature and mild primary hypothyroidism received subcutaneous injections of either recombinant human growth hormone or placebo in diluent that contained glycerol and m-cresol as a preservative. While he was receiving the study drug, myalgia developed and serum creatine kinase values were elevated. Both resolved when injections were stopped and recurred when injections of diluent alone were given. The myalgia and elevated creatine kinase activity were apparently caused by a component of the diluent.