Cheek Cells Research Articles

The Fatty Acid Profile of Buccal Cheek Cell Phospholipids Is a Noninvasive Marker of Long-Chain Polyunsaturated Fatty Acid Status in Piglets

The fatty acid pattern of cheek cell phospholipids has been proposed as a noninvasive marker of long-chain polyunsaturated fatty acid (PUFA) status. However, the cheek cell phospholipid fatty acid pattern has been compared only with that of plasma and erythrocytes. The objective of this study was to assess the extent to which the fatty acid profile of cheek cell phospholipids reflects that of tissue phospholipids. Piglets (n = 31; 6 d old) were fed five formula diets differing in total fat and fatty acid composition. After 14 d of consuming the assigned diets, cheek cell plasma, erythrocyte, liver, muscle, adipose tissue, retina and brain samples were collected for determination of the phospholipid fatty acid patterns. There were significant correlations between the cheek cell phospholipid content of most PUFA and the content of these fatty acids in tissue phospholipids (r = 0.509-0.951, P < 0.01). The cheek cell phospholipid content of most of the PUFA, except 20:4(n-6), reflected that of other tissue phospholipids as well as, or nearly as well as the contents of plasma and/or erythrocyte phospholipids. The correlations between the 22:6(n-3) contents of cheek cell, plasma, or erythrocyte phospholipids and those of brain and retina phospholipids were relatively poor (r = 0.596-0.737, P < 0.001). We conclude that the fatty acid pattern of cheek cell phospholipid can be used as a noninvasive marker of PUFA status, but it is not a better index than the pattern of plasma or erythrocyte phospholipids, particularly for assessing the fatty acid pattern of organs with slow fatty acid incorporation and/or turnover rates.

Association between a serotonin transporter promoter region polymorphism and mood response during tryptophan depletion.

This study investigated the relationship between depressive symptom response during tryptophan (TRP) depletion and a functional polymorphism of the promoter region of the serotonin (5-HT) transporter gene (SLC6A4).(1) Forty-three subjects in remission from a major depressive episode who underwent TRP depletion were genotyped. DNA was extracted from blood lymphocytes or from cheek cells.(2) The two common alleles are designated long (l) and short (s). Depressive symptoms were measured with the 25-item Hamilton Depression Rating Scale (HDRS).(3) There was a significant association between the l homozygous genotype and the depressive response to TRP depletion, with a significant main effect of time (F = 8.763, df = 3, 38, P = <0.001), and time x l homozygous allele interaction (F = 3.676, df = 3, 38, P = 0.02). Individuals whose genotype predicted increased 5-HT transporter activity may be more susceptible to depressive changes in response to transient 5-HT perturbations. The use of endophenotypic markers for affective disorders such as the mood response to TRP depletion may facilitate studies of complex genetic traits such as depression by decreasing its heterogeneity.

Capillary-based fully integrated and automated system for nanoliter polymerase chain reaction analysis directly from cheek cells

A miniaturized, integrated and automated system based on capillary fluidics has been developed for nanoliter DNA analysis directly from cheek cells. All steps for DNA analysis, including injecting aqueous reagents and DNA samples, mixing the solutions together, thermal cell lysis, polymerase chain reaction (PCR), transfer and injection of PCR product, separation, sizing and detection of those products are performed in a capillary-based integrated system. A small amount of cheek cells collected by a plastic toothpick is directly dissolved in the PCR cocktail in a plastic vial or mixed on-line with a small volume of PCR cocktail (125 nl) in the capillary. After thermal cell lysis and PCR in a microthermal cycler, the DNA fragments are mixed with DNA size standards and transferred to a micro-cross for injection and separation by capillary gel electrophoresis. Programmable syringe pumps, switching valves, multiposition and freeze–thaw valves are used for microfluidic control in the entire system. This work establishes the feasibility of performing all the steps of DNA analysis from real samples in a capillary-based nanoliter integrated system.

High-throughput polymerase chain reaction analysis of clinical samples by capillary electrophoresis with UV detection.

Routine genetic analysis of large numbers of individuals by polymerase chain reaction (PCR) using capillary electrophoresis is often restricted by the low throughput of standard protocols and the tedious sample preparation process. Here, we demonstrate that capillary electrophoresis with UV detection can be used in PCR-based DNA analysis starting from clinical samples without purification or complicated sample manipulation. After PCR reaction using cheek cells, blood, or HIV-1 gag DNA, the reaction mixtures were injected into a capillary array either on-line or off-line by base stacking. The use of multiplexed absorption detection and the elimination of any purification steps both before and after PCR reaction can potentially provide significant benefits compared to current methods for DNA analysis with regard to time, cost, and labor.

