Assessment of intake of caffeine in random population in Riyadh and its levels in some food by HPLC

This study involves 160 Saudi females of a mean age of 23±3.7 years who regularly consumed coffee and soft drinks. The highest percentage of participants were reported to consume less than 300 mg of caffeine daily (42.5%) and the highest level of caffeine consumption (more than 2800 mg/day) was calculated among 7.5% of the participants. The highest percentages of participants consuming caffeine were detected in coffee consumers followed by soft drink consumers (93.75% and 90.63% respectively), while the least percentage was detected in tea consumers (45.63%). The mean of consumed caffeine from coffee was the highest value (1599±416.7) compared with the caffeine content in other foods. In relation to anthropometric measurements, there were no significant correlations between them and the level of caffeine consumption except weight. There was a significant correlation between the decrease in body weight and the increase in amount of caffeine consumed (p<0.05). On the other hand, there was no significant correlation between the hours of sleep and food consumption, and the amount of caffeine consumed (p<0.05). There was, however, a high correlation between the employment status of the participant and the caffeine consumption (p> 0.05).


Introduction
Humans have consumed caffeine since the Stone Age (Escohotado, 1999).Caffeine is a bitter white crystalline xanthine alkaloid that acts as a psychoactive stimulant drug and a mild diuretic (speeds up urine production) in humans and other animals (Armstrong, 2002).Caffeine was discovered by a German chemist, Friedrich Ferdinand Runge, in 1819.The disparity in experience and effects between the various natural caffeine sources could be due to the fact that plant sources of caffeine also contain widely varying mixtures of other xanthine alkaloids, including the cardiac stimulants theophylline and theobromine and other substances such as polyphenols which can form insoluble complexes with caffeine (Balentine et al., 1998).
Caffeine is found in varying quantities in the beans, leaves, and fruit of over 60 plants, where it acts as a natural pesticide that paralyzes and kills certain insects feeding on the plants (Nathanson, 1984); and as an inhibitor of seed germination of other nearby coffee seedlings (Baumann and Gabriel, 1984), thus giving it a better chance of survival.It is most commonly consumed by humans in the form of infusions extracted from the beans of the coffee plant and the leaves of the tea bush, as well as from various foods and drinks containing products derived from kola nut or cacao.Other sources include yerba mate, guarana berries, and the Yaupon Holly (Matissek, 1997).
The world's primary source of caffeine is the coffee bean (the seed of the coffee plant).Caffeine content in coffee varies widely depending on the type of coffee bean and on the method of preparation (Stavric, 1988).Beans within a given bush can show variations in caffeine concentration.In general, one serving of coffee ranges from 40 milligrams, for a single shot (30 milliliters) of Arabica variety espresso, to about 100 milligrams for a cup (120 milliliters) of drip coffee.Generally, dark-roast coffee has less caffeine than lighter roasts because the roasting process reduces the bean's caffeine content (Lecos, 1984 andBruce, 1986).Arabica coffee normally contains less caffeine than the robusta variety (Stavric, 1988).Coffee also contains trace amounts of theophylline, but no theobromine.
Tea is another common source of caffeine.Although tea contains more caffeine than coffee, a typical serving contains much less, as tea is normally brewed much weaker.Besides the strength of the brew, growing conditions, processing techniques and other variables also affect caffeine content.Certain types of tea may contain more caffeine than others.In general, tea contains small amounts of theobromine and slightly higher levels of theophylline than coffee.Preparation and many other factors have a significant impact on tea, and color is a very poor indicator of caffeine content.Teas like the pale Japanese green tea gyokuro, for example, contain far more caffeine than much darker teas like lapsang souchong, which has very little (Food Research International, 1996).
Caffeine is also a common ingredient of soft drinks such as cola, originally prepared from kola nuts.Soft drinks typically contain about 10 to 50 milligrams of caffeine per serving (Haskell et al., 2007).Chocolate derived from cocoa contains a small amount of caffeine.In addition to its caffeine content, the weak stimulant effect of chocolate may also be due to a combination of theobromine and theophylline (Smit et al., 2004).Chocolate contains too little of these compounds per reasonable serving to create an effect in humans.A typical 28 gram serving of a milk chocolate bar has approximately as much caffeine as a cup of decaffeinated coffee (Smith, 2002).
Caffeine is also packed in the form of tablets, with the claim that using caffeine of pharmaceutical quality improves mental alertness and is used by students during the exams period.It is also used by people who work or drive for long hours (Ivan, 2008).
Caffeine is a central nervous system and metabolic stimulant (Nehlig et al., 1992) and is used both recreationally and medically to reduce physical fatigue and restore mental alertness when unusual weakness or drowsiness occurs.Caffeine stimulates the central nervous system first at higher levels, resulting in increased alertness and wakefulness, faster and clearer flow of thought, increased focus, and better general body coordination; and later at the spinal cord level at higher doses (Bolton, 1981).Caffeine is completely absorbed by the stomach and small intestine within 45 minutes of ingestion.After ingestion it is distributed throughout all tissues in the body and then eliminated by first-order kinetics (Newton et al., 1981).Caffeine can also be ingested rectally, evidenced by the formulation of suppositories of ergotamine tartrate and caffeine for the relief of migraine (Graham, 1954); and chlorobutanol and caffeine for the treatment of hyperemesis (Brødbaek and Damkier, 2007).
The half-life of caffeine (the time required for the body to eliminate one-half of the total amount of caffeine consumed at a given time) varies widely among individuals and depends on factors such as age, liver function, pregnancy, some concurrent medications, and the level of enzymes in the liver needed for caffeine metabolism.In healthy adults, caffeine's half-life is approximately 3 to 4 hours.In women taking oral contraceptives this is increased to 5 to 10 hours (Meyer et al., 1991), and in pregnant women the half-life is roughly 9 to 11 hours (Ortweiler et al., 1985).Caffeine can accumulate in individuals with severe liver disease, increasing its half-life up to 96 hours (Bolton, 1981).In infants and young children, the half-life may be longer than in adults, while the half-life in a newborn baby may be as long as 30 hours.Other factors such as smoking can shorten caffeine's halflife (Springhouse, 2005).
Caffeine is metabolized in the liver by the cytochrome P 450 oxidase enzyme system into three metabolic dimethylxanthines, which each have their own effects on the body: Paraxanthine (84%), has the effect of increasing lipolysis, leading to elevated glycerol and free fatty acid levels in the blood plasma, Theobromine (12%) dilates blood vessels and increases urine volume and Theophylline (4%) relaxes smooth muscles of the bronchi, and is used to treat asthma.Each of these metabolites is further metabolized and then excreted in the urine (Fisone et al., 2004).
Taking into account all of these various factors, the objectives of this study are to assess the intake of caffeine in a random sample of female population in Riyadh, determine caffeine levels in some foods by HPLC, propose guidelines that may aid practitioners in advising their population, and provide recommendations for future research.