New predictive test for high blood pressure

Researchers at CSIRO Health Sciences and Nutrition (Adelaide, Australia) have developed a new test for high blood pressure that is based on the measurement of Na+ uptake in cheek cells. Human cheek cells stained to show stages of their maturation cycle. Kindly provided by Ted McMurchie (CSIRO, Adelaide, Australia). A simple mouth-wash is all that is required of subjects to be tested, and the assay has both diagnostic and predictive potential.

Cheek cell phospholipids in human infants: a marker of docosahexaenoic and arachidonic acids in the diet, plasma, and red blood cells

Assessment of essential fatty acid status requires collection of blood or adipose tissue samples. However, these invasive techniques cannot always be used in studies involving infants, young children, or subjects from whom it is difficult to obtain blood. A body tissue that is easy to access is the buccal mucosa (cheek cells). The objective was to investigate the degree to which fatty acids of cheek cells reflect the fatty acid content of plasma, red blood cells, and the diet. Thirty-one infants aged 12 mo were enrolled. Five infants were fed human milk and 26 infants received formulas that provided a wide range of arachidonic acid and docosahexaenoic acid (DHA) intakes. Cheek cells were collected on a small piece of gauze by gently swabbing the inside of the cheek 3 times. Lipids were extracted from the gauze and the phospholipid fatty acid content of the cheek cells was determined. Cheek cell DHA and arachidonic acid in phospholipids were significantly correlated with DHA and arachidonic acid in plasma [r = 0.61 (P < 0.001) and r = 0.37 (P <0.05), respectively], red blood cells [r = 0.58 (P < 0.001) and r = 0.37 (P < 0.05), respectively], and the diet [r = 0.65 (P < 0.001) and r = 0. 51 (P < 0.01), respectively]. Given these correlations and the ease and noninvasive nature of this technique, cheek cell fatty acids may serve as a marker of the essential fatty acid content, especially of DHA and arachidonic acid, in plasma, red blood cells, and the diet.

Effects of Dietary Fish Oil Supplementation on the Phospholipid Composition and Fluidity of Cell Membranes from Human Volunteers

Background: Membrane fluidity is an important aspect of cellular physiology which may be manipulated by diet. Methods: We studied the effect of dietary fish oil on the membrane composition of erythrocytes and cheek cells, and on membrane fluidity of erythrocytes as assessed by fluorescence recovery after photobleaching (FRAP). Healthy volunteers received a daily supplement of fish oil (930 mg EPA, 630 mg DHA) for 42 days. Results: The intervention reduced the ratio of n-6 to total fatty acid in the phospholipid fraction of erythroctye membranes but the n-3 fraction remained stable and the ratio of cholesterol to phospholipid increased. The level of EPA and DHA in cheek cells increased significantly during the intervention period. The mean diffusion coefficient of the fluorescent probe in erythrocyte membranes increased from 7.2 ± 0.7 × 10^–9 cm²/s at the start to 9.8 ± 0.5 × 10^–9 cm²/s after 21 days. Membrane fluidity remained higher than the initial value 42 days after withdrawal of the supplement. Fish oil also reduced platelet aggregation in response to ADP but there was no effect on plasma lipid profiles. Conclusion: We conclude that n-3 fatty acids influence erythrocyte membrane composition at relatively low levels of supplementation by a mechanism which does not necessarily involve an increase in the level of EPA or DHA present in the membrane.

Olive Oil Supplementation in Healthy Adults: Effects in Cell Membrane Fatty Acid Composition and Platelet Function

Ten healthy volunteers were given a daily supplement of 30 g olive oil for 6 weeks in order to evaluate how it would affect cell membrane composition and ultimately platelet function. Fasting blood and cheek cell samples were taken before commencing the study, after 21 and 42 days of supplementation and also at 30 days after finishing the supplement (washout). C18:1n-9 was significantly increased (p < 0.01) in platelet and cheek cell phospholipids. Erythrocytes were not good markers for C18:1n-9 intake and no significant change was found in this tissue. There was a small nonsignificant decrease in platelet phospholipid 20:4n-6 after the supplementation, in accordance with previously published results. C18:1n-9 did not persist in platelet membranes after the volunteers stopped consuming the olive oil supplement, but in erythrocytes a significant increase (p < 0.05) was found after the washout period. None of these changes in fatty acid composition in the different tissues were related to significant changes in serum cholesterol-related variables or in clotting factors or adenosine diphosphate-induced platelet aggregation.

Alternative, Noninvasive Tissues for Quantitative Screening of Mutant Mitochondrial DNA

Since the molecular basis of the first mitochondrial DNA (mtDNA) disorder, Leber hereditary optic neuropathy (LHON), was established (1), mtDNA mutations have become increasingly more recognized as an important cause of genetic disease. The most common mutations identified are A3243G, A8344G, T8993G/C, G11778A, and large mtDNA deletions. The A3243G mutation accounts for 80% of patients with MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes) (2), and the A8344G mutation accounts for 80% of patients with MERRF (myoclonic epilepsy and ragged-red fibers) (3)(4). Substitution of T8993 with G or C causes NARP (neuropathy, ataxia, retinitis pigmentosa) (5)(6), and the G11778A mutation is responsible for >50% of patients with LHON (1). MtDNA deletions and rearrangements are found in Kearns–Sayre syndrome and chronic progressive external ophthalmoplegia patients. Most pathogenic mtDNA mutations are heteroplasmic, with normal and mutant DNA coexisting in the same cell or tissue. The phenotypic expression of a pathogenic mutation depends on tissue-specific energy requirements (7). Accordingly, age of onset, tissues involved, and clinical severity vary greatly between individuals, even within a given family, depending on the proportions of mutant and normal mtDNA. Direct mtDNA mutation analysis allows for identification of asymptomatic relatives who harbor the mutant mtDNA. Prediction of future disease expression is assisted by quantitative determination of the proportion of mutant mtDNA in the relevant tissues. DNA-based molecular diagnosis of mtDNA disorders has been routinely performed on blood or biopsied skeletal muscle (8)(9). Because of variation in percentage of mutant mtDNA among different tissues, mutant mtDNA may not be detectable in blood. Nevertheless, it is difficult to justify obtaining muscle biopsies from multiple asymptomatic at-risk family members. In this setting, the examination of alternative tissues such as cheek cells or hair follicles is especially relevant. Studies of MELAS mutant heteroplasmy …

Genetic factors associated with altered sodium transport in human hypertension: a twin study.

1. Na+/H+ antiporter/exchange activity (NHE) in human cheek epithelial cells was assessed in 288 female twins and siblings. The genetic contribution of factors to NHE activity was assessed in 128 matched twin pairs (76 monozygotic (MZ); 52 dizygotic (DZ)). 2. There was a small reduction in NHE with age and body mass index. The significant correlations (+/-their standard error (SE)) within MZ and DZ pairs of twins were 0.54 +/- 0.08 and 0.26 +/- 0.13, respectively, implying that genetic factors accounted for 54% of the variance in age-adjusted NHE. There was no cross-sectional relationship between NHE and measures of blood pressure. Based on within-pair differences, however, there was a weak negative association (r = 0.22; P < 0.05) between mean arterial pressure and NHE. 3. It remains to be determined whether NHE in cheek cells is associated with blood pressure tracking over time in young females.

Stimulation of human cheek cell Na+/H+ antiporter activity by saliva and salivary electrolytes: amplification by nigericin.

Proton-dependent, ethylisopropylamiloride (EIPA)-sensitive Na+ uptake (Na+/H+ antiporter) studies were performed to examine if saliva, and ionophores which alter cellular electrolyte balance, could influence the activity of the cheek cell Na+/H+ antiporter. Using the standard conditions of 1 mmol/l Na+, and a 65:1 (inside:outside) proton gradient in the assay, the uniport ionophores valinomycin (K+) and gramicidin (Na+) increased EIPA-sensitive Na+ uptake by 177% (p < 0.01) and 227% (p < 0.01), respectively. The dual antiporter ionophore nigericin (K(+)-H+) increased EIPA-sensitive Na+ uptake by 654% (p < 0.01), with maximal Na+ uptake achieved by 1 min and at an ionophore concentration of 50 mumol/l, with an EC50 value 6.4 mumol/l. Pre-incubation of cheek cells with saliva or the low molecular weight (MW) components of saliva (saliva activating factors, SAF) for 2 h at 37 degrees C, also significantly stimulated EIPA-sensitive Na+ uptake. This stimulation could be mimicked by pre-incubation with 25 mmol/l KCl or K(+)-phosphate buffer. Pre-incubating cheek cells with SAF and the inclusion of 20 mumol/l nigericin in the assay, produced maximum EIPA-sensitive Na+ uptake. After pre-incubation with water, 25 mmol/l K(+)-phosphate or SAF, with nigericin in all assays, the initial rate of proton-gradient dependent, EIPA-sensitive Na+ uptake was saturable with respect to external Na+, with Km values of 0.9, 1.7, and 1.8 mmol/l, and Vmax values of 13.4, 25.8, and 31.1 nmol/mg protein/30 sec, respectively. With 20 mumol/l nigericin in the assay, Na+ uptake was inhibited by either increasing the [K+]o in the assay, with an ID50 of 3 mmol/l. These results indicate that nigericin can facilitate K+i exchange for H+o and the attending re-acidification of the cheek cell amplifies 22Na+ uptake via the Na+/H+ antiporter. The degree of stimulation of proton-dependent, EIPA-sensitive Na+ uptake is therefore dependent, in part, on the intracellular [K+]i.

HUMAN BUCCAL EPITHELIAL CELLS AS A POTENTIAL BIOCHEMICAL PREDICTOR OF ESSENTIAL HYPERTENSION: IDENTIFICATION OF KEY CELLULAR PROCESSES

1. Human cheek cell Na+/H+ antiporter activity (measured as the rate of proton-dependent 22Na+ uptake) was determined in seven normotensive (NT) and four hypertensive (HT) subjects following preincubation of cheek cells with a low molecular weight fraction isolated from NT saliva together with the ionophore, nigericin. 2. Cheek cells preincubated in this manner exhibited greater Na+/H+ antiporter activity with the mean values being 4.2 nmol Na+.mg protein.5 min for the NT group and 1.7 for the HT group. 3. It is possible that stimulation of Na+ transport is due to cellular accumulation of K+ ions during preincubation which, in the presence of the K+/H+ selective ionophore, nigericin, can cause cellular reacidification promoting further 22Na+ uptake via the Na+/H+ antiporter.

Cheek cell membrane fluidity measured by fluorescence recovery after photobleaching and steady-state fluorescence anisotropy

Membrane fluidity of human cheek cells was determined using fluorescence recovery after photobleaching (FRAP) and steady-state fluorescence anisotropy. The FRAP data showed that the lateral diffusion coefficient (D) and mobile fraction (%R) of lipid in the plasma membrane of control cells were 2.01 x 10(-9) cm2/sec and 54.25%, respectively. Trypsin treatment increased D and %R to 6.4 x 10(-9) cm2/sec and 72.15%. In contrast, the anisotropy (r) for control cells was 0.270 which remained unchanged by trypsin treatment. The results show that diffusion of lipids in the plane of the membrane is restricted by trypsin-sensitive barriers.

Osmotic and other properties of isolated human cheek epithelial cells.

This study describes some biological properties of human cheek (buccal epithelial) cells, isolated by mouth wash. Yields ranged from 6.5 to 20.6 x 10(6) cells, with a mean (+/- SE) of 12.2 +/- 4.2 x 10(6) cells, which gave 0.55 +/- 0.01 x 10(6) cells/mg protein. Vital stain exclusion was similar in cells isolated in either water (89 +/- 2%) or 250 mM sucrose (87 +/- 3%). From our measurements of cell volume and electrolyte content, we estimated intracellular Na+ and K+ concentrations to be between 0.3-0.5 and 7.4-13.0 mM, respectively. In 22 adult subjects, basal, prickle, intermediate, and superficial cells represented 0.3 +/- 1.4, 51 +/- 2.4, 26 +/- 0.9, and 22.7 +/- 1.8%, respectively, of the total sample. Cheek cells exhibited a low endogenous rate of oxygen consumption, which was stimulated by glucose or succinate and inhibited by KCN or NaF. Cheek cells were osmotically stable in a wide range of media, including water. However, they exhibited shrinkage and collapse in hypertonic media, particularly polyethylene glycol.

Characterization of Na(+)-H+ antiporter activity associated with human cheek epithelial cells.

Na+ transport activity was characterized in human cheek epithelial cells obtained from normotensive adult subjects. The cells were isolated using a mouth-wash procedure and assayed for Na+ uptake using a radioactive (22Na+) rapid filtration assay. Cheek cells displayed proton-dependent Na+ uptake activity that was dependent on the magnitude of the externally directed proton gradient measured using the fluorescent probe 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein to determine intracellular pH. Amiloride, ethylisopropylamiloride (EIPA), 5-(N,N-dimethyl)-amiloride, 5-(N-methyl-N-isobutyl)-amiloride (MIA), and 5-(N,N-hexamethylene)-amiloride (NNHA) all inhibited proton-dependent Na+ uptake, with MIA, EIPA, and NNHA being the most potent. The Michaelis constant (Km) for extracellular Na+ was 5.7 mM, while the maximum velocity for Na(+)-H+ antiporter activity was 4.3 nmol Na+.mg protein-1.30s-1. The Km for intracellular H+ was 0.17 microM, with a Hill coefficient of 0.7. Stimulation by ouabain and inhibition by bumetanide of cheek cell proton-dependent Na+ uptake indicated only relatively low activities of Na(+)-K(+)-ATPase and Na(+)-K(+)-2Cl- cotransport, respectively. These results are consistent with the presence of Na(+)-H+ antiporter activity in cheek cells. Cheek cells therefore provide a convenient, relatively noninvasive source of tissue for examining Na(+)-H+ antiporter activity in human subjects.