Data collection
The study was conducted on healthy women who regularly visited the Central Riyadh hospital and Al-yamamah hospital, Riyadh, Kingdom of Saudi Arabia.The data was collected from a sample of one hundred sixty Saudi people, out of which the average age was determined to be 23±4 through a self-administered questionnaire.In addition to the information about their socioeconomic characteristics, the participants were asked to indicate the quantity of their daily consumption of caffeine containing foods and drinks.Questions concerning demographics, medical history, lifestyle habits (e.g.smoking, illicit drug use, caffeine), environmental and occupation exposures, and psychosocial stresses, were included in the questionnaire.

Detection of caffeine consumption
To measure caffeine consumption, a 7day recalls method was used, in which the participants recorded both the type and amount of all beverages consumed during the preceding 7 days.Female participants were questioned about the type (i.e.caffeinated or decaffeinated coffee, tea, soft drinks, cocoa drinks, milk chocolate, and dark chocolate) and amount (i.e.number of cups, glasses, or ounces per day and/or week) of caffeine consumed during the various time periods.Also, the questionnaire included other types of food.Caffeine intake was converted to exact amounts in mg, based on estimates reported in several previous studies (Wilcox et al., 1988;Clausson andGranath, 2002 andDerbyshire andAbdula, 2008) and various nutrition tables.For each time period, the total amount of caffeine was calculated by adding the amounts consumed through all food products containing caffeine.

Determination of caffeine
58 samples of foods and beverages containing caffeine (as shown table 1a and  1b) were analyzed by using highperformance liquid chromatography (SHIMADZU HPLC) with a UV Detector (λ 273nm).This method provides separation by utilizing a non-polar stationary phase chemically bonded to an inert support in a column through which a polar mobile phase passes.The mobile phase consisted of 60 % ammonium acetate (005M), 40 % methanol ((HPLC GRADE) COLUMN: RESTICpinnacle II C18 5U (150X4.6mmcat=9214565) (AOAC, 16 th ed., 1990)).Compounds (including caffeine) in the study samples which have different polarities, are distributed between the stationary and mobile phase differently, and are thus eluted from the column at different times.As caffeine exhibits ultraviolet absorption, a variable wavelength detector was used to measure elution from the column.
To prepare the samples from carbonated beverages, they were decarbonated by sonicating for 5 to 10 minutes and then diluted with ammonium acetate buffer.For coffee and tea samples, 1 g of each was weighed into a 100 ml volumetric flask, dissolved by adding boiling water, cooled and diluted to the mark.The sample was filtered through the sample clarification kit prior to injection.
The HPLC was set up for analysis and the buffer was set up on channel A and the methanol on channel B. The solvents were degassed under vacuum for 30 minutes before use.The variable wavelength detector was turned on 15 minutes before the first injection.After equilibrating the column with the mobile phase for 30 minutes, 10 ul of the standard mixture was injected.As caffeine has an approximate retention time of 5.4 minutes and with the instrument set to the relative percent mode, the area of caffeine peak was calculated and then used to quantitate its component in the sample.

Statistical analyses
Results with high measures of associations (OR >2.0) or overall P-values 0.05 were presented with corresponding 95% CI.All analyses were performed using STATA software (Stata Corp., College Station, TX, USA).

Results
Tables 1a and 1b show the results of measurement of caffeine contents in foods and beverages using HPLC.Among the coffees, it can be seen that Turkish coffee contains the highest amount of caffeine (2905.5 mg) followed by Starbucks coffee (1516.7 mg), while the coffee with chocolate (Rabai) contains the least amount of caffeine (129.3 mg).Among chocolate beverages, chocolate milk ((Al-Othman Agri.Prod & Proc.Co.) (NADA)) contains the highest amount of caffeine (79.1 mg) followed by Chocolate milk (Al-Marai) (67.6 mg) and chocolate milk (Korenta) (22.4 mg).Among chocolates, M&Mُ s is the ones contain the highest amount of caffeine (466.5 mg) followed by Maltesers (288.6 mg) and the least amount was found in Tola Nest chocolate (33 mg).The soft drink with the highest amount of caffeine was Power Play drink (331 mg) followed by Boom-Boom drink (306.7 mg), while the drink which had the least amount of caffeine is Pepsi-Cola (Al-Jomaih) (90 mg).Among the teas, green tea (leaf) contains the highest amount of caffeine (308 mg) followed by red tea (leaf) (281 mg), while green tea (Lipton) had the least amount of caffeine (49.3 mg).Table 2 shows the percentage of participants consuming different amounts of caffeine as recorded through a 7-day recall (mg/day).The highest percentage of participants was reported to consume less than 300 mg of caffeine daily (42.5%) followed by 30% of participants consuming between 300 and 800 mg of caffeine daily.The highest level of caffeine consumption (more than 2800 mg/day) was found in 7.5% of participants.There was a significant negative correlation between the number of participants consuming caffeine and the amount of caffeine consumed; with one exception that was recorded at the highest level of caffeine consumption (7.5% consumed more than 2800 mg daily).Table 4 shows the correlation between the amount of caffeine consumption (mg/day) and general characteristics of participants, and the 95% confidence interval of caffeine consumption for different groups of study.
There was no correlation between the mean age (23±4) of the studied population and the amount of caffeine consumed (p>0.05).With regard to anthropometric measurements (height, weight, chest circumference, waist circumference, and hip circumference), there was no correlation between them and the level of caffeine consumption except for weight.On the other hand, there was a significant negative correlation between the decrease in body weight and the increase in the amount of caffeine consumed (p<0.05).There was also a significant negative correlation between the employment status of the participants and the amount of caffeine consumed (p< 0.05).On the other hand, there was no correlation between the hours of sleep and the amount of caffeine consumed (p<0.05).

Table 1a . Caffeine content in 23 foods and beverages measured by HPLC.
Caffeine content was measured in the different foods by HPLC as described in Materials and Methods.Results are shown as mean ± SEM and are the average of 2 determinations.

Table 1b . Caffeine content in 35 foods and beverages measured by HPLC.
Caffeine content was measured in the different foods by HPLC as described in Materials and Methods.Results are shown as mean ± SEM and are the average of 2 determinations.