Depressed cheek cell sodium transport in human hypertension.

Na+ transport activity was measured in cheek cells from untreated hypertensive subjects and age-matched normotensive controls identified from a blood pressure screening program. Cheek cells were isolated by a simple mouth wash procedure and Na+ transport activity was measured as the proton-dependent uptake of 22Na+ using a rapid filtration assay. The rate of Na+ uptake was about 45% lower in hypertensive subjects and this difference persisted in a follow up study 2 years later involving those subjects who remained untreated for their hypertension. The proton independent Na+ uptake was also reduced by about 46% in the hypertensive group. The increase in the rate of cheek cell Na+ transport with increasing transcellular proton gradient values was also significantly lower in hypertensive subjects. The reduced cheek cell Na+ transport observed in hypertensive subjects may indicate decreased activity of the Na+/H+ antiporter and/or changes in the ion permeability properties of the cheek cell plasma membrane in the hypertensive state. This novel assay provides a biochemically based method for discriminating between normotensive and hypertensive subjects and makes use of tissue which can be obtained in a relatively non-invasive manner.

The relationship between salivary growth factors, electrolytes and abnormal sodium transport in human hypertension.

We have previously shown cheek cell Na+/H+ antiporter activity to be reduced in human hypertensives. We have now examined the relationship between abnormal antiporter activity and a variety of salivary factors. Total protein concentration and amylase activity were higher in hypertensives, but salivary flow rate and epidermal growth factor, transforming growth factor-alpha, calcium, and magnesium concentrations were not significantly different between hypertensives and normotensives. The lowered cheek cell Na+/H+ antiporter activity in those hypertensives with diastolic BP greater than 95 mmHg was accompanied by lowered salivary Na+/H+ ratios. In borderline hypertensives (diastolic BP between 90 and 95 mmHg), the Na+/H+ ratio was reduced to a similar extent to that seen in those hypertensives with a diastolic BP above 95 mmHg, however the cheek cell antiporter activity was not reduced, suggesting that these two differences are not related in a simple fashion in all hypertensives. It is concluded that it is unlikely that differences in salivary growth factors explain the lowered cheek cell Na+/H+ antiporter activity observed in human hypertension. Our findings indicate that salivary electrolyte composition may be related to cheek cell Na+/H+ antiporter activity and these parameters may be altered in hypertension.

Variability in lipids isolated from human cheek cells

Major lipid classes were examined in human cheek cell preparations isolated 3 or 7 days apart. In 13 of 14 subjects, samples isolated 7 days apart had less phospholipid and a higher ratio of sterol + nonpolar lipid to phospholipid than samples isolated 3 days apart. It was concluded that frequency of cheek cell collection is one factor that contributes to variability noted in studies of human cheek cell lipids.

Human cheek cell [formula omitted] antiporter activity in essential hypertension: Use as a novel predictive test

Human cheek cell [formula omitted] antiporter activity in essential hypertension: Use as a novel predictive test

Cheek cell fatty acids as indicators of dietary lipids in humans.

Analysis of cheek cell lipids has been suggested as a noninvasive method for monitoring the fatty acid composition of diets in humans. In a pilot study conducted to determine the validity of the method, cheek cell samples were collected from subjects consuming a low fat (20% of calories) diet consisting of fatty acids with either a 1.0 or 0.3 P/S ratio. Neither total lipid nor polar lipid fatty acids in cheek cells consistently reflected the P/S ratio of the diets. However, there were trends, particularly in the nonpolar lipids, suggesting that cheek cell fatty acid ratios might be useful for monitoring the fatty acid composition of the diets. The diet with the higher P/S ratio (1.0 vs 0.3) consistently resulted in cheek cell lipids with lower ratios of 18:1/saturated fatty acids and greater 18:2/20:4, 18:2/18:1 and 18:2/18:0 fatty acid ratios